Study On Multifunctional Nanoparticles For The Molecular Diagnosis And Therapeutics Of Hepatocellular Carcinoma Based On Theranostics

Human hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Chemotherapy is one of the main treatment methods in clinic. However, Chemotherapy usually met the following two problems:a) the common dosage was based on the average dosage level of patients which might result in the failure of HCC therapy; b) the evaluation of the treatment was carried out after a treatment cycle, which mighy missed the best time of the treatment.Recently, theranostics which combined cancer diagnosis and therapeutics in a single platform, received great attention in cancer treatment. Theranostics offered several advantages including the assessment of the biodistribution and accumulation of drugs at target sites noninvasively, the visualization of drug distribution and drug release at the target site, the optimization of formulation which relied on triggered drug release, and the real-time monitoring the therapeutic responses. So, theranostics therapy showed great potential in the individualized therapy and the real-time monitoring the treatment of HCC patients.In this study, the multifunctional carriers were designed and prepared to achieve the individualized therapy of HCC based on theranostics. Firstly, the polymeric nanoparticles were selected as the theranostics carriers. The novel three block polymer PLA-PEG-PLL which combined drug-loading, long circulation time and easily modification functions and two block polymer PLH-PEG-Biotin which combined pH-sensitivity, long circulation time and active modification functions were designed and synthesized. Then, the HCC therapeutic drugs Paclitaxel (PTX) and sorafenib were employed as the model drug; Magnetic Resonance Imaging (MRI) was employed as the diagnosis method; vascular endothelial growth factor (VEGF), Alpha-fetoprotein (AFP) and vascular endothelial growth factor receptor (VEGFR) which were overexpressed in tumor microenvironment were selected as the delivery target; there multifunctional polymeric nanoparticles, including VEGF antibody modified PTX-loaded polymeric nanoparticles (TPN), AFP antibody modified Gd/PTX-loaded polymeric nanoparticles (TGPN) and pH-sensitive VEGFR antibody modified Gd/PTX-loaded polymeric nanoparticles (TPTN) were prepared by self-assembly method. At last, the basic properties, in vitro release profiles, cell cytotoxicity, cell uptake properties, in vitro MRI properties, in vivo MRI properties, in vivo anti-tumor ability and tissue safety were evaluated.Above all, this article designed and synthesized two novel polymers, PLA-PEG-PLL and PLH-PEG-biotin, and prepared three kinds of multifunctional polymeric nanoparticles based on theranostics to achieve the target therapy, treatment monitoring, individualized therapy of HCC. The main study methods and results were showed as follows:1. Synthesis and characterization of multifunctional materialsTo fabricate multifunctional theranostic polymeric nanoparticles, the multifunctional polymer were firstly synthesized by polymer-polymer conjugation method which was first proposed by our group. Polymer-polymer conjugation is a novel multi-block polymers synthesis method that conjugated different polymer blocks by reactions between the terminal active groups of different blocks. The synthesis procedure was relatively simple without complex synthesis steps compared to traditional polymerization method. The synthesized multi-block polymers could have more relatively precise molecular weight of each block than that synthesized by traditional polymerization. Otherwise, different single block could be selected according to the expected functions of the multi-block polymers. Any single block that contained functional active groups for the conjugation could be used in this novel synthesis method. This facility and availability of different blocks indicated that multi-block polymers with various blocks or different block numbers could be synthesized theoretically by polymer-polymer conjugation.1.1Synthesis and characterization of PLA-PEG-PLLThe carboxyl group of PLA-COOH reacted with the amino group of NH2-PEG-NH2to obtain PLA-PEG-NH2. Then, the amino group of PLA-PEG-NH2reacted with the carboxyl group ofPLL(Cbz)-COOH to obtain PLA-PEG-PLL(Cbz). After the deprotection of amino groups of PLA-PEG-PLL(Cbz), PLA-PEG-PLL was successfully synthesized. PLA-PEG-PLL combined drug loading, long circulating in vivo, and easy for modification functions. The structures of products were verified by NMR. GPC results showed that the number-average molar mass of PLA, PLA-PEG and PLA-PEG-PLL was13691,16013and20441Da and the molecular weight distribution (Mw/Mn) was1.427,1.277and1.358, respectively. These results indicated the successfully synthesis of PLA-PEG-PLL by polymer-polymer conjugation. The critical micelle concentration of PLA-PEG-PLL was determined using pyrene as an extrinsic probe and the critical micelle concentration of the PLA-PEG-PLL was7.70×10-3mg/mL. Based on PLA-PEG-PLL, two modified polymer PLA-PEG-PLL-DTPA and PLA-PEG-PLL-Biotin were synthesized by amide reaction for Gd loading and antibodies modification, respectively.1.2Synthesis and characterization of PLH-PEG-BiotinThe carboxyl group of PLA-COOH reacted with the amino group of Biotin-PEG-NH2to obtain PLH-PEG-Biotin. The structures of PLH-PEG-Biotin were verified by NMR. The acid-base titration results showed that PLH-PEG-biotin exhibited great buffering capacity with a buffering zone within pH5-7, which could induce the solubility change of PLH from the blood (pH=7.4) to the tumor tissue (pH=5～7).2. Preparation and characterization of TPNMolecular therapy was the hot spot in recent years. It could increase the therapeutic effect of HCC and decrease the toxicity in the treatment. Thus, the VEGF antibody modified PTX-loaded polymeric nanoparticles (TPN) were prepared for the molecular therapy of HCC. PTX HPLC detection method was firstly established and the detection method reach the methodological study requirements. The TPN were prepared by solvent diffusion method. The morphology of TPN had spherical or ellipsoidal shapes without agglomeration. The particle size, zeta potential, EE%and DL%of TPN were203.6±4.10nm,20.76±0.34mv,86.23±1.79%and1.42±0.03%, respectively. In the release studies in vitro, compared to Taxol(?), TPN showed a controlled release profile and fitted Higuchi equation:Q=8.0449t1/2-0.5721, R=0.9678. The results of cell cytotoxicity studies showed that TPN (IC50=0.392±0.012μM) had significantly higher cytotoxicity compared to Taxol(?)(IC50=3.554±0.233μM) and non-target TPN (IC50=1.510±0.099μM)(P＜0.01), which indicated that TPN had good in vitro anti-tumor activity. In cell uptake studies, the cell uptake of FITC-labeled TPN was both time and concentration dependence. The cell uptake rates of FITC-labeled TPN were significantly higher than FITC-labeled non-target TPN at different incubation concentrations (P＜0.01). The results of in vivo tumor growth inhibition studies showed that TPN could significantly inhibit the growth of tumors compared to NS, Taxol(?), and non-target TPN (P＜0.01), which indicated that TPN had good in vivo anti-tumor activity. According to these results, TPN was an excellent drug carrier for the molecular therapy of HCC.3. Preparation and characterization of TGPNThe achieve theranostic and individualized therapy of HCC, the diagnositic method should be added to the target therapy therapy carrier. Thus, the AFP antibody modified Gd/PTX-loaded polymeric nanoparticles (TGPN) was prepared for the theranostic therapy of HCC. The TGPN were prepared by solvent diffusion method. The morphology of TGPN had spherical or ellipsoidal shapes without agglomeration. The particle size, zeta potential, EE%and DL%of TPN were147.50±4.7nm,24.45±1.04mv,88.76±1.64%and1.59±0.06%, respectively. In the release studies in vitro, compared to Taxol(?), TGPN showed a controlled release profile and fitted Weibull equation:lnln[1/(1-Q/100)]=0.696ltt-6.5891, R=0.9533. The results of cell cytotoxicity studies showed that TGPN had higher cytotoxicity compared to Taxol(?) and non-target TGPN in24h (P＜0.05), which indicated that TGPN had good in vitro anti-tumor activity. In cell uptake studies, the cell uptake of FITC-labeled TGPN was both time and concentration dependence. The cell uptake rates of FITC-labeled TGPN were significantly higher than FITC-labeled non-target TGPN at different incubation concentrations in AFP-positive HepG2cells in0.5h (P＜0.01). While, the cell uptake rates did not showed any difference in AFP-nagetive B16cells, which proved the increased cell uptake was induced by AFP antibodies. The results of in vitro MRI studies showed that TGPN had high relaxivity (25.388nM-1s-1) which was5.18time to Magnevist(?)(4.9mM-1s-1). The results of in vivo MRI studies showed that the enhance signal area (AUC) of TGPN was87.6time to Magnevist(?) and the highest signal enhancement was2.94time to Magnevist(?). Meanwhile, the diagnosis time of TGPN was greatly prolonged from less than1h (Magnevist(?)) to more than6h. There results indicated that TGPN had good in vivo diagnosis ability. The results of in vivo tumor growth inhibition studies showed that TGPN could significantly inhibit the growth of tumors compared to NS, and Taxol(?)(P＜0.01), which indicated that TGPN had good in vivo anti-tumor activity. According to these results, TGPN had excellent HCC diagnosis and treatment ability, which showed great potential in the theranostic and individualized therapy of HCC.4. Preparation and characterization of TPTNThe controlled and responsive release in the tumor area is important to increase the diagnostic sensitivity and therapeutic efficacy of HCC. Base on this consideration, the pH-sensitive VEGFR antibody modified Gd/PTX-loaded polymeric nanoparticles (TPTN) was prepared for the theranostic therapy of HCC. Sorafenib HPLC detection method was firstly established and the detection method reach the methodological study requirements. The TPTN were prepared by solvent diffusion method. The morphology of TPTN had spherical or ellipsoidal shapes without agglomeration. The particle size, zeta potential, EE%and DL%of TPN were181.4±3.4nm,14.95±0.60mv,95.02±1.47%and2.38±0.04%, respectively. TPTN showed good stability in the presence of10%serum in2-4h. In the release studies in vitro, TPTN showed pH-sensitive drug release profile. The release rate in the release media (pH=5.0) was significantly higher than in the release media (pH=7.4)(P＜0.01). The results of cell cytotoxicity studies showed that TPTN had similar cytotoxicity after incubated in pH5.0for1h compared to sorafenib silution, which indicated that TPTN had good in vitro anti-tumor activity. In cell uptake studies, the cell uptake of FITC-labeled TPTN was both time and concentration dependence. The cell uptake rates of FITC-labeled TPTN were significantly higher than FITC-labeled PTN at different incubation concentrations in VEGFR-positive HepG2cells (P＜0.01). The results of in vitro MRI studies showed that TPTN had high relaxivity (17.300mM-1s-1) which was3.53time to Magnevist(?)(4.9mM-1s-1). In MRI studies in vivo, after administration of TPTN, the tumor area showed visual brighter image than the surrounding tissue and the boundary of tumor tissue could be clearly demarcated. Meanwhile, the diagnosis time of TPTN was greatly prolonged from less than1h (Magnevist(?)) to more than1.5h. There results indicated that TPTN had good in vivo diagnosis ability. The results of in vivo tumor growth inhibition studies showed that TPTN could significantly inhibit the growth of tumors compared to NS, sorafenib solution and PTN (P＜0.01), which indicated that TPTN had good in vivo anti-tumor activity. The results of histological evaluation studies showed that TBN groups did not have visible difference compared to the control in different organs, which indicated that TBN was a safe carrier. According to these results, TPTN could achieve the target release of drug and imaging agent at tumor area to increase the diagnostic sensitivity and therapeutic efficacy. TPTN was a promising theranostic carrier for the individualized therapy of HCC.Thus, this study synthesized novel multi-block materials and fabricated theranostic nanoparticles for the theranostic treatment of HCC. The theranostic nanoparticles combined pH-sensitivity, long circulating time, drug/imaging agent co-loading and active targeting functions showed great potential in target therapy, treatment monitoring, individualized therapy of HCC. All in all, the theranostic nanoparticles provided a new concept, new tool and new strategy to theranostic tumor therapy.