Construction Of Injectable Hydrogel Vaccine And Application For Postoperative Immunotherapy Of Triple-negative Breast Cancer

Breast cancer is the most common malignant tumor among women worldwide,with the triple-negative subtype being highly invasive,prone to recurrence and metastasis after surgery,and insensitive to targeted and endocrine therapies,and lack of specific therapeutic approaches.Triple-negative breast cancer is characterized by a higher mutation burden and increased immune cell infiltration,making it an ideal candidate for immunotherapy.Therapeutic tumor vaccines,as an emerging immunotherapy method,are considered one of the potential therapeutic strategies for TNBC because of their strong specificity,minimal side effects,and ability to form long-term immune memory.However,there are currently no breast cancer tumor vaccines applied in clinical settings,mainly because the response rate of tumor vaccines was generally low.The two key issues are:1.The short retention time of antigens in the body leads to insufficient immune stimulation;2.The inability to successfully deliver antigens to dendritic cells（DCs）in the lymph nodes,hence failing to present antigens to induce T cell activation.Therefore,extending the vaccine’s retention time in the body and efficiently delivering it to dendritic cells in lymph nodes are the challenges in vaccine research.This study developed a new type of injectable hydrogel vaccine for preventing postoperative recurrence and metastasis of TNBC.The vaccine,named NIGel-Vax（Nano-in-Gel Vaccine）,consists of an injectable hydrogel loaded with antigen nanoparticles.The injectable hydrogel was made from Oxidized dextran（ODEX）and4arm-PEG-ONH2,which could protect the antigen from premature degradation,prolong the antigen’s residence time in the body,and achieve sustained immune activation,thus regulating the vaccine’s release kinetics over time.The nanoparticles were assembled from antigen proteins and the polymeric adjuvant PEI-4BImi-Man.Our previous work found that PEI-4BImi was a STING polymeric agonist,which could serve as an adjuvant for vaccines to promote the activation of DCs.Based on this,we modified PEI-4BImi with mannose to obtain PEI-4BImi-Man,which could mediate antigen internalization by binding to mannose receptors on the DC surface,enhancing DC targeting,improving antigen presentation,and enhancing T cell immune responses,thus spatially regulating the targeted delivery of antigens.By loading the nanoparticles into the injectable hydrogel,we aim to achieve synergistic temporal and spatial control of the vaccine,activate specific immune responses,and establish long-term immune memory,offering a new therapeutic approach for preventing postoperative recurrence and metastasis of TNBC.The experimental results are as follows:1.Synthesis and characterization of NIGel-Vax.We initially modified PEI-4BImi with mannose to obtain PEI-4BImi-Man and assembled the protein antigen with PEI-4BImi-Man in a 1:1 mass ratio to form nanoparticles（Nano-OVA）with a hydration particle size of approximately 110 nm.The hydrogel,composed of 40%oxidized ODEX and 4arm-PEG-ONH₂,was prepared by mixing in a 2:1 mass ratio via oxime linkage reaction.By adjusting the total mass ratio of ODEX to 4arm-PEG-ONH₂ in the hydrogel,we synthesized hydrogels with different mass ratios of 4%,6%,and 8%,achieving degradation and release times of 7-27 days,and demonstrated good injectability of the hydrogel through rheological testing.Scanning electron microscopy revealed that the average pore size of the hydrogel decreased from 20 micrometers to 6micrometers as the mass ratio increased.Finally,the antigen nanoparticles were loaded into the hydrogel to create NIGel-Vax.2.Degradation,antigen release,lymph node drainage,and immune cell infiltration of NIGel-Vax in vivo.The OVA protein,labeled with the Cy5 fluorescent dye,was used to prepare NIGel-Vax,which was then administered to mice via subcutaneous injection.The release kinetics of the vaccine and the drainage of the antigen to the lymph nodes were observed through in vivo fluorescence imaging.The results indicated that NIGel-Vax could act as an antigen reservoir,prolonging the release time of the antigen in the body,and the duration of sustained antigen release was extended with an increase in the hydrogel’s mass ratio.The fluorescent signal of the antigen in the lymph nodes demonstrated that Nano-OVA could accumulate over time within the lymph nodes,and Nano-OVA infiltrate into the lymph nodes effectively.Additionally,flow cytometry analysis showed that NIGel-Vax could recruit immune cells such as DCs,and there was no significant increase in pro-inflammatory cytokine levels within the gel,indicating the high safety profile of NIGel-Vax.3.The immunostimulatory effects of NIGel-Vax on DCs in vitro and in vivo.The capacity of PEI-4BImi-Man and Nano-OVA to significantly enhance the secretion of IFN-βby DC2.4 cells was confirmed using the ELISA method,indicating the inherent immunostimulatory activity of PEI-4BImi-Man.Flow cytometry further substantiated that the mannose-modified Nano-OVA significantly increased endocytosis efficiency through interaction with mannose receptors on the DCs’surface.In in vivo experiments,comparing the DC activation effects of different mass ratios of NIGel-Vax（4%,6%,8%）,traditional aluminum adjuvant,and Nano-OVA,it was found that 8%NIGel-Vax exhibited the best performance in activating DCs and promoting cellular and humoral immune responses.These findings highlight the importance of sustained antigen stimulation in eliciting effective immune responses.4.Using OVA as a model antigen,the therapeutic efficacy and immunological analysis of the NIGel-Vax vaccine were evaluated in the B16-OVA tumor model.The experiment compared the tumor inhibition effects of traditional aluminum adjuvant,Nano-OVA,4%NIGel-Vax,6%NIGel-Vax,and 8%NIGel-Vax in the B16-OVA tumor model.The results showed that the NIGel-Vax formulations with long-term release characteristics（6%and 8%）significantly outperformed the other groups in terms of tumor suppression,particularly the 8%NIGel-Vax group,which achieved a tumor inhibition rate of 93%on day 26.Furthermore,in the 8%NIGel-Vax group,the number of activated CD8⁺T cells in the peripheral blood significantly increased,and the proportion of CD4⁺and CD8⁺T cells as well as activated DCs in the tumor tissue also increased.Furthermore,an increase in the proportion of effector and central memory T cells in the spleen indicated the establishment of an immune memory effect by NIGel-Vax.ELISPOT assays further confirmed the significantly higher antigen-specific immune responses in the 8%NIGel-Vax group compared to the other groups.These results indicate that 8%NIGel-Vax with a longer sustained release period was more effective in tumor suppression.5.The therapeutic effect and immunological analysis of NIGel-Vax in a 4T1breast cancer postoperative model.A triple-negative breast cancer postoperative recurrence model was established using the 4T1 cell line.Personalized antigens were extracted from the resected tumor cells to prepare NIGel-Vax/4T1.The experimental results demonstrated that NIGel-Vax/4T1 showed the best anti-tumor effect compared to the aluminum adjuvant,achieving a tumor inhibition rate of 92%on day 22,and two mice were cured.Moreover,NIGel-Vax/4T1 significantly increased the proportion of effector T cells in peripheral blood and elevated the levels of IL-2 and IFN-γin the immune microenvironment,indicating its effectiveness in activating immune responses.Analysis of lung metastasis revealed almost no pulmonary metastatic foci in the NIGel-Vax treatment group,suggesting that NIGel-Vax effectively inhibits distant metastasis of breast cancer.TROP2 is overexpressed in various tumors,including TNBC.The TROP2 protein was used as a tumor-associated antigen to prepare the NIGel-Vax/TROP2 vaccine aimed at TNBC.The study established a postoperative recurrence model of breast cancer using the 4T1 cell line that expresses TROP2,to evaluate the effect of NIGel-Vax/TROP2 in the 4T1 tumor postoperative recurrence model.The results showed that NIGel-Vax/TROP2 effectively inhibited the growth of 4T1 tumors,achieving a 96.7%tumor inhibition rate and a 50%cure rate,with high treatment safety.Flow cytometry analysis and ELISPOT assays confirmed that NIGel-Vax/TROP2 significantly activated specific T-cell responses,and tumor rechallenge experiments verified that NIGel-Vax/TROP2 established an immune memory effect.Through the research presented in this paper,we developed an injectable hydrogel vaccine delivery system,NIGel-Vax,and demonstrated that its long-term release significantly enhances cellular immune responses and tumor suppression effects.Utilizing personalized tumor protein antigens or TROP2 protein antigens,NIGel-Vax achieved a tumor inhibition rate of over 90%and a cure rate of 33%in the postoperative treatment of TNBC,successfully establishing an anti-tumor immune memory effect.This study provides guidance for understanding the mechanism of tumor vaccines and developing new postoperative treatment strategies for TNBC.