Development of New Xanthan-Aldehyde/Gelatin Nanogels for Enhancement of Ibuprofen Transdermal Delivery: In-Vitro/Ex-Vivo/In-Vivo Evaluation

Abstract

The aim of this study was to prepare nanogels based on gelatin and xanthan-aldehyde for the enhancement of ibuprofen transdermal delivery. Firstly, the process of formulating nanogels using the reaction of Schiff’s base was optimized using experimental designs. Secondly, the structural characterization of nanogels was performed using laser particle size, zetometry, FTIR (Fourier Transform Infrared Spectroscopy), XRD (X-Ray Diffraction), SEM (scanning electron microscopy), and thermogravimetric analysis. Finally, the evaluation of pharmacological characteristics and formulation therapeutic efficacy were achieved using in vitro dissolution kinetics, ex vivo transdermal diffusion studies, and an evaluation of in vivo anti-inflammatory activity. The results of the experimental plan show that the formulations containing a ratio of 15:10 ibuprofen/polymer and a ratio of 1:2 gelatin/xanthan-aldehyde with a gelling time of 2 h exhibited the best results; the formulations showed a mean diameter of 179.9 ± 6.2 nm, a polydispersity index of 0.193, which confirms monodispersed particles, a zeta potential of 24.7 mV, denoting a high degree of particle stability, and an encapsulation rate of 93.78%. The FTIR spectroscopy analysis showed the formation of imine function in the nanogel, and scanning electron microscopy showed the globular and porous form of the formulation. The incorporation of ibuprofen into nanogels improved their in vitro dissolution kinetics and ex vivo transdermal diffusion. The incorporation of nanogels into a patch system for its in vivo anti-inflammatory activity has shown excellent efficiency with a percentage of edema inhibition at a dose of 25 mg and 50 mg of 38.77 ± 1.6% and 82.03 ± 9.03%, respectively, while the commercial reference gel presented inhibition values at a dose of 25 mg and 50 mg of 10.61 ± 1.71% and 37.03 ± 11.43%, respectively. Thus, the innovative pharmaceutical form of ibuprofen offers a promising model for enhancing drug bioavailability and therapeutic effects while reducing adverse effects.

1. Introduction

Inflammation can be considered as either a secondary symptom or a disease for which various substances of natural or synthetic origin are utilized in treatment. Non-steroidal anti-inflammatory drugs (NSAIDs) are considered the most effective pharmacological approach for alleviating pain and inflammation by inhibiting the cyclo-oxygenase (COX)-2 enzyme, which plays a key role in the biosynthesis of prostaglandins [1]. Ibuprofen is one of the most widely used NSAIDs for the treatment of fever, pain, migraine, and articular rheumatism. In addition, ibuprofen is rapidly absorbed into the body and has a short half-life, which requires repeated administration [2].

The most widely used galenic form for the delivery of ibuprofen uses the oral route; in fact, this medication form highlights the pharmacological properties of this bioactive molecule; nevertheless, the greatest disadvantage of this galenic form remains the direct contact between ibuprofen and gastric mucosa [3,4,5]. As for all anti-inflammatory drugs, digestive disorders remain the most important side effect. The first step is the preparation of gelatin and xanthan-aldehyde nanogels and the optimization of their elaboration parameters using an experiment plan.

Transdermal drug administration offers advantages such as ease of access, a large surface area, and non-invasive delivery while leveraging the skin’s circulatory and lymphatic networks [6]. Therefore, transdermal administration is important for drugs that may cause systemic adverse effects, such as anti-inflammatory drugs.

In the field of health, the design and preparation of nanogels have attracted a great deal of interest in pharmaceutical applications because of their adjustable three-dimensional chemical and physical structure, their good mechanical properties, their high water content, and their biocompatibility [7]. The amphiphilic nature of nanogels allows them to incorporate highly hydrophobic drugs through various interactions, i.e., hydrophobic interactions, van der Waals forces, and electrostatic interactions [8,9], in order to alter their physicochemical and pharmacological properties. An active drug insoluble in an aqueous medium can be solubilized by its incorporation into a micro/nanosystem, thus helping to improve the bioavailability and therapeutic effect of active drugs [10,11,12]. The nanogel encapsulation of highly hydrophobic molecules such as progesterone [13], ketorprofen [14,15], luliconazole [16] and dexamethasone [17] has already been successfully performed.

The contribution of this work is the development of innovative nanogels based on xanthan-aldehyde and gelatin for the enhancement of ibuprofen transdermal delivery. For this purpose, an innovative system for allowing transdermal delivery with the same or better capacities than the oral route is proposed in this work. The first step is the preparation of gelatin and xanthan-aldehyde nanogels and the optimization of their elaboration parameters using a plan of experiments. The second step is the structural characterization of nanogels using laser particle size, zetometry, Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XRD), scanning electron microscopy (SEM), and thermogravimetric analysis. The final step is the evaluation of pharmacological characteristics and formulation therapeutic efficacy using in vitro dissolution kinetics, ex vivo transdermal diffusion studies, and an evaluation of in vivo anti-inflammatory activity.

2. Materials and Methods

2.1. Materials

Ibuprofen was offered by the pharmaceutical group EL KENDI (Algiers, Algeria); the gelatin, sodium tetraborate, and Span80 were purchased from Sigma-Aldrich, France. Hexane, ethanol, and other chemicals were all of pharmaceutical grade and were purchased from Biochem Chemopharma, France. Xanthan-aldehyde (with an aldehyde content in the order of 0.15 mol/g) was synthesized in the Chemical Engineering Laboratory using an oxidation reaction using sodium periodate. The modified natural Xanthan Gum was offered by the pharmaceutical group SAIDAL (Algeria).

2.2. General Procedure for the Formulation of Nanogels

Nanogel formation was based on the cross-linking of two macromolecules; the first is gelatin, carrying amine functions, and xanthan-aldehyde, which is a polymer rich in aldehyde functions. The Schiff’s base reaction between the amino (NH₂) groups of gelatin and the aldehydes (CHO) groups of xanthan-aldehyde allows the formation of an imine bond (C=N) between the two macromolecules [18,19,20,21]. The reaction of Schiff’s base is given in Figure 1.

For the preparation of the nanogels, an aqueous solution of xanthan-aldehyde (10%) was prepared in a 0.1 M sodium tetraborate solution. A 10% gelatin solution was prepared in distilled water at 40 °C; then, an adequate amount of ibuprofen was dispersed in this solution using an ultrasonic bath (Branson 2800, St. Louis, MO, USA). The two suspensions prepared were mixed in different proportions to obtain a 10 mL final volume of polymer suspension. The resulting mixture was dispersed in 20 mL of cyclohexane containing 40 mg of Span80, and the resulting system was ultrasonically homogenized to produce an inverse mini-emulsion. The mini-emulsion was agitated to allow for the gelling of nanodroplets and the encapsulation of ibuprofen in the nanogels. The resulting nanogels were separated by centrifugation using a centrifuge (Sigma 3-30K, Osterode am Harz, Germany). After three rinses, the nanogel was recovered and freeze-dried.

2.3. Optimization of the Formulation Process

For the optimization of the nanogel formulation process, a full factorial experimental design was used. A complete factorial design using two levels, three factors, and a point in the center was chosen. The three factors studied were the ratio of ibuprofen/polymer (X1, with a high level (15:10) and a low level (5:10)), the ratio of gelatin/xanthan-aldehyde (X2, with a high level (2:1) and a low level (1:2)), and the gelling time (X3, with a high level (8 h) and a low level (2 h)). The number of experiments was calculated using the following equation:

N = L^K + C

(1)

where N is the number of experiments, L is the number of levels, K is the number of factors, and C is the number of points in the center. Then, the total number of tests was of the order of 2³ + 1 = 9 tests. The test matrix is given in

Table 1. Matrix obtained using the complete factorial design.

Factors Tests	X1		X2		X3
	Level	Value	Level	Value	Level	Value
Test 1	+1	15:10	+1	2:1	+1	8 h
Test 2	−1	5:10	+1	2:1	+1	8 h
Test 3	+1	15:10	−1	1:2	+1	8 h
Test 4	−1	5:10	−1	1:2	+1	8 h
Test 5	+1	15:10	+1	2:1	−1	2 h
Test 6	−1	5:10	+1	2:1	−1	2 h
Test 7	+1	15:10	−1	1:2	−1	2 h
Test 8	−1	5:10	−1	1:2	−1	2 h
Test 9	0	10:10	0	1:1	0	5 h

.

The studied answers were Y1: encapsulation rate (ER) in %; Y2: mean diameter (MD) in nm; Y3: polydispersity index (PI); Y4: zeta potential (ZP) in mV.

The dependence of each experimental response, $Y_i$, on its factors was modeled by applying the following equation [22,23,24]:

$$Y_i={\beta }_0+{\sum }_{i=1}^n\beta ixi+{\sum }_{i=1}^n{\sum }_{j=i+1}^n\beta ijxixj+\epsilon$$

(2)

where ${\beta }_0$ is the constant term, ${\beta }_i$ and ${\beta }_{ii}$ are the regression coefficients, and $\epsilon$ is the error.

The experimental results were analyzed using MODDE 6 software (Umetrics, Sweden, 2001).

2.4. Characterization of Nanogels

2.4.1. DLS Analysis (Mean Diameter and Polydispersity Index)

The granulometry (mean diameter and polydispersity index) of the obtained nanogels was measured using the dynamic scattering of light. Measurements were made with a Horiba brand DLS device (SZ-100, Tokyo, Japan). Hydrodynamic diameters were calculated from the autocorrelation function of light intensity diffused by particles. The software used was Next Spec version 1.0.0.1. All samples were diluted with distilled water before being placed in the apparatus; a quartz cell with four smooth faces was used. All measurements were taken at 25 °C [25].

2.4.2. Zeta Potential

The rate of particle movement under the influence of an external oscillating electric field was measured with a Horiba Zetameter (SZ-100, Kyoto, Japan). All samples were diluted and placed in a 6 mm gold plating cell. All measurements were taken at 25 °C.

2.4.3. Encapsulation Rate

A total of 10 mg of dry nanogel was dispersed in 5 mL of ethanol and placed in an ultrasound bath for 15 min to extract the entire encapsulated ibuprofen inside the nanogel. Then, the suspensions were filtered, and their absorbance was measured at 264 nm using a UV–visible spectrophotometer (Hitachi U-5100, Ibaraki, Japan). The encapsulation rate was calculated using an ibuprofen calibration curve, previously performed under the same operating conditions using standard solutions of concentrations ranging from 0.5 mg/mL to 0.15 mg/mL. The encapsulation rate was calculated using the following formula:

$$ER={\displaystyle \frac{m_{exp}}{m_{int}}} \ast 100$$

(3)

where ER, $m_{exp}$, and $m_{int}$ are the encapsulation rate in %, the experimental mass in mg, and the initial mass in mg (theoretical, which varies depending on the test studied: 0.15, 0.1, and 0.05 mg), respectively.

2.5. Structural Analysis of Nanogels

2.5.1. FTIR Analysis

The FTIR spectra of xanthan-aldehyde, ibuprofen, gelatin, and the prepared nanogels were performed using a brand-name FTIR-8900 spectrometer (Shimadzu, Shanghai, China) over a wavenumber range of 400 and 4000 cm⁻¹. The analyzed samples were prepared as KBr pellets.

2.5.2. X-Ray Analysis

X-ray diffractograms of xanthan-aldehyde, ibuprofen, gelatin, and the prepared nanogels were obtained using a Philips PW 3710 X-ray diffractometer (Cambridge, MA, USA). The tube was operating at 45 kV and 9 mA.

2.5.3. SEM Analysis

The analysis was performed using a type of scanning electron microscope QUANTA-400 FEI (Hillsboro, OR, USA). The sample was placed directly on a carbon pellet; an LFD detector was used in this analysis.

2.6. In Vitro Dissolution Kinetics

The dissolution tests for the nanogels and free ibuprofen were carried out using a dissolutest with a pallet system of the brand Erweka (Gif sur Yvette, France). Briefly, 250 mg of dried nanogels or pure ibuprofen powder were incorporated into a vessel containing 500 mL of phosphate buffer at pH 7.4; the pallet rotation speed was set at 50 rpm, and the temperature was set at 37 °C. After each 30 min interval, a 5 mL sample was taken and replaced by an equivalent volume of phosphate buffer; the samples were filtered using a 0.2 µm porosity syringe filter. The ibuprofen content in each sample was determined using UV–visible spectrophotometry at 264 nm and the previously established calibration curve. The total time of dissolution kinetics was 120 min.

The mechanism and release kinetics of the drug were derived by adjusting the dissolution curves to different mathematical models: zeroth order, first order, the model of Higuchi [26], and the Korsmeyer-Peppas equation [27]. To describe release behavior, the best fit was established by choosing the correlation coefficient closest to 1. The equations of the mathematical models used are given in

Table 2. Mathematical models used.

Mathematical Model	Equation
Zeroth-order equation	${\displaystyle \frac{M_t}{M_{\infty }}}=k_{0}t$ (4)
First-order equation	${\displaystyle \frac{M_t}{M_{\infty }}}={-k}_{1}t$ (5)
Higuchi equation	${\displaystyle \frac{M_t}{M_{\infty }}}=k_H{t}^{1/2}$ (6)
Korsmeyer-Peppas equation	${\displaystyle \frac{M_t}{M_{\infty }}}=k_{Kp}{t}^n$ (7)

.

$M_t/M_{\mathrm{\infty }}$ is the fraction of drug released at time t, $k_0$ is the zeroth-order release constant, $k_1$ is the first-order release constant, $k_H$ is the Higuchi rate constant, and $k_{Kp}$ is the kinetic constant of the Korsmeyer-Peppas model. n is less than 0.5 for a Fickian diffusion, and n is between 0.5 and 0.1 for a non-Fickian diffusion; n = 1 denotes the zeroth order, corresponding to the case II version; a number higher than 1 is used for the super case II mechanism [27,28].

2.7. Ex Vivo Transdermal Diffusion Study

To study the ex vivo diffusion of the nanogels, a Franz cell with freshly prepared skin was used. Wistar rats weighing about 150 g were anesthetized, their abdomens were shaved, and the skin was retrieved after dissection. The removed skin was placed in a diffusion cell, and the dermal side of the skin was brought into contact with the liquid (phosphate buffer), thermostated at 37 °C, which moisturizes and maintains the physiological integrity of the skin explant. The diffusion kinetics of encapsulated ibuprofen in the nanogels was compared to free ibuprofen; for this purpose, a 15% ibuprofen solution was prepared in PEG400 (comparative solution). Nanogels or ibuprofen solutions were gently deposited on the upper side (epidermal) of the explant. The study was conducted for 6 h, and 0.5 mL volumes were taken each hour. The collected samples were filtered by a 0.2 µm syringe filter and analyzed using UV–visible spectroscopy at 264 nm to determine the amount of ibuprofen that passed through the skin [29,30,31]. In order to better understand the diffusion kinetics of ibuprofen through the skin, several parameters were determined. After plotting the ibuprofen permeation time cumulative curve, we determined the slope in the linear region that corresponds to steady-state flux ($J_{ss}$) (its unit is mg.cm⁻².h⁻¹) and its intersection with the x-axis that corresponds to lag-time ($L_{gt}$) (its unit is the time). In addition, we calculated the steady-state permeability coefficient ($K_{app}$) (its unit is cm.h⁻¹) using the following equation:

$$K_{app}={\displaystyle \frac{J_{ss}}{C_d}}$$

(8)

where $C_d$ is the concentration of ibuprofen in the nanogels applied to the skin in the donor compartment of the Franz cell.

2.8. In Vivo Anti-Inflammatory Activity

2.8.1. Patch Preparations for In Vivo Study

Patches measuring 3 cm long and 3 cm wide were prepared (Figure 2). First, a hypoallergenic tissue was impregnated with a bioadhesive glue, and then the rehydrated nanogels were placed in the patch center on a surface of 1 cm². For this purpose, patches containing two doses of ibuprofen were prepared (patches containing 25 mg of ibuprofen and 50 mg of ibuprofen).

2.8.2. Animal Material and Ethics Statement

The in vivo anti-inflammatory activity was determined using the carrageenan-induced paw edema in the rat assay [32].

Ethical approval for the animal studies was obtained from the Experimental station of Saad Dahleb University, Blida 1, Blida, Algeria, and this study was carried out in strict accordance with the ethical standards in animal experimentation set by EU Directive 2010/63/EU [33].

The in vivo anti-inflammatory activity was investigated in 30 Wistar-type female rats, with weights ranging from 120 g to 150 g. These animals were obtained from the Pasteur Institute of Algiers (IPA, Algiers, Algeria). The animals were placed in polypropylene cages where they had free access to water and food. The animals were given an adaptation period of 7 days. Prior to the anti-inflammatory study, the animals were kept under fasting conditions (no food only) for 18 h.

2.8.3. Experimental Design and Inflammation Induction

The 30 rats were randomly divided into 5 groups of 6 rats each; the experimental design of this study is given in

Table 3. Experimental design of in vivo anti-inflammatory activity.

N°	Groups	Treatments
1	Negative control	No treatment
2	Commercial gel	25 mg of ibuprofen (Commercial gel)
3	Commercial gel	50 mg of ibuprofen (Commercial gel)
4	Nanogel patch	25 mg of ibuprofen (Nanogel patch)
5	Nanogel patch	50 mg of ibuprofen (Nanogel patch)

. All rats were shaved in the dorsal region to apply the different pharmaceutical forms studied (gels and patches).

After drug application to the rats of groups 2, 3, 4, and 5 (at time T0), the animals were returned to the metabolic cages.

At time T0 + 30 min, the rats of the 5 groups received 0.025 mL of carrageenan each using injection under the plantar fascia of the left hind leg.

At time T0 + 4 h, the rats were sacrificed by cervical dislocation under anesthesia, and their right and left hind legs were cut at the joint; then, they were weighed on an analytical scale. The results were expressed as the variation in paw weight (mg), calculated as the difference from the non-injected paw’s weight [34].

The percentage increase in leg weight (percentage of edema) was calculated using the following formula:

$$\mathrm{Percentage} \mathrm{of} \mathrm{edema}={\displaystyle \frac{\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{g} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{l}\mathrm{e}\mathrm{f}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{g} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}{\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{g} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}}\times 100$$

The difference in the control and the carrageenan-treated paw weights served as an index of reduction edema. The calculation for the percentage reduction in edema in treated rats compared to controls is

$$\mathrm{Percentage} \mathrm{of} \mathrm{edema} \mathrm{reduction}={\displaystyle \frac{\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{d}\mathrm{e}\mathrm{m}\mathrm{a} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{d}\mathrm{e}\mathrm{m}\mathrm{a} \mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}{\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{e}\mathrm{d}\mathrm{e}\mathrm{m}\mathrm{a} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}}}\times 100$$

2.8.4. Statistical Analysis

Each calculated value is presented as mean ± SEM (n = 6 rats; one-way ANOVA followed by post-hoc Bonferroni test) compared with the different groups). If p > 0.05, there was a significative difference; if p < 0,05, there was a non-significative difference.

3. Results and Discussion

3.1. Formulation Process of Ibuprofen Nanogels

The experimental design results matrix is given in

Table 4. Experimental design results matrix. The optimum test of the experimental design studied, was test number 7.

Tests	Factor 1	Factor 2	Factor 3	MD (nm)	P.I	ZP (mV)	E.R (%)
1	+1	+1	+1	161.8	1.276	32	30.06
2	−1	+1	+1	209.6	0.289	13.8	49.96
3	+1	−1	+1	208.4	0.326	31.5	84.79
4	−1	−1	+1	200.6	0.342	26.3	47.91
5	+1	+1	−1	140.6	0.402	32.3	55.55
6	−1	+1	−1	217.3	0.315	45.1	67.05
7	+1	−1	−1	179.9	0.193	24.7	93.78
8	−1	−1	−1	216.5	0.359	41	60.22
9	0	0	0	192.2	0.885	24.5	64.57

. The optimum test of the experimental design studied, allowing for the best encapsulation rate, was test number 7, which corresponds to a ratio of 15:10 (ibuprofen/polymer) and a ratio of 1:2 (gelatin/xanthan-aldehyde), with a gelling time of 2 h. The collected data were analyzed using MODDE 6 software to find a relationship between the factors (X1, X2, and X3) and the different responses.

The obtained mathematical models allowed us to predict answers for the proposed values of the factors; the significance of the obtained values depends on the accuracy of the obtained models. The different mathematical models of our process are given in

Table 5. Mathematical models.

Responses	Mathematical Models	p Value	Model Significance	R2	Q2
Encapsulation rate (%)	Y = 61.54 + 4.88X1 − 10.51X2 − 7.98X3 − 12.73X1X2 − 0.63 X1X3 − 2.66X2X3	0.028	Significant (p < 0.05)	0.991	0.876
Mean diameter (nm)	Y = 191.87 − 19.16X1 − 9.51X2 +3.26X3 − 11.96X1X2 + 9.16X1X3 + 0.11X2X3	0.016	Significant (p < 0.05)	0.995	0.822
Polydispersity index	Y = 0.13 + 2.98X1 + 0.43X2 + 0.84X1X2 + 0.11X1X3 + 0.75X2X3	0.042	Significant (p < 0.05)	0.786	0.295
Zeta potential (mV)	Y = 30.13 − 0.71X1 − 0.03 X2 − 4.93X3 + 2.06X1X2 + 6.56 X1X3 − 2.96X2X3	0.189	Not significant (p > 0.05)	0.932	0.433

. According to the p values obtained using ANOVA analysis, all mathematical models were statistically significant (p < 0.05) except zeta potential, which was not significant (p > 0.05).

The correlation coefficients (R2) and prediction coefficients (Q2) for the three responses (encapsulation rate, mean diameter, and zeta potential) were excellent. The prediction of the encapsulation rate and mean diameter were good, whereas the prediction of the zeta potential and polydispersity index remained very difficult. The correlation coefficients for the four responses (encapsulation rate, mean diameter, polydispersity index, and zeta potential) were 0.991, 0.995, 0.786, and 0.932, respectively. The prediction coefficients for the four responses (encapsulation rate, mean diameter, polydispersity index, and zeta potential) were 0.876, 0.822, 0.295, and 0.433, respectively.

3.1.1. Effects of Factors on Responses

• Encapsulation rate (ER)

Histograms representing the effect of the factors on the encapsulation rate are given in Figure 3A. The gelatin/xanthan-aldehyde ratio factors and time negatively influenced the encapsulation rate, unlike the ibuprofen/polymer ratio. The interactions between factors negatively influenced the encapsulation rate.

• Mean diameter (MD)

Histograms representing the effect of the factors on the mean diameter are given in Figure 3B. The ibuprofen/polymer ratio factors and the gelatin/xanthan-aldehyde ratio negatively influenced the mean diameter, whereas time had a positive influence. The interaction between the ibuprofen/polymer ratio with the gelatin/xanthan-aldehyde ratio was influenced negatively, and the interaction between the ibuprofen/polymer ratio and time was influenced positively; the interaction between the gelatin/xanthan-aldehyde ratio and time is almost zero.

• Polydispersity index

Histograms representing the effect of the factors on the polydispersity index are given in Figure 3C. All factors and their interactions positively influenced the polydispersity index.

• Zeta potential (ZP)

Histograms representing the effect of the factors on zeta potential are given in Figure 3D. The time factor and the ibuprofen/polymer ratio negatively influenced the zeta potential, whereas the influence of the gelatin/xanthan-aldehyde ratio was zero. The interactions between the ibuprofen/polymer ratio with time and the gelatin/xanthan-aldehyde ratio positively influenced the zeta potential, whereas the interaction of the gelatin/xanthan-aldehyde ratio with time had a negative influence.

3.1.2. Modeling and Prediction of Results

• Encapsulation rate

The contours in Figure 4 (left) represent the encapsulation rate prediction of ibuprofen in the nanogels. For a fixed value of the time factor X3 (Figure 4A), the encapsulation rate decreases when factor X2 (the gelatin/xanthan-aldehyde ratio) decreases and factor X3 (the ibuprofen/polymer ratio) increases. For a fixed value of factor X2, the gelatin/xanthan-aldehyde ratio (Figure 4B), the encapsulation rate increases when factor X1 (ibuprofen/polymer ratio) increases and factor X3 (time) decreases. For a fixed value of factor X1, the ibuprofen/polymer ratio (Figure 4C) and the encapsulation rate decrease when factor X3 (time) and factor X2 (the gelatin/xanthan-aldehyde ratio) decrease.

The ibuprofen/polymer ratio positively influenced the encapsulation rate. Indeed, a 15:10 ratio allows for a greater amount of the active substance (Ibuprofen) to be introduced into the reactor compared to a 5:10 ratio. Consequently, during the reaction between the two polymers, the encapsulation of the active substance was enhanced.

The gelatin/xanthan-aldehyde ratio negatively influenced the encapsulation rate. A 1:2 ratio provides better encapsulation of ibuprofen than a 2:1 ratio because the polycation gelatin (protein) contains more amine groups (NH2) than the aldehyde groups present in the polysaccharide (xanthan-aldehyde). Therefore, more polysaccharide than protein is required to promote the reaction between the two polymers and achieve a higher encapsulation rate.

Time negatively influenced the encapsulation rate. A reaction time of 2 h was sufficient for the formation of the nanogels. However, a reaction time of 8 h promoted the irreversible adsorption of the bioactive molecule (ibuprofen) onto the protein (gelatin), causing the ibuprofen to remain permanently bound to the protein and preventing its release.

• Mean diameter

The contours in Figure 4 (right) represent nanogel mean diameter prediction. For a fixed value of factor X1, time (Figure 4A), the mean diameter decreases when the two factors X1 (the ibuprofen/polymer ratio) and X2 (the gelatin/xanthan-aldehyde ratio) decrease. For a fixed value of factor X2, the gelatin/xanthan-aldehyde ratio (Figure 4B), the mean diameter decreases when factors X1 (ibuprofen/polymer ratio) and X3 (time) decrease. For a fixed value of factor X1, the ibuprofen/polymer ratio (Figure 4C), the mean diameter increases when factor X3 (time) increases and factor X2 (the gelatin/xanthan-aldehyde ratio) decreases.

The ibuprofen/polymer ratio negatively influenced the average diameter of the nanogels. A 15:10 ratio allows for a greater amount of the active substance (Ibuprofen) to be introduced into the reactor compared to a 5:10 ratio. Since the nanogel is formed by the reaction between the two polymers, a smaller amount of polymers in the reactor resulted in nanogels with smaller diameters (a lower average diameter). In contrast, reactors with higher polymer concentrations produced nanogels with larger sizes (greater average diameters).

The gelatin/xanthan-aldehyde ratio negatively influenced the average diameter because more polysaccharide than protein is needed to promote the reaction between the two polymers and obtain nanogels with a larger average diameter.

Time positively influenced the average diameter since a longer reaction time allows the polymers to react for a longer period, enabling the nanogels to grow and reach larger sizes (greater average diameters).

• Polydispersity index

The contours in Figure 5 (left) represent nanogel polydispersity index prediction. For a fixed value of factor X3, time (Figure 5A), the polydispersity index increases proportionally to the two factors X1 (the ibuprofen/polymer ratio) and X2 (the gelatin/xanthan-aldehyde ratio). For a fixed value of factor X2, the gelatin/xanthan-aldehyde ratio (Figure 5B), the polydispersity index increases when factor X1 (ibuprofen/polymer ratio) and factor X3 (time) increase. For a fixed value of factor X1, the ibuprofen/polymer ratio (Figure 5C), the polydispersity index increases when both factors X3 (time) and X2 (gelatin/xanthan-aldehyde ratio) increase.

The three factors studied during this optimization all had a very slight positive influence on the polydispersity index, which is practically negligible. The polydispersity index of nanogels depends more on factors that stabilize the emulsion between the two aqueous phases (the dispersed phase that will form the nanogel) and the organic phase (the dispersing phase, which serves as the mold for nanogel formation). Since this step was identical in all experiments (the proportions between the aqueous and organic phases, the concentration of the surfactant Tween 80, and the homogenization system), the polydispersity indices obtained were similar.

• Zeta potential

The contours in Figure 5 (right) represent nanogel zeta potential prediction. For a fixed value of factor X3, time (Figure 5A), the zeta potential decreases when the two factors X1 (the ibuprofen/polymer ratio) and X2 (the gelatin/xanthan-aldehyde ratio) decrease.

For a fixed value of factor X2, the gelatin/xanthan-aldehyde ratio (Figure 5B), the zeta potential decreases when factor X1 (ibuprofen/polymer ratio) and factor X3 (time) decrease. For a fixed value of factor X1, the ibuprofen/polymer ratio (Figure 5C), the zeta potential decreases when factor X3 (time) decreases and factor X2 (gelatin/xanthan-aldehyde) increases.

The zeta potentials of all the nanogels were positive and similar because gelatin is a polycation with positive charges, unlike xanthan-aldehyde, which carries no charges. Among the three components forming the nanogel (gelatin, xanthan-aldehyde, and ibuprofen), the only charged compound influencing the zeta potential is gelatin (there is no competition between the charges of the different components). Therefore, it is logical that factors X1 and X2 did not influence the zeta potential of the nanogels. However, the reaction time significantly impacted the zeta potential of the nanogels because a longer reaction time between the aldehyde groups of xanthan-aldehyde and the amine groups of gelatin reduces the number of amine groups in the nanogel. Consequently, this decreases its zeta potential, which is directly linked to NH³⁺ charge.

3.2. Structural Characterization of Nanogels

3.2.1. FTIR Spectroscopy Analysis

The infrared spectra of ibuprofen, gelatin, xanthan-aldehyde, and the nanogels are given in Figure 6.

Table 6. Information obtained from FTIR analysis.

Substance	Wavenumber (cm−1)	Functional Group	Description	Refs.
Ibuprofen	1461	C=C	Characteristic peak of the benzene double bonds (C=C)	[35,36]
	1722	C=O	Characteristic peak of the carboxylic acid group
Xanthan-aldehyde	1749	C=O	Peak characteristic of the aldehyde group
	3480	O-H	Peak characteristic of the alcohol group
	2913	C-H	Peak characteristic of the C-H group
	1230–1000	C-O and C-O-O	Peak characteristic of C-O and C-O-O groups
	1439	O-H	Peak characteristic of the O-H group in glucose molecules
Gelatin	1553	NH2	Peak characteristic of the amine group (NH2)
	1650	C=O	Peak characteristic of the amide group I.
Nanogels	1686 (new)	C=N	New peak characteristic of the imine group formed from Schiff’s base reaction
	1749 (disappeared)	C=O	Disappearance of the peak characteristic of the aldehyde group (C=O) from xanthan-aldehyde
	1553 (decreased)	NH2	Decrease in peak intensity characteristic of the amine group (NH2) from gelatin

presents a summary of the information extracted from the analyses obtained using FTIR.

For the infrared spectrum of pure ibuprofen, the two main characteristic peaks of this drug were observed, with the first one being the carboxylic acid group and the second being the benzene double bonds C=C. For the infrared spectrum of xanthan-aldehyde, we observed a peak at 1749 cm⁻¹, characteristic of the aldehyde group (C=O), a peak between 3600–3000 cm⁻¹, characteristic of the alcohol group (O-H), a peak 2913 cm⁻¹, characteristic of the C-H group, peaks within 1230–1000 cm⁻¹, characteristic of groups C-O and C-O-O, and a peak at 1439 cm⁻¹, characteristic of the group O-H in glucose molecules. For the infrared spectrum of gelatin, we observed a peak at 1650 cm⁻¹, characteristic of amide group I, and a peak at 1553 cm⁻¹, characteristic of the amine group (NH2).

For the spectrum of the nanogels, we detected the appearance of a new peak at 1686 cm⁻¹, characteristic of the imine group C=N, and the disappearance of the peak at 1749 cm⁻¹, characteristic of the aldehyde group (C=O) contained in xanthan-aldehyde; in addition, we observed a decrease in peak intensity at 1553 cm⁻¹, characteristic of the amine group (NH2) contained in gelatin. The results obtained for the infrared spectrum of nanogels include the formation of imine groups from Schiff’s base reaction between the amino groups (NH2) of gelatin and the aldehyde groups (CHO) of xanthan-aldehyde [35,36]. The chemical structure of the resulting nanogel after the reaction between gelatin and xanthan-aldehyde is given in Figure 7.

3.2.2. X-Ray Diffraction Analysis (XRD)

X-ray diffractograms of the two polymers used—xanthan-aldehyde and gelatin—pure ibuprofen, and the nanogels are given in Figure 8. Figure 8A shows an X-ray diffractogram of pure ibuprofen. The drug has a crystalline structure; the characteristic peaks of the crystalline phases of ibuprofen are located in the 2-theta region between 3 and 25. The intensity of its peaks is very high and exceeds 70,000. The crystallinity of ibuprofen is affected by its synthesis method. During the synthesis of an organic molecule, the obtained product is recovered using crystallization and then purified by recrystallization. Furthermore, ibuprofen is an arylpropionic acid for which the chemical structure promotes the formation of crystals due to interactions between the molecules. Hydrogen bonds and van der Waals interactions contribute to stabilizing the crystalline structure. On the other hand, both polymers have the characteristics of amorphous materials, with a wide shoulder in the diffractogram. An intensity of 18,000 for xanthan-aldehyde (Figure 8B) and 22,000 for gelatin (Figure 8C) were observed, respectively. Figure 8D shows the diffractogram of the nanogels, the disappearance of the great majority of characteristic crystalline phases of ibuprofen, and a low peak intensity not exceeding 20,000. The diffractogram confirms the encapsulation of the drug in the polymeric matrix. The decrease in ibuprofen crystallinity in nanogels permits the prediction of an improvement of its solubility in aqueous media and thus improves its absorption and bioavailability in the body. Indeed, the decrease in the crystallinity of organic drugs permits the improvement of their solubility in aqueous media and thus improves their absorption and bioavailability in the body [37,38,39,40,41,42].

3.2.3. Scanning Electron Microscopy Analysis

Figure 9 shows the microphotography (post-drying) of the nanogels. The nanosystems observed in the micrograph have diameters ranging from 96.41 to 513.07 nm. These nanosystems are agglomerates of nanogels. During the drying process, the solid powder forms as multiple nanogels adhere together, creating agglomerates.

The observed nanogels have a porous structure and a more or less globular shape that recalls the spherical shape of a gelled nanodroplet. This porous aspect allows us to predict that nanogels have the ability to absorb liquids and deform.

3.3. Results of In Vitro Dissolution Kinetics

The results of the in vitro free ibuprofen dissolution kinetics and time-dependent ibuprofen release from the nanogels are given in Figure 10 (at pH 7.4 and 37 °C). We observe a very rapid dissolution increase in the first 30 min; then, this dissolution increases more slowly for the nanogels and free ibuprofen. Free ibuprofen has a dissolution rate of 27.34 ± 2.170%, which corresponds to a concentration of 0.020 ± 0.001 mg/L; this value is close to the ibuprofen dissolution found in the literature (0.021 mg/L in water at 25 °C).

The dissolution of ibuprofen contained in the nanogels increases very rapidly to a value of 95,524 ± 3906% after 120 min. The obtained values show that the prepared nanogels have the capacity to release the entire encapsulated active substance; this characteristic is indispensable for an effective delivery system. The incorporation of ibuprofen in nanogels allows for an improvement in its in vitro dissolution in a very important way; this dissolution increased from 27.34 ± 2.170% to 95.524 ± 3.906% after 120 min at a pH of 7.4 and at a temperature of 37 °C. Previous work has shown that ibuprofen is an organic molecule that is practically insoluble in water. Some of the nanosystems proposed in the bibliography allowed for an improvement in the dissolution of ibuprofen but without ever reaching a nearly total dissolution after 120 min, as in our case using our nanogels [43,44,45].

The modeling of the dissolution kinetics using four mathematical pharmacokinetic models showed that the dissolution is close to the kinetics described by the Korsmeyer-Peppas model (

Table 7. Mathematical modeling results of in vitro dissolution kinetics of free ibuprofen and nanogels (correlation coefficients and model parameters).

Formulation	Zeroth Order		First Order		Higuchi		Korsmeyer-Peppas
	R2	K0	R2	K1	R2	KH	R2	KKP	N
Nanogel	0.507	0.597	0.187	0.021	0.801	8.365	0.893	0.926	2.210
Free ibuprofen	0.517	0.176	0.253	0.016	0.810	2.466	0.859	0.687	1.599

). The correlation coefficient R2 = 0.893, the dissolution constant KKP = 0.926, and the value of the diffusion exponent n = 2.210. This value of n is greater than 1, so the diffusion of ibuprofen from the nanogels does not follow a Fickian diffusion but rather a super case II transport mechanism, indicating a mechanism dominated by polymer relaxation or erosion.

3.4. Study of Ex Vivo Transdermal Diffusion

The curves of the ex vivo transdermal diffusion kinetics using the Franz cell for free ibuprofen and nanogels are given in Figure 11 and

Table 8. Ex vivo diffusion parameters of free ibuprofen and nanogels.

Permeation Parameters	Nanogels	Ibuprofen Solution
Steady-state flux (mg.cm−2.h−1)	0.378 ± 0.065	0.076 ± 0.007
Lag time (h)	0.121 ± 0.011	0.119 ± 0.086
Steady-state permeability coefficient (cm.h−1)	0.450 ± 0.077	0.070 ± 0.009
Amount of ibuprofen permeated after 6 h (mg.cm−2)	2.412 ± 0.370	0.466 ± 0.067
Percentage of ibuprofen permeated after 6 h (%)	84.953 ± 13.030	16.417 ± 2.345

.

The diffusion follows a more or less linear trajectory after six hours of application; the nanogels allowed for an 84.953 ± 13.03% diffusion of the total ibuprofen encapsulated through the skin, but the ibuprofen solution allowed for only the diffusion of 16.417 ± 2.345%.

The incorporation of ibuprofen into the nanogels improved its ex vivo transdermal diffusion significantly; the permeation of ibuprofen through the abdominal skin of rats increased from 0.466 ± 0.067 mg.cm⁻² to 2.412 ± 0.37 mg.cm⁻² after 6 h at pH 7.4 and 37 °C.

The estimation of the scattering parameters of ibuprofen through the skin showed a significant increase in the steady-state flux and steady-state permeability coefficient. Indeed, the steady-state flux of ibuprofen changed from 0.076 ± 0.007 mg.cm⁻².h⁻¹ to 0.378 ± 0.065 mg.cm⁻².h⁻¹ after its incorporation into the nanogels, and the steady-state permeability coefficient changed from 0.07 ± 0.009 cm.h⁻¹ to 0.450±0.077 cm.h⁻¹. On the other hand, the lag time remained practically the same for the nanogels and ibuprofen solution; these values are 0.121 ± 0.011 h and 0.119 ± 0.086 h, respectively.

3.5. Results of In Vivo Anti-Inflammatory Activity

The in vivo anti-inflammatory activity assessment was performed using carrageenin-induced leg edema, which increased significantly, with a value of 118.79 ± 17.74% for the rats in the negative control group. The results of increased edema are shown in Figure 12A. The percentages of edema inhibitions for other groups compared to the negative control are given in the histograms of Figure 12B.

The percentages of edema increase for the commercial gel at a dose of 25 mg and 50 mg is 106.17 ± 17.17% and 74.79 ± 23.09%, respectively. The percentages of edema increase for the patch at a dose of 25 mg and 50 mg are 72.72 ± 3.00% and 21.34 ± 7.55%, respectively.

The percentages of edema inhibition for the commercial gel at a dose of 25 mg and 50 mg are 10.61 ± 1.71% and 37.03 ± 11.43%, respectively. The percentages of edema inhibition for the innovative patch at a dose of 25 mg and 50 mg are 38.77 ± 1.6% and 82.03 ± 9.03%, respectively.

In general, the increase in edema for rats treated with conventional commercial gel is greater than for rats treated with innovative nanogel-based patches (p < 0.05).

4. Conclusions

This work allowed us to successfully develop a new innovative pharmaceutical form of nanogel using experimental designs. The physicochemical analysis permitted us to determine nanogel characteristics, such as the encapsulation rate, mean diameter, polydispersity index, and zeta potential. The FTIR spectroscopy analysis showed the formation of imine function in the nanogel, and XRD analysis showed that the nanogel reduces ibuprofen crystallinity; SEM informed us about the globular and porous form of the formulation. The in vivo, ex vitro, and in vivo tests demonstrated a good release of the active substance and a good diffusion through the skin with better therapeutic efficacy. The innovative formulation of a patch based on the nanogel evidences better performance than traditional gels; this pharmaceutical form is very promising for the transdermal delivery of bioactive molecules, serving as a model for drug delivery in different therapeutic classes to improve their bioavailability and therapeutic effects at the same time as decreasing their adverse effects on the human body.