Studies On The Preparation, Antitumor Effect, Bio-safety And Tumor Angiogenesis Targeting Of Doxorubicin-loaded Cationic Liposomes

N-trimethyl chitosan(TMC), a2-NH2partly quaternized derivative of chitosan(CS), is widely used as a drug absorption enhancer and sustained-release vector. In thetreatment of tumour, the tumour vessels are considered as important targets. Amongvarious vehicles for antitumor drug delivery, cationic liposomes (CLs) have beenreported to accumulate in tumor vascular endothelial cells (VECs) to show highlyselective targeting ability. Therefore, chemotherapeutic agent-loaded CLs areconsidered as new therapeutic preparations to enhance the treatment efficacy that notonly lower the toxicity, but also increase the target ability of chemotherapeutic agent.Doxorubicin(DOX) is a broad-spectrum anthracycline used in clinics to treat solidtumour, such as breast cancer, thyroid cancer, lung cancer, ovarian cancer and sarcoma,et al. However, DOX shows high toxicity and can cause such side effects as nausea,vomitting and bone marrow depression, especially, the dose-dependent cardiotoxicity.Liposomes are considered as one of effective vehicles to lower the side effects of DOXwith targeting and sustained release properties.In our study, DOX-loaded liposomes (DOXL) were prepared followed by TMCcoating. The anti-tumour efficacy, bio-safety in vitro and in vivo of the vehicles andtumor angiogenesis targeting in vitro and in vivo of TMC-coated DOXL were studied tolay experimental basis for a new preparation of DOX for clinical application.In the first part, TMC with different DQ was synthesized by four-step method andthe structure was confirmed by IR while DQ was determined by¹H-NMR. The resultshowed that the characteristic absorption peak of TMC has been significantly changedin IR analysis, indicating that TMC has been successfully synthesized and the DQ of thesynthesized TMC was25%(TMC20),38%(TMC40) and59%(TMC60) by1H-NMRanalysis, respectively. Film dispersion method was used in the preparation of blankTMC-coated liposomes and ammonium sulfate gradient method was applied in thepreparation of DOXL and TMC-coated DOXL. The formulation and preparationprocess of TMC-coated DOXL were optimized and the quality, such as morphology,size, Zeta potential and drug release of the liposomes was evaluated in vitro. The resultsshowed that the liposomes were obtained with spherical shape, narrow size distribution,positive Zeta potential and sustained release property in vitro, which provided the material basis for the further studies.In the second part, a solid tumor, formed by implantation of murine hepatomacells (H22) into mice, as tumor model, the tumor inhibition rate and tumor histologicalsections stained by HE of TMC-coated DOXL group were researched compared withthose of free DOX and DOXL group. It was found that with the DQ increase of TMC,the positive surface charge of TMC-coated DOXL was enhanced accordingly, which ledto the enhancement of anti-tumor efficacy in vivo. Among them, TMC60-coated DOXLshowed the highest tumor inhibition rate of71.4%with significant difference (P<0.05)when compared with that of free DOX (42.5%) and un-coated DOXL (34.7%).In the third part, the bio-safety of blank TMC-coated liposomes was evaluated tolay foundation on the application in the tumor-targeting vehicles. The cytotoxicity ofTMC as liposomal coating materials with various concentrations and DQ was evaluatedon mouse fibroblasts cell L-929by MTT assay through calculating the relative growthrate (RGR), and the extent of toxicity was classified according to the evaluation criteriaof United States Pharmacopoeia. The cell proliferation rate of TMC at concentration of0.02%and0.05%(w/w) were both higher than80%, even the concentration of TMC20was increased to0.2%(w/w), cell proliferation rate was still higher than80%showingnon-toxicity. However, with the increase of concentration and DQ, cytotoxicity of TMCwas increased accordingly. The acute toxicity in mice of TMC60-coated liposomes by ivand ip was studied and LD₅0was obtained. The intravenous stimulation, injectionanaphylaxis and hemolysis were investigated as well. The results showed that the LD₅0and95%confidence limit of TMC60-coated liposomes by iv and ip was224.1(178.8-302.6)mg/kg and342.3(269.7-534.2)mg/kg, respectively. Meanwhile,TMC60-coated liposomes showed no intravenous stimulation, injection anaphylaxis andhemolysis at the testing concentration.In the forth part, the targeting of tumor angiogenesis of TMC-coated DOXL wasstudied in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) as thecell model, the vascular targeting ability of TMC-coated DOXL was evaluated in vitro.It was found that with the DQ increase of TMC, the positive surface charge ofTMC-coated DOXL was enhanced accordingly, which led to the significant increase ofDOX uptake by HUVECs in vitro. Especially, TMC-coated DOXL showed bettertargeting ability to the nuclei compared with free DOX and DOXL, which could furtherenhance the efficacy of DOX in vivo. The mice H₂2tumor model was established by animal transplanted tumor experiment. The in vivo fluorescence in tumor tissue wasinvestigated through the tail vein injection of fluorescein isothiocyanate conjugateddextran (FITC-Dextran). The shape and arrangement conditions of tumor angiogenesiswere observed qualitatively and relative density of tumor angiogenesis was determinedquantitatively. In the mice given TMC-coated DOXL, the tumor angiogenesis had goodshape, uniform arrangement and little vascular branches, while the tumor angiogenesisof the other treatment groups showed more distorted shape, uneven thickness and anode having multiple vascular branches. By comparing the adhesion amount ofFITC-Dextran in the tumor tissue of the different groups, the vascular density ofTMC-coated DOXL was lower than that of the free drug group and un-coatedDOXL(P<0.05). The results further proved that TMC-coated DOXL could significantlyreduce the density of angiogenesis and had notable anti-tumor angiogenesis effect.In summary, the whole experiment studied the preparation, anti-tumor efficacy andtumor angiogenesis targeting in vitro and in vivo of TMC-coated DOXL. The bio-safetyof blank TMC-coated liposomes was also investigated in vitro and in vivo. As a result,the experiment can provide experimental basis for the widened use of TMC and a newpromising preparation of DOX for clinical application.