Construction And Evaluation Of A Microenviron- Ment Responsive Nano Drug Delivery System For Ovarian Cancer In Vitro

Objective: A dual responsive nano-drug delivery system of matrix metalloproteinase and ATP was constructed in the microenvironment of ovarian cancer.Doxorubicin hydrochloride(doxorubicin hydrochloride,DOX)embedded in C?G base pairs of DNA double helix structure,to improve its curative effect in the treatment of ovarian cancer,reduce the side effects of traditional chemotherapy.Methods: 1.A method for direct fluorescence analysis of DOX was established.2.Preparation of tumor microenvironment responsive micelles.With Matrix metalloproteinases-9(MMP-9)substrate specificity of cracking of polypeptide(GPQGIAGQR)as a connecting arm,connecting Polyethylene glycol(PEG)and ATP responsiveness Aptamer(apt)complementary DNA(complementary DNA and c DNA),and connect again Polycaprolactone(PCL)apt hybrid complementary,PCL-NDAPeptide-PEG was synthesized by self-assembly of MMP-9 and ATP responsive polymers in aqueous solution.The nano-micelles PCL-DNA/Dox-Peptide-PEG were prepared by embedding adriamycin hydrochloride into the C?G base pairs of DNA double helix structures.ATP-responsive nanomicelle PCL-DNA/Dox-PEG was also prepared.The characterization(particle size,potential,surface morphology,etc.),drug loading and drug release behavior of the nanoparticles were analyzed.3.CCK-8 method was used to detect the survival ability of mice ovarian epithelial tumor cells treated with nano-micelles.4.Confocal microscopy was used to localize and analyze the uptake of the nanomicelles by ID-8 cells.5.The uptake of nano-micelles by ID-8 cells was quantitatively analyzed by flow cytology.6.The apoptosis of ID-8 cells was analyzed by TUNEL assay.Results: 1.DOX concentration within the range of 0.08-4.93?M was linearly related to the fluorescence intensity,R2=0.998,indicating that the method was simple,accurate and met the experimental requirements.2.Nano-micelles were prepared by Peptide reaction.The MMP-9 substrate polypeptide was linked with PEG and c DNA,complemented with apt linked with PCL,and embedded with DOX to self-assemble into a double-responsive NM PCL-DNA/DoxPeptide-PEG.Single responsive NM PCL-DNA/Dox-PEG was prepared by the same method.The particle sizes of the two nanoparticles were evenly distributed and similar in size,and the drug loading binding rate was consistent with the drug release behavior.3.CCK-8 vectors with different concentrations showed no difference in the proliferation of ID-8 cells,P=0.099>0.05,R2=0.305.The cell survival rate of the vector on ID-8 cells was 79.9±1.0%.DOX preparations with different concentrations had significant statistical significance for the survival rate of ID-8 cells,P=0.000 < 0.001,R2=0.976.Comparison of cytotoxicity by group: ATP&MMP Group ? ATP Group> Free DOX Group.4.Confocal microscopy was performed to observe the significant difference in DOX uptake of ID-8 cells in different treatment methods,P=0.000 < 0.001,R2=0.996,that is,for ID-8 cell DOX uptake in Free DOX Group > ATP&MMP Group > ATP Group.5.The average fluorescence intensity of intracellular DOX in different groups was significantly different by flow cytological analysis,P=0.000< 0.001,R2=0.962,that is,for ID-8 cell DOX intake:Free DOX Group > ATP&MMP Group > ATP Group.The results were consistent with confocal microscopy.6.The apoptosis of ID-8 cells under different treatment methods was significantly different by TUNEL assay(P=0.000<0.001,R2=0.996).Pairwise comparison result showed that the ATP&MMP group was higher than the ATP group and the Free DOX group,and the P value was the same,P=0.000<0.001,while the ATP group was slightly higher than the Free DOX group,but there was no statistic difference between the two groups,P=0.570>0.05.Conclusion: The self-assembled double-responsive NM(PCL-DNA/Dox-Peptide-PEG)has good particle size,stability,no obvious cytotoxicity and anti-tumor effect,and is expected to have good tumor tissue permeability and less toxic and side effects of normal organs.In vivo evaluation of this dual responsive delivery system for ovarian cancer microenvironment was based on experiments.The dual responsive NM PCLDNA/Dox-Peptide-PEG may be one of the treatment methods for ovarian cancer.