Influence of Mucoadhesive Polymers on Physicochemical Features and Biocompatibility of Collagen Wafers

Abstract

The aim of this study was to develop and characterize some freeze-dried wafers based on collagen and two mucoadhesive polymers, namely, hydroxypropyl methylcellulose (HPMC) and Carbomer 940 (CBM). The wafers were obtained by lyophilization of the corresponding hydrogels, which were evaluated by circular dichroism in order to investigate mucoadhesive polymers' influence on collagen's secondary structure. The obtained freeze-dried wafers were characterized by FT-IR spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), contact angle measurements, and water uptake capacity. Furthermore, biocompatibility assessment was performed by evaluating the impact of freeze-dried wafer extracts on cell viability, morphology, and migration capacity. Circular dichroism showed more significant changes in the secondary structure of collagen associated with the addition of Carbomer 940. The FT-IR spectra displayed specific peaks for collagen and the two mucoadhesive polymers. SEM images illustrated a microporous structure for both collagen and Carbomer 940, while HPMC displayed a more sheet-like structure. The addition of HPMC increased the thermal stability of collagen, while Carbomer 940 had a negative impact on the samples' thermal stability. Contact angle measurements and water uptake capacity showed good hydrophilicity of the wafers. Except for CBM 100%, all samples supported the viability of human fibroblasts and did not have any inhibitory effect on cell migration capacity, demonstrating good biocompatibility, which is an essential attribute in developing drug delivery supports intended for mucosal applications.

Keywords: biocompatibility; carbomer; collagen; hydroxypropyl methylcellulose; wafer.

1

Freeze-drying diagram.

2

CD spectra of (a) COL-HPMC hydrogels and (b) COL-CBM hydrogels.

3

FT-IR spectra of COL 100%, HPMC 100%, and COL-HPMC samples.

4

FT-IR spectra of COL 100%, CBM 100%, and COL-CBM samples.

5

Overlay of FT-IR spectra monitored in the 1770–1580 cm^–1 range (Amide I) for (a) COL-HPMC and (b) COL-CBM samples.

6

SEM images (100× magnification) of the prepared freeze-dried wafers: (a) COL-HPMC1, (b) COL-HPMC2, (c) COL-HPMC3, (d) COL 100%, (e) HPMC 100%, (f) CBM 100%, (g) COL-CBM1, (h) COL-CBM2, and (i) COL-CBM3.

7

TGA thermograms of (a) COL-HPMC and (b) COL-CBM samples.

8

Comparative plot of mean contact angle values obtained for freeze-dried wafers.

9

Water uptake capacity (g/g) of collagen/HPMC-based wafers throughout the 72 h test interval.

10

Water uptake capacity (g/g) of collagen/CBM-based wafers throughout the 72 h test interval.

11

XTT assay showing the viability of human adult fibroblasts after 24, 48, and 72 h of incubation in the presence of freeze-dried wafer extracts (*p < 0.05, **p < 0.01, ***p < 0.001 versus control at each time point; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus COL at each time point).

12

In vitro scratch test showing the impact of collagen-based freeze-dried wafers on fibroblast motility. Upper panel: phase-contrast microscopy showing the scratched area at 0 and 16 h later (magnification 5×); lower panel: the quantification of covered area as percentage of the initial scratched area where positive and negative controls are serum and serum-free medium (*p < 0.05, **p < 0.01, ***p < 0.001 versus the positive control; ^## p < 0.01 versus COL).

13

Images of fluorescence-based staining of the actin cytoskeleton (green fluorescence) and nucleus (blue). For evaluation of cell density, images are shown in the upper panels of each time point, with lower magnification (bar = 200 μm), while the cytoskeleton organization and F-actin stress fibers are shown in the lower panels with higher magnification (bar = 50 μm).

14

Time-dependent assessment of chemotactic migration of human adult fibroblasts. The lower panel illustrates the migration index at 3 h versus 6 h.