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WO2019034597A1 - Polymersomes et nanoréacteurs sensibles à la force; procédés les utilisant - Google Patents

Polymersomes et nanoréacteurs sensibles à la force; procédés les utilisant Download PDF

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WO2019034597A1
WO2019034597A1 PCT/EP2018/071904 EP2018071904W WO2019034597A1 WO 2019034597 A1 WO2019034597 A1 WO 2019034597A1 EP 2018071904 W EP2018071904 W EP 2018071904W WO 2019034597 A1 WO2019034597 A1 WO 2019034597A1
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methacrylate
acrylate
acid
ethyl
poly
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Omar RIFAIE GRAHAM
Nico Bruns
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Adolphe Merkle Institute University of Fribourg
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Adolphe Merkle Institute University of Fribourg
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    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • 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
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
    • 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

Definitions

  • the encapsulation of horseradish peroxidase enabled the reaction of luminol with hydrogen peroxide to yield a luminescence producing species similar to the marine bioluminescence.
  • the same nano-reactors were employed to catalyze the formation of a polyacrylamide gel when force was applied. Insights into the change of permeability of supramolecular networks upon force are provided. These systems are useful for drug delivery, as nanoreactors and for the selective release of curing agents for 3D printing, or fragrances.
  • Amphiphilic block copolymers can mimic the polarity of phospholipids and can self-assemble into a variety of defined structures such as polymersomes, which are hollow polymer vesicles. 3 Moreover, the chemical structure of polymers allows the incorporation of chemical motives which can add functionality to polymersome membranes. 4 Thus, functionalized polymersomes are ideal candidates to imitate the extraordinary responses of cells to a variety of stimuli such as light, 5 redox potentials, 6 pH, 7 temperature 8 or magnetism. 9 A possible response is the liberation of compounds from the inner pool of the polymersomes. If (bio)catalysts are enclosed into polymersomes, nanoreactors can be obtained. Such nanoreactors can be used to gate chemical reactions when the appropriate stimulus is applied. 10
  • Double stranded (ds) DNA is stabilized by the formation of hydrogen bonded base pairs.
  • Single stranded DNA is a highly hydrophilic molecule, because its outer phosphate units are charged and because the unpaired nucleobases can form hydrogen bonds to water.
  • the inner part of the double helix becomes hydrophobic and water only interacts with the macromolecule by its outer phosphate units. 17
  • stable adenine-thymine base pairs can be formed in hydrophobic matrices but are hindered when the nucleobases are hydrated. 18
  • formation of hydrogen bonds between complementary nucleobases represents a switch in polarity.
  • nucleobases can act as mechanophores that allow switching between a hydrophobic paired state and a more hydrophilic unpaired state by mechanical forces. Even though nucleobase and nucleobase mimic containing polymers have been described and are interesting due to their complementarity and self-assembly properties, 23"34 neither their switchable polarity nor their mechanically responsiveness have been exploited in polymeric systems.
  • amphiphilic block copolymers that carry complementary nucleobases in their hydrophobic block are self-assembled into force- responsive polymersomes and are used, in some embodiments, to switch bioluminescent reactions on and off by mechanical forces.
  • force is applied to the polymersome membranes
  • force is transduced to the hydrogen bonds between the complementary base pairs. This caused the disruption of the interactions and the transient exposure of nucleobases to the hydrophobic matrix.
  • a change of permeability was exploited for the capture and release of substances into and from the polymersomes.
  • biotransformations are carried out in the polymersome nanoreactors in such embodiments.
  • biocatalytic nanoreactors are controlled by mechanical forces. Bioluminescent reactions were carried out to mimic the marine glow of dinoflagellates. This work shows how the incorporation of complementary supramolecular motives can change the permeability of polymeric materials.
  • mechanical forces the compositions of the invention can be subjected to include, but are not limited to, mechanical agitation, turbulent mixing, shearing, mechanical perturbation, a pressure gradient, a pressure increase, buoyancy, drag force, lift force, compression, and a deformation force or any combination thereof.
  • mechanical agitation turbulent mixing, shearing, mechanical perturbation, a pressure gradient, a pressure increase, buoyancy, drag force, lift force, compression, and a deformation force or any combination thereof.
  • a polymersome-containing composition comprising: a first amphiphilic block copolymer having a first functional group; a second amphiphilic block copolymer having a second functional group complementary to the first functional group; wherein below a force threshold the first functional group and second functional group are hydrogen bonded, and wherein the hydrogen bonding is disrupted at or above the force threshold.
  • each of the first amphiphilic block copolymer and second amphiphilic block copolymer have a hydrophilic block comprising one or more of a) the following polymers: a poly(alkyl) oxide, poly(alkalene oxides), polyamines, polyacrylic acid, polymethacrylic acid, polyacrylamide, polymethylacrylamide, polyaminoacid, polynucleic acid, polysaccharide, poly(2-alkyloxazoline, peptoid, poly-beta-aminoacid, polyorthoester, poly(phosphate) and/or polyethers, and/or b) one or more of the following monomers: ethylene imine, ethylene oxide, ⁇ -glutamic acid, acrylic acid, 2- methyloxazoline, 2-(methacryloyloxy)-ethyl-phosphorylcholine, 2-hydroxyethyl methacrylate, 2-acrylamido-2-methyl-1 -propanes
  • the first amphiphilic block copolymer and second amphiphilic block copolymer each, independently, have a hydrophobic block comprising repeat units derived from one or more of hexyl methacrylate, succinic acid, 1 ,4-butanediol, 3-hydroxybutanoic acid, 3-hydroxpentanoic acid, butyrolactone, valerolactone, terephthalic acid, ethylene glycol, 1 ,4-butanediol, 1 ,3-propanediol, naphthalene dicarboxylic acid, 4-hydroxybenzoic acid, methyl methacrylate, caprolactone, dioxanone, lactic acid, glycolic acid, butadiene, dimethyl siloxane, propylene sulfide, styrene, pentafluorostyrene, isoprene, ethylene, vinyl chloride, vinyl fluoride, vinyl acetate
  • the first functional group and second functional group are complementary nucleobases.
  • the first functional group and the second functional group are derived from complementary pairs of the following functional groups: adenine, thymine, uracil, guanine, cytosine, adenosine, thymidine, deoxythymidine, uridine, deoxyuridine, guanosine, deoxy guanosine, cytidine, deoxycytidine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, thymidine monophosphate, thymidine diphosphate, thymidine triphosphate, guanine monophosphate, guanine diphosphate, guanine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, diaminotriazine, diacyldiaminopyridine, 6-ferrocenyl uracil, flavin thymine
  • the first functional group is present in an amount from about 1 to about 20 mole percent based upon the total amount of monomer repeating units in the hydrophobic block of the first amphiphilic block copolymer
  • the second functional group is present in an amount from about 1 to about 20 mole percent based upon the total amount of monomer repeating units in the hydrophobic block of the second amphiphilic block copolymer.
  • the first functional group is present in an amount from about 5 to about 10 mole percent based upon the total moles of the hydrophobic block of the first amphiphilic block copolymer, and wherein the second functional group is present in an amount from about 5 to about 10 mole percent based upon the total moles of the hydrophobic block of the second amphiphilic block copolymer.
  • the first functional group is present in an amount within 5 mole percent based on the total mole percent of the second functional group in the composition.
  • the composition further includes an encapsulated material located within the polymersome.
  • the encapsulated material is one or more of a drug, a small organic molecule, a fluorescent dye, a dye, a carbohydrate, a protein, a polypeptide, an amino acid, a nucleic acid, a nucleotide, a polynucleotide, an aptamer, DNA or RNA segments (RNA or DNA of either natural or synthetic origin, including recombinant RNA and DNA, antisense RNA, RNA interference, and small interfering RNA or combinations), a lipid, a polysaccharide, an antibody, an antibody fragment, a therapeutic agent, an epitope for biological receptors or other ligands, an enzyme, a polymer, an ion, a polymerization initiator, a monomer for polymerization, a reducing agent, a catalyst, and a chain transfer agent.
  • a drug a small organic molecule
  • a fluorescent dye a dye
  • a dye a carbohydrate
  • a protein a poly
  • a process for absorption or release of substances comprising the steps of: obtaining the composition as described herein; and subjecting the composition to a sufficient amount of mechanical force such that the at least a portion of the encapsulated material is expelled from or absorbed by the polymersome.
  • the mechanical force is one or more of mechanical agitation, turbulent mixing, shearing, mechanical perturbation, a pressure gradient, a pressure increase, buoyancy, drag force, lift force, compression, and a deformation force.
  • the first functional group and second functional group are hydrogen bonded prior to subjecting the composition to sufficient mechanical force such that at least a portion of the encapsulated material is absorbed into the polymersome, and wherein the hydrogen bonding is disrupted upon application of the shear force.
  • the first functional group and second functional group are hydrogen bonded prior to subjecting the composition to sufficient shear such that at least a portion of the encapsulated material is expelled from the polymersome, and wherein the hydrogen bonding is disrupted upon application of the mechanical force.
  • a biological or non-biological catalyst is encapsulated to produce a nanoreactor system which is activated upon mechanical stimulation.
  • the catalyst is an enzyme.
  • the nanoreactor system can catalyze biotransformations, and wherein the catalyst is horseradish peroxidase, hemoglobin, laccase, myoglobin, tyrosinase, glucose oxidase, phosphorylase, glycosyltransferase, acyltransferase, glycosidase, cellulase, amylase, xylanase, chitinase, hyaluronidase, lipase, protease, peptidase, decarboxylase, aldolase, dehydratase, racemase, epimerase, isomerase, ligase, synthase, acyl CoA synthase, chitinase, nylon hydrodase, bilirubin oxidase, soybean peroxidase, coprinus cinereus peroxidase, poly(ethylene glycol) modified hematin, beta-hematin
  • the nanoreactor system can catalyze an oxidoreduction reaction.
  • the nanoreactor system can catalyze oxidation or reduction reactions, and wherein the catalyst is a peroxidase, an oxidoreductase, a heme containing enzyme, haemoglobin, cytochromes, laccases, molecule based on heme or a polymer functionalized heme.
  • the catalyst is a peroxidase, an oxidoreductase, a heme containing enzyme, haemoglobin, cytochromes, laccases, molecule based on heme or a polymer functionalized heme.
  • the nanoreactor system can catalyze a luminescent reaction, and wherein the catalyst is a luciferase, a peroxidase, a heme containing enzyme, haemoglobin, cytochromes, molecule based on heme or a polymer functionalized heme.
  • the catalyst is a luciferase, a peroxidase, a heme containing enzyme, haemoglobin, cytochromes, molecule based on heme or a polymer functionalized heme.
  • the nanoreactor can catalyze a polymerization reaction, and wherein the catalyst is horseradish peroxidase, hemoglobin, laccase, myoglobin, tyrosinase, glucose oxidase, phosphorylase, glycosyltransferase, acyltransferase, glycosidase, cellulase, amylase, xylanase, chitinase, hyaluronidase, lipase, protease, peptidase, decarboxylase, aldolase, dehydratase, racemase, epimerase, isomerase, ligase, synthase, acyl CoA synthase, chitinase, nylon hydrodase, bilirubin oxidase, soybean peroxidase, coprinus cinereus peroxidase, poly(ethylene glycol) modified hematin, beta-he
  • the polymer is a crosslinked network.
  • the nanoreactor can catalyze a colorimetric reaction.
  • the enzyme is an oxidoreductase, transferase, hydrolase, polymerase, lyase, isomerase or ligase.
  • the reaction is oxidation of 3-amino-9- ethylcarbazole (AEC), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), pyrogallol, 3,3',5,5'-tetramethylbenzidine (TMB), or arginine.
  • AEC 3-amino-9- ethylcarbazole
  • ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid
  • TMB 3,3',5,5'-tetramethylbenzidine
  • arginine arginine
  • FIG. 1 illustrates complementary nucleobase-containing amphiphilic block copolymers self-assembly into force-responsive polymersomes
  • FIG. 2 illustrates that nucleobase-functionalized polymersomes change permeability upon force application, wherein A. shows release of sodium fluorescein from 5% functionalized polymersomes ) when passed through a syringe needle into a syringe, 5% functionalized polymersomes which were intermittently stimulated ( ⁇ ), non-stimulated 5% functionalized vesicles ( ⁇ ) and non-functionalized polymer vesicles (A), B.
  • FIG. 3 illustrates reversible capture and release of cargo from force- responsive polymersomes, wherein A. shows fluorescence emission spectra of empty polymer vesicles (bottom black line), fluorescence emission spectrum of 5% functionalized polymersomes after sodium fluorescein capture (step increase), fluorescence increase after stimulation by passing the suspension through a syringe needle into a syringe during 21 minutes, B. shows sodium fluorescein release after external capture by 5% functionalized polymersomes stimulated for 21 minutes ( ⁇ ), and non-stimulated throughout the same period ( ⁇ ), and C. is a TEM micrograph of 5% functionalized polymersomes after stimulation. The scale bar represents 200 nm;
  • FIG. 4 illustrates bioluminescence biomimicry through a reaction of N- (4-Aminobutyl)-N-ethylisoluminol (ABEI) with 2-butanone peroxide catalyzed by HRP-filled 5% functionalized nanoreactors, wherein A. shows photographs of luminescence formation after an initial permeation of the substrates into the nanoreactors, the luminescence is quenched after mild stirring with a needle. Photographs were successively taken every 5 syringe passes. Luminescence increases upon force stimulation, B. shows stimulation was stopped in the previously force stimulated suspension. Photographs were taken every 30 seconds, C. shows the system was reinitiated upon force stimulation.
  • ABEI N- (4-Aminobutyl)-N-ethylisoluminol
  • FIG. 5 illustrates polyacrylamide gel formation by HRP-filled nanoreactors, with polyacrylamide gel formed after 1 minute in a sonicator bath (left), non-stimulated nanoreactor suspension in polymerization solution after 24 hours (right);
  • FIG. 6 illustrates synthesis of poly(ethylene glycol)-i -(poly(hexyl methacrylate)-co-poly(pentafluorophenyl methacrylate)), wherein A. shows an amphiphilic block copolymer consisting of a hydrophilic block (PEG) and a hydrophobic block which is a randomly distributed copolymer. The ratio of 2 and HMA were varied to render polymers with different degrees of functionalization, and B. shows a GPC elugram in THF showing the chain extension from PEG right to form a diblock copolymer with a proportion of PHMAPPFMA 95:5 left; [0041 ] FIG.
  • A shows an amphiphilic block copolymer consisting of a hydrophilic block (PEG) and a hydrophobic block which is a randomly distributed copolymer. The ratio of 2 and HMA were varied to render polymers with different degrees of functionalization
  • B. shows a GPC elugram in THF showing
  • FIG. 7 illustrates a post-polymerization functional ization scheme, wherein A. shows a polymer batch containing a defined number of activated ester groups (PHMAPPFMA 99:1 ) is split and functionalized reacted with an amine functionalized adenine, an amine functionalized thymine and with hexylamine. This process renders polymers with similar molecular weights and number of functional groups, B. shows the amide formation was followed by FT-IR. With the formation of an amide bond at 1668-1544 cm “1 , the disappearance of the PFMA activated ester at 1774 cm “1 was observed. The aliphatic esters of the poly(hexyl methacrylate) motives at 1727 cm "1 were taken as a reference to normalize the spectra;
  • FIG. 8 illustrates TEM micrographs of polymersomes containing different functional groups (M) at varying molar ratios, being either hexyl methacrylamide (HMAm) or adenine and thymine (A T), wherein A. shows polymersomes with a proportion of HMA:A T 99:1 .
  • the scale bar corresponds to 500 nm
  • B. shows polymersomes containing a proportion of HMA:A/T of 95:5.
  • the scale bar corresponds to 200 nm
  • C. shows polymersomes with a proportion of HMA:A T 90:10.
  • the scale bar corresponds to 100 nm
  • D. shows polymersomes with a proportion of HMA:A T 85:15.
  • the scale bar corresponds to 500 nm
  • E. shows polymersomes with a proportion of HMA:A/T 80:20.
  • the scale bar corresponds to 200 nm
  • F. shows polymersomes with a proportion of HMA:HMAm 99:1 .
  • the scale bar corresponds to 500 nm
  • G. shows polymersomes with a proportion of HMA:HMAm 95:5.
  • the scale bar corresponds to 200 nm
  • H. shows polymersomes with a proportion of HMA:HMAm 90:10.
  • the scale bar corresponds to 100 nm
  • I. shows polymersomes with a proportion of HMA:HMAm 85:15.
  • the scale bar corresponds to 200 nm
  • J. shows polymersomes with a proportion of HMA:HMAm 80:20.
  • the scale bar corresponds to 500 nm;
  • FIG. 9 illustrates calibration curves for the calculation of sodium fluorescein encapsulation in polymersomes, wherein A. shows a calibration curve for the calculation of the total encapsulated sodium fluorescein in polymersomes, B. shows a calibration curve for the calculation of the maximum fluorescence expected upon total release of sodium fluorescein from polymersomes; and [0044]
  • Amphiphilic block copolymers which can self-assemble into polymersomes were synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization.
  • RAFT reversible addition fragmentation chain transfer
  • amphiphilic block copolymers include at least one hydrophilic block and at least one hydrophobic block.
  • the hydrophilic block comprises two or more, same or different, monomers such as, but not limited to, a) monomers that form polymers such as poly(alkalene oxides), polyamines, polyacrylic acids, polymethacrylic acids, polyacrylamides, polymethylacrylamides, polyaminoacids, polynucleic acids, polysaccharides, poly(2-alkyloxazolines), peptoids, poly-beta-aminoacids, polyorthoesters, poly(phosphates) and polyethers; and/or b) monomers such as, but not limited to, ethylene imine, ethylene oxide, ⁇ -glutamic acid, acrylic acid, 2-methyloxazoline, 2-(methacryloyloxy)-ethyl-phosphorylcholine, 2- hydroxyethyl methacrylate, 2-acrylamido-2-methyl-1 -propanesulfonic acid, (3- acrylamidopropyl)trimethylammonium, 3-(
  • Preferred polymers for the hydrophilic block include poly(alkaline oxides), for example, polyethylene glycol (PEG) polypropylene glycol (PPG), polybutylene glycol (PBG), Pluronic ® type polymers, and derivatives thereof and the like.
  • Poly(etheylene glycol) is the preferred hydrophilic block in one embodiment.
  • the hydrophilic block is functionalized.
  • polyethylene glycol when polyethylene glycol is utilized, it can be functionalized with 4, cyano-4- (phenylcarbonothioylthio) pentanoate.
  • cyano-4- (phenylcarbonothioylthio) pentanoate One example procedure is set forth hereinbelow.
  • Other suitable methods can be utilized to provide a functionalized hydrophilic polymer used to form the hydrophobic block of the amphiphilic block copolymer as desired.
  • Various monomers can be utilized to form the backbone of the hydrophobic block, for example acrylates, such as but not limited to, hexyl methacrylate (HMA), succinic acid, 1 ,4-butanediol, 3-hydroxybutanoic acid, 3- hydroxpentanoic acid, butyrolactone, valerolactone, terephthalic acid, ethylene glycol, 1 ,4-butanediol, 1 ,3-propanediol, naphthalene dicarboxylic acid, 4- hydroxybenzoic acid, methyl methacrylate, caprolactone, dioxanone, lactic acid, glycolic acid, butadiene, dimethyl siloxane, propylene sulfide, styrene, pentafluorostyrene, isoprene, ethylene, vinyl chloride, vinyl fluoride, vinyl acetate, alkylacrylamide, N-tert-butoxymethyl
  • Preferred polymers for the hydrophobic block include derivatives of polyesters, poylcarbonates, polyacrylates, polyetherglycols, propylene oxide, polyacrylamides polyamines, polyacrylic acid, polymethacrylic acid, polyacrylamides, polymethylacrylamides, polyaminoacids, polynucleic acids, polysaccharides, poly(2-alkyloxazolines), peptoids, poly-beta-aminoacids, polyorthoesters, poly(phosphates), and polyethers.
  • Polymethacrylates are preferred in one embodiment.
  • the hydrophobic block can be of a random or controlled distribution form, but is preferably random in one embodiment.
  • at least one monomer is utilized that provides an activated ester that can be substituted to include a functional group.
  • Suitable functional groups include a nucleobase such as, but not limited to, adenine, thymine, uracil, guanine, cytosine, adenosine, thymidine, deoxythymidine, uridine, deoxyuridine, guanosine, deoxyguanosine, cytidine, deoxycytidine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, thymidine monophosphate, thymidine diphosphate, thymidine triphosphate, guanine monophosphate, guanine diphosphate, guanine triphosphate, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, diaminotriazine, diacyldiaminopyridine, 6-ferrocenyl uracil, flavin thymine, succinimide, di
  • the functional groups can be incorporated into the hydrophobic block at different proportions in order to provide a desired force-responsive polymersome.
  • the mole percent thereof can vary.
  • the functional group is incorporated in an amount from about 1 to about 20 mole percent, desirably from about 3 to about 15 mole percent and preferably from about 5 to about 10 mole percent based on the total moles of the hydrophobic block. These ranges are particularly preferred when functional groups including adenine and thymine are utilized.
  • polymers having complementary functional groups are mixed together having substantially the same degree of functionalization.
  • the degree of functionalization can vary by up to 5 percent or up to 10 percent in various embodiments.
  • one amphiphilic block copolymer containing 5 percent functionalization e.g. adenine
  • an amphiphilic block copolymer containing 5 percent functionalization e.g. thymine.
  • the amphiphilic block copolymer can be formed via reversible addition fragmentation-transfer (RAFT) polymerization utilizing a suitable chain transfer agent.
  • RAFT reversible addition fragmentation-transfer
  • a specific example is set forth below wherein synthesis of poly(ethylene glycol)-b-(poly(hexyl methacrylate)-co-poly(pentaflurophenyl methacrylate)) is described. The procedure can be modified by those of ordinary skill in the art to synthesize other amphiphilic block copolymers.
  • post polymerization modification can be utilized to incorporate the desired functional group by reacting a functional group-containing compound therewith. Specific procedures for incorporating thymine and adenine-containing amines are set forth hereinbelow. Other functional groups can be utilized in analogous procedures or modification of the methods as known to ordinary skill in the art.
  • Force-responsive polymersomes are formed by combining complementary functional group-containing amphiphilic block polymers, with an equivalent degree of functional ization.
  • the complementary polymers are mixed, preferably at equimolar concentrations, and self-assembled into polymersomes utilizing film hydration.
  • the desired amphiphilic block copolymers having a functional group incorporated into the hydrophobic block are dissolved together in a suitable solvent.
  • the solvent is slowly evaporated, such as using a rotary evaporator in order to yield a thin film.
  • purified water such as MILLI-Q® water is added to the vessel which is then placed on an ultrasound bath for a suitable period of time, such as about 30 minutes to about 6 hours.
  • an encapsulated material is to be located within the polymersome, said material is also added in a suitable amount to the vessel.
  • the resulting polymersomes can be extruded, such as through a track-etched polycarbonate membrane.
  • the polymersomes can also be dialysed with water, preferably purified water for a period of time, such as about three days in one embodiment. The water can be exchanged at intervals, such as once each day.
  • the polymersomes can be further purified, such as from encapsulated material and micelles by size exclusion chromatography using water as the mobile phase. Spin diafiltration or centrifugation can be used in other embodiments.
  • encapsulating material is present during self-assembly of the polymersomes and encapsulated therein to at a self-quenching concentration during vesicle formation.
  • encapsulating materials can be utilized, including but not limited to, therapeutic agents, curing agents, fragrances, drugs, small organic molecules, fluorescent dyes, dyes, carbohydrates, proteins, polypeptides, amino acids, nucleic acids, nucleotides, polynucleotides, aptamers, DNA or RNA segments (RNA or DNA of either natural or synthetic origin, including recombinant RNA and DNA, antisense RNA, RNA interference, and small interfering RNA or combinations), lipids, polysaccharides, antibodies, antibody fragments, therapeutics, epitopes for biological receptors or other ligands, enzymes, polymers, ions, polymerization initiators, monomers for polymerization, reducing agents, catalysts, chain transfer agents or combinations
  • a poly(ethylene glycol) (PEG) macro-chain transfer agent (1 ) ) was employed as the hydrophilic block to which hydrophobic monomers were polymerized forming a diblock copolymer.
  • the hydrophobic block consisted of a random copolymer of hexyl methacrylate (HMA) and pentafluorophenyl methacrylate (PFMA) (2) yielding poly(ethylene glycol)-i - poly(hexyl methacrylate-co-pentafluorophenyl methacrylate) (PEG-£>-P(HMA- co-PFMA)) (3).
  • (2) contains an activated ester which can be substituted by amine containing chemical motives, such as modified nucleobases. 4
  • the random copolymerization allowed the even distribution of functional groups across the hydrophobic block of these polymers.
  • (2) was copolymerized at different proportions in the hydrophobic block, ranging from 1 to 20 mol% (FIG. 8).
  • Gel permeation chromatography measurements (GPC) showed polymers with narrow molecular weight distributions (FIG. 6), as expected for a well-controlled RAFT polymerization.
  • the concentration of nucleobase pairs in the hydrophobic leaflet was systematically varied between 5 and 20% to study the influence of the composition towards the response of the membranes. As it was envisioned that the size of the particles could affect the response to force, the size of the particles was homogenised for this set of experiments. Therefore, the polymersomes were extruded through track-etched membranes and fractionised by size exclusion columns. Permeability changes in response to forces were assessed by the release of sodium fluorescein. Polymersomes containing approximately 5% functional groups in the hydrophobic leaflet showed the highest release. After 250 syringe needle passes (250 aspirations and ejections) 45% of the content was released.
  • This value indicates that the release of compounds across the membrane of the polymersomes is due to the application of force, and excludes that the permeation of compounds is due to an intrinsic increased polarity by the incorporation of nucleobases to the hydrophobic leaflet. Moreover, this threshold value indicates that the hand driven syringe experiments were carried out well above the forces needed to activate the nucleobase-functionalized polymersomes (i.e. 71 ml_ min ⁇ 1 ).
  • Force-responsive biocatalytic nanoreactors were prepared by encapsulation of horseradish peroxidase (HRP) into force-responsive polymersomes during vesicle formation.
  • HRP horseradish peroxidase
  • HRP can catalyze the reaction of pyrogallol with hydrogen peroxide to form purpurogallin and water.
  • purpurogallin is a yellow to brown compound, its formation can be followed by UV-vis measurements.
  • HRP-filled polymersomes were added to a solution of hydrogen peroxide and pyrogallol. An initial permeation of the substrates catalyzed the formation of purpurogallin which stopped after 15 minutes.
  • the HRP-filled polymersomes were mechanically activated by repeated aspiration and release through a syringe needle into a syringe.
  • the frequency was of 15.71 aspirations and ejections per minute for a period of 70 minutes.
  • UV-vis measurements were recorded (FIG. 2C).
  • Purpurogallin was only produced when force was applied.
  • the reaction was interrupted as soon as the force stimulation was stopped. The reaction could be reinitiated by stimulating again the dispersion with force. The latter observation shows that the enzyme was not released from the polymersomes during the force experiments, which is further proof that the polymersomes remained stable.
  • non-functionalized HRP-filled polymersomes did not show catalytic activity when stimulated under the same conditions.
  • Force-responsive nanoreactors can be used in applications to trigger hydrogel formation on demand, e.g. as needed for subcutaneous drug-delivery applications where a liquid drug formulation is injected and then jellifies under the skin to form a depot for retarded and long-lasting drug release.
  • Most gel- forming drug delivery formulations are based on temperature-responsive polymers or on the formation of physical crosslinks.
  • a covalent crosslinking method would have the advantage that the formed hydrogel is more stable and long-lasting, thus potentially allowing for a drug-release for longer time periods.
  • HRP is known to initiate free radical polymerizations of vinyl monomers in the presence of hydrogen peroxide. Force-responsive nanoreactors could therefore be used to cure monomer mixtures in response to mechanical agitation, e.g. for the formation of hydrogels on demand.
  • HRP-containing nucleobase-functionalized nanoreactors were introduced in a water solution of the monomer acrylamide, the crosslinker bisacrylamide, hydrogen peroxide, and the mediator 2,4- pentanedione. The solution was purged with argon and was then introduced into an ice-cold ultrasound bath. Formation of a gel was observed within a period of 1 minute (FIG. 5).
  • SLA stereolithography
  • the thickness of the cured polymer depends of factors such as the duration of exposure to light, the scan speed, the depth of light penetration, and the intensity of the power source, all of which ultimately depend on the energy of the UV light.
  • the efficiency of the light-curing step can be limited by the introduction of pigments which withdraw energy destined to the polymerization process. In many occasions a UV post- curing step is required.
  • Inkjet printing eliminates the need for the separation of waste ink from the printer head.
  • 3D inkjet printing is generally based on the use of solid microparticles which are printed together with a liquid that binds them together.
  • a drawback of inkjet printing is that the printed liquid will determine the chemical and physical properties of the printed device, as many polymer glues are biologically toxic and cannot be used for tissue scaffold fabrication.
  • Another limitation is the optical transparency of the finished products, as an incomplete binding of the liquid with the powder particles will limit transparency due to light scattering. And last, the roughness of the object will also depend of the binding efficiency which may be an advantage or limitation depending on the application.
  • Inkjet printing is based on the application of a pressure pulse onto a nozzle by a piezo device which we envisioned would produce sufficient force to activate our force responsive polymersomes.
  • HRP horseradish peroxidase
  • luminol When left unperturbed again, the luminescence decayed in 120 minutes and could be reinitiated another time when the polymersomes were stimulated again.
  • AEBI AEBI
  • luminol can permeate through the nucleobase-functionalized polymersome membranes even in the absence of forces.
  • the permeability of luminol increases when the reaction mixture is stimulated with force.
  • the solubility of the compounds in water may affect the permeability through the membranes.
  • not all the nucleobases of the system form base pairs before stimulation. More water soluble compounds will have a higher tendency to interact with exposed nucleobases than compounds with lower water solubility.
  • less water soluble compounds will need higher quantities of exposed nucleobases to permeate through the membranes.
  • isoluminol does not permeate through the membrane of the force-responsive nanoreactors, even when force was applied. This observation is in line with literature reports that isoluminol does not cross through cell membranes while luminol is a membrane-permeable luminescent probe. 39 40 Therefore, the membranes show selectivity to certain compounds, which could be used to alter the substrate specificity of biotransformations.
  • Hexylamine (99%), and 4-dimethylaminopyridne (DMAP) (99%) were purchased from Acros Organics and were used as received.
  • Hydrogen peroxide (35%) was purchased from Reactolab SA and was used as received.
  • Deuterated solvents (D2O, CDC , DMSO- cfe) were purchased from Cambridge Isotope Laboratories, Inc.
  • M ill iQ water was obtained from Purelab Flex II (Veolia water system) at 18.2 ⁇ using an LC208 purification pack.
  • Pentafluorophenyl methacrylate was synthesized modifying a procedure described by Theato and coworkers. 4 2,6-Lutidine (12.96 ml, 0.1 1 1 mol) and pentafluorophenol (20 g, 0.108 mol) were jointly dissolved in 185 mL DCM and cooled at 0 °C. Methacryloyl chloride (1 1 .53 mL, 0.1 18 mol) was added drop by drop and the mixture was stirred at 0 °C for a period of 3 h. Then, the reaction was allowed to warm to room temperature and was stirred overnight.
  • RAFT Reversible addition fragmentation-transfer
  • CTA chain transfer agent poly(ethylene glycol) 4-cyano- 4-(phenylcarbonothioylthio) pentanoate
  • the purified monomers were added to the AIBN and CTA solution, and the mixture was bubbled for 1 hour with argon.
  • the reaction was initiated by heating the solution at 90 °C under an argon atmosphere.
  • the reaction was ended after 2 hours by exposing the solution to atmospheric oxygen and precipitated in a 60 % methanol / water mixture.
  • the polymer suspension was then centrifuged at 5000 g for 30 minutes at 0 °C (Thermo Scientific, Heraeus Megafuge 16R, TX-400 x 400 mL Swinging Bucket Rotor).
  • the reaction was stirred at room temperature for 48 hours and was quenched with 82 mL water followed by the evaporation of solvents in vacuo.
  • the resulting solid was dissolved in 1 L of a 1 :1 DCM/water mixture.
  • the organic phase was washed 3 times with M ill iQ water and was then evaporated to dryness.
  • the resulting oil was dissolved in 200 mL of a 2 M HCI solution in methanol and let to react overnight.
  • the desired product precipitated throughout the reaction and was purified in the same way as 3-(adenine-9-yl) propylamine yielding a white solid (3.65 g, 31 %).
  • M n M n PEG +— 170.25 g mol +— M n x
  • c being the 2 methylene protons adjacent to the ester group of poly(hexyl methacrylate) of poly(hexyl methacrylate) (3.82 - 4.15 ppm)
  • d being the 2 methylene protons adjacent to the amide groups of either poly(3- (adenine-9-yl)propyl methacrylamide), poly(3-(thymine-1 -yl) methacrylamide), or poly(hexyl methacrylamide) (2.82 - 3.30 ppm).
  • the polymersomes were further purified from remaining free dye and micelles by size exclusion chromatography on Sepharose 2B using M ill iQ water as the mobile phase.
  • Spectral scans were taken every 10 injections obtaining a maximum fluorescence emission at 512 nm (excitation wavelength: 494 nm; excitation slit width: 5; emission wavelength range: 450 nm - 650 nm; emission slit width: 5; emission scanning speed: 600 nm min "1 ). If bubbles were observed in the cuvette, the sample was gently shaken to avoid interference with the fluorescence measurements.
  • F Fluorescence emission at 512 nm
  • Fmax Calculated maximum fluorescence at 512 nm.
  • UV-Vis spectral scans from 300 to 600 nm were taken every 100 syringe pass cycles until a total of 1 100. At a frequency of 15.71 cycles per minute, this corresponds to a total time of 70 min.
  • absorbance was recorded at 420 nm every 13.6 s with an integration time of 1 s for a period of 70 minutes.
  • Luminol + hydrogen peroxide Unless otherwise state 100 mg (0.56 mmol) of luminol were weighted together with 100 mg (0.45 mmol) of p- iodophenol. Thereafter, 10 mL of 0.1 M Tris-HCI buffer (pH 8.5) together with 3 mL of 0.4 M NaOH were added to obtain a stock solution that would be used for the luminescence reaction (final pH 9.74). 1 mL of this stock solution was added to a glass test tube. To this solution, 0.5 mL of a 403 nmol ml "1 hydrogen peroxide solution was added. Finally, 40 ⁇ of a polymersome suspension was added.
  • the final concentration of the reagents was 20.9 ⁇ ml -1 luminol, 16.8 ⁇ ml -1 p-iodophenol, and 193.8 nmol ml "1 hydrogen peroxide.
  • the concentration of HRP was 1 nmol ml "1 in the final suspension.
  • the reactions were carried out at room temperature in a dark room. They were documented with a Nikon D7100 camera, equipped with a Nikon AF-S Micro NIKKOR 60mm 1 :2:8 G ED lens.
  • Photographs were taken with dimensions of 4800 pixels width x 3200 pixels height, horizontal resolution of 300 dpi, vertical resolution of 300 dpi, F-stop f/4, exposure time of 8 seconds, ISO speed 1250, focal length of 60 mm, and maximum aperture of 3.2.
  • Test tube and camera were placed in a distance of 25 cm by means of a clamp affixed to a scaffold and a tripod.
  • Polymersomes were mechanically stimulated with a syringe and syringe needle as described above, and photographs were taken every 5 or 10 syringe passes. When the reaction mixture was not mechanically stimulated, photographs were recorded every 30 or 120 s.
  • Syringe aspiration does not work for this reaction, because it constantly introduces air into the reaction mixture. Oxygen inhibits radical polymerization reactions.
  • To stimulate nucleobase-functionalized nanoreactors they were ultrasonicated for 5 min in an ice-cold sonicator bath (Sonoswiss SW3) at an ultrasonic intensity of 4.1 W cm -2 .
  • the progress of the reaction mixture was constantly assessed by turning the reaction flask and observing when the solution gelled. In the case of non- functionalized nanoreactors, they were introduced in the sonicator bath for a period of 2 h. For comparison, reaction mixtures were also left at room temperature without ultrasonication for a period of 24 h.
  • UV-Vis measurements were carried out on an Analytik Jena Specord 50 Plus spectrophotometer. Fluorescence spectroscopy measurements were carried out with a Varian Cary Eclipse Fluorescence Spectrometer and the temperature was controlled at 20 °C with a Varian Cary Single Cell Peltier Accessory. NMR Spectroscopy was carried out at 297.2 K on a Bruker Avance DPX 400 spectrometer at frequencies 400.19 MHz for 1 H nuclei and 100.63 MHz for 13 C nuclei. The spectra were referenced internally with residual solvent peaks.
  • compositions of the present invention encompass all possible combinations of the components, including various ranges of said components, disclosed herein. It is further noted that the term “comprising” does not exclude the presence of other elements. However, it is to also be understood that a description of a product or composition comprising certain components also discloses a product consisting of said components. Similarly, it is also to be understood that a description of a process comprising certain steps also discloses a process consisting of the steps.
  • thermoresponsive diblock copolymer assemblies 12 Kessel, S., Urbani, C. N. & Monteiro, M. J. Mechanically driven reorganization of thermoresponsive diblock copolymer assemblies in water. Angewandte Chemie International Edition 50, 8082-8085 (201 1 ).

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Abstract

Les propriétés de fusion de l'ADN induites mécaniquement ont été utilisées pour obtenir des membranes labiles du point de vue de la force. Des paires de nucléobases ont été utilisées en tant que mécanophores. Des copolymères à blocs amphiphiles complémentaires fonctionnalisés avec adénine et thymine ont été auto-assemblés en polymersomes. Les nucléobases ont formé des liaisons hydrogène qui ont été rompues lors d'une stimulation de force. L'exposition des nucléobases déconnectées à la matrice hydrophobe des membranes conduit à un changement de perméabilité qui autorise l'échange de molécules hydrosolubles à travers la matrice polymère. De plus, l'encapsulation de peroxydase de raifort a permis la réaction du luminol avec du peroxyde d'hydrogène pour produire une espèce produisant une luminescence similaire à la bioluminescence marine. De plus, les mêmes nanoréacteurs ont été utilisés pour catalyser la formation d'un gel de polyacrylamide lorsqu'une force a été appliquée. Des aperçus du changement de perméabilité de réseaux supramoléculaires en réponse à une force sont prévus. Ces systèmes sont utiles pour l'administration de médicaments, en tant que nanoréacteurs et pour la libération sélective d'agents durcisseurs pour l'impression 3D ou de parfums.
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CN112574415A (zh) * 2020-12-09 2021-03-30 吉林大学 一种活性氧响应性材料及其制备方法与应用
CN112656763A (zh) * 2020-12-29 2021-04-16 吉林大学 一种基于剪切力响应的载药纳米胶束的制备方法
WO2022222495A1 (fr) * 2021-04-21 2022-10-27 中国科学院深圳先进技术研究院 Polymère de type à réponse ultrasonore, nanoparticules préparées à partir de celui-ci, procédé de préparation associé et application correspondante
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CN119371591A (zh) * 2024-10-21 2025-01-28 西北工业大学 一种荧光单链聚合物纳米粒子的“一步法”制备方法

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WO2022222495A1 (fr) * 2021-04-21 2022-10-27 中国科学院深圳先进技术研究院 Polymère de type à réponse ultrasonore, nanoparticules préparées à partir de celui-ci, procédé de préparation associé et application correspondante
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