Synthesis, Tumor-targeting And Anti-tumor Mechanism Studies Of CRGD-conjugated SiRNA

Introduction:RNAi technology, with siRNAs as its key component, is based on natural intracellular mechanisms and holds great potential as novel therapeutic strategy for a broad range of diseases-from genetic disorders to cancer and viral infection. To become widely applied in clinics, the technology has to address several challenges, including stability of siRNA molecules in vivo, bio-distribution to intended tissues/organs, and potential non-specific (e.g. off-target and immunostimulatory) effects. While stability and specificity of siRNAs in vivo has been achieved using various chemical modifications, in vivo delivery of therapeutic molecules remains the biggest obstacle, due to their large size (-13kDa) and strong negative charge. Numerous approaches have been explored with varying success, including assembly of siRNAs into nanoparticles with cationic polymers and lipids, integration into exosomes, complexation with peptide containing antibodies and nucleic acid binding domains, as well as siRNA conjugation with peptide transduction domain, cell-specific aptamer, receptor-specific ligands, such as cholesterol, alpha-tocopherol, lauric acid, receptor-specific agonist, and cell growth factor (or its peptide analogue).In the early years of developing RNAi therapeutics, a challenge to the promise of RNAi as a therapeutic modality was delivery-i.e., getting the siRNA into the right body organs and cells so that it could trigger the RNAi mechanism. Therefore, selecting the appropriate drug delivery system is the most important in application of siRNA molecular in clinical treatment, which can improve the targeting and transfection efficiency of siRNA molecular, as well as maximize the efficacy and reduce side effects.Integrin αvβ3plays important role in angiogenesis and tumor metastasis, and its expression is significantly up-regulated in tumor blood vessels, as well as in invasive tumor cells of many cancer types (but not in cells of quiescent endothelium and normal tissues). Well characterized high affinity of cancer-related integrin avβ3to arginine-glycine-aspartate (RGD) peptide has prompted the use of RGD as ligand for tumor targeting liposomes. In a recent study, siRNA conjugated to cyclic RGD (cRGD) was demonstrated to selectively enter cells expressing αvβ3integrin (ligand’s receptor). We explored further the potentials of cRGD conjugated siRNAs through additional cell culture and whole animal studies.Hypoxia, activation of oncogene and induction of metabolic enzyme plays an important role in the early formation of tumors, which promote the dormant period broken. Then a series of angiogenic factors are increased significantly and angiogenesis. The formation of the new functional microvascular derived from the preexisting blood vessel is called angiogenesis, which is induced by the tumor, and subsequently satisfied all the nutrients and oxygen that tumor needs for growth and metastasis. Both the endothelial cells and the tumor cells are responsible for this pathological process, in which lots of pro-angiogenic factors and the endogenous angiogenesis inhibitors secreted by them are playing important roles. Once the scale loses its balance, the tumor swells. Without the nutrition, blood and oxygen supplied by microvascular, tumor growth stopped at a diameter of about1-2mm. As a test target for new cRGD-siRNA constructs we chose VEGFR2gene. Vascular endothelial growth factors (VEGFs) and corresponding receptors (VEGFRs) participate in regulation of blood vessel development from precursor cells during early embryogenesis and in formation of new blood vessels from pre-existing vessels at later stages. In solid tumors, VEGF is mainly produced by cancer cells, and binding of VEGF to VEGFR2activates multiple cellular pathways important for tumor angiogenesis. Therefore, effective delivery of functional VEGFR2cRGD-siRNAs was expected to inhibit angiogenesis and, consequently, progression of tumors in vivo.In our study, we developed a novel tumor targeting delivery systerm for efficient receptor-mediated siRNA delivery with independent intellectual property rights, simple process and easy industrialization:cRGD-siRNA molecule, which can mediate siRNA into tumor vascular endothelial cells and most tumor cells via systemic delivery, and play the biological functions of RNAi.Objectives:1. To synthesize the cRGD-siRNA conjugate molecule, and identify the structure of purified cRGD-siRNA molecules.2. To investigate the cytotoxicity, specifically cell targeting and silencing of cRGD-siRNA in vitro3. To investigate the anti-angiogenesis of cRGD-siRNA in zebrafish4. To investigate the bio-distribution of cRGD-siRNA (Cy5labled) in vivo5. To investigate the inhibition of tumor growth via RNAi of cRGD-siRNA in the A549-luc+NSCLC tumor xenograft mouse model. And investigate the anti-cancer action mechanism, as well as the toxicity after long-term treatment with cRGD-siRNA.Methods:1. To prepare cRGD-siRNA molecules, cyclic RGD was covalently conjugated to the3’-end of siRNA sense strand using thiol-maleamide linker. The purity of conjugated cRGD-siRNA was determined by HPLC. The molecular weight of cRGD-sense strand siRNA was characterized by Mass-spectrometric analysis.2. Specificity/cellular toxicity of cRGD-siRNA in vitro was assessed using CCK-8. To demonstrate ability of cRGD-siRNA to enter integrin αvβ3expressing cells, we labeled molecules with Cy5and monitored fluorescence patterns for both cell types αvβ3receptor positive and negative) using confocal laser scanning microscopy. We tested the ability of cRGD-siRNAs to down-regulate gene expression in the absence of a transfection reagent invitro. Cells were cultured under standard conditions for a further48h before being examined by RT-qPCR. For western blot cells were cultured for a further72h. The statistical significances were measured by ANOVA. LSD or Dunnett T3test was chosen depend on homogeneity of variances or not for multiple comparison.3. Wild-type and Tg (flkl; eGFP) Zebrafish were used for in vivo model systems. Normotrophic Zebrafish embryos were chosen at about5h post-fertilization (hpf) under the dissecting microscope and20embryos/well were seeded in6-well plate. cRGD-siRNA (zVEGFR2,100μM,2nl), zVEGFR2(100μM,2nl), cRGD(2mg/ml,2nl) or ddH2O (2nl) were delivered into5hpf zebrafish embryos through microinjection. After incubated at28.5℃for24h blood-vessels were detected using Stereo Fluorescence Microscope and for72h were detected using confocal microscopy. The relative fluorescent intensity was then applied to confirm the specific anti-angiogenic activity of cRGD-siRNA in vivo. Z-stack projection of confocal laser microscopy images was processed to gray images using Adobe Photoshop7.0software. Then the gray images were scanned using Image J software, and the resulting values reflected fluorescent intensity of blood vessels in Tg (flkl; eGFP) Zebrafish. Final relative fluorescent intensities were expressed as percentage of the ddH2O treated control group.4. Bio-distribution of cRGD-conjugated vs. non-conjugated chemically stabilized siRNAs in vivo was examined by whole animal bioluminescence imaging using the IVIS Spectrum (Xenogen) system upon intravenous injection of the molecules into the tumor-bearing mice. The easily visualizable tumors originated from luciferase-expressing A549-luc cells injected prior to the main experiment, and1nmole of Cy-5labeled cRGD-siRNA (-0.7mg/kg) was injected when tumor volumes reached40-50mm3. Intense fluorescent signal was detected30min and24hours after injection in tumors of mice and ex vivo images of tumors and organs excised from mice after24h injected with Cy5-labeled cRGD-siRNA. And Cy-5labeled cRAD-siRNA was chosen as the negative control.5. BALB/c nude mice (female,4-6weeks,-20g) were inoculated subcutaneously on the right back with5×106A549luciferase-expressing cells (A549-luc). When tumor volume reached40-60mm, the animals were randomized into different groups for treatment testing. Mice bearing A549-luc tumors were treated with1nM cRGD-siRNAl (～-0.753mg/kg; n=8),1nM cRGD-siRNA2(-0.753mg/kg; n=5),1nM cRAD-siRNA2(-0.753mg/kg; n=5), cRGD alone (-0.045mg/kg; n=5),1nM cRGD-sip-actin (-0.753mg/kg; n=5), cRGD-control siRNA (-0.753mg/kg; n=5) or saline (n=10) by intravenous injections in150μl volumes every two days for a total of six injections. Tumor volumes were measured with a caliper the day before injection and calculated using the formula:Volume=1/2×length×(width)2, where length represented the longest tumor diameter and width represented the shorted tumor diameter. The growth curves were plotted as the mean tumor volume±SD (standard deviation). Luciferase signal was measured by whole animal bioluminescence imaging at indicated time points. Data normalized to saline treated animals. Animals were euthanized2days after the last treatment and the tumors were excised and preserved in liquid nitrogen for further analysis. Total RNA was isolated from tumor tissues and the mRNA expression levels of VEGFR2in vivo were determined by real time reverse transcription PCR. Proteins were seperated from tumor tissues and analyzed by western blot for the detection of VEGFR2protein expression level. Levels of the cytokines IFN-a, IFN-y, IL-6and IL-12in the serum of treated mice were determined by ELISA. The density of micro-vessels (CD31positive) in tumors was analyzed by immuno-histochemical. Tissue sections were processed for TUNEL analysis using the In situ cell death detection kit-POD (Roche) as a measure of apoptosis. The statistical significances were measured by ANOVA.Results:1. Identify the structure of purified cRGD-siRNA moleculesIn our study, we obtained cRGD-siRNAl (mouse VEGFR2), cRGD-siRNA2(mouse VEGFR2), cRAD-siRNA (mouse VEGFR2), cRGD-control siRNA (scrambled sequence for control), cRGD-siRNAl (human VEGFR2), cRGD-siRNA2(human VEGFR2), cRAD-siRNA2(human VEGFR2). Schematic diagram of cRGD-siRNA conjugate molecule was shown in Figure1A. The purity of conjugated cRGD-siRNA was determined as above90%by HPLC. The molecular weight of cRGD-sense strand siRNA was characterized as of7975.3(calculated) and7977.9(determined) by Mass-spectrometric analysis. Resolved in20%SDS-PAGE, cRGD-siRNA was showed retarded shift than that of non-conjugated one.2. Functions of cRGD-siRNA in vitro(1) Following24hour incubation at37℃, survival rate of cells treated with targeting cRGD-siRNA and control siRNA (cRGD-NC-siRNA) were96.5%and96.0%respectively, compared to75.2%(P=0.000)of cells treated with conventional cationic liposome (Lipofectamine2000)/siRNA complex(2) Most HUVEC cells treated with Cy5-cRGD-siRNA produced strong cytoplasmic fluorescence, and no signal was detected when cells were treated with integrin av(33specific antibody or cRGD prior to incubation with Cy5-cRGD-siRNA. However, Cy5-labled cRGD-siRNA was taken up by HUVEC cells seemed unaffected by pre-blocked cRAD. In addition, Cy5-labled cRAD-siRNA can’t enter into HUVEC cells.(3) qPCR analysis demonstrated that unassisted (no transfection reagent used) delivery of cRGD-siRNA and transfection of siRNA using cationic lipid reagent resulted in comparable gene silence (80%for cRGD-siRNA vs.75%for siRNA/Lipofectamine2000) in HUVEC cells, P=0.022. On the protein level, cRGD-siRNAl yielded80%efficient knockdown of VEGFR2in HUVEC cells vs.70%achieved by siRNA1/Lipofectamine2000, P=0.522. And we also had the same pharmacological effects for cRGD-siRNA2, about66%(P=0.000) target knockdown at the mRNA (qPCR) and almost75%(P=0.009) at the protein (Western blotting) levels.3. Anti-angiogenesis of cRGD-siRNA in zebrafishZebrafish embryos expressed integrin αvβ3at48hpf. The major vessels, including the dorsal aorta and the ventral veins, are fully developed by24hpf. Angiogenic vessels, including the ISVs, develop between24and72hpf. Therefore, we chose these two time points to initiate and terminate drug treatment.24hpf after treatment, no significant difference of the trunk vascular development between cRGD-siRNA and siVEGFR2(zebrafish) injected embryos was observed by Stereo Fluorescence Microscope. However, the only impaired angiogenesis in cRGD-siRNA injected embryos at72hpf was found, the value of relative fluorescence about48.8% (P=0.000). In the course of observation (24-72hpf), the zebrafish develops rapidly from an embryo within chorioallantoic membrane to a larva. If the general development was blocked, it will result in embryo death or severe developmental defects such as trunk mal-formation and loss of posterial structure.4. Bio-distribution of cRGD-siRNA (Cy5labled) in vivoIntense fluorescent signal was detected30min after injection and lasted for at least24hours in tumors of mice. No signal was detected in mice injected with non-cRGD conjugated Cy-5labeled siRNA or Cy-5labeled cRAD-siRNA. Ex vivo images of tumors and organs excised from mice injected with Cy5-labeled siRNAs confirmed the initial whole animal imaging observations, and the significant enhancement of fluorescent signal coming from tumors of mice injected with cRGD-conjugated vs. non-conjugated molecules and cRAD-siRNA.5. Biological functions and anti-cancer mechanisms of cRGD-siRNA in vivoTreatments were made every two days for a total of six times, and subsequent tests and measurements were conducted at various time points. Analysis of the excised tumor samples two days upon the last injection (day17) revealed about50.9%(P=0.000) target knockdown at the mRNA (qPCR) and almost57.8%(P=0.000) at the protein (Western blotting) levels for cRGD-siRNA1. VEGFR2knockdown, in its turn, resulted in stalled progression of tumor growth (P=0.000) and>90%(P=0.000) reduction of tumor originating luciferase signal. And we also had the same pharmacological effects for cRGD-siRNA2, about45%(P=0.000) target knockdown at the mRNA (qPCR) and almost65%(P=0.000) at the protein (Western blotting) levels, as well as in reduction of tumor volume (P=0.000) and about70%reduction of tumor originating luciferase signal (P=0.005). The results further proved that gene silencing efficiency is correlated with siRNA sequence-specific. The TUNEL assay demonstrated significantly higher number of TUNEL-positive nuclei in tumors of cRGD-siRNA treated mice than in animals treated with cRAD-siRNA and saline. In addition, immuno-histochemical analysis showed that the number of micro-vessels (CD31positive) in VEGFR2cRGD-siRNA treated tumors was significantly reduced comparing to that in tumors of animals treated with cRAD-siRNA and saline, which might result from reduction of VEGFR2. No significant difference in the levels of both cytokines was observed between the test and control treated animals at6hours and24hours upon initial treatment. In addition, unchanged ALT and creatinine serum levels indicated that cRGD-siRNA is well tolerated (P=0.890, P=0.930), producing no significant renal and liver toxicity. Finally, no morbidity or weight loss in mice was observed following the16day treatment with cRGD-siRNA and corresponding controlsConclusion:1. Cyclic RGD was covalently conjugated to the3’-end of siRNA sense strand usingthiol-maleamide linker. We have succeeded in making cRGD-siRNA molecule.2. The result of CCK-8indicated that superior specificity of cRGD-siRNA non-invasive treatment.3. The imagines of confocal laser scanning microscopy showed that ability of cRGD-siRNA to enter integrin αvβ3expressing cells, demonstrating the receptor mediated nature and specificity of the delivery. The RT-qPCR and Western Blot data demonstrate the ability of siRNA conjugated to cRGD to enter integrin αvβ3expressing cells and to generate targeted gene knockdown as efficiently as with assistance of a conventional transfection reagent.4. Our study demonstrated that cRGD-siRNA, targeting VEGFR2, inhibited angiogenesis and significantly knockdown the target gene by integrin αvβ3receptor-mediated in zebrafish.5. The introduction of targeting RGD ligand plays a major role in accumulating the siRNA to the tumor.6. Covalently conjugated cRGD-siRNA molecules engineered here showed target ability to the specific tissue and gene silence in vivo. And the tumor growth inhibition was mediated by the reduction of VEGFR2protein expression directly or indirectly. The anti-angiogenesis may attribute to the specific uptake of cRGD-siVEGFR2mediated by αvβ3receptor overexpressed on the new vascular endothelial cells and tumor cells. 7. There was no immunogenicity and renal and liver toxicity after short time treatment with cRGD-siRNA and indicated well tolerated.8. cRGD-siRNA has the potential for use in targeted cancer therapy.