Synthesis and characterization of polyaminoacidic polycations for gene delivery

doi:10.1016/j.biomaterials.2005.09.027

Biomaterials

Volume 27, Issue 9, March 2006, Pages 2066-2075

https://doi.org/10.1016/j.biomaterials.2005.09.027 Get rights and content

Abstract

The properties as non viral gene vector of a protein-like polymer, the α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) were exploited after its derivatization with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) as molecule bearing a cationic group, in order to obtain stable polycations able to condense DNA. PHEA was firstly functionalized with aminic pendant groups by reaction with ethylenediamine (EDA) obtaining the α,β-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)-d,l-aspartamide (PHEA-EDA) copolymer. We demonstrated that polymer functionalization degree is easily modulable by varying reaction conditions, so allowing to produce two PHEA-EDA derivatives at different molar percentage of amine groups. Subsequently, the condensation reaction of PHEA-EDA copolymers with CPTA yielded α,β-poly(N-2-hydroxyethyl)(2-[3-(trimethylammonium chloride)propylamide]-amidoethylcarbamate)-d,l-aspartamide (PHEA-EDA-CPTA) polycation derivatives.

In vitro studies were carried out to evaluate polycations ability to complex DNA and to protect it from nuclease degradation. Obtained results demonstrated the good ability of our new PHEA polycationic derivatives, PHEA-EDA-CPTA, to complex and condense genomic material, neutralizing its anionic charge even at very low polycation/DNA weight ratio.

Finally, PHEA-EDA-CPTA polycations were characterized by in vitro cytotoxicity studies to evaluate their effects on the viability of HuH-6 human hepatocellular carcinoma cells by MTS assay. No cytotoxicity was evidenced by both polycationic derivatives after 48 h of incubation at all tested concentrations.

Introduction

Gene therapy allows the selective introduction of genetic material into specific cells of a patient, thus curing a wide range of diseases by delivering a missing gene or a functional substitute of a defective gene [1].

Due to its degradation by serum nuclease, naked DNA has a low serum half-life and moreover, since it possesses high molecular weight and polyanionic nature, it is not able to cross cell membrane [2]; for this reason the in vivo transport of genetic materials needs a suitable delivery system [3].

A variety of gene transfer systems are currently employed to insert therapeutics gene into somatic cells; they are divided into viral vectors (or biological systems) and non-viral vectors.

It is well know that viral vectors, despite to their high transfection efficiency, have a lot of safety problems, related to the risk of an immune or inflamed response; moreover further improvements of viral gene vectors achieved a difficult and expensive production in a broad scale [4], [5].

For all these motives non-viral gene delivery vectors such us water-soluble polycations are preferable [6], [7]. Due to their versatility, synthetic polycations offer the possibility to modify their characteristics according to specific functional and biological needs; in fact, they should be safe, easy to produce in large quantities with high reproductibility and acceptable cost, and chemically derivatizable in order to introduce in their structure an active targeting group [8], [9]. Besides, they are able to form complexes with DNA based on electrostatic interactions [10], [11], thus allowing its entrance into cells, whose membrane presents a negative charge.

Moreover, polycations should be non-toxic [3], able to interact reversibly with DNA forming stable complexes and protecting DNA against degradation during biodistribution process and small enough to extravasate in order to deliver efficiently gene material inside the cells [1].

Since few years, our research group is oriented towards the synthesis of new DNA delivery systems, such as non-toxic water-soluble polycations.

In this context, the aim of this work was to synthetize a non-toxic non-viral gene vector starting from a protein-like polymer, the α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) [12].

PHEA is a highly water-soluble synthetic polymer possessing many favourable properties that allowed its proposition for biomedical applications. In fact, it is biocompatible, not cytotoxic and haemolytic, not antigenic and not immunogenic [12], [13]. The large number of hydroxylic groups in PHEA backbone allow to link, via chemical bonds, many molecules of different molecular weight. Many studies showed the possibility of using PHEA as a drug carrier in the synthesis of macromolecular prodrugs [14], [15].

The PHEA–EDA–CPTA polycation synthesis have been realized, from starting PHEA, in two steps: firstly, PHEA reacted with bis(4-nitrophenyl)carbonate (4-NPBC) and ethylenediamine (EDA) to functionalize the polymer with pendant amine groups; secondly, PHEA–EDA copolymers, reacted with 3-(Carboxypropyl)trimethyl-ammonium chloride (CPTA), yielding the complete derivatization of pendant amine groups, thus obtaining two new polycations of PHEA at different molar percentage of positive charge. Characterization and in vitro cytotoxicity studies were carried out to evaluate the potential of these polycations to act as gene delivery carrier.

Section snippets

Materials and methods

PHEA was prepared and purified according to the previously reported procedure [12]. Spectroscopic data (FT-IR and ¹H-NMR) were in agreement with previous results [13]: ¹H-NMR[D₂O]: δ 2.83 (m, 2 H, –CH–CH₂–CO–NH–), 3.37 (t, 2 H, –NH–CH₂–CH₂–OH), 3.66 (t, 2 H,–CH₂–CH₂–OH), 4.73 br (m, 1 H, –NH–CH–CO–CH₂–).

PHEA average molecular weight was 41.1 KDa ( $M_{w} / M_{n} = 1.68$ ) based on PEO/PEG standards, measured by size exclusion chromatography (SEC).

3-CPTA and hydroxybenzotriazole (HOBT) were purchased from Aldrich

Synthesis of PHEA–EDA–CPTA polycations

Two polycationic copolymers of PHEA containing different amount of a quaternary amine were synthetized in very high yields (95%). The activation reaction of hydroxyl groups of PHEA with 4-NPBC followed by the reaction with EDA, allowed the derivatization of polymer chains with pendant primary amine groups, (obtaining the PHEA–EDA copolymers; as reported in Scheme 1) that easily react with a low molecular weight carboxylic acid carrying a positive charge moiety (like

Conclusions

The use of bis(4-nitrophenyl)carbonate as activating agent of hydroxylic groups of PHEA showed to be a very efficient method to easily functionalize polymeric backbone with ethylenediamine. Two copolymers (PHEA–EDA) at different functionalization degrees (15 and 37 mol%) have been obtained just varying the activation times. Consecutively, two new polycationic derivatives have been synthesized by coupling the primary amine groups of PHEA–EDA with carboxypropyltrimethylammonium chloride (CPTA), in

Acknowledgements

Authors thank MIUR for funding.

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Synthesis and characterization of polyaminoacidic polycations for gene delivery

Abstract

Introduction

Section snippets

Materials and methods

Synthesis of PHEA–EDA–CPTA polycations

Conclusions

Acknowledgements

J Control Release

Adv Drug Deliv Rev

Biotechnol Adv

J Control Release

J Control Release

Polymer

Eur J Pharm Biopharm

Carbohydr Polym

Int J Pharm

J Control Release

Eur J Cancer

Biomaterials