Development of antibiotic and debriding enzyme-loaded PLGA microspheres entrapped in PVA-gelatin hydrogel for complete wound management

Figures & data

Table I. Preliminary studies for selection of gelatin-PVA blend H1, H2, and H3 with 10/90, 20/80, 30/70 weight ratios, respectively.

S. No	Studies	Methodology	Results
1.	Infra-red spectroscopy	FTIR of PVA, gelatin and PVA-gelatin Hydrogel were taken (Schimadzu FTIR-8400S, Tokyo, Japan)	FTIR of hydrogel showed the peak due to C = O stretching of the gelatin at 1680 cm^–1 shifted to 1760 cm^–1 in the PVA-gelatin blend. No peaks were observed at 1680 cm^–1 indicating esterification of all the free carboxylic groups of gelatin in the blend.
2.	Scanning Electron Microscopy	The surface morphology was investigated by scanning electron microscopy (Leo, VP-435, Cambridge, UK).	Pure PVA hydrogel showed porous morphology. PVA-gelatin blend showed lamellar appearance and an apparent preferential orientation in ratio of 20/80 (H2), but on further increase it became less ordered.
3.	Water vapor transmission rate (WVTR)	WVTR was measured using the upright wet cup method as described by ASTM E96.	The PVA-gelatin films showed “wet” WVTR characteristics that are close to that of intact skin. H1 and H2 had almost similar transpiration rates at 1308.59 g/m^2/24 h and 1386.71 g/m^2/24 h, respectively. H3 transmitted water vapor significantly faster at 1445.31 g/m^2/24 h.
4.	Hemocompatibility	The hemocompatibility tests were carried out broadly on the basis of ASTM standard.	H2 was (20/80) found to be highly hemocompatible with % hemolysis less than 5% as compared to other blend ratios.
5.	Water uptake capacity	A conventional gravimetric procedure was followed	Swelling ratio for PVA-gelatin blends H1, H2, and H3 were found to be 1.24 ± 0.23, 1.28 ± 0.33, and 1.32 ± 0.38, respectively.
6.	Diffusion coefficient	A diaphragm cell was used to measure the diffusion coefficient	The diffusion coefficient of drugs (GM and STP) through the membrane was found to be 1.42 × 10 ^− 5 cm^2/s and 1.35 × 10 ^− 5 cm^2/s, respectively, in the case of H2.

Figure 1. Scanning electron micrographs of microspheres; a) STP-loaded PLGA microspheres; b) GM-loaded PLGA microspheres; c) drug-loaded PLGA microspheres on day 12 at high magnification (STP); d) drug-loaded PLGA microspheres on day 12 at high magnification (GM); e) drug-loaded MS after 21 days at high magnification (STP); f) drug-loaded MS after 21 days at high magnification (GM).

Figure 2. 3D surface curve for the effect of selected variables for desirability in terms of maximum entrapment and particle size in range.

Figure 3. Multiphase hydrogel: (a) hydrogel film; (b) photomicrograph of hydrogel film; (c) SEM of microsphere-loaded PVA-gelatin hydrogel.

Figure 4. Cumulative release of gentamicin (GM) from PLGA microspheres (▪) and serratiopeptidase (STP) from PLGA microspheres (+); GM incorporated into the PVA-gelatin hydrogel matrix GPH (♦); STP incorporated into the PVA-gelatin hydrogel matrix SPH (-); pure STP and GM loaded into hydrogels (▴) and (×), respectively (results are given as M ± SD (p < 0.05), n = 3, where n is the number of repeat experiments conducted).

Table II. Release behavior of drugs in phosphate buffer (pH 7.4) from hydrogel formulations.

Formulation code	First order		Higuchi		Ritger-Peppas
	k	R^2	k	R^2	n	R^2
GPH	2.252	0.978	17.74	0.984	0.446	0.982
SPH	2.203	0.957	17.99	0.967	0.431	0.960
GH	0.057	0.881	10.62	0.991	0.282	0.979
SH	0.057	0.907	10.47	0.996	0.271	0.981

*K, Release rate constant; R², coefficient of determination; n, release exponent.

Table III. Effect of formulation and standard preparation on % wound contraction and epithelialization period on excision wound model in mice.

Groups	Post-Wounding Days
	4	8	12	13	14	15	16	17	18	E.P
I-H2 (control) (without any drug)	53.34 ± 0.62	75.03 ± 0.54	83.34 ± 0.69	89.41 ± 0.85	94.12 ± 0.62	95.81 ± 0.84	96.08 ± 0.83	98.87 ± 0.65	99.67 ± 0.47	18
II-PDH hydrogel containing pure drug STP and GM)	55.39 ± 0.32	86.96 ± 0.45	93.68 ± 0.43	96.32 ± 0.65	97.9 ± 0.61	99.4 ± 0.73	100 ± 0.64	–	–	16
III-DPMH Multi-phase hydrogel containing PLGA microspheres of STP and GM)	65.89 ± 0.42	89.01 ± 0.49	100 ± 0.92	–	–	–	–	–	–	12

n = 6 albino mice per group;, tabular value represents Mean ± S.D; E.P-Epithelialization Period.

Figure 5. Photographs of wounds 4, 8, and 12 days after surgery illustrating the changes in wound contraction and epithelialization with TS of mice skin on the twelfth day in all groups stained with hematoxylin and eosin at 40 x (A) control; (B) serratiopeptidase and gentamicin in hydrogel; (C) serratiopeptidase and gentamicin PLGA microspheres in hydrogel.