**3. Results and Discussion**

## *3.1. Preparation and Physicochemical Characterization of Hyt-Loaded Nanoparticles (Hyt NPs)*

OA, the most common musculoskeletal disease in the elderly population, involves the inflammatory immune response at both local (joint site) and systemic levels leading to severe articular joint pain and reduced joint mobility. To date, local anti-inflammatory treatment is usually insufficient because of their short intra-articular half-lives, while systemic administration is associated with more adverse events [42–44]. Chitosan-based nanoparticles have been extensively used as ideal drug carriers for wide range of biomedical applications due to their good compatibility and degradability [45,46].

In this work, Hyt-loaded nanoparticles (Hyt NPs) were successfully produced by the ionic gelation method, using tripolyphosphate (TPP) as the crosslink. Although the ionic gelation process represents a simple and robust route to obtain chitosan NPs in aqueous medium and under mild conditions, the optimal process parameters were determined to achieve NPs with high drug loading and narrow polydispersity index (PDI). Indeed, the ratio of chitosan/TPP, the chitosan concentration, and the concentration of the encapsulated drug could interfere with the NP size and size distribution during NP formation.

Results of polymer ratio on particle size and size distribution, polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE%) of nine batches of Hyt NPs studied are summarized in Table 2. Particle size of the prepared formulations was in a nanometric range varying between 510.14 ± 13.21 nm (CS:TPP 1:1) and 137.56 ± 3.13 nm (CS:TPP 10:1) demonstrating that the size of the nanoparticles depends greatly on the ratio of CS to TPP. This behavior is achieved by the interaction of the phosphate charged groups of TPP with the –NH3 <sup>+</sup> groups within the CS structure. Indeed, as the amount of TPP increases the particle size decreases because of the increment in the cross-linking of CS macromolecules mediated by TPP, leading to a minimum particle size at 10:1 CS:TPP ratio.

**Table 2.** Effect of chitosan concentration and chitosan/TPP ratio on the size (hydrodynamic diameter), polydispersity index (PDI), zeta potential (ZP), and encapsulation efficiency (EE) of Hyt-loading nanoparticles (Hyt NPs). The Hyt concentration was kept constant at 10 mg.


Note: In the same column, value with the same subscript letter (a–c) were not significantly different (*p* > 0.05). Data were mean of three replications ± standard deviation (SD).

Moreover, all formulations present a narrow size distribution and high positive surface charge indicating their better stability to aggregation due to the repulsive forces exerted by the positive surface charge. As reported in Table 2, the EE of Hyt NPs enhanced with an increase in CS:TPP ratio ranging between 18.31 ± 1.23% of 1:1 and 74.18 ± 3.16% of 10:1. This could likely be attributed to the number of crosslinking units associated with different TPP concentrations [47]. Moreover, the reduction in nanoparticle size obtained with 10:1 CS:TPP ratio resulted in increment of space for drug encapsulation.

According to the above results, the chitosan and TPP ratio of 10:1 was chosen for further study as the obtained Hyt NPs showed the highest EE with acceptable particle size and distribution.

Figure 1 shows representative images of: size (1A), zeta potential (1B) distribution, screenshot of nanoparticles tracking analysis video (NTA, 1C), and measurements (1D) of Hyt-NPs synthetized in the optimal condition.

#### *3.2. In Vitro Hydrogel Formulation (Hyt@tgel) and Hyt Release*

To obtain a sustained and localized drug delivery of Hyt at body temperature, different amounts of Hyt NPs were dispersed into injectable hydrogels composed of 20 wt% of Pluronic F127 and 1 wt% Hyaluronic acid (Hyt@tgel). According to Young-seok et al. [33] the Hyt@tgel formulation was optimized to reduce the Pluronic F-127 concentration needed to obtain gelation at body temperature. Moreover, the hydrophobic interaction between acetyl groups on HA and methyl groups on Pluronic could enhance the mechanical strength of the resulting hydrogel at temperatures above the critical gelation temperature (CGT). As shown in Figure 2A, the addition of different concentrations of Hyt-NPs (1, 5, and 10 mg) did not significantly affect the Hyt@tgel gelation temperature, suggesting that hydrogel structure organization was maintained after nanoparticles dispersion. These results are in agreement with previous studies at the same Pluronic concentrations [48,49]. The gelation time of the Hyt@tgel at 35 ◦C was slightly increased by Hyt NPs incorporation (Figure 2B). In particular, the presence of high nanoparticle concentrations increases the gelation time by 0.5 min (10 mg, Hyt@tgel10) and 0.2 min (5 mg, Hyt@tgel5) with respect to Hyt@tgel alone (10.6 min). Long in vivo gelation time, in fact, can cause nanoparticle loss by diffusion into the surrounding tissue. On the contrary, gelation that occurs too quickly could lead to

clogging of the injection needle resulting in incomplete administration. Based on suitable gelation time and temperature, further analyses were conducted only on the Hyt@tgel10 sample. As shown in Figure 2C, Hyt@tgel10 demonstrated easy injectability through hypodermic needles at room temperature, while when the temperature increases at 35 ◦C, the extrusion needs the application of an extra force due to the increase in the viscosity. When the gel concentration reached the critical gelation concentration, the Hyt@tgel10 passed from an aqueous solution to a gel as the temperature was increased from 4 to 35 ◦C as demonstrated by the inversion test tube (Figure 2D). Hyt@tgel10 exhibited a viscous flowable form at low temperature becoming a semi-solid gel after incubation at temperature higher than 30 ◦C. This behavior was confirmed by the measure of viscosity as a function of temperatures (Figure 2E).

There is a substantial body of evidence that encapsulation enhances the bioactivity of compounds improving their stability in aqueous medium and increasing upon the delivery at the target site. Chen et al. demonstrated the ability of chitosan microspheres dispersed in a thermally responsive chitosan hydrogel to load anti-inflammatory drugs. After injection into the knee joints of OA rabbits, drugs were released for more than 7 days in a controlled manner [50]. According to Wang et al., curcumin-loaded HA/chitosan nanoparticles exhibited a good sustained-release property leading to inflammation and cartilage apoptosis inhibition acting on the NF-κB pathway [51].

**Figure 2.** Characterizations of Hyt@tgel. Gelation temperature (**A**) and gelation time at 37 ◦C (**B**) of different hydrogel compositions (Hyt@tgel, Hyt@tgel1, Hyt@tgel5, and Hyt@tgel10). Representative photographs of the Hyt@tgel10 syringeability (**C**) and inverted test tube (**D**) obtained at 4 and 35 ◦C. Phenol red was added to facilitate hydrogel monitoring. (**E**) Solution viscosity measurement of the Hyt@tgel10 as a function of temperature. (**F**) Cumulative Hyt release from Hyt NPs and Hyt@tgel10 in phosphate buffer saline (PBS) after 24 h (and eight days). Six different experiments were conducted, and the results expressed as the mean of the values obtained (mean ± SD).

Chitosan nanoparticles have been recognized as a useful drug delivery tool in OA for their ability to prolong the drug retention time. To evaluate the sustained release properties of Hyt-NPs, an in vitro drug release study of Hyt from Hyt-NPs and from HYt@tgel10 was carried out using dialysis membrane against phosphate buffer saline (PBS). As shown in Figure 2F, the in vitro release of Hyt by chitosan NPs exhibited a fast drug release rate with 41% of Hyt released within the first hour, with the majority of the release occurring during the initial 2 days (75%). On the contrary, Hyt release rate from Hyt@tgel10 significantly slowed down (*p* < 0.05) with only 10% of Hyt released after 1 h, followed by a prolonged Hyt release up to 1 week. The slow Hyt release from the hydrogel could be attributed to the densely packed inter-micellar structure due to the presence of HA. Moreover, the highly packed super-molecular structure could reduce the diffusion coefficients inside of the hydrogel leading to a prolonged drug release.

Physical stability of nanoparticles in Hyt@tgel was investigated at 4 and 25 ◦C over 14 days by measuring size and PDI. As reported in Table 3, Hyt NPs were stable when stored at both low and room temperatures without a significant increase in particle size and PDI. Moreover, the drug encapsulation efficiency, assessed in parallel, demonstrated no decrease in the Hyt retention rate over the 14-day period confirming the protective effect of chitosan nanoparticles on biomolecules.


**Table 3.** Particle size and entrapment efficiency studies of Hyt@tgel10 before and after 14-day storage.

Note: Data were expressed as mean ± standard deviation, *n* = 3.

Taken together, the physicochemical behavior of Hyt@tgel10 is consistent with a potential use as a device to be injected through a syringe, because the sol-to-gel transition temperature is between room temperature and physiological temperature.
