Study On Silk Protein-mediated CGas-STING Immunotherapy And Tumor Imaging

Tumor metastasis is the main reason why cancer is difficult to overcome nowadays.Advanced molecular imaging technology has great potential in noninvasively monitoring the pathological state of early tumor progression and metastasis.“Integration of diagnosis and treatment”refers to diagnosis and treatment in a narrow sense.In a broad sense,diagnosis includes laboratory diagnosis,imaging diagnosis,pathological diagnosis,and molecular diagnosis and treatment,which integrate tumor imaging,targeted drug delivery,in vivo tracking,The multifunctional nano-diagnostic agent that integrates drug treatment and prognosis monitoring has become a research hotspot in the medical field.It can accurately diagnose the condition in real time and perform treatment simultaneously,monitor the curative effect in real time,and adjust the dosage regimen at any time,which is conducive to achieving the best treatment.Effective,reduce toxic side effects,and achieve precise treatment of malignant and metastatic tumors.This approach brings new hope for tumor treatment.Magnetic Resonance Imaging（MRI）is one of the most effective clinical imaging diagnostic methods.It has the advantages of non-invasive,no radiation damage,high soft tissue resolution,multi-core and multi-parameter imaging,arbitrary layer scanning and so on.For the central nervous system,neurovascular imaging is the preferred method,especially in small tumors that cannot be identified by other detection methods.Because it can provide high-resolution soft tissue anatomical images,MRI has become an important tool for clinical imaging.It is estimated that nearly 70%of MRI examinations use contrast agents to improve image contrast,and this proportion is still expanding.Contrast agent（CA）has a dual function:acquiring high-resolution images and reducing the time required for image acquisition.In addition,the new materials can help the early detection of lesions and accurately deliver targeted drugs to tumor tissues.The NMR measurement signal is caused by the spin motion of the nucleus.According to its principle,contrast agents can be divided into two types,namely longitudinal relaxation（T1）contrast agents and transverse relaxation（T2）contrast agents.T1contrast agents are usually paramagnetic elements,such as gadolinium ions and manganese ions,which can effectively shorten the spin lattice relaxation time and enhance the longitudinal and transverse relaxation rate（r1,r2）of water molecules.However,these contrast agents are tissue non-specific,have a short metabolic time in the body,and have potential cell and tissue toxicity.Innate immunity is the first line of defense for organisms against pathogen infection.Some researchers have pointed out that the DNA in the cytoplasm is recognized by cyclicGMP-AMP synthase（cGAS）,initiate the combination of cyclicGMP-AMP（cGAMP）and stimulator of interferon（STING）.Finally,it stimulates the release of type I interferon（Interferon,IFN-I）.Recent studies have shown that Mn²⁺is an activator of cGAS,which can induce conformational changes of cGAS protein and activate the cGAS-STING signaling pathway to produce an immune response.As a new STING agonist,Mn²⁺has shown great application potential in enhancing anti-tumor and anti-viral immune response.Silk sericin（SS）of the silkworm is a water-soluble protein and a natural moisturizing factor for the skin.However,its poor mechanical properties limit the use of sericin alone.In order to improve the stability of the sericin matrix,it must be mixed or cross-linked with other polymers to improve its mechanical properties.In recent years,silk protein has attracted the attention of many researchers in enhancing biocompatibility and reducing the cytotoxicity of materials.Low molecular weight sericin peptides or sericin hydrolysates are used in cosmetics,care products,health care products and medicines.High molecular weight sericin is mainly used for cartilage tissue engineering,it has good biodegradability,biocompatibility and low immunogenicity.Natural silk fibers have been used as sutures for biomedical applications and scaffolds for tissue engineering,which can induce the production of pro-inflammatory cytokines and increase the phagocytosis of cells.At the same time,SS can promote cell proliferation and increase the cell viability of different cell lines.Due to the beneficial properties of SS,sericin is being explored more and more in the field of biomedicine.In recent decades,Gd and Mn chelates have occupied most of the market for magnetic resonance contrast agents.The rapid development of nanotechnology has brought new and important opportunities for the development of magnetic resonance contrast agents.Many researchers have combined contrast agents with a variety of tumor treatment methods to prepare and improve nano-formulations to improve their targeted anti-tumor ability,relaxation efficacy,safety and stability.Integrated medicine for diagnosis and treatment is the current main research hotspot and the future development trend.The following research results have been achieved:1.Preparation and application of SS@GAH-GdCl₃It is of great significance to develop gadolinium ion contrast agents for magnetic resonance imaging with p H response and tumor targeting capabilities.Sericin Gadolinium Contrast Agent（SS@GAH-GdCl₃）is obtained by covalently cross-linking sericin and Gadolinium Acetate（GAH）through Schiffer’s base reaction,then it is prepared by electrostatic adsorption with gadolinium chloride（GdCl₃）.Zeta potential detection results show that SS@GAH-GdCl₃can naturally respond to changes in p H and freely realize the transition from the negative surface potential of p H 7.5 to the positive surface potential of p H 5.8.The T1-weighted MR image shows that under a 3.0T magnetic field,SS@GAH-GdCl₃has a higher T1 relaxivity in a p H 5.8 than p H 7.4.Compared with the commercial contrast agent Magnevist,the relaxation rate of SS@GAH-GdCl₃is 4.32 times higher.At the same time,the T1-weighted MR image of the tumor shows that SS@GAH-GdCl₃can target the tumor and gather around the tumor tissue to achieve up to 2 hours of MR imaging on the tumor.More importantly,SS@GAH-GdCl₃has good cell compatibility,there is no leakage of gadolinium ions,and it will not cause hemolysis,inflammation,tissue damage and changes in serum biochemical indicators and body weight.Therefore,SS@GAH-GdCl₃has great potential as an MRI contrast agent for p H response and tumor targeting functions.2.Preparation and application of Mn-CREKA-CTLA-4-SSThe design principle of this experiment is due to the fact that1-（3-Dimethylaminopropyl）-3-ethylcarbodiimide hydrochloride（EDC）and N-Hydroxy sulfosuccinimide sodium salt（sulfo-NHS）can react with carboxylate（-COOH）to form a stable N-hydroxysuccinimide ester,and further can react with primary amine（-NH2）to make an amide bond between the carboxy and amino.So we first combine manganese acetate with EDC and sulfo-NHS,and then connect with CREKA,CTLA-4and SS.Thus,Mn-CREKA-CTLA-4-SS（MCCS）contrast agent can be prepared.The pentapeptide protein CREKA（Cys-Arg-Glu-Lys-Ala）has a very high affinity for fibronectin and its complexes,which can promote MCCS to recognize and respond to tumor tissues in situ or metastasis,and bind to tumors,Mn and cytotoxic T lymphocyte-associated antigen-4（CTLA-4）stimulates the cGAS-STING signaling pathway to produce an immune response,and then kill tumor cells.T1-weighted imaging relaxation data showed that compared to the control group magnevist,the relaxation rate of the MCCS group was nearly 3 times higher.T1-weighted MR images of tumors in tumor-bearing mice show that MCCS can directly target tumors and gather around tumor tissues,enhancing the visualization of tumor tissues.3.MCCS for tumor treatment functionIncubating MCCS（50μg Mn）with adenocarcinoma human alveolar basal epithelial cells A549-Luc-GFP,about 60%of lung cancer cells undergo apoptosis after48 hours.Western blot experiment results showed that after MCCS and tumor cells were incubated together,autophagy and apoptosis related proteins（LC3Blight chain 3 B,LC3B）,Bcl-2 related X protein（Bax）and cysteinylaspartate specific proteinase-3（Caspase3）up-regulated,nuclear factor-p65（p65）and B-cell lymphoma-2（Bcl-2）were down-regulates,as well as,there is a significant accumulation of ROS.Secondly,MCCS also up-regulated the expression of cGAS-STING related proteins Interferon-β（IFN-β）,interferonregulatory factor 3（IRF3）,phospho-IRF3（P-IRF3）and STING in tumor cells,but is also related to the autophagy and apoptosis pathways.In human lymphocyte experiments,MCCS up-regulated 78.10%CD8-positive effector T cells and down-regulated regulatory T cells by 13.30%,indicating that it significantly activates the cellular immune system.In in vivo experiments,MCCS activated the proliferation of immune cells such as CD8-positive effector T cells and CD80-positive T cells in the spleen and blood of mice,and the expression of interferon-γ（IFN-γ）and granzyme.MCCS has good cell compatibility in normal mice,without causing hemolysis,inflammation,tissue damage,changes in serum biochemical indicators and blood routines.While achieving tumor targeting and enhancing MRI imaging,MCCS effectively killed tumor cells,providing important theoretical basis and guidance for the development of new integrated biomaterials for tumor diagnosis and treatment.