CN106310230A - Preparing method and application of oral insulin transport system in layer-by-layer self-assembly structure - Google Patents
Preparing method and application of oral insulin transport system in layer-by-layer self-assembly structure Download PDFInfo
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- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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Abstract
The invention discloses a preparing method for oral insulin transport system in layer-by-layer self-assembly structure comprising the steps of separating out insulin nano ball with electropositivity by salting out method under the condition of low pH, and successively packing the insulin nano ball using polygalacturonic acid (PGLA) with electronegativity and chitosan (CS) with electropositivity by layer-by-layer self-assembly method according to the electrical property and electrostatic force of materials. The oral insulin transport system in the invention is characterized by nature, low toxicity, natural metabolism and degradation and excellent biocompatibility, and has small partical size, strong small-intestine absorption rate, high drug loading ratio and excellent stability. The system can quickly and potently exert the hypoglycemic effect of insulin and also obviously and quickly control the blood glucose level in a long-term with the potential of diabetes treatment.
Description
Technical field
The invention belongs to biological medicine field of material technology.Being administered orally more particularly, to a kind of LBL self-assembly structure
Insulin transports preparation method and the application of system.
Background technology
The number that diabetes are suffered from the whole world is about 2.8 hundred million, and wherein 5～10% is type i diabetes, and about 80～90% is II type
Diabetes.Type i diabetes is fallen ill 0～4 years old also known as juvenile diabetes, the patient that suffers from of 50%.Type i diabetes is by siberian crabapple
System disorder makes B cell death cause, and during patient's first attack, B cell has lost 70%-90%, after morbidity 1～2
Year, B cell was the most dead, and patient's amount of insulin secretion is zero.Type ii diabetes is typically fallen ill 35～40 years old.Some trouble
In person's body, insulin even produces too much, but the action effect of insulin is poor, and insulin the most in the patient is a kind of phase
To shortage.Insulin is the necessary medicine of type i diabetes, is also that type ii diabetes people is at necessary curative in ill late period
Thing.
Although insulin preparation is constantly updated in recent years, there are the different types of insulin preparation confession patients such as long-acting, fugitive
Select, but, injection remains and insulin is inputted mode in the patient.On the one hand, insulin injection can cause patient
The discomforts such as fear, pain, research shows that some Type II diabetics can postpone the use time of insulin due to this discomfort,
Long term injections also can cause skin problem, increase Infection probability；On the other hand, insulin injection circulation style in vivo also with
Naturally secretion is different, and for comparing, insulin injection can avoid the first pass effect of liver, relatively increases peripheral vascular
Insulin concentration, is comparatively fast gathered in kidney and discharges.Therefore, the research that transports of the non-intruding approach of exploitation insulin becomes in recent years
Carry out the research emphasis in this field.Transporting approach compared to multiple Noninvasive insulin, oral route transports insulin to be had
One big advantage, it is most like with what self secreted that the i.e. oral insulin transported circulates approach in vivo, the oral insulin transported
Stay longer can be assembled in liver, then discharge through kidney, the concentration distribution at the most each position and circulation path and nature
Secretion insulin the most similar, this for avoid or reduce complication, increase insulin medication effect the most extremely important.This
Outward, oral insulin transports system and also has taking convenience, the advantage such as is easily accepted by patients, and therefore, the oral of insulin transports
System is that current insulin transports one of study hotspot.Also the para-insulin being the most potential entrance clinical practice is defeated
Fortune mode.
At present, the research that oral insulin transports focuses primarily upon this one side of increase transport efficiency.Can grind this kind of
Study carefully and be divided three classes: prevent digestive enzyme degraded, promote that intestinal mucosa attachment, promotion penetrate enteric epithelium.Wherein, promote that particle passes on intestinal
The approach of chrotoplast is the problem that much research endeavours to capture, and current achievement is broadly divided into four classes, i.e. enters system by M cell
Uniting and circulate, targeting enters system by Goblet cell and circulates, and opens enteric epithelium by endocytosis entrance enterocyte thin
The compact siro spinning technology of intercellular, makes particle pass intercellular substance and enters system circulation.Some research combines comprehensively and promotes to promote insulin
The mode of pick-up rate, the insulin transport system of use had both promoted mucoadhesive, and using nano material to transport insulin again can
By M cell, to be also equipped with compact siro spinning technology or the promotion endocytosis opening between enterocyte simultaneously.Existing insulin mouth
Fortune of admitting defeat system common materials mainly has chitosan (chitosan), alginate (alginate), glucosan (dextran) etc.
Natural material, also has a polymer through the nontoxic synthetic of U.S. FDA certification such as PLGA, PEG, or the inorganic material such as silicon.
Additionally, chitosan (chitosan), PEG, some targeting factor also can serve as the surface modification of system, play increase little
The effect of intestinal absorption rate.Wherein chitosan (chitosan) have electropositive, intestinal mucosa tack, can open between small intestine epithelium tight
The effect of close connection is the most clearly studied and is repeatedly verified.
Existing drug oral transport architectural study be usually taken selection natural material preparation transport carrier reach low toxicity or
Nontoxic purpose.But, by existing research ignore a bit, natural material, in addition to having low toxicity character, also has latent
Medical functions, i.e. natural material has the potentiality of the dual-use function being provided simultaneously with conveying material and medicine.Indian is early
Just diabetes were divided into before B.C. 200 years two classes that are that caused by heredity and that caused by drinking and eating irregularly.For centuries,
There is the experience using plant treatment diabetes in each state.Ethnobotany research research shows that at least 800 kinds of plants have anti-
The potentiality of diabetes.In recent decades, along with the research development of chemical purification technique and bio-science field, medicinal plants
In effective ingredient be accurately separated purification, also have hundreds of kind of hypoglycemic active substance of plant and chemical constitution thereof to obtain really
Fixed, the most amount of activated material blood sugar lowering ability is also elucidated in the mechanism of action of cell aspect with it.Therefore, carry out from planting
Active substances is chosen the preparation of Nantural non-toxic material and transports carrier, make insulin transport system itself simultaneously and there is blood sugar lowering merit
The research of energy, has great importance.
Summary of the invention
The technical problem to be solved is to have control blood glucose and electronegative natural poly-by exploring application
The chitosan of compound polygalacturonic acid and positively charged alternately assembles structure layer by layer, and steady to the size of this structure, shape, structure
Qualitative, drug release rate, insulin transport efficiency, cytotoxicity, body absorption are studied with aspects such as drug effects.Gained is layer by layer
The oral insulin of self-assembled structures transports system with polygalacturonic acid as stock, and building-up process is without Organic substance and poisonous
Material, possesses the potentiality being applied to treating diabetes.
The oral insulin that it is an object of the invention to provide a kind of LBL self-assembly structure transports the preparation method of system.
Another object of the present invention is to provide the oral insulin of described LBL self-assembly structure and transports the application of system.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The oral insulin of a kind of LBL self-assembly structure transports the preparation method of system, the most at low ph conditions, makes
Separate out positively charged insulin nano ball with salting out method, according to the electrical of material and electrostatic force, apply LBL self-assembly
Method uses electronegative polygalacturonic acid (PGLA) and electropositive chitosan (CS) Islets element nanosphere successively respectively.
First the present invention screens plant origin, has the polymer of hypoglycemic activity, obtains having glycemic control function, band
The polygalacturonic acid (PGLA) of negative electricity, combined with the natural copolymer chitosan of positively charged (CS), prepare LBL self-assembly
The oral insulin of structure transports system (LBL).This system have natural, low toxicity, can natural metabolism degraded, good biocompatibility
Advantage, this system particle diameter is little, has stronger intestinal absorption rate, and carrying drug ratio is high, good stability, it is possible to quick and potent
Performance insulin blood sugar lowering effect, it is also possible to control blood sugar level significantly, quickly, in a long time, possess and be applied to glycosuria
The potentiality of sick treatment.
Specifically, as preferably can embodiment, the oral insulin of above-mentioned LBL self-assembly structure transports system
Preparation method, comprises the steps:
S1. electropositive insulin nano ball is prepared
Under 10～20 DEG C of environment, insulin is dissolved in dilute hydrochloric acid solution, adds NaCl and be allowed to final concentration and reach
0.5～0.7M, solution stirring 0.4～0.6h, so centrifugal that to precipitate；
The most electronegative polygalacturonic acid (PGLA) wraps up
The precipitation of step S1 adds polygalacturonic acid (PGLA) solution, be stirred for 0.4～0.6h, ultrasonic 0.8～
1.5min, continues stirring 0.4～0.6h, so centrifugal that to precipitate；
The most electropositive chitosan (CS) wraps up
The precipitation of step S2 adds chitosan (CS) solution, stirs 0.4～0.6h, is centrifuged to obtain precipitation；
The most repeatedly it is repeated in step S2 and step S3, is i.e. alternately added PGLA solution and CS solution wraps up, obtain
The auto polymerization nanosphere of the different numbers of plies, the oral insulin of the LBL self-assembly structure of the i.e. different numbers of plies transports system.
Wherein it is preferred to, step S1 insulin is 4～6mg/ml with the mass volume ratio of dilute hydrochloric acid solution.
It is highly preferred that the mass volume ratio of step S1 insulin and dilute hydrochloric acid solution is 5mg/ml.
Preferably, ambient temperature described in step S1 is 15 DEG C.
Preferably, the pH of dilute hydrochloric acid solution described in step S1 is 1.0～1.4.
It is highly preferred that the pH of dilute hydrochloric acid solution described in step S1 is 1.1.
Preferably, the final concentration of 0.6M of NaCl described in step S1.
Preferably, described in step S1, the time of stirring is 0.5h.
Preferably, centrifugal described in step S1 is 4000～6000rpm to be centrifuged 2～4min.
It is highly preferred that centrifugal described in step S1 is that 5000rpm is centrifuged 3min.
Preferably, the pH of polygalacturonic acid described in step S2 (PGLA) solution is 3～5.
It is highly preferred that the pH of polygalacturonic acid described in step S2 (PGLA) solution is 4.
Preferably, the preparation method of step S2 polygalacturonic acid (PGLA) solution is: by polygalacturonic acid (PGLA)
Being dissolved in dilute hydrochloric acid solution, add NaCl, make NaCl final concentration reach 0.5～0.7M, pH is 3～5.
Wherein it is preferred to, described polygalacturonic acid (PGLA) is 4～6mg/ with the mass volume ratio of dilute hydrochloric acid solution
ml。
Preferably, described polygalacturonic acid (PGLA) is 5mg/ml with the mass volume ratio of dilute hydrochloric acid solution.
Preferably, the final concentration of 0.6M of NaCl in described polygalacturonic acid (PGLA) solution.
Preferably, the pH of described polygalacturonic acid (PGLA) solution is 4.
Furthermore it is preferred that the time of stirring described in step S2 is 0.5h.
Preferably, ultrasonic described in the step S2 time is 1min.
Preferably, the time continuing stirring described in step S2 is 0.5h.
Preferably, centrifugal described in step S2 is 4000～6000rpm to be centrifuged 2～4min.
It is highly preferred that centrifugal described in step S2 is that 5000rpm is centrifuged 3min.
Preferably, the preparation method of chitosan described in step S3 (CS) solution is: chitosan (CS) is dissolved in dilute hydrochloric acid
In solution, adding NaCl solid, make NaCl final concentration reach 2～4M, pH is 3～5.
Wherein it is preferred to, described chitosan (CS) is 4～6mg/ml with the mass volume ratio of dilute hydrochloric acid solution.
It is highly preferred that the mass volume ratio of described chitosan (CS) and dilute hydrochloric acid solution is 5mg/ml.
Preferably, the final concentration of 3M of NaCl in described chitosan (CS) solution.
Preferably, the pH of described chitosan (CS) solution is 4.
Preferably, described in step S3, the time of stirring is 0.5h.
Preferably, centrifugal described in step S3 is 4000～6000rpm to be centrifuged 2～4min.
It is highly preferred that centrifugal described in step S3 is that 5000rpm is centrifuged 3min.
Furthermore it is preferred that precipitation and the mass volume ratio of polygalacturonic acid (PGLA) solution described in step S2 be 0.8～
1.2g:100ml.
It is highly preferred that the mass volume ratio of precipitation and polygalacturonic acid (PGLA) solution is 1g described in step S2:
100ml。
Preferably, described in step S3, the mass volume ratio of precipitation and chitosan (CS) solution is 4～6g:100ml.
It is highly preferred that the mass volume ratio of precipitation and chitosan (CS) solution is 5g:100ml described in step S3.
It addition, the oral insulin of the LBL self-assembly structure prepared by said method transports system, and in system
Application in terms of standby oral insulin medicament, the most all within protection scope of the present invention.
LBL self-assembly structure is a kind of to apply storeroom positive and negative charge captivation to carry out the nano-particle assembled, Ke Yitong
Crossing the assembling number of plies and the size controlling reaction times control nano-particle, its size is relative compared with the nano-particle of other structure
Less.So, it is believed that LBL self-assembly structure is that research small size transports system and carries out the research that drug oral transports
The resulting structure of effect.Simultaneously as what the charge attraction power that its structure is dependent between material maintained, usual pH value is to material
Stability have stronger impact, this can make this structure that the release of medicine is had pH Irritability.
The present invention, first in the environment of low pH, high salt, makes insulin solutions separate out insulin nano ball；Then application is poly-
Galacturonic acid material with positive electricity electronegative with two kinds of difference of chitosan, Islets element nanosphere layer by layer, form 4 layers of LBL
Structure.This system has nano level particle diameter, spherical profile, and positively charged, has higher intestinal surface-clinging ability.
The present invention transport system selection and design from source, ensure that it has extremely low toxicity, this transport system be by
Prepared by the main component polygalacturonic acid of plant cell wall, polygalacturonic acid has the merit slowing down intestinal absorption glucose
Can, this makes system not only toxicity low, and in the application for the treatment of diabetes, the system that transports can be made inherently to have disease
Therapeutic effect.This is the once innovation in drug delivery field, i.e. applies natural, to have treatment function material to prepare
Drug delivery system, makes medicine-carried conveying system reach therapeutical effect from many aspects, possesses multiple disease therapeuticing effect.Meanwhile,
During synthetic system, do not apply organic solvent or toxicant, it is to avoid system produces toxicity.
The present invention is in the characterization research transporting system, and Ramon's spectrum tables of data phaneroplasm system the most successfully synthesizes, TEM image with
Dynamic scattering analysis all shows that particle has less particle diameter, and this will help particle by M cell through enteric epithelium, enter machine
Body blood circulation.Meanwhile, particle positively charged, its carried charge and relatively large (25mV), electropositive particle is easily with electronegative
Mucous membrane of small intestine attracts attachment mutually, promotes that it is absorbed by enteric epithelium, also helps to maintain the stability of particle.In TEM image indistinctly
The package structure layer by layer of visible particles, this is similar to the structure in other synthesis LBL architectural study.Additionally, thermogravimetric analysis shows,
In LBL system, material significantly reduces the thermal decomposition speed of insulin to the parcel of insulin.
The release in vitro research transporting system is shown by the present invention, and system is insulin releasing speed in the environment of pH=3-5
Rate is relatively slow, relatively fast in the intestinal environment insulin releasing speed of neutral pH, and this character also makes LBL transport system to possess
Bigger application potential.The low ph conditions of stomach and pepsic degraded are in oral protein drug design and practice process
One important difficult problem, builds pH and senses system, slow down insulin releasing in gastric environment, increases insulin in intestinal environment
Release is one of approach solving this difficult problem.The LBL4 diagram of system that the present invention builds reveals at pole low ph conditions and neutral pH ring
Quick release under border, the release at a slow speed (pH=3-5) under the conditions of moderate acid.The insulin of LBL4 is low to be discharged interval and enters
After food, the pH value of stomach is consistent, and can speculate that LBL4 system is administered orally the most afterwards is probably the way promoting medicine pick-up rate
Footpath.
The SD rat that the present invention also uses STZ to induce sets up type i diabetes rat model, separately verifies its ligation intestinal segment pair
LBL4 transports the amount of insulin of middle absorption and oral insulin to transport the blood glucose after system dynamic.Wherein, ImmunohistochemistryResults Results shows
Showing and take 1h after LBL4 system, duodenal segment just has significant absorption of insulin.This absorption rate is for oral drugs
For, relatively fast.Matching with this result, oral LBL4 has quick hypoglycemic effect, 1h after oral, blood glucose
I.e. it is down to the 40% of initial blood glucose value.Compared with similar research, the vivo efficacy of LBL4 system demonstrate faster drug absorption,
Drug effect time and hypoglycemic effect faster, this is a big advantage of LBL4 system.
In sum, the LBL of present invention synthesis transports system and successfully synthesizes, and is the spheroid of Nano Particle, and surface band is just
Electricity, has high thermal stability and pH Irritability；In vivo in research, show intestinal absorption speed faster, faster blood sugar lowering
Reaction and more significant hypoglycemic effect, possess research further and the value of practical application.
The method have the advantages that
The present invention is successfully prepared a kind of material polygalacturonic acid with plant origin insulin mouth as matrix material
Fortune of admitting defeat system LBL4, refers to alternately at Nanometer Insulin outer wrapping 4 strata galacturonic acid and the nanometer system of chitosan, body
System's synthesis, according to the positive and negative electrical attraction power of storeroom, makes material self assembles become nanometer and transports system, transports pancreas for oral
Island element.
Used by the present invention, polygalacturonic acid, chitosan are all the polymer of natural origin, make this system have natural, low
Poison, can natural metabolism degraded advantage.And building-up process does not has the participation of organic solvent and noxious substance, the material used
It is the polymer of natural origin, it is ensured that the biocompatibility of system.
This system particle diameter is little, has and increases the probability absorbed by enteric epithelium, and surface positively charged promotion particle is with electronegative
Enteric epithelium adsorb mutually, strengthen the characteristic such as particle stability, these character can help LBL system to enter machine from oral route
This system of body blood circulation can enter blood circulation through small intestine epithelium, transports system diameter little (less than 200nm), absorbance
Higher.
It addition, this system can possess higher carrying drug ratio, have stronger intestinal absorption rate, in vivo in research, energy
Enough quick and potent performance insulin blood sugar lowering effects, it is also possible to control blood sugar level significantly, quickly, in a long time, tool
The standby potentiality being applied to treating diabetes.
Accompanying drawing explanation
Fig. 1 is that LBL particle synthesizes schematic diagram.
Fig. 2 is the sign of LBL particle；A: Raman spectrum, is followed successively by insulin, PGLA, chitosan, 1 layer of LBL from top to bottom
Particle, 4 layers of LBL particle.B: Nanometer Insulin and the potential value of LBL4, a is the current potential of Nanometer Insulin, and b is 4 layers of LBL system
Current potential.C:TEM outward appearance, is the LBL system of parcel 1,2,3,4 layer material the most successively, and in figure, black scale represents
100nm。
Fig. 3 is thermogravimetric analysis；Grey filled lines represents the mass change situation that insulin raises with temperature, and solid black lines represents
The mass change situation that LBL 4 particle raises with temperature.
Fig. 4 is insulin UV Absorption standard curve.
Fig. 5 is LBL4 particle insulin releasing curve in simulation stomach, intestinal buffer liquid.
Fig. 6 is the insulin release after LBL4 hatches 2h in different pH buffer systems.
Fig. 7 is that cell viability is affected by transporting system concentration.
Fig. 8 be type i diabetes model foundation and modeling Mus compare with the build of normal mice.
Fig. 9 is blood glucose and Avoirdupois monitoring result during the modeling of SD rat；In A figure square for matched group, other is shaped as building
Module；In B figure, black line is Experimental modeling group, and grey lines is matched group.
Figure 10 is daily drink and food-intake monitoring result during the modeling of SD rat；In figure, black line is Experimental modeling group, ash
Colo(u)r streak is matched group.
Figure 11 is the situation of type i diabetes feeding rats LBL4 system 1h, 2h metaduodenum and ileal absorption insulin；
In figure, black scale represents 100 μm.
Figure 12 is that after type i diabetes rat limosis is administered orally LBL4, blood glucose is dynamic.
Detailed description of the invention
Further illustrate the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus are the examination of the art routine
Agent, method and apparatus.
Unless stated otherwise, following example agents useful for same and material are commercial.
Experiment material used in following example is as follows:
(1) experiment reagent:
Polygalacturonic acid (PGLA), chitosan, Pluronic 188, bovine insulin is purchased from Sigma, and STZ chain is urinated
Assistant rhzomorph, is purchased from Aladdin biochemical technology company limited.It is public that pancreatin, DMEM in high glucose culture medium, non essential amino acid are Gibco
Department's product；Newborn calf serum is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.；96/24 hole polystyrene cell is cultivated
Plate is Corning Corning Incorporated of U.S. product.
(2) experimental apparatus:
Sigma32184 High speed refrigerated centrifuge, Japan's HITACHI 7650 transmission electron microscope, Jintan City of Jiangsu Province
Medical Instruments factory 78-1 magnetic stirring apparatus, Japan's Nikon microscope, Britain MalvernZEN3600, Japan's Olympus optics falls
Putting microscope, H.J.Y company of France LabRAM Aramis micro-Raman spectroscopy, Switzerland Mettler, TGA/DSC 1 synchronizes heat
Analyser.
(3) experimental cell: Human colon adenocarcinoma cell line Caco-2, obtains in pharmaceutical university of Guangdong Province.
(4) laboratory animal: SD rat, is purchased from Nanfang Medical Univ's Experimental Animal Center.
The oral insulin of embodiment 1 LBL self-assembly structure transports preparation and the sign of system (LBL)
1, the preparation of LBL particle
The synthetic method of LBL particle is as shown in Figure 1.
(1) under 15 DEG C of environment, 5mg insulin is dissolved in the dilute hydrochloric acid solution of 1ml pH 1.1, adds NaCl
Solid, makes NaCl final concentration reach 0.6M, solution stirring 0.5h, and 5000rpm is centrifuged 3min, obtains precipitation；
(2) precipitation of step (1) adds polygalacturonic acid (PGLA) solution of pH=4, NaCl 0.6M, is stirred for
0.5h, ultrasonic 1min, continue stirring 0.5h, 5000rpm and be centrifuged 3min, obtain precipitation；
The preparation method of polygalacturonic acid (PGLA) solution of described pH=4, NaCl 0.6M is: by polygalacturonic
Acid (PGLA) is dissolved in 1ml dilute hydrochloric acid solution, adds NaCl solid, makes NaCl final concentration reach 0.6M, pH=4；
(3) precipitation step (2) obtained adds in chitosan (CS) solution of pH=4, NaCl 3M, stirs 0.5h, from
The heart, obtains precipitation；
The preparation method of chitosan (CS) solution of described pH=4, NaCl 3M is: chitosan (CS) is dissolved in 1ml dilute
In hydrochloric acid solution, add NaCl solid, make NaCl final concentration reach 3M, pH=4；
(4) repeatedly it is repeated in step (2) and step (3), is alternately added PGLA solution and chitosan solution wraps up,
Obtaining the auto polymerization nanosphere of the different number of plies, the oral insulin of the LBL self-assembly structure of the i.e. different numbers of plies transports system.
2, the sign of LBL particle
(1) Raman spectrum detection
By the raw material PGLA of system synthesis, chitosan, insulin, and the LBL systems of 1 layer, 4 layers parcel are completely dry
Dry, take suitable sample and be placed in the groove of sample loading plate, use coverslip compacting, make sample consolidation and there is flat surface, use
Raman spectrometer detects.Raman spectrum can detect the composition of particle to a certain extent.
Result shows: insulin is at 639cm-1, 853cm-1, 3313cm-1-SH ,-COOH ,-NH is demonstrated respectively at three3Three
The vibration of individual key.These three mark peak the most all comes across in the Ramon's spectrum of LBL1, LBL4, can confirm that there is islets of langerhans in end-product
The existence of element.PGLA and chitosan have abundant-OH (429cm-1, 421cm-1), in the wave spectrum of LBL1 Yu LBL4, also have-
Appearance (the 429cm at OH peak-1, 446cm-1), containing polysaccharide and insulin in this explanation LBL1 Yu LBL4, grain can be it was initially believed that
Son successfully synthesizes (Fig. 2 A).
(2) Zeta potential detection
Respectively each take turns synthesis after, take the 100 μ l detection for current potential.Nano particle diameter and Zeta potential detection make
(ZEN3600, Malvern equipment, Britain) is measured with Zetasizer nanoanalysis instrument.In particle size determination, sample is dissolved in ultrapure
In water, minimum measurement 180s.Instrument will be measured repeatedly automatically, and according to correlation analysis, result is obtained hydrodynamics particle diameter.
During Zeta potential measures, sample is dissolved in 0.1mM KCl, uses automatic mode to measure.All of measurement is in triplicate.
Fig. 2 B is respectively Nanometer Insulin and the Zeta potential of LBL4 particle.Result shows, Nanometer Insulin slightly positive electricity
Being 1.9 ± 0.01mV, LBL4 particle positively charged, its potential value is about 13.39 ± 0.01mV.Show Nanometer Insulin and LBL4's
Electrically it is consistent with expection, shows that the outer layer of LBL4 is fully wrapped up by chitosan simultaneously, make the positive charge that particle band is stronger.
(3) transmission electron microscope observing
Respectively each take turns synthesis after, take 100 μ l for outward appearance and the detection of particle diameter.The suspension of particle is dropped in and is covered with carbon
On the copper mesh of film, stand 5min, blot excessive moisture with filter paper, stand 30min, treat that it parches completely.Use HITACHI
7650 transmission electron microscopes, electron accelerating voltage is 300kV, respectively at 1x104Again, 2x104Under amplification again, observe particle shape
State.
Fig. 2 C is the TEM profile of particle, and the nm-class insulin particles after parcel polymer is rounded.Wrap up 1,2 respectively
Seen from the image of layer, the insulin of spheroidal is wrapped in flocculent structure, forms the form of similar grape cluster.Along with integument
The increase of number, particle diameter is continuously increased.The membrane structure that in figure, the color of visible polysaccharide formation is shallower is distributed in particle periphery.Parcel
During to the 4th layer, LBL4 particle forms the ball of about 200nm, the most independent existing form, but still has to a certain degree between particle
Involve.Simultaneously, it is seen that particle is the result wrapped up by multilayer film, and from outside to inside, color deepens successively.
(4) thermogravimetric analysis
The nano-particle LBL4 of parcel to four layers is dried process, uses Switzerland Mettler, TGA/DSC1 to synchronize heat
Analyser, under n 2 atmosphere, measurement scope is 30 DEG C to 900 DEG C, and programming rate is 10 DEG C/min.
Fig. 3 illustrates LBL4 system with insulin at N2Thermogravimetric curve under atmosphere, heating-up temperature from room temperature to 800 DEG C,
Programming rate is 10 DEG C/min.It is observed that the weightlessness of insulin has two intervals, it is 30-100 DEG C and 200-400 respectively
℃；The weightlessness of LBL4 system has three intervals, 30-100 DEG C, 200-300 DEG C and 750-800 DEG C.First interval, 300-100
DEG C corresponding is the weight loss of insulin and LBL4 system about 3%, and this is likely to the hydrone being absorbed by system or combining
Loss cause.Second interval, 200-400 DEG C corresponding is the mass loss of insulin about 70%, major part insulin
In this interval decomposed；Corresponding, LBL4 system about loses the quality of 20% in this temperature range, part insulin, chitosan,
PGLA is in this interval decomposed.Owing to LBL4 has stronger intermolecular hydrogen bonding and electrostatic attraction, compared with insulin group, this
It is later that the weight loss stage starts, and the degree of mass loss is the lightest.3rd interval, 750-800 DEG C of LBL4 system starts
Mass loss process for the third time.In this temperature range, each composition of LBL4 system decomposes further, not only includes sugar in polysaccharide
The dehydration of ring, also includes the dehydration of the group such as carboxyl, acetyl group, and the decomposition of part carbon skeleton
(5) entrapment efficiency (encapsulation ratio) measures
First insulin standard curve is drawn.It is respectively configured insulin solutions (0.1mg/ml, the 0.01mg/ of variable concentrations
Ml, 0.005mg/ml, 0.001mg/ml, 0.0005mg/ml, 0.0001mg/ml), use Germany Perkin Elmer Lambda
25 ultraviolet spectrophotometers measure its ultra-violet absorption spectrum between 200～300nm.And it is bent to draw standard according to its absworption peak
Line, the linear fit equation of calculated curve and degree of fitting.
High speed centrifuge is used to use high speed centrifuge to be centrifuged the particle suspension of the Islets finally synthesized element,
11000rpm/min, 10 DEG C, 30min.Take supernatant vacuum hang inspissation contracting after, application ultraviolet spectrophotometer measure its insulin inhale
Receive spectrum, and according to its conversion insulin content of light absorption value at the wavelength drawing standard curve.
The envelop rate of insulin calculates according to below equation with carrying drug ratio:
Result is as follows: insulin ultra-violet absorption spectrum in dilute HCl solution shows, is respectively provided with at 205nm with 275nm
Absworption peak, and it is positioned at absworption peak at 205nm higher than at 275nm more than 10 times, insulin absworption peak at 205nm is suitableeer
It is suitable for the pattern detection of low concentration.From 10-4In the range of mg/ml to 1mg/ml, insulin ultraviolet absorption value at 205nm in
Semi-parabolic type.The sample insulin concentration related to due to this research is relatively low, chooses 10-4Mg/ml and 10-2Purple between mg/ml
Outer absorption value draws standard curve, it is seen that it has linear relationship, and equation of linear regression is y=18.641x+0.0019, its phase
Close sex index R2=0.9967 (Fig. 4).
Through the compound experiment of three times, statistical data, the envelop rate of LBL4 is 55.21 ± 4%, and medicine carrying efficiency is 60.73
± 6%.
(6) drug release rate measures
The PBS of configuration 0.1M HCl and pH=6.8, is separately added into 200 μ l particle solutions in 10ml buffer,
Ice bath, is placed on shaking table, rocks gently with 50r/min.Respectively at 0.5h, 1h, 2h, 3h, 4h sample 700 μ l, centrifugal, take supernatant
Detect for insulin content, i.e. use ultraviolet spectrophotometer, measure its absorbance, and the conversion of establishing criteria calibration curve.
Each delivery systme is repeated 3 times, and collects data for statistical computation.
As depicted in figures 5 and 6, the result of extracorporeal releasing experiment shows, LBL4 system has in the hydrochloric acid solution of pH=1
Certain slow releasing function, between 0.5h to 5h, its burst size there is no significant difference.In the PBS of pH=6.8, LBL4 body
System shows to dash forward releases pattern, when 1h, reaches to discharge maximum.Relatively equivalent LBL4 release in two kinds of systems can be sent out
Existing, system has certain pH Irritability, and this character transports the demand of system and matches, i.e. at gastric environment with oral insulin
In not uelralante, thus get up to protect insulin not by pepsic destruction, discharge in intestinal environment, beneficially islets of langerhans
Element is absorbed by enteric epithelium.Speculating that the source of this pH sensing character is likely due at low ph conditions, material charging property is relatively strong,
Having stronger captivation between material can make the particle by electrostatic interaction self assembly more stable.And under conditions of neutral ph,
Active force between particle layer Rotating fields is more weak, structure loosely, so uelralante faster.
LBL4 particle is hatched the insulin releasing situation of 2h in different pH systems and is shown, interval at pH value 3-5, LBL4 body
The speed being uelralante is the slowest, and its burst size is relatively low has statistical significance (p < 0.05).And in pH value is 2 or close
During property, the rate of release of its insulin is very fast.After feed, stomach pH is about about 3, and therefore, this insulin transports system
The effect taken the most afterwards is better than effect on an empty stomach.
Embodiment 2 cell experiment
1, experimental technique
(1) cell is cultivated
Caco-2 cell line is provided by Guangdong Pharmaceutical University.Cell is after in culture bottle, enrichment culture is to 80%, with 5
The density in 000/ hole is seeded on 96 orifice plates, cultivates 1,2 days, to carry out subsequent experimental.Its cell culture condition is: new containing 20%
Raw Ox blood serum, the DMEM in high glucose culture medium of non essential amino acid, 37 DEG C, 5.0%CO2。
(2) system toxicity is transported
The system that transports taking various dose respectively adds in the Caco-2 cell cultivated, and after 24h, uses mtt assay to survey
Amount cell survival rate.I.e. inoculation Caco-2 cell on 96 well culture plates, 5 000/hole, after cultivating 24h, sucking-off culture medium,
Add serum-free medium, and the insulin of variable concentrations transports system, after cultivating 24h, add the MTT solution of 5mg/ml
20ul/ hole.Continue to cultivate 4h, sucking-off culture medium, add DMSO and dissolve the first a ceremonial jade-ladle, used in libation of intracellular formation.Low-speed oscillation on shaking table
10min, in microplate reader reading, wavelength 510nm.
2, experimental result
Insulin transports system and is assessed by mtt assay in the bio-toxicity of cell aspect.Result is as shown in Figure 7.
The LBL4 adding variable concentrations in the serum-free medium of Caco-2 cell transports system (0 μ g/ml, 125 μ g/
Ml, 250 μ g/ml, 500 μ g/ml, 1000 μ g/ml), result display cell viability is also not affected by the biggest impact, and cell Rate activity is all
It is maintained at more than 90%.Wherein, 500 μ g/ml, 1000 μ g/ml medicine to cell cultivate for, concentration is the most relatively large.Right
Medicine within toxic medicament, 100 μ g/ml i.e. can reach the cell lethality of 50%.And LBL4 system reaches at upper dose
During 1000 μ g/ml, cell viability level is still about 100%, it can be seen that, LBL4 has extremely low cytotoxicity, bio-compatible
Property is the highest.
The Study of cytotoxicity of embodiment 3 experiment in vivo system
1, type i diabetes mouse model builds
(1) method
Buy 6 week old SD rats, adapt to environment one week.Taking 3 is matched group, and remaining is experimental group.Rat Fast one night,
Measuring blood glucose, use the citrate buffer solution ice bath lucifuge before the injection preparation STZ solution of pH=4.4, concentration is 15mg/ml.
Experimental group rat is carried out lumbar injection by the dosage of 50mg/kg.
Self-modeling rose the same day, measured rat body weight, feed and amount of drinking water every day.Within 3rd day, the 7th day, use Roche vigor type
Blood glucose meter monitoring blood glucose.The rat relatively low to blood glucose, supplements injection STZ (30mg/kg).
After one week, (after hungry 3h), blood sugar concentration reaches more than 13mmol/L on an empty stomach, is classified as the ill Mus of diabetes.
Type i diabetes becomes mould rat be grouped by fasting glucose average, keeps often organizing the mean blood glucose of rat without significance difference
Different.
(2) result
As shown in Figure 8, using male SD rat, fasting 16 hours, lumbar injection STZ, dosage is 50mg/kg, after one week
Detection blood glucose, the fasting blood sugar of the continuous 4 days SD rat more than 13mmol/L is considered as being modeled as the type i diabetes rat of merit.
Injection medicine is after 12 days, and the mould rat that becomes of STZ injection group has obvious difference with the build of control rats.
From the point of view of glucose monitor data (Fig. 9 A), after injection STZ the 3rd day, after Rat Fast one night, the blood of STZ injection group
Sugar has been significantly higher than matched group the most.In experimental group, having 1 rat blood sugar value is 12.6mmol/L, remaining STZ injection group rat serum
Sugar value is all at more than 20mmol/L, and the 6th, 9 days, trend did not changed.It can be assumed that except blood glucose value is that this of 12.6mmol/L is big
Outside Mus, all model successfully, be modeled as power and reach 92%.
After first 1 day of STZ of injection to injection STZ, the rat body weight of the 9th day, drinking-water, the Monitoring Data of food-intake show (figure
9B, Figure 10), STZ injection group without statistical discrepancy, is noted in the injection STZ data of first 1 day with the body weight of matched group, drinking-water, food-intake
Penetrate first 1 day and be also completely superposed to the variation tendency injecting the same day.Rising the same day to injection STZ, it is poor that body weight, drinking-water, food-intake occur
Different, i.e. matched group body weight constantly increases, and drinking-water and food-intake are without notable change；STZ injection group body weight is without growth trend, amount of drinking water
Increase sharply about 7 times to injection, food-intake increase about 50%.
2, Immunohistochemical Method observes the intestinal villi absorption to system
(1) method
Taking 3 SD rats suffering from type 1 diabetes, before experiment, at fasting one night, can't help water.Taking 1 is matched group, gavage 300 μ
L normal saline, taking 2 is experimental group, gavage LBL4 aqueous solution 300 μ l.By matched group after gavage 2 hours, experimental group was respectively
Within 1 hour, 2 hours after gavage, use pentobarbital sodium (0.05mg/kg) anesthesia, after abdominal part is exposed, cut 5cm ileum and 5cm
Colon, uses normal saline to clean respectively.After cutting intestinal segment, rat put to death by injection excess anaesthetic.
(2) result
Figure 11 is that the type i diabetes rat at fasting one night fills and feeds the duodenum of 1h and 2h and exempting from of ileum after LBL4 system
Epidemic disease group result.
Owing to experiment uses the hungry type i diabetes rat at fasting one night, the medicine of gavage is that insulin transports body
Being the aqueous solution of LBL4, medicine can pass through stomach faster and enter 12 fat intestinal.From the point of view of the coloration result of matched group, research
Without the insulin of self secretion in the type i diabetes Mus intestinal villus applied.From the point of view of the coloration result of Duodenal villi,
Fill after feeding 1h, with regard to visible obvious absorption of insulin phenomenon in duodenal fine hair, visible a small amount of suction in the fine hair of ileum
Receive.Visible LBL4 just can be rapidly absorbed at duodenal segment.Fill after feeding 2h, the intravillous islets of langerhans of duodenum, ileum
Element is the most clearly visible.
3, Composition analyzed type i diabetes rat limosis administering effect in medicine body
(1) method
Type i diabetes rat is run out of grain 12h, keeps drinking-water unobstructed.The nanometer being respectively provided with blank group and the present invention carries
Powder subgroup 2 groups, often group type i diabetes rat is no less than 3, takes normal saline and LBL4 system (oral insulin respectively
Amount is 50IU/kg), in 0h, 1h, 2h, 3h, 5h, 7h, 9h, 17h, 22h survey blood glucose.Give food during 17h, measure returning of blood glucose
The situation of liter.
(2) result is as shown in Figure 12.
Diabetic model rats is after oral LBL4 (50IU/kg) system, and from 1h, becoming occurs significantly declining in blood glucose
Gesture, touches the bottom in 3h, close to the 40% of experiment initial stage blood sugar level.After reaching blood glucose minimum point, glucostasis is in relatively low
Level, matched group blood glucose engenders downward trend.During 17h, experimental group is little with matched group difference.After feeding, experimental group with
Matched group blood glucose synchronizes.
It addition, compared with the research that other oral insulin transports system, effect aspect in vivo, this research synthesis
LBL4 has the most rapid-action, effect outstanding feature.1h after taking medicine, is significantly reduced to initial blood glucose value by blood glucose
40%, compared with matched group now, the blood sugar level of LBL4 group is the 50% of matched group.This result absorbs with intestinal villus
Result is consistent, all indicates infiltration rate this feature fast, rapid-action of LBL4 particle.
In sum, successful Application insulin nano ball of the present invention, polygalacturonic acid, chitosan successfully synthesize layer by layer
The oral insulin of self assembly transports system, and self-assembling nanoparticles heat stability is higher than free insulin.Wrap up 4 layer materials
It is positively charged that LBL system has more independent structure, less particle diameter (less than 200nm), surface.Envelop rate is 55.21 ±
4%, medicine carrying efficiency is 60.73 ± 6%, has higher Drug loading capacity.Extracorporeal releasing experiment shows, at the hydrochloric acid of pH=1.1
In solution, LBL system has obvious controlled-release function, and in the PBS of pH=6.8, LBL diagram of system reveals to dash forward releases mould
Formula, it is believed that LBL system has pH response characteristic, under the conditions of insulin releasing speed at low ph conditions is slightly slower than high pH
Rate of release.In vivo it was found that, this transports the control of hyperglycemia that system can be notable and long-acting, after the 1h that takes medicine i.e.
Occur that blood glucose declines, touch the bottom in 3h, in 10h, possess the ability of regulating and controlling blood sugar.
Claims (10)
1. the oral insulin of a LBL self-assembly structure transports the preparation method of system, it is characterised in that first at low pH
Under the conditions of, use salting out method to separate out positively charged insulin nano ball, according to the electrical of material and electrostatic force, application layer
Layer self-assembly method uses electronegative polygalacturonic acid and electropositive chitosan Islets element nanosphere successively respectively.
Preparation method the most according to claim 1, it is characterised in that comprise the steps:
S1. under 10～20 DEG C of environment, insulin is dissolved in dilute hydrochloric acid solution, adds NaCl and be allowed to final concentration and reach
0.5～0.7M, solution stirring 0.4～0.6h, so centrifugal that to precipitate；
S2. the precipitation of step S1 adds polygalacturonic acid solution, is stirred for 0.4～0.6h, ultrasonic 0.8～1.5min, continues
Stirring 0.4～0.6h, so centrifugal that to precipitate；
S3. the precipitation of step S2 adds chitosan solution, stirs 0.4～0.6h, is centrifuged to obtain precipitation；
The most repeatedly it is repeated in step S2 and step S3, is i.e. alternately added polygalacturonic acid solution and chitosan solution is carried out
Parcel, the oral insulin of the LBL self-assembly structure obtaining the different number of plies transports system.
Preparation method the most according to claim 2, it is characterised in that step S1 insulin and the mass body of dilute hydrochloric acid solution
Long-pending ratio is 4～6mg/ml.
Preparation method the most according to claim 2, it is characterised in that the pH of dilute hydrochloric acid solution described in step S1 be 1.0～
1.4。
Preparation method the most according to claim 2, it is characterised in that described in step S2, the pH of polygalacturonic acid solution is
3～5.
Preparation method the most according to claim 2, it is characterised in that the preparation method of step S2 polygalacturonic acid solution
For: being dissolved in dilute hydrochloric acid solution by polygalacturonic acid, add NaCl, make NaCl final concentration reach 0.5～0.7M, pH is
3～5.
Preparation method the most according to claim 2, it is characterised in that the preparation method of chitosan solution described in step S3
For: dissolving the chitosan in dilute hydrochloric acid solution, add NaCl solid, make NaCl final concentration reach 2～4M, pH is 3～5.
Preparation method the most according to claim 2, it is characterised in that precipitation and polygalacturonic acid solution described in step S2
Mass volume ratio be 0.8～1.2g:100ml；Described in step S3, the mass volume ratio of precipitation and chitosan solution is 4～6g:
100ml。
9. the oral insulin of the LBL self-assembly structure prepared according to the arbitrary described method of claim 1～8 transports body
System.
10. the oral insulin of LBL self-assembly structure described in claim 9 transports system and is preparing oral insulin medicament side
The application in face.
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Cited By (3)
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| CN107296960A (en) * | 2017-05-25 | 2017-10-27 | 华南师范大学 | A kind of oleanolic acid nano oral system for having inhibitory action to insulin resistance |
| CN110522921A (en) * | 2018-05-24 | 2019-12-03 | 广东省医疗器械研究所 | A kind of insulin with insulin capturing function transports the synthetic method of system |
| CN118197634A (en) * | 2024-05-16 | 2024-06-14 | 简阳市人民医院 | Personnel health management method and system based on blood glucose monitoring |
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| CN105078890A (en) * | 2015-08-18 | 2015-11-25 | 江南大学 | Method for preparing multi-layer bio-based vesica capable of releasing insulin |
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| CN105078890A (en) * | 2015-08-18 | 2015-11-25 | 江南大学 | Method for preparing multi-layer bio-based vesica capable of releasing insulin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107296960A (en) * | 2017-05-25 | 2017-10-27 | 华南师范大学 | A kind of oleanolic acid nano oral system for having inhibitory action to insulin resistance |
| CN107296960B (en) * | 2017-05-25 | 2020-09-15 | 华南师范大学 | Oleanolic acid nano oral system with inhibition effect on insulin resistance |
| CN110522921A (en) * | 2018-05-24 | 2019-12-03 | 广东省医疗器械研究所 | A kind of insulin with insulin capturing function transports the synthetic method of system |
| CN118197634A (en) * | 2024-05-16 | 2024-06-14 | 简阳市人民医院 | Personnel health management method and system based on blood glucose monitoring |
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