**1. Introduction**

The dynamics of medical research around the world are being directed towards developing new materials to solve and remedy various health problems. In recent years, dentistry, a part of the medical field, has been seeking to solve a series of problems related to the prevention of dental caries, periodontal disease and the treatment of bone tissue loss [1]. It is known that periodontitis (periodontal disease) is a chronic inflammatory condition of the gingiva and the tissues supporting teeth on their arches (gingiva, periodontal ligament, alveolar bone) [2,3]. The World Health Organization considers periodontal disease as one of the most common diseases of the oral cavity, statistically affecting three quarters of the world's population.

The treatment of periodontal disease is complex and generally aims to slow down the evolution of the disease. In the early stages of the disease, local treatment is instituted [3], which is generally antimicrobial and consists of: scaling, which helps remove tartar and bacteria from the tooth surface and under the gingiva; and the use of antibiotics.

The development of polymer science and polymer characterization methods has led to the use of natural and synthetic polymers to produce polymeric materials in the form of microparticles to improve the prevention, diagnosis and treatment of injured tissues.

Among the natural polymers, chitosan, pectin, alginate, starch and dextran are used to prepare microparticles loaded with different drugs to treat periodontal diseases [4–6]. Another important polysaccharide, hyaluronic acid, which is used in the cosmetic industry, prepares the scaffold for tissue engineering for drug delivery, disease diagnoses and biomedical imaging; it represents an ideal candidate to obtain polymeric materials for the dental field [7–10]. The use of polymeric microparticles in dentistry has been increasing due to the excellent properties of both polymeric materials (good surface, biological and mechanical properties, low manufacturing cost, simple synthesis methods) as well as of microparticles (high surface/volume ratio, good accessibility to the site of action due to their size, stability in biological fluids, drug transport capacity, decreased frequency and intensity of adverse effects) [11,12]. Polymer-based microparticles can be achieved through several processes, the most important of which is polymerization. Among the polymerization methods used to obtain spherical microparticles (bulk polymerization, precipitation polymerization, dispersion polymerization, emulsion polymerization, etc.), aqueous suspension polymerization can be used because of the technical and economic advantages it confers. Since in suspension polymerization a small number of reagents and simple reaction equipment are used, the purification of the resulting products takes place by simple methods and the cost of obtaining the products is low; thus, it can be concluded that this method is suitable for obtaining polymeric materials with special properties that can be successfully applied both in the field of environmental protection and in the medical or pharmaceutical fields [13–15].

The main aim of this work is to develop polymeric materials in the form of porous microparticles with special architectures that are obtained by combining simple, economical and environmentally friendly methods that combine the properties of synthetic polymers (chemical, mechanical and thermal stability) with those of natural polymers (biocompatibility, biodegradability, non-toxicity) and have potential applications in the dental field. The choice of sodium hyaluronate, a derivative of hyaluronic acid, as a natural component in the production of the hybrid microparticles was based on the following considerations: hyaluronic acid is an essential component of the periodontal ligament matrix; it plays important roles in cell migration, adhesion and differentiation by binding proteins and cell receptors; and it has been studied as a metabolite or diagnostic marker of inflammation in gingival crevicular fluid [16,17]. Thus, by grafting hyaluronic acid onto the surface of precursor microparticles, it was intended to obtain a system for the delivery of chemotherapeutic agents (antimicrobial and anti-inflammatory drugs) to treat dental diseases. For obtaining polymer–drug systems, the biological active principle chosen was metronidazole, which is a bactericide that can be administered orally together with other antibiotics with an increased efficacy in treating periodontal disease.

#### **2. Materials and Methods**

#### *2.1. Materials*

All reagents used in the preparation and characterization of the precursor/hybrid microparticles were purchased from Sigma-Aldrich (Schnelldorf, Germany). Glycidyl methacrylate (GMA) and hydroxyethyl methacrylate (HEMA) were distilled before use under reduced pressure to remove the inhibitor. Dimethacrylic monomers [ethylene glycol dimethacrylate (EGDMA), diethylene glycol dimethacrylate (DEGDMA) and triethylene glycol dimethacrylate (TEGDMA)], the initiator [benzoyl peroxide (BOP)], porogenic agent [butyl acetate], poly(vinyl alchohol) (PVA, M<sup>w</sup> = 67,000 g/mol, degree of hydrolysis, 88%), gelatine, NaCl, sodium hyaluronate (HA), HBr, NaOH, glacial acetic acid, Crystal violet, n-heptane, methanol, ethanol and metronidazole (M<sup>w</sup> = 171.064 g/mol) are of analytical grade and were used as received.

#### *2.2. Methods*

2.2.1. Synthesis of Precursor Microparticles

The precursor microparticles based on GMA, HEMA and EGDMA, DEGDMA or TEGDMA denoted AE, AD and AT were synthesized by the suspension polymerization technique in a 250 cm<sup>3</sup> cylindrical reactor fitted with mechanical stirrer, thermometer and reflux condenser. The reaction mixture is formed by two phases:


The copolymerization reaction was conducted under a nitrogen atmosphere for 8 h at 78 ◦C and 1 h at 90 ◦C with a stirring rate of 400 rpm. After the copolymerization reactions were completed, the precursor microparticles were separated by decantation and washed with hot water. Then, to remove traces of residual monomers and porogenic agent, the precursor microparticles were extracted with methanol or ethanol in a Soxhlet apparatus.

#### 2.2.2. Synthesis of Hybrid Microparticles

The hybrid microparticles denoted AEHA, ADHA and ATHA were prepared by grafting the sodium hyaluronate to the epoxy groups situated on the surface of the precursor microparticles. A known quantity of precursor microparticles was immersed in a solution of various concentrations of sodium hyaluronate (0.2–1%) obtained by dissolving the polysaccharide in water with the addition of NaOH (pH = 7–10). The reaction took place at temperatures between 30 and 60 ◦C over a period of time ranging between 2 and 10 h. After the aforementioned time, the hybrid microparticles were filtered off, washed with water to remove unreacted polysaccharide and NaOH and then dried under a vacuum at 40 ◦C for 24 h.

#### 2.2.3. Infrared Spectroscopy

The precursor/hybrid microparticles were characterized by FTIR spectroscopy using a Bruker Vertex FT-IR Spectrometer at a resolution of 2 cm−<sup>1</sup> in the range of 4000–400 cm<sup>1</sup> by KBr pellet technique. In order to obtain FT-IR spectra, 0.03 g the precursor/hybrid microparticles or HA were mixed and ground with potassium bromide.

#### 2.2.4. Epoxy Group Content

The epoxy group content was determined by ASTM D1652 (standard test method for epoxy content of epoxy resins) [18]. This method consisted of direct titration with a standard solution of HBr in glacial acetic acid. Experimental values of epoxide equivalent weight (*EEW*) were determined using the following equation:

$$EEW = \frac{100 \cdot w}{N \cdot V} \left(\text{mol} \cdot \text{g}^{-1}\right) \tag{1}$$

where *w*—grams of precursor/hybrid microparticles; *N*—normality of the HBr in acetic acid (mol·L −1 ); and *V*—volume of HBr solution used for titration (mL).

#### 2.2.5. Thermogravimetric Analysis (TGA)

The thermal behavior of the precursor/hybrid microparticles (4 mg of sample) was performed at a heating rate of 10 ◦C·min−<sup>1</sup> in nitrogen atmosphere, using a Mettler Toledo TGA 851 Derivatograph.

#### 2.2.6. Scanning Electron Microscopy (SEM)

The surface morphology of the precursor/hybrid microparticles was analyzed with a Quanta 200 environmental scanning electron microscope at 25 kV.

#### 2.2.7. Atomic Force Microscopy (AFM)

AFM images for the precursor/hybrid microparticles were performed using a Scanning Probe Microscope Solver Pro-M platform (NT-MDT, Moscow, Russia) with a rectangular silicon cantilever NSG 10 and 203 kHz oscillation frequency, in air at ambient temperature (23 ◦C). The latest version of NT-MDT NOVA software was used for the analysis and calculation of the microparticle surface characteristic parameters (arithmetic mean deviation of the surface (*Sa*); root-mean-square deviation of the surface (*Sq*); surface skewness (*Ssk*); and surface kurtosis (*Sku*)) and AFM imaging. The equations of the threedimensional roughness parameters used in the current study are presented below [19]:

$$S\_a = \frac{1}{MN} \sum\_{j=1}^{N} \sum\_{i=1}^{M} |z(\mathbf{x}\_i, \mathbf{y}\_j) - \overline{z}| \tag{2}$$

$$S\_q = \left[\frac{1}{MN} \sum\_{j=1}^{N} \sum\_{i=1}^{M} |z(\mathbf{x}\_i, y\_j) - \overline{z}|^2\right]^{1/2} \tag{3}$$

$$S\_{sk} = \frac{1}{MNS\_q^3} \sum\_{j=1}^{N} \sum\_{i=1}^{M} |z(\mathbf{x}\_{i\prime} y\_j) - \overline{z}|^3 \tag{4}$$

$$S\_{ku} = \frac{1}{MNS\_q^4} \sum\_{j=1}^{N} \sum\_{i=1}^{M} |z(x\_{i\prime}y\_i) - \overline{z}|^4 \tag{5}$$

where *N*—number of points along of scan line; *M*—number of lines; *Sq*—the root mean square roughness; and *z*—the height of each point of coordinates *x<sup>i</sup>* and *x<sup>j</sup>* .

Additionally, shape and elongation factors are two important parameters in pore structure analysis and can be calculated with the following equations:

$$f\_{\text{shape}} = \frac{4 \cdot \pi \cdot A}{1.064 \cdot P^2} \tag{6}$$

$$f\_{elongation} = \frac{D\_{\min}}{D\_{\max}}\tag{7}$$

where *A*—pore area; *P*—pore perimeter; *Dmin*—minimum Feret diameter; and *Dmax* maximum Feret diameter.

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

The number average diameter (D) was obtained using a WingSALD 7001 laser diffraction particle size analyzer (UK). The measurements were performed by suspending the precursor microparticles or hybrid microparticles in methanol (non-solvent). The experimental data were recorded and processed using WingSALD software.

#### 2.2.9. Specific Parameters for the Characterization of the Morphology of Porous Structure

The morphology of the porous structure of the precursor/hybrid microparticles can be characterized using the following parameters: porosity (*P*, *%*), pore volume (*PV*, mL·g −1 ) and specific surface area (*Ssp*, m<sup>2</sup> ·g −1 ). The pore volume and the porosity of the precursor/hybrid microparticles were calculated as follows:

$$PV = \frac{1}{\rho\_{ap}} - \frac{1}{\rho\_{sp}} \tag{8}$$

$$\%P = 100 \cdot \left(1 - \frac{\rho\_{ap}}{\rho\_{sp}}\right) \tag{9}$$

where *<sup>ρ</sup>ap*—apparent density (g·cm−<sup>3</sup> ); and *<sup>ρ</sup>sp*—skeletal density (g·cm−<sup>3</sup> ).

The apparent and skeletal densities of precursor/hybrid microparticles were measured by pycnometric methods with mercury and n-heptane, respectively [20] and were calculated with the following equations:

$$\rho\_{ap} = \frac{m\_1}{V\_P - (m\_3 - m\_2)/\rho\_{Hg}}\tag{10}$$

$$\rho\_{sp} = \frac{m\_1}{V\_f - (m\_s - m\_4)/h} \tag{11}$$

where *m1*—mass of the sample (precursor/hybrid microparticles) (g); *VP*—volume of the pycnometer (cm<sup>3</sup> ); *Vf*—volume of the volumetric flask (cm<sup>3</sup> ); *m2*—mass of the pycnometer with the sample (g); *m3*—mass of the pycnometer with mercury and the sample (g); *ρHg* density of mercury (g·cm−<sup>3</sup> ); *m4*—mass of the volumetric flask with the sample (g); *mS* mass of the volumetric flask with the sample and n-heptane(g); and *h*—density of n-heptane (g·cm−<sup>3</sup> ).

The specific surface area was determined by dynamic vapor sorption using the Brunauer, Emmet and Teller (BET) method [21] and the sorption–desorption curves recorded for the precursor/hybrid microparticles. Sorption–desorption isotherms were registered using the fully automated gravimetric analyzer IGAsorp produced by Hiden Analytical, Warrington (UK).

#### 2.2.10. Swelling Studies

The swelling capacities of the precursor/hybrid microparticles were determined in aqueous solution at pH 1.2 and 5.5, respectively, using the gravimetric method. A known amount of dried precursor/hybrid microparticles (0.2 g) was immersed in 10 mL aqueous solution at 25 ◦C. At a certain period of time ranging between 10 and 1440 min, the precursor/hybrid microparticles were removed, centrifuged at 500 rpm for 10 min and weighed. The swelling capacity of the precursor/hybrid microparticles was calculated using the following equation:

$$S\_w \left( \% \right) = \frac{w\_S - w\_d}{w\_d} \cdot 100 \tag{12}$$

where *wS*—amount of swollen precursor/hybrid microparticles (g); and *wd*—amount of dry precursor/hybrid microparticles (g).

#### 2.2.11. Bach Adsorption Studies

The adsorption of metronidazole on precursor/hybrid microparticles was investigated in a batch system. Metronidazole adsorption was realized as follows: 0.1 g of precursor/hybrid microparticles of known moisture were introduced in 50 mL conical flasks filled with 10 mL metronidazole solution with various initial concentrations (0.25–1 mg·mL−<sup>1</sup> ). The conical flasks were placed in a thermostatic shaker bath (Memmert M00/M01, Germany) and shaken at 180 rpm and 25, 30 and 40 ◦C for different periods of time ranging from 10 to 1440 min. After the specified period of time, the precursor/hybrid microparticles were removed quantitatively from the metronidazole solution by centrifugation at 1000 rpm for 10 min. The concentration of metronidazole in the supernatant solution before and after adsorption was determined using a UV–VIS spectrophotometer (UV–VIS SPEKOL 1300, Analytik Jena, Jena, Germany) at a wavelength of 277 nm based on the calibration curve obtained with various drug solutions of known conditions. The amounts of metronidazole at equilibrium, *q<sup>e</sup>* (mg·g −1 ), and at any time, *q<sup>t</sup>* (mg·g −1 ), were calculated from the following equations:

$$q\_{\varepsilon} = \frac{(\mathbb{C}\_0 - \mathbb{C}\_{\varepsilon}) \cdot V}{w} \tag{13}$$

$$q\_t = \frac{(\mathbb{C}\_0 - \mathbb{C}\_t) \cdot V}{w} \tag{14}$$

where *C*0—initial concentration of metronidazole solution (mg·g −1 ); *Ce*—concentration of metronidazole at equilibrium (mg·g −1 ); *Ct*—concentration of metronidazole at any time (mg·g −1 ); *V*—volume of drug solution (L); and *w*—amount of precursor/hybrid microparticles (g).

#### 2.2.12. Drug Release Studies

In vitro drug release studies were carried out as follows: 100 mg of the drug-microparticle systems were introduced in 10 mL of buffer solution of pH = 1.2 (stimulated gastric solution) at 37 ◦C, over a period of 8 h, under gentle shaking (50 rpm) using a thermostatic shaker bath (Memmert M00/M01). Very small volumes of the release medium (1 µL) were collected with microsyringes at different intervals of time. The amount of metronidazole was determined spectrophotometrically (Nanodrop ND100, Wilmington, DE, USA) at a wavelength of 277 nm using a calibration curve.

#### **3. Results and Discussion**

#### *3.1. Synthesis of Precursor/Hybrid Microparticles*

The synthesis of the hybrid microparticles took place in two steps.

In the first step, precursor microparticles based on glycidyl methacrylate, hydroxyethyl methacrylate and dimethacrylic monomers (EGDMA, DEGDMA and TEGDMA) were synthesized using the suspension polymerization technique. The reaction to obtain the precursor microparticles (AE, AD and AT) is shown in Figure 1.

**Figure 1.** Schematic representation of the reaction to obtain precursor microparticles (AE, AD or AT).

Table 1 shows the experimental conditions required for the synthesis of precursor microparticles.



From Table 1 it can be seen that suspension polymerization produces high-yield microparticles, regardless of the type of crosslinker used. Similar results were observed for grafting chitosan onto three-dimensional networks based on glycidyl methacrylate and dimethacrylic esters when the grafting reaction occurred directly in the suspension polymerization process [13].

In the second step, hybrid microparticles were synthesized by grafting sodium hyaluronate to the epoxy groups located on the surface of the precursor microparticles; the reaction was carried out in a basic medium under gentle stirring and in a nitrogen atmosphere (Figure 2).

**Figure 2.** Schematic representation of the reaction to obtain hybrid microparticles (AEHA, ADHA or ATHA).

The amount of grafted HA was determined gravimetrically and calculated using the following relationship:

$$Q\left(\%\right) = \frac{W\_1 - W\_0}{W\_2} \cdot 100\tag{15}$$

where *W*0—the initial amount of microparticles (g); *W*1—the amount of grafted microparticles after purification (g); and *W*2—the initial amount of HA (g).

#### Optimization of the Grafting Reaction

The optimal conditions for obtaining hybrid microparticles with the highest degree of HA grafting were determined by changing only one reaction parameter (HA concentration, temperature, pH, reaction time) while keeping the other parameters constant. Thus, the influences of the mentioned reaction parameters on the grafting degree of HA are shown in Figures 3 and 4.

**Figure 3.** (**a**) The influence of HA concentration on amount of grafted polymer (T = 35 ◦C, t = 6 h, pH = 9); and (**b**) the influence of temperature on amount of grafted polymer (C<sup>P</sup> = 0.6%, t = 6 h, pH = 9).

**Figure 4.** (**a**) The influence of the reaction time on the amount of grafted polymer (C<sup>P</sup> = 0.6%, T = 50 ◦C, pH = 9); and (**b**) the influence of the pH of the HA solution on amount of grafted polymer (C<sup>P</sup> = 0.6%, T = 50 ◦C, t = 6 h).

From the graphical representations presented above, it can be seen that:


The chemical structure of the crosslinker used to obtain the precursor microparticles also influences the grafting yield. As can be seen, the amount of grafted HA increases in the following order: ADHA < AEHA < ATHA, so the highest amount of grafted HA is recorded in the ATHA hybrid microparticles that were obtained in the presence of TEGDMA as a crosslinker. Thus, increasing the chain length between the two methacrylic groups leads to the formation of microparticles with larger mesh sizes, thus allowing a better interaction between the two reaction partners (precursor microparticles and sodium hyaluronate). A special case is the ADHA microparticles that were obtained in the presence of DEGDMA as a crosslinking agent. In this case, the lower amount of grafted HA is probably due to the more compact structure of the microparticles, which is the result of the complexity of the crosslinking polymerization reaction, a reaction that is often accompanied by internal cyclisation processes.

In conclusion, the most favorable conditions for the synthesis of hybrid AEHA, ADHA and ATHA microparticles are as follows: the concentration of the HA solution = 0.6%, T = 50 ◦C, t = 6 h, pH = 9, and the ratio of the amount of microparticles: HA = 1:0.6 (g·g −1 ).
