This study explored strategies for increasing the encapsulation efficiency, modulating the release kinetics, and improving the transfection ability of DNA loaded into PLGA particles. Two fabrication modifications were utilized to accomplish these goals: (1) chemical conjugation of poly-L-lysine to the termini of PLGA polymer to provide more favorable interactions between the polymer and DNA, and (2) the method of agitation (sonication or homogenization) in the fabrication process.
One of the benefits of sonication as an agitation source is the ability to fabricate particles that are both relatively small (submicron) and uniform. Unfortunately, sonication usually results in considerable DNA loss during encapsulation (> 80%, ) and a substantial burst release within the first 24 hours. These unfavorable characteristics were overcome by the incorporation of PLGA-PLL into particles during fabrication. Blending five weight percent of PLGA-PLL/PLGA polymer resulted in a substantial reduction in plasmid loss. As more PLGA-PLL was incorporated into the particles, a corresponding reduction in DNA loss during encapsulation was observed. In addition, the magnitude of the initial burst release of DNA within the first 24 hours from the particles was controlled by addition of PLGA-PLL. While lower blends of PLGA-PLL/PLGA (5% and 10%, wt/wt) maintained a burst similar to PLGA alone (>40% within the first 24 hrs), higher blends of PLGA-PLL/PLGA (25%, 50%, and 100%; wt/wt) drastically reduced the burst release (33±4%, 22±9%, and 11±4% respectively). It is hypothesized that the diffusion of DNA from the emulsified droplets during particle hardening is slowed by electrostatic interactions with positively charged PLL. At eight weeks of incubation, a second release was observed. This was most likely due to the degradation of polymer by hydrolysis. The presence of primary amines in PLGA-PLL may also accelerate hydrolysis, and subsequently, DNA release in particles. This may explain the increase in release rates observed in particles as the blend of PLGA-PLL increases from 25% to 100% despite similar DNA loadings. Both the increase in loading and the delay in release kinetics correlated with the surface charge of the particles: particles became more positively charged as increasing amounts of PLGA-PLL were incorporated. Interestingly, particles incorporating as little as 25% PLGA-PLL/PLGA (wt/wt) achieved close to maximal DNA loading.
Homogenization is another commonly used method of agitation for fabricating DNA-loaded particles [8
]. In this approach, emulsions were created through high speed mixing (24,000 RPM) of DNA/polymer in the presence of a surfactant. In comparison with sonication, producing submicron particles by homogenization required a higher concentration of surfactant (5% vs. 1%) and the use of a co-solvent. Even with these changes, on average, particles fabricated with homogenization were slightly larger than those produced by sonication (~450 nm vs. ~280 nm). One advantage of this technique was that considerably less DNA was left unencapsulated during fabrication (~50%). The differences in unencapsulated DNA between the two methods may be explained by (1) the slight increase in particle size (which yields better loading), and (2) the manner in which DNA associates within the emulsion during particle formation by the specific energy sources. Another possible contributing factor in enhanced DNA encapsulation efficiency and more thorough distribution in particles was the presence of the co-solvent, TFE, which resulted in faster hardening and hindered DNA diffusion out of the particle during fabrication by homogenization. It was also demonstrated that when a relatively small amount of PLGA-PLL/PLGA (10%, wt/wt) was incorporated into the particles fabricated by homogenization, the loss of DNA during loading was completely eliminated. Thus, independent of agitation method, incorporation of PLGA-PLL within the formulation results in a higher loading of plamsid DNA within the particles. Both agitation methods resulted in 30–50% of total DNA in the supercoiled isoform after encapsulation.
Particles fabricated by homogenization had slower, more linear, DNA-release profiles than particles fabricated by sonication. There are two possible reasons behind the differences in the release profiles. The first is differences in area-to-volume ratio. A more speculative explanation may be differences in the distribution of DNA within the particle. If homogenization indeed results in a more uniform distribution of DNA throughout the particles, then it would take longer for the dispersed molecules to diffuse out of the particles. Particles with 10% PLGA-PLL/PLGA (wt/wt) incorporated demonstrated the same relative DNA-release profile as particles without PLGA-PLL. Increases above 10% were not explored because maximum loading was achieved with this fabrication method.
The functionality of particles were examined by transfecting COS cells in tissue culture. In these studies it was observed that the particles fabricated using sonication were able to transfect mammalian cells; the extent of transfection depended on the blend of PLGA-PLL/PLGA (wt/wt) incorporated into the particles. Transfection occurred even with changes in DNA isoform during fabrication. Further studies are needed to address the reliability of DNA incorporated in particles. The differences in transfection efficiency between the sonicated formulations can be explained by the release kinetics and cell culture model. The particles were incubated on the cells for 24 hours, removed, rinsed, and replaced with new medium. Two days later, luciferase expression was evaluated; transgene expression correlated with DNA released between 1–3 days (). In the formulations where less PLGA-PLL was incorporated (which resulted in a larger burst), the majority of DNA may have been released prior to internalization into the cells (< 1 day). Poor transfection with lower weight percentages of PLGA-PLL/PLGA may also be a result of the magnitude of negative charge on the surface of the particles. illustrates that 25% PLGA-PLL/PLGA (wt/wt) and greater a reduction in negative surface charge, which results in much more favorable electrostatic interactions with the cell membrane, facilitating internalization into the cells. Cationic charge on the surface of microparticles has demonstrated increased uptake of particles in both monocytes and dendritic cells [18
]. Particles fabricated with homogenization did not transfect cells under the conditions investigated. One explanation for their failure to transfect cells may be that an inadequate amount of DNA was delivered to the cell to generate transfection after three days. In addition, barriers of internalization such as surface charge and particle size may also have been contributing factors.
The use of cationic polymers to condense plasmid DNA for transfection of mammalian cells has been well studied. More recently, this interaction has been explored to enhance the transfection ability of biodegradable particles. For example, Capan et al. have encapsulated DNA previously complexed with PLL into PLGA microparticles and demonstrated the ability to control release of these complexes by using different molecular weight polymers or changing the surfactant concentration [19
]. The cationic surfactant cetylrimethylammonium bromide (CTAB) was shown effective for absorbing DNA onto the surface microparticles [12
]. In this strategy, the concentration of CTAB used during fabrication dictates both the loading and the amount of DNA released within the first 24 hours. Similarly, PLL has been able to change the surface charge when used as a emulsifier in the fabrication PLGA particles [22
]. Plasmid DNA absorbed on PLL coated polystyrene nanoparticles has been shown to transfect dendritc cells [24
]. Others have utilized surface adsorption of DNA by conjugating another common transfection cationic polymer, polyethyleneimine (PEI), to PLGA [13
]. In these studies, the release profile was dictated by the molecular weight of PEI and whether it was branched or linear in structure [25
]. A concern with coating the surface of particles with cationic polymers is cellular toxicity. At high coating concentrations of CTAB or PEI, membrane toxicity was observed in cells [26
The grafting of poly(L-lysine) to PLGA has been previously reported as a gene delivery carrier [27
]. In this approach, the modified polymer complexed with plasmid DNA was used to form nano-sized micelles. The studies presented in this paper differ in that the particles formed were a depot from which DNA is released over a short and long time scale.
Utilizing the same electrostatic interactions, but in a different molecular format, this study presents a new platform for controlled release of DNA. Unlike other strategies, which are limited by the manufacturer’s selection of molecular weight polymers, controlling the magnitude of the burst release was accomplished relatively easily, by simply changing the amount of PLGA-PLL incorporated into the particles. An additional benefit of this method is that incorporation of PLGA-PLL dramatically improves the loading efficiency of plasmid DNA. These particles may prove valuable in providing a tunable range of release profiles to explore that may be specific for a given application. For example, a robust burst release may be necessary when trying to initiate an immune response, whereas slower, more persistent release may be desirable for a plasmid encoding a growth factor for tissue regeneration. Alternatively, the superposition of both release profiles provides different therapeutic delivery ranges which may ultimately lead to the desired response. Clinically, it is envisioned that these particles would be administered orally, intranasally, or injected at a local tissue site. To date, the modified polymer has not been tested with other hydrophilic anionic agents for delivery, but it is anticipated that such agents will exhibit similar characteristics (enhanced loading and control over release profile) to what was observed with DNA.