The Combination Of Targeting Nanoparticale With C-erbB2 Antisense Oligodeoxynucleotide Used For The Targeting Research Of Breast Cancer

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 ¹²⁵I 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 ¹²⁵I-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 125 I -EGF. 2. Distribution of ¹²⁵I-EGF-BSA targeting nano-carrier in bearing breast cancer nude mouseComparing the distributions of ¹²⁵I-EGF-BSA targeting nano-carriers and BSA nano-carriers in bearing cancer SK-BR3 nude mouse respectively.3. Targeting performance of c-erbB2 targeting nanoparticles loading ¹²⁵I–ASODN on breast cancer SK-BR3C-erbB2 ASODN was labeled for ¹²⁵I by chloramine T method,and ASODN labeled for ¹²⁵I 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 ¹²⁵I-ASODN targeting nano-carriers and ¹²⁵I-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 radionuclide Observed 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 ¹²⁵I-BSANP with 125 I 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 ¹²⁵I-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 ¹²⁵I 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, ¹²⁵I-EGF-BSANP had good stability in neutral solution at room temperature within 24 h, and ¹²⁵I not falling. Serum stability, there was no significant(P>0.05)between the groups of 1h, 6h, 12h and 24 h 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, ¹²⁵I-EGF-BSANP had good stability in serum, at room temperature within 24h, ¹²⁵I not falling.3. Distribution of ¹²⁵I-EGF-BSA targeting nano-carrier in bearinbreast cancer nude mouseRadioactivity ratio of tumor/serum and tumor/muscle of the ¹²⁵I-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 ¹²⁵I-BSA nano-carrier was similar to ¹²⁵I-EGF-BSA targeting nano-carrier,but the radioactivity ratio of tumor/serum and tumor/muscle of ¹²⁵I-BSA nano-carrier was relatively low in the same point of time.Analysis of variance was done to ¹²⁵I-EGF-BSA targeting nano-carriers, ¹²⁵I-BSA nano-carriers and ¹²⁵I-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 ¹²⁵I-EGF-BSA targeting nano-carrier and ¹²⁵I-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 ¹²⁵I-EGF-BSA targeting nano-carriers and ¹²⁵I-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 ¹²⁵I-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 ¹²⁵I-EGF-BSA targeting nano-carrier, 125 I-BSA nano-carrier and¹²⁵I-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 125 I-EGF-BSA targeting nano-carrier, ¹²⁵I-BSA nano-carrier and ¹²⁵I-BSA (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 ¹²⁵I-EGF-BSA targeting nano-carrier, ¹²⁵I-BSA nano-carrier and ¹²⁵I-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 ¹²⁵I-EGF-BSA targeting nano-carrier, ¹²⁵I-BSA nano-carrier and ¹²⁵I-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 ¹²⁵I-EGF-BSA targeting nano-carrier, ¹²⁵I-BSA nano-carrier and ¹²⁵I-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.¹²⁵I-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. 3. Targeting performance of c-erbB2 targeting nanoparticles loading ¹²⁵I–ASODN on breast cancer SK-BR3The 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.