Bioactive Proteins-directed Facile Fabrication Of Novel High-performance Nanoprobes With Good Biocompatibility For Cancer Theranostics

Part1 Facile preparation of hyaluronic acid and transferrin co-modified Fe₃O₄ nanoparticles with inherent biocompatibility for dual-targeting magnetic resonance imaging of tumors in vivoObjective Clinical diagnosis of malignant tumors using nanoprobes needs severe acquirement in the aspects of sensitivity and biocompatibility.Integrating dual-targeting strategy and selection of human-inherent elements and molecules as raw materials shows great potential in the development of biosafe and sensitive nanoplatform.To carry out the proposed design,we constructed a biocompatible dual-targeting MR imaging nanoprobe based on Fe₃O₄ nanoparticles?NPs?co-modified with inherently innoxious hyaluronic acid?HA?and transferrin?Tf?.Methods In current work,we synthesized Fe₃O₄@HA NPs using HA as both template and targeting molecules via a one-step co-precipitation method at room temperature.Then the prepared Fe₃O₄@HA NPs were further modified with Tf to generate dual-targeting Fe₃O₄@HA@Tf NPs at room temperature.The biosafety of the proposed Fe₃O₄@HA@Tf NPs was demonstrated in vitro and in vivo through cell viability assay,intravital metabolism and biochemical analysis respectively.The cellular uptake and blocking tests were performed to study the tumor targeting ability of the developed nanoprobes.Results Low cytotoxicity and excellent biocompatibility of the nanoprobes were confirmed by the cell viability assay,intravital metabolism and biochemical analysis.The uptake test of Hela cells and MR imaging of tumor-bearing mice exhibited the desirable tumor-targeting ability of the dual-targeting nanoparticles.ConclusionWe employed bio-safe HA and Tf to co-modify the non-toxic Fe₃O₄ NPs to obtain a dual-targeting Fe₃O₄@HA@Tf NPs,which possess the advantages of dual-targeting ability,excellent biocompatibility,facile synthesis procedure and mild condition.More importantly,the developed nanoplatform provides a novel strategy based on integrating dual-targeting strategy and selection of human-inherent elements and molecules as raw materials for the fabrication of high sensitive and biocompatible nanoplatform for clinical tumor-targeted imaging in vivo.Part2 Mimicking drug-substrate interaction: A smart bio-inspired technology for the fabrication of theranostic nanoprobesObjective Versatile bio-inspired strategies are urgently needed to fabricate high-performance nanoprobes for biomedicine application.Here,we wanted to estimate the feasibility for mimicking drug-substrate interaction strategy to construct a high-performance nanoplatform for theranostic application via imitating the disinfection process of potassium hypermanganate?KMn O4?.Methods The bio-inspired manganese dioxide?MnO₂?nanoparticles were fabricated using bovine serum albumin?BSA?as both the template and the reducing agent under mild conditions via mimicking the disinfection process of KMn O4 and then were characterized systematically.The cytotoxicity and biocompatibility of proposed BSAMnO₂ nanoparticles?BM NPs?were comprehensively estimated though cellular MTT assay,metabolism in vivo,weight monitoring,histological H&E staining and biochemical indicators analysis.MR imaging of normal mice and tumor-bearing mice with BSA-MnO₂ nanoparticles were conducted to exhibit the targeted MR imaging capacity of the kidney and tumor,respectively.To expand the function of BSA-MnO₂ nanoplatform,two FDA approved therapeutic reagents,indocyanine green?ICG?and paclitaxel?PTX?were loaded on BM NPs in a facile and energy-saving mixing method to generate two theranostic nanoprobes BSA-MnO₂-ICG?BMI?and BSA-MnO₂-PTX?BMP?for MR imaging-guided photothermal ablation and chemotherapy of tumors,respectively.To demonstrate the universality of the mimicking drug-substrate interaction strategy,another two bioactive proteins including ovalbumin?OVA?and transferrin?Tf?were also employed as substrates to synthesis nanoprobes.Then the corresponding characterizations and MR imaging of normal mice were also carried out.Results The fabricated BM NPs possessed sub-10 nm size,superior monodispersity,favorable water solubility,impressive T1 relaxivity of 7.9 m M-1s-1 and admirable biocompatibility.The BMI and BMP NPs also exhibited impressive MR imaging-guided photothermal ablation and chemotherapy of tumors,respectively.Encouragingly,the T1 relaxivity of BMI NPs could reach as high as 70.6 m M-1s-1,which is one of the highest values in all available T1 MR imaging contrast agents.The developed multifunctional nanoprobe not only serves as a versatile MR imaging nanoprobe for tumor and renal imaging,but also as a platform for integrating therapeutic strategies towards tumors.The proposed strategy could be easily extended to the fabrication of nanoprobes using other functional proteins such as ovalbumin?OVA?and transferrin?Tf?.Conclusion The nanoparticles synthesized by mimicking the disinfection of KMn O4 not only possessed several excellent chemical properties and impressive MR imaging of the kidney and tumor,but also could be expanded to the theranostics of tumor with high performance by loading therapeutic agents.More importantly,the strategy of mimicking drug-substrate interaction for fabricating multifunctional nanoprobes can be easily promoted to the other bioactive molecules,which could constructing many more valuable theranostics for biomedicine.Part3 Antigen-directed fabrication of ovalbumin-ICG nanovaccine with ultrahigh antigen loading efficacy for dendritic cell stimulation/tracking and imaging-guided photothermal-immnunotherapy of cancerObjective To overcome the drawbacks of currently available approaches for building multifunctional nanovaccines in the aspects of low yield,limited antigen loading efficiency,tedious manufacturing process,uncertain composition and structure,and the risk of systemic toxicity of carriers,we showed an antigen-directed synthesis strategy for fabrication of a high-performance multifunctional nanovaccine with imaging-guided phothemal-immunotherapy capacity in an extremely facile way.Methods A nanovaccine was elegantly constructed by simple mixing of ovalbumin?OVA?as a model antigen and FDA-approved indocyanine green?ICG?at room temperature based on hydrophobic interaction between them and then were characterized systematically.The cytotoxicity and immune stimulation to dendritic cells of proposed OVA-ICG nanovaccine were comprehensively estimated though cellular MTT assay,cellular phagocytose,cytokines detection and flow cytometry assay.The photothermal therapy in vitro was demonstrated through the assays of photothermal performance and photothermal ablation against tumor cells.Then OVA-ICG nanovaccine was applied to the in vivo anti-tumor therapy to estimate the synergetic photothermal-immunotherapy effect.The tumor prevention ability was assessed by the means of pre-immunization with OVA-ICG nanovaccine and then challenging with tumor cells.The dendritic cells labeled with OVA-ICG nanovaccine were subcutaneously planted to show the immune tracing ability of OVA-ICG nanovaccine.Results The OVA-ICG nanovaccine exhibits as high as 80.8% of antigen loading efficiency,impressive water solubility and high reproducibility.The smart multifunctional nanvaccine not only enables remarkable dendritic cell?DC?-labeling and stimulation as well as effective photothermal ablation against melanoma cells in vitro,but also could be utilized for sensitive near-infrared fluorescent DC tracking and admirable primary tumor ablation together with reliable immunotherapy against residual tumors in vivo.In addition,OVA-ICG nanovaccine possesses a robust tumor prevention effect that endows the immunized mice with an impressing tumor suppression capability towards transplanted tumors.Conclusion In this work,we proposed the antigen-directed synthesis strategy for constructing multifunctional nanovaccine for the first time.Compared with previous reported multifunctional nanovaccines,OVA-ICG nanovaccine is the simplest immunotherapy-based multifunctional theranostics agent and enjoys unique superiority in the aspects of simple synthetic protocols,nontoxic reagents,outstanding antigen loading efficiency and high reproducibility.The proposed antigen-directed synthesis strategy opens up a new way for building high-performance multifunctional nanovaccines in a green and facile way,and shows great potential in fabrication of other specific nanovaccines using various antigens and theranostics agents.