Phenylboronic Acid Modified Dendrimers For Cytosolic Delivery Of Biomacromolecules

Biomacromelcules based pharmaceutics have achieved huge success in the past decades,such as gene therapy and gene editing.However,inefficient delivery of nucleic acid,native proteins and peptides into target cells hinders their application.In Chapter 1,we summarized the definition,categories and applications of biomacromelcules and methods of cytosolic delivery.We propose strategies to overcome the main obstacles in cytosolic delivery of nucleic acid and proteins.Cationic polymers were widely used as gene vectors.However,polymer-based gene transfection reagents are still involved with several challenges.One of the most important problems is the correlation between the deliver efficacy and cytotoxicity.High molecular weight polymers are reported with relatively high delivery efficacy but serious cytotoxicity due to the excess positive charges on the polymers.However low molecular weight polymers with minimal cytotoxicity have weak delivery efficacy.In Chapter 2,We present a facile strategy to prepare high efficient polymers with low transfection toxicity.Dedrimers with unique properties were used as a representative polymer.Small dendrimers were clustered into 100 nm nanostructures via phenylboronic acid modification and self-assembled materials enable efficient DNA and siRNA delivery.The synthesized materials can disassemble into small dendrimers in acidic conditions thus exerting significantly less cytotoxicity.Further structurefunction relationship studies reveal that both the phenyl group and boronic acid group play essential roles in the self-assembly and gene delivery processes and demonstrate the potential application of phenylboronic acid in biomacromelcules cytosolic delivery.We provide a facile strategy in the development of efficient and biocompatible gene vectors based on low molecular weight polymers.Cytosolic delivery of protein and peptide is of great importance for biotherapeutics and basic biological research,but is challenging due to the lack of efficient and safe transduction technologies.The problems facing protein and peptide delivery are manifold and much complicated than gene or chemical drugs delivery including the uncertainty of protein charge,large molecular weight and the easy of degradation.Cationic polymer,liposomes and nanoparticles are widely used as protein and peptide carriers for cytosolic delivery.However,challenges still remain regarding the low efficient cytosolic delivery of native proteins and peptides.One of the main obstacles is the limited binding sites on proteins.Previous approaches for solving the problem usually need the fusion of proteins with protein transduction domains,or supercharged molecular tags for binding with vehicles such as polymers and nanoparticles.These approaches still possess some limitations such as limited transduction efficacy,the need of covalent modification on proteins,or complicated synthesis,which will reduce protein bioactivity and unexpected safety concerns.In Chapter 3,a boronate-rich dendrimer was rationally designed with robust efficacy for intracellular delivery of native proteins and peptides.The phenylboronic acid-conjuated dendrimer could bind with both negatively and positively charged proteins and was efficient in the delivery of 13 proteins and 6 peptides into cytosol of living cells.The enzymes and other functional proteins kept their bioactivites after cytosolic delivery.By comparing the protein delivery efficiency of several phenylboronic acid analogue materials,we confirmed the essential role of boronic and phenyl groups in binding protein and cytosolic delivery.The significances of this study providing a novel strategy to design polymers with high generality for protein delivery because the polymer could bind proteins of different molecular weights and isoelectric points via a combination of ionic interactions,boronate-nitrogen coordination and cation-? interactions.More importantly,the dendrimer allows the efficient delivery of Cas9 ribonucleoprotein targeting multiple genome loci of different types of cell lines,which defines a useful material for the delivery of genome editing tools in a wide range of biomedical applications in the future.Considering the proteins diversity and how to improve the complex stability and cytosolic delivery efficiency,there is an urgent need to provide multiple strategies.Polyphenols which are the most common and widespread constituents in plants and human diets have strong interaction with proteins.The phenomenon has been sufficiently investigated and widely used for biomedical applications.In Chapter 4,we proposed a novel strategy to deliver proteins of different molecular sizes and isoelectric points by specific recognitions between natural polyphenols such as green tea catechins and boronic acid containing polymers.Various proteins were decorated with natural polyphenols via non-covalent hydrogen-bond,hydrophobic interactions or reversible dynamic covalent bonds.The polyphenols increase the binding affinity between proteins and phenylboronic acid containing polymers.The strategy allows proteins form stable complexes with the designed polymers,promote the endosome escape and cytosolic delivery efficiency.After intracellular delivery,the bound proteins can release from the complex in acidic environments due to pHsensitive of catechol-boronated esters,which maintains the proteins bioactivity.In Chapter 5,we drawed the conclusions that phenylboronic acid modified dendrimers have great potential to solve the key issues in biomacromelcules cytosolic delivery with high efficiency and low cytotoxicity.The results in these studies are essential for rational design of polymers for intracellular delivery of biomacromelcules such as nucleic acid,proteins and peptides.The strategies will promote the development of biomacromolecule based biotechnologies and therapeutics.