Preparation and Characterization of Cationic PLA-PEG Nanoparticles for Delivery of Plasmid DNA
© to the authors 2009
Received: 4 March 2009
Accepted: 6 May 2009
Published: 21 May 2009
The purpose of the present work was to formulate and evaluate cationic poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) nanoparticles as novel non-viral gene delivery nano-device. Cationic PLA-PEG nanoparticles were prepared by nanoprecipitation method. The gene loaded nanoparticles were obtained by incubating the report gene pEGFP with cationic PLA-PEG nanoparticles. The physicochemical properties (e.g., morphology, particle size, surface charge, DNA binding efficiency) and biological properties (e.g., integrity of the released DNA, protection from nuclease degradation, plasma stability, in vitro cytotoxicity, and in vitro transfection ability in Hela cells) of the gene loaded PLA-PEG nanoparticles were evaluated, respectively. The obtained cationic PLA-PEG nanoparticles and gene loaded nanoparticles were both spherical in shape with average particle size of 89.7 and 128.9 nm, polydispersity index of 0.185 and 0.161, zeta potentials of +28.9 and +16.8 mV, respectively. The obtained cationic PLA-PEG nanoparticles with high binding efficiency (>95%) could protect the loaded DNA from the degradation by nuclease and plasma. The nanoparticles displayed sustained-release properties in vitro and the released DNA maintained its structural and functional integrity. It also showed lower cytotoxicity than Lipofectamine 2000 and could successfully transfect gene into Hela cells even in presence of serum. It could be concluded that the established gene loaded cationic PLA-PEG nanoparticles with excellent properties were promising non-viral nano-device, which had potential to make cancer gene therapy achievable.
KeywordsCationic PLA-PEG nanoparticles (DNA-PLA-PEG-NPs) Gene therapy Nanoprecipitation method Non-viral gene vector
Gene therapy is a rapidly advancing field with enormous potential to treat vital human diseases such as cancer and inherited genetic disorders fundamentally. The development of potent gene transfer systems that can deliver foreign genes efficiently and safely into target cells is of crucial importance for successful gene therapy. The non-viral vector, possessing significantly low safety risks and convenient preparation in large quantities easily and inexpensively, has been suggested as an alternative to viral vector . Therefore, to develop a safe and effective nonviral vector system is an urgent matter. Among non-viral vectors, cationic gene delivery polymers include poly(ethylenimine) (pEI), poly(2-dimethylaminoethyl methacrylate) (pDMAEMA), and poly-l-lysine (pLL) have been frequently studied. Their gene binding and condensation capacities as well as their in vitro and in vivo transfection properties have been reported in recently ample literatures. These polymers are, however, non-degradable and there is consequently a risk that accumulation in the body occurs, in particular after repeated administration. Further, most of these cationic polymers show some cytotoxicity likely due to adverse interactions with membranes resulting in loss of cytoplasmic proteins, in permeabilization of cellular membranes and collapse of the membrane potential . Consequently, there is a need for biodegradable gene delivery polymers. It’s clear that the potential advantages of biodegradable carriers are their reduced toxicity (provided that degradation leads to non-toxic products) and avoidance of accumulation of the polymer in the cells. Moreover, the degradation of the polymer can be used as a tool to release the plasmid DNA into the cytosol. Recently, the use of nanoparticles prepared with biocompatible and biodegradable poly (d l-lactide-co-glycolide) (PLGA) or poly (d l-lactide) (PLA) polymers have attracted much attention due to their favorable physicochemical characteristics in terms of safety, stability, the relative ease of large-scale production, and lack of intrinsic immunogenicity that make them suitable candidates for gene delivery application [3, 4]. These biodegradable polymers undergo bulk hydrolysis thereby providing sustained delivery of the therapeutic agent depending on the polymer molecular weight and copolymer . The degradation products, lactic acid and glycolic acid, are removed from the body through citric acid cycle. The degradation time of PLGA/PLA can be altered from days to years by varying the polymer molecular weight, the lactic acid to glycolic acid ratio in copolymer. And recent studies demonstrated rapid escape of PLGA nanoparticles from the endo-lysosomal compartment into cytosol following their uptake . Therefore, PLGA/PLA nanoparticles might be a promising and suitable candidate for gene delivery application.
Although all features makes PLGA/PLA attractive to many researchers involved in DNA delivery,it should be noticed that simple PLGA/PLA nanoparticles shows, indeed, several drawbacks as an ideal gene delivery system . First, because of the large size and hydrophilic character of DNA, encapsulation of plasmid DNA in hydrophobic PLGA/PLA nanoparticles is a challenge. Second, the rate of DNA release from PLGA/PLA nanoparticles is often too slow, and thus also subsequent antigen production in case of DNA vaccination, which may prevent an optimal immune response. Third, PLGA/PLA nanoparticles tend to bind serum protein (such as albumin) in systemic application due to the hydrophobic surface of PLGA/PLA nanoparticles, which lead to opsonization and clearance by the reticuloendothelial system (RES), limiting their therapeutic applications. Based on these considerations, it’s necessary to modify the structure of PLGA/PLA in order to improve its hydrophilicity, the gene loading efficiency, release behavior, and stability both in vitro and in vivo.
Polyethylene glycol (PEG) is a commonly used modifier, which is expected to be a good candidate as the soluble polymeric modifier in organic synthesis or a pharmacological polymer due to its high hydrophilicity, low cytotoxicity and high cell permeability . Reportedly, the PLA and PLGA nanoparticles with either adsorbed or grafted PEG layers exhibit prolonged blood circulation times and reduced uptake by the reticuloendothelial system in comparison with their uncoated counterparts . It has been proved that PEG corona could diminish non-specific interactions with serum proteins and decrease the uptake of cationic polymer/DNA complex by the macrophages in the liver and spleen leading to an increased blood circulation time, which ultimately improve the transfection efficiency of polycationic polymers. Furthermore, the hydrophilicity of PLA nanoparticles could be improved after modification with PEG, and the affinity between polymers and DNA would be accordingly enhanced which is favorable to load gene into the nanoparticles. In addition, the microenvironment formed by PEG is beneficial to the activity of protein or gene agents in storage or administration. In addition, although PEGylated PLA or PLGA polymer have come out for a while as drug delivery systems [10, 11], there are few reports on gene delivery systems [12, 13]. Therefore the feasibility and suitability of PEGylated PLA or PLGA polymer as gene delivery system remains to be adequately investigated and addressed.
Usually plasmid DNA is encapsulated into PLGA/PLA particles using the common water-in-oil-water (W/O/W) double emulsion/solvent evaporation method in order to achieve a better protection of plasmid and a more precise control of the release process [14, 15]. However, the double emulsion/solvent evaporation method can not guarantee the integrity of DNA under ultra-sonication or high speed homogenization, which is necessary in the encapsulation process to obtain smaller particle size [16, 17]. One strategy to solve the problem is to adsorb the plasmid DNA onto the surface of cationic PLA/PLGA particles which are modified to display a positively charged surface by inclusion of cationic surfactants such as cetyltrimethyl ammonium bromide (CTAB) in formulations [18, 19]. It was reported that the cationic gene loaded particles substantially improved the immune responses generated by DNA, both in mouse and macaque models .
Based on these considerations, the main goal of the present work was to develop a novel cationic PEGylated PLA (PLA-PEG) nanoparticles and explore its applicability and feasibility as a nonviral vector for gene transport. For this purpose, cationic PLA-PEG nanoparticles with appropriate positive charge and particle size was prepared applying an easily performed nanoprecipitation technique and using CTAB as a cationic surfactant and Tween 80 as a cosurfactant. The cationic gene loaded PLA-PEG nanoparticles was then obtained by adsorbing plasmid DNA onto the surface of cationic PLA-PEG nanoparticles through electrostatic interactions. The physicochemical properties and biological properties of the nanoparticles were investigated and characterized, respectively.
Materials and Methods
Poly(ethylene glycol)-block-polylactide copolymer (PLA-PEG, PEG average Mn 3,000 Da, PLA average Mn 50,000 Da, synthesized by school of chemistry of Shandong University). Cetyltrimethyl ammonium bromide (CTAB) was purchased from Amresco (Amresco, China). pEGFP-N1 was provided by Zhejiang University (China). PicoGreen®dsDNA reagent was obtained from Molecular Probes (Invitrogen, USA). Agarose was purchased from BIO-WEST (Spain). Goldview was obtained from Beijing Saibaisheng Biological Engineering Co. (Beijing, China). DNase I enzyme was obtained from Beijing Yinfeng Century Scientific Develop Co., Ltd (Beijing, China). MTT (3-[4, 5-dimehyl-2-thiazolyl]-2, 5-diphenyl-2H- tetrazolium bromide) were purchased from Sigma-Aldrich (China). Lipofectamine 2000 was from Invitrogen (USA). Hela cell line was obtained from American Type Culture Collection (ATCC, USA). All the other chemicals and reagents used were of analytical purity grade or higher, obtained commercially.
Preparation of Cationic PLA-PEG Nanoparticles
Cationic PLA-PEG nanoparticles (abbreviated as PLA-PEG-NPs below) was prepared by the nanoprecipitation method  according to the optimized formulation. Typically, accurately weighed (20 mg) PLA-PEG was dissolved into 5 mL acetone under sonication. The resulting polymer solution was slowly (30 mL/h) injected by a micro-syringe pump (KDS 100, USA) into 20 mL magnetically stirring (600 rpm, RCT basic stirrer, IKA, Germany) mixed surfactant solution containing CTAB (0.1%, w/v) and Tween 80 (0.2%, w/v) and agitated for 8 h at room temperature until complete evaporation of the organic solvent. The entire dispersed system was then centrifuged (Beckman, Fullerton, CA) at 15,000 rpm, 4 °C for 30 min. The pellet was re-suspended in Milli-Q water and washed three times to remove the redundant surfactant. Finally it was re-suspended in phosphate buffered saline (PBS, pH 7.4) solution followed by filtered through 0.45 μm nitrocellulose membrane (Millipore) filter and was stored at 4 °C until use.
Preparation and Optimization of Gene Loaded Cationic PLA-PEG Nanoparticles
Adsorption of Plasmid DNA onto PLA-PEG-NPs
The gene loaded cationic PLA-PEG nanoparticles (abbreviated as DNA-PLA-PEG-NPs below) were obtained by means of electrostatic attraction between the anionic plasmid DNA and the blank cationic nanoparticles. Briefly, the report gene pEGFP solution was added into PLA-PEG-NPs at a fixed weight ratio (PLA-PEG: DNA, w/w). The mixed liquor was kept for 20 min at room temperature and the resultant DNA-PLA-PEG-NPs were directly used for further study.
Agarose Gel Electrophoresis of DNA/Nanoparticles Complex
Complex formation between nanoparticles and DNA was analyzed by agarose gel electrophoresis. The gels were prepared with 0.8% (w/v) agarose in 20 mL TAE buffer (40 mM Tris, 40 mM Acetic acid, 1 mM EDTA, pH 8.5) containing 2 μL goldview as stains. A fixed amount (1 μg) of DNA was incubated with various amounts of PLA-PEG-NPs in 100 μL of PBS (pH = 7.4) (the weight ratio of PLA-PEG and DNA is 10:1, 50:1, 100:1, 150:1, 20:1, 500:1, respectively). The resultant DNA-PLA-PEG-NPs and control plasmid DNA were applied to gel electrophoresis at a constant 90 V for 25 min. After the electrophoresis, images were obtained using UV transilluminator and Multimage™ Light Cabinet (Alpha Imagers EC, Alpha Innotech Corporation) to show the location of DNA.
Determination of DNA Binding Efficiency
Morphology, Particle Size and Zeta Potential of Nanoparticles
The morphology of PLA-PEG-NPs and DNA-PLA-PEG-NPs was examined under transmission electron microscope (TEM, JEM-1200EX, Japan). Samples were prepared by placing a drop of nanoparticle suspension onto a copper grid and air-dried, following negative staining with one drop of 2% aqueous solution of sodium phosphotungstate for contrast enhancement. The air-dried samples were then directly examined under the transmission electronic microscope.
The average particle size, size distribution, and zeta potential of the nanoparticles were measured by photon correlation spectroscopy (PCS) using Zetasizer 3000 (Malvern Instruments, Malvern, England). All measurements were carried out in triplicates. The average particle size was expressed in volume mean diameter and the reported value was represented as mean ± SD (n = 3).
Stability Test of the DNA-PLA-PEG-NPs Against DNase I
Protection of plasmid DNA from nucleases is one of the most important properties for efficient gene delivery both in vitro and in vivo. To test whether DNA-PLA-PEG-NPs can protect the loaded plasmid DNA from nucleases digestion, the results of DNase I mediated digestion was evaluated using agarose gel electrophoresis . In brief, 100 μL of DNA-PLA-PEG-NPs containing 1 μg of DNA were, respectively, incubated with different amounts of DNase I (0.1, 0.2 and 0.4 U/μg DNA) in DNase I /Mg2+ digestion buffer (50 mM, Tris–HCl, pH 7.6, and 10 mM MgCl2). Naked DNA (1 μg) was treated with DNase I at 0.1 U/μg DNA as a reference. The suspension was incubated in shaking water bath (100 rpm) for 30 min at 37 °C. After that, the enzymatic digestion reaction was terminated with 5 μL EDTA solution (0.5 M, pH 8.0) for 10 min at room temperature. To asses the integrity of DNA loaded in the nanoparticles, it was dissociated from the cationic nanoparticles by adding heparin solution , an anionic glycosaminoglycan, at final concentration of 1% (w/v) and the suspension was then incubated in shaking water bath (100 rpm) for 3 h at 37 °C. The configuration of plasmid DNA in the nanoparticles after extraction was analyzed by gel electrophoresis with untreated naked DNA as a reference. The samples were applied to a 0.8% (w/v) agarose gel in TAE buffer as described above.
The Plasma Stability Investigation of DNA-PLA-PEG-NPs
To examine resistance of DNA-PLA-PEG-NPs to DNA degradation in plasma, 25 μL of plasmid DNA solution and DNA-PLA-PEG-NPs were, respectively, incubated with 25 μL of 20% human plasma for 1 h in a 37 °C incubator. Immediately following the incubation, nucleases were inactivated with 3 μL of EDTA solution (0.5 M, pH 8.0) for 10 min at room temperature. Thereafter, the plasma treated samples together with untreated DNA-PLA-PEG-NPs and DNA were all applied to 0.8% (w/v) agarose gel as described above.
The In Vitro Release of DNA-PLA-PEG-NPs
The Stability of Plasmid DNA in the Release Medium
During the prolonged in vitro drug release, the biomacromolecule drug may be degraded, and this may affect in vitro drug release studies [13, 25]. Therefore, it is essential to select a suitable drug release medium to protect the plasmid DNA from degradation during in vitro drug release period. Based on physiological environment and the result of preliminary tests, the mixed solution containing phosphate buffered saline solution at pH 7.4 (100 mM) and 150 mM NaCl solution was selected as the release medium for the DNA-PLA-PEG-NPs. The stability of plasmid DNA in the release medium in the duration of in vitro release was investigated at first. Briefly, 200 μL DNA solution (10 μg/mL) were diluted with isovolumic release medium mentioned above in Eppendorf® tubes and then shaken horizontally in water bath at 37 °C and 100 rpm. Separate tubes were used for each data point. At predetermined time intervals (2 h, 5 h, 8 h, 12 h, 24 h, 2 days, 4 days, 7 days, 10 days, 15 days, 20 days), the samples were withdrawn and the concentration of DNA was determined by the PicoGreen fluorimetric assay mentioned above. The experiments were repeated three times and all measurements were collected in triplicates. The profile of percent content of DNA versus time in release medium was drawn.
Measurement of In Vitro DNA Release
The profiles of in vitro DNA release from DNA-PLA-PEG-NPs were measured over 20 days using separate samples for each time point according to the following operations. Typically, 200 μL of DNA-PLA-PEG-NPs (DNA concentration: 10 μg/mL) were diluted with isovolumic release medium in Eppendorf®tubes and then shaken horizontally in water bath at 37 °C and 100 rpm. At predetermined time intervals, the tubes were withdrawn and centrifugated at 15,000 rpm for 30 min, the supernatants were collected for analysis. The amount of the released DNA was evaluated by the PicoGreen fluorimetric assay. Background readings were corrected using the centrifugation supernatants from blank nanoparticles.
Integrity Determination of Plasmid DNA After Release
The molecular form of DNA (often referred to as “topology”) has been demonstrated to affect the transfection efficiency in vitro and in vivo . Supercoiled plasmid was believed to be the most efficient form for cell transfection and it was established that the order of transfection efficiency was supercoiled > relaxed > linear . Fabrication conditions such as sonication, lyophilization, and change of pH were reported previously to decrease the supercoiled content. It is well known that the hydrolysis of PLA may substantially decrease the pH in PLA nanoparticles, which potentially change the topology structure of DNA and decrease the biological activity of DNA accordingly. Therefore, it was crucial to examine the plasmid structural and functional integrity following release from nanoparticles.
Structural Integrity Determination
Structural integrity of plasmid DNA released from nanoparticles at selective time points was evaluated by agarose gel electrophoresis. To determine plasmid stability during release, the samples released at several time points (i.e. 2 h, 8 h, 24 h, 4 days, 10 days, 15 days and 20 days) along with control untreated plasmid DNA were applied to 0.8% agarose gel in TAE buffer. Band separation for topological structure of plasmid DNA was observed after gel electrophoresis.
Functional Integrity Determination
The in vitro transfection experiment was performed to investigate the functional integrity of released DNA. Hela cells (ATCC) were seeded into 24-well plates at a density of 1 × 105 cells/well 24 h prior to transfection and 1 μg of DNA of was used per well (n = 6 wells per formulation). An aliquot of untreated DNA solution and supernatants of nanoparticles in vitro release at different time points after moderate concentration (1 day, 10 days, 20 days, equivalent to 1 μg DNA) were complex with Lipofectamine 2000 according to the manufacturer’s instructions . After incubation for 4 h at 37 °C in 5% CO2 incubator, the original incubation media was replaced with 1 mL of complete medium and cells were incubated sequentially until 24 h post transfection. The percentage of fluorescent cells (defined as cells having fluorescence levels above that of untreated control cells) presented in every 10,000 cells counted by the flow cytometry was calculated. Transfection experiments were performed in triplicates.
Cell Viability Test of DNA-PLA-PEG-NPs
OD570(samples) represented measurements from the cells treated with samples and OD570(control) from the untreated cells.
All measurements were collected in triplicates and experiments were repeated three times. Values were expressed as mean ± standard deviation. Unpaired Student’st-test was used to assess statistical significant differences (p < 0.05) between the group means.
In Vitro Transfection Assays of DNA-PLA-PEG-NPs
The transfection activity of DNA-PLA-PEG-NPs was evaluated in Hela cell lines using plasmid DNA, encoding enhanced green fluorescence protein (EGFP) as reporter gene in the transfection studies. The cells were seeded into 24-well plates at a density of about 1 × 105 cells per well in 1 mL of RPMI 1640 culture medium with 10% FBS, 24 h prior to transfection. At a confluence level of 70–80%, cells were washed twice with PBS, and, respectively, incubated with 500 μL of media (with or without 10% FBS) containing 1 μg of DNA in transfection vectors at 37 °C. Lipofectamine 2000 (Invitrogen) was used as positive control, and the formulation of Lipofectamine/DNA complex was carried out according to the manufacturer’s protocols. 1 μg of plasmid DNA was used as negative control. The cells were incubated with the vectors for 4 h, in the presence or absence of serum medium. The transfection media was then replaced with 1 mL of fresh complete culture media, and the cells were incubated sequentially until 24 h post transfection. Detection of expression of EGFP was carried out using an inverted fluorescent microscope with an attachment for fluorescent observation (OLYMPUS, ZX71, Japan) and the picture was captured using a 400× objective. Transfection experiments were performed in triplicates.
Results and Discussions
Formation of PLA-PEG-NPs
Formation of DNA-PLA-PEG-NPs
Physicochemical Characterization of PLA-PEG-NPs and DNA-PLA-PEG-NPs
Protection of DNA-PLA-PEG-NPs Against DNase I
Stability of DNA-PLA-PEG-NPs in Plasma
In Vitro Release of DNA-PLA-PEG-NPs
The in vitro drug release property is one of the important characteristics of nanoparticles.
Integrity Investigation of the DNA Released from DNA-PLA-PEG-NPs
Biological activity of DNA released from DNA-PLA-PEG-NPs (n = 3)
Untreated DNA/lipofectamine complex
Released DNA/lipofectamine complex
Transfection efficiency (%)
51.01 ± 2.43
49.36 ± 2.83
46.95 ± 1.68
43.41 ± 2.36*
Evaluation of the Cytotoxicity of DNA-PLA-PEG-NPs
In Vitro Transfection Studies
In the present work, cationic biodegradable nanoparticles based on PEGylated PLA copolymers was formulated using the mild and simple performed nanoprecipitation technique. Report gene, pEGFP was adsorbed onto the surface of the cationic PLA-PEG-NPs through electrostatic interactions, leading to the gene loaded nanoparticles (DNA-PLA-PEG-NPs). The obtained DNA-PLA-PEG-NPs with high binding efficiency (95.36%), positive surface charge (+16.8 mV) and small particle size (128.9 nm) showed sustained-release of DNA in vitro within 20 days. Adsorption and release from DNA-PLA-PEG-NPs did not alter structural and functional integrity of plasmid DNA. Moreover, DNA adsorbed onto DNA-PLA-PEG-NPs was efficiently protected from nuclease degradation and could remain relatively stable in plasma. It was less toxic than commercial Lipofectamine 2000 and safer to Hela cell. And it could successfully transfer plasmid EGFP into Hela cells, what’s more, the transfection activity was not diminished dramatically by the serum in transfection medium which surpassed the commercial cationic liposome based reagent, Lipofectamine 2000. It could be anticipated that the established DNA-PLA-PEG-NPs in current study was a promising nonviral gene delivery system used in gene therapy. It has a potential to alleviate the acute toxicity effect of other vectors and prolong the circulation time in vivo. Our further study will focus on the optimization of the formulation for higher transfection efficiency and the in vivo performance of the DNA-PLA-PEG-NPs as gene delivery system will also be conducted.
The Project was Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.
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