CN1757398A - Method for preparing inorganic salt nanometer particles containing and packing traditional Chinese medicine and its application - Google Patents
Method for preparing inorganic salt nanometer particles containing and packing traditional Chinese medicine and its application Download PDFInfo
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Abstract
An inorganic salt nano-particle containing the encapsulated Chinese-medicinal components is prepared through preparing the inorganic salt nano-particles containing Chinese-medicinal components, encapsulating the active components of Chinese medicine and encapsulating by bioactive substance. It can be used as the carrier for transferring the bioactive substance or as the adjuvant of vaccine.
Description
Technical field
The present invention is the transmission system of a kind of medicine or vaccine.Especially in the transmission system of medicine or vaccine, provide a kind of preparation method and application thereof that contains and seal the inorganic salt nano particles of Chinese medicine ingredients.
Background technology
Nanotechnology is 21 century one of the revolutionary technology of tool, is a boundary science technology interdisciplinary.It is the crystallization of multi-door subjects such as biology, medical science, materialogy, chemistry, physics.Nanotechnology is as a kind of space platform technology, and its scope has related to many subjects and technical field.Especially aspect medical domain, pharmaceutics development and biological engineering and the medical science equipment revolutionary breakthrough being arranged, become one of focus in medical science, biomedicine and the study of pharmacy.
Nanotechnology relates to the development of the development technology of medical material, Medical Equipment, pharmaceutics in medical research.The preparation of nanoparticle and application then are to have the break-through point of hope in the pharmaceutics and have great market and using value.Nanoparticle typically refers to size particle (flat its energy: " Nano medication and nano-carrier system " in the 100nm-1000nm scope in pharmaceutics research.Chinese Journal of New Drugs 11 the 1st phases of volume of ground in 2002, numbering 1003-3734 (2002) 01-0042-05).
In biomedical sector, utilize nanometer or micron particle as diagnostic tool, improve the sensitivity and the correctness of diagnosis, thereby reach early stage, quick and accurately diagnosis in conjunction with computer technology.Be the healing of tumor disease offer help (Borm PJ, Kreyling W.J Nanosci Nanotechnol.2004 May; 4 (5): 521-31.Toxicological hazards of inhaled nanoparticles-potential implications for drugdelivery).Simultaneously, because the diagnostic kit of nanometer preparation is convenient to storage and transportation, thereby reduced diagnostic fees usefulness.Being particularly suitable for extensive on-the-spot EPDML generaI investigation and backwoodsman hospital uses.
Nanoparticle has following advantage as drug delivery system in materia medica and pharmaceutics:
1, nanoparticle is particularly suitable as various types of pharmaceutical carriers because of its size, physicochemical property and surface characteristic, and forms the transmission system of medicine by different combinations.Because its size belongs to subcellular fraction, sub-micrometer range, in vivo by the efficient of cellular uptake than the big 15-250 of micron particle doubly and can pass inferior mucous layer, and micron only rests on the mucosa top layer.Nanoparticle can also pass blood brain barrier effectively, thereby the transmission medicine enters brain, increases the concentration of medicine at brain, effectively treats brain diseases.For example cerebroma can not well can not get effective treatment (Lisa Brannon-Peppas and James O.Blanchette.Adv Drug DelivRev.2004 Sep 22 by blood brain barrier owing to medicine exactly; 56 (11): 1649-59.Nanoparticle and targeted systems for cancer therapy. and Kreuter J.J Nanosci Nanotechnol.2004 May; 4 (5): 484-8.Influence of thesurface properties on nanoparticle-mediated transport of drugs to the brain.).
2, some nanoparticle is can be biodegradable.Carry medicine when nanometer and enter in the body,, guaranteed drug concentrations thereby medicine continues release along with the degraded of nanoparticle, improved the action time of medicine, reduce dosage, alleviated the toxic and side effects of medicine, reduced the discomfort reaction that patient causes because of taking medicine.
3, nanoparticle can directedly be carried medicine, especially can directedly carry antitumor drug, behind intravenous injection nanometer antitumor drug, find that medicine accumulates in around the tumor, improved medicine antitumor characteristic, reduced dosage, can obviously reduce the reaction of patient the toxic and side effects of medicine.The function of this targeted medicine of nanoparticle has improved the medicine local concentration, has increased the curative effect of medicine, be particularly suitable for treatment (the Lockman PR of brain and ocular disease, Mumper RJ, Khan MA, Allen DD.Drug Dev Ind Pharm.2002 Jan; 28 (1): 1-13.Nanoparticle technology for drugdelivery across the blood-brain barrier.).
4, improve the stability of macromolecular drug.Especially peptide class and protein drug.This class medicine is synthesized in the nanoparticle, can protect medicine when oral, not destroyed and protect medicine not by enzymatic degradation (Gavini E by the low pH value environment of gastrointestinal, Chetoni P, Cossu M, Alvarez MG, SaettoneMF, Giunchedi P.Eur J Pharm Biopharm.2004 Mar; 57 (2): 207-12.PLGA microspheres forthe ocular delivery of a peptide drug, vancomycin using emulsification/spray-drying as thepreparation method:in vitro/in vivo studies. and Morishita M, Takayama K.NipponRinsho.2002 Sep; 60Suppl 9:516-21.The design of oral insulin delivery systemsbased on smart hydrogels).
5, discover that recently not only can targeting transmission medicine can also transmit medicine to specific tissue and cell with nanoparticle as the medicine of carrier arrives specific organelle, further improve bioavailability of medicament (Takeuchi H, Yamamoto H, Kawashima Y.Adv Drug Deliv Rev.2001Mar 23; 47 (1): 39-54.Mucoadhesive nanoparticulate systems for peptide drugdelivery.).
Nanoparticle occupies more and more important position in the development of drug delivery and pharmaceutics and development, the medicine that will become potentialization soon carries system.Especially along with the increase of the demand and the market proportion of biologics, the demand of Nano medication transmission system will improve thereupon.
At present in drug delivery system the nanoparticle of common research have: solid lipid nanoparticle of forming by fatty acid, aliphatic alcohol and phospholipid; Nanosphere or nanometer bundle by bioabsorbable polymer material preparations such as polylactic acid, the poly-third glue fat, poly-chitose, gelatin; The nanoparticle of forming by the copolymer of polylactic acid and Polyethylene Glycol or poly-chitose and derivant thereof.Its synthetic method has polymerization; Supercritical technology; The cohesion dispersion method; The high pressure homogenization method; The fusion dispersion method; Solvent evaporated method and emulsifying/solvent diffusion method.At present China the nanoparticle of normal research lipid nanometer, nanocapsule, nanosphere and Nano capsule are arranged.Wherein liposome nano granule is the most a kind of (flat its energy, " present situation of nano-drug preparation is with following " 2002-09-03, a Chinese Medicine of research both at home and abroad; And Maitra, et al.United State Patent 6,555,376.July 24,2002: and Bell, etal.United State Patent 635,5271.March, 2002; And Fulton et al.United StatePatent 5,266,205.Nov, 1993).
Though synthetic medicine of nanotechnology and vaccine delivery system have very big progress in recent years, but still have some important obstacles, make nanoparticle stagnate aspect the industrialization as drug delivery system.The problem that exists has (Rao GC, Kumar MS, Mathivanan N, Rao ME.Pharmazie.2004 Jan at present; 59 (1): 5-9.Nanosuspensions as the most promising approach innanoparticulate drug delivery systems.):
At first, the raw material of most of nanoparticles is organic compound or derivatives thereofs, the building-up process complexity.Change the structure of macromolecular drug with meeting in the bonded preparation process of macromolecular drug, thereby reduce its activity.For example, PLGA with the bonded process of pharmaceutical grade protein in can lose about 30% protein active.
Secondly, some nanoparticles in vivo can not biodegradation, thereby influences the slow release and the controlled-release function of entrained medicine, and because the gathering in vivo of particle might produce certain side effect.
The 3rd, most of nanoparticle building-up process complexity, raw material mostly is some organic macromolecule materials again, thereby the poor repeatability between each batch product, the size of particle, surperficial physicochemical characteristic and concentration are criticized in difference very big difference between the product, be difficult to the suitability for industrialized production of control nanoparticle, bring very big obstacle for the industrialization of nanoparticle.The liposome nanometer is modal.
The 4th, there is certain side effect in some particle carrier system, and the mechanism of the side effect of its generation it be unclear that.For example, with the carrier of liposome as influenza vaccines, facial paralysis rate occurred frequently appears in clinical report, forbids in the European market at present.
The 5th, the polymer nanoparticle is after repeatedly taking, and is slow because of its biodegradation rate, may bring out the general toxic reaction, and may the toxigenicity metabolite in degradation process.
The 6th, the nanoparticle of macromolecule organic can't carry out autoclave sterilization, can only sterilize with gamma ray.This also has influence on the application of nanoparticle at human body.
The 7th, do not find still that at present a kind of method can produce lipid nanometer particle and effectively controlling cost on a large scale.
The 8th, it is poor that solid lipid nanoparticle and medicine combine rear stability, and medicine very easily decomposes from nanoparticle, thereby influences the envelop rate of medicine.
Still not having a kind of nanometer transmission system so far formally is applied to clinical.
Summary of the invention:
The objective of the invention is to: the objective of the invention is to: mostly be solid lipid nanoparticle at the nanoparticle that in materia medica and pharmaceutics, adopts at present, and a series of problems of existing in using in preparation with as drug delivery system of nanocapsule, nanosphere and Nano capsule, provide a kind of in vivo can biodegradation and contain and seal preparation method and the application thereof of the inorganic salt nano particles of Chinese medicine ingredients and bioactive substance.
The object of the present invention is achieved like this: a kind of preparation method that contains and seal the inorganic salt nano particles of Chinese medicine ingredients, it is characterized in that: at first preparation contains the inorganic salt nano particles of Chinese medicine ingredients: with 0.01～1.0 mole saline solution, 0.01～1.0 moles phosphoric acid solution, after the solution of the active substance of Chinese medicine mixed by 1: 1: 1, at room temperature stirred 48 hours, 4 ℃ of ambient temperatures, centrifugal 15 minutes of 8500rpm, removal contains the supernatant of free Chinese medicine active substance solution, use the deionized water suspended particles, supersound process is 15 minutes again, concentration at 5mg/ml forms the inorganic salt particle that contains the Chinese medicine ingredients active substance, and its size dimension is 100nm～800nm; On inorganic salt nano particles, seal the active substance of Chinese medicine then, detailed process is: the nanoparticle of pharmaceutically active substance is with after middle pharmaceutically active substance mixed by 1: 10 in will containing under the room temperature, concentration by 10mg/ml is suspended in pH value 7.0,0.01 in the phosphate buffer of mole, on shaking table, shook under 4 ℃ 2 hours, and formed the nanoparticle of pharmaceutically active substance in sealing; Last in sealing the inorganic salt of pharmaceutically active substance receive outside the particle reuse bioactive substance and seal, detailed process is: the bioactive substance of getting with previous step the middle pharmaceutically active substance equivalent of sealing in rapid, add in the phosphate buffer that has been suspended with the nanoparticle of sealing middle pharmaceutically active substance, 4 ℃ are continued down to shake on shaking table 2 hours, under 4 ℃, centrifugal three times of 8500rpm each 15 minutes, obtains the inorganic salt nano particles that contains and seal Chinese medicine ingredients and bioactive substance.
The inorganic salt nano particles that contains and seal Chinese medicine ingredients and bioactive substance that obtains should be stored in 4 ℃ or freeze-dried back.
Described saline solution is 0.01～1.0 mole a manganese chloride with the deionized water preparation, or magnesium chloride, or calcium chloride solution; The active substance of described Chinese medicine is the monomer polysaccharide of Chinese medicine or the active substance of complex; Described bioactive substance solution is to contain protein, or fat-soluble small-molecule drug, or water miscible small-molecule drug, or the antibody class medicine, or the solution of polypeptide drug, or gene genetic active matter liquid.
The monomer active polysaccharide material of described Chinese medicine is a Cordyceps polysaccharide, or pachyman, or astragalus polysaccharides, or glycyrrhizic acid, or lycium barbarum polysaccharide; The complex active substance of described Chinese medicine is a Cordyceps, or Poria, or the Radix Astragali, or Radix Glycyrrhizae, or the crude extract of Fructus Lycii.
A kind of application that contains and seal the inorganic salt nano particles of Chinese medicine ingredients is characterized in that: as carrier transfer bioactive substance treatment disease, or as vaccine adjuvant.
Described bioactive substance is meant protein, or fat-soluble small-molecule drug, or water miscible small-molecule drug, or polypeptide drug, and described vaccine is meant protein, or polypeptides matter, or antibody, or the gene genetic active matter.
The mode of transmitting bioactive substance comprises mucosal drug delivery or by being administered systemically.
Described mucosal drug delivery is meant the nasal mucosa administration, or vagina administration, or oral administration; Described being administered systemically is meant lumbar injection, or intravenous injection, or intramuscular injection.
The invention has the advantages that: because the calcium phosphate or the inorganic salt nano particless such as magnesium phosphate or manganese phosphate that form in preparation process are easy to inter-adhesive, dispersivity, the Surface Physical Chemistry characteristic needs to improve, adopt the inorganic salt nano particles of the active substance parcel back formation of Chinese medicine not only can change ionic surface electrode, improve their dispersion rate, and improved the envelop rate of biological active matter confrontation inorganic salt nano particles; Effective ingredient in the Chinese medicine (compositions such as polysaccharide, saponin) itself has certain physiological function, in case with after inorganic salt nano particles combines, can make nanoparticle become the nanometer portable object with certain biological function from pure portable object; Simultaneously, the nanoparticle of formation biodegradation in vivo.
The present invention be advantageous in that: not only preparation contains and to seal the technology of inorganic salt nano particles of Chinese medicine ingredients simple, and the stability of product is high, different batches batch between difference minimum, be easy to industrialization.
Description of drawings
Fig. 1: the illustraton of model that contains and seal the inorganic salt nano particles of Chinese medicine ingredients;
Fig. 2: the technological process of preparation method that contains and seal the inorganic salt nano particles of Chinese medicine ingredients;
Fig. 3: the stereoscan photograph that contains and seal the inorganic salt nano particles of Chinese medicine ingredients;
Fig. 4: the size distribution that contains and seal the inorganic salt nano particles of Chinese medicine ingredients;
Fig. 5: norvancomycin is in the extracorporeal releasing test result;
Fig. 6: the concentration of norvancomycin in aqueous humor;
Fig. 7: Chinese medicine inorganic salt nano particles and methazolamide compare in conjunction with the effectiveness of back with the intraocular pressure lowering of Pai Liming;
Fig. 8: contain and the inorganic salt nano particles of sealing Chinese medicine ingredients is carried the transmission electron microscope photo that the oral back of amifostine is absorbed by gastrointestinal tract cell;
Fig. 9: contain and the inorganic salt nano particles of sealing Chinese medicine ingredients is carried oral back 4 hours content in different tissues of amifostine.
In Fig. 1, Fig. 2: 1, inorganic salt nano particles, 2, the active substance of Chinese medicine, 3, bioactive substance, 4, saline solution, 5, phosphoric acid solution, 6, the nanoparticle that contains middle pharmaceutically active substance, 7, agitator, 8, high speed centrifuge, 9, the ultrasonic disruption instrument, 10, shaking table, 11, freezer dryer.
The specific embodiment
Preparation before the preparation
Saline solution: with the calcium chloride of 0.01～1.0 mole of deionized water preparation (or magnesium chloride, or manganese chloride solution).
Phosphoric acid solution: with the phosphoric acid solution of 0.01～1.0 mole of deionized water preparation.
Bioactive substance solution: the ratio in every 100ml phosphate buffer+1mg norvancomycin prepares 1% norvancomycin solution.
The active substance solution of Chinese medicine: prepare the active substance solution of Chinese medicine lycium barbarum polysaccharide in the every 100ml normal saline+ratio of 1mg lycium barbarum polysaccharide.
PH value is that 7.0,0.01 moles phosphate buffer can outsourcing.
Fig. 1 is the illustraton of model that contains the Chinese medicine inorganic salt nano particles, as seen from Figure 1, inorganic salt nano particles 1 inside in the model is the active substance 2 of Chinese medicine, be encapsulated with the active substance 2 of one deck Chinese medicine in the periphery of the inorganic salt nano particles 1 of the active substance 2 that contains Chinese medicine again, seal bioactive substance 3 at last again.What present embodiment reflected is the Chinese medicine inorganic salt nano particles that contains lycium barbarum polysaccharide, and what select in inorganic salt nano particles 1 inside and periphery all is lycium barbarum polysaccharide, and the bioactive substance 3 that outermost layer is sealed is norvancomycin.
Fig. 2 seals the technological process that the preparation method of the inorganic salt nano particles that contains Chinese medicine ingredients relates to.As seen from Figure 2, preparation process is as follows:
A) inorganic salt of pharmaceutically active substance was received particle during preparation contained:
With active substance 2 solution of the phosphoric acid solution 5 of 4,0.01～1.0 mole of 0.01～1.0 mole saline solution, Chinese medicine by mix at 1: 1: 1 place agitator 7 after, at room temperature stirred 48 hours, 4 ℃ of ambient temperatures, is centrifugal 15 minutes of 8500rpm through high speed centrifuge 8 at rotating speed, removal contains the supernatant of free active substance solution, use the deionized water suspended particles, passed through ultrasonic disruption instrument 9 supersound process 15 minutes again, the inorganic salt particle 6 of pharmaceutically active substance in the concentration formation of 5mg/ml contains, its size dimension is 100nm～800nm;
B) on receiving particle, seals inorganic salt middle pharmaceutically active substance:
Under the room temperature, the nanoparticle of pharmaceutically active substance is with after active substance 2 solution of Chinese medicine mixed by 1: 10 in will containing, and presses in the phosphate buffer of 7.0,0.01 moles of concentration suspension pH values of 10mg/ml, on shaking table 10, shook 2 hours under 4 ℃, form the nanoparticle of pharmaceutically active substance in sealing;
C) inorganic salt of pharmaceutically active substance is received outside the particle reuse bioactive substance and is sealed in sealing:
Get with the b step in the bioactive substance of active substance equivalent of Chinese medicine, add in the phosphate buffer that has been suspended with the nanoparticle of sealing middle pharmaceutically active substance, 4 ℃ are continued down to shake on shaking table 10 2 hours, under 4 ℃, is centrifugal three times of 8500rpm through high speed centrifuge 8 at rotating speed, each 15 minutes, obtain the inorganic salt nano particles that contains and seal Chinese medicine ingredients and bioactive substance.
The inorganic salt nano particles that contains and seal Chinese medicine ingredients and bioactive substance that obtains should be stored in 4 ℃ or by freezer dryer 11 freeze-dried backs.
In concrete preparation process, described saline solution is 0.01～1.0 mole a manganese chloride with the deionized water preparation, or magnesium chloride, or calcium chloride solution; The active substance of described Chinese medicine is the monomer polysaccharide of Chinese medicine or the active substance of complex, and they comprise Cordyceps polysaccharide, or pachyman, or astragalus polysaccharides, or glycyrrhizic acid, or lycium barbarum polysaccharide and comprise Cordyceps, or Poria, or the Radix Astragali, or Radix Glycyrrhizae, or the crude extract of Fructus Lycii; Described bioactive substance solution is to contain protein, or fat-soluble small-molecule drug, or water miscible small-molecule drug, or the antibody class medicine, or the solution of polypeptide drug, or gene genetic active matter liquid.
After preparation finishes, can calculate the concentration of bioactive substance in the nanoparticle, concrete grammar is: use the deionized water suspended particles, detect the concentration of free bioactive substance, calculate the concentration of bioactive substance in the nanoparticle, detect the size and the distribution of particle with Backman Counter N5 Plus.The inorganic salt nano particles stereoscan photograph that contains and seal Chinese medicine ingredients is referring to Fig. 3, contain and the size distribution of inorganic salt nano particles of sealing Chinese medicine ingredients referring to Fig. 4.
Because the saline solution that adopts is a calcium chloride in the present embodiment, the Chinese medicine active substance solution that adopts is the active substance solution of Chinese medicine lycium barbarum polysaccharide, the bioactive substance solution that adopts is 1% norvancomycin solution, its preparation be the inorganic salt nano particles that a kind of norvancomycin that contains the Chinese medicine lycium barbarum polysaccharide is sealed.
Norvancomycin is a kind of peptide medicament, is used for the treatment of endophthalmitis clinically, and this disease may cause nonvolatil ocular damage even blind.Endophthalmitis is caused by antibacterial and fungal infection that usually effectively medicine is an optical fundus injection norvancomycin.The norvancomycin gastrointestinal absorption is very poor, and the systemic injection administration causes serious side effects, and the optical fundus drug administration by injection is the most effective application method at present, but that technical operation requires is high, and patient is often because painful and refusal medication, thereby causes the permanent damage of eyes.In addition, the common bacteria of ocular infection is a staphylococcus, norvancomycin is antistaphylococcic specific drug, but norvancomycin is not used for the staphy lococcus infection of eye as yet at present, reason is that its molecular weight is big, the hydrophilic height can be discharged from very soon when being used for the eyes topical and can not effectively pass cornea.Norvancomycin is as a kind of effective anti-eye bacterial infection medicine, because of its drug delivery problem can not be given full play to drug effect like this.This invention adds that with inorganic salt nano particles the Chinese medicine Fructus Lycii combines with norvancomycin, can directly splash into eyes, has increased the concentration of medicine in aqueous humor, has prolonged the holdup time of medicine in eyes, has eliminated the misery that patient causes because of the optical fundus injection.Norvancomycin in extracorporeal releasing test result and the concentration in aqueous humor respectively referring to Fig. 5, Fig. 6.
Present embodiment prepares the inorganic salt nano particles that contains and seal Chinese medicine ingredients that methazolamide is sealed according to method and the step of embodiment 1, the difference of it and embodiment 1 only is: the Chinese medicine complex active substance of selection is: the crude extract of Fructus Lycii, the bioactive substance of selection is: methazolamide.
In the experiment of result of use, we have selected rabbit to be divided into two groups, under identical external environment condition, the inorganic salt nano particles that contains and seal Chinese medicine ingredients, the Pai Liming that adopt methazolamide to seal respectively pass through the eye mucosal drug delivery, effectiveness for their intraocular pressure lowering compares, comparing result as shown in Figure 7, by the contrast, as can be seen: effectiveness of the present invention with have remarkable advantages with reference to drugs compared Pai Liming.
Embodiment 3: contain the application of the inorganic salt nano particles that the amifostine of Chinese medicine Ganoderma seals
Crude extract with Ganoderma is the complex active substance of Chinese medicine, is bioactive substance with the amifostine, according to the preparation method of embodiment 1, obtains the inorganic salt nano particles that a kind of amifostine that contains the Chinese medicine ganoderan is sealed,
Oral administration is that most convenient patient Yu also is the most frequently used medication, but a lot of medicines, and especially bio-pharmaceutical is because the effect of the sour environment of gastrointestinal system and digestive enzyme and can't be oral can only be leaned on drug administration by injection.Amifostine is a kind of cytoprotective medicament, and the U.S. FDA approved can be used for cancer patient, in particular for the chemotherapy and radiation patient, and the protection normal cell wound of not being killed.Amifostine is generally intravenous injection once a day at present, for cancer patient has caused inconvenience and painful.Crude extract with Ganoderma is the complex active substance of Chinese medicine, with the amifostine bioactive substance, according to the preparation method of embodiment 1, obtains the inorganic salt nano particles that a kind of amifostine that contains the Chinese medicine ganoderan is sealed, and just can be taken orally.So not only alleviated patient misery and inconvenience, reduced side effect, and ganoderan (Lentinan) has enhancing immunity, increases the secretion of Gamma interferon and strengthens the function of natural killer cell (NK), makes the effect of amifostine improve greatly.In the experiment, we select mouse is experimental subject, carries the oral back of amifostine by the transmission electron microscope photo of the tree-shaped aixs cylinder cellular uptake of gastrointestinal tract (referring to Fig. 8) and the oral back 4 hours content (referring to Fig. 9) in different tissues by the Chinese medicine inorganic salt nano particles.
Embodiment 4: the schistosomiasis monoclonal anti-idio-typic antibody NP 30 is sealed the application of the inorganic salt nano particles of Chinese medicine astragalus polysaccharide
We select rabbit, and they are divided into 5 groups, and injection contains the injection of following ingredient respectively: 1, blank; 2, the polysaccharide component in the Chinese medicine astragalus; 3, NP30 is prepared into blood fluke vaccine; 4, the polysaccharide component+NP30 in the Chinese medicine astragalus is prepared into blood fluke vaccine; 5, the inorganic salt nano particles that contains the polysaccharide component in the Chinese medicine astragalus of the present invention's preparation is the NP30 blood fluke vaccine of transmission system.
Inorganic salt nano particles is combined transmission system as vaccine with polysaccharide component in the Chinese medicine astragalus; be prepared into blood fluke vaccine with the schistosomiasis monoclonal anti-idio-typic antibody NP 30 again; in rabbit schistosomicide model; we find to compare with control animals inorganic salt nano particles and add polysaccharide component in the Chinese medicine astragalus and NP30 antigen as vaccine; can improve TH1 cellular immunization and the humoral immune reaction of animal; obviously strengthen host's protective effect; worm reduction rate significantly improves (referring to table 1), and liver blood trematodiasis ovum egg reduction rate is compared with matched group also obvious reduction (referring to table 2).
Table 1 host adult worm reduction rate chart (%)
| | 0±0.21 |
| The | 0±0.19 |
| NP30 Ag | 23.1±0.14 |
| The NP30+ Radix Astragali | 33.4±3.6 |
| NP30+ inorganic salt nanometer+Radix Astragali | 53.5±4.7 |
Table 2 host worm's ovum egg reduction rate chart (%)
| | 0±0.35 |
| The | 0±0.21 |
| NP30 Ag | 30.5±0.33 |
| The NP30+ Radix Astragali | 42.1±6.3 |
| NP30+ inorganic salt nanometer+Radix Astragali | 55.7±2.1 |
Astragalus polysaccharides is a kind of promoter during as adjuvant, can strengthen host's t cell immune response.In the middle of prescription body polysaccharide with after inorganic salt nano particles combines, make vaccine become particle state from the solvent state, thereby increased the speed of lymphocyte and tree-shaped aixs cylinder cellular uptake vaccine, excite host's immunoreation to accelerate, and because nanoparticle is in the intravital degraded voluntarily of host, antigen continue to discharge and to make the antigenic activation that is subjected to that host immune system do not stop, thereby immunoreation obtains prolonging and strengthening, and host antigen infection ability again significantly improves (referring to table 3 and table 4).
Table 3: schistosomicide antigen-specific antibodies concentration (ug/ml)
| The immunity back time (week) | Contrast | The Radix Astragali | NP30Ag | The NP30+ Radix Astragali | NP30+ nanometer+Radix Astragali |
| 8 | 0 | 100 | 200 | 840 | 1280 |
| 16 | 0 | 160 | 360 | 1000 | 1640 |
Table 4: IFN-concentration u (pg/ml)
| The immunity back time (week) | Contrast | The Radix Astragali | NP30Ag | The NP30+ Radix Astragali | NP30+ nanometer+Radix Astragali |
| 8 | 9 | 20 | 50 | 140 | 290 |
| 16 | 5 | 25 | 50 | 160 | 392 |
The foregoing description that this case provides not is that the present invention is carried out a kind of unique restriction, in actual applications, especially as carrier transfer bioactive substance treatment disease, or during as vaccine adjuvant, bioactive substance should comprise protein, or fat-soluble or soluble small molecular medicine, or polypeptide drug, described vaccine is meant protein or polypeptides matter, or antibody, or the gene genetic active matter.Their transfer mode should comprise mucosal drug delivery or by being administered systemically.Described mucosal drug delivery is meant the nasal mucosa administration, or vagina administration, or oral administration; Described being administered systemically is meant lumbar injection, or intravenous injection, or intramuscular injection.
Claims (8)
1, a kind of preparation method that contains and seal the inorganic salt nano particles of Chinese medicine ingredients is characterized in that:
A) preparation contains the inorganic salt nano particles of Chinese medicine ingredients:
After 0.01～1.0 mole saline solution, 0.01～1.0 mole the solution of active substance of phosphoric acid solution, Chinese medicine mixed by 1: 1: 1, at room temperature stirred 48 hours, 4 ℃ of ambient temperatures, centrifugal 15 minutes of 8500rpm, removal contains the supernatant of free Chinese medicine active substance solution, uses the deionized water suspended particles, and supersound process is 15 minutes again, concentration at 5mg/ml forms the inorganic salt particle that contains the Chinese medicine ingredients active substance, and its size dimension is 100nm～800nm;
B) on inorganic salt nano particles, seal the active substance of Chinese medicine:
The nanoparticle of pharmaceutically active substance is with after middle pharmaceutically active substance mixed by 1: 10 in will containing under the room temperature, concentration by 10mg/ml is suspended in pH value 7.0,0.01 in the phosphate buffer of mole, on shaking table, shook 2 hours under 4 ℃, form the nanoparticle of pharmaceutically active substance in sealing;
C) inorganic salt of pharmaceutically active substance is received outside the particle reuse bioactive substance and is sealed in sealing:
Get with the b step in the bioactive substance of the middle pharmaceutically active substance equivalent sealed, add in the phosphate buffer that has been suspended with the nanoparticle of sealing middle pharmaceutically active substance, 4 ℃ are continued down to shake on shaking table 2 hours, under 4 ℃, centrifugal three times of 8500rpm, each 15 minutes, obtain the inorganic salt nano particles that contains and seal Chinese medicine ingredients and bioactive substance.
2, the preparation method that contains and seal the inorganic salt nano particles of Chinese medicine ingredients according to claim 1 is characterized in that: the inorganic salt nano particles that contains and seal Chinese medicine ingredients of acquisition is stored in 4 ℃ or freeze-dried back.
3, the preparation method that contains and seal the inorganic salt nano particles of Chinese medicine ingredients according to claim 1 is characterized in that: described saline solution is 0.01～1.0 mole a manganese chloride with the deionized water preparation, or magnesium chloride, or calcium chloride solution; The active substance of described Chinese medicine is the monomer polysaccharide of Chinese medicine or the active substance of complex; Described bioactive substance solution is to contain protein, or fat-soluble small-molecule drug, or water miscible small-molecule drug, or the antibody class medicine, or the solution of polypeptide drug, or gene genetic active matter liquid.
4, the preparation method that contains and seal the inorganic salt nano particles of Chinese medicine ingredients according to claim 3, it is characterized in that: the monomer active polysaccharide material of described Chinese medicine is a Cordyceps polysaccharide, or pachyman, or astragalus polysaccharides, or glycyrrhizic acid, or lycium barbarum polysaccharide; The complex active substance of described Chinese medicine is a Cordyceps, or Poria, or the Radix Astragali, or Radix Glycyrrhizae, or the crude extract of Fructus Lycii.
5, claim 1 or the 2 described application that contain and seal the inorganic salt nano particles of Chinese medicine ingredients is characterized in that: as carrier transfer bioactive substance treatment disease, or as vaccine adjuvant.
6, according to the described application of sealing the inorganic salt nano particles that contains Chinese medicine ingredients of claim 5, it is characterized in that: described bioactive substance is meant protein, or fat-soluble small-molecule drug, or water miscible small-molecule drug, or polypeptide drug, described vaccine is meant protein, or polypeptides matter, or antibody, or gene genetic active matter.
7, according to the described application that contains and seal the inorganic salt nano particles of Chinese medicine ingredients of claim 5, it is characterized in that: the mode of transmitting bioactive substance comprises mucosal drug delivery or by being administered systemically.
8, according to the described application that contains and seal the inorganic salt nano particles of Chinese medicine ingredients of claim 7, it is characterized in that: described mucosal drug delivery is meant the nasal mucosa administration, or vagina administration, or oral administration; Described being administered systemically is meant lumbar injection, or intravenous injection, or intramuscular injection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2953646A4 (en) * | 2013-02-05 | 2017-02-15 | 1Globe Health Institute LLC | Biodegradable and clinically-compatible nanop articles as drug delivery carriers |
| CN106727985A (en) * | 2016-11-29 | 2017-05-31 | 浙江美保龙生物技术有限公司 | A kind of porcine parvnvirus living vaccine dilution |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP5021149B2 (en) * | 2000-10-09 | 2012-09-05 | スリーエム イノベイティブ プロパティズ カンパニー | Medical aerosol formulation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2953646A4 (en) * | 2013-02-05 | 2017-02-15 | 1Globe Health Institute LLC | Biodegradable and clinically-compatible nanop articles as drug delivery carriers |
| US11001840B2 (en) | 2013-02-05 | 2021-05-11 | 1Globe Health Institute Llc | Biodegradable and clinically-compatible nanoparticles as drug delivery carriers |
| CN106727985A (en) * | 2016-11-29 | 2017-05-31 | 浙江美保龙生物技术有限公司 | A kind of porcine parvnvirus living vaccine dilution |
| CN106727985B (en) * | 2016-11-29 | 2020-09-22 | 浙江美保龙生物技术有限公司 | Diluent for porcine parvovirus live vaccine |
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| CN100435784C (en) | 2008-11-26 |
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