Injectable Hydrogel Loaded With Paclitaxel And Epirubicin To Prevent Postoperative Recurrence Of Breast Cancer

Objective:An injectable hydrogel system co-loaded with paclitaxel(PTX)nanoparticles and epirubicin(EPB)was prepared,and the efficacy of the system in preventing postoperative recurrence of breast cancer was investigated.Methods:In this study,we firstly loaded PTX into triblock copolymer poly(ε-caprolactone)-polyethylene glycol-poly(ε-caprolactone)(PCL-PEG-PCL,PCEC)to prepare drug-loaded nanoparticles(PPNPs).Their particle size,Zeta potential,dispersion index(PDI)and stability were detected by dynamic light scattering(DLS),and their morphologies were observed by transmission electron microscopy(TEM).The drug loading rate and encapsulation rate were analyzed by chromatography(HPLC).Meanwhile,we prepared hyaluronic acid-tyrosine conjugate(HA-Tyr)by dialysis,and verified its successful preparation by hydrogen nuclear magnetic resonance spectroscopy(1HNMR).Then,a local injectable hydrogel system(PPNPs/EPB@HA-Gel)was constructed by compounding HA-Tyr,paclitaxel nanoparticles,and epirubicin.The in vitro drug release properties of paclitaxel nanoparticles(PPNPs),paclitaxel nanoparticles and epirubicin-loaded hyaluronic acid hydrogel system(PPNPs/EPB@HA-Gel)were analyzed by dialysis.The in vitro cytotoxicity of blank PCEC nanoparticles(Blank-NPs)and blank PCEC nanoparticle hydrogel system(Blank-NPs@HA-Gel)was investigated by methylthiazolyl tetrazolium(MTT)method.Flow cytometry was used to analyze the apoptosis induction of different treatment groups on 4T1 breast cancer cells.The anti-proliferative effects of different treatment groups on 4T1 breast cancer cells were compared by scratch test,and the drug uptake of different treatment systems was observed by fluorescence microscope.The degradability of the hydrogel was evaluated by dynamic observation of the remaining amount of the hydrogel after subcutaneous injection of Blank-NPs@HA-Gel,and its biocompatibility was verified by analyzing the results of hematoxylin-eosin staining(HE staining)of the implanted tissue.In the in vivo study,BALB/c mice 4T1 breast cancer postoperative models were divided into 7 groups:(1)normal saline group(Control),(2)blank nanogel group(Blank-NPs@HA-Gel),(3)Free double drug group(Free PTX/EPB),(4)Epirubicin hydrogel group(EPB@HA-Gel),(5)Paclitaxel nanoparticle gel group(PPNPs@HA-Gel),(6)Double-drug hydrogel-loaded postoperative group(PPNPs/EPB@HA-Gel),(7)Double-drug-loaded hydrogel without surgery(PPNPs/EPB@HA-Gel(no operation)).After intervening the mouse models with the above different treatments,the weight and tumor volume of the mice were dynamically observed,and the survival time was recorded to draw a survival curve.After the observation period,the mice were euthanized,the tumors and the main organs of the heart,liver,spleen,lung and kidney were collected for HE staining to evaluate the systemic toxicity of different administration systems.Tumor tissues were additionally stained with Ki-67 fluorescent staining and terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL)staining to evaluate the efficacy of the hydrogel system against postoperative recurrence of breast cancer.Results:The particle size and Zeta potential of PPNPs analyzed by DLS were 27.96±0.14 nm and-3.27±0.64 m V,and the PDI was 0.175±0.004,while the particle size and Zeta potential of Blank-NPs were 28.16±0.71 nm and-0.71±1.45 m V,respectively,and the PDI was 0.234±0.004.The drug loading rate(DL)of PPNPs was 10.81±0.43%,and the encapsulation efficiency was 90.09±3.59%.Both Blank-NPs and PPNPs were spherical and uniformly distributed by TEM.DLS also confirmed the good stability of PPNPs under different conditions including temperature,media and p H.The ~1HNMR results showed that HA-Tyr had doublets of aromatic protons at chemical shifts of 6.7ppm and 7.1 ppm,indicating that Tyr was successfully coupled to the HA backbone.The results of in vitro release experiments showed that free paclitaxel exhibited a burst-release effect,while both paclitaxel nanoparticles(PPNPs)and drug-loaded hydrogels exhibited a sustained-release effect.The MTT results indicated that the blank nanocarriers and hyaluronic acid hydrogels were nontoxic,and PTX and EPB had synergistic antitumor effects.Apoptosis experiments showed that the nanoparticle/hydrogel hybrid system had good biocompatibility,and PTX and EPB had synergistic antitumor effects.Scratch experiments showed that PTX/EPB and PPNPs/EPB@HA-Gel could significantly inhibit the migration of tumor cells,while the hydrogel carrier did not significantly reduce the synergistic effect of PTX and EPB;cell uptake experiments showed that tumor cells had better targeted uptake of nanomedicines.In vivo biocompatibility experiments showed that HA-Gel had degradability and good biocompatibility.In the mouse breast cancer model,we could find that the PPNPs/EPB@HA-Gel group not only inhibited the growth of the primary tumor in the non-operated mice,but also effectively inhibited the tumor recurrence in the postoperative group.Encapsulation of chemotherapeutic drugs in nano-hydrogel systems also effectively mitigated systemic toxicity.Furthermore,compared with EPB@HA-Gel or PPNPs@HA-Gel,PPNPs/EPB@HA-Gel confirmed the synergistic antitumor effect of the two drugs on this model.Ki-67 and TUNEL results further confirmed the above results.Conclusion:In this study,we successfully prepared a hyaluronic acid(HA)-based injectable hydrogel system(PPNPs/EPB@HA-Gel)co-loaded with paclitaxel(PTX)nanoparticles and epirubicin(EPB).In vitro studies showed that the drug-loaded hydrogel can inhibit the proliferation and migration of tumor cells,and had sustained release effect,degradability and good biocompatibility.In vivo studies found that PPNPs/EPB@HA-Gel stably released the encapsulated drugs in a mouse model after breast cancer surgery,achieved long-term inhibition of tumor recurrence,and effectively prolonged the survival of mice after breast cancer surgery.Systemic toxicity was relatively reduced.