CN105288594A - Growth factor porous micro-sphere compound system coated by injectable hydrogel - Google Patents
Growth factor porous micro-sphere compound system coated by injectable hydrogel Download PDFInfo
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- CN105288594A CN105288594A CN201510863496.XA CN201510863496A CN105288594A CN 105288594 A CN105288594 A CN 105288594A CN 201510863496 A CN201510863496 A CN 201510863496A CN 105288594 A CN105288594 A CN 105288594A
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Abstract
The invention relates to the preparation of a growth factor porous micro-sphere compound system coated by an injectable hydrogel. The preparation is characterized by comprising a first component, namely HA-ADH sol obtained by cross-linking of hyaluronic acid (HA) and adipic dihydrazide (ADH), and polylactic acid (PLLA) porous micro-spheres which are carried with growth factors and loaded in the first component. The compound system is capable of degrading and slowly releasing the growth factors under physiological conditions, so that the slow releasing of the growth factors in a body is realized, and the problems that the growth factors are released too fast and an action period is too short are solved, thereby reaching the effect of neural restoration.
Description
Technical field
The invention belongs to nervous tissue's reparation field of biomedical materials, be specifically related to a kind of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel.
Background technology
Peripheral nerve defection and to repair be one of disease of the puzzlement numerous people in the world always, how caused by cross-section wound, burn or degenerative disease etc.Broken ends of fractured bone N/D can be divided into damage for peripheral nerve injury and the broken ends of fractured bone has defect two class.Damage for N/D between the broken ends of fractured bone can adopt adventitia to coincide, and bundle film coincide and waits operation method to carry out identical reparation.And the reparation of the peripheral nerve defection of long section and reconstruction remain one of clinical difficult problem, have had a strong impact on the quality of life of patient.Since eighties of last century, utilized inanimate catheter treatment peripheral nerve defection to become study hotspot, the non-degradable material taking silica gel as representative, due to its prolonged stay original place, can bring out the inflammatory reaction of surrounding tissue, and a large amount of cicatrixs also can be caused to produce.Although be that the Biodegradable nerve conduit material of representative can reach the effect similar with nerve autograft with polylactic acid, but then can not support neuranagenesis for the neurologic defect of long section, and because the implantation of foreign body inevitably produces inflammatory reaction, the success rate of the operation of impact and postoperative recovery.This may be owing to lacking the cause of the internal matrix such as somatomedin as supporting tissue, is therefore very important at the material of the built-in promotion nerve growth of nerve trachea and reparation.
Nerve growth factor (NGF) is a kind of nerve growth regulatory factor with neurotrophic and short enation double biological function.Research shows, NGF has great expression in nerve injury place, and it all has certain facilitation to the propagation of neurocyte, neuronic regeneration and differentiation etc.Basic fibroblast growth factor (bFGF) is also called corium somatomedin, to neuroectodermal cell, there is biologic activity widely, cell life microenvironment can be improved, promote the regeneration of injured nerve, be neurotrophic and regeneration factor, have the title of " cell activating agent ".Long felt through people finds, use in conjunction nerve growth factor and basic fibroblast growth factor can promote the division growth of neural stem cell, increases the ratio of Differentiation of Neural Stem Cells.But compared with the half-life of bFGF10h, the biological half life of NGF is too short, and easily lose activity by the impact of other materials, limit its scope of application.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of wrapped up by injection aquagel somatomedin microsphere hybrid system, preparation method and application.This hybrid system can be degraded in physiological conditions and slowly release somatomedin, achieves somatomedin slow releasing in vivo, solves the problem that growth factor release is too fast, the action period is too short, thus reaches the effect of CO2 laser weld., in order to solve above technical problem, the technical solution used in the present invention is:
A kind of preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel, it comprises makes the first component with the crosslinked HA-ADH colloidal sol obtained of hyaluronic acid (HA) and adipic dihydrazide (ADH), and polylactic acid (PLLA) porous microsphere that be loaded with somatomedin of load in the first component.
By such scheme, described in be loaded with somatomedin in polylactic acid (PLLA) porous microsphere of somatomedin mass percent be 4wt%-12.5wt%.
By such scheme, described somatomedin is nerve growth factor and basic fibroblast growth factor bFGF, and its mass ratio is 8.5-11.5:1.
By such scheme, HA-ADH colloidal sol compares for 1:1.25-5.75 with polylactic acid (PLLA) the porous microsphere mixing quality being loaded with somatomedin.
By such scheme, described HA-ADH colloidal sol be by hyaluronic acid (HA) and adipic dihydrazide (ADH) in acid condition and under the activation of EDC/NHS activator 0-4 DEG C be obtained by reacting obtain.
By such scheme, described hyaluronic acid and the mass ratio of adipic dihydrazide are 1:2-6, and described acid condition is pH is 4.5-5.75.
By such scheme, described in be loaded with polylactic acid (PLLA) porous microsphere of somatomedin average diameter be 2 ± 1 μm.
By such scheme, described polylactic acid (PLLA) porous microsphere being loaded with somatomedin is the solution containing somatomedin being added with porogen and emulsifying agent is interior aqueous phase, take PLLA as oil phase, be outer aqueous phase with the solution containing emulsifying agent, use W/O/W multi-emulsion method to obtain emulsion, then stir, leave standstill solidification, centrifugal collecting precipitation, washing, lyophilizing obtains.
A kind of preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel is as follows:
(1) hyaluronic acid (HA) and adipic dihydrazide (ADH) are obtained HA-ADH colloidal sol in acid condition and under the activation of EDC/NHS activator;
(2) be interior aqueous phase to be added with the solution containing NGF and bFGF component of porogen and emulsifying agent, with polylactic acid (PLLA) for oil phase, be outer aqueous phase with the solution containing PVA, W/O/W multi-emulsion method is used to obtain emulsion, then stir, leave standstill solidification, centrifugal collecting precipitation, washing, lyophilizing obtains growth factor-loaded polylactic acid (PLLA) porous microsphere;
(3) HA-ADH colloidal sol above-mentioned steps obtained and growth factor-loaded polylactic acid (PLLA) porous microsphere stir.
By such scheme, the W/O/W multi-emulsion method of described step (2) is: be dissolved in by PLLA in dichloromethane, obtain oil phase, then join be added with porogen and emulsifying agent containing in the aqueous phase of somatomedin, stirring or forming W/O/W type emulsion under ultrasonic condition, and then the outer aqueous phase added wherein containing emulsifying agent, high-speed stirred a period of time becomes emulsion.Use W/O/W multi-emulsion method, obtain growth factor-loaded polylactic acid (PLLA) porous microsphere, hydrophilic somatomedin can be avoided to be attached to microsphere surface to continuous phase seepage, cause the prominent problem released.
By such scheme, in described step (2), the envelop rate of somatomedin is 55-65wt%.
By such scheme, step (1) response time is 18-24h.
By such scheme, the freeze-drying time in step (2) is 24-48h, and hardening time is 80-120min.
By such scheme, step (3) whipping temp is 0-4 DEG C.
By such scheme, in step (2) aqueous phase, polylactic acid (PLLA) is 1:0.67-0.89 with the amount ratio of porogen, polylactic acid (PLLA) is 1:1-1.33 with the amount ratio of emulsifying agent, and in outer aqueous phase, polylactic acid (PLLA) is 1:0.53-0.71 with the amount ratio of emulsifying agent.
By such scheme, described step (2) porogen is sodium carbonate (Na
2cO
3); Emulsifying agent is polyvinyl alcohol (PVA), and PVA relative number average molecular weight is 35000-50000.
Beneficial effect of the present invention:
1. the slow-releasing of somatomedin
It is growth factor-loaded that polylactic acid is made porous microsphere by the present invention, improve drug loading and the stability of somatomedin, then by this medicine carrying porous microsphere load in HA-ADH colloidal sol, after being used HA-ADH colloidal sol to wrap up, the somatomedin microsphere hybrid system of being wrapped up by injection aquagel obtained, on the one hand: gel state can be changed under human physiological environment, thus, to porous polylactic acid microball, there is good parcel and fixation, on the other hand, in physiological conditions, polylactic acid molecule chain generation chemolysis becomes micromolecule, its growth-factor medication comprised discharges into body fluid gradually in the process, starting stage drug release is slow, along with the rapid degraded of later stage polylactic acid, drug release rate constantly increases.The even slow releasing of medicine can be realized thus, can avoid and direct contact of organizing and cause adhesion and prominently to release simultaneously.
The concertedness of 2.NGF and bFGF
Nerve growth factor (NGF) is one of most important bioactive substance in nervous system, all has important biological action to aspects such as neural normal development and promotion regeneration.Basic fibroblast growth factor (bFGF) is also called corium somatomedin, to neuroectodermal cell, there is biologic activity widely, cell life microenvironment can be improved, promote the regeneration of injured nerve, be neurotrophic and regeneration factor, have the title of " cell activating agent ".Research shows, use in conjunction nerve growth factor and basic fibroblast growth factor can promote the division growth of neural stem cell, increases the ratio of Differentiation of Neural Stem Cells.But compared with the half-life of bFGF10h, the biological half life of NGF is too short, and easily lose activity by the impact of other materials, limit its scope of application.Complex microsphere system of the present invention can realize the slow Stable Release of these two kinds of somatomedin, overcome the NGF somatomedin half-life short and subject to the shortcoming of chemical depletion, can reach better than the effect being used alone NGF and better promoting defect CO2 laser weld based on the synergy of nerve growth factor and basic fibroblast growth factor.
3. the protectiveness of CO2 laser weld process
Hyaluronic acid gel as adhesive operating theater instruments in operation, in intra-operative protective tissue, and can provide a security tool abdicating space and moving tissue to operation; Post operation, at operative site, namely this hydrogel becomes adhesive surgery implant, ensures that injured nerve separates with other tissue surfaces, provides a barrier preventing postoperative intestinal adhesion.Hyaluronic acid is as desirable nature moisturizing factor, and firmly can keep the moisture of 98% after surgery, the reparation for nerve provides good microenvironment.Hyaluronic parcel makes polylactic acid medicine carrying porous microsphere dispersed, avoids microsphere and contacts with the direct of injured nerve, medicaments uniformity is discharged slowly.
4. good biocompatibility
Hyaluronic acid (HA) is as a kind of mucopolysaccharide be extensively present in body fluid and tissue; be considered to the polysaccharide of a kind of packing space, rock-steady structure, painting confluent monolayer cells and Cell protection; its Main Function is fiber and Membrane protein conformation between stabilized cell, and therefore highly purified HA can be applied to the sensitive part such as nerve and eyes.After crosslinked with ADH, with the HA fluid-phase ratio applied separately, add the viscoelasticity of HA and better rheological property, extend the holdup time in the tissue, and good biocompatibility can be kept, simple HA hydrogel mechanical property can be avoided low simultaneously, comparatively fragile, the problem of easily subsiding in vivo.
Polylactic acid (PLLA) is as a kind of high molecular polymer non-immunogenicity, also be a kind of there is comparatively excellent biocompatibility and biodegradable, finally being degraded to carbon dioxide and water in vivo, is a kind of desirable biodegradation material that can be used for human body.
5. the simple and feasible prepared of system
The preparation process of hyaluronic acid gel and polylactic acid medicine carrying porous microsphere hybrid system is comparatively simple, and reaction temperature is all at room temperature, and reaction condition comparative maturity, has stronger operability.Use W/O/W multi-emulsion method, avoid hydrophilic somatomedin and be attached to microsphere surface to continuous phase seepage, cause the prominent problem released.
6. the broad applicability of this system
The somatomedin porous microsphere hybrid system of this hyaluronic acid parcel not only can be applicable to the reparation of peripheral nerve defection, by porous polylactic acid microball load different pharmaceutical, is widely used in ophthalmology, otorhinolaryngology disease etc.This drug-supplying system is expected to solve that peripheral nerve injury repair process Chinese medicine action time is short, effect system unstable, postoperative tissue adhesion and recover the problem such as slow.
Accompanying drawing explanation
Fig. 1 is the HA-ADH colloidal sol nuclear magnetic spectrum of the embodiment of the present invention 1;
Fig. 2 be the present invention's somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel prepare schematic diagram;
The outer weight-loss ratio curve chart of average body of a kind of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel that Fig. 3 provides for embodiment of the present invention 1-5;
Fig. 4 is the NGF average cumulative release rate curve chart of the somatomedin porous microsphere hybrid system that embodiment 1-5 provided by the invention a kind is wrapped up by injection aquagel.
Detailed description of the invention
For a better understanding of the present invention, set forth content of the present invention further below in conjunction with embodiment, but content of the present invention, be not only confined to the following examples.
Embodiment 1
A kind of preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel
(1) under 0-4 DEG C of condition, be dissolved in by 0.5gHA in 100ml ultra-pure water, magnetic agitation adds 2gADH after fully dissolving, and then adds 0.8gEDC and 0.7gNHS, and 1mol/lHCl regulates pH to 5.0, and reaction 20h, dialyses and obtain HA-ADH colloidal sol;
(2) PLLA of 1.5g is dissolved in 20mL dichloromethane, then joins NGF, bFGF (NGF4.5mg/ml containing 1g sodium carbonate and 1.5gPVA; BFGF is 0.5mg/ml) in aqueous phase, stirring or forming O/W type emulsion under ultrasonic condition, then add the outer aqueous phase of the 26.7%PVA of 3ml wherein, after high-speed stirred a period of time one-tenth emulsion, be transferred to by emulsion in beaker, 300rpm stirs 80min solidification.Centrifugal collecting precipitation also retains supernatant aqueous phase, wash three times and retain cleaning mixture, then lyophilizing precipitation 24h;
(3), at 0-4 DEG C, HA-ADH colloidal sol 0.35g above-mentioned steps prepared and growth factor-loaded PLLA porous microsphere 1.25g stirs and get final product.
The structural characterization of HA-ADH colloidal sol
By the hydrogel adjusted to ph 7.4 that above-mentioned steps (1) obtains, to changing gel state into completely, lyophilization 24-48h is for subsequent use to be placed in 37 DEG C of thermostat water baths (simulation human physiological environment).
The hydrogel sample of getting after 6-10mg lyophilization is dissolved in the deuterated heavy water of 500 μ l, carries out nucleus magnetic hydrogen spectrum detection.Nuclear magnetic spectrum is shown in Fig. 1.Nuclear magnetic spectrum shows: the peak at δ 1.70 place belongs to the amino hydrogen in a place in Fig. 1, the peak at δ 1.40 (Fig. 1 c place) and 1.90 (Fig. 1 b place) places belongs to the hydrogen on methylene respectively, δ 2.90-δ 3.60 place is for containing hyaluronic structure peak, be containing hyaluronic mark, prove that ADH and HA is successfully cross-linked to form amide.
The sign of polylactic acid medicine carrying microballoons envelop rate
Above-mentioned supernatant aqueous phase and cleaning mixture are merged, by the content of enzyme-linked immunosorbent assay somatomedin, by analysis: sample envelop rate of the present invention is 58%.
The detection of polylactic acid medicine carrying microballoons TEM, DOS
Get above-mentioned reactant liquor (before centrifugal) 2ml and carry out TEM, DOS detection, the diameter of polylactic acid medicine carrying microballoons of the present invention is 2.5 μm.
A kind of release amount of medicine of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
On superclean bench, insert aseptic 5ml centrifuge tube by after the sample lyophilization of above-mentioned preparation, add 3ml phosphate buffer (pH=7.4), 37 DEG C of rotating speeds are cultivate in the isothermal vibration incubator of 60r/min.Get lixiviating solution at 1d, 3d, 6d, 9d, 12d, 15d respectively, and add the PBS buffer of equivalent, by enzyme-linked immunosorbent assay, detect the Cumulative release amount of somatomedin, in 15d, somatomedin Cumulative release amount is 68%.
A kind of degraded of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
Get system prepared by 1ml above-mentioned (3) to be placed in 15ml centrifuge tube, add 3mlPBS buffer (pH=7.4), and isothermal vibration (37 DEG C, 60r/min) degraded in shaking bath.Degradation time is respectively 1d, 3d, 6d, 9d, 12d, 15d etc.After corresponding degradation time, take out sample, milli-Q water, detect weight-loss ratio.After testing, sample of the present invention weight-loss ratio in 15d is 71%.
A kind of cytotoxicity of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel detects (PC12 cell)
(1) experiment be divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group.With card punch, above-mentioned material is made 96 orifice plate pore sizes, ultraviolet sterilization, put into 96 orifice plates for subsequent use, often group 5 is parallel.
(2) the PC12 cell that the phase of taking the logarithm grows, adjustment concentration is 2 × 10
4individual/ml, every hole 100 μ l adds in (1) in 96 orifice plates, with the cell culture fluid of sky in contrast.5%CO
2, 37 DEG C cultivate 72h.
(3) measure at 24h, 48h, 72h respectively, concrete grammar is as follows: every hole adds 10 μ lCCK-8 solution, continues to cultivate 4h, measures each hole OD value at 450nm place, calculate the survival rate of cell in incubator.
CCK-8 experimental result
Embodiment 2
A kind of preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel
(1) under 0-4 DEG C of condition, be dissolved in by 1.0gHA in 100ml ultra-pure water, magnetic agitation adds 2.0gADH after fully dissolving, and then adds 1.6gEDC and 1.4gNHS, and 1mol/lHCl regulates pH to 4.5, and reaction 19h, dialyses and obtain HA-ADH colloidal sol;
(2) PLA of 2.25g is dissolved in 20mL dichloromethane, then joins NGF, bFGF (NGF7.875mg/ml containing 2.0g sodium carbonate and 3.0gPVA; BFGF is 0.75mg/ml) in aqueous phase, stirring or forming O/W type emulsion under ultrasonic condition, then add the outer aqueous phase of the 26.7%PVA of 6ml wherein, after high-speed stirred a period of time one-tenth emulsion, be transferred to by emulsion in beaker, 300rpm stirs 100min solidification.Centrifugal collecting precipitation also retains supernatant aqueous phase, wash three times and retain cleaning mixture, then lyophilizing precipitation 36h;
(3), at 0-4 DEG C, HA-ADH colloidal sol 0.85g above-mentioned steps obtained and growth factor-loaded PLLA porous microsphere 1.98g stirs and get final product.
The structural characterization of HA-ADH colloidal sol
By the hydrogel adjusted to ph 7.4 that above-mentioned steps (1) obtains, be placed in 37 DEG C of thermostat water baths to changing gel state into completely, lyophilization 24-48h is for subsequent use.
The structure of characterizing method to HA-ADH colloidal sol of reference example 1 characterizes.Nuclear-magnetism result shows successfully to generate amide.
The sign of polylactic acid medicine carrying microballoons envelop rate
Above-mentioned supernatant aqueous phase and cleaning mixture are merged, by the content of enzyme-linked immunosorbent assay somatomedin, sample envelop rate of the present invention is 60%.
The detection of polylactic acid medicine carrying microballoons TEM, DOS
Get above-mentioned reactant liquor (before centrifugal) 2ml and carry out TEM, DOS detection, the diameter of polylactic acid medicine carrying microballoons of the present invention is 2.7 μm.
A kind of release amount of medicine of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
On superclean bench, insert aseptic 5ml centrifuge tube by after the sample lyophilization of above-mentioned preparation, add 3ml phosphate buffer (pH=7.4), 37 DEG C of rotating speeds are cultivate in the isothermal vibration incubator of 60r/min.Get lixiviating solution at 1d, 3d, 6d, 9d, 12d, 15d respectively, and add the PBS buffer of equivalent, by enzyme-linked immunosorbent assay, detect the Cumulative release amount of somatomedin, in 15d, somatomedin Cumulative release amount is 64%.
A kind of degraded of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
Get system prepared by 1ml above-mentioned (3) to be placed in 15ml centrifuge tube, add 3mlPBS buffer (pH=7.4), and isothermal vibration (37 DEG C, 60r/min) degraded in shaking bath.Degradation time is respectively 1d, 3d, 6d, 9d, 12d, 15d etc.After corresponding degradation time, take out sample, milli-Q water, detect weight-loss ratio.After testing, sample of the present invention weight-loss ratio in 15d is 69%.
A kind of cytotoxicity of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel detects (PC12 cell)
(1) experiment be divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group.With card punch, above-mentioned material is made 96 orifice plate pore sizes, ultraviolet sterilization, put into 96 orifice plates for subsequent use, often group 5 is parallel.
(2) the PC12 cell that the phase of taking the logarithm grows, adjustment concentration is 2 × 10
4individual/ml, every hole 100 μ l adds in (1) in 96 orifice plates, with the cell culture fluid of sky in contrast.5%CO
2, 37 DEG C cultivate 72h.
(3) measure at 24h, 48h, 72h respectively, concrete grammar is as follows: every hole adds 10 μ lCCK-8 solution, continues to cultivate 4h, measures each hole OD value at 450nm place, calculate the survival rate of cell in incubator.
CCK-8 experimental result
Embodiment 3
A kind of preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel
(1) under 0-4 DEG C of condition, be dissolved in by 1.0gHA in 100ml ultra-pure water, magnetic agitation adds 3.0gADH after fully dissolving, and then adds 1.6gEDC and 1.4gNHS, and 1mol/lHCl regulates pH to 5.5, and reaction 21h, dialyses and obtain HA-ADH colloidal sol;
(2) PLA of 1.5g is dissolved in 20mL dichloromethane, then joins NGF, bFGF (NGF9.5mg/ml containing 1g sodium carbonate and 1.5gPVA; BFGF is 1mg/ml) in aqueous phase, stirring or forming O/W type emulsion under ultrasonic condition, then add the outer aqueous phase of the 26.7%PVA of 3ml wherein, after high-speed stirred a period of time one-tenth emulsion, be transferred to by emulsion in beaker, 300rpm stirs 110min solidification.Centrifugal collecting precipitation also retains supernatant aqueous phase, wash three times and retain cleaning mixture, then lyophilizing precipitation 30h;
(3), at 0-4 DEG C, HA-ADH0.87g hydrogel above-mentioned steps obtained and growth factor-loaded PLLA porous microsphere 1.13g stir and get final product.
The structural characterization of HA-ADH colloidal sol
By the hydrogel adjusted to ph 7.4 that above-mentioned steps (1) obtains, be placed in 37 DEG C of thermostat water baths to changing gel state into completely, lyophilization 24-48h is for subsequent use.
The structure of characterizing method to HA-ADH colloidal sol of reference example 1 characterizes.Nuclear-magnetism result shows successfully to generate amide.
The sign of polylactic acid medicine carrying microballoons envelop rate
Above-mentioned supernatant aqueous phase and cleaning mixture are merged, by the content of enzyme-linked immunosorbent assay somatomedin, sample envelop rate of the present invention is 65%.
The detection of polylactic acid medicine carrying microballoons TEM, DOS
Get above-mentioned reactant liquor (before centrifugal) 2ml and carry out TEM, DOS detection, the diameter of polylactic acid medicine carrying microballoons of the present invention is 1.9 μm.
A kind of release amount of medicine of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
On superclean bench, insert aseptic 5ml centrifuge tube by after the sample lyophilization of above-mentioned preparation, add 3ml phosphate buffer (pH=7.4), 37 DEG C of rotating speeds are cultivate in the isothermal vibration incubator of 60r/min.Get lixiviating solution at 1d, 3d, 6d, 9d, 12d, 15d respectively, and add the PBS buffer of equivalent, by enzyme-linked immunosorbent assay, detect the Cumulative release amount of somatomedin, in 15d, somatomedin Cumulative release amount is 69%.
A kind of degraded of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
Get system prepared by 1ml above-mentioned (3) to be placed in 15ml centrifuge tube, add 3mlPBS buffer (PH=7.4), and isothermal vibration (37 DEG C, 60r/min) degraded in shaking bath.Degradation time is respectively 1d, 3d, 6d, 9d, 12d, 15d etc.After corresponding degradation time, take out sample, milli-Q water, detect weight-loss ratio.After testing, sample of the present invention weight-loss ratio in 15d is 75%.
A kind of cytotoxicity of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel detects (PC12 cell)
(1) experiment be divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group.With card punch, above-mentioned material is made 96 orifice plate pore sizes, ultraviolet sterilization, put into 96 orifice plates for subsequent use, often group 5 is parallel.
(2) the PC12 cell that the phase of taking the logarithm grows, adjustment concentration is 2 × 10
4individual/ml, every hole 100 μ l adds in (1) in 96 orifice plates, with the cell culture fluid of sky in contrast.5%CO
2, 37 DEG C cultivate 72h.
(3) measure at 24h, 48h, 72h respectively, concrete grammar is as follows: every hole adds 10 μ lCCK-8 solution, continues to cultivate 4h, measures each hole OD value at 450nm place, calculate the survival rate of cell in incubator.
CCK-8 experimental result
Embodiment 4
A kind of preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel
(1) under 0-4 DEG C of condition, be dissolved in by 0.5gHA in 100ml ultra-pure water, magnetic agitation adds 3gADH after fully dissolving, and then adds 1.2gEDC and 1.35gNHS, and 1mol/lHCl regulates pH to 5.0, and reaction 23h, dialyses and obtain HA-ADH colloidal sol;
(2) PLLA of 2.25g is dissolved in 20mL dichloromethane solution, then joins NGF, bFGF (NGF14.375mg/ml containing 2.0g sodium carbonate and 2.25gPVA; BFGF is 1.25mg/ml) in aqueous phase, stirring or forming O/W type emulsion under ultrasonic condition, then add the outer aqueous phase of the 26.7%PVA of 4.5ml wherein, after high-speed stirred a period of time one-tenth emulsion, be transferred to by emulsion in beaker, 300rpm stirs 120min solidification.Centrifugal collecting precipitation also retains supernatant aqueous phase, wash three times and retain cleaning mixture, then lyophilizing 48h;
(3), at 0-4 DEG C, HA-ADH colloidal sol 0.39g above-mentioned steps obtained and growth factor-loaded PLLA porous microsphere 2.05g stirs and get final product.
The structural characterization of HA-ADH colloidal sol
By the hydrogel adjusted to ph 7.4 that above-mentioned steps (1) obtains, be placed in 37 DEG C of thermostat water baths to changing gel state into completely, lyophilization 24-48h is for subsequent use.
The structure of characterizing method to HA-ADH colloidal sol of reference example 1 characterizes.Nuclear-magnetism result shows successfully to generate amide.
The sign of polylactic acid medicine carrying microballoons envelop rate
Above-mentioned supernatant aqueous phase and cleaning mixture are merged, by the content of enzyme-linked immunosorbent assay somatomedin, sample envelop rate of the present invention is 62%.
The detection of polylactic acid medicine carrying microballoons TEM, DOS
Get above-mentioned reactant liquor (before centrifugal) 2ml and carry out TEM, DOS detection, the diameter of polylactic acid medicine carrying microballoons of the present invention is 1.5 μm.
A kind of release amount of medicine of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
On superclean bench, insert aseptic 5ml centrifuge tube by after the sample lyophilization of above-mentioned preparation, add 3ml phosphate buffer (pH=7.4), 37 DEG C of rotating speeds are cultivate in the isothermal vibration incubator of 60r/min.Get lixiviating solution at 1d, 3d, 6d, 9d, 12d, 15d respectively, and add the PBS buffer of equivalent, by enzyme-linked immunosorbent assay, detect the Cumulative release amount of somatomedin, in 15d, somatomedin Cumulative release amount is 71%.
A kind of degraded of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
Get system prepared by 1ml above-mentioned (3) to be placed in 15ml centrifuge tube, add 3mlPBS buffer (pH=7.4), and isothermal vibration (37 DEG C, 60r/min) degraded in shaking bath.Degradation time is respectively 1d, 3d, 6d, 9d, 12d, 15d etc.After corresponding degradation time, take out sample, milli-Q water, detect weight-loss ratio.After testing, sample of the present invention weight-loss ratio in 15d is 69%.
A kind of cytotoxicity of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel detects (PC12 cell)
(1) experiment be divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group.With card punch, above-mentioned material is made 96 orifice plate pore sizes, ultraviolet sterilization, put into 96 orifice plates for subsequent use, often group 5 is parallel.
(2) the PC12 cell that the phase of taking the logarithm grows, adjustment concentration is 2 × 10
4individual/ml, every hole 100 μ l adds in (1) in 96 orifice plates, with the cell culture fluid of sky in contrast.5%CO
2, 37 DEG C cultivate 72h.
(3) measure at 24h, 48h, 72h respectively, concrete grammar is as follows: every hole adds 10 μ lCCK-8 solution, continues to cultivate 4h, measures each hole OD value at 450nm place, calculate the survival rate of cell in incubator.
CCK-8 experimental result
Embodiment 5
A kind of preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel
(1) under 0-4 DEG C of condition, be dissolved in by 0.25gHA in 100ml ultra-pure water, magnetic agitation adds 1.0gADH after fully dissolving, and then adds 0.4gEDC and 0.35gNHS, and 1mol/lHCl regulates pH to 5.0, and reaction 24h, dialyses and obtain HA-ADH colloidal sol;
(2) PLLA of 0.75g is dissolved in 20mL dichloromethane, then joins NGF, bFGF (NGF2.125mg/ml containing 0.5g sodium carbonate and 0.75gPVA; BFGF is 0.25mg/ml) in aqueous phase, stirring or forming O/W type emulsion under ultrasonic condition, then add the outer aqueous phase of the 26.7%PVA of 1.5ml wherein, after high-speed stirred a period of time one-tenth emulsion, be transferred to by emulsion in beaker, 300rpm stirs 90min solidification.Centrifugal collecting precipitation also retains supernatant aqueous phase, wash three times and retain cleaning mixture, then lyophilizing precipitation 40h;
(3), at 0-4 DEG C, HA-ADH colloidal sol 0.19g above-mentioned steps obtained and growth factor-loaded PLLA porous microsphere 0.65g stirs and get final product.
The structural characterization of HA-ADH colloidal sol
By the hydrogel adjusted to ph 7.4 that above-mentioned steps (1) obtains, be placed in 37 DEG C of thermostat water baths to changing gel state into completely, lyophilization 24-48h is for subsequent use.
The structure of characterizing method to HA-ADH colloidal sol of reference example 1 characterizes.Nuclear-magnetism result shows successfully to generate amide.
The sign of polylactic acid medicine carrying microballoons envelop rate
Above-mentioned supernatant aqueous phase and cleaning mixture are merged, by the content of enzyme-linked immunosorbent assay somatomedin, sample envelop rate of the present invention is 59%.
The detection of polylactic acid medicine carrying microballoons TEM, DOS
Get above-mentioned reactant liquor (before centrifugal) 2ml and carry out TEM, DOS detection, the diameter of polylactic acid medicine carrying microballoons of the present invention is 2.9 μm.
A kind of release amount of medicine of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
On superclean bench, insert aseptic 5ml centrifuge tube by after the sample lyophilization of above-mentioned preparation, add 3ml phosphate buffer (pH=7.4), 37 DEG C of rotating speeds are cultivate in the isothermal vibration incubator of 60r/min.Get lixiviating solution at 1d, 3d, 6d, 9d, 12d, 15d respectively, and add the PBS buffer of equivalent, by enzyme-linked immunosorbent assay, detect the Cumulative release amount of somatomedin, in 15d, somatomedin Cumulative release amount is 65%.
A kind of degraded of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel characterizes
Get system prepared by 1ml above-mentioned (3) to be placed in 15ml centrifuge tube, add 3mlPBS buffer (pH=7.4), and isothermal vibration (37 DEG C, 60r/min) degraded in shaking bath.Degradation time is respectively 1d, 3d, 6d, 9d, 12d, 15d etc.After corresponding degradation time, take out sample, milli-Q water, detect weight-loss ratio.After testing, sample of the present invention weight-loss ratio in 15d is 72%.
A kind of cytotoxicity of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel detects (PC12 cell)
(1) experiment be divided into four groups: PLLA, HA-ADH, HA-ADH/PLLA/NGF/bFGF, blank group.With card punch, above-mentioned material is made 96 orifice plate pore sizes, ultraviolet sterilization, put into 96 orifice plates for subsequent use, often group 5 is parallel.
(2) the PC12 cell that the phase of taking the logarithm grows, adjustment concentration is 2 × 10
4individual/ml, every hole 100 μ l adds in (1) in 96 orifice plates, with the cell culture fluid of sky in contrast.5%CO
2, 37 DEG C cultivate 72h.
(3) measure at 24h, 48h, 72h respectively, concrete grammar is as follows: every hole adds 10 μ lCCK-8 solution, continues to cultivate 4h, measures each hole OD value at 450nm place, calculate the survival rate of cell in incubator.
CCK-8 experimental result
Claims (10)
1. the preparation of a somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel, it is characterized in that: it comprises and is cross-linked with hyaluronic acid and adipic dihydrazide the HA-ADH colloidal sol obtained and makes the first component, and the porous polylactic acid microball that be loaded with somatomedin of load in the first component.
2. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, it is characterized in that: described somatomedin is nerve growth factor and basic fibroblast growth factor bFGF, and its mass ratio is 8.5-11.5:1; The described mass percent being loaded with somatomedin in the porous polylactic acid microball of somatomedin is 4wt%-12.5wt%.
3. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: HA-ADH colloidal sol is 1:1.25-5.75 with the mass ratio of the porous polylactic acid microball being loaded with somatomedin.
4. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: described HA-ADH colloidal sol be by hyaluronic acid and adipic dihydrazide under the activation of acid condition and EDC/NHS activator 0-4 DEG C be obtained by reacting; Described hyaluronic acid and the mass ratio of adipic dihydrazide are 1:2-6, and described acid condition is pH is 4.5-5.75.
5. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: described in be loaded with the porous polylactic acid microball of somatomedin average diameter be 2 ± 1 μm.
6. the preparation of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, it is characterized in that: the described porous polylactic acid microball being loaded with somatomedin is the solution containing somatomedin being added with porogen and emulsifying agent is interior aqueous phase, take PLLA as oil phase, be outer aqueous phase with the solution containing emulsifying agent, use W/O/W multi-emulsion method to obtain emulsion, then stir, leave standstill solidification, centrifugal collecting precipitation, washing, lyophilizing obtains.
7. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: step is as follows:
(1) by hyaluronic acid and adipic dihydrazide in acid condition and under the activation of EDC/NHS activator 0-4 DEG C be obtained by reacting HA-ADH colloidal sol;
(2) be interior aqueous phase to be added with the solution containing NGF and bFGF component of porogen and emulsifying agent, take polylactic acid as oil phase, be outer aqueous phase with the solution containing PVA, W/O/W multi-emulsion method is used to obtain emulsion, then stir, leave standstill solidification, centrifugal collecting precipitation, washing, lyophilizing obtains growth factor-loaded porous polylactic acid microball;
(3) HA-ADH colloidal sol above-mentioned steps obtained and growth factor-loaded porous polylactic acid microball stir.
8. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, it is characterized in that: described W/O/W multi-emulsion method is: be dissolved in by PLLA in dichloromethane, obtain oil phase, then join be added with porogen and emulsifying agent containing in the aqueous phase of somatomedin, stirring or forming W/O/W type emulsion under ultrasonic condition, and then the outer aqueous phase added wherein containing emulsifying agent, high-speed stirring mixes emulsion;
In described step (2), the envelop rate of somatomedin is 55-65wt%.
9. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: the response time of described step (1) is reaction 18-24h;
Freeze-drying time described in step (2) is 24-48h, and hardening time is 80-120min;
Step (3) whipping temp is 0-4 DEG C;
In step (2) aqueous phase, the amount ratio of polylactic acid and porogen is 1:0.67-0.89, and the amount ratio of polylactic acid and emulsifying agent is 1:1-1.33, and in outer aqueous phase, the amount ratio of polylactic acid and emulsifying agent is 1:0.53-0.71.
10. the preparation method of somatomedin porous microsphere hybrid system of being wrapped up by injection aquagel according to claim 1, is characterized in that: described step (2) porogen is sodium carbonate; Emulsifying agent is polyvinyl alcohol, and PVA relative number average molecular weight is 35000-50000.
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