Abstract
The recent advances in genetic engineering demand the development of conceptually new methods to prepare and identify efficient vectors for the intracellular delivery of different nucleotide payloads ranging from short single-stranded oligonucleotides to larger plasmid double-stranded circular DNAs. Although many challenges still have to be overcome, polymers hold great potential for intracellular nucleotide delivery and gene therapy. We here develop and apply the postpolymerization modification of polyhydrazide scaffolds, with different degree of polymerization, for the preparation of amphiphilic polymeric vehicles for the intracellular delivery of a circular plasmid DNA. The hydrazone formation reactions with a mixture of cationic and hydrophobic aldehydes proceed in physiologically compatible aqueous conditions, and the resulting amphiphilic polyhydrazones are directly combined with the biological cargo without any purification step. This methodology allowed the preparation of stable polyplexes with a suitable size and zeta potential to achieve an efficient encapsulation and intracellular delivery of the DNA cargo. Simple formulations that performed with efficiencies and cell viabilities comparable to the current gold standard were identified. Furthermore, the internalization mechanism was studied via internalization experiments in the presence of endocytic inhibitors and fluorescence microscopy. The results reported here confirmed that the polyhydrazone functionalization is a suitable strategy for the screening and identification of customized polymeric vehicles for the delivery of different nucleotide cargos.
Highlights
The transfection of genetic material to the interior of the cells continues to be a key challenge for chemical biology and biomaterials science.[1−10] great progress has been made in recent years, the potential of gene therapy remains fairly underexploited.[8]
The hydrazone bio-orthogonal connection has been confirmed as an excellent and compatible chemical motif for the conjugation of chemical functions in different biomolecular templates.[31,74−80] In this regard, we have recently reported the use of the postpolymerization functionalization of polyhydrazide polymers to afford in situ activated amphiphilic polymers for the delivery of small interfering RNA.[63]
The parent polyhydrazides were incubated with a mixture of one cationic and one hydrophobic aldehyde in H2O/ DMSO (1:1) at 60 °C
Summary
The transfection of genetic material to the interior of the cells continues to be a key challenge for chemical biology and biomaterials science.[1−10] great progress has been made in recent years, the potential of gene therapy remains fairly underexploited.[8]. In most of these examples, the monomer composition strongly influences the final structure of the products after the polymerization reaction.[72,73] The hydrazone bio-orthogonal connection has been confirmed as an excellent and compatible chemical motif for the conjugation of chemical functions in different biomolecular templates.[31,74−80] In this regard, we have recently reported the use of the postpolymerization functionalization of polyhydrazide polymers to afford in situ activated amphiphilic polymers for the delivery of small interfering RNA (siRNA).[63] it is well-known that the intracellular nucleotide delivery mediated by synthetic nonviral vectors is strongly dependent on the nature and the size of the transported cargo.[6,81]
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