| Triple-negative breast cancer is characterized to possess poor prognosis and easily metastasized to brain,lung or bone,which urge to develop a long-acting drug release platform for inhibition of its growth and metastasis.In recent years,tumor immunotherapy research has progressed rapidly,and it has achieved relatively ideal therapeutic effects in the clinic,so it has received widespread attention.Toll Like Receptor(TLR)7/8 agonists have attracted particular attention due to its potent anti-tumor properties and its capacity in reversing tumor immunosuppressive microenvironment.Resiquimod(R848),as a TLR7/8 agonist that can trigger both innate immunity and acquired immune response,which implied tempting potential in utility for anti-tumor immunotherapy.However,most of the dosage forms of R848 are solution preparations,which leads to limited response and often accompanied by systemic side effects during treatment.With the intention of sustainable retention of R848 in the pathologic tumor site,so as for potentiating an adequate anti-tumor microenvironment,it is crucial to develop a sustained-release dosage formulation with excellent biocompatibility.Therefore,a microsphere(Ms)-hydrogel(Gel)composite carrier R848@Ms-Gel,which can release R848 in situ through long-acting and sustained-release manner,was designed and prepared in this study.It is envisioned to accomplish persistent treatment of tumors and cultivate an anti-tumor immune microenvironment for maximized inhibition of the primary tumor growth and preventing the potential tumor metastasis.(1)Preparation and characterizations of R848 polylactic acid-glycolic acid microspheres(R848@Ms)Mixture of R848 and polylactic acid-glycolic acid copolymer(PLGA)in dichloromethane was titrated into high-speed stirred polyvinyl alcohol(PVA-124)to prepare R848@Ms through emulsification.The morphologies of R848@Ms were observed by optical microscope and scanning electron microscope(SEM).The results showed that R848@Ms was spherical and uniformly distributed microparticle with average diameter of approximate44.6 μm.The drug loading and encapsulation efficiency of R848@Ms were determined by high performance liquid chromatography(HPLC)to be 1.65% and 75.90%,respectively.(2)Preparation and characterizations of R848@Ms-hydrogel(R848@Ms-Gel)The four-arm polyethylene glycol acrylate(4-arm PEG-AA)was used as the matrix material for reaction with the crosslinking agent of dithiothreitol(DTT),and the double bond of 4-arm PEG-AA was conjugated with the sulfhydryl group of DTT to yield hydrogel network.Finally,the drug-loaded microspheres and the hydrogel precursor were mixed to form drug-loaded microspheres-hydrogel(R848@Ms-Gel)under photoinitiator and UV photocatalysis.Rheometer was employed to conduct rheological test for analysis of the mechanical properties of the yielded hydrogel.The degradation behavior of hydrogel and the release behavior of R848 were investigated in a simulated humoral environment in vitro.The results showed that the hydrogel can degrade in about 2-3 weeks,and R848 can be sustainably released from R848@Ms-Gel up to 2-3 weeks.Furthermore,biodistribution study by IVIS approved sustainable release at the dosage site for about 20 days.The results of cytotoxicity experiment and living dead cell staining experiment verified the excellent safety properties of our proposed depot of R848@Ms-Gel.(3)Evaluation of the anti-tumor efficacy of R848@Ms-Gel against the primary tumors and metastasisBal b/c mice with subcutaneous tumor was used for evaluation of our proposed immunostimulatory depot for in vivo treatment of triple-negative breast cancer based on 4T1 breast cancer cells.PBS,Free R848 and R848@Ms-Gel were administered by subcutaneous injection next to the tumor about seven days after the tumor was implanted.After the different treatments,blood was taken from the eyes of mice at different time points to detect the levels of immune factors in the serum of mice.It was found that the R848@Ms-Gel group could up-regulate the above immune factors IL-6,IL-12 and IFN-γ.To observe continuously the mouse body weight and tumor growth,and found that the R848@Ms-Gel group can achieve superior anti-tumor efficacy compared with control groups.In addition,using 4T1 cells to construct a lung metastasis model to observe the effect of R848@Ms-Gel in inhibiting tumor metastasis,it was found that the R848@Ms-Gel group had a better inhibitory effect on the lung invasion of 4T1 tumors.In summary,the synthesized R848@Ms-Gel immune hydrogel has appreciable in situ long-acting sustained-release function and biocompatibility,and has potent tumor suppressing ability,which provides important idea for the immunotherapy of triple-negative breast cancer. |
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