Intracellular delivery of nano-formulated antituberculosis drugs enhances bactericidal activity

Tuberculosis kills more people worldwide than any other infectious disease. Treatment requires multiple drug therapy administered over long periods (6 – 24 months). The emergence of multidrug-resistant strains is a major problem, and with few new drugs in the pipeline, a novel modus operandi is urgently required. Solid drug nanoparticles (SDNs), a new development in nanomedicine, offer a fresh therapeutic approach. Here, we show that SDNs are more effective (50-fold) at killing pathogenic mycobacteria than aqueous forms of the same drug and can target mycobacteria internalised by macrophages, where bacilli reside. We demonstrate synthesis of dual and triple drug loaded SDNs, facilitating combination tuberculosis therapy. Our results suggest that by employing SDNs of existing antibiotics, it may be possible to improve drug delivery and therefore reduce drug dosage to lessen side effects and ﬁ ght drug resistance.


Introduction
The use of nanoparticles (NPs) for drug delivery systems is an expanding area of research (Vadakkan et al. 2013;Pedrosa et al. 2014;Choudhary and Kusum Devi 2015;Patel et al. 2013). The antimicrobial properties of some NPs are well documented (Donnellan immunodeficiency virus (HIV) (Mcdonald et al. 2014;Williams et al. 2015).
Solid drug nanoparticles (SDNs) are a novel development in nanotechnology (Mcdonald et al. 2014). They are not a drug carrier system; they are nanosized amorphous particles, constructed of active pharmaceutical ingredients (APIs) (Siccardi et al. 2016). There are many advantages to creating excipient stabilised nanoparticulate versions of FDA-approved therapeutics, for example, they use existing APIs, thus saving time and the exorbitant costs involved in novel drug development and testing, they increase bioavailability, and they may offer an opportunity to lower treatment dose.
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), kills up to 2 million people each year with one third of the world's population estimated to have latent TB (WHO 2016). Drug-resistant strains and the susceptibility to TB infection of HIV patients heighten this global crisis, compounded by the development of very few drugs in the past 40 years (WHO 2014;WHO 2015;WHO 2016). Treatment periods are very long (6-24 months) requiring multiple drug administration, which is highly expensive and often unsuccessful because of patient noncompliance. Current treatment typically involves an initial fourdrug regimen: rifampicin (RIF), isoniazid (INH), pyrazinamide (PZA), and ethambutol (WHO 2016;Pandey and Ahmad 2011).
Herein, we describe the manufacture of anti-TB SDNs and hypothesise that the delivery of existing antibiotics in the form of SDNs would enhance toxicity to mycobacteria and facilitate delivery to intracellular compartments due to colocalisation following phagocytic uptake.

Experimental Section
Aqueous drug preparation Rifampicin and INH were purchased from Sigma-Aldrich ® (Poole, UK) and prepared (and stored) following product documentation. Stock solutions were stored in tin-foil wrapped bottles at 4°C. From these stock solutions, appropriate serial dilutions were made in Middlebrook 7H9 broth for the desired concentrations.

SDN manufacture
Solid drug nanoparticles of first line TB drugs were prepared similarly to Mcdonald et al. (2014) as outlined later. The incorporation of a fluorescent dye (1,1 0 -dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD)) to confer a fluorescent signal to each particle was included in some incidences for imaging purposes. Both freeze and spray drying methods were used, essentially producing the same NP dispersions, while spray drying only was used to scale up the samples as freeze drying could only produce relatively small quantities (up to 10 mg).
Preparation of emulsion-spray-dried 53% TRIPLE drug powders containing RIF, INH, PZA (TRIPLE SDN) A mass of 51.2-mg PZA, 20-mg RIF, and 8.8-mg INH were weighed directly into a sample vial. A volume of 1-mL DCM was then added and the powders mixed (inverted and vortexed). Stock solutions of Kollicoat ® Protect (22.5 mg/mL in water) and Hyamine ® (22.5 mg/mL in water) were dissolved. The aqueous stock solutions were added to the 1-mL API mix in DCM in the ratio 267:0.44 (mL) plus 0.89-mL water. The final solid mass ratio was therefore 34% PZA, 13% RIF : 6% INH : 40% Kollicoat ® Protect : 7% Hyamine ® in a 1:4 DCM to water mixture (total volume 5 mL, 53% combined API). The sample was emulsified using a Hielscher UP400S ultrasonic processor equipped with H7 Probe at 100% output (22 W) for 15-20 sec. Immediately after emulsification, the sample was spray dried on a benchtop spray dryer (BUCHI Mini-290) using an air-atomizing nozzle and compressed air as the drying gas. Spray drying process conditions were 5 mL/min solution flow rate and 65°C outlet temperature (120°C inlet temperature). The orange powder was collected and stored in the dark at room temperature.
Preparation of emulsion-spray-dried 52% TRIPLE drug powders containing RIF, INH, PZA labelled with 1% of the dye DiD (TRIPLE SDN + DiD) Preparation followed the same procedure as described previously; however, a stock solution of 53-mg/mL DiD was prepared. A mass of 50.7-mg PZA, 19.8-mg RIF, and 8.7-mg INH were weighed directly into a sample vial. A volume of 97-mL DCM was then added and the powders mixed. To this, 0.03 mL of DiD stock was added. The aqueous stock solutions were added to the 1-mL API mix in DCM in the ratio 267:0.44 (mL) plus 0.89-mL water. The final solid mass ratio was therefore 33.7% PZA, 13% RIF : 6% IZH : 1% DiD : 40% Kollicoat ® Protect : 7% Hyamine ® in a 1:4 DCM to water mixture (total volume 5 mL, 52% combined API, 1% DiD).
Preparation of emulsion-spray-dried 30% DUAL drug powders containing RIF, INH Preparation followed the same procedure as described previously for the TRIPLE SDN; however, 40-mg RIF and 20-mg INH were weighed directly into a sample vial. Stock solutions of Kollicoat Protect ® (45 mg/mL in water) and Hyamine ® (45 mg/mL in water) were dissolved. The aqueous stock solutions were added to the 1-mL API mix in DCM in the ratio 5.34-mL Kollicoat Protect ® : 0.88-mL Hyamine ® plus 1.78-mL water. The final solid mass ratio was therefore 20% RIF : 10% IZH : 60% Kollicoat Protect ® : 10% Hyamine ® in a 1:4 DCM to water mixture (total volume 5 mL, 30% combined API).
Powder X-ray diffraction RIF SDNs were stored at 25°C in their powder, monolith form for 1 month at 25°C. Analysis was carried out by powder X-ray diffraction (p-XRD). p-XRD measurements were collected in transmission mode on solid monolith samples held on a thin Mylar film in aluminium well plates on a Panalytical X'Pert PRO MPD instrument with X'PERT OPERATOR INTERFACE (version 1.0b) software.

SDN preparation and characterisation
All SDNs were sub sampled under sterile conditions and kept in the dark at ambient temperature until use. SDNs were diluted in sterile 2% (vol/vol) foetal calf serum (prepared in sterile distilled water) at a concentration of 1 mg/mL and vortexed and then placed on ice. SDNs were then diluted in Middlebrook 7H9 broth supplemented with 10% (vol/vol) Middlebrook ADC enrichment (albumin/dextrose/catalase (Becton Dickinson, UK)), 0.2% (vol/vol) glycerol, 2-μg/mL mycobactin J (7H9). Agglomeration status, Zeta potential, and hydrodynamic sizes were analysed by dynamic light scattering (DLS) using plastic disposable cuvettes at an SDN concentration of 100 μg/mL using a Zetasizer Nano-ZS instrument (Malvern Instruments Ltd., UK). Each sample was measured in triplicate.

Screening the antimicrobial properties of SDNs
The antimicrobial properties of SDNs and aqueous drugs were screened using the previously described assay (Donnellan et al. 2015). Briefly, Map K10/GFP was cultured in 7H9K + until the exponential stage of growth. Sterile, black, 96 well plates (Nunc, Thermo Fisher) were seeded with Map K10/GFP (2 × 10 6 CFU/100 μL/well). The bacteria were then exposed to SDNs and aqueous drugs in 7H9 at concentrations ranging from 1.875 ng/mL to 4 μg/mL (final 200 μL volume per well) and incubated for up to 7 days at 37°C with medium agitation (250 rpm) in the dark. Blank wells, medium only (no bacteria, no SDN/drug) and SDN/drug (no bacteria) were included as controls to check for SDN/drug interference. Fluorescence was measured using a GloMax ® Multi+ plate reader (Promega, UK) (Ex/Em 490/510-570 nm). Bacterial fluorescence was monitored immediately after SDN/drug addition (day 0), then at the same time each day for 7 days. All experiments were performed in triplicate, a minimum of three times, under sterile conditions.

Data analysis
Results expressed in arbitrary fluorescent units against time (days) were transformed and normalised to obtain a standard dose response relationship for comparison between SDNs and aqueous RIF using a data analysis protocol for screening the efficacy of antimycobacterial substances as previously described (Donnellan et al. 2015), where G is the fluorescent units, G 0 is the bacterial growth at time 0, β is the growth rate (per day), and t is the time (days).

Phage amplification assay
Detection and enumeration testing was carried out according to Rees and Botsaris (2012). Briefly, to perform the phage amplification assay, cell culture samples (200 μL) was mixed with 800-μL Middlebrook 7H9 broth supplemented with ADC and CaCl 2 (2 mmol/L). Samples (1 mL) containing mycobacteria were mixed with 1 × 10 8 mycobacteriophage D29 (100 μL) and incubated for 1 h. Remaining extracellular phage were then inactivated using a virucide treatment (100 μL 10 mmol/L ferrous ammonium sulphate for 6 min). The virucide was then neutralised by dilution using 5-mL Middlebrook 7H9 broth, and the phage-infected cells were plated in a lawn of fast growing Mycobacterium smegmatis (1 mL, 10 7 cfu/mL) using soft agar (0.8% w/v). Lysis of the infected cells at the end of the lytic cycle leads to the formation of plaques in the lawn of M. smegmatis. Because each plaque formed represents one mycobacterial cell in the original sample the assay can be used to enumerate the mycobacterial cultures (data reported as pfu/mL).

Cytotoxicity testing
Cytotoxicity of the SDNs on macrophages was assessed using the Alamar Blue ® assay, a cell viability test by measuring mitochondrial activity. Macrophages (murine macrophage cell line J744A.1) were cultured in 75cm 2 T5 flasks in complete medium (RPMI 1640 (Thermo Fisher Scientific, UK) supplemented with 10% heat inactivated foetal calf serum, 100-μg/mL streptomycin, 100-IU/mL penicillin, 0.1-mmol/L nonessential amino acid, 1-mmol/L sodium pyruvate solution) and incubated at 37°C, 5% CO 2 until 95% confluent. Cells were harvested using a gentle scraping method under sterile conditions and resuspended in 6 mL of RPMI, centrifuged at 850 g for 5 min then seeded at a density of 5×1 0 4 cells/well into flat-bottomed, cell culture 96 well plates in a total volume of 100 μL per well. The plates were incubated at 37°C, 5% CO 2 for 24 h. SDN/drugs at a concentration range up to 8 μg/mL (each concentration was prepared in triplicate treatment groups) were added and incubated at 37°C, 5% CO 2 for a further 24 h. Positive (macrophages and 0.1% Triton (to indicate 100% of lysis of cells)) and negative (macrophages and medium only) controls were included in each plate. Following incubation, the supernatant was removed, cells were washed twice with 200 μL of PBS, and 0.1 mg/mL of Alamar Blue ® reagent was added to each well and incubated for a final 4 h at 37°C, 5% CO 2 , whereby fluorescence was read at an ex wavelength of 560 nm and an em wavelength of 590 nm using a SpectraMax M5 plate reader (USA). To determine if any SDN/drug interference occurred, the 96 well plate was centrifuged at 850 g for 2 min, and the supernatant was removed from each well and transferred into a fresh plate and reread.

Infection of macrophages with pathogenic Mycobacterium
Macrophages were detached from flasks using TrypLE ™ and centrifuged at 850 g for 2 min before seeding into eight well chambered Permanox ® slides (1 × 10 5 cells/well in a final volume of 200 μL) and incubated for 24 h at 37°C, 5% CO 2 . Supernatant was removed from chambers, and cells were washed twice with 200-μL PBS. Map K10/GFP was cultured as described previously, and 10 mL of bacterial culture (in the exponential phase of growth) was centrifuged at 3090 g for 20 min and resuspended in 1 mL of PBS. The bacterial cell suspension was then centrifuged for 5 min at 670 g to remove any residual antibiotic (from the 7H9) and resuspended in 1 mL of PBS and passed through a Gilson tip several times to reduce clumping. Macrophages were infected with mycobacteria at a multiplicity of infection (MOI) of 30:1, 10:1, and 5:1 in complete medium (without streptomycin or penicillin) at a final volume of 200 μL per chamber (each MOI was performed in triplicate) and incubated for 4 h at 37°C, 5% CO 2 . Supernatant was removed, and each chamber was washed twice with 200 μLo fP B St o remove any residual/surface bound mycobacteria before fixing in 4% paraformaldehyde for 1 h. Control Intracellular delivery of nano-formulated anti-TB drugs Samantha Donnellan et al.
chambers were subject to the same procedures and contained macrophages and medium only.

Imaging the uptake of SDNs by macrophages
Macrophages were prepared as outlined previously. SDNs with DiD incorporated were added to eight wellchambered Permanox ® slides in complete medium (without streptomycin or penicillin) at a concentration range of 10-20 μg/mL in 200-μL final volume (each concentration performed in triplicate) to Map K10/GFPinfected and uninfected macrophages, which were incubated for a further 4 h at 37°C, 5% CO 2 . Supernatant was removed, and each chamber was washed twice with 200 μL of PBS to remove any residual/surface bound SDNs and/or mycobacteria before fixing using 4% paraformaldehyde for 1 h. Control chambers contained macrophages and medium only and macrophages with fluorescent polystyrene bead NPs in medium (Molecular Probes, USA)).

SDN manufacture and screening
Solid drug nanoparticles of the first line TB antibiotics; RIF, RIF + INH (DUAL) and RIF + INH + PZA (TRIPLE) were synthesised and characterised via either an emulsion template freeze dried or emulsion spray dried method described previously (Giardiello et al. 2016;Zhang et al. 2008). Drug loading, size, and Zeta potential of the tested SDNs (in Middlebrooks 7H9 broth) are provided in Tables 1a and 1b. We also adapted two of the compounds (RIF and TRIPLE) to contain DiD (Table 1a), for imaging purposes. The drug/drug ratio of the loading was chosen to try and reflect what is used clinically (TRIPLE SDN) but with respect to maximising excipients wherever possible. A series of drug ratios were initially produced, and the drug combination that was the most reproducible was selected for this study. Excipients were required to stabilise the prespray of freeze drying, generally more excipient resulted in smaller particles (confirmed by DLS) by preventing particle growth and agglomeration. Specific excipients Intracellular delivery of nano-formulated anti-TB drugs Samantha Donnellan et al.
(e.g., Pluronic F68 ® ) were chosen based on their stability, determined by DLS. The Z-Average diameter (nm) readings for the SDNs with DiD were larger than SDNs without the incorporated DiD dye, suggesting that those with the DiD dye either agglomerated more readily, or the dye incorporation increased the individual particle size (Table 1b). No statistical difference of the Zeta potential or dispersity was observed (Table 1b). Stability tests were conducted to confirm that RIF was stable by employing p-XRD. Relative to crystalline nonformulated RIF, RIF SDN was shown to be amorphous via p-XRD. Samples were stored for 4 weeks at 25°C and were shown to have remained amorphous over this period and not regressed back to their crystalline forms ( Figure S1).
To investigate the bactericidal activity of the SDN formulations, as a surrogate for Mtb, we employed a green fluorescent protein (GFP) reporter strain of Mycobacterium avium subsp. paratuberculosis (Map K10/GFP) (Harris et al. 2002) permitting viability to be monitored using fluorescence. The antimycobacterial properties of the SDN formulations and aqueous preparations of RIF and INH (solubilised per product instructions) were assessed using the previously described assay (Donnellan et al. 2015). Viable Map K10/GFP (2×10 6 cfu/mL) was exposed to SDNs and aqueous drugs ranging in concentrations (0.001875-1.33 μg/mL) for 7 days. Following exposure to the SDNs the growth of Map K10/GFP was inhibited in a dose-dependent and time-dependent manner, but differing in efficacy between SDN types. The growth rates of Map K10/GFP were expressed as the change in fluorescence per day (β) (Donnellan et al. 2015) and plotted against drug concentration using the data modelling software GRAPHPAD (GraphPad, Prism 5 ® ). For each compound, rather than comparing the total mass concentration (for which mass includes polymer and/or surfactant), the molar equivalent of drug loading for all three SDNs was used (Fig. 1(A)). The IC 50 was calculated for each compound: RIF SDN 0.02 μg/mL, DUAL SDN 0.17 μg/mL, and TRIPLE SDN 0.02 μg/mL; RIF aqueous 1.06 μg/mL, INH aqueous did not show an effect and therefore could not be calculated (potency ranking: RIF SDN = TRIPLE SDN > DUAL SDN > RIF aq > INH aq).
All compounds, except INH, induced a dosedependent and time-dependent inhibition of Map K10/GFP growth (Fig. 1(A)). RIF, the backbone of TB therapy (Maartens and Wilkinson 2007;Longo et al. 2015;Laurenzo and Mousa 2011), as an SDN (containing 30% drug) was more inhibitory than aqueous RIF ( Fig. 1(B)). Figure 1(B) demonstrates that by fitting the data using a three-parameter sigmoidal model, the RIF SDN exhibited improved potency (as demonstrated by the net reduction in control growth rate, i.e., the compound's kill rate) as evidenced by a 50-fold reduced IC 50 value (0.02 μg/mL compared with 1.06 μg/mL) observed for the aqueous formulation. The efficacy between compounds at different time points (from day 0 to day 7) shows that RIF can also elicit a faster kill rate of Map K10/GFP when the drug is in the SDN form compared with a conventional aqueous solution (Table S1a,b). The INH formulated as the DUAL SDN, containing 10% INH and 20% RIF (Table 1a) or as an aqueous formulation, had little impact on the growth of Map as previously reported (Fig. 1(A)) (Williams et al. 1999;Krishnan et al. 2009). Although effective at killing Mtb and M. bovis, this prodrug requires activation via the product of the katG gene (catalase-peroxidase). This gene is found in Map (Granger et al. 2004) but may be down regulated in the K10/GFP strain, suggesting that the INH content contributed a relatively small inhibitory effect on mycobacteria growth (Fig. 1(A)). The TRIPLE SDN, which contained 34% PZA, 13% RIF, and 6% Hydrodynamic diameter (Z-average), Zeta potential, and polydispersity index (PdI) of the SDN panel was assessed using DLS. Data expressed as mean ± SEM (n = 6) (temperature: 25°C, dispersant: Middlebrooks 7H9 broth, concentration 100 μg/mL, pH 6.9, equilibrium time: 30 sec). With or without 1,1 0dioctadecyl-3,3,3 0 ,3 0 -tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD).
Intracellular delivery of nano-formulated anti-TB drugs Samantha Donnellan et al.
INH (Table 1a) was as potent as the RIF SDN (IC 50 0.02 μg/mL). PZA is activated at an acidic pH during inflammation, and if phagocytosed by the macrophages, the SDN would be in an acidic environment. However, assessment of Map growth was performed in neutral conditions, which may suggest that this component of the SDN was relatively inactive and RIF exerted the dominant effect.

Infected macrophage SDN uptake
The next stage was to determine whether the SDNs could traffic to mycobacteria residing in macrophages. Firstly, we checked that the SDNs and aqueous RIF were not toxic to healthy macrophages (using the Alamar Blue ® assay). The SDNs and drugs tested did not significantly affect cell viability (P > 0.05 ( Figure S2)). We used fluorescence imaging to demonstrate uptake of both Map K10/GFP and SDNs and to determine if they colocalised to the same intracellular location of the cell. Macrophages were seeded (5 × 10 4 cells/well) and infected with mycobacteria at an MOI of 5:1, 10:1, and 30:1, then treated with SDNs incorporating DiD. Control experiments included macrophages only, macrophages treated with nontoxic, fluorescent 20 nm NP beads, and macrophages infected with Map K10/GFP only (Fig. 2(A-D)). Washing removed extracellular mycobacteria/SDNs/NPs beads prior to imaging. Figure 2(E-F) illustrates the uptake of fluorescent versions of the RIF and TRIPLE SDNs by macrophages. Z-stacks confirmed that RIF SDNs were distributed throughout the cell, both within the cytoplasm and nucleus ( Figure S3a-g). The TRIPLE SDNs, being larger particles, appear to be agglomerated (consistent with the DLS data) with limited uptake by cells observed (Fig. 2(F)), which has been previously reported (Clift et al. 2008). Overall, it appears that SDNs remained as full, intact particles once engulfed by macrophages.
To determine localisation relative to mycobacteria, the fluorescent SDNs were administered to Map K10/GFP-infected macrophages. Figure 3(A) illustrates macrophages infected with Map K10/GFP (MOI of 10) treated with 20 μg/mL of RIF SDN. Both SDNs and mycobacteria were observed to be intracellular. To consolidate this finding, it was repeated with a lower SDN concentration and Z-stack images taken (Fig. 3(B)). The same test was carried out for the TRIPLE SDN ( Fig. 3(C,D)). The mycobacteria and SDN appear to be colocalised for both SDN types, at least within the same 200-nm pixel area (the limit of image resolution). Because of the distance between GFP and far-red excitation (Ex)/emission (Em) peaks, wavelength crosstalk was unlikely to have occurred during the imaging. However, GFP and far-red signal crosstalk were tested for and found not to be occurring ( Figure  S4a,b). Therefore, we are confident that colocalisation of SDNs and mycobacteria does occur within macrophages.

SDN versus aqueous drug intracellular potency
Our next objective was to determine the antimicrobial effects of the SDN within the infected macrophages. A preliminary study was carried out to compare the killing of Map K10/GFP by RIF SDN or aqueous RIF, within macrophages. Infected macrophages (MOI 1:1, 10:1 and 30:1) were exposed to each treatment for 24 h then lysed, and a phage amplification assay (Rees and Botsaris 2012) was performed. The results demonstrated that the mycobacteria were being killed by the drugs within the macrophages and that at an MOI of 10:1 and 30:1, RIF SDN was more potent to intracellular mycobacteria over the 24-h period than its aqueous counterpart (Fig. 4). Although an exciting initial result, further repeats in an intracellular model is required to consolidate this finding.

Conclusions
The main aims of novel drug discovery for TB are to lower effective drug dose and shorten the treatment period, which will consequently decrease costs and likelihood of resistance. This work aimed to address these factors. By working with FDA-approved drugs (RIF, INH, and PZA), excipients from the Inactive Ingredients Database (Kollicoat ® protect, Hyamine ® , and Pluronic F68 ® ) and synthesising formulations of these drugs at the nano-scale, this approach may avoid the vast costs and timescales normally associated with novel drug development. Although SDNs would be viewed as novel drugs by the Medicines and Healthcare Products Regulatory Agency, as all the products are FDA approved, progression to clinical studies may be fast tracked. There are ongoing clinical trials of lopinavir and Efavirenz SDNs to treat HIV (Eudract number 2013-004913-41), thus showing the feasibility and potential of using SDNs to treat human diseases (Giardiello et al. 2016).
In summary, we have presented evidence, which shows that a nano-formulation of RIF is more effective (50-fold) than the conventional aqueous drug at killing a pathogenic Mycobacterium in vitro. We have verified that mycobacteria-infected macrophages can engulf SDNs, and both the drug and bacilli colocalise within macrophages. Nano-dispersions of drugs could be a more effective way of killing pathogens (Zhang et al. 2008;Choudhary and Kusum Devi 2015). Furthermore, we have demonstrated that we can synthesise dualloaded and triple-loaded SDNs of FDA-approved anti-TB first line drugs, which would allow simultaneous delivery of the drugs in the multidrug treatment regime. Targeting drugs to macrophages, where the bacilli reside, is an important consideration in treating both active and latent TB infections because it can increase antimicrobial efficiency (Wang et al. 2014;Alexandru-flaviu and Cornel 2014). Our studies suggest that SDNs could offer an alternative, effective strategy for treating TB. This has important implications as it offers the potential to lower the dose and shorten the duration of treatment. In turn, this could lead to a reduction in treatment side effects and slow down the emergence of multidrug-resistant strains. Maintaining concentrations in target cells, with lower systemic concentrations, may help mitigate drug-to-druginteractions (a major problem faced when treating HIV and TB-coinfected patients) through lower hepatic exposure. The application of SDNs requires further investigation, but has the potential to become invaluable in treating infectious diseases worldwide.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article.
Figure S1 RIF SDN stability test over 4 weeks at 25°C. Figure S2 SDN/drug cytotoxicity to macrophages after 24 hr incubation. Figure S3 Separated Z-stacks (a-f) and resorted Z-stacks of macrophages following 4 hr incubation with 10 μg/mL RIF SDN (g). Figure S4 (a&b) 'Crosstalk test' Table S1a Difference in bacteria growth between days when treated with RIF SDN. Table S1b Difference in bacteria growth between days when treated with RIF aqueous.