High-Molecular-Weight Polyethyleneimine Conjuncted Pluronic for Gene Transfer Agents

Abstract

Development of non-viral vectors for treatment of diseases such as genetic disorders, inflammation or cancer remains an important goal for gene therapy. However, gene therapy is experimental and has not been proven to be very successful in clinical trials. The primary hurdle to the clinical success of gene therapy has been the absence of carriers (vectors) that can deliver DNA and RNA into target cells safely and efficiently. Basically, there are two kinds of gene delivery vectors, viral and nonviral. Viral vectors are very efficient in delivering plasmids into mammalian cells, however, their usefulness is limited due to immunogenicity, low loading capacity and insertional mutagenesis. 1) Nonviral gene delivery systems based on cationic liposomes and polymers have recently received increasing attention, although the gene transfection efficiency is still lower than viral vector systems, there are some unique advantages for practical use such as easy manipulation, stability, low cost, safety and their induction of a less immune response. Polycations have emerged as efficient transfection vectors and are able to condense DNA into nanocomplexes suitable for endocytic uptake. Among the polycationic polymers, polyethylenimine (PEI), has shown high transfection efficiency both in vitro and in vivo, 1,2) probably due to its stable complex structure which is appropriate for being taken by cells; the following "proton sponge" effect from PEI facilitates the escape of the complex from an endosomal compartment. The gene delivery efficiency and cytotoxicity of PEI vary according to its molecular weight. The high density of primary, secondary and tertiary amino groups of high molecular weight PEI (HMW PEI), may partially account for the high transfection efficiency of PEI-based complexes. On the other hand, the huge amount of cationic charge frequently results in a rather high toxicity, 3,4) which can be regarded as one of the major factors limiting its use in vivo. Another problem of using polycation DNA complexes is their poor solubility. 5) They may immediately precipitate out of a solution when being prepared at a higher concentration. Many in vivo gene administrations are limited by the volume of a solution. To improve its biocompatibility, PEI has been modified by human serum albumin, 6) dextran, 7) or hydrophilic copolymers such as poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA) 8,9) and poly(ethylene glycol) (PEG).10-12) Such hydrophilic and biocompatible polymers can improve the solubility of the complexes, minimize the aggregation of particulates and reduce their interaction with proteins in the physiological fluid. The less cytotoxic PEI derivatives, however, showed reduced gene transfection efficiency compared to those unmodified due to decreased cellular association and internalization. 13,14) Amphiphilic copolymers-Pluronic, poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO x -b-PPO y -b-PEO x triblock copolymer) that self-assemble to form micelles in aqueous solution were used as additiv...

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

“…containing serial dilutions of PP/DNA or PEI complexes at various concentrations (4,6,8,16,24, and 32 mg/mL). After 24 hours of incubation, 20 µL of CellTiter 96 ® AQ ueous One Solution Reagent (Promega) was added to each well and the cells were further incubated for 1-4 hours.…”

“…24 Naked DNA was digested when treated with DNase I (Figure 6, Lane 1). The other two complexes could encapsulate DNA completely and protect it from digestion by DNase I (Figure 6, Lane 3).…”

“…26 However, strong cationic charges of the complexes are often cytotoxic. 24 Changes in the charge of the complexes upon addition of the cationic copolymers were further characterized by measuring the zeta potential of various w/w ratios. Figure 8 shows zeta potentials of the complexes according to the w/w ratio.…”

Dual-degradable disulfide-containing PEI–Pluronic/DNA polyplexes: transfection efficiency and balancing protection and DNA release

“…Interestingly, addition of Pluronic P123 or P85 to the PEI-based polyplexes significantly enhanced pDNA (plasmid DNA) cellular uptake, nuclear translocation, and gene expression in several cell lines as a result of the binding of Pluronic with the cellular membrane and activation of cell-signaling pathways. 30,31 Few references so far have proposed that the conjugation of Pluronic with PEI-SS (disulfide-containing PEI) via ester hydrolysis bonds may achieve efficient transfection and a balance between protection and efficient DNA release.…”

Synthesis of Mannosylated Polyethylenimine and Its Potential Application as Cell-Targeting Non-Viral Vector for Gene Therapy

“…The net positive charge of the copolymer/DNA complex plays a major role in determining the extent of its interaction with the negatively charged phospholipid surface of the cell membrane, which can have a significant impact on the gene transfection efficiency [17]. Strong cationic charges on the complexes, however, can result in cytotoxic activity [18]. Figure 3B indicated that the Man-PEI25k/DNA complexes had a low cationic charge density on the surface (when they had an N/P ratio in the range of 1-30), which was compared with that of the unmodified PEI25k/DNA complexes.…”