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Self-assembled nanoparticles based on the c(RGDfk) peptide for the delivery of siRNA targeting the VEGFR2 gene for tumor therapy

The clinical application of small interfering RNA (siRNA) has been restricted by their poor intracellular uptake, low serum stability, and inability to target specific cells. During the last several decades, a great deal of effort has been devoted to exploring materials for siRNA delivery. In this study, biodegradable, tumor-targeted, self-assembled peptide nanoparticles consisting of cyclo(Arg–Gly–Asp–d–Phe–Lys)-8–amino–3,6–dioxaoctanoic acid–β–maleimidopropionic acid (hereafter referred to as RPM) were found to be an effective siRNA carrier both in vitro and in vivo. The nanoparticles were characterized based on transmission electron microscopy, circular dichroism spectra, and dynamic light scattering. In vitro analyses showed that the RPM/VEGFR2-siRNA exhibited negligible cytotoxicity and induced effective gene silencing. Delivery of the RPM/VEGFR2 (zebrafish)-siRNA into zebrafish embryos resulted in inhibition of neovascularization. Administration of RPM/VEGFR2 (mouse)-siRNA to tumor-bearing nude mice led to a significant inhibition of tumor growth, a marked reduction of vessels, and a down-regulation of VEGFR2 (messenger RNA and protein) in tumor tissue. Furthermore, the levels of IFN-α, IFN-γ, IL-12, and IL-6 in mouse serum, assayed via enzyme-linked immunosorbent assay, did not indicate any immunogenicity of the RPM/VEGFR2 (mouse)-siRNA in vivo. In conclusion, RPM may provide a safe and effective delivery vector for the clinical application of siRNAs in tumor therapy.


Introduction
Double-stranded, small interfering RNA (siRNA)-induced gene silencing through the inhibition of specific messenger RNA (mRNA) translation, also known as RNA interference, has been utilized for quite some time. 1 siRNA has attracted intense interest due to its promising therapeutic effects in various diseases, such as neuronal diseases, infectious diseases, and various cancers. 2,3 However, siRNA technology still faces a series of obstacles before it can be applied in a clinical setting, related to issues such as poor pharmacokinetics profiles 2 due to degradation by nucleases in the serum, poor cellular uptake, rapid elimination, and the inability to target specific cell types. Therefore, designing carriers that can effectively deliver specific siRNAs to targeted tissues represents a great challenge and is the subject of intense research. Many nonviral carriers that can self-assemble into supramolecular complexes have been designed for siRNA delivery to date. For example, liposomes, lipoplexes, stable nucleic acid lipid particles, cationic polymers, and peptides have been employed to protect siRNAs from undesirable degradation during the transfection process. 4 Additionally, these carriers have been modified with different targeting ligands, such as the Arg-Gly-Asp (RGD) peptide, 5 folic acid, 6 transferrin protein, 7 and antibodies, 8 to increase their targeting ability. The RGD peptide and structurally related compounds [9][10][11][12][13][14] are the best-studied ligands that belong to the integrin ligand group. [15][16][17][18] Because these ligands specifically bind to the integrin receptor, which is overexpressed in the endothelial cells of the tumor neovasculature, 19 when applied in vivo, an 8-amino-3,6-dioxaoctanoic acid (PEG)β-maleimidopropionic acid (MAL) hydrophilically modified, specific integrin αvβ3 receptor-targeted small cyclopeptide c(RGDfk) could lead to the accumulation of siRNA in tumors, resulting in tumor targeting.
Inhibition of angiogenesis, which blocks the supply of nutrition to and waste discharge from tumors, results in inhibition of the growth, invasion, and metastasis of tumors and has been widely applied in antitumor studies. 20,21 Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, plays a vital role in the angiogenic process by binding to the specific VEGF receptor 2 (VEGFR2, also known as KDR/Flk-1), a tyrosine kinase receptor, which then activates downstream signaling pathways and results in the proliferation and migration of endothelial vessels, consequently promoting angiogenesis and vascular growth. [22][23][24][25][26] Therefore, inhibition of VEGFR2 mRNA expression in new vessels is an effective method of tumor therapy.
In the present study, RPM was found to self-assemble into nanoparticles (NPs) that could be used for efficient siRNA delivery. We examined the characteristics of the NPs and validated their function by studying the gene-silencing effects of RPM/VEGFR2-siRNA both in vitro and in vivo. We achieved two levels of targeting: targeted binding to the integrin αvβ3 receptor, which is overexpressed in new vessels, via the ligand cyclo(RGD-d-Phe-Lys) (c[RGDfk]) and gene pathway selectivity via the siRNA oligonucleotide. To our knowledge, this is the first study to show that the modified small cyclopeptide c(RGDfk) has the ability to self-assemble and can effectively deliver siRNA to targeted tissue sites. The following siRNA sequences were used in the in vitro experiments: anti-human VEGFR2 siRNA (sense strand, 5′-GGUAAAGAUUGAUGAAGAAdTdT-3′, and antisense strand, 3′-dTdTCCAUUUCUAACUACUUCUU-5′); and scramble siRNA, referred to as control siRNA (sense strand, 5′-CCUGGAGAAUCAGACGACAAGUAUU-3′, and antisense strand, 3′-GGACCUCUUAGUCUGCUGUUCA UAA-5′). The following siRNA sequences were employed in the in vivo experiments: anti-mouse VEGFR2 siRNA, which was 2′-o-methyl sugar modified (sense strand, 5′-CGGAGAAGAAUGUGGUUAAdTdT-3′, and antisense strand, 3′-dTdTGCCUCUUCUUACACCAAUU-5′); a n t i -z e b r a f i s h V E G F R 2 s i R N A ( s e n s e s t r a n d , 5′-CUGAAAACAAUGUUGUGAAdTdT-3′, and antisense strand, 3′-dTdTGACUUUUGUUACAACACUU-5′); and control siRNA (mouse, zebrafish) (sense strand, 5′-CGUGAUUGCGAGACUCUGAdTdT-3′, and antisense strand, 3′-dTdTGCACUAACGCUCUGAGACU-5′). Indodicarbocyanine-5 (Cy5)-labeled siRNA (siRNA-Cy5) and all of the abovementioned siRNAs were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, People's Republic of China). The siRNA-Cy5 was synthesized on the solid support using Cy5-phosphoramidite. Standard coupling conditions for synthesis of Cy5 labeling was carried out at the 5′-end of the guide (antisense) strand of the molecules, which formed a phosphodiester linkage. The siRNA-Cy5 was only used for the targeting research in vitro and biodistribution in vivo.

Materials and methods
synthesis of rPM and raPM RPM (96.8% purity) and RAPM (96.9%) were produced and purified by Peptides International, Inc. RPM consisted of c(RGDfk) peptide, PEG, and MAL, while RAPM consisted of c(RADfk) peptide, PEG, and MAL.

Preparation of rPM NPs and rPM/sirNa complexes
Lyophilized RPM powder was dissolved in double-distilled water (ddH 2 O) at a concentration of 2 mg/mL, and the siRNAs were dissolved in RNAse-free ddH 2 O at a concentration of 20 μM. For siRNA loading, RPM was added to the siRNA solution in different proportions (Table 1) to obtain the optimal mixture. After gentle mixing, the solution was incubated at room temperature for 30 minutes prior to the next steps.

characterizations of rPM NPs and complexes
The particle size and zeta potential of the RPM NPs and RPM/siRNA complexes were both measured by dynamic light scattering (DLS) using a Malvern Zetasizer (Nano-ZS90; Malvern Instruments Ltd., Malvern, UK). Samples were prepared as described in the Preparation of RPM NPs and RPM/siRNA complexes section. The volume ratio of International Journal of Nanomedicine 2014:9 submit your manuscript | www.dovepress.com Dovepress Dovepress 3511 rPM nanoparticles for siVegFr2 delivery in vitro and in vivo RPM (2 mg/mL, 750 μL) and siRNA (20 μM, 50 μL) was 1.5:1; the detection volume of both solutions was 1 mL; and the final concentration of siRNA in RPM/siRNA group was 100 nM. The morphology of the NPs and the NP complexes were observed using a transmission electron microscope ([TEM] JEM-100CXII; JEOL, Tokyo, Japan). About 20 μL of each sample was placed on the carbon-coated copper grids respectively, negatively stained with 2% (w/v) phosphotungstic acid, and then dried at room temperature. The secondary structures of the complexes were detected based on circular dichroism spectra (using a JASCO J-810 spectropolarimeter; Jasco, Inc., Eastern, Md, Japan) and scanned at 0.1 nm intervals from 250 to 190 nm (0.1 mm quartz cuvette). The detection volume was 1 mL for both groups. The ellipticity measurements are presented as the mean residue ellipticity ([θ] in degrees cm 2 dmol −1 ). gel retardation assay NP complexes were prepared at different ratios. All the samples contained 2 μL of siRNA (20 μM) and 1 μL, 2 μL, 3 μL, and 4 μL of RPM (2 mg/mL) were used, respectively. The complexes mixed with DNA loading buffer (6×) were then electrophoresed in a 2% agarose gel containing 0.5 μg/mL ethidium bromide. Electrophoresis was performed at 80 mV for 30 minutes, and the resulting gels were photographed under ultraviolet illumination. Naked siRNA was used as a control.

cell culture
Human umbilical vein endothelial cells (HUVECs) (American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in DMEM supplemented with 10% FBS and a 1% antibiotic-antimycotic solution. The human non-small-cell lung cancer A549 cell line (ATCC) and the luciferase-A549 cell line (a generous gift from the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science) were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% FBS and a 1% antibiotic-antimycotic solution. The cells were incubated at 37°C in a 5% CO 2 atmosphere. The cells for all experiments were in logarithmic growth phase.

Fluorescence-activated cell sorting analysis
To quantitatively examine the cellular uptake level of RPM/ siRNA-Cy5, fluorescence-activated cell sorting (FACS) analysis was used. HUVECs were incubated with different RPM/siRNA complexes ( The assays were conducted in triplicate.
NP complex-mediated gene silencing in vitro

Intracellular sirNa transfection
HUVECs were seeded into a six-well plate and incubated for 24 hours, until the cells were 60% confluent. The samples were then transfected with RPM/siVEGFR2, RPM/control siRNA, RPM NPs alone, or siVEGFR2 alone using the Lipo2000 reagent. The final volume was 1 mL and the final concentration of siRNAs was 100 nM. Untreated cells were employed as a blank control. The cellular levels of VEGFR2 mRNA were measured after 48 hours, and VEGFR2 protein levels were measured after 72 hours through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot analyses, respectively.

Western blot analysis
Transfected cells were collected, and Western blot analysis was conducted as described previously. 28

In vivo angiogenesis assay in zebrafish
Flk-1-GFP transgenic and wild-type zebrafish were provided by the Department of Cell Biology of Southern Medical University, Guangzhou, People's Republic of China. The zebrafish were raised according to the methods of Westerfield. 29  Then the gray images were analyzed using Image J software. The zebrafish were anesthetized using 0.02% tricaine mesylate prior to observation.

In vivo analyses in tumor-bearing nude mice
human non-small-cell lung cancer xenograft tumor model Saline, RPM/control siRNA, and RPM/siVEGFR2 were administered via intravenous injection in 100 μL volumes (RPM: 60 μL, 5 mg/mL; siRNAs: 40 μL, 50 μM) once every 3 days at a dose of 2 nmol of siRNAs per mouse, and the treatment was repeated six times. Tumor luminescence was detected on day 0 and day 20 to monitor the development of the tumors using the In Vivo Imaging Systems Spectrum imaging system (Xenogen Corporation-Caliper, Ala-meda, CA, USA) following intraperitoneal injection of luciferin (Promega Corporation, Madison, WI, USA) at a dose of 150 mg/kg per mouse. Tumor volume and mouse weight were monitored prior to injection. The tumor volume was calculated using the following formula: The curves were plotted as the mean tumor volume ± standard deviation. The animals were euthanized 2 days after the last treatment, and the tumors were excised and preserved in liquid nitrogen.

In vivo localization of NP complexes with sirNa-cy5
For the tissue distribution analysis, the successful model animals were randomly divided into three groups (N=3). A single dose of saline, RPM/siRNA-Cy5 (0.2 nmol), RAPM/ siRNA-Cy5 (0.2 nmol), or naked siRNA-Cy5 (0.2 nmol) was injected via the tail vein in 40 μL volumes (RPM: 30 μL, 2 mg/mL; siRNA-Cy5: 10 μL, 20 μM). The subsequent biodistribution was monitored at 10 minutes, 0.5 hours, 3 hours, 6 hours, and 24 hours after administration using the IVIS Spectrum imaging system at the appropriate wavelength (siRNA-Cy5: λ ex =640 nm, λ em =680 nm). The mice were sacrificed after the last time point, and the tumors and major organs were excised and imaged. The uptake level of siRNA-Cy5 in different organs was quantified by doing region of interest.

Detection of VegFr2 mrNa and protein expression in tumor tissue
Total RNA was extracted from the tumor tissues, and RT-qPCR was performed using the same protocol described above. The following primer pairs were used in this experiment: VEGFR2 sense, 5′-AGAATGCGGGCTCCTGACTA-3′, and antisense, 5′-CCATGCTCAGTGTCTCTGACA-3′; and 18s sense, 5′-CCTGGATACCGCAGCTAGGA-3′, and antisense, 5′-GCGGCGCAATACGAATGCCCC-3′. The expression of the 18s gene was used as an internal reference. The VEGFR2 protein expression level was detected through Western blot analysis.

Immunohistochemical analysis
The tumors were frozen and cut into 6 μM sections after being embedded in Tissue-Tek ® optimum cutting temperature medium (Sakura Finetek Japan, Tokyo, Japan). The sections were fixed in cold 100% acetone for 10 minutes, then stored at −20°C. Prior to analysis, the sections were removed and allowed to acclimate to room temperature. The sections were then washed with PBS and incubated in endogenous peroxidase (3% H 2 O 2 ) for 10 minutes, followed by 1% normal goat serum in PBS for 30 minutes at room temperature. A rat anti-mouse CD31 antibody (1:50; BD Biosciences) was added, and the sections were incubated overnight at 4°C. The sections were subsequently washed three times in PBS, followed by incubation with rabbit anti-rat IgG-biotin ([1:100 dilution] Wuhan Boster Biological Technology, Ltd, People's Republic of China) at 37°C for 1 hour, after which they were washed with PBS again and incubated with peroxidase-conjugated streptavidin ([1:100 dilution] Proteintech Group, Inc., Chicago, IL, USA). The chromogen DAB Kit (Boster) and hematoxylin were used to stain vessels and nuclei. A fluorescence microscope (DM4000B; Leica Microsystems, Wetzlar, Germany) was employed to obtain photographs.

Mouse cytokine and interferon response
At 6 and 24 hours after administration of RPM NPs, RPM/ siVEGFR2, and saline, respectively, serums were collected and assayed to detect mouse IFN-α, IFN-γ, IL-12, and IL-6 using a quantitative enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, Inc., San Diego, CA, USA) according to the manufacturer's instructions. The absorbance was read using a Multiskan TM MK3 microplate reader (Thermo Fisher Scientific) at 450 nm.
renal and liver toxicity analysis RPM/siRNA-and saline-treated animals were sacrificed 2 days after the last injection, and serum was collected and isolated for the measurements of alanine aminotransferase (ALT) and creatinine using an Aeroset automated analyzer (Abbott Diagnostics, Berkshire, UK) according to the manufacturer's instructions.

statistical analysis
Statistical analysis of the data was performed through one-way analysis of variance (ANOVA) (SPSS software, version 13.0; SPSS Inc., Chicago, IL, USA). The results are expressed as the mean ± standard error, and P0.05 was considered statistically significant. All statistical tests were two-sided.

Preparation and characterizations of NPs
The mechanism of NP assembly is presented in Figure 1, which was speculated according to the basic characteristics that were detected by TEM, CD spectra and DLS. Through intermolecular hydrogen bonding interactions, with the β-sheet and random coil secondary structure, RPM molecules self-assembled into spherical NPs and c(RGDfk)s were on the surface of NPs for the targeting of αvβ3 receptors. siRNAs in the solution were encapsulated in the NP cavities or "trapped" in the gap of molecules due to the positively charged arginine on the c(RGDfk)s and electropositive carbonyl carbon and hydrogen atoms on the maleimide.
The TEM images presented in Figure 2A (inset images) show that the RPM NPs and RPM/siRNA complexes were round in shape, and their diameters were less than 100 nm. The particle size of the RPM NPs ranged from 91.28 to 122.4 nm, while the size of the RPM/siRNA complexes ranged from 105.7 to 141.8 nm (Table 2). Both types of NPs exhibited a narrow size distribution ( Figure 2A) and possessed a negative charge ( Table 2) (zeta potentials of −6.16 and −14.6 mV, respectively). Furthermore, Figure  2B shows a mixed secondary structure of the NPs including β-sheet and random coil, with the β-sheet accounting for the majority.

Protection of the sirNas encapsulated in rPM
The result of gel retardation assay shows that the RPM/ siRNA NPs clearly lagged behind the naked siRNA, which may be due to the disparities in their molecular weights. And with increasing the amount of RPM, more siRNAs were trapped in the NPs ( Figure S1A). When the ratio reached 1.5:1, there was no difference of band intensity between naked siRNA and RPM/siRNA NPs ( Figure S1B). The serum stability test demonstrated that the naked siRNA was nearly completely degraded after 4 hours of incubation in the serum. In contrast, siRNA was still present in the RPM/ siRNA group after 12 hours ( Figure 2C).

Intracellular distribution of sirNa
When the volume ratio reached 1.5:1, siRNA-Cy5 aggregated around the nucleus, and this phenomenon was not observed above or below this ratio ( Figure 3A). Therefore, this ratio was used in the subsequent experiments as the desired ratio between RPM and the siRNAs. High-definition differential interference contrast images clearly show that siRNA-Cy5s were distributed in the cell cytoplasm ( Figure 3B). The result of FACS analysis, shown in Figure S2A, was in accordance  with the result of confocal microscopy images. Although no difference was found in the percentage of positive cells in different ratios ( Figure S2B), when the volume ratio reached 1.5:1, the fluorescence intensity of siRNA-Cy5 in the cells was the strongest ( Figure S2C).
The HUVECs were incubated and maintained as indicated in the methods. As shown in Figure 4A signal from siRNA-Cy5 in the cytoplasm was weak, and puncta formed after 1 hour; however, after 6 hours of incubation, the fluorescence signal became strong and dispersed ( Figure 4B), while the HUVECs treated with anti-integrin αvβ3 antibody for 30 minutes before the incubation of RPM/ siRNA-Cy5 ( Figure 5A), or treated with RAPM/siRNA-Cy5 ( Figure 5B), showed an extremely weak fluorescent signal for siRNA-Cy5 in the cytoplasm. cytotoxicity As shown in Figure 6A, there was no difference in cell viability between the RPM NP, RPM/siRNA, and untreated groups, while the cell viability observed in the Lipo2000/ siRNA group was approximately 50%, which was significantly different from the other groups.

In vitro gene-silencing efficiency
RT-qPCR analysis demonstrated that transfection of siRNAs using RPM and a cationic lipid reagent resulted in comparable (~38% for RPM/siRNA versus ~39% for Lipo2000/siRNA) gene knockdown in HUVECs following treatment for 48 hours, and no significant difference was observed between other groups ( Figure 6B). Total protein was collected for Western blot analysis after 72 hours, and VEGFR2 protein expression was markedly reduced in the RPM/siRNA and Lipo2000/ siRNA groups (Figures 6C and 6D), by approximately 29% and 36%, respectively. Taken together, these data demonstrate the ability of the RPM/siRNA to enter the integrin αvβ3-expressing cells and induce targeted gene knockdown efficiently.  In vivo gene silencing in zebrafish In zebrafish, hypogenetic vessels or a lack of vessels can be clearly observed because the normal vessels formed in the zebrafish express strong green fluorescent protein (GFP). As shown in Figure 7A, strong GFP fluorescence was observed in the ddH 2 O-treated group, while the embryos treated with the RPM/siRNA failed to completely form dorsal longitudinal anastomotic vessels and intersegmental vessels. The fluorescence statistics for each group revealed that RPM/ siRNA could have an antiangiogenic effect ( Figure 7B).

Tumor targeting
Intense fluorescence accumulated in the tumors after 0.5 hours in the RPM/siRNA-Cy5 group, reflecting the presence of siRNA-Cy5 in the blood pool, and this accumulation lasted for at least 24 hours after injection. However, no fluorescence was detected in the tumors of mice that were injected with naked siRNA-Cy5 and RAPM/siRNA-Cy5 ( Figure 8A). Fluorescence was also detected in the kidneys and livers of the mice, and ex vivo images corroborated the findings of the whole-animal imaging analysis ( Figure 8B). The statistic of fluorescence intensity of siRNA-Cy5 in different organs showed that siRNA-Cy5s mainly accumulated in the kidneys, then livers and tumors ( Figure S3); however, there was a significant difference of fluorescence intensity in tumors between the RPM/siRNA-Cy5 group and other groups, which proved the tumor-targeting ability of RPM (**P0.01, ## P0.01 [**refers that there is a extreme difference between the RPM/ siRNA-Cy5 group and naked siRNA-Cy5 group. ## refers that there is an extreme difference between the RPM/siRNA-Cy5 group and RAPM/siRNA-Cy5 group]).

In vivo antitumor activity
Twenty days after the first injection, no difference was observed in the body weights of the mice between the three groups ( Figure 9A). An obvious tumor growth was  found in the saline-and RPM/control siRNA-treated mice, and there was no significant difference between these two groups, which indicated that RPM/control siRNA did not have specific antitumor effects. Compared to the saline-and RPM/control siRNA-treated mice, the tumor volume was effectively inhibited in the mice treated with RPM/siRNA (*P<0.05, **P<0.01) ( Figure 9B). Distinct attenuation of the tumor bioluminescence intensity was observed only in the RPM/siRNA group ( Figure 9C).
To determine whether the anti-tumor effect of the RPM/ siRNA was due to immune effects, the levels of IFN-α, IFN-γ, IL-12, and IL-6 in the serum were determined at 6 and 24 hours after administration. The results showed that there were no differences in the cytokine or interferon responses of the groups ( Figure 10A). In addition, unchanged ALT and creatinine serum levels indicated that RPM/siRNA is well tolerated, producing no significant renal and liver toxicity ( Figure 10B). Anti-tumor effect of RPM/siRNA was also determined at the gene level; a highly significant difference was observed in the levels of VEGFR2 mRNA between the tumors of the RPM/siRNA group and the other groups ( Figure 10C). Furthermore, Figure 10D shows that the VEGFR2 protein level was markedly reduced in the RPM/siRNA group.
Additionally, immunohistochemistry was performed to detect vessels in the tumors, and the density of vessels observed in the RPM/siRNA group was clearly lower than in the other two groups, as shown in Figure 10E.

Discussion
In this study, we have described a targeted type of NP and demonstrated that these NPs could effectively carry siRNA into tumors, resulting in effective gene silencing both in vitro and in vivo. First, we intuitively selected the optimal ratio of RPM and siRNA via confocal laser scanning microscopy and FACS analysis: ratios below the ideal ratio did not cause sufficient transfection effects, possibly due to a lack of RPM, while ratios above the ideal ratio were not effective due to competitive binding of the receptors by the RPM NPs and RPM/siRNA. Based on TEM and dynamic light scattering (DLS) results, a unimodal distribution was present in the RPM NP and RPM/siRNA groups. TEM showed that the NPs were spherical particles, and no distinct aggregation was observed, which may have been due to the secondary structure of the NPs. It had been proved that a helical structure can induce membrane fusion, even at physiological pH, which would be expected to induce aggregation. 30,31 However, the In vivo localization of nanoparticle complexes with sirNa-cy5. Notes: (A) In vivo distribution of sirNa-cy5 in nude mice bearing xenografted luciferase-a549 tumor at the different time points after intravenous injection of rPM/ sirNa-cy5, raPM/sirNa-cy5, or naked sirNa-cy5. Whole-animal bioluminescent imaging was performed to detect luciferase-expressing a549 cells. (B) after 24 hours, ex vivo imaging of tumor and organs excised from BalB/c nude mice. saline was used as the blank control. Images acquired using IVIs spectrum imaging system (Xenogen corporation-caliper, ala-meda, ca, Usa) with appropriate wavelength (sirNa-cy5: λ ex =640 nm, λ em =680 nm). all images were scaled to the same minimum and maximum color values. Abbreviations: c(raDfk), cyclo(arg-ala-asp-d-Phe-lys); c(rgDfk), cyclo(arg-gly-asp-d-Phe-lys); cy5, indodicarbocyanine-5; Mal, β-maleimidopropionic acid; Peg, 8-amino-3,6-dioxaoctanoic acid; raPM, c(raDfk)-Peg-Mal; rPM, c(rgDfk)-Peg-Mal; sirNa, small interfering rNa; sirNa-cy5, cy5-labeled sirNa. secondary structure of the RPM NPs consisted of a β-sheet and a random coil. Because TEM was used to measure the dehydrated diameter of the NPs, while the laser particle size analyzer determined their hydrodynamic diameter, there was a slight difference in size between the results of the TEM and DLS analyses, as previously reported. [32][33][34] The zeta potentials of the RPM NPs and RPM/siRNA were negative. In general, negatively charged particles are taken up less efficiently by cells, and they are therefore not an obvious choice for use as siRNA carriers, though they have occasionally been employed for siRNA delivery. 31,35 The level of cellular uptake of NPs is always determined by three crucial factors: the zeta potential, 36 particle size, 37 and receptor-mediated 38 internalization. Cationic NPs can effectively combine with siRNA through electrostatic attractions, whereas a small particle size is beneficial in facilitating the entry of NPs into cells through endocytosis. A targeting ligand is also required to enhance the cellular uptake of NPs, especially anionic NPs. In the present investigation, receptor-mediated internalization may have played a major role in the cellular uptake of NPs. The siRNA-Cy5s transfected into cells using RPM NPs were distributed throughout the cytoplasm and exhibited strong fluorescence. However, the fluorescence intensity of the siRNA-Cy5s in the cytoplasm was obviously reduced in cells that were initially incubated with anti-integrin αvβ3 antibody for 30 minutes;

A B
International Journal of Nanomedicine 2014:9 submit your manuscript | www.dovepress.com Dovepress Dovepress 3521 rPM nanoparticles for siVegFr2 delivery in vitro and in vivo  this may have been due to the competitive binding of the anti-integrin αvβ3 antibody and the RPM/siRNA-Cy5 to the integrin αvβ3 receptor. Extremely weak fluorescence in the RAPM/siRNA-Cy5 control group further demonstrated that the RPM NPs could attach to the cytomembrane and serve as an effective siRNA delivery system for integrin αvβ3-positive cells, which implies that an active transport process was used for siRNA uptake.
Due to their negative charge, our NPs did not act like cationic NPs, which often result in high cytotoxicity, especially in NPs with high zeta potentials, due to electrostatic interactions with the negatively charged glycocalyx on the cell membranes. 39 An obvious cytotoxic effect of Lipo2000/siRNA was observed, which is commonly proposed to be due to the inhibition of adenosine triphosphate (ATP) enzyme activity 40 and to involve the protein kinase C (PKC) pathway 41 in cells. In contrast, negligible cytotoxicity was observed for our NPs, indicating the biocompatibility of the RPM NPs and demonstrating the tremendous advantages of a safe and effective siRNA delivery system. Stability of the siRNAs in serum is another indispensable condition for the clinical use of siRNAs. Due to the increased molecular weight associated with encapsulation in the RPM NPs during the self-assembly process, the RPM/siRNA complexes exhibited a lag in the gel retardation assay compared to naked siRNA. The RPM NPs protected the siRNAs from being degraded; the PEG-MAL hydrophilic modification of the NPs as well as their negative surface charge inhibits the adsorption of proteins present in serum. These results provide a theoretical basis for the in vivo application of RPM NPs in the near future.
When gene knockdown was examined in vitro, the RPM/ siRNA showed a robust gene-silencing effect at both the mRNA and protein levels in HUVECs. The data also demonstrated that the RPM/siRNA displayed efficient release of the siRNAs into the cytoplasm. However, the mechanism by which the siRNAs are released from the NPs is not clear, and we speculate that dilution in the cytoplasm is the main reason for this phenomenon, 42 resulting in disassociation of the RPM/siRNA and release of the siRNA. The results of the PT-qPCR and Western blot analyses indicated that off-target effects can be ignored. In summary, RPM is a useful tool for the in vitro delivery of siRNAs. Based on the analyses described above, we next evaluated the antiangiogenic effects of RPM/siRNAs in vivo using  Abbreviations: alT, alanine aminotransferase; cD31, platelet endothelial cell adhesion molecule-1 (also known as cluster of differentiation 31); control sirNa, scramble sirNa; elIsa, enzyme-linked immunosorbent assay; gaPDh, glyceraldehyde-3-phosphate dehydrogenase; mrNa, messenger rNa; qPcr, quantitative polymerase chain reaction; rPM, cyclo(arg-gly-asp-d-Phe-lys)-8-amino-3,6-dioxaoctanoic acid-β-maleimidopropionic acid; sirNa, small interfering rNa; VegFr2, vascular endothelial growth factor receptor 2.   zebrafish. It is worth noting that naked siRNA also exhibited antiangiogenic effects, similar to reports showing that injection of naked siRNA into early zebrafish embryos can lead to gene silencing through double-stranded RNA-mediated interference if the targeted gene plays a critical role during the earlier stages of development. 43,44 However, a significant difference was still observed between the RPM/siRNA and naked siRNA groups ( ## P0.01), demonstrating the specific, targeted gene-silencing effect of the RPM/siRNA in later developmental stages in embryos ( ## indicates the extremely significant difference between RPM/siRNA group and naked siRNA group).
We next evaluated the tumor-targeting effect of the RPM/ siRNA in vivo. siRNA-Cy5 accumulated in tumors within 0.5 hours, and the fluorescence intensity of the siRNA-Cy5 became stronger as time elapsed. Two explanations for this phenomenon were considered. First, the delivery of the drugs to a specific site is based on the molecular recognition of an appropriate ligand. 45 The c(RGDfk) peptide specifically attaches to integrin αvβ3, which is expressed on newly formed blood vessels. Thus, the RPM/siRNA might rapidly redistribute the siRNA-Cy5 from the blood pool to tumors and immobilize the NPs within the tumors. 46 Second, passive targeting may also play a role in enhancing the accumulation of siRNA-Cy5 in the tumor. Previous reports have demonstrated that particles with an appropriate diameter (200 nm) can effectively target tumors due to size-dependent properties and the enhanced permeation and retention effect of the tumor. 47 Additionally, the negative charge of the NPs decreases nonspecific interactions, leading to their clearance from the circulation, thus improving the passive targeting of tumors, and the binding of the ligand and receptor might consequentially contribute to a longer retention time in the tumor's interstitial space and an improved enhanced permeation and retention effect. Moreover, no fluorescence was detected in the tumors of mice that were injected with naked siRNA-Cy5 or RAPM/siRNA-Cy5, which further proved the tumor-targeting effect of RPM NPs. It was also noted that, 24 hours after administration of the RPM/siRNA-Cy5, the kidneys of the mice still showed strong fluorescence, indicating that the RPM/siRNA-Cy5 were undergoing renal excretion.
No reduction of body weight and renal or liver toxicity were observed in the RPM/siRNA group compared to the saline-treated group, further demonstrating that the RPM NPs exhibited no toxicity. The tumor growth curves and the downregulation of VEGFR2 mRNA and protein, as well as the decreased density of vessels, further verified the gene-and receptor-targeting effects of the RPM/siRNA. Therefore, the inhibition of vessel formation resulted in insufficient nutrition and efficiently suppressed tumor development, causing a marked reduction of the luciferase signal originating from the tumor.
Recent reports have suggested that the administration of double-stranded siRNA might induce an innate immune response, leading to nonspecific antitumor effects. 48,49 Hence, IFN-α, IFN-γ, IL-12, and IL-6 were tested. The results demonstrated that neither the RPM/siRNA nor blank RPM NPs activated the innate immune response, indicating that specific gene knockdown was achieved with the RPM/siRNA.

Conclusion
We have demonstrated that RPM could self-assemble into NPs, and DLS showed that these NPs presented a narrow size distribution. TEM analyses revealed that the NPs possessed a spherical shape. In vitro assays indicated that the NPs showed good biocompatibility and negligible cytotoxicity. Due to the receptor-mediated pathway involved, the RPM/siRNA complexes exhibited effective, targeted cellular uptake and resulted in significant gene silencing in HUVECs. Moreover, no bodily toxicity or innate immune response was induced in tumor-bearing mice. Treatment with RPM/siRNA resulted in marked inhibition of tumor growth, effective gene knockdown, and antiangiogenic effects in the tumors. Collectively, RPM represents a novel delivery system for siRNAs and a promising tool for cancer therapy. The International Journal of Nanomedicine is an international, peerreviewed journal focusing on the application of nanotechnology in diagnostics, therapeutics, and drug delivery systems throughout the biomedical field. This journal is indexed on PubMed Central, MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine, Journal Citation Reports/Science Edition, EMBase, Scopus and the Elsevier Bibliographic databases. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.