Development and Characterization of Lipid Nanoparticles prepared by Miniemulsion Technique

Development and Characterization of Lipid Nanoparticles prepared by Miniemulsion Technique Clara Patrícia Andrade Lopes Instituto Superior Técnico, Universidade de Lisboa, Portugal clara.cpal@gmail.com Abstract: In the present work a MCFA SLNs and NLCs based on a combination of MCFA and natural oil were successfully prepared by sonication technique and employing a nonionic surfactant. The two anti-tb drugs, rifampicin and pyrazinamide, are entrapped into obtained lipid nanoparticles. The developed particles were characterized in terms of particle size, polydispersity index, zeta potential, morphology and encapsulation efficiency. Mean particle size of all formulations ranged between 69 ± 5 nm to 601 ± 100 nm, showing a suitable size for oral administration. The zeta potential obtained was negatively enough to ensure a good physical stability of the particles. Morphological studies by TEM showed spherical to oval SLNs and RIF-NLCs with well-defined periphery. The rifampicin encapsulation efficiency ranges between 67 ± 7% and 85 ± 5%, while pyrazinamide encapsulation efficiency ranges between 14 ± 8% to 29±15%. In conclusion, despite being essential several further studies to ensure the efficacy of obtained lipid nanoparticles, the results from the present work pose a strong argument for lipid nanoparticles as a promising strategy for the oral delivery of anti-tb drugs. Keywords: Oral administration; Solid lipid nanoparticles; Rifampicin; Pyrazinamide Introduction Apart from some particular situations, the oral route is the first choice for drug administration. This preference is related with its easy access and non-invasive nature, which improves patient compliance and, therefore, facilitates treatments. However, the poor water solubility of several drug molecules and/or the risk of degradation throughout the gastrointestinal tract, turn into impossible their oral administration. In this perspective, efforts have been done in order to improve the oral bioavailability of poorly water-soluble drugs, by means of developing new colloidal delivery carriers. Among these systems are lipid nanoparticles, which have been showing promising results. The main reason for this is related with the wellknown conception that lipids promote oral drug absorption, because they undergo the same physiological mechanisms of food lipid digestion [1]. Lipid nanoparticles also offer unique properties such as small size, large surface area, increased drug loading and stability (especially for lipophilic drugs), 1

possibility of controlled drug release, no toxicity and high bioavailability [2]. Therefore, the first objective of the present work was to perform the production, characterization and study of a MCFA SLNs and MCFA/natural oil NCLs prepared by sonication and employing a nonionic surfactant. The second objective of the project was to study the obtained SLNs and NCLs as an alternative system to improve the oral delivery of two anti-tb drugs, rifampicin and pyrazinamide. Materials Rifampicin and Pyrazinamide were purchased from Sigma-Aldrich. The Hexadecane 99% (Mw= 226.44 g/mol; ρ= 0.773 g/ml) was obtained from Sigma- Aldrich. Water from a Millipore Milli-Q ultrapure water purification unit was used. Methods Preparation of Lipid Nanoparticle Lipid nanoparticles were prepared by ultrasonication and simple magnetic stirring methods. Both aqueous and lipid phases are separately prepared before mixing. The aqueous phase is composed of water ( 80.0% w/w), non-ionic surfactant ( 8.0% w/w) and hexadecane ( 3.5% w/w). Six formulations of empty SLNs was prepared ranging lipid content: SLN_1 represents the formulation with the lowest lipid phase content and SLN_6 represents the formulation with the highest lipid phase content. For NLC formulations was used a combination of natural oil with the MCFA. The content of bioactive compounds incorporated in lipid nanoparticles are shown at Table 1. Table 1 - Composition % (w/w) of bioactive compounds loaded to lipid nanoparticles. Formulation β RIF PYZ 2RIF_SLN - 0.06-2RIF_NLC - 0.06-4RIF_SLN - 0.11-4RIF_NLC - 0.11-7RIF_SLN - 0.20-7RIF_NLC - 0.20-4PYZ_SLN - - 0.11 4PYZ_NLC - - 0.11 β10-sln 0.3 - - β10-nlc 0.3 - - In detail, the lipids are melted in a warm water bath above their melting points. Different temperatures conditions were tested up than 50ºC. When the MCFA or the MCFA/natural oil combination was fully melt a hot aqueous solution with nonionic surfactant and hexadecane preheated at the same conditions was added. A hot coarse o/w was obtained under magnetic stirring at 750 rpm for 1min and was subjected to sonication using a probe (MS72) sonicator (Bandelin, Germany) during 5 minutes. Hot o/w emulsion was allowed to cool to room temperature under magnetic stirring at 750 rpm to obtain the lipid nanoparticles. After 2h, the samples of each formulation were stored both in a refrigerator at 4ºC and at room temperature. Some aqueous samples of lipid nanoparticles were fast frozen under 80 C in a deep freeze for 5h in ultra-low refrigerator. Then, the samples were moved 2

to the freeze-drier (Christ Alpha 1-2 LD) during 48h in order to get powder of lipid nanoparticles. For active agents-loaded lipid nanoparticles, the active agent was added to the lipid phase, 15 minutes earlier the addition of aqueous solution. Lipid Nanoparticles Characterization Particle Size Zetasizer Nano ZS, Malvern Instruments (Malvern, UK) was used to measure the size of lipid nanoparticles produced. Particle size measurements were made in disposable cells at 25 C, by non-invasive back scatter, with dynamic light scattering detected at an angle of 173. 1 ml of samples were used and 1:10 dilutions were performed to avoid multiple scattering phenomenon. All measurements are performed in triplicate with 20 runs per measurement. Zeta Potential Zeta potential measurements were also performed using a Malvern Zetasizer Nano ZS, Malvern Instruments (Malvern, UK) at 25ºC. The samples were diluted 1:10 in ultrapure Milli-Q water and placed at a folded capillary cell (DTS1060) where an alternating voltage of ±150mV was applied. All measurements are performed in triplicate with 20 runs per measurement. Particle Morphology Morphological observations were performed using a transmission electron microscope (TEM). 20 µl of samples previous analysed by DLS was deposited over a carbon covered TEM copper grid and then dried at room temperature. TEM images were collected using a microscope H-8100 Hitachi, operated with 200 kv of acceleration voltage incorporated with a CCD MegaView II bottom-mounted camera. Encapsulation Efficiency An indirect method was used to determine the encapsulation efficiency using the following equation: EE% = TD FD TD 100 where TD is total drug weighted at the beginning and FD is the free drug dissolved in aqueous medium. The amount of active agents in the supernatant was determined from its absorption using a double beam UV VIS spectrophotometer (Model U-2000, Hitachi) and 1cm quartz cells. The measures of absorbance were done at 333 nm for rifampicin and at 548 nm for β-carotene. Pyrazinamide was analysed based on the formation of a coloured complex between the drug and sodium nitroprusside at alkaline ph with a mixing ratios of 4:1, which could be measured at 495nm. The solution of sodium nitroprusside at alkaline ph was prepared mixing a solution of 2% sodium nitroprusside in milli-q water with a 2N sodium hydroxide solution in a ratio of 1:1 [3]. Calibration curves of rifampicin, pyrazinamide and β-carotene were obtained by measuring solutions with known concentrations of the active compounds dissolved in milli-q water. Samples were estimated in triplicates. Resulting calibration curves and respective equations are represented in Figure 1-3. 3

Figure 1 - Rifampicin calibration curve Figure 2 - β-carotene calibration curve Figure 3 - Pyrazinamide calibration curve Results and Discussion Study of Fabrication Parameters The greatest advantage of ultrasonication is the easily production of SLNs without using organic solvents. The droplet size is governed by energy dissipation, temperature, surfactant and lipid concentration and other factors. Nonionic surfactant was employed, providing a steric hindrance, avoiding emulsion droplets from coming close to each other and thus preventing flocculation and coalescence. It was tested the influence of different parameters, such as applied lipid amount, temperature and sonication step on mean particle size expressed as Z-average mean size (Z-ave), polydispersity index (PDI) and zeta potential (ZP) (Table 2, Figure 4 and Figure 5) The lipid nanoparticles obtained are suitable carrier systems for the incorporation of different bioactive compounds intended for oral administration, since mean particle size of all formulations ranged from 69 ± 5 nm to 601 ± 100 nm and the great part of the formulations revealed a particle size less than 400 nm, which easily cross intestinal cells [4]. Also, it was obtained a lot of formulations with a particle size less than 200 nm, which is an even better result since they will remain invisible to the reticulo-endothelial system (RES) and keep on circulation system over a prolonged period of time [5]. The Zeta Potential obtained was negatively enough to ensure a good physical stability of the particles, indicating that the nonionic surfactant is a good surfactant for lipid nanoparticles produced with this particular formulation. In general, the formulations show a PDI higher than 0.1, indicating that none are monodisperse. 4

Table 2 - Physicochemical characteristics of empty SLN at different conditions (mean ± SD, n = 6). T (ºC) A B C D Non-sonicated Sonicated Formulation Z-Ave Z-Ave PdI ZP (mv) (d.nm) (d.nm) PdI ZP (mv) SLN_1 601 ± 100 0.70 ± 0.08-29.6 ± 0.6 146 ± 2 0.18 ± 0.05-25.8 ± 0.7 SLN_2 250 ± 6 0.56 ± 0.03-27.5 ± 0.2 130 ± 9 0.18 ± 0.02-27.8 ± 1.5 SLN_3 224 ± 28 0.46 ± 0.11-28.8 ± 0.1 105 ± 1 0.18 ± 0.02-27.8 ± 0.8 SLN_4 284 ± 19 0.87 ± 0.09-30.4 ± 0.8 115 ± 1 0.18 ± 0.04-29.4 ± 0.7 SLN_5 249 ± 16 0.70 ± 0.07-29.3 ± 0.3 121 ± 3 0.15 ± 0.02-28.6 ± 0.6 SLN_6 326 ± 6 0.85 ± 0.09-32.4 ± 0.3 121 ± 1 0.18 ± 0.05-28.1 ± 1.2 SLN_1 299 ± 8 0.55 ± 0.06-31.0 ± 0.4 139 ± 2 0.17 ± 0.03-27.2 ± 2.0 SLN_2 262 ± 13 0.44 ± 0.07-29.9 ± 0.5 134 ± 23 0.18 ± 0.02-25.1 ± 1.0 SLN_3 231 ± 25 0.54 ± 0.16-28.3 ± 0.6 121 ± 25 0.20 ± 0.04-30.3 ± 0.7 SLN_4 255 ± 8 0.47 ± 0.09-30.0 ± 0.8 122 ± 10 0.17 ± 0.04-28.6 ± 0.6 SLN_5 411 ± 22 0.51 ± 0.15-26.5 ± 0.2 122 ± 11 0.19 ± 0.02-27.2 ± 0.9 SLN_6 330 ± 55 0.45 ± 0.35-35.8 ± 0.8 109 ± 19 0.18 ± 0.04-31.7 ± 0.9 SLN_1 515 ± 140 0.52 ± 0.13-30.6 ± 0.6 141 ± 1 0.14± 0.03-28.4 ± 0.8 SLN_2 155 ± 4 0.26 ± 0.01-28.3 ± 0.7 126 ± 7 0.15± 0.02-26.8 ± 0.9 SLN_3 140 ± 1 0.20 ± 0.01-29.3 ± 0.6 97 ± 30 0.23 ± 0.09-28.9 ± 1.0 SLN_4 196 ± 2 0.30 ± 0.03-31.5 ± 0.2 119 ± 1 0.13 ± 0.02-29.2 ± 0.6 SLN_5 149 ± 1 0.29 ± 0.02-29.9 ± 0.5 120 ± 1 0.17 ± 0.05-28.5 ± 3.9 SLN_6 179 ± 1 0.25 ± 0.01-25.5 ± 0.7 122 ± 2 0.18 ± 0.01-28.2 ± 0.7 SLN_1 333 ± 50 0.41 ± 0.03-30.0 ± 0.5 139 ± 1 0.11 ± 0.02-26.6 ± 0.6 SLN_2 93 ± 7 0.30 ± 0.08-29.1 ± 0.6 87 ± 12 0.21 ± 0.02-22.7 ± 0.7 SLN_3 144 ± 44 0.87 ± 0.05-12.3 ± 0.6 105 ± 1 0.23 ± 0.03-28.4 ± 0.6 SLN_4 90 ± 3 0.46 ± 0.03-29.3 ± 0.3 69 ± 5 0.19 ± 0.03-29.4 ± 0.7 SLN_5 99 ± 2 0.49 ± 0.05-34.7± 1.5 97 ± 30 0.23 ± 0.09-28.9 ± 1.0 SLN_6 121 ± 2 0.40 ± 0.02-30.6 ± 0.3 126 ± 7 0.19 ± 0.03-26.0 ± 0.2 Table 2 shows a notorious size reduction of non-sonicated particles with the increase of temperature, while PDI and ZP values remain with non-significant variation. These results are expected since in general, the increase of temperature usually decreases the viscosity of the lipid and aqueous phases and hence the efficiency of the agitation and mixture is increased. In other words, increasing the fusion temperature of the lipids results in smaller droplet/particle sizes [6]. From analysis of non-sonicated samples of a, c and d in Figure 4, it seems evident that particle size of SLNs prepared with the lowest concentration of MCFA are larger when compared with other SLNs. In addition, PDI of these samples are also high. However, ZP appears not be affected as well as all features considered of lipid particles obtained by sonication. As PDI of non-sonicated SLNs produced with lowest lipid content are also very high, the Ostwald ripening effects could be an explanation for their larger sizes. 5

Figure 4- Z-ave and PDI of empty SLNs with different content of MCFA (SLN_1 represents the lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest temperature (a) to highest temperature (d). Figure 5 - Zeta Potential of empty SLNs with different content of MCFA (SLN_1 represents the lowest lipid phase content and SLN_6 represents the highest lipid phase content), ranging from a lowest temperature (a) to highest temperature (d). 6

At Figure 4, it is clear the effect of sonication in both Z-ave and PDI of MCFA SLNs. All sonicated SLNs show a mean Z- ave lower than 150 nm. Despite of this significant effect on particle size, ultrasonication does not appear to play an essential role in physical stability of SLNs, since ZP is very similar between nonsonicated and sonicated SLNs, although there is an insignificant decrease in absolute ZP value (Figure 5). The reduction effect of sonication at particle size is also visible by macroscopic aspect. It is possible to see at Figure 6 that nonsonicated SLNs dispersion has a milky aspect, while sonicated SLNs dispersion are more clear and bluish. From literature, usually colloidal dispersions with droplet sizes between 20 100 nm are transparent or bluish, while colloidal dispersions with larger droplets sizes up to 500 nm has a milky aspect [7]. Figure 6 - Macroscopic aspect of empty SLNs: (left) - non-sonicated sample; (right) sonicated sample. Examining particle size distribution of SLN_1b_II before and after lyophilisation (Figure 7), it is possible to realize the appearance of a second particles population larger than 1 µm in reconstituted lyophilised sample. This larger population could be result of aggregation phenomenon, indicating physical instability of SLNs. However, another explanation for these results could be an insufficient stirring force/time applied on resuspension of lyophilised SLNs in milli-q water. To explore this hypothesis, it could be done a comparative study between different forces used to resuspension of lyophilised SLNs. In addition, it could be also investigated the combination of a cryoprotector previously to lyophilisation since it was found that cryoprotective agents preserve the physicochemical properties of SLNs and decrease SLNs aggregation. [8]. An example of a macroscopic milky-like colloidal SLNs dispersion stored at room temperature was also analysed. The mean particle size remained lower than 400 nm, although there was an increasing on the size (Figure 8). Since PDI and particle size remain relatively low, homogeneously sized SLNs displayed a satisfactory long-term stability. The shape and surface morphology of empty SLNs after seven months were also analysed by TEM and the resulting images are represented in Figure 9. All the particles were found to be smoothly spherical or oval in shape with a welldefined periphery. 7

Figure 7 - Particle Size Distribution by Intensity of SLN_1b_II before and after lyophilisation. Figure 8 - Particle Size Distribution by Intensity of SLN_4b_I after one day and 7 months after production. Figure 9 - TEM image of SLN_4b_I after 7 months. Bioactive Compounds Loaded SLNs and NLCs β-carotene Comparing with rifampicin and pyrazinamide loaded-particles, β-carotene shows the highest percentage of encapsulation efficiency (Table 3). This result was predictable, since β-carotene is the most lipophilic of these three active compounds. Also, since β-carotene is less soluble in oil then in fat, the %EE of β10- SLN is higher than β10-nlc. Rifampicin As expected RIF-NLCs samples are larger than RIF-SLNs (Table 3). In addition, RIF loaded particles show a reduction in the electrical charge at the surface to mean values below -20 mv. These results could be taken as an indication that RIF is entrapped in the lipid matrix. Due to the high solubility of RIF in the lipid core, the encapsulation efficiency was good enough for all the formulations, ranging between 67 ± 7% and 85 ± 5%. RIF-NLCs also exhibit a spherical shape, which indicates drug loading is not lead to morphological changes (Figure 10). From literature, it was found two studies involving the encapsulation of RIF in lipid nanoparticles. In first one, RIF was loaded into Compritol ATO 888 SLNs fabricated by a modified microemulsion technique. The particles sizes obtained were 141 ± 13nm, ZP value was 3.5 ± 0.8mV and %EE was 65 ± 3% [9]. In second one, RIF was encapsulated in group with pyrazinamide and isoniazid in a stearic acid SLNs produced by emulsion solvent diffusion with a %EE of 51 ± 5% [10]. Perhaps due to RIF was been encapsulated combined with other anti-t 8

Table 3 - Physicochemical characteristics of the loaded lipid nanoparticles (mean ± SD, n = 3). Formulation Z-Ave ZP EE PDI (nm) (mv) (%) β10-sln 281 ± 19 0.34 ± 0.03-29 ± 5 95 ± 5 β10-nlc 328 ± 65 0.28 ± 0.06-25 ± 3 83 ± 6 2RIF_SLN 341 ± 78 0.45 ± 0.12-20.3 ± 2.2 74 ± 10 2RIF_NLC 391 ± 210 0.62 ± 0.10-18.5 ± 1.3 67 ± 7 Figure 10 - TEM image of rifampicin loaded NLC. 4RIF_SLN 84 ± 1 0.23 ± 0.01-17.8 ± 0.7 69 ± 1 4RIF_NLC 180 ± 90 0.36 ± 0.08-19.5 ± 0.9 69 ± 1 7RIF_SLN 181 ± 31 0.34 ± 0.03-19.2 ± 1.3 79 ± 7 7RIF_NLC 215 ± 5 0.28 ± 0.03-18.8 ± 0.6 85 ± 5 4PYZ-SLN 217 ± 87 0.31 ± 0.08-21.0 ± 0.7 14 ± 8 4PYZ-NLC 313 ± 214 0.34 ± 0.17-22.7 ± 2.4 29 ± 15 Figure 11 - TEM image of rifampicin loaded NLC. drugs the %EE was significant lower than results described in this work. Pyrazinamide The results of Z-ave, PDI, ZP and %EE of pyrazinamide loaded lipid nanoparticles are represented in Table 3. The %EE value obtained range from 14 ± 8% for PYZ-SLN to 29±15% for PYZ-NLC. These low results are expected since pyrazinamide is the most hydrophilic compound in that study. Thus, pyrazinamide partitioned between the melted lipid and aqueous phase. Since pyrazinamide is one of anti-tb drugs administered in current chemotherapy, in further studies could be investigated the combine incorporation of pyrazinamide with other anti-tb drugs such as rifampicin, isoniazid or ethambutol. Furthermore, the process of cooling from high to room temperature could be optimized in order to control crystallization processes and consequently the encapsulation efficiency. Pyrazinamide TEM images revealed nanoparticles with poorly spherical shapes. It was also reported the presence of aggregates, indicating important alterations on PYZ loaded particles structure (Figure 11). Conclusion MCFA SLNs and NCLs based on MCFA and natural oil were successfully prepared by sonication technique. This technique was simple, reproducible, prepared nanoparticles without the need of organic solvents or any sophisticated instruments and has the potential to easily scale up for large scale production. Also, rifampicin and pyrazinamide are successful incorporated 9

in obtained lipid nanoparticles. Therefore, despite must be done several further studies to ensure the efficacy of obtained lipid nanoparticles as drug delivery system for oral route, these initial results are very promising. References [1] Muchow M, Maincent P, Muller RH (2008) Lipid nanoparticles with a solid matrix (SLN, NLC, LDC) for oral drug delivery. Drug Dev. Ind. Pharm. 34, 1394 405. [2] Müller RH, Mäder K, Gohla S (2000) Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. Eur. J. Pharm. Biopharm. 50, 161 77. [3] Gurumurthy P, Nair NG, Sarma GR (1980) Methods for the estimation of pyrazinamide and pyrazinoic acid in body fluids. Indian J. Med. Res. 71, 129 34. [4] Hyuk Suh W, Suslick S, Stucky D, Suh Y-H (2009) Nanotechnology, nanotoxicology, and neuroscience. Pro Neurobiol 87, 133 170. [5] Bhandari R, Kaur IP (2013) Pharmacokinetics, tissue distribution and relative bioavailability of isoniazid-solid lipid nanoparticles. Int. J. Pharm. 441, 202 12. [6] Parhi R, Suresh P (2012) Preparation and characterization of solid lipid nanoparticles-a review. Curr. Drug Discov. Technol. 9, 2 16. [7] Sadtler V, Galindo-alvarez JM, Bégué EM, Gemico P, Université C, Cnrs-ens-upmc UMR, Supérieure EN (2008) Low Energy Emulsification Methods for Nanoparticles Synthesis. [8] Schwarz C, Mehnert W (1997) Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles. Int. J. Pharm. 157, 171 179. [9] Singh H, Bhandari R, Kaur IP (2013) Encapsulation of Rifampicin in a solid lipid nanoparticulate system to limit its degradation and interaction with Isoniazid at acidic ph. Int. J. Pharm. 446, 106 11. [10] Pandey R, Sharma S, Khuller GK (2005) Oral solid lipid nanoparticle-based antitubercular chemotherapy. Tuberculosis (Edinb). 85, 415 20. 10