(M1330-02-13) Preparation and Optimization of Piperine Nanoparticles for Its Intranasal Delivery

Purpose: Piperine, a natural compound found in piper retrofractum and piper nigrum, has potential therapeutic effects on central nervous system (CNS) diseases such as Alzheimer's disease ^[1], Parkinson's disease ^[2], Epilepsy^[3] and FXTAS ^[4]. However, its low aqueous solubility (0.04 mg/mL) limits its brain uptake and pharmacological investigations at higher doses. Although nanoparticle formulation and nasal route of administration has been tried to increase the aqueous solubility of piperine and the brain uptake of other drugs, respectively, there was no investigation on intranasal delivery of piperine. This project was designed aiming to significantly increase the solubility of piperine up to 50 mg/mL by formulating it into nanoparticles for its intranasal delivery to the mice.

Methods: Nanoparticles of piperine were prepared using flash nanopricipitation via a Four-stream Multi-inlet Vortex Mixer (MIVM) system with the ethanolic solution containing piperine and Eudragit L100-55 and aqueous solution containing poloxamer 188. Preliminary experiments were conducted to identify the key factors affecting the nanoparticle formation and its subsequent concentration by solvent evaporation for intranasal delivery. Optimization of piperine nanoparticles was conducted using DoE method aiming to minimize particle size and maximize encapsulation efficiency. The optimized nanoparticles were characterized by particle size, polydispersity index, zeta potential, encapsulation efficiency, morphology under Transmission Electron Microscopy, characteristic peaks in Fourier-transform infrared spectroscopy (FTIR), and in-vitro release of piperine.

Results: ANOVA for Quadratic Model for particle size (Y1) and encapsulation efficiency (Y2) are shown in Table 1, and the 3D surface plots and contour plots for particle size (Y1) and encapsulation efficiency (Y2) as shown in Figure 1. Concentration of Eudragit L100-55 in ethanolic solution (X1), aqueous solution to ethanolic solution volume ratio (X2) and Poloxamer 188 to piperine ratio (X3) were identified as the key factors for the piperine nanoparticle preparations with 15 mg/mL Eudragit L100-55, volume ratio between aqueous solution and ethanolic solution of 2.8 to 1, and 1.67 to 1 as the ratio between Poloxamer 188 to piperine as the optimized parameters. The optimized piperine nanoparticles were prepared by 15 mg/mL of Eudragit L100-55 and 4.5 mg/mL of piperine in ethanolic solution and 2.68 mg/mL of Poloxamer 188 in water injected at 6 mL/min and 16.8 mL/min , respectively, and characterized with a particle size of 170.11±1.64 nm, polydispersity index of 0.27±0.01, zeta potential of -43.71±5.11 mv and an encapsulation efficiency of 92.49±1.92%. Further FTIR analyses of Eudragit L100-55, Poloxamer 188, piperine, their physical mixture and freeze dried piperine particles indicated the disappearance of characteristic peaks from piperine after its encapsulation into nanoparticles. The initial dissolution rate of piperine nanoparticles in pH 6.5 sodium phosphate buffer was 0.2 mg/min, which is 10 times of that from piperine suspension (0.02 mg/min), while the accumulated amount of piperine released from its nanopartocles at 24 hours was 4.91 times of that from its suspension as shown in Figure 2. After solvent evaporation of the optimized piperine nanoparticles, the final concentration of piperine could reach to 52.31 ± 0.9 mg/mL.

Conclusion: Formulating piperine into nanoparticles using flash nanopricipitation followed by solvent evaporation could significantly improve its solubility up to 52.3 mg/mL for its intranasal delivery.

References: 1. Wang, C., Cai, Z., Wang, W., Wei, M., Kou, D., Li, T., Yang, Z., Guo, H., Le, W., & Li, S. (2019). Piperine attenuates cognitive impairment in an experimental mouse model of sporadic Alzheimer's disease. J Nutr Biochem, 70, 147-155.
https://doi.org/10.1016/j.jnutbio.2019.05.009
2. Shrivastava, P., Vaibhav, K., Tabassum, R., Khan, A., Ishrat, T., Khan, M. M., Ahmad, A., Islam, F., Safhi, M. M., & Islam, F. (2013). Anti-apoptotic and anti-inflammatory effect of Piperine on 6-OHDA induced Parkinson's rat model. J Nutr Biochem, 24(4), 680-687.
https://doi.org/10.1016/j.jnutbio.2012.03.018
3. Ren, T., Hu, M., Cheng, Y., Shek, T. L., Xiao, M., Ho, N. J., Zhang, C., Leung, S. S. Y., & Zuo, Z. (2019). Piperine-loaded nanoparticles with enhanced dissolution and oral bioavailability for epilepsy control. Eur J Pharm Sci, 137, 104988.
https://doi.org/10.1016/j.ejps.2019.104988
4. Verma, A. K., Khan, E., Mishra, S. K., Jain, N., & Kumar, A. (2019). Piperine modulates protein mediated toxicity in fragile X-associated tremor/ataxia syndrome through interacting expanded CGG repeat (r (CGG) exp) RNA. ACS chemical neuroscience, 10(8), 3778-3788.

Acknowledgements: General Research Fund from University Grant Council of Hong Kong SAR( Reference number: 1145732).

Table 1. (a) Response Surface Quadratic Model for particle size (Y1). (b) Response Surface Quadratic Model for encapsulation efficiency (Y2).

Figure 1. Diagrams of the 3D surface and contour plot showing effect of (a) factor 2 (X2) and factor 3 (X3), (b) factor 1 (X1) and factor 3 (X3), and (c) factor 1 (X1) and factor 2 (X2) on particle size (Y1). Diagrams of the 3D surface and contour plot showing effect of (d) factor 2 (X2) and factor 3 (X3), (e) factor 1 (X1) and factor 3 (X3), and (f) factor 1 (X1) and factor 2 (X2) on encapsulation efficiency (Y2)

Figure 2. In-vitro release profiles of piperine nanoparticles and unformulated piperine in phosphate buffer at 37 °C for 24h. Each point represents mean ± S.D. (n = 3).