| Purpose To synthesize a p H-responsive dextran-based doxorubicin-loaded nanodrug and evaluate its antitumor performance.Method1.Drug preparation and properties evaluation:(1)Prepare DEX-Br and MGMA and synthesize DPP via a one-step ATRP.Then Analyze its chemical and physical properties using ~1H NMR and GPC.(2)To obtain DOXDT prodrug through reaction between DPP and doxorubicin hydrochloride(DOX)and then characterized by 1H NMR.(3)DOXDT micelles are prepared from prodrugs and Lip@DOX is obtained using the commercial liposome DOX preparation method.The morphology and size of DOXDT are measured by transmission electron microscope(TEM)and dynamic light scattering particle size analyzer(DLS).(4)Detect the release curve of DOXDT at different p H values.2.In vitro experiment:(1)Culture 4T1 and He La cells.(2)MTT and live/dead stain test to measure DOXDT cytotoxicity.(3)Cell fluorescence imaging quantitatively study the cell uptake of DOXDT.(4)Establish a 4T1 multicellular spheroids model(MCS)to simulate solid tumors to explore the penetration ability of DOXDT.3.In vivo experiment:(1)Establish 4T1 tumor-bearing mouse model.(2)Routine blood test to evaluate the blood compatibility of DOXDT.(3)Near-infrared imaging to study the in vivo distribution of Lip@DOX and DOXDT.(4)Four control groups were set up by injecting saline,free DOX,Lip@DOX and DOXDT.Monitor body weight,tumor volume and tumor weight.Compare the H&E staining of tissue sections of major organs(heart,liver,spleen,lung,kidney),and H&E,TUNEL,DAPI staining of tumor in different group to evaluate the antitumor ability and toxicity of DOXDT.Result1.Drug preparation and properties evaluation:(1)DPP was successfully prepared and~1H NMR showed characteristic peaks of MGMA.GPC showed a polymer dispersibility index of 1.8.(2)The ~1H NMR spectrum of DOXDT showed the characteristic peaks of hydrazone bonds.(3)TEM showed that DOXDT micelles are uniformly distributed and spherical with a diameter of 26.90±2.01 nm.DLS showsed that DOXDT has a hydradynamic diameter of 32.4nm and remains stable for 7 days,carrying a negative Zeta potential.(4)The DOXDT release amount was 72.43%,28.97%,15.71%at p H 5.0,6.8,and 7.4,respectively.2.In vitro experiment:(1)MTT showed that the viability of 4T1 and He La cells in the DPP group at 72h was still greater than 80%,and the viability of the DOXDT group was only about 30%.The ratio of red and green fluorescence in the DOXDT and free DOX groups at 24h in the live-death staining test was similar.(2)CLSM observed the red fluorescence of DOX in the cells of DOXDT and free DOX group at2h,and the intracellular fluorescence intensity of DOXDT group increased with time.(3)The cell uptake rate of DOXDT group reached 91.65%within 6 hours measured by flow cytometry.(4)The red fluorescence intensity of each layer of 4T1 MCS in the DOXDT group was stronger than that in the Lip@DOX group.3.In vivo experiment:(1)There was no obvious abnormality in blood routine of DOXDT group.(2)The fluorescence signal of tumor and major organs in DOXDT group was stronger than that in free DOX group.(3)DOXDT group has the smallest tumor volume increase and weight,and no significant decrease in body weight at the end of experiment.H&E and TUNEL staining of tumor tissues in this group indicated the widest range of cell apoptosis.The strongest DOX red fluorescence can be seen under tumor DAPI staining,and no obvious cell apoptosis can be seen in organ H&E staining.Conclusion1.DOXDT has good structural stability and biological safety.The negative Zeta potential would enhance its stability in blood circulation and prolong blood circulation time.2.DOXDT exhibits selective drug release behavior regulated by p H value.The releasing velocity can accelerate under the acidic tumor microenvironment and achieve tumor targeting effect.3.Compared with liposomal doxorubicin,DOXDT shows good tumor permeability,selective drug release performance in vivo and in vitro,exhibiting stronger anti-tumor activity and less damage to normal tissues and organs. |
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