Tumor Cell Derived-microparticles Packaged Chemotherapeutic Drugs In Tumor Therapy

Objectives: Microparticles are cell membrane fragments with a diameter of100-1000nanometres, which shed by cells in response to various stimuli such asphysiological and artificial factor. Tumor cells release chemotherapeutic drugsencapsulating microparticles after incubated with chemotherapeutic drugs. Tumorcells derived microparticles can be used as vectors deliver chemotherapeutic drugs totumor site，to find a novel way of tumor therapy.Method:1In vitro experimentMTX (10μgml^-1,30μgml^-1,50μgml^-1) was added to H22tumor cells （1×10⁷） for40minand then treated with UBV. After isolation24h later, MTX-encapsulating MPs wereincubated with3×10⁵H22tumor cells. The death of tumor cells was observed at timepoint of24h.2Treatment of tumor model1)subcutaneous tumor model: BALB/c mice were inoculated with2×10⁵H22cells.MPs-loaded MTX were injected i.v. daily for9days when tumors reach to5×5mm3insize. Record the volumes of the tumors and body weights every day, the mice werekilled at day14, detected ALT and creatinine in peripheral blood.2)myosarcoma model: BALB/c mice were i.m. injected with5×10⁴H22cells at legmuscle,2μgml^-1MTX was added to4×10⁷H22cells in5ml culture medium and theprepared MTX loaded MPs were then administered to one mouse per packaged MPs. On day3after the inoculation, MPs were i.v. injection once per day for10days, andrecord the tumor weight and body weights, after that, sacrifice the mice, detected ALTand creatinine in peripheral blood.3) Hepato-carcinoma ascites model:1×10⁵H22murine hepato-carcinoma tumor cells（BALB/c background） were i.p. injected into BALB/c mice. On day5after theinoculation,2μgml^-1MTX was added to4×10⁷H22cell in5ml culture medium toprepare MTX packaging MPs. The MPs were i.p. injection once per day for10days,after that the mice were fed for the long-term survival study.4) Ovary cancer model:8-week-old nude mice were i.p. injected with1×10⁷A2780cells, On day10after the inoculation, Cisplatin (300μgml^-1) or Paclitaxel(100μgml^-1)combined Cisplatin (320μgml^-1) were added to4×10⁷A2780cells to prepare drugspackaging MPs, the MPs were i.p. injection once per day for7days, after20days,additional treatment was administered once per day for7days. The mice were killedon day30, then move out the tumor and take photoes.3Fluorescence microscope detectingMPs are accumulated in solid tumor site after i.v. injection. MPs isolated from2mgml^-1MTX treated4×10⁷H22cells and then stained with PKH26. PKH26（red）-conjugated MPs were injected to H22subcutaneous tumor-bearing mouse. After4h, tumor tissues were used for the analysis by fluorescence microscope.4MPs countingA flow cytometry-based method was used to count the number of MPs.1×10⁷H22cells treated with10μgml^-1,50μgml^-1MTX, after centrifugation, the MPs weresuspended with PBS that was prefiltered through0.1μm filter and passed through1μmfilter to further exclude background noise or nonspecific events，The MPs mixedevenly with3μm （LB-30） latex beads with a known number. For flow cytometricanalysis,0.8μm deep-blue dyed-latex beads （L1398, Sigma） were first used for gatingand voltage adjustment, as such beads are fluorescent and can be detected on FL4 channel. When the mixture was analysed by flow cytometry, each LB30bead formeda dot in the gate of the large-size population. If10,000counts of LB30were collected,the number of MP can be calculated with formula: N=10,000×（MPs%/beads%）.5HPLCThe concentration of chemotherapeutic drug in MPs was measured by HPLC.1×10⁸H22cells treated with0,50,100,200,400,800μgml^-1MTX, then isolated MPs, andmembranes of MPs damaged by NP-40lysis buffer, and then detected the drugconcentration using High Performance Liquid Chromatograph.Result:1MPs derived from different concentration of MTX treated H22cells result indifferent killing effect, and stronger killing effect together with increasing of MTXconcentration.2After9days therapy, the subcutaneous tumor and myosarcoma volume are smallerthan control，meanwhile, both the weight, Creatinine, and ALT levels are normal. Inaddition, abdominal ascites mouse treated with MP survives longer than the control,and ovary cancer mouse tumor volume are smaller than the control.3Mps stained with PKH26and injected from tail vein,4hours later, sacrificed themouse, tumor tissue analysis by fluorescence microscope, MPs are accumulated insolid tumor site after i.v. injection.4As many as7×10⁵MPs detected from10μgml^-1MTX treated1×10⁷H22cells, and1×10⁶MPs from50μgml^-1MTX treated1×10⁷H22cells.5MPs isolated from1×10⁷H22cells contain the concentration of chemotherapydrugs is one percent of the chemotherapeutic drugs which used to treat the tumorcells.Conclusions: MPs packaged with chemotherapeutic drugs is a novel drug deliverystrategy with potential clinical application and without typical side effect.