A liposomal composition comprising GHK-cu, a method for preparing thereof, and its use as a component of a cosmetic formulation

Abstract

The object of the present invention is a liposomal composition comprising the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex, wherein said complex is encapsulated in lipid carrier A comprising 19 to 39 wt % of the sodium salt of 1,2-distearoyl-sn-glycero-3- phosphoglycerol (DSPG-Na), 29 to 42 wt % of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 24 to 41 wt % of cholesterol, or in lipid carrier B comprising 16 to 21 wt % of N- [carbonyl-methoxy(polyethylene glycol)-2000]-1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000), 48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and 16 to 36 wt % of cholesterol. A further object of the invention is a method for preparing the liposomal composition of the invention and its use as a component of a cosmetic formulation.

Description

[0001] A liposomal composition comprising GHK-Cu, a method for preparing thereof, and its use as a component of a cosmetic formulation

[0002] The object of the present invention is a liposomal composition comprising the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex, a method for preparing said composition, and its use as a component of a cosmetic formulation.

[0003] Cosmetically active compounds, both lipophilic and hydrophilic, have difficulty reaching the deeper layers of the skin, which significantly reduces their effectiveness. One of cosmetically active compounds that naturally occurs in the human body and exhibits many beneficial properties is the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex. This complex, among others, reduces wrinkles and discoloration, takes part in wound healing processes, protects skin cells from ultraviolet radiation, as well as improves skin elasticity and firmness [1]. GHK-Cu is a hydrophilic compound, which makes its penetration through the lipophilic stratum corneum of the epidermis significantly limited. Suitable carriers can improve the penetration of this compound through the skin barriers. Such carriers are liposomes, vesicles made of a phospholipid bilayer. The encapsulation of a cosmetically active compound inside liposomes, which resemble cell membranes in their structure, may contribute to its more effective delivery to the deeper layers of the skin, thus increasing its effectiveness [2].

[0004] Various lipids are available, both natural and synthetic, which are used to prepare liposomes. Importantly, liposomes can vary greatly depending on their lipid composition. The lipid composition affects various parameters, including size, shape, homogeneity, stability, permeability or hardness. All these parameters may successively affect the effectiveness of GHK- Cu encapsulation, as well as the subsequent release of the cosmetically active compound from the inside of the carriers, which is crucial for its potential use in cosmetic products. In addition, the way in which the carriers described herein are prepared significantly affects their physicochemical parameters, and the effectiveness with which appropriate compounds are encapsulated inside them. Only the appropriate selection of a lipid composition and a method of carrier preparation provides suitable nanocapsules which effectively encapsulate GHK-Cu.

[0005] So far, it has not been possible to select such a lipid composition and such a method of carrier preparation to enclose the copper tripeptide in liposomes at an average rate greater than 33%. M. Dymek et al. confirmed that it was possible to encapsulate the GHK-Cu tripeptide inside liposomes consisting of lecithin, cholesterol and dicetyl phosphate, and lecithin, cholesterol and stearylamine, usingthe thin-film hydration method, and the encapsulation efficiency determined by them ranged from 0 to 33% depending on the lipid composition, the concentration of GHK-C used and the total lipid content. In this case, chloroform was applied to form liposomes, and the preparation procedure included a step of extrusion through a polycarbonate membrane for homogenization and size reduction [4]. In their studies, X. Wang et al. prepared GHK-Cu containing liposomes consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2- dioleyl-sn-glycero-3-phospho-(1 '-rac-glycerol) (DOPG) and cholesterol, using a similar procedure, but they did not check encapsulation efficiency [5]. With the thin-film hydration method, S. Erdem et al. obtained systems for which the encapsulation rate was 6, 33 and 27% for Epicuron 200SH / cholesterol, Epicuron 100H / cholesterol and 1 ,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC) / cholesterol compositions, respectively [3].

[0006] Liposomal carrier-GHK-Cu systems known so far have been obtained by the thin-layer hydration method. This method required the use of hazardous and toxic solvents, such as chloroform, methanol or diethyl ether. Importantly, the thin-film hydration method allows to obtain only multilamellar vesicles (MLVs) with a diameter of 400 to 1000 nm and is characterised by the polydispersity of the resulting carriers [6,7]. In order to obtain nanocapsules with smaller diameters which show increased accumulation of a transported compound in the stratum corneum of the epidermis, epidermis and dermis [8], an additional homogenization and size reduction step is required. Considering a potential subsequent use of formed liposomal carriers with GHK-Cu in cosmetic products for skin applications, the thin-film hydration method has numerous limitations.

[0007] In turn, studies have shown that attempts to obtain liposomal carrier-GHK-Cu systems by the ethanol injection method are not successful for all types of lipids. For example, liposomes obtained from 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol by the ethanol injection method were characterised by the lack of stability. During studies, no encapsulation of the GHK-Cu complex was observed within liposomes composed of, among others, the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na) / 1 ,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC).

[0008] For the composition in which the sodium salt of 1 ,2-distearoyl-sn-glycero-3- phosphoglycerol (DSPG-Na) (a synthetic lipid, negatively charged, consisting of two saturated fatty acid chains with 18 carbon atoms) was replaced with the chloride salt of 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP) (a synthetic cationic lipid, with the same chain length as DSPG-Na, but unsaturated), no formation of nanocapsules was observed. In contrast, for a composition in which, instead of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC) (a synthetic neutral lipid, consisting of saturated fatty acids with 16 carbon atoms), 1 ,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) (a synthetic neutral lipid, unlike DPPC being an unsaturated lipid with a longer carbon chain) was introduced, the formation of carriers was observed, but encapsulation efficiency was only about 3%. The results of these experiments confirm that there is a need to develop effective methods for preparing liposomal compositions containing GHK-Cu.

[0009] The object of the present invention is a liposomal composition comprising the tri peptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex, wherein said complex is encapsulated in lipid carrier A comprising:

[0010] 19 to 39 wt % of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na),

[0011] 29 to 42 wt % of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and

[0012] 24 to 41 wt % of cholesterol, or in lipid carrier B comprising:

[0013] 16 to 21 wt % of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000),

[0014] 48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and

[0015] 16 to 36 wt % of cholesterol.

[0016] Preferably, the composition is characterised in that lipid carrier A consists of:

[0017] 19 to 39 wt % of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na),

[0018] 29 to 42 wt % of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and

[0019] 24 to 41 wt % of cholesterol.

[0020] Preferably, the composition is characterised in that lipid carrier B consists of:

[0021] 16 to 21 wt % of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000),

[0022] 48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and

[0023] 16 to 36 wt % of cholesterol.

[0024] Preferably, the composition is characterised in thatthe concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier A, determined by capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP- MS / MS) is at least 28 ± 7 wt %.

[0025] Preferably, the composition is characterised in thatthe concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier A is from 28 ± 7 wt % to 37 ± 2 wt %.

[0026] Preferably, the composition is characterised in thatthe concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier A is 37 ± 2 wt %.

[0027] Preferably, the composition is characterised in thatthe concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier B, determined by capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP- MS / MS) is at least 23 ± 5 wt %. Preferably, the composition is characterised in thatthe concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier B is from 23 ± 5 wt % to 42 ± 8 wt %.

[0028] Preferably, the composition is characterised in thatthe concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier B is 42 ± 8 wt %.

[0029] A further object of the invention is a method for preparing a liposomal composition comprising the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex comprising the following steps:

[0030] (a) a mixture of lipids constituting lipid carrier A, comprising:

[0031] 19 to 39 wt % of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 29 to 42 wt % of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and

[0032] 24 to 41 wt % of cholesterol, or a mixture of lipids constituting lipid carrier B, comprising:

[0033] 16 to 21 wt % of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE-PEG2000),

[0034] 48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and

[0035] 16 to 36 wt % of cholesterol, is dissolved in ethanol and the resulting solution is kept at a temperature of at least 50°C to obtain a lipid phase and then

[0036] (b) the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex is dissolved in water or in a sucrose solution to obtain an aqueous phase,

[0037] (c) the aqueous phase is heated to a temperature higher than the lipid phase temperature,

[0038] (d) during intensive stirring, the lipid phase is injected into the aqueous phase, and the resulting solution is kept at a temperature higher than the lipid phase temperature in step (a), after which the resulting solution is gradually cooled down to ambient temperature.

[0039] Preferably, the method is characterised in that the temperature of dissolving lipid carrier A or B in ethanol does not exceed the ethanol boiling point.

[0040] Preferably, the method is characterised in that the concentration of the tripeptide glycyl- L-histidyl-L-lysine-copper (GHK-Cu) complex in the aqueous phase in step (b) is from 0.005 wt % to 0.1 wt %. Preferably, the method is characterised in that the volume ratio of the lipid phase from step (a) to the aqueous phase from step (b) is from 1 :6 to 1 :2.

[0041] Preferably, the method is characterised in that the volume ratio of the lipid phase from step (a) to the aqueous phase from step (b) is 1 :2.

[0042] Preferably, the method is characterised in that the aqueous phase in step (c) is heated to a temperature of at least 55°C, preferably 70°C.

[0043] Preferably, the method is characterised in that the temperature of the aqueous phase in step (c) does not exceed the ethanol boiling point.

[0044] Preferably, the method is characterised in that the injection in step (d) is made when the aqueous phase is shaken.

[0045] Preferably, the method is characterised in that in step (d) the resulting solution is kept at the injection temperature for at least 30 minutes, and then at a temperature of 60°C for at least 90 minutes.

[0046] A further object of the invention is a cosmetic formulation containing a liposomal composition according to the invention.

[0047] Preferably, the cosmetic formulation is characterised in that it comprises not more than 10.0 wt % of the liposomal composition, preferably not more than 2.0 wt %.

[0048] Preferably, the cosmetic formulation is characterised in that it is a formulation for external use.

[0049] Preferably, the cosmetic formulation is characterised in that the formulation for external use is selected from the group comprising: gel, cream, ointment, serum, O / W (oil / water) emulsions.

[0050] A further object of the present invention is the use of the liposomal composition of the present invention as a component of the cosmetic formulation.

[0051] Increasing the effectiveness of the GHK-Cu complex enclosed in the liposomal composition according to the invention allows the concentration of this complex to be reduced while maintaining the same level of effectiveness, thus affecting the efficiency of production and the safety of cosmetic products. The more substance that can be encapsulated, the more effectively raw materials can be used, which can lead to reduced production costs for a potential cosmetic.

[0052] The liposomal composition and the method for preparing thereof, being the object of the invention, allow for the preparation of liposomes that encapsulate GHK-Cu at a greater or similar rate to that achieved by the previously known compositions proposed in the literature, and using a much simpler method. The method according to the invention allows for the elimination of toxic solvents. Instead, ethanol is used, which, in addition to being non-toxic, can additionally be included in cosmetic products in a specific amount to act, among others, as a preservative or permeation promoter. The use according to the present invention is therefore more environ mentally friendly. The liposomal composition of the invention is stable at a temperature of 4°C for at least two weeks. Also, these liposomes do not disintegrate during heat treatment, which may significantly affect their long-term storage. In addition, their size is suitable for use in cosmetic products (<200 nm), since, according to the current state of knowledge, it should ensure an increased accumulation of a transported compound in the stratum corneum of the epidermis, as well as in epidermis and dermis [8]. Obtaining nanocapsules of the expected small size directly, as a result of forming, eliminates the need for additional processing steps, such as homogenisation and size reduction. Thus, it is possible to reduce the costs of carrying out the process. The use according to the present invention may improve the efficiency of production and increase the safety of cosmetics. Furthermore, the liposomes are characterised by high monodispersity and stability in suspension, as evidenced, in turn, by low polydispersity index values (<0.3) and high absolute zeta potential values (>30 mV).

[0053] The object of the invention is clarified in the following examples, which do not limit its scope.

[0054] Example 1 - Composition comprising GHK-Cu, DSPG-Na, DPPC, cholesterol

[0055] The following was weighed and dissolved in 1.0 mL of ethanol:

[0056] 4.8 mg of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 4.4 mg of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 3.1 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been reached, ethanol dissolved lipids were injected into the prepared aqueous phase using a glass syringe. The resulting solution was incubated at a temperature of 70°C for 30 minutes, then for 90 minutes at a temperature of 60°C. Subsequently, the resulting liposomal suspension was kept for 30 minutes at room temperature. Liposome-GHK-Cu systems with a mean hydrodynamic diameter of 152 ± 4 nm, a mean polydispersity index of 0.29 ± 0.01 and a mean Zeta potential of -35.4 ± 0.4 mV were obtained, which was verified with the use of 10 independent product batches. The values were determined using the dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 . Table 1 . DLS Analysis Conditions.

[0057] The mean efficiency of GHK-Cu encapsulation in the liposomes with the proposed lipid composition (n = 10), obtained as in the presented example, is 37 ± 2%. This parameter was determined using capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) directly from the entire reaction mixture. The mean encapsulation efficiency was determined using the equation below and then converted to the concentration of the encapsulated complex, assuming that a GHK-Cu concentration of 0.1 mg mL-1was 100%. The amount of the active ingredient can also be represented as the amount of encapsulated GHK-C in the entire volume of the post-reaction suspension. cGHK — Cu = %GHK - Cu ■ 0.1 xGHK — Cu = cGHK - Cu - V

[0058] Where:

[0059] • %GHK — Cu - mean percentage of GHK-Cu encapsulation in liposomes [%]

[0060] • A of63Cu+signal in liposomes - sum of areas of signals coming from63Cu+ions in liposomes

[0061] • A of63Cu+signals - sum of areas of all63Cu+signals in an electropherogram

[0062] • n- number of measurements

[0063] • cGHK — C - mean concentration of encapsulated GHK-Cu [mg / mL] • xGHK — Cu-the amountof encapsulated GHK-C in the entire volume of the post-reaction suspension

[0064] • V - total volume of the suspension Table 2 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) amount of encapsulated complex in the entire volume of the suspension.

[0065] Table 2. Average encapsulation efficiency (effectiveness) values.

[0066] The exact parameters of CE-ICP-MS / MS are compiled in Table 3.

[0067] Table 3. CE-ICP-MS / MS parameters. The stability of the process was monitored using the same technique and the compositions were shown to be stable and the encapsulation rate preserved (± 1%) after 2 weeks of storage of the finished systems at 4 C. Cycles of freezing for 16 hours at -20 °C and of thawing for 30 minutes at 50°C cause a slight increase in the encapsulation rate, without significantly affecting the parameters of the liposomes - the liposomes remain stable during heat treatment.

[0068] Example 2- Composition comprising GHK-Cu, DSPG-Na, DPPC, cholesterol

[0069] The following was weighed and dissolved in 1.0 mL of ethanol:

[0070] 4.8 mg of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 4.4 mg of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 6.2 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been rached, ethanol dissolved lipids were injected into the prepared aqueous phase using a glass syringe. The resulting solution was incubated at70°C for 30 minutes, and then for 90 minutes at 60°C. Subsequently, the resulting liposomal suspension was left for 30 minutes at room temperature. Liposome-GHK-Cu systems with a mean hydrodynamic diameter of 106.2 ± 0.5 nm, a mean polydispersity index of 0.11 ± 0.01 and a mean Zeta potential of -51 .1 ± 0.6 mV were obtained, which was verified with the use of 10 independent product tranches. The values were determined usingthe dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 .

[0071] The mean efficiency of GHK-Cu encapsulation in the liposomes with the proposed lipid composition (n = 10), obtained as in the presented example, is 34 ± 2%. This parameter was determined using capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) directly from the entire reaction mixture (CE-ICP-MS / MS parameters are compiled in Table 3).

[0072] Table 4 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) amount of encapsulated complex in the entire volume of the suspension (determined in the same manner as in Example 1).

[0073] Table 4. Average encapsulation efficiency (effectiveness) values. The stability of the process was monitored using the same technique and the compositions was shown to be stable and the encapsulation rate maintained (± 1 %) after 2 weeks of storage of the finished systems at 4°C.

[0074] Example 3- composition comprising GHK-Cu, DSPG-Na, DPPC, cholesterol

[0075] The following was weighed and dissolved in 1.0 mL of ethanol:

[0076] 4.8 mg of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 4.4 mg of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 4.5 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been reached, ethanol dissolved lipids were injected into the prepared aqueous phase using a glass syringe. The resulting solution was incubated at70°C for 30 minutes, and then for 90 minutes at 60°C. Subsequently, the resulting liposomal suspension was left for 30 minutes at room temperature. Liposome-GHK-Cu systems with a mean hydrodynamic diameter of 140 ± 2 nm, a mean polydispersity index of 0.04 ± 0.01 and a mean Zeta potential of -61 .8 ± 0.6 mV were obtained, which was verified with the use of 10 independent product tranches. The values were determined usingthe dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 .

[0077] The mean efficiency of GHK-Cu encapsulation in the liposomes with the proposed lipid composition (n = 10), obtained as in the presented example, is 33 ± 2%. This parameter was determined using capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) directly from the entire reaction mixture (CE-ICP-MS / MS parameters are compiled in Table 3).

[0078] Table 5 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) amount of encapsulated complex in the entire volume of the suspension (determined in the same manner as in Example 1).

[0079] Table 5. Average encapsulation efficiency (effectiveness) values. The stability of the process was monitored using the same technique and the compositions was shown to be stable and the encapsulation rate maintained (± 1 %) after 2 weeks of storage of the finished systems at 4°C.

[0080] Example 4- composition comprising GHK-Cu, DSPG-Na, DPPC, cholesterol

[0081] The following was weighed and dissolved in 1.0 mL of ethanol:

[0082] 2.1 mg of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 4.4 mg of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 4.4 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been reached, ethanol dissolved lipids were injected into the prepared aqueous phase using a glass syringe. The resulting solution was incubated at70°C for 30 minutes, and then for 90 minutes at 60°C. Subsequently, the resulting liposomal suspension was left for 30 minutes at room temperature. Liposome-GHK-Cu systems with a mean hydrodynamic diameter of 123 ± 2 nm, a mean polydispersity index of 0.09 ± 0.01 and a mean Zeta potential of -52.5 ± 1.4 mV were obtained, which was verified with the use of 10 independent product tranches. The values were determined usingthe dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 .

[0083] The mean efficiency of GHK-Cu encapsulation in the liposomes with the proposed lipid composition (n = 10), obtained as in the presented example, is 28 ± 7%. This parameter was determined using capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) directly from the entire reaction mixture (CE-ICP-MS / MS parameters are compiled in Table 3).

[0084] Table 6 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) amount of encapsulated complex in the entire volume of the suspension (determined in the same manner as in Example 1 ).

[0085] Table 6. Average encapsulation efficiency (effectiveness) values. The stability of the process was monitored using the same technique and the compositions was shown to be stable and the encapsulation rate maintained (± 1 %) after 2 weeks of storage of the finished systems at 4°C.

[0086] Example 5- Composition comprising GHK-Cu, DSPG-Na, DPPC, cholesterol

[0087] The following was weighed and dissolved in 1.0 mL of ethanol:

[0088] 2.6 mg of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 4.7 mg of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 4.0 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been reached, ethanol dissolved lipids were injected into the prepared aqueous phase using a glass syringe. The resulting solution was incubated at70°C for 30 minutes, and then for 90 minutes at 60°C. Subsequently, the resulting liposomal suspension was left for 30 minutes at room temperature. Liposome-GHK-Cu systems with a mean hydrodynamic diameter of 133 ± 1 nm, a mean polydispersity index of 0.08 ± 0.02 and a mean Zeta potential of -53.7 ± 0.8 mV were obtained, which was verified with the use of 10 independent product tranches. The values were determined usingthe dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 .

[0089] The mean efficiency of GHK-Cu encapsulation in the liposomes with the proposed lipid composition (n = 10), obtained as in the presented example, is 35 ± 5%. This parameter was determined using capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) directly from the entire reaction mixture (CE-ICP-MS / MS parameters are compiled in Table 3).

[0090] Table 7 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) amount of encapsulated complex in the entire volume of the suspension (determined in the same manner as in Example 1 ).

[0091] Table 7. Average encapsulation efficiency (effectiveness) values. The stability of the process was monitored using the same technique and the compositions was shown to be stable and the encapsulation rate maintained (± 1 %) after 2 weeks of storage of the finished systems at 4°C.

[0092] Example 6- Composition comprising GHK-Cu, DSPG-Na, DPPC, cholesterol

[0093] The following was weighed and dissolved in 1.0 mL of ethanol:

[0094] 4.3 mg of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 5.0 mg of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 3.0 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been reached, ethanol dissolved lipids were injected into the prepared aqueous phase using a glass syringe. The resulting solution was incubated at70°C for 30 minutes, and then for 90 minutes at 60°C. Subsequently, the resulting liposomal suspension was left for 30 minutes at room temperature. Liposome-GHK-Cu systems with a mean hydrodynamic diameter of 137 ± 3 nm, a mean polydispersity index of 0.12 ± 0.02 and a mean Zeta potential of -61 .2 ± 1 .1 mV were obtained, which was verified with the use of 10 independent product tranches. The values were determined usingthe dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 .

[0095] The mean efficiency of GHK-Cu encapsulation in the liposomes with the proposed lipid composition (n = 10), obtained as in the presented example, is 31 ± 4%. This parameter was determined using capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) directly from the entire reaction mixture (CE-ICP-MS / MS parameters are compiled in Table 3).

[0096] Table 8 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) amount of encapsulated complex in the entire volume of the suspension (determined in the same manner as in Example 1 ).

[0097] Table 8. Average encapsulation efficiency (effectiveness) values. The stability of the process was monitored using the same technique and the compositions was shown to be stable and the encapsulation rate maintained (± 1 %) after 2 weeks of storage of the finished systems at 4°C.

[0098] Example 7 - composition comprising GHK-Cu, DSPE-PEG2000, HSPC, cholesterol

[0099] The following was weighed and dissolved in 1.0 mL of ethanol:

[0100] 1.5 mg of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000), 4.4 mg of hydrogenated soybean phosphatidylcholine (HSPC) and 1.1 mg of cholesterol. The mixture was sonicated for 4 minutes at a temperature of 50°C and maintained at a temperature of 50°C until the injection was made. 2 mL of an aqueous solution containing 0.2 mg of GHK-Cu (aqueous phase) was placed in an incubator set to 70°C with a shaker function set at 1000 rpm. After the set temperature had been reached, ethanol dissolved lipids were injected into the prepared aqueous phase usinga glass syringe. The resulting solution was incubated at 70°C for 30 minutes, and then for 90 minutes at 60°C. Subsequently, the resulting liposomal suspension was left for 30 minutes at room temperature. Liposome-GHK- Cu systems with a mean hydrodynamic diameter of 180 ± 2 nm, a mean polydispersity index of 0.13 ± 0.01 and a mean Zeta potential of -50.0 ± 1.3 mV were obtained, which was verified with the use of 10 independent product tranches. The values were determined using the dynamic light scattering (DLS) technique with a Zetasizer Nano ZS, Malvern Panalytical, UK. Specific conditions of the analysis are compiled in Table 1 .

[0101] DLS measurements for the compositions of Examples 7-8 were carried out in the same way as for Examples 1-6 using the method set out in Example 1.

[0102] CE-ICP-MS / MS measurements for the composition of Examples 7-8 were carried out in the same way as in Examples 1-6 using the method set out in Example 1 .

[0103] Table 9 compiles the determined values of (1 ) mean encapsulation efficiency (effectiveness), (2) mean encapsulated complex concentration and (3) the amount of encapsulated complex in the entire volume of the suspension.

[0104] Table 9. Average encapsulation efficiency (effectiveness) values. The stability of the process was monitored using the same technique and the compositions were shown to be stable and the encapsulation rate maintained (± 1 %) after 2 weeks of storage of the finished systems at 4 C. Cycles of freezing for 16 hours at -20 °C and of thawing for 30 minutes at 50°C cause a slight increase in the encapsulation rate, without significantly affecting the parameters of the liposomes - the liposomes remain stable during heat treatment.

[0105] Example 8- Composition comprising GHK-Cu, DSPE-PEG2000, HSPC, cholesterol

[0106] The lipid preparation process was performed in the same way as in Example 7, except that the lipid carrier was prepared by weighing and dissolving the following in 1 .0 of mL ethanol: 1 .5 mg of

[0107] N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE-PEG2000), 4.4 mg of hydrogenated soybean phosphatidylcholine (HSPC) and 3.3 mg of cholesterol.

[0108] The obtained liposome composition was characterized by a GHK-Cu encapsulation rate of about 23%.

[0109] References:

[0110]

[0001] Pickart L., Margolina A. Regenerative and Protective Actions of the GHK-Cu Peptide in the Light of the New Gene Data, International Journal of Molecular Sciences 2018; 19(7):1987

[0111] [2] Ahmadi Ashtiani H.R., Bishe P., Lashgari N.-A., Nilforoushzadeh, M. A., Zare S. Liposomes in Cosmetics, Liposomes in Cosmetics 2016; 3(3):e65815

[0112] [3] Erdem S., Turkoulu M. Glycyl-L-Histidyl-L-Liysine-Cu(2+) loaded liposome formulations, Marmara Pharmaceutical Journal 2010,14: 91 -97

[0113] [4] Dymek M, Olechowska K, Hqc-Wydro K, Sikora E. Liposomes as Carriers of GHK-Cu Tripeptide for Cosmetic Application, Pharmaceutics. 2023, 18;15(10):2485

[0114] [5] Wang X., Liu B., Xu Q., Sun H., Shi M., Wang D., Guo M., Yu J., Zhao C., Feng B. GHK-Cu- liposomes accelerate scald wound healing in mice by promoting cell proliferation and angiogenesis, Wound Repair and Regeneration 2017, 25: 270-278 [6] Goik U., Zat^ska-Zytka I., Pietrzycka A. Liposomes as carriers for the delivery of active substances to the skin, Engineering of Biomaterials 2015, 130:27-39

[0115] [7] Shah S., Dhawan V., Holm R., Nagarsenker M.S., Perrie Y. Liposomes: Advancements and innovation in the manufacturing process, Advanced Drug Delivery Reviews 2020; 154-155, 102- 122

[0116] [8] Verma D.D., Verma S., Blume G., Fahr A. Particle size of liposomes influences dermal delivery of substances into skin, International Journal of Pharmaceuticals 2003; 258, 141-151

Claims

Claims1. A liposomal composition comprising the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex, wherein said complex is encapsulated in lipid carrier A comprising:19 to 39 wt % of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG- Na),29 to 42 wt % of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and24 to 41 wt % of cholesterol, or in lipid carrier B comprising:16 to 21 wt % of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE-PEG2000),48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and16 to 36 wt % of cholesterol.

2. The composition of claim 1 , characterised in that lipid carrier A consists of:19 to 39 wt % of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG- Na),29 to 42 wt % of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and24 to 41 wt % of cholesterol.

3. The composition of claim 1 , characterised in that lipid carrier B consists of:16 to 21 wt % of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE-PEG2000),48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and16 to 36 wt % of cholesterol.

4. The composition of any one of claims 1-3, characterised in that the concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier A, determined by capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS) is at least 28 ± 7 wt %.

5. The composition of any one of claims 1-4, characterised in that the concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier A is from 28 ± 7 wt % to 37 ± 2 wt %.

6. The composition of any one of claims 1-5, characterised in that the concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier A is 37 ± 2 wt %.

7. The composition of any one of claims 1-6, characterised in that the concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier B, as determined by capillary electrophoresis combined with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS / MS), is at least 23 ± 5 wt %.

8. The composition of any one of claims 1-7, characterised in that the concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier B is from 23 ± 5 wt % to 42 ± 8 wt %.

9. The composition of any one of claims 1-8, characterised in that the concentration of the encapsulated GHK-Cu complex in the composition comprising lipid carrier B is 42 ± 8 wt %.

10. A method for preparing the liposomal composition comprising the tripeptide glycyl-L- histidyl-L-lysine-copper (GHK-Cu) complex comprising the following steps:(a) a mixture of lipids constituting lipid carrier A comprising:19 to 39 wt % of the sodium salt of 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG-Na), 29 to 42 wt % of 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and24 to 41 wt % of cholesterol, or a mixture of lipids constituting lipid carrier B comprising:16 to 21 wt % of N-[carbonyl-methoxy(polyethylene glycol)-2000]-1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine (DSPE-PEG2000),48 to 63 wt % of hydrogenated soybean phosphatidylcholine (HSPC) and16 to 36 wt % of cholesterol. is dissolved in ethanol and the resulting solution is kept at a temperature of at least 50°C to obtain a lipid phase and then(b) the tripeptide glycyl-L-histidyl-L-lysine-copper (GHK-Cu) complex is dissolved in water or in a sucrose solution to obtain an aqueous phase,(c) the aqueous phase is heated to a temperature higher than the lipid phase temperature,(d) during intensive stirring, the lipid phase is injected into the aqueous phase, and the resulting solution is kept at a temperature higher than the lipid phase temperature in step (a), after which the resulting solution is gradually cooled down to ambient temperature.

11. The method of claim 10, characterised in that the temperature of dissolving lipid carrier A or B in ethanol does not exceed the ethanol boiling point.

12. The method of claim 10, characterised in that the concentration of the tripeptide glycyl-L- histidyl-L-lysine-copper (GHK-Cu) complex in the aqueous phase in step (b) is from 0.005 wt % to 0.1 wt %.

13. The method of any one of claims 10-12, characterised in that the volume ratio of the lipid phase from step (a) to the aqueous phase from step (b) is from 1 :6 to 1 :2.

14. The method of any one of claims 10-13, characterised in that the volume ratio of the lipid phase from step (a) to the aqueous phase from step (b) is 1 :2.

15. The method of any one of claims 10-14, characterised in that the aqueous phase in step (c) is heated to a temperature of at least 55°C, preferably 70°C.

16. The method of any one of claims 10-15, characterised in that the temperature of the aqueous phase in step (c) does not exceed the ethanol boiling point.

17. The method of any one of claims 10-16, characterised in that the injection in step (d) is made when the aqueous phase is shaken.

18. The method of any one of claims 10-17, characterised in that in step (d) the obtained solution is kept at the injection temperature for at least 30 minutes, and then at a temperature of 60°C for at least 90 minutes.

19. A cosmetic formulation comprising the liposomal composition defined in any one of claims 1-9.

20. The cosmetic formulation of claim 19, characterised in that it comprises not more than 10.0 wt % of the liposomal composition, preferably not more than 2.0 wt %.

21. The cosmetic formulation of any one of claims 19-20, characterised in that it is a formulation for external use.

22. The cosmetic formulation of any one of claims 19-21 , characterised in that the formulation for external use is selected from the group comprising: gel, cream, ointment, serum, O / W (oil / water) emulsions.

23. The use of the liposomal composition of any one of claims 1 -9 as a component of the cosmetic formulation.

Images