Tumor Cell Derived-microparticles Packaged Chemotherapeutic Drugs in Tumor Therapy

Author :XuPingWei
Tutor:HuangBo
School :Huazhong University of Science and Technology
CLC :R730.54
TYPE :Masters thesis
Download the PDF Full Text:http://www.topresearch.org/showinfo-171-677768-0.html
Year:2012
Abstract:
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 (10gml-1,30gml-1,50gml-1) was added to H22tumor cells 1107
for40minand then treated with UBV. After isolation24h later, MTX-encapsulating MPs wereincubated
with3105H22tumor cells. The death of tumor cells was observed at timepoint of24h.2Treatment of
tumor model1)subcutaneous tumor model: BALB/c mice were inoculated with2105H22cells.MPsloaded MTX were injected i.v. daily for9days when tumors reach to55mm3insize. 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
with5104H22cells at legmuscle,2gml-1MTX was added to4107H22cells 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:1105H22murine hepato-carcinoma tumor cells BALB/c
background were i.p. injected into BALB/c mice. On day5after theinoculation,2gml-1MTX was
added to4107H22cell 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 with1107A2780cells, On day10after the
inoculation, Cisplatin (300gml-1) or Paclitaxel(100gml-1)combined Cisplatin (320gml-1) were
added to4107A2780cells 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
treated4107H22cells and then stained with PKH26. PKH26red-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.1107H22cells treated with10gml-1,50gml-1MTX, after centrifugation, the MPs
weresuspended with PBS that was prefiltered through0.1m filter and passed through1mfilter to
further exclude background noise or nonspecific eventsThe MPs mixedevenly with3m LB-30
latex beads with a known number. For flow cytometricanalysis,0.8m 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.1108H22cells treated
with0,50,100,200,400,800gml-1MTX, then isolated MPs, andmembranes of MPs damaged by NP40lysis 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 controlmeanwhile, 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 as7105MPs detected from10gml1MTX treated1107H22cells, and1106MPs from50gml-1MTX treated1107H22cells.5MPs
isolated from1107H22cells 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.