Microspheres Encapsulating Combretastatin A4 Phosphate (CA4P) For Tumor Therapy

Combretastatin A4(CA4)is a cis stibene isolated from the African shrub,Combretum caffrum.And it is known to be one of the most active compounds of tubulin inhibitors.Combretastatin A4 phosphate(CA4P)is the water-soluble phosphate pro-drug of CA4 and its anti-tumor mechanisms mainly include tubulin polymerization inhibition and anti vascular effect.CA4P selectively binding to microtubulin,preventing the formation of mitotic spindle to block tumor cells divisionThe results of pharmacokinetic experiments showed the quite short half-life(～30min)and the quick elimination rate from the body.These drawbacks cause frequent dosing,increasing the suffering of patients and negative patient compliance,which significantly limits its application.Therefore,it is beneficial to develop a sustained release preparation of CA4P with relatively long duration of action and sustained activity.This article innovatively presents the drug delivery system of reverse micelle-in-microspheres.We adopts the amphiphilic copolymers of PELA to encapsulate CA4P,thus forming reverse micelles(CA4P-RM)through self-assembly.And then we use PLGA to encapsulate reverse micelles by emulsion-solvent evaporation.This novel composite Nano-in-Micro system(CA4P-MS)has not only achieved the sustained release of CA4P.but also solved the problem of the poor encapsulation effiency of conventional microspheres for water-soluble small molecules.The specific research work is divided into the following three parts1、The amphiphilic copolymers of PELA was synthesized by ring-opening polymerization.The results of 1H NMR and GPC proved the success of synthetic PELA with specific molecular weight.The DSC analysis results showed the excellent compatibility between PELA and PLGA.We established the method of content determination for CA4P and observed the physical form,particle sizes of CA4P-RM and CA4P-MS.CA4P-RM displayed the discrete and spherical morphology with a narrow size distribution.CA4P-MS had spherical outline and smooth and intense surface devoid of any obvious defects.The analysis results of XRD and CLSM proved that CA4P was distributed wildly throughout the whole microspheres matrix.Taking the encapsulation efficiency(EE)as the test indicator,we performed the single factor tests and the orthogonal design to opitimize the conditions for the process:the molecular weight of PLGA(20k),LA/GA ratio(50:50),the feed mass of PELA/PLGA(w/w)(1:1),the homogenization speed(4k rpm);the affecting degree towards EE:LA/GA ratio>homogenization speed>feed mass of PELA/PLGA>molecular weight of PLGA2、We simulated the normal physiological environment and invesigated the in vitro release and degradation of microspheres.The typical triphasic profile of CA4P-MS was observed with:an initial burst release phase,a lag phase and a sustained release phase.Besides,we inspected the physical form of CA4P-MS during different release phases.Over the release time,noticeable changes occurred including surface morphology,particle roundness and dispersibility.We examined the changes of swelling rate during the different release phases and the results were considered to be an important corroborative evidence for the explanation of the release behaviour and degradation.In addition,we determined the changes of surface morphology,particle sizes,EE and in vitro release profile under different storage temperatures(room temperature and 4℃).And the results proved that the lyophilized CA4P-MS had quite stable property and were easy to preserve,which made a lot of sense for industrial production3、The cytotoxicity of CA4P,blank MS and CA4P-MS toward S180 cells were determined by the MTT assay.The results showed that the blank MS exhibited low cytotoxicity and excluded the influence of polymers.Compared with CA4P,CA4P-MS evidently increased inhibiting tumor effects.The IC50 values of CA4P and CA4P-MS were 0.19 and 0.098μg/mL.The in vivo tumor inhibition demonstrated that the tumor sizes rapidly increased in CA4P solution group when administration terminated.While the CA4P-MS group provided continuous tumor suppression.In addition,no mice died and a steady increase of mice body weight was observed,which suggested the safety for its in vivo application.The pharmacokinetics results showed that the plasma drug concentration of CA4P solution group increased sharply and dropped rapidly during each administration period,while the CA4P-MS group maintained at a relatively stable level.The results of immunostaining of CD31 and Ki67 indicated that CA4P-MS had not only antiangiogenic effects but also synergistic anti-tumor.