Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible

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Highly Efficient and Safe Delivery of VEGF siRNA by BioReducible Fluorinated Peptide Dendrimers for Cancer Therapy Xiaojun Cai, Haofang Zhu, Yanmei Zhang, and Zhongwei Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16689 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 24, 2017

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ACS Applied Materials & Interfaces

Highly Efficient and Safe Delivery of VEGF siRNA by Bio-Reducible Fluorinated Peptide Dendrimers for Cancer Therapy

Xiaojun Cai, Haofang Zhu, Yanmei Zhang, Zhongwei Gu*

College of Materials Science and Engineering, Nanjing Tech University, 30 South Puzhu Road, Nanjing 211816, PR China

ACS Paragon Plus Environment

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Abstract： RNA interference (RNAi) have a great promise in treating various acquired and hereditary diseases. However, it remains highly desirable to develop new delivery system to circumvent complex extra- and intracellular barriers for successful clinical translation. Here, we report on a versatile polymeric vector, bio-reducible fluorinated peptide dendrimers (BFPD), for efficient and safe siRNA delivery. In virtue of skillfully integrated all of the unique advantages of reversible crosslinking, fluorination and peptide dendrimers, this novel vector can surmount almost all extra- and intracellular barriers associated with local siRNA delivery through highly improved physiological stability and serum resistance, significantly increased intratumoral enrichment, cellular internalization, successful facilitation of endosomal escape and cytosolic siRNA release. BFPD polyplexes, carrying siVEGF, demonstrated excellent VEGF silencing efficacy (~65%) and a strong capability for inhibiting HeLa cell proliferation. More importantly, these polyplexes showed superior performance in long-term enrichment in the tumor sites and had a high level of tumor growth inhibition. Furthermore, these polyplexes not only exhibited excellent in vivo anti-tumor efficacy, but demonstrated superior biocompatibility, compared with LPF2000, both in vivo and in vitro. These findings indicate that BFPD is an efficient and safe siRNA delivery system and has remarkable potential for RNAi-based cancer treatment. Keywords: RNA interference, cancer therapy, fluorination, reversible cross-linking, peptide dendrimers, extracellular and intracellular barriers


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1. Introduction RNA interference (RNAi) is an important mechanism that regulates gene expression in eukaryotic cells via sequence-specific degradation of mRNA.1-3 Because of its high specificity, significant effects, minor side effects and ease of synthesis, the RNAi machinery has been widely regarded as a promising modality for treating different kinds of acquired and hereditary diseases.4-10 In particular, several clinical trials using RNAi are currently ongoing.11-12 However, the clinical translation of RNAi has been hampered by the poor bioavailability of siRNA stemming from its high susceptibility to enzymatic hydrolysis, rapid elimination from the circulatory

system,

off-target

effects,

poor

cellular

uptake,

as

well

as

potential

immunogenicity.13-16 Therefore, effective, safe and clinically suitable systems that can avoid siRNA degradation and clearance, promote its accumulation at tumor sites, as well as facilitate its cytosolic siRNA delivery are highly desirable to ensure the broad clinical application of RNAi.17-19 In recent years, a large number of viral and non-viral carriers have been developed for siRNA delivery.20-22 Viral vectors feature high gene silencing efficacy, but have inherent safety concerns, which greatly restrict their use clinically.23 For this reason, non-viral vectors including inorganic nanoparticles,24,25 liposomes26,27 and polymers28-31 have been widely investigated as alternatives to viral carriers. Among these, polymeric vectors are particularly attractive since they provide potential opportunities to address the intrinsic deficiencies of siRNA via forming polyplexes with siRNA.32,33 However, in spite of these advantages, the in vivo therapeutic efficacy of existing polymeric vectors is still far from what is required clinically, presumably due to their poor ability to surmount complex extra- and intracellular barriers.34,35 Nonspecific interactions, disassembly and attacks by endogenous nucleases are critical extracellular barriers


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to systemic delivery of siRNA polyplexes and normally lead to serious aggregation and rapid elimination from the circulatory system,36 premature release and degradation of siRNA cargoes.37,38 Another major extracellular barrier to efficient RNAi stems from the need for active recognition and entry of siRNA polyplexes into target cells, otherwise massive nonspecific internalization of the siRNA polyplexes can take place.39,40 Furthermore, even when successfully internalized into the target cells, siRNA polyplex must still avoid endosomal entrapment and allow efficient release of siRNA cargoes into the cytoplasm.41,42 Even more problematic, any of these barriers could become the Achilles' heel in siRNA delivery process, which would remarkably undermine or even totally eliminate the treatment effect of the siRNA polyplex.35 To circumvent these critical barriers, polymers have been modified with various moieties such as hydrophobic components,43,44 targeting ligands/cell penetrating peptides,45-47 acidcleavable linkers/acid-protonating groups,48,49 or stimulus-sensitive linkages50,51 to improve physiological stability, cellular uptake, facilitate endosomal escape and cytoplasmic traffic, disassembly, as well as siRNA cargo release. Despite the individual success of some of these strategies in overcoming certain barriers, generating a single system that integrates whole of the rationally designed components into one vehicle to overcome all of these crucial obstacles, has been synthetically challenging. Thus, the rational design and facile synthesis of versatile polymeric vectors, with many advanced features that could cooperatively circumvent various extra- and intracellular barriers, is currently a major challenge for improving the therapeutic efficacy of RNAi therapies. Unfortunately, research into such versatile polymeric vectors is rarely reported. We recently developed a novel kind of polymeric vector, bio-reducible fluorinated peptide dendrimer (BFPD), via reversible crosslinking of fluorinated generation two poly(L-lysine)


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dendrimers.52 The unique advantages of reversible crosslinking, fluorination and peptide dendrimers were skillfully integrated into this novel vector, which exhibits many advanced features, including excellent physiological stability, superior serum resistance, rapid and efficient cellular uptake, endosomal escape, cytosolic traffic, nuclear entry, intracellular unpacking and cargo release. As a result, BFPD not only showed superior gene transfection efficacy, but offered considerable in vivo gene transfection efficacy as well. Encouraged by these advanced features and results, we hypothesize that BFPD may be capable of addressing nearly all of the extra- and intracellular barriers associated with local siRNA delivery and may have remarkable potential for use in RNAi based cancer therapy (Scheme 1). In the current study, BFPD was used as an siRNA delivery vehicle to examine its potential for RNAi-based cancer therapy. VEGF served as the target gene in our work, since it is the most critical regulator of tumor-induced angiogenesis and plays a crucial role in tumor growth.53-55 The capabilities of BFPD to circumvent complex extraand intracellular barriers for successful siVEGF delivery and VEGF silencing were systematically evaluated. The in vivo and in vitro therapeutic efficacy and biocompatibility of BFPD were also investigated in detail. The effect of various BFPD formulations on the biological efficacy of BFPD polyplexes was also assessed.


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2. Experiments Chemicals. Boc-Lys(Boc)-OH, N-hydroxybenzotriazole (HOBt), N,N,N′,N′-TetramethylO-(1H-benzotriazol-1-yl) uronium hexafluorophosphate (HBTU) were obtained from GL Biochem. Trifluoroacetic acid (TFA) and N-Ethyldiisopropylamine (DIPEA) were obtained from Asta Tech Pharmceutical. (3-aminopropyl)-triethoxysilane, heptafluorobutyric anhydride (HFBA), Di(N-succinimidyl) 3,3'-Dithiodipropionate (DSP), reduced glutathione (GSH), DLbuthionine sulfoximine (BSO), Bafilomycin A1, Sucrose, Methyl-β-cyclodextrin (mβCD) were obtained from Sigma-Aldrich. Chlorpromazine, Genistein, Wortmannin, and Dynasore were obtained from Aladdin Reagent Company. LipofectamineTM2000 (LPF2000) was obtained from Invitrogen.

siRNA

targeting

GAUUAUGCGGAUCAAACCUtt-3’),

VEGF scrambled

mRNA siRNA

(siVEGF, (siN.C,

sense:

5’-

sense:

5’-

AUCGGAGCAGUC-GUAAUGCtt-3’), Cy5- and FAM-labeled siRNA were synthesized by Guangzhou RiboBio Life Science Co. The control sequence did not match any human genome sequences. The SsoFast EvaGreen supermix and iScript cDNA Synthesis Kit were acquired from Bio-Rad (Bio-Rad, USA). DEPC water and RIPA buffer were obtained from Beyotime Institute of Biotechnology (China). CCK-8 was obtained from Dojindo Laboratories (Japan). Rabbit IgG against human VEGF was obtained from Epitomics (USA). Goat anti-rabbit IgG HPR was ordered from Santa Cruz Biotechnology. Enhanced chemiluminescence (ECL) was obtained from Thermo Scientific. All other reagents were of analytic grade. De novo synthesis of bio-reducible fluorinated peptide dendrimers (BFPD). The bioreducible fluorinated peptide dendrimers (BFPD) were synthesized as described previously with a minor modification.52 In Brief, polyhedral oligomeric silsesquioxane (POSS) core-based generation two poly(L-lysine) dendrimers (G2-Lys) were first synthesized via a divergent


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approach in anhydrous DMF with HOBt and HBTU served as condensation reagent and DIPEA served as acid-binding reagent, then deprotected by TFA. The deprotected G2-Lys was dispersed in distilled water, followed by adjusted to pH 7.4 by sodium bicarbonate solution, then dialyzed exhaustively in distilled water for 48 h and collected after lyophilization. Afterwards, the amino group ended G2-Lys and HFBA with a molar ratio of G2-Lys to HFBA of 1:12.8 were mixed in CH3OH. The mixture was maintained at RT for 2 days, then dialyzed exhaustively in PBS and water, and collected after lyophilization. The obtained FG2-Lys were mixed with appropriate amount of DSP dissolved in DMSO. Three molar ratios of DSP to FG2-Lys (2:1, 1:1 and 1:2) were utilized to optimize the synthesis of BFPD. The mixture was maintained at RT overnight, then dialyzed exhaustively in PBS and water, and collected after lyophilization. The successful synthesis of POSS, G1-Lys, G2-Lys, FG2-Lys and BFPD were monitored and demonstrated by 1

H NMR (Bruker Advance II, Bruker), Electrospray ionization time-of-flight (Waters Q-TOF

Premier, Waters), Elemental analysis (vario EL III, Elementar), Gel permeation chromatography (Waters1515, Waters). Formation and characterization of BFPD polyplexes. BFPD (1 mg mL-1) were mixed with siRNA (0.1 mg mL-1) at a certain BFPD/siRNA weight ratio in water (DEPC-treated), followed by pipetting for 25 second and maintained at RT for half an hour before use. Gel retardation assay was performed to investigate the binding affinity of BFPD to siRNA. BFPD polyplexes carrying 200 ng siRNA with weight ratios ranging from 0 to 3.24 were first loaded onto 1% agarose gel, then electrophoresed at 90 V for 10 min. The gels were stained with ethidium bromide (EB) and visualized by a Bio-Rad ChemiDoc XRS+UV illuminator. For EB exclusion assay, the freshly prepared BFPD polyplexes with weight ratios ranging from 1.62 to 6.48 were firstly incubated with EB for 60 min prior to fluorescence intensity measurement by


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microplate reader. The excitation and emission wavelength of EB is 510 and 590 nm, respectively. The mixture of siRNA/EB and pure EB solution was individually served as positive and negative control. The siRNA complexing efficacy (%) was defined as, siRNA complexing efficacy (%) = [1-(F-FEB) / (F0-FEB)] x 100, where F0, FEB and F respectively represent the fluorescence intensity of siRNA/EB, pure EB and siRNA/EB/BFPD solution. The size and ζpotential of the newly formed BFPD polyplexes with weight ratios ranging from 2 to 8 were determined by using dynamic light scattering (NANO ZSPO, Malvern). Morphologies of BFPD12 polyplexes with a weight ratio of 6 were observed by AFM (MFP-3D-BIO, USA) and TEM (FEI Tecnai GF20S-TWIN, USA). For AFM measurement, a drop of BFPD-12 polyplexes solution was dripped onto a mica plate, and dried in air for one day. Physiological stability of BFPD polyplex. The physiological stability of BFPD polyplex was evaluated through measurement the changes in polyplex size, siRNA condensation ability as well as siRNA release profiles following a 12 h incubation in the presence of 10 times volume of PBS (pH 7.0) supplemented with 10% FBS and 5 µM GSH. The variations in particle size of BFPD polyplexes were monitored by DLS every 2 h. For siRNA condensation ability assay, after 4 h of incubation, BFPD-12 and BFPD-21 polyplexes carrying 200 ng siRNA were first loaded onto 1% agarose gel, then electrophoresed at 90 V for 10 min. Naked siRNA and BFPD polyplexes without any treatment were used as control. For siRNA release profile analysis, FAM-siRNA carrying BFPD polyplexes were taken and centrifuged for half an hour at 13,000 rpm. The FAM-siRNA concentration in 50 µL supernatant was measured by microplate reader. The excitation and emission wavelength of FAM-siRNA is 480 and 520 nm, respectively. The amount of siRNA released from the polyplexes was calculated according to the equation: siRNA released (%) = (F1-F2) / (F0-F2) × 100%, where F0 was the fluorescence of the freshly prepared


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BFPDs polyplexes carrying 2 µg FAM-siRNA in 1 mL PBS (pH 7.0) supplemented with 10% FBS and 5 µM GSH, F1 was the fluorescence of siRNA in supernatant, F2 was the fluorescence induced by PBS (pH 7.0) supplemented with 10% FBS and 5 µM GSH. Cytotoxicity assay. HeLa cells were maintained in DMEM containing 10% FBS. For cytotoxicity assay, HeLa cells (5 x 103 cells per well) were seeded in 96-well plates. After 24 h incubation, the medium was replaced by three kinds of BFPD polyplexes carrying 100 nM siN.C in 0.5 mL serum-free DMEM. After incubation for another 4 h, the medium was replaced by complete DMEM medium and the cells were cultured for another 44 or 68 h. The medium was refreshed with PBS twice, then 10 µL of CCK8 was added and cultured for another 2 h. The optical density (OD) of each well was determined at 450 nm using a microplate reader. Cell viability was measured as: Cell viability (%) = (OD sample) / (OD control) x 100%. Free siRNA and LPF2000 polyplexes served as control. Cellular uptake and intracellular trafficking of BFPD polyplexes. To visualize the cellular internalization of BFPD polyplexes, HeLa cells at a density of 1 x 104 cells per dish were cultured in culture dishes for 24 h. The medium was replaced by serum-free DMEM and BFPD polyplexes carrying 60 nM FAM-siRNA were added with a weight ratio of 6. Following 4 h incubation, the cells were rinsed twice with heparin (20 U mL-1) containing PBS, the cellular uptake of BFPD polyplexes was visualized and recorded by CLSM (Leica TCS SP5, Germany). To investigate the effect of siRNA concentration on the cellular uptake level of BFPD polyplexes, HeLa cells (1 x 105 cells per well) were seeded into 24-well plates and cultured overnight. Then BFPD-12 polyplexes carrying 20, 40, 60, 80 or 100 nM FAM-siRNA in 500 µL serum-free DMEM were added with a weight ratio of 6. After incubation for another 4 h, the cells were washed twice with heparin (20 U mL-1) containing PBS, then digested with trypsin,


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re-dispersed in 300 µL PBS for flow cytometry analysis (FACSAria, BD, USA). LPF2000 polyplexes carrying 60 nM FAM-siRNA served as control. To evaluate the effect of BFPD formulation and BFPD/siRNA weight ratio on the uptake level of BFPD polyplexes, HeLa cells (1 x 105 cells per well) were seeded in 24-well plates and cultured overnight. Then the cells were exposed to various BFPD polyplexes accompanied with different weight ratios ranging from 2 to 8 for 4 h. Afterwards, the cells were collected according to the same procedures described above and analyzed by flow cytometer. To reveal the mechanisms about the cellular uptake of BFPD polyplexes, cellular internalization study was conducted either with various endocytic inhibitors or at 4 °C. In brief, HeLa cells were pre-treated with various endocytic inhibitors including wortmannin (50 nM), chlorpromazine (10 µg mL-1), genistein (200 µg mL-1), methyl-βcyclodextrin (50 µM), and dynasore (80 µM) for 30 min, then BFPD-12 polyplexes carrying 60 nM siRNA were added with a weight ratio of 6 and cultured at 37 °C for 4 h. Afterwards, the cells were collected for FACS analysis. To observe the intracellular trafficking of BFPD polyplexes, HeLa cells (1 x 104 cells per dish) were seeded on glass-bottomed dishes and cultured overnight. After 4 h of incubation with BFPD polyplexes carrying 60 nM Cy5-siRNA with a weight ratio of 6, the cells were washed twice with heparin (20 U mL-1) containing PBS, stained with Lysotracker Green (50 nM), and visualized by CLSM. The fluorescent probes of Lysotracker Green and Cy5-siRNA were excited at 495 nm and 633 nm, respectively. Redox-responsive-induced disassembly and siRNA release of BFPD polyplexes. The redox-responsive-induced disassembly and siRNA release of BFPD polyplexes were investigated through measurement the changes in polyplex size, morphology, as well as siRNA release profiles following 6 h incubation at 37 °C in the presence of 10 mM GSH. The effect of reversible-crosslinking on VEGF silencing efficacy of BFPD polyplexes was examined in a GSH


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inhibition experiment. In brief, HeLa cells (3 × 104 cells per well) were seeded in 24-well plates and cultured overnight prior to the addition of BSO (200 µM), a GSH-depleting agent. After 24 h incubation, the cells were exposed to 0.5 mL serum-free BFPD-12 polyplexes carrying 60 nM siRNA for 4 h. Afterwards, the cells were cultured in complete culture medium for another 44 h for VEGF silencing. The culture medium was collected for VEGF ELISA assay according to the same procedures described above. LPF2000 polyplexes served as control. In vitro VEGF silencing and anti-tumor efficacy of BFPD polyplexes. The in vitro VEGF silencing efficacy of BFPD polyplexes was evaluated by ELISA, qRT-PCR, and Western bloting analysis. In brief, HeLa cells (5 × 104 cells per well) were seeded in 24-well plates and cultured overnight before the addition of BFPD polyplexes carrying 60 nM siVEGF. After 4 h of incubation, the cells were cultured in complete culture medium for another 44 h for VEGF silencing. The medium and cells were collected for ELISA and qRT-PCR analysis, respectively. In qRT-PCR analysis, total RNA was extracted from transfected cells using the Trizol reagent. One micrograms of total RNA were transcribed into cDNA using a commercial iScript™ cDNA Synthesis Kit and qRT-PCR analysis were conducted using SsoFast EvaGreen® Supermix. Data were collected and analyzed using CFX Manager Software. GAPDH and blank cells served as control. The VEGF mRNA levels were normalized to the GAPDH expression and finally normalized to the blank cells. The sequence of the VEGF forward primer was 5’TTTCTGCTGT-CTTGGGTG-3’, and the reverse primer was 5’-CTCGATTGGATGGCAGTA3’. The sequence of the GAPDH forward sequence was 5’-AC-CCAGAAGACTGTGGATGG3’, and the reverse primer was 5’-TTCAGCTCAGGGATGACCTT-3’. For Western Bloting analysis, transfected cells were lysed with RIPA buffer supplemented with a protease inhibitor. 40 µg of total protein were electrophoresed and further analyzed using a previously reported


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procedure.36 The membranes were visualized using an enhanced ECL system and the protein expression levels were normalized to actin protein expression levels. In Vivo fluorescence imaging of BFPD polyplexes. Animal experiments were conducted following the ethics of Sichuan University and National Institute of Health Guide for the Care and Use of Laboratory animals. BALB/c nude mice (20 ± 2 g) were purchased from National Rodent Laboratory Animal Resources and maintained under SPF (specific pathogen free) conditions. 1 × 106 HeLa cells in 0.1 mL of PBS were subcutaneous injected into the right upper flanks of mice to establish the xenograft tumor model. Tumor volume was measured by caliper across its shortest and longest diameters, calculated by the following equation: Volume (mm3) = 0.5 × length × width2. For in vivo fluorescence imaging, BFPD polyplexes carrying 1 nmol Cy5siRNA were intratumorally administered into mice when the tumor grew to around 100 mm3. At 3 h, 6 h, 24 h, 48 h and 72 h, the mice were imaged by CRi Maestro EX in-vivo imaging system with 649 nm excitation and 680 nm emission. Afterwards, the mice were dissected, and the tumor tissues were collected for quantifying the Cy5-siRNA fluorescence intensity in tumor tissues. Results were analyzed by CRI Maestro measurement software. The Cy5-siRNA distribution within the tumor tissues were observed by CLSM according to a previously reported method.36 In Vivo anti-tumor efficacy of BFPD polyplexes. When the tumor grew to around 50 mm3, the mice were divided into six groups with five mice in each group and treated by intratumoral injection of PBS, BFPD-12/siN.C, LPF2000/siVEGF, BFPD-11/siVEGF, BFPD12/siVEGF, and BFPD-21/siVEGF polyplexes (2.5 nmol siVEGF per mouse) every other day for a total of eleven times. The volume of tumor and weight of body were measured every other day.


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After 22 days treatment, all mice were killed and the excised tumors were immediately photographed and stored at -80 °C for PCR and Western bloting analysis. Immunohistochemistry and in situ cell death detection. After 22 days treatment, all mice were killed and the tumor tissues were collected for immunohistochemistry and in situ cell death detection. For immunohistochemistry analysis, the paraffin-embedded tumor tissues were first cut at 5 µm thickness, then de-paraffinized, re-hydrated, and quenched with 3% H2O2 for 10 min prior to antigen retrieval by trypsinization. Afterwards, sections were blocked and incubated overnight at 4 °C with primary antibody, then treated with second antibody for another half an hour. After washing with PBS twice, sections were incubated with HRP-conjugated streptavidin at 37 °C for 30 min. Sections were stained with hematoxylin, dehydrated and observed in a microscope. For TUNEL assay, sections were first treated with proteinase K at 37 °C for 15 min, then washed twice with PBS, followed by treatment with TUNEL reaction mixture at 37 °C for 1 h. After rinsing with PBS twice, sections were mounted with anti-fade mounting solution to avoid fluorescence photo-bleaching. All sections were imaged using inverted optical microscopy. The integrated optical density (IOD) value was calculated by using an Image-Pro plus 6.0 software. TUNEL-positive cells were quantified in ten randomly selected fields (× 400) per slice and represented as percentages of the total number of tubular cells. Statistical Analysis. Variance analysis was analyzed using the unpaired Student’s t test and p value 2.16 (Figure 1C). This suggests that both BFPD-11, BFPD-12 and BFPD-21 are capable of condensing siRNA, but BFPD-11 and BFPD-12 are more potent in siRNA complexing, probably due to their relative high surface charge ratios (27 and 29 mV) and high molecular weight (36.8 and 24.2 kDa) compared to BFPD-21, which are 16 mV and 14.3 kDa, respectively. Such observations were further confirmed by a quantitative EB exclusion assay. For


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instance, the siRNA complexing efficacy was only 68% for BFPD-21 at a weight ratio of 3.24, but was as high as 92% and 89% for BFPD-11 and BFPD-12, respectively (Figure 1D). The polyplex size and ζ- potential of BFPD polyplexes were measured at various BFPD/siRNA weight ratios. As shown in Figure 2A and B, an increase in BFPD/siRNA weight ratio led to a notably decreased particle size and increased ζ-potential. At weight ratios ≥6, all BFPD polyplexes had an average diameter of ∼120 nm, a PDI of 0.185, and a positive ζ-potential of ∼25 mV. These values proved to be more suitable for passive tumor targeting via the EPR effect and to be more efficient for cellular uptake via clathrin-mediated endocytosis pathways.58 The TEM (Figure 2C) and AFM (Figure 2D) images show that BFPD-12 polyplexes have a classic spherical structure, with a mean diameter around 70 ± 10 nm, which was slightly smaller than that measured by DLS (120 nm). The difference in size characteristics among the three methods was mainly ascribed to the water evaporation required for TEM and AFM sample preparation.59 Excellent physiological stability for transportation across the complex physiological milieu. As mentioned previously, physiological stability is a key factor in siRNA polyplexes, playing a crucial role in pharmacokinetics and the capability to protect siRNA from enzymatic hydrolysis. Therefore, whether BFPD polyplexes can transport across complex physiological milieu to efficiently deliver siRNA to target tissues was systematically examined. During this process, siRNA polyplexes are faced with numerous barriers, including massive dilution, serum attack, stimuli-responsive triggered carrier degradation and polyplex dissociation. In order to evaluate stability under simulated physiological milieu, BFPD polyplexes were incubated with a 10-fold volume of PBS (pH 7.4) containing 10% FBS and 5 µM GSH. As depicted in Figure 3A, BFPD-11 and BFPD-12 polyplexes demonstrated superior stability as seen by their consistent diameters following a 12 h incubation, while BFPD-21 polyplexes showed limited stability with


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a particle size that increased gradually from 115 to 280 nm during the 12 h incubation. In addition, the BFPD-21 polyplexes allowed a notable amount of siRNA migration during electrophoresis (Figure 3B) following a 4 h incubation. The siRNA release profiles (Figure 3C) further confirmed the superior stability of BFPD-11 and BFPD-12 polyplexes, as well as the limited stability of BFPD-21 polyplexes. For instance, only less than 6% siRNA was released from the BFPD-11 and BFPD-12 polyplexes during a 4 h incubation, however about 16% siRNA was released from the BFPD-21 polyplexes during this same time frame. More remarkable difference was detected at 12 h. By this point, the BFPD-21 polyplexes had released ~26% siRNA while the BFPD-11 and BFPD-12 polyplexes had still released 90% (Figure 3D) following a 72-h incubation; significantly higher than that seen with LPF2000 polyplexes (∼60%). Efficient cellular internalization, endosomal escape, cytosolic disassembly and siRNA release to circumvent complex intracellular barriers. With lots of optimal physiochemical


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properties, including appropriate particle size, zeta potential, surface morphology, as well as excellent physiological stability, BFPD polyplexes may be accompanied by a high level of intratumoral enrichment. However, efficient tumor accumulation alone is insufficient to enable successful RNAi, since they still have to face more complex intracellular barriers, including cellular internalization, endosomal/lysosomal escape, cytoplasm disassembly and siRNA release. More importantly, any of these barriers could become the Achilles' heel in siRNA delivery process, which would significantly undermine or even totally eliminate the treatment effect of RNAi. Therefore, the capabilities of BFPD polyplexes to circumvent these critical barriers were probed. First, the uptake profiles and pathways of BFPD polyplexes were examined in HeLa cells by CLSM and flow cytometry analysis. FAM-labeled siRNA was used to highlight their cellular localization. As depicted in Figure 4A, after 4-h incubation, extensive BFPD polyplexes not only entered into the cells but trafficked into the cytoplasm, suggesting a rapid and efficient cellular uptake due to the unique feature of fluorination, which not only improved the affinity of BFPD polyplexes to cell membrane, but also increased their ability to cross the membrane. Hence, the uptake level of the BFPD polyplexes was close to, or even higher than, that of LPF2000 polyplexes. In addition, the uptake level of BFPD polyplexes was siRNA dose-dependent. An increase in siRNA concentration led to a notably increased uptake level (percentage of FAMsiRNA positive cells), which peaked at the siRNA concentration of 60 nM (Figure 4B). At this concentration, BFPD-12 polyplexes achieved their optimal cellular uptake efficacy of 96%, which was notably higher than that of LPF2000 polyplexes (82%). It is worth noting that the uptake profiles of BFPD polyplexes were also weight ratio and BFPD formulation-dependent, as shown in Figure 4C with a fixed siRNA concentration of 60 nM. BFPD-11 and BFPD-12


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polyplexes achieved their optimal uptake level of 93% and 96%, respectively, at BFPD/siRNA weight ratios of 4 and 6, which were appreciably higher than that of the BFPD-21 polyplexes (72%) even at a BFPD/siRNA weight ratio of 8. The limited uptake level of BFPD-21 polyplexes may be related to their poor stability, which may lead to a premature disassembly of polyplexes and a gradual increase in particle size during incubation, consequently impeding efficient cellular uptake. The uptake pathway is closely related to the intracellular fate, which ultimately dominates RNAi efficacy. As such, the mechanisms underlying the uptake of BFPD polyplexes were illustrated. As depicted in Figure 4D, majority of BFPD polyplexes were enter cells via energy-dependent and clathrin-mediated endocytosis (CME) pathways, as the uptake level was reduced by 62% at 4 °C and 54% in the presence of CME inhibitor chlorpromazine, respectively. The caveolae inhibitor, genistein and mβCD, as well as the clathrin and caveolae inhibitor, dynasore also exerted an appreciable inhibitory effect, while the macropinocytosis inhibitor, wortmannin had negligible inhibitory effect, indicating that CME and caveolae, rather than macropinocytosis are involved in the uptake of BFPD polyplexes. As depicted in Figure 4D, BFPD polyplexes were mainly endocytosed via CME pathways. These pathways usually lead to endosomal entrapment and lysosomal degradation of the siRNA cargo,61 resulting in low RNAi efficacy unless they are able to trigger effective endosomal escape.62 To validate this, CLSM was performed to visualize the co-localization of the Cy5siRNA-containing BFPD polyplexes and Lysotracker-Green-stained endosomes/lysosomes. As depicted in Figure 5, in spite of the fact that a small amount of BFPD-11 and BFPD-12 polyplexes (red fluorescence) were still co-localized with endosomes/lysosomes (green fluorescence) following a 4 h incubation, the majority of them had successfully trafficked into the cytoplasm, suggesting that they had efficiently bypassed endosomal entrapment due to the


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fluorination effect. Noteworthy, the BFPD-21 polyplexes experienced a severe endosomal entrapment, primarily owing to their rapidly intracellular disassembly and siRNA cargo release following cellular uptake. Overall, the premature disassembly and siRNA cargo release of polyplexes not only impeded efficient cellular uptake but also endosomal escape. These results further substantiate the importance of physiological stability in influencing the cellular uptake and trafficking of siRNA polyplexes. Following efficient cellular uptake, endosomal/lysosomal escape, inefficient polyplex disassembly and siRNA cargo release still represent limiting steps for subsequent posttranscriptional gene silencing.38 One key BFPD design was the disulfide-cleavage mediated polyplex disassembly to facilitate siRNA release. In order to demonstrate this, BFPD polyplexes were subjected to 10 mM GSH for a predetermined period of time to track the disassembly and siRNA release profiles through measurement of changes in particle size, morphology and amount of siRNA released. As depicted in Figure 6A, the polyplex size of BFPD-11, BFPD-12 and BFPD-21 polyplexes increased dramatically from 108, 105 and 116 nm to 508, 540 and 1040 nm, respectively, within 3 h, implying disulfide-cleavage result in the transformation from compact polyplexes to loose polyplexes, which even increased to above 1986, 2183 and 2383 nm, respectively, within 6 h, suggesting the thorough dissociation of the polyplexes, which was in accordance with the TEM results (Figure 6B) showing that the particle size of BFPD-12 and BFPD-21 were about 1500-2000 nm following a 6 h incubation. More importantly, the polyplex dissociation led to a remarkable siRNA release (Figure 6C), in which all BFPD polyplexes released about 95% siRNA following a 6 h incubation. Overall, these results highlight the possibility of disulfide-cleavage mediated dissociation of BFPD polyplexes and siRNA release in the cytoplasm since high GSH levels (5~10 mM) were present in the cytoplasm as essential


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cellular components. The crucial role of disulfide-cleavage in regulating polyplex dissociation and siRNA release was further verified by a transfection experiment in the presence of BSO, a GSH-depleting agent.63 As depicted in Figure 6D, BSO treatment had a negligible effect on the VEGF silencing efficacy of LPF2000 polyplexes, while significantly decreasing the VEGF silencing efficacy of BFPD-12 polyplexes, which again substantiated the importance of disulfide cleavages in BFPD polyplexes during VEGF silencing, as well as the fact that regulation of siRNA release through polyplexes dissociation is critical to efficient RNAi. The significantly improved VEGF silencing efficacy and inhibition effect on cancer cell proliferation. Encouraged by the excellent results in overcoming the complex extra- and intracellular barriers against RNAi, we speculated that BFPD polyplexes would be accompanied by improved VEGF silencing efficacy and antitumor effect. To demonstrate this, HeLa cells were incubated with different BFPD polyplexes carrying 60 nM siVEGF for two days, and VEGF gene silencing was analyzed by qRT-PCR, Western Bloting and ELISA. As depicted in Figure 7A, free siVEGF and BFPD polyplexes carrying siN.C showed negligible VEGF silencing efficacy, while BFPD polyplexes carrying siVEGF exhibited remarkably improved VEGF silencing efficacy, suggesting that BFPD polyplexes can efficiently inhibit VEGF expression in a highly sequence-specific manner. In addition, BFPD-11 and BFPD-12 polyplexes evoked the greatest inhibition effect of VEGF expression, achieving 58 ± 2.3% and 65 ± 1.5%, respectively, which was comparable or even slightly higher than that of LPF2000 polyplexes (60 ± 2.2%). Consistent with the qRT-PCR results, the VEGF protein expression levels measured by western blot (Figure 7B) and ELISA assay (Figure 7C) further confirmed the superior VEGF silencing efficacies of BFPD-11 and BFPD-12 polyplexes which was notably higher than that of BFPD-21 polyplexes. The relatively poor VEGF silencing efficacy of BFPD-21 polyplexes can


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be ascribed to their limited stability, poor uptake level and weak capability in mediating endosomal escape. The inhibition of VEGF expression in tumor cells suppressed cellular proliferation.59 The significantly improved VEGF silencing efficacy by BFPD polyplexes may have resulted in elevated suppression of cellular proliferation. To demonstrate this, after 4 h of transfection, HeLa cells were cultured in complete DMEM medium for another 44 or 68 h, then treated with CCK8 to determine cell viability. No significant inhibition of cellular proliferation was detected for treatment with free siVEGF, while BFPD polyplexes carrying siVEGF exerted a significant inhibitory effect on cellular proliferation in a BFPD formulation-dependent manner (Figure 7D). For instance, after 72 h treatment, the viability of HeLa cells treated with BFPD-21, BFPD-11 and BFPD-12 polyplexes was reduced to 75%, 65%, and 51%, respectively and the proliferation of HeLa cells were significantly inhibited. This suggests that both siVEGF and BFPD formulation played roles in inhibiting cellular proliferation in HeLa cells. In addition, LPF2000 polyplexes induced a marked inhibitory effect on cellular proliferation. The percentage of living cells following three day’s treatment with LPF2000 polyplexes was 42%, which was appreciably lower compared to the percentage of living cells after treatment with BFPD polyplexes. The superior inhibitory effect of the LPF2000 polyplexes was partially ascribed to their inherent cytotoxicity as evidenced in Figure 3D. In the light of the above results (Fig. 3 to Fig. 7), BFPD appears to be a versatile siRNA delivery vehicle, which not only can transport siRNA across the complex physiological milieu, but can also circumvent multiple intracellular barriers, thus highly improving the silencing efficacy of VEGF and the inhibition of cancer cellular proliferation. Superior serum resistance, rapid and efficient cellular internalization enables longterm enrichment within the intratumoral sites. According to the results of the cell


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experiments, it was hypothesized that BFPD polyplexes could have great potential for RNAi based cancer therapy. BFPD polyplexes were intratumorally injected into mice in order to explore their preliminary application for in vivo siRNA delivery and cancer treatment. The enrichment behavior of siRNA polyplexes within tumors play a key role in the ultimate therapeutic efficacy, thus these parameters were monitored after BFPD polyplexes, carrying Cy5-siRNA, were administered intratumorally to a mouse bearing HeLa tumor xenografts. As depicted in Figure 8A, the fluorescence signals (Cy5-siRNA) of BFPD-11 and BFPD-12 polyplexes were only visible in tumor sites between 3 and 72 h with no significant reduction, while there was a slight reduction in the fluorescence signal of the BFPD-21 polyplexes and a remarkable, time-dependent, reduction in the fluorescence signal of the LPF2000 polyplexes, were no longer visible 72 h post-injection. Consistent with this observation, the ex vivo semiquantitative fluorescence intensity of tumor tissues also indicated that almost all of the BFPD-11 and BFPD-12 polyplexes were located in the tumor sites, while the LPF2000 and BFPD-21 polyplexes were rapidly excreted by the mice (Figure 8B). The long-term enrichment behavior of BFPD-11 and BFPD-12 polyplexes may be related to their excellent physiological stability, while the relative short enrichment time of BFPD-21 and LPF2000 polyplexes is likely related to their limited stability in vivo, caused by the relatively weak siRNA complexing efficacy and high surface charges, respectively. In order to confirm this hypothesis, siVEGF transfection was conducted in the presence of 30% FBS. As depicted in Figure 8C, the VEGF silencing efficacy of BFPD-11 and BFPD-12 polyplexes was hardly affected by serum, suggesting that those polyplexes have superior serum resistance, a key requirement for enabling long-term enrichment in the tumor sites. The VEGF silencing efficacy of BFPD-21 polyplexes was slightly reduced by serum, implying that these polyplexes undergo a


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certain degree of dissociation via counter polyion exchange reactions due to relatively weak siRNA complexing efficiency, which in turn leads to premature siRNA release. The released siRNA cannot traffic into the cells and undergoes rapid degradation by nuclease or elimination from the tumor sites via the lymphatic system. Moreover, the VEGF silencing efficacy of the LFP2000 polyplexes was remarkably reduced by serum. VEGF expression in the LPF2000 polyplexes increased dramatically from 269 ± 16 to 659 ± 37 pg/mL in the presence of 30% FBS, implying unsatisfactory serum resistance due to high surface charges,64 thus they aggregated rapidly and formed large aggregates in the tumor sites, largely hindering their tumor penetration and resulting in their continual elimination from the mice via the lymphatic system. In addition to superior serum resistance, the significantly enhanced cell internalization of BFPD11 and BFPD-12 polyplexes was also a main reason for their long-term enrichment in the tumor sites, seen as the CLSM images of tumor tissue sections (Figure 8D). On the contrary, LPF2000 polyplexes with limited serum resistance and inefficient cellular internalization profiles were easily clean up by the lymphatic system. BFPD polyplexes carrying siVEGF significantly promoted the antitumor effect following intratumoral injection. The long-term intratumoral enrichment behavior, significantly improved VEGF silencing efficacy and inhibitory effect on cancer cell proliferation of BFPD-11 and BFPD-12 polyplexes will potentially enhance their RNAi efficacy in cancer treatment. To validate it, the antitumor effect of intratumoral administration of BFPD polyplexes carrying siVEGF was examined in mice with HeLa tumor xenografts. As illustrated in Figure 9A, compared to PBS (tumor volume of 592 ± 81%), LPF2000 and BFPD-21 polyplexes slightly inhibited tumor growth, leading to a relative tumor volume of 358 ± 42% and 310 ± 28%, respectively, on the 22rd day. Conversely, delivery of siVEGF with BFPD-11 and BFPD-12 led


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to a remarkable inhibition on tumor growth, with relative tumor volume of 205 ± 31% and 105 ± 20%, respectively, on the 22rd day. It is noteworthy that administration of BFPD-12 polyplexes containing scrambled siRNA could not suppress tumor growth, suggesting that the anti-tumor effect was siRNA sequence-specific. The antitumor effect presented in Figure 9B was consistent with the inhibition curves in Figure 9A. To probe whether the inhibition of tumor growth by BFPD polyplexes was associated with the VEGF gene silencing, the excised tumor tissues were collected for VEGF mRNA and protein analyses. As depicted in Figure 9C, after treatment with BFPD-11, BFPD-12, BFPD-21, and LPF2000 polyplexes, the levels of VEGF mRNA levels were reduced 52.0 ± 7.1%, 65 ± 3.2%, 33 ± 4.3%, and 25 ± 2.4% reduction, respectively, compared to the levels in tumors treated with saline. VEGF protein expression displayed a consistent silencing efficacy in each group. As depicted in Figure 9D, only a slight down-regulation in VEGF protein expression was observed after administration of BFPD-21 and LPF2000 polyplexes, while a remarkable inhibition of VEGF protein expression occurred after treatment with BFPD-11 and BFPD-12 polyplexes, indicating that the remarkable anti-tumor effect of BFPD-11 and BFPD-12 polyplexes was due to the efficient VEGF silencing. Furthermore, it is worth noting that in addition to significant antitumor effects, BFPD polyplexes also showed excellent biocompatibility, as indicated by the fact that no pathological changes was noted between mice treated with BFPD polyplexes carrying siVEGF or siN.C during the experimental period, as depicted in Figure 9E. Comparatively, a notable reduction in body weight, combined with serious inflammatory reaction and necrosis focus were observed in mice treated with LPF200 polyplexes, suggesting the high toxicity of LPF2000 polyplexes due to high surface charges.


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It’s well-known that the VEGF-mediated angiogenesis is particularly important in tumor growth and metastasis, therefore, the significantly retarded tumor growth by BFPD-11 and BFPD-12 polyplexes may be closely relate to the inhibition of new blood vessel formation. To demonstrate this, the microvessel density (MVD) in BFPD polyplexes treated tumor tissues was analyzed using immunohistochemical staining assay. As depicted in Figure 10A and B, tumors treated with siVEGF polyplexes showed a significant reduction in MVD, suggesting that siVEGF polyplexes can inhibit the formation of microvessels. Furthermore, the MVD varied depending on the siRNA formulations, BFPD-11 and BFPD-12 polyplexes carrying siVEGF demonstrated the highest inhibitory effect on the formation of microvessels, consistent with their high antitumor efficacy (Figure 9). Decreased tumor angiogenesis is usually accompanied by an increase in tumor apoptosis, thus, the high inhibitory effect on tumor angiogenesis by BFPD-11 and BFPD-12 polyplexes may also result in a high level of tumor apoptosis. As expected, treatment with BFPD-11 and BFPD-12 polyplexes carrying siVEGF resulted in the highest level of tumor apoptosis (40 ± 2.18% and 45 ± 2.11%) compared to than that of BFPD-21 (20 ± 1.25%) and LPF2000 (27 ± 1.16%) polyplexes (Figure 10C). 4. Conclusions In summary, we designed and developed a versatile polymeric vector, bio-reducible fluorinated peptide dendrimers (BFPD), that can surmount almost all extra- and intracellular barriers associated with local siRNA delivery was developed by improving physiological stability and serum resistance, promoting intratumoral enrichment, and cellular internalization, as well as facilitating endosomal/lysosomal escape and reduction-triggered cytoplasm siRNA release. BFPD polyplexes afford excellent VEGF gene silencing efficacy (~65%) and have a strong ability to inhibit HeLa cell proliferation. Furthermore, BFPD polyplexes showed superior


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performance in long-term enrichment in the tumor sites and significantly retarded tumor growth. More importantly, BFPD polyplexes not only showed remarkable in vivo anti-tumor efficacy but superior biocompatibility compared with LPF2000 both in vivo and in vitro. These findings indicate that BFPD polyplexes represent an efficient and safe siRNA delivery vehicle and have remarkable potential for RNAi-based cancer treatment. ASSOCIATED CONTENT Supporting Information The synthesis routes for BFPD, Fig. S1 and Fig. S2 are shown in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]. Notes: The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (51133004, 81361140343, 81621003, and 51503131), the Joint Sino-German Center for Research Promotion (GZ905), the International Science and Technology Cooperation Program of China (2015DFE52780). REFERENCES (1)

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(51) Han, L.; Zhao, J.; Zhang, X.; Cao, W.; Hu, X.; Zou, G.; Duan, X.; Liang, X. J. Enhanced siRNA Delivery and Silencing Gold-Chitosan Nanosystem with Surface Charge-Reversal Polymer Assembly and Good Biocompatibility ACS NANO 2012, 6, 7340-7351. (52) Cai, X.; Jin, R.; Wang, J.; Yue, D.; Jiang, Q.; Wu, Y.; Gu, Z. Bioreducible Fluorinated Peptide Dendrimers Capable of Circumventing Various Physiological Barriers for Highly Efficient and Safe Gene Delivery. ACS Appl. Mater. Interfaces 2016, 8, 5821-5832. (53) Pittella, F.; Miyata, K.; Maeda, Y.; Suma, T.; Watanabe, S.; Chen, Q.; Christie, R. J.; Osada, K.; Nishiyama, N.; Kataoka, K. Pancreatic cancer therapy by systemic administration of VEGF siRNA contained in calcium phosphate/charge-conversional polymer hybrid nanoparticles. J. Controlled Release 2012, 161, 868-874. (54) Chen, J.; Tian, H.; Dong, X.; Guo, Z.; Jiao, Z.; Li, F.; Kano, A.; Maruyama, A.; Chen, X. Effective Tumor Treatment by VEGF siRNA Complexed with Hydrophobic Poly(Amino Acid)-Modified Polyethylenimine. Macromol. Biosci. 2013, 13, 1438-1446. (55) Kim, H. A.; Nam, K.; Kim, S. W. Tumor targeting RGD conjugated bio-reducible polymer for VEGF siRNA expressing plasmid delivery. Biomaterials 2014, 35, 7543-7552. (56) Liu, H.; Wang, Y.; Wang, M.; Xiao, J.; Cheng, Y. Fluorinated poly(propylenimine) dendrimers as gene vectors. Biomaterials 2014, 35, 5407-5413. (57) Yang, J.; Zhang, Q.; Chang, H.; Cheng, Y. Surface-Engineered Dendrimers in Gene Delivery. Chem. Rev. 2015, 115, 5274-5300, (58) Wang, T.; Upponi, J. R.; Torchilin, V. P. Design of multifunctional non-viral gene vectors to overcome physiological barriers: Dilemmas and strategies. Int. J. Pharm. 2012, 427, 3-20.


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Scheme 1. Schematic illustration for the preparation of BFPD polyplexes as well as their excellent performance to circumvent all of the complex extra- and intracellular barriers.


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Figure 1. Binding affinity of (A) BFPD-11, (B) BFPD-12, and (C) BFPD-21 to siRNA at different BFDP/siRNA weight ratios, ranging from 0 to 3.24, demonstrated by gel retardation assay. (D) Complexing efficacy of BFPD-11, BFPD-12 and BFPD-21 to siRNA at different BFPD/siRNA weight ratios, ranging from 0 to 6.48, quantified by EB exclusion assay (n = 3).


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Figure 2. (A) Particle Size and (B) Zeta Potential of BFPD-11, BFPD-12 and BFPD-21 polyplexes at different BFPD/siRNA weight ratios, ranging from 2 to 8, measured by DLS. (C) TEM and (D) AFM images of BFPD-12 polyplexes at a weight ratio of 6.


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Figure 3. (A) Changes in particle size, (B) siRNA binding affinity, and (C) siRNA release profiles of BFPD polyplexes (weight ratio = 6) following a 12 h incubation at 37°C in the presence of a 10-fold volume of PBS (0.2 M, pH, 7.0) supplemented with 10% FBS and 5 µM GSH (n = 3). *p < 0.05, **p < 0.01, BFPD-21 polyplexes vs BFPD-12 polyplexes at the same time point. (D) Cell viability of HeLa cells following a 48 or 72 h incubation with various BFPD polyplexes (weight ratio = 8) carrying 100 nM scramble siRNA was quantified using the CCK8 assay (n = 5). **p < 0.01, LPF2000 polyplexes vs PBS at the same time point.


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Figure 4. (A) CLSM images of cellular uptake of FAM-siRNA containing BFPD polyplexes (weight ratio = 6) in HeLa cells after 4 h of incubation. (B) Cellular uptake level of FAMsiRNA-containing BFPD-12 polyplexes (weight ratio = 6) in HeLa cells after 4 h of incubation at various siRNA concentrations (BFPD-12/siRNA weight ratio fixed at 6) (n = 3). *p < 0.05, **p < 0.01, uptake level of BFPD-12 polyplexes at the siRNA concentration of 60 nM vs that of at the other concentrations. (C) Cellular uptake level of FAM-siRNA-containing BFPD polyplexes in HeLa cells after 4 h of incubation at various BFPD formulations and weight ratios (siRNA dose fixed at 60 nM) (n = 3). **p < 0.01, uptake level of BFPD-21 polyplexes vs that of BFPD-12 polyplexes at the same weight ratios. (D) Cellular uptake level of FAM-siRNA-containing BFPD-12 polyplexes (weight ratio = 6) in HeLa cells after 4 h of incubation in the presence of various endocytic inhibitors or at 4 °C (n = 3). *p < 0.05, **p < 0.01, uptake level of BFPD-12 polyplexes without any treatment vs that of treated with various endocytic inhibitors or at 4 °C.


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Figure 5. CLSM images of intracellular trafficking of Cy5-siRNA-containing BFPD-11, BFPD12 and BFPD-21 polyplexes in HeLa cells after 4 h of incubation. All BFPDs polyplexes were prepared at a weight ratio of 6. The endosomes/lysosomes were stained with Lysotracker Green. The scale bars correspond to 25 µm.


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Figure 6. (A) Changes in particle size (n = 3), (B) morphologies, and (C) siRNA release profiles of BFPDs polyplexes (weight ratio = 6) after 6 h of incubation with 10 mM GSH. **p

Highly Efficient and Safe Delivery of VEGF siRNA by Bioreducible

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