Construction Of Enzyme-responsive Drug Delivery System And Its Application In Tumor Treatment In Vitro And In Vivo

Objective:To design a drug delivery system which responses to MMP,2 in order to improve the ability of drug penetration into tumor tissue,the efficacy of antitumor drugs and to reduce the side effects on normal tissues and organs.Methods:1.The method of analysizing doxorubicin in vitro was established by UV-Vis spectrophotometry.2.The MMP-2 enzyme-responsive tumor targeted drug delivery system based on hydrophilic hyaluronic acid(HA)and spherical PAMAM was chemically conjugated through the MMP-2-sensitive linker Gly-PLGLAG-Cys.MMP-2 non-responsive nanoparticles HA-PAMAM were directly synthesized through the reaction between the aldehyde group of HA and the amino group of the PAMAM.The surface morphology(TEM),particle size(DLS),Zeta potential(DLS),drug-loading and drug-release behavior and the responsive ability to MMP-2 of the carriers were investigated.Examine the viability of the A549 cells(MMP-2 were over-expressed)and MCF7 cells(MMP-2 were low-expressed)by CCK-8 method after being treated with different praparations for 48h;4.Investigate cellular uptake of nano preparations with different treatment by flow cytometry;5.Establish the 3D tumor spheroids and evaluate the effect on the penetration of drugs to A549 tumor spheroids by laser scanning confocal microscopy;Investigated the inhibitory effect of the preparations with different treatment on the growth of tumor spheroids.6.Establish A549 tumor-bearing mice to evaluate the targeting effect,anti-tumor effect and the safety of the drug delivery system in vivo.Results:1.The method to determine DOX was established by UV-Vis spectrophotometer.The method was simple,accurate and sensitive,which meets the requirements of the experiment.2.Using the MMP-2 substrate polypeptide(Gly-PLGLAG-Cys)as the linker,the DOX-containing PAMAM was successfully conjugated with the hydrophilic polysaccharide HA,which could response to the tumor microenvironment.Meanwhile,HA-PAMAM was successfully prepared without the MMP-2-sensitive linker.Both of the prepared nanocarriers were spherical in shape,with the same particle size and drug loading and release behavior.In addition,by CTAB turbidity method,HA-pep-PAMAM was able to cleave the substrate polypeptide incubated with MMP-2,and then HA was removed,releasing the small-sized PAMAM.3.For A549 cells which have higher expression level of MMP-2,HA-pep-PAMAM/DOX preparations in different conditions had no significant difference in cell proliferation.For MMP-2 low-expressed MCF7 cells,the cytotoxicity of MMP-2-pretreated HA-pep-PAMAM/DOX preparations was significantly higher than that of HA-pep-PAMAM/DOX and HA-PAMAM/DOX,both of them couldn't degrade into small particles.4.Flow cytometry results showed that the cell uptake of HA-pep-PAMAM/DOX on A549 and MCF7 cells was significantly higher after being pretreated with MMP-2,indicating the responsive ability to the enzyme of HA-pep-PAMAM/DOX.5.Laser scanning confocal microscope results showed that HA-pep-PAMAM/DOX had a strong effect on the penetration of DOX into the 3D tumor spheroids after incubating with enzyme for 4h and 24h.The results of tumor growth inhibition by different preparations showed that the structure of the tumor spheroids were loose and cells on the edge were dead under the action of doxorubicin.Meanwhile,the suppression effects of HA-pep-PAMAM/DOX was slightly stronger than HA-PAMAM/DOX group.6.Compared with ICG solution and PAMAM/ICG,HA-pep-PAMAM/ICG and HA-PAMAM/ICG showed better ability of tumor targeting and accumulation in tumor sites.For the anti-tumor effect,compared with PBS group and HA-pep-PAMAM group,HA-pep-PAMAM/DOX group could effectively inhibit the growth of tumor in vivo.Furthermore,compared with DOX solution,the antitumor effect of HA-pep-PAMAM/DOX was more significant,and the tumor targeting ability of the drug delivery system also reduce the side effects and toxicity of free doxorubicin on the heart and liver.Conclusion:HA-pep-PAMAM/DOX drug delivery system enhanced the ability of DOX penetration into tumor tissue,the efficacy of antitumor drugs and reduced the side effects on normal tissues and organs.The enzyme-responsive delivery system has a good proepect on the research of targeted tumor therapies.