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Fine tuning of core-shell structure of hyaluronic acid/cellpenetrating peptides/siRNA nanoparticles for enhanced gene delivery to macrophages in anti-atherosclerotic therapy Yi Zhao, Zhiyu He, Hai Gao, Haoyu Tang, Jianhua He, Qing Guo, Wenli Zhang, and Jianping Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00501 • Publication Date (Web): 11 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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Fine tuning of core-shell structure of hyaluronic acid/cell-penetrating peptides/siRNA nanoparticles for enhanced gene delivery to macrophages in anti-atherosclerotic therapy Yi Zhao†‡, Zhiyu He†§⊥, Hai Gao‡, Haoyu Tang⊥, Jianhua He‡, Qing Guo‡, Wenli Zhang*,‡, and Jianping Liu*,‡ ‡

Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, P. R.

China §

School of Materials Science and Engineering, Key Laboratory for Polymeric Composite

and Functional Materials of Ministry of Education, Sun Yat-sen University, Guangzhou 510275, P. R. China ⊥

Department of Materials Science and Engineering, and Institute for NanoBioTechnology,

Johns Hopkins University, Baltimore, MD 21218, USA

ABSTRACT Hyaluronic acid (HA)-coated LOX-1 specific siRNA-condensed cell-penetrating peptides (CPPs) nanocomplexes were developed for targeted gene delivery to macrophages and suppression of lipid accumulation. The HA coating facilitated the accumulation of nanoparticles at leaky endothelium overexpressing CD44 receptors and was further degraded by hyaluronidase (HAase) intra plaques for exposing the naked CPPs nanocomplexes (NCs) and achieving the ultimate location into macrophages. The surface coating of HA was verified by the increased particle size, inverted zeta potential, and TEM images.

The

targeting

mechanism

was

studied

on

the

established

injured

endothelium-macrophages co-culture system, which revealed that modification of higher molecular weight HA and higher HA coating density on NCs, termed as NPs-3, improved

ACS Paragon Plus Environment

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the intracellular uptake of nanoparticles by macrophages. Macrophages internalized NCs via caveolae-mediated endocytosis pathway. Moreover, NPs-3 exhibited better cellular drug efficacy in preventing macrophage-derived foam cells formation than other preparations. Compared with NCs, HA decoration showed enhanced atherosclerotic lesions targeting efficiency, proven by results from ex vivo imaging. Furthermore, atheroprotective efficacy study in apoE-deficient mice showed that NPs-3 had the best potent efficacy, which was demonstrated by the fewest atherosclerotic lesions sizes and lipid accumulation, the lowest macrophage infiltration, and the lowest expression of monocyte chemoattractant protein-1 (MCP-1), respectively. Collectively, the HA-coated CPPs nanocomplexes were promising nanocarriers for efficient macrophages-targeted gene delivery and anti-atherogenic therapy.

KEYWORDS: cell-penetrating peptides, small interfering RNA, hyaluronic acid, atherosclerosis, active targeting, HAase-sensitive


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1. INTRODUCTION Atherosclerosis, characterized by the build-up of fatty plaques within the artery walls, is a major cause of death worldwide.1,2 Monocyte-derived macrophages play a pivotal role in the atherogenic process as the modulators of lipid metabolism and inflammatory responses.3,4 Aberrant cholesterol accumulation in macrophages due to the uptake of oxidized low density lipoprotein (oxLDL) results in foam cells formation, activates atherogenic inflammatory cascade, and eventually causes arterial damage and plaque rupture. Therefore, the intracellular lipid deposition of macrophages is pursued as therapeutic target to prevent atherosclerosis. Scavenger receptors (SRs)-mediated uptake of oxLDL into macrophages is being recognized as critical to lipid accumulation within the intima. Several SRs for oxLDL have been identified in macrophages, such as cluster of differentiation 36 (CD36), scavenge receptor type-A (SR-A) and lectin-like oxLDL receptor-1 (LOX-1).5,6 Although CD36 and SR-A are generally recognized as the principal contributors to cholesterol uptake in macrophages, conflicting results were reported regarding to the antiatherosclerotic efficiency of SR-A and CD36 knockout.7-9 LOX-1, an up-regulated receptor in activated macrophages is correlated with tissue factor expression and apoptosis, indicating the involvement of LOX-1 in initial stages of atherosclerosis and plaque instability.10-13 Overexpression of LOX-1 was shown to cause lipid deposition in the coronary arteries of apolipoprotein E (apoE)-deficient mice, and less pronounced development of atherosclerotic plaques was observed in LDL receptor-deficient mice with genetic deletion of LOX-1.14,15 Considering the important roles of LOX-1 in the pathogenesis of atherosclerosis, down-regulating the LOX-1 level in macrophages is appealing in atherosclerosis therapy. Small interfering RNAs (siRNA) with the capability to silence the expression of targeted protein is tremendously attractive for disease treatment. Blocking the expression of LOX-1 on macrophages via LOX-1 specific siRNA might be a promising strategy for



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atherosclerosis intervention. Nevertheless, naked siRNA is unfavorable for systemic delivery because of its inherent limitations such as negative charge, large molecular weight, instability, rapid elimination, poor cellular internalization and less tissue selectivity.16 To overcome the limitations, engineered vehicles have been designed as delivery carriers for stable

encapsulation,

protection,

and

intracellular

trafficking.17

Among

them,

cell-penetrating peptides (CPPs) are one of the most popular and efficient vectors for intracellular transport. CPPs are a class of diverse peptides, typically with 5-30 amino acids, and can be taken up by cells via multiple pathways, such as direct translocation across the membrane bilayer and endocytosis-mediated uptake.18,19 Kang et al. incorporated oligonucleotide into lipid-peptide hybrid nanoparticles with CPPs further modification to facilitate cellular uptake and transfection efficiency.20 Qi et al. employed cell-penetrating magnetic nanoparticles, which were surface modified by protamine peptide, for highly efficient delivery and intracellular imaging of siRNA in cancer cells.21 However, compelling evidences manifested that CPPs had strong non-specific binding because other molecules can attach onto them and lead to the binding to non-targeted cells. As a result, this issue is probably one of the major drawbacks for the use of CPPs as delivery vehicles in vivo.22 Therefore, alternative strategies are needed to promote cell-specific delivery, such as further modification with an appropriate target ligand to elicit cell surface binding.23 Cell adhesion molecule CD44 is overexpressed on various cancer cells and impaired endothelium in atherosclerotic lesions.24,25 The primary endogenous ligand of CD44 is hyaluronic acid (HA), a naturally existing mucopolysaccharide composed of tandem disaccharide repeats of β-1,4-D-glucuronic acid-β-1,3-D-N-acetylglucosamine. As reported, HA-decorated nanocarriers could efficiently evade recognition by reticuloendothelial system (RES), owing to the similar molecular structure to polyethylene glycol (PEG).26,27 Moreover, HA is biodegradable, non-toxic, nonimmunogenic and non-inflammatory, allowing it to be widely applied in anticancer drug delivery.28-30 Zhang et al. designed HA-modified chitosan nanoparticles with paclitaxel-D-α-tocopherol succinate prodrug


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loaded and showed that the nanoparticles could actively target CD44-overexpressed cancer cells and efficiently reverse the multidrug resistance of treated cells.31 Moreover, HA-decorated gold nanoparticles and superparamagnetic iron oxide nanoparticles were developed and applied in cancer diagnosis and hyperthermia therapy.32,33 Apart from the wide application of HA in cancer therapy, the up-regulation of CD44 at inflammatory sites of atherosclerotic lesions and the intrinsic attributes of HA make it appealing to utilize HA for plaque imaging.34-36 However, barely research was reported on HA-modified nanocarriers to achieve atherosclerotic lesions-targeted gene delivery. In view of the increased permeability of impaired endothelium and wide availability of hyaluronidase (HAase) in extracellular matrix of plaque, we developed HA-coated CPPs/siRNA nanoparticles (NPs) and hypothesized that the delivery mechanism of NPs from blood to macrophages is an ingenious multistage-targeting process. At the initial stage, in virtue of the HA coating, NPs preferentially target atherosclerotic lesions by adhering to the CD44 on the impaired endothelial cells. Subsequently, NPs translocated across the permeable endothelium via the gap between injured endothelial cells, a process that was similar to enhanced permeation and retention effect (EPR). The capping HA layer would be degraded by HAase within plaque at the subsequent stage, allowing the exposure of naked siRNA-loaded CPPs nanocomplexes (NCs) to foam cells.37 Then, siRNA could be efficiently delivered into foam cells via the uptake pathways of CPPs (Scheme 1). Noteworthy, part of the nanoparticles might be internalized by endothelial cells when interaction with CD44. We mainly focused on the translocation of nanoparticles via intercellular gap of endothelial cells and the final uptake by macrophages for gene delivery to macrophages and anti-atherosclerotic therapy.



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Scheme 1. Illustration of HA-coated CPPs/siRNA nanoparticles for targeted gene delivery to macrophages in atherosclerotic plaques.

In summary, we constructed siRNA-condensed CPPs nanocomplexes with HA coating via electrostatic adsorption for effective siRNA delivery into macrophages within the atherosclerotic plaques. The physicochemical properties, such as particle size, zeta potential, stability test, and particle morphology were elaborately characterized. Furthermore, in vitro cell viability, cellular uptake behavior, internalization mechanisms, and in vitro gene silencing efficacy were assessed. The anti-atherosclerotic efficacies involving intracellular lipid dispositions and DiI-oxLDL uptake were also investigated. In vivo studies, including atherosclerotic

lesions

targeting

property

and

atheroprotective

comprehensively examined in apoE-deficient mice.


efficacy

were

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2. EXPERIMENTAL SECTION 2.1. Materials. Penetratin peptide (RQIKIWFQNRRMKWKK) was chemically synthesized by Nanjin Taiye Biopharmaceuticals Co., Ltd. (Jiangsu, China). Non-targeted control siRNA (siNonsense), Cy3-siRNA (the 5’-end of the sense strand in siNonsense was conjugated with Cy3 dye), Cy5-siRNA (the 5’-end of the sense strand in siNonsense was conjugated with Cy5 dye), human LOX-1 siRNA (5’-AGGACGGUUCUCCUUUGAU dTdT-3’ and 3’-dTdT UCCUGCCAAGAGGAAACUA-5’), and mouse LOX-1 siRNA (5’-GUGGCCAGUUACUACAAAU

dTdT-3’

and

3’-dTdT

CACCGGUCAAUGAUGUUUA-5’) were obtained from Guangzhou RiboBio Co., Ltd. (Shenzhen, China). Sodium hyaluronic acid (molecular weights 8 kDa and 200 kDa) was purchased

from

Freda

Biochem

Co.,

Ltd.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium

(Shandong,

China).

Oil

red

O,

bromide

(MTT),

hyaluronidase

(HAase), 4’,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich (USA). OxLDL and DiI-oxLDL were purchased from Yiyuan Biotech. Co. Ltd (Guangzhou, China). Human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes were kindly donated by Atherosclerosis Research Centre, Nanjing Medical University (Nanjing, China). All the other chemical reagents were of analytical grade. 2.2. Preparation and characterization of nanoparticles. For NCs formation, CPPs were mixed with siRNA at a desired N/P ratio. After vortexing for 200 seconds, the nanocomplexes were incubated at room temperature for 30 min. HA-coated nanoparticles (NPs) prepared with different HA molecular weight (Mw) and coating density were termed accordingly as NPs-1 (Mw 8 kDa), NPs-2 (Mw 200 kDa, low coating density), and NPs-3 (Mw 200 kDa, high coating density). In order to prepare the aforementioned NPs, NCs dispersion obtained above was dropwise added to HA solution with different Mw or concentration under vigorous stirring for 10 min and further incubated at room temperature for 1 h with gentle agitation. The excessive HA was removed by centrifugation at 50000 rpm for 1 h. The amount of HA coated on the nanoparticles was calculated by subtracting



the amount in the supernatants from the initial amount added to the mixture. The amount of excessive HA in the supernatants after centrifugation was quantified by the hexadecyltrimethyl ammonium bromide (CTAB) turbidimetric method.38 Briefly, 50 µL of HA standard solutions (0.1-2.0 mg/mL) or supernatants were added into a 96-well plate, and all the samples were incubated with 50 µL of sodium acetate buffer (0.2 M, pH 6) at 37 °C for 10 min. Then, 100 µL of CTAB (10 mM) in 2% sodium hydroxide was added to the mixture and incubated at 37 °C. After shaking the plate with a vortex, the absorbance of the precipitated complex was read within 10 min against the blank at 570 nm using a microplate reader (Biotek ELx800, USA). The particle size and zeta potential of the nanosystems were determined using a dynamic light scattering (DLS) analyzer (Zetasizer 3000 HAS, Malvern Instruments, UK). Serum stability test and RNase A sensitivity assay were performed to evaluate the effect of nanoparticles on protecting siRNA from serum and RNase A degradation. Briefly, the naked siRNA and various nanoparticles (NCs, NPs-1, NPs-2, NPs-3) were incubated with 50% final concentration of fetal bovine serum (FBS) or RNase A solution (final working concentration 10 µg/mL) at 37 °C for the prearranged time intervals. To release siRNA from nanoparticles, heparin (1%, w/v) was added to the above samples and incubated at room temperature for 10 min. Finally, all the samples were analyzed by 1% agarose gel electrophoresis at 90 V for 30 min in 1× Tris-Borate-EDTA (TBE) buffer solution. Heparin, the highly charged anionic polymer, could release gene from the complex. This is probably due to the great linear negative charge density of heparin, which is much greater than gene.39 The shape and morphology of nanoparticles were observed by transmission electron microscopy (TEM, H-7650, Hitachi High-Technologies Corporation, Japan). A drop of the nanoparticle suspension was dipped onto a 200-mesh copper grid coated with carbon film, and the excess solution was drawn off using filter paper. Samples were negatively stained with freshly prepared phosphotungstic acid aqueous solution (2%, w/v) for approximately 2 min at room temperature. The grid was allowed to air-dry thoroughly prior to imaging.


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2.3. Injured endothelium-macrophages co-culture. HUVECs and human THP-1 monocytes were cultured in endothelial basal medium supplemented with growth supplements and RPMI 1640, respectively. The cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. The passage number of HUVECs used in this study was 4-7. HUVECs were cultured on a polyester membrane (0.4 µm in pore size, diameter 12 mm, 1.1 cm2 of cell growth area) in Costar Transwell 12 wells/plate (Corning). The changes of the trans-endothelial electrical resistance (TEER) values representing the tightness of the cell monolayers were measured with an electrical resistance meter (Millicell®-Electrical Resistance System, Millipore). The cells were used for experiments until forming an endothelial monolayer (steady TEER values in the range of 300-400 Ω×cm2). THP-1 cells were seeded in the basolateral chambers of transwell and differentiated into macrophages by stimulation with phorbolmyristate acetate (100 ng/mL) for 72 h. In order to accelerate endothelial dysfunction, cells were treated with TNF-α (10 ng/mL) for 12 h.40 2.3.1. CD44 expression analysis. The expression of CD44 receptors on HUVECs was analyzed by flow cytometry and confocal laser scanning microscopy (CLSM). For flow cytometry analyses, the cells were washed three times with PBS and incubated 30 min with the CD44 primary antibody and then 30 min in the dark with the FITC-conjugated secondary antibody. Then, HUVECs were washed with cold PBS and analyzed by flow cytometry (MACSQuant, Miltenyi Biotec, Gladbach, Germany). For the microscopic analyses, after incubation with the CD44 primary antibody and FITC-conjugated secondary antibody, the cells were washed three times with cold PBS and fixed in 4% (w/v) paraformaldehyde for 15 min.36 Then, the cells were stained by DAPI and imaged by CLSM (LSM700, Carl Zeiss, Germany). As a negative control, the CD44 expression level in HUVECs without TNF-α stimulation was also assayed. 2.4. Cell viability assay. The cytotoxicities of various nanoparticles on HUVECs and THP-1 derived macrophages were evaluated using the MTT method. Specifically, HUVECs



and THP-1 derived macrophages were treated with various preparations for 24 h. After washing, 200 µL of MTT solution (0.5 mg/mL in PBS) was added to the cells for another 4 h, and then discarded and replaced by 150 µL of dimethyl sulfoxide. The optical densities were measured at 570 nm by the microplate reader (Biotek ELx800, USA). Untreated cells were used as control and cell viability was calculated as a percentage relative to the control cells. 2.5. Transport investigation. HUVECs were cultured on a polyester membrane and were used for experiments until TEER values higher than 300 Ω×cm2. The media in the apical chembers was replaced by fresh media with test samples. After 4 h of incubation, the basolateral medium was collected. The Cy3-siRNA concentration was determined and the apparent permeability coefficient (Papp, cm/s) was calculated.41,42 To investigate the permeation ability of various test samples on injured endothelium, experments were also performed with a procedure to stimulate cell monolayer with TNF-α for 12 h. The influence of free HA on Cy3-siRNA permeation was also evaluated after free HA (10 mg/mL) was added to the apical chamber for 1 h prior to the addition of nanoparticles. 2.6. Cellular uptake study. To assess the intracellular delivery of siRNA, fluorescence labeled siRNA (Cy3-siRNA) was loaded into nanoparticles as previously described. After adding HAase (2 mg/mL) to the lower compartment of transwell chambers, various nanoparticles diluted by medium were added into the upper compartment, and incubated with cells at 37 °C for 4 h. Then, THP-1 derived macrophages were rinsed with PBS and harvested for the analysis by flow cytometry. Cellular uptake of the nanoparticles was also visualized by fluorescence microscope (IX 71, Olympus, Japan). After 4 h of incubation with different formulations, THP-1 derived macrophages were washed with PBS and fixed by 4% paraformaldehyde. Afterward, cell nuclei were labeled by DAPI before the fluorescence imaging. The untreated cells were used as a negative control. To investigate whether the specific targeting ability of NPs was mediated by CD44 receptors on the injured endothelium, cells were pre-incubated with free HA (10 mg/mL) for 1 h before the addition


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of nanoparticles.40 2.7. Endocytic Pathways study. For blocking the energy-dependent endocytosis, THP-1 derived macrophages were cultured at 4 °C instead of 37 °C. For ATP depletion, the cells were treated with sodium azide (1 mg/mL) for 1 h previous to the incubation with NCs for 4 h at 37 °C. To investigate the influence of uptake inhibitors on uptake pathway, the cells were pre-incubated with different endocytosis inhibitors (amiloride 13 µg/mL, chlorpromazine hydrochloride 10 µg/mL, and genistein 54 µg/mL) for 1 h at 37 °C.43 The amount of intracellular fluorescence intensity was measured by flow cytometry. Cellular uptake of the fluorescent nanoparticles were also observed by fluorescence microscope. Positive control cells were incubated with fresh medium, instead of inhibitor solution. 2.8. In vitro gene silencing efficiency assay. The gene silencing efficiency was assessed by quantitative real-time PCR (qRT-PCR). After construction of the injured HUVECs and THP-1 derived macrophages co-culture system, HAase was added in the basolateral chambers and various samples were added in the apical chambers, respectively. After 4 h of incubation, the medium was exchanged for fresh medium. After 48 h of incubation, THP-1 derived macrophages were harvested, and the mRNA was extracted with TriZol reagent (Invitrogen, Grand Island, NY) according to the manufacturer's protocol and reverse transcribed into cDNA by reverse transcription Kit (GenePharm, Shanghai, China). Real-time PCR reactions were carried out on an ABI Step One Plus Real-Time PCR systems (Applied Biosystems, Foster City, USA) with SYBR Master Mix (GenePharm, Shanghai, China).43 The relative LOX-1 expression was analyzed by normalizing with the endogenous glyceraldehydes-phosphate dehydrogenase (GAPDH) levels.. 2.9. Intracellular lipid disposition stained by oil Red O. After transfection with various formulations for 48 h, the cells were washed with PBS and stimulated by oxLDL (80 µg/mL) for 12 h. Thereafter, the cells were washed with PBS for three times and fixed in 4% paraformaldehyde for 15 min. After being rinsed in PBS for three times, the cells were



stained with oil red O working solution for 1 h in darkness at room temperature, washed with 60% isopropanol, and then morphologically evaluated by microscope.44 2.10. Uptake of DiI-oxLDL. Cells were transfected with various preparations for 48 h and washed with PBS, followed by incubation with DiI-oxLDL (80 µg/mL) for another 12 h at 37 °C. Then, the cells were washed again, fixed with 4% paraformaldehyde, labeled with DAPI, and finally visualized by fluorescence microscope.44 The cells treated with DiI-oxLDL were set as positive control. 2.11. Animal studies. 2.11.1. Induction of atherosclerosis in apoE-deficient mice. All the animal experiments were approved by Institutional Animal Care and Use Committee of China Pharmaceutical University. Five-week-old male apoE-deficient mice were provided by Qinglong Mountain Animal Center (Nanjing, China) and fed with a high-cholesterol diet (21% fat and 0.15% cholesterol) for 16 weeks to develop atherosclerotic lesions. Mice that did not feed with high-cholesterol diet were set as control. The mice were imaged with high-frequency ultrasound (HF-US, VEVO® 2100, VisualSonics Inc.) using the MS400 probe over aortic roots. The mice were subjected to euthanasia and aortas were dissected from the mice to further validate the development of atherosclerotic lesions in the model mice by naked eye and H&E staining. 2.11.2. Ex vivo imaging. The mice were administrated with Cy5-siRNA loaded nanoparticles (1 mg/kg Cy5-siRNA) through the tail veins. Meanwhile, free Cy5-siRNA was also injected into atherosclerotic mice as a control. At 4 h post injection, the mice were sacrificed and different tissues including aorta, liver, heart, spleen, lung, and kidney were carefully collected and imaged with IVIS optical imaging system equipped with an excitation bandpass filter at 630 nm and an emission at 670 nm. 2.11.3. In vivo pharmacodynamic study. Six apoE-deficient model mice were sacrificed as the control group, and the remaining mice were randomized into 6 groups (n = 6) and received saline, free siRNA, NCs, NPs-1, NPs-2, and NPs-3 (1.5 mg/kg siRNA, two


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injections per week) for 12 weeks via tail veins injection, respectively. Mice were maintained on a high-cholesterol diet during the 12-week treatment. Aortic roots were harvested, embedded in optimal cutting temperature compound, and serial-sectioned at a thickness of 6 µm onto glass slides. For morphometric analysis of lesions, the slides were stained with H&E. In order to detect neutral lipid, oil red O staining was performed as previously described.45 Monocyte chemoattractant protein 1 (MCP-1) immunostaining was performed using anti-mouse MCP-1 antibody and imaged under a fluorescent microscope. Immunohistochemistry was adopted to analyze the macrophage infiltration. Firstly, endogenous peroxidase activity was deterred with 3% hydrogen peroxide in methanol, and nonspecific antibody reactivity was quenched by incubating with 5% goat serum in 0.01% Triton X-100 buffer for 1 h. Subsequently, the specimens were incubated for 12 h at 4 °C with anti-mouse CD68 primary antibody, for 1 h with secondary antibody, for 5 min with 3,3′-diaminobenzidine tetrahydrochloride, and finally for 5 min with hematoxylin. Microscopic images of aortic root sections were digitized, and macrophage recruitment was quantified with Image Pro Plus 6.0 software. 2.11.4. Safety studies. To assess hepatic toxicity, muscle damage, and renal toxicity of different formulations, the blood was collected and analyzed for alanine transaminase (ALT), aspartate transaminase (AST), creatine kinase (CK), and creatinine (CRE). Main organs (heart, liver, spleen, lung, and kidney) were processed for H&E staining. 2.12. Statistical analysis. All values were expressed as mean ± SD. Statistical analysis was performed with One-way ANOVA or t-test by GraphPad Prism version 5.0 for Windows (GraphPad Software, USA). Significance was reported as *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001. 3. RESULTS AND DISCUSSION



3.1. Preparation and characterization of nanoparticles. Before coating HA, CPPs were used to condense the siRNA. Agarose gel electrophoresis was carried out to investigate whether siRNA was retarded completely by CPPs. When the N/P of CPPs/siRNA reached 4/1, almost all siRNA was complexed and there was no free siRNA band observable (Figure 1B). In order to research the influence of HA molecular weight and coating density on targeting ability, three kinds of HA coated nanoparticles were fabricated in this study. The amount of HA absorbed on the NPs-1, NPs-2 and NPs-3 was quantified by CTAB method and was approximately 0.51, 0.25 and 0.43 mg, respectively. The mean particle size of NCs was 124.9 ± 2.4 nm (Figure 1A), whereas the addition of HA increased the mean diameter to about 140.7 ± 2.2 nm, 141.3 ± 2.1 nm, 153.1 ± 1.9 nm for NPs-1, NPs-2, and NPs-3, respectively. The zeta potential shifted from positive values (21.4 ± 1.2 mV) for naked NCs to negative values about -22.2 ± 1.4 mV, -22.1 ± 1.3 mV, and -30.5 ± 1.6 mV for NPs-1, NPs-2, NPs-3, respectively, due to the carboxylic negative residues in the HA coating. The increased particle size and inversed zeta potential confirmed the presence of HA on the surface of NCs. The encapsulation efficiency (EE) of siRNA before and after the coating of HA was measured. As depicted in Figure S1, HA coating did not influence the EE of siRNA. Thus, HA would not compete with siRNA binding to CPPs in the present study.


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Figure 1. (A) Gel retardation assay of NCs at various N/P ratio. (B) Particle size and zeta potential of CPPs/siRNA nanocomplexes (NCs), HA (Mw 8 kDa)-coated NCs (NPs-1), HA (Mw 200 kDa, low coating density)-coated NCs (NPs-2), and HA (Mw 200 kDa, high coating density)-coated NCs (NPs-3) (n = 6). (C) Serum stability and RNase A sensitivity assay of naked siRNA, NCs, NPs-1, NPs-2 and NPs-3 in 50% final concentration of FBS or RNase A solution (final working concentration 10 µg/mL) at 37 °C for the prearranged time intervals. (D) TEM images of different formulations.

The nonspecific interactions between gene delivery vectors and serum components could lead to siRNA dissociation and subsequent degradation by nucleases. Agarose gel electrophoresis was performed to confirm the serum stability of the nanoparticles (Figure 1C). The results demonstrated that the naked siRNA band disappeared completely after 12 h in 50% FBS. However, the siRNA encapsulated in nanoparticles showed high stability and remained relatively intact during 36 h in 50% FBS, indicating that the nanoparticles could efficiently protect siRNA from degradation in the serum. Another important factor affecting siRNA delivery for efficient gene silencing therapy is the enzymatic degradation. Naked siRNA was quickly degraded when exposed to the RNase A. On contrary, siRNA in nanoparticles was well protected against the nuclease. The morphological characteristics of different nanoparticles were visualized by TEM as shown in Figure 1D. NCs appeared as dispersed black spots, matching the size measured from DLS. NPs were decorated by a gray rim and revealed as a clear core-shell structure, which was absent in NCs, suggesting that HA was coated on NCs surface. 3.2. CD44 expression analysis. An impaired endothelium-macrophages co-culture system was developed in transwell chambers to mimic the in vivo permeable endothelial barrier of atherosclerotic plaques. Long-term culture of HUVECs produced an endothelial monolayer with a TEER value of greater than 300 Ω×cm2. A continuous ring feature of



occluding proteins around cell boundaries supports the formation of an intact endothelial monolayer with tight junctions. The monolayer becomes highly permeable when stimulated with TNF-α, as evidenced by reduced TEER and disruption of intercellular junctional structures (Figure S2A). For the aim to identify the effect of TNF-α on the receptor expression and the establishment of injured endothelium, the CD44 receptor levels of HUVECs were evaluated by flow cytometry and CLSM. The results showed that activated HUVECs exhibited significantly enhanced CD44 expression after TNF-α stimulation (Figure 2A, B).

Figure 2. (A) Flow cytometry analysis of CD44 expression in HUVECs with or without


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TNF-α (10 ng/mL) stimulation for 12 h. (B) Confocal microscopy images of CD44 expression in HUVECs in the absence or presence of TNF-α (10 ng/mL) for 12 h (at 600× magnification). Cells were incubated with CD44 primary antibody and FITC-conjugated secondary antibody (shown in green). Cell nuclei were stained with DAPI (shown in blue). (C) Viabilities of HUVECs and THP-1 derived macrophages after incubation with different nanoparticles (NCs, NPs-1, NPs-2, NPs-3) with an LOX-1 specific siRNA concentration of 200 nM (n = 6) for 24 h. The untreated cells were set as control. (D) Papp values of different preparations across the cell monolayer in the trans-endothelial transport study (n = 6, **** p

cell-penetrating

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