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WO2014200441A1 - Nanoparticules de conversion-élévation fluorescentes de cœur-coquille pour photoactivation de multiples biomolécules - Google Patents

Nanoparticules de conversion-élévation fluorescentes de cœur-coquille pour photoactivation de multiples biomolécules Download PDF

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WO2014200441A1
WO2014200441A1 PCT/SG2014/000281 SG2014000281W WO2014200441A1 WO 2014200441 A1 WO2014200441 A1 WO 2014200441A1 SG 2014000281 W SG2014000281 W SG 2014000281W WO 2014200441 A1 WO2014200441 A1 WO 2014200441A1
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ucns
ucn
nir
cells
cell
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Yong Zhang
Muthu Kumara Gnanasammandhan JAYAKUMAR
Akshaya BANSAL
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National University of Singapore
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/77—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals
    • C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
    • C09K11/7772—Halogenides
    • C09K11/7773—Halogenides with alkali or alkaline earth metal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/008—Two-Photon or Multi-Photon PDT, e.g. with upconverting dyes or photosensitisers
    • 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
    • A61K47/6923—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 an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • 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
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media

Definitions

  • nanoparticles as engineered non-viral vectors have potential, but gene delivery via nanoparticles has two niggling issues: efficient delivery of genes and controlled expression. Therefore, a need exists for improved nanoparticles and methods of making and using them for, inter alia, gene therapy.
  • the present invention provides improved nanoparticles and methods of making and using them ⁇ e.g., for gene therapy) that overcome problems associated with existing viral and non- viral delivery systems.
  • FIG. 1A shows fluorescence emission peaks for NIR to UV UCNs.
  • FIG. IB is an MTS assay for the effect of varying concentrations of NIR to UV UCNs on cell viability of B16F0 cells.
  • FIG. 1C shows the expression of STAT-3 in B16F0 cells after treatment with NIR-to-UV UCNs loaded with caged STAT-3 siRNA with (grey) and without (black) NIR irradiation.
  • FIG. 2A shows the fluorescence emission spectrum for NIR to Visible UCNs.
  • FIG. 2B shows the effect of NIR to visible UCNs on the viability of B16F0 cells.
  • FIG. 2C shows the effect of TPPS2a on the viability of B16F0 cells.
  • FIG, 2D shows STAT3 knockdown with and without PCI.
  • FIG. 3A is a schematic showing the structure of core-shell UCNs and its various coatings.
  • FIG. 3B is a transmission electron micrograph of NIR-to-UV core.
  • FIG. 3C is a transmission electron micrograph of NIR-to-UV /Vis core-shell UCNs.
  • FIG. 3D shows the fluorescence emission spectrum of NIR-to-UV UCN core.
  • FIG. 3E shows the fluorescence emission spectrum of NIR-to-UV /Vis core-shell UCN.
  • FIG. 3F shows the fluorescence emission spectrum of NIR-to-UV Vis Core-shell UCNs.
  • FIG. 3 is substantially idential to FIG. 24.
  • FIG. 4A is a transmission electron micrograph of mesoporous silica coated NIR-to-UV Vis core shell UCNs. The inset shows a magnified image showing the mesopores.
  • FIG. 4B shows the cumulative percentage release of siRNA (grey) and TPPS-2a (black) from UCNs over time.
  • FIG. 4C shows the fluorescence stability of NIR-to-UV Vis core shell UCN exposed to different pH conditions. Black bars denote the UV emission peak at 350 nm and the grey bars denote the visible emission peak at 410 nm.
  • FIGs. 4A, 4B, and 4C are substantially idential to FIGs. 24G, 26A, and 30, respectively.
  • FIG. 5 A shows the cytotoxicity of B 16FO cells exposed to different combinations of UCNs, TPPS-2a and NIR.
  • FIG. 5B shows the phototoxicity of B16FO cells exposed to different durations of 980 nm NIR laser.
  • FIG. 5C shows the toxicity of zebrafish embryos exposed to different concentrations of UCNs (heart rate @ 72 hpf).
  • FIG. 5D shows the percentage hatching rate of zebrafish embryos.
  • FIGs. 5E-5G are optical micrographs of embryos at 24 hours.
  • FIGs. 5H-5J are optical micrographs of embryos at 72 hours.
  • the embryos in FIGs. 5E and 5H were exposed to 0 ug/ml of UCNs.
  • the embryos in FIGs. 5F and 51 were exposed to 50 ug/ml of UCNs.
  • the embryos in FIGs. 5G and 5 J were exposed to 100 ug/ml of UCNs.
  • FIG. 6A shows ROS production by core-shell UCNs after irradiation with NIR and determined by APF.
  • FIGs. 6B and 6C show fluorescence imaging of normal cells (FIG. 6B) and cells treated with UCNs+TPPS-2a with NIR irradiation (FIG. 6C) after incubation with Image-IT® LIVE Green Reactive Oxygen Species Detection reagent to detect the presence of singlet oxygen. Cells were counterstained with DAPI. Scale bar: 50 ⁇ .
  • FIG. 6D shows photoactivation of caged siRNA by core-shell UCNs after NIR irradiation and determined by UV-Vis absorbance spectrophotometry.
  • FIGs. 6E and 6F are a comparison of the photoactivation of TPPS-2a (FIG. 6E) and caged siRNA (FIG. 6F) by core-shell UCNs when the molecules loaded onto the UCNs are just incubated together.
  • FIGs. 6G-6N show cellular distribution of UCNs (FIGs. 6G-6J) and their redistribution after 10 mins of NIR irradiation (FIGs. 6K-6N). Concanavalin was used to stain the cell membrane in FIGs. 6G and 6K, and DAPI was used to stain the nucleus in FIGs. 6H and 61.
  • UCN fluorescence is shown in FIGs. 6J and 6M, and FIGs. 6J and 6N are the respective merged images. Scale bar: 50 ⁇ .
  • FIGs. 6B and 6C are substantially identical to FIGs. 26D and 26E.
  • FIG. 7A is a schematic showing simultaneous activation of endosomal escape and uncaging of DNA siRNA by NIR-to-UV/Vis core-shell UCNs for enhanced and controlled gene expression/knockdown.
  • FIG. 7B shows cellular uptake of UCNs into B16F0 cells with and without NIR irradiation (PO.05 when compared to control).
  • FIG. 7C shows the percentage of STAT-3 expression in B16F0 cells exposed to different combinations of UCNs [U], siRNA [S], and TPPS-2a [T] (PO.05 for
  • FIGs. 8A-8I show GFP expression in H-226 without any transfection (FIGs. 8A-8C) or transfected with UCNs loaded with caged GFP (FIGs. 8D-8F) or UCNs co- loaded with TPPS-2a and caged GFP (FIGs. 8G-8I) and transplanted into adult zebrafish and irradiated using a NIR laser.
  • FIGs. 8J-8L show in vivo imaging of zebrafish injected with TPPS-2a and caged GFP loaded UCNs and activated using a NIR laser for 8 mins. Scale bar: 2mm.
  • FIG. 9 shows the fluorescence emission spectrum of UCNs uptaken by embryos.
  • FIGs. 1 OA- 10C show the morphology of zebrafish at 24, 48 and 72 hpf.
  • FIG. 1 OA shows the normal development of control zebrafish at 24, 48 and 72 hpf.
  • FIG. 10B shows the normal development of zebrafish microinjected with UCNs loaded with morpholino and TPPS2a but not irradiated with NIR.
  • FIG. IOC shows no tail morphology at 24, 48 and 72 hpf seen in zebrafish embryos microinjected with UCNs loaded with morpholino and irradiated with NIR (980 nm) for 8 mins at 5 hpf.
  • FIGs. 11 A-l 1C show the distribution of UCNs in zebrafish.
  • FIG. 11 A shows the control embryo (no UCN).
  • FIG. 1 IB shows the distribution of UCNs in a no tail embryo. These embryos were microinjected with UCNs co-loaded with no tail photomorpholino and TPPS2a and irradiated with NIR for 8 mins at 5 hpf.
  • FIG. 11C shows the distribution of UCNs in a normal zebrafish. The specimen was microinjected with UCNs co-loaded with no tail photomorpholino and TPPS2a but was not irradiated, resulting in normal development.
  • FIG. 12 is a Z-stack image of zebrafish microinjected with UCNs.
  • FIG. 13 is a figure showing the percentage of no tail and normal
  • FIG. 14 is a TPPS2a excitation curve with TPPS2a structure inset.
  • FIG. 14 is substantially identical to FIG. 24F.
  • FIG. 15 shows the rise in temperature of water and DMEM with increasing durations of NIR exposure.
  • FIG. 16 shows the zeta potential of mesoporous silica-coated core-shell NIR-to-UV/Vis UCNs.
  • FIG. 17 shows in vivo imaging of UCNs in adult zebrafish.
  • FIGs. 18A-18D show fluorescence microscopy of cancer cells injected with cancer cells transfected with UCNs.
  • FIG. 18A shows control fish injected with non- transfected cancer cells.
  • FIG. 18B shows fish injected with UCNs loaded with GFP plasmid and TPPS2a without NIR irradiation.
  • FIG. 18C shows fish injected with UCNs loaded with GFP plasmid and irradiated with NIR.
  • FIG. 18D shows fish injected with UCNs co-loaded with GFP plasmid and TPPS2a and irradiated with NIR Scale bar: 2 mm.
  • FIGs. 19A-C show characterization of UCNs.
  • FIGs. 19A and 19 B are transmission electron micrographs of NIR-to-UV UCN core (FIG. 19A) and after coating with a layer of mesoporous silica (FIG. 19B).
  • FIG. 19C shows the fluorescence emission spectrum of NIR-to-UV UCNs.
  • FIG. 20A provides FTIR spectra of NaYF4: Yb/Tm UCN core and UCN core coated with a layer of mesoporous silica.
  • FIG. 20B is a bar graph showing
  • FIGs. 21 A-21H summarizes toxicity data.
  • FIG. 21 A is a bar graph of cell ciability of ZFL cells exposed to different concentrations of UCNs.
  • FIG. 21 B shows the percentage hatching rate of zebrafish embryos.
  • FIGs. 21C-21E are optical micrographs of the embryos exposed to UCNs at 24 hrs (scale bar: 200 ⁇ ).
  • FIGs. 21F-21H are optical micrographs of the embryos exposed to UCNs at 48 hrs (scale bar: lmm).
  • UCN concentration was 0 ⁇ g/ml for FIGs. 21 C and 2 IF, 50 ⁇ g/ml for FIGs. 21 D and 21 G and 100 ⁇ g/ml for FIGs. 21 E and 21 H.
  • FIGs. 22A-22N summarize photomorpholino studies.
  • FIG. 22A is a bar graph showing percentage increase in absorbance due to photolysis of
  • FIG. 22B is a bar graph of comparison of the percentage of no tail embryos across different samples (*P ⁇ 0.05 between control and the sample exposed to UCNs and activated with NIR).
  • FIGs. 22C- 22H show the morphology of zebrafish injected with UCNs loaded with morpholinos and irradiated with NIR (980 nm) for 8 mins.
  • FIGs. 22C-22H show control embryos (FIGs. 22C and 22F), embryos microinjected with UCNs but without irradiation (FIGs. 22D and 22G) and embryos microinjected with UCNs and irradiated with NIR (FIGs.
  • FIGs. 22I-22N show GFP expression in H-226 transfected with UCNs loaded with caged GFP and transplanted into adult zebrafish.
  • FIGs. 22I-22K show control fish without NIR irradiation.
  • FIGs. 22L-22N show test fish with NIR irradiation for 8 mins. Scale bar: 1 mm.
  • FIGs. 23A-23D are micrographs of in vivo imaging of zebrafish embryos using UCNs.
  • FIG. 23 A shows the control embryo without UCN injection.
  • FIG. 23 B shows a no tail embryo with injected UCNs.
  • FIG. 23 C shows three-dimensional Z- stack images of an embryo microinjected with UCNs. Scale bar: 200 ⁇ .
  • FIG. 23D shows in vivo imaging of UCNs in adult zebrafish. 20 ⁇ 1 of UCNs (1 mg/mL) was injected intra-peritoneally and imaged using an animal imaging system equipped with a 980 nm NIR laser.
  • FIGs. 24A-24G provide an overview of UCNs.
  • FIG. 24 A is a schematic showing the structure of core-shell UCNs and its various coatings.
  • FIGs. 24B and 24C are transmission electron micrographs (TEM) of NIR-to-UV core (FIG. 24B) and NIR- to-UV/Vis core-shell UCNs (FIG. 24C).
  • FIG. 24D shows the fluorescence emission spectrum of NIR-to-UV UCN core (inset shows the total fluorescence of the
  • FIG. 24E shows the fluorescence emission spectrum of NIR-to-UV /Vis core-shell UCNs (inset shows the total fluorescence of the nanoparticles in a cuvette) when irradiated with NIR at 980 nm.
  • FIG. 24F shows the absorbance spectrum of TPPS2a with its structure inset.
  • FIG. 24G shows mesoporous silica coated NIR-to-UV/Vis core shell UCNs.
  • FIGs. 25A-25C provide bar graphs showing the effect of varying
  • TPPS2a concentrations of TPPS2a on the viability of B16F0 cells (FIG. 25 A), phototoxicity of B 16FO cells exposed to different durations of 980 nm NIR laser (FIG. 25B), and cytotoxicity of B16F0 cells exposed to different combinations of UCNs, TPPS2a and NIR (UCNs alone, TPPS2a alone, UCN+TPPS2a, UCN+NIR, TPPS2a+NIR and UCN+TPPS2a+NIR).
  • FIGs. 26A-G provide line graphs, bar graphs and micrographs on studies with morpholinos.
  • FIG. 26A shows the cumulative percentage release of
  • FIG. 26B shows absorbance readings at 260 nm of photomorpholinos incubated with and without UCNs post NIR irradiation. Increase in absorbance indicates the increase in
  • FIG. 26C shows ROS production by UCNs (TPPS2a loaded) after irradiation with NIR and determined by APF.
  • FIG. 26D shows fluorescence images after incubation with Image-IT® LIVE Green Reactive Oxygen Species detection reagent of untreated cells.
  • FIG. 26E shows cells treated with UCNs+TPPS2a post NIR irradiation of 8 mins at a power density of 2.8 W/crn" Cells were counterstained with DAPI (scale bar: 50 ⁇ ).
  • FIGs. 26F and 26G show distribution of UCNs before (FIG. 26F) and after (FIG. 26G) NIR irradiation (scale bar: 5 ⁇ ; UCNs: red (periphery in F, G); DAPI: blue (circular or oval staining). More diffuse pattern shows cytosolic release after endosomal escape.
  • FIGs. 27A-C provide bar graphs of fluorescence intensity of UCNs in B16F0 cell suspension with and without TPPS2a at normal temperature and 4°C 24 hours after irradiation with NIR at 980 nm (FIG. 27A), and percentage STAT-3 expression (FIG. 27B) and cell viability (FIG. 27C) of B16F0 cells exposed to different combinations of UCNs, TPPS2a, morpholinos and NIR.
  • FIGs. 28A-28E show the effect of STAT-3 knockdown in a murine model of melanoma.
  • FlGs. 28C-28E are representative gross photos of a mouse from Groups 1-3, respectively. Scale bar: 1 cm; * p ⁇ 0.05 between Group 1 and Groups 2 and 3; # p ⁇ 0.05 between Group 2 and Group 3-
  • FIGs. 29A-29E provide bar graphs and micrographs of expression of STAT- 3 in tumor tissues from Groups 1-3 analyzed by ELISA after harvesting (FIG. 29A), hemolytic activity of different concentrations of UCNs in mice blood (FIG. 29B), and imaging of UCNs in tumor tissue sections with DAPI (FIG. 29C) and UCN
  • FIG. 29E is a merged image of FIGs. 29C and 29D.
  • Scale bar 50 ⁇ ; * p ⁇ 0.05 between Group 1 and Groups 2 and 3; # p ⁇ 0.05 between Group 2 and Group 3.
  • FIG. 30 is a bar graph of UV (black) and visible (grey) fluorescence of core shell UCNs with varying pH.
  • FIG. 31 is a bar graph of siRNA and TPPS2a co-stability. To determine whether co-loading of siRNA and TPPS2a would affect the stability of siRNA, siRNA and TPPS2a were incubated together for 24 hours and the absorbance of siRNA was measured at time points of 0, 2, 4, 6, and 24 hours. The absorbance value was almost stable, indicating that the co-loading with TPPS2a does not affect its stability.
  • FIG. 32 is a bar graph illustrating functional integrity of morpholinos post ROS exposure.
  • morpholinos were subjected to ROS produced by TPPS-2a. This was done in two ways: the TPPS-2a was excited by a visible laser to produce ROS or the TPPS-2a was excited through UCNs by a NIR laser. In both cases, it was observed that there was no change in the efficiency of morpholinos in knocking down STAT-3, as shown in FIG. 32.
  • FIGs. 27b and 32 are substantially identical. In FIG. 27b, the graph represents %age expression, while in FIG. 32, the graph represents %age knockdown (100 - %age expression).
  • FIG. 33 is a bar graph of UCN fluorescence in cells after overnight incubation of UCNs with cells and before NIR irradiation.
  • FIG. 34 is a bar graph of UCN fluorescence in the supernatant measured 24 hours post irradiation. From FIG. 34, it can be seen that the UCN fluorescence is lower (lesser UCN concentration of UCNs) in the supernatant in wells incubated with TPPS2a loaded UCNs as compared to wells incubated with UCNs alone. This indicates that cells in the former case expel lesser UCNs, possibly because of better endosomal escape (through PCI) and thus higher concentration of UCNs in the cytoplasm, as opposed to greater concentration of UCNs in the endocytic vesicles in the latter.
  • PCI endosomal escape
  • FIGs. 35A-35C provide micrographs of brightfield images of B16F0 cells.
  • FIG. 35A shows the control.
  • FIG. 35B shows cells transfected with UCN+Morpholinos and activated with NIR.
  • FIG. 35C shows cells transfected with
  • FIGs. 36A-36D provide micrographs and line graphs of tumors at day 12 in mice treated with Morpholino loaded UCNs (without NIR) (FIG. 36A), NIR alone (FIG. 36B), UCNs loaded with Morpholino and TPPS2a but without NIR irradation (FIG. 36C) and NIR irradiation alone (FIG. 36D).
  • FIGs. 36E and 36F show the tumor volumes from FIGs. 36A-36D (FIG. 36E) and bodyweight across 12 days (FIG. 36F). Scale bar: 1cm. From FIGs. 36A-36F, it can be seen that the progression in tumor volume was similar for all control groups across the duration of the study. This highlights the specific nature of this therapy, since the different components of the therapy do not individually result in a therapeutic effect. A combination of the above results in a marked decrease in tumor progression (shown in FIGs. 27A-27C).
  • FIGs. 37A-37B provide transmission electron micrographs of NIR-to-Vis UCN core (FIG. 37A) and after mesoporous silica coating (FIG. 37B).
  • FIG. 38 is a graph of the fluorescence emission spectrum of NIR-to-Vis UCNs.
  • FIG. 28 is substantially identical to FIG. 2A.
  • FIG. 39 is a schematic showing the comparison between natural nanoparticle delivery process and enhanced nanoparticle delivery through photochemical internalization.
  • FIGs. 40A-40D illustrate the drop in ABDA fluorescence intensity showing the production of singlet oxygen with increase in dose of NIR irradiation (FIG. 40 A), and fluorescence imaging of normal cells (FIG. 40B) and cells treated with
  • FIG. 40 is substantially identical to FIGs. 6B, 6C, 26D and 26E.
  • FIG. 41 is a bar graph comparison of the cell viability of cells loaded with Paclitaxel loaded UCNs. with and without photochemical internalization.
  • the invention provides a composition comprising an upconversion nanoparticle (UCN) configured for near infrared (NIR) light excitation and ultraviolet (UV) light emission, visible light emission, or UV and visible light emission.
  • UPN upconversion nanoparticle
  • An "upconversion nanoparticle” is nanometer-sized particle adapted for anti-Stokes emission of light.
  • a UCN has a host lattice of ceramics (such as LaF 3 , YF 3 , Y 2 0 3 , LaP0 4 , NaYF 4 , Y 2 0 2 S, Gd 2 0 2 S, La 2 0 2 S, YOF, Y 3 OCl 7 , YF 3 , GdF 3 , BaYF 5 , BaY 2 F 8 , YSi 2 0 5 , YGa0 3 , and NaGdF 4 ) doped with one or more trivalent lanthanides (such as Yb 3+ , Er 3+ , Tm 3+ , Tb + , Eu 3+ , Sm 3+ , Ho 3+ , and Dy 3+ ).
  • UCNs can be substantially solid or porous and may be substantially spherical or have other shapes, such as oblong spheroid, rectangular, rod, hexagonal, et cetera. In more particular embodiments, the UCNs are substantially spherical. In particular
  • the host lattice is NaYF 4 , such as ⁇ - NaYF 4 .
  • the host lattice is doped with Yb 3+ , Er 3+ , Tm 3+ , or a combination thereof, such as Yb 3+ and Er 3+ or Yb 3+ and Tm 3+ .
  • UCNs in some embodiments, have a "core-shell" structure, where the core and shell of the UCN are ceramics doped with one or more lanthanides. In different embodiments, the core and the shell of the UCN may have the same lanthanides or different lanthanides.
  • the core and shell of the UCN are ceramics doped with two or more different lanthanides to produce different emission peaks (e.g., UV or visible).
  • the core is doped with Yb 3+ and Er 3+ or Yb 3+ and Tm 3+ .
  • the shell is doped with Yb 3+ and Er 3+ .
  • the core is doped with Yb 3+ and Tm 3+ and the shell is doped with Yb and Er .
  • Numerous shells can be used, and core-shell UCNs need not be limited to a single shell on the core, as UCNs with multiple shells, to emit multiple wavelengths, are encompassed by the present invention.
  • the UCN has 1, 2, 3, 4, or more shells. Individual shells can contain more than one doping— with the same or a different lanthanide(s)— to enhance the fluorescence intensity and quantum yield. In certain embodiments, multiple shells have the same doping, e.g., shells 1 and 3 or 2 and 3 in a UCN with 3 or more shells. In some embodiments, the UCN has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or more emission peaks.
  • the core of the UCNs provided by the invention have an average diameter of less than about 30 nm, e.g., about 17, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nm.
  • UCNs comprising a shell and functionalizeable outer layer can have an average size, for example, of about 50 nm to about 180 nm (e.g., about 40 nm to about 200 nm).
  • the average size of the UCN is about: 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.
  • the average size of the UCN is less than about 100 nm, e.g., about 60 nm to about 100 nm.
  • UCNs consonant with the present invention can be substantially uniform in size (monodipserse) or non-uniform in size (polydisperse)— e.g. about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% polydispersity (100 x polydispersity index).
  • the UCNs are substantially monodisperse, e.g. , with less than about: 40, 35, 30, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 10, 5, 1% polydispersity.
  • UCNs provided by the invention have, in certain embodiments, a primary excitation wavelength of about 980 nm +/- about 80 nm; e.g., an excitation peak of about 890, 900, 920, 940, 960, 980, 1000, 1020, 1040, 1060, or 1080 nm.
  • an excitation wavelength of about 915nm can be used, e.g., to lower any heating effect on cells or tissues.
  • emission spectra for UCNs can be tuned throughout the UV and visible spectrum for particular applications, e.g., from about 300 nm to about 700 nm, such as about 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, or 700 nm.
  • emission peaks include peaks at 350nm, 410nm, e.g., both 350 and 410 nm.
  • the UCN can have multiple emission peaks, e.g. , in the UV and/or visible spectra.
  • the core and shell have different emission spectra, while in other embodiments, they may have the same emission spectra.
  • the core has one or more UV emission peaks and the shell has one or more visible light emission peaks.
  • the shell has one or more UV emission peaks and the core has one or more visible light emission peaks.
  • UCNs according to the invention include a
  • the functionalizeable outer layer which improve stability of UCNs in physiological solutions, and also allow for surface functionalization, e.g., association, such as adsorption or conjugation (covalent (e.g. , N-Hydroxysuccinimide (NHS) and ethyl(dimethylaminopropyl) carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC) chemistry), ionic, et cetera), of a payload, such as a bioactive molecule or amphiphilic photosensitizer.
  • the functionalizable outer layer is an amorphous layer, such as an amorphous silica coating.
  • the functionalized outer layer is a "mesoporous outer layer.”
  • the mesoporous outer layer increases the solubility of UCNs in an aqueous solution, as well as optionally provides a substrate for attachment of a payload, such as a photosensitizer and/or a bioactive molecule, such as a caged bioactive molecule.
  • the mesoporous outer layer is a silica.
  • UCNs comprising, e.g., contained within, functionalized outer layers are encompassed by the invention.
  • Exemplary functionalizable outer layers for use in the invention include amorphous silica coating, silane-PEG coating, polymer coating, dendrimer coating, citric acid ligand exchange, et cetera.
  • UCNs can include additional outer layers, such as titanium oxide ⁇ e.g., for use in radiotherapy), but typically the outermost layer is a functionalizable outer layer, such as a mesoporous outer layer. So, for example, where a UCN includes a titanium oxide layer, such as the next to most outer layer, the UCN still includes a functionalizable outer layer, such as silica.
  • UCNs provided by the invention can include multiple outer layers, such as an amorphous outer layer and an outermost mesoporous outer layer— e.g., an amorphous silica coating and an outermost mesoporous silica coating.
  • Functionalizable outer layers such as a mesoporous outer layer, are about 4 nm in thickness; e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10 nm in thickness, or more. Pore sizes can be varied by modifying the coating procedure.
  • the average pore size for the mesoporous outer layer is about: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0 nm. in more particular embodiments the average pore size for the mesoporous outer layer is about 1.5nm to about 2.5nm, e.g., about 2.0 nm.
  • the UCNs provided by the invention can further comprise one or more compounds associated with ⁇ e.g. adsorbed or bound (covalently or non-covalently)) the functional izeable outer layer.
  • One or more of a variety of molecules can be associated with the functionalizeable outer layer for, e.g. delivery to a cell or tissue, and/or to facilitate uptake or delivery of the UCN and any associated compounds.
  • a molecule in a composition with a UCN is an amphiphilic photosensitizer.
  • Amphiphilic photosensitizers for use in the present invention are capable of light-induced release of reactive oxygen species (ROS), such as singlet oxygen, and include meso- tetraphenylporphine with two sulfonate groups on adjacent phenyl rings (TPPS2a) or Al(III) phthalocyanine disulfonate chloride (adjacent isomer) (AlPcS 2 a)-
  • ROS reactive oxygen species
  • the amphiphilic photosensitizer is adsorbed to a mesoporous outer layer of the UCN.
  • Amphiphilic photosensitizers can be used in the methods provided by the invention to facilitate release of UCNs from intracellular compartments, such as endosomes, e.g., when UCNs are contacted with cells for uptake by an endosomal pathway (e.g. , clatharin-mediated endocytosis, caveolae, macropinocytosis,
  • an endosomal pathway e.g. , clatharin-mediated endocytosis, caveolae, macropinocytosis,
  • the working range of such photosensitisers is about 0.1 to 1 ⁇ g/ml.
  • the release rate of TPPS2a from the UCNs is about 50-70% over 72 hours, which makes the effective concentration of exposure about 0.8-1.2 ⁇ g/mg.
  • the concentration of UCNs usually used is about 500 ⁇ g/ml (e.g., about 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ⁇ ), thus, the effective concentration of TPPS2a to which cells are exposed is about 0.4-0.6 ⁇ g/ml.
  • any suitable delivery mechanism for UCNs is encompassed by the invention, including gene guns, tissue-site administration (such as injection), as well as administration of moieties comprising the UCNs, such as cells (e.g., stem cells, fibroblasts, and combinations thereof) containing UCNs (by any means), e.g. , administering UCN-containing cells to a tissue.
  • tissue-site administration such as injection
  • moieties comprising the UCNs, such as cells (e.g., stem cells, fibroblasts, and combinations thereof) containing UCNs (by any means), e.g. , administering UCN-containing cells to a tissue.
  • the compound associated with a UCN is a bioactive molecule.
  • a "bioactive molecule” in this invention includes molecules that elicit a biological effect, and includes nucleic acids, plasmids (including expression plasmids), morpholinos or siRNAs for knocking down gene expression, proteins (e.g., growth factors or other signaling proteins, hormones, as well as immunoglobulins and immunoglobulin-like molecules), peptides, amino acids, neurotransmitters, non-peptide hormones, coenzymes, vitamins, as well as small molecule drugs (e.g., organic or inorganic), and combinations of any of the foregoing.
  • Exemplary small molecule drugs include, for example, chemotherapeutic agents, including anti-neoplastics, such as diterpenes, which include taxanes, such as paclitaxel (ChemID CID 36314) and docetaxel (Chem ID CID 148124), as well as their various salts, esters, or derivatives (such as conjugates).
  • chemotherapeutic agents for use consonant with the invention include alkylating agents, such as, nitrogen mustards (e.g. mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan, and their salts, esters, and
  • nitrosoureas e.g N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin, and their salts, esters, and derivatives
  • tetrazines e.g dacarbazine, mitozolomide and temozolomide, and their salts, esters, and derivatives
  • aziridines e.g.
  • thiotepa mytomycin and diaziquone (AZQ), and their salts, esters, and derivatives
  • cisplatins and derivatives e.g., cisplatin, carboplatin and oxaliplatin, and their salts, esters, and derivatives
  • non-classical alkylating agents e.g. procarbazine and
  • anti-folates such as methotrexate and pemetrexed, and their salts, esters, and derivatives
  • fluoropyrimidines such as fluorouracil and capecitabine and their salts, esters, and derivatives
  • deoxynucleoside analogues such as cytarabine, gemcitabine, decitabine, Vidaza, fiudarabine, nelarabine, cladribine, clofarabine and pentostatin and their salts, esters, and derivatives
  • thiopurines such as thioguanine and mercaptopurine and their salts, esters, and derivatives
  • anti-microtubule agents including vinca alkaloids (such as vincristine, vinblastine, vinorelbine, vindesine, and vinflunine and their salts, esters, and derivatives) and taxanes, as described above); topoisomerase inhibitors (such as irinotecan, topotecan,
  • the bioactive molecule is a caged bioactive molecule.
  • a "caged" bioactive molecule is an inactive form of the bioactive molecule where the bioactive molecule is attached or linked to a caging group, e.g. a photolabile caging group that is subject to photoactivation— i.e., the caging group is photolabile.
  • Exemplary caging molecules include l-(4,5-dimethoxy-2-nitrophenyl) diazoethane, NPE [ 3 -(l-(2-Nitrophenyl)Ethyl) Ester, Disodium Salt)], CNB [a-Carboxy-2- Nitrobenzyl Ester], ATFB, [SE (4-azido-2,3,5,6-tetrafluorobenzoic acid, succinimidyl ester)], CMNB [(5-Carboxymethoxy-2-Nitrobenzyl) Ether, Dipotassium Salt], DMNB [4,5-Dimethoxy-2-Nitrobenzyl ester], et cetera.
  • the UCNs and UCN-containing compositions provided by the invention can be used in a variety of methods that make up additional aspects provided by the invention.
  • the invention provides methods of visualizing a biological tissue.
  • the methods entail contacting the tissue with an upconversion nanoparticle (UCN) configured for near infrared (NIR) light activation and ultraviolet (UV) light emission, visible light emission, or UV and visible light emission, exposing the tissue to NIR light; and detecting UV light, visible light, or UV and visible light emitted from the UCN.
  • the visualizing of the tissue is non-destructive.
  • the invention provides methods of photochemical internalization of a UCN. These methods include the steps of contacting a cell with an upconversion nanoparticle (UCN) configured for near infrared (NIR) light activation and ultraviolet (UV) light emission, visible light emission, or UV and visible light emission, wherein the UCN has a mesoporous outer layer with an adsorbed amphiphilic photosensitizer and exposing the cell to NIR light to release the UCN to the cytosol of the cell.
  • the photochemical internalization is non-destructive to the cell.
  • the cell comprises a photosensitive ion channel and in more particular embodiments, the photosensitive ion channel is Channelrhodopsin.
  • the photosensitive ion channel is stimulateable by light with the wavelength of an emission peak of the UCN.
  • the methods further include detecting UV light, visible light, or UV and visible light emitted from the UCN.
  • the UCN comprises a caged bioactive molecule adsorbed to the mesoporous outer layer of the UCN.
  • the invention provides methods of delivering a target compound to a cell.
  • the methods include the steps of contacting the cell with an upconversion nanoparticle (UCN) configured for near infrared (NIR) light activation and ultraviolet (UV) light emission, visible light emission, or UV and visible light emission, where the UCN has a mesoporous outer layer with an adsorbed target compound (optionally, in some embodiments, further including an adsorbed
  • UCN upconversion nanoparticle
  • NIR near infrared
  • UV ultraviolet
  • the target compound is a bioactive molecule (such as a chemotherapeutic agent).
  • the bioactive molecule is a caged bioactive molecule, e.g., releasable by light with the wavelength of an emission peak of the UCN.
  • the caged biomolecule is released substantially simultaneously with the activation of the amphipillic photosensitizer.
  • the invention provides methods of modulating (e.g.
  • nucleic acid DNA, RNA, mRNA, gene, siRNA, or morpholino
  • methods include the steps of exposing a cell contacted with an upconversion
  • the invention also provides methods of modulating protein expression and/or activity in a cell, where the bioactive molecule modulates protein expression and/or activity, e.g. by binding or association with a target protein.
  • the cell is an isolated cell.
  • the isolated cell is administered to a tissue.
  • the tissue is in a multicellular organism.
  • the multicellular organism is a chordate, such as a zebrafish, or a vertebrate, such as a mammal, such as a human.
  • the invention provides methods of treating cancer or reducing tumor volume in a subject in need thereof. These methods entail
  • chemotherapeutic agent (as described above), e.g., on a functionalized outer layer, such as a mesoporous outer layer).
  • the UCN-containing composition can be administered systemically (e.g., intravenously) or at the site of a tumor and, optionally, the method can entail the uncaging of a caged chemotherapeutic agent and/or photochemical internalization (e.g., with enhanced endosomal release) of the chemotherapeutic agent as described herein.
  • the chemotherapeutic agent is a taxane, such as paclitaxel.
  • the cancer is a solid tumor, in other embodiments, the caner is a hematological cancer.
  • the cancer is melanoma.
  • a "subject" is a mammal, including primates (e.g., humans or monkeys), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species.
  • suitable subjects include, but are not limited to, human patients (e.g., a human with, suspected of having, a cancer).
  • the subject can be at any stage of development, including prenatal, perinatal, infant, toddler, child, young adult, adult, middle-aged, or geriatric.
  • UCNs upconversion nanoparticles
  • NIR near-infrared
  • the nanoparticles are synthesized in a core-shell format making them have multiple emission peaks in the UV and Visible range.
  • UCNs are excited by NIR light which has excellent tissue penetration properties since the absorption of NIR by the tissue components is very minimal. Since it has excellent penetration, UCNs can be activated in deep tissues for photoactivation of different molecules.
  • NIR light is very safe for use when compared to UV light.
  • the UCNs show excellent photostability, chemical stability and thermal stability.
  • the synthesized core-shell UCNs were used for simultaneous activation of endosomal escape and gene expression/knockdown by using deep penetrating NIR irradiation.
  • Endosomal escape was achieved by photochemical internalization (PCI) by using the visible emission of UCNs for activating a photosensitiser TPPS-2a that can disrupt the endosomal membrane and deliver the nanoparticles to the cytosol, thereby enhancing the intracellular uptake of UCNs.
  • Control of gene expression was achieved by using the UV emission of UCNs to activate photocaged nucleic acids to make them functional only at the intended site.
  • Gene expression can be photo-controlled by caging transcriptional activators/plasmid for gene expression or short interfering RNAs (siR As) for RNA interference (RNAi) with a light sensitive molecule— dubbed "photocaging”— that renders the nucleic acid (NA) non-functional.
  • RNAs short interfering RNAs
  • photocaging RNA interference
  • Photocaging is commonly done by modifying certain base pairs or chemical bonds.
  • UV light ⁇ ⁇ 350 nm
  • the photocaging modification is destroyed-termed “uncaging”— rendering the NA functional.
  • DMNPE l-(4,5-Dimethoxy-2-nitrophenyl) diazoethane
  • PCI Photochemical Internalization
  • PS amphiphilic photosensitizers
  • TPPS-2a meo-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings
  • UV/Visible light required for activation of caged nucleic acids/TPPS2a has very low tissue penetration capabilities and thus limits these techniques to in vitro use.
  • NIR light is ideal for photoactivation as it has very good tissue penetrating capabilities and is very safe for in vivo use.
  • NIR light cannot be used directly for photoactivation, and hence a system is required to convert NIR light to different wavelengths in the UV/Visible range for efficient deep-tissue photoactivation.
  • upconversion nanoparticles known as upconversion nanoparticles have the ability to convert near- infrared (NIR) to ultraviolet (UV) or visible light via an anti-Stokes emission.
  • UCNs have host lattices of ceramics (LaF 3 , YF 3 , Y 2 0 3 , LaP0 4 , NaYF 4 ) and doped with trivalent lanthanides (Yb 3+ , Er 3+ , Tm 3+ ).
  • Upconversion fluorescence can be generated using inexpensive, commercial continuous wave laser diodes, is exceptionally photostable with low photodamage to cells and proteins.
  • UCNs have the ability to be activated in deep tissues due to absence of upconversion property in biological molecules and the penetration capability of NIR light, thus enabling photoactivation of molecules and long-term live imaging in deep tissues.
  • UCNs for enhanced endosomal escape through PCI and photoactivation of caged NA.
  • Special Mesoporous core-shell UCNs that could emit in both UV and visible ranges were synthesized for these purposes.
  • Both the core and the shell are P-NaYF 4 crystallines with the core doped with Ytterbium (Yb) and Thulium (Tm) and the shell doped with Yb and Erbium (Er) to achieve UV and visible emissions, respectively.
  • Caged nucleic acids (plasmid DNA or siRNA) and TPPS-2a were loaded onto the mesoporous silica layer.
  • the enhanced intracellular delivery of caged nucleic acids and controlled gene expression/knockdown by NIR photoactivation were studied in vitro in B16F0 cells and in vivo in Zebrafish.
  • the core-shell UCNs had excellent control over deep tissue activation of TPPS 2a and caged nucleic acids. This novel technique is not limited to activate caged nucleic acids and photosensitizers but can be used for a wide range of other applications which requires deep tissue photoactivation.
  • NIR-to-UV UCNs was used to check if it can be used efficiently for nucleic acid delivery and for photocontrollable gene expression.
  • Photoiabile groups such as 4,5-dimethoxy-2-nitroacetophenone (DMNPE) can be cleaved from 'Caged' DNA or siRNA due to the UV emission (FIGs. 1 A-IC) of such nanoparticles upon excitation with NIR at 980nm.
  • FIG. 3A shows a diagrammatic representation of the core-shell UCN with various coatings.
  • FIGs. 3B and 3C show TEM images of the core and core shell UCNs, respectively. From these images we can see that the nanoparticle core is below 30 nm in size and there is a slight increase in size with the shell.
  • the core of the nanoparticles is Yb/Tm doped NaYF 4 , which emits in the UV- Blue range as can be seen from FIG. 3D.
  • the shell of the UCNs is Yb/Er doped and emits in the visible (green) range (FIG. 3E), and the core shell UCNs emit across the entire range from UV to blue and green in the visible range (FIG. 3F).
  • the core shell nanoparticles were coated with a mesoporous silica layer (FIG. 4A) in order to improve the solubility in aqueous solutions and to enable loading of biomolecules and other chemicals on to their surface.
  • a mesoporous silica layer (FIG. 4A)
  • biomolecules like siRNA and other chemicals such as photosensitisers (in this case TPPS-2a) could be loaded on to these core shell UCNs efficiently and released in a controlled manner
  • TPPS-2a photosensitisers
  • FIG. 4B shows a cumulative release profile for UCNs co-loaded with siRNA and TPPS-2a. From the graph, we can see that the release is not immediate but takes place over several hours.
  • TPPS-2a works by producing reactive oxygen species
  • the first method employed APF (Amino phenyl Fluorescein) dye to detect ROS production by TPPS-2a in solution.
  • a solution containing TPPS-2a loaded UCNs and APF was exposed to NIR at 980 nm for increasing durations of time and fluorescence response (at 515 nm) was recorded. From FIG. 6A, we can see that with increasing irradiation time, the amount of ROS produced increased indicating that the emissions from UCNs are sufficient to induce ROS production by TPPS-2a.
  • the second method used the Image-iT LIVE Reactive Oxygen Species (ROS) Kit to determine whether TPPS-2a loaded onto the core shell UCNs was capable of producing ROS in cells.
  • the assay is based on 5-(and-6)- carboxy-2',7'- dichlorodihydrofluorescein diacetate (carboxy- H2DCFDA), a reliable fluorogenic marker for ROS in live cells.
  • carboxy- H2DCFDA dichlorodihydrofluorescein diacetate
  • ROS production the reduced fluorescein compound is oxidized and emits bright green fluorescence.
  • TPPS-2a when excited, produces ROS in cells (seen in green). The staining is not very dark, which is in keeping with the theory that the production of ROS is localized and minimal. Also, the ROS production is indeed because of the TPPS-2a and not an artifact because in the negative control (with cells only, FIG. 6B), no staining was observed.
  • ROS Image-iT LIVE Reactive Oxygen Species
  • FIG. 6J shows the distribution of core shell UCNs in B16F0 cells at time 0 and 10 mins after irradiation with NIR, respectively. From FIG. 6 J, we can see that the UCNs are present in clumps and are not very well dispersed inside the cells. However, only 10 mins after irradiation, we can see a marked change in the distribution of UCNs. In FIG.
  • the UCNs When the cells are irradiated with NIR at 980 nm, the UCNs emit UV and visible light. Visible light at 413 nm causes the TPPS- 2a to become activated, upon which it produces reactive oxygen species (ROS). This localized production of ROS causes the disruption of the walls of the endosomal vesicles, causing the contents of the endosomes to be released into the cytoplasm. Simultaneously, the UV emission of the UCNs results in the uncaging of the siRNA. In this way, core shell UCNs, by the virtue of their emissions, can be used for targeted and enhanced delivery of nucleic acids. The mechanism is illustrated in the schematic given in FIG. 7A. We further proceeded to show that this mechanism works in vitro and in vivo.
  • ROS reactive oxygen species
  • FIGs. 18A-18D microscopy as shown in FIGs. 18A-18D. It can be seen that the delivery of co-loaded UCNs without NIR activation does not show significant GFP expression indicating the successful caging of plasmids, thus enabling control over the whole process.
  • UCNs ability to activate photo-morpholinos in zebrafish embryos was tested by microinjecting UCNs loaded with No-tail morpholinos.
  • the No- tail morpholino knocks down the ntla gene, which is responsible for the development of tail in zebrafish.
  • the UCNs injected embryos were irradiated with UV light or NIR light at 5 hpf and the development of the embryos were monitored over a period of 72 hours.
  • the embryos injected with UCNs and irradiated with a NIR laser showed no-tail morphology, whereas the embryos without NIR irradiation developed normally as seen in FIGs. 1 OA- IOC.
  • NIR irradiated samples had lower phototoxicity when compared to samples exposed to conventional UV light.
  • the UCNs were also distributed well in the embryos, as seen in FIGs. 11 A-l 1C and 12A and 12B.
  • concentration of TPPS-2a used for loading was determined according to these values such that the concentration that the cells were exposed to was below 0.8 ⁇ g/mL.
  • nucleic acids like plasmids/siRNA and small molecules like TPPS-2a can be efficiently co-loaded on to mesoporous silica coated UCNs. Cumulative release profiles for siRNA and TPPS-2a showed a gradual release over several hours, ensuring that nucleic acids and TPPS-2a are delivered efficiently to the cells.
  • P-NaY 4.7 F4:Yb25,Tm 03 core was synthesized using thermal decomposition method and then purified and dispersed in cyclohexane. Briefly, 0.78 M of YC1 3 , 0.20 M of YbCl 3 , and 0.2 M of ErCl 3 was taken in a 50 ml three-necked flask and heated till dryness. Then, 6ml of Oleic Acid and 15ml of 10-Octadecene was added and the solution was heated till 150°C.
  • the solution was cooled to 50°C and the previously synthesized NaYF 4 :Yb,Tm core was added and the resulting mixture was heated to 110°C for 30 minutes to remove cyclohexane. Once the cyclohexane was removed, the solution was cooled to 50°C and 0. lg of NaOH and 0.1482 g of NH 4 F in 5ml methanol each were added. Subsequently, the solution was heated to 110°C for 15 minutes and then degassed at the same temperature for next 20 minutes. The solution was then heated at 300°C under argon atmosphere for 1 hour, cooled to room
  • a second coating of mesoporoous silica was done.
  • 2.6 ml of 30% NH 4 OH, 13 mL ethanol, 260uL TEOS (Tetraethyl orthosilicate) and 104uL CI 8 TMS (Octadecyltrimethoxysilane 90%) were added and shaken for 6 hours.
  • the resulting homogeneous white solution was dried in hot-air oven at 60°C overnight and then subsequently calcinated at 500°C in a furnace for 6 hours. The dried powder was then milled and subsequently dissolved in deionized water.
  • NCI-H226 cells and B16-F0 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in DMEM culture medium (Invitrogen) supplemented with 10 % FBS (Invitrogen), 100 units/mL of penicillin and 100 ⁇ g/mL of streptomycin, and maintained in a humidified, 5 % carbon dioxide (C0 2 ) atmosphere at 37°C.
  • ATCC American Type Culture Collection
  • FBS Invitrogen
  • C0 2 carbon dioxide
  • B 16F0 cells were treated with different conditions and then incubated for 24 hours before being assayed for cell viability using CellTiter 96 ® AQ ueo us One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) as per manufacturer's instructions.
  • B 16F0 cells were incubated with 0.5 mg/mL of UCNs loaded with TPPS-2a overnight. The excess nanoparticles were then washed off the cells and the cells were irradiated using a 980 nm NIR laser. The ROS generated in the cells was detected using an Image-iT LIVE Reactive Oxygen Species (ROS) Kit (Molecular Probes, OR, USA) as per manufacturer's instruction. The cells were also counterstained with DAPI and imaged using a confocal laser-scanning microscope (Nikon CI Confocal, Nikon, Tokyo, Japan) specially fitted with a CW 980 nm laser excitation source (Opto-Link Corp., Hong Kong). Endosomal release of UCNPs
  • Bl 6F0 cells were incubated with TPPS-2a loaded UCNs in a 96 well plate and different columns were irradiated at 4 time points (6, 8, 10 and 12 hrs, respectively) with NIR at 980 nm for 8 mins per well.
  • 4 other columns contained cells incubated with UCNs without any TPPS-2a. These columns were also irradiated with NIR at time points of 6, 8, 10 and 12 hours.
  • the wells were washed thoroughly and cells trypsined.
  • the cells from each of the 8 columns were taken in different cuvettes, i.e., 4 cuvettes containing cells that had been irradiated at 6, 8, 10, 12 hrs, respectively, with TPPS-2a and 4 without.
  • B16F0 cells were plated on a 24 well plate. After overnight incubation, TPPS-2a loaded UCNPs were added to the test and control wells. After 8 hours of incubation, the media was removed and the cells were incubated with O.lmg/mL Concanavalin A-Alexa Fluor 488 in culture medium and 0.01 mg/mL DAPI for 30 min at 37°C. They were then washed thrice with PBS and replenished with fresh culture medium. The cells were then imaged using a confocal microscope (Nikon CI Confocal, Nikon, Tokyo, Japan) (UCNs, the cell stained with Concanavalin A and nuclei stained with DAPI).
  • Concanavalin emission was pseudo-coloured to red to distinguish from the green emission of UCNs. Merging of the two images showed localization of UCNPs in the cells.
  • Another set of wells were irradiated with a CW 980 nm NIR laser for 8 mins and was imaged similarly after 10 mins.
  • Example 2 Mesoporous Silica Coated Upconversion Nanocrystals for Near Infrared Light-Triggered Control of Gene Expression in Zebrafish
  • This example demonstrates the use of nanoparticles as carriers and nanotransducers for light controlled gene knockdown in Zebrafish embryos and light controlled gene expression in adult Zebrafish.
  • This example includes data presented in Example 1 , as well as new data.
  • the no tail gene, involved in mesoderm patterning and notochord formation was successfully knocked down in Zebrafish embryos and GFP expression using UCNs was demonstrated in cancer cells injected into adult Zebrafish.
  • the absence of photobleaching and low autofluorescence of these nanoparticles allowed for background free NIR based imaging in both the embryos and adult fish.
  • UCNs were synthesized with a NaYF 4 core and codoped with Yb 3+ (25%) and Tm 3+ (0.3%) using the method as previously reported.
  • YC1 3 , YbCl 3 and TmCl 3 were taken in the ratio mentioned above and mixed with 6 ml of Oleic acid and 15 ml of Octadecene and heated to 160°C to form a homogenous solution. Then, a solution of 0.4 mmol sodium hydroxide (0.1 g) and 0.25 mmol ammonium fluoride (0.148 g) in 10 ml of methanol was slowly added and stirred for 30 min. Methanol was then
  • FTIR spectroscopy study was performed by KBr-disc pellet method using Shimadzu IRPrestige-21 model spectrometer (Shimadzu Corporation, Kyoto, Japan). With an agate mortar, sample was thoroughly ground with some KBr salt that acts as a non-absorbing matrix and background in the disc pellet preparation. The ground mixture was then pressed using a Mini Hand Press MHP-1 (Shimadzu Corporation, Kyoto, Japan) to produce highly transparent KBr-disc pellets. FTIR spectra of these discs containing the samples were recorded by averaging 45 scans at a resolution of 4 cm 1 and drawn in transmittance mode. This was performed for the UCN core as well as mesoporous silica coated UCNs to confirm presence of mesoporous silica coating.
  • ZFL cells were grown in complete growth medium at 28°C without carbon- di-oxide.
  • the media consisted of 50% L-15 medium, 35 % DMEM high-glucose medium and 15 % Ham's F12 medium supplemented with O.Olmg/ml bovine insulin, 50 ng/ml mouse EGF, 5% heat-inactivated fetal bovine serum and 0.5% Trout Serum.
  • the Academic were treated with different concentrations of UCNs and then incubated for 24 hours before being assayed for cell viability using CellTiter 96 ® AQ ue0 us One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) as per manufacturer's instructions.
  • photomorpholino duplex were resuspended in saline. About 120 embryos was injected with 300 ng of UCNs loaded with the photomorpholino duplex, 60 with the UCNs loaded with a scrambled sequence and another 50-60 were untreated. This was done immediately post fertilization. At 2 hpf, all embryos except one group injected with UCNs loaded with photomorpholino, were irradiated with NIR at 2.8W/cm 2 for 8 mins. The embryos were kept in E3 medium at 28°C and the medium was replenished twice a day. The morphology of the embryos was observed at 24, 48 and 72 hpf for the no tail phenotype.
  • Wild-type adult zebrafishes and zebrafish embryos were obtained from Institute of Biochemistry & Cell Biology, Shanghai, China and maintained according to the protocol approved by the Institutional Animal Care and Use Committee, Hefei University of Technology.
  • Healthy zebrafish embryos were collected immediately after spawning from the aquarium. Ten embryos were taken for each concentration of nanoparticle exposure and they were thoroughly rinsed three times using E3 medium. The embryos were then incubated in a 24-well microplate containing different concentrations of UCNs in E3 medium for 72 hours in 28°C. All the experiments were done in triplicate. The hatching rate of the embryos was noted. The embryos were also imaged at 24 and 72 hpf using a Nikon microscope.
  • Zebrafish embryos were placed on a petridish and imaged using a fluorescence confocal microscope (Nikon CI Confocal, Nikon, Tokyo, Japan) modified with a 980 nm NIR laser.
  • the adult zebrafishes were imaged in an iBox® SpectraTM Small Animal Imaging System (UVP, LLC, CA, USA) equipped with a 980 nm NIR laser.
  • UVP iBox® SpectraTM Small Animal Imaging System
  • NIR to UV UCNs were synthesized, with a P-NaYF 4 crystalline structure doped with Ytterbium (Yb 3+ ) and Thulium (Tm 3+ ). These UCNs were about 20 nm in diameter (FIG. 19 A) before coating with mesoporous silica. After coating, the size increased to about 30 nm (FIG. 19B). This coating was done to improve the aqueous solubility of the UCN core (previously in cyclohexane), to improve physiological stability and safety for biological use. These nanoparticles could be excited by NIR at 980 nm and emitted in the UV and blue visible ranges (FIG. 19C).
  • FTIR was performed for both the UCN core and mesoporous silica coated particles to ascertain whether the particles had indeed been coated. Characteristic FTIR spectra were observed for both the core and the coated particles (FIG. 20A). This confirmed the presence of the mesoporous coating on the UCNs. After coating, the mesoporous silica coated UCNs were subjected to a photostability test to determine whether their upconverted UV emission remains stable over the irradiation duration (8 mins) used in subsequent experiments. From FIG. 20B, it can be seen that up to 16 mins of irradiation with NIR, the UV emission of the UCNs remains stable, indicating excellent photostability of these particles.
  • FIG. 21 A depicts the effect of increasing UCN concentrations on the viability of ZFL cells (Zebrafish liver).
  • the toxicity was negligible even at 0.5 mg/ml nanoparticle concentration.
  • One of the ways in which the developmental toxicity of a foreign material is tested in Zebrafish is by analyzing the affect that material has on the hatching rate of the embryos. If the hatching rate is retarded, the material can be deemed to be toxic.
  • incubation with UCNs did not affect the hatching rate (FIG. 2 IB). Nearly 100% of embryos hatched even at the highest UCN concentration (0.1 mg/ml). In addition, incubation with these nanoparticles did not cause any
  • the no tail gene was chosen primarily because of its role in early development and the startling phenotype resulting from its knockdown. When this gene is knocked down early in the development, the resulting embryos are formed without a 'tail' causing them to take on a stunted appearance with a poorly developed caudal region.
  • a photomorpholino duplex directed against this gene was used. This duplex consists of a sense strand containing a photolabile group in its backbone (e.g. between or on nucleotides 10 and 1 1 in SEQ ID NO: 1) and an antisense strand that is unmodified.
  • RNA interference RNA interference
  • the sequence is GATCGTCGGATTATCTCAAG (SEQ ID NO: 1) and was obtained from GeneTools LLC.
  • RNAi RNA interference
  • the sense strand gets cleaved, releasing the antisense strand and resulting in RNAi-mediated knockdown of this gene.
  • mass UV irradiation is undesirable and with our UCNs, we wanted to ascertain whether NIR induced, localized UV production (by these UCNs) could be used for efficient knockdown.
  • FIGs. 22C and 22D show normal development, the no tail phenotype is evident in FIG. 22E.
  • FIGs. 22F-22H show embryos from the same groups at 72 hpf. Again, the no tail phenotype was evident in the test group as the embryo appears stunted with a poorly developed caudal region. This led to the conclusion that NIR to UV emission of these UCNs could be used effectively for photocontrolled gene knockdown in Zebrafish embryos.
  • caged EGFP enhanced green fluorescent protein
  • UCNs were used for photocontrolled expression of this gene.
  • 'Caging' of a plasmid involves modifying it with a photolabile caging group that prevents it from getting expressed in the cell. Upon irradiation with light of a suitable wavelength, usually UV, the photolabile caging group gets released, thereby allowing the plasmid to get expressed again.
  • a suitable wavelength usually UV
  • FIGs. 22I-22N shows the comparison of GFP expression between the control and test animals. It can be seen that the test group (FIGs.
  • FIGs. 23 A and 23B show Zebrafish embryos at 72 hpf for control (untreated) and test embryos, respectively. It could be seen that UCNs were distributed throughout the body in the test group and that there was no signal in the control group. This showed that the signal was due to UCNs alone and not due to background. Z-stack imaging confirmed that these nanoparticles were distributed throughout the body and not present on the surface alone (FIG. 23C).
  • Example 3 Near Infrared Light Based Nano-Platform Boosts Endosomal Escape and Controls Gene Knockdown In-vivo
  • TPPS2a (meso-tetraphenylporphine with two sulfonate groups on adjacent phenyl rings), a photosensitiser used in PCI (photochemical internalization)
  • PCI photochemical internalization
  • photomorpholino in this case anti STAT3
  • the data in this example includes data presented in Example 1 as well as new data.
  • the core shell UCNs were coated with a layer of mesoporous silica and then co-loaded with TPPS2a and photomorpholinos.
  • TPPS2a is a photosensitiser that absorbs maximally at 413 nm and is used for photochemical internalization. It should not be confused with photosensitisers used for PDT ⁇ e.g. Merocyanine 540, Zinc phthalocyanine et cetera), where the purpose of the photosensitiser is to cause cell death.
  • the photomorpholino loaded is a duplex consisting of a sense photomorpholino and an antisense morpholino hybridized together. Upon irradiation with UV light, the sense photomorpholino gets cleaved, resulting in the release of the antisense
  • RNAi RNAinterference
  • TPPS2a and photomorpholino duplex loaded UCNs enter the cell by endocytosis. When these cells are irradiated with NIR at 980 nm, the UCNs emit both UV and visible light simultaneously. The visible light at 413 nm causes TPPS2a to become activated upon which it produces reactive oxygen species (ROS) locally.
  • ROS reactive oxygen species
  • FIG. 24A shows a diagrammatic representation of this core-shell UCN with various coatings.
  • Both the core and the shell are P-NaYF 4 crystalline structures with the core doped with Ytterbium (Yb ) and Thulium (Tm ) and the shell doped with Yb and Erbium (Er ) to achieve multiple UV and visible emissions respectively.
  • Yb Ytterbium
  • Tm Thulium
  • Er Er
  • FIGS. 24B and 24C show transmission electron microscope (TEM) images of the core and core-shell UCNs respectively. These images illustrated that the nanoparticle core was below 30 nm in size with a slight increase in size due to the shell. This size was suitable for endosomal uptake. Although the core alone could emit in the UV and blue regions (FIG. 24D), emission at 413 nm needed for TPPS2a excitation was absent. The core shell UCNs however, had multiple UV and visible emission peaks (FIG. 24E), two of which coincided with the photomorpholino and TPPS2a absorption maxima respectively (FIG. 24F). The insets in FIGs. 24D and 24E show the visible fluorescence of the NIR to UV core and NIR to UV-Vis core-shell UCNs when irradiated with an NIR laser at 980 nm.
  • TEM transmission electron microscope
  • the core-shell nanoparticles were coated with a mesoporous silica layer (FIG. 24G) in order to improve their solubility in aqueous solutions and enable loading of molecules onto their surface.
  • the mesoporous silica coating has a pore size of about 2 nm. This coating also significantly increased the surface area, which was suitable for loading nucleic acids and photosensitisers.
  • UCNs stand for mesoporous silica-coated NIR-to-UV/ Visible core-shell UCNs.
  • UCN+TPPS2a, UCN+NIR, TPPS2a+NIR and UCN+TPPS2a+NIR was tested and it was found that in all 6 cases the cell death was minimal (FIG. 25C).
  • the concentration of UCNs used was 500 g/mL, TPPS 2a- 0.7 ⁇ g/mL and irradiation was done using a CW 980 nm NIR laser at a power density of 2.8 W/cm 2 for 8 min.
  • FIG. 26A is a diagrammatic representation of FIG. 26A.
  • TPPS2a did not affect the stability of the photomorpholino (FIG. 31) since the absorbance of the nucleic acid remained unchanged even after 24 hours of co-incubation with TPPS2a.
  • the ROS produced by TPPS2a did not affect the functional integrity of the nucleic acid (FIG. 32).
  • FIGs. 26F and 26G show the distribution of core-shell UCNs loaded with TPPS2a in the same field of cells at time 0 and 10 mins after irradiation with NIR respectively. Initially the UCNs are present in clumps and not very well dispersed inside the cells. However, only 10 mins after irradiation, we could see a marked change in the distribution of UCNs, indicating improved endosomal release.
  • Nanoparticle-based gene therapy faces debilitating hurdles like poor endosomal escape and limited control over gene expression.
  • a unique solution was provided to address existing limitations by developing a nano-platform, which could utilize highly penetrating NIR light for photoactivation. This is believed to be the first report of using such a system for simultaneous gene delivery, photo- controlled gene expression and photochemical internalization in-vitro and in-vivo with negligible toxicity and additional background free imaging capabilities.
  • B16F0 cells were purchased from the American Type Culture Collection (ATCC). All chemicals for nanoparticle synthesis and surface coating like Yttrium chloride, Thulium chloride, Ytterbium chloride, N-[3-
  • AEAPTMS trimethoxysilylpropyl]ethylenediamine
  • acetic acid and cyclohexane were purchased from Sigma-Aldrich (Singapore).
  • Photo-morpholinos were purchased from Gene Tools, LLC, USA.
  • TPPS2a was purchased from PCI Biotech, Oslo, Norway.
  • CellTiter 96 ® AQ ue0 us One Solution Cell Proliferation Assay for cytotoxicity testing was purchased from Promega, Madison, WI, USA.
  • Image-iT LIVE Reactive Oxygen Species (ROS) Kit was purchased from Molecular Probes, OR, USA. Thermo
  • the solution was cooled to 50°C and the previously synthesized NaYF 4 :Yb,Tm core was added and the resulting mixture was heated to 110°C for 30 minutes to remove cyclohexane. Once the cyclohexane was removed, the solution was cooled to 50°C and O.lg of NaOH and 0.1482 g of NH4F in 5ml methanol each were added. Subsequently, the solution was heated to 110°C for 15 minutes and then degassed at the same temperature for next 20 minutes. The solution was then heated at 300°C under argon atmosphere for 1 hour, cooled to room temperature, purified and redispersed in cyclohexane.
  • [001S0] Mesoporous coating of the synthesized core-shell UCNs were done by calcination method. Briefly, the UCNs were coated with a silica layer as follows: 1ml of Igepal CO-520 and 18.4ml of cyclohexane were added to 1.6ml of O.05M UCNs, homogenized under ultrasonication. To the solution, 160ul of 30% Nh40H and 40ul of TEOS (Tetraethyl orthosilicate) was added and shaken for 2 days. After 2 days, the resulting silica coated UCNs were purified using acetone and Ethanol. Subsequently, a second coating of mesoporoous silica was done.
  • TEOS Tetraethyl orthosilicate
  • B 16F0 were grown in DMEM culture medium supplemented with 10% FBS, 100 units/mL of penicillin and 100 ⁇ g/mL of streptomycin, and maintained in a humidified, 5% carbon dioxide (C02) atmosphere at 37°C.
  • B16F0 cells were treated with different conditions and then incubated for 24 hours before being assayed for cell viability using an MTS assay as per manufacturer's instructions.
  • B 16F0 cells were incubated with 0.5 mg/mL of UCNs loaded with TPPS2a overnight. The excess nanoparticles were then washed off the cells and the cells were irradiated using a 980 nm NIR laser. The ROS generated in the cells was detected using an Image-iT LIVE Reactive Oxygen Species (ROS) kit as per manufacturer's instruction. The cells were also counterstained with DAPI and imaged using a confocal laser-scanning microscope (Nikon CI Confocal, Nikon, Tokyo, Japan) specially fitted with a CW 980 nm laser excitation source (Opto-Link Corp., Hong Kong). All images were taken using the same gain and pixel dwell (30 ⁇ ).
  • ROS Image-iT LIVE Reactive Oxygen Species
  • B16F0 cells were incubated with mesoporous silica coated core-shell UCNs either loaded with TPPS2a or without.
  • One set of the above said sample was incubated at 4°C and another at 37°C. After 8 hours of incubation, all the samples were irradiated with a 980 nm CW NIR laser for 8 mins. The samples were then incubated overnight at the respective temperatures. The cells were washed thrice to remove the UCNs present in the supernatant and the surface of the cells.
  • the cells were then trypsinized and the fluorescence emission of the UCNs was recorded using a Hitachi F-500 fluorescence spectrophotometer (Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with an NIR continuous wave laser with emission at 980 nm (Photonitech (Asia) Pte. Ltd., Singapore).
  • B16F0 cells were plated on a 24 well plate. After overnight incubation, TPPS2a loaded UCNs were added to the test and control wells. After 8 hours of incubation, the media was removed and the cells were incubated with 0.1 mg/mL Concanavalin A-Alexa Fluor 488 in culture medium and 0.01 mg/mL DAPI for 30 min at 37°C. They were then washed thrice with PBS and replenished with fresh culture medium. The cells were then imaged using a confocal microscope (Nikon CI Confocal, Nikon, Tokyo, Japan) (UCNs, the cell stained with Concanavalin A and nuclei stained with DAP I).
  • Concanavalin emission was pseudo-coloured to red to distinguish from the green emission of UCNs. Merging of the two images showed localization of UCNs in the cells.
  • Another set of wells was irradiated with a CW 980 nm NIR laser for 8 mins and was imaged similarly after 10 mins.
  • B16F0 cells in which STAT3 is aberrantly activated were incubated with UCNs loaded with double stranded (sense and antisense) anti STAT3 photomorpholino in 2 columns (3 wells/ column) of a 96 well plate and UCNs co-loaded with
  • medetomidine (1 mg/kg body weight).
  • mice implanting 3 x 10 6 B16-F0 cells suspended in 100 of serum-free DMEM in the lower flanks of the mice. Six days after inoculation of tumor cells, the mice were randomly divided into different groups. Each groups were then injected with 100 ⁇ of the following intra-tumorally. Group 1 - Saline, Group 2 - UCNs loaded with anti- STAT 3 photo morpholinos, Group 3 - UCNs co-loaded with TPPS2a and anti-STAT 3 photo morpholinos.
  • Laser treatment was performed on groups 2 and 3, 6 hours after injection by irradiating the tumor region with a CW 980 nm laser (EINST Technology Pte Ltd, Singapore) at a laser power density of 300 mW/cm 2 and exposure time of 40 min.
  • a second dose of the above PDT treatment was repeated three days following the first.
  • Tumor size and body weight was measured thrice a week.
  • mice were chosen randomly from each group and euthanized.
  • the tumors 50 mg each
  • the homogenate was centrifuged at 12,000 rpm for 20 min.
  • the supernatant was taken and used for further analysis.
  • Expression of STAT-3 was measured using ELIS A.
  • the tumors were harvested and snap frozen using liquid nitrogen. They were cryosectioned at 10- ⁇ thickness onto slides and fixed using paraformaldehyde. The sections were then counter-stained with DAPI and the fluorescence of DAPI and UCNS was imaged using a fluorescence confocal microscope (Nikon CI Confocal, Nikon, Tokyo, Japan).
  • rRBCs were washed with PBS thrice and subjected to 25 ⁇ volumetric dilutions in PBS to achieve 4% blood content (by volume).
  • Different concentrations of mesoporous silica coated UCNs were diluted in saline. Equal volume of blood and the UCN solution was mixed together and the mixture was incubated at 37°C for 1 h to allow for the interactions between rRBC and UCNs. After incubation, the mixture was centrifuged at 4000 rpm for 5 min and the supernatant was transferred into a 96-well microplate. The haemoglobin release was measured spectrophotometrically by measuring the absorbance of the samples at 576 nm using a microplate reader. Two control groups were provided for this assay: untreated rRBC suspension (as negative control), and rRBC suspension treated with 0.1% Triton-X (as positive control). Each assay was performed in triplicates.
  • Example 4 Light-activated endosomal escape using upconversion nanoparticles for enhanced delivery of drugs
  • PCI Photochemical Internalization
  • This example describes the use of Upconversion nanoparticles (UCNs) as a transducer for activation of the photosensitizer, TPPS 2a.
  • NIR light has good tissue penetrating ability and thus enables PCI in greater depths.
  • biocompatible upconversion nanoparticles were synthesized with a mesoporous silica coating. These UCNs activated TPPS 2a efficiently in solution and in cells. Paclitaxel, an anti-cancer drug was used as a model drug and was loaded into the mesoporous silica coating. B16F0 cells transfected with drug-loaded UCNs and irradiated with NIR showed significantly higher nanoparticle uptake and in turn higher cell death caused by the delivered drug. This technique can be used to enhance the delivery of any therapeutic molecule and thus increase the therapeutic efficiency considerably.
  • Yb/Er (Ytterbium/Erbium) doped NaYF 4 upconversion nanoparticles were synthesized in a one pot process and they were then coated with a mesoporous silica layer as reported previously in Qian HS, Guo HC, Ho PC-L, Mahendran R, Zhang Y. Mesoporous-Silica-Coated Up-Conversion Fluorescent Nanoparticles for Photodynamic Therapy. Small. 2009;5:2285-90.
  • the silica coating because of its stability in physiologic solutions, reduces the risk of a toxic affect due to leaching of lanthanide ions in to the body and also allows for controlled surface functionalization.
  • the UCNs were characterized by measuring size and zeta potential using Malvern Nano ZS (Zeta Sizer). TEM images of NIR-to-UV UCNs were recorded on a JEOL 201 OF TEM and fluorescence emission spectrum of the same was acquired on a SpetraPro 2150i fluorescence spectrometer equipped with a commercial 980 nm NIR laser.
  • the solution can be irradiated directly with light in the 375-450 nm range. Following this, the solution was irradiated till 8 mins, 2 mins at a time and fluorescence measured each time i.e. at 0, 2, 4, 8 mins.
  • ABDA had a characteristic fluorescence at 431nm, with production of singlet oxygen, more and more ABDA is consumed and this results in the decline in fluorescence. The fluorescence values at 431 run were noted and a graph of fluorescence intensity versus irradiation time was plotted.
  • ROS reactive oxygen species
  • the cells were then subjected to irradiation by 980 nm NIR laser irradiation for 8 min before they were washed thrice with plain HBSS. Immediately after that, fluorescent images of carboxy-H2DCFDA, and DAPI stainings on the cells were promptly captured by excitation at 488, and 408 nm respectively using a confocal laser- scanning microscope (Nikon C 1 Confocal, Nikon, Tokyo, Japan).
  • FIG. 37 shows a TEM image of the Yb/Er UCN core (FIG. 37A), UCN with silica coating (FIG. 37B) and UCN core with a mesoporous silica coating (FIG. 37C).
  • the particles are uniform in size and are less than lOOnm.
  • PCI is a technique that can be used to overcome this problem.
  • a mild photosensitizer TPPS2a that localizes in the endosomes was identified. Its excitation range is 375-450 nm, which falls in the emission range of the UCNs being used.
  • TPPS2a photoensitizer
  • Its excitation range is 375-450 nm, which falls in the emission range of the UCNs being used.
  • PCI could be integrated with the system.
  • a schematic below portrays the difference between normal UCN delivery and delivery in the presence of PCI.
  • TPPS2a and UCNs When cells incubated with TPPS2a and UCNs are irradiated with NIR, the UCNs emit visible light, which excites the photosensitizer embedded in the walls of the endosomal vesicles. Upon excitation, TPPS2a produces ROS, which cause the disruption of the endosomal vesicle thereby enhancing the release of the UCNs into the cytoplasm.
  • FIGs. 40B-40D Image it Green live staining in FIGs. 40B-40D is an indicator of ROS production by TPPS2a in cells (upon excitation). It can be seen that TPPS2a when excited does indeed produce ROS as can be seen by the green staining in FIG. 40D. The staining is not very dark which is in keeping with the theory that the production of ROS is localized. Also, the ROS production is because of the TPPS2a and not an artifact because in the negative control (with cells only), there is no staining. Also, in the second control (with TPPS2a but without excitation/irradiation), the staining is minimal.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • This concept applies to all aspects of this application, including elements of a composition of matter and steps of method of making or using the compositions.

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Abstract

La présente invention concerne, entre autres, des nanoparticules de conversion-élévation (UCN) configurées pour l'excitation de lumière infrarouge proche (NIR) et l'émission de lumière ultraviolette (UV), l'émission de lumière visible ou l'émission d'UV et de lumière visible ainsi que les compositions les contenant. La présente invention concerne également des procédés de fabrication et d'utilisation de ces UCNs, par exemple pour la distribution et la photoactivation de composés cibles, de tissus biologiques d'imagerie ainsi que des procédés de traitement, par exemple pour des cancers, tels qu'un mélanome, en distribuant des agents chimiothérapeutiques, tels que des taxanes.
PCT/SG2014/000281 2013-06-14 2014-06-13 Nanoparticules de conversion-élévation fluorescentes de cœur-coquille pour photoactivation de multiples biomolécules Ceased WO2014200441A1 (fr)

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