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Niacinamide works by a unique mechanism of controlling pigmentation, by restricting the transfer of melanosome from melanocyte to keratinocyte, while most of the other skin lightening actives work by inhibiting the enzyme tyrosinase and therefore interfere with the synthesis of melanin. Various patents have been filed for the skin benefits of niacinamide. The skin benefits mostly covered are treatment of pigmentation, antiaging, moisturization, slimming, antiacne, hypoallergenic benefits, and for niacinamide synergistic benefits with sunscreens. Some patents have been issued and some are in the application stage. Niacinamide is one of the safe and effective ingredients used for over three decades in cosmeceuticals. Niacinamide has been studied for various skin benefits. Niacinamide influence cutaneous pigmentation by down-regulating transfer of melanosomes from the melanocytes to the keratinocytes[1]. Studies suggest that niacinamide has no effect on tyrosinase activity, melanin synthesis or melanocyte number in a monolayer culture system[2]. Alternatively, the authors found that niacinamide down-regulated the number of melanosomes transferred from melanocytes to keratinocytes by 35 to 68% in a co culture model system[3]

Liposomes range in size from 0.025 micrometres (extremely small liposomes) to several micrometres (large liposomes). They are categorised based on how many double layers there are[4,5]. The primary liposome type for skin delivery of substances, according to the majority of authors, is LUV. Researchers are attempting to increase the permeability of the vesicles by adding edge activators in addition to optimising liposome size (EA).

Since Tweens and bile salts such sodium cholate (SC) increase membrane fluidity and thus deformability, they are preferred to Span or other EAs[6]. EA's physical characteristics in UDL enable alteration of the liposome's structure at a reduced energy cost, speeding up the lipid bilayer's rearrangement.

The procedure used to generate and characterise conventional liposomes can be used to prepare and characterise other types of liposomes. In contrast to normal liposomes, which have lesser skin permeation, ethosomes are another type of liposome that were designed to increase skin permeation. These vesicles are made of phospholipids, ethanol, and water[7]. Ethosomes' durability, encapsulation effectiveness, and capacity to penetrate the stratum corneum have all improved[8]. Because ethanol gives nanoparticles flexibility, ethosomes can penetrate deeper skin layers. Liposomes made with phospholipids, EA, and menthol are known as mentosomes[9]. Menthol is a permeation enhancer that alters vesicle organization[10]. Thin-film hydration is a method that can be used to create these liposomes¹¹.

Preparation of liposome suspension with niacinamide: Liposome suspension was prepared with a phospholipid available under brand name Pro-lipo Neo69, supplied by Lucas Meyer cosmetics, France. Liposome suspension formula Niacinamide (water soluble active) Pro-lipo NeoR [lecithin (22%) in propanediol] Water Aqueous solution of niacinamide was made and added to a specified quantity of Phospholipid, mixed and homogenized to obtain the liposome suspension. While formulating a cream with the liposome, required quantity of the liposome suspension was added to the cream base at 400C with slow mixing. The phospholipid ratio for formulating the liposome suspension was calculated by using the formula 1part of niacinamide X solubility factor of niacinamide X 0.11 (Min Phospholipid level) - Eq1 1 part of niacinamide X solubility factor of niacinamide X 0.43(Max Phospholipid level) - Eq 2 The phospholipid factor, 0.11 and 0.43 is unique to the phospholipid composition derived by the ingredient supplier. Niacinamide aqueous solubility was considered as 40% based on the study on its solubility and crystallization at various storage temperatures. At 40% solubility, the solubility factor of niacinamide will be 100/40 = 2.5. From this data minimum and maximum ratio of phospholipid, for effective liposome formation, was calculated using eq 1and 2. 1 X 2.5 X 0.11 (Minimum phospholipid ratio) = 0.275 1 X 2.5 X 0.43(Minimum phospholipid ratio) = 1.075 For our study phospholipid ratios selected were 0.275, 0.55, 0.75 and 1.075. Liposome suspension was obtained by dissolving 1 part of niacinamide with 2.5 parts of water and the solution was added to 0.275, 0.55, 0.75 and 1.075 parts of phospholipid respectively and homogenized at 5000 RPM for 5 minutes using homogenizer polytron PT 1600E. Characterization of liposomes: The liposome suspension was characterized as such and in diluted form (1:5). Samples were diluted with dust-free ultra-pure water. Zeta potential, conductivity, particle size and PDI were measured at 25°C and at 90° scattering angle. Surface morphology: Surface morphology of the liposome suspension as such and in diluted form (1:5) with ultra-pure water was studied using SEM, model Joel-5400. This is to evaluate the effect of dilution on the surface morphology of the liposomes. The sample was taken in an aluminum stabu (Plate). The stabu was insulated with a carbon tape and placed in the instrument. Vacuum was applied and SEM photographs were taken. Entrapment value: The liposomal suspension was diluted to 400gm with water to yield 1.5% niacinamide both as entrapped and non-entrapped form. 5 ml of the diluted sample was centrifuged at 20000 RPM. After one hour the supernatant liquid was drawn and analyzed for niacinamide, from which entrapment value was calculated, since supernatant liquid will be niacinamide that is un-entrapped. The phospholipid ratio that gave higher entrapment value was introduced in the cream base for further studies.

Characterization of liposomes:of liposomes:

Four liposome suspensions of niacinamide were prepared as per the method discussed and were characterized, both in diluted and undiluted form, for zeta potential, conductivity, Particle size and PDI. The results obtained are given below.

Zeta potential:

The results of the zeta potential measurements of the un-diluted and the diluted liposome suspensions are given in table 20a and 20b respectively. From the results it was observed that with increase in lipid levels from 0.275 to 1.075 the zeta potential increased from -10.92 to -3.27. Whereas, in diluted samples as lipid level was increased from 0.275 to 1.075, zeta potential increased from -51.4 to -40, but the variation in the case of undiluted samples was not statistically significant. Zeta potential increased by 10 times on dilution of the sample. The Zeta potential of undiluted and diluted samples was significantly different. The zeta potential of undiluted sample was found to be significantly influenced by phospholipid levels used.

Figure -2 Effect of phospholipid level and sample concentration of liposomes on zeta potential

Conductivity:

The results of the conductivity measurements of the un-diluted and the diluted liposome suspensions are given in table 21a and 21b respectively. It was observed that the conductivity of undiluted and diluted samples was significantly different. However, phospholipid levels had no significant effect on conductivity in both undiluted and diluted samples. There was about 0.5 fold decrease in conductivity in diluted samples compared to undiluted samples.

Figure-3: Effect of phospholipid level and sample concentration of liposomes on conductivity

Particle size:

The results of the particle size measurements of the un-diluted and the diluted liposome suspensions are given in table 22a and 22b respectively. It was observed that the particle size of undiluted and diluted samples was significantly different. The particle sizes of undiluted sample were found to be significantly influenced by the quantity of lipid used and ranged from 650nm to 950 nm with the increase of phospholipid levels from 0.275 to 1.075. In diluted sample the particle size ranged from 243 nm to 264 nm and phospholipid levels had no significant effect on particle size of diluted samples.

PDI:

The results of the PDI measurements of the un-diluted and the diluted liposome suspensions are given in table 23a and 23b respectively. It was observed that the PDI of undiluted sample was found to be significantly increased by 2.5 folds with the increase in lipid levels from 0.275 to 1.075. The PDI ranged from 0.4 to 1 in undiluted samples. PDI of diluted samples were found to be in a range of 0.25 to 0.27 and were not significantly influenced by phospholipid levels.

Figure -4: Effect of phospholipid level and sample concentration of liposomes on Particle size

Figure -5: Effect of phospholipid level and sample concentration of liposomes on PDI
From the results of zeta potential, conductivity, particle size and PDI, it was observed that liposomes in two different concentrations exhibit differences in the properties. Results were consistent in the diluted form indicating the importance of sample preparation to determine the physico-chemical properties of dispersed system using dynamic light scattering technique.

Surface morphology:

The SEM image of liposomal suspension in un-diluted and diluted form is shown in figure 6. It was observed that the particle size and shape of liposomes in both the samples were found to be similar. This is in contrary to particle size data obtained through Malvern zetasizer.

Undiluted                                                     Diluted
Figure -6 SEM image of niacinamide liposomal suspension

The results can be explained through the principle of differential light scattering (DLS) technique. DLS is a technique that measures Brownian motion and correlate to the particle size. The particles have to be suspended in a liquid. The fluctuation in the intensity of the scattered light is detected and digitally processed through a correlator. The data is further analyzed and information on particle size and zeta potential are generated. The sample preparation is critical to get reliable results.

Malvern in their technical note has discussed on the sample requirements to obtain reliable results [12-14]. The optical properties, particle size and PDI are important factors affecting the zeta potential data. The samples have to be optically clear.

Technical literature from nano Composix[15-16] explains that in a highly conductive sample, the movement of conductive ions can lead to electrode polarization and degradation and therefore can lead to inconsistent Zeta potential values.

In the present study SEM pictures of both diluted and concentrated samples were similar. However, they showed different characterization parameter. The concentration of the sample that had altered conductivity, and optical properties, were important contributors to get different results. The concentrated samples in the experiment scatter more light that gave inconsistent particle size data with high PDI. The diluted samples scatter light within the detection limits and therefore the results were consistent. It is also seen in the diluted samples conductivity decreased significantly. This can be an important factor for obtaining zeta potential data. Conventional equipment for measuring particle size using Dynamic light scattering has been upgraded regularly. The non-invasive back scatter (NIBS) technology, of Malvern[17-19] is an upgraded technology that can measure wide range of concentration of the samples.

Entrapment value: The results of the effect of phospholipid level on entrapment value of the liposomal suspensions are given in figure 25. It was observed that the entrapment value of liposomes with phospholipid levels 0.275, 0.55, 0.75 and 1.025 were 66.3%, 67.75%, 65.6% and 71.87% respectively. The phospholipid level at 1.025 gave directionally higher entrapment value and therefore the liposomes made with phospholipid at the level of 1.025 was taken for further studies.

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