Synthesis and Characterization of Low Molecular Weight Linear Polyethylenimines for Gene Delivery

Abstract

The therapeutic application of commercially available polyethylenimines (e.g., PEI 25 kD) is marred by substantial toxicity. The benefit of using lower molecular weight polyethylenimines (<25 kD) has not been fully explored because of limited availability and high molecular heterogeneity (e.g., degree of branching) among the available few. We have synthesized a series of low molecular weight linear polyethylenimines (LPEI; 2.5-25 kD) with a single synthesis scheme to minimize molecular heterogeneity. DNA formulation with the newly synthesized linear polyethylenimines resulted in the formation of stable nanoparticles (100-150 nm) of positive zeta potential. Addition of these nanoparticles onto COS-1 and HEK 293 cell cultures led to transgene expression the efficiency and cytotoxicity of which varied with the LPEI size. The lowest molecular weight LPEI (LPEI 2.5 kD) gave the smallest level of gene expression and did not exert any cytotoxicity. The transfection activity exponentially increased with higher molecular weight LPEIs reaching maximal level with 7.5 kD LPEI and was accompanied with some cytotoxicity. The transfection activity of 7.5 kD LPEI was equal to that of the higher molecular weight LPEIs including 25 kD LPEI, but caused less cytotoxicity. To achieve high transfection efficiency without substantial increase in cytotoxicity, we cross-linked LPEI 3.6 kD with a biodegradable linkage to form a multi-block copolymer (BD3.6K) of approximately 8 kD. The multi-block copolymer, BD3.6K, expressed 20-fold higher transfection activity than that of the monomer block and produced significantly lower cytotoxicity than 25 kD PEI in vitro. Following intravenous administration, plasmid/BD3.6K complexes elicited significant gene transfer in lungs, while complexes prepared with monomer block did not yield discernable transfection activity. The transfection efficiency of the systemically administered plasmid/BD3.6K complexes was 2.5-times and 70-times higher than that of linear and branched 25 kD PEI, respectively. Transfection complexes prepared with BD3.6K exhibited better tolerability than complexes prepared with 25 kD PEIs. These results demonstrate that: (1) the lower molecular weight linear polyethylenimines (<10 kD) are more suitable for gene delivery than the commercially available higher molecular weight polyethylenimines (25 kD) and (2) the cross-linking of the non-toxic low molecular weight polyethylenimines via biodegradable linkage is a viable approach to improving PEI transfection efficiency without significantly increasing the cytotoxicity.

Synthetic tumor networks for screening drug delivery systems

“…Express-In is a commercially available polymer based transfection reagent that has been shown to produce very high transfection activity in a variety of cell types in vitro [39]. PPC is a polymeric delivery system that has been shown to efficiently deliver plasmid in vivo [40] and has been tested in clinically for the delivery of IL-12 plasmid in ovarian cancer patients with recurrent platinum resistant ovarian cancer [41–42].…”

Impact of Cationic Charge Density and PEGylated Poly(Amino Acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 2: DNA Protection, Stability, Cytotoxicity, and Transfection Efficiency

“…The cells were then supplemented with 10% FBS, incubated for 48 h, and gene transfection efficiency was examined through luciferase protein expression as a function of N:P ratios. COS‐1 cells were used due to their ability to grow rapidly with an approximate population‐doubling time of 18 h. COS cells have been used extensively in pDNA transfection using a number of different vectors, including PEI, DEAE, and lipid transfection …”

Evaluation of a Cationic Poly(β-hydroxyalkanoate) as a Plasmid DNA Delivery System

“…For a polymer to serve as an effective plasmid DNA delivery agent, it must interact with the nucleic acid. Among nonviral delivery systems, this is most often accomplished through electrostatic interactions. − PHON was synthesized to contain accessible protonable tertiary amines, which, at physiological pH, would provide a polycationic nature to the polymer (Figure ). Polymers of this type have been shown to electrostatically bind and condense polyanionic nucleic acids such as plasmid DNA. − The majority of polyamines such as poly( l -lysine) investigated as DNA-complexing agents or transfection vectors contain amines at the termini of short, conformationally flexible side chains. , Other polyesters containing amines such as poly(β-amino esters) and poly(4-hydroxy- l -proline esters) possess cationic amines within the polymer main chain. , …”

“…Over the past two decades, the field of nonviral gene delivery has developed as a means to overcome these obstacles through the utilization of materials with a wide array of useful properties. Nonviral delivery systems have been composed of cationic lipids, − polypeptides, − polyethyleneimine, − polysaccharides, − and polyesters. − We have previously reported the synthesis of PHON, which is a modified member of the poly(β-hydroxyalkanoate) (PHA) family of polymers . These materials are produced by a wide range of microorganisms as a form of energy storage. , PHON was designed to possess several properties seen in a variety of polymers used in applications requiring interaction with nucleic acids.…”

“…Over the past two decades, the field of nonviral gene delivery has developed as a means to overcome these obstacles through the utilization of materials with a wide array of useful properties. Nonviral delivery systems have been composed of cationic lipids, [4][5][6] polypeptides, [7][8][9] polyethyleneimine, [10][11][12] polysaccharides, [13][14][15] and polyesters. [16][17][18] We have previously reported the synthesis of PHON, which is a modified member of the poly(β-hydroxyalkanoate) (PHA) family of polymers.…”