Rifampicin Nanoprecipitation using Flow Focusing Microfluidic Device

Microfluidic devices have successfully been applied to produce many kinds of particles in the micro [1-3] and nanoscale range [4,5]. Microfluidics have applications in the pharmaceutical, cosmetic and personal care areas, as well as in chemical processing including paints and coatings, food industry among others [6-8]. In pharmaceutical applications, particularly, microfluidic devices have been applied to synthesize poorly water-soluble drugs with additives in low concentrations, improving the bioavailability by size reduction and morphology changes, while maintaining chemical composition and therapeutic effects [9-12]. Microfluidic devices are utilized to synthesize nanoparticles of organic and inorganic materials such oxides, metals, semiconductors, polymers, including different types of drugs. In the case of pharmaceutical products, there are examples such as liposomes [13-15], niosomes [12], PLGA-PEG copolymer nanoparticles [16] and some poorly soluble drugs that have successfully been produced in microfluidic systems.


Introduction
Microfluidic devices have successfully been applied to produce many kinds of particles in the micro [1][2][3] and nanoscale range [4,5]. Microfluidics have applications in the pharmaceutical, cosmetic and personal care areas, as well as in chemical processing including paints and coatings, food industry among others [6][7][8]. In pharmaceutical applications, particularly, microfluidic devices have been applied to synthesize poorly water-soluble drugs with additives in low concentrations, improving the bioavailability by size reduction and morphology changes, while maintaining chemical composition and therapeutic effects [9][10][11][12]. Microfluidic devices are utilized to synthesize nanoparticles of organic and inorganic materials such oxides, metals, semiconductors, polymers, including different types of drugs. In the case of pharmaceutical products, there are examples such as liposomes [13][14][15], niosomes [12], PLGA-PEG copolymer nanoparticles [16] and some poorly soluble drugs that have successfully been produced in microfluidic systems.
The antisolvent precipitation technique is one of the mechanisms applied to obtain nanoparticles and can be improved by using microfluidic devices [17], leading to particles with a smaller nucleation size [18]. Some examples of nanoprecipitation using different kinds of devices can be found in the literature, such as amorphous cefuroxime axetil prepared by nanoprecipitation to enhance the dissolution rate, using a Y-junction micro reactor [19] and a T-junction micro reactor [20]. In that work it was observed that the total fluid flow rate, the antisolvent to solvent flow rate ratio and the cefuroxime concentration influence the particle sizes. Danazol [21] and calcium atorvastatin [22] are other examples of pharmaceutical ingredients processed in micro reactor, resulting in particles in nanoscale range, with low polidispersity and improved dissolution rate.
Hydrocortisone for ophthalmic delivery was also processed in a commercial micro reactor with Y-junction geometry with good results [23]. The study involved micro reactor with different microchannel diameters and inlet angles. It was observed that smaller microchannels diameters as well as the sharper inlet angles resulted in a reduction of the particle sizes. In the same way, the total flow rate and anti-solvent/solvent ratio affect the particle size. The particle sizes varied from 80 to 450 nm, with the particles having an amorphous profile and the results indicate that the drug dissolution rate is improved if compared with the dissolution rate of raw hydrocortisone. In another work [24], the authors compare the microfluidic nanoprecipitation (a bottom-up method) with the wet milling process (top-down method). It was observed that both methods lead to particles in nanoscale range, improving the bioavailability. However, nanoprecipitation requires low energy consumption and is considered a simple process, while wet milling produces a nanosuspension with more physical stability.
In this work, we present a study of the rifampicin synthesis in a microfluidic device, using the anti-solvent nanoprecipitation process. Rifampicin (C 43 H 58 N 4 O 12 ) is an antibiotic commonly used in Tuberculosis and Hansen disease treatments. However, rifampicin presents a large variability in its bioavailability, due mainly to its two polymorphic forms. Because of its low solubility, rifampicin is administered in high concentrations causing serious side effects [25]. Rifampicin is categorized in class II in the biopharmaceutical classification system (BCS), in which drugs are characterized for their low solubility and high permeability [26,27]. Some methods have been used to increase the rifampicin BCS Class, such as amorphization [28], pH modification and particle size reduction [29] or rifampicin encapsulation in liposomes [30], biodegradable polymers [31]. Although these efforts showed promising results, these techniques utilize additives that are not approved by health organizations and usually cause an increasing in the production costs.
processes was used for the study presented here [32,33]. The device has a flow-focusing geometry with 45° angle between the inlets to enhance the diffusion process. The particle sizes and morphology of rifampicin were evaluated while observing the influence of fluid flow, anti-solvent to solvent flow rates, the presence of surfactants and rifampicin concentrations. The nanoparticles were analyzed by techniques of Dynamic Light Scattering (DLS) to measure particle sizes and Field Emission Gun Scanning Electron Microscopy (FEG-SEM) to observe the morphology. Fourier Transform Infrared Spectroscopy analysis was used to observe the chemical composition (FTIR) and X-ray Diffraction (XRD) to analyze the crystallinity. Differential Scanning Calorimetry (DSC) and dissolution tests were used to analyze the rifampicin nanoparticles behavior as compared to raw rifampicin.

Microfluidic device fabrication
Microscope glass slides were used to obtain the microfluidic devices using standard photolithographic and wet etching procedures. The glasses were patterned with hydrofluoridric acid solution combined with hydrochloric acid to improve the etching process (HF:HCl:H 2 O-1:1:3). Chromium films with a thickness of 50 nm, obtained by sputtering, were used as masking material for etching the microchannels. To seal the channels with a cover glass, we used UV glue diluted in 50% pure acetone. Access holes were patterned with diamond burs and brass tubing's used for liquid inlet and outlet was glued with epoxy resin. The flow focusing geometry was patterned to obtain the hydrodynamic flow focusing phenomena. The angle formed between each of the side channels and the main channel is 45° degrees (Figure 1a). The side channels, where the anti-solvent flows, have a width of 110 ± 1µm and the main channel, where the rifampicin solution flows, has 100 ± 1 µm in width. The microchannels have 80 µm + 1 in depth and the diffusion region has 45 + 0.5 mm in length. The glass microfluidic system microfabricated is shown in Figure 1b.

Rifampicin solution and nanoprecipitation process
Rifampicin was dissolved in methanol in three different concentrations, and different flow rates were tested (Q RIF ). De-ionized water flow (Q WATER ) was used as anti-solvent and was introduced in the side micro channels. The Tween 20 was used as surfactant and we also observed the influence of the flow rate ratio R, which is defined as Q WATER /Q RIF . Syringe pumps, PHD 4400 (Harvard Aparatus), were used for controlling the fluid flows. The flow focusing behavior on the microchannel was observed using an optical microscope (Coleman, XTB-2T).
Statistical data processing using a two-factor experiment with repeated measures at central points was applied to study the influence of the parameters settings in the particle size and polydispersity. The parameters are expressed in Particle size: The size measurement was made in a Dynamic Light Scattering apparatus (Delsa TM Nano C Particle Analyzer, Beckman Coulter). The rifampicin was maintained as a suspension and the DLS analyzer performed six measurements on each sample.

Sample preparation:
We filtered and dried the rifampicin nanosuspensions for analysis presented on sequence. Rifampicin nanosuspensions were filtered in a hydrophilic membrane with 0.22 µm in pore (Millipore Ind.) with a vacuum system. The membrane was maintained in a vaccum desiccator per 24 h to dry the sample and obtain the rifampicin powder. We decided also to use a condition without surfactant to observe chemical and physical parameters, and the process conditions: the flow rate ratio between water and rifampicin flow (R) is equal to 10, resulting in particle sizes of 690 nm (Table 1).

Morphology:
The nanoparticles morphology was observed by FEG-SEM (Quanta 3D model, FEI).The membrane with nanoparticles was fixed on the FEG-SEM stub using cooper double-sided adhesive tape and sputtered with 20 nm of Au coating. The thickness was defined through the sputtering deposition rate, in our case 5 nm/min. Chemical composition: The Fourier Transform Infrared Spectroscopy analysis (NICOLET 6700 FTIR, Thermo Scientific) was performed to evaluate the chemical composition of the rifampicin processed in the micro-channel system. FTIR analysis was carried out in a range of 500 to 5000 cm -1 with spectral resolution of 2 cm -1 , using the potassium bromide pellet method, described elsewhere [34].

Water
Physical characteristics: The crystalline structure was examined using wide X-ray Diffraction (XRD, XRD-6000, and Shimadzu) to observe the crystalline profile changes on the rifampicin processed in microfluidic channels. Data were collected over an angular range from 5 to 50º (2θ) in continuous scan mode using a step size of 0.02°. The generator was set to 40 kV and 30 mA.
Thermal analysis: Thermograms were performed using a Differential Scanning Calorimeter (DSC), Model 822 e (Mettler Toledo). Samples were weighed in aluminum crimped pans. The parameters used for analysis were: heating rate 10°C/min, temperature range 25°C to 300°C with nitrogen purge (100 mL/min) and pan partially covered.
Dissolution rate: Dissolution rate tests were performed in a dissolution apparatus (Shaker 430, Nova Etica). The speed and temperature of the bath were set at 100 rpm and 37.0°C ± 0.5°C, respectively. A PBS buffer solution with pH of 7.4 was employed as the dissolution medium. Rifampicin samples, both the commercial and the rifampicin nanoparticles, with 6 mg of mass were added to different vessels containing 100 mL of the dissolution medium. A 3 mL aliquot was taken each time at specific time intervals (2,5,10,15,20,25,30,35,40,45, 50, 60, 90, 120 and 180 minutes) and the concentration of samples was obtained by absorbance measurement in an UV spectrophotometer (U-2000, UV Hitachi) at 333 nm.
As a reference, a sample of raw rifampicin (pure drug) was characterized with the same techniques applied to the rifampicin nanoparticle obtained in microfluidic process (FEG-SEM, FTIR, DSC, XRD and dissolution tests) and the results are discussed below. Commercial Rifampicin was diluted with distilled water and filtered in the same way as the nanoparticles. The initial average size of raw rifampicin was 42 µm.

Results and Discussion
Flow focusing and particle sizes Figure 2, presents the images of microfluidic device in two different flow rate ratios and their respective particle size measurement. For the examples shown below, the initial rifampicin concentration is 50 mg/ mL. In (a) the anti-solvent to solvent ratio R is equal to 10, resulting in a 690 nm rifampicin particle size. In (b), the ratio is equal to 50 and resulted in a 160 nm rifampicin particle size. In these examples, we fixed the water flow rate, Q WATER , to 200 µL/min. As expected, for higher R values, small particle sizes are obtained. However, this microfluidic system allows a maximum R value of 50, since for higher values, the flow focusing is no longer achieved.

Factorial analysis
Under the conditions used in this work, it was possible to obtain rifampicin nanoparticles in a range from 100 nm to 1120 nm and polidispersity below 0.3. In figure 3, we can see the surface analysis response showing the influence on the particle size of each selected parameters used in the experiments. It is possible to observe that the increase in total fluid flow combined with the increase of Q WATER /Q RIF ratio causes a reduction in the particle size.
Higher rifampicin concentration results in larger particles, while higher surfactant concentration reduces the particle sizes. By varying the concentration of surfactant in the range of 0-1% by volume of water, there is a reduction in particle size from 800 nm to 250 nm approximately, considering an anti-solvent to solvent flow ratio of    Figure 5 shows micrographs of rifampicin nanoparticles obtained with a FEG-SEM. In this analysis the rifampicin was maintained in the membrane used previously to filter rifampicin. In figure 5a, a micrograph of raw rifampicin is shown and in figure 5b rifampicin processed in microfluidic devices is presented (measured size of 250 nm). For this analysis we utilized Rifampicin in high initial concentration (50 mg/mL), and the microfluidic system was operated with a water flow rate (Q water ) of 200 µL/min and R of 50. We observed that the particles obtained from the microfluidic process have a spherical shape as compared to commercial raw rifampicin.

Chemical composition
FTIR analysis was performed to evaluate the chemical composition of the raw rifampicin and the microfluidic processed rifampicin nanoparticles. The corresponding FTIR spectra are presented in figure 6. The FT-IR spectra curves suggest that there was no chemical composition change, because rifampicin nanoparticles have the same absorption peaks as raw rifampicin.

X-ray diffraction analysis
In figure 7, a XRD profile is presented comparing the raw rifampicin with rifampicin processed in the microfluidic device. Raw rifampicin shows characteristic peaks at 19.50°, 14.35° and 13.65°. As presented in the article by Agrawal et al. [27], commercial rifampicin is usually a mixture of crystal forms I (presenting peaks at 14.35° and 13.65°) and II (19.5°), and amorphous material. Observing the curve for rifampicin processed in microfluidic system, the absence of peaks is verified, revealing that this material has a significant reduction in crystallinity and is mostly amorphous. In many pharmaceuticals applications, size reduction is combined with crystal amorphization, since amorphous particles have higher solubility compared to crystalline particles, even for particles on nanoscale range [34].

Thermal analysis
The DSC profile of raw rifampicin showed two peaks at 190°C and 260°C, which correspond to the melting point and decomposition of the raw rifampicin, respectively (Figure 8a). In contrast, in the DSC thermal profile of rifampicin nanoparticles, we can see slight bands confirming the amorphous state of these particles (Figure 8b). Although we could observe some noises on the DSC profile of rifampicin nano, we could see that there are marked differences between DSC profiles. The XRD analysis presented before helped us maintain this argument.

Dissolution tests
The dissolution profiles of raw rifampicin and the nanoparticles (690 nm) in phosphate buffer solution are compared in figure 9. In ten minutes, the rifampicin nanoparticles reached 50% drug dissolution against 30% of the raw rifampicin in the same period. The green line on the graphic highlights the period of 45 minutes stipulated by US with flow ratio R (Q WATER /Q RIF ) on the rifampicin particle sizes. The water flows (Qwater) used in our experiments do not provide significant differences in particle sizes (around 50 nm). The ratio between water and rifampicin flow, combined with rifampicin initial concentration causes a relevant influence on the particle sizes.  Pharmacopeia a for rifampicin dissolution tests [35]. In this point, we observed that 80% of nano rifampicin is dissolved in contrast of 60% of raw rifampicin. The results indicate that rifampicin nanoparticles provide an improved dissolution rate compared to raw rifampicin.

Conclusion
In this work, a glass micro fluidic device was employed to produce rifampicin nano-suspension with a relative control over particle size and morphology. The nanoprecipitation process in our device leads to rifampicin nanoparticles with sizes in a range varying from 100 nm up to 1120 nm. When comparing processed with unprocessed rifampicin, we observed that the chemical characteristics were maintained, as can be seen from the FTIR analysis, however, the particles morphology becomes amorphous. The change to amorphous profile was confirmed  by DSC and XRD analysis. The dissolution profile was evaluated in a buffer solution, and we observed that rifampicin nanoparticle has the dissolution rate enhanced compared to raw rifampicin. In conclusion, the rifampicin nano-precipitation in micro fluidic device offers a continuous controlled process to obtain rifampicin nanoparticles.