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Article Cite This: ACS Omega 2019, 4, 7524−7532
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Controllably Biodegradable Hydroxyapatite Nanostructures for Cefazolin Delivery against Antibacterial Resistance Muhammad Usman Munir,*,†,‡ Ayesha Ihsan,*,‡ Ibrahim Javed,§ M. Tayyab Ansari,∥ Sadia Z. Bajwa,‡ Syed Nasir Abbas Bukhari,† Arsalan Ahmed,⊥ M. Zubair Malik,# and Waheed S. Khan*,‡ †
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Aljouf 72388, Saudi Arabia National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, Faisalabad 38000, Pakistan § ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia ∥ Faculty of Pharmacy, Bahauddin Zakariya University, Multan 60000, Pakistan ⊥ Interdisciplinary Research Centre in Biomedical Materials, COMSATS Institute of Information Technology, Lahore 54000, Pakistan # Department of Pharmaceutics, College of Pharmacy, University of Sargodha, Sargodha 40100, Pakistan
ACS Omega 2019.4:7524-7532. Downloaded from pubs.acs.org by 95.181.176.125 on 04/24/19. For personal use only.
‡
ABSTRACT: Multidrug resistance (MDR) is a global threat posed by continuously evolving microbial resistance against currently available antimicrobial agents. In this study, we synthesized hydroxyapatite-based porous nanocarriers with pH-dependent biodegradation, using cefazolin (CFZ) as cargo drug against MDR E. coli, S. aureus, and P. aeruginosa. Oval-shaped porous hydroxyapatite nanoparticles (opHANPs) were synthesized via core−shell method. Field emission scanning electron microscopy revealed that the average length and width of opHANPs were found to be ∼90 and ∼110 nm, respectively with monodispersed size and morphology. The encapsulation efficiency (EE) of CFZ was observed to be dependent on the initial concentration of the drug (EE, 41.37−92.40% with 300−2000 μg/mL of CFZ). Brunauer−Emmett− Teller specific surface area and pore width of opHANPs were 166.73 m2/g and 3.3 nm, respectively, indicating hierarchal pore distribution. The pH-responsive drug release was observed from CFZ-loaded opHANPs (CFZ@opHANPs). An enhanced drug-releasing behavior was observed at lower pH (4.5, 2.5, and 1.5). The study of release kinetics revealed that at pH 7.4, drug release is due to anomalous diffusion, while at lower pH, the drug release followed fickian diffusion model. Cytotoxic and hemolytic studies showed biocompatibility of CFZ@opHANPs with HepG2 and red blood cells. The growth kinetic study and colony-forming unit assay showed the superior antibacterial potential of CFZ@opHANPs, in contrast to carrier or CFZ alone, against MDR E. coli, S. aureus, and P. aeruginosa strains.
1. INTRODUCTION Bone and joint infections (BJIs) and degenerative disorders like osteoarthritis, osteomyelitis, rheumatoid arthritis, bone injury, and spinal problems cost around 250$ billion worth of treatment every year and influence countless individuals over the world.1 Bacterial strains responsible for BJIs include MDR methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (P. aeruginosa), and extended spectrum β lactamase (ESBL) positive Escherichia coli.2,3 Irrational use of antibiotics has led to the evolution of microbial pathogens to develop resistance against currently marketed antibacterial agents.4−7 In addition, BJIs need long-term treatment with antibiotics that may lead to severe gastrointestinal (GI) side effects and poor patient compliance.8 Recent literature reports the emergence of superbugs, resistant against even the most advanced antibacterial agents available, i.e., carbapenems that are usually reserved for hard-to-treat microbial infections.9 It grows a global concern for MDR. © 2019 American Chemical Society
Multiple classes of antibiotics including cephalosporins have been reported inefficient to cure the MDR bacterial infections. ESBLs are the most dominant and common strains isolated from the patients infected with MDR bacteria.10 Almost 80% of the E. coli strains are ESBL-positive.11 Cefazolin (CFZ) is a first-generation broad spectrum cephalosporin effective against Gram-positive microbes like Staphylococcus aureus, Staphylococcus epidermidis, and streptococci, but face resistance against MRSA and Gram-negative bacteria like P. aeruginosa and E. coli.12,13 The route of administration of CFZ is parenteral as its absorption from GI tract is insufficient. Moreover, it exhibits poor pharmacokinetic properties, i.e., short half-life (1.8 and 2 h after IV and IM Received: February 26, 2019 Accepted: April 16, 2019 Published: April 24, 2019 7524
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Figure 1. Schematic illustration of synthesis of sole and cefazolin-loaded opHANPs.
Figure 2. SEM images at (A) 8500× and (B) 30 000 ×. (C) Length and width of opHANPs and (D) XRD pattern of opHANPs.
2. RESULTS AND DISCUSSION ESBL-generating microbes are posing the threat of resistance from last century, and thus carbapenems were turned into the drugs of choice for medical microbiologists. Due to irrational use of drugs, carbapenem-resistant enterobacteriaceae (CRE) has emerged and resisted all of the accessible treatments.19 Similarly, P. aeruginosa was impervious to all tried antiinfection agents, while E. coli was found to be ESBL-positive.20 All of this emergence of MDR cases requires either new therapeutic strategy or drug-delivery systems to prevent and combat the drug resistance in bacteria. 2.1. Morphology, Size, and Phase Purity. The ovalshaped porous HANPs (opHANPs) were prepared by the core−shell method (Figure 1) and were characterized for morphology, size, phase purity, porosity with surface area, and drug−material interaction. The morphology of opHANPs was studied by scanning electron microscopy (SEM). Morphology of the NPs appeared to be oval and porous. Figure 2A,B presents the SEM images at low and high magnification. It can
administration, respectively), low bioavailability, and high protein binding around 74−86%. This study presents the design of porous hydroxyapatite nanoparticles (HANPs) with CFZ as loaded drug cargo against MDR bacteria. HA materials have demonstrated the ability of carrying drug cargo, sustained drug release, and targeted delivery.14,15 Compositional and structural resemblance to bone and tailorable biodegradability of HA are the key factors that can make them useful in design of targeted drug carriers against arthritic, dental, and orthopedic infections.16−18 In BJIs, the pH of bone tissues is dropped to acidic pH from normal pH (7.45) of bone matrix attributable to bacterial strike. We fine-tuned the CFZ loading and pH-controlled release from HANPs. We further observed biocompatibility and pharmacological potential of CFZ with HANPs against MRSA, E. coli, and P. aeruginosa. 7525
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Figure 3. (A) Nitrogen physisorption isotherms, BJH pore size distribution (B) adsorption (C) desorption, (D) FTIR spectra of (i) opHANPs, (ii) CFZ, and (iii) CFZ@opHANPs.
delayed desorption curve in the isotherm has shown that the pores are organized and consistent.28,29 It can be suggested that these interconnected pores have played a vital role in sustained and delayed release of the drugs. FTIR was used to study the chemical integrity of the drug after loading into opHANPs. We observed the distinctive peaks of hydroxyapatite as stretching and bending vibrations of phosphate groups and hydroxyl groups, as shown in Figure 3D.30 These peaks were intact in the empty and CFZ@ opHANPs. The prominent spectral peaks of CFZ were observed at 3418 cm−1 (stretching bands of −NH), at 3228 cm−1 bands (stretching of =CH), at 1760 cm−1 (stretching of carboxylate), and at 1670 cm−1 (stretching of C = O of amide). The bands at 1240, 1185, and 1064 cm−1 represent the −CN stretching, while the band at 1540 cm−1 is due to the stretching of secondary amide −NH.31 The characteristics peaks of CFZ were intact in CFZ@opHANPs, presenting chemical stability of CFZ inside opHANPs. The textural parameters of N2 sorption analysis are specified in Table 1.
be seen that opHANPs have pores with interrelated network that can be responsible for high drug loading. The opHANPs have average length and width of 90 and 110 nm, respectively, calculated by ImageJ software with oval profile plugin as shown in Figure 2C. Phase purity of opHANPs was studied with powder X-ray diffraction (PXRD) (Figure 2D), and representative peaks of hydroxyapatite material were observed at 30−35°.21 The strong diffraction peaks at 25.78, 29.46, 31.69, 32.82, 33.25, and 33.79° correspond to (002), (102), (210), (211), (112), and (300) planes of hydroxyapatite particles (HAP), respectively.22 These diffraction peaks matched with the standard data of HAP (JCPDS file: 090432). By using the Scherrer formula, the average crystallite size of opHANPs was estimated to be ∼55 nm.23 2.2. Porosity and FTIR Pattern. The porous properties including specific surface area (SSA), pore size, and pore volume were determined by N2 adsorption/desorption isotherms (Figure 3A). According to the IUPAC nomenclature, opHANPs have revealed type IV isotherm with H3 hysteresis loop.24 Brunauer−Emmett−Teller (BET) SSA of opHANPs was found to be 166.73 m2/g with pore width of 3.3 nm, indicating tapered pore size distribution. In the type IV isotherm, the mesoporous walls of nanoparticles followed the same path during initial adsorption as the subsequent part of a type-II isotherm that represented pore condensation. The term pore condensation denotes that the gas is condensed in a pore to liquid-like phase and this occurs at a pressure “p” less than the saturation pressure po of the bulk liquid.25 We observed that opHANPs had higher SSA than previously reported hydroxyapatite materials.26,27 Furthermore, the larger SSA of opHANPs may be due to smaller crystallite size of the nanoparticles, as suggested by XRD. The extensive and broader hysteresis loop indicates the presence of bottle-necked pores. This was also supported by the Barrett−Joyner−Halenda (BJH) adsorption and desorption dv/dw pore volume as shown in Figure 3B,C. It indicates a pore diameter of 9.8 nm during adsorption while a pore width of 3.2 nm during desorption, resulting in delayed desorption. Additionally, the
Table 1. N2 Adsorption/Desorption Parameters BET surface area (m2/g)
BJH adsorption pore diameter (nm)
BJH desorption pore diameter (nm)
cumulative surface area of pores (m2/g)
pore volume (cm3/g)
166.73
9.8
3.2
256.6
0.555
2.3. Encapsulation Efficiency. To determine the influence of initial drug concentration on encapsulation efficiency (EE), different concentrations of CFZ were loaded in NPs. We observed that EE increased as the amount of drug was increased. EE (%) was determined to be 41.37% ± 2.78, 53.26% ± 3.24, 79.85% ± 2.91, and 92.40% ± 1.85 for drug concentrations of 300, 500, 1000, and 2000 μg/mL, respectively. This enhanced encapsulation of drug in NPs can be related to the porous structure of opHANPs. Drug EE 7526
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Figure 4. (A) CFZ release from CFZ@opHANPs at different pH values; (B) hemolytic analysis.
Figure 5. Cytotoxic assay: (A) Untreated HepG2 cell line, (B) blank opHANPs, (C) CFZ@opHANPs. Cell viability assays of (D) controls and (E) CFZ@opHANPs at different concentrations.
(%) for CFZ@opHANPs was better compared to previously reported hydroxyapatite nanoparticles.32−34 2.4. In Vitro Drug Release. Figure 4A presents the percent cumulative release of CFZ from CFZ@opHANPs at different pH values. The pH of bone tissues (7.45) falls to acidic pH due to microbial attack. The release of drug from opHANPs was dependent on pH. It was studied by placing the CFZ@opHANPs in the dissolution mediums of pH values 7.4, 4.5, 2.5, and 1.5. At pH 7.4, there was sustained release of the drug compared to the drug release at pH values 4.5, 2.5, and 1.5. High drug release at lower pH can be explained due to acid labile structure of hydroxyapatite. It was proposed that the acid delicate stuff of opHANPs will benefit drug release at lower pH, comparable to pH of infected bone tissues, and combating MDR in bacteria. Kinetics of drug release revealed that opHANPs have followed the Higuchi model, i.e., diffusioncontrolled and zero-order kinetics at pH 7.4. Furthermore, release exponent (n) for CFZ@opHANPs was following anomalous diffusion as governed by the Korsmeyer−Pappas model. Conversely, the drug release from opHANPs had followed fickian diffusion at lower pH values of 4.5, 2.5, and 1.5 based on Korsmeyer−Pappas model. 2.5. In Vitro Hemolytic Analysis. Red blood cells (RBCs) and plasma proteins are the first physiological components that drug NPs face upon in vivo administration. The hemolytic interaction of opHANPs was studied by incubating them with RBCs. The results (Figure 4B) presented no hemolysis of the RBCs that were exposed to the empty or CFZ-loaded
opHANPs. The hemolysis ratio (Z) less than 2 is considered as nonhemolytic according to ASTM 756-00 standards.35 The hemcompatibility of opHANPs primarily contributed to their resemblance with the native bone composition and structure.36 Additionally, the lower density of surface-charged sites on opHANPs prevents hemolytic interaction with the RBCs membrane.37 Highly protein bound drugs have obstacle to deliver desired therapeutic action at the target site. The plasma proteins also have the capability to bind with NPs by opsonization, i.e., the drug carrier removal from the body by reticuloendothelial system.38 A study reported that compared to hydrophilic drug carriers including hydroxyapatite materials, quick opsonization is seen in hydrophobic carriers due to greater adsorption ability of blood proteins on such materials surface.39,40 By considering this point, the hydrophilic nature of opHANPs will hinder the protein binding to its surface. Accordingly, we assumed that CFZ protein binding will also be reduced in the form of CFZ@ opHANPs. 2.6. Cytotoxic Study. The biocompatibility of prepared formulations was evaluated by cytotoxic screening on human liver cells (HepG2). The optical images of HepG2 cells treated with opHANPs and CFZ@opHANPs are shown in Figure 5A−C, representing that no morphological change regarding necrotic injury was observed. Our experimental results shown in Figure 5D were in agreement with Sun et al., who reported 80% cells viability after treating HepG2 cells with hydroxyapatite materials.41 Figure 5E presents the cytotoxicity in a 7527
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Figure 6. Kinetics of bacterial growth: (A) MRSA, (B) E. coli, and (C) P. aeruginosa. (D) Colony-forming unit assay of CFZ@opHANPs against MDR SA, E. coli, and P. aeruginosa. (E) CFU time profile for bacterial killing. The sole media and media with inoculum are negative and positive controls, respectively.
the surface of HA materials and positioned on the crystal faces (ac or bc). The “C” site is substantial in calcium ions and involves binding to the acidic groups or negatively charged surfaces. Thus, opHANPs have excellent adhesion and adsorption for the negatively charged surfaces.45 It is suggested that there are electrostatic interactions between positively charged binding site on CFZ@opHANPs surface and negatively charged surface of the bacteria.46 Gottenbos et al. reported the declined growth of bacteria on the materials surface with better adhesion strength.47 Such adsorptive interaction of CFZ@opHANPs leads to disruption of bacterial cell wall. Empty opHANPs failed to wipe out the microbes (Figure 6). We used broth dilution and CFU assays to determine antibacterial activities of CFZ@opHANPs. The growth kinetics of MRSA, E. coli, and P. aeruginosa can be seen in Figure 6A−C. Figure 6D,E presents the results of colonyforming unit (CFU) assay and the time needed to kill the bacteria. It was seen that, with CFZ@opHANPs, the lower concentrations of CFZ were effective in inhibiting the bacterial growth in broth dilution assay. Positive control (without NPs or drug) and empty opHANPs have shown bacterial growth up to 4 × 106 and 4 × 105 CFU, respectively, supporting that empty opHANPs are only acting as drug carrier and have no native antibacterial activity. It is notable that as the concentration of the drug increased, the number of bacterial
low dose-dependent manner. The IC50 for CFZ@opHANPs against human liver cells was found to be 57.5 μg/mL. 2.7. MIC Determination by Agar Well Diffusion Method. Different dilutions of CFZ were initially screened for the minimum inhibitory concentration (MIC) via testing against MRSA, E. coli, and P. aeruginosa. MRSA was found to be moderately resistant (MIC; 6000 μg/mL), while E. coli and P. aeruginosa were found to be highly resistant (MIC: 25 000 μg/mL) that was already beyond the recommended dose.42 We performed this step to validate the resistance pattern of bacterial strains. 2.8. MIC Determination by Broth Dilution Method. The broth dilution method was used to further confirm the MIC of CFZ. In the case of MRSA, it was 1000 μg/mL, but for E. coli and P. aeruginosa, no clear solution was observed, indicating high resistance. MIC experiments from broth dilution and agar well diffusion methods confirmed the resistance of used strains against CFZ. In our study, CFZ@ opHANPs suppressed the growth of bacteria in selective mode with MIC ranging from 0.012 to 0.1135, 0.08 to 0.215, and 0.04 to 0.175 μg/mL for MRSA, E. coli, and P. aeruginosa, respectively. These results were worthy compared with the previous reports.43,44 2.9. Antibacterial Activity. The final objective of this study was to combat ESBL producing MRSA, E. coli, and P. aeruginosa, with CFZ@opHANPs. There is “C” binding site on 7528
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colonies decreased.48,49 Our findings suggest that opHANPs can be used as prospective drug-delivery carrier system against antimicrobial-resistant strains because of efficient antibiotic delivery and biocompatibility.
opHANPs, and CFZ3@opHANPs, respectively. The mixtures were stirred for 3 h. After that CFZ-loaded opHANPs were collected as pellet by centrifugation at 15 000 rpm for 20 min. The physical adsorption of CFZ on the CFZ-loaded nanoparticles was decreased by washing the white precipitates with deionized water and then drying in a vacuum oven. 4.3. Estimation of Minimum Inhibitory Concentration (MIC) of CFZ. Agar well diffusion and broth dilution methods were used to estimate the MIC of CFZ against MRSA, E. coli, and P. aeruginosa. The initial concentration of MRSA, E. coli, and P. aeruginosa used for antibacterial study was 105 CFU/ mL, which was measured by the plate-count method. It had increased to 109, 1010, and 1010 CFU/mL for MRSA, E. coli, and P. aeruginosa, respectively, over 48 h incubation at 37 °C. A stock solution of CFZ (20 mg/mL) was prepared in water and used to make further dilutions. Different dilutions from the stock were rushed in the wells (40 μL) of agar plates that were inoculated with culture. The plates were incubated at 37 °C overnight and zone of inhibition was measured to calculate MIC. In the second method, bacterial cultures were grown in the nutrient broth up to the exponential growth stage. Macfarland solution was used as standard to match the turbidity. The microbial suspension (10 μL) from saturated growth solution was inoculated in broth (9 mL), and different concentrations of CFZ were included in the broth. The tubes were incubated at 37 °C overnight in a shaker incubator. The lowest dilution giving no notable growth of bacteria was considered as MIC. The positive and negative controls of bacterial culture and media without inoculum were additionally performed. 4.4. Characterization. Field emission scanning electron microscopy (JSM-7500F, Jeol, Japan) was used to examine the morphology of opHANPs. For SEM imaging, the samples were prepared by placing a drop of sample on a wafer of silicon. Porosity and SSA were calculated by N2 adsorption/desorption analysis (Micromeritics ASAP 2020). Subsequent degassing estimations were obtained by N2 adsorption/desorption at 77 K. SSA was calculated with the help of BET using adsorption information at a relative pressure (p/p°) range of 0−0.95. The BJH model was applied to estimate the pore size and volume distribution from desorption isotherm. The drug−particle interaction was studied by FTIR spectroscopy (Bruker-α). FTIR scan of sole and drug-encapsulated opHANPs was recorded (4000−500 cm−1). X-ray diffraction (XRD) tops were recorded at room temperature utilizing a D8 Advance diffractometer (Bruker) with Cu Kα radiation (λ = 0.1540 nm) over the scanning interim (2θ) range of 10−60°. 4.5. Encapsulation Efficiency Calculations. Indirect method was used to calculate the encapsulation efficiency of CFZ-loaded opHANPs. CFZ@opHANPs were centrifuged at 15 000 rpm for 15 min, and the supernatant was collected and quantified for unentrapped drug via a UV−vis spectrophotometer at λmax (298 nm). The encapsulation efficiency (%) of CFZ@opHANPs was estimated by the following equation. These experiments were performed in triplicate.
3. CONCLUSIONS In conclusion, oval-shaped porous HANPs were synthesized by core−shell method with porous morphology. High drug loading was seen due to increased surface area and porosity. These opHANPs were found to be a potential candidate for MDR strains of MRSA, E. coli, and P. aeruginosa with good biocompatibility and nontoxic skills. The ability to release the drug in pH-responsive fashion makes opHANPs for superlative use in BJIs. In BJIs, the pH of bone tissues is dropped from normal pH (7.45) of bone matrix to acidic pH attributable to bacterial strike. The degradation of opHANPs in acidic environment assists the CFZ release to destroy the bacteria from infected bone fraction. Compared to controls, the CFZ@ opHANPs showed many folds better antibacterial activities, due to the small size and the cationic nature of opHANPs. The action mechanism for CFZ@opHANPs to kill the bacteria was supposed to be membrane disruption. In summary, this study proposed inorganic opHANPs as effective nanomaterial to combat MDR in bacteria and future possibility to deliver therapeutic cargo to infections at bone or cartilaginous tissues. 4. MATERIALS AND METHODS Calcium acetate hydrate, ethylene glycol, ethanol, phosphoric acid, and calcium nitrate tetra hydrate were procured from Acros Organics. Ammonium hydroxide solution was purchased from Merck, Germany. Diammonium hydrogen phosphate was bought from Scharlau Chemie, Spain. Muller Hinton agar media and sodium bicarbonate were obtained from Merck, Germany, and Fisher Scientific Company, New Jersey, respectively. These materials were of analytical grade. Cefazolin was obtained as gift sample from local source. The clinical bacterial cultures of MRSA, E. coli, and P. aeruginosa were obtained from National Institute for Biotechnology and Genetic Engineering Faisalabad, Pakistan. 4.1. Synthesis of Oval-Shaped Porous Hydroxyapatite Nanoparticles. Oval-shaped porous HANPs were synthesized based on core−shell strategy by modifying the reported method.50 First, solutions 1 and 2 were prepared by adding ethylene glycol (EG, 10 mL) in equal quantity to calcium acetate (0.3 M) and sodium bicarbonate (0.5 M), respectively. These two solutions were stirred for 3 h and the clear solution gradually became turbid, which was a sign of the development of calcium carbonate (CaCO3) nanoparticles. Phosphoric acid (H3PO4, 0.025 M, 1.25 mL) solution was added dropwise into the solution of CaCO3 nanoparticles. After 6 h of stirring, the nanocomposites were separated through centrifugation at 12 000 rpm for 8 min and washed three times with deionized water. The as-prepared composite was treated with acetic acid solution (35 mM, 10 mL) for the evacuation of carbonate core and to get hydroxyapatite with a porous shell structure in the form of opHANPs. The settled opHANPs were washed with water to remove acetic acid residues and dried in vacuum at 60 °C overnight. 4.2. Preparation of CFZ-Loaded opHANPs. CFZ was dissolved in aqueous solution at desired concentrations (10, 20, and 40 mM). To these solutions, 10 mg of opHANPs was added separately and named CFZ1@opHANPs, CFZ2@
EE(%) =
total drug − unentrapped drug × 100 total drug
4.6. In Vitro Release of the Drug and Dissolution Kinetics. Dialysis tube technique was used to study the in vitro drug release from CFZ@opHANPs at 37 ± 0.5 °C and 50 rpm. The drug release was studied at different pH values to 7529
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ately mixed with 1% solution of CFZ@opHANPs, and then incubated for 45 min at 37 °C. Negative control (unexposed samples), positive control (1% SDS), and vehicle control (distilled water) were used as test controls. The incubated blood samples were centrifuged at 5000 rpm for 5 min, and the absorbance of supernatant was observed at 540 nm using NanoDrop (Thermo Scientific).60 Comparative to the untreated cells, percent hemolysis was calculated. 4.9. Statistical Analysis. All of the experiments were performed in triplicates, and the results are reported as mean ± standard deviation (SD). To compare the results of antibacterial activity and cytotoxicity assay, ANOVA and Student’s t-test were applied to calculate significance (p) level, while the confidence interval was 95%.
determine the pattern of release profile of the drug. Four types of phosphate buffers with pH values 7.4, 4.5, 2.5, and 1.5 were prepared and used as dissolution medium. The sample was drawn at specified time intervals, and sink conditions were maintained by replacing with fresh medium. The absorbance of these samples was done at 298 nm, and the obtained values were put in standard curve equation to get cumulative drug release profile. The dissolution profiles of CFZ@opHANPs were further studied with the model-dependent and model-independent tactics.51 Model-dependent study included zero-order, firstorder, Higuchi, and Korsmeyer−Pappas models, while modelindependent analysis included estimation of f1 (difference factor) and f 2 (similarity factor). DDSolver software (extension of MS Excel) was used for model analysis. The factors f1 and f 2 calculate percent error and percent similarity between two curves. Typically, if the value of f1 is 50, then it certifies the similarity in two data sets of drug release.52 4.7. Antibacterial Activity of CFZ@opHANPs. Antimicrobial activity of empty as well as CFZ@opHANPs was estimated according to the standard broth microdilution method, agar well diffusion, and CFU assays. Specified bacteria were grown in the broth nutrient medium until phase of exponential growth is achieved. The bacterial turbidity was determined by measuring the optical density (OD) of bacterial suspension and matched with standard McFarland solution. OD quantification and measurement of bacterial concentration are synonymous to each other.53 The bacterial culture (10 μL) was mixed with broth (9 mL), and different concentrations of CFZ in the context of CFZ@opHANPs were mixed with inoculated broth. These tubes were incubated at 37 °C overnight in a shaking incubator. After every 24 h for 120 h, OD of the bacterial density was determined at 595 nm via an Elisa multiplate reader.54−56 Nutrient broth alone and cultureinoculated broth were used as negative and positive controls, respectively. CFU assay was performed to further affirm the outcomes. Inoculated broth with different concentrations of CFZ@opHANPs was streaked on nutrient agar plate, and CFU was calculated after 48 h of incubation. 4.8. Cytotoxicity Studies. This study was conducted to assess the safety of opHANPs, using HepG2 (human liver cancer, ATCC HB-8065TM) cell lines.57 The cells were cultured and retained in DMEM having 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 mg/mL streptomycin, and 100 U/mL penicillin. The incubator was maintained at 37 °C and CO2 (5%). SRB (sulforhodamine B) assay was used to estimate the cytotoxicity of CFZ@ opHANPs.58 Briefly, the cells were seeded at the confluency of 10 000 cells/well and exposed to drug-loaded opHANPs for 24 h. Untreated cultures were used as negative control. After incubation, 50% trichloroacetic acid (TCA) was used to fix the cultures and further incubated for 1 h at 4 °C. All of the samples were washed with deionized water and dried in air. SRB solution (0.4%) was used to stain the cells for 30 min, washed with 1% acetic acid, and dried whole night. Olympus IMT-2 inverted stereomicroscope armed with digital camera was used to take photographs of dried samples. For the evaluation of cell viability assay, the method reported by Sun et al. was used.59 4.8.1. In Vitro Hemolysis Study. Blood was collected from healthy individuals with informed consent, diluted appropri-
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (M.U.M.). *E-mail:
[email protected] (A.I.). *E-mail:
[email protected] (W.S.K.). ORCID
Muhammad Usman Munir: 0000-0003-0740-6081 Ibrahim Javed: 0000-0003-1101-5614 Sadia Z. Bajwa: 0000-0002-4755-0267 Funding
The funding for this work was provided by internal department. Notes
The authors declare no competing financial interest.
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REFERENCES
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