Effect of Ratio Span 60 - Cholesterol on the Characteristic of Niosomes Vitamin D3

 

Audia Triani Olii¹*, Akhmad Kharis Nugroho², Ronny Martien², Sugeng Riyanto²

¹Faculty of Pharmacy, Universitas Muslim Indonesia, Makassar, Indonesia.

²Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia.

 

ABSTRACT:

Vitamin D3 (Cholecalciferol) is a fat-soluble vitamin that is claimed to be an ingredient added to food and beverage products such as cheese and milk. However, due to its unstable nature, it causes vitamin D3 to be degraded in the product. Niosomes are a vesicle drug delivery system formed by nonionic surfactants and cholesterol that can protect both hydrophilic and lipophilic compounds by entrapping them in their vesicle system. Span 60 is the surfactant most often used to form niosomes and cholesterol because it is known to have a higher entrapment ability than other nonionic surfactants. Using Design-Expert version 10 with the Simplex Lattice Design (SLD) model, eight formulas with varying concentrations of Span 60 cholesterol were obtained. SLD analysis results showed a negative interaction (interaction coefficient -301.35) between Span 60 cholesterol on particle size so that the Span 60 Cholesterol mixture reduced the size of niosome particles. However, cholesterol has a more significant positive effect (coefficient value + 279.45) on niosome particle size than Span 60 (coefficient value + 243.20), which means a formula with a higher amount of cholesterol causes a larger particle size. Contrary to zeta potential, Span 60 shows a more significant negative effect (coefficient value -47.54) than cholesterol (coefficient value-29.25), which means, Formula with a higher number of Span 60 causes the zeta potential to be more negative.

 

 

INTRODUCTION:

Vitamin D3 is a fat-soluble vitamin that is always associated with bone health. Still, now it is known that vitamin D3 affects several health conditions such as cardiovascular disorders, diabetes, asthma, to cancer^1,2.

 

Research shows that the main problem related to vitamin D3 deficiency, which occurs in about 30 - 50% of the entire world population, is caused by inadequate vitamin D3 due to many factors. Vitamin D3 derived from food and beverages or supplements can only provide about 10-20% of vitamin D3. Low stability of vitamin D3 becomes an obstacle when formulated into food products and supplements^1,3–5.

 

 

 

Niosome analog vesicle systems of liposomes formed from nonionic surfactants and cholesterol can entrap both hydrophilic or lipophilic with a size range of nm 10 - 1000 nm^6–9. Besides increasing the bioavailability of drugs with poor solubility, another advantage of niosomes is that they protect the drug from degradation that may occur before the drug reaches its target¹⁰. Various factors can affect the characteristics of the niosome including the type and amount of surfactant and cholesterol used and the method of making niosomes¹¹. The type of nonionic surfactant that is most often used to form niosomes is Span 60 which is stabilized by the addition of some cholesterol^12,13. The function of adding cholesterol is to improve the bilayer density, affecting the thickness and permeability of the niosome. Also, cholesterol causes the size of the vesicles more bigger, the possibility of increasing the efficiency of drug absorption in the niosome is also greater^14–17.

 

Particle size, particle size distribution, and zeta potential of niosomes are parameters that significantly affect stability, solubility, release velocity, and bioavailability of vesicles ^5,18–20. Nanoparticles with a small size below 200 nm are often used for oral delivery systems considering long transit times in the gastrointestinal tract ⁵. Zeta potential ranging from -41.7 and -58.4mV are good for electrostatic stabilization in the niosome system ⁶. In general, zeta potential above 30mV (absolute value) in a system such as niosomes is considered stable due to the repulsion between particles in the system²¹. It should be understood that, however, niosomes are forming from the nonionic surfactants. It will show a negative charge due to the adsorption of OH^‑ from water to the water-lipid interface⁵.

 

In this research, niosomes formed by Span 60 and cholesterol. Span 60 is a surfactant whose HLB value is 4.7 and CPP 0.5-1, and the phase transition temperature is 53^oC. Based on several studies, Span 60 can form the most optimal niosome^{15, 18, 22–25}. The aim of this study is to determine the effect of ratio Span 60 and cholesterol on the characteristics of niosome vitamin D3.

 

MATERIAL AND METHODS:

Material:

Vitamin D3, ethanol dan metanol purchased from Sigma-Aldrich (USA), Span 60, cholesterol purchased from Croda Europe Ltd (UK), aqua deion. (Indonesia).

Methods.

1.     Preparation of niosomes:

Niosomes were prepared by a thin layer hydration method8,22. Span 60, cholesterol, and vitamin D3 are dissolved in 10mL ethanol in a round bottom flask. Ethanol is then removed under vacuum with a rotary evaporator at 60oC and 150rpm for 30 minutes until a thin layer is formed on the wall. The thin layer was dried in a desiccator for 24hours, then hydrated with 10 mL aqua deion and sonicated for 30 minutes. The optimation formula using Design-Expert version 10.0.1 with the Simplex Lattice Design (SLD) model.

Tabel 1: Simplex Lattice Design of noisome preparation using 10 mg of vitamin D3

Formulation	Span 60: cholesterol (mole ratio)
R1	75:125
R2	150:50
R3	100:100
R4	125:75
R5	50:150
R6	100:100
R7	150:50
R8	50:150

 

2.     Characterization of niosomes vitamin D3:

Morphology characterized using Transmission Electron Microscopy (TEM), while the particle size, polydispersion index, and potential zeta were measured using Horiba SZ-100 particle size analyzer. Entrapment Eficiency (EE) of niosomes vitamin D3 was determined by HPLC 8. Niosomes vitamin D3 in etanol centrifuged at 4oC, 15.000 rpm for 30 minutes and than concentration of vitamin D3 in supernatan determined using HPLC analisys, EE was calculated by the following equation5:

           Vitamin D3 used in preparation – vitamin D3 in supernatan

% EE ----------------------------------------------------------------------- x100

                                   vitamin D3 used in preparation

RESULTS AND DISCUSSION:

Morphology and visual appearance of noisome vitamin D3

The analysis morphology of niosomes using TEM both in formulas with or without vitamin D3 show spherical vesicles. The visual appearance of niosome is opaque dispersion with turbidity that decreases after sonication. This is because the particle size of niosomes affects the dispersion's turbidity 26.

  

(a)                                            (b)

Figure 1: TEM image (a) Morfologi niosom Span 60: Cholesterol: vitamin D3, (b) Morfologi niosom Span 60: Cholesterol

Particle size, polydispersion index, and potential zeta

Results of characteristic from the eighth Formula using Design-Expert version 10.0.1 with the Simplex Lattice Design model (Table 2.) shows that interaction between Span 60 and cholesterol with niosome particle size means is a negative interaction (interaction coefficient -301.35), which means that the mixture of Span 60 cholesterol decreases the size of niosome particles (Tabel 3). However, either cholesterol or Span 60 each has a positive influence on particle size. Cholesterol has a more significant positive effect (coefficient value + 279.45) on niosome particle size than Span 60 (coefficient value + 243.20), which means a formula with a higher amount of cholesterol causes a larger particle size (Figure 2). The size of niosome particles depends on the composition of vesicle-forming surfactants and cholesterol. More lipophilic with longer the alkyl chain surfactant will produce niosomes with large size. Span 60 is a lipophilic surfactant with a large CPP value, between 0.5 - 1. This makes the Span 60 niosome quite large. As for cholesterol, the higher the amount in niosomes will cause the particle size to be larger. This is because cholesterol molecules place themselves between the surfactant's tail in the bilayer, making the bilayer more thickness and the niosome becomes larger. Although there is also a theory that states that when the amount of cholesterol is lowered, the size of the niosome particles is larger because the bilayer's hydrophilicity increases. Increasing in hydrophilicity causes increased surface free energy and water entering the vesicle's core21,27,28. The method of preparing niosomes might be the cause of the varied effect of cholesterol on particle size, same as in the case of liposomes, where the effect of cholesterol on particle size can vary depending on the method and type of phospholipid29.

Similar to particle size, the results of the analysis of potential zeta responses from the eight formulas illustrate that the interaction between span 60 and cholesterol on zeta potential is negative (interaction coefficient -58.98). This means that the mixture of Span 60 and cholesterol makes zeta potential of niosom more negative and illustrates that the possibility of aggregate in the system decreases. Surfactants have a more significant effect (coefficient value-47.54) than cholesterol with a coefficient of -29.25. This is indicated that the more Span 60 in formulas, the more negative zeta potential of niosome (Table 3).

The polydispersion index and % EE showed insignificant results. This indicates that the ratio of Span 60 and cholesterol in the niosome formulation of vitamin D3 did not significantly affect both the polydispersion index and % EE.

 

Tabel 2. Characteristic of Niosomes vitamin D3

Run	Span 60 (µmol)	Cholesterol (µmol)	size particles (nm)	polydispersion index	Zeta potential (mV)	%EE
1	75	125	253	0.339	-38.6	94.647
2	150	50	230.5	0.461	-54.8	95.389
3	100	100	176.2	0.165	-55.3	95.558
4	125	75	163.8	0.318	-60.7	91.018
5	50	150	276.5	0.269	-27.5	97.085
6	100	100	190.4	0.381	-50.7	94.488
7	150	50	255	0.376	-37	94.449
8	50	150	281.5	0.283	-34.2	97.079

 

Tabel 3. Statistical analisys software 

Respons	Equation
Size	Y=243.20(A)+279.45(B)-301.35(AB)-379.07(AB)(A-B)
Zeta potensial	Y=-47.54(A)-29.25(B)-58.98(AB)

(a)

(b)

Figure 2: Effect of Span 60 cholesterol ratio on particle size (a), the effect of Span 60 cholesterol ratio on zeta potential (b)

CONCLUSION:

In this study, the comparison of the concentration of Span 60 and cholesterol as niosome forming material greatly influences the particle size and zeta potential of niosomes. The results showed that an increase in the amount of cholesterol in the Formula caused a rise in niosome particle size and an increase in the number of Span 60, causing the zeta potential of the niosome to become more negative. However, at the same ratio between Span 60 and cholesterol, the niosomes particles' size can be reduced, and zeta potential becomes more negative.

 

ACKNOWLEDGMENT:

Financial support from the Ministry of Research, Technology, and Higher Education of Indonesia (Kemenristek Dikti) through a doctoral dissertation research grant program.

 

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Accepted on 03.01.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5551-5554.