WO2023245293A1 - A nanoparticle architecture for combined in vivo stealth and selective targeting - Google Patents

A nanoparticle architecture for combined in vivo stealth and selective targeting Download PDF

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Publication number
WO2023245293A1
WO2023245293A1 PCT/CA2023/050868 CA2023050868W WO2023245293A1 WO 2023245293 A1 WO2023245293 A1 WO 2023245293A1 CA 2023050868 W CA2023050868 W CA 2023050868W WO 2023245293 A1 WO2023245293 A1 WO 2023245293A1
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nanoparticle
targeting
nanoparticles
molecules
core
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PCT/CA2023/050868
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French (fr)
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Alexandre MOQUIN
Sam Alexandre SELMANI
Newsha KOUSHKI
Soufiane CHENITE
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Oligo Medic Inc.
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Publication of WO2023245293A1 publication Critical patent/WO2023245293A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides

Definitions

  • nanoparticle surface architecture comprising shielding molecules and targeting ligands.
  • Encapsulation of active pharmaceutical ingredients serves to prolong their circulation time in vivo, enable their penetration through various biological barriers (epithelial barrier, cellular membrane, nucleus, etc.), protect them from chemical/enzymatic degradation and overcome resistance mechanisms due to increased drug efflux mechanisms in most cancers. Side effects of drugs are commonly caused by off-target delivery.
  • the nanoparticles physical (size, charge, density, shape, hydrophilicity/hydrophobicity) and chemical (surface chemistry) properties will affect their blood circulation properties and into which organs and to what extent they accumulate. This biodistribution is mainly governed by the biological identity of the nanoparticles which is created as the nanoparticles interact with serum proteins. The identity of the surface-adsorbed proteins will direct how the nanoparticles interact with the cells and tissues encountered by the nanoparticles as they enter the blood circulation. This biological identity of the nanoparticles can hinder the chemical aspects of the nanoparticles and reduce the ability of the targeting moieties to efficiently bind their receptors.
  • nanoparticle physicochemical properties is prime to reduce as much as possible the risk of protein opsonization which hides the targeting moiety and causes rapid clearance of the nanoparticles by the mononuclear phagocytotic system (a.k.a. the reticuloendothelial system) and off-target delivery.
  • Protein opsonization will cause the nanoparticles to be detected by circulating macrophages and Kupffer cells in the hepatic tissue which will ultimately lead to a reduction in circulating nanoparticles and to liver accumulation.
  • This accumulation in the Mononuclear Phagocytotic system (MPS) also known as the reticuloendothelial system is responsible for the reduced tumor delivery efficiency.
  • U.S. 7348424 teaches the preparation of phosphorylcholine-modified polysaccharides wherein the aldehyde derivative-containing compound obtained by the oxidative ring-opening reaction of glycerophosphorylcholine is added to a polysaccharide containing amino groups as well as a new polysaccharide having phosphorylcholine groups obtained from this manufacturing method.
  • Lysosome burst is a mechanism by which a particle internalized by endocytosis may escape into the cytosol.
  • Particles containing substantial protonable species such as amines can achieve lysosome burst by the “proton sponge” effect.
  • endosomes enter the lysosome pathway by activating proton pumps and acidifying its interior.
  • Protonable species absorb these protons and contribute to the ingress of water by osmosis which leads to swelling and eventual burst.
  • Nanoparticles can take advantage of this mechanism to swell and thereby release their therapeutic payload.
  • Lipid nanoparticles are excellent transfection systems adequate for certain applications such as mRNA vaccines but suffer from poor biodistribution and extensive liver accumulation.
  • Receptor-mediated transcytosis is a mechanism by which essential molecules are actively transported to the brain. The pathway can be exploited to enable drug delivery to the brain.
  • Circulation time is another factor severely limiting the effectiveness of several drugs.
  • Certain endogenous proteins and molecules have long circulation times thanks to recycling mechanisms.
  • Albumin and transferrin are examples making use of the megalin-cubilin complex of endocytic receptors to achieve renal readsorption.
  • Albumin also makes use of the FcRn receptor to be recycled from macrophage endocytosis.
  • the enhanced permeability and retention (EPR) effect is a mechanism used by macromolecular compounds, such as nanoparticles, to passively target tumor tissue. Due to the hyperpermeability of the neovasculature at tumor sites, the high molecular- weight nanoparticles can extravasate through the leaky blood vessels and accumulate in the tumor tissue. Additionally, inadequate lymphatic drainage leads to the retention of these accumulating nanoparticles at the tumor sites.
  • the EPR effect was previously thought to be one of the key mechanisms for targeted cancer therapy, however today its importance has been widely debated. As of date, there is growing evidence that the EPR effect is relatively small in human tumor tissue compared to in vital organs. Furthermore, evidence suggests that conjugated anticancer drugs on a nanocarrier results in a decreased accumulation in tumors compared to with the free drug.
  • ADCs Antibody-drug conjugates
  • ADCs consist of monoclonal antibodies specific to surface antigens on certain tumor cells, and a highly cytotoxic payload linked via chemical linkers.
  • ADCs combine the high specificity, and antitumor activity of monoclonal antibodies which are not very potent, with highly potent and cytotoxic anticancer agents that cannot be administered by themselves. Once internalized in the tumor cell, the ADC is transported to an endosome and/or lysosome where it is degraded and the anticancer agent is released, leading to rapid cell death.
  • ADCs have some challenges associated with their use.
  • nanoparticle complex composition bearing protective moieties and presenting surface ligands.
  • a nanoparticle comprising a core and a surface endowed with targeting chemical moieties and shielding molecules enhancing the accessibility of the targeting chemical moieties, grafted to the nanoparticle surface in a chemically reversible way.
  • the nanoparticle comprises a core bearing a surface of amine groups; and zwitterionic molecules grafted on the surface of the core through imine bonding with the amine groups, wherein the imine bonding is reversible in acidic environment.
  • the targeting chemical moiety is a biological ligand.
  • the shielding molecules are grafted on the nanoparticle surface via a pH-reversible bond.
  • the shielding molecules are zwitterionic.
  • the shielding molecules are zwitterionic and grafted on the nanoparticle surface via a pH-reversible bond.
  • the pH-reversible bond is an imine bond.
  • the zwitterionic molecules are glyceraldehydephosphorylcholine or glyceraldehyde-sulfobetaine.
  • the shielding molecules are polyethylene glycol molecules terminated at a first end with an imine-forming aldehyde or benzaldehyde moiety, and terminated at a second end with a shielding moiety.
  • the shielding molecules are terminated at the second end by a hydroxyl, a carboxyl, or a methyl group.
  • the shielding molecules are glyoxylate, glyceraldehyde phosphate, or betaine aldehyde.
  • the core comprises a polyelectrolyte complexation of a polyanion and a polycation, or a polyampholyte.
  • the core comprises a polyglucosamine.
  • the polyanion is dextran sulfate, carboxylated chitosan, chondroitin sulfate, alginate or hyaluronic acid.
  • the polyanion is dextran sulfate with an average molecular weight above 500 kDa.
  • the polyanion is a polyphosphate.
  • the polyphosphate is from a DNA molecule, an mRNA molecule, or a siRNA molecule.
  • the polycation is a polysaccharide derivative bearing quaternary ammonium groups.
  • the polycation is chitosan derived with quaternary ammonium groups.
  • the polycation is a polysaccharide derived with tertiary ammonium groups.
  • the polycation is chitosan derived with quaternary ammonium groups or tertiary ammonium groups and the polyanion is dextran sulfate, wherein the core is prepared by polyelectrolyte complexation between chitosan and dextran sulfate in the presence of a ionic gelation agent.
  • the ionic gelation agent is sodium tri polyphosphate.
  • a fraction of the surface of the core are endowed with a linker.
  • the linker is a clickable group.
  • the clickable group is dibenzocyclooctyne (DBCO).
  • the ligand is grafted to the core surface or the clickable group via a spacer.
  • the ligand is an aptamer, a small molecule, a polysaccharide, a peptide, a protein, a monoclonal antibody, an antibody fragment, or a nanobody.
  • the ligand is folic acid, a NOAM targeting peptide, a RGD peptide targeting avp3 integrin, a tumor-associated antigen targeting peptide, a HER-2 targeting peptide, a HER-2 or EGFR targeting antibody, an EGFR targeting aptamer, transferrin, insulin, or hyaluronic acid.
  • the spacer is polyethylene glycol (PEG) or a spacing peptide.
  • the ligand binds to cell membrane receptors.
  • the ligand binds to a soluble protein.
  • the nanoparticle encompassed herein further comprises a therapeutic payload encapsulated by the core or covalently bonded to the core.
  • the therapeutic payload is a small molecule, a protein, a peptide, DNA, RNA, an aptamer, an antibody, mRNA, sgRNA, miRNA, or siRNA.
  • the antibody is a monoclonal antibody, a nanobody, a humanized antibody or an antibody fragment.
  • the nanoparticle encompassed herein is formulated for an administration to a patient to deliver a therapeutic cargo.
  • the nanoparticle encompassed herein is formulated for an administration to a patient to deliver a contrast agent for imaging a tissue or diagnosing a disease.
  • composition comprising the nanoparticle defined herein and a carrier.
  • Fig. 1 illustrates fluorescent micrographs of SKOV-3 ovarian cancer cells treated with quaternary-ammonium-modified chitosan nanoparticles (QNP) with different surface modifications, either imine-phosphorylcholine (NP-PC) or a combination of amide-PEGl .5kDa-triazole-Folic acid and imine-phosphorylcholine (NP- PC/FA).
  • QNP quaternary-ammonium-modified chitosan nanoparticles
  • NP-PC imine-phosphorylcholine
  • NP- PC/FA imine-phosphorylcholine
  • Rhodamine-isothiocyanate was used as a covalent fluorescent marker attached to dextran sulfate to label nanoparticles.
  • Scale bar 200 pm in micrographs for 20x optical magnification
  • Scale bar 25 pm for 8x digital magnification from 20x image.
  • Fig. 2 illustrates hydrodynamic diameters and zeta-potential of quaternary ammonium-modified chitosan nanoparticles (QNP) labeled with CFTM750 dye (left), and after attachment of phosphorylcholine groups on the surface through imine bonds (right).
  • QNP quaternary ammonium-modified chitosan nanoparticles
  • QNP quaternary-ammonium chitosan nanoparticles
  • Fig. 4 illustrates nanoparticle as encompassed herein accumulation in clearance organs.
  • Fig. 5 illustrates hydrodynamic diameters and zeta-potential of quaternary- ammonium-modified chitosan nanoparticles (QNP) labeled with CFTM750 dye (QNP), and after reacting surface amines with three amounts of NHS-PEG1 ,5kDa-DBCO (low, medium, or high) and an excess of glyceraldehydephosphorylcholine. Nanoparticles were then “clicked” with azide-PEG-conjugated folic acid (FA). After surface functionalization, nanoparticles were rinsed thoroughly by dialysis on a membrane with a cut-off of 12-14 kDa. Graphs show the intensity weighted size distribution (hydrodynamic diameter) from quadruple measurements measured by DLS (Malvern Zetasizer NanoZS).
  • Fig. 8 illustrates relative fluorescence emission from brain, liver and lung tissues from nude mice bearing SKOV-3 tumors treated with one intravenous injection of quaternary-ammonium chitosan nanoparticles coated with increasing amounts of folic acid or just PEG. All formulations were also treated with glyceraldehyde-PC to bind available surface amines. Cy7 excitation and emission filters were used (excitation infrared Epi luminescence BioRad ChemiDoc MP system; emission 835/50nm Filter).
  • Fig 9 illustrates immunohistochemistry micrograph from mouse lung tissue.
  • the 5 pm thick slice was stained with CD31-FITC (eBioscience, cat No. 11-0311-82, 1in1000, green), HER2-AF594 (Cell Signaling, cat No. 2165S, 1 in200, DAPI.
  • NP-FA- CF750 with high density folic acid were administered intravenously in female SCID nude mice with SKOV3 xenograft grown for 28 days. The organs were collected 24h post administration.
  • Fig. 10 illustrates confocal micrograph from 3 areas in the lung tissue presented in Fig. 9 showing accumulation of NP-FA high inside HER-2 positive SKOV- 3 cells.
  • FIG. 11 illustrates immunohistochemistry micrograph from SKOV-3 tumor bearing mouse liver tissue.
  • the 5 pm thick slice was stained with CD31-FITC (eBioscience, cat No. 11-0311-82, 1 in1000), HER2-AF594 (Cell Signaling, cat No. 2165S, 1in200), DAPI.
  • NP-FA-CF750 with high density folic acid were administered intravenously in female SCID nude mice with SKOV3 xenograft grown for 28 days. The organs were collected 24h post administration.
  • Fig. 12 illustrates ex vivo fluorescence quantification from liver tissue at 24h, 48h and 72 hours post intravenous administration.
  • Fig. 13 illustrates fluorescence signal over 7 days incubation in sterile serum containing culture media.
  • Fig. 14 illustrates immunohistochemistry micrograph from healthy mouse liver tissue. Liver samples were labeled with DAPI, CD68 and FOLR1.
  • FOLR1-FITC antibody (ThermoFisher, cat No. FOLR1-FITC) was diluted 1 :100 and CD68-eFluor 660 antibody (ThermoFisher, cat No. 50-0681-82) was diluted 1 :500 in blocking buffer.
  • Fig. 15 illustrates cell viability of A549 lung cancer cells exposed for 24h to polyethyleneimine (Mw 25,000 Da) with increasing phosphorylcholine conjugation through imine bonds.
  • the molar ratio of glyceraldehyde-phosphorylcholine/surface amine was varied from 0 to 4 equivalent.
  • the glyceraldehyde- phosphorylcholine/PEI nanoparticles were added after 2h reaction or 96h reaction. With longer reaction times more PC groups get attached resulting in enhanced biocompatibility.
  • a nanoparticle comprising a surface endowed with targeting chemical moieties, and shielding molecules enhancing the accessibility of the targeting chemical moieties, grafted to the nanoparticle surface in a chemically reversible way.
  • the targeting chemical moieties is intended to encompass a molecule binding with high affinity and high selectivity with a target of interest.
  • the targeting chemical moiety is a biological ligand such as for example an aptamer, a small molecule, a peptide, a protein, a monoclonal antibody, an antibody fragment, or a nanobody.
  • the ligand is folic acid, a NOAM targeting peptide, a RGD peptide targeting avp3 integrin, a tumor- associated antigen targeting peptide, a HER-2 targeting peptide, a HER-2 or EGFR targeting antibody, an EGFR targeting aptamer, transferrin, insuline, or hyaluronic acid.
  • phosphorylcholine moieties as biomimetic groups onto the surface of preassembled nanoparticles containing primary amines on their surfaces.
  • the phosphorylcholine groups prevents non selective cellular uptake of nanoparticles.
  • the covalent formed bond between the phosphorylcholine group and the amine is an imine which is reversible in an acidic environment (such as the tumor microenvironment and the lysosomal compartment).
  • the detachment of the zwitterion in a tumor microenvironment exposes the positive surface charge of the nanoparticles which can promote cell association onto the surface of cells which are negatively charged and thus promotes internalization.
  • the detachment of the imine-zwitterionic groups allows the amines of the nanoparticles to buffer the protons leading to the proton sponge effect and enabling the lysosomal swelling/rupture escape mechanism.
  • the functional moieties are best taken from the class of highly hydrophilic molecules such as zwitterionic molecules.
  • the hydration layer thus created forms a barrier impeding interaction of the nanoparticle with its biological environment thereby conferring it stealth properties.
  • a fraction of surface amines can be used as sites to graft ligands with an affinity for a desired cell membrane receptor.
  • the ligand can be grafted directly or better through a spacer such as polyethylene glycol (PEG) or a spacing peptide.
  • PEG polyethylene glycol
  • Ligands thus grafted onto the nanoparticle enable internalization into specific cells via receptor-mediated endocytosis or other modes of cell uptake (e.g. cell penetrating peptides).
  • Transformation of the endosome into a lysosome is accompanied by influx of protons, causing a cleavage of the imine bond and liberating surface amines.
  • nanoparticles as drug delivery systems. These nanoparticles are designed to be administered to a patient intravenously, orally, by inhalation or by other methods.
  • the architecture involves a core nanoparticle bearing surface amines, protective functional moieties bearing an aldehyde, and optional surface ligands targeting suitable cell membrane receptors.
  • the protective functional moiety is an aldehyde bearing zwitterion such as glyceraldehyde-phosphorylcholine or glyceraldehydesulfobetaine.
  • the core nanoparticle is constituted by polyelectrolyte complexation of a polycation and a polyanion, optionally with the assistance of small linking molecules and optionally incorporating a therapeutic payload.
  • the polyanion is a polysaccharide taken from dextran sulfate, chondroitin sulfate, sodium alginate, or hyaluronic acid.
  • the polyanion is a polyphosphate taken for example and not limited to, from DNA, mRNA, siRNA, etc.
  • a small linking molecule or ionic gelation agent such as tripolyphosphate (TPP) is used to complex the polyanion with the polycation.
  • TPP tripolyphosphate
  • the polycation is a polysaccharide derivative bearing quaternary ammonium groups and primary amines.
  • the polycation is chitosan.
  • the therapeutic payload is a small molecule.
  • the therapeutic payload is a biomacromolecule such as for example but not limited to, a protein, a peptide, DNA, RNA, mRNA, sgRNA, miRNA, or siRNA.
  • the nanoparticle is formed with excess polycation and results in a positive surface charge.
  • the polyanion used is dextran sulfate with an average molecular weight above 500 kDa.
  • a fraction of surface amines are endowed with a linker grafted by amidation and presenting a “clickable” group such as di benzocyclooctyne (DBCO).
  • DBCO di benzocyclooctyne
  • the nanoparticles are coated with the glyceraldehyde-phosphorylcholine which binds to the remaining unreacted surface amines.
  • a ligand modified to bear an azide group is “clicked” on the spacer.
  • Fig. 1 provides fluorescent micrographs of SKOV-3 ovarian cancer cells treated with Quaternary-ammonium-modified chitosan nanoparticles (QNP) with different surface modifications, either iminephosphorylcholine (NP-PC) or a combination of amide-PEGl ,5kDa-triazole-Folic acid and imine-phosphorylcholine (NP-PC/FA).
  • QNP Quaternary-ammonium-modified chitosan nanoparticles
  • NP-PC Quaternary-ammonium-modified chitosan nanoparticles
  • NP-PC/FA iminephosphorylcholine
  • Rhodamine-isothiocyanate was used as a covalent fluorescent marker attached to dextran sulfate to label nanoparticles.
  • Nanoparticles with zwitterionic surface moieties are not uptaken by the SKOV-3 human ovarian cancers cells after 24h incubation in vitro (see Fig. 1).
  • Modified-chitosan nanoparticles were prepared by polyelectrolyte complexation between modified-chitosan and dextran sulfate in the presence of a ionic gelation agent (sodium tripolyphosphate). Chitosan modified with 40-60% quaternary ammonium groups demonstrated improved solubility in neutral pH at physiological salt concentrations. A near-infrared fluorescent dye (CFTM750 dye) was covalently attached to modified chitosan to track biodistribution of nanoparticles.
  • CFTM750 dye near-infrared fluorescent dye
  • Figs. 3 and 4 demonstrate how conjugation of phosphorylcholine moieties on the primary amines through imine-bond formation, strongly reduces the accumulation of the nanoparticles in the tissues of the reticuloendothelial system (RES) that play an important role in the clearance of particles in circulation.
  • the quaternary-ammonium- modified chitosan nanoparticles have mucoadhesive properties explaining their accumulation in lung tissues.
  • the phosphorylcholine groups added at the surface alleviate the mucoadhesive properties as observed by the strong reduction in lung accumulation of the QNP-iminePC (Fig. 3).
  • Nanoparticles prepared with a 50/50 mixture of quaternary-ammonium-modified chitosan and quaternary-modified-chitosan also substituted at 25-30% with phosphorylcholine groups show a biodistribution pattern in between that of QNP and QNP-iminePC, with reduced accumulation in the lung tissues compared to QNP but higher than that of QNP-iminePC for which the PC groups were added in excess after the nanoparticles were assembled.
  • SKOV-3 metastasis have already been reported in lung tissue (Baker et al., 2018, Clinical & Experimental Metastasis, 35:691-705), brain (Baker et al., 2018, Clinical & Experimental Metastasis, 35:691-705), and liver tissue (Baker et al., 2018, Clinical & Experimental Metastasis, 35:691-705; Long et al., 2022, Front. Oncol., 12, 884554; Yang et al., 2022, Cancer Gene Ther).
  • HER2-positive cells were detected in the histological slices (Fig. 9 for lungs and Fig. 11 for liver) from the mice which developed SKOV-3 tumors. We analyzed the organs from mice 1 (top) and 3 (bottom) of the NP-FA high density.
  • Nanoparticles were distributed homogeneously through the lung tissue.
  • the fluorescence signal from the nanoparticles can be observed as single puncti or diffuse patches which overlap with the HER-2 signal.
  • confocal microscopy was used and 3D reconstructed images were produced to confirm the intracellular distribution of the nanoparticles in SKOV-3 cells found in the lung tissues.
  • CD31 also known as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31 ), is considered a tumor angiogenesis- associated molecule which is expressed by SKOV-3 cells and other tumor distant metastases in many malignant cancers.
  • PECAM-1/CD31 platelet endothelial cell adhesion molecule-1
  • HER-2 positive SKOV-3 cells were also observed, but the cells were localized in between two lobes (Fig. 11 ) and had not uptaken NP-FA, which were unable to come into contact with these cells.
  • nanoparticles coated with the reversible antifouling agent and a targeting agent had distributed throughout the vascularized organs and could be detected at varying levels inside each organs.
  • microscopic assessment of the localization of the nanoparticles demonstrated that the nanoparticles were homogeneously distributed in the organs and appeared internalized in the cells known to express the receptor specific for the targeting agent.
  • nanoparticles The potential for the nanoparticles to be cleared out of the organs was assessed.
  • the nanoparticles were administered to healthy mice and tracked the extent of accumulation in the liver over 3 days. It was observed a decrease over time of the fluorescence signal from the tissue which was independent from the degradation of the fluorescent dye (CF 750) as seen in Fig. 13.
  • the fluorescence signal from the NP-FA labeled with CF750 dye can be observed both as puncti and as larger structures. These larger structures occur upon the cellular internalization which causes the accumulation of the nanoparticles within a micrometric structure (cell).
  • An anti-CD68 antibody was used to reveal the macrophages in the tissue (Kupffer cells) which are scavenger cells.
  • the signal from the larger structures of NP-FA overlap with the signal from the CD68 positive cells and there are not larger structures which are not also positive for CD68, meaning that the nanoparticles do not accumulate in hepatocytes. Over the 3 days it can observed a decrease of the puncti which either are removed by the Kupffer cells or are flushed out of the organ.
  • PEI Polyethyleneimine
  • PEI Because of their high cationic charge densities, PEI demonstrate high toxicities to cells. 25kDa polyethyleneimine led to 100% cell death at concentrations as low as 30 pg/mL. Upon the premixing of PEI with amounts corresponding to 50%, 100%, 150%, 200% or 400% alleviated the toxicity, even after 2h reaction. When the reagents were premixed for longer times (96h for 2 equivalent) a higher functionalization degree was reached which led to reduced toxicity of the PEI- iminePC on the lung cells in vitro (see Fig. 15).
  • Nanoparticles were prepared using quaternary ammonium-substituted chitosan having a molecular weight >100 kDa (viscosity 30-100 cps) and a substitution degree with the trimethylammonium chloride groups between 40-60%. Nanoprecipitates were obtained by mixing Quaternary ammonium chitosan with dextran sulfate in the presence of the ionic gelation agent tripolyphosphate at a final wt/wt/wt ratio of 6.5:1 :0.5 between the polycation:polyanion:ionic gelation agent, respectively. The three species were solubilized prior to the nanoprecipitation in phosphate buffer saline at pH 7.4.
  • the excess polycation was separated from the nanoprecipitates using a tangential flow filtration system with a size cut-off of 0.05 pm.
  • the excess polycation can also be separated from the nanoprecipitates by centrifugation at high centrifugal speeds (40,000 g) as long as the pellet can be resuspended in physiological buffers (PBS, citrate buffer with saline, etc.).
  • the isolated nanoparticles were functionalized with a bifunctional linker made from poly(ethylene glycol) having at one end a succinimide group which reacts with the primary amines on the surface of the nanoparticles and at the other end a di benzocyclooctyne (DBCO) group which can react, through copper-free “click” chemistry, with azide bearing molecules.
  • This bifunctional linker can be of variable length depending on the number of ethylene glycol repeating units with molecular weights varying between 532 ⁇ Mw ⁇ 5000 Da.
  • the excess unreacted PEG-DBCO can be cleared by dialysis after the reaction is terminated using a dialysis membrane with a molecular weight cut-off (MWCO) at least 5-fold larger than the molecular weight of the linker.
  • MWCO molecular weight cut-off
  • Glyceraldehyde-phosphorylcholine (GAPC) were added in excess compared to the remaining available primary amines and reacted for 12-24h.
  • the excess free GAPC was removed by dialysis using a 12-14 kDa cut-off membrane. It is also possible to use a hollow-fiber tangential flow filtration (TFF) system to separate the nanoparticles from the free unattached small molecules (GAPC and PEG linker).
  • TFF hollow-fiber tangential flow filtration
  • the targeting moiety having an azide group (N3) such as folic acid conjugated to an amino-PEG3-azide bifunctional linker, was mixed with the nanoparticles allowing the copper-free azide-alkyne cycloaddition, also known as “click” reaction, to take place (Agard, Prescher, & Bertozzi, 2004). This reaction is known to be fast, high yielding and necessitates only mild- conditions, resulting in almost quantitative yields of biocompatible and stable triazoles.
  • the “clickable” groups DBCO and N3 can be conjugated on the targeting group or the nanoparticle surface, interchangeably.

Abstract

It is provided a means of endowing the surface of nanoparticles with a combination of targeting and shielding moieties, wherein the shielding molecules enhance the selective accessibility of the targeting moieties and are reversibly grafted.

Description

A NANOPARTICLE ARCHITECTURE FOR COMBINED IN VIVO
STEALTH AND SELECTIVE TARGETING
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is claiming priority from U.S. Provisional Application No. 63/366,878 filed June 23, 2022, the content of which is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
[0002] It is provided a nanoparticle surface architecture comprising shielding molecules and targeting ligands.
BACKGROUND
[0003] Encapsulation of active pharmaceutical ingredients serves to prolong their circulation time in vivo, enable their penetration through various biological barriers (epithelial barrier, cellular membrane, nucleus, etc.), protect them from chemical/enzymatic degradation and overcome resistance mechanisms due to increased drug efflux mechanisms in most cancers. Side effects of drugs are commonly caused by off-target delivery.
[0004] The nanoparticles physical (size, charge, density, shape, hydrophilicity/hydrophobicity) and chemical (surface chemistry) properties will affect their blood circulation properties and into which organs and to what extent they accumulate. This biodistribution is mainly governed by the biological identity of the nanoparticles which is created as the nanoparticles interact with serum proteins. The identity of the surface-adsorbed proteins will direct how the nanoparticles interact with the cells and tissues encountered by the nanoparticles as they enter the blood circulation. This biological identity of the nanoparticles can hinder the chemical aspects of the nanoparticles and reduce the ability of the targeting moieties to efficiently bind their receptors. The selection of nanoparticle physicochemical properties is prime to reduce as much as possible the risk of protein opsonization which hides the targeting moiety and causes rapid clearance of the nanoparticles by the mononuclear phagocytotic system (a.k.a. the reticuloendothelial system) and off-target delivery.
[0005] Protein opsonization will cause the nanoparticles to be detected by circulating macrophages and Kupffer cells in the hepatic tissue which will ultimately lead to a reduction in circulating nanoparticles and to liver accumulation. This accumulation in the Mononuclear Phagocytotic system (MPS) also known as the reticuloendothelial system is responsible for the reduced tumor delivery efficiency.
[0006] The use of surfactants or highly hydrated molecules as surface moieties on nanoparticles has been suggested to reduce unwanted protein opsonization. Polyethylene glycol has been widely used in that effect on many types of nanoparticles (e.g. inorganic, polymeric, lipidic). The wide use of polyethylene glycol in consumer goods (e.g. cosmetics) has led to the development of immune sensitivity to PEG in certain individuals. Zwitterions are another type of highly hydrated molecule which can prevent protein opsonization. Zwitterions, such as phosphorylcholine are present on the surface of all mammalian cells as it is the polar head of phospholipids (phosphatidylcholine) which are the main component of the plasma membrane. Although it is small in terms of molecular weight, it has a high capacity to attract water molecules, thus creating the amphiphilicity allowing for the self-assembly of phosphatidylcholine into bilayer membranes.
[0007] U.S. 7348424 teaches the preparation of phosphorylcholine-modified polysaccharides wherein the aldehyde derivative-containing compound obtained by the oxidative ring-opening reaction of glycerophosphorylcholine is added to a polysaccharide containing amino groups as well as a new polysaccharide having phosphorylcholine groups obtained from this manufacturing method.
[0008] Lysosome burst is a mechanism by which a particle internalized by endocytosis may escape into the cytosol. Particles containing substantial protonable species such as amines can achieve lysosome burst by the “proton sponge” effect. Briefly, endosomes enter the lysosome pathway by activating proton pumps and acidifying its interior. Protonable species absorb these protons and contribute to the ingress of water by osmosis which leads to swelling and eventual burst. Nanoparticles can take advantage of this mechanism to swell and thereby release their therapeutic payload.
[0009] Some highly protonated species such as branched polyethyleneimine have been proposed as delivery systems due to their strong proton sponge effect leading to swelling and efficient lysosome burst and payload release. They have also been employed in active targeting strategies. However, their toxicity profile limits their applicability. [0010] Lipid nanoparticles are excellent transfection systems adequate for certain applications such as mRNA vaccines but suffer from poor biodistribution and extensive liver accumulation.
[0011] Crossing the blood-brain barrier remains a considerable challenge in drug delivery. Receptor-mediated transcytosis is a mechanism by which essential molecules are actively transported to the brain. The pathway can be exploited to enable drug delivery to the brain.
[0012] Circulation time is another factor severely limiting the effectiveness of several drugs. Certain endogenous proteins and molecules have long circulation times thanks to recycling mechanisms. Albumin and transferrin are examples making use of the megalin-cubilin complex of endocytic receptors to achieve renal readsorption. Albumin also makes use of the FcRn receptor to be recycled from macrophage endocytosis.
[0013] The enhanced permeability and retention (EPR) effect is a mechanism used by macromolecular compounds, such as nanoparticles, to passively target tumor tissue. Due to the hyperpermeability of the neovasculature at tumor sites, the high molecular- weight nanoparticles can extravasate through the leaky blood vessels and accumulate in the tumor tissue. Additionally, inadequate lymphatic drainage leads to the retention of these accumulating nanoparticles at the tumor sites. The EPR effect was previously thought to be one of the key mechanisms for targeted cancer therapy, however today its importance has been widely debated. As of date, there is growing evidence that the EPR effect is relatively small in human tumor tissue compared to in vital organs. Furthermore, evidence suggests that conjugated anticancer drugs on a nanocarrier results in a decreased accumulation in tumors compared to with the free drug.
[0014] Antibody-drug conjugates (ADCs) consist of monoclonal antibodies specific to surface antigens on certain tumor cells, and a highly cytotoxic payload linked via chemical linkers. ADCs combine the high specificity, and antitumor activity of monoclonal antibodies which are not very potent, with highly potent and cytotoxic anticancer agents that cannot be administered by themselves. Once internalized in the tumor cell, the ADC is transported to an endosome and/or lysosome where it is degraded and the anticancer agent is released, leading to rapid cell death. However, despite their targeting nature, ADCs have some challenges associated with their use. There have been instances of the payload being released from the tumor cell after internalization as well as, instances of extracellular release of the payload in the tumor tissue. This limitation, termed the bystander killing effect, not only compromises the anticancer activity of these agents, but the drug is then taken up by, and kills the surrounding/bystander cells.
[0015] It is thus highly desired to be provided with improved means to deliver active pharmaceutical ingredients or therapeutic nanoparticles.
SUMMARY
[0016] It is provided a nanoparticle complex composition bearing protective moieties and presenting surface ligands.
[0017] In an embodiment, it is provided a nanoparticle comprising a core and a surface endowed with targeting chemical moieties and shielding molecules enhancing the accessibility of the targeting chemical moieties, grafted to the nanoparticle surface in a chemically reversible way.
[0018] In an embodiment, the nanoparticle comprises a core bearing a surface of amine groups; and zwitterionic molecules grafted on the surface of the core through imine bonding with the amine groups, wherein the imine bonding is reversible in acidic environment.
[0019] In an embodiment, the targeting chemical moiety is a biological ligand.
[0020] In a further embodiment, the shielding molecules are grafted on the nanoparticle surface via a pH-reversible bond.
[0021] In another embodiment, the shielding molecules are zwitterionic.
[0022] In an embodiment, the shielding molecules are zwitterionic and grafted on the nanoparticle surface via a pH-reversible bond.
[0023] In an embodiment, the pH-reversible bond is an imine bond.
[0024] In an embodiment, the zwitterionic molecules are glyceraldehydephosphorylcholine or glyceraldehyde-sulfobetaine. [0025] In another embodiment, the shielding molecules are polyethylene glycol molecules terminated at a first end with an imine-forming aldehyde or benzaldehyde moiety, and terminated at a second end with a shielding moiety.
[0026] In a further embodiment, the shielding molecules are terminated at the second end by a hydroxyl, a carboxyl, or a methyl group.
[0027] In an embodiment, the shielding molecules are glyoxylate, glyceraldehyde phosphate, or betaine aldehyde.
[0028] In another embodiment, the core comprises a polyelectrolyte complexation of a polyanion and a polycation, or a polyampholyte.
[0029] In an embodiment, the core comprises a polyglucosamine.
[0030] In another embodiment, the polyanion is dextran sulfate, carboxylated chitosan, chondroitin sulfate, alginate or hyaluronic acid.
[0031] In a further embodiment, the polyanion is dextran sulfate with an average molecular weight above 500 kDa.
[0032] In another embodiment, the polyanion is a polyphosphate.
[0033] In an embodiment, the polyphosphate is from a DNA molecule, an mRNA molecule, or a siRNA molecule.
[0034] In another embodiment, the polycation is a polysaccharide derivative bearing quaternary ammonium groups.
[0035] In an embodiment, the polycation is chitosan derived with quaternary ammonium groups.
[0036] In another embodiment, the polycation is a polysaccharide derived with tertiary ammonium groups.
[0037] In an embodiment, the polycation is chitosan derived with quaternary ammonium groups or tertiary ammonium groups and the polyanion is dextran sulfate, wherein the core is prepared by polyelectrolyte complexation between chitosan and dextran sulfate in the presence of a ionic gelation agent. [0038] In another embodiment, the ionic gelation agent is sodium tri polyphosphate.
[0039] In an embodiment, a fraction of the surface of the core are endowed with a linker.
[0040] In another embodiment, the linker is a clickable group.
[0041] In a further embodiment, the clickable group is dibenzocyclooctyne (DBCO).
[0042] In an embodiment, the ligand is grafted to the core surface or the clickable group via a spacer.
[0043] In an embodiment, the ligand is an aptamer, a small molecule, a polysaccharide, a peptide, a protein, a monoclonal antibody, an antibody fragment, or a nanobody.
[0044] In an embodiment, the ligand is folic acid, a NOAM targeting peptide, a RGD peptide targeting avp3 integrin, a tumor-associated antigen targeting peptide, a HER-2 targeting peptide, a HER-2 or EGFR targeting antibody, an EGFR targeting aptamer, transferrin, insulin, or hyaluronic acid.
[0045] In a further embodiment, the spacer is polyethylene glycol (PEG) or a spacing peptide.
[0046] In another embodiment, the ligand binds to cell membrane receptors.
[0047] In another embodiment, the ligand binds to a soluble protein.
[0048] In an embodiment, the nanoparticle encompassed herein further comprises a therapeutic payload encapsulated by the core or covalently bonded to the core.
[0049] In a further embodiment, the therapeutic payload is a small molecule, a protein, a peptide, DNA, RNA, an aptamer, an antibody, mRNA, sgRNA, miRNA, or siRNA.
[0050] In another embodiment, the antibody is a monoclonal antibody, a nanobody, a humanized antibody or an antibody fragment.
[0051] In an embodiment, the nanoparticle encompassed herein is formulated for an administration to a patient to deliver a therapeutic cargo. [0052] In an embodiment, the nanoparticle encompassed herein is formulated for an administration to a patient to deliver a contrast agent for imaging a tissue or diagnosing a disease.
[0053] It is also provided a composition comprising the nanoparticle defined herein and a carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0054] Reference will now be made to the accompanying drawings.
[0055] Fig. 1 illustrates fluorescent micrographs of SKOV-3 ovarian cancer cells treated with quaternary-ammonium-modified chitosan nanoparticles (QNP) with different surface modifications, either imine-phosphorylcholine (NP-PC) or a combination of amide-PEGl .5kDa-triazole-Folic acid and imine-phosphorylcholine (NP- PC/FA). After 24h the media was changed, the cells were rinsed thoroughly and a mixture of calcein and Hoechst were used to label viable cells and cell nuclei, respectively. Rhodamine-isothiocyanate was used as a covalent fluorescent marker attached to dextran sulfate to label nanoparticles. Scale bar = 200 pm in micrographs for 20x optical magnification, Scale bar = 25 pm for 8x digital magnification from 20x image.
[0056] Fig. 2 illustrates hydrodynamic diameters and zeta-potential of quaternary ammonium-modified chitosan nanoparticles (QNP) labeled with CF™750 dye (left), and after attachment of phosphorylcholine groups on the surface through imine bonds (right).
[0057] Fig. 3 illustrates lung accumulation of quaternary-ammonium chitosan nanoparticles (QNP) after intravenous administration, measured 24h post-injection (n=3 per condition).
[0058] Fig. 4 illustrates nanoparticle as encompassed herein accumulation in clearance organs.
[0059] Fig. 5 illustrates hydrodynamic diameters and zeta-potential of quaternary- ammonium-modified chitosan nanoparticles (QNP) labeled with CF™750 dye (QNP), and after reacting surface amines with three amounts of NHS-PEG1 ,5kDa-DBCO (low, medium, or high) and an excess of glyceraldehydephosphorylcholine. Nanoparticles were then “clicked” with azide-PEG-conjugated folic acid (FA). After surface functionalization, nanoparticles were rinsed thoroughly by dialysis on a membrane with a cut-off of 12-14 kDa. Graphs show the intensity weighted size distribution (hydrodynamic diameter) from quadruple measurements measured by DLS (Malvern Zetasizer NanoZS).
[0060] Fig. 6 illustrates tumor volumes measured from day 6 to day 28 postinoculation of 2.5x10s SKOV-3 cells subcutaneously in the flank of SCID nude mice. Volumes (V) were estimated by measuring the length (L) and width (W) of the tumors by Vernier Caliper, V = (W2xL)/2.
[0061] Fig. 7 illustrates accumulation of quaternary-ammonium modified chitosan nanoparticles coated with increasing amounts of folic acid or just PEG in SKOV-3 tumors (n=3 per group) 24h after intravenous administration. Left panel - fluorescence emission from tumor tissues using Cy7 excitation and emission filters (excitation infrared Epi luminescence BioRad ChemiDoc MP system; emission 835/50nm Filter). Nanoparticles were covalently labeled with CF™750 dye (from Biotium) which is a near-infrared dye with optimal excitation and emission wavelengths of 755/777nm, respectively. Exposure time t=0.400s. Tumors framed in red showed signs of vascularization as noted by the pathologist. Right panel - Histogram of photon quantification from the different groups normalized to the autofluorescence from the tissue (PBS group) and the absorbance at A=755 nm of the nanoparticles administered.
[0062] Fig. 8 illustrates relative fluorescence emission from brain, liver and lung tissues from nude mice bearing SKOV-3 tumors treated with one intravenous injection of quaternary-ammonium chitosan nanoparticles coated with increasing amounts of folic acid or just PEG. All formulations were also treated with glyceraldehyde-PC to bind available surface amines. Cy7 excitation and emission filters were used (excitation infrared Epi luminescence BioRad ChemiDoc MP system; emission 835/50nm Filter). Nanoparticles were covalently labeled with CF™750 dye (from Biotium) which is a near- infrared dye with optimal excitation and emission wavelengths of 755/777nm, respectively. Exposure times from left to right: t=3.400s, 0.227s, 0.225s.
[0063] Fig 9 illustrates immunohistochemistry micrograph from mouse lung tissue. The 5 pm thick slice was stained with CD31-FITC (eBioscience, cat No. 11-0311-82, 1in1000, green), HER2-AF594 (Cell Signaling, cat No. 2165S, 1 in200, DAPI. NP-FA- CF750 with high density folic acid were administered intravenously in female SCID nude mice with SKOV3 xenograft grown for 28 days. The organs were collected 24h post administration.
[0064] Fig. 10 illustrates confocal micrograph from 3 areas in the lung tissue presented in Fig. 9 showing accumulation of NP-FA high inside HER-2 positive SKOV- 3 cells.
[0065] Fig. 11 illustrates immunohistochemistry micrograph from SKOV-3 tumor bearing mouse liver tissue. The 5 pm thick slice was stained with CD31-FITC (eBioscience, cat No. 11-0311-82, 1 in1000), HER2-AF594 (Cell Signaling, cat No. 2165S, 1in200), DAPI. NP-FA-CF750 with high density folic acid were administered intravenously in female SCID nude mice with SKOV3 xenograft grown for 28 days. The organs were collected 24h post administration.
[0066] Fig. 12 illustrates ex vivo fluorescence quantification from liver tissue at 24h, 48h and 72 hours post intravenous administration.
[0067] Fig. 13 illustrates fluorescence signal over 7 days incubation in sterile serum containing culture media.
[0068] Fig. 14 illustrates immunohistochemistry micrograph from healthy mouse liver tissue. Liver samples were labeled with DAPI, CD68 and FOLR1. FOLR1-FITC antibody (ThermoFisher, cat No. FOLR1-FITC) was diluted 1 :100 and CD68-eFluor 660 antibody (ThermoFisher, cat No. 50-0681-82) was diluted 1 :500 in blocking buffer.
[0069] Fig. 15 illustrates cell viability of A549 lung cancer cells exposed for 24h to polyethyleneimine (Mw 25,000 Da) with increasing phosphorylcholine conjugation through imine bonds. The molar ratio of glyceraldehyde-phosphorylcholine/surface amine was varied from 0 to 4 equivalent. At 2 equivalent the glyceraldehyde- phosphorylcholine/PEI nanoparticles were added after 2h reaction or 96h reaction. With longer reaction times more PC groups get attached resulting in enhanced biocompatibility.
DETAILED DESCRIPTION
[0070] In accordance with the present description, there is provided a means of endowing the surface of nanoparticles with a combination of targeting and shielding moieties, wherein the shielding molecules enhance the selective accessibility of the targeting moieties and are reversibly grafted.
[0071] It is thus provided a nanoparticle comprising a surface endowed with targeting chemical moieties, and shielding molecules enhancing the accessibility of the targeting chemical moieties, grafted to the nanoparticle surface in a chemically reversible way.
[0072] In an embodiment, the targeting chemical moieties is intended to encompass a molecule binding with high affinity and high selectivity with a target of interest.
[0073] In an embodiment, the targeting chemical moiety is a biological ligand such as for example an aptamer, a small molecule, a peptide, a protein, a monoclonal antibody, an antibody fragment, or a nanobody. In an embodiment, the ligand is folic acid, a NOAM targeting peptide, a RGD peptide targeting avp3 integrin, a tumor- associated antigen targeting peptide, a HER-2 targeting peptide, a HER-2 or EGFR targeting antibody, an EGFR targeting aptamer, transferrin, insuline, or hyaluronic acid.
[0074] It is provided reversible conjugation of phosphorylcholine moieties as biomimetic groups onto the surface of preassembled nanoparticles containing primary amines on their surfaces. The phosphorylcholine groups prevents non selective cellular uptake of nanoparticles. The covalent formed bond between the phosphorylcholine group and the amine is an imine which is reversible in an acidic environment (such as the tumor microenvironment and the lysosomal compartment). The detachment of the zwitterion in a tumor microenvironment exposes the positive surface charge of the nanoparticles which can promote cell association onto the surface of cells which are negatively charged and thus promotes internalization. In the lysosomal compartment the detachment of the imine-zwitterionic groups allows the amines of the nanoparticles to buffer the protons leading to the proton sponge effect and enabling the lysosomal swelling/rupture escape mechanism.
[0075] It is described herein a means of endowing the surface of amine-bearing nanoparticles with functional moieties or as described herein “anti-fouling” moieties to protect them from non-specific protein adsorption and inhibit their internalization in undesired cells all the while enabling efficient internalization in desired cells. [0076] The functional moieties are grafted onto the nanoparticles via reversible bonding with a fraction of surface amines. The imine bond has good stability at physiological pH of about 7.4.
[0077] The functional moieties are best taken from the class of highly hydrophilic molecules such as zwitterionic molecules.
[0078] The hydration layer thus created forms a barrier impeding interaction of the nanoparticle with its biological environment thereby conferring it stealth properties.
[0079] A fraction of surface amines can be used as sites to graft ligands with an affinity for a desired cell membrane receptor. The ligand can be grafted directly or better through a spacer such as polyethylene glycol (PEG) or a spacing peptide.
[0080] Ligands thus grafted onto the nanoparticle enable internalization into specific cells via receptor-mediated endocytosis or other modes of cell uptake (e.g. cell penetrating peptides).
[0081] Transformation of the endosome into a lysosome is accompanied by influx of protons, causing a cleavage of the imine bond and liberating surface amines.
[0082] Liberated surface amines become available for protonation by influxing protons, buffering the lysosomal pH and causing swelling from accompanying water molecules and chloride ions.
[0083] Swelling of the lysosome ultimately leads to lysosome rupture, liberating the nanoparticle and its therapeutic payload into the cytosol.
[0084] The present description relates to nanoparticles as drug delivery systems. These nanoparticles are designed to be administered to a patient intravenously, orally, by inhalation or by other methods.
[0085] In accordance with the present description, there is provided a family of nanoparticle architectures capable of holding a therapeutic payload and delivering said payload into intracellular spaces, or interacting selectively with targets of interest in physiological circulation. [0086] In an embodiment, the architecture involves a core nanoparticle bearing surface amines, protective functional moieties bearing an aldehyde, and optional surface ligands targeting suitable cell membrane receptors.
[0087] In an embodiment, the protective functional moiety is an aldehyde bearing zwitterion such as glyceraldehyde-phosphorylcholine or glyceraldehydesulfobetaine.
[0088] In an embodiment, the core nanoparticle is constituted by polyelectrolyte complexation of a polycation and a polyanion, optionally with the assistance of small linking molecules and optionally incorporating a therapeutic payload.
[0089] In an embodiment, the polyanion is a polysaccharide taken from dextran sulfate, chondroitin sulfate, sodium alginate, or hyaluronic acid.
[0090] In another embodiment, the polyanion is a polyphosphate taken for example and not limited to, from DNA, mRNA, siRNA, etc.
[0091] In an embodiment, a small linking molecule or ionic gelation agent such as tripolyphosphate (TPP) is used to complex the polyanion with the polycation.
[0092] In an embodiment, the polycation is a polysaccharide derivative bearing quaternary ammonium groups and primary amines.
[0093] In another embodiment, the polycation is chitosan.
[0094] In an embodiment, the therapeutic payload is a small molecule.
[0095] In another embodiment, the therapeutic payload is a biomacromolecule such as for example but not limited to, a protein, a peptide, DNA, RNA, mRNA, sgRNA, miRNA, or siRNA.
[0096] In an embodiment, the nanoparticle is formed with excess polycation and results in a positive surface charge.
[0097] In an embodiment, the polyanion used is dextran sulfate with an average molecular weight above 500 kDa. [0098] In an embodiment, a fraction of surface amines are endowed with a linker grafted by amidation and presenting a “clickable” group such as di benzocyclooctyne (DBCO).
[0099] In an embodiment, the nanoparticles are coated with the glyceraldehyde-phosphorylcholine which binds to the remaining unreacted surface amines.
[00100] In an embodiment, a ligand modified to bear an azide group is “clicked” on the spacer.
[00101] As exemplified, Fig. 1 provides fluorescent micrographs of SKOV-3 ovarian cancer cells treated with Quaternary-ammonium-modified chitosan nanoparticles (QNP) with different surface modifications, either iminephosphorylcholine (NP-PC) or a combination of amide-PEGl ,5kDa-triazole-Folic acid and imine-phosphorylcholine (NP-PC/FA). After 24h the media was changed, the cells were rinsed thoroughly and a mixture of calcein and Hoechst were used to label viable cells and cell nuclei, respectively. Rhodamine-isothiocyanate was used as a covalent fluorescent marker attached to dextran sulfate to label nanoparticles.
[00102] Nanoparticles with zwitterionic surface moieties are not uptaken by the SKOV-3 human ovarian cancers cells after 24h incubation in vitro (see Fig. 1). The covalent conjugation of folic acid onto the surface of nanoparticles, even in the presence of imine-phosphorylcholine groups, allows the nanoparticles to be internalized by SKOV-3 cells which express folic acid receptor.
[00103] The effect of surface modification with phosphorylcholine on the biodistribution of quaternary ammonium chitosan nanoparticles in healthy mice was further investigated,
[00104] Modified-chitosan nanoparticles were prepared by polyelectrolyte complexation between modified-chitosan and dextran sulfate in the presence of a ionic gelation agent (sodium tripolyphosphate). Chitosan modified with 40-60% quaternary ammonium groups demonstrated improved solubility in neutral pH at physiological salt concentrations. A near-infrared fluorescent dye (CF™750 dye) was covalently attached to modified chitosan to track biodistribution of nanoparticles. [00105] Hydrodynamic diameters and zeta-potential of quaternary-ammonium- modified chitosan nanoparticles (QNP) labeled with CF™750 dye (left), and after attachment of phosphorylcholine groups on the surface through imine bonds (right) are shown in Fig. 2. Nanoparticles were rinsed using a tangential flow filtration system with a cut-off of 0.05 pm. Graphs show the intensity weighted size distribution (hydrodynamic diameter) from quadruple measurements measured by DLS (Malvern Zetasizer NanoZS). Nanoparticles were administered in 9 week old C57BL/6 mice by intravenous.
[00106] (IV) tail vein injection (5 mL/kg) volumes were adjusted according to individual mouse body weight. 24 hrs after injection, mice were euthanized for organs collection (liver, lung, kidneys, spleen, brain, heart). Whole body perfusion was performed with ice cold saline to remove blood from organs. Fluorescence analysis was conducted using infrared Epi Illumination with a Cy7 filter Aex = 755- 777 nm; Aem = 810-860 nm. The exposure time was adjusted automatically to prevent saturation of the detector.
[00107] As seen in Fig. 3, a strong reduction in the lung accumulation (12-fold) was observed for QNP which were surface-modified with glyceraldehydephosphorylcholine (GAPC) to form imine bonds with amine groups on the NP surfaces (QNP-iminePC) compared to native QNP. Nanoparticles made from a mixture of quaternary-ammonium-modified chitosan and quaternary-ammonium- modified chitosan with permanent phosphorylcholine groups (after reduction of the imine bond to form a secondary amine bond) identified as (Q+Q- PC)NP were also tested. The (Q+Q-PC)NP group had size and surface charge of DH =231.6 ± 4.9 nm, Pdl = 0.119 ± 0.01 , and ^=+12.8 ± 1 .0 mV.
[00108] The use of the glyceraldehyde-phosphorylcholine moiety to coat QNP reduced the accumulation at 24h of QNP in the liver and spleen tissue, which are organs of the mononuclear phagocytosis system (see Fig. 4).
[00109] Figs. 3 and 4 demonstrate how conjugation of phosphorylcholine moieties on the primary amines through imine-bond formation, strongly reduces the accumulation of the nanoparticles in the tissues of the reticuloendothelial system (RES) that play an important role in the clearance of particles in circulation. The quaternary-ammonium- modified chitosan nanoparticles have mucoadhesive properties explaining their accumulation in lung tissues. The phosphorylcholine groups added at the surface alleviate the mucoadhesive properties as observed by the strong reduction in lung accumulation of the QNP-iminePC (Fig. 3). Nanoparticles prepared with a 50/50 mixture of quaternary-ammonium-modified chitosan and quaternary-modified-chitosan also substituted at 25-30% with phosphorylcholine groups show a biodistribution pattern in between that of QNP and QNP-iminePC, with reduced accumulation in the lung tissues compared to QNP but higher than that of QNP-iminePC for which the PC groups were added in excess after the nanoparticles were assembled.
[00110] After demonstrating the effectiveness of the imine-PC groups to i) selectively internalize in folate receptor (FR) expressing cells when folic acid is concomitantly attached to the surface of the nanoparticles and ii) to reduce undesired accumulation in clearance organs, the biodistribution of fluorescently- labeled modified chitosan nanoparticles were evaluated following intravenous (IV) injection in the subcutaneous SKOV-3 ovarian cancer xenograft mouse model with a single dose at a single time point.
[00111] Higher accumulation of quaternary ammonium nanoparticles functionalized with folic acid in organs such as the brain, the liver and the lungs tissues correlate with the more advanced state of the tumors was observed as seen in Fig. 7.
[00112] Histological analyses by immunofluorescence and H&E staining of the tumor and the organs presented in Fig. 8 were done to identify if the correlation of high accumulation of nanoparticles in more advanced tumors and accumulation in secondary tissues such as the brain, the lungs and the liver could be explained because of SKOV- 3 metastasis in these organs. An anti-HER2 antibody was used to visualize SKOV-3 cells in the organs as well as CD31 antibody to confirm the vascularization of the tumors (human HER2 proteins are overexpressed at the surface of SKOV-3 cells administered).
[00113] SKOV-3 metastasis have already been reported in lung tissue (Baker et al., 2018, Clinical & Experimental Metastasis, 35:691-705), brain (Baker et al., 2018, Clinical & Experimental Metastasis, 35:691-705), and liver tissue (Baker et al., 2018, Clinical & Experimental Metastasis, 35:691-705; Long et al., 2022, Front. Oncol., 12, 884554; Yang et al., 2022, Cancer Gene Ther). [00114] In lung and liver tissue histological slices, HER2-positive cells were detected in the histological slices (Fig. 9 for lungs and Fig. 11 for liver) from the mice which developed SKOV-3 tumors. We analyzed the organs from mice 1 (top) and 3 (bottom) of the NP-FA high density.
[00115] Nanoparticles (NP-FA) were distributed homogeneously through the lung tissue. The fluorescence signal from the nanoparticles can be observed as single puncti or diffuse patches which overlap with the HER-2 signal. To confirm that the nanoparticles were inside the SKOV-3 cells, confocal microscopy was used and 3D reconstructed images were produced to confirm the intracellular distribution of the nanoparticles in SKOV-3 cells found in the lung tissues.
[00116] Interestingly, SKOV-3 cells which migrated in secondary organs such as the lung, were positive for HER-2 and CD31. CD31 , also known as platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31 ), is considered a tumor angiogenesis- associated molecule which is expressed by SKOV-3 cells and other tumor distant metastases in many malignant cancers.
[00117] In the liver, HER-2 positive SKOV-3 cells were also observed, but the cells were localized in between two lobes (Fig. 11 ) and had not uptaken NP-FA, which were unable to come into contact with these cells.
[00118] The nanoparticles coated with the reversible antifouling agent and a targeting agent had distributed throughout the vascularized organs and could be detected at varying levels inside each organs. In detail microscopic assessment of the localization of the nanoparticles demonstrated that the nanoparticles were homogeneously distributed in the organs and appeared internalized in the cells known to express the receptor specific for the targeting agent.
[00119] The potential for the nanoparticles to be cleared out of the organs was assessed. The nanoparticles were administered to healthy mice and tracked the extent of accumulation in the liver over 3 days. It was observed a decrease over time of the fluorescence signal from the tissue which was independent from the degradation of the fluorescent dye (CF 750) as seen in Fig. 13.
[00120] In the liver, the fluorescence signal from the NP-FA labeled with CF750 dye can be observed both as puncti and as larger structures. These larger structures occur upon the cellular internalization which causes the accumulation of the nanoparticles within a micrometric structure (cell). An anti-CD68 antibody was used to reveal the macrophages in the tissue (Kupffer cells) which are scavenger cells. The signal from the larger structures of NP-FA overlap with the signal from the CD68 positive cells and there are not larger structures which are not also positive for CD68, meaning that the nanoparticles do not accumulate in hepatocytes. Over the 3 days it can observed a decrease of the puncti which either are removed by the Kupffer cells or are flushed out of the organ.
[00121] Polyethyleneimine (PEI) are polycationic macromolecules composed of branched ethyleneimines with the following structures having a high density of primary amines at their extremities:
[00122] Because of their high cationic charge densities, PEI demonstrate high toxicities to cells. 25kDa polyethyleneimine led to 100% cell death at concentrations as low as 30 pg/mL. Upon the premixing of PEI with amounts corresponding to 50%, 100%, 150%, 200% or 400% alleviated the toxicity, even after 2h reaction. When the reagents were premixed for longer times (96h for 2 equivalent) a higher functionalization degree was reached which led to reduced toxicity of the PEI- iminePC on the lung cells in vitro (see Fig. 15).
EXAMPLE I
Nanoparticles preparation for biodistribution study with folate receptor expressing cancerous cells SKOV-3
[00123] Nanoparticles were prepared using quaternary ammonium-substituted chitosan having a molecular weight >100 kDa (viscosity 30-100 cps) and a substitution degree with the trimethylammonium chloride groups between 40-60%. Nanoprecipitates were obtained by mixing Quaternary ammonium chitosan with dextran sulfate in the presence of the ionic gelation agent tripolyphosphate at a final wt/wt/wt ratio of 6.5:1 :0.5 between the polycation:polyanion:ionic gelation agent, respectively. The three species were solubilized prior to the nanoprecipitation in phosphate buffer saline at pH 7.4.
[00124] The excess polycation was separated from the nanoprecipitates using a tangential flow filtration system with a size cut-off of 0.05 pm. The excess polycation can also be separated from the nanoprecipitates by centrifugation at high centrifugal speeds (40,000 g) as long as the pellet can be resuspended in physiological buffers (PBS, citrate buffer with saline, etc.). [00125] The isolated nanoparticles were functionalized with a bifunctional linker made from poly(ethylene glycol) having at one end a succinimide group which reacts with the primary amines on the surface of the nanoparticles and at the other end a di benzocyclooctyne (DBCO) group which can react, through copper-free “click” chemistry, with azide bearing molecules. This bifunctional linker can be of variable length depending on the number of ethylene glycol repeating units with molecular weights varying between 532 < Mw < 5000 Da.
[00126] A 3-fold excess of succinimide-PEGl ,5kDa-DBCO was added relative to the targeted degree of substitution, because the amidation reaction competes with hydrolysis of the succinimide ester groups. For smaller species such as the 0- length bifunctional linker NHS-DBCO, a smaller ratio can be added as the reactivity is higher due to the higher mobility. The successful conjugation of the DBCO onto the nanoparticles’ surfaces can be confirmed by UV-vis spectrophotometry as the absorbance peak at 308 nm should remain after rinsing in a dialysis tube.
[00127] The excess unreacted PEG-DBCO can be cleared by dialysis after the reaction is terminated using a dialysis membrane with a molecular weight cut-off (MWCO) at least 5-fold larger than the molecular weight of the linker.
[00128] Glyceraldehyde-phosphorylcholine (GAPC) were added in excess compared to the remaining available primary amines and reacted for 12-24h. The excess free GAPC was removed by dialysis using a 12-14 kDa cut-off membrane. It is also possible to use a hollow-fiber tangential flow filtration (TFF) system to separate the nanoparticles from the free unattached small molecules (GAPC and PEG linker).
[00129] The targeting moiety having an azide group (N3), such as folic acid conjugated to an amino-PEG3-azide bifunctional linker, was mixed with the nanoparticles allowing the copper-free azide-alkyne cycloaddition, also known as “click” reaction, to take place (Agard, Prescher, & Bertozzi, 2004). This reaction is known to be fast, high yielding and necessitates only mild- conditions, resulting in almost quantitative yields of biocompatible and stable triazoles. The “clickable” groups DBCO and N3 can be conjugated on the targeting group or the nanoparticle surface, interchangeably.
[00130] After rinsing off the surface conjugation reagents by dialysis, nanoparticles were collected and their size and surface charges were measured by dynamic light scattering (DLS) and electrophoresis (zeta-potential). The results are presented in Fig. 5.
[00131] The hydrodynamic diameters increased slightly as the PEG-FA was attached to the surfaces of the nanoparticles.
[00132] Cells from SKOV-3, a human ovarian serous cystadenocarcinoma cell line, were implanted (2.5x10s cells suspended in 50 pL of serum-free RPMI-1640 medium supplemented with 50 pL Matrigel) subcutaneously in the right flank of CB17 SCID female mice (6-8 weeks old, from Charles River Laboratories), for xenograft tumor establishment. Tumor growth was monitored until tumors reached a size allowing significant tumor sampling (400-500 mm3). Mice were randomly distributed between the 5 treatment groups. Fig. 6 presents the evolution of tumor volume over time for each group.
[00133] While the present disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations and including such departures from the present disclosure as come within known or customary practice within the art and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1 . A nanoparticle comprising a core and a surface endowed with: a) targeting chemical moieties; and b) a shielding molecules enhancing the accessibility of the targeting chemical moieties, grafted to the nanoparticle surface in a chemically reversible way.
2. The nanoparticle of claim 1 , wherein the targeting chemical moiety is a biological ligand.
3. The nanoparticle of claim 1 or 2, wherein the shielding molecules are grafted on the nanoparticle surface via a pH-reversible bond.
4. The nanoparticle of any one of claims 1-3, wherein the shielding molecules are zwitterionic.
5. The nanoparticle of any one of claims 1-4, wherein the shielding molecules are zwitterionic and grafted on the nanoparticle surface via a pH-reversible bond.
6. The nanoparticle of claim 3 or 5, wherein the pH-reversible bond is an imine bond.
7. The nanoparticle of any one of claims 4-6, wherein the zwitterionic molecules are glyceraldehyde-phosphorylcholine or glyceraldehyde-sulfobetaine.
8. The nanoparticle of any one of claims 4-7, wherein the shielding molecules are polyethylene glycol molecules terminated at a first end with an imine-forming aldehyde or benzaldehyde moiety, and terminated at a second end with a shielding moiety.
9. The nanoparticle of claim 8, wherein the shielding molecules are terminated at the second end by a hydroxyl, a carboxyl, or a methyl group.
10. The nanoparticle of any one of claims 4-9, wherein the shielding molecules are glyoxylate, glyceraldehyde phosphate, or betaine aldehyde.
1 1. The nanoparticle of any one of claims 1-10, wherein the core comprises a polyelectrolyte complexation of a polyanion and a polycation, or a polyampholyte.
12. The nanoparticle of claim 11 , wherein the core comprises a polyglucosamine.
13. The nanoparticle of claim 11 , wherein the polyanion is dextran sulfate, carboxylated chitosan, chondroitin sulfate, alginate or hyaluronic acid.
14. The nanoparticle of claim 13, wherein the polyanion is dextran sulfate with an average molecular weight about 500 kDa.
15. The nanoparticle of claim 11 , wherein the polyanion is a polyphosphate.
16. The nanoparticle of claim 16, wherein the polyphosphate is from a DNA molecule, an mRNA molecule, or a siRNA molecule.
17. The nanoparticle of claim 11 , wherein the polycation is a polysaccharide derivative bearing quaternary ammonium groups.
18. The nanoparticle of claim 17, wherein the polycation is chitosan derived with quaternary ammonium groups.
19. The nanoparticle of claim 11 , wherein the polycation is a polysaccharide derived with tertiary ammonium groups.
20. The nanoparticle of claim 11 , wherein the polycation is chitosan derived with quaternary ammonium groups or tertiary ammonium groups and the polyanion is dextran sulfate, wherein the core is prepared by polyelectrolyte complexation between chitosan and dextran sulfate in the presence of a ionic gelation agent.
21. The nanoparticle of claim 20, wherein the ionic gelation agent is sodium tri polyphosphate.
22. The nanoparticle of any one of claims 1-21 , wherein a fraction of the surface of the core are endowed with a linker.
23. The nanoparticle of claim 22, wherein the linker is a clickable group.
24. The nanoparticle of claim 23, wherein the clickable group is dibenzocyclooctyne (DBCO).
25. The nanoparticle of claim 2, wherein the ligand is grafted to the core surface or the clickable group via a spacer.
26. The nanoparticle of claim 2 or 25, wherein the ligand is an aptamer, a small molecule, a polysaccharide, a peptide, a protein, a monoclonal antibody, an antibody fragment, or a nanobody.
27. The nanoparticle of claim 2 or 25, wherein the ligand is folic acid, a NCAM targeting peptide, a RGD peptide targeting avp3 integrin, a tumor-associated antigen targeting peptide, a HER-2 targeting peptide, a HER-2 or EGFR targeting antibody, an EGFR targeting aptamer, transferrin, insuline, or hyaluronic acid.
28. The nanoparticle of claim 25, wherein the spacer is polyethylene glycol (PEG) or a spacing peptide.
29. The nanoparticle of any one of claims 2 and 25-28, wherein the ligand binds to cell membrane receptors.
30. The nanoparticle of any one of claims 2 and 25-28, wherein the ligand binds to a soluble protein.
31. The nanoparticle of any one of claims 1-30, further comprising a therapeutic payload encapsulated by the core or covalently bonded to the core.
32. The nanoparticle of claim 31 , wherein the therapeutic payload is a small molecule, a protein, a peptide, DNA, RNA, an aptamer, an antibody, mRNA, sgRNA, miRNA, or siRNA.
33. The nanoparticle of claim 32, wherein the antibody is a monoclonal antibody, a nanobody, a humanized antibody or an antibody fragment.
34. The nanoparticle of any of the claims 1-33, formulated for an administration to a patient to deliver a therapeutic cargo.
35. The nanoparticle of any one of the claims 1-34, formulated for an administration to a patient to deliver a contrast agent for imaging a tissue or diagnosing a disease.
36. A composition comprising the nanoparticle of any one of claims 1-35 and a carrier.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5527528A (en) * 1989-10-20 1996-06-18 Sequus Pharmaceuticals, Inc. Solid-tumor treatment method
US20210244668A1 (en) * 2016-08-01 2021-08-12 The Brigham And Women`S Hospital, Inc. Particles for Delivery of Proteins and Peptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5527528A (en) * 1989-10-20 1996-06-18 Sequus Pharmaceuticals, Inc. Solid-tumor treatment method
US20210244668A1 (en) * 2016-08-01 2021-08-12 The Brigham And Women`S Hospital, Inc. Particles for Delivery of Proteins and Peptides

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DENG XINGYUE, ZHAO JING, LIU KAIWEN, WU CHAO, LIANG FEI: "Stealth PEGylated chitosan polyelectrolyte complex nanoparticles as drug delivery carrier", JOURNAL OF BIOMATERIALS SCIENCE. POLYMER EDITION., VSP, UTRECHT., NL, vol. 32, no. 11, 24 July 2021 (2021-07-24), NL , pages 1387 - 1405, XP093124161, ISSN: 0920-5063, DOI: 10.1080/09205063.2021.1918043 *
DONGYING ZHAO, ET AL.: "Biomedical Applications of Chitosan and Its Derivative Nanoparticles", POLYMERS, vol. 10, no. 4, 1 April 2018 (2018-04-01), pages 1 - 17, XP055654841, DOI: 10.3390/polym10040462 *
JIN QIAO, DENG YONGYAN, CHEN XIAOHUI, JI JIAN: "Rational Design of Cancer Nanomedicine for Simultaneous Stealth Surface and Enhanced Cellular Uptake", ACS NANO, AMERICAN CHEMICAL SOCIETY, US, US , XP093124157, ISSN: 1936-0851, DOI: 10.1021/acsnano.8b07746 *
JIN, Q. ET AL.: "Zwitterionic drug nanocarriers : A biomimetic strategy for drug delivery", COLLOIDS AND SURFACES B: BIOINTERFACES, vol. 124, 2014, pages 80 - 86, XP055466189, DOI: 10.1016/j.colsurfb.2014.07.013 *
LOIOLA, L.M.D. ET AL.: "Shielding and stealth effects of zwitterion moieties in double- functionalized silica nanoparticles", JOURNAL OF COLLOID AND INTERFACE SCIENCE, vol. 553, 2019, pages 540 - 548, XP085779113, DOI: 10.1016/j.jcis.2019.06.044 *
SIEW, A. ET AL.: "Enhanced Oral Absorption of Hydrophobic and Hydrophilic Drugs Using Quaternary Ammonium Palmitoyl Glycol Chitosan Nanoparticles", MOL. PHARMACEUTICS, vol. 9, 2012, pages 14 - 28, XP009167275, DOI: 10.1021/mp200469a *
SUN LU, WEI HUA, ZHANG XIANSHUO, MENG CHAO, KANG GUIYING, MA WEI, MA LIWEI, WANG BAOYAN, YU CUIYUN: "Synthesis of polymeric micelles with dual-functional sheddable PEG stealth for enhanced tumor-targeted drug delivery", POLYMER CHEMISTRY, ROYAL SOCIETY OF CHEMISTRY, CAMBRIDGE, vol. 11, no. 27, 14 July 2020 (2020-07-14), Cambridge , pages 4469 - 4476, XP093124153, ISSN: 1759-9954, DOI: 10.1039/D0PY00653J *
XIAO DONG, JIA HUI‐ZHEN, ZHANG JING, LIU CHEN‐WEI, ZHUO REN‐XI, ZHANG XIAN‐ZHENG: "A Dual‐Responsive Mesoporous Silica Nanoparticle for Tumor‐Triggered Targeting Drug Delivery", SMALL, WILEY, HOBOKEN, USA, vol. 10, no. 3, 1 February 2014 (2014-02-01), Hoboken, USA, pages 591 - 598, XP093124155, ISSN: 1613-6810, DOI: 10.1002/smll.201301926 *

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