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In vitro Efficacy of Free and Nanoparticle Formulations of Gallium(III) meso-tetraphenylporphyrine Against Mycobacterium avium and Mycobacterium abscessus and Gallium Biodistribution in Mice Seoung-ryoung Choi, Bradley E. Britigan, Barbara Switzer, Traci Hoke, David Moran, and Prabagaran Narayanasamy Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b01036 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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Molecular Pharmaceutics

In vitro Efficacy of Free and Nanoparticle Formulations of Gallium(III) meso-tetraphenylporphyrine Against Mycobacterium avium and Mycobacterium abscessus and Gallium Biodistribution in Mice Seoung-ryoung Choi1, Bradley E. Britigan1,2,3, Barbara Switzer2,3, Traci Hoke1,2,3, David Moran1and Prabagaran Narayanasamy1,*

1

Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA 2

Department of Internal Medicine, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA 3

Research Service, Veterans Affairs Medical Center-Nebraska Western Iowa, Omaha, Nebraska, USA.

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract. The nontuberculous mycobacterial (NTM) pathogens, M. avium complex (MAC) and M. abscessus, can result in severe pulmonary infections. Current antibiotics confront significant challenges for treatment of these NTM infections due to emerging multidrug-resistance. Thus, development of new antibiotics targeted against these agents is needed. We examined the inhibitory activities of Ga(NO3)3, GaCl3, gallium meso-tetraphenylporphyrin (GaTP) and gallium nanoparticles (GaNP) against intra- and extracellular M. avium and M. abscessus. GaTP, an analogue of natural heme, inhibited growth of both M. avium and M. abscessus with MICs in Fefree 7H9 media of 0.5 and 2 µg/mL, respectively. GaTP was more active than Ga(NO3)3 and GaCl3. Ga(NO3)3 and GaCl3 were not as active in Fe-rich media compared to Fe-free media. However, GaTP was much less impacted by exogenous Fe, with MICs against M. avium and M. abscessus of 2 and 4 µg/mL, respectively, in 7H9 OADC media (Fe rich). Confocal microscopy showed that GaNP penetrates the M. avium cell wall. As assessed by determining colony forming units, GaNP inhibited the growth of NTM growing in THP-1 macrophages up to 15 days after drug-loading of the cells, confirming a prolonged growth inhibitory activity of the GaNP. Biodistribution studies of GaNP conducted in mice showed that intraperitoneal injection is more effective than intramuscular injection in delivering Ga(III) into lung tissue. GaTP exhibits potential as a lead compound for development of anti-NTM agents that target heme-bound iron uptake mechanisms by mycobacteria and inhibit growth by disrupting mycobacterial iron acquisition/utilization.

Keywords: Nontuberculous mycobacteria, Mycobacterium avium, Mycobacterium abscessus, Gallium nanoparticle, Biodistribution


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Introduction Nontuberculous mycobacteria (NTM) cause life-threatening pulmonary and extrapulmonary infections. These often occur in patients with some form of immunocompromised state (e.g. AIDS) or abnormal lung structure function (e.g. COPD or cystic fibrosis), but can occur in otherwise normal patients, for example as a consequence of prosthetic devices contaminated with NTM. NTM infections may be caused by both slow growing mycobacteria (e.g. Mycobacterium avium complex (MAC) and M. kansasii) and more rapidly growing mycobacteria (e.g. M. abscessus, M. chelonae, M. fortuitum).1 MAC is the most frequent cause of pulmonary and disseminated NTM infection in immunosuppressed patients. Pulmonary MAC infection most commonly occurs in the lungs of people with intact immune systems, but abnormal lung structure or function.2 In immunosuppressed patients involving some aspect of T cell mediated immunity MAC may result in disseminated disease involving organs, such as the CNS, gastrointestinal tract, spleen and the bone marrow.2 M. abscessus primarily infects lungs, and skin, but it can also infect the CNS.3-4 In addition, it is a life-threatening pulmonary pathogen in patients with cystic fibrosis that appears to be increasing in frequency.5 NTM, like M. tuberculosis (M.tb), have evolved to grow and replicate within host macrophages.6 In general, to be pathogenic bacteria must acquire iron from the host to meet their metabolic and growth needs.7-8 Most bacterial pathogens possess highly efficient iron uptake mechanisms.; 1) Heme, iron-containing porphyrin, from host hemoproteins is the most abundant source of iron.9 Bacterial pathogens either degrade heme to acquire free iron or use it as a cofactor for bacterial hemoproteins after acquisition of heme. 2) High affinity iron chelators, called siderophores, secreted by bacteria compete with the host iron-binding glycoproteins lactoferrin and transferrin for local ferric iron. 3) A selective group of pathogens possesses membrane receptors that



recognize the host iron-binding proteins lactoferrin and transferrin and confer the ability of the bacteria to remove iron from these glycoproteins. 10 Similarly, intracellular pathogens like M.tb, M. abscessus and MAC need to acquire iron for survival.11-12. However these intracellular pathogens do not have direct access to extracellular sources of iron. Instead, they must access intracellular sources of host iron. Mycobacteria synthesize high affinity iron-chelating siderophores, such as mycobactin, carboxymycobactin and exochelin, to capture iron from the host.8, 12 Siderophore production has been shown to be required for optimal growth of mycobacteria in macrophages.13 Mycobacteria also have a separate mechanism to acquire iron from heme.14 Given the critical requirement for iron for growth and metabolism, bacterial iron acquisition has been identified as a potential target for novel antimicrobial agents. Gallium-based complexes and compounds have received increasing attention as a means to interrupt iron acquisition and/or utilization. Since Ga(III) is physicochemically similar to Fe(III) in terms of electron configuration and coordination chemistry, Ga(III) is able to bind many iron-dependent proteins and chelating agents (including heme) with similar affinity to iron. Inability to distinguish Ga(III) from Fe(III), results in Ga(III) binding to iron binding sites in bacterial proteins. This disrupts essential iron-dependent biological processes that rely on the redox properties of iron (reduction of Fe(III) to Fe(II)) because Ga(III) cannot be reduced to Ga(II) under biological conditions.15 Ga(NO3)3 is FDA approved for the treatment of hypercalcemia of malignancy and known to inhibit bone resorption and increase calcium content of bone.16-18 Ga(III) has been shown to be an effective antimicrobial agent against broad spectrum of bacterial species.19-20 For example, Ga(NO3)3 exhibits antimicrobial activity against Francisella sp., Pseudomonas


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aeruginosa, Acinetobacter baumannii and mycobacteria within mononuclear phagocytes by disrupting Fe acquisition.11, 21-24 Recently, heme-mediated iron acquisition has been targeted for the development of porphyrincomplexed agents. Many mycobacteria, Gram-positive and Gram-negative bacteria possess efficient heme transport mechanisms for iron uptake. Thus, metalloporphyrins are potential candidates. Porphyrin analogues - complexed with a non-iron metal - have a great advantage for targeted drug delivery and antibacterial drug due to the similarity of heme transporters. Gallium, indium, and manganese meso-tetra-4-sulphonatophenyl porphyrins exhibited inhibitory activity against Gram-negative Y. enterocolitica, Gram-positive S. aureus, and M. smegmatis with low MICs in iron-limited growing conditions.25 In contrast, high MICs were observed without the metals, indicating the porphyrin alone lacked antibacterial activity.25 Gallium protoporphyrins also showed antimicrobial activity against A. baumannii and M. abscessus.26-27 Nanoparticles offer several advantages to traditional drug delivery methods, providing efficient and targeted delivery and a sustained release of drug to infection sites. In previous studies, we found that Ga(III) nanoparticles exhibited greater growth inhibition of pathogens residing in macrophages

compared

to

free

Ga(III).19

Nanoparticles

encapsulating

Ga

meso-

tetraphenylporphyrin (GaTP), which is structurally similar to heme, were shown to inhibit the growth of both HIV and mycobacteria (both M.tb and M. smegmatis) residing within MDMs or THP-1 macrophages.20, 28 These nanoparticles released Ga(III) over 15 days at levels sufficient to inhibit mycobacterial growth.19, 29-30 31 Since iron is also an essential cofactor for the growth of M. abscessus and M. avium,32 in the present work, we examined the potential of Ga nanoparticles and GaTP for the treatment of



NTM (MAC and M. abscessus) in vitro and studied the biodistribution of Ga(III) following Ga nanoparticle administration in mice. Materials and Methods Reagents. Gallium(III) meso-tetraphenylporphyrine chloride (GaTP) was purchased from Frontier Scientific (Logan, Utah, USA). Fluorescein isothiocyanate, GaCl3, and Ga(NO3)3 were purchased from ACROS (New Jersey, USA). Polyvinyl alcohol (PVA) was purchased from MP Biomedicals, LLC (Illkirch, France). Difco™ Middlebrook 7H9 broth and BBL™ Middlebrook OADC Enrichment media were purchased from BD (Sparks, MD, USA). Tryptic soy agar (TSA) and 7H10 agar plates were purchased from Remel Inc. (Lenexa, KS). RPMI 1640, L-glutamine, HEPES, sodium pyruvate, and fetal bovine serum were purchased from HyClone (Logan, UT). Fe-free 7H9 medium was prepared as described previously.11 Mycobacterial Strains. Mycobacterium avium Chester (ATCC® 700898™) from human and (ATCC® 35719™) from chicken, and M. abscessus (ATCC® 19977™) were purchased from ATCC. Clinical isolates of M. abscessus and M. avium were provided by the Clinical Pathology/Microbiology Laboratory at Nebraska Medicine, Omaha, NE. All NTMs were cultured at 37°C in Fe-free 7H9 medium for 2-3 days prior to use in MIC and macrophage infection experiments. Preparation and characterization of nanoparticles containing Ga(III). F127 block copolymer was used to prepare nanoparticles containing GaTP. A single emulsion method was used to formulate nanoparticles (GaNP). GaTP (20 mg) and F127 (20 mg) were dissolved in CH2Cl2 (2 mL) at room temperature and stirred overnight. The organic solution was slowly added to 4 mL of 1% polyvinyl alcohol (PVA) with vortexing. This was followed by sonication


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(Sonic Dismembrator Model 500, Fisher Scientific) on ice (amplitude 40%, 1 minute pulse with 3 minute resting on ice, 5 cycles). The sonicated mixture was added to 1% PVA (20 mL) and stirred overnight at room temperature to remove CH2Cl2. The nanoparticles were washed with distilled water three times using a centrifuge (20,000 x g, 30 min). Finally, the nanoparticles were suspended in water containing trehalose (5% w/v, Sigma-Aldrich), lyophilized and stored at –80°C until used. Determination of the minimum inhibitory concentrations. Broth serial dilution methods were used to determine the MICs of GaTP, GaCl3, Ga(NO3)3 and Ga nanoparticles against M. avium and M. abscessus. The desired mycobacterial species (5 x 105 CFU/mL) was added to each microtitration plate well of Fe-free 7H9 media

11

or the media supplemented with 10% OADC

containing serial dilutions of the antimicrobial agents. The plate was incubated for 7-8 days and 2-3 days for M. avium and M. abscessus, respectively. MICs were determined by OD600 or resazurin reduction assay.33 Measurement of drug uptake by M. avium-infected or non-infected THP-1 macrophages. THP-1 cells (7.5 × 105 per well) were differentiated in RPMI 1640 containing 10% FBS, phorbol myristate acetate (PMA, 100 nM), 10 mM sodium pyruvate, 50 µg/mL gentamicin and 10 mM HEPES (pH 7.0) at 37°C in 5% CO2 humidified atmosphere for 2 days. The cells were then “rested” in media without PMA for 1 day at 37°C. After washing with PBS buffer, THP-1 macrophages or M. avium infected THP-1 macrophages (MOI = 10, 1 hour) were treated with 300 µM Ga nanoparticles or GaTP in RPMI 1640 containing 1% fetal bovine serum for up to 24 h. The Ga(III) content of the THP-1 cells was then determined at 1, 4, 8 and 24 h incubation. Adherent cells were washed with PBS buffer three times and collected by treating them with



0.25% trypsin/EDTA at the designated incubation time. The viable cells were counted and centrifuged at 500 × g for 10 min. The supernatants were discarded. Cell pellets were sonicated in 200 µL of methanol and centrifuged at 21,000 × g at 4 °C for 15 min. Ga(III) in methanol was then quantified by UV spectroscopy (A588nm) and normalized to the number of viable cells. Determination of M. avium and M. abscessus growth inside macrophages. The differentiation of THP-1 cells and primary monocytes (7.5 x 105 cells/well/mL in a tissue culture 24 well plate) was carried out as described.29 For the post-infection treatment study, the macrophages were incubated with M. avium (MOI=10, 1 hour) or M. abscessus (MOI=1, 1 hour) in RPMI1640 media containing 1% FBS (no gentamicin) at 37°C in a 5% CO2 humidified atmosphere. After washing with PBS, the infected cells were further incubated for 24 h, followed by treatment with gallium nanoparticles or free drug (300 µM) in RPMI 1640 medium containing 1% FBS for 24 h at 37°C. The nanoparticle loaded-macrophages were washed with PBS and incubated for the desired number of days with media change every 2 days. The cells were then lysed using 0.05% SDS at desired days post-infection for determination of colony forming units (CFU). The lysed cells were centrifuged at 14,000 × g for 15 min and the pellets were resuspended, serially diluted in sterile 7H9 and plated onto 7H10 agar plates (M. avium) or TSA agar plates (M. abscessus) with colonies counted later. Total protein concentrations from the supernatant were determined using the BCA assay for normalization. For the study in which the macrophages were loaded with drug prior to infection, THP-1 macrophages or monocyte-derived macrophages (MDM) were treated with 300 µM Ga nanoparticle or GaTP for 24 h. The treated cells were infected with M. avium (MOI = 10, 1 hour) or M. abscessus (MOI = 1, 1 hour) in RPMI media containing 1% FBS (no gentamicin) at days


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5, 10 and 15 following free drug or nanoparticle treatment. After infections, the cells were washed with PBS to remove extracellular M. avium or M. abscessus and then placed in media containing gentamicin. The cells were then lysed at the designated days using 0.05% SDS for determination of CFU. Measurement of drug uptake by M. avium. M. avium (from human) were grown in 7H9 containing OADC until OD600 reached ~0.4 and then incubated with 20 µg/mL of Ga nanoparticles for defined time periods (1-8 hour). The culture was washed with PBS 5-7 times by centrifuging at 1000 × g for 5 min. The pellet was resuspended in methanol and sonicated. GaTP amounts were determined by UV spectroscopy (A588nm). Visualization of Ga (III) nanoparticle uptake by M. avium. M. avium (from human) were grown in 7H9 containing OADC until OD600 reaches ~0.5 and then incubated with DAPI (10 µg/mL) at room temperature for 30 min. After several washings with PBS by centrifugation (14,000 × g, 15 min), the mycobacteria were incubated with EtBr-labeled Ga nanoparticles (supporting information) at 37°C for 1 h. The culture was centrifuged and washed with PBS at 4,000 × g for 10 min, 6 times at 4°C. The pellet was resuspended in PBS and 5 µL of the M. avium suspension was trapped between a glass slide and a coverslip. Nanoparticle uptake by M. avium was visualized using confocal imaging using a Zeiss LSM 710 laser scanning microscope (Carl Zeiss, Inc. Thornwood, NY. USA). Biodistribution of Ga(III) in mice. Balb/cJ mice were administered 10 mg/kg GaM (Mannoseconjugated GaNP), 10 mg/kg Ga(NO3)3 or 2 mg/kg GaTP at designated days via intramuscular or intraperitoneal injection. Ga(NO3)3 was dissolved in sterilized PBS and GaTP was suspended in 20% DMSO in sterilized PBS. GaM was suspended in sterilized PBS. Mice were dissected to



determine distribution of Ga(III) in organs. Organs were washed with PBS buffer and homogenized in methanol using a tissue homogenizer (Omni international, GA, USA). After centrifuging homogenized samples at 14,000 x g for 20 min at 4°C, the supernatants were analyzed using ICP for Ga(III) as previously described.19 Statistical analysis. All experiments were performed in triplicate, and data are the mean ± SEM of triplicate experiments (n = 3). Statistically significant variance (p) for collected data was determined by one-way or two-way Anova from GraphPad Prism 6.0 (GraphPad Software, Inc. La Jolla, CA).

Results Table 1. Minimum inhibitory concentration of gallium (III) compounds against NTM and effect of iron availability.

MIC M. avium (µg/mL) M. abscessus (µg/mL) Media Fe free 7H9 7H9 OADC Fe free 7H9 7H9 OADC GaTP 0.25 - 0.5 2 1-2 4a b GaCl3 4 >1024 (16) 16 >1024 Ga(NO3)3 4-8 >1024 (32)b 32 >1024 TPP >128 >128 >128 >128 a MIC90, bMIC50. 7H9 OADC contains 40 µg/mL ferric ammonium citrate. TPP: tetraphenyl porphyrin. Human M. avium (ATCC® 700898™), M. abscessus (ATCC® 19977™).

M. avium and M. abscessus are more susceptible to gallium meso-tetraphenylporphyrin (GaTP) than gallium salts. It has been shown that Ga(NO3)3 inhibits the growth of intracellular and extracellular bacteria, including M.tb, M. abscessus and P. aeruginosa, by disrupting microbial iron metabolism.11,

22, 34

We investigated if a hemin mimetic GaTP is an effective

antimicrobial agent against M. avium and M. abscessus under iron-free and in standard ironreplete 7H9 media (7H9 supplemented with OADC) and compared the results to those obtained


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with GaCl3 and Ga(NO3)3 (Table 1). In the iron-free medium (no OADC), each of the three tested gallium compounds inhibited growth of both mycobacterial species with MICs of 0.250.5, 4 and 4-8 µg/mL against M. avium and 1-2, 16 and 32 µg/mL against M. abscessus for GaTP, GaCl3 and Ga(NO3)3, respectively (Table 1). The MIC values for the three gallium compounds were ~4-8 times lower for M. avium than M. abscessus (Table 1). In 7H9 supplemented with OADC, which contains 40 µg/mL ferric ammonium citrate GaTP showed significant growth inhibition of M. avium, with a MIC of 2 µg/mL, and M. abscessus, with MIC of 4 µg/mL. In contrast, the two gallium salts (GaCl3 and Ga(NO3)3) did not completely inhibit growth of M. avium and M. abscessus at concentrations up to 1024 µg/mL (Table 1). However, GaCl3 and Ga(NO3)3 inhibited M. avium growth by 50% at 16 and 32 µg/mL respectively (Table 1). Likewise, M. abscessus growth was reduced by GaCl3 and Ga(NO3)3 by 50% at 16 and 32 µg/mL, respectively (Table 1). Tetraphenylporphyrin (TPP) without gallium did not inhibit the growth of either NTM in iron-free media (Table 1). These results suggest that both mycobacteria are more susceptible to GaTP than other gallium salts and that the growth inhibitory effect of GaTP requires the presence of Ga. Since GaTP shows significant bacterial growth inhibition under both iron limited and iron-replete growth conditions, disruption of iron uptake via a hememediated transporter in M. avium and M. abscessus may be a more efficient way35 to develop anti-mycobacterial agents than targeting mycobacterial siderophore-mediated iron uptake.



Figure 1. Dose response curves of the effect of three different gallium compounds in the presence and absence of two different Fe(III) sources, Fe(NO3)3 or hemin, on the growth of M. avium (human strain) or M. abscessus in vitro. M. avium (panels A-C) or M. abscessus (Panels D-F) were inoculated into Fe free 7H9 to which in some cases was added either 50 or 100 µg/mL Fe(NO3)3 or hemin. Growth inhibition of the mycobacterial species was determined (expressed as the percentage of OD600 measured in the presence of the gallium compound relative to its absence) in the presence of increasing concentrations of Ga(NO3)3 (Panels A and D), GaTP (gallium tetraphenylporphyrin, Panels B and E), or Ga nanoparticles of GaTP (GaNP, Panels C and F). Results shown are the mean +/- SEM of 3 determinations.

The growth inhibition of M. avium and M. abscessus by gallium compounds is rescued by exogenous Fe(III) and hemin. We previously reported that Ga(NO3)3 inhibits growth of intracellular and extracellular M. abscessus and this growth inhibition is reversed by exogenous Fe(NO3)3 supplementation.27 In the present work, we extended this work to M. avium and compared the results to those observed with M. abscessus. Two Ga compounds were initially tested - Ga(NO3)3 and GaTP. We also tested whether there was a difference in the ability of Fe(NO3)3 and hemin to reverse the growth inhibitory effect of these Ga compounds.


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As shown in Figure 1, M. avium was significantly less susceptible to Ga(NO3)3 when growing in Fe(NO3)3 -supplemented medium with a >60 fold higher MIC (Figure 1A). Exogenous Fe(III) also reversed the ability of Ga(NO3)3 to inhibit M. abscessus growth (Figure 1D). Interestingly, Ga(NO3)3 inhibition of both M. avium and M. abscessus under iron-free conditions was also partially reversed by the addition of hemin (Figures 1A and 1D). The protoporphyrin mimic, GaTP, showed more potent growth inhibitory effects than Ga(NO3)3 against M. avium, with MICs of 1 and 2 µg/mL in media supplemented with 50 and 100 µg/mL Fe(NO3)3, respectively (Figure 1B). Furthermore, the addition of 50 µg/mL hemin did not completely rescue the growth of M. avium inhibited by GaTP. The MIC under those conditions was 2 µg/mL. Although the MIC was 8-fold higher than the MIC in iron-free condition, GaTP still exerted strong inhibitory activity against M. avium. The inhibitory effect of GaTP was also tested in medium supplemented with excess Fe (III) (7H9 + OADC + 100 µg/mL Fe(NO3)3, Figure 1B). Although GaTP did not completely inhibit the growth of M. avium at up to 8 µg/mL in the presence of excess Fe (III), the growth was reduced by 50% at concentration of 1 µg/mL. The observed results suggest that M. avium may prefer to use a heme-mediated transporter as an iron source. GaTP potently inhibited (MIC of ~1-2 µg/mL) against M. abscessus under iron-free condition (Table 1 and Figure 1E). Growth of M. abscessus was fully reversed by the addition of each of the two Fe(III) sources (Figure 1E). The inhibitory effect of GaTP against M. abscessus was more dramatically affected than M. avium by iron-rich conditions (Figure 1E versus 1B). Nanoparticles of GaTP inhibit mycobacterial growth. Given the above data, the effect of encapsulating GaTP into nanoparticles (GaNP) as a means to enhance anti-mycobacterial activity



against M. avium and M. abscessus was tested. This was carried out in both iron-free and iron replete media (Figure 1C and 1F). GaNP was prepared from F127 polymer and GaTP using an emulsification-evaporation method and characterized using DLS as described in the methods section. The size of the nanoparticles was 273.3 ± 0.7 nm with PDI 0.136 ± 0.012 and the ζ potential was +23.2 ± 0.8. When the susceptibility of M. avium and M. abscessus to GaNP was tested (Figure 1C and 1F), GaNP was not as effective as the free GaTP in inhibiting either mycobacterial species, regardless of whether the testing was done in iron-free or iron-replete media.

Figure 2. Growth curves of M. avium in Fe-free or Fe-rich 7H9 media. Fe-free 7H9 (+) medium was supplemented with 50 µM of the Fe source indicated and M. avium growth measured as OD600 over 15 days. Data are the means ± standard error for n = 3 at each time point. *P < 0.0001.

Fe (III) sources do not increase the growth rate but prolong exponential growth of M. avium. Growth inhibition studies by gallium compounds above led us to investigate the iron source preference of M. avium, as such data could assist in identifying a specific iron-uptake mechanism to target in the development of anti-mycobacterial agents. Thus, growth curves were determined by culturing M. avium in media supplemented with three different iron sources, Fe(NO3)3, ferric ammonium citrate and Fe-protoporphyrin (hemin) as shown in Figure 2 and S1. Interestingly, the M. avium growth rate did not vary among the iron sources. There was no significant difference in the maximum growth rates between iron salts and hemin. Instead, when


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cultured in iron-rich media M. avium exhibited a more prolonged exponential growth phase resulting in a larger bacterial population at stationary phase.

Table 2. MICs of clinical isolates of M. abscessus and M. avium. MIC (µg/ml) MIC (µg/ml) M. abscessus M. avium GaTP Ga(NO3)3 GaTP Ga(NO3)3 UNMC 1362 4 32 UNMC 1587 8 16 UNMC 1374 128 64 UNMC 1623 4 16 UNMC 1423 4 64 UNMC 1643 1 8 UNMC 1477 8 64 UNMC 2638 4 64 -

GaTP inhibits clinical isolates of M. abscessus and M. avium. GaTP inhibited growth of both ATCC strains (ATCC® 700898™ and ATCC® 19977™, Table 1) that utilize heme as an iron source. These results prompted us to investigate the effect of GaTP against clinical isolates of both NTMs. Clinical strains of M. abscessus and M. avium were tested with growth inhibition determined in Fe-free 7H9 medium supplemented with increasing concentration of GaTP or Ga(NO3)3 (Table 2). GaTP inhibited the growth of clinical isolates of M. avium at 1-8 µg/mL and M. abscessus at 4-8 µg/mL, except the UNMC 1374 strain. However, Ga(NO3)3 displayed 4-16 fold higher MICs against both clinical isolates than GaTP. The results support the ability of both NTM to use heme as a major iron source. Interestingly, UNMC 1374 strain is more resistant to GaTP and we are currently investigating the mechanism responsible.



Figure 3. Confocal micrographs (A) and Quantification (B) of Ga(III) nanoparticle uptake by M. avium. 20 µg of Ethidium-labeled GaTP containing nanoparticles (GaNP, red) were incubated with M. avium. They were then stained with DAPI (blue). The amount of GaNP uptake by M. avium was then measured using UV spectroscopy. Data represents the means ± standard error for n = 3 at each time point.

Gallium nanoparticles penetrate and inhibit the growth of M. avium. We hypothesized that GaNP would penetrate and disrupt iron metabolism of M. avium similar to M. tuberculosis (importance of PPE surface proteins for heme utilization)35 and Pseudomonas aeruginosa (report on inhibition of hemophore against heme uptake and its x-ray structure with metal complexes).36 To test this, we prepared ethidium (Et)-conjugated F127 polymer to study its fluorescence on binding to DNA. EtGaNP (Et-labeled nanoparticle containing GaTP) was formulated using a sonication method (Supporting information). As shown in Figure 3A, enhanced ethidium staining from EtGaNPs was observed inside bacteria and also found to be co-localized with DAPI within M. avium, indicating that the GaNP penetrated the pathogen and ethidium was bound to DNA. We also measured GaNP uptake by M. avium (OD600 = 0.4, Fe-free 7H9) as a function of time and found that most of the GaNP (20 µg) resided within M. avium by 8 hours (Figure 3B).


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Figure 4. GaNP uptake by noninfected- and infected-THP-1 macrophages. A) THP-1 macrophages were treated with different concentrations of GaNP (100-500 µM) and incubated for designated times. The cells were harvested and the amount of gallium at each time point was measured and normalized to the number of viable cells. Data represents the means ± standard error for n = 3 at each time point. B) M. avium-infected THP-1 macrophages were treated with GaNP (300 µM). The cells were harvested and the amount of gallium at each time point was measured and normalized to the number of viable cells. Data represent the means ± standard error for n = 3 at each time point.

GaNP uptake by noninfected and M. avium-infected THP-1 macrophages. Drug nanoparticle formulations have the potential to increase and maintain more sustained drug levels within macrophages. Therefore, we determined GaNP uptake by infected- and noninfected THP-1 macrophages. First, we tested various concentrations of GaNP to profile uptake by THP-1 macrophages. The uptake increased in a time- and concentration-dependent manner, reaching maximum uptake at 8 hours of incubation (Figure 4). Slow release of GaNP from the noninfected macrophages was observed after it peaked at 8 hours. The THP-1 macrophages contained ~58 and 28 µg GaNP/million cells at 8 hours when treated with 500 and 300 µM GaNP, showing 22% and 17% uptake efficiency, respectively. On the other hand, GaNP uptake by M. aviuminfected macrophages continued to increase over the entire 24 h period of study. At 24 hours, approximately 50% higher uptake of GaNP was seen in the infected macrophages compared to noninfected macrophages treated with equal concentration of GaNP. The infected macrophages demonstrated higher uptake at 1 and 4 h with 72 and 60% more uptake, respectively, compared to the corresponding macrophages, while only 20% higher uptake was observed at 8 h. The mechanism whereby this higher uptake occurred is unclear. Gallium nanoparticle-treated macrophages resist mycobacterial infection and inhibit the growth of intracellular M. avium and M. abscessus within macrophages. We previously reported the inhibition of M. abscessus growth residing within macrophages by Ga(NO3)3 and Ga protoporphyrin and found that Ga protoporphyrin to be 20 times more effective than Ga(NO3)3 in inhibiting M. abscessus growth in THP-1 macrophages.27 Correspondingly, we compared the



ability of Ga nanoparticles (GaNP)19 to inhibit the growth of M. avium and M. abscessus occurring within THP-1 macrophages or MDMs. In the first set of experiments (Figure 6), macrophages were treated with GaNP or the free drug GaTP before infection. At defined time periods (5, 10, and 15 days) after incubation of the THP-1 cells with GaNP or GaTP, the cells were infected with one of the two mycobacteria species. Twenty four hours later the cells were harvested and mycobacterial CFU determined. At the first time point after THP-1 cell incubation with the drugs tested (5 days) and beyond (days 10 and 15), GaTP failed to inhibit growth of M. avium (both human and chicken strains) or M. abscessus (Figs 6A 6B, and 6C). The lack of effect of GaTP on mycobacterial growth is likely due to our previous finding that macrophages do not retain sufficient GaTP to inhibit mycobacterial growth.29-30 In contrast, GaNP significantly inhibited M. abscessus and M. avium growth within macrophages up to 15 days after the cells were loaded with the compound (Figure 6A-C), indicating prolonged resistance of GaNP-pretreated macrophages to infection and implying prolonged retention of the nanoparticles and sustained release of biologically active Ga(III). These data suggest that during active infection GaNP could help treat the infection by eliminating the cycle of infection of newly arriving mononuclear phagocytes that had taken up GaNP. It also raises the possibility of GaNP as prophylaxis for patients at high risk of infection with M. avium or M. abscessus, such as those with CF.

Figure 6. Growth inhibition of intracellular mycobacteria in GaNP- and GaTP-pretreated macrophages. MDM (panels A and C) or THP-1 cells (Panel B) were incubated with either vehicle, GaNP, or GaTP for 24 hours and then


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washed. At defined time points after drug loading, the cells were infected with A) M. avium (MOI = 10) from chicken, B) M. avium (MOI = 10) from human, or C) M. abscessus (MOI = 1). After 24 hours of infection, the cells were harvested and mycobacterial CFU determined. Anova was used to calculate significant differences (GraphPad Prism 6.0). In each case GaNP provided more sustained inhibition of mycobacterial growth. The data shown are the mean ± SEM, performed in triplicate. *p

meso-tetraphenylporphyrine Against ... - ACS Publications

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