Integrin ?v?6-targeted Immunoliposomes:A Novel Approach For Tumor-Specific Drug Delivery And Therapy In Colon Carcinoma

BackgroundColon cancer is the third most common cancer and the fourth leading cause of cancer-related deaths worldwide.Although surgery remains the prefcerred treatment.5-fluorouracil(5-FU)-based adjuvant chemotherapy is the conventional care for stage?(lymph node-positive)patients,which can reduce mortality by 25%compared with surgery alone and greatly improve the five-year survival rate.However.because the response rate of chemotherapy is only 10%to 20%.the treatment of advanced and metastatic cases remains a challenging problem.Therefore.it is urgent to explore novel therapeutic strategies for colon cancer treatment to overcome chemotherapeutic drug resistance and to improve chemotherapeutic efficacy.Liposomes are small artificial vesicles of spherical shape that can be created from cholesterol and natural non-toxic phospholipids just like the cell membrane.Due to their hydrophobic and hydrophilic character,liposomes could be used as drug delivery systems by encapsulating chemical drugs.Initially,liposomes were not used for drug carriers,but for the research of the biomembrane system.With the deep study and development of the liposomal preparation process,liposomes become ideal drug delivery systems because of its biofilm properties,low toxicity and no immunogenicity characteristics,which can reduce the dose and the side effects of chemical drugs and improve the therapeutic effect.More and more liposomal chemotherapeutic agents have been well applied for tumor therapy in clinic.But in vivo,albumin,antibodies and opsonin in the circulation can destroy the structure of liposomes and affect its stability,resulting in the leakage of internal encapsulated drugs.As a result,the agent is not able to achieve the desired effect in tumor therapy.Sterically stabilized liposomes with a polymeric PEG coating can obviously improve the stability of liposomes and lower reticuloendothelial system(RES)uptake,which prolong circulation times and lead to higher amount of drug in the blood.Additionally.this kind of liposomes are able to promote the tumor accumulation of chemical drugs because tumor tissues always have increased permeability of capillary.However,these liposomes passively interact with tumor cells,in vitro or in vivo,resulting in nonspecific drug release that leads to the eventual diffusion of the drugs into some normal tissues or cells rather than tumors.Immunoliposomes.which are conjugated with monoclonal antibodies(mAb),are capable of both drug delivery and molecular targeting.By combining the specific targeting properties of mAbs and drug delivery advantages of' liposomes,immunoliposomes are a promising approach for the targeted delivery of' anticancer drugs to tumors.To date,various tumor-associated antigens have been validated as targets for antibody-based immunoliposomes in cancer therapeutics.Immunoliposomes targeting CD30.HER2/neu.EGFR.and VEGFR.which are expressed on various tumor cell types,have been developed and thoroughly characterized.On the basis of encouraging preclinical data and advances in large scale production processes,EGFR-specific immunoliposomes are already being used in clinical trials.Integrin ?v?6 is a subtype of integrin that is expressed exclusively on the surfaces of epithelial cells and is a receptor for extracellular matrix proteins.Integrin?v?6 expression is upregulated during embryogenesis.oncogenesis,and epithelial repair,where as it is generally undetectable in healthy epithelial tissues.In colon cancer,integrin ?v?6 is specifically expressed in tumor tissues and is rarely present in tissues adjacent to the tumor.In addition,we previously reported that integrin ?v?6 was associated with colon cancer pathology,malignancy,and TNM stage and could act as a prognostic indicator in aggressive colon carcinomas.Our research previously confirmed that integrin ?v?6 contributed to chemotherapeutic resistance in colon cancer;integrin ?v?6 protected colon cancer cells from 5-FU-induced growth inhibition and apoptosis.Unsurprisingly,the exclusive expression of integrin ?v?6 and its influential effects in colon cancer make it a novel therapeutic target for colon cancer treatment.Integrin ?v?6 has previously been employed as a clinical biomarker for early cancer detection.Moreover,integrin ?v?6 signaling axis blocking,agents have also been designed to exploit this receptor as a therapeutic target for cancer treatment.However,research concerning integrin av(36-targeted drug delivery for colon cancer chemotherapy has not been reported.In the current study,we describe the design,preparation,and characterization of integrin ?v?6-targeted immunoliposomes,and explore their antitumor efficiency against colon cancer in vitro and in tumor xenograft models using 5-FU-loaded integrin ?v?6-targeted immunoliposomes.Part 1 Preparation and Characterization of Integrin ???6-targeted ImmunoliposomesObjectiveThis study was to design a novel method for preparation of the integrin?v?6-targeted immunoliposomes and evaluate its characteristic.MethodsFirstly,PEG-liposomes were prepared using reverse rotary evaporation.Then integrin ?v?6-specific antibody was coupled to the activated DSPE-PEG2000-NH2 in liposomes using the cross-linker in order to get the integrin ?v?6-targeted immunoliposomes.The morphologies of liposomes were examined by transmission electron microscopy(TEM).The mean particle size and distribution of liposomes,as well as zeta-potential values,were determined by Laser particle size analyzer.5-FU-loaded integrin avp6-targeted immunoliposomes was prepared by the same method,then the encapsulation efficiency and in vitro release rate were examined by the spectrophotometer.ResultsThe morphology of liposomes and integrin ?v?6-targeted immunoliposomes were spherical or ellipsoidal.The mean particle size of integrin ?v?6-targeted immunoliposomes was 405.1±2.74 nm,which was greater than that of liposomes(345.5±2.21 nm).The zeta potential of the liposomes was-23.77±2.35mV.After mAb conjugation,the zeta potential further decreased to a value of-8.18±1.83 mV.The 5-FU encapsulation efficiencies of the liposomes and immunoliposomes were 78.43%±2.23%and 75.95%±2.30%.respectively.Integrin ?v?6-targeted immunoliposomes and liposomes had similar trends of 5-FU release in vitro,and could reach over 90%and 50%.Integrin ?v?6-targeted immunoliposomes showed no greatly sensitivity to different pHs on the release of 5-FU.Conclusions The method for preparation of integrin ?v?6-targeted imunoliposomes that integrin ?v?6-specific antibody was coupled to the activated DSPE-PEG2000-NH2 in liposomes using the cross-linker was proved feasible in this part.Moreover,the 5-FU loaded integrin ?v?6-targeted immunoliposomes had an ideal encapsulation efficiencies and in vitro release rate.Part 2 Integrin ?v?6-targeted Immunoliposomes Targeting in Colon Cancer Cells and Antitumor Efficiency in vitroObjectiveThis study was to investigate the uptake of integrin ?v?6-targeted immunoliposomes in integrin av(36 expressing colon cancer cells,and explore the potential mechanism of its improved anticancer efficacy in vitro.Methods1.The integrin ?v?6 expression level in HT-29 and SW480?6 colon cancer cells was assessed by flow cytometry.2.Liposomes containing coumarin-6 were prepared by the same method in part one.The fluorescence microscope and flow cytometry were used to test cellular uptake and internalization of liposomes in colon cancer cells.3.The cell Counting Kit-8 assay(CCK-8)was performed to determine the administration drug dose and the cytotoxicity of 5-FU-loaded integrin?v?6-targeted immunoliposomes on colon cancer cells.4.Apoptosis of colon cancer cells was detected by flow cytometry.5.The cytosol lysate was extracted using the cytochrome C apoptosis assay kit by the method described in the manuscript and the Cytochrome C release was analyzed by Western blotting.The levels of caspase-3/9.cleaved caspase-3 and cleaved PARP were also determined using western blotting.A caspase activity assay was carried out using a caspase fluorometric assay kit following the instructions provided by the manufacturer.Results1.Results of flow cytometry showed that both HT-29 and SW480(36 cells obviously expressed integrin ?v?6.whereas HT-29 treated with ?6-siRNA and wild type SW480 had low or negative integrin ?v?6 expression.2.For both integrin avP6 positive colon cancer cells HT-29 and SW480?6,the fluorescence images indicated that the fluorescence intensities of integrin?v?6-targeted immunoliposomes were significantly greater than the liposomes.However,in comparison with HT-29 and SW480?6 cells.fluorescence in av(36-suppressed HT-29 or ?v?6-negative SW480 cells was significantly weaker after ?v?6-targeted immunoliposomes+coumarin-6 treatment.3.The integrin ?v?6-targeted immunoliposomes+5-FU inhibited the cellular growth more greatly than liposomes+5-FU for both HT-29 and SW480?6 cells(P<0.01).However,inhibition between these two types of liposomes was almost undistinguishable in the ?v?6-negative SW480 cells.The integrin ?v?6-targeted immunoliposomes produced an obvious decrease in 5-FU IC50 values relative to that of liposomes.4.In the integrin av(36-targeted immunoliposomes groups,the apoptotic rate of HT-29 and SW480P6 cells was 21.91±1.62%and 26.18±2.43%.which was higher than that of non-target liposomes(14.53±0.99%and 16.61±1.23%)and free 5-FU(8.99±1.00%and 12.77±1.21%).(P<0.01).5.Western blot showed in HT-29 and SW480?6 cells,cytosolic cytochrome C level was evidently higher in the integrin ?v?6-targeted immunoliposome group than the liposome group(P<0.01).Consequently,the integrin ?v?6-targeted immunoliposomes group had higher cleaved caspase-3 and cleaved PARP levels compared with the liposome group.In the meantime,caspase-9 and caspase-3 activities of both HT-29 and SW480f36 cells were hilgher in integrin?v?6-targeted immunoliposomes groups than liposome groups(P<0.05).ConclusionsThe integrin ?v?6-targeted immunoliposomes could enhance the cellular internalization compared with liposomes,which probably resulted in improved anticancer efficacy in colon cancer cells.In addition,the integrin ?v?6-target immunoliposomes were capable of reducing chemo resistance by facilitating Cytochrome C-Caspase-9/3 mitochondrial apoptotic pathway.Part 3 Integrin ?v?6-targeted Immunoliposomes Targeting in Colon Cancer and Antitumor Efficiency in vitroObjectiveThis part of study was to investigate the tumor targeted delivery and antitumor efficiency of integrin ?v?6-targeted immunoliposomes in a nude mouse tumor-xenograft model of colorectal carcinoma.Methods1.BALB/C female nude mice were subcutaneously implanted with HT-29 or SW480?6 human colon cancer cells at a final concentration of 1×107/200 mL to set up tumor-xenograf-t model of colorectal carcinoma.2.Chose the mice of which the tumor size was larger than 100 mm3,and randomly assigned to three groups and injected intravenously through tail veins with a dosage of 5-FU.liposomes+5-FU,or integrin ?v?6-targeted immunoliposoMes+5-FU with the 5-FU final concentration of 20mg/kg.The mice were sacrificed at the indicated time points after the drug was administered i.v.to collect tissue samples.The tissues(heart,liver,spleen,lung,kidney,and tumor)were removed,washed,weighed,and homogenized in physiologic saline.5-FU was extracted using ethylacetate.The amount of 5-FU in tissues was analyzed by the HPLC assay.3.Mice with tumor volumes of approximately 50 mm3 were selected and randomly assigned to three treatment groups.Free 5-FU,5-FU loaded liposomes,or integrin ?v?6-targeted immunoliposomes were intravenously injected twice a week at a 5-FU dosage of 20 mg/kg.Blank immunoliposomes or physiologic saline(N.S.)were also injected as controls.The tumor size and body weight were measured every other day during the treatment period.After 3 weeks,mice were sacrificed and the tumors were dislodged and weighed.4.Mice were sacrificed,and sections of tumor tissues in each group were prepared.The TUNEL assay was carried out using an In Situ Cell Death Detection Kit according to the manufacturer's instructions.Results1.HPLC assay showed that the 5-FU concentrations were high in heart,liver,and kidney tissues after free 5-FU injection.Both liposomal formulations decreased the 5-FU concentration in the heart,liver,and kidney.In tumor tissues,integrin?v?6-targeted immunoliposomes and liposomes had a higher 5-FU concentration than free 5-FU.However.the 5-FU concentration for integrin ?v?6-targeted immunoliposomes in the tumor steadily increased and climaxed after 12 hours,whereas 5-FU concentrations for liposomes decreased as time progressed.In addition,the AUCO-24 values of integrin ?v?6-targeted immunoliposomes in tumor tissues were 39.28±0.80 mg/g*h.higher than that of liposomes(10.42±0.62 mg/g*h).2.The antitumor effect was indicated by tumor growth.In comparison with free 5-FU,both liposomal 5-FU formulations obviously suppressed tumor growth during the treatment period on both HT-29 models and SW480?6 models.No antitumor effects were observed in the N.S.and blank immunoliposome groups.Moreover.the tumor suppression of integrin ?v?6-targeted immunoliposomes+5-FU was significantly stronger than liposomes+5-FU.Meanwhile,the average tumor weight in mice treated with integrin avp6-targeted immunoliposomes+5-FU was approximately 3-fold lower than that in mice treated with liposomes+5-FU.3.An obvious loss of body weight was observed in mice treated with 5-FU compared with mice treated with 5-FU-loaded liposomes or immunoliposomes.Body weight gain of liposomes and immunoliposomes groups that was similar.4.The TUNEL assay showed that compared with liposomes+5-FU,integrin avp6-targeted immunoliposomes+5-FU induced apoptosis in tumor cells by a factor of 1.5-to 1.7-fold.ConclusionsThe antitumor efficacy of integrin ?v?6-targeted immunoliposomes was significantly superior to that of liposomes,which was probably caused by increased drug accumulation in tumor issues.The body weight was additionally observed to evaluat the side effects.Although body weight changes in immunoliposome and liposomes groups were unobvious.which was most likely due to their similar tissue biodistributions and pharmacokinetic properties in vivo,integrin avp6-targeted immunoliposomes may be more inclined to reduce the side effects in the long run,as a result of the target drug accumulation in cancer cells.