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Bilayered Nanoparticles with Sequential Release of VEGF Gene and Paclitaxel for Restenosis inhibition in Atherosclerosis Xiaowei Zhu, Hongzhi Xie, Xiaoyu Liang, Xuanling Li, Jianwei Duan, Yongxia Chen, Ziying Yang, Chao Liu, Cuiwei Wang, Hailing Zhang, Quan Fang, Hongfan Sun, Chen Li, Yongjun Li, Chun Wang, Cunxian Song, Yong Zeng, and Jing Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08312 • Publication Date (Web): 27 Jul 2017 Downloaded from http://pubs.acs.org on July 31, 2017

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Bilayered Nanoparticles with Sequential Release of VEGF Gene and Paclitaxel for Restenosis inhibition in Atherosclerosis Xiaowei Zhu

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, Hongzhi Xie2#, Xiaoyu Liang1, Xuanling Li1, Jianwei Duan1, Yongxia Chen1,

Ziying Yang1, Chao Liu 1, Cuiwei Wang1, Hailing Zhang1, Quan Fang2, Hongfan Sun1, Chen Li1, Yongjun Li3, Chun Wang1,4, Cunxian Song1, Yong Zeng2*, Jing Yang1* 1

Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese

Academy of Medical Science, Tianjin 300192, China 2

Peking Union Medical College Hospital, Beijing 100730, China

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Department of Vascular Surgery, Beijing Hospital, Beijing 100730, China

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Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455,

USA # Contribute equally *Corresponding author: Prof Yong Zeng and Prof Jing Yang Email address: [email protected] [email protected] Mobile: 008613389060566 Fax: 0086 022 87891191

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Abstract Complete re-endothelialization followed by inhibition of smooth muscle cell proliferation is considered an effective therapeutic option to prevent restenosis. We have designed poly(lactide-co-glycolide)-loaded bilayered nanoparticles (NPs) with the ability to sequentially release VEGF-encoding plasmids from the outer layer and paclitaxel (PTX) from the core to promote endothelial regeneration while also prevent restenosis. Comparing with conventional NPs, which release VEGF plasmid and PTX simultaneously, we expect that the bilayered NPs could release the VEGF plasmid more rapidly followed by a delayed release of PTX, resulting in efficient VEGF gene transfection, which ideally could promote re-endothelialization while inhibit excessive smooth muscle cell growth. Indeed, in the present study, we have observed efficient gene transfection using a model plasmid as well as cell growth attenuation in vitro using Chinese Hamster Ovary (CHO) cells. Therapeutic efficacy of the bilayered NPs on restenosis was further evaluated in vivo using a rabbit model of atherosclerosis. The bilayered NPs were administered locally via balloon angioplasty to the injured aortic wall through perfusion. Twenty-eight days after NPs administration, rabbits treated with the bilayered NPs exhibited rapid re-endothelialization and inhibition of restenosis as demonstrated by histological analysis. Increased level of VEGF and decreased level of C-reactive protein (CRP), a biological marker that closely related to atherosclerosis, were also observed from animals treated with the bilayered NPs, implicating ameliorated atherosclerosis. Our results suggest that the VEGF plasmid/ PTX loaded bilayered NPs exert a beneficial impact on atherosclerotic restenosis by sequentially releasing VEGF and PTX in vivo. Keywords:

restenosis, re-endothelialization, atherosclerosis, balloon reperfusion, nanoparticles

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Introduction Cardiovascular disease (CVD) is the leading cause of global death1. Currently, interventional procedures, including balloon angioplasty, percutaneous transluminal coronary angioplasty (PTCA) and stent implantation are some routinely employed therapeutic options2,3, although patients often develop restenosis following these procedures4,5. Migration of vascular smooth muscle cells (SMCs) to the intimal space of blood vessels is a major cause of restenosis6. Paclitaxel (PTX), one of the antiproliferative drugs discovered in the past few decades7, is routinely prescribed to prevent restenosis for patients with CVD that have undergone surgical procedures8,9. Previous studies have shown that anti-proliferative agents such as PTX and sirolimus could reduce SMCs proliferation and the risk of restenosis. Indeed, drug-eluting stents (DES) that release PTX or sirolimus have been developed to inhibit the pathological processes of restenosis10-12. However，the mechanical exfolication by stenting could cause endothelial cell dysfunction. PTX release may also hamper healing of the vascular wall by excessive inhibition of vascular endothelial cell (ECs) growth13,14, resulting in late stage stent thrombosis. Restenosis often occurs at the sites of vascular endothelial injury, where platelets aggregate and the processes of endothelial healing are compromised15,16. While many researchers have focused their effort on reducing SMCs proliferation and migration, poor ECs recovery that is caused by treatments such as PXT is also detrimental. As a result, an ideal treatment for restenosis is to inhibit SMC proliferation after complete reformation of the endothelium, which is composed of the vascular endothelial cells. ECs often growth as monolayers and their characteristics are regulated by several growth factors.

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Vascular endothelial growth factor (VEGF) has been shown to directly control ECs migration and proliferation by increasing FGF-2 (fibroblast growth factor-2, which enhances ECs proliferation and vascular permeability while decreases the level of transforming growth factor-1(TGF-1), which has the function to alleviate vascular endothelial injury reactions, promote vascular endothelial repair, inhibit formation of atheromatous plaque17,18. Moreover, multiple roles of VEGF in vascular reformation have also been reported, including suppressing SMCs proliferation, enhancing ECs survival, inhibiting thrombosis and local inflammatory processes within the vessel wall19. Therefore, combining VEGF with PTX could be a more effective treatment for restenosis, because VEGF promotes endothelial healing while PTX inhibits excessive SMC growth. Previously, we have developed a stent that co-elutes both VEGF plasmids and PTX. We have observed that one month after stent implantation in the coronary artery of pigs, the NPs-eluting stent resulted in complete re-endothelialization and restenosis inhibition20. However, 30-40% of critical vascular lesions, such as lesions in branch sites or small arteries, cannot be surgically operated with vascular stent21. Considering the limitations of stenting, we then explored an approach of local administration of bilayered NPs via a perfusion balloon22 that directly injects to the intra-vascular site (Figure 1). The double-layered design of NPs, which contains the VEGF plasmids in the shell and PTX in the core, enables sequential release of the two different therapeutic mediators as desired. Moreover, the bilayered nanoparticles could also be administered less invasively compared to stents, by direct injection to the intra-vascular site using a catheter. We have characterized the properties of the bilayer NPs in detail and evaluated the in vivo efficacy of the bilayered NPs delivered via local perfusion in a rabbit atherosclerotic

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model. 2. Methods 2.1 Materials The VEGF 165 plasmid was a gift from the lab of Beijing Hospital (Beijing, China). PTX (99% purity) was purchased from Chongqing Meilian Company (Chongqing, China). Poly(lactic acid-co-glycolic acid) (PLGA 50:50) was purchased from Birmingham Polymer Co. (AL, USA). Polyvinyl alcohol (PVA; molecular weight: 30,000–70,000) and 3% pentobarbital sodium salt were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The purified anti-human Fc antibody, hIgG standard, Bio–anti-Fc, HRP-avidin, TMB （3, 3 ' , 5 , 5'-tetramethylbenzidine） and TMB stopping solution were purchased from eBioscience (San Diego, CA, USA). Lipofectamine 2000 Transfection Reagent was purchased from Invitrogen (Carlsbad, CA, USA). All other chemicals were analytical reagent grade and were purchased from Tianjin No. 1 Chemical Reagent Factory (Tianjin, China). Fogarty catheters were purchased from Cordis Corporation (Bridgewater Township, NJ, USA). 2.2 Preparation of bilayered nanoparticles (NPs) The bilayered NPs were prepared according to the following 2-step protocol. First, PTX was encapsulated in the core of NPs. PLGA (50 mg) and PTX (30 mg) were dissolved in 1 ml of dichloromethane. The solution was injected into 5 mL of 1% polyvinyl alcohol (PVA) aqueous solution and ultrasound (35W) for 5 min and further stirring 3 hours at room temperature till the organic solvent was evaporated completely to yield NPs with PTX excapsulated. The PTX NPs were washed thoroughly with distilled water three times before they were lyophilized over 24h.

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To load plasmid DNA to the shell of the bilayered NPs, 10 mg of gene plasmid and the lyophilized PTX NPs, prepared in the first step, were added in 1 mL of distilled water and mixed with 2 mL of dichloromethane solution containing 50 mg of PLGA and stirred using a homogenizer for 15 min. The resulting emulsion was added into 15 mL of 1% aqueous PVA and stirred vigorously with a homogenizer to form the bilayered NPs. The particles were washed thoroughly with distilled water three times, lyophilized over 24 h (-50oC, 100mT), then sterilized with Cobat-60 gamma irradiation at 2.5 kGy for 48h. The dose and duration of irradiation has been previously shown to cause no damage to DNA. Sterilized NPs were stored at 4 oC before use. The PTX loaded PLGA nanoparticles (PTX NPs), VEGF plasmid DNA (human VEGF165 expression plasmid) loaded PLGA nanoparticles (VEGF NPs), Fc plasmid DNA (human IgG Fc expression plasmid) loaded PLGA nanoparticles (Fc NPs) and Blank nanoparticles (Blank NPs) were also prepared according to the above protocol. 2.3 Characterization of bilayered NPs The size, size distribution, and zeta (z) potential of the prepared NPs were measured by dynamic laser light scattering (Zeta PALS analyser; Brookhaven Instruments Corp., Holtsville, NY, USA). NPs were suitably diluted in distilled water. The concentration of NPs was kept at 200 µg/ml. The average values were calculated from at least 3 measurements performed on each sample. Zeta potential measurement was based on the Laser Doppler Electrophoresis (LDE) method after diluting the samples in distilled water. Particle morphology was determined by transmission electron microscopy (TEM) (Tecnai-F20; FEI Company, Eindhoven, The Netherlands). Freeze-dried NPs were dispersed in distilled water,

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deposited on a copper grid, dried by blotting with tissue paper and examined by TEM. 2.4 Plasmid and PTX loading and encapsulation efficiency 5 mg of lyophilized NPs was dissolved in 2 mL of chloroform. Plasmid was extracted from chloroform by TE buffer (10 mM Tris-HCl 1 mM EDTA pH 8.0) and quantified by measuring optical absorbance at 260 nm using a spectrophotometer. All experiments were repeated three times. To quantify the content of PTX, lyophilized NPs were dissolved in chloroform, mixed with the mobile phase of high performance liquid chromatography (HPLC), nitrogen stream was used to evaporate chloroform until clear solution was obtained. The amount of PTX in the lyophilized NPs was determined by HPLC. The HPLC assay was performed on a reverse phase Diamond C18 column (inner diameter 150 *4.6 mm, pore size 5 µm) (Dikma Technologies Co. Ltd., Beijing, China). The mobile phase was a mixture of acetonitrile and water (50:50, v/v) with a flow rate of 1.0 mL/min. The column effluent was detected at 227 nm with an UV detector. All experiments were repeated three times. Plasmid and PTX encapsulation efficiencies were defined as the ratio of the amount detected in the NPs to the amount used for NPs preparation. 2.5 Elements composition of NPs In order to demonstrate that bilayered NPs were successfully prepared, scanning electron microscope (SEM) was used to analyze the elements composition of NPs. To stick the conductive adhesive on a glass slide, a small amount of nanoparticle was dispersed on the conductive adhesive, washing ears ball forward blowing nanoparticles, SEM was used to analyze elements composition after spraying gold on the nanoparticles.

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2.6 Release kinetics of DNA and PTX from NPs in vitro To determine release of DNA, NPs suspension in PBS buffer was placed in a double-chamber diffusion cell at 37 oC. A Millipore hydrophilic polyvinylidene fluoride membrane was placed between the two chambers and buffer of receiving chamber was replaced with fresh distilled water every 24 h. Plasmid concentration in the collected buffer solution was analyzed using a spectrophotometer at the 260 nm wavelength. The quantity of PTX was determined by HPLC following the previous protocol. Three samples were tested in each experimental group. 2.7 Gene transfection with bilayered NPs Chinese hamster ovary (CHO) cells were cultured overnight in 24-well plates at 1×105 cells/well with fetal bovine serum (FBS)-free DMEM medium. Different NPs suspension of 20 µg/ml were added to the 24-well plate for transfection, including blank NPs, Fc NPs, PTX NPs, bilayered NPs, Fc NPs+ PTX NPs and medium control group. After 24 and 48 h, the cell culture supernatant was collected. Human IgG Fc expression levels in cell culture medium were determined by sandwich ELISA following the manufacturer’s instructions (eBioscience Inc). The optical density of each well at 450 nm was determined and Fc concentrations were calculated based on the standards. For each experimental group, five samples were measured. 2.8 Cell growth inhibition by NPs CHO cells were cultured in 96-well plates with 1×103 cells/well. Different NPs and full DMEM medium solution were added at the same time. The concentration of NPs suspension was 20 µg/mL in the blank NPs, Fc NPs, PTX NPs, bilayered NPs, Fc NPs+ PTX NPs and medium control group. Cell viability was evaluated by MTT assay every day for 7 days following the standard protocol (Beyotime C). 40 µL of MTT (5 mg/mL) was added to each well and cell

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culture was continued for another 2 h, followed by the addition of 75 µL of lysis buffer to prepare the culture for reading in an OD570 spectrometer. The viable cell numbers were determined based on standards for known cell number culture wells in triplicate. For each experimental group, five samples were measured and each experiment was repeated three times. 2.9 Cellular uptake study in vitro To better clarify and visualize cell uptake of the bilayered nanoparticles, we employed coumarin 6 (a hydrophobic fluorophore which resembles PTX in solubility) and CY7 labeled OVA(OVA-CY7) (a fluorescently-labeled protein to resemble plasmid in size and solubility), and the following experiments have been performed. Firstly, coumarin 6 and OVA-CY7 loaded bilayered NPs were prepared in the same way as the bilayered NPs loaded with VEGF plasmid and PTX. Coumarin 6 (hydrophobic) and OVA-CY7 (43 kd) were employed here to represent PTX (hydrophobic) and VEGF plasmid (45 kd), respectively. Qualitative analysis was performed by confocal laser scanning microscopy (CLSM) using the human umbilical vein endothelial cells (HUVEC). In brief, HUVECs were seeded onto 35 mm glass bottom culture dish (5×104 cells per dish) and incubated in epithelial cell media (ECM) (1 mL/dish) in a 5% (v/v) carbon dioxide atmosphere incubator for 24 h. Cells were treated with coumarin-6- loaded nanoparticles or OVA-CY7-loated nanoparticles or bilayered nanoparticles loaded with both coumarin 6 and OVA-CY7 at a concentration of 200 µg/mL for 3 h. The cells were then rinsed three times with PBS and fixed with 300 µL 4% paraformaldehyde for 20 min before stained with 300 µL DAPI. Cells were examined by CLSM (Zeiss 410, Jena, Germany) and analyzed using the imaging software Fluoview FV500.

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2.10 Atherosclerotic rabbit model with balloon injuries and perfusion of NPs for restenosis treatment All the animal experiments including surgical procedures were performed in compliance with the Guiding Principles for the Care and Use of Laboratory Animals of the Peking Union Medical College (Beijing, China). New Zealand white rabbits (2.3-2.7 kg, male or female) were purchased from the Institute of Laboratory Animal Sciences at the Chinese Academy of Medical Sciences (Peking Union Medical College, Beijing, China). All the rabbits were fed with a cholesterol-rich diet (1% cholesterol, 5% yolk, 5% lard and 89% standard diet) for four weeks after their injuries, all the animals had free access to water at all times. Two weeks before surgery, rabbits were kept in a facility with constant temperature and humidity and a light cycle from 8:00 a.m. to 8:00 p.m. After being anesthetized with 3% pentobarbital sodium salt (30 mg/kg of body weight), all the rabbits received the following balloon-induced injuries: Femoral artery and the abdominal aorta were dissected. Injury in the aortic wall performed by inserting a 5F Fogarty catheter through a right femoral artery cut-down. The balloon injury model was created by inflating the balloon to a pressure of 10 atm, and moving the balloon retrograde by 100 mm three times for 15 sec each. The balloon catheter was then removed. Rabbits with balloon injuries were randomly divided into five experiment groups (n=8/each group). A site 5-10 mm below the renal artery was selected as the NPs delivery site. The size of the balloon catheter (Cordis Corporation) was selected according to the outer diameter of the abdominal aorta. The balloon catheter was located at the labeled site of the abdominal aorta. The

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perfusion balloon, GENIE CatheterTM (Acrostak Corporation, Geneva, Switzerland), is a new local drug delivery catheter that can deliver various liquid therapeutic agents into arteries by pressure. Rabbits with balloon injuries were randomly divided into five experiment groups (n=8 each group). Blank NPs, VEGF NPs, PTX NPs, bilayered NPs and saline were delivered to the injury site using the GENIE CatheterTM, with a perfusion pressure of 2-3 atm over 5 min. After delivering NPs, the GENIE CatheterTM and 5-French sheath were removed. The proximal end of the femoral artery was ligatured. Penicillin (80 MU) was intramuscularly administrated daily for three days after surgery. After surgery, rabbits were fed followed by 6 weeks of regular-cholesterol chow diet. 2.11 The healing of vascular endothelium observed by OCT Euthanasia of rabbits was performed after 28 days of treatment so that histological and morphological analyses could be performed. Before euthanasia, the endothelium was visualized using optical coherence tomography (OCT). In addition to this, results of OCT about the healing of vascular endothelium could indirectly demonstrated that whether bilayered NPs could release VEGF plasmid and PTX sequentially. OCT acquisition was performed with a commercially available system (M3 OCT system, Light Lab Imaging). The image wire (ImageWire, Light Lab Imaging) was positioned distal to the region of interest with an over-the-wire OCT catheter (Helios OBC, Light Lab Imaging) that had been placed in the artery over a conventional guidewire. Then, the OCT catheter was withdrawn from the distal to proximal end of the artery. Automated pullback was performed at 1.5 mm/s while blood was removed by continuous infusion of Lactated Ringer’s solution at 0.5-1.5 mL/s using a power injector. After OCT was completed, euthanasia was performed and artery samples were retrieved. A portion of each

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specimen taken from each rabbit was fixed in 10% formalin for histological analysis. 2.12 VEGF and CRP expression and histological analysis of arterial tissue A computerized coronary angiography analysis system (X-ray digital tablet imaging system FD 10; Philips Healthcare, Amsterdam, The Netherlands) was used to determine the amount of stenosis, the proliferative index, and maximal intimal thickness. Part of the artery was perfused with saline, frozen in liquid nitrogen, sectioned into 5 µm slices, and stained with hematoxylin-eosin (H&E). All slices of arterial tissue were examined by optical microscopy (CKX41-A32PH; Olympus Co., Tokyo, Japan). The lumen area and neointimal area were measured using computerized planimetry (Spot Advanced 4; SPOT Imaging Solutions, Sterling Heights, MI, USA). In order to test VEGF and C-reaction protein (CRP) expression of artery, paraffin section of the samples were immersed in xylene and dehydrated through graded concentrations of ethanol. After that, sections in citrate buffer were heated by a microwave oven for two circles of 15 min each. Endogenous peroxidase was blocked with H2O2 solution in methanol for 30 min. Nonspecific binding was blocked by incubating the sections for 30 min with blocking buffer. After washed with PBS for 5 min, VEGF or CRP primary antibodies for section were incubated for 30 min in 4 oC. After washing three times by using PBS, sections were incubated with the biotinylated secondary antibodies in 37 oC for 15 min, and washed with PBS three times. After incubated with streptavidin-HRP in 37 oC for 15min and washed three times, monitoring the colour development, then envision system was used for visualization of expression of VEGF or CRP. 2.13 Statistical analysis

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For each experimental group, data was presented as mean±SD. Statistical significance for each variable was estimated by a two-way analysis of variance (ANOVA) or a t-test, wherever appropriate. 3. Results and discussion 3.1 Characterization of bilayered NPs First, PTX NPs were prepared which served as the cores of the bilayered NPs (Fig. 2A). After freeze drying, PTX NPs were dispersed in solution containing plasmids, then mixed with PLGA dichloromethane solution with a homogenizer, during which process the plasmids were loaded in PLGA outer layer as shell of bilayered NPs (Fig. 2B). The cores of bilayered NPs were uniform in size (average: 176.97 ± 0.32 nm), with a polydispersity index of 0.059 and mean zeta potential of -2.39±0.57 mV. The average diameter of the bilayered NPs was 306.53 ±16.16 nm (Fig. 2C) with a polydispersity index value of 0.209. The size of bilayered NPs was controlled by the power and duration during ultrasonication. The mean zeta-potential of bilayered NPs was -6.01 ± 0.19 mV. NPs are uniquely suited to reperfusion balloon administration due to their small size, uniformed dispersion, and permeability through the arterial walls. The sizes of NPs are critical in determining the therapeutic outcome of NP-based therapies. NPs with diameter ranging between 120-300 nm are able to penetrate the intimal layers of blood vessels23. For the present study, the average diameter of the bilayered NPs also falls within this range. TEM was used to examine the morphology of the bilayered NPs (Fig. 2D, E and insert). NPs were uniform and spherical in shape, with an evident double-layered structure, shown by dark core regions and lighter outer layers.

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We designed bilayer gene and drug co-delivery system, which loaded gene by double emulsion methods, which is a classic method of the preparation of nanoparticles. Through adjusting the concentration of the water phase, oil phase and the ratio of water phase and oil phase, high encapsulation efficiency of gene nanoparticle can was obtained24. Cumulative PTX and DNA were loaded into bilayered NPs with a PTX entrapment rate of 92% and a DNA entrapment rate of 98%. The PTX content was 28.58% (weight percentage) and the VEGF DNA content was 4.67% (weight percentage). As shown in Figure A, the chromatographic peak of PTX standard was test at time 2.733 min, and as shown in Figure B, there was the chromatographic peak at 2.735 min of release liquid of NPs which demonstrated that the PTX have been successfully loaded in PLGA (supplementary material Figure S1.)

3.2 Elements composition of NPs As shown in Table 1 and Figure 3, there were C, H, O, N elements existed in PTX-NPs and existence of N indicated that PTX was successfully loaded in PLGA. In addition to these elements, P was also detected in the bilayered NPs. Since P is the unique elements of the DNA plasmids, this proves that the plasmid/PTX-loated bilayered NPs were successfully prepared. 3.3 Release of DNA and PTX from single and bilayered NPs in vitro It has been reported in Xu's study that double-walled microspheres consisting of a poly (d, l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly (lactic acid) (PLA) shell could be fabricated via the precision particle fabrication technique25,26. Consistently, we designed the bilayered NPs containing a core region that was encapsulated with PTX and another polymer layer containing Fc (a model plasmid) or VEGF plasmids for sequential release using the same

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method. By encapsulating the Fc-encoding plasmid in the outer layer of the NPs, we expect the Fc gene to be released first for ECs transfection followed by a delayed PTX release that inhibits cell proliferation without interfering with the transfection process. Our in vitro results using CHO cells confirmed the sequential release of Fc plasmids followed by PTX (Figure 4). Thus, single-layered NPs containing either plasmid or PTX were prepared as controls and the releasing profiles of plasmid and PTX were determined using a mixture of these two types of NPs. The separately loaded DNA plasmid and PTX were released almost simultaneously with similar kinetics during the first 5 days (Fig. 4A). Sequential release was not observed. For bilayered NPs, plasmids were released rapidly at the beginning of the observation period and over 70% was released in 20 days (Fig. 4B). The release kinetics of plasmids from single-layered and bilayered NPs were similar, and this is because that the plasmids were encapsulated in the outer layer of the bilayered NPs, which similar to the single-layered NPs, was in contact to aqueous buffer and could be released readily. In contrast to the single-layered NPs, there was a considerable delay in the release of PTX from the bilayered NPs, especially during the first 5 days, during which almost none of the loaded PTX was released in the first two days (Fig. 4B). After 30 days, only 50% of PTX loaded in the NPs had been released. This delayed release of PTX was likely due to the fact that PTX was loaded only in the core (the inner compartment) of the bilayered NPs. These results demonstrated that the design of bilayered NPs with spatially segregated plasmids in the outer layer and PTX in the core was crucial in achieving sequential release of plasmid and PTX. Furthermore, the release rates of both plasmids and PTX could be further fine-tuned by adjusting the thickness of the polymer shell or by using different polymers with variable degradation

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property. 3.4 Gene transfection and viability of NP-treated cells To examine the biological effect of the sequential release of plasmid and PTX for gene transfection and effects on cell growth, a human IgG Fc expression plasmid was loaded as a reporter in the outer layer of bilayered NPs with PTX in the core. After co-cultured with NPs, Fc protein expression was detected by ELISA after 24 h in CHO cells that treated with NPs, and the expression level remained relatively high after 48 h compared to cells treated with Lipofectamine 2000 (a standard cell transfection reagent; Fig. 4C). In addition, there was no significant deference of the protein expression between Fc NPs and bilayered NPs after incubating for 24 h. However, the expression Fc protein of bilayered NPs was obviously higher than Fc NPs at 48 h because that the PTX in the core began to release to influence growth of cells, which furthermore demonstrated that bilayered NPs could release gene and PTX step by step and suggested that the bilayered NPs were successfully prepared. No expression of Fc was detected in blank NPs, PTX NPs and medium control groups. Cell viability was quantified by MTT assay, which allowed us to observe the potential inhibitory effect of Fc/PTX NPs on cell growth (Fig. 4D). As expected, no evident cytotoxicity could be observed from the Fc/PTX bilayered NPs treated group in the first 24 h. A sustained inhibition of cell growth was observed after 48 h following bilayered NPs administration and lasted several days beyond, resulting from delayed PTX release. On the contrary, cells treated with single-layered PTX NPs or a mixture of single-layered PTX NPs exhibited immediate cell death during the first 24 h, likely because of the more rapid PTX release. These results suggested that sequential plasmid and PTX release could be achieved by the bilayered NPs which led to

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transgene expression with delayed inhibition of cell proliferation in vitro. 3.5 Cellular uptake study in vitro Average particle size ranges between 100 and 300 nm is considered facilitative of cell endocytosis23. We have already demonstrated in Fig. 2C that the bilayered NPs falls within this range. For in vitro cell uptake experiments, in order to visualize cellular uptake of the nanoparticles, nanoparticles containing either coumarin-6, a hydrophobic green fluorphore similar to PTX, or CY7-labelled OVA, a red fluorescence protein that resembles the size of plasmids, or both in a bilayered arrangement (CY7-OVA in the outer layer and coumarin-6 in the core) were prepared. As shown in Fig.5, nanoparticles containing either coumarin-6 (shown in green) or OVA-CY7 (shown in red) were both readily uptaken by the vascular endothelial cells HUVECs. Similarly, cells treated with the bilayered nanoparticles containing both coumarin-6 and OVA-CY7 also exhibited evidence fluorescence signals corresponding to coumarin-6 and OVA-CY7 within the cytoplasm, demonstrative of successful endocytosis of the bilayered nanoparticles by the vascular endothelial cells in vitro. 3.6 Bilayered NPs promoted re-endothelialization in vivo Since a more effective way to decrease the risk of restenosis would be to accelerate endothelium healing followed by inhibition of SMC proliferation, we have designed the bilayered NPs that contain the VEGF gene in the outer layer and PTX in the NPs core as illustrated in Fig 1. In this design, the VEGF-encoding plasmids loaded in the outer layer were expected to be released first to increase vascular VEGF expression that has been shown to stimulate endothelial growth and healing. PTX, which was encapsulated within the core, was released afterwards, to suppress SMC proliferation and reduce restenosis.

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To evaluate this hypothesis, we performed balloon angioplasty in atherosclerotic rabbits to cause injury to the coronary endothelium, followed by local administration of the bilayered NPs loaded with VEGF plasmids and PTX. For controls, rabbits were treated with single-layered NPs loaded with either VEGF plasmids or PTX. The vascular endothelium of the atherosclerotic rabbits was inspected with OCT before euthanasia 28 days after surgery. Suppression of endothelialization and proliferation of SMC were observed in the PTX NPs treated and blank NPs groups (Fig. 6 C, D, E, F). It was found that the endothelia of rabbits that were treated with VEGF NPs or the bilayered NPs were healed completely, which was not the case in other groups (Fig. 6 G, H, I, J, K). Complete regeneration of the endothelia was also expected to modulate vascular tension, inhibit

thrombosis

formation

and

SMCs

proliferation

by

enhancing

nitric

oxide

generation27.These results indicate that local VEGF gene delivery mediated by the NPs was responsible for re-endothelialization and that through the delayed release of PTX, the bilayered NPs prevented PTX from hindering endothelia repair. The differences observed in rabbits treated with the VEGF plasmid containing NPs and NPs with no VEGF plasmid suggest sequential release of plasmids and PTX showed in vivo. 3.7 Bilayered NPs reduced restenosis in atherosclerotic rabbits Representative photomicrographs of angiographic and morphologic cross sections from the different experimental groups were shown in Fig 7. The photomicrographs of animals treated with blank NPs and saline control group were much the same. As a result, only representative photomicrographs of the blank NPs group were presented here. The amount of stenosis in the bilayered NP group was 7.4% ± 8.2% with a proliferative index of 0.13 ± 0.022. Maximal intimal thickness was 0.08 ± 0.097. The amount of stenosis in the VEGF NP group was 12.5% ±

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8.6% with a proliferative index of 0.19 ± 0.023 and a maximal intimal thickness of 0.12 ± 0.099. These results of the other groups were Saline = 33.5% ± 22.8%, 0.43 ± 0.025, 0.18± 0.097; Blank NP group = 33.5% ± 15.2%, 0.3 ± 0.02, 0.22 ± 0.028; PTX NP group = 23.1% ± 14.6%, 0.26 ± 0.015, 0.18 ± 0.077. Restenosis (characterized by the amount of stenosis, the proliferative index, and the maximal intimal thickness) was significantly reduced in the bilayered NPs and VEGF NPs groups, and there were significant differences in therapeutic effect (P < 0.05) compared to control groups. Average values were improved for the bilayered NPs compared to the VEGF NPs, but the difference was not statistically significant. Since anti-restenosis effects were observed from both VEGF NPs and the bilayered NPs groups, it suggested that VEGF gene therapy have played a major role during the initial endothelial healing period. The rapid healing process of the endothelia also provides a natural barrier that inhibits leukocyte and platelet adhesion, which prevent late thrombosis formation28,29. While the VEGF NPs group which exhibited enhanced vascular endothelia regeneration, the PTX NPs group only showed decreased SMC proliferation. As expected, the bilayered NPs group showed both vascular endothelia regeneration and inhibition of SMCs proliferation. 3.8 VEGF expression in target tissue To confirm the success of VEGF gene delivery, VEGF protein expression levels in the target blood vessels of different treatment groups were measured (Fig. 8A). VEGF level in the arterial walls of the bilayered NP group was 40.6% ± 17.5%, which was comparable to VEGF expression in normal animal artery. In the VEGF NPs group, it was 42.9% ± 11%; but in the saline, Blank NPs, and PTX NPs groups, the VEGF protein expression rate was 25.7% ± 3.3%, 29.5% ± 8.3%, and 30.5% ± 12.1% respectively. These results proved that transfecting the VEGF

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gene into tissue resulted in VEGF protein expression, which was responsible for improved vascular endothelia healing. 3.9 C-reactive protein (CRP) expression in target tissue The expression level of CRP is considered an atherosclerotic indicator of inflammatory activity, which is closely related to the severity of atherosclerosis30. It was found that the CRP level was the lowest in the bilayered NP group (Fig. 8B), shown as 42.3% ± 8.6% as compared to 65.7% ± 12.6% in the saline group, 61.7% ± 18.5% in the Blank NP group, 58.2% ± 15% in the PTX NP group, and 47.9% ± 9.86% in the VEGF NP group. The significant reduction of CRP expression observed in the bilayered NPs treated group further indicate ameliorated atherosclerotic inflammation and alleviated symptoms of atherosclerosis. 4 Conclusion In summary, we report here a bilayered VEGF/PTX NPs capable of local and sequential release of a VEGF-encoding plasmid and PTX. The results of the particle size analysis showed facile and rapid infiltrating ability of the bilayered NPs into the blood vessel. Results of in vitro release and cytotoxic experiments indicated that bilayered NPs enable sequential release of plasmids and PTX, which attenuates cell proliferation. In vivo experiments further demonstrated a “two-step action” of the bilayered NPs in coronary atherosclerosis recovery, shown as complete endothelia regeneration after 1 month bilayered NPs administration with limited proliferation of the SMCs. Furthermore, normalization of the atherosclerotic indicator, CRP, has been observed, also suggesting a positive role of the bilayered NPs in atherosclerosis recovery. In summary, our data proved to be encouraging, nevertheless, future studies are required to evaluate long-term therapeutic effect of the bilayered NPs beyond 28 days.

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Description of Supporting Information: As shown in supporting information Figure A, the chromatographic peak of PTX standard was test at time 2.733 min, and as shown in Figure B, there was the chromatographic peak at 2.735 min of release liquid of NPs which demonstrated that the PTX have been successfully loaded in PLGA (supplementary material Figure S1). Acknowledgement These studies were supported by grants from the National Natural Science Foundation of China (81271706, 31300794), the Tianjin Research Foundation Advanced Technology Program (17JCZDJC37400, 16JCQNJC13900, 17JCYBJC29100 and 15RCGFSY00146 ), CAMS Innovation Fund for Medical Sciences (2017-I2M-1-021).

References [1] Roger, VL.; Go, AS.; Lloyd-Jones, DM.; Adams, RJ.; Berry, JD.; Brown, TM.; Carnethon, MR.; Dai, S.; de Simone, G.; Ford, ES.; Fox, CS.; Fullerton, HJ.; Gillespie, C.; Greenlund, KJ.; Hailpern, SM.; Heit, JA.; Ho, PM.; Howard, VJ.; Kissela, BM.; Kittner, SJ.; Lackland, DT.; Lichtman, JH.; Lisabeth, LD.; Makuc, DM.; Marcus, GM.; Marelli, A.; Matchar, DB.; Mc Dermott, MM.; Meiqs, JB.; Moy, CS.; Mozaffarian, D.; Mussolino, ME.; Nichol, G.; Paynter, NP.; Rosamond, WD.; Sorlie, PD.; Stafford, RS.; Turan, TN.; Turner, MB.; Wong, ND.; Wylie-Rosett, J. Heart Disease and Stroke Statistics--2011 Update: a Report from the American Heart Association. Circulation 2011, 123, e18-e209. [2] Buechel, R.; Stirnimann, A.; Zimmer, R.; Keo, H.; Groechenig, E. Drug-eluting Stents and Drug-coated Balloons in Peripheral Artery Disease. Vasa 2012, 41, 248-261 [3] Quang, MN.; Kruger, B.; Wenning, M.; Kruger, CD.; Tokmak, F.; Kisters, K.; Wunsch, A.;

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Schenker, P.; Viebahn, R.; Kramer, BK. Autotransplantation for the Treatment of Severe Renal Artery Stenosis in a Solitary Kidney after Repeated Percutaneous Transluminal Angioplasty: a Case Report. Clin Nephrol 2012, 78, 418-422. [4] Hofma, SH.; Brouwer, J.; Velders, MA.; van't Hof, AW.; Smits, PC.; Quere, M.; de Vries, CJ.; van Boven, AJ. Second-generation Everolimus-eluting Stents Versus First-generation Sirolimus-eluting Stents in Acute Myocardial Infarction. 1-year Results of the Randomized XAMI (XienceV Stent vs. Cypher Stent in Primary PCI for Acute Myocardial Infarction) Trial. J Am Coll Cardiol 2012, 60, 381-387. [5] Wohrle, J. Drug-coated Balloons for Coronary and Peripheral Interventional Procedures. Curr Cardiol Rep 2012, 14, 635-641. [6] Tarantini, G.; Barioli, A.; Facchin, M.; Frigo, AC.; Napodano, M.; Buja, P.; Damico, G.; lliceto, S.; Isabella, G. Six-year Clinical Outcomes of First-generation Drug-eluting Stents: a Propensity-matched Analysis. Coron Artery Dis 2013, 24, 440-448. [7] Grube, E.; Silber, S.; Hauptmann, KE.; Mueller, R.; Buellestfeld, L.; Gerckens, U.; Russell, ME. TAXUS I: Six- and Twelve-month Results from a Randomized, Double-blind Trial on a Slow-release Paclitaxel-eluting Stent for de novo Coronary Lesions. Circulation 2003, 107, 38-42. [8] Lee, JH.; Kim, SJ.; Park, SI.; Ko, YG.; Choi, D.; Hong, MK.; Jang, Y. Development of a New Hybrid Biodegradable Drug-eluting Stent for the Treatment of Peripheral Artery Disease, Biomed Res Int 2016, 2016, 1-7. [9] Flueckiger, A.; Strahm, Y.; Billinger, M.; Meier, P.; Mettler, D.; Studer, U.; Schaffner, T.; Hess, OM. Intimal Proliferation and Restenosis in Paclitaxel-eluting Stents with

22



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Aminoparylene as Carrier Substance in Swines, J Invasive Cardiol 2009, 21, 128-132. [10] Garg, S.; Serruys, PW. Coronary Stents: Current Status. J Am Coll Cardiol 2010, 56, S1-42. [11] Iqbal, J.; Gunn, J.; Serruys, PW. Coronary Stents: Historical Development, Current Status and Future Directions. Br Med Bull 2013, 106, 193-211. [12] Garg, S.; Serruys, PW. Coronary Stents: Looking Forward. J Am Coll Cardiol 2010, 56, S43-78. [13] Sun, D.; Zheng, Y.; Yin, T.; Tang, C.; Yu, Q.; Wang, G. Coronary Drug-eluting Stents: from Design Optimization to Newer Strategies. J Biomed Mater Res A 2014, 102,1625-1640. [14] Joner, M.; Finn, AV.; Farb, A.; Mont, EK.; Kolodgie, FD.; Ladich, E.; Kutys, R.; Skorija, K.; Gold, HK.; Virmani, R. Pathology of Drug-eluting Stents in Humans: Delayed Healing and Late Thrombotic Risk. J Am Coll Cardiol 2006, 48, 193-202. [15] Pasceri, V.; Pelliccia, F.; Pristipino, C.; Roncella, A.; Irini, D.; Varveri, A.; Biscigia, A.; Speciale, G. Clinical Effects of Routine Postdilatation of Drug-eluting Stents. Catheter Cardiovasc Interv 2014, 83, 898-904. [16] Byrne, RA.; Cassese, S.; Windisch, T.; King, LA.; Joner, M.; Tada, T.; Mehilli, J.; Pache, J.; Kastrati, A. Differential Relative Efficacy between Drug-eluting Stents in Patients with Bare Metal and Drug-eluting Stent Restenosis; Evidence in Support of Drug Resistance: Insights from the ISAR-DESIRE and ISAR-DESIRE 2 trials. Eurointervention 2013, 9, 797-802. [17] Santos, SC.; Miquel, C.; Dominques, I.; Calado, A.; Zhu, Z.; Wu, Y.; Dias, S. VEGF and VEGFR-2 (KDR) Internalization is Required for Endothelial Recovery during Wound Healing. Exp Cell Res 2007, 313, 1561-1574. [18] Lazarous, DF.; Shou, M.; Stiber, JA.; Hodge, E.; Thirumurti, V.; Goncalves, L.; Unger, EF.

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Page 24 of 34

Page 25 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Adenoviral-mediated Gene Transfer Induces Sustained Pericardial VEGF Expression in Dogs: Effect on Myocardial Angiogenesis. Cardiovasc Res 1999, 44, 294-302. [19] Bachmann, BO.; Luetjen-Drecoll, E.; Bock, F.; Wiegand, SJ.; Hos, D.; Dana, R.; Kruse, FE.; Cursiefen, C. Transient Postoperative Vascular Endothelial Growth Factor (VEGF) -Neutralisation Improves Graft Survival in Corneas with Partly Regressed Inflammatory Neovasculation. Br J Ophthalmol 2009, 93, 1075-1080. [20] Yang, J.; Zeng, Y.; Zhang, C.; Chen, YX.; Yang, Z.; Li, Y.; Leng, X.; Kong, D.; Wei, XQ.; Sun, HF.; Song, CX. The Prevention of Restenosis in vivo with a VEGF Gene and Paclitaxel Co-eluting Stent. Biomaterials 2013, 34, 1635-1643. [21] Schneider, M.; Wiebe, W.; Hraska, V.; Zartner, P. Coronary Interventions in Congenital Heart Diseases: from Preterm to Young Adult Patients. J Interv Cardiol 2013, 26, 287-294. [22] Guzman, LA.; Labhasetwar, V.; Song, C.; Jang, Y.; Lincoff, AM.; levy, R.; Topol, EJ. Local Intraluminal Infusion of Biodegradable Polymeric Nanoparticles. A Novel Approach for Prolonged Drug Delivery after Balloon Angioplasty. Circulation 1996, 94, 1441-1448. [23] Xu, Q.; Xia, Y.; Wang, CH.; Pack, DW. Monodisperse Double-walled Microspheres Loaded with Chitosan-p53 Nanoparticles and Doxorubicin for Combine Gene Therapy and Chemotherapy.

J Controlled Release 2012, 163, 130-135.

[24] Yang, J., Zeng, Y., Li, YJ., Song, CX., Zhu, WL., Guan, H., Li, XH. Intravascular Site-specific Delivery of a Therapeutic Antisense for the Inhibition of Restenosis[J]. Eur J Pharm Sci 2008, 35, 427-434. [25] Xu, Q.; Leong, J.; Chua, QY.; Chi, YT.; Chow, PK.; Pack, DW.; Wang, CH. Combined Modality Doxorubicin-based Chemotherapy and Chitosan-mediated p53 Gene Therapy

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using Double-walled Microspheres for Treatment of Human Hepatocellular Carcinoma. Biomaterials 2013, 34, 5149-5162. [26] Rome, JJ.; Shayani, V.; Fluqelman, MY.; Newman, KD.; Farb, A.; Virmani, R.; Dichek, DA. Anatomic Barriers Influence the Distribution of in vivo Gene Transfer into the Arterial Wall: Modeling with Microscopic Tracer Particles and Verification with a Recombinant Adenoviral Vector. Arterioscler Thromb 1994, 14, 148-161. [27] Schinzari, F.; Tesauro, M.; Cardilo, C. Vascular Hyperpolarization in Human Physiology and Cardiovascular Risk Conditions and Disease. Acta Physiol 2017, 219, 124-137. [28] Kipshidze, N.; Dangas, G.; Tsapenko, M.; Moses, J.; leon, MB.; Kutryk, M.; Serruys, P. Role of the Endothelium in Modulating Neointimal Formation: Vasculoprotective Approaches to Attenuate Restenosis after Percutaneous Coronary Interventions. J Am Coll Cardiol 2004, 44, 733-739. [29] Ando, H.; Ishii, H.; Yoshikawa, D.; Uetani, T.; Amano, T.; Murohara, T. Disruption of Atherosclerotic Neointima as a Cause of Very Late Stent Thrombosis after Bare Metal Stent Implantation. Am J Cardiol 2012, 109, 448-449. [30] Koenig, W. High-sensitivity C-reactive Protein and Atherosclerotic Disease: from Improved Risk Prediction to Risk-guided Therapy. Int J Cardiol 2013, 168, 5126-5134.

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Figure Legends

Figure1. Illustration of local infused bilayered NPs to inhibit restenosis in atherosclerotic animal model. (A) Healthy vessel. (B) Atherosclerotic plaque in a vessel. (C) Bilayered NPs infused at the site of vessel injury. (D) Step I: VGEF gene transfection (E) VEGF protein promotes rapid re-endothelialization in the period immediately after injury. (F) Step II: PTX inhibits smooth muscle cell proliferation in the second period.

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Figure 2. Characterization of bilayered NPs in vitro. (A) Schematic illustration of the structures of core and (B) bilayered NPs. (C) Size distribution of bilayered NPs and their cores. (D) TEM images of PTX-NPs and (E) bilayered NPs.

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Table 1 Elements composition and quantity percentage Blank-NPs

PTX-NPs

VEGF-NPs

Bilayered-NPs

C

69.83

70.19

67.36

71.76

O

30.17

26.19

30.35

25.47

3.63

2.19

2.91

0.11

0.09

N P

Figure 3. Elements of different NPs and the quality percentage. (A) Blank-NPs; (B) PTX-NPs; (C) PGL3-NPs; (D) Bilayered-NP

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Figure 4. In vitro release, gene expression an cytotoxicity of bilayered NPs. Gene plasmid and PTX release in vitro, gene expression and cytotoxicity of bilayered NPs. (A) Release of Fc NPs and PTX NPs in vitro. (B) Release of Fc/PTX bilayered NPs in vitro. The release behavior of single layer NPs ( Fc NPs and PTX NPs ) compare with bilayered NPs were different, the release of Fc plasmid and PTX can be observed from the first day, in bilayered NPs experimental group, FC NPs was tested in first day, but for PTX, it can be detected from two days later. (C) Transfection of the Fc/PTX bilayered NPs was tested by ELISA in comparison with single-layered NPs. (D) The growth curve of CHO cells in the presence of Fc/PTX bilayered NPs in comparison with single-layer NPs. Cell viability was determined by the MTT assay. 29


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Figure 5. Cellular uptake of different NPs. Nanoparticles containing either coumarin-6 (shown in green) or OVA-CY7 (shown in red) were both readily uptaken by the vascular endothelial cells HUVECs.

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Figure 6. Representative photos of re-endothelization after the infusion of different NP groups, observed under OCT 28 days after surgery(On the left of photos are the results of H&E staining, on the right of photos are the results of OCT). (A), (B) Healthy vessel, (C), (D) Blank NPs infused vessel. (E), (F) PTX NPs infused vessel. (G), (H) VEGF NPs infused vessel. (I), (J), (K) Bilayered NPs infused vessel.

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Figure 7. Representative images of H&E-stained artery cross-sections 28 days after treatments. (A) Normal. (B) Blank NPs. (C) PTX NPs. (D) VEGF NPs. (E) Bilayered NPs (40×), quantitative analysis of percent area stenosis, proliferation index and maximal intimal thickness 28 days after treatments. (F) Percentage of stenosis. (G) Proliferation index. (H) Maximum intimal thickness. **P < 0.05 vs. saline and blank NPs group.

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Figure 8. VEGF and CRP expression. (A) Quantitative analysis of VEGF expression. **P < 0.05 vs. saline and blank NPs group. (B) The positive expression rate of CRP. **P < 0.05 vs. saline and blank NPs group.

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Graphic for manuscript

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Bilayered Nanoparticles with Sequential Release ... - ACS Publications

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