Design of an injectable multi-functional hydrogel as mini-invasive delivery carrier for the pH-controlled release of human lactoferrin in the treatment of chronic skin wounds

Chronic skin wounds represent one of the leading concerns faced by the healthcare systems creating a burden on the economy and society. The major cause hindering their successful regeneration is the presence of a persistent inflammation state and bacterial invasion. During the last decades, many different types of wound dressings have been used in the treatment/regeneration process. This work aims to develop an innovative injectable multi-stimuli responsive hydrogel to enhance the regeneration of damaged tissues through the smart release of an innate immune system protein, i.e., human lactoferrin (hLF). An amphiphilic poly(ether-urethane) (PEU) was first synthesized by reacting Poloxamer® 407 macrodiol, 1,6-hexamethylene diisocyanate and 1,4-cyclohexane dimethanol and then, functionalized through the green plasma treatment introduce alkaline pH-responsive carboxylic acid groups. The obtained thermo- and alkaline-pH responsive polymer was then chemically characterized through infrared spectroscopy (IR) and Size Exclusion Chromatography, confirming the successful synthesis of a high molecular weight PEU (Mw=57.4 vs. 10.4 kDa) through the appearance of the characteristic IR peaks of urethane bonds and the absence of evidence of plasma-induced degradation. Exposed functional -COOH groups were quantified to be 1018 units/g of polymer by Toluidine Blue O assay. Hydrogels were then prepared at 15% w/V polymeric concentration and loaded with hLF (1 mg/mL) in the sol state (i.e., at 4°C). Protein encapsulation did not affect hydrogel thermo-responsiveness and gelation kinetics as verified through rheology, with a gelation temperature of 16.7 and 16.4 °C for hLF-unloaded and loaded formulations. Subsequently, hydrogel responsiveness to alkaline pHs was assessed through in vitro hLF release tests against buffers at 5, 7, 9, and 11 pH values. The quantification of released hLF demonstrated the hydrogel suitability to achieve a pH sensitive release mechanism (e.g., released hLF turned out to be 3.3%±0.4%, 4.6±0.2%, 5.9%±0.7% and 9.0%±0.9% after 6h of incubation at pH 5,7, 9 and 11, respectively. Afterwards, the preservation of its anti-inflammatory and antibacterial properties was in vitro assessed by exploiting human dermal fibroblast (hDF) 2D culture assays. First, hLF-treated hDFs showed cell metabolic viability percentages higher than the control after 5 days of culture, thus suggesting hLF capability to promote cell proliferation. Moreover, such evidences were further confirmed through the cell cycle assay, giving higher cell percentages in the proliferative phase for hLF-treated cells compared to control cells. Afterwards, the in vitro investigated hLF antimicrobial properties evidenced weak bactericidal activity against Escherichia coli and Staphylococcus aureus, but considerable bacteriostatic properties, thus suggesting their potential in combination with stronger antimicrobial molecules. On the other hand, hLF anti-inflammatory and pro-regenerative properties were tested on inflammation-induced hDFs. The DNA quantification assay confirmed hLF capability to promote cell proliferation under an inflammatory state with DNA amounts quantified to be 24.50 ±6.18 μg/mL and 18.61± 3.27 μg/mL for hDFs untreated and treated with hLF for 5 days. Lastly, hLF capability to reduce inflammation was assessed through the evaluation of gene expression using primers specific for TNF-α, IL-6,IL-1β, and IL10 cytokines and Collagen type III and GAPDH. [1] Laurano et al. 2021 Bioactive Materials, 6, 3013-3024