CN110251412A - The preparation method of rhIL-12(p70) liposome and products thereof purposes - Google Patents

The preparation method of rhIL-12(p70) liposome and products thereof purposes Download PDF

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Publication number
CN110251412A
CN110251412A CN201910570541.0A CN201910570541A CN110251412A CN 110251412 A CN110251412 A CN 110251412A CN 201910570541 A CN201910570541 A CN 201910570541A CN 110251412 A CN110251412 A CN 110251412A
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rhil
liposome
preparation
skin
added
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熊盛
卢加
谢秋玲
刘忠
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Guangdong Jida Genetic Pharmaceutical Engineering Research Center Co Ltd
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Guangdong Jida Genetic Pharmaceutical Engineering Research Center Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14—Liposomes; Vesicles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/004—Aftersun preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/82—Preparation or application process involves sonication or ultrasonication
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying

Abstract

The invention belongs to technical field of liposome preparation, and in particular to the preparation method of rhIL-12(p70) liposome and the purposes for obtaining product.RhIL-12(p70) is added in the substances such as soybean lecithin and cholesterol and rhIL-12(p70) liposome is made by the present invention, rhIL-12(p70) after liposomal encapsulated is applied to comparatively fast enter deep skin in skin care item, it can be very good to play its antiallergy after skin absorbs, enhancing is immune, repair the effect of light injury, it is injury-free to maintain epidermis skin, tyrosinase related protein2 in basal layer of epidermis chromatophore is avoided or reduced to be activated, to reduce the melanin transfer generated after tyrosine activation to skin holostrome, the evaporation of moisture can also be reduced simultaneously, to reach whitening, moisturizing, it is wrinkle resistant, except the effect of scar, enter the good application prospect of high-end cosmetic display for rhIL-12(p70).

Description

The preparation method of rhIL-12(p70) liposome and products thereof purposes
Technical field
The invention belongs to technical field of liposome preparation, and in particular to the preparation of rhIL-12(p70) liposome Method and the purposes for obtaining product, specifically 2 liposome of recombinant interleukin-1 is preparing the application in skin care item.
Background technique
Interleukin 12 (IL-12) is initially referred to as natural killer cells stimulating factor (NKSF) or cytotoxicity lymph Cell maturation factors (CLMF), mainly by the monocyte activated and other kinds of cell (Dendritic Cells, B cell, in Property granulocyte and horn cell) generate, be a kind of heterodimeric cytokine.IL-12 was found in 1992, found her Begin to show that IL-12 has strong antiviral and anti-tumor effect, is known as the core that immune system exercises antiviral and antitumor Regulatory factor is also known as the key protein matter of body natural immune system.
In recent years, discovery IL-12 has rush to the DNA damage of epidermal keratinocytes confluent monolayer cells caused by ultraviolet light (in involve long wave) Into its repair.IL-12 mainly has the following aspects to the effect of skin: 1) IL-12 can reverse ultraviolet light and aging Caused skin lesion, IL-12 reduce damage of the ultraviolet light to epidermal dna by activating the repair mechanism of DNA；2) purple UV radiation can lead to the immunosuppressive action of skin；3) IL-12 Transdermal absorption passes through bright Han Shi to corium and subcutaneous tissue The cell-mediated T lymphocyte that can activate corium promotes it to generate V- interferon, and V- interferon can inhibit fiber female thin Born of the same parents' proliferation, inhibits proline hydroxylase required for collage synthesis, and collagen is prevented to generate.
In conclusion IL-12 has triple role to skin, it can by the prevention of activation DNA repair mechanism or mitigate Epidermis caused by ultraviolet light or aging, DNA damage mitigate cutaneous immunisation caused by ultraviolet light or aging and inhibit, and prevent collagen fine The hyperplasia of dimension promotes the hydrolysis of collagenous fibres, removes wrinkle, the function of scar to have.So as it can be seen that since IL-12 is tieed up It is without damage to have protected normal epidermis, has avoided or reduced tyrosinase related protein2 in basal layer of epidermis chromatophore and is activated, to subtract The melanin transfer generated after few tyrosine activation is to skin holostrome, to play the whitening function of skin, meanwhile, it can be with The moisture evaporation for reducing skin, avoids or reduces the apoptosis of keratinocyte, reduces the secretion of elastase, so that elasticity is hard Albumen maintains normal condition, increases skin elasticity, reduces the appearance of wrinkle.Therefore, more and more IL-12 are applied to In cosmetics.
Patent document CN102641217A discloses a kind of application of interleukin 12 in cosmetics, is intended to mention Skin immune function controlling, antiallergy, the cosmetic additive agent for repairing skin injury can be enhanced for one kind.However IL-12 is molecular weight For the macromolecular substances of 70KD, directly adding use can not be such that skin absorbs, and be unable to reach due effect.
Summary of the invention
In order to overcome defect existing in the prior art, the purpose of the present invention is to provide a kind of combining human interleukins The preparation method of plain -12 liposomes, encapsulates rhIL-12(p70) using liposome, and protection recombined human is white thin Born of the same parents interleukin -12 prevent it from interfering with each other with other substances in formula, and avoid the contact with extraneous factor, reduce by wind A possibility that dry, can also avoid the degradation of rhIL-12(p70) and go bad, improve the stability of itself, and drop Its low irritation to skin.
The present invention provides a kind of preparation methods of rhIL-12(p70) liposome, comprising the following steps:
The Chinese hamster ovary celI for stablizing expression rhIL-12 is carried out fed-batch cultivation through bioreactor by S1, is taken in culture Clear liquid is chromatographed through weak cation exchange respectively, and heparin affinity chromatography separation obtains rhIL-12；
S2 takes soybean lecithin, cholesterol to be dissolved in chloroform, is ultrasonically treated 8~12min, the ultrasound input power It is 40%, obtains mixed liquor I；
Ether and chloroform are added into the mixed liquor I that step S2 is obtained by S3, and the PBS buffer solution that pH is 6.8 is added dropwise, and surpass To not stratified, the ultrasound input power is 40% for sonication, obtains w/o type lotion；
The w/o type lotion that step S3 is obtained is evaporated under reduced pressure S4 under the conditions of 50 DEG C of waters bath with thermostatic control, obtains O/W type lotion；
The O/W type lotion that S5 obtains step S4 carries out high-pressure homogeneous 15~25min, and every 5min is 1 circulation, filters, The additive amount that rhIL-12 described in the rhIL-12 that step S1 is collected is added is 70-100ug/ml, It is subsequently added into protein protectant, 18~22min is incubated under conditions of 37 DEG C, excipient mixing packing is added, it is pre- in -40 DEG C Freeze 2h, be freeze-dried under conditions of vacuumizing for 24 hours, in 4 DEG C save to get.
Further, the solid-to-liquid ratio of soybean lecithin, cholesterol and chloroform is 4g:1g in the step S2: 200ml。
Further, the volume ratio of ether, chloroform and PBS buffer solution is 3:1:2 in the step S3.
Further, the volume ratio of chloroform and the chloroform in step S3 is 2:1 in the step S2.
Further, the concentration of PBS buffer solution is 0.02mol/L in the step S3.
Further, the pressure of the step S5 mesohigh homogeneous is 10000~15000Pa.
Further, the filtering in the step S5 be pass sequentially through 1.0um, 0.6um, 0.45um and 0.22um filter membrane into Row filtering.
Further, the protein protectant in the step S5 is that the bovine serum albumin(BSA) that mass concentration is 0.1% is molten The volume ratio of liquid, the protein protectant and rhIL-12(p70) is 10:1.
Further, the excipient in the step S5 is (1.5~2) by trehalose and glyceroglycolipid in mass ratio: 1 group At the mass ratio of the excipient and rhIL-12(p70) is 2:1.
In addition, the present invention also provides the preparation methods of the rhIL-12(p70) liposome to be prepared RhIL-12(p70) liposome preparing the purposes in skin care item.
RhIL-12(p70) provided by the invention is by the recombination of expressing cho cell fermentation supernatant purification HIL-12, host cell are preferably Chinese hamster ovary cell CHO-S.Recombined human produced by the present invention is white thin The average grain diameter of -12 liposome of born of the same parents' interleukin is 100~300nm, preferably 200nm.The rhIL-12(p70) liposome can The form for thinking freeze-drying prods, further includes mannitol.
The preparation method of rhIL-12(p70) liposome provided by the invention is the load using lipid as IL-12 The transdermal absorption factor of IL-12 can be improved by the IL-12 of the encapsulating of liposome for body, while can also enhance the anti-mistake of IL-12 Quick, enhancing is immunized, repairs the effect of light injury, extends action time, reduces the additive amount of IL-12 in skin care item.
In short, compared with prior art, the preparation method of rhIL-12(p70) liposome provided by the invention has There is following advantage:
(1) preparation method of rhIL-12(p70) liposome provided by the invention is using liposome to recombination HIL-12 is encapsulated, and rhIL-12(p70) is protected, and prevents itself and other substances in skin care item formula Interfere with each other, and avoid the contact with extraneous factor, reduce a possibility that being air cured, recombinant human leucocyte can also be avoided The degradation of interleukin -12 and rotten, improves the stability of itself, and reduce its irritation to skin.
(2) recombinant human leucocyte made from the preparation method of rhIL-12(p70) liposome provided by the invention The transdermal absorption factor of -12 liposome of interleukin is high, can greatly promote the absorption and utilization of skin, more advantageous combining human interleukins Element -12 plays antiallergy, and enhancing is immune, repairs the effect of light injury.
Detailed description of the invention:
Fig. 1 is the transdermal absorption factor figure of rhIL-12(p70) liposome.
Specific embodiment
The present invention is further described below by way of specific embodiment, the present invention is not limited only to following embodiment.In this hair In bright range or the contents of the present invention are not being departed from, in spirit and scope, the change that carries out to the present invention is combined or replaced It changes, will be apparent to the person skilled in the art, and be included within the scope of the present invention.Makeup of the invention Product ingredient is conventional commercially available ingredient.
Embodiment 1, a kind of preparation method of rhIL-12(p70) liposome
The Chinese hamster ovary celI for stablizing expression rhIL-12 through 5L bioreactor fed-batch cultivation, is taken culture supernatant by S1 Liquid is chromatographed through weak cation exchange respectively, and heparin affinity chromatography separation obtains rhIL-12；
S2 takes soybean lecithin, cholesterol to be dissolved in chloroform, the soybean lecithin, cholesterol and chloroform Solid-to-liquid ratio is 4g:1g:200ml, is ultrasonically treated 8~12min, and the ultrasound input power is 40%, obtains mixed liquor I；
Ether and chloroform are added into the mixed liquor I that step S2 is obtained by S3, and it is 6.8 that pH, which is added dropwise, and concentration is The PBS buffer solution of 0.02mol/L, the volume ratio of the ether, chloroform and PBS buffer solution are 3:1:2, in the step S2 The volume ratio of chloroform in chloroform and step S3 is 2:1, and to not stratified, the ultrasound input power is ultrasonic treatment 40%, obtain w/o type lotion；
The w/o type lotion that step S3 is obtained is evaporated under reduced pressure S4 under the conditions of 50 DEG C of waters bath with thermostatic control, obtains O/W type lotion；
The O/W type lotion that step S4 is obtained is carried out high-pressure homogeneous 15 under conditions of pressure is 10000~15000Pa by S5 ~25min, every 5min are 1 circulation, pass sequentially through 1.0um, 0.6um, 0.45um and 0.22um filter membrane and are filtered, and step is added The additive amount of rhIL-12 described in the rhIL-12 that rapid S1 is collected is 70-100ug/ml, then plus Entering mass concentration is 0.1% bovine serum albumin solution, the volume of the protein protectant and rhIL-12(p70) Than for 10:1, excipient mixing packing is added in 18~22min of incubation under conditions of 37 DEG C, and the excipient and recombined human are white The mass ratio of cytokine -12 is 2:1, and the excipient is (1.5~2) by trehalose and glyceroglycolipid in mass ratio: 1 group At, in -40 DEG C of pre-freeze 2h, be freeze-dried under vacuum-pumping conditions for 24 hours, in 4 DEG C save to get.
Embodiment 2, a kind of preparation method of rhIL-12(p70) liposome
The Chinese hamster ovary celI for stablizing expression rhIL-12 through 5L bioreactor fed-batch cultivation, is taken culture supernatant by S1 Liquid is chromatographed through weak cation exchange respectively, and heparin affinity chromatography separation obtains rhIL-12；
S2 takes 1.0g soybean lecithin, 0.25g cholesterol to be dissolved in 50ml chloroform, is ultrasonically treated 10min, described super Vocal input power is 40%, obtains mixed liquor I；
75ml ether and 25ml chloroform are added into the mixed liquor I that step S2 is obtained by S3, and it is 6.8 that pH, which is added dropwise, concentration For the PBS buffer solution of 0.02mol/L, for ultrasonic treatment to not stratified, the ultrasound input power is 40%, obtains w/o type lotion；
The w/o type lotion that step S3 is obtained is evaporated under reduced pressure S4 under the conditions of 50 DEG C of waters bath with thermostatic control, obtains O/W type lotion；
The O/W type lotion that S5 obtains step S4 is that 12000Pa carries out high-pressure homogeneous 20min in pressure, and every 5min is 1 Circulation, passes sequentially through 1.0um, 0.6um, 0.45um and 0.22um filter membrane and is filtered, and the recombination that step S1 is collected is added Human Interleukin-12, the additive amount of the rhIL-12 are 800ug/ml, and being subsequently added into mass concentration is 0.1% ox blood The volume ratio of pure protein solution, the protein protectant and rhIL-12(p70) is 10:1, in 37 DEG C of condition Lower incubation 20min is added excipient mixing and dispenses, and the mass ratio of the excipient and rhIL-12(p70) is 2:1, The excipient is made of in mass ratio for 1.8:1 trehalose with glyceroglycolipid, in -40 DEG C of pre-freeze 2h, under vacuum-pumping conditions Freeze-drying for 24 hours, in 4 DEG C save to get.
Embodiment 3, a kind of skin cream containing rhIL-12(p70) liposome
The formula of the skin cream is as shown in table 1:
A kind of skin cream containing rhIL-12(p70) liposome of table 1
By the formula of table 1: respectively by A phase and B heat phase to 80 DEG C, in the case where starting homogeneous stirring, A being added to B phase In, after emulsification uniformly, C phase is added after reducing temperature, is cooled to 40 DEG C of addition D phases, discharges after mixing evenly.
Embodiment 4, a kind of facial mask liquid containing rhIL-12(p70) liposome
The formula of the facial mask liquid is as shown in table 2:
A kind of facial mask liquid containing rhIL-12(p70) liposome of table 2
By the formula of table 2: being first pre-mixed B phase, be heated to 80 DEG C, adjust pH with triethanolamine, be gradually added preheating A phase constituent, stir evenly, after cooling, gradually add C phase constituent, stir evenly.
The stability test of test example one, rhIL-12(p70) liposome
1, test material:
The rhIL-12(p70) liposome that embodiment 2 is prepared.
2, test method:
2.1, study on the stability:
The recombinant human interleukin 12 liposome that embodiment 2 is prepared is dissolved in water, by rhIL-12 aqueous solution in temperature It spends at 4,25,30,40 DEG C, closed placement under the conditions of relative humidity 75%.0 after placement, 1,2, March.It is surveyed respectively with Elisa method The content of recombinant human interleukin-12 in liposome and aqueous solution is determined, with combining human interleukins in liposome at 0 month and aqueous solution Element -12 is 100%, other each time rhIL-12(p70) contents are made comparisons therewith, obtain combining human interleukins Plain -12 contents change over time percentage.
Detailed process is as follows for sandwich ELISA method:
(1) Human IL-12 p70 Antibo is diluted with coating buffer (carbonate buffer solution of pH9.6,0.1M) (CatalogNumber:MAB611-500) is used for coated antibody to 2 μ g/ml, is added in 96 hole elisa Plates with 100 holes μ l/, 4 DEG C Overnight.
(2) detain dry coating buffer, with PBST (phosphate buffer+polysorbas20 of pH7.4,0.1M, polysorbas20 it is final concentration of Percent by volume 0.05%) it is added in 96 hole elisa Plates with 300 holes μ l/, board-washing 1 time, each 3min.
(3) by the standard items diluted (Recombinant Human IL-12 standard items be purchased from R&D company, article No.: 219-IL) with 2 times of gradient dilutions of the PBST of the 1%BSA containing mass volume ratio, dilution range 0.12ng/L-7.8ng/L；It is to be measured Sample dilutes 10000 times with the PBST of the 1%BSA containing mass volume ratio, is added in plate with 100 holes μ l/, and 37 DEG C are placed 1 hour.
(4) with PBST cleaning solution with 300 hole μ l/ board-washing 3 times, each 3min.
(5) with the secondary antibody diluent of the 5%BSA containing mass volume ratio (2g BSA+40ml PBST) by biotin labeling Human IL-12Biotinylated Antibody (Catalog#BAF219) is diluted to 0.1 μ g/ml, is added to 100 holes μ l/ In plate, 37 DEG C are placed 1 hour.
(6) dry microwell plate is detained, 1x PBST is added, 300 holes μ l/ stand 3min, wash 5 times.
(7) be added HRP label Streptavidin (1:1000 dilution: Solarbio | article No.: S9170), 100 holes μ l/, It is diluted with 1x PBS or 1xPBST.37 DEG C of incubation 45min.
(8) dry microwell plate is detained, washing lotion is added, 300 holes μ l/ stand 3min, wash 5 times.
(9) TMB chromogenic substrate (green skies P0209) is added, 100 holes μ l/ are protected from light, and are incubated at room temperature 5-30min.
(10) 20%H is added2SO4It terminates, 100 holes μ l/ are normal from indigo plant flavescence, please be light if greening or color change are uneven Sheet frame is hit in tapping, is mixed well.
(11) OD450, OD630 are detected in 30min, OD value=OD450-OD630 does standard curve by standard items, counts Calculate the content of rhIL-12 in sample.Each sample does 3 multiple holes when detection, is averaged.
2.2, the measurement of liposome encapsulation:
It is centrifuged using ultrafiltration, after liposome is separated with free drug, free drug is contained with indirect method Amount is measured.This test of separation method selection ultrafiltration of liposome is divided rhIL-12 liposome and free rhIL-12 From.
The pre-treatment of ultra-filtration centrifuge tube takes ultra-filtration centrifuge tube capacity for 4ml, molecular cut off 100KD, ultrapure at 4 DEG C Soaked overnight in water.Ultrapure water used when immersion is discarded, fresh ultrapure water is added in ultra-filtration centrifuge tube and is placed in high speed Washing is centrifuged in centrifuge, is centrifuged 15min in 3000rpm.It is discarded after centrifugation ultrapure in ultra-filtration centrifuge tube Simultaneously fresh ultrapure water is added in water, this process is repeated 3 times, spare.
The ultrafiltration of rhIL-12 liposome is centrifugated, and is taken the liposome of 300ul rhIL-12, is diluted to 3.0ml with PBS It is added in ultra-filtration centrifuge tube afterwards.It after sample stable equilibrium, is placed in supercentrifuge and is centrifuged, be centrifuged in 3000rpm 20min retains lower layer's ultrafiltrate, spare.
Penetration ration of the rhIL-12 to ultrafiltration membrane: precision measures the rhIL-12 reference solution that concentration is 50ug/ml and is Each 4 parts of 0.15ml, 0.50ml, 1.5ml, distilled water is added in ultra-filtration centrifuge tube after being diluted to 3.0ml.It is centrifuged in 3000rpm 20min retains lower layer's ultrafiltrate, is measured according to the above method to rhIL-12 content.
The amount of rhIL-12/addition rhIL-12 Zong Liang ╳ 100% in Penetration ration (%)=ultrafiltrate.
The rhIL-12 of various concentration is about (93.05 ± 5.33) % to the average Penetration ration of ultrafiltration membrane after measured, shows to put down It is the super of 100KD that the recombined human rhIL-12 protein molecular that equal relative molecular weight is 70KD, which most can penetrate molecule interception, Filter membrane.
2.3, the encapsulation rate of ultrafiltration measurement liposome:
Ultrafiltrate after taking liposome ultrafiltration to be centrifugated, measures the content (C trip) of rhIL-12, to dissociate rhIL-12's Content separately takes liposomal samples 300ul, and chloroform 900ul is added, and shake well is uniformly mixed, and is centrifuged in 12000rpm 20min takes supernatant and is diluted with distilled water, measures the content (C is total) of rhIL-12, the total content of rhIL-12, by following public affairs Formula calculates the encapsulation rate of liposome:
Encapsulation rate (EN)=(total-C trip of C)/C Zong ╳ 100%
Physical stability, this test are measured physical stability parameter using centrifugation-spectrophotometry.Precision is inhaled RhIL-12 liposomal samples 1mL is taken, 3500rpm/min in centrifuge tube is set and is centrifuged 10min, liposome and centrifugation before taking centrifugation Each 0.3mL of upper liquid afterwards, respectively plus PBS (pH6.8) is diluted to 5.0mL, using PBS as blank, measures it at Yu Bochang 500nm Absorbance (absorbance value that liposome centrifugation front and back measures is respectively A0, A), computational stability parameter value.KE=(A0-A)/A0 × 100% value is smaller, illustrates that liposome is more stable.
2.4, Determination of biological activity:
The natural killer cells NK92mi cell of the people Chlorambucil patient applied flexibly is surveyed purchased from Chinese food Drug assay research institute: NK-92mi uses culture medium A lpha MEM supplemented with 12%fetal bovine Serum, 12%horse serum, 1%penicillin/streptomycin, 0.2mM inositol, 0.02mM folic Acid, and 0.1mM 2-mercaptoethanol are cultivated spare.
(1) NK92mi cell grows to logarithmic growth phase, and piping and druming is uniformly dispersed, and is resuspended in fresh complete medium, adjusts Whole cell density is to 1 × 106A cell/ml is inoculated with 96 porocyte culture plates.It is inoculated with 100 hole μ l/ of volume.
(2) 37 DEG C, 5%CO2, it is incubated overnight under conditions of saturated humidity.
(3) 4 times of gradient dilution standard items and sample are pressed with complete medium dilution.50 holes μ l/ are added in 96 orifice plates, and 37 DEG C It is incubated for culture.
(4) with the IFN-Y's in eBiosciences88-7317 kit method detection culture supernatant after 24-48 hours Content.
(5) 4 parametric methods are used, are mapped with the logarithm of IL-12 concentration to sample light absorption value, according to the ED50 of each test sample (test sample concentration when i.e. IFN-Y content is maximum concentration half), is calculated as follows the potency of sample to be tested.
The ED50 of sample to be tested potency (IU/MG)=standard items potency X standard items ED50/ test specimen
The above-mentioned rhIL-12 liposome prepared by embodiment 2 is placed at 4,25,30,40 DEG C of temperature respectively, is relatively wet Closed placement under the conditions of degree 75%.0 after placement, investigate within 1,3,5,10,30,60,90 days its encapsulation rate, physical stability, activity.
3, test result:
3.1, study on the stability result: the result shows that, under the conditions of 40 DEG C of temperature, relative humidity 75%, place 3 months, Changes of contents of the recombinant human interleukin 12 in liposome complex is little, and in recombinant human interleukin 12 aqueous solution Middle content reduces, it was demonstrated that after liposomal encapsulated, hence it is evident that improve the stability of recombinant human interleukin 12.
3.2, the measurement result of liposome encapsulation it is as shown in table 3.
3.3, the measurement result of the encapsulation rate of liposome is as shown in table 4.
3.4, the results are shown in Table 5 for Determination of biological activity.
The encapsulation rate (%) of rhIL-12 liposome is investigated under 3 different condition of table
0 day 1 day 3 days 5 days 10 days 30 days 60 days 90 days
4℃ 85.20 84.00 81.45 78.25 78.93 74.65 69.25 64.07
25℃ 85.20 85.15 78.83 74.85 73.22 64.76 57.38 45.45
30℃ 85.20 84.25 76.05 69.83 63.22 41.35 29.95 21.88
40℃ 85.20 85.05 72.35 66.92 60.13 37.55 23.15 18.76
The physical stability of rhIL-12 liposome investigates (%) under 4 different condition of table
0 day 1 day 3 days 5 days 10 days 30 days 60 days 90 days
4℃ 1.08 1.55 1.52 2.34 2.61 3.88 3.56 3.68
25℃ 1.08 1.23 1.13 1.46 1.53 2.01 6.12 6.68
30℃ 1.08 1.43 2.65 4.58 11.88 24.33 32.66 39.35
40℃ 1.08 2.05 3.55 9.98 10.76 25.12 35.02 43.12
The biological activity (× 10 of rhIL-12 liposome under 5 different condition of table5IU/ml)
0 day 1 day 3 days 5 days 10 days 30 days 60 days 90 days
4℃ 4.22 4.05 3.94 4.11 3.78 3.58 3.88 3.78
25℃ 4.22 4.68 3.78 3.98 3.84 3.35 3.12 3.05
30℃ 4.22 3.45 3.23 2.89 2.55 1.88 1.63 1.14
40℃ 4.22 3.95 3.22 2.65 2.73 1.18 1.26 0.98
The percutaneous absorbability of test example two, rhIL-12(p70) liposome
1, test material:
The rhIL-12(p70) liposome that embodiment 2 is prepared.
2, test method:
2.1, prepared by skin: 12 Wistar female mices, and mouse is broken after neck puts to death, belly wool is cut off, separates abdomen skin Skin removes subcutaneous fascia and fat, puts -20 DEG C of ice of people after being wrapped up with aluminium foil until without muddiness with physiological saline repeated flushing Case refrigeration is spare.
2.2, Transdermal absorption measures: same mouse mouse skin being cut into 2 pieces of suitable size, infuses human physiology salt in receiving chamber Water receiving liquid 6.87mL, mouse skin is fixed between the diffuser casing and receiving chamber of skin permeation test in vitro device, and stratum corneum side is to expansion Room is dissipated, liquid level is contacted with skin inner layer completely, is respectively charged into the combining human interleukins of equivalent in two diffusion cells respectively The rhIL-12(p70) liposome that plain 12 aqueous solutions and embodiment 2 are prepared, sample-adding start to count after balancing 30min When, electromagnetic constant-temperature blender is opened, 37 DEG C ± 2 DEG C of temperature, the stirring of 100r/min constant speed takes in reception tank respectively at 1-12h Sample 0.3mL, while adding 0.3mL physiological saline and draining bubble.It is saturating that different time in two reception tanks is measured using ELISA method Protein content in skin liquid calculates skin permeation rate；Using relative molecular weight in high performance liquid chromatography measurement each sample.
The calculating of skin permeation rate: skin permeation rate=(CnVn+ Σ CkVk)/m × 100%
In formula: the protein concentration (mg/mL) that n-th of sample point of Cn-measures；All samplings before n-th of sample point of Ck- The corresponding protein concentration (mg/mL) measured of point；Ck-receiving liquid volume (mL)；All sample point phases before n-th of sample point Corresponding sample volume (mL)；The quality (mg) of protein in m-raw sample.
3, test result:
Test result is as shown in Figure 1.
The prevention and repair of test example three, the skin care item of liposome containing rhIL-12(p70) to light injury
1, test material:
Skin cream containing rhIL-12(p70) liposome made from embodiment 3.
2, test method:
Volunteer men and women each 24 for choosing health, are randomly divided into 3 groups, respectively control group, comparative example group and examination Test group, in which: control group is physiological saline group, and comparative example group is by the recombinant human interleukin-in the skin cream of embodiment 3 12 liposomes replace with skin cream made from rhIL-12(p70), and test group is to contain recombined human made from embodiment 3 The skin cream of interleukin 12 liposome.Subject takes position of leaning forward, and takes back normal skin region, and cleaning skin is dried, Three regions, each region 4cm × 4cm are divided into, luminous intensity is tested in xenon lamp preheating after five minutes, and light source generates 280~400nM Ultraviolet light, after optical filter, light source output is stablized, and light is uniform.Physiological saline, the comparative example of control group are drawn with cotton swab The skin cream about 1g of group and test group is applied to respectively in corresponding region, is spontaneously dried, and after half an hour, subject receives artificial mould Quasi- solar attachment Solar Light 601 irradiates, exposure dose 80mJ.cm-2.Examination is observed after 3 times repeatedly, 24 hours and 72 hours Test result.
3, test result:
After 24 hours, the erythema rate in control group region is 76.2%, and the erythema rate in comparative example group region is 50.4%, test The erythema rate in group region is 28.5%, and erythema is light.After 72 hours, part subject's control group region and comparative example group region The appearance desquamation of skin, and the intact no furfur phenomenon of test group area skin.

Claims (10)

1. a kind of preparation method of rhIL-12(p70) liposome, which comprises the following steps:
The Chinese hamster ovary celI for stablizing expression rhIL-12 is carried out fed-batch cultivation through bioreactor by S1, takes culture supernatant It is chromatographed respectively through weak cation exchange, heparin affinity chromatography separation obtains rhIL-12；
S2 takes soybean lecithin, cholesterol to be dissolved in chloroform, is ultrasonically treated 8~12min, obtains mixed liquor I；
Ether and chloroform are added into the mixed liquor I that step S2 is obtained by S3, and pH is added dropwise for 6.8 PBS buffer solution, at ultrasound Reason obtains w/o type lotion to not stratified；
The w/o type lotion that step S3 is obtained is evaporated under reduced pressure S4 under the conditions of 50 DEG C of waters bath with thermostatic control, obtains O/W type lotion；
The O/W type lotion that S5 obtains step S4 carries out high-pressure homogeneous 15~25min, and every 5min is 1 circulation, filters, is added The additive amount of rhIL-12 described in the rhIL-12 that step S1 is collected is 70-100ug/ml, then Protein protectant is added, 18~22min is incubated under conditions of 37 DEG C, excipient mixing packing is added, in -40 DEG C of pre-freezes 2h, be freeze-dried under conditions of vacuumizing for 24 hours, in 4 DEG C save to get.
2. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step The solid-to-liquid ratio of soybean lecithin, cholesterol and chloroform is 4g:1g:200ml in rapid S2.
3. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step The volume ratio of ether, chloroform and PBS buffer solution is 3:1:2 in rapid S3.
4. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step The volume ratio of chloroform and the chloroform in step S3 is 2:1 in rapid S2.
5. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step The concentration of PBS buffer solution is 0.02mol/L in rapid S3.
6. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step The pressure of rapid S5 mesohigh homogeneous is 10000~15000Pa.
7. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step Filtering in rapid S5 is to pass sequentially through 1.0um, 0.6um, 0.45um and 0.22um filter membrane to be filtered.
8. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step Protein protectant in rapid S5 is the bovine serum albumin solution that mass concentration is 0.1%.
9. the preparation method of rhIL-12(p70) liposome as described in claim 1, which is characterized in that the step Suddenly the excipient in S5 is (1.5~2) by trehalose and glyceroglycolipid in mass ratio: 1 forms.
10. the preparation method of the rhIL-12(p70) liposome as described in claim 1~9 is any is prepared RhIL-12(p70) liposome is preparing the purposes in skin care item.
CN201910570541.0A 2019-06-27 2019-06-27 The preparation method of rhIL-12(p70) liposome and products thereof purposes Pending CN110251412A (en)

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Application publication date: 20190920