Development and Optimization of Chitosan-Hydroxypropyl Methylcellulose In Situ Gelling Systems for Ophthalmic Delivery of Bupivacaine Hydrochloride

: The aim of this study was the development and optimization of chitosan and hydroxypropyl methylcellulose (HPMC) in situ gelling systems, loaded with bupivacaine hydrochloride for topical ocular administration. This study is based on the properties of two polymers: chitosan, which has mucoadhesive action and is a pH-sensitive polymer, but also the cellulose derivative hydroxypropyl methylcellulose, a thermosensitive polymer which has mucoadhesive properties and increases the viscosity of systems. The analysis and optimization of in situ gelling systems were performed based on an experimental design and response surface methodology. The following formulation parameters were considered: X 1 = chitosan concentration (0.5%, 1%), X 2 = HPMC E 5 LV concentration (2%, 5%) and X 3 = Chitosan/HPMC E 5 LV ratio (1/1, 2/1). In addition, the parameters to be optimized were represented by the contact angle (CA ( ◦ )), viscosity and cumulative percentage of bupivacaine hydrochloride released in vitro. The results indicate that the designed in situ gelling systems are suitable for bupivacaine prolonged ophthalmic release and overcome the principal disadvantages of the liquid’s ocular formulations. An immediate therapeutic effect corresponding to ocular anesthetic installation was assured in the ﬁrst stage: burst bupivacaine release. In the second phase, the gradual drug release was assured for over 6 h. This drug release proﬁle, together with the corresponding rheological proﬁle and a collection of superﬁcial properties for good ocular adhesion balanced with an adequate hydrophilic character, assured the desired quality of the attributes for the proposed systems. The system, based on chitosan 1%, HPMC E 5 LV 5% and a 1/1 polymer ratio, could be a solution for the proposed formulation of in situ gelling colloidal systems, since the viscosity of the system was within the range of the optimal viscosity of the eye, and the amount of bupivacaine hydrochloride released after 6 h was the highest at 69.55%. Author Contributions: Conceptualization, L.P., T.I. and M.V.G.; methodology, L.P. and M.V.G.; experiments, L.P. and T.I.; in vitro drug release experiments, L.P., M.V.G., C.-E.D.-P. and R.P.; writing— preparation, T.I., L.P. and R.P.; writing—review and L.P., R.P. and M.V.G.;


Introduction
The human eye is a complex system, so the administration of drugs to the eye is in continuous research. Studies are designed to develop modern drug delivery systems that increase the delivery of the active pharmaceutical ingredient to the ocular system [1], such as inserts [2,3], contact lenses [4], hydrogels [5], nanostructured lipid carriers, nanoparticles, liposomes [1] and so on. Certain systems may cause discomfort to the patient in the eye system, and in some situations, the method of administration is invasive. Instead, topical ocular administration is a simple method that can be performed at home without the need for medical care, thus improving patient adherence to the treatment [1].
Chitosan is a biopolymer with many useful properties for ophthalmic use; it is biocompatible, biodegradable, non-toxic and has its own antibacterial and antifungal actions [6]. Chitosan has mucoadhesive action on the mucous membranes, enhances permeability

Materials
For the preparation of colloidal gelling systems in situ with chitosan and HPMC E 5 LV, the following substances were used: a chitosan base with an average Mw of 190,000-310,000 and DD 75-85% (Sigma-Aldrich, Saint Louis, MO, USA), hydroxypropyl methylcellulose grade E 5 LV Premium (Loba Chemie, Mumbai, India) and bupivacaine hydrochloride (Sigma-Aldrich, Saint Louis, MO, USA). The other reagents and solvents used (1% acetic acid solution, a 10% NaOH solution, distilled water and sodium chloride) had analytical purity.

Preparation of In Situ Gelling Systems with Chitosan and HPMC E 5 LV
Polymeric chitosan solutions (0.5% and 1%) were prepared by weighing the appropriate amount of chitosan (0.5 g and 1 g) and dissolving in 100 mL of 1% acetic acid by continuous homogenization for 1 h at room temperature.
Colloidal solutions of HPMC E 5 LV (2% and 5%) were obtained by dissolving the appropriate amount (2 g and 5 g) in about 80 mL of distilled water heated to 80-85 • C under continuous stirring, followed by ice cooling. It was created up to the mark with distilled water in a 100 mL volumetric flask [13].
The samples were prepared and coded according to Table 1 by varying the polymer concentrations and the ratio between them. Briefly, a volume of chitosan was measured in a vessel over which the polymeric solution of HPMC E 5 LV was gradually added under continuous stirring. Then, the equivalent of a 0.9% sodium chloride (NaCl) solution was added, and stirring was continued until complete dissolution of the salt. The equivalent of 0.25% bupivacaine hydrochloride was weighed and incorporated into the polymeric mixture of chitosan and HPMC E 5 LV and then titrated with a 10% sodium hydroxide (NaOH) solution to a pH of 5 to 5.5, measured using a Mettler Toledo pH meter (Switzerland) GmbH (Im Langacher 44, 8606 Greifensee, Switzerland).

Determination of Gelling Capacity for Colloidal Chitosan and HPMC E 5 LV Systems
The gelling capacity was determined with a water bath GFL-1012 with a thermostat using previously prepared samples from the colloidal solutions and simulated tear fluid (STF) with a pH of 7.4.
The gelling capacity for the formulated systems was evaluated by adapting the method described by Sheshala et al. (2019) [13]. In test tubes containing 5 mL STF (pH 7.4 at 35 • C), 1 mL of the sample was added. Then, they were placed on the water bath GFL-1012 with a thermostat. The gelling capacity was determined by visual inspection of the degree of rigidity of the gels formed as well as their stability over time. To facilitate visual observation of the gel, a drop of Congo red dye was added to each tube.

Determination of Contact Angle Values
The contact angle of the in situ gelling systems was determined at 23 • C (storage temperature) and 35 • C (physiological eye temperature) by the sessile drop technique [29]. Evaluation of the contact angle was performed with the following materials: samples from the previously prepared polymer systems with chitosan and HPMC E 5 LV, glass slides, a Hamilton sampling syringe and the CAM 101 sphygmomanometer-goniometer.
The working technique involved displaying a drop of the sample to be analyzed on the surface of a glass slide and processing its image by the Young-Laplace method. For each sample, the measurements were replicated 5 times.

Evaluation of Surface Tension
The determination of the surface tension (ST = γ LG ) as an indicator of the strength of the intermolecular bonds in the fluid system was performed using the "pendant drop method". The same materials were used to evaluate the surface tension as when determining the contact angle.
The working technique differed from that presented in the section for the contact angle by shooting the image of the drop just before detachment from the needle of the Hamilton syringe. The droplet profile was analyzed based on the Young-Laplace equation, determining the surface tension at the air-droplet interface. For each sample, 5 measurements were performed.

Rheological Studies on Colloidal Systems with In Situ Gelling Based on Chitosan and HPMC E 5 LV
Rheological studies for the previously prepared chitosan and HPMC E 5 LV formulations were performed at 35 • C using a multi-visc rotary viscometer, to which a thermostat (ThermoHaake P5 Ultrathermostat) was attached to ensure a constant working temperature.
The viscosity values, as a parameter to be optimized for the prepared formulations, were considered to be those obtained at a shear rate of 20 rpm, corresponding to the ocular physiological conditions [30,31].

In Vitro Drug Release Kinetics Studies from Colloidal Systems with Chitosan and HPMC E 5 LV
In vitro drug release studies from chitosan-and HPMC E 5 LV-based in situ colloidal gelling systems were performed using a Hanson Vision ® G2 Classic 6TM Dissolution Tester.
For these determinations, 5 mL of each colloidal system with the previously prepared chitosan and HPMC E 5 LV were used, and Visking dialysis bags (from Sigma-Aldrich, Saint Louis, MO, USA) made of cellulose were used as a semipermeable membrane (type: 20/32 inches, thickness: 0.020 mm).
The experiments were performed at 35 • C, and the rotational speed of the blades was set at 50 rpm. As a release medium, a phosphate buffer solution with a pH of 7.4 (500 mL volume) was used. At predetermined time intervals over a period of 6 h, volumes of 5 mL of the release medium were taken and replaced with equivalent volumes of the phosphate buffer maintained at 35 • C.
The concentration of bupivacaine hydrochloride in each sample was determined spectrophotometrically by recording the absorbance at 263 nm wavelengths. Using the calibration curve (A 1cm 1% = 14.051), the cumulative percentage of the drug substance released after each time interval was calculated.
Data from in vitro release studies on the colloidal systems used were evaluated using the mathematical models of Higuchi (Equation (1)) and Korsmeyer-Peppas (Equation (2)) to evaluate the release mechanism of the active pharmaceutical ingredient bupivacaine hydrochloride: where M t is the amount of the substance released over time, M ∞ is the total amount of the drug substance in the system under analysis, M t M ∞ is the fraction of the drug substance released at each time point, k H is the Higuchi constant, k KP is the Korsmeyer-Peppas kinetic constant and n is the release exponent.

Optimization of Colloidal Systems with In Situ Gelling Based on Chitosan and HPMC E 5 LV Using Response Surface Analysis
As was previously mentioned, response surface methodology is a statistical method suitable for multi-factorial experiments, providing a relationship between different parameters for optimal operating conditions.
The response of the system is represented graphically in three dimensions, with highlighting of the interactions between the formulation factors [32].
The 3D representation of the responses of the system designed in this study was made using OriginPro 8.5.1 software (OriginLab ® ).

Results
Polymer mixtures of chitosan and HPMC E 5 LV were prepared by varying the concentration and ratio of the polymers in order to increase the ocular bioavailability of bupivacaine hydrochloride.
The design of the experimental matrix for colloidal systems in this study included a factorial plan with three factors (X1, X2 and X3) and two levels of variation (lower and upper) as shown in Table 2, and the three independent variables were as follows: • X1: chitosan concentration (0.5% or 1%); • X2: HPMC E 5 LV concentration (2% or 5%); • X3: (chitosan / HPMC ratio of 1/1 or 2/1). For the in situ gelling systems obtained, the following parameters were identified and evaluated: a contact angle of 35 • C, viscosity at a shear rate of 20 rpm and cumulative percentage of bupivacaine hydrochloride released in vitro.
Clear, colorless, homogeneous colloidal solutions free of suspended particles were obtained. Increasing the concentration of HPMC E 5 LV from 2% to 5% did not affect the transparency of the formulations. A pH of the samples between 5 and 5.5 ensured the solubility of the components and their stability within the limits of ocular tolerability.
The gelling capacity was coded as follows: (−) not gelled; (+) gelled after a few minutes and quickly despaired; (++) gelled quickly and remained undispersed for several hours; and (+++) gelled instantly and remained undispersed for a long time [33]. The results of the gelling capacity assessment for the designed systems are shown in Table 3.
Sample 2 (chitosan 1%, HPMC E 5 LV 2%) and sample 5 (chitosan 1%, HPMC E 5 LV 5%), both with a polymer ratio of 1/1, gelled instantly in a few seconds and remained undispersed for more than 6 h. In addition, sample 7 (chitosan 1%, HPMC E 5 LV 2%) and sample 8 (chitosan 1%, HPMC E 5 LV 5%), with a polymer ratio of 2/1, had immediate gelation. Visual analysis of the formulations in which Congo red was added ( Figure 1) showed the presence of gelling on contact with the simulated tear fluid. In the case of samples 2, 5, 7 and 8, gelation occurred rapidly in a few seconds, resulting in a gel with high stability. For the other samples, gelation did not occur as quickly, but after gel formation, all samples had high stability. This phenomenon was most likely because they had the highest concentration of chitosan of all the formulations analyzed. Chitosan is a pH-sensitive polymer, with the changing pH of the formulations upon contact with tear fluid in the simulated electrostatic repulsion occurring between the polymer chains to promote crosslinking. Not only was the degree of viscosity of the gel important, but the speed with which the gel was formed was also important. The faster the system gelled, the more it stayed in contact with the mucosa and the harder it was to remove with the tear fluid secreted by the eye system as a method of defending the body against external factors. The contact angle values for the polymer preparations were intended to evaluate their ability to display on the ocular surface. Table 4 presents the experimental data obtained after analysis of the droplets from the samples taken, captured with the help of the CAM 101 tensiometer-goniometer at 23 • C and at 35 • C. Table 4. Synthesis of experimental data resulting from CA ( • ) evaluation for the polymer systems, analyzed at 23 • C and 35 • C. The recorded quantitative parameters were the average of the two values of the contact angle to the left and to the right CA (M), the volume of the drop (V) (µL) and the interfacial tension (γ SL ) (mN/m) at 23 • C and 35 • C.
The wettability of the polymeric solutions of chitosan and HPMC E 5 LV analyzed at 23 • C proved to be satisfactory for all 8 samples, an aspect highlighted by the values of the average contact angle (CA (M) ( • )) between 37.67 • ± 3.49 and 52.89 • ± 4.96, respectively.
It was noted that formulations 2 and 5, containing 1% chitosan and different concentrations of HPMC E 5 LV, had the highest CA ( • ) values, while formulations 4 and 6, incorporating concentrations of only 0.5% chitosan and different concentrations of HPMC E 5 LV and maintaining the same ratio between polymers, had the lowest CA ( • ) values in the entire series of samples analyzed.
The characteristic CA ( • ) data for the analyzed in situ gelling colloidal systems showed lower values compared with those obtained for the polymer solutions at 23 • C for most of the samples in the analyzed series, as can be seen in Figure 2, and the p value obtained through the ANOVA test was 0.6999. As an exception, samples 4 (0.5% chitosan, 2% HPMC E 5 LV), 6 (0.5% chitosan, 5% HPMC E 5 LV) and 8 (1% chitosan, 5% HPMC E 5 LV), all having a ratio between the polymers of 2/1, showed higher values for CA ( • ) at 35 • C. In system 7, although the chitosan/HPMC E 5 LV ratio was 2/1, a lower value of the contact angle was found in the physiological conditions (35 • C) than in the non-physiological ones (23 • C). For this colloidal system formulation with in situ gelling, the concentration of the chitosan biopolymer was at its maximum level (1%). For all eight analyzed samples, the optimal values of the surface tension γ LG between 37.32 ± 2.79 mN/m and 43.99 ± 1.05 mN/m were obtained, corresponding to volumes for the drops between 9.23 ± 0.34 µL and 10.82 ± 0.43 µL, respectively. Additionally, a direct proportional relationship between the volume of the sample droplets to be analyzed and the value of the surface tension γ LG was noticed.
The values obtained for the determination of γ LG for polymeric systems with in situ gelling based on chitosan and HPMC E 5 LV at 35 • C were between 38.93 ± 0.64 mN/m and 41.35 ± 0.71 mN/m (Table 5). It was observed that, for the systems evaluated at 35 • C, most γ LG values were lower than those resulting from the analysis at 23 • C (Figure 3), and the p value obtained by the ANOVA test was 0.0666. Table 5. Synthesis of experimental data resulting from the evaluation of the surface tension γ LG for the polymeric systems analyzed at 23 • C and 35 • C.    At the same time, the γ LG values close to those of the tears determined a minimal disturbing effect on the tear film, increasing the degree of comfort and compliance of the patient.
It is concluded that by corroborating the results obtained from the experiments evaluating the gelling capacity and surface properties, namely the contact angle and the surface tension, that system 2, system 5 and system 7 had the properties and characteristics of an appropriate quality.
The viscosity values for the colloidal systems with in situ gelling of chitosan and HPMC E 5 LV corresponding to a shear rate of 20 rpm are shown in Table 6.  Figure 5 shows the evolution of the viscosity under the influence of the shear rate. It was observed that there was a decrease in the viscosity of the colloidal systems with an increasing shear rate for all eight systems analyzed. By applying the ANOVA test, we obtained a p value of less than 0.05.
Carrying out experiments at 35 • C had dual roles: to simulate the physiological ocular temperature and to favor the initiation of the gelling process, with an impact on the rheological behavior of the formulations.
The polymer with a major influence on the viscosity of the designed systems was HPMC E 5 LV. Cellulose derivatives of the HPMC type are often used as agents for increasing the viscosity of ophthalmic preparations. As can be seen from Table 6, the highest viscosity values recorded at 20 rpm were observed in colloidal systems 5 (70.36 cP), 7 (62.32 cP) and 8 (119.87 cP), and they also had high concentrations of polymers in the composition. Table 7 shows the cumulative percentages of bupivacaine hydrochloride released after 120 min, 240 min and 360 min, along with the 3 formulation variables (X1: concentration percentage of chitosan; X2: concentration percentage of HPMC E 5 LV; and X3: polymer ratio for chitosan/HPMC E 5 LV). The colloidal gelling in situ systems released more than 56% of the drug substance after 2 h, and after 6 h, they released between 61.7% (system 7) and 83.1% (system 1) of the drug substance. The dynamics of bupivacaine hydrochloride release from the formulations under analysis over 6 h is shown in Figure 6. The highest amount of the drug released after 6 h (83.198%) and was recorded in the case of system 1 (0.5% chitosan, 2% HPMC E 5 LV and polymer ratio 1/1). This was followed by system 3 (0.5% chitosan, 5% HPMC E 5 LV and polymer ratio 1/1), in which the amount of bupivacaine hydrochloride released after the same interval of 6 h was 82.291%. The cumulative percentage of the drug released in vitro from the eight colloidal systems is plotted against time in Figure 7.  In vitro drug release studies looked at the release profile of bupivacaine hydrochloride from chitosan-and HPMC E 5 LV-based in situ gelling colloidal systems. The analyzed formulations showed a prolonged release over a time interval of 6 h ( Figure 6) with a p value less than 0.05. This result corresponds to the initial formulation hypothesis and may ensure an increase in the efficacy of the drug at the site of action [34]. Colloidal system 1 with 0.5% chitosan, 2% HPMC E 5 LV and a polymer ratio of 1/1 showed the highest cumulative bupivacaine release percentage, exceeding 83%. The system with the lowest percentage of anesthetic released after 6 h (61.77%) was system 7, with 1% chitosan, 2% HPMC E 5 LV and a polymer ratio of 2/1. Basically, the percentage of the drug given decreases in the order of 1 > 3 > 2 > 4 > 5 > 8 > 6 > 7. Figure 7 indicates that the colloidal systems shown had similar kinetic profiles. The p value after using the ANOVA test on the data obtained was 0.0179.
In Table 8, the recorded values for the determination coefficient R 2 and the release exponent n are shown, which were determined using the mathematical models mentioned above, together with the fraction of bupivacaine hydrochloride M t M ∞ released at each time point t.  Table 9 summarizes all the responses of the colloidal systems that followed optimization, summarizing the data obtained for the parameters to be optimized Y 1 -Y 3 . Y 1 represents the values obtained after measuring the contact angle (CA) at 35 • C, Y 2 defines the viscosity of the systems at 35 • C, and Y 3 represents the percentage of bupivacaine hydrochloride released from the colloidal systems with chitosan and HPMC E 5 LV after 6 h. The three independent variables were X 1 (chitosan concentration), X 2 (HPMC E 5 LV concentration) and X 3 (chitosan/HPMC E 5 LV polymer ratio). Table 9. Synthesis of the results obtained at the optimization of the formulation of the colloidal systems with in situ gelling, with gels analyzed containing bupivacaine hydrochloride in a polymeric matrix of chitosan and HPMC E 5 LV. Regarding the optimization of colloidal systems with gelling in situ, from the point of view of the contact angle (CA ( • )), as indicator of hydrophilicity, wetting and mucoadhesivity which was considered the parameter (response variable), it was intended that its value at 35 • C, the ocular physiological temperature, to fall within the range of hydrophilicity, or more precisely, to have a value lower than 90 • (Figure 8a-c). Moreover, the values of this quality parameter were in a relatively narrow range: 40-60 • . This fact illustrates a uniformity of the formulations, with the chosen composition leading to values of this indicator located around a central optimum. Higher values of the CA ( • ) parameter were evident for high concentrations of HPMC E 5 LV, as a result of or in relation to the chitosan biopolymer (Figure 8a-c). Figure 8. Highlighting in 3D the evolution of the Y (CA ( • )) response parameter as a function of the formulation variables (a) X 1 (chitosan concentration) and X 2 (HPMC E 5 LV concentration); (b) X 1 (chitosan concentration) and X 3 (chitosan/HPMC E 5 LV ratio); and (c) X 2 (HPMC E 5 LV concentration) and X 3 (chitosan/HPMC E 5 LV ratio).

Formulation Variables
The variation of the contact angle values according to X 1 (chitosan concentration) and X 3 (chitosan/HPMC E 5 LV ratio) (Figure 8b) shows a high "plateau" of values for high concentrations of chitosan (1%) and for a ratio of the two polymers of 2/1 in favor of chitosan, respectively.
The appearance of the response surface of the contact angle parameter according to the variables X 2 (HPMC E 5 LV concentration) and X 3 (chitosan/HPMC E 5 LV polymer ratio) (Figure 8c) was similar to that in Figure 8a. This supports a similar contribution of the two polymers to the variation of the contact angle parameter. However, high values of CA ( • ) were obtained for a chitosan/HPMC E 5 LV ratio at a higher level (2/1).
The viscosity response parameter was evaluated at a shear rate of 20 rpm and analyzed from the point of view of the response surfaces, depending on the formulation factors (Figure 9a-c). High viscosity values were recorded for high polymer concentration levels (Figure 9a). . Highlighting in 3D the evolution of the Y (viscosity) parameter according to the formulation variables (a) X 1 (chitosan concentration) and X 2 (HPMC E 5 LV concentration); (b) X 1 (chitosan concentration) and X 3 (chitosan/HPMC E 5 LV ratio); and (c) X 2 (HPMC E 5 LV concentration) and X 3 (chitosan/HPMC E 5 LV ratio).
It should be noted that at the higher level of the chitosan concentration (1%), high values of viscosity for the solutions were obtained, even if the level of the polymer HPMC E 5 LV was lower (2%), an example in this respect being system 2 (with a viscosity of 47.6 cP at 20 rpm).
There was a prevalence of the influence of chitosan over the other formulation variables and in the variability of the viscosity of the colloidal systems with in situ gelling being analyzed.
High viscosity values were obtained in system 8, specifically 119.87 cP (colloidal system with 1% chitosan, 5% HPMC E 5 LV and a 2/1 chitosan/HPMC E 5 LV ratio). This is not beneficial for the performance of the system, taking into account that agglomerations of the preparation at the site of action may occur or inadequate display, resulting in either local irritation with acceleration of the washout phenomenon or inadequate availability for absorption of the substance drugs from the polymer matrix.
Regarding the optimization of bupivacaine hydrochloride release, system 1 and system 3, which differed only in the concentration of HPMC E polymer 5 LV, were the formulations that led to the maximum percentages of bupivacaine hydrochloride released after 6 h (83.19% and 82.29%, respectively). However, this observation must also be corroborated with the results of these systems from the point of view of the superficial and rheological properties (Figure 10a-c). Figure 10. Highlighting in 3D the evolution of the Y parameter (percentage of bupivacaine hydrochloride) according to the formulation variables (a) X 1 (chitosan concentration) and X 2 (HPMC E 5 LV concentration); (b) X 1 (chitosan concentration) and X 3 (chitosan/HPMC E 5 LV ratio); and (c) X 2 (HPMC E 5 LV concentration) and X 3 (chitosan/HPMC E 5 LV ratio).
As a result, the proposed range was "increased" to be optimal for the percentage of the drug given after 6 h, meaning a value of at least 70% (Figure 10c).

Discussion
In this study, the aim was to formulate ophthalmic systems with in situ gelling in order to increase the ocular bioavailability of bupivacaine hydrochloride by combining chitosan, a pH-sensitive and mucoadhesive polymer, with HPMC E 5 LV, a thermosensitive polymer with the property of increasing the viscosity, with lubricating and mucoadhesive actions.
We set out to design colloidal systems with in situ gelling based on chitosan and HPMC E 5 LV as potential vehicles, which would undergo a phase transition under the action of two existing factors in the tear fluid: a physiological pH of 7.4 and a physiological temperature of 35 • C. An optimal in situ gelation system should be able to undergo phase transition immediately after contact with the ocular surface, ensuring the remainder of the drug substance without causing discomfort to the patient [35].
HPMC E 5 LV is a thermosensitive polymer, but the solutions showed a phase transition at high temperatures. The process takes place gradually with an increasing temperature in four phases, according to Bajwa et al. [36]. In the first phase, a superficial increase of the gelling takes place at temperatures between 10 • C and 40 • C following the cleavage of the intramolecular bonds, increasing the mobility of the polymer chains. At temperatures up to 56 • C, there is a decrease in gelling due to the existence of a rarefied network resulting from the process of separation of the polymeric structures. Reaching 56 • C determines the existence in the solution of an increased number of molecular fractions whose interaction results in a gel with an elastic structure influenced relatively little by the subsequent increase in temperature [36].
The sodium chloride (0.9%) of the compositions of the formulations had an isotonizing role and decreased the transition temperature of HPMC E 5 LV. The addition of sodium chloride influences the temperature at which the onset of gelation occurs, according to Almeida et al. [37]. Chloride ions have a small radius and a high charge density, with the ability to compete for water molecules and reduce the hydrogen bonds between the water and the polymer. As a result, a relatively small number of water molecules remains available for polymer solvation, which favors hydrophobic associations between HPMC chains at low temperatures [38].
By evaluating the contact angle in the optimization study of the ophthalmic systems with in situ gelling based on chitosan and HPMC E 5 LV, the profile of the liquid droplets was investigated, with emphasis on the impact on a hydrophobic surface. The corneal epithelium is known to have a lipophilic character, to which the mucin layer is added on its surface with a protective role, which explains the hydrophobicity of this tissue.
For the colloidal systems of chitosan and HPMC E 5 LV, CA values ( • ) lower than 90 • were obtained, which shows the hydrophilic character and the increased wetting capacity of the analyzed mixtures.
Surface tension is a key parameter in optimizing ophthalmic formulations. The administration of ocular preparations with a surface tension lower than that characteristic of tears can cause the destabilization of the tear film and its dispersion in the form of drops [39].
The surface tension at the air-tear film interface has a physiological value between 40 and 46 mN/m, ensuring the stability and breakup time of the tear film [40]. The results of the γ LG determinations at 23 • C and 35 • C were within the physiological limits.
For the polymeric mixtures of chitosan and HPMC E 5 LV analyzed at the ocular physiological temperature, the γ LG values were lower than those obtained at 23 • C, an aspect that is consistent with the statements of Han et al., according to which γ LG decreases with an increasing temperature [41].
Another important aspect to mention is the influence of γ LG on the volume of the drop. It is known that volumes between 5 and 15 µL have been proven effective in drug release, and the volume of a drop influences the amount of the active pharmaceutical ingredient administered and its maintenance for as long as possible in the eye [42]. The volumes of the droplets in the samples analyzed in this study fell within the aforementioned range, which prevented the onset of the immediate washout phenomenon.
The rate of blinking of a normal, healthy eye varies between 6 and 30 blinks/min, with a reported average of 22 blinks/min when the eye is relaxed [30,31]. Rheological studies follow the influence of the formulation factors and working temperature on the flow behavior of colloidal systems with chitosan and HPMC E 5 LV [43].
Akinosho et al. specified that the methyl and hydroxypropyl groups in the composition of HPMC determine how a colloidal dispersion after gelling can be described as a polymeric network or a gel with different degrees of rigidity, respectively [44]. According to the statements presented by Sandri et al., the increase of the temperature determines the partial dehydration of the polymeric chains of HPMC, favoring the hydrophobic interactions and the cross-linking between them [45].
In the first phase, there is a rapid release, known in the literature as the "burst effect", followed by a prolonged release for 6 h. This two-stage process corresponds to a rapid initial onset of local anesthetic action of bupivacaine hydrochloride in ocular surgery, followed by a gradual analgesic action maintained for a longer period of time, a beneficial aspect in postoperative pain management [46,47].
The polymer matrix has a significant influence on the percentage and rate of drug release [34]. Thus, an increase in the concentrations of the polymers caused a decrease in the rate of release of the bupivacaine hydrochloride, which is consistent with the statements of Ghosal et al. [48]. A high concentration of polymers causes the formation of a polymer network resistant to the action of the release medium (phosphate buffer solution pH: 7.4), thus reducing the amount of bupivacaine hydrochloride released. Compared with the previously mentioned statements, Chanaj-Kaczmarek et al. stated that the addition of chitosan decreases the permeability of the polymer network [49].
For the approached study, the drug release kinetics were analyzed using the Korsmeyer-Peppas mathematical model, for which the values of the coefficient of determination R 2 exceeded the value of 0.98. The values of the yield exponent n within the same model varied between 0.205 and 0.426. In this case, the release mechanism depended on the migration of water in the polymer matrix and the diffusion of the drug substance, a fact also confirmed by Song et al. [50].
For the optimization of the contact angle, a concentration of 1% chitosan and a higher chitosan/HPMC E 5 LV ratio (2/1) were favorable for appropriate hydrophilic or wetting properties, but the HPMC E 5 LV concentration must be at a lower level (2%) to compensate for hydrophobia induced by chitosan. These conditions were met by samples 7 and 8.
The values of the viscosity located in the middle zone of the variation range 13.72-119.87 cP, namely between 60 cP and 70 cP, can be considered the optimal values of the systems. The colloidal systems that correspond to these values are system 5 (1% chitosan, 5% HPMC E 5 LV, 1/1 chitosan/HPMC E 5 LV ratio) and system 7 (1% chitosan, 2% HPMC E 5 LV, 2/1 chitosan/HPMC E 5 LV ratio). So far, only substances soluble at an acidic pH have been analyzed with this type of system, because the natural polymer chitosan is only soluble in an acidic pH.

Conclusions
This study shows the potential of chitosan in the formulation of colloidal systems with in situ gelling by associating it with a cellulose derivative, namely HPMC sort E 5 LV, in order to increase the ocular residence time of bupivacaine hydrochloride. This study was consolidated upon an experimental plan that aimed to optimize the formulation parameters and their impact on the system responses.
The predominantly hydrophilic behavior of the formulations, expressed by CA ( • ) values lower than 90 • , together with the γ LG values located in physiological limits determined a good watering capacity of the corneal surface. Regarding the viscosity parameter evaluated at 20 rpm, high values of it were recorded in systems with a chitosan concentration at a higher level (1%).
In vitro release studies of bupivacaine hydrochloride for the designed systems indicated similar kinetic profiles and a two-stage release of the active substance: an initial rapid release or "burst effect" followed by a gradual release for 6 h. The analysis of the obtained data led to the selection of the Korsmeyer-Peppas model as the mathematical model that characterized the kinetics of the drug release, with the values of the coefficient of determination R 2 exceeding 0.98.
The in situ gelling capacities of the designed colloidal systems were mainly influenced by the independent variable X 1 , represented by the chitosan concentration, such that the systems with a chitosan biopolymer concentration at a higher level (1%) showed a rapid phase transition in a few seconds following contact with the simulated tear fluid.
It can be concluded from the optimization analysis, in terms of the surface properties, rheological profiles and kinetic profiles, that the system with 1% chitosan, 5% HPMC E 5 LV and the chitosan/HPMC E 5 LV 1/1 ratio demonstrated a performance level that can be considered a promising alternative to the prolonged release of bupivacaine hydrochloride to the eye. The developed in situ gelling systems can be further used as delivery platforms to overcome low ocular bioavailability of the drugs. Funding: This paper was financially supported by the "Carol Davila" University of Medicine and Pharmacy through Contract No. CNFISFDI-2021-0300 and RDI capability consolidation at the institutional level of the multidisciplinary research teams involved in the sustainability of UMFCD priority research directions.