Cell Penetrating Polymers Containing Guanidinium Trigger Apoptosis in Human Hepatocellular Carcinoma Cells unless Conjugated to a Targeting N-Acetyl-Galactosamine Block

Abstract

A series of 3-guanidinopropyl methacrylamide (GPMA)-based polymeric gene delivery vehicles were developed via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymers have been evaluated for their cellular internalization ability, transfection efficiency, and cytotoxicity. Two homopolymers: P(GPMA), P(GPMA), were synthesized to study the effect of guanidium polymer length on delivery efficiency and toxicity. In addition, an N-acetyl-d-galactosamine (GalNAc)-based hydrophilic block was incorporated to produce diblock polymers, which provides a neutral hydrophilic block that sterically protects plasmid-polymer complexes (polyplexes) from colloidal aggregation and aids polyplex targeting to hepatocytes via binding to asialoglycoprotein receptors (ASGPRs). Polyplexes formed with P(GPMA) (x = 20, 34) homopolymers were shown to be internalized via both energy-dependent and independent pathways, whereas polyplexes formed with block polymers were internalized through endocytosis. Notably, P(GPMA) polyplexes enter cells very efficiently but are also very toxic to human hepatocellular carcinoma (HepG2) cells and triggered cell apoptosis. In comparison, the presence of a carbohydrate block in the polymer structures reduced the cytotoxicity of the polyplex formulations and increased gene delivery efficiency with HepG2 cells. Transfection efficiency and toxicity studies were also carried out with HEK 293T (human embryonic kidney) cells for comparison. Results showed that polyplexes formed with the P(GPMA) homopolymers exhibit much higher transfection efficiency and lower toxicity with HEK 293T cells. The presence of the carbohydrate block did not further increase transfection efficiency in comparison to the homopolymers with HEK 293T cells, likely due to the lack of ASGPRs on the HEK 293T cell line. This study revealed that although guanidinium-based polymers have high membrane permeability, their application as plasmid delivery vehicles may be limited by their high cytotoxicity to certain cell types. Thus, the use of cell penetrating structures in polyplex formulations should be used with caution and carefully tailored toward individual cell/tissue types.

Guanidinium-Functionalized Interpolyelectrolyte Complexes Enabling RNAi in Resistant Insect Pests

“…25 It has been demonstrated that pGPMA has a modest capacity to deliver plasmid DNAs to nuclei, though toxicity was observe in one cell line. 36 However, studies in other cell culture systems have demonstrated negligible toxicity for similar polymers, 37−40 suggesting that any toxicity may be confined to certain cell types or configuration of IPECs. Use of pGPMA-dsRNA IPECs also addresses the alkalinity and high RNase activity in insect guts: lumen pH ranges from 10 to 11, 41 where commonly used tertiary amine-containing nucleic acid carriers become deprotonated, leading to IPEC dissociation.…”

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

“…34,36,85 Regarding the guanidinium functional polymers, previous studies of other research groups showed contradictory results of temperatureindependent and temperature-dependent uptake, respectively. 38,70 This inconsistency is also known for guanidinium-containing peptides, 86 indicating that there are other factors additional to the type of functional group determining the way of internalization and should therefore be considered for novel polymers.…”

“…Relatively low molar mass guanidiniumbearing poly(methacrylamides) (DP of 20) offered transfection efficiency of about 50% of that of jetPEI in HEK293T cells in serum free medium and 48 h post transfection. 70 On the other hand, a guanidinium-bearing polymethacrylate with an approximately twofold higher number average molar mass (25 kg mol À1 ) compared to PGPAm 89 and a 42.4 kDa poly-arginine exhibited lower transfection efficiency than PDMAEMA in COS-7 cells and serum-free medium. 38 In another study a similar poly-arginine showed transfection efficiency comparable to lipofectamine in mixed cortical cells.…”

Synthetic materials at the forefront of gene delivery

“…Glycan, protein and antibody conjugates. N-acetyl-D-galactosamine pendants have been incorporated in guanidinium polymers to enhance targeting and reduce toxicity in the transfection of HepG2 cells 159 . Similar behaviour has been observed in poly(glycoamidoamines) and PEI derived polymers that incorporate carbohydrate (D -glucaric acid) co-monomers 160 .…”