EP4702059A1 - N-cadherin targeting chimeric peptides for inhibiting restenosis - Google Patents

Abstract

Novel chimeric peptides comprise an N-cadherin binding domain for binding N-cadherin, which is expressed on the surface of cells, in particular smooth muscle cells, and a fibronectin- binding domain, which binds surrounding extracellular matrix and a binding site for fibronectin. The chimeric peptides can be loaded on to nanoparticles, suitably degradable polar hydrophobic ionic polyurethane (D-PHI) nanoparticles for delivery, which can be incorporated into coatings for medical devices, including stents and balloons.

Description

N-CADHERIN TARGETING CHIMERIC PEPTIDES FOR INHIBITING RESTENOSIS

CROSS-REFERENCE TO RELATED APPLICATIONS AND DOCUMENTS

[0001] This application claims priority to United States provisional patent application number 63/462,427, filed April 27, 2023, the contents of which are incorporated herein by reference. This application also comprises a sequence listing in electronic form which is also incorporated in its entirety.

TECHNICAL FIELD

[0002] This disclosure relates to chimeric peptides and inhibiting restenosis, particularly in the context of angioplasty, stenting, and bypass grafting.

BACKGROUND OF THE ART

[0003] Heart-related complications, including stroke and other cardiovascular disease, are a leading cause of death globally. Coronary artery disease or coronary heart disease are caused by a build-up of plaque in the coronary artery leading to narrowing and hardening of the artery. Patients with occluded arteries are treated with angioplasty, stenting, or bypass grafting. Drug eluting stents and balloons are the current standard of treatment to reverse vessel occlusion, with stents being used more in coronary artery disease, and balloon angioplasty used to treat peripheral artery disease (PAD). These strategies are limited by restenosis, thrombosis, and graft failure. In-stent restenosis is a common complication of treatment, and in many cases is treated with balloon angioplasty to relieve the occlusion within the stent without introducing another stent. The current generation of drug-eluting stents can reduce restenosis, but the pharmacological agents they release (such as paclitaxel) indiscriminately induce cell death at the site of application. While the newer generation of “- limus” drugs including Everolimus and Zotarolimus may have superior efficacy and safety compared to earlier pharmacological agents, there remains a need for alternative treatment options. While killing smooth muscle cells (SMCs) may have a beneficial effect of reducing neointima formation, it can destabilize existing plaques, and killing endothelial cells (ECs) prevents them from creating an anti-thrombotic lining for the vessel wall, increasing the risk of thrombosis. In-stent thrombosis is more common in patients with drug eluting stents compared to bare metal stents. Drug-eluting stents reduce early restenosis by inhibiting SMC proliferation and migration, but because current drugs are cytotoxic they impair endothelial cell (EC) repair, causing late stent thrombosis. BRIEF SUMMARY

[0004] In one aspect, there is provided a chimeric peptide that includes an N-cadherin binding domain and a domain that binds a component of the extracellular matrix, wherein the chimeric peptide inhibits smooth muscle cell migration.

[0005] In various embodiments, the domain that binds a component of the extracellular matrix comprises an amino acid sequence selected from the group consisting of:

LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10);

LTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 11);

KLTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 12);

KPSYGFGGHNSVDFEEDTLPKV (SEQ ID NO: 13);

ETVTIVEDTRPKLVFHFDNNEPKVE (SEQ ID NO: 14);

KLKSQLVKRK (SEQ ID NO: 15);

KNGRYSISR (SEQ ID NO: 16);

KVGKSPPVR (SEQ ID NO: 17);

KTFGKMKPR (SEQ ID NO: 18);

STMMSRSHKTRSHHV (SEQ ID NO: 19);

AAYLEQLN (SEQ ID NO: 20);

GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 21); and WREPSFCALS (SEQ ID NO: 22); or a functional fragment or variant thereof, preferably the amino acid sequence LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10) or a functional fragment or variant thereof.

[0006] Also provided is a chimeric peptide that includes an N-cadherin binding domain and a fibronectin-binding domain, preferably a fibronectin binding domain derived from a bacterial source, more preferably from the species Staphylococcus aureus, in some embodiments, an amino acid sequence selected from: LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10); LTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 11); KLTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 12); and KPSYGFGGHNSVDFEEDTLPKV (SEQ ID NO: 13).

[0007] In some embodiments, the N-cadherin binding domain comprises the HAV binding motif, preferably LRAHAVDING (SEQ ID NO: 23). In some embodiments, the chimeric peptide includes a N-cadherin binding domain comprising or consisting of an amino acid sequence selected from CLRAHAVDING (SEQ ID NO: 24) and a fibronectin binding domain comprising or consisting of LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10). In one embodiment, there is provided a chimeric peptide having a structure shown in the figures.

[0008] Also provided is a nanoparticle that includes a chimeric peptide as provided herein. The nanoparticle may comprise, consist or consist essentially of a Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI), and the chimeric peptide. The nanoparticle may have alternating layers of polymer and the chimeric peptide.

[0009] Also provided is a method of making a nanoparticle as provided herein that includes depositing alternating layers of a cationic polymer, suitably poly-L-lysine or poly-L- histidine, and the chimeric peptide.

[0010] In one embodiment, there is provided coating for a medical device that includes a chimeric peptide or a nanoparticle as provided herein. The coating may further include polyethylene oxide (PEO), which may have a molecular weight of between about 50,000 and about 200,000. In some embodiments, the coating is a composite coating comprising a peptide-containing layer that includes the chimeric peptide and nanoparticles for application to a surface of the medical device and a protective layer for application over the peptide- containing layer, preferably wherein the protective layer is or includes PEO, polyethylene glycol (PEG) or hyaluronic acid.

[0011] The chimeric peptide, the nanoparticle or the coating may be for used in inhibiting migration of smooth muscle cells (SMCs).

[0012] Also provided is a medical device (e.g. a stent or angioplasty balloon) that includes a coating as provided herein. The medical device may include a protective layer as described; in one embodiment, the average weight of the protective coating per square millimeter is between about 0.1 pg/mm² and about 1 pg/mm², preferably about 0.5 pg/mm² and/or wherein at least 80%, at least 90% or at least 99% of the protective coating dissolves within 4 minutes under physiological conditions.

[0013] Also provided is a method for inhibiting deposition of thrombotic material on an implantable medical device for implantation in a patient in need thereof (e.g. for the treatment of atherosclerosis or restenosis), by applying a coating as provided herein to a surface of the medical device. In some embodiments, the method includes applying the peptide-containing layer by a pH-driven coating method and/or applying the protective layer by spray coating, ultrasonic spray coating, spin-coating, dip-coating, or micropipetting. [0014] Also provided is a method for treating restenosis in a subject, comprising implanting a medical device as provided herein in a blood vessel of the subject. In some embodiments, the restenosis has occurred after angioplasty or vascular stent placement In some embodiments, the subject has coronary artery disease, coronary heart disease, peripheral artery disease, or dysfunctional arteriovenous (AV) fistula. In some embodiments, the blood vessel is a coronary artery, peripheral artery, or AV fistula.

[0015] Also provided is a nanoparticle for peptide delivery comprising, consisting or consisting essentially of a Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI).

[0016] Also provided are compositions for peptide delivery that include nanoparticles as provided herein and optionally a carrier, the nanoparticles having an average radii between about 20 nm and about 150 nm, preferably between about 30 nm and about 110 nm, and/or a polydispersity index of less than about 0.1 as measured by Dynamic Light Scattering (DLS), preferably less than about 0.05 as measured by DLS.

[0017] Also provided is a medicament that includes a therapeutically effective amount of a chimeric peptide as provided herein and a nanoparticle capable of binding the chimeric peptide under physiological conditions. The chimeric peptide and the nanoparticle may be for administration sequentially or concurrently.

[0018] Also provided is a method of treating restenosis in a subject by administering a therapeutically effective amount of a chimeric peptide, a composition or a medicament as provided herein to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 illustrates N-cadherin targeting peptide design and sequences according to embodiments of the present invention; chimeric peptides include a fibronectin binding domain derived from staph, aureaus FnbpA.

[0020] Figure 2 shows N-cadherin targeting chimeric peptide inhibited migration of rodent SMCs without reducing viability or inducing apoptosis. Per Example 1 , scratch-wounded SMCs were treated with N-cadherin targeting peptides or scrambled control peptides and imaged after 24h for MOVAS (A) or 48h for RNISMC (B). The area of the wound gap was subtracted from and divided by the initial gap area to give a percentage of the wound that had closed. For viability measurements, MOVAS (C) or RNISMC (D), were treated with N-cadherin targeting chimeric peptides or scrambled control peptides and allowed to proliferate for 24h. Metabolic activity was assessed by colorimetric change of WST-1 , measured as absorbance at 450nm, with background at 610nm subtracted. Apoptosis was assessed by staining for cleaved caspase 3 in MOVAS treated with N-cadherin targeting peptides or scrambled controls (E). Scrambled control peptide data were pooled. Two-way ANOVA tests were used to determine statistical significance between multiple conditions. Data shown are means ± SEM, n>3 biological replicates.

[0021] Figure 3 shows N-cadherin targeting chimeric peptide inhibited the polarization of wound-edge SMCs. Per Example 1 , MOVAS were scratched and treated with 500 pg/mL short, cyclic or N-cadherin targeting chimeric peptide for 6h. A) Cells were fixed and stained for y-tubulin to label the MTOC, indicated by the white arrow. B) The position of the MTOC was determined relative to a centreline through the nucleus parallel to the wound gap for each cell per field of view. C) The percentage of polarized cells per field of view are shown with means ± SEM in n>6 biological replicates. Scrambled control peptide data were pooled. Two- way ANOVA with multiple comparisons were used to determine statistical significance, * p<0.05.

[0022] Figure 4 shows N-cadherin targeting chimeric peptide inhibited migration of human SMCs, but not ECs, and did not impact viability. Per Example 1 , scratch-wounded HASMCs (A) or HUVECs (B) were treated with N-cadherin targeting chimeric peptide or scrambled control peptide and imaged after 48h. The area of the wound gap was subtracted from and divided by the initial gap area to give a percentage of the wound that had closed. For viability measurements, HASMCs (C) or HUVECs (D) were treated with N-cadherin targeting chimeric peptide or scrambled control peptide and allowed to proliferate for 24h. Metabolic activity was assessed by colorimetric change of WST-1 , measured as absorbance at 450nm, with background at 610nm subtracted. Scrambled control peptide data were pooled. Two-way ANOVA tests were used to determine statistical significance between multiple conditions. Data shown are means ± SEM, n>3 biological replicates.

[0023] Figure 5 shows N-cadherin targeting chimeric peptide was co-localized in vitro with fibronectin and was found in vivo in balloon-injured rat carotid arteries. Per Example 1 , RNISMCs were treated for 24 hours with TAMRA-labelled N-cadherin targeting chimeric peptide (A) or scrambled control peptide (B). Cells were fixed and stained with DAPI (nuclei), phalloidin (actin) and anti-fibronectin and imaged by confocal microscopy. Rat carotid arteries were balloon-injured and infused with TAMRA-labelled N-cadherin targeting chimeric peptide (C) or phosphate buffered saline (PBS) only (D) for 5 minutes and allowed to recover for 24h. Vessels were perfusion-fixed with paraformaldehyde and prepared en face for confocal microscopy. [0024] Figure 6 shows N-cadherin targeting chimeric peptide inhibited intimal thickening in rat carotid arteries without impairing re-endothelialization. Per Example 1 , carotid arteries were subject to balloon catheter denudation of the endothelium then the carotids were locally infused with N-cadherin targeting chimeric or scrambled control peptides for 5 minutes. Animals were allowed to recover for 1 or 2 weeks, then the carotids were perfusion-fixed and artery sections stained with H&E for histological analysis (A), and Intimal-to-medial ratio was calculated (B). For scrambled control and N-cadherin peptide n=6 at 1 week and n=7 and 9 respectively for 2 week intimal thickening experiments. C) 2 weeks following injury and peptide treatment, animals were injected with Evan’s Blue dye to measure re-endothelialization, and photomicrographs of stained vessels were used to measure blue stained and unstained areas. D) The percentage of unstained area, a measure of endothelial cell coverage, was compared between groups. N=6 for scrambled control and 5 for N-cadherin targeting peptide treated groups. Data shown are means ± SE, *p<0.05.

[0025] Figure 7 shows scrambled control peptides did not impact cell migration, viability or polarization. Per Example 1 , MOVAS cells were treated with scrambled control peptides, which had identical amino acid residues as the corresponding active N-cadherin mimetic peptides, but in a randomized sequence. In MOVAS, migration measured via scratch wounding (A), viability measured by WST-1 (B), or polarization measured by MTOC position (C) were assessed following treatment with peptides or an equal volume of vehicle (DMSO). In RNISMCs, migration and viability were assessed in the identical manner. Data are means ± SEM, n>3 biological replicates.

[0026] Figure 8 shows treatment of injured vessels with N-cadherin targeting chimeric peptide did not impact cell proliferation. Per Example 1 , A) Cross-sections of injured carotid arteries treated with peptides were stained for Ki67 as a marker of proliferative cells (arrows). B) Positive and negative stained cells were counted in each anatomical layer of the vessel and used to calculate the percentage cells positive for Ki67. For scrambled control treated n=3, for N-cadherin peptide treated n=3 for 1 week and 5 for 2 week experiments. Data are means ± SEM.

[0027] Figure 9 shows a schematic for the synthesis of Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI), per Example 2.

[0028] Figure 10 shows the fabrication process D-PHI particles via emulsion inversion polymerization, per Example 2b. [0029] Figure 11 shows a typical distribution of particle radii, analyzed using dynamic light scattering (DLS) methods (DelsaMax Pro), before and after subsequent centrifugation steps, per Example 2c. Stock Nanoparticles: solution of nanoparticles in MilliQ prior to any centrifugation. Post 2K RCF: stock: Solution of particles after 5 minutes of centrifuging at 2000 RCF. Post 20k RCF (x5): Solution of particles after 5 minutes of centrifuging at 2000 RCF and 5 minutes at 4°C at 20 000 RCF 5 times. Subsequent 20k RCF: Solution of particles analyzed after subsequent 20 000 RCF centrifugations.

[0030] Figure 12 shows a representative graph from the NanoSight nanoparticle tracking analysis (NTA)analysis of a stock of D-PHI Nanoparticle solution per Example 2c. The graph displays the size of the particles detected as well as the concentration of each particle size. Total concentration included accounting for dilution in sample preparation.

[0031] Figure 13 represents the image of D-PHI nanoparticles (NPs) obtained using transmission and scanning electron microscopy (TEM and SEM), per Example 2c. All scale bars are 500 nm. A) Typical TEM image of D-PHI NPs. Particles ranged from 100-300 nm in diameter. C) Zoomed TEM image of a single D-PHI NPs. B) Representative SEM image of D- PHI NPs. D) Close up SEM image of individual D-PHI Nanoparticles.

[0032] Figure 14 shows A) STEM Image of D-PHI PU nanoparticles taken at 100 OOOx magnification as measured on a Hitachi S-5200; B) Average diameter and zeta potential of blank and coated particles as measured on the DelsaMax Pro Zeta Potential DLS Analyzer.

[0033] Figure 15 shows a compilation of DLS data of D-PHI NPs overtime, as discussed in Example 2f. Three different temperatures were used to store the NPs: CT - 4 °C, RT - 23 °C and AT - 60 °C. N = 3 with n=3 technical replicates.

[0034] Figure 16 shows A) Relative metabolic activity and DNA content of A10 cells as a % of growth medium (negative) control after 24-hour exposure to D-PHI nanoparticles per Example 2d, values shown are the mean ±SE. N=3, n=3; and B) Relative hemolysis of RBCs when exposed to D-PHI nanoparticles as a % of Triton x-100 hemolytic control, per Example 2e. Values represent the mean ± standard deviation. N=3, n=3.

[0035] Figure 17A shows a schematic of the structure of the model N-cadherin peptide according to an embodiment of the present invention, and as discussed in Example 3a.

[0036] Figure 17B shows a structure of a chimeric peptide according to an embodiment.

[0037] Figure 18 shows a schematic of how to generate various embodiments of peptide- coated nanoparticles, with or without multilayering to optimize peptide loading. A) depicts peptide coating using the base nanoparticle formulation. B) depicts peptide loading using optimized base nanoparticles. C) shows a charge-driven approach for peptide loading utilizing a cationic polymer to facilitate a single peptide coating layer. D) shows a multi-layer peptide loading approach using alternative layers of poly-L-lysine and peptide to increase loading potential.

[0038] Figure 19 shows representative images of single layer D-PHI nanoparticles (A-C) and fluorescently labelled chimeric n-cadherin peptide alone (D-F) taken by the FluoView FV3000 using the 640 nm channel and differential interference contrast technique, per Example 3d. Images were taken within 48 hrs of preparation. In F, the white circle indicates a region where no peptide was detected, and the black arrows denote potential peptide aggregates.

[0039] Figure 20 shows A) The size and zeta potential of single and multilayer particles analyzed using the DelsaMax Pro (Beckman Coulter), coupled with a DLS and phase analysis light scattering apparatus. Experiments were conducted with N=3, n=3 replicates, the error bars represent standard deviation on the mean. B) Merged differential interference contrast and laser scanning confocal microscopy images of uncoated D-PHI nanoparticles. C) Merged differential interference contrast and laser scanning confocal microscopy images of D-PHI nanoparticles after being coated with peptide tagged with a TAMRA fluorophore. The scale bar is 15 pm. Figure 20 is discussed in Example 3d and Example 3e.

[0040] Figure 21 shows A) Graph of fluorescence intensity of chimeric N-cadherin peptide ranging from 0.1 - 500 pg/mL; and B) Representative image of an optimized standard curve generated for quantifying the fluorescence of the chimeric N-cadherin peptides in solution from 0.08 - 1 pg/mL. n= 2 N= 3, per Example 3i. Error bars represent standard deviation.

[0041] Figure 22 shows A) Representative graph of reverse phase HPLC analysis of chimeric N-cadherin in release media (PBS); and B) Standard curve of triplicate injections of various amounts of peptide, from 1 .25 - 20 pg N=3, n=3, per Example 3i. Error bars represent standard deviation.

[0042] Figure 23 shows the fluorescent chimeric N-cadherin peptide release study in PBS per Example 3f. A) is the early release in hours where 100% free peptide passes the membrane within 1 hour whereas the peptide on the loaded particles has a delayed release; and B) is a graph that shows that it requires 3 days for release of 100% of the peptide from the nanoparticles. N=3, n=3, Error bars represent standard deviation. * p < 0.05, ** p < 0.001. [0043] Figure 24 shows the relative metabolic activity and DNA content of MOVAS cells as a percentage of growth medium control after 24-hour exposure to D-PHI nanoparticles, per Example 3g. Values shown are the mean ± standard deviation. N=3, n=3.

[0044] Figure 25 shows a representative well in a 24 well plate used in a scratch assay per Example 3h. The shaded portions represents the areas where cells are. The white represents regions where the cell monolayer has been disrupted by scratching a line using a 200 pL pipette tip. The squares represents the four regions where the wound areas were assessed or the four technical replicates per experiments.

[0045] Figure 26 shows A) Different concentrations of peptide delivered on D-PHI NPs were used in a scratch assay with mouse vascular aortic smooth muscle cells cultured in cell growth media N=3, n=4, error bars represent standard deviation; and B) The scratch assay was repeated with free peptide and peptide delivered on D-PHI nanoparticles. The scratch assay performed with mouse vascular aortic smooth muscle cells cultured in cell growth media at pH 7.4, 37°C. N=3, n=4. Error bars represent standard deviation, * P< .05. This is discussed in Example 3h.

[0046] Figure 27 shows optimization of poly-L-lysine (PLL) concentration required to coat the nanoparticles resulting in optimal particle size and zeta potential based on surface chemistry, avoiding aggregation and cytotoxic effects on cells. Size and zeta potential measurements were evaluated by DLS. PLL concentration of 0.25 mg/mL was selected to fully coat the nanoparticles and avoid aggregation.

[0047] Figure 28 shows PLL-coated nanoparticles at the selected design criteria (0.25 mg/mL PLL) did not impact cell viability. MOVAS cells were treated with nanoparticles fabricated with varying concentrations of PLL and cell viability was measured via cleaved caspase-3 apoptosis marker quantification indicative of apoptosis (A), and viability measured by WST-1 (B).

[0048] Figure 29 shows a representative image of entrapped peptide and particles under multiple layers of polyethylene oxide (PEO) coating, per Example 4g. A) shows the view from the top, focused on the plane containing peptide, as indicated by the yellow line in the cross- sectional images. B) shows a cross-sectional view from the east side of the top view. C) Cross-sectional view from the south side of the top view.

[0049] Figure 30 shows an image of dip coated glass slides with PEO, as discussed in Example 4a. [0050] Figure 31 shows a representative chromatogram of an isocratic flow rate. The peptide peak is overlapping with a second peak, correlating to DMSO, as discussed in Example 3i.

[0051] Figure 32 shows A) Relative metabolic activity and DNA content of MOVAS cells as a % of growth medium control after 24-hour exposure to D-PHI nanoparticles. Values shown are the mean ±standard deviation. 3 independent experiments were conducted with 3 technical replicates. One-way ANOVA was used to analyze differences in conditions (* p< 0.05 relative to all other samples). B) shows relative hemolysis of isolated human red blood cells when exposed to D-PHI nanoparticles as a % of Triton x-100 hemolytic control. Values represent the mean ±standard deviation. 3 independent experiments were conducted with 2 technical replicates. A one-way ANOVA was used to analyze differences in conditions.

[0052] Figure 33 shows an ultrasonic spray coated fabrication technique that can be used to deposit thin films of PEO, as per Example 4c.

[0053] Figure 34 shows typical images of the cross-section of high molecular weight PEO (Mw 100000) deposited onto a glass slide per Example 4a. Scale bars are 10 pm. Left Image: The spin coating process was repeated ten times to generate a 14.90 pm layer of PEO. Right Image: The spin coating process was done once to generate a 1 .51 pm layer of PEO.

[0054] Figure 35 shows three different blends of PEO tested to investigate the effects of molecular weight on the dissolution time per Example 4e. The lower molecular weight PEO immediately lost 50% of its mass at the minute mark while the high molecular weight blend had a prolonged and delayed dissolution. The blend of the two PEOs allows for a more gradual release of the polymer coating. N= 3, n= 3. Error bars represent standard deviation.

[0055] Figure 36 shows a representative cross-sectional scanning electron microscopy image of PEO spray coated onto a flat substrate, per Example 4f. The scale bar represents 5 pm. 22 x 22 mm glass cover slips were coated using the ultrasonic spray coating fabrication technique ten times. The average coating weight was 4.19 mg.

[0056] Figure 37 shows dissolution results from Example 4e. PEO dissolution for blends of PEO with varying molecular weight was tracked by measuring the percent weight loss over time. The ultrasonic spraying fabrication method was optimized to deposit 5 thin layers of PEO that was optimized to have 80% polymer retention to prevent premature loss of the peptide and nanoparticle at the 2-minute time point, which is considered an average balloon angioplasty tracking time. N=3, n=3. Error bars represent the standard deviation. [0057] Figure 38 shows the relative metabolic activity and DNA content of MOVAS cells as a percentage of growth medium (negative) control after 24-hour exposure to different concentrations of 100 000 Mw PEO, per Example 4f. N=3, n=3. Error bars represent standard deviation.

[0058] Figure 39 shows delivery of fluorescent-tagged chimeric N-cadherin targeting peptide to balloon-injured rat carotid arteries per Example 5. Fluorescence intensity of the peptide tag was quantified in three locations from the lumen of the artery: through the internal elastic lamina (IEL), through the first layer of smooth muscle cells (SMCs), and through the second layer of smooth muscle cells. The addition of D-PHI nanoparticles to 500 pg/mL of N- cadherin peptide increased the measured fluorescence intensity of the delivered peptide in the IEL and first layer of SMCs. The delivery of the peptide-nanoparticle mixture from a coated balloon further increased the amount and the depth of the peptide deposited in the tissue.

[0059] Figure 40 shows the results of VE-cadherin staining in HUVECs treated with N- cadherin targeting peptide per Example 6. Cells were grown in transwells with 0.4pm pore membranes. The top chamber media replaced with 5 pM FITC-dextran and N-cadherin peptide or scrambled control. Media in the abluminal (bottom) chamber was sampled into a 96-well dish and luminescence at 530nm read by plate reader. FITC-dextran concentration calculated against a standard curve. Data are means ± SEM, n=3 experimental replicates.

[0060] Figure 41 shows the results of staining for cleaved caspase 3 (CC3) as a marker of apoptotic cells in cross-sections of rat carotid arteries 1wk post-injury and peptide treatment. Positive and negative stained cells were counted in each anatomical layer of the vessel and used to calculate the percentage cells positive for CC3. For scrambled control treated (black) n=3, for N-cadherin peptide treated (red) n=3 for 1 week and Data are means ± SEM.

[0061] Figure 42 shows the results of staining for CC3 as a marker of apoptosis in RNISMCs treated for 24 hours with N-cadherin targeting peptide.

[0062] Figure 43 shows the results of staining for CC3 as a marker of apoptosis in HUVECs treated for 24 hours with N-cadherin targeting peptide.

[0063] Figure 44 shows the results of VE-cadherin staining in HUVECs treated with N- cadherin targeting peptide. Quantification of VE-cadherin staining fluorescence intensity was calculated by integrated density on images taken under 10x objective lens magnification and normalized to F-actin. n=6 experimental replicates per condition. Data are means ± SEM. [0064] Figure 45 shows transcript expression of inflammation markers in HUVECs treated with N-cadherin targeting peptide. RNA was extracted from HUVECs treated with N-cadherin targeting peptide for 24h. RT-qPCR was for ICAM1 , VCAM1 markers of endothelial cell adhesion, and THBD (thrombomodulin).

DETAILED DESCRIPTION

[0065] In one embodiment, this disclosure provides novel peptides having multiple domains: an N-cadherin mimetic domain to bind N-cadherin, a protein expressed on the surface of cells, and a fibronectin-binding domain modified from bacterial (staph, aureus) fibronectin binding protein 1 , which binds surrounding extracellular matrix.

[0066] N-cadherin is a protein that belongs to a family of transmembrane proteins that play fundamental roles in mediating cell-cell adhesion as well as guiding tissue organization as well as morphogenesis [C. M. Niessen, D. Leckband, and A. S. Yap, “Tissue organization by cadherin adhesion molecules: Dynamic Molecular and Cellular Mechanisms of Morphogenetic Regulation,” Physiol. Rev., vol. 91 , no. 2, pp. 691-731 , Apr. 2011 , doi: 10.1152/physrev.00004.2010; R. S. Krauss, F. Cole, U. Gaio, G. Takaesu, W. Zhang, and J. S. Kang, “Close encounters: Regulation of vertebrate skeletal myogenesis by cell-cell contact,” J. Cell Sci., vol. 118, no. 11 , pp. 2355-2362, Jun. 2005, doi: 10.1242/jcs.02397.]. These proteins form junctions that are believed to play an important anchoring role in connecting the actin cytoskeleton to the cell membrane at sites between apposing cells [Z. Sun, A. R. Parrish, M. A. Hill, and G. A. Meininger, “N-cadherin, A Vascular Smooth Muscle Cell-Cell Adhesion Molecule: Function and Signaling for Vasomotor Control,” Microcirculation, vol. 21 , no. 3, pp. 208-218, Apr. 2014, doi: 10.1111/micc.12123.].

[0067] SMCs are the dominant cell type found in restenotic lesions, a consequence of directional migration of SMCs from the media to the intima. A selective therapy targeted at SMCs therefore offers a considerable advantage over the current treatment options by reducing SMC neointima formation, but allowing EC regeneration to cover the vessel wall to re-establish favourable hemostasis. As demonstrated in the Examples, provided herein are N- cadherin mimetic peptides that inhibit SMC migration, but spare EC migration and viability. These peptides can be administered in vivo to balloon-injured carotid arteries to reduce neointimal formation.

[0068] Migration of SMCs is a main contributor to restenosis. SMCs are activated following mechanical injury and EC denudation that occurs during balloon catheter usage and stenting. The asymmetrical distribution of N-cadherin engagement triggers SMCs to undergo directional migration. While this mechanism is likely to be adaptive in wound-healing situations, it becomes mal-adaptive after vascular injury and SMCs form a neointimal hyperplasia that restricts the lumen diameter and blocks blood flow. Of the total amount of SMCs that end up forming the neointima, half have migrated from the medial layer, and half arise as a result of proliferation from the cells that have migrated. Thus, migration is a pivotal early process that underlies restenosis. By limiting the ability of SMCs to migrate at the earliest timepoints postinjury, their proliferation in the intima is avoided. As demonstrated in the Examples, N-cadherin mimetic peptides inhibited SMC migration and reduced polarization without impacting viability.

[0069] N-cadherin is upregulated following experimental injury in vivo and in vitro. [Jones, M., Sabatini, P. J. B., Lee, F. S. H., Bendeck, M. P. & Langille, B. L. N-cadherin upregulation and function in response of smooth muscle cells to arterial injury. Arteriosclerosis, Thrombosis, and Vascular Biology 22, 1972-1977 (2002).] N-cadherin localizes to posterior-lateral borders in wound-edge SMCs, an asymmetrical distribution essential for polarization of the microtubule organizing centre (MTOC) to the leading edge of the migrating cell [Sabatini, P. J. B., Zhang, M., Silverman-Gavrila, R., Bendeck, M. P. & Langille, B. L. Homotypic and endothelial cell adhesions via N-cadherin determine polarity and regulate migration of vascular smooth muscle cells. Circ. Res. 103, 405-412 (2008).] MTOC polarization is a hallmark of directional cell migration and governs microtubule assembly and protein delivery to the leading edge. N-cadherin is also delivered to lamellipodial protrusions of migrating SMCs, where it functions to fuse protrusions and seal macropinocytotic vesicles [Sabatini, P. J. B., Zhang, M., Silverman-Gavrila, R. V. & Bendeck, M. P. Cadherins at cell-autonomous membrane contacts control macropinocytosis. Journal of Cell Science 124, 2013-2020 (2011).] N-cadherin mimetic peptides administered in soluble form have been used in cell culture media to block or interfere with cell migration and/or proliferation. These N-cadherin mimetic peptides contain an HAV amino acid motif which forms a binding partner for a tryptophan residue on endogenous N-cadherin.

[0070] ECs also express N-cadherin and bind to adjacent N-cadherin molecules on underlying SMCs. EC denudation, or intimal thickening during the formation of fatty streaks, has the potential to disrupt these contacts, inducing an asymmetrical distribution of N-cadherin in SMCs at the edge of the media, which could stimulate the directional migration of SMCs to the intima.

[0071] Endothelial denudation and vessel wall mechanical injury from balloon angioplasty activate VSMCs to undergo directional migration to vessel interior. It has been demonstrated that following balloon injury in vivo or scratch wounding in vitro, SMCs upregulate N-cadherin [M. Jones, P. J. B. Sabatini, F. S. H. Lee, M. P. Bendeck, and B. L. Langille, “N-cadherin upregulation and function in response of smooth muscle cells to arterial injury,” Arterioscler. Thromb. Vase. Biol., vol. 22, no. 12, pp. 1972-1977, 2002.] However, because contacts between cells have been disrupted by denudation or wounding, this leaves the leading edge of the cell free, and sets up an asymmetric distribution of N-cadherin at the cell periphery. The asymmetry of N-cadherin distribution causes the polarization of the microtubule organizing centre, a hallmark of directional cell migration.

[0072] The chimeric peptides as provided herein include an N-cadherin binding domain to engage unbound N-cadherin at the leading edge of migrating SMCs to artificially establish contact symmetry and inhibit their migration. The peptides are chimeric, because they also contain a binding site for fibronectin, which allows them to bind to the sub-endothelial fibronectin-rich matrix exposed after endothelial denudation. Whereas SMC cell-cell contacts are made with N-cadherin, endothelial cell-cell contacts are made with VE- cadherin. Engaging N-cadherin with mimetic peptides provides a cell type-selective therapeutic strategy to inhibit polarization and directional migration of SMCs without negatively impacting ECs.

[0073] In some embodiments, the peptide sequence of the N-cadherin binding domain is not particularly restricted. Suitable sequences may be known to those of skill in the art - see for example E. Williams, G. Williams, B. J. Gour, O. W. Blaschuk, and P. Doherty, “A novel family of cyclic peptide antagonists suggests that N-cadherin specificity is determined by amino acids that flank the HAV motif,” J. Biol. Chem., vol. 275, no. 6, pp. 4007-4012, 2000, doi: 10.1074/jbc.275.6.4007, incorporated herein by reference in its entirety or functional fragments or variations thereof.

[0074] Table 1 discloses short chain cyclic N-cadherin peptides that have the HAV motif that has been identified to have a prominent role in N-cadherin binding specificity [per E. Williams et al. cited in the preceding paragraph.]

[0075] Table 1. Representative short chain cyclic N-cadherin peptides, with HAV motif, used as references for the design of the novel chimeric N-cadherin targeting peptide.

[0076] In some embodiments, the peptide sequence of the fibronectin-binding domain is not particularly restricted, and suitable sequences may be known to those of skill in the art. Such sequences may be found e.g. in K.C. Ingham et al. “Interaction of Staphylococcus aureus fibronectin-binding protein with fibronectin: affinity, stoichiometry, and modular requirements”. J. Biol. Chem. 279(41):42945-53, 2004.

[0077] In some embodiments, the ECM-binding domain has or includes an amino acid sequence selected from Table 2.

[0078] Table 2. ECM binding domains. [0079] In some embodiment, the chimeric peptide comprises a fibronectin- binding domain from the species Staphylococcus aureus, more preferably comprising or consisting of an amino acid sequence selected from LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10); LTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 11); KLTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 12); and KPSYGFGGHNSVDFEEDTLPKV (SEQ ID NO: 13) or a functional fragment or variant thereof.

[0080] In one embodiment, the fibronectin-binding domain comprises an amino acid sequence LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10).

[0081] In one embodiment, a chimeric peptide as provided herein has a peptide sequence: CLRAHAVDINGKLTGQYDKNLVTTVEEEYDS (SEQ ID NO: 25) (structure shown in Figure 17B.)

[0082] As detailed in the Examples, treating scratch-wounded SMCs with N-cadherin targeting peptide inhibited migration and reduced polarization of wound-edge cells. The peptide co-localized with fibronectin. Importantly, neither EC viability nor migration was impacted by peptide treatment.

[0083] Further, a chimeric peptide prepared according to an embodiment persisted for 24 hours after transient delivery in a balloon-injured rat carotid artery. Treatment with the N- cadherin targeting chimeric peptide reduced intimal thickening in balloon-injured rat carotid arteries at 1 and 2 weeks after injury, while re-endothelialization of injured vessels after 2 weeks was unimpaired by peptide treatment.

[0084] The chimeric design of the peptide and nanoparticle carrier system combine to form a unique system, the advantages of which can include: enabling processing of the peptide to form composites within films that can be processed on the surfaces of devices such as drug eluting balloons, stents and catheters; allowing for delayed and controlled release of the peptides to inhibit vascular smooth muscle cell migration; allowing a high affinity for the particles enabling concentration of peptide on the nanoparticle; and enabling the preparation of simple delivery vehicles that do not denature the peptide.

Nanoparticles

[0085] In one embodiment, the chimeric peptides as provided herein are loaded on nanoparticles for delivery. In one embodiment, the nanoparticle delivery vehicle may be a nanoparticle know in the art as being suitable for in vivo delivery and susceptible to coating with the chimeric peptides as provided herein without or with limited denaturation or loss of peptide function.

[0086] However, in one embodiment, the nanoparticles are formed of a Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI). As detailed in the Examples, degradable biocompatible D-PHI nanoparticles are demonstrated to carry the peptide and deliver it in a manner that does not denature the peptide or cause it to lose function. Further, once the peptide is released, the particles themselves have no detectable toxic effects on endothelial or smooth muscle cells.

[0087] As used herein “degradable” references that the nanoparticle contains hydrolyzable groups and can be resorbed via either solvation, oxidation, hydrolysis or a combination, catalyzed by enzymes and/or acids.

[0088] The nanoparticles used herein are suitably biodegrade, i.e. they degrade under physiological conditions. This may be contrasted with "non-biodegradable" biomaterials that undergo less than 1 % degradation within the first month of implantation in a subcutaneous rat implant model such as that reported on by McBane JE, Sharifpoor S, Kuihua C, Labow RS, and Santerre JP, Biodegradation and in vivo biocompatibility of a degradable, polar/hydrophobic/ionic polyurethane for tissue engineering applications, Biomaterials. 32, 6034-44 (2011).

[0089] D-PHI materials and methods of their preparation can be found in International patent application PCT/CA2015/000461 , publication no. WO2016/023102, the contents of which are incorporated herein by reference in their entirety.

[0090] Polyurethanes are synthetic block copolymers characterized by the presence of a urethane linkage created in a condensation reaction (step-growth polymerization). Polyurethanes can be linear, branched, or cross-linked. Polyurethanes are copolymers and contain two repeating segments; a hard segment of the polyurethane (the isocyanate), which endows the biomaterial with mechanical strength and a soft segment (the polyol), which provides flexibility. The soft and hard segments can microphase separate to form soft and hard phases; these phases provide the polymer with both flexibility and strength. The combination of segments manifests itself in the bulk material composition and surface microstructure. The differences in polarity of the hard and soft segments affect the hydrophilic- hydrophobic balance of the biomaterial. Furthermore, the soft segments are mobile and will optimize their location to minimize the free energy at the surface of the material. The copolymer structure and the composition of its monomers provide a polyurethane with its unique in vivo properties and biocompatibility.

[0091] In accordance with one aspect of the present invention, polyurethanes undergo biodegradation in vivo due to their chemical composition and the presence of hydrolytic esterases in the body and their biodegradation tendencies can be exploited to design specific biodegradation profiles. Suitably, monomers and other degradation byproducts can be selected such that they are not cytotoxic.

[0092] In one aspect, the D-PHI material comprises, consists essentially of, or consists of, the reaction product of: (a) at least one polar non-ionic macromer component; and one or both of: (b) at least one anionic component; and (c) at least one hydrophobic component. Suitably, in some embodiments, the molar ratio of (a) to (b) and (c) combined being at least about 1 :10; the molar ratio of (b) to (c) being between about 20: 1 and 1 : 100; and the maximum combined number of components (a),(b) and (c) in the polymer being 9. In one embodiment, the molar ratio of (a) to (b) and (c) combined is at least 1 :>20.

[0093] In various embodiments, the molecular weight of each polar non-ionic macromer component of (a) is between about 400 and about 5000, about 500 and about 5000, about 1000 and about 5000, about 1000 and about 4000, about 1000 and 4500 or about 1500 and about 4500. In various embodiments, the molecular weight of each anionic component (b) is between about 50 and about 1000 and the molecular weight of each hydrophobic component (c) is between about 50 and about 1000.

[0094] As used herein, the term “polar non-ionic macromer” is a molecule of greater than about 400 molecular weight but less than about 5000, with a segment of the molecule that contains repeat monomer units, and at least two polar functional groups, and at least two reactive groups which can be readily covalently coupled with similar type groups (e.g. vinyl groups) to yield a material. In one embodiment, the polar non-ionic macromer is generated with molecules containing isocyanate groups. The molecules do not contain free ionic function but may be precursors to said ionic function upon hydrolysis of protecting groups within the precursors. Said protecting groups may be used to couple covalent or non-covalent drugs, biological agents (peptides, or other pendent molecules), or contribute to the ionic content of cured polymers.

[0095] In one embodiment, the macromer is the reaction product of at least one isocyanate compound, at least one polyol terminated with hydroxyl or amine groups and at least one vinyl coupling agent. [0096] In various embodiments, the isocyanate compound may be Diisocyanatomethane; 1 ,4-Diisocyanatobutane; 1 ,1-Diisocyanatohexane; Hexamethylene diisocyanate (1 ,6- Diisocyanatohexane); Octamethylne diisocyanate (1 ,8-Diisocyanatooctane); 1 ,1-

Diisocyanatoethane; 1 ,1-Diisocyanatobutane; 2,2-Dimethylpentane-1 ,5-diyl diisocyanate; 1 ,6- Diisocyanato-2,2,4-trimethylhexane; Trimethyl hexamethylene diisocyanate; Lysine diisocyanate; 1 ,1-Diisocyanatocyclohexane; Isophorone diisocyanate (5-lsocyanato-1- (isocyantomethyl)-1 ,3,3-trimethylcyclohexane); 4,4’-Methylenebis(cyclohexyl isocyanate; L- Lysine ethyl ester diisocyanate; 1 ,3-Phenylene diisocyanate; 1 ,4-Phenylene diisocyanate; 2- (diisocyanatomethyl)furan; Toluene 2,4-diisocyanate (2,4-Diisocyanatotoluene, 4-Methl-m- phenylene diisocyanate, Tolylene 2,4-diisocyanate); Tolylene 2,5-Diisocyanate; Tolylene 2,6- Diisocyanate; m-Xylylene diisocyanate (1 ,3-Bis(isocyanatomethyl)benzene); 2,2- Diisocyanato-2,3-dihydro-1 H-indene; 2,4,6-Trimethyl-1 ,3-phenylene diisocyanate; 1 ,5- Naphthalene diisocyanate; a,a,a’,a’-Tetramethyl-1 ,3-xylylene diisocyanate (1 ,3,-Bis(1- isocyanato-1-methylethyl)benzene); 4,4’-Methylenebis(phenyl isocyanate) (4,4’-MDI, Bis(4- isocyanatophenyl)methane); 4,4’-Oxybis(phenyl isocyanate); 3,3’-Dimethyl-4,4’-Biphenylene Diisocyanate; 3,3’-Dimethyldiphenylmethane-4,4’-diisocyanate; 2,4,6-T riisopropyl-m- phenylene diisocyanate; 3,3’-Dimethoxy-4,4’-biphenylene diisocyanate; 3,3’- (Tetrafluoroethane-1 ,2-diyl) bisphenyl diisocyanate; 1 ,1 ,1 ,2,2,3,3,4,4-nonafluoro-7,7- diisocyanato-heptane; Tetraisocyanatosilane, Bis(1 ,1-dimethylethoxy)diisocyanato-silane; 1 ,1 ,3,3-tetraisocyanato-1 ,3-dimethyl disiloxane; 2-Propenoic acid, 2-methyl-, 3- (triisocyanatosilyl)propyl ester; 2-Propenoic acid, 2-methyl-, 4-[3-

(triisocyanatosilyl)propyl]phenyl ester; 1 ,2,2-Triisocyanatobutane, Hexamethylene diisocyanate-biuret; Hexamethylene diisocyanate isocyanurate; 2,2'-Methylenebis[6-(o- isocyanatobenzyl)phenyl] diisocyanate; 2,4,6-trioxotriazine-1 ,3,5(2H,4H,6H)-triyl)tris(methyl- m-phenylene) isocyanate; Poly[methylene(polyphenyl) isocyanate]; Poly(hexamethylene diisocyanate); Polycarbonate-based diol MDI terminated Prepolymer; Polyether based diol HDI terminated Prepolymer; Polypropylene glycol), tolylene 2,4-diisocyanate terminated (isocyanate ~ 3.6 wt.%); Polycarbonate-based diol PPDI terminated Prepolymer; or Poly(1 ,4- butanediol)tolylene 2,4-diisocyanate terminated (1.9 wt. % isocyanate).

[0097] In various embodiments, the at least one polyol terminated with hydroxyl or amine groups comprises polyethylene oxide; polypropylene oxide; polytetramethylene oxide; polyisobutylene; polybutadiene; polyethylene adipate; polytetramethylene adipate; polycaprolactone; polydimethylsiloxane; polycarbonate; polysiloxane; polyethylene-butylene; polyester; polyether sulfone; polyurethane; polyurea; polyamide; polyalkylene oxide; polyvinyl derivatives; polypeptide derivatives; polysaccharide derivatives; polyehtylenebutylene; 4- butanediol; ethylene diamine; 4,4’ methylene, bis(2-chloroaniline); ethylene glycol; hexanediol; butane diol; ethylene diamine; hexamethylene diamine; hexamethylene dicarboxylic acid; lysinate; hexane diol; 2,5 diaminobenzenesulfonic acid; 4,4’diamino 2,2’- biphenyl disulfonic acid; 1 ,3-diamino 2-hydroxypropane; N-(2-aminoethyl)-3-aminopropane sulfonate; dihydroxy vinyl derivatives; dihydroxy diphenylsulfone; hexamethylene diol; 1 ,5 pentanediol; 2,2-dimethyl-1 ,3 propanediol; 1 ,2-diamino-2 methylpropane; 3,3,-diamino-N- methyldipropylamine; 1 ,4 diaminobutane; 1 ,7 diaminoheptane; 1 ,8 diaminooctane; glutaryl dichloride; or adipoyl dichloride

[0098] In various embodiments, the at least one vinyl coupling agent may be a vinyl alcohol, an alkyl amine with vinyl groups, a vinyl amine, hydroxypropyl (meth)acrylate, 2,3- di hydroxy propyl (meth)acrylate, 1 ,4-butanediol monoacrylate, (poly)ethylene glycol mono(meth)acrylate, 3-aminopropyl vinyl ether, or 2-hydroxyethyl methacrylate (HEMA).

[0099] In one embodiment, the macromer is the reaction product of poly(hexamethylene carbonate) diol, lysine diisocyanate and 2-hydroxyethyl methacrylate.

[0100] In various embodiments, the anionic component is a vinyl monomer with mono acid function such as methacrylic acid, vinyl phosphoric acid, styrene sulphonic acid or the like; vinyl monomers with di-acids such as itaconic acid, maleic acid or the like; or vinyl monomers with tri-acids such as tricarballylic acid, tricarboxylic acid or the like. The anionic component may also be a methacrylic acid derivative; 2-(methacryloyloxy)ethyl phosphate; 2(methacryloyloxy)ethyl succinate, [3-(methacryloylamino)propyl]trimethyl ammonium chloride; or 2-(methacryloyloxy)ethyl]trimethylammonium methyl chloride. The methacrylic acid derivative may be an amino-acid derivative. The anionic component may be methacrylic acid.

[0101] In one embodiment, the hydrophobic component is an alkyl methacrylate, wherein the alkyl chain is linear or branched, saturated or unsaturated, and wherein the number of carbons is less than 12. In various embodiments, the hydrophobic component may be methyl, propyl, butyl, iso-butyl or t-butyl methacrylate; or styrene. The hydrophobic component may be methyl methacrylate.

[0102] In one embodiment, the polymer material is synthesized using a free radical initiator. The free radical initiator may be selected from diacyl peroxides, peroxy esters, dialkyl peroxides, dialkyl peroxydicarbonates, tert-alkylhydroperoxides, and ketone peroxides. In various embodiments, the free radical initiator is dibenzoyl peroxide, diisobutyrul peroxide, t- butyl peracetate, dicumyl peroxide, di-sec-butyperoxydicarbonate, diphenylmethanone, methyl ethyl ketone peroxide, benzyl peroxide or 1 ,1 ’-azobis(cyclohexanecarbonitrile). [0103] To coat the D-PHI nanoparticles with the chimeric N-cadherin peptide, the peptide is suitably solubilized in a suitable solvent and combined with the nanoparticles. E.g. the peptide can be solubilized, suitably to a concentration of between about 25 mg/mL and about 100 mg/mL in dimethyl sulfoxide (DMSO). In one embodiment, 5 pL of the peptide solution was added to 120 pL of D-PHI nanoparticles (particle concentration was 8 x 10⁸ particles per mL in ultrapure water.) In some embodiments, a nanoparticle concentration between 8 x 10⁸ particles and 1 .5 x 10¹⁰ per mL in ultrapure water can be used.

[0104] As illustrated in the Examples, in some embodiments, a pH Driven Coating methodology is employed, which utilizes a change in the pH of the solution to direct the interactions between the polyelectrolytes and the particles of interest [S. E. Burke and C. J. Barrett, “pH-responsive properties of multilayered poly(L-lysine)/hyaluronic acid surfaces,” Biomacromolecules, vol. 4, no. 6, pp. 1773-1783, 2003.; J. H. Choi etal., “Influence of pH and Surface Chemistry on Poly(l -lysine) Adsorption onto Solid Supports Investigated by Quartz Crystal Microbalance with Dissipation Monitoring,” J. Phys. Chem. B, vol. 119, no. 33, pp. 10554-10565, Aug. 2015.] Suitably, two polyelectrolytes of opposite charge are employed to perform a multi layer coating on D-PHI nanoparticles.

[0105] The chimeric peptide-loaded nanoparticles can be applied to a surface of a biomedical article. Methods of application can include e.g. spraying, dipping, micropipetting, or imprinting.

[0106] Suitably, the peptide delivery nanoparticles as provided herein are further provided with a protective coating, in one embodiment, a protective polymer coating, which suitably dissolves in accordance with the timing of balloon tracking and inflation. In some embodiments, this protective coating is formed of polyethylene glycol (PEG), polyethylene oxide (PEO) or hyaluronic acid (HA).

[0107] PEG and PEO are widely used in the pharmaceutical and biomedical industry due to their bioresorbable properties, “non-fouling” and protein resistant and cellular resistant properties. They are US FDA-approved and are widely popular because of their tunable properties and well-established safety profile. PEG or PEO have high structural flexibility, biocompatibility, amphiphilicity, are devoid of steric hinderances, and have a high hydration capacity. In the drug eluting balloon applications, un-crosslinked PEO has a relatively fast dissolution time which is favorable for drug release. This has the potential to be optimized by adjusting and combining different molecular weight blends of the polymer to achieve a desired effect [0108] The process of balloon angioplasty starts with the introduction of a guidewire that is manipulated such that it leads to the treatment site. Once the guidewire is in place, the balloon catheter is then tracked down along the guidewire to the treatment site which takes approximately 1-2 minutes in a standard procedure. When the balloon catheter is in the correct position, the balloon is inflated to deliver the therapeutic payload, which typically takes between 1 to 4 minutes. As detailed in the Examples, in one embodiment there is provided herein a PEO coating that dissolves in accordance with the timing of balloon tracking and inflation.

[0109] In some embodiments, protective coatings may be deposited using any suitable method know in the art. As will be apparent to a person of skill in the art, a coating may cover all or a portion of a surface of a medical device. In some embodiments, the coating is deposited by dip coating or spray coating. In a preferred embodiment, the protective coating is deposited by ultrasonic spray coating.

[0110] Reference may be made to a peptide-containing layer comprising the chimeric peptide and a protective layer. While reference is made to a layer, as will be understood by a person of skill in the art (and with reference to the Examples), a layer of a given composition can be applied in a single step or may be built up by sequential application of thinner layers to yield a final layer having e.g. a desired thickness or weight.

[0111] In some embodiments, the PEG or PEO has a molecular weight of between about 50,000 and 200,000, preferably about 100,000. As detailed in the Examples, the polymer coating may be deposited in multiple thin layers. As detailed in the Examples, 5 layers of thinly coated PEO on a nylon (a common balloon catheter material) substrate were able to generate a dissolution profile that retained over 80% of its mass after 2 minutes. This coating adherence, simulated in PBS release buffer at physiological conditions, coincides well with the projected timeline of protection during balloon tracking and full release during balloon inflation. A change of substrate to latex (an alternative balloon catheter material) had no significant effect on the dissolution profile of the coating. Furtherthis advantageous dissolution profile was retained when D-PHI nanoparticles according to the present invention were incorporated into the coating at clinically useful levels.

Biomedical articles and implants

[0112] The peptides and loaded nanoparticles as provided herein can suitably be used as coatings for biomedical articles. In some embodiments, the biomedical articles are temporarily or permanently implantable biomedical articles. [0113] Such, biomedical articles can include e.g. cardiac assist devices, tissue engineering polymeric scaffolds and related devices, cardiac replacement devices, cardiac septal patches, wound dressings, intra aortic balloons, peripheral artery angioplasty balloons, percutaneous cardiac assist devices, extra-corporeal circuits, A-V fistula, dialysis components (tubing, filters, membranes, etc.), aphoresis units, membrane oxygenator, cardiac by-pass components (tubing, filters, etc.), pericardial sacs, contact lenses, cochlear ear implants, sutures, sewing rings, cannulas, contraceptives, syringes, o-rings, bladders, penile implants, drug delivery systems, drainage tubes, a pacemaker e.g. pacemaker lead insulators, heart valves, blood bags, coatings for implantable wires, catheters, vascular stents, angioplasty balloons and devices, bandages, heart massage cups, tracheal tubes, mammary implant coatings, artificial ducts, craniofacial and maxillofacial reconstruction applications, ligaments, fallopian tubes, biosensors and bio-diagnostic substrates.

[0114] Still further, biomedical devices can include (without limiting the foregoing) a stent, a stent graft, a synthetic vascular graft, a heart valve, a vascular prosthetic filter, a defibrillator, a patent foramen oval septal closure device, a vascular clip, a vascular aneurysm occluder, a hemodialysis graft, a hemodialysis catheter, an atrioventricular shunt, an aortic aneurysm graft device, a venous valve, a suture, a vascular anastomosis clip, an indwelling venous catheter, an indwelling arterial catheter, a vascular sheath and a drug delivery port.

[0115] Occlusive vascular diseases including atherosclerosis, restenosis and bypass graft failure are leading causes of morbidity and mortality. Drug eluting stents and balloons are the current standard of treatment to reverse vessel occlusion, with stents being used more in coronary artery disease, and balloon angioplasty used to treat peripheral artery disease (PAD). In-stent restenosis is a common complication of treatment, and in many cases is treated with balloon angioplasty to relieve the occlusion within the stent without introducing another stent. Drug eluting balloons enable targeted drug delivery, while avoiding implanting a permanent foreign object. A review of the properties of drug-eluting balloons and cardiac stents and treatment methods employing the same (including limitations associated with known devices and techniques) can be found e.g. in M. Bukka et al., Drug-eluting balloon: design, technology and clinical aspects. 2018 Biomed. Mater. 13 032001. In one embodiment, there is provided methods of treating to reverse vessel occlusion in a patient in need thereof using a stent or balloon having a coating that includes a chimeric peptide as provided herein, in one embodiment, chimeric peptide loaded nanoparticles as provided herein. Treatments may also be preventative to inhibit the onset of vessel occlusion/restenosis in a subject at risk thereof, and references to “treating” or “treatment”, should be understood to include preventative treatments, unless the context clearly indicates otherwise. [0116] Drug-eluting balloons may be formed of various materials, including, such as cross-linked polyethylene, polypropylene, polyamides, polyimides, polyesters like polyethylene tetrapthalate (PET) and poly ethylene (naphthalene dicarboxylate), latex, and nylon. Similarly, stents may be formed of metals and polymers known to those of skill in the art, including nylon and polyurethane.

Inhibition of Smooth Muscle Cells and Sequestering of Free Peptide

[0117] Without wishing to be bound by a theory, the Examples provide evidence that the D-PHI particles described herein can function to inhibit restenosis in two ways: by delivering the N-cadherin binding peptide, but also by sequestering free peptide in solution to bind to the tissues. Thus, while in some embodiments, treatments are provided in the form of nanoparticles coated with peptide, which optionally may be coated on an implantable or implanted device, in other embodiments the nanoparticles and peptides described herein may be administered separately, in some embodiments, concurrently e.g. in a single composition, which can include a suitable carrier, or in close temporal proximity, but as separate compositions.

[0118] All documents referenced herein are incorporated by reference, however, it should be appreciated that any patent, publication, or other disclosure material, in whole or in part, that is incorporated by reference herein is incorporated only to the extent that the incorporated material does not conflict with definitions, statements, or other disclosure material set forth in this disclosure. As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting material incorporated herein by reference.

[0119] It will be understood that numerous modifications thereto will appear to those skilled in the art. Accordingly, the above description and accompanying drawings should be taken as illustrative of the invention and not in a limiting sense. It will further be understood that it is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims. EXAMPLES

[0120] Example 1 : N-cadherin targeting chimeric peptide inhibits vascular smooth muscle cell migration and intimal thickening without affecting endothelial cell repair in balloon-injured carotid arteries

[0121] All statistical analyses were performed with Prism 9 software (GraphPad). A value of p<0.05 was considered statistically significant. Student’s t-tests were used for pair-wise comparisons. Two-way ANOVA with a Tukey correction for multiple analyses were used for comparisons between multiple groups where appropriate. Where indicated, multiple fields of view from each well were considered technical replicates and averaged together. Values from separate wells were then considered biological replicates for analysis. For intimal:medial ratio calculation, three measurements were made along each injured carotid artery specimen and these measurements were averaged for each animal, then the overall mean was calculated. Sample sizes were: 6 animals per group for 1 week intimal thickening; 7 scrambled control and 9 N-cadherin targeting chimeric peptide treated animals for 2 week intimal thickening; 6 and 5 animals respectively for scrambled control and N-cadherin targeting peptide treatment to measure re-endothelialization, 3 animals per condition for Ki67 staining at 1 week; and 3 and 5 animals for scrambled control and N-cadherin targeting peptide treated animals for Ki67 at 2 weeks.

Example 1a: Materials and Methods

N-cadherin targeting peptides

[0122] Several putative N-cadherin targeting peptides were designed, and three versions chosen for in vitro testing (Fig. 1). The chimeric peptide included a fibronectin-binding domain based on the Fnbpl from Staph. Aureus bacteria and an N-cadherin binding domain based on the “HAV” amino acid motif responsible for homotypic cadherin binding. “Short” (linear) and “cyclic” versions were also designed lacking the fibronectin domain, but maintaining the N- cadherin mimetic element and HAV binding motif. The short and cyclic peptide sequences were derived from examples in the literature, for the purpose of comparison to the novel matrix anchoring configuration provided herein. Each peptide was paired with a scrambled control version in which the N-cadherin binding domain contained the identical amino acids in a randomized sequence. Additionally, 5-carboxytetramethylrhodamine (TAMRA) fluorescent tags were added to peptides in some experiments to allow for visualization by fluorescence microscopy. For experiments not requiring this, untagged versions of these peptides were used. Peptides were synthesized to a purity >95% and were provided as lyophilized powder. Peptides were dissolved in pure dimethylsulfoxide (DMSO) to a concentration of 100 mg/mL before being diluted in cell culture media for in vitro administration, or in phosphate buffered saline (PBS) for use in vivo.

Cell Culture

[0123] Immortalized mouse vascular arterial smooth muscle cells (MOVAS) (ATCC, CRL- 2797) were cultured in Dulbecco’s modified eagle medium (DMEM) (Life Technologies, 11885084, Carlsbad CA) with 10% fetal bovine serum (FBS) (Thermo-Fisher Scientific, 12483020, Waltham MA) and 2% penicillin-steptomycin (P/S) (Thermo-Fisher Scientific, 15140122). Rat neointimal smooth muscle cells (RNISMC) were isolated from the intima of balloon-injured male Sprague-Dawley rat carotid arteries as previously described [Hou G, Mulholland D, Gronska MA, Bendeck MP. Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury. Am J Pathol. 2000;156(2):467-476.] and cultured in DMEM with 10% FBS and 2% P/S. Human umbilical vein endothelial cells (HUVEC) and human arterial smooth muscle cells (HASMC) were purchased from Lonza (Basel, Switzerland) and cultured in EGM-2 or SmGm-2 media supplemented with the included bullet kit according to the manufacturer’s instructions. RNISMC, HUVEC and HASMC were used at passage 2-10 and MOVAS cells were used at passage 20-50.

[0124] For scratch wound assays, cells were plated at a seeding density of 150,000 cells I mL in 24-well dishes which had been coated with 20 pg/mL fibronectin (Roche, 10838039001 , Basel, Switzerland) for 1 hour at 37°C and washed once with PBS (ThermoFisher Scientific, 14190144). Cells were incubated for 24h, scratched with a 200 pL pipette tip guided along a straight edge, washed with PBS, and media replaced. Short, cyclic, or N-cadherin targeting chimeric peptides were diluted from 100 mg/mL in DMSO, by addition to DMEM with 0.1% FBS and 2% P/S to final concentrations of 125-500 pg/mL. Cells were treated for 24h (MOVAS) or 48h (RNISMC, HASMC and HUVEC) and imaged to visualize the scratch wound area. The wound gap was manually traced, and the area measured using Nikon NIS Elements software. The wound gap area was subtracted from, and then divided by, the initial area of the wound to give a final measurement for percentage of wound area that had closed.

Immunostaining

[0125] Cells were fixed in 4% paraformaldehyde (or 100% ice cold methanol for y-tubulin) for 10 minutes and permeabilized in 0.2% triton X-100 for 10 minutes at room temperature. Cells were blocked in 0.2% BSA for 1 hour and stained overnight with primary antibodies: anti- N-cadherin (rabbit-anti-mouse, Abeam 18207, 1 :800), anti-y-tubulin (rabbit-anti-mouse, Abeam ab11317, 1 :800), anti-fibronectin (rabbit-anti-mouse, Abeam ab23750, 1 :1000), anti- cleaved-caspase-3 (rabbit-anti-mouse Cell Signalling Technologies 9661 , 1 :1000). All antibodies used were polyclonal and raised in rabbit against mouse epitopes. Cells were washed thrice with PBS and stained with DAPI (Roche 10236276001), phalloidin 488 or 568 (Invitrogen A12379, A12380), and/or secondary antibodies: goat-anti-rabbit Alexa488 (Invitrogen A-11008), goat-anti-rabbit Alexa568 (Invitrogen A-11011) at 1 :1000. Coverslips with cells were washed and mounted on slides in Prolong Gold antifade mounting media (Invitrogen P10144). Images were obtained using a laser-scanning confocal microscope (Olympus FV3000).

Cell polarity measurement

[0126] For MTOC polarization experiments, cells were scratch-wounded and treated with 500 pg/mL short, cyclic, or chimeric N-cadherin targeting peptides, or their respective scrambled controls, then 6h post-injury, cells were fixed in ice-cold methanol for 10 minutes. Cells were stained for y-tubulin to visualize the MTOC. Cells at the wound-edge were imaged by confocal microscopy using a 60x oil immersion objective lens to generate fields of view containing 4-10 wound-edge cells. Cells were grouped into two categories based on the position of the MTOC relative to the wound gap and the nucleus. A virtual line was drawn through the centre of each nucleus parallel to the wound gap. Cells with an MTOC in front of this line were considered polarized, and those with an MTOC behind this line were considered non-polarized. At least 20 fields of view were analyzed from at least 3 separate wells for each condition.

Animal surgery

[0127] Carotid balloon-injury surgery was performed on Sprague-Dawley rats weighing 350-400g (Charles River) by inflating and withdrawing a 2F embolectomy catheter (Edwards) three times through the carotid artery [Hou G, Mulholland D, Gronska MA, Bendeck MP. Type VIII collagen stimulates smooth muscle cell migration and matrix metalloproteinase synthesis after arterial injury. Am J Pathol. 2000; 156(2) :467-476.; Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circulation Research. 1994;75(3):539-545.]

[0128] Following balloon-injury, 500 pg/mL of N-cadherin targeting chimeric or scrambled control peptide dissolved in 100pL of PBS, also containing heparin at 50 lU/mL, was infused through the arteriotomy near the site of injury, and the vessel segment was held closed by metal clamps for 5 minutes to allow incubation of the chimeric peptide in the carotid segment, following which the clamps were released, and blow flow restored. Animals were allowed to recover for 24h (for peptide residency experiments), 1 or 2 weeks (for neointima formation experiments), and then sacrificed by C02 asphyxiation and bilateral thoracotomy. Animals were immediately perfusion-fixed with 4% paraformaldehyde for 5 minutes at 120 mmHg, through a needle inserted into the heart, to preserve vessel physiological shape and structure for histological or immunofluorescence microscopy. Carotid segments were excised and processed into cross-sections 4 pm thick for mounting on slides, histological staining with hematoxylin and eosin, and imaging by conventional brightfield microscopy (Nikon Eclipse Ci upright microscope). Luminal, intimal and medial areas were measured (Nikon NIS Elements software) by manually selecting each area landmarked by cells and elastic lamellae. Luminal area was determined by measuring the circumference and applying the formula for area of a circle (A=irr2, C=2irr) to correct for vessel deformation during processing. These measurements were used to calculate specific areas for each arterial layer, and the intimal- to-medial ratio.

[0129] Separate groups of animals and carotid segments were harvested 24h after injury, and the carotids opened longitudinally and imaged en face, for assessment of fluorescent peptide binding with confocal microscopy (Olympus FV3000). To measure re- endothelialization, rats were injected with 0.5 mL of 5% Evan’s Blue dye dissolved in sterile PBS through the tail vein. At 30 minutes following dye administration, animals were sacrificed and perfusion-fixed as described. Vessel segments were prepared en face and imaged using a dissection microscope (Nikon). Using Imaged software, unstained area was divided by total vessel area and multiplied by 100% to determine the percentage of the intimal surface with an intact endothelial cell barrier. Cross-sections of carotid arteries were also stained for Ki67 to assess cell proliferation. The total number of cells, and the total positive for Ki67, were counted in the intimal and medial layers, and the percentage of Ki67 positive cells was calculated. Rat carotid arteries were excluded from analysis in the event of one of the following criteria being met: premature death of the animal, artefactual dissociation of the intima.

Example 1b. N-cadherin targeting chimeric peptide inhibited migration of rodent SMCs

[0130] Three versions of N-cadherin mimetic peptides, including the chimeric N-cadherin targeting peptide with a matrix-binding domain, were designed (Fig. 1) and synthesized by solid phase peptide synthesis (Lifetein, Inc). To determine the effects of these peptides on cell migration, two types of rodent SMCs (MO AS, RNISMC), and human SMC and EC (HASMC, HUVEC) cell lines were cultured in 24-well dishes to confluency and scratched with a pipette tip along a straightedge to create a wound gap. Cells were washed and treated with N- cadherin targeting peptides, scrambled control peptides, or vehicle for 24 hours. In MOVAS cells, the chimeric peptide significantly reduced wound closure at 250 and 500 pg/mL compared to the scrambled control treated cells (Fig. 2A). In RNISMCs, the chimeric N- cadherin targeting peptide significantly reduced wound closure at 250 or 500 pg/mL (Fig. 2B) There were no statistically significant differences in wound closure in cultures treated with the different scrambled control peptides, which were each tested individually (Fig. 7A). The data from these scrambled control groups were pooled for ease of comparison in Fig 2.

Example 1c. N-cadherin targeting peptides did not impact viability or apoptosis of rodent SMCs

[0131] MOVAS or RNISMC were cultured in 96-well dishes and treated with N-cadherin peptides, scrambled control peptides, or vehicle for 24 hours. A WST-1 assay was performed to assess viability, measuring cell metabolism as a proxy for the amount of viable cells present in each well. In particular, for viability assays, cells were plated at 10,000 cells/well in fibronectin-coated (20 pg/mL) 96-well plates and cultured for 24h. Cells were treated with peptides for 24h before adding water-soluble tetrazolium salts (WST-1) (Roche, 05015944001) to assess metabolic activity by changing colour in response to NAD(P)H oxidation, which was recorded by measuring absorbance at 450 and 610 nm in a plate-reader (PerkinElmer EnSpire Multimode Plate Reader).

[0132] There were no statistically significant differences in viability in MOVAS or RNISMC treated with N-cadherin peptides compared to controls (Fig. 2C, 2D). Apoptosis was measured by staining MOVAS cells for cleaved caspase 3 after 24h of peptide treatment at 500 pg/mL. There was no significant change in the proportion of CC3+ cells in the peptide-treated groups compared with the controls (Fig. 2E). Each different scrambled control peptide was tested individually and differences were not statistically significant (Fig. 7B, 7E) so the data from these groups were pooled for ease of comparison in Fig. 2.

Example 1d. N-cadherin targeting chimeric peptide inhibited the polarization of woundedge SMCs

[0133] To determine whether cell polarity of wound-edge migrating VSMCs was inhibited by N-cadherin peptides, MOVAS cells were cultured to confluency in 24-well dishes and scratch-wounded. Cells were treated with 500 pg/mL N-cadherin targeting peptides or scrambled controls for 6 hours, then fixed and stained for y-tubulin, a marker for the microtubule organizing centre (MTOC) (Fig. 3A). The position of the MTOC was analyzed relative to the centreline of the nucleus, indicated by a line drawn through the centre of the nucleus parallel to the wound gap (Fig. 3B), and cells in which the MTOC was forward of this centreline relative to the direction of migration were counted as polarized. The N-cadherin targeting chimeric peptide significantly reduced the number of polarized wound-edge cells by 21.7% (Fig. 3C). The scrambled peptides did not significantly affect cell polarization (Fig. 7C), so data from the scrambled control groups was pooled for ease of comparison in figure 3. Example 1e. N-cadherin targeting chimeric peptide inhibited migration of human SMCs but not human ECs, and did not impact viability of either human cell type

[0134] HASMC were subject to scratch wounding, then treated with N-cadherin targeting chimeric peptide or scrambled control peptide for 48 hours. In HASMC, the N-cadherin targeting chimeric peptide significantly reduced wound closure at a dose of 500 pg/mL (Fig. 4A). In HUVEC, there was no impact of N-cadherin targeting chimeric peptide treatment on cell migration (Fig 4B). There were no statistically significant effects of N-cadherin targeting chimeric peptides on the viability of either HASMC or HUVEC (Fig. 4C & 4D).

Example 1f. N-cadherin targeting chimeric peptide co-localized in vitro with fibronectin, and was retained in vivo in balloon-injured carotid arteries

[0135] To determine whether the N-cadherin targeting chimeric peptide could bind with fibronectin, RNISMC were plated on fibronectin-coated coverslips, treated with 100 pg/mL TAMRA-labelled N-cadherin targeting or scrambled peptide, and stained to visualize fibronectin by immunofluorescence microscopy. Significant co-localization of fluorescent peptide and fibronectin was observed, regardless of whether the N-cadherin binding domain was active (Fig. 5A) or scrambled (Fig. 5B). To assess binding of peptide to damaged vessel wall in vivo, the carotid arteries in male Sprague-Dawley rats were injured using a balloon catheter to denude the endothelium. Immediately after balloon injury, the common carotid was clamped at both ends and 100 pL of PBS with 500 pg/mL TAMRA-labelled chimeric peptide was instilled locally for 5 minutes. After this, the clamps were removed, and blood flow was restored. 24 hours later, binding of the TAMRA-labelled peptide was confirmed by confocal microscopy of en face vessel segments (Fig. 5C), whereas PBS-only control treated vessels lacked any red fluorescence (Fig. 5D). Autofluorescence of the elastic lamellae was evident as green signal, and Z-stacks to visualize tissue in three dimensions revealed that the N- cadherin targeting peptide was localized on the intimal surface of the vessel, as well as within the top layers of the media.

Example 1g. N-cadherin targeting chimeric peptide inhibited intimal thickening in injured rat carotid arteries, without impairing re-endothelialization.

[0136] Carotid arteries were injured using a balloon catheter to denude the endothelium. This disrupted the cell adhesions between EC and SMCs, and triggered migration of SMCs to the intima. Immediately following balloon injury, the common carotid artery segment was clamped and infused with N-cadherin targeting chimeric or scrambled control peptide at a concentration of 500 pg/mL and a volume of 100 pl for 5 minutes, followed by removal of the clamps and restoration of blood flow, closure of the surgical incisions, and the rats were allowed to recover. At 1- or 2-weeks after injury, the carotid arteries were perfusion-fixed, excised and paraffin embedded, and cross-sections were stained with hematoxylin and eosin to visualize cells and tissue layers. There was a marked reduction in intimal thickening in the vessels treated with the N-cadherin targeting chimeric peptide compared to those treated with scrambled control peptide, which was apparent at both 1 and 2 weeks after balloon injury (Fig. 6A). Measurement of the ratio of intimal:medial area revealed significant reductions in N- cadherin targeting chimeric peptide-treated vessels compared to scrambled control peptide- treated vessels (63.8% reduction at 1 week; 32.4% reduction at 2 weeks) (Fig. 6B). Cell proliferation was assessed by immunostaining for Ki67, which is expressed in proliferating cells (Fig. 8A). The percentage of medial cells proliferating was low (~1%) at 1 and 2 weeks after balloon injury. Intimal cell proliferation was highest at 1 week (~30%), then declined to ~7% at 2 weeks post injury. There were no significant differences in the percentage of proliferating cells in the intima or media comparing N-cadherin targeting and scrambled control peptide treated rats at one or two weeks after balloon injury. (Fig. 8B).

[0137] Re-endothelialization was measured by staining with Evan’s Blue dye, which binds albumin in the circulation and stains artery segments lacking an endothelium, but not those with an intact endothelial barrier. Balloon injured carotid arteries from rats treated with scrambled control peptide and the N-cadherin targeting chimeric peptide are shown in figure 6C. There was no significant difference in the percentage of endothelialized intimal surface comparing vessels treated with N-cadherin targeting chimeric peptide and the scrambled control peptide (Fig. 6D). This suggests that the N-cadherin targeting peptide did not impair the endothelial healing response.

Example 2: D-PHI Nanoparticles

[0138] Figure 9 shows a schematic for the synthesis of Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI) used in the synthesis of nanoparticles prepared and characterized according to this Example.

[0139] All data was analyzed using GraphPad Prism 7 with a one-way or two-way ANOVA and a Tukey’s multiple comparisons post hoc test, p < 0.05 was considered statistically significant. The data was also evaluated to have a normal distribution using the Shapiro-Wilk test and equal variance using the Welch’s test for unequal variances.

Example 2a: Divinyl Oligomer Synthesis

[0140] DVO was synthesized through reacting hydroxyethyl methacrylate (HEMA) (Sigma Aldrich), poly (hexamethylene carbonate) diol (PCN) (UBE Industries) and lysine diisocyanate (LDI) (Kyowa Yuka Co.) in a 2.01 :1 .00:2.00 ratio. This isocyanate reaction was catalyzed using dibutyltin dilaurate (DBDL) at 10.97 mg per gram of LDI (Sigma Aldrich). To carry out the reaction, water was removed from the starting monomers. To remove water and contaminants, LDI and HEMA were distilled under vacuum, while PCN was degassed overnight prior to the reaction. In the first step of the reaction, PCN and LDI were reacted in a 1 :2 ratio with DBDL for 4 hours at 45-50°C. HEMA was then added to the mixture and the reaction continued for another 18 hours or until there were no isocyanates that could be detected. Residual isocyanate activity was measured using an isocyanate titration [S. Sharifpoor, R. S. Labow, and J. P. Santerre, “Synthesis and characterization of degradable polar hydrophobic ionic polyurethane scaffolds for vascular tissue engineering applications,” Biomacromolecules, vol. 10, no. 10, pp. 2729-2739, 2009, doi: 10.1021/bm9004194; E. Mathieu, K. G. Battiston, J. E. Mebane, L. Davidson, E. J .Suuronen, J. P. Santerre, and R. S. Labow, “Characterization of a degradable polar hydrophobic ionic polyurethane with circulating angiogenic cells in vitro,” J. Biomater. Sci. Polym. Ed., vol. 25, no. 11 , pp. 1159-1173, 2014, doi: 10.1080/09205063.2014.923367.]. The molecular weight was calculated from NMR spectra by comparing the integration of the CH3 peak associated with the terminal HEMA and the integration of the CH3 peak associated with the LDI.

Example 2b: Emulsion Inversion Polymerization (EIP) of D-PHI Nanoparticles

[0141] Tween-20^TM (Sigma) was added at a 2:1 molar ratio of surfactant to total monomer, irrespective the number of vinyl groups, in the fabrication process.

[0142] As shown in Figure 10, DVO (150 mg) was mixed with methyl methacrylate (MMA) (Sigma Aldrich) and methacrylic acid (MAA) (Sigma Aldrich) in a 1 :5:15 ratio, respectively, to make the oil phase. After 8 hours of stirring, benzophenone (Sigma Aldrich) was added to the mixture at 0.03 mol/per vinyl group. The mixture was left to stir for 30 minutes, which ensured that the benzophenone was well incorporated with the monomers.

[0143] Polyethylene glycol sorbitan monolaurate (Tween-20^TM) (BioShop) was used as the surfactant in this emulsion process. The total amount of Tween-20 ^TM used in each reaction was based on a molar ratio of 2 to 1 (surfactant to total amount of monomers). Half of the surfactant was added to the monomer mixture (the oil phase) and the second half was added to 100 mL of Milli-Q grade water (the aqueous phase) at pH 10. After stirring the oil phase for 30 minutes, the stir rate was adjusted to 400 rpm and 10 mL of the aqueous phase was added to the oil phase by dropwise addition via gravity using a 21 -gauge needle without a stopper, over a period of 15 minutes. Once the aqueous phase was fully added to the mixture, the beaker was transferred to a fume hood and was exposed to UV light. The UV light used was the FireEdge UV LED curing system (FE300, Phoseon Technology) and the mixture was exposed at 100% intensity for 45 minutes under constant stirring at 400 rpm. Afterwards, the solution was collected and stored in a fridge at 4°C and light protected. Example 2c: Isolation of Nanoparticles

[0144] The nanoparticle solution was spun down for 5 minutes at 2000 RCF (relative centrifugal force), which collected particles with a diameter larger than 300 nm in the resultant pellet. The pellet was discarded, and the supernatant collected in 1.5 mL Eppendorf tubes was then centrifuged at 20 000 RCF for 10 minutes at 4°C. After centrifugation, the supernatant was removed and replaced with Milli-Q grade water. The mixture was vortexed and pipetted up and down until the mixture was homogenous. This process (i.e., centrifuging at 20 000 RCF and resuspending) was repeated four more times to wash away excess surfactant from the solution and remove smaller particles (< 100 nm). After the last centrifugation, the particles were collected as a homogenous nanoparticle stock and concentrated to approximately 1x10¹¹ particles/mL, by resuspending multiple pellets of the nanoparticles into the same solution.

Example 2c: Characterization

(i) Scanning Transmission Electron Microscopy (STEM; FEI Quanta 250 FEG) was used to characterize individual particle size. The stock nanoparticle solution was diluted down until the solution was transparent to the naked eye. Multiple 1 pL beads of the diluted solution were added onto a lacey carbon type A, 300 mesh, copper grids from Ted Pella. The lacey grids were placed inside of a protective holder and placed in a fume hood to air dry and keep free of air borne contaminants. Once dry, these grids were analyzed.

[0145] STEM and SEM images, in Figure 13, show D-PHI nanoparticles with average diameters between 100-300 nm showing good agreement with the sizes characterized by the DelsaMax Pro system (Figure 11) and the NanoSight system (Figure 12). The D-PHI nanoparticles appeared to be spherical in shape as shown in the close-up STEM and SEM images. Aggregates of D-PHI Nanoparticles can be seen in both Figure 13-A and Figure 13- B

[0146] (ii) Dynamic Light Scattering (DLS; Delsa Max Pro System) was used to analyze the nanoparticle distribution, average size and charge and its changes throughout the experimentation process. The sample of interest for the DLS analysis was first diluted until it was transparent to the eye using Milli-Q grade water. For samples used in cell culture, phosphate buffered saline (PBS) was used as the diluent. Once diluted, the sample was transferred into a cuvette for analysis.

[0147] Prior to centrifugation filtration of the D-PHI NPs, the nanoparticles analyzed through DLS had an approximate radius of 150 nm and high polydispersity, as seen in Figure 11 . After centrifugation at 2 000 RCF and collecting the supernatant, the average radii of the particles shifted to 60 nm, which indicated the removal of larger particles (with radii > 150 nm) from the solution. The supernatant was then centrifuged at 20 000 RCF and resuspended in MilliQ grade water five times. This process removed excess amounts of surfactant, which could negatively impact cell viability. This removal was indicated by the increase in the average radii of the analyzed nanoparticles in (Figure 11). When this solution was spun down, the supernatant was collected and analyzed through DLS. When analyzed through DLS, a comparable mixture of the surfactant only in MilliQ grade was detected to have micelles with 7 nm in diameter.

[0148] The final suspension of D-PHI nanoparticles was quite monodisperse and had a polydispersity of 0.043, as measured through DLS.

[0149] (iii) NanoSight (NTA3000) was used to quantify the concentration of the stock particle solution and the size of the particles. Stock solutions or samples were diluted with MilliQ grade water prior to analysis. The dilution step was different for the different samples and was adjusted to yield approximately 60-100 particles detected per frame on the NanoSight system, which correlates to roughly 10⁷-10¹⁰ particles/mL. Before the sample was added, either Milli-Q grade water or PBS, depending on which solvent was used to dilute the preparation, was flowed through the system to flush out any possible contaminants. The flow rate of the system, during analysis was set such that particles crossed the analysis frame every 10 seconds, which was optimal for the software particle tracking. The temperature was set to 21 °C for analysis. For analysis, three 30-second videos were taken for each sample, and the particle tracking analysis software evaluated the size and the concentration based on the average of these videos.

[0150] Subsequent analysis using the NanoSight machine, as seen in Figure 12, detected particle concentrations in the 1x10¹¹ magnitude after accounting for dilution.

Example 2d: In Vitro Cytotoxicity Assay of D-PHI Nanoparticles

[0151] Vascular Smooth Muscle Cells (A10 Cells) were cultured in 96 well plates. Cells were cultured for 24 hours in growth medium (Dulbecco’s Modified Eagles Medium containing 1 g/L D-glucose, L-glutamine and 110 mg/L sodium pyruvate (Gibco), supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin-Streptomycin (Gibco)) until the cells generated a confluent monolayer. Once confluent, the media was exchanged for the testing conditions. In all the assays, growth medium was used as a negative control whereas dimethyl sulfoxide (DMSO) (Sigma Aldrich) at 5% were used as a positive control. The cell viability was assessed for a dose response of D-PHI nanoparticles (0.05x10¹⁰, 0.25 x10¹⁰, 1 x10¹⁰, 4 x10¹⁰ particles/mL). After 24 hours of exposure to each condition, the media in each well was aspirated and 100 pL of growth media at 37 °C, supplemented with 10pL of WST-1 reagent (Roche), was added to the well. Once all the media in the wells had been exchanged, the well plates were incubated for 1 hour in a 37 °C incubator with a humid, 5% CO₂ atmosphere. After incubation, the absorbance of the media was analyzed at 450 nm in a microplate reader (EnVision Multimode Microplate Reader, Perkin Elmer).

[0152] The media was then completely removed, and cell monolayers were washed twice by carefully adding 100 pL of PBS and then aspirating it. The cell lysate solution (TNE Buffer; 50 mM Tris-HCI (pH 7.4), 100 mM NaCI, 0.1 mM EDTA) (Sigma Aldrich) was then added to the wells and left on ice for 1 hour. After an hour, the cell lysate solution was pipetted up and down and the tip of a 200 pL pipette tip was used to scratch the surface of the well, helping to ensure that all the cells from the well were collected in the solution. The solution was then collected and stored in 1.5 mL Eppendorf tubes. DNA dye was prepared by mixing 10 mL of TNE Buffer with 10 pL of 1 mg/ml_ Hoechst Dye. This solution was vortexed for 30 seconds to ensure that it was homogenous. For each well to be analyzed, 100 pL of DNA dye was placed into a well in a black well, clear bottom 96-well plate (VWR). 10 pL of each sample was added to separate wells containing the DNA dye. When possible, aluminum foil was used to cover the wells to prevent loss in fluorescence. For each 96-well plate, a standard curve of known DNA concentrations was included in duplicates. Like the wells containing samples, 100 pL of DNA dye and 10 pL of TNE Buffer was placed into a well for each concentration. After, known amounts of DNA standard (0, 2, 4, 6, 8, or 10 pL) with a concentration of 19.48 ng/mL were added to separate wells containing the DNA dye and TNE buffer mixture. The fluorescence of the well was analyzed using a microplate reader (EnVision Multimode Microplate Reader, Perkin Elmer), with an excitation filter with a wavelength of 355 nm and an emission filter with a wavelength of 460 nm. The standard curve was plotted first and then the DNA content of the samples were quantified using the standard curve.

[0153] Cytotoxic effects of the D-PHI nanoparticles were evaluated using the WST-1 assay that measured the cell proliferation and cell viability through the assessment of the mitochondrial activity in the cells. The assay was conducted with a positive control (5% DMSO in growth media), a negative control (growth media only) and reagent blanks (well plates without any cells).

[0154] As seen in Figure 16A, no significant difference was observed between the D-PHI nanoparticle-treated groups and the media-only treated groups. This indicates that the particles have no impact on cell proliferation and metabolism, as measured by WST-1 assay.

Example 2e: Hemolysis Assay

[0155] Human whole blood was collected in citrate buffered tubes. PBS was added to the citrate buffer-treated human whole blood at 1 :1 ratio and centrifuged at 5000 rpm for 5 min. Supernatant was discarded and replaced with an equal amount of PBS. This step of centrifugation and re-suspension in PBS was repeated at least 5 times. The purified red blood cells were diluted in equal amount of PBS and kept on ice. 100 pL of the purified red blood cell solution was combined with 400 pL of the PBS loaded with nanoparticles. As the hemolytic response could change with the concentration of particles, the hemolysis assay was performed as a dose response of the nanoparticles (0.05 x10¹⁰, 0.25 x10¹⁰, 1 x10¹⁰, 4 x10¹⁰ particles/mL). Triton X-100 at a concentration of 0.1 g/100 mL was used as a control to cause hemolysis. This was incubated at 37°C for 30 minutes under gentle shaking conditions. After incubation, the samples were centrifuged at 4000 rpm for 5 minutes. 100 pL of the supernatant was removed and absorbance was read at 570 nm in a microplate reader to analyze relative hemoglobin content which was calculated as a percentage of the Triton X-100 hemolytic control. This experiment was conducted with three different blood donors in duplicate. Average values and standard deviations were reported. As seen in Figure 16B, there was no significant difference observed in the release of hemoglobin between the D-PHI nanoparticle-treated groups and the PBS-treated control.

Example 2f: Stability and Storage of D-PHI Nanoparticles

[0156] To determine the stability of the nanoparticles, known concentrations of D-PHI nanoparticles were stored in 10ml_ scintillation vials under three different storage conditions: Cold Temperature at 4 °C (CT), Room Temperature at 23 °C (RT) and Accelerated Temperature at 60 °C (AT). 100 pL of the sample was taken and analyzed under DLS methods to check for particle size and population distribution at specific time points of incubation: 0 days, 2 days, 1 week, 4 weeks and 8 weeks. Three experiments (N=3) were conducted on separate days with three technical replicates (n=3) and the aggregate data was used for analysis.

[0157] As seen in Figure 15, particle degradation was only evident in the AT condition from Week 4 and onwards. At the 4-week time point, the polydispersity of the particles increased, and the peak of the particle distribution decreased, suggesting that instability was observed.

[0158] The Arrhenius equation:

Accelerated Aging Time ( /1T) = Desired (RT^/AA where AAF = Accelerated Aging Factor, AAT = Accelerated Aging Time, Desired (RT) = Desired real-time = 1 year (generally), Q10 = An Aging Factor for 10°C increase or decrease in temperature = 2 (conventionally accepted rate for a first order chemical reaction, TAA = Accelerated Aging Temperature (°C), TRT = Ambient Temperature (°C), approximates that a 10°C increase in temperature doubles the rate of degradation. Based on the equation, one week at the accelerated aging temperature of 60°C projects three months at room temperature (23°C) and approximately 1 year at a colder temperature of 4°C. Based on the data in Figure 15, the nanoparticle suspension can be stored at 4°C for up to a year without any significant changes to the particle population.

Example 2g: Nanoparticle Variations

[0159] When the divinyl oligomer was taken excluded from the monomers, no particles were detected using DLS analysis methods. The solution itself was not cloudy, signifying that there were no particles that were refracting lights in the solution. A monomer ratio of 1 :20:39 (DVO: MAA: MMA) was also tested and could be seen to generate particles in the same range. MAA was also exchanged for 2-(Dimethylamine)ethyl methacrylate (DMAEMA; Sigma- Aldrich). Results showed that it was possible to generate particles of the same size with a slightly positive zeta potential.

Example 3: Delivery of Chimeric Peptides using D-PHI Nanoparticles

[0160] D-PHI nanoparticles loaded with chimeric peptides of the present invention were prepared and characterized. The fabrication of D-PHI nanoparticles was as described in detail in Example 2. Particles can be prepared with single layer or multiple layers of proteins, peptides, or other agents.

[0161] All data was analyzed using GraphPad Prism 7 with a one-way or two-way ANOVA and a Tukey’s multiple comparisons post hoc test, p < 0.05 was considered statistically significant. The data was also evaluated to have a normal distribution using the Shapiro-Wilk test and equal variance using the Welch’s test for unequal variances.

Example 3a: Preparation of N-cadherin Peptide Solution

[0162] A peptide according to an embodiment of the present invention, including an N- cadherin binding segment, and a matrix binding segment used in this Example is shown in Figure 17A and B. A scrambled peptide, that had identical amino acids in the N-cadherin segment in a randomized incorrect order, was also designed as a control. The purity of the peptide was assessed through the combination of high performance liquid chromatography (HPLC) and mass spectrometry. The N-Cadherin peptide used had a molecular weight of 3482 Da and an isoelectric point of 4.58. The lyophilized peptide was stored at -20°C until needed. When first taken out of storage, the lyophilized peptides were dissolved in DMSO to yield stock concentrations of 50 mg/mL, aliquoted and then stored at -20°C. [0163] A fluorescent version of the peptides was made by attaching a 5-TAMRA (5- Carboxytetramethylrhodamine) to the C- terminus of the peptide. This fluorescent tag has a peak excitation wavelength of 546 nm and a peak emission wavelength of 579 nm.

Example 3b: Single Coating of peptide on D-PHI Nanoparticles

[0164] To coat the D-PHI nanoparticles with the chimeric N-cadherin peptide, the peptide was first solubilized to a concentration of 25 mg/mL, 50 mg/mL, or 100 mg/mL in DMSO (Sigma Aldrich). 5 pL of the peptide solution was added to 120 pL of D-PHI nanoparticles (particle concentration was 8 x 10⁸ particles per mL in ultrapure water (water that contained only H₂O with a resistivity of 18.2 MO. cm, TOC < 10 ppb and bacterial count < 10 CFU/mL)). This solution was then diluted in cell growth media prior to use in cell assays or ultrapure water for characterization under DLS or laser scanning confocal microscopy.

Example 3c: Specific Layer by Layer Coating Approach

[0165] As seen in Figure 18, two polyelectrolytes of opposite charge were used to perform a multi layer coating on D-PHI nanoparticles. 400 pL of Poly-L-Lysine (PLL M.W.: 4 000 - 15 000 by viscosity) (Sigma Aldrich P6516-500MG) solution, at a concentration of 4 mg/mL and diluted Milli-Q water at pH 11 (Sigma-Aldrich), was added to a 1.5 mL Eppendorf tube. The PLL solution was placed on the shaker for 30 minutes to allow time for the PLL to take on an alpha-helical structure induced by the pH. 1 mL of D-PHI nanoparticles at 1.5x10¹⁰ particles per mL, diluted with Milli-Q water at pH 7, were added to the Eppendorf tube. The solution was returned to the shaker for a further 30 minutes to allow time for PLL coating on the nanoparticle surface. 12.5 pL of Chimeric N-Cadherin peptide, at a concentration of 20 mg/mL and diluted in Milli-Q water, was added to the Eppendorf tube and was mixed on the shaker for 1 hour. The pH of the solutions was monitored using a SymPHony Benchtop pH meter (VWR) with a standard Ag/AgCI electrode (ThermoFisher part number: 9102BNWP).

Example 3d: Laser Scanning Confocal Microscopy (LCSM) and Differential Interference Contrast (DIC) Microscopy

[0166] Stock solutions of uncoated, PLL coated, or peptide coated D-PHI nanoparticles were diluted to a concentration of 1x10⁸ particles per mL in ultrapure grade water and then pipetted as 0.5 pL drops onto the glass slides. The stock solution of peptide coated D-PHI nanoparticles were made the same day and were used to create samples within an hour. These samples were then left to air dry overnight inside of a covered box with holes on the side in a fume hood. A coverslip was then placed on top of the area where the solution of particles was placed. Imaging was carried out within 48 hrs of sample preparation on an Olympus FluoView FV3000. [0167] Solutions of D-PHI nanoparticles, at a concentration of 1x10⁸ particles per ml_, and fluorescently labelled chimeric N-cadherin peptide, at a concentration of 500 pg per ml_, in ultrapure grade water were prepared and placed onto glass slides for analysis using laser scanning confocal microscopy technique. A laser (wavelength of 561 nm) was used to illuminate the sample and emission from the sample was detected from 575-675 nm. The samples were also analyzed using differential interference contrast technique [P. O. Bayguinov, D. M. Oakley, C. C. Shih, D. J. Geanon, M. S. Joens, and J. A. J. Fitzpatrick, “Modern Laser Scanning Confocal Microscopy,” Curr. Protoc. Cytom., vol. 85, no. 1 , pp. 1- 39, Jul. 2018, doi: 10.1002/cpcy.39.; A. S. Stender, “Performing spectroscopy on plasmonic nanoparticles with transmissionbased nomarski-type differential interference contrast microscopy,” J. Vis. Exp., vol. 148, no. e59411 , pp. 1-10, Jun. 2019, doi: 10.3791/59411 ; S. Y. Lee, G. W. Kim, and J. W. Ha, “Differential interference contrast microscopy imaging of single gold nanospheres beyond the quasi-static limit,” Chem. Phys. Lett., vol. 676, pp. IOS- 111 , 2017, doi: 10.1016/j.cplett.2017.03.053], The first row of images as seen in Figure 19 (A- C) display D-PHI nanoparticles without any peptide. The particles under DIC microscopy appear to range in size between 200 - 300 nm.

[0168] The second row in Figure 19 (D-F) are images of the N-cadherin peptide on its own. In Figure 19-D, the peptide showed up as red in the image. In the same image, there were two red artifacts that were likely the result of aggregated peptide. The DIC image (Figure 19-E), had two small black dots which indicated that there were particulate in the sample. The overlapped image (Figure 19-F), demonstrated that the red artifacts from the fluorescent image and the black dots from the DIC image coincided.

[0169] Figure 20-C is an image of the fluorescent peptide coated D-PHI nanoparticles. The LSCM images in Figure 20-C confirmed the coating of D-PHI nanoparticles with the chimeric N-cadherin peptide. As seen in Figure 20-C, the merged view of both the fluorescence image and the DIC microscopy image, there was a red corona of protein around the particles and virtually no florescent imaging in between the particles. As the peptide was fluorescently labelled with a TAMRA tag (with a peak excitation wavelength of 546 nm and a peak emission wavelength of 579 nm), it showed up red in the fluorescent image, whereas the differential interference contrast images helped to identify the particles or aggregates in the sample. Figure 20-B was an image of particles alone. The particles in the plane of focus showed up as white dots on the sample (those that appear black are out of the plane of focus) as they created a region in which the light waves were altered and this generated interference [Z. U. Aydm, T. Ozyiirek, ■ Bii§ra Keskin, and T. Baran, “Effect of chitosan nanoparticle, QMix, and EDTA on TotalFill BC sealers’ dentinal tubule penetration: a confocal laser scanning microscopy study,” Odontology, vol. 107, pp. 64-71 , 1234, doi: 10.1007/s10266-018-0359-0; S. M. Abdel-Hafez, R. M. Hathout, and O. A. Sammour, “Tracking the transdermal penetration pathways of optimized curcumin-loaded chitosan nanoparticles via confocal laser scanning microscopy,” Int. J. Biol. Macromol., vol. 108, pp. 753-764, 2018, doi: 10.1016/j.ijbiomac.2017.10.170], There was no fluorescence detected in the sample as no peptides were added into this solution.

[0170] Figure 19-F indicates that the peptide had the potential to aggregate.

Example 3e: Dynamic Light Scattering Characterization

[0171] DLS (DLS; Delsa Max Pro System) was used in this work to analyze the nanoparticle distribution, average size and charge, and any changes to the particles throughout the peptide coating processes. The sample of interest for the DLS analysis was first diluted until it was transparent to the eye using ultrapure water. If the sample was going to be used in cell culture, PBS at pH 7.4 was used rather than ultrapure water, as the diluent. Once diluted, the sample was transferred into the device’s cuvette for analysis.

[0172] The coating of the nanoparticles was verified by DLS as well as LSCM as seen in Figure 20 [P. Verma and S. K. Maheshwari, “Preparation of Sliver and Selenium Nanoparticles and Its Characterization by Dynamic Light Scattering and Scanning Electron Microscopy.,” J. Microsc. Ultrastruct., vol. 6, no. 4, pp. 182-187, 2018, doi: 10.4103/JMAU.JMAU_3_18; A. C. G. Weiss, K. Kruger, Q. A. Besford, M. Schlenk, K. Kempe, S. Forster, and F. Caruso, “In Situ Characterization of Protein Corona Formation on Silica Microparticles Using Confocal Laser Scanning Microscopy Combined with Microfluidics,” ACS Appl. Mater. Interfaces, vol. 11 , no. 2, pp. 2459-2469, 2019, doi: 10.1021/acsami.8b14307], Unmodified D-PHI nanoparticles were approximately 175 nm in diameter with a zeta potential of around -85 mV. Following the coating procedures described above, the resultant particles were shown to have significant changes in both size and zeta potential (P<.05). The size of the detected particles with PLL coating resulted in a size increase to 230 nm and an inversion of the zeta potential to +80 mV. Unmodified nanoparticles and PLL-coated nanoparticles increased in size following addition of peptide (approximately 260 nm and 300 nm, respectively), and showed changes in zeta potential (-13 mV and -21 mv, respectively).. Quantification of the chimeric N-cadherin is discussed further below at 3i with reference to Figures 21 and 22.

Example 3f: Peptide Release Kinetics from the D-PHI Nanoparticle

[0173] The release of fluorescent chimeric N-cadherin peptide from D-PHI nanoparticles was quantified using dialysis [A. Bernkop-Schnurch, A. Malkawi, A. Jalil, I. Nazir, B. Matuszczak, and R. Kennedy, “Self-emulsifying drug delivery systems: Hydrophobic drug polymer complexes provide a sustained release in vitro,” Mol. Pharm., vol. 17, no. 10, pp. 3709-3719, 2020, doi: 10.1021/acs.molpharmaceut.0c00389], Free peptide in solution and peptide coated D-PHI nanoparticles in solution (25 pg of peptide in PBS or 6.33 pg of D-PHI Nanoparticles coated with the same amount of peptide), were added into a dialysis bag (Biotech-Grade Cellulose Ester Dialysis Tubing, 100 000. MWCO, 16mm/10mm; 33ft by Spectra Por) [S. D’Souza, “A Review of In Vitro Drug Release Test Methods for Nano-Sized Dosage Forms,” Adv. Pharm., vol. 2014, pp. 1-12, 2014, doi: 10.1155/2014/304757], This dialysis bag was then placed into PBS at pH 7.4. The experimental set up was kept within a humidity chamber which was maintained at 37°C, with a relative humidity of 100%, and kept under constant shaking on a ThermoFisher company shaker. The release media was sampled at specific times (2 min, 5 min, 10 min, 30 min, 1-hour, 2-hour, 3-hour, 4-hour, 5-hour, 6-hour, day 1 , day 2, day 3, day 4 and day 6) and the removed volume was replaced with the equivalent amount of PBS at pH 7.4. The sampled release media was aliquoted into a 96-well clear bottom plate (Corning® 96 Well Black Polystyrene Microplate P/N: CL S3603) in 100 pL volumes. This was then analyzed using a plate reader (EnVision Multimode Microplate Reader, Perkin Elmer) , as described in Example 2d.

[0174] The quantification of peptide released from D-PHI nanoparticles during a 6-day drug release study is shown in Figure 23. The 100 000 molecular weight cut off of the dialysis bag was verified by sampling the release media and analyzing it using DLS methods (DelsaMax Pro, Beckman Coulter). No particles were detected in any of the tested release media indicating no particles passing through the membrane. All the experiments were conducted three times and each experiment had three technical replicates.

[0175] When a solution of peptide in free solution was placed inside the dialysis bag, the solution inside and outside of the dialysis bag reached equilibrium (100% release) by 2 hours (Figure 23-A). When the peptide was coated onto the surface of D-PHI nanoparticles, the time to reach equilibrium (100% release) was prolonged (P<.05). When looking at the profile of the accumulated release of the peptide, there was still an initial burst release that occurred in the first hour when in PBS at a pH of 7.4. After the first hour, there was a steady linear release up to 90% over the course of six hours (Figure 23-A). The total cumulative release of the peptide was monitored until all the peptides were released. The release reached 100% by the third day (Figure 23-B).

Example 3g: In Vitro Cytotoxicity Assay

[0176] Cytotoxic effects of the D-PHI nanoparticles were evaluated against Mouse Aortic Smooth Muscle Cells (MOVAS). MOVAS were cultured in 96 well plates. Cells were cultured for 24 hours in growth medium (Dulbecco’s Modified Eagles Medium containing 1 g/L D- glucose, L-glutamine and 110 mg/L sodium pyruvate (Gibco), supplemented with 10% Fetal Bovine Serum (Gibco) and 1% Penicillin-Streptomycin (Gibco)) until the cells generated a confluent monolayer. Once confluent, the media was exchanged for the testing conditions. In the cytotoxicity assays, growth media and reagent blanks (well plates without any cells) were used as a negative control. As the fabrication of D-PHI nanoparticles required the use of Tween-20 ^TM, as a surfactant, this experiment used Tween-20 ^TM at 1% concentration (10pl_/ml_) diluted in the cell media as a positive control to induce cell lysis and death. Cells were exposed to experimental and control conditions for 24 hours, at which point they were tested for cell metabolic activity using the WST-1 assay, as described in Example 2d. Briefly, the cells were incubated with growth media containing 10% WST-1 reagent (Roche). After 1 hour of incubation, the absorbance of the media was measured.

[0177] Once the WST-1 assay was completed, the cells were lysed with lysis buffer (TNE Buffer) to collect the DNA for analysis. Hoechst dye was added to the samples to quantify the DNA content. The cell viability was assessed at different concentrations of D-PHI nanoparticles (0.05x10¹⁰, 0.25 x10¹⁰, 1.00 x10¹⁰, 4.00 x10¹⁰ particles/mL). Higher concentrations of the D-PHI nanoparticles were also tested to attempt to determine the maximum number of particles that could be used to deliver therapeutics. A concentration of 4x10¹⁰ particles per mL reflects the highest concentration that could be produced by the emulsion inversion fabrication method as described in Example 2b. As seen in Figure 24, no significant difference was observed between the D-PHI nanoparticle-treated groups.

Example 3h: Scratch Wound Assay

[0178] MO AS cells were seeded at a density of 5x10⁴ cells per plate and cultured for 24 hours at which point they were confluent. A 200 pL pipette tip was then used to scratch two lines perpendicular to each other from one edge of the well to the opposite edge in each well along a straightedge, which made 2 wound gaps (each approximately 0.89 mm in width) that the cells closed by migrating. The wound area was measured and monitored then processed using Imaged software to quantify the area without cells. The wound area (region without cells) was defined manually in the software. 24 hours after wounding, the cell monolayerwas imaged again and the percent wound area closed was calculated by dividing the wound area after the 24-hour incubation by the original wound area. These experiments were all repeated three times with four separate regions that are distinct were evaluated as technical repeats in each well (Figure 25).

[0179] The efficacy and dose dependence of the active peptide coupled with DPHI nanoparticles were tested in the first series of experiments (Figure 26-A). After wounding, the cell monolayer was treated with 4 X 10⁸ DPHI nanoparticles per mL, which were coated with the following: Scrambled control peptide 500 pg/ml (a non-binding version of the N-cadherin peptide with the binding sequence scrambled); or Active N-cadherin binding peptide at concentrations ranging from 100-5. When treated with the scrambled control peptide coupled DPHI NPs, MOVAS g/mL cells migrated to close 90% of the wound at 24 hours (Fig. 26A). There was no significant difference in wound closure when cells were treated with DPHI NPs coated with either 100 or 250 ug/mL of active peptide. However, treatment with NPs coated with 500 pg/mL of active peptide resulted in a significant decrease in wound closure to 72%.

[0180] The second experiment (Fig. 26B) was designed to assess the efficacy of the N- cadherin targeting peptide solubilized alone in culture media, vs. coated on DPHI NPs as described in paragraph 157. Cells were wounded and imaged as described in paragraph 156. Cells were treated as follows: Vehicle (5pL/mL DMSO, the same concentration used to solubilize the peptide); DPHI NPs at 4 x 10⁸ particles per ml_; Scrambled Peptide alone (500 pg/mL); Active Peptide Alone (500 pg/mL); Scrambled Control Peptide (randomized N- cadherin binding domain) coated on DPHI NPs (500 pg/mL); and Active N-cadherin targeting Peptide coated on DPHI NPs (500 pg/mL). Treatment with Vehicle resulted in 80% wound closure after 24 hours. Treatment with DPHI NPs alone, or NPs coated with scrambled peptide did not significantly affect wound closure. Treatment with active N-cadherin peptide, either free in solution or administered on coated nanoparticles, significantly inhibited wound closure (P<.05). The results of the second scratch assay (Figure 26-B) also demonstrated that, when the active peptide was delivered on the surface of the D-PHI nanoparticles compared to the free peptide in solution, there was no significant change in peptide activity (P>0.99).

Example 3i: Quantification of the Chimeric N-cadherin

(i) Fluorescence Assay:

[0181] Chimeric N-cadherin peptide was first diluted in dimethyl sulfoxide (DMSO) at 100 mg/mL and then further diluted in PBS. Dilutions from 0.1 - 500 pg/mL were made and 100 pL aliquots of each concentration were added in triplicate to a black well, clear-bottom 96- well plate (Corning® 96 Well Black Polystyrene Microplate P/N: CL S3603). This was then analyzed using the parameters identified in Example 2d for the fluorescence plate reader (EnVision Multimode Microplate Reader, Perkin Elmer). Figure 21-A shows the graph of fluorescence intensity for peptide concentrations from 0.1 - 500 pg/mL. As seen in the graph, concentrations above 100 pg/mL were out of the linear range and should not be used for quantification. No fluorescence was detected for concentrations of the peptide below 0.08 pg/mL. The optimized standard curve was used, which included peptide concentrations from 0.08 - 1 pg/mL, seen in Figure 21-B.

[0182] For quantification of the fluorescent variant of chimeric N-cadherin peptides, 2 x 100 pL of sample were transferred to a 96-well plate (Corning® 96 Well Black Polystyrene Microplate P/N: CL S3603) and fluorescence intensity was measured against two standard calibration curves, made by diluting the free peptide solution in PBS from 0.08 - 1 g/mL, on the same microplate using a fluorescence reader (EnVision Multimode Microplate Reader, Perkin Elmer, USA). Measurements were performed at the following wavelengths: AEx = 570 nm, AEm = 610 nm, to align with the specific fluorescent marker (5- TAMRA (5- Carboxytetramethylrhodamine)) attached onto the peptide which has a peak excitation wavelength of 546 nm and a peak emission wavelength of 579 nm. Fluorescent substances and samples were protected from light whenever possible throughout experiments. Chimeric N-cadherin content of the samples was also determined using a Waters HPLC system with Waters 600 EF pump, a 4.6mm x 250 mm Kinetex 100-5 C18 column (Phenomenex, Torrance, CA), and a Waters 2996 photodiode array detector.

(ii) High performance liquid chromatography (HPLC)

[0183] A gradient method was developed to resolve the chimeric N-cadherin peptide- related release products, using a binary mobile phase of acetonitrile (HPLC grade) (Fischer Scientific Company) with 0.1% v/v trifluoroacetic acid (TFA) (mobile phase B) and water (Ultrapure Grade) with 0.1% v/v TFA (mobile phase C). During the method development, a 4.6mm x 250 mm Kinetex 100-5 C18 column (Phenomenex, Torrance, CA) was used. For the purposes of optimization, the peptide was first dissolved in DMSO at a concentration 100 mg/mL before diluting to 1 mg/mL in the mobile phase initially being passed through the HPLC system when the injection was conducted. An isocratic gradient of both mobile phases with a flow rate of 1 mL/min was first used to see under what conditions the peptide would pass the column. Using the gradient method, the peptide peaks retention time was observed to be 7- minutes with a well-defined peak area that could be quantified as seen in Figure 22-A

[0184] Calibration Curves were obtained using solutions with 0.125, 0.25, 0.5 and 1 mg/mL of peptide. Two injections, N-cadherin peptide with solubilized in DMSO and DMSO only, were conducted using an isocratic flow with 100% mobile phase C. Each sample injection was 20 pL. Chromatograms were analyzed at 235 nm. With both injections, only one peak formed which had the same peak area. When the same injections were performed with 100% mobile phase B, two peaks appeared in the chromatogram when the peptide in DMSO was injected (Figure 31), while only one peak appeared when only DMSO was injected.

Example 4: Controlled Coating Release from a Balloon Catheter

[0185] All data was analyzed using GraphPad Prism 7 with a one-way or two-way ANOVA and a Tukey’s multiple comparisons post hoc test, p < 0.05 was considered statistically significant. The data was also evaluated to have a normal distribution using the Shapiro-Wilk test and equal variance using the Welch’s test for unequal variances. All experiments included 3 technical replicates and at least 3 repeat experiments, which used different polymer batches to check for consistency.

Example 4a: Fabrication of Thin Layers of Polyethylene Oxide

[0186] To deposit layers of polyethylene-oxide onto different substrates, a spin-coating method was utilized. High molecular weight polyethylene-oxides (Mv 100,000, Sigma Aldrich, CAS #: 25322-68-3, Mv 200,000, Sigma Aldrich, CAS #: 25322-68-3) was dissolved in methanol at 1-10% weight by volume (g/100mL) of methanolm (HPLC Grade; Caledon). This mixture was heated to 50°C and stirred for 4 hours at 120 rpm, with a viscosity of 0.015 Pas at 50°C measured on a Viscotester iQ (ThermoFisher; measuring geometry: CC10 DIN/Ti). The substrate to be coated was affixed onto the spin-coater (Specialty Coating Systems, Spin Coater model P6700), and brought up to 2000 rpm. While spinning, 200pL of the polymer mixture was deposited in the middle of the substrate using a P200 Pipette (ThermoFisher). The substrate was spun more than 60 seconds, allowing for the methanol to fully evaporate.

[0187] One round of coating produced a 1.51 pm thin film on the glass substrate, which translated to approximately 2.06 mg of polyethylene-oxide deposited across the surface of the glass slide. Additional rounds of coatings were performed on the same samples. Each additional round of coating was shown to proportionally increase both the weight and thickness added to the substrate. Figure 34 shows representative SEM images of the cross-section of the PEO thin film coated onto glass slides.

(i) Dip Coating Polyethylene Oxide

[0188] A solution of 10% weight by volume (g/100 mL) of 100 000 Mw polyethylene oxide (PEO) dissolved in methanol (HPLC grade; Fisherbrand) was prepared. The polymer solution was heated to 50°C and allowed to stir overnight (> 8 hours). Once the mixture was homogenous and all the PEO had dissolved, the solution was used for dip coating. 50 mL of the polymer solution was transferred to a 100 mL beaker. A KSV NIMA Dip Coater (NanoScience Instruments) was used to dip samples in and out of the polymer solutions. Five different draw speeds (25, 50, 100, 500, 1000 mm/min) were tested to see the effect on polymer deposition on glass slides. Draw speeds 100 mm/min and less resulted in a visually even coating of polyethylene oxide. Draw speeds of 500 mm/min and above resulted in uneven and rougher to the touch coatings, as seen in Figure 30.

Example 4b: Preparation of Nylon and Latex Substrates

[0189] Nylon and latex substrates were prepared and affixed onto glass microscope slides (75 mm by 25 mm). Thin nylon sheets (0.001” thick, McMaster-Carr: 8539K199) and latex sheet (1/16” thick, McMaster-Carr: 6856K13) were cut into 25 mm by 25mm squares. Glue (Krazy Glue by Grand and Toy) was then applied to one side of the cut-out materials, ensuring that all edges had glue. This was then placed at one end of a glass microscope slide. These samples were then coated and used as representative materials for subsequent experiments.

Example 4c: Ultrasonic Spraying

[0190] Figure 33 shows a schematic for the ultrasonic spray coated fabrication technique.

[0191] High molecular weight PEG was dissolved in methanol at a 2% weight to volume ratio or 2 gram of polymer in 100 mL of solvent. This mixture was heated to 50°C and stirred for 1 hour at 120 rpm, with a viscosity of 0.015 Pas at 50°C measured on a Viscotester iQ (ThermoFisher; measuring geometry: CC10 DIN/Ti). The substrate to be coated was affixed inside the ultrasonic sprayer (PRISM 400 Benchtop Ultrasonic Spray Coating System by Ultrasonic Systems Inc.). Once the polymer solution was homogenous, it was loaded into the ultrasonic sprayer and a coating protocol was loaded in the spray-programmer. This coating protocol could be repeated several times to produce different coating thicknesses.

[0192] Using the ultrasonic spray coating protocol above, it was possible to generate thin coats of PEO blends using 100,000 and 200,000 Mw PEO onto different substrates like the representative image in Figure 36.

Example 4d: Scanning Electron Microscopy

[0193] PEO coated glass slides were prepared for scanning electron microscopy. Glass slides were first submerged in liquid nitrogen and then cracked into several small pieces. Small pieces with a straight edge were affixed at a 90-degree angle onto aluminum stubs using carbon tape and hot glue. Other fragmented pieces were also affixed flat onto the aluminum stubs (Electron Microscopy Sciences) using carbon tape (Ted Pella). These samples were then coated with a visible layer of platinum and then analyzed using a Hitachi FlexSEM 1000 Scanning Electron Microscope.

[0194] A representative cross sectional scanning electron microscopy image of the PEO thin film on a glass slide can be seen in Figure 36.

Example 4e: Dissolution Test

[0195] Substrates were prepared using the protocol described in Example 4b, and were coated with 10 layers of PEO using the methods described in Example 4c. Substrates were weighed prior to and post the coating process. Then they were attached onto a holder and submerged for specific times (0, 0.5, 1 , 2, 3 and 5 minutes) to capture the material loss over time. PBS at 37°C was used as the release media and was stirred at 50 rpm. Once out of the release media, the samples were air dried in a protective case for 48 hours under a fume hood. When fully dry, they were then weighed, and the percent and absolute loss of the coating was quantified.

[0196] Three different molecular weight blends of PEO (low molecular weight (M.W. = 4 000), high molecular weight (M.W. = 100 000) and a 50%/50% blend of both) were spun coated onto glass slides and the coating’s dissolution was tested using the protocol described above. The results of the dissolution testing are provided in Figure 35. In comparison to the other two blends, the coating made of 4 000 molecular weight PEO (low molecular weight blend) experienced a rapid loss in mass. The coating immediately lost 50% of its mass within 1 minute. On the other hand, the coating of 100 000 Mw PEO retained more than 80% of its mass until the 4th minute demonstrating a delayed dissolution profile. When a 50%/50% blend of both PEO polymers was tested, the coating lost about 20% of its mass within the first minute and gradually dissolved over the course of the next 4 minutes.

[0197] 5 layers of thinly coated PEO were able to generate a dissolution profile that retained over 80% of its mass after 2 minutes, as seen in Figure 37. This experiment was repeated with latex, an alternative balloon catheter material, and with D-PHI nanoparticles (approximately 0.79 mg) contained in the first deposited layer onto the substrates, fabricated as described in Example 2, incorporated into the polymer coating. The change in substrate and the incorporation of particles had no significant effect on the dissolution profile of the coating when compared to the original nylon substrate.

Example 4f: Cytotoxicity test (Cell Proliferation and Viability Assay)

[0198] Mouse aortic smooth muscle cells (MOVAS) were cultured in 96 well plates. Cells were cultured for 24 hours in growth medium (Dulbecco’s Modified Eagles Medium containing 1 g/L D-glucose, L-glutamine and 110 mg/L sodium pyruvate (Gibco), supplemented with 10% Fetal Bovine Serum (Gibco) and 1 % Penicillin-Streptomycin (Gibco)) until the cells generated a confluent monolayer. Once confluent, the media was exchanged for the testing conditions. In all the assays, growth medium was used as a negative control while Tween-20 ^TM (Bio Shop) or DMSO (Sigma Aldrich) at 10% were used as a positive control. Solutions of 2%, 1%, 0.2% and 0.02% 100 000 Mw PEO weight by volume (g/100 mL) in media were used as the testing conditions.

[0199] After 24 hours of exposure, the cell media was changed again for media supplemented with 10% WST-1 assay. The following controls were used: a positive control (10% Tween-20 ^TM in growth media), a negative control (growth media only) and reagent blanks (well plates without any cells). The well plates were then incubated for an hour and then the absorbance of the media was analyzed. The media was then completely removed, and cell monolayers were washed twice by adding 100 pL of PBS and then aspirating it. The cell lysate solution (TNE Buffer; 50 mM Tris-HCI (pH 7.4), 100 mM NaCI, 0.1 mM EDTA) (Sigma Aldrich) was then added to the wells and left on ice for 1 hour. The cell solution was isolated, collected, and then quantified for DNA content using Hoechst Dye and a known standard curve.

[0200] Based on the data in Figure 36, the average weight of PEO coating per square millimeter was 8.65 pg/mm². A cell monolayer cultured in a 96 well plate (with a growth area of 0.32 cm²) was expected to be exposed to about 277 pg of PEO. The 2% weight by volume concentration used in the cytotoxicity tests resulted in 4 mg of PEO in each well. As seen in Figure 38, even at the highest concentration, the PEO did not have any significant impact on the cell monolayers’ metabolic activity and proliferation, as measured by WST-1 and DNA quantification. The dilutions of this stock solution, from 50% to 1%, were also not found to have any cytotoxic effect on the cell monolayer, as measured by WST-1 and DNA quantification.

Example 4g: Delivering Peptide Coated Nanoparticles on Balloon Catheter Substrates

[0201] A solution of peptide coated D-PHI nanoparticles was generated using the protocol from Example 3. A nylon substrate was prepared and fixed onto a glass slide according to Example 4b. The nanoparticle solution was then pipetted in 10 pL aliquots onto the nylon substrate and allowed to air dry. The nylon substrate was then coated with 100 000 Mw PEO according to the protocol in Example 4c. The PEO was deposited to protect the loss of nanoparticles during the time it takes to track a balloon to the treatment site during a balloon angioplasty. A glass slide was then placed over the coated region and the sample was viewed under laser scanning confocal microscopy, as seen in Figure 29. The image looked at the edge of an area in which fluorescently tagged peptide, which showed up in red, was pipetted onto the nylon substrate. To the right and below the image, there are z-stack (cross-sectional) images of the area of interest. These z-stack images indicated that the peptide was generally localized on one plane.

Example 5: Delivery of Fluorescent-tagged Chimeric Peptide to Balloon-Injured Rat Carotid Arteries

[0202] With reference to Figure 39, fluorescent-tagged chimeric N-cadherin targeting peptide (see Example 3a) at concentrations of either 10 or 500 pg/mL, and NPs (see Example 2) were diluted in PBS and mixed together by rotation for one hour, then were administered to balloon-injured rat carotid arteries for 5 minutes and held within the injured segment by tying off each end with suture. Or, peptide and nanoparticles were coated onto balloon catheters and delivered by inflation and rotation. (Briefly, balloon catheters were prepared by coating layers onto a deflated latex balloon in the following order: a hydrophilic spacer layer (PEO), a NCAD mimetic peptide-coated D-PHI nanoparticles layer, and a PEO sacrificial coating, and then dried.) The arteries were then flushed with PBS, fixed with 4% paraformaldehyde, dissected and prepared en face for imaging by laser-scanning confocal microscopy. With reference to Figure 39, fluorescence intensity of the tagged peptide was quantified for each set of images in three locations from the lumen of the artery: through the internal elastic lamina (IEL), through the first layer of smooth muscle cells (SMCs), and through the second layer of smooth muscle cells. The addition of D-PHI nanoparticles to 500 pg/mL of N-cadherin peptide significantly increased the measured fluorescence intensity of the delivered peptide in the IEL and first layer of SMCs as compared to 500 ug/mL of N-cadherin peptide alone. Controls of PBS alone and NPs with a low concentration of peptide (10 pg/mL) established a lack of background fluorescence for the solution and nanoparticles. The delivery from a coated balloon further enhanced the deposition in the tissue and fluorescence measured. This Example provides evidence that NPs can sequester free peptide in solution to bind to the tissues and that it can be delivered effectively by coated balloon.

Example 6: Permeability of human endothelial cell line to N-cadherin targeting peptide

[0203] Transwell inserts with 0.4 pm pores were coated with 20 pg/mL of fibronectin for 1 h, seeded with HUVECs a density of 100,000/cm², and incubated for 3 days. The media in the top chamber was replaced with phenol-free DMEM containing 500pg/mL of N-cadherin targeting peptide or scrambled control as well as 5 pM FITC-Dextran, and the media in the bottom (abluminal) chamber was replaced with phenol-free DMEM. 100 pL of abluminal chamber media was sampled at 0.5h, 3h and 24h into a 96-well dish and luminescence read by plate reader with excitation of 495 nm and emission at 530 nm. Concentration of FITC- dextran was quantified by interpolation on a standard curve. Results are shown in Figure 40.

Example 7: In vivo and in vitro apoptosis after N-cadherin targeting peptide treatment

[0204] Carotid artery tissues from balloon-injured and peptide-treated rats at 1 week postinjury were cross-sectioned with 4 pm thickness and stained by immunohistochemistry for cleaved caspase 3 (CC3) as a marker for apoptosis. Deparaffinization with stepwise ethanol baths was followed by permeabilization in 0.2% triton, blocking in 0.2% BSA, and incubation with primary anti-CC3 antibody (CST #9664). Positively stained cells were counted as a percentage of total cells in sections from three distinct locations along each injured vessel segment for each specimen. Results are shown in Figure 41.

[0205] HUVECs and rat neointimal smooth muscle cells (RNISMCs) isolated and cultured from balloon-injured rat carotid arteries were plated on glass coverslips in 24-well dishes for 24h and treated with 500 pg/mL of N-cadherin targeting peptide or scrambled control for 24h. Cells were fixed with 4% paraformaldehyde and stained with DAPI, phalloidin, and anticleaved caspase 3 as a marker for apoptosis (CST #9664). CC3-positive cells were counted as a percentage of total cell nuclei. Results are shown in Figures 42 and 43.

[0206] These results show that the N-cadherin targeting peptide does not increase baseline levels apoptosis of vascular smooth muscle or endothelial cells when administered in vitro or in vivo compared to the scrambled control. This provides evidence of lack of cell toxicity and safety of the peptide.

Example 8: Endothelial cell junctions

[0207] HUVECs plated on coverslips were treated for 24h with 500 pg/mL N-cadherin targeting peptide or scrambled control, fixed with 4% paraformaldehyde, and stained with DAPI, phalloidin, and anti-VE-cadherin to visualize cell junctions (Abeam ab33168). Cells were imaged by laser-scanning confocal microscopy (Olympus FV3000). Images collected with 10x objective were used for quantification of total fluorescence intensity of VE-cadherin normalized to intensity of filamentous actin. Images taken with 60x objective were taken for qualitative observation. Quantification results are shown in Figure 44. This shows that the N-cadherin targeting peptide does not disrupt intercellular adhesions between endothelial cells, and therefore does not disrupt the endothelial permeability barrier (confirming results shown in figure 40). Taken together, this data suggests the peptide will not disrupt the permeability barrier in endothelial cells lining the blood vessels, and will not disrupt the establishment of a new barrier in the regenerated endothelium after angioplasty.

Example 9: Markers of endothelial activation (inflammation and thrombosis)

[0208] HUVECs plated in 6-well dishes were treated for 24h with 500 pg/mL N-cadherin targeting peptide or scrambled control and RNA was extracted (Qiagen RNeasy Plus 74134). RNA was reverse transcribed to cDNA (Qiagen RT kit 205111) and qPCR performed (BioRad CFX384) to measure expression of transcripts for intercellular adhesion molecule 1 (ICAM1), vascular endothelial cell adhesion molecule 1 (VCAM1) and thrombomodulin (THBD). Expression was quantified by AACt method and normalized to two housekeeping genes phosphoglycerate kinase 1 (PGK1) and TATA-binding protein (TBP). Results are shown in Figure 45. These results show that treatment with the peptide does not adversely affect endothelial cell functions in inflammation or thrombosis, as the adhesion markers and thrombomodulin are commonly surveyed and upregulated with endothelial injury and inflammation.

Claims

WHAT IS CLAIMED IS:

1 . A chimeric peptide comprising an N-cadherin binding domain and a domain that binds a component of the extracellular matrix, wherein the chimeric peptide inhibits smooth muscle cell migration.

2. The chimeric peptide of claim 1 , wherein the domain that binds a component of the extracellular matrix comprises an amino acid sequence selected from the group consisting of:

LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10);

LTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 11);

KLTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 12); KPSYGFGGHNSVDFEEDTLPKV (SEQ ID NO: 13); ETVTIVEDTRPKLVFHFDNNEPKVE (SEQ ID NO: 14); KLKSQLVKRK (SEQ ID NO: 15);

KNGRYSISR (SEQ ID NO: 16);

KVGKSPPVR (SEQ ID NO: 17);

KTFGKMKPR (SEQ ID NO: 18);

STMMSRSHKTRSHHV (SEQ ID NO: 19);

AAYLEQLN (SEQ ID NO: 20);

GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 21); and WREPSFCALS (SEQ ID NO: 22); or a functional fragment or variant thereof, preferably the amino acid sequence LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10) or a functional fragment or variant thereof.

3. A chimeric peptide comprising an N-cadherin binding domain and a fibronectin- binding domain, preferably a fibronectin binding domain derived from a bacterial source, more preferably from the species Staphylococcus aureus, more preferably comprising an amino acid sequence selected from LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10); LTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 11); KLTGQYDKNLVTTVEEEYDSK (SEQ ID NO: 12); and

KPSYGFGGHNSVDFEEDTLPKV (SEQ ID NO: 13); and most preferably comprising an amino acid sequence LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10).

4. The chimeric peptide of any one of claims 1 to 3, wherein the N-cadherin binding domain comprises the HAV binding motif, preferably comprising LRAHAVDING (SEQ ID NO: 23).

5. The chimeric peptide of claim 4, comprising a N-cadherin binding domain comprising or consisting of an amino acid sequence selected from CLRAHAVDING (SEQ ID NO:

24) and a fibronectin binding domain comprising or consisting of LTGQYDKNLVTTVEEEYD (SEQ ID NO: 10).

6. The chimeric peptide of any one of claims 1 to 5 having the structure:

7. A nanoparticle comprising the chimeric peptide of any one of claims 1 to 6.

8. The nanoparticle of claim 7, wherein nanoparticle comprises, consists or consists essentially of a Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI), and the chimeric peptide.

9. The nanoparticle of claim 7 or 8, wherein the nanoparticle comprises alternating layers of polymer and the chimeric peptide.

10. A method of making the nanoparticle of any one of claims 7 to 9 comprising depositing alternating layers of a cationic polymer, optionally poly-L-lysine or poly-L-histidine, preferably poly-L-lysine, and the chimeric peptide.

11. A coating for a medical device comprising the chimeric peptide of any one of claims 1 to 6 or a nanoparticle of any one of claims 7 to 9.

12. The coating of claim 11 , further comprising polyethylene oxide (PEO), wherein preferably the PEO has a molecular weight of between about 50,000 and about 200,000.

13. The coating of claim 11 or 12, wherein the coating is a composite coating comprising a peptide-containing layer comprising the chimeric peptide and nanoparticles for application to a surface of the medical device and a protective layer for application over the peptide-containing layer, preferably wherein the protective layer comprises PEO, polyethylene glycol (PEG) or hyaluronic acid, preferably PEO according to claim 12.

14. The chimeric peptide of any one of claims 1 to 6, the nanoparticle of any one of claims 7 to 9 or the coating of any one of claims 11 to 13 for use in inhibiting migration of smooth muscle cells (SMCs).

15. A medical device, optionally a stent or angioplasty balloon, comprising a coating according to any one of claims 11 to 13.

16. The medical device of claim 15, wherein the coating is a coating according to claim 13, wherein the protective layer comprises or consists of PEO according to claim 12.

17. The medical device of claim 16, wherein the average weight of the protective coating per square millimeter is between about 0.1 pg/mm² and about 1 pg/mm², preferably about 0.5 pg/mm² and/or wherein at least 80%, at least 90% or at least 99% of the protective coating dissolves within 4 minutes under physiological conditions.

18. A method for inhibiting deposition of thrombotic material on an implantable medical device for implantation in a patient in need thereof, comprising: applying a coating of any one of claims 11 to 13 to a surface of the medical device.

19. The method of claim 18, wherein the method comprises applying the peptide- containing layer by a pH-driven coating method and/or applying the protective layer by spray coating, ultrasonic spray coating, spin-coating, dip-coating, or micropipetting.

20. The method of claim 18 or 19, wherein the implantable medical device is for the treatment of atherosclerosis or restenosis.

21. A method for treating restenosis in a subject, comprising implanting the medical device of any one of claims 15 to 17 in a blood vessel of the subject.

22. The method of claim 21 , wherein the restenosis occurs after angioplasty or vascular stent placement.

23. The method of claim 21 or 22, wherein the subject has coronary artery disease, coronary heart disease, peripheral artery disease, or dysfunctional arteriovenous (AV) fistula.

24. The method of any one of claims 21 to 23, wherein the blood vessel is a coronary artery, peripheral artery, or AV fistula.

25. A nanoparticle for peptide delivery comprising, consisting or consisting essentially of a Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI).

26. A composition for peptide delivery comprising, consisting or consisting essentially of nanoparticles according to claim 25 and, optionally a carrier, the nanoparticles having an average radii between about 20 nm and about 150 nm, preferably between about 30 nm and about 110 nm, and/or a polydispersity index of less than about 0.1 as measured by Dynamic Light Scattering (DLS), preferably less than about 0.05 as measured by DLS.

27. A medicament comprising: a therapeutically effective amount of a chimeric peptide as defined in any one of claims 1 to 6 and a nanoparticle capable of binding the chimeric peptide under physiological conditions.

28. The medicament of claim 27, wherein the nanoparticle is the nanoparticle as defined in claim 25.

29. The medicament of claim 27 or 28, wherein the chimeric peptide and the nanoparticle are for administration sequentially.

30. A method of treating restenosis in a subject comprising administering a therapeutically effective amount of the chimeric peptide of any one of claims 1 to 6, the composition of claim 26 or the medicament of any one of claims 27 to 29 to a subject in need thereof.