Hydrindantin

The following enzymes and organic/inorganic
chemicals were purchased from Sigma-Aldrich: premade ninhydrin solution
(consisting of 2% ninhydrin, hydrindantin in dimethyl sulfoxide (DMSO)
and lithium acetate buffer pH 5.2), Bradford reagent (consisting of
Coomassie Brilliant Blue G-250 dye, methanol, and phosphoric acid),
α-naphthol, sodium hydroxide (NaOH), 200 proof ethanol (EtOH),
bromine (Br₂; ACS reagent), urea. Hydrochloric acid (HCl),
manufactured by EMD Millipore were purchased from Fisher Scientific.
Additionally, polyethylene film or PEF (plastic wrap) was purchased
from Price Chopper. Water used in all of the experiments was ultrapure
(18.2 MΩ·cm) water from PURELAB Flex, an ELGA water purification
system.

HR-ADM samples were air dried overnight, weighed (21–25 mg), and rinsed in 0.9% saline solution. Samples (n = 3 donors) were then enzymatically digested (6 hours, 37°C water bath) in a collagenase type 1A (6.65 U/mL final enzyme solution; Sigma, St. Louis, Mo.) and thermolysin (15 U/mL final enzyme solution; Sigma, St. Louis, Mo.) solution in tricine buffer (pH 7.5). The filtered extract was mixed with ninhydrin (0.016 g/1 mL solution)–hydrindantin (0.0024 g/1 mL solution) (Sigma, St. Louis, Mo.) in ethylene glycol monoethyl solution and 4 N sodium acetate buffer (pH 5.5) that reacts with the released amino acids, producing a deep purple color proportional to the amount of peptides released. The standard curve was established with l-leucine (stock solution 2.0 mg/mL; Sigma, St. Louis, Mo.) and sample absorbances were read at 570 nm. The controls were crosslinked^{28 (link)} and denatured dermis samples. Unprocessed dermal tissue was crosslinked (16 hours, room temperature) with 0.025% glutaraldehyde (Sigma, St. Louis, Mo.) solution, followed by a 2-hour rinse step to remove residual glutaraldehyde. The denatured condition (representing harsh chemical processing) was prepared by crosslinking as above and then boiling (at 100°C) the rinsed samples for 5 minutes. These samples were digested as stated above, reacted, and read at 570 nm.

Low-molecular-weight chitosan (molecular weight: 20 000–50 000 Da, degree of deacetylation ≥ 90%; Zhejiang Aoxing Biotechnology Co. Ltd., China) and high-molecular-weight chitosan (molecular weight: 310 000–375 000 Da, degree of deacetylation ≥ 75.0%; Sigma-Aldrich, Ireland) were used as the matrix material of nanoparticles with glacial acetic acid as the solvent of chitosan (Merck, Germany). Tween 80 (Fisher Scientific, UK), span 80 (Merck, Germany) and ethanol absolute for analysis (Merck, Germany) were used as additives. Lithium acetate anhydrous (ACROS Organics™, USA), ninhydrin and hydrindantin (Sigma-Aldrich, USA) were used in quantification assay of chitosan. Lactose monohydrate (Sorbolac 400; Meggle, Germany) was used as the microparticulate carrier with polyethylene glycol 3000 (PEG3000; Merck, Germany) as the stabilizer.