*3.2. Structural Characterization*

#### 3.2.1. FTIR Spectroscopy

FTIR spectroscopy was used to highlight HA grafting on precursor microparticles. The infrared spectra of precursor/hybrid microparticles are presented in Figure 5 as well as in Figures S1 and S2.

**Figure 5.** The infrared spectra of AT and ATHA microparticles.

The characteristic absorption bands of AE, AD and AT microparticles are located at: 3452, 3482 and 3515 cm−<sup>1</sup> , characteristic of valence vibrations νO-H belonging to the HEMA monomer; 2991–2997 cm−<sup>1</sup> and 2953 cm−<sup>1</sup> are specific to the symmetric and asymmetric stretching vibrations of the -CH3, >CH<sup>2</sup> and >CH- groups; 1633, 1634 and 1637 cm−<sup>1</sup> are attributed to the >C=C< bond; 1730 cm−<sup>1</sup> is characteristic of the >C=O bond of the ester group, which is present in all the chemical structures of the three types of precursor microparticles; 1481 and 1484 cm−<sup>1</sup> are specific to the bending vibration of the methylene group (δCH2); and 907 cm−<sup>1</sup> is attributed to the stretching vibrations of the epoxy groups.

The appearance of new absorption bands at 1559, 1539, 1367 and 1341 cm−<sup>1</sup> indicates the presence in the structures of the hybrid microparticles (AEHA, ADHA and ATHA) of the carboxylate group characteristic of the polysaccharide.

In addition, by comparing the corresponding areas of AE, AD and AT microparticles at wavenumbers 3450, 1152 and 907 cm−<sup>1</sup> with the specific areas of similar absorption bands for AEHA, ADHA and ATHA microparticles, the following can be observed:


Based on the data obtained from the FTIR spectra, it can be concluded that the grafting reaction of HA on the surface of the precursor microparticles took place successfully.

#### 3.2.2. Dimensional Analysis of Precursor/Hybrid Microparticles

In the case of suspension polymerization, the size and size distribution of the microparticles are influenced by various parameters: the shape of the reaction vessel, the type of stirrer, the stirring speed, the temperature, the chemical structure of the crosslinker or the thermodynamic quality of the porogenic agent used.

The particle size distributions as well as the diameter values of the precursor/hybrid microparticles analyzed using laser diffractometry are shown in Figure S3 and Table 2, respectively.


**Table 2.** Diameters of precursor/hybrid microparticles.

As can be seen from Table 2, the precursor microparticles are micrometric in size, their diameter being influenced by the chemical structure of the crosslinker, i.e., they increase with increasing chain length between the methacrylic groups in the crosslinking agent. Additionally, the hybrid microparticles have larger diameters than the precursor microparticles, leading to the idea that HA has reacted with the epoxy groups to form a layer on the surface covering these microparticles, generating a core-shell structure.

#### 3.2.3. Thermogravimetric Analysis

Thermogravimetric studies were carried out to obtain additional information about the precursor/hybrid microparticles.

Table 3 shows the thermogravimetric characteristics of the precursor/hybrid microparticles, namely: the degradation steps, temperature range for each degradation step, residual mass, activation energy (*Ea*) and reaction order (*n*).


**Table 3.** Thermogravimetric characteristics of precursor/hybrid microparticles.

The thermal behavior of precursor microparticles and sodium hyaluronate is characterized by three stages of thermal decomposition. The first degradation step between 65 and 135 ◦C (HA), 180–260 ◦C (AE), 188–222 ◦C (AD) and 142–154 ◦C (AT) is characterized by weight losses of 6.20% (HA), 15% (AE), 9.88% (AD) and 6.32% (AT), which are associated in the case of precursor microparticles with the loss of solvents retained in the crosslinked mesh of their structure. The second stage of degradation occurs in the temperature ranges of 225–263 ◦C (HA), 270–375 ◦C (AE), 249–349 ◦C (AD) and 212–308 ◦C (AT) and is characterized by the highest amount of weight loss: 38.16% (HA), 73.59% (AE), 76.21% (AD) and 38.37% (AT). In this stage, the breakage of the labile bonds occurs first, followed by the destruction of the crosslinked network by the cleavage of the macromolecular chains. The third stage of degradation is in the temperature ranges: 309–510 ◦C (HA), 380–411 ◦C (AE), 349–439 ◦C (AD) and 381–442 ◦C (AT) with mass losses of 18.21% (HA), 6.46% (AE), 12.34% (AD) and 31.76% (AT).

In the case of the hybrid microparticles, the presence of sodium hyaluronate leads to a slight increase in thermal stability compared to that of the precursor microparticles. The thermal degradation of the microparticles occurs in three steps for AEHA and ADHA microparticles and in four steps for ATHA microparticles. Additionally, as in the case of the precursor microparticles, the second degradation step is characterized by the highest amount of weight loss: 61.27% (AEHA), 67.9% (ADHA) and 28.55% (ATHA). The fourth degradation step specific only to the ATHA microparticles located in the temperature range 361–434 ◦C is characterized by a weight loss of 19.80%.

The kinetic parameters (activation energy and reaction order) for each thermal decomposition step were determined using the Urbanovici–Segal integral method [22]. If we consider for comparison the second degradation step, which is the step characterized by the highest weight loss, it can be observed that the activation energies for the precursor microparticles have close values (179 kJ·mol−<sup>1</sup> (AE), 171 kJ·mol−<sup>1</sup> (AD) and 173 kJ·mol−<sup>1</sup>

(AT)), so the chemical structure of the crosslinker does not influence the way the microparticle degradation takes place. In the case of the hybrid microparticles, however, the activation energies are different (194 kJ·mol−<sup>1</sup> (AEHA), 174 kJ·mol−<sup>1</sup> (ADHA), 239 kJ·mol−<sup>1</sup> (ATHA)) leading to the idea that HA grafting to epoxy groups produces polymeric materials with different chemical structures and thermal stabilities depending on the degree of grafting of the polysaccharide.

## 3.2.4. Determination of Epoxy Groups

Since hybrid microparticles are obtained by grafting HA to the epoxy groups found in the precursor microparticle structure, it is important to determine their content in the microparticle structure before and after the grafting reaction. The HBr-glacial acetic acid titrimetric method was chosen for the determination of the epoxy groups. The reaction between halogenated acids and the epoxide group results in the opening of the three-atom ring and the formation of a hydroxyl functional group. Table 4 shows the results of the titrimetric method for the determination of the epoxy groups.


**Table 4.** The values of epoxy groups obtained theoretically and experimentally by titration.

From Table 4, it can be seen that the theoretical values obtained for the epoxy groups are higher than those obtained experimentally by titration. By titration, only the epoxide groups can be determined, which are accessible to the HBr reaction, especially those on the surface and those on the layers which are very close to the surface, which partly explains these differences. Another factor to be taken into account is the difference in the chemical composition of the copolymers compared to the starting monomers. For AEHA, ADHA and ATHA microparticles, the number of epoxy groups is reduced due to HA grafting by the ring-opening reaction of the epoxy groups found on the microparticle surface or in the surface layers. However, a small percentage of these epoxy groups remain unreacted, probably due to their reduced accessibility of HBr. From the data in Table 4, it can be seen that grafting was best performed on AT microparticles, the results of which are also in agreement with the amount of grafted HA calculated by the gravimetric method.

#### *3.3. Morphological Characterization*

#### 3.3.1. Scanning Electron Microscopy

The size, shape and surface morphology of the synthesized polymeric materials were analyzed using scanning electron microscopy. Figures 6 and 7 show micrographs of the precursor/hybrid microparticles, and for easy comparison, SEM images were taken at the same magnification (×5000 for surface structures (small images) and ×500 for microparticle overview (large images)).

**Figure 6.** SEM micrographs of precursor microparticles.

**Figure 7.** SEM micrographs of hybrid microparticles.

The SEM micrographs show that by the chosen synthesis method, spherical particles of micrometric dimensions are obtained and that the surface morphology is influenced by the chemical structure of the crosslinking agent used. Thus, as the alkyl chain between the two methacrylic groups increases, precursor microparticles with a more pronounced porous structure and a rougher surface are obtained. The interaction of the precursor microparticles with sodium hyaluronate results in hybrid microparticles that retain their spherical shape, but the surface morphology changes due to the deposition of a polymer layer on the surface of the precursor microparticles, confirming once again that the grafting reaction of the polysaccharide to the epoxy groups has taken place.

#### 3.3.2. Atomic Force Microscopy

Atomic force microscopy was also used to investigate the surface morphology of the precursor/hybrid microparticles, obtaining information on surface roughness, pore size and geometry. AFM images for AE and AEHA microparticles are shown in Figure 8 as an example.

The AFM images correlate well with those from the scanning electron microscopy, revealing differences in surface morphology between the precursor and hybrid microparticles. Table 5 shows the values of the parameters characteristic of microparticle surfaces.

From the data in Table 5, it can be seen that grafting HA onto precursor microparticles has the effect of decreasing the surface roughness, but also the size of existing pores on the surface. The negative values of Ssk, a statistical parameter that gives us information about the degree of asymmetry of the distribution of heights on the surface [23], indicate that the two types of microparticles analyzed show porous structures. Additionally, Sku values lower than three confirm that the microparticles present irregular surfaces with various roughness. All these observations reinforce and confirm the conclusions drawn from the SEM analysis. Shape and elongation factors are two important parameters, with which pore structures can be analyzed [24]. The values of these parameters shown in Table 5 indicate that the precursor/hybrid microparticles have elliptical-shaped pores with an irregular outline.

**Figure 8.** AFM images (2D) and cross-section profile of AE and AEHA microparticles.


**Table 5.** Surface profile parameters for precursor/hybrid microparticles.

3.3.3. Specific Parameters for Characterizing the Morphology of Porous Structures

The introduction of a pore-forming substance, known as a porogenic agent or diluent, into the organic phase of the reaction system specific to suspension polymerization leads to the formation of permanently heterogeneous structures, i.e., structures containing pores after drying. A highly effective porogenic agent should not react during polymerization but should remain within the microparticle structure until the end of the reaction. When it is removed by extraction, the sites occupied by the porogen become the pores of the crosslinked networks.

Table 6 shows the values of the specific parameters determined to characterize the morphology of the precursor/hybrid microparticles.


**Table 6.** Porosity parameters of precursor/hybrid microparticles.

From the data presented in Table 6, it can be seen that the pore volume and porosity of the hybrid microparticles increase with the increasing alkyl chain, except for the AD microparticles, whose values decrease. When DEGDMA is used as a crosslinking agent, probably during the crosslinking radical polymerization process, a decrease in the apparent reactivity of the double pendant groups occurs due to steric factors, resulting in the appearance of internal cyclizations and thus the formation of microparticles with more compact structures characterized by lower values of both porosity and pore volume. In the case of the AEHA, ADHA and ATHA microparticles, decreases in pore volume and porosity values compared to those of precursor microparticles are observed, which is due to HA grafting to the epoxy groups on the microparticle surface. Through the grafting reaction, HA coats part of the pores or penetrates into the pores, decreasing their size. This is confirmed by the information obtained by the AFM method as well as by the SEM micrographs. The decrease in pore size was also observed by the AFM method as follows:


It is also observed that the specific surface area values are higher for hybrid microparticles. This can be explained by the fact that Ssp was determined by the dynamic vapor sorption method, and hybrid microparticles due to their hydrophilic structure have a higher capacity to absorb water than precursor microparticles. Additionally, the higher values of the specific surface are due to the fact that the hybrid microparticles have smaller pore sizes than the corresponding precursor microparticles.

The morphology of the pore structures is influenced by the structure and concentration of the monomers, the nature of the porogenic agent and in particular the amount of porogenic agent used. Thus, Figure 9 shows graphical representations of the specific surface area and pore volume values, depending on the amount of porogenic agent used to obtain the AT and ATHA microparticles.

**Figure 9.** The influence of the amount of porogenic agent on the specific surface area (**a**) and the pore volume (**b**) for AT and ATHA microparticles.

From Figure 9, it can be seen that microparticles with higher porosity structures and more specific surface values are obtained when the amount of porogen agent is increased. For this reason, for the preparation of the precursor microparticles, the dilution was chosen to be 0.6.

#### *3.4. Swelling Capacity of Precursor/Hybrid Microparticles in Aqueous Media*

Graphical representations of the degree of swelling of precursor/hybrid microparticles versus time in aqueous media with different pH values are shown in Figure 10.

**Figure 10.** Time dependence of the degree of swelling in pH = 1.2 (**a**) and pH = 5.5 (**b**) for precursor/hybrid microparticles.

Figure 10 shows that the swelling process is carried out in three stages. In the first stage, the rapid absorption of aqueous solutions of different pH values into the structure of the precursor/hybrid microparticles takes place. The second stage is characterized by a slower absorption, and in the third stage the equilibrium of the swelling process is reached. For AE, AD and AT microparticles, an equilibrium is reached after 960 min and for hybrid microparticles, an equilibrium is reached after 840 min.

The swelling degree of precursor and hybrid microparticles determined with Equation (12) depends on the pore structure and the presence of HA on the microparticle surface. Thus, the degree of swelling for precursor microparticles is not influenced by the pH value of the swelling medium and increases in the order SW,AD < SW,AE < Sw,AT, similar to the increase in the specific surface values (Table 6). Thus, AT microparticles characterized by a high specific surface area (160 m<sup>2</sup> ·g −1 ) show the highest degree of swelling. In the case of the hybrid microparticles, it is observed that the degree of swelling is higher than that corresponding to the precursor microparticles. This is explained by the presence of HA on the surface of the hybrid microparticles, which is a hydrophilic polymer having several -OH groups in its structure. The swelling degrees of the hybrid microparticles in pH = 1.2 are lower than in pH = 5.5. This behavior can be explained by the fact that the COO- groups of the HA molecule in the acidic medium are transformed into COOH groups, thus reducing the electrostatic repulsion between them, and consequently the polymer matrix swells less.

Two mathematical models were used to describe the swelling mechanism in media with different pH values, namely:

1. The second-order kinetic model using the equations [25]:

$$\frac{t}{SR} = \frac{1}{K\_{\text{S}} \cdot \text{S}\_{eq}^{2}} + \frac{t}{\text{S}\_{eq}}\tag{16}$$

$$SR\left(g \cdot g^{-1}\right) = \frac{W\_t - W\_0}{W\_0} \tag{17}$$

$$\mathcal{S}\_{\varepsilon q} \left( \mathbf{g} \cdot \mathbf{g}^{-1} \right) = \frac{W\_{\varepsilon q} - W\_0}{W\_0} \tag{18}$$

where *W0*, *W<sup>t</sup>* and *Weq* are the amount of precursor/hybrid microparticles at time *t* = 0, *t* = *t* and at equilibrium, respectively [26]. The straight-line plots of *t*/*SR* versus *t* gave the slope of *Seq* and intercept *KS*.

2. Korsmeyer–Peppas model. The linear form of the Korsmeyer–Peppas equation [27] is given as:

$$
\ln \text{F} = \ln \text{K} + \text{n} \cdot \text{Int} \tag{19}
$$

where *F* = *Mt*/*M*∞, *Mt*—the amount of water uptake at time t; *M*∞—the amount of water uptake at time approaching infinity; *K*—the swelling rate constant; and *n*—diffusion exponent characteristic for the transport mechanism. The values of *K* and *n* were determined from the linear plots of *lnF* versus *lnt*.

The values of the kinetic parameters obtained by applying the two models are shown in Table 7.


**Table 7.** Kinetic parameters of the swelling process in various aqueous solutions.

From the data in Table 7, it can be seen that there is a good correlation between the experimental (*Sexp*) and calculated (*Seq*) values and the correlation coefficient *R* <sup>2</sup> values are greater than 0.997. These results suggest that the swelling mechanism of precursor/hybrid microparticles in aqueous media with different pH values follows the second-order kinetic model. The values of n were in the range 0.141–0.242, indicating that the most likely swelling mechanism is Fickian.

#### *3.5. Metronidazole Adsorption and Release Studies*

In order to achieve an optimal system with a controlled drug release, the influence of the following parameters must be taken into account: pH; contact time; temperature; and initial drug concentration.

Since metronidazole dissolves in acidic pH, the adsorption process of metronidazole on the precursor/hybrid microparticles was performed from an aqueous solution with pH = 1.2.

Additionally, the effect of contact time is very important for assessing the affinity of precursor/hybrid microparticles for the model drug. Figure 11 shows the influence of contact time on the adsorption capacity of metronidazole on precursor/hybrid microparticles for a metronidazole concentration of 0.5 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>g</sup>·mL−<sup>1</sup> at a temperature of 25 ◦C.

For precursor microparticles, the contact time for reaching the equilibrium is 720 min, while for hybrid microparticles, the equilibrium is reached at 600 min. Above these contact time values, the amount of drug adsorbed on the precursor/hybrid microparticles remains constant. The shorter time to reach equilibrium indicates a better affinity of the hybrid microparticles for metronidazole compared to that of the precursor microparticles.

Temperature is another important parameter to be taken into account when adsorbing drugs on different polymeric supports, with metronidazole adsorption studies being carried out at 25, 30 and 35 ◦C (Figure 12a).

**Figure 12.** Influence of temperature (**a**) and the initial concentration of metronidazole (**b**) on the adsorption capacity of the drug onto precursor/hybrid microparticles.

Analyzing the graphical representation in Figure 12a, it can be seen that the adsorption of the drug is favored by increasing temperature, an effect that is absolutely expected since the process is of a diffusional nature, causing an increase in the degree of swelling and thus in the diffusion rate of metronidazole into the pores of the precursor/hybrid microparticles.

Increasing the concentration of the drug has the effect of increasing the rate of adsorption. It was also observed that drug adsorption was achieved in higher amount in the case of hybrid microparticles compared to that of precursor microparticles (Figure 12b). This phenomenon is explained by the presence of sodium hyaluronate, which has the role of enhancing the hydrophilicity of the microparticles, leading to a higher degree of

swelling and consequently to the adsorption of a higher amount of the drug. In the case of metronidazole adsorption, in an acidic pH mainly physical interactions take place, such as hydrogen bonding between the OH group of metronidazole and the -COOH and -OH groups of the hybrid microparticle structure. The greatest amount of immobilized drug was obtained in the case of the ATHA hybrid microparticles.

In-depth studies of the adsorption process were carried out considering two physicochemical aspects, namely: the adsorption equilibrium by means of adsorption isotherms, which quantify the interaction between the drug and the support; and adsorption kinetics, which can explain the mechanism of drug adsorption on the solid supports.

#### 3.5.1. Adsorption Equilibrium Studies

For efficient polymer–drug systems, it is important to know how the adsorbate (drug solution) and adsorbent (precursor/hybrid microparticles) interact. For this purpose, the description of the adsorption equilibrium of metronidazole on precursor/hybrid microparticles was performed using the mathematical models of Langmuir, Freundlich, Dubinin–Radushkevich (two-parameter models), Sips and Khan (three-parameter models) isotherms.

The nonlinear forms of the isotherms used can be written as follows:

	- Langmuir isotherm [28]:

$$q\_{\varepsilon} = \frac{q\_m \cdot K\_L \cdot \mathbf{C}\_{\varepsilon}}{1 + K\_L \cdot \mathbf{C}\_{\varepsilon}} \tag{20}$$

• Freundlich isotherm [29]:

$$q\_{\varepsilon} = \mathbb{K}\_{\mathcal{F}} \cdot \mathbb{C}\_{\varepsilon}^{\frac{1}{n\_f}} \tag{21}$$

• Dubinin–Radushkevich isotherm [30]:

$$q\_{\varepsilon} = q\_m \exp\left(-K\_D \cdot \epsilon\_D^2\right) \tag{22}$$

where *q<sup>e</sup>* is the metronidazole amount adsorbed at equilibrium (mg·g −1 ); *q<sup>m</sup>* is the maximum adsorption capacity (mg·g −1 ); *K<sup>L</sup>* is the Langmuir constant that reflects the affinity between the adsorbate and the adsorbent (L·g −1 ); *K<sup>F</sup>* is the adsorption capacity for a unit's equilibrium concentration (L·g −1 ); 1/n<sup>F</sup> is a constant that suggests the favorability and capacity of the adsorbent–adsorbate system; ε is the Polanyi potential; and *K<sup>D</sup>* is the constant which is related to the calculated average sorption energy *<sup>E</sup>* (kJ·mol−<sup>1</sup> ). The constant *K<sup>D</sup>* can give the valuable information regarding the mean energy of adsorption by the equation:

$$E = \left(-2K\_D\right)^{1/2} \tag{23}$$

	- Sips isotherm [31]:

$$q\_{\varepsilon} = \frac{q\_{m} \cdot K\_{\text{S}} \cdot \mathbb{C}\_{\varepsilon}^{n\_{\text{S}}}}{1 + K\_{\text{S}} \cdot \mathbb{C}\_{\varepsilon}^{n\_{\text{S}}}} \tag{24}$$

• Khan isotherm [32]:

$$q\_{\varepsilon} = q\_m \frac{b\_K \cdot \mathbb{C}\_{\varepsilon}}{(1 + b\_K \cdot \mathbb{C}\_{\varepsilon})^{n\_K}} \tag{25}$$

where *<sup>K</sup><sup>S</sup>* is the Sips constant (L·mg−<sup>1</sup> ); *n<sup>S</sup>* is the Sips model exponent; *b<sup>K</sup>* is the Khan model constant; and *n<sup>K</sup>* is the Khan model exponent.

Sips and Khan isotherms represent the combined features of the Langmuir and Freundlich isotherm equations. Thus, in the case of a low adsorbent concentration, the Sips isotherm is reduced to the Freundlich isotherm, while at high adsorbate concentrations, it shows the characteristics of the Langmuir isotherm [33] Additionally, if *n<sup>K</sup>* = 1, Equation (24) can be simplified to the Langmuir isotherm equation, whereas if *nK*·*C<sup>e</sup>* >> 1, Equation (25) can be approximated by the Freundlich type isotherm equation [32].

The isotherm model plots of metronidazole adsorption onto precursor/hybrid microparticles are illustrated in Figure 13, while the parameters and the statistical error functions values (*R* <sup>2</sup> and χ 2 ) are presented in Tables 8 and 9.

**Figure 13.** The results of nonlinear fits of Langmuir (dash line); Freundlich (solid line); Dubinin– Radushkevich (short dot line) (**a**); Sips (dash dot line); and Khan (short dash line). (**b**) Isotherm for metronidazole adsorption on precursor/hybrid microparticles at T = 35 ◦C.




**Table 9.** Two- and three-parameter isotherm values for adsorption of metronidazole onto AEHA, ADHA and ATHA microparticles.

From the analysis of the data presented in Tables 8 and 9, it can be seen that:


#### 3.5.2. Kinetic Studies

In order to investigate the mechanism of metronidazole adsorption on precursor/hybrid microparticles, the experimental data were interpreted using four mathematical models, namely: the Lagergren model (pseudo-first order kinetic model), the Ho model (pseudo-second order kinetic model), the Elovich model and the Weber–Morris intraparticle diffusion model. The nonlinear forms of the Lagergren (Equation (26)) [34], Ho (Equation (27)) [35] and Elovich models (Equation (28)) [36], as well as the linear form of the Weber–Morris model (Equation (29)) [37] are written below:

$$q\_t = q\_\varepsilon \left(1 - e^{-k\_1 t}\right) \tag{26}$$

$$q\_t = \frac{k\_2 \cdot q\_\varepsilon^2 \cdot t}{1 + k\_2 \cdot q\_\varepsilon \cdot t} \tag{27}$$

$$q\_t = \frac{1}{\beta} \ln(1 + \mathfrak{a} \cdot \mathfrak{k} \cdot t) \tag{28}$$

$$q\_t = k\_{\rm id} \cdot t^{0.5} + \mathcal{C}\_i \tag{29}$$

where k<sup>1</sup> is the rate constant of the pseudo-first order model (min−<sup>1</sup> ); k<sup>2</sup> is the rate constant of the pseudo-second order model (g·mg−<sup>1</sup> ·min−<sup>1</sup> ); α is the initial adsorption rate (mg·g −1 · min−<sup>1</sup> ); <sup>β</sup> is the desorption constant (g·mg−<sup>1</sup> ); kid is the intraparticle diffusion rate constant (g·mg−<sup>1</sup> ·min−0.5); and *<sup>C</sup><sup>i</sup>* is the constant that gives information about the thickness of the boundary layer.

Figure 14a,b presents the nonlinear plots of the Lagergren, Ho and Elovich models as well as the straight-line plots of the Weber–Morris model in case of metronidazole adsorption (Cmetronidazole = 1 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>g</sup>·mL−<sup>1</sup> ) on the precursor/hybrid microparticles at 35 ◦C.

**Figure 14.** Lagergren (solid line), Ho (dash line), Elovich (dot line) models (**a**) and Weber–Morris intraparticle diffusion model (**b**) for metronidazole adsorption on precursor/hybrid microparticles (Cmetronidazole = 1 <sup>×</sup> <sup>10</sup>−<sup>3</sup> <sup>g</sup>·mL−<sup>1</sup> , T = 35 ◦C).

The kinetic parameters obtained from the Lagergren, Ho, Elovich and Weber–Morris models are presented in Tables 10 and 11.


**Table 10.** Kinetic parameters for adsorption of metronidazole onto AE, AD and AT microparticles.

**Table 11.** Kinetic parameters for adsorption of metronidazole onto AEHA, ADHA and ATHA microparticles.


From the data presented in Tables 10 and 11, it can be seen that the calculated adsorption capacity values based on the first-order kinetic model are very close to the experimental values for metronidazole adsorption on the precursor/hybrid microparticles. The values of the rate constant k<sup>1</sup> increase with increasing temperatures, indicating a higher adsorption rate of the drug at higher temperatures. It is also observed that values of R<sup>2</sup> are very close to unity and are associated with low values of χ 2 , showing that the first-order kinetic model describes the experimental data quite well. These results suggest that the adsorption of metronidazole on precursor/hybrid microparticles is of a physical nature. By applying the second-order kinetic model, it can be seen that the values of qe,calc are not as close to the values of qe,exp as obtained in the case of applying the first-order kinetic model. The relatively high values of R<sup>2</sup> associated with the high values of χ 2 indicate that the second-order kinetic model does not describe the experimental data well in the case of metronidazole adsorption on the precursor/hybrid microparticles. Additionally, the value of the rate constant k<sup>2</sup> increases with increasing temperatures, again asserting that the rate of drug adsorption is higher at higher temperatures. The lower values of R<sup>2</sup> were correlated with higher values of χ <sup>2</sup> obtained for metronidazole adsorption on precursor/hybrid microparticles; hence, the application of the Elovich model provides a further argument that the metronidazole adsorption is not chemical in nature.

Additionally, from Tables 10 and 11, it can be seen that the Ci2 values increase with increasing temperatures, thus indicating increasing boundary layer thickness associated with decreasing external mass transfer and increasing internal mass transfer. The highest values of Ci2 were obtained for hybrid microparticles, confirming that they are good adsorbents. The results obtained by applying the Weber–Morris model lead us to the conclusion that intraparticle diffusion is not the only process influencing the adsorption rate.

#### 3.5.3. Thermodynamic Studies

The adsorbent performance of the precursor/hybrid microparticles was also demonstrated by thermodynamic studies. For this purpose, the following thermodynamic parameters were calculated: Gibbs free energy changes (∆*G*), enthalpy change (∆*H*) and entropy change (∆*S*).

The values of ∆*H* and ∆*S* were estimated using Van't Hoff equation [38]:

$$
\ln K = \frac{\Delta S}{R} - \frac{\Delta H}{RT} \tag{30}
$$

where *K*—the Langmuir adsorption equilibrium constant obtained at different temperature values [39]; *<sup>R</sup>*—the ideal gas constant (8.314 J·mol−<sup>1</sup> ·K−<sup>1</sup> ); and *T*—temperature in Kelvin. The ∆*G* value was calculated using the thermodynamic equation:

$$
\Delta G = \Delta H - T \cdot \Delta S \tag{31}
$$

According to the data in the literature, the values of ∆*H* and ∆*G* can provide information about the type of adsorption process [40]. The linear plot of lnK versus 1/*T* gives us the thermodynamic parameters of the adsorption process of metronidazole on precursor/hybrid microparticles, and their values are shown in Table 12.


**Table 12.** Thermodynamic parameters.

From the data presented in Table 12, it can be seen that:


#### 3.5.4. Release Studies

The goal of the research was to obtain a microparticulate system capable of the controlled/sustained release of the adsorbed drug. For this reason, after loading the precursor/hybrid microparticles with metronidazole, kinetic release studies were performed. Release studies have been conducted for the precursor–drug microparticle and respectively for the hybrid–drug microparticle systems containing the highest amount of the included drug. The release profiles are represented in Figure 15.

**Figure 15.** Metronidazole release profiles from precursor/hybrid microparticles (pH = 1.2).

From the graphical representations, it can be seen that the release process of metronidazole from the precursor microparticles occurs at a higher rate than that for the hybrid microparticles.

The interpretation of the metronidazole release kinetics from precursor/hybrid microparticle–drug systems was performed using three mathematical models:

• Higuchi model [41]:

$$Q\_t = k\_H \cdot t^{0.5} \tag{32}$$

• Korsmeyer–Peppas model [27]:

$$\frac{M\_{\rm f}}{M\_{\infty}} = k\_r \cdot t^n \tag{33}$$

• Baker–Lansdale model [42]:

$$\frac{3}{2}\left[1-\left(1-\frac{M\_{\rm{f}}}{M\_{\infty}}\right)^{\frac{2}{3}}\right]-\frac{M\_{\rm{f}}}{M\_{\infty}}=k\_{\rm{BL}}\cdot t\tag{34}$$

where *Qt*—the amount of drug released at time t; *kH*—the Higuchi dissolution constant; *Mt*/*M*∝—the fraction of drug released at time t; *kr*—the release rate constant that is characteristic for drug–polymeric interactions; *n*—the diffusion exponent that is characteristic for the release mechanism; and *kBL*—the release constant.

The values of the release parameters are presented in Table 13.



The rate constants obtained by applying the three kinetic models indicate that the release rate of metronidazole from the precursor microparticles is higher than that for the hybrid microparticles. The different amounts of drug released can be explained by the physical interactions of metronidazole with the functional groups belonging to the chemical structure of the hybrid microparticles. In the case of precursor microparticles, the drug is retained in a larger quantity on the surface and for this reason can be released at a higher rate.

The value of the diffusion exponent n calculated on the basis of the Korsmeyer–Peppas model further argues that:


Similar results have been found in the literature for other microparticulate systems. For example, in case of microparticles based on gelatin and poly(ethylene glycol) coated with ethyl cellulose, the metronidazole release rates and transport parameters have suggested the non-Fickian mechanism [43]. Additionally, the release kinetics of the metronidazole from the hydrogel containing crosslinked chitosan microparticles best fit the Higuchi model [44].

#### **4. Conclusions**

By aqueous suspension polymerisation, three series of porous microparticles were obtained based on GMA, HEMA and one of the following crosslinking agents: EGDMA, DGDMA and TEGDMA. By the grafting reaction of sodium hyaluronate to the existent epoxy groups on the surface of AE, AD and AT microparticles, hybrid porous microparticles were obtained.

Precursor/hybrid microparticles were structurally characterized by appropriate techniques: FTIR spectroscopy, epoxy groups content, thermogravimetric analysis, dimensional analysis, and grafting degree of HA. From a morphological point of view the precursor/hybrid microparticles were characterized by: scanning electron microscopy, atomic force microscopy, and specific parameters for the characterization of the morphology of porous structures.

The information from the data acquired using the above-mentioned techniques showed that spherical microparticles of micrometer size with different surface morphologies depending on the synthesis conditions were obtained by suspension polymerization, and the grafting reaction of HA on the surface of the precursor microparticles in a basic medium was a success.

The swelling ability of precursor/hybrid microparticles in aqueous media with different pH values was studied, and the mechanism by which the swelling of precursor/hybrid microparticles in aqueous solutions with different pH values occurred is Fick-type and follows the second-order kinetic model.

The adsorptive performance of the precursor/hybrid microparticles has been shown by kinetic, thermodynamic and equilibrium studies. The experimental data obtained in the case of the metronidazole adsorption on precursor/hybrid microparticles were described using the nonlinear forms of Langmuir, Freundlich, Dubinin–Radushkevich, Sips and Khan isotherms. Adsorption isotherms demonstrate that the adsorption of metronidazole on precursor/hybrid microparticles occurs according to a monolayer adsorption.

To explain the mechanism of metronidazole adsorption on precursor/hybrid microparticles, the experimental data were modelled using four kinetic models, namely: first-order kinetic model, second-order kinetic model, Elovich model and Weber–Morris intraparticle diffusion model. The first-order kinetic model describes the experimental data quite well for metronidazole adsorption on both precursor and hybrid microparticles.

The release kinetics reflect that the release mechanism of metronidazole is a Fick-type diffusion mechanism in the case of precursor microparticles, while in the case of hybrid microparticles, it is a complex mechanism characteristic of anomalous or non-Fickian diffusion.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14194151/s1, Figure S1: The infrared spectra of AE and AEHA microparticles; Figure S2: The infrared spectra of AD and ADHA microparticles; Figure S3: Particle size distributions of precursor/hybrid microparticles.

**Author Contributions:** Conceptualization, A.I.G. and S.R.; methodology, S.R., A.I.G. and S.V.; software, A.I.G.; validation, S.R., S.V. and M.P.; formal analysis, A.I.G.; investigation, A.I.G. and S.R.; data curation, A.I.G., S.R. and S.V.; writing—original draft preparation, A.I.G.; writing—review and editing, S.V. and M.P.; visualization, S.V.; supervision, M.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**

