DC3-Decorated Polyplexes for Targeted Gene Delivery into Dendritic Cells

Abstract

Dendritic cells (DCs) are a family of specialized antigen presenting cells (APCs) that detect antigens and initiate a wide spectrum of immune responses against them. These characteristics make them promising candidates for immunotherapy manipulations. In this study we designed and synthesized DC-targeted block copolymers composed of linear polyethylenimine (PEI) conjugated to hydrophilic polyethylene glycol (PEG) installed with a DC-targeting peptide (DC3, primary sequence FYPSYHSTPQRP). Two different conjugation procedures (basic and modified) were employed to synthesize the DC3-PEG-b-PEI and the control SCRM-PEG-b-PEI (with a scrambled DC3 peptide sequence) by one-pot synthesis, in two steps. In the first, basic conjugation procedure, PEG with N-hydroxysuccinimide (NHS) ester and maleimide (MAL) groups (NHS-PEG-MAL, 3.5 kDa) was first coupled to linear PEI (25 kDa) via the NHS group to yield the intermediate MAL-PEG-b-PEI, that was then conjugated to N-terminus-cysteine harboring peptides DC3 or SCRM via the MAL double bond to yield the final DC3-PEG-b-PEI or SCRM-PEG-b-PEI copolymers, respectively. In the second, modified conjugation procedure, Fmoc-cysteine harboring DC3 or SCRM peptides were first conjugated to NHS-PEG-MAL via the MAL group followed by coupling to linear PEI via the NHS functional group. Fmoc cleavage yielded the same final product as in the basic procedure. The modified conjugation procedure was capable of yielding block copolymers richer with peptides, as determined by (1)H NMR analysis. Self-assembly of DC3-PEG-b-PEI copolymers and DNA molecules yielded nanosized polyion complexes (polyplexes), with lower surface charge and limited cytotoxicity when compared to the PEI building block. Significant transfection efficiency of the DC-targeted polyplexes by murine dendritic DC2.4 cells was observed only in DC3-PEG-b-PEI/DNA polyplexes synthesized by the modified conjugation procedure. These polyplexes, with higher peptide-load, showed greater transfection capability in DC2.4 cells relative to the control nontargeted SCRM-PEG-b-PEI/DNA polyplexes, but not in endothelial cells. The transfection efficiency was comparable to or higher than that of the PEI/DNA positive control. The results indicate that PEGylated-PEI polyplexes show significant transfection efficiency into DCs when decorated with DC3 peptide, and are attractive candidates for immunotherapy via DCs.

Formation of functional super-helical assemblies by constrained single heptad repeat

“…10 , the condensed DNA could be detected after a prolong incubation period whereas degradation of bare DNA was completed within a few minutes. The efficient condensation and superior protection of the DNA by SHR-FLLF peptide comprised of hydrophobic and aromatic blocks with a single lysine residue may help to delineate the role of positive charges and other non-covalent interactions towards DNA condensation and eventually may lead to the design of efficient peptide-based transfection agents having less positive charge density, an essential requirement for transfecting agents with lower cytotoxicity 49 .…”

Multifunctional Immunoadjuvants for Use in Minimalist Nucleic Acid Vaccines

“…A block copolymer based on linear PEI conjugated to PEG, installed with a DC-targeting peptide (sequence: FYPSYHSTPQRP), was used to prepare pDNA polyplexes. Uptake was significantly enhanced in the DC2.4 cell line, resulting in a higher efficiency of gene transfer [ 203 ].…”

Polymers for DNA Vaccine Delivery

“…39 A similar strategy was employed by the same group in PEI modification with a DC targeting peptide (DC3). 159 Another study incorporated mannose and deoxycholic acid dual modified PEI into polyelectrolyte multilayer assembly films for cutaneous delivery of DNA vaccines. 160 Finally, modification of low MW PEI (600 Da) with cationic cell-penetrating Tat peptide was also effective in intracellular delivery of DNA vaccine and eliciting humoral and cellular immune responses after s.c. injection.…”