FORMULATION AND CHARACTERIZATION OF THERMOSENSITIVE CHITOSAN HYDROGEL LOADED WITH ERLOTINIB: FULL FACTORIAL DESIGN OF RELEASE DATA

Erlotinib is a Tyrosine Kinase Inhibitor (TKI) that is utilized in the treatment of various types of tumors expressing the epidermal growth factor receptors (EGFR) protein. However, it is characterized as a drug with poor solubility in water and a high affinity for blood plasma proteins. According to the biopharmaceutical classification system (BCS), Erlotinib falls under class two, where its solubility acts as the rate-limiting step for its bioavailability. In systemic administration, Erlotinib exhibits a bioavailability of approximately 60%, but it also presents several systemic side effects, which can be severe in certain cases due to its poor solubility in water and its strong binding to plasma proteins. To address the issue of low bioavailability, we pursued an efficient approach to enhance the solubility of Erlotinib. nanoparticle formation using the non-solvent method, and dynamic light scattering (DLS) and in vitro dissolution studies were employed to characterize the Erlotinib nanoparticles (ETB-NPs). These studies also produced favorable outcomes, demonstrating the successful improvement of Erlotinib's solubility through nanoparticle formation. Following the solubility enhancement, the drug was formulated into a local injectable hydrogel for targeted delivery. This formulation exhibited significant improvements in the release of Erlotinib. The solubility enhancement and localized administration of Erlotinib within the tumor microenvironment facilitated controlled release and contributed to the observed enhancements. The chitosan hydrogel used as the carrier system for Erlotinib was thoroughly characterized using various studies, including viscosity measurements, pH determination, and in vitro release studies, all of which yielded positive results.


Introduction
Erlotinib HCL (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazoline-4amine) is a member of TKIs, specifically a quinazoline derivative that acts as an ATP competitor by binding to EGFR-TK.It was approved by the FDA in 2004 as a monotherapy for the treatment of advanced non-small cell lung cancer (NSCLC).In 2005, it received approval for the treatment of metastatic pancreatic carcinoma in combination with Gemcitabine, and in 2013, it was approved as a first-line treatment for NSCLC.(Pandey et al., 2019;Uddin etal, 2020;Mandal et al., 2016).
Although Erlotinib demonstrates good efficacy in combating cancer, it is associated with several side effects due to its poor solubility in water and its high affinity for binding with blood plasma proteins.According to the biopharmaceutical classification system, Erlotinib falls into category two, indicating limited bioavailability resulting from its poor aqueous solubility (S 0 = 12.46 μM), It has a high lipophilicity with a log P value of 2.75 and a protonation constant of log K=5.33.(Erdoğar et al., 2021;Pandey et al., 2019;Lee et al., 2021;Salehi et al., 2021;Honeywell et al., 2020;Betriu et al., 2021).Various strategies have been employed to enhance the solubility of Erlotinib (ETB).These approaches can be categorized as physical or chemical methods and encompass techniques such as particle size reduction, crystal engineering, salt formation, solid dispersion, and complexation (Salehi et al., 2021).These approaches have been widely utilized to improve the solubility of poorly watersoluble drugs (Honeywell et al., 2020;Betriu et al. , 2021;Pandey et al., 2019;Zhang et al., 2019).
Nanocrystals preparation is one of the newest physical strategies to improve the dissolution rate of poorly water-soluble materials (Mostafa et al. , 2022;Amin et al. , et al., 2023).According to the Noyes-Whitney equation, the particle size reduction will result in an increased surface area to volume ratio which increases the solubility and dissolution rate of badly soluble drugs (Mostafa et al. , 2022).Improvement of the solubility of low soluble highly permeable molecules will subsequently enhance the bioavailability and efficacy of such drugs (Merisko-liversidge and Liversidge, 2008;Amin et al., 2023;Wang et al., 2018;Hassan et al., 2022;Pandey et al., 2019).
Nanocrystals can be prepared using either the top-down or bottom-up method (Betriu et al., 2021;Zhang et al., 2019).In the top-down technique, larger particles are mechanically comminuted into smaller nanoparticles using a high-energy mill.This method requires a significant amount of energy input, consuming long time, and may introduce contamination.On the other hand, the bottom-up approach involves the precipitation of nanoparticles from dissolved drug molecules.This method yields better product output by achieving a homogeneous particle distribution with lower energy input.It also utilizes cost-effective equipment and occurs at ambient temperatures and atmospheric pressure, thereby preserving the stability of the processed materials (Betriu et al., 2021;Amin et al., 2023;L. Zhang et al., 2019;Hassan et al., 2022;Elkarim et al. , 2022).To ensure the production of stable nanoparticles, it is essential to coat the particle's surface with a suitable stabilizer.This coating serves multiple purposes, including preventing particle growth and aggregation during preparation and storage.Additionally, it contributes to the enhancement of solubility and dissolution properties of the nanoparticles (Betriu et al., 2021;Amin et al., 2023;L. Zhang et al., 2019;Elkarim et al. , 2022).
Hydrogels are composed of polymers that can be crosslinked to form a network structure with a high water-absorbing capacity.(A.M. Mohammed et al., 2020a;Zheng et al., 2019).Thermosensitive hydrogels exhibit a transition from a gel to a sol state upon temperature changes.These hydrogels can contract upon injection, entrapping the loaded content and controlling its release.(Merisko-liversidge & Liversidge, 2008;Rani et al., 2022;Hassan et al., 2022;Elkarim & El-shenawy, 2022;A. M. Mohammed et al., 2020a;Du et al., 2021).Chitosan polymer is biocompatible and biodegradable, making it well-tolerated by the body and capable of gradual degradation, eliminating the need for additional removal surgeries (Zheng et al., 2019;Zheng et al., 2019;Du et al., 2021;Balo et al., 2010).Chitosan hydrogel provides protection to drugs against degradation and inactivation, especially in harsh physiological environments.The gel matrix acts as a barrier, shielding the drug from enzymatic degradation or pH-related instability, improving its stability and bioavailability (Balo et al., 2010;A. M. Mohammed et al., 2020b;Du et al., 2021).
Chitosan hydrogel can encapsulate drugs within its gel matrix, enabling controlled release pattern.The porous structure of the hydrogel allows for diffusion of drugs at a controlled rate, leading to sustained drug release over an extended period (Balo et al., 2010;A. M. Mohammed et al., 2020a;Du et al., 2021;Balo et al., 2010).Moreover, Chitosan hydrogel allows for localized drug delivery by direct application to the target site.This is particularly advantageous for treatment of localized diseases (Balo et al., 2010;Du et al., 2021;Dora et al., 2017).
Various formulations have been developed by researchers to enhance the solubility and bioavailability of Erlotinib.These include the utilization of complexing agents such as α-cyclodextrin, β-cyclodextrin, HP-β-cyclodextrin, and phospholipids (Tóth et al., 2016;Mandal et al., 2016;Dora et al., 2017;Uddin & Ju, 2020 ) .Additionally, nano preparations have been employed to improve the solubility of Erlotinib ( Mandal et al., 2016;Fathi et al., 2017;Pandey et al., 2019;Moradpour & Barghi, 2019;L. Zhang et al., 2019;Pandey et al., 2019;).However, these approaches have certain limitations.Some of these limitations may be associated with factors like drug loading capacity, stability, or potential toxicity.For example, the use of chemical solvents or multiple stabilizers in these formulations may pose risks to human health.It is essential to consider these limitations and explore alternative strategies to address them while developing Erlotinib formulations with improved solubility and bioavailability.
The primary objective of this study was to enhance the solubility of Erlotinib (ETB) by developing stable Erlotinib Nanoparticles (ETB-NPs) without the use of excessive stabilizers and excipients.This was achieved by reducing particle size and increasing the surface area of the drug, leading to improved solubility and dissolution.Span-stabilized nanoparticles were prepared using the solvent anti-solvent technique, where dimethyl sulfoxide (DMSO)/Methyl alcohol (1:4 ratio) served as the solvent and water acted as the anti-solvent.The nanocrystals obtained were subjected to various characterizations, including particle size, solubility, in vitro dissolution, and dissolution kinetics.Additionally, the study focused on formulating ETB-NPs in chitosan hydrogel to achieve a local, controlled, and targeted therapeutic effect.The chitosan hydrogel formulation was evaluated for organoleptic properties, viscosity, drug content, in vitro drug release, and dissolution kinetics.

Preparation of ETB-nanoparticles
Erlotinib nanoparticles (NPs) were prepared by solvent/anti-solvent precipitation technique.In this method, dimethylsulphoxide/methanol mixture (water miscible organic solvent) is considered as the solvent.While as, water was utilized as anti-solvent.Span 80 was utilized as a surface stabilizer.The required amounts of ETB (10 mg) and span80 (5mg) were dissolved in 10 ml of solvent mixture (1:4, v/v ratio) of DIMSO/methanol in a test tube under sonication for 3 minutes at room temperature until a clear solution was obtained.The produced solution was then slowly added dropwise into 50 ml of distilled water by the aid of a syringe (over 20 minutes) under continuous stirring at room temperature using an overhead magnetic stirrer (Daihan Scientific Co., Korea) adjusted at 1000 rpm.To ensure evaporation of the organic solvents, the mixture was stirred for additional 2 hrs.To obtain pure NPs, the produced nano suspension was centrifuged, using high speed centrifuge (Mumbai, India), at 10000 rpm for 30 minutes.The supernatant was discarded, and the precipitated nanoparticles were washed twice with distilled water and dried under vacuum (Zeamil Horyzont Co., Poland) at 50° C for 24 h.The modified ETB-NPs were stored in a desiccator until further use (Mostafa et al., 2022;Gadhiya et al., 2021;Amin et al., 2023;Hassan et al., 2022).

Size measurement
The size, size distribution, and polydispersion index (PDI) were measured at room temperature using Zetasizer (Malvern Zetasizer 300 HAS, Malvern Instruments, UK).Practically, 3 ml of an aqueous solution of each sample (prepared in PBS) was adjusted at 25 C and then allowed to expose to a laser beam of ≈633 nm at a scattering angle of ≈90°.The measurements were carried out in triplicate for each sample.The obtained results were presented as the average of the three measurements ±SD, whereas each measurement was run ≈20 times (with a≈10 s duration) (H. A. Mohammed et al., 2019;Shariare et al., 2018;Okafor and Aigbavboa, 2019).

Determination of Saturation Solubility
Generally, the saturation solubility of materials depends mainly on the temperature and the properties of the dissolution medium.However, below a size of approximately 1-2 µm, the saturation solubility is also a function of particle size (Pressure et al., 2015) .The saturation solubility of pure drug, physical mixture, and Span stabilized nanoparticles were determined in phosphate buffer (pH, 6.8).Typically, an excess amount of each sample was added to 10 ml of the buffer in screw glass tubes which were shacked at 50 rpm for 48 hrs in a thermostatically controlled water bath shaker fixed at 37 o C. Afterwards, samples were centrifuged at 10000 rpm at 4 o C and the drug concentration in supernatants was then analyzed spectrophotometrically (Shimadzu Co., Japan) at max 334 nm (Agrawal et al., 2004) .

Determination of the Production Efficiency (PE)
PE of the modified ETB-NPs was determined by dissolving a known amount (5 mg) of completely dried nanoparticles powder in methanol/DMSO and diluted with phosphate buffer (pH 7.4).The drug concentration was measured spectrophotometrically at 334 nm.The production efficiency of ETB nanocrystals was calculated using equation 1 (Mothilal et al., 2014).PE % = Weight of ETB in NPs /Total weight of NPs × 100 eq (1)

In vitro Dissolution Studies
The in vitro dissolution profiles of either native ETB or ETB-NPs were performed using USP dissolution apparatus type II (Dissolution test apparatus, SR II, 6 flasks, paddle type, Hanson research Co., USA).Typically, 10 mg of either pure drug or an equivalent amount of the modified drug NPs was dispersed in 900 ml of PBS (pH 7.4) which was stirred at 100 rpm and maintained at 37±1 °C for 2 hrs.At specified interval times, a liquate of 10 ml samples was taken, filtered using filter tip, and replaced immediately with an equal volume of fresh dissolution medium to maintain the dissolution volume constant.The drug concentration was determined spectrophotometrically (Shimadzu 1601, Japan) at max 334 nm.Each experiment was done three times and the results were presented graphically as the cumulative percent of dissolved drug against time.

Kinetic treatment of ETB-NPs dissolution data
The in vitro dissolution data were analyzed using various kinetic models, including zero order, first order, Higuchi diffusion, and Korsmeyer-Peppas models ((A.M. Mohammed et al., 2020b;Mostafa et al., 2022) as presented in Tables 1 and 2. The objective was to determine the most suitable dissolution model that accurately describes the drug dissolution pattern .The selection of the model was based on the coefficient of determination (R 2 ) values as recommended by Burnham and Anderson ((Y.Zhang et al., 2010) Amin, Osman, et al., 2023;Mostafa et al., 2022;Rani et al., 2022;Paarakh et al., 2018).
Table 1.Kinetic equations for different kinetic models (zero order, first order, Higushi diffusion and Korsmeyer-Peppas (A.M. Mohammed et al., 2020b) Where Qt is the quantity of the drug dissolved at time t, K is the dissolution rate constant, and n is the diffusional exponent that characterizes the best fitted release mechanism (Y.Zhang et al., 2010).

Preparation of chitosan hydrogel loaded ETB-NPs
Different chitosan/Pluronic F127 hydrogel systems were constructed according to the previously reported method (Abdellatif et al., 2022;Berger et al., 2005).Practically, a 1.8% w/v chitosan solution was prepared using 0,1M acetic acid as solvent.To obtain a solution mixture of Pluronic/β-GP Also, 10% w/v pluronic solution and 35% w/v β-GP solution were prepared using distilled water.Then, the aqueous solution was added gradually to chitosan solution at 0 •C for 15 min until the hydrogel formation.For comparison, a separate hydrogel system was prepared according to the previous procedure but without the addition of Pluronic solution or without addition of β-GP solution.The composition of different hydrogel systems was presented in Table 3.The drug loading was achieved by incorporating unmodified ETB and ETB-NPs at a concentration of 1% w/v into the chitosan solution before crosslinking by gentle stirring until complete dissolution.Then, the prepared unmodified ETB and ETB-NPs loaded hydrogel systems were stored in the refrigerator at 8 •C until further experiments.A one-gram sample was prepared, containing either unmodified ETB or ETB-NPs equivalent to 1 mg of ETB.To dissolve the sample, an appropriate quantity of a mixture of dimethyl sulfoxide (DMSO) and ethyl alcohol was used.The resulting solution was then subjected to filtration using a Whatman filter paper to remove any impurities or solid particles.(Lee et al., 2021).The obtained filtrate was further diluted with distilled water to an appropriate concentration.The drug content was determined using a UV-Visible Spectrophotometer at 334 nm.

Visual Inspection (Organoleptic Properties)
The prepared systems underwent visual examination in both sol and gel forms to assess their purity, homogeneity, fluidity, and presence of phase separation.

pH Determination
The pH values of the prepared hydrogel systems were determined using an Ama Digital pH meter (Ama Co., Germany).The probe of the pH meter was immersed directly into the samples, enabling straightforward and accurate measurement of the pH values of the hydrogel systems.

Viscosity Measurement
An Ostwald U tube capillary viscometer was used to evaluate the viscosity of the produced chitosan hydrogel solutions at room temperature (25±1°C).In the viscometer, the liquid is briefly added till the level reaches the target.In order for the viscometer to reach the proper temperature, it is positioned vertically in a thermostatic bath.Once the liquid level is just over the mark, the sample volume is adjusted and the liquid is sucked or blown into the other arm.When the pressure or suction is removed, the time for the liquid to fall from the top mark to the bottom mark is recorded (H. A. Mohammed et al., 2019;LLOYD, 1958).Chitosan hydrogels' viscosity was assessed at 37°C using a Brookfield DV-III ultra-viscometer and a T-bar spindle (T-D 94) spinning at 50 rpm (Abdellatif et al., 2022).

Syringeability and injectability
The ability of dosage form to be easy injectable is very important issue.Noteworthy. the injectable system should have a suitable consistency in order to be released through the syringe needle into skin layers (Ramstack et al., 2002) .Therefore, the injectability test was carried out by filling a syringe with the hydrogel solution at room temperature and then allowed to be injected into a meat sample by the aid of finger pressure (Ramstack et al., 2002).

Sol-Gel Transition Temperature Measurement
To determine the gelation temperature (Tgel), the cold solutions of various hydrogel systems were heated on a water bath.The temperature range used for heating ranged from 8 to 40°C, and the heating process was conducted at a constant rate (1°C /min) with continuous stirring at a rate of shear (30 rpm).During the heating process, the gelation of the hydrogel systems was indicated by the point at which the magnetic bar within the solution stopped to move.At this point, the temperature was recorded as the gelation temperature (Tgel) for each respective hydrogel system (Balo et al., 2010).

In Vitro Drug Release Studies
Design of Experiment (DOE) was employed as a statistical technique to investigate and comprehend the influence of various factors, both individually and in combination, on the responses or outcomes of interest, with the aim of optimizing these responses and analyzing the factors under study.Examining each factor separately can be a laborious and time-consuming task.However, by utilizing a full factorial design, these limitations can be overcome as it allows for the simultaneous optimization of multiple factors, thereby reducing the total number of experiments required to achieve process optimization (Seyed Shahabadi & Reyhani, 2014;Arena et al., 2007;Amin, Amin, et al., 2023).
In the current study, the primary and interactive effects of two experimentally studied factors, namely "Drug " and "Chitosan Hydrogel Formula," were investigated using model equations designed by a three-level full factorial design.The predicted results obtained from the full factorial design model exhibited high values of R 2 , and the significance value ( P-value) indicating agreement with the experimental data and confirming the appropriateness of the full factorial design model (Mishra & Singh, 2010;Shokri, 2018).Moreover, to study the effects of the independent variables, namely "Drug " and "Chitosan Hydrogel Formula," on the response variable "% release" and determine the optimal formula, a three-level two-factor full factorial design was implemented using JMP® (version 16, SAS, USA).JMP® was utilized to statistically analyze the results and visualize the findings, enabling a comprehensive exploration of the data (Shokri, 2018;Amin et al., 2023;Rostami et al., 2019) Practically, Eppendorf tubes containing 0.5 ml samples were prepared, consisting either 0.5 mg of un-modified ETB or ETB-NPs.These samples were then incubated at 37°C for 1 hour to facilitate gelation.After gel formation, the gels were exposed to 1 ml of phosphate buffer solution at pH 7.4, maintained at 37°C and shaken at 50 rpm.(Mccarthy et al., 2021;A. M. Mohammed et al., 2020a).At specific time intervals, 0.5 ml samples were withdrawn from the system, and an equivalent volume of fresh buffer solution was added to maintain sink conditions.To determine the amount of drug released, a newly developed UV-spectrophotometric method was employed for simultaneous analysis of ETB.Each measurement was performed in triplicate, and the obtained data were reported as means ± standard deviation (SD).(A.M. Mohammed et al., 2020a).

Kinetic treatment of ETB-NPs dissolution data
The in vitro dissolution data were analyzed using various kinetic models, including zero order, first order (Y.Zhang et al., 2010), Higuchi diffusion, and Korsmeyer-Peppas models (A.M. Mohammed et al., 2020b ;Mostafa et al., 2022).The objective was to determine the most suitable dissolution model that accurately describes the drug dissolution pattern.The selection of the model was based the coefficient of determination (R 2 ) values as recommended by Burnham and Anderson (Y.Zhang et al., 2010;Amin et al., 2023;Mostafa et al., 2022).Table 1 and 2 represent the utilized kinetic equations.

Preparation of ETB-NPs
Particle size, polydispersity index (PDI) and surface charge (zeta potential) are considered as the most important parameters for characterization the prepared nanocrystals.Particle size is considered as a reflective mirror for physicochemical properties such as physical stability, dissolution pattern, saturation solubility, and bioavailability.Also, pharmacokinetic parameters and therapeutic efficacy of the particular drug may be affected by the heterogeneity of size distribution (Janga et al., 2012).Figure 2 showed the size and PDI of the modified ETB-NPs. the results indicated the successful formation of ETB nanoparticles via the anti-solvent method.The relatively uniform particle size and shape, with a low PDI of 0.13 and 100% intensity for ETB with an average size of 230 nm, indicates that the nanoparticles are relatively uniform in size and shape, which is desirable for pharmaceutical applications.

3.2.In vitro drug dissolution
Figure 3 display the in vitro dissolution profile of ETB and ETB-NPs.The results show that the formulated nanoparticle achieved a drug dissolution rate of approximately 100% in 105 minutes in either phosphate buffer(pH7.4) or distilled water.This represents a 7-fold increase in drug release compared to pure ETB, which only achieved a drug release of 14.4% in distilled water.In phosphate buffer (PH 7.4), ETB-NPs exhibited a 22-fold increase in drug release, with approximately 100 % drug dissolution within 105 minutes, compared to only 4.68% drug dissolution for pure ETB.The significant enhancement in dissolution can be attributed to the particle size reduction of ETB-NPs to the molecular level, which increases the surface area.
During a 60-minute period, ETB-NPs exhibited a 67% release rate, whereas pure ETB demonstrated only an 8.6% release rate in distilled water, resulting in an eight-fold increase in dissolution.In Phosphate buffer (pH7.4),ETB-NPs showed a 68% release rate, while pure ETB exhibited only a 17% release rate, resulting in a four-fold increase in dissolution.The significant enhancement of ETB solubility, achieved via NPs formation, may have important implication for their potential use as drug delivery systems.The high surface area to volume ratio of nanoparticles can increase the dissolution rate of poorly soluble drugs, resulting in faster onset of action and improved therapeutic efficacy (Shariare et al., 2018).

3.3.Kinetic treatment of dissolution data profile of ETB and ETB-NPs
The dissolution data of ETB were analyzed using four kinetic models: zeroorder, first-order, Peppas and Higuchi models.The analysis was performed separately for two different dissolution media: distilled water and phosphate buffer (pH 7.4), the obtained results were tabulated in (Tables 4 and 5).
Whether in distilled water or phosphate buffer (pH7.4), the dissolution of ETB followed the zero order kinetic model, as indicated by the high values of determination coefficient R 2 , which was obtained from the analysis (Tables 4 and 5).Zero order Regarding ETB-NPs, the dissolution mechanism, whether in distilled water or phosphate buffer (pH 7.4), was described by the Korsmeyer-Peppas model Figure 5. Furthermore, the Fickian constant 'n' was calculated using the Korsmeyer-Peppas model, which helps distinguish different release processes.In conclusion, the approach employed to enhance the solubility of ETB played a significant role in the dissolution mechanism, as evidenced by the results presented in When quantitatively evaluating drug dissolution characteristics and comparing dissolution profiles using model-dependent approaches, it is crucial to select an appropriate model for fitting the data.Various statistical criteria are commonly employed to assess the goodness of fit of a model in this context.
Coefficient of determination (R 2 ) measures the proportion of the data variation that can be explained by the model (Y.Zhang et al., 2010)., namely R 2 , was widely utilized for the evaluation of drug dissolution kinetic models and the comparison of dissolution profiles in model-dependent approaches.
Table 4 presents an analysis of the dissolution behavior of ETB-NPs in distilled water and phosphate buffer (pH 7.4) using different kinetic models.The results indicate a strong fit of the Korsmeyer-Peppas model to the dissolution data, as evidenced by high R 2 values of 0.9805 in distilled water and 0.9705 in phosphate buffer.These findings confirm the successful evaluation of ETB-NPs dissolution behavior using the Korsmeyer-Peppas kinetic model.The improved solubility of ETB resulting from nanoparticle formation is likely responsible for the favorable results observed in the dissolution kinetics model.
The dissolution behavior of ETB was examined in both distilled water and phosphate buffer (pH 7.4) using various kinetic models.The results indicate that the zero-order kinetic model adequately describe the dissolution of ETB in both media.This conclusion is supported by the values of R 2 .In distilled water, the R 2 value of 0.6978 suggests a reasonable fit to the zero-order kinetic model.In phosphate buffer (pH 7.4), the fit to the zero-order kinetic model is weaker, as indicated by the lower R 2 value of 0.2067.Overall, these results suggest that the zero-order kinetic model provide a reasonable description of the dissolution behavior of ETB in both distilled water and phosphate buffer (pH 7.4).It is worth noting that the fair fit observed may be attributed to the low aqueous solubility of ETB.
Table 5 displays the results for ETB-NPs in distilled water, with DE (dissolution efficiency) of 0.558, MDT (mean dissolution time) of 48.113, MRT (mean residence time) of 35.867, RD (relative dissolution) of 0.561, and VDT (variance of dissolution time) of 1298.916.Notably, DE show relatively high value, while (MDT) and (MRT) exhibit low values compared to the other parameters in the table.These findings can be attributed to the significant improvement in ETB solubility resulting from the formation of nanoparticles, as well as the favorable dissolution rate observed for ETB-NPs in distilled water Furthermore, when considering the dissolution of ETB-NPs in phosphate buffer (pH 7.4), the value of (DE) was relatively high, measuring 0.577.On the other hand, the values of (MDT), (MRT), (RD), and (VDT) were comparatively low, with values of 47, 34.572, 0.570, and 1259.75, respectively.These positive outcomes can be attributed to the favorable solubility of ETB-NPs in phosphate buffer (pH 7.4).
In contrast, when considering the dissolution of ETB (unmodified) in either distilled water or phosphate buffer (pH 7.4), the following values were observed.Comparing these values with the corresponding values for ETB-NPs, it becomes apparent that the aqueous solubility of ETB is significantly poorer when dissolved in distilled water.This is evidenced by the low value of (DE), as well as the high values of (MDT), (MRT), and (RD).kinetic data of dissolution profile of ETB&ETBNPs was illustrated in figures (4&5).

Preparation of ETB-loaded Chitosan Hydrogels
The drug loading was achieved by incorporating unmodified ETB and ETB-NPs at concentration of 1% w/v into chitosan solution before crosslinking by stirring at 500rpm for 30 minutes to ensure uniform drug distribution.

Characterization of the prepared pure chitosan hydrogels 3.5.1.1. Visual inspection (organoleptic properties)
Before and after the gelation process, the sensory qualities of the chitosan samples were assessed visually.The results showed that there were no lumps or signs of phase separation in either the liquid or gel states, and that all of the formulations had a uniform and homogeneous texture.The liquid was transparent and colourless, whereas the gel had a whitish, hazy appearance.

pH determination
The produced chitosan hydrogels for PL F127 and β-GP were found to have a pH range of 6.9 to 7.3, as shown in the Table 6.This pH range shows that the injectable suitability of all the formulations.Injectable hydrogels should have a pH between 6.5-7.4 in order to prevent cell damage, according to earlier study (Gupta P et al. ,2002).

Viscosity measurements
The produced hydrogels' viscosity measurements in both the sol and gel stages are shown in Table 6.Because water is the main ingredient in all of the formulations, the results show that all hydrogel solutions have a comparable level of viscosity.For instance, the gel state viscosities for CH/ -GP, CH/PL F127, and CH/ -GP (35%)/ PL F127 (10%) were 114 Cp, 117.8, and 153 Cp, respectively.The increased interaction between the crosslinkers and the chitosan solution after mixing may be the cause of the higher viscosity in the gel state.On the other hand, the sol state viscosities for CH/ -GP (35%), CH/PL F127 (10%), and CH/ β-GP (35%/PL F127 (10%), respectively, were determined to be 110 Cp, 137.2 Cp, and 148.3.The CH/ β-GP (35%)/ PL F127 (10%) exhibited the maximum viscosity, measuring at 148.3.

Syringeability and injectability
For injectable hydrogels to guarantee a specific level of syringeability and injectability, it is significant to note that the sol-gel transition phenomenon is a fundamental prerequisite.The cross-linking agents used were either 35% -GP, 10% PL F127, or 35% -GP-10% PL F127.With light to moderate finger pressure, these solutions may be smoothly and easily extruded from a 5 mm syringe, yielding a consistent 0.5 cm of solution in 5-10 seconds (Table 6).These results indicate that these solutions are suitable for syringe injection testing with meat samples which comes with patient compliance.

Sol-gel transition temperature measurement
All of the synthesized chitosan solutions underwent a sol-to-gel transition in response to temperature changes.The temperature at which gelation takes place for each formulation is displayed in Table 6.It is thought that the mechanisms underlying these sol-to-gel transitions involve a switch in the equilibrium from monomer to micelle, development of the micelle, or expansion of the micelle coupled with an increase in the number of aggregations brought on by hydrophobic or attractive forces (Liu D-M et al.2001;Jeong B et al., 2012).

In vitro drug release studies
Design of experiments (DOE) facilitates the simultaneous assessment of the impact of drug and chitosan hydrogel formula as well as their actual significance on the % of drug release within seven days.Table 7 describes the full factorial design.Investigation of the effect of drug and chitosan hydrogel formula revealed the significant effect of drug on % of drug release within seven days (P-value=0.00008)(Figure 6), while chitosan hydrogel formula had statistically insignificant effect on % of release (P-value=0.57)(Figure 7).The interactive effect of drug and chitosan hydrogel formula was insignificant (P-value= 0.98), the experimental data had a high degree of correlation with the predicted response, the % of release actual Vs. the predicted plot had an R 2 of 1 and a P-value of 0.0002, The statistical factorial approach optimized the response using the desirability function.The desirability is a mathematical method used to spot the optimized formulation.The optimization was set for drug (ETB-NPs).
From factorial design we have selected ETB-NPs as drug dosage form exhibited the maximum % of release, Regarding ETB-NPs, there were notable variations in the percentage of drug release within one week among different formulas.Specifically, formula no. 2 exhibited a substantial release of 91.26%, whereas formula no. 1 resulted in a 30% release rate.Interestingly, formula no. 3, which is the reverse of the previous statement, showed a release percentage of 27.76%.In contrast, when considering ETB by itself, the release percentages from different formulas showed distinct patterns.Formula no. 2 had a significantly lower release rate of 2.65%, while formula no. 1 yielded a moderately higher release of 1.75%.Lastly, formula no. 3 had the lowest release percentage for ETB at 0.17%.The results showed that the percentage of drug release was the highest from chitosan-pluronic F127 (104% drug release was achieved within 10 days for ETB-NPs but only 4.5% drug release was obtained within 30 days for native ETB), this attributed to the great enhancement of solubility of ETB by nanoparticle formation.On the other hand, the percentage of drug release were the smallest from chitosan/B-Glycerophosphate/Pluronic F127 (F3).Furthermore, the controlled release of drugs from nanoparticles can also prolong the duration of action and reduce the frequency of dosing, which can enhance patient compliance and reduce the potential for side effects.

Kinetic analysis
The release data of ETB were analyzed using four kinetic models: zero-order, first-order, Peppas, and Higuchi models.The analysis was performed separately for three different chitosan hydrogel formula F1, F2 and F3 (Figures 9 and 10 as well as Table 8 and 9).
The release of ETB from chitosan hydrogel formula (F1) followed the Korsmeyer-Peppas mechanism, Higuchi model in case of F2 and Korsmeyer-Peppas mechanism in case of F3, as indicated by the high value of coefficient of determination (R 2 ) obtained from the analysis (Figure 9).In case of ETB-NPs release from F1 followed to Kerseymere-Peppas mechanism, followed Higushi in case of F2 while from F3 it was followed zero order kinetic mechanism (Figure 10).Table 8 presents an analysis of the release behavior of ETB and ETB-NPs from different chitosan hydrogel systems using various kinetic models.For ETB-NPs in formula F1, the results indicate a strong fit of the Korsmeyer-Peppas model to the release, as evidenced by high R 2 value of 0.997.These finding confirm the successful evaluation of ETB-NPs release behavior using the Korsmeyer-Peppas kinetic model.Regarding the release mechanism of ETB-NPs from formula F2, it was observed that the release mechanism follows Higushi diffusion model, as indicated from the values of R 2 value of 0.9968 (Table 8).However, in the case of ETB-NPs release from hydrogel F3, the values of R 2 indicate a good fit of the zero-order kinetic model to the release, with value of 0.9932.
On the other hand, the release behavior of unmodified ETB from different gel formula was investigated.The results indicated that Higushi diffusion model adequately describes the release in formula F1, with R 2 value of 0.9864.Similarly, in formula F2, the Higushi diffusion model is also suitable for describing the release of ETB, with R 2 value of 0.9767.But, in the case of F3, the release of drug follows the Korsmeyer-Peppas model, as indicated from the highest value of R 2 (0.9918).
Table 9 displays the parameters for characterizing release data profile of ETB and ETB-NPs from different chitosan hydrogel systems.An analysis was conducted to examine the release behavior of ETB-NPs from various chitosan hydrogel systems using different kinetic models.The results obtained were as follows: For F1: The DE and MDT were 0.54, and 11, respectively.Regarding gel F2, the values of DE, and MDT, were 0.666, 3.27, respectively.Whiles as in the case of F3, the values were 0.39, and 8.26, respectively.From the obtained results it could be concluded that the release from F2 demonstrated the highest DE and the smallest MDT, VDT, and MRT, compared with the other two investigated gel formula.The obtained results come in a good agreement with results of in vitro release studies.These findings can be attributed to the significant improvement in ETB solubility resulting from the formation of nanoparticles.Additionally, the favorable dissolution rate observed for ETB-NPs in distilled water contributes to these results.
Regarding the release of unmodified ETB from different chitosan hydrogel systems, the following values were obtained: In the case F1, the dissolution efficiency DE was 0.022, mean dissolution time (MDT) was 5.4, the relative dissolution (RD) was 0.92.whiles as in the case of F2, the results showed that the values of DE, MDT, and RD were 0.034, 4.032, and 1.11, respectively.Also, the obtained values in the case of F3 were 0.013, 8.439, and 0.23, respectively.As a conclusion, among these systems, F2 exhibited the highest dissolution efficiency and relative dissolution and the lowest MDT compared with either F1 and F3.
By taking F2 as a selected formula in order to compare between the modified and the unmodified Drug, the results which was presented in table 8 showed that the obtained very low DE value (0.034) of pure drug from F2 compared with that of ETB-NPs (0.666) can be attributed to the poor aqueous solubility of ETB.Also, the limited solubility of ETB may hinder its dissolution and subsequent release from the chitosan hydrogel systems, leading to lower DE values compared to ETB-NPs, which increased the drug surface area and consequently improved solubility due to nanoparticle formation.

Conclusion
To enhance the solubility of ETB, we employed a highly effective strategy involving the formation of nanoparticles.Simultaneously, we aimed to address the systemic side effects of ETB by implementing a localized delivery method using an injectable hydrogel.Chitosan hydrogel emerged as the ideal platform for precisely delivering ETB, accommodating both unmodified ETB and ETB-NPs.Our research findings underscored that the most substantial drug release was achieved through the nanoparticle formation process, outperforming the release rate of traditional ETB delivery.Notably, these modified nanoparticles had an average size of approximately 230 nm.In conclusion, these results emphasize the promising potential of ETB in cancer treatment, offering enhanced safety and a reduction in undesirable side effects

Figure 2 .
Figure 2. The size and size distribution of the modified ETB nanocrystal.

Figure 3 .
Figure 3.The dissolution profiles for ETB and ETB-NPS in distilled water (a); and in Phosphate buffer (PH.7.4) (b); also, it represents bar plot for comparison between the effect of distilled water and phosphate buffer (PH.7.4|) on the release data profiles of ETB and ETB-NPs (c), and bar plot comparison between the release data profiles of ETB and ETB-NPs in distilled water and phosphate buffer (d).

Figure 4 .Figure 5 .
Figure 4. a Kinetic treatment of dissolution data profile of ETB in different media plotted according to zero order kinetic mechanism b: Kinetic treatment of dissolution data profile of ETB in different media plotted according to first order kinetic mechanism c: Kinetic treatment of dissolution data profile of ETB in different media plotted according Korsmeyer-Peppas mechanism d: Kinetic treatment of dissolution data profile of ETB in different media plotted according Higushi kinetic mechanism

Figure 6 .Figure 8
Figure 6.a:Regression plot b:Actual by predict plot c: Prediction profiler d: Interaction plot

Figure 8 .
Figure 8. a: ETB-NPS release data profile from different hydrogel systems b: ETB release data profile from different hydrogel systems c: ETB and ETB-NPs release data profile from (F2) hydrogel system d: bar plot for comparison of release data profile of ETB and ETB-NPs from different chitosan hydrogel systems.

Figure 9 .
Figure 9. Kinetic treatment of release data profile of ETB from different chitosan hydrogel systems plotted according to zero order (a), first order (b), Korsmeyer-Peppas (c), and Higushi kinetic mechanism (d).

Figure 10 :
Figure 10: Kinetic treatment of release data profile of ETB-NPs from different chitosan hydrogel systems plotted according to zero order (a), first order (b), Korsmeyer-Peppas (c), and Higushi kinetic mechanism (d).

Table 4 .
kinetic data treatment for dissolution of ETB and ETB-NPs in different media

Table 4 Table 5 .
Parameters for characterizing dissolution curve of ETB and ETB-NPs in different media Dissolution Efficiency (DE); Mean Dissolution Time (MDT) ; Mean Retention Time (MRT); Relative Dispersion (RD); and Variance of Dissolution Time (VDT).

Table 6 .
The physicochemical properties (drug content, pH, viscosity, gelation point, and syringeability) of the modified chitosan hydrogel systems

Table 7 .
Full factorial design run matrix and the obtained responses: drug and Chitosan hydrogel formula are the studied factors, while the %Release is the response

Table 8 :
kinetic treatment for ETB and ETB-NPs release data profile from different chitosan hydrogel systems

Table 9 .
parameters for characterizing release data profile of ETB and ETB-NPs from different chitosan hydrogel systems Relative Dispersion (RD); and Variance of Dissolution Time (VDT).