Epidermal Growth Factor Receptor-Mediated Targeting Gene Therapy Delivery System Of Breast Cancer

Besides surgery,radiotherapy and chemotherapy,gene therapy is potentialto be another way for cancer therapy.It may play an important role in preventing cancer from recurrence and metastasis.However,the main issueremains to be resolved is lack of an efficient targeting gene transfer system.Therefore,it is very urgent to develop an efficient targeting gene deliveryvector.Since overexpression of EGFR has been frequently found in a widespectrum of human tumors of epithelial origin,we have attempted to construct efficient EGFR-mediated gene delivery vectors.It will provide new technologies and practical guidance for targeting cancer gene therapy.Albumin nano-carrier is of high efficiency,stability and non-toxic. Albumin nano-carrier mediated by the epidermal growth factor receptor (EGFR) increased the intake and the targeting of cancer cells to drugs, antibodies,genes carried by the EGF coupled nano-carrier.This study was designed to prepare the nano-carrier of high stability and high targeting loading target gene to achieve better targeting in the diagnosis and treatment of cancer.Epidermal growth factor receptor(EGFR) involved in activation of a series of complicated cell signaling pathway,and widely expressed in the epithelial,stromal and nerve tissue.There was one or more of the EGFR family receptor over expression and(or) mutations in many malignant tumors.The expression rate of the over expression and(or) mutations in various tumors was different,and it was closely related with the tumor's differentiation,malignancy and infiltration,radiotherapy and chemotherapy sensitivity,drug resistanc and prognosis.EGFR family was considered an important anti-cancer therapy molecular target.Albumin is of safety,non-toxicity and non-immunogenicity.It has outstanding biocompatibility and can be biodegraded.The nanoparticles prepared with albumin may improve the stability of drugs,antibodies,gene, and the ability to enter cells.According to the characteristics of nano-carrier itself and the pathophysiology changes in the body when tumors occurred,then we could produced nano-carrier with the characteristics of targeting EGFR,which could achieve specific targeting role in the body environment.The overexpression of EGFR in some cancer cells are prevalent while its quantities in normal breast tissue were very low.So the epidermal growth factor(EGF) wsa conjunct to albumin nanoparticle and then targeting nanoparticle loading gene,drugs and protein was prepared Consequently,its advantages were as follows:Firstly,the combinantion of EGF with its receptors on breast carcinoma cells can increase the aggregation of drug-carrying nanoparticle on the membrane of carcinoma cells,then expands the concentration difference between in and our of cells thus help the carcinoma cells uptake and raise the ratio of target/non-target;Secondly,the specific binding of receptor to its ligand can promote the carcinoma cells phagecytosis of nanoparticle and increase the velocity and quantity entering carcinoma cells,thus raising its binding velocity and quantity of the load of drugs,antibodies,gene to the target.This study was designed to use EGF-BSA nano-carrier to increase the stability and targeting of the loading gene,and avoid the degradation by nucleic acid.Method:1.Preparation of BSA nano-carrier and EGF-BSA nano-carrier and identification of radionuclideBovine serum albumin nanoparticles(BSANP) were prepared by twice ultrasonic emulsification and volatilization technology.The particle diameter and their distribution were determined with laser particle size analyzer.Shapes were observed on transmission electron microscope. Iodogen was used for ¹³¹I labeling to BSANP.Purified by Sephadex G25 column chromatography,labeling yield,specific activity and radiochemical purity were tested by thin-layer chromatography.The radiochemical purity of ¹³¹I-BSANP at ambient temperature and incubated in fresh human serum at 37℃was determined for research on its stability.BSANP and EGF were connected by chemical technology. EGF-BSANP was separated and purified with Sephadex G50 column,and its particle diameter and distribution were determined.The linkage situation of EGF-BSANP was identified by SDS-PAGE.The linking quantity of EGF on EGF-BSANP was measured with ¹³¹â… -EGF.2.Distribution of 131I-EGF-BSA targeting nano-carrier in bearing breast cancer nude mouseComparing the distributions of ¹³¹â… -EGF-BSA targeting nano-carriers and BSA nano-carriers in bearing cancer SK-BR3 nude mouse respectively.3.Intake and retention of human breast cancer SK-BR3 cells to epidermal growth factor receptor targeting nanoparticles loading c-erbB2 antisense oligodeoxyribonucleotideC-erbB2 ASODN was labeled for ¹³¹â… by chloramine T method,and ASODN labeled for ¹³¹â… with radionuclide were entrapped with EGF targeting nanoparticle by packing.Measuring the the uptake and retention rates in human breast cancer cells SK-BR3 and the distribution in bearing human breast cancer nude mouse of ¹³¹â… -ASODN targeting nano-carriers and ¹³¹â… -ASODN in the various organs in vivo.Results:1.Preparation of BSA nano-carrier and EGF-BSA nano-carrier and identification of radionuclide1.1 Preparation of BSA nano-carrier and identification of radionuclideObserved on transmission electroscope,the nanoparticles were spherical and of uniformity.90%of the BSANP particle diameters determined with laser particle size analyzer were smaller than 38 nm with an average of 34 nm.Labeling yield of ¹³¹â… -BSANP with ¹³¹â… was 92.3%±3.9%and specific activity,(3.1±0.7) MBq/μg.The first radioactive peak after separated and purified with Sephadex G25 column was ¹³¹â… -BSANP.Radiochemical purity(average more than 80%)subtly declined after 24h.Incubated in fresh human serum at 37℃,radiochemical purity determined by paper chromatography at 4h(average more than 85%)was slightly lower than that at 1h.Labeling yield of BSANP with ¹³¹â… stored at-20℃save 1d later was (89.7±6.4)%;15d later,(86.2±5.7)%;30d later,(84.3±5.2)%;90d later,(81.2±5.5)%.1.2 Preparation and Identification of EGF-BSA nano-carrier SDS-PAGE displayed that the BSANP strip was near to a Marker 68kU while EGF-BSANP near to Marker 75kU.Linkage rate of EGF and BSANP in five groups with different quantities of EGF ranged from 92.3% to 98.9%.Stability at room temperature,there was no significant(P＞0.05)between the groups of 1h,6h,12h and 24h at the same time,while the radiochemical purity difference was significant (P＜0.05)with the group within 24 h.Regression analysis was done for the F value of time 30.46 in a linear radiation-related,and linear regression equation for Y=99.37-3.451X,R² =0.942,n=3.The results showed that, ¹³¹â… -EGF--BSANP had good stability in neutral solution at room temperature within 24h,and ¹³¹â… not falling.Serum stability,there was no significant(P＞0.05)between the groups of lh,6h,12h and 24h at the same time,while the radiochemical purity difference was significant (P＜0.05)with the group within 24 h.Regression analysis was done for the F value of time 87.98 in a linear radiation-related,and linear regression equation for Y= 99.86-4.156X,R² to 0.981,n= 3.The results showed that, ¹³¹â… -EGF-BSANP had good stability in serum,at room temperature within 24h,¹³¹â… not falling.4.Distribution of ¹³¹â… -EGF-BSA targeting nano-carrier in bearinbreast cancer nude mouseRadioactivity ratio of tumor/serum and tumor/muscle of the ¹³¹â… -EGF-BSA targeting nano-carrier gradually rose and up to its peak at 2h and then subtly declined.The ratio trend of tumor/serum and tumor/muscle of ¹³¹â… -BSA nano-carrier was similar to ¹³¹â… -EGF-BSA targeting nano-carrier,but the radioactivity ratio of tumor/serum and tumor/muscle of ¹³¹â… -BSA nano-carrier was relatively low in the same point of time.Analysis of variance was done to ¹³¹â… -EGF-BSA targeting nano-carriers,¹³¹â… -BSA nano-carriers and ¹³¹â… -BSA(non-nano):among the three drug groups,MS= 3.203,F=14.86,R²=0.725,p＜0.01,the tumor/serum ratio was of significant difference among the three drug groups.Analysis of variance was done to tumor/muscle ratio of three drug groups:MS=15.814,F=6.443,R²=0.403,p= 0.005,p＜0.01,the tumor/muscle ratio was of significant difference among the three drug groups.T-test was done to tumor/serum ratio between ¹³¹â… -EGF-BSA targeting nano-carrier and ¹³¹â… -BSA(non-nano):Levene's variance-test:F=1.336,p= 0.26,variance at the same level,t=3.467,P=0.02,p＜0.05,the difference was significant.T-test was done to tumor/serum ratio between ¹³¹â… -EGF-BSA targeting nano-carriers and ¹³¹â… -BSA vector:Levene's variance-test:F=3.373, p=0.08,variance at the same level,t=2.405,p=0.025,p＜0.05,the difference was significant.Therefore,the tumor/serum ratio of ¹³¹â… -EGF-BSA targeting nano-carrier was higher than the other two drug groups,and the difference was significant.Variance analysis was done to the distribution of cancer among ¹³¹â… -EGF-BSA targeting nano-carrier,¹³¹â… -BSA nano-carrier and¹³¹â… -BSA (non-nano):among the three drug groups,MS=171.994,F=107.499,R²= 0.889,P＜0.01,the differences of the tumor distribution was significant among three drug groups.Variance analysis was done to the distribution of ¹³¹â… -EGF-BSA targeting nano-carrier,¹³¹â… -B SA nano-carrier and ¹³¹â… -B SA(non-nano) in the liver:among the three drug groups,MS=11.594,F=4.561,R²=0.608,P=0.019, p＜0.05,the differences of the three drugs in the liver was significant.Variance analysis was done to the distribution of ¹³¹â… -EGF-BSA targeting nano-carrier,¹³¹I-BSA nano-_carrier and ¹³¹â… -BSA(non-nano) in the lung:among the three drug groups,MS-14.059,F=8.102,R²=0.623,P=0.02, p＜0.05,the differences of the three drugs in the lung was significant.Variance analysis was done to the distribution of ¹³¹â… -EGF-BSA targeting nano-carrier,¹³¹â… -BSA nano-carrier and ¹³¹â… -BSA(non-nano) in the spleen:among the three drug groups,MS=9.870,F=6.164,R²=0.758,P= 0.006,p＜0.01,the differences of the three drugs in the spleen was significant.Variance analysis was done to the distribution of ¹³¹â… -EGF-BSA targeting nano-carrier,¹³¹â… -BSA nano-carrier and ¹³¹â… -BSA(non-nano) in the kidney:among the three drug groups,MS=32.187,F=17.672,R²=0.757,P＜0.01,the differences of the three drugs in the kidney was significant.¹³¹â… -EGF-BSA targeting nano-carrier was of targeting activity to SK-BR3 human breast cancer tumor in nude mouse,while there were less distribution in the liver,spleen,lungs and other normal organs,above all,the new nano-carrier had better targeting and lower side effects.5.Intake and retention of human breast cancer SK-BR3 cells to epidermal growth factor receptor targeting nanopartieles loading c-erbB2 antisense oligodeoxyribonueleotideThe ratio trend of tumor/serum and tumor/muscle of ASODN targeting nano-carrier was similar to,but the radioactivity ratio of tumor/serum and tumor/muscle of ASODN group was relatively low in the same point of time.T-test was done to tumor/serum ratio between ASODN targeting nano-carrier and ASODN group:Levene's variance-test:F = 67.207,variance at the same level,t=5.092,p＜0.05,the difference was significant.T-test was done to tumor/muscle ratio between ASODN targeting nano-carrier and ASODN group:Levene's variance-test:F=32.92, variance at the same level,t=9.477,p＜0.01,the difference was significant. The ratio of tumor/serum and tumor/muscle of ASODN targeting nano-carrier was higher than ASODN group,and the difference was significant.T-test was done to the distribution of ASODN targeted nano-carrier and ASODN in the tumor:Levene's variance-test:F=34.958,variance at the same level,t=5.242,p＜0.01,the difference of the distribution in tumor was significant. T-test was done to the distribution of ASODN targeted nano-carrier and ASODN in the liver:Levene's variance-test:F=20.896,variance at the same level,t=16.347,p＜0.01,the difference of the distribution in the liver was significant.T-test was done to the distribution of ASODN targeted nano-carrier and ASODN in the spleen:Levene's variance-test:F=19.344,variance at the same level,t=18.992,p＜0.01,the difference of the distribution in the spleen was significant.T-test was done to the distribution of ASODN targeted nano-carrier and ASODN in the kidney:Levene's variance-test:F=8.999,variance at the same level,t=1.909,p=0.069,p＞0.05,the difference of the distribution in the kidney was no significant.ASODN's target and non-target ratio coule be improved and its stability and targeting in vivo could be increased by using EGF targeting nano-carrier in bearing breast cancer nude mouse.Conclusion:As bovine serum albumin itself has some good physical and chemical properties,the EGF targeting nano-carrier made by using BSA was the ideal size and good stability.Besides the general nature of nano-carrier,the EGF-BSA nano-carrier was obviously superior to other general nano-carriers.By study the targeting of C-erbB2 combined with EGF-BSA nano-carrier in SK-BR3 breast cancer cells,and it was proved that the EGF-BSA nano-carrier produced in our research to be a new stable and strong targeting carrier.