Manuscrito 2 – Nanoemulsion, more than simple drug delivery systems in

Mycobacterium tuberculosis

Priscila Cristina Bartolomeu Halicki1,2; Gabriela Hädrich3, Daniela Fernandes Ramos2, Tatiane Silveira Coelho2, Laís Andrade Ferreira2, Cristiana Lima Dora3, Pedro Eduardo Almeida da Silva#1,2

1 _{Programa de Pós-Graduação em Biotecnologia – Universidade Federal de Pelotas} (UFPel)

2

Núcleo de Pesquisa em Microbiologia Médica (NUPEMM) – Universidade Federal do Rio Grande (FURG)

3

Laboratório de Nanotecnologia Aplicada à Saúde, Programa de Pós-Graduação em Ciências da Saúde – Universidade Federal do Rio Grande (FURG)

Corresponding author: Pedro Eduardo Almeida da Silva ([email protected])

Abstract

The long duration of the tuberculosis (TB) treatment combined with toxic effects can hamper patient life-style and induces patient non-compliance and microbial resistance. New strategies using nanotechnology represent a new tool for the therapy. This study aimed to evaluate the in vitro antimycobacterial activity of a nanoemulsion (NE) containing rifampicin (RIF-NE) and its excipients. Using the hot solvent diffusion method associated with phase inversion technique was possible to prepare a NE containing 500 µg/mL of rifampicin (RIF) with average size of 25 nm. The antimycobacterial activity was evaluated by Resazurin Microtiter Assay, against three Mycobacterium tuberculosis strains: two susceptible and a multidrug resistant (MDR). The minimum inhibitory concentration of RIF-NE was equal to free RIF for the susceptible strains, while RIF-NE was able to increase the susceptibility of MDR strain. Despite the encapsulated RIF does not have superior activity to free RIF against to susceptible strains of M. tuberculosis, it was observed that the components of the NE does not seem to interfere in the in vitro activity of RIF. Interesting, castor oil and PEG 660 stearate, excipients of NE, showed antimycobacterial effect, being responsible for unloaded-NE activity for all strains. They may be considered promising components as adjuvants in formulating other lipid-carrier to be used in TB treatment, since they have similar activities for strains with different susceptibility profiles.

Introduction

The basic scheme of treatment of tuberculosis (TB), whose main drugs are isoniazid (INH), rifampicin (RIF), pyrazinamide and ethambutol, achieves a cure rate of approximately 95%, but lasts at least six months. Since 1993 the TB is considered an emergency global public health, mainly due to the co-infection TB-HIV, bacillus dormancy stage, emergence of resistant strains, toxicity of the treatment and its pharmacokinetic interactions with other drugs (1).

Currently, there are some candidates for new anti-TB drugs in advanced study phases, such as sutezolide (PNU-100480), SQ109 and pretomanide (PA-824), as well as two new drugs (bedaquiline and delamanid) and new treatment regimens (2); however, these alternatives they have not been effective enough to control TB. Thus, it grows the search for new therapeutic alternatives that, while not antimicrobial per

se, are able to enhance the activity of the drugs already used to treat the disease (3).

Several types of carriers have been developed for carrying anti-TB drugs (4,5,6). Among the latest technological tools, we highlight a variety of nanostructures, such as lipid nanocarriers, which are good candidates in the administration of drugs to be biocompatible and biodegradable (7). These delivery systems are able to increase the bioavailability of drugs in vivo and may lead to dose and treatment time reduction and, therefore, the reduction of side effects and systemic toxicity, increasing patient compliance to therapy (8).

The nanoemulsions (NE) are low-viscosity liquids, consisting of two immiscible liquids stabilized by surfactants, with a diameter between 10 and 500 nm (9,10). The NE is stable sterically systems that can be produced on large scale and allows encapsulation of lipophilic compounds (11,12). Therefore, this study aimed to evaluate the in vitro antimycobacterial activity of a nanoemulsion containing RIF (RIF- NE) and its excipients.

Materials and Methods

Preparation of nanoemulsions containing rifampicin: Castor oil and 12- hydroxystearic acid-polyethyleneglycolcopolymer (PEG-660 stearate/Solutol HS15®) were purchase from Sigma-Aldrich (St. Louis, EUA) and hydrogenated soybean lecithin (Phospholipon 80®) were obtained from Lipoid (Steinhausen, Switzerland). The NEs were prepared by hot solvent diffusion method associated with phase inversion temperature technique, previously described by Dora et al. (2012), using

castor oil as the oil phase and PEG-660 stearate and hydrogenated soybean lecithin as surfactant and co-surfactant (12), respectively. The components are described in Table 1. A mixture containing RIF, castor oil and lecithin diluted in 5mL acetone/ethanol (60:40, v/v) at 60 °C was added to an aqueous phase containing PEG-660 stearate at 82 °C under magnetic stirring (700 rpm). Subsequently, the formulations were kept under magnetic stirring at room temperature until its cooling. The organic solvents were evaporated under low pressure (23 mbar) until complete elimination to give a final volume of 20 ml. The final nanoemulsion were filtered using filter 8 microns. Before performing evaluation tests of antimicrobial activity, the formulations were sterilized using a 0.22 µM filter and transferred to sterile tubes. From the aliquots were performed sterility tests of each formulation in LB medium and incubating at 37 °C for 72 hours.

Table 1. Composition of RIF-NE formulation prepared by hot solvent diffusion associated with phase inversion technique.

Nanoemulsion PEG-660 stearate (µg/mL) Castor oil (µg/mL) Lecithin (µg/mL) RIF (µg/mL) RIF-NE 15000 7500 1000 500 UN-NE 15000 7500 1000 -

Size, polydispersity index and zeta potential: The average diameter and polydispersity index (PDI) of the formulations were determined by dynamic light scattering using the equipment Zetasizer Nano Series (Malvern Instruments, Worcestershire, UK). The light scattering measurements were performed at 90° at 25 °C. The hydrodynamic radius was determined using the Stokes-Einstein equation R= (kBT/6phD), where kB is the Boltzmann constant, T is the temperature, D is the diffusion constant and h is the medium viscosity. Zeta potential was determined in the same equipment, by laser-Doppler anemometry. The measures were performed at 25 °C after appropriate dilution of the samples in 1mM NaCl.

Analysis of rifampicin content in nanoemulsion: An initial scan for the choice of the appropriate wavelength for the measurements was performed and it was chosen value of 337 nm for detection of RIF. The amount of encapsulated RIF was determined by UV-visible spectrophotometry after complete dissolution of the formulations in methanol (11).

Validation of analytical method by UV/Vis spectroscopy: The analytical methodology was evaluated for parameters as specificity, linearity, limit of quantification (LQ) and detection (LD), precision and accuracy, as described in

International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (2005) (13). The measurements

were performed in a spectrophotometer (UV/VIS Spectrophotometer Perkin-Elmer, Massachusetts, USA) using a quartz cuvette with 1 cm of optical path, and detection at 337 nm. The curve was shown to be linear in the ranges of evaluated concentrations and LD and LQ were 2.7512 and 0.9079 µg/mL, respectively (Table 2). These data indicate that the methods are sufficiently sensitive for determining the total content of RIF in the formulation. In relation to the accuracy, the recovery values vary between 71 and 116% (Table 3).

Table 2. Results of linear regression analysis of the calibration curve data. Parameters Spectrophotometry (UV/Vis)

Concentration (µg/mL) 2.5 - 15

Equation of straight line y = 0,0289x + 0,0392

r² 0.9999

LD (µg/mL) 0.9079

LQ (µg/mL) 2.7512

Table 3. Recovery values.

Concentration level Presumed content of RIF (µg/mL) Average concentration of RIF (µg/mL) Recovery Low 7.0 7.1 71% Medium 10.0 9.0 90% High 13.0 11.6 116%

Strains and inoculum: Antimycobacterial activity of NE was evaluated against three

Mycobacterium tuberculosis strains: a susceptible clinical isolate (Strain 1), a MDR

clinical isolate, with mutations in katG (S315T) and rpoB (S531L) genes (Strain 2) and a pan-susceptible H37Rv (ATCC 27294), which were cultured in Ogawa-Kudoh, for up to 14 days at 37 °C. For the evaluation of antimicrobial activity and interaction between the compounds, a bacterial suspension was prepared in sterile distilled water according to 1.0 McFarland scale (3,2 x 106 UFC/mL). The inoculum was

prepared by diluting the bacterial suspension at a ratio of 1:20 in 7H9 medium (Middlebrook 7H9 Broth) (14).

Resazurin Microtiter Assay (REMA): By REMA method was performed antimycobacterial activity of the compounds: castor oil, PEG-660 stearate, soybean lecithin, free RIF, RIF-NE and an unloaded-NE (UN-NE). In a 96-well microplate were conducted a serial microdilution (1:2) of 100 µL of each compound to be evaluated in 100 µL of 7H9 medium supplemented with 10% OADC (Oleic acid Albumin Dextrose Catalase). The concentrations ranging from 234.4 to 3.66 µg/mL for CO; 250 to 0.49 µg/mL for LEC; 468.8 to 7.32 µg/mL for PEG; and 2048 to 0.06 µg/mL for free RIF. For NE, concentrations range from 5875 µg/mL and 5.8 µg/mL for UN-NE and between 6000 µg/mL and 0.7 µg/mL for RIF-NE. At the end of microdilution was added 100 µL of standard inoculum and in each plate were added sterility and growth controls. After seven days of plate’ incubation at 37 °C was added 30 µL of resazurin 0.02%, which was used as indicator of cell viability (14). The MIC was defined as the lowest concentration of compound capable of inhibiting bacterial growth.

Evaluation of the interaction between the compounds of the UN-NE and free RIF: The interaction between NE excipients and RIF was performed at MIC, ½ MIC and ¼ MIC, based in the proportional concentrations of each excipient in RIF-NE, by Resazurin drugs combination microtiter assay (REDCA) (15).

Results

By hot solvent diffusion method associated with phase inversion temperature technique, it was possible to formulate an NE containing approximately 500 µg/mL RIF with the following chemical-physical characteristics: approximate size of 25 nm and PDI equal to 0.18. Moreover, the negative zeta potential (-8.22 mV) indicated that stabilization was obtained by the steric effect produced by the presence of polyethylenoglycol chains of PEG-660 stearate at droplet surface (Table 4). The UN- NE showed similar chemical-physical characteristics from Hädrich et al., (2015)(16).

Table 4. Physical and chemical characteristics of RIF-NE. Formulation Size (nm) PDI Zeta potential (mV) RIF amount (µg/mL) Recovery (%) RIF-NE 25,75 ± 0,01 0,18 ± 0,01 -8,22 ± 4,59 488,8 ± 22,7 97,7% ± 4,51

The MIC of free RIF and RIF-NE for the susceptible M. tuberculosis strains was 0.25 µg/ml for strain 1 and 0.5 µg/ml for H37RV, while for strain 2 was 1.024 and 7.8 µg/ml, respectively, for free RIF and RIF-NE (Table 5).

Although RIF encapsulation has not been able to increase its in vitro activity against the susceptible strains, the RIF-NE increase the susceptibility of MDR strain, more than 130 times. These results indicate that antimicrobial activity was presumably of the NE. Thus, the antimycobacterial activity of UN-NE and its compounds, separately, was also evaluated (Table 5).

Table 3. Minimum inhibitory concentration of each compound. Value for UN-NE is the total of components (castor oil + lecithin + PEG-660 stearate) in proportion to the concentrations used in the formulation. Values for RIF-NE corresponding to the content of encapsulated RIF.

MIC (µg/mL)

Free RIF RIF-NE UN-BR Castor oil Lecithin PEG-660 stearate Strain 1 0.25 0.25 183.1 > 234.4 >250 234.4 Strain 2 1024 7.8 367.2 > 234.4 >250 468.8 H37RV 0.5 0.5 367.2 > 234.4 >250 468.8

UN-NE and PEG-660 stearate showed antimycobacterial activity, while lecithin and castor oil were not active up to evaluated concentrations, demonstrating that UN- NE and RIF-NE activity may be related to action of each constituent separately or together (Table 6).

Table 4. MIC of NEs and proportional concentrations of each compound in the MIC. TOTAL = Castor oil + Lecithin + PEG-660 stearate + RIF.

MIC (µg/mL)

TOTAL Proportional concentrations

Castor oil Lecithin PEG-660 stearate RIF

UN-NE Strain 1 183.1 58.6 7.8 117.2 - Strain 2 367.2 117.2 15.6 234.4 - H37Rv 367.2 117.2 15.6 234.4 - RIF-NE Strain 1 11.7 3.7 0.5 7.3 0.25 Strain 2 375 117.2 15.6 234.4 7.8 H37Rv 23.4 7.3 1 14.6 0.5 Discussion

All excipients used in the formulation of these studies are approved for internal use by the Food and Drug Administration (FDA) (17). With those reported in vivo results, these NE can be considered safe for oral administration. Hädrich et al. (2015) (16) evaluated the anti-inflammatory activity of NE containing quercetin, with the same basic constitution of NE evaluated in our study. In the control group, the rats treated with the UN-NE showed no damage to the liver and kidneys, or in neutrophils, lymphocytes and monocytes.

In spite of excipients used in the formulation of nanocarriers facilitate the preparation and use of certain substances and, moreover, protect them from degradation, it is important to emphasize that they are not inert. In the formulation, the excipients must be considered as important as the drug, since they may influence the absorption rate and consequently the bioavailability of the encapsulated compound (18).

The castor oil, which is used in the oil phase of NE, has excellent ability to solubilize hydrophobic compounds (11,12). Although this compound has antifungal and antimicrobial activity reported, it showed no antimycobacterial activity until the concentration of 234.4 µg/mL. (19).

The soybean lecithin used in the organic phase is a co-surfactant from natural origin with amphoteric character and about 70% of its composition is phosphatidylcholine. Due to the lipophilic character of lecithin, the use of cosurfactantes in the formulation is recommended, such as PEG-660 stearate, to

promote stabilization of the system. PEG is widely utilized in drug delivery and nanotechnology that was firstly described to have “stealth” properties. In particular, the stealth effect is due to the formation of a dense hydrophilic barrier of PEG chains on the surface of the carrier, thereby reducing interactions with the reticular- endothelial system. In addition, pegylation increases the hydrodynamic size of drug delivery systems and consequently decreases their clearance from the body (20).

PEG-660 stearate, when evaluated separately, also showed antimycobacterial activity for all the strains, but in concentrations higher than the MIC concentrations contained in the MIC of UN-NE, different from castor oil (Table 6). This excipient is a non-ionic surfactant, having a lipophilic region consisting of mono and diesters of polyglycols chains of 12-hydroxystearic acid. It also has a hydrophilic region consisting of approximately 30% free polyethylene glycol. There are no reports about the antimicrobial activity of the PEG-660 stearate, however, other non-ionic surfactants have been shown antimycobacterial activity and the suggested mechanism of action is related to the bacterial membrane fluidization (21,22). Analyzing the different susceptibility profiles of strains, it is important to emphasize that the strain 2 has mutations in the katG gene (S315T) and rpoB gene (S531L), which confer high levels of resistance to INH and RIF, respectively (23). Therefore, there seems no cross-resistance of castor oil with INH and RIF, showing a possible role of this compound on other targets than the rpoB and katG genes, since it was active against the MDR strain.

It is important to take into consideration the contribution of each component of the formulation in bacterial inhibition, since the MIC PEG-660 stearate is close to its concentration in the MIC of UN-NE. This result may also explain the activity of RIF- NE against the MDR strain (MIC = 7.8 µg/mL) in a drug concentration less than the MIC of free RIF (MIC = 1024 µg/mL). Despite the RIF-NE increase the susceptibility of the strain 2, the inhibition of growth is not due to the activity of RIF, but due to the activity of the excipients from NE, separately or combined.

It is known that the encapsulation of drugs is able to prevent their degradation and enhance their pharmacological activity (24), however, in vitro, RIF-NE did not show superior activity to free RIF against M. tuberculosis strains evaluated. Non potentiation of pharmacological in vitro activity of RIF after its encapsulation led to the evaluation of the interaction between the isolated excipients from UN-NE and free RIF in order to determine whether there would be an antagonistic relationship,

preventing a better action of RIF to your target. There was no interaction between the compounds, so the activity of RIF remains the same, even when combined with the UN-NE excipients separately.

Even the RIF-NE not presenting a different activity of free RIF in vitro, we cannot rule out the possibility of it show positive results in vivo, mainly related to its bioavailability. Sanzhakov et al. (2014) (25) reported the increased peak concentration of RIF in rat plasma after its packaging in lipid-based nanoparticles and oral administration.

Conclusions

Thus, despite the encapsulated RIF does not have superior activity to RIF free front to susceptible strains of M. tuberculosis, it was observed that the components of the NE does not seem to interfere in the in vitro activity of RIF. The NE are delivery systems able to reduce drug degradation after oral administration and increase its bioavailability, therefore, further studies are needed to evaluate the activity of encapsulated RIF in vivo, since we cannot ignore its potentiation in biological systems.

Added to this, the excipients used in the formulation of the NE of this study showed different activities against M. tuberculosis when evaluated separately. The PEG 660 stearate showed antimycobacterial action and may be considered a promising component as adjuvants in formulating other lipid-carrier to be used in TB treatment. This requires synergistic activity studies between components and other anti-TB drugs to assess their adjuvant effect.

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