polyacrylic acid-coated iron oxide nanoparticles for targeting drug resistance in mycobacteria

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Article

Polyacrylic acid coated iron oxide nanoparticlesfor targeting drug resistance in mycobacteria

Priyanka Shivaji Padwal, Rajdip Bandyopadhyaya, and Sarika MehraLangmuir, Just Accepted Manuscript • DOI: 10.1021/la503808d • Publication Date (Web): 06 Nov 2014

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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

*Corresponding Authors: [email protected]

[email protected]

AUTHOR INFORMATION

Corresponding Authors

* Phone Nos.: (91 22) 2576 7209, Fax: (91 22) 2572 6895, Email: [email protected]

* Phone Nos.: (91 22) 2576 7221, Fax: (91 22) 2572 6895, Email: [email protected]

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ABSTRACT

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 low intracellular

drug concentration. Thus, development of agents which can enhance the effectiveness of drugs

used in TB treatment and bypass the efflux mechanism is very crucial. In this study, we present a

new nanoparticle based strategy for enhancing the efficacy of existing drugs. To that end, we

have developed polyacrylic 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 four times higher growth inhibition, compared to rifampicin alone. This is because,

combination of PAA-MNP and rifampicin results upto three times 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 accumulation of EtBr significantly and also minimizes EtBr

efflux in direct proportion to PAA-MNP concentration. Our results thus illustrate that addition of

PAA-MNP with rifampicin may bypass the innate drug resistance mechanism of M. smegmatis.

This generic strategy is also found successful for other anti-TB drugs, like isoniazid and

fluoroquinolones (e. g. norfloxacin); only when stabilized, coated nanoparticles (like 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.

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Keywords: drug resistance, tuberculosis, mycobacterium, magnetite nanoparticles, rifampicin,

accumulation, efflux

INTRODUCTION

Discovery of antibiotics in 1942 was a breakthrough in the treatment of bacterial infections.

However, use of antibiotics is now compromised by the emergence of many multidrug resistant

bacteria.1 Drug resistance develops mainly by four mechanisms, namely, formation of defence

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 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. 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, 5, 6

In addition, mycobacterial

genome encodes several putative efflux pumps responsible for drug efflux. Drug efflux is

responsible for 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 dosing frequency.9, 10, 11, 12, 13, 14, 15

Metallic nanoparticles such as silver and oxides of

metals, including zinc and titanium, themselves are antimicrobial nanomaterials.8, 16

When these

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nanoparticles are used along with antibiotics, an additive or synergistic effect is observed on

bacterial growth inhibition.17, 18

However, a major drawback of using antibacterial nanoparticles

is the danger of acquiring resistance against these nanoparticles themselves.19

Further, 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

various biomedical applications.21, 22

In addition, they can be easily functionalized to interact

with biological systems and can be utilized in tissue specific release of therapeutic agents.23, 24, 25

Although various nanoparticles have been utilized against drug resistance, active efflux of

drugs by efflux pumps still 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 drug 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 polyacrylic 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 multi-drug resistant (MDR) M. tuberculosis. Thus, it

can be used as a ‘surrogate’ screen to test new anti-TB drugs.26

Uptake of nanoparticles or

rifampicin alone by M. smegmatis, and their individual effect on growth of cells was compared

with that of a combination of rifampicin and PAA-MNP. To elaborate the role of nanoparticles

on drug transport, real time accumulation and efflux studies of a fluorescent tracer ethidium

bromide were carried out, both in presence and absence of PAA-MNP. This is the first study

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which modulates the efflux mechanism, to enhance efficacy of anti-TB drugs, by non-toxic 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.

EXPERIMENTAL SECTION

Materials: Sodium salt of polyacrylic acid (molecular weight: 2100), iron (III) acetyl acetonate

(Fe(C5H7O2)3, [Fe(acac)3]), 2-pyrrolidone (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). Deionised water (Millipore-Milli-Q) was used in all the experiments. All

chemicals were of 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) media at 37˚C and

180 rpm. After autoclaving, the media was supplemented with 10% (v/v) ADC (5g albumin, 2g

glucose and 0.85g NaCl in 100ml distilled water) which was initially filter-sterilized. Plating was

performed in Luria-Bertani (LB) media (Hi-media) at 37˚C.

Synthesis of polyacrylic acid coated magnetite nanoparticles (PAA-MNP): PAA-MNP have been

synthesised 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-bottom flask

(equipped with a stirrer and a condenser), containing 20 ml of 2-pyrrolidone (boiling point:

245˚C). Nitrogen was purged for 30 min through this mixture to remove any dissolved oxygen.

This mixture was subsequently heated up to 210˚C for 30 min. Further, refluxing at 245˚C was

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carried out for 40 min, under vigorous mechanical stirring. The resultant dispersion was then

cooled down 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 ether/acetone. The washed particles were dispersed in water and

dialyzed for 1 day using 12500 kDa cutoff cellulose membranes and finally, filtered through 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 JEOL-JEM 2100F, operated at 200 KV. X-ray

diffractometry (XRD) of PAA-MNP was done using a PANalytical X’Pert Pro (Philips

PW3040/60) provided with CuKα radiation. Magnetic measurements were made using

superconducting quantum interference device-vibrating sample magnetometers, SQUID–VSM

(Quantum design, USA) at room temperature. For XRD and VSM study, few milligrams of

PAA-MNP were dried at 80˚C. Zeta potential was measured in Zeta Sizer NanoS (Malvern

Instruments), equipped with a 4.0 mW, solid state He-Ne laser of wavelength 633 nm, set at

room temperature. 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

mid-log phase, corresponding to OD600 of 0.5. PAA-MNP were 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 10000 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

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4˚C. The pellet was then washed 5 to 6 times with PBS to remove the primary fixative and post

fixed with secondary fixative (osmium tetraoxide) for 2 h at 4˚C. The samples were again

washed 5 to 6 times with PBS and then dehydrated with various percentage 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 thickness 60 nm were cut,

mounted on 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 were added to mid-log phase cells at varying concentration

range (4-64 µg/ml) and incubated at 37˚C, 180 rpm. After 4 h of incubation, extraction of

nanoparticles from bacterial cultures was performed. The culture was centrifuged at 5000 g for

10 min, such that only the micron sized cells settled down and separated as pellet. The cells were

further concentrated and lysed. M. smegmatis have a complex cell envelope 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 the 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 mid-log phase (OD600 of 0.5). Cultures were diluted in PBS to have 106

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cells/ml (McFarland No. 0.5 standard). Aliquots of 0.1 ml of diluted cultures were transferred to

each well of 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 combination of rifampicin and PAA-MNP on 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 sub inhibitory concentration of 8 µg/ml (b) nanoparticles at

concentrations of 8 µg/ml, 16 µg/ml and 32 µg/ml, (c) combination of rifampicin at 8 µg/ml,

with increasing concentration of nanoparticles (8 µg/ml, 16 µg/ml and 32 µg/ml). In all cases,

OD600 was measured spectrophotometrically (NanoPhotometer-IMPLEN), at specific time

intervals. OD600 for blank drug and nanoparticle solution is negligible, as compared to that of the

bacterial solution. Further, colony forming units per ml (cfu/ml) was determined for the samples

which were exposed to drug and nanoparticles at 0, 4 and 24 h using the drop count method.29

1

ml of the sample at each time point were centrifuged to remove media, antibiotic and

nanoparticles; then washed twice with sterile PBS and re-suspended in 1 ml of sterile PBS.

Finally, 10 fold serial dilutions were made in sterile PBS. Subsequently, dilutions were chosen

which gave approximately 3 to 30 cfu/10 µl drop. 5-6 drops of 10 µl volume of the chosen

dilution were placed on LB agar plate and the plate was incubated at 37˚C for 48 h. Cfu/drop was

counted and cfu/ml for each condition was compared to control conditions. Relative cfu/ml at 4 h

(ratio of cfu/ml at 4 h to cfu/ml at 0 h) and at 24 h (ratio of cfu/ml at 24 h to cfu/ml at 0 h) for the

respective conditions were obtained.

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Intracellular rifampicin quantification in M. smegmatis: M. smegmatis cells were grown till they

reach OD600 of 0.5. They were further concentrated to OD600 of 5. These cells were subjected to

rifampicin at 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 with control condition

(without any treatment). Cells were incubated with these conditions for 1 h, at 37˚C and 180

rpm. After 1 h of incubation, the cells were centrifuged at 10000 rpm at 4˚C for 10 min and were

washed twice with distilled water (Note: all washing steps 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 supernatant was further processed. 500 µl of supernatant was vacuum dried

at room temperature. Dried residue obtained was reconstitutes in 250 µl of acetonitrile-methanol

(1:2 v/v) mixture and sonicated for 5 min. Finally, the mixture was centrifuged at 10000 rpm for

5 min. From the supernatant, 0.5 µl sample was injected for LCMS analysis (Agilent 6550

iFunnel liquid chromatograph-quadrupole time-of-flight mass spectrometer (Q-TOF LC/MS)).

Mass spectrometer was operated in positive ion mode by using dual Agilent jet stream

electrospray ionization (dual AJS ESI) source. Chromatographic separation was obtained by

Agilent ZORBAX rapid resolution high definition SB-C18 threaded column (2.1 mm id x 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 the flow rate 0.3

ml/min, with injection volume of 0.5 µl. Quantification was done by comparing with standard

curve and repeated for 3 biological replicates. The results were expressed in terms of ng of

rifampicin, per mg dry cell weight.

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EtBr accumulation and efflux by Semi-automated fluorometric method: This was performed by

modification of previous semi-automated fluorometric method.30, 31

Briefly, M. smegmatis

cultures were grown in M7H9 medium supplemented with ADC at 37˚C, until they reached mid-

log phase corresponding to OD600 of 0.5. Cultures were then centrifuged at 13000 rpm for 3 min;

pellet was washed with PBS (pH 7.4) and finally re-suspended in PBS and PBS containing

nanoparticles, at varying concentrations ranging from, 1 µg/ml to 155 µg/ml. EtBr was added at

a concentration of 3 µg/ml (less than MIC1/2) to the cellular suspension. 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 nm and 585 nm as excitation and emission wavelengths,

respectively. For efflux assay, EtBr was added at 3 µg/ml concentration (less than MIC1/2) to the

cell and the cells were incubated at 37˚C and 180 rpm in a shaker, to get maximum accumulation

of EtBr. After an hour of incubation, EtBr loaded cells were centrifuged at 13000 rpm for 3 min,

at 4˚C, and were re-suspended in PBS and PBS containing PAA-MNP. Aliquots of 200 µl were

transferred to 96 well plate and EtBr efflux was measured by acquiring the fluorescence as

mentioned above. Efflux of EtBr from cells loaded with only ethidium bromide was defined as

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, RFU data at each time-

point were divided by its initial value. Assays were performed for 3 biological replicates. Assay

was validated by using references such as, carbonyl cyanide m-chlorophenylhydrazone (CCCP)

and reserpine (RES), which are known efflux pump inhibitors.

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

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PAA-MNP. Microscopy was done at excitation and emission wavelengths of 530 nm and 590

nm, respectively. Finally, 10 µl of cell suspension after 1 h of accumulation and 1 h of efflux was

placed in between two cover slips. The cover slip with cell suspension was kept inverted and

focused from bottom. For quantification of the level of EtBr accumulation and efflux in presence

of nanoparticles, intensity plots were obtained and compared with control. Intensity was

calculated for each cell and the average intensity of about 100 cells was plotted.

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 based on previous studies in our laboratory.27, 32

From amongst

different coating agents (like carboxymethyl cellulose, oleic acid, citric acid, dextran, PAA etc.),

PAA was chosen for MNP synthesis due to the following advantages – resulting monodisperse,

coated nanoparticles of small mean diameter, non-aggregated dispersed state, stability of the

aqueous dispersion of PAA-MNP and finally its cell viability against human liver cell line

(HepG2), which are all desirable characteristics for therapeutic use.33

In addition, PAA-MNP are

smaller in size and well dispersed than uncoated magnetite nanoparticles, and thus have a higher

specific surface area, making them more suitable for adsorption and internalization into cells.

Average particle size of PAA-MNP obtained from transmission electron microscopy (TEM) is 9

nm with a standard deviation of 2 nm (Figures 1a and 1b). Electron diffraction pattern of the

selected area of PAA-MNP (insert in Figure 1a) indicates that the sample is polycrystalline. X-

ray diffraction (XRD) confirms it is magnetite (Fe3O4), with an inverse cubic spinel structure

(Figure 1c). Magnetization versus applied magnetic field curve from superconducting quantum

interference device-vibrating sample magnetometer (SQUID-VSM) indicates that the

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nanoparticles are super-paramagnetic at room temperature (Figure 1d), as desired in many

therapeutic applications. PAA-MNP have a large negative zeta potential value at pH 7 (absolute

value above 30 mV), which implies that coated particles have a high negative surface charge,

and are hence highly stable in aqueous solution.

Figure 1. Synthesis and characterization of PAA-MNP. (a) Low magnification TEM image, inset

shows the selected area electron diffraction pattern (b) High magnification TEM image, inset shows high

resolution TEM (HRTEM) image of a single spherical particle, (c) XRD pattern, and (d) magnetization

versus applied magnetic field of PAA-MNP.

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Uptake of PAA-MNP by M. smegmatis

TEM of unstained ultrathin cell sections was used to examine the relative location of the

nanoparticle and cell. Figures 2a and 2b show sections of control cells (without nanoparticle

treatment) and nanoparticle-treated cells, respectively. Micrographs of cells incubated for 4 h

with 16 µg/ml nanoparticles demonstrate that, in presence of particles, most of the cells are intact

without any membrane damage (Figure 2b). Further, some particles are attached to the

mycobacterial cell surface (Figure 2b). Enlarged image from Figure 2b shows that some particles

are also internalized by the cells. Selected area electron diffraction pattern (SAED) of control

cells (Figure 2c) is compared with that of PAA-MNP treated cells (Figure 2d). SAED pattern in

case of nanoparticle treated cells is in the form of rings, indicating the presence of many

individual nanoparticles.

To quantitate the amount of internalized nanoparticles, we performed inductively coupled

plasma atomic emission spectroscopy (ICP-AES) of Fe content in lysates of cells incubated with

PAA-MNP at 37˚C and 4˚C. After 4 h of incubation at 37˚C, uptake of nanoparticles is seen to

be directly proportional to the original administered concentration of nanoparticles added

externally in the dispersion (Figure 2e). At 37˚C both processes, adsorption as well as

internalization of nanoparticles, occur simultaneously, while, at 4˚C nanoparticles interact with

the membrane, but do not transport across the membrane. Thus, internalization of nanoparticles

can be obtained from the difference between total nanoparticles uptaken at 37˚C and

nanoparticles adsorbed by the cells at 4˚C. Comparison of ICP-AES analysis of 16 µg/ml

nanoparticle concentration at 4˚C and 37˚C reveals that 50% of the nanoparticles are adsorbed on

the cell surface and 50% are internalized by the cells (Figure 2f).

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Thus, microscopy and ICP-AES data present clear evidence of uptake of nanoparticles by

bacterial cells. Charge on particle surface plays an important role in membrane interaction and

internalization.34

PAA coated nanoparticles are anionic in nature due to surface carboxylate

groups of PAA. Increased uptake of anionic nanoparticles in comparison to neutral nanoparticles

has been reported earlier in human carcinoma cell lines.35

Previous studies suggest that,

electrostatic interaction between anionic nanoparticles and cationic sites present on the cell

membrane may contribute to the uptake of anionic nanoparticles.35, 36

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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; magnified region of area enclosed in dotted black square clearly showing the attachment of

nanoparticles with cell membrane (white dotted circle), small black arrows represent nanoparticles and

white triangle shows the cell membrane, (c) SAED from Figure 2a, (d) SAED from Figure 2b, (e)

quantitative uptake of nanoparticles by ICP-AES technique at 37˚C after 4 h of incubation in presence of

varying extracellular nanoparticle concentration, (f) adsorption (4˚C) versus total uptake (37˚C) of PAA-

MNP at concentration 16 µg/ml after 4 h of incubation.

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Inhibition of bacterial growth by synergistic action of PAA-MNP and rifampicin

Minimum inhibitory concentration (MIC) of rifampicin (a first line anti-TB 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 continuous exposure of treatment (drug,

nanoparticle or both combined) on mid-log phase cells; growth kinetics was evaluated by

measuring the optical density at 600 nm (OD600). In addition, colony forming unit (cfu) count

was used to determine the viability of treated cells, when grown on fresh (treatment free) media.

Cell viability was recorded in terms of relative cfu/ml at two representative time points, after 4 h

(short term) and 24 h (long term) of treatment, respectively. Relative cfu/ml at 4 h and 24 h is

normalized with respect to that at 0 h.

Figure 3a presents the growth kinetics of M. smegmatis in 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 are not toxic to

cells even at 256 µg/ml (Figure S1). Biocompatibility of PAA-MNP observed by us is consistent

with that reported in literature for malignant as well as non-cancerous 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 non-toxic in itself, PAA-MNP enhanced the antibacterial effect of

rifampicin, as shown in Figures 3a and 3b. Figure 3a represents the growth kinetics of M.

smegmatis in presence of a sub-inhibitory 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

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inhibition is observed for a combination of rifampicin and nanoparticles. Growth is reduced by

as much as 89% for rifampicin with 32 µg/ml of nanoparticles. Note that, growth profile in

presence of rifampicin alone at 32 µg/ml is similar to that in presence of a combination of 8

µg/ml of rifampicin and 32 µg/ml of PAA-MNP. Thus, PAA-MNP displays a synergistic effect

with rifampicin, in direct proportion to administered dose.

We also evaluated 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 µg/ml or 16 µg/ml of 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 < 0.05), both with respect to control and rifampicin

treated cells (Figure 3b). We also note that, for this particular combination, the relative cfu/ml is

approximately equal to 1.0, which denotes complete growth inhibition. Cell viability was also

analyzed after 24 h of exposure. A remarkable decrease in the proliferation of cells is seen for

treatment with rifampicin and rifampicin together with nanoparticles at 8, 16 and 32 µg/ml

respectively, as compared to control (Figure 3b). Moreover, rifampicin with either 16 or 32

µg/ml nanoparticles decreased the cfu/ml even more than that in the presence of rifampicin

alone. Furthermore, in cells exposed to rifampicin and 32 µg/ml of nanoparticles simultaneously

for 24 h, the relative cfu/ml is approximately equal to 0.5. This suggests that, growth is not only

inhibited, but cell survival is also affected.

We note that, enhancement of antibiotic efficacy by the use of metal oxide nanoparticles has

been shown earlier.8 For example, a concentration dependent increase in the antibacterial effect

against Staphylococcus aureus and Escherichia coli was observed when ciprofloxacin was used

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in the presence of zinc oxide (ZnO) nanoparticles.18

However, the mechanism behind this

enhancement in terms of effect of nanoparticles on drug transport is not addressed in literature.

In anticipation that the observed synergy might be due to increased intracellular antibiotic

concentration, we also determined the intracellular rifampicin concentration, both in presence

and absence of nanoparticles.

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Figure 3. Enhancement of rifampicin efficacy by PAA-MNP on growth of M. smegmatis. (a) Growth

kinetics (b) relative colony forming units per ml (cfu/ml) at 4 h and 24 h of M. smegmatis in presence of

only rifampicin (represented as RIF), only PAA-MNP (represented as NP) and combination of both

rifampicin and PAA-MNP (represented as RIF + NP). (↓) indicates point of addition of different

dispersions. * indicates statistical significance (p-value < 0.05 by student’s t-test) with respect to control

and # indicates statistical significance (p-value < 0.05 by student’s t-test) with respect to rifampicin at 8

µg/ml. Black line shows relative cfu/ml of 1.0 and grey dotted line shows relative cfu/ml of 0.5; (c)

Intracellular rifampcin levels as quantified by HRLCMS quantification in presence of varying

extarcellular rifampicin concentrations and combination of rifampicin (8µg/ml) with increasing

concentration (8µg/ml, 16µg/ml, and 32µg/ml) of PAA-MNP.

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PAA-MNP increases intracellular rifampicin concentration in M. smegmatis

For the quantification of intracellular rifampicin in cells, we have developed a rapid detection

method based on high resolution liquid chromatography, coupled to tandem mass spectrometry

(HR-LCMS). Representative chromatograms and mass spectra of rifampicin standards are shown

in Figures S2a and S2b, respectively. A standard curve for rifampicin in a mixture of acetonitrile

and methanol is also presented (Figure S2c). Specimen chromatograms and mass spectra of

rifampicin extracted from cells treated with either rifampicin or with a combination of rifampicin

and PAA-MNP are presented in Figure S3, respectively.

Rifampicin uptake increases with increase in exogenous rifampicin concentration (Figure 3c).

Moreover, when rifampicin is supplemented with nanoparticles, accumulation of rifampicin

increases with nanoparticle concentration. On exposing cells to an exogenous rifampicin

concentration of 8 µg/ml for an hour, intracellular levels of around 20 ng of rifampicin per mg

dry cell weight were achieved. This intracellular level of rifampicin was increased by 1.5-3 times

when rifampicin was supplemented with nanoparticles (Figure 3c). Note that, intracellular

rifampicin concentration achieved in presence of only 8 µg/ml rifampicin when supplemented

with 32 µg/ml nanoparticles, is similar to that in presence of a high rifampicin concentration of

32 µg/ml, when rifampicin is given alone. Thus, decrease in MIC of rifampicin in presence of

nanoparticles can be correlated with higher intracellular rifampicin levels.

In general, expression of antibiotic efflux pumps reduces the intracellular drug concentration,

thereby making the antibiotic ineffective. Rifampicin is one such antibiotic which is eliminated

from wild type mycobacteria by efflux.38

In presence of efflux pump inhibitors, MIC of

rifampicin is reduced due to higher intracellular drug concentration.39

The enhanced intracellular

rifampicin concentration observed in the presence of PAA-MNP can thus be due to reduced

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efflux of rifampicin. Thus, we next investigate the kinetics of drug transport in presence of

various nanoparticle concentrations. We employ a method based on transport of a fluorescent

tracer, which allows monitoring the drug accumulation and efflux process in real time.

Effect of PAA-MNP on net intracellular EtBr accumulation

To investigate the effect of PAA-MNP on kinetics of drug-transport in and out of M.

smegmatis, we used ethidium bromide (EtBr), a fluorescent tracer molecule. EtBr emits a weak

fluorescence outside the bacterial cells and becomes strongly fluorescent when it enters inside

cells.40

EtBr transport in M. smegmatis occurs by a relative balance between entry into cells by

diffusion, and exit by efflux pumps.39

Uptake of many hydrophobic antibiotics including

rifampicin and fluoroquinolones occurs by diffusion, similar to EtBr.6, 41

Further, EtBr is also

known to be effluxed out by putative drug efflux pumps in M. smegmatis.5, 39, 42

It can thus be

used as an efflux pump substrate to mimic drug efflux. Moreover, for the assessment of drug

transport in real time, a semi-automated fluorometric method based on ethidium bromide (EtBr),

is usually employed in different organisms, including M. smegmatis.30, 31, 40, 43

EtBr is used at a

concentration of only 3 µg/ml (MIC of EtBr being 7 µg/ml) in all our experiments, so that it does

not affect cell viability.

EtBr accumulation increases in presence of PAA-MNP

Figure 4a illustrates the kinetics of EtBr accumulation in presence of varying concentrations of

nanoparticles. Known efflux pump inhibitors, carbonyl cyanide m-chloro phenyl hydrazone

(CCCP, MIC 20 µg/ml) and reserpine (RES, MIC 256 µg/ml) are used as positive controls to

validate the assay. Accumulation of EtBr is lowest in the control group (without any treatment).

However, on addition of nanoparticles, EtBr accumulation increases significantly. As the

extracellular nanoparticle increase from 1 µg/ml to 155 µg/ml, there is a progressive increase in

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net EtBr accumulation (Figure 4a). Similar increase is observed in the presence of CCCP and

reserpine. Accumulation of EtBr reaches a steady state within 1 h of incubation. Normalized end

point relative fluorescence unit (RFU) values [(final RFU– initial RFU)/ initial RFU] are plotted

in Figure 4b. Steady state levels of EtBr accumulated inside the cells in presence of 155 µg/ml

PAA-MNP are comparable to that achieved in presence of 10 µg/ml of CCCP and 64 µg/ml of

reserpine. As a negative control, experiments were performed with uncoated nanoparticles and

polyacrylic acid only without nanoparticles, which did not show any effect on EtBr accumulation

(Figure S4).

EtBr efflux decreases in presence of PAA-MNP

Our results demonstrate that, accumulation of EtBr is higher in presence of nanoparticles and

known efflux pump inhibitors. Thus, to determine if nanoparticles are interfering with the drug

efflux process, we monitor the effect of nanoparticles on EtBr efflux itself. Cells are incubated

with EtBr for 1 h to achieve maximum EtBr accumulation, following which the EtBr preloaded

cells are exposed to EtBr free PBS, both with and without the nanoparticles. Efflux of EtBr from

cells leads to decrease in intracellular fluorescence, which is measured as a function of time

(Figure 4c). The normalized relative fluorescence value at the end of 60 min is compared in

Figure 4d. Nanoparticles at concentrations of 1 µg/ml and 2 µg/ml do not have any significant

impact on efflux kinetics. In contrast, a remarkable effect on efflux inhibition is seen in presence

of more than 4 µg/ml of nanoparticles. Moreover, higher the concentration of nanoparticles,

stronger is the efflux inhibition. Thus, PAA-MNP inhibits the efflux in a concentration

dependent manner from 4 µg/ml to 155 µg/ml. Similar inhibition is detected in presence of,

either CCCP (2.5, 5 and 10 µg/ml) or RES (32 and 64 µg/ml), with respect to control (without

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any treatment). Highest level of efflux inhibition is achieved when nanoparticles are used at a

concentration of 155 µg/ml. This is comparable to efflux inhibition in presence of 64 µg/ml RES.

Microscopic confirmation of the observed effect of PAA-MNP on EtBr transport

Finally, confocal microscopy is used to confirm the increased net EtBr accumulation in

presence of nanoparticles. Figure 5a presents images of cells subjected to EtBr accumulation and

post-efflux in presence of various PAA-MNP concentrations, respectively. The fluorescence

emitted by cells increases in presence of nanoparticles, demonstrating the enhanced

accumulation of EtBr. Complete efflux of EtBr was seen in control cells, whereas intracellular

fluorescence of EtBr increases prominently in presence of nanoparticles. Further, this emitted

fluorescence by each cell is recorded, and the average intensity per cell is plotted as a function of

nanoparticles (Figures 5b and 5c). Intensity plots demonstrate a dose dependent effect for the

tested nanoparticle concentrations (4 µg/ml to 32 µg/ml) on EtBr transport. This confirms that,

both accumulation as well as efflux of EtBr is dependent on nanoparticle concentration.

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Figure 4. Kinetics of EtBr transport in presence of PAA-MNP. (a) EtBr accumulation in M.

smegmatis in presence of PAA-MNP (referred to as NP), and known efflux pump inhibitors, CCCP and

reserpine (RES), (b) comparison of EtBr accumulation between PAA-MNP, CCCP and RES by using

normalized RFU value at the end of 60 min, (c) Kinetics of EtBr efflux in M. smegmatis in presence of

increasing concentration (1 µg/ml to 155 µg/ml) of PAA-MNP, CCCP and RES, and, (d) comparison of

amount of EtBr retained inside the cells at the end of 1 h of efflux (using RFU value at the end of 60 min),

between, PAA-MNP, CCCP and RES.

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Figure 5. Role of PAA-MNP on transport of EtBr by confocal microscopy. (a) Confocal images of

accumulation and post-efflux of EtBr in presence of increasing concentration of PAA-MNP. The phase

contrast images post-efflux are also presented, (b) Intensity plots based on, accumulation of EtBr in

presence of PAA-MNP, and (c) Intensity plots based on, amount of EtBr remaining inside the cells after

efflux. The intensity values are calculated from confocal microscopy images.

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A generic effect of PAA-MNP on other anti-tuberculosis drugs

To generalize, we determine if this approach of using PAA-MNP in combination with

rifampicin can be extended and used with other anti-TB drugs. We investigate the growth

inhibition in presence of a combination of nanoparticles with isoniazid, another first line anti-TB

drug. Combination of PAA-MNP with isoniazid shows higher growth inhibition than isoniazid

alone at same drug concentration (Figure S5a). Moreover, growth inhibition increases with

increase in nanoparticle concentration. This effect is also confirmed by counting the cfu/ml at 4 h

and 24 h (Figure S5b). A four-fold reduction in MIC of isoniazid is observed in presence of

PAA-MNP. Similar synergistic effect on growth inhibition is seen in a combination of

norfloxacin (a model fluoroquinolone) and PAA-MNP. MIC of norfloxacin is reduced by four

folds in presence of PAA-MNP.

Rationale for enhanced drug potency in presence of nanoparticles

Figure 6 demonstrates a likely mechanism for the synergy observed while administering a

combination of drug and nanoparticles, and its relation with drug transport. In absence of

nanoparticles, due to intrinsic resistance mechanisms, there is lower drug uptake possibly due to

efflux of the drug by efflux pumps. Thus, the bacteria is able to resist a higher administered drug

concentration, due to lower intracellular levels. We have shown that, on addition of

nanoparticles, its upake in M. smegmatis increases with extracellular nanoparticle concentration.

The latter blocks drug efflux, thus increasing the intracellular drug concentration. The

mechanism is further elucidated by real time transport kinetics on EtBr. In presence of PAA-

MNP, EtBr efflux reduces and accumulation enhances. Furthermore, efflux inhibition increases

with increase in PAA-MNP concentration (Figure S6a). Therefore, accumulation of EtBr also

correlates linearly with intracellular nanoparticle concentration (Figure S6b). As a result of this

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enhanced intacellular drug level in presence of PAA-MNP, the drug is able to kill the bacteria

even at a subinhibitory concentration of 8 µg/ml. Thus, a synergistic effect on growth inhibition

is observed in presence of a combination of PAA-MNP with drug, and the extent of synergism is

proportional to the concentration of nanoparticles.

PAA-MNP can inhibit drug efflux either by inhibiting the efflux pump directly, or by

interfering in the efflux process, thus, making the pumps dysfunctional. We present a few

possibilities which could lead to efflux inhibition. One of the possible reasons could be

decreased membrane potential in presence of PAA-MNP. Another possibility could be

impairment of driving force required for efflux pumps, in order to efflux the drug outside the

cell. Our preliminary work suggests that membrane potential is reduced in cells treated with

PAA-MNP (data not shown). These mechanisms of efflux inhibition are found in case of proton

ionophores, such as CCCP, which destroy the membrane potential by eliminating the proton

gradient, thus disrupting the driving force for efflux. Since, magnetite nanoparticles have the

ability to interact with proteins 44

and bacterial cell membrane, there is a high possibility that,

they interact with efflux pump proteins on mycobacterial membrane or block the outer channel

of efflux pump, and thus inhibit drug efflux. Finally, nanoparticles can also act as a competitive

inhibitor and compete with antibiotic as a substrate for efflux pumps. In order to investigate the

detailed mechanism of nanoparticles in inhibiting the efflux process, further study is underway.

This is the first study which explains the observed synergy of drug and nanoparticles by proving

that drug efflux is reduced.

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Figure 6. Proposed hypothesis for synergy. Schematic showing the probable mechanism of action of

PAA-MNP, in enhancing the anti-TB drug efficacy.

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CONCLUSIONS

Current study demonstrates that PAA-MNP with an average diameter of 9 nm, are taken up by

M. smegmatis, in direct proportion to the extracellular concentration of nanoparticles and are

non-toxic to the cells themselves. However, nanoparticles are found to interfere with the drug

transport process. In presence of PAA-MNP, efflux inhibition of drug increases by three times

leading to a similar increase in drug accumulation as compared to that without the nanoparticles.

As a result, we obtained a four times increased susceptibility of M. smegmatis to rifampicin,

merely on addition of PAA-MNP. Consequently, rifampicin is effective even at a sub-inhibitory

concentration of 8 µg/ml when administered along with PAA-MNP, compared to its MIC of 32

µg/ml when administered alone. These findings thus suggest the possibility of utilizing this

synergy between PAA-MNP and rifampicin, in overcoming intrinsic drug resistance of

mycobacteria. Through transport studies of EtBr (tracer), we have established for the first time

that the reduced efflux of drug in presence of nanoparticles is the primary cause of observed

synergy in bacterial growth inhibition. Moreover, inhibition of efflux and enhanced drug

accumulation by PAA-MNP is not specific to a particular drug, since similar results for growth

inhibition are obtained when nanoparticles are used in combination with other drugs like,

isoniazid or norfloxacin. Thus, this strategy of using a combination of nanoparticles and drug, to

inhibit bacterial growth, can be generally used for a broad range of anti-TB drugs. While, future

work is underway to understand the molecular mechanism behind efflux inhibition, this route of

reducing efflux by nanoparticles may open further avenues in countering antibiotic resistance of

other bacterial strains too.

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ASSOCIATED CONTENT

Supporting information: Percentage viability of bacteria in presence of PAA-MNP,

chromatograms, mass spectra and standard curve required for intracellular rifampicin

measurements and graphs showing the relation between uptaken nanoparticles with efflux and

accumulation of EtBr are available as supporting information. This material is available free of

charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENT

We would like to thank Sophisticated Analytical Instrumental Facility (SAIF) of Indian Institute

of Technology Bombay for providing FEG-TEM and HR-LCMS facilities. We also thank

Department of Metallurgical Engineering and Material Science, IIT Bombay (for XRD

measurements); Physics Department, IIT Bombay (for providing the VSM facility) and Industrial

Research and Consultancy Centre (IRCC), IIT Bombay (for providing the ultra microtome, VSM

and confocal microscopy facilities). We thank the support of Chettiannan Ravikumar, Minal

Patkari and Kusum Saini, research scholars, IIT Bombay for their useful discussions and help in

initial stages of experiments.

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Table of Contents graphics:

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polyacrylic acid-coated iron oxide nanoparticles for targeting drug resistance in mycobacteria

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