CN108498786A - Carry the slow release nano-particle and its preparation method and application of osteoprotegerin - Google Patents
Carry the slow release nano-particle and its preparation method and application of osteoprotegerin Download PDFInfo
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Abstract
The present invention relates to biomedical sectors, and in particular to a kind of slow release nano-particle and its preparation method and application carrying osteoprotegerin.The present invention provides a kind of slow release nano-particle carrying osteoprotegerin, the nanoparticle is prepared by the raw material containing osteoprotegerin, maleimide polyethylene glycol polycaprolactone and polycaprolactone.The present invention also provides a kind of preparation methods for the slow release nano-particle carrying osteoprotegerin; it is using osteoprotegerin, maleimide polyethylene glycol polycaprolactone and polycaprolactone and the mixture of organic solvent formation as oil phase; using the aqueous solution of water-soluble polymer as water phase, it is ultrasonically treated to obtain.Osteoprotegerin is encapsulated in structure nano drug-carrying controlled release system in nanoparticle by the present invention; enable osteoprotegerin from nanoparticle with certain rate local slow release; the concentration for the treatment of can be reached in this way, osteoprotegerin is also can avoid in vivo by protease hydrolytic, improve bioavilability.
Description
Technical field
The present invention relates to biomedical sectors, and in particular to a kind of slow release nano-particle and preparation method thereof carrying osteoprotegerin
And application.
Background technology
The incidence of calcific aortic disease (Calcific aortic valve disease, CAVD) is in old age
Ascendant trend year by year is presented in population, current main therapeutic modality is aortic valve replacement operation, but existing artificial valve
Film displacement after there is bleeding, calcification decline, durability is poor the problems such as.With deepening continuously to CAVD study of incident mechanism,
Some researches show that CAVD is a kind of active lesion mistake being related to the pathological changes such as inflammation, lipid-metabolism, cell differentiation, matrix remodeling
Journey, while also new therapy target is provided for the treatment of CAVD.
Osteoprotegerin (Osteoprotegerin, OPG) is a kind of soluble secreting type glycoprotein, is tumor necrosis factor
One of receptor superfamily member.OPG contestables prevent the nuclear factor Kb receptor activations factor (RANK) and PANK ligands
(RANKL) it combines, can adjust, protect vascular endothelial cell, inhibit blood vessel and the differentiation of valve interstitial cell exception skeletonization sample,
The relative or absolute deficiency of secretion is likely to be the major reason for leading to valvular calcification.But due to free OPG easy quilts in vivo
Proteasome degradation, stability is poor, and half-life period is shorter, it is difficult in the concentration for locally reaching treatment.
Nano medicament carrying system is a series of general name of grain sizes in nano level novel small drug delivery system, is to utilize nanometer skill
Using natural or synthetic high molecular material as carrier, the drug that manufactured grain size is 1~1000nm together with drug conveys art
System, the drug-loading system have the remarkable advantages such as targeting, slow release, carrier material be biodegradable.
Invention content
For this purpose, the problem of technology solved by the invention, is：Protein medicaments are in vivo easily by protease in the prior art
Degradation, half-life period is shorter, in the concentration for being locally extremely difficult to treatment.
The present invention provides a kind of slow release nano-particles carrying osteoprotegerin, and the nanoparticle is by containing osteoprotegerin, horse
The raw material for coming acid imide-polyethylene glycol-polycaprolactone and polycaprolactone is prepared.
The present invention also provides a kind of preparation methods for the slow release nano-particle carrying osteoprotegerin, and it includes following step：
The mixing that osteoprotegerin, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent are formed
Object is as oil phase, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain.
Specifically, the present invention proposes following technical solution.
The present invention provides a kind of slow release nano-particles carrying osteoprotegerin, and the nanoparticle is by containing osteoprotegerin, horse
The raw material for coming acid imide-polyethylene glycol-polycaprolactone and polycaprolactone is prepared.
Preferably, for the slow release nano-particle of the load osteoprotegerin, wherein the osteoprotegerin is 0.5-3 weight
Part, preferably 0.5-2 parts by weight；Maleimide-the polyethylene glycol-polycaprolactone is 4000-8000 parts by weight, preferably
4000-6000 parts by weight；The pla-pcl is 12000-24000 parts by weight, preferably 12000-18000 parts by weight.
Preferably, for the slow release nano-particle of the load osteoprotegerin, wherein the nanoparticle is protected by containing bone
Element, phosphatide, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone raw material be prepared.
Preferably, for the slow release nano-particle of the load osteoprotegerin, wherein the osteoprotegerin is 0.5-3 weight
Part, preferably 0.5-2 parts by weight；The phosphatide is 5000-15000 parts by weight, preferably 5000-10000 parts by weight；The horse
It is 4000-8000 parts by weight, preferably 4000-6000 parts by weight to carry out acid imide-polyethylene glycol-polycaprolactone；It is described poly- interior
Ester is 12000-24000 parts by weight, preferably 12000-18000 parts by weight.
Preferably, for the slow release nano-particle of the load osteoprotegerin, wherein the nanoparticle passes through following methods system
It is standby to obtain：
The mixing that osteoprotegerin, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent are formed
Object is as oil phase, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain.
Preferably, for the slow release nano-particle of the load osteoprotegerin, wherein the nanoparticle passes through following methods system
It is standby to obtain：
(1) osteoprotegerin and phosphatide are mixed to get osteoprotegerin-phosphatide complexes；
(2) by osteoprotegerin-phosphatide complexes, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic
The mixture that solvent is formed, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain as oil phase.
Preferably, for the slow release nano-particle of the described load osteoprotegerin, wherein the molecular weight of the polycaprolactone is
40000~50000.
Preferably, for the slow release nano-particle of the load osteoprotegerin, wherein the maleimide-polyethylene glycol-
The molecular weight of polycaprolactone is 10000~20000, and the molecular weight of the polyethylene glycol segment is 1000~2000.
The present invention provides a kind of preparation methods of the slow release nano-particle carrying osteoprotegerin, and it includes following step：
The mixing that osteoprotegerin, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent are formed
Object is as oil phase, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain.
Preferably, for the preparation method of the slow release nano-particle of the load osteoprotegerin, wherein it includes following steps
Suddenly：
(1) osteoprotegerin and phosphatide are mixed to get osteoprotegerin-phosphatide complexes；
(2) by osteoprotegerin-phosphatide complexes, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic
The mixture that solvent is formed, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain as oil phase.
Preferably, for the preparation method of the slow release nano-particle of the load osteoprotegerin, wherein the oil phase and water phase
Volume ratio be 1:2~6, preferably 1:4~5.
Preferably, for the preparation method of the slow release nano-particle of the load osteoprotegerin, wherein the water-soluble polymeric
Object is selected from polyvinyl alcohol, polysorbate or fatty acid sorbitan, preferably polyvinyl alcohol.
Preferably, for the preparation method of the slow release nano-particle of the load osteoprotegerin, wherein the organic solvent choosing
From in dichloromethane, chloroform or acetone, preferably dichloromethane.
The slow release nano-particle for carrying osteoprotegerin or the preparation-obtained nanoparticle of the above method of the present invention are in nanometer
Application in medicine carrying material.
Advantageous effect obtained by the present invention is：
The present invention uses biodegradable material, and osteoprotegerin is encapsulated in nanoparticle, enables osteoprotegerin from nanometer
Grain in certain rate in the release of local slow, the concentration for the treatment of can be reached, also can avoid osteoprotegerin in vivo by
Protease hydrolytic improves the bioavilability of drug.The obtained slow release nano-particle for carrying OPG inhibits in certain degree
Calcification process, and show stronger anticalcium effect.
Description of the drawings
Fig. 1 is that the slow release nano-particle of load osteoprotegerin in embodiment one prepares schematic diagram；
Fig. 2 is the slow release nano-particle particle diameter distribution schematic diagram that osteoprotegerin is carried in embodiment one；
Fig. 3 is the slow release nano-particle Zeta schematic diagrames that osteoprotegerin is carried in embodiment one；
Fig. 4 is the slow release nano-particle transmission electron microscope schematic diagram that osteoprotegerin is carried in embodiment one；
Fig. 5 is the In-vitro release curves schematic diagram for the slow release nano-particle that osteoprotegerin is carried in embodiment one；
Fig. 6-1 is original cuiture rat bone marrow mesenchymal stem cells after one day；
Fig. 6-2 is original cuiture rat bone marrow mesenchymal stem cells after three days；
Fig. 6-3 is original cuiture rat bone marrow mesenchymal stem cells after five days；
Fig. 6-4 is original cuiture rat bone marrow mesenchymal stem cells after seven days；
Fig. 6-5 is the rat bone marrow mesenchymal stem cells of second generation culture；
Fig. 6-6 is the rat bone marrow mesenchymal stem cells of third generation culture；
Fig. 7-1 is the schematic diagram that rat bone marrow mesenchymal stem cells express CD29, wherein abscissa represents fluorescence signal phase
To intensity, ordinate represents cell number；
Fig. 7-2 is the schematic diagram that rat bone marrow mesenchymal stem cells express CD90, wherein abscissa represents fluorescence signal phase
To intensity, ordinate represents cell number；
Fig. 7-3 is the schematic diagram that rat bone marrow mesenchymal stem cells express CD34, wherein abscissa represents fluorescence signal phase
To intensity, ordinate represents cell number；
Fig. 7-4 is the schematic diagram that rat bone marrow mesenchymal stem cells express CD45, wherein abscissa represents fluorescence signal phase
To intensity, ordinate represents cell number；
Fig. 8 is the cytotoxicity of the obtained nanoparticle for carrying osteoprotegerin of embodiment one, control group and blank nanoparticle
Result schematic diagram；
Fig. 9-1 is the calcium tubercle Alizarin red staining result schematic diagram that calcification induces liquid group (DAG)；
Fig. 9-2 is the calcium tubercle Alizarin red staining result schematic diagram of blank nanoparticle group；
Fig. 9-3 is the calcium tubercle Alizarin red staining result signal of the nanoparticle group of the load protection element prepared by embodiment one
Figure；
Fig. 9-4 is the calcium tubercle Alizarin red staining result schematic diagram of Normal group；
Figure 10 be embodiment one prepare it is obtained carry the nanoparticle group of osteoprotegerin, blank nanoparticle group, control group and
Schematic diagram of the DAG groups to doped calcium in rat bone marrow mesenchymal stem cells osteogenic differentiation process, wherein * represents P ＜ 0.05, * * *
Represent P ＜ 0.001.
Specific implementation mode
Fig. 1 is that the slow release nano-particle of load osteoprotegerin in embodiment one prepares schematic diagram, will include osteoprotegerin, Malaysia acyl
The raw material of imines-polyethylene glycol-polycaprolactone and pla-pcl obtains carrying the sustained release of osteoprotegerin by emulsion-solvent evaporation method
Osteoprotegerin is encapsulated in nanoparticle by nanoparticle, the present invention, makes osteoprotegerin in nanoparticle with part under certain rate
Slowly release, can reach the concentration for the treatment of, also can avoid osteoprotegerin in vivo by protease hydrolytic, improve drug
Bioavilability.
Fig. 2 is the slow release nano-particle particle diameter distribution schematic diagram that osteoprotegerin is carried in embodiment one, figure it is seen that institute
The grain size of the nanoparticle of preparation is 133nm, and dispersity index (PDI) is 0.131.
Fig. 3 is the slow release nano-particle Zeta schematic diagrames that osteoprotegerin is carried in embodiment one, from figure 3, it can be seen that Zeta is electric
Position is -27.8mV.
Fig. 4 is the slow release nano-particle transmission electron microscope schematic diagram that osteoprotegerin is carried in embodiment one, it can be seen from the figure that receiving
The grain of rice is rounded, and size rule is evenly distributed, has no phenomenon adhered to one another.
Fig. 5 is the In-vitro release curves schematic diagram for the slow release nano-particle that osteoprotegerin is carried in embodiment one, from release profiles
As can be seen that at release initial stage, OPG is released with faster speed from nanoparticle, but does not occur phenomenon of burst release.24 hours
OPG Cumulative release amounts reach 52%, later slow release, are discharged up to 89% until the 7th day OPG is accumulative.
Fig. 6-1 is the rat bone marrow mesenchymal stem cells of original cuiture, it can be seen from the figure that just inoculated cell is in circle
Shape suspends in the medium.
Fig. 6-2 is rat bone marrow mesenchymal stem cells after cultivating three days, it can be seen from the figure that after culture three days, portion
Divide cell to start adherent growth, is in spindle shape.
Fig. 6-3 is rat bone marrow mesenchymal stem cells after cultivating five days, it can be seen from the figure that after culture 5 days, big portion
Divide cell adherent, forms cell colony and simultaneously gradually extend outwardly.
Fig. 6-4 is rat bone marrow mesenchymal stem cells after cultivating seven days, it can be seen from the figure that after culture 7 days, cell
With certain direction arrangement, to show swirl shape.
Fig. 6-5 is the rat bone marrow mesenchymal stem cells of second generation culture, and after carrying out second pass generation, cell growth is prosperous
It contains, form is uniform, is in spindle shape adherent growth.
Fig. 6-6 is the rat bone marrow mesenchymal stem cells of third generation culture, and after carrying out third time passage, cell is in spindle shape
Adherent growth.
Fig. 7-1 is the schematic diagram of rat bone marrow mesenchymal stem cells expression CD29, wherein abscissa represents fluorescence signal phase
To intensity, ordinate represents cell number, and FCM analysis instrument is used to be measured, it can be seen from the figure that rat marrow
Mescenchymal stem cell being capable of high expression CD29.
Fig. 7-2 is the schematic diagram of rat bone marrow mesenchymal stem cells expression CD90, wherein abscissa represents fluorescence signal phase
To intensity, ordinate represents cell number, and FCM analysis instrument is used to be measured, it can be seen from the figure that rat marrow
Mescenchymal stem cell being capable of high expression CD90.
Fig. 7-3 is the schematic diagram of rat bone marrow mesenchymal stem cells expression CD34, and Fig. 7-4 is that rat marrow mesenchyma is dry thin
The schematic diagram of cellular expression CD45 can be seen that rat bone marrow mesenchymal stem cells from above-mentioned two figure and not express or extremely low expression
CD34 and CD45.
Fig. 8 is the cytotoxicity of the obtained nanoparticle for carrying osteoprotegerin of embodiment one, control group and blank nanoparticle
Result schematic diagram, it can be seen from the figure that be added carry osteoprotegerin nanoparticle after, by 12h, for 24 hours with the culture of 48h,
It is compared with control group PBS, the cell growth of the nanoparticle group of blank nanoparticle and load osteoprotegerin and the shadow for being not affected by nanoparticle
It rings, has no significant difference (P ＞ 0.05) between each group, illustrate that nanoparticle has no effect on the increasing of rat bone marrow mesenchymal stem cells
It grows, i.e., nanoparticle is to rat bone marrow mesenchymal stem cells nontoxicity.
Fig. 9-1 and Fig. 9-2 is respectively the calcium tubercle Alizarin red staining result schematic diagram of DAG groups and blank nanoparticle group, from
Above-mentioned two figure, which can be seen that, has there is a large amount of calcium tubercle, and assembles agglomerating, wherein black portions represent alizarin red dye it is red
The part of color.
Fig. 9-3 is the alizarin calcium tubercle element red colouring result signal of the nanoparticle group of the load protection element prepared by embodiment one
Figure illustrates that carrying bone protects it can be seen from the figure that the nanoparticle group for carrying osteoprotegerin has the calcium tubercle being dispersed in, but negligible amounts
The nanoparticle of shield element inhibits calcification process to a certain extent, wherein black portions are that alizarin red dyes red part.
Fig. 9-4 is the calcium tubercle Alizarin red staining result schematic diagram of Normal group, it can be seen from the figure that having no calcium knot
Section.
Figure 10 be embodiment one prepare it is obtained carry the nanoparticle group of osteoprotegerin, blank nanoparticle group, control group and
Schematic diagram of the DAG groups to doped calcium in rat bone marrow mesenchymal stem cells osteogenic differentiation process, wherein it can be seen from the figure that
There are significant difference (P ＜ compared with the nanoparticle group for carrying osteoprotegerin in the calcium ion concentration of osteogenic induction the 7th day, DAG groups
0.05), after culture the 14th day, the calcium ion concentration of DAG groups and blank nanoparticle group and the nanoparticle group phase for carrying osteoprotegerin
Than there is significant significant difference (P ＜ 0.001), after culture 21 days, the calcium ion concentration of each group continues growing, but carries bone and protect
The calcium ion concentration for protecting the nanoparticle group of element is significantly lower than DAG groups and blank nanoparticle group (P ＜ 0.001), illustrates to carry protection element
Nanoparticle group have stronger anticalcium effect.
As described above, the present invention provides a kind of slow release nano-particles carrying osteoprotegerin, by containing osteoprotegerin, horse
The raw material for coming acid imide-polyethylene glycol-polycaprolactone and polycaprolactone is prepared.
In a kind of currently preferred specific implementation mode, the osteoprotegerin is 0.5-3 parts by weight, preferably 0.5-
2 parts by weight；Maleimide-the polyethylene glycol-polycaprolactone is 4000-8000 parts by weight, preferably 4000-6000 weight
Part；The pla-pcl is 12000-24000 parts by weight, preferably 12000-18000 parts by weight.
In the preferred embodiments of the disclosure, the present invention provides it is a kind of carry osteoprotegerin slow release nano-particle,
It is prepared by the raw material containing osteoprotegerin, phosphatide, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone.
In a kind of currently preferred specific implementation mode, wherein the osteoprotegerin is 0.5-3 parts by weight, preferably
For 0.5-2 parts by weight；The phosphatide is 5000-15000 parts by weight, preferably 5000-10000 parts by weight；The maleimide
Amine-polyethylene glycol-polycaprolactone is 4000-8000 parts by weight, preferably 4000-6000 parts by weight；The pla-pcl is
12000-24000 parts by weight, preferably 12000-18000 parts by weight.
Preferably, the molecular weight of the polycaprolactone is 40000~50000, preferably 45000.
Preferably, the molecular weight of the maleimide-polyethylene glycol-polycaprolactone is 10000~20000, preferably
12000, the molecular weight of the polyethylene glycol segment is 1000~2000, preferably 2000.
The present invention provides a kind of preparation methods carrying osteoprotegerin, and it includes following step：By osteoprotegerin, Malaysia acyl
The mixture that imines-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent are formed is as oil phase, by water-soluble polymer
Aqueous solution as water phase, be ultrasonically treated to obtain.
In a kind of preferred embodiment of the present invention, the present invention provides a kind of slow release nanometers carrying osteoprotegerin
The preparation method of grain, it includes following step:
(1) osteoprotegerin and phosphatide are mixed to get osteoprotegerin-phosphatide complexes；
(2) by osteoprotegerin-phosphatide complexes, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic
The mixture that solvent is formed, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain as oil phase.
Preferably, the organic solvent is selected from dichloromethane, chloroform or acetone, preferably dichloromethane, described
Water-soluble polymer is selected from polyvinyl alcohol, polysorbate or fatty acid sorbitan, preferably polyvinyl alcohol.
Preferably, the organic solvent is 0.75~2.5ml, preferably 0.75-2ml.
Preferably, the volume ratio of the oil phase and water phase is 1:2~6, preferably 1:4~5, more preferably 1:4.
In a kind of currently preferred specific implementation mode, in step (1), after osteoprotegerin is mixed with phosphatide,
Also include that will be placed on the step of being dried in freeze drier, wherein the cryogenic temperature of freeze drier is -55~-85 DEG C,
Drying time is 1~5 hour.
In more preferably a kind of specific implementation mode of the present invention, in step (1), after being freeze-dried, also wrap
The step of containing vacuum drying, wherein the vacuum drying time is 20-30h.
The average grain diameter of the preparation-obtained nanoparticle for carrying osteoprotegerin of the present invention is 133~148nm, the nanoparticle
Dispersity index be 0.117~0.210.
Preferably, the encapsulation rate of osteoprotegerin is 65~75% in the nanoparticle, the 7 of osteoprotegerin in the nanoparticle
Its burst size is 85~96%, preferably 89~96%.
The preparation-obtained slow release nano-particle of the present invention, in part with certain rate slow release, to a certain extent
Can prevent calcification, and osteoprotegerin is encapsulated in nanoparticle, can to avoid osteoprotegerin by internal protease hydrolytic,
Improve the bioavilability of drug.
The effect of phosphatide used in the present invention is, is combined with osteoprotegerin, can reduce the water-soluble of osteoprotegerin
Property, make it have hydrophobic property.
The equipment and analysis that the manufacturer to raw material and equipment used in the present embodiment and product analysis use below
Method is described as follows, wherein the chemical substance do not indicate be conventional reagent the pure rank of chemistry.
Wherein, the information of the raw material used in embodiment is as follows,
Soybean lecithin is purchased from Shanghai Taiwei Pharmaceutical Co., Ltd.
Polyvinyl alcohol (PVA) is purchased from Aladdin reagent (China) Co., Ltd, product identification：P105128
The tert-butyl alcohol is purchased from Sigma-Aldrich, product identification：471712
Polycaprolactone (PCL) is purchased from Sigma-Aldrich, product identification：704105
Maleimide-polyethylene glycol-polycaprolactone (MAL-PEG-PCL) is purchased from the auspicious auspiciousness limited public affairs of biotechnology in Xi'an
Department, product identification：RP0160885
Triton x-100 (Triton X-100) is purchased from SIAMA companies of the U.S., product identification：X-100
Recombined human osteoprotegerin (OPG) is purchased from PEPROTECH companies of the U.S., product identification：450-14
Fetal calf serum (FBS) is purchased from Hyclone companies of the U.S., product identification：RB35935
0.25% trypsase-EDTA is purchased from Gibco companies of the U.S., product identification：1760553
TransDetectTMCell Counting Kit (CCK) are purchased from Beijing Quanshijin Biotechnology Co., Ltd, production
Product are numbered：FG701-01
Alizarin red dye liquor is purchased from Beijing Suo Laibao companies, product identification：G1452
Osteoprotegerin (OPG) detection kit is purchased from Cloud-Clone companies of the U.S.
CD34 antibody is purchased from Abcam companies, product identification：ab187284
CD45, CD29, CD90 antibody are purchased from Biolegend companies
β-phosphoglycerol, vitamin C and dexamethasone are purchased from Sigma Co., USA
QuantiChromTMCalcium Assay Kit are purchased from BioAssy Systems companies of the U.S.
Other reagents such as dichloromethane, dimethyl sulfoxide (DMSO) (DMSO) are purchased from Xilong Chemical Co., Ltd
Experimental facilities used in embodiment is as follows：
Isothermal vibration device：SHA-BA, Changzhou Lang Yue instrument manufacturings Co., Ltd
Freeze drier：FD-1A-50, Beijing Bo Yikang laboratory apparatus Co., Ltd
Transmission electron microscope：JEM-2100, Japan
Electronic balance：BSA124S, Sai Duolisi scientific instrument Co., Ltd
Multi-function microplate reader：VARIOSKAN, Thermo Fischer Scient Inc. of the U.S.
Magnetic stirring apparatus：DF-101S, Ke Xi Instrument Ltd. of Jintan City
Ultracentrifuge：OptimaTML-100KUltracentrifuge, Beckman Kurt commerce and trade company of the U.S.
Laser granulometry：PSA NANO2590, Malvern company of Britain
CO2Cell incubator：HERACELL 150i, Thermo Fischer Scient Inc. of the U.S.
Ultraviolet specrophotometer：UV-9600, Beijing North divide Rayleigh Analytical Instrument Co., Ltd
Ultrasonic cell disruptor：II D of SCIENTZ-, NingBo XinZhi Biology Science Co., Ltd
Flow cytometer：FACS Calibur, U.S. company BD
Inverted microscope：Co., Ltd in AE2000, Motic
Embodiment one
1. carrying the preparation of OPG nanoparticles
(1) preparation of osteoprotegerin (OPG)-phosphatide complexes
1ugOPG is dissolved in sterile tri-distilled water and adjusts a concentration of 1ug/ml, 10mg soybean lecithins is accurately weighed and is dissolved in
In the 1ml tert-butyl alcohols, then by 1ml 1ug/ml OPG aqueous solutions and 1ml 10mg/ml soybean lecithins/t-butanol solution liquid relief
Device is fully blown and beaten uniformly, is evacuated after 2 hours in -60 DEG C of first pre-freezes in freeze drier 22 hours dry, obtains OPG- phosphatide
Compound.
(2) preparation of OPG nanoparticles is carried
It is prepared using emulsion-solvent evaporation method and carries OPG nanoparticles.It is accurate weigh 4mg maleimides-polyethylene glycol oneself
Lactone (MAL-PEG-PCL) and 12mg polycaprolactones (PCL) are dissolved in 1.5ml dichloromethane, then again that step (1) is prepared
OPG- phosphatide complexes be dissolved in wherein formed oil phase (O), 6ml 2%PVA aqueous solutions be used as water phase (W), by oil phase (O) slowly
It is added dropwise in water phase (W), is placed in ultrasonic cup with 500 turns/min stirrings 1min on magnetic stirring apparatus, 60W under condition of ice bath
Ultrasonic 2min forms colostrum (O/W), and colostrum, which is then placed in stirring 4.5h on magnetic stirring apparatus, fully volatilizes dichloromethane, then adds
Enter 100ul Triton X-100 to continue to stir the nanoparticle that 30min obtains carrying OPG.
Blank nanoparticle is prepared using blank phosphatide complexes, and OPG is not contained, other operating procedures are as above.
2. carrying the characterization of OPG nanoparticles
2.1Zeta current potentials and grain size
Take 1ml nanoparticles suspension and 4ml tri-distilled waters to mix well, with laser granulometry to the grain size of nanoparticle,
Zeta potential and dispersity index (PDI) are investigated.
Testing result is as shown in Figures 2 and 3, figure it is seen that the nanometer particle size prepared is 133nm, PDI is
0.131, from figure 3, it can be seen that Zeta potential is -27.8mV.
2.2 transmission electron microscope observation nanoparticle surface topographies
It draws the diluted nanoparticle suspensions of 10ul to be added dropwise on copper mesh, naturally dry is placed on 2% phosphotungstic acid negative staining
Penetrate observed under electron microscope nanoparticle surface topography.
Nanoparticle surface topography is as shown in figure 4, as can be seen from Figure 4：Nanoparticle is rounded, and size rule is evenly distributed,
Have no phenomenon adhered to one another.
The measurement of 2.3 encapsulation rates
It takes the nanoparticle suspension of preparation appropriate, supernatant is taken after low-temperature and high-speed centrifugation (32000rpm, 20min, 4 DEG C), survey
Determine free OPG contents in supernatant and is denoted as WTrip, by dimethyl sulfoxide (DMSO) (DMSO) and nanoparticle suspension mixing, keep nanoparticle completely molten
Total OPG contents are measured after solution is denoted as WAlways, above-mentioned OPG contents pass through enzyme linked immunosorbent assay, nanoparticle encapsulation rate
(entrapment efficiency, EE) calculation formula is：EE=(WAlways-WTrip)/WAlways× 100%
By one obtained nanoparticle of embodiment using enzyme linked immunosorbent assay, the encapsulation rate for obtaining nanoparticle is
75%.
2.4 carry OPG nanoparticle extracorporeal releasing characteristics
It takes suitable nanoparticle suspension, low-temperature and high-speed to centrifuge (32000rpm, 20min, 4 DEG C), discards supernatant liquid, precipitation is used
5mlPBS disperses to be placed on isothermal vibration device again with 37 DEG C, 60rpm sustained oscillations, defined time point (6h, 12h,
For 24 hours, 2d, 3d, 5d, 7d) (32000rpm, 20min, 4 DEG C) is centrifuged again, supernatant is taken, is provided with enzyme-linked immunosorbent assay
OPG contents in time point supernatant calculate OPG cumulative release amounts.
The OPG contents in above-mentioned defined time point detection supernatant, draw OPG Cumulative release profiles, as shown in Figure 5.From
Release profiles can be seen that at release initial stage, and OPG is released with faster speed from nanoparticle, but it is existing not occur burst release
As.OPG Cumulative release amounts reach 52% within 24 hours, later slow release, are discharged up to 89% until the 7th day OPG is accumulative.
3. carry OPG nanoparticles to rat bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells,
BMSCs influence)
Separation, the culture of 3.1BMSCs
3 weeks size male SD rats (coming from medical college of University Of Nanchang animal center) are put to death using cervical dislocation,
75% ethyl alcohol impregnates 10min, detaches rat femur and shin bone under aseptic condition, extra muscle is removed with eye scissors, cuts stock
The epiphysis end of bone and shin bone is extracted with 5ml syringes containing 10% fetal calf serum (FBS), 100U/ml penicillin, 100ug/ml chains
The culture medium of mycin rinses ossis, and the marrow gone out is blown and beaten mixing, stands 5min, collects the cell suspension in addition to precipitation,
It centrifuges (1000rpm, 5min), discards supernatant, with complete medium again cell dispersion, fully blow and beat mixing and be placed on new training
It supports in bottle, in 37 DEG C, 5%CO2It is cultivated in incubator, full dose changes liquid within the 4th day, changes liquid every 2 days later 1 time.
It is observed using inverted microscope, as in Figure 6-1, just inoculated cell is rounded, suspends in the medium.
To the 3rd day, part cell started adherent growth for culture, is in spindle shape, sees Fig. 6-2.5th day, most cells were adherent, shape
Extend outwardly at cell colony and gradually, sees Fig. 6-3.7th day, cell confluency to 70% or more, cell was arranged with certain direction
Row, show swirl shape, see Fig. 6-4.Cell growth is vigorous after passage, and form is uniform, is in spindle shape adherent growth, sees Fig. 6-5
And 6-6.
3.2 Flow cytometry BMSCs surface antigen expressions
It is 1 × 10 to take the sterile PBS cell dispersions of eugonic 3rd generation BMSCs and adjust cell density6/ ml is drawn
In 500ul to EP pipes, into each EP pipes be added 10ul fluorescein isothiocynates (FITC) mark CD29, CD90, CD45 and
The CD34 antibody of 10ulPE labels, using homotype IgG antibody as Isotype control, 4 DEG C are protected from light incubation 30min, with the sterile PBS of 500ul
In being detected on flow cytometer after cleaning 3 times.
Shown in FCM analysis result figure 7-1 to 7-4, as can be seen that BMSCs high expression from Fig. 7-1 and Fig. 7-2
CD29 and CD90, and from Fig. 7-3 and Fig. 7-4 as can be seen that BMSCs is not expressed or extremely low expression CD34 and CD45.
3.3 carry detection of the OPG nanoparticles to cytotoxicity
The influence for carrying OPG nanoparticles and being proliferated to BMSCs is detected with CCK methods, takes the nanoparticle suspension prepared appropriate, low temperature
It discards supernatant after high speed centrifugation (32000rpm, 20min, 4 DEG C), is disperseed again with the sterile PBS of 2ml, then use 0.22um sterile
It is spare after membrane filtration.Experimental setup blank nanoparticle group (NL-OPG-NPs) carries OPG nanoparticles group (OPG-NPs), is simple
PBS control group, blank control group.Take eugonic 3rd generation BMSCs with 1 × 104The density of/ml is seeded in 96 well culture plates
On, it is inoculated with 100ul cell suspending liquids per hole, is placed in 37 DEG C, 5%CO2After being cultivated for 24 hours in incubator, it is added in corresponding hole
100ulPBS is only added in 10ulNL-OPG-NPs, OPG-NPs, PBS, blank well, respectively at 12h, for 24 hours, the training that more renews after 48h
10ulCCK detection liquid is simultaneously added in nutrient solution, continues to be incubated the absorbance that 2h is placed on each hole at measurement 450nm on enzyme mark detector
Value.
CCK is that a kind of cell Proliferation based on water-soluble tetrazolium salts and cytotoxicity detection reagent, tetrazolium salts can be by lines
Dehydrogenase in plastochondria be reduced to soluble orange-yellow formazan , formazans quantity and living cells quantity it is proportional, i.e. color
More deeply feeling, clear-cells proliferation is faster, and cytotoxicity is smaller, and cell quantity is more.Pass through from figure 8, it is seen that nanoparticle is added
12h, for 24 hours, the culture of 48h compared with control group, NL-OPG-NPs and OPG-NPs groups cell growth is simultaneously not affected by nanoparticle
It influences, no difference of science of statistics (P between each group>0.05), and over time, the absorbance of each group is consequently increased, explanation
Nanoparticle has no effect on the proliferation of BMSCs, and nanoparticle is to BMSCs nontoxicitys.
3.4 carry the external anticalciumization detection of OPG nanoparticles
Mesenchymal stem cell (BMSCs) is that there are have height self-renewal capacity and multi-lineage potential in marrow
Stem cell, can directed differentiation be the tissues such as bone, cartilage, muscle, ligament, with calcification induction liquid (high glucose medium containing DMEM,
10%FBS, 100U/ml penicillin, 100ug/ml streptomysins, 10mmol/L β-phosphoglycerol, 50 μ g/ml vitamin Cs, 100nM
Dexamethasone, hereinafter referred to as DAG) BMSCs is can induce to osteoblast differentiation.Take the load OPG nanoparticles (OPG- prepared
NPs it) is discarded after centrifuging (32000rpm, 20min, 4 DEG C) with blank nanoparticle (NL-OPG-NPs) appropriate suspension, low-temperature and high-speed
Clearly, disperseed again with the sterile PBS of 2ml, it is spare after then 0.22um sterilised membrane filters being used to filter.Experimental setup DAG groups, OPG-NPs
Group, NL-OPG-NPs groups, Normal group, first 3 groups are respectively 40ul PBS, OPG-NPs, NL-OPG-NPs and 500ul calcification
Induce the mixing liquid of liquid, Normal group that 540ul complete mediums are only added.By eugonic 3rd generation BMSCs with 2 ×
104The density of/ml is seeded in 24 well culture plates, is placed in 37 DEG C, 5%CO2It is cultivated in incubator.When cell fusion reaches 60%-
When 70%, carefully culture medium in cultivation plate hole is siphoned away, above-mentioned each group mixing is added to each hole after fully being rinsed with sterile PBS
Liquid.Culture medium is replaced weekly 1 time.7d, 14d, 21d after Osteoinductive differentiation evaluate calcification situation.
3.4.1 Alizarin red staining
Culture plate is cleaned with PBS first, 4% paraformaldehydes of 500ul then are added to each hole, fixes 30min at room temperature,
500ul alizarin red dye liquors are added per hole for the abundant rinsed cleans of PBS, dye 5min, after PBS rinsed cleans under inverted microscope
Observe calcium tubercle coloring.
For calcium tubercle staining conditions as shown in Fig. 9-1 to Fig. 9-4, there is a large amount of calcium tubercle in DAG groups and NL-OPG-NPs groups
(see Fig. 9-1 and 9-2), and assemble agglomerating, and the visible calcium tubercle being dispersed in of OPG-NPs groups, negligible amounts (see Fig. 9-3), normally
Control group has no calcium tubercle (see Fig. 9-4), illustrates that OPG-NPs inhibits calcification process to a certain extent, wherein black portions
To be dyed red part by alizarin red.
3.4.2 doped calcium detects
For quantitative detection each group calcium concentration, 500ul 0.6N HCL, 37 DEG C of overnight incubations are added to each hole.Next day collects each
Group leaching liquor, centrifuges (2500rpm, 5min), collects supernatant.With reference to calcium immue quantitative detection reagent box (QuantiChromTM
Calcium Assay Kit) specification, 5ul supernatants are added dropwise to 96 well culture plate hole bottoms, are then added again into every hole
200ul detect working solution, at room temperature place 5min after on enzyme mark detector measure 630nm at each hole absorbance value.
Each group doped calcium situation indicated by the calcium ion concentration extracted, as shown in Figure 10.Osteogenic induction the 7th
It, the calcium ion concentration of DAG groups has significant difference (P compared with OPG-NPs groups<0.05).To the 14th day, DAG groups and NL-
The calcium ion concentration of OPG-NPs groups obviously increases, and has significant significant difference (P compared with OPG-NPs groups<0.001).To
21 days, the calcium ion concentration of each group continued growing, and wherein DAG groups, NL-OPG-NPs groups and OPG-NPs group calcium ion concentrations are bright
It is aobvious to increase, but OPG-NPs group calcium ion concentrations are still significantly lower than DAG groups and NL-OPG-NPs groups (P<0.001).1-14 days,
No difference of science of statistics (P between OPG-NPs groups calcium ion concentration and Normal group>0.05), at the 21st day, OPG-NPs groups and
Normal group calcium ion concentration differs greatly (P<0.001) it, but with DAG groups and NL-OPG-NPs groups compares, OPG-NPs groups are also
It is to show stronger anticalcium effect.
Embodiment two
1. carrying the preparation of OPG nanoparticles
(1) preparation of osteoprotegerin (OPG)-phosphatide complexes
0.5ugOPG is dissolved in sterile tri-distilled water and adjusts a concentration of 1ug/ml, it is molten accurately to weigh 5mg soybean lecithins
In the 0.5ml tert-butyl alcohols, then 1ml 1ug/mlOPG aqueous solutions and 1ml 10mg/ml soybean lecithins/t-butanol solution are moved
Liquid device is fully blown and beaten uniformly, is evacuated after 1 hour in -55 DEG C of first pre-freezes in freeze drier 20 hours dry, obtains OPG- phosphorus
Fat complexes.
(2) preparation of OPG nanoparticles is carried
It is prepared using emulsion-solvent evaporation method and carries OPG nanoparticles.It is accurate weigh 6mg maleimides-polyethylene glycol oneself
Lactone (MAL-PEG-PCL) and 15mg polycaprolactones (PCL) are dissolved in 1ml dichloromethane, then again will be prepared by step (1)
OPG- phosphatide complexes, which are dissolved in, wherein forms oil phase (O), and 5ml 2%PVA aqueous solutions are slowly dripped as water phase (W), by oil phase (O)
It adds in water phase (W), is placed in ultrasonic cup with 500 turns/min stirrings 0.5min on magnetic stirring apparatus, 60W under condition of ice bath
Ultrasonic 2min forms colostrum (O/W), and colostrum, which is then placed in stirring 4h on magnetic stirring apparatus, fully volatilizes dichloromethane, adds
50ul Triton X-100 continue to stir the nanoparticle that 30min obtains carrying OPG.
Blank nanoparticle is prepared using blank phosphatide complexes, and OPG is not contained, other operating procedures are as above.
Two preparation-obtained nanoparticle of embodiment carries out grain according to the method for embodiment one using laser granulometry
The measurement of diameter, Zeta potential and dispersity index (PDI), measurement result are：Zeta potential is -21.7mV, dispersity index
(PDI) it is 0.117, grain size 131.5nm.
Two preparation-obtained nanoparticle of embodiment is carried out to the test of encapsulation rate according to the method described in embodiment one,
As a result：Encapsulation rate is 65%.
Two preparation-obtained nanoparticle of embodiment is released into experiment in vitro according to the method progress described in embodiment one, as a result
For：The burst size accumulation of 7th day osteoprotegerin reaches 96% or so.
Embodiment three
1. carrying the preparation of OPG nanoparticles
(1) preparation of osteoprotegerin (OPG)-phosphatide complexes
2ugOPG is dissolved in sterile tri-distilled water and adjusts a concentration of 1ug/ml, 8mg soybean lecithins is accurately weighed and is dissolved in
In the 0.8ml tert-butyl alcohols, then by 1ml 1ug/mlOPG aqueous solutions and 1ml 10mg/ml soybean lecithins/t-butanol solution liquid relief
Device is fully blown and beaten uniformly, is evacuated after 3 hours in -70 DEG C of first pre-freezes in freeze drier 25 hours dry, obtains OPG- phosphatide
Compound.
(2) preparation of OPG nanoparticles is carried
It is prepared using emulsion-solvent evaporation method and carries OPG nanoparticles.It is accurate weigh 5mg maleimides-polyethylene glycol oneself
Lactone (MAL-PEG-PCL) and 18mg polycaprolactones (PCL) are dissolved in 1.75ml dichloromethane, then again that step (1) is made
Standby OPG- phosphatide complexes, which are dissolved in, wherein forms oil phase (O), and 8ml 2%PVA aqueous solutions are slow by oil phase (O) as water phase (W)
Slowly it is added dropwise in water phase (W), is placed in ultrasonic cup with 500 turns/min stirrings 1.5min on magnetic stirring apparatus, condition of ice bath
Lower 60W ultrasounds 2min forms colostrum (O/W), and colostrum, which is then placed in stirring 6h on magnetic stirring apparatus, fully volatilizes dichloromethane,
200ul Triton X-100 are added to continue to stir the nanoparticle that 30min obtains carrying OPG.
Blank nanoparticle is prepared using blank phosphatide complexes, and OPG is not contained, other operating procedures are as above.
Three preparation-obtained nanoparticle of embodiment carries out grain according to the method for embodiment one using laser granulometry
The measurement of diameter, Zeta potential and dispersity index (PDI), measurement result are：Zeta potential is -19.2mV, dispersity index
(PDI) it is 0.203, grain size 123.9nm.
Three preparation-obtained nanoparticle of embodiment is carried out to the test of encapsulation rate according to the method described in embodiment one,
As a result：Encapsulation rate is 69%.
Three preparation-obtained nanoparticle of embodiment is released into experiment in vitro according to the method progress described in embodiment one, as a result
For：The burst size accumulation of 7th day osteoprotegerin reaches 92% or so.
Example IV
1. carrying the preparation of OPG nanoparticles
(1) preparation of osteoprotegerin (OPG)-phosphatide complexes
3ugOPG is dissolved in sterile tri-distilled water and adjusts a concentration of 1ug/ml, 15mg soybean lecithins is accurately weighed and is dissolved in
In the 1.5ml tert-butyl alcohols, then by 1ml 1ug/mlOPG aqueous solutions and 1ml 10mg/ml soybean lecithins/t-butanol solution liquid relief
Device is fully blown and beaten uniformly, is evacuated after 5 hours in -80 DEG C of first pre-freezes in freeze drier 30 hours dry, obtains OPG- phosphatide
Compound.
(2) preparation of OPG nanoparticles is carried
It is prepared using emulsion-solvent evaporation method and carries OPG nanoparticles.It is accurate weigh 8mg maleimides-polyethylene glycol oneself
Lactone (MAL-PEG-PCL) and 24mg polycaprolactones (PCL) are dissolved in 2ml dichloromethane, then again will be prepared by step (1)
OPG- phosphatide complexes, which are dissolved in, wherein forms oil phase (O), and 10ml 2%PVA aqueous solutions are slow by oil phase (O) as water phase (W)
It is added dropwise in water phase (W), is placed in ultrasonic cup with 500 turns/min stirrings 3min on magnetic stirring apparatus, 60W under condition of ice bath
Ultrasonic 2min forms colostrum (O/W), and colostrum, which is then placed in stirring 8h on magnetic stirring apparatus, fully volatilizes dichloromethane, adds
300ul Triton X-100 continue to stir the nanoparticle that 30min obtains carrying OPG.
Blank nanoparticle is prepared using blank phosphatide complexes, and OPG is not contained, other operating procedures are as above.
The preparation-obtained nanoparticle of example IV carries out grain according to the method for embodiment one using laser granulometry
The measurement of diameter, Zeta potential and dispersity index (PDI), measurement result are：Zeta potential is -12.9mV, dispersity index
(PDI) it is 0.150, grain size 147.6nm.
The preparation-obtained nanoparticle of example IV is carried out to the test of encapsulation rate according to the method described in embodiment one,
As a result：Encapsulation rate is 66%.
The preparation-obtained nanoparticle of example IV is released into experiment in vitro according to the method progress described in embodiment one, as a result
For：The burst size accumulation of 7th day osteoprotegerin reaches 93% or so.
Embodiment two to the preparation-obtained slow release nano-particle of example IV all has stronger anticalcium effect.
In conclusion the present invention utilizes biodegradable material, osteoprotegerin is encapsulated in nanoparticle, osteoprotegerin is made
Can from nanoparticle with certain rate local slow release, the present invention it is obtained carry osteoprotegerin slow release nanometer
Grain, the in vitro release rate of osteoprotegerin is up to 85~96% in 7 days, and the obtained slow release nano-particle for carrying OPG is in certain degree
On inhibit calcification process, and show stronger anticalcium effect.
The above is only the preferred embodiment that the present invention is implemented, and not does limitation in any form to the present invention, all
The modifications, equivalent substitutions and improvements etc. done within the spirit and principles in the present invention are required to the protection included in the present invention
Within the scope of.
Claims (14)
1. a kind of slow release nano-particle carrying osteoprotegerin, which is characterized in that the nanoparticle is by containing osteoprotegerin, Malaysia acyl
The raw material of imines-polyethylene glycol-polycaprolactone and polycaprolactone is prepared.
2. nanoparticle according to claim 1, wherein the osteoprotegerin is 0.5-3 parts by weight, preferably 0.5-2 weights
Measure part；Maleimide-the polyethylene glycol-polycaprolactone is 4000-8000 parts by weight, preferably 4000-6000 parts by weight；
The pla-pcl is 12000-24000 parts by weight, preferably 12000-18000 parts by weight.
3. nanoparticle according to claim 1, wherein the nanoparticle is by containing osteoprotegerin, phosphatide, maleimide
The raw material of amine-polyethylene glycol-polycaprolactone and polycaprolactone is prepared.
4. nanoparticle according to claim 3, wherein the osteoprotegerin is 0.5-3 parts by weight, preferably 0.5-2 weights
Measure part；The phosphatide is 5000-15000 parts by weight, preferably 5000-10000 parts by weight；The poly- second of the maleimide-two
Alcohol-polycaprolactone is 4000-8000 parts by weight, preferably 4000-6000 parts by weight；The pla-pcl is 12000-24000
Parts by weight, preferably 12000-18000 parts by weight.
5. according to claim 1-4 any one of them nanoparticles, wherein the nanoparticle is prepared by the following method：
The mixture that osteoprotegerin, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent are formed is made
It is ultrasonically treated to obtain using the aqueous solution of water-soluble polymer as water phase for oil phase.
6. nanoparticle according to claim 3 or 4, wherein the nanoparticle is prepared by the following method：
(1) osteoprotegerin and phosphatide are mixed to get osteoprotegerin-phosphatide complexes；
(2) by osteoprotegerin-phosphatide complexes, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent
The mixture of formation is as oil phase, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain.
7. according to claim 1-6 any one of them nanoparticles, wherein the molecular weight of the polycaprolactone be 40000~
50000。
8. according to claim 1-8 any one of them nanoparticles, wherein the maleimide-polyethylene glycol-polycaprolactone
Molecular weight be 10000~20000, the molecular weight of the polyethylene glycol segment is 1000~2000.
9. the preparation method of any one of the claim 1-8 nanoparticles, which is characterized in that it includes following step：
The mixture that osteoprotegerin, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent are formed is made
It is ultrasonically treated to obtain using the aqueous solution of water-soluble polymer as water phase for oil phase.
10. preparation method according to claim 9, wherein it includes following step：
(1) osteoprotegerin and phosphatide are mixed to get osteoprotegerin-phosphatide complexes；
(2) by osteoprotegerin-phosphatide complexes, maleimide-polyethylene glycol-polycaprolactone and polycaprolactone and organic solvent
The mixture of formation is as oil phase, using the aqueous solution of water-soluble polymer as water phase, is ultrasonically treated to obtain.
11. preparation method according to claim 9 or 10, wherein the volume ratio of the oil phase and water phase is 1:2~6, it is excellent
It is selected as 1:4~5.
12. according to claim 9-11 any one of them preparation methods, wherein the water-soluble polymer is selected from polyethylene
Alcohol, polysorbate or fatty acid sorbitan, preferably polyvinyl alcohol.
13. according to claim 9-12 any one of them preparation methods, wherein the organic solvent selected from dichloromethane,
Chloroform or acetone, preferably dichloromethane.
What 14. claim 1-8 any one of them nanoparticle or claim 9-13 any one of them methods were prepared
Application of the nanoparticle in nano drug-carrying material.
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