| Cancer is one of the most serious diseases that threaten the human's health,for which the incidence and mortality of colorectal cancer(CRC)rank third among all of the cancer,and show an upward trend nowadays.Due to the symptoms on CRC is ambiguous and hard to be distinguished at the early stage,nearly half of the patients have suffered the tumor metastasis once diagnosis,and the 5-year survival rate of patients on metastatic CRC is only around 10%.Chemotherapy is one of the most important methods for the treatment of CRC.especially for those patients who are bearing with metastatic CRC and cannot accept a surgical resection or radiotherapy.However,the therapeutic efficacy of single chemotherapeutic drug is often limited and may cause multidrug resistance,tumor tolerance and recurrence,therefore,combinational stragegy is widely applied for the first-line treatment of CRC.Among that,oxaliplatin(OXA)and irinotecan(IRI)combinational scheme is recommended inthe "Guidelines for clinical treatment of CRC''by Comprehensive National Cancer Network(NCCN).Although the combination on OXA and IRI can both act with DNA of the cells with different mechanisms and hence generate the synergistic effect,there are series of defects in both drugs.such as low drug tolerance,non-specific biodistribution,multidrug resistance and unexpected pharmacokinetics,which may cause serious damage to normal tissue when killing tumor cells.Additionally,the combination of both drugs will further aggravate the toxic side effects of the body,and hence limit its clinical applications.On the other hand,traditional approach in clinic is just simply a cocktail,and thus the release of drugs in viv ocan not be kept in control.Due to the different pharmacokinetics of each drug,different drug ratios may play a synergistic,additive,or even antagonistic effect,and thus their combination may lead to the uncertainty of therapeutic effect.Consequetly,it would becruical to build a noval strategy to improve the therapeutic effect on CRC and reduce the side effects of the two drugs.Drug delivery system(DDS)provides a good opportunity to overcome the disadvantages of the chemotherapeutics,and it has become a hot topic in pharmaceutical field to delivery or co-delivery the drugs by the proper nanocarriers.There are three strategies to achieve the combination therapy by DDS,first is "cocktail strategy",for which one free drug is combined with another drug loaded in nanocarriers;second is"mixture strategy",for which both drugs are delivered by separate nanocarriers;third is"co-encapsulation strategy",for which both drugs are co-encapsulated in the same nanocarrier.During the delivery process of the drugs in vivo,the ratios of both drugs in the specified site would be random in both cocktail and mixture strategies,while the ratio can be fixed in co-encapsulation strategy and thus an optimal synergistic effect can be achieved.Lipid-based nanocarriers are one kind of the fundamental carriers for building the DDS with the liposomes and lipid emulsions(LEs)as typical representatives.Lipid-based nanocarriers have desired safety,satisfied biocompatibility and biodegradability,and many liposomal and lipid emulsions' products have been approved in clinics,showing great application prospects.In this study,in order to improve the therapeutic efficay of CRC and reduce the side effects of chemotherapeutics,liposomes and LEs are selected as the nanocarriers and two types DDS co-encapulated with OXA and IRI were built.The enhanced therapeutic effect of CRC was realized by controlling the release of two kinds of drugs in vivo and effectively delivery of the two drugs into the tumor cells synchronously.Furthermore,the differences in therapeutic efficacy betwen mixture strategy and co-encapulation strategy in DDS were further explored and analyzed,which would provide theoretical basis on the design of drug co-delivery systems.The main contents and results in this study are listed as follows:PART ONE:preparation,characterization and evaluation of OXA and IRI co-loaded liposomesChapter 1.Determination method of OXA and IRI were established by HPLC and UV-Vis,respectively.Both drugs showed good linear relationships under determinated concentrations,and the precisions and recoveries could meet the requirements.The encapsulation efficacy of OXA in liposomes was established by both dialysis and Sephadex G25 methods,aslo the encapsulation efficacy of IRI was established by Sephadex G25 method.Both drugs could be well seprated from the liposomes and the recoveries could meet the requirements.Chapter 2.The synergistic effect of OXA and IRI was verified by both Gaddum model and CI index methods,and both drugs have maximum synergistic effect at OXA/IRI molar-ratios from 1:1 to 1:1.5.OXA/IRI co-loaded liposomes were prepared by ethanol injection followed with ammonium sulfate gradient method,with particle size less than 200 nm,PDI less than 0.2,and well-formed spherical shape.In vitro release study showed that the release behavior of the two drugs include the initial burst release and subsequent sustained release of the release of the two parts,and co-loaded liposomes can better control the molar-ratios of two drugs within the optimal synergistic ranges.Chapter 3.In vitro cytotoxicity study showed that co-loaded liposomes had stronger cell killing ability.In vivo antu-tumor activity experiment was investigated in CT-26 tumor bearing mice,and co-loaded liposomes can significantly enhance the anti-tumor activity compared with mixture solution and mixture liposomes(p<0.05).Near-infrared fluorescence imaging was carried out after the mice were treated with different DiR formulaitons,and the results showed that liposomes could increase the accumulation of drugs in the tumor site.By labeling liposomes with Rhodamine B and FITC as fluorescent dyes,the co-delivery ability of the liposomes were evaluated through in vitro cell uptake and in vivo cryo-sections observation,the results showed that co-loaded liposomes had higher co-delivery efficiency than mixture formulations.Additionally,body weight of tumor bearing mice after administrated with co-loaded liposomes were obviously higher that of mixture solutions,as well as abnormal morphology of the mice could be seen in heart,lung and spleen tissue by HE staining for mixture solution group,indicated that liposomes have superior safety and can decrease the side effects of the drugs.PART TWO:preparation,characterization and evaluation of OXA and IRI co-loaded LEsChapter 4.Oxaliplatin phophoslipid complex(OPPC)and irinotecan phophoslipid complex(IPPC)was successfully formulated,and the optimal procedure was investigated.The prepared OPPC and IPPC exhibited good stability within measured 7 days.OPPC and IPPC were then characterized by DSC curve,in which the endothermic and exothermic peaks were significantly changed compared with the free drug,proving the successful formulation of the complexes.Chapter 5.OXA and IRI co-loaded LEs was successfully prepared by drug-phophoslipid complex technique,and the the optimal formulation is:MCT selected as the oil phase and the proportion of MCT 5%;Pluronic F68 2%;glycerol 2%and oleic acid 0.125%,respectively;emulsification temperature at 60?;homogenization pressure at 1000 bar for15 cycles.The prepared co-loaded LEs have particle size around 100-120 nm,PDI less than 0.2,zeta potential around-20 mV,also with good stability.Long-term stability study showed that the co-loaded LEs could remain stable without obvious particle size changes after stored under 4 degrees for three months.In vitro release study showed that LEs could control the sustained release of drugs,and the cumulative release of both drugs in both single and co-loaded LEs was more than 80%and 70%within 48 h,respectively.Comparative release behaviors of both drugs indicate that LEs can control the simultaneous release of two drugs and achieve the optimal synergy.Chapter 6.In vitro cytotoxicity study showed that blank LEs had desired safety,although co-loaded LEs had lower cell viability than mixture solution and mixture LEs,no statistical difference existed(p>0.05).In vivo anti-tumor activity experiment showed that co-loaded liposomes can significantly enhance the anti-tumor activity compared with mixture solution and mixture LEs(p<0.05),additionally,LEs could reduce the side effects of the mixtue solution.Near-infrared fluorescence imaging also verified that LEs could increase the accumulation of drugs in the tumor site and sustain the metabolism of drugs.By labeling liposomes with Dil and DiO as fluorescent dyes,the co-delivery ability of LEs were evaluated through in vitro cell uptake and in vivo cryo-sections observation,the results also showed that co-loaded LEs had superior co-delivery efficiency than mixture formulations,herein generate the optimal synergistic effect. |
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