3.1 γ-PGA characterization
Previous to use the γ-PGA to prepare the nanofibers mats, the polymer was characterized by RMN-1H spectra to assure its purity. The RMN-1H spectra show the chemicals displacements that correspond to γ-PGA (Fig. 1), the proton for α-CH at 4.2 ppm, and β-CH2 has two signals at 2.16, 1.9 ppm, and γ-CH2 at 2.37 ppm 15.
3.2 Presence of γ-PGA in the PVA nanofibers
The presence of γ-PGA in nanofiber mats was determinate by FT-IR spectra (Fig. 2). In the PVA case, the existence of O-H stretching in the region at 3298 cm-1 and the methyl groups at 2940 y 2898 cm-1, they overlap with the γ-PGA presence and the acetyl group C-O characteristic to this polymer at 1090 cm-1 16. The increase of the γ-PGA can observe the corresponding band for amine group N-H stretching at 1590 cm-1, and 1640 cm-1 for PVA/g-PGA 5 y PVA/g-PGA 10 , In 3307 cm-1 found the band attributed to The OH stretching mode. The C-O stretching vibration at 1222 cm-1 and the corresponding C-N stretching to 1132 cm-1, the peak to corresponding to the aliphatic side for γ-PGA are at 3073 and 2932 cm-1 15. The molecular weight of γ-PGA obtains through to the biosynthesis with Bacillus licheniformis ATTC9945a strain shows an Mw=243,023 g/mol (Fig S1).
3.3 Morphology
Highly hydrolyzed PVA is difficult to electrospun due to its high surface tension. Previously, we have overcome the problem by blending, partially hydrolyzed, and low molecular weight PVA with highly hydrolyzed and larger molecular weight PVA. We think that the first polymer provides better mechanical properties and stability to the solvent. In contrast, the second polymer improves the electrospinnability of the blended polymer solution12a.
The morphology and diameter of the electrospun nanofibers of PVA were analyzed before and after the crosslink, CGA encapsulation processes (Fig 2S). SEM morphological characterization of nanofibers shows that using a 10 % solution of PVA can form fibers easily, at the nanometric size order. The obtained nanofiber had an average diameter of 277 nm, and they are flawless and smooth, verifying that the elaboration conditions were optimum. Once, the nanofiber mats were submitted to crosslink by a glutaraldehyde vapor process, and the fiber diameter was increased until 298 nm. However, this increase in the fiber diameter was not appreciative, conserving the particular large surface area of the nanofibers (Fig. 3).
The inclusion of γ-PGA in the PVA solution has a critical effect on nanofibers average diameter in both regular and after crosslinked. The addition of γ-PGA at 5 reduces the average size diameter of the nanofibers at 163 nm and, after crosslinked them, suffer an increase up to 221 nm (Fig. S3). Once, the nanofibers were crosslinked shows an entanglement. Both the fiber diameter increment and entanglement could be related to the absorption of glutaraldehyde vapor inside the nanofiber structure. Nevertheless, the diameter increment was not apparent in comparison to the diameter without any crosslinking treatment. Hence, we can conclude that glutaraldehyde vapor treatment is an effective method to crosslink PVA blended with other polymers.
Figure 3 shows the SEM micrograph for nanofibers PVA /γ-PGA 10. These nanofibers have a size average of 119 nm. The fibers have a homogenous surface, and they are defect-free. The rise of γ-PGA into the PVA solution had a notorious effect on the average diameter of the electrospun nanofibers. It was observed as the concentration of γ-PGA increase; there was a reduction in the average diameter. These reductions could be attributed to the strong hydrogen bonding among OH groups of PVA and NH2 groups of γ-PGA. However, the charge density of the polymer solution is a crucial factor in obtaining defect-free and thinner diameter fibers17. γ-PGA is a natural polyelectrolyte, and as such, its density charge is high. So, increasing the concentration of γ-PGA in the PVA solution is expected to obtain thinner fiber. As well, because of the increase in the conductivity of the polymer solution18.
After crosslinked PVA /γ-PGA 10 with glutaraldehyde, again, there is an increase of average diameter up to 148 nm, which confirms the bonding among polymer chains. Likely, the absorption of glutaraldehyde vapors during the polymer electrospun.
3.4 In vitro release of ACG from fibers of PVA and PVA /γ-PGA.
In this work, the cumulative release in vitro of GCA nanofibers phosphate buffer, pH 4.8, was studied, and the percentage of the accumulative GCA release against time was plotted. As it is shown for many other systems, typically consisted of biphasic release profiles, beginning with a burst release stage and then follow by sustained release phase in the time (Fig. 4). The presence of the burst phase could be due to the release of free GCA molecules and GCA incorporated into the network nanofibers through non-inclusion interactions. Moreover, the release of CGA inside of the nanofibers occurs through slower dissociation, and diffusion processes should be related to the sustained release phase. As can see in Fig. 4, after the one h, the release of CGA from PVA, PVA /γ-PGA 5, and PVA /γ-PGA 10 electrospun mats reached 6, 28, and 66%, respectively. Similarly, the maximal CGA diffused out to the three systems' buffer solution is achieved after 72 h with values of 36, 65, and 82 %, respectively. The presence of the γ-PGA has a positive effect by facilitating the CGA release from the polymer spun mats. Such influence has explained by the existence of repulsive negative charges, which at pH 4.8 are located in the carboxylic acid groups of both γ-PGA and CGA18b. A second possibility could be attributed to strong hydrogen bonding among inter and intra-chain OH groups of PVA that limit the diffusion of small molecules, as is the CGA. On the other hand, γ-PGA is a linear natural polyelectrolyte that acts as porogenic material. Wich, likely form small micrometric pores by increasing the surface area to facilitate the CGA release from the fiber mats (Table S1)19.
Drug release kinetics and mechanism is a fundamental aspect to describe the main properties and characteristics of a carrier system. Several kinetic models have applied to study drug release from different systems. Usually, the release process of drug molecules from electrospun fibers may be described with zero-order, pseudo-first-order kinetic, or pseudo-second-order kinetic equations20.
Herein, the in vitro release of CGA from the electrospun PVA and PVA/γ-PGA) nanofiber mats was fitted with pseudo-first kinetics order21.
Where K1 Is the rate constant obtained by the pseudo-first-order equation, t is the time, qe, and qt are amount release at the equilibrium time and amount release at any time, respectively. The k1 value can be obtained from the slope of the linear plot.
It was found that with the above kinetic model is appropriate for describing the kinetic release process of CGA from the electrospun fiber mats. Figure 5 shows the plots of ln(qe-qt) vs. t0.65 for the release of CGA at pH 4.8 and 37 ⁰C conditions. As can see in all the graphics, a clean straight line was obtained.
Table 1 shows the calculated values of K1, been 0.29, 0.33, and 0.11 for PVA/γ-PGA 10, PVA/γ-PGA 5, respectively. In the three cases, the lineal correlation coefficient (R2) is very high and quite similar.
Table 1. Rates constants (K1) and correlation coefficient R2 of PVA, PVA/γ-PGA 5, and PVA/γ-PGA 10.
Sample
|
K1
(h-1)
|
R2
Pseudo-first-order
|
PVA/γ-PGA 10
|
0.29
|
0.96
|
PVA/γ-PGA 5
|
0.33
|
0.99
|
PVA
|
0.11
|
0.98
|
From the above information values of the constant velocity K1, several observations can be drawn. The lowest value of this constant is for the PVA fiber mats, which could indicate that the release of CGA takes a longer time to reach saturation equilibrium, showing the burst effect. Meanwhile, the value of K1 for the PVA/γ-PGA 10 and PVA/γ-PGA 5 fiber mats are very similar, meaning that the presence of γ-PGA influences the release of CGA by a diffusion effect and both cases reach the saturation in less time than of PVA fiber mats.
Alternatively, the Peppas 22 and Weibull23 models were applied to obtain more information regarding the type of diffusion mechanism.
Where: qt and qT represent the concentration of ACG released at any time and the total amount of this compound loaded in the fibers, t is the release time, k represents a constant kinetic, n is the exponent that shows the mechanism of the liberation (equation 2), and finally α corresponds to a scale factor, and β is a form factor in equation (3). The exponent n below 0.5 in the Peppas model represents a drug control release driven by a diffusion effect, and for the exponent β lower than 0.75 in the Wiebull model (Fig. 6).
Figure 6 shows the plots of qt/qT vs. t for the release of CGA in the fiber mats at pH 4.8 for Peppas and Weibull models. The simulation result indicates that PVA, PVA/γ-PGA 10, and PVA/γ-PGA 5 have a good fitting to the drug control release of Peppas model with a fair regression coefficient (R2), being the best for the PVA system and slightly worst for the PVA/γ-PGA system. Otherwise occurs with the Weibull model, it found that simulation results of the PVA system unfit, but an excellent fitting was observed for PVA/γ-PGA 10 and suitable for PVA/γ-PGA 5. Again these data indicate a drug release mechanism to be predominantly diffusion-controlled with exponent n ranging from 0.19 to 0.2120b, 24, where the investigated formulation and processing variables did not alter the drug release mechanism (Table 2). The unfit data of the release of CGA from the PVA electrospun fiber mats in the Weibull model can be attributed to the percentage of the released drug, which is lower than 50 %. Introducing a small modification of 0.5 for 1 in the equation of this model shows an excellent fit to the experimental data (R2, 99). Therefore, this model limits its application in systems with a drug release percentage of less than 50 % (Fig. S4).
Table 2. Kinetics models parameters of CGA released from loaded PVA, PVA/γ-PGA 5, and PVA/γ-PGA 10 fiber mats.
Sample
|
Peppas Model
|
Weibull Model
|
Ƞ
|
K
|
R2
|
α
|
β
|
R2
|
PVA/γ-PGA 10
|
0.21±0.04
|
0.28±0.05
|
0.84
|
0.22±0.05
|
0.42±0.05
|
0.92
|
PVA/γ-PGA 5
|
0.19±0.04
|
0.24±0.05
|
0.78
|
0.23±0.06
|
0.30±0.06
|
0.83
|
PVA
|
0.21±0.02
|
0.13 ± 0.01
|
0.92
|
-
|
-
|
-
|