CN106943336B - PRP/phospholipid liposome transdermal preparation and preparation method thereof - Google Patents
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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- A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/81—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions involving only carbon-to-carbon unsaturated bonds
- A61K8/8129—Compositions of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an alcohol, ether, aldehydo, ketonic, acetal or ketal radical; Compositions of hydrolysed polymers or esters of unsaturated alcohols with saturated carboxylic acids; Compositions of derivatives of such polymers, e.g. polyvinylmethylether
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Abstract
The invention discloses a preparation method of a PRP/phospholipid liposome transdermal preparation, which comprises the following steps: (1) dissolving polyvinyl alcohol in PBS, sequentially adding soybean lecithin, cholesterol and methyl p-hydroxybenzoate, stirring, and swelling overnight; then carrying out ultrasonic treatment to prepare phospholipid liposome; (2) carrying out gradient centrifugation on autologous whole blood to obtain concentrated PRP; (3) injecting PRP into the phospholipidosome, blowing and beating uniformly, and then incubating to obtain the product. In the PRP/phospholipid liposome transdermal preparation, the phospholipid liposome is used as a carrier of the PRP, and the active substance GFs which is continuously released by the PRP is combined with the liposome in a non-covalent bond, so that the penetration of the two to the skin is enhanced, and the active substance GFs is left between the epidermis and the dermis to avoid systemic adverse reaction. The liposome double-layer membrane wraps PRP and released GFs, is isolated from the external environment, and can greatly improve the stability of active substance GFs, so that the active substance can exert efficacy for a long time.
Description
Technical Field
The invention relates to the technical field of liposome preparations, in particular to a PRP/phospholipid liposome transdermal preparation and a preparation method thereof.
Background
In modern society, people are confronted with increasingly severe working and living pressure, environmental pollution and the like, which can not accelerate the aging of the skin of people, and therefore, the skin protection and the repair of the damaged skin are particularly needed.
The traditional skin care mostly adopts fine chemical raw materials, and the skin care products are only limited to physical (friction stripping), chemical (corrosion covering) and other effects, so that the fundamental problem of the skin cannot be effectively solved, and the aging problem of the skin cannot be solved. With the improvement of living standard and the rapid development of science and technology, cell science is favored by people in the field of dermatology, and functional cosmetics are widely concerned. Wherein, biological preparation and bioactive extract have become a main direction for developing functional cosmetics, such as coenzyme Q10 liposome product with anti-aging effect, VE liposome product with anti-aging effect, and V with whitening effectCLiposome products, and the like.
The recombinant human epidermal growth factor is a peptide consisting of 53 amino acid residues, has various biological activities, has the efficacies of resisting aging, repairing epidermis, smoothing wrinkles, fading colored patches, moistening and the like, is known as 'beauty factor', is taken as a cosmetic raw material by bioactive substances such as the recombinant growth factor sold in the market and the like, but has overhigh price, and is researched and found that the recombinant Growth Factor (GF) has side effects such as tumor generation and the like because the dosage is far higher than the normal physiological concentration.
Platelet Rich Plasma (PRP) is high concentration platelet plasma obtained by centrifuging autologous whole blood, can release various Growth Factors (GFs) that promote healing of body tissues, and can avoid immunogenic reactions, contamination, infection, and the like as an autologous source, and thus PRP is widely used in the fields of plastic surgery, sports medicine, skin treatment, and the like as a new technology. However, PRP does not penetrate through the surface layer of the skin into the interior, and its application alone in the treatment of skin wounds does not significantly promote wound healing, probably because GFs has a too short half-life or because it is degraded and inactivated by a large amount of hydrolytic enzymes in the wound area at an early stage. Therefore, an ideal PRP vector is needed to prolong GFs half-life and improve PRP therapeutic effect.
The liposome is a spheroid which is formed by one or more layers of concentric lipid bilayer membranes formed by dispersing amphiphilic substances mainly including phospholipid in water. Liposomes have a number of advantages as drug carriers, such as: the liposome can encapsulate both fat-soluble drugs and water-soluble drugs; reduction of allergic and immune reactions; delay the release and reduce the elimination speed in vivo; the coated medicine can be effectively protected, and the bioavailability is improved; change the distribution of the medicine in the body, and can carry out targeted medicine release and the like.
However, the challenge in preparing liposomes is to select the appropriate liposome composition and preparation. Since the properties of liposomes, such as stability, encapsulation efficiency, onset time, in vivo circulation time, bioavailability, toxic and side effects, are closely related to the composition of liposomes, which is closely related to the drug to be encapsulated, it is an urgent problem to select what ingredients to form a liposome preparation with good quality.
At present, PRP is mainly prepared into gel for use in clinical application, and the research of wrapping PRP with phospholipid liposome has not been reported.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a PRP/phospholipid liposome transdermal preparation and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, a method for preparing a PRP/phospholipid liposome transdermal formulation is provided, comprising the steps of:
(1) dissolving polyvinyl alcohol (PVA) in PBS, sequentially adding soybean lecithin, Cholesterol (CHO) and methyl p-hydroxybenzoate, stirring, and swelling overnight; then carrying out ultrasonic treatment to prepare phospholipid liposome;
(2) carrying out gradient centrifugation on autologous whole blood to obtain concentrated PRP;
(3) and (3) injecting the PRP prepared in the step (2) into the phospholipidosome prepared in the step (1), blowing and beating uniformly, and then incubating to obtain the PRP/phospholipid lipidosome transdermal preparation.
In step (1), the pH of the PBS is 5.0.
In the step (1), the adding amount ratio of the polyvinyl alcohol, the soybean lecithin, the cholesterol and the methyl p-hydroxybenzoate is (25-35): (80-100): (10-20): 1; preferably, the addition ratio of the polyvinyl alcohol, the soybean lecithin, the cholesterol and the methyl p-hydroxybenzoate is 30: 90: 15: 1.
in the step (1), the ultrasonic treatment time is 350s, and specifically comprises the following steps: turn off for 2s every 5s on, cycle 50 times.
In the step (2), the autologous whole blood gradient centrifugation specifically comprises: centrifuging at a primary speed of 200g for 10min, and collecting the supernatant; the rotating speed of the secondary centrifugation is 400g, and the centrifugation time is 10 min; the supernatant was discarded.
In step (3), PRP was injected into phospholipid liposomes at 10% (v/v).
In the step (3), the incubation time is 1 h.
In a second aspect of the present invention, there is provided a PRP/phospholipid liposome transdermal preparation prepared by the above preparation method.
In a third aspect of the invention, there is provided the use of a PRP/phospholipid liposome transdermal formulation as described above for the preparation of a skin care cosmetic.
The technical scheme has the following beneficial effects:
in the PRP/phospholipid liposome transdermal preparation prepared by the invention, the active substance GFs which takes the phospholipid liposome as the carrier of the PRP and is continuously released by the PRP is bonded with the liposome by non-covalent bonds, thereby enhancing the penetration effect of the two on the skin and keeping the active substance GFs between the epidermis and the dermis so as to avoid systemic adverse reaction. The liposome double-layer membrane wraps PRP and released GFs, is isolated from the external environment, and can greatly improve the stability of active substance GFs, so that the active substance can exert efficacy for a long time. In addition, the phospholipid liposome also has the function of moisturizing. Therefore, the PRP/phospholipid liposome transdermal preparation can be used in the fields of medical cosmetology, surgical plastic and the like.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1: the PRP/phospholipid liposome transdermal formulation prepared in example 1;
FIG. 2: the PRP/phospholipid liposome transdermal formulation prepared in comparative example 1.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
As described in the background, PRP cannot penetrate through the skin surface and enter the skin, and the active substance GFs released by PRP is inherently unstable, sensitive to light, heat and PH, and easily inactivated, so that an ideal PRP carrier is needed to prolong GFs half-life and improve the effect of PRP. Based on the above, the invention provides a PRP/phospholipid liposome transdermal preparation and a preparation method thereof.
In one embodiment of the present application, there is provided a method for preparing a PRP/phospholipid liposome transdermal formulation, comprising the steps of:
(1) dissolving polyvinyl alcohol (PVA) in PBS, sequentially adding soybean lecithin, Cholesterol (CHO) and methyl p-hydroxybenzoate, stirring, and swelling overnight; then carrying out ultrasonic treatment to prepare phospholipid liposome; the adding amount ratio of the polyvinyl alcohol, the soybean lecithin, the cholesterol and the methyl p-hydroxybenzoate is (25-35): (80-100): (10-20): 1;
(2) carrying out gradient centrifugation on autologous whole blood, namely carrying out primary centrifugation at a rotation speed of 200g for 10min, and taking a supernatant; the rotating speed of the secondary centrifugation is 400g, and the centrifugation time is 10 min; discarding the supernatant to obtain concentrated PRP;
(3) and (3) injecting the PRP prepared in the step (2) into the phospholipidosome prepared in the step (1), blowing and beating uniformly, and then incubating to obtain the PRP/phospholipid lipidosome transdermal preparation.
To form a good quality PRP/phospholipid liposome transdermal formulation, it is critical to find a film-forming material that is well compatible with PRP, so that it is well encapsulated and does not leak. According to the invention, a large number of researches and experiments are carried out, and PRP, polyvinyl alcohol, soybean lecithin, cholesterol and methyl p-hydroxybenzoate in specific weight proportions can be prepared into a PRP/phospholipid liposome transdermal preparation with excellent quality within a certain ultrasonic time, wherein the PRP serving as a medicinal active ingredient has high encapsulation rate and good dissolution, the skin permeation capability of the obtained transdermal preparation is obviously enhanced, and the bioavailability is obviously improved.
The stability and transdermal effect of liposomes are related to factors such as temperature, pH, ionic strength, etc. in addition to their composition. Polyvinyl alcohol can be used for improving the stability of the liposome on the one hand, and can form a space barrier between liposome particles to generate repulsive force so as to prevent aggregation and fusion between the liposomes; on the other hand, the polyvinyl alcohol can increase the viscosity of the liposome, and the addition amount of the polyvinyl alcohol needs to be regulated and controlled in the preparation process, so that the proper concentration of the polyvinyl alcohol is controlled. Cholesterol has great influence on the volume of the liposome, the structure of the lipid bilayer and the transdermal effect of the liposome. Depending on the interaction of the phospholipid liposomes with skin lipids and the fluidity of the phospholipid bilayer. The addition of a suitable amount of cholesterol is beneficial to enhancing the percutaneous absorption of PRP and GFs encapsulated by liposome. In the process of preparing the liposome, the addition amounts of the polyvinyl alcohol, the cholesterol, the soybean lecithin and the methylparaben are optimized and inspected, and the result shows that the addition amount ratio of the polyvinyl alcohol, the soybean lecithin, the cholesterol and the methylparaben is (25-35): (80-100): (10-20): 1, the prepared phospholipidosome has better encapsulation effect on PRP; and when the adding amount ratio of the polyvinyl alcohol, the soybean lecithin, the cholesterol and the methyl p-hydroxybenzoate is 30: 90: 15: 1, the prepared phospholiposome has optimal encapsulation effect on PRP.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1: preparation of PRP/phospholipid liposome transdermal preparation
(1) Preparation of phospholipid liposomes
0.3g of polyvinyl alcohol (PVA) was weighed out and dissolved in PBS (pH 5), and 0.9g of soybean lecithin (PG90), 0.15g of Cholesterol (CHO) and 10mg of methylparaben were added in this order, stirred overnight, and then sonicated with a sonicator. 4 '10' (2 s per 5s switch on, 50 cycles), 6 '15' (2 s per 5s switch on, 75 cycles), the phospholipid liposomes were prepared.
(2) Preparation of PRP:
gradient centrifugation to obtain rat PRP: centrifuging a certain amount of rat blood (added with sodium citrate glucose solution) at 200g for 10min, collecting upper layer plasma, centrifuging at 400g for 10min twice, and discarding supernatant to obtain concentrated PRP.
(3) Preparation of PRP/phospholipid liposome transdermal preparation
PRP was pipetted into the liposomes (10%, v/v), pipetted evenly and incubated for 1h (pH 6.4).
Example 2: preparation of PRP/phospholipid liposome transdermal preparation
(1) Preparation of phospholipid liposomes
0.3g of polyvinyl alcohol (PVA) was weighed out and dissolved in PBS (pH 5), and 0.6g of soybean lecithin (PG90), 0.3g of Cholesterol (CHO) and 10mg of methylparaben were added in this order, stirred overnight, and then sonicated with a sonicator. Sonication time 2' 5 "(2 s off for 5s each, 25 cycles) to prepare phospholipid liposomes.
(2) Preparation of PRP:
gradient centrifugation to obtain rat PRP: centrifuging a certain amount of rat blood (added with sodium citrate glucose solution) at 200g for 10min, collecting upper layer plasma, centrifuging at 400g for 10min twice, and discarding supernatant to obtain concentrated PRP.
(3) Preparation of PRP/phospholipid liposome transdermal preparation
PRP was pipetted into the liposomes (10%, v/v), pipetted evenly and incubated for 1h (pH 6.4).
Example 3: preparation of PRP/phospholipid liposome transdermal preparation
(1) Preparation of phospholipid liposomes
0.3g of polyvinyl alcohol (PVA) was weighed out and dissolved in PBS (pH 5), and 1.2g of soybean lecithin (PG90), 0.3g of Cholesterol (CHO) and 10mg of methylparaben were added in this order, stirred overnight, and then sonicated with a sonicator. The sonication time was 6' 15 "(2 s off for 5s each, cycle 75 times), and phospholipid liposomes were prepared.
(2) Preparation of PRP:
gradient centrifugation to obtain rat PRP: centrifuging a certain amount of rat blood (added with sodium citrate glucose solution) at 200g for 10min, collecting upper layer plasma, centrifuging at 400g for 10min twice, and discarding supernatant to obtain concentrated PRP.
(3) Preparation of PRP/phospholipid liposome transdermal preparation
PRP was pipetted into the liposomes (10%, v/v), pipetted evenly and incubated for 1h (pH 6.4).
Comparative example 1: preparation of PRP/phospholipid liposome transdermal preparation
(1) Preparing phospholipid liposome:
dissolving 0.3g of polyvinyl alcohol, 0.9g of soybean lecithin, 0.15g of cholesterol and 10mg of methylparaben in an appropriate amount of chloroform, removing the solvent by rotary evaporation under reduced pressure at 30 ℃, putting the mixture in a vacuum drying oven for vacuum drying overnight (completely removing the solvent), adding 10ml of PBS (pH 5), and carrying out water bath at 40 ℃; sonication 4 '10')
(2) Preparation of PRP:
gradient centrifugation to obtain rat PRP: centrifuging a certain amount of rat blood (added with sodium citrate glucose solution) at 200g for 10min, sucking upper layer plasma from the position close to the interface of plasma and red blood cells, centrifuging at 400g for 10min twice, and discarding supernatant to obtain concentrated PRP.
(3) Preparation of PRP/phospholipid liposome transdermal preparation
PRP was pipetted into the liposomes (10%, v/v), pipetted evenly and incubated for 1h (pH 6.4).
The PRP/phospholipid liposome transdermal preparations prepared in example 1 and comparative example 1 were observed, and the results are shown in fig. 1 and 2, respectively. In fig. 1, which shows the PRP/phospholipid liposome transdermal preparation prepared in example 1, phospholipid spherical particles (spherical particle diameter of about 30nm to 50nm) can be aggregated around platelets, as shown in fig. 1. The enlargement at the upper left corner shows that the phospholipid globules are embedded in the platelet membrane and the phospholipid liposomes encapsulate the platelets.
FIG. 2 is a PRP/phospholipid liposome transdermal preparation prepared in comparative example 1, and it can be seen from FIG. 2 that platelets and phospholipid spheres (40nm-80nm) are not uniformly distributed, and it can be seen from the enlarged view of the upper right corner of FIG. 2 that there are fewer phospholipid liposome particles aggregated around the platelets, and the phospholipid liposome spheres are not successful in forming a package on the platelets.
Test example 1: PRP/phospholipid liposome and determination of PRP in vitro release GFs rate and activity
Enzyme-linked immunosorbent assay is adopted to determine the PRP/phospholipid liposome and the PRP release GFs rate. Using PRP, the PRP/phospholipid liposomes prepared in example 1 of the present invention and comparative example 1, the release amount of GFs was measured by ELISA method as a time-varying curve.
The GFs activity was measured by endothelial cell proliferation and migration assay. PRP, PRP/phospholipid liposomes prepared according to example 1 and comparative example 1 of the present invention were added to 24-well serum-cultured plates of the same amount of adherent endothelial cells and cultured for 14 days, and counted every 4 days.
The proliferation assay results were consistent with GFs release kinetics, i.e., the PRP group promoted the most cell proliferation within 4d, followed by inventive example 1 and the lowest comparative example 1. After 4 days, the cells proliferated most in the example 1 group of the present invention and the cells proliferated least in the PRP group in the comparative example 1, indicating that the PRP/phospholipid liposome can prolong GFs half-life and release GFs slowly.
Test example 2: stability study
The PRP/phospholipid liposomes prepared in example 1 and comparative example 1 of the present invention were stored at-20 ℃ for 6 months, and sampled and tested at 0, 1, 2, 3, and 6 months, respectively. And (5) observing the change conditions of indexes such as content, particle size distribution and the like before and after the test sample.
As a result, the PRP/phospholipid liposome prepared in example 1 has no obvious change in each index during the standing process of the accelerated test of 6 months, which shows that the PRP/phospholipid liposome prepared in example 1 of the invention has stable quality.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A preparation method of a PRP/phospholipid liposome transdermal preparation is characterized by comprising the following steps:
(1) dissolving polyvinyl alcohol in PBS, sequentially adding soybean lecithin, cholesterol and methyl p-hydroxybenzoate, stirring, and swelling overnight; then carrying out ultrasonic treatment to prepare phospholipid liposome;
(2) carrying out gradient centrifugation on autologous whole blood to obtain concentrated PRP;
(3) injecting the PRP prepared in the step (2) into the phospholipidosome prepared in the step (1), blowing and beating uniformly, and then incubating to prepare a PRP/phospholipid lipidosome transdermal preparation;
wherein in the step (1), the adding amount ratio of the polyvinyl alcohol, the soybean lecithin, the cholesterol and the methyl p-hydroxybenzoate is 25-35: 80-100: 10-20: 1.
2. the method of claim 1, wherein the PBS has a pH of 5.0 in step (1).
3. The method of claim 1, wherein the polyvinyl alcohol, the soybean lecithin, the cholesterol, and the methylparaben are added in a ratio of 30: 90: 15: 1.
4. the preparation method according to claim 1, wherein in the step (1), the time for the ultrasonic treatment is 350s, and specifically comprises: turn off for 2s every 5s on, cycle 50 times.
5. The preparation method according to claim 1, wherein in the step (2), the autologous whole blood gradient centrifugation is specifically: centrifuging at a primary speed of 200g for 10min, and collecting the supernatant; the rotating speed of the secondary centrifugation is 400g, and the centrifugation time is 10 min; the supernatant was discarded.
6. The method according to claim 1, wherein in the step (3), the PRP is injected into the phospholiposome at 10%.
7. The method according to claim 1, wherein the incubation period in the step (3) is 1 hour.
8. A PRP/phospholipid liposome transdermal preparation prepared by the preparation method according to any one of claims 1 to 7.
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| WO2013007308A1 (en) * | 2011-07-14 | 2013-01-17 | Garbin Stefania | Platelet-rich plasma composition for tissue repair and regeneration |
| WO2014126931A1 (en) * | 2013-02-15 | 2014-08-21 | Victor Steven | Stable platelet- rich-plasma compositions and methods of use |
| CN104644522A (en) * | 2015-01-21 | 2015-05-27 | 北京弘润天源生物技术有限公司 | Transdermal beautifying combined preparation and kit as well as preparation method of combined preparation |
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| WO2013007308A1 (en) * | 2011-07-14 | 2013-01-17 | Garbin Stefania | Platelet-rich plasma composition for tissue repair and regeneration |
| WO2014126931A1 (en) * | 2013-02-15 | 2014-08-21 | Victor Steven | Stable platelet- rich-plasma compositions and methods of use |
| CN104644522A (en) * | 2015-01-21 | 2015-05-27 | 北京弘润天源生物技术有限公司 | Transdermal beautifying combined preparation and kit as well as preparation method of combined preparation |
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