Antimicrobial resistance is a growing problem [1
]. The emergence of intracellular bacterial infections and acquired resistance of pathogenic microbes pose significant challenges for many antimicrobial agents [2
]. Since the 1980s, flouroquinolones have been used in clinical practice [3
], and they have contributed to major advances in the medical treatment of gram negative bacterial infections as frontline drugs [2
]. Active efflux from prokaryotes as well as eukaryotic cells strongly modulates the activity of this class of antibiotics [4
]. Thus, the intracellular actions of flouroquinolones are often sub-optimal [7
] due to continued efflux [5
]. This contributes to failure of conventional fluoroquinolones therapies as a result of decreased accumulation and poor retention of the antibiotics inside the cells [8
]. Other factors limiting the success and clinical use of fluoroquinolones like ciprofloxacin include their bitter taste in solution, and rapid renal clearance, in which a minimum of 70% of the oral dose is excreted unchanged in the urine. Moreover, frequent administration of ciprofloxacin is associated with numerous side-effects [10
]. In order to achieve successful treatment, antibiotics must fulfill a series of criteria, including the ability to penetrate and be retained by the cell, the capacity to reach the intracellular target, and the display of activity against bacteria residing in the intracellular environment [13
]. On the other hand, due to the deficiency in new antibacterial agents, there is considerable interest in restoring the activity of older and conventional antimicrobials [14
The use of safe and efficient delivery systems, capable of delivering therapeutic agents in an adequate concentration within the appropriate intracellular compartment is an ultimate goal in enhancing therapeutic effect. It is also a promising strategy in overcoming microbial resistance [15
]. The encapsulation of antibiotics in carriers could avoid antibiotic efflux and enhance the drugs’ intracellular retention, since delivery systems like nanoparticles are not substrates of the efflux pump proteins [17
]. Moreover, encapsulation of antibiotics improves their pharmacokinetics by increasing serum half-life [2
]. Nanoparticles can be phagocytose by host phagocytes containing intracellular microbes. Once inside host phagocytes, the antibiotic–nanoparticle delivery system could release high dose of the antibiotic to eliminate the intracellular microbes before developing resistance [18
Many studies have reported the increased antimicrobial activity of ciprofloxacin conjugated nanoparticles [21
]. Likewise, decreased antibiotic resistance and increase antibacterial activity of ciprofloxacin was reported in the presence of Zinc Oxide nanoparticles [24
]. It is anticipated that the use of nanoparticles-based drug delivery systems will continue to improve treatment of bacterial infections and multidrug-resistant microbes [19
]. However, no studies have been conducted on the potential of ciprofloxacin encapsulated cockle shells-calcium carbonate (aragonite) nanoparticles (CSCCAN), to enhance the efficacy of the drug. The cockle shells (Anadara granosa
), which is available in abundance, is often considered a waste and it is a cheap protein source [25
]. Moreover, calcium carbonate has been used for controlled delivery of biomolecules due to it biodegradability, biocompatibility, porous nature and simple bulk-scale preparation [26
]. A porous aragonite calcium carbonate nanoparticles loaded with gentamicin sulfate with controlled released property have been successfully used in osteomyelitis treatment [28
]. Thus, calcium carbonate nanoparticles are expected to also enhance the efficacy of ciprofloxacin.
The continuous assembly of engineered nanoparticles as drug carrier system necessitates a comprehensive understanding of their potential toxicity [29
]. Despite many reports on the toxicity of nanomaterials, the precise association between engineered nanoparticles and the immune system have not been broadly studied [29
]. Macrophages are the key players in the innate immune response that phagocytose large foreign particles or endocytose biological molecules and tiny materials. Furthermore, engulfment of foreign materials renders macrophages more activated to complete the task of instigating and inducing the adaptive immune responses by discharging a variety of pro-inflammatory cytokine [32
]. A critical indicator of nanoparticles toxicity is its ability to provoke an unwanted immune response in a biological system. Additionally, understanding the biological response to nanoparticles at the sub cellular level is crucial and can present further evidence on the interaction between nanomaterials and cells. It is thus essential to understand the immunogenic potential of the CSCCAN with respect to pro-inflammatory protein production.
3. Materials and Methods
3.1. Reagents, Chemicals and Media
Polysorbate-Tween 80 (Thermo Fisher Scientific, Waltham, MA, USA), glyceride (Sigma-Aldrich Co., St. Louis, MO, USA), ciprofloxacin (LKT Laboratories, St. Paul, MN, USA), dulbecco’s modified eagle’s medium (Sigma-Aldrich Co.), foetal bovine serum (Sigma-Aldrich Co.), mueller–hinton agar (Difco Becton Dickinson, Sparks, MD, USA), mueller–hinton broth (Difco Becton Dickinson), ethanol (Joseph Mills Denaturants, Liverpool, UK), phosphate buffer saline (Sigma-Aldrich Co.), dimethylsulfoxide (Sigma-Aldrich Co.), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) dye (Naclai tesque, Inc., Kyoto, Japan), penicillin and streptomycin antibiotics (i-DNA Biotecnology(M) Sdn Bhd, Kuala Lumpur, Malaysia), Access RT-PCR System (Promega Corp., Madison, WI, USA), lipopolysaccharides from Salmonella enterica serotype enteritidis (Sigma-Aldrich Co.), RNA isolation kit (RBC Bioscience Corp., Xiandin Dist., New Taipei, Taiwan), BrdU cell proliferation assay kit (Biovision Inc., Milpitas, CA, USA), agarose LE, analytical grade (Promega Corp.), SYBR Safe DNA gel stain (Invitrogen, Waltham, MA, USA), loading dye and DNA ladder (i-DNA Biotechnology (M) Sdn Bhd).
3.2. Bacterial Strain
Salmonella Typhimurium ATCC 14208 was obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). S. Typhimurium ATCC 14028 were grown and maintained on Tryptic Soy agar (TSA) (Sigma-Aldrich Co.).
3.3. Top-Down Synthesis of Cockle Shells Calcium Carbonate Nanoparticles
The synthesis of the nanoparticles was achieved via oil-in-water (O/W) microemulsions using a high pressure homogenizer (HPH) [71
]. The microemulsion samples were prepared by mixing the surfactant and co-surfactant (Tween-80 and glycerol) into 20 mL oil in a glass reactor followed by addition of the 65 mL deionized water. The mixed solutions were stirred for 30 min until they became transparent. The nanoparticles were first prepared by suspending 2 gram of dry cockle shells-calcium carbonate powder (CSCCP) into the formulated oil-in-water (O/W) microemulsion, and moderately stirred for 10 min at 1000 rpm to form a cockle shells calcium carbonate suspension. The formulated suspension was sucked through the fluid opening tube of HPH for pre-milling at low pressures of 200 and 500 bars for three cycles each. After pre-milling, the suspension was collected at the HPH outlet and was again passed through the HPH fluid inlet at a high pressure of 1500 bars for 25 homogenizing cycles to get the desired fine particles. The final particles suspension was filtered and oven dried at 95 °C for 24 h [26
3.4. Drug Loading and Encapsulation
An optimized aqueous solution of ciprofloxacin (3 mg/mL) was added into the CSCCAN suspension (50 mg/mL). The formulation was mechanically agitated overnight at 200 rpm at room temperature using a laboratory multi-hotplate stirrer (Witeg, Wise Stir SMHS, Witeg Labortechnik GmbH, Wertheim, Germany). Then it was centrifuged at 15,000 rpm for 15 min. The ciprofloxacin–cockle shell-derived calcium carbonate (aragonite) nanoparticles (C-CSCCAN) were freeze-dried [50
The ciprofloxacin loading and encapsulation efficiency were analyzed by calculating the difference between the total drug (Wt
) and the free encapsulated drug (Wf
) in the nanoparticles supernatant per nanoparticles weight. The residual quantity of free encapsulated ciprofloxacin remaining in the supernatant was determined by computing the optical density at 291 nm [73
], on a micro-titer plate reader (TECAN Safire, Tecan Austria GmbH, Grödig, Austria) [74
]. A calibration curve of standard ciprofloxacin solution was used to obtain ciprofloxacin concentration (R2
= 0.9823, Y
+ 1.4894). Data were given as an average measurement of three independent values. Drug loading contents (LC%) and encapsulation efficiency (EE%) of the nanoparticle were calculated as previously described [75
= the total weight of drug used, Wf
= the weight of non-encapsulated free drug, and Wnp = the weight of the nanoparticle.
= the total weight of drug, and Wf
= the weight of non-encapsulated free drug.
3.5. Transmission Electron Microscopy
The shape and particle size of CSCCAN and C-CSCCAN were analyzed using Transmission Electron Microscopy (TEM) (Hitachi H-7100, Tokyo, Japan). Samples were mixed with 95% alcohol then sonicated for 45 min. The colloidal drop from each sample were loaded on carbon coated copper grids, placed on a filter paper, and dried at room temperature for 1 h. TEM measurements were carried out at 150 kilovolts [76
3.6. Field Emission Scanning Electron Microscope
The surface morphology of nude CSCCAN and C-CSCCAN were observed with Field Emission Scanning Electron Microscope (FESEM) (Model 100, Perkin Elmer, 710 Bridge port Avenue, Shelton, CT, USA), equipped with an energy-dispersive X-ray spectroscopy. The samples were individually prepared on aluminum stubs and coated with gold under argon atmosphere using a sputter coater. The FESEM observations were performed at 200 kilovolts.
3.7. ζ Potential
The particle surface charge was measured using Malvern Zetasizer Nano (Malvern Instruments, Worcestershire, UK), which measures the electrophoretic mobility of particles in an electrical field, which is then converted into ζ potential. The measurement was performed by injecting the samples into the cells of the zetasizer and run at room temperature. The process was done three times and the average was taken to determine the ζ potentials.
3.8. X-ray Powder Diffraction
The purity and crystalline properties of the CSCCAN, C-CSCCAN and free ciprofloxacin powders were investigated using X-ray Powder Diffraction (XRD) (Shimadzu XRD-6000, Yokohama, Kanagawa, Japan). Cross-section of the samples was taken and placed on a quartz plate for exposure to Cu Kα radiation of wavelength λ = 1.5406 Å. The samples were then examined at room temperature over a 2θ range of 4°–50°, with sampling intervals of 0.02° 2θ and a scanning rate of 0.6°/min.
3.9. In Vitro Biocompatibility Assays
3.9.1. Cell Culture
Macrophage J774A.1 cells were purchased from the American Type Culture Collection (ATCC). They were maintained as semi-adherent cell cultures at 37 °C in a humidified atmosphere (5% CO2) in Dulbecco modified Eagle’s minimal essential medium (DMEM, 25 mM glucose) supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 100 µg/mL each of penicillin and streptomycin.
3.9.2. RNA Extraction and RT-PCR
Macrophages J774A.1 cells were seeded at a density of 5 × 105
cells/well in 2 mL medium of 6-wells culture plates and grown overnight. The cells were then washed with PBS and treated with different concentrations of CSCCAN in culture medium suspension (100–3.125 µg/mL) for 3 h. Bacterial lipopolysacchride treated cells served as a positive control, and untreated cells served as a standard control for RT-PCR analysis. After exposure to nanoparticles, cells were collected, extracted and analyzed for mRNA expression of IL-1β cytokine. The pro-inflammatory cytokine, IL-1β, was evaluated and β-actin was used as the control. The total RNA was extracted using the RBC Bioscience Total RNA Isolation kit, according to the manufacturer’s instructions. RNA Quantification and purity was determined using NanoDrop Spectrophotometer. Primer sequences were designed on the NCBI website with accession number: NM_008361.3 and product size: 645 for IL-1β and β-actin with accession number: NM_013458.5 and product size: 470. The primers (Table 2
) were supplied by (First Base Laboratories Sdn Bhd, Kuala Lumpur, Malaysia). Reverse transcription-PCR were performed according to the Access Reverse Transcriptase System Protocol in a sensquest labcycler under the following conditions: 45 °C for 45 min, 94 °C for 2 min, 94 °C for 30 s, 65 °C for 1 min, 72 °C for 2 min, and 68 °C for 7 min for a total of 40 cycles. The same cycling conditions were used for β-actin with the exception of annealing temperature (59 °C for 1 min). The PCR amplification products were analyzed by gel electrophoresis on a 0.1% agarose gel, stained with SYBR Safe DNA Gel Stain, and fluoresce with ultraviolet source.
3.9.3. MTT—Viability Assay
For cytocompatibility study, macrophages J774A.1 were split by mechanical scraping and cell suspensions were seeded in 96-well micro-titer plate at a density of 10,000 cells/well and incubated for 24 h. During each experiment, the media was removed and the cells were cultured with 100 µL of different concentrations of C-CSCCAN, CSCCAN and ciprofloxacin in culture medium (100 to 6.25 µg/mL), or of the control (culture medium only). The experiment was conducted in triplicates, and the optical densities were measured at 570 nm in a micro-titer plate reader [50
]. The cell viability was computed using the following equation:
Cell Viability (%) = Atest/Acontrol × 100
is the optical density of the cells incubated with the different treatments and Acontrol
is the optical density of the cells incubated with the culture medium only (negative control). The cytotoxicity was determined from the average of three replicate tests and results were expressed as mean ± standard deviation.
3.9.4. BrdU (ELISA) Genotoxicity Assay
The cell genotoxicity and quantitative determination of DNA synthesis in cells was evaluated based on the incorporation of BrdU into the synthesized DNA of a proliferating cell [67
]. Macrophages J774A.1 cells were seeded into 96-well micro-titer plate at density at a density of 1 × 104
cells per well and incubated for 24 h. The cells were then washed with PBS and treated with different concentrations of C-CSCCAN, CSCCAN and ciprofloxacin in culture medium suspension (100–3.125 µg/mL). Cells incubated with culture medium only (untreated cells) were regarded as negative control. After the cell incubation for 24 h, the medium was removed and the BrdU labeling assay was performed according to the manufacturer’s instructions (Bio Vision Incorporated, Milpitas, CA, USA). The color intensity absorbance directly correlated to the amount of synthesized DNA, which also reflects the number of proliferating cells. The BrdU incorporation was determined by analyzing cells treated compared to controls and the absorbance was measured at 450 nm using a micro-titer plate reader [67
]. The data were determined from the average of three replicate tests and results were expressed as mean ± standard deviation.
3.10. In Vitro Antibacterial Susceptibility Test
The bacterial stock solution were diluted according to the method in Clinical and Laboratory Standards Institute (CLSI) [77
3.10.1. Preparation of Drugs Stock Solutions
A stock solution of C-CSCCAN suspensions and ciprofloxacin dispersion (pH 7.4) at concentration of 1 mg/mL was prepared in 10% DMSO.
3.10.2. Disc Diffusion Susceptibility Assay
Susceptibility of C-CSCCAN and ciprofloxacin against S.
Typhimurium were determined by disc diffusion method described previously [78
], with little modifications. This method was performed in Muller Hinton agar media. A single colony of S.
Typhimurium was grown overnight in MHB on a rotary shaker (250 rpm) at 35 °C. Inoculums were prepared by diluting the overnight cultures to a 106
colony-forming units/mL (CFU/mL) suspension according to the turbidity of 0.5 McFarland standards. Then, 100 µL bacterial suspensions were inoculated to the prepared MHA plates and spread all over in one direction. Sterile paper disc (6 mm in diameter) was placed on the MHA plates and impregnated with 10 µL of C-CSCCAN formulation and CSCCAN (100 µg/mL). Ciprofloxacin (100 µg/mL) and DMSO was correspondingly prepared and served as a positive and negative control respectively. After 37 °C incubation for 24 h, the diameters of inhibition zone were measured in millimeters. The data obtained from three replicate tests were expressed as mean ± SD.
3.11. Statistical Analysis
All statistical analyses were performed using Minitab statistical software (Minitab Inc., State College, PA, USA). All experiments were performed in triplicate. Values were expressed as mean ± standard deviation. Comparisons of treatment effects and statistical significant differences between groups were determined using one-way analysis of variance (ANOVA) (for MTT and BrdU Assay) and Student’s independence t-test (for disc diffusion assay). A value of p < 0.05 was regarded significant unless indicated otherwise.