Calcium Carbonate Nanodrug Delivery System For Improving Colon Cancer Immunotherapy

Colon cancer is currently the most deadly malignant tumor affecting the digestive tract and poses a serious threat to human health.Unfortunately,there is a lack of efficient treatments for this type of cancer.However,the emergence of immunotherapy has brought new hope for colon cancer treatment.Despite this,the poor tumor target capability and lack of specificity would limit the therapeutic effect of immune modulators and cause serious side effects.Additionally,monotherapy alone is often insufficient to achieve ideal therapeutic effects,due to the multiple immunosuppressive signals in tumor sites.Therefore,it has become essential to improve drug target capability and co-delivery as a key clinical cancer treatment strategy.Nano-drug delivery systems provide a promising new approach to immunotherapy of colon cancer.These systems have unique physical and chemical properties that allow for better drug absorption,protect the drugs they contain,and deliver them directly to the tumor.This targeted delivery system improves the bioavailability of the drugs,while reducing the amount of drug distribution in non-targeted tissues.This,in turn,reduces the risk of drug toxicity and side effects.However,they have some limitations,such as delayed drug release after uptake by lysosomes and poor tumor retention ability.Calcium carbonate（CaCO₃）is a commonly used material in nano-drug delivery systems due to its biocompatibility and acid-sensitivity.By using tumor-targeted delivery strategies and taking advantage of the acidic tumor microenvironment（TME）in tumor tissue,CaCO₃ nano-drug delivery systems can rapidly release drugs in the tumor microenvironment and promote efficient lysosomal escape,resulting in more effective tumor treatment.The nano drug delivery system has the ability to achieve multiple drugs simultaneously,which can improve the efficacy of colon cancer immunotherapy.The inhibition of immune cells in the TME is a crucial factor that affects the occurrence and development of colon cancer.Studies have shown that drugs like imiquimod（R837）and nitric oxide（NO）can regulate the polarization of M2-type macrophages and the perfusion capacity and integrity of tumor vessels,respectively.This can promote the anti-tumor effect of macrophages and tumor drug delivery.Drugs like galunisertib and IL-15 can also relieve the inhibitory effect of TGF-βon natural killer（NK）cells and activate the anti-tumor effect of NK cells.The right combination of drugs can significantly improve the efficiency of immunotherapy.However,the nano drug delivery system still has some limitations in tumor immunotherapy,such as the untimely release of drugs after endocytosis and poor tumor retention ability.This study aimed to construct two CaCO₃-based nano drug delivery systems to improve the effectiveness of immunomodulatory drug delivery.The systems were designed to activate the anti-tumor response of immune cells,reverse the immunosuppressive TME,and enhance the therapeutic effect of immunomodulatory drugs on colon cancer.The research in this thesis mainly includes the following research contents:1.Preparation and in vitro characterization of R837/Arg@CaM.To solve the issue of delayed release of intracellular drug delivery systems,we developed a lysosomal acid-responsive CaCO₃ nanoparticle drug delivery system（R837/Arg@CaM）for co-delivery of R837 and L-arginine（Arg）.We first prepared the lipid bilayer containing R837 and the core containing Arg and CaCO₃ liposomes and then fused them with the CT26 membrane by extrusion to obtain R837/Arg@CaM.A nanoparticle size analyzer determined the particle size andζ-potential of R837/Arg@CaM.The morphology and elemental composition of R837/Arg@CaM were examined by transmission electron microscopy.The membrane protein integrity and in vitro drug release were also evaluated.The results indicated that R837/Arg@CaM was successfully prepared with a 125.5±2.1 nm particle size and aζ-potential of-1.9±0.2 m V.R837/Arg@CaM has an accelerated release rate of R837 and Arg in acidic solutions.At the cytological level,we evaluated the lysosome escape,polarization,and production of NO of M2 macrophages by R837/Arg@CaM.The results indicated that R837/Arg@CaM had good lysosomal escape ability.R837/Arg@CaM could polarize M2-phenotype macrophages to M1-phenotype and enhance the level of NO in macrophages that enhanced anti-tumor capability.These results indicated that R837/Arg@CaM is an ideal drug carrier for tumor immunotherapy.2.In vivo anti-tumor effect of R837/Arg@CaMIn the present study,we constructed the CT26-tumor-bearing mouse model and examined the in vivo distribution and tumor vascular regulation of R837/Arg@CaM by an IVIS system and immunofluorescence method.The effect of R837/Arg@CaM on immune infiltration and tumor growth inhibition in a subcutaneous colon cancer model was also investigated.Moreover,the anti-tumor effect of R837/Arg@CaM combined with chemotherapy drugs or immune blockade therapy was investigated.The results indicated that R837/Arg@CaM could be passively targeted to the tumor sites.By infusing the CT26 membrane,it could improve the tumor target ability of R837/Arg@CaM.The administration of R837/Arg@CaM improved the tumor vascular perfusion ability,increased the coverage of tumor perivascular cells,and effectively relieved the tumor hypoxia situation.The results indicated that R837/Arg@CaM could polarize tumor-associated macrophages（TAMs）to M1-phenotype,and promote the infiltration,proliferation,and activation of CD8⁺T cell,significantly inhibiting the CT26-tumor growth.Better tumor inhibition was acquired when R837/Arg@CaM was combined with chemotherapeutics gemcitabine（Gem）and immune checkpoint inhibitors PD-L1 antibodies.3.Preparation and in vitro characterization of Gal/IL-15@CaLN.To improve the poor tumor retention ability,we constructed a tumor acidic microenvironment response CaCO₃nano-drug delivery system（Gal/IL-15@CaLN）.Gal/IL-15@CaLN consisted of a liposome that was encapsulated with CaCO₃ and sodium alginate adsorbed on its surface for the co-encapsulation of the TGF-βreceptor kinase inhibitor of galunisertib and IL-15.We examined its physicochemical properties and regulatory effects on NK cells.The results indicated that Gal/IL-15@CaLN had a particle size of 124.7±6.0 nm and aζ-potential of-26.8±1.2 m V with good in vitro stability.The acidic buffer（p H≤6.8）would trigger Gal/IL-15@CaLN with nanoscale aggregating to micrometer scale with an accelerated release rate of galunisertib and IL-15.The cytological results indicated that Gal/IL-15@CaLN could efficiently promote the expression of activation receptors and enhance the cytotoxicity of NK cells.The expression of p-STAT5 and p-m TOR increased after the treatment of Gal/IL-15@CaLN,while p-SMAD2 and TGF-βRI decreased.These results indicated that Gal/IL-15@CaLN exhibited TME-responsiveness and the activation of NK cells.4.In vivo immune regulation and anti-tumor inhibition of Gal/IL-15@CaLN.We examined the in vivo distribution of Gal/IL-15@CaLN,anti-tumor immunity,and tumor growth inhibition by an IVIS system on CT26 subcutaneous tumor model.The results showed that Gal/IL-15@CaLN could effectively deliver galunisertib and IL-15 to tumor sites and be retained in the tumor for over 120 h.On the one hand,Gal/IL-15@CaLN promoted the proliferation and activation of NK cells in the tumor sites of immune-deficient mice,and inhibited 70.7%of tumor growth in BALB/c nude mice.The antitumor effect of Gal/IL-15@CaLN was also examined on immunocompetent mice with the presence of T cells and NK cells.The results indicated that Gal/IL-15@CaLN could simultaneously promote the proliferation and activation of NK cells and CD8⁺T cells,boost innate NK cell immunity and adaptive T cell immunity,and enhance the tumor inhibition rate to 82.4%.Herein,we focused on CaCO₃ nano-drug delivery system realizing the combinational therapy strategy of R837 with Arg and galunisertib with IL-15.The in vitro and in vivo results indicated that R837/Arg@CaM exhibited enhanced tumor-targeting ability and intelligent drug delivery characteristics.R837/Arg@CaM could promote the perfusion ability and integrity of tumor vessels,activate anti-tumor immune effects,and achieve good therapeutic effects on colon cancer treatment.Gal/IL-15@CaLN was successfully constructed with tumor microenvironment-responsiveness and enhanced tumor retention capability.Gal/IL-15@CaLN could promote the proliferation and activation of NK cells,and enhanced the inhibition of colon cancer.The research provides new strategies for clinical cancer therapy with good application prospects.