Summary
Introduction
What Are Nanoparticles?
Preparation and Characterization of Nanoparticles
Unique Properties of Ultra Small Nanoparticles
Stability of Nanoparticles
Activity of Nanoparticles
Small Positively Charged
Nanoparticles for Hair Care
Conclusions
References

Summary
Nanoparticles are small lipid vesicles formed by a monolayer of phospholipids. Whereas liposomes are typical carriers for hydrophilic substances, nanoparticles are the ideal delivery system to transport and protect lipophilic agents.

In our laboratory, we have developed a method to prepare very small nanoparticles encapsulating different agents of cosmetic and pharmaceutical interest (Tretinoin, Retinol, Vitamin E Acetate, UV-Filters, Fragrance). The technique of high pressure homogenization at 1200 bar using a microfluidizer yields a 100% encapsulation of the oil in defined vesicles.

The vesicle size has a great influence on the optical appearance of the nanoparticle dispersion. Preparations of particles with diameters of less than 60 nm are transparent dispersions of oil in water. These small nanoparticles show unique additional physical properties and offer new application possibilities.

Our data show that nanoparticles are very stable and have a high affinity to the stratum corneum. Therefore, an enhanced bioavailability of the encapsulated material to the skin is achieved.

We have also developed a nanoparticle delivery system to target the vesicles to hair. For that purpose, we have dotted the nanoparticle shell with cationic molecules thus producing a positively charged surface. Our experiments show that positively charged nanoparticles loaded with UV-filters have an almost one hundred fold higher affinity to hair than negatively charged particles.

Introduction

Lipid vesicles were first described by Dr. Alec Bangham in 1965 [ 1 ]. He had observed that handshaken phospholipid dispersions in water form multilamellar spherical structures. These vesicles, soon named liposomes, consist of an aqueous cavity encapsulated by one (Figure 1) or more lipid bilayer membranes. Since these early investigations, more than 20 years have past till the first cosmetic products containing liposomes appeared on the market (NIOSOMES from Lanc?me and CAPTURE from Dior, 1986). However, only three years later, more than one hundred different liposomal formulations could be found.

Figure 1. Comparison of the structure of liposomes and nanoparticles formed by soy phospholipids.

Liposomes are used to carry and protect hydrophilic agents. Water soluble agents are enclosed into liposomes if they are present during the preparation. However, some part of the material always remains in the outer phase. In addition to water soluble substances, also amphiphilic and lipophilic substances can be loaded into liposomes to some extent. Amphiphilic molecules stick to the membrane whereas lipophilic substances can be incorporated into the hydrophobic part of the bilayer. Usually such molecules have a negative influence on the stability of the liposomes.

In contrast to liposomes, nanoparticles are the ideal carrier system to transport and protect lipophilic agents.

What Are Nanoparticles?
Nanoparticles are small lipid vesicles in the range of nanometers. The best way to characterize them is to compare them with liposomes and emulsions. Liposomes and nanoparticles are of comparable size. Both occur in the range from 20 to 1000 nm in diameter. Whereas liposomes are composed of one or more bilayer membranes, nanoparticles are formed by a single layered shell (Figure 1). Liposomes are filled with water and therefore are typical carriers for hydrophilic substances. On the other side, nanoparticles are filled with oil and lend themselves ideally as carriers for lipophilic agents.

Nanoparticles can also be described as a submicron emulsion of oil in water stabilized by a natural emulsifier. These emulsions are well accepted and used as delivery system for parenteral drug administration [ 2, 3 ].

Preparation and Characterization of Nanoparticles
High pressure homogenization using a microfluidizer is a sophisticated technology to prepare lipid vesicles such as liposomes and nanoparticles [ 4 ]. The method is easy to scale up and yields reproducible results. The homogenizer has a specially designed interaction chamber. In this chamber, the stream of the premixed components is first divided and then combined again at a particular angle. At this point, high shear and cavitation forces form the lipid vesicles at a pressure of up to 1200 bar.

The technique of high pressure homogenization yields in a 100% encapsulation of dispersed oil into the vesicles.

Table 1. Correlation of particle size with concentrations of lecithin and oil

Usually, multiple cycles through the interaction chamber are necessary to obtain a homogenous product. The mean droplet size and the size distribution are the main parameters to characterize nanoparticle preparations. They can be determined by photon correlation spectroscopy or by means of electron microscopy of samples prepared by freeze fracture.

The core of the particles can contain a wide variety of different cosmetic oils (triglycerides, jojoba oil, borage oil, wheat germ oil, macadamia nut oil) and lipophilic agents (vitamin A palmitate, vitamin E acetate, retinol, tretinoin, UV filters, fragrances). The chemical stability of these ingredients (against oxidation) can be enhanced by their encapsulation into nanoparticles [ 5 ].

Nanoparticle preparations can contain up to 40% of oil. The vesicle size is influenced by many parameters. Most important are homogenization pressure, concentration and type of lecithin, concentration and type of oil and the solvent concentration in the water phase [ 6 ]. Very small particles can only be achieved at a high ratio of phospholipid to oil. Table 1 correlates the phospholipid concentrations of nanoparticle preparations and the encapsulated oil volumes depending on the size of the vesicles. Preparations consisting of high concentrations of phospholipids and oil are very viscous and unstable and therefore not suitable as cosmetic raw materials.

Unique Properties of Ultra Small Nanoparticles

The vesicle size has a great influence on the optical appearance of the nanoparticle dispersions. Preparations of particles with diameters of 200 nm or more are white even in diluted dispersions. Preparations containing particles of 100 nm appear opaque. A further reduction of the particle size to below 60 nm results in clear transparent dispersions of oil in water. These preparations offer new application possibilities in hair care preparations or transparent hydrogel formulations. In addition, a very high bioavailability of the encapsulated material to skin and hair is obtained.

Figure 2. Stability of a nanoparticle preparation containing 3% vitamine E acetate and 1% vitamine A palmitate at different temperatures.

As a result of the very small particle size of transparent preparations, we observed a retarded crystallization of molten lipids. A nanoparticle dispersion prepared from molten hydrogenated peanut oil (melting point 35°C) remains a liquid submicron oil-in-water emulsion (determined by differential scanning calorimetry) even after the preparation was stored for several weeks at 4°C. As with the phenomenon of the supercooled melt, we observed the presence of supersaturated solution of an UV-filter encapsulated in nanoparticles.

Stability of Nanoparticles
Nanoparticles are very stable dispersions of oil in water. These emulsions are stabilized by a negative zeta potential which prevents droplet coalescence upon random collisions of particles. The instability of nanoparticles is measured as an increase in particle size determined by photon correlation spectroscopy. At high temperature and high particle concentration, the vesicles start to fuse. An increase of the mean particle size can then be measured. Figure 2 shows the stability of a nanoparticle preparation at different temperatures.

The overall negative charge (zetapotential - 30 mV) on the surface of our particles results in repulsion forces stabilizing the preparation. The strength of these forces is strongly reduced when ions are present. Even low concentrations of salt (50 mM) result in a quick increase of particle size. Ions and positively charged polymers must therefore be avoided in cosmetic preparations containing nanoparticles.

Activity of Nanoparticles
The most important property of lipid vesicles based on phospholipids is their affinity to the stratum corneum. A large number of investigations provide clear evidence that vesicles, such as liposomes, exert a pronounced influence on the epidermis [ 7, 8, 9 ]. In an review [ 10 ], Mezei summarizes clinical investigations which clearly demonstrate that topical applications of drugs, such as corticosteroids, antifungals, local anesthetics and retinoids, encapsulated in liposomes result in increased concentrations of the agents in the epidermis and dermis compared to conventional formulations. On the other hand, the systemic concentrations of these drugs (plasma, liver and spleen) are reduced compared to the controls. These results prove that liposomes are suitable vehicles for a selective drug delivery in the skin.

Nanoparticles have a structure similar to liposomes and can therefore perform in a similar way (Table 2).

Table 2. Performance of lipid vesicles on the skin

Figure 3. Effect of nanoparticles containing derivatives of vitamines E and A on skin humidity determined with Corneometer CM 820. The products (xanthan gum gel 20% nanoparticles and xanthan gum gel as control) were applied twice daily on the forearm of 20 volunteers over a period of 14 days.

Figure 3 demonstrates the influence of nanoparticles on skin humidity. The application of a gel containing nanoparticles loaded with vitamin A and E derivatives enhances the skin humidity compared to the controls. The effect is statistically significant and proves that these lipid vesicles interact with the stratum corneum. The increase of skin humidity is due to the high waterbinding capacity of the phospholipids which form the nanoparticles. Similar beneficial effects are also obtained regarding skin roughness by topical application of nanoparticles (data not shown). However, the main goal of the treatment is to improve the bioavailability of the applied vitamins to the skin. It is evident that the nanoparticles penetrate into the top layers of the stratum corneum. There they fuse with skin lipids and the active agents (vitamins) are released.

The beneficial properties of these vitamin derivatives have been investigated by in vitro experiments (Figure 4). Mouse fibroblast cells were cultured in serum free medium containing different concentrations of nanoparticles loaded with vitamin A palmitate and vitamin E acetate. A pronounced growth stimulation of these skin cells could be determined with increasing concentrations of these lipid vesicles indicating their nutritional and protective value.

Small Positively Charged Nanoparticles for Hair Care

Figure 4. Growth stimulation of mouse fibroblast cells with nanoparticles in a serum free medium. The cells were cultured adherent in tissue flasks at different concentrations of the nanoparticles. The nanoparticle preparation was sterilized by filtration (0.1µm) and contained 0.6% phospholipids, 0.6% carrier oil, 0.3% vitamine E acetate and 0.1% vitamine A palmitate.

The formulation of lipophilic substances in hair care products is unsatisfactory. Conventional oil in water emulsions used to deliver lipophilic agents to hair and scalp leave hair feeling sticky and greasy. In addition, only a poor affinity of the substances to hair is usually observed. In contrast, nanoparticle preparations with a vesicle size of less than 50 nm are transparent and do not feel greasy. These particles represent a new delivery system to the scalp. The natural phospholipids are well tolerated emulsifiers which enhance the penetration of the active agents. Thus the encapsulation of lipophilic agents in nanoparticles is a very promising galenic novelty which is easily applicable for the treatment of disorders like alopecia, dandruff or sunburn.

We have developed a positively charged delivery system to target encapsulated agents to hair. For that purpose, we have dotted the shell of nanoparticles with cationic molecules to get a positive zetapotential [ 11 ]. In our in vitro experiments, we have encapsulated Uvinul T 150® (UV-B filter) as active agent. We observed an almost one hundred fold higher affinity of Uvinul T 150® to hair from positively charged particles compared to negatively charged particles (Figure 5). The highly improved substantivity to hair through positively charged nanoparticles can also be obtained with other loadings, such as UV-A sunscreens, vitamins or colors.

Figure 5. 0.5 g human hair was treated according to the protocol and incubated for 5 minutes with 100 ml of two different preparations (positively and negatively charged nanoparticles) containing 6% of UV-B filter Uvinul T-150®. Bar A shows the amount of UV filter which could be extracted after treatment with negatively charged particles. Bar B shows the value after treatment with positively charged particles. Control C shows the value for untreated hair.

Conclusions
Phospholipids from soy have successfully been used to prepare small vesicular carriers for topical application of hydrophilic (liposomes) and lipophilic (nanoparticles) agents. The technique of high pressure homogenization permits the industrial production of high quality vesicle dispersions for cosmetic and dermatological use.

Particle size, surface charge and payload determine the properties of the preparation and their application.

Liposomes and nanoparticles have become an indispensable component of today's advanced personal care products and have acquired a permanent place in cosmetic formulations. The phospholipids forming these carriers enhance the penetration of the active agents into the stratum corneum and therefore increase their bioavailability. At the same time, lecithin is also an excellent skin softening and moisturizing agent itself. Furthermore, sensitive compounds can be protected with these structures.

The preparation of very small nanoparticles offers new application possibilities of the established carrier system. Lipophilic ingredients become water-dispersible in transparent formulations and have a very high affinity to the stratum corneum.

Other modifications of nanoparticles such as the inversion of surface charge, allow the design of new consumer products for hair care.

Further scientific work in the field of liposomes and nanoparticles will generate new properties of these vesicles for commercial applications in advanced dermatological products.

References:
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10 Mezei, M. Biodisposition of liposome-encapsulated active ingredients applied on the skin. In O. Braun-Falco, H. C. Korting and H. I. Maibach, eds, Griesbach Conference on Liposome Dermatics, Heidelberg: Springer-Verlag, Berlin, 206-214, 1992

11 Z?lli, F., Suter, F. and Birman, M. Cationic nanoparticles: A new system for the delivery of lipophilic UV-filters to hair. Drug & Cosmetic Industry, 4, 46-48, 1996