Preparation Of Environmentally Responsive Polydopamine Nanocomposites And Their Applications In Multimodal Therapy Of Thyroid Carcinoma

Background and objectives:As the most common endocrine malignant tumor,the incidence rate of thyroid carcinoma is on the rise all over the world.Although surgery is the preferred treatment for most patients with thyroid carcinoma,there are still many situations where surgery cannot completely solve the problem:refractory thyroid carcinoma,represented by advanced differentiated thyroid carcinoma and anaplastic thyroid carcinoma,cannot be cured through surgery.When some postoperative patients experience recurrence or distant metastasis,they are no longer able to undergo surgery again.Patients with poor physical condition or concurrent malignant tumors cannot tolerate surgical trauma,etc.The existing methods of chemotherapy,radiation therapy,and TSH suppression therapy can only serve as adjuvant therapy and cannot fundamentally solve the problem of eliminating the lesion,which only prolongs the patient’s diseased survival.Before using targeted therapy,it is necessary to clarify the type of gene mutation in the patient,and the toxic side effects are significant,making it impossible for patients to use it for a long time.Immunotherapy is still in the clinical trial stage,and its long-term efficacy and stability still need to be investigated.Therefore,there is an urgent need to explore and develop new treatment technologies represented by nanotechnology to provide new choices for the radical treatment of thyroid carcinoma.Multi-functional nanocomposites can precisely solve this problem,achieving high sensitivity and strong specificity in imaging thyroid carcinoma by loading imaging function-related probes and tumor-specific responsive components,and then intelligently combining multimodal treatment components to achieve genuine integration of treatment and imaging,even dynamic visualization guidance for treatment.Polydopamine has been successfully applied in various nanocomposites due to its excellent physical and chemical properties,outstanding photothermal conversion performance,and good biocompatibility.The surface or internal modification and loading are no longer limited to the simple superposition of different components.The challenge is to cleverly design the combination effect of multiple components without affecting the function of a single component,and achieve mutual complementarity.In addition,the lack of specificity in recognizing tumor cells by nanocomposites has always been a challenge.Designing nanocomposites that can be responsive to the tumor microenvironment can alleviate these problems and achieve artificially controllable precision therapy to a certain extent by adding components that can respond to external stimuli.Based on the above clinical issues and research bottlenecks,this paper designs two polydopamine-based nanocomposites based on multiple responses to tumor microenvironment and external stimuli and explores their applications in imaging and multimodal therapy,which provides new ideas for nano therapy modes for thyroid carcinoma.Methods:1.Polyamine nanoparticles（PDA NPs）were synthesized using the traditional solution oxidation method.c Cp G oligonucleotides（ODNs）were attached to the surface of PDA NPs by Schiff base reaction,and Cp G ODNs were loaded onto the spherical structure through a hybridization reaction with c Cp G ODNs.Subsequently,doxorubicin（DOX）can not only be inserted into the double helix structure of ds DNA but also directly adsorb onto the surface of PDA NPs throughπ-πstacking,successfully preparing a multifunctional nanocomposite:PDA-ds Cp G-DOX NP.Basic physicochemical properties and photothermal conversion ability were investigated,and the loading efficiency and release efficiency of Cp G ODNs and DOX under different stimulation conditions were measured.In vitro experiments validated the cytotoxicity and synergistic therapeutic effects,while in vivo experiments further investigated the fluorescence imaging and photothermal imaging capabilities,synergistic therapeutic effects,and biosafety of applications.2.Hollow mesoporous polydopamine nanoparticles（HMPDA NPs）were synthesized by optimizing the conditions of the soft template method.Cu-HMPDA NPs were synthesized by introducing copper into HMPDA NPs in different valence states through the conjugation between metal cations andπbonds within polydopamine under the redox environment.Subsequently,F127 and photosensitizer Ce6 were further loaded to synthesize CPCF NPs.Characterize its physicochemical properties,measure the activities of three types of enzymes:catalase,peroxidase,and glutathione peroxidase,and evaluate their dual responsiveness to the tumor microenvironment and external laser.Further in vivo and in vitro experiments were conducted to investigate the synergistic therapeutic effects of chemodynamic therapy,chemotherapy,photodynamic therapy,and mild photothermal therapy,and validate the non-iron-induced ferroptosis treatment strategy for tumors.Results:1.PDA-ds Cp G-DOX NPs are spherical with uniform morphology and good dispersion,with a general size of 92.6±10.0 nm in diameter and an average hydrodynamic diameter of 180.13 nm.Under optimal conditions,the conjugation efficiency of Cp G ODNs is 77.86%,and the loading amount of DOX reaches 1.32 mg DOX/mg PDA ds Cp G NPs.PDA-ds Cp G-DOX NPs exhibit good colloidal stability,thermal stability,and structural stability during temperature change,with a photothermal conversion efficiency of 29.43%.The release efficiency of DOX at p H5.0（18.5%）is much higher than that at p H 7.4（4.0%）.After introducing 808 nm NIR irradiation,a 10.91%increase in DOX release was achieved in an environment with p H 5.0,fully demonstrating that PDA-ds Cp G-DOX NPs have dual responsiveness to p H and NIR irradiation.The dual imaging function provides important support for selecting the optimal treatment timing for in vivo experiments.PDA-ds Cp G-DOX NPs have a triple treatment mode of photothermal therapy,chemotherapy,and immunotherapy.When used in combination,the tumor inhibition rate is close to 100%,and there is no significant biological toxicity.2.Sunflower-shaped CPCF NPs have the characteristics of good dispersion and uniformity,with a general size of 237.1±21.6 nm in diameter and an average hydrodynamic diameter of 262.73 nm.The loading efficiency of Ce6 is as high as89.94%.CPCF NPs,as catalase-like enzymes,also have strong catalytic ability in slightly acidic environments at p H 6.5.The V_max and K_m as peroxidase-like enzymes are9.09×10^-8 M s^-1and 40.36 m M,respectively,which exhibits a strong affinity for H₂O₂.As a type of glutathione peroxidase,the enzyme activity gradually increases with the decrease in p H.When introducing 808 nm NIR,CPCF NPs exhibit excellent photothermal performance with a photothermal conversion efficiency of 35.96%.When introducing a 650 nm laser,CPCF NPs exhibit excellent ¹O₂ production performance due to their multi-enzyme activities combined with the laser responsiveness of the photosensitizer Ce6.Both in vivo and in vitro experiments have confirmed the effectiveness of the multimodal collaborative treatment strategy,with a tumor inhibition rate of 92.30%in the comprehensive treatment group.CPCF NPs have the characteristics of producing a large amount of ROS and consuming GSH.The introduction of dual lasers enhances the reaction efficiency,breaks the antioxidant defense effect of tumor cells,significantly inhibits the expression of GPX4,exacerbates lipid peroxidation,and enhances ferroptosis.In addition,CPCF NPs have good biocompatibility,and the related therapeutic procedures are safe and feasible in vivo.Conclusions:1.A multifunctional nanocomposite PDA-ds Cp G-DOX NP has been synthesized,which combines fluorescence imaging,photothermal imaging,and multimodal therapy functions（including photothermal therapy,chemotherapy,and immunotherapy）.The novel loading method significantly improves the loading efficiency of Cp G ODNs and DOX.In the treatment of thyroid carcinoma,PDA-ds Cp G-DOX NPs exhibit excellent responsiveness to both internal and external stimuli,and their flexible and controllable release kinetics enable them to achieve higher treatment selectivity under the combined stimulation of the acidic tumor microenvironment and exogenous NIR.PDA-ds Cp G-DOX NPs with good photothermal conversion performance effectively increased the temperature of the tumor area in response to the stimulation of NIR.The temperature-responsive deconvolution of Cp G ODNs in nanocomposites not only stimulates and enhances immune activation in the body,but also further promotes the release of DOX,thereby synergistically enhancing the chemotherapy effect.In addition,the composite’s dual imaging function helps track the distribution in the body,providing important support for selecting the optimal treatment timing.PDA-ds Cp G-DOX NPs integrate the triple treatment modes of photothermal therapy,chemotherapy,and immunotherapy,presenting a synergistic effect of 1+1+1>3,providing an excellent reference solution for developing precise and efficient thyroid carcinoma treatment.2.A multifunctional nanocomposite CPCF NP with a sunflower structure was constructed,which exhibits dual responsiveness to the tumor microenvironment（lower p H,higher H₂O₂ and GSH）and exogenous laser（808 nm NIR and 650 nm laser）.Based on the first experiment,structural optimization and improvement were carried out on the polydopamine nano substrate,using hollow mesoporous polydopamine nanoparticles as carriers,which have a larger specific surface area and more active groups,significantly improving the drug loading efficiency.In a redox synthesis system,copper ions with different valence states(Cu⁺and Cu²⁺)jointly fill and modify the hollow and mesoporous structures.The mutual conversion of Cu⁺,Cu²⁺,and elemental copper in the tumor microenvironment endows CPCF NPs with three types of enzyme activities:catalase,peroxidase,and glutathione peroxidase,providing strong support for efficient CDT.Due to the excellent catalase-like activity of CPCF NPs,the enhanced PDT mode under continuous oxygen supply enhances the overall anti-tumor effect.In addition,a mild photothermal treatment mode（maximum temperature<45°C）was adopted to reduce damage to surrounding normal tissues.CPCF NPs achieve a cascaded multimodal synergistic therapeutic function of chemodynamic therapy,chemotherapy（copper ion cytotoxicity）,photodynamic therapy,and mild photothermal therapy,as well as dual-mode imaging function of fluorescence imaging and photothermal imaging.By examining the consumption of GSH,changes in mitochondrial membrane potential,degree of lipid peroxidation,and expression of glutathione peroxidase 4,it was confirmed that the mechanism of CPCF NPs killing tumor cells involves ferroptosis.This non-iron-induced ferroptosis treatment strategy for tumors provides valuable guidance for the development of related nanocomposites in the future and also provides a new choice for a mild,safe,and efficient nano therapy mode for thyroid carcinoma.