Statin Targeted Treatment Against Intimal Hyperplasia Using Unique Chitosan-PLGA Nanoparticles

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Peters, Gloria Grace Poland, Maleen Cabe, Chanpreet Kaur, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4601140/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: Statins have pleiotropic effects, including reducing intimal hyperplasia (IH). Using unique nanoparticle (NPs), we hypothesized chitosan-functionalized polymeric NPs loaded with simvastatin (SL-cNPs) would: 1) readily associate with endothelial cells (ECs) and vascular smooth muscle cells (VSMCs); 2) affect EC and VSMC function; and 3) reduce IH compared to systemic simvastatin. Methods Human aortic ECs and VSMCs were cultured with SL-cNPs tagged with fluorescent tracer. Association of SL-cNPs was assessed by immunostaining and flow cytometry. The effect of SL-cNPs, empty cNPs (E-cNPs) and free simvastatin on cells was determined using RT-qPCR for RhoA and RhoB. Carotid artery balloon injured rats were treated intraoperatively with intraluminal saline, E-cNPs, low or high dose SL-cNPs; or with pre- and post-operative oral simvastatin plus intraoperative intraluminal saline or low dose SL-cNPs. Rats were euthanized (day 14) and IH was quantified. Results SL-cNPs readily associated with ECs and VSMCs. Low and high dose SL-cNPs induced significant increases in EC and VSMC RhoA gene expression. High dose SL-cNPs induced a significant increase in EC RhoB expression, while free simvastatin, low and high dose SL-cNPs significantly increased RhoB expression in VSMCs. In vivo , oral simvastatin plus intraluminal SL-cNPs significantly reduced IH compared to controls. Conclusion cNPs can be used as a novel vehicle to locally deliver statins to vascular cells. Although only the combination of oral simvastatin and SL-cNPs effectively reduced IH, different routes of delivery and/or concentration of SL-cNPs may allow for a more robust effect on IH prevention. chitosan poly-lactic-co-glycolic acid nanoparticles intimal hyperplasia peripheral arterial disease statins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Restenosis secondary to intimal hyperplasia (IH) after percutaneous transluminal balloon angioplasty (PTA) for peripheral arterial disease (PAD) remains an ongoing challenge that leads to reintervention and poor patient outcomes [ 1 – 4 ]. Depending on the location and type of intervention, secondary occlusion ranges from 20–60% [ 5 ]. To help mitigate this important clinical problem, understanding the pathophysiology of IH is crucial. IH is triggered by endothelial cell (EC) denudation and vascular smooth muscle cell (VSMC) injury [ 3 , 6 ]. The endothelial damage then stimulates the production of proinflammatory molecules and activation of circulatory monocytes that initiate excessive neointima formation through VSMC migration, VSMC proliferation, and excessive extracellular matrix deposition [ 3 , 6 – 8 ]. While the application of drug eluting systems, such as paclitaxel coated balloons and stents, was a major advance in the prevention of IH for coronary artery occlusive disease, this technology remains controversial in PAD management [ 9 – 12 ] [ 4 ]. Therefore, the lack of effective treatments for the prevention of IH in PAD after PTA represents an important gap in our knowledge. Statins are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG CoA) reductase, blocking a rate-limiting step in the mevalonate/cholesterol synthesis pathway [ 13 ]. In addition to reducing cholesterol levels, inhibition of HMG-CoA also prevents the production of isoprenoid intermediates. This in turn disrupts the signaling of small G proteins, including Ras and Rho, that are post-translationally modified by isoprenoids. The inhibition of isoprenoid intermediates largely enable statins known beneficial pleiotropic effects, including reducing IH [ 14 – 16 ]. For example, statins have been demonstrated to: 1) reduce VSMC migration and proliferation [ 17 ]; 2) attenuate vascular inflammation by reducing leukocyte adhesion and trans-endothelial migration [ 18 – 22 ]; and 3) accelerate reendothelialization by mobilizing, differentiating and improving survival of resident and circulating endothelial progenitor cells [ 23 ]. In animal models, oral simvastatin has been shown to reduce IH by 25% [ 24 ]. Unfortunately, in the clinical realm, statins are not tolerated in nine percent of patients due to negative-off target effects [ 17 ]. Furthermore, systemic delivery of statins has not proven to be highly effective in preventing IH due to statins’ low solubility, rapid metabolism and low bioavailability [ 25 ]. Because of these issues, research has focused on improving in vivo drug delivery through the use of localized therapies. Currently, to maximize statins’ pleiotropic effects while minimizing systemic toxicity, studies are investigating the use of statins loaded into drug delivery carriers, including nanoparticles (NPs) [ 25 , 26 ]. While NPs have demonstrated the ability to increase the solubility, stability and absorption of statins, no FDA approved statin-loaded NP for clinical use currently exists [ 25 ]. Poly-lactic-co-glycolic acid (PLGA) is an FDA approved biodegradable polymer that can be formed into NPs that release incorporated agents in a controlled, localized fashion. Recent preclinical studies have investigated polymeric NPs in cancer and cardiovascular research, with one study investigating pitavastatin eluting stents in coronary artery disease [ 25 , 27 , 28 ]. While this study demonstrated promise towards IH treatment with nanotechnology, variability in NP formulations now raises questions concerning the optimal delivery vehicle(s) for IH and its effect on VSMC and EC physiology. Chitosan, a naturally occurring biopolymer derived from crustacean shells, is polycationic. Studies have indicated coating NPs with positively-charged chitosan improves adhesion and solubility compared to PLGA alone, thereby optimizing release of embedded agents [ 29 , 30 ]. The purpose of this study was to determine the feasibility and therapeutic efficacy of simvastatin-loaded chitosan-PLGA NPs (SL-cNPs) on IH in a rat carotid artery injury model. Given that many PAD interventions require balloon angioplasty without stent placement, this study focuses on an intraluminal therapy that would not require the placement of a stent. We hypothesized that SL-cNPs would: 1) readily associate with ECs and VSMCs in vitro ; 2) affect EC and VSMC function; and 3) reduce IH compared to systemic simvastatin in vivo . Methods Materials Human aortic ECs, human VSMCs, and corresponding cell culture media were purchased from Cell Applications, Inc (San Diego, CA). RNA extraction was performed utilizing QIAGEN RNA extraction kits (Germantown, MD). All PCR primers were from Applied Biosystems (Waltham, MA). For nanoparticle synthesis, ester-terminated poly(lactic-co-glycolic) acid (PLGA 85:15) was obtained from LACTEL (Birmingham, AL). Dichloromethane (DCM), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), poly(vinyl alcohol) (PVA, 31,000–50,000 Da, 87–89% hydrolyzed), and chitosan (low molecular weight (50,000-190,000 Da, 75%-85% deacetylated) were purchased from Sigma-Aldrich (St. Louis, MO). Simvastatin was obtained from Cayman Chemical (Ann Arbor, MI). 1,1’-dioactadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) was from Thermofisher Scientific (Waltham, MA). Simvastatin for rat chocolate-flavored treats was purchased from Fisher Scientific (Hampton, NH) and sent to Bio-Serv (Flemington, NJ) where the rodent specific oral simvastatin chocolate-flavored treats were manufactured (4 milligrams simvastatin/treat). Nanoparticle Synthesis Chitosan functionalized PLGA nanoparticles (cNPs) were prepared by oil-in-water single emulsion, using commercially available polymer (85:15, viscosity 0.55–0.75) as previously described [ 30 ]. Chitosan stock solution was made by dissolving chitosan powder in 1% acetic acid to form 1% ( w/ v) and diluted 1:1 with PVA to make solutions. PLGA polymer was first dissolved in an organic solvent (0.6mL MeCN and 0.4mL DCM) and then added dropwise into an aqueous solution containing 5% PVA and 0.5% chitosan (6mL) under vigorous vortexing. The emulsion was formed by sonication (sonication amplitude 70%) using an ultrasonic processor (GE130PB, Cole-Parmer, Vernon Hills, IL) for 10 rounds of 30 seconds on and 30 seconds off. After sonication, the emulsified mixture was added to a 1 L beaker containing PVA solution (0.5% PVA and 0.5% chitosan (45mL)) and stirred overnight to allow evaporation of the organic solvent. The cNPs were collected and washed with diH 2 O with a Sorvall RC-5B centrifuge, (refrigerated, superspeed). Washed cNPs were dispersed in 2% sucrose in diH 2 O, frozen at -80°C, freeze-dried (Edwards K4 Modulyo Freeze Dryer) and stored at a -20°C in a desiccator. Lipophilic tracer DiD with excitation/emission wavelength at 646/663nm (0.3% w/w , Invitrogen) was loaded into cNPs by addition to the organic phase. Drug loaded cNPs (SL-cNPs) were made with the addition of simvastatin (4% w/w drug loading) to the organic phase; empty cNPs (E-cNPs) did not receive drug but did receive DiD. Nanoparticle Characterization Hydrodynamic diameter (size, nm) and polydispersity index (PDI) were determined with dynamic light scattering, and zeta potential (mV) was calculated after determining electrophoretic mobility, using a Zetasizer Nano ZS90 (Malvern Panalytical, Westborough, MA). cNPs were suspended in deionized water (0.1 mg/ml) and transferred to a disposable polystyrene cuvette or capillary cell. Suspension was equilibrated for 3 minutes in the cuvette and measured at 90° angle. Each measurement was an average of four separate, consecutive measurements. Drug loading was quantified using high performance liquid chromatography (HPLC). Briefly, cNPs were solubilized in DMSO (4 mg/ml), and compounds were separated on a reversed-phase C18 column with a mobile phase of 70:30 acetonitrile: formic acid (0.05N). Simvastatin was detected by UV absorbance at 240 nm, and integrated peak areas were extrapolated to an external standard curve. Cell Culture ECs (passage 3–10) and VSMCs (passage 3–5) were plated in 6-well plates or 25 cm 2 flasks. Cells were maintained in an incubator at 95% O 2 /5% CO2, 37 o C. Cell viability was determined with the Trypan blue exclusion assay using the Countess cell counter (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Only cells with > 90% viability were used. Cell Staining Autoclaved 0.5mm thickness glass coverslips were placed in 6-well plates (Fisher Scientific, Hampton, NJ). ECs and VSMCs were individually seeded to 50% confluence. A stock solution of 2 mg/mL cNPs encapsulated with DiD was made using EC or VSMC basal media. Cells were washed once with PBS, and then treated with either basal media or treatment media (0.2mg cNPs) for 30 minutes. Media was then removed, cells washed and fresh basal media was then added to the cells and incubated for 24 hours. The cells were fixed and stained with DAPI (4’,6- Diamidino-2-Phenylindole, Dihydrochloride) and Alexa Fluor 488 Phalloidin (Invitrogen, Carlsbad, CA) per the manufacturer’s instructions. Imaging was performed using a fluorescent microscope. The experiment was repeated three times. Uptake of cNPs was determined by visualization of DiD fluorescence (excitation 646 nm, emission 664nm). Flow Cytometry ECs or VSMCs were seeded to 50% confluence in 25cm 2 flasks. The treatment media was prepared by making a stock solution of DiD encapsulated cNPs in basal media, as previously described [ 30 ]. Cells were treated with either basal media or treatment media (0.2mg cNPs) for 30 minutes. The media was then replaced with fresh basal media and incubated for 24 hours. The cells were then harvested and placed into flow cytometry tubes. Greater than 92% viability was confirmed with a trypan blue assay. Flow cytometry was performed by the Loyola University of Chicago Flow Cytometry Core Facility. Acquisition was performed on a 4-Laser LSRFortessa from BD Biosciences using BD FACSDiva Software (version 9.0). Analysis was performed with FlowJo Software (version 10.9.0) from BD Biosciences (Franklin Lakes, NJ). In vitro Evaluation of SL-cNP Function Cells were treated with basal media or basal media containing E-cNPs (0.2mg), free simvastatin (10µM), low-dose SL-cNPs (0.2mg) or high-dose SL-cNPs (2mg). After 30 minutes, treatment solutions were removed and replaced with fresh basal media. After 24 hours, total RNA was extracted from the cells. cDNA was generated and quantitative real-time PCR performed using human-specific primers to RHOA or RHOB [ 31 ]. For each sample, GAPDH was used as a reference control. Rat Carotid Artery Balloon Injury Model All animal protocols were approved by Loyola University of Chicago IACUC and Edward Hines, Jr Veteran Affairs Hospital IACUC. Age and weight-matched male Sprague-Dawley rats were purchased at 17–20 weeks at 400 grams from Envigo (Indianapolis, IN). In total, 6 experimental groups (n = 6–9 animals for each group) received intraluminal balloon injury using a 2 French Fogarty Catheter (5 atmospheres/5 minutes, Medline, Northfield, IL) in the common carotid artery. The experimental groups consisted of: intraluminal normal saline (control), oral simvastatin plus intraluminal saline, cNPs without simvastatin (E-cNPs), intraluminal low-dose SL-cNPs (0.2mg), intraluminal high-dose SL-cNPs (2mg) or oral simvastatin plus low-dose intraluminal SL-cNPs. To induce carotid artery balloon injury, the left superior thyroid, occipital and the distal external carotid arteries were ligated. The internal and common carotid arteries were clamped, an arteriotomy was made at the proximal external carotid artery, and the Fogarty Catheter was inserted and inflated at 5 atm for 5 minutes. After intraluminal balloon injury was performed, the common carotid artery received an intraluminal infusion of either normal saline (control group), SL-cNPs or E-cNPs via a 27-gauge blunted needle inserted into the arteriotomy. The infusion was administered gently under pressure and held in place for 30 minutes. Animals receiving oral simvastatin were pretreated with simvastatin (10mg/kg/day) via chocolate-flavored treats for 1 week prior to surgery and for 14 days postoperatively. Rats receiving oral simvastatin required food rationing to ensure the complete ingestion of treat. Normal chow was provided ad libitum for the other experimental groups. All rats received water ad libitum. On postoperative day 14, animals were euthanized and perfusion fixed with formalin. Right and left carotid arteries were harvested, preserved in formalin, and sectioned and stained with hematoxylin and eosin. Morphometric analysis was performed using Motic Images Plus 3.0 (Kowloon Bay, Kowloon, Hong Kong) using intimal-medial area ratio defined as intima/(intima + media). Statistical Analysis Statistical analysis for RhoA and RhoB mRNA content and intimal hyperplasia was analyzed using two-way ANOVA with p < 0.05 being considered significant. Results Characteristics of E-NPs and SL-NPs Hydrodynamic diameters of SL-cNPs and E-cNPs were 182.45 ± 1.38 nm and 179.63 ± 1.32nm, respectively (Fig. 1 a). PDI and zeta potential of SL-cNPs were 0.12 ± 0.019 and + 11.49 ± 5.59. PDI and zeta potential of E-cNPs were 0.098 ± 0.009 and + 4.49 ± 0.35 (Fig. 1 b & 1 c). No significant differences existed between SL-cNPs and E-cNPs. Encapsulation efficiency of cNPs was 71.78% ± 2.86% (actual drug loading/theoretical drug loading x 100%). Actual drug loading of simvastatin in cNPs was 2.77% w/w . We utilized 0.2mg of SL-cNPs as low-dose (effectively locally administering 5.5 \(\mu\) g simvastatin) and 2mg SL-cNPs as high-dose (effectively locally administering 55 \(\mu\) g simvastatin). Cumulative drug release was determined previously and approached 100% at 100 hours [ 30 ]. SL-cNPs readily associate with ECs and VSMCs ECs and VSMCs exposed to SL-cNPs demonstrated increased red fluorescence in immunostaining (Fig. 2 b, 2 f) and increased allophycocyanin (APC) intensity in flow cytometry (Fig. 2 d, 2 h). Analysis demonstrated SL-cNP uptake in ECs was 79.6% (78-82.8%) while VSMCs uptake of SL-cNPs was 46.4% (43.4–49.4%). There was no significant difference between total number of untreated compared to treated ECs (45589 ± 1864 vs 48501 ± 77 cells, respectively) or VSMCs (52367 ± 863 vs 55549 ± 3266 cells, respectively). Untreated ECs and VSMCs are demonstrated in Figs. 2 a, 2 e, 2 c and 2 g. SL-cNPs increase RhoA and RhoB mRNA content in ECs and VSMCs ECs and VSMCs cultured in the presence of E-cNPs demonstrated no significant difference in expression of RhoA or RhoB compared to control cells (Fig. 3 a, 3 b). ECs cultured in the presence of low-dose SL-cNPs demonstrated a 1.28-fold increase in RhoA mRNA expression while high-dose SL-cNPs demonstrated a 1.57-fold increase compared to control ECs ( p < 0.05, Fig. 3 a). High-dose SL-cNPs demonstrated increased RhoA mRNA expression compared to free simvastatin and low-dose SL-cNPs (1.34 and 1.27-fold increase, respectively, p < 0.05). Only high-dose SL-cNP demonstrated increased RhoB expression, with a 1.20 and 1.26-fold increase in RhoB mRNA compared to control and low-dose SL-cNP, respectively ( p < 0.05). Low-dose SL-cNP and free simvastatin expression of RhoB was not significantly different compared to control. RhoA mRNA expression was significantly increased in VSMCs cultured in the presence of low or high dose SL-cNPs (1.42 and 1.61-fold increase, respectively, Fig. 3 b). RhoB expression was significantly increased after VSMC treatment with free simvastatin, low dose and high dose SL-cNPs (2.68, 4.26, and 4.17-fold increases, respectively). Low dose and high dose SL-cNPs demonstrated increased expression of RhoB compared to VSMCs treated with free simvastatin (1.59 and 1.55-fold increases, respectively). Oral simvastatin and SL-cNPs reduce IH following carotid artery balloon injury Mean ± SEM IH ratios were as follows: control animals 0.440 ± 0.04, intraluminal empty-cNPs 0.416 ± 0.05, oral simvastatin 0.35 ± 0.03, low dose intraluminal SL-cNP 0.377 ± 0.03, high dose intraluminal 0.41 ± 0.04, and oral + SL-cNPs 0.34 ± 0.03. Representative images of IH are presented in Fig. 4 a- 4 e. Only oral simvastatin with the addition of low dose intraluminal SL-cNPs significantly reduced IH by 23% ( p < 0.05) compared to the untreated control group. The data is summarized in Fig. 5 . Discussion Restenosis secondary to IH after PTA for PAD remains an ongoing challenge that decreases long-term patency rates and limb preservation [ 1 – 3 ]. Although the clinical use of FDA approved paclitaxel coated balloons and stents has represented a major advance in the prevention of IH, particularly in coronary artery disease, there remains controversy regarding their benefit in PAD lesions below the knee [ 9 – 12 ]. Therefore, the purpose of this study was to demonstrate feasibility of a novel intraluminal treatment, SL-cNPs, to reduce IH after PTA. We found SL-cNPs: 1) are readily taken up by ECs and VSMCs in vitro ; 2) affect VSMC and EC function as demonstrated by compensatory increases in gene expression in vitro ; and 3) reduce IH when used in combination with oral simvastatin in vivo . To our knowledge, the use of statin loaded, chitosan functionalized nanoformulations to limit IH formation after balloon injury is novel. Clinical studies since the late 1990s demonstrated statins have beneficial pleiotropic effects independent of lowering cholesterol levels. These studies have demonstrated statins reduce major adverse cardiovascular events, have anti-inflammatory properties, improve PAD symptoms and increase circulatory endothelial progenitor cells [ 17 ]. At the cellular level, statins interfere with IH by decreasing the local inflammatory response (i.e. reducing leukocyte EC transmigration), inhibiting VSMC migration from the adventitial layer to intimal layer, and improving reendothelialization by increasing endothelial progenitor cell survival [ 8 , 17 , 32 ]. Nevertheless, systemic delivery of statins has not proven to be highly effective in preventing IH due to statin’s low solubility, rapid metabolism and low bioavailability [ 25 ]. More recently, novel drug delivery mechanisms, such as microparticles, micelles and NPs, have been developed to prevent IH [ 24 , 25 , 27 , 28 , 33 ]. Of these carriers, FDA approved polymeric NPs, such as PLGA-NPs, are being considered the new frontier of medicine given their ability to provide precise targeting, sustained drug release and improved bioavailability [ 25 , 26 , 30 , 31 , 34 ]. Furthermore, by modifying the surface composition of the nanomaterial, NPs provide new strategies for passive or active targeting [ 25 ]. The variability in NP formulation now raises questions concerning the optimal drug delivery vehicle(s) for IH and its effect on VSMC and EC physiology. Of the various formulations of PLGA-NPs, we chose to utilize chitosan functionalized PLGA-NPs due to chitosan’s inherent beneficial properties. First, chitosan is polycationic, which increases the bioadhesion and solubility of NPs compared to PLGA alone [ 29 , 30 ]. Second, chitosan increases the stability, bioavailability and controlled release of the encapsulated drug. Third, chitosan contains inherent antimicrobial and anti-inflammatory properties [ 29 , 35 ]. cNPs have also been shown to have satisfactory biocompatibility with normal developing cells and have been safely utilized in oral, eye, cutaneous and transdermal applications [ 29 , 30 ]. Furthermore, chitosan has also been used in the surgical field with applications in surgical sutures, wound dressings, defect fillers and tissue-engineering scaffolds, demonstrating its wide range/ease of use [ 36 ]. Regarding the use of simvastatin within our cNP formulation, previous studies have demonstrated systemic and local delivery of simvastatin significantly reduces IH [ 24 ]. We elected to utilize a low (0.2mg) and high dose (2mg) SL-cNPs as this would provide local delivery of 5.5 µg or 55 µg simvastatin, respectively. We elected to assess varying doses of simvastatin that were significantly reduced to systemic therapy (10mg/kg/day) to minimize any potential negative off-target side effects while maximizing therapeutic efficacy [ 17 , 37 ]. Furthermore, we proceeded with the low dose SL-cNP as it is similar to the dose we used in a previous study[ 24 ]. Our in vitro results demonstrate SL-cNPs associate with ECs and VSMCs and have the potential to reduce IH. To assess SL-cNPs cellular effect on ECs and VSMCs and its potential to reduce IH, we assessed the relative gene expression of RhoA and RhoB in vitro . We elected to assess gene expression as previous studies have demonstrated statin’s inhibition of isoprenoid intermediates leads to a compensatory increase in constitutive RhoA and inducible RhoB mRNA expression [ 31 , 38 ]. RhoA expression is particularly relevant to our study as statin’s inhibition of RhoA is a suspected pleiotropic effect that leads to IH reduction. For example, RhoA, a small GTP binding protein, interacts with Rho-kinase to promote vascular contraction and VSMC migration by phosphorylating myosin light chain and myosin phosphatase, target subunit 1 [ 39 ]. RhoA/Rho-kinase is also implicated in reducing nitric oxide synthase gene expression, an important mediator of EC and VSMC function [ 40 ]. RhoA expression also positively modulates the expression of endothelial adhesion molecules, such as P-and E-selectins, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, that are important for leukocyte-endothelial adhesion and transmigration [ 21 , 22 ]. Therefore, inhibition of RhoA is suspected to reduce IH by inhibiting VSMC migration and the activation of a vascular inflammatory milieu. In our study, we found low and high dose SL-cNPs significantly increased the expression of RhoA in ECs and VSMCs. Only high dose SL-cNPs increased RhoB expression in ECs, while RhoB expression was increased in VSMCs treated with low or high dose SL-cNPs. Furthermore, SL-cNPs demonstrated a more robust effect on RhoA and RhoB mRNA expression compared to free simvastatin alone. This suggest SL-cNPs have the potential to reduce IH through its effect on RhoA and given the more robust effect on gene expression compared to free simvastatin, have greater therapeutic efficacy in the acute period (30 minutes). Although our in vivo results did not demonstrate a significant effect of oral simvastatin, low or high dose SL-cNPs alone, the combination of low dose SL-cNPs and oral simvastatin was effective in reducing IH. The compound effect of SL-cNPs and oral simvastatin suggests further investigation is necessary to assess the optimal dose and/or route of administration that would enhance SL-cNPs inhibitory effect on IH. For example, a previous study using a porcine coronary model demonstrated a pitavastatin-loaded NP eluting stent significantly reduced IH when 20 µg of pitavastatin was utilized per stent [ 28 ]. In our study, we examined the use of intraluminal administration of 5.5µg or 55 µg of simvastatin loaded in cNPs for 30 minutes, with subsequent removal. Although 30-minute duration appears to be a feasible mechanism to administer SL-cNPs, we expect a different route of administration with prolonged exposure to the intimal, medial or adventitial arterial wall may demonstrate a more profound effect on IH. Another consideration is the use of a higher dose of SL-cNPs. When assessing systemic simvastatin, in a previous study, we found a significant reduction of IH by 25% when oral simvastatin was used in isolation [ 24 ]. In this study, oral simvastatin did not have a statistically significant impact, although there was a 20% reduction in IH. We expect the effect of simvastatin may vary among animals. In conclusion, although systemic simvastatin can reduce IH, targeted delivery mechanisms have the potential to maximize statins pleiotropic benefits. The current study is primarily one of feasibility and it does have limitations. One limitation includes the delivery mechanism of SL-cNPs. Our results cannot be compared to the previous statin-NP formulations as we assessed a temporary intraluminal treatment while the previous study utilized a permanent NP-coated stent. We elected to test our hypothesis with a short-term treatment approach as clinically, this treatment would be ideal after balloon angioplasty. Another limitation includes the lack of documented localization of SL-cNPs to the arterial wall. Although we cannot confirm residence of the SL-cNPs within the arterial wall, we expect SL-cNP association given the direct delivery of localized intraluminal treatment and reduced IH. Future directions include optimizing delivery of cNPs. This can be accomplished by using different sizes, concentrations of SL-cNPs and examining different delivery routes of SL-cNPs, including luminal and periadventitial administration. Given the release kinetics of SL-cNPs, we expect statins have a profound effect in the acute period, resulting in decreased IH. Conclusion In conclusion, for IH prevention, chitosan functionalized nanoformulations are feasible to use as a novel vehicle to locally deliver statins to vascular cells. Future studies should evaluate the optimal size, concentration, delivery and residence time of SL-cNPs to maximize statin’s effect on IH prevention. Declarations Ethics approval All animals were treated ethically during the course of this study. All animal protocols were approved by Loyola University of Chicago IACUC and Edward Hines, Jr Veteran Affairs Hospital IACUC. Consent for Publication All authors agreed on the content and explicitly consented to the submission of this manuscript for publication. Availability of Data and Materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding This study was awarded the 2022 Eastern Vascular Society Research Seed Grant, with funding provided by Boston Scientific. Author Ashley A. Peters (formerly Ashley A. Penton) received the award and funding. Author Contributions All authors contributed to the study conception and design. Nanoparticle material preparation was performed by Maleen Cabe, Chanpreet Kaur and Kelly Langert. Other material preparation was performed by Ashley A. Peters, Xuerong Wang, Kristopher Maier and Vivian Gahtan. Data collection was performed by Ashley A. Peters and Gloria Grace Poland. Data analysis was performed by Ashley A. Peters and Kristopher Maier. The first draft of the manuscript was written by Ashley A. Peters and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Acknowledgements This study was awarded the 2022 Eastern Vascular Society Research Seed Grant, with funding provided by Boston Scientific. The author Ashley A. Peters received the award. We thank Dr. David J. Rademacher at Loyola University Chicago's Core Imaging Facility for performing transmission electron microscopy and the Loyola Flow Cytometry Core Facility (RRID:SCR_025109), especially Bert Ladd, for their assistance with the flow cytometry data analysis. Author’s Information Ashley A. Peters ORCID ID: 0000-0002-7457-9786 Data Availability Statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Collins TC, Beyth RJ. Process of care and outcomes in peripheral arterial disease. Am J Med Sci. 2003;325(3):125–34. Desai SS, et al. Outcomes after endovascular repair of arterial trauma. J Vasc Surg. 2014;60(5):1309–14. Schillinger M, Minar E. Restenosis after percutaneous angioplasty: the role of vascular inflammation. Vasc Health Risk Manag. 2005;1(1):73–8. Beckman JA, Schneider PA, Conte MS. Advances in Revascularization for Peripheral Artery Disease: Revascularization in PAD. Circ Res. 2021;128(12):1885–912. Deglise S, Bechelli C, Allagnat F. Vascular smooth muscle cells in intimal hyperplasia, an update. Front Physiol. 2022;13:1081881. Serrano CV Jr., et al. Coronary angioplasty results in leukocyte and platelet activation with adhesion molecule expression. Evidence of inflammatory responses in coronary angioplasty. J Am Coll Cardiol. 1997;29(6):1276–83. Lemson MS, et al. Intimal hyperplasia in vascular grafts. Eur J Vasc Endovasc Surg. 2000;19(4):336–50. Wang G, et al. Origin and differentiation of vascular smooth muscle cells. J Physiol. 2015;593(14):3013–30. Hata Y, et al. Similar one-year primary patency rates of common femoral artery angioplasty alone when performed utilizing drug-coated versus noncoated balloons for the treatment of peripheral artery disease. Vasc Med. 2022;27(4):380–1. Ipema J, et al. editors. 's Choice - Drug Coated Balloon Angioplasty vs. Standard Percutaneous Transluminal Angioplasty in Below the Knee Peripheral Arterial Disease: A Systematic Review and Meta-Analysis. Eur J Vasc Endovasc Surg, 2020. 59(2): pp. 265–275. Liao CJ, et al. Orchid drug-coated balloon versus standard percutaneous transluminal angioplasty for the treatment of femoropopliteal artery disease: 12-month result of the randomized controlled trial. Vascular. 2022;30(3):448–54. Zeller T, et al. Paclitaxel-coated balloons for femoropopliteal peripheral arterial disease: final five-year results of the IN.PACT Global Study. EuroIntervention. 2022;18(11):e940–8. Gonzalez L, Helkin A, Gahtan V. Dyslipidemia Part 2: Review of Dyslipidemia Treatment in Patients With Noncoronary Vascular Disease. Vasc Endovascular Surg. 2016;50(2):119–35. Alnaeb ME, et al. Statins and peripheral arterial disease: potential mechanisms and clinical benefits. Ann Vasc Surg. 2006;20(5):696–705. Coppola G, Novo S. Statins and peripheral arterial disease: effects on claudication, disease progression, and prevention of cardiovascular events. Arch Med Res. 2007;38(5):479–88. Daskalopoulou SS, et al. Peripheral arterial disease: a missed opportunity to administer statins so as to reduce cardiac morbidity and mortality. Curr Med Chem. 2005;12(4):443–52. Penton A et al. Beyond Cholesterol Reduction-Statin Pleiotropy and Peripheral Arterial Disease , in Statins-From Lipid-Lowering Benefits to Pleiotropic Effects . 2023, IntechOpen. Diomede L, et al. In vivo anti-inflammatory effect of statins is mediated by nonsterol mevalonate products. Arterioscler Thromb Vasc Biol. 2001;21(8):1327–32. Schlaepfer DD, et al. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994;372(6508):786–91. Dimmeler S, et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest. 2001;108(3):391–7. Honjo M, et al. Statin inhibits leukocyte-endothelial interaction and prevents neuronal death induced by ischemia-reperfusion injury in the rat retina. Arch Ophthalmol. 2002;120(12):1707–13. Wojciak-Stothard B, Williams L, Ridley AJ. Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol. 1999;145(6):1293–307. Walter DH, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation. 2002;105(25):3017–24. Helkin A, et al. Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury. Vasc Endovascular Surg. 2019;53(5):379–86. Montelione N et al. Tissue Engineering and Targeted Drug Delivery in Cardiovascular Disease: The Role of Polymer Nanocarrier for Statin Therapy. Biomedicines, 2023. 11(3). Nenna A et al. Polymers and Nanoparticles for Statin Delivery: Current Use and Future Perspectives in Cardiovascular Disease. Polym (Basel), 2021. 13(5). Chu J, et al. An atorvastatin calcium and poly(L-lactide-co-caprolactone) core-shell nanofiber-covered stent to treat aneurysms and promote reendothelialization. Acta Biomater. 2020;111:102–17. Tsukie N, et al. Pitavastatin-incorporated nanoparticle-eluting stents attenuate in-stent stenosis without delayed endothelial healing effects in a porcine coronary artery model. J Atheroscler Thromb. 2013;20(1):32–45. Jafernik K et al. Chitosan-Based Nanoparticles as Effective Drug Delivery Systems-A review. Molecules, 2023. 28(4). Yang F et al. Chitosan/poly(lactic-co-glycolic)acid Nanoparticle Formulations with Finely-Tuned Size Distributions for Enhanced Mucoadhesion. Pharmaceutics, 2022. 14(1). Langert KA, Goshu B, Stubbs EB Jr.. Attenuation of experimental autoimmune neuritis with locally administered lovastatin-encapsulating poly(lactic-co-glycolic) acid nanoparticles. J Neurochem. 2017;140(2):334–46. Greenwood J, Mason JC. Statins and the vascular endothelial inflammatory response. Trends Immunol. 2007;28(2):88–98. Zhao C, et al. Periadventitial Delivery of Simvastatin-Loaded Microparticles Attenuate Venous Neointimal Hyperplasia Associated With Arteriovenous Fistula. J Am Heart Assoc. 2020;9(24):e018418. Yu J et al. Polymeric Drug Delivery System Based on Pluronics for Cancer Treatment. Molecules, 2021. 26(12). Friedman AJ, et al. Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens. J Invest Dermatol. 2013;133(5):1231–9. Wang L et al. Poly(lactic-co-glycolic) acid/nanohydroxyapatite scaffold containing chitosan microspheres with adrenomedullin delivery for modulation activity of osteoblasts and vascular endothelial cells. Biomed Res Int, 2013. 2013: p. 530712. Ward NC, Watts GF, Eckel RH. Statin Toxic Circ Res. 2019;124(2):328–50. Von Zee CL, et al. Increased RhoA and RhoB protein accumulation in cultured human trabecular meshwork cells by lovastatin. Invest Ophthalmol Vis Sci. 2009;50(6):2816–23. Sugimoto M, Yamanouchi D, Komori K. Therapeutic approach against intimal hyperplasia of vein grafts through endothelial nitric oxide synthase/nitric oxide (eNOS/NO) and the Rho/Rho-kinase pathway. Surg Today. 2009;39(6):459–65. Ming XF, et al. Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol. 2002;22(24):8467–77. Hawes BE, et al. Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem. 1995;270(29):17148–53. Nan X, et al. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway. Proc Natl Acad Sci U S A. 2015;112(26):7996–8001. Chu T, et al. Atorvastatin Reduces Accumulation of Vascular Smooth Muscle Cells to Inhibit Intimal Hyperplasia via p38 MAPK Pathway Inhibition in a Rat Model of Vein Graft. Arq Bras Cardiol. 2020;115(4):630–6. Sakamoto K, et al. Fluvastatin prevents vascular hyperplasia by inhibiting phenotype modulation and proliferation through extracellular signal-regulated kinase 1 and 2 and p38 mitogen-activated protein kinase inactivation in organ-cultured artery. Arterioscler Thromb Vasc Biol. 2005;25(2):327–33. Supplementary Files floatimage1.jpeg Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4601140","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323463251,"identity":"aa5ba388-5f08-42b5-b3e6-9582769c1b19","order_by":0,"name":"Ashley A. Peters","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDADNvbmgw8+gBlEqU9gYODjOZZsOAOkhZlYLXISOWbSPCAOIS38/WcPPi78YZPHJpFjIG3za5s8HzMD44ePObi1SNzISzaekZBWzMbzrMA4t++2YRszA7PkzG14rLnBA3RPwuHENvbkDcm5PbcZgVrYmHnxaJE/f8b8N1gLQ4LBYcue2/YEtRgcyDFjBmvhSDFsZvhxO5GgFkOgX6R50tIS24CBzNjbcDu5jZmxGa9f5M6fPfiZx8YmcX578/EfP/7ctgUyDn74iM/7DDxIbMY2MNmATz2aFoY/BBSPglEwCkbBiAQApiRQHzJLlIAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-7457-9786","institution":"Loyola University Chicago","correspondingAuthor":true,"prefix":"","firstName":"Ashley","middleName":"A.","lastName":"Peters","suffix":""},{"id":323463252,"identity":"631ec012-0540-4c70-b305-87781486cbad","order_by":1,"name":"Gloria Grace Poland","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Gloria","middleName":"Grace","lastName":"Poland","suffix":""},{"id":323463253,"identity":"f403df26-0721-485e-a380-87691179efdb","order_by":2,"name":"Maleen Cabe","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Maleen","middleName":"","lastName":"Cabe","suffix":""},{"id":323463254,"identity":"0db0074b-d916-486a-81aa-b09b6633973d","order_by":3,"name":"Chanpreet Kaur","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Chanpreet","middleName":"","lastName":"Kaur","suffix":""},{"id":323463255,"identity":"9f985806-67dd-43a3-8841-c9d7aa84b456","order_by":4,"name":"Kelly Langert","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Langert","suffix":""},{"id":323463256,"identity":"69c7f439-0c0a-4fcb-b4bc-93b12243685c","order_by":5,"name":"Kristopher Maier","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Kristopher","middleName":"","lastName":"Maier","suffix":""},{"id":323463257,"identity":"878732e1-6e9f-4e07-a536-3c476ab85ee1","order_by":6,"name":"Vivian Gahtan","email":"","orcid":"","institution":"Loyola University Chicago","correspondingAuthor":false,"prefix":"","firstName":"Vivian","middleName":"","lastName":"Gahtan","suffix":""}],"badges":[],"createdAt":"2024-06-18 15:54:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4601140/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4601140/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61442052,"identity":"94a4bde4-7606-4c96-b52b-739e513dd98f","added_by":"auto","created_at":"2024-07-30 20:29:18","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":550158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of SL-cNPs and E-cNPs. \u003c/strong\u003eSize, PDI and zeta potential (charge) were determined for SL-cNPs and E-cNPs (1a-c, respectively). Data shown are the mean ± SD, using 3 separate NP lots (analyzed using Student’s T-test, with \u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e as significant). Transmission electron microscopy was utilized to visualize the SL-cNPs and E-cNPs at 3000x and 5000x magnification (d).\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/550cfe9119183073509997ce.jpg"},{"id":61442054,"identity":"e38f5674-b494-4cd3-9e08-06139d196a0a","added_by":"auto","created_at":"2024-07-30 20:29:18","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":714260,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSL-cNPs encapsulated with DiD lipophilic tracer readily associate with ECs and VSMCs. \u003c/strong\u003eHuman ECs (a-d) or VSMCs (e-h) were cultured with basal media without SL-cNPs (a, c, e, g) or with 0.2mg SL-cNPs (b, d, f, h) for 30 minutes. For immunostaining (a, b, e, f), fixed cells were labeled with DAPI (blue) or Alexa Fluor 488 Phalloidin (green). Red fluorescence signifies DiD encapsulated cNPs (outlined with red circle, b, f). For flow cytometry (c, d, g, h), cells were harvested and dispersed into flow cytometry tubes with basal media. SL-cNP positive cells elicited an emission wavelength at 664nm. ECs and VSMCs treated with cNPs demonstrated up take as shown by cNP positive cells within the gating (d, h).\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/dcc7b198fa2643431438c7a6.jpg"},{"id":61442057,"identity":"c0e7846e-ea0b-4bc8-82c8-51e0ef5f1b88","added_by":"auto","created_at":"2024-07-30 20:29:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":196227,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSL-cNPs enhance RhoA and RhoB mRNA content in EC and VSMC cultures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConfluent ECs or VSMCs were cultured in basal media without or with E-cNPs, free simvastatin, low dose or high dose SL-cNPs for 30 minutes and then washed. Relative changes in RhoA and RhoB mRNA content were quantified by RT-qPCR. Data shown are means ± SEM (\u003cem\u003en\u003c/em\u003e = 3) and are expressed as GAPDH-normalized fold-changes. \u003cstrong\u003e*\u003c/strong\u003e \u003cem\u003ep \u0026lt;0.05 compared to control ,\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e #\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e p \u0026lt; 0.05 compared to low dose SL-cNPs, \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e§ \u003c/strong\u003e\u003c/em\u003e\u003cem\u003ep \u0026lt; 0.05 compared to free simvastatin, \u003c/em\u003estatistical analysis performed using\u003cem\u003e \u003c/em\u003eANOVA.\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/af6c84849649d4bd942ef275.jpg"},{"id":61442055,"identity":"8cf39888-186b-4727-82f2-09016accc14c","added_by":"auto","created_at":"2024-07-30 20:29:18","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":796749,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotomicrograph of representative cuts of intimal hyperplasia after balloon injury of carotid arteries \u003c/strong\u003eRats were sacrificed 14 days after balloon injury and carotid arteries were harvested, perfused, fixed, sectioned and stained with hematoxylin and eosin. Utilizing autofluorescence, intimal hyperplasia was measured using intimal-medial area ratios. A. No statin control, b. cNPs empty, c. oral simvastatin, d. intraluminal SL-cNPs, e. intraluminal SL-cNPs and oral simvastatin. Arrows indicate areas of intimal hyperplasia.\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/be32c666ecbab0f46aadf433.jpg"},{"id":61442385,"identity":"d3c2c380-13f6-42b1-8e78-7abda8007d5c","added_by":"auto","created_at":"2024-07-30 20:37:18","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":87428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBox \u0026amp; whiskers plot of intimal-medial ratios\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOral simvastatin and intraluminal administration of low dose SL-cNP significantly reduced intimal hyperplasia compared to control rats and rats receiving E-cNPs. Control (n = 8), E-cNP (n=7), low dose SL-cNP (n=7), high dose SL-cNPs (n=8), oral simvastatin (n=9), oral + SL-cNP (n=7). \u003cstrong\u003e+\u003c/strong\u003e, demonstrating mean.\u003c/p\u003e\n\u003cp\u003e*, \u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e, ANOVA.\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/0f07e6e6ad9539963343e312.jpg"},{"id":62722273,"identity":"8588773a-fe3a-40a2-9efe-620e60b199be","added_by":"auto","created_at":"2024-08-18 16:32:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2926545,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/07cdb3f7-aad7-49b1-ae75-0fa4803b60a2.pdf"},{"id":61442384,"identity":"7fec67dd-7159-49ee-b084-e028859db33a","added_by":"auto","created_at":"2024-07-30 20:37:18","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":129141,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4601140/v1/0d85c02775c6606a5a51efb9.jpeg"}],"financialInterests":"","formattedTitle":"Statin Targeted Treatment Against Intimal Hyperplasia Using Unique Chitosan-PLGA Nanoparticles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRestenosis secondary to intimal hyperplasia (IH) after percutaneous transluminal balloon angioplasty (PTA) for peripheral arterial disease (PAD) remains an ongoing challenge that leads to reintervention and poor patient outcomes [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Depending on the location and type of intervention, secondary occlusion ranges from 20\u0026ndash;60% [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. To help mitigate this important clinical problem, understanding the pathophysiology of IH is crucial. IH is triggered by endothelial cell (EC) denudation and vascular smooth muscle cell (VSMC) injury [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The endothelial damage then stimulates the production of proinflammatory molecules and activation of circulatory monocytes that initiate excessive neointima formation through VSMC migration, VSMC proliferation, and excessive extracellular matrix deposition [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While the application of drug eluting systems, such as paclitaxel coated balloons and stents, was a major advance in the prevention of IH for coronary artery occlusive disease, this technology remains controversial in PAD management [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, the lack of effective treatments for the prevention of IH in PAD after PTA represents an important gap in our knowledge.\u003c/p\u003e \u003cp\u003eStatins are competitive inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG CoA) reductase, blocking a rate-limiting step in the mevalonate/cholesterol synthesis pathway [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition to reducing cholesterol levels, inhibition of HMG-CoA also prevents the production of isoprenoid intermediates. This in turn disrupts the signaling of small G proteins, including Ras and Rho, that are post-translationally modified by isoprenoids. The inhibition of isoprenoid intermediates largely enable statins known beneficial pleiotropic effects, including reducing IH [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For example, statins have been demonstrated to: 1) reduce VSMC migration and proliferation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]; 2) attenuate vascular inflammation by reducing leukocyte adhesion and trans-endothelial migration [\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]; and 3) accelerate reendothelialization by mobilizing, differentiating and improving survival of resident and circulating endothelial progenitor cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In animal models, oral simvastatin has been shown to reduce IH by 25% [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Unfortunately, in the clinical realm, statins are not tolerated in nine percent of patients due to negative-off target effects [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, systemic delivery of statins has not proven to be highly effective in preventing IH due to statins\u0026rsquo; low solubility, rapid metabolism and low bioavailability [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Because of these issues, research has focused on improving \u003cem\u003ein vivo\u003c/em\u003e drug delivery through the use of localized therapies. Currently, to maximize statins\u0026rsquo; pleiotropic effects while minimizing systemic toxicity, studies are investigating the use of statins loaded into drug delivery carriers, including nanoparticles (NPs) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While NPs have demonstrated the ability to increase the solubility, stability and absorption of statins, no FDA approved statin-loaded NP for clinical use currently exists [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePoly-lactic-co-glycolic acid (PLGA) is an FDA approved biodegradable polymer that can be formed into NPs that release incorporated agents in a controlled, localized fashion. Recent preclinical studies have investigated polymeric NPs in cancer and cardiovascular research, with one study investigating pitavastatin eluting stents in coronary artery disease [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. While this study demonstrated promise towards IH treatment with nanotechnology, variability in NP formulations now raises questions concerning the optimal delivery vehicle(s) for IH and its effect on VSMC and EC physiology. Chitosan, a naturally occurring biopolymer derived from crustacean shells, is polycationic. Studies have indicated coating NPs with positively-charged chitosan improves adhesion and solubility compared to PLGA alone, thereby optimizing release of embedded agents [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The purpose of this study was to determine the feasibility and therapeutic efficacy of simvastatin-loaded chitosan-PLGA NPs (SL-cNPs) on IH in a rat carotid artery injury model. Given that many PAD interventions require balloon angioplasty without stent placement, this study focuses on an intraluminal therapy that would not require the placement of a stent. We hypothesized that SL-cNPs would: 1) readily associate with ECs and VSMCs \u003cem\u003ein vitro\u003c/em\u003e; 2) affect EC and VSMC function; and 3) reduce IH compared to systemic simvastatin \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eHuman aortic ECs, human VSMCs, and corresponding cell culture media were purchased from Cell Applications, Inc (San Diego, CA). RNA extraction was performed utilizing QIAGEN RNA extraction kits (Germantown, MD). All PCR primers were from Applied Biosystems (Waltham, MA).\u003c/p\u003e \u003cp\u003eFor nanoparticle synthesis, ester-terminated poly(lactic-co-glycolic) acid (PLGA 85:15) was obtained from LACTEL (Birmingham, AL). Dichloromethane (DCM), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), poly(vinyl alcohol) (PVA, 31,000\u0026ndash;50,000 Da, 87\u0026ndash;89% hydrolyzed), and chitosan (low molecular weight (50,000-190,000 Da, 75%-85% deacetylated) were purchased from Sigma-Aldrich (St. Louis, MO). Simvastatin was obtained from Cayman Chemical (Ann Arbor, MI). 1,1\u0026rsquo;-dioactadecyl-3,3,3\u0026rsquo;,3\u0026rsquo;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) was from Thermofisher Scientific (Waltham, MA).\u003c/p\u003e \u003cp\u003eSimvastatin for rat chocolate-flavored treats was purchased from Fisher Scientific (Hampton, NH) and sent to Bio-Serv (Flemington, NJ) where the rodent specific oral simvastatin chocolate-flavored treats were manufactured (4 milligrams simvastatin/treat).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle Synthesis\u003c/h2\u003e \u003cp\u003eChitosan functionalized PLGA nanoparticles (cNPs) were prepared by oil-in-water single emulsion, using commercially available polymer (85:15, viscosity 0.55\u0026ndash;0.75) as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Chitosan stock solution was made by dissolving chitosan powder in 1% acetic acid to form 1% (\u003cem\u003ew/\u003c/em\u003ev) and diluted 1:1 with PVA to make solutions. PLGA polymer was first dissolved in an organic solvent (0.6mL MeCN and 0.4mL DCM) and then added dropwise into an aqueous solution containing 5% PVA and 0.5% chitosan (6mL) under vigorous vortexing. The emulsion was formed by sonication (sonication amplitude 70%) using an ultrasonic processor (GE130PB, Cole-Parmer, Vernon Hills, IL) for 10 rounds of 30 seconds on and 30 seconds off. After sonication, the emulsified mixture was added to a 1 L beaker containing PVA solution (0.5% PVA and 0.5% chitosan (45mL)) and stirred overnight to allow evaporation of the organic solvent. The cNPs were collected and washed with diH\u003csub\u003e2\u003c/sub\u003eO with a Sorvall RC-5B centrifuge, (refrigerated, superspeed). Washed cNPs were dispersed in 2% sucrose in diH\u003csub\u003e2\u003c/sub\u003eO, frozen at -80\u0026deg;C, freeze-dried (Edwards K4 Modulyo Freeze Dryer) and stored at a -20\u0026deg;C in a desiccator. Lipophilic tracer DiD with excitation/emission wavelength at 646/663nm (0.3% \u003cem\u003ew/w\u003c/em\u003e, Invitrogen) was loaded into cNPs by addition to the organic phase. Drug loaded cNPs (SL-cNPs) were made with the addition of simvastatin (4% \u003cem\u003ew/w\u003c/em\u003e drug loading) to the organic phase; empty cNPs (E-cNPs) did not receive drug but did receive DiD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle Characterization\u003c/h2\u003e \u003cp\u003eHydrodynamic diameter (size, nm) and polydispersity index (PDI) were determined with dynamic light scattering, and zeta potential (mV) was calculated after determining electrophoretic mobility, using a Zetasizer Nano ZS90 (Malvern Panalytical, Westborough, MA). cNPs were suspended in deionized water (0.1 mg/ml) and transferred to a disposable polystyrene cuvette or capillary cell. Suspension was equilibrated for 3 minutes in the cuvette and measured at 90\u0026deg; angle. Each measurement was an average of four separate, consecutive measurements.\u003c/p\u003e \u003cp\u003eDrug loading was quantified using high performance liquid chromatography (HPLC). Briefly, cNPs were solubilized in DMSO (4 mg/ml), and compounds were separated on a reversed-phase C18 column with a mobile phase of 70:30 acetonitrile: formic acid (0.05N). Simvastatin was detected by UV absorbance at 240 nm, and integrated peak areas were extrapolated to an external standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture\u003c/h2\u003e \u003cp\u003eECs (passage 3\u0026ndash;10) and VSMCs (passage 3\u0026ndash;5) were plated in 6-well plates or 25 cm\u003csup\u003e2\u003c/sup\u003e flasks. Cells were maintained in an incubator at 95% O\u003csub\u003e2\u003c/sub\u003e/5% CO2, 37\u003csup\u003eo\u003c/sup\u003eC. Cell viability was determined with the Trypan blue exclusion assay using the Countess cell counter (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. Only cells with \u0026gt;\u0026thinsp;90% viability were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell Staining\u003c/h2\u003e \u003cp\u003eAutoclaved 0.5mm thickness glass coverslips were placed in 6-well plates (Fisher Scientific, Hampton, NJ). ECs and VSMCs were individually seeded to 50% confluence. A stock solution of 2 mg/mL cNPs encapsulated with DiD was made using EC or VSMC basal media. Cells were washed once with PBS, and then treated with either basal media or treatment media (0.2mg cNPs) for 30 minutes. Media was then removed, cells washed and fresh basal media was then added to the cells and incubated for 24 hours. The cells were fixed and stained with DAPI (4\u0026rsquo;,6- Diamidino-2-Phenylindole, Dihydrochloride) and Alexa Fluor 488 Phalloidin (Invitrogen, Carlsbad, CA) per the manufacturer\u0026rsquo;s instructions. Imaging was performed using a fluorescent microscope. The experiment was repeated three times. Uptake of cNPs was determined by visualization of DiD fluorescence (excitation 646 nm, emission 664nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow Cytometry\u003c/h2\u003e \u003cp\u003eECs or VSMCs were seeded to 50% confluence in 25cm\u003csup\u003e2\u003c/sup\u003e flasks. The treatment media was prepared by making a stock solution of DiD encapsulated cNPs in basal media, as previously described [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cells were treated with either basal media or treatment media (0.2mg cNPs) for 30 minutes. The media was then replaced with fresh basal media and incubated for 24 hours. The cells were then harvested and placed into flow cytometry tubes. Greater than 92% viability was confirmed with a trypan blue assay. Flow cytometry was performed by the Loyola University of Chicago Flow Cytometry Core Facility. Acquisition was performed on a 4-Laser LSRFortessa from BD Biosciences using BD FACSDiva Software (version 9.0). Analysis was performed with FlowJo Software (version 10.9.0) from BD Biosciences (Franklin Lakes, NJ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro Evaluation of SL-cNP Function\u003c/h2\u003e \u003cp\u003eCells were treated with basal media or basal media containing E-cNPs (0.2mg), free simvastatin (10\u0026micro;M), low-dose SL-cNPs (0.2mg) or high-dose SL-cNPs (2mg). After 30 minutes, treatment solutions were removed and replaced with fresh basal media. After 24 hours, total RNA was extracted from the cells. cDNA was generated and quantitative real-time PCR performed using human-specific primers to \u003cem\u003eRHOA\u003c/em\u003e or \u003cem\u003eRHOB\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For each sample, \u003cem\u003eGAPDH\u003c/em\u003e was used as a reference control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRat Carotid Artery Balloon Injury Model\u003c/h2\u003e \u003cp\u003e All animal protocols were approved by Loyola University of Chicago IACUC and Edward Hines, Jr Veteran Affairs Hospital IACUC. Age and weight-matched male Sprague-Dawley rats were purchased at 17\u0026ndash;20 weeks at 400 grams from Envigo (Indianapolis, IN). In total, 6 experimental groups (n\u0026thinsp;=\u0026thinsp;6\u0026ndash;9 animals for each group) received intraluminal balloon injury using a 2 French Fogarty Catheter (5 atmospheres/5 minutes, Medline, Northfield, IL) in the common carotid artery. The experimental groups consisted of: intraluminal normal saline (control), oral simvastatin plus intraluminal saline, cNPs without simvastatin (E-cNPs), intraluminal low-dose SL-cNPs (0.2mg), intraluminal high-dose SL-cNPs (2mg) or oral simvastatin plus low-dose intraluminal SL-cNPs. To induce carotid artery balloon injury, the left superior thyroid, occipital and the distal external carotid arteries were ligated. The internal and common carotid arteries were clamped, an arteriotomy was made at the proximal external carotid artery, and the Fogarty Catheter was inserted and inflated at 5 atm for 5 minutes. After intraluminal balloon injury was performed, the common carotid artery received an intraluminal infusion of either normal saline (control group), SL-cNPs or E-cNPs via a 27-gauge blunted needle inserted into the arteriotomy. The infusion was administered gently under pressure and held in place for 30 minutes. Animals receiving oral simvastatin were pretreated with simvastatin (10mg/kg/day) via chocolate-flavored treats for 1 week prior to surgery and for 14 days postoperatively. Rats receiving oral simvastatin required food rationing to ensure the complete ingestion of treat. Normal chow was provided ad libitum for the other experimental groups. All rats received water ad libitum. On postoperative day 14, animals were euthanized and perfusion fixed with formalin. Right and left carotid arteries were harvested, preserved in formalin, and sectioned and stained with hematoxylin and eosin. Morphometric analysis was performed using Motic Images Plus 3.0 (Kowloon Bay, Kowloon, Hong Kong) using intimal-medial area ratio defined as intima/(intima\u0026thinsp;+\u0026thinsp;media).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis for RhoA and RhoB mRNA content and intimal hyperplasia was analyzed using two-way ANOVA with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 being considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacteristics of E-NPs and SL-NPs\u003c/h2\u003e \u003cp\u003eHydrodynamic diameters of SL-cNPs and E-cNPs were 182.45\u0026thinsp;\u0026plusmn;\u0026thinsp;1.38 nm and 179.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32nm, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). PDI and zeta potential of SL-cNPs were 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.019 and +\u0026thinsp;11.49\u0026thinsp;\u0026plusmn;\u0026thinsp;5.59. PDI and zeta potential of E-cNPs were 0.098\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 and +\u0026thinsp;4.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb \u0026amp; \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). No significant differences existed between SL-cNPs and E-cNPs. Encapsulation efficiency of cNPs was 71.78% \u0026plusmn; 2.86% (actual drug loading/theoretical drug loading x 100%). Actual drug loading of simvastatin in cNPs was 2.77% \u003cem\u003ew/w\u003c/em\u003e. We utilized 0.2mg of SL-cNPs as low-dose (effectively locally administering 5.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eg simvastatin) and 2mg SL-cNPs as high-dose (effectively locally administering 55 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eg simvastatin). Cumulative drug release was determined previously and approached 100% at 100 hours [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSL-cNPs readily associate with ECs and VSMCs\u003c/h2\u003e \u003cp\u003eECs and VSMCs exposed to SL-cNPs demonstrated increased red fluorescence in immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) and increased allophycocyanin (APC) intensity in flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). Analysis demonstrated SL-cNP uptake in ECs was 79.6% (78-82.8%) while VSMCs uptake of SL-cNPs was 46.4% (43.4\u0026ndash;49.4%). There was no significant difference between total number of untreated compared to treated ECs (45589\u0026thinsp;\u0026plusmn;\u0026thinsp;1864 vs 48501\u0026thinsp;\u0026plusmn;\u0026thinsp;77 cells, respectively) or VSMCs (52367\u0026thinsp;\u0026plusmn;\u0026thinsp;863 vs 55549\u0026thinsp;\u0026plusmn;\u0026thinsp;3266 cells, respectively). Untreated ECs and VSMCs are demonstrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSL-cNPs increase RhoA and RhoB mRNA content in ECs and VSMCs\u003c/h2\u003e \u003cp\u003eECs and VSMCs cultured in the presence of E-cNPs demonstrated no significant difference in expression of RhoA or RhoB compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). ECs cultured in the presence of low-dose SL-cNPs demonstrated a 1.28-fold increase in RhoA mRNA expression while high-dose SL-cNPs demonstrated a 1.57-fold increase compared to control ECs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). High-dose SL-cNPs demonstrated increased RhoA mRNA expression compared to free simvastatin and low-dose SL-cNPs (1.34 and 1.27-fold increase, respectively, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). Only high-dose SL-cNP demonstrated increased RhoB expression, with a 1.20 and 1.26-fold increase in RhoB mRNA compared to control and low-dose SL-cNP, respectively (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). Low-dose SL-cNP and free simvastatin expression of RhoB was not significantly different compared to control.\u003c/p\u003e \u003cp\u003eRhoA mRNA expression was significantly increased in VSMCs cultured in the presence of low or high dose SL-cNPs (1.42 and 1.61-fold increase, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). RhoB expression was significantly increased after VSMC treatment with free simvastatin, low dose and high dose SL-cNPs (2.68, 4.26, and 4.17-fold increases, respectively). Low dose and high dose SL-cNPs demonstrated increased expression of RhoB compared to VSMCs treated with free simvastatin (1.59 and 1.55-fold increases, respectively).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOral simvastatin and SL-cNPs reduce IH following carotid artery balloon injury\u003c/h2\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM IH ratios were as follows: control animals 0.440\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, intraluminal empty-cNPs 0.416\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, oral simvastatin 0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, low dose intraluminal SL-cNP 0.377\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, high dose intraluminal 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, and oral\u0026thinsp;+\u0026thinsp;SL-cNPs 0.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03. Representative images of IH are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee. Only oral simvastatin with the addition of low dose intraluminal SL-cNPs significantly reduced IH by 23% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the untreated control group. The data is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eRestenosis secondary to IH after PTA for PAD remains an ongoing challenge that decreases long-term patency rates and limb preservation [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although the clinical use of FDA approved paclitaxel coated balloons and stents has represented a major advance in the prevention of IH, particularly in coronary artery disease, there remains controversy regarding their benefit in PAD lesions below the knee [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Therefore, the purpose of this study was to demonstrate feasibility of a novel intraluminal treatment, SL-cNPs, to reduce IH after PTA. We found SL-cNPs: 1) are readily taken up by ECs and VSMCs \u003cem\u003ein vitro\u003c/em\u003e; 2) affect VSMC and EC function as demonstrated by compensatory increases in gene expression \u003cem\u003ein vitro\u003c/em\u003e; and 3) reduce IH when used in combination with oral simvastatin \u003cem\u003ein vivo\u003c/em\u003e. To our knowledge, the use of statin loaded, chitosan functionalized nanoformulations to limit IH formation after balloon injury is novel.\u003c/p\u003e \u003cp\u003eClinical studies since the late 1990s demonstrated statins have beneficial pleiotropic effects independent of lowering cholesterol levels. These studies have demonstrated statins reduce major adverse cardiovascular events, have anti-inflammatory properties, improve PAD symptoms and increase circulatory endothelial progenitor cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. At the cellular level, statins interfere with IH by decreasing the local inflammatory response (i.e. reducing leukocyte EC transmigration), inhibiting VSMC migration from the adventitial layer to intimal layer, and improving reendothelialization by increasing endothelial progenitor cell survival [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Nevertheless, systemic delivery of statins has not proven to be highly effective in preventing IH due to statin\u0026rsquo;s low solubility, rapid metabolism and low bioavailability [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. More recently, novel drug delivery mechanisms, such as microparticles, micelles and NPs, have been developed to prevent IH [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Of these carriers, FDA approved polymeric NPs, such as PLGA-NPs, are being considered the new frontier of medicine given their ability to provide precise targeting, sustained drug release and improved bioavailability [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Furthermore, by modifying the surface composition of the nanomaterial, NPs provide new strategies for passive or active targeting [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The variability in NP formulation now raises questions concerning the optimal drug delivery vehicle(s) for IH and its effect on VSMC and EC physiology. Of the various formulations of PLGA-NPs, we chose to utilize chitosan functionalized PLGA-NPs due to chitosan\u0026rsquo;s inherent beneficial properties. First, chitosan is polycationic, which increases the bioadhesion and solubility of NPs compared to PLGA alone [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Second, chitosan increases the stability, bioavailability and controlled release of the encapsulated drug. Third, chitosan contains inherent antimicrobial and anti-inflammatory properties [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. cNPs have also been shown to have satisfactory biocompatibility with normal developing cells and have been safely utilized in oral, eye, cutaneous and transdermal applications [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, chitosan has also been used in the surgical field with applications in surgical sutures, wound dressings, defect fillers and tissue-engineering scaffolds, demonstrating its wide range/ease of use [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Regarding the use of simvastatin within our cNP formulation, previous studies have demonstrated systemic and local delivery of simvastatin significantly reduces IH [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. We elected to utilize a low (0.2mg) and high dose (2mg) SL-cNPs as this would provide local delivery of 5.5 \u0026micro;g or 55 \u0026micro;g simvastatin, respectively. We elected to assess varying doses of simvastatin that were significantly reduced to systemic therapy (10mg/kg/day) to minimize any potential negative off-target side effects while maximizing therapeutic efficacy [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Furthermore, we proceeded with the low dose SL-cNP as it is similar to the dose we used in a previous study[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e results demonstrate SL-cNPs associate with ECs and VSMCs and have the potential to reduce IH. To assess SL-cNPs cellular effect on ECs and VSMCs and its potential to reduce IH, we assessed the relative gene expression of RhoA and RhoB \u003cem\u003ein vitro\u003c/em\u003e. We elected to assess gene expression as previous studies have demonstrated statin\u0026rsquo;s inhibition of isoprenoid intermediates leads to a compensatory increase in constitutive RhoA and inducible RhoB mRNA expression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. RhoA expression is particularly relevant to our study as statin\u0026rsquo;s inhibition of RhoA is a suspected pleiotropic effect that leads to IH reduction. For example, RhoA, a small GTP binding protein, interacts with Rho-kinase to promote vascular contraction and VSMC migration by phosphorylating myosin light chain and myosin phosphatase, target subunit 1 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. RhoA/Rho-kinase is also implicated in reducing nitric oxide synthase gene expression, an important mediator of EC and VSMC function [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. RhoA expression also positively modulates the expression of endothelial adhesion molecules, such as P-and E-selectins, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, that are important for leukocyte-endothelial adhesion and transmigration [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, inhibition of RhoA is suspected to reduce IH by inhibiting VSMC migration and the activation of a vascular inflammatory milieu. In our study, we found low and high dose SL-cNPs significantly increased the expression of RhoA in ECs and VSMCs. Only high dose SL-cNPs increased RhoB expression in ECs, while RhoB expression was increased in VSMCs treated with low or high dose SL-cNPs. Furthermore, SL-cNPs demonstrated a more robust effect on RhoA and RhoB mRNA expression compared to free simvastatin alone. This suggest SL-cNPs have the potential to reduce IH through its effect on RhoA and given the more robust effect on gene expression compared to free simvastatin, have greater therapeutic efficacy in the acute period (30 minutes).\u003c/p\u003e \u003cp\u003eAlthough our \u003cem\u003ein vivo\u003c/em\u003e results did not demonstrate a significant effect of oral simvastatin, low or high dose SL-cNPs alone, the combination of low dose SL-cNPs and oral simvastatin was effective in reducing IH. The compound effect of SL-cNPs and oral simvastatin suggests further investigation is necessary to assess the optimal dose and/or route of administration that would enhance SL-cNPs inhibitory effect on IH. For example, a previous study using a porcine coronary model demonstrated a pitavastatin-loaded NP eluting stent significantly reduced IH when 20 \u0026micro;g of pitavastatin was utilized per stent [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, we examined the use of intraluminal administration of 5.5\u0026micro;g or 55 \u0026micro;g of simvastatin loaded in cNPs for 30 minutes, with subsequent removal. Although 30-minute duration appears to be a feasible mechanism to administer SL-cNPs, we expect a different route of administration with prolonged exposure to the intimal, medial or adventitial arterial wall may demonstrate a more profound effect on IH. Another consideration is the use of a higher dose of SL-cNPs. When assessing systemic simvastatin, in a previous study, we found a significant reduction of IH by 25% when oral simvastatin was used in isolation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In this study, oral simvastatin did not have a statistically significant impact, although there was a 20% reduction in IH. We expect the effect of simvastatin may vary among animals. In conclusion, although systemic simvastatin can reduce IH, targeted delivery mechanisms have the potential to maximize statins pleiotropic benefits.\u003c/p\u003e \u003cp\u003eThe current study is primarily one of feasibility and it does have limitations. One limitation includes the delivery mechanism of SL-cNPs. Our results cannot be compared to the previous statin-NP formulations as we assessed a temporary intraluminal treatment while the previous study utilized a permanent NP-coated stent. We elected to test our hypothesis with a short-term treatment approach as clinically, this treatment would be ideal after balloon angioplasty. Another limitation includes the lack of documented localization of SL-cNPs to the arterial wall. Although we cannot confirm residence of the SL-cNPs within the arterial wall, we expect SL-cNP association given the direct delivery of localized intraluminal treatment and reduced IH. Future directions include optimizing delivery of cNPs. This can be accomplished by using different sizes, concentrations of SL-cNPs and examining different delivery routes of SL-cNPs, including luminal and periadventitial administration. Given the release kinetics of SL-cNPs, we expect statins have a profound effect in the acute period, resulting in decreased IH.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, for IH prevention, chitosan functionalized nanoformulations are feasible to use as a novel vehicle to locally deliver statins to vascular cells. Future studies should evaluate the optimal size, concentration, delivery and residence time of SL-cNPs to maximize statin\u0026rsquo;s effect on IH prevention.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals were treated ethically during the course of this study. All animal protocols were approved by Loyola University of Chicago IACUC and Edward Hines, Jr Veteran Affairs Hospital IACUC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agreed on the content and explicitly consented to the submission of this manuscript for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was awarded the 2022 Eastern Vascular Society Research Seed Grant, with funding provided by Boston Scientific. Author Ashley A. Peters (formerly Ashley A. Penton) received the award and funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Nanoparticle material preparation was performed by Maleen Cabe, Chanpreet Kaur and Kelly Langert. Other material preparation was performed by Ashley A. Peters, Xuerong Wang, Kristopher Maier and Vivian Gahtan. Data collection was performed by Ashley A. Peters and Gloria Grace Poland. Data analysis was performed by Ashley A. Peters and Kristopher Maier. The first draft of the manuscript was written by Ashley A. Peters and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was awarded the 2022 Eastern Vascular Society Research Seed Grant, with funding provided by Boston Scientific. The author Ashley A. Peters received the award. We thank Dr. David J. Rademacher at Loyola University Chicago\u0026apos;s Core Imaging Facility for performing transmission electron microscopy and the Loyola Flow Cytometry Core Facility (RRID:SCR_025109), especially Bert Ladd, for their assistance with the flow cytometry data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAshley A. Peters ORCID ID: 0000-0002-7457-9786\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCollins TC, Beyth RJ. Process of care and outcomes in peripheral arterial disease. Am J Med Sci. 2003;325(3):125\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDesai SS, et al. Outcomes after endovascular repair of arterial trauma. J Vasc Surg. 2014;60(5):1309\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchillinger M, Minar E. Restenosis after percutaneous angioplasty: the role of vascular inflammation. Vasc Health Risk Manag. 2005;1(1):73\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBeckman JA, Schneider PA, Conte MS. Advances in Revascularization for Peripheral Artery Disease: Revascularization in PAD. Circ Res. 2021;128(12):1885\u0026ndash;912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeglise S, Bechelli C, Allagnat F. Vascular smooth muscle cells in intimal hyperplasia, an update. Front Physiol. 2022;13:1081881.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerrano CV Jr., et al. Coronary angioplasty results in leukocyte and platelet activation with adhesion molecule expression. Evidence of inflammatory responses in coronary angioplasty. J Am Coll Cardiol. 1997;29(6):1276\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLemson MS, et al. Intimal hyperplasia in vascular grafts. Eur J Vasc Endovasc Surg. 2000;19(4):336\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang G, et al. Origin and differentiation of vascular smooth muscle cells. J Physiol. 2015;593(14):3013\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHata Y, et al. Similar one-year primary patency rates of common femoral artery angioplasty alone when performed utilizing drug-coated versus noncoated balloons for the treatment of peripheral artery disease. Vasc Med. 2022;27(4):380\u0026ndash;1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIpema J, et al. editors. 's Choice - Drug Coated Balloon Angioplasty vs. Standard Percutaneous Transluminal Angioplasty in Below the Knee Peripheral Arterial Disease: A Systematic Review and Meta-Analysis. Eur J Vasc Endovasc Surg, 2020. 59(2): pp. 265\u0026ndash;275.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao CJ, et al. Orchid drug-coated balloon versus standard percutaneous transluminal angioplasty for the treatment of femoropopliteal artery disease: 12-month result of the randomized controlled trial. Vascular. 2022;30(3):448\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeller T, et al. Paclitaxel-coated balloons for femoropopliteal peripheral arterial disease: final five-year results of the IN.PACT Global Study. EuroIntervention. 2022;18(11):e940\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGonzalez L, Helkin A, Gahtan V. Dyslipidemia Part 2: Review of Dyslipidemia Treatment in Patients With Noncoronary Vascular Disease. Vasc Endovascular Surg. 2016;50(2):119\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlnaeb ME, et al. Statins and peripheral arterial disease: potential mechanisms and clinical benefits. Ann Vasc Surg. 2006;20(5):696\u0026ndash;705.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoppola G, Novo S. Statins and peripheral arterial disease: effects on claudication, disease progression, and prevention of cardiovascular events. Arch Med Res. 2007;38(5):479\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaskalopoulou SS, et al. Peripheral arterial disease: a missed opportunity to administer statins so as to reduce cardiac morbidity and mortality. Curr Med Chem. 2005;12(4):443\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePenton A et al. \u003cem\u003eBeyond Cholesterol Reduction-Statin Pleiotropy and Peripheral Arterial Disease\u003c/em\u003e, in \u003cem\u003eStatins-From Lipid-Lowering Benefits to Pleiotropic Effects\u003c/em\u003e. 2023, IntechOpen.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiomede L, et al. In vivo anti-inflammatory effect of statins is mediated by nonsterol mevalonate products. Arterioscler Thromb Vasc Biol. 2001;21(8):1327\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchlaepfer DD, et al. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994;372(6508):786\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimmeler S, et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase/Akt pathway. J Clin Invest. 2001;108(3):391\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHonjo M, et al. Statin inhibits leukocyte-endothelial interaction and prevents neuronal death induced by ischemia-reperfusion injury in the rat retina. Arch Ophthalmol. 2002;120(12):1707\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWojciak-Stothard B, Williams L, Ridley AJ. Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering. J Cell Biol. 1999;145(6):1293\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalter DH, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation. 2002;105(25):3017\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHelkin A, et al. Intraluminal Delivery of Simvastatin Attenuates Intimal Hyperplasia After Arterial Injury. Vasc Endovascular Surg. 2019;53(5):379\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontelione N et al. Tissue Engineering and Targeted Drug Delivery in Cardiovascular Disease: The Role of Polymer Nanocarrier for Statin Therapy. Biomedicines, 2023. 11(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNenna A et al. Polymers and Nanoparticles for Statin Delivery: Current Use and Future Perspectives in Cardiovascular Disease. Polym (Basel), 2021. 13(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu J, et al. An atorvastatin calcium and poly(L-lactide-co-caprolactone) core-shell nanofiber-covered stent to treat aneurysms and promote reendothelialization. Acta Biomater. 2020;111:102\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsukie N, et al. Pitavastatin-incorporated nanoparticle-eluting stents attenuate in-stent stenosis without delayed endothelial healing effects in a porcine coronary artery model. J Atheroscler Thromb. 2013;20(1):32\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJafernik K et al. Chitosan-Based Nanoparticles as Effective Drug Delivery Systems-A review. Molecules, 2023. 28(4).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang F et al. Chitosan/poly(lactic-co-glycolic)acid Nanoparticle Formulations with Finely-Tuned Size Distributions for Enhanced Mucoadhesion. Pharmaceutics, 2022. 14(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLangert KA, Goshu B, Stubbs EB Jr.. Attenuation of experimental autoimmune neuritis with locally administered lovastatin-encapsulating poly(lactic-co-glycolic) acid nanoparticles. J Neurochem. 2017;140(2):334\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreenwood J, Mason JC. Statins and the vascular endothelial inflammatory response. Trends Immunol. 2007;28(2):88\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao C, et al. Periadventitial Delivery of Simvastatin-Loaded Microparticles Attenuate Venous Neointimal Hyperplasia Associated With Arteriovenous Fistula. J Am Heart Assoc. 2020;9(24):e018418.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J et al. Polymeric Drug Delivery System Based on Pluronics for Cancer Treatment. Molecules, 2021. 26(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedman AJ, et al. Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens. J Invest Dermatol. 2013;133(5):1231\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L et al. \u003cem\u003ePoly(lactic-co-glycolic) acid/nanohydroxyapatite scaffold containing chitosan microspheres with adrenomedullin delivery for modulation activity of osteoblasts and vascular endothelial cells.\u003c/em\u003e Biomed Res Int, 2013. 2013: p. 530712.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWard NC, Watts GF, Eckel RH. Statin Toxic Circ Res. 2019;124(2):328\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVon Zee CL, et al. Increased RhoA and RhoB protein accumulation in cultured human trabecular meshwork cells by lovastatin. Invest Ophthalmol Vis Sci. 2009;50(6):2816\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSugimoto M, Yamanouchi D, Komori K. Therapeutic approach against intimal hyperplasia of vein grafts through endothelial nitric oxide synthase/nitric oxide (eNOS/NO) and the Rho/Rho-kinase pathway. Surg Today. 2009;39(6):459\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMing XF, et al. Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells. Mol Cell Biol. 2002;22(24):8467\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHawes BE, et al. Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem. 1995;270(29):17148\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNan X, et al. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway. Proc Natl Acad Sci U S A. 2015;112(26):7996\u0026ndash;8001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChu T, et al. Atorvastatin Reduces Accumulation of Vascular Smooth Muscle Cells to Inhibit Intimal Hyperplasia via p38 MAPK Pathway Inhibition in a Rat Model of Vein Graft. Arq Bras Cardiol. 2020;115(4):630\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakamoto K, et al. Fluvastatin prevents vascular hyperplasia by inhibiting phenotype modulation and proliferation through extracellular signal-regulated kinase 1 and 2 and p38 mitogen-activated protein kinase inactivation in organ-cultured artery. Arterioscler Thromb Vasc Biol. 2005;25(2):327\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"chitosan, poly-lactic-co-glycolic acid, nanoparticles, intimal hyperplasia, peripheral arterial disease, statins","lastPublishedDoi":"10.21203/rs.3.rs-4601140/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4601140/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eStatins have pleiotropic effects, including reducing intimal hyperplasia (IH). Using unique nanoparticle (NPs), we hypothesized chitosan-functionalized polymeric NPs loaded with simvastatin (SL-cNPs) would: 1) readily associate with endothelial cells (ECs) and vascular smooth muscle cells (VSMCs); 2) affect EC and VSMC function; and 3) reduce IH compared to systemic simvastatin.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eHuman aortic ECs and VSMCs were cultured with SL-cNPs tagged with fluorescent tracer. Association of SL-cNPs was assessed by immunostaining and flow cytometry. The effect of SL-cNPs, empty cNPs (E-cNPs) and free simvastatin on cells was determined using RT-qPCR for RhoA and RhoB. Carotid artery balloon injured rats were treated intraoperatively with intraluminal saline, E-cNPs, low or high dose SL-cNPs; or with pre- and post-operative oral simvastatin plus intraoperative intraluminal saline or low dose SL-cNPs. Rats were euthanized (day 14) and IH was quantified.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSL-cNPs readily associated with ECs and VSMCs. Low and high dose SL-cNPs induced significant increases in EC and VSMC RhoA gene expression. High dose SL-cNPs induced a significant increase in EC RhoB expression, while free simvastatin, low and high dose SL-cNPs significantly increased RhoB expression in VSMCs. \u003cem\u003eIn vivo\u003c/em\u003e, oral simvastatin plus intraluminal SL-cNPs significantly reduced IH compared to controls.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003ecNPs can be used as a novel vehicle to locally deliver statins to vascular cells. Although only the combination of oral simvastatin and SL-cNPs effectively reduced IH, different routes of delivery and/or concentration of SL-cNPs may allow for a more robust effect on IH prevention.\u003c/p\u003e","manuscriptTitle":"Statin Targeted Treatment Against Intimal Hyperplasia Using Unique Chitosan-PLGA Nanoparticles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 20:29:14","doi":"10.21203/rs.3.rs-4601140/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"460975d8-8efe-4cd5-baf3-9c7b5c40b5a5","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-18T16:24:13+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-30 20:29:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4601140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4601140","identity":"rs-4601140","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}