[go: up one dir, main page]

WO2021055467A1 - Orally administrable nano-medicine for viral diseases - Google Patents

Orally administrable nano-medicine for viral diseases Download PDF

Info

Publication number
WO2021055467A1
WO2021055467A1 PCT/US2020/051061 US2020051061W WO2021055467A1 WO 2021055467 A1 WO2021055467 A1 WO 2021055467A1 US 2020051061 W US2020051061 W US 2020051061W WO 2021055467 A1 WO2021055467 A1 WO 2021055467A1
Authority
WO
WIPO (PCT)
Prior art keywords
ivm
nanoparticle
cells
nps
ivermectin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2020/051061
Other languages
French (fr)
Inventor
Shanta Dhar
Bapurao SURNAR
Dushyanth JAYAWEERA
Sylvia Daunert
Sapna DEO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Miami
Original Assignee
University of Miami
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Miami filed Critical University of Miami
Publication of WO2021055467A1 publication Critical patent/WO2021055467A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • C07K17/08—Peptides being immobilised on, or in, an organic carrier the carrier being a synthetic polymer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This disclosure relates generally to orally administrable nanoparticle for treating and preventing viral infection, specifically ZIKA and coronavirus infections, in particular humans with COVID 19.
  • the disclosure provides for a controlled release polymeric nanoparticle comprising a maleimide functional group on the nanoparticle surface, and a hydrophobic drug inside the nanoparticle.
  • Methods for using the orally administrable nanoparticle are also provided for treating and preventing viral disease.
  • ZIKV Zika virus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 viral strain is highly transmittable and infects respiratory tissue via the SARS-CoV-2 spike protein targeting the angiotensin-converting enzyme. SARS-CoV-2 infection causes flu-like symptoms as well as more severe respiratory issues and death by respiratory failure.
  • biodegradable nanoparticles provide a steady delivery of ivermectin, in order to maintain an appropriate level in the body and following oral administration can cross the intestinal epithelial barrier and enter the blood stream.
  • IVM delivery via the and because IVM is delivered in a synthetic nanoparticle IVM accumulates at safe levels in the blood has the ability to target non-structural 1 protein of ZIKV.
  • the disclosed ivermectin-loaded, orally administrable, biodegradable nanoparticle can also be used to treat coronavirus, acting through the inhibition of the SARS-CoV-2 spike protein and targeting of the angiotensin-converting enzyme.
  • the nanoparticle delivered ivermectin inhibits nuclear transport activities mediated through proteins such as the importin a/b1 heterodimer.
  • the disclosed ivermectin packaged nanoparticle serves as a less toxic, more potent oral therapeutic, that decreases viral entry into cells and reduce overall viral load, both of which are keys to lowering disease transmission rates.
  • This invention provides reagents, pharmaceutical formulations, and methods for treating or preventing coronavirus infection in humans, and particularly COVID-19 infection.
  • a controlled release polymeric nanoparticle comprising a maleimide functional group on the nanoparticle surface, and a hydrophobic drug such as ivermectin inside the nanoparticle is disclosed.
  • a controlled release targeted polymeric nanoparticle comprising a FcRn binding domain that binds to a target cell or tissue; a hydrophobic drug encapsulated in the nanoparticle; and a targeting ligand with -SH functionality which can react at the nanoparticle surface with the maleimide functional group.
  • the nanoparticle described herein is a dry formulation comprising trehalose and/or sucrose. Further, the nanoparticle is a frozen formulation of nanoparticles comprising trehalose and/or sucrose. In the preferred embodiment the nanoparticle contains the hydrophobic drug ivermectin.
  • the polymeric nanoparticle comprises poly(lactide-co- glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymer that further comprises a poly(lactic-co-glycolic acid) (PLGA) core.
  • the FcRn binding domain targets tissue of the gastrointestinal tract.
  • RNA virus is Zika virus that is targeted with a nanoparticle that encapsulates ivermectin.
  • the nanoparticle comprises poly(lactide- co-glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymer.
  • the FcRn binding domain targets tissue of the gastrointestinal tract following oral administration of the nanoparticle.
  • the ivermectin is released at a therapeutic dose over a sustained period of time.
  • the method can be used to treat an individual who is or has been infected with a MERS virus, a Dengue virus, a hepatitis virus, a West nile fever virus, or an Ebolavirus.
  • RNA virus comprising administering the ivermectin containing nanoparticle to an individual is or has been infected with the SARS- COV-2 virus that results in a COVID-19 infection.
  • the FcRn binding domain targets tissue of the respiratory epithelia and the nanoparticle targets ACE2-expressing cells in the lungs.
  • Figure 1 (A) FcRn binds to Fc-lvermectin-NPs in an acidic environment, NPs transcytose across the epithelial-cell barrier and gets released at physiological pH of blood. (B) Synthetic strategy of PLGA-b-PEG-Mal. (C) GPC traces of the polymers. (D) Synthesis of IVM loaded NPs using nanoprecipitation. (E) Incorporation of targeting antibody Fc on the NPs. [00018]
  • Figure 2 (A) Characterization of IVM-loaded NPs by DLS and percent loading and encapsulation efficiency by HPLC.
  • B Characterization of targeted Fc-conjugated NPs by DLS and determination of Fc conjugation efficiency by the bicinchoninic acid assay (BCA).
  • Figure 3 (A) A cartoon of transcytosis of Fc-NP across the trans-epithelial barrier derived from Caco-2 cells. (B) Formation of tight junction was confirmed by measuring the trans- epithelial electrical resistance (TEER) with a Millicell-ERS (Millipore) and intact TEER after addition of NPs. (C) Expression of tight junction protein zonula occludens-1 (ZO-1). Quantification of NPs in the apical, basolateral sides of the barrier (D) in the absence and (E) in the presence of external Fc.
  • TEER trans- epithelial electrical resistance
  • Millicell-ERS Millicell-ERS
  • ZO-1 tight junction protein zonula occludens-1
  • Figure 5 (A) Biodistribution of IVM, NT-OH-IVM-NP, and T-Fc-IVM-NP in intestine and blood of Balb/c female mice after 24 h of post administration by oral gavage. (B) FcRn expression level in mice intestinal tissue by western blotting. (C) Cytokine expression in the blood plasma of the IVM or its NP-treated mice. (D) H & E staining of major tissue after treatment with saline or T-Fc-IVM-NP.
  • FIG. 1 A) Release of IVM from NT-Mal-IVM-NPs at pH 7.4 and 6.5 at 37 °C.
  • B Cellular toxicity of ivermectin and ivermectin-loaded NPs by performing mitochondrial respiration profiles of Caco-2 cells in presence targeted and non-targeted NPs by using Seahorse analyzer and MitoStress assay. Oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-p- trifluoromethoxyphenylhydrazone, an ionophore; Rotenone, an inhibitor of mitochondrial complex I; and Antimycin A, an inhibitor of mitochondrial complex III.
  • C Comparison of maximal respiration, basal respiration, ATP production, and coupling efficiency from the Mitostress assay mentioned in B.
  • Figure 7 NS1 expression level in HEK293T cells after treatment with NPs by (A) Western blotting and (B) immunofluorescence. Scale bar: 10 pm.
  • Figure 8 Temperature-dependent stability of (A) NT-Mal-NP and (B) NT-Mal-IVM- NP by analyzing the diameter and zeta potential over the course of 1 month.
  • C Stability of NT- Mal-IVM-NP alone and with cryoprotectants over the course of three 1 h freeze-thaw cycles (freezing at -80 °C).
  • D Stability of NT-Mal-IVM-NP stored at -80 °C both alone and with cryoprotectants, measured at 15 timepoints over the course of 180 days.
  • E Stability of NT-Mal- IVM-NP after freeze drying at -50 °C and 2 Pa and reconstitution in nanopure water.
  • F Comparison of inhibition of NS1 expression in HEK293T cells after treatment with freshly prepared NPs, dried powder, and the NPs stored at -80 °C for 180 days.
  • Figure 9 (A) Formation of placental barrier-like tight junction in JEG-3 cells as confirmed by measuring the TEER and intact TEER after addition of NPs. (B) Quantification of NPs in the apical side, inside the cells, and the basolateral sides of the barrier. (C) Expression of ZO-1by immunofluorescence. (D) Cellular toxicity of ivermectin and the NPs by performing mitochondrial respiration profiles of JEG-3 cells using MitoStress assay.
  • Oligomycin ATP synthase inhibitor
  • FCCP-carbonyl cyanide-pthfluoromethoxyphenylhydrazone an ionophore
  • Rotenone an inhibitor of mitochondrial complex I
  • Antimycin A an inhibitor of mitochondrial complex III.
  • the cells were treated with the articles for 24 h at a concentration of 10 mM with respect to ivermectin.
  • Figure 10 (A) 1 H NMR and (B) 13C NMR of MAL-NHS in CDCI3.
  • Figure 11 LC-MS-ESI of MAL-NHS.
  • Figure 12 (A) 1 H NMR and (B) 13C NMR of PLGA-b-PEG-NH2 in CDCI 3 .
  • Figure 13 (A) 1 H NMR and (B) 13C NMR of PLGA-b-PEG-MAL in CDCI 3 .
  • Figure 14 DLS histograms of (A) Mal-NP, (B) Mal-IVM10-NP, (C) Mal-IVM20-NP, (D) Mal-IVM30-NP, (E) Mal-IVM40-NP and (F) Mal-IVM50-NP in nanopure water at 37° C.
  • Figure 15 Zeta potential (mV) of (A) Mal-NP, (B) Mal-IVM10-NP, (C) Mal-IVM20- NP, (D) Mal-IVM30-NP, (E) Mal-IVM40-NP and (F) Mal-IVM50-NP in nanopure water at 37° C.
  • Figure 16 TEM images of (A) NT-Mal-NP and (B) NT-Mal-IVM-NP stained with 4% of uranyl acetate.
  • Figure 17 (A) DLS histograms of (A) NT-Mal-NP, (B) T-Fc-NP, (C) NT-Mal-IVM-NP, and (D) T-Fc-IVM-NP in nanopure water at 37° C.
  • Figure 18 Zeta potential (mV) of (A) NT-Mal-NP, (B) T-Fc-NP, (C) NT-Mal-IVM-NP, and (D) T-Fc-IVM-NP in nanopure water at 37° C.
  • Figure 19 (A) Diameters, (B) Zeta potentials of NT-Mal-QD-NP and T-Fc-QD-NP. (C) Fc conjugation efficiency of targeted NPs by the bicinchoninic acid assay (BCA). (D) TEM images of T-Fc-QD-NP (unstained).
  • Figure 20 Quantification of QD (Cd) loaded NPs in the (A) apical and (B) basolateral sides of the endothelial cell barrier.
  • ALT Alanine aminotransferase
  • AST Aspartate Aminotransferase
  • Figure 23 In vitro efficacy of (A) ivermectin, (B) NT-OH-IVM-NP, (C) NT-Mal-IVM- NP, and (D) T-Fc-IVM-NP in Caco-2 cells by the MTT assay. (E) IC50 values of the articles in the Caco-2 cells after treatment for 72 h.
  • Figure 24 Comparison of FcRn expression level in Caco-2, HEK293, and JEG-3 cells by western blotting.
  • Figure 25 Morphological comparison of NT-Mal-IVM-NP and NT-Mal-IVM-NP with sucrose after 180 days by TEM.
  • Figure 26 A) Targeted Fc-lvermectin-NPs in the acidic gut lumen bind to FcRn receptors, allowing NPs to transcytose across the epithelial cell barrier and release at the physiological pH of blood.
  • IVM delivered via T-Fc-IVM-NPs shows the ability to (1) decrease ACE2 receptor levels, (2) decrease SARS-CoV-2 spike protein levels, and (3) decrease levels of the nuclear transport proteins Importin a and b1 , which leads to (4) an increase in the antiviral activity of infected cells.
  • Figure 27 (A) Western blot showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 2 h or 4 h at a concentration of 10 mM with respect to ivermectin, the media was changed, and further incubated for 22 h or 20 h.
  • Immunofluorescence staining showing expression of (B) ACE2 and spike (C) in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 pM with respect to IVM.
  • Figure 28 (A) Western blot showing expression of ACE2 in A549 adenocarcinomic alveolar basal epithelial cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 24 h at a concentration of 10 pM with respect to IVM. (B) Western blot showing dose-dependent decrease in ACE2 expression after treatment with varying concentrations of T-Fc-IVM-NPs with respect to IVM in A549 cells.
  • FIG. 29 (A) Schematic representation of ACE2 expression and pseudovirus infection in HEK293T cells. Efficacy of T-Fc-IVM-NP showing inhibition of both ACE2 and pseudovirus uptake under (B) therapeutic and (C) preventative settings as measured by plate reader. Confocal microscopy images revealing the changes in the expression of red-tagged ACE2 receptor on the cell membrane and mNeonGreen pseudovirus accumulation in the nucleus following the treatment of T-Fc-IVM-NP under (D) therapeutic and (E) preventative settings.
  • Figure 30 Confocal microscopy images revealing the changes in the expression of red-tagged ACE2 receptor on the cell membrane and mNeonGreen pseudovirus accumulation in the nucleus following the treatment of T-Fc-IVM-NP under (A) therapeutic and (B) preventative settings in primary small airway epithelial human HSAEC cells.
  • FIG. 31 (A) Schematic representation of the how IVM delivered through T-Fc- IVM-NP inhibits IMP a and b1.
  • Figure 32 (A) Cellular toxicity of IVM and IVM-loaded NPs as measured by mitochondrial respiration profiles in spike protein-expressing HEK293T cells using the Sea horse analyzer and MitoStress assay: oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, an ionophore; rotenone, an inhibitor of mitochondrial complex I; and antimycin A, an inhibitor of mitochondrial complex III.
  • B Basal respiration and ATP production from the MitoStress assay.
  • C Expression of cytokines IL-6, IL- 1b, and TNFa in the media of spike protein-expressing HEK293T cells treated with IVM, NT- IVM-NP, or T-Fc-IVM-NP.
  • Figure 33 Characterization of IVM NPs (A) for diameter by DLS, (B) zeta potential, (C) percent loading and %EE of IVM in NT-NPs, and (D) percent loading, %EE of IVM and %Fc conjugation in T-Fc-NPs.
  • Figure 34 Uptake kinetics of IVM, NT-IVM-NPs, or T-FC-IVM-NPs in HEK293T cells.
  • Figure 35 Cytotoxicity of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs in HEK293T cells as determined by the MTT assay.
  • Figure 36 Densitometric analyses of Western blots showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs.
  • Cells were treated with the articles for 2 h or 4h at a concentration of 10 mM with respect to ivermectin, the media was changed, and further incubated for 22 h or 20 h. Analyses were performed by ImageJ.
  • Figure 37 (A) Western blot showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IV, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 24h at a concentration of 10 pm with respect to ivermectin. (B) Densitometric analyses of Western blots showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expression spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs.
  • Figure 38 Western blot showing increased expression of ACE2 HEK293T cells upon treatment with Ang II at a varied concentration.
  • Figure 39 (A) Western blot showing expression in A549 adenocarcinomic alveolar basal epithelial cells transfected with plasmid expressing spike protein with and without treatment of IV, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4h at a concentration of 10 pm with respect to IVM. (B) Densitometric analyses of Western blots showing dose-dependent decrease in ACE2 expression after treatment with varying concentrations of T-Fc-IVM-NPs with respect to IVM in A549 cells.
  • Figure 40 Effects of IVM and IVM-loaded NPs on mitochondrial complex IV and complex V activity in spike protein expressing HEK293T cells.
  • Figure 41 (A) Variation of hydrodynamic diameter of nano-ivermectin and (B) TEMs of nano-ivermectin obtained using the ultrasound-assisted reprecipitation method (aqueous medium) and (C) (acidic conditions of acetate buffer pH 5).
  • Figure 42 Change of (A) diameter and (B) zeta potential with volume of ethanolic ivermectin during the time growth of nanomaterials. Change of (C) diameter and (D) zeta potential with the pH of the suspension before and after the addition of a buffer.
  • Figure 43 (A) Absorbance spectra of bulk ivermectin and two nano-ivermectin suspensions aged for 24 hours, and (B) bulk ivermectin and nano-ivermectin suspensions aged for 24, 48, and 72 hours. [00060] Figure 44: Release profile of nano-ivermectin in PBS buffer pH 7.4.
  • Figure 45 Schematic of oral delivery of nanoparticles.
  • Figure 46 Schematic of orally deliverable clinically approved anti-viral drug loaded nanoparticles.
  • This disclosure relates generally to orally administrable nanoparticle for treating and preventing viral infection, specifically ZIKA and coronavirus infections, in particular humans with COVID 19. Methods for using the orally administrable nanoparticle are also provided for treating and preventing viral disease
  • the term "about” is understood as within a range of normal tolerance in the art, for example, within two standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,
  • nanoparticle refers to a particle of matter that is between 1 and 100 nanometers (nm) in diameter.
  • nanoparticle refers to a particle of matter that is between 1 and 100 nanometers (nm) in diameter.
  • Nanoparticles occur widely in nature and are objects of study in many sciences such as chemistry, physics, geology and biology. Nanoparticles can also be synthesized and the production of nanoparticles with specific properties is an important branch of nanotechnology.
  • Nanoparticles can be part of nanotechnology-based delivery systems and are capable of transporting their contents across cellular membranes to deliver a specific message for the execution of a biological activity or function. Further, nanovesicles possess unique flow properties that endow them with an amended bioavailability. Nanovesicles have gained a particular importance due to their ability to enhance permeation rates of cargos such as drugs, through resistant biological membranes, mainly the skin barrier.
  • vesicle/cargo nanoscale system contributes to a major improvement of several pharmacokinetic properties of the cargo, including and not limited to, the solubility properties, controlled release, and milieu sensitivity (pH and type of medium).
  • solubility properties including and not limited to, the solubility properties, controlled release, and milieu sensitivity (pH and type of medium).
  • milieu sensitivity pH and type of medium.
  • maleimide-mediated methodologies are widely used in bioconjugation.
  • the name is a contraction of maleic acid and imide, the - C(0)NHC(0)- functional group.
  • Maleimides functional groups can be used in the preparation of targeted therapeutics, assemblies for studying proteins in their biological context, protein-based microarrays, or proteins immobilization.
  • Maleimides can be used in targeted drug therapies to bind and help deliver compounds in the body.
  • maleimides also describes a class of derivatives of the parent maleimide where the NH group is replaced with alkyl or aryl groups and that the substituent can comprise a wide number of molecules.
  • a controlled release targeted polymeric nanoparticle comprising: a FcRn binding domain that binds to a target cell or tissue; a hydrophobic drug encapsulated in the nanoparticle; and a targeting ligand with -SH functionality which can react at the nanoparticle surface with the maleimide functional group.
  • nanovesicle is synonymous with nanoparticle.
  • nanovesicles with the surprising technical effect of being composed solely of drug molecules, as nanodrugs, in a novel configuration where the drug molecules are self-carriers. This system introduces a novel area different from the norm wherein the nanovesicles are typically composed of phospholipid molecules.
  • the nanoparticles, serving as nanovesicles, are assemblies of drugs, thus, allow the delivery of an enhanced number of drug therapeutics without the need of a carrier or vehicle avoiding the problems related to the low loading capacity and unknown metabolism or degradation of the carrier in the body.
  • the nanodrugs disclosed herein can be efficiently implemented in aqueous medium in absence of any toxic organic solvent, which is highly significant for biological applications.
  • the nanovesicles, as described herein are composed of a potent antiviral drug in a nanoscale platform that can be widely applied to target various flaviviruses, and treat life-threatening viral infections.
  • the nanovesicles are composed of antiviral drug molecules with amphiphilic structures and cumulative hydrophobic property.
  • amphiphilic property of the drug is beneficial for topical deliveries, and eliminates the need for the use of penetration enhancers such as surfactants that are added to the nanovesicles suspensions.
  • the nanoparticle is a dry formulation comprising trehalose or sucrose.
  • the nanoparticle is a frozen formulation of nanoparticles comprising trehalose or a frozen formulation of nanoparticles comprising sucrose.
  • the polymeric nanoparticle comprises poly(lactide-co-glycolide)-b- polyethyleneglycol (PLGA-b-PEG) block copolymer that can further comprise a poly(lactic-co- glycolic acid) (PLGA) core.
  • PLGA poly(lactic-co- glycolic acid)
  • nanoparticles are advantageous as they can target specific tissue.
  • the FcRn binding domain targets tissue of the gastrointestinal tract.
  • the cargo of nanoparticles can have unknown effects on developing fetuses it is critical that some drug or antiviral containing nanoparticles not cross the placental barrier. Consistent with this, in one aspect of the current disclosure the nanoparticle described herein does not cross the placental barrier.
  • nanovesicles described herein are prepared from ivermectin drug for its highly efficient antiviral activity against flaviviruses, including but not limited to Yellow Fever, Dengue, and Zika Viruses. Also, disclosed is the use of nanovesicles containing ivermectin for use in treating COVD-19.
  • ivermectin for use in treating COVD-19.
  • One of skill in the art will further understand that the disclosed nanovesicles can be adapted or even used as disclosed for virus with similar origins.
  • the nanovesicle disclosed herein includes ivermectin within the nanoparticle.
  • Ivermectin inhibits viral protein replication, more specifically in relation to the inhibition of importin a/b that is included in several nuclear transport processes. Ivermectin use, historically has been limited as the potent action of the drug is only observed at high concentrations of micromolar range, resulting in toxic effects (i.e., cell toxicity).
  • ivermectin is part of a nanodrug overcomes this limitation and has the surprising technical effect lowering the effective dosage of ivermectin drug required to be effective, consequently avoiding toxicity and manifestation of side effects.
  • the innovative nanoivermectin-based formulation described herein drastically increases the therapeutic window, and can eventually eliminate the necessity for a frequent dosing of the drug.
  • the formulation of self-assembled nano ivermectin addresses the issue ivermectin solubility. Overcoming the "solubilizing" ivermectin issue is a significant technical effect, allowing use of the drug to treat viral infections.
  • RNA virus Zika virus (ZIKV).
  • the ZIKV causes microcephaly as well as a spectrum of neurologic problems including seizures in newborn babies and Guillain-Barre syndrome in adults.
  • the ultimate scale and impact of ZIKV remain to be determined as the severe abnormalities recognized at birth only represent the tip of the iceberg.
  • FDA Food and Drug Administration
  • This work identified more than 20 agents that decreased ZIKV infection in HuH-7 cells.
  • the most potent were ivermectin (IVM), mycophenolic acid (MPA), and daptomycin.
  • the current disclosure describes a new formulation of ivermectin as well as use of the nanoparticle for treating ZIKV.
  • the disclosed nanoparticle may also be used to treat related diseases in which ivermectin is effective, but its use may be limited by toxicity issues when administered in a non-nanoparticle formulation.
  • Targeted nanoparticles such as those disclosed herein can differentially deliver drugs to the site of interest in the body to improve the therapeutic index of drugs.
  • the Polymeric NPs of poly(lactide-co-glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymers designed and described herein are especially promising as drug delivery vehicles.
  • the core shell structure of polymeric NPs allows them to encapsulate and carry poorly water-soluble drugs such as IVM resulting long circulation half-life for the drug, release drugs at a sustained rate, and be functionalized with targeting ligands to modulate delivery method to target specific regions.
  • IVM poorly water-soluble drugs
  • the nanoparticles encapsulate ivermectin. And can comprise a poly(lactide-co-glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymer.
  • the FcRn binding domain targets tissue of the gastrointestinal tract.
  • the nanoparticles described herein can be are administered orally although one of skill in the art will understand the nanoparticles can be administered in alternative routes, including but not limited to intravenous.
  • the disclosed nanoparticle is further advantageous as the ivermectin payload is released at a therapeutic dose over a sustained period of time. Consistent with this, the nanoparticle can be loaded with alternative drugs, allowing for sustained release of alternative drugs, compounds, or therapeutics.
  • RNA virus in a further aspect of the disclosure is a method of treating an RNA virus, comprising administering the nanoparticle of any one of claims 1-11 to an individual infected with a single strand RNA virus.
  • the individual is or has been infected with the SARS- COV-2 virus that has presently or previously resulted in a COVID-19 infection.
  • the method disclosed herein can also be used as a prophylactic measure.
  • the FcRn binding domain targets tissue of the respiratory epithelia and can the nanoparticle can target ACE2- expressing cells.
  • COVID-19 a disease caused by a novel coronavirus strain SARS-CoV-2 is highly transmittable and infects respiratory tissue, and can cause flu-like symptoms as well as more severe respiratory issues and death by respiratory failure.
  • SARS-CoV-2 virus surface spike protein interacts with angiotensin-converting enzyme 2 (ACE2) receptors in the lung and facilitates the entry of virus into host cells, and much of the tissue damage done is actually a product of the immune response and resulting inflammation.
  • ACE2 angiotensin-converting enzyme 2
  • the disclosed IVM nanoformulation allows the therapeutic to be gradually released into the blood-stream, which maintains its level in the blood about the minimum effective therapeutic dose while keeping it below the maximum tolerated dose.
  • the NP was constructed using poly(lactide-co-glycolide)-b-poly(ethylene glycol)-maleimide (PLGA-b-PEG-MAL) polymer, and was targeted to the gut epithelial barrier for crossover into the bloodstream by covalent attachment of an Fc immunoglobulin fragment, which binds to receptors on epithelial cells in the gut lumen (Figure 26).
  • ACE2 which is present in high quantities in respiratory epithelia, allows for viral entry and infection of the lung and alveolar cells.
  • ACE2-expressing cells in the lung are involved in key processes such as blood pressure regulation and interferon production, and SARS-CoV-2 binding to this receptor can impede on those processes, making it an important target to reduce viral infection.
  • nanoparticle delivered IVM effectively decreases levels of viral spike protein as well as cellular levels of ACE2.
  • the disclosed IVM-loaded nanoparticles are engineered to contain a bound Fc immunoglobulin anti-body fragment to target FcRn receptors on gut epithelial cells, which will allow for transcytosis of orally delivered nanoparticles into the bloodstream and potential accumulation at respiratory epithelial cells, which are particularly affected by SARS-CoV-2 ( Figure 26).
  • the disclosed nanoparticle can be loaded with alternative therapeutics, including but not limited to, alternative drugs, chemicals, or antibodies.
  • the NPs disclosed herein can be further used to treat a wide range of viral infections.
  • the ivermectin nanoparticle can be used to treat an individual is or has been infected with a MERS virus.
  • the ivermectin nanoparticle can be used to treat an individual who is or has been infected with a Dengue virus.
  • the ivermectin nanoparticle can be used to treat an individual who is or has been infected with a hepatitis virus.
  • the ivermectin nanoparticle can be used to treat individual who is or has been infected with a West nile fever virus or an Ebolavirus.
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim.
  • any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features.
  • Polyethylene glycol (H2N-PEG2000-NH2) was procured from JenKem Technology, Bachem. Deuterated solvents, CDCI3 and DMSO-d6 were purchased from Cambridge Isotope Laboratories Inc. Regenerative cellulose membrane Amicon Ultra centrifugal 100 kDa filters were purchased from Merck Millipore Ltd. Strata C18-T columns (catalog number 8B-S004- EAK) were purchased from Phenomenex. Copper grids for transmission electron microscopy (TEM) were purchased from Electron Microscopy Sciences. Qdot. 705 ITKTM Amino (PEG) Quantum Dots (catalog number Q21561MP) and ProLong.
  • Gold anti-fade reagent with 4', 6- diamidino-2-phenylindole (DAPI) were purchased from Life Technologies.
  • Trans-well system polycarbonate (0.4-pm pore size, 12-well plates) were purchased from Corning, Lowell, MA.
  • the tight junction antibody ZO-1 (catalog number ab59720) was purchased from Abeam.
  • Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (catalog number A11008) was procured from Invitrogen, ThermoFisher Scientific.
  • Phosphate buffered saline (1X PBS) was purchased from Gibco (reference number 10010-023).
  • Goat serum was obtained from Sigma Aldrich (catalog number G9023).
  • Glutamine, penicillin/streptomycin trypsin-EDTA solution, HEPES buffer (1 M in water), and sodium pyruvate were procured from Sigma Life Sciences.
  • Dulbecco's Modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies.
  • Mouse monoclonal IgG, Fc-Rn (A-6) (Catalog number SC-393064) was purchased from Santa Cruz Biotechnology.
  • Zika virus NS1 antibody (EA88) catalog number. MA5-24583) was purchased from Invitrogen.
  • Flag-tagged Zika NS1 plasmid (Catalog number 79641) was procured from Addgene.
  • Native human IgG FC fragment protein (catalog number Ab90285) was procured from Abeam.
  • Ammonium persulfate (Catalog number 161-0180), tris/glycine/SDS buffer (Catalog number 161-0732), SDS-PAGE gel preparation kit TGX stain- freeTM fast castTM acrylamine 10% (Catalog number 161-0182), and ClarityTM western ECL substrate (Catalog number 170-5060) were purchased from Bio-Rad Inc.
  • Beta-actin antibody (Catalog number ab8226), nitrocellulose membrane (catalog number 88018), and tween-20 was purchased from Fisher Bioreagents.
  • High-performance liquid chromatography (HPLC) analyses were made on an Agilent 1200 series instrument equipped with a multi-wavelength UV-visible and a fluorescence detector. Cells were counted using Countess Automated Cell Counter procured from Invitrogen. TEM images were acquired using a JEOL JEM-1400 equipped with a Gatan Orius SC 200D CCD digital camera with a magnification of 80K. Inductively coupled plasma mass spectrometry (ICP-MS) studies were performed on an Agilent 7900 ICP-MS instrument. Mitochondrial bioenergetics assays were performed on XFe96 Extracellular Flux Analyzer (Agilent Seahorse Biosciences).
  • TEER measurements were performed on a Millicell.
  • ERS-2 Voltohmmeter Instrument (Catalog number MERS00002) purchased from Millipore. Confocal microscopy images were obtained using an Olympus FluoView FV3000. Mouse organ images were captured using a Zeiss Stemi 2000-CS stereoscope fitted with a CL-1500 ECO SteREO light source.
  • Human epithelial colorectal adenocarcinoma cells (Caco-2) cells were procured from ATCC. These cells were grown in Eagle's Minimum Essential Medium (DMEM) along with 20% fetal bovine serum. Cell cultures were maintained in a humidified cell culture incubator at 37°C and with 5% CO2. Transfectable derivative of human embryonic kidney 293 (HEK293T) cells were procured from ATCC. Cells were grown in Eagle's Minimum Essential Medium (DMEM) along with 10% fetal bovine serum. Cell cultures were maintained in a humidified cell culture incubator at 37°C and with 5% CO2. Human placental choriocarcinoma (JEG-3) cells were procured from ATCC. Cells were grown in Dulbecco Modified Eagle Medium (DMEM) along with 10% fetal bovine serum. Cell cultures were maintained in a humidified cell culture incubator at 37°C and with 5% C02.
  • DMEM Eagle's Minimum Essential Medium
  • JEG-3 Human placental chorio
  • 6-amino hexanoic acid (1 g, 7.62 mmol) and maleic anhydride (0.78 g, 80 mmol) were dissolved in 7.5 mL of acetic acid.
  • the reaction mixture was stirred for 1 h at room temperature until a white color product precipitated.
  • Formed Mal- acid (0.8 g, 3.8 mmol) and Nhydroxysuccinimide (0.48 g, 4.1 mmol) were dissolved in 5 mL of dry DMF. The reaction mixture was cooled to 0°C with stirring.
  • NH2-PEG-NH2 (1.0 g, 0.5 mmol), PLGA-COOH (0.825 g, 0.16 mmol), and DMAP (0.022 g, 0.2 mmol) were dissolved in dry CH2CI2 (12mL). This reaction mixture was cooled to 0 °C along with stirring. DCC (0.036 g, 0.17 mmol) was dissolved in CH2CI2 (1 mL) and added drop wise to the reaction vessel. The mixture was then warmed to room temperature and stirred overnight. Later on, precipitated DCU was filtered out and the resulting mixture was in a 1:1 mixture of cold diethyl ether: methanol (50 mL). This was repeated 5 times.
  • PLGA-b-PEG-Mal 5 mg mL-1 and ivermectin (1 mg mL-1) was made in 1 mL of DMF. The solution was added dropwise to 10 mL of Dl water with constant stirring (900 RPM) at room temperature and stirred for 2h. Nanoparticles (NPs) were washed 3 times with nanopure water with amicon ultracentrifugation filtration membranes with a molecular weight cutoff of 100 kDa (2800 rpm, 4°C). Formed NPs were suspended in water and stored at 4°C.
  • NP size (diameter, nm), PDI, and surface charge (zeta potential, mV) were obtained from three independent measurements.
  • NP solution was diluted with water, and 4% uranyl acetate added into the solution to stain the NPs.
  • the NP mixture was vortexed and dropped into a copper grid and dried overnight at room temperature using a JEOL JEM-1400 equipped with a Gatan Orius SC 200D CCD digital camera.
  • Nanoparticles (5 mg/mL with respect to total polymer) were diluted 100 times using nanopure water.
  • the NP solution (1 mL) was mixed with a 4% solution of uranyl acetate solution (5 pL) and vortexed.
  • the solution was filtered with a 0.45-micron filter and ⁇ 20 pL was dropped on a dark side of the copper grid and allowed to dry for 24 hours in desiccator at room temperature.
  • TEM images were recorded using JEOL JEM-1400 instrument.
  • cytotoxicity of ivermectin, NT-OH-IVM-NP, and T-Fc-IVMNP was tested in Caco- 2 cells using an MTT assay.
  • Cells were plated (3000 cells/well) in a 96-well plate and allowed to grow overnight. Media was changed and increasing concentrations of each article was added. Media was aspirated and fresh media added, and cells further incubated for an additional 48 h, after which 20 pL/well MTT was added (5 mg/mL Stock in PBS) and incubated for 5 h in order for MTT to be reduced to purple formazan. Media was removed and cells lysed with 100 pL of DMSO.
  • IC50 is the concentration of agent that reduces cell growth by 50% under experimental conditions and is the average from at least three independent measurements that were reproducible and statistically significant.
  • oligomycin (10 mM), FCCP (10 mM) and antimycin-A/rotenone mixture (10 mM each) were made in seahorse media.
  • Port A was filled with 20 mI_ of oligomycin, port B with 22 mI_ of FCCP and port C with 25 mI_ of antimycin A/rotenone to a final concentration of 0.1 mM in each well.
  • the cartridge was calibrated for pH and 02. After calibration, the experiment plate was run where 3 measurements were recorded for basal OCR and after addition of each reagent. The media was aspirated and 20 mI_ of RIPA buffer was added to each well and incubated for 10 mins at 37°C. Further BCA assays were performed to obtain protein normalized OCR values.
  • Caco-2 cells were plated in trans-well plate with a density of 50,000 cells/well on the apical side in 500 mI_ of DMEM (with 20% FBS) media. On the basal side, 1 mL of fresh media was added, and cells were grown up to 9 days. Before the addition of compounds, the integrity of the monolayer was monitored by TEER (transepithelial electrical resistance) using Epithelial Volt-ohmmeter. Media was replenished every two days. On the ninth day, 2 pg/mL of IgG-Fc (Fc) fragment was added in order to find out the targeting ability of the Fc-targeting nanoparticles.
  • TEER epithelial electrical resistance
  • ivermectin, NT-OH-IVM-NP, and T-Fc-IVM-NP were added to the cells with a concentration of 20 pg/mL, with respect to ivermectin, and were incubated for 12 h.
  • Apical and basal media were collected in eppendorf tubes and dissolved in 2 mL of acetonitrile.
  • 10 pg/mL of ivermectin was added to the collected media. This mixture was sonicated for 20 mins followed by centrifugation at 5000 rpm for 10 mins. From the precipitated debris, supernatant was gently collected.
  • Strata C18-T columns were activated by passing 1 mL of methanol and water through the filter in sequence.
  • the collected supernatant was passed through the activated column in order to get rid of remaining debris and impurities.
  • the column was washed with 1-2 mL of 5% methanol in order to remove the impurities.
  • NT-OH-IVM-NP and T-Fc-IVM-NP added with concentration of 20 pg/mL with respect to ivermectin and incubated for 12 h.
  • the cells were washed with PBS (1X) 3 times and fixed with 4% paraformaldehyde for 1 hour at 37 °C. After performing 3 washings, cells were permeabilized using 0.1% Triton-X100 for 10 min at 37 °C. The cells were washed with 1X PBS 3 times and blocked with 1% goat serum in 1X PBS for 12 h.
  • mice Female balb/c albino mice were used to understand the ivermectin and T-Fc-IVM- NPs distribution and toxicity after oral administration in vivo.
  • NT-OH-IVM-NP served as a non- targeted control.
  • Animals were divided into four groups, of three animals: Group 1- Saline, Group 2- Ivermectin, Group 3- NT-OH-IVM-NP, and Group 4- T-Fc-IVM-NP. Before oral dosage, animals were fasted for 8 h. Animals in each group received saline treatment, ivermectin, targeted NPs, or nontargeted NPs via oral gavage.
  • the dose of nanoparticle was 40 mg/kg with respect to ivermectin weight. Animals were sacrificed after 24 h and organs harvested. Blood (-200 pl_) was collected in heparinized tubes via cardiac puncture. Perfusion was performed with 1X PBS for 10 min with a flow rate of 7 mL/min. Collected blood was centrifuged to collect blood plasma. Organs were weighed and homogenized using a dounce homogenizer and collected in 2 mL of acetonitrile.
  • ivermectin peak 20 pg/mL was added to the crushed tissues and to the blood plasma and the mixture sonicated for 20 mins followed by centrifugation at 5000 rpm for 10 mins and supernatant collected.
  • Strata C18-T columns were activated by passing 1 mL of methanol and water through the filter in sequence. Supernatant was passed through the activated column for purification and the column washed with 1-2 mL of 5% methanol in order to remove the impurities.
  • the membrane was incubated at 4 °C overnight for primary antibody (FcRn and b-actin) and washed 5 times with TBST buffer and incubated with appropriate secondary antibodies at room temperature for 1 h. Membranes were washed five times with TBST buffer and developed using ECL. Images were taken using a BioRad ChemiDocTM imaging system and bands quantified using ImageJ software.
  • Enzyme-linked immunosorbent assay (ELISA) to determine immunogenic effect
  • the levels of pro-inflammatory cytokines IL-1 b, IL-6, and TNF-a in plasma were determined using ELISA kits following the manufacturer’s protocol. Briefly, 100 pL/well of coating buffer with capture antibody was added to the 96-well ELISA plate and the plate sealed overnight at 4°C. Buffer was aspirated from the wells and cells washed 3 times with 300 pL/well of wash buffer. After the last wash, the plate was inverted and blotted on absorbent paper to remove any residual buffer. Wells were blocked with 200 pL/well of assay diluent and incubated at room temperature for 1 h. The diluent was aspirated from the wells and the wells washed.
  • the reagent was aspirated from wells, and substrate solution (100 mI_) was added to each well and the plate sealed and incubated for 30 minutes at room temperature in the dark. Stop solution was added to each well and the absorbance was recorded at 450 nm.
  • master reaction mix 80 mI_ of AST Assay Buffer, 2 mI_ of AST Enzyme Mix, 8 mI_ of AST Developer and 10 mI_ of AST Substrate
  • T i nitial Absorbance at initial time, T i nitial , was (A450)i nitiai , and at the end the final time point, T finai , was at (A450) finai .
  • the absorbance was measured at 450 nm at the initial time.
  • the AST activity of a sample was determined by the following equation:
  • ALT Assay Plasma was used to determine ALT activity. Using Cayman’s ALT Assay Kit Measurement of the ALT activity was carried out by monitoring NADH oxidation rate in a coupled reaction system employing lactate dehydrogenase (LDH). The oxidation of NADH to NAD+ is accompanied by a decrease in absorbance at 340 nm. Under circumstances in which the ALT activity is rate limiting, the rate decrease is directly proportional to the ALT activity in the sample. Substrate (150 pL), 20 pL of Cofactor, and 20 pL of sample were added to each well of a 96 well plate and incubated at 37°C for 15 min. The reaction was initiated by the addition of 20 pL of ALT initiator and absorbance recorded immediately at 340 nm once every minute for five minutes. The change in absorbance (DA340) per minute was determined using the following equation.
  • DA /min A340 (Time 2) - A340 (Time 1 )/ Time 2 (min) - Time 1 (min)
  • the reaction rate at 340 nm was determined using the NADH extinction coefficient of 4.11 mM 1 .
  • ALT and H aspartate aminotransferase
  • Agar plates (1.5%) containing 100 pg/mL ampicillin were prepared and streaked with Flag-tagged Zika NS1 and control plasmid. Plates with NS1 and control plasmid were incubated at 37°C and 30°C respectively for 16 h. Single colonies were inoculated in 5 mL of LB broth media containing 100 pg/mL ampicillin for primary culture and incubated at 37°C with shaking for 16 hours. Plasmid isolation using Midiprep kit
  • NS1 and control plasmids were isolated using Qiagen Midiprep plasmid isolation kit. Overnight grown bacterial culture was harvested by centrifuging at 4500 rpm for 20 minutes at 4°C. Supernatant was discarded and the bacterial pellet dissolved in 4 mL of buffer P1 and mixed. Buffer P2 (4 mL) was added and mixed thoroughly by vigorously inverting the tube 4-6 times followed by incubation at room temperature for 5 minutes. Bbuffer P3 (4 mL) was mixed thoroughly by vigorously inverting the tube 4-6 times and incubated on ice for 15 minutes. Tubes were centrifuged at 4500 rpm for 30 minutes at 4°C.
  • HEK293T cells (0.3 x 106) were seeded overnight in 6-well plates with 10% FBS containing DMEM medium. Cells were transfected with 2 pg of Flag-tagged Zika NS1 plasmid and an empty vector using turbofectin (turbofectin: DNA-3:1) in Opti-MEM medium. Six hours post-transfection, medium was removed and replaced with fresh medium and incubated for additional 6 h. Cells were treated with 10 mM concentration of ivermectin (IVM), T-Fc-IVM-NP and NT-OH-IVM-NP for 6 h, cells lysed and total cell lysates (60 pg) resolved on a 4-20% gradient gel.
  • IVM ivermectin
  • Proteins were transferred to PVDF membrane and probed with Anti-flag (1:1000) and b-actin (1:1000) antibody overnight at 4°C, washed with TBST and probed with HRP- conjugated anti-mouse secondary antibody (1:2000) for 1 h at room temperature.
  • Membranes were developed using SuperSignal west pico chemiluminescence substrate (Thermo Scientific).
  • HEK293T cells were plated on coverslips in a 12 well plate at a density of 20,000 cells/well in 1 mL of DMEM (with 10% FBS) media.
  • Cells were transfected with 2 pg of Flag- tagged Zika NS1 plasmid (addgene# 79641) and an empty vector using turbofectin (turbofectin: DNA-3:1) in Opti-MEM medium. After 6 h post transfection, medium was removed and replaced with fresh medium and incubated for additional 6 hours.
  • Cells were treated with ivermectin (IVM), T-Fc-IVM-NP, or NT-OH-IVM-NP at a concentration of 10 pM IVM for 6 h.
  • Cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at 37°C. Cells were permeabilized using 0.1% Triton-X100 for 10 min at 37°C, washed with 1X PBS 3 times and blocked with 1% goat serum in 1X PBS for 12 h. Cells were treated with the respective primary antibody (Anti-flag (1 :1000) in 1% goat serum containing 1X PBS for 12 h at 4°C in humidified chambers. Cells were washed three more times with 1 % goat serum containing 1X PBS, appropriate secondary antibodies (Alexa 488 conjugated anti-mouse antibody) added along with DAPI and incubated for 1 hour at room temperature.
  • Primary antibody Anti-flag (1 :1000
  • NPs were prepared for the temperature dependent stability using 5 mg/mL PLGA- PEG-Mal alone (for Mal-NP) or using 5 mg/mL PLGA-PEG-Mal polymer and 1 mg/mL ivermectin (for Mal-IVM-NP). Solutions were stirred for 2 hours, then filtered using Amicon filtration (100 MWCO) at 2800 RPM. Three 1 mL solutions of both Mal-NP and Mal-IVMNP were prepared, and after initial DLS measurements of size and zeta potential were taken, the solutions were stored at 4°C, room temperature, and 37°C.
  • Mal-IVM-NPs were prepared in a similar fashion as described above, but with starting concentrations as 10 mg/mL PLGA-PEG-Mal polymer and 2 mg/mL ivermectin.
  • NPs were mixed with the cryoprotectants sucrose and trehalose in NP:cryoprotectant ratios of 1:0.1 , 1 :0.5, 1 :1, and 1 :2.
  • Final volumes of each sample were 400 pL, and each contained the same amounts of nanoparticles.
  • DLS measurements (size and zeta potential) of the samples were taken prior to placing samples -80 °C and measurements of size and zeta potential captured every hour. This was repeated for 3 cycles, each of which had the NPs at -80°C for one hour.
  • NPs must be dried and made into powdered form in order to eventually be packed into capsules to serve as a viable antiviral treatment.
  • Mal-IVM-NPs were produced as described above, with 10 mg/mL PLGA-PEG-Mal polymer and 2 mg/mL ivermectin. NPs were dried, with and without the cryoprotectants, using low pressure and temperature. Sucrose and trehalose were added in 1:2 NP:cryoprotectant ratios, and a third sample contained NPs alone. NPs were dried at approximately -50 °C and 2 Pa using a VirTis Benchtop K Freeze Dryer. Powdered NPs were reconstituted in 1 mL nanopure water. Size and zeta potential measurements were taken, and data suggested that NPs dried in the presence of sucrose displayed the best results with slightly increased size.
  • JEG-3 cells were plated in transwell plate at a density of 50,000 cells/well on the apical side in 500 pL of DMEM (with 10% FBS) media. On the basal side, 1 mL of fresh media was added and cells grown for up to 9 days. Prior to the addition of compounds, the integrity of the monolayer was monitored by TEER (transepithelial electrical resistance) using Epithelial Volt-ohmmeter.
  • TEER epithelial electrical resistance
  • ivermectin, NT- OH-IVM-NP, or T-Fc-IVMNP were added to the cells at a concentration of 20 pg/mL with respect to ivermectin, and were incubated for 12 h.
  • Apical and basal media were collected in eppendorf tubes and dissolved in 2 mL of acetonitrile.
  • 10 pg/mL of ivermectin was added to the collected media, the mixture sonicated for 20 min followed by centrifugation at 5000 rpm for 10 min and the supernatant gently collected.
  • ZO-1 tight junction protein
  • Bacterial agar plates (1.5%) containing 100 pg/mL ampicillin were prepared and streaked with SARS-CoV-2 spike protein plasmid and incubated at 37 °C for 16 h. A single colony from the plate was inoculated in 5 mL of LB broth media containing 100 pg/mL ampicillin for primary culture. The culture was incubated at 37°C in an incubator shaker for 16 h. Spike protein plasmid was isolated using Qiagen Midiprep plasmid isolation kit. Bacterial culture grown overnight was harvested by centrifuging at 1900g for 20 min at 4 °C.
  • the supernatant was discarded and the bacterial pellet was dissolved in 4 mL of buffer P1 and mixed properly, buffer P2 added and tubes mixed thoroughly incubated at room temperature for 5 min.
  • Buffer P3 (4 mL) was mixed thoroughly by vigorously inverting the tube four to six times and incubated on ice for 15 min followed by centrifugation at 1900g for 30 min at 4 °C.
  • the Qiagen midi column was equilibrated with 4 mL of buffer QBT and bacterial supernatant was loaded onto the column, and the column allowed to empty by gravity flow.
  • the plasmid was eluted from the column by using QF buffer and isopropanol used to precipitate the plasmid.
  • the plasmid was dissolved in water and the purity and quantification of plasmid measured using Nanodrop.
  • HEK293T cells, HeLa cells, and A549 cells (1 A ⁇ 105) were seeded in 6-well plates in 10% FBS-containing DMEM medium and were incubated overnight.
  • Cells were transfected with 2 pg of SARS-CoV-2 spike protein plasmid using turbofectin (turbofectin: DNA-3:1) in Opti- MEM medium.
  • turbofectin turbofectin: DNA-3:1
  • Cells were treated with 10 mM concentration of IVM, NT-IVM-NP, and T-Fc-IVM-NP for 24 hours.
  • the articles in the same concentration were added to cells for 2, 4, and 6 h, followed by incubation of cells in normal media for up to 24 hours.
  • HEK293T cells (1 A ⁇ 105) were seeded in 6-well plates in 10% FBS-containing DMEM medium and incubated overnight. Cells were transfected with 2 pg of SARS-CoV-2 spike protein plasmid using the transfection reagent turbofectin (turbofectin: DNA-3:1) in Opti- MEM medium. Six hours post-transfection, media was removed and replaced with fresh media and incubated for additional 6 hours. IVM and its nanoformulations were added to cells at 10 pM concentrations with respect to IVM to cells for 2, 4, and 6 h, followed by incubation of cells in normal media up to 24 hours total.
  • turbofectin turbofectin: DNA-3:1
  • HEK293T cells, HeLa cells, and A549 cells were each plated on coverslips in separate 12-well plates at a density of 20,000 cells/well in 1 mL of DMEM (with 10% FBS) media.
  • Cells were transfected with 2 pg of SARS-CoV-2 spike protein plasmid and an empty vector using turbofectin (turbofectin: DNA-3:1) in Opti-MEM medium.
  • turbofectin turbofectin: DNA-3:1
  • Opti-MEM medium Six hours post transfection, medium was removed and replaced with fresh medium and incubated for additional 6 hours.
  • Cells were treated with IVM, NT-IVM-NP, or T-Fc-IVM-NP at a concentration of 10 pM with respect to IVM for 6 h.
  • HEK293T cells were seeded in 6 well plate and transfected with 2pg of plasmid using turbofectin.
  • Cell were treated with ivermectin, NT-IVM-NP and T-Fc-IVMNP for 4 h at a concentration of 10 mM, medium was removed, and cells were kept for additional 20 hours.
  • RNA was extracted by harvesting cells with trypsin and lysed with buffer RLT. Ethanol (70%,) was added to the cell lysate , lysates transferred to RNeasy mini spin columns and centrifuged for 1 minute at 8000 rpm.
  • Reverse transcription from each sample was carried out using 1 pg of RNA using iScript Reverse Transcription Supermix.
  • Real time PCR reaction was performed using SsoAdvanced Universal SYBR® Green Supermix in a 20 pL reaction.
  • Beta-actin was used as an internal control and data analyzed using the comparative Ct value and expressed as fold change 2 DD0T .
  • the forward and reverse primer sequence was
  • 5OCAGTACGCCATGTAACGGA3’ (SEQ ID NO: 1) and 5’CGTGGAGGAGCTCAAAGGAC 3’ (SEQ ID NO: 2) respectively for spike gene.
  • the primer for human ACE2 gene was purchased from Sino Biological Inc (catalogue number: HP100185).
  • the primer sequence for b-actin gene was: Forward 5’ GCATCCTCACCCTGAAGTAC 3’ (SEQ ID NO: 3) and reverse 5’GATAGCACAGCCTGGATAGC 3’ (SEQ ID NO: 4).
  • ACE2-Red reporter assay The Angiotensin Converting Enzyme 2 (ACE2)-Red reporter assay and Pseudo SARS-CoV-2 Green Reporter assay were performed with the goal of increasing ACE2 expression in HEK293T cells and A549 cells and observing the effects of IVM-loaded nanoparticle treatment on ACE2 expression and SARS-CoV-2 virus cell entry. These effects were measured through fluorescence imaging using two different methods of treatment: a preventive method, in which IVM and IVM nanoformulation treatment preceded pseudovirus infection in the cells, and a therapeutic method, in which IVM and IVM nanoformulation treatment followed pseudovirus infection in the cells.
  • the preventive method’s aim was to decrease ACE2 levels initially through IVM treatment so that the rate at which pseudoviruses entered cells would decrease.
  • the therapeutic method aimed to interrupt ACE2 and pseudovirus binding and show a lowering in pseudovirus infection presence after the treatment.
  • HEK293T cells were detached using a standard trypsinization protocol and then counted. Cells were prepared for plating in an 8-well live cell imaging chamber at 20,000 cells per well, using 100 pl_ of media for each well. These cells were maintained in a single test tube at 500,000 cells per ml_, ready to be mixed with the viral transduction reaction.
  • an ideal NP system for oral delivery of IVM needs to have pH stability, intestinal absorption, and epithelium crossing ability, and should also demonstrate high IVM loading capacity and controlled release of the drug. It is also critical that orally delivered NP transports the payload efficiently from the intestine to the blood stream.
  • the neonatal Fc receptor (FcRn) mediates immunoglobulin G (IgG) transport across the polarized epithelial barriers.
  • FcRn is expressed at a level that is closely similar to fetal expression in the apical region of epithelial cells in the small intestine and diffuse throughout the colon in adulthood.
  • FcRn binds to the Fc portion of IgG following a pH-driven pathway; acidic pH of ⁇ 6.5 promotes binding of Fc to FcRn and physiological pH of ⁇ 7.4 releases the Fc from the FcRn binding pocket (Figure 1 A).
  • Figure 1B a biodegradable PLGA polymer based platform
  • the linkers on the polymer are comparatively acid resistant to provide stability to the NPs under acidic pH in the stomach when administered via oral route (Figure 1B). All monomers and polymers were characterized by NMR spectroscopy ( Figures 10-13).
  • the polymers were also analyzed by gel permeation chromatography (GPC) demonstrating purity and monodisperse distribution (Figure 1C).
  • GPC gel permeation chromatography
  • Figure 1C monodisperse distribution
  • the morphology of IVM loaded NPs was determined by transmission electron microscopy (TEM) confirming spherical, homogeneous particle population ( Figure 16). Ivermectin concentrations in the NPs were quantified using high performance liquid chromatography (HPLC). As IVM feed was increased from 10% to 50%, the NP size increased from ⁇ 60 nm to 140 nm. The NPs gave a very stable surface charge of nearly around -25.0 mV. The NPs with feed of 10% to 30% showed PDI -0.21 which suggested the formation of monodisperse particles. The 40% and 50% showed higher PDI of -0.45 indicating formation of higher aggregates.
  • the NPs with NH2 upon interaction with Fc showed similar size (-60 nm) and zeta potential ( ⁇ -12 mV) as NTMal-NPs.
  • the Fc-SH attached NPs showed increased in size ( ⁇ 88 nm) and ( ⁇ 22 mV). This increase is due to the covalent attachment of the Fc-SH on the NT Mal-NPs.
  • NP transport ability across the Caco-2 monolayer were determined by quantifying IVM in the apical (AP) and basolateral (BL) side media using HPLC (Figure 3A). Caco-2 cells were plated in a transwell plate on the apical side. On the basolateral side, 1 mL of media was added and the cells were grown up to 9 days. Before the addition of NPs, the monolayer integrity was checked by measuring the trans-epithelial resistance (TEER) indicating TEER values of >800 W/cm2 on day 9 ( Figure 3B, top). Media was replenished once every two days.
  • TEER trans-epithelial resistance
  • T-Fc-IVM-NP or NT-OH-IVM-NP was added to the apical side of the barrier and incubated for 12 h.
  • NT-OH-NP constructed using PLGA-b-PEG-OH polymer was used as a non-targeted control since -Mai containing NPs can interact with biological thiols or other reactive groups.
  • Addition of NPs did not change the tight junction function of the epithelial barrier as evident from intact TEER ( Figure 3B, bottom) and the expression of tight junction protein zonula occludens-1 (ZO-1) ( Figure 3C). Media from apical and basolateral sites were collected and stored at room temperature.
  • T-Fc-IVM- NPs were found at a much higher concentration in the basolateral side compared to the non- targeted NT-OH-IVM-NPs ( Figure 3D).
  • mice were used to evaluate the distribution properties of T-Fc-NP after oral administration.
  • NT-OH-NP was used as a non-targeted control. Animals were divided in three groups, each group containing three animals. Group assignments were: Group 1-Saline, Group 2-NT-OH-QD-NP, and Group 3-T-Fc-QD-NP. Animals in each group received saline, targeted, or non-targeted NPs via oral gavage. The dose of NP was 50 mg/kg with respect to total polymer. After 24 h, around 300 pL of blood was collected in heparinized tubes via cardiac puncture. Collected blood was centrifuged to collect blood plasma.
  • T-Fc-IVM-NP The percentage IVM accumulation for T-Fc-IVM-NP was found to be around 12%, 9%, 5%, 5%, and 60% in duodenum, jejunum, ileum, colon, and blood, respectively ( Figures 5A, and 21) 24 h post administration via oral gavage. Analyses of the intestinal tissue samples by Western Blot indicated significant expression of FcRn thus confirming that the targeted NPs utilize these receptors to get associated with the intestinal tissue (Figure 5B). T-Fc-IVM-NPs did not have any effect on serum proinflammatory markers IL1 -b, IL-6, and TNF-a suggesting that these NPs do not induce immunogenicity (Figure 5C).
  • T- Fc-IVM-NP has the potential to cross the intestine epithelial barrier to reach the blood to attack Zika virus, whereas free ivermectin might be disrupting the barrier due to its toxicity on the epithelial cells at the barrier.
  • the toxicity data was further confirmed by performing conventional MTT assay.
  • T-Fc-IVM-NP inhibits NS1 expression.
  • ZIKV is a single stranded RNA virus which encodes for three structural proteins (C, PrM or M, and E) and seven non-structural (NS) proteins NS1 , NS2A, NS2B, NS3, NS4A,
  • NS4B The NS1 protein is involved in viral replication, immune evasion, and pathogenesis of the host cells. NS1 is also a major antigenic marker for viral infection and is regarded as a potential therapeutic target for antiviral drug discovery. Earlier studies have suggested that ivermectin inhibits ZIKV infection. The current study evaluated whether T-Fc- IVM- NP inhibits the expression of NS1 protein. NS1 protein was expressed in HEK293T cells and demonstrated that T-Fc-IVM-NPeffectively inhibited NS1 expression, 0.08 relative to 1.05 for NS1 plasmid alone (Figure 7A). These results were further confirmed using immunofluorescence (Figure 7B). Under the in vitro settings using HEK293 cells, no significant differences were observed between free ivermectin and when delivered with targeted NP due the fact that HEK293 cells do not have significant FcRn expression (Figure 24).
  • Example 7 Dry Formulation of IVM-Loaded NPs (ZIKA)
  • the IVM-loaded TFc IVM-NPs must be made into powder form to be able to be packed into a capsule and to serve as a viable treatment option for ZIKV.
  • Formulation optimization studies were performed using NT-Mal-IVM-NP. Our studies involved freeze-drying the NPs in the presence of cryoprotectants to ensure their size and stability are maintained in powder form. Sucrose and trehalose were identified as potential cryoprotectants. These optimization studies were carried out under different conditions as discussed below.
  • NP ratios were set as 0.1 :1 , 0.5:1 , 1:1 , and 2:1 , as well as control samples with only NP (0:1 ). Size and zeta potential of mixtures of NP and cryoprotectant were tested both after initial mixture and after 1 , 2, and 3 freeze-thaw cycles from -80 °C to room temperature. Each cycle lasted approximately an hour. The tests revealed that both sucrose and trehalose in ratios of 1:1 or 2:1 offered the best protection to NP size and zeta potential, and so these two concentrations of both cryoprotectants were chosen for the long-term storage of NPs at -80 °C.
  • NPs with cryoprotactants maintain size better, and overall, the cryoprotectants in higher concentrations (2:1 cryoprotectant: NP ratio) were chosen for testing of the drying process (Figure 8C). IVM levels in the NPs were assessed using HPLC; it was found the levels did not show significant change after 180 days of long term storage.
  • cryoprotectant NP ratio of 1 :1 and 2:1 ratios are the best for dry formulation. These NPs maintained the size ⁇ 65 nm and a negative zeta potential.
  • Example 8 Transport of IVM loaded NPs across an in vitro Placental Barrier.
  • the placental barrier is built from various types of cell layers which allows for diffusion of different substances between the maternal and fetal circulatory systems. For this reason, the placental barrier is known as the leakiest barrier allowing small molecules to pass through between the mother and the fetus. This leaky nature of the placental barrier is a major reason for doctors’ reluctance towards prescribing medicines for chronic diseases such as hyperlipidemia and ZIKV infection to pregnant patients.
  • the fate of IVM and IVM loaded NPs were studied. NP transport ability across the placental carcinoma JEG-3 monolayer was assessed by quantifying IVM in the apical and basolateral side media using HPLC.
  • JEG-3 cells were plated in a trans-well plate on the apical side. On the basolateral side, media was added and the cells were grown up to 9 days. Before the addition of IVM or NPs, the monolayer integrity was checked by measuring the TEER indicating a value of >900 W/cm2 on day 9 ( Figure 9A). Media was replenished once every two days. On the ninth day, IVM, NT-OH-IVM- NP, or T-Fc-IVM-NP was added to the apical side of the barrier and incubated for 12 h. The TEER values and tight junction protein, ZO-1 expression confirmed that the addition of the articles did not damage the placental barrier ( Figures 9A and 9C).
  • IVM alone was able to cross the membrane significantly compared to the T-Fc-IVM-NPs and the NT-OH-IVMNPs ( Figure 9C).
  • NPs can be useful for the treatment pregnant patient without affecting the infant.
  • Cellular toxicity of IVM was compared with T-Fc-IVMNP in JEG-3 cells by studying basal respiration, maximal respiration, ATP production, extra cellular acidification. These studies indicated that at the same concentration, IVM completely disrupts cellular respiration of the cells which form the placental barrier while when ivermectin is loaded into T-Fc-NPs, it doesn’t show such toxicity (Figure 9D).
  • Example 9 Ivermectin Nanoformulation Has the Ability to Reduce ACE2 and Spike Protein Expression.
  • IVM-loaded PLGA-b-PEG-MAL nanoparticles were synthesized by following a nanoprecipitation method.
  • the nanoparticles were characterized using dynamic light scattering (DLS), and were found to have sizes around 60-70 nm and zeta potential around -30 mV for nanoparticles with 20% feed of IVM ( Figure 33).
  • IVM loading was quantified using HPLC ( Figure 33).
  • Fc immunoglobulin fragment targeting moiety was attached using thiolene chemistry, creating the targeted T-Fc-IVM-NPs. Conjugation of the Fc fragment was confirmed and quantified through a bicinchoninic acid (BCA) assay.
  • BCA bicinchoninic acid
  • SARS-CoV-2 is a positive sense single-stranded RNA virus and one of the most crucial components of its structure is the surface spike protein that al-lows it to enter and infect cells.
  • a logical method to model the conditions of viral infection in vitro and study the expression of the spike protein is to transfect cells using a plasmid expressing spike protein. This would mimic infectious conditions and allow for measurements of NP-delivered IVM’s ability to inhibit viral spike protein without requiring the construction of pseudoviruses or other technologies.
  • Spike protein of SARS-CoV- 2 interacts with ACE2 receptors on human cells, thereby infecting the host.
  • HEK293T human embryonic kidney epithelial cells were transfected with a plasmid containing the SARS-CoV-2 viral spike protein. These cells were subsequently treated with IVM, a non- targeted IVM-loaded nanoparticle, NT-IVM-NP, made from an PLGA-b-PEG-OH polymer, and the T-Fc-IVM-NPs. Treatments were with 10 mM of free IVM or the nanoformulations with respect to IVM for a period of 4 h followed by incubation for 20 h.
  • IVM nanoformulations showed significantly higher accumulation in the blood. These studies together suggest the superior ability of IVM nanoparticle to reduce spike and ACE2 compared to free IVM. Our studies also revealed that the HEK293T cells have low basal ACE2 expression.
  • NT-IVM-NP also decreased ACE2 mRNA expression, but this decrease was less as compare to T-IVM-NP.
  • SARS-CoV-2 spike protein binds to the ACE2 receptor on host cells and initiates a cascade of steps for cell fusion and viral uptake into the host cells.
  • Our results indicate that the T-IVM-NP might inhibit viral uptake or entry to the host cells by inhibiting spike and ACE2 expression.
  • EK1C4 a lipopeptide, targeting spike protein inhibited SARS-CoV-2 membrane fusion and viral entry into the host cells.
  • T-Fc-IVM-NP T-Fc-IVM-NP in two other ACE2-expressing epithelial cell lines, A549 adenocarcinomic alveolar basal epithelial cells and HeLa malignant epithelial cells, was also assessed to study the potential impact of the therapeutic on ACE2 and spike protein expression in lung cells and other epithelia that may be infected by SARS-CoV-2.
  • ACE2 expression was found to decrease after treatment with IVM and the IVM nanoformulations, and the largest decrease in expression was seen after treatment with the IVM-loaded nanoparticles (Figure 28A, Figure 39A for quantification).
  • the A549 cells were treated with increasing doses of T-Fc-IVM-NPs, and the western blot revealed a dose-dependent effect of the nanoformulation on ACE2 expression (Figure 28B, Figure 39B for quantification).
  • treatment with T-Fc-IVM-NP showed a de-crease in the expression of both spike protein and ACE2, and the more evident decrease appeared to be in cells treated with the nanoparticles rather than free IVM ( Figure 28C and Figure 39C for quantification).
  • immunofluorescence staining in A549 cells revealed a drop in expression of ACE2 following treatment with IVM, NT-IVM-NPs, and T-Fc-IVM-NPs ( Figure 28D).
  • Example 10 Pseudo-virus Inhibition Study.
  • Fluorescent green surface ACE2-expressing HEK293T cells were treated using two different approaches, a therapeutic approach and a preventative approach, which varied in terms of the order of T-Fc-IVM-NP treatments and the Pseudo SARS-CoV-2 exposure (Figure 29A).
  • the therapeutic approach in which cells were exposed to pseudo SARS-CoV-2 followed by T-Fc-IVM-NP treatment, resulted in significant decreases in the levels of both ACE2 and pseudovirus ( Figure 29B).
  • the preventative approach in which T-Fc-IVM-NP treatment preceded pseudovirus exposure with the goal of preventing uptake, there were decreases in both ACE2 express expression and pseudovirus uptake (Figure 29C).
  • Example 11 A Potential Mechanism of Action of T-Fc-IVM-NP [000162] Though the specific mechanism by which the re-leased IVM could inhibit the replication of the SARS-CoV-2 virus and expression of spike protein is yet to be determined, a possibility could be through the inhibition of the nuclear transport activities mediated through proteins such as importin (IMP) a/b1 heterodimer, as IVM was previously shown to inhibit a similar interaction between IMRa/b1 ( Figure 31 A) and the human immunodeficiency virus-1 (HIV-1) integrase protein.
  • IMP a/b1 heterodimer is a key nuclear transport protein and is believed to play a role in transporting viral proteins to the nucleus of infected cells.
  • IMP a and b1 work through the recognition of nuclear localization signals on proteins, and IMP a and b1 have previously been associated with the nuclear transport of other viral proteins such as HIV-1 integrase and dengue virus non-structural protein 5 (NS5).
  • IMP a and b1 transport of viral proteins to the nucleus allows proteins such as dengue virus’ NS5 protein to diminish cells’ antiviral responses by impacting mRNA splicing and immune signaling. Therefore, investigating the IVM-loaded nanoparticle’s potential inhibitory effect on IMP a and b1 is key to fully characterizing the therapeutic’s antiviral properties.
  • Example 12 Mitochondrial Functions and Inflammation of Spike-infected Cells and Effects of T-Fc-IVM-NP
  • SARS-CoV-2 has been found to impact host mitochondrial functions through ACE2 regulation and open-reading frames that can allow for increased viral replication and evasion of host cell immunity.23
  • the mitochondrial effects of spike protein expression and the mitochondrial toxicity of treatment using IVM and its nanoformulations was tested using a Mitostress assay in HEK293T cells transfect-ed with spike plasmid. Initially, spike protein ex pression within the HEK293T cells was found to slightly impact mitochondrial bioenergetics through the decrease of basal and maximum respiration as well as ATP production (Figure 33). This effect was further compounded by treatment with free IVM, which more significantly decreased these three metrics and led to further mitochondrial dysfunction.
  • the T-Fc-IVM-NPs serve as a far more helpful treatment compared to free IVM by not impeding mitochondrial respiration or causing inflammation, and the IVM-loaded nanoparticles in fact allow for treated cells to regain some respiration and ATP production capacity that was initially lowered by spike protein expression and the infection in general.
  • Solubility was achieved by preparing nano-ivermectin vesicles following an optimized reprecipitation method, wherein the drug was dissolved in a good solvent (ethanol) at millimolar concentration and its ethanolic solution (in small increments) was added to a poor solvent (water) to allow the precipitation of the nanovesicles.
  • the synthesis was assisted with ultrasound (ultrasonication bath) for a better size-control of the nanomaterials, that was achieved by the action of acoustic cavitations to promote intermolecular interactions.
  • the disclosed nanovesicles were analyzed using materials characterization techniques, optical properties, in addition to the release studies at the physiological pH.
  • nanovesicles are based on a spontaneous self-assembly mechanism that is governed in this case by hydrophobic and hydrogen bonding interactions.
  • the formation of nanovesicles is represented in Figure 41 C; the self-assembly of ivermectin amphiphilic molecules into nanovesicles is demonstrated under aqueous conditions, to overcome the undesirable hydrophobe-water interactions.
  • the self-assembly mechanism has been applied to produce various nanodrugs, only restricted in majority to the domain of anticancer application.
  • the procedure is different and unique, where the mechanism relates to the organization of a double-tailed surfactant under aqueous conditions contributing to the formation of nanovesicles-based drugs.
  • the disclosed nanoparticle has a hydrophilic head of the structure that faces the aqueous medium while the hydrophobic part is directed towards the interior of the vesicle, where a small volume of water/ethanol is contained.
  • the thickness of the shell is mainly determined by the interfacial tension, showing the following dimensions: total size of the shell, 133 nm; the dark interior shell, 27 nm ( Figure 41 C).
  • Zeta potential measurements were conducted to determine the electrostatic surface potential and evaluate the stability of the nanosuspensions.
  • Zeta potential (CE5) value was -24 mV (average zeta potential values of duplicate syntheses, 3 records per synthesis) with pH of the medium being 7.35.
  • the synthesized nanovesicles present an acceptable stability and exhibit minimal level of agglomeration considering that the zeta potential value is close to the limit value (-30 mV) that is globally accepted as the normal value reflecting the stability of nanosuspensions.
  • the presence of this high surface charge contributes to an electrostatic repulsion among the vesicles and causes a decrease in the level of agglomeration.
  • Ivermectin vesicles might have redistributed and stabilized after a sufficient time of growth or aging. Zeta potential measurements studies were also performed on ivermectin nanosuspensions prepared using different volumes of ethanolic solutions ( Figure 42B). The nanosuspensions formulated with 50 mI_ and 100 mI_ (zeta potential magnitude higher or equal to 30 mV) were more stable than the nanosuspension formulated with 150 mI_ volume. In fact, the nanosuspensions generated with 100 mI_ had the most constant response during the growth of nanoparticles, which indicates that the use of this volume is optimal for the formulation of long term-stored suspensions.
  • the evaluation of the surface charge of these nanovesicles reflects the optimal interaction of nanomaterials with the biological target, as well as the bioavailability of these materials.
  • the pH stability of these nanoformulations was also performed by resuspending the aqueous suspension of nanovesicles in different pH buffers ( Figures 42C and 42D).
  • the effect of pH on the nanoformulations in terms of size and stability is dictated by two factors: the first is the pKa of ivermectin molecule (pka value is around 6.5) and the second is the presence of ions that originate from the buffers.
  • the pka of ivermectin had its heavy effect on the size of nanomaterials that showed a tremendous increase in size after adding phosphate buffers of pH 7 and 7.8.
  • the presence of ions caused a drop in zeta potential values by almost 15 mV magnitude, especially when using buffers of pHs 5 and 7.
  • the distinctive effect of those two buffers is that pH 5 buffer is an acetate buffer where the acetate ions might have some hydrogen bonding interactions with ivermectin molecules, leading to an increase in size and decrease of zeta potential magnitude.
  • the pH 7 phosphate buffer which caused the noticeable changes in size and zeta potential values, has a pH that is slightly higher than the pka value of ivermectin.
  • the ivermectin molecule exists in its ionic form and its solubility in aqueous system increases.
  • an uncontrolled precipitation and unstable formulation were observed as a result of the pH increase during the suspension of the nanomaterials in the buffer.
  • the long-term storage of the nanovesicles was also assessed after two months at 25°C from their preparation in aqueous medium, which is important for the potential use of this compound as a pharmaceutical product.
  • the fabricated nanovesicles have shown a long-shelf life (at least 2 months, (84 nm size)) and a high colloidal stability (-32 mV), an advantage that was observed in difference to what conventional liposomes present.
  • Nanomaterials usually exhibit optical properties that lie between atomic and bulk properties.31 Their properties different from their bulk counterpart as a result of the inter- and intra-molecular interactions that contribute to the formation of the nanomaterials. It is informative to understand the changes of optical properties for nano-ivermectin and the type of molecular arrangement that controlled the formation of nanovesicles. For this purpose, nano ivermectin suspensions were synthesized in duplicate under aqueous conditions using the reprecipitation method, then left to age for 24 hours (Figure 43A).
  • the bulk of ivermectin was prepared following a similar procedure in ethanol, and both bulk and nano-ivermectin were compared at the same concentration.
  • the conjugated-diene chromophore of ivermectin has an absorbance maximum at 245 nm.32
  • Both nanosuspensions were consistent in terms of optical properties, with absorbance ranging between 220 and 260 nm.
  • the nanosuspensions showed a decrease in the molar absorptivity in comparison to the bulk material, as a result of the presence of intermolecular electronic interactions upon aggregation.
  • the decrease of the cross-section of nano-ivermectin exposed to the light can also contribute to the decrease in absorbance value.
  • nanosuspensions presented more defined shoulders in the absorbance spectra, suggesting the presence of multiple excited states.
  • Our previous studies have shown size stability of nano-ivermectin suspensions (for 100 -, pL ethanolic ivermectin formulations) at various aging periods (or growth time) ( Figures 42A and 42B).
  • the optical measurements confirmed the stability as well of the nanosuspensions, that were synthesized and left to age for 24, 48, and 72 hours (Figure 43B).
  • the properties of the nanosuspensions were similar indicating consistency in the synthesis method (same average size of 80 nm) and stability of the suspensions over time.
  • the nanosuspensions were synthesized in duplicate using the optimized reprecipitation method, with an amount of 100 -, pL of 1 mM ivermectin drug. After 24 hours, the nanomaterials were suspended in PBS and dialyzed against PBS buffer pH 7.4. Nano-ivermectin showed first a burst release of 20% of its molecules or entities, suggesting that those molecules were not strongly bound to the assembled nanoscale aggregates.
  • the nanovesicles kept then a slower and sustainable release for a long period of time, about 220 hours.
  • the general profile of the release/degradation rate implies a maintained efficacy of nano-ivermectin for several days. This is advantageous because it reflects a controlled release of the therapeutic agents under physiological conditions.
  • the examples herein demonstrate that a synthetic nanoparticle can cross the intestinal epithelial barrier when administered via oral route and distribute in the blood at a considerable concentration. It is further disclosed that the plasma concentration of ivermectin can be increased significantly when delivered with this synthetic nanoparticle. Further, the toxicity of ivermectin on epithelial cells can be lowered by entrapment inside the polymeric nanoparticles. The examples demonstrate that Ivermectin-loaded NPs demonstrated stability over time, particularly at 4°C and even at room temperature.
  • NT-Mal-IVMNP when combined with cryoprotectant such as sucrose or trehalose in higher concentrations, showed maintenance of both size and zeta potential across multiple freeze-thaw cycles and over several months at -80°C.
  • cryoprotectant such as sucrose or trehalose in higher concentrations

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nanotechnology (AREA)
  • Epidemiology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

This disclosure relates generally to orally administrable nanoparticle for treating and preventing viral infection, specifically ZIKA and coronavirus infections. Methods for using the orally administrable nanoparticle are also provided for treating and preventing viral disease.

Description

Orally Administrable Nano-medicine for Viral Diseases
PRIORITY STATEMENT
[0001] This application claims the benefit of United States provisional application serial number 62/901,077, filed on September 16, 2019, which is incorporated by reference herein in its entirety.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates generally to orally administrable nanoparticle for treating and preventing viral infection, specifically ZIKA and coronavirus infections, in particular humans with COVID 19. The disclosure provides for a controlled release polymeric nanoparticle comprising a maleimide functional group on the nanoparticle surface, and a hydrophobic drug inside the nanoparticle. Methods for using the orally administrable nanoparticle are also provided for treating and preventing viral disease.
BACKGROUND OF THE INVENTION
[0003] Since 2015, Zika virus (ZIKV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus resulting in COVID-19, have significantly disrupted daily life. The spread of ZIKV infection across the USA and various countries in the last three years has had a direct impact on the U.S. health care system and caused international concerns as well. The ultimate impact of ZIKV infection remains to be understood. Currently, there are no therapeutic or vaccine options available to protect those infected by ZIKV.
[0004] Similarly, COVID-19, poses unique challenges to domestic and international public health. The SARS-CoV-2 viral strain is highly transmittable and infects respiratory tissue via the SARS-CoV-2 spike protein targeting the angiotensin-converting enzyme. SARS-CoV-2 infection causes flu-like symptoms as well as more severe respiratory issues and death by respiratory failure.
[0005] Antiviral treatment options for ZIKV and SARS-COV-2 remain elusive and irradication efforts to date have largely proved unsuccessful. As a result society continues to be unprepared to address the health, economic, and social disruptions associated with current, and possible future, viral outbreaks. The current disclosure provides a viable route to overcome these challenges [0006] The food and drug administration-approved drug ivermectin (IVM) has long been shown to be both anti-helmintic agent and a potent inhibitor of viruses such as Yellow Fever Virus. The current disclosure reveals the use of an IVM, packaged in a synthetic nanoparticle as a viable agent for the prevention of ZIKV transmission. The disclosed, biodegradable nanoparticles provide a steady delivery of ivermectin, in order to maintain an appropriate level in the body and following oral administration can cross the intestinal epithelial barrier and enter the blood stream. IVM delivery via the and because IVM is delivered in a synthetic nanoparticle IVM accumulates at safe levels in the blood has the ability to target non-structural 1 protein of ZIKV.
[0007] The disclosed ivermectin-loaded, orally administrable, biodegradable nanoparticle can also be used to treat coronavirus, acting through the inhibition of the SARS-CoV-2 spike protein and targeting of the angiotensin-converting enzyme. The nanoparticle delivered ivermectin inhibits nuclear transport activities mediated through proteins such as the importin a/b1 heterodimer. The disclosed ivermectin packaged nanoparticle serves as a less toxic, more potent oral therapeutic, that decreases viral entry into cells and reduce overall viral load, both of which are keys to lowering disease transmission rates.
SUMMARY OF THE DISCLOSURE
[0008] This invention provides reagents, pharmaceutical formulations, and methods for treating or preventing coronavirus infection in humans, and particularly COVID-19 infection. [0009] In one aspect is a controlled release polymeric nanoparticle comprising a maleimide functional group on the nanoparticle surface, and a hydrophobic drug such as ivermectin inside the nanoparticle is disclosed. In a further aspect is a controlled release targeted polymeric nanoparticle comprising a FcRn binding domain that binds to a target cell or tissue; a hydrophobic drug encapsulated in the nanoparticle; and a targeting ligand with -SH functionality which can react at the nanoparticle surface with the maleimide functional group.
[00010] The nanoparticle described herein is a dry formulation comprising trehalose and/or sucrose. Further, the nanoparticle is a frozen formulation of nanoparticles comprising trehalose and/or sucrose. In the preferred embodiment the nanoparticle contains the hydrophobic drug ivermectin. In yet another aspect the polymeric nanoparticle comprises poly(lactide-co- glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymer that further comprises a poly(lactic-co-glycolic acid) (PLGA) core. As disclosed herein, the FcRn binding domain targets tissue of the gastrointestinal tract. [00011] In another aspect is a method of treating an individual infected with an RNA virus, comprising administering the ivermectin containing nanoparticle to an individual infected with the virus. In one embodiment the RNA virus is Zika virus that is targeted with a nanoparticle that encapsulates ivermectin. In another embodiment the nanoparticle comprises poly(lactide- co-glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymer.
[00012] In one embodiment of the disclosure the FcRn binding domain targets tissue of the gastrointestinal tract following oral administration of the nanoparticle.
[00013] In yet another embodiment the ivermectin is released at a therapeutic dose over a sustained period of time.
[00014] In a further embodiment the method can be used to treat an individual who is or has been infected with a MERS virus, a Dengue virus, a hepatitis virus, a West nile fever virus, or an Ebolavirus.
[00015] In a further aspect is a method of treating an RNA virus, comprising administering the ivermectin containing nanoparticle to an individual is or has been infected with the SARS- COV-2 virus that results in a COVID-19 infection. In one embodiment the FcRn binding domain targets tissue of the respiratory epithelia and the nanoparticle targets ACE2-expressing cells in the lungs.
[00016] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
BRIEF DESCRIPTION OF THE DRAWINGS [00017] Figure 1: (A) FcRn binds to Fc-lvermectin-NPs in an acidic environment, NPs transcytose across the epithelial-cell barrier and gets released at physiological pH of blood. (B) Synthetic strategy of PLGA-b-PEG-Mal. (C) GPC traces of the polymers. (D) Synthesis of IVM loaded NPs using nanoprecipitation. (E) Incorporation of targeting antibody Fc on the NPs. [00018] Figure 2: (A) Characterization of IVM-loaded NPs by DLS and percent loading and encapsulation efficiency by HPLC. (B) Characterization of targeted Fc-conjugated NPs by DLS and determination of Fc conjugation efficiency by the bicinchoninic acid assay (BCA).
[00019] Figure 3: (A) A cartoon of transcytosis of Fc-NP across the trans-epithelial barrier derived from Caco-2 cells. (B) Formation of tight junction was confirmed by measuring the trans- epithelial electrical resistance (TEER) with a Millicell-ERS (Millipore) and intact TEER after addition of NPs. (C) Expression of tight junction protein zonula occludens-1 (ZO-1). Quantification of NPs in the apical, basolateral sides of the barrier (D) in the absence and (E) in the presence of external Fc.
[00020] Figure 4: NP absorption and biodistribution in mice. Biodistribution of QD labeled non-targeted NT-OH-NP (A) and targeted T-Fc-NP (B) after oral administration to Balb/c Albino mice. Data are mean percent injected dose (%ID) and %ID per gram of tissue ± SD (n = 3 mice per group). (C) Localization of the NPs in the duodenum after oral administration.
[00021] Figure 5: (A) Biodistribution of IVM, NT-OH-IVM-NP, and T-Fc-IVM-NP in intestine and blood of Balb/c female mice after 24 h of post administration by oral gavage. (B) FcRn expression level in mice intestinal tissue by western blotting. (C) Cytokine expression in the blood plasma of the IVM or its NP-treated mice. (D) H & E staining of major tissue after treatment with saline or T-Fc-IVM-NP.
[00022] Figure 6. (A) Release of IVM from NT-Mal-IVM-NPs at pH 7.4 and 6.5 at 37 °C. (B) Cellular toxicity of ivermectin and ivermectin-loaded NPs by performing mitochondrial respiration profiles of Caco-2 cells in presence targeted and non-targeted NPs by using Seahorse analyzer and MitoStress assay. Oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-p- trifluoromethoxyphenylhydrazone, an ionophore; Rotenone, an inhibitor of mitochondrial complex I; and Antimycin A, an inhibitor of mitochondrial complex III. (C) Comparison of maximal respiration, basal respiration, ATP production, and coupling efficiency from the Mitostress assay mentioned in B.
[00023] Figure 7: NS1 expression level in HEK293T cells after treatment with NPs by (A) Western blotting and (B) immunofluorescence. Scale bar: 10 pm.
[00024] Figure 8: Temperature-dependent stability of (A) NT-Mal-NP and (B) NT-Mal-IVM- NP by analyzing the diameter and zeta potential over the course of 1 month. (C) Stability of NT- Mal-IVM-NP alone and with cryoprotectants over the course of three 1 h freeze-thaw cycles (freezing at -80 °C). (D) Stability of NT-Mal-IVM-NP stored at -80 °C both alone and with cryoprotectants, measured at 15 timepoints over the course of 180 days. (E) Stability of NT-Mal- IVM-NP after freeze drying at -50 °C and 2 Pa and reconstitution in nanopure water. (F) Comparison of inhibition of NS1 expression in HEK293T cells after treatment with freshly prepared NPs, dried powder, and the NPs stored at -80 °C for 180 days.
[00025] Figure 9: (A) Formation of placental barrier-like tight junction in JEG-3 cells as confirmed by measuring the TEER and intact TEER after addition of NPs. (B) Quantification of NPs in the apical side, inside the cells, and the basolateral sides of the barrier. (C) Expression of ZO-1by immunofluorescence. (D) Cellular toxicity of ivermectin and the NPs by performing mitochondrial respiration profiles of JEG-3 cells using MitoStress assay. Oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-pthfluoromethoxyphenylhydrazone, an ionophore; Rotenone, an inhibitor of mitochondrial complex I; and Antimycin A, an inhibitor of mitochondrial complex III. The cells were treated with the articles for 24 h at a concentration of 10 mM with respect to ivermectin.
[00026] Figure 10: (A) 1 H NMR and (B) 13C NMR of MAL-NHS in CDCI3.
[00027] Figure 11 : LC-MS-ESI of MAL-NHS.
[00028] Figure 12: (A) 1 H NMR and (B) 13C NMR of PLGA-b-PEG-NH2 in CDCI3.
[00029] Figure 13: (A) 1 H NMR and (B) 13C NMR of PLGA-b-PEG-MAL in CDCI3.
[00030] Figure 14: DLS histograms of (A) Mal-NP, (B) Mal-IVM10-NP, (C) Mal-IVM20-NP, (D) Mal-IVM30-NP, (E) Mal-IVM40-NP and (F) Mal-IVM50-NP in nanopure water at 37° C. [00031] Figure 15: Zeta potential (mV) of (A) Mal-NP, (B) Mal-IVM10-NP, (C) Mal-IVM20- NP, (D) Mal-IVM30-NP, (E) Mal-IVM40-NP and (F) Mal-IVM50-NP in nanopure water at 37° C. [00032] Figure 16: TEM images of (A) NT-Mal-NP and (B) NT-Mal-IVM-NP stained with 4% of uranyl acetate.
[00033] Figure 17: (A) DLS histograms of (A) NT-Mal-NP, (B) T-Fc-NP, (C) NT-Mal-IVM-NP, and (D) T-Fc-IVM-NP in nanopure water at 37° C.
[00034] Figure 18: Zeta potential (mV) of (A) NT-Mal-NP, (B) T-Fc-NP, (C) NT-Mal-IVM-NP, and (D) T-Fc-IVM-NP in nanopure water at 37° C.
[00035] Figure 19: (A) Diameters, (B) Zeta potentials of NT-Mal-QD-NP and T-Fc-QD-NP. (C) Fc conjugation efficiency of targeted NPs by the bicinchoninic acid assay (BCA). (D) TEM images of T-Fc-QD-NP (unstained).
[00036] Figure 20: Quantification of QD (Cd) loaded NPs in the (A) apical and (B) basolateral sides of the endothelial cell barrier.
[00037] Figure 21 : Biodistribution of ivermectin, NT-OH-IVM-NP, T-Fc-IVM-NP after oral administration to Balb/c albino mice. Data are mean %ID per gram of tissue ± SD (n = 3 mice per group).
[00038] Figure 22: Alanine aminotransferase (ALT) and Aspartate Aminotransferase (AST) levels from the blood plasma of BALB/c mice (n=3 in each group) treated with single dose of articles (at a dose of 40 mg/kg with respect to ivermectin) via oral gavage for 24 h.
[00039] Figure 23: In vitro efficacy of (A) ivermectin, (B) NT-OH-IVM-NP, (C) NT-Mal-IVM- NP, and (D) T-Fc-IVM-NP in Caco-2 cells by the MTT assay. (E) IC50 values of the articles in the Caco-2 cells after treatment for 72 h.
[00040] Figure 24: Comparison of FcRn expression level in Caco-2, HEK293, and JEG-3 cells by western blotting. [00041] Figure 25: Morphological comparison of NT-Mal-IVM-NP and NT-Mal-IVM-NP with sucrose after 180 days by TEM.
[00042] Figure 26: A) Targeted Fc-lvermectin-NPs in the acidic gut lumen bind to FcRn receptors, allowing NPs to transcytose across the epithelial cell barrier and release at the physiological pH of blood. (B) IVM delivered via T-Fc-IVM-NPs shows the ability to (1) decrease ACE2 receptor levels, (2) decrease SARS-CoV-2 spike protein levels, and (3) decrease levels of the nuclear transport proteins Importin a and b1 , which leads to (4) an increase in the antiviral activity of infected cells.
[00043] Figure 27: (A) Western blot showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 2 h or 4 h at a concentration of 10 mM with respect to ivermectin, the media was changed, and further incubated for 22 h or 20 h. Immunofluorescence staining showing expression of (B) ACE2 and spike (C) in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 pM with respect to IVM. (D) Real time PCR showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4 h at a concentration of 10 pM with respect to ivermectin, media was removed and kept for additional 20 h.
[00044] Figure 28: (A) Western blot showing expression of ACE2 in A549 adenocarcinomic alveolar basal epithelial cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 24 h at a concentration of 10 pM with respect to IVM. (B) Western blot showing dose-dependent decrease in ACE2 expression after treatment with varying concentrations of T-Fc-IVM-NPs with respect to IVM in A549 cells. (C) Western blot showing expression of ACE2 in HeLa malignant epithelial cells transfected with plasmid expressing spike protein with and with-out treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 24 h at a concentration of 10 pM with respect to IVM. (D) Immunofluorescence staining showing expression of ACE2 in A549 cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 24 h at a concentration of 10 pM with respect to IVM.
[00045] Figure 29: (A) Schematic representation of ACE2 expression and pseudovirus infection in HEK293T cells. Efficacy of T-Fc-IVM-NP showing inhibition of both ACE2 and pseudovirus uptake under (B) therapeutic and (C) preventative settings as measured by plate reader. Confocal microscopy images revealing the changes in the expression of red-tagged ACE2 receptor on the cell membrane and mNeonGreen pseudovirus accumulation in the nucleus following the treatment of T-Fc-IVM-NP under (D) therapeutic and (E) preventative settings.
[00046] Figure 30: Confocal microscopy images revealing the changes in the expression of red-tagged ACE2 receptor on the cell membrane and mNeonGreen pseudovirus accumulation in the nucleus following the treatment of T-Fc-IVM-NP under (A) therapeutic and (B) preventative settings in primary small airway epithelial human HSAEC cells.
[00047] Figure 31 : (A) Schematic representation of the how IVM delivered through T-Fc- IVM-NP inhibits IMP a and b1. (B) West-ern blots showing expression of IMP a and b1 in the cells transfected with plasmid expressing spike protein and 2 h, 4 h, or 6 h of article treatment followed by incubation in normal media up to 24 h. (C) Effect of importazole on HEK293T cells by Western blot. Effects of importazole on ACE2 and pseudovirus uptake under (B) therapeutic and (C) preventative settings as measured by plate reader.
[00048] Figure 32: (A) Cellular toxicity of IVM and IVM-loaded NPs as measured by mitochondrial respiration profiles in spike protein-expressing HEK293T cells using the Sea horse analyzer and MitoStress assay: oligomycin, ATP synthase inhibitor; FCCP-carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, an ionophore; rotenone, an inhibitor of mitochondrial complex I; and antimycin A, an inhibitor of mitochondrial complex III. (B) Basal respiration and ATP production from the MitoStress assay. (C) Expression of cytokines IL-6, IL- 1b, and TNFa in the media of spike protein-expressing HEK293T cells treated with IVM, NT- IVM-NP, or T-Fc-IVM-NP.
[00049] Figure 33: Characterization of IVM NPs (A) for diameter by DLS, (B) zeta potential, (C) percent loading and %EE of IVM in NT-NPs, and (D) percent loading, %EE of IVM and %Fc conjugation in T-Fc-NPs.
[00050] Figure 34: Uptake kinetics of IVM, NT-IVM-NPs, or T-FC-IVM-NPs in HEK293T cells.
[00051] Figure 35: Cytotoxicity of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs in HEK293T cells as determined by the MTT assay.
[00052] Figure 36: Densitometric analyses of Western blots showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 2 h or 4h at a concentration of 10 mM with respect to ivermectin, the media was changed, and further incubated for 22 h or 20 h. Analyses were performed by ImageJ.
[00053] Figure 37: (A) Western blot showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IV, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 24h at a concentration of 10 pm with respect to ivermectin. (B) Densitometric analyses of Western blots showing expression of ACE2 and spike protein in HEK293T cells transfected with plasmid expression spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM-NPs. (C) Immunofluorescence staining showing expression of ACE2 in HEK293T cells transfected with plasmid expressing spike protein with and without treatment of IVM, NT-IVM-NPs, or T-Fc-IVM- NPs. Cells were Cells were treated with the articles for 24h at a concentration of 10 pm with respect to IVM.
[00054] Figure 38: Western blot showing increased expression of ACE2 HEK293T cells upon treatment with Ang II at a varied concentration.
[00055] Figure 39: (A) Western blot showing expression in A549 adenocarcinomic alveolar basal epithelial cells transfected with plasmid expressing spike protein with and without treatment of IV, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4h at a concentration of 10 pm with respect to IVM. (B) Densitometric analyses of Western blots showing dose-dependent decrease in ACE2 expression after treatment with varying concentrations of T-Fc-IVM-NPs with respect to IVM in A549 cells. (C) Densitometric analyses of Western blot showing expression of ACE2 and spike in HeLa malignant epithelial cells transfected with plasmid expressing after treatment of IV, NT-IVM-NPs, or T-Fc-IVM-NPs. Cells were treated with the articles for 4h at a concentration of 10 pm with respect to IVM.
[00056] Figure 40: Effects of IVM and IVM-loaded NPs on mitochondrial complex IV and complex V activity in spike protein expressing HEK293T cells.
[00057] Figure 41 : (A) Variation of hydrodynamic diameter of nano-ivermectin and (B) TEMs of nano-ivermectin obtained using the ultrasound-assisted reprecipitation method (aqueous medium) and (C) (acidic conditions of acetate buffer pH 5).
[00058] Figure 42: Change of (A) diameter and (B) zeta potential with volume of ethanolic ivermectin during the time growth of nanomaterials. Change of (C) diameter and (D) zeta potential with the pH of the suspension before and after the addition of a buffer.
[00059] Figure 43: (A) Absorbance spectra of bulk ivermectin and two nano-ivermectin suspensions aged for 24 hours, and (B) bulk ivermectin and nano-ivermectin suspensions aged for 24, 48, and 72 hours. [00060] Figure 44: Release profile of nano-ivermectin in PBS buffer pH 7.4.
[00061] Figure 45: Schematic of oral delivery of nanoparticles.
[00062] Figure 46: Schematic of orally deliverable clinically approved anti-viral drug loaded nanoparticles.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[00063] This disclosure relates generally to orally administrable nanoparticle for treating and preventing viral infection, specifically ZIKA and coronavirus infections, in particular humans with COVID 19. Methods for using the orally administrable nanoparticle are also provided for treating and preventing viral disease
1. Definitions
[00064] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the claimed invention belongs. The terminology used herein is for describing particular embodiments only and is not intended to be limiting of the claimed invention. All technical and scientific terms used herein have the same meaning.
[00065] The following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings known or understood by those having ordinary skill in the art are also possible, and within the scope of the claimed invention. All publications, patent applications, patents, and other references mentioned or discussed herein are expressly incorporated by reference in their entireties. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[00066] As used herein, the singular forms "a," "and," and "the" include plural references, unless the context clearly dictates otherwise or is otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
[00067] As used herein, the term "or" means, and is used interchangeably with, the term "and/or," unless context clearly indicates otherwise.
[00068] As used herein, the term "including" means, and is used interchangeably with, the phrase "including but not limited to."
[00069] As used herein, the term "such as" means, and is used interchangeably with, the phrase "such as, for example" or "such as but not limited."
[00070] Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example, within two standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%,
3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.
[00071] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,
41 , 42, 43, 44, 45, 46, 47, 48, 49, or 50.
2. Nanotechnology-based delivery systems
[00072] In one aspect of the current disclosure is a controlled release polymeric nanoparticle comprising a maleimide functional group on the nanoparticle surface, and a hydrophobic drug inside the nanoparticle. As used herein a “nanoparticle” refers to a particle of matter that is between 1 and 100 nanometers (nm) in diameter. One of skill in the art will understand that the properties of nanoparticles are different than larger particles of the same substance and that a large fraction of the material within a nanoparticle lies within a few atomic diameters from the surface.
[00073] Nanoparticles occur widely in nature and are objects of study in many sciences such as chemistry, physics, geology and biology. Nanoparticles can also be synthesized and the production of nanoparticles with specific properties is an important branch of nanotechnology. [00074] Nanoparticles can be part of nanotechnology-based delivery systems and are capable of transporting their contents across cellular membranes to deliver a specific message for the execution of a biological activity or function. Further, nanovesicles possess unique flow properties that endow them with an amended bioavailability. Nanovesicles have gained a particular importance due to their ability to enhance permeation rates of cargos such as drugs, through resistant biological membranes, mainly the skin barrier.
[00075] One of skill in the art will understand that the vesicle/cargo nanoscale system contributes to a major improvement of several pharmacokinetic properties of the cargo, including and not limited to, the solubility properties, controlled release, and milieu sensitivity (pH and type of medium). Thus, an ample of biological applications benefited from the use of nanovesicles as diagnostic tools, and therapeutics carriers. For example, different designs (unilamellar and multilamellar, liposomes, niosomes, transferosomes, spherical assemblies, polymersomes and chemical composition of nanovesicles (lipid-, peptide-, polymer-based nanovesicles) were used for improvement of the penetration efficiency through biological membrane and delivery of therapeutic agents such as drugs and photosensitizers to cure medical conditions and diseases.
[00076] One of skill in the art will understand that maleimide-mediated methodologies are widely used in bioconjugation. The name is a contraction of maleic acid and imide, the - C(0)NHC(0)- functional group. Maleimides functional groups can be used in the preparation of targeted therapeutics, assemblies for studying proteins in their biological context, protein-based microarrays, or proteins immobilization. Maleimides can be used in targeted drug therapies to bind and help deliver compounds in the body. One of skill in the art will further understand that maleimides also describes a class of derivatives of the parent maleimide where the NH group is replaced with alkyl or aryl groups and that the substituent can comprise a wide number of molecules.
[00077] In another aspect is a controlled release targeted polymeric nanoparticle comprising: a FcRn binding domain that binds to a target cell or tissue; a hydrophobic drug encapsulated in the nanoparticle; and a targeting ligand with -SH functionality which can react at the nanoparticle surface with the maleimide functional group.
[00078] As used herein, the term “nanovesicle” is synonymous with nanoparticle. Disclosed herein are nanovesicles with the surprising technical effect of being composed solely of drug molecules, as nanodrugs, in a novel configuration where the drug molecules are self-carriers. This system introduces a novel area different from the norm wherein the nanovesicles are typically composed of phospholipid molecules. The nanoparticles, serving as nanovesicles, are assemblies of drugs, thus, allow the delivery of an enhanced number of drug therapeutics without the need of a carrier or vehicle avoiding the problems related to the low loading capacity and unknown metabolism or degradation of the carrier in the body. [00079] In one aspect the nanodrugs disclosed herein can be efficiently implemented in aqueous medium in absence of any toxic organic solvent, which is highly significant for biological applications. The nanovesicles, as described herein are composed of a potent antiviral drug in a nanoscale platform that can be widely applied to target various flaviviruses, and treat life-threatening viral infections.
[00080] In another aspect the nanovesicles are composed of antiviral drug molecules with amphiphilic structures and cumulative hydrophobic property. One of skill in the art will understand that the amphiphilic property of the drug is beneficial for topical deliveries, and eliminates the need for the use of penetration enhancers such as surfactants that are added to the nanovesicles suspensions.
[00081] In one aspect, the nanoparticle is a dry formulation comprising trehalose or sucrose. In another aspect the nanoparticle is a frozen formulation of nanoparticles comprising trehalose or a frozen formulation of nanoparticles comprising sucrose.
[00082] One of skill in the art will understand, that unique design of the nanoparticles described herein allows for inclusion of a wide variety of drugs including, but not limited to antivirals. In one aspect the hydrophobic drug ivermectin is part of the nanoparticle. In yet another aspect, the polymeric nanoparticle comprises poly(lactide-co-glycolide)-b- polyethyleneglycol (PLGA-b-PEG) block copolymer that can further comprise a poly(lactic-co- glycolic acid) (PLGA) core.
[00083] One of skill in the art will understand that nanoparticles are advantageous as they can target specific tissue. In one aspect of the current disclosure, the FcRn binding domain targets tissue of the gastrointestinal tract. Further, because the cargo of nanoparticles can have unknown effects on developing fetuses it is critical that some drug or antiviral containing nanoparticles not cross the placental barrier. Consistent with this, in one aspect of the current disclosure the nanoparticle described herein does not cross the placental barrier.
2. ZIKA VIRUS (ZIKV)
[00084] The nanovesicles described herein are prepared from ivermectin drug for its highly efficient antiviral activity against flaviviruses, including but not limited to Yellow Fever, Dengue, and Zika Viruses. Also, disclosed is the use of nanovesicles containing ivermectin for use in treating COVD-19. One of skill in the art will further understand that the disclosed nanovesicles can be adapted or even used as disclosed for virus with similar origins. It is further understood that the disclosed technology can be used to with a wide range of therapeutics beyond ivermectin, including, but not limited to antivirals, chemotherapies, and antibodies [00085] The nanovesicle disclosed herein includes ivermectin within the nanoparticle. One of Ivermectin inhibits viral protein replication, more specifically in relation to the inhibition of importin a/b that is included in several nuclear transport processes. Ivermectin use, historically has been limited as the potent action of the drug is only observed at high concentrations of micromolar range, resulting in toxic effects (i.e., cell toxicity). The current disclosure in which ivermectin is part of a nanodrug overcomes this limitation and has the surprising technical effect lowering the effective dosage of ivermectin drug required to be effective, consequently avoiding toxicity and manifestation of side effects. The innovative nanoivermectin-based formulation described herein drastically increases the therapeutic window, and can eventually eliminate the necessity for a frequent dosing of the drug. Further, the formulation of self-assembled nano ivermectin addresses the issue ivermectin solubility. Overcoming the "solubilizing" ivermectin issue is a significant technical effect, allowing use of the drug to treat viral infections.
[00086] In another aspect is a method of treating an individual infected with an RNA virus, comprising administering the nanoparticle containing ivermectin to an individual infected with an RNA virus. In one aspect the RNA virus is Zika virus (ZIKV).
[00087] The ZIKV causes microcephaly as well as a spectrum of neurologic problems including seizures in newborn babies and Guillain-Barre syndrome in adults. The ultimate scale and impact of ZIKV remain to be determined as the severe abnormalities recognized at birth only represent the tip of the iceberg. To address the immediate need for therapy against ZIKV, a collection of Food and Drug Administration (FDA)-approved anti-viral drugs were tested for the ability to inhibit ZIKV. This work identified more than 20 agents that decreased ZIKV infection in HuH-7 cells. Among these drug candidates, the most potent were ivermectin (IVM), mycophenolic acid (MPA), and daptomycin. Consistent with this, the current disclosure describes a new formulation of ivermectin as well as use of the nanoparticle for treating ZIKV. One of skill in the art will understand that the disclosed nanoparticle may also be used to treat related diseases in which ivermectin is effective, but its use may be limited by toxicity issues when administered in a non-nanoparticle formulation.
[00088] Consistent with this, the concentration of IVM at which inhibitory effects on ZIKV infection can be achieved might cause toxic effects when applied to human population.
Targeted nanoparticles such as those disclosed herein can differentially deliver drugs to the site of interest in the body to improve the therapeutic index of drugs. The Polymeric NPs of poly(lactide-co-glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymers designed and described herein are especially promising as drug delivery vehicles. [00089] The core shell structure of polymeric NPs allows them to encapsulate and carry poorly water-soluble drugs such as IVM resulting long circulation half-life for the drug, release drugs at a sustained rate, and be functionalized with targeting ligands to modulate delivery method to target specific regions. Here, we report a controlled released targeted NP with the ability to supply slow therapeutic dose of IVM over a prolonged period when administered via oral route.
[00090] Consistent with this, in a further aspect of the disclosure, the nanoparticles encapsulate ivermectin. And can comprise a poly(lactide-co-glycolide)-b-polyethyleneglycol (PLGA-b-PEG) block copolymer. In a further aspect the FcRn binding domain targets tissue of the gastrointestinal tract. The nanoparticles described herein can be are administered orally although one of skill in the art will understand the nanoparticles can be administered in alternative routes, including but not limited to intravenous. The disclosed nanoparticle is further advantageous as the ivermectin payload is released at a therapeutic dose over a sustained period of time. Consistent with this, the nanoparticle can be loaded with alternative drugs, allowing for sustained release of alternative drugs, compounds, or therapeutics.
3. SARS-COV-2 (COVID19)
[00091] In a further aspect of the disclosure is a method of treating an RNA virus, comprising administering the nanoparticle of any one of claims 1-11 to an individual infected with a single strand RNA virus. In one embodiment the individual is or has been infected with the SARS- COV-2 virus that has presently or previously resulted in a COVID-19 infection. The method disclosed herein can also be used as a prophylactic measure. In one aspect the FcRn binding domain targets tissue of the respiratory epithelia and can the nanoparticle can target ACE2- expressing cells.
[00092] COVID-19, a disease caused by a novel coronavirus strain SARS-CoV-2 is highly transmittable and infects respiratory tissue, and can cause flu-like symptoms as well as more severe respiratory issues and death by respiratory failure.
[00093] The SARS-CoV-2 virus surface spike protein interacts with angiotensin-converting enzyme 2 (ACE2) receptors in the lung and facilitates the entry of virus into host cells, and much of the tissue damage done is actually a product of the immune response and resulting inflammation.
[00094] There are currently at least 15 completed clinical trials conducted around the world to search for various therapeutics to combat COVID-19 infection with no appreciable success. Many of the experimental therapeutics are meant only to rescue patients in severe respiratory distress or those already undergoing intubation and mechanical ventilation, rather than patients at an early to moderate stage of infection. Herein, disclosed is an orally administrable Ivermectin (IVM)-loaded nanoparticle (NP) and its ability to lower the expression of the ACE2 receptor and the SARS-CoV-2 spike protein.
[00095] The disclosed IVM nanoformulation allows the therapeutic to be gradually released into the blood-stream, which maintains its level in the blood about the minimum effective therapeutic dose while keeping it below the maximum tolerated dose. The NP was constructed using poly(lactide-co-glycolide)-b-poly(ethylene glycol)-maleimide (PLGA-b-PEG-MAL) polymer, and was targeted to the gut epithelial barrier for crossover into the bloodstream by covalent attachment of an Fc immunoglobulin fragment, which binds to receptors on epithelial cells in the gut lumen (Figure 26).
[00096] As disclosed herein, a surprising technical effect of the described nanoparticle that it not only reduces levels of proteins that contribute to the virus' infectious nature, but to also exploits mechanisms to prevent viral entry into cells in the first place.
[00097] ACE2, which is present in high quantities in respiratory epithelia, allows for viral entry and infection of the lung and alveolar cells. ACE2-expressing cells in the lung are involved in key processes such as blood pressure regulation and interferon production, and SARS-CoV-2 binding to this receptor can impede on those processes, making it an important target to reduce viral infection. As further disclosed in the examples, nanoparticle delivered IVM effectively decreases levels of viral spike protein as well as cellular levels of ACE2. These surprising technical effects suggest nanoparticle IVM is therapeutic in part due to the controlled delivery afforded by the disclosed orally administrable NP. NP-mediated delivery of IVM allows the drug to be gradually released into the bloodstream at an effective therapeutic dose while keeping it below the maximum tolerated dose. In particular, the disclosed IVM-loaded nanoparticles are engineered to contain a bound Fc immunoglobulin anti-body fragment to target FcRn receptors on gut epithelial cells, which will allow for transcytosis of orally delivered nanoparticles into the bloodstream and potential accumulation at respiratory epithelial cells, which are particularly affected by SARS-CoV-2 (Figure 26).
[00098] Furthermore, the Centers for Disease Control and Prevention (CDC) recently suggested that the pregnant population are at a higher risk for severe complications from COVID-19 as compared to non-pregnant people, and that adverse pregnancy outcomes can happen among pregnant people with COVID-19. As a whole, pregnant women are more likely to be admitted to intensive care units and put on mechanical ventilators than are nonpregnant women, placing the fetus at increased risk as well. Thus, it is important to develop therapeutics that can be provided as a method of care to pregnant women without affecting the fetus. As disclosed herein, the IVM containing NP is unable to cross the placental barrier thereby providing the additional benefit of treating pregnant individuals without harming the fetus. [00099] One of skill in the art will understand that the disclosed nanoparticle can be loaded with alternative therapeutics, including but not limited to, alternative drugs, chemicals, or antibodies. As such, the NPs disclosed herein can be further used to treat a wide range of viral infections. In one embodiment the ivermectin nanoparticle can be used to treat an individual is or has been infected with a MERS virus. In an alternative embodiment the ivermectin nanoparticle can be used to treat an individual who is or has been infected with a Dengue virus. In yet another embodiment the ivermectin nanoparticle can be used to treat an individual who is or has been infected with a hepatitis virus. In yet another embodiment the ivermectin nanoparticle can be used to treat individual who is or has been infected with a West nile fever virus or an Ebolavirus.
[000100] Having described the invention in detail and by reference to specific aspects and/or embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention can be identified herein as particularly advantageous, it is contemplated that the present invention is not limited to these particular aspects of the invention. Percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated invention.
[000101] The invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements and/or features, certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements and/or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms "comprising" and "containing" are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
EXAMPLES
Materials
[000102] All chemicals noted herein were used as received without further purification unless otherwise mentioned. Ivermectin, N, N'-dicyclohexylcarbodiimide (DCC), 4- dimethylaminopyridine (DMAP), N-hydroxysuccinimide (NHS), 6-aminohexanoic acid, maleic anhydride, N,N-diisopropylethylamine (DIPEA), sucrose, and D-(+)-trehalose dihydrate were purchased from Sigma-Aldrich. Acid terminated poly(DL-lactide-coglycolide) (PLGA-COOH) of inherent viscosity dL/g, 0.15 to 0.25 was purchased from Durect LACTEL. absorbable Polymers. Polyethylene glycol (H2N-PEG2000-NH2) was procured from JenKem Technology, Bachem. Deuterated solvents, CDCI3 and DMSO-d6 were purchased from Cambridge Isotope Laboratories Inc. Regenerative cellulose membrane Amicon Ultra centrifugal 100 kDa filters were purchased from Merck Millipore Ltd. Strata C18-T columns (catalog number 8B-S004- EAK) were purchased from Phenomenex. Copper grids for transmission electron microscopy (TEM) were purchased from Electron Microscopy Sciences. Qdot. 705 ITK™ Amino (PEG) Quantum Dots (catalog number Q21561MP) and ProLong. Gold anti-fade reagent with 4', 6- diamidino-2-phenylindole (DAPI) were purchased from Life Technologies. Trans-well system polycarbonate (0.4-pm pore size, 12-well plates) were purchased from Corning, Lowell, MA. The tight junction antibody ZO-1 (catalog number ab59720) was purchased from Abeam. Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (catalog number A11008) was procured from Invitrogen, ThermoFisher Scientific. Phosphate buffered saline (1X PBS) was purchased from Gibco (reference number 10010-023). Goat serum was obtained from Sigma Aldrich (catalog number G9023). Glutamine, penicillin/streptomycin trypsin-EDTA solution, HEPES buffer (1 M in water), and sodium pyruvate were procured from Sigma Life Sciences. Dulbecco's Modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Life Technologies. Mouse monoclonal IgG, Fc-Rn (A-6) (Catalog number SC-393064) was purchased from Santa Cruz Biotechnology. Zika virus NS1 antibody (EA88) (catalog number. MA5-24583) was purchased from Invitrogen. Flag-tagged Zika NS1 plasmid (Catalog number 79641) was procured from Addgene. Native human IgG FC fragment protein (catalog number Ab90285) was procured from Abeam. Ammonium persulfate (Catalog number 161-0180), tris/glycine/SDS buffer (Catalog number 161-0732), SDS-PAGE gel preparation kit TGX stain- freeTM fast castTM acrylamine 10% (Catalog number 161-0182), and Clarity™ western ECL substrate (Catalog number 170-5060) were purchased from Bio-Rad Inc. Beta-actin antibody (Catalog number ab8226), nitrocellulose membrane (catalog number 88018), and tween-20 was purchased from Fisher Bioreagents.
Instruments
[000103] 1H and 13C NMR spectra were recorded on 400 MHz Bruker NMR spectrometer.
Gel permeation chromatographic (GPC) analyses were performed on a Shimadzu LC20-AD prominence S4 liquid chromatographer equipped with a refractive index detector and water columns; molecular weights calculated using a conventional calibration curve constructed from narrow polystyrene standards using DMF as an eluent at a temperature of 40°C. Dynamic light scattering (DLS) measurements were carried out using a Malvern Zetasizer Nano ZS system. Distilled water was purified by passage through a Millipore Milli-Q Biocel water purification system (18.2 MW) containing a 0.22 pm filter. Absorbance analyses were performed on a Bio- Tek Synergy HT microplate reader. High-performance liquid chromatography (HPLC) analyses were made on an Agilent 1200 series instrument equipped with a multi-wavelength UV-visible and a fluorescence detector. Cells were counted using Countess Automated Cell Counter procured from Invitrogen. TEM images were acquired using a JEOL JEM-1400 equipped with a Gatan Orius SC 200D CCD digital camera with a magnification of 80K. Inductively coupled plasma mass spectrometry (ICP-MS) studies were performed on an Agilent 7900 ICP-MS instrument. Mitochondrial bioenergetics assays were performed on XFe96 Extracellular Flux Analyzer (Agilent Seahorse Biosciences). TEER measurements were performed on a Millicell. ERS-2 Voltohmmeter Instrument (Catalog number MERS00002) purchased from Millipore. Confocal microscopy images were obtained using an Olympus FluoView FV3000. Mouse organ images were captured using a Zeiss Stemi 2000-CS stereoscope fitted with a CL-1500 ECO SteREO light source.
Methods (ZIKA)
Cell Culture
[000104] Human epithelial colorectal adenocarcinoma cells (Caco-2) cells were procured from ATCC. These cells were grown in Eagle's Minimum Essential Medium (DMEM) along with 20% fetal bovine serum. Cell cultures were maintained in a humidified cell culture incubator at 37°C and with 5% CO2. Transfectable derivative of human embryonic kidney 293 (HEK293T) cells were procured from ATCC. Cells were grown in Eagle's Minimum Essential Medium (DMEM) along with 10% fetal bovine serum. Cell cultures were maintained in a humidified cell culture incubator at 37°C and with 5% CO2. Human placental choriocarcinoma (JEG-3) cells were procured from ATCC. Cells were grown in Dulbecco Modified Eagle Medium (DMEM) along with 10% fetal bovine serum. Cell cultures were maintained in a humidified cell culture incubator at 37°C and with 5% C02.
Synthesis of MAL-NHS
[000105] 6-amino hexanoic acid (1 g, 7.62 mmol) and maleic anhydride (0.78 g, 80 mmol) were dissolved in 7.5 mL of acetic acid. The reaction mixture was stirred for 1 h at room temperature until a white color product precipitated. The product was dispersed in toluene and evaporated under high vacuum to remove remaining acetic acid (Yield = 89%). Formed Mal- acid (0.8 g, 3.8 mmol) and Nhydroxysuccinimide (0.48 g, 4.1 mmol) were dissolved in 5 mL of dry DMF. The reaction mixture was cooled to 0°C with stirring. DCC (0.87 g, 4.1 mmol) was dissolved in DMF (2 mL) and added to the reaction vessel. The mixture was warmed to room temperature and stirred for 12 h. Precipitated DCU was filtered out and DMF removed under high vacuum. Crude product was purified using column chromatography (3% methanol in CH2CI2). Yield = 76 %. Melting point = 63-68 °C. 1H NMR (CDCI3, 400 MHz): d 6.69 [s, 2 H (CH=CH)], 3.55 [t, 2 H (NCH2)], 2.83 [m, 2 H (CH2CH2)], 2.60 [t, 2 H (CH2CO)], 1.79 [m, 2H (CH2CH2)], 1.61 [m, 2H (CH2CH2)] ppm. 13C NMR (CDCI3, 400 MHz): d 173.2, 170.7, 168.8, 133.2, 132.3, 30.4 and 30.2.
Synthesis of PLGA-b-PEG-NH2
NH2-PEG-NH2 (1.0 g, 0.5 mmol), PLGA-COOH (0.825 g, 0.16 mmol), and DMAP (0.022 g, 0.2 mmol) were dissolved in dry CH2CI2 (12mL). This reaction mixture was cooled to 0 °C along with stirring. DCC (0.036 g, 0.17 mmol) was dissolved in CH2CI2 (1 mL) and added drop wise to the reaction vessel. The mixture was then warmed to room temperature and stirred overnight. Later on, precipitated DCU was filtered out and the resulting mixture was in a 1:1 mixture of cold diethyl ether: methanol (50 mL). This was repeated 5 times. The resulting solid was centrifuged at 5000 rpm for 5 min. The resulting solid was dried under high vacuum and stored. Yield = 51%. 1 H NMR (CDCI3, 400 MHz): d 5.21 [m, 1 H (OCHCH3C(0)], 4.82 [m, 2 H (OCH2C(0)], 3.64 [s, 1.7 H (OCH2)], 1.58 [m, 3.2 H (CH3CH)] ppm. 13C NMR (CDCI3, 400 MHz): d 164.5, 161.6, 65.7, 64.2, 56.0, and 11.9. GPC Molecular weight: Mn = 17,500, PDI= 1.12 in DMF. Synthesis of PLGA-b-PEG-MAL:
[000106] PLGA-b-PEG-NH2 (0.5 g, 0.06 mmol) and MALNHS (0.04 g, 0.13 mmol) dissolved in 10 mL of dry CH2CI2 along with stirring. Diisopropylethylamine (0.06 g, 0.13 mmol) was added to the reaction mixture and stirred for 20 hours. Afterwards, CH2CI2 was concentrated to 2 mL under vacuum and precipitated in 1 : 1 mixture of cold diethyl ether and methanol three times. Precipitated solid product was dried and stored (yield: 72 %). 1 H NMR (CDCI3, 400 MHz): d 6.45 [s, 2 H (CH=CH)], 5.23 [m, 1 H (OCHCH3C(0)], 4.83 [m, 2 H (OCH2C(0)], 3.67 [s,1.77 H (OCH2)], 1.58 [m, 3.1 H (CH3CH)] ppm. 13C NMR (CDCI3, 400 MHz): d 183.8, 169.2, 166.4, 70.5, 60.8, and 16.6. GPC Molecular weight: Mn = 17,600, PDI= 1.12 in DMF.
Nanoparticle (NP) Formation
[000107] A solution of PLGA-b-PEG-Mal (5 mg mL-1) and ivermectin (1 mg mL-1) was made in 1 mL of DMF. The solution was added dropwise to 10 mL of Dl water with constant stirring (900 RPM) at room temperature and stirred for 2h. Nanoparticles (NPs) were washed 3 times with nanopure water with amicon ultracentrifugation filtration membranes with a molecular weight cutoff of 100 kDa (2800 rpm, 4°C). Formed NPs were suspended in water and stored at 4°C. NP size (diameter, nm), PDI, and surface charge (zeta potential, mV) were obtained from three independent measurements. For TEM studies, NP solution was diluted with water, and 4% uranyl acetate added into the solution to stain the NPs. The NP mixture was vortexed and dropped into a copper grid and dried overnight at room temperature using a JEOL JEM-1400 equipped with a Gatan Orius SC 200D CCD digital camera.
Sample Preparation for TEM
[000108] Nanoparticles (5 mg/mL with respect to total polymer) were diluted 100 times using nanopure water. The NP solution (1 mL) was mixed with a 4% solution of uranyl acetate solution (5 pL) and vortexed. The solution was filtered with a 0.45-micron filter and ~20 pL was dropped on a dark side of the copper grid and allowed to dry for 24 hours in desiccator at room temperature. TEM images were recorded using JEOL JEM-1400 instrument.
Release Kinetics of Ivermectin from NPs
[000109] Release of ivermectin from the nanoparticles was evaluated in PBS (pH = 7.4) and PBS of pH = 6.0 (pH was adjusted using concentrated 0.1 N HCI) to mimic the physiological conditions of colon (pH = 6.0) and blood (pH = 7.4). Nanoparticle solutions were prepared by diluting 4 times with nanopure water and 200 pL of diluted solution was added to dialysis tubes (Thermo Scientific, MWCO = 10,000). Dialysis tubes were submerged in respective buffer solutions and kept in a shaking incubator at 37 °C for up to 96 h. For the first 6h, buffer was replenished every hour with fresh buffer and buffer changed every 12 h. Samples were collected at predetermined time points, dissolved in acetonitrile and analyzed by HPLC for remaining IVM concentrations. The release of the IVM was then determined. HPLC studies were carried out using an Agilent 1200 series instrument to quantify the amount of ivermectin. Dialysis solution (20 pL) from the dialysis tube in acetonitrile (0.1% trifluoroacetic acid) was injected into the HPLC. A 10:70:20 water:acetonitrile:methanol solution was used as a mobile phase in the Zorbax C18 columns. A detection wavelength of 268 nm was used for in the experiments. Ivermectin concentration remaining in the samples was quantified by HPLC using ivermectin standards, and values subtracted from the concentration of IVM that was initially added to the dialysis tubes, to calculate the amount of IVM released at the various time points.
Cell Viability Assays:
[000110] The cytotoxicity of ivermectin, NT-OH-IVM-NP, and T-Fc-IVMNP was tested in Caco- 2 cells using an MTT assay. Cells were plated (3000 cells/well) in a 96-well plate and allowed to grow overnight. Media was changed and increasing concentrations of each article was added. Media was aspirated and fresh media added, and cells further incubated for an additional 48 h, after which 20 pL/well MTT was added (5 mg/mL Stock in PBS) and incubated for 5 h in order for MTT to be reduced to purple formazan. Media was removed and cells lysed with 100 pL of DMSO. In order to homogenize the formazan solution, plates were subjected to 10 min of gentle shaking and absorbance at 550 nm measured with a background reading at 800 nm. Control values were set to 100% of cell viability. Cytotoxicity data (where appropriate) was fitted to a sigmoidal curve and a three parameters logistic model used to calculate the IC50, which is the concentration of chemotherapeutics causing 50% inhibition in comparison to untreated controls. Mean IC50 is the concentration of agent that reduces cell growth by 50% under experimental conditions and is the average from at least three independent measurements that were reproducible and statistically significant.
Mitostress Assay
[000111] Different parameters of mitochondrial respiration such as basal respiration, maximal respiration, coupling efficiency, and spare respiratory capacity were investigated using seahorse XFe96 Analyzer. One day prior to the assay, XF sensor cartridges were hydrated using 200 pL of XF calibrant buffer and kept at 37°C incubator without CO2 overnight. Caco-2 cells were plated at a density of 20,000 cells per well in 80 pl_ DMEM media (with 20% FBS) and the plate kept at room temperature for 1 hour followed by incubation at 37°C with 5% CO2 for 3 h. Fresh media (130 mI_) was added to a total of 180 mI_ per well and incubated for 16 h. Media was aspirated and various compounds ( i.e 10 mM ivermectin) added to a final volume in each well of 180 mI_. Cells were incubated for additional 24 h; before conducting the Mitostress assay, Seahorse media (XF Assay Medium Modified DMEM) was reconstituted with glucose (1.8 mg/mL), sodium pyruvate (1%) and L-glutamine (1%) and adjusted to pH 7.4 using 0.1 N HCI. Cells were washed thrice with freshly prepared seahorse medium and incubated at 37°C in a non-CC>2 incubator for 1 h. Cartridge ports were added with various inhibitors. Stocks of oligomycin (10 mM), FCCP (10 mM) and antimycin-A/rotenone mixture (10 mM each) were made in seahorse media. Port A was filled with 20 mI_ of oligomycin, port B with 22 mI_ of FCCP and port C with 25 mI_ of antimycin A/rotenone to a final concentration of 0.1 mM in each well. The cartridge was calibrated for pH and 02. After calibration, the experiment plate was run where 3 measurements were recorded for basal OCR and after addition of each reagent. The media was aspirated and 20 mI_ of RIPA buffer was added to each well and incubated for 10 mins at 37°C. Further BCA assays were performed to obtain protein normalized OCR values.
In vitro epithelial barrier experiment
[000112] Caco-2 cells were plated in trans-well plate with a density of 50,000 cells/well on the apical side in 500 mI_ of DMEM (with 20% FBS) media. On the basal side, 1 mL of fresh media was added, and cells were grown up to 9 days. Before the addition of compounds, the integrity of the monolayer was monitored by TEER (transepithelial electrical resistance) using Epithelial Volt-ohmmeter. Media was replenished every two days. On the ninth day, 2 pg/mL of IgG-Fc (Fc) fragment was added in order to find out the targeting ability of the Fc-targeting nanoparticles. After two hours, ivermectin, NT-OH-IVM-NP, and T-Fc-IVM-NP were added to the cells with a concentration of 20 pg/mL, with respect to ivermectin, and were incubated for 12 h. Apical and basal media were collected in eppendorf tubes and dissolved in 2 mL of acetonitrile. To spike the peak for ivermectin, 10 pg/mL of ivermectin was added to the collected media. This mixture was sonicated for 20 mins followed by centrifugation at 5000 rpm for 10 mins. From the precipitated debris, supernatant was gently collected. Meanwhile, Strata C18-T columns were activated by passing 1 mL of methanol and water through the filter in sequence. The collected supernatant was passed through the activated column in order to get rid of remaining debris and impurities. The column was washed with 1-2 mL of 5% methanol in order to remove the impurities. The ivermectin from the column was collected in 2 mL of methanol and quantified using HPLC (wavelength = 268 nm at 21.01).
Immunofluorescence
[000113] In order to confirm the formation of the epithelial barrier by Caco-2 cells, immunostaining of tight junction protein (ZO-1) was performed. Caco-2 cells were plated in trans-well plate with density of 50000 cells/well on apical side in 500 pl_ of DMEM (with 20% FBS) media. On the basal side, 1 mL of fresh media was added, and cells were grown up to 9 days. Before addition of the compounds, the monolayer integrity was monitored by TEER (Trans epithelial electrical resistance) using Epithelial Voltohmmeter. Media was replenished once in every two days. On the ninth day 2 pg/mL of IgG-Fc (Fc) fragment was added in order to found out the targeting ability of the Fc targeting nanoparticles. After two hours, NT-OH-IVM-NP and T-Fc-IVM-NP added with concentration of 20 pg/mL with respect to ivermectin and incubated for 12 h. The cells were washed with PBS (1X) 3 times and fixed with 4% paraformaldehyde for 1 hour at 37 °C. After performing 3 washings, cells were permeabilized using 0.1% Triton-X100 for 10 min at 37 °C. The cells were washed with 1X PBS 3 times and blocked with 1% goat serum in 1X PBS for 12 h. Cells were treated with the respective primary antibody (ZO-1 antibody, #40-2200) in 1% goat serum containing 1X PBS for 12 h at 4 AaC in humidified chamber. After washing the cells for three more times with 1 % goat serum containing 1 X PBS, the respective secondary antibody (Alexa 488 conjugated anti-chicken antibody, ab150169) solution in 1% goat serum containing 1X PBS was added along with DAPI and incubated for 1 hour at room temperature. Cells were washed three times with 1% goat serum containing 1X PBS. The membrane was gently removed and kept on glass slides and coverslipped using mounting solution (n-propyl gallate, Tris and glycerol in nanopure water, pH = 8.0). Confocal images were recorded using an Olympus FluoView FV3000 confocal microscope using 405/460 nm for DAPI and 488/510 nm for Alexa488.
Biodistribution of QD loaded NPs:
[000114] Biodistribution properties of NT-OH-QD-NP and T-Fc-QD-NP along with saline were determined in balb/c female mice weighing ~22 g. Three mice per group (n=3) were used in these studies. Before oral dosage, animals were fasted for 8 h. NP (50 mg/kg) was administered via oral gavage. Saline (100 mI) of saline was injected via oral gavage as control. After 24 h, around 300 pL of blood was collected in heparinized tubes via cardiac puncture. Animals were perfused using 1X PBS for 10 min with a flow rate of 7 mL/min. Blood was centrifuged to collect blood plasma. After 24 h, animals were sacrificed, and organs were harvested. Biodistribution was followed by performing ICP-MS on digested organs and plasma. Before digestion, portions of the intestine, including the duodenum, jejunum, Ilium and colon, were stored at -80 °C to use for further studies. The organs were weighed and digested in aquaregia (all organs were dissolved in 3 mL except liver; liver was dissolved in 10 mL) and the amount of the QD was determined using ICP-MS. The percentage of NP in the blood was calculated by taking into consideration that blood constitutes 7% of body weight and plasma constitutes 55% of blood volume.
Immunofluorescence Studies of Duodenum:
[000115] To study T-Fc-QD-NP co-localization in the duodenum FcRn antibody was used. Slides with tissue sections (5 pm) were de-paraffinized in xylenes followed and rehydrated in graded ethanol (100% to 10%). Brain sections were baked in 10 mM sodium citrate buffer (Target Retrieval Solution, Citrate pH 6, Dako, USA) for 15 min for antigen retrieval using Biocare decloaking chamber (# DC2012; Biocare Medicals, USA) and then maintained at a sub boiling temperature for 10 min. Blocking was performed in 5% goat serum in phosphate buffered saline-Tween 100 (PBST) for 1 h at room temperature. Primary antibody (Fc-Rn, SC-393064) was applied at 4 AaC in a humidified chamber for overnight incubation, with final concentrations of 1:1000 in 1% PBST. Secondary antibody (Goat Anti-mouse; #ab ab6785) was applied after 5 TBST washes and incubated for 1 h with a final concentration of 1 :2000 in 1% goat serum in PBST. Coverslips were mounted on glass slides and covered with nail polish. Confocal images were recorded using an Olympus FluoView FV3000 confocal microscope using 405/705 nm for QD and 488/510 nm for Alexa488. As seen from the images, the T-Fc-QD-NPs showed significantly higher accumulation in the duodenum over NT-OH-QD-NPs.
Biodistribution of IVM loaded NPs:
[000116] Female balb/c albino mice were used to understand the ivermectin and T-Fc-IVM- NPs distribution and toxicity after oral administration in vivo. NT-OH-IVM-NP served as a non- targeted control. Animals were divided into four groups, of three animals: Group 1- Saline, Group 2- Ivermectin, Group 3- NT-OH-IVM-NP, and Group 4- T-Fc-IVM-NP. Before oral dosage, animals were fasted for 8 h. Animals in each group received saline treatment, ivermectin, targeted NPs, or nontargeted NPs via oral gavage. The dose of nanoparticle was 40 mg/kg with respect to ivermectin weight. Animals were sacrificed after 24 h and organs harvested. Blood (-200 pl_) was collected in heparinized tubes via cardiac puncture. Perfusion was performed with 1X PBS for 10 min with a flow rate of 7 mL/min. Collected blood was centrifuged to collect blood plasma. Organs were weighed and homogenized using a dounce homogenizer and collected in 2 mL of acetonitrile. To spike the ivermectin peak, 20 pg/mL of ivermectin was added to the crushed tissues and to the blood plasma and the mixture sonicated for 20 mins followed by centrifugation at 5000 rpm for 10 mins and supernatant collected.
Strata C18-T columns were activated by passing 1 mL of methanol and water through the filter in sequence. Supernatant was passed through the activated column for purification and the column washed with 1-2 mL of 5% methanol in order to remove the impurities. The ivermectin from the column was collected in 2 mL of methanol and quantified using HPLC (wavelength = 268 nm at 21.01 ). The percentage of ivermectin in the blood was calculated by taking into consideration that blood constitutes 7% of body weight and plasma constitutes 55% of blood volume.
Western blot of intestine tissue:
[000117] The stored intestinal tissues from the above study were homogenized and collected in RIPA buffer and incubated for 30 mins on ice. Protein fractions were collected from digested tissues by centrifugation at 10,000 rpm for 20 min. Tissue proteins were quantified using a bicinchoninic acid (BCA) assay. Protein (60 pg ) was used in each well along with 1X Laemmli sample buffer and electrophoresis performed at 100 mV for 2 h. Resolved proteins were transferred onto a PVDF membrane at 50 mV and 4°C for 2 h. The membrane was blocked for 1h in blocking buffer (5% skim milk powder in TBST. The membrane was incubated at 4 °C overnight for primary antibody (FcRn and b-actin) and washed 5 times with TBST buffer and incubated with appropriate secondary antibodies at room temperature for 1 h. Membranes were washed five times with TBST buffer and developed using ECL. Images were taken using a BioRad ChemiDoc™ imaging system and bands quantified using ImageJ software.
Enzyme-linked immunosorbent assay (ELISA) to determine immunogenic effect
The levels of pro-inflammatory cytokines IL-1 b, IL-6, and TNF-a in plasma were determined using ELISA kits following the manufacturer’s protocol. Briefly, 100 pL/well of coating buffer with capture antibody was added to the 96-well ELISA plate and the plate sealed overnight at 4°C. Buffer was aspirated from the wells and cells washed 3 times with 300 pL/well of wash buffer. After the last wash, the plate was inverted and blotted on absorbent paper to remove any residual buffer. Wells were blocked with 200 pL/well of assay diluent and incubated at room temperature for 1 h. The diluent was aspirated from the wells and the wells washed. Standards (100 mI_) in assay diluent, as well as the samples (50 mI_ of plasma sample + 50 mI_ of assay diluent), were added to the appropriate wells. The plate was sealed and incubated for 2 h at room temperature. Solutions were aspirated from the wells and the wells washed. Detection antibody (100 mI_; IL-1 b, IL-6, and TNF-a) diluted in assay diluent was added to each well, the plate sealed and incubated for 1 h at room temperature. Enzyme reagent (100 mI_) was added to each well after the last wash. The plate was sealed and incubated at room temperature for 30 min. The reagent was aspirated from wells, and substrate solution (100 mI_) was added to each well and the plate sealed and incubated for 30 minutes at room temperature in the dark. Stop solution was added to each well and the absorbance was recorded at 450 nm.
Aspartate Aminotransferase (AST) Activity Assay
Plasma was used to determine AST activity as an indicator of liver function. Samples and standards were studied in duplicates. Serum samples were used to determine the AST levels. From each sample, 50 mI_ of serum was added to a 96-well plate along with 50 mI_ glutamate standards at concentrations of 0, 2, 4, 6, 8, and 10 nmol/well prepared in AST assay buffer. To each well, 100 mI_ of master reaction mix (80 mI_ of AST Assay Buffer, 2 mI_ of AST Enzyme Mix, 8 mI_ of AST Developer and 10 mI_ of AST Substrate) was added and incubated for 5 min at 37 °C in the dark. Absorbance at initial time, T initial, was (A450)initiai, and at the end the final time point, Tfinai, was at (A450)finai. The absorbance was measured at 450 nm at the initial time.
Calculated the change in absorbance from T initial toTfinai for the samples.
DA450 = (A450)final - (A450)initial
The amount of generated glutamate using standard curve determined for above obtained DA450 (B).
The AST activity of a sample was determined by the following equation:
AST Activity (milliunit/mL) = B/Reaction Time * V
Where B = Amount (nmole) of glutamate generated between nitiai and Tfinai- Reaction Time (min) = Tfinai — T initial V = sample volume (mL) added to well
Alanine Aminotransferase Colorimetric Activity (ALT) Assay: [000118] Plasma was used to determine ALT activity. Using Cayman’s ALT Assay Kit Measurement of the ALT activity was carried out by monitoring NADH oxidation rate in a coupled reaction system employing lactate dehydrogenase (LDH). The oxidation of NADH to NAD+ is accompanied by a decrease in absorbance at 340 nm. Under circumstances in which the ALT activity is rate limiting, the rate decrease is directly proportional to the ALT activity in the sample. Substrate (150 pL), 20 pL of Cofactor, and 20 pL of sample were added to each well of a 96 well plate and incubated at 37°C for 15 min. The reaction was initiated by the addition of 20 pL of ALT initiator and absorbance recorded immediately at 340 nm once every minute for five minutes. The change in absorbance (DA340) per minute was determined using the following equation.
DA /min = A340 (Time 2) - A340 (Time 1 )/ Time 2 (min) - Time 1 (min)
ALT activity was determined using following formula = (AA34o/min x 0.21 mL)*4.11 mM 1 x 0.02 mL
The reaction rate at 340 nm was determined using the NADH extinction coefficient of 4.11 mM 1.
The hepatotoxicity was monitored by measuring the levels of alanine aminotransferase (ALT) and (H) aspartate aminotransferase (AST) in the blood plasma. AST and ALT levels were not affected by treatment.
H&E imaging:
Harvested organs (brain, liver, lungs, kidney, and spleen) were snap frozen and post-fixed in 4% PFA for 48 hours. Section (5 pm) were stained with hematoxylin (H) and eosin (E) and scanned with images captured using the Olympus VS120 image analysis system (Olympus America Inc., Philadelphia, PA).
Bacterial culture for Zika NS1 and control plasmid
[000119] Agar plates (1.5%) containing 100 pg/mL ampicillin were prepared and streaked with Flag-tagged Zika NS1 and control plasmid. Plates with NS1 and control plasmid were incubated at 37°C and 30°C respectively for 16 h. Single colonies were inoculated in 5 mL of LB broth media containing 100 pg/mL ampicillin for primary culture and incubated at 37°C with shaking for 16 hours. Plasmid isolation using Midiprep kit
NS1 and control plasmids were isolated using Qiagen Midiprep plasmid isolation kit. Overnight grown bacterial culture was harvested by centrifuging at 4500 rpm for 20 minutes at 4°C. Supernatant was discarded and the bacterial pellet dissolved in 4 mL of buffer P1 and mixed. Buffer P2 (4 mL) was added and mixed thoroughly by vigorously inverting the tube 4-6 times followed by incubation at room temperature for 5 minutes. Bbuffer P3 (4 mL) was mixed thoroughly by vigorously inverting the tube 4-6 times and incubated on ice for 15 minutes. Tubes were centrifuged at 4500 rpm for 30 minutes at 4°C. Qiagen midi columns were equilibrated with 4 mL of buffer QBT and bacterial supernatants loaded. Plasmid was eluted using QF buffer and Isopropanol to precipitate the plasmid. Plasmids were dissolved in autoclaved water and the purity and quantification of plasmid was checked via Nanodrop.
NS1 Protein expression in HEK293T cells
[000120] HEK293T cells (0.3 x 106) were seeded overnight in 6-well plates with 10% FBS containing DMEM medium. Cells were transfected with 2 pg of Flag-tagged Zika NS1 plasmid and an empty vector using turbofectin (turbofectin: DNA-3:1) in Opti-MEM medium. Six hours post-transfection, medium was removed and replaced with fresh medium and incubated for additional 6 h. Cells were treated with 10 mM concentration of ivermectin (IVM), T-Fc-IVM-NP and NT-OH-IVM-NP for 6 h, cells lysed and total cell lysates (60 pg) resolved on a 4-20% gradient gel. Proteins were transferred to PVDF membrane and probed with Anti-flag (1:1000) and b-actin (1:1000) antibody overnight at 4°C, washed with TBST and probed with HRP- conjugated anti-mouse secondary antibody (1:2000) for 1 h at room temperature. Membranes were developed using SuperSignal west pico chemiluminescence substrate (Thermo Scientific).
NS1 Expression by Immunofluorescence
[000121] HEK293T cells were plated on coverslips in a 12 well plate at a density of 20,000 cells/well in 1 mL of DMEM (with 10% FBS) media. Cells were transfected with 2 pg of Flag- tagged Zika NS1 plasmid (addgene# 79641) and an empty vector using turbofectin (turbofectin: DNA-3:1) in Opti-MEM medium. After 6 h post transfection, medium was removed and replaced with fresh medium and incubated for additional 6 hours. Cells were treated with ivermectin (IVM), T-Fc-IVM-NP, or NT-OH-IVM-NP at a concentration of 10 pM IVM for 6 h. Cells were washed with PBS and fixed with 4% paraformaldehyde for 1 h at 37°C. Cells were permeabilized using 0.1% Triton-X100 for 10 min at 37°C, washed with 1X PBS 3 times and blocked with 1% goat serum in 1X PBS for 12 h. Cells were treated with the respective primary antibody (Anti-flag (1 :1000) in 1% goat serum containing 1X PBS for 12 h at 4°C in humidified chambers. Cells were washed three more times with 1 % goat serum containing 1X PBS, appropriate secondary antibodies (Alexa 488 conjugated anti-mouse antibody) added along with DAPI and incubated for 1 hour at room temperature. Cells were washed and membranes gently removed and kept on glass slides and covered with coverslips using mounting solution (n-propyl gallate, Tris and glycerol in nanopure water, pH = 8.0). Confocal images were captured using an Olympus FluoView FV3000 confocal microscope using 405/460 nm for DAPI and 488/510 nm for Alexa488.
Temperature-Dependent Stability:
[000122] NPs were prepared for the temperature dependent stability using 5 mg/mL PLGA- PEG-Mal alone (for Mal-NP) or using 5 mg/mL PLGA-PEG-Mal polymer and 1 mg/mL ivermectin (for Mal-IVM-NP). Solutions were stirred for 2 hours, then filtered using Amicon filtration (100 MWCO) at 2800 RPM. Three 1 mL solutions of both Mal-NP and Mal-IVMNP were prepared, and after initial DLS measurements of size and zeta potential were taken, the solutions were stored at 4°C, room temperature, and 37°C. Every 3 days, solutions were removed from their respective locations, allowed to equilibrate for approximately 15 minutes, and the size and zeta potential measurements taken and repeated for up to 36 days. Measurements were discontinued early for the 37 °C samples as the sample displayed obvious signs of deviating from the normal size and zeta potential of the nanoparticles.
Dry Formulation:
(i) Freeze-thaw
[000123] Mal-IVM-NPs were prepared in a similar fashion as described above, but with starting concentrations as 10 mg/mL PLGA-PEG-Mal polymer and 2 mg/mL ivermectin. In addition to the control sample (NP alone), NPs were mixed with the cryoprotectants sucrose and trehalose in NP:cryoprotectant ratios of 1:0.1 , 1 :0.5, 1 :1, and 1 :2. Final volumes of each sample were 400 pL, and each contained the same amounts of nanoparticles. DLS measurements (size and zeta potential) of the samples were taken prior to placing samples -80 °C and measurements of size and zeta potential captured every hour. This was repeated for 3 cycles, each of which had the NPs at -80°C for one hour.
(ii) long-term [000124] Long-term storage of the Mal-IVM-NPs at -80°C with and without cryoprotectants was used to gauge the ability of the cryoprotectants to maintain the NP stability (based on size and zeta potential) over an extended period of time. Mal-IVM-NPs were produced as described above, with 10 mg/mL PLGA-PEG-Mal polymer and 2 mg/mL ivermectin. Because the previous testing revealed that the higher concentrations of cryoprotectants maintained size and zeta potential of the NPs best, only samples consisting of NPs alone and in the presence of sucrose and trehalose, in ratios of 1 : 1 and 1 :2 (NP:cryoprotectant ratio) were used. The total volume of each sample was 50 pL. Sixteen total vials of each sample were produced, because the testing was expected to run for up to 6 months. Size and zeta potential measurements were taken after 1 , 2, 3, 4, 5, 6, 7, 15, 30, 45, 60, 75, 90, 120, 150, and 180 days.
[000125] From previous testing, it was observed that nanoparticles often gave more consistent size and zeta potential measurements when allowed to thaw for a slightly longer period of time. This allowed the NPs in solution, regardless of whether cryoprotectants were present or absent, to stabilize at room temperature and properly equilibrate. Thus, during the long-term storage testing, NPs were allowed to thaw for between 30 and 45 min, to ensure the temperature of the solution reached close to room temperature.
(iii) drying process
[000126] NPs must be dried and made into powdered form in order to eventually be packed into capsules to serve as a viable antiviral treatment. Mal-IVM-NPs were produced as described above, with 10 mg/mL PLGA-PEG-Mal polymer and 2 mg/mL ivermectin. NPs were dried, with and without the cryoprotectants, using low pressure and temperature. Sucrose and trehalose were added in 1:2 NP:cryoprotectant ratios, and a third sample contained NPs alone. NPs were dried at approximately -50 °C and 2 Pa using a VirTis Benchtop K Freeze Dryer. Powdered NPs were reconstituted in 1 mL nanopure water. Size and zeta potential measurements were taken, and data suggested that NPs dried in the presence of sucrose displayed the best results with slightly increased size.
Placental Barrier Formation:
[000127] JEG-3 cells were plated in transwell plate at a density of 50,000 cells/well on the apical side in 500 pL of DMEM (with 10% FBS) media. On the basal side, 1 mL of fresh media was added and cells grown for up to 9 days. Prior to the addition of compounds, the integrity of the monolayer was monitored by TEER (transepithelial electrical resistance) using Epithelial Volt-ohmmeter. Media was replenished every two days and on the ninth day, ivermectin, NT- OH-IVM-NP, or T-Fc-IVMNP were added to the cells at a concentration of 20 pg/mL with respect to ivermectin, and were incubated for 12 h. Apical and basal media were collected in eppendorf tubes and dissolved in 2 mL of acetonitrile. As an internal standard, 10 pg/mL of ivermectin was added to the collected media, the mixture sonicated for 20 min followed by centrifugation at 5000 rpm for 10 min and the supernatant gently collected. Strata C18-T columns were activated by passing 1 mL of methanol and water through the filter in sequence. Columns were washed with 1-2 mL of 5% methanol in order to remove the impurities and ivermectin from the column was collected in 2 mL of methanol and quantified using HPLC (wavelength = 268 nm at 21.01). In order to confirm the formation of the epithelial barrier, immunostaining of tight junction protein (ZO-1) was performed. Caco-2 cells were plated in trans-well plate with density of 50000 cells/well on apical side in 500 pL of DMEM (with 20% FBS) media as described above.
SARS-CoV-2 Spike Protein Plasmid Purification
[000128] Bacterial agar plates (1.5%) containing 100 pg/mL ampicillin were prepared and streaked with SARS-CoV-2 spike protein plasmid and incubated at 37 °C for 16 h. A single colony from the plate was inoculated in 5 mL of LB broth media containing 100 pg/mL ampicillin for primary culture. The culture was incubated at 37°C in an incubator shaker for 16 h. Spike protein plasmid was isolated using Qiagen Midiprep plasmid isolation kit. Bacterial culture grown overnight was harvested by centrifuging at 1900g for 20 min at 4 °C. The supernatant was discarded and the bacterial pellet was dissolved in 4 mL of buffer P1 and mixed properly, buffer P2 added and tubes mixed thoroughly incubated at room temperature for 5 min. Buffer P3 (4 mL) was mixed thoroughly by vigorously inverting the tube four to six times and incubated on ice for 15 min followed by centrifugation at 1900g for 30 min at 4 °C. The Qiagen midi column was equilibrated with 4 mL of buffer QBT and bacterial supernatant was loaded onto the column, and the column allowed to empty by gravity flow. The plasmid was eluted from the column by using QF buffer and isopropanol used to precipitate the plasmid. The plasmid was dissolved in water and the purity and quantification of plasmid measured using Nanodrop.
ACE2 and Spike Protein expression by Western Blot
[000129] HEK293T cells, HeLa cells, and A549 cells (1 A~ 105) were seeded in 6-well plates in 10% FBS-containing DMEM medium and were incubated overnight. Cells were transfected with 2 pg of SARS-CoV-2 spike protein plasmid using turbofectin (turbofectin: DNA-3:1) in Opti- MEM medium. Six hours post-transfection, media was removed and replaced with fresh media and samples incubated for additional 6 hours. Cells were treated with 10 mM concentration of IVM, NT-IVM-NP, and T-Fc-IVM-NP for 24 hours. In a separate experiment to test the time dependent effects of free IVM and IVM-NPs, the articles in the same concentration were added to cells for 2, 4, and 6 h, followed by incubation of cells in normal media for up to 24 hours.
Cells were lysed and the total cell lysate (30 pg) resolved on a 4-20% gradient gel. Proteins were transferred to PVDF membranes and probed with HA-tag (1:1000), ACE2 (1 :1000), and b- actin (1:1000) antibodies overnight at 4°C. After 3 TBST washes, the membrane was probed with HRP-conjugated secondary antibody(1 :2000) for 1 h at room temperature. The membrane was washed 5 times with TBST and developed using Super Signal west pico- chemiluminescence substrate (Thermo Scientific). Densitometric analysis of western blots was performed using ImageJ software.
Importin Proteins expression by Western Blot
[000130] HEK293T cells (1 A~ 105) were seeded in 6-well plates in 10% FBS-containing DMEM medium and incubated overnight. Cells were transfected with 2 pg of SARS-CoV-2 spike protein plasmid using the transfection reagent turbofectin (turbofectin: DNA-3:1) in Opti- MEM medium. Six hours post-transfection, media was removed and replaced with fresh media and incubated for additional 6 hours. IVM and its nanoformulations were added to cells at 10 pM concentrations with respect to IVM to cells for 2, 4, and 6 h, followed by incubation of cells in normal media up to 24 hours total. Cells were lysed and the total cell lysate (30 pg) was resolved on a 4-20% gradient gel. Proteins were transferred to PVDF membrane and probed with IMPa (1 :1000), IMRb1 (1 :1000), and b-actin (1:1000) antibodies overnight at 4°C. Membranes were washed and probed with HRP-conjugated secondary antibody (1:2000) for 1 h at room temperature. The membrane was washed 5 times with TBST and developed using Super Signal west pico-chemiluminescence substrate (Thermo Scientific). Densitometric analysis of western blots was performed using ImageJ software.
ACE2 Expression by Immunofluorescence
[000131] HEK293T cells, HeLa cells, and A549 cells were each plated on coverslips in separate 12-well plates at a density of 20,000 cells/well in 1 mL of DMEM (with 10% FBS) media. Cells were transfected with 2 pg of SARS-CoV-2 spike protein plasmid and an empty vector using turbofectin (turbofectin: DNA-3:1) in Opti-MEM medium. Six hours post transfection, medium was removed and replaced with fresh medium and incubated for additional 6 hours. Cells were treated with IVM, NT-IVM-NP, or T-Fc-IVM-NP at a concentration of 10 pM with respect to IVM for 6 h. Cells were washed with PBS (1X) 3 times and fixed with 4% paraformaldehyde for 1 h at 37°C. Cells were washed again and permeabilized using 0.1% Triton-X100 for 10 min at 37°C. The cells were washed with 1X PBS and blocked with 1% goat serum in 1X PBS for 12 h. Cells were treated with the respective primary antibody in 1% goat serum containing 1X PBS) for 12 h at 4°C in a humidified chamber. Respective secondary antibody (Alexa 488 conjugated antibody) solution in 1% goat serum containing 1X PBS was added along with DAPI and incubated for 1 hour at room temperature. Cells were washed and the membrane gently removed and kept on glass slides and covered with coverslips using mounting solution (n-propyl gallate, Tris and glycerol in nanopure water, pH = 8.0). Confocal images were recorded using an Olympus FluoView FV3000 confocal microscope using 405/460 nm for DAPI and 488/510 nm for Alexa488.
RT-qPCR
[000132] HEK293T cells were seeded in 6 well plate and transfected with 2pg of plasmid using turbofectin. Cell were treated with ivermectin, NT-IVM-NP and T-Fc-IVMNP for 4 h at a concentration of 10 mM, medium was removed, and cells were kept for additional 20 hours. RNA was extracted by harvesting cells with trypsin and lysed with buffer RLT. Ethanol (70%,) was added to the cell lysate , lysates transferred to RNeasy mini spin columns and centrifuged for 1 minute at 8000 rpm. Flow-throughs were discarded and 700 pl_ of buffer RW1 was added to the mini spin column and centrifuged for 1 for minute at 8000 rpm. Buffer RPE (500 mI_) was added to the mini spin column and centrifuged for 1 minute at 8000 rpm. RNA was recovered from mini spin column using RNase-free water. Purity and concentration of RNA was checked using Nanodrop.
[000133] Reverse transcription from each sample was carried out using 1 pg of RNA using iScript Reverse Transcription Supermix. Real time PCR reaction was performed using SsoAdvanced Universal SYBR® Green Supermix in a 20 pL reaction. Beta-actin was used as an internal control and data analyzed using the comparative Ct value and expressed as fold change 2DD0T. The forward and reverse primer sequence was
5OCAGTACGCCATGTAACGGA3’ (SEQ ID NO: 1) and 5’CGTGGAGGAGCTCAAAGGAC 3’ (SEQ ID NO: 2) respectively for spike gene. The primer for human ACE2 gene was purchased from Sino Biological Inc (catalogue number: HP100185). The primer sequence for b-actin gene was: Forward 5’ GCATCCTCACCCTGAAGTAC 3’ (SEQ ID NO: 3) and reverse 5’GATAGCACAGCCTGGATAGC 3’ (SEQ ID NO: 4). Pseudo SARS-CoV-2 Reporter Assay and Virus Uptake:
[000134] The Angiotensin Converting Enzyme 2 (ACE2)-Red reporter assay and Pseudo SARS-CoV-2 Green Reporter assay were performed with the goal of increasing ACE2 expression in HEK293T cells and A549 cells and observing the effects of IVM-loaded nanoparticle treatment on ACE2 expression and SARS-CoV-2 virus cell entry. These effects were measured through fluorescence imaging using two different methods of treatment: a preventive method, in which IVM and IVM nanoformulation treatment preceded pseudovirus infection in the cells, and a therapeutic method, in which IVM and IVM nanoformulation treatment followed pseudovirus infection in the cells. The preventive method’s aim was to decrease ACE2 levels initially through IVM treatment so that the rate at which pseudoviruses entered cells would decrease. In contrast, the therapeutic method aimed to interrupt ACE2 and pseudovirus binding and show a lowering in pseudovirus infection presence after the treatment. a. Pseudo SARS-CoV-2 Uptake by Plate Reader [000135] HEK293T cells and A549 cells were detached using a standard trypsinization protocol and then counted. Cells were prepared for plating in a 96-well plate at 50,000 cells per well, using 100 pl_ of media for each well. These cells were maintained in a single test tube at 500,000 cells per ml_, ready to be mixed with the viral transduction reaction. For each well with a transduction reaction, using the Red ACE2 Reporter kit (C1100R), 5 mI_ of the fluorescent ACE2 BacMam was mixed with 0.6 mI_ of the 500 mM sodium butyrate (SB) solution and 44.4 mI_ of the complete culture media for the HEK293T cells, for a total volume of 50 mI_. Next, the tube containing cells and tube containing the transduction reaction mix were mixed and kept for 5 minutes at room temperature. 150 mI_ of this mixture was added to each well of the 96-well plate. This plate was covered to protect from light, and kept for 30 minutes at room temperature. Cells were then incubated for 24 hours under standard cell growth conditions. Then cells were treated with IVM, NT-IVMNPs, and T-Fc-IVM-NPs at a concentration of 10 mM with respect to IVM. These treatments were kept for 24 hours, after which media was removed. In a separate experiment to test the time-dependent effects of free IVM and IVM-NPs, the articles in the same concentration were added to cells for 2, 4, and 6 h, followed by incubation of cells in normal media up to 24 h total. Then, 100 mI_ of media containing 8 x 107 viral genes of pseudo-SARS- CoV-2 were added to each well from the stock solution of 2 x 1010 viral genes per mL (C1110G). Cells were incubated for 1 hour at normal incubation conditions (37 °C, 5% C02), after which media was changed to remove the pseudovirus and replaced with fresh media containing the 2mM sodium butyrate. The cells were then incubated for 12 hours in the dark, under normal cell growth conditions. Prior to fluorescence reading, the cells were washed (2 times, PBS) gently. Finally, 100 pL of PBS was added and the florescence levels were measured using plate reader measurements at 506/517 (Ex/Em) and 558/603 (Ex/Em). [000136] This described procedures details the preventative method, but the therapeutic option was also explored, in which the after the pseudovirus addition, IVM and IVM-loaded nanoparticle treatments. Besides this change in order, the rest of the protocol was kept the same. b. Pseudo SARS-CoV-2 Uptake by Confocal Microscopy [000137] HEK293T cells were detached using a standard trypsinization protocol and then counted. Cells were prepared for plating in an 8-well live cell imaging chamber at 20,000 cells per well, using 100 pl_ of media for each well. These cells were maintained in a single test tube at 500,000 cells per ml_, ready to be mixed with the viral transduction reaction. For each well with a transduction reaction, using the Red ACE2 Reporter kit (C1100R), 5 mI_ of the fluorescent ACE2 BacMam was mixed with 0.6 mI_ of the 500 mM sodium butyrate (SB) solution and 44.4 mI_ of the complete culture media for the HEK293T cells, for a total volume of 50 mI_. Next, 150 mI_ of this mixture was added to each well. This chamber was covered to protect from light, and kept for 30 minutes at room temperature. Cells were then incubated for 24 hours under standard cell growth conditions. Then cells were treated with IVM, NT-IVM-NPs, and T-Fc-IVM-NPs at a concentration of 10 mM with respect to IVM. These treatments were kept for 24 hours, after which media was removed.
[000138] In a separate experiment to test the time-dependent effects of free IVM and IVM- NPs, the articles in the same concentration were added to cells for 2, 4, and 6 h, followed by incubation of cells in normal media up to 24 h total. Then, 100 mI_ of media containing 8 x107 viral genes of pseudo-SARS-CoV-2 were added to each well from the stock solution of 2 x 1010 viral genes per mL (C1110G). Cells were incubated for 1 hour at normal incubation conditions (37 °C, 5% C02), after which media was changed to remove the pseudovirus and replaced with fresh media containing the 2mM sodium butyrate. The cells were then incubated for 12 hours in the dark, under normal cell growth conditions. Prior to imaging, the cells were washed (2 times, PBS) gently. Finally, 100 mI_ of phenol red free media was added and images were obtained using an Olympus FluoView FV3000 confocal microscope.
EXAMPLE 1 Development of Orally Administrable Nanoparticle for Ivermectin Delivery.
[000139] We focused on biocompatible targeting strategies, FDA-approved polymer components, and the use of an FDA-approved drug IVM to provide an efficient and safer therapeutic platform for ZIKV. A number of factors need to be considered to design and optimize a nano-formulation of ivermectin, which can be administered orally for Zika infected patients: (i) the efficacy will depend on the stability of the NP under acidic pH of the stomach, NP capacity to show intestinal absorption, and ability of the NP to cross the intestinal epithelium to reach the circulatory system, (ii) a NP needs to have an appropriate pharmacokinetic (PK) profile that can result in a concentration of IVM in the circulation that is safe to the individual while resulting in an effective therapeutic dose, (iii) a controlled fashion to fit the window of effectiveness over a prolonged period of time, thus eliminating the need of repeated dosing, and (iv) for oral formulation, stability of the NP under acidic pH requires that these NPs are engineered with acid-resistant chemical linkages. Considering the above-mentioned criterion, an ideal NP system for oral delivery of IVM needs to have pH stability, intestinal absorption, and epithelium crossing ability, and should also demonstrate high IVM loading capacity and controlled release of the drug. It is also critical that orally delivered NP transports the payload efficiently from the intestine to the blood stream. The neonatal Fc receptor (FcRn) mediates immunoglobulin G (IgG) transport across the polarized epithelial barriers. FcRn is expressed at a level that is closely similar to fetal expression in the apical region of epithelial cells in the small intestine and diffuse throughout the colon in adulthood. FcRn binds to the Fc portion of IgG following a pH-driven pathway; acidic pH of <6.5 promotes binding of Fc to FcRn and physiological pH of ~7.4 releases the Fc from the FcRn binding pocket (Figure 1 A). Few recent studies demonstrated that Fc-decorated nano-vehicles show enhanced crossing of the intestinal barrier into the blood stream when administered orally. Disclosed herein is a biodegradable PLGA polymer based platform (Figure 1B). The linkers on the polymer are comparatively acid resistant to provide stability to the NPs under acidic pH in the stomach when administered via oral route (Figure 1B). All monomers and polymers were characterized by NMR spectroscopy (Figures 10-13). The polymers were also analyzed by gel permeation chromatography (GPC) demonstrating purity and monodisperse distribution (Figure 1C). First, we carried out encapsulation of IVM in PLGA-b-PEG-Mal polymer. In this study, we used 10, 20, 30, 40, and 50 percent feed of IVM with respect to the polymer. The NPs were synthesized by nanoprecipitation method (Figure 1 D). The drug loaded NPs were characterized by dynamic light scattering (DLS) for size and surface charge (Figure 2A, 14, and 15). We observed an increase in the size of NT-Mal-IVM-NP as percent feed of IVM was increased (Figure 2A). The morphology of IVM loaded NPs was determined by transmission electron microscopy (TEM) confirming spherical, homogeneous particle population (Figure 16). Ivermectin concentrations in the NPs were quantified using high performance liquid chromatography (HPLC). As IVM feed was increased from 10% to 50%, the NP size increased from ~ 60 nm to 140 nm. The NPs gave a very stable surface charge of nearly around -25.0 mV. The NPs with feed of 10% to 30% showed PDI -0.21 which suggested the formation of monodisperse particles. The 40% and 50% showed higher PDI of -0.45 indicating formation of higher aggregates. These studies established that the most well-defined and stable NPs can be achieved by IVM feed up to 30%. [000140] Polyclonal IgG Fc fragments were covalently conjugated on top of the NT-Mal-NPs using thiolene chemistry. 2-lminothiolane (Traut's reagent) was used to modify the NH2 groups of Fc fragments (FcNH2) with thiol groups (Fc-SH). Fc-SH was incubated with NPs for conjugation overnight at 4°C (Figure 1 E). As control, NPs with NH2 were allowed to react with the Fc fragment. T-Fc-NPs were purified via centrifugation and characterized by DLS (Figures 2B, 17, and 18). The NPs with NH2 upon interaction with Fc showed similar size (-60 nm) and zeta potential (~ -12 mV) as NTMal-NPs. The Fc-SH attached NPs showed increased in size (~ 88 nm) and (~ 22 mV). This increase is due to the covalent attachment of the Fc-SH on the NT Mal-NPs. Determination of Fc conjugation efficiency by the bicinchoninic acid assay (BCA assay) indicated high conjugation efficiency of -60% for empty NPs and -40% for 20% IVM feed loaded NPs (Figure 2B).
Example 2: Transport of T-Fc-IVM-NPs Across an in vitro Intestinal Epithelial Barrier Model. (ZIKA)
[000141] Previous studies supported that Caco-2 cells express human FcRn and human b2- microglobulin. NP transport ability across the Caco-2 monolayer were determined by quantifying IVM in the apical (AP) and basolateral (BL) side media using HPLC (Figure 3A). Caco-2 cells were plated in a transwell plate on the apical side. On the basolateral side, 1 mL of media was added and the cells were grown up to 9 days. Before the addition of NPs, the monolayer integrity was checked by measuring the trans-epithelial resistance (TEER) indicating TEER values of >800 W/cm2 on day 9 (Figure 3B, top). Media was replenished once every two days. On the ninth day, T-Fc-IVM-NP or NT-OH-IVM-NP was added to the apical side of the barrier and incubated for 12 h. In this study, NT-OH-NP constructed using PLGA-b-PEG-OH polymer was used as a non-targeted control since -Mai containing NPs can interact with biological thiols or other reactive groups. Addition of NPs did not change the tight junction function of the epithelial barrier as evident from intact TEER (Figure 3B, bottom) and the expression of tight junction protein zonula occludens-1 (ZO-1) (Figure 3C). Media from apical and basolateral sites were collected and stored at room temperature. Cells on the apical side were washed with PBS, trypsinized, and centrifuged to have a pellet. The targeted T-Fc-IVM- NPs were found at a much higher concentration in the basolateral side compared to the non- targeted NT-OH-IVM-NPs (Figure 3D).
[000142] These data were also confirmed by incorporating PLGA-coated quantum dots into the T-Fc and NT-Mal NP be generating T-Fc-QD-NP and NT-Mal-QD-NPs (Figure 19). Incorporation of QD allowed us to determine NP concentration in the apical medium and basolateral media by more sensitive and quantitative Inductively coupled plasma mass spectrometry (ICP-MS) by analyzing cadmium. These data further confirmed transport of the targeted NPs across the epithelial barrier (Figure 20). We also carried out experiments where free IgG Fc was added to block the FcRn receptors before adding the NPs. Blocking of the receptors resulted reduced transcytosis of the T-Fc-IVM-NPs across the barrier (Figure 3E).
EXAMPLE 3: In Vivo Distribution of T-Fc-NP after Oral Administration (Zika)
[000143] Balb/c Albino mice were used to evaluate the distribution properties of T-Fc-NP after oral administration. NT-OH-NP was used as a non-targeted control. Animals were divided in three groups, each group containing three animals. Group assignments were: Group 1-Saline, Group 2-NT-OH-QD-NP, and Group 3-T-Fc-QD-NP. Animals in each group received saline, targeted, or non-targeted NPs via oral gavage. The dose of NP was 50 mg/kg with respect to total polymer. After 24 h, around 300 pL of blood was collected in heparinized tubes via cardiac puncture. Collected blood was centrifuged to collect blood plasma. Animals were sacrificed and the major organs were harvested. Organs were weighed and digested in aqua regia and amount of the QD was determined using ICP-MS. The percent NP in the blood was calculated by taking into consideration that blood constitutes 7% of body weight and plasma constitutes 55% of blood volume. Biodistribution was followed by performing ICP-MS on digested organs and plasma.
[000144] This study indicated that the targeted NPs were able to cross the intestine barrier effectively and reach the blood stream. After 24 h, -65% of injected NP was distributed in the blood and -24% was still distributed in different parts of intestine (Figure 4A). As demonstrated in Figure 4A, for the NT-Mal-NPs, significantly small amount of NP was measured in the organs. By contrast, a large amount of T-Fc-NP was measured in the major organs indicating that these NPs entered the systemic circulation after oral administration and reached several organs known to express FcRn. [000145] These results indicate that the targeted nanoparticles when administered orally would be able to cross the intestinal barrier effectively and distribute in the blood to deliver ivermectin in the blood to fight against ZIKA virus. Analyses of duodenum by immunostaining for FcRn and QD from the NPs indicated that the presence of targeted-NPs in the duodenum at a much higher concentration when compared to the non-targeted NPs (Figure 4C).
Example 4: Comparison of Biodistribution of Ivermectin and T-IVMNP when Delivered Orally. (ZIKA)
[000146] A comparison of biodistribution pattern of IVM with its non-targeted and targeted NP constructs indicated that accumulation of IVM or NT-IVM-NP was not very significant in duodenum, jejunum, ileum, colon and blood after oral administration in normal female mice (Figure 5A). When IVM was delivered using T-Fc-IVM-NP, a significant amount of this drug was found in the intestinal tissue as well as in the blood thus indicating that IVM is able to cross the intestinal barrier into the blood only if it is delivered with a targeted nanoparticle via oral route (Figure 5A). The percentage IVM accumulation for T-Fc-IVM-NP was found to be around 12%, 9%, 5%, 5%, and 60% in duodenum, jejunum, ileum, colon, and blood, respectively (Figures 5A, and 21) 24 h post administration via oral gavage. Analyses of the intestinal tissue samples by Western Blot indicated significant expression of FcRn thus confirming that the targeted NPs utilize these receptors to get associated with the intestinal tissue (Figure 5B). T-Fc-IVM-NPs did not have any effect on serum proinflammatory markers IL1 -b, IL-6, and TNF-a suggesting that these NPs do not induce immunogenicity (Figure 5C). Further, no evident pathological changes such as inflammation, necrosis, or cellular abnormalities were observed in H & E sections of the major organs such as brain, heart, lung, liver, spleen and kidney after treatment with T-Fc-IVM- NPs (Figure 5D). Analyses of Alanine aminotransferase (ALT) and Aspartate Aminotransferase (AST) levels from blood plasma from the NP treated animals revealed that these targeted NPs do not induce any liver toxicity (Figure 22).
Example 5: In Vitro Properties of Nanoparticle-Delivered IVM (ZIKA)
[000147] Controlled release of ivermectin from the nanoparticles was observed and the nanoparticles continued to release ivermectin for an extended period of greater than 72 h at pH of 7.4 (Figure 6A). At pH 6.5, the release of the drug followed slightly faster kinetics, but even after 72h, only -60% of ivermectin was released (Figure 6A). These data indicated that the nanoparticles are controlled release delivery vehicles and these particles will be able to release ivermectin in the blood after oral administration making this system suitable for ZIKA infection treatment. Cellular toxicity of ivermectin was compared with ivermectin-loaded T-Fc-IVMNP in Caco-2 cells by studying several parameters of respiration which included overall oxygen consumption rate (OCR) by the cells, basal respiration, maximal respiration, ATP production, extra cellular acidification. We have also used empty T-Fc-NP, NT-Mal-NP, and ivermectin loaded non-targeted NT-Mal-IVM-NP as controls. These studies indicated that at the same concentration, ivermectin completely disrupts cellular respiration while when ivermectin is loaded into T-Fc-NPs, it doesn’t show such toxicity (Figure 6B). These studies indicated that T- Fc-IVM-NP has the potential to cross the intestine epithelial barrier to reach the blood to attack Zika virus, whereas free ivermectin might be disrupting the barrier due to its toxicity on the epithelial cells at the barrier. The toxicity data was further confirmed by performing conventional MTT assay. Comparison of IC50 value of T-Fc-IVM-NPs with that of ivermectin in Caco-2 cells indicated that nanoparticle-delivered ivermectin has much less toxicity compared to free ivermectin (Figure 23).
T-Fc-IVM-NP inhibits NS1 expression.
[000148] ZIKV is a single stranded RNA virus which encodes for three structural proteins (C, PrM or M, and E) and seven non-structural (NS) proteins NS1 , NS2A, NS2B, NS3, NS4A,
NS4B, and NS5). The NS1 protein is involved in viral replication, immune evasion, and pathogenesis of the host cells. NS1 is also a major antigenic marker for viral infection and is regarded as a potential therapeutic target for antiviral drug discovery. Earlier studies have suggested that ivermectin inhibits ZIKV infection. The current study evaluated whether T-Fc- IVM- NP inhibits the expression of NS1 protein. NS1 protein was expressed in HEK293T cells and demonstrated that T-Fc-IVM-NPeffectively inhibited NS1 expression, 0.08 relative to 1.05 for NS1 plasmid alone (Figure 7A). These results were further confirmed using immunofluorescence (Figure 7B). Under the in vitro settings using HEK293 cells, no significant differences were observed between free ivermectin and when delivered with targeted NP due the fact that HEK293 cells do not have significant FcRn expression (Figure 24).
Example 6: Temperature Dependent Stability of IVM-Loaded NPs (ZIKA)
[000149] Stability studies for a month showed that both NT-Mal-NP and NT-Mal-IVM-NP maintained their size and zeta potential best when stored at 4 °C (Figures 8A and 8B). When stored at room temperature, NT-Mal-NP became unstable (Figure 8A), NTMal-IVM-NP remained stable at this temperature for a month (Figure 8B). The nanoparticles became unstable only a few days when stored at 37 °C as evident from increase in size and decrease in zeta potential (Figure 8A and B); however, the NPs containing IVM stayed in well- defined form for a longer period compared to the NPs without IVM even at this temperature.
Example 7: Dry Formulation of IVM-Loaded NPs (ZIKA)
[000150] The IVM-loaded TFc IVM-NPs must be made into powder form to be able to be packed into a capsule and to serve as a viable treatment option for ZIKV. Formulation optimization studies were performed using NT-Mal-IVM-NP. Our studies involved freeze-drying the NPs in the presence of cryoprotectants to ensure their size and stability are maintained in powder form. Sucrose and trehalose were identified as potential cryoprotectants. These optimization studies were carried out under different conditions as discussed below.
[000151] (i) Multiple Freeze-Thaw Cycle Testing: The cryoprotectant: NP ratios were set as 0.1 :1 , 0.5:1 , 1:1 , and 2:1 , as well as control samples with only NP (0:1 ). Size and zeta potential of mixtures of NP and cryoprotectant were tested both after initial mixture and after 1 , 2, and 3 freeze-thaw cycles from -80 °C to room temperature. Each cycle lasted approximately an hour. The tests revealed that both sucrose and trehalose in ratios of 1:1 or 2:1 offered the best protection to NP size and zeta potential, and so these two concentrations of both cryoprotectants were chosen for the long-term storage of NPs at -80 °C.
[000152] (ii) Long-term Storage of NPs at -80 °C: Testing was conducted to identify the amount of time for which the IVM-loaded NPs, with and without the presence of cryoprotectants, could maintain size and zeta potential while stored at -80 °C. Since the 1:1 and 2:1 ratios offered the best protection to NPs in previous testing, cryoprotectants and NPs were mixed in 1:1 and 2:1 ratios (as well as control samples of NP alone) and stored at -80 °C. Size and zeta potential values were recorded ranging from 1 day to 180 days. NPs with cryoprotactants maintain size better, and overall, the cryoprotectants in higher concentrations (2:1 cryoprotectant: NP ratio) were chosen for testing of the drying process (Figure 8C). IVM levels in the NPs were assessed using HPLC; it was found the levels did not show significant change after 180 days of long term storage.
[000153] (iii) Drying of NPs: IVM-loaded NPs alone, as well as the NPs with cryoprotectants in the 2:1 ratios were freeze-dried at approximately -50 °C and 2 Pa and then reconstituted in nanopure water and tested to see if size and zeta potential were maintained. After mixing and gentle vortexing, size and zeta potential measurements were taken, and the NPs showed signs of aggregation. After further attempts, the reconstitution of the nano nanoparticles freeze-dried in the presence of sucrose (2:1 ratio) resulted the best nanoparticles with slightly increased size. The HPLC data shows that the amount of loaded cargo exhibited slight decrease after drying the NPs with cryoprotectants. These results indicated that cryoprotectant: NP ratio of 1 :1 and 2:1 ratios are the best for dry formulation. These NPs maintained the size ~65 nm and a negative zeta potential. The long term storage of NPs at -80 °C revealed that the size and stability was initially maintained both for the NPs alone as well as the NP with cryoprotectant. After around 45 to 75 days, the NPs without the cryoprotectants deviate from the other samples. The NPs with cryoprotect ants continued to stay stable marinating the diameter around 65-75 nm, the NP without cryoprotectant continued to aggregate and destabilize (Figure 8D).
[000154] Morphological analyses by TEM of NT-Mal-IVM-NP and NT-Mal-IVM-NP with sucrose (1 :2) after storing at -80 °C for 180 days indicated that the NPs without cryoprotectant do not maintain their integrity, the NPs which were stored with sucrose demonstrated spherical well defined NPs (Figure 25). During the drying process at -50 °C and 2 Pa, some NP aggregation occurred. The powdered form of the NPs alone or mixed with cryoprotectant was difficult to reconstitute in nanopure water. NPs mixed with sucrose in 2:1 ratio showed the closest size compared to the normal size of NT-Mal-IVM-NP (Figure 8E). Freeze dried NT- MAL-IVM-NPs and the NP which were stored at -80 °C for 180 days with trehalose demonstrated NS1 inhibition (Figure 8F). IVM encapsulated in the NP platform is able to retain its property after 6 months of freeze storage and freeze dried (Figure 8F).
Example 8: Transport of IVM loaded NPs across an in vitro Placental Barrier.
[000155] In pregnant women, the mother and fetus are separated by the placental barrier.
This barrier is built from various types of cell layers which allows for diffusion of different substances between the maternal and fetal circulatory systems. For this reason, the placental barrier is known as the leakiest barrier allowing small molecules to pass through between the mother and the fetus. This leaky nature of the placental barrier is a major reason for doctors’ reluctance towards prescribing medicines for chronic diseases such as hyperlipidemia and ZIKV infection to pregnant patients. By building an in vitro placental barrier, the fate of IVM and IVM loaded NPs were studied. NP transport ability across the placental carcinoma JEG-3 monolayer was assessed by quantifying IVM in the apical and basolateral side media using HPLC. JEG-3 cells were plated in a trans-well plate on the apical side. On the basolateral side, media was added and the cells were grown up to 9 days. Before the addition of IVM or NPs, the monolayer integrity was checked by measuring the TEER indicating a value of >900 W/cm2 on day 9 (Figure 9A). Media was replenished once every two days. On the ninth day, IVM, NT-OH-IVM- NP, or T-Fc-IVM-NP was added to the apical side of the barrier and incubated for 12 h. The TEER values and tight junction protein, ZO-1 expression confirmed that the addition of the articles did not damage the placental barrier (Figures 9A and 9C). IVM alone was able to cross the membrane significantly compared to the T-Fc-IVM-NPs and the NT-OH-IVMNPs (Figure 9C). NPs can be useful for the treatment pregnant patient without affecting the infant. Cellular toxicity of IVM was compared with T-Fc-IVMNP in JEG-3 cells by studying basal respiration, maximal respiration, ATP production, extra cellular acidification. These studies indicated that at the same concentration, IVM completely disrupts cellular respiration of the cells which form the placental barrier while when ivermectin is loaded into T-Fc-NPs, it doesn’t show such toxicity (Figure 9D).
Example 9: Ivermectin Nanoformulation Has the Ability to Reduce ACE2 and Spike Protein Expression.
[000156] IVM-loaded PLGA-b-PEG-MAL nanoparticles (IVM-NPs) were synthesized by following a nanoprecipitation method. The nanoparticles were characterized using dynamic light scattering (DLS), and were found to have sizes around 60-70 nm and zeta potential around -30 mV for nanoparticles with 20% feed of IVM (Figure 33). IVM loading was quantified using HPLC (Figure 33). Fc immunoglobulin fragment targeting moiety was attached using thiolene chemistry, creating the targeted T-Fc-IVM-NPs. Conjugation of the Fc fragment was confirmed and quantified through a bicinchoninic acid (BCA) assay. SARS-CoV-2 is a positive sense single-stranded RNA virus and one of the most crucial components of its structure is the surface spike protein that al-lows it to enter and infect cells. Thus, a logical method to model the conditions of viral infection in vitro and study the expression of the spike protein is to transfect cells using a plasmid expressing spike protein. This would mimic infectious conditions and allow for measurements of NP-delivered IVM’s ability to inhibit viral spike protein without requiring the construction of pseudoviruses or other technologies. Spike protein of SARS-CoV- 2 interacts with ACE2 receptors on human cells, thereby infecting the host. To test the therapeutic abilities of the T-Fc-IVM-NPs against both ACE2 and the viral spike proteins, HEK293T human embryonic kidney epithelial cells were transfected with a plasmid containing the SARS-CoV-2 viral spike protein. These cells were subsequently treated with IVM, a non- targeted IVM-loaded nanoparticle, NT-IVM-NP, made from an PLGA-b-PEG-OH polymer, and the T-Fc-IVM-NPs. Treatments were with 10 mM of free IVM or the nanoformulations with respect to IVM for a period of 4 h followed by incubation for 20 h. Incubation time and dosing were decided based on the uptake kinetics and the IC50 values for the articles in HEK293T cells (Figure 34 for MTT assay data and Figure 35 for uptake data). [000157] Western blot data also revealed that the expression of the spike protein and ACE2 in the HEK293T cells was signific significantly decreased by IVM nanoformulations (Figures 27A and 36 for quantification) but not by free IVM (Figures 27A and 27B). These results were further confirmed by immunofluorescence studies demonstrating the ability of T-Fc-IVM-NP to reduce ACE2 and spike protein levels (Figure 27B and 27C). A decrease in spike and ACE2 expression in HEK293T cells when cells were treated with IVM for 24h was observed (Figure 30A and 30B). Similarly, a decrease in spike and ACE2 expression at 24 h was demonstrated by immunofluorescence (Figure 30C). At 4 h treatment, we observed differential effect of IVM and IVM nanoformulations. However, 24 h treatment did not show any difference in IVM and IVM-NPs. This may explain that IVM-loaded nanoparticles are able to be taken up into cells more at earlier time points than free IVM and exert their effects. Furthermore, we have shown that bioavailability of IVM nanoformulations is more than free IVM. In balb/c mice free IVM showed only 20% of injected dose in the blood. Importantly, IVM nanoformulations showed significantly higher accumulation in the blood. These studies together suggest the superior ability of IVM nanoparticle to reduce spike and ACE2 compared to free IVM. Our studies also revealed that the HEK293T cells have low basal ACE2 expression.
[000158] This was further confirmed by treating cells with increased concentration of angiotensin II (ANG II) and documented that such treatment increases the ACE2 levels in these cells (Figure 38). We also evaluated the effect of free IVM and its formulation on Spike and ACE2 gene expression at transcriptional level (Figure 27C). HEK293T cells transfected with spike plasmid were treated with 10 /M of IVM and its formulation for 4 h and real time PCR was carried out. It was observed that T-IVM-NP significantly inhibited Spike mRNA expression. Free IVM and NT-IVM-NP did not have significant inhibitory effect on Spike mRNA expression. Similarly, we observed that ACE2 mRNA expression was decreased by T-IVM-NP. NT-IVM-NP also decreased ACE2 mRNA expression, but this decrease was less as compare to T-IVM-NP. Taken together, our data suggest that, at some cellular context, T-IVM-NP is more effective than free IVM and NT-IVM-NP at transcriptional level with respect to Spike and ACE2 gene expression. SARS-CoV-2 spike protein binds to the ACE2 receptor on host cells and initiates a cascade of steps for cell fusion and viral uptake into the host cells. Our results indicate that the T-IVM-NP might inhibit viral uptake or entry to the host cells by inhibiting spike and ACE2 expression. EK1C4, a lipopeptide, targeting spike protein inhibited SARS-CoV-2 membrane fusion and viral entry into the host cells.
[000159] The efficacy of the T-Fc-IVM-NP in two other ACE2-expressing epithelial cell lines, A549 adenocarcinomic alveolar basal epithelial cells and HeLa malignant epithelial cells, was also assessed to study the potential impact of the therapeutic on ACE2 and spike protein expression in lung cells and other epithelia that may be infected by SARS-CoV-2. In the A549 cells, ACE2 expression was found to decrease after treatment with IVM and the IVM nanoformulations, and the largest decrease in expression was seen after treatment with the IVM-loaded nanoparticles (Figure 28A, Figure 39A for quantification). Accordingly, the A549 cells were treated with increasing doses of T-Fc-IVM-NPs, and the western blot revealed a dose-dependent effect of the nanoformulation on ACE2 expression (Figure 28B, Figure 39B for quantification). Similarly, in HeLa cells, treatment with T-Fc-IVM-NP showed a de-crease in the expression of both spike protein and ACE2, and the more evident decrease appeared to be in cells treated with the nanoparticles rather than free IVM (Figure 28C and Figure 39C for quantification). Furthermore, immunofluorescence staining in A549 cells revealed a drop in expression of ACE2 following treatment with IVM, NT-IVM-NPs, and T-Fc-IVM-NPs (Figure 28D). The results from both cell lines upon treatment with IVM and IVM nanoformulations indicate that treatment with the IVM-loaded nano-particle may be able to both decrease SARS- CoV-2 spike protein ex-pression as well as prevent more viral entry to the cells by decreasing ACE2 expression.
[000160] Although the free IVM and nanoparticle-encapsulated IVM both significantly decreased spike protein levels in the HEK293T cells and showed clear effects against ACE2 and spike protein in A549 and HeLa cells, this is likely because the cells in the two-dimensional in vitro assay did not contain the FcRn receptors to which the targeted nanoparticles attach to, so these nanoparticles were likely not taken up into the cells in higher amounts than the free drug. However, oral delivery of the T-Fc-IVM-NPs in animal models will certainly yield higher delivery into the bloodstream than free IVM, and will result in lower toxicity, increased IVM concentrations inside cells, and thus increased spike protein and ACE2 inhibition. In addition, as with previous experiments, a more pronounced effect on ACE2 and spike protein expression may have been seen for IVM-loaded nanoparticles compared to free IVM if shorter treatment times such as 2 h or 4 h were used in these studies, as nanoparticle uptake would have far exceeded free IVM uptake in a short-er period of time. The inhibition of both ACE2 and the viral spike protein in a variety of cell lines shows that the IVM-loaded nanoparticle will be effective in treating many different cell types that may be infected. A decrease in viral replication and overall viral load is essential to reducing disease transmission, so in addition to preventing spread within an individual, the therapeutic would also serve to de-crease the overall viral replication to lower the odds of an individual passing the disease on to others. This IVM-loaded nanoparticle shows that both goals can be accomplished using a single therapeutic, which will make future treatments more feasible and effective.
Example 10: Pseudo-virus Inhibition Study.
[000161] In order to increase the basal expression of ACE2 in HEK293T cells and thus more clearly observe the effects of T-Fc-IVM-NP on ACE2 expression and SARS-CoV-2 virus cell entry, we utilized a combined mNeonGreen ACE2 reporter and Psuedo SARS-CoV-2 assay under both therapeutic and preventative settings (Figure 29A). This two-step assay allows for the nuclei of transduced cells to fluoresce green if surface ACE2 was bound by the pseudovirus, helping reveal whether the viral particles’ binding was decreased due to treatment with the T-Fc- IVM-NPs. Fluorescent green surface ACE2-expressing HEK293T cells were treated using two different approaches, a therapeutic approach and a preventative approach, which varied in terms of the order of T-Fc-IVM-NP treatments and the Pseudo SARS-CoV-2 exposure (Figure 29A). The therapeutic approach, in which cells were exposed to pseudo SARS-CoV-2 followed by T-Fc-IVM-NP treatment, resulted in significant decreases in the levels of both ACE2 and pseudovirus (Figure 29B). In the preventative approach, in which T-Fc-IVM-NP treatment preceded pseudovirus exposure with the goal of preventing uptake, there were decreases in both ACE2 express expression and pseudovirus uptake (Figure 29C). In addition, the effects of the articles were monitored via confocal microscopy, which also revealed de-creases in the red and green fluorescence in the cells after treatment with T-Fc-IVM-NP treatment, indicating a decrease in both ACE2 expression and Pseudo SARS-CoV-2 uptake, respectively under the therapeutic (Figure 29D) and preventative (Figure 29E) approaches. The decrease in green fluorescence in cells’ nuclei after the therapeutic approach, in which pseudovirus particles had already entered cells prior to T-Fc-IVM-NP treatment, showed that this nanoformulation may also be effective in de-creasing the expression of the viral proteins once they are inside the cells. This assay was also per-formed in normal human small airway epithelial cells (HSAEC) which are known to get infected by SARS-COV-2 (Figure 30). It is evident that the nano particles can decrease the pseudo virus levels in both preventative and therapeutic settings. These data demonstrated the ability of the nanoparticles not only to treat SARS-CoV-2 infection, but to lower ACE2 expression significantly in cells and prevent virus uptake, viral protein expression, cell-to-cell spread, and thus overall disease transmission.
Example 11: A Potential Mechanism of Action of T-Fc-IVM-NP [000162] Though the specific mechanism by which the re-leased IVM could inhibit the replication of the SARS-CoV-2 virus and expression of spike protein is yet to be determined, a possibility could be through the inhibition of the nuclear transport activities mediated through proteins such as importin (IMP) a/b1 heterodimer, as IVM was previously shown to inhibit a similar interaction between IMRa/b1 (Figure 31 A) and the human immunodeficiency virus-1 (HIV-1) integrase protein. The IMP a/b1 heterodimer is a key nuclear transport protein and is believed to play a role in transporting viral proteins to the nucleus of infected cells. IMP a and b1 work through the recognition of nuclear localization signals on proteins, and IMP a and b1 have previously been associated with the nuclear transport of other viral proteins such as HIV-1 integrase and dengue virus non-structural protein 5 (NS5). IMP a and b1 transport of viral proteins to the nucleus allows proteins such as dengue virus’ NS5 protein to diminish cells’ antiviral responses by impacting mRNA splicing and immune signaling. Therefore, investigating the IVM-loaded nanoparticle’s potential inhibitory effect on IMP a and b1 is key to fully characterizing the therapeutic’s antiviral properties. To more closely study the potential inhibitory activity of IVM and IVM nano-formulations on IMP a/b1 activity, we conducted a time- dependent treatment study in spike protein-expressing HEK293T cells. Cells were treated with IVM and its nanoformulations for periods of 2, 4, or 6 h, and then incubated in normal media up to 24 h, and the western blot revealed that the IVM-loaded nanoparticles showed increased ability to inhibit spike protein, particularly after the 4 h treatment. In addition, treatment with IVM- loaded nanoparticles for 4 h led to the inhibition of IMP b1 , whereas treatment for 6 h resulted in decreases in both IMP a and b1 (Figure 6B). These results suggest that the IVM-loaded nanoparticles can inhibit importin activity and thus decrease the amount of viral mate-rial being transported to the nucleus, lowering over-all viral replication and spread. The results may also indicate that uptake of nanoparticle-delivered IVM into cells may occur more quickly than the uptake of free IVM, as the effects of free IVM on IMP a and b1 expression only begin to be seen at the 6 hour timepoint. Previous results show that the effects of IVM and IVM-loaded nanoparticles balance out when treatments are kept for 24 h. However, this initial discrepancy in the inhibitory effect of the IVM-loaded nanoparticles versus free IVM indicates that the nanoparticles are more likely to be taken up by cells, as compared to the hydrophobic nature of free IVM that may lower its ability to enter cells and exert its effects. This may allow the nanoparticle-delivered IVM to more effectively prevent IMP a/b1 nuclear transport of viral proteins. In our studies, we observed that T-Fc-IVM-NP released IVM has a greater efficiency in inhibiting both IMPa and IMP b1 compared to free IVM or NT-IVM-NP (Figure 31 B). To\time dependent studies demonstrated that an incubation time of 4 h is most efficient in bringing such inhibition when the cells are treated with T-Fc-IVM-NP (Figure 31 B). To probe whether the inhibition of IMP a/b1 has any effect on the pseudo virus assay, which was used to understand activity of IVM nanoparticle, we carried out studies using importazole, an IMP b1 inhibitor. The effects of importazole at varied concentration in IMP a/b1 in HEK293T cells indicated that this inhibitor can inhibit the expression of IMP b1 at a concentration of ~20 mM (Figure 31 C). Importantly, by inhibiting IMP b1 expression, we did not observe any decrease in uptake of pseudovirus protein (Figure 30D). Thus, these studies indicated that T-Fc-IVM-NP has ability to have combinatorial effects by inhibiting ACE2, spike, and IMP a/b1 on the treated cells.
Example 12: Mitochondrial Functions and Inflammation of Spike-infected Cells and Effects of T-Fc-IVM-NP
[000163] SARS-CoV-2 has been found to impact host mitochondrial functions through ACE2 regulation and open-reading frames that can allow for increased viral replication and evasion of host cell immunity.23 The mitochondrial effects of spike protein expression and the mitochondrial toxicity of treatment using IVM and its nanoformulations was tested using a Mitostress assay in HEK293T cells transfect-ed with spike plasmid. Initially, spike protein ex pression within the HEK293T cells was found to slightly impact mitochondrial bioenergetics through the decrease of basal and maximum respiration as well as ATP production (Figure 33). This effect was further compounded by treatment with free IVM, which more significantly decreased these three metrics and led to further mitochondrial dysfunction. However, the NT- IVM-NP and T-Fc-IVM-NP treatments did not lead to further decreases in basal respiration, maximum respiration, and ATP production over what had already been caused by spike protein, indicating nanoparticle-delivered IVM is less toxic and may even slightly reverse the toxic effects of spike protein on mitochondria (Figure 33). The improvement of these indicators of mitochondrial health supports the use of the therapeutic nanoparticle over free IVM as an antiviral agent to treat SARS-CoV-2 infection. Seeing as the therapeutic nanoparticle increased respiration and ATP production close to the levels of normal untreated cells, future time- dependent treatments using IVM-loaded NPs may show that higher doses of T-Fc-IVM-NPs allow for a full increase of mitochondrial health back up to the levels of control cells (Figure 33A and 33B). The studies also indicated that the spike doesn’t have any activity on the complex IV and complex V activity (Figure 40).
[000164] Furthermore, we analyzed the levels inflammatory markers such as I L- 1 b , IL-6, and TNFa before and after infection and treatment in HEK293T cells. We did not observe any change in these inflammatory markers (Figure 31 C). The aim of these studies into the effects of spike protein infection and IVM nanoformulation treatment on the oxidative stress and inflammation that cells face was to examine whether the IVM-loaded nanoparticles could mitigate the mitochondrial dysfunction caused by SARS-CoV-2 infection. The oxidative stress faced by infected patients undoubtedly con-tributes to the progression of their symptoms, and an ideal therapeutic to treat SARS-CoV-2 must not only lower viral entry into cells and the expression of viral proteins, but also avoid adding to the mitochondrial dysfunction that infected cells already face. In this regard, the T-Fc-IVM-NPs serve as a far more helpful treatment compared to free IVM by not impeding mitochondrial respiration or causing inflammation, and the IVM-loaded nanoparticles in fact allow for treated cells to regain some respiration and ATP production capacity that was initially lowered by spike protein expression and the infection in general.
EXAMPLE 13: Assembly of Nanovesicles
[000165] Solubility was achieved by preparing nano-ivermectin vesicles following an optimized reprecipitation method, wherein the drug was dissolved in a good solvent (ethanol) at millimolar concentration and its ethanolic solution (in small increments) was added to a poor solvent (water) to allow the precipitation of the nanovesicles. The synthesis was assisted with ultrasound (ultrasonication bath) for a better size-control of the nanomaterials, that was achieved by the action of acoustic cavitations to promote intermolecular interactions. The disclosed nanovesicles were analyzed using materials characterization techniques, optical properties, in addition to the release studies at the physiological pH.
[000166] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were initially used to estimate the size and morphology of the nanodrugs at neutral pH.
Measurements revealed an average size of 76 nm (average of duplicate samples, 3 records each) for nano-ivermectin after 24 hours from its synthesis (Figure 41 A); and 13 nm as shown in the TEM micrographs (Figure 41 B).
[000167] The formation of nanovesicles is based on a spontaneous self-assembly mechanism that is governed in this case by hydrophobic and hydrogen bonding interactions. The formation of nanovesicles is represented in Figure 41 C; the self-assembly of ivermectin amphiphilic molecules into nanovesicles is demonstrated under aqueous conditions, to overcome the undesirable hydrophobe-water interactions. The self-assembly mechanism has been applied to produce various nanodrugs, only restricted in majority to the domain of anticancer application. [000168] In the disclosed case, the procedure is different and unique, where the mechanism relates to the organization of a double-tailed surfactant under aqueous conditions contributing to the formation of nanovesicles-based drugs. The disclosed nanoparticle has a hydrophilic head of the structure that faces the aqueous medium while the hydrophobic part is directed towards the interior of the vesicle, where a small volume of water/ethanol is contained. The thickness of the shell is mainly determined by the interfacial tension, showing the following dimensions: total size of the shell, 133 nm; the dark interior shell, 27 nm (Figure 41 C). Zeta potential measurements were conducted to determine the electrostatic surface potential and evaluate the stability of the nanosuspensions. Zeta potential (CE5) value was -24 mV (average zeta potential values of duplicate syntheses, 3 records per synthesis) with pH of the medium being 7.35. The synthesized nanovesicles present an acceptable stability and exhibit minimal level of agglomeration considering that the zeta potential value is close to the limit value (-30 mV) that is globally accepted as the normal value reflecting the stability of nanosuspensions. The presence of this high surface charge contributes to an electrostatic repulsion among the vesicles and causes a decrease in the level of agglomeration.
[000169] Concentration- and pH- variation were monitored versus time of nanovesicles aging to further understand the formation of vesicles-like aggregates (Figure 42). The data showed that the hydrodynamic diameter is similar for the 50 pl_ and 100 -,pL volumes of ethanolic solutions (Figure 42A); however, the volume of 150 mI_ (higher number of moles of ivermectin) resulted in a larger size of vesicles that was monitored during 24 hours and 48 hours of growth time. The hydrodynamic diameter produced from the 150 -,pL volume levels off with the 50 -,pL and 100 pL-suspensions after 72 hours. Ivermectin vesicles might have redistributed and stabilized after a sufficient time of growth or aging. Zeta potential measurements studies were also performed on ivermectin nanosuspensions prepared using different volumes of ethanolic solutions (Figure 42B). The nanosuspensions formulated with 50 mI_ and 100 mI_ (zeta potential magnitude higher or equal to 30 mV) were more stable than the nanosuspension formulated with 150 mI_ volume. In fact, the nanosuspensions generated with 100 mI_ had the most constant response during the growth of nanoparticles, which indicates that the use of this volume is optimal for the formulation of long term-stored suspensions. The evaluation of the surface charge of these nanovesicles reflects the optimal interaction of nanomaterials with the biological target, as well as the bioavailability of these materials. The pH stability of these nanoformulations was also performed by resuspending the aqueous suspension of nanovesicles in different pH buffers (Figures 42C and 42D). The effect of pH on the nanoformulations in terms of size and stability is dictated by two factors: the first is the pKa of ivermectin molecule (pka value is around 6.5) and the second is the presence of ions that originate from the buffers. The pka of ivermectin had its heavy effect on the size of nanomaterials that showed a tremendous increase in size after adding phosphate buffers of pH 7 and 7.8. In contrast, the presence of ions caused a drop in zeta potential values by almost 15 mV magnitude, especially when using buffers of pHs 5 and 7. The distinctive effect of those two buffers is that pH 5 buffer is an acetate buffer where the acetate ions might have some hydrogen bonding interactions with ivermectin molecules, leading to an increase in size and decrease of zeta potential magnitude. The pH 7 phosphate buffer, which caused the noticeable changes in size and zeta potential values, has a pH that is slightly higher than the pka value of ivermectin. At a pH higher than the pKa, the ivermectin molecule exists in its ionic form and its solubility in aqueous system increases. Thus, an uncontrolled precipitation and unstable formulation were observed as a result of the pH increase during the suspension of the nanomaterials in the buffer. In this context, the long-term storage of the nanovesicles was also assessed after two months at 25°C from their preparation in aqueous medium, which is important for the potential use of this compound as a pharmaceutical product. The fabricated nanovesicles have shown a long-shelf life (at least 2 months, (84 nm size)) and a high colloidal stability (-32 mV), an advantage that was observed in difference to what conventional liposomes present. Thus, the hydrophobic interactions among ivermectin molecules are proved to be stronger than the interactions with water molecules. These formulations show, under similar conditions, a better performance in comparison to lipid-based vesicles, which are reported to destabilize during the formulation process, and are more prone to problems related to their physicochemical stability.29
[000170] Optical properties of nano-ivermectin were also evaluated, taking into consideration that these properties might be size-dependent as it is often observed in the case of organic nanomaterials.30 Nanomaterials usually exhibit optical properties that lie between atomic and bulk properties.31 Their properties different from their bulk counterpart as a result of the inter- and intra-molecular interactions that contribute to the formation of the nanomaterials. It is informative to understand the changes of optical properties for nano-ivermectin and the type of molecular arrangement that controlled the formation of nanovesicles. For this purpose, nano ivermectin suspensions were synthesized in duplicate under aqueous conditions using the reprecipitation method, then left to age for 24 hours (Figure 43A). The bulk of ivermectin was prepared following a similar procedure in ethanol, and both bulk and nano-ivermectin were compared at the same concentration. The conjugated-diene chromophore of ivermectin has an absorbance maximum at 245 nm.32 Both nanosuspensions were consistent in terms of optical properties, with absorbance ranging between 220 and 260 nm. However, the nanosuspensions showed a decrease in the molar absorptivity in comparison to the bulk material, as a result of the presence of intermolecular electronic interactions upon aggregation. The decrease of the cross-section of nano-ivermectin exposed to the light can also contribute to the decrease in absorbance value. Moreover, the nanosuspensions presented more defined shoulders in the absorbance spectra, suggesting the presence of multiple excited states. Our previous studies have shown size stability of nano-ivermectin suspensions (for 100 -,pL ethanolic ivermectin formulations) at various aging periods (or growth time) (Figures 42A and 42B). The optical measurements confirmed the stability as well of the nanosuspensions, that were synthesized and left to age for 24, 48, and 72 hours (Figure 43B). The properties of the nanosuspensions were similar indicating consistency in the synthesis method (same average size of 80 nm) and stability of the suspensions over time.
[000171] A continuous dialysis of the nanosuspensions in a phosphate-buffered saline PBS solution (pH 7.4) was also performed to evaluate the release profile, more precisely the degradation profile of the nanovesicles (Figure 44). The nanosuspensions were synthesized in duplicate using the optimized reprecipitation method, with an amount of 100 -,pL of 1 mM ivermectin drug. After 24 hours, the nanomaterials were suspended in PBS and dialyzed against PBS buffer pH 7.4. Nano-ivermectin showed first a burst release of 20% of its molecules or entities, suggesting that those molecules were not strongly bound to the assembled nanoscale aggregates. The nanovesicles kept then a slower and sustainable release for a long period of time, about 220 hours. Overall, the general profile of the release/degradation rate implies a maintained efficacy of nano-ivermectin for several days. This is advantageous because it reflects a controlled release of the therapeutic agents under physiological conditions.
[000172] The examples herein demonstrate that a synthetic nanoparticle can cross the intestinal epithelial barrier when administered via oral route and distribute in the blood at a considerable concentration. It is further disclosed that the plasma concentration of ivermectin can be increased significantly when delivered with this synthetic nanoparticle. Further, the toxicity of ivermectin on epithelial cells can be lowered by entrapment inside the polymeric nanoparticles. The examples demonstrate that Ivermectin-loaded NPs demonstrated stability over time, particularly at 4°C and even at room temperature. The NT-Mal-IVMNP, when combined with cryoprotectant such as sucrose or trehalose in higher concentrations, showed maintenance of both size and zeta potential across multiple freeze-thaw cycles and over several months at -80°C. The inability of T-Fc-NP to cross the placental barrier indicates that this delivery vehicle can be used to pregnant population.
[000173] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present invention that falls within the prior art can be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they can be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
[000174] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the present invention, as defined in the following claims

Claims

WHAT IS CLAIMED IS:
Claim 1. A controlled release polymeric nanoparticle wherein the nanoparticle is porous and has a surface, comprising: i) a maleimide functional group on the nanoparticle surface, and ii) a hydrophobic drug inside the nanoparticle.
Claim 2. A controlled release nanoparticle capable of being targeted to a cell or tissue, comprising: i) a FcRn binding domain that binds to a target cell or tissue; ii) a hydrophobic drug encapsulated in the nanoparticle; and ill) a targeting ligand with -SH functionality which can react at the nanoparticle surface with the maleimide functional group.
Claim 3. The nanoparticle of claim 1 or 2, wherein the nanoparticle is a dry formulation comprising trehalose.
Claim 4. The nanoparticle of claim 1 or 2, wherein the nanoparticle is a dry formulation comprising sucrose.
Claim 5. The nanoparticle of claim 1 or 2, wherein the nanoparticle is a frozen formulation of nanoparticles comprising trehalose.
Claim 6. The nanoparticle of claim 1 or 2, wherein the nanoparticle is a frozen formulation of nanoparticles comprising sucrose.
Claim 7. The nanoparticle of any one of claims 1-6, wherein the hydrophobic drug is ivermectin.
Claim 8. The nanoparticle of any one of claims 1-7, wherein the polymeric nanoparticle comprises poly(lactide-co-glycolide)-/b-polyethyleneglycol (PLGA-b-PEG) block copolymer.
Claim 9. The nanoparticle of claim 8, further comprising a poly(lactic-co-glycolic acid) (PLGA) core.
Claim 10. The nanoparticle of any one of claims 2-6, wherein the FcRn binding domain targets tissue of the gastrointestinal tract.
Claim 11. A method of treating an individual infected with an RNA virus, comprising administering the nanoparticle of any one of claims 1-10 to an individual infected with the RNA virus.
Claim 12. The method of claim 11 , wherein the RNA virus is Zika virus.
Claim 13. The method of claim 11 , wherein the nanoparticles encapsulate ivermectin.
Claim 14. The method of claim 11 , wherein the nanoparticle comprises poly(lactide-co- glycolide)-/b-polyethyleneglycol (PLGA-b-PEG) block copolymer.
Claim 15. The method of claim 11 , wherein the FcRn binding domain targets tissue of the gastrointestinal tract.
Claim 16. The method of claim 11 , wherein the nanoparticles are administered orally.
Claim 17. The method of claim 11 , wherein the ivermectin is released at a therapeutic dose over a sustained period of time.
Claim 18. A method of treating an individual infected with an RNA virus, comprising administering the nanoparticle of any one of claims 1-10 to an individual infected with a single strand RNA virus.
Claim 19. The method of claim 18, wherein the individual is or has been infected with a SARS virus.
Claim 20. The method of claim 19, wherein the SARS virus is a SARS-COV-2 virus.
Claims 21. The method of claim 19, wherein the SARS-COV-2 virus results in a COVID-19 infection.
Claim 22. The method of claim 18, wherein a FcRn binding domain targets tissue of the respiratory epithelia.
Claim 23. The method of claim 18, wherein the nanoparticle targets ACE2-expressing cells.
Claim 24. The method of claim 18, wherein the individual is or has been infected with a
MERS virus.
Claim 25. The method of claim 18, wherein the individual is or has been infected with a Dengue virus.
Claim 26. The method of claim 18, wherein the individual is or has been infected with a hepatitis virus.
Claim 27. The method of claim 18, wherein the individual is or has been infected with a West nile fever virus.
Claim 28. The method of claim 18, wherein the individual is or has been infected with an Ebolavirus.
PCT/US2020/051061 2019-09-16 2020-09-16 Orally administrable nano-medicine for viral diseases Ceased WO2021055467A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201962901077P 2019-09-16 2019-09-16
US62/901,077 2019-09-16

Publications (1)

Publication Number Publication Date
WO2021055467A1 true WO2021055467A1 (en) 2021-03-25

Family

ID=74884330

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2020/051061 Ceased WO2021055467A1 (en) 2019-09-16 2020-09-16 Orally administrable nano-medicine for viral diseases

Country Status (1)

Country Link
WO (1) WO2021055467A1 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022129417A1 (en) * 2020-12-16 2022-06-23 Medincell Methods and compositions for the prophylactic treatment of sars-cov-2 virus (covid-19)
WO2022217966A1 (en) * 2021-04-15 2022-10-20 苏州大学 Nano-trapping agent that inhibits sars-cov-2
WO2023283106A1 (en) * 2021-07-07 2023-01-12 Codagenix Inc. Deoptimized sars-cov-2 variants and methods and uses thereof
CN115737595A (en) * 2022-11-24 2023-03-07 深圳先进技术研究院 ACE2 engineered cell membrane drug-loaded nanoparticle, and preparation method and application thereof
EP3989981A4 (en) * 2020-02-07 2023-12-27 Topelia Australia Pty Ltd Products of manufacture and methods for treating, ameliorating or preventing coronavirus infection
WO2024254428A3 (en) * 2023-06-07 2025-05-01 The Research Foundation For The State University Of New York Bioactive telodendrimer nanodrug for immune modulation and drug delivery

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100303723A1 (en) * 2006-11-20 2010-12-02 Massachusetts Institute Of Technology Drug delivery systems using fc fragments
WO2017075369A1 (en) * 2015-10-30 2017-05-04 Pfizer Inc. Therapeutic nanoparticles comprising a therapeutic agent and methods of making and using same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100303723A1 (en) * 2006-11-20 2010-12-02 Massachusetts Institute Of Technology Drug delivery systems using fc fragments
WO2017075369A1 (en) * 2015-10-30 2017-05-04 Pfizer Inc. Therapeutic nanoparticles comprising a therapeutic agent and methods of making and using same

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3989981A4 (en) * 2020-02-07 2023-12-27 Topelia Australia Pty Ltd Products of manufacture and methods for treating, ameliorating or preventing coronavirus infection
WO2022129417A1 (en) * 2020-12-16 2022-06-23 Medincell Methods and compositions for the prophylactic treatment of sars-cov-2 virus (covid-19)
WO2022217966A1 (en) * 2021-04-15 2022-10-20 苏州大学 Nano-trapping agent that inhibits sars-cov-2
WO2023283106A1 (en) * 2021-07-07 2023-01-12 Codagenix Inc. Deoptimized sars-cov-2 variants and methods and uses thereof
CN115737595A (en) * 2022-11-24 2023-03-07 深圳先进技术研究院 ACE2 engineered cell membrane drug-loaded nanoparticle, and preparation method and application thereof
WO2024254428A3 (en) * 2023-06-07 2025-05-01 The Research Foundation For The State University Of New York Bioactive telodendrimer nanodrug for immune modulation and drug delivery

Similar Documents

Publication Publication Date Title
WO2021055467A1 (en) Orally administrable nano-medicine for viral diseases
Verma et al. Vitamin B12 functionalized layer by layer calcium phosphate nanoparticles: A mucoadhesive and pH responsive carrier for improved oral delivery of insulin
Yang et al. Recent advances in the application of vitamin E TPGS for drug delivery
Yuan et al. Stearic acid-g-chitosan polymeric micelle for oral drug delivery: in vitro transport and in vivo absorption
Lakkadwala et al. Biodistribution of TAT or QLPVM coupled to receptor targeted liposomes for delivery of anticancer therapeutics to brain in vitro and in vivo
Mao et al. Chylomicron-pretended nano-bio self-assembling vehicle to promote lymphatic transport and GALTs target of oral drugs
Ruttala et al. Facile construction of bioreducible crosslinked polypeptide micelles for enhanced cancer combination therapy
Song et al. Erythrocyte-biomimetic nanosystems to improve antitumor effects of paclitaxel on epithelial cancers
Wang et al. Low-molecular-weight protamine-modified PLGA nanoparticles for overcoming drug-resistant breast cancer
Lee et al. Development of pH-responsive organic-inorganic hybrid nanocomposites as an effective oral delivery system of protein drugs
JP5539993B2 (en) Nanocarriers for drug delivery
Zhou et al. Redox-triggered activation of nanocarriers for mitochondria-targeting cancer chemotherapy
Wang et al. Surface-modified nanoerythrocyte loading DOX for targeted liver cancer chemotherapy
Bian et al. Awakening p53 in vivo by D-peptides-functionalized ultra-small nanoparticles: Overcoming biological barriers to D-peptide drug delivery
Li et al. Bioinspired peptosomes with programmed stimuli-responses for sequential drug release and high-performance anticancer therapy
Shukla et al. Multifunctional hybrid nanoconstructs facilitate intracellular localization of doxorubicin and genistein to enhance apoptotic and anti-angiogenic efficacy in breast adenocarcinoma
Liu et al. Hierarchically stimuli-responsive nanovectors for improved tumor penetration and programed tumor therapy
Zhang et al. Dual-responsive doxorubicin-loaded nanomicelles for enhanced cancer therapy
BRPI0715299A2 (en) Method for preparing polymeric nanoparticles, Method for preparing a micellar cup, Reconstitutable polymer micelles, Bioactive polymeric nanoparticle composition, Method for providing a patient a medicament and Method for preparing polymeric nanoparticle compositions
Duan et al. Vincristine-loaded and sgc8-modified liposome as a potential targeted drug delivery system for treating acute lymphoblastic leukemia
Sun et al. High loading of hydrophobic and hydrophilic agents via small immunostimulatory carrier for enhanced tumor penetration and combinational therapy
US20230107937A1 (en) Zwitterionic polypeptide and derivative thereof and nanodrug based thereon
Han et al. Free paclitaxel-loaded E-selectin binding peptide modified micelle self-assembled from hyaluronic acid-paclitaxel conjugate inhibit breast cancer metastasis in a murine model
Shi et al. Tumor microenvironment targeting with dual stimuli-responsive nanoparticles based on small heat shock proteins for antitumor drug delivery
Zhang et al. A PEGylated alternating copolymeric prodrug of sulfur dioxide with glutathione responsiveness for Irinotecan delivery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20866532

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20866532

Country of ref document: EP

Kind code of ref document: A1