Impact Of Molecular Rigidity On The Gene Delivery Efficiency Of Core-shell Tecto Dendrimers

Gene therapy has become one of the most effective treatment methods for malignant tumors,cardiovascular diseases and other genetic diseases.The construction of safe and efficient gene delivery vectors is the key to the successful implementation of gene therapy.In recent years,non-viral vectors(liposomes,cationic polymers,etc.)have attracted widespread attention due to their higher biological safety and lower immunoreactivity than viral vectors.However,there are still huge challenges in developing safe gene vectors with high transfection efficiency.Among many non-viral vectors,polyamidoamine(PAMAM)dendrimers have been favored in recent years due to their unique three-dimensional structure,positive charge and easy surface functionalization.The literature shows that the gene delivery efficiency of PAMAM can be enhanced by surface modification(such as target ligand modification),increasing generation number and adjusting internal structure(such as encapsulating gold nanoparticles).The higher the PAMAM generation number is,the better gene delivery efficiency is obtained,while the greater the synthesis cost and material defects are.In recent years,high-generation-like core-shell tecto dendrimers(CSTD)synthesized by supramolecular host-guest chemistry have been used as substitution of high-generation PAMAM for gene delivery.The enhanced gene delivery efficiency of encapsulated gold nanoparticles are postulated to be that gold nanoparticles help maintain a three-dimensional structure of dendrimer to endow enough gene binding sites.However,the effect of molecular rigidity on gene delivery still needs further investigation.In addition,another new member of the dendrimers family,phosphorous dendrimers have been gradually applied in biomedical fields,such as gene delivery and tumor therapy,due to their superior biocompatibility,highly uniform molecular weight and prior rigidity caused by their own molecular skeleton.Based on these,in this study we constructed two kinds of CSTDs with different rigidity using PAMAM(generation 3,G3)and phosphorous dendrimers(generation 2.5,G2.5)as the core and the same PAMAM(G3)as the shell,respectively,to explore the effect of molecular rigidity on gene delivery efficiency and enrichment degree in tumor site.Firstly,the phosphorus dendrimers of generation 2.5(P-G2.5)with aldehyde termini were used as cores,and the G3 PAMAM dendrimers as shells were chemically assembled onto the P-G2.5dendrimer surface to form the P-G2.5/G3 CSTD with rigid aromatic backbone cores.At the same time,G3-CD/Ad-G3 CSTD with flexible core architectures were synthesized with G3 PAMAM as both core and shell via the?-CD and Ad supramolecular assembly.Two types of CSTDs were combined with p DNA through electrostatic adsorption to form vectors/p DNA complexes.Then,CSTDs with different rigid cores and the two vectors/p DNA complexes were evaluated by various characterization techniques.Subsequentially,the cytocompatibility of two vectors and the vector/p DNA complexes,cellular uptake and intracellular localization were evaluated by in vitro cell assay.The gene transfection efficiency of the two vectors was evaluated by green fluorescent protein(EGFP)expression assay and luciferase(Luc)expression assay.Finally,two different rigid core-shell tecto dendrimers were chelated with ^99mTc,and then multifunctional modification was performed to obtain ^99mTc-vector-DOTA-Cy5.5-PS.The effect of material rigidity on enhanced permeability and retention effect(EPR)on material accumulation at tumor site was investigated by using SPECT/fluorescence dual-mode imaging.We show that the developed P-G2.5/G3 CSTDs with rigid aromatic backbone cores display better DNA binding ability,more enhanced cellular uptake of the vector/p DNA polyplexes,and 4.1and 1.7 times higher expression of Luc and EGFP,respectively than G3-CD/Ad-G3 CSTDs with flexible core architectures,showing a stronger ability to penetrate into tumors.Overall,we reveal herein that the increase of molecular rigidity of the vectors can help enhance the gene delivery efficiency and the ability of tumor penetration,providing a new insight for future development of enhanced non-viral gene delivery.