Preparation Of Drug Loaded Radiation-sensitive Liposomes And Their Antitumor Effect Study

As a standard therapeutic mode of various kinds of cancers, the comprehensivetherapeutic method, chemoradiotherapy, was restricted in terms of the applicationscope and the therapeutic effects by its toxic and side effects. As a kind of deliverysystem for antineoplastic agents, liposomes had been confirmed by means of theclinical efficacy and reduced the toxic and the side effects of the antineoplastic agentssignificantly. But for some reasons, such as the release and the absorption of theagents loaded in them, liposomes loading agents could not achieved much bettertherapeutic effects we had expected compared with the ordinary prepared agents. Sowe were lead in the research of developing a series of new radiation-sensitiveliposomes with radiation and ionizing radiation methods as the control switches ofthem. The radiation-sensitive liposomes loading the antineoplastic agents wereexpected to integrate radiotherapy and chemotherapy in time and space from theperspective of the therapeutic modes and then form a novel synchronous real-timechemoradiotherapy mode for anti-tumor treatment. In the process of that treatmentmode, the radiation-sensitive liposomes loading the antineoplastic agents would makeuse of their advantages as the drug delivery systems, have the tumors as theirtherapeutic targets passively, and reduce the toxic and the side effects of the agents.Furthermore, those liposomes could also modulate the release of the agents loaded inthem initiatively, increase the concentrations of the agents in the tumor tissues and thesafety margins by three-dimensional conformal or intensity modulated irradiation soas to improve the therapeutic efficacy and reduce the systemic toxic and side effectsof the agents. There were five parts of this topic. The first part was about directed synthesis of theradiation-sensitive compounds1,2-Bis[10-(2',4'-hexadienoyloxy)decanoyl-]sn-glycero-3-Phosphatidylcholine (SorbPC), Di-(1-hydroxylundecyl) diselenide (DHD) and12-(2-(10-carboxyd-ecyl)-di-selanyl) dodecanoic acid (CDA). Their radiation sensiti-vity were detected by UV spectrophotometry(SorbPC) and infrared spectroscopy(DHD,CDA). The experimental results showed that SorbPC had the most obviousradiation sensitivity. Absorption peak of SorbPC changed after exposed to only0.2Gydoses of X-rays and decreased with increasing doses of X-rays. A new absorptionpeak appearred after DHD and CDA exposed to more than15Gy doses of X-rayswhich was shown in infrared spectrum and increased with increasing doses of X-rays.The second part included the synthesis and characterization of Dox-loadedradiation-sensitive liposome and evaluation of they release effect after exposed todifferent dose of X-rays in vitro. Film dispersion method was used to synthesize threekinds of liposomes with corresponding radiation-sensitive compounds. Dox wasselected as a model drug. After optimized formulation, phospholipid concentrationwas determined to be40mg·mL^-1and the molar ratio of the phospholipid andcholesterol was12:1, molar ratio of phospholipid/cholesterol/radiation-sensitivecompounds was12:1:6/1.8/2.64(Dox-SorLip, Dox-DhdLiP and Dox-CdaLip). Wedetected the content of selenium in Dox-DhdLip of Dox-CdaLip by ICP andcalculated the content of DHD and CDA in liposome which could indirect proof themand other membranes could together constitute the structure of the lipid membrane.Contrast the encapsulation efficiency and drug release after exposed to X-rays, pHgradient method was selected for the drug-loaded. UV spectrophotometry, HPLC andfluorescence quenching methods were established for the detection of encapsulationefficiency, drug loading, accumulation percentage of drug release after exposed toX-rays and stability of liposome. The accuracy and specificity of those methods wereaccepted. After all, the average encapsulation efficiency of Dox-loadedradiation-sensitive liposome was about90%and their average particle size was about180nm, zeta potential was about4mV. Dox-loaded radiation-sensitive liposome wasslightly unstable after90days stored at4°C, but showed better stability in fetal calf serum,1640medium and DMEM culture medium after24hours storage. In the test ofDox releasing after exposed to different dose of X-rays, Dox-SorLip showed a betterresult.74.3%of Dox was release after exposed to1Gy dose of X-rays, whileradiation exposure up to4Gy, the cumulative release rate could reach88.3%. Itsrelease profiles in vitro could be expressed by Ritger-Peppas equation(Lny=0.37752Lnx-0.97598, Adj. R²=0.90514). The Dox releasing of Dox-DhdLip andDox-CdaLip need a higher dose of X-rays. Exposing to the15Gy of X-rays, drugrelease rate of Dox-CdaLip reached66.3%, and its release profiles in vitro could beexpressed by Higuchi equation (y=0.17397x1/2-0.01453, Adj. R²=0.91396). Exposingto the30Gy of X-rays, drug release rate of Dox-CdaLip reached52.6%. Its releaseprofiles in vitro could be expressed by Ritger-Peppas equation(Lny=0.3571Lnx-1.19346, Adj. R²=0.90360).The third part mainly included pharmacokinetic studies of Dox-loadedradiation-sensitive liposomes which were carried out in SD rats. The results showedthat Dox, Dox-SorLip, Dox-DhdLip and Dox-CdaLip rapid distributionand slowlyeliminated after injected from tail vein and its fit for the three compartment mode. Theplasma concentration less than0.02mg·L^-1in Dox solution group after injection4hand and could not be detected. In Dox-SorLip group, its able to be detected at8h.Cmax cal, t_1/2α, AUC and CL of Dox, Dox-SorLip, Dox-DhdLip and Dox-CdaLipgroup calculated by Kinetica were7.006,19.005,9.412and20.896mg·L^-1;0.059,0.119,0.187and0.234mg·L^-1;0.891,4.765,3.890, and3.814mg·L^-1;2694.32,503.64,616.92and629.21. The peak plasma concentration of radiation-sensitiveliposome group was significantly higher than the Dox solution group and also withhigher bioavailability and lower clearance rate.In the fourth Part, the pharmacodynamic effects of Dox-loaded radiation-sensitiveliposome were investigated by HepG2cell and mouse model with transplanted H22tumor. The results showed that within6-48h, HepG2cell toxicity of Dox,Dox-SorLip, Dox-DhdLip and Dox-CdaLip showed significant time-dependentchanges and reach the peak at48h time point. Dox-SorLip, Dox-DhdLip andDox-CdaLip group showed slightly lower cytotoxicity compared with Dox solution group at6h point, but higher at24h and36h point. When combined with ionizingradiation cytotoxicity of each group in HepG2showed a similar trend, but thedifference was Dox solution group showed slightly higher cytotoxicity at6h pointand no obvious difference in other time point. In pharmacokinetics study, comparingwith the negative control group, the tumor volume and tumor weight growth began toslow down after treatment two days, showing a good treatment effect (p<0.05). Whencombined ionizing radiation treatment, tumor suppressor was enhanced (p<0.05) andtumor inhibition rate reached to61%in Dox-SorLip group which was the highest. Thedrug concentration in heart and tumor showed a clear distinction between Doxsolution group and radiation-sensitive liposome group. Radiation-sensitive liposomesignificantly reduced the drug concentration in heart and increased in tumor whichwill reduce the cardiac toxicity of the drug and improved the therapeutic effect.The fifth part mainly included toxicological studies of Dox-loaded radiation-sensitive liposomes. MTT assay was used to detect the cytotoxicity of SorbPC, DHDand CDA on HL-7702. Selecting Dox-CdaLip as the tested drugs carried out acutetoxicity test, bone marrow micronucleus test and chromosomal aberration test in rats.Cytotoxicity test results showed that SorbPC, of DHD, the CDA had no significantcytotoxic in the1-2000μg·mL^-1dose range in the24h and48h experimental group.The acute toxicity test results showed that The LD₅₀of Dox was13.52mg·kg^-1inmice,95%confidence interval was12.70-14.40mg·kg^-1. The LD₅₀of Dox-CdaLipwas19.27mg·kg^-1,95%confidence interval was18.02-20.62mg·kg^-1. Dox-CdaLip reduced the toxicity and improved the safety of the Dox.Chromosome aberration test in mice showed that the chromosome aberration ratewas10.8%in Dox group. The incidence of chromosomal aberrations of Dox-CdaLipwas7.4%,5.6%and3.4%in three different dose groups (9.64,4.82, and2.41mg·kg^-1), which was significantly lower than Dox group (p<0.05). The result indicatedthat the Dox-CdaLip could reduce the incidence of chromosome aberration in micecaused by Dox.Micronucleus test in mice showed that the micronucleus rate was21.6‰in Doxgroup. The micronucleus rates of Dox-CdaLip were14.2‰10.7‰and7.4‰in three different dose groups (9.64,4.82and2.41mg·kg^-1) which was significantly lowerthan Dox group (p<0.05), the result indicated that Dox-CdaLip could significantlyreduce micronucleus incidence in mice caused by Dox.In summary, SorLip, DhdLip and CdaLip, these three types of radiation-sensitivecompounds were safe and nontoxic. In certain prepared ratios, the radiation-sensitiveliposomes could be prepared successfully with the radiation-sensitive materials abovementioned, have the relative stabilities on the physical and the chemical properties,and achieve the release controls of the antineoplastic agents by irradiation. Comparedwith the free ones, the agents loaded in the radiation-sensitive liposomes performedbetter in the aspects of the plasma peak concentration, the half-life time and thebioavailability in vivo. The anti-tumor effect of Dox-loaded radiation-sensitiveliposomes performed slightly better than the free drug group in anti-tumorexperiments in vivo. When combined with radiation, tumor growth inhibitory effect ofDox-loaded radiation-sensitive liposomes was better than the free drug group. But itwas not significant in tumor inhibition rate which was based on tumor weight. Thismight be due to a shorter course of treatment and large individual differences inanimals. All the above were just preliminary study on anti-tumor effect ofradiation-sensitive liposomes. Further study would be carried on, including therelationship between radiation dose/dose rate and the membrane structureconformational change of radiation-sensitive liposomes, the distribution ofradiation-sensitive liposome in the tissues and organs, the mechanisms of anti-tumoreffects and so on.