Construction Of Hyperbranched Macromolecules-based Multifunctional Nanoplatforms For Gene Therapy Application

The rapid development of molecular biology and nanotechnology has greatly promoted the application of nanomaterials in biomedical field,which plays a key role in gene delivery,photothermal therapy?PPT?,drug delivery and medical imaging,etc.In order to realize the widely application of nanotechnology in disease treatment,the exploitation of safe,stable and efficient functional nanomaterials has become a critical goal.A variety of nanomaterials,including but not limited to liposome,hyperbranched polymers and nature polymers have become a hot topic in current research.Thereinto,the unique physicochemical property and structural features of hyperbranched polymers can not only provide theoretical foundation for the construction of advanced functional hyperbranched polymers,but also provide new insights into the discovery of new methods for the treatment of disease.In order to optimize the biocompatibility of hyperbranched polymers?Generation five Poly?amidoamine?dendrimers?G5.NH2?or polyethylene imine?PEI??,enhance gene transfection efficiency of vectors and improve cancer-killing effects of materials,in this thesis,hyperbranched polymers were employed as a platform to construct a series of hyperbranched polymers-based nanomaterials for biological therapies of diseases.The specific contributions of this thesis can be outlined as follows:?1?RGD peptide-modified dendrimer-entrapped gold nanoparticles loaded with h BMP-2 p DNA for gene therapy in mesenchymal stem cells;?2?Efficient delivery of therapeutic si RNA into glioblastoma cells using multifunctional dendrimer-entrapped gold nanoparticles for gene therapy;?3?Dendrimer-modified Mo S₂ nanoflakes as a platform for combinational gene silencing and photothermal therapy of tumors;?4?Multifunctional PEI-entrapped gold nanoparticles enable efficient delivery of therapeutic si RNA for cancer gene therapy.The systematic research of prepared functional hyperbranched polymers will be implemented;on the one hand,we aggressively explore the potential of dendrimer-based nanopaticles in multiple gene therapy,even in photothermal therapy.On the other hand,we try to develop low-cost hyperbranched polymers instead of dendrimers,and provide new ideas and developing direction for the preparation of novel multifunctional nanomaterials.The main contents and results of this dissertation are are shown below:1)In chapter 2,RGD peptide-modified dendrimer-entrapped gold nanoparticles were prepared according to the following progress: Generation 5 poly?amidoamine?dendrimers modified with RGD via a poly?ethylene glycol??PEG?spacer and with PEG monomethyl ether were used as templates to entrap gold nanoparticles?Au NPs?with the molar feeding ratio of Au/G5.NH2 = 25 via sodium borohydride reduction chemistry.The formed Au NPs inside of Au DENPs displayed a similar spherical shape with an uniform size distribution around 1.9-2.1 nm.Under the N/P ratio of 1 or above,p DNA was completely compressed by Au DENPs.Therefore,N/P ratios between 1 5 were selected for the following gene transfection studies.For the gene transfection in stem cells by using Au DENPs/EGFPLuc p DNA polyplexes,the result of Luc activity assay revealed that the K4 vector possessed the highest gene transfection efficiency when compared with the other four vectors at an N/P ratio of 2.5.In the meanwhile,the result of EGFP gene expression was in agreement with the Luc activity assay.Whereafter,the data of h BMP-2 gene delivery efficiency,alkaline phosphatase activity,osteocalcin secretion,calcium deposition and von Kossa staining assays of Au DENPs/h BMP-2 p DNA polyplexes was evaluated.Our results reveal that the stem cell gene delivery efficiency is largely dependent on the composition and the surface functionality of the dendrimer-based vectors.The coexistence of RGD and Au NPs rendered the designed dendrimeric vector with specific stem cell binding ability likely via binding of integrin receptor on the cell surface and improved three-dimensional conformation of dendrimers,which is beneficial for highly efficient and specific stem cell gene delivery applications.It is proved that the developed RGD functionalized Au DENPs are able to transfect the h BMP-2 gene to h MSCs safely and efficiently to enable osteogenic differentiation of the h MSCs.2)Based on chapter 2,chapter 3 chose two different Au DENPs possessing higher gene transfection efficiency as delivery vectors to complex with therapeutic si RNAs?VEGF si RNA ?Bcl-2 si RNA?.The gene transfection efficiency and specific gene silencing property of Au DENPs/si RNA polyplexes was studied.The results demonstrated that Au DENP/si RNA polyplexes were in the nanoscale size range??200 nm?and with a surface potential of 20 m V,indicating that the Au DENP/si RNA polyplexes prepared may be considered as suitable transfection materials to cross the cell membrane.Under different dendrimer concentrations?0 3000 n M?and different N/P ratios?2.5 15?,the cell viability was shown to be concentration-dependent and N/P ratio-dependent,respectively.However,even under the highest Au DENP concentration and when ender the highest N/P ratio,more than 70% of the cells were still alive,showing outstanding cytocompatibility.The results of si RNA transfection expriment proved that at an N/P ratio of 10,Au DENPs/si RNA polyplexes displayed the strongest fluorescence intensity,and RGD-modified materials showed higher gene transfection efficiency than non-targeted ones.The western blot data in vitro and in vivo demonstrated that in terms of the Au DENPs/si RNA polyplexes,low levels of gene expression were observed at an N/P ratio of 10,indicating the successful gene silencing.Furthermore,the RGD modification rendered the Au DENPs with more than 70% gene silencing efficiency.All the results revealed that with the good cytocompatibility of Au DENP/si RNA polyplexes and a high efficiency transfection capacity of the polyplexes,therapeutic si RNAs may be complexed with the RGD-modified Au DENPs for gene therapy applications.3)The research in chapter 2 and chapter 3 testified that dendrimers were a kind of safe and efficienct gene delivery vector.Nevertheless,previous work indicated that individual treatment via gene therapy was difficult to guarantee the complete killing of cancer cells,and only inhibited the proliferation of cancer cells to a certain extent.The unique advantages such as high specific surface area,enhanced surface adsorption property,good biocompatibility and high photothermal conversion efficacy make Mo S₂ nanoflakes have the potential to be used as a safe and efficient photothermal agent.In this work,Mo S₂ nanoflakes synthesized via a hydrothermal approach were modified with G5 dendrimers partially functionalized with lipoic acid via disulfide bond.The formed G5-Mo S₂ nanoflakes displayed good colloidal stability,photothermal stability and biocompatibility,and possessed superior photothermal conversion efficiency?47.8%?,which showed great damage to cancer cells.With the dendrimer surface amines on their surface,the G5-Mo S₂ nanoflakes were capable of delivering Bcl-2?B-cell lymphoma-2?si RNA to 4T1 cells with excellent transfection efficiency,inducing 47.3% of Bcl-2 protein expression inhibition.Moreover,in vitro combinational PTT and gene therapy data indicated that cells treated with the G5-Mo S₂/Bcl-2 si RNA polyplexes under laser irradiation have a viability of 21.02%,which was much lower than other groups of single mode PTT treatment?45.8%?or single mode of gene therapy?68.7%?.Moreover,the super efficacy of combinational therapy in vivo was further proved that tumors were almost completely destroyed,and the survival rate of mice was about 100%.These results suggested that the designed G5-Mo S₂ nanoflakes possess good biocompatibility and blood compatibility,which can realize the combinational gene silencing and PTT of tumors.4)Considering of the high-cost of dendrimers against the industrialization production of final products,chapter 5 attempt to use PEI as vector,in which functionalized modifications can be performed.In this work,PEI was partially linked with PEG and a PEGylated RGD peptide via covalent linkage,which was used as templates to entrap gold nanoparticles?Au NPs?with the molar feeding ratio of Au/G5.NH2 = 25 via sodium borohydride reduction chemistry.The formed Au NPs inside of Au PENPs had a relatively narrow size distribution with a nanoscale size range of 2.5 2.9 nm and exhibited a spherical or semi-spherical shape,showing good stability and biocompatibility.The gene transfer data demonstrated that the developed Au PENPs had the highest gene delivery efficiency at an N/P ratio of 10.The western blot data in vitro and in vivo demonstrated that Au PENPs/si RNA polyplexes inhibited the expression of homologous protein,thus inducing specific gene silencing.This study demonstrated that Au PENPs were able to effectively condense si RNA and delivered si RNA to cancer cells overexpressing ?v?3 integrin,inducing the gene silencing to knockdown the target protein expression.