Preparation and Characterization of Mucoadhesive Nanoemulsion containing Piperine for Nasal Drug Delivery System

 

Deepika Yadav¹*, Avijit Mazumder², Roop K Khar¹

¹B. S. Anangpuria Institute of Pharmacy, Faridabad Haryana.

²Institute of Pharmacy, Noida Institute of Engg. and Technology, Greater Noida.

 

ABSTRACT:

Piperine a bio active constituent isolated from pepper (Piper nigrum and Piper longum L.) is a naturally occurring alkaloid have validated for several health effects and valuable therapeutic effects. However, its biological applications are limited due to its poor aqueous solubility. This emphasizes on the development of new drug delivery system for piperine to improve its in-vivo bioavailability. The present study reports the development and characterization of mucoadhesive nanoemulsion (MNE) containing piperine. MNE formulations were prepared using titration method and characterized in relation to appearance, globule size, zeta potential, thermodynamic stability testing, Ex-vivo evaluation and in-vitro drug permeation study. The MNE of piperine have small globule size (˂ 150nm) and positive zata potential. The spherical surface was confirmed from TEM. pH of MNE was compatible with nasal administration. In vitro release of MNE system in nasal mucosal membrane demonstrated prompt and effective release with more than 75 % of drug release in 4 h.

 

 

 

INTRODUCTION:

Plants have a long-standing history of use in medicinal preparation because of their reported pharmacological properties¹. The pepper (Piper nigrum and Piper longum L.) belonging to the botanical family of piperaceae accounts for about 35% of the spice trade of the world. It contains 5 -9% of alkaloids, piperine is the major one responsible for pungency and biting taste to it². Piperine is already been reported for various therapeutic activities like antidepressant³, anticancer⁴, antioxidant⁵, antinflammatory⁶, antihypertensive, anticonvulsant⁷, hepatoprotective⁸ and many more.

 

Currently the most interesting group of drug delivery vehicles are nanoemulsions.  They can be prepared by various method which have been exhaustively reviewed and reported in various papers⁹.

 

Nanoemulsions are not form spontaneously, because external shear has to applied to rupture large droplet into smaller one. They are mainly oil-in-water or water-in-oil type stabilized using appropriate surfactant¹⁰. The size of particles in nanoemulsion fluctuates from 10 to 1000 nm¹¹. Nanoemulation base formulations are the area of interest nowadays due to properties like high physical stability, bioavailability and optical transparency¹². The nanoemulsion have unique feature in the area of drug delivery that they can also act as efficient carriers for bioactives of medicinal plants, enabling administration by different routes.

 

In this sense, the objective of this study was to develop Mucoadhesive nanoemulsion containing piperine. The formulations were prepared using titration method and characterized in relation to appearance, globule size, zeta potential, thermodynamic stability testing, Ex-vivo evaluation and in-vitro drug permeation study. 

 

MATERIAL AND METHODS:

Chemicals and reagents:

Piperine was purchased from Sigma-Aldrich (St Louis, MO, USA), Oleic acid and Polyethylene sorbitan monolaurate was supplied by S. D. Fine Chemicals, India. Polyxy-35-castor oil (Cremophor EL) of Gattefosse, France and Purified water was used.

 

METHODS:

Preparation of nanoemulsion:

Piperine nanoemulsion were prepared by titration method using Oleic acid as as oil, carbitol as cosurfactant and tween 20 as surfactant and purified water as continuous phase. Oil phase were mixed with Smix of a particular ratio, Oil and Smix ratio (0-3:3-0) were taken in various ratios (1-9:9-1) and finally titrated with purified water. Water was added to drug loaded internal phase in dropwise manner under continuous stirring. The compositions which are optically clear have been evaluated further by constructing pseudo ternary phase diagrams.

 

Preparation of mucoadhesive nanoemulsion:

Mucoadhesive nanoemulsions of piperine were prepared by addition of mucoadhesive polymer (showing maximum strength, ref section of selection of mucoadhesive agent) such as chitosan (Dodane et al., 1999) to optically clear nanoemulsion. The mucoadhesive Nanoemulsions were prepared by first preparing a nanomulsion of the drug using minimum volume of external phase and then adding the required volume of concentrated polymer solution to it such that the required final concentration. Piperine Mucoadhesive nanoemulsion were prepared as described under Piperine nanoemulsion preparation and chitosan was added in a concentration of 0.25%/0.50%/1.0% w/v with continuous stirring for 30 minutes.

 

Characterization of nanoemulsion:

Globule size and zeta potential determination:

The Globule size determination of Piperine loaded nanoemulsion were determined using photon correlation spectroscopy (PCS) with in-built Zetasizer (model: Nano ZS, Malvern instruments, UK). Measurement conditions for globule size were optimized by measuring globule size for the dispersions of different dilutions. The dilution of the nanoemulsion in water was made in such a way that the integrity of the globules were maintained with sufficient inter particle space and minimal multiple light scattering during measurement.

 

Malvern Zeta sizer Nano ZS was used to measure the zeta potential of the globules based on the electrophoresis and electrical conductivity of the formed nanoemulsion. The electrophoretic mobility (µm/s) of the particles was converted to the zeta potential by in-built software based on Helmholtz-Smoluchowski equation. Measurements were performed using small volume disposable zeta cell.

 

Transmission electron microscopy (TEM):

TEM is used as a tool to study the morphology and structure of the delivery systems. The images were taken by Tecnai200 with CCD camera operating at 200kV (Philips Instruments, Holland) and capable of point to point resolution. Nanoemulsion were diluted in de-ionized water (1 in 10 dilution). To measure the morphology and size distribution, a drop of sample was placed onto a 300-MEh copper grid coated with carbon. Approximately 2 min after deposition, the grid was tapped with filter paper to remove surface water and air-dried. Negative staining was performed using a droplet of 0.5% w/v phosphotungstic acid.

 

Thermodynamic stability testing of drug loaded nanoemulsions:

To check that the nanoemulsions were stable, the drug loaded nanoemulsions were subjected to thermodynamic stability testing¹³, which comprises of heating cooling cycle, freeze thaw cycle and centrifugation tests. Physical stability was continuously monitored over the period of time. Various aspects like phase separation, turbidity etc. at room temperature were observed.

·       Freeze thaw cycle:

Selected nanoemulsions were kept in deep freezer (at -20°C) for 24h. After 24h the nanoemulsions were removed and kept at room temperature. The thermodynamically stable nanoemulsions returned to their original form within 2-3 minutes. 2-3 such cycles were repeated.

·       Centrifugation studies:

Nanoemulsions after freeze thaw cycle were subjected to centrifugation studies where they were made to undergo centrifugation for 30 minutes at 5,000rpm in a centrifuge. The stable formulations did not show any phase separation or turbidity.

·       Heating cooling cycle:

Nanoemulsions were kept at 37±0.5°C for 24 hrs. After that the nanoemulsions were kept at room temperature. The stable nanoemulsion should not show any sign of turbidity, cracking, creaming during the entire cycle.

 

Ex vivo evaluation:

The Ex vivo evaluation was done by using rd-loaded nanoemulsion formulations by using CLSM (Confocal Laser Scanning Microscope)¹⁴. The (Rhodamine) Rd was added to the during the preparation stages of nanoemulsion and mucoadhesion. The studies were performed in triplicate. In order to evaluate the retention, capacity of Rd-Nanoformulations into the nasal epithelium, prepared specimen was washed with normal saline 3 time on every 5 minutes and then these specimens were directly mounted, mucosal side up, on a glass slide and examined without further tissue processing by CLSM (Olympus FluoView FV 1000, Hamburg, Germany). Samples were excited with green helium neon 543nm laser beam. Images were taken employing a 20× oil objective, assembled in an integral image processor and displayed on a digital video monitor. To confirm the penetration of Rd-Nanoformulations, stacks of serial 4.4m optical sections were captured along the Z-axis.

 

In vitro drug permeation study:

In-Vitro permeation studies through Goat nasal mucosa was performed using an automated Transdermal Diffusion Cell Sampling System¹⁵ (SFDC6, LOGAN Inst, NJ, USA). Separated nasal mucosa samples were mounted into the diffusion cells (area 0.75 cm²: effective diffusion area 0. 636 cm²) equilibrated at 32± 0.2^oC for 8-10 hrs.  PBS pH 6.4 + 30% ethanol were used for piperine loaded nanoemulsion.  0.1ml or 0.12ml of nanoemulsion was placed in the donor compartment along with 0.1ml of diffusion media. Recipient compartment containing recipient medium was stirred with Teflon coated magnetic bead. Aliquot (500μl) was withdrawn from the receptor compartment of vertical cell at predetermined time intervals and analyzed. Each sample removed was replaced by an equal volume diffusion media. Study was carried for a period of 8 h, during which the drug in receiver chamber (µg/ml) across the Goat nasal membrane calculated at each sampling point. The formulations were studied in triplicate for diffusion studies. The release kinetics of diffusion was studied by calculating the regression coefficient for zero order, Higuchi’s equation, first order equations, Hixson-crowell cube root law, Korsmeyer-peppas equation.  The diffusion coefficients and flux were also determined.

 

RESULTS AND DISCUSSION:

Nanoemulsions of piperine was characterized for their appearance, globule size, zeta potential, drug content, pH, viscosity, and transmittance, and the results were recorded in Table 1 and 2. Nanoemulsion formulations have globules in less than 150nm. Low polydispersity index values suggest narrow size distribution. ZP were lesser than -8.0 mV indicating stability against globule-globule aggregation. The pH of the formulations was found in the range of pH 4.5 to 6, which is compatible with nasal mucosa. Viscosities of the developed formulation were recorded in the range of 100-200cps. The percentage transmittance of nanoemulsion formulations was found to be more than 99% and shows that the prepared PNE are isotropic in nature. TEM images (Figure 1) are in agreement with the globule size distribution measured by PCS (DLS) (Figure 2).

 

Table 1: Characterization of Developed Piperine Nanoemulsion Formulations:

* Values are represented as mean ± SD, n=3;

 

Figure 1: TEM images for Piperine Loaded nanoemulsion and mucoadhesive nanoemulsion.

 

Table 2: Final Compositions and Characterization of Piperine Nanoemulsion formulations Selected for In-vitro permeation studies:

 

Figure 2: Globular Size distribution for Piperine Loaded nanoemulsion and mucoadhesive nanoemulsion.

 

Table 3: Comparative results of the various parameters calculated from In-vitro permeation profile of piperine nanoemulsion

 

 

Figure 3: In-vitro permeation release profile of piperine from PNE1, PNE 8, PNE15 and PNE 21

 

Table 4: Comparative results of the various parameters calculated from In-vitro permeation profile of piperine mucoadhesive nanoemulsion

 

 

Figure 4: In-vitro permeation release profile of piperine from PNE1, PMNE (CH 0.25%), PMNE (CH 0.50%) and PMNE (CH 1%)

 

Figure 5: CLSM image of nanoemulsion and mucoadhesive nanoemulsion of piperine

 

In- vitro permeation studies across Goat nasal mucosa were performed to compare the release of drug from selected nanoemulsion formulations of piperine (e.g. PNE 1, PNE 8, PNE 15, and PNE 21) and all having the same quantity of drug. In vitro permeation across nasal mucosa was highest in formulation PNE1 and lowest for PNE 8 (Figure 3 and Table 3). The formulation PNE15 and PNE21 showed an intermediate permeation profile. The nasal permeation profile of PNE1 was significantly different when compared with that of PNE26 (P <0 .05). The significant difference in piperine permeation between nanoemulsion formulations was probably due to the mean size of internal phase droplets, which were significantly smaller in nanoemulsions. The maximum release in PNE1 could be due to having the lowest droplet size and lowest viscosity of all the nanoemulsions.

 

The highest permeate formulation PNE 1 from piperine screened formulation was prepared as mucoadhesive nanoemulsion and evaluation for three different level of chitosan concentration (e.g. for Piperine PNE1, PMNE0.25%, PMNE 0.50%, PMNE 1.0%) for the selected formulation and measure the permeability parameters across nasal mucosa. Results indicate that chitosan increases the permeability of the piperine in linear manner along with shortening of initial drug release time (Figure 4 and Table 4). In order to elucidate the disposition of Nanoemulsions in the Nasal mucosa, we examined cross-sections of the Nasal mucosa by CLSM. The confocal images of different cross-sections of the goat Nasal mucosa post washing with buffer solution exposed to the Rd-Nanoemulsions. Qualitative assessment of confocal images revealed intense red colored fluorescent areas located in between and inside the mucosal cells.  Due to the mucoadhesive nature, it was observed that mucoadhesive formulation PMNE shows more red colored intense areas as compared to non-mucoadhesive nanoemulsion PNE as shown in Figure 5.

 

CONCLUSION:

Nanoemulsions and mucoadhesive nanoemulsions were successfully prepared by titration method. Piperine nanoemulsion have very small glouble size (<100nm) and negative zeta potential. While, mucoadhesive nanoemulsion have small glouble size (<150nm) and positive zeta potential. The spherical surface of nanoemulsions and mucoadhesive nanoemulsions was confirmed from TEM. pH of nanoemulsions and mucoadhesive nanoemulsions is compatible with nasal fluid and viscosity of nanoemulsions and mucoadhesive nanoemulsions is suitable nasal administration. In vitro release of nanoemulsion and mucoadhesive nanoemulsion system in nasal mucosal membrane demonstrated prompt and effective release with more than 85 % of drug release in 4 h.

 

ACKNOWLEDGEMENT:

This project has been funded by Department of Science and Technology (DST), New Delhi for providing financial grant under Woman Scientist Scheme.

 

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Received on 03.11.2019           Modified on 15.02.2020

Accepted on 27.04.2020         © RJPT All right reserved