| BackgroundIn recent years, cancer has been one of the fatal diseases that severely threaten human being’s health. Effective prevention of cancer has become a main concern for medical providers and researchers worldwide. Current conventional treatments for cancer include surgery, radiotherapy and chemotherapy, while the treatment effect is limited with adverse reaction and injury to human body. Recently, novel molecular targeted anti-tumor drugs have been applied as a main approach for tumor treatment with the characteristics of high specificity, less toxic and side effects and good tolerance.Epidermal growth factor receptor (EGFR), belonging to epidermal growth factor family, is a ubiquitously-expressed transmembrane receptor that possesses intrinsic protein tyrosine kinase activity. Previous study showed that abnormal expression of EGFR gene could activate genes related with tumor proliferation and differentiation and played a critical role in the occurrence and development of tumor. The EGFR tyrosine kinase inhibitors (EGFR-TKI) have become an important direction for the tumor-related drug development. Nowadays, drugs targeting EGFR receptors are mainly divided into three categories, including small-molecule targeted drugs, monoclonal antibodies and cancer vaccine, such as Cetuxima and Panitumumab. With the wide use of tumor-targeting drugs, severe adverse reaction and drug tolerance have been the challenges for the treatment of cancer. The development of novel tumor-targeting drug becomes more and more urgent.After searching the Clinical Trials.gov and International Clinical Trials Registry Platform (ICTRP), no clinical registration on siRNA drugs targeted EGFR was found. Hence, it’s valuable and innovative to develop siRNA drugs targeted EGFR by using modern biological technology.Previous study showed that αvβ3 receptor was high-expressed in cell surface of some malignant tumors, while barely expressed in normal cells at stable stage and tissues. Hence, cRGD-siEGFR can be synthesized based on the specific binding of cRGD and αvβ3 and entering into the cell through receptor mediation. It’s valuable and promising to develop novel molecular-targeted anti-tumor drugs that can target inside the tumor cell and silence EGFR expression specifically.In this study, cRGD peptide was covalently attached to the end of sense strand of specific silencing EGFR siRNA through low-molecular-weight PEG for the synthesis of cRGD-siEGFR. The effect of specific silencing EGFR, tumor targeting, anti-tumor growth, toxic and side effect and immune stimulation reaction were observed. Immunohistochemistry and TUNEL analysis were performed on tumor tissue slice from treated tumor-bearing mice to verify the inhibition of cRGD-siEGFR to tumor cell proliferation, induction to tumor cell apoptosis and the toxic and side effect on organs and tissues. This study also explored the feasibility and shortcomings for the using of cRGD-siEGFR as a novel molecular-targeted anti-tumor drug.Objectives1. To screen siRNA sequences with effective silencing EGFR mRNA expression and backbone modifications;2. To synthetize cRGD-siEGFR and analyze its structure and purity;3. To study the in vitro plasma stability of cRGD-siEGFR;4. To study the cell targeting, cytotoxicity and the function of silencing EGFR mRNA specifically of cRGD-siEGFR in vitro;5. To investigate tumor targeting and distribution of metabolites of cRGD-siRNA in tumor-bearing mice;6. To establish tumor-bearing mice models for the function investigations on anti-tumor growth of cRGD-siRNA and underlying mechanisms (gene silencing efficiency and tumor cells apoptosis);7. To perform animal studies on the immunogenicity and the effects on liver and kidney function of cRGD-siRNA.Methods1. EGFR siRNA sequence verification and backbone modificationsThe relative expression of EGFR mRNA was tested by RT-qPCR. Inorder to inprove the stability, reduce the immunogenicity and off-target effect, EGFR siRNA sequence was modified.2. Synthesis of cRGD-siEGFRTo prepare cRGD-siRNA molecules, cyclic RGD was covalently conjugated to the 5’-end of siRNA sense strand using thiol-maleamide linker. The molecular weight of cRGD-sense strand siRNA was characterized by Mass-spectrometric analysis. The purity of conjugated cRGD-siRNA was determined by HPLC.3. Plasma stability of cRGD-siEGFRFive microliter of 20 uM cRGD-siRNA was mixed with 5 ul of mouse serum and incubated at 37℃ for 0,12,24,36 and 48 h. Aliquotswere taken at each of the time points and subjected to electrophoresis in 1.2% non-denaturing agarose gels.4. Study on biological functions of cRGD-siEGFR in vitroSpecificity/cellular toxicity of cRGD-siRNA in vitro was assessed using CCK-8. To demonstrate ability of cRGD-siRNA to enter integrin αvβ3 expressing cells, we labeled molecules with Cy5 and monitored fluorescence patterns for both cell types (αvβ3 receptor 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 further 48 h before being examined by RT-qPCR. For western blot cells were cultured for a further 72 h. The uptake ability was determined by Flow cytometry. The cell proliferation was detected by CCK-8 and EDU. The apoptosis was examined by Flow cytometry.5. Tumor targeting of cRGD-siRNA and its distribution of metabolites in vivoBio-distribution of cRGD-conjugated vs. non-conjugated chemically stabilized siRNAs in vivo was examined by whole animal bioluminescence imaging using the I VIS Spectrum (Xenogen) system upon intravenous injection of the molecules into the tumor-bearing mice.1 nmole of Cy-5 labeled cRGD-siRNA (-0.7 mg/kg) was injected when tumor volumes reached 150 mm3. Intense fluorescent signal was detected 12h,24h,48h and 72h after injection in tumors of mice and ex vivo images of tumors and organs excised from mice after 72h injected with Cy5-labeled cRGD-siRNA. And Cy-5 labeled nake siRNA was chosen as the negative control.6. Tumor tissue targeting of cRGD-siRNA Nude mice (female,4-6 weeks,-20 g) were inoculated subcutaneously on the right back with 5×106 U87 MG or Hela cells. When tumor volume reached 150 mm3, the animals were randomized into different groups for treatment testing. Mice bearing U87 MG tumors were injected with cRGD-siEGFR-Cy5 and EGFR siRNA-Cy5, respectively. Mice bearing Hela tumors were injected with cRGD-siEGFR-Cy5 Animals were euthanized 24h after treatment and the tumors were excised and preserved in liquid nitrogen for further analysis.7. Biological activity of antitumor of cRGD-siRNANude mice (female,4-6 weeks,～20 g) were inoculated subcutaneously on the right back with 5×106 U87 MG cells (A549-luc). When tumor volume reached 150 mm3, the animals were randomized into different groups for treatment testing. Mice bearing U87 MG tumors were injected with cRGD-siEGFR 7 times continuous (48h interval). A:saline, B:cRGD-siNC (5 nmol/20g), C:cRGD-siVegfr2 (1.5 nmol/20g), D:cRGD-siEGFR (1.5 nmol/20g), E:cRGD-siVegfr2(1.5 nmol/20g)+ cRGD-siEGFR (1.5 nmol/20g), F:cRGD-siEGFR (5 nmol/20g). 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). Animals were euthanized 3 days 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 VEGFR2 in vivo were determined by real time reverse transcription PCR. Proteins were seperated from tumor tissues and analyzed by western blot for the detection of VEGFR2 protein expression level. The density of micro-vessels (CD31 positive) 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.8. Immunogenicity and liver and kidney toxicity of cRGD-siEGFRLevels of the cytokines IFN-a, IFN-y, IL-6 and IL-12 in the serum of treated mice were determined by ELISA. The statistical significances were measured by ANOVA.Results1. EGFR siRNA sequence verification and backbone modificationsAfter screening and verification, good silencing was found in EGFR siRNA sequence (sense strand:5’-CAA AGU GUG UAA CGG A AUA dTdT-3’; antisense strand:5’-UAU UCC GUU ACA CAC UUUG dTdT-3’) with the silence efficiency over 80%. The stability of EGFR siRNA was obviously improved without decreasing the silence efficiency by using backbone modifications that three bases on the both ends of sense strand and antisense strand were modified by methoxyl.2.Synthesis of cRGD-siEGFRLC-MS results showed that the molecular mass of synthetized cRGD-siEGFR was same with theoretical molecular mass. RP-HPLC results showed that the purity of cRGD-siEGFR reached 88.0%.3. Plasma stability of cRGD-siEGFRNaked EGFR siRNA had worse stability in plasma, mostly being degraded after 12 h and absolutely degraded after 24 h. siRNA had best stability by using the third backbone modifications method (three bases on the both ends of sense strand and antisense strand were modified by methoxyl), a few siRNA being not degraded after 36h; The most stable molecular structure was cRGD-siRNA, only few being degraded after 48 h.4. Study on biological functions of cRGD-siEGFR in vitro(1) CCK-8 experiment results showed that cRGD-siRNA was of very low toxicity. When the concentration of cRGD-siRNA reached 1500nM, the cell viability was still at 95.56%. When the concentration reached 2000nM, the cell viability was still at 91.92%.(2) Confocal microscopy was used to observe the specific targeting of cRGD-siEGFR to U87 MG. cRGD-siEGFR could deliver EGFR siRNA into cell well except naked siRNA. After αvβ3 receptor was specifically blocked by RAD, cRGD-siEGFR was failed to enter in cells, which suggested that cRGD-siEGFR could specifically target tumor cells with high-expressed αvβ3 receptor, such as U87 MG cell line.(3) RT-qPCR results showed that compared with the control group, EGFR mRNA in those groups transfected with 100 nM and 200nM cRGD-siEGFR had lower silence efficacy (97.45% and 84.90%, respectively), with no statistical significance (P=0.809 and P=0.073, respectively). Compared with the control group, EGFR mRNA in those groups transfected with 500nM,800nM, 1000nM and lipo 2000 /siRNA had decreased significantly (47.39%,21.93%,13.34% and 18.57%, respectively,P<0.01). In terms of the amount of transfection, the silence efficacy of cRGD-siEGFR was increased with the rising of concentration, but when reached 1000nM, the silence efficacy was higher than lipo 2000/siRNA group with statistical difference (P=0.034).(4) Western Blot results showed that compared with control group, EGFR protein expression in the naked siRNA group and cRGD-siNC group had no statistical differences (P> 0.05); EGFR protein expression was significantly lower in the cRGD-siEGFR group (26.47%) and lipo 2000/siRNA group (15.04%) than in the control groups (P< 0.01). Those results suggested that cRGD-siEGFR could effectively silence EGFR protein expression in U87 MG cell.(5) Flow cytometry results showed that naked siRNA barely enter into U87 MG cell, with the Cy5 positive rate 1.27% and fluorescence intensity 11.67, which was consistent with the results of confocal microscopy. Compared with naked siRNA-Cy5 group, U87 MG had better uptake ability to cRGD-siEGFR-cy5 and lipo2000/ siRNA-cy5, the positive uptake rate was 97.97%,98.68% and 98.58%, respectively and the fluorescence intensity was 347.00,1145,00 and 3133.67, respectively. The uptake ability was increased with the rising of the dose of administration. The uptake amount of cRGD-siEGFR-cy5 with the dose of 400nM was 4 times as much as group with 100nM dose. Compared with lipo2000/siRNA-cy5 group, U87 MG had lower uptake amount to cRGD-siEGFR-cy5, accounting for 35 per cent of that in lipo2000/ siRNA-cy5 group.(6) CCK-8 experiment results showed that cRGD-siEGFR with different concentration (400 nM,600 nM and 800 nM) could inhibit the cell proliferation of U87 MG at 48 h (77.11±10.19%,61.13±12.20% and 44.52±7.35%, respectively) and 72 h (68.60±9.23%,52.11±8.21% and 33.74±6.84%). Statistical significance was found as compared with the control group (P<0.01). The results of EDU experiment were consistent with those in CCK-8 experiment.(7) Flow cytometry results showed cRGD-siEGFR with different concentration (400 nM,600 nM and 800 nM) could induce apoptosis of U87 MG cells with the apoptosis rate (26.59±5.87)%, (45.23±6.99)% and (55.28±7.49)%, respectively. Compared with the control and negative groups, there was significant difference(P< 0.05).5. Tumor targeting of cRGD-siRNA and its distribution of metabolites in vivoThe results of in vivo imaging of nude mice showed that cRGD-siEGFR could specifically target tumor location after the tail intravenous injection of cRGD-siEGFR (1nmol, Cy5-labeled) to mice. At 12h and 24h, a large number of Cy5 fluorescent expressions was found in tumor location, and also observed in kidney tissue, few in liver tissue. However, when injected with naked siRNA (Cy5-labeled), no Cy5 fluorescent expression was found in tumor location at 12h and 24h, high expression was found in kidney and liver tissues. After dissection, consistent results were found as compared the images of organs and tissues in vivo of naked mice, both of the results showing that cRGD-siEGFR could specifically target tumor tissue. Further observation showed that Cy5 fluorescent expression in tumor and kidney was decreased gradually at 24h, no fluorescent expression found in liver at 48h; at 72h, no distribution of naked siRNA (Cy5-labeled) was seen in tumor. After dissection, consistent results were found as compared the images of organs and tissues of naked mice in vivo, showing that cRGD-siEGFR had good stability. High Cy5 fluorescent expression was found in tumor at 48h and only few was found at 72h, showing that the main metabolism was kidney, and the second one was liver.6. Tumor tissue targeting of cRGD-siRNAAfter the tail intravenous injection of naked siRNA and cRGD-siEGFR (1 nmol), cRGD-siEGFR could permeate into tumorous stroma via tumor vessel, while naked siRNA was failed to enter in tumor. In normal tissue without expression of αvβ3 receptor and Hela tumor tissue, cRGD-siEGFR failed to reach tumor location. Further in vivo study on naked mice confirmed that cRGD-siEGFR could specifically target tumor with high-expressed αvβ3 receptor and permeate into tumorous stroma.7. Biological activity of antitumor of cRGD-siRNAThe mice were administrated continuously 7 times via tail intravenous injection, with the interval of 48h. The tumor volume and weight were measured before injection. On the third day after the last administration, the tumor volume and weight were measured again. The tumor was removed after the mice were killed, being washed with normal saline, absorbed the water, taking photos and being weight. Using statistical methods analyzed the changes of tumor volume and weight of nude mice, the results showed that only high-dose cRGD-siEGFR (5 nmol/20g) could inhibit tumor growth, and low-dose cRGD-siVegfr2 (1.5 nmol/20g) and cRGD-siEGFR (1.5nmol/20g) had the tendency of inhibition, but lack of significant difference.RT-qPCR and Western Blot results showed that cRGD-siEGFR (1.5nmol) group, cRGD-siVegfr2 (1.5nmol) combined with group cRGD-siEGFR (1.5nmol) group and cRGD-siEGFR (5nmol) group could obviously decreased EGFR mRNA and protein expressions in tumor tissues, and cRGD-siEGFR (5nmol) was the strongest with 50% silencing efficiency. Tunel staining results demonstrated that high-dose cRGD-siEGFR (5 nmol/20g) could obviously induce apoptosis of tumor cells, and low-dose cRGD-siVegfr2 (1.5 nmol/20g) and cRGD-siEGFR (1.5nmol/20g) had some inhibition effects.8. Immunogenicity and liver and kidney toxicity of cRGD-siEGFRElisa and blood biochemical test results showed that no significant difference in IFN-a, IFN-γ, IL-6, IL-12 creatinine and alanine aminotransferase was found between the cRGD-siRNA group and control group (P> 0.05), suggesting low immunogenicity and toxicity of cRGD-siEGFR. Pathological section results further confirmed the low toxicity of cRGD-siRNA. Even though the mice were administrated 7 times with high dose (5nmol), no obvious toxicity to organs was found.ConclusionThe cRGD peptide can specifically direct conjugated siRNA molecules inside the αvβ3-expressing cells, resulting in the effective knockdown of selected. We then demonstrated that cRGD-siRNA conjugates biodistribute in mice to the engrafted tumors after systemic intravenous delivery in vivo. Delivery of the cRGD-siRNAs to the tumors resulted in the specific and effective down-regulation of targeted genes. Finally, we showed that downregulation of EGFR in mouse tumors resulted in slower tumor growth. Taken together, the obtained results provide a strong proof-of-concept for the potential use of cRGD conjugation strategy in targeting siRNA molecules to solid tumors and, in particular, for the use of cRGD-siEGFR as anticancer therapeutic. |
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