Synthesis,Characterization,and In Vitro Evaluation Of Poly(β-amino Ester)as Potential PDNA Carrier

The unique properties of polymers have performed an essential contribution to the drug delivery system by providing an outstanding platform for the delivery of therapeutics macromolecules and genes.Poly(β-amino ester)s(PβAE)s are substantially getting interest as efficient and biodegradable cationic polymeric carriers.They are considered the most potent alternative to viral vectors,because of their hydrophilic behaviors,self-assembled nanoparticle formulations,and excellent transfection performances.PβAEs exhibit dynamic kinetic profiles and tunable charge density,which can be modulated by binding it with the diverse library of functional conjugates.A parallel screening strategy is an effective approach to synthesize different compositional and structural PβAEs that fulfill the multiple requirements of targeted delivery.These intriguing attributes allow PβAEs to have customized therapeutic functions such as excellent encapsulation capacity,high stability,and stimuli-responsive release.The transfection efficiency of non-viral vectors especially polymer is significantly governed by physicochemical properties.The manipulation of physicochemical properties such as molecular weight and formulation determinants are important parameters to be optimized when developing cationic polymeric vehicles.Decreasing the molecular weight of the cationic polymer will lead to a decrease in the protection of the condensed nucleic acids,and cause less cellular uptake,subsequently low transfection efficiency.More detailed information about PβAE-pDNA complex formation and its size/charge,colloidal stability,interactions with blood components,and aggregation is required to develop an efficient vehicle that achieves maximum gene transfection.In this context,we first tried to control PβAE’s molecular weights without modifying its chemical compositions for enhanced gene transfection.The importance of various critical formulation determinants were investigated to increase the reproducibility and efficiency of poly(β-amino esters)(PβAEs)as a safe and potential nanocarrier.Among the various of PβAEs reported in the literature,has been shown to have the most diversified properties for DNA delivery,and thereby PβAE-447 was selected in this work to illustrate the detailed synthetic evaluation of the PβAEs and its application in the preparation of its nanoparticles for the DNA carriers.PβAE-447 is one of the most studied and promising PβAE due to its higher transfection activity.Initially,we applied two synthesis strategies to formulate a library of molecular weight variant PβAE-447 without changing its chemical composition and then assessed their utility as a gene carrier.In one approach,the morphology of the polymers was changed with the modulation of reaction temperature and low viscous polymers were obtained at low temperature.In a second approach,high molecular weight PβAE-447 was obtained at a monomer stoichiometric ratio of 1:1 over a wide range of monomers ratio,while the molecular weight was decreased when the feed ratio diverged from unity.The molecular weights but not the structures were effectively controlled by adjusting the ratio between diacrylate and amine monomers during the synthesis.The PβAE-447 based nanoparticles(NPs)had a diameter of 145.85 ± 31.55 nm along with the zeta potential of 11.28 ± 1.72 mV.The nanoparticles formulated with high molecular weight showed a higher level of gene expression and low cytotoxicity compared to PEI.The results infer that optimizing the molecular weight could be a general approach to modify functionality and would assist to design non-viral vectors with significantly enhanced gene transfection properties.Then high molecular weight PβAE-447 was further characterized,aimed to identify the influence of some key parameters in the formulation process.Initially;high molecular weight PβAE-447 was characterized for aqueous solubility,swelling capacity,proton buffering ability,and cytotoxicity study before nanoparticles formulation.Interestingly,the polymer-supported higher cell viability(above 80%)than the Polyethylenimine(PEI)(18%)at 100 μg/mL.PβAE-447 complexed with plasmid DNA(pDNA)generated nanocarriers of 184 nm hydrodynamic radius(+7.42 mV Zeta potential)for cell transfection.Three different cell lines(HEK-293,BEAS-2B,and A549)were transfected with PEGylated and lyophilized PβAE-447/pDNA complexes.PEGylated PβAE-447 nanoparticles showed transfection efficiencies of 51%,66%,and 22%in HEK-293,BEAS-2B,and A549 cell lines respectively.While the transfection efficiencies of lyophilized PβAE-447 nanoparticles in HEK-293,BEAS-2B,and A549 were 49%,67%,and 21%,respectively.PEGylated and lyophilized PβAE-447 nanoprticles demonstrated better transfection efficiency than PEI.The outcomes toward A549 cells(above 66%)showed the highest transfection efficiency compared to the other cell lines.Innovative-engineered nanostructures are at the cutting edge of nanobiotechnology and nanomedicine.Meticulous synthesis,characterization of physicochemical properties,and the manifestation of biological or chemical moieties on the surface of materials make tailored nanostructures suitable for a number of biomedical applications.After material synthesis,the detailed particles characterization is most important because the physicochemical properties of the material could have a major effect on their biological properties.In the past,there have been many studies conducted to understand PβAEs architecture or optimization to formulate nanoparticles with desired characteristics.These studies;however,focused on a single variable for example controlled release,while in reality,PβAE based nano-complexes face a wide spectrum of bio-physicochemical interactions.The results of this study conclude that the future of nanomedicine depends on the rational design of nanomaterials with controllable physicochemical properties to govern their interactions with biological systems in a predictable manner.Meanwhile,the therapeutic efficacy of various nanomaterials is affected primarily by their composition,however,in the case of cationic nanomaterials,additional physicochemical properties such as size,zeta potential,surface chemistry,the dispersion medium,and ability to agglomerate play a vital role in determining their performances.This study explored valuable information that tailored PβAE-447 nanoparticles exhibit better interactions,insensitivity to the aqueous environment,and superior functionality.Altogether,these results suggested that detailed and thorough understandings of nano-bio interactions are necessary to identify favorable physicochemical properties of various nanomaterials.