Amniotic membrane derived nanoparticles and methods of making and using the same

Abstract

Disclosed are nanoparticles, pharmaceutical compositions comprising the nanoparticles, methods of generating the nanoparticles, methods of using the nanoparticles, methods of reducing ischemic injury to a cell, methods of treating ischemic injury in a tissue in a subject, methods of delivering a payload molecule to a target tissue, and kits comprising the nanoparticles. Compositions coated with the disclosed nanoparticles and methods of generating the same are also disclosed herein.

Description

Atty. Dkt. No. 650053.01258AMNIOTIC MEMBRANE DERIVED NANOPARTICLES AND METHODS OF MAKING AND USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application No.63 / 747,490 that was filed January 21, 2025, the entire contents of which are hereby incorporated by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable.SEQUENCE LISTING

[0003] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named ‘“650053 01258. xml” which is 22,920 bytes in size and was created on January 5, 2026. The sequence listing is electronically submitted via Patent Center and is incorporated by reference herein in its entirety.BACKGROUND

[0004] Myocardial infarction (MI), primarily caused by coronary artery blockage, accounts for approximately 42% of cardiovascular-related deaths in the United States [1]. The mortality rate of MI continues to rise due to an aging population, tobacco use, sedentary lifestyles, and increasing rates of obesity, diabetes, and metabolic syndrome [2-4], Current MI treatments include medications, such as ACE inhibitors, beta blockers, and vasodilators, as well as surgical interventions like stenting, bypass surgery, ventricular assist devices, and heart transplants [5-9], While current therapies have successfully curtailed heart injury, many patients still progress to cardiac hypertrophy and heart failure following their initial MI due to myocardial ischemia / injuries and limited regenerative potential of adult cardiomyocytes to repair the massive loss of heart tissue

[0010] ,

[0005] Controlled delivery systems for MI therapy offer a promising approach by precisely targeting therapeutic agents — such as drugs, genes, or cells — to specific sites, enhancing efficacy and minimizing side effects [11-14], Nanoparticles (NPs) play a key role in this field, offering sophisticated mechanisms for myocardial treatment and regeneration [15-18], Due to their nanoscale dimensions and engineered surface properties, NPs can encapsulate or attach therapeutic agents, protecting them from degradation and allowing for tunable drug-loading and release kinetics. This enables sustained and controlled delivery of the therapeutic payload.Atty. Dkt. No. 650053.01258Additionally, NPs can be internalized by cardiomyocytes and other relevant cell types, promoting efficient intracellular delivery' while avoiding endosomal entrapment [19-22],

[0006] Various types of NPs, including lipid, polymeric, metal, silica, and hybrid NPs. have emerged as innovative delivery systems for heart-related applications. Although early-stage studies have shown promising results, none have yet received approval from the U.S. Food and Drug Administration (FDA) for clinical use in cardiac therapies [23, 24], Major challenges include low delivery efficiency in heart, cardiac toxicity, poor retention time, and induced inflammation, exemplified by zinc oxide or nickel NPs [25, 26], The complexity of heart repair, involving processes like apoptosis, angiogenesis, inflammation, and fibrosis, further complicates these challenges. Efforts to improve delivery efficiency and safety, enhance myocardial homing and engraftment, extend circulation half-life and retention time, and optimize the therapeutic effects of NPs are ongoing [24. 27-32], Accordingly, there is a need in the art for improved nanoparticles.SUMMARY

[0007] In an aspect of the current disclosure, nanoparticles are provided. In some embodiments, the nanoparticles comprise at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing. In some embodiments, the nanoparticles comprise hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90), or a fragment of each of the foregoing proteins. In some embodiments, the nanoparticles comprise decellularized amniotic membrane. In some embodiments, the nanoparticle further comprises a payload. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the polynucleotide comprises an siRNA, an mRNA, a polynucleotide comprising a gRNA and / or a Cas protein, optionally, the polynucleotide may comprise YAP, LMNB2, LMNB1, LMNA, E2F6, E2F7, E2F8, TGF-beta related genes, or any combination thereof.Atty. Dkt. No. 650053.01258

[0008] In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, the method of nanoparticles are crosslinked. In some embodiments, the amniotic membrane is human amniotic membrane. In some embodiments, the nanoparticles further comprise a payload. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the nanoparticles comprise at least one protein selected from hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90). or a fragment of at least one of the foregoing proteins. In some embodiments, the nanoparticles have a mean diameter of about 100 nm to about 150 nm. In some embodiments, the nanoparticles have a mean diameter of about 120 nm or about 117 nm. In some embodiments, the nanoparticles have a positive zeta potential. In some embodiments, the nanoparticles have a zeta potential of about 1 to about 10 mV or about 8.5 mV.

[0009] In an aspect of the present disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing. In some embodiments, the nanoparticles comprise hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90). or a fragment of each of the foregoing proteins. In some embodiments, the nanoparticles comprise decellularized amniotic membrane. In some embodiments, the nanoparticle further comprises a payload. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrastAtty. Dkt. No. 650053.01258dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the polynucleotide comprises an siRNA, an mRNA, a polynucleotide comprising a gRNA and / or a Cas protein. The polynucleotide may comprise YAP, LMNB2, LMNB 1, LMNA, E2F6, E2F7, E2F8, TGF-beta related genes, or any combination thereof. In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, the nanoparticles are crosslinked. In some embodiments, the amniotic membrane is human amniotic membrane. In some embodiments, the nanoparticles further comprise a payload. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the nanoparticles comprise at least one protein selected from hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing proteins. In some embodiments, the nanoparticles have a mean diameter of about 100 nm to about 150 nm. In some embodiments, the nanoparticles have a mean diameter of about 120 nm or about 117 nm. In some embodiments, the nanoparticles have a positive zeta potential. In some embodiments, the nanoparticles have a zeta potential of about 1 to about 10 mV or about 8.5 mV.

[0010] A method of generating nanoparticles, the method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the digested milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate the nanoparticles. In some embodiments, decellularizing amniotic membrane comprises contacting the amniotic membrane with at least one detergent for a sufficient amount of time to decellularize the amniotic membrane. In some embodiments, the atAtty. Dkt. No. 650053.01258least one detergent comprises a non-ionic detergent or an ionic detergent or both a non-ionic detergent and an ionic detergent. In some embodiments, digesting the milled lyophilized decellularized membrane comprises pre-treating the milled lyophilized decellularized membrane in an aqueous solution in contact with an acid. In some embodiments, digesting the milled lyophilized decellularized membrane comprises contacting the milled lyophilized decellularized membrane with a protease for a sufficient amount of time to generate an aqueous solution comprising the digested milled lyophilized decellularized membrane. In some embodiments, in desolvating the aqueous solution comprises contacting the aqueous solution with a polar aprotic solvent. In some embodiments, the polar aprotic solvent comprises or consists of acetone. In some embodiments, the method further comprises crosslinking the nanoparticles. In some embodiments, crosslinking the nanoparticles comprises contacting the aqueous solution with a crosslinking agent. In some embodiments, the crosslinking agent comprises or consists of glutaraldehyde. In some embodiments, the method further comprises adding a payload to the aqueous solution comprising the digested milled lyophilized decellularized membrane. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the polynucleotide comprises an siRNA, a polynucleotide comprising a gRNA and / or a Cas protein.

[0011] In an aspect of the current disclosure, method are provided. In some embodiments, the methods comprise contacting a cell with nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing. In some embodiments, the nanoparticles comprise hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90), or a fragment of each of the foregoing proteins. In some embodiments, the nanoparticles comprise decellularized amniotic membrane. In some embodiments, the nanoparticles further comprise a payload. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadoliniumAtty. Dkt. No. 650053.01258contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the polynucleotide comprises an siRNA, an mRNA, a polynucleotide comprising a gRNA and / or a Cas protein, optionally, the polynucleotide may comprise YAP, LMNB2, LMNB1, LMNA, E2F6, E2F7, E2F8, TGF-beta related genes, or any combination thereof. In some embodiments, the cell is a vertebrate cell. In some embodiments, the cell is mammalian cell. In some embodiments, the cell is a canine, feline, equine, or bovine cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell of the heart, lungs, liver, pancreas, brain, spinal cord, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen. In some embodiments, the cell is a cardiomyocyte.

[0012] In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, the method of nanoparticles are crosslinked. In some embodiments, the amniotic membrane is human amniotic membrane. In some embodiments, the nanoparticles further comprise a payload. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In some embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the nanoparticles comprise at least one protein selected from hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing proteins. In some embodiments, the nanoparticles have a mean diameter of about 100 nm to about 150 nm. In some embodiments, the nanoparticles have a mean diameter of about 120 nm or about 117 nm. In some embodiments, the nanoparticles have a positive zeta potential. In some embodiments, the nanoparticles have a zeta potential of about 1 to about 10 mV or about 8.5 mV. In some embodiments, the cell is a vertebrate cell. In some embodiments,Atty. Dkt. No. 650053.01258the cell is mammalian cell. In some embodiments, the cell is a canine, feline, equine, or bovine cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell of the heart, lungs, liver, pancreas, brain, spinal cord, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen. In some embodiments, the cell is a cardiomyocyte.

[0013] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods comprise administering a pharmaceutical composition comprising nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing, to a subject. In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, administering comprises administering a therapeutically effective amount of the nanoparticles to a subject in need thereof.

[0014] A method of reducing ischemic injury to a cell, the method comprising contacting the cell with an effective amount of a nanoparticle comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90). or a fragment of at least one of the foregoing, wherein the nanoparticles comprise a payload that reduces ischemic injury. In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, the cell is mammalian cell. In some embodiments, the cell is a canine, feline, equine, or bovine cell. In some embodiments, the cell is a primate cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell of the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen. In some embodiments, the cell is a cardiomyocyte.Atty. Dkt. No. 650053.01258

[0015] A method of treating ischemic inj ury in an organ of a subj ect in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing, to the subject to treat the ischemic injury in the subject, wherein the pharmaceutical composition comprises nanoparticles comprising a payload that reduces ischemic injury. In some embodiments, the nanoparticles are produced by a method compnsing decellulanzing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, administering comprises intravenous administration. In some embodiments, administering comprises local administration to the organ. In some embodiments, the organ comprises the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.

[0016] In an aspect of the current disclosure, methods of delivering a pay load to an anatomical region are provided. In some embodiments, the methods comprise administering nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90). or a fragment of at least one of the foregoing to the anatomical region. In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, the anatomical region comprises the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen. In some embodiments, the payload comprises a dye, a therapeutic, or both a dye and a therapeutic. In some embodiments, the dye comprises a contrast dye. In some embodiments, the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye. In some embodiments, the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide. In someAtty. Dkt. No. 650053.01258embodiments, the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator. In some embodiments, the polynucleotide comprises an siRNA, a polynucleotide comprising a gRNA and / or a Cas protein.

[0017] In an aspect of the current disclosure, kits are provided. In some embodiments, the kits comprise a nanoparticle nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing to the anatomical region. In some embodiments, the nanoparticles are produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane. In some embodiments, the kits comprise a pharmaceutical composition comprising the nanoparticles. In some embodiments, the kits further comprise instructions for using the kit in any of the methods disclosed herein.

[0018] In an aspect of this disclosure, compositions coated with the disclosed nanoparticles are provided.

[0019] In an aspect of this disclosure, methods of generating a coated composition are provided. In some embodiments, the methods comprise applying the nanoparticles of this disclosure to the composition to generate the coated composition.BRIEF DESCRIPTION OF THE FIGURES

[0020] FIGs. 1A, IB, 1C, ID, IE, and IF show characterization of AMPs: (A) Schematic representation of the structure of HAM; (B) H&E staining of HAM (top) and DAM (bottom), showing complete cellular removal and preservation of the matrix structure in DAM; (C) SEM image of AMPs; (D) Size distribution of AMPs based on SEM images; (E) Particle size distribution of AMPs measured by DLS, showing an average size of 117.78 ± 51.4 nm; (F) Zeta potential of AMPs, measured at 8.53 ± 6.3 mV.

[0021] FIGs.2A, 2B, and 2C show characterization of ICG release and particle degradation of AMPs: (A) Fluorescence signal decay of AMPs incubated for 42 days at 37°C, with samples in water (top) and phosphate-buffered saline (PBS, bottom). Both conditions showed a gradual and consistent decrease in fluorescence intensity7over time; (B) Indocyanine Green (ICG) release rate from AMPs over the 42-day incubation period, demonstrating the controlled release of ICG; (C)Atty. Dkt. No. 650053.01258Weight loss profile of AMPs during the 42-day incubation, indicating the gradual degradation of AMPs over time.

[0022] FIGs.3A, 3B, and 3C show uptake and compatibility of AMPs in human iPSC-derived cardiomyocytes: (A) Representative confocal microscopy images of iPSC-derived cardiomyocytes co-cultured with Texas Red-labeled AMPs for 72 hours and 18 days. Cells are stained for troponin T (cTnT), Ki67, and DAPI to visualize cardiomyocyte markers and cell proliferation; (B) Quantification of AMP localization in the nucleus and cytoplasm of iPSC-cardiomyocytes after 72 hours of co-culture. showing the distribution of AMPs within the cells; (C) Percentages of Ki67+ proliferating iPSC-cardiomyocytes after 72 hours and 18 days of coculture with AMPs, indicating the effect of AMPs on cardiomyocyte proliferation. Scale bar: 20 pm. Results are expressed as mean ± SEM.

[0023] FIGs. 4A and 4B show that AMP retention does not affect survival of human iPSC-cardiomyocytes: (A) Representative confocal images of iPSC-derived cardiomyocytes cocultured with Texas Red-labeled AMPs for 72 hours and 18 days. Cells are stained for troponin T (cTnT), Cleaved Caspase-3, and DAPI to assess cardiomyocyte survival and apoptosis; (B) Quantification of the percentage of Cleaved Caspase-3+ apoptotic iPSC-cardiomyocytes after 72 hours and 18 days of co-culture with AMPs, showing no significant effect on cell survival. Scale bar: 20 pm. Results are expressed as mean ± SEM.

[0024] FIGs. 5A, 5B, and 5C show intramyocardial distribution and retention of AMPs: (A) In situ NIR fluorescence imaging of mice to visualize the biodistribution of free ICG (top) and ICG / AMPs (bottom) over a 14-day period following administration. (B) ROI analysis performed using Livingimage software to quantify the fluorescence intensity of ICG and ICG / AMPs in heart. (C) Normalized fluorescence intensify of free ICG and ICG / AMPs, as quantified by the ROI algorithm applied to the data shown in panel (A).

[0025] FIGs. 6A, 6B, 6C, 6D, and 6E show heart function by echocardiography evaluation: (A) Representative echocardiography of mouse two weeks post AMP intramyocardial delivery. Baseline echocardiography w as measured prior to the injection. n=5 mice. Left ventricle function quantified by echocardiographic analysis of (B) ejection fraction (EF%), (C) fractional shortening (FS%), (D) left ventricle internal diameter end diastole (LVIDd, mm), and (E) left ventricle volume end diastole (LVVd, pL). Statistical significance was calculated using two-tailed unpaired Student’s / -test. Results are presented as mean ± SEM.

[0026] FIGs. 7A, 7B, and 7C show' Histological analysis of heart following AMP injection: Gross view (left column), 4x H&E image (middle column), and 20x magnified view of theAtty. Dkt. No. 650053.01258highlighted rectangular region in the 4x image (right column) are shown for: (A) normal mouse hearts in the control group, (B) hearts injected with ICG, and (C) hearts injected with AMPs.

[0027] FIG. 8 shows Table 1 - which provides an analysis of the components of the disclosed nanoparticles. To identify the individual components in the AMPs, samples were cleaned using the PreOmics Phoenix kit according to the manufacturer’s directions. Samples were then dissolved in 2% acetonitrile 0.1% formic acid and analyzed on a Thermo Scientific Orbitrap Fusion Lumos MS. MS data was analyzed using the Proteome Discoverer 2.4 (Thermo Scientific, Waltham, MA) platform and protein identifications were filtered to include only those proteins identified by two or more unique peptides identified and ranked as high confidence.

[0028] FIG. 9 shows a schematic of an exemplary process for generating the disclosed nanoparticles.

[0029] FIG. 10 shows a schematic of decellularization, fabrication, and exemplary uses of the disclosed nanoparticles.

[0030] FIGs. 11A and 11B show (A) SEM and (B) FTIR analysis of HAM, DAM, and AMP coating.

[0031] FIGs. 12A, 12B, 12C, 12D, and 12E show (A) AFM characterization, (B) contact angle measurements, (C) quantification of surface RMS roughness, (D) Young’s modulus, and (E) contact angle values for 1-, 2-, and 3-layer AMP-coated surfaces compared with uncoated surface.

[0032] FIGs. 13A and 13B show (A) HUVECs on AMP and DAM surfaces demonstrated increased proliferation and migration relative to standard culture plates. (B) RNA-seq revealed that both surfaces altered HUVEC gene expression, with DAM inducing broader global changes and AMP coating producing a more targeted profile that may favor endothelialization and integration.

[0033] FIGs. 14A, 14B, 14C, 14D, and 14E show gene expression levels of (A) CXCL10, (B) ILip, (C) IL6, (D) TNFa. and (E) MMP3 in LPS-stimulated ECs cultured on 3-layer AMP-coated surfaces, DAM, or standard culture plates, with normal (unstimulated) ECs included as baseline controls.

[0034] FIGs. 15A and 15B show (A) Immunoblot analysis of IL6 expression in LPS-stimulated ECs cultured on 3-layer AMP-coated surfaces or standard culture plates, with normal (unstimulated) ECs serving as baseline controls. (B) Immunofluorescence staining of IL6 and MMP3 in the same treatment groups, illustrating protein-level changes in response to LPS stimulation and AMP coating.Atty. Dkt. No. 650053.01258

[0035] FIGs. 16A, 16B, and 16C show (A) Schematic overview of AMP coating. Surface properties of (B) uncoated and (C) AMP-coated ePTFE grafts demonstrate that uniform AMP coating converts the graft surface from hydrophobic to hydrophilic and limites platelet adhesion (yellow square) compared with uncoated ePTFE.

[0036] FIGs. 17A, 17B, and 17C show (A) Custom reactor with continuous flow for shear testing; (B) Fluorescence signal change before and after AMP coating on ePTFE graft under shear stress; (C) AMP coating showed slow long-term retention beyond 6 months.

[0037] FIGs. 18A, 18B, and 18C show HUVECs were seeded on uncoated ePTFE grafts (top) and AMP-coated ePTFE grafts (bottom) under static culture for (A) 1 day and (B) 5 days, then exposed to continuous shear stress for 3 days using a custom bioreactor (C). (D) TEM shows well-formed cell junctions (yellow arrows) in AMP-coated grafts, indicating improved endothelial integrity.

[0038] FIGs.19A, 19B, and 19C show (A) After 1 month of implantation in pig carotid arteries, (B) ultrasound and (C) gross examination showed that AMP-coated grafts maintained patent lumens (top, black arrows), whereas uncoated grafts exhibited lumen narrowing and marked NIH (bottom, light gray arrows).DETAILED DESCRIPTION

[0039] Disclosed herein are nanoparticles, pharmaceutical compositions comprising the nanoparticles, methods of generating the nanoparticles, methods of using the nanoparticles, methods of reducing ischemic injury to a cell, methods of treating ischemic injury in a tissue in a subject, methods of delivering a payload molecule to a target tissue, kits comprising the nanoparticles, compositions coated with the disclosed nanoparticles, and methods of coating compositions with the disclosed nanoparticles.Nanoparticles

[0040] In an aspect of the current disclosure, nanoparticles are provided. In some embodiments, the nanoparticles comprise at least one protein selected from hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or fragments thereof. The nanoparticles may comprise one, two, three, four, five, six, seven, or all eight of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90), or fragments thereof. The nanoparticles may compriseAtty. Dkt. No. 650053.01258hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90) or fragments thereof.

[0041] Hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90) may have the amino acid sequences SEQ ID NOs: 1-8, respectively, or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or more, to SEQ ID NOs: 1-8, respectively.

[0042] The nanoparticles may comprise decellularized amniotic membrane, e.g., human decellularized amniotic membrane.

[0043] The nanoparticles may further comprise a payload, which is a molecule or plurality of molecules (i. e. , a plurality of molecules with different identities, a plurality of molecules with the same identity, or both) that is substantially or completely contained within the nanoparticles.

[0044] The payload molecules may be, e.g., a dye, a therapeutic, or both a dye and a therapeutic.

[0045] The dye may comprise a pigmented or fluorescent dye, e.g., indocyanine green (ICG), or may comprise a dye used as a contrast material for imaging, e.g., tomography (e.g., CT scanning) or magnetic resonance imaging (MRI). The contrast dye may comprise an iodinated contrast dye or a gadolinium-based contrast dye.

[0046] The therapeutic may comprise a small molecule pharmaceutical, a polynucleotide, or a polypeptide. The small molecule pharmaceutical may comprise, e.g., an ACE inhibitor, a beta blocker, or a vasodilator. Exemplary therapeutics comprise drugs, growth factors, or signaling modulators such as DNAs and RNAs aimed at enhancing specific biological outcomes, such as promoting tissue regeneration, reducing inflammation, or targeting pathological pathways. The encapsulation efficiency, stability, and controlled release of these small molecules within the nanoparticle system will be systematically evaluated to ensure optimal therapeutic delivery and efficacy in vitro and in vivo.

[0047] The polynucleotide may comprise an RNA or a DNA or a combination of an RNA or DNA. The polynucleotide may comprise an RNA / DNA hybrid or a peptide nucleic acid. The polynucleotide may encode a polypeptide, e.g., a Cas protein, e.g., Cas9. The polynucleotide may encode a therapeutic polypeptide or a guide RNA (gRNA).

[0048] The polynucleotides may comprise sequences encoding growth factors, cell cycle regulators, or signaling modulators aimed at enhancing specific biological outcomes, such asAtty. Dkt. No. 650053.01258promoting tissue regeneration, reducing inflammation, or targeting pathological pathways. The polynucleotides may comprise sequences encoding proteins including, e.g., YAP, LMNB2, LMNB1, LMNA, E2F6, E2F7. E2F8, TGF-beta related genes. The sequences for YAP, LMNB2, LMNB1, LMNA, E2F6, E2F7, and E2F8 may comprise or consist of SEQ ID NOs: 9-15, respectively, or a sequence with at least 80% identity, at least 81% identity, at least 82% identity, at least 83% identity, at least 84% identity', at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or more to SEQ ID NOs: 9-15, respectively.

[0049] In some embodiments, the nanoparticles are made by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, desolvating the aqueous solution, and crosslinking the components of the aqueous solution to generate nanoparticles from the decellularized amniotic membrane.

[0050] Decellularizing the amniotic membrane may comprise contacting the amniotic membrane with at least one detergent for a sufficient amount of time to decellularize the amniotic membrane. The at least one detergent may be a non-ionic detergent or an ionic detergent or both a non-ionic detergent and an ionic detergent. An exemplary non-ionic detergent is Triton X-100, and an exemplary’ ionic detergent is sodium dodecyl sulfate (SDS).

[0051] Milling the decellularized membrane may comprise reducing the decellularized membrane to small particles, e.g., about 0.1 nm to about 1 mm in diameter using any suitable physical process, e.g., grinding yvith a mill, e.g., a Thomas Wiley® Mini Cutting Mill (Thomas Scientific, Swedesboro, NJ).

[0052] Milling the decellularized membrane may comprise reducing the decellularized membrane to small particles, for example, about 0.1 nm to about 1 mm in diameter, using any suitable physical size-reduction process, such as grinding with a mill, for example, a Thomas Wiley® Mini Cutting Mill (Thomas Scientific, Swedesboro, NJ). In addition to milling, alternative techniques capable of achieving the desired particle size may include cryogenic milling, ball milling, homogenization, high-shear mixing, ultrasonication, or other mechanical fragmentation methods, yvhich may be used alone or in combination, yvith optional postAtty. Dkt. No. 650053.01258processing steps such as sieving, filtration, or centrifugation to obtain particles within a desired size range.

[0053] Digesting the milled lyophilized decellularized membrane may comprise pre-treating the milled lyophilized decellularized membrane in an aqueous solution in contact with an acid, e.g., a strong acid, e.g., hydrochloric acid (HC1). Further exemplary acids may comprise acetic acid, peracetic acid, or formic acid. The concentration of the acid may be, e.g., about 0.1M to about IM, or more, or about 1 M. Digesting the milled lyophilized decellularized membrane may further comprise contacting the milled lyophilized decellularized membrane with a protease for a sufficient amount of time to generate an aqueous solution comprising the digested milled lyophilized decellularized membrane. The protease may be, e.g., pepsin. Additional exemplary proteases may comprise trypsin, collagenase, or proteinase K.

[0054] Desolvating the aqueous solution may comprise contacting the aqueous solution with a polar aprotic solvent, e.g., acetone or ethanol. The polar aprotic solvent may, in some embodiments, be acetone, e.g., at about a 7: 1 ratio, about a 6: 1 ratio, about a 5: 1 ratio, about a 4: 1 ratio, about a 3:1 ratio, about a 2:1 ratio, or about a 1:1 ratio of acetone to aqueous solution. In some embodiments, the ratio of acetone to aqueous solution is about 3:1. Desolvation may be performed under stirred conditions.

[0055] The nanoparticles may further be crosslinked, e.g., by contacting the nanoparticles with a crosslinking agent, e.g., an aldehyde, e.g., glutaraldehyde. Additional crosslinking agents may include genipin, carbodiimides (e.g., l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), epichlorohydrin, formaldehyde, or citric acid.

[0056] The nanoparticles may have a mean diameter of about 100 nm to about 150 nm, e g., about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm, about 106 nm, about 107 nm, about 108 nm, about 109 nm, about 110 nm, about 111 nm, about 112 nm, about 113 nm, about 114 nm, about 115 nm, about 116 nm. about 117 nm, about 118 nm, about 119 nm, about 120 nm, about 121 nm, about 122 nm, about 123 nm, about 124 nm, about 125 nm, about 126 nm, about 127 nm, about 128 nm, about 129 nm, about 130 nm, about 131 nm, about 132 nm, about 133 nm, about 134 nm, about 135 nm, about 136 nm, about 137 nm, about 138 nm, about 139 nm, about 140 nm, about 141 nm, about 142 nm, about 143 nm, about 144 nm, about 145 nm, about 146 nm, about 147 nm, about 148 nm, about 149 nm, about 150 nm, or any value therein inclusive of the endpoints, or about 120 nm, or about 117 nm.

[0057] The nanoparticles may have a positive zeta potential, e.g., about 1 to about 10 mV, e.g., about 1 mV, about 1.5 mV, about 2 mV, about 2.5 mV, about 3 mV, about 3.5 mV. about 4 mV,Atty. Dkt. No. 650053.01258about 4.5 mV, about 5 mV, about 5.5 mV, about 6 mV, about 6.5 mV, about 7 mV, about 7.5 mV, about 8 mV, about 8.5 mV, about 9 mV, about 9.5 mV, about 10 mV, or any subrange or value therein inclusive of the endpoints, or about 8.5 mV.Pharmaceutical compositions

[0058] In an aspect of the current disclosure, pharmaceutical compositions comprising the disclosed nanoparticles are provided. The pharmaceutical compositions may comprise a therapeutically effective amount of the disclosed nanoparticles. As used herein, a “therapeutically effective amount7’ or an “effective amount” refers to an amount of the nanoparticles that is effective for effecting at least one change in the target, e g., a target may comprise a cell, tissue, organ, or subject. In one example, a therapeutically effective amount of the nanoparticles for treatment of ischemic injury in a tissue in a subject is an amount that improves at least one sign or symptom of the ischemic injury in the tissue from the subject.

[0059] The pharmaceutical compositions may comprise additional therapeutics that are not incorporated as a payload in the disclosed nanoparticles.

[0060] The inert ingredients and manner of formulation of the pharmaceutical compositions are conventional. The usual methods of formulation used in pharmaceutical science may be used here. The activity of the compounds employed in the compositions and methods disclosed herein are not believed to depend greatly on the nature of the composition, and, therefore, the compositions can be chosen and formulated primarily or solely for convenience and economy.

[0061] As one skilled in the art will also appreciate, the formulation can be prepared with materials (e.g., actives excipients, carriers (such as cyclodextrins), diluents, etc.) having properties (e.g., purity) that render the formulation suitable for administration to humans. Alternatively, the formulation can be prepared with materials having purity and / or other properties that render the formulation suitable for administration to non-human subjects, but not suitable for administration to humans.

[0062] In some embodiments, the pharmaceutical compositions are formulated for administration through any suitable route, e.g., intravenous administration, intrathecal administration, intrarectal administration, oral administration, intranasal administration, intraosseous administration, subcutaneous administration, intramuscular administration, or intratumoral administration.Atty. Dkt. No. 650053.01258Methods of generating nanoparticles

[0063] In an aspect of the current disclosure, methods of generating nanoparticles are provided. In some embodiments, the methods comprise decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the digested milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate the nanoparticles. The methods may further comprise crosslinking the nanoparticles.Methods of using the nanoparticles

[0064] In an aspect of the current disclosure, methods of using the disclosed nanoparticles are provided. In some embodiments, the disclosed nanoparticles are administered to a subject or are contacted to a cell, tissue, or organ.

[0065] The cell may be any cell, e.g., a vertebrate cell, a mammalian cell, a canine cell, a feline cell, an equine cell, a bovine cell, an ovine cell, a porcine cell, a rodent cell (e.g., murine cell or rat cell), an avian cell, or primate cell, e.g., a human cell.

[0066] The cell may be a cell of the heart, lungs, liver, pancreas, brain, spinal cord, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, spleen. The cell may be a cardiomyocyte.Methods of reducing ischemic injury to a cell, organ, or tissue

[0067] In an aspect of the current disclosure, methods of reducing ischemic injury to a cell are provided. In some embodiments, the methods compnse contacting the cell with an effective amount of the disclosed nanoparticles, wherein the nanoparticles comprise a pay load that reduces ischemic injury. The methods may include methods of reducing ischemic injury' to a tissue or an organ.

[0068] Exemplary payload molecules that reduce ischemic injury comprise drugs, growth factors, or signaling modulators such as DNAs, RNAs, and peptides aimed at enhancing specific biological outcomes, such as promoting tissue regeneration, reducing inflammation, enhancing cellular function, or targeting pathological pathways. The encapsulation efficiency, stability, and controlled release of these small molecules within the nanoparticle system will be systematically evaluated to ensure optimal therapeutic delivery, efficacy, and safety in vitro and in vivo. Genome editing elements such as Cas9 and guide RNAs may be used as a payload to treat ischemic injuries.Atty. Dkt. No. 650053.01258Methods of delivering a payload molecule to an anatomical region

[0069] The inventors delivered the disclosed nanoparticles comprising a fluorescent dye as a payload to the left ventricle of animals. Surprisingly, the inventors observed that the nanoparticles continued to localize to the ventricle with minimal diffusion into the body cavity at D14 post administration (FIG. 5A, bottom panel). Thus, the disclosed nanoparticles are effective when used to target a payload molecule to be delivered to a particular anatomical region and promote extended contact of the nanoparticles comprising the pay load molecule with the anatomical region.

[0070] Accordingly, in an aspect of the current disclosure, methods of delivering a payload molecule to an anatomical region are provided. In some embodiments, the methods comprise administering the disclosed nanoparticles comprising a payload molecule. The anatomical region may comprise the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.Methods of treating ischemic injury in an organ in a subject in need thereof

[0071] In an aspect of the current disclosure, methods of treating ischemic injury in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of the pharmaceutical compositions of this disclosure to the subject to treat the ischemic injury in the subject, wherein the pharmaceutical compositions comprise nanoparticles comprising a payload that reduces ischemic injury.

[0072] In some embodiments, administration comprises intravenous administration, intrathecal administration, intrarectal administration, oral administration, intranasal administration, intraosseous administration, subcutaneous administration, intramuscular administration, intratumoral administration. In some embodiments, administration comprises local administration to a particular organ, e.g., the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.Kits, Systems, and Platforms

[0073] In an aspect of the current disclosure, kits, systems, and platforms are provided. In some embodiments, the kits, systems, or platforms, comprise the disclosed nanoparticles or the disclosed pharmaceutical compositions and, optionally, instructions for using the kits, systems, or platforms for performing the disclosed methods.Atty. Dkt. No. 650053.01258

[0074] The present invention is described herein using several definitions, as set forth below and throughout the application.Coated Compositions

[0075] The disclosed nanoparticles may be used to coat a composition, e.g., a vascular graft (FIGs. 11-19). Accordingly, compositions coated with the disclosed nanoparticles are described herein along with methods of generating such coated compositions. In some aspects, the nanoparticles may be applied as coatings to various substrates to impart beneficial properties, such as enhanced biocompatibility, improved endothelialization, and anti-inflammatory effects.

[0076] In some embodiments, a composition may be coated with the nanoparticles of any one of the embodiments described herein. The nanoparticle coating may provide a bioactive surface that supports cellular attachment and function while maintaining hemocompatibility.

[0077] In some embodiments, the composition may be coated with at least one layer of the nanoparticles, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more layers. In some aspects, the composition may be coated with two layers of the nanoparticles. In some embodiments, the composition may be coated with three or more layers of the nanoparticles. The particle density’ may increase proportionally with the number of coating layers, with particles evenly distributed across the surface.

[0078] In some embodiments, the composition may comprise at least one primer layer underneath the coating of the nanoparticles, e.g., 1, 2, 3. 4, 5, 6. 7, 8, 9, or 10 primer layers. The primer layer may enhance adhesion of the nanoparticle coating to the underlying substrate and improve coating stability. In some aspects, the at least one primer layer may comprise a polydopamine layer. In some aspects, the at least one primer layer may comprise a poly 1,8-octamethylene citrate (POC) layer. In some embodiments, the at least one primer layer may comprise both a poly dopamine layer and a POC layer.

[0079] In some embodiments, the at least one primer layer may comprise a first primer layer comprising the polydopamine layer and a second primer layer comprising the POC layer, wherein the second primer layer is located on top of the first primer layer. In some aspects, the nanoparticle coating may be located on top of the second primer layer. This layered configuration may provide enhanced coating stability and adhesion to the underlying substrate.

[0080] In some embodiments, the composition may comprise a medical device. In some aspects, the medical device may comprise a vascular graft. The nanoparticle coating may enhanceAtty. Dkt. No. 650053.01258endothelialization of the vascular graft and reduce complications such as thrombosis and neointimal hyperplasia.

[0081] In some embodiments, the composition may comprise a plastic. In some aspects, the plastic may comprise polytetrafluoroethylene (PTFE), such as expanded PTFE (ePTFE). The nanoparticle coating may convert the plastic graft surface from hydrophobic to hydrophilic, which may support endothelial attachment and function.Methods of Generating Coated Compositions

[0082] In some embodiments, a method of generating a coated composition may comprise applying the nanoparticles of any one of the embodiments described herein to the composition to generate the coated composition.

[0083] In some embodiments, applying the nanoparticles may comprise contacting the composition with a solution of the nanoparticles. In some aspects, the solution of the nanoparticles may be an aqueous solution when contacted to the composition. The nanoparticles may be dissolved or suspended in another non-aqueous solvent.

[0084] In some embodiments, the solution of the nanoparticles may comprise about 0.1 mg / ml to about 100 mg / ml of the nanoparticles, e.g., about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml. about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml, about 31 mg / ml, about 32 mg / ml, about 33 mg / ml, about 34 mg / ml, about 35 mg / ml. about 36 mg / ml, about 37 mg / ml, about 38 mg / ml, about 39 mg / ml. about 40 mg / ml, about 41 mg / ml, about 42 mg / ml, about 43 mg / ml, about 44 mg / ml, about 45 mg / ml, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, about 55 mg / ml, about 56 mg / ml, about 57 mg / ml, about 58 mg / ml, about 59 mg / ml, about 60 mg / ml, about 61 mg / ml, about 62 mg / ml, about 63 mg / ml, about 64 mg / ml, about 65 mg / ml, about 66 mg / ml, about 67 mg / ml, about 68 mg / ml, about 69 mg / ml, about 70 mg / ml, about 71 mg / ml, about 72 mg / ml, about 73 mg / ml, about 74 mg / ml, about 75 mg / ml, about 76 mg / ml, about 77 mg / ml, about 78 mg / ml, about 79 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml,Atty. Dkt. No. 650053.01258about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, about 94 mg / ml, about 95 mg / ml, about 96 mg / ml, about 97 mg / ml, about 98 mg / ml, about 99 mg / ml, about 100 mg / ml, or any subrange or value therein. In some aspects, the solution may comprise about 50 mg / ml of the nanoparticles.

[0085] In some embodiments, the solution of the nanoparticles may further comprise POC. In some aspects, the POC may comprise about 1% to about 10% POC, e g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, or any subrange or value therein. In some embodiments, the POC may comprise about 5% POC. The inclusion of POC in the nanoparticle solution may enhance coating stability7and uniformity.

[0086] In some embodiments, the method may comprise applying at least one primer layer prior to applying the nanoparticles. The primer layer may increase surface reactivity and enhance adhesion of the nanoparticle coating. In some aspects, the at least one primer layer may comprise a poly dopamine layer. In some aspects, the at least one primer layer may comprise a poly 1,8-octamethylene citrate (POC) layer.

[0087] In some embodiments, the method may comprise applying a first primer layer comprising the polydopamine layer and a second primer layer comprising the POC layer, wherein the second primer layer is located on top of the first primer layer. In some aspects, the composition may be subjected to oxygen plasma activation prior to applying the poly dopamine layer to increase surface reactivity. In some aspects, the polydopamine layer may be generated by incubating the composition in a dopamine solution. In some aspects, the POC layer may be applied by immersing the composition in a POC prepolymer solution and curing.

[0088] In some embodiments, the method may further comprise pretreating the composition prior to applying the primer layer or nanoparticle coating. In some aspects, pretreating may comprise rinsing the composition with deionized water and / or treating with ethanol. In some aspects, the composition may be dried under nitrogen prior to plasma activation.Further Definitions

[0089] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0090] As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “aAtty. Dkt. No. 650053.01258substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0091] As used herein, “about”, “approximately,” “substantially.” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0092] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0093] The phrase "such as” should be interpreted as "for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0094] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g, “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or B or “A and B.”

[0095] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3Atty. Dkt. No. 650053.01258members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth.

[0096] The modal verb "may" refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0097] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.Exemplary embodiments1. Nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing.2. The nanoparticles of embodiment 1, wherein the nanoparticles comprise hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90), or a fragment of each of the foregoing proteins.3. The nanoparticles of embodiment 1 or 2, wherein the nanoparticles comprise decellularized amniotic membrane.4. The nanoparticles of any one of the preceding embodiments, wherein the nanoparticle further comprises a pay load.5. The nanoparticles of embodiment 4, wherein the payload comprises a dye, a therapeutic, or both a dye and a therapeutic.6. The nanoparticles of embodiment 5, wherein the dye comprises a contrast dye.7. The nanoparticles of embodiment 6, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.8. The nanoparticles of embodiment 5, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.Atty. Dkt. No. 650053.012589. The nanoparticles of embodiment 8, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.10. The nanoparticles of embodiment 8, wherein the polynucleotide comprises an siRNA, an mRNA, a polynucleotide comprising a gRNA and / or a Cas protein, optionally, the polynucleotide may comprise YAP, LMNB2, LMNB1, LMNA, E2F6, E2F7, E2F8, TGF-beta related genes, or any combination thereof.11. Nanoparticles produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane.12. The nanoparticles of embodiment 11, wherein the method of nanoparticles are crosslinked.13. The nanoparticles of embodiment 11 or 12, wherein the amniotic membrane is human amniotic membrane.14. The nanoparticles of any one of embodiments 11-13, wherein the nanoparticles further comprise a payload.15. The nanoparticles of embodiment 14, wherein the payload comprises a dye, a therapeutic, or both a dye and a therapeutic.16. The nanoparticles of embodiment 15, wherein the dye comprises a contrast dye. 17. The nanoparticles of embodiment 16, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.18. The nanoparticles of embodiment 15, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.19. The nanoparticles of embodiment 18, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.20. The nanoparticles of any one of embodiments 11-19, wherein the nanoparticles comprise at least one protein selected from hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing proteins.21. The nanoparticles of any one of the preceding embodiments, wherein the nanoparticles have a mean diameter of about 100 nm to about 150 nm.Atty. Dkt. No. 650053.0125822. The nanoparticles of any one of the preceding embodiments, wherein the nanoparticles have a mean diameter of about 120 nm or about 117 nm.23. The nanoparticles of any one of the preceding embodiments, wherein the nanoparticles have a positive zeta potential.24. The nanoparticles of any one of the preceding embodiments, wherein the nanoparticles have a zeta potential of about 1 to about 10 mV or about 8.5 mV.25. A pharmaceutical composition comprising the nanoparticles of any one of the preceding embodiments.26. A method of generating nanoparticles, the method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the digested milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate the nanoparticles.27. The method of embodiment 26, wherein decellularizing amniotic membrane comprises contacting the amniotic membrane with at least one detergent for a sufficient amount of time to decellularize the amniotic membrane.28. The method of embodiment 27, wherein the at least one detergent comprises a nonionic detergent or an ionic detergent or both a non-ionic detergent and an ionic detergent.29. The method of any one of embodiments 26, wherein digesting the milled lyophilized decellularized membrane comprises pre-treating the milled lyophilized decellularized membrane in an aqueous solution in contact with an acid.30. The method of embodiment 26 or 29, wherein digesting the milled lyophilized decellularized membrane comprises contacting the milled lyophilized decellularized membrane with a protease for a sufficient amount of time to generate an aqueous solution comprising the digested milled lyophilized decellularized membrane.31. The method of any one of embodiments 26-39, wherein desolvating the aqueous solution comprises contacting the aqueous solution with a polar aprotic solvent.32. The method of embodiment 31, wherein the polar aprotic solvent comprises or consists of acetone.33. The method of any one of the embodiments 26-32, wherein the method further comprises crosslinking the nanoparticles.34. The method of embodiment 33, wherein crosslinking the nanoparticles comprises contacting the aqueous solution with a crosslinking agent.Atty. Dkt. No. 650053.0125835. The method of embodiment 34, wherein the crosslinking agent comprises or consists of glutaraldehyde.36. The method of any one of embodiments 26-35. wherein the method further comprises adding a payload to the aqueous solution comprising the digested milled lyophilized decellularized membrane.37. The method of embodiment 36, wherein the payload comprises a dye, a therapeutic, or both a dye and a therapeutic.38. The method of embodiment 37, wherein the dye comprises a contrast dye.39. The method of embodiment 38, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.40. The method of embodiment 37, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.41. The method of embodiment 40, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.42. The method of embodiment X40, wherein the polynucleotide comprises an siRNA, a polynucleotide comprising a gRNA and / or a Cas protein.43. A nanoparticle made by the method of any one of embodiments 26-42.44. A method comprising contacting a cell with the nanoparticles of any one of embodiments 1-24.45. The method of embodiment 44, wherein the cell is a vertebrate cell.46. The method of embodiment 45, wherein the cell is mammalian cell.47. The method of embodiment 46, wherein the cell is a canine, feline, equine, or bovine cell.48. The method of embodiment 45, wherein the cell is a primate cell.49. The method of embodiment 48, wherein the cell is a human cell.50. The method of any one of embodiments 44-49, wherein the cell is a cell of the heart, lungs, liver, pancreas, brain, spinal cord, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.51. The method of any one of embodiments 44-50. wherein the cell is a cardiomyocyte.52. A method comprising administering the pharmaceutical composition of embodiment 25 to a subject.53. The method of embodiment 52, wherein administering comprises administering a therapeutically effective amount of the nanoparticles to a subject in need thereof.Atty. Dkt. No. 650053.0125854. A method of reducing ischemic injury to a cell, the method comprising contacting the cell with an effective amount of the nanoparticles of any one of embodiments 1-24, wherein the nanoparticles comprise a payload that reduces ischemic injury.55. The method of embodiment 54, wherein the cell is mammalian cell.56. The method of embodiment 55, wherein the cell is a canine, feline, equine, or bovine cell.57. The method of embodiment 55, wherein the cell is a primate cell.58. The method of embodiment 57, wherein the cell is a human cell.59. The method of any one of embodiments 54-58, wherein the cell is a cell of the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.60. The method of any one of embodiments 59, wherein the cell is a cardiomyocyte.61. A method of treating ischemic injury in an organ of a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 25 to the subject to treat the ischemic injury in the subject, wherein the pharmaceutical composition comprises nanoparticles comprising a payload that reduces ischemic injury.62. The method of embodiment 61, wherein administering comprises intravenous administration.63. The method of embodiment 61, wherein administering comprises local administration to the organ.64. The method of any one of embodiments 61-63, wherein the organ comprises the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.65. A method of delivering a payload to an anatomical region, the method comprising administering the nanoparticles of any one of embodiments 1-24 to the anatomical region.66. The method of embodiment 65, wherein the anatomical region comprises the heart, lungs, liver, pancreas, brain, spinal cord, peripheral nervous system, muscle, kidneys, bladder, eyes, stomach, intestines, skin, ovaries, testes, endocrine system, lymphatic system, or spleen.67. The method of embodiment 65 or 66, wherein the payload comprises a dye, aAtty. Dkt. No. 650053.01258therapeutic, or both a dye and a therapeutic.68. The method of embodiment 67, wherein the dye comprises a contrast dye.69. The method of embodiment 68, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.70. The method of embodiment 67, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.71. The method of embodiment 70, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.72. The method of embodiment 70, wherein the polynucleotide comprises an siRNA, a polynucleotide comprising agRNA and / or a Cas protein.73. A kit comprising the nanoparticle of any one of embodiments 1-24 or the pharmaceutical composition of embodiment 25.74. The kit of embodiment 73, further comprising instructions for using the kit in any of the methods disclosed herein.75. A composition coated with the nanoparticles of any one of embodiments 1-24. 76. The composition of embodiment 75, wherein the composition is coated with at least one layer of the nanoparticles.77. The composition of embodiment 75, wherein the composition is coated with two layers of the nanoparticles.78. The composition of embodiment 75, wherein the composition comprises at least one primer layer underneath the coating of the nanoparticles.79. The composition of embodiment 78, wherein the at least one primer layer comprises:(a) a polydopamine layer; or(b) a poly 1,8-octamethylene citrate (POC) layer.80. The composition of embodiment 78, wherein the at least one primer layer comprises a first primer layer comprising the polydopamine layer and a second primer layer comprising the POC layer, wherein the second primer layer is located on top of the first primer layer.81. The composition of embodiment 80, wherein the nanoparticle coating is located on top of the second primer layer.82. The composition of any one of embodiments 75-81, wherein the composition comprises a medical device.Atty. Dkt. No. 650053.0125883. The composition of embodiment 82, wherein the medical device comprises a vascular graft.84. The composition of any one of embodiments 75-83, wherein the composition comprises a plastic.85. The composition of embodiment 82, wherein the plastic comprises polytetrafluoroethylene (PTFE).86. A method of generating a coated composition, the method comprising applying the nanoparticles of any one of embodiments 1-24 to the composition to generate the coated composition.87. The method of embodiment 86, wherein applying the nanoparticles comprises contacting the composition with a solution of the nanoparticles.88. The method of embodiment 87. wherein the solution of the nanoparticles is an aqueous solution.89. The method of embodiment 86 or 87, wherein the solution of the nanoparticles comprises about 0.1 mg / ml to about 100 mg / ml of the nanoparticles.90. The method of any one of embodiments 86-89, wherein the solution of the nanoparticles further compnses POC.91. The method of embodiment 90, wherein the POC comprises about l%to about 10% POC.92. The method of embodiment 91, wherein the POC comprises about 5% POC.93. The method of any one of embodiments 86-92. wherein the method comprises applying at least one primer layer prior to applying the nanoparticles.94. The method of embodiment 93, wherein the at least one primer layer comprises (a) a polydopamine layer; or(b) a poly 1,8-octamethylene citrate (POC) layer.95. The method of embodiment 94, wherein the method comprises applying a first primer layer comprising the polydopamine layer and a second primer layer comprising the POC layer, wherein the second primer layer is located on top of the first primer layer.EXAMPLES

[0098] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Atty. Dkt. No. 650053.01258Example 1 - Transforming Human Amniotic Membrane into Nanoparticles: A Novel Approach for Cardiac Delivery Systems

[0099] The article Stellpflug ci al. Transforming Human Amniotic Membrane into Nanoparticles: An Approach for Cardiac Delivery Systems” ACS Applied Biomaterials, 2025, is incorporated by reference herein in its entirety.

[0100] Heart diseases, such as coronary artery disease, myocardial infarction, and heart failure, remain leading causes of mortality worldwide. Despite advances in therapies, such as pharmacological treatments and surgeries, these approaches mainly manage symptoms and do not address limited cardiac regeneration. The adult heart's limited ability to repair itself, due to the restricted proliferation of cardiomyocytes, complicates the replacement of damaged tissue and leads to cardiac hypertrophy and heart failure. Therefore, therapies targeting tissue regeneration are urgently needed.

[0101] Nanoparticles (NPs) offer a promising solution for sustained, targeted delivery of therapeutic agents in myocardial regeneration. In this study, the inventors developed amniotic membrane-derived nanoparticles (AMPs) from decellularized human amniotic membrane. AMPs exhibited optimal size, positive surface charge, stability, and a sustained release profile, making them ideal for biomedical applications. Their efficient encapsulation of fluorescein dye demonstrated both effective drug loading and potential for non-invasive imaging. AMPs showed excellent biocompatibility with minimal cytotoxicity and no adverse inflammatory responses. Additionally, they retained and localized more effectively than free agents, highlighting their potential for targeted cardiac drug delivery. These findings suggest that AMPs offer great promise for clinical applications in cardiovascular medicine, with favorable properties supporting their use in treating chronic cardiac conditions.

[0102] Introduction

[0103] Heart diseases encompass coronary' artery disease (CAD), myocardial infarction (MI), and heart failure, remain the leading causes of mortality worldwide, imposing a significant burden on healthcare systems and drastically reducing patients' quality of life [1-4],

[0104] Despite significant advances in therapies such as pharmacological treatments, interventional procedures, coronary artery bypass surgery7, and heart transplantation, these approaches primarily focus on managing symptoms and prolonging patient survival [5-8], While effective in reducing the immediate impact of heart injury, these treatments do not address the fundamental issue of limited cardiac regeneration [9-13], The adult heart has a minimal capacity for self-repair due to the restricted proliferative ability of cardiomyocytes, making it difficult toAtty. Dkt. No. 650053.01258replace the substantial loss of cardiac tissue caused by severe heart injuries. Consequently, many patients develop cardiac hypertrophy and progress to heart failure, highlighting the need for therapies that target heart tissue regeneration and repair

[0014] ,

[0105] Recent research has focused on developing cost-effective, less invasive heart therapies through controlled deliver}7systems that precisely target therapeutic agents — such as drugs, genes, or cells — while enhancing efficacy, promoting heart regeneration, and reducing side effects [15-18], Nanoparticles (NPs) play a critical role in advancing these treatments, with an emphasis on designing targeted delivery systems that offer high specificity, biodegradability, biocompatibility, and low toxicity [19-22], Due to their nanoscale dimensions and engineered surface properties, NPs can encapsulate or attach therapeutic agents, protecting them from degradation and allowing for tunable drug-loading and release kinetics. This enables sustained, precise delivery of the therapeutic payload. Additionally, NPs can be efficiently taken up by cardiomyocytes and other relevant cells, ensuring effective intracellular delivery while avoiding issues like endosomal entrapment [23-26],

[0106] Various ty pes of NPs, including lipid, polymeric, metal, silica, and hybrid NPs, have emerged as innovative delivery systems for heart-related applications. Although early-stage studies have shown promising results, none have yet received approval from the U.S. Food and Drug Administration (FDA) for clinical use in cardiac therapies [27, 28], Major challenges include low delivery efficiency in heart, cardiac toxicity, poor retention time, and induced inflammation, exemplified by zinc oxide or nickel NPs [29, 30], The complexity of heart repair, involving processes like apoptosis, angiogenesis, inflammation, and fibrosis, further complicates these challenges. Efforts to improve delivery efficiency and safety, enhance myocardial homing and engraftment, extend circulation half-life and retention time, and optimize the therapeutic effects ofNPs are ongoing [28, 31-36],

[0107] The human amniotic membrane (HAM), the innermost layer of the placenta, is a thin, semi-transparent membrane rich in essential components such as collagen (e.g., types I, III, IV, V, and VII), non-collagenous proteins (e.g., fibronectin, laminin, and nidogen), and growth factors [e.g., Vascular Endothelial Growth Factor (VEGF), Transforming Growth Factor-P (TGF-P), and basic Fibroblast Growth Factor (b-FGF)] [37-39], HAM's unique biological properties — anti-inflammatory, low immunogenicity, and anti-fibrotic — make it an ideal biomaterial for a range of clinical applications, including ophthalmology, abdominal and plastic surgery, dental procedures, and w ound healing [40-44] .Atty. Dkt. No. 650053.01258

[0108] In the inventors’ laboratory, the inventors have developed an effective method for decellularizing the human amniotic membrane (HAM), producing a decellularized amniotic membrane (DAM) that retains essential extracellular matrix (ECM) components but removes all the cellular and antigen components

[0045] , The inventors’ previous work has demonstrated DAM's effectiveness as a wound dressing for various tissue injuries, including those in the oral cavity, bone, liver, and muscle, where it reduces inflammation and promotes wound healing and tissue regeneration [45-47], Additionally, DAM has been successfully used in fabricating small vascular grafts, which have shown excellent material stability, biocompatibility, and long-term patency in vascular transplantation surgery

[0048] ,

[0109] In addition to the inventors’ lab's efforts, recent studies have demonstrated the potential of DAM as a therapeutic strategy for heart treatment and regeneration. DAM has been developed into injectable hydrogels for direct delivery’ into infarcted heart tissue and as patches for stem cell transport in myocardial infarction repair [49-51], Both approaches have shown promise in improving heart function and reducing infarct size. Given DAM's proven ability’ to reduce inflammation, promote tissue healing and regeneration, and its excellent biocompatibility', the inventors believe DAM is an ideal foundational biomaterial for developing nano-carriers for cardiac drug delivery.

[0110] This study aims to develop NPs derived from DAM, referred to as AMPs, and evaluate their potential as a delivery’ platform for cardiac applications. The research will focus on characterizing the morphology and physical properties of AMPs, assessing their in vitro release kinetics, and evaluating their compatibility with cardiac cells. Additionally’, in vivo studies will examine cardiac cell uptake, retention, and safety7to determine the biocompatibility7and therapeutic potential of AMPs. These evaluations will provide insights into the feasibility of AMPs as a NP-based platform for cardiac delivery and therapies.

[0111] 2. Materials and Methods

[0112] 2.1. HAM decellularization and digestion

[0113] Fresh HAMs (Fig. 1A) were obtained from healthy donors through the tissue bank at the Medical College of Wisconsin (MCW). The HAMs were decellularized using 1% Triton X-100 and 0.1% sodium dodecyl sulfate (SDS) solutions (Sigma- Aldrich, St. Louis, MO), following the inventors’ previously established protocol to prepare DAMs [45, 46, 48], After thorough washing with distilled water, the DAMs were lyophilized at -80 °C using a Freeze-Dryer SystemAtty. Dkt. No. 650053.01258(Cole-Parmer, Vernon Hills, IL), and subsequently ground into a fine powder using a Thomas Wiley® Mini Cutting Mill (Thomas Scientific, Swedesboro, NJ).

[0114] The DAM powder was further digested according to the inventors' previous protocol [52-54], Powders were pre-treated with hydrochloric acid (HC1) solution (1 g of DAM powder per 100 ml of IM HC1) at 4 °C for 48 hours. The solution was then heated to 45 °C and digested with 15% w / w pepsin (Sigma-Aldrich) for another 48 hours. Once the majority of the tissue was fully digested, acetone (Sigma- Aldrich) was gradually added at a 1:1 v / v ratio to precipitate proteins and remove salts. After centrifugation, the resulting pellet was collected and resuspended in distilled w aler by vortexing, creating the DAM 'stock' solution. The protein concentration of this solution was measured using the Pierce™ bicinchoninic acid assay (BCA assay, ThermoFisher Scientific, Waltham, MA) and concentrated to 330 pg / ml.

[0115] 2.2. AMP fabrication

[0116] AMPs were synthesized from the DAM 'stock' solution according to the inventors’ previously established methods [53, 54], To allow for in vitro and in vivo detection, Indocyanine Green (ICG) dye (Sigma- Aldrich) was incorporated into the DAM 'stock' solution at 10% w / w of the total protein content. Acetone was then gradually added to the ICG and DAM mixture at a 3: 1 volume ratio (acetone to ICG / DAM solution) using a syringe pump, with a flow rate of 1 ml / min, while stirring at 500 RPM at room temperature. The resulting particles were crosslinked by adding 0.5% glutaraldehyde (Sigma- Aldrich) and stirring for 30 minutes at room temperature. The ICG-encapsulated AMPs were then washed three times with distilled water by centrifugation, and the resulting pellet was redispersed in distilled water by sonication using a Branson® Sonifier 150 (Brookfield, CT). For cell culture experiments, texas red dye-encapsulated AMPs were prepared following the same procedure. The final AMPs were freeze-dried at -80 °C for further experimental characterization or long-term storage.

[0117] 2.3. Characterization of AMP morphology and surface property

[0118] Scanning Electron Microscopy (SEM): To analyze the surface morphology and size of AMPs, the particles were first dissolved in water and sonicated to ensure dispersion. The resulting suspension was placed onto a carbon disc and allowed to air-dry at room temperature. Once dried, the samples were coated with gold using a sputter coater and imaged with a JEOL JSM-6510LV Series Scanning Electron Microscope.Atty. Dkt. No. 650053.01258

[0119] Zetasizer measurement: The hydrodynamic diameter and zeta potential (ZP, Q of the AMPs were measured using a Zetasizer 3000 dynamic light scattering (DLS) system. AMPs were suspended in 1 mL of Milli-Q water, sonicated on ice for 3 minutes to ensure uniform dispersion, and analyzed at 25°C. Zeta potential was calculated using the Smoluchowski approximation, optimized for proteins. Each sample underwent five measurements (n=5), and the collected data were analyzed using Microsoft Excel.

[0120] 2.4. Characterization of in vitro degradatation and release rate of AMP

[0121] Lyophilized AMPs (ICG-encapsulated) were weighed, and 5 mg of the powder was dissolved in 1 mL of either Milli-Q water or phosphate-buffered saline (PBS. pH 7.4) in eppendorf tubes. The samples were incubated at 37°C in the dark to prevent light-induced degradation, with measurements taken every 7 days for a total of 42 days. At each time point, the tubes were centrifuged, and the supernatant was completely removed and collected for protein and fluorescence analysis. To maintain the experimental conditions, 1 mL of fresh Milli-Q water or PBS was replaced to the tubes for continued incubation.

[0122] In vitro degradation rate: To assess the in vitro degradation rate, 100 pL of supernatant was collected weekly (n = 4 per time point) and the protein concentration was measured using a BCA assay, following the manufacturer's protocol. The degradation rate of the AMPs was calculated as follows: In vitro degradation rate (%) = [total protein loss at each time point (mg) / initial dry weight of the sample (mg)] x 100%. This calculation provided a quantitative measure of protein degradation over time, expressed as a percentage relative to the initial sample weight.

[0123] ICG release rate: To assess the ICG release rate from AMPs, particle samples (n = 4 per time point) were collected weekly during particle incubation by centrifugation and the remaining pellet in each tube was analyzed for fluorescent intensity using an IVIS Spectrum CT imaging system (PerkinElmer, MA, USA) with ICG-specific excitation / emission settings (745 / 840 nm). Fluorescence was quantified in Radiant Efficiency [(p / s / cm2 / sr) / (pW / cm2)] for a direct measure of ICG release. After imaging, the pellet was resuspended in fresh solution and incubated at 37°C for continuous monitoring.

[0124] The ICG release rate (%) was calculated as: [(NFo - NFT) / NFo]x100%, where NFT is the normalized fluorescence at each time point, and NFo is the initial fluorescence. Min / Max normalization adjusted for autofluorescence from non-ICG encapsulated AMPs, ensuring accurate tracking throughout.Atty. Dkt. No. 650053.01258

[0125] 2.5. Human Pluripotent Stem Cell (iPS) culture and cardiomyocyte differentiation

[0126] CiPSOOl-13 iPSCs generously donated by Drs. Kevin Bersell and Dan Roden (Vanderbilt University) were maintained in mTESR 1 medium (Stem Cell Technologies, Aylesbury', United Kingdom) supplemented with 1% Antibiotic-Antimycotic (Uife Technologies, Brown Deer, WI) on BD hESC-qualified Matrigel (Coming, Coming, NY). Cells were passaged every 5-6 days using ReLeSR dissociation reagent (Stem Cell Technologies) according to the manufacturer’s protocol. Then, 10 mM of Rho-associated kinase inhibitor (ROCK) Y27632 (EMD, Burlington, MA) was added to the medium for the first 24 hours after passaging. Human iPSCs were incubated at 37°C in 5% CO2 and maintained in 2 mL mTESR 1 medium with changes every 48 hours until the day of passage.

[0127] At all pluripotent and differentiative stages, cells were maintained at 37°C in 5% CO2. CiPS001-13 human iPSCs were split using StemPro Accutase dissociation reagent (Life Technologies) and seeded onto 6-well plates coated with growth-factor-reduced (GFR) Matrigel. Cells were expanded in mTESR 1 medium for 4 days, during which time the medium was replaced at 24 and 72 hours and allowed to reach 75-85% confluency. On different! ation-day 0 (subsequently denoted dO), differentiation was induced by exchanging the medium for RPMI 1640 containing 2% B-27 without insulin (Life Technologies) including 1% Antibiotic-Antimycotic (Life Technologies), supplemented with 7.5 mM GSK3 inhibitor CHIR99021 (Selleck, Houston, TX) to induce differentiation. After precisely 48 hours, medium was replaced with 2 mL RPMI / B27- containing 5 mM WNT inhibitor IWR-1 (Sigma- Aldrich) and incubated for 48 hours. On d4, medium was replaced with 2 mL fresh RPMI / B27-, followed on d6 by changing to RPMI / B27 with insulin. Contracting cells appeared between d7-d9. To enrich the cardiomyocyte population, on dl2 medium was exchanged for RPMI / B27+ without glucose containing 5 mM sodium D-lactate.

[0128] 2.6. Co-culture of AMPs and iPSC-cardiomyocytes

[0129] On day 15 of differentiation, iPSC-derived cardiomyocytes were seeded onto coverslips at a density' of 80,000 cells per coverslip and cultured in RPMI / B27+ medium. On day 22, AMPs (Texas red dye-encapsulated) w ere prepared at a concentration of 20 pg / mL (based on dry particle weight) in RPMI / B27+ medium and introduced to the cardiomyocyte cultures. After a 24-hour incubation at 37°C with 5% CO2, the AMP-containing medium was replaced with freshAtty. Dkt. No. 650053.01258RPMI / B27+ medium. The co-cultures were maintained for an additional 18 days, with medium changes every' 4-5 days to support optimal cell health and viability.

[0130] 2.7. Immunocytochemistry staining of cells

[0131] On days 25 and 40 of differentiation, iPSC-derived cardiomyocytes on coverslips w ere prepared for immunocytochemistry staining. The cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and permeabilized with 0.5% Triton X-100 for 10 minutes, followed by blocking with 3% Bovine Serum Albumin (Sigma-Aldrich) at room temperature for 1 hour. Primary antibodies were applied overnight at 4°C in blocking buffer, including mouse anti-Cardiac Troponin T (Invitrogen, Carlsbad. CA, 1 :400), rabbit anti-Ki67 (Abeam, Cambridge, United Kingdom, 1:400), and rabbit anti-Cleaved Caspase-3 (Cell Signaling, Danvers, MA, 1:400). After PBS washes, secondary antibodies (1:400 in PBS) were added and incubated for 1 hour at room temperature. Cell nuclei were stained with 1 pg / mL DAPI (4',6'-diamidino-2-phenylindole; Sigma-Aldrich) following additional PBS washes. Images were obtained using a Nikon Al laser scanning confocal microscope (Keyence. Osaka. Japan) and analyzed with Fiji software.

[0132] 2.8. Intramyocardial delivery of AMPs

[0133] All animal protocols were approved by the Institutional Animal Care and Use Committees (IACUC) at the Medical College of Wisconsin. C57BL / 6 mice (8-10 weeks old, both male and female) were used for intramyocardial injection. The mice were anesthetized with 2% inhaled isoflurane, intubated, ventilated, and positioned in a right lateral decubitus position. A left thoracotomy was performed to expose the heart. Experimental groups include:

[0134] AMP injection group: AMPs (ICG-encapsulated) were dissolved in saline at a concentration of 40 pg / mL (based on the dry weight of ICG / AMPs) and injected into three sites around the left ventricular wall using a modified Hamilton needle (10 pL per site, totaling 30 pL per mouse). After the injections, the pectoral muscles and skin were closed using 6-0 sutures.

[0135] ICG injection group: Free ICG dissolved in saline (concentration at 100 uM) was injected into three sites around the left ventricular wall using a modified Hamilton needle (10 pL per site, totaling 30 pL per mouse). After the injections, the pectoral muscles and skin were closed using 6-0 sutures.

[0136] Control group: Healthy mice with no intramyocardial injections were used as controls.Atty. Dkt. No. 650053.01258

[0137] Each group consisted of three mice (n = 3). F oilowing the procedure, the skin was closed using horizontal mattress sutures with 5-0 silk. The mice were then placed on a heating pad and closely monitored for any signs of complications or abnormal behavior.

[0138] Evaluation of AMPs retention and heart function

[0139] AMP retention monitoring: To monitor AMP retention, near-infrared (NIR) imaging was conducted at specific intervals after intramyocardial injection of either free ICG or AMPs. Imaging time points included 0.5 hours, 2 hours, 8 hours, and 1, 3, 5, 7, and 14 days post-injection. Mice were positioned supine on the imaging stage, and fluorescence images were obtained using the IVIS Spectrum CT system with excitation / emission settings at 745 / 840 nm, binning set to (M) 8, a field of view (FOV) of 13.2, f-stop of 2, and a high sensitivity level. Fluorescence intensities were analyzed by creating regions of interest (ROIs) over the heart using the Livingimage® software's ROI Toolkit, applying an auto-ROI function with specific parameters (Threshold % = 40, Lower Limit = 1.0, Minimum Size = 20). To ensure accuracy, fluorescence values for each animal were normalized against background autofluorescence.

[0140] Heart function by Echocardiography: Echocardiography was performed using a VisualSonics 3100 high-frequency ultrasound imaging system for baseline prior to injections in the heart and at 2 weeks after injections. Mice were lightly anesthetized with isofl urane via a nose cone (1.0-1.5%). An MX550D transducer operating at 30-40 mHz was used to obtain parasternal long-axis, short-axis and apical four-chamber views. Short-axis views in M(otion)-mode were used to assess left ventricular internal diameter (LVID), LV anterior wall (LVAW) thickness and LV posterior wall (LVPW) thickness at end-diastole (d) and end-systole (s) at the mid-ventricular level. Long-axis views in B(rightness)-mode assessed left ventricular internal area (LVA) and length (L) at end-diastole and end-systole. Left ventricular systolic function was calculated by the following: % fractional shortening (FS) calculated as [(LVIDd-LVIDs) / LVIDd]*100; % fractional area change (FAC) calculated as [(LVAd-LVAs)ZLVA d]xl00; and % ejection fraction (EF) calculated as [(end-diastolic volume (EDV)-end-systolic volume(ESV)] / EDV, where volumes were estimated in B-mode calculated as 4;i / 3xL / 2x[LVAHr(L / 2)]. In addition, global LV function was assessed by calculating the myocardial performance index (MPI) as (isovolumic contraction time+isovolumic relaxation time) / ej ection time. Time intervals were obtained from pulsed Doppler waveforms of mitral valve inflow and aortic valve outflow obtained from apical four-chamber views.Atty. Dkt. No. 650053.01258

[0141] Histology evaluation of heart: Two weeks post-injection, the mice were euthanized, and their hearts were collected for histological analysis. The harvested hearts were fixed in 10% buffered formalin, followed by dehydration through a graded series of ethanol concentrations. The tissues were then cleared using xylene and subsequently embedded in paraffin. Longitudinal sections of the paraffin-embedded hearts were prepared and stained with hematoxylin and eosin (H&E) (Sigma- Aldrich) for histological evaluation.

[0142] Statistical Analysis

[0143] Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using ANOVA (SAS 9.0) to evaluate differences between groups, followed by Holm-Sidak post-hoc tests for pairwise comparisons where significant ANOVA results were observed. For assessing correlations, the Pearson correlation coefficient was applied to parametric data, while the Spearman rank-order correlation was used for nonparametric data. A p-value of less than 0.05 was considered statistically significant for all analyses.

[0144] 3. Results

[0145] 3.1. Morphology, size, and surface property of AMP

[0146] After decellularization, the DAM was confirmed to be completely cell-free compared to HAM, while preserving its extracellular matrix structure (Fig. IB). Processing DAM into AMPs yielded small, spherical particles with an average diameter of 50.27 ± 30.5 nm, as shown in SEM images (Fig. 1C). The SEM analysis of particle size distribution (Fig. ID) revealed a broad range of sizes, with the majority measuring under 100 nm. When lyophilized AMPs were resuspended and sonicated in distilled water, dynamic light scattering (DLS) measured an average size of 117.78 ± 51.4 nm (Fig. IE), reflecting the typical differences between nanoparticle sizes in dry and hydrated states. The zeta potential of resuspended AMPs in water was 8.53 ± 6.3 mV, indicating the surface charge (Fig. IF).

[0147] ICG release rate and AMP stability

[0148] ICG release rate: Over a 42-day incubation at 37°C in both water and PBS, a gradual and steady decrease in fluorescence signal was observed (Fig. 2A), suggesting a sustained release of ICG from the AMPs. By the end of the incubation period, approximately 56% of the initialAtty. Dkt. No. 650053.01258ICG remained in the water-incubated samples, while around 53% persisted in the PBS samples (Fig. 2B), indicating a relatively stable ICG release profile from AMPs.

[0149] Particle stability: The weight loss analysis (Fig. 2C) over a 42-day period suggests that the AMPs exhibit gradual degradation, reflecting controlled material stability under physiological conditions. The slight but steady decrease in weight indicates a stable degradation profile, which aligns with the observed release of ICG.

[0150] Impact of AMP on cardiomyocyte proliferation and survival

[0151] To assess the impact of AMPs on proliferation and apoptosis of cardiomyocytes in vitro, the inventors performed co-culture experiments using Texas Red-conjugated AMPs with human iPSC-derived cardiomyocytes. Cardiomyocyte proliferation was evaluated by staining with the proliferation marker Ki67 (Fig. 3A). Immunocytochemistry analysis revealed successful internalization of AMPs by iPSC-cardiomyocytes, with localization observed in both the nucleus and cytoplasm (Fig. 3B). Among AMP-positive CMs, more than 60% of CMs have cytoplasmic AMPs while about 30% of CMs have AMPs localized in both cytoplasm and nucleus (Fig. 3B). Quantification of Ki67-positive cells indicated a slight, non-significant increase in cardiomyocyte proliferation after AMP treatment for short term (3-days) and long term (18 days), suggesting a potential mild proliferative effect (Fig.3C).

[0152] To examine AMP effects on cardiomyocytes apoptosis, the inventors assessed cleaved caspase-3 expression, a marker of apoptosis, through immunocytochemistry (Fig. 4A). Results showed no significant difference in cleaved caspase-3 expression following AMP short- and longterm treatment, indicating that AMPs did not induce apoptosis in iPSC-cardiomyocytes (Fig. 4B). Together, those results suggest that AMP administration to cardiomyocytes is does not cause detrimental effects on survival and proliferation.

[0153] Intramyocardium distribution and retention of AMPs

[0154] ICG dye: Following ICG dye injection, ICG initially localized in the left ventricle of the heart and gradually dispersed into the abdominal cavity, as evidenced by a secondary fluorescence signal below the heart (Fig. 5 A, top; green circle). Within the first 0.5 to 2 hours post-injection, the fluorescence signal within the heart shifted medially and laterally from its original position, suggesting a dynamic redistribution of ICG within the circulatory system. AAtty. Dkt. No. 650053.01258marked decline in signal intensity was observed after this period, with fluorescence nearly undetectable by day 3, indicating complete systemic clearance of the ICG dye.

[0155] AMPs: Following injection, AMPs displayed strong localization within the left ventricle, with minimal diffusion into the abdominal cavity (Fig. 5 A, bottom). Over a 14-day period, the signal intensity from AMPs decreased gradually and maintained a detectable, albeit reduced, fluorescence signal even at the 14-day mark, indicating prolonged retention within the heart.

[0156] To quantitatively evaluate retention, regions of interest (ROIs) were carefully defined for each animal to isolate fluorescence signals from the heart while minimizing background interference (Fig. 5B). A consistent ROI methodology w as applied across all time points, enabling accurate measurements of maximum fluorescence intensity throughout the study (Fig. 5C). Analysis of the normalized data revealed that fluorescence in the AMP injection group was significantly higher than in the free ICG group at all time points, demonstrating prolonged retention of AMPs within the heart compared to free ICG.

[0157] Heart function and morphology after AMP injection

[0158] Echocardiography evaluation: Two weeks following the intramyocardial injection of AMPs in mice, echocardiographic assessments (Fig. 6A) revealed no significant changes in heart function. Specifically, no alterations were observed in the anatomy, ejection fraction (Fig. 6B), or fractional shortening (Fig. 6C), all of which remained within normal ranges, indicating preserved cardiac function. Furthermore, measurements of the left ventricular internal diameter (Fig. 6D) and volume at end-diastole (Fig. 6E) were unchanged post-injection, further supporting the absence of structural or functional impairments. These findings suggest that the delivery of AMPs does not induce any detrimental effects on cardiac health, highlighting their potential as a safe and effective platform for therapeutic applications in disease treatment.

[0159] Histology of hearts: Two weeks after the intramyocardial injection, hematoxylin and eosin (H&E) staining was performed on heart to assess potential injection-related tissue damage. Minor tissue necrosis was observed in both ICG group (Fig. 7B) and AMP injection group (Fig.7C) when compared to healthy heart tissue (Fig. 7A). However, it is unclear whether this necrosis was caused by the needle puncture or the injected substances. Notably, there was no evidence of myocardial infarction or significant muscle damage in either group, indicating that the injections did not result in severe cardiac injury’.Atty. Dkt. No. 650053.01258

[0160] Discussion

[0161] NPs hold significant promise for diagnosing and treating ischemic heart diseases by facilitating targeted intracellular delivery and sustained release of therapeutic agents [55-57], However, their clinical efficacy is often hindered by challenges such as limited retention time within the myocardium and potential cardiac toxicity7[29, 30], which collectively diminish their therapeutic impact. Addressing these limitations requires strategies to enhance NP retention within cardiac tissue and improve their biocompatibility [28, 31-36], ensuring safer and more effective treatment outcomes.

[0162] In this study, the inventors developed a novel method for creating NPs from DAM, referred to as AMPs. This method is based on a previously established protocol for generating bone-derived NPs from porcine bone [53, 54], demonstrating the adaptability of the approach to different tissue ty pes. AMPs were designed to be versatile carriers for encapsulating therapeutic agents. As a proof of concept, a fluorescent dye was encapsulated within the AMPs for dual purposes: (1) enabling fluorescence-guided tracking to evaluate the in vitro release kinetics of the AMPs and (2) facilitating enhanced localization and visualization of the AMPs in real time, both in vitro and in vivo. This innovative approach highlights the potential of AMPs as multifunctional platforms for therapeutic delivery and monitoring.

[0163] The size and surface charge of NPs are factors that influence their circulation, cellular uptake, biodistribution, and half-life [58-62], In this study, AMPs exhibited a spherical morphology with an average size of 50.27 ± 30.5 nm in their dry state, as observed by SEM, and 117.78 ± 51.4 nm upon resuspension in water, as measured by DLS. The observed increase in hydrodynamic diameter is consistent with previous findings for bone-derived NPs

[0053] , likely resulting from particle swelling or mild aggregation under wet conditions.

[0164] The zeta potential of the AMPs was measured at 8.53 ± 6.3 mV, indicating a mildly positive surface charge. This is advantageous for cellular uptake, as positively charged particles interact effectively with negatively charged cell membrane components, including phospholipids, proteins, and glycans. Such electrostatic interactions can facilitate endocytosis, improving the potential for targeted therapeutic delivery

[0063] , However, the relatively low positive zeta potential may compromise AMP stability, making them susceptible to aggregation under physiological conditions. This instability could negatively impact colloidal stability, bioavailability, and therapeutic efficacy [64, 65], To address this challenge, optimizing AMP stability7through surfaceAtty. Dkt. No. 650053.01258modifications or the incorporation of stabilizing agents could enhance their performance and reliability in drug delivery applications.

[0165] In addition to particle size and surface properties, the material stability and sustained release profile of NPs are crucial for optimizing their therapeutic efficacy and safety as drug delivery systems. The AMPs developed in this study exhibited a slow and sustained degradation and release profile, retaining over 50% of the encapsulated ICG for up to 42 days in both water and PBS. This gradual release suggests that AMPs can maintain therapeutic levels over extended periods, making them suitable for applications that require prolonged efficacy, such as extended imaging or targeted drug delivery to tissues. The predictable release behavior allows for precise control over dosage and release duration, enhancing the therapeutic potential of AMPs for stable, long-term, and targeted drug delivery in clinical applications.

[0166] The inventors’ study also evaluated the biocompatibility of AMPs for cardiac applications by assessing their effects on cardiomyocyte proliferation, apoptosis, and retention in heart tissue. In co-culture with iPSC-derived cardiomyocytes, AMPs were successfully internalized, with localization observed in both the nucleus and cytoplasm — key for NPs designed to deliver intracellular therapeutic agents. A slight, non-significant increase in Ki67-positive cardiomyocytes was observed, suggesting a mild, controlled proliferative effect without disrupting normal cell cycle dynamics. Additionally, cleaved caspase-3 expression analysis show ed no increase in apoptosis following AMP exposure, further confirming their compatibility7with cardiac cells. These results highlight AMPs as a safe and promising platform for cardiac therapies, offering the potential to promote controlled cell proliferation while maintaining cell viability' and preventing apoptosis.

[0167] To assess the retention and safety of AMPs in the myocardium, the inventors injected them into the left ventricle of mouse hearts and compared their performance with free ICG dye to evaluate their potential for targeted cardiac therapies. Following injection, free ICG dye rapidly diffused from the myocardium into the abdominal cavity, with fluorescence intensity sharply declining within 2 hours and completely disappearing by day73. This signal diffusion and rapid clearance indicate the limited retention and short half-life of free dyes or drugs in cardiac tissue, which poses a challenge for applications that require prolonged imaging or sustained therapeutic effects.

[0168] In contrast, AMPs exhibited significantly improved retention in the left ventricle, with minimal diffusion to other organs. The fluorescence signal remained detectable for up to 14 days, indicating a controlled, localized release within the myocardium. This prolonged retentionAtty. Dkt. No. 650053.01258suggests that AMPs offer greater stability and reduced off-target effects compared to free dyes, making them a promising option for precise and effective cardiac-targeted therapies.

[0169] Echocardiography and histological analyses further confirmed that both AMPs and free ICG injections exhibited minimal impact on heart function and structure change following injection. Importantly, there was no evidence of myocardial damage or impairment in cardiac performance, underscoring the biocompatibility' of AMPs and their suitability for long-term delivery’ applications.

[0170] Overall, the inventors’ findings support the potential of AMPs as a biocompatible and promising nanoplatform for cardiac delivery and therapies. The demonstrated safety' and effectiveness of AMPs as nanovehicles for intracellular and intramyocardial delivery' position them as strong candidates for both therapeutic and imaging applications. Their ability to retain and release encapsulated agents within heart tissue over extended periods highlights their potential for treating chronic cardiac conditions that require sustained intervention.

[0171] However, a current limitation of AMPs is their lack of specificity in targeting cardiomyocytes, which may reduce their therapeutic efficiency, especially in systemic cardiac delivery’. Future research will focus on enhancing AMP targeting to infarcted myocardium by functionalizing them with heart-specific targeting molecules or utilizing non-invasive physical cues to improve localization. Further optimization of AMP synthesis and functionalization is also essential to maximize their potential in both diagnostic and therapeutic applications. While the initial results are promising, it is crucial to assess the long-term safety and biocompatibility' of AMPs. Addressing these challenges will be pivotal in advancing AMP-based delivery systems, ensuring their safe and effective use in clinical cardiovascular medicine.

[0172] Conclusion

[0173] In conclusion, this study successfully transformed DAM into nanosized AMPs with desirable properties, including optimal size, positive surface charge, particle stability, and a sustained release profile. These characteristics make AMPs highly promising for various biomedical applications. The efficient encapsulation of fluorescein dye within the AMPs not only- confirmed their ability to load therapeutic agents effectively but also demonstrated their potential for non-invasive imaging, which is essential for real-time monitoring in clinical settings.

[0174] The AMPs exhibited excellent biocompatibility, showing minimal cytotoxicity' and no signs of adverse inflammatory' responses or tissue damage within the cardiac environment.Atty. Dkt. No. 650053.01258Additionally, they demonstrated superior retention and localization compared to free therapeutic agents, emphasizing their potential as a reliable platform for cardiac drug delivery'.

[0175] These findings suggest that AMPs provide exceptional intracellular and intramyocardial compatibility, positioning them as highly suitable for clinical applications in cardiovascular medicine. The combination of favorable biocompatibility, prolonged retention, and sustained release capabilities further supports their clinical translation potential for treating chronic cardiac conditions.1. Salzillo, C., et al.. Cardiovascular Diseases in Public Health: Chromosomal Abnormalities in Congenital Heart Disease Causing Sudden Cardiac Death in Children. Medicina (Kaunas), 2024. 60(12).2. Kim. Y, et al.. Gene Therapy in Cardiovascular Disease: Recent Advances and Future Directions in Science: A Science Advisory From the American Heart Association. Circulation, 2024. 150(23): p. e471-e480.3. Ismaiel, A. and D.L. Dumitrascu, Cardiovascular Risk in Fatty’ Liver Disease: The Liver- Heart Axis-Literature Review. Front Med (Lausanne), 2019. 6: p. 202.4. 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[0176] In the foregoing description, it will be readily apparent to one skilled in the art that var ing substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use ofAtty. Dkt. No. 650053.01258such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and / or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0177] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency betw een a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.References for Example 11. 2022 Heart Disease & Stroke Statistical Update Fact Sheet Global Burden of Disease.American Heart Association, 2022.2. Bougouin, W., et al., Incidence of sudden cardiac death after ventricular fibrillation complicating acute myocardial infarction: a 5-year cause-of-death analysis of the FAST- MI 2005 registry. Eur Heart J, 2014. 35(2): p. 116-22.3. Huber. C.A., et al., Post-myocardial Infarction (MI) Care: Medication Adherence for Secondary Prevention After MI in a Large Real-world Population. Clin Ther, 2019. 41(1): p. 107-117.4. Weber, B., et al., Association of inflammatory disease and long-term outcomes among young adults with myocardial infarction: the Mass General Brigham YOUNG-MI Registry. Eur J Prev Cardiol, 2022. 29(2): p. 352-359.5. Kattel, S., et al., Motivation, Perception, and Treatment Beliefs in the Myocardial Infarction Genes (MI-GENES) Randomized Clinical Trial. J Genet Conns. 2017. 26(5): p. 1153-1161.6. Erlinge, D., et al., Rapid endovascular catheter core cooling combined with cold saline as an adjunct to percutaneous coronary intervention for the treatment of acute myocardial infarction. The CHILL-MI trial: a randomized controlled study of the use of central venous catheter core cooling combined with cold saline as an adjunct to percutaneous coronary’ intervention for the treatment of acute myocardial infarction. J Am Coll Cardiol, 2014. 63(18): p. 1857-65.7. Golikov. A.P. and V.A. Riabinin, [Current trends in the treatment and outcome of acute myocardial infarction (MI)]. Klin Med (Mosk). 2000. 78(5): p. 19-21.8. Inamdar, A. A. and A.C. Inamdar, Heart Failure: Diagnosis, Management and Utilization.J Clin Med, 2016. 5(7).9. Doenst, T., et al., PCI and CABG for Treating Stable Coronary Artery Disease: JACC Review Topic of the Week. J Am Coll Cardiol, 2019. 73(8): p. 964-976.10. Mersmann, J., et al., Measure for measure-determination of infarct size in murine models of myocardial ischemia and reperfusion: a systematic review. Shock, 2011. 35(5): p. 449- 55. 'Atty. Dkt. No. 650053.01258Golomb, G., et al., Controlled-release drug delivery) of diphosphonates to inhibit bioprosthetic heart valve calcification: release rate modulation with silicone matrices via drug solubility and membrane coating. J Pharm Sci, 1987. 76(4): p. 271-6.Kiaie, N., et al., Targeted and Controlled Drug Delivery to aRat Model of Heart Failure Through a Magnetic Nanocomposite. Ann Biomed Eng, 2020. 48(2): p. 709-721.Chen, W.C., et al.. Controlled dual delivery of fibroblast growth factor-2 and Interleukin-10 by heparin-based coacervate synergistically enhances ischemic heart repair.Biomaterials, 2015. 72: p. 138-51.Takakura, S.. et al., The Successful Use of Nitroglycerin for Uterine Hyperstimulation with Fetal Heart Rate Abnormality Caused by a Controlled-Release Di noprostone Paginal Delivery System (PROPESS): A Case Report. Medicina (Kaunas). 2021. 57(5).Mehany, H.M., et al., Potential of chitosan nanoparticle / fluoride nanocomposite for reducing the toxicity of fluoride an in-vivo study on the rat heart functions: Hematopoietic and immune systems. Int J Biol Macromol, 2022. 216: p. 251-262.Ong. S.B., et al., Nanoparticle delivery) of mitoprotective agents to target ischemic heart disease. Future Cardiol, 2017. 13(3): p. 195-198.Fan, C., et al., Nanoparticle-Mediated Drug Delivery) for Treatment of Ischemic Heart Disease. Front Bioeng Biotechnol, 2020. 8: p. 687.Ribeiro, S., Nanotechnology: Change of heart on nanoparticle risks. Nature, 2012.490(7418): p. 37.Aoki, K. and N. Saito, Biodegradable Polymers as Drug Delivery Systems for Bone Regeneration. Pharmaceutics, 2020. 12(2).Dorati. R., et al., Biodegradable Scaffolds for Bone Regeneration Combined with Drug-Delivery Systems in Osteomyelitis Therapy. Pharmaceuticals (Basel), 2017. 10(4).Ogay, V., et al., Progress and Prospects of Polymer-Based Drug Delivery Systems for Bone Tissue Regeneration. Polymers (Basel), 2020. 12(12).Xinluan, W., et al., Systemic drug delivery systems for bone tissue regeneration- a mini review. Curr Pharm Des, 2015. 21(12): p. 1575-83.Park, J.H., et al., Biomimetic nanoparticle technology) for cardiovascular disease detection and treatment. Nanoscale Horiz, 2020. 5(1): p. 25-42.Fredman. G., et al., Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice. Sci Transl Med, 2015. 7(275): p. 275ra20.Nagarajan, M., et al., Exposure to zinc oxide nanoparticles (ZnO-NPs) induces cardiovascular toxicity and exacerbates pathogenesis - Role of oxidative stress and MAPK signaling. Chem Biol Interact, 2022. 351: p. 109719.Magaye, R.R., et al., Acute toxicity) of nickel nanoparticles in rats after intravenous injection. Int J Nanomedicine, 2014. 9: p. 1393-402.Saludas, L., et al., Heart tissue repair and cardioprotection using drug delivery systems. Maturitas, 2018. 110: p. 1-9.Kamaly, N., et al., Targeted Interleukin-10 Nanotherapeutics Developed with a Microfluidic Chip Enhance Resolution of Inflammation in Advanced Atherosclerosis. ACS Nano, 2016. 10(5): p. 5280-92.Gorain, B., et al.. Carbon nanotube scaffolds as emerging nanoplatform for myocardial tissue regeneration: A review of recent developments and therapeutic implications.Biomed Pharmacother, 2018. 104: p. 496-508.Sperling, R.A.. et al.. Biological applications of gold nanoparticles. Chem Soc Rev, 2008.37(9): p. 1896-908.Adokoh, C.K., et al., Development and characterization of functionalized glyco thiolate capped gold nanoparticles for biological applications. RSC Med Chem, 2020. 11(2): p.283-292.Sanchez-Gaytan, B.L., et al., HDL-mimetic PLGA nanoparticle to target atherosclerosis plaque macrophages. Bioconjug Chem, 2015. 26(3): p. 443-51.Atty. Dkt. No. 650053.01258Hopkinson, A., et al., Proteomic analysis of amniotic membrane prepared for human transplantation: characterization of proteins and clinical implications. J Proteome Res, 2006. 5(9): p. 2226-35.Elkhenany. H., et al., Applications of the amniotic membrane in tissue engineering and regeneration: the hundred-year challenge. Stem Cell Res Ther, 2022. 13(1): p. 8.Niknejad, H._ et al., Properties of the amniotic membrane for potential use in tissue engineering. Eur Cell Mater, 2008. 15: p. 88-99.Tettelbach, W.H., et al., Outcomes for Medicare debridement intervals receiving dehydrated human amnion / chorion membrane. J Wound Care, 2022. 31 (10): p. 886. Tacktill, J.Z., et al., Wound repair, safety, and functional outcomes in reconstructive lower extremity foot and ankle surgery using a dehydrated amnion / chorion allograft membrane. Int Wound J, 2022.Mueller, S.M., et al., Pain reduction by dehydrated human amnion / chorion membrane allograft in nondiabetic leg ulcers might be an early indicator of good response: A case series. Dermatol Ther, 2020. 33(4): p. el3587.Sanders, R. J. and S. J. Annest, Amnion membrane improves results in treating neurogenic thoracic outlet syndrome. J Vase Surg Cases Innov Tech, 2018. 4(2): p. 163-165.Mohan, R.. A. Bajaj, and M. Gundappa, Human Amnion Membrane: Potential Applications in Oral and Periodontal Field. J Int Soc Prev Community Dent, 2017. 7(1): p. 15-21.Li, W., ct al., Investigating the Potential of Amnion-Based Scaffolds as a Barrier Membrane for Guided Bone Regeneration. Langmuir, 2015. 31(31): p. 8642-53.Li. W.. et al., Polymer-integrated amnion scaffold significantly improves cleft palate repair. Acta Biomater, 2019.Wang. B., W. Li. and J. Harrison, An Evaluation of Wound Healing Efficacy of a Film DressingMade from Polymer-integrated Amnion Membrane. Organogenesis, 2020. 16(4): p. 126-136.Wang, B., et al., Developing small-diameter vascular grafts with human amniotic membrane: long-term evaluation of transplantation outcomes in a small animal model. Biofabrication, 2023. 15(2).Francisco, J.C.. et al., Decellularized Amniotic Membrane Scaffold as a Pericardial Substitute: An In Vivo Study. Transplant Proc, 2016. 48(8): p. 2845-2849.Henry. J. J.D., et al., Development of Injectable Amniotic Membrane Matrix for Postmyocardial Infarction Tissue Repair. Adv Healthc Mater. 2020. 9(2): p. el900544. Khorramirouz, R., et al., Evaluating the efficacy of tissue-engineered human amniotic membrane in the treatment of myocardial infarction. Regen Med. 2019. 14(2): p. 113-126. Li. W.. et al., A comparative study’ on fabrication techniques of gelable bone matrix derived from porcine tibia. J Biomed Mater Res B Appl Biomater, 2021.Stellpflug. A., et al., From bone to nanoparticles: development of a novel generation of bone derived nanoparticles for image guided orthopedic regeneration. Biomater Sci, 2024.Binsalamah, Z.M., et al., Intramyocardial sustained delivery of placental growth factor using nanoparticles as a vehicle for delivery in the rat infarct model. Int J Nanomedicine, 2011. 6: p. 2667-78.Paul. A., et al., A nanobiohybrid complex of recombinant baculovirus and Tat DNA nanoparticles for delivery of Ang-1 transgene in myocardial infarction therapy.Biomaterials, 2011. 32(32): p. 8304-18.Oduk, Y., et al., VEGF nanoparticles repair the heart after myocardial infarction. Am J Physiol Heart Circ Physiol, 2018. 314(2): p. H278-H284.Agarwal, R.. et al.. Effect of shape, size, and aspect ratio on nanoparticle penetration and distribution inside solid tissues using 3D spheroid models. Adv Healthc Mater. 2015. 4(15): p. 2269-80.Atty. Dkt. No. 650053.0125854. Brown, T.D., et al., Effect of Nanoparticle Composition, Size, Shape, and Stiffness on Penetration Across the Blood-Brain Barrier. ACS Biomater Sci Eng, 2020. 6(9): p. 4916- 4928.55. Guo. H., G. Stan, and Y. Liu, Nanoparticle separation based on size-dependent aggregation of nanoparticles due to the critical Casimir effect. Soft Matter, 2018. 14(8): p. 1311-1318.56. Horechyy, A., et al., Nanoparticle assembly under block copolymer confinement: The effect of nanoparticle size and confinement strength. J Colloid Interface Sci, 2020. 578: p. 441-451.57. Kang, J.H., J. Clio, and Y.T. Ko, Investigation on the effect of nanoparticle size on the blood-brain tumour barrier permeability: by in situ perfusion via internal carotid artery in mice. J Drug Target, 2019. 27(1): p. 103-110.58. Forest. V. and J. Pourchez, Preferential binding of positive nanoparticles on cell membranes is due to electrostatic interactions: A too simplistic explanation that does not take into account the nanoparticle protein corona. Mater Sci Eng C Mater Biol Appl, 2017. 70(Pt l): p. 889-896.59. Fatima, H., et al., A Review of Multifunction Smart Nanoparticle based Drug Delivery Systems. Curr Pharm Des. 2022. 28(36): p. 2965-2983.60. Ruiz-Hernandez, E., A. Baeza, and M. Vallet-Regi. Smart drug delivery through DNA / magnetic nanoparticle gates. ACS Nano, 2011. 5(2): p. 1259-66.Example 2 - Amniotic Membrane-Derived Nanocoatings for Improved Endothelialization and Inflammation Control in Small-Diameter Vascular Grafts

[0178] Background: Endothelial migration and control of inflammation are critical for vascular repair and implant integration, yet current biomaterial coatings provide limited support for these processes. The human amniotic membrane (HAM) contains bioactive factors, but its fragile structure restricts broader applications.

[0179] Objective / Approach: To overcome these limitations, the inventors developed nanoparticles derived from HAM (AMPs) and applied them as a novel nanocoating to evaluate their effects on endothelial function .

[0180] Methods: Coatings were characterized by SEM. AFM, and contact angle analysis. Functional assays using human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs) assessed migration and inflammatory7activation in response to lipopolysaccharide (LPS).

[0181] Results: AMP nanocoating markedly7enhanced endothelial migration, closing scratch wounds nearly twice as fast as controls. Upon LPS stimulation, AMP-coated cells showed significantly reduced expression of pro-inflammatory mediators (IL-6, TNF-a, IL-1 , CXCL10) and matrix metalloproteinase-3 (MMP3), confirmed at both mRNA and protein levels.Atty. Dkt. No. 650053.01258

[0182] Conclusion: HAM-derived nanoparticles provide a stable nanocoating that simultaneously promotes endothelial migration and attenuates inflammatory activation. This dual functional effect offers a novel and scalable strategy to improve vascular repair and may support future applications in vascular grafts, stents, and tissue engineering.

[0183] Vascular endothelial cells (ECs), which form a dynamic monolayer lining the interior of blood vessels, play a central role in maintaining vascular homeostasis and coordinating responses to injury’ and inflammation [1-7], Under physiological conditions, ECs maintain vascular homeostasis and tissue perfusion by releasing vasoactive mediators that regulate vascular tone, oxidative stress, and angiotensin II activity, while also preserving a non-thrombogenic and selectively permeable barrier by limiting leukocyte adhesion, inhibiting platelet aggregation, and controlling vascular permeability [8-15], In response to pathological stimuli such as pro-inflammatory cytokines, oxidized low-density lipoprotein (LDL), and abnormal mechanical forces, ECs transition from a quiescent to an activated state through two distinct phases, initiating inflammation to recruit immune cells and subsequently restoring the tissue barrier to promote repair [16, 17],

[0184] In the early phase of EC activation triggered by injury or inflammatory stimuli, ECs rapidly upregulate adhesion molecules and secrete pro-inflammatory cytokines and chemokines to recruit immune cells to the injury site [17-21], In the later phase, cytokines such as tumor necrosis factor-alpha (TNF-a) and interleukin-1 (IL-1) activate gene expression and protein synthesis in ECs, leading to the production of adhesion molecules and chemokines that promote leukocyte recruitment and initiate repair processes such as barrier restoration, angiogenesis, and tissue remodeling; however, if inflammation persists, it can result in endothelial dysfunction, abnormal vessel grow th, and chronic inflammation, ultimately impairing wound healing [3, 22-29] . Especially in chronic wounds and conditions like diabetic ulcers, radiation-induced damage, and sepsis, ECs exhibit persistent activation, barrier disruption, impaired nitric oxide signaling, and reduced angiogenesis, all of which contribute to ongoing inflammation, delayed healing, and fibrosis [30-34],

[0185] Due to the central role of ECs in initiating inflammation and supporting wound healing, restoring endothelial homeostasis has emerged as a key therapeutic target for resolving chronic inflammation and promoting tissue regeneration

[0019] , Therapies that target EC dysfunction are increasingly recognized as promising strategies to improve healing in chronic wounds by reducing inflammation, stabilizing vascular integrity, and enhancing angiogenesis [19, 35-43], By shifting the wound environment from a pro-inflammatory state to one that supports repair andAtty. Dkt. No. 650053.01258regeneration, the ECs emerge as a critical and versatile target in both wound healing and regenerative medicine [19, 41, 44, 45],

[0186] Current therapies targeting endothelial dysfunction include anti-cytokine agents, immunomodulators, endothelial stabilizers, and stem cell therapy. Anti-cytokine agents such as etanercept (a tumor necrosis factor-alpha (TNF-a) receptor fusion protein) and tocilizumab (an interleukins (IL-6) receptor-blocking antibody), reduce vascular inflammation but are limited in wound healing due to concerns about immune suppression and the need for timely administration [46-48], Broad-spectrum agents such as corticosteroids and non-steroidal anti-inflammatory drugs lack EC specificity and may impair tissue repair [49-51], Endothelial stabilizers, including sphingosine- 1 -phosphate (SIP) analogs, angiopoietin-1 mimetics, and statins, help support vascular integrity, though their clinical translation is challenged by dosing difficulties and context-dependent effects [52-58], Stem cell therapies using mesenchymal stromal cells (MSCs) or endothelial progenitor cells (EPCs) promote endothelial repair but are limited by issues of cell sourcing, consistency, immune compatibility’, and survival [59-61],

[0187] Given the limitations of current therapies, future strategies should focus on resolving inflammation and restoring barrier function of ECs to reestablish vascular homeostasis and promote sustained wound healing, particularly in chronic or complex inflammatory conditions.

[0188] The human amniotic membrane (HAM), a thin membrane from the innermost layer of the placenta, has emerged as a promising biological material due to its unique structural and biochemical properties, being rich in collagens (types I, III, IV, V, and VI), non-collagenous glycoproteins (such as fibronectin and laminin), and various growth factors (including vascular endothelial growth factor (VEGF), transforming growth factor- (TGF-0), and basic fibroblast growth factor (b-FGF)) that play key roles in angiogenesis, wound healing, and tissue regeneration [62-64], Due to its special anti-inflammatory, anti-fibrotic, antimicrobial, and pro-regenerative properties, along with its low cost, abundance, minimal ethical concerns, and immunoprivileged nature [65-69], HAM has been extensively used as a biological wound dressing in a variety of clinical applications, including bums, chronic wounds (e.g., diabetic, arterial, and venous ulcers), comeal reconstruction, and the repair of abdominal wall and dural defects [70-77],

[0189] While HAM has shown significant clinical benefits in wound healing, its therapeutic use remains largely limited to topical membrane dressings for localized injuries, such as bums, ulcers, and exposed wounds, due to its inherently thin, fragile structure, and limitation in size [78, 79], These physical constraints pose challenges for consistent handling, scalability, andAtty. Dkt. No. 650053.01258reproducibility, especially when treating large, irregular, or deep tissue defects. Its delicate nature also hinders integration with more robust biomaterials or devices, restricting its broader use in complex wound care and regenerative applications. Additionally, HAM in membrane form is incompatible with systemic or injectable delivery, which is essential for addressing diffuse conditions like sepsis, chronic inflammation, or multi-organ injury [80-84], These limitations highlight the need for next-generation HAM-derived formulations that preserve its bioactivity while enabling more versatile, scalable, and clinically adaptable delivery options.

[0190] Recently, the inventors’ lab developed an innovative technique to convert native tissues into protein-based nanoparticles (NPs), with successful application to both bone and HAM. These tissue-derived NPs preserve key extracellular matrix (ECM) proteins from native tissue, exhibit uniform and nano-scale sizes, preserve bioactivity, and show excellent biocompatibility for diverse biomedical applications. Notably, these NPs can serve as versatile drug delivery systems for both local and systemic use, overcoming key limitations associated with native tissue formats. Moreover, their small size and excellent dispersibility allow for easy integration into other materials for surface functionalization, composite fabrication, and implant coatings, enabling broad applications in targeted therapy, biomedical imaging, and regenerative medicine.

[0191] The goal of this study is to develop HAM-derived nanoparticles (AMPs) as a bioactive and clinically translatable coating material for vascular applications. The inventors will engineer and thoroughly characterize AMP surface coatings, evaluate their effects on endothelial cells with a focus on migration, endothelialization. and anti-inflammatory responses, and assess their performance on small-diameter ePTFE grafts. Specifically, the inventors will determine whether AMP-coated grafts maintain stable surface coating under flow, promote sustained endothelialization, and reduce thrombosis.

[0192] Materials and Methods

[0193] HAM decellularization and AMP fabrication

[0194] HAM decellularization: Fresh HAMs were obtained from healthy donors through the tissue bank of the Medical College of Wisconsin (MSC). HAM decellularization and AMP fabrication were performed according to the inventors’ previously established protocols [85, 86], Briefly, HAMs were decellularized with a solution containing 1% Triton X-100 and 0.1% SDS (Sigma-Aldrich), followed by extensive washing with distilled water to remove residual detergents. The decellularized HAM (DAM) was then lyophilized at -80 °C using a freeze-dryer (Cole-Parmer). The lyophilized DAM was ground into a fine powder using a Thomas Wiley®Atty. Dkt. No. 650053.01258Mini Cutting Mill (Thomas Scientific), and subsequently digested in a solution of 15% pepsin in 0.1 N HC1 (pH = 2) under constant stirring at 45 °C for 48 hours to obtain the DAM digest.

[0195] AMP fabrication: To fabricate AMPs. acetone was added dropwise to the DAM digest at a 3:1 volume ratio (acetone: DAM digest) under continuous stirring at 500 RPM at room temperature. The resulting suspension was centrifuged, and the precipitated particles were washed three times with distilled water. The final pellet was resuspended in distilled water and sonicated using a Branson® Sonifier 150 to achieve a uniform dispersion. The resulting solution was lyophilized to produce dry AMP powders [65, 66],

[0196] AMP coating on plate:

[0197] AMP powder was dissolved in ultra-pure distilled water (Milli-Q, Millipore) at a concentration of 50 pg / mL. The suspension was sonicated on ice using a Branson® Sonifier 150 to ensure complete dispersion of the particles.

[0198] 1-layer AMP coating: Muscovite mica discs (12 mm diameter; Sigma- Aldrich) were placed in individual wells of a sterile 12-well plate. A total of 1 mL of AMP solution was added to each well to fully cover the mica surface and allowed to air dry completely at room temperature under sterile conditions, forming a uniform single-layer coating.

[0199] 2-layer AMP coating: After the first layer had dried completely, a second 1 mL aliquot of AMP solution was applied and again allowed to air dry under the same conditions.

[0200] 3-layer AMP coating: Following complete drying of the second layer, a third 1 mL aliquot of AMP solution was added and dried to complete the triple-layer coating process.

[0201] Surface characterization of AMP coating

[0202] Scanning Electron Microscopy (SEM): To evaluate surface morphology, lyophilized HAM and DAM membranes, along with 1-, 2-, and 3-layer AMP-coated mica discs, were sputter-coated with a thin layer of gold. The samples were then imaged using a JEOL JSM-6510LV scanning electron microscope to visualize surface topography and structural features of the membranes and AMP coatings.

[0203] Atomic force microscopy (AFM): All samples were measured under ambient conditions using a JPK NanoWizard 4 AFM system in quantitative imaging (QI) mode. Topography profiles and the corresponding Young's modulus maps were acquired using a RTES PA-300 probe (Bruker) with a spring constant of 40 N / m, a resonant frequency of 300 kHz,Atty. Dkt. No. 650053.01258and a tip radius of 8 nm. For each surface type, i.e., uncoated mica and mica coated with 1, 2, or 3 layers of AMP, three independent samples were prepared and measured. For each sample, three randomly selected 2x2 pm2regions were scanned to obtain both topography and stiffness maps using JPK data processing software. Specifically, surface roughness was quantified as the root mean square (RMS) roughness calculated by the following equation:"

[0205] Where Rq is RMS roughness, n is the number of data points, and z; is the height deviation of the i-th point from the mean line.

[0206] Young's modulus was calculated using Hertz model:

[0208] Where F is the indentation force, E is Young’s modulus, v is Poisson’s ratio, R is the probe tip radius, and 5 is the indentation depth. Each region was scanned in 128x128 pixels. For each surface type, data were collected from a total of nine regions (3 samplesx3 regions per sample), resulting in approximately 140,000 data points (3x3x128x128). Surface roughness and Young’s modulus were calculated for each scanned region, and values were averaged for all 9 regions of each sample type. Young's modulus of nanoparticles was analyzed through MATLAB based on the morphology and Young's modulus maps. Nanoparticle locations were identified by applying the height threshold at the interface between nanoparticles and substrate. The corresponding Young’s modulus data were extracted from the same location identified in nanoparticle morphology to investigate average modulus and standard deviation.

[0209] Contact angle measurement: Surface hydrophobicity was assessed using contact angle measurements using 1 x PBS (n = 3 for each surface type). For each measurement, a 10 pL droplet of 1 x PBS was deposited onto the sample surface. Side view' images of the droplet were captured at 40x magnification using an Aven Mighty Scope with MicroViewer DMC software. Contact angles were then quantified using Drop Shape Analysis plugin in ImageJ

[0087] ,

[0210] HUVECs culture and RNA extraction:Atty. Dkt. No. 650053.01258

[0211] Prepare DAM and AMP-coated plates: To prepare DAM-coated plates, lyophilized DAM was trimmed into circular discs with a diameter of 1.6 cm to fit the bottom of each well in a 12-well culture plate. Each DAM disc was carefully placed to fully cover the bottom surface of a well. To prepare AMP-coated plates, a 3-layer AMP coating was applied as previously described. Briefly, AMP powder was dissolved in ultra-pure distilled water at a concentration of 50 pg / mL and sonicated on ice using a Branson® Sonifier 150 to ensure complete dispersion. For each coating layer, 1 mL of the AMP solution was pipetted into a well of 12-well culture plate to fully cover the bottom surface and allowed to air dry at room temperature. This process was repeated two additional times to create a uniform 3-layer AMP coating. Following coating, both DAM-and AMP-coated plates were sterilized by soaking in 70% ethanol for 20 minutes. The wells were then rinsed three times with sterile phosphate-buffered saline (PBS) to remove residual ethanol and ensure sterility before further use.

[0212] Cell culture: Human umbilical vein endothelial cells (HUVECs, Lonza), at passage 3, were seeded onto the AMP-coated, DAM-coated, and plate control wells at a seeding density of IxlO5cells / well. The cells were cultured in endothelial growth medium (Lonza) under static conditions in a humidified atmosphere of 5% CO2 at 37°C. The culture medium was refreshed every two days to support optimal cell growth. The culture was maintained for a total of 7 days for subsequent analysis.

[0213] RNA extraction: After 7 days of cell culture, total RNA was extracted from cells in the AMP-coated, DAM-coated, and plate control groups using the RNeasy Plus Mini Kit (Qiagen), following the manufacturer’s protocol. The purified RNA was assessed for quantity and quality before sequencing.

[0214] RNA sequencing (RNA-Seq) was performed by Novogene Corporation Inc. (Sacramento, CA). Briefly, RNA-seq libraries were prepared using the KAPA Stranded RNA-Seq Library Prep Kit (Illumina) and sequenced by the NovaSeq 6000 platform (Illumina), generating paired-end reads. The raw reads were trimmed to remove 5' and 3' adaptor sequences using the Cutadapt software. The cleaned reads were then aligned to the human reference genome (hg38) using Hisat2 software. Transcript abundances were quantified with StringTie, and the expression levels of genes and transcripts were reported as fragments per kilobase of transcript per million mapped reads (FPKM). Data were further processed and filtered using the Ballgown R package to identify differentially expressed genes across experimental groups [88, 89],

[0215] Anti-inflammatory evaluation of AMP coating using LPS-stimulated HUVECsAtty. Dkt. No. 650053.01258

[0216] LPS-treated ECs culture: To induce inflammation, HUVECs (Lonza; passages 3-6) were treated with LPS (50 pg / mL; E. coli O11LB4) for 24 h. After washing and detachment, LPS-treated cells were seeded onto AMP-coated plates (AMP group), DAM-coated plates (DAM group), or uncoated plates (LPS control) at 30,000 cells / cm2Untreated HUVECs on uncoated plates served as the baseline group. Cells were cultured under static conditions in a humidified atmosphere of 5% CO2 at 37°C for 5 days with medium changes every 3 days. All experiments were performed in triplicate (n=3).

[0217] QRT-PCR: After 5 days of culture, total RNA was extracted from HUVEC using TRIzol™ Reagent (Invitrogen) following the manufacturer’s instructions. For each sample, 500 ng of total RNA was reverse transcribed into cDNA using the iScript Reverse Transcription Supermix (Bio-Rad) according to the manufacturer's protocol. The resulting cDNA was diluted five-fold, and qRT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) on a CFX Connect Real-Time System (Bio-Rad). Gene expression levels were normalized to the endogenous control GAPDH gene. Primer sequences used for qRT-PCR analysis are listed in the supplementary7data.

[0218] Immunoblotting: After 5 days of culture, cells from different groups were lysed in RIP A buffer (50 mM Tns-HCl, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) supplemented with a protease inhibitor cocktail (cOmplete™, EDTA-free, Roche). Protein concentrations were measured, and 50 pg from each sample was separated by SDS-PAGE using 12% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad). Proteins were transferred to PVDF membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Membranes were blocked for 1 h at room temperature in 3% nonfat dry milk prepared in TBST (20 mM Tris-HC1, 150 mM NaCl, 0.1% Tween-20), followed by overnight incubation at 4°C with primary antibodies on a shaker. The following primary antibodies were used: IL-6 (Invitrogen, #P620, 1 :2000) and GAPDH (Cell Signaling Technology7, #5174, 1 :5000). After three washes with TBST, membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies: anti-rabbit IgG (Invitrogen, #31460, 1:10000) or anti-mouse IgG (Invitrogen, #62-6520, 1:10000). Signals were detected using Clarity™ Max ECL Western Blotting Substrate (Thermo Fisher Scientific) and visualized on a ChemiDoc™ XRS system (Bio-Rad). Densitometric quantification of protein bands was performed using ImageJ software.

[0219] Immunofluorescence staining: After 5 days of culture, cells from different groups were washed twice with DPBS and fixed in 4% paraformaldehyde for 15 min at room temperature. Slides were then washed three times with DPBS (5 min each) and permeabilized with 0.25%Atty. Dkt. No. 650053.01258Triton X-100 in DPBS for 15 min at room temperature. To prevent nonspecific binding, cells were blocked with 20% goat serum and 3% BSA for 1 h at room temperature. Subsequently, cells were incubated overnight at 4°C with the indicated primary antibodies. After three washes with PBST (0.1% Tween-20 in PBS, 5 min each), cells were incubated for 1 h at room temperature in the dark with the appropriate fluorophore-conjugated secondary antibodies. Nuclei were counterstained with DAPI (if applicable). Following three final washes with PBST, slides were mounted using an antifade mounting medium and imaged using an ECHO Revolve Motorized Fluorescence Microscope.

[0220] 2.6. AMP coating of ePTFE grafts and coating stability under shear stress

[0221] AMP coating of ePTFE grafts: Commercial 6-mm inner diameter (ID) ePTFE grafts (BD, USA) were cut into 3-cm segments, rinsed with deionized (DI) water, and pretreated in 90% ethanol for 24 h. Grafts were then dried under nitrogen and subjected to oxygen plasma activation for 1 h to increase surface reactivity. Immediately after plasma treatment, grafts were incubated in a dopamine HCl solution (2 mg / mL in 10 mM Tris-HCl, pH 8.5) for 24 h at room temperature to generate a reactive polydopamine primer layer. For the first coating layer, grafts were fully immersed in 10% (w / v) poly (1,8-octamethylene citrate) (POC) prepolymer (dissolved in 100% ethanol) and cured at 100 °C for 48 h. After cooling and rinsing, a second AMP-containing layer was applied by circulating an AMP solution (50 mg / mL in 5% POC) through the graft lumen at 40 °C for 48 h to allow uniform AMP incorporation. Grafts were thoroughly washed with DI water to remove unbound material. To ensure continuous and stable AMP coverage across the entire luminal surface, a final thin AMP / POC layer was applied by static luminal filling and incubated under the same conditions. Coated grafts were rinsed, dried under sterile airflow, and stored at room temperature until use.

[0222] Evaluation of coating stability under shear stress: To assess coating stability, coated grafts were mounted in a perfusion chamber and exposed to physiological shear stress (10 mL / min, 37 °C) using PBS perfusion for 6 months and graft surfaces were examined by fluorescent imaging to assess coating retention.

[0223] EC durability under shear stress

[0224] HUVECs (passage 3-6) were seeded onto uncoated and AMP-coated ePTFE grafts at a density of -30,000 cells / cm2and cultured under static conditions for 3 days at 37 °C and 5% CO2Atty. Dkt. No. 650053.01258to allow initial cell attachment. After static culture, grafts were transferred to a closed-loop perfusion bioreactor system and perfused with endothelial growth medium at a flow rate of 10 mL / s. maintaining physiological temperature (37 °C) and pH. Perfusion was continued for 3 days to evaluate endothelial durability under sustained shear stress. Cell attachment and viability were tested by the Live / Dead fluorescence staining according to the manufacturer’s instructions.

[0225] In vivo hemocompatibility evaluation in pigs

[0226] As a proof of concept, the inventors assessed the in vivo hemocompatibility of AMP-coated ePTFE grafts using a Yorkshire pig carotid artery implantation model. AMP-coated grafts were implanted into the carotid arteries and compared directly to clinically used unmodified ePTFE grafts (Becton, Dickinson and Company, BD; inner diameter = 6 mm). Both graft types were implanted under identical surgical conditions and harvested after 1 month to evaluate graft patency, thrombosis, endothelialization, and overall hemocompatibility7.

[0227] Results:

[0228] AMP fabrication and characterization of AMP coating

[0229] SEM: After decellularization, the DAM scaffolds exhibited effective removal of cellular components while preserving the native collagen architecture, as confirmed by SEM imaging (Fig. 1 A). To generate nanoparticles, DAMs were lyophilized, milled into a fine powder, enzymatically digested to break down extracellular matrix components, and then precipitated to obtain AMPs. AMP-coated surfaces were prepared by applying one, two. or three coating layers (50 pg / mL each), followed by air-drying. SEM analysis revealed that particle density increased proportionally with the number of coating layers, with particles evenly distributed across the surface (Fig. 1A).

[0230] FTIR Analysis: To evaluate compositional and structural changes during the conversion of HAM into AMPs, the inventors performed FTIR spectroscopy (Fig. IB). Comparative spectra of native HAM, digested DAM, and final AMPs revealed a progressive loss of key functional groups throughout processing. The broad N-H / O-H stretch (-3300 cm '). typically associated with hydroxyl and amine groups in amino acids and proteins, and the C-H stretch (-2950related to aliphatic side chains in proteins and lipids, both showed significant decreases across the processing stages. Protein-associated amide peaks, including amide I (1600-1700 cm '). amide II (1500-1600 cm '). and amide III (1200-1300 cm '), wereAtty. Dkt. No. 650053.01258progressively attenuated in DAM and AMPs, indicating the loss or disruption of protein secondary' structure and peptide backbone integrity. Additionally, the C-0 stretching band (-1040 cm '). commonly attributed to carbohydrates such as polysaccharides and glycoproteins, was substantially reduced in the final AMPs, suggesting degradation or removal of carbohydrate components during processing.

[0231] AFM characterizations: AFM analysis (Fig. 2A) revealed that AMP-coated surfaces (1-, 2-, and 3-layer) exhibited significantly higher RMS roughness (Fig. 2C) and lower Young’s modulus (Fig. 2D) compared to uncoated controls (p < 0.001). The RMS roughness of uncoated samples was 0.74 ± 0.13 nm, whereas AMP coatings increased roughness to 3.21 ± 0.79 nm (1-layer), 4.04 ± 2.91 nm (2-layer), and 4.72 ± 2.50 nm (3-layer). Despite these increases, no statistically significant differences in RMS roughness were observed among the three AMP coating thicknesses. Similarly, the Young’s modulus of uncoated mica was 24.49 ± 16.26 GPa. which was significantly reduced in AMP-coated samples to 4.24 ± 0.98 GPa (1-layer), 4.70 ± 2.87 GPa (2-layer), and 3.83 ± 0.60 GPa, with no significant differences among coating thicknesses.

[0232] Contact angle testing: Contact angle measurements demonstrated significant differences in surface hydrophobicity between uncoated and AMP-coated samples (p < 0.001; Fig. 2B). Uncoated samples exhibited the highest contact angle (67.67 ± 5.47°), indicating greater hydrophobicity7. In contrast, AMP-coated surfaces showed markedly reduced contact angles of 50.39 ± 1.41° (1-layer), 47.96 ± 2.52° (2-layer), and 47.12 ± 4.71° (3-layer), with no statistically significant differences among the coated groups (Fig. 2E). These results indicate that AMP coatings substantially increase surface hydrophilicity compared to the uncoated mica surface.

[0233] Endothelial activity and function on AMP coating

[0234] AMP coating showed similar endothelial function as DAM: To test whether AMP coatings preserve the key endothelial-supportive functions of DAM, HUVECs were cultured at equal densities on 3-layer AMP coated surface, DAM, and standard culture plate. HUVECs on AMP and DAM surfaces both showed increased cell proliferation and migration relative to standard culture plates (Fig. 3 A). After 7 days, RNA sequencing demonstrated that AMP coatings effectively replicated the endothelial-supportive properties of DAM. HUVECs on AMP coating showed gene expression patterns similar to those on DAM, with largely overlap in significantly upregulated and downregulated genes (Fig. 3B).Atty. Dkt. No. 650053.01258

[0235] AMP coating attenuates LPS-induced inflammation

[0236] To evaluate the protective efficacy of AMP coating against endothelial dysfunction. LPS-induced ECs were cultured on 3-layer AMP coated surface, DAM, and standard culture plate and compared with normal ECs. LPS exposure caused a significant increase in the expression of CXCL10 (Fig. 4A), IL-ip (Fig. 4B), IL-6 (Fig. 4C), TNF-a (Fig. 4D), and MMP3 (Fig. 4E), confirming a strong inflammatory response. In contrast, LPS-induced ECs on AMP-coated surfaces exhibited substantially attenuated cytokine induction, with expression levels remaining close to baseline (<2-fold).

[0237] Immunoblot (Fig. 5A) and immunofluorescence analyses (Fig. 5B) further confirmed lower IL-6 and MMP3 protein expression in LPS-induced ECs were cultured on 3-layer AMP coated surface, supporting the anti-inflammatory and cytoprotective roles of AMPs. These results suggest that AMP coating may help stabilize endothelial homeostasis by limiting NF-KB-dependent transcriptional activation and subsequent inflammatory' pathways.

[0238] AMP coating stability on ePTFE grafts

[0239] The inventors have established a coating protocol including cleaning, plasma activation, and dopamine priming, followed by two-layer POC coating (Fig. 6A). SEM of uncoated ePTFE showed fibrous structure (Fig. 6B) and AMP coating uniformly covered the fibrous, porous structure (Fig. 8C). AMP coating also converted the graft surface from hydrophobic to hydrophilic. Both uncoated and AMP-coated grafts exhibited minimal platelet adhesion, indicating that AMP surface preserves hemocompatibility.

[0240] To access coating stability under shear stress, Texas Red-labeled AMPs were uniformly coated to ePTFE grafts and exposed to continuous PBS flow (10 mL / min, 37 °C) (Fig. 7A). Fluorescence monitoring showed a gradual decrease in signal (Fig. 7B), indicating slow AMP degradation with coating stability' maintained for more than six months (Fig. 7C)

[0241] EC durability under shear stress

[0242] HUVECs were seeded at equal densities on uncoated and AMP-coated ePTFE grafts under static culture for 5 days and both showed initial EC attachment (Fig. 8A,B). After 3 days of culture under continuous flow in a bioreactor 8, uncoated grafts showed poor EC retention (Fig.8C,D, top), whereas AMP-coated grafts preserved substantially more viable ECs and exhibitedAtty. Dkt. No. 650053.01258well-defined junctions on TEM (yellow arrows; Fig. 8C,D, bottom), indicating improved endothelial durability under physiological shear.

[0243] In vivo hemocompatibility evaluation in pigs

[0244] As a proof of concept, the inventors evaluated the in vivo hemocompatibility of AMP-coated ePTFE grafts by implanting them into the carotid arteries of Yorkshire pigs, using uncoated ePTFE grafts (ID=6mm) on the contralateral side as controls (Fig. 9A). After 1 -month of implantation, ultrasound imaging showed that AMP -coated grafts maintained clear and open lumens (Fig. 9B, middle), whereas uncoated grafts exhibited lumen narrowing (Fig. 9B, right). Gross examination confirmed that AMP -coated grafts (Fig. 9C, top, black arrows) remained open lumen, while uncoated grafts (Fig. 9C, bottom, white arrows) displayed pronounced NIH (yellow arrows) extending from the proximal anastomosis to mid-graft.

[0245] Conclusion

[0246] Systematic material characterization confirmed that AMPs can be reliably fabricated from DAM and applied as uniform nanoscale coatings that increase surface roughness, reduce stiffness, and enhance hydrophilicity — features that support endothelial attachment and function. AMP-coated surfaces promoted endothelial proliferation, migration, and homeostatic gene regulation comparable to DAM, while effectively attenuating LPS-induced inflammatory responses at both the transcriptional and protein levels. When applied to ePTFE grafts, AMP coatings were stable under prolonged shear stress, maintained hemocompatibility, and significantly improved endothelial retention and junctional integrity during flow exposure. Importantly, in a porcine carotid implantation model, AMP-coated grafts preserved luminal patency and reduced neointimal hyperplasia relative to uncoated controls. Together, these results establish AMPs as a robust, durable, and biologically active coating technology capable of enhancing vascular graft integration and mitigating complications associated with synthetic small-diameter grafts.

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Claims

1. Atty. Dkt. No. 650053.01258CLAIMS1. Nanoparticles comprising at least one of hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing.

2. The nanoparticles of claim 1, wherein the nanoparticles comprise hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, and heat shock protein 90 (HSP 90), or a fragment of each of the foregoing proteins.

3. The nanoparticles of claim 1 or 2, wherein the nanoparticles comprise decellularized amniotic membrane.

4. The nanoparticles of claim 1, wherein the nanoparticle further comprises a payload.

5. The nanoparticles of claim 4, wherein the payload comprises a dye, a therapeutic, or both a dye and a therapeutic.

6. The nanoparticles of claim 5, wherein the dye comprises a contrast dye.

7. The nanoparticles of claim 6, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.

8. The nanoparticles of claim 5, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.

9. The nanoparticles of claim 8, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.

10. The nanoparticles of claim 8, wherein the polynucleotide comprises an siRNA, an mRNA, a polynucleotide comprising a gRNA and / or a Cas protein, optionally, the polynucleotide may comprise YAP, LMNB2, LMNB1, LMNA, E2F6, E2F7, E2F8, TGF-beta related genes, or any combination thereof.

11. Nanoparticles produced by a method comprising decellularizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularizedAtty. Dkt. No. 650053.01258membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate nanoparticles from decellularized amniotic membrane.

12. The nanoparticles of claim 11, wherein the method of nanoparticles are crosslinked.

13. The nanoparticles of claim 11 or 12, wherein the amniotic membrane is human amniotic membrane.

14. The nanoparticles of claim 11, wherein the nanoparticles further comprise a pay load.

15. The nanoparticles of claim 14, wherein the payload comprises a dye, a therapeutic, or both a dye and a therapeutic.

16. The nanoparticles of claim 15, wherein the dye comprises a contrast dye.

17. The nanoparticles of claim 16, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.

18. The nanoparticles of claim 15, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.

19. The nanoparticles of claim 18, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.

20. The nanoparticles of claim 11, wherein the nanoparticles comprise at least one protein selected from hemoglobin subunit beta, hemoglobin subunit alpha, hemoglobin subunit gamma, serum albumin, chorionic somatomammotropin hormone 2, cytoplasmic actin, fibronectin, or heat shock protein 90 (HSP 90), or a fragment of at least one of the foregoing proteins.

21. The nanoparticles of claim 1 or 11 , wherein the nanoparticles have a mean diameter of about 100 nm to about 150 nm.

22. The nanoparticles of claim 21, wherein the nanoparticles have a mean diameter of about 120 nm or about 117 nm.Atty. Dkt. No. 650053.0125823. The nanoparticles of claim 1 or 11, wherein the nanoparticles have a positive zeta potential.

24. The nanoparticles of claim 23, wherein the nanoparticles have a zeta potential of about 1 to about 10 mV or about 8.5 mV.

25. A pharmaceutical composition comprising the nanoparticles of any one of the preceding claims.

26. A method of generating nanoparticles, the method comprising decellul arizing amniotic membrane, lyophilizing the decellularized membrane, milling the lyophilized decellularized membrane, digesting the milled lyophilized decellularized membrane, preparing an aqueous solution comprising the digested milled lyophilized decellularized membrane, and desolvating the aqueous solution to generate the nanoparticles.

27. The method of claim 26, wherein decellularizing amniotic membrane comprises contacting the amniotic membrane with at least one detergent for a sufficient amount of time to decellularize the amniotic membrane.

28. The method of claim 27, wherein the at least one detergent comprises a non-ionic detergent or an ionic detergent or both a non-ionic detergent and an ionic detergent.

29. The method of claim 26, wherein digesting the milled lyophilized decellularized membrane comprises pre-treating the milled lyophilized decellularized membrane in an aqueous solution in contact with an acid.

30. The method of claim 29, wherein digesting the milled lyophilized decellularized membrane comprises contacting the milled lyophilized decellularized membrane with a protease for a sufficient amount of time to generate an aqueous solution comprising the digested milled lyophilized decellularized membrane.

31. The method of claim 26. wherein desolvating the aqueous solution comprises contacting the aqueous solution with apolar aprotic solvent.

32. The method of claim 31, wherein the polar aprotic solvent comprises acetone.Atty. Dkt. No. 650053.0125833. The method of claim 26, wherein the method further comprises crosslinking the nanoparticles.

34. The method of claim 33, wherein crosslinking the nanoparticles comprises contacting the aqueous solution with a crosslinking agent.

35. The method of claim 34. wherein the crosslinking agent comprises or consists of glutaraldehyde.

36. The method of claim 26, wherein the method further comprises adding a payload to the aqueous solution comprising the digested milled lyophilized decellularized membrane.

37. The method of claim 36, wherein the payload comprises a dye, a therapeutic, or both a dye and a therapeutic.

38. The method of claim 37, wherein the dye comprises a contrast dye.

39. The method of claim 38, wherein the contrast dye comprises an iodinated contrast dye or a gadolinium contrast dye.

40. The method of claim 37, wherein the therapeutic comprises a small molecule pharmaceutical, a polynucleotide, or a polypeptide.

41. The method of claim 40, wherein the small molecule pharmaceutical comprises an ACE inhibitor, a beta blocker, or a vasodilator.

42. The method of claim 40, wherein the polynucleotide comprises an siRNA, or a polynucleotide comprising agRNA and / or a Cas protein.

43. A nanoparticle made by the method of any one of claims 26-42.

44. A method comprising contacting a cell with the nanoparticles of claim 1 or 11.

45. A method comprising administering the pharmaceutical composition of claim 25 to a subject.Atty. Dkt. No. 650053.0125846. A method of reducing ischemic injury to a cell, the method comprising contacting the cell with the nanoparticles of claim 1, wherein the nanoparticles comprise a payload that reduces ischemic injury’.

47. A method of treating ischemic injury in an organ of a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 25 to the subject to treat the ischemic injury in the subject, wherein the pharmaceutical composition comprises nanoparticles comprising a payload that reduces ischemic injury.

48. A kit comprising the nanoparticle of claim 1 or the pharmaceutical composition of claim 25.

49. The kit of claim 48, further comprising instructions for using the kit in any of the methods disclosed herein.

50. A composition coated with the nanoparticles of claim 1 or 11.

51. The composition of claim 50, wherein the composition is coated with at least one layer of the nanoparticles.

52. The composition of claim 51, wherein the composition is coated with two layers of the nanoparticles.

53. The composition of claim 50, wherein the composition comprises at least one primer layer underneath the coating of the nanoparticles.

54. The composition of claim 53. wherein the at least one primer layer comprises:(a) a polydopamine layer; or(b) a poly 1,8-octamethylene citrate (POC) layer.

55. The composition of claim 53. wherein the at least one primer layer comprises a first primer layer comprising the polydopamine layer and a second primer layer comprising the POC layer, wherein the second primer layer is located on top of the first primer layer.

56. The composition of claim 55, wherein the nanoparticle coating is located on top of the second primer layer.Atty. Dkt. No. 650053.0125857. The composition of claim 50, wherein the composition comprises a medical device.

58. The composition of claim 57, wherein the medical device comprises a vascular graft.

59. The composition of claim 50, wherein the composition comprises a plastic.

60. The composition of claim 59. wherein the plastic comprises polytetrafluoroethylene (PTFE).

61. A method of generating a coated composition, the method comprising applying the nanoparticles of claim 1 or 11 to the composition to generate the coated composition.

62. The method of claim 61, wherein applying the nanoparticles comprises contacting the composition with a solution of the nanoparticles.

63. The method of claim 62, wherein the solution of the nanoparticles is an aqueous solution.

64. The method of claim 61, wherein the solution of the nanoparticles comprises about 0.1 mg / ml to about 100 mg / ml of the nanoparticles.

65. The method of claim 61, wherein the solution of the nanoparticles further comprises poly 1,8-octamethylene citrate (POC).

66. The method of claim 65, wherein the POC comprises about 1% to about 10% POC.

67. The method of claim 66, wherein the POC comprises about 5% POC.

68. The method of claim 61, wherein the method comprises applying at least one primer layer prior to applying the nanoparticles.

69. The method of claim 68, wherein the at least one primer layer comprises(a) a polydopamine layer; or(b) a poly 1,8-octamethylene citrate (POC) layer.

70. The method of claim 69, wherein the method comprises applying a first primer layer comprising the poly dopamine layer and a second primer layer comprising the POC layer, wherein the second primer layer is located on top of the first primer layer.

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