The Preparation And Properties In Vitro Of A New Type Of Immuneliposome For Breast Cancer

Doxorubicine (DOX) is a kind of common antitumor drugs belonging to the cyclenon-specific drug with a strong inhibition on RNA.The antineoplastic spectrum isbroader,because of the toxicity is higher, the long-term use can make the body resistance,resulting in its curative effect and application being limited by greatly.Liposome as a newtype of pharmaceutical preparations, it can not only reduce the side effects of drugs on thebody, but improve the drug pharmacokinetic distribution with slow-release controlledrelease, but the biggest drawback is the lack of active targetion of tumors.Immunoliposome is a new research areas in recent years, It is the antibodies or ligandsconnecting or bonding on the liposome by polymer (such as polyethylene glycolderivatives), takeing advantage of the specific targetion of antibodies or ligands,combiningwith the tumor cell surface antigens or receptors. Drugs are transferred to the lesion,immuneliposome has some the advantages such as the stronger targeting, smaller sideeffects, longer half-life and larger drug loading capacity.About20-30%of breast cancer possesses the phenomenon of overexpression of human epidermal growth factor2,HER2is gene encoding a protein of tyrosine kinase activity, itcan strengthen the kinase activity and promote cell division, proliferation andtransformation. The expression level of gene in the adult normal tissue islow.overexpression of HER2protein appeared in the early stages of breast cancer, raisingthe metastatic potential of breast cancer cells. In ovarian cancer and cancer of thestomach cancer cells have different degrees of high expression. In recent years,trastuzumab,monoclonal antibody have been preparated according to the structure ofHER2, it can resistance the HER2receptor and have a strong affinity and specificity, byblocking the human epidermal growth factor on the HER2to produce antitumor effect.providing a new way for the treatment of breast cancer. trastuzumab (Herceptin) has beenapprovaled in October1998, by the United States.Liposomes as drug carrier, trastuzumab is bonged on the surface of doxorubicineliposome by DSPE-PEG-NHS, doxorubicine liposome is preparated by phosphatidylcholine, cholesterol, adriamycin, and MPEG2000-DSPE by thin-film ultrasonic method a,meanwhile, trastuzumab is connected with DSPE-PEG-NHS through chemical bondconnection,doxorubicine liposome would connect with TMAB-(PEG-DSPE) n, Constanttemperature shocking5h at4℃condition, to get doxorubicine immuneliposomes（DOXIML）.This paper determined the number of active amino groups is85, when n (DSPE-PEG-NHS): n (TMAB)=10:1.the number of active amino groups in reaction is7,respectively.Doxorubicine immunoliposome(DOX IML) was separation by Sephadex G-150, and then the BCA method is used to determine the content of TMAB,three differentantibody levels of doxorubicine immuneliposome are preparated(antibody levels37,83,108μg·mg-1, the concentration of DOX being10μg·mL-1), and the nature of the invitro study is carrying out,HER2positive cells is AU565cells, MDA-MB-231cells isnegative on HER2, through three different antibody concentration of DOX IML laserconfocal and flow cytometry test, the antibody concentration of DOX IML is83μg·mg-1,the cellular uptake is86.8%, the capacity of invading cells and cell toxicity are secondonly to doxorubicine. By the study of properties in vitro on doxorubicine, doxorubicineliposome, DOX IML, DOX IML has stronger targeting, the capacity of invading cells and cell toxicity are higher than doxorubicine liposome. This paper also study size change ofcoating drug before and after and bonding TMAB before and after: before and aftercoating doxorubicin,the size of doxorubicin liposome has no change, before and afterbonding TMAB,the size of DOX IML has no change. The encapsulation rate ofdoxorubicine liposome was89.85%, drug-loading rate being8.03%, the charge being-26mv,the study of drug release and stability in vitro. DOX IML has slow-release andcontrolled release,the cumulative release dosage within12h was40%, with the timegoing,cumulative release dosage almost balances at72h, the cumulative release dosagewas79%within72h. the stability of DOX IML is good under4℃. Under25℃,with thetime going, the particle size was more and more bigger, the color changed from red to darkred accompanying a large amount of precipitation.