Elsevier

Acta Biomaterialia

Volume 10, Issue 2, February 2014, Pages 670-679
Acta Biomaterialia

Contribution of hydrophobic/hydrophilic modification on cationic chains of poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) amphiphilic co-polymer in gene delivery

https://doi.org/10.1016/j.actbio.2013.09.035Get rights and content

Abstract

Nanoparticles (NPs) assembled from amphiphilic polycations have been certified as potential carriers for gene delivery. Structural modification of polycation moieties may be an efficient route to further enhance gene delivery efficiency. In this study two electroneutral monomers with different hydrophobicities, 2-hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA), were incorporated into the cationic poly(dimethylamino ethyl methacrylate) (PDMAEMA) side-chains of amphiphilic poly(ε-caprolactone)-graft-poly(dimethylamino ethylmethacrylate) (PCD) by random co-polymerization, to obtain poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl methacrylate) (PCD-HEMA) and poly(ε-caprolactone)-graft-poly(dimethylamino ethyl methacrylate-co-2-hydroxyethyl acrylate) (PCD-HEA). Minimal HEA or HEMA moieties in PDMAEMA do not lead to statistically significant changes in particle size, zeta potential, DNA condensation properties and buffering capacity of the naked NPs. However, the incorporation of HEMA and HEA lead to reductions and increases, respectively, in the surface hydrophilicity of the naked NPs and NPs/DNA complexes, which was confirmed by water contact angle assay. These simple modifications of PDMAEMA with HEA and HEMA moieties significantly affect the gene transfection efficiency on HeLa cells in vitro: PCD-HEMA NP/DNA complexes show a much higher transfection efficiency than PCD NPs/DNA complexes, while PCD-HEA NPs/DNA complexes show a lower transfection efficiency than PCD NP/DNA complexes. Fluorescence activated cell sorter and confocal laser scanning microscope results indicate that the incorporation of hydrophobic HEMA moieties facilitates an enhancement in both cellular uptake and endosomal/lysosomal escape, leading to a higher transfection efficiency. Moreover, the process of endosomal/lysosomal escape confirmed in our research that PCD and its derivatives do not just rely on the proton sponge mechanism, but also on membrane damage due to the polycation chains, especially hydrophobic modified ones. Hence, it is proved that hydrophobic modification of cationic side-chains is a crucial route to improve gene transfection mediated by polycation NPs.

Graphical abstract

2-Hydroxyethyl methacrylate (HEMA) and 2-hydroxyethyl acrylate (HEA) were respectively used to modify a kind of amphiphilic cationic nanoparticles to change their external hydrophilicity. The hydrophilic/hydrophobic modifications significantly influenced the gene transfection efficiency of these nanoparticles in vitro, due to different cellular uptake and endosomal escape efficacy.

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Introduction

There are high expectations that gene therapy can cure some diseases caused by genetic disorders. Due to the poor stability of naked nucleic acids in the bloodstream, vehicles for efficient gene delivery are required [1], [2]. Polycations, a major class of non-viral gene carriers, have been reported to have good potential for gene delivery, because of their efficiency in condensing negatively charged nucleic acids into stable nanosized complexes via strong electrostatic interaction, and low immunogenicity [3]. However, the lower gene delivery efficiency of polycations is a crucial obstacle to their application as gene carriers.

In order to improve the gene delivery efficiency different strategies for the structural modification of polycations have been studied to promote cellular uptake [4], [5], [6], escape from the endosomal compartment [7], [8] and release of the payload at target intracellular locations [9], [10], such as targeted modification [11], [12], [13], [14], blocking or grafting polycations onto hydrophobic polymers [15], [16], [17], stimuli-triggered conformation transformation [18], using lipid-enveloped hybrid nanoparticles (NPs) [19], [20], etc. Recently the configuration of cationic moieties on the surface of polymer NPs were shown to be a major influencing factor. It was reported that NPs with cationic groups such as guanidinium were internalized by cells via transcytosis, while NPs with imidazole and tertiary amine groups were internalized via the endocytosis [21]. Similarly, research on linear poly(β-amino ester)s showed that amine-containing end-capped polymers were much more efficient than the corresponding acrylate-terminated forms, in terms of both uptake and transfection, even though there were minimal differences between acrylate- and amine-terminated polymers in terms of DNA retardation in gel electrophoresis, NP size, NP zeta potential, polymer buffering capacity and cytotoxicity [22]. An odd–even law was recently revealed in research on N-substituted polyaspartamides (PAsp), in that PAsp with even numbered repeating aminoethylene units in their side-chains showed appreciably high buffering capacity and facilitated endosomal escape without severe cytotoxicity [23]. Thus it can be seen that minimal differences in cationic polymer structure could affect gene delivery remarkably. However, configuration adjustments on polycation moieties have until now only focused on cationic groups, although the relationship between the structure of cationic moieties and gene transfection is far from understood.

Among the polycations used as gene carriers, poly(dimethylamino ethyl methacrylate) (PDMAEMA) has been widely studied because of its outstanding ability to condense DNA stably in aqueous solution [24] and its unique mechanism of escape from lysosomes [25], [26], [27]. PDMAEMA can be readily structurally modified by controlled radical polymerization [28], [29], [30]. Our previous work confirmed that NPs assembled from polycaprolactone (PCL) grafted PDMAEMA (PCL-g-PDMAEMA) could further enhance DNA and siRNA delivery efficiency compared with PDMAEMA homopolymer and branched polyethylenimine (PEI) (Mw 25 kDa) [31], [32], [33]. Therefore, the present study aimed to optimize the structure to further improve the functions of PCL-g-PDMAEMA as a gene carrier and to study the structural contribution of PDMAEMA cationic side-chains to gene delivery efficiency. 2-Hydroxyethyl acrylate (HEA) and 2-hydroxyethyl methacrylate (HEMA), two monomers extensively used in constructing biomaterials for tissue engineering and drug delivery [34], [35], were randomly introduced into the PDMAEMA side-chains of PCL-graft-PDMAEMA by co-polymerization of DMAEMA with HEA or HEMA. It was expected that the random HEMA or HEA units would disturb the conformation of the cationic PDMAEMA chains and affect gene delivery efficiency.

Section snippets

Materials

γ-(2-Bromo-2-methylpropionate)-ε-caprolactone (BMPCL) was synthesized by methods reported previously [36], [37], [38]. Phenylcarbinol and ε-caprolactone (Sigma–Aldrich) were dried over calcium hydride for 24 h at room temperature and distilled under reduced pressure. Copper (I) bromide, stannous octanoate, branched PEI (Mw 25 kDa), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA), 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and N,N-dimethylaminoethyl methacrylate were purchased from

Synthesis and characterization of amphiphilic polycations

In order to study the contributions of different moieties in PDMAEMA chains to gene delivery PCD, PCD-HEMA, and PCD-HEA were synthesized by combining ring-opening polymerization and atom transfer radical polymerization (as shown in Scheme 1). All the amphiphilic polycations used in this paper were synthesized from the same macroinitiator with 70 U of ε-caprolactone and 4 U of ATRP initiating monomer, as listed in Table 1. Polymer structures were confirmed by 1H NMR and gel permeation

Conclusions

To reveal how the structure of cationic chains affects gene delivery two electroneutral monomers based on the amphiphilic cationic polymer PCD with different hydrophobicities, HEMA and HEA, synthesized by combining ring-opening polymerization and atom transfer radical polymerization, were incorporated into the PDMAEMA side-chains of PCD by random co-polymerization, to obtain PCD-HEMA and PCD-HEA. The gene delivery characteristics and functions of PCD, PCD-HEMA and PCD-HEA self-assembled NPs

Acknowledgements

This project was supported by a grant from the National High Technology Research and Development Program of China (863) (2012AA022501) and the National Natural Science Foundation of China (31271073 and 31101000).

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