CN104587450A - Canine interferon alpha particle compound as well as preparation method and application thereof - Google Patents
Canine interferon alpha particle compound as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a canine interferon alpha particle compound CaIFN alpha-NPa and a preparation method thereof. The compound comprises a gamma-polyglutamic acid-phenylalanine shell and canine interferon alpha wrapped in the shell and is formed by assembling the gamma-polyglutamic acid-phenylalanine shell and the canine interferon alpha through a self-polymerization process. The invention also discloses application of the canine interferon alpha particle compound in the preparation of canine antiviral drugs and an antiviral composition. The canine interferon alpha particle compound is high in wrapping rate, free of toxic and side effects and good in biocompatibility, can be used for retarding enzymatic degradation to realize the drug effect slow-release effect, is simple and easy to operate, low in cost and suitable for large-scale production, and has favorable application prospect.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, be specifically related to a kind of dog interferon alpha particle composites and preparation method thereof and application and a kind of antiviral composition.
Background technology
At present, the raising quantity of dog is increasing, but the sickness rate of dog disease, particularly virosis constantly raises, and usually causes the death of dog, causes spirit and loss economically to the mankind.Dog interferon alpha is due to its antiviral activity and being used widely in treatment dog viroid disease widely.But existing dog interferon alpha preparation commercially needs administration every day, and 2-4 week is a course for the treatment of.Administration every day and long cycle are not only the misery of disease dog, too increase and raise main spirit and financial burden, also need a large amount of medicines simultaneously.This present situation is mainly because existing dog interferon alpha activity time is short.Therefore, the long-acting dog interferon alpha preparation of research and development is badly in need of.
In prior art, a rarely seen patent [publication number: CN 101411868A] discloses a kind of preparation method of dog interferon long-acting injection dosage form.But have employed third friendship fat/co-glycolide in this preparation method, non-degradable in vivo, poor biocompatibility.And introduce the organic reagent that the bio-toxicity such as dichloromethane, polyvinyl alcohol is larger in this technology, very large hidden danger has been buried in the application of the residual drug of organic reagent.Meanwhile, this preparation method complex process, expend time in length, not only makes preparation cost high, and preparation process may make dog interferon alpha degrade or active reduction.Gamma-polyglutamic acid-, i.e. γ-PGA, it is a kind of amino acid polymer of Microbe synthesis, degradation in vivo can become glutamic acid, therefore have no side effect, good biocompatibility [leaf seapeak. the preparation of gamma-polyglutaic acid-CDDP complex and antitumor activity [D] thereof. Shanghai: East China Normal University .2007].Studies have reported that polyglutamic acid is effective pharmaceutical carrier, there is the effect such as circulation time [the synthesis and characterization of cisplatin-loaded strengthening pharmaceutically active, slow releasing medicine, prolong drug, EGFR-targeted biopolymer and in vitroevaluation for targeted delivery [J] .Xu Geng, etc.Journal of Biomedical Materials Research Part A2012,100,2839-2848.].But γ-PGA self hydrophilic is strong, cannot form shell mechanism, need link on the carboxyl of γ-PGA by the method for chemosynthesis by medicine in aqueous phase, therefore strong to the selectivity of medicine, do not possess versatility; The activity of chemical reactions at the same time process to medicine especially protein drug has very large damage.
On the other hand, in prior art, dog interferon alpha activity time is short in the market, and dog interferon alpha preparation needs administration every day, and dosage period is long.
At present at drug world, due to the superiority of nano material, the research of nano-medicament carrier is got more and more.Nano-medicament carrier refers to the carrier of particle diameter at 10-1000nm, Nano medication after nano-medicament carrier drug loading has many advantages, comprise improve medicine trap and stability, improve pharmaceutical properties and targeting, drug treating time extends, curative effect increases, toxic and side effects is little etc. [research of nano-medicament carrier and clinical practice [J]. Jin Lixia. Chinese Tissue Engineering Study and clinical rehabilitation .2010,8:1429-1432].If but Nano medication is too little, urine will be become through the filtration of kidney and be discharged to external; On the contrary, if be greater than 400nm will rejection system identify and be disposed in body, therefore, Nano medication size must between 4-400nm [leaf seapeak. the preparation of gamma-polyglutaic acid-CDDP complex and antitumor activity [D] thereof. Shanghai: East China Normal University .2007].Patent [publication number: CN 101411868A] discloses a kind of preparation method of dog interferon long-acting injection dosage form, and the granule of gained is microsphere, is far longer than 400nm.But in the prior art, Nano medication mostly is people's medicines such as antitumor drug, yet there are no the report that it is applied to dog class antiviral drugs.
Summary of the invention
For overcoming above defect, the present invention proposes a kind of long-effect active dog interferon alpha particle composites.Nano material has remarkable superiority at drug world, but only has size to be only effective Nano medication between 4-400nm.The present invention proposes a kind of dog interferon alpha particle composites CaIFN α-NPs, its particle diameter, between 100-300nm, is one effective dog class antiviral drugs Nano medication.
The present invention, by hydrophilic γ-PGA and hydrophobic phenylalanine ethyl ester (i.e. PAE), is connected by amido link dehydration condensation, thus changes the hydrophilic of γ-PGA, obtains having amphipathic PGA-PAE material.The present invention is by PGA-PAE material and dog interferon alpha aqueous solution, amphipathic due to PGA-PAE material, auto polymerization forms the shell mechanism of external hydrophilic and inner hydrophobic in aqueous, by dog interferon alpha parcel wherein, obtain dog interferon alpha particle composites, i.e. CaIFN α-NPs.
CaIFN α-NPs of the present invention is made up of PGA-PAE shell and the dog interferon alpha be wrapped in shell.Shell can alleviate the degraded of enzyme to dog interferon alpha, extends its metabolism time in vivo, and slow releasing dog interferon alpha, reaches long-acting object simultaneously, thus improve dog interferon alpha activity time in prior art short, need the defects such as frequent injection.
CaIFN α-NPs material therefor safety non-toxic of the present invention, and preparation condition is gentle, technique is simple and quick, overcomes above-mentioned deficiency of the prior art.This preparation method is also applicable to multi-medicament simultaneously, has versatility, has expanded the application of γ-PGA as pharmaceutical carrier.
The present invention proposes a kind of dog interferon alpha particle composites CaIFN α-NPs, it dog interferon alpha comprising gamma-polyglutamic acid--phenylalanine shell and PGA-PAE shell and be wrapped in described shell.
Wherein, described gamma-polyglutamic acid--phenylalanine shell and PGA-PAE shell are formed by amido link dehydrating condensation by gamma-polyglutamic acid-and phenylalanine ethyl ester.Wherein, described gamma-polyglutamic acid--phenylalanine shell is Biodegradable material.Gamma-polyglutamic acid-is a kind of amino acid polymer of Microbe synthesis, and molecular weight is 50,000-100 ten thousand, is made up of natural amino acids glutamic acid, degradation in vivo can become glutamic acid.Therefore have no side effect, good biocompatibility.Studies have reported that polyglutamic acid is effective pharmaceutical carrier, there is the effect such as circulation time strengthening pharmaceutically active, slow releasing medicine, prolong drug.Phenylalanine is also a kind of essential amino acids.Therefore the shell be made up of PGA-PAE material can be degraded into aminoacid in vivo, safety non-toxic, good biocompatibility.Meanwhile, shell can slow down the degraded of enzyme to dog interferon alpha, and extend its metabolism time in vivo, slow releasing dog interferon alpha, reaches long-acting object, thus improve dog interferon alpha activity time in prior art short, need the defects such as frequent injection.By comparison, in prior art, patent [publication number: CN 101411868A] have employed third friendship fat/co-glycolide, non-degradable in vivo, poor biocompatibility.
Wherein, described dog interferon alpha particle composites CaIFN α-NPs particle diameter, between 100-300nm, is nano-scale medicine.Nano medication refers to the system of particle diameter after the use nano-carrier drug loading of 10-1000nm, there are many advantages, comprise trap and the stability of raising medicine, improve pharmaceutical properties and targeting, drug treating time prolongation, curative effect increases, toxic and side effects is little.
Wherein, described dog interferon alpha particle composites CaIFN α-NPs wraps up dog interferon alpha by PGA-PAE material and forms, and encapsulation ratio is very high, reaches more than 80%.Namely, when PGA-PAE material and dog interferon alpha are mixed and made into dog interferon alpha particle composites, the dog interferon alpha of more than 80% can be wrapped up by PGA-PAE material, avoids the waste of dog interferon alpha, decrease the consumption of dog interferon alpha, reduce production cost.By comparison, in prior art, such as patent [publication number: CN101411868A] discloses a kind of preparation method of dog interferon long-acting injection dosage form, and gained dog interferon microspherulite diameter is below 40 μm, and encapsulation ratio only has about 30%.
Wherein, the formation of described dog interferon alpha particle composites CaIFN α-NPs is formed by the auto polymerization process assembling of described gamma-polyglutamic acid--phenylalanine shell and dog interferon alpha.Because PGA-PAE material has amphipathic i.e. gamma-polyglutamic acid--phenylalanine shell simultaneously with hydrophilic group and hydrophobic group, when being dissolved in the PGA-PAE droplets of material in dimethyl sulfoxide DMSO and being added to dog interferon alpha aqueous solution, described material amphipathic make hydrophilic group outside, hydrophobic group/lipophilic group is interior, form spheroidal particle structure, dog interferon alpha is wrapped in grain structure inside simultaneously.This process is auto polymerization, and operation is simple, and cost is low, easy large-scale production.By comparison, in prior art, such as patent [publication number: CN 101411868A] introduces the larger organic reagent of the bio-toxicity such as dichloromethane, polyvinyl alcohol in preparation process, and very large hidden danger has been buried in the application of the residual drug of organic reagent.Meanwhile, this preparation method complex process, expend time in length, not only makes preparation cost high, and preparation process may make dog interferon alpha degrade or active reduction.And the present invention is in particulate production; dog interferon alpha is dissolved in water phase buffer solution; only introduce the DMSO relatively gentle, toxicity is low when wrapping up; after parcel completes instantaneously; namely by the centrifugal DMSO that goes out; preparation is simple fast, greatly reduces preparation cost, protects again the biological activity of dog interferon alpha largely.
Wherein, described dog interferon alpha comprises free dog interferon alpha or merges dog interferon alpha.Interferon is that a class can the infection of viral interference and the cytokine that copies, and be divided into a few class, wherein interferon-ALPHA is widely used because of the biological function of its wide spectrum and studies.Dog interferon alpha has antiviral effect, is therefore widely used in the virosis for the treatment of dog class.In a specific embodiment, the sequence of described dog interferon alpha is as shown in SEQ ID No:1.In another embodiment, the sequence of described dog interferon alpha is as shown in SEQ ID No:2.
Present invention also offers the preparation method of dog interferon alpha particle composites and CaIFN α-NPs, comprise the following steps:
(1) gamma-polyglutamic acid--phenylalanine sheathing material is prepared: get gamma-polyglutamic acid-and be dissolved in pure water, be made into 2%m/v solution.Regulate pH to 3.0, degrade in 98 DEG C of water-baths, ice bath immediately after degraded terminates.PH to 7.0 is regulated after cooling.Add water soluble polypeptide condensing agent 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and EDC.I successively, and phenylalanine ethyl ester hydrochlorate.37 DEG C, under 210rpm condition, react 24h.Reaction system is centrifugal, obtain white precipitate.Abandon supernatant, by resuspended for precipitation pure water rear centrifugal.Repeat 2 times.White precipitate is dried.By dmso solution precipitation, obtain PGA-PAE material.
(2) the auto polymerization reaction of gamma-polyglutamic acid--phenylalanine shell and dog interferon alpha: PGA-PAE droplets of material is added in isopyknic dog interferon alpha solution, obtains white emulsion.
(3) dog interferon alpha particle composites is obtained: by the centrifugal 15min of white emulsion 16000rpm, abandon supernatant, precipitation pure water is resuspended, recentrifuge.Repeat 2 times.To lyophilizing be precipitated, obtain dog interferon alpha particle composites CaIFN α-NPs of the present invention.
In step (1), degradation time is 3-15min, and preferably, degradation time is 11min.The mol ratio of described gamma-polyglutamic acid-and EDC.I is 1: 2.The mol ratio of described gamma-polyglutamic acid-and phenylalanine ethyl ester is 1: 0.60-1: 0.98, and preferably, mol ratio is 1: 0.82.
The concentration of the PGA-PAE material that step (1) obtains is 20-40mg/ml, and preferably, concentration is 40mg/ml.
Present invention also offers the application of dog interferon alpha particle composites CaIFN α-NPs in preparation dog antiviral drugs.Wherein, described CaIFN α-NPs is made up of PGA-PAE shell and the dog interferon alpha be wrapped in shell.PGA-PAE shell can slow down the degraded of enzyme to dog interferon alpha, extends its metabolism time in vivo, and slow releasing dog interferon alpha, reaches long-acting object simultaneously.Experiment proves; CaIFN α-NPs of the present invention has antiviral activity; can protect Madin-Darby canine kidney(cell line) (MDCK) not by the infection of vesicular stomatitis virus (VSV), simultaneously compared to common dog interferon alpha, CaIFN α-NPs of the present invention has long-acting antiviral activity.Therefore, CaIFN α-NPs of the present invention can be applicable to make dog antiviral drugs.
Another object of the present invention is to provide a kind of antiviral composition, is used for the treatment of dog virosis, and it contains described dog interferon alpha particle composites.Described compositions comprises pharmaceutical composition.Can prepare according to approach well known.One or more pharmaceutically acceptable excipient and/or adjuvant can be comprised, comprise various diluent well known in the art, lubricant, wetting agent, adhesive, disintegrating agent, fluidizer etc.Can be made into applicable any dosage form and comprise liquid dosage form, solid dosage forms, semisolid dosage form etc., comprise ordinary preparation, slow releasing preparation, targeting preparation etc.Described dog interferon alpha particle composites or described compositions can with unit dose administrations, and route of administration comprises intestinal or non-bowel, comprise oral, injection etc.Can be used alone or be combined with other drug.Described dog interferon alpha particle composites content is in the composition 0.1 ~ 99%.In a specific embodiment, described compositions comprises mannitol and dog interferon alpha, and its content is than being 50mg/ml mannitol, 1mg/mlCaIFN α-NPs.
Beneficial effect of the present invention comprises: CaIFN α-NPs of the present invention is made up of PGA-PAE shell and the dog interferon alpha be wrapped in shell, its advantage comprises: first, the shell of PGA-PAE material composition has degradability, safety non-toxic, good biocompatibility, avoids the toxic and side effects because carrier organism poor stability causes.The second, CaIFN α-NPs proposed by the invention is formed by PGA-PAE material and dog interferon alpha auto polymerization, and preparation condition is gentle, overcomes the defect that preparation process in prior art introduces poisonous organic reagent.Operation is simple, and cost is low, easy large-scale production.Three, PGA-PAE shell is very high to the encapsulation ratio of dog interferon alpha, avoids the waste of dog interferon alpha, decreases the consumption of dog interferon alpha, reduces production cost.4th, CaIFN α-NPs particle diameter proposed by the invention is between 100-300nm, for effective Nano medication, advantages such as there is trap and the stability of raising medicine, improve target-oriented drug, drug treating time extends, curative effect increases, toxic and side effects is little, overcome the defect of micronized drug simultaneously, reduce the clearance rate of body.5th, shell can slow down the degraded of enzyme to dog interferon alpha, and extend its metabolism time in vivo, slow releasing dog interferon alpha, reaches long-acting object, thus improves the defects such as dog interferon alpha in prior art needs frequent injection, activity time short.Experiment proves, CaIFN α-NPs proposed by the invention has long-acting antiviral activity, and therefore, CaIFN α-NPs of the present invention can be made into dog antiviral drugs, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture that parcel merges the dog interferon alpha particle composites CaIFN α-NPs of dog interferon alpha.
Fig. 2 is the transmission electron microscope picture of the dog interferon alpha particle composites CaIFN α-NPs of the free dog interferon alpha of parcel.
Fig. 3 is the In-vitro release curves of dog interferon alpha particle composites CaIFN α-NPs of the present invention.
Fig. 4 is the long-acting antiviral activity of dog interferon alpha particle composites CaIFN α-NPs of the present invention.
Detailed description of the invention
In conjunction with following specific embodiments and the drawings, the present invention is described in further detail, and protection content of the present invention is not limited to following examples.Under the spirit and scope not deviating from inventive concept, the change that those skilled in the art can expect and advantage are all included in the present invention, and are protection domain with appending claims.Implement process of the present invention, condition, reagent, experimental technique etc., except the following content mentioned specially, be universal knowledege and the common practise of this area, the present invention is not particularly limited content.
The synthesis of embodiment 1 PGA-PAE material
Take 0.1g gamma-polyglutamic acid-, be dissolved in 5ml pure water, be made into the PGA solution of 2%.The pH of PGA solution is regulated to be 3.0 with the hydrochloric acid of 1mol/L.PGA solution is put into 98 DEG C of water-baths to degrade 11min, ice bath cooling immediately after degraded terminates.PH to 7.0 is regulated subsequently with the sodium hydroxide solution of 1mol/L.Add 0.297g EDC.I and 0.146g phenylalanine ethyl ester successively.To shaking table, 37 DEG C of 210rpm react 24h.By centrifugal for reaction system 12000rpm 1min after reaction terminates, precipitation pure water is resuspended.Repeated centrifugation 2 times.White precipitate is dried.Weigh, by precipitation dmso solution, be made into 40mg/ml, obtain PGA-PAE material.
Embodiment 2 wraps up the preparation of the dog interferon alpha particle composites CaIFN α-NPs merging dog interferon alpha
Get the PGA-PAE material that 1ml is above-mentioned, be slowly added drop-wise to 1ml 0.25mg/ml and merge in dog interferon alpha aqueous solution, mixing obtains white emulsion.By centrifugal for emulsion 16000rpm 15min.Precipitation is separated with supernatant.Precipitation 2ml pure water is resuspended, 16000rpm recentrifuge 15min.Precipitation is separated with supernatant, will precipitate lyophilizing, and obtain CaIFN α-NPs.
Wherein, the sequence of dog interferon alpha is merged as shown in SEQ ID No:1.
Merge dog interferon alpha for checking whether wrapped in granule and calculate the encapsulation ratio merging dog interferon alpha, by CaIFN α-NPs 4% sodium dodecyl sulfate solution cracking.Because CaIFN α-NPs leans on the amphipathic auto polymerization of PGA-PAE material to form, and dodecyl sodium sulfate is surfactant, can simultaneously in conjunction with hydrophilic radical and lipophilic group, so sodium dodecyl sulfate solution by CaIFN α-NPs cracking, can discharge parcel dog interferon alpha wherein.Do not wrapped in PGA-PAE material if merge dog interferon alpha, then can not precipitate with granule time centrifugal, namely be present in supernatant.Be attached to PGA-PAE material surface if merge dog interferon alpha and be centrifuged in first time is centrifugal, then the resuspended second time of precipitation pure water centrifugal after, fusion dog interferon alpha should be dissolved in the supernatant of secondary centrifuging.CaIFN α-NPs lysate and centrifugal supernatant is analyzed with SDS-PAGE.
3 repeated experiments, each encapsulation ratio and drug loading as shown in table 1 below.Wherein encapsulation ratio is the quality of the fusion dog interferon alpha wrapping in granule interior and the ratio merging dog interferon alpha gross mass, and drug loading is the quality of the fusion dog interferon alpha wrapping in granule interior and the ratio of CaIFN α-NPs gross mass.As seen from Table 1, the encapsulation ratio of CaIFN α-NPs is very high, reaches more than 80%.
The encapsulation ratio of table 1 CaIFN α-NPs and drug loading
Embodiment 3 wraps up the preparation of the dog interferon alpha particle composites CaIFN α-NPs of free dog interferon alpha
Get the PGA-PAE material that 1ml is above-mentioned, be slowly added drop-wise to 1ml 0.4mg/ml and dissociate in dog interferon alpha aqueous solution, mixing obtains white emulsion.By centrifugal for emulsion 16000rpm 15min.Precipitation is separated with supernatant.Precipitation 2ml pure water is resuspended, 16000rpm recentrifuge 15min.Precipitation is separated with supernatant, will precipitate lyophilizing, and obtain CaIFN α-NPs.
Wherein, the sequence of free dog interferon alpha is as shown in SEQ ID No:2.
For verifying whether free dog interferon alpha is wrapped in granule and calculate the encapsulation ratio of free dog interferon alpha, by CaIFN α-NPs 4% sodium dodecyl sulfate solution cracking.Because CaIFN α-NPs leans on the amphipathic auto polymerization of PGA-PAE material to form, and dodecyl sodium sulfate is surfactant, can simultaneously in conjunction with hydrophilic radical and lipophilic group, so sodium dodecyl sulfate solution by CaIFN α-NPs cracking, can discharge parcel free dog interferon alpha wherein.If free dog interferon alpha is not wrapped in PGA-PAE material, then can not precipitate with granule time centrifugal, namely be present in supernatant.If free dog interferon alpha is attached to PGA-PAE material surface and is centrifuged in first time is centrifugal, then the resuspended second time of precipitation pure water centrifugal after, free dog interferon alpha should be dissolved in the supernatant of secondary centrifuging.CaIFN α-NPs lysate and centrifugal supernatant is analyzed with SDS-PAGE.
3 repeated experiments, each encapsulation ratio and drug loading as shown in table 2 below.Wherein encapsulation ratio is the quality of the free dog interferon alpha wrapping in granule interior and the ratio of free dog interferon alpha gross mass, and drug loading is the quality of the free dog interferon alpha wrapping in granule interior and the ratio of CaIFN α-NPs gross mass.As seen from Table 2, the encapsulation ratio of CaIFN α-NPs is very high.
The encapsulation ratio of table 2 CaIFN α-NPs and drug loading
The sign of embodiment 4 two kinds of CaIFN α-NPs
Two kinds of CaIFN α-NPs that the parcel got respectively in 0.1g embodiment 2 and embodiment 3 merges dog interferon alpha and the free dog interferon alpha of parcel are dissolved in 10ml ultra-pure water, be added drop-wise on copper mesh, dry 10ulCaIFN α-NPs solution.After oven dry, copper mesh 1% ammonium molybdate solution is dyeed, contaminated in rear ultra-pure water and rinsed.Transmission electron microscope observing is used after oven dry.
Parcel merges the transmission electron microscope results of the CaIFN α-NPs of dog interferon alpha as Fig. 1.As seen from the figure, CaIFN α-NPs is in regular spherical.
Get the CaIFN α-NPs solution that above-mentioned parcel merges dog interferon alpha, analyze the size of CaIFN α-NPs with Malvern laser particle size analyzer.Result shows that the particle diameter of CaIFN α-NPs is 165nm.
The transmission electron microscope results of the CaIFN α-NPs of the free dog interferon alpha of parcel is as Fig. 2.As seen from the figure, CaIFN α-NPs is equally in regular spherical.
Get above-mentioned parcel to dissociate the CaIFN α-NPs solution of dog interferon alpha, analyze the size of CaIFN α-NPs with Malvern laser particle size analyzer.Result shows that the particle diameter of CaIFN α-NPs is 160nm.
The detection of the antiviral activity of embodiment 5 two kinds of CaIFN α-NPs
Few cells pathological changes is adopted to suppress method: to be inoculated in by Madin-Darby canine kidney(cell line) MDCK in 96 orifice plates, 10000, every hole cell, the parcel adding 4 doubling dilutions after treating cell attachment respectively merges two kinds of CaIFN α-NPs of dog interferon alpha and free dog interferon alpha, and each dilution factor does 8 repetitions.At CO
2cultivate 24 hours in incubator.Supernatant discarded, adds the blister type oral herpes virus VSV of 100TCID50.Set up negative control group (only add CaIFN α-NPs, do not add virus), positive control (only add virus, do not add CaIFN α-NPs), blank group (do not add CaIFN α-NPs, do not add virus) simultaneously.At CO
2cultivate 24 hours in incubator.Observation of cell pathological changes situation.Half cell can be protected from the most highly diluted multiple of interferon of viral infection, be tiring of interferon.Experimental result shows, pathological changes appears in positive controls cell, and illustrate that this is tested VSV used and really can infect MDCK, namely this test macro is effective.The cell state of negative control group is identical with blank group, illustrates that CaIFN α-NPs of the present invention does not have toxic and side effects to cell.
Two kinds of parcels merge cell in the experimental group higher with the CaIFN α-NPs concentration of the free dog interferon alpha of parcel of dog interferon alpha and all do not occur pathological changes, illustrate that CaIFN α-NPs of the present invention can protect MDCK not by the infection of VSV.By Reed-Muench method experiment with computing group, i.e. the tiring of CaIFN α-NPs of the present invention.Wherein, parcel merges tiring as (2.9 ± 1.5) × 10 of the CaIFN α-NPs of dog interferon alpha
5u/mg, the CaIFN α-NPs of the free dog interferon alpha of parcel tire as (1.4 ± 0.6) × 10
6u/mg, visible, the present invention wrap up merge dog interferon alpha with the CaIFN α-NPs of the free dog interferon alpha of parcel, there is antiviral activity.
The extracorporeal releasing experiment of embodiment 6 two kinds of dog interferon alpha particle composites
That gets that above-mentioned two kinds of parcels merge dog interferon alphas is dissolved in 10% fresh mice serum with the CaIFN α-NPs of the free dog interferon alpha of parcel respectively, be placed in 37 DEG C to hatch, get 80ul sample at regular intervals, wherein 40ul directly freezes in-20 DEG C of preservations, is the total content of this time point dog interferon alpha; The centrifugal 15min of another 40ul 16000rpm, supernatant separates with precipitation, and supernatant freezes in-20 DEG C of preservations, is the dog interferon alpha content that this time point discharges.The content of dog interferon alpha in each sample is finally analyzed with western-blot.The ratio of the dog interferon alpha sometime in some supernatant and the total content of dog interferon alpha is the releasing ratio of dog interferon alpha in this time point CaIFN α-NPs.
The In-vitro release curves of CaIFN α-NPs is as 32.As seen from the figure, along with the prolongation of time, two kinds of dog interferon alphas all can slow releasing, within 30 days, releases the medicine of more than 80%.
The long-acting antiviral activity of embodiment 7 two kinds of CaIFN α-NPs
Be inoculated in by Madin-Darby canine kidney(cell line) MDCK in 96 orifice plates, add above-mentioned two kinds of dog interferon alpha particle composites of 4 doubling dilutions after treating cell attachment respectively, each dilution factor does 8 repetitions.At CO
2different time is cultivated in incubator.Supernatant discarded, adds the blister type oral herpes virus VSV of 100TCID50.At CO
2cultivate 24 hours in incubator.Observation of cell pathological changes situation.Half cell can be protected from the most highly diluted multiple of interferon of viral infection, be the antiviral activity of interferon.The antiviral activity of each time period two kinds of CaIFN α-NPs is calculated by Reed-Muench method.As shown in Figure 4, along with the prolongation in processing time, two kinds of CaIFN α-NPs have antiviral activity to experimental result, and active rising.The dog interferon alpha slow releasing wrapped up in it is described, plays antiviral activity.Therefore CaIFN α-NPs has long-acting antiviral activity.
Embodiment 8 comprises the antiviral activity of the compositions of two kinds of CaIFN α-NPs
Taking the above-mentioned two kinds of CaIFN α-NPs of 1mg is respectively dissolved in 0.5ml deionized water, separately gets 50mg mannitol, is dissolved in 0.5ml deionized water; The two is mixed, obtains the compositions of two kinds of CaIFN α-NPs and mannitol, wherein all containing the CaIFN α-NPs of 1mg/ml and the mannitol of 50mg/ml.During the compositions prepared by the present embodiment is used for embodiment 4, embodiment 5, embodiment 6 test, all obtain and embodiment 4, embodiment 5, result that embodiment 6 is similar, show the compositions energy slow releasing medicine of two kinds of CaIFN α-NPs, there is long-acting antiviral effect, thus may be used for treatment dog virosis.
Claims (11)
1. a dog interferon alpha particle composites, is characterized in that, described dog interferon alpha particle composites is CaIFN α-NPs, and it comprises gamma-polyglutamic acid--phenylalanine shell and is wrapped in the dog interferon alpha in described shell.
2. dog interferon alpha particle composites as claimed in claim 1, it is characterized in that, described gamma-polyglutamic acid--phenylalanine shell is formed by amido link dehydrating condensation by gamma-polyglutamic acid-and phenylalanine ethyl ester.
3. dog interferon alpha particle composites as claimed in claim 2, it is characterized in that, described gamma-polyglutamic acid-is fermentable gained, and its molecular weight is 50,000-100 ten thousand.
4. dog interferon alpha particle composites as claimed in claim 1, is characterized in that, described dog interferon alpha comprises free dog interferon alpha or merges dog interferon alpha.
5. dog interferon alpha particle composites as claimed in claim 1, is characterized in that, described dog interferon alpha particle composites is formed by the assembling of auto polymerization process by gamma-polyglutamic acid--phenylalanine shell and dog interferon alpha; Described gamma-polyglutamic acid--phenylalanine shell is simultaneously with hydrophilic group and hydrophobic group, and the outside hydrophobic group of hydrophilic group inwardly forms grain structure, and described dog interferon alpha is wrapped in grain structure inside.
6. dog interferon alpha particle composites as claimed in claim 1, it is characterized in that, the particle diameter of described complex is 100-300nm.
7. dog interferon alpha particle composites as claimed in claim 1, it is characterized in that, described encapsulation ratio reaches more than 80%.
8. a preparation method for dog interferon alpha particle composites, is characterized in that, described method comprises the steps:
(1) getting gamma-polyglutamic acid-is dissolved in pure water, is made into 2%m/v solution; Regulate pH to 3.0, degrade in 98 DEG C of water-baths, ice bath immediately after degraded terminates; PH to 7.0 is regulated after cooling; Add water soluble polypeptide condensing agent EDC.I and phenylalanine ethyl ester hydrochlorate successively; 37 DEG C, under 210rpm condition, react 24h; Reaction system is centrifugal, obtain white precipitate; Abandon supernatant, by resuspended for precipitation pure water rear centrifugal; White precipitate is dried; By dmso solution precipitation, obtain gamma-polyglutamic acid--phenylalanine and PGA-PAE material;
(2) gamma-polyglutamic acid--phenylalanine and PGA-PAE droplets of material are added in isopyknic dog interferon alpha solution, obtain white emulsion;
(3) by centrifugal for white emulsion 16000rpm, abandon supernatant, precipitation pure water is resuspended, centrifugal, lyophilizing, obtains dog interferon alpha particle composites as claimed in claim 1.
9. preparation method as claimed in claim 8, it is characterized in that, in described step (1), degradation time is 3-15min; The mol ratio of gamma-polyglutamic acid-and EDC.I is 1: 2; The mol ratio of gamma-polyglutamic acid-and phenylalanine ethyl ester is 1: 0.60-1: 0.98; The concentration of the PGA-PAE material obtained is 20-40mg/ml.
10. the application of dog interferon alpha particle composites as claimed in claim 1 in preparation dog antiviral drugs.
11. 1 kinds of antiviral compositions, is characterized in that, described compositions is containing, for example dog interferon alpha particle composites according to claim 1.
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| CN106282279A (en) * | 2016-08-25 | 2017-01-04 | 安徽九川生物科技有限公司 | A kind of canine recombinant interferon-ALPHA standard substance, its preparation method and titration method |
| CN112079912A (en) * | 2020-09-25 | 2020-12-15 | 广州源博医药科技有限公司 | High-activity canine alpha interferon recombinant protein and preparation method and application thereof |
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| CN104587450B (en) | 2017-01-11 |
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