Self-assembled Nanoparticle Complexes RPM/siRNA With C(RGDfk) Peptide For The Tumor Targeting Therapy

Introduction:Double-stranded, small interfering RNA (siRNA)-induced gene silencing through the inhibition of specific mRNA translation, also known as RNA interference (RNAi), has been utilized for quite some time. SiRNA has attracted intense interest due to its promising therapeutic effects in various diseases, such as neuronal diseases, infectious diseases and various cancers. 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 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 represent a great challenge and is the subject of intense research. Many non-viral 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 (SNALPs), cationic polymers and peptides have been employed to protect siRNAs from undesirable degradation during the transfection process. Additionally, these carriers have been modified with different targeting ligands, such as the Arg-Gly-Asp (RGD) peptide, folic acid, transferring and antibodies, to increase their targeting ability. The RGD peptide and structurally related compounds are the best studied ligands that belong to the integrin ligand group. Because these ligands specifically bind to the integrin receptor, which is over-expressed in the endothelial cells of the tumor neovasculature, when applied in vivo, a PEG-MAL hydrophilically modified, specific integrin-α vβ3 receptor-targeted small cyclopeptide (RPM) 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 has been widely applied in anti-tumor studies. 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. 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-α vβ3 receptor, which is overexpressed in new vessels, via the ligand c(RGDfk) and gene pathway selectivity via the siRNA oligonucleotide. To our knowledge, this is the first study to show that the small cyclopeptide RPM has the ability to self-assemble and can effectively deliver siRNA to targeted tissue sites.Objectives:1. To prepare the RPM nanoparticles and RPM/siRNA complexes, then study their characterizations.2. To investigate specifically cell targeting, the distribution, the cytotoxicity and silencing of RPM/siRNA complexes in vitro.3. To investigate the anti-angiogenesis of RPM/siRNA complexes in zebrafish.4. To investigate the tumor targeting and bio-distribution of RPM/siRNA-Cy5 complexes in vivo.5. To investigate the anti-tumor effect and the action mechanism of RPM/siRNA complexes in the A549-luc+NSCLC tumor xenograft mouse model. And investigate the toxicity after long-term treatment with RPM/siRNA complexes.Methods:1. Preparation of RPM/siRNA complexes, and their characterizationsRPM was produced and purified by PEPTIDES INTERNATIONAL (Louisville, KY, USA), RPM was added to the siRNA solution for incubation to get the RPM/siRNA complexes. The morphology of the nanoparticles and the nanoparticle complexes were observed using a transmission electron microscope. The particle size and zeta potential of the RPM/siRNA complexes were determined using a ZetaPals z-potential and particle size analyzer. The secondary structures of the complexes were detected based on circular dichroism (CD) spectra. At the same time the gel retardation and the serum stability was studied.2. The functions of RPM/siRNA complexes in vitroConfocal laser scanning microscope was used to observe how RPM/SiRNA-Cy5 distributed in different cells. The FACS analysis was used to quantitatively examine the cellular uptake level of RPM/siRNA-cy5, and to get the best proportion of RPM and siRNA. Cytotoxicity was assessed using the CCK-8 assay according to the manufacturer’s instructions. The cellular levels of VEGFR2 mRNA were measured after 48 h, and VEGFR2 protein levels were measured after 72 h through quantitative reverse transcription-PCR and western blot analyses, respectively. The statistical significances were measured by ANOVA. LSD or Dunnett T3 test was chosen depend on homogeneity of variances or for multiple comparison.3. In vivo angiogenesis assay in zebrafishTg{flk-1:GFP) and Wild-type Zebrafish were used for in vivo model systems. Normotrophic Zebrafish embryos were chosen at about 10 h post-fertilization (hpf) under the dissecting microscope and 20 embryos/well were seeded in 6-well plate. RPM/siRNA complexes (2 nl), RPM/ControlsiRNA complexes (2 nl), zVEGFR2 (2 nl) or ddH2O (2nl) were delivered into 10 hpf zebrafish embryos through microinjection. After incubated at 28.5℃ for 72 h, the blood vessel formation and associated fluorescence intensity were examined via confocal microscopy. The relative fluorescent intensity was then applied to confirm the specific anti-angiogenic activity of RPM/siRNA complexes in zebrafish. Confocal laser microscopy images were processed to gray images using Adobe Photoshop 7.0 software. Then the gray images were analyzed using Image J software. Final relative fluorescent intensities were expressed as percentage of the ddH2O treated control group.4. In vivo localization of nanoparticle complexes RPM/siRNA-Cy5After the administration of RPM/SiRNA-Cy5 nanoparticle complexes, or naked SiRNA-Cy5, the in vivo distribution of the SiRNA-Cy5 in the tumor-bearing mice was examined by whole animal imaging using the IVIS Spectrum (Xenogen) system. When the tumor volume reached 50 mm3, the tumor was detected by intraperitoneal injection of luciferin (Promega) at a dose of 150 mg/kg per mouse, then a single dose of RPM/SiRNA-Cy5 nanoparticle complexes (contain lnmol SiRNA-Cy5), or naked SiRNA-Cy5 (1 nmol) was injected via the tail vein. The subsequent biodistribution was monitored at different time point after administration using the IVIS Spectrum Imaging System at the appropriate wavelength (SiRNA-Cy5:λ ex=640nm, λ em =680 nm). The mice were sacrificed after the last time point, and the tumors and major organs were excised and imaged.5. The investigation of the anti-tumor effect and the action mechanism of RPM/ siRNA complexes in the A549-luc+NSCLC tumor xenograft mouse modelLuciferase-A549 cells (1×106) were subcutaneously injected into the right shoulder of the mice. Tumor volumes were measured with a caliper, and the tumor volume was calculated using the following formula:Volume=1/2×length× width2. When the tumor volume reached 50 mm3, the animals were randomly divided into three different groups for testing. Saline, RPM/ControlsiRNA and RPM/siVEGFR2 were administered via intravenous injection in 100μl volumes (RPM:60μl,5mg/ml; siRNAs:40μl,50μM) once every 3 days at a dose of 2 nmol of siRNAs per mouse, and the treatment was repeated 6 times. Tumor volume and mouse weight were monitored prior to injection. Tumor luminescence was detected on Day 0 and Day 20 to monitor the development of the tumors using the IVIS Spectrum Imaging System (Xenogen) following intraperitoneal injection of luciferin (Promega) at a dose of 150 mg/kg per mouse. The curves are plotted as the mean tumor volume±s.d. The statistical significances were measured by ANOVA. At 6 h and 24 h after administrated with RPM, RPM/SiVEGFR2 and Saline respectively, serums were collected and assayed to detect mouse IFN-a, IFN-y, IL-12 and IL-6 using a quantitative enzyme-linked immunosorbent assay (ELISA) kit (Bender Med Systems) according to the manufacturer’s instructions. After the last injection, serum were collected and isolated for the measurements of ALT (alanine aminotransferase) and creatinine using automated analyzer (Aeroset) according to the manufacturer’s instructions. At the same time, the tumors were excised and preserved in liquid nitrogen for the extraction of RNA and protein. The density of micro-vessels (CD31 positive) in tumors was analyzed by immuno-histochemical. The statistical significances were measured by ANOVA.Results:1. The characterizations of RPM/siRNA complexesThe RPM/siRNA complexes were prepared via a easy way, and the mechanism of nanoparticle assembly is presented in Figl-1. The characterizations of RPM/ siRNA complexes were studied:The TEM images show that the RPM/siRNA complexes were round in shape, and their diameters were less than 100 nm. The particle size of the RPM/siRNA complexes ranged from 105.7 to 141.8 nm, and exhibited a narrow size distribution and possessed a negative charge. Furthermore, the CD shows a mixed secondary structure of the NPs including β-sheet and random coil, with the P-sheet accounting for the majority. The serum stability test demonstrated that the naked siRNA was nearly completely degraded after 4 h of incubation in the serum. In contrast, siRNA was still present in the RPM/siRNA group after 12 h.2. The functions of RPM/siRNA complexes in vitro(1) As shown in the result of confocal laser scanning, SiRNA-Cy5s were distributed in the cytoplasm and around the nuclei in both the HUVECs and A549 cells after incubation with RPM/SiRNA-Cy5 for 6 h, while the HUVECs that treated with anti-integrinavβ3 antibody for 30 min before the incubation of RPM/SiRNA-Cy5 or treated with RAPM/SiRNA-Cy5 showed extremely weak fluorescent signal for SiRNA-Cy5 in the cytoplasm. The result of FACS analysis demonstrated that when the volume ratio reached 1.5:1, the fluorescence intensity of SiRNA-Cy5 in the cells was the strongest.(2) There was no difference in cell viability between the RPM NPs, 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.(3) RT-qPCR analysis demonstrated that transfection of siRNAs using RPM and a cationic lipid reagent resulted in comparable (-38% for RPM/siRNA vs.-39% for Lipo2000/siRNA) gene knockdown in HUVECs following treatment for 48 h, and no significant difference was observed between other groups. Total protein was collected for western blot analysis after 72 h, and VEGFR2 protein expression was markedly reduced in the RPM/siRNA and Lipo2000/siRNA groups, by approximately 29% and 36% in the RPM/siRNA and Lipo2000/siRNA groups, respectively.3. The anti-angiogenesis effect of RPM/siRNA complexes in zebrafishAt 72 hpf, we used the confocal laser scanning to see the anti-angiogenesis effect of RPM/siRNA complexes 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). Zebrafish embryos expressed integrin αvβ3 at 48 hpf. The major vessels, including the dorsal aorta and the ventral veins, are fully developed by 24 hpf. Angiogenic vessels, including the ISVs, develop between 24 and 72 hpf. Therefore, we chose to initiate and terminate drug treatment at 72 hpf. Strong GFP fluorescence was observed in the ddH2O-treated group, while the embryos treated with the RPM/siRNA failed to completely form dorsal longitudinal anastomotic vessels (DLAVs) and intersegmental vessels (ISVs), and the value of relative fluorescence about 51.4%, significant difference was observed between those two groups (P< 0.05). And the fluorescence statistics for each group revealed that RPM/siRNA could have an anti-angiogenic effect.4. Tumor targeting and bio-distribution of RPM/siRNA (Cy5 labled) complexes in vivoA single dose of saline, RPM/SiRNA-Cy5, naked SiRNA-Cy5 was injected via the tail vein in 40μl volumes. The subsequent biodistribution was monitored at 10 min,0.5 h,3 h,6 h and 24 h after administration using the IVIS Spectrum Imaging System (Xenogen) at the appropriate wavelength (SiRNA-Cy5:λex=640 nm, λ em=680 nm). Intense fluorescence accumulated in the tumors after 0:5 h in the RPM/siRNA-Cy5 group, reflecting the presence of SiRNA-Cy5 in the blood pool, and this accumulation lasted for at least 24 h after injection. However, no fluorescence was detected in the tumors of mice that were injected with naked siRNA-Cy5. 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.5. Biological functions and anti-cancer mechanisms of RPM/siRNA complexes in vivo(1) Saline, RPM/ControlsiRNA and RPM/siVEGFR2 were administered via intravenous injection in 100μl volumes (RPM:60μl,5mg/ml; siRNAs:40μl,50μM) once every 3 days at a dose of 2 nmol of siRNAs per mouse, and the treatment was repeated 6 times. Twenty days after the first injection, there was obvious tumor growth in the saline- and RPM/ControlsiRNA-treated mice, and no significant difference was observed between these two groups, which indicated that RPM/ControlsiRNA did not have specific anti-tumor effects. Compared to the saline-and RPM/ControlsiRNA-treated mice, the tumor volume was effectively reduced in the mice treated with RPM/siRNA (*p<0.05,**p<0.01). Distinct attenuation of the tumor bioluminescence intensity was observed only in the RPM/siRNA group. Moreover, there was no difference in the body weights of the mice in the three groups.(2) And a highly significant difference was observed in the levels of VEGFR2 mRNA between the tumors of the RPM/siRNA (siVEGFR2) group (-23%) and the other groups (P=0.000<0.05). Furthermore, the result of western blot showed that the VEGFR2 protein level was markedly reduced in the RPM/siRNA group (-43%), which has a significant difference with other groups.(3) In addition, mmunohistochemistry analysis showed that the density of micro-vessels (CD31 positive) in RPM/siRNA (VEGFR2) complexes treated tumors was clearly lower comparing to that in tumors of animals treated with RPM/ControlsiRNA and saline, which might result from the reduction of VEGFR2 gene. To determine whether the anti-tumor effects of the RPM/siRNA were due to immune effects, the levels of EFN-a, IFN-γ, IL-12 and IL-6 in the serum were determined at 6 and 24 h after administration. The results showed that there were no differences in the cytokine or interferon responses of the groups (P>0.05). In addition, unchanged ALT and creatinine serum levels indicated that RPM/siRNA is well tolerated, producing no significant renal and liver toxicity (P>0.05). Finally, no morbidity or weight loss in mice was observed following the 20 day treatment with RPM/siRNA (VEGFR2) complexes and corresponding controls.Conclusion:1. RPM and siRNA can self-assembled into nanopartical complexes via a easy way, and these RPM/siRNA complexes were serum stable.2. The imagines of confocal laser scanning microscopy showed that ability of RPM/siRNA complexes to enter integrin αvβ3 expressing cells, demonstrating the receptor mediated nature and specificity of the delivery. The RT-qPCR and Western Blot data demonstrate the ability of RPM/siRNA complexes to generate targeted gene knockdown efficiently.3. There is no significantly cellular toxicity of RPM/siRNA in vitro4. RPM/siRNA complexes can significantly inhibited angiogenesis in zebrafish by integrin αvβ3 receptor-mediated way.5. After the administration of RPM/siRNA complexes via the tail vein, RPM/siRNA complexes can target tumor specifically in vivo. RPM/siRNA complexes can lead to efficient gene silencing. Future more, can inhibit angiogenesis in tumor.6. RPM/siRNA complexes can significantly inhibit tumor growth, the tumor growth inhibition was mediated by the reduction of VEGFR2 gene expression and the inhibition of angiogenesis.7. There was no significantly immunogenicity and renal and liver toxicity after short time treatment with RPM/siRNA complexes and indicated well tolerated.