Different-Length Hydrazone Activated Polymers for Plasmid DNA Condensation and Cellular Transfection

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.

Supramolecular Recognition and Selective Protein Uptake by Peptide Hybrids

“…The protein‐binding units were located at the interface of the hydrophilic/hydrophobic domains to minimize the impact on the secondary structure. Peptide synthesis was accomplished by a solid‐phase approach using orthogonal protecting groups (Figure S1 in the Supporting Information) . The key step was the selective cleavage of the methyltrityl (Mtt) groups of the two lysines.…”

“…Peptide synthesis was accomplished by a solid-phase approach using orthogonal protecting groups (Figure S1 in the Supporting Information). [50,51] The key step was the selective cleavage of the methyltrityl (Mtt) groups of the two lysines.Mtt removal proceeded in weakly acidic conditions and the reactive alkoxyamines were then coupled" on resin" for the subsequent incorporation of aldehydes or ketones (R 2 , R 3 ). The peptidew as terminatedw ith either af luorophore (TAMRA), the steroid dexamethasone or by simple acetylation (R 1 ,F igure 1C).…”

Glycosylated Cell‐Penetrating Peptides (GCPPs)

“…The results reported herein highlight the potential of the glycosylationo fp enetratingp eptides to modulatetheir activity. [17][18][19][20][21][22][23][24][25] Interesting penetrating properties were further discovered in different naturala nd artificial structures, such as polyprolines, [26] guanidinyl glycosides, [27,28] b-peptides, [29] peptiden ucleic acids (PNAs), [30] nonpeptidic guanidinylated dendritic structures, [31] self-assembled nanofibers, [32,33] synthetic polymers, [34][35][36][37] supramolecular structures, [38][39][40] and polydisulfides. [14] These studies also showed that oligolysines were less effective than that of the oligoarginines analogues, [15] and triggered the development of synthetic penetrating oligoarginines.…”

Where in the Cell Is our Cargo? Methods Currently Used To Study Intracellular Cytosolic Localisation

“…The development of the next generationo ft herapeutics, such as proteins, nucleic acids and antibodies, or their analogues, [1][2][3][4][5][6] has the potentialt or evolutionise chemical biology and medicine. However,t he transport of these large, hydrophilic and labile biomolecules constitutes ag reat challenge in comparison with traditional small-molecule therapies.…”