Reduction-Responsive Nucleic Acid Delivery Systems To Prevent In

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Reduction-Responsive Nucleic Acid Delivery Systems to Prevent In-Stent Restenosis in Rabbits Weijie Ye, Yiming Chen, Wenxiong Tang, Na Zhang, Zhonghao Li, Zunjing Liu, Bingran Yu, and Fu-Jian Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08544 • Publication Date (Web): 25 Jun 2019 Downloaded from pubs.acs.org on July 26, 2019

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ACS Applied Materials & Interfaces

Reduction-Responsive Nucleic Acid Delivery Systems to Prevent In-Stent Restenosis in Rabbits Weijie Yea,†, Yiming Chenb,†, Wenxiong Tanga, Na Zhangb, Zhonghao Lia, Zunjing Liua,*, Bingran Yub,*, and Fu-Jian Xub,* aDepartment

of Neurology, China-Japan Friendship Hospital, Beijing, 100029, China Lab of Biomedical Materials of Natural Macromolecules (Beijing University of Chemical Technology), Ministry of Education, Beijing Laboratory of Biomedical Materials, Beijing University of Chemical Technology, Beijing, 100029, China bKey

†Both

authors contributed equally to this work. *Corresponding author. Tel.: 8610-64421243 E-mail address: [email protected] (Z. Liu); [email protected] (F.-J. Xu); [email protected] (B. Yu)

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ABSTRACT Cardiovascular and cerebrovascular ischemic diseases seriously affect human health. Endovascular stent placement is an effective treatment but always leads to in-stent restenosis (ISR). Gene-eluting stent, which combines gene therapy with stent implantation, is a potential method to prevent ISR. In this study, an efficient gene-eluting stent was designed based on one new nucleic acid delivery system to decrease the possibility of ISR. The reduction-responsive branched nucleic acid vector (SKP) with low cytotoxicity was first synthesized via ring-opening reaction. The impressive in vitro transfection performances of SKP were proved using luciferase reporter, enhanced green fluorescent protein plasmid, and vascular endothelial growth factor plasmid (pVEGF). Subsequently, SKP/pVEGF complexes were coated on the surfaces

of

pre-treated

clinical

stents

to

construct

gene-eluting

stents

(S-SKP/pVEGF). Anti-restenosis performance of S-SKP/pVEGF was evaluated via implanting stents into rabbit aortas. S-SKP/pVEGF could lead to the localized upregulation of VEGF proteins, improve the progress of re-endothelialization, and inhibit the development of ISR in vivo. Such efficient pVEGF-eluting stent with responsive nucleic acid delivery systems is very promising to prevent in-stent restenosis of cerebrovascular diseases. KEYWORDS: in-stent restenosis, endothelialization, gene therapy, polycation, VEGF

2


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1. INTRODUCTION Cardiovascular and cerebrovascular ischemic diseases are the main causes of disability and mortality.1,2 Endovascular stent placement is one suitable and effective treatment for ischemic disease caused by arteriostenosis.3 However, as a foreign body, implanted stent will induce platelet aggregation and lead to acute thrombosis events. At the same time, exposed stents can lead to disordered proliferation of endothelial cells (ECs), causing in-stent restenosis (ISR).4-7 The complete ECs can act as a nature barrier to control material exchange between vessel lumen and surrounding tissue, which plays an important role in the healing process of impaired vasculars walls.8,9 Re-endothelialization after stent implantation can prevent stents from contacting blood flow, promote the release of various active factors that can inhibit thrombosis and inflammation, and reduce the incidence of ISR consequently.10-12 Therefore, improving re-endothelialization after stent implantation is a potential therapeutic target. Normally, drug-eluting stents are limited to use in the treatment of cerebral arterial stenosis due to intracranial hemorrhage.13,14 To solve this problem, gene-eluting stents have attracted much attention.15-18 Vascular endothelial growth factor (VEGF) is one of the most effective factor that stimulates re-endothelialization.19 VEGF gene has been used on gene-eluting stents for accelerating endothelial cells proliferation and reducing ISR.20,21 To ensure effective transfection of plasmids, a safe and efficient delivery system is necessary. Due to flexibility and easy preparation of polymers, 3



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polycationic systems as the major non-viral vectors draw much more attention.22 Compared

with

linear

polycation,

branched

polycation

has

one

unique

three-dimensional branched structure which could benefit gene transfection performance.23-26 In addition, branched polycations are easily prepared and have abundant terminal functional groups for further modification.27,28 Introducing biodegradable chemical bonds into polycation can not only decrease cytotoxicity but also accelerate the release of nucleic acids within cells to promote transfection performances.29,30 Biodegradable branched nucleic acid vectors used on gene-eluting stents are still absent. Their potential excellent property may impart gene-eluting stents with good performances to prevent ISR. In this study, one new reduction-responsive nucleic acid vector (SKP) with disulfide bonds was synthesized via ring-opening reaction of disulfide bond-modified lysine (SK) (Figure 1). Numerous hydroxyl groups induced by ring-opening reaction can contribute to low cytotoxicity and good blood compatibility.22,31 SKP/pVEGF (VEGF plasmid) complexes could be coated on the pre-treated clinical stent to produce the pVEGF-eluting stent (S-SKP/pVEGF). In vivo re-endothelialization and anti-restenosis performance of S-SKP/pVEGF were evaluated via implanting stents into rabbit abdominal aortas. It was expected that such well-defined gene-eluting stent could provide the effective treatment of vascular in-stent restenosis.

2. EXPERIMENTAL SECTION 2.1. Materials. D-lysine hydrochloride (98%), di-tert-butyl dicarbonate (98%), 4


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cystamine

dihydrochloride

(99%),

1,6-hexanediamine

(99%),

dicyclohexylcarbodiimide (DCC, 98%), 1-hydroxybenzotriazole (HOBT, 99%), trifluoroacetic acid (TFA, 99%), triethylamine (TEA, 99.5%), dithiothreitol (DTT, 98%), Trometamol (Tris, 99.5%) and 1,3,5-triglycidyl isocyan urate (TGIC, 98%) were purchased from Energy Chemical Co. (Shanghai, China). Ethylenediamine (ED, 98%), branched polyethyleneimine (PEI, Mw ≈ 25 kDa), deuterium oxide (D2O), dimethyl sulfoxide-d6 (DMSO-d6), Rhodamine 123 (99%), dopamine hydrochloride (99%), Triton X-100 and 4′,6-diamidino-2-phenylindole (DAPI) were bought from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Endothelial cell medium (ECM) was obtained from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA). Human umbilical vein endothelial cell line (HUVEC) was obtained from Procell Life Science&Technology Co. Ltd. (Hubei, China). Radioimmunoprecipitation assay (RIPA) buffer and cell counting kit-8 (CCK-8) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). YOYO-1 was purchased from Thermo Fisher Scientific Inc. (USA). Anti-VEGF-165a, anti-VEGFA, anti-CD31 [JC/70A] and anti-β-actin were bought from Abcam plc. (Cambridge, UK). Aspirin was purchased from Bayer HealthCare Manufacturing S.r.l. (Milano, Italy). 10% xylazine hydrochloride was obtained from Jilin Huamu Co. (Jilin, China). Apollo 3.0 × 8.0 mm stainless steel stents were purchased from Shanghai MicroPort Scientific Co. (Shanghai, China). Mirror polished 316L stainless steel (Steel, Φ21.5 mm, thickness:0.2 mm) was bought from Dongguan Kailu Co. (Guangdong, China). 2.2. Preparation of D-Lys-Boc. D-lysine hydrochloride (5.00 g, 0.027 mol) and 5



di-tert-butyl dicarbonate (7.17 g, 0.033 mol) were dissolved in 120 mL of H2O/THF mixture (v:v = 1:1), and NaHCO3 (6.90 g, 0.082 mol) was added into the solution. The mixture was stirred at 25 °C for 12 h, and di-tert-butyl dicarbonate (7.17 g, 0.033 mol) was added into the reaction solution. The mixture was stirred at 25 °C for another 12 h. The final solution was evaporated to remove THF and washed with n-hexane. Then, pH value of the solution was adjusted to 3-4 using 1 M HCl. Finally, the solution was extracted with CH2Cl2, dried over MgSO4, and evaporated to get D-Lys-Boc. 1H NMR (400 MHz, DMSO-d6) δ 1.37 (s, 18H, Me), 1.21-1.66 (m, 6H, CH2), 2.87 (s, 2H, CH2), 3.79 (s, 1H, CH), 6.76 (m, 1H, NH), 6.97 (d, 1H, NH). 2.3. Preparation of SK and CK. D-Lys-Boc (4.25 g, 0.012 mol), cystamine dihydrochloride (1.29 g, 0.006 mol), TEA (1.68 mL, 0.012 mol), DCC (2.49 g, 0.012 mol) and HOBT (1.65g, 0.012 mol) were dissolved in 120 mL of ethyl acetate. The mixture was stirred violently at 25 °C for 72 h and centrifuged to get the supernate. The supernate was washed with NaHCO3 solution (pH = 8-9) and HCl solution (pH = 3-4) three times respectively, dried over MgSO4, and evaporated to get the faint yellow solid. The faint yellow solid was then dissolved in 5 mL of CH2Cl2. 5 mL of TFA was added into the solution, and the mixture was stirred at room temperature for 30 min. The reaction solution was poured into isopropyl ether to precipitate SK as a white solid. The precipitate was collected by filtration, washed with isopropyl ether and dried in vacuo. The synthesis of CK was similar as SK by using D-Lys-Boc and 1,6-hexanediamine. 1H NMR of SK (400 MHz, D2O): δ 4.00 (t, 2H, e), 3.70-3.50 (m, 4H, g), 3.02 (t, 4H, a), 2.91 (m, 4H, f), 1.94 (m, 4H, d), 1.73 (m, 4H, c), 1.48 (4H, m, 6


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b). 1H NMR of CK (400 MHz, D2O) δ 3.85 (t, 2H, e), 3.15 (m, 4H, a), 2.92 (t, 4H, f), 1.81 (m, 4H, d), 1.65 (m, 4H, c), 1.45 (m, 4H, b), 1.36 (m, 4H, g), 1.26 (m, 4H, h). 2.4. Preparation of SKP and CKP. SK (0.60 g, 0.69 mmol) and TGIC (0.27 g, 0.92 mmol) were dissolved in 1 mL of DMSO, and polymerization was conducted at 45 °C for 48 h. Then, 470 μL of ED was added to the mixture and further stirred at 45 °C for 24 h. Finally, crude products were purified by using dialysis (MWCO 1000) prior to lyophilization. The corresponding product was denoted by SKP. CKP was synthesized by ring-opening reaction of CK (0.57 g, 0.69 mmol) and TGIC (0.27 g, 0.92 mmol) using similar methods. 2.5. Characterization of Materials. The chemical structures and molecular weights of polycations were characterized by nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC), respectively. The chirality and optical activities of SKP and CKP were characterized by circular dichroism (CD) spectrometer. The details were available in Supporting Information. The ability of condensing DNA, in vitro biodegrability of polycation and particle sizes and zeta potentials of polycation/pRL-CMV (pDNA) complexes were evaluated as described in our previous work.31,32 The details were available in Supporting Information. 2.6. Biocompatibility Assay. The biocompatibility assays of polycations were evaluated with hemolysis and cell viability assays, which were described in Supporting Information. 2.7. In Vitro Characterizations with Reporter Genes. For evaluating in vitro 7



transfection assay, the reporter genes pRL-CMV (pDNA) and enhanced green fluorescent protein plasmid (pEGFP-N1) were used. The details were described in Supporting Information. 2.8. In Vitro Transfection Assay with pVEGF. Western blot assay and cell proliferation assay were used to assess VEGF expressions using pVEGF as described in our earlier work.33 The details were described in Supporting Information. 2.9. Preparation and Characterizations of S-SKP/pVEGF. The coating of polydopamine (PDA) was deposited on mirror polished 316L stainless steel (Steel, Φ21.5 mm) through dip-coating. In brief, dopamine hydrochloride was dissolved in Tris buffer (1.25 mg/mL) at the final concentration of 2 mg/mL. Then steel substrates were immersed into the mixed solution. After deposition for 24 h at 37 °C, the specimens were washed by distilled water three times to remove loosely adsorbed polymers, named as S-PDA. To construct S-SKP/pVEGF, SKP/pVEGF solution (with 0.05 mg/mL of pVEGF) was prepared at the weight ratio of 30. S-PDA was immersed into different volumes of SKP/pVEGF solution for 1 h, rinsed with distilled water three times. In addition, S-PDA was immersed into 1 mL of pVEGF solution (0.05 mg/mL) for 1 h, rinsed with distilled water three times to construct S-pVEGF. The characterizations of S-SKP/pVEGF were described in Supporting Information. 2.10. Cell Attachment and Proliferation on Surfaces. SKP/pVEGF (0.05 mg/mL of pVEGF) complexes solution was prepared at the weight ratio of 30. The details were described in Supporting Information. 8


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2.11. Animal Model. All animal studies were approved by the Animal Care & Welfare Committee of China-Japan Friendship Hospital. A total of 30 Japanese white rabbits were randomly divided into three groups: clinical stent (n = 10), S-SKP/pNC (n = 10), and S-SKP/pVEGF (n = 10). The details were described in Supporting Information. 2.12. In Vivo Gene Expression. Expression of VEGF protein was detected by western blot and immunohistochemistry staining. The details were described in Supporting Information. 2.13. In Vivo Therapeutic Effects of S-SKP/pVEGF. In vivo therapeutic effects were detected by scanning electron microscope (SEM) and histological analysis.4,34 The details were described in Supporting Information. 2.14. Statistical Analysis. Student’s t-test and one-way ANOVA were used. The details were described in Supporting Information.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of SKP/pDNA Complexes. The detailed synthesis of SKP based on the ring-opening reaction of SK is shown in Figure 1. SK with disulfide bond was synthesized using the amidation of cystamine and D-lysine. In addition, CK without disulfide bond as the control was prepared using the amidation of 1,6-hexanediamine and D-lysine. 1H NMR spectra of SK and CK were shown in Figure S1 which could prove their successful synthesis. SKP was synthesized by the ring-opening reaction of SK with four amine groups and TGIC 9



with three epoxy groups. To terminate the reaction, SKP was end-capped by the addition of excess ethylenediamine. CKP, which did not contain disulfide bonds, was synthesized as the control. The GPC curves of typical SKP and CKP were shown in Figure 2(a) and Figure S2. SKP (Mn = 15575 g/mol, polymer dispersity index (PDI) = 1.52) and CKP (Mn = 15875 g/mol, PDI = 1.60) were prepared. Characteristic proton signals of SKP and CKP were much broader than the corresponding peaks of SK and CK (Figure S1), indicating the successful synthesis of SKP and CKP. CD spectra of SKP and CKP shown in Figure S3 confirmed that SKP and CKP have similar chiral structures. Polycationic nucleic acid delivery systems require the efficient condensation of pDNA via electrostatic interactions to form polycation/pDNA complexes with appropriate sizes and surface charges. In this study, agarose gel electrophoresis, particle size, zeta potential measurements, and atomic force microscopy (AFM) imaging were employed to evaluate the DNA condensation capability of SKP and CKP. As shown in Figure 2(b), SKP and CKP could compact pDNA completely at the weight ratio of 0.8-1.0. The particle sizes of complexes with various weight ratios, which gradually decreased with the increase of weight ratios (Figure 2(c)), ranged from 100 to 200 nm, indicating that they were suitable for cellular internalization.35 The zeta potentials of SKP/pDNA and CKP/pDNA ranged from 30 to 43 mV with various weight ratios. With the increase of weight ratios, abundant positive charges compressed pDNA, leading to smaller and tighter nanoparticles. The AFM images of

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typical SKP/pDNA and CKP/pDNA complexes at a weight ratio of 30 were shown in Figure 2(d). SKP and CKP compacted pDNA into uniform nanoparticles. Degradability is essential for nucleic acid delivery systems. Degradation can not only reduce cytotoxicity, but also benefit nucleic acid release. SKP possesses disulfide bonds, which can be degraded in a reductive environment. As shown in Figure 2(a), the molecular weight of SKP in a reductive environment of DTT decreased significantly after 30 min, confirming the degradability of SKP. By contrast, the molecular weight of CKP in a reductive environment did not change, as shown in Figure S3. Agarose gel electrophoresis (Figure 2(b)) provided the direct evidence of the facilitated releases of pDNA by reduction-responsive degradation. In comparison to pristine SKP, SKP in a reductive environment could not efficiently compact pDNA, which demonstrated that reductive responsiveness could facilitate the releases of pDNA. AFM imaging was also used to characterize degraded complexes, as shown in Figure 2(d). The degraded SKP/pDNA resulted in the formation of unstable nanoparticles. 3.2. Biocompatibility Assays. The blood biocompatibility of nucleic acid delivery systems is also essential for in vivo applications. The possible severe hemolysis from polycations might result in thrombosis. The hemolysis ratios of SKP and CKP were much lower than that of branched PEI (25 kDa, the golden standard of nonviral gene vectors20), as shown in Figure S4(a, b). The hemolysis ratios of SKP and CKP were below 5%, even at a concentration of 2 mg/mL while the hemolysis ratio of branched PEI was approximately 60%. Moreover, the morphologies of red blood cells (RBCs) 11



in different groups, as shown in Figure S4(c), illustrated that SKP and CKP could not cause structural damages to RBCs, which was opposite to branched PEI. The cytotoxicities of SKP and CKP were also evaluated. SKP/pDNA and CKP/pDNA complexes did not exhibit obvious cytotoxicity at weight ratios of 10-40 as shown in Figure 3(a). By contrast, branched PEI (25 kDa) demonstrated significantly higher cytotoxicity, even at its optimal N/P ratio of 15 in HUVECs (see below). SKP and CKP possess plenty of hydroxyl species from the ring-opening reaction. Rich hydroxyl groups could shield part of cytotoxicity caused by positive charges.20,36 In addition, CKP/pDNA complexes showed slightly higher cytotoxicity than SKP/pDNA complexes at higher weight ratios. The degradation behavior of SKP may reduce cytotoxicity.25,26 In vitro characterizations with reporter genes. The subsequent transfection performances of SKP and CKP at various weight ratios were first analyzed with the reporter gene pDNA, as shown in Figure 3(b), where branched PEI at its optimal N/P ratio was taken as control. The transfection performance of branched PEI at various N/P ratios was shown in Figure S5, and the optimal N/P ratio of branched PEI was 15. The optimal weight ratio of SKP and CKP was 30 in HUVECs, and SKP demonstrated higher optimal transfection efficiencies than CKP. Biocleavable disulfide bonds of SKP promoted the release of pDNA and increased gene transfection efficiency. To further confirm the transfection performance of SKP, EGFP expression was evaluated using another reporter gene pEGFP-N1. After transfection, Figure S6 12


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showed the representative images of EGFP expression mediated by SKP and CKP at the optimal weight ratio of 30. The percentages of EGFP-positive cells for SKP and CKP in HUVECs were 20% and 7%, respectively. SKP showed significantly more EGFP-positive HUVECs than CKP, further confirming that the degradation of SKP could enhance gene transfection efficiency. The good cellular internalization ability of complexes is also necessary for effective gene transfection. Fluorescently YOYO-1-labeled pDNA was utilized to evaluate cellular uptake. The fluorescence images of HUVECs treated with SKP/pDNA and CKP/pDNA complexes at the weight ratio of 30 were shown in Figure 3(c). The pDNA labeled with YOYO-1 is shown in green and nucleus stained with DAPI is shown in blue. The internalization ratios of SKP/pDNA and CKP/pDNA complexes were determined by flow cytometry, as shown in Figure 3(d). The percentages of fluorescence-positive cells treated with SKP/pDNA and CKP/pDNA complexes in HUVECs were 64% and 62%, respectively. The similar cellular uptake ratios of SKP and CKP indicated that the introduction of disulfide bonds had few effects on internalization. 3.3. In Vitro Transfection Assay with pVEGF. Based on above results, SKP demonstrated degradability, low cytotoxicity and good gene transfection efficiency. Thus, SKP was selected as the delivery system of pVEGF. VEGF could attenuate restenosis by accelerating the proliferation of endothelial cells. VEGF expression in HUVECs was evaluated by western blot assays (Figure 4(a)). The reporter gene pDNA was used as negative control plasmid named pNC. HUVECs treated with 13



SKP/pVEGF demonstrated the increased VEGF expression compared with the cells treated with SKP/pNC or control. The overexpression of VEGF could promote the proliferation of HUVECs. As shown in Figure 4(b), the cell proliferation assays demonstrated that pVEGF mediated by SKP could more effectively promote cell proliferation than pNC mediated by SKP. This might provide possibility for the using of SKP/pVEGF on gene-eluting stents. 3.4. Characterization of SKP/pVEGF Complexes Coated Stents. SKP/pVEGF complexes, which contain abundant amino groups, can be readily introduced to various surfaces with the assistance of a mussel-inspired PDA adhesive layer via electrostatic interaction, Schiff base reaction and Michael addition reaction.37 SKP/pVEGF complexes were coated on the surfaces of PDA-pretreated clinical stents to construct gene-eluting stents (S-SKP/pVEGF). In order to easily characterize the surface functionalization of clinical stent, flat 316L stainless steel (named as Steel), which is used to make clinical stents, was selected for in vitro characterization. As shown in Figure S7, the fluorescence intensity of pVEGF in excess YOYO-1 dye solution at wavelength of 513 nm was used to separately estimate the amount of pVEGF on the surfaces of S-SKP/pVEGF under different conditions. The amount of pVEGF on S-SKP/pVEGF surfaces was positively related to the pVEGF amount in the SKP/pVEGF solution (Figure 5(a)), and did not change significantly when the amount of pVEGF on the S-SKP/pVEGF surface reached 17.32 μg/cm2. As shown in Figure S8(a) and Figure 5(b), after PDA coating, the surface zeta potential of S-PDA became -17.71 mV from -13.97 mV of pristine steel. After SKP/pVEGF coating, steel 14


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surface zeta potential changed to be positive, which confirmed the successful introduction of SKP/pVEGF with positive charges. Surface zeta potential of S-SKP/pNC was similar to that of S-SKP/pVEGF (Figure S8(a)). S-pVEGF as the control was prepared pVEGF solution instead of SKP/pVEGF solution, where pVEGF was labeled with YOYO-1. Compared with the fluorescence image of S-SKP/pVEGF (Figure 5(c)), the corresponding fluorescence image of S-pVEGF showed that negatively-charged pVEGF could not efficiently be coated on the S-PDA surface in the absence of SKP (Figure S8(b)). X-ray photoelectron spectroscopy (XPS) analysis revealed a clear enhancement of the N 1s signal in the S-PDA surface compared with Steel surface (Figure S8(c, d)), confirming the successful immobilization of PDA layer on Steel surface. Compared with S-PDA surface, the N 1s signal was further enhanced, and the P 2p signal clearly appeared in S-SKP/pVEGF surface as shown in Figure S8(e) and Figure 5(d), also confirming the successful immobilization of SKP/pVEGF complexes. The surface morphologies of S-PDA and S-SKP/pVEGF were characterized using AFM. As shown in Figure S9, the PDA thickness of S-PDA was approximately 40 nm. After coating SKP/pVEGF on S-PDA surface, the layer thickness of S-SKP/pVEGF reached approximately 70 nm (Figure 5(e)). In addition, uniform spherical nanoparticles were observed on the surface of S-SKP/pVEGF, confirming that SKP/pVEGF complexes were still stable after being coated on S-PDA surface. The release of pVEGF from S-SKP/pVEGF surface was significant for the in vivo application. As shown in Figure S10, quantitative results showed that most pVEGF 15



(about 75%) could be released during 36 hours. Effective release could be beneficial to early vessel re-endothelialization and the inhibition of excessive EC proliferation. As implants are in direct contact with tissues and blood in practical clinical applications, it is important to evaluate hemocompatibility of S-SKP/pVEGF. Figure 5(f) showed that the hemolysis ratios of Steel and S-SKP/pVEGF were both below 5%. As shown in Figure 5(g), the supernatants of RBCs suspensions were clear after incubation with Steel and S-SKP/pVEGF. Figure 5(h) showed the corresponding optical images of RBCs after different treatments. RBCs did not display an obvious morphological transformation after incubation with Steel and S-SKP/pVEGF. These results demonstrated that S-SKP/pVEGF has good hemocompatibility, similar to SKP (Figure S4). 3.5. Cell Adhesion and Proliferation on The Surface of S-SKP/pVEGF. Cell attachment was the first stage of cell adhesion. In this part, cell attachment was first investigated as the interaction between cells and substrates for optimizing the amount of SKP/pVEGF complexes on the surfaces. As shown in Figure 6(a), a 4-hour culture of HUVECs revealed that cells were easily to adhere on the surface of S-SKP/pVEGF with the pVEGF amount of 8.74 μg/cm2. Under such loading amount of SKP/pVEGF, the proliferation of HUVECs cultured on the S-SKP/pVEGF surface was significantly faster than that on Steel, S-PDA and S-SKP/pNC surfaces (Figure 6(b) and Figure S11(a)) during 72 h culture. The fluorescence images shown in Figure 6(c) and Figure S11(b) revealed the similar tendency with cell counts. These results indicated that

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S-SKP/pVEGF can improve the proliferation of HUVECs, which is suggestive of a great potential to improve re-endothelialization. 3.6. In Vivo Gene Expression on S-SKP/pVEGF. In vivo animal experiments were conducted to evaluate the re-endothelialization-promoting effects of S-SKP/pVEGF using rabbit abdominal aorta injury model. Clinical stent was used as control. All modified stents were successfully implanted without adverse reactions. The angiography images (Figure 7(a)) revealed that there was no collapse or blocking in the abdominal aorta after stent placement. In order to characterize in vivo VEGF expression mediated by S-SKP/pVEGF, stented arterial tissues at the 1st week were analyzed by western blot. As shown in Figure 7(b), there was more VEGF protein expression in the S-SKP/pVEGF group than S-SKP/pNC and clinical stents, confirming that S-SKP/pVEGF could realize pVEGF transfection in vivo. The similar results were obtained by VEGF immunohistochemical analysis (Figure 7(c)). The S-SKP/pVEGF group exhibited intense staining of VEGF at the 1st week. No significant differences were observed in VEGF immunohistochemical staining among clinical stent, S-SKP/pNC and S-SKP/pVEGF at the 4th week, which confirmed to the previous report.38 These results demonstrated that S-SKP/pVEGF could realize pVEGF gene transfection in vivo at the 1st week (which could accelerate early re-endothelialization) and non-function at the 4th week (which might avoid the excessive endothelial cell proliferation and ISR). 3.7. Re-endothelialization Assay. After stent implantation, the progress of re-endothelialization was observed by SEM and CD31 immunohistochemical staining 17



(Figure 8(a, b)). At the 1st week, there were only several cells attached on the clinical stent surfaces and the surfaces of S-SKP/pNC were partially covered with cells. Suitable positive charges on the surface may promote cell attachment. More importantly, the surfaces of S-SKP/pVEGF were almost completely covered by cells, because VEGF could further enhance ECs proliferation. At the 4th week, the surfaces of all three groups were completely covered by cells. However, ECs on the surface of S-SKP/pVEGF were more regular and smoother than those on clinical stent and S-SKP/pNC surfaces. Immunohistochemical staining for CD31 was another important characterization of re-endothelialization. As shown in Figure 8(b), the luminal surfaces were completely covered by CD31-positive cells in the S-SKP/pVEGF group in comparison with the clinical stent and S-SKP/pNC groups. These data indicate that re-endothelialization could be promoted with S-SKP/pVEGF and might further prevent in-stent restenosis. 3.8. Evaluation of Anti-Restenotic Effect. The extent of in-stent restenosis was evaluated by histological analysis. Haematoxylin and eosin (H&E) staining of cross sections was shown in Figure 9(a). There was no significant difference among three groups in neointimal area, media area, neointimal area/media area (N/M) ratio and neointimal stenosis at the 1st week as shown in Figure 9(b) and Figure S12. At the 4th week, the neointimal area of S-SKP/pVEGF (0.453 ± 0.070 mm2) is significantly smaller than those of clinical stent (0.839 ± 0.043 mm2) and S-SKP/pNC (0.815 ± 0.078 mm2) (Figure 9(c)). In addition, both N/M ratio and neointimal stenosis of S-SKP/pVEGF were the smallest among three groups. The media areas of three 18


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groups were still no significant differences (Figure S12). In vivo studies revealed that S-SKP/pVEGF could effectively prevent in-stent restenosis.

4. CONCLUSION In this study, one novel reduction-responsive branched nucleic acid vector (SKP) was

successfully

synthesized

via

ring-opening

reaction.

Due

to

its

reduction-responsive property, SKP showed better gene transfection performances than CKP without biodegradable chemical bonds. SKP/pVEGF complexes were successfully coated on pre-treated clinical stents (S-SKP/pVEGF). In vitro studies confirmed that S-SKP/pVEGF could accelerate cell proliferation on the stent surface. Furthermore, the in vivo animal data in rabbits demonstrated that S-SKP/pVEGF could realize local overexpression of VEGF at the initial implant period, promote the progress of re-endothelialization, and finally alleviate in-stent restenosis. This study provides a promising approach for the design of a degradable pVEGF-eluting stent that effectively improves endothelialization and reduce restenosis after stent implantation.

ASSOCIATED CONTENT Supporting Information Experimental section, 1H NMR spectra of SK, CK, SKP and CKP, GPC curves of CKP, CD spectra of SKP, and CKP, hemolysis assay of RBCs treated with SKP, CKP 19



and PEI, transfection efficiencies of PEI/pRL-CMV at different N/P ratios in HUVECs, representative images of EGFP expression mediated by SKP, CKP and PEI in HUVECs, fluorescence quantitation of pVEGF, surface zeta potentials of S-PDA and S-SKP/pNC, fluorescence images of S-pVEGF surface, XPS and P 2p XPS spectra of Steel and S-PDA and coating heights and AFM images of S-PDA, cumulative pVEGF release curve of S-SKP/pVEGF, cell counts attached onto S-PDA surface, rhodamine123 fluorescence staining of HUVECs cultured on S-PDA surface, histological analysis of media area, N/M ratio neointimal stenosis and statistical analysis were shown in Supporting Information.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (Z. Liu). *E-mail: [email protected] (F.-J. Xu). *E-mail: [email protected] (B. Yu). Author Contributions Z. L., B. Y. and F-J. X. conceived the experiments, planned synthesis, designed the research project. W. Y. and Y. C. performed the experiments, analyzed the data and wrote the manuscript. W. T. helped to perform the stent implantation in vivo. N. Z. and Z. L. helped to analyze the results and wrote the manuscript. All the authors commented on the manuscript and have given approval to the final version of the manuscript. 20


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Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS W. Ye and Y. Chen contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant numbers 31771045, 51733001 and 21875014), and Fundamental Research Funds for the Central Universities (PYBZ1808 and XK1802-2).

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Figure 1. Schematic illustration of the preparation of S-SKP/pVEGF and its in vivo implantation process in rabbit.

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Figure 2. (a) GPC curves of SKP treated with DTT at different time points. (b) Electrophoretic mobility retardation assay of pDNA complexes with SKP and CKP at various weight ratios. (c) Particle sizes and zeta potentials of SKP/pDNA and CKP/pDNA complexes at various weight ratios. (d) AFM images of typical SKP/pDNA and CKP/pDNA complexes in neutral and reductive environments.

29



Figure 3. (a) Cytotoxicities of polycation/pDNA complexes at different weight ratios in HUVECs. (b) In vitro gene transfection efficiencies of polycation/pDNA complexes at various weight ratios in HUVECs in comparison with that mediated by branched PEI (25 kDa, at its optimal N/P ratio of 15) (mean ± SD, n = 3, *p < 0.05). (c) Fluorescence images and (d) flow cytometry analysis plots of HUVECs treated with SKP/pDNA, CKP/pDNA and PEI/pDNA complexes at the respective optimal weight ratio or N/P ratio, where the YOYO-1-labeled pDNA was shown in green, and the DAPI-labeled nuclei were shown in blue.

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Figure 4. (a) Western blot analysis of related VEGF protein expressions after 72 h transfection. (b) Cell counts after transfection from 0 to 3 d (mean ± SD, n = 3, *p < 0.05).

31



Figure 5. (a) pVEGF amounts on the surfaces of S-SKP/pVEGF under various volumes of SKP/pVEGF solution (with 0.05 mg/mL pVEGF). (b) Surface zeta potentials of Steel and S-SKP/pVEGF. (c) Fluorescence image of S-SKP/pVEGF surface, where the YOYO-1-labeled pVEGF was shown in green. (d) P 2p core-level spectrum of S-SKP/pVEGF. (e) Coating height and AFM image of S-SKP/pVEGF. (f) Hemolysis ratio of RBCs treated with Steel and S-SKP/pVEGF, and (g, h) images of RBCs treated with Steel and S-SKP/pVEGF, where Triton X-100 and PBS were used as positive and negative controls.

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Figure 6. (a) Cell counts attached onto S-SKP/pVEGF surfaces with different amounts of SKP/pVEGF complexes after 4 h culture. (b) Cell densities attached onto Steel, S-SKP/pNC and S-SKP/pVEGF surfaces after 4-72 h culture. (c) Rhodamine123 fluorescence staining of HUVECs cultured on Steel, S-SKP/pNC and S-SKP/pVEGF surfaces after 4-72 h culture.

33



Figure 7. (a) Typical angiography images of clinical stent, S-SKP/pNC and S-SKP/pVEGF before and after stent implantation. Red rectangles show the sites of stent implantation. (b) Western blot analysis of rabbit arterial segments treated with clinical stent, S-SKP/pNC and S-SKP/pVEGF after 1 week implantation (mean ± SD, n = 3, *p < 0.05). (c) Immunohistochemical staining for VEGF of rabbit arterial segments treated with clinical stent, S-SKP/pNC and S-SKP/pVEGF at the 1st week and 4th week after implantation. The area surrounded by red line represents the position of stents. Black arrows highlighted the overexpression of VEGF.

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Figure 8. (a) Representative SEM images and (b) immunohistochemical staining for CD31 of rabbit arterial segments treated with clinical stent, S-SKP/pNC and S-SKP/pVEGF at the 1st week and 4th week after implantation. Black arrows highlighted the incomplete places of endangium and red arrows highlighted the complete places of endangium.

35



Figure 9. (a) Typical optical photographs of H&E stained cross section slices of rabbit femoral arteries treated with clinical stent, S-SKP/pNC and S-SKP/pVEGF at the 1st week and 4th week. The areas surrounded by red line represent the parts of neointimal area. Histological analysis of neointimal area at the 1st week (b) and 4th week (c) (mean ± SD, n = 3, *p < 0.05).

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Table of Contents

37


Reduction-Responsive Nucleic Acid Delivery Systems To Prevent In

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