"LIPOSOMES LOADED WITH FULLERENE AND PROCESS FOR THETR PREPARATION"

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The present -invention relates to liposomes loaded with fullerene and the process for their preparation.

Liposomes exhibit a structure similar to the one of the cell membranes. It is well known that liposomes are bi-layer phospholipid spherical aggregates that are formed in aqueous solutions as a consequence of a mechanical reorganization of the bimolecu- lar layers in which phospholipids spontaneously organize when dispersed in an aqueous solution. The liposome vesicles may consist of a single membrane phospholipid double layer (SUV-Small Unilamellar Vesicles) or two or more membrane phospholipid double layers (MLV-Multilayer Lipid Vesicles) having their polar heads oriented toward the exterior and toward the inner aqueous layers of the vesicle. Liposomes have been used in the past in the pharmaceutical field, nowadays they find an extensive use in the cosmetic industry. The interest of said industry in the use of liposomes is due to the fact that liposomes may constitute highly effective systems for the delivery of a broad spectrum of hydro- and lipo-soluble active principles. The hy- drosoluble materials may be contained into the liposome inner aqueous spaces and electrostatically bound to the lipid double layer surface. On the other hand the lipo- soluble materials are retained within the lipid double layer or are entrapped, as minute droplets, within the liposome aqueous zones. The use of liposomes as pharmaceutical and cosmetic active principle carriers has some advantages i.e. a higher treatment efficiency, a reduction of the active principle to be administered, an active principle protection against its deterioration as well as a prolonged release time thereof. At present there are various methods for the production of liposomes. EP 678295 de- scribes a more advantageous process with respect to those more widely diffused in the prior art, the latter having, among the main drawbacks, the difficulty, if not impossibil-

ity, of "anchoring" the active principles to lhe liposomes. The process of EP 678295 is more effective for the production of liposomes containing active principles with a high encapsulating capacity. The above process of EP 678295 comprises:

1) dissolution of the active principle(s) in a solvent;

2) addition of the phospholipid to the above solution;

3) possible elimination of the solvent if this is undesired in the final product;

4) addition to the complex thus obtained of a hydrophilic medium, in particular an alcohol, to allow the organization of the molecules of this complex into a multilamellar structure organized in a series of bimolecular layers;

5) treatment of the complex to obtain it in the desired form.

As already mentioned above, in order to obtain liposomes, it is sufficient to add an aqueous solution to the phospholipid/active principle complex, whose volume is calculated in relation to the concentration of the active principle to be obtained in the liposomal solution.

It is also known that fullerene is a cluster consisting of 60 carbon atoms which is obtained from graphite crystals treated with a high energy laser. Fullerene contains 30 C=C double bonds, it is an extremely stable compound, insoluble in water, but soluble in some organic solvents, in particular toluene. It is also known that fullerene has excellent antioxidant capacities. Its use in particular applicative fields, for example der- matocosmetology, however, is inhibited by the extremely poor solubility (or also dis- persibility) in an aqueous environment.

Liposomes loaded with fullerene have now been found, and this forms a first object of the present invention, the above liposomes being present in aqueous solution and/or dispersion.

A simpler method than that described in EP 678295 has also been found, which allows the above liposomes loaded with fullerene to be obtained.

A second object of the present invention therefore relates to a process for the preparation of liposomes loaded with fullerene, which comprises: a) dissolving fullerene in a solvent; b) adding the phospholipid to the solution obtained in step (a) and mixing until the complete disappearance of the fullerene; c) eliminating the solvent, thus obtaining the phospholipid/fullerene complex; d) diluting the phospholipid/fullerene complex with water thus obtaining liposomes loaded with fullerene, the above fullerene being directly bound to the liposome membrane.

The term "phospholipids" refers to any phospholipid or combinations of phospholipids capable of forming liposomes. Phosphatidylcholines (PC), comprising those of a natu- ^' ral origin (vegetable or animal) and/or those partially or totally synthetic, with a varying lipid chain length and with varying saturation degrees, are preferably used for the present invention. Among phospholipids of a vegetable origin, soya lecithin can be mentioned; among those of an animal original lecithin ex ovo and cerebrosides should be remembered.

The preferred solvent which can be used in step (a) is toluene, a relatively low-boiling solvent with excellent solvent capacities towards fullerene.

In step (b), the phospholipid is added in solid form, usually in powder form. The mixture is kept under stirring until the almost total complexing of the fullerene. The com- plexing of fullerene is easily monitored by visibly observing the disappearance of the typical black colour of free fullerene.

In step (c) the solvent used in step (a) is eliminated using well-known techniques, for example by means of atmospheric distillation or, more preferably, at reduced pressure. At the end of step (c) the phospholipid/fullerene complex is in the form of a solid. The fullerene content in the phospholipid/fullerene complex depends on the type of phospholipid used. The fullerene content in the complex normally varies from 0.1% to 1%

by weight.

Step (d) consists in dilution with water (or physiological solution) of the phosphol- ipid/fullereήe complex obtained at the end of step (c). A gel is thus obtained, whose viscosity obviously depends on the quantity of water added. The quantity of water (or physiological solution) used in step (d) is such as to obtain liposomes in a concentration of 10% to 80% (preferably from 20% to 70%) with respect to the liposomes- water sum. In this step, it is preferable to use the lowest possible quantity of water. Possible dilutions will be effected during the use of the liposomal solution. In step (d) of the present invention, it is preferable to also add a preserving system suitable for inhibiting the growth of micro-organisms, thus obtaining a microbiologi- cally stable product.

The liposomes loaded with fullerene of the present invention show (see experimental part) a high skin absorption and excellent anti-oxidizing capacity, much higher than Vitamin C and Vitamin E. These properties can be exploited in biological systems, in particular in dermocosmetic compositions.

The present invention also relates to the use of liposomes complexed with fullerene of the present invention in the preparation of dermatological compositions suitable for reducing the formation of free radicals.

The following examples are provided for a better understanding of the present invention.

EXAMPLES

Preparation of the liposome loaded with fullerene

A solution is prepared at 0.2% (w/w) of pure Fullerene in Toluene (10% w/w), by dissolving it under stirring and, if necessary, also agitating it. When the solution is complete, the phospholipid (89.8% w/w) is added a little at a time and the solution is mixed until a homogeneous compound is obtained. The toluene is evaporated under a suction hood for at least 2 hours, or in any case until

there is no longer the characteristic odour of the solvent.

Phospholipid of Fullerene is thus obtained from which the related liposome is prepared.

Deionized water (46.8% w/w) is mixed a little at a time with the phospholipid previously obtained (50% w/w) under stirring and the mixture is then agitated for at least 5 minutes until a homogeneous paste is obtained. Finally, the preserving system (about 3% w/w) previously dissolved in propylene glycol, is added. The percentage of preservative guarantees the growth inhibition of micro-organisms which is included in the acceptability range for considering the product microbiologically stable. Once all the preservatives have been added, the mixture is agitated until a homogeneous product is obtained.

DETERMINATION IN VITRO OF THE SKIN ABSORPTION OF THE LIPOSOMES LOADED WITH FULLERENE Materials and methods

1. Skin membranes:

Pigskin membranes treated and selected as specified below, were used:

Intestinal epithelium of fresh swine from animals selected from about 130-160 days of live and 100 kg of weight.

2. Characteristics of the samples tested: Three types of samples were tested:

The tests were carried out using a physiological solution as carrier (NaCl 0.9% w/w), thus diluting the three samples of phospholipids (to show that there were no interferences in the method), free fullerene and fullerene complexed in phospholipids. The last two samples were thus obtained wherein the only difference consisted in the presence of fullerene in and not in liposomes, but with the same concentration of Fullerene. The samples were tested at a final concentration of Fullerene equal to 0.1, mg/ml.

3. Instrumentation

Four Franz-type thermostat-regulated cells were used, specifically constructed in glass with a surface of 20 mm, equal to an exchange surface of 12.56 cm ² , containing a volume of liquid receiver of 31 ml.

The thermostat-regulation system was obtained by means of the continuous circulation of fluid through a thermostat/cryostat system at 37° ± 1°C.

4. Analytical determination

The analytical determination of the permeated active principles was effected by removing an aliquot of liquid receiver at pre-established time intervals and determining the quantity in mg/ml of Fullerene by HPLC.

5. Parameters tested

Absorption, i.e. quantity absorbed/permeated expressed as μg/ml of Fullerene passed into the liquid receiver. The determination was effected directly using the value of Fullerene permeated by the membrane to the liquid receiver.

6. Procedure and results

10 g of each solution of phospholipids, free Fullerene and Fullerene in a liposomal suspension at 0.1% (w/w) of Fullerene, accurately dosed, were distributed on the surface of the membranes, positioned in the Franz-type cell permeation system. The whole mixture was expanded on all the surface of the skin membrane, covering it, and forming an upper layer of a few millimeters, extremely compact and homogeneous (a simple operation as it is a liquid).

** A cell was prepared for each sample, for a total of 3 cells. ** The three cells, containing the liquid receiver, were placed in their support and adequately thermostat-regulated, thus initiating the comparative test. ** Samples of liquid receiver were removed at intervals of 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and analyzed according to- the method described above.

** The results arc schematically indicated in figure 1.

The above figure 1 shows the difference of absorption in vitro of the acth'e principles forming the liposomal solution of Fullerene complexed in phospholipids and the same in free form, over a period of 8 hours. The differences in absorption are evident, all in favour of the solution of liposomes charged with Fullerene. An increase in the bioavailability of Fullerene thus complexed is evident, whereas that in free form is not at all bioavailable.

It should be pointed out, moreover, that the liposomes loaded with Fullerene proved to be hydrodispersible, contrary to fullerene. This characteristic is due to the complexing with phospholipids and, in addition to a greater specific absorption, also leads to a greater release with time and consequently a greater bioavailability with time. COMMENTS ON SKIN ABSORPTION TESTS

The diffusion cells have a volume of 31 ml. The passage of the active principles from the solutions to the liquid receiver stops when a concentration equilibrium is obtained between the residual active principles in the solutions containing Fullerene and the liquid receiver situated beyond the skin membrane; i.e. when the 10 g of solution plus the 31 g of liquid receiver have an equal concentration.

If there is a concentration of 0.1 g/Kg of Fullerene in the solution, this means that in 10 g of solution used, there will be a total quantity of Fullerene equal to 1/1000 x 10, i.e. 0.01 g, which correspond to 10 mg. These 10 mg will permeate the liquid receiver until they find a concentration equilibrium with the liquid donor, said equilibrium will therefore be reached at the following concentration: 10/(10+31) = 10/41 = 0.244 mg/rril. In other words, the maximum theoretical value which will be obtained in the permeate will not exceed a concentration of 244 μg/ml of Fullerene. In the test with Fullerene complexed in phospholipids, the maximum concentration of permeate is obtained, equal to 103 μg/ml, after 8 hours. _. This corresponds to 42.21% of the theoretical maximum obtainable.

In the free form, on the contrary, no type of absorption is observed after 8 hours. From an analysis if the results indicated in Figure 1 , the following conclusions can be made:

1) there is a substantial permeation difference between Fullerene complexed or non-complexed with phospholipids. This leads us to deduce that there is a correlation between phospholipids and free Fullerene and this correlation can be attributed to a complexing process which modifies its release and bioavailability with time. It should be noted that the complexing of Fullerene with phospholipids forms a more hydrodis- persible complex (liposomes). This diversity considerably influences the release of ^■ Fullerene and consequently its absorption, increasing the bioavailability and at the same time also increasing the release time.

2) From figure 1 a greater absorption of the complexed Fullerene is evident, if compared with that in free form. With phospholipids, Fullerene proves to be more greatly absorbed in the system and above all has a more prolonged release with time. This greater-efficiency of the system is undoubtedly due to the greater hydrodispersi- bility obtained with the complexing which increases the bioavailability. DETERMINATION BY MEANS OF SPECTROPHOTROMETRIC ANALYSIS OF THE ANTIOXIDIZING CAPACITY OF LIPOSOMES LOADED WITH FULLERENE.

Free radials are "waste" products which are naturally formed during cellular metabolic processes using oxygen for producing energy (oxidation).

The destructive action of free radicals is directed in particular towards the fats which form cellular membranes (liperoxidation) and is one of the causes of cellular aging. When lipids are attacked by radicals (for example, superoxide anion, hydroxyl, nitric oxide ...) lipidic radicals (L) are formed, which can react with oxygen forming per- oxylic radicals, which attract a hydrogen atom from the adjacent lipidic molecules thus generating new lipidic radicals (see reactions 1. to 3,). The last step of this chain of re-

actions (4.) requires the encounter of two radicals and takes place with a very low probability with respect to the previous passages.

1. LH + OH- -* L + H ₂ O

2. L + O ₂ → LOO-

3. LOO' + LH → L ^• + LOOH

4. L- + L- → L-L

This series of reactions therefore produces a large quantity of lipidic peroxides, whose polar part (peroxide group) can move through the aqueous phase breaking the integrity of the membrane.

The organism naturally defends itself from free radicals by producing endogenic antioxidants such as SOD, catalasi and glutathione. These molecules neutralize the action of the radicals, providing them with the electrons that are missing. The antioxidants, however, can also come from the outside, from natural sources, such as, for example, food.

The main antioxidants normally used in the dermocosmetic field are α-tocoferol and vitamin C. α-tocoferol is the. main natural liposoluble antioxidant. Its biological functions consist in contrasting, in synergy with glutathione, the peroxidation of fatty acids on a cellular level. It is in fact capable of terminating the chain of reactions which produce peroxides donating a hydrogen atom to the lipidic peroxides. In this way, tocoferol and tocoferoxyl radicals are produced, which are more stable and are neutralized by the action of Vitamin C, with the subsequent regeneration of Vitamin E. Vitamin C is a natural hydrosoluble antioxidant and intervenes during the peroxidation of cellular fats contrasting the formation of free radicals, comprising tocoferoxyls, as mentioned above.

Materials, methods and procedures 1. Materials ** Fullerene C ₆₀ (99.9+%) (MER Corp.)

In this study the antioxidant activity of fullerene complexed in liposomes, was tested. The sample of fullerene in liposomic complexes was obtained according to what is described above. An aqueous solution of 0.1% is prepared of liposomes . loaded with fullerene (corresponding to a degree of pure fullerene of 0.002 mg/ml. ** Vitamin C (Roche)

An aqueous solution of Vitamin C 0.002 mg/ml is prepared. ** Vitamin E (Basf) An aqueous solution of Vitamin E 0.002 mg/ml is prepared.

2. Analytical determination and instrumentation

• KIT for the determination of lipidic hydroperoxides (SIGMA).

This kit allows the lipidic peroxides in organic solvents within a range of 1-16 nano- moles per reaction volume, to be determined. It contains the following reagents:

- Organic Peroxide Color Reagent, phial containing 480 μmoles of BHT (butyl- hydroxytoluene) and 15 μmoles of xylenol orange. The solution is reformed using methanol at 90%.

- Ferrous Ammonium Sulfate Reagent, 25 mM of iron ammonium sulfate in 2.5 M of sulfuric acid.

- Tert-butyl Hydroperoxide, aqueous solution at 70%

• Spectrophotometer within the UV-visible range

3. Methods

The method used in this analysis allows a quantitative determination to be made of the lipidic hydroperoxides contained in organic solvents and is based on the fact that the peroxides convert the Fe ²⁺ ion into Fe ³⁺ at pH acid values. The Fe ³⁺ ions subsequently form a coloured compound with xylenol orange which can be observed with the spectrophotometer reading at 560 nm. The reaction which takes place is as follows: Fe ²⁺ + R-OOH -> Fe ³⁺ + RO- + OH Fe ³⁺ + XO → Fe ³⁺ -XO (coloured compound)

Wherein: XO = xylcnol orange R = H or lipidic group.

4. Procedures

The procedure envisages preparing a standard calibration curve using increasing concentrations of tert-Buty] hydroperoxide incubated with a reagent which consists of xy- lenol orange 125 μM and butyl-hydroxytoluene (BHT) 4 mM dissolved in methanol at 90%. BHT has an antioxidant effect and prevents an excessive peroxidation which would give false results. Iron ammonium sulfate 25 mM dissolved in a 2.5 M solution of sulfuric acid is added to this solution of reagents. The samples tested (described in the materials) were all incubated with the same concentration of t-BuOOH (160 μg/μl) which corresponds to one of the points of the calibration curve, and with the addition of the same quantity of xylenol orange reagent used for the standards. After half an hour of incubation at 25 ⁰ C, the standards and samples are read at 560 nm at the spectrophotometer subtracting the blank (methanol at 90%).

On the basis of the calibration line obtained by reading the various standards of t- BuOOH, the Gary WinUV program automatically determines the concentration in μg/μL of lipidic hydroperoxides contained in the various samples read at the spectrophotometer. ,

5. Results and conclusions

The results of the analyses on the three samples described above are indicated in Table 1 and Figure 2.

TABLE l

Various considerations can be made from observing these results. The absorbance read at the spectrophotometer for each sample varies in relation to the concentration of lipidic peroxides contained in the sample being tested: the greater the quantity of peroxides present in the solution analyzed, the greater will be the absorbance value read. By incubating the same quantity of t-Butyl-hydroperoxide with the various principles studied, it is possible to verify their possible antioxidant activity, evaluating their capacity to reduce the concentration of peroxides present in the solution.

From the results, it can be seen that, with the same concentration, liposomes loaded with fullerene have a much higher antioxidant effect, i.e. inhibition of the release of free radicals, with respect to Vitamin E and Vitamin C.

This antioxidant capacity of fullerene can be exploited in biological systems, as fullerene complexed in liposomes can permeate the cellular plasmatic membrane.