Sustained DNA Release Research Articles

Gelation Properties and Drug Release Kinetics of a Thermoreversible Hydrogel as a Drug Carrier

Pluronic F-127 (PF-127) is a thermoreversible hydrogel that is a promising candidate for drug delivery. This hydrogel has a unique property that transforms its phase between liquid and gel as the temperature changes. PF-127 can be explored as a potential drug delivery system to treat diseases due to its low toxicity and controllable, sustained drug release. This study aims to determine the optimized relationship between concentration and temperature for successful PF-127 gelation and to characterize its drug release kinetics. PF-127 powder was dissolved in a saline solution to make concentrations ranging from 10% to 30%. The temperature was increased gradually to determine the precise temperature at which each PF-127 solution at a different concentration turned to gel. Concentrations under 16% did not turn to gel at any range of temperature. From 20% to 24% concentrations, PF-127 was a liquid at room temperature, while 26% to 30% concentrations were in a gel state. To characterize the drug release kinetics of PF-127, calf thymus DNA was loaded into PF-127. It showed sustained DNA release over a prolonged period of time as opposed to an immediate burst release. Sustained release kinetics shown in our study is preferable because it may steadily release a drug at an optimal concentration, preventing toxicity from a high drug concentration while maintaining therapeutic potential. The outcomes of this study elucidate a broad application of PF-127 as a potential drug carrier.

Controlled pVEGF delivery via a gene-activated matrix comprised of a peptide-modified non-viral vector and a nanofibrous scaffold for skin wound healing

Regulating cell function and tissue formation by combining gene delivery with functional scaffolds to create gene-activated matrices (GAMs) is a promising strategy for tissue engineering. However, fabrication of GAMs with low cytotoxicity, high transfection efficiency, and long-term gene delivery properties remains a challenge. In this study, a non-viral DNA delivery nanocomplex was developed by modifying poly (D, L-lactic-co-glycolic acid)/polyethylenimine (PLGA/PEI) nanoparticles with the cell-penetrating peptide KALA. Subsequently, the nanocomplex carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF) was immobilized onto a polydopamine-coated electrospun alginate nanofibrous scaffold, resulting in a GAM for enhanced skin wound healing. The nanocomplex exhibited much lower cytotoxicity and comparable or even higher transfection efficiency compared with PEI. The GAM enabled sustained gene release and long-tern transgene expression of VEGF in vitro. In an excisional full-thickness skin wound rat model, the GAM could accelerate wound closure, promote complete re-epithelization, reduce inflammatory response, and enhance neovascularization, ultimately enhancing skin wound healing. The current GAM comprising a low-toxic gene delivery nanocomplex and a biocompatible 3D nanofibrous scaffold demonstrates great potential for mediating long-term cell functions and may become a powerful tool for gene delivery in tissue engineering. Statement of significanceGene delivery is a promising strategy in promoting tissue regeneration as an effective alternative to growth factor delivery, but the study on three-dimensional gene-activated scaffolds remains in its infancy. Herein, a biodegradable nanofibrous gene-activated matrix integrating non-viral nanoparticle vector was designed and evaluated both in vitro and in vivo. The results show that the nanoparticle vector provided high transfection efficiency with minimal cytotoxicity. After surface immobilization of the nanocomplexes carrying plasmid DNA encoding vascular endothelial growth factor (pVEGF), the nanofibrous scaffold enabled sustained DNA release and long-term transgene expression in vitro. In a rat full-thickness skin wound model, the scaffold could accelerate wound healing. This innovative gene-activated matrix can be a promising candidate for tissue regeneration.

(3-Aminopropyl)-4-methylpiperazine End-capped Poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)-based Multilayer Films for Gene Delivery

Biodegradable polyelectrolyte surfaces for gene delivery were created through electrospinning of biodegradable polycations combined with iterative solution-based multilayer coating. Poly(β-amino ester) (PBAE) poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) end-capped with 1-(3-aminopropyl)-4-methylpiperazine was utilized because of its ability to electrostatically interact with anionic molecules like DNA, its biodegradability, and its low cytotoxicity. A new DNA release system was developed for sustained release of DNA over 24 h, accompanied by high exogenous gene expression in primary human glioblastoma (GB) cells. Electrospinning a different PBAE, poly(1,4-butanediol diacrylate-co-4,4'-trimethylenedipiperidine), and its combination with polyelectrolyte 1-(3-aminopropyl)-4-methylpiperazine end-capped poly(1,4-butanediol diacrylate-co-4-amino-1-butanol)-based multilayers are promising for DNA release and intracellular delivery from a surface.

Polypseudorotaxanes of pegylated α-cyclodextrin/polyamidoamine dendrimer conjugate with cyclodextrins as a sustained release system for DNA

Nonviral gene delivery suffers from a number of limitations including short transgene expression times and low transfection efficiency. In this study, we examined whether polypseudorotaxanes (PPRXs) of polyethylene glycol (PEG, molecular weight: 2,000)-grafted α-cyclodextrin (α-CyD)/polyamidoamine dendrimer conjugate (PEG-α-CDE) with CyDs have the potential for the novel sustained release systems for plasmid DNA (pDNA). The PEG-α-CDE/pDNA complex formed PPRXs with α-CyD and γ-CyD solutions, but not with β-CyD solution. In the PEG-α-CDE/CyDs PPRX systems, 20.6mol of α-CyD and 11.8mol of γ-CyD were involved in the PPRXs formation with one PEG chain by α-CyD and γ-CyD, respectively, consistent with in the PEG-dendrimer/CyDs systems. PEG-α-CDE/pDNA/α-CyD PPRX and PEG-α-CDE/pDNA/γ-CyD PPRX formed hexagonal and tetragonal columnar channels in the crystalline phase, respectively. In addition, the CyDs PPRX provided the sustained release of pDNA from PEG-α-CDE complex with pDNA at least 72h in vitro. The release of pDNA from CyDs PPRX retarded as the volume of dissolution medium decreased. Furthermore, the PEG-α-CDE/γ-CyD PPRX system showed sustained transfection efficiency after intramuscular injection to mice at least for 14days. These results suggest that the PEG-α-CDE/CyD PPRX systems are useful for novel sustained DNA release systems.

Potential Use of Polypseudorotaxanes of Pegylated Polyamidoamine Dendrimer with Cyclodextrins as Novel Sustained Release Systems for DNA

In this study, we demonstrated the potential use of polypseudorotaxanes (PPRXs) of polyethylene glycol (PEG, molecular weight: 2000)-grafted polyamidoamine dendrimer (PEG-dendrimer) with cyclodextrins (CyDs) as novel sustained release systems for plasmid DNA (pDNA). PEG-dendrimer/pDNA complex formed PPRXs with α-CyD and γ-CyD solutions, but not with β-CyD solution. In the PEG-dendrimer/CyDs PPRXs systems, 17.9 mol of α-CyD and 8.8 mol of γ-CyD were involved in the PPRXs formation with one PEG chain by α-CyD and γ-CyD, respectively. In addition, the CyDs PPRX formation provided the sustained release of pDNA from PEG-dendrimer complex with pDNA at least 72 h in vitro. In addition, the release of pDNA from CyDs PPRX retarded as the dissolution medium volume decreased. These results suggest that the PEG-dendrimer/CyD PPRX systems can work as a sustained DNA release system, and the PPRX formation with CyDs may be useful as a sustained drug delivery technique for other pegylated polymers.

Formulation and characterization of naked DNA and complexed DNA loaded polymer films

Sustained release of DNA from polymeric films is of considerable interest for enhanced and prolonged gene therapy. This report describes the detailed studies of the formulation of naked plasmid DNA (pDNA) and complexed DNA-incorporated polymer films and in vitro release of the DNA. The effect of hydrophilic polymers (PEG, HA) also studied to modulate the release of pDNA and complexed DNA (lipoplex) from slow biodegradable polymer (PCL) films. The polymer system consists of a biodegradable semi-crystalline polymer (PCL) blended with a hydrophilic polymer such as poly (ethylene glycol) (PEG) or hyaluronic acid (HA). For the release of pDNA (naked DNA), a burst effect was always seen, and the addition of HA and PEG did not suppress the burst release of pDNA from PCL films. For complexed pDNA (lipoplex), the release was slow, but it could be accelerated using additives such as PEG or HA. The transfection efficiency of the complexed DNA and the naked pDNA was determined in vitro using COS 7cells to evaluate the bioactivity of the released DNA. Transfection was observed from released lipoplexes samples from PCL/HA film. Overall, this work suggested that these polymeric DNA delivery systems are promising for the local sustained release of DNA from implanted films.

Polysaccharide/polyaminoacid composite scaffolds for modified DNA release

In this work composite polymeric films or sponges, based on hyaluronic acid (HA) covalently crosslinked with α,β-poly(N-2-hydroxyethyl)(2-aminoethylcarbamate)- d, l-aspartamide (PE), have been prepared and characterized as local gene delivery systems. In particular, HA/PE scaffolds have been loaded with PE/DNA interpolyelectrolyte complexes, employing PE as a macromolecular crosslinker for HA and as a non-viral vector for DNA. In vitro studies showed that HA/PE films and sponges have high compatibility with human dermal fibroblasts and they give a sustained DNA release, whose trend can be easily tailored by varying the crosslinking ratio between HA and PE. Electrophoresis analysis and transfection studies on B16-F10 cells revealed that DNA is released as a complex with PE and it retains its bioactivity.

Hyaluronic acid and chitosan-DNA complex multilayered thin film as surface-mediated nonviral gene delivery system

Sustained release of DNA from the surface of materials represents a promising approach to combine the gene therapy and implantable biomaterials. The nonviral chitosan-DNA complexes were incorporated into the multilayer via layer-by-layer deposition with hyaluronic acid (HA). The UV–vis spectroscopy and atomic force microscopy (AFM) results showed the successful construction of the nonviral complex contained multilayers. The complexes were releasable in physiological condition and a sustained release manner was gained when the multilayer was crosslinked. The cell viability test and the gene transfection assay showed that the natural polyelectrolyte-based nonviral complex incorporated multilayer not only had good cytocompatibility, but also possessed the in vitro gene transfection ability. This kind of surface-mediated nonviral complex incorporated multilayer may have great potential in the localized and controlled delivery of DNA in biomedical implants and tissue engineering application.

Lysine-based peptide-functionalized PLGA foams for controlled DNA delivery

Due to its hydrophobicity and negatively charged surfaces, PLGA-based scaffolds have encountered problems in controlled-release and tissue engineering applications. The effects of charge modification of PLGA micro-porous foams on DNA delivery and DNA transfection are investigated herein. Tailor-designed l-lysine peptides (K4 and K20) were employed to modify the surface charge of PLGA foams using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide cross linkers and the effects of charge modification of PLGA were examined in three main aspects: DNA adsorption, DNA release properties and DNA transfection. Successful conjugation of peptide and DNA adsorption were verified by X-ray photoelectron spectroscopy. A plasmid encoding bone morphogenetic protein-2 (BMP2) was used throughout the current study and the results indicate that adsorption capacity and release behavior of DNA were highly dependent on the charge properties of the foam surfaces. The release rates of DNA from the K4- and K20-functionalized foams are more sustainable as compared to the blank foam. As a result, the sustained release of DNA from modified foams led to negligible cytotoxicity and sustained expression of DNA which is favorable for DNA delivery and tissue engineering application. Furthermore, the ease of fabrication and modification of PLGA foams makes it a promising DNA delivery device.

Calcium phosphate‐DNA nanoparticle gene delivery from alginate hydrogels induces in vivo osteogenesis

There is a significant need for improved therapy for bone regeneration. The delivery of recombinant bone morphogenetic proteins has been approved for clinical use to promote osteogenesis, but still has limitations such as expense, degradation of the proteins in vivo and difficulties retaining protein at the site of injury. Localized gene delivery is a promising alternative therapy, as it would allow sustained expression of specific osteoinductive growth factors by cells near the damaged site. We have engineered an injectable system for localized, sustained nonviral gene delivery from alginate hydrogels containing preosteoblastic cells and calcium phosphate-DNA nanoparticles. The nanoparticles utilized in this report are stable, on the order of 100 nm, and have a high DNA incorporation efficiency (>66%). When the nanoparticles were incorporated in alginate hydrogels, sustained release of DNA was observed. Furthermore, MC3T3-E1 preosteoblast cells exhibited the capacity to form bony tissue in as little as two and half weeks when mixed with DNA nanoparticles encoding for BMP-2 into the alginate hydrogels and injected subcutaneously in the backs of mice. This injectable, minimally invasive gene delivery system may be efficacious in bone regeneration applications.

PLGA/chitosan composites from a combination of spray drying and supercritical fluid foaming techniques: New carriers for DNA delivery

The use of poly( D, L-lactic- co-glycolic acid) for DNA delivery application is limited by its negative surface charge and acidic degradation products. The motivation of the present work was to study the effects of chitosan incorporation into PLGA foams on DNA delivery. PLGA/chitosan composite foams loaded with luciferase plasmid were fabricated by a combination of spray drying and supercritical CO 2 foaming techniques. The resultant composite foams showed good morphology and chitosan was found to be poorly crystallized in the PLGA matrixes. The composite foams exhibited a sustained release of DNA (5–9weeks) with decreasing release rate upon increasing content of chitosan. With this encapsulation technique, it was also observed that the integrity of plasmid was well maintained. Moreover, cell culture results proved that the bioactivity of plasmid released from all foams was well maintained and the incorporation of chitosan in foams helps increase cell adhesion and maintain high cell viability. Therefore, it can be concluded that PLGA/chitosan composite foams fabricated by combining spray drying and supercritical CO 2 foaming are promising in DNA delivery applications.

Diclofenac sodium delivery to the eye: In vitro evaluation of novel solid lipid nanoparticle formulation using human cornea construct

Solid lipid nanoparticles (SLNs) were prepared with a combination of homolipid from goat (goat fat) and phospholipid, and evaluated for diclofenac sodium (DNa) delivery to the eye using bio-engineered human cornea, produced from immortalized human corneal endothelial cells (HENC), stromal fibroblasts and epithelial cells CEPI 17 CL 4. Encapsulation efficiency was high and sustained release of DNa and high permeation through the bio-engineered cornea were achieved. Results obtained in this work showed that permeation of DNa through the cornea construct was improved by formulation as SLN modified with phospholipid.

Fabrication and characterization of PLGA/HAp composite scaffolds for delivery of BMP-2 plasmid DNA

The objective of this study is to construct complex of DNA and PLGA/HAp composite scaffold for bone tissue engineering. Naked DNA has low transfection efficiency so DNA loaded chitosan particles are used nowadays in gene delivery due to their high transfection efficiency, but unfortunately this is accompanied by strong immunological reaction of cells. In order to preserve the advantage of polymeric particles and reduce immunological effect at the same time, a new DNA release system is developed which makes possible sustained DNA release with negligible immunological effects. Poly (lactide-co-glycolide) (PLGA)/Hydroxylapatite (HAp) composite scaffolds with different HAp contents (0%, 5% and 10%) are fabricated by an electrospinning method and DNA is incorporated into the scaffolds in 3 ways (i.e. naked DNA, encapsulation of DNA/chitosan nanoparticles into scaffolds after fiber fabrication by dripping, and encapsulation of DNA/chitosan nanoparticles into scaffold by mixing with PLGA/HAp solution before fiber fabrication). All the scaffolds are characterized by SEM, XRD, DSC and GC-MS and the results show that the scaffolds are non-woven, nano- to micro-fibered membrane structures composed predominantly of PLGA with amorphous dispersion of HAp nanoparticles inside polymeric matrix. In vitro release tests were carried out on 9 different scaffolds to check the effects of HAp contents and the encapsulation ways of DNA on the release properties. These effects are also tested by human marrow stem cells (hMSCs) by comparing their cell attachment ability, cell viability and DNA transfection efficiency. It is demonstrated that the addition of HAp nanoparticles increased the release rate of DNA for both naked and encapsulated DNA. Cell culture experiments show that the scaffolds with encapsulated DNA/chitosan nanoparticles have higher cell attachment, higher cell viablility and desirable transfection efficiency of DNA. The observations show that DNA/chitosan nanoparticles encapsulated PLGA/HAp composite scaffold is promising for use in bone regeneration.

A novel PEGylation of chitosan nanoparticles for gene delivery

CS (chitosan) has emerged as a promising non-viral vector for gene delivery because of its ability to form complexes with pDNA (plasmid DNA) and enhance its transport across cellular membranes through endocytosis. Complexes of CS and pDNA may improve transfection efficiency; however, they are not capable of sustained DNA release and prolonging gene transfer. In order to achieve prolonged delivery of CS-DNA complexes, we prepared CS NP (nanoparticle) and CS-DNA complexes. alpha-Methoxy-omega-succinimidylpoly(ethylene glycol) was then conjugated to the surface of CS-DNA complexes using an active ester scheme; finally, the potential of PEGylation [poly(ethylene glycol)ylation] of CS NP as a non-viral gene-delivery vector to transfer exogenous genes in vitro and in vivo were examined. Electrophoretic analysis suggested that CS NPs could protect the DNA from nuclease degradation. The pDNA carried by CS NPs could enter and be expressed in HepG2 cells. However, the transfection efficiency was very low and the highest dose of DNA transferred was 1.6 microg. The transfection activities of CS-DNA-PEG were preserved and a higher dose (2.4 microg) of pDNA was transferred. This indicated that the transfection efficiency of the PEGylated complexes had been improved. In vivo experiments also showed that CS-DNA-PEG complexes mediated higher gene expression in tissues than did CS-DNA complexes, and that gene expression in tumours induced by CS-DNA-PEG complexes was the highest of all. These results suggested that PEGylation of CS-DNA complexes improves non-viral gene delivery in vitro or in vivo and has the potential to deliver therapeutic genes directly into hepatoma tissues.

Tunable Film Degradation and Sustained Release of Plasmid DNA from Cleavable Polycation/Plasmid DNA Multilayers under Reductive Conditions

The controllable and sustained release of DNA from the surfaces of biomaterials or biomedical devices represents a new method for localized gene delivery. We report the synthesis of a novel polycation containing disulfide bonds in its backbone and the fabrication of polycation/plasmid DNA multilayered thin films by layer-by-layer assembly. The films are very stable during preparation and in storage, however, they gradually degrade and release the incorporated DNA when incubated in PBS buffer containing dithiothreitol (DTT), which results from the degradation of a disulfide-contained polymer under reductive conditions. The film degradation rate and DNA release rate can be tuned by the concentration of reducing agent. This approach will be useful in gene therapy and tissue engineering by controlled administration of therapeutic DNA deposited on the surface of implantable biomedical devices or tissue engineering scaffolds.

Thiolated Chitosan/DNA Nanocomplexes Exhibit Enhanced and Sustained Gene Delivery

Thiolated chitosan appears to possess enhanced mucoadhesiveness and cell penetration properties, however, its potential in gene-drug delivery remains unknown. Herein, we report on a highly effective gene delivery system utilizing a 33-kDa thiol-modified chitosan derivative. Thiolated chitosan was prepared by the reaction with thioglycolic acid. Nanocomplexes of unmodified chitosan or thiolated chitosan with plasmid DNA encoding green fluorescenct protein (GFP) were characterized for their size, zeta potential, their ability to bind and protect plasmid DNA from degradation. The transfection efficiency of thiolated chitosan and sustained gene expression were evaluated in various cell lines in vitro and in Balb/c mice in vivo. Thiolated chitosan-DNA nanocomplexes ranged in size from 75 to 120 nm in diameter and from +2.3 to 19.7 mV in zeta potential, depending on the weight ratio of chitosan to DNA. Thiolated chitosan, CSH360, exhibited effective physical stability and protection against DNase I digestion at a weight ratio>or=2.5:1. CSH360/DNA nanocomplexes induced significantly (P<0.01) higher GFP expression in HEK293, MDCK and Hep-2 cell lines than unmodified chitosan. Nanocomplexes of disulphide-crosslinked CSH360/DNA showed a sustained DNA release and continuous expression in cultured cells lasting up to 60 h post transfection. Also, intranasal administration of crosslinked CSH360/DNA nanocomplexes to mice yielded gene expression that lasted for at least 14 days. Thiolated chitosans condense pDNA to form nanocomplexes, which exhibit a significantly higher gene transfer potential and sustained gene expression upon crosslinking, indicating their great potential for gene therapy and tissue engineering.

Release of DNA from dendriplexes encapsulated in PLGA nanoparticles

Biodegradable PLGA particles of less than 1 μm can encapsulate DNA and DNA–dendron complexes (dendriplexes) providing sustained DNA release for transfecting cells in gene delivery. Two polylysine-based dendrons prepared by solid state peptide synthesis were used to condense pRedN-1 DNA (7.5 kbp), a fluorescent protein vector. The dendrons had 16 free surface amino groups attached to seven lysine groups, bound to a lipid core, one containing three C 18 chains and the other a single C 10 chain. Increased lipophilicity and molar charge ratios are key factors in producing compact and reproducible dendriplexes, shown by the hydrodynamic diameter which is of the order of 800 nm (p.d. > 0.5) at a 2:1 molar charge ratio, a value which decreases to around 200 nm at a 5:1 charge ratio. At lower charge ratios the dendriplexes are negative and have a zeta potential in order of −18 mV. As the ratio increases (5:1, 10:1) the complexes bear a positive potential (13 ± 2 mV). This suggests that at the 2:1 ratio the DNA is not fully condensed. The DNA was radiolabelled with 35S dCTP (deoxycytidinetriphosphate) with the removal of the un-incorporated radiolabelled nucleotides. The encapsulation efficiency of dendriplexes in PLGA particles is higher than that for uncomplexed DNA. When the results are normalised for DNA content and particle surface area, complexation of the DNA was found to decrease release rate.

Sustained release of PEG-g-chitosan complexed DNA from poly(lactide-co-glycolide)

Chitosan and its derivatives have emerged as promising gene-delivery vehicles because of their capability to form polyplexes with plasmid DNA and enhance its transport across cellular membranes through endocytosis. Evidently, polyplexes of chitosan and DNA significantly improve transfection efficiency; however, these polyplexes are not capable of sustained DNA release and, thus, prolong gene transfer. In order to achieve prolonged delivery of DNA/chitosan polyplexes, we have formulated microspheres by physically combining poly(ethylene glycol)-grafted chitosan (PEG-g-CHN) with poly(lactide-co-glycolide) (PLGA) using a modified conventional in-emulsion solvent evaporation method. Electrophoretic analysis of materials released from these microspheres suggests the presence of PEG-g-CHN complexed DNA and these microspheres are capable of sustained release of DNA/PEG-g-CHN for at least 9 weeks. The rate of DNA release can be modulated by varying the amount of PEG-g-CHN. The release products from these microspheres are bioactive and show enhanced transfection in vitro over DNA released from conventional PLGA microspheres containing no PEG-g-CHN. In vivo experiments also show that these microspheres are capable of achieving gene transfer in a rat hind limb muscle model.

Controlled release of plasmid DNA from biodegradable scaffolds fabricated using a thermally-induced phase-separation method

Highly porous poly(D, L-lactic-co-glycolic acid) (PLGA) scaffolds were fabricated by a thermally-induced phase-separation (TIPS) method to deliver plasmid DNA in a controlled manner. A variety of TIPS parameters directly affecting pore structures and their interconnectivities of the scaffold, such as polymer concentration, solvent/non-solvent ratio, quenching methods and annealing time, were systematically examined to explore their effects on sustained release behaviors of plasmid DNA. Plasmid DNA was directly loaded into the inner pore region of the scaffold during the TIPS process. By optimizing the parameters, PLGA scaffolds releasing plasmid DNA over 21 days were successfully fabricated. DNA release profiles were mainly affected by the pore structures and their interconnectivities of the scaffolds. Plasmid DNA released from the scaffolds fully maintained its structural integrity and showed comparable transfection efficiency to native plasmid DNA. These biodegradable polymeric scaffolds capable of sustained DNA release can be potentially applied for various tissue engineering purposes requiring a combined gene delivery strategy.

CNS gene transfer mediated by a novel controlled release system based on DNA complexes of degradable polycation PPE-EA: a comparison with polyethylenimine/DNA complexes

Nonviral gene delivery systems based upon polycation/plasmid DNA complexes are quickly gaining recognition as an alternative to viral gene vectors for their potential in avoiding immunogenicity and toxicity problems inherent in viral systems. We investigated in this study the feasibility of using a controlled release system based on DNA complexed with a recently developed polymeric gene carrier, polyaminoethyl propylene phosphate (PPE-EA), to achieve gene transfer in the brain. A unique feature of this gene delivery system is the biodegradability of PPE-EA, which can provide a sustained release of DNA at different rates depending on the charge ratio of the polymer to DNA. PPE-EA/DNA complexes, naked DNA, and DNA complexed with polyethylenimine (PEI), a nondegradable cationic polymer known to be an effective gene carrier, were injected intracisternally into the mouse cerebrospinal fluid. Transgene expression mediated by naked DNA was mainly detected in the brain stem, a region close to the injection site. With either PPE-EA or PEI as a carrier, higher levels of gene expression could be detected in the cerebral cortex, basal ganglia, and diencephalons. Transgene expression in the brain mediated by PPE-EA/DNA complexes at an N/P ratio of 2 persisted for at least 4 weeks, with a significant higher level than that produced by either naked plasmid DNA or PEI/DNA at the 4-week time point. Furthermore, PPE-EA displayed much lower toxicity in cultured neural cells as compared to PEI and did not cause detectable pathological changes in the central nervous system (CNS). The results established the potential of PPE-EA as a new and biocompatible gene carrier to achieve sustained gene expression in the CNS.