| Cancer is the leading threaten diseases to the health of developed countries human life and the second leading cause of death in developing countries.In recent years,with the acceleration of population growth and aging,and the neglect of healthy lifestyle,the incidence of cancer has being increased.Researchers found that the key to cure tumors is existing in eliminating tumor stem cells which were the root cause of tumor and postoperative recurrence.With the gradual development of the medical level,the tumor treatments are also improving,and currently,the extensive research is to combine the anticancer drugs with microspheres drug delivery system technology.Multi-functional microspheres drug delivery system to some certain extent,can overcome the drug resistance and selectively delivery drugs to tumor tissue which reduces the toxicity to normal tissues.They can achieve long-term antitumor effect through sustained drug release,thus becoming a new method for the treatment of tumor related diseases.As a new chemotherapy drug paclitaxel(PTX)has shown significant antitumor efficacy and prevention of multidrug resistant against various malignant tumors.However,the pharmacological mechanism of antitumor drugs(PTX)is more complex,and there is very little research focused on the in vivo anti-effect and mechanism towards gliomas and liver tumors.The purpose of this paper is to prepare a novel morphological PTX loaded poly(lactide-co-glycolide)(PLGA)microspheres and then evaluate their drug capacity and drug release properties,as well as the in vitro and in vivo anti-tumor effect of drug loaded microsphere.Furthermore,the drug release and tumor cell proliferation inhibition mechanism of the drug release system were systematically studied to provide experimental basis for further tumor therapy application.In this paper,different glycolide(GA)/lactide(LA)proportion of PLGA polymers were synthesized by ring opening polymerization method using high purity LA and GA.Than their crystallization behaviors,thermal properties,and in vitro degradation and cytotoxicity performance were systematic studied.Thereafter,PLGA(LA/GA=75/25)was selected as the drug carrier for the drug-loaded microspheres preparation and anti-tumor research.The novel morphological PTX-PLGA-MS were prepared using double emulsion method based on solvent evaporation as previously described with modification.The internal water phase ABC solution and oil phase PTX and PLGA DCM solution were prepared and formed as homogeneous emulsion.Then,the resultant emulsion was transferred into external water phase PVA solution under magnetic stirring,and was continuously stirred to remove the solvent.The solidified drug-loaded MS were obtained through centrifuging,washing and lyophilizing.FT-IR and ~1H NMR observations indicated that PTX was successfully encapsulated into PLGA-MS.SEM and SR-?CT graphs showed that the smooth PTX-PLGA-MS were smooth and spherical in shape with internal sporadic porosity.While,the porous PTX-PLGA-MS exhibited microporous surface and porous internal structures.In vitro release analysis showed that the 48 h cumulative free PTX release amount was more than 88%.While,the porous PTX-PLGA-MS showed initial slow release,then they achieved a rapid sustained drug release.A systematic evaluation including:cell cytotoxicity test,flow cytometry,fluoroimmunoassay,and western blot analysis were performed to evaluate the antitumor effect of porous PTX-PLGA-MS.The antitumor results showed that the porous PTX-PLGA-MS displayed stronger anti-tumor activity in a dose-and time-dependent manner.The porous PTX-PLGA-MS with better sustained drug release effect could achieve long-time inhibition potency to the tumor cell proliferation and little cytotoxicity to normal cells with low dose.Results indicated that the PTX released from porous PTX-PLGA-MS can arrest U251 cell division in G2/M phase and promote the microtubules polymerization and stability,thus disturbing the stable cell division and showing a prolongation of tumor cell apoptosis.Additionally,The cells in free PTX and porous PTX-PLGA-MS treated groups showed a higher expression of Bax and Cyclin B1 than that of control group.Therefore,the activation of pro-apoptotic signaling and stabilization of microtubules are thought to be the principal mechanisms for the cytotoxic action of PTX drugs in inhibiting motility and invasion of U251 cells.To determine the in vivo therapeutic efficacy of porous PTX-PLGA-MS,the tumor xenograft models were created by subcutaneously inoculating freshly harvested HepG2 cells in BALB/c-nu mice.When the average tumor volume reached a value,the mice were randomly divided into four groups(smooth and porous PTX-PLGA-MS,free PTX,saline).The intratumoral injection cycle of porous PTX-PLGA-MS were set as 12 days(released drug amount for 88%),while the saline and free PTX were injected every four days.After injections,the tumor volumes and mice weight were recorded every four days,and at the end of the experiment,the excised tumors were photographed and weighted.Then the tumor tissues were evaluated by hematoxylin and eosin(HE)and TUNEL staining,Quantitative Real-Time PCR(qRT-PCR)and Western-blot analysis.In vivo antitumor results showed that the porous PTX-PLGA-MS displayed better tumor suppression effect than free PTX and smooth PTX-PLGA-MS.The porous PTX-PLGA-MS can sustainably and rapidly release drugs to the tumor cells.The in vivo cell apoptosis and cycle related gene and protein expression results of porous PTX-PLGA-MS were consistent with their in vitro results.Additionally,different from the toxic side effects of free PTX group,the porous PTX-PLGA-MS showed superior anti-tumor effect and lower toxic side effects.The developed porous PTX-PLGA-MS with microporous surface and porous internal structure achieve remarkable and safe efficient purpose of cancer therapy.At the same time,the drug release process in the body and the in vivo anti-tumor mechanism of the porous PTX-PLGA-MS should be further researched. |
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