CN101897956A

CN101897956A - External use recombinant human neuropeptide Y nanoemulsion and preparation method thereof

Info

Publication number: CN101897956A
Application number: CN2010102257657A
Authority: CN
Inventors: 杨永鹏; 朱晓亮; 董萍; 丁克祥; 左夏林; 丁宇
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-07-13
Filing date: 2010-07-13
Publication date: 2010-12-01
Anticipated expiration: 2030-07-13
Also published as: CN101897956B

Abstract

The invention provides an external use recombinant human neuropeptide Y nanoemulsion and a preparation method thereof, ensuring that the recombinant human neuropeptide Y is protected from influence caused by temperature, illumination, oxygen concentration and the like, and the bioactivity of the recombinant human neuropeptide Y can be maintained for long term. External administration of the recombinant human neuropeptide Y nanoemulsion can avoid the side effect of injection to patients, and avoid the problem that oral administration can be easily degraded by gastrointestinal proteolytic enzyme and subjected to first-pass effect. Furthermore, due to the small size of the nanoemulsion, viscosity and high dispersibility of the nanoemulsion prolong the contact time with skin, so that the recombinant human neuropeptide Y can easily penetrate skin tissue to overcome the defects of small percutaneous absorption and low biological utilization of the traditional transdermal preparation. Moreover, the method for preparing the recombinant human neuropeptide Y nanoemulsion has the advantages of reliability, easy operation, simple preparation process, easy practical operation without special instrument or equipment, environmental pollution reduction, low carbon, environmental protection and wide market prospect.

Description

Recombinant human nerve peptide Y nano-emulsion of a kind of external preparation for skin and preparation method thereof

Technical field

The present invention relates to the biological medicine healthcare field, relate to recombinant human nerve peptide Y nano-emulsion of a kind of external preparation for skin and preparation method thereof especially.

Background technology

Along with the raising of people's material life, a lot of people begin to pursue the demand on the higher level, and the high-level demand of people's psychology has just been satisfied in beauty and shaping.The present situation of beauty and shaping is that a hundred flowers blossom, makes rapid progress, and its development in future has a extensive future especially.With regard to today, a hundred flowers blossom, be exactly that people constantly grope on the means of promoting the physical beauty, weed out the old and bring forth the new.

Breast is one of women's symbol and bodily form standard of beauty, and along with the development of human civilization, breast enlargement is in vogue day by day, and new method continues to bring out.The enlarge the bosom century-old history of art of review, be used for breast enlargement material passed through liquid breast prosthesis, solid-state breast prosthesis and the development of this three phases of autologous tissue, appearance correspondingly injection augmentation mammaplasty, prosthese enlarge the bosom and autologous fat these the three kinds popular now means of enlarging the bosom of enlarging the bosom.I, injection augmentation mammaplasty: because the injection augmentation mammaplasty injection is liquid, can be compatible with bodily tissue, can not take out fully after going wrong, no matter what take out, remaining 5% and 50% can have injury to health equally, just the degree size of injury.In addition, the complication that injection augmentation mammaplasty often occurs together has 1. infection, shows as heating, inflammation; 2. local scleroma, breast diminish; 3. Zhu She material has the migration phenomenon; 4. might cause breast carcinoma; 5. local dull pain; 6. local muscle contractile function infringement.II, implanting prosthetic: advantage is one-time-reach-place, if problem can be taken out fully, shortcoming is to operate on, and otch is arranged, and has rejection, feel hardening, breast deformation.Implanting prosthetic neither be lifelong, and fracture phenomena can appear in prosthese after general 12 years to 15 years, needs to change.III, autologous fat transplantation: its advantage is not have the edge of a knife, does not have rejection; Shortcoming is to adopt people's requirement of this method more fat, and certain fat be arranged, and multiple injection just can finish, and has 10% people the enclosed mass phenomenon can occur, causes and the difficult difference of breast carcinoma.The fatty survival rate of general autologous transplanting if injection volume is excessive, fatty bland necrosis can occur about 50%, cause breast further to infect, even the mammary gland necrosis, also occurs fatty fibrosis easily, promptly local scleroma phenomenon.

Make a general survey of in the existing various shaping bust-forming technology, autologous fat transplantation is subjected to people's attention.Autologous fat derives from autologous tissue, and histocompatibility is good, does not have irritated reaction, it can remove the characteristics that unnecessary fat is filled depression in addition, makes total body fat rationally " allotment ", has shown its powerful advantages as the soft tissue filler.Though the fat after transplanting at present still exists absorbance height, the unsettled shortcoming of curative effect, has accumulated many experiences and viewpoint in clinical.

It is with a long history to use autologous fat granule transplantation treatment tissue defect and dysplasia.Neuber successfully carried out fatty free grafting in 1893.Autologous fat granule injection transplantation treatment small-breast disease promptly in the abundant position of body fat, by liposuction technique, is drawn the lipochondrion of some and size, and injection transplantation is in breast or the mammary gland post gap behind rinsing, purification; Transplant the rear udder attachment volume and increase, elasticity increases, and feel and profile are improved; Simultaneously, deduct the fat that pile up at other positions, reinvent the bodily form.Therefore, the autologous fat granule is transplanted mamaplasty will become emerging developing direction of plastic surgery.In addition, the autologous fat source is abundant, and it is easy to draw materials, and histocompatibility is better than artificial organ succedaneum and allosome foreign material, and no immunological rejection is ideal tissue filling material.Simultaneously, the liposuction breast enlargement can deduct the fat that pile up at other positions, and the dresser body curve is reinvented the bodily form.Adopt injection transplantation have wound light, painful less, leave over the little advantage of cicatrix, clinical practice is more and more widely.

But because fatty tissue is relatively poor to the tolerance of ischemia and wound, autologous fat granule transplanting success is lower at present, and survival rate is not high after transplanting.Owing to the generation reason for the part complication is not still understood, operation improperly can cause severe complications.As blind, central nervous system injury, infection, hematoma, granuloma, wrinkling, numb, injection back part scleroma, liquefaction, necrosis, fibrous capsuleization, edema etc.Aspect the complication of autologous fat transplantation breast enlargement generation, clinical practice shows that the liquefaction of fat absorbance of mamaplasty is up to more than 30%, and a large amount of autologous fat transplantations can also produce cyst and fibrocalcification, form scleroma, to the certain influence of having checked oneself of mammary gland tumor.Simultaneously, injection shot is big, and the normal both central necrotic that easily forms liquefies.Therefore, it is prudent that autologous fat transplantation is used for the mamaplasty needs, should be by the doctor's operation that possesses knack.If have a kind of method or technology can reach the effect of mamaplasty by the propagation of autologous fat, the defective that just can avoid autologous fat transplantation to bring: relatively poor to the tolerance of ischemia and wound as fatty tissue, autologous fat granule transplanting success is lower, it is not high to transplant the back survival rate, can avoid autologous fat transplantation to produce cyst and fibrocalcification again, form scleroma etc.

U.S. scientist developed a kind of medicine that shifts body part fat recently in 2007.Find that in research this medicine can help fat is transferred to other positions of health, such as chest to mouse.This research is based on neuropeptide tyrosine.Neuropeptide tyrosine (Neuropeptide Y, abbreviation NPY) neurotransmitter of forming by 36 aminoacid, the C end has a tyramine amide residue in its peptide molecule structure, most important to keeping the neuropeptide tyrosine biological activity, N holds a tyrosine residue, then with stable neuropeptide tyrosine structure with to combine neuropeptide Y receptor closely related.Npy gene duplicates and is delivered to teleneuron and coexists as orthosympathetic secretion vesicle with norepinephrine through axon by transcribing, translate to finish in neurocyte.Neuropeptide Y receptor then belongs to the g protein coupled receptor family member, and its receptor subtype mediation information conduction is all relevant with the G albumen coupling.Neuropeptide tyrosine must be by combining back its due biological action of competence exertion after synthetic and release with neuropeptide Y receptor, equally, neuropeptide Y receptor also only could finally be mobilized or suppress Ca2+ to discharge by suppressing adenylate cyclase activity with inhibition G albumen (Gi) coupling after the effect of neuropeptide tyrosine part.Be that neuropeptide tyrosine is being subjected to the former effect of some stimulation back quickening, is strengthening synthetic and secretion and release, and the synthetic and secretion of urging adrenal gland's releasing factor, thyroliberin, luteotropic hormone, growth hormone etc. rapidly by the performance of neuropeptide tyrosine specific receptor, and directly affect various biological functions such as human vas contraction, hypertension, trophic behavior, hormone secretion, cardiovascular function, regulate body temperature, biorhythm, stress, sexual behaviour and emotion.

The leader of this research, the medical center physiology of Georgetown University and biophysics are that professor Zuo Fei Asia says that " in the U.S., 60% people suffers from obesity in various degree.Two kinds of different viewpoints are arranged in this field, a kind ofly think that fat reason is to eat the food that is rich in sugar and fat too much, ' comfortable ' food just oneself is liked the thing of eating in other words.Another kind thinks that pressure can be causeed fat.Such as people are when sick, and they tend to bear more pressure, and these pressure have expedited the emergence of the intravital hormone of people, and hormone has caused obesity.”

Researcher attempts explaining the root of fat this problem, they pass through the research in 4 years, found a pressure can promote the mechanism of weight increase, this mechanism can explain that the body weight (calculating according to the calorie that absorbs) that the people's that those have pressure for a long time weight increase should increase than them will more reason.Research group has been observed mouse suffers from the long-term pressure of all kinds in waste Yezhong influence, for example have stood in ice-cold cesspool the inside in one hour every day in the time limit in one two week, perhaps fight with the mouse that have to struggle against, they give the experimental mouse feeding according to the menu of a normal menu and a higher fatty acid high sugar in experiment simultaneously.Final result is: the animal that is stressed of the normal menu of feeding does not have weight increase, but the pressure animal that has of the higher fatty acid menu of feeding has increased body weight greatly--with in contrast the group those do not have the animal of pressure to compare, although eat the same food, the fat of increase is 2 times of the latter.If " considering the evolution advantage; this is just found highly significant: if you can be at the difficult period deposit fats; you just stock and can be converted into energy carrying out next fight, " Zuo Feiya says that " same mechanism may also can take place on mankind.The tension-causing factor of long-term accumulation, for example with your superior's dissension, perhaps look after an ill child for a long time, perhaps be in always " highway indignation disease " (promptly since traffic congestion etc. former thereby cause irritable and angry), this state of long delays just might develop into the amplifier of high calorie foods ".

Further research is sought the chemical mechanism that pressure is causeed fat from molecular level exactly.Two participants of the main force that researcher has found this mechanism to have an effect: neuropeptide tyrosine and receptor Y2 thereof.Certainly, they are all relevant with pressure.But research in the past thinks that they more may be to have an effect at brain.In order optionally to increase the fat of the mouse in their test, research worker is beaten neuropeptide tyrosine at a specific zone--abdominal part.They find that neuropeptide tyrosine is upset in press process, cause the mouse abdominal part to begin to pile up fat, the obesity and the metabolism syndrome of apple shape occur.The fat abdomen area that all appears at experimental mouse, and with scientist in the past to the understanding of neuropeptide different be that neuropeptide tyrosine is worked in fatty tissue, rather than works in brain.This means that by adjusting the CONCENTRATION DISTRIBUTION of parts of body neuropeptide tyrosine, people can optionally control the growth of fat at parts of body.And on the other hand, neuropeptide tyrosine will be had an effect also must be by means of the help of neuropeptide Y 2 receptor.By blocking-up Y2R, neuropeptide tyrosine will no longer be had an effect, thereby in this local fat-reducing effect that produces.

Research worker is said, the fastest clinical practice of these discoveries is beauty treatment and plastic surgery operations again; Over the long term, then can control metabolism syndrome better, this syndrome has been gathered increases the risk factor that patient brings out heart disease, apoplexy and diabetes probability.According to a research of the Center for Disease Control subsidy in 2004, nearly 6,000 ten thousand Americans in 2000 are subjected to the influence of metabolism syndrome, and wherein quite a few is finally causeed fat.The countless different experiment that this 4 years interior-excess are executed has confirmed that minimum it is feasible on one's body mouse; Nearest vectoring information has then illustrated monkey also a similar mechanism on one's body.Zuo Feiya says that " by the control to neuropeptide tyrosine and Y2R, we can reduce the fat of mouse abdominal part, can also reduce the fat on their livers and the skeletal muscle, and help opposing, glucose intolerance, blood pressure and the inflammation of control insulin.The effect of blocking-up Y2R may can play same effect to the mankind, but needs more research to prove it.I think that our research can help people to lose weight, and also can contribute to whole life sciences simultaneously ".

" targeted ground increases fat and thickens operation to recovering facial youth, plentiful breast, buttocks and lip; and facial shaping again is very useful; " the associate professor plastic surgery Si Tefen of hospital of Georgetown University says, " utilize the injection of those tests in this research can allow fat transplantation become possibility; and cost is cheap relatively, also can not cause nonvolatil rejection.The result that giving neuropeptide tyrosine can increase target ground increase fat blocking-up Y2R has eliminated partial fat.This is the first intact mechanism that is described to, can motionless operation and increase effectively or eliminate fat." certainly, said here " fat shifts " " fat is eliminated " is a kind of indirect transfer and elimination, increases the neuropeptide tyrosine of some body parts that is:, makes it to accept the excess energy of health; The Y2R effect of the target site of blocking-up fat-reducing simultaneously makes it no longer to accept superabundant fats, and the fat of having piled up will consume under the situation that can not get replenishing very soon.This discovery not only has very big effect for fat-reducing, also tool is very helpful for the operation of filling tissue in face-lifting and the prosthesis, such as breast enlargement.Research worker finds, after the bead that will comprise neuropeptide tyrosine injected the skin of mouse or monkey, it is fat that mouse and monkey will become.This technology will provide new method for fat-reducing, liposuction, face's rejuvenation operation and breast enlargement art, and may substitute various face-liftings and prosthesis in future.For the woman who needs breast enlargement and breast reconstruction, this can well improve the graceful curve of their health.

Generally speaking, the operation by to neuropeptide tyrosine and receptor Y2 thereof both can weaken the negative effect that is caused by obesity: such as heart disease and diabetes etc.; Can help fat is transferred to other positions such as chest, the buttocks etc. of health again, not only give and want that the Ms who likes to be beautiful again brings good news, and this achievement in research also will provide new means for plastic surgeon by the healthy way fat-reducing.

But neuropeptide tyrosine is degraded in vivo easily as a kind of polypeptide drugs, and the half-life is shorter, necessary frequent drug administration, and this brings constant to the patient.Therefore, developing corresponding neuropeptide tyrosine preparation and administering mode is the challenge that pharmaceutics is faced.Neuropeptide tyrosine polypeptide drugs poor stability, this structure with polypeptide drugs itself is relevant, and is also relevant with administering mode with the neuropeptide tyrosine polypeptide formulations.

1. cause the unsettled self reason of polypeptide

(1) deacylated tRNA amine reaction: in the reaction of deacylated tRNA amine, the hydrolysis of Asn/Gln residue forms Asp/Glu.The deacylated tRNA amine reaction of non-enzymatic catalysis, relevant with the structure of environmental condition and polypeptide.Raising pH value, elevated temperature all will help the carrying out of deacylated tRNA amine reaction.Amide group in-Asn-Glu-structure is facile hydrolysis more, and the amide group that is positioned at molecular surface is also than intramolecular amide group facile hydrolysis.

(2) oxidation: the main cause of the easy oxidation of polypeptide solution has two kinds, and the one, the pollution of peroxide is arranged in the solution, the 2nd, the spontaneous oxidation of polypeptide.In all amino acid residues, the easiest oxidations such as Met, Cys and His, Trp, Tyr.Partial pressure of oxygen, temperature and buffering solution are also all influential to oxidation.

(3) hydrolysis: the peptide bond facile hydrolysis fracture in the polypeptide.Participate in the peptide bond form by Asp than other peptide bond more easy fracture, especially Asp-Pro and Asp-Gly peptide bond.

(4) form wrong disulfide bond: between the disulfide bond or between disulfide bond and the sulfydryl exchange takes place and to form wrong disulfide bond, cause tertiary structure to change and loss of activity.

(5) racemization: except that Gly, the alpha-carbon atom of all amino acid residues all is a chirality, easily under base catalysis racemization takes place.The easiest generation racemization of Asp residue wherein.

(6) β-elimination: β-elimination is meant the elimination of group on the β carbon atom in the amino acid residue.Residues such as Cys, Ser, Thr, Phe, Tyr all can pass through β-elimination degraded.β-elimination easily takes place under alkaline PH, and temperature and metal ion are also influential to it.

(7) degeneration, absorption, gathering or precipitation: degeneration generally all is relevant with the destruction of three dimensional structure and secondary structure.At the often easier generation chemical reaction of denatured state polypeptide, activity is difficult to recover.In the polypeptide degenerative process, at first form intermediate.Usually the dissolubility of intermediate is low, is easy to assemble, and forms aggregation, and then forms macroscopic precipitation.Proteinic surface adsorption is another problem that runs in its storage, the use, can be adsorbed on pipe surface as IL-2 when pouring into, and causes loss of activity.

2. route of administration is to the influence of polypeptide drugs

Absorption is meant that skin or mucosa that medicine sees through human body enter into interstice, are back to sanguimotor process through blood capillary or lymphatic capillary.Because of the medicine-feeding part difference, the internal organs of flowing through after causing the drug absorption process and entering circulation are also different.This is the importance that medication preparation will be considered.

(1) intravenous injection medicine: the intravenous injection medicine then directly enters blood circulation, but medicine need and play a role through pulmonary circulation and body circulation tremulous pulse arrival target site, has serious adverse and needs frequent drug administration cause patient's misery.

(2) oral administration: the polypeptide drug molecular weight is big, and is fat-soluble poor, is difficult to see through biomembrane, generally can only drug administration by injection.But drug administration by injection, especially those need the medicine of frequent drug administration, to patient Lai Shuoshi and inconvenience thereof.Therefore be necessary to carry out the non-injection administration approach research of polypeptide.But under the normal condition, most of peptide medicaments seldom or can not be through gastrointestinal absorption.Its main cause has: 1. polypeptide molecular weight is big, and is fat-soluble poor, is difficult to by the biomembrane barrier; 2. exist a large amount of peptidohydrolases and proteolytic enzyme degradable polypeptide in the gastrointestinal tract; 3. easily eliminated after absorbing by liver; 4. there are chemistry and conformation instability problem.Focusing on of research overcomes preceding two obstacles at present, both how to have improved biomembrane permeability and these two aspects of opposing proteasome degradation of polypeptide.Using absorption enhancer to improve the biomembrane permeability is the main method that adopts in the research at present.Big quantity research shows that also absorption enhancer has polypeptid specificity.Other method that improves the biomembrane permeability also has: 1. polypeptide is connected with VB12, by receptor-mediated absorption; 2. use the fatty acid modifying polypeptide, improve fat-soluble.The approach that overcomes polypeptide oral absorption enzyme has: 1. use the PEG modified polypeptide, the opposing enzymolysis; 2. use enzyme inhibitor; 3. use microparticle formulation; 4. applying nano grain preparation; 5. applying biological cohesive particle.Diameter is that the PEG nanoparticle of 100nm can major part be absorbed in intestinal, breaks through enzyme barrier and film barrier.United states drug development company uses the oral formulations that the Technosphere technology prepares calcitonin, reaches 26% at the intravital absolute bioavailability of Canis familiaris L..

Outside the division ring spore bacterium (cyclic peptide), yet there are no the clinical practice report of polypeptide oral formulations so far.It is said that the calcitonin oral formulations of Cortecs company has entered the clinical research of III phase, be expected to be called first real polypeptide oral formulations.

(3) rectally: research is arranged in the presence of 5-methoxyl group sodium salicylate, insulin absorbs the approach that the back arrives systemic circulation by rectum.Cut thoracic duct behind the rectally and get lymphatic vessel, measure insulin in lymph fluid and the blood, relatively, amount of insulin reduces a lot in the blood when having complete thoracic duct, and content is very high in the lymph fluid, proves that insulin mainly enters blood circulation through lymphsystem after rectum absorbs.

(4) nasal-cavity administration: the nasal cavity position exists abundant blood capillary and lymphatic vessel, and the nasal membrane epithelium closely links to each other with blood vessel wall, and the gap is bigger between epithelial cell, and capillary endothelium has the space.The basement membrane of all blood capillaries is leakage hole shape.Though the venule inner membrance is complete, its basement membrane and smooth muscle cell are highly porous property.Medicine can have dual mode to pass mucosa, and the one, by the water solublity intercellular substance; The 2nd, by the lipid carrier passage in the mucosa.Because the diameter of intercellular substance is generally 0.4-0.8nm, has only diameter can not freely pass through intercellular substance greater than the biomolecule of 0.7nm.This has higher permeability and is beneficial to drug absorption, medicine can directly enter blood circulation, avoid liver first-pass effect to compare with gastrointestinal tract, the pH value at nasal membrane place and enzyme are little to polypeptide, protein destructiveness, and low-molecular-weight medicine reaches easily to be absorbed and enters blood circulation.To the big polypeptide of molecular weight religion,, under suitable absorption enhancer helps, also can be absorbed, but bioavailability is lower as calcitonin, insulin etc.The mode of nasal-cavity administration has intranasal administration method and spray delivery method, adopts back one method can obtain relative high bioavailability.

(5) pulmonary administration: the sorbent surface of lung amasss 140m ², blood flow reaches 5000ml/min, and proteinase activity is lower with respect to gastrointestinal tract, does not have liver first-pass effect.Alveolar wall is better than capillary wall permeability.

(6) dosing eyes: the eye conjunctiva contains abundant capillary network and abundant lymphatic capillary net, dosing eyes and subcutaneous absorb rapidly the same with intramuscular injection, can avoid liver first-pass effect, ocular tissue compares with other tissue or organ, and is more insensitive for immunoreation.

3. improve the approach of polypeptide stability

(1) rite-directed mutagenesis: cause the unsettled residue of polypeptide or introduce the residue that can increase polypeptide stability by the genetic engineering means replacement, can improve the stability of polypeptide.

(2) chemical modification: the chemical modification method of polypeptide is a lot, and studying maximum is that PEG modifies.PEG is a kind of water-soluble high-molecular compound, and degradable is nontoxic in vivo.PEG with can improve heat stability after polypeptide combines, the degraded of opposing protease reduces antigenicity, the half-life in the extension body.But select suitable method of modifying and control degree of modification body constitution or improve the protozoa activity.

(3) additive:,, can improve the stability of polypeptide as saccharide, polyhydric alcohol, gelatin, aminoacid and some salt by adding additive.Sugar and polyhydric alcohol force more hydrone to be centered around around the protein under low concentration, thereby have improved the stability of polypeptide.In freeze-drying process, above-mentioned substance can also replace water and stablize the native conformation of polypeptide with polypeptide formation hydrogen bond, but also can improve the vitrification point of freeze-dried products.This external surfactants such as SDS, Tween, Pluronic can prevent polypeptide surface adsorption, gathering and precipitation.

(4) lyophilizing: series of chemical that polypeptide takes place such as deacylated tRNA amine, β-elimination, hydrolysis etc. all need water to participate in, and water can also be as the mobile phase of other reactant.In addition, water content reduces the denaturation temperature rising that can make polypeptide.Therefore, lyophilizing can improve the stability of polypeptide.

(5) other: the transformation of dosage form also is the direction of peptide medicament preparation research.Using microgranule, nano-emulsion system is one of direction in the present polypeptide drugs research as carrier.At present more external biotechnological pharmaceutics companies cooperate with relevant research company's utilization advantage separately, and research and development make fast progress.

Though the non-injection administration research to polypeptide obtains some progress in recent years, the difficulty that faces is still a lot.The mucosa transmission of nearly all polypeptide drugs all needs penetration enhancer, and its kind is numerous and diverse, and the problem of existence is: how to reduce its stimulation, whether life-time service influences epithelial integrity and how to improve functional component by problems such as biomembranes.

Along with development of biology, peptide medicament comes into one's own day by day, and especially the development of gene recombination technology makes that these medicines can extensive, industrialization production.Yet peptide medicament is subjected to certain restriction in actual applications, this is because peptide medicament is degraded rapidly by various protease easily, and the half-life is short, usually adopt vein frequent drug administration mode, use inconvenient, peptide medicament passes a little less than the ability of biological barrier simultaneously, and these are because the weak diffusion and the low partition coefficient of peptide medicament.In order to address these problems, for the safe and effective intravenously administrable and the topical of peptide medicament, people urgently are desirable to provide a kind of new drug-loading system.

The nano-emulsion carrier system of Chu Xianing has good application prospects in recent years.Nano-emulsion claims that also (microemulsion ME), is made up of oil phase, water, surfactant and cosurfactant microemulsion, as long as the composition of four phases is suitable, can forms homogeneous transparent or shows slightly opalescent liquid, is thermodynamic stable system.Can be divided into oil-in-water (O/W) type, Water-In-Oil (W/O) type and doubly-linked ideotype nano-emulsion by structure.Because nano-emulsion has good topical and transdermal characteristic as carrier, so since the nineties in 20th century, nano-emulsion becomes the focus of pharmaceutics research as the research of transdermal drug delivery system.The nano-emulsion particle diameter is little, has good transdermal and stability, and its transdermal drug delivery system is better than common Emulsion.A lot of characteristics that nano-emulsion possessed make it be fit to very much make preparation capable of permeating skin, its particle diameter Xiao Yi by skin absorbs, have good skin sense of touch, vision, practical function etc.Therefore, the nano-emulsion carrier system is being the vehicle that very big development prospect is arranged aspect the transdermal external preparation, the decentralized photo of nano-emulsion can be written into functional component, can keep higher functional component concentration, improve the functional component Concentraton gradient between nano-emulsion and skin, can increase the stability and the percutaneous rate of functional component greatly.

And the nano-emulsion system is as the carrier of medicine, be that a kind of novel medicament is carried and the hierarchy of control, its mechanism of sealing with release is by active substance being wrapped in nanometer emulsion droplet inside or the nanometer emulsion droplet, utilize the small size characteristics of nanometer emulsion droplet, make peptide medicament penetrate tissue gap and absorbed by cell, and then can also can pass through the biomembrane barrier by the blood capillary of human body.And the nano-emulsion carrier can improve the stability and the bioavailability of peptide medicament, is the good transport agent of polypeptide and protein medicaments, and special advantages is arranged.

Summary of the invention

The objective of the invention is to be improved, innovate, recombinant human nerve peptide Y nano-emulsion of a kind of external preparation for skin and preparation method thereof is provided at shortcoming that exists in the background technology and problem.The neuropeptide tyrosine molecular weight is big, the hydrophilic height, and the half-life is short, and is fat-soluble poor, can not pass biomembrane effectively during local application, and adopt the intravenously administrable approach at present, has serious adverse and patient misery.Oral administration also easily by gastrointestinal Proteolytic enzyme enzymatic degradation, bioavailability is low and difficult by biomembrane etc., its Clinical Application is very limited, and deliver the system famine of polypeptide drug at present, press for the new drug administration carrier of exploitation to satisfy the demands.For this reason, for the route of administration of peptide medicament, Chinese scholars is devoted to the research and development of non-injection administration dosage form always.

The present invention can improve the stability of recombinant human nerve peptide Y greatly; Improve the biological activity of recombinant human nerve peptide Y, prolong half-life does not need frequent drug administration; Changed traditional drug administration carrier of neuropeptide tyrosine peptide medicament, convenient, and reduced risk and by the side effect of its generation; The viscosity of nano-emulsion in addition, polymolecularity has prolonged the time of contact of medicine and mucosa, makes medicine pass through skin barrier better, can keep treatment concentration for a long time, the bioavailability height.

The recombinant human nerve peptide Y nano-emulsion of a kind of external preparation for skin of the present invention, liquid-in-liquid dispersions system by recombinant human nerve peptide Y, trehalose, isopropyl myristate, sad capric acid polyethyleneglycol glyceride, polyglycereol-3-dioleate, distilled water are formed is characterized in that being made by following weight percentages:

Recombinant human nerve peptide Y 0.0003%～0.0006%

Trehalose 1%～3%

Isopropyl myristate 5%～15%

Sad capric acid polyethyleneglycol glyceride 25%～32%

Polyglycereol-3-dioleate 7%～12%

Distilled water 45%～52%

The total weight percent of above-mentioned raw materials is 100%.

The recombinant human nerve peptide Y nano-emulsion of external preparation for skin of the present invention is characterized in that being made by the raw material of following preferred weight percent:

Recombinant human nerve peptide Y 0.0005%

Trehalose 2%

Isopropyl myristate 10%

Sad capric acid polyethyleneglycol glyceride 30%

Polyglycereol-3-dioleate 10%

Distilled water 47.9995%

The total weight percent of above-mentioned raw materials is 100%.

Another object of the present invention provides a kind of preparation method of recombinant human nerve peptide Y nano-emulsion of external preparation for skin:

1. recombinant human nerve peptide Y aqueous solution preparation: according to formula ratio recombinant human nerve peptide Y and trehalose are dissolved in distilled water, with this solution as water (I);

2. accurately take by weighing the isopropyl myristate of formula ratio with analytical balance, place another to be labeled as oil phase (II) through knowing among the conical flask of decontamination and with marking pen;

3. according to the sad capric acid polyethyleneglycol glyceride (S) that has designed in advance: the ratio of polyglycereol-3-dioleate (C)=3: 1, accurately take by weighing the sad capric acid polyethyleneglycol glyceride and the polyglycereol-3-dioleate of formula ratio respectively, and the two raw material is placed the 3rd through among the clean conical flask of decontamination; Add a cover, and this conical flask is placed rapidly on the liquid flash mixer, fully mix homogeneously, make it to form emulsifying agent/co-emulsifier (S/C) mixture, behind the 3-5min, the closing liquid flash mixer, take off conical flask, and with the clear S/C mixture (III) that is labeled as of marking pen;

4. according to oil phase (II): S/C mixture (III): the ratio of water (I)=10%: 40%: 50%, the phase (I) of fetching water respectively earlier, S/C mixture (III) place the 4th through among the clean conical flask of decontamination;

5. after above-mentioned water (I), S/C mixture (III) two liquid phases fully being mixed, again under 25 ℃ of conditions of room temperature or in the natural room temperature, put it into and shake in the ultrasonator also about ultrasonic 2min, perhaps under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1Rotating speed magnetic agitation 5min, then, in this container, directly add oil phase (II) again, and with whole system in ultrasonator about ultrasonic 5min, perhaps under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1Rotating speed magnetic agitation 20min;

6. close ultrasonator or time constant-temperature magnetic stirring apparatus, take off conical flask, observe its outward appearance limpid in this way colourless, fluidity and good dispersion, obviously visible opalescence person is arranged, be the recombinant human nerve peptide Y nano-emulsion of external preparation for skin;

7. the recombinant human nerve peptide Y nano-emulsion of this external preparation for skin is sub-packed in rapidly among the lucifuge glass container of different size, adds a cover rapidly, packing, and place 4 ℃ of airtight preservations of refrigerator to get final product.

The principles of formulating prescriptions of the present invention:

The liquid-in-liquid dispersions system that the recombinant human nerve peptide Y nano-emulsion of external preparation for skin of the present invention is made up of neuropeptide tyrosine, isopropyl myristate, sad capric acid polyethyleneglycol glyceride, polyglycereol-3-dioleate, distilled water.

The polypeptide that neuropeptide tyrosine is made up of 36 aminoacid, it belongs to pancreatic polypeptide family, and molecular weight is 4215.Its N end and C end respectively have a tyrosine residue and tyramine amide residue, and the acyl groupization of C end is most important to the biological activity of neuropeptide tyrosine, the tyrosine residue that N holds and the tertiary structure of stable neuropeptide tyrosine and to combine neuropeptide Y receptor closely related.X-ray crystal structure pictorial display neuropeptide tyrosine has 2 mutual antiparallel helical regions, the spiral of 1 proline rich and 1 alpha-helix, and 2 helical regions all have the ionized characteristics of both sexes, borrow hydrophobic bond to keep its stable tertiary structure between 2 spirals.The biological activity of neuropeptide tyrosine just disappears when certain factor causes the molecule tertiary structure to change.It is distributed widely in striatum, corpus amygdaloideum, nucleus solitarius, hypothalamus, Hippocampus, diencephalon and the spinal cord etc. of nervus centralis, and in the sympathetic ganglionic cell such as superior cervical ganglion of peripheral nervous tissue, stellate ganglion, ganglia coeliaca.Neuropeptide tyrosine also is present in tissue, organ and the body of gland in addition, in skeletal muscle, blood vessel, the heart, liver, spleen, lung, adrenal gland, thyroid and submaxillary gland, and less in pancreas and the kidney.

Neuropeptide tyrosine can act on hypothalamus, has appetite strengthening, changes the organism metabolism environment, activates the lipoprotein lipase of fatty tissue, promotes the effects such as synthetic and storage of fat.The central nervous system is the important moderator of trophic behavior, and the position of regulating energy metabolism focuses mostly at hypothalamus.Neuropeptide tyrosine is a kind of very strong appetite stimulator in the hypothalamus, and human body energy absorption, storage and consumption and the energy balance are played an important role.

Neuropeptide tyrosine can help fat is transferred to other positions of health, such as chest.Neuropeptide tyrosine can help the health depot fat.The bead that research worker is made neuropeptide tyrosine is implanted under the skin of macaque, found that, has all grown fat around the bead.Neuropeptide tyrosine helps cosmetic surgery, as allows on aging people's cheek round out etc.The control neuropeptide tyrosine will provide new means for plastic surgeon, as " can transfer to chest or buccal to the fat of buttocks ".And then well improve the graceful curve of health.Compare with traditional autologous fat transplantation, the complication of this method can be still less.

The neuropeptide tyrosine medicine is subjected to certain restriction in actual applications, and this is because peptide medicament is degraded rapidly by various protease easily, and the half-life is short.We add trehalose in prescription for this reason, and it has non-specific protective effect to biomacromolecule, and it can protect the structure of peptide medicament effectively, make peptide medicament avoid damage under abnormal conditions, have improved neuropeptide tyrosine greatly in external stability.To being described below of trehalose.

Trehalose (trehalose, a-D-glucopyranosyl-a-glucopyranoside) be the irreducibility disaccharidase that forms by the hemiacetal group condensation by two glucosyl group molecules, extensively distribute at occurring in nature, especially those have the organism of stronger anti-dehydration, these organisms even still can survive under the situation of having lost health 99% moisture.Wigger separation first from the Clavicipitaceae of rye (Secale cereale L.) in 1832 obtains trehalose.Because the trehalose chemical property is stable, safety non-toxic, sweet taste is moderate and can protect tissue and the macromolecular function and the activity of organism under poor environments such as high temperature, freezing, dry dehydration, hyperosmosis, makes trehalose in fields such as food, medical industry, cosmetics and agriculturals wide application prospect be arranged.

Trehalose has non-specific protective effect to organism and biomacromolecule; it can protect cell membrane and proteinic structure effectively; make organism under abnormal conditions; still keep moistening in the cell as high temperature, drying, hyperosmosis, when freezing, prevent that cell from causing the loss and the cells injury of nutrient because of dehydration.And external trehalose has the characteristic of stabilate film and protein structure equally.Its mechanism of action has following several theory.1. water substitutes theory: around the protein in the organism, nucleic acid, lipid and other macromolecular substances, all surrounded by water membrane and protecting.This layer moisture film is the indispensable basis of 26S Proteasome Structure and Function of keeping these materials.In dry run, trehalose can the place of water molecule, forms hydrogen bond with protein and membrane lipid, replaces the hydrogen bond that is formed by hydrone, thus the fusion and the proteinic degeneration of inhibition membrane lipid.2. glassy state theory: trehalose has higher glass transition temperature, exists with the glassy state form easily.When biomolecule is dry, trehalose closely encases adjacent molecule, form a kind of structurally with the similar carbohydrate glass of glass ice, its diffusion coefficient is very low, molecular motion and molecule degeneration are very faint, can make biomolecule keep certain space structure.The formation of glassy state not only can prevent the generation of ice crystal in the refrigerating process, the structure of all right stable protein.3. preferential exclusion theory: saccharide can be under dry and the solution condition protein to be played certain stable and protective effect.Trehalose is the same with some other micromolecule saccharide, not direct and protein structure interacts, but preferentially combine with water, make in their solvated layers from protein molecule and get rid of to come out, cause proteinic solvated layer reduced radius, molecular structure more is becoming tight close, and conformation is more stable, thereby resists the III that influences of extraneous extreme environment.Because aquation volume ratio sucrose, the maltose of trehalose are big 2.5 times, therefore, the size-exclusion effect is more obvious, just can realize proteinic preferential exclusion under low concentration, produces the stablizing effect stronger than other disaccharidase.4. studies show that of number of mechanisms synergism theory: Crowe etc., simple glassy state forms the protective effect that can not bring into play trehalose, only could bring into play the protective effect of trehalose when glassy state forms synergism with the hydrone vicarious function better.

Trehalose has the function of unique protection biomolecule; be the specially good effect protective agent and the histiocytic stabilizing agent of biomolecule; it will be the medical bio goods; kept dry, transportation and use as skin, organ, recombinant human albumen, isolated cells and the tissue etc. of blood products, enzyme, vaccine, vaccine, virus, hormone, cell membrane, monoclonal antibody, drug-loaded liposome, antiserum, the required storage of surgical operation bring great convenience, and have all inaccessible protection effect of protective agent of other kind.

So the present invention forms recombinant human nerve peptide Y with nano-emulsion as drug administration carrier.Can be wrapped in recombinant human nerve peptide Y in the emulsion droplet nuclear, make recombinant human nerve peptide Y avoid the influence to neuropeptide tyrosine stability such as PH, temperature, illumination, oxygen concentration, trehalose can be used as the stabilizing agent of recombinant human nerve peptide Y in the prescription in addition, has guaranteed that long-distance transportation recombinant human nerve peptide Y still can keep quite high activity.So under dual protection, recombinant human nerve peptide Y biological activity can well be protected.

Analysis and Identification of the present invention:

1. naked eyes outward appearance of the present invention is observed: the recombinant human nerve peptide Y nano-emulsion outward appearance of external preparation for skin refrigerant colourless, fluidity and good dispersion, obviously visible opalescence person is arranged.

2. nano-emulsion size of the present invention, measure of spread: adopt the Ma Erwen assay method, the recombinant human nerve peptide Y nano-emulsion 2ml of bark fetching skin external injects the test cup, measures the size and the distribution of nano-emulsion particle by particle size analyzer.The mean diameter of nano-emulsion particle is that particle size distribution is between 10～100nm below 100nm.

3. the evaluation of nano-emulsion type of the present invention: adopt staining to identify that the cardinal principle of the type of nano-emulsion is to judge the type of nano-emulsion by tonyred (oiliness dyestuff) and methylene blue (water-soluble dye) red or blue diffusion speed in nano-emulsion: water-soluble dye in the diffusion of O/W nano-emulsion soon, because O/W nano-emulsion continuous phase is water, water-soluble dye is faster than spreading at oil phase at water, as a same reason, oil-soluble dyes are fast in the diffusion of W/O nano-emulsion.Recombinant human nerve peptide Y nano-emulsion through identifying external preparation for skin of the present invention is the O/W nano-emulsion.

4. stability of the present invention is identified: the recombinant human nerve peptide Y nano-emulsion thermodynamic stability of the present invention's preparation is good, and the influence of hands external environment is little, can protect the biological activity of recombinant human nerve peptide Y well.Nano-emulsion itself of the present invention in addition has antiseptic property, does not need to add antiseptic, good antiseptic power is just arranged, long shelf-life.

1. to the stability experiment under condition of different temperatures: the recombinant human nerve peptide Y nano-emulsion product of bark fetching skin external, place glass ground joint flat bottle, placed 0 ℃, 4 ℃, 25 ℃, 40 ℃, 60 ℃, 80 ℃ temperature simultaneously following 30 days, by sampling in 0,5,10,15,30 day.Through measuring index of correlation, the present invention's stability under condition of different temperatures is fine.

2. to the stability experiment of illumination: the recombinant human nerve peptide Y nano-emulsion product of bark fetching skin external, place glass ground joint flat bottle, placed under daylight lamp and the natural light Continuous irradiation simultaneously 30 days, by sampling in 0,5,10,15,30 day.Through measuring index of correlation, the present invention is stable fine to light.

3. high speed centrifugation experiment: the recombinant human nerve peptide Y nano-emulsion product of bark fetching skin external, and place high speed centrifuge supporting centrifuge tube, with 10000-16000rpmmin ^-1The centrifugal 30min of speed, do not find the recombinant human nerve peptide Y nano-emulsion layering of external preparation for skin, illustrate that the recombinant human nerve peptide Y nano-emulsion of external preparation for skin is stable.

4. the bioactive test of neuropeptide tyrosine: respectively according to preparation method of the present invention liquid preparation preparation method that prepare and traditional, under identical condition, the neuropeptide tyrosine of biologically active of the same race is mixed with the recombinant human nerve peptide Y nano-emulsion and the neuropeptide tyrosine liquid preparation of external preparation for skin respectively, under identical storage condition, measure the neuropeptide tyrosine activity respectively respectively at placing 30 days front and back, found that, the neuropeptide tyrosine of the biological activity of the neuropeptide tyrosine in the recombinant human nerve peptide Y nano-emulsion of external preparation for skin in the common liq preparation illustrates that the present invention can keep the biological activity of neuropeptide tyrosine peptide better.

In sum, the present invention has following beneficial effect:

1. the present invention's usefulness nano-emulsion system is as the carrier of medicine, be that a kind of novel medicament is carried and the hierarchy of control, its mechanism of sealing with release is by active substance being wrapped in nanometer emulsion droplet inside or the nanometer emulsion droplet, utilize the small size characteristics of nanometer emulsion droplet, make peptide medicament penetrate tissue gap and absorbed by cell, and then can also can pass through the biomembrane barrier by the blood capillary of human body.And the nano-emulsion carrier can improve the stability and the bioavailability of peptide medicament, is the good transport agent of polypeptide and protein medicaments, and special advantages is arranged.

2. the recombinant human nerve peptide Y nano-emulsion stability of external preparation for skin of the present invention improves greatly.The present invention forms recombinant human nerve peptide Y nano-emulsion with nano-emulsion as carrier, can be wrapped in recombinant human nerve peptide Y in the emulsion droplet nuclear, makes recombinant human nerve peptide Y avoid the influence to it such as PH, temperature, illumination, oxygen concentration.So thermodynamic stability of the present invention is good, can improve its stability significantly, prolong its effect duration greatly.

3. the present invention has improved the bioavailability of recombinant human nerve peptide Y.The present invention has changed the drug administration carrier of NPY peptide medicament, and reduced the risk that intravenous injection brought and by the side effect of its generation, the viscosity of nano-emulsion of the present invention, polymolecularity has prolonged the time of contact of medicine and mucosa, make medicine can pass through biological barrier better, can keep treatment concentration for a long time, the bioavailability height.

4. the trehalose in the recombinant human nerve peptide Y nano-emulsion prescription of external preparation for skin of the present invention can be used as the stabilizing agent of neuropeptide tyrosine bioactive substance, guaranteed that the long-distance transportation neuropeptide tyrosine still can keep quite high activity, reduced the storage and the cost of transportation of neuropeptide tyrosine greatly.

5. the recombinant human nerve peptide Y nano-emulsion of the present invention preparation, its dispersibility, flowability, uniformity, stability, percutaneous absorbability, increase with the area of contact skin, common influence such as prolong action time, the effect of the recombinant human nerve peptide Y nano-emulsion of the external preparation for skin of the present invention that makes will be more obvious.

6. the recombinant human nerve peptide Y biological activity in the recombinant human nerve peptide Y nano-emulsion of the external preparation for skin of the present invention's preparation can be significantly improved.Nanoemulsions-liquid dispersion of forming by isopropyl myristate, sad capric acid polyethyleneglycol glyceride, polyglycereol-3-dioleate, distilled water, can be wrapped in recombinant human nerve peptide Y in the nanometer emulsion droplet nuclear, make recombinant human nerve peptide Y avoid the influence to neuropeptide tyrosine stability such as PH, temperature, illumination, oxygen concentration, trehalose can be used as the stabilizing agent of recombinant human nerve peptide Y in the prescription of the present invention in addition, has guaranteed that long-distance transportation recombinant human nerve peptide Y still can keep quite high activity.So under dual protection, recombinant human nerve peptide Y biological activity can well be protected.

7. safety is improved.Though the non-injection administration research to polypeptide obtains some progress in recent years, the difficulty that faces is still a lot.The mucosa transmission of nearly all polypeptide drugs all needs penetration enhancer, and its kind is numerous and diverse, has stimulating mucosal and the problem that influences the skin integrity.And the product of the present invention preparation does not need to add penetration enhancer and just has osmosis, and the safety of application is improved.

And preparation composition of the present invention simplify, with low cost, preparation technology is simple, practical operation is easy, preparation efficiently fast, does not need special instruments and equipment, save raw material, reduce environmental pollution, low-carbon environment-friendly.

Preparation points for attention of the present invention:

1. product used container of when preparation must strictly according to the rules clean and sterilization, and preferably adopt strong acid to soak back water and dash, clear water washes clean again, dry with distilled water immersion the back, places after the baking oven high-temperature sterilization drying standby again; In addition, when carrying out the product preparation, used container all will be paid special attention to prevent to contain impurity or pollutions such as metal ion and microorganism, in order to avoid influence product quality and stability.

2. recombinant human nerve peptide Y is that Dinke auspicious professor's neuropeptide tyrosine research team designs synthetic functional component voluntarily, its design and synthetic very difficult, therefore, should in time link up in when preparation and to get in touch with the fourth professor, synthetic and prepare so that this seminar can finish the recombinant human nerve peptide Y of q.s.

3. prepare in strict accordance with above-mentioned dosage and method.When carrying out this product preparation, should add according to above-mentioned flow sequence, its additive capacity must require accurate, and, in the interpolation of other raw material, could add a kind of raw material down after must waiting a kind of raw material of adding fully to dissolve again,, influence the preparation of whole nano-emulsion system in order to avoid occur muddyly.

4. when preparation external recombinant human nerve peptide Y nano-emulsion, need select and definite S/C mass ratio (km value), and definite S/C mass ratio (km value) 1,2,3,4, be to carry out the oily biphase binary liquid test of titration water and last definite by ability after the pseudo-ternary phase diagram analysis, only, be only comparatively ideal km value determining and calculating the km value that obtains maximum nano-emulsion zone.Above-mentioned preparation method determines that by testing Km value has been 3, but actual when carrying out the product preparation, surfactant (S) and cosurfactant in this ratio still must be after mixing also will be more abundant mixing, prepare effect and quality in order to avoid influence.

5. because the proportion or the different relation of density of liquid solution, when carrying out the product preparation, the capacity of selected preparation container must be a bit larger tham the product volume of actual preparation, as prepare the 100g product, need to select the preparation container of 150-200mL, so both be convenient to each composition and fully mixed and dissolve, and also be convenient to ultrasonic concussion or stirring and evenly mixing, and be unlikely to overflow outward.

6. though blank nano-emulsion itself has the characteristics of certain bacteria growing inhibiting, other interpolation antiseptic is not arranged in this formula for a product, so when carrying out the product preparation, except that the preparation container must carry out strictness cleaning and sterilization, also should be during concrete the preparation in strict accordance with rule of operation, under aseptic condition and according to the sterile working, require to operate as far as possible, avoid this nano-emulsion sample to be polluted, influence product quality.

7. preparing the sample of finishing must pack with lucifuge, and places shady and cool dry ventilation lucifugal place to preserve as far as possible, keep fire away or thermal source.In addition, after product uses at ordinary times, need not place low temperature or refrigerator to preserve, but must seal tightly, be positioned over natural room temperature and keep in Dark Place and get final product.

8. this product may have zest to skin, please suspends use immediately as using the position significant reaction to occur; In actual use, be sure not this product is done into ophthalmic; Skin is injured, have wound or redness must not use, use the back red and swollen if any continuing, and should stop using; Baby and child must not use; The skin sensitivity person should do the skin sensitivity property testing before using, and tests completely without irritative response and can use.

9. recombinant human nerve peptide Y is a peptide matters, so-20 ℃ of left and right sides cold preservations of these solid feed needs are comparatively suitable, face with facing and join, and each amount of preparation should not be too big, and is behind the preparation recombinant human nerve peptide Y aqueous solution, now with the current as far as possible, as too many or too much for use, should place 2-8 ℃ of refrigerator short-term to preserve.

10. recombinant human nerve peptide Y nano-emulsion muddiness can occur after putting into refrigerator, but recovers can form nano-emulsion voluntarily after the room temperature, does not influence use.

Description of drawings

Fig. 1. the abduction delivering of recombinant human nerve peptide Y.

Fig. 2. the affinitive layer purification of recombinant human nerve peptide Y.

Fig. 3. the Western Blot that recombinant human nerve peptide Y purification is given birth to identifies.

Fig. 4. a kind of preparation technology's flow process and technology path of recombinant human nerve peptide Y nano-emulsion of external preparation for skin.

The specific embodiment

Embodiment 1: for immunogenicity and not high the stablizing of purity that overcomes the animal origin neuropeptide tyrosine, the NPY gene order that this research team includes in earlier stage according to Gene bank, design this gene upstream and downstream sequence, cDNA through the synthetic NPY of PCR reaction, recombinate with the pET28a+ carrier then, screening positive clone and dna sequence analysis identify that dna sequence analysis shows that clone's dna fragmentation is a people NPY gene.And institute's cloned genes 36 aminoacid of encoding altogether, molecular weight is 4.2KD, reaches 100% with NPY gene order homology among the GenBank.

The preparation of reorganization neuropeptide tyrosine

1 material and method

1.1 material

E.coli BL21 (DE3), the preservation bacterium that contains the correct recombiant plasmid pET28a-NPY of order-checking preserve by this chamber; Albumen marker is available from TaKaRa company; Mouse-anti people Anti-6 * His antibody, the anti-mice IgG of horseradish peroxidase (HRP) labelled goat all purchase in sky root biochemical technology company limited; Nickel ion affinity chromatograph prepacked column and chemiluminescence colour reagent are purchased the company in QIAGEN; Protein electrophoresis instrument, electrotransfer instrument (Bio-Rad company); The ultrasonic cell pulverization machine of cientz-IID is a Ningbo Xin Zhike device institute product; Other agents useful for same is homemade analytical reagent.

1.2 method

1.2.1 the abduction delivering of recombined human NPY fusion rotein

Pick the preservation bacterium that contains recombiant plasmid pET28a-NPY on a small quantity with inoculating loop, line on LB (Kan+) flat board is inverted overnight incubation for 37 ℃.Next day, picking list bacterium colony was inoculated in 5ml LB (Kan+) fluid medium, and 37 ℃ of shaken cultivation are spent the night.Get overnight incubation bacterium liquid 500 μ l next day and be inoculated in the 50ml selectivity LB fluid medium, shaken cultivation to OD600 value reaches 0.6.Add IPTG after drawing 1ml, final concentration is 1mmol/L, and 37 ℃ are continued inducing culture 4h.Get add before the IPTG and after each 1ml of bacterium liquid, the centrifugal 5min of 12000rpm collects bacterial sediment, and adds 50 μ l distilled water in precipitation, add 2 * SDS-PAGE sample-loading buffer, 50 μ l behind the mixing again, mixing, boiling water bath 5min, the centrifugal 10min of 12000rpm, each sample is got 20 μ l and is splined on electrophoresis on 15% the SDS-PAGE glue, after finishing, gets electrophoresis Coomassie brilliant blue R250 dyeing 2h, decolouring then, Taking Pictures recording result.

1.2.2 the release of inclusion body

According to the above, induce scale in the LB fluid medium expansion of shaking dress 500ml in the bottle of 2000ml.With abduction delivering bacterium liquid, 4 ℃ with the centrifugal 15min of 5000rpm, collects bacterial sediment; Add 25mlPBS, blow and beat abundant suspension bacterial sediment, 4 ℃ of centrifugal 30min of 12000rpm, supernatant discarded is washed bacterial sediment 3 times according to this repeatedly; Add cell pyrolysis liquid 25ml, piping and druming evenly suspends bacterial sediment; With the bacterial sediment suspension put-20 ℃ freezing, melt in room temperature again, so multigelation is 3 times; The 4 ℃ of centrifugal antibacterial freeze thawing of 12000rpm liquid 30min, supernatant discarded adds ultrasonic degradation liquid 25ml in precipitation, the piping and druming mixing; Put on ice antibacterial freeze thawing liquid is carried out supersound process, 10min/ time, processing time 30min; After the supersound process, in 4 ℃ of centrifugal 30min of 12000rpm, supernatant discarded is precipitated as bacterial cell cracking precipitate.

1.2.3 the purification of inclusion body

In bacterial cell cracking precipitate, add the inclusion body purification liquid 25ml that contains 4mol/L carbamide, the piping and druming mixing, room temperature leaves standstill 30min, and the centrifugal 30min of 12000rpm 3 times repeatedly, promptly obtains the inclusion body precipitate.

1.2.4 the cracking of inclusion body

In the inclusion body precipitate, add inclusion body lysate 25ml, the piping and druming mixing, room temperature leaves standstill 6h, makes the abundant cracking of inclusion body, and the centrifugal 30min of 2000rpm collects supernatant, is the inclusion body lysate.

1.2.5 the renaturation of Denatured protein

The inclusion body lysate supernatant of collecting is joined in the bag filter of handling, tighten the bag filter two ends; Bag filter integral body is immersed in protein renaturation liquid, under 4 ℃ of conditions, dialyses; Urea concentration in the renaturation solution successively decreases successively from 7mol/L-1mol/L, and 3-4h dialyses in each urea concentration gradient renaturation solution.The each replacing before the different urea concentration renaturation solutions need at 4 ℃ of centrifugal 30min of 12000rpm, add the sample sucking-off in the bag filter in the bag filter to supernatant again, tightens again and dialyses; After dialysis finishes, sample is taken out, 4 ℃ of centrifugal 30min of 12000rpm add bag filter with supernatant again, and 20h dialyses in the PBS of 4 ℃ of pre-coolings; Take out the good sample of dialysis, 4 ℃ of centrifugal 30min of 12000rpm collect supernatant and are renaturation product.-70 ℃ of preservations.

1.2.6 the purification of renaturation product

1. go up sample: renaturation product slowly is added on has used in the equilibrated nickel ion affinity chromatograph prepacked column of PBS buffer, the used flow velocity of application of sample will be controlled in the 1mL/min; 2. flushing: the PBS washing is removed not conjugated protein, and irrigation flow rate should be controlled in the 5mL/min; 3. eluting: respectively with contain 50,100,150mmol/L imidazoles PBS buffer solution elution fusion rotein, flow velocity 5mL/min.Get 20 μ l behind the purification and be splined on electrophoresis on 15% the SDS-PAGE glue.

1.2.6 purified product Western blot detects

After getting purification afterproduct 20 μ l and being splined on 15% separation gel SDS-PAGE electrophoresis, electrotransfer on the film, carries out Western blot engram analysis to the nitrocellulose filter.The one anti-mouse-anti people Anti-6 * His of being (diluting at 1: 1000), two resist for the sheep anti-mouse igg antibody of horseradish peroxidase (dilution in 1: 5000), adopt the chemoluminescence method development, expose on the X-ray sheet.

2 results

2.1 induce pET28a-NPY to express NPY result

The NPY that pET28a-NPY expresses has 36 amino acid whose fusion rotein of non-NPY at the N end, the tag (His-tag) that 6 histidine are wherein arranged, destination protein NPY is made up of 36 aminoacid, owing to after the 36th amino acid whose base of coding NPY, and then inserted a termination codon during design NPY primer, therefore, the NPY of pET28a-NPY expression is the 36th aminoacid of destination protein NPY at last aminoacid of C end.Be that the complete fusion rotein that pET28a-NPY expresses is made up of 72 aminoacid, molecular weight is approximately less than 10KD.PET28a-NPY induces through IPTG and the results are shown in Figure 1, has tangible induced protein to express in the position that is lower than 10KD, and NPY reorganization bacterium does not almost have expression before IPTG induces.

2.2 the evaluation behind the fusion rotein purification

NPY albumen contain 150mmol/L imidazoles PBS buffer can be fully by eluting, carry out 15% SDS-PAGE electrophoresis respectively and identify (Fig. 2).

2.3 the Western Blot result of purifying protein

Develop with chemoluminescence method, X-ray sheet exposure back visible one clear band shows that destination protein obtains effective expression and purification, shows as Fig. 3.

We utilize the DNA recombinant technique to clone the NPY gene, and with its be expressed among the DE3 after the pET28a carrier is connected.Promptly with BL21 (DE3)/pET228a+ as expression system.The pET-28a expression vector contains His-Tag oligo-histidine chain at N end, and therefore, the fusion rotein that gives expression to can be expediently under multiple situation and carried out purification economically.Our designed NPY fusion rotein N end is the non-destination protein that 36 aminoacid are formed, and wherein comprises a His-tag, and purpose NPY albumen thereafter also has 36 aminoacid.Be that the fusion rotein that pET28a-NPY expresses is made up of 72 aminoacid altogether, about the about 10KD of molecular weight.Carry out western blot with 6 * his-tag antibody and identify, showing has a large amount of fusion rotein to produce.Its molecular weight conforms to the NPY fusion rotein.

Embodiment 2: the recombinant human nerve peptide Y nano-emulsion technology of preparing of external preparation for skin

1) preparation primary raw material: (1) blank nano-emulsion raw material and source: 1. isopropyl myristate (IPM): for moistening prompt chemical reagent company limited or Shanghai Chemical Reagent Co., Ltd., Sinopharm Group product in Shanghai, content 〉=98%), raw material is bought phone 021-52511155.2. sad capric acid polyethyleneglycol glyceride (Labrasol): be French GATTEFOSSE company product, the agent: Shanghai gloomy monarch's chemical pharmaceutical adjuvant company limited, raw material is bought phone 021-51611173.3. polyglycereol-3-dioleate (Plurol Oleique CC497): be French GATTEFOSSE company product, the agent: Shanghai gloomy monarch's chemical pharmaceutical adjuvant company limited, raw material is bought telephone number 021-51611173.4. distilled water: laboratory self-control.

(2) main functional component and this sample raw material sources in the recombinant human nerve peptide Y nano-emulsion of external preparation for skin: neuropeptide tyrosine (this seminar designs voluntarily, synthesizes and preparation).

2) main equipment of preparation and software: the container of analytical balance, electronic balance, liquid flash mixer, time constant-temperature magnetic stirring apparatus, ultrasonic concussion instrument, quartz ampoule glass aqua bi-distilling apparatus, certain specification, flask, beaker, suction pipe, burette etc.In addition, also have analysis software Origin 7.0 pattern analysis systems (U.S. Microcal company).

3) complete formula is formed and accurate consumption (100g is an example with preparation): the prescription of neuropeptide tyrosine nano-emulsion is formed and consumption: isopropyl myristate 10g, sad capric acid polyethyleneglycol glyceride 30g, polyglycereol-3-dioleate 10g, trehalose 2g, neuropeptide tyrosine aqueous solution 48g amounts to 100g.

4) the concrete preparation method of recombinant human nerve peptide Y nano-emulsion and implementation example (* is an example with preparation 100g, enlarges preparation as needs, can the rest may be inferred):

1. recombinant human nerve peptide Y aqueous solution preparation: 0.0005g neuropeptide tyrosine and 2g trehalose are dissolved in the 47.9995g distilled water.With this solution as water 50g (I).

2. accurately take by weighing the isopropyl myristate of 10g weight with analytical balance, place another to be labeled as oil phase (II) 10g through knowing among the 50mL conical flask of decontamination and with marking pen.

3. according to the sad capric acid polyethyleneglycol glyceride (S) that has designed in advance: the ratio of polyglycereol-3-dioleate (C)=3: 1, accurately take by weighing the sad capric acid polyethyleneglycol glyceride of 30g and 10g polyglycereol-3-dioleate respectively, and the two raw material is placed among the clean conical flask of 100mL of the 3rd process decontamination; Add a cover, and this conical flask placed rapidly on the liquid flash mixer, open the liquid flash mixer, and it is transferred to the II shelves, fully mix homogeneously makes it to form emulsifying agent/co-emulsifier (S/C) mixture, behind the 3-5min, the closing liquid flash mixer takes off conical flask, and with clear S/C mixture (III) 40g that is labeled as of marking pen.

4. according to oil phase (II): S/C mixture (III): the ratio of water (I)=10%: 40%: 50%, fetch water respectively earlier phase (I) 50g, S/C mixture (III) 40g place among the 4th the clean conical flask of the 200mL through decontamination.

5. after above-mentioned water (I), S/C mixture (III) two liquid phases fully being mixed, again under 25 ℃ of conditions of room temperature or in the natural room temperature, put it into and shake in the ultrasonator also about ultrasonic 2min, perhaps under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1Rotating speed magnetic agitation 5min, then, in this container, directly add oil phase (II) 10g again, and with whole system in ultrasonator about ultrasonic 5min, perhaps under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1Rotating speed magnetic agitation 20min.

6. close ultrasonator or time constant-temperature magnetic stirring apparatus, take off the 200mL conical flask, observe its outward appearance limpid in this way colourless, fluidity and good dispersion, obviously visible opalescence person is arranged, be the recombinant human nerve peptide Y nano-emulsion of 100g external preparation for skin.

7. the recombinant human nerve peptide Y nano-emulsion of this 100g external preparation for skin is sub-packed in rapidly among the lucifuge glass container of different size, adds a cover rapidly, packing, and place 4 ℃ of airtight preservations of refrigerator to get final product.

8. according to International or National or enterprise's relevant criterion and requirement, stochastic sampling from above-mentioned packing product, at first adopt the distribution of particles and the size of this nano-emulsion sample of Ma Erwen instrument detecting, after meeting the quality standard of nano-emulsion, according to the specific requirement of national departments concerned, the system that carries out index of correlation detects again; After waiting to judge or estimating it and meet national relevant regulations and standard and authentication code fully, can be used as promptly that launch is sold or special-purpose in the beauty parlor.

Embodiment of the present invention only is the description that preferred implementation of the present invention is carried out; be not that design of the present invention and scope are limited; under the prerequisite that does not break away from design philosophy of the present invention; engineers and technicians make technical scheme of the present invention in this area various modification and improvement; all should fall into protection scope of the present invention; the technology contents that the present invention asks for protection all is documented in claims.

Claims

1. the recombinant human nerve peptide Y nano-emulsion of an external preparation for skin, liquid-in-liquid dispersions system by recombinant human nerve peptide Y, trehalose, isopropyl myristate, sad capric acid polyethyleneglycol glyceride, polyglycereol-3-dioleate, distilled water are formed is characterized in that being made by following weight percentages:

Recombinant human nerve peptide Y 0.0003%～0.0006%

Trehalose 1%～3%

Isopropyl myristate 5%～15%

Sad capric acid polyethyleneglycol glyceride 25%～32%

Polyglycereol-3-dioleate 7%～12%

Distilled water 45%～52%

The total weight percent of above-mentioned raw materials is 100%.

2. according to the recombinant human nerve peptide Y nano-emulsion of the described external preparation for skin of claim 1, it is characterized in that by following

Stating the raw material of preferred weight percent makes:

Recombinant human nerve peptide Y 0.0005%

Trehalose 2%

Isopropyl myristate 10%

Sad capric acid polyethyleneglycol glyceride 30%

Polyglycereol-3-dioleate 10%

Distilled water 47.9995%

The total weight percent of above-mentioned raw materials is 100%.

3. according to the recombinant human nerve peptide Y nano-emulsion of claim 1 and the described external preparation for skin of claim 2, it is characterized in that the preparation method of the recombinant human nerve peptide Y nano-emulsion of described external preparation for skin may further comprise the steps:

6. close ultrasonator or time constant-temperature magnetic stirring apparatus, take off conical flask, observe its outward appearance limpid in this way colourless, fluidity and good dispersion, obviously visible opalescence person is arranged, be a kind of recombinant human nerve peptide Y nano-emulsion of external preparation for skin;

7. the recombinant human nerve peptide Y nano-emulsion of this a kind of external preparation for skin is sub-packed in rapidly among the lucifuge glass container of different size, adds a cover rapidly, packing, and place 4 ℃ of airtight preservations of refrigerator to get final product.

4. according to the recombinant human nerve peptide Y nano-emulsion of the prepared a kind of external preparation for skin that goes out of claim 3, it is characterized in that the emulsion droplets particle diameter of the recombinant human nerve peptide Y nano-emulsion of described external preparation for skin is 10～100nm.