| BackgroundColorectal carcinoma,whose mortality rate is the fourth in all deaths because of cancers,is the third most common cancer in the world.And it's incidence rate is also rising at the increase speed of 4%,which is two times as the average level of the world,in our country every year.Although the chemotherapy adjusting the surgery, the core means to treat colorectal cancers today,can prolong the survival time of patients in a certain extent,it has the defects such as poor drug targeting,drug side effects,et al.Therefore,it is particularly necessary to research and develop new types of anticancer drugs with the guide of target therapy strategy.The drugs enwraped in liposome carriers for cancer chemotherapy,with its surface composed by natural cholesterol and phospholipid which help drugs have the characteristic of passive targeting,is just an ideal drug-carrying system for the reason that the biodegradable drugs system will not resulting in lipid accumulation in vivo,and the drugs have long-time effect by slow liberation.Further more,the immunoliposome known as the liposome tagged with monoclonal antibodies or fragments of antibodies,which has the both characteristics of liposome's advantages and antibody targeting,has became the one of the most conductions for carrying out targeting therapy. HER2,a member of epidermal growth factor receptor family,is an 185KD trans-membrane protein encoded by the human proto-oncogene her2/neu.When it becomes the heterologous dimer together with other family members,HER2 can strongly induce the internal membrane tyrosine kinase activation which can stimulate the intracellular signaling cascade reaction.Then the active downstream signaling molecules ultimately stimulate cells' proliferation,development,differentiation, migration and tumor formation.HER2 over-expression was found to be closely associated with a variety of tumors' formation,development and distant transformation.Thus,the aims of the cancer treatment and keeping malignant tumor cells from proliferation may be reached by the way of blocking HER2 protein downstream signaling system.Trastuzumab,a monoclonal humanized antibody against HER2 developed by U.S.GeneTech Limited Company,is mainly used in adjuvant chemotherapy for metastatic breast cancer patients with HER2 over-expression in the cancer tissues.And many clinical experiments have showed that chemotherapies combined with trastuzumab for breast cancer treatments have significantly better efficacy than simple chemotherapy.It is found that HER2 is also over-expressed in colon cancer cells,and the targeting therapy to this point may be a new way to deal with colon cancer.Therefore,the immunoliposomes encapsulating chemical drugs tagged with trastuzumab can treat colon cancer with two different tumor treatment strategies theoretically.Paclitaxel which was discovered in recent years is one of the great hopeful anticancer drugs.It is proved that Paclitaxel can cure types of human malignant tumors by keeping the stability of cells' microtubules and inhibiting the caryomitosis of cells in vivo.However,it has always been a critical problem of limitation in clinical application to select the suitable dosage form for paclitaxel due to its physical characteristic of poor water-soluble.The use of mixed solution composed of cremophor EL and absolute alcohol as the solvent overcomes the disadvantage of poor water-soluble characteristic of paclitaxel,but it will result in serious acute allergic encephalitis(AEE)caused by cremophor EL or strong drug toxicity caused by the dissolution of plasticizers in PVC packaging sometimes,and these restrict the clinical application of paclitaxel too.Using liposome as the carrier for paclitaxel can not only effectively overcome the inadequacies of the current dosage forms,but also can achieve targeted releasing of drugs and reduce the toxicity on normal tissue or organ by modifying liposome with antibodies.Based on the researches mentioned above,our research is going to prepare immunoliposomal paclitaxel tagged with trastuzumab F(ab')2,and explore a new means of treating colon cancer by researching the anti-tumor activities of this drug to colon cancer cells—HT29.Note:This research "The Killing Effect of Immunoliposomal Paclitaxel on Colon Cancer"(Item No.04105743)is aided financially by Natural Science Foundation of Guangdong Province.ObjectivesTo explore the appropriate way to prepare immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 in the laboratories,and to establish the method based on the colon cancer therapy which takes the immunoliposome as chemical drugs carrier by compare the killing effect to HT29 cells in vitro with using different drugs: immunoliposomal paclitaxel,liposomal paclitaxel and Trastuzumab.MethodsPreparation of immunoliposomal paclitaxel tagged with trastuzumab F(ab')2:①First,change theα-hydroxy group of polyethylene glycol 2000(PEG2000)with amino group by ion exchange method,then make out theα-amino terminal PEG-2000 phospholipid derivative(α-amino PEG2000-DSPE)by linking distearoylphosphatidylethanolamine to theω-hydroxy of PEG2000 with theα-amino being protected;②β-maleimido group modified PEG2000-DSPE (MP-PEG2000-DSPE),which is ready to link to the antibodies by hydrosulfide groups,is prepared by the adequate reaction betweenα-amino PEG2000-DSPE and 1,2-disteaoryl-sn-glycero-3-phosphatidylethanoleamine(MPS)in a mixed organic solvent;③Liposomal paclitaxel are made by reversal-phase evaporation method and ultrasound dispersion method after hydrogenated soybean phosphatidylcholine, cholesterol,PEG-DSPE,MP-PEG-DSPE and pacltaxel are mixed uniformly in a molar ratio in the rotatory evaporator;④Tastuzmab F(ab')2,which is ready to link to the surface of the liposomes,is obtained by cutting the J hinge at the side of Fc side of the antibodies with pepsin;⑤Immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 is obtained by linking Tastuzmab F(ab')2 to MP-PEG-DSPE in the membrane of the liposomes at a neutral pH,room temperature and vacuum environment;⑥The fluorescent immunoliposomes and lipoosomes are prepared by adding the fluorescer NBD-DSPE (6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]labled DSPE)to the materials during the preparation process of immunoliposomes and liposomes respectively;⑦The encapsulation efficiency of the immunoliposomes is estimated by RP-HPLC,and its stability is evaluated through comparing the encapsulation efficiency of immunoliposomes suspension laid for different times;⑧The figures of immunoliposomes are observed by TEM and their size distribution is measured by graphics software;⑨The immune activity of F(ab')2 fragment and the releasing of paclitaxel encapsulated in the two types of liposomes will be evaluated under the fluorescence microscopy after the fluorescent immunoliposomes or lipoosomes are incubated together with HT29 cells.The killing effect of immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 on HT29 cells in vitro:①The HT29 cells in log phase,whose cell density is 5×103/ml,are cultured in four 96-well tissue culture plates for 72 hours in a 37℃,5%CO2 incubator;②In each plate HT29 cells are divided into immunoliposome group,liposome group and monoclonal antibody group according to the difference in drug treatment,and in each group the cells are further divided into seven clusters by different drug(paclitaxel or trastuzumab)dosages,which have the equal dose of drug every three wells;③HT29 cells in the four tissue culture plates continue to be cultured in the 37℃,5%CO2 incubator for 8 hours,16 hours,24 hours and 32 hours;④The death status of different drug groups are observed under optical microscopy, and the optical density(OD)values of the wells are measured by MTT assay;⑤The killing rate of drugs is calculated through particular formulation,and the influence of types,dosage,action time of drugs and the interaction among them was analyzed by multi-way classification ANOVA.ResultsThe immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 which is synthesized in this experiment have an uniform size distribution with the average size of 210nm,and 91.37%of their size are smaller than 200nm;The encapsulation efficiency of this fresh immunolipsomes is 89.23%while it deceases to 84.42%with a small decreasing degree of 4%when the immunoliposomes are kept in a 4℃environment for 30 days;The immunoliposomes,which are recognized earlier by the HT29 cells,begin to be taken up by cells just from the 10th minute after drugs administration by observing through the fluorescence microscopy.And the number of the taken immunoliposomes,which is larger than that of the liposomes,is increasing along with the length of the incubate time with HT29 cells.The killing effect of immunoliposome group is obviously superior to other two groups when using the equal dosage of drugs for the same time.And the best killing effect of 98.58%is in the immunoliposome group(32h,400μg/ml paclitaxel)while the worst killing effect of 0.17%is in the trastuzumab group(16h,43.75μg/ml trastuzumab);It has the significant difference of the killing effect caused by types of drugs(F=5751.178,P＜0.001),action time of drugs(F=342.119,P＜0.001), concentration of drugs(F=4302.472,P＜0.001),interaction between types and action time of drugs(F=58.370,P＜0.001),interaction between types and concentration of drugs(F=912.283,P＜0.001),interaction between concentration and action time of drugs(F=43.985,P＜0.001),and interaction among the three factors(F=10.042,P＜0.001);It also has the significant difference of the killing effect between every two drugs by LSD law multiple comparisons,and the immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 has the strongest killing effect in all the drugs.Conclusions①The immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 prepared by the method in this article has the appropriate size,size distribution,encapsulation efficiency and stability for experimental requirements,and there is no serious leakage of the drugs as its solution has been stored at 4℃for 30 days;②The immunoliposome is superior to liposome at the identification capability of recognizing HT29 cells under the same condition;③The killing effect is affected by types,concentration and action time of the drugs;④The immunoliposomal paclitaxel tagged with trastuzumab F(ab')2 has the strongest killing effect in all of the three drugs with the equal drug dosage and the same action time;⑤Trastuzumab also can kill HT29 cells,and this effect may be related to the expression level of HER2 in the cells' surface. |
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