Interferon alpha-2 b slow-release oral patch and preparation method thereof
阅读说明：本技术 干扰素α-2b缓释口腔贴片及制备方法 (Interferon alpha-2 b slow-release oral patch and preparation method thereof ) 是由 徐寒梅 沈子龙 薛建鹏 于 2021-08-17 设计创作，主要内容包括：本发明提供干扰素α-2b缓释口腔贴片及其制备方法,采用复乳-溶剂挥发法或减压蒸馏制备纳米颗粒并将其冻干粉与辅料压制成口腔粘贴片。以聚乳酸-羟基乙酸共聚物(PLGA)为纳米颗粒载体,将干扰素α-2b包裹于内部,提高干扰素α-2b稳定性,并且能够控制干扰素α-2b缓慢释放,延长血浆半衰期。所制得纳米粒颗粒圆整,分散均匀,性质稳定,冻干后复溶性好,体外释放符合长效制剂特征,粒径分布主要在50-1000nm,体内半衰期延长至120-600h,包封率70％-90％左右,干扰素α-2b缓释口腔贴片载药量为8-45μg。将纳米颗粒冻干后与辅料羟丙甲纤维素、羧甲基纤维素钠、卡波姆、硬脂酸镁等均匀混合压制成片剂,可应用于口腔贴片的缓释给药。(The invention provides an interferon alpha-2 b sustained-release oral patch and a preparation method thereof, which adopts a multiple emulsion-solvent volatilization method or reduced pressure distillation to prepare nano particles, and the freeze-dried powder and auxiliary materials are pressed into an oral sticking tablet. Polylactic acid-glycolic acid copolymer (PLGA) is used as a nanoparticle carrier, and interferon alpha-2 b is wrapped inside, so that the stability of the interferon alpha-2 b is improved, the slow release of the interferon alpha-2 b can be controlled, and the half-life period of blood plasma is prolonged. The prepared nanoparticles are round, uniform in dispersion, stable in property, good in redissolution after freeze-drying, and in vitro release conforms to the characteristics of a long-acting preparation, the particle size distribution is mainly 50-1000nm, the in vivo half-life period is prolonged to 120-600h, the encapsulation rate is about 70-90%, and the drug-loading rate of the interferon alpha-2 b sustained-release oral patch is 8-45 mu g. The nano particles are freeze-dried, and then uniformly mixed with auxiliary materials such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, magnesium stearate and the like to be pressed into tablets, and the tablets can be applied to sustained-release drug delivery of oral patches.)
1. A nanoparticle characterized by: the nanoparticle has a bilayer structure comprising:
(a) an outer layer of chitosan bearing a positive charge; and
(b) a biodegradable polymer inner layer surrounded by an outer layer;
the solid interferon alpha-2 b is uniformly distributed in the inner layer of the polymer.
2. The nanoparticle of claim 1, wherein: the inner layer of the biodegradable polymer contains polylactic acid-glycolic acid copolymer, polyvinyl alcohol and sodium alginate.
3. The nanoparticle according to claim 1 or 2, wherein: the particle size of the nano particles is 50-1000 nm; and/or the nanoparticles have a PDI of 0.1 to 0.3; and/or the thickness of the chitosan outer layer is 12-22 nm.
4. The nanoparticle according to any one of claims 1 to 3, wherein: the preparation method comprises the following steps:
1) dispersing interferon alpha-2 b in sodium alginate solution with the mass volume ratio of 0.00001-5.0000% to serve as an internal water phase, mixing the internal water phase with an organic phase, and performing ultrasonic emulsification to form water-in-oil colostrum;
2) injecting the primary emulsion into an external water phase, fully and uniformly mixing, and then carrying out ultrasonic emulsification to form a water-in-oil-in-water double emulsion solution, wherein the external water phase also contains polyvinyl alcohol and Ca2+And chitosan;
3) and (3) completely volatilizing the organic solvent of the multiple emulsion solution, and freeze-drying to form the nano-particles.
5. A method of preparing nanoparticles, comprising: the method comprises the following steps:
1) dispersing interferon alpha-2 b in sodium alginate solution with the mass volume ratio of 0.00001-5.0000% to serve as an internal water phase, mixing the internal water phase with an organic phase, and performing ultrasonic emulsification to form water-in-oil colostrum;
2) injecting the primary emulsion into an external water phase, fully and uniformly mixing, and then carrying out ultrasonic emulsification to form a water-in-oil-in-water double emulsion solution, wherein the external water phase also contains polyvinyl alcohol and Ca2+And chitosan;
3) and (3) completely volatilizing the organic solvent in the multiple emulsion solution, and freeze-drying to form the nano-particles.
6. The method for producing nanoparticles according to claim 5, characterized in that: the preparation process of the organic phase comprises the following steps: dissolving a solid polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase; and/or complete volatilization of the organic solvent of the multiple emulsion solution comprises low-speed stirring or reduced pressure distillation at room temperature.
7. The method for preparing nanoparticles according to claim 6, characterized in that: the external water phase consists of 0.0001-20.000% (w/v) PVA, 0.00005-0.15000% (w/v) chitosan solution and 0.0001-0.18(mol/L) Ca2+The solution is formed after mixing.
8. The method for producing nanoparticles according to claim 7, characterized in that: the organic solvent comprises one or a mixture of dichloromethane, ethyl acetate and acetone; and/or the volume ratio of the organic phase to the internal aqueous phase is (2-40): 1, the volume ratio of the primary emulsion to the external water phase is 1 (3-20).
9. A pharmaceutical formulation characterized by: comprising the nanoparticle of any one of claims 1 to 4.
10. The pharmaceutical formulation of claim 9, wherein: the dosage form of the pharmaceutical preparation is an oral patch.
11. The pharmaceutical formulation of claim 10, wherein: comprises the nanoparticles of any one of claims 1-4 and a pharmaceutical excipient, wherein the pharmaceutical excipient comprises a filler, an adhesive and a lubricant.
12. The pharmaceutical formulation of claim 11, wherein: the mass ratio of the nano particles to the pharmaceutic adjuvant is 4: 1; and/or the total weight ratio of the filling agent in the pharmaceutical excipients to the oral patch is 2:25, the total weight ratio of the adhesive agent to the oral patch is 2:25, and the total weight ratio of the lubricant to the oral patch is 1: 500.
13. The pharmaceutical formulation of claim 12, wherein the filler comprises starch or mannitol, and/or the adhesive comprises one or more combinations of carbomer, carboxymethylcellulose, sodium carboxymethylcellulose; and/or the lubricant comprises magnesium stearate or talc.
14. A method for preparing a pharmaceutical formulation according to any one of claims 9 to 13, wherein the formulation is prepared by mixing and tabletting the nanoparticles according to any one of claims 1 to 4 with a pharmaceutical excipient in a certain ratio, wherein the pharmaceutical excipient is a mixture comprising a filler, an adhesive and a lubricant.
15. Use of the nanoparticle of any one of claims 1-4 for the preparation of a pharmaceutical formulation.
16. The use of claim 15, wherein the pharmaceutical formulation is an oral patch.
Technical Field
The invention relates to the field of biological medicines, in particular to an interferon alpha-2 b sustained-release oral patch and a preparation method thereof.
Background
With the continuous development of biotechnology, biological drugs, especially polypeptide protein drugs, are continuously developed, and have very good and unique curative effects in clinical applications, such as insulin, interferon, bevacizumab, combretacept, hepatitis b vaccine, and the like. However, most polypeptide protein drugs have short half-life, poor stability, easy deterioration and poor gastrointestinal absorption, and the clinical application of the drugs needs frequent injection administration due to the reasons, so that the pain of patients and complications are caused, and the clinical application of the drugs is hindered. It is a complex and difficult task to formulate stable, safe and efficient pharmaceutical preparations.
The interferon is a protein induced by human cells infected by viruses, and has antiviral, anti-cell proliferation, immunoregulation and antitumor effects. Interferon is classified into alpha, beta and gamma interferon according to its antigen specificity and structure. Among them, there are three types of interferon-alpha.
In 1980, active human alpha-type interferon protein molecules were successfully expressed in escherichia coli in the united states, and the biological activity and clinical efficacy of the protein molecules were verified to be the same as those of natural interferon.
The alpha-interferon produced by the genetic engineering technology is widely applied to clinic and has definite clinical curative effect when being used for treating various viral diseases and malignant tumors. It is mainly used for treating hepatitis, upper respiratory tract infection, tumor, rheumatoid arthritis, etc. caused by various virus infections. However, since interferon is inactivated orally, it is usually administered by intramuscular injection or subcutaneous injection in clinic, and its half-life in blood is short, and it is rapidly eliminated or metabolized, so that a large dose and frequent injection are required. Usually 200-. Therefore, the research of improving the stability of interferon, prolonging the half-life of interferon in blood and using a painless and noninvasive administration manner is very critical and urgent.
2. Summary of the invention
One of the purposes of the present invention is to provide a nanoparticle containing interferon alpha-2 b with a double-layer structure, which not only has a good slow release effect to realize a function of releasing a drug for a long time, but also increases the drug intake and transport because the outer layer of the nanoparticle has a positive charge to facilitate transmembrane movement of the nanoparticle. The nanoparticles can be mixed with pharmaceutical adjuvants to make into pharmaceutical preparations. The invention also provides a preparation method of the nano-particles.
The oral administration is the most acceptable administration method due to the characteristics of simplicity and no pain, and the second aim of the invention is to provide an oral patch prepared from interferon alpha-2 b nano-particle freeze-dried powder and a preparation method thereof.
The present disclosure provides a nanoparticle having a double-layer structure, including:
(a) an outer layer of chitosan bearing a positive charge; and
(b) a biodegradable polymer inner layer surrounded by an outer layer;
the solid interferon alpha-2 b is uniformly distributed in the inner layer of the polymer;
the chitosan in the interferon alpha-2 b nanoparticles can increase the charge of the nanoparticles, so that the nanoparticles are positively charged, and are favorable for being combined with negatively charged cell membranes, and further favorable for transmembrane movement of the nanoparticles, thereby increasing the intake and transport of interferon alpha-2 b drugs.
In an embodiment, the inner biodegradable polymer layer comprises a polylactic acid-glycolic acid copolymer, polyvinyl alcohol, and sodium alginate.
The main component of the inner polymer layer in this disclosure is polylactic-co-glycolic acid (PLGA), also known as lactide-glycolide, poly-glycolide, etc. It is made up by using glycolic acid (or called glycolic acid) and lactic acid through polymerization, and is a biodegradable high-molecular material. The polylactic acid-glycolic acid copolymer material selected in the invention not only has the slow release function, but also has no toxicity to human body after being degraded. On the other hand, the outer layer of the nanoparticle disclosed by the disclosure is a chitosan layer, and the inner layer of the nanoparticle is a biodegradable polymer inner layer, wherein the chitosan layer on the outer layer and the polymer on the inner layer both have a slow release effect, so that the drug is slowly released. Specifically, the outer chitosan can be hydrolyzed in blood and degraded by in vivo enzymes so as to be metabolized, the PLGA of the inner layer can be slowly degraded at the body temperature, and because the PLGA layer under the body temperature condition is relatively soft, the inner wrapping drug is exchanged to the blood under the action of osmotic pressure, and the drug is slowly released by the degradation of the PLGA layer after the osmotic pressure is balanced, so that the characteristic of releasing the drug for a long time is achieved. The nanoparticles achieve the purpose of releasing the drug for a long time by means of the matching of the inner layer and the outer layer.
The interferon alpha-2 b nano-particles prepared by the invention can prolong the in vivo half-life of the interferon alpha-2 b, reduce clearance rate, immunogenicity and antigenicity, reduce administration frequency and improve the tolerance of patients.
In embodiments, the weight of the interferon alpha-2 b in the solid state is 0.1 to 0.5 wt% of the total weight of the nanoparticle;
in a preferred embodiment, the weight of the interferon alpha-2 b in solid state is 0.15 to 0.4 wt% of the total weight of the nanoparticle. More preferably, the weight of the interferon alpha-2 b in the solid state is 0.2 to 0.3 wt% of the total weight of the nanoparticle.
In a preferred embodiment, the nanoparticles have a particle size of 50 to 1000 nm; and/or the nanoparticles have a PDI of 0.1 to 0.3;
the particle size of the interferon PLGA nano-particles is 50-1000nm, and the preferred scheme is 200-400nm, compared with 30-micron PLGA microspheres disclosed in CN1308034C, the particle size is smaller, the particle size range is favorable for the drug-loaded nano-particles to be absorbed through an oral mucosa to play a drug effect, meanwhile, the PLGA nano-particles encapsulation rate can reach 70-90%, and the PLGA microspheres encapsulation rate disclosed in CN1308034C is only 40%.
The interferon oral patch disclosed by the invention adopts PLGA nano-particles to deliver the medicament, and a comparison experiment is carried out with the interferon oral patch disclosed by CN1513548A, and the result shows that the oral patch disclosed by the invention obviously prolongs the action time in a medicament body and improves the relative bioavailability of the medicament.
In a preferred embodiment, the thickness of the chitosan outer layer is 12-22 nm.
The invention provides a preparation method of the nano-particles, which comprises the following steps:
1) dispersing interferon alpha-2 b in a sodium alginate solution to serve as an internal water phase, mixing the internal water phase with an organic phase, and performing ultrasonic emulsification to form water-in-oil colostrum;
2) injecting the primary emulsion into an external water phase, fully and uniformly mixing, and then carrying out ultrasonic emulsification to form a water-in-oil-in-water double emulsion solution, wherein the external water phase also contains polyvinyl alcohol and Ca2+And chitosan;
3) and (3) completely volatilizing the organic solvent in the multiple emulsion solution, and freeze-drying to form the nano-particles.
Wherein the sodium alginate in the step 1) has the effect of assisting dissolution and dispersion, can promote the dissolution and dispersion of samples, can be used as a surfactant to promote the formation of colostrum with an inner water phase, and can also be used as a surfactant to promote the formation of Ca in an outer water phase2+The reaction forms gel to help the medicine to be wrapped in the nano particles, the encapsulation efficiency is improved, and the nano particles are easy to disperse after being freeze-dried.
In a preferred embodiment, the organic phase is prepared by: dissolving a solid polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase; and/or complete volatilization of the organic solvent of the multiple emulsion solution comprises low-speed stirring or reduced pressure distillation at room temperature.
In a preferred embodiment, the specific preparation method of the nanoparticles comprises the following steps:
(1) preparing a polylactic acid-glycolic acid copolymer solution: dissolving a solid polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase;
(2) preparing an interferon alpha-2 b dispersion solution: the interferon alpha-2 b is uniformly dispersed in a mass-to-volume ratio of
0.00001-5.0000% (w/v) sodium alginate solution to form an inner water phase;
(3) preparing colostrum: fully mixing the internal water phase and the organic phase, and then carrying out ultrasonic emulsification to form W/O primary emulsion;
(4) preparing multiple emulsion: injecting the colostrum into a solution containing certain concentration of polyvinyl alcohol and Ca2+Mixing with chitosan external water phase, mixing, and ultrasonic emulsifying to obtain W/O/W compound emulsion;
(5) nano-particles: completely volatilizing the organic solvent of the W/O/W multiple emulsion, and then carrying out freeze drying treatment.
In a preferred embodiment, the external aqueous phase consists of 0.0001% -20.000% (w/v) PVA, 0.00005% -0.15000% (w/v) chitosan solution, 0.0001-0.18(mol/L) Ca2+Mixing the solution to form;
the organic solvent comprises one or a mixture of dichloromethane, ethyl acetate and acetone; and/or the volume ratio of the organic phase to the internal aqueous phase is (2-40): 1, the volume ratio of the primary emulsion to the external water phase is 1: (3-20);
in a preferred embodiment, the volume ratio of the organic phase to the inner aqueous phase is 5:1 and the volume ratio of colostrum to the outer aqueous phase is 1: 7.
In a preferred embodiment, the ultrasonic condition in the step 3) is 10-1000w, 1-60min ultrasonic; the ultrasonic condition for the multiple emulsion formation in the step 4) is 10-1000w, and the ultrasonic treatment is carried out for 1-60 min.
In a preferred embodiment, in step 5), the double emulsion solution is stirred at a low speed at 4 ℃ or vacuum rotary evaporated at room temperature until the organic solvent is completely volatilized. The stirring speed is 1000-5000rpm, the stirring time is 4-12 hours, or the rotary evaporation temperature is 25 ℃, and the rotary evaporation time is 30-60 minutes; the freeze drying treatment steps are as follows: adding the nano particles into a mannitol solution containing 5-7% (m/v), pre-freezing, and freeze-drying for 48h to obtain white powder with uniform texture.
The present disclosure provides a pharmaceutical preparation comprising the interferon alpha-2 b nanoparticles. The interferon alpha-2 b nano-particles can be prepared into different formulations according to requirements, and can play the roles of prolonging the in vivo half-life period of the interferon alpha-2 b, reducing clearance rate, immunogenicity and antigenicity, reducing administration frequency and improving tolerance of patients. For example, it can be prepared into, for example, interferon α -2b injection, interferon α -2b eye drop, interferon α -2b nose drop, interferon α -2b oral preparation or other pharmaceutically acceptable dosage forms as required.
In a preferred embodiment, the dosage form of the pharmaceutical formulation is an oral patch.
In a preferred embodiment, the pharmaceutical preparation comprises nanoparticles and pharmaceutic adjuvants, wherein the pharmaceutic adjuvant comprises a filler, an adhesive and a lubricant, and the mass ratio of the nanoparticles to the pharmaceutic adjuvants is (4-3): (1-2).
In a preferred embodiment, the mass ratio of the nanoparticles to the pharmaceutical excipients is 4: 1; and/or, based on the total weight of the oral patch, the total weight ratio of the filling agent in the pharmaceutical excipients to the oral patch is 2:25, the total weight ratio of the adhesive agent to the oral patch is 2:25, and the total weight ratio of the lubricant to the oral patch is 1: 500; the oral patch is a tablet with a diameter of 5-10 mm, a thickness of 1-3 mm and a weight of 30-200 mg. The tablet has good adhesion, good release, and moderate dissolution rate.
In a preferred embodiment, the freeze-dried white powder and the oral patch auxiliary materials are mixed at a ratio of 4:1, and the mixture is directly tableted by a dry method to obtain the sustained-release oral patch which has the width of 6 mm, the thickness of 2 mm and the weight of about 50 mg and has good adhesion and good drug release.
In a preferred embodiment, the filler comprises starch or mannitol, and/or the adhesive comprises one or more combinations of carbomer, carboxymethylcellulose, sodium carboxymethylcellulose; and/or the lubricant comprises magnesium stearate or talc; and/or the nano particles are selected from a white freeze-dried powder form formed by freeze-drying with a freeze-drying protective agent.
In a preferred embodiment, the present disclosure provides a method of preparing a pharmaceutical formulation formed from nanoparticles: the tablet is prepared by mixing and tabletting nano particles and pharmaceutic adjuvants according to a certain proportion, wherein the pharmaceutic adjuvants are a mixture containing a filling agent, an adhesive and a lubricant.
In a preferred embodiment, the present disclosure provides the use of said nanoparticles for the preparation of a pharmaceutical formulation.
In a preferred embodiment, the present disclosure provides the use of said nanoparticles for the preparation of an oral patch.
When the oral patch prepared above acts, the interferon alpha-2 b nanoparticles can be released from the tablet into the blood for slow release. According to the results of pharmacokinetic studies, the administration in this way can improve the half-life of interferon in vivo and release it for a long time, thus reducing the administration frequency of the drug and better improving the compliance of patients.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) the interferon alpha-2 b nano-particles prepared by the invention have simple and stable process, high feasibility and industrialization, are round in shape, have a double-layer structure, smooth surface and good fluidity, have the PDI of 0.1-0.3, the particle size of 50-1000nm and the entrapment rate of about 70-90 percent, and have in-vitro drug release performance conforming to the characteristics of long-acting preparations; on the other hand, the characteristic that the outer layer of the nanoparticle has positive charges is favorable for being combined with a cell membrane with negative charges, and further favorable for transmembrane movement of the nanoparticle, so that the absorption and the transportation of interferon alpha-2 b drugs are increased.
(2) The oral patch provided by the invention uses a biodegradable material as a carrier, coats interferon alpha-2 b to prepare nanoparticles, and further prepares the nanoparticles into the oral patch, and the nanoparticles are small in particle size and stable in dispersion, so that the oral patch provided by the invention can increase the stability of the drug, increase the half-life period of the drug and prolong the action time of the drug, thereby reducing the administration times and dosage, improving the curative effect of the drug, and being safer and more convenient by using an oral administration mode. On the other hand, the interferon alpha-2 b is prepared into the sustained-release oral patch, so that the first-pass effect of gastrointestinal tracts is avoided, the action time of the medicine is prolonged, the curative effect and the economic benefit of the medicine are improved, the influence of enzymes on the interferon is reduced, the pain of a patient caused by injection is avoided, and the prepared tablet has strong adhesive capacity, good release and moderate dissolution rate.
Drawings
FIG. 1 is a scanning electron micrograph of the interferon alpha-2 b nanoparticles of the present invention;
FIG. 2 is a graph showing the particle size distribution of interferon alpha-2 b nanoparticles according to the present invention;
FIG. 3 is a ZETA potential distribution diagram of interferon alpha-2 b nano-particles of the invention;
FIG. 4 shows the stability change of the interferon alpha-2 b nanoparticles of the present invention;
FIG. 5 is a report of 24, 48 hour biological activity of interferon alpha-2 b nanoparticles of the present invention;
FIG. 6 is a tabular representation of data for pharmacokinetics of buccal and sublingual administration of the interferon alpha-2 b sustained release oral patch of the present invention;
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described. For purposes of this disclosure, the following terms are defined as follows.
The interferon alpha-2 b stock solution is provided by Xiamen Tebao bioengineering Co., Ltd, and has a concentration of 9.52mg/ml and an activity of 1.8 × 108IU/mg。
Example 1
Preparation of interferon alpha-2 b slow-release oral patch
(1) Preparation of polylactic-co-glycolic acid (PLGA) solution
100mg of polylactic acid-glycolic acid copolymer (50: 50) was dissolved in 2ml of methylene chloride to form an organic phase (0.05 g/ml);
(2) preparation of interferon alpha-2 b dispersion solution
Uniformly dispersing 20 mu l of interferon alpha-2 b stock solution in 250 mu l of sodium alginate solution with the mass-volume ratio of 0.003% (w/v) to form an internal water phase;
(3) preparation of colostrum
Injecting the inner water phase into the organic phase, and performing ultrasonic emulsification under the ultrasonic condition of 10-1000W for 60min to form primary emulsion (W/O); the volume ratio of the organic phase to the internal aqueous phase is 7.5: 1;
(4) preparation of multiple emulsions
The colostrum thus prepared was injected into a container containing 10ml of a solution containing 0.5% PVA (w/v), 0.045mol/LCaCl20.01% (W/v) of chitosan in the external water phase, and carrying out ultrasonic emulsification for 60min under the ultrasonic condition of 10-1000W (the ultrasonic condition is the same as the above), so as to emulsify the chitosan into multiple emulsion (W/O/W); the volume ratio of the primary emulsion to the external water phase is 1: 5;
(5) formation of nanoparticles
And distilling the multiple emulsion at room temperature under reduced pressure for 1 h. The organic solvent is removed.
(6) Freeze drying
The interferon alpha-2 b nano-particle solution and a 5% mannitol solution are mixed evenly, pre-frozen at 20 ℃ for 2h, pre-frozen at 80 ℃ for about 24h, and freeze-dried in a freeze dryer for 48 h.
The obtained nano-particle freeze-dried powder is white loose powder, has good redissolution effect, uniform particle dispersion and good stability. The obtained interferon alpha-2 b nano-particles have round shape, good fluidity, average particle size of 216nm, PDI of about 0.12 and encapsulation rate of 90 percent, and the in vitro drug release performance conforms to the characteristics of long-acting preparations.
(7) Preparation of oral Patches
The interferon alpha-2 b nano-particle freeze-dried powder is mixed with auxiliary materials such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, mannitol, magnesium stearate and the like according to the mass ratio of 4:1 for tabletting, and tablets with the width of 6 mm, the thickness of 2 mm and the weight of 50 mg are obtained.
Example 2
Preparation of interferon alpha-2 b slow-release oral patch
(1) Preparation of polylactic acid-glycolic acid copolymer (PLGA) solution
80mg of polylactic acid-glycolic acid copolymer (50: 50) was dissolved in 2ml of methylene chloride to form an organic phase (0.04 g/ml);
(2) preparation of interferon alpha-2 b dispersion solution
20 μ l of stock solution of interferon alpha-2 b was uniformly dispersed in 125 μ l of sodium alginate solution at a mass to volume ratio of 0.00001% (w/v) to form an internal aqueous phase.
(3) Preparation of colostrum
Injecting the inner water phase into the organic phase, and performing ultrasonic emulsification for 1min under the ultrasonic condition of 10-1000W to form primary emulsion (W/O); the volume ratio of the organic phase to the internal aqueous phase is 13.5: 1;
(4) preparation of multiple emulsions
The colostrum thus prepared was injected into a container containing 6ml of a solution containing 20% PVA (w/v), 0.0001mol/LCaCl2Emulsifying with 0.00005% (W/v) chitosan in external water phase under ultrasonic condition of 10-1000W for 1min (the ultrasonic condition is the same as above) to obtain multiple emulsion (W/O/W); the volume ratio of the primary emulsion to the external water phase is 1: 3;
(5) formation of nanoparticles
The multiple emulsion is stirred at the temperature of 4 ℃ and the speed of 1500rpm/min until the organic solvent is completely volatilized.
(6) Freeze drying
The interferon alpha-2 b nano-particle solution and 7.5 percent (m/v) mannitol solution are mixed evenly, pre-frozen at the temperature of-20 ℃ for 2h and pre-frozen at the temperature of-80 ℃ for about 24h, and freeze-dried in a freeze dryer for 48 h.
The obtained nano-particle freeze-dried powder is white loose powder, has good redissolution effect, uniform particle dispersion and good stability. The obtained interferon alpha-2 b nano-particles have round shapes, good fluidity and an average particle size of 92 nm. The PDI is about 0.2, the encapsulation rate is 75%, and the in vitro release performance accords with the characteristics of a long-acting preparation.
(7) Preparation of oral Patches
Mixing the interferon alpha-2 b nano particle freeze-dried powder, a filling agent, an adhesive and a lubricant according to a mass ratio of 4:1, mixing and tabletting, wherein the total weight ratio of the filling agent to the oral patch in the pharmaceutical excipients is 2:25, the total weight ratio of the adhesive to the oral patch is 2:25, and the total weight ratio of the lubricant to the oral patch is 1: 500; the oral patch is a tablet with a diameter of 5-10 mm, a thickness of 1-3 mm and a weight of 30-200 mg. The tablet has good adhesion, good release, and moderate dissolution rate.
The filler is starch or mannitol, and the adhesive comprises one or more of carbomer, carboxymethyl cellulose and sodium carboxymethyl cellulose; the lubricant is magnesium stearate or talcum powder.
Example 3
Preparation of interferon alpha-2 b slow-release oral patch
(1) Preparation of polylactic-co-glycolic acid (PLGA) solution
160mg of polylactic acid-glycolic acid copolymer (50: 50) was dissolved in 2ml of methylene chloride to form an organic phase (0.08 g/ml);
(2) preparation of interferon alpha-2 b dispersion solution
Uniformly dispersing 50 mu l of interferon alpha-2 b stock solution in 125 mu l of sodium alginate solution with the mass-volume ratio of 0.006 percent (w/v) to form an internal water phase;
(3) preparation of colostrum the inner water phase is injected into the organic phase, and ultrasonic emulsification is carried out for 2min under the ultrasonic condition of 10-1000W to form colostrum (W/O); the volume ratio of the organic phase to the internal aqueous phase is 11.5: 1;
(4) preparation of multiple emulsions
The prepared colostrum was injected into a container containing 10ml of 0.0001% PVA (w/v), 0.045mol/LCaCl20.01% (W/v) of chitosan in the external water phase, and carrying out ultrasonic emulsification for 2min under the ultrasonic condition of 10-1000W (the ultrasonic condition is the same as the above), so as to emulsify the chitosan into multiple emulsion (W/O/W); the volume ratio of the primary emulsion to the external water phase is 1: 7;
(5) formation of nanoparticles
And distilling the multiple emulsion at room temperature under reduced pressure for 1 h. The organic solvent is removed.
(6) Freeze drying
The interferon alpha-2 b nano-particle solution and a 5% mannitol solution are mixed evenly, pre-frozen at 20 ℃ for 2h, pre-frozen at 80 ℃ for about 24h, and freeze-dried in a freeze dryer for 48 h.
The obtained nano-particle freeze-dried powder is white loose powder, has good redissolution effect, uniform particle dispersion and good stability. The obtained interferon alpha-2 b nano-particles have round shapes, good fluidity and average particle size of 312 nm. The PDI is about 0.2, the encapsulation rate is 75%, and the in vitro release performance accords with the characteristics of a long-acting preparation.
(7) Preparation of oral Patches
The interferon alpha-2 b nano-particle freeze-dried powder is mixed with auxiliary materials such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, mannitol, magnesium stearate and the like according to the volume ratio of 4:1 for tabletting, and tablets with the width of 6 mm, the thickness of 2 mm and the weight of 50 mg are obtained.
Example 4
Preparation of interferon alpha-2 b slow-release oral patch
(1) Preparation of polylactic-co-glycolic acid (PLGA) solution
100mg of polylactic acid-glycolic acid copolymer (50: 50) was dissolved in 4ml of methylene chloride to form an organic phase (0.05 g/ml);
(2) preparation of interferon alpha-2 b dispersion solution
Uniformly dispersing 50 mu l of interferon alpha-2 b stock solution in 100 mu l of sodium alginate solution with the mass-volume ratio of 0.003% (w/v) to form an internal water phase;
(3) preparation of colostrum the inner water phase is injected into the organic phase, and ultrasonic emulsification is carried out for 2min under the ultrasonic condition of 10-1000W to form colostrum (W/O); the volume ratio of the organic phase to the internal aqueous phase is 40: 1;
(4) preparation of multiple emulsions
The colostrum thus prepared was injected into a container containing 10ml of 1% PVA (w/v), 0.13mol/LCaCl20.01% (W/v) of chitosan in the external water phase, and carrying out ultrasonic emulsification for 2min under the ultrasonic condition of 10-1000W (the ultrasonic condition is the same as the above), so as to emulsify the chitosan into multiple emulsion (W/O/W); the volume ratio of the primary emulsion to the external water phase is 1: 7;
(5) formation of nanoparticles
And distilling the multiple emulsion at room temperature under reduced pressure for 1 h. The organic solvent is removed.
(6) Freeze drying
The interferon alpha-2 b nano-particle solution and a 5% mannitol solution are mixed evenly, pre-frozen at 20 ℃ for 2h, pre-frozen at 80 ℃ for about 24h, and freeze-dried in a freeze dryer for 48 h.
(7) Preparation of oral Patches
The interferon alpha-2 b nano-particle freeze-dried powder is mixed with auxiliary materials such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, mannitol, magnesium stearate and the like according to the volume ratio of 4:1 for tabletting, and tablets with the width of 6 mm, the thickness of 2 mm and the weight of 50 mg are obtained.
Example 5
Preparation of interferon alpha-2 b slow-release oral patch
(1) Preparation of polylactic-co-glycolic acid (PLGA) solution
100mg of polylactic acid-glycolic acid copolymer (50: 50) was dissolved in 2ml of methylene chloride to form an organic phase (0.05 g/ml);
(2) preparation of interferon alpha-2 b dispersion solution
Uniformly dispersing 20 mu l of interferon alpha-2 b stock solution in 980 mu l of sodium alginate solution with the mass-volume ratio of 0.003% (w/v) to form an internal water phase;
(3) preparation of colostrum the inner water phase is injected into the organic phase, and ultrasonic emulsification is carried out for 2min under the ultrasonic condition of 10-1000W to form colostrum (W/O); the volume ratio of the organic phase to the internal aqueous phase is 2: 1;
(4) preparation of multiple emulsions
The colostrum thus prepared was injected into a container containing 10ml of 20% PVA (w/v), 0.045mol/LCaCl20.01% (W/v) of chitosan in the external water phase, and carrying out ultrasonic emulsification for 2min under the ultrasonic condition of 10-1000W (the ultrasonic condition is the same as the above), so as to emulsify the chitosan into multiple emulsion (W/O/W); the volume ratio of the primary emulsion to the external water phase is 1: 7;
(5) formation of nanoparticles
And distilling the multiple emulsion at room temperature under reduced pressure for 1 h. The organic solvent is removed.
(6) Freeze drying
The interferon alpha-2 b nano-particle solution and a 5% mannitol solution are mixed evenly, pre-frozen at 20 ℃ for 2h, pre-frozen at 80 ℃ for about 24h, and freeze-dried in a freeze dryer for 48 h.
(7) Preparation of oral Patches
The interferon alpha-2 b nano-particle freeze-dried powder is mixed with auxiliary materials such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, mannitol, magnesium stearate and the like according to the volume ratio of 4:1 for tabletting, and tablets with the width of 6 mm, the thickness of 2 mm and the weight of 50 mg are obtained.
Example 6
Preparation of interferon alpha-2 b slow-release oral patch
(1) Preparation of polylactic-co-glycolic acid (PLGA) solution
100mg of polylactic acid-glycolic acid copolymer (50: 50) was dissolved in 4ml of methylene chloride to form an organic phase (0.04 g/ml);
(2) preparation of interferon alpha-2 b dispersion solution
Uniformly dispersing 50 mu l of interferon alpha-2 b stock solution in 100 mu l of sodium alginate solution with the mass-volume ratio of 0.003% (w/v) to form an internal water phase;
(3) preparation of colostrum the inner water phase is injected into the organic phase, and ultrasonic emulsification is carried out for 2min under the ultrasonic condition of 10-1000W to form colostrum (W/O); the volume ratio of the organic phase to the internal aqueous phase is 40: 1;
(4) preparation of multiple emulsions
The colostrum thus prepared was injected into a container containing 10ml of 1.75% PVA (w/v), 0.18mol/LCaCl20.15% (W/v) of chitosan in the external water phase, and carrying out ultrasonic emulsification for 2min under the ultrasonic condition of 10-1000W (the ultrasonic condition is the same as the above), so as to emulsify the chitosan into multiple emulsion (W/O/W); the volume ratio of the primary emulsion to the external water phase is 1: 5;
(5) formation of nanoparticles
And distilling the multiple emulsion at room temperature under reduced pressure for 1 h. The organic solvent is removed.
(6) Freeze drying
The interferon alpha-2 b nano-particle solution and a 5% mannitol solution are mixed evenly, pre-frozen at 20 ℃ for 2h, pre-frozen at 80 ℃ for about 24h, and freeze-dried in a freeze dryer for 48 h.
(7) Preparation of oral Patches
The interferon alpha-2 b nano-particle freeze-dried powder is mixed with auxiliary materials such as hydroxypropyl methylcellulose, sodium carboxymethylcellulose, carbomer, mannitol, magnesium stearate and the like according to the volume ratio of 4:1 for tabletting, and tablets with the width of 6 mm, the thickness of 2 mm and the weight of 50 mg are obtained.
The interferon alpha-2 b nanoparticles prepared according to the methods of examples 1-6 have the following structures and properties:
FIG. 1 is a scanning electron microscope image of interferon alpha-2 b nanoparticles. As can be seen from fig. 1, the nanoparticle has a double-layer structure, and the nanoparticle has a double-layer structure including: (a) an outer layer of chitosan bearing a positive charge; and (b) a biodegradable polymeric inner layer surrounded by an outer layer; the solid interferon alpha-2 b is uniformly distributed in the inner layer of the polymer; the weight of the solid interferon alpha-2 b is 0.1-0.5% of the total weight of the nano-particles.
FIG. 2 is a graph of the particle size distribution of nanoparticles prepared according to the methods of examples 1-3, with a mean value of about 300nm and a specific distribution interval of 50nm to 1000nm, which facilitates penetration of the nanoparticles across the oral mucosa into the blood stream for therapeutic and prophylactic purposes.
The thickness of the chitosan outer layer is detected to be 12-22 nm.
And (3) charge detection: the charge of the outer layer was monitored using an antopa lite size r 500 particle size analyzer according to the Electrophoretic Light Scattering (ELS)/cmPALS principle to a charge of 3.5 mv. The outer layer of the nano-particle prepared by the invention has positive charges, and can be fused with a negatively charged cell membrane, so that transmembrane movement is carried out, and the nano-particle enters a blood vessel to generate an effect. FIG. 3 is a ZETA potential distribution diagram of interferon alpha-2 b nano-particles of the invention;
interferon alpha-2 b nanoparticle stability test
The prepared interferon alpha-2 b nano-particles are subjected to particle size detection at the same time of 1-7 days, the nano-particles are placed at 4 ℃, and whether aggregation or degradation occurs is observed. The particle size of the particle is detected by an Antopa LiteSIZER 500 particle size analyzer according to the Dynamic Light Scattering (DLS) principle, the PDI value is 0.1-0.3, and the dispersibility is good.
FIG. 4 shows the stability change of the interferon alpha-2 b nanoparticles of the present invention; it is shown in fig. 4 that the interferon alpha-2 b nanoparticles have good stability at 4 ℃ and no obvious aggregation or degradation phenomenon.
Example 7
Interferon alpha-2 b nano particle in vitro activity determination experiment
The interferon alpha-2 b nanoparticle powder thus prepared was used as a method for measuring the in vitro activity of the interferon alpha-2 b nanoparticle of this time by the second method (reporter gene method) in the interferon biological activity measurement method 3523, the fourth pharmacopoeia of China, 2020 edition.
Second method reporter Gene method (for type I Interferon)
The method comprises the steps of transfecting plasmids containing an interferon stimulation response element and a luciferase gene into HEK293 cells, constructing a cell line HEK293 puro ISRE-Luc as a biological activity determination cell, activating the interferon stimulation response element through signal transduction after I-type interferon is combined with a receptor on a cell membrane, starting the expression of luciferase, positively correlating the expression quantity with the biological activity of the interferon, and determining the luminous intensity of the human cell lysate and a luciferase substrate after adding the human cell lysate and the luciferase substrate, so as to determine the biological activity of the I-type interferon.
Assay method
HEK293 puro ISRE-Luc cells were grown adherently in complete medium. According to the following steps: 4 passages, 2-3 times per week, and growth in complete medium. Removing culture medium from cultured cells, washing with PBS for 1 time, digesting, collecting cells, preparing into 3.5 × 10 per l m l culture medium5～4.5×105A cell suspension of individual cells. Transferring the prepared standard solution and the interferon alpha-2 b nanoparticle solution into a 96-well cell culture plate which can be used for cell culture and chemiluminescence enzyme-labeling instrument measurement, adding 100 mu l of the prepared standard solution and the prepared interferon alpha-2 b nanoparticle solution into each well, and then inoculating the cell suspension into the same 96-well cell culture plate, wherein each well contains 100 mu l of the prepared cell suspension. Culturing at 37 deg.C and 5% carbon dioxide for 18-24 hr. Carefully sucking up the supernatant in a 96-well cell culture plate, adding human cell lysate and luciferase substrate according to the specification of a luciferase reporter gene detection kit, measuring the luminous intensity by using a chemiluminescence enzyme-linked immunosorbent assay, and recording the measurement result.
The results were calculated according to the following formula:
in the formula PrThe biological activity of the standard substance is IU/ml;
Dspre-diluting for the sample;
Drpre-diluting the standard substance by multiple times;
Esthe dilution factor is half effective amount of the standard substance for the test substance;
Eris the dilution multiple of half effective amount of the standard.
The experiment respectively determines the biological activity of the interferon alpha-2 b nano-particles and the standard substance within 24 hours and 48 hours, and the result of figure 5 shows that the interferon alpha-2 b nano-particles still have the biological activity of the interferon within a long time after freeze-drying and re-dissolving. Therefore, the interferon alpha-2 b still has biological activity after being prepared into the nano-particles, and the biological function of the nano-particles can not be influenced after being prepared into the nano-particles.
Example 8
Pharmacokinetic experiment of interferon alpha-2 b oral patch
24 rats were randomly divided into A, B, C, D groups of 6 rats each, and were normally kept for one week without water deprivation for fasting for 12 hours before administration.
The interferon alpha-2B injection with the same dosage as the sublingual injection is injected subcutaneously in the group A, the interferon alpha-2B oral mucosa adhesive sheet l sheet (without nanoparticles adhered to oral buccal mucosa) in the group B, the nano-particle interferon alpha-2B oral mucosa adhesive sheet l sheet (adhered to oral buccal mucosa) is respectively administered to the group C (rats are anesthetized), and the nano-particle interferon alpha-2B oral mucosa adhesive sheet l sheet (adhered to the sublingual buccal mucosa) in the group D.
The preparation method of the group B interferon alpha-2B oral mucosa adhesive sheet comprises the following steps: adding 100mg mannitol into 100 μ l interferon solution as excipient and freeze-drying protective agent, freeze-drying to obtain lyophilized powder, mixing with tabletting adjuvant, and directly tabletting. CD group is the experimental group of the present invention (prepared according to the methods of examples 1-6);
blood collection points (0h, 1h, 2h, 3h, 5h, 7h, 9h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, 96h, 120h, 144h and 168h) in group ABC, blood collection points (0h, 0.08h, 0.25h, 0.5h, 0.75h, 1h, 1.5h, 2h, 3h, 5h, 7h, 12h, 24h, 48h, 72h, 120h and 168h) in group D, orbital blood collection of 0.2mL, EDTA-K2 anticoagulation, centrifugation at 12000r/min for 5min, plasma separation and storage in a refrigerator at minus 80 ℃ are set after administration. The plasma sample processing method is prepared according to the standard curve and used for subsequent ELISA detection, and the experimental result is processed by winolin to obtain corresponding pharmacokinetic data. A, B, C groups were appropriately supplemented with anesthetic in the experiment.
The results show that the interferon alpha-2B oral patch in the A group is completely metabolized within about 12 hours when being injected subcutaneously, the interferon alpha-2B oral patch in the B group is completely metabolized within about 24 hours, and the interferon alpha-2B oral patches in the C, D two groups of nano-particle interferon alpha-2B oral patches can still detect the interferon alpha-2B in blood after 96 hours. The relative bioavailability of the interferon alpha-2B oral patch in the group B is about 1.5 percent, the relative bioavailability of the nano-interferon alpha-2B oral mucosa adhesive sheet (adhered to buccal mucosa) in the group C is about 6 percent, and the relative bioavailability of the nano-interferon alpha-2B oral mucosa adhesive sheet (adhered to sublingual buccal mucosa) in the group D is about 13 percent. Therefore, the relative bioavailability of C, D two groups of nano-particle interferon alpha-2B oral mucosa adhesive tablets is 4-10 times that of B group of interferon alpha-2B oral patches. Therefore, the nano particles in the oral patch enter blood through the oral mucosa route and slowly release the medicine, so that the purposes of prevention and treatment can be achieved.
The interferon alpha-2 b sustained-release oral patch has the following advantages: 1. can not only directly activate interferon receptors in oral mucosa and lymphatic circulation system to prevent virus invasion, but also enter blood into the whole body to enhance the immunity of the whole body. 2. The action time of the interferon alpha-2 b slow-release oral patch is greatly prolonged to about 240 hours. 3. The oral administration is convenient, the raw materials are safe, and the production is convenient. Figure 6 is a tabular representation of data for pharmacokinetics of interferon alpha-2 b sustained release buccal patch and sublingual administration.