Preparation Of Targeted Liposomes Co-loaded With Paclitaxel And Itraconazole And Evaluation Of Their Antitumor Effects In Vitro

Colon cancer is a malignant tumor that occurs in the colon.With high risk of tumor metastasis and recurrence,colon cancer has became one of the leading causes of cancer-related deaths.At present,combined chemotherapy is an important strategy for the treatment of colon cancer.Paclitaxel（PTX）is a first-line clinical chemotherapeutic drug,widely used in the treatment of various cancer types.Itraconazole（ITA）is a broad-spectrum antifungal drug.In recent years,it has been found that ITA could exert a synergistic anti-tumor activity with PTX through inhibiting tumor cell proliferation and angiogenesis.However,the clinical application of these two drugs suffers from multiple problems,such as low solubility,poor oral absorption,and accumulated toxicity in vivo.In this paper,a PTX/ITA-loaded targeting liposome was prepared,which promoted tumor cell uptake of both drugs and inhibited cell proliferation and migration.This article includes the following four parts:1.Establishment of PTX and ITA in vitro analysis methodsThis paper established a dual-wavelength high performance liquid chromatography（HPLC）detection method for PTX and ITA.The method had good detection specificity for PTX and ITA,and had a good linear relationship in the concentration range of 1.0-500.0μg/m L.The intra-day and inter-day precision RSD values were both less than 2%,and the recovery rate reached 98%-102%.Therefore,the detection method established in this paper met the analytical requirements and could be used for quantification of PTX and ITA in liposomes.The drugs could be separated from liposomes through centrifugation at 4000 rpm for 20 minutes,which can be used for the detection of liposome encapsulation efficiency.2.Verification of the synergistic effect of PTX and ITA on inhibiting tumor progression in vitroIn this paper,MTT method was used to investigate the toxicity of combined administration of PTX and ITA on mouse colon cancer cells（CT26）.The scratch test was used to investigate the inhibitory effect of combined therapy on cell migration.The results showed that the survival rate of CT26 cells treated with 0.5μM PTX and 5.66μM ITA decreased to 50%of that in controls.The synergy index was 0.75（less than 1）,indicating that PTX and ITA had a positive synergistic effect on inhibiting cell proliferation.Furthermore,the combined therapy of PTX and ITA also exhibited an enhanced inhibitory effect on tumor cell migration,compared with single drug treatment.3.Preparation of PTX/ITA-loaded liposomesIn this paper,PTX/ITA-loaded liposomes were prepared by ethanol injection method and thin film hydration method.The effects of PC content,drug-to-lipid ratio and PC/Chol on encapsulation rate were detected.We finally used film hydration method to prepare PTX/ITA-loaded liposomes（P/I-Lip）.An orthogonal design was used to optimize the liposome formulation.According to the range analysis,the degree of influence of the three factors on the encapsulation rate was ranked as PC content>Drug-to-lipid ratio>PC/Chol.The optimal formulation was 1.5%of PC content,1:50 of Drug-to-lipid ratio and 15:1 of PC/Chol.Targeted liposomes（FA-P/I-Lip）were prepared by adding DSPE-PEG₂₀₀₀-FA with a molar ratio of 8%to the formulation.The particle size of FA-P/I-Lip was 195.2 nm and the Zeta Potential is-7.67 m V.The distribution of liposomes was uniform and the appearance is round.The encapsulation rates of PTX and ITA were 93.47%and 92.58%,respectively.The liposomes could increase the solubility of PTX and ITA to 29 and 84 times,respectively.FA-P/I-Lip had a sustained-release effect on both drugs within 72 hours.The stability of liposomes was good after stored at 4℃for 2 months.4.Evaluation of in vitro anti-tumor effect of FA-P/I-liposomesThis paper verified cell uptake,cytotoxicity and apoptosis-inducing ability of FA-P/I-Lip on cell level.In the cell uptake experiment,27.7%of the folate-modified targeted liposomes were taken up by the cells,which was about 1.65 times higher than the normal liposome group.Free folic acid could significantly reduce the cellular uptake rate of the targeted liposomes,indicating that the uptake of targeted liposomes by cells was mediated by folate receptors.The results of MTT assay showed that blank liposomes had no obvious toxicity to CT26 cells.Compared with the combination of PTX and ITA,the cytotoxicity of P/I-Lip group was significantly increased（p<0.05）.Besides,the cytotoxicity of FA-P/I-Lip was further enhanced compared with that of P/I-Lip group（p<0.05）.The results of apoptosis analysis showed that the average apoptosis percentage of FA-P/I-Lip group was19.01%,which was 2 times higher than that of P/I-Lip group..In summary,this article explored the analysis methods of PTX and ITA,prepared a targeted liposome delivery system that can carry two drugs at the same time.In vitro results demonstrated that the targeted liposomes had a good biosafety and improved the efficiency of drug delivery.The anti-tumor activity of the combined therapy provided more options for the combined therapy of colon cancer.