Plasmids, that are round double-stranded DNA molecules, have the power to copy independently of the chromosomal DNA inside host cells, offering a steady genetic setting for exogenous genes. [1] In genetic engineering, plasmids are indispensable gene vectors. They’re used for cloning and amplifying particular DNA sequences, discovering in depth functions in gene cloning, expression, supply, and regulation research. [2] Notably, plasmids have numerous functions in gene remedy. As an illustration, in cell therapies akin to CAR-T, [3] TCR-T, [4] UCAR-T, [5] and adeno-associated virus gene remedy, [6] plasmids perform as elementary supplies for setting up viral vectors and delivering goal genes. Within the manufacturing of mRNA vaccines, plasmids function transcription templates. [7] Moreover, in DNA vaccine manufacturing, plasmids are the ultimate product. [8], [9] Subsequently, the environment friendly supply of plasmids is essential for these functions, impacting each gene expression effectivity and the protection and efficacy of biotechnological merchandise.
Regardless of the quite a few benefits, current plasmid supply methods are fraught with substantial limitations. For instance, bodily strategies akin to electroporation, microinjection, and the gene gun necessitate refined tools, which may trigger mobile harm and thereby disrupt the conventional physiological state of the cells. [10] Organic strategies (e.g., viral-mediated transfection) provide excessive transfection effectivity, however pose potential security dangers, together with the oncogenicity and immunogenicity of viral vectors. [11], [12] Chemical strategies regularly utilized in analysis embody calcium phosphate co-precipitation and cationic liposome transfection, [13], [14], [15] which face unavoidable cytotoxicity and infrequently contain the aggregation of enormous quantities of plasmids inside a single provider. The ensuing points, akin to plasmid entanglement or advanced construction formation, can affect their stability and correct expression in host cells. Moreover, in gene remedy functions, the exact supply of particular genes to particular cell sorts is regularly important, [16] which could be difficult for bulk plasmid supply.
Rational plasmid encapsulation methods are instrumental in enhancing key parameters of supply, akin to security, stability, and expression effectivity. In latest instances, DNA-based mineralization and self-assembly methods have emerged as more and more outstanding and quickly evolving approaches. Nonetheless, most of those strategies depend on disordered meeting and fail to use the innate guiding position of the DNA spine. Impressed by conventional metal-organic coordination chemistry, [17], [18] the rising metal-phenolic networks (MPNs) have garnered substantial consideration and supplied new insights into plasmid supply methods. [19], [20], [21] MPNs as hybrid organic-inorganic programs, mix the favorable biocompatibility of pure polyphenols with the coordinative exercise of metallic ions. [22], [23], [24] Metallic ions can act as cross-linking brokers between polyphenol molecules, permitting quickly self-assembling of polyphenols and metallic ions into molecular cross-linked networks to kind nanoparticles, [25] vesicles, [26] composite hydrogels or scaffold supplies. [27] Nonetheless, the variety and purposeful variability of metallic ions and polyphenols, particularly inside the intricate ternary system that includes DNA, pose substantial challenges to their utilization as carriers for macromolecular nucleic acids akin to plasmids. These challenges embrace points associated to precursor choice, advanced synthesis situations, and materials instability.
To handle these limitations, the present research introduces an revolutionary DNA (plasmid)-templated in situ self-assembly technique for setting up MPN-pDNA complexes, as illustrated in Scheme 1. By using low-toxicity zinc ions (Zn2+) and extremely biocompatible polyphenols, this strategy achieves plasmid encapsulation via the design of metal-phenolic coordination websites and exact management of their ratios. This methodology of fixedly encapsulating plasmids considerably reduces the disorderly aggregation and entanglement of DNA strands, thereby enhancing the steadiness of the plasmids and minimizing potential cell harm and toxicity. Moreover, the theoretical evaluation of the chemical self-assembly mechanism of phenolic ligands signifies that the self-assembly means of the Zn-polyphenol@pDNA (ZP@p) advanced is achieved via non-covalent interactions between Zn and the plasmid bases. This non-covalent interplay is advantageous in sustaining the structural integrity of the plasmid throughout meeting and intracellular launch, thereby facilitating environment friendly launch inside the host cells, bettering the transfection effectivity and reaching exact gene expression. This novel plasmid encapsulation technique gives a safer, extra environment friendly, and controllable methodology for plasmid supply, paving the best way for development in DNA nanobiotechnology and MPN chemistry.