The Construction And Application Of Protein Carrier Based Diagnostic And Theraputic System

With the continuous progress of nanotechnology and imaging methods,diagnostic and theraputic system,as a treatment strategy chich covers personalized medicine,molecular imaging and so on,has stronger influence on treatment of diseases.In the process of designing and constructing of diagnostic and theraputic system,how to combine each functional part organically to achieve synergistic and efficient treatment output,and then meet the needs of practical application is still the key and difficult points.The carrier systems,which can integrate the functions of other parts as a"plugbroad",play a vital role in the diagnostic and theraputic.Among various carrier systems,protein carrier system with natural gre at biocompatibility,specific binding ability and surface functionalization modification ability,has great research value in the construction of an efficient diagnostic and theraputic system.Human serum albumin(HSA),as the most carrier characteristic o f protein in serum,which could combine various hydrophobic small molecular drugs,inorganic nanoparticles,imaging molecules with their various binding domains,has been widely applied in building multifunctional diagnostic and theraputic system.Not only simply transport to the loads,HSA could be an amplifier when combining paramagnetic metal ions in its magnetic resonance property.Thus will enabl e the construction of magnetic resonance imaging(MRI)-mediated biosensor platform and efficient theranostics system,and then deepen our understanding of disease from molecular level for developing more effective treatment strategies.Hemoglobin(Hb),which widely exists in organisms,is a natural molecular carrier of metalloporphyrin and plays a unique role in energy transfer and molecular transport.The binding characteristics of heme-free hemoglobin(ApoHb)with porphyrin compounds can not only enhance the water solubility of porphyrins and enable its biological application,but also effectively improve rela ted properties by preventing intermolecular aggregation of porphyrins.Besides,the robust surface chemical modification of Hb can integrate multiple molecular functions,and facilitate the design and construct of efficient theranostics system.In this thesis,based on the unique carrier characteristics of the above two proteins,and combined the advantages of MRI technology and photodynamic therapy(PDT),we successfully constructed several kinds of diagnostic and theraputic systems with high sensitivity and high selectivity for the detection or/and treatment of disease.Details are as follows:(1)In chapter 2,we constructed a HSA-paramagnetic metal manganese ions(HSA-Mn)system with longitudinal relaxation rate amplification ability,which pH responsiveness was explored,and found that could significantly improve the sensitivity of molecular MRI.In addition,through introducing a ¹O₂ responsible polymer molecules m PEG-PAsp-IM,we fabricated a ¹O₂responsible HSA-Mn magnetic resonance probe system,which has application value on MRI-mediated drug transportation and PDT.Otherwise,through experiments,we found that the effectively longitudinal relaxation rate amplification ability of HSA to Mn(II)was highly depend on the free state of Mn(II).This conclusion has important reference value for the construction of probe system based on protein MR amplification effect.(2)In chapter 3,combining the pH-responsive MR amplification of HSA-Mn system with the noninvasive in situ detection advantages of MRI technology,we developed a HSA-Mn hydrogel capsule,which overcame the poor patient compliance,limited penetration depth and unsafe problems of existed methods,and is the first successfully application for in situ noninvasive MRI detection of gastric pH.Taking advantage of the size selectivity of hydrogel capsule to the different-sized molecules,the novel MRI probe could effectively resist the gastric interferences,such as pepsin and mucin.In the meantime,the pH sensibility of HSA-Mn system significantly improved the sensitivity of molecular MRI,which achieved excellent resolution in the detection of abnormal gastric pH(>3)in vitro and in vivo.Hence,the HSA-Mn hydrogel capsule with great biocompatibility could be expected to be a potent tool for in situ gastric pH detection in future clinical application.(3)In chapter 4,based on the Hb,we prepared a novel heme-free hemoglobin carrier(ApoHb),which provided an efficient strategy to the problem of poor water-solubility and limited biological application of porphyrin compounds.In exploring the combination characteristic of ApoHb and porphyrin compounds,we found protoporphyrin and several kinds of metal porphyrin compounds can form stable protein complexes,thus provided the possibility for the further develo pment of the diagnostic and theraputic system.The molecular docking simulation and the calculation of binding constants confirmed that the binding form was efficient and orderly,which provides a basis for further biological applications.In addition,we also prepared an iron/zinc mixed substituted hemoglobin and found that it was also photosensitive,which may open a new avenue to construct a multifunctional system based on natural hemoglobin as a carrier.(4)In Chapter 5,we designed a tumor PDT system whi ch is based on the unique photosensitive properties of synthetic zinc porphyrin-substituted hemoglobin(Zn PHb).This protein complex was relatively stable under physiological conditions.By comparing with the commonly used protein carrier HSA,we found tha t the obtained protoporphyrin zinc substituted hemoglobin(Zn PH b)can effectively resist the interference of physiological hydrophobic molecules.In addition,we found the loaded Zn Pp IX can effectively reduce the heme oxygenase-1(HO-1)activity of tumor cells,which could increase the PDT effect synergistically by reduce the production of bilirubin.Besides,we found the targeted molecule modification of folic acid on protein surface could enhance the intake and retention ability in tumor cells.Thus provi ded the possibility to realize precise treatment.Finally,we applied the prepared two protein complexes in PDT experiments of 4T1 subcutaneous tumor in mice.It was found that the two protein complexes could significantly inhibit the growth of tumor witho ut side effects on other organs and tissues.Besides,the fluorescence property of Zn Pp IX itself facilitated the trace of distribution in the tumors.Hence,our works indicated that the ApoHb,as a novel protein carrier,could build efficient protein nano theranostic system for cancer therapy.