The Power of Shielding: Low Toxicity and High Transfection Performance of Cationic Graft Copolymers Containing Poly(2-oxazoline) Side Chains

Abstract

We show the potential of oligo(2-ethyl-2-oxazoline) (Ox)-shielded graft copolymers of (2-aminoethyl)-methacrylate and N-methyl-(2-aminoethyl)-methacrylate for pDNA delivery in HEK cells. For the effect of grafting density and side chain length concerning improved transfection properties through the concept of shielding to be investigated, copolymers were synthesized via the macromonomer method using a combination of cationic ring opening polymerization and reversible addition-fragmentation chain transfer polymerization to vary the degree of grafting (DG = 10 and 30%) as well as the side chain degree of polymerization (DP = 5 and 20). Investigations of the polyplex formation, in vitro flow cytometry, and confocal laser scanning microscopy measurements on the copolymer library revealed classical shielding properties of the Ox side chains, including highly reduced cytotoxicity and a partial decrease in transfection efficiency, as also reported for polyethylene glycol shielding. In terms of the transfection efficiency, the best performing copolymers (A- g-Ox(10) and M- g-Ox(10)) revealed equal or better performances compared to those of the corresponding homopolymers. In particular, the graft copolymers with low DG and side chain DP transfected well with over 10-fold higher IC values. In contrast, a DG of 30% resulted in a loss of transfection efficiency due to missing ability for endosomal release, and a side chain DP of 20 hampered the cellular uptake.

Nanocarrier‐Mediated Cytosolic Delivery of Biopharmaceuticals

“…The reported methods to reduce the toxicity of the polycationic materials include PEGylation, [ 67 ] reduction of molecular weight, [ 63c ] and controlling ring‐opening polymerization of lactones with tertiary amino‐alcohols. [ 63b,c ] Furthermore, conjugation of cholesterol, [ 68 ] cyclodextrin, [ 69 ] oligo(2‐ethyl‐2‐oxazoline), [ 70 ] fluorobenzene, [ 71 ] and alkyl chains [ 72 ] have also proven to reduce toxicity. Another method known as charge reversal maintains the negative charge of the vectors in circulation to avoid clearance and causes the vectors to become positively charged upon entrapment in the endosomes.…”

Tuning of endosomal escape and gene expression by functional groups, molecular weight and transfection medium: a structure–activity relationship study

“…A reduced uptake of pDNA in the presence of growth medium was also observed by other groups. 87,88 In the presence of serum, the cationic charged polyplexes tend to interact with negatively charged proteins, leading to aggregation and therefore reduced uptake. 6,89,90 It could also be assumed that the interaction with extracellular matrix components such as heparan sulfate proteoglycans is less pronounced due to competition with serum proteins, so that less pDNA can be uptaken.…”

Bottlebrush copolymers for gene delivery: influence of architecture, charge density, and backbone length on transfection efficiency

“…34,35 Several POx bottlebrush-based transfection materials have been reported, however, in all cases POx was used for its biocompatible properties to reduce toxicity and shield the cationic moieties, rather than to introduce charge into the polymer. [36][37][38] POx has been grafted onto a linear cationic polymer backbone to shield charge in several instances or a macromonomer has been prepared and copolymerized with cationic monomers.…”