The study described here was designed to determine the effect of certain processing parameters on electrospun fiber diameter distribution, PEI-HA release kinetics, and transfection efficiencies of pDNA released from electrospun coaxial fiber mesh scaffolds incorporating pDNA and PEI-HA, a non-viral gene delivery vector. Coaxial electrospinning has thus far not been employed for delivery of pDNA, and factors influencing the formation of coaxial fiber meshes and their release properties are largely unknown. The experimental plan was formulated with the goal of establishing parameters that allowed for the formation of coaxial electrospun fiber meshes and determining if the examined values of these parameters could dictate the release kinetics of pDNA and r-PEI-HA, as well as the associated transfection efficiencies. Just as the process of electrospinning is dependent on the interaction of multiple factors, including the dielectric properties of the solvents used [30
], flow rates of polymer solutions during extrusion [32
], the electric potential and quantity of charge circulating through the electrospinning circuit [33
], and the distance between the needle and collecting plate [34
]; the coaxial electrospinning process has a similar set of complex governing interactions.
In the experimental design implemented here, pDNA was incorporated within the core polymer (PEG) solution, whereas r-PEI-HA was pulverized and dissolved within the sheath polymer (PCL) solution. We had previously described experiments where a set of coaxial electrospun fiber mesh scaffolds were fabricated based on a full factorial design using parameters similar to those described in this study [29
]. These common parameters included sheath (PCL) Conc., core (PEG) Conc., and (PEG) MW. It was observed that the range of the parameters tested had to be limited significantly to allow the coaxial electrospinning of polymer solutions incorporating the cationic gene delivery vector and anionic pDNA, thus limiting the versatility of the coaxially electrospun groups. Furthermore, despite the formation of a stable Taylor cone during fabrication of the fiber meshes for all the formulations, the coaxial fibers showed a greater distribution of fiber diameters, as shown in and discussed in Section 3.1. Previously, DNA has been incorporated into uniaxial electrospun fibers by Luu et al. [11
], and the fibers obtained had a significant variation in fiber diameters. Both these observations suggest that the inclusion of highly charged moieties, such as a cationic gene delivery vector and anionic pDNA, significantly affect the electrospinning properties of polymer jets.
The assessed parameters showed a similar effect of increasing sheath polymer concentration and core polymer molecular weight on fiber diameter, as was previously observed in the absence of the vector and plasmid. The average fiber diameter increased with the increase in sheath (PCL) Conc. and PEG MW. In addition, an increase in the concentration of PEG, pDNA and r-PEI-HA also caused a similar increase in the average fiber diameter. To determine if all fibers within the mesh were truly coaxial, we immersed them in PBS for 7 days. Analysis showed a significant number of fibers across various groups that completely dissolved in a period of 7 days, which suggested that these fibers were composed predominantly of PEG and were prevalent in various size ranges within different groups. However, there was also a significant and larger population of fibers that did not change in prevalence across subgroups of fiber diameters, suggesting that these fibers were indeed coaxial.
As one of the goals of this experiment, we attempted to characterize the release of incorporated r-PEI-HA from the sheath of coaxial fibers. The direct release of pDNA could not be monitored in this case, as r-PEI-HA significantly decreased binding of pDNA to dyes such as PicoGreen or ethidium bromide during complex formation. Hence, the release of r-PEI-HA was monitored via fluorescence. In the case of uniaxial electrospinning, Luu et al. [11
] observed that most of the burst release occurred at 2 hrs, after which pDNA release decreased precipitously. Similar to observations made by Luu et al., r-PEI-HA contained within the sheath fibers of the present study displayed a burst release within 24 hrs after immersion in PBS. However, in the fibers fabricated here, there was also a significant amount of r-PEI-HA released between days 2 to 10, ranging from 1.75 ± 0.39 to 6.30 ± 1.60% of theoretical loading. Only the loading concentration of pDNA (and subsequently that of r-PEI-HA, which was increased to maintain a constant N:P ratio among groups) appeared to significantly influence the release kinetics of r-PEI-HA. Some of the burst release observed here could be attributed to the dissolution of fibers made predominantly from PEG as described above.
The release kinetics were also significantly influenced by the location of PEI-HA and DNA within the coaxial fibers. The initial design of the experiments described here attempted to electrospin r-PEI-HA/pDNA complexes entirely within the core of the coaxial fibers by preassembling the complexes before mixing them with the core polymer solution. To accommodate for the solubility of PEI-HA/pDNA complexes, dextran (instead of PEG) was used as a core polymer and the optimal viscosity for spinning was attained at concentrations noted in the table in the supplemental section (Supplemental Table 1
). However, the core polymer containing the complexes showed negligible release of PEI-HA/pDNA complexes (Supplemental Figure 1
). Although we could not determine the cause behind the absence of release of PEI-HA/pDNA complexes, a possible reason could be that the interactions between the core and sheath polymer limited the incorporation of the PEI-HA/pDNA complexes within the coaxial fibers.
Some of the coaxial fiber mesh scaffold groups in the study where PEI-HA was incorporated within the polymer sheath and pDNA was incorporated within the fiber core showed greater than 100% cumulative release over the duration of the study. Although, during the fabrication of the scaffolds, the r-PEI-HA and pDNA were loaded such that the N:P ratio between them was constant at 7.5:1, it is feasible that the ratio at which r-PEI-HA and pDNA were released was not constant over the duration of the experiment. The r-PEI-HA/pDNA release values were calculated using a calibration curve generated from known concentrations of r-PEI-HA/pDNA in solution (constant N:P ratio of 7.5:1), with the assumption that there was no significant difference between the fluorescence of r-PEI-HA and r-PEI-HA/pDNA complexes of differing N:P ratios. However, it was found that calibration curves generated by measuring the fluorescence corresponding with known concentrations of r-PEI-HA alone (N:P ratio of 1:0) and r-PEI-HA/pDNA complexes (N:P ratio of 7.5:1) in solution were significantly different from each other, as illustrated in Supplemental Figure 2
. The inability of the detection method employed in the release study to differentiate between free r-PEI-HA and r-PEI-HA/pDNA complexes of different N:P ratios in solution was a limitation of the study and taken together with the differences in fluorescence for a given concentration of free r-PEI-HA versus r-PEI-HA/pDNA complexes, may account for the greater than 100% cumulative release observed for some groups.
The temporal differences between peaks of r-PEI-HA/pDNA complex release () and maximum EGFP expression in CRL 1764 cell lines () further suggests a potential variation in the ratios of r-PEI-HA and pDNA in the duration of the release. In general, EGFP expression could be more directly correlated to the release of pDNA rather than r-PEI-HA. However, pDNA release could not be directly detected in this experimental design and is a limitation associated with this study. However, transfection efficiencies with scaffolds containing r-PEI-HA were significantly higher than with those containing pDNA alone, suggesting that the presence of r-PEI-HA in the fibers did enhanced transfection, relative to pDNA alone in the fibers. Transfection efficiency seemed to be most influenced by core polymer parameters; PEG MW and concentration. Changing PEG concentration from low to high values decreased the observed transfection of cells. The decrease in transfection could be related to a potential decrease in the amount of pDNA released due to the increase in PEG concentration, as has been observed in other controlled release systems with proteins and peptides [35
]. The lower release observed in other studies has been attributed to an increase in the matrix density of the polymer holding the bioactive molecule of interest. However, pDNA release was not directly measured in the present study, so the effects of PEG Conc. on pDNA release are not known in the context of this study.
A similar phenomenon can be expected when the core polymer MW is increased. The increase in PEG MW, however, caused an increase in transfection, which is counter to expectations. However, this effect can be attributed to a number of factors. Increase in the MW of PEG has been reported to increase condensation of pDNA in the presence of NaCl, which is present within the core fiber [36
]. pDNA with a more compact structure would potentially have less retention within the coaxial fiber, which could lead to an increase in release and subsequently in transfection. Furthermore, any interaction between the r-PEI-HA and pDNA within the coaxial fiber may also influence release kinetics, due to a differential degree of condensation of pDNA after its interaction and complexation with r-PEI-HA.
We observed a significant effect of PEG Conc. and MW on transfection efficiencies at days 14 and 21. There were significant differences in transfection between groups up to day 21. However, after day 21 there were no significant differences in transfection between groups. It can be surmised that the observed transfection efficiency is dependent on the core polymer properties, i.e., molecular weight and concentration. Further changing the amount of r-PEI-HA loaded within the sheath fibers, which in turn would affect the N:P ratio at which complexes are formed, may give additional insight into the change in release kinetics of r-PEI-HA and the effect on pDNA transfection efficiency.
Lastly, cells directly seeded onto the fabricated coaxial fiber mesh scaffolds showed successful expression of EGFP, and this expression was significantly higher than that observed on meshes containing pDNA alone. The increase in EGFP expression in meshes containing both PEI-HA and pDNA suggests that, despite separating the pDNA and the gene delivery vector (r-PEI-HA) in different components of the coaxial fibers, the pDNA and r-PEI-HA are able to form complexes, be it inside or outside of the coaxial fibers, which are able to transfect cells with a greater efficiency than released pDNA alone.