Development And Application Of A Protein Delivery System Based On Crosslinked Spherical Nucleic Acid

In recent years,with the rapid development of the field of biopolymer drug development,protein drugs with characteristics such as high activity,high specificity,low toxicity,and clear biological function have become a research hotspot in the biomedical field.However,due to the disadvantages of large molecular weight,poor permeability,instability,and difficulty in achieving controlled release,protein drugs face huge challenges in clinical application.Based on the limitations of proteins,researchers have developed various protein drug delivery strategies.Based on different transmembrane mechanisms,such as liposome-like liposomes,inorganic nanoparticles,polymers,responsive polymers,and other carrier delivery strategies.These methods have their own advantages,but the carriers have significant cell toxicity,complex preparation,and cannot estimate the damage to the body after entering the body.There are also defects in carrier-free delivery strategies that have been developed.For example,the carrier-free delivery process of physical methods can damage the cell membrane,which can affect the protein structure and activity.Therefore,it is particularly necessary to build a simple,efficient,and highly safe carrier-free protein drug delivery strategy.Protein spherical nucleic acids（Pro SNAs）are a type of spherical nucleic acid with a protein core and a DNA shell.It has the advantage of the same natural structure as SNAs and participates in receptor-mediated endocytosis on the cell surface to promote protein transfection.More importantly,the protein core of Pro SNAs retains its biological activity.Although Pro SNAs have many advantages,their efficiency is still too low compared to protein delivery systems that rely on carriers,and they cannot meet people’s pursuit of high-efficiency protein delivery.Therefore,some researchers have studied the method of increasing the density of modified DNA to improve the uptake efficiency of proteins,which has been proven to be effective.However,too much DNA modification can cause steric hindrance and may inhibit the activity of functional proteins in the center.At the same time,the number of modification sites on the surface of the central protein is limited,so DNA quantity cannot be increased without limit.Nano-grid structures such as hydrogels and ultra-thin nanofibers have good biocompatibility.Their unique hydrophilic and three-dimensional structures make molecules easy to diffuse and attach to cells.Researchers have confirmed that nano-grid structures of a certain size can promote the entry of nanoparticles into cells.Based on this,this paper proposes a universal carrier-free protein delivery system and designs a nanostructure structure with good biocompatibility and high cell permeability.This structure is centered around spherical nucleic acids,which are cross-linked into a larger nanostructure using DNA base pairing principles,and we have achieved controlled release of protein drugs within tumor cells.The main research contents of the paper are:（1）Construction and characterization of LOX X-SNAs nanoparticles.Based on the excellent cell permeability of protein spherical nucleic acids,LOX-Pro SNAs with LOX as the central protein were constructed,and DNA was cross-linked to the LOX surface via thiol-maleimide reaction.Based on the nanostructure structure to promote nanoparticle delivery strategies,particles centered around lactate oxidase spherical nucleic acids were self-assembled using DNA base pairing principles,and the morphology and size of LOX X-SNAs particles were characterized through atomic force microscopy experiments and hydration particle size measurement experiments.（2）Study on the cellular uptake performance of LOX X-SNAs nanoparticles and quantitative measurement of lactate in living cells.The uptake rate of nanoparticles by cells is affected by factors such as size and charge.We synthesized various sizes of nanostructures by controlling the chemical stoichiometric ratio of LOX SNAs and DNA cross-linking sequences.Several nanostructure were delivered into cells to systematically investigate their delivery efficiency and mechanisms.Then,lactate was quantitatively measured in vitro and in vivo by utilizing the reaction of lactate and its oxidoreductase to generate H₂O₂and acetone.The H₂O₂fluorescence indicator was used to indirectly measure lactate content,and the hydrolysis lactate performance of LOX X-SNAs was explored.Finally,high-efficiency delivery of proteins without carriers and visualization of lactate in living cells were achieved.By comparing the lactate signal-to-noise ratio in cells detected by X-SNAs and SNAs,we found that X-SNAs significantly increased the lactate detection sensitivity of LOX in cells.It showed better lactate detection performance than SNAs.（3）SA-SNAs nanoparticles for cell delivery of protein drugs and controlled drug release in tumor cells in response to stimulation.Due to the high-efficiency cell delivery potential of X-SNAs nanoparticles,we designed DNA cross-linking sequences to assemble RNAse H1 protein drugs（X-SNAs-H1）inside X-SNAs nanoparticle using protein-DNA force.At the same time,we designed DNA sequences to induce the structures to rupture in response to high-expressed survivin m RNA in tumor cells,leading to the release of RNAse H1 and achieving controlled release of protein drugs in tumor cells.The therapeutic effects of X-SNAs-H1 were systematically evaluated in tumor cells and normal cells,demonstrating that SA-SNAs-H1 can specifically respond in tumor cells and control the release of protein drugs.Because we insert survivin antisense oligonucleotide sequences（survivin ASO,promoting cancer cell death）in the DNA cross-linked sequence,we evaluated the therapeutic effect of SA-SNAs H1 in tumor cells through controlled experiments,and found that the dual effects of survivin ASO and RNAse H1 protein drugs accelerated tumor cell death.In conclusion,we successfully constructed a new type of nanostructure particle for carrier-free protein drug delivery system,which has good biocompatibility and high-efficiency cell delivery efficiency.This system promotes disease treatment based on protein delivery.Besides,by delivering particles into cells,we achieved quantitative detection of metabolites in living cells.This is important to visually monitor intracellular lactate content and use lactate for early assessment of related diseases.More importantly,LOX X-SNAs have better detection performance than LOX SNAs,providing a new method for quantifying intracellular tumor characteristic metabolites using its oxidase.At the same time,we loaded tumor treatment protein drugs into cells using this particle and achieved specific response and controlled release of drugs in tumor cells.The successful development of this nanoparticle provides a new pathway for the preparation of green biomedical materials and has an important driving effect on disease treatment based on protein delivery.