CN106880512A - People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications and its preparation and application - Google Patents
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications and its preparation and application Download PDFInfo
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Abstract
The invention discloses a kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications, by mass percentage including following components：People's umbilical cord MSC serum-free medium activated proteins composition 5~12%, shitosan 20~65%, sodium tripolyphosphate 1~15%, hyaluronic acid 20~65%, water≤5%.Its preparation method is, the stem cell serum-free culture supernatant with various active cell factor is coated using CS nanoparticles preparation technology first, and then it is lyophilized, then make the active amino on CS nanoparticles surface that covalent coupling reaction occur with the carboxyl on HA surfaces, product of the present invention is obtained.After the product freeze-dried powder aquation, it is directly added into cosmetics, crease-resistant moisturizing can be played and delay cell senescence to act on, the product has long quality guarantee period.
Description
Technical field
The invention belongs to stem cell medium cosmetic field, it is related to a kind of people's umbilical cord MSC free serum cultures of HA modifications
Liquid CS nano-granule freeze-dried powders, have further related to the preparation method of the freeze-dried powder, and its application in cosmetics.
Background technology
Stem cell beauty is earliest the application in plastic surgery, and the more stem cell species applied in plastic surgery
It is mescenchymal stem cell, epidermal stem cells, endothelial progenitor cell and PECTORAL LIMB SKELETON etc..
Traditionally so-called stem cell cosmetology, typically all direct injection stem cell injection.And this injection is dry thin
What the source of born of the same parents was mainly extracted from the embryo of miscarriage, so immunological rejection can be caused unavoidably, and exist certain
Ethics problem.Even with autologous stem cells, but exogenous albumen can be introduced in the culture amplification procedure of stem cell
(hyclone), easily causes immunological rejection.
Human mesenchymal stem cell (mesenchymal stem cell, MSC) secretes basic fibroblast life in culture
The factor (bFGF) long, hEGF (EGF), VEGF (VEGF), insulin-like growth factor-i
And the growth factor that is regenerated in interior various promotion Skin Cells of HGF (HGF) (IGF-1).These have biology
The growth factor of activity can be effectively promoted the metabolism of Skin Cell；Promote extracellular hyaluronic acid, glycoprotein etc. big
The synthesis and secretion of molecule, strengthen the hydrophily of skin；Promote growth, differentiation and the reparation of epidermal cell, make aging death
Cell is able to supplement in time；Cuticula reparation is can speed up simultaneously, accelerates the new old cell of substrate to substitute, skin is returned to young appearance
State.
The human mesenchymal stem cell culture supernatant of liquid is very short in the normal temperature holding time, Porcine HGF easy in inactivation, i.e.,
Make to be to remove moisture by Freeze Drying Technique to make freeze-dried powder, it is also difficult to be directly appended in the cosmetics of various formulations, because
This greatly limit its application in cosmetic field.
The content of the invention
First purpose of the invention is to provide a kind of people umbilical cord MSC serum-free medium CS nanoparticles of HA modifications and freezes
Powder, solves liquid limited this bottleneck of stem cell culture supernatant storage life, while solving cell growth in stem cell culture supernatant
Factor active preserves problem.
Second object of the present invention is to provide above-mentioned people's umbilical cord MSC serum-free cell culture medium CS nano-granule freeze-dried powders
Preparation method.
Third object of the present invention provides answering for above-mentioned people's umbilical cord MSC serum-free cell culture medium CS nano-granule freeze-dried powders
With solving the problems, such as that existing beauty employment umbilical cord MSC nutrient solutions are difficult to be directly added into cosmetics.
First technical scheme of the present invention is that a kind of people's umbilical cord MSC serum-free mediums CS of HA modifications receives
Grain of rice freeze-dried powder, by mass percentage including following components：People's umbilical cord MSC serum-free medium activated proteins composition 5~12%,
CS (shitosan) 20~65%, TPP (sodium tripolyphosphate) 1~15%, HA (hyaluronic acid) 20~65%, water≤5%, the above is each
The quality summation of component is 100%.
Second technical scheme of the present invention is that people's umbilical cord MSC serum-free mediums CS of above-mentioned HA modifications receives
The preparation method of grain of rice freeze-dried powder, comprises the following steps：
Step 1, prepares load people's umbilical cord MSC serum-free medium CS nanoparticles；
During people umbilical cord MSC serum-free mediums are added into CS acid solutions, and TPP is added, room temperature reaction.Received reacted
Grain of rice suspension is centrifuged, gained washing of precipitate, freeze-drying, obtains loading people's umbilical cord MSC serum-free medium CS nanoparticles.
Step 2, prepares load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications；
Carboxyl in HA is activated, load people's umbilical cord MSC serum-free medium CS nanoparticles are then added to work
Reacted in HA after change, take reacted nanosuspension, be centrifuged, gained washing of precipitate simultaneously freeze-drying obtains final product HA modifications
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders.
Wherein, the preparation method of people's umbilical cord MSC serum-free mediums is in step 1：Neonatal umbilical cord is taken, by intravenous blood
Mark washes clean；Removal epidermis, two arteries and a vein blood vessel, tissue is shredded, and adds the DMEM/F12 containing FBS
Medium culture, obtains primary human umbilical cord mesenchymal stem cells；When cell fusion degree is 80%-90%, Trypsin Induced
Passage culture, takes passage cell, continues to cultivate, and as cell fusion degree 80%-90%, abandons the culture supernatant containing FBS,
PBS washed cells, add the DMEM/F12 culture mediums of serum-free to continue to cultivate 72 hours, collect serum-free medium.
Shitosan (CS) is called chitosan, soluble chitin and poly glucosamine etc., and its chemical entitled β-
(1-4) -2-amino-2-deoxy-D-Glucose, is product obtained from deacetylate after the treatment of chitin concentrated base.Shell gathers
Sugar is rare alkaline polysaccharide in natural polymer, and water insoluble and organic solvent dissolves in the diluted acid of pH ＜ 6.5, in diluted acid
In, its Glucoamino is converted into R-NH3 +, form polycation gel solution.Shitosan have excellent biodegradability,
Low toxicity, good biocompatibility, therefore it is highly suitable for biological medicine and cosmetic industry.
The present invention prepares CS nanoparticles using ionic cross-linking, using the sodium tripolyphosphate (TPP) having no adverse reaction to CS
Carry out ion induction gelation and form nanoparticle.Under agitation, TPP solution is added in the acid solution of CS, by negatively charged
Phosphate anion and CS strands on the protonated amino of positively charged there is intramolecular and intermolecular cross-linking gelation, just can be fast
Fast-growing is into nanoparticle.
The macromolecule polysaccharide that hyaluronic acid (HA) is made up of D-Glucose aldehydic acid and N-acetyl-glucosamine, is optimal guarantor
One of wet composition.Under certain pH value condition, hyaluronic acid is negatively charged, and CS nanoparticles are positively charged, between the two can
Enough attracted each other by electrostatic force, make CS nanoparticles that there is good moisture-keeping function.
The features of the present invention is also resided in：
Preferably, the mass ratio of activated protein is 2 in CS and people's umbilical cord MSC serum-free mediums in step 1:1~10:1,
The mass ratio of CS and TPP is 2:1~5:1, CS molecular weight is 2-30 ten thousand.
Preferably, in CS acid solutions described in step 1 be used for dissolve CS acid be formic acid, acetic acid, propionic acid, lactic acid, malic acid,
Citric acid, ascorbic acid, oxalic acid, succinic acid, malonic acid, aliphatic acid, pyruvic acid, glutaric acid, tartaric acid, aspartic acid, bigcatkin willow
Acid or glycolic in one or more.
Preferably, parameter of noncentricity is in step 1：2~8 DEG C of centrifuging temperature, 10~60min of centrifugation time, centrifugal force 1.0 ×
103~1.0 × 105g。
Preferably, the molecular weight of HA is CS nanometers of 8-30Kda, HA and load people's umbilical cord MSC serum-free mediums in step 2
The mass ratio of grain is 1:1~5:1.
Preferably, the activated carboxylic method of HA is in step 2, sub- using 1- ethyls -3- (3- dimethylamino-propyls)-carbon two
Amine (EDC) and N-hydroxy-succinamide (NHS) are activated to the carboxyl in HA, then adjust pH to 7.5,37 DEG C of incubations.
Preferably, freeze-drying parameter is in step 1 and step 2：± 2 DEG C of -60 DEG C of pre-freezing temperature, pre-freeze time 6h with
On, cryogenic temperature -50~-56 DEG C, vacuum≤80mT, freeze-drying time 24-36h.
3rd technical scheme of the present invention be, above-mentioned people's umbilical cord MSC serum-free cell culture medium CS nanoparticles
Application of the freeze-dried powder in cosmetics.When preparing cosmetics, CS nanometers of people's umbilical cord MSC serum-free mediums that above-mentioned HA is modified
After grain freeze-dried powder aquation, it is directly added into cosmetics.The freeze-dried powder can directly make an addition to common emulsion, creams, Essence, solidifying
The cosmetics of various formulations such as glue.
The beneficial effects of the invention are as follows：
(1) the stem cell culture supernatant used by the present invention is free serum culture supernatant, reduces stem cell for beauty
Present in immunological rejection.
(2) CS nanoparticles preparation technology is used by the stem cell serum-free culture supernatant with various active cell factor
Cladding, so it is lyophilized, and the shelf-life is 3 years, far above the shelf-life of existing human mesenchymal stem cell, solves supernatant
The short problem of storage life.
(3) with the carboxyl on HA surfaces there is covalent coupling reaction in the active amino on CS nanoparticles surface, and HA modifications CS is obtained
Nanoparticle, so not only solves the problem of CS nanoparticles stability difference, while it is efficient to introduce cosmetics in CS nanoparticles
NMF HA.
(4) CS used by the present invention can drop as a kind of natural alkaline polysaccharide with good biocompatibility and biology
Xie Xing, security is higher, with low cost, and nanoparticle is prepared using CS, and reaction condition is gentle, process is simple, low cost.
(5) the stem cell serum-free culture supernatant after being coated through CS nanoparticles can directly make an addition to emulsion, creams, elite
The cosmetics of various formulations such as liquid, gel, have widened its application in cosmetic field.
Brief description of the drawings
Fig. 1 is the preparation method flow of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications of the present invention
Figure；
Fig. 2 MSC-CS-NPs and HA-MSC-CS-NPs 48h Flow cytometry results；
Fig. 3 MSC-CS-NPs and HA-MSC-CS-NPs 72h Flow cytometry results；
Fig. 4 MSC-CS-NPs and HA-MSC-CS-NPs 48h and 72h normal live cells numbers account for TCS percentage；
Fig. 5 MSC-CS-NPs and HA-MSC-CS-NPs 48h and 72h viable apoptotic cell numbers account for TCS percentage；
Fig. 6 Rz and Ra result (comparing of product zone, check plot and initial value) compare figure.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description, but the present invention is not limited to
These implementation methods.
Embodiment 1
Prepare a kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modification, reference picture 1, its method is such as
Under：
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium
Human umbilical cord mesenchymal stem cells are extracted：Under aseptic condition, 5~10cm of neonatal umbilical cord is taken, put umbilical cord preserving fluid, 4
DEG C storage (time be no more than 8 hours).Rinsed with D-Hanks liquid until intravenous without bloodstain.Removal epidermis, two arteries
And a vein blood vessel, tissue shear is broken to about 1mm3/ block, adds DMEM culture mediums (containing the dual anti-) culture containing 10%FBS
In about one week, condition of culture is 37 DEG C, saturated humidity, 5%CO2, you can obtain primary human umbilical cord mesenchymal stem cells.
Collect serum-free medium：During cell fusion degree 80%, trypsin digestion and cell Secondary Culture takes the thin of 6 generations
Born of the same parents continue to cultivate.When cell fusion degree 80%, the culture supernatant containing 10%FBS is abandoned, PBS (pH=7.0) washed cell three times,
Add the DMEM culture mediums of serum-free to continue to cultivate 72 hours, collect serum-free medium.
Step (2), ionic cross-linking prepares load people's umbilical cord MSC serum-free medium CS nanoparticles
The CS powder 25mg that molecular weight is 20,000 are weighed, is dissolved in the 25mL acetums that mass fraction is 1%, be made into matter
Amount fraction is 0.1% CS solution, and then ultrasound removing bubble crosses 0.45 μm of water system filter membrane, obtain the bright CS solution of clarification.
2.5mL people's umbilical cord MSC serum-free mediums that activated protein concentration is 998 μ g/mL are slowly dropped in CS solution, are stirred
10min, obtains mixed liquor.During 0.2% TPP solution 5mL dropped into above-mentioned mixed liquor, room temperature reaction 30min.Wherein CS with
The mass ratio of TPP is 2.5:1, CS is 10 with the mass ratio of activated protein in people's umbilical cord MSC serum-free mediums:1.
By reacted nanoparticle suspension high speed centrifugation, 4 DEG C of centrifuging temperature, centrifugation time 10min, centrifugal force 1.0 ×
105g.Then centrifugation gained precipitation carries out freeze-drying with water washing is distilled 3 times, and freeze-drying parameter is：Pre-freezing temperature -60
DEG C ± 2 DEG C, more than pre-freeze time 6h, cryogenic temperature -50~-56 DEG C, vacuum≤80mT, freeze-drying time 24h.After freeze-drying
People's umbilical cord MSC serum-free medium CS nanoparticles must be loaded.
Step (3), HA activation；
It is the HA aqueous solution 50mL that the HA concentration of 8KDa is 0.05% to prepare molecular weight, adds 1- ethyls -3- (3- diformazan ammonia
Base propyl group)-carbodiimide (EDC) 137.5mg, N-hydroxy-succinamide (NHS) 165mg is added after it is completely dissolved,
It is 7.5,37 DEG C of incubation 1h to be adjusted to pH with 0.1M NaOH.
Step (4), prepares load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications
After load people's umbilical cord MSC serum-free medium CS nanoparticles that step (2) is obtained are added to step (3) activation
In HA, the mass ratio that feeds intake of HA and CS nanoparticles is 1:1.Be stirred at room temperature reaction 2h, take reacted nanosuspension, at a high speed from
The heart, parameter of noncentricity is：4 DEG C of centrifuging temperature, centrifugation time 30min, centrifugal force 1.0 × 104g.Gained precipitation distillation water washing
10 times, freeze-drying is then carried out, freeze-drying parameter is：± 2 DEG C of -60 DEG C of pre-freezing temperature, more than pre-freeze time 6h, freezing temperature
- 50~-56 DEG C of degree, vacuum≤80mT, freeze-drying time 24h.The people umbilical cord MSC serum-frees training of HA modifications is obtained final product after freeze-drying
Nutrient solution CS nano-granule freeze-dried powders.
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of the HA modifications that the present embodiment is obtained are clear by determining
Wash in CS nanoparticle liquid and the content of TPP determines the content of the TPP being crosslinked in centrifuged supernatant, detection method uses light splitting
Photometry (GB/T13171-1997), the inventory of TPP is 10mg, and that be crosslinked is 6.4mg.Obtained by the Percentage bound for detecting HA
Know, the Percentage bound of HA and CS nanoparticles is 36.0%, wherein, human umbilical cord mesenchymal stem cells serum-free medium activated protein into
Divide 5.5%CS55.2%, TPP14.1%, HA21.9%, water 3.3%.
Embodiment 2
A kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications are prepared, its method is as follows：
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium, and specific method is with embodiment 1.It is wherein different
Part is that cell fusion degree is 85% when collecting serum-free medium.
Step (2), ionic cross-linking prepares load people's umbilical cord MSC serum-free medium CS nanoparticles
The CS powder 20mg that molecular weight is 200,000 are weighed, is dissolved in the 10mL citric acid solutions that mass fraction is 4.0%, matched somebody with somebody
Into the CS solution that mass fraction is 0.2%, then ultrasound removing bubble crosses 0.45 μm of water system filter membrane, obtains the bright CS of clarification
Solution.9.9mL people's umbilical cord MSC serum-free mediums that activated protein concentration is 1015 μ g/mL are slowly dropped in CS solution,
Stirring 20min, obtains mixed liquor.During 0.2% TPP solution 2.5mL dropped into above-mentioned mixed liquor, room temperature reaction 30min.Wherein
The mass ratio of CS and TPP is 4:1, CS is 2 with the mass ratio of activated protein in people's umbilical cord MSC serum-free mediums:1.
By reacted nanoparticle suspension high speed centrifugation, 4 DEG C of centrifuging temperature, centrifugation time 30min, centrifugal force 1.0 ×
104g.Then centrifugation gained precipitation carries out freeze-drying with water washing is distilled 5 times, and freeze-drying parameter is：Pre-freezing temperature -60
DEG C ± 2 DEG C, more than pre-freeze time 6h, cryogenic temperature -50~-56 DEG C, vacuum≤80mT, freeze-drying time 30h.After freeze-drying
People's umbilical cord MSC serum-free medium CS nanoparticles must be loaded.
Step (3), HA activation；
It is the HA aqueous solution 100mL that 15KDaHA concentration is 0.14% to prepare molecular weight, adds 1- ethyls -3- (3- diformazan ammonia
Base propyl group)-carbodiimide (EDC) 700mg, N-hydroxy-succinamide (NHS) 840mg is added after it is completely dissolved, use
It is 7.5,37 DEG C of incubation 5h that 0.1M NaOH is adjusted to pH.
Step (4), prepares load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications
After load people's umbilical cord MSC serum-free medium CS nanoparticles that step (2) is obtained are added to step (3) activation
In HA, the rate of charge of HA and CS nanoparticles is 4.7:1.Be stirred at room temperature reaction 10h, take reacted nanosuspension, at a high speed from
The heart, parameter of noncentricity is：4 DEG C of centrifuging temperature, centrifugation time 60min, centrifugal force 1.0 × 103g.Gained precipitation distillation water washing 3
It is secondary, freeze-drying is then carried out, freeze-drying parameter is：± 2 DEG C of -60 DEG C of pre-freezing temperature, more than pre-freeze time 6h, freezing temperature
- 50~-56 DEG C of degree, vacuum≤80mT, freeze-drying time 30h.The people umbilical cord MSC serum-frees training of HA modifications is obtained final product after freeze-drying
Nutrient solution CS nano-granule freeze-dried powders.
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of the HA modifications that the present embodiment is obtained are clear by determining
Wash in CS nanoparticle liquid and the content of TPP determines the content of the TPP being crosslinked in centrifuged supernatant, detection method uses light splitting
Photometry (GB/T13171-1997), the inventory of TPP is 5mg, and that be crosslinked is 3.3mg.Detect that the Percentage bound of HA is learnt, HA
It is 42.3% with the Percentage bound of CS nanoparticles, wherein, human umbilical cord mesenchymal stem cells serum-free medium activated protein composition
10.8%, CS 21.3%, TPP3.5%, HA63.2%, water 1.3%.
Embodiment 3
A kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications are prepared, its method is as follows：
Step (1), prepares human umbilical cord mesenchymal stem cells serum-free medium, and specific method is with embodiment 1.It is wherein different
Part is that cell fusion degree is 90% when collecting serum-free medium.
Step (2), ionic cross-linking prepares load people's umbilical cord MSC serum-free medium CS nanoparticles
The CS powder 50mg that molecular weight is 300,000 are weighed, is dissolved in the 10mL tartaric acid solutions that mass fraction is 0.3%, matched somebody with somebody
Into the CS solution that mass fraction is 0.5%, then ultrasound removing bubble crosses 0.45 μm of water system filter membrane, obtains the bright CS of clarification
Solution.10.5mL people's umbilical cord MSC serum-free mediums that activated protein concentration is 950 μ g/mL are slowly dropped in CS solution,
Stirring 30min, obtains mixed liquor.During 0.18% TPP solution 5.5mL dropped into above-mentioned mixed liquor, room temperature reaction 30min.Its
The mass ratio of middle CS and TPP is 5:1, CS is 5 with the mass ratio of activated protein in people's umbilical cord MSC serum-free mediums:1.
By reacted nanoparticle suspension high speed centrifugation, 4 DEG C of centrifuging temperature, centrifugation time 60min, centrifugal force 1.0 ×
103g.Then centrifugation gained precipitation carries out freeze-drying with water washing is distilled 10 times, and freeze-drying parameter is：Pre-freezing temperature -60
DEG C ± 2 DEG C, more than pre-freeze time 6h, cryogenic temperature -50~-56 DEG C, vacuum≤80mT, freeze-drying time 36h.After freeze-drying
People's umbilical cord MSC serum-free medium CS nanoparticles must be loaded.
Step (3), HA activation；
It is the HA aqueous solution 150mL that 30KDaHA concentration is 0.1% to prepare molecular weight, adds 1- ethyls -3- (3- diformazan ammonia
Base propyl group)-carbodiimide (EDC) 750mg, N-hydroxy-succinamide (NHS) 900mg is added after it is completely dissolved, use
It is 7.5,37 DEG C of incubation 10h that 0.1M NaOH is adjusted to pH.
Step (4), prepares load people's umbilical cord MSC serum-free medium chitosan nano freeze-dried powders of HA modifications
After load people's umbilical cord MSC serum-free medium CS nanoparticles that step (2) is obtained are added to step (3) activation
In HA, the rate of charge of HA and CS nanoparticles is 2.5:1.Be stirred at room temperature reaction 24h, take reacted nanosuspension, at a high speed from
The heart, parameter of noncentricity is：4 DEG C of centrifuging temperature, centrifugation time 10min, centrifugal force 1.0 × 105g.Gained precipitation distillation water washing 6
It is secondary, freeze-drying is then carried out, freeze-drying parameter is：± 2 DEG C of -60 DEG C of pre-freezing temperature, more than pre-freeze time 6h, freezing temperature
- 50~-56 DEG C of degree, vacuum≤80mT, freeze-drying time 36h.The people umbilical cord MSC serum-frees training of HA modifications is obtained final product after freeze-drying
Nutrient solution CS nano-granule freeze-dried powders.
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of the HA modifications that the present embodiment is obtained are clear by determining
Wash in CS nanoparticle liquid and the content of TPP determines the content of the TPP being crosslinked in centrifuged supernatant, detection method uses light splitting
Photometry (GB/T13171-1997), the inventory of TPP is 10mg, and that be crosslinked is 6.8mg.Detect that the Percentage bound of HA is learnt,
The Percentage bound of HA and CS nanoparticles is 28.6%, wherein, human umbilical cord mesenchymal stem cells serum-free medium activated protein composition
8.8%, CS44.1%, TPP6.0%, HA37.8%, water 3.3%.
According to the method described above, raw material components or adjusting process parameter are changed within the scope of the present invention, and HA modifications can be obtained
People's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders, and can make an addition to cosmetics.
Properties to people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA of the invention modifications are examined
Survey, sample sets are the HA-MSC-CS-NPs that embodiment 1 is prepared, control group uses the MSC-CS-NPs modified without HA,
Other technological parameters are identical with embodiment 1.
(1) particle diameter and particle diameter distribution
MSC-CS-NPs the and HA-MSC-CS-NPs particle diameter distributions of table 1 and PDI
| Group | Nanoparticle | Particle diameter distribution/nm | PDI |
| Control group | MSC-CS-NPs | 185.3±25.8 | 0.289±0.041 |
| Sample sets | HA-MSC-CS-NPs | 209.5±14.9 | 0.125±0.032 |
As it can be seen from table 1 the MSC-CS-NPs particle diameters slightly increase tendency after being modified through HA, but PDI indexes are obvious
Reduce (PDI is polydispersity index, and its size represents the width of particle diameter distribution), illustrate the particle diameter distribution ratio of HA-MSC-CS-NPs
Control group is more homogeneous, and system also tends to stabilization.
(2) flow cytometer investigates increasing of the people's umbilical cord MSC free serum culture CS nano-granule freeze-dried powders of HA modifications to cell
Grow power.
It is target cell to use l cell L929, contrasts control group --- people's umbilical cord MSC serum-free mediums CS
Nano-granule freeze-dried powder (MSC-CS-NPs), sample sets --- the people umbilical cord MSC serum-free medium CS nanoparticles of HA modifications are freezed
Powder (HA-MSC-CS-NPs) is in culture 48h and 72h, the influence to L929 cell proliferation activities.
As shown in Figure 2,3, on the scatter diagram of bivariate flow cytometer, left lower quadrant was represented for X-axis and Y-axis institute's generation
The parameter of table is normal live cells in the cell of double-negative reaction, and right lower quadrant is represented and is positive for the parameter representated by X-axis
Reaction, the cell negative for parameter representated by Y-axis is viable apoptotic cell, and right upper quadrant is represented for X-axis and Y
Parameter representated by axle is late apoptic and dead cell in the cell of double sun reactions, and left upper quadrant is represented for representated by Y-axis
Parameter be positive, the cell negative for parameter representated by X-axis is fragment and damaging cells.
As shown in Figure 4,5, compare with control group MSC-CS-NPs, HA-MSC-CS-NPs 48h after the addition, it is normal living thin
Born of the same parents' number is the 92.51% of sum, and control group is only 88.95%, and 3.56 percentage points are higher by than control group；And viable apoptotic cell
Number for sum 4.00%, control group is 8.30%, it is lower than control group go out 4.30 percentage points.
Compare with control group MSC-CS-NPs, HA-MSC-CS-NPs 72h after the addition, normal live cells number is sum
96.13%, control group is only 93.05%, and 3.08 percentage points are higher by than control group；And viable apoptotic cell number is sum
2.38%, control group is 3.87%, it is lower than control group go out 1.49 percentage points.
From this result can be seen that MSC-CS-NPs in introduce HA after, compared with unmodified nanoparticle, not only improve
Promotion cel l proliferation, while also having the effect for delaying Apoptosis.
(3) the anti-ageing reparation work(that the people umbilical cord MSC free serum culture CS nano-granule freeze-dried powders of HA modifications are used in cosmetics
Effect is evaluated.
People's umbilical cord MSC free serum culture CS nano-granule freeze-dried powders (HA-MSC-CS-NPs) that HA of the invention is modified is added
It is added in face cream, control group is entered using two face cream using the face cream without HA-MSC-CS-NPs to 48 women
Row measure of merit.The composition of HA-MSC-CS-NPs face cream is shown in Table 2.
The HA-MSC-CS-NPs face cream component lists of table 2
The preparation method of face cream is：, to after 80 DEG C, B phases are slowly added to A phases for A phases, B heat phases, stir 15min, add C
10min, homogeneous 5min (4000r/min) are stirred after phase, (slow, 1 DEG C/min of rate of temperature fall or so, stirs speed during cooling for cooling
Rate must not exceed 500r/min) to 35 DEG C of addition D phases, 5min is stirred, 30 DEG C of addition E phases are cooled to, go out after low rate mixing 5min
Material.
Study subject：The female volunteers of 48 ages without skin sensitivity history at 37~55 years old, volunteer during test
Random side face uses reference product --- and the substrate face cream without HA-MSC-CS-NPs is (except without HA-MSC-CS-
NPs, other compositions are identical with HA-MSC-CS-NPs face cream compositions), other side uses HA-MSC-CS-NPs skin cares
Frost, sooner or later each daily using once, continuous to use 8 weeks, volunteer is unusable except test product and reference product during test
Any skin care item in addition.In product using preceding, using 4 weeks and 8 weeks after, skin fast optical imaging is carried out to both sides canthus
(FOITS), and to picture it is analyzed, Rz represents skin mean roughness, Ra represents skin arithmetic average roughness.Statistics side
Method uses ANOVA, conspicuousness："+" represents significant difference (p≤0.05)；"-" represents non-limiting difference (p ＞ 0.05), knot
Fruit sees Fig. 6 and Biao 3.
Rz the and Ra statisticses of table 3
In the efficacy test, 48 subjects none have immunological rejection or sense of discomfort.
Found out by Fig. 6 and Biao 3, compared with initial value, using after 4 weeks, the Rz and Ra of product zone are reduced product respectively
6.2% and 11.1%, the Rz and Ra of check plot reduce 1.6% and 7.0% respectively, product using after 8 weeks, the Rz of product zone and
Ra reduces 9.6% and 20.6% respectively, and the Rz and Ra of check plot reduce 3.8% and 13.4% respectively, compare with initial value,
After product is used 4 weeks and 8 weeks, product area Rz (skin mean roughness) and Ra (skin arithmetic average roughness) have aobvious
Sex differernce, control zone Rz (skin mean roughness) and Ra (skin arithmetic average roughness) difference that there are no significant are write, is said
The bright face cream containing HA-MSC-CS-NPs has the effect for significantly improving skin roughness and texture, i.e., with anti-aging
Effect.
Claims (10)
1. people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders that a kind of HA is modified, it is characterised in that by mass percentage
Including following components：People's umbilical cord MSC serum-free medium activated proteins composition 5~12%, shitosan 20~65%, tripolyphosphate
Sodium 1~15%, hyaluronic acid 20~65%, water≤5%, the quality summation of above each component is 100%.
2. a kind of preparation of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications as claimed in claim 1
Method, it is characterised in that comprise the following steps：
Step 1, prepares load people's umbilical cord MSC serum-free medium CS nanoparticles；
During people umbilical cord MSC serum-free mediums are added into CS acid solutions, and TPP is added, room temperature reaction；By reacted nanoparticle
Suspension is centrifuged, gained washing of precipitate, freeze-drying, obtains loading people's umbilical cord MSC serum-free medium CS nanoparticles；
Step 2, prepares load people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications；
Carboxyl in HA is activated, after load people's umbilical cord MSC serum-free medium CS nanoparticles then are added into activation
HA in react, take reacted nanosuspension, be centrifuged, gained washing of precipitate and freeze-drying, obtain final product people's navel of HA modifications
Band MSC serum-free medium CS nano-granule freeze-dried powders.
3. the preparation side of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA according to claim 2 modification
Method, it is characterised in that the preparation method of the MSC serum-free mediums of people's umbilical cord described in step 1 is：Neonatal umbilical cord is taken, will be quiet
Bloodstain washes clean in arteries and veins；Removal epidermis, two arteries and a vein blood vessel, tissue is shredded, and is added containing FBS
DMEM/F12 medium cultures, obtain primary human umbilical cord mesenchymal stem cells；When cell fusion degree is 80%-90%, pancreas egg
White enzymic digestion passage culture, takes passage cell, continues to cultivate, and as cell fusion degree 80%-90%, abandons the training containing FBS
Supernatant is supported, PBS washed cells add the DMEM/F12 culture mediums of serum-free to continue to cultivate 72 hours, collect serum-free medium.
4. people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders that HA according to claim 2 is modified, its feature exists
In the mass ratio of activated protein is 2 in CS described in step 1 and people's umbilical cord MSC serum-free mediums:1~10:1, CS and TPP
Mass ratio be 2:1~5:1, CS molecular weight is 2-30 ten thousand.
5. the preparation side of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA according to claim 2 modification
Method, it is characterised in that parameter of noncentricity is described in step 1：2~8 DEG C of centrifuging temperature, 10~60min of centrifugation time, centrifugal force
1.0×103~1.0 × 105g。
6. the preparation side of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA according to claim 2 modification
Method, it is characterised in that the molecular weight of HA described in step 2 is that 8-30Kda, HA and load people's umbilical cord MSC serum-free mediums CS receive
The mass ratio of the grain of rice is 1:1~5:1.
7. the preparation side of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA according to claim 2 modification
Method, it is characterised in that the activated carboxylic method of HA described in step 2 is, using 1- ethyls -3- (3- dimethylamino-propyls)-carbon
Diimine (EDC) and N-hydroxy-succinamide (NHS) are activated to the carboxyl in HA, then adjust pH to 7.5, and 37 DEG C incubate
Educate.
8. the preparation side of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA according to claim 2 modification
Method, it is characterised in that freeze-drying parameter described in step 1 and step 2 is：± 2 DEG C of -60 DEG C of pre-freezing temperature, pre-freeze time
More than 6h, cryogenic temperature -50~-56 DEG C, vacuum≤80mT, freeze-drying time 24-36h.
9. the preparation side of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA according to claim 2 modification
Method, it is characterised in that in CS acid solutions described in step 1 be used for dissolve CS acid be formic acid, acetic acid, propionic acid, lactic acid, malic acid,
Citric acid, ascorbic acid, oxalic acid, succinic acid, malonic acid, aliphatic acid, pyruvic acid, glutaric acid, tartaric acid, aspartic acid, bigcatkin willow
Acid or glycolic in one or more.
10. a kind of people's umbilical cord MSC serum-free medium CS nano-granule freeze-dried powders of HA modifications as claimed in claim 1 are being made up
Application in product, it is characterised in that when preparing cosmetics, by the freeze-dried powder aquation after, be directly added into cosmetics.
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