Preparation And Pharmacodynamic Study Of Gemcitabine And Docetaxel Co-loaded Liposomal Nanocomplexes

The research content of this topic mainly includes the following three parts:(1)Preparation,characterization,and in vitro evaluation of gemcitabine and docetaxel co-loaded lipid nanocomplexes.The dehydration condensation of GEM with HA was conducted in the DMSO solvent environment under the action of catalyst DCC and DMAP,and a one-step synthesis of the gemcitabine-hyaluronic acid prodrug conjugate HA-GEM,characterized by 1H-NMR and FT-IR.The chemical structure showed that the compound was successfully synthesized,the modification rate was 11.83%,and the mass ratio was 18.61%and the drug loading was relatively high.At the same time,DTX cationic liposome was prepared by thin-film hydration method using DOTAP and cholesterol as carrier materials.The TEM photographs showed that the prepared DTX-CLs were well-rounded with good dispersibility.The drug loading measured by HPLC method was 6.15.± 0.08%,the encapsulation efficiency is 85.98 ± 1.59%,particle size is 149.9 ± 4.6 nm,PDI is 0.19 ± 0.02,zeta potential is 33.3 ± 4.6 mV,surface potential is high,and solution stability is good.Because the prepared HA-GEM prodrug has good water solubility and exists as a polyanion macromolecule in solution,HA-GEM and DTX-CLs are incubated in aqueous solution with slow stirring followed by incubation.Finally,the GEM and DTX co-loaded Combo NCs were successfully fabricated.The size of Combo NCs increased to 186.7 ± 5.3 nm,PDI 0.21 ± 0.03,zeta potential to-31.1 ± 5.1 mV,and it was evident from TEM images that the liposome surface was covered by HA-GEM polymer layer,which proved Combo NCs were successfully prepared and the particle size distribution was uniform.The in vitro release of DTX and GEM was measured by dynamic membrane dialysis and HPLC.The results showed that both drugs released slowly under neutral conditions of pH =7.4,GEM and DTX only released 18.6 ± 2.5%and 37.1 ± 2.6%respectively in the first 4 hours.When pH was 5.0,hyaluronidase was added to simulate the intracellular lysosomal environment,the release of the two drugs was significantly increased,and the cumulative release rate at 72 h reached 81.9 ± 2.3%and 88.1 ± 2.3%,respectively,indicating that Combo NCs have a low pH and enzyme double environmental response release characteristics.Hemolysis experiments showed that even at a high concentration of 500 ?g/mL,the blank carrier had a hemolysis rate of only 3.8 ± 0.6%,indicating good blood compatibility and suitable for intravenous injection.(2)In vitro studies of gemcitabine and docetaxel co-loaded lipid nanocomplexes.In view of the fact that Combo NCs enter the cell with CD44 active targeting,two different breast cancer cells,MCF-7 and MDA-MB-231,were selected as the study subjects.CD44 was labeled on the cell surface by immunofluorescence and flow cytometry was performed.Quantitative analysis results showed that MDA-MB-231 cells CD44 positive expression rate was 10.85 times higher than MCF-7.The results of cell uptake and competitive inhibition experiments showed that the MDA-MB-231 cells had higher fluorescence uptake of Combo NCs,and the fluorescence intensity was greatly decreased after co-incubation with free HA,demonstrating that Combo NCs enter the cell through CD44 active targeting while competitive inhibition of MCF-7 cells was not significant,probably because CD44 expression was not as high as that of MDA-MB-231 cells.CCK-8 cytotoxicity experiments showed that the cytotoxicity of Combo NCs was the strongest,the IC50 at 24 hours was 0.67 ?g/mL,the IC50 at 48 hours was only 0.17 ?g/mL,and the antitumor activity in vitro was significant.At the same time,the synergistic index of the combination of DTX and GEM drugs was analyzed.The CI value was<0.5 both in the formulation and in the free drug form,showing a very strong synergistic effect.Apoptosis of MDA-MB-231 cells was measured by flow cytometry using Annexin V-FITC/PI double staining and the percentage of apoptosis of Combo NCs reached 80 ± 5.12%,which was higher than that of other free drugs and preparations.These results are consistent with cytotoxicity experiments.Due to the high metastasis of MDA-MB-231 cells,in vitro wound healing assay was used to examine the inhibitory effects of each experimental group on cell migration.The results showed that the scratch width of Combo NCs was 0.89 ± 0.07 times that of 0 h.Tumor cells had the most significant metastatic effect,whereas free GEM had no significant anti-rmetastatic effect compared to the control group.The cell cycle and mRNA expression results showed that Combo NCs greatly increased the number of S phase in MDA-MB-231 cells,and by adjusting the dCK/CDA balance,compared with the blank control group,the expression of CDA was decreased to 0.09 times,and at the same time The expression of dCK increased to 1.36 times.Overall,the ratio of dCK/CDA reached 15.3 times that of the blank group.The increase was significant,which promoted the accumulation of active metabolites of GEM and enhanced the efficacy.(3)In vivo studies of gemcitabine and docetaxel co-loaded lipid nanocomplexes.In this section,an animal model of tumor-bearing nude mice was established by subcutaneously injecting cell suspensions using human triple negative breast cancer cell line MDA-MB-231.The tumor bearing nude mice were used for the following experiments.DiR-loaded cationic liposomes(DiR-CLs)and HA-modified DiR-loaded lipid nanocompolexes(HA/DiR-CLs NCs)were prepared using DiR near-infrared fluorescent probes instead of drugs.In vivo and ex vivo fluorescence imaging results were obtained.The HA/DiR-CLs NCs showed strong tumor targeting in vivo.The fluorescence intensity at the tumor site was 4.3 times that of the free DiR group,while the fluorescence intensity of the DiR-CLs group was the lowest,0.3 times that of the free DiR group.In vivo tumor inhibition rate and histopathology experiments showed that Combo NCs did not reduce the body weight of nude mice and had no obvious toxicity to normal organs and tissues,but the body weight of nude mice injected with free Combo decreased significantly.After the end of the chemotherapy cycle,the liver and kidneys were damaged to some extent,indicating that free DTX and GEM are not suitable for direct administration.TUNEL in situ apoptosis staining and Ki67 immunohistochemistry were used to further study the proliferation and apoptosis of tumor cells after administration.The results showed that the tumor cells in Combo NCs group had the highest degree of apoptosis,reaching 24.7 ± 2.1%and the lowest level of proliferation,reaching only 9.2 ± 1.8%.