| Objective:Colorectal cancer(CRC)is a health concern due to its high morbidity and mortality.Over the past 40 years,use of 5-FU as the main chemotherapeutic agent has significantly improved the prognosis of patients with advanced CRC.However,the drug side effects and adverse reactions due to the lack of chemotherapeutic selectivity of traditional chemotherapy agents,as well as the inherent MDR(multidrug resistance)of cancer cells,limit their efficacy.In recent years,it has been found that triptolide has high efficiency and broad-spectrum antitumor activity,and can reverse multidrug resistance of tumors.However,the clinical application of triptolide is seriously limited by narrow therapeutic window and side effects.LA67 is a new antitumor agent with expanded therapeutic window,which is obtained by structural modification of triptolide.Previous studies have shown that the toxicity of LA67 is significantly lower than triptolide,and it has anti-melanoma activity at low concentration.However,due to its poor water solubility,LA67 is difficult to be injected intravenously.Therefore,development of a drug delivery system for intravenous infusion of LA67 is important.In the past two decades,polymeric micelles have been ideal carriers of antitumor drugs.In addition to helping with solubility,polymeric micelles can also improve drug stability,control drug release,optimize drug distribution,prolong blood circulation time,and passively target cancer cells via the EPR(enhanced permeability and retention)effect,thus reducing side effects and improving therapeutic effects.To improve solubility and further increase therapeutic efficacy and reduce adverse drug reactions,the polymer micelles containing the new antitumor drug LA67(LA67-loaded polymeric micelles,LA67-PMs)were prepared by a film hydration method.The physicochemical properties of LA67-PMs were investigated,and the antitumor activity of this formulation against C26(Colon26)cancer cells line was evaluated in vitro and in vivo with LA67 as a control.Methods:1.LA67-PMs were prepared by a film hydration method,and the physicochemical properties were characterized by observing their appearance,electron microscope morphology,particle size,Zeta potential,encapsulation efficiency,loading capacity,reconstitution stability and in vitro release.2.In vitro antitumor evaluation,the uptake of C6-loaded polymeric micelles(C6-PMs)by C26 cells was investigated using C6(coumarin-6)labeled polymeric micelles.In addition,the ability of LA67-PMs to inhibit the proliferation and migration of C26 cells and induce the apoptosis of tumor cells was further investigated using free LA67 as the control.3.In vivo antitumor evaluation,the inhibition effect of LA67-PMs on tumor growth and distant metastasis was investigated,and the potential mechanisms of these effects were further investigated,including induction of apoptosis,inhibition of cell proliferation and neovascularization.Finally,the effect of LA67-PMs on the survival rate of tumor-bearing mice was investigated.Results:1.LA67-PMs were completely dissolved in the aqueous solution,forming a transparent and homogeneous solution.Under TEM,they were spherical or quasi-spherical in appearance and nearly uniform in size.The average particle size of LA67-PMs was 17.88 ± 0.84 nm,the PDI(Polymer dispersity index)was 0.145 ± 0.02,and the zeta potential was-1.72 ± 0.36 mv.Encapsulation rate and drug loading were94.84±1.09% and 1.8±0.01%,respectively.The resolution stability experiment showed that the particle size and PDI of LA67-PMs solution did not change significantly within 7 days,and the stability was good.In vitro release experiments showed that LA67-PMs were released slowly and continuously,and the release behavior was most consistent with Weibull sustained release model.2.The results of a confocal laser scanning microscope showed that the intracellular uptake efficiency of C6-PMs was significantly higher than that of free C6.The results of flow cytometry showed that the fluorescence intensity of C6-PMs uptake by C26 cells was significantly increased,and the C6-loaded micelles could enhance the uptake of C6.Cellular uptake assay suggested that LA67-PMs were more capable of delivering drugs to cancer cells than free LA67,thus facilitating drug accumulation in tumors.Cell counting kit 8 assay results show that the cell viability decreased significantly after incubation with LA67-PMs for 48 or 72 h.Moreover,the inhibitory effect of LA67-PMs on C26 cell proliferation was more significant than that of free LA67 at the same concentration and time of administration.The IC50 of 48 h administration time of LA67-PMs was 21.38 ng/ml.Treatment with blank PMs at concentrations that would contain LA67 at concentrations ranging from 1 to 100 ng/ml resulted in cell viability of 90%,which indicated that the blank PMs exerted minimal cytotoxicity in C26 cells.These results show that PMs are safe drug delivery carriers.To evaluate the impact of LA67-PMs on migration of C26 cells,wound healing assay was performed.The results showed that LA67-PMs could effectively inhibit the migration of C26 cells,and thus significantly reduce the degree of scratch healing.In addition,LA67-PMs induced apoptosis more effectively than free LA67 and blank micelles had little effect on apoptosis.3.By measuring the tumor volume and weight,it was found that LA67-PMs could effectively inhibit the growth of tumor,and the inhibitory effect of LA67-PMs was stronger than that of free LA67.By dynamic observation of the body weight of mice,it was found that the body weight of the LA67 group was significantly decreased,but no significant weight loss was observed in the other three groups,indicating that blank PMs had a low toxicity and could reduce the toxicity of LA67 in vivo.The results of inhibition of metastasis by LA67-PMs showed that liver metastasis was observed in the saline group,the blank PMs group and the free LA67 group,while no obvious abnormity was observed in the liver tissue of the LA67-PMs group.These results suggested that LA67-PMs were more effective in inhibiting distant metastasis than free LA67.The TUNEL assay,CD31 assay and Ki-67 assay were used to observe the induction of apoptosis,inhibition of cell proliferation and neovascularization.The results showed that LA67-PMs could induce cell apoptosis,inhibit neovascularization and cell proliferation,and were more effective than free LA67.This may be the potential mechanism of inhibiting tumor growth,which needs further systematic studies.Finally,the effect of LA67-PMs on the survival rate of tumor-bearing mice was observed.The results showed that the median survival time of LA67-PMs group was significantly longer than that of free LA67 group,the saline group and blank PMs group.Conclusion:LA67-PMs had good physicochemical properties and were easy to prepare.Moreover,they could effectively inhibit the growth and distant metastasis of colon cancer.This paper provides theoretical support for the further research and development of LA67,and LA67-PMs may be a promising formulation for treatment of advanced colon cancer. |
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