Design,Synthesis And Application Of Prussian Blue Nanoparticles In Tumor Multimodal Imaging And Hyperthermia Therapy

Cancer has become a major public health problem in the world with increasing incidence.Therapies for cancer include traditional open surgery,radiotherapy,chemotherapy and immunotherapy,but the side effects such as trauma,toxicity and drug-resistance caused by these treatments have led to the search for better minimally invasive/noninvasive treatments and therapeutic implications for cancer patients.With the rapid development of nanotechnology,the integration of cancer diagnosis and treatment has received widespread attention.Among them,the multi-modality imaging based on nanomaterials and physical therapy of tumors have drawn great attention.As an effective way to treat tumors,hyperthermia therapy can heat the tumor area to reach the effective treatment temperature by physical energy?such as light,ultrasound,microwave,radiofrequency,etc.?for a certain period of time.Upon the difference in temperature tolerance ability between normal tissues and tumor cells,hyperthermia therapy is currently the fifth largest therapy after surgery,radiotherapy,chemotherapy and immunotherapy.Prussian blue?PB?is a FDA-approved antidote for the clinical treatment of radiotoxins such as thallium and therefore has good biocompatibility and safety.PB nanoparticles possess magnetic properties and photo-thermal conversion properties,so that it has potential in the field of biomedical applications.In this thesis,the controlled synthesis of PB nanoparticles and its application in multimodal imaging and hyperthermia of tumors were mainly studied.First of all,we optimized the synthesis of PB nanoparticles by adjusting the ratio of raw materials and reaction process,etc.,Then,we screened out the appropriate PB nanoparticles.Upon the magnetic properties and photo-thermal conversion properties of PB nanoparticles with good stability,the effect of magnetic resonance imaging?MRI?/photoacoustic imaging?PA?dual-mode imaging and photo-thermal therapy on tumor were studied,which provided a scientific basis for the imaging and treatment of tumors.The pore diameter and structure of the PB nanoparticles with good stability were then designed by the defect selection etching method.Finally,we explored its application as a drug delivery system in focused ultrasound synergistic therapy.The main research contents and conclusions are summarized as follows:?1?Background and Purpose:Prussian blue nanoparticles possess magnetic properties and photo-thermal conversion properties,so that it has potential applications in the field of biomedicine.For therapeutic purposes,the nanoparticles must through intravenous injection into the body which requires the nanoparticles remain stable in the physiological environment.The current strategy to solve this problem is mainly through the layers of coating or post-modification methods,so that PB in the physiological environment to maintain good stability.However,these strategies have some shortcomings such as complicated process and difficult preparation,which hinders the further clinical application of PB.In response to the above problems,this chapter proposed"one-pot method"to prepare PB nanoparticles with controlled and stable properties.Methods:Through the study of the ratio of synthetic raw materials,preparation conditions and other factors,further characterization;and the advantages of this method of bulk synthesis was studied.Subsequently,its multi-modality imaging and photothermographic properties were verified by in vitro and in vivo experiments.Results:By researching the influencing factors such as the ratio of raw materials,preparation conditions and so on,PB nanoparticles with the size of⁷0 nm,high stability and controllability in physiological environment were finally obtained.This strategy allows for the bulk preparation of PB nanoparticles,which can stably exist in physiological environment for at least 90 days,and can maintain stable performance in various environments?different temperatures and different pHs?and is easy to be preserved,thereby being beneficial for clinical transformation applications.Then the toxicity of PB nanoparticles in mice was studied.The results showed that injecting PB nanoparticles into mice for 90 days did not cause obvious damage to the blood or organs of mice,as well as systemic toxicity,suggesting it was suitable for intravenous application in the field of biomedicine.PB nanoparticles have strong absorption in the near infrared region?600-900 nm?,excellent photo-thermal conversion(molar extinction coefficient of 4.7×10¹⁰ M^-1cm^-1)and high photothermal conversion efficiency??:36.4%?and photo-thermal stability.As an example of this,this chapter also studied the dual-mode MRI and PA magnetic resonance imaging and photoacousti imaging and photo-thermal therapy of PB nanoparticles on tumors.The results showed PB can be used as T₁ MRI contrast agent,with longitudinal relaxation rate of r₁=0.1665mM^-1S^-1 and lateral relaxation rate of r₂=0.2699 mM^-1S^-1,enabling dual-mode MR/PA imaging both in vitro and in vivo,The PB nanoparticles possess high efficiency with photo-thermal treatment.Conclusions:Therefore,this work successfully solved the dispersion stability of PB nanoparticles in physiological environment with a simple and effective one-pot method and is expected to promote the application of PB nanoparticles as theranostic platforms in the biomedical field.?2?Background and purpose:High-intensity focused ultrasound?HIFU?synergist can effectively solve the problems of high power and long-term ablation of potential damage to the normal tissue on the acoustic channel.However,the current HIFU synergists require external excitation to generate bubbles and then enhance ultrasound imaging of the tumor and HIFU synergistic treatment.However,they could not achieve ultrasound imaging for diagnosis and location guidance of the tumor before HIFU treatment,resulting in low efficiency of diagnosis and treatment of cancer.To solve this problem,on the basis of the work in the previous chapter,the work in this chapter creatively proposes the preparation of controlled-pore mesoporous Prussian blue nanoparticles by using"defect selective etching method",which solves the problem that biological macromolecules drugs may degrade and easily lose activities when transported in vivo.Methods:Firstly,the influencing factors of"defective selective etching"were studied,the conditions for the construction of macroporous PB were discussed,and the enzyme-loaded enzyme was used to verify its in vitro and in vivo catalytic performance and ultrasonic imaging performance.Finally,the in vitro and in vivo efficacy experiments of HIFU were studied.Results:First of all,the influencing factors of"defect selective etching method"were studied,and PB nanoparticles with controlled pore diameter were successfully prepared.The macroporous Prussian blue nanoparticles?mPBs?with a pore of 3.0¹5.0 nm and a diameter of about 65 nm were screened out by high-performance screening with high dispersion and good stability.The mPBs nanoparticles have high specific surface area(70.530m²g^-1)and a large pore size,which realizes the loading and protection of biological macromolecular drugs.The catalase?CAT?loading amount is163?g/mg and the encapsulation efficiency reaches 74%.Next,oxygen is generated in situ by CAT-catalyzed hydrogen peroxide?H₂O₂?at the tumor site by intravenously delivering CAT-loaded mPBs?CAT@mPBs?to the tumor site for enhanced ultrasound imaging,and focused ultrasound synergistic therapy of liver cancer.The results showed that CAT@mPB nanoparticles can react with low concentration of H₂O₂ to generate a lot of oxygen bubbles in vitro,which can be used as a good contrast agent and enhance ultrasound imaging in vitro and in vivo.In addition,hemolysis tests and in vitro and in vitro toxicity tests demonstrated good biocompatibility and safety of CAT@mPB nanoparticles.In vitro and in vivo HIFU ablation experiments observed that CAT@mPB can be used as a drug delivery carrier in situ enhancing ultrasound imaging of the tumor,in situ ultrasound imaging guided HIFU synergistic effect.Conclusions:This strategy generates oxygen by in situ decomposition of H₂O₂ by catalyzing the high concentration of H₂O₂ in the tumor microenvironment,which not only realizes the ultrasound imaging and localization of the tumor before treatment,but also can guide subsequent multiple HIFU synergistic effects and is expected to solve the problem of HIFU synergism agents,and improve HIFU tumor diagnosis and treatment efficiency.