Article pubs.acs.org/Langmuir
Polyacrylic Acid-Coated Iron Oxide Nanoparticles for Targeting Drug Resistance in Mycobacteria Priyanka Padwal, Rajdip Bandyopadhyaya,* and Sarika Mehra* Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India S Supporting Information *
ABSTRACT: The emergence of drug resistance is a major problem faced in current tuberculosis (TB) therapy, representing a global health concern. Mycobacterium is naturally resistant to most drugs due to export of the latter outside bacterial cells by active efflux pumps, resulting in a low intracellular drug concentration. Thus, development of agents that can enhance the effectiveness of drugs used in TB treatment and bypass the efflux mechanism is crucial. In this study, we present a new nanoparticle-based strategy for enhancing the efficacy of existing drugs. To that end, we have developed poly(acrylic acid) (PAA)coated iron oxide (magnetite) nanoparticles (PAA-MNPs) as efflux inhibitors and used it together with rifampicin (a first line anti-TB drug) on Mycobacterium smegmatis. PAA-MNPs of mean diameter 9 nm interact with bacterial cells via surface attachment and are then internalized by cells. Although PAA-MNP alone does not inhibit cell growth, treatment of cells with a combination of PAA-MNP and rifampicin exhibits a synergistic 4-fold-higher growth inhibition compared to rifampicin alone. This is because the combination of PAA-MNP and rifampicin results in up to a 3-fold-increased accumulation of rifampicin inside the cells. This enhanced intracellular drug concentration has been explained by real-time transport studies on a common efflux pump substrate, ethidium bromide (EtBr). It is seen that PAA-MNP increases the accumulation of EtBr significantly and also minimizes the EtBr efflux in direct proportion to the PAA-MNP concentration. Our results thus illustrate that the addition of PAA-MNP with rifampicin may bypass the innate drug resistance mechanism of M. smegmatis. This generic strategy is also found to be successful for other anti-TB drugs, such as isoniazid and fluoroquinolones (e.g., norfloxacin), only when stabilized, coated nanoparticles (such as PAA-MNP) are used, not PAA or MNP alone. We hence establish coated nanoparticles as a new class of efflux inhibitors for potential therapeutic use.
■
INTRODUCTION The discovery of antibiotics in 1942 was a breakthrough in the treatment of bacterial infections. However, the use of antibiotics is now compromised by the emergence of many multi-drug-resistant bacteria.1 Drug resistance develops mainly by four mechanisms, namely, the formation of defense enzymes to degrade or convert the antibiotic to an inactive metabolite, genetic alteration of the target site on which the antibiotic acts, altered permeability of the cellular membrane that leads to restricted uptake of the antibiotic to the target, and forced efflux of the antibiotic from cytosol by efflux pumps present on the bacterial cell.2 Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the highly chronic bacterial infections and a leading killer worldwide. The emergence of drug resistance in mycobacteria is a major limitation in tuberculosis treatment.3 Mycobacterium displays intrinsic resistance to many drugs by decreasing drug uptake and increasing drug efflux. Its unique cell wall structure acts as a permeability barrier to various drugs.4−6 In addition, the mycobacterial genome encodes several putative efflux pumps responsible for drug efflux. Drug efflux is responsible for © 2014 American Chemical Society
reduced drug accumulation, which in turn leads to drug resistance.4,5,7 Thus, there is an urgent need to develop agents that can help the drug to bypass these mechanisms. Nanoparticles have been explored to overcome bacterial drug resistance in various ways.8 They act as effective drug-delivery carriers and help in increasing drug bioavailability and reducing the dosing frequency.9−15 Metallic nanoparticles such as silver and oxides of metals, including zinc and titanium, themselves are antimicrobial nanomaterials.8,16 When these nanoparticles are used along with antibiotics, an additive or synergistic effect is observed upon bacterial growth inhibition.17,18 However, a major drawback of using antibacterial nanoparticles is the danger of acquiring resistance to these nanoparticles themselves.19 Furthermore, the toxicity of these nanoparticles limits their in vivo use.19,20 In contrast, magnetite (Fe3O4) nanoparticles are biocompatible, and many of them are currently marketed or are under clinical investigation for Received: September 24, 2014 Revised: November 5, 2014 Published: November 6, 2014 15266
dx.doi.org/10.1021/la503808d | Langmuir 2014, 30, 15266−15276
Langmuir
Article
various biomedical applications.21,22 In addition, they can be easily functionalized to interact with biological systems and can be utilized in the tissue-specific release of therapeutic agents.23−25 Although various nanoparticles have been utilized against drug resistance, the active efflux of drugs by efflux pumps remains a major challenge in overcoming mycobacterial drug resistance. To counter this, we have developed a novel nanoparticle-based strategy. Furthermore, to understand the mechanism of action of nanoparticles, we explore a systematic three-way effect in which we use either drugs or nanoparticles individually and also their combination on bacterial growth. We address this effect through carefully designed kinetic measurements of both accumulation and efflux, which have not been elucidated earlier. Thus, we use a combination of poly(acrylic acid)-coated magnetite nanoparticles (PAA-MNP), along with the first line anti-TB drug, rifampicin (RIF), against the intrinsic resistance of Mycobacterium smegmatis. We use M. smegmatis, which is a close homolog of M. tuberculosis and is found to display a profile similar to multidrug-resistant (MDR) M. tuberculosis. Thus, it can be used as a “surrogate” screen to test new anti-TB drugs.26 The uptake of nanoparticles or rifampicin alone by M. smegmatis and their individual effect on the growth of cells was compared to that of a combination of rifampicin and PAAMNP. To elaborate the role of nanoparticles on drug transport, real-time accumulation and efflux studies of a fluorescent tracer, ethidium bromide, were carried out in both the presence and absence of PAA-MNP. This is the first study that modulates the efflux mechanism to enhance the efficacy of anti-TB drugs by nontoxic metal oxide nanoparticles. To generalize this strategy to other anti-TB drugs, we have substituted rifampicin with isoniazid (another first-line anti-TB drug) or norfloxacin (model fluoroquinolone), thereby addressing the challenge of drug resistance across a spectrum of drugs.
■
ether/acetone. The washed particles were dispersed in water and dialyzed for 1 day using 12 500 kDa cutoff cellulose membranes and finally filtered through a 0.22 μm filter (Millex-GP) before use. Characterization of PAA-MNP. To obtain the size and morphology of PAA-MNP, transmission electron microscopy (TEM) was carried out in a JEOL-JEM 2100F operated at 200 kV. X-ray diffractometry (XRD) of PAA-MNP was carried out using a PANalytical X’Pert Pro (Philips PW3040/60) provided with Cu Kα radiation. Magnetic measurements were made using superconducting quantum interference device−vibrating sample magnetometers, SQUIDVSM (Quantum Design, USA), at room temperature. For the XRD and VSM study, a few milligrams of PAA-MNP was dried at 80 °C. The zeta potential was measured in a Zeta Sizer NanoS (Malvern Instruments), equipped with a 4.0 mW solid-state He−Ne laser of wavelength 633 nm set at room temperature. The Smoluchowski equation was used to calculate the potential values of nanoparticles. Uptake of PAA-MNP by M. smegmatis Using Transmission Electron Microscopy. M. smegmatis cultures were grown in M7H9 medium supplemented with ADC at 37 °C until they reached the midlog phase, corresponding to OD600 = 0.5. PAA-MNP was added to this culture at a concentration of 16 μg/mL and incubated at 37 °C, 180 rpm for 4 h. The culture was then centrifuged at 10 000 rpm for 10 min, and the pellet was washed twice with distilled water and used for further processing. The pellet was primarily fixed with 2% glutaraldehyde overnight at 4 °C. The pellet was then washed five to six times with PBS to remove the primary fixative and postfixed with secondary fixative (osmium tetraoxide) for 2 h at 4 °C. The samples were again washed five to six times with PBS and then dehydrated with various percentages of ethanol (50, 70, 85, 95%, and absolute ethanol, respectively), followed by 1:1 ethanol and propylene oxide and finally with 100% propylene oxide, each for 5 min. Subsequently, infiltration and embedding were performed in Araldite. Finally, ultrathin sections of 60 nm thickness were cut, mounted on a Formvar-coated copper grid, and observed under TEM (JEOL-JEM 2100F). Uptake of PAA-MNP by M. smegmatis Using Inductively Coupled Plasma−Atomic Emission Spectroscopy (ICP-AES). PAA-MNP was added to mid-log-phase cells over a varying concentration range (4−64 μg/mL) and incubated at 37 °C, 180 rpm. After 4 h of incubation, an extraction of nanoparticles from bacterial cultures was performed. The culture was centrifuged at 5000g for 10 min such that only the micrometer-sized cells settled down and separated as a pellet. The cells were further concentrated and lysed. M. smegmatis has a complex cell envelope, and hence to lyse the cells, mechanical bead beating was performed by using 0.1 mm zirconium beads (Unigenetics). To the cell lysate, concentrated HCl was added to release Fe from nanoparticles. Finally, supernatant after centrifugation was used for ICP-AES analysis for Fe content. Determination of Minimum Inhibitory Concentration (MIC). Minimum inhibitory concentrations (MIC) of rifampicin, ethidium bromide (EtBr), carbonyl cyanide m-chlorophenylhydrazone (CCCP), reserpine (RES), isoniazid, and norfloxacin were determined by the broth microdilution method, according to clinical and laboratory standards institute (CLSI) guidelines.28 Briefly, M. smegmatis mc2155 cultures were grown in M7H9 medium, supplemented with ADC at 37 °C, until they reached the mid-log phase (OD600 = 0.5). Cultures were diluted in PBS to have 106 cells/mL (McFarland No. 0.5 standard). Aliquots of 0.1 mL of diluted cultures were transferred to each well of a 96-well plate containing 0.1 mL of each agent whose MIC is to be determined, at various concentrations prepared by dilution in M7H9 medium. The plates were incubated at 37 °C for 48 h. MIC was identified as the lowest concentration of the compound that inhibited visible growth. Effect of the Combination of Rifampicin and PAA-MNP on the Growth of M. smegmatis. M. smegmatis cells were grown in M7H9 medium supplemented with ADC at 37 °C and 180 rpm. When the cultures reached an OD600 of 0.5 (mid-log phase), they were exposed to each of the following three conditions: (a) rifampicin at a subinhibitory concentration of 8 μg/mL, (b) nanoparticles at concentrations of 8, 16, and 32 μg/mL, and (c) a combination of
EXPERIMENTAL SECTION
Materials. The sodium salt of poly(acrylic acid) (molecular weight 2100), iron(III) acetyl acetonate (Fe(C5H7O2)3, [Fe(acac)3]), 2pyrrolidone (C4H7NO), carbonyl cyanide m-chlorophenylhydrazone (CCCP), and norfloxacin were purchased from Sigma-Aldrich Corp. (USA). Rifampicin, reserpine, ethidium bromide (EtBr), and isoniazid were purchased from Hi-Media (India). Deionized water (Millipore Milli-Q) was used in all of the experiments. All chemicals were reagent grade and used without further purification. Strain, Media, and Culture Conditions. Mycobacterium smegmatis mc2155 was used for the experimental studies. It was cultured in Middlebrook 7H9 (M7H9, Hi-media) medium at 37 °C and 180 rpm. After being autoclaved, the medium was supplemented with 10% (v/v) ADC (5 g of albumin, 2 g of glucose, and 0.85 g of NaCl in 100 mL of distilled water) that was initially filter-sterilized. Plating was performed in Luria-Bertani (LB) media (Hi-media) at 37 °C. Synthesis of Poly(acrylic acid)-Coated Magnetite Nanoparticles (PAA-MNP). PAA-MNP have been synthesized by the thermal decomposition route.27 Briefly, we added 0.9 mmol of iron(III) acetyl acetonate (Fe(C5H7O2)3, [Fe(acac)3]) and 0.4 mmol of PAA to a round-bottomed flask (equipped with a stirrer and a condenser) containing 20 mL of 2-pyrrolidone (boiling point 245 °C). Nitrogen purged this mixture for 30 minutes to remove any dissolved oxygen. This mixture was subsequently held at 210 °C for 30 min. Furthermore, refluxing at 245 °C was carried out for 40 min under vigorous mechanical stirring. The resultant dispersion was then cooled to room temperature and a 5:1 volume ratio of diethyl ether/acetone was added to separate out the particles. To remove any unreacted PAA, particles were washed three times with Milli-Q water and diethyl 15267
dx.doi.org/10.1021/la503808d | Langmuir 2014, 30, 15266−15276
Langmuir
Article
Figure 1. Synthesis and characterization of PAA-MNP. (a) Low-magnification TEM image, with the inset showing the selected-area electron diffraction pattern. (b) High-magnification TEM image, with the inset showing a high-resolution TEM (HRTEM) image of a single spherical particle. (c) XRD pattern. (d) Magnetization vs applied magnetic field of PAA-MNP. rifampicin at 8 μg/mL, with an increasing concentration of nanoparticles (8, 16, and 32 μg/mL). In all cases, OD600 was measured spectrophotometrically (NanoPhotometer-IMPLEN) at specific time intervals. OD600 for a blank drug and nanoparticle solution is negligible, as compared to that of the bacterial solution. Furthermore, colony-forming units per milliliter (cfu/mL) were determined for the samples that were exposed to drug and nanoparticles for 0, 4, and 24 h using the drop count method.29 The sample (1 mL) at each time point was centrifuged to remove media, antibiotics, and nanoparticles and then washed twice with sterile PBS and resuspended in 1 mL of sterile PBS. Finally, 10-fold serial dilutions were made in sterile PBS. Subsequently, dilutions were chosen that gave approximately 3 to 30 cfu/10 μL drop. Five to six drops of 10 μL volume of the chosen dilution were placed on an LB agar plate, and the plate was incubated at 37 °C for 48 h. The cfu/drop was counted, and the cfu/mL for each condition was compared to control conditions. Relative cfu/mL values after 4 h (ratio of cfu/mL at 4 h to cfu/mL at 0 h) and after 24 h (ratio of cfu/mL at 24 h to cfu/ mL at 0 h) for the respective conditions were obtained. Intracellular Rifampicin Quantification in M. smegmatis. M. smegmatis cells were grown until they reached OD600 = 0.5. They were further concentrated to OD600 = 5. These cells were subjected to rifampicin at a concentration of 8 μg/mL and combinations of rifampicin (8 μg/mL) with 8, 16, and 32 μg/mL nanoparticle concentrations, respectively. These were compared to the control condition (without any treatment). Cells were incubated under these conditions for 1 h at 37 °C and 180 rpm. After 1 h of incubation, the
cells were centrifuged at 10 000 rpm at 4 °C for 10 min and were washed twice with distilled water. (Note that all washing steps were performed at 4 °C.) Finally, the pellet was lysed by adding 0.1 M glycine HCl (pH 3) and kept overnight at room temperature. Samples were centrifuged, and the supernatant was further processed. The supernatant (500 μL) was vacuum dried at room temperature. The dried residue obtained was reconstituted in 250 μL of an acetonitrile− methanol (1:2 v/v) mixture and sonicated for 5 min. Finally, the mixture was centrifuged at 10 000 rpm for 5 min. From the supernatant, a 0.5 μL sample was injected for LCMS analysis (Agilent 6550 iFunnel liquid chromatograph−quadrupole time-of-flight mass spectrometer (Q-TOF LC/MS)). The mass spectrometer was operated in positive ion mode by using a dual Agilent jet stream electrospray ionization (dual AJS ESI) source. Chromatographic separation was obtained with an Agilent ZORBAX rapid resolution high-definition SB-C18 threaded column (2.1 mm id × 50 mm, 1.8 μm). The mobile phase consisted of (A) 100% water acidified with 0.1% formic acid and (B) 90% acetonitrile acidified with 0.1% formic acid and 10% water. For sample injection, the mobile phase composition used was A (0%) and B (100%) for 3 min at a flow rate of 0.3 mL/min with an injection volume of 0.5 μL. Quantification was done by comparing with a standard curve and was repeated for three biological replicates. The results were expressed in terms of ng of rifampicin/mg of dry cell weight. EtBr Accumulation and Efflux by the Semiautomated Fluorometric Method. This was performed by a modification of the previous semiautomated fluorometric method.30,31 Briefly, M. 15268
dx.doi.org/10.1021/la503808d | Langmuir 2014, 30, 15266−15276
Langmuir
Article
Figure 2. Uptake of PAA-MNP by M. smegmatis. TEM images of (a) M. smegmatis cells without nanoparticles (control). (b) Sections of cells incubated for 4 h with PAA-MNP at a concentration of 16 μg/mL with the magnified region of the area enclosed by the dotted black square clearly showing the attachment of nanoparticles to the cell membrane (white dotted circle), where small black arrows represent nanoparticles and white triangle shows the cell membrane. (c) SAED from panel a. (d) SAED from panel b. (e) Quantitative uptake of nanoparticles by the ICP-AES technique at 37 °C after 4 h of incubation in the presence of a varying extracellular nanoparticle concentration. (f) Adsorption (4 °C) versus total uptake (37 °C) of PAA-MNP at a concentration of 16 μg/mL after 4 h of incubation. EtBr Accumulation and Efflux by Fluorescence Microscopy. Confocal laser scanning microscopy (Olympus IX81 FV500) was performed to study EtBr accumulation and efflux in the presence of PAA-MNP. Microscopy was carried out at excitation and emission wavelengths of 530 and 590 nm, respectively. Finally, 10 μL of a cell suspension after 1 h of accumulation and 1 h of efflux was placed in between two coverslips. The coverslip with the cell suspension was kept inverted and focused from the bottom. For a quantification of the level of EtBr accumulation and efflux in the presence of nanoparticles, intensity plots were obtained and compared to the control. The intensity was calculated for each cell, and the average intensity of about 100 cells was plotted.
smegmatis cultures were grown in M7H9 medium supplemented with ADC at 37 °C until they reached mid-log phase corresponding to OD600 = 0.5. Cultures were then centrifuged at 13 000 rpm for 3 min; the pellet was washed with PBS (pH 7.4) and finally resuspended in PBS and PBS containing nanoparticles at varying concentrations ranging from 1 to 155 μg/mL. EtBr was added to the cellular suspension at a concentration of 3 μg/mL (less than that of MIC1/2). Aliquots of 200 μL were transferred to 96-well plates (black polysorp, Nunc). EtBr accumulation was measured by measuring the fluorescence in a microplate reader (spectramax multimode M5, Molecular Devices) at 37 °C using 530 and 585 nm as the excitation and emission wavelengths, respectively. For the efflux assay, EtBr was added to the cell at a 3 μg/mL concentration (less than that of MIC1/2), and the cells were incubated at 37 °C and 180 rpm in a shaker to obtain the maximum accumulation of EtBr. After an hour of incubation, EtBr-loaded cells were centrifuged at 13 000 rpm for 3 min at 4 °C and were resuspended in PBS and PBS containing PAA-MNP. Aliquots of 200 μL were transferred to a 96-well plate, and EtBr efflux was measured by acquiring the fluorescence as mentioned above. The efflux of EtBr from cells loaded with only ethidium bromide was defined as the control, and all other conditions were compared to this control. The relative fluorescence unit (RFU) obtained for all efflux data were plotted as relative RFU, where the RFU at each time point was divided by its initial value. Assays were performed for three biological replicates. The assay was validated by using references such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine (RES), which are known efflux pump inhibitors.
■
RESULTS AND DISCUSSION Nanoparticle (PAA-MNP) Synthesis and Characterization. PAA-MNP has been synthesized by the thermal decomposition route. PAA-coated magnetite nanoparticles were chosen on the basis of previous studies in our laboratory.27,32 From among different coating agents (such as carboxymethyl cellulose, oleic acid, citric acid, dextran, PAA, etc.), PAA was chosen for MNP synthesis because of the following advantages, which are all desirable characteristics for therapeutic use:33 monodisperse coated nanoparticles of small mean diameter, a nonaggregated dispersed state, the stability of the aqueous dispersion of PAA-MNP, and finally its cell 15269
dx.doi.org/10.1021/la503808d | Langmuir 2014, 30, 15266−15276
Langmuir
Article
cationic sites present on the cell membrane may contribute to the uptake of anionic nanoparticles.35,36 Inhibition of Bacterial Growth by the Synergistic Action of PAA-MNP and Rifampicin. The minimum inhibitory concentration (MIC) of rifampicin (a first-line antiTB drug) in M. smegmatis was found to be 32 μg/mL. We examined the individual effects of only rifampicin, only nanoparticles, and the combined presence of both rifampicin and nanoparticles on the growth of M. smegmatis. To interpret the effect of the continuous exposure of treatment (drug, nanoparticle, or both) on mid-log phase cells, the growth kinetics was evaluated by measuring the optical density at 600 nm (OD600). In addition, a colony-forming unit (cfu) count was used to determine the viability of treated cells when grown on fresh (treatment free) media. The cell viability was recorded in terms of the relative cfu/mL at two representative time points after 4 h (short term) and 24 h (long term) of treatment, respectively. The relative cfu/mL at 4 and 24 h is normalized with respect to that at 0 h. Figure 3a presents the growth kinetics of M. smegmatis in the presence of only nanoparticles at 8, 16, and 32 μg/mL. At these concentrations, nanoparticles alone did not have any effect on bacterial growth. Moreover, broth microdilution demonstrates that PAA-MNP is not toxic to cells even at 256 μg/mL (Figure S1). The biocompatibility of PAA-MNP observed by us is consistent with that reported in the literature for malignant as well as noncancerous fibroblast cells.21,37 According to standard toxicological and pharmacological tests, magnetite nanoparticles have also been proven to be biocompatible and safe for human use.22 However, despite being nontoxic in itself, PAA-MNP enhanced the antibacterial effect of rifampicin, as shown in Figure 3a,b. Figure 3a represents the growth kinetics of M. smegmatis in the presence of a subinhibitory rifampicin concentration (8 μg/mL) and a combination of rifampicin with varying nanoparticle concentrations. When rifampicin is administered alone, cells exhibit only 42% growth inhibition. A concentration-dependent increase in growth inhibition is observed for a combination of rifampicin and nanoparticles. Growth is reduced by as much as 89% for rifampicin with 32 μg/mL nanoparticles. Note that the growth profile in the presence of rifampicin alone at 32 μg/mL is similar to that in the presence of a combination of 8 μg/mL rifampicin and 32 μg/mL PAA-MNP. Thus, PAA-MNP displays a synergistic effect with rifampicin in direct proportion to the administered dose. We also evaluated the cell viability to understand the ability of cells to multiply, once the stress due to drug alone or in combination with nanoparticles was removed. After 4 h of treatment, rifampicin alone or rifampicin in combination with either 8 or 16 μg/mL nanoparticles, respectively, did not have any significant effect on cell viability as compared to control cells (without any treatment). In contrast, rifampicin with nanoparticles at a concentration of 32 μg/mL decreased the cfu/mL significantly (p value
