**Ground Calcium Carbonate as a Low Cost and Biosafety Excipient for Solubility and Dissolution Improvement of Praziquantel**

**Ana Borrego-Sánchez 1,2,\*, Rita Sánchez-Espejo 1, Beatrice Albertini 3, Nadia Passerini 3, Pilar Cerezo 2, César Viseras 1,2 and C. Ignacio Sainz-Díaz <sup>1</sup>**


Received: 6 September 2019; Accepted: 12 October 2019; Published: 14 October 2019

**Abstract:** Calcium carbonate is an abundant mineral with several advantages to be a successful carrier to improve oral bioavailability of poorly water-soluble drugs, such as praziquantel. Praziquantel is an antiparasitic drug classified in group II of the Biopharmaceutical Classification System hence characterized by high-permeability and low-solubility. Therefore, the dissolution rate is the limiting factor for the gastrointestinal absorption that contributes to the low bioavailability. Consequently, the therapeutic dose of the praziquantel must be high and big tablets and capsules are required, which are difficult to swallow, especially for pediatric and elderly patients. Mixtures of praziquantel and calcium carbonate using solid-solid physical mixtures and solid dispersions were prepared and characterized using several techniques (X-ray diffraction differential scanning calorimetry, thermogravimetric analysis, scanning electron microscopy, laser diffraction, Fourier transform infrared and Raman spectroscopies). Solubility of these formulations evidenced that the solubility of praziquantel-calcium carbonate interaction product increased in physiological media. *In vitro* dissolution tests showed that the interaction product increased the dissolution rate of the drug in acidic medium. Theoretical models were studied to understand this experimental behavior. Cytotoxicity and cell cycle studies were performed, showing that praziquantel-calcium carbonate physical mixture and interaction product were biocompatible with the HTC116 cells, because it did not produce a decrease in cell viability or alterations in the cell cycle.

**Keywords:** praziquantel; calcium carbonate; schistosomiasis; bioavailability; solubility; cytotoxicity

#### **1. Introduction**

Calcium carbonate, CaCO3, being a low cost material, with high surface area, excellent safety, biocompatibility and biodegradability, is a well-documented excipient in pharmaceutical solid dosage forms, mainly used as diluent [1]. Recently, it has also been demonstrated that calcium carbonate can successfully act as hydrophilic porous carrier to improve the oral bioavailability of low water soluble drugs [2–5]. Functionalization with hydroxypropyl-β-cyclodextrin [6], enzymatic macromolecules [7], or incorporation in polymeric hydrogels [8–11] can further enhance the ability of calcium carbonate to improve oral delivery of several biomolecules, including proteins [12] and anti-cancer drugs [13]. As recently reviewed, most of the successfully designed calcium carbonate carriers have been

prepared by emulsion techniques or chemical precipitation of the carbonate micro/nanoparticles [14]. However, precipitation of calcium carbonate micro/nanoparticles is difficult to reproduce and scale up of the procedure is most of times challenging, avoiding the clinical use of these carriers. Moreover, detailed understanding of biosafety and *in vivo* degradation of the new calcium carbonate particles would require preclinical studies. An interesting alternative is to overcome these challenges by using normalized pharmaceutical excipients grades of calcium carbonate as drug delivery carriers [1].

Praziquantel (PZQ) is the drug of choice in the treatment of schistosomiasis [15], being included in the WHO Model List of Essential Drug for the treatment of adults and children [16]. Schistosomiasis affects approximately 210 million people, causing 200,000 deaths every year. Moreover, it is widely extended, mainly in 78 developing countries in the tropics and subtropics, although at least 92% of people who need treatment for schistosomiasis live in Africa and, is the second of the most prevalent disease (after malaria) affecting African children. PZQ is classified in group II of the Biopharmaceutical Classification System (BCS) and hence characterized by high permeability and low solubility [17]. Therefore, the dissolution rate is the limiting factor for the gastrointestinal absorption that contributes to the low oral bioavailability. Moreover, absorbed amounts of PZQ suffer an extensive first-pass metabolism [18], leading to administration of high-dosed dosage forms, which are difficult to swallow, especially for pediatric patients [19].

Enhancing the water solubility and the dissolution rate is critical to increase the oral bioavailability of PZQ. Several studies and strategies have been carried out to improve PZQ water solubility, such as incorporation into liposome vectors [20], preparation of solid dispersions of the drug with different excipients such as clay minerals [21], β-cyclodextrins [22–24], polyvinyl-pyrrolidone [25–27], polyethylene glycols [28–30], sodium starch glycolate [31] and preparation of dispersible granules [14]. In addition, other techniques have been used to increase the solubility of PZQ such as co-grinding with several excipients [32], melt granulation and ultrasonic spray congealing [27], and the transition in a new crystalline polymorph by milling [19]. More recently, our previous paper evidenced conformational changes of PZQ in association with calcium carbonate after a solvent evaporation process [33]. This interaction product revealed the formation of modifications of the pristine racemic drug attributable to either polymorph B [19] or disordered pseudoracemate solid phases [33].

Given these premises, the first aim of this work was to verify if PZQ solid state modification within calcium carbonate solid dispersion is due to the solvent evaporation process or to drug-carrier interactions that would happen during a mere blending procedure. Thus, the physico-chemical properties of the physical mixture were compared to those of the interaction product. Further, we explored the possibilities of calcium carbonate/PZQ systems for the improvement of biopharmaceutical properties of the drug. In particular, solubility and dissolution profiles of PZQ from interaction product were compared to the corresponding physical mixture, and the results explained by using theoretical modeling. In addition, we investigated the *in vitro* cytotoxicity and cell cycle studies of calcium carbonate/PZQ systems.

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

#### *2.1. Materials*

PZQ drug was kindly donated by Fatro S.p.A. (Bologna, Italy). Ground Calcium Carbonate Calcitec Pure PH V/40S (GCC) was purchased from Mineraria Sacilese (Sacile, Italy). This kind of calcium carbonate is used in the pharmaceutical industry and was accepted in the latest edition of the European and United States Pharmacopoeias. The GCC finds also employment in the food industry and in industries where is requested a low content of heavy metals. Ethanol of 96% of purity was used as solvent.

#### *2.2. Preparation of the PZQ and GCC Physical Mixture*

Physical mixtures (PM) of PZQ and GCC (1:5 *w*/*w*) were prepared blending both solids in an agate mortar of 100 mm of diameter at room temperature for 5 min.

#### *2.3. Preparation of the PZQ and GCC Interaction Product (IP)*

GCC was dispersed in 1 L of ethanolic solution of PZQ under magnetic stirring at room temperature for 24 h, so drug/GCC ratio was 1:5 *w*/*w* in order to ensure the complete interaction between PZQ and GCC. After 24 h, the solvent was evaporated with rotary evaporator (Rotary evaporator Buchi® R II, Flawil, Switzerland) at 40 ◦C and reduced pressure. The solid residue was dried in a desiccator and then it was pulverized.

#### *2.4. Solid State Characterization of Calcium Carbonate*/*Praziquantel Systems*

#### 2.4.1. X-ray Diffraction (XRD)

An X-Pert Pro® diffractometer (Marvel Panalytical, Madrid, Spain) with the CuKα radiation was used for performing powder X-ray diffraction. The powder samples were scanned in the range of 4◦–70◦ of the 2θ angle, steps were of 0.008 of 2θ and the counting time was of 10.16 sec/step. The diffraction results were analyzed with the XPOWDER® software version 2004 [34].

#### 2.4.2. Thermal Analysis

Differential scanning calorimetric analysis (DSC) and thermogravimetric analysis (TGA) were performed with a mod. TGA/DSC1 calorimeter (Mettler Toledo, Barcelona, Spain) equipped with a sensor and FRS5 microbalance (precision 0.1 μg) and FP89 software package. Samples were heated in air atmosphere at 5 ◦C/min in the in the 30–200 ◦C temperature range for DSC and 30–420 ◦C temperature range for TGA.

#### 2.4.3. Scanning Electron Microscope (SEM)

Microphotographs of the samples were performed using a Hitachi S-510 scanning electron microscope (voltage 25 kV, secondary electron images) (Hitachi Scientific Instruments Ltd., Tokyo, Japan). The samples were mounted on adhesive carbon paper, fixed with colloidal gold and metallized with gold in two orientations (20–30◦). The images were captured digitally using the program attached to the microscope (ScanVision, Version 1.2).

#### 2.4.4. Particle Size Analysis

The particle size distribution of the solid sample materials suspended in liquid medium was analyzed with Laser Light Diffraction technology. The equipment used was a Mastersizer 2000LF from Malvern Panalytical Instruments (Madrid, Spain) consisting of HYDRO MU Malvern manual liquid sample dispersion unit and Malvern HYDRO 2000Up minimum volume liquid sample dispersion unit.

#### 2.4.5. Fourier Transform Infrared and Raman Spectroscopies

Fourier Transform Infrared Spectroscopy (FTIR) spectra were recorded with a 6200 spectrophotometer (JASCO, Pfungstadt, Germany) in the range 4000–600 cm−<sup>1</sup> with a 0.5 cm−<sup>1</sup> resolution and a well-plate sampler along with the Spectra Manager II software.

Raman spectra were recorded using a JASCO NRS-5100 Micro-Raman dispersive spectrophotometer in the range 3500–800 cm−<sup>1</sup> with a 6.48 cm−<sup>1</sup> resolution, with laser light source VIS-NIR with red diode at 785 nm with 500 mW of power (Torsana Starbright) refrigerated by air and the KnowItAII JASCO for Raman software.

#### *2.5. Solubility Studies*

The solubility of PZQ as well as the physical mixture PM PZQ-GCC and the interaction product IP PZQ-GCC were studied separately in two media: an acidic medium of HCl 0.001 M and a simulated intestinal fluid (SIF) without enzymes with a buffer at pH 6.8. The solubility of PZQ was calculated by placing a supersaturated solution of the pristine drug, specifically 30 mg in 10 mL in each of the media. The supersaturated solution was stirred in a thermostatic bath for 72 h at 37 ◦C. After 72 h, it was centrifuged, and the supernatant was filtered and measured on high-performance liquid chromatography (HPLC). Obtaining by means of the HPLC the amount of dissolved drug that corresponds to its solubility. This experiment was repeated 6 times. In the same way this procedure was applied for the PM and the IP PZQ-GCC.

#### *2.6. Dissolution Studies*

PZQ-GCC physical mixture or PZQ-GCC interaction product (210 mg) were encapsulated in double zero (00) gelatin capsules, corresponding to 35 mg of PZQ and 175 mg of GCC in each capsule. As well as, 35 mg of PZQ were encapsulated as reference. The obtained capsules were subjected to sink conditions dissolution tests using the official Pharmacopoeia USP apparatus 2 for the dissolution test of oral solid dosage forms (Sotax AT7, S). This apparatus is equipped with a rotation system type palettes and sinkers, piston pump for the automatic sampling at scheduled times and collector of fractions. The measurements were performed at 37 ◦C and 150 rpm in 1 L of medium. Two separate dissolution media were studied: an acidic medium of HCl 0.001 M (simulated stomach) and a SIF medium without enzymes with a buffer at pH 6.8 (simulated intestine). Aliquots of 5 mL were collected from the dissolution test, filtered through 0.45 μm Millipore® (S) membranes and analyzed by HPLC for drug content. Volumes of 5 mL of fresh dissolution medium were replaced after each sampling to maintain the volume constant. At least three replicates for each sample were assayed.

#### *2.7. HPLC Analysis*

Drug analysis was performed using a 1260 Infinity II Agilent HPLC system (Santa Clara, CA, USA) equipped with quaternary pump, autosampler, column oven and UV-VIS diode-array spectrophotometer. The stationary phase was a Kromasil® C18 column, 5 <sup>μ</sup>m, 250 <sup>×</sup> 4.6 mm (Teknokroma, Barcelona, Spain) and the mobile phase was a mixture of H2O and CH3CN (35:65 *v*/*v*). The flow rate was set at 0.8 mL/min with an injection volume of 10 μL. A spectrophotometer detector at a 225 nm wavelength was used and the run time for each analysis was 5 min. Data were recorded and analyzed by using software LC Open LAB HPLC 1260 (Agilent). The response of the analytical method was linear in the concentration range 5–100 mg/L of PZQ in both media, resulting in correlation coefficients of 1 (both in HCl 0.001 M and SIF).

#### *2.8. Computational Methods*

The molecular model of PZQ was extracted from previous calculations [35] and the rest of components were placed by hand. The Compass force field (FF) [36] based on empirical interatomic potentials was used within the Discover program of the Materials Studio package [37]. This FF was used previously to describe PZQ molecules and crystal structure with satisfactory results [35,38]. An atomic interactions cut-off of 18.5 Å was used for calculating Van der Waals and Coulomb interactions.

#### *2.9. Cell Culture*

Cell viability tests and study the cell cycle profiles were performed for observing a possible production of cell death in a tumor line of cells derived from colorectal carcinoma, called HCT116. This tumor line of HCT116 colorectal carcinoma cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco Invitrogen, Dublin, Ireland) supplemented 10% with decomplemented Heat-Inactivated Fetal Bovine Serum (FBS) (Gibco Invitrogen), with glutamax (BioWhittaker, Cologne,

Germany) at 1% and with Penicillin/Streptomycin (BioWhittaker) at 1%. The cell culture was kept in an incubator at 37 ◦C and 5% of CO2.

#### *2.10. Cytotoxicity Studies*

Firstly, PZQ and GCC samples were prepared relying on the administered drug amount in animals as antiparasitic. In such way, the amount of the mineral was five times more than that of PZQ. To do this, we prepared an intermediate dilution in dimethylsulfoxide (DMSO) at a concentration of 100 mM and then another dilution at 10 mM. The physical mixture and the interaction product PZQ-GCC were studied using the same procedure.

To perform the proliferation assays by Alamar Blue, about 10,000–20,000 cells/well of HCT116 were seeded in 96-well plates, in a final volume of 200 μL of DMEM medium using several concentrations (100 μM, 20 μM, 4 μM, 800 nM, 160 nM, 32 nM and 6.4 nM) for each sample studied (PZQ, GCC, PM and IP PZQ-GCC). Cells were incubated at 37 ◦C, 5% CO2 for 48 h. After the incubation time, 10 μL per well of the PrestoBlue cell Viability Reagent (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) was added, incubating for 15 min. After this period, fluorescence was measured at 535–90 nm in a Tecan reader (Männedorf, Switzerland). This reagent is based on resazurin compound, which works as an indicator of viability when it is reduced by living cells and presents a colour change from blue (dead cells) to pink (living cells).

#### *2.11. Cell Cycle Studies*

The cells were cultured in 24 well plates at a concentration of 250,000 cells/well in 500 μL and treated with the previously selected PZQ, GCC, PM and IP PZQ-GCC samples, at increasing concentrations of 0.8, 4, 20 and 100 μM for 48 h. Dead cells (apoptotic and necrotic) were detected based on staining with propidium iodide following the protocol described [39]. Briefly, the cells after the corresponding treatments were collected and washed with 2 mL of phosphate buffered saline (PBS) at 4 ◦C and fixed with 100 μL of PBS and 900 μL of 70% ethanol on ice for 5 min. After washing with PBS, they were resuspended in 250 μL of PBS and another 250 μL of a DNA extraction solution (0.2 M Na2HPO4, 0.1M C6H8O7, pH 7.8) and incubated at 37 ◦C for 10 min. The supernatant was removed and 200 μL of the staining solution was added (8 μL propidium iodide (1 mg/mL) and 2 μL RNAse 100 (μg/mL)), incubating the samples for 10 min at 37 ◦C in the dark. Fluorescence was measured in the FL2 detector of the FACScalibur cytometer (Becton Dickinson & Co., Franklin Lakes, NJ, USA) and the analysis of the sub-G1 population (population of dead cells: necrotic and apoptotic) was done using the BD CellQuest software v1.0.2 (BD, Biosciences).

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

#### *3.1. Solid State Characterization of Calcium Carbonate*/*Praziquantel Systems*

PZQ and GCC (1:5) solid dispersions obtained by solvent evaporation evidenced conformational changes of the pristine drug solid state [33]. Recently, a number of papers [19,32,40] have elucidated that the mechanochemical activation of PZQ, via neat grinding or comilling PZQ with different polymers (Povidone, Copovidone, Crospovidone and Sodium Starch Glycolate) enabled the transformation of the original PZQ polymorphic form into a new polymorphic variety of racemic PZQ or into a drug amorphous state.

In this study, the properties of PZQ-GCC physical mixture (produced by blending) were compared to those of the PZQ-GCC interaction product (obtained by solvent evaporation). A solid state characterization of the samples studied was performed by X-ray diffraction (Figure S1), differential scanning calorimetric analysis (Figure S2), thermogravimetric analysis (Figure S3), scanning electron microscope (Figure S4), Fourier Transform Infrared and Raman Spectroscopies (Figure S5) and particle size analysis. The results demonstrated that PZQ-GCC physical mixture did not show any chemical-physical change with respect to the pure drug. On the contrary, in the PZQ-GCC interaction

product multiple changes were observed, as already described in a previous work [33]. The detailed results and analyses of the solid state characterization of the samples studied (PZQ, GCC, PM and IP) are provided in the Supplementary Material.

#### *3.2. Solubility Studies*

The solubility tests showed that the IP PZQ-GCC increased the solubility of the pure drug from 0.50 mg/mL to 1.42 mg/mL in an acid aqueous medium with 0.001 M HCl (pH = 3), due to its interaction with the GCC and the solubility of the GCC in acidic medium. On the contrary, the PM PZQ-GCC showed a small and unimportant increase in the solubility of the drug (Table 1). The results in a SIF medium (pH = 6.8) revealed that the IP PZQ-GCC increased the solubility of PZQ from 0.45 mg/mL to 0.73 mg/mL; while the PM showed a slight increase in the solubility of PZQ from 0.45 mg/mL to 0.50 mg/mL, which was not considered notable. Therefore, the solubility of the IP PZQ-GCC improves in acid and SIF media with respect to the PZQ and PM. This increase is much higher at pH 3 (Table 1).

**Table 1.** Solubility values of praziquantel (PZQ), physical mixture (PM) of PZQ-GCC and interaction product (IP) of PZQ-GCC in acid and simulated intestinal fluid (SIF) media (mean values ± standard deviation; *n* = 0.07).


#### *3.3. Dissolution Studies*

In order to explore the *in vitro* bioavailability of PZQ, dissolution tests in both media simulating the gastric and the intestinal fluids, were performed for the pristine drug and the combinations of PZQ with GCC (IP and PM) (Figure 1).

PZQ-GCC physical mixture presented a similar behavior to the pristine PZQ in both dissolution media (Figure 1a,b), while the IP showed a completely different behavior at pH 3 with respect to pH 6.8. Interaction product PZQ-GCC showed an increase in the dissolution rate in acid medium (Figure 1a) and a strong decrease in SIF (Figure 1b). These results were concordant with solubility tests results, where the solubility of IP PZQ-GCC is higher in acid medium than in SIF. This behaviour may be correlated to the different solubility of GCC at acidic and neutral pH and to structural changes observed in IP: as GCC is soluble at low pH, its interaction at molecular level with water and PZQ favoured the dissolution and solubility of the drug. Likewise, the PZQ-GCC interaction at pH 6.8, where the carbonate is completely insoluble, negatively affected the solubilisation of the PZQ molecules.

**Figure 1.** Drug release (% *w*/*w*) profiles from the PZQ and the PM in 0.001 M HCl at pH = 3 (**a**) and in a SIF medium at pH = 6.8 (**b**) in sink conditions; (mean values ± standard deviation; *n* = 5).

#### *3.4. Modeling Approach*

In order to explain the dissolution behavior of the PZQ-GCC systems, small models of PZQ molecules with the GCC and water molecules were created (Figure 2). In the PM, the carbonyl groups of PZQ are at the same side of the molecule as a *syn* conformer, whereas in IP the proportion of the *anti* conformer is higher [38]. The geometry of these models was optimized with Compass FF calculations, using the SPC water model (Figure 2c,d). In both models the carbonate anion is coordinated with the Ca2<sup>+</sup> cation *d*(Ca ... OCO) = 1.88–1.91 Å and at least one water molecule is between the Ca2<sup>+</sup> cation and a carbonyl group forming a hydrogen bond with the corresponding carbonyl group *d*(HOH ... O=C) = 1.84–1.88 Å. In the *syn* conformer, both water molecules are coordinating the Ca2<sup>+</sup> cation and the water H atoms *d*(HOH ... Ca) = 2.27 Å (Figure 2c).

**Figure 2.** Possible complexes of PZQ molecule conformer *syn* (**a**) and *anti* (**b**) with hydrated Ca2<sup>+</sup> carbonates clusters, and optimized complexes of *syn* (**c**) and *anti* (**d**) PZQ (non-bonding distances are included in Å). The C, Ca, H, N, and O atoms are in grey, green, white, blue, and red color.

In the *anti* conformer, one water molecule is coordinating the Ca2<sup>+</sup> cation, d(O ... Ca) = 2.27 Å, and one carbonyl O atom d(CO ... H) = 2.22 Å leaving the another carbonyl group free in the opposite side of the molecule to form an additional carbonate complex. The conformer *syn* can form only one complex with the hydrated Ca2<sup>+</sup> cation (Figure 2). This could explain the higher dissolution process of the *anti* form of PZQ and hence the higher solubility of IP PZQ-GCC.

#### *3.5. Cytotoxicity Studies*

*In vitro* cytotoxicity tests in the HCT116 cell line showed that all the PZQ and GCC pure concentrations (6.4 nM–100 μM) tested provided viability values slightly lower than the untreated cells (control). Therefore, PZQ and GCC pure samples can be considered biocompatible toward HCT116 cell lines (Figure 3). In the IP and PM PZQ-GCC products similar results were found, whose biocompatibility across the range of concentrations was evaluated and a trend similar to the control was observed and slightly positive with respect to the pure samples (PZQ and GCC) in cell growth. Therefore, it is notable that IP and PM PZQ-GCC products have a biocompatible behaviour, so the interaction between PZQ and GCC had a positive effect in the cell viability (Figure 3).

**Figure 3.** Cell viability from the studied samples after 48 h of treatment. (Control: untreated cells in complete medium; mean values ± standard error; *n* = 8).

#### *3.6. Cell Cycle Studies*

A study of the cell cycle by means of propidium iodide was carried out to study the cell cycle corresponding to the Sub-G1 phase of the cells in contact with our samples (Figure 4). Once observed through cytotoxicity studies that the samples tested did not cause cell death, a cell cycle study was carried out to check whether the compounds affected any phase of the cell cycle despite not affecting proliferation. In the upper part of the figure the control tests can be observed: an example of the cell cycle of healthy untreated cells, an example of cells treated with DMSO, where it is observed that DMSO does not affect the cell and also an example of cells treated with etoposide, an antineoplastic drug that damages cells. In this last example, the cell cycle of the control cells is severely affected and induces death to 64.3% of the cell population.

**Figure 4.** Cell cycle of the HCT116 line treated with the sample studies. The percentages indicate the number of cells in Sub-G1 (apoptotic or necrotic).

The studied samples showed that they do not affect in any case the cellular cycle of the cells, only in some cases at high concentrations can the cell death increase slightly, although not in a noteworthy way (Figure 4).

#### **4. Conclusions**

Interaction products obtained with PZQ and GCC increased the solubility of PZQ in physiological media, particularly in acid medium. *In vitro* dissolution tests evidenced that the interaction product increased the dissolution rate of the drug in acidic medium. *In vitro* cytotoxicity and cell cycle studies were performed, showing neither the PZQ-GCC physical mixture nor the interaction product produced cellular damage in any of the studied concentrations. Therefore, the samples studied did not produce cell death or alteration in the cell cycle and were biocompatible with the HTC116 cells.

As a general conclusion, the use of low cost GCCimprove solubility and dissolution rates of PZQ, without producing cytotoxicity or alterations in the cell cycle of HTC116 cells. This allows exploring the use of GCC as an interesting technological strategy for a drug administration in a more effective biopharmaceutical and clinical way ofPZQ. Also providing the great advantage that the design and development of the PZQ-GCC new formulations would not increase the final cost of the drug, overcoming a great challenge due to the population which the treatment is destined and the high prevalent of the neglected tropical disease.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/10/533/s1, Section 1: Solid State Characterization of Calcium Carbonate/Praziquantel Systems. Figure S1: Powder X-ray diffraction (XRD) patterns of praziquantel (PZQ), ground calcium carbonate (GCC), physical mixture (PM) of PZQ-GCC and interaction product (IP) of PZQ-GCC. Figure S2: DSC curves of PZQ, GCC, PM and IP. Figure S3: TGA profiles of the GCC, IP, PM, and PZQ solids, Figure S4: SEM micropictures of GCC, pristine PZQ, PM and IP. Figure S5: FTIR (a), and Raman (b) spectra of PZQ, GCC, IP and PM.

**Author Contributions:** Conceptualization, B.A., N.P., C.V. and C.I.S.-D.; methodology, A.B.-S., C.V. and C.I.S.-D.; software, A.B.-S. and C.I.S.-D.; formal analysis, A.B.-S. and R.S.-E.; investigation, A.B.-S. and R.S.-E.; resources, P.C., C.V. and C.I.S.-D.; writing—original draft preparation, A.B.-S.; writing—review and editing, A.B.-S., B.A., N.P., P.C., R.S.-E., C.V. and C.I.S.-D.; supervision, A.B.-S., C.V. and C.I.S.-D.; project administration, A.B.-S.; funding acquisition, P.C., C.V. and C.I.S.-D.

**Acknowledgments:** The authors are thankful to Fatro S.p.A. for donating the praziquantel drug and to Xtrem Biotech, specially Ignacio Molina and Sara Torres, for their contributions with the cytotoxicity and cell cycle experiments. We also acknowledge for financial support the MINECO, for projects FIS2016-77692-C2-2-P and CGL2016-80833-R, and the Andalusian government, for project RNM1897.

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

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Resorbable Beads Provide Extended Release of Antifungal Medication: In Vitro and In Vivo Analyses**

**Yung-Heng Hsu 1, Huang-Yu Chen 2, Jin-Chung Chen 3, Yi-Hsun Yu 1, Ying-Chao Chou 1, Steve Wen-Neng Ueng 1,\* and Shih-Jung Liu 1,2,\***


Received: 15 September 2019; Accepted: 22 October 2019; Published: 24 October 2019

**Abstract:** Fungal osteomyelitis has been difficult to treat, with first-line treatments consisting of implant excision, radical debridement, and local release of high-dose antifungal agents. Locally impregnated antifungal beads are another popular treatment option. This study aimed to develop biodegradable antifungal-agent-loaded Poly(d,l-lactide-*co*-glycolide) (PLGA) beads and evaluate the in vitro/in vivo release patterns of amphotericin B and fluconazole from the beads. Beads of different sizes were formed using a compression-molding method, and their morphology was evaluated via scanning electron microscopy. Intrabead incorporation of antifungal agents was evaluated via Fourier-transform infrared spectroscopy, and in vitro fluconazole liberation curves of PLGA beads were inspected via high-performance liquid chromatography. When we implanted the drug-incorporated beads into the bone cavity of rabbits, we found that a high level of fluconazole (beyond the minimum therapeutic concentration [MTC]) was released for more than 49 d in vivo. Our results indicate that compression-molded PLGA/fluconazole beads have potential applications in treating bone infections.

**Keywords:** fluconazole; orthopedic infection; Poly(d,l-lactide-*co*-glycolide) beads; sustained release

#### **1. Introduction**

Fungal osteomyelitis is an uncommon disease presenting significant challenges to orthopedic surgeons. The severity of a fungal infection is associated with the immune status of individuals and the fungal species. A large number of fungal infections have been reported in both immunocompromised and immunocompetent individuals [1]. Fungal infections typically result from three routes: direct inoculation, hematogenous spreading, or contiguous spreading [2,3]. In certain conditions, including long-term antibiotic use, infections of *Candida*, *Aspergillus*, and other common fungi are characterized by the formation of a biofilm that resists antifungal treatment, thus further strengthening the infection [4]. Certain fungal infections can exclusively be treated with antifungal agents [5]. However, most patients are difficult to treat via radical debridement. General principles for treatment of both bacterial osteomyelitis and fungal infections include excision of all nonviable tissue, the use of orthopedic hardware, radical debridement, and local drug delivery with an effective concentration of antifungal/antibiotic agents [6,7].

The most common fungi causing bone and joint infections are *Candida* spp., and *Candida albicans* in particular [8]. Fluconazole is a first-generation triazole antifungal agent commonly used to treat *Candida albicans* infections [9]. Furthermore, amphotericin B sodium deoxycholate effectively inhibits biofilm formation in multiple *Candida* infections [10]. Treatment of *Candida* osteomyelitis involves surgical debridement and long-term administration of antifungal agents. The guidelines of the Infectious Diseases Society of America (IDSA) for the treatment of *Candida* osteomyelitis suggest the administration of fluconazole for 6–12 months [11]. Currently, antifungal-agent-containing beads or polymethyl methacrylate (PMMA) spacers are popular treatment methods for fungal osteomyelitis or periprosthetic joint fungal infections [4]. The beads or spacers delivered to bone tissue display sustained long-term release of antifungal agents at an effective concentration. However, the potential risk of irritation in host tissues combined with the non-biodegradable nature of PMMA may limit application in osteomyelitis treatment, thus warranting surgical excision. Kweon et al. reported that adding 10 g poragen to antifungal-loaded bone cement (ALBC) containing 200 mg amphotericin B decreases the compressive strength of PMMA beads and thus limits their use for implant fixation [12]. Sealy et al. showed poor release dynamics of fluconazole in ALBC [13]. Furthermore, Goss et al. reported that amphotericin B could not be eluted through PMMA bone cement [14]. An ideal drug delivery system should provide adequate antifungal concentrations at the target site, offer a slow and sustained release of an antimicrobial over an extended period, and be biodegradable so that a second operation is not needed.

Biodegradable antifungal-agent-loaded beads possess advantages over conventional PMMA beads in four ways. First, biodegradable beads provide high concentrations of antifungal agents for the extended time needed to completely treat the particular orthopedic infection. Second, variable biodegradability from weeks to months permits various types of infections to be treated. Third, the biodegradable vehicles degrade eventually, and surgical removal of the beads is not required. Fourth, the biodegradable beads dissolve gradually and the soft tissue or bone defect slowly fills with tissue, it is thus not necessary for bone/tissue reconstruction [15].

This current study developed biodegradable antifungal-agent-loaded vehicles for a long-term drug release. We utilized a compression-molding method to fabricate fluconazole-incorporated Poly(d,l-lactide-*co*-glycolide) (PLGA) beads and assessed drug release dynamics in vitro and in vivo. Among the various polymeric materials available to develop local drug release systems, PLGA is promising, owing to its degradable polymer that facilitates long-term drug delivery at high doses to the target region [16,17]. Moreover, this material has been certified for clinical use owing to its nontoxic nature and minimum inflammatory effects.

After molding, bead morphology was evaluated via scanning electron microscopy (SEM), and intrabead antifungal drug incorporation was assessed via Fourier-transform infrared (FTIR) spectroscopy. In vitro fluconazole liberation curves of PLGA beads were inspected via high-performance liquid chromatography (HPLC), and in vivo fluconazole release was investigated by implanting the drug-incorporated beads into the bone cavity of rabbits. We found that a level of fluconazole greater than the minimum therapeutic concentration (MTC) was released for more than 49 d in vivo, indicating that compression-molded PLGA/fluconazole beads are a promising candidate for the treatment of bone infections.

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

#### *2.1. Fabrication of Poly(*d*,*l*-lactide-co-glycolide) PLGA*/*Amphotericin B and PLGA*/*Fluconazole Beads*

All materials utilized in this study, including PLGA (50:50), amphotericin B, and fluconazole, were acquired from Sigma-Aldrich (St. Louis, MO, USA).

PLGA/amphotericin B and PLGA/fluconazole beads were prepared using a laboratory-scale compression-molding system equipped with an isothermal oven with a temperature range of 25 ◦C to 300 ◦C. Beads of two different polymer:drug ratios (6:1 and 4:1) and three sizes (3, 5, and 8 mm) were fabricated. PLGA, amphotericin B, and fluconazole at predetermined weights were first mixed using a dry mixer and placed in molds (Figure 1) customized for this study. Table 1 lists the composition of beads of different sizes. The mold, along with the mixture, was then compressed at 700 MPa and placed in an isothermal oven at 65 ◦C for sintering for 1.5 h. Pure PLGA beads were simultaneously prepared as a control.

**Figure 1.** Mold used for compression molding of the antifungal-agent-containing beads.



#### *2.2. In Vitro Analysis of Amphotericin B and Fluconazole Release*

In vitro release of amphotericin B and fluconazole from the biodegradable beads was assessed via an elution method. PLGA/amphotericin B and PLGA/fluconazole beads were placed in test tubes (*N* = 3) containing 1 mL buffered solution at 37 ◦C. The tubes were deposed in an isothermal oven for 24 h, and the eluent was harvested and substituted with fresh solution (1 mL). The process was carried out in duplicate for 40 d. Amphotericin B and fluconazole levels in the harvested eluents were quantified via HPLC, carried out using a Hitachi L-2200 System (Hitachi Medical Systems, Tokyo, Japan). All experiments were performed in triplicate (*N* = 3).

#### *2.3. FTIR Spectrometry*

The thermal stability of fluconazole was determined via FTIR spectrometry to evaluate whether the chemistry and orientation of material structures varied with temperature. FTIR analysis was conducted using a Nicolet iS5 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) at a resolution of 4 cm−<sup>1</sup> (32 scans). The drugs were compressed as KBr discs, and spectra were recorded over 400–4000 cm<sup>−</sup>1. Fluconazole was considered stable if no obvious structural changes occurred due to temperature. The FTIR spectra of pure PLGA beads were compared with those of fluconazole-loaded PLGA beads.

#### *2.4. Determination of the Water Contact Angle*

The water contact angles of the beads were measured on a contact angle measurement device (First Ten Angstroms, Portsmouth, VA, USA) (*N* = 3).

#### *2.5. Cell Culture*

Cytotoxicity of fluconazole-loaded PLGA beads was examined via a Cell Counting Kit-8 (CCK-8) assay (Sigma-Aldrich, St. Louis, MO, USA) for cell viability, in accordance with the manufacturer's

instructions. Eluents harvested at 1, 2, 3, 7, and 14 d were placed in 96-well culture plates. Human fibroblasts obtained from foreskins of patients (1–3 years of age) undergoing surgery were seeded (1 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well) in Dulbecco's Modified Eagle's Medium (DMEM) at 37 ◦C and 5% CO2/95% for 48 h. Cell viability was monitored via the CCK-8 assay and quantified using an ELISA reader.

#### *2.6. In Vivo Animal Study*

All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Chang Gung University (CGU107-275, Approved 19 March 2019), and all experimental animals were provided care in accordance with the regulations of the Ministry of Health and Welfare of Taiwan under the supervision of a licensed veterinarian.

Four adult New Zealand white rabbits (Animal Health Research Institute, Panchiao, Taiwan) weighing approximately 3.5 ± 0.3 kg were enrolled in the experiment. The rabbits were housed in individual cages in a temperature- and light-controlled room, with ad libitum access to standard rabbit chow and sterilized drinking water. All animals were administered general anesthesia via inhalation of isoflurane through a vaporizer (Matrx, Pompano Beach, FL, USA) in a plastic box (40 cm × 20 cm × 28 cm). Anesthesia was maintained during the entire surgical procedure via mask inhalation of isoflurane.

After rabbits were sedated, the right femoral sites were depilated, washed with soft soap, and treated aseptically with 70% ethanol directly before the surgical procedure. The other site of the animals was covered with a sterile blanket. Under aseptic conditions, the middle/third region of the right femur was dissected via an anterolateral approach. A bone defect (5.0 <sup>×</sup> 10.0 mm2) was induced at the right femoral middle site, and a polymethylmethacrylate (PMMA) spacer was initially inserted. The wound was closed with 3-0 Vicryl sutures (Johnson & Johnson, New Brunswick, NJ, USA). After 2 weeks, the PMMA spacer was surgically excised and fluconazole-impregnated PLGA cylindrical beads (5.0 <sup>×</sup> 6.0 mm2) were placed into the right femoral bone cavity (Figure 2). The wound was closed in a layer-by-layer manner. In vivo drug concentrations were determined by sampling specimens (surgical site fluid) with aspirates obtained on days 1, 2, 3, 7, and 14. After 2 weeks, owing to difficulty aspirating fluid from the surgical site, we harvested tissue around the bead area on days 21, 28, 35, 42, and 49. Cylindrical specimens of tissue surrounding the beads were centrifuged, and the plasma was sampled and stored at −80 ◦C until analysis. In vivo fluconazole concentrations in the tissue samples were determined via HPLC. All samples were diluted with phosphate-buffered saline and assessed in accordance with the assay standard curve. A calibration curve was generated for each set of measurements (correlation coefficient > 0.99). Blood samples were obtained from the marginal ear vein using a syringe to determine serum aspartate transaminase (AST) levels and antibiotic concentrations after implanting the fluconazole-impregnated beads. AST levels were determined using the IDEXX Catalyst DX system (Westbrook, ME, USA), while fluconazole concentrations were determined via HPLC. Data thus obtained were used to assess the liver function of rabbits during experiments.

**Figure 2.** Images of the surgical procedure. (**A**) The right femoral site was depilated and sterilized. (**B**) a bone cavity was formed (5.0 <sup>×</sup> 10.0 mm2); (**C**) a polymethylmethacrylate spacer was placed into the bone cavity; (**D**) after 2 weeks, fluconazole-impregnated Poly(d,l-lactide-*co*-glycolide) cylindrical beads (5.0 <sup>×</sup> 6.0 mm2) were placed into the right femoral bone cavity.

#### **3. Results**

#### *3.1. Characterization of Fabricated Poly(*d*,*l*-lactide-co-glycolide) PLGA*/*Amphotericin B and PLGA*/*Fluconazole Beads*

Amphotericin B- and fluconazole-containing beads of 3, 5, and 8 mm were prepared through compression molding (Figure 3). To confirm that the molding temperature did not deactivate the drugs, a thermal stability test for fluconazole was carried out using FTIR spectroscopy. As shown in Figure 4, amphotericin B and fluconazole remained intact at 70 ◦C, indicating that a temperature of 65 ◦C is optimal for compression molding.

**Figure 3.** Photographs of the fabricated drug-loaded beads. The white cylindrical beads are fluconazole-impregnated Poly(d,l-lactide-*co*-glycolide) (PLGA) beads. The yellow cylindrical beads are amphotericin B-impregnated PLGA beads.

**Figure 4.** Effect of molding temperature on (**A**) amphotericin B and (**B**) fluconazole stability determined by Fourier-transform infrared spectroscopy.

Water contact angles determined herein are shown in Figure 5. While the pure PLGA beads exhibited hydrophobic properties (water contact angle of 97.38◦), antifungal drug-containing beads were hydrophilic (all angles were less than 70◦). In addition, the water contact angle of PLGA/fluconazole beads decreased with an increase in the drug content of beads, primarily owing to the hydrophilic nature of fluconazole.

**Figure 5.** Water contact angles for pure Poly(d,l-lactide-*co*-glycolide) and drug-loaded beads of different polymer:drug ratios and sizes.

To confirm successful incorporation of amphotericin B and fluconazole in the beads, FTIR spectra of drug-loaded beads were compared with those of pure PLGA beads (Figure 6). The new absorption peak at 1600 cm−<sup>1</sup> might be attributable to the C=N bonds of amphotericin B and fluconazole [18,19]. Enhanced absorption at 1670–1780 cm−<sup>1</sup> corresponded to C=O bonds, primarily owing to supplementation with antifungal drugs, and an absorbance peak of approximately 1270 cm−<sup>1</sup> may have resulted from C–F bond enhancement in loaded drugs. The FTIR spectra indicate that the antifungal drugs were successfully incorporated into PLGA beads.

**Figure 6.** Fourier-transform infrared spectra of (**A**) pure Poly(d,l-lactide-*co*-glycolide) (PLGA) and amphotericin B/PLGA beads (**B**) pure Poly(d,l-lactide-*co*-glycolide) (PLGA) and fluconazole/PLGA beads.

#### *3.2. In Vitro Release Dynamics of Poly(*d*,*l*-lactide-co-glycolide) PLGA*/*Amphotericin B and PLGA*/*Fluconazole Beads*

Figure 7 illustrates the release dynamics of amphotericin B and fluconazole from antifungal-drug-loaded beads with different polymer:drug ratios (6:1 and 4:1). Drug release was slightly less than 1% of the total release of amphotericin B. Triphasic liberation curves were generated, displaying blast release on the first day, followed by gradual elution on days 2–23, and accelerated drug release on days 23 and 10 in beads measuring 3, 5, and 8 mm, respectively. Beads with a

higher drug loading ratio (i.e., polymer:drug = 4:1) generally exhibited greater antifungal drug concentrations in the eluent. Figure 8 shows the release dynamics of PLGA/fluconazole beads of different sizes. Larger beads exhibited an earlier accelerated release of fluconazole than smaller beads. All antifungal-drug-embedded beads exhibited sustained release of fluconazole for more than 30 d.

**Figure 7.** In vitro release curves of amphotericin B from the drug-loaded beads.

**Figure 8.** In vitro release curves of fluconazole from drug-loaded beads with various polymer:drug ratios. (**A**). The accumulated release of beads of different sizes with 4:1 polymer:drug ratios. (**B**). The accumulated release of beads of different sizes with 6:1 polymer:drug ratios.

Cytotoxicity analysis was performed using CCK-8 assays to measure cell viability. The eluent from 5 mm beads with a 4:1 polymer:drug ratio was analyzed. Figure 9 shows that cell viability was reduced on day 1, probably owing to burst release of fluconazole, thereby potentially affecting cell proliferation. Thereafter, PLGA/fluconazole beads showed no signs of cytotoxicity.

**Figure 9.** Cell viability of the fluconazole-incorporated beads (\* *p* < 0.05).

#### *3.3. In Vivo Drug Release*

Owing to the poor release dynamics of amphotericin B, only PLGA/fluconazole beads were selected for in vivo analysis of drug release. Figure 10 shows drug concentrations as measured in bone cavity tissue and blood. Fluconazole-embedded beads displayed long-term fluconazole release (beyond the MTC) for more than 49 d in vivo. Furthermore, blood drug concentration was significantly lower than that in bone tissue.

**Figure 10.** In vivo release curves of fluconazole from the drug-loaded beads.

Blood AST levels were within the physiological range (Figure 11) of 33–99 (U/L) [20].

**Figure 11.** Estimation of blood aspartate transaminase (AST) levels.

#### **4. Discussion**

Treatment of fungal osteomyelitis is more complicated than that of chronic osteomyelitis. Local antibiotic release is important in treating chronic osteomyelitis; however, the use of antifungal-loaded bone cement beads to treat fungal osteomyelitis remains controversial, mainly owing to the inconsistent release dynamics of antifungal drugs from the PMMA beads. While some studies have reported the successful eradication of fungal osteomyelitis or periprosthetic joint infections [21], others have yielded conflicting results. During production of PMMA beads containing amphotericin B or fluconazole, covalent crosslinkage may result in poor drug release from the bone cement [13,14]. The potential risk of irritation of host tissues and the non-biodegradable nature of PMMA potentially limit its applications in treating osteomyelitis, and therefore, surgical excision is often performed as an alternative. This study reports the successful production of antifungal-agent-loaded PLGA beads with amphotericin B or

fluconazole using the compression-molding method. In vitro analysis revealed that the elution rate of amphotericin-B-loaded PLGA beads with different ratios did not exceed 1% for 28 d (Figure 7). Moreover, amphotericin-B-loaded PLGA beads displayed poor sustained drug release. However, the fluconazole-loaded PLGA beads with different ratios displayed an elution rate above 90% for 28 d. Based on these results, we selected PLGA/fluconazole beads for in vivo analysis.

Few studies have evaluated the efficacy of antifungal-impregnated PLGA carriers [22–24], although the release profile of amphotericin B with an organic solvent was shown to be promising. In the present study, PLGA/amphotericin B beads showed poor release dynamics [25]. The temperature during compression molding was increased up to 65 ◦C, which is also the temperature used for polymerization of amphotericin-impregnated PMMA beads, before sintering for 1.5 h. The polymerization process may have facilitated covalent linkages in amphotericin-impregnated PMMA beads; however, the actual mechanism resulting in the poor release profile of amphotericin-impregnated PLGA beads remains unclear. Nonetheless, poor release was not observed with fluconazole-impregnated PLGA beads.

Biodegradable PLGA/fluconazole beads released antifungal drugs at high concentrations for over 49 d, thus controlling the bone infection. The present work is the first study to develop biodegradable antifungal beads using a compression-molding technique without the use of organic solvents and to evaluate the sustained release of high and local fluconazole concentrations in vivo. Owing to the absence of organic solvents during bead preparation, these fluconazole/PLGA beads are potentially applicable for clinical use for the treatment of fungal infections in bone tissue. The present results show that most drugs were absorbed by the surrounding tissues, while the systemic drug concentration remained low (Figure 10).

Fluconazole is a first-generation triazole antifungal agent used to treat fungal infections through both oral and parental routes. Fluconazole is metabolized in the kidneys, unlike other azoles metabolized in the liver. Adverse effects have been commonly reported with use of long-term fluconazole therapy [26], and although none of these have been severe or life-threatening [19], liver damage has been observed in some cases [27,28]. In a previous study, elevation of liver transaminases was more common than liver damage, and the incidence of treatment termination owing to elevated liver enzymes was 0.7% [28]. In the present study, hepatoxicity potentially resulting from fluconazole in the blood during the treatment period was assessed upon local administration at high doses. Blood samples were collected from the marginal ear vein to quantify blood AST levels after each biopsy. Louie et al. reported an optimal fluconazole therapeutic concentration of not more than 10 mg/kg [29]. In the present study, fluconazole was used at 10 mg/kg (a cylindrical bead containing 35 mg fluconazole/3.5 kg body weight), and blood AST levels were within the physiological range in all the New Zealand white rabbits used in the study. These results indicate that fluconazole-impregnated PLGA beads are safe (Figure 11).

PLGA is one of the most suitable biodegradable polymeric materials for synthesizing drug delivery devices and for tissue engineering [30–32]. The material is biocompatible and biodegradable, exhibits a wide range of erosion times, and is mechanically tunable. Most importantly, PLGA is an FDA-approved polymer used extensively for controlled release of small-molecule drugs, proteins, and other macromolecules. Therefore, we selected PLGA as the carrier material for antifungal fluconazole-containing beads.

Drug release from a biodegradable carrier generally occurs in three stages: primary blast, diffusion-dominated elution, and degradation-dominated release [15]. After compression molding, most drugs are dispersed into the volume of the PLGA/fluconazole beads; however, certain drug formulations on the particle surface may lead to an initial drug release burst, followed by controlled drug release by diffusion and other factors. Relatively constant slow elution of the antifungal agents was thus observed. Finally, PLGA/fluconazole beads swell owing to water uptake during elution, thus damaging the polymer matrix and forming openings for antifungal release. The rate of fluconazole release thus accelerated accordingly.

Sustained local release of high levels of antifungals contributes to infection control. Louie et al. reported that the minimum inhibitory concentration median (MIC50) in a designated fluconazole mid-resistant infection was 64–128 μg/mL. In a fluconazole-susceptible strain of *Candida*, the median MIC was 0.5 μg/mL [29], and the concentration of released fluconazole was greater than 128 μg/mL. These results demonstrate that biodegradable antifungal-embedded beads can release high concentrations of fluconazole (well beyond the MIC50) for more than 49 d (Figure 10). Furthermore, FTIR analysis suggests that bead-embedded drug formulations remain stable during the molding process.

Although the current study has generated promising preliminary data, some limitations should be noted. First, we used a non-infected animal model, and therefore it is unclear whether the antifungal beads might perform differently in infected tissue. Further evaluation of the antifungal agent-embedded PLGA copolymer beads in an animal model of fungal infection is necessary to address this limitation. Second, despite the experimental data showing that 80% of the drug was released at the end of the study, it would have been faster given larger amounts of liquid. The sink condition analysis is needed to ensure the drug was released freely. Third, although no obvious sign of inflammation was observed in the in vivo test, the influence of controlled release of drugs and carriers on the local irritation should be further examined. Finally, the relevance of the present findings to patients with bone infections remains unclear and warrants further investigation. We intend to further explore these topics in future studies.

#### **5. Conclusions**

This study is the first to perform in vitro and in vivo analyses of drug release dynamics in the bone cavity from compression-molded antifungal-incorporated PLGA beads prepared without organic solvents. Biodegradable PLGA/fluconazole beads released high concentrations of antifungals for over 49 d at the target site, while the antifungal agent blood concentration remained low. In summary, the compression-molded PLGA/fluconazole beads that we describe here have potential applications for treating bone infections.

**Author Contributions:** Y.-H.H., S.W.-N.U., and S.-J.L.; methodology, Y.-H.H., H.-Y.C., J.-C.C., and Y.-H.Y.; validation, Y.-H.H., S.W.-N.U., and S.-J.L.; formal analysis, Y.-H.H., H.-Y.C., J.-C.C.; investigation, Y.-C.C.; resources, Y.-H.H. and S.-J.L.; data curation, Y.-H.H., H.-Y.C., J.-C.C., and S.-J.L.; writing—original draft preparation, Y.-H.H.; writing—review and editing, Y.-H.H. and S.-J.L.; visualization, Y.-H.Y. and Y.-C.C.; supervision, S.W.-N.U. and S.-J.L.; project administration, Y.-H.H., H.-Y.C., and J.-C.C.; funding acquisition, S.W.-N.U. and S.-J.L.

**Funding:** This work was supported by the Ministry of Science and Technology, Taiwan (Contract No. 107-2221-E-182-017) and the Chang Gung Memorial Hospital (Contract No. CMRPD2H0032).

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

#### **References**


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