Abstract
Cationic polymers have been widely studied for non-viral gene delivery due to their ability to bind genetic material and to interact with cellular membranes. However, their charged nature carries the risk of increased cytotoxicity and interaction with serum proteins, limiting their potential in vivo application. Therefore, hydrophilic or anionic shielding polymers are applied to counteract these effects. Herein, a series of micelle-forming and micelle-shielding polymers were synthesized via RAFT polymerization. The copolymer poly[(n-butyl acrylate)-b-(2-(dimethyl amino)ethyl acrylamide)] (P(nBA-b-DMAEAm)) was assembled into cationic micelles and different shielding polymers were applied, i.e., poly(acrylic acid) (PAA), poly(4-acryloyl morpholine) (PNAM) or P(NAM-b-AA) block copolymer. These systems were compared to a triblock terpolymer micelle comprising PAA as the middle block. The assemblies were investigated regarding their morphology, interaction with pDNA, cytotoxicity, transfection efficiency, polyplex uptake and endosomal escape. The naked cationic micelle exhibited superior transfection efficiency, but increased cytotoxicity. The addition of shielding polymers led to reduced toxicity. In particular, the triblock terpolymer micelle convinced with high cell viability and no significant loss in efficiency. The highest shielding effect was achieved by layering micelles with P(NAM-b-AA) supporting the colloidal stability at neutral zeta potential and completely restoring cell viability while maintaining moderate transfection efficiencies. The high potential of this micelle-layer-combination for gene delivery was illustrated for the first time.
Highlights
In the last decades, the development of non-viral nanocarriers for the delivery of genetic material has seen great progress [1, 2], not least because of the urgent need for effective vaccines [3,4,5]
Considering nanomedicine in general, functional amphiphilic block copolymers have gained increasing attention due to their ability to self-assemble into higher ordered structures such as micelles or vesicles in aqueous solutions [14,15,16,17,18]. Different compositions of these polymeric micelles have been investigated for gene delivery, utilizing a cationic polymer block within their shell to form complexes with genetic material [19, 20], whereas another approach uses cationic-hydrophilic block copolymers to incorporate the genetic material inside a polyion complex (PIC) with a neutral shell [14, 21]
Polymer synthesis and characterization The diblock and triblock copolymers were synthesized via sequential reversible-addition fragmentation chain transfer (RAFT) polymerizations with purification after each block synthesis step (Fig. 1A). (Propanoic acid) yl butyl trithiocarbonate (PABTC) was used as the chain transfer agent (CTA), since it is suitable to control the polymerization of acrylates and acrylamides [57]
Summary
The development of non-viral nanocarriers for the (targeted) delivery of genetic material has seen great progress [1, 2], not least because of the urgent need for effective vaccines [3,4,5]. Considering nanomedicine in general, functional amphiphilic block copolymers have gained increasing attention due to their ability to self-assemble into higher ordered structures such as micelles or vesicles in aqueous solutions [14,15,16,17,18] Different compositions of these polymeric micelles have been investigated for gene delivery, utilizing a cationic polymer block within their shell to form complexes with genetic material (polyplexes) [19, 20], whereas another approach uses cationic-hydrophilic block copolymers to incorporate the genetic material inside a polyion complex (PIC) with a neutral shell [14, 21]. Compared to polyplexes of cationic homopolymers, micelles have been demonstrated to increase the stability of polyplexes and the delivery efficiency [22,23,24,25], which is, among others, related to an increased interaction of the hydrophobic block with cellular membranes [26]
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