CN114177278A - Liposome preparation - Google Patents

Liposome preparation Download PDF

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CN114177278A
CN114177278A CN202111212295.5A CN202111212295A CN114177278A CN 114177278 A CN114177278 A CN 114177278A CN 202111212295 A CN202111212295 A CN 202111212295A CN 114177278 A CN114177278 A CN 114177278A
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weight
phospholipid
cholesterol
liposome
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郭志栋
黄桂华
单如梦
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Shandong Duomeikang Biomedical Co ltd
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Abstract

Disclosed are liposomes comprising a phospholipid, cholesterol, and a recombinant type III humanized collagen, wherein the weight ratio of the phospholipid to the weight of the cholesterol is 3-7.5:1 and the weight ratio of the recombinant type III humanized collagen to the weight of the phospholipid and the cholesterol is 1: 5-15.

Description

Liposome preparation
FIELD
The present disclosure relates generally to the field of formulations, and more particularly, to liposomes.
Background
Collagen is a large group of molecules consisting of various glycoproteins, the major protein component of connective tissue, and accounts for approximately 25% of the total protein in the human body. To date, at least 30 more genes encoding collagen chains have been discovered, the different collagen chains differing from each otherIn combination, at least 16 or more collagen molecules can be formed. The 16 different types of collagen molecules are mostly formed by twisting 3 alpha chains, and are shaped like rods. Some of them consist of 3 identical alpha chains, e.g. [ alpha ]1]3Constituting a homogeneous trimer, and some of them consisting of 3 different alpha chains, e.g. [ alpha ]1]2α2Or alpha1α2α3A heterogeneous trimer is composed. The size of the alpha chain typically varies from 600 to 1000 amino acids. New collagen chains and collagen types are being discovered continuously. In terms of molecular structure, each collagen peptide chain is mainly composed of Gly-X-Y (X, Y is any amino acid residue except Gly) triplets repeatedly, the unique structure is necessary for forming a collagen fiber high-grade structure, the excellent biocompatibility and the low immunogenicity of the collagen are determined, and the collagen protein is widely applied to the industries of medicines, health care products and cosmetics.
SUMMARY
In one aspect, the disclosure relates to a liposome comprising a phospholipid, cholesterol, and a recombinant type III humanized collagen, wherein the weight ratio of the phospholipid to the weight of the cholesterol is 3-7.5:1, and the weight ratio of the recombinant type III humanized collagen to the weight of the phospholipid and the cholesterol is 1: 5-15.
In another aspect, the present disclosure relates to a gel comprising a liposome described in the present disclosure and a gel matrix.
In yet another aspect, the present disclosure relates to a method of preparing a liposome, comprising:
dissolving phospholipid and cholesterol in alcohol, and evaporating to remove the alcohol to obtain lipid film;
mixing the recombinant III type humanized collagen, tween and distilled water to obtain a mixed solution; and
mixing and hydrating the lipid membrane with the mixed solution, filtering to obtain the liposome,
wherein the weight ratio of the phospholipid to the cholesterol is 3-7.5:1, and the weight ratio of the recombinant type III humanized collagen to the phospholipid and the cholesterol is 1: 5-15.
Brief description of the drawings
Figure 1 shows the effect of drug lipid ratio on recombinant type III humanized collagen liposomes particle size, Entrapment Efficiency (EE) and Drug Loading (DL);
figure 2 shows the effect of phospholipid to cholesterol ratio on recombinant type III humanized collagen liposome particle size, Entrapment Efficiency (EE) and Drug Loading (DL);
figure 3 shows the effect of tween 80 concentration on recombinant type III humanized collagen liposome particle size, Entrapment Efficiency (EE) and Drug Loading (DL);
figure 4 shows the effect of hydration time on recombinant type III humanized collagen liposome particle size, Entrapment Efficiency (EE) and Drug Loading (DL);
FIG. 5 shows the water loss rate versus time curve for a gel in a silica gel dryer; and
fig. 6 shows the water loss rate versus time curve for a gel in a dryer with a relative humidity of 43%.
Detailed description of the invention
In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth.
Throughout this specification and the claims which follow, unless the context requires otherwise, the words "comprise", "comprising", and "have" are to be construed in an open, inclusive sense, i.e., "including but not limited to".
As used in this disclosure and the appended claims, a singular reference of an element without a numerical designation includes a plural reference unless the context clearly dictates otherwise.
Reference throughout the specification to "one embodiment," "an embodiment," "in another embodiment," or "in certain embodiments" means that a particular reference element, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
It should be understood that, as used in the specification of the present disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a lyophilized powder comprising "recombinant humanized collagen" includes one recombinant humanized collagen, or two or more recombinant humanized collagens.
Definition of
In the present disclosure, the term "liposome" refers to a microvesicle formed by encapsulating an active ingredient within a lipid bilayer.
In the present disclosure, the term "recombinant humanized collagen" refers to a full-length or partial amino acid sequence fragment encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of human collagen.
In the present disclosure, the term "type III" refers to recombinant collagen encoded based on the collagen COL3a1 gene or a partial gene.
In the present disclosure, the term "phospholipid" refers to a lipid containing a phosphoric acid.
In the present disclosure, the term "phosphoglyceride" refers to glycerol-3-phosphate, wherein the other two hydroxyl groups of glycerol molecule are esterified by fatty acid, and the phosphate group can be esterified by various small molecular compounds with different structures to form various phosphoglycerides.
In the present disclosure, the term "sphingomyelin" refers to sphingosine-or dihydrosphingosine-containing phospholipids.
In the present disclosure, the term "hydration" means that the substance dissolves in water.
Detailed Description
In one aspect, the disclosure relates to a liposome comprising a phospholipid, cholesterol, and a recombinant type III humanized collagen, wherein the weight ratio of the phospholipid to the weight of the cholesterol is about 3-7.5:1, and the weight ratio of the recombinant type III humanized collagen to the weight of the phospholipid and the cholesterol is about 1: 5-15.
In certain embodiments, the weight of phospholipid to weight of cholesterol is about 7.5: 1.
In certain embodiments, the weight of phospholipid to weight of cholesterol is 7.5: 1.
In certain embodiments, the weight ratio of recombinant type III humanized collagen to the weight of phospholipids and cholesterol is about 1: 10.
In certain embodiments, the weight ratio of recombinant type III humanized collagen to the weight of phospholipids and cholesterol is about 1: 10.
In certain embodiments, illustrative examples of phospholipids that can be used in the present disclosure include, but are not limited to, phosphoglycerides and sphingomyelin.
In certain embodiments, illustrative examples of phosphoglycerides that can be used in the present disclosure include, but are not limited to, Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidylglycerol (PG), glycerophosphatidic acid (PA), soybean phospholipids, and egg yolk lecithin.
In the present disclosure, yolk lecithin is extracted from yolk, has properties of low price, neutral charge, chemical inertness, and is a main phospholipid component of cell membranes, has good biocompatibility and no immunogenicity, when the yolk lecithin is dispersed in an aqueous phase, hydrophobic tails of molecules tend to gather together to avoid the aqueous phase, while a hydrophilic head is exposed to the aqueous phase to form a closed vesicle with a bimolecular structure, and water-soluble recombinant type III humanized collagen is wrapped in the aqueous phase in the vesicle, thereby preparing the recombinant type III humanized collagen liposome.
In the present disclosure, cholesterol is one of important components in biological membranes, which does not form a lipid bilayer structure by itself, but can be incorporated into phospholipid membranes in a high concentration manner to play a role in regulating the fluidity of the membrane structure.
In another aspect, the present disclosure relates to a gel comprising a liposome described in the present disclosure and a gel matrix.
In certain embodiments, illustrative examples of gel bases that can be used in the present disclosure include, but are not limited to, carbomer, sodium carboxymethylcellulose (CMC-Na), Hydroxypropylmethylcellulose (HPMC), methylcellulose, chitosan, and sodium alginate.
In certain embodiments, the gel further comprises tween.
In certain embodiments, tween is present in an amount of from about 0% to about 4% by weight based on the weight of the gel.
In certain embodiments, tween is present in an amount of 0% to 4% by weight based on the weight of the gel.
In certain embodiments, tween is present in an amount of about 0.5% to about 2% by weight based on the weight of the gel.
In certain embodiments, tween is present in an amount of 0.5% to 2% by weight, based on the weight of the gel.
In the present disclosure, the liposome membrane is a dynamic membrane, the phospholipid molecules continuously exchange positions, and the liposome particles spontaneously aggregate and precipitate, so that the morphological structure of the liposome is unstable. Tween 80 as a nonionic surfactant can inhibit aggregation of nanoparticles to a certain extent, and therefore, tween 80 was selected as a stabilizer.
In certain embodiments, the phospholipids and cholesterol are present in an amount of about 0.6% to about 1.5% by weight, based on the weight of the gel.
In certain embodiments, the phospholipids and cholesterol are present in an amount of 0.6 to 1.5% by weight, based on the weight of the gel.
In certain embodiments, a gel comprises a liposome as described in the present disclosure, carbomer 940, triethanolamine, glycerin, methylparaben, phenoxyethanol, and purified water.
In certain embodiments, the gel comprises 50% by weight of liposomes described in the present disclosure, 0.5% by weight of carbomer 940, 0.4% by weight of triethanolamine, 15% by weight of glycerol, 0.1% by weight of methylparaben, 0.5% by weight of phenoxyethanol, and 33.5% by weight of purified water, based on the total weight of the gel.
In yet another aspect, the present disclosure relates to a method of preparing a liposome, comprising:
dissolving phospholipid and cholesterol in alcohol, and evaporating to remove the alcohol to obtain lipid film;
mixing the recombinant III type humanized collagen, tween and distilled water to obtain a mixed solution; and
mixing and hydrating the lipid membrane with the mixed solution, filtering to obtain the liposome,
wherein the weight ratio of the phospholipid to the cholesterol is about 3-7.5:1 and the weight ratio of the recombinant type III humanized collagen to the weight of the phospholipid and the cholesterol is about 1: 5-15.
In certain embodiments, illustrative examples of alcohols that can be used in the present disclosure include, but are not limited to, ethanol.
In certain embodiments, liposomes can be obtained by filtration using a filter membrane.
In certain embodiments, tween is at a concentration of about 10%.
In certain embodiments, tween is at a concentration of about 3%.
In certain embodiments, the hydration time is about 15 minutes to about 30 minutes.
In certain embodiments, the hydration time is from 15 minutes to 30 minutes.
In certain embodiments, the hydration time is about 30 minutes.
In certain embodiments, the hydration time is 30 minutes.
Hereinafter, the present disclosure will be explained in detail by the following examples in order to better understand various aspects of the present application and advantages thereof. It should be understood, however, that the following examples are not limiting and are merely illustrative of certain embodiments of the present disclosure.
Examples
The reagents and equipment used in the examples of the present disclosure are conventional and commercially available. For example:
TABLE 1 common instrumentation for preparation of recombinant type III humanized collagen liposome gel
Figure BDA0003309365540000061
Figure BDA0003309365540000071
Note that: other laboratory tools include beakers, glass rods, droppers, thermometers, and the like.
TABLE 2 pharmaceutical and reagent for recombinant human collagen liposome gel
Figure BDA0003309365540000072
Figure BDA0003309365540000081
Figure BDA0003309365540000091
Example 1: screening of prescription composition
150mg of egg yolk lecithin, 20mg of cholesterol, 5mL of absolute ethyl alcohol and 5mL of collagen hydration solution (which consists of 1mL of collagen, 1.5mL of 10% Tween 80 and 2.5mL of distilled water). The basic preparation process of the thin film evaporation method comprises the following steps: 150mg of egg yolk lecithin and 20mg of cholesterol were precisely weighed and dissolved in 5mL of absolute ethanol. And (3) removing the ethanol by reduced pressure rotary evaporation (50 ℃, the rotating speed is 2) until the ethanol is completely volatilized to form a uniform film. Adding 5mL of hydration solution dissolved with collagen at normal temperature, performing rotary hydration until the film is completely separated to form milky suspension liquid, and performing rotary evaporation at 40 ℃ for 30 min.
Example 2: screening of drug-to-lipid ratio
The dosage of other components in the basic prescription in example 1 was fixed, the basic preparation process was used to prepare recombinant type III humanized collagen liposome (rhc-III-Lips), the drug-lipid ratio (i.e., the ratio of recombinant type III humanized collagen to phospholipid and cholesterol) was changed (the drug-lipid ratio was 1:5, 1:10, 1:15, respectively), and the effect of the drug-lipid ratio on the evaluation index was examined using the particle size, the Entrapment Efficiency (EE), and the drug loading capacity (DL) as evaluation indexes, and the results are shown in table 3 and fig. 1.
TABLE 3 screening results of drug-to-lipid ratio
Figure BDA0003309365540000092
As can be seen from fig. 1, the encapsulation efficiency tends to increase and then decrease with the increase of the lipid dosage, the drug loading rate decreases with the increase of the carrier material, and the particle size tends to decrease. Therefore, the medicine-fat ratio is selected to be 1: 5-15.
Example 3: screening for phospholipid to cholesterol ratio
The amount of other components in the basic recipe in example 1 was fixed, the basic preparation process was used to prepare a recombinant type III humanized collagen liposome (rhc-III-Lips), the ratio of phospholipid to cholesterol (3: 1, 5:1, and 7.5:1, respectively) was changed, the particle size, Encapsulation Efficiency (EE), and drug loading capacity (DL) were used as evaluation indices, and the influence of the evaluation indices on the ratio of phospholipid to cholesterol was examined, and the results are shown in table 4 and fig. 2.
TABLE 4 screening of phospholipid to Cholesterol ratios
Figure BDA0003309365540000101
As can be seen from fig. 2, the encapsulation efficiency and particle size tended to decrease and then increase as the phospholipid/cholesterol ratio increased. The cholesterol can adjust the fluidity of the lipid bilayer and enhance the anti-coagulation capacity of the lipid bilayer, but when the cholesterol is used in an excessive amount, the fluidity of the liposome is increased, and the medicament is easy to leak, so the ratio of the phospholipid to the cholesterol is selected to be 3-7.5: 1.
Example 4: screening of Tween 80 dosage
The amount of other components in the basic prescription of example 1 was fixed, the recombinant humanized collagen type III liposome (rhc-III-Lips) was prepared by the basic preparation process, the amount of tween 80 (0, 1, 2, 3, 4, respectively) was changed, the particle size, the Entrapment Efficiency (EE) and the drug loading rate (DL) were used as evaluation indices, and the effect of the amount of tween 80 on the evaluation indices was examined, and the results are shown in table 5 and fig. 3.
TABLE 5 screening results for Tween 80 dosage
Figure BDA0003309365540000102
Figure BDA0003309365540000111
From the results shown in fig. 3, it can be seen that when the amount of tween 80 is 0%, the liposome is layered after being placed for 2 hours, which is very unstable, and the encapsulation efficiency tends to increase and decrease with the increase of the amount of tween 80, so the amount of tween 80 is 1% to 4%.
Example 5: screening of hydration temperature in preparation process of recombinant type III humanized collagen liposome (rhc-III-Lips)
When the liposome is prepared by adopting a film dispersion method, the hydration temperature needs to be higher than the phase transition temperature of phospholipid, the fluidity of a lipid film is increased, and the medicine can better enter an internal water phase so as to increase the encapsulation efficiency. The phase transition temperature of egg yolk lecithin was 28 ℃. In a certain temperature range, the encapsulation efficiency of the liposome is increased along with the increase of the hydration temperature. Therefore, it is preferable to select a higher hydration temperature within a certain range. Because the recombinant III type humanized collagen (rhc-III) is thermolabile, the theory shows that the highest heat-resistant temperature is 40 ℃, and in the preliminary experiment, the recombinant III type humanized collagen (rhc-III) solution is placed in a water bath condition at 40 ℃ for 60min, and the content of the recombinant III type humanized collagen is not obviously changed. Thus, in the present disclosure, the hydration temperature was fixed at 40 ℃ and no screening was performed.
Example 6: screening of hydration time in preparation process of recombinant type III humanized collagen liposome (rhc-III-Lips)
The components and the dosage of the prescription in example 1 were fixed, the basic preparation process was used to prepare the recombinant type III humanized collagen liposome (rhc-III-Lips), the hydration time in the basic preparation process was changed (15, 30, 45min, respectively), the particle size, the Entrapment Efficiency (EE) and the drug loading rate (DL) were used as evaluation indexes, and the influence of the hydration time on the evaluation indexes was examined, and the results are shown in table 6 and fig. 4.
TABLE 6 hydration time screening results
Figure BDA0003309365540000112
From the results in fig. 4, it can be seen that the encapsulation efficiency, drug loading and particle size tended to decrease with increasing hydration time, and the decrease was evident after 30min, with hydration time being selected from 15min to 30min in view of time cost.
Example 7: reproducibility test of three samples of recombinant type III humanized collagen liposomes (rhc-III-Lips)
Three batches of samples were prepared repeatedly according to the screened recipe and preparation process, examined according to the method under the quality standard item, and the feasibility of the recipe composition and the stability of the preparation process were evaluated, and the results are shown in table 7.
TABLE 7 results of reproducibility test of three batches of recombinant type III humanized collagen liposomes (rhc-III-Lips) formulation process
Quality inspection index Batch No. 20200914-1 Batch No. 20200914-2 Batch No. 20200914-3
Appearance character Milky white suspension Milky white suspension Milky white suspension
Particle size (nm) 273.53±2.10 261.20±9.54 248.80±2.71
PDI 0.156±0.001 0.142±0.004 0.139±0.007
Electric potential (mV) -8.03±0.44 -8.52±0.48 -8.28±0.25
pH 6.22±0.02 6.28±0.01 6.32±0.04
Encapsulation efficiency (%) 81.43±2.45 85.22±1.72 82.93±1.93
Drug loading (%) 6.13±0.17 6.34±0.12 6.24±0.14
Experimental results show that the recombinant III type humanized collagen liposome (rhc-III-Lips) has reasonable prescription composition and stable preparation process.
Example 8: screening of gel matrices
Commonly used aqueous gel bases include carbomer, Hydroxypropylmethylcellulose (HPMC), methylcellulose, sodium carboxymethylcellulose (CMC-Na), chitosan, sodium alginate, and the like. According to the concentration commonly used by various gel matrixes, 0.3%, 0.5% and 1% carbomer (needing to adjust the pH by triethanolamine), 1%, 3% and 5% sodium carboxymethylcellulose (CMC-Na), 0.5%, 1% and 1.5% chitosan (dissolved by 1% acetic acid) and 0.5%, 1% and 1.5% hydroxypropyl methyl cellulose (HPMC) are respectively prepared.
And (3) screening the types of the gel matrix by taking the appearance, pH, uniformity, viscosity and spreadability of the gel matrix as evaluation indexes. The results are shown in Table 8.
TABLE 8 screening results for gel matrix type and concentration
Figure BDA0003309365540000131
Figure BDA0003309365540000141
As can be seen from the results in Table 8, the high concentration of CMC-Na is good in formability, but poor in spreadability, and the surface is permeable to water after 5% of CMC-Na is left for one day; the chitosan is insoluble in water and needs to be dissolved by 1% acetic acid, so that the pH of the gel matrix is low, and the peracid gel matrix can stimulate the skin; the low-concentration HPMC has poor formability and high fluidity, and the high-concentration HPMC is difficult to swell, and even if the HPMC swells for 24 hours, gel lumps still exist and are difficult to stir and disperse uniformly; the carbomer gel matrix has good formability, is easy to spread and has no greasy feeling, and after the pH is adjusted by triethanolamine, the pH is close to the pH of normal human skin, so that the carbomer gel matrix is suitable for skin administration. A viscosity of 0.5% carbomer matrix is preferred, and therefore, 0.5% carbomer may be selected as the gel matrix.
Example 9: screening of the type and amount of pH modifier
Aqueous carbomer solutions are acidic and have a pH of about 3.0. When neutralized with alkali, the molecular chain which is originally coiled is stretched, so that the volume is increased, the viscosity is gradually increased, a colorless and transparent semisolid gel is formed when the concentration is higher, and the carbomer gel with proper consistency is selected according to clinical application and actual needs. The most commonly used pH adjusters are triethanolamine and sodium hydroxide. Sodium hydroxide is strong alkali, and has large irritation to skin; triethanolamine is a weak base, has mild properties and small irritation to skin, so the triethanolamine is selected as a pH regulator of the gel. The amount of triethanolamine was selected according to the group shown in Table 9 using the appearance, pH, viscosity and spreadability as evaluation indexes, and the results are shown in Table 10.
TABLE 9 formulation composition for screening triethanolamine dosage (total 100g)
Figure BDA0003309365540000142
TABLE 10 results of triethanolamine dosage screening
Figure BDA0003309365540000151
From the results of Table 10, the gels of each group have high viscosity, good formability and a semisolid state, and can be well adhered to the skin; from the pH results, the pH of the 0.2% and 0.3% group gels was below 6, acidic; the 0.4% group gel is weakly acidic, and related researches show that the weakly acidic environment is favorable for healing wounds; the 0.5% group gel had a pH of 7.02, near neutral; the pH of the 0.6% group gel is slightly higher than 7, considering that the amine substances in the triethanolamine can stimulate the skin, and the triethanolamine with too high concentration can not be used, therefore, the triethanolamine is selected to be used in an amount of 0.4% by comprehensively considering the stimulation of the gel matrix to the skin and the influence of the gel matrix to the wound healing condition.
Example 10: screening of humectant type and amount
The moisturizer commonly used in the gel is mainly glycerin, propylene glycol, Polyethylene glycol 400 (PEG 400), hyaluronic acid, etc., which achieve moisturization by enhancing the water absorption and water binding capacity of the stratum corneum of the skin. Glycerol is also called as glycerol, the molecule of glycerol contains hydroxyl groups, hydrogen bonds can be formed with water molecules, water is firmly locked, the moisture near the wound is kept, the wound healing can be promoted, and high-concentration glycerol can absorb the water in the skin to generate burning sensation, so that the common concentration is 5-15%, and the glycerol is widely applied due to low price and easy acquisition; propylene glycol also has good moisture retention, but studies have found that it has some skin carcinogenicity, thus, propylene glycol is avoided; PEG400 has effects of keeping moisture and lubricating, and can be used as a skin membrane forming agent to form a membrane on skin surface after application to lock skin moisture; hyaluronic acid is a water-soluble polymer which is excellent in moisture retention and widely used in cosmetics, but many studies have shown that hyaluronic acid has an effect of promoting wound healing, and hyaluronic acid is not selected as a humectant in the present preparation in order to avoid its effect on subsequent pharmacodynamic studies in rats. Since a single humectant can not achieve various purposes such as water locking, moisture retention and moisture absorption, in practical applications, a plurality of humectants are often used in combination, and therefore, in this example, the moisture retention of glycerin (5%, 10% and 15%), PEG400 (5%, 10% and 15%) and glycerin-PEG 400 (fixed total concentration is 10%, and combination ratio is 2:8, 5:5 and 8:2) with different concentrations are studied.
Reference is made to the method of investigating the moisture retention of cosmetic products (water loss rate measurement method) in terms of water lossThe loss rate was an evaluation index. Weighing the culture dish dried to constant weight by an in vitro weighing method and recording the weight as M1Weighing appropriate amount of gel matrix containing different moisturizers, respectively placing into a culture dish, and weighing as M2Placing the culture dish containing gel matrix in a drier with relative humidity of 43% and a silica gel drier respectively, taking out and weighing at the time of placing for 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h and 48h, and recording as MtThe water loss rate was calculated according to the following formula, and the specific dosage is shown in Table 11.
Water loss rate [ (M)2-Mt)/(M2-M1)]×100％
TABLE 11 screening of formula dosages for humectant type and dosage
Figure BDA0003309365540000161
The results of the moisture loss rate of gels containing varying types and concentrations of moisturizers in a silica gel dryer at a relative humidity of 43% are shown in fig. 5 and 6.
As can be seen from fig. 5 and 6, the higher the glycerin concentration, the better the moisturizing effect; compared with glycerin and PEG400 with the same concentration, glycerin has better moisturizing effect; when the glycerin and the PEG400 are compounded, the water loss rate is still higher than that of glycerin with the same concentration, and the glycerin is selected to be used as the humectant by combining the results of all the formulas, wherein the dosage is 15 percent by weight.
Example 11: determination of the type and amount of preservative
Preservatives are often added to gels to prevent microbial spoilage of higher moisture gels, and the preservatives included in wound-care preparations should meet the two basic requirements of being non-irritating and preserving.
In the development stage of the preparation, the bacteriostatic activity of the preparation is measured by using a bacteriostatic efficacy test method so as to guide the determination of the type and the dosage of the preservative in the preparation, and in order to ensure the medication safety, the dosage of the preservative is the minimum effective amount. The single preservative needs higher concentration to achieve the preservative effect, and the compounding of the preservative can expand the antibacterial broad spectrum. Phenoxyethanol has good antibacterial ability, but has a general antibacterial effect on gram-positive bacteria and fungi. The methylparaben has strong inhibiting effect on fungi, has a certain effect on gram-positive bacteria and has weak effect on gram-negative bacteria, so that the aims of increasing antibacterial spectrum, low dosage and high bacteriostasis can be achieved by matching phenoxyethanol and methylparaben. The lowest effective amount of the phenoxyethanol and the methylparaben is determined by consulting the prior art, and finally 0.5 percent of phenoxyethanol and 0.1 percent of methylparaben are selected as the preservative.
Example 12: screening of preservative dissolution conditions in blank gel matrix preparation process
The water solubility of the methylparaben is poor, the solubility of the methylparaben is high in glycerin, the phenoxyethanol is slightly soluble in water but can be freely mixed with the glycerin, and in order to ensure that the methylparaben is completely dissolved and the phenoxyethanol is uniformly dispersed in the gel matrix to achieve a better anti-corrosion effect, therefore, the methylparaben, the phenoxyethanol and the glycerin in the formula amount are mixed together, on a magnetic stirrer, the shortest time required by the complete dissolution of the methylparaben is taken as an evaluation index, the fixed rotating speed is 300rpm, and the optimal dissolving temperature is screened; fixing the temperature and screening the optimal rotating speed. The results are shown in tables 12 and 13.
TABLE 12 screening of methylparaben dissolution times at various temperatures
Figure BDA0003309365540000171
TABLE 13 results of methylparaben dissolution time screening at different speeds
Figure BDA0003309365540000181
From the experimental results shown in Table 12, the dissolution rate of methyl hydroxybenzoate increases with the increase of temperature, and when the heating temperature is 80 ℃, the dissolution time of methyl hydroxybenzoate is greatly shortened, and the methyl hydroxybenzoate can be completely dissolved in 6.67 min. From the results of the experiment shown in Table 13, the dissolution rate of methylparaben increased with the increase of the rotation speed. Finally determining the dissolving condition of the methyl hydroxybenzoate by comprehensively considering time and economic cost: the temperature was 80 ℃ and the rotational speed was 300 rpm.
Example 13: sterilization method and sterilization condition screening in blank gel matrix preparation process
Since the recombinant type III humanized collagen (Rhc-III) is a biomacromolecule drug and is sensitive to heat, a filtration sterilization method is adopted, namely a 0.22 mu m filter membrane is used for filtration and then is added into a gel matrix. Therefore, the sterilization method screening is mainly aimed at the sterilization of the gel matrix. Sterilizing the gel matrix by flowing steam sterilization (100 deg.C, 30min) and autoclaving (121 deg.C, 20min), (115 deg.C, 30min), comparing the appearance, viscosity, pH and spreadability of the gel matrix before and after sterilization, and screening out the most suitable sterilization method and sterilization conditions. The results are shown in Table 12.
TABLE 14 Change in Properties of gel base before and after Sterilization
Figure BDA0003309365540000182
Figure BDA0003309365540000191
As can be seen from table 14, the gel matrix after steam sterilization was passed through, the viscosity decreased by about 5.17Pa · s, and there was no significant change in pH; after autoclave sterilization (115 ℃, 30min), the viscosity is reduced by about 21.64 pas and the pH is reduced by 0.09; after autoclaving (121 ℃, 20min), the viscosity decreased by about 22.25 pas and the pH decreased by 0.07. It can be seen that the flow-through steam sterilization has minimal effect on the viscosity of the gel matrix, and although autoclaving may reduce the viscosity of the gel, it does not have much effect on the skin adhesion of the gel, so autoclaving (115 ℃, 30min) was selected to sterilize the gel matrix.
Example 14: reproducibility test of blank gel matrix
The prescription is obtained and the preparation process is as follows by screening the prescription and the process of the blank gel:
1. the prescription composition is as follows: 0.5% carbomer as gel matrix, 0.4% triethanolamine as pH regulator, 15% glycerin as humectant, and 0.1% methyl hydroxybenzoate and 0.5% phenoxyethanol as antiseptic.
2. The preparation process comprises the following steps: (1) firstly, dissolving a prescription amount of carbomer 940 for later use; (2) the prescribed amounts of methylparaben, phenoxyethanol and glycerol were weighed out and heated under magnetic stirring (80 ℃, 300rpm) to give a colorless, transparent, homogeneous solution. Adding the mixed solution into a carbomer solution, and uniformly mixing; (3) dropwise adding triethanolamine in a prescribed amount into the solution while stirring to adjust the pH value to form semisolid gel; and (4) sterilizing by hot-pressing sterilization method (115 deg.C, 30min), and cooling to obtain blank gel.
Preparing three batches of blank gels according to the prescription and the process, and inspecting the appearance, the viscosity, the pH, the spreadability and the like of the gel to determine the stability of the prescription and the process. The results are shown in Table 15.
TABLE 15 results of three blank gel reproducibility tests
Figure BDA0003309365540000201
The results of the experiments in Table 15 show that the three batches of blank gel matrix formulations and processes are stable.
Example 15: preparation of recombinant type III humanized collagen liposome (rhc-III-Lips) gel
Determining the dosage of the recombinant type III humanized collagen liposome (rhc-III-Lips) in the recombinant type III humanized collagen liposome (rhc-III-Lips) gel according to the content of the collagen in the recombinant type III humanized collagen liposome (rhc-III-Lips). The formula and preparation process of the recombinant type III humanized collagen liposome (rhc-III-Lips) gel are determined according to the formula of the blank gel matrix, and the results are as follows.
1. Prescription
The formulation of the recombinant type III humanized collagen liposome (rhc-III-Lips) gel is shown in Table 16.
TABLE 16 formulation of recombinant type III humanized collagen liposomes (rhc-III-Lips) gels
Figure BDA0003309365540000202
Figure BDA0003309365540000211
2. Preparation process
(1) The carbomer 940 is first dissolved in a prescribed amount of purified water to form a viscous, homogeneous solution for use.
(2) Weighing the prescribed amount of methylparaben, phenoxyethanol and glycerol in a 25mL small beaker, heating for 7min under magnetic stirring (80 ℃, 300rpm) to obtain a transparent colorless uniform solution, and cooling to room temperature for later use.
(3) And (3) adding the solution in the item (2) into the carbomer solution in the item (1), uniformly stirring, and dropwise adding triethanolamine while stirring to adjust the pH until a thick gel matrix is formed.
(4) Sterilizing the gel matrix by autoclaving. The sterilization conditions are as follows: the temperature is 115 deg.C, and the time is 30 min.
(5) Cooling the sterilized matrix to below 40 deg.C, adding recombinant type III humanized collagen liposome (rhc-III-Lips) suspension sterilized by microporous membrane filtration, and stirring to obtain recombinant type III humanized collagen liposome (rhc-III-Lips) gel.
Example 16: reproducibility test of recombinant type III humanized collagen liposome (rhc-III-Lips) gel
Three batches were prepared according to the recipe and preparation process of example 15, examined according to the methods under the quality standard terms, and the feasibility of the recipe composition and the stability of the preparation process were evaluated. The results are shown in Table 17.
TABLE 17 results of process reproducibility test of three batches of recombinant type III humanized collagen liposomes (rhc-III-Lips) gel formulations
Figure BDA0003309365540000212
Figure BDA0003309365540000221
Experimental results show that the formula of the recombinant III type humanized collagen liposome (rhc-III-Lips) gel is reasonable, and the preparation process is stable.
In the present disclosure, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
From the foregoing it will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications or improvements may be made by those skilled in the art without departing from the spirit and scope of the disclosure. Such variations and modifications are intended to fall within the scope of the appended claims of this disclosure.

Claims (10)

1. A liposome comprising a phospholipid, cholesterol, and a recombinant type III humanized collagen, wherein the ratio of the weight of the phospholipid to the weight of the cholesterol is 3-7.5:1, and the ratio of the weight of the recombinant type III humanized collagen to the weight of the phospholipid and the cholesterol is 1: 5-15.
2. The liposome of claim 1, wherein the phospholipid is selected from the group consisting of phosphoglycerides and sphingomyelin.
3. The liposome of claim 2, wherein the phosphoglyceride is selected from Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidylglycerol (PG), glycerophosphatidic acid (PA), soybean phospholipid, egg yolk lecithin or any mixture thereof.
4. A gel comprising the liposome of any one of claims 1 to 4 and a gel matrix.
5. The gel of claim 4, wherein the gel matrix is selected from carbomer, sodium carboxymethylcellulose (CMC-Na), Hydroxypropylmethylcellulose (HPMC), methylcellulose, chitosan, sodium alginate, or any mixture thereof.
6. The gel of claim 4 or 5, wherein the gel further comprises tween, preferably in an amount of 1 to 4% by weight based on the weight of the gel.
7. A gel according to any one of claims 4 to 6 wherein the phospholipid and cholesterol are present in an amount of from 0.6% to 1.5% by weight, based on the weight of the gel.
8. A method of preparing a liposome comprising:
dissolving phospholipid and cholesterol in alcohol, and evaporating to remove the alcohol to obtain lipid film;
mixing the recombinant III type humanized collagen, tween and distilled water to obtain a mixed solution; and
mixing and hydrating the lipid membrane with the mixed solution, filtering to obtain the liposome,
wherein the weight ratio of the phospholipid to the cholesterol is 3-7.5:1, and the weight ratio of the recombinant type III humanized collagen to the phospholipid and the cholesterol is 1: 5-15.
9. The method of claim 8, wherein the tween is at a concentration of 10% by volume.
10. The method of claim 8 or 9, wherein the hydration time is from 15 minutes to 30 minutes.
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