CN104644545A - Controlled-release and slow-release silk fibroin gel preparation for treating inner ear disease - Google Patents
Controlled-release and slow-release silk fibroin gel preparation for treating inner ear disease Download PDFInfo
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Abstract
The invention relates to a preparation for treating an inner ear distance, in particular to a controlled-release and slow-release silk fibroin gel preparation for treating an inner ear disease. The controlled-release and slow-release silk fibroin gel preparation for treating the inner ear disease provided by the invention comprises a main preparation body, wherein the main preparation body comprises a gel-state carrier, and a medicine dispersed or adsorbed in the carrier; the medicine is a hormone medicine for treating the inner ear disease; and the carrier is silk fibroin gel. The controlled-release and slow-release silk fibroin gel preparation for treating the inner ear disease is relatively long in medicine release time, and is free of effect on hearing after being used, safe and harmless.
Description
Technical field
The present invention relates to a kind of preparation for the treatment of disease of inner ear, particularly relate to a kind of controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear.
Background technology
In recent years, by tympanum (IT), hormone medicine is more and more applied with the scheme for the treatment of sudden deafness (SSNHL) to internal ear administration, contains from first-line treatment to multiple clinical fields such as the first aids of refractory sudden deafness (RSSNHL).Compared to Formulations for systemic administration mode, IT injection can not only make perilympha contain the medicine of higher concentration, and enhances blood flow and the ion gradient of cochlea, and these all contribute to better recovering audition.But, the IT administration of clinicing aspect water-soluble hormones medicine is limited by very large, and reason is that some drugs by the mucosa absorption of tympanum, or may be flowed away from tympanum by pharyngotympanic tube, thus can not contact with round window membrane (RWM) fully, and then enter internal ear by round window membrane.Round window membrane leads to the main barrier of internal ear from tympanum, its absorb the drug number directly determine the drug level of interior in ear.The medicine of direct injection by intratympanic mucosa absorption, or may be flowed away from tympanum by pharyngotympanic tube, only has minute quantity can be absorbed by round window membrane.Because drug diffusion is limited to internal ear, so independent injection of tympanum can not give the medicine of internal ear abundance.Repeatedly or can continue medication in clinical to maintain internal ear drug concentration, namely implant the conduit of company's pump at tympanum, thus utilize full-implantation type microtubular to connect the successive administration of Micropump.Compared to repeatedly injecting, pump administration is the effect that can reach continuous drug delivery.But implantable micropump is not approved clinically widely, reason inserts and remove process to bring damage to body tissue.
The degradable biomaterial being subject to extensive concern recently controls sustained drug release, can as the another kind of scheme except multiple injection and embedded type micropump.Company both domestic and external and research unit just at the multiple synthesis of active research or natural material, with set up a kind of can the internal ear medicament slow release controlled release system of clinical practice.Its basic conception is that be placed in round window membrane surrounding, material can stick on round window membrane, and the treatment molecule of sustained release embedding is to internal ear by degradation material embedding treatment molecule.
Fibroin albumen is a kind of native protein polymer separated from domestic silkworm silk, there are all characteristics required for tissue repair and drug delivery vehicle, comprise biodegradability, biocompatibility, stablize medicine embedding, material forms multiformity and processing simple, without the need to organic solvent etc.Research has confirmed the clinical practice feasibility of injection Silk fibroin gel.Fibroin albumen is by the biomaterial that FDA ratifies, so it is safe that silk extract gel is applied to clinical.These characteristics make Silk fibroin gel can as pharmaceutical carrier delivering drugs to internal ear, by the time of staying of IT prolong drug at tympanum, reduce the drug wastage caused because flowing into pharyngotympanic tube, but, how medicine and Silk fibroin gel being formed stable controlled release fertilizer preparation, is still a difficult problem.
Because above-mentioned defect, the design people, actively in addition research and innovation, to founding a kind of controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear, make it have more value in industry.
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide a kind of pharmaceutical release time longer, on the controlled release fertilizer Silk fibroin gel preparation of audition without impact, safe and harmless treatment disease of inner ear.
The controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear of the present invention, comprise formulation mass, the carrier that described formulation mass comprises gel state and the medicine disperseing or be adsorbed in carrier, described medicine is the hormone medicine for the treatment of disease of inner ear, and described carrier is Silk fibroin gel.
Concrete, described Silk fibroin gel is made by induction plastic mode with silk fibroin protein solution.
Concrete, described induction plastic mode comprises pH value and changes method, sonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H
2o
2(hydrogen peroxide) blending method and low-molecular-weight PEG (Polyethylene Glycol) blending method.
Preferably, described induction plastic mode is low-molecular-weight PEG (Polyethylene Glycol) blending method.
Concrete, described formulation mass is mixed afterwards by inducing plastic mode to make with silk fibroin protein solution in form of an aqueous solutions or with insoluble microspheres form by described medicine.
Concrete, in described formulation mass, the concentration of fibroin albumen is 1-30%.
Preferably, in described formulation mass, the concentration of fibroin albumen is 7.5-15%.
Concrete, described disease comprises prunus mume (sieb.) sieb.et zucc. Ni Ershi disease, sudden deafness (SSNHL), Meniere disease, sensorineural hearing loss and Autoimmune Inner Ear Disease (AEID), described hormone medicine comprise in dexamethasone, betamethasone, hydrogenation Bo Nisong, methyl meticortelone, desoxycortone, 11-desoxycortone, 18-H-11-desoxycortone, beclometasone, triamcinolone acetonide and its chemical synthetic derivative wherein one or more.
The present invention also provides a kind of preparation method of controlled release fertilizer Silk fibroin gel preparation of above-mentioned treatment disease of inner ear, comprises the following steps:
1) mix: the hormone medicine for the treatment of disease of inner ear is mixed homogeneously with silk fibroin protein solution in form of an aqueous solutions or with insoluble microspheres form, obtains drug suspension;
2) plastic: by drug suspension to induce the mode of plastic, make formulation mass.
Further, described induction plastic mode comprises pH value change method, sonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H
2o
2(hydrogen peroxide) blending method and low-molecular-weight PEG (Polyethylene Glycol) blending method.
Further, in described blend step, hormone medicine also carries out coating process before mixing homogeneously with silk fibroin protein solution with insoluble microspheres form, and described coating process comprises the following steps:
A) hormone medicine microsphere is suspended in low concentration silk fibroin protein solution, mix homogeneously, and supersound process a period of time is with the agglomerating microsphere of scatter-gather;
B) solution step a) obtained, through vibration, centrifugal, go supernatant, washing and drying after, obtain the hormone medicine microsphere being coated with fimbrin.
Be coated with the hormone medicine microsphere of multilamellar fimbrin if need obtain, repeat step a-b.
The controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear of the present invention, its using method comprises two kinds, one is gel in-situ, one is pre-plastic, gel in-situ is the same with the plastic mode of pre-plastic, only exist different in concrete occupation mode, both distinctive points are, the injection or implant internal ear after plastic in vitro of pre-plastic, and gel in-situ is whole space drug suspension being injected into REN, then form the semisolid gel of convenient form, contact the surface of round window membrane to greatest extent; The posture that pre-plastic does not need patient to keep certain, thus more convenient, but shortcoming more difficultly under high concentration to be injected by fine needle.
By such scheme, the present invention at least has the following advantages: medicine of the present invention, as controlled release fertilizer preparation, can treat disease of inner ear.Injectable silk fibroin gel is as pharmaceutical carrier, controlled (the Wang X of fibroin albumen-PEG solution gelation time height, Partlow B, Liu J, et al.Injectable silk-polyethylene glycolhydrogels.Acta Biomater.2015; 15 (12): 51-61); The preparation scheme of gel in-situ can reach the standard of Zero order release; Pharmaceutical release time at least can reach 10 days; And on audition without impact.Simultaneously as pharmaceutical carrier, fibroin albumen-PEG gel safety non-toxic, degraded are slowly.Illustrate that injectable fibroin-PEG gel is a kind of pharmaceutical carrier of effective and safe, can be used for hormones slow releasing pharmaceutical treatment disease of inner ear.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of description, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope result figure of fibroin-PEG-mDEX gel in the embodiment of the present invention six;
Fig. 2 is the extracorporeal releasing test result figure of fibroin-PEG-mDEX gel in the embodiment of the present invention seven;
Fig. 3 is the concentration results figure of DEX in perilympha under different time points in the embodiment of the present invention eight;
Fig. 4 is that in the embodiment of the present invention eight, sham operated rats and round window membrane injection fibroin-PEG-mDEX gel group affect comparison diagram to the threshold of audibility;
Fig. 5 is the tissue slice figure of Cavia porcellus tympanum and internal ear sample in the embodiment of the present invention eight;
Fig. 6 is rhodamine in the embodiment of the present invention eight-phalloidin basement membrane fluorescence staining result figure;
Fig. 7 is biodegradation figure in fibroin-PEG-mDEX gelinite in the embodiment of the present invention eight.
Detailed description of the invention
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment one
The controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear of the present invention, comprise formulation mass, the carrier that formulation mass comprises gel state and the medicine disperseing or be adsorbed in carrier, medicine is the hormone medicine for the treatment of disease of inner ear, and carrier is Silk fibroin gel.Wherein, Silk fibroin gel is made by induction plastic mode with silk fibroin protein solution; Induction plastic mode comprises pH value and changes method, sonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H
2o
2(hydrogen peroxide) blending method and low-molecular-weight PEG (Polyethylene Glycol) blending method.
Hormone medicine is divided into water soluble drug and poorly water soluble drugs, and thus, formulation mass is mixed afterwards by inducing plastic mode to make with silk fibroin protein solution in form of an aqueous solutions or with insoluble microspheres form by medicine.Consider that medicine needs long-acting release, the medicament forms of optimization is poorly water soluble drugs, more optimized choice microsphere state poorly water soluble drugs, and poorly water soluble drugs can make microspheres form by means such as spraying dry.Consider effect of long-acting release, pharmaceutical dosage form can do further pretreatment, and fibroin albumen is smeared on the microsphere top layer by medicine, can more effective promotion medicine slow release effect in gel degradation process.Poorly water soluble drugs suspends in a suitable solvent with microspheres form, and induce plastic after then mixing with silk fibroin protein solution, suitable solvent is including, but not limited to water, organic solvent, solubilizing agent, viscosifier and penetrating agent.
Different according to the consumption of different pharmaceutical, in drug solution, the scope of drug level is 0.5%-15% (w/v), and wherein, the concentration of low carrying capacity is 0.5-2%; Middle carrying capacity: 2-5%; Does is high carrying capacity concentration 5-15? %.And the concentration of fibroin albumen is 1-30% in formulation mass; In formulation mass, the concentration of fibroin albumen is 7.5-15%.
The controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear of the present invention, adaptable disease of inner ear comprises the multiple disease of inner ear such as prunus mume (sieb.) sieb.et zucc. Ni Ershi disease, sudden deafness (SSNHL), Meniere disease, sensorineural hearing loss and Autoimmune Inner Ear Disease (AEID); Corresponding, the hormone medicine of use comprise in dexamethasone, betamethasone, hydrogenation Bo Nisong, methyl meticortelone, desoxycortone, 11-desoxycortone, 18-H-11-desoxycortone, beclometasone and triamcinolone acetonide and its chemical synthetic derivative wherein one or more.
The controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear of the present invention, its drug effect position is internal ear, pharmaceutical preparation is gel state, covers on i-coch round window membrane and surrounding, by REN injection of medicine preparation in cochlea and/or can pass through injection of tympanum; Its using method comprises two kinds, one is gel in-situ, one is pre-plastic, both distinctive points are, the injection or implant internal ear after plastic in vitro of pre-plastic, and gel in-situ is whole space drug suspension being injected into REN, then forms the semisolid gel of convenient form, contact the surface of round window membrane to greatest extent.When pharmaceutical dosage form is gel in-situ, inject tympanomastoid notch, microsyringe administration when solution state including, but not limited to ordinary syringe and small syringe needle.
The controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear of the present invention, use gel in-situ according to low-molecular-weight PEG (Polyethylene Glycol) blending method, its drug mechanism is: after injection, fibroin albumen-PEG (Polyethylene Glycol) mixed liquor fills the whole space of REN, then form the semisolid gel of convenient form, contact the surface of round window membrane to greatest extent; Along with gel long-term adhesion is on round window membrane, sustained drug discharges and enters the effect that internal ear reaches disease therapy.Along with the release of medicine, gel is easily degraded by proteases in vivo, and catabolite is polypeptide and aminoacid.Degradation time is learnt by experiment, and within the 10th day, start degraded, when 21 days, medicinal part only has a small amount of residual gel to exist, and at least can reach 10 days between the control time slack simultaneously finding medicine.
Embodiment two
The invention provides a kind of preparation method for the treatment of the controlled release fertilizer Silk fibroin gel preparation of disease of inner ear, comprise the following steps:
1) mix: the hormone medicine for the treatment of disease of inner ear is mixed homogeneously with silk fibroin protein solution in form of an aqueous solutions or with insoluble microspheres form, obtains drug suspension;
2) plastic: by drug suspension to induce the mode of plastic, make formulation mass.
Wherein, induce plastic mode to comprise pH value and change method, sonic oscillation method, electrophoresis method, HRP (horseradish peroxidase)-H
2o
2(hydrogen peroxide) blending method and low-molecular-weight PEG (Polyethylene Glycol) blending method.
The operating procedure of not isogeneous induction plastic mode is as follows, and comparing result is as shown in table 1:
A) Silk fibroin gel is prepared in pH value change
Get the silk fibroin protein solution of 10mL 5% (w/v) with the small beaker of 20mL, regulate solution PH to be 5.2 with NaH2PO4-Na2HPO4 buffer, in overnight at room temperature, slow plastic.
B) sonic oscillation prepares Silk fibroin gel
The silk fibroin protein solution of 5mL5% (w/v) is got with the test tube of 10mL, under JYD-900 Intelligent supersonic cell disruptor is under 500w power, carries out supersonic oscillations 30-60sec (second), then small beaker is placed in room temperature 25 DEG C and forms gel.
C) electrophoresis method prepares Silk fibroin gel.
Electrode immerses 8% (w/v) silk fibroin water solution, and be energized under 25 volts of unidirectional currents 3min (minute).Because silk fibroin protein solution water content is high, can be hydrolyzed in galvanization, produce oxygen and hydrogen, so carry out along with energising, there is bubble in two electrodes.There is plastic phenomenon in anode position simultaneously.
D) HRP (horseradish peroxidase)-H2O2 (hydrogen peroxide) plastic
HRP freeze-dried powder joins in deionized water and forms concentration is 1000U/ml solution, after get appropriate HRP solution and add in 3% (w/v) silk fibroin protein solution, in modulation solution, HRP final concentration is 10U/ml.Then in every milliliter of silk fibroin protein solution, add 10 μ l hydrogenperoxide steam generators, reach the hydrogen peroxide final concentration of 165nM, after mixing, prepare plastic.
E) low-molecular-weight PEG (Polyethylene Glycol) plastic
80% (w/w) PEG400 (PEG400) solution of 15% (w/v) silk fibroin protein solution mixing same volume, hatches plastic at 37 DEG C.
The different Silk fibroin gel preparation method of table 1 compares
Learnt by the primary structure after X-diffraction checks various method plastic, its primary structure is Silk II.Silk II crystal structure content is the most important factor determining the mechanical strength of fibroin albumen biomaterial, degradation rate and medicament slow release controlled release properties, therefore Silk fibroin gel without differences on material property of preparing of known above distinct methods accordingly.Because the clinical practice of low-molecular-weight PEG (Polyethylene Glycol) plastic method is more convenient, controlled, safety non-toxic, therefore in following embodiment, induce the method for plastic all to adopt fibroin albumen-PEG gel method, other method repeats no more.In addition, it should be pointed out that when adopting low-molecular-weight PEG (Polyethylene Glycol) blending method to prepare gel, also above-mentioned hormone medicine can be mixed with Polyethylene Glycol, or mix with fibroin albumen and Polyethylene Glycol simultaneously.
Embodiment three
The invention provides a kind of preparation method for the treatment of the controlled release fertilizer Silk fibroin gel preparation of disease of inner ear, be applied to the preparation of hormon class pharmaceutical preparation:
A) fibroin albumen-PEG-mDEX (micronized dexamethasone, dexamethasone microsphere) preparation
A certain amount of mDEX microsphere is suspended in 15% (w/v) silk fibroin water solution of 0.5ml, and 0.5ml 80% (w/w) PEG400 (PEG400) solution with rear suspension liquid and same volume mixes rear plastic.
B) fibroin albumen-PEG-DA (dexamethasone acetate) gel preparation
Aseptic DA (dexamethasone acetate) solution mixs homogeneously the medicine carrying capacity reaching certain with the silk fibroin water solution of high concentration, fibroin final concentration is 15% (w/v), rear plastic mixed with 80% (w/w) PEG400 (PEG400) solution of same volume.
C) fibroin albumen-PEG-MPS (dexamethasone sodium phosphate) gel preparation
Aseptic DSP (dexamethasone sodium phosphate) solution mixs homogeneously the medicine carrying capacity reaching certain with the silk fibroin water solution of high concentration, fibroin final concentration is 15% (w/v), rear plastic mixed with 80% (w/w) PEG400 (PEG400) solution of same volume.
Because the drug effect of different pharmaceutical is different, consumption is different, the present invention is difficult to, to its detailed description of illustrating one by one, thus, in subsequent embodiment, only be described in detail with the example that is prepared as of fibroin albumen-PEG-mDEX.
Embodiment four
The invention provides a kind of preparation method for the treatment of the controlled release fertilizer Silk fibroin gel preparation of disease of inner ear, prepare for fibroin albumen-PEG-mDEX:
A) mDEX-fimbrin preparation: approximately the mDEX of 20mg is suspended in 0.1% (w/v) silk fibroin protein solution of 1ml.Slowly vibrate under suspension room temperature 2min.Then the ultrasonic about 1min of microsphere suspension liquid, with the microsphere that scatter-gather is agglomerating.Solution centrifugal 2min (14000r.p.m) after vibration 2min again, to remove upper strata silk fibroin protein solution.Microsphere washes 2 times with 1ml, vibration 1min, centrifugal.Microsphere after washing obtains with nitrogen drying.Above coating step can repeatedly, until obtain desirable coating layer thickness and corresponding drug release rate.
B) fibroin albumen-PEG-mDEX gel preparation: the mDEX of obtained fimbrin is suspended in the high concentration silk fibroin water solution of 0.5ml, fibroin final concentration is 15% (w/v), and 0.5ml80% (w/w) PEG400 (PEG400) solution with rear suspension liquid and same volume mixes rear plastic.
Should be noted that: above-mentioned steps b) can be used alone to prepare the fibroin albumen-PEG-mDEX gel without fimbrin, also a) can combine with step, preparation has the fibroin albumen-PEG-mDEX gel of fimbrin.
Embodiment five
The invention provides a kind of preparation method for the treatment of the controlled release fertilizer Silk fibroin gel preparation of disease of inner ear, prepare for fibroin albumen-PEG-mDEX, consistent with embodiment four of its method, its distinctive points is:
The concentration of fibroin albumen is adjusted to 1-30%, but fibroin final concentration is when being 1%, because gel strength is lower, medicine carrying is poor, the rate of release of medicine is very fast, when fibroin final concentration is 30%, because gel strength is higher, medicine carrying is higher, more remarkable to the adsorption of medicine, the rate of release of medicine is comparatively slow, and therefore, the concentration being preferably fibroin albumen in formulation mass is 7.5-15%.
Embodiment six
The invention provides a kind of controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear, adopt the preparation method preparation in embodiment four, adopt SEM (scanning electron microscopy) to carry out morphological examination to fibroin-PEG-mDEX gel subsequently.Test method is:
It is standby by embodiment four directions legal system that fibroin albumen-PEG gel (7.5%w/v) embeds mDEX (0%, 1.5% carrying capacity), and the solution that 200 μ L prepare is instilled 24 orifice plates, and under being placed on 37 DEG C of environment, hatching is until solution plastic.Orifice plate immerses in the glass beaker of 1000ml pure water, and slowly stir more than 24 hours, period replaces 4 pure water, by washed gel freeze overnight lyophilizing 48 hours at-80 DEG C.Sample is observed by SEM.
As shown in Figure 1, observe morphology by SEM, PEG removes after washing from gel, causes the gel be left to be porous spongy.As shown in figs. ia-1 c, do not embed the scanning electron microscope result figure of the fibroin-PEG gel of mDEX under being respectively 20,5 and 1 μm of amplifications, the fibroin-PEG gel not embedding mDEX has loose structure, inside has the hole of the mutual UNICOM of about 100 μm.As shown in Fig. 1 D-1F, the scanning electron microscope result figure of the fibroin-PEG gel of mDEX is embedded under being respectively 20,5 and 1 μm of amplifications, fibroin-PEG the gel having embedded mDEX then presents similar porous laminated structure, and particulate mDEX (1-5 μm) is then embedded in the Medium Culture of fibroin albumen.As shown in Fig. 1 G-1I, the scanning electron microscope result figure of the insoluble microsphere of mDEX water do not embedded under being respectively 20,5 and 1 μm of amplifications, only self-metering mDEX microgranule is the same with the particle size be embedded in Silk fibroin gel, and this shows that the process that Silk fibroin gel is formed does not affect the size of mDEX microgranule and dispersibility.
Embodiment seven
The invention provides the controlled release fertilizer Silk fibroin gel preparation of the treatment disease of inner ear described in embodiment four, the analysis of experiments result of controlled release in vitro, slow release effect, fibroin albumen-PEG-mDEX gel process for preparing is consistent with the method provided in embodiment four, distinctive points is that the drug loading of medicine is different, and mDEX medicine microspheres is not through fimbrin simultaneously.Extracorporeal releasing test is as follows:
Fibroin albumen-PEG-mDEX gel, is divided into low dosage (0.5%) and high dose (2.5%) according to the difference of its drug loading mDEX, for the difference of plastic mode, is divided into gel in-situ group and pre-plastic group.Wherein, for gel in-situ group, the fibroin albumen-PEG-mDEX solution of 5 μ l, is then hatched for 37 DEG C, plastic in 30 minutes to the microcentrifugal tube of 2-ml by 25G needle injection.For pre-plastic group, the fibroin albumen-PEG-mDEX solution contained in syringe first hatched plastic under the environment of 37 DEG C in 30 minutes, in 2-ml microcentrifugal tube, then inject the gel (not being with syringe needle) of 5ml.When experiment starts, each Guan Zhongjun adds 1ml PBS, the buffer of PH=7.4, and all centrifuge tubes vibrate at 37 DEG C.1st, 6,24 hours, 2,3,4,5,6,7 days, extract the release medium of 0.95ml to a blank tube, then store at 4 DEG C, detect for LC/MS/MS.In former centrifuge tube, the PBS of 0.95ml is supplemented again, the buffer of PH 7.4 under constant temperature.
Result as shown in Figure 2, wherein Fig. 2 A is under different time, 4 kinds of fibroin albumen-PEG-mDEX gels are at release medium (PBS, pH 7.4) in the comparison and detection result figure of DEX concentration (μ g/ml), Fig. 2 B is under different time, the cumulative release amount comparison diagram of 4 kinds of fibroin albumen-PEG-mDEX gel DEX in 144h.As shown in Figure 2 A, drug loading be 0.5% fibroin albumen-PEG-mDEX gel demonstrate the process of fast release at initial period, reach all rapidly maximum concentration value (about 4 μ g/ml) under the pattern of gel in-situ and pre-plastic during 2h, after 6h, be then reduced to baseline value rapidly.When gel drug loading is 2.5%, the sustained release time of DEX high concentration level is more of a specified duration, wherein under gel in-situ pattern, the concentration of DEX changes in 96h in 5-10 μ g/ml, 1.6 μ g/ml are then dropped at 144h, under preformation rubber moulding formula, in 72h, the concentration of DEX changes at 5-10 μ g/ml, drops to 2.2 μ g/ml during 96h.When the drug loading of mDEX is 0.5%, gel in-situ pattern and be respectively 38% and 25% (Fig. 2 B) with the cumulative release amount of preformation rubber moulding formula 2h.When mDEX drug loading is 2.5%, gel in-situ and pre-plastic nearly all reach Zero order release, and after 144h, its release rate is respectively close to 44% and 30% (Fig. 2 B)
Embodiment eight
The invention provides the controlled release fertilizer Silk fibroin gel preparation of the treatment disease of inner ear described in embodiment four, the analysis of experiments result of controlled release in vivo, slow release effect.Fibroin albumen-PEG-mDEX gel process for preparing is consistent with the method provided in embodiment four, and distinctive points is that drug loading is different with fibroin albumen concentration, and in body, pharmacokinetic trial method is as follows:
A) ear operation:
Method:
In fibroin-PEG-mDEX gel, fibroin albumen concentration is 7.5% (w/v), and mDEX carrying capacity is 0.5% and 2.5% (w/v), studies in vivo test.In matched group 0.5% and the preparation of 2.5%mDEX solution be suspended in 10mM PBS with mDEX, prepared by PH7.4.The sterile working that whole process is carried out at aseptic operating platform.Guinea pig intraperitoneal injection 1% pentobarbital sodium anesthesia (35mg/kg)., to keep body temperature, subcutaneous injection 1% lignocaine local anaesthesia, by surgical exposure otic capsule after ear, otic capsule gets out the duck eye that diameter is 3mm, sees REN with clear to need animal to be placed on (38 DEG C) on electric warming pad in whole operation process.To instil on round window membrane the mDEX suspension (matched group) of 10 μ l or fibroin-PEG-mDEX mixing suspension with tuberculin syringe (27G syringe needle), after injecting, Cavia porcellus is fixed this posture and is about 30min, to wait for solution plastic, hole suture also stamps dental cement sealing.
B) sampling and DEX Concentration Testing
Method: Cavia porcellus is divided into 2 groups (50): fibroin-PEG-mDEX gel group (sample time: 1h, 1,4,7,10,14 day) and mDEX matched group (sample time: 1,3,6,12h).Each sample point is 5 Cavia porcelluss (3 gel groups and 2 matched groups) sample analysis.The extraction of cerebrospinal fluid (CSF), overhead rear median line otch, peel off Musclar layer, expose cerebellomedullary cistern, and microsyringe extracts CSF20 μ l.The cardiac puncture that is extracted as of blood sample extracts, and blood sample is transferred to the centrifuge tube centrifugal 5min (3500rpm/s) that add heparin.Subsequently the serum (about 100 μ l are only every) of supernatant is moved in blank pipe.For avoiding cerebrospinal fluid to pollute during perilympha sampling, adopting and extracting in vitro.Cochlea is separated from temporal bone after experimental animal general anesthesia.Open a duck eye at cochlea collar, extract perilymph (5-7 μ l) with microsyringe pump.Preserve under placing-80 DEG C of environment before all samples detects.
Result:
After the round window membrane injection fibroin-PEG-mDEX gel and mDEX solution (matched group) of Cavia porcellus, to the concentration timing sampling detection of DEX in the perilympha of Cavia porcellus, CSF (cerebrospinal fluid) and plasma sample.As shown in Figure 3, for fibroin-PEG-mDEX gel and mDEX solution that drug loading is 0.5% and 2.5%, the comparison diagram of the DEX concentration discharged in perilympha under different time points, the comparison diagram of Fig. 3 A to be the result figure of 0-200h, Fig. 3 B be partial enlargement in 0-30h region.As seen from the figure, in matched group, perilymph DEX concentration 1h just reaches peak value (13.47 ± 6.02 μ g/ml), and be then down to 2.984 ± 1.33 μ g/ml when 6h, 12h just drops into below detection line.DEX carrying capacity is in the fibroin-PEG-mDEX gel group of 0.5%, and in perilymph, DEX concentration 1h is 8.06 ± 4.56 μ g/ml, 12h is 2.70 ± 0.97 μ g/ml, illustrates that the DEX of this group is longer than matched group for release time.When DEX carrying capacity rises to 2.5%, in fibroin-PEG-mDEX group, DEX significantly increased in time of internal ear and concentration, and perilymphatic DEX detectable concentration 1h is 25.71 ± 11.50 μ g/ml, 4d is that 6.46 ± 2.89 μ g/ml, 10d reach 0.26 ± 0.15 μ g/ml.After topical, DEX is very low in the concentration of whole body, and 1h after fibroin-PEG-mDEX (2.5%) group injection of middle ear, detects that in CSF and blood plasma the concentration of DEX is 0.23 ± 0.10 and 0.032 ± 0.014 μ g/ml respectively.To compare the concentration in perilympha, reduce about 110 times and 800 times.Then DEX cannot be detected after 2h.Fibroin-PEG-mDEX the group of low carrying capacity (0.5%) also all cannot detect DEX at 1h in CSF and blood plasma.These results show that fibroin-PEG gel slow release release DEX in cochlea reaches more than 10 days.
C) auditory brainstem response assessment (ABR)
Method:
For evaluating the safety of fibroin-PEG-mDEX gel and apply the impact of generation in audition surgical procedures, to be tested by ABR at 4 time points and assess audition function: operation consent, postoperative 1 day, postoperative 4 days and postoperative 14 days.12 Cavia porcelluss are divided into 2 groups: operative control group (not adding medicine, n=6) and fibroin-PEG-mDEX gel group (n=6).TDT-II recording system (Alachua, FL, USA) under specific frequency (4K, 8K, 16K, 24K) is utilized to analyze ABR.Lumbar injection 1% pentobarbital (35mg/kg) anesthesia test animal.Experimental animal is placed on (38 DEG C) on electric blanket, is in soundproof room and tests.Subcutaneous implantation 3 needle electrodes are to measure brainstem activity: one is implanted in ear rear (reference electrode), and one is implanted in skull summit (working electrode), an offside at back leg (ground electrode).Evoked ptential is filtered at 100-300Hz by band filter, average 512 times.In each given frequency, tone burst intensity starts at 90dB SPL, and progressively 5dB weakens, until reach threshold value.The threshold value of ABR is positioned at the reproduced wave band III of the minimum intensity that can collect.
Result:
The situation that in experiment, threshold value changes with tone burst Strength Changes as shown in Figure 4 A.In operative control group, audition function is not all affected (Fig. 4 B) under all time points (preoperative, 1,4,14 day) and any frequency.In fibroin-PEG-mDEX high dose (2.5%) gel group (Figure 4C), 4kHz and 8kHz frequency band audition does not change, and does not occur the situation that threshold value promotes.At 16K and 24K frequency band, compared to preoperative threshold level, after gel injection, audition has transient decline by a small margin, and in 1 day, threshold value rises 5-15dB SPL (39.2 ± 6.6vs, 30.8 ± 5.8at 16k Hz; 45.0 ± 7.1vs, 34.2 ± 6.6at 24k Hz, p<0.01).The phenomenon that this threshold of audibility promotes started at the 4th day to weaken (37.5 ± 5.2vs, 30.8 ± 5.8at 16k Hz; 41.7 ± 7.5vs, 34.2 ± 6.6at 24k Hz, p<0.01), disappear (34.2 ± 5.8vs, 30.8 ± 5.8at 16k Hz on the 14th day completely; 35.8 ± 5.8vs, 34.2 ± 6.6at 24k Hz, p>0.05), illustrate that the impact of gel injection on audition function is temporary transient, fade away along with the degraded of gel and the release of medicine.
D) Histological assessment
Method:
After experimental animal is lethal, sharp separation cochlea also pours into the PBS containing 4% paraformaldehyde, is then immersed in fixative 4 DEG C and spends the night.In order to obtain whole organ of Corti, the careful under the microscope dissection of basement membrane and organ of Corti separately.Rhodamine-phalloidin (Rhodamine phalloidin, 1:100) contaminates tragus cell, to recognize cochlear hair cell.Every cochlea is respectively at Leica TCS SPE test under microscope.The cross section counting of every cochlea 3 random selecting 200 μm.Active hair cell percentage with active hair cell number divided by total hair cell number gained (competent cell number adds inner hair cells).The preparation of cochlea section be cochlea and middle ear are immersed 0.1M EDTA 14 days with decalcification, and along the section of cochlea longitudinal axis position, slice thickness is 7 μm, and H & E dyes, observation by light microscope.
Result:
Post operation starts to carry out Histological evaluation to middle ear and internal ear on the 10th day.The inflammation focusing on may occurring at tympanum, REN, round window membrane, tympanic canal analyzed.Assess the impact that fibroin-PEG-mDEX gel produces organ of Corti and spiral ganglion simultaneously.From result, in all groups, all there is not obvious extensive inflammation reaction (hydrops, edema, fibrosis etc.).The tympanic canal that round window membrane closes on there will be a small amount of inflammatory cell and hemocyte, but from experimental group (fibroin-PEG-mDEX gel, Fig. 5 B) and the inflammatory cell number of matched group (ear is untreated, Fig. 5 A) compare, its numerical value does not also have significant difference.Be compared to matched group (mDEX solution group, Fig. 5 C), gel delivery group (Fig. 5 D) does not produce any change on size, morphology and ganglionic cell number.
By the integral surface histology of microscopic examination cochlea, after studying injection fibroin-PEG-mDEX gel, inside and outside hair cell produces the probability of loss.Quantitative counting analysis in table 2 shows, treatment group and blank group are all found to have lost some outer hair cells, but do not present significant difference (p<0.01).As shown in Figure 6, then there is not any change (A: matched group in the inner hair cells of all groups; B: treatment group).
The percentage ratio (n=3) of the 10th day internal hair living cells and outer knit cell after fibroin albumen-PEG-mDEX gel injected by table 2
E) fibroin-PEG-mDEX gel degraded in vivo
Method:
Gel injects 1h, puts to death animal after 4,7,10,14,21 days, obtains otic capsule.Open otic capsule and expose cochlea, remained in the fibroin-PEG-mDEX gel on REN by microscopic examination.
Result:
After separation otic capsule, we monitor the biodegradability of fibroin-PEG-mDEX gel by timing microscopic examination.As shown in Figure 7, to represent respectively after injected gel 1 hour, 4 days, 7 days, 10 days, 14 days and the situation of 21 days fibroin-PEG-mDEX gel degradations, red arrow instruction be REN position and residual gel.Can find, after injected gel, 1h can observe the stable gel state of white at REN, within the 10th day, sees that the volume of gel changes, and gel starts degraded, until the 14th day gel volume reduces always, in REN, substantially can't see gel when the 21st day remained.
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (10)
1. treat the controlled release fertilizer Silk fibroin gel preparation of disease of inner ear for one kind, it is characterized in that: comprise formulation mass, the carrier that described formulation mass comprises gel state and the medicine disperseing or be adsorbed in carrier, described medicine is the hormone medicine for the treatment of disease of inner ear, and described carrier is Silk fibroin gel.
2. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 1, is characterized in that: described Silk fibroin gel is made by induction plastic mode with silk fibroin protein solution.
3. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 2, is characterized in that: described induction plastic mode comprises pH value and changes method, sonic oscillation method, electrophoresis method, horseradish peroxidase-hydrogen peroxide blending method and low molecular poly blending method.
4. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 3, is characterized in that: described induction plastic mode is low molecular poly blending method.
5. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 2, is characterized in that: described formulation mass is mixed afterwards by inducing plastic mode to make with silk fibroin protein solution in form of an aqueous solutions or with insoluble microspheres form by described medicine.
6. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 1, is characterized in that: in described formulation mass, the concentration of fibroin albumen is 1-30%.
7. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 6, is characterized in that: in described formulation mass, the concentration of fibroin albumen is 7.5-15%.
8. the controlled release fertilizer Silk fibroin gel preparation for the treatment of disease of inner ear according to claim 1, is characterized in that: described hormone medicine comprise in dexamethasone, betamethasone, hydrogenation Bo Nisong, methyl meticortelone, desoxycortone, 11-desoxycortone, 18-H-11-desoxycortone, beclometasone, triamcinolone acetonide and its chemical synthetic derivative wherein one or more.
9. a preparation method for the controlled release fertilizer Silk fibroin gel preparation of the treatment disease of inner ear according to any one of claim 1-6, is characterized in that: comprise the following steps:
1) mix: the hormone medicine for the treatment of disease of inner ear is mixed homogeneously with silk fibroin protein solution in form of an aqueous solutions or with insoluble microspheres form, obtains drug suspension;
2) plastic: by drug suspension to induce the mode of plastic, make formulation mass.
10. preparation method according to claim 9, is characterized in that: in described blend step, hormone medicine also carries out coating process before mixing homogeneously with silk fibroin protein solution with insoluble microspheres form, and described coating process comprises the following steps:
A) hormone medicine microsphere is suspended in low concentration silk fibroin protein solution, mix homogeneously, and supersound process a period of time is with the agglomerating microsphere of scatter-gather;
B) solution step a) obtained, through vibration, centrifugal, go supernatant, washing and drying after, obtain the hormone medicine microsphere being coated with fimbrin.
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| PCT/CN2015/075019 WO2015144056A1 (en) | 2014-03-27 | 2015-03-25 | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
| US15/129,246 US10533037B2 (en) | 2014-03-27 | 2015-03-25 | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
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| CN110812335A (en) * | 2019-12-04 | 2020-02-21 | 苏州大学 | Silk fibroin micro-nano particle sustained-release preparation loaded with hydrophobic drug and preparation method thereof |
| CN110917400A (en) * | 2019-12-05 | 2020-03-27 | 中山大学 | Nano-hybrid silk fibroin hydrogel and preparation method and application thereof |
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