**1. Introduction**

Osteoarthritis (OA) is a chronic inflammatory degenerative disease affecting millions of people worldwide. OA leads to cartilage deterioration, inflammation of the synovial membrane, and subchondral bone sclerosis due to abnormal bone remodeling caused by an overproduction of enzymes degrading the extracellular matrix [1–3]. This disease considerably reduces the quality of life for patients and is associated with pain, transient morning stiffness, and crepitus felt in a joint on moving it [4–6]. A large body of evidence

**Citation:** Valentino, A.; Conte, R.; De Luca, I.; Di Cristo, F.; Peluso, G.; Bosetti, M.; Calarco, A. Thermo-Responsive Gel Containing Hydroxytyrosol-Chitosan Nanoparticles (Hyt@tgel) Counteracts the Increase of Osteoarthritis Biomarkers in Human Chondrocytes. *Antioxidants* **2022**, *11*, 1210. https://doi.org/10.3390/ antiox11061210

Academic Editors: Li Liang and Hao Cheng

Received: 20 May 2022 Accepted: 19 June 2022 Published: 20 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

supports the involvement of inflammation and reactive oxygen species (ROS) production by chondrocytes in OA cartilage [7–9].

To date, numerous pharmacological and non-pharmacological therapies have been developed for the management of OA. However, the current therapies are limited to palliative care and do not exclude the necessity of surgical intervention [10,11]. Under alternative or adjuvant therapeutic schemes, regular dietary intake of natural functional foods containing polyphenols and other phytochemicals such as fruits, vegetables, whole grains, legumes, and olive oil [12–17] has been associated to a protective role in chronic disease prevention such as OA because of their anti-inflammatory and antioxidant properties [18].

Among all, Hydroxytyrosol (Hyt), a polyphenol found mainly in olive oil and raw olives, exerts strong antioxidant activity (as a potent radical scavenger and metals chelator) and acts as an anti-inflammatory as well as antithrombotic, antitumor, antimicrobial, and neuroprotective agent [19–22].

Fucelli et al. demonstrated the ability of Hyt to reduce inflammatory markers, such as Cyclooxygenase-2 (COX2) and Tumor Necrosis Factor alfa (TNF-α) and reduces oxidative stress on a mouse model of systemic inflammation [23]. Pre-treatment of Balb/c mice with Hyt (40 and 80 mg/Kg b.w.) prevented all lipopolysaccharide-induced effects and decreased oxidative stress. In another study, Cetrullo et al. demonstrated that Hyt inhibits the inflammatory response in vascular endothelial cells, macrophages, and monocytes [24]. Furthermore, Hyt reduces oxidative stress and damage, exerts pro-survival and antiapoptotic actions, and favorably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features [25].

However, the amount of Hyt obtained through its natural sources' consumption is considerably lower than the recommended daily intake able to exert its claimed healthpromoting properties [26]. Furthermore, the local therapeutic concentration of Hyt following oral administration is limited due to its poor bioavailability and enzyme degradation. Encapsulation of Hyt could be a functional alternative strategy to preserve its the biological activity and to ensure controlled release of the latter increasing the residence time inside the joint. Chitosan biopolymer has been extensively used as a matrix for the encapsulation of a wide range of natural products due to its beneficial properties including biodegradability, biocompatibility, and low cost [27]. Moreover, the ionic gelation method allows to obtain drug-loaded chitosan nanoparticles with a controlled size and satisfactory encapsulation capacity, protecting polyphenols from enzymatic oxidation or degradation. However, polymeric particles present some significant limitations such as initial burst release, escape from the joint's cavity, and in vivo rejection.

To overcome the above-mentioned drawbacks, in the present work, a localized drug delivery platform containing a combination of Hyt-loading chitosan nanoparticles (Hyt-NPs) and in situ forming hydrogel have been developed to derive the benefits of both hydrogels and nanoparticles. This thermo-sensitive formulation, based on Pluronic F-127 (F-127), hyaluronic acid (HA), and Hyt-NPs (called Hyt@tgel) presents the unique ability to be injected in a minimally invasive way into a target region as a freely flowing solution. When the temperature rises near the body temperature of 37 ◦C, hydrogel has in situ sol-to-gel transition accommodating the shape to the geometry of the treated area.

HA, a naturally polysaccharide, represents one of the largest components of the extracellular matrix of articular cartilage and plays an important endogenous role in the protection of articular cartilage decreasing the gene expression of inflammatory cytokines. Moreover, HA degrade ECM enzymes with stimulating in vitro chondrocytes proliferation, and chondrogenesis by directing mesenchymal stromal cells (MSCs) differentiation and increasing type 2 collagen production [28–30]. Although HA represents a conventional treatment in knee OA management, several lines of clinical evidence have questioned the effectiveness of such therapies due to HA prompt in vivo degradation mediated by hyaluronidases and oxidative stress [31,32]. To prolong HA residence time and confer optimized product functionality, Pluronic F-127 (F-127) consisting of hydrophilic poly (ethylene oxide) (PEO) and hydrophobic poly (propylene oxide) (PPO) (PEO-PPO-PEO) was added. Moreover, reported by Young-seok Jung and colleagues [33], the addition of high-molecular-weight HA (Mw: ~1000 kDa) increases the mechanical strength of thermosresponsive hydrogel hindering the interactions between water and poloxamer molecules due to HA-assisted inter-micellar packing. Starting from the results obtained in the work of Young-seok Jung, nanocomposite hydrogel formulations (Hyt@tgels) were optimized to ensure gelation around 37 ◦C, as well as allowing Hyt release exerting antioxidant and antiinflammatory activity on an in vitro induced inflammatory environment mimicking OA.

Based on the above, the developed platform may serve as both a Hyt delivery system and as a tissue engineering scaffold to stimulate the regeneration of a lesioned tissue and to prevent chondrocytes senescence providing an alternative and potentially more effective loco-regional approach to manage OA.
