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Doxycycline-encapsulated solid lipid nanoparticles as promising tool against Brucella melitensis enclosed in macrophage: a pharmacodynamics study on J774A.1 cell line

Doxycycline-encapsulated solid lipid nanoparticles as promising tool against Brucella melitensis... Background: Brucellosis is a zoonotic disease caused by Brucella species. It has been estimated that more than 500,000 new cases of Brucellosis occur annually all around the world. Relapse of the disease is one of the most important challenges. The most important reason for the relapse of brucellosis is the survival of the bacteria inside the macrophages, which makes them safe from the immune system and disrupts drug delivery mechanism. Objectives: The present study was performed to assess the effects of Doxycycline-loaded Solid Lipid Nanoparticles (DOX-SLN) on the Brucella melitensis inside macrophages. Methods: DOX-SLN was prepared using double emulsion method. The technological characterization of DOX-SLN, including particle size, zeta potential, polydispersity index (PDI), drug loading and encapsulation efficiency were used. Fourier-transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC) were used to assess the interactions between Nanoparticles (NPs) components and crystalline form of doxycycline. Moreover, the effect of DOX-SLN on the bacteria were compared with that of the doxycycline using various methods, including well diffusion, Minimum Inhibitory Concentration (MIC), and investigation of their effects on murine macrophage-like cells cell line J774A.1. Results: The means of particle size, zeta potential, PDI, drug loading and encapsulation efficiency were 299 ± 34 nm, − 28.7 ± 3.2 mV, 0.29 ± 0.027, 11.2 ± 1.3%, and 94.9 ± 3.2%, respectively. The morphology of NPs were spherical with a smooth surface. No chemical reaction was occurred between the components. Doxycycline was located within NP matrix in its molecular form. The DOX-SLN significantly decreased the microbial loading within macrophages (3.5 Log) in comparison with the free doxycycline. Conclusions: Since the DOX-SLN showed better effects on B. melitensis enclosed in macrophages than the free doxycycline, it is recommended to use it for treating brucellosis and preventing relapse. Keywords: Brucella melitensis, Doxycycline, Solid lipid nanoparticle, Relapse * Correspondence: Mohammad.arabestani@gmail.com Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran Brucellosis Research Center, Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 2 of 12 Background stability, increase bioavailability of drug, effective pass from Brucellosis is a zoonotic disease caused by different biological barriers, reduce dose and frequency of drug ad- Brucella strains following direct or indirect contact of ministration, fast identification by phagocytic system, con- human with infected animals or dairy products [1]. The trolled release of drug, specific targeting, slow release of most common cause of the disease is Brucella melitensis, drug in a long-term period, protect drugs against pharma- which is endemic in Middle East countries, including Iran. cologic and toxicological effects, prevent early breakdown Brucella is an intracellular bacterium [2]. The phagocyt- of drug molecules, prevent immune response, increase drug osis activity of polymorphonuclear and monomorphonuc- retention in tissue, minimize resistance to drug, improve lear cells such as macrophages plays an important role in therapeutic index of drug, minimize drug toxicity, and im- the disease treatment [3]. Upon entering the body, about prove treatment efficiency. 90% of the bacteria were killed by neutrophils, monocytes, Despite its partial limitations, the followings can be and macrophages. However, some of them survive inside mentioned as the most significant features of SLNs: pro- macrophages and find a place to proliferation within the tecting drugs from chemical and enzymatic decomposi- cell [1, 4]. tions as well as pharmacologic and toxicological effects, The World Health Organization (WHO) suggests the physical and biotical stability, hydrophilic and hydropho- combination of gentamicin and doxycycline for brucellosis bic drug loading capacity, easy production, no need for treatment [5]. The combination of doxycycline and strepto- organic solvents in synthesis, ability to carry two active mycin has also been suggested to prevent the disease agents simultaneously, increase drug efficiency, easy relapse. The combination of doxycycline and rifampin for sterilization, small diameter, administration via different 45 days is the best known treatment for Brucellosis because routes, biodegradability trait, increase bioavailability of of having no side effects and its acceptability by patients. drug, effective pass from biological barriers, reduce dose However, this treatment has been less effective with about and frequency of drug administration, fast identification 15% failure in different types of the disease [6]. by phagocytic system, controlled release of drug, specific Despite the significant number of new antibiotics, treat- targeting, slow release of drug in a long-term, prevent early ment of intracellular pathogens is still a major challenge breakdown of drug molecules, prevent immune response, [7]. To date, no antibiotic therapy has been reported to increase drug retention in tissue, minimize resistance to eradicate Brucella intracellular infections. The main chal- drug, improve therapeutic index of drug, minimize drug lenge in intracellular chemotherapy is the design and toxicity and improve treatment efficiency [11, 12]. development of a carrier system for antibiotics that can be Given the above, the present study was carried out to effectively endocytosed by phagocytic cells and release develop a new therapeutic mechanism for the treatment drugs. Targeted drug delivery system can be considered as of brucellosis in order to enhance efficacy of antibiotics. an appropriate tool to overcome such problems [8]. The therapeutic efficacy of doxycycline-loaded SLNs in One of the most promising strategies to prevent the chronic infections of B. melitensis was assessed in cellular disease recurrence is using the antibiotic-loaded nanocar- models and experimental conditions. riers. Nowadays, with the production of pharmaceutical NPs, unique characteristics can be achieved, which will Methods result in increased performance of drugs and diversity of Materials their forms. Hydrogenated palm oil, (Softisan 154 or S154) a gift from These carrier systems have to be non-toxic and have Condea (Witten, Germany), and stearic acid (SA, Sigma sufficient capacity to take the drug. In addition, they Aldrich, USA) were the lipids used in this study. Water should have the possibility of controlling drug release. soluble surfactants used were polioxiethylene-20-sorbitan Solid lipid nanoparticles (SLNs) as a carrier system have monooleate (Tween 80- Sigma Aldrich, USA), Poloxa- been investigated for many applications. These drug mer407 (Pluronic F127, Sigma Aldrich, USA). Furthermore, delivery systems provide controlled release of medicines lipo-soluble surfactants used were Sorbitanmonooleate that they carry and increase the chemical stability of (Span 80, Sigma Aldrich, USA), and soy lecithin them [9, 10]. (Sigma-Aldrich). Doxycycline hyclate (Sigma-Aldrich). In spite of negligible limitations, the most important Water used in our study was distilled twice and purified properties of SLNs are protecting drugs from chemical by an appropriate filter. and enzymatic decompositions, physical stability, hydro- philic and hydrophobic drug loading capacity, easy pro- Preparation of NPs duction, no need for organic solvents in synthesis, ability Solid-lipid NPs (SLNs) were synthesized using the above to simultaneous carry two active agents, increase drug mentioned chemicals and double emulsion/melt disper- efficiency, ease of sterilization, small diameter, adminis- sion technique based on the study conducted by Luana tration via different routes, biodegradability and biotic Becker Peres [13]. The first emulsion contained palm oil, Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 3 of 12 Poloxumer, distilled water, and doxycycline antibiotic. In literature. In this step, 10 mg of prepared NPs was added brief, firstly, 0.6 g of palm oil was warmed to 60 °C (the to 10 ml distilled water and vortexed to become boiling point) in Ben Murray. Then 60 mg Poloxamer homogenous. Then, the solution was centrifuged at 4 °C and 30 mg doxycycline were added to the melted palm for 20 min at 1500 rpm. The supernatant was investigated oil and mixed on the magnetic stirring (60 °C and 150 using spectrophotometer (2100 UV, USA) at a wavelength rpm) for 5 min. In the following step, 0.5 ml hot distilled of 270 nm. The drug concentration was determined using water was added to the mixture. Mixture was sonicated the standard curve which have been previously depicted using sonicator (skymen, China) during 60 s at 45% based on various concentrations of the drug [14, 15]. The amplitude (20 W) to obtain the first emulsion. (W1/O). amount of loaded and encapsulated drug was obtained In the next step, 30 mg tween-80 was added to the pre- using the following equations: pared first emulsion and made homogenous using ultra- sonic device (Bandelin Sonopuls, Berlin, Germany) at initial drug amount−free drug amount Entrapment Efficiency EEðÞ % ¼  100 45% amplitude (20w) at a regular pulse rhythm (10s on initial drug amount and 5 s off) for 60s to the second emulsion (W1/O/W2) initial drug amount−free drug amount be provided. The prepared second emulsion was gently Drug Loading DLðÞ % ¼  100 initial lipid amount added to 30 ml of cold distilled water (4 °C) under the condition of magnetic starring (5 min) to stabilize SLN. The amount of doxycycline contained in NPs was in- Materials required for each formulation are presented vestigated using HPLC equipped (SY-8100, China) with in Table 1. Finally, doxycycline-loaded solid lipid nano- UV detector at a wavelength of 270 nm. The mobile particles (DOX-SLN) was separated using the high speed phase was a mixture of ethanol (50%) and water (50%), centrifuge (37,000 g for 15 min) and washed using steril- the pump was of SY-8100 type, injector loop was at 772 ized distilled water three times. Almost 500 mg of each PSI, and the column was of C-18 type (4.6 X 250 mm) is formulation was added to 1 ml glycerin. The prepared run at a flow rate of 1 mL/min. In this step, 10 mg of the samples were lyophilized at − 80 °C using a vacuum sample alongside 10 ml of water and ethanol mixture pump (Christ, china) with a condenser flow. For being were mixed and vortexed using sonication for 10 min to usable in biologic studies on bacteria and cell line, ly- become homogenous. Then, it was centrifuged at 1500 ophilized NPs transformed in solution and sterilized rpm for 20 min and the supernatant was purified using using 450 nm filters [14]. 220 nm filters. A linear calibration curve with a good correlation coefficient (r = 0.9990) for concentrations in Characteristics of NPs (particle size, polydispersity index, a range from 1 μg/ml to 250 μg/ml was achieved. and zeta potential) Particle size, Polydispersity Index, and Zeta Potential were Physical and chemical stability of NPs determined by Dynamic Light Scattering (DLS) technique The stability of NPs loaded with doxycycline was assessed performed by Zetasizer Nano ZS 3600 (Malvern Instru- in regular time intervals. After passing the NPs cross 450 ments, Worcestershire, United Kingdom) device. The men- nm filters, particle size, zeta potential and PDI were deter- tioned factors were measured twice; firstly, immediately mined using nano Zetasizer device in the time intervals of after preparation, and secondly, after lyophilization [13]. 1 h, 24 h, 48 h, and also 1, 2, 4, 8, and 12 months after the preparation. At the same times, the concentration of Morphology doxycycline in NPs was determined using HPLC [14]. Field emission scanning electronic microscope (FE-SEM) was used for assessing the morphological characteristics In vitro release experiment of NPs. To do so, 10 mg lyophilized DOX-SLN was added The lyophilized samples which were exactly weighted to 1 ml distilled water and then 2 μl of this suspension was and enclosed in a dialysis bag (cut-off 12,000, Dialysis placed on a glass surface. Since the suspension was dried, tubing, Sigma Chem. Co., Missouri, USA) were placed in it was covered by a thin gold layer to avoid electrostatic a 40 ml release medium on the magnetic starring of 100 charging during examination and analyzed using FE-SEM rpm at 37 °C. At predetermined time intervals, 0.5 ml of (TSCAN, Czech Republic) [15]. the medium was sampled and its doxycycline content was investigated using HPLC. For comparing the results Investigation of capsulated and loaded drug of this set of experiments with those of free doxycycline, For determining how much the doxycycline have been the same procedure was conducted in which free doxy- loaded and encapsulated in the synthesized NPs, an indir- cycline were enclosed in the dialysis bags and placed in ect method, i.e. spectrophotometer, and a direct one, i.e. the same medium. Then at the same time intervals sev- high-performance liquid chromatography (HPLC), were eral samples were obtained from the medium and inves- employed in accordance with guidelines recommended in tigated. It should be noted that after each sampling from Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 4 of 12 Table 1 Materials and technological parameters of Various formulation sample Lipid (mg) Lipid surfactant Water surfactant Water added PS (nm) PDI Zeta potential EE (%) DL (%) (ML, Mg) (ML, Mg) (ML) (mV) Before lyophilization SLN1 without doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 251 ± 15 0.277 ± 0.021 −31.6 ± 1.4 95.4 ± 3.5 11.3 ± 1.1 SLN 1 with doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 265 ± 23 0.284 ± 0.012 −29.3 ± 2.1 SLN 2 without doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 456 ± 21 0.429 ± 0.032 −11.6 ± 4.4 93.7 ± 5.3 8.9 ± 2.3 SLN 2 with doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 465 ± 23 0.344 ± 0.012 −14.3 ± 3.5 SLN 3 without doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 358 ± 39 0.536 ± 0.019 −9.6 ± 1.7 94.1 ± 7.1 9.2 ± 3.8 SLN 3 with doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 375 ± 42 0.591 ± 0.010 −11.3 ± 2.6 SLN 4 without doxycycline Stearic acid (1200) Lecithin (60) Tween 80 (3) 50 491 ± 25 0.447 ± 0.033 −18.6 ± 1.4 97.4 ± 4.5 10.7 ± 1.4 SLN 4 with doxycycline Stearic acid (1200) Lecithin (60) Tween 80 (3) 50 415 ± 59 0.494 ± 0.093 −19.3 ± 1.2 After lyophilization SLN 1 without doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 294 ± 27 0.281 ± 0.044 −30.7 ± 2.04 94.9 ± 3.2 11.2 ± 1.3 SLN 1 with doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 299 ± 34 0.290 ± 0.027 −28.7 ± 3.2 SLN 2 without doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 491 ± 33 0.499 ± 0.042 −12.6 ± 3.5 93.9 ± 4.7 8.2 ± 1.9 SLN 2 with doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 471 ± 34 0.412 ± 0.012 −14.3 ± 3.5 SLN 3 without doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 378 ± 28 0.591 ± 0.053 −17.6 ± 2.1 91.3 ± 2.7 8.8 ± 3.1 SLN 3 with doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 399 ± 39 0.612 ± 0.125 −15.3 ± 1.7 SLN 4 without doxycycline stearic acid (1200) Lecithin (60) Tween 80 (3) 50 511 ± 45 0.493 ± 0.325 −17.8 ± 1.7 96.1 ± 3.9 9.9 ± 1.7 SLN 4 with doxycycline stearic acid (1200) Lecithin (60) Tween 80 (3) 50 575 ± 39 0.524 ± 0.987 −16.3 ± 2.2 PS particle size, PDI polydispersity index, EE entrapment efficiency, DL drug loading Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 5 of 12 the medium, the new and fresh medium with the same MIC was determined by visual examination of the turbid- amount was added [14]. ity and culturing the contents of the wells. The well with no bacterial growth was regarded as the minimum inhibi- Fourier-transform infrared spectroscopy and differential tory concentration [20]. scanning calorimetry In the present study, a set of experiments was also con- In vitro cytotoxicity ducted to discover any possible interaction between doxy- MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo- cycline and lipid compounds contained in DOX-SLN. lium bromide) Assay test was performed using the MTT FTIR analysis was performed to investigate such a possi- assay kit (Kiazist, Iran). The contents of the kit were as bility in a temperature range from 400 °C to 4000 °C [16]. follows; MTT reagent, solubilizer, 2 Chanel reservoir, and DSC analysis was carried out to determine that the 96-Well clean plate. In this step, 1 × 10 J774A.1 were compound was at crystalline or amorphous form. The counted by trypan blue staining method and added to device was calibrated using indium for melting point and DMEM medium culture contained 10% FBS (fetal bovine fusion heat. The heating rate was adjusted at 10 °C/min serum) and 1% penicillin–streptomycin and placed in for the range of 20 to 400 °C. About 5 mg of DOX-SLN 96-well cell culture plates. After 24 h of incubation, the samples which were freeze-dried were placed on an DMEM culture medium was removed and DOX-SLN, free aluminum plate to be analyzed. Moreover, a blank plate doxycycline, and free SLN (blank sample) at various con- was used as the reference for each sample. For each sam- centrations (25, 50, 100, 200, 400, 800 μl) alongside ple, the experiment was repeated three times [17]. DMEM contained 10% FBS were added and incubated for 24 h. Several positive control samples which contained no Bacterial strains and cell line drug were also provided. All experiments were repeated For conducting in vitro experiments, the B. melitensis three times. Next, all cell were washed by FBS to all drugs M16 (obtained from Razi Vaccine and Serum Research and remained polymers be removed and 150 μloffresh Institute, Iran) was used. For the culture of the bacteria, DMEM free of Fetal Bovine Serum was added to each brucella agar, tryptic soy broth (TSB) and tryptic soy well. Then, 20 μl of MTT Assay reagent was added to each agar (TSA) in the presence of 5% CO at 37 °C was used. well except the well associated with negative control The cell line of J774A.1(obtained from Pasteur Institute, sample. All plates were incubated at 37 °C in the presence Iran) was employed for the cell culture study. Moreover, of 5% CO for 3–4 h. After this period of time, 100 μl for the culture of cell, Dulbecco’s Modified Eagle’s media solubleizer was added to each well and mixed well using (DMEM) medium with 10% FBS and 1% penicillin and Orbital Shaker for 15 min to formazan particles be totally streptomycin (pen-strep) was utilized in the presence of saluted. A 96-well ELISA plate reader was used to meas- 5% CO at 37 °C [18, 19]. ure the absorption at 570 nm. The viability of the cells was calculated based on the degree of absorption of the posi- Antibiotic sensitivity test under in vitro condition tive control group (100% alive) [21]. Well diffusion and minimum inhibitory concentrations (MIC) tests were carried out in accordance with the In vitro infection assay guidelines of CLSI [20]. Each well was filled in by 100 μl The efficiency of DOX-SLN in comparison with free of DOX-SLN and free doxycycline with various concen- doxycycline to kill intra-cellular brucella was assessed trations (50–25 and 12.5 μg/ml) and incubated at 37 °C using J774A.1 cells. Macrophages were cultured in in the presence of 5% CO for 24 h, 48 h and 72 h. After 24-cell plates since 24-36 h before the infection. After these time intervals, inhibition zone diameter was mea- reaching 90% confluence in each well and increasing the sured for each well and used as a basis for analyzing the number of cells up to 5000 per well, 5 × 10 B. melitensis antibacterial performance of DOX-SLN. (the 1:100 ratio of cell to bacteria) were added to each For performing the MIC test, sterilized 96-well cell cul- well and incubated for 1 h. After phagocytosis, the culture ture plates were employed. All samples were sonicated to medium was removed and replaced by a fresh culture be homogenized before the test. The first concentration of medium containing 50 μg/ml Gentamicin and incubated NPs with the lowest antimicrobial concentration in the for 1 h for killing of extracellular bacteria. Next, the cul- well diffusion test was selected. A series of concentrations ture medium was removed again and replaced by fresh was provided from them and 100 μl of each concentration culture medium containing 10% FBS and then incubated. was added to the wells of the micro plates. Next, 100 μlof Twenty four hours after the infection, the cells were Muller Hinton broth was added to all wells. Moreover, washed by DMEM culture medium twice. Later again, 5 μl of the bacteria solution was also added to all wells 100 μl of DMEM containing 10% FBS was added to each except the control one. The microplates were incubated at well and different dilutions (25, 50, and 100 μl) of 37 °C in the presence of 5% CO for 24h,48h,and 72h. DOX-SLN, free doxycycline, and free SLN (blank) were 2 Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 6 of 12 added to infected cells, and incubated for different time FE-SEM microscopy interval, including 24, 48, and 72 h, at 37 °C in the pres- The results of DOX-SLN-1 morphology analysis by ence of 5% CO . Subsequently, the culture medium was FE-SEM is shown in Fig. 1. As seen, the majority of parti- removed and cells were washed by FBS twice. For the de- cles were spherical and had smooth surface with a homo- termination of intracellular bacterial load, cells were lysed geneous polydispersity. The mean diameter obtained by in 250 μLof0.1%TritonX-100™, and after being diluted FE-SEM is significantly smaller than that obtained by PS, were cultured on the Brucella agar culture medium. Col- which is due to evaporating the SLN matrix during the ony forming units (CFUs) were counted after 24, 48, and SEM sample preparation at oven-drying process [23]. 72 h of incubation at 37° in the presence of CO [10, 22]. FTIR analysis Statistical analysis of data The Fourier transform infra-red (FTIR) spectra of doxy- The Analysis of Variance (ANOVA) test was used to inves- cycline, palm oil and SLN-DOX are presented in Fig. 2. tigate the difference between the treatments. The Dunnett As shown in Fig. 2, loading doxycycline into the SLN test was another statistical analysis performed for compar- nanoparticles cause shielding the absorption peaks of drug ing groups. The confidence level was regarded as 95% and − 1 at 400–1700 cm , but three characteristic absorption P < 0.05 was considered statistically significant. peaks of doxycycline are still observed at 938, 993 and − 1 1041 cm in the corresponding positions of SLN-DOX. Comparing the spectra of palm oil and SLN-DOX nano- Results particles shows that main absorption peaks of palm oil at Properties of the NPs − 1 1472, 1741 and 2918 cm appeared in the SLN-DOX The mean diameter of NPs in all formulations was nanoparticle spectra. 405 nm, which is relatively large for therapeutic objec- tives in the present study. However, in the optimum formulation (DOX-SLN-1), the mean diameter and PDI of the NPs were 299 ± 34 nm and 0.29 ± 0.027, re- DSC analysis spectively. The means of zeta potential for all formu- To investigate the recrystallization and melting behav- lations was − 17.57 mV and for the DOX-SLN-1 was ior of SLN nanoparticles, DSC thermograms of DOX, − 28.7 mV (Table 1). palm oil, physical mixture and SLN-DOX nanoparticles are obtained (Fig. 3). DSC thermogram of palm oil shows a melting process at 63 °C. The melting points of Drug loading and encapsulation efficiency physical mixture and SNL-DOX nanoparticles were The amount of doxycycline loaded on SLN in different similar to that of palm oil. However, a small change in formulations was between 8.2 and 11.3%. The amount of the melting process of the pail oil in the physical mix- encapsulation was in the range of 91.3 to 97.4%. For ture with the drug is observed which is in agreement DOX-SLN-1, the amounts of loaded and encapsulated with the previous studies [24]. In the DSC thermograms doxycycline were 11.2% ± 1.3 and 94.9% ± 3.2, respect- of DOX, a sharp endothermic peak was observed at ively (Table 1). 230 °C. Tracking this point for other compounds shows a small melting point for physical mixture and NPs stability SLN-DOX. It is interesting that no any significant Particle size, PDI and zeta potential were assessed at 0, change was observed in the endothermic peak positions 1st, 2nd, 4th, 8th, and 12th month of synthesis (Table 2). of DOX, physical mixture and SLN-DOX. The absence The size of NPs was nearly constant until the 8th month of a sharp melting peaks of DOX-SLN thermogram and the size difference was not significant. After 12 suggest that no free dox crystal remained in the months, the size of NPs showed a 10.7% increase in SLN-DOX or DOX molecules are stabilized in the pail diameter (from 299 to 320 nm). oil matrix [25]. Table 2 Technological characteristics of the DOX-SLN formulation: average diameter, polydispersity index and zeta potential during stability study, (Means ± SD, n =3) Technological parameters Time (months) 0 1248 12 Average diameter nm (±SD) 299±34 301±34 300±34 305±34 311±34 320±34 Polydispersity index 0.290 ±0.027 0.292±0.012 0.295±0.031 0.299±0.024 0.316±0.017 0.401±0.045 Zeta potential (mV ± SD) −28.7±3.2 −29.2±2.9 −24.1±3.6 −31.9±4.0 −21.9±1.8 −26.2±2.7 Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 7 of 12 Fig. 1 Field emission scanning electronic microscope images of DOX-SLN Fig. 2 FTIR spectra of doxycycline, palm oil and DOX-SLN Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 8 of 12 Fig. 3 DSC thermograms of doxycycline, palm oil + doxycycline (physical mixture), palm oil and DOX-SLN Drug release than DOX-SLN-1in well diffusion test and MIC. These The profile of drug release (in vitro condition, pH = 7.4, PBS observations were predictable because in both methods, buffer) is shown in Fig. 4. The results demonstrated that the the bacteria were in direct contact with the drug. After rate of free doxycycline release was higher than that of 72 h of incubation, the diameter of halo of growth inhib- DOX-SLN-1, suggesting that DOX-SLN-1 is capable to con- ition was gradually increased, which indicates the slow trol the release of encapsulated doxycycline (P = 0.04). release of the drug from DOX-SLN-1. Antibacterial studies Toxic effects of NPs on cell The results of well diffusion and MIC indicated in The toxicity of different concentrations of DOX- Table 3. doxycycline showed better antibacterial effects SLN-1 and free SLN on cells is shown in Fig. 5.Cells Fig. 4 In vitro release profiles of doxycycline from the DOX-SLN formulation in pH = 7.4 phosphate buffer (n = 3). Free doxycycline was used as control Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 9 of 12 Table 3 Results of well diffusion and MIC test Methods Antibacterial activity Well Diffusion Zone of inhibition (mm) in three time (h) and three concentration(μg/ml) Doxycycline DOX-SLN Free SLN (blank) (50-25-12.5 μg/ml) (50-25-12.5 μg/ml) (50-25-12.5 μg/ml) 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 35 30 22 35 30 22 35 30 22 21 19 15 26 24 20 31 30 22 0 0 0 0 0 0 0 0 0 MIC MIC value (μg/ml) doxycycline Dox-SLN free SLN (blank) 24 h 24 h 48 h 72 h 24 h 48 h 72 h 0.5 25 12.5 6.25 - - - with different concentrations of free doxycycline, after treating with different concentrations of doxycyc- DOX-SLN, and free SLN (blank) were incubated at line, DOX-SLN, and free SLN are indicated in Table 4. 37 °C and the presence of 5% of CO . Moreover, the same cells were incubated in the culture medium as Discussion the positive controls (without any treatment). All ex- Chronic infectious diseases such as brucellosis impose a periments were repeated three times. Cell viability considerable economic burden on societies. In addition, was assessed using the MTT assay method. Absorp- since the bacteria are inside the cell, they are protected tion was measured at 570 nm. The basis of compari- from the body immune system but antibiotics that are sons was 100% viability of positive controls. presented in extracellular environment [21]. Therefore, it The studied concentrations were much higher than is necessary to develop drug delivery systems to achieve the concentrations needed to treat infected macrophages better treatment of intracellular infections. The aim of the under in vitro conditions. present study was to investigate the effect of DOX-SLN on intra-Cellular B. melitensis in order to develop a more Intracellular infection study effective and consistent drug delivery system for bacteria The results of intracellular infection study showed that inside the macrophages. The results indicated that DOX-SLN-1 was able to reduce the number of colonies DOX-SLN is significantly more effective in reducing the to as low as 3.5 log which was significantly lower than number of bacteria than free Doxycycline (P =0.01), sug- 5.4 log colonies obtained after the treatment by free gesting that the use of NPs resulted in the continuous and doxycycline (P = 0.01). The number of colonies grown consistent presence of drug at the target site. Fig. 5 The effect of NPs on J774A.1 cells Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 10 of 12 Table 4 In vitro efficacy of DOX-SLN compared to free doxycycline against B. melitensis inside J774A.1 cells Treatment Concentration (μg/ml) 100 50 25 Mean CFUs ± SEM Log CFUs reduction Mean CFUs ± SEM Log CFUs reduction Mean CFUs ± SEM Log CFUs reduction Doxycycline 5.4 ± 0.03 0.9 5.6 ± 0.01 0.7 6.1 ± 0.04 0.2 DOX-SLN 3.5 ± 0.07 2.8 3.9 ± 0.03 2.4 3.6 ± 0.01 2.7 Free SLN (blank) 6.3 ± 0.01 0 6.3 ± 0.01 0 6.3 ± 0.01 0 Negative Control 6.3 ± 0.02 0 6.3 ± 0.02 0 6.3 ± 0.02 0 Brucella spp. are facultative intracellular pathogens physical stability of the nanoparticle. Increasing the amount that mainly infect and replicate inside liver and spleen surfactant resulted in increase of zeta potential. As the cells or the mononuclear phagocytic system (MPS). After double emulsion method is a 2-step method, so two types capturing by these cells, the organism presents an excellent of surfactant were used, accordingly the zeta number ob- model for escaping to remain safe from regular phagosome tained in the present study is rationale [13, 27]. maturation process. After ingestion, most of them are in Nowadays, many different NPs are used in order to cell vacuoles retaining late endosomal/lysosomal markers drug delivery. The levels of drug loading and encapsulat- (LAMP-1 positive) which will eventually be killed while a ing for different NPs depends on materials used for their few of these vacuoles survive by avoiding lysosomal fusion. synthesis and methods of preparation [31]. The drug Brucella lipopolysaccharide surface and cyclic beta-1, loading and encapsulating efficiency in the present study 2-glucan (which are essential for bacterial survival and rep- were 11.2% ± 1.3 and 94.9% ± 3.2, respectively; which lication, from lipid rafts to phagosomes) has been suggested was similar to those of other studies. D Liu et al. have to play a role in the control of the phagosomal maturation. produced a nanoparticle using modified emulsion/solv- Anyway, those vacuoles that successfully evade lysosomal ent evaporation method and phospholipids for loading fusion are capable of interacting with ER exit sites. After- the hydrophilic diclofenac sodium. Similar to the present ward, they fuse to ER to generate ER-derived replicative study, they applied two methods of spectrophotometer Brucella-containing vacuoles which acidification become and HPLC for assessing the rate of drug loading and en- substantial to trafficking to the ER and subsequent intracel- capsulation. They reported the rate of drug loading and lular replication of the bacteria [26]. encapsulation as 6.5 and 72.9%, respectively, which is The obtained diameter (299 ± 34 nm) for DOX-SLN is similar to the results of the present study [27]. Other an appropriate size to be phagocytosed by phagocytes. factors able to affect encapsulation efficiency is the The results of the present study demonstrated that as amount of surfactant used during the preparation of the sonication time increases, the size of nanoparticle NPs [14, 32]. decreases. The results are in line with those of Liu et al. The double emulsion/melt dispersion technique is more [27]. The present study also demonstrated that the size effective in loading and encapsulation of doxycycline in NPs of SLN increased after drug loading. In the other words, than other techniques such as the modified solvent removal the size of DOX-SLN is larger than free SLN. Moreover, method, the hot homogenization and ultrasonication the size of nanoparticle increased after lyophilization, method, and the high shear homogenization-ultrasonication which is consistent with the findings of a study carried method [33–35]. out by Chantaburanan et al. [16]. Severino et al. pre- In the current study, after 12 months, the size of NPs pared SLN using high pressure homogenization method showed a 10.7% increase in diameter (from 299 to 320 nm). and obtain nanoparticle of 439.5 nm in size [28]. Chetoni This can be because of agglomeration of NPs. However, no et al. applied 80 nm NPs in eye drops [14]. significant change was observed for PDI and zeta potential. ThemeanPDIofthe NPsinour studywere0.29±0.027. These levels of alteration can be acceptable because the Since a PDI value lower than 0.3 has been reported as an NPs were used to assess their effects on the macrophages ideal index, the size distribution of NPs was therefore satis- in experimental conditions. factory [15]. In the present study, it was revealed that as the In the study carried out by Dong et al., the stability of time of homogenization using ultrasonic increased, the size NPs was investigated in a 6-month time period and it of NPs tended to smaller and more homogenous, which is was concluded that the NPs size and PDI became larger, in line with those of previous studies, such as Ding et al. while no change was observed in the zeta potential and and Marquele-Oliveira et al. [29, 30]. loading rate [27]. Chetoni et al. also loaded tobramycin The mean value of zeta potential for the DOX-SLN in SLN and observed no change in nanoparticle character- was − 28.7, which is good enough to prevent NPs to be istics for a time period of 12 months, while after 24 months, agglomerated and formed large colloids. This resulted in thesizeofNPs increasedfrom80nmto120nm [14]. Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 11 of 12 The release time of doxycycline from DOX-SLN in the FTIR: Fourier-transform infrared spectroscopy; HPLC: High-performance liquid chromatography; MIC: Minimum Inhibitory Concentration; MTT: 3-(4,5- current study was almost 72 h. However, other studies have Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NPs: Nanoparticles; reported different release times which is mainly due to the PDI: Polydispersity index; SLN: Solid Lipid Nanoparticles; TSA: Tryptic soy agar; use of different methods for producing NPs [14, 15, 29]. TSB: Tryptic soy broth; WHO: World Health Organization Most studies have outlined that the release time of Acknowledgments drug from nanoparticle is higher than those of their con- The authors would like to acknowledge the Vice Chancellor of Hamadan trols, which is due to the slow release of drug from lipid University of Medical Sciences for the funding and support of the study and Hamedan Science and Technology Park. matrix. Moreover, previous studies have demonstrated that as the size of nanoparticle decreases the release Funding time also decreases, which is due to the increase in the This study has been adapted from a PhD. thesis at Hamadan University of Medical Sciences (Project No. 9510146066). surface area of smaller particles [17, 36]. Imbuluzqueta et al. utilized the gentamycin loaded in Availability of data and materials PLGA for killing B. melitensis phagocytosized by macro- The data can be accessible to the interested researchers by the corresponding authors on reasonable request. phages, which had a lower efficacy than DOX-SLN syn- thesized in the present study [26]. Another study Authors’ contributions conducted by Seleem et al. used doxycycline loaded in SMH and MA designed the study. SMH contributed in the experimental studies, and drafting the work. GHR performed the analysis of the data. FJ and SS Nanoplexes for killing B. melitensis in J774A.1 cells. contributed in the cell culture. RA and AF designed the nanoparticles. All They observed no significant difference between the effi- authors read and approved the final manuscript. cacies of the drug loaded in NPs and the corresponding Ethics approval and consent to participate free drug [22]. The results are in contradiction with This study was approved by the ethics committee of Hamadan University of those of the present study, which can be due to the rapid Medical Sciences (No: IRUMSHA. REC. 1395066). release of the drug from the NPs; the release time re- Consent for publication ported by that study was 15 h which is significantly Not applicable lower than 72 h we observed in the current study. There were a number of limitations to our study in- Competing interests The authors declare that they have no competing interests. cluding: studying on B. melitensis was very difficult due to its pathogenic nature. Because we had many tests and Publisher’sNote errors to set up an appropriate nanoparticle, the dur- Springer Nature remains neutral with regard to jurisdictional claims in ation of the project was long. This project needs many published maps and institutional affiliations. equipment, as all of them were not centralized in one Author details place, the work became very hard. Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. Department of Clinical Biochemistry, Faculty of Medicine, Hamadan Conclusions University of Medical Sciences, Shahid fahmideh street, Park Mardome, Thedoubleemulsionmethod issuitabletoencapsulation Hamadan, IR, Iran. Department of Virology, Faculty of Medicine, Hamadan of DOX as a hydrophilic drug so that particle size, zeta University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. Department of Anatomical Sciences, Faculty of Medicine, potential, and PDI were desirable for our objectives. The Hamadan University of Medical Sciences, Shahid fahmideh street, Park time required for the complete release of drug and there- Mardome, Hamadan, IR, Iran. Dental Research Center, School of Dentistry,, fore its antibacterial effectiveness was 72 h. The effects of Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. Department of Biostatistics, School of Health, DOX-SLN on the bacteria enclosed in macrophages was Hamadan University of Medical Sciences, Shahid fahmideh street, Park significantly higher than the free DOX. This can be attrib- Mardome, Hamadan, IR, Iran. Brucellosis Research Center, Hamadan uted to the phagocytosis of NPs by macrophages and slow University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. continuous release of drug. The application of these NPs can be considered as a promising tool for treating intracel- Received: 17 January 2019 Accepted: 27 February 2019 lular bacteria, particularly B. melitensis and preventing the relapse of the disease. References Suggestions that can help researchers interested in this 1. de Figueiredo P, Ficht TA, Rice-Ficht A, Rossetti CA, Adams LG. Pathogenesis topic: using multiple-drug combination that are routinely and immunobiology of brucellosis: review of Brucella–host interactions. Am J Pathol. 2015;185(6):1505–17. used to treat brucellosis, investigating nanoparticle’s effect 2. Musallam I, Abo-Shehada M, Hegazy Y, Holt H, Guitian F. Systematic review and toxicity in in vivo conditions, utilizing other nanopar- of brucellosis in the Middle East: disease frequency in ruminants and ticles and comparing its effect on B. melitensis with SLN. humans and risk factors for human infection. Epidemiol Infect. 2016;144(4): 671–85. Abbreviations 3. Byndloss MX, Tsolis RM. 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Doxycycline-encapsulated solid lipid nanoparticles as promising tool against Brucella melitensis enclosed in macrophage: a pharmacodynamics study on J774A.1 cell line

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Biomedicine; Medical Microbiology; Drug Resistance; Infectious Diseases
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Abstract

Background: Brucellosis is a zoonotic disease caused by Brucella species. It has been estimated that more than 500,000 new cases of Brucellosis occur annually all around the world. Relapse of the disease is one of the most important challenges. The most important reason for the relapse of brucellosis is the survival of the bacteria inside the macrophages, which makes them safe from the immune system and disrupts drug delivery mechanism. Objectives: The present study was performed to assess the effects of Doxycycline-loaded Solid Lipid Nanoparticles (DOX-SLN) on the Brucella melitensis inside macrophages. Methods: DOX-SLN was prepared using double emulsion method. The technological characterization of DOX-SLN, including particle size, zeta potential, polydispersity index (PDI), drug loading and encapsulation efficiency were used. Fourier-transform infrared spectroscopy (FTIR) and Differential scanning calorimetry (DSC) were used to assess the interactions between Nanoparticles (NPs) components and crystalline form of doxycycline. Moreover, the effect of DOX-SLN on the bacteria were compared with that of the doxycycline using various methods, including well diffusion, Minimum Inhibitory Concentration (MIC), and investigation of their effects on murine macrophage-like cells cell line J774A.1. Results: The means of particle size, zeta potential, PDI, drug loading and encapsulation efficiency were 299 ± 34 nm, − 28.7 ± 3.2 mV, 0.29 ± 0.027, 11.2 ± 1.3%, and 94.9 ± 3.2%, respectively. The morphology of NPs were spherical with a smooth surface. No chemical reaction was occurred between the components. Doxycycline was located within NP matrix in its molecular form. The DOX-SLN significantly decreased the microbial loading within macrophages (3.5 Log) in comparison with the free doxycycline. Conclusions: Since the DOX-SLN showed better effects on B. melitensis enclosed in macrophages than the free doxycycline, it is recommended to use it for treating brucellosis and preventing relapse. Keywords: Brucella melitensis, Doxycycline, Solid lipid nanoparticle, Relapse * Correspondence: Mohammad.arabestani@gmail.com Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran Brucellosis Research Center, Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 2 of 12 Background stability, increase bioavailability of drug, effective pass from Brucellosis is a zoonotic disease caused by different biological barriers, reduce dose and frequency of drug ad- Brucella strains following direct or indirect contact of ministration, fast identification by phagocytic system, con- human with infected animals or dairy products [1]. The trolled release of drug, specific targeting, slow release of most common cause of the disease is Brucella melitensis, drug in a long-term period, protect drugs against pharma- which is endemic in Middle East countries, including Iran. cologic and toxicological effects, prevent early breakdown Brucella is an intracellular bacterium [2]. The phagocyt- of drug molecules, prevent immune response, increase drug osis activity of polymorphonuclear and monomorphonuc- retention in tissue, minimize resistance to drug, improve lear cells such as macrophages plays an important role in therapeutic index of drug, minimize drug toxicity, and im- the disease treatment [3]. Upon entering the body, about prove treatment efficiency. 90% of the bacteria were killed by neutrophils, monocytes, Despite its partial limitations, the followings can be and macrophages. However, some of them survive inside mentioned as the most significant features of SLNs: pro- macrophages and find a place to proliferation within the tecting drugs from chemical and enzymatic decomposi- cell [1, 4]. tions as well as pharmacologic and toxicological effects, The World Health Organization (WHO) suggests the physical and biotical stability, hydrophilic and hydropho- combination of gentamicin and doxycycline for brucellosis bic drug loading capacity, easy production, no need for treatment [5]. The combination of doxycycline and strepto- organic solvents in synthesis, ability to carry two active mycin has also been suggested to prevent the disease agents simultaneously, increase drug efficiency, easy relapse. The combination of doxycycline and rifampin for sterilization, small diameter, administration via different 45 days is the best known treatment for Brucellosis because routes, biodegradability trait, increase bioavailability of of having no side effects and its acceptability by patients. drug, effective pass from biological barriers, reduce dose However, this treatment has been less effective with about and frequency of drug administration, fast identification 15% failure in different types of the disease [6]. by phagocytic system, controlled release of drug, specific Despite the significant number of new antibiotics, treat- targeting, slow release of drug in a long-term, prevent early ment of intracellular pathogens is still a major challenge breakdown of drug molecules, prevent immune response, [7]. To date, no antibiotic therapy has been reported to increase drug retention in tissue, minimize resistance to eradicate Brucella intracellular infections. The main chal- drug, improve therapeutic index of drug, minimize drug lenge in intracellular chemotherapy is the design and toxicity and improve treatment efficiency [11, 12]. development of a carrier system for antibiotics that can be Given the above, the present study was carried out to effectively endocytosed by phagocytic cells and release develop a new therapeutic mechanism for the treatment drugs. Targeted drug delivery system can be considered as of brucellosis in order to enhance efficacy of antibiotics. an appropriate tool to overcome such problems [8]. The therapeutic efficacy of doxycycline-loaded SLNs in One of the most promising strategies to prevent the chronic infections of B. melitensis was assessed in cellular disease recurrence is using the antibiotic-loaded nanocar- models and experimental conditions. riers. Nowadays, with the production of pharmaceutical NPs, unique characteristics can be achieved, which will Methods result in increased performance of drugs and diversity of Materials their forms. Hydrogenated palm oil, (Softisan 154 or S154) a gift from These carrier systems have to be non-toxic and have Condea (Witten, Germany), and stearic acid (SA, Sigma sufficient capacity to take the drug. In addition, they Aldrich, USA) were the lipids used in this study. Water should have the possibility of controlling drug release. soluble surfactants used were polioxiethylene-20-sorbitan Solid lipid nanoparticles (SLNs) as a carrier system have monooleate (Tween 80- Sigma Aldrich, USA), Poloxa- been investigated for many applications. These drug mer407 (Pluronic F127, Sigma Aldrich, USA). Furthermore, delivery systems provide controlled release of medicines lipo-soluble surfactants used were Sorbitanmonooleate that they carry and increase the chemical stability of (Span 80, Sigma Aldrich, USA), and soy lecithin them [9, 10]. (Sigma-Aldrich). Doxycycline hyclate (Sigma-Aldrich). In spite of negligible limitations, the most important Water used in our study was distilled twice and purified properties of SLNs are protecting drugs from chemical by an appropriate filter. and enzymatic decompositions, physical stability, hydro- philic and hydrophobic drug loading capacity, easy pro- Preparation of NPs duction, no need for organic solvents in synthesis, ability Solid-lipid NPs (SLNs) were synthesized using the above to simultaneous carry two active agents, increase drug mentioned chemicals and double emulsion/melt disper- efficiency, ease of sterilization, small diameter, adminis- sion technique based on the study conducted by Luana tration via different routes, biodegradability and biotic Becker Peres [13]. The first emulsion contained palm oil, Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 3 of 12 Poloxumer, distilled water, and doxycycline antibiotic. In literature. In this step, 10 mg of prepared NPs was added brief, firstly, 0.6 g of palm oil was warmed to 60 °C (the to 10 ml distilled water and vortexed to become boiling point) in Ben Murray. Then 60 mg Poloxamer homogenous. Then, the solution was centrifuged at 4 °C and 30 mg doxycycline were added to the melted palm for 20 min at 1500 rpm. The supernatant was investigated oil and mixed on the magnetic stirring (60 °C and 150 using spectrophotometer (2100 UV, USA) at a wavelength rpm) for 5 min. In the following step, 0.5 ml hot distilled of 270 nm. The drug concentration was determined using water was added to the mixture. Mixture was sonicated the standard curve which have been previously depicted using sonicator (skymen, China) during 60 s at 45% based on various concentrations of the drug [14, 15]. The amplitude (20 W) to obtain the first emulsion. (W1/O). amount of loaded and encapsulated drug was obtained In the next step, 30 mg tween-80 was added to the pre- using the following equations: pared first emulsion and made homogenous using ultra- sonic device (Bandelin Sonopuls, Berlin, Germany) at initial drug amount−free drug amount Entrapment Efficiency EEðÞ % ¼  100 45% amplitude (20w) at a regular pulse rhythm (10s on initial drug amount and 5 s off) for 60s to the second emulsion (W1/O/W2) initial drug amount−free drug amount be provided. The prepared second emulsion was gently Drug Loading DLðÞ % ¼  100 initial lipid amount added to 30 ml of cold distilled water (4 °C) under the condition of magnetic starring (5 min) to stabilize SLN. The amount of doxycycline contained in NPs was in- Materials required for each formulation are presented vestigated using HPLC equipped (SY-8100, China) with in Table 1. Finally, doxycycline-loaded solid lipid nano- UV detector at a wavelength of 270 nm. The mobile particles (DOX-SLN) was separated using the high speed phase was a mixture of ethanol (50%) and water (50%), centrifuge (37,000 g for 15 min) and washed using steril- the pump was of SY-8100 type, injector loop was at 772 ized distilled water three times. Almost 500 mg of each PSI, and the column was of C-18 type (4.6 X 250 mm) is formulation was added to 1 ml glycerin. The prepared run at a flow rate of 1 mL/min. In this step, 10 mg of the samples were lyophilized at − 80 °C using a vacuum sample alongside 10 ml of water and ethanol mixture pump (Christ, china) with a condenser flow. For being were mixed and vortexed using sonication for 10 min to usable in biologic studies on bacteria and cell line, ly- become homogenous. Then, it was centrifuged at 1500 ophilized NPs transformed in solution and sterilized rpm for 20 min and the supernatant was purified using using 450 nm filters [14]. 220 nm filters. A linear calibration curve with a good correlation coefficient (r = 0.9990) for concentrations in Characteristics of NPs (particle size, polydispersity index, a range from 1 μg/ml to 250 μg/ml was achieved. and zeta potential) Particle size, Polydispersity Index, and Zeta Potential were Physical and chemical stability of NPs determined by Dynamic Light Scattering (DLS) technique The stability of NPs loaded with doxycycline was assessed performed by Zetasizer Nano ZS 3600 (Malvern Instru- in regular time intervals. After passing the NPs cross 450 ments, Worcestershire, United Kingdom) device. The men- nm filters, particle size, zeta potential and PDI were deter- tioned factors were measured twice; firstly, immediately mined using nano Zetasizer device in the time intervals of after preparation, and secondly, after lyophilization [13]. 1 h, 24 h, 48 h, and also 1, 2, 4, 8, and 12 months after the preparation. At the same times, the concentration of Morphology doxycycline in NPs was determined using HPLC [14]. Field emission scanning electronic microscope (FE-SEM) was used for assessing the morphological characteristics In vitro release experiment of NPs. To do so, 10 mg lyophilized DOX-SLN was added The lyophilized samples which were exactly weighted to 1 ml distilled water and then 2 μl of this suspension was and enclosed in a dialysis bag (cut-off 12,000, Dialysis placed on a glass surface. Since the suspension was dried, tubing, Sigma Chem. Co., Missouri, USA) were placed in it was covered by a thin gold layer to avoid electrostatic a 40 ml release medium on the magnetic starring of 100 charging during examination and analyzed using FE-SEM rpm at 37 °C. At predetermined time intervals, 0.5 ml of (TSCAN, Czech Republic) [15]. the medium was sampled and its doxycycline content was investigated using HPLC. For comparing the results Investigation of capsulated and loaded drug of this set of experiments with those of free doxycycline, For determining how much the doxycycline have been the same procedure was conducted in which free doxy- loaded and encapsulated in the synthesized NPs, an indir- cycline were enclosed in the dialysis bags and placed in ect method, i.e. spectrophotometer, and a direct one, i.e. the same medium. Then at the same time intervals sev- high-performance liquid chromatography (HPLC), were eral samples were obtained from the medium and inves- employed in accordance with guidelines recommended in tigated. It should be noted that after each sampling from Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 4 of 12 Table 1 Materials and technological parameters of Various formulation sample Lipid (mg) Lipid surfactant Water surfactant Water added PS (nm) PDI Zeta potential EE (%) DL (%) (ML, Mg) (ML, Mg) (ML) (mV) Before lyophilization SLN1 without doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 251 ± 15 0.277 ± 0.021 −31.6 ± 1.4 95.4 ± 3.5 11.3 ± 1.1 SLN 1 with doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 265 ± 23 0.284 ± 0.012 −29.3 ± 2.1 SLN 2 without doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 456 ± 21 0.429 ± 0.032 −11.6 ± 4.4 93.7 ± 5.3 8.9 ± 2.3 SLN 2 with doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 465 ± 23 0.344 ± 0.012 −14.3 ± 3.5 SLN 3 without doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 358 ± 39 0.536 ± 0.019 −9.6 ± 1.7 94.1 ± 7.1 9.2 ± 3.8 SLN 3 with doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 375 ± 42 0.591 ± 0.010 −11.3 ± 2.6 SLN 4 without doxycycline Stearic acid (1200) Lecithin (60) Tween 80 (3) 50 491 ± 25 0.447 ± 0.033 −18.6 ± 1.4 97.4 ± 4.5 10.7 ± 1.4 SLN 4 with doxycycline Stearic acid (1200) Lecithin (60) Tween 80 (3) 50 415 ± 59 0.494 ± 0.093 −19.3 ± 1.2 After lyophilization SLN 1 without doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 294 ± 27 0.281 ± 0.044 −30.7 ± 2.04 94.9 ± 3.2 11.2 ± 1.3 SLN 1 with doxycycline Palm oil (600) Lecithin (60) Tween 80 (6) 50 299 ± 34 0.290 ± 0.027 −28.7 ± 3.2 SLN 2 without doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 491 ± 33 0.499 ± 0.042 −12.6 ± 3.5 93.9 ± 4.7 8.2 ± 1.9 SLN 2 with doxycycline Stearic acid (600) Span 80 (6) Poloxamer407 (60) 100 471 ± 34 0.412 ± 0.012 −14.3 ± 3.5 SLN 3 without doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 378 ± 28 0.591 ± 0.053 −17.6 ± 2.1 91.3 ± 2.7 8.8 ± 3.1 SLN 3 with doxycycline Palm oil (1200) Span 80 (6) Tween 80 (3) 100 399 ± 39 0.612 ± 0.125 −15.3 ± 1.7 SLN 4 without doxycycline stearic acid (1200) Lecithin (60) Tween 80 (3) 50 511 ± 45 0.493 ± 0.325 −17.8 ± 1.7 96.1 ± 3.9 9.9 ± 1.7 SLN 4 with doxycycline stearic acid (1200) Lecithin (60) Tween 80 (3) 50 575 ± 39 0.524 ± 0.987 −16.3 ± 2.2 PS particle size, PDI polydispersity index, EE entrapment efficiency, DL drug loading Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 5 of 12 the medium, the new and fresh medium with the same MIC was determined by visual examination of the turbid- amount was added [14]. ity and culturing the contents of the wells. The well with no bacterial growth was regarded as the minimum inhibi- Fourier-transform infrared spectroscopy and differential tory concentration [20]. scanning calorimetry In the present study, a set of experiments was also con- In vitro cytotoxicity ducted to discover any possible interaction between doxy- MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo- cycline and lipid compounds contained in DOX-SLN. lium bromide) Assay test was performed using the MTT FTIR analysis was performed to investigate such a possi- assay kit (Kiazist, Iran). The contents of the kit were as bility in a temperature range from 400 °C to 4000 °C [16]. follows; MTT reagent, solubilizer, 2 Chanel reservoir, and DSC analysis was carried out to determine that the 96-Well clean plate. In this step, 1 × 10 J774A.1 were compound was at crystalline or amorphous form. The counted by trypan blue staining method and added to device was calibrated using indium for melting point and DMEM medium culture contained 10% FBS (fetal bovine fusion heat. The heating rate was adjusted at 10 °C/min serum) and 1% penicillin–streptomycin and placed in for the range of 20 to 400 °C. About 5 mg of DOX-SLN 96-well cell culture plates. After 24 h of incubation, the samples which were freeze-dried were placed on an DMEM culture medium was removed and DOX-SLN, free aluminum plate to be analyzed. Moreover, a blank plate doxycycline, and free SLN (blank sample) at various con- was used as the reference for each sample. For each sam- centrations (25, 50, 100, 200, 400, 800 μl) alongside ple, the experiment was repeated three times [17]. DMEM contained 10% FBS were added and incubated for 24 h. Several positive control samples which contained no Bacterial strains and cell line drug were also provided. All experiments were repeated For conducting in vitro experiments, the B. melitensis three times. Next, all cell were washed by FBS to all drugs M16 (obtained from Razi Vaccine and Serum Research and remained polymers be removed and 150 μloffresh Institute, Iran) was used. For the culture of the bacteria, DMEM free of Fetal Bovine Serum was added to each brucella agar, tryptic soy broth (TSB) and tryptic soy well. Then, 20 μl of MTT Assay reagent was added to each agar (TSA) in the presence of 5% CO at 37 °C was used. well except the well associated with negative control The cell line of J774A.1(obtained from Pasteur Institute, sample. All plates were incubated at 37 °C in the presence Iran) was employed for the cell culture study. Moreover, of 5% CO for 3–4 h. After this period of time, 100 μl for the culture of cell, Dulbecco’s Modified Eagle’s media solubleizer was added to each well and mixed well using (DMEM) medium with 10% FBS and 1% penicillin and Orbital Shaker for 15 min to formazan particles be totally streptomycin (pen-strep) was utilized in the presence of saluted. A 96-well ELISA plate reader was used to meas- 5% CO at 37 °C [18, 19]. ure the absorption at 570 nm. The viability of the cells was calculated based on the degree of absorption of the posi- Antibiotic sensitivity test under in vitro condition tive control group (100% alive) [21]. Well diffusion and minimum inhibitory concentrations (MIC) tests were carried out in accordance with the In vitro infection assay guidelines of CLSI [20]. Each well was filled in by 100 μl The efficiency of DOX-SLN in comparison with free of DOX-SLN and free doxycycline with various concen- doxycycline to kill intra-cellular brucella was assessed trations (50–25 and 12.5 μg/ml) and incubated at 37 °C using J774A.1 cells. Macrophages were cultured in in the presence of 5% CO for 24 h, 48 h and 72 h. After 24-cell plates since 24-36 h before the infection. After these time intervals, inhibition zone diameter was mea- reaching 90% confluence in each well and increasing the sured for each well and used as a basis for analyzing the number of cells up to 5000 per well, 5 × 10 B. melitensis antibacterial performance of DOX-SLN. (the 1:100 ratio of cell to bacteria) were added to each For performing the MIC test, sterilized 96-well cell cul- well and incubated for 1 h. After phagocytosis, the culture ture plates were employed. All samples were sonicated to medium was removed and replaced by a fresh culture be homogenized before the test. The first concentration of medium containing 50 μg/ml Gentamicin and incubated NPs with the lowest antimicrobial concentration in the for 1 h for killing of extracellular bacteria. Next, the cul- well diffusion test was selected. A series of concentrations ture medium was removed again and replaced by fresh was provided from them and 100 μl of each concentration culture medium containing 10% FBS and then incubated. was added to the wells of the micro plates. Next, 100 μlof Twenty four hours after the infection, the cells were Muller Hinton broth was added to all wells. Moreover, washed by DMEM culture medium twice. Later again, 5 μl of the bacteria solution was also added to all wells 100 μl of DMEM containing 10% FBS was added to each except the control one. The microplates were incubated at well and different dilutions (25, 50, and 100 μl) of 37 °C in the presence of 5% CO for 24h,48h,and 72h. DOX-SLN, free doxycycline, and free SLN (blank) were 2 Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 6 of 12 added to infected cells, and incubated for different time FE-SEM microscopy interval, including 24, 48, and 72 h, at 37 °C in the pres- The results of DOX-SLN-1 morphology analysis by ence of 5% CO . Subsequently, the culture medium was FE-SEM is shown in Fig. 1. As seen, the majority of parti- removed and cells were washed by FBS twice. For the de- cles were spherical and had smooth surface with a homo- termination of intracellular bacterial load, cells were lysed geneous polydispersity. The mean diameter obtained by in 250 μLof0.1%TritonX-100™, and after being diluted FE-SEM is significantly smaller than that obtained by PS, were cultured on the Brucella agar culture medium. Col- which is due to evaporating the SLN matrix during the ony forming units (CFUs) were counted after 24, 48, and SEM sample preparation at oven-drying process [23]. 72 h of incubation at 37° in the presence of CO [10, 22]. FTIR analysis Statistical analysis of data The Fourier transform infra-red (FTIR) spectra of doxy- The Analysis of Variance (ANOVA) test was used to inves- cycline, palm oil and SLN-DOX are presented in Fig. 2. tigate the difference between the treatments. The Dunnett As shown in Fig. 2, loading doxycycline into the SLN test was another statistical analysis performed for compar- nanoparticles cause shielding the absorption peaks of drug ing groups. The confidence level was regarded as 95% and − 1 at 400–1700 cm , but three characteristic absorption P < 0.05 was considered statistically significant. peaks of doxycycline are still observed at 938, 993 and − 1 1041 cm in the corresponding positions of SLN-DOX. Comparing the spectra of palm oil and SLN-DOX nano- Results particles shows that main absorption peaks of palm oil at Properties of the NPs − 1 1472, 1741 and 2918 cm appeared in the SLN-DOX The mean diameter of NPs in all formulations was nanoparticle spectra. 405 nm, which is relatively large for therapeutic objec- tives in the present study. However, in the optimum formulation (DOX-SLN-1), the mean diameter and PDI of the NPs were 299 ± 34 nm and 0.29 ± 0.027, re- DSC analysis spectively. The means of zeta potential for all formu- To investigate the recrystallization and melting behav- lations was − 17.57 mV and for the DOX-SLN-1 was ior of SLN nanoparticles, DSC thermograms of DOX, − 28.7 mV (Table 1). palm oil, physical mixture and SLN-DOX nanoparticles are obtained (Fig. 3). DSC thermogram of palm oil shows a melting process at 63 °C. The melting points of Drug loading and encapsulation efficiency physical mixture and SNL-DOX nanoparticles were The amount of doxycycline loaded on SLN in different similar to that of palm oil. However, a small change in formulations was between 8.2 and 11.3%. The amount of the melting process of the pail oil in the physical mix- encapsulation was in the range of 91.3 to 97.4%. For ture with the drug is observed which is in agreement DOX-SLN-1, the amounts of loaded and encapsulated with the previous studies [24]. In the DSC thermograms doxycycline were 11.2% ± 1.3 and 94.9% ± 3.2, respect- of DOX, a sharp endothermic peak was observed at ively (Table 1). 230 °C. Tracking this point for other compounds shows a small melting point for physical mixture and NPs stability SLN-DOX. It is interesting that no any significant Particle size, PDI and zeta potential were assessed at 0, change was observed in the endothermic peak positions 1st, 2nd, 4th, 8th, and 12th month of synthesis (Table 2). of DOX, physical mixture and SLN-DOX. The absence The size of NPs was nearly constant until the 8th month of a sharp melting peaks of DOX-SLN thermogram and the size difference was not significant. After 12 suggest that no free dox crystal remained in the months, the size of NPs showed a 10.7% increase in SLN-DOX or DOX molecules are stabilized in the pail diameter (from 299 to 320 nm). oil matrix [25]. Table 2 Technological characteristics of the DOX-SLN formulation: average diameter, polydispersity index and zeta potential during stability study, (Means ± SD, n =3) Technological parameters Time (months) 0 1248 12 Average diameter nm (±SD) 299±34 301±34 300±34 305±34 311±34 320±34 Polydispersity index 0.290 ±0.027 0.292±0.012 0.295±0.031 0.299±0.024 0.316±0.017 0.401±0.045 Zeta potential (mV ± SD) −28.7±3.2 −29.2±2.9 −24.1±3.6 −31.9±4.0 −21.9±1.8 −26.2±2.7 Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 7 of 12 Fig. 1 Field emission scanning electronic microscope images of DOX-SLN Fig. 2 FTIR spectra of doxycycline, palm oil and DOX-SLN Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 8 of 12 Fig. 3 DSC thermograms of doxycycline, palm oil + doxycycline (physical mixture), palm oil and DOX-SLN Drug release than DOX-SLN-1in well diffusion test and MIC. These The profile of drug release (in vitro condition, pH = 7.4, PBS observations were predictable because in both methods, buffer) is shown in Fig. 4. The results demonstrated that the the bacteria were in direct contact with the drug. After rate of free doxycycline release was higher than that of 72 h of incubation, the diameter of halo of growth inhib- DOX-SLN-1, suggesting that DOX-SLN-1 is capable to con- ition was gradually increased, which indicates the slow trol the release of encapsulated doxycycline (P = 0.04). release of the drug from DOX-SLN-1. Antibacterial studies Toxic effects of NPs on cell The results of well diffusion and MIC indicated in The toxicity of different concentrations of DOX- Table 3. doxycycline showed better antibacterial effects SLN-1 and free SLN on cells is shown in Fig. 5.Cells Fig. 4 In vitro release profiles of doxycycline from the DOX-SLN formulation in pH = 7.4 phosphate buffer (n = 3). Free doxycycline was used as control Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 9 of 12 Table 3 Results of well diffusion and MIC test Methods Antibacterial activity Well Diffusion Zone of inhibition (mm) in three time (h) and three concentration(μg/ml) Doxycycline DOX-SLN Free SLN (blank) (50-25-12.5 μg/ml) (50-25-12.5 μg/ml) (50-25-12.5 μg/ml) 24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h 35 30 22 35 30 22 35 30 22 21 19 15 26 24 20 31 30 22 0 0 0 0 0 0 0 0 0 MIC MIC value (μg/ml) doxycycline Dox-SLN free SLN (blank) 24 h 24 h 48 h 72 h 24 h 48 h 72 h 0.5 25 12.5 6.25 - - - with different concentrations of free doxycycline, after treating with different concentrations of doxycyc- DOX-SLN, and free SLN (blank) were incubated at line, DOX-SLN, and free SLN are indicated in Table 4. 37 °C and the presence of 5% of CO . Moreover, the same cells were incubated in the culture medium as Discussion the positive controls (without any treatment). All ex- Chronic infectious diseases such as brucellosis impose a periments were repeated three times. Cell viability considerable economic burden on societies. In addition, was assessed using the MTT assay method. Absorp- since the bacteria are inside the cell, they are protected tion was measured at 570 nm. The basis of compari- from the body immune system but antibiotics that are sons was 100% viability of positive controls. presented in extracellular environment [21]. Therefore, it The studied concentrations were much higher than is necessary to develop drug delivery systems to achieve the concentrations needed to treat infected macrophages better treatment of intracellular infections. The aim of the under in vitro conditions. present study was to investigate the effect of DOX-SLN on intra-Cellular B. melitensis in order to develop a more Intracellular infection study effective and consistent drug delivery system for bacteria The results of intracellular infection study showed that inside the macrophages. The results indicated that DOX-SLN-1 was able to reduce the number of colonies DOX-SLN is significantly more effective in reducing the to as low as 3.5 log which was significantly lower than number of bacteria than free Doxycycline (P =0.01), sug- 5.4 log colonies obtained after the treatment by free gesting that the use of NPs resulted in the continuous and doxycycline (P = 0.01). The number of colonies grown consistent presence of drug at the target site. Fig. 5 The effect of NPs on J774A.1 cells Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 10 of 12 Table 4 In vitro efficacy of DOX-SLN compared to free doxycycline against B. melitensis inside J774A.1 cells Treatment Concentration (μg/ml) 100 50 25 Mean CFUs ± SEM Log CFUs reduction Mean CFUs ± SEM Log CFUs reduction Mean CFUs ± SEM Log CFUs reduction Doxycycline 5.4 ± 0.03 0.9 5.6 ± 0.01 0.7 6.1 ± 0.04 0.2 DOX-SLN 3.5 ± 0.07 2.8 3.9 ± 0.03 2.4 3.6 ± 0.01 2.7 Free SLN (blank) 6.3 ± 0.01 0 6.3 ± 0.01 0 6.3 ± 0.01 0 Negative Control 6.3 ± 0.02 0 6.3 ± 0.02 0 6.3 ± 0.02 0 Brucella spp. are facultative intracellular pathogens physical stability of the nanoparticle. Increasing the amount that mainly infect and replicate inside liver and spleen surfactant resulted in increase of zeta potential. As the cells or the mononuclear phagocytic system (MPS). After double emulsion method is a 2-step method, so two types capturing by these cells, the organism presents an excellent of surfactant were used, accordingly the zeta number ob- model for escaping to remain safe from regular phagosome tained in the present study is rationale [13, 27]. maturation process. After ingestion, most of them are in Nowadays, many different NPs are used in order to cell vacuoles retaining late endosomal/lysosomal markers drug delivery. The levels of drug loading and encapsulat- (LAMP-1 positive) which will eventually be killed while a ing for different NPs depends on materials used for their few of these vacuoles survive by avoiding lysosomal fusion. synthesis and methods of preparation [31]. The drug Brucella lipopolysaccharide surface and cyclic beta-1, loading and encapsulating efficiency in the present study 2-glucan (which are essential for bacterial survival and rep- were 11.2% ± 1.3 and 94.9% ± 3.2, respectively; which lication, from lipid rafts to phagosomes) has been suggested was similar to those of other studies. D Liu et al. have to play a role in the control of the phagosomal maturation. produced a nanoparticle using modified emulsion/solv- Anyway, those vacuoles that successfully evade lysosomal ent evaporation method and phospholipids for loading fusion are capable of interacting with ER exit sites. After- the hydrophilic diclofenac sodium. Similar to the present ward, they fuse to ER to generate ER-derived replicative study, they applied two methods of spectrophotometer Brucella-containing vacuoles which acidification become and HPLC for assessing the rate of drug loading and en- substantial to trafficking to the ER and subsequent intracel- capsulation. They reported the rate of drug loading and lular replication of the bacteria [26]. encapsulation as 6.5 and 72.9%, respectively, which is The obtained diameter (299 ± 34 nm) for DOX-SLN is similar to the results of the present study [27]. Other an appropriate size to be phagocytosed by phagocytes. factors able to affect encapsulation efficiency is the The results of the present study demonstrated that as amount of surfactant used during the preparation of the sonication time increases, the size of nanoparticle NPs [14, 32]. decreases. The results are in line with those of Liu et al. The double emulsion/melt dispersion technique is more [27]. The present study also demonstrated that the size effective in loading and encapsulation of doxycycline in NPs of SLN increased after drug loading. In the other words, than other techniques such as the modified solvent removal the size of DOX-SLN is larger than free SLN. Moreover, method, the hot homogenization and ultrasonication the size of nanoparticle increased after lyophilization, method, and the high shear homogenization-ultrasonication which is consistent with the findings of a study carried method [33–35]. out by Chantaburanan et al. [16]. Severino et al. pre- In the current study, after 12 months, the size of NPs pared SLN using high pressure homogenization method showed a 10.7% increase in diameter (from 299 to 320 nm). and obtain nanoparticle of 439.5 nm in size [28]. Chetoni This can be because of agglomeration of NPs. However, no et al. applied 80 nm NPs in eye drops [14]. significant change was observed for PDI and zeta potential. ThemeanPDIofthe NPsinour studywere0.29±0.027. These levels of alteration can be acceptable because the Since a PDI value lower than 0.3 has been reported as an NPs were used to assess their effects on the macrophages ideal index, the size distribution of NPs was therefore satis- in experimental conditions. factory [15]. In the present study, it was revealed that as the In the study carried out by Dong et al., the stability of time of homogenization using ultrasonic increased, the size NPs was investigated in a 6-month time period and it of NPs tended to smaller and more homogenous, which is was concluded that the NPs size and PDI became larger, in line with those of previous studies, such as Ding et al. while no change was observed in the zeta potential and and Marquele-Oliveira et al. [29, 30]. loading rate [27]. Chetoni et al. also loaded tobramycin The mean value of zeta potential for the DOX-SLN in SLN and observed no change in nanoparticle character- was − 28.7, which is good enough to prevent NPs to be istics for a time period of 12 months, while after 24 months, agglomerated and formed large colloids. This resulted in thesizeofNPs increasedfrom80nmto120nm [14]. Hosseini et al. Antimicrobial Resistance and Infection Control (2019) 8:62 Page 11 of 12 The release time of doxycycline from DOX-SLN in the FTIR: Fourier-transform infrared spectroscopy; HPLC: High-performance liquid chromatography; MIC: Minimum Inhibitory Concentration; MTT: 3-(4,5- current study was almost 72 h. However, other studies have Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NPs: Nanoparticles; reported different release times which is mainly due to the PDI: Polydispersity index; SLN: Solid Lipid Nanoparticles; TSA: Tryptic soy agar; use of different methods for producing NPs [14, 15, 29]. TSB: Tryptic soy broth; WHO: World Health Organization Most studies have outlined that the release time of Acknowledgments drug from nanoparticle is higher than those of their con- The authors would like to acknowledge the Vice Chancellor of Hamadan trols, which is due to the slow release of drug from lipid University of Medical Sciences for the funding and support of the study and Hamedan Science and Technology Park. matrix. Moreover, previous studies have demonstrated that as the size of nanoparticle decreases the release Funding time also decreases, which is due to the increase in the This study has been adapted from a PhD. thesis at Hamadan University of Medical Sciences (Project No. 9510146066). surface area of smaller particles [17, 36]. Imbuluzqueta et al. utilized the gentamycin loaded in Availability of data and materials PLGA for killing B. melitensis phagocytosized by macro- The data can be accessible to the interested researchers by the corresponding authors on reasonable request. phages, which had a lower efficacy than DOX-SLN syn- thesized in the present study [26]. Another study Authors’ contributions conducted by Seleem et al. used doxycycline loaded in SMH and MA designed the study. SMH contributed in the experimental studies, and drafting the work. GHR performed the analysis of the data. FJ and SS Nanoplexes for killing B. melitensis in J774A.1 cells. contributed in the cell culture. RA and AF designed the nanoparticles. All They observed no significant difference between the effi- authors read and approved the final manuscript. cacies of the drug loaded in NPs and the corresponding Ethics approval and consent to participate free drug [22]. The results are in contradiction with This study was approved by the ethics committee of Hamadan University of those of the present study, which can be due to the rapid Medical Sciences (No: IRUMSHA. REC. 1395066). release of the drug from the NPs; the release time re- Consent for publication ported by that study was 15 h which is significantly Not applicable lower than 72 h we observed in the current study. There were a number of limitations to our study in- Competing interests The authors declare that they have no competing interests. cluding: studying on B. melitensis was very difficult due to its pathogenic nature. Because we had many tests and Publisher’sNote errors to set up an appropriate nanoparticle, the dur- Springer Nature remains neutral with regard to jurisdictional claims in ation of the project was long. This project needs many published maps and institutional affiliations. equipment, as all of them were not centralized in one Author details place, the work became very hard. Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. Department of Clinical Biochemistry, Faculty of Medicine, Hamadan Conclusions University of Medical Sciences, Shahid fahmideh street, Park Mardome, Thedoubleemulsionmethod issuitabletoencapsulation Hamadan, IR, Iran. Department of Virology, Faculty of Medicine, Hamadan of DOX as a hydrophilic drug so that particle size, zeta University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. Department of Anatomical Sciences, Faculty of Medicine, potential, and PDI were desirable for our objectives. The Hamadan University of Medical Sciences, Shahid fahmideh street, Park time required for the complete release of drug and there- Mardome, Hamadan, IR, Iran. Dental Research Center, School of Dentistry,, fore its antibacterial effectiveness was 72 h. The effects of Hamadan University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. Department of Biostatistics, School of Health, DOX-SLN on the bacteria enclosed in macrophages was Hamadan University of Medical Sciences, Shahid fahmideh street, Park significantly higher than the free DOX. This can be attrib- Mardome, Hamadan, IR, Iran. Brucellosis Research Center, Hamadan uted to the phagocytosis of NPs by macrophages and slow University of Medical Sciences, Shahid fahmideh street, Park Mardome, Hamadan, IR, Iran. continuous release of drug. The application of these NPs can be considered as a promising tool for treating intracel- Received: 17 January 2019 Accepted: 27 February 2019 lular bacteria, particularly B. melitensis and preventing the relapse of the disease. References Suggestions that can help researchers interested in this 1. de Figueiredo P, Ficht TA, Rice-Ficht A, Rossetti CA, Adams LG. Pathogenesis topic: using multiple-drug combination that are routinely and immunobiology of brucellosis: review of Brucella–host interactions. Am J Pathol. 2015;185(6):1505–17. used to treat brucellosis, investigating nanoparticle’s effect 2. Musallam I, Abo-Shehada M, Hegazy Y, Holt H, Guitian F. Systematic review and toxicity in in vivo conditions, utilizing other nanopar- of brucellosis in the Middle East: disease frequency in ruminants and ticles and comparing its effect on B. melitensis with SLN. humans and risk factors for human infection. Epidemiol Infect. 2016;144(4): 671–85. Abbreviations 3. Byndloss MX, Tsolis RM. 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