CN110123842A - A kind of excretion body slow-releasing system and its construction method and application - Google Patents
A kind of excretion body slow-releasing system and its construction method and application Download PDFInfo
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Abstract
The present invention relates to a kind of excretion body system based on hyaluronic acid and memebrane protein CD44 combination realization CD44 positive excretion body sustained release and its construction method and applications.CD44 positive excretion body is effectively combined in the material constructed by hyaluronic acid by the present invention, and then realizes the permanent release of excretion body.Method provided by the invention can effectively solve the problems, such as excretion body, and residence time is short in vivo, improves the therapeutic efficiency of excretion body.
Description
Technical field
The invention belongs to field of biotechnology, more particularly, to a kind of excretion body slow-releasing system and its construction method with answer
With.
Background technique
Excretion body is to steep inclusion bodies inside eukaryocyte to be discharged into extracellular nanoscale with what cell membrane fusion was formed more
Vesica, diameter are 30-100nm.In human body, almost all of cell can all secrete excretion body；Meanwhile various body fluid and blood
In have the distribution of excretion body.Excretion body includes the substances such as gene, protein and the phosphatide of mother cell, can be with recipient cell
The cell membrane fusion of born of the same parents, and these substances are passed into recipient cell, and then exert one's influence to recipient cell.In addition, excretion body
Surface inherits the memebrane protein of part mother cell, has the ability of identification specific cell.The excretion body of different cell origins
The type of middle internal substance and surface membrane protein has very big difference, and therefore, these excretion body surfaces reveal function abundant.Mesh
Before until largely research report confirm excretion body very important effect is played in the physiology course of body.For example, outer
Secreting body can be used as the promoter of immune response, and play the effect of antigen presentation person；Excretion body withers in the cell of sequencing
It dies, play function in revascularization, inflammation and blood coagulation.Excretion body is also closely bound up with disease.Part cancerous swelling oncocyte point
The excretion body secreted can transmit oncogene to periphery cell.For example, the Malignant glioma cells of the mankind would generally express shortening
And the endothelial growth factor receptor EGFRvIII of tumorigenesis, and EGFRvIII can lack EGFRvIII to periphery by excretion body
Glioma cell transmitted, lead to transmitting of the carcinogen to target cell；Equally, excretion body is also in pathogen in cell
Between transmittance process in play effect.And the relationship between excretion body and related disease also provides for the diagnosis of these diseases
New foundation.
At the same time, excretion body function abundant makes it obtain extensive research and application in the treatment of disease,
Middle use most is exactly extensively source of human stem cell excretion body.In recent years, numerous research reports confirm that stem cell mainly passes through paracrine
Mechanism promotes the reparation and regeneration of tissue damage, and excretion body contains greatly as the main undertaker of stem cell paracrine mechanism
The substances such as gene and the albumen of mother cell are measured, therefore source of human stem cell excretion body is likewise supplied with certain stem-cell therapy
Potentiality.2006, J.Ratajczak et al. confirmed that the excretion body of derived from embryonic stem cells can be by the mRNA in embryonic stem cell
And albumen is transferred to hematopoietic progenitor cells, causes reprograming for hematopoietic progenitor cells.Source of human stem cell has also been established in their research
Excretion body can change the basis of cell fate and then transmitting Self substances to recipient cell.Researches show that dry including multipotency
The excretion body of the cells such as cell, mescenchymal stem cell secretion, which has, to be promoted cell Proliferation, migration, inhibits cellular inflammation reaction, is thin
The functions such as born of the same parents' protection.And stem cell excretion body to the positive influence of cell in reparation, the regeneration for macroscopically promoting tissue damage
And inhibit the further lesion of tissue.Currently, the excretion body of source of human stem cell it is verified that kidney, liver, heart,
There is good therapeutic effect in the damage of kinematic system and the ischemia injury of Various Tissues.Compared with stem-cell therapy, do
The use of cell origin excretion body avoids the safety that stem cell transplantation faces and is in fashion, and it uses, stores and transports more
It is convenient.Therefore, the treatment method based on stem cell excretion body has great potential substitution stem-cell therapy.Currently, based on dry thin
The extracellular treatment technology for secreting body, which has begun to clinical application, to be converted.Another application mode of the excretion body in disease treatment
It is the nano-carrier as drug, realizes the treatment of disease.Excretion body is the natural nano substance transmitting carrier of body secretes.With
Artificial synthesized nano-medicament carrier is compared, and excretion body has unique advantage: the substance of excretion body first is secreted by cell,
The problems such as avoiding the bio-toxicity of artificial synthesized carrier material；Secondly, the outer membrane of excretion body is from cell membrane, it is easier to
With cell membrane fusion, drug effectively is delivered to cell interior；Again, the excretion body surface EDS maps of different cells type it is abundant
Surface protein, these albumen assign excretion body multiplicity function, such as Immune privilege, specific recognition etc..Meanwhile excretion body
Surface can be surface modified by molecular biology or physics, chemical means, assign the more functions of excretion body.Example
Such as, the excretion body in immature dendritic cells source has low immunogenicity, therefore is widely used as the nano-carrier of drug.
Nerve cell is targeted group RVG modification to dendritic cells by molecular biology method by Lydia Alvarez-Erviti et al.
On the memebrane protein Lamp2b of excretion body, and using the siRNA of these excretion bodies load BACE1, demonstrates these excretion bodies and ending
Grow the potentiality in the silent disease treatment in sea.Yanhua Tian et al. equally passes through molecular biology method for tumor-targeting group
IRGD is modified to surface of dendritic cells, and loads adriamycin, improves the curative effect of excretion body carrying medicament treatment tumour.
So far, excretion body main mode of giving in disease treatment is intravenous injection or locally injecting.But
Relevant research report display, the excretion body of intravenous injection enters mainly is enriched in liver and spleen in the mammalian body, and by
Rapid metabolization, residence time in vivo are no more than 24 hours；The excretion body of locally injecting equally exists the residence time and short asks
Topic.The problem has seriously affected the therapeutic efficiency of excretion body.In current research report, excretion body will be realized effective to disease
The raising for the treatment of cost must be caused by a large amount of multiple injections to maintain its effective concentration by treating.In consideration of it, how real
Existing excretion body is to realize the critical issue of excretion body treatment, but do not have at present in the permanent resident and release of site of action
The method of effect solves the above problems.
Summary of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of excretion body sustained releases
System and its construction method and application.
The present invention proposes a kind of based on hyaluronic acid and memebrane protein CD44 combination realization CD44 positive excretion body sustained release
Method.CD44 is a kind of cell surface glycoprotein, and distribution is extremely extensive, including stem cell, tumour cell, endothelial cell etc.
Numerous kinds cell surface is all distributed.Containing the binding domain of hyaluronic acid in the structure of CD44, hyaluronic acid can be incorporated into
On strand.Therefore, the excretion body of the CD44 positive can be effectively combined in the material constructed by hyaluronic acid, and then be realized
The permanent release of excretion body.Method provided by the invention can effectively solve the problems, such as excretion body, and residence time is short in vivo, mentions
The therapeutic efficiency of high excretion body.
The purpose of the present invention can be achieved through the following technical solutions:
First aspect present invention: a kind of excretion body sustained release based on hyaluronic acid Yu CD44 Interaction between membrane proteina is provided
The construction method of system, mainly comprises the steps that
1) it, by the excretion body of certain method separation and Extraction CD44 positive, and mixes, is prepared into biocompatible media
Certain density excretion body suspension；
2), the hyaluronic acid of certain molecular weight or hyaluronic acid decorated object are dissolved in biocompatible media；
3), the solution in step 1) and step 2) is sufficiently mixed uniformly；
4) sustained release of load C D44 positive excretion body, is prepared by certain method using the mixed solution in step 3)
System, the slow-releasing system includes solution, hydrogel, porous support or membrane material.
In an embodiment of the invention, in step 1), the excretion body of the CD44 positive mainly includes stem cell
Source excretion body (such as embryonic stem cell induces multi-potent stem cell, mescenchymal stem cell), swells at dendritic cells source excretion body
Oncocyte source excretion body etc..
In an embodiment of the invention, in step 1), the separating and extracting process of the excretion body of the CD44 positive
It mainly include supercentrifugation, ultrafiltration, size exclusion chromatography and immunomagnetic beads method, preferably supercentrifugation.
In an embodiment of the invention, in step 1), the excretion body of the CD44 positive is in excretion body suspension
Concentration range be 1 × 105/mL–1×1013/ mL, preferably 1 × 1010/mL–1×1012/mL。
In an embodiment of the invention, in step 1), the biocompatible media includes distilled water, physiology salt
Water, physiological buffer and cell culture medium.
In an embodiment of the invention, in step 2), the range of number-average molecular weight of the hyaluronic acid is 1KDa-
6000KDa。
In an embodiment of the invention, in step 2), the range of number-average molecular weight of the hyaluronic acid decorated object
For 5KDa -6000KDa.
In an embodiment of the invention, in step 2), the hyaluronic acid decorated object is in following substance
It is one or more of: the hyaluronic acid of esters of acrylic acid base group modification, the hyaluronic acid of methyl acrylic ester base group modification, end group
The hyaluronic acid of functional polyethylene glycol modification, the more aldehyde radical hyaluronic acids of oxidation, catechol base group modification hyaluronic acid,
The hyaluronic acid of aminoderivative modification, the hyaluronic acid of mercapto derivatives modification, maleimide derivatives are modified transparent
Matter acid etc..
In an embodiment of the invention, in step 2), the biocompatible media includes distilled water, physiology salt
Water, physiological buffer and cell culture medium.
In an embodiment of the invention, in step 4), when the slow-releasing system is solution, preparation load C D44 sun
The buffer solutions of property excretion body method particularly includes:
Solution in step 1) and step 2) is sufficiently mixed uniformly, hyaluronic acid solution is obtained.Wherein, hyaluronic acid or
The mass concentration range of bright matter acid modifier is 0.001%-20%, preferably 0.1%-8%.
In an embodiment of the invention, in step 4), when the slow-releasing system is hydrogel, load C D44 is positive
The sustained-release hydrogel of excretion body includes the sustained release photocrosslinkable hydrogel and load C D44 positive excretion of load C D44 positive excretion body
The sustained release in-situ injection hydrogel of body.
Wherein, the sustained release photocrosslinkable hydrogel of load C D44 positive excretion body is prepared method particularly includes:
Certain density hyaluronic acid decorated object and photoinitiator are completely dissolved in biocompatible media, then with
Excretion body suspension is sufficiently mixed, and irradiates certain time using the light source of certain wavelength, preparation load C D44 positive excretion body delays
Release photocrosslinkable hydrogel.
In this method, hyaluronic acid decorated object mainly includes the hyaluronic acid of esters of acrylic acid base group modification, metering system
The hyaluronic acid of esters of gallic acid base group modification；The mass concentration range of hyaluronic acid decorated object is 0.5%-10%, preferably 2%-
5%；Photoinitiator includes I2959, TPO or EosinY, and corresponding excitation wavelength is respectively 365nm, 365nm, 524nm；Light
Source exposure time range is 10s -30min, preferably 1min -10min.
Wherein, the sustained release in-situ injection hydrogel of load C D44 positive excretion body is prepared method particularly includes:
Certain density hyaluronic acid decorated object is dissolved in biocompatible media, and outstanding with CD44 positive excretion body
Liquid is sufficiently mixed；Another component solution of hydrogel is dissolved in identical biocompatible media, two groups of solution are distinguished
It is added in the syringe of double-component injection device, is injection moulded and is infused in situ to get to the sustained release of load C D44 positive excretion body
Jetting gel.
In this method, hyaluronic acid decorated object includes:
A, more aldehyde radical hyaluronic acids are aoxidized, corresponding another component includes H2N(CH2)nNH2(n is positive integer, n >=1),
Water soluble chitosan, end group are the single-stranded polyethylene glycol of amino or multi-arm polyethylene glycol, gelatin, polylysine, collagen etc.；
B, the hyaluronic acid of catechol base group modification, corresponding another component include sodium metaperiodate, hydrogen peroxide
Etc. small molecules oxidant；
C, aminoderivative modification hyaluronic acid, corresponding another component include glutaraldehyde, Geniposide, end group be aldehyde
The single-stranded polyethylene glycol of base or multi-arm polyethylene glycol etc.；
D, the hyaluronic acid of mercapto derivatives modification, corresponding another group is divided into maleimide, modified by vinyl
Water soluble polymer, including water soluble chitosan, carboxymethyl cellulose, gelatin, alginic acid, glucan, heparin, chondroitin sulfate
Plain, single-stranded or multi-arm polyethylene glycol, polymethylacrylic acid etc.；
E, the hyaluronic acid of maleimide derivatives modification, corresponding another group is divided into mercapto-modified high water solubility
Molecule, including water soluble chitosan, carboxymethyl cellulose, gelatin, alginic acid, glucan, heparin, chondroitin sulfate, it is single-stranded or
Multi-arm polyethylene glycol, polymethylacrylic acid etc..
In an embodiment of the invention, in step 4), when the slow-releasing system is membrane material, load C D44 is positive outer
Secrete the sustained release membrane material of body the preparation method comprises the following steps:
Hyaluronic acid or hyaluronic acid decorated object are dissolved in biocompatible media, it is then mixed with excretion body suspension
It closes, then is sufficiently mixed with the biocompatible media of another water-soluble high-molecular material；Above-mentioned solution is uniformly applied to bottom plate
On, then negative is placed in the drying box of certain temperature, until evaporating completely, the membrane material on bottom plate is removed.
In the above method, water soluble polymer includes chitosan, alginic acid, glucan, carboxymethyl cellulose, heparin, sulphur
Aching and limp ossein and their modifier etc.；
Albumen and polypeptide, including collagen, gelatin, polyaminoacid and their modifier etc.；
Artificial synthesized high molecular material, including polyethylene glycol, multi-arm polyethylene glycol, polymethylacrylic acid, polymethyl
Amide, polypyrrole alkanone and their modifier.
In the above method, the range of drying temperature is 4 DEG C -80 DEG C, preferably 40 DEG C.
In the above method, bottom plate can use polytetrafluoroethylene (PTFE) material.
Second aspect of the present invention: provide based on the construction method that foregoing invention first aspect provides obtain based on hyalomitome
The excretion body slow-releasing system of acid and CD44 Interaction between membrane proteina.
The slow-releasing system of load C D44 positive excretion body includes solution, hydrogel, porous support or membrane material.
Wherein, the buffer solutions of load C D44 positive excretion body are as follows:
It is resulting mixed after CD44 positive excretion body, hyaluronic acid or hyaluronic acid decorated object, biocompatible media mixing
Close solution, wherein the mass concentration range of hyaluronic acid or bright matter acid modifier is 0.001%-20%, preferably 0.1%-
8%.
Wherein, the sustained release photocrosslinkable hydrogel of load C D44 positive excretion body are as follows:
After hyaluronic acid decorated object, photoinitiator, CD44 positive excretion body, biocompatible media mixing, institute after illumination
Obtain object.
Hyaluronic acid decorated object mainly includes the hyaluronic acid of esters of acrylic acid base group modification, methyl acrylic ester group
The hyaluronic acid of modification；The mass concentration range of hyaluronic acid decorated object is 0.5%-10%, preferably 2%-5%；It is light-initiated
Agent includes I2959, TPO or EosinY, and corresponding excitation wavelength is respectively 365nm, 365nm, 524nm；Light source irradiation time
Range is 10s -30min, preferably 1min -10min.
Wherein, the sustained release in-situ injection hydrogel of load C D44 positive excretion body are as follows:
Aquogel system one is obtained after hyaluronic acid decorated object, CD44 positive excretion body, biocompatible media mixing；Separately
One component solution, which is dissolved in identical biocompatible media, obtains aquogel system two, aquogel system one and water gel
It is gains after two injection mouldings.
Hyaluronic acid decorated object includes:
A, more aldehyde radical hyaluronic acids are aoxidized, corresponding another component includes H2N(CH2)nNH2(n is positive integer, n >=1),
Water soluble chitosan, end group are the single-stranded polyethylene glycol of amino or multi-arm polyethylene glycol, gelatin, polylysine, collagen etc.；
B, the hyaluronic acid of catechol base group modification, corresponding another component include sodium metaperiodate, hydrogen peroxide
Etc. small molecules oxidant；
C, aminoderivative modification hyaluronic acid, corresponding another component include glutaraldehyde, Geniposide, end group be aldehyde
The single-stranded polyethylene glycol of base or multi-arm polyethylene glycol etc.；
D, the hyaluronic acid of mercapto derivatives modification, corresponding another group is divided into maleimide, modified by vinyl
Water soluble polymer, including water soluble chitosan, carboxymethyl cellulose, gelatin, alginic acid, glucan, heparin, chondroitin sulfate
Plain, single-stranded or multi-arm polyethylene glycol, polymethylacrylic acid etc.；
E, the hyaluronic acid of maleimide derivatives modification, corresponding another group is divided into mercapto-modified high water solubility
Molecule, including water soluble chitosan, carboxymethyl cellulose, gelatin, alginic acid, glucan, heparin, chondroitin sulfate, it is single-stranded or
Multi-arm polyethylene glycol, polymethylacrylic acid etc..
Wherein, the sustained release membrane material of load C D44 positive excretion body are as follows: hyaluronic acid or hyaluronic acid decorated object, CD44 are positive
After excretion body, water-soluble high-molecular material, biocompatible media mixing, resulting membrane material after smearing, being dry.
Wherein, water soluble polymer includes chitosan, alginic acid, glucan, carboxymethyl cellulose, heparin, chondroitin sulfate
Element and their modifier etc.；
Albumen and polypeptide, including collagen, gelatin, polyaminoacid and their modifier etc.；
Artificial synthesized high molecular material, including polyethylene glycol, multi-arm polyethylene glycol, polymethylacrylic acid, polymethyl
Amide, polypyrrole alkanone and their modifier.
Third aspect present invention: provide based on foregoing invention second aspect provide based on hyaluronic acid and CD44 memebrane protein
The application of the excretion body slow-releasing system of interaction, including apply below:
1, the application on arthritis treatment related drugs is being prepared；
2, the application on preparation skin repair related drugs；
3, the application on preparation congealed shoulder treatment related drugs；
4, the application on preparation treatment cerebral ischemic damage related drugs；
5, as the application of tissue renovation material.
Compared with prior art, the present invention has the following advantages and beneficial effects:
1) the invention discloses the control method for releasing that a kind of CD44 expresses positive excretion body, can effectively overcome current outer
It secretes excretion body in body treatment and is difficult to resident for a long time in vivo and the problem of site of action persistently effectively functions, be excretion
Body treatment provides a kind of completely new method.
2) most cells can all express CD44 memebrane protein in human body, therefore the excretion body of most cells secretion is all
CD44 is positive, therefore the use scope of method of the invention is very extensive；
3) implementation of this method includes the building for loading the material of excretion body, these materials are highly suitable as tissue and repair
Multiple materials'use, this method are highly suitable for regenerative medicine field and are applied.
4) material preparation method used in this method is simple, low in cost, and the scope of application is very extensive.
Detailed description of the invention
Attached drawing 1: the curve graph that excretion body is discharged from photo-crosslinking hyaluronic acid gel.
Attached drawing 2: excretion body is released from hyaluronic acid-gelatin membrane material release profiles and its with carboxymethyl cellulose-gelatin membrane material
To one's heart's content condition compares.
Attached drawing 3: living imaging fluorescence intensity changes over time figure.
Attached drawing 4: the ICRS histological score of mouse arthritis treatment effect.
Attached drawing 5: the range of motion of mouse.
Attached drawing 6: the capsula articularis humeri thickness of mouse.
Attached drawing 7: different time points, the volume of nude mouse tumor.
Attached drawing 8: under specific time, mouse back wound area.
Specific embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1:
Load the preparation of mesenchymal stem cell source excretion body (BMSC-Exos) hyaluronic acid solution
The extraction of BMSC-Exos: it is filled between taking the cell of 5 bottles of T75 to grow the people's bone marrow in the 5th generation that area is 60%-70%
Matter stem cell, the serum free medium for replacing 15mL continue culture 2 days.These culture mediums are collected, and are sub-packed in 10 pipe 50mL's
In centrifuge tube.Respectively 300g, 2,000g, 10,000g revolving speed under be respectively centrifuged 20min, take out cell, cell in culture medium
The impurity such as fragment and organelle.Subsequent 100,000 lower centrifugation 1h, and supernatant is sucked, every pipe retains the lower layer of 200 μ L or so
Liquid.Excretion body is identified by the immunoblotting of transmission electron microscope and surface protein.
Then, it disperses the excretion body of 200 μ L in the physiological buffer of 1ml.It is measured by nanoparticle analyzer outer
The concentration for secreting excretion body in body suspension is 1 × 1011/mL.0.1g hyaluronic acid (molecular weight: 1300KDa) is accurately weighed, it is sufficiently molten
Solution is in the physiological buffer of 9mL.Excretion body suspension is sufficiently mixed with hyaluronic acid solution, be vortexed 10-20s of concussion, obtains
Load the hyaluronic acid solution of mesenchymal stem cell source excretion body.
Embodiment 2:
Load induces multi-potent stem cell the photocrosslinkable hydrogel excretion body slow-releasing system of source excretion body (iPS-Exos)
Building
People induces the culture of human pluripotent stem cells (iPSCs) and the extraction and identification of excretion body
In one layer of embryonic stem cell matrigel of culture dish middle berth (ESC-Qualified BD Matrigel, BDSparks, MD, USA), iPSC is moved into the culture dish, mTeSR1 serum free medium is added
(StemCell Vancouver, BC, Canada), and in incubator (37 DEG C, 5%CO2, saturated humidity) and training
It supports, collects the culture medium replaced daily.Culture medium is centrifuged 30 points by 0.22 micron pore size membrane filtration and 4 DEG C of 10000g
Clock removes cell fragment；Using the super filter tube of 100KD number-average molecular weight, it is centrifuged in (3500g, 15min) retention concentration supernatant
Excretion body obtains excretion body concentrate；Concentrate is transferred to 30% sucrose/heavy water density pad (1.210g/cm3), 4 DEG C
100000g is centrifuged 210 minutes, collects bottom 5ml sucrose/heavy water density pad, PBS dilution is added, 100KD number can be retained by being transferred to
In the ultra-filtration centrifuge tube of average molecular weight, 4 DEG C of 3500g are centrifuged 15min；PBS is washed 3 times, final fixed with PBS by subsequent experimental requirement
Hold to certain volume, obtain excretion body suspension iPS-Exos, packing is saved to -80 DEG C.
The form of iPS-Exos is observed by transmission electron microscope (TEM).Load sample copper mesh (aperture 2nm) is fixed on bracket,
20 μ L samples are added drop-wise on copper mesh, after being stored at room temperature 3 minutes, are sucked liquid with filter paper in copper mesh side, 3% phosphorus tungsten is added dropwise
30 μ L of acid solution carries out negative staining (room temperature, 5 minutes) to sample.Negative staining liquid is sucked with filter paper, copper mesh is transferred to transmitted electron and is shown
Under micro mirror, excretion volume morphing is observed.
Said extracted is induced multi-potent stem cell into source excretion body (iPS-Exos), is scattered in the physiological saline of 1mL.
It is 1 × 10 by the concentration that nanoparticle analyzer measures the excretion body of extraction12/mL.According to the method synthesizing methyl third of document
The hyaluronic acid (HAMA, number-average molecular weight 250KDa) of olefin(e) acid ester modification.The I2959 for accurately weighing the HAMA and 1mg of 100mg is molten
Solution uniformly mixes dispersion with the iPS-Exos suspension of 1mL in the physiological saline of 9mL, by the solution.500 μ L mixed liquors are taken, are added
Enter into Teflon mould, irradiates 3min using the LED light source of 365nm, prepare the sustained-release hydrogel of iPS-Exos.It utilizes
The modulus of shearing that rheometer measures the hydrogel is 3KPa.
The hydrogel material of the load iPS-Exos of above-mentioned preparation is immersed in 2mL physiological buffer, and is placed in 37
DEG C isoperibol in.The buffer for impregnating gel is collected daily, while the fresh buffer of 2mL is added.Utilize nanoparticle
The concentration of excretion body in analysis-e/or determining buffer, and then measure the release conditions of iPS-Exos.Experimental result shows (attached drawing
1), within 1 month detection time limit, it can detecte the release of excretion body approximately linear；After one month, the release of excretion body is imitated
Rate is the 60% of total amount.The experimental result confirms that it is effective for a long time that the positive excretion body of CD44 expression may be implemented in method of the invention
Release.
Embodiment 3:
Load the building of the hyaluronic acid membrane material of derived from embryonic stem cells excretion body (ES-Exos)
Source excretion body (iPS-Exos) is induced multi-potent stem cell by what embodiment 2 was extracted, is scattered in the distilled water of 1mL
In, it is 1 × 10 by the concentration that nanoparticle analyzer measures excretion body suspension10/mL.Accurately weighing number-average molecular weight is
The hyaluronic acid 0.05g and gelatin 0.05g of 130KDa is dissolved in the distilled water of 9mL, and is mixed with iPS-Exos suspension.Then
It takes the 500 above-mentioned solution of μ L to be uniformly applied on polytetrafluoroethylene (PTFE) bottom plate, bottom plate is placed in 40 DEG C of thermostatic drying chamber, it is dry
For 24 hours, hyaluronic acid-gelatin membrane material of load iPS-Exos is obtained.
Further, the carboxymethyl cellulose-for preparing load iPS-Exos identical with above-mentioned membrane material after the same method is bright
Glue film.Two films are soaked in respectively in the physiological buffer of 2mL.Separated in time measures the excretion body in physiological buffer
Content.Experimental result shows (attached drawing 2), and in 2 days release time, 90% excretion body is discharged from carboxymethyl-gelatin film
Into the liquid of surrounding, and only 30% or so excretion body is discharged from hyaluronic acid-gelatin film.The result sufficiently proves,
The sustained release of CD44 positive excretion body can be effectively realized using method of the invention.
Embodiment 4:
Load resident situation of the hyaluronic acid solution of BMSC-Exos in rat joint cavity
The working solution for taking the DiI fluorescent dye of 5uL, is added in the BMSC-Exos suspension of 2mL, is incubated at 37 DEG C
15min.Then, the excretion body marked using size exclusion chromatograph post separation DiI.Referring to the method building load DiI of embodiment 1
The hyaluronic acid solution of the BMSC-Exos of label.
10 SD rats are taken, stochastic averagina is divided into two groups.It is loaded to the 200 μ L of Injection in knuckle articular cavity of first group of rat
The hyaluronic acid solution of the BMSC-Exos of DiI label, is marked to the 200 μ L DiI containing equivalent of Injection in knuckle articular cavity of second group of rat
The excretion body suspension of note.The intracavitary fluorescent emission of every rat articular is detected in particular point in time using small animal living body imager
Number of photons.Experimental result shows (attached drawing 3), and fluorescent emission number of photons decays very in articular cavity in simple injection excretion body group
Fastly, any fluorescent emission is substantially not detectable in 3 days；And inject fluorescence in the hyaluronic acid solution group articular cavity of load excretion body
Decaying slowly, still can detecte fluorescent emission at 7 days.The result proves that method of the invention can be effective real in vivo
The sustained release of existing excretion body.
Embodiment 5:
Utilize the Osteoarthritis of the hyaluronic acid solution treatment rat of load BMSC-Exos
20 SD rats are taken, the osteoarthritis of rat is established using ligamentaum cruciatum intercept method.Meanwhile referring to embodiment
Method in 1 prepares the hyaluronic acid solution of 10mL load BMSC-Exos.Above-mentioned SD rat is divided into four groups, first group of pass
Save the hyaluronic acid solution of 500 μ L of intracavitary administration load BMSC-Exos, second group of 500 μ L equivalent BMSC- of intraarticular injection
The suspension of Exos；The hyaluronic acid solution of 500 isoconcentration of third group intraarticular injection；4th group of 500 μ L of intraarticular injection
Physiological saline.
After injecting 4 weeks, the mouse in above-mentioned every group is put to death, extracts knee joint sample.Decalcification, slice are fixed to sample
And H&E dyeing processing, the case where observing the knee cartilage of every mouse in every group, and according to international chondrology can cartilage
Repair tissue standards of grading carry out histological score.Experimental result shows (attached drawing 4), loads the hyaluronic acid of BMSC-Exos
The histological score of the mouse knee joint position of solution processing is significantly higher than other two groups, and the knee joint position of these mouse
Cartilage degradation situation is weaker；And the histological score for injecting the mouse knee joint position of excretion body or hyaluronic acid merely is similar,
There is significantly missing, cartilage matrix diacrisis in knee cartilage layer in this two groups, but cartilage degradation situation is slightly good
In control group.The above results show, method of the invention can effectively realize that excretion body in the sustained release of articular cavity, and then improves outer
Secrete the therapeutic efficiency of body.
Embodiment 6:
Utilize the congealed shoulder of the hyaluronic acid solution prevention rat of load iPS-MSC-Exos
The mescenchymal stem cell (iPS-MSC) for the iPS induction differentiation that the growth area of 5 bottles of T75 is 60%-70% is taken, more
Change the MSC culture medium culture of 15mL serum-free 2 days into.These culture mediums are collected, and go out to train by the centrifugation of 300g and 2000g
Support the cell and cell fragment in base.Then, above-mentioned culture medium is concentrated into 5mL using the super filter tube of 100KDa.Finally, will
Above-mentioned concentrate is divided into 5 points, is added separately in qEV size exclusion chromatograph column, collects the later mobile phase 1mL of 3min, obtains
iPS-MSC-Exos.Finally the excretion body of extraction is identified using transmission electron microscope and surface protein immunocy trace.
The above-mentioned excretion body suspension of 200 μ L is taken to be scattered in the physiological buffer of 1mL.Method preparation referring to embodiment 1 is negative
Carry the hyaluronic acid solution of iPS-MSC-Exos.20 SD rats are taken, the elbow joint and shoulder of the fixed rat of Tendon Suture are utilized
Shoulder blade constructs congealed shoulder animal model.After constructing animal model, above-mentioned SD rat is divided into four groups, in first group of articular cavity
The hyaluronic acid solution of 200 μ L load BMSC-Exos is injected weekly；200 μ L equivalent BMSC- are injected in second group of articular cavity weekly
The suspension of Exos；The hyaluronic acid solution of 200 μ L isoconcentrations is injected in third group articular cavity weekly；4th group of intraarticular injection
The physiological buffer of 200 μ L.After continuous injection 5 weeks, the shoulder joint sample of every mouse is extracted.Above-mentioned each group mouse is tested first
In, the range of motion of every mouse.Experimental result shows (attached drawing 5) that injection loads the hyaluronic acid solution of excretion body
The range of motion of mouse is 130 degree or so, substantially close to the range of motion of normal rat；Simple injection excretion body group
Range of motion be 110 degree or so；And inject merely hyaluronic acid range of motion and control group it is close.Then,
Joint capsule sample is extracted, the thickness of transmission electron microscope observing joint capsule is utilized.The experimental result of transmission electron microscope is shown (attached drawing 6), is infused
The joint capsule thickness for penetrating the hyaluronic acid solution group of load excretion body is obviously thinner than other groups.In summary experimental result, this hair
Bright method can effectively prevent the generation of congealed shoulder, meanwhile, being loaded using hyaluronic acid and be sustained excretion body can be significant
Promote the effect of excretion body treatment.
Embodiment 7:
The anticancer effect evaluation of the hyaluronic acid gel of the ES-Exos of load package taxol (PTX)
The physiological buffer liquor of 5 μM of PTX is prepared, and disperses freshly extd ES-Exos with the solution, is placed in 37 DEG C
Lower incubation 2h.Then, it carries out ultrafiltration three times using physiological buffer to wash, and with size exclusion chromatograph column purification load PTX's
ES-Exos.Measuring excretion body to wrap up the concentration of PTX by high performance liquid chromatography is 2 μM.
The hyaluronic acid that the number-average molecular weight of 0.5g is 4000KDa is taken, 0.1% aqueous solution is configured to, reference literature
Method preparation aoxidizes the hyaluronic acid of more aldehyde radicals.It takes hyaluronic acid 0.2g to be dissolved in 5mL physiological buffer, while taking 0.2g
Water soluble chitosan (carboxymethyl chitosan) be dissolved in the physiological buffer of 4mL, and the ES- of above-mentioned package PTX is added
Exos suspension 1mL, is sufficiently mixed.Above-mentioned two solution is added separately in two syringes of double-component injection device.
36 nude mices are taken, to the mixture of oxter implantation human breast cancer cell and matrigel, are cultivated 2 weeks, are formed subcutaneous swollen
Tumor.Mouse is divided into 3 groups: first groups at random and wraps up PTX to the above-mentioned load of mouse subcutaneous tumor adjacent margins injection 0.2mL
ES-Exos oxidized hyaluronic acid-aquagel precursor solution, and gel in-situ；Second group to mouse subcutaneous tumor portion
The physiological buffer of the injection of position edge 0.5mL PTX containing equivalent and ES-Exos；Third group direct injection physiological buffer.The 2nd,
7,14,28 days when every group take out three mouse respectively, extract tumor tissues, measure the volume of tumour.Experimental result shows (attached
Fig. 7), the 2nd day when injection PTX excretion body group gross tumor volume be less than injection load excretion body hydrogel group；And by the 7th day
When, hydrogel group is substantially better than excretion body group to the inhibitory effect of tumour；At the 28th day, the tumor tissues volume of hydrogel group is aobvious
It writes and is less than other two groups.Above-mentioned the results show method of the invention, which can be realized effectively, carries the slow of medicine CD44 positive excretion body
It releases, extends its residence time in vivo, improve therapeutic efficiency.
Embodiment 8:
Utilize hyaluronic acid-gelatin film treatment rat skin injury of load iPS-Exos
Referring to hyaluronic acid-gelatin film of the method building load iPS-Exos in embodiment 3.15 SD rats are taken,
The skin of back position of mouse constructs the Round scalp defect surface of a wound that symmetrical 4 diameters are 2cm.The of every mouse
Hyaluronic acid-gelatin film of one skin wound position covering load iPS-Exos, and carry out suture fixation；Second skin
The excretion body physiological saline suspension of wound site smearing 0.2mL iPS-Exos containing equivalent；It smears at third skin wound position
The physiological saline of 0.2mL.Within the reparation phase, the area of the rat skin surface of a wound is measured every three days, and calculates healing efficiency.Experiment
(attached drawing 8) as the result is shown, the healing rate using hyaluronic acid-gelatin film process skin wound of load iPS-Exos are obvious
Better than other two groups.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.
Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general
Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability
Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention
Within protection scope.
Claims (10)
1. a kind of construction method of excretion body slow-releasing system, which comprises the following steps:
1), separation and Extraction CD44 positive excretion body, and mixed with biocompatible media, it is prepared into excretion body suspension；
2), hyaluronic acid or hyaluronic acid decorated object are dissolved in biocompatible media；
3), the solution in step 1) and step 2) is sufficiently mixed uniformly；
4) slow-releasing system of the mixed solution preparation load C D44 positive excretion body in step 3), the slow-releasing system packet, are utilized
Include solution, hydrogel, porous support or membrane material.
2. a kind of construction method of excretion body slow-releasing system according to claim 1, which is characterized in that the CD44 is positive
Excretion body includes source of human stem cell excretion body, dendritic cells source excretion body, tumour cell source excretion body.
3. a kind of construction method of excretion body slow-releasing system according to claim 1, which is characterized in that the CD44 is positive
Concentration range of the excretion body in excretion body suspension is 1 × 105/mL–1×1013/ mL, preferably 1 × 1010/mL–1×1012/
mL。
4. a kind of construction method of excretion body slow-releasing system according to claim 1, which is characterized in that the hyaluronic acid
Modifier is selected from one or more of following substance: hyaluronic acid, the methyl acrylic ester of esters of acrylic acid base group modification
The polyethyleneglycol modified hyaluronic acid of hyaluronic acid, the end group functional of base group modification, the more aldehyde radical hyaluronic acids of oxidation, adjacent benzene two
The hyaluronic acid of phenolic group modification, the hyaluronic acid of aminoderivative modification, the hyaluronic acid of mercapto derivatives modification, Malaysia
The hyaluronic acid of imide derivative modification.
5. a kind of construction method of excretion body slow-releasing system according to claim 1, which is characterized in that the slow-releasing system
When for solution, the buffer solutions of load C D44 positive excretion body are prepared method particularly includes:
Solution in step 1) and step 2) is sufficiently mixed uniformly, hyaluronic acid solution is obtained.Wherein, hyaluronic acid or bright matter
The concentration range of sour modifier is 0.001%-20%, preferably 0.1%-8%.
6. a kind of construction method of excretion body slow-releasing system according to claim 1, which is characterized in that the slow-releasing system
For load CD44 positive excretion body sustained release photocrosslinkable hydrogel when, preparation method are as follows: draw hyaluronic acid decorated object with light
Hair agent is completely dissolved in biocompatible media, is then sufficiently mixed with excretion body suspension, is irradiated using light source, preparation load
The sustained release photocrosslinkable hydrogel of CD44 positive excretion body；
Hyaluronic acid decorated object mainly includes the hyaluronic acid of esters of acrylic acid base group modification, methyl acrylic ester base group modification
Hyaluronic acid；The concentration range of hyaluronic acid decorated object is 0.5%-10%, preferably 2%-5%；Photoinitiator includes
I2959, TPO or EosinY, corresponding excitation wavelength are respectively 365nm, 365nm, 524nm；Light source exposure time range is
10s -30min, preferably 1min -10min.
7. a kind of construction method of excretion body slow-releasing system according to claim 6, which is characterized in that the slow-releasing system
For load CD44 positive excretion body sustained release in-situ injection hydrogel when, method particularly includes: hyaluronic acid decorated object is dissolved
It is sufficiently mixed in biocompatible media, and with CD44 positive excretion body suspension；Another component solution of hydrogel is dissolved
In identical biocompatible media, two groups of solution are added separately in the syringe of double-component injection device, are injected into
Type is to get the sustained release in-situ injection hydrogel for arriving load C D44 positive excretion body；
Hyaluronic acid decorated object includes:
A, more aldehyde radical hyaluronic acids are aoxidized, corresponding another component includes H2N(CH2)nNH2It is (n is positive integer, n >=1), water-soluble
Property chitosan, end group be amino single-stranded polyethylene glycol or multi-arm polyethylene glycol, gelatin, polylysine, collagen；
B, the hyaluronic acid of catechol base group modification, corresponding another component include that sodium metaperiodate, hydrogen peroxide etc. are small
Molecular oxygen agent；
C, aminoderivative modification hyaluronic acid, corresponding another component include glutaraldehyde, Geniposide, end group be aldehyde radical
Single-stranded polyethylene glycol or multi-arm polyethylene glycol；
D, mercapto derivatives modification hyaluronic acid, corresponding another group be divided into maleimide, modified by vinyl it is water-soluble
Property macromolecule, including water soluble chitosan, carboxymethyl cellulose, gelatin, alginic acid, glucan, heparin, chondroitin sulfate, list
Chain or multi-arm polyethylene glycol, polymethylacrylic acid；
E, the hyaluronic acid of maleimide derivatives modification, corresponding another group is divided into mercapto-modified water-soluble high score
Son, including it is water soluble chitosan, carboxymethyl cellulose, gelatin, alginic acid, glucan, heparin, chondroitin sulfate, single-stranded or more
Arm polyethylene glycol, polymethylacrylic acid.
8. a kind of construction method of excretion body slow-releasing system according to claim 1, which is characterized in that the slow-releasing system
When for membrane material, the sustained release membrane material of load C D44 positive excretion body the preparation method comprises the following steps:
Hyaluronic acid or hyaluronic acid decorated object are dissolved in biocompatible media, then mixed with excretion body suspension, then
Biocompatible media with another water-soluble high-molecular material is sufficiently mixed；Above-mentioned solution is uniformly applied on bottom plate, so
Negative is set in drying box afterwards, until evaporating completely, the membrane material on bottom plate is removed；
The water soluble polymer selects one or more of following substance:
Chitosan, alginic acid, glucan, carboxymethyl cellulose, heparin, chondroitin sulfate and their modifier；
Albumen and polypeptide, including collagen, gelatin, polyaminoacid and their modifier；
Artificial synthesized high molecular material, including polyethylene glycol, multi-arm polyethylene glycol, polymethylacrylic acid, polymethacrylamide,
Polypyrrole alkanone and their modifier.
9. a kind of excretion body slow-releasing system, which is characterized in that it is outer based on hyaluronic acid and CD44 Interaction between membrane proteina
Body slow-releasing system, including solution, hydrogel or membrane material are secreted,
The buffer solutions of load C D44 positive excretion body are as follows: CD44 positive excretion body, hyaluronic acid or hyaluronic acid decorated object, life
Resulting mixed solution after the mixing of object media compatibility, wherein the concentration range of hyaluronic acid or bright matter acid modifier is
0.001%-20%, preferably 0.1%-8%；
The sustained release photocrosslinkable hydrogel of load C D44 positive excretion body are as follows: hyaluronic acid decorated object, photoinitiator, CD44 are positive outer
After secreting body, biocompatible media mixing, gains after illumination；
The sustained release in-situ injection hydrogel of load C D44 positive excretion body are as follows: hyaluronic acid decorated object, CD44 positive excretion body, life
Aquogel system one is obtained after the mixing of object media compatibility；Another component solution is dissolved in identical biocompatible media and obtains
To aquogel system two, gains after aquogel system one and the injection moulding of aquogel system two；
The sustained release membrane material of load C D44 positive excretion body are as follows: hyaluronic acid or hyaluronic acid decorated object, CD44 positive excretion body, water
After soluble macromolecular material, biocompatible media mixing, resulting membrane material after smearing, being dry.
10. the application of excretion body slow-releasing system as claimed in claim 9, which is characterized in that including applying below:
1), the application on arthritis treatment related drugs is being prepared；
2), the application on preparation skin repair related drugs；
3), the application on preparation congealed shoulder treatment related drugs；
4), the application on preparation treatment cerebral ischemic damage related drugs；
5), as the application of tissue renovation material.
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| CN115317401A (en) * | 2022-05-26 | 2022-11-11 | 西安博鸿生物技术有限公司 | Skin care composition and preparation method and application thereof |
| CN116036310A (en) * | 2023-01-13 | 2023-05-02 | 广东医科大学附属医院 | Succinylated chitosan modified exosome and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2766205A1 (en) * | 1997-07-16 | 1999-01-22 | Inst Nat Sante Rech Med | Antigen-loaded vesicles derived from tumour cells or antigen-presenting cells |
| CN106913583A (en) * | 2017-04-20 | 2017-07-04 | 深圳市赛欧细胞生物科技有限公司 | The preparation method and application of human mesenchymal stem cell source excretion body biologically active agents |
-
2018
- 2018-02-09 CN CN201810136419.8A patent/CN110123842B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2766205A1 (en) * | 1997-07-16 | 1999-01-22 | Inst Nat Sante Rech Med | Antigen-loaded vesicles derived from tumour cells or antigen-presenting cells |
| CN106913583A (en) * | 2017-04-20 | 2017-07-04 | 深圳市赛欧细胞生物科技有限公司 | The preparation method and application of human mesenchymal stem cell source excretion body biologically active agents |
Non-Patent Citations (1)
| Title |
|---|
| 白啸: "硫酸软骨素基可注射水凝胶的制备及其作为骨修复支架的研究", 《中国博士学位论文全文数据库，医药卫生科技辑，E080-8，2008年第01期》 * |
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