Palm oil (Softisan 154 or S154), a gift from Condea (Witten, Germany), and stearic acid (Merck, Germany) were the lipids used in this study. Surfactants include the following: Poloxamer407 (Sigma Aldrich, St. Louis, MI, United States) and polioxiethylene-20-sorbitan monooleate (tween 80- Sigma Aldrich, St. Louis, MI, United States), lipo-soluble surfactants including Span 80 (Sigma Aldrich, St. Louis, MI, United States) and lecithin (Sigma Aldrich, St. Louis, MI, United States), doxycycline (Sigma Aldrich, St. Louis, MI, United States), and hydroxychloroquine sulfate (Sigma Aldrich, St. Louis, MI, United States). Sodium borohydride (NaBH4) (Merck, Germany), tellurium (Sigma Aldrich), cadmium salt (Scharlau, Spain), thioglycolic acid (TGA) (Merck, Germany), and distilled water were used throughout the study.

500 mg of NaBH₄ powder is added to a 50-ml balloon on a magnetic stirrer with 7 ml of distilled water. Then during the flow of argon gas, 90 mg of tellurium powder was added to the solution. Initially, because the mixture inside the balloon reacts strongly with oxygen, the color of the solution turns purple, but with the flow of argon gas, the purple color disappears and the solution becomes colorless. 2 h later, the white sodium tetraborate powder precipitates. Next, cadmium salt solution in a 250-ml flask to which thioglycolic acid (TGA) was added was prepared. NaHTe solution is added drop-wise to the cadmium salt. The nanoparticles begin to form and grow in the balloon when the Te²⁻ and Cd²⁺ ions combine. To grow the clusters, the balloon is placed in a water bath at 90°C (Nguyen et al., 2019).

The nanoparticle was synthesized by using the double emulsion/melt dispersion technique. Different percentages of lipids, surfactants, and drugs were tested to achieve the optimal formulation. First emulsions included palm oil (1,200 Mg) or stearic acid, poloxamer407 (120 Mg), soy lecithin, distilled water, and antibiotics. Briefly, palm oil or stearic acid were heated at 70°C (5°C above the melting point) and 75°C (5°C above the melting point) using a ben murray, respectively. At this time, poloxamer407, soy lecithin, doxycycline (50 Mg) and hydroxychloroquine (75 Mg) antibiotics were added to the melted oil. It was mixed for 5 min under magnetic stirring (60°C and 150 rpm). Then, 1 ml heated distilled water was added to the mixture and homogenized. To obtain the first emulsion, the mixture was sonicated by using a sonicator (skymen, China) at 45% amplitude (20 W) for 60 s (W1/O). In the following step, 20 ml heated tween-80 was added to the first emulsion and the mixture was homogenized using an ultrasonic device (Bandelin Sonopuls, Berlin, Germany) at 45% amplitude (20 w) at a regular pulse rhythm (10 s on and 5 s off) for 60 s to the second emulsion (W1/O/W2) be provided. The second emulsion was added drop-wise to cold distilled water (4°C) containing 100 µl cadmium-telluride quantum dot (5 nm) under the magnetic stirring condition (5 min) to stabilize the synthesized nanoparticles. The exact same conditions were done for preparation of free SLN (solid lipid nanoparticles without drug). Materials and conditions for each formulation are presented in Table 1. Finally, the synthesized nanoparticles was separated using a high-speed centrifuge (35,000 rpm for 15 min) and washed three times using distilled water.

500 mg of each formulation was added to 1 ml glycerin and lyophilized using a vacuum pump (Christ, China) with a condenser flow at −80°C. To use lyophilized nanodrugs in biological studies on bacteria, the nanoparticles were first dissolved in distilled water and then sterilized by using a 450-nm filter (Black Peres et al., 2016; Hosseini et al., 2019a).

The mean particle size, polydispersity index (PDI), and zeta potential of solid lipid nanoparticles loaded with doxycycline and hydroxychloroquine (DOX-HCQ-SLN) were measured at 25°C by using the dynamic light scattering (DLS) technique performed using a zetasizer nano ZS 3600 (Malvern Instruments, Worcestershire, United Kingdom) device. Each reported value was an average of three measurements. The mentioned factors were measured in two stages, first, immediately after preparation, and in the second stage, after lyophilization.

It is necessary to obtain the maximum adsorption wavelength of purchased drugs, in order to evaluate the efficiency of encapsulation and loading of drugs. For this purpose, 5 mg of each drug was weighed and dissolved in 5 ml of their solvent (distilled water for doxycycline and ethanol for hydroxychloroquine). The volume of 1 ml of this solution was increased to 10 ml with double distilled water (stock prepared: 100 μg/ml), and then serial dilutions of the stock solution were prepared using distilled water and antibiotic solvent. The absorbance (OD) of the samples was measured using a UV–vis spectrophotometer (UV-2100, Spectrum Technologies, Fort Worth, TX, United States) at wavelengths of 200–400 nm at 5 nm intervals and the maximum wavelength was recorded. Dilutions of 0–45 μg/ml (at 5 μg/ml intervals) were prepared in double distilled water using the prepared stock (10 mg of drug in 2 ml of solvent). Then the absorbance of each sample at maximum wavelength was read using a UV–vis spectrophotometer (Iorhemen et al., 2017). Then, the standard curve of each drug was drawn based on the obtained data.

To evaluate how much drug is loaded and encapsulated in the synthetic nanoparticle, direct and indirect methods were used. In the indirect method, 10 mg of the synthesized nanoparticle was dissolved in 10 ml of distilled water and completely vortexed to become homogeneous. 2 ml of the prepared suspension was centrifuged at 15,000 rpm for 30 min at 4°C, and this was done three times to make the supernatant completely clear. After centrifugation, the supernatant absorbance (OD) was read using a UV–vis spectrophotometer at wavelengths of 270 and 343 nm for doxycycline and hydroxychloroquine, respectively. The absorbance (OD) obtained was entered into the standard curve (previously plotted for drugs), and the amount of encapsulation and loading was calculated using the following formula (Gupta et al., 2016).

In the direct method, the amount of doxycycline and hydroxychloroquine contained in NPs was investigated by HPLC equipped (SY-8100, China) with a UV detector at a wavelength of 270 and 343 nm. The mobile phase was a mixture of water (50%) and ethanol (50%), the pump was of SY-8100 type, the injector loop was at 772 PSI, and the column was of C-18 type (4.6 × 250 mm), run at a flow rate of 1 ml/min. In this step, 10 mg of the sample alongside 10 ml of water and ethanol mixture were mixed and vortexed using sonication for 10 min to become homogenous. Then, it was centrifuged at 1,500 rpm for 20 min and the supernatant was purified using 220-nm filters (Chen et al., 2019). A linear calibration curve with a good correlation coefficient (r ² = 0.9990) for concentrations in a range from 1 μg/ml to 250 μg/ml was achieved. E n t r a p m e n t   E f f i c i e n c y   E E ( % ) = i n i t i a l   d r u g   a m o u n t   −   f r e e   d r u g   a m o u n t   i n i t i a l   d r u g   a m o u n t × 100 , D r u g   L o a d i n g   D L ( % ) = i n i t i a l   d r u g   a m o u n t   −   f r e e   d r u g   a m o u n t i n i t i a l   l i p i d   a m o u n t × 100 .

The thermal behavior of the optimal formulation and its components were evaluated using the device (METTLER TOLEDO—DSC 1). 5–10 mg of lyophilized powder of optimal formulation and each of its components including doxycycline, hydroxychloroquine, palm oil, and physical mixture of drug and lipid was examined in the temperature range of 20–400°C (5 k/min) and under nitrogen gas (80 ml/min), separately (Müsellim et al., 2018).

In order to investigate the chemical structure of the samples, the synthesized nanoparticle lyophilized powder (optimal formulation), and the nanoparticle components were mixed separately with potassium bromide (KBr) simultaneously and converted into compact discs using a hydraulic compressor. Spectroscopy was performed by FTIR spectroscopy (Perkin Elmer, Waltham, MA, Spectrum 400) in middle-range IR (4,000–400 cm⁻¹) (Müsellim et al., 2018).

Release was performed using a dialysis bag (with fine pores of 2.5 nm and molecular weight of 12,000–14,000 Daltons). The membrane was soaked twice in distilled water 12 h before use. Phosphate buffer with pH: 7.4 at 37°C was used as the release medium. Ten milligram of lyophilized powder of the optimal formulation was dispersed in 2 ml of double distilled water and placed in the bag, and both ends of the bag were sealed. Similarly, 10 mg of both free drugs were placed in 2 ml of double distilled water in a separate bag. The bags were placed separately in the release medium (60 ml), and the lids of the containers containing the bag and buffer were closed to prevent water vapor from escaping. Each vessel was then placed in a shaker incubator (37°C, 100 rpm). At constant intervals (0, 1, 2, 4, 20, 40, 60, 80, 100, 120, and 140 h), 2 ml was removed from the medium and immediately replaced with the same volume of release medium to maintain the sink condition. Drug released from the synthesized nanodrug was measured using a UV–vis spectrometer at the maximum wavelength of each drug (Yu et al., 2019).

The stability of the optimal formulation in suspension was evaluated for the short term (1 week in terms of appearance and 1 month in terms of appearance and physicochemical properties) and long term (6–12 months in terms of appearance and physicochemical properties). In the short term, at intervals of 24, 48, 72, 96, 120, and 144 h, the samples were inspected visually. At this stage, formulation without sediment formation, drug leakage, and accumulation were considered as stable in the short term. To evaluate the stability of the optimal formulation over a long period of time, physical appearance and physicochemical parameters (particle size, PDI, and zeta potential) with respect to zero time over a long period (2–12 months) were evaluated at refrigerator temperature (5°C) (Kalpana and Devi Rajeswari, 2018).

A field emission scanning electronic microscope (Fe-SEM) (MIRA3, TESCAN Company, Brno, and Czech Republic) was used to examine the morphology of the nanoparticles. 2 mg of lyophilized nanoparticles were added to 2 ml of distilled water and sonicated. Then 2 μl of the suspension was transferred to a microscope slide and allowed to dry, covered with a thin layer of gold, and evaluated (Brodusch et al., 2018).

For in vitro and in vivo tests, Brucella melitensis 16M (purchased from Razi Vaccine and Serum Research Institute, Iran) was used. Brucella agar, TSB (Tryptic Soy Broth), and TSA (Tryptic Soy Agar) were used for bacterial culture at 37°C and 5% of CO₂. The J774-A1 mouse macrophage cell line (purchased from the Pasteur Institute of Iran, Biology Bank) was employed for the cell culture study. Dulbecco’s Modified Eagle cell culture Medium (DMEM) was used with 10% FBS. Male Wistar rats (6–8 weeks old) weighing 250 (±30) gr were purchased from Hamadan University of Medical Sciences.

The well diffusion method and minimum inhibitory concentration of nanoparticles and free drug were performed according to CLSI guidelines. Concentrations of 12.5, 25, and 50 μg/ml were prepared from drugs and nanodrugs. 0.5 McFarland suspension of Brucella melitensis were cultured on Mueller-Hinton agar plates using sterile swabs. In the following step, Wells 8 mm in size were created in culture media by using a sterile Pasteur pipette and each well was filled in by 100 μl of nanodrugs and free drug and incubated at 37°C for 24, 48, and 72 h. After these time intervals, bacterial growth around the wells was measured for each well and used as a basis for analyzing the antibacterial performance of nanodrugs (Hosseini et al., 2017).

96-well flat-bottomed plates were used to determine the minimum concentration of drug and nanodrug that had the ability to inhibit bacterial growth. The first concentration which was used was the lowest amount of antibiotics and nanodrugs obtained in the well diffusion test. 100 μl was added to each well from different dilutions. Then 100 μl of mueller hinton broth culture medium was added to each well, and finally, 5 μl of 0.5 McFarland bacterial suspensions was added to all wells and incubated for 24, 48, and 72 h at 37°C. After this time, to determine the minimum concentration, it was first examined visually, and in wells where bacteria had not grown, it was considered as the minimum inhibitory concentration (Espinel-Ingroff et al., 2017).

The MTT kit (3- (4, 5-Dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) was used to evaluate the cytotoxicity of the synthesized nanodrug according to the instructions of the kit and Mouse monocyte-macrophage cells J774A.1 (ATCC TIB-67; BALB/c Mouse, hematopoietic, macrophage-like. 1 × 10⁴ cells were added to each well containing DMEM (Gibco Product, Carlsbad, CA) enriched by 10% of FBS and 1% of penicillin–streptomycin antibiotics and incubated for 24 h at 37°C and 5% of CO₂. After this period, the medium was removed from the wells and 100 μL of Free doxycycline, Free hydroxychloroquine, DOX-SLN, HCQ-SLN, and Free SLN (blank sample) at various concentrations (25, 50, 100, 200, 400, and 800 μg/ml) alongside DMEM containing 10% FBS were added and incubated for 24 h.

The treated cells were washed with PBS, and 150 μl of fresh DMEM medium without fetal bovine serum was added to the wells again. 10 μl of MTT assay reagent was added and the culture medium was removed after 3–4 h of incubation, and then 200 μl of DMSO was added to each well and placed on a shaker at 100 RPM for 20 min to dissolve the formazan particles. Optical absorption of cell-containing plates was evaluated at 570 nm. The number of viable cells in each well was evaluated by positive control comparison (Sánchez-López et al., 2016; Kumar et al., 2018; Fernandes et al., 2021).

24-cell culture plates (Corning Inc., Corning, NY, United States) were used to investigate the effect of nanoparticles on Brucella melitensis. 10⁵ cells were added to each well containing DMEM medium with 10% of FBS and incubated for 24 h. Then, cells were infected with Brucella melitensis in log phase at a 1:100 ratio of cell to bacteria and incubated for 1 h to bacterial phagocytosis. The cells were washed three times with DMEM medium containing 50 μg/ml gentamicin antibiotic (Sigma Product Co.) to remove bacteria that were non-phagocyted or attached to the cell wall. Different dilutions of nanoparticles and drug (25, 50, and 100 μl) along with DMEM medium with 10% FBS were added to the cells and incubated for 24, 48, and 72 h at 37°C and 5% of CO₂. After these times, the cells were washed using PBS twice. The cells were lysed with 250 μl of 0.1% Triton X-100 in order to determine the number of bacteria in the macrophages. 10 serial dilution was prepared from this suspension, and cultured on Brucella agar medium. The colony-forming unit (CFU) of bacteria was numbered and recorded after 24, 48, and 72 h of culture (Hosseini et al., 2019b; Hosseini et al., 2020).

Rats were infected with 1 ml of 1.5 × 10⁶ CFUs of Brucella melitensis M16 by intraperitoneal injection. Animal studies were performed in two phases of acute and chronic brucellosis. Rats were divided into eight groups after 10 days of bacterial injection (acute phase) and 5 weeks after injection (chronic phase). All experiments were done according to the guidelines for maintenance, surveillance, and usage of laboratory animals published by the National Institute of Health United State (NIH publication No. 85-23, revised 1985). Moreover, the study was approved by the ethics committee of the Hamadan University of Medical Sciences (No: IRUMSHA. REC.1399.736). Three and five rats were considered in each group, for acute and chronic phases of the disease, respectively. The dose of injection was based on rat weight. Study groups included the following: 1) control group (infected without treatment), 2) DOX-SLN-treated group (2.5 mg/kg), 3) HCQ-SLN-treated group (6.5 mg/kg), 4) DOX-HCQ-SLN–treated group (4.5 mg/kg), 5) free hydroxychloroquine–treated group (6.5 mg/kg), 6) free doxycycline–treated group (2.5 mg/kg), 7) free doxycycline and free hydroxychloroquine–treated group (4.5 mg/kg), and 8) drug-free nanoparticle-treated group. In the acute phase of brucellosis, three doses of drug and nanoparticle were administered every other day (days 11, 13, and 15 after bacterial injections). In the chronic phase of brucellosis, ten doses of drugs and nanoparticle were administered intraperitoneally 5 weeks after bacterial injection (once daily).

One day after the last injection of the drug and nanodrugs, the rats were euthanized. The liver and spleen were extracted and weighted in observance of sterile conditions. Tissues were homogenized using sterile physiological serum. Different dilutions were prepared from the suspension and cultured on Brucella agar medium and incubated for 4 days at 37°C with 5% of CO₂. The number of bacterial colonies that had grown was then counted and recorded (Hosseini et al., 2019b; Ghaderkhani et al., 2019).

Due to the fact that our data are non-parametric, Kruskal–Wallis test was used to compare the number of bacterial colonies grown between groups in the acute and chronic stages. All statistical tests were performed at the 0.05 level of confidence.

Depending on the lipid and surfactant used, the duration of homogenization, and strength of the ultrasound probe, the properties of nanoparticles differ (Table 1). The mean size and optimum PDI for the nanoparticles used for the rest of the study were 214.3 ± 25 and 0.385 ± 0.02, respectively. The zeta potential of the optimum nanoparticle was −18.7 mV (Figures 1, 2).

The encapsulation rate of doxycycline and hydroxychloroquine in the synthesized nanoparticles was 95.8 and 92.5%, respectively. The mean drug load in different formulations was 12.79%. In optimum formulation, the amount loaded was 17.7% (Table 2).

The results of electron microscopy showed that the synthesized nanoparticles were morphologically spherical and had a smooth surface with homogeneous polydispersity (Figure 3).

DSC analysis was used to investigate the melting behavior of DOX-HCQ-SLN. Thermograms of DOX-HCQ-SLN, palm oil, free doxycycline, free hydroxychloroquine, and [free doxycycline and free hydroxychloroquine (physical mixture)], shown in Figure 4. As shown in Figure 4, the DSC thermogram shows the melting process of palm oil at 63°C. The melting points of the physical mixture and DOX-HCQ-SLN contained cadmium-telluride quantum dots are similar to the melting point of the palm oil. In the DSC thermograms of doxycycline and hydroxychloroquine, a sharp endothermic peak was seen at 230 and 250°C. While these points in other compounds show a small melting point for physical mixture and DOX-HCQ-SLN. Note that no any significant change was observed in the endothermic peak positions of doxycycline, hydroxychloroquine, physical mixture, and DOX-HCQ-SLN. The absence of a sharp melting peak of DOX-HCQ-SLN thermogram suggests that no free drug crystal remained in the synthesized nanoparticles.

The FTIR spectra of doxycycline, hydroxychloroquine, palm oil, and DOX-HCQ-SLN are shown in Figure 5. According to Figure 5, it can be seen that DOX-HCQ-SLN absorption peaks are consistent with the basic ingredients of nanoparticles (palm oil, antibiotics, and surfactant), and new absorption peaks were not seen indicating a new functional group or bond. However, a small change in the oil melting process was observed in the physical mixture.

Examination of stability of nanoparticles at different times showed that the synthesized optimum formulation was still stable after 6 months. After 9 months, the synthesized nanoparticles did not change in terms of color and turbidity. However, an increase in PDI was observed. The size also changed from 214.3 to 258.3 (20.53%) Table 3.

The release test was performed for Free DOX, Free HCQ, and DOX-HCQ-SLN over a period of 120 h (pH: 7.4, PBS buffer). In the first 20 h, there was a very rapid release in free DOX and free HCQ (about 60% of the total drug). During this time, only 10% of the drug was released from DOX-HCQ-SLN. Overall, it took 100 h for 82% of the drug to be released from DOX-HCQ-SLN (Figure 6).

The results of toxicity study showed that up to a concentration of 100 μg/ml of various formulations had no toxic effect on cells. In this study, there was no significant difference between synthesized nanoparticles with free drug in terms of effect on cells (p > 0.5). Free SLN, which included palm oil, poloxamer407, and Tween 80, had no toxic effect on cells up to 400 mg/ml (Figure 7). The dose used in antibacterial studies in in vitro and in vivo conditions was much lower than the concentrations used in the toxicity study.

Different concentrations of free drugs and nanodrugs were used in this experiment. Well diffusion and MIC findings showed that there was no significant difference between nanodrugs and free drug in terms of inhibiting bacterial growth (p > 0.5). Free DOX and Free HCQ performed better than DOX-HCQ-SLN in the first 24 h. Bacterial inhibition in 72 h after treatment was approximately the same for DOX-HCQ-SLN and Free drugs. The minimum inhibitory concentrations of DOX-HCQ-SLN and free drugs were close after 72 h and there was no statistically significant difference (p > 0.05, Figure 8).

The results of cell treatment with different concentrations of free drugs and nanodrugs showed that DOX-HCQ-SLN had the greatest effect on bacteria enclosed macrophages. The use of 100 μg/ml reduced the number of bacterial colonies to 3.5 log compared to the control group, which reduced it by 6.5 log. Comparison of treatment groups showed that there was a significant difference between DOX-HCQ-SLN and DOX-SLN in reducing the number of bacteria with Free DOX-HCQ and Free DOX (p < 0.5). Importantly, Free HCQ and HCQ-SLN alone had little effect on reducing the number of bacteria (Table 4). The entry of the bacteria and labeled nanoparticles into the cell is shown in Figure 9.