Self-assembled carboxymethyl poly (L-histidine) coated PbAE/DNA complexes as non-viral gene delivery system

Jijin Gu, Xiao Wang, Hongliang Xin, Yanzuo Chen , Xinyi Jiang, Xianyi Sha*, Xiaoling Fang*




Compared to viral vectors, non-viral gene vectors are easier to synthesize, perform stably, and have low cytotoxicity and low immunogenicity, etc. Biomaterials coated polymer/DNA complexes are developed as a novel and efficient non-viral gene delivery system. Our interest has been focused on polyanion/polycation/plasmid ternary complexes system, which is able to avoid changing variety of biophysical properties of the biomaterials and the corresponding polymer/DNA complexes with covalent linkage, based on physical electrostatic coating. As a promising approach, electrostatic coatings biomaterials such as poly (γ-glutamic acid) (γ-PGA), hyaluronic acid (HA) and oligonucleotides are involved to modify polycation/plasmid binary complexes for facilitating gene delivery in vitro and in vivo recently. A common characteristic among these polymers is high charge density at physiological pH, which can interact with the cationic polymers causing the reduction of positively charge of the complexes.
In the present study, we introduced pH-sensitive carboxymethyl poly (L-histidine) (CM-PLH) and Poly (β-amino ester) (PbAE) as functional biomaterials to form a CM-PLH/PbAE/DNA core–shell ternary complexes system (TCS) in light of electrostatic adsorbed coatings for efficient gene delivery and transfection. PbAE, a promising pH-sensitive and biodegradable polycation for gene delivery, which is easy to be prepared via the Michael addition of primary amine or secondary amine to diacrylate, can condense and protect DNA from degradation, enabling efficient cellular uptake and subsequent intracellular endosomal escape. Using two monomers, namely, 5-amino-1-pentanol and 1, 4-butanediol diacrylate, amine-terminated PbAE was synthesized by Michael addition, which was characterized by 1H-NMR. (Fig.1). Capped functional groups greatly affected the transfection efficiencies (Fig.2). The chemical structures of poly (β-amino-ester) s, including main-chain structure and side-chain structure, contained secondary, tertiary amines and ester bonds and there was no primary amine in the main chain. PbAE/DNA binary complexes were prepared by mixing PbAE with plasmid DNA, self-assembling in 25 mM sodium acetate buffer solution at pH 5.2 (NaAc buffer). Plasmid DNA protection upon polyplex formation was evaluated by agarose gel retardation electrophoresis assay. MTT colorimetric assay was used to investigate the cell viability of the PbAE/DNA complexes after 4 h in HEK293 and B16-F10. The transfection efficiency assay of the complexs was compared with DNA–Lipoplex in vitro. PbAE could efficiently condense negatively charged DNA at low mass ratio (Fig.4 A). Meanwhile, PbAE had shown lower positive charge, reducing the cytotoxicity in many cell lines (Fig.5) and still maintained high transfection efficiency (Fig.6A).
Poly (L-histidine) (PLH) is a peptide with rich imidazole groups. Because of its poor water solubility at physiological pH conditions, some polar carboxymethyl side chains are introduced to modify PLH, yielding pH-sensitive carboxymethyl poly (L-histidine) (CM-PLH) (Fig.3). On one hand, it is able to avoid chemical reactions changing various biophysical properties of the carrier materials and the corresponding polymer/DNA complexes with covalent linkage to affect the carrier and DNA binding, reducing the shortcomings of the transfection efficiency. On the other hand, CM-PLH negative charge shielding of the positive charge on the PbAE/DNA complexes surface was employed to restrain the undesirable interactions, maintain vector stability in serum and enhanced the systemic duration. In addition, CM-PLH contains a large number of pH-sensitive imidazole groups, which can protect the plasmid from lysosomal degradation through "proton sponge effect" and improve the transfection efficiency without cytotoxicity and agglutination of erythrocytes. The preparation of the TCS was performed, self-assembling in 25 mM sodium acetate buffer solution at pH 5.2. The TCS kept stable nano-size, good condensation capacity (Fig.4B) and low toxicity (Fig.5), even provided a higher transgene expression over the binary complexes (PbAE/DNA without CM-PLH) (Fig.6B) and transfected up to 90% in HEK293 and 60% in B16-F10 in vitro (Fig.7). The ternary complexes significantly enhanced their cellular uptake and endosomal escape which were proved by the result that TCS could evade the endosomal lumen and localize in the nucleus of treated cells visualized under Fluorescence Confocal Microscopy (FCM). The aforementioned results indicated that CM-PLH with pH-sensitive imidazole groups played an important role in enhancing the endosomal escape and transfection efficiency of the TCS (Fig.8). Consequently, TCS exhibited significant improvements in transfection efficiencies with comparison to non-modified PbAE/DNA, highlighting their functional usefulness. Our approach and the gene delivery system fabrication could potentially be useful for effective gene delivery and therapies to targeted cells.
In summary, the key issue to efficient gene delivery remained cellular internalization and endosomal escape of the pDNA, particularly in vivo. As expected, the addition of CM-PLH markedly decreased the toxicity of PbAE/DNA complexes with high tansfection efficiency in HEK293 and B16-F10 compared to the commercial transfection reagent Lipofectamine2000. And the ternary complexes presented higher gene transfection efficiency in vitro and in vivo than the binary complexes. Because of weaker positive charge on the surface, the internalization efficiency of YOYO-1-labeled ternary complexes in the B16-F10 cells was lower than that of the binary complexes as determined in vitro. But the efficiency of ternary complexes delivered plasmid DNA was time-dependent endocytosis. Confocal microscope results showed that complexes were trapped in the lysosomal compartment, while ternary complexes have stronger escape ability from endosomes compared with the binary complexes.

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