CN106310230B - A kind of oral insulin of LBL self-assembly structure transports the preparation method and application of system - Google Patents
A kind of oral insulin of LBL self-assembly structure transports the preparation method and application of system Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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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
- A61K9/5073—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 having two or more different coatings optionally including drug-containing subcoatings
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Abstract
The invention discloses the preparation methods that a kind of oral insulin of LBL self-assembly structure transports system, first at low ph conditions, positively charged insulin nano ball is precipitated using salting out method, according to the electrical property and electrostatic force of material, electronegative polygalacturonic acid (PGLA) and electropositive chitosan (CS) successively Islets element nanosphere are used respectively using self-assembly method layer by layer.Oral insulin of the invention, which transports system, has the advantages that natural, low toxicity, can natural metabolism degradation, good biocompatibility, the system partial size is small, with stronger small intestine absorptivity, and carrying drug ratio is high, stability is good, performance insulin hypoglycemic effect that can be quick and potent, can also it is significant, quickly, control blood glucose level in a long time, have the potentiality applied to treating diabetes.
Description
Technical field
The invention belongs to biological medicine field of material technology.More particularly, to a kind of the oral of LBL self-assembly structure
Insulin transports the preparation method and application of system.
Background technique
The number that diabetes are suffered from the whole world is about 2.8 hundred million, wherein 5~10% be type-1 diabetes mellitus, about 80~90% be II type
Diabetes.Type-1 diabetes mellitus is also known as adolescent diabetes, and 50% patient fell ill at 0~4 years old.Type-1 diabetes mellitus is by siberian crabapple
Caused by system disorder keeps islet B cell dead, islet B cell has lost 70%-90% when patient's first attack, and after the onset 1~2
Year, islet B cell was all dead, and patient's amount of insulin secretion is zero.Type-2 diabetes mellitus was generally fallen ill at 35~40 years old.Some trouble
Insulin even generates excessive in person's body, but the function and effect of insulin are poor, therefore the insulin of patient's body is a kind of phase
To shortage.Insulin is the necessary therapeutic agent of type-1 diabetes mellitus and type-2 diabetes mellitus people in illness advanced stage necessary medicine
Object.
Although insulin preparation is constantly updated in recent years, there is the different types of insulin preparation such as long-acting, short-acting for patient
Selection, still, injection are still by the mode of insulin input patient's body.On the one hand, insulin injection can cause patient
The discomforts such as fear, pain, research shows that some Type II diabetic can use the time due to this uncomfortable postponement insulin,
Long term injections can also cause skin problem, increase Infection probability；On the other hand, the circulation style of insulin injection in vivo also with
Naturally secret it is different, compare for, insulin injection can avoid the first pass effect of liver, relative increase peripheral blood vessel
Insulin concentration is comparatively fast gathered in kidney and is discharged.Therefore, the research that transports for developing the non-intruding approach of insulin becomes in recent years
Carry out the research emphasis in this field.Approach is transported compared to a variety of Noninvasive insulin, transporting insulin by oral route has
One big advantage, i.e., the oral insulin transported recycle approach in vivo and secrete most like, the oral insulin transported with itself
Stay longer can be assembled in liver, then be discharged through kidney, in vivo the concentration distribution at each position and circulation path and nature
The insulin of secretion is the most similar, this is all extremely important for avoiding or reducing complication, increase insulin medication effect.This
Outside, oral insulin transports system also and has many advantages, such as and is convenient to take, is easily accepted by patients, therefore, the oral of insulin transports
System is that current insulin transports one of research hotspot.It is also that the para-insulin into clinical application most potential in recent years is defeated
Fortune mode.
Currently, the research that oral insulin transports, which focuses primarily upon, increases transport efficiency this aspect.It can be ground this kind of
Study carefully and be divided into three classes: preventing digestive ferment degradation, promote intestinal mucosa attachment, promote to penetrate enteric epithelium.Wherein, particle is promoted to pass through on intestines
The approach of chrotoplast is that many researchs are endeavoured the problem of capturing, and current achievement is broadly divided into four classes, i.e., is entered by M cell and be
System circulation, targeting enter system circulation by Goblet cell, and it is thin to enter enterocyte opening enteric epithelium by encytosis
The close connection of intercellular makes particle pass through space between cells and enters system circulation.Some researchs combine comprehensively to be promoted to promote insulin
The mode of pick-up rate, the insulin transport system used not only promotes mucoadhesive, but also transporting insulin using nano material can
With by M cell, while being also equipped with the close connection between opening enterocyte or promoting encytosis.Existing insulin mouth
Admit defeat and transports system common materials mainly and have chitosan (chitosan), alginate (alginate), glucan (dextran) etc.
Natural material also has PLGA, PEG etc. to authenticate the inorganic material such as nontoxic artificial synthesized polymer or silicon by U.S. FDA.
In addition, chitosan (chitosan), PEG, certain targeting factors also all can serve as the surface modification of system, it is small to play increase
The effect of intestinal absorption rate.Wherein chitosan (chitosan) has tight between electropositive, intestinal mucosa adhesion, openable small intestine epithelium
The effect of close connection has all obtained clearly research and has verified repeatedly.
Existing drug oral transport architectural study be usually taken selection natural material preparation transport carrier reach low toxicity or
Nontoxic purpose.However, being a little by what existing research was ignored, natural material also has latent other than having less toxic property
Medical functions, that is, natural material has the potentiality for the dual function for being provided simultaneously with conveying material and therapeutic agent.Indian is early
Diabetes are just divided into two class as caused by heredity and as caused by feeding desorder before B.C. 200 years.For centuries,
There is the experience using plant treatment diabetes in each state.Ethnobotany research is research shows that at least 800 kinds of plants are with anti-
The potentiality of diabetes.In recent decades, as the research of chemical purification technique and bio-science field continues to develop, medicinal plant
In effective component be accurately separated purification, also there are several hundred kinds of hypoglycemic active substance of plant and its chemical structure to obtain really
Fixed, part of hypoglycemic ability of active material is also set forth with it in the mechanism of action of cell level.Therefore, carry out from plant
The preparation of Nantural non-toxic material is chosen in active substances and transports carrier, while making insulin transport system itself there is hypoglycemic function
The research of energy, has great importance.
Summary of the invention
The technical problem to be solved by the present invention is to have control blood glucose and electronegative natural poly- by exploring application
It closes object polygalacturonic acid and replaces assembling structure layer by layer with positively charged chitosan, and is steady to the size of this structure, shape, structure
Qualitative, drug release rate, insulin transport efficiency, cytotoxicity, body absorption and drug effect etc. are studied.Gained is layer by layer
The oral insulin of self-assembled structures transports system using polygalacturonic acid as basic material, and synthesis process is without organic matter and toxic
Substance has the potentiality applied to treating diabetes.
The object of the present invention is to provide the preparation methods that a kind of oral insulin of LBL self-assembly structure transports system.
The oral insulin that another object of the present invention is to provide the LBL self-assembly structure transports the application of system.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method that the oral insulin of LBL self-assembly structure transports system makes first at low ph conditions
Positively charged insulin nano ball is precipitated with salting out method, according to the electrical property and electrostatic force of material, using LBL self-assembly
Method uses electronegative polygalacturonic acid (PGLA) and electropositive chitosan (CS) successively Islets element nanosphere respectively.
The present invention screens plant origin, the polymer with hypoglycemic activity first, obtains having the function of glycemic control, band
The polygalacturonic acid (PGLA) of negative electricity is combined with positively charged natural copolymer chitosan (CS), prepares LBL self-assembly
The oral insulin of structure transports system (LBL).The system has natural, low toxicity, can natural metabolism degradation, good biocompatibility
The advantages of, the system partial size is small, has stronger small intestine absorptivity, and carrying drug ratio is high, stability is good, can be quick and potent
Performance insulin hypoglycemic effect, can also it is significant, quickly, control blood glucose level in a long time, have applied to glycosuria
The potentiality of disease treatment.
Specifically, as preferred implementable solution, the oral insulin of above-mentioned LBL self-assembly structure transports system
Preparation method includes the following steps:
S1. electropositive insulin nano ball is prepared
Under 10~20 DEG C of environment, insulin is dissolved in dilute hydrochloric acid solution, NaCl is added and is allowed to final concentration and reach
0.5~0.7M, solution stir 0.4~0.6h, are centrifuged to obtain precipitating；
S2. electronegative polygalacturonic acid (PGLA) package
Polygalacturonic acid (PGLA) solution is added in the precipitating of step S1, is stirred for 0.4~0.6h, and ultrasound 0.8~
1.5min continues 0.4~0.6h of stirring, is centrifuged to obtain precipitating；
S3. electropositive chitosan (CS) package
Chitosan (CS) solution is added in the precipitating of step S2, stirs 0.4~0.6h, is centrifuged to obtain precipitating；
S4. it is repeated in step S2 and step S3 repeatedly, that is, is alternately added PGLA solution and CS solution is wrapped up, obtain
The oral insulin of different layers of auto polymerization nanospheres, i.e., different layers of LBL self-assembly structures transports system.
Wherein it is preferred to which the mass volume ratio of step S1 insulin and dilute hydrochloric acid solution is 4~6mg/ml.
It is highly preferred that the mass volume ratio of step S1 insulin and dilute hydrochloric acid solution is 5mg/ml.
Preferably, environment 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, the time of stirring described in step S1 is 0.5h.
Preferably, centrifugation described in step S1 is that 4000~6000rpm is centrifuged 2~4min.
It is highly preferred that centrifugation described in step S1 is 5000rpm centrifugation 3min.
Preferably, the pH of polygalacturonic acid (PGLA) solution described in step S2 is 3~5.
It is highly preferred that the pH of polygalacturonic acid described in step S2 (PGLA) solution is 4.
Preferably, step S2 polygalacturonic acid (PGLA) solution the preparation method comprises the following steps: by polygalacturonic acid (PGLA)
It is dissolved in dilute hydrochloric acid solution, adds NaCl, NaCl final concentration is made to reach 0.5~0.7M, pH is 3~5.
Wherein it is preferred to which the mass volume ratio of the polygalacturonic acid (PGLA) and dilute hydrochloric acid solution is 4~6mg/
ml。
Preferably, the mass volume ratio of the polygalacturonic acid (PGLA) and dilute hydrochloric acid solution is 5mg/ml.
Preferably, the final concentration of 0.6M of NaCl in polygalacturonic acid (PGLA) solution.
Preferably, the pH of polygalacturonic acid (PGLA) solution is 4.
Furthermore it is preferred that the time of stirring described in step S2 is 0.5h.
Preferably, the time ultrasonic described in step S2 is 1min.
Preferably, the time for continuing stirring described in step S2 is 0.5h.
Preferably, centrifugation described in step S2 is that 4000~6000rpm is centrifuged 2~4min.
It is highly preferred that centrifugation described in step S2 is 5000rpm centrifugation 3min.
Preferably, chitosan (CS) solution described in step S3 the preparation method comprises the following steps: chitosan (CS) is dissolved in dilute hydrochloric acid
In solution, NaCl solid is added, NaCl final concentration is made to reach 2~4M, pH is 3~5.
Wherein it is preferred to which the mass volume ratio of the chitosan (CS) and dilute hydrochloric acid solution is 4~6mg/ml.
It is highly preferred that the mass volume ratio of the chitosan (CS) and dilute hydrochloric acid solution is 5mg/ml.
Preferably, the final concentration of 3M of NaCl in chitosan (CS) solution.
Preferably, the pH of chitosan (CS) solution is 4.
Preferably, the time of stirring described in step S3 is 0.5h.
Preferably, centrifugation described in step S3 is that 4000~6000rpm is centrifuged 2~4min.
It is highly preferred that centrifugation described in step S3 is 5000rpm centrifugation 3min.
Furthermore it is preferred that described in step S2 precipitating and polygalacturonic acid (PGLA) solution mass volume ratio be 0.8~
1.2g:100ml.
It is highly preferred that the mass volume ratio of precipitating and polygalacturonic acid (PGLA) solution described in step S2 is 1g:
100ml。
Preferably, the mass volume ratio of precipitating and chitosan (CS) solution described in step S3 is 4~6g:100ml.
It is highly preferred that the mass volume ratio of precipitating and chitosan (CS) solution described in step S3 is 5g:100ml.
In addition, the oral insulin of LBL self-assembly structure prepared by the above method transports system, and its making
Application in terms of standby oral insulin medicament, also all within protection scope of the present invention.
LBL self-assembly structure is the nano particle that positive and negative charge attraction is assembled between a kind of application material, Ke Yitong
The assembling number of plies and size of control reaction times control nano particle are crossed, size is opposite compared with the nano particle of other structures
It is smaller.So, it is believed that LBL self-assembly structure is to study small size to transport the research that system progress drug oral transports
The resulting structure of effect.Simultaneously as its structure is maintained by the charge attraction power between material, usual pH value is to material
Stability have stronger influence, this can make this structure have pH irritability to the release of drug.
The present invention makes insulin solutions that insulin nano ball be precipitated first under low pH, environment with high salt；Then application is poly-
Galacturonic acid and the negatively charged material with positive electricity of two kinds of difference of chitosan, Islets element nanosphere, forms 4 layers of LBL layer by layer
Structure.The system has nanoscale partial size, spherical shape, and positively charged, intestines surface-clinging ability with higher.
The present invention transports the selection of system and design ensure that from source its with extremely low toxicity, this transport system be by
Prepared by the main component polygalacturonic acid of plant cell wall, polygalacturonic acid has the function for slowing down intestinal absorption glucose
Can, this makes system, and not only toxicity is low, but also in the application for the treatment of diabetes, can make to transport system inherently has to disease
Therapeutic effect.This is the primary innovation in drug delivery field, i.e., using substance preparation natural, with treatment function
Drug delivery system makes medicine-carried conveying system reach therapeutic effect from many aspects, has multiple disease therapeuticing effect.Meanwhile
During synthetic system, organic solvent or toxicant are not applied, are avoided system and are generated toxicity.
The present invention in the characterization research for the system that transports, Ramon's spectrum statistics indicate that system successfully synthesizes, TEM image with
Dynamic scattering analysis all shows that particle has lesser partial size, this will help particle to pass through enteric epithelium by M cell, into machine
Body blood circulation.Meanwhile particle is positively charged, carried charge and relatively large (25mV), electropositive particle easily with it is electronegative
Mucous membrane of small intestine mutually attracts attachment, it is promoted to be 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 studies.In addition, thermogravimetric analysis shows
Material significantly reduces the rhermal decomposition rate of insulin to the package of insulin in LBL system.
The present invention is to the release in vitro for the system that transports studies have shown that system insulin releasing speed in the environment of pH=3-5
Rate is relatively slow, relatively fast in the intestines environment insulin releasing rate of neutral pH, this property also makes LBL transport system to have
Bigger application potential.The low ph conditions of stomach and the degradation of pepsin are in the design of oral protein drug and practice process
One important problem, building pH incude system, slow down insulin releasing in gastric environment, increase insulin in intestines environment
Release is to solve one of the approach of this problem.The LBL4 system that the present invention constructs is shown in 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 low release section of the insulin of LBL4 with into
The pH value of stomach is consistent after food, can speculate that it may be to promote a way of drug pick-up rate that LBL4 system is taken orally after feed
Diameter.
The present invention also establishes type-1 diabetes mellitus rat model using the SD rat that STZ is induced, and separately verifies it and ligatures intestinal segment pair
LBL4 transports the amount of insulin of middle absorption and oral insulin transports the blood glucose dynamic after system.Wherein, ImmunohistochemistryResults Results are aobvious
Show that 1h after taking LBL4 system, duodenal segment just have significant absorption of insulin.This absorption rate is for oral drugs
For, it is relatively fast.It matches with this result, taking orally LBL4 has quickly hypoglycemic effect, the 1h after oral, blood glucose
It is down to the 40% of initial blood glucose value.With it is similar research compared with, the vivo efficacy of LBL4 system show faster drug absorption,
Faster drug effect time and hypoglycemic effect, this is a big advantage of LBL4 system.
It is successfully synthesized in conclusion the LBL that the present invention synthesizes transports system, is the sphere of Nano Particle, surface band is just
Electricity has high thermal stability and pH irritability；In vivo in research, faster intestinal absorption rate, faster hypoglycemic are shown
Reaction and more significant hypoglycemic effect have the value of further research and practical application.
The invention has the following advantages:
The present invention is successfully prepared a kind of using the substance polygalacturonic acid of plant origin as the insulin mouth of basis material
Admit defeat and transport system LBL4, refers to the nanometer system for alternately wrapping up 4 strata galacturonic acids and chitosan outside Nanometer Insulin, body
Positive and negative electrical attraction power of system's synthesis according to storeroom, makes material self assembles become nanometer and transports system, transports pancreas for oral
Island element.
Polygalacturonic acid, chitosan used in the present invention are all natural polymer, have this system natural, low
Poison, can natural metabolism degradation the advantages of.And there is no the participation of organic solvent and noxious material in synthesis process, used material
It is natural polymer, ensure that the biocompatibility of system.
The system partial size is small, has and increases the probability that is absorbed by enteric epithelium, the positively charged promotion particle in surface with it is negatively charged
Enteric epithelium mutually adsorb, enhancing characteristics, these properties such as particle stability can help LBL system to enter machine from oral route
The body blood circulation system can pass through small intestine epithelium and enter blood circulation, and it is small (being less than 200nm) to transport system diameter, absorptivity
It is higher.
In addition, the system can have higher carrying drug ratio, have stronger small intestine absorptivity, and in vivo in research, energy
Enough quick and potent performance insulin hypoglycemic effects, can also it is significant, quickly, control blood glucose level in a long time, have
The standby potentiality for being applied to treating diabetes.
Detailed description of the invention
Fig. 1 is that LBL particle synthesizes schematic diagram.
Fig. 2 is the characterization 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: the potential value of Nanometer Insulin and LBL4, a are the current potential of Nanometer Insulin, and b is 4 layers of LBL system
Current potential.C:TEM appearance is successively the LBL system of 1,2,3,4 layer material of package from left to right, and black scale represents in figure
100nm。
Fig. 3 is thermogravimetric analysis；Grey filled lines indicate insulin with the raised quality change situation of temperature, and solid black lines indicate
4 particle of LBL is with the raised quality change situation of temperature.
Fig. 4 is insulin UV Absorption standard curve.
Fig. 5 is insulin releasing curve of the LBL4 particle in simulation stomach, intestinal buffer liquid.
Fig. 6 is that LBL4 is incubated for the insulin release after 2h in different pH buffer systems.
Fig. 7 is that cell viability is influenced by system concentration is transported.
Fig. 8 is the foundation of type-1 diabetes mellitus model and models mouse compared with the figure of normal mice.
Blood glucose and Avoirdupois monitoring result during Fig. 9 is the modeling of SD rat；Rectangular in A figure is control group, and other shapes are to build
Mould group；Black line is Experimental modeling group in B figure, and grey lines are control group.
Daily drink and food-intake monitoring result during Figure 10 is the modeling of SD rat；Black line is Experimental modeling group, ash in figure
Colo(u)r streak is control group.
The case where Figure 11 is type-1 diabetes mellitus feeding rats LBL4 system 1h, 2h metaduodenum and ileal absorption insulin；
Black scale represents 100 μm in figure.
Figure 12 is that type-1 diabetes mellitus rat limosis takes orally blood glucose dynamic after LBL4.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
Experimental material used in following embodiment is as follows:
(1) experiment reagent:
Polygalacturonic acid (PGLA), chitosan, Pluronic 188, bovine insulin are purchased from Sigma Corporation, STZ chain urine
Rhzomorph is helped, Aladdin biochemical technology Co., Ltd is purchased from.Pancreatin, DMEM in high glucose culture medium, nonessential amino acid are Gibco public affairs
Take charge of product；Newborn calf serum is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd.；96/24 hole polystyrene cell culture
Plate is U.S. Corning Corning Incorporated product.
(2) laboratory apparatus:
Sigma32184 high speed freezing centrifuge, Japanese 7650 transmission electron microscope of HITACHI, Jintan City, Jiangsu Province
Medical Instruments factory 78-1 magnetic stirring apparatus, Japanese Nikon microscope, Britain MalvernZEN3600, Japanese Olympus optics fall
Microscope, French H.J.Y company LabRAM Aramis micro-Raman spectroscopy are set, Switzerland Mettler, TGA/DSC 1 synchronizes heat
Analyzer.
(3) experimental cell: Human colon adenocarcinoma cell line Caco-2 is obtained in pharmaceutical university, Guangdong Province.
(4) experimental animal: SD rat is purchased from Nanfang Medical Univ's Experimental Animal Center.
The oral insulin of 1 LBL self-assembly structure of embodiment transports the preparation and representation of system (LBL)
1, the preparation of LBL particle
The synthetic method of LBL particle is as shown in Fig. 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, and solution stirs 0.5h, and 5000rpm is centrifuged 3min, must precipitate；
(2) polygalacturonic acid (PGLA) solution of pH=4, NaCl 0.6M is added in the precipitating of step (1), is stirred for
0.5h, ultrasonic 1min continue to stir 0.5h, and 5000rpm is centrifuged 3min, must precipitate；
Polygalacturonic acid (PGLA) solution of the pH=4, NaCl 0.6M the preparation method comprises the following steps: by polygalacturonic
Sour (PGLA) is dissolved in 1ml dilute hydrochloric acid solution, adds NaCl solid, NaCl final concentration is made to reach 0.6M, pH=4；
(3) precipitating for obtaining step (2) is added in chitosan (CS) solution of pH=4, NaCl 3M, stirs 0.5h, from
The heart must precipitate；
Chitosan (CS) solution of the pH=4, NaCl 3M the preparation method comprises the following steps: that chitosan (CS) is dissolved in 1ml is dilute
In hydrochloric acid solution, NaCl solid is added, NaCl final concentration is made to reach 3M, pH=4；
(4) it is repeated in step (2) and step (3) repeatedly, is alternately added PGLA solution and chitosan solution is wrapped up,
Different layers of auto polymerization nanospheres are obtained, i.e., the oral insulin of different layers of LBL self-assembly structures transports system.
2, the characterization of LBL particle
(1) Raman spectrum detects
The LBL system of the raw material PGLA of system synthesis, chitosan, insulin and 1 layer, 4 layers of package is completely dry
It is dry, it takes appropriate sample to be placed in the groove of sample loading plate, is suppressed using coverslip, make sample consolidation and there is flat surface, use
Raman spectrometer detection.Raman spectrum can detect the ingredient of particle to a certain extent.
As the result is shown: insulin is in 639cm-1, 853cm-1, 3313cm-1- SH ,-COOH ,-NH are shown at three respectively3Three
The vibration of a key.These three mark peaks also all come across LBL1, and in the Ramon's spectrum of LBL4, can be confirmed has pancreas islet in final product
The presence of element.There is-OH (429cm abundant in PGLA and chitosan-1, 421cm-1), in the wave spectrum of LBL1 and LBL4, also have-
Appearance (the 429cm at the peak OH-1, 446cm-1), this illustrates to be initially believed that grain containing polysaccharide and insulin in LBL1 and LBL4
Son successfully synthesizes (Fig. 2A).
(2) Zeta potential detects
Respectively after each round synthesis, detection of the 100 μ l for current potential is taken.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, 180s is at least measured.Instrument is multiple by automatic measurement, and result is obtained hydrodynamics partial size according to correlation analysis.
In Zeta potential measurement, sample is dissolved in 0.1mM KCl, is measured using automatic mode.All measurements are in triplicate.
Fig. 2 B is respectively the Zeta potential of Nanometer Insulin and LBL4 particle.The result shows that Nanometer Insulin slightly positive electricity
For 1.9 ± 0.01mV, LBL4 particle is positively charged, and potential value is about 13.39 ± 0.01mV.Show Nanometer Insulin and LBL4
Electrically it is consistent with expection, while shows that the outer layer of LBL4 is sufficiently wrapped up by chitosan, makes the stronger positive charge of particle band.
(3) transmission electron microscope observing
Respectively after each round synthesis, detection of the 100 μ l for appearance and partial size is taken.The suspension drop of particle is being covered with carbon
On the copper mesh of film, 5min is stood, excessive moisture is blotted with filter paper, stands 30min, parched completely to it.Use HITACHI
7650 transmission electron microscopes, electron accelerating voltage 300kV, respectively at 1x104Again, 2x104Under amplification factor again, particle shape is observed
State.
Fig. 2 C is the TEM shape of particle, and the nm-class insulin particles after wrapping up polymer are rounded.1,2 are wrapped up respectively
The insulin of the visible spheroidal of image of layer is wrapped in flocculent structure, forms the form of similar grape cluster.With wrapping layer
Several increases, partial size are continuously increased.The shallower membrane structure of the color that visible polysaccharide is formed in figure is distributed in particle periphery.Package
When to the 4th layer, LBL4 particle forms the ball of about 200nm, existing to be relatively independent form, but still has to a certain degree between particle
Involve.Simultaneously, it is seen that particle is to be wrapped up by multilayer film as a result, from outside to inside, color successively deepens.
(4) thermogravimetric analysis
Nano particle LBL4 by package to four layers is dried, and synchronizes heat using Switzerland Mettler, TGA/DSC1
Analyzer, under n 2 atmosphere, measurement range are 30 DEG C to 900 DEG C, and heating rate is 10 DEG C/min.
Fig. 3 illustrates LBL4 system and insulin in N2Thermogravimetric curve under atmosphere, heating temperature from room temperature to 800 DEG C,
Heating rate is 10 DEG C/min.It is observed that there are two sections for the weightlessness of insulin, it is 30-100 DEG C and 200-400 respectively
℃；LBL4 system it is weightless there are three section, 30-100 DEG C, 200-300 DEG C and 750-800 DEG C.First section, 300-100
DEG C corresponding is the weight loss of insulin Yu LBL4 system about 3%, this is likely to the hydrone for being absorbed by system or being combined
Loss caused by.Second section, it is the mass loss of insulin about 70%, most of insulin that 200-400 DEG C corresponding
In this interval decomposed；Corresponding, LBL4 system about loses 20% quality in this temperature range, part insulin, chitosan,
PGLA is in this interval decomposed.Since LBL4 has stronger intermolecular hydrogen bonding and electrostatic attraction, compared with insulin group, this
The weight loss stage starts later, and the degree of mass loss is also relatively light.Third section, 750-800 DEG C of LBL4 system start
Third time mass loss process.In this temperature range, each ingredient of LBL4 system is further decomposed, and not only includes sugar in polysaccharide
The dehydration of ring also includes the dehydration of groups and the decomposition of part carbon skeleton such as carboxyl, acetyl group
(5) entrapment efficiency (encapsulation ratio) measures
Insulin standard curve is drawn first.Insulin solutions (0.1mg/ml, the 0.01mg/ of various concentration is respectively configured
Ml, 0.005mg/ml, 0.001mg/ml, 0.0005mg/ml, 0.0001mg/ml), use German Perkin Elmer Lambda
25 ultraviolet specrophotometers measure its ultra-violet absorption spectrum between 200~300nm.And standard song is drawn according to its absorption peak
Line, the linear fit equation and degree of fitting of calculated curve.
The particle suspension of the Islets finally synthesized element is centrifuged using supercentrifuge using supercentrifuge,
11000rpm/min, 10 DEG C, 30min.After taking supernatant vacuum to hang inspissation contracting, its insulin is measured using ultraviolet specrophotometer and is inhaled
Spectrum is received, and according to its light absorption value conversion insulin content at the wavelength for drawing standard curve.
The encapsulation rate of insulin calculates according to the following formula with carrying drug ratio:
As a result as follows: ultra-violet absorption spectrum of the insulin in dilute HCl solution is shown, is respectively provided in 205nm and 275nm
Absorption peak, and be located at the absorption peak at 205nm and be higher than at 275nm 10 times or more, absorption peak of the insulin at 205nm is more suitable
It is suitable for the pattern detection of low concentration.From 10-4In the range of mg/ml to 1mg/ml, ultraviolet absorption value of the insulin at 205nm is in
Semi-parabolic type.Since the sample insulin concentration that this research is related to is lower, 10 are chosen-4Mg/ml and 10-2Purple between mg/ml
Outer absorption value draws standard curve, it is seen that it is with linear relationship, equation of linear regression y=18.641x+0.0019, phase
Close sex index R2=0.9967 (Fig. 4).
By compound experiment three times, statistical data, the encapsulation rate of LBL4 is 55.21 ± 4%, drug-loading efficiency 60.73
± 6%.
(6) drug release rate measures
The PBS buffer solution for configuring 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, is shaked gently with 50r/min.700 μ l are sampled respectively at 0.5h, 1h, 2h, 3h, 4h, centrifugation takes supernatant
It is detected for insulin content, that is, uses ultraviolet specrophotometer, measure its absorbance, and establishing criteria calibration curve converts.
Each delivery systme is repeated 3 times, and is collected data and is calculated for counting.
As depicted in figures 5 and 6, extracorporeal releasing experiment the result shows that, LBL4 system has in the hydrochloric acid solution of pH=1
Certain slow releasing function, for 0.5h between 5h, burst size has no significant difference.In the PBS buffer solution of pH=6.8, LBL4 body
System shows burst release mode, when 1h, reaches release maximum value.Comparing release of the equivalent LBL4 in two kinds of systems can send out
Existing, system has certain pH irritability, and this property matches with the demand that oral insulin transports system, i.e., in gastric environment
In do not discharge insulin, to get up to protect insulin by the destruction of pepsin, not discharge in intestines environment, be conducive to pancreas islet
Element is absorbed by enteric epithelium.Speculate this pH induction property source may be since at low ph conditions, material charging property is stronger,
Between material there is stronger attraction the particle by electrostatic interaction self assembly can be made more stable.And under conditions of neutral ph,
Interstructural active force is weaker layer by layer for particle, structure loosely, so discharging insulin faster.
The insulin releasing situation that LBL4 particle is incubated for 2h in different pH systems shows in the section pH value 3-5, LBL4 body
The rate of system's release insulin is most slow, and burst size is lower to have statistical significance (p < 0.05).And in pH value is 2 or is close
When property, the rate of release of insulin is very fast.After feed, stomach pH is about 3 or so, and therefore, which transports system
The effect taken after feed is better than effect on an empty stomach.
2 cell experiment of embodiment
1, experimental method
(1) cell culture
Caco-2 cell line is provided by Guangdong Pharmaceutical University.Cell is Multiplying culture is to after 80% in culture bottle, with 5
The density in 000/ hole is seeded on 96 orifice plates, is cultivated 1,2 days, to carry out subsequent experimental.Its cell culture condition are as follows: new containing 20%
The DMEM in high glucose culture medium of raw cow's serum, nonessential amino acid, 37 DEG C, 5.0%CO2。
(2) system toxicity is transported
Take the system that transports of various dose to be added in the Caco-2 cell cultivated respectively, for 24 hours after, surveyed using mtt assay
Measure cell survival rate.It is inoculated with Caco-2 cell i.e. on 96 well culture plates, 5 000/hole, after culture for 24 hours, culture medium is sucked out,
The insulin that serum free medium and various concentration is added transports system, and after cultivating for 24 hours, the MTT solution of 5mg/ml is added
The hole 20ul/.Continue to cultivate 4h, culture medium is sucked out, DMSO is added and dissolves the first a ceremonial jade-ladle, used in libation formed into the cell.The low-speed oscillation on shaking table
10min is read, wavelength 510nm in microplate reader.
2, experimental result
Insulin transports system and is assessed in the bio-toxicity of cell level by mtt assay.As a result as shown in Fig. 7.
The LBL4 that various concentration is added 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), cell viability and it is not affected by too big influence as the result is shown, cell Rate activity is all
It is maintained at 90% or more.Wherein, for the drug of 500 μ g/ml, 1000 μ g/ml for cell culture, concentration is relatively large.It is right
Drug within toxic medicament, 100 μ g/ml is the cell lethality that can reach 50%.And LBL4 system reaches in upper dose
When 1000 μ g/ml, cell viability level is still 100% or so, it can be seen that, LBL4 has extremely low cell toxicant, bio-compatible
Property is very high.
3 experiment in vivo of embodiment --- the Study of cytotoxicity of system
1, type-1 diabetes mellitus mouse model constructs
(1) method
6 week old SD rats are bought, are adapted to environment one week.3 are taken as control group, remaining is experimental group.One night of Rat Fast,
Measure blood glucose, using pH=4.4 citrate buffer solution before the injection ice bath be protected from light prepare STZ solution, concentration 15mg/ml.
Experimental group rat is injected intraperitoneally by the dosage of 50mg/kg.
It is risen on the day of self-modeling, measures rat body weight, feed and amount of drinking water daily.Use Roche vigor type within 3rd day, the 7th day
Blood glucose meter monitors blood glucose.To the lower rat of blood glucose, supplement injection STZ (30mg/kg).
After a week, (after hungry 3h) blood sugar concentration reaches 13mmol/L or more on an empty stomach, is classified as diabetes illness mouse.
Type-1 diabetes mellitus is grouped at mould rat by fasting blood-glucose mean value, keeps the blood glucose mean of every group of rat without significance difference
It is different.
(2) result
As shown in figure 8, fasting 16 hours, STZ, dosage 50mg/kg is injected intraperitoneally, after a week using male SD rat
Blood glucose is detected, SD rat of the continuous 4 days fasting blood sugars greater than 13mmol/L is considered as the type-1 diabetes mellitus rat for being modeled as function.
After injection drug 12 days, the figure at mould rat and control rats of STZ injection group has apparent difference.
From the point of view of glucose monitor data (Fig. 9 A), inject after STZ the 3rd day, after one night of Rat Fast, the blood of STZ injection group
Sugar has just been significantly higher than control group.In experimental group, having 1 rat blood sugar value is 12.6mmol/L, remaining STZ injection group rat serum
Sugar value is all in 20mmol/L or more, and the 6th, 9 day, trend did not changed.It can be assumed that except blood glucose value is big for this of 12.6mmol/L
It outside mouse, all models successfully, modeling success rate reaches 92%.
From injection STZ before 1 day to injection STZ after the 9th day rat body weight, drinking-water, food-intake monitoring data show (figure
9B, Figure 10), the weight of STZ injection group and control group, drinking-water, food-intake before injecting STZ 1 day data without statistical discrepancy, note
The first 1 day variation tendency to the injection same day is penetrated also to be completely coincident.It was risen to the injection STZ same day, weight, drinking-water, food-intake appearance are poor
Different, i.e. control group weight constantly increases, and drinking-water and food-intake are without significant changes；STZ injection group weight is without growth trend, amount of drinking water
7 times or so to increase sharply to before injecting, food-intake increase about 50%.
2, absorption of the Immunohistochemical Method observation intestinal villi to system
(1) method
3 SD rats for suffering from type 1 diabetes are taken, at one night of fasting before testing, can't help water.Taking 1 is control group, 300 μ of stomach-filling
L physiological saline, taking 2 is experimental group, 300 μ l of stomach-filling LBL4 aqueous solution.By control group 2 hours after stomach-filling, experimental group difference
It 1 hour after stomach-filling, is anaesthetized using yellow Jackets (0.05mg/kg) within 2 hours, after abdomen is exposed, cuts 5cm ileum and 5cm
Colon is cleaned using physiological saline respectively.After cutting intestinal segment, injects excessive anaesthetic and put to death rat.
(2) result
Figure 11 is that the type-1 diabetes mellitus rat at one night of fasting feeds the duodenum of 1h and 2h and exempting from for ileum after LBL4 system
Epidemic disease group result.
Due to testing the hungry type-1 diabetes mellitus rat using one night of fasting, the drug of stomach-filling is that insulin transports body
It is the aqueous solution of LBL4, drug can pass through faster stomach and enter 12 rouge intestines.From the point of view of the coloration result of control group, research
Insulin in applied type-1 diabetes mellitus mouse intestinal villus without itself secretion.From the point of view of the coloration result of Duodenal villi,
After feeding 1h, just visible apparent absorption of insulin phenomenon in duodenal villus, visible a small amount of suction in the villus of ileum
It receives.It can be seen that LBL4 can be rapidly absorbed in duodenal segment.After feeding 2h, the intravillous pancreas islet of duodenum, ileum
Element is clearly visible.
3, Composition analyzed in drug body --- type-1 diabetes mellitus rat limosis administering effect
(1) method
Type-1 diabetes mellitus rat is run out of grain 12h, keeps drinking-water unobstructed.Blank control group is respectively set and nanometer of the invention carries
2 groups of powder subgroup, every group of type-1 diabetes mellitus rat is no less than 3, takes physiological saline and LBL4 system (oral insulin respectively
Amount is 50IU/kg), in 0h, 1h, 2h, 3h, 5h, 7h, 9h, 17h, 22h survey blood glucose.Food is given when 17h, measures returning for blood glucose
Rise situation.
(2) result is as shown in Fig. 12.
Diabetic model rats are after oral LBL4 (50IU/kg) system, and from 1h, blood glucose significant decline occurs and becomes
Gesture touches the bottom in 3h, close to the 40% of experiment initial stage blood glucose level.After reaching blood glucose minimum point, glucostasis is in lower
Level, control group blood glucose gradually appear downward trend.When 17h, experimental group and control group difference are little.After feeding, experimental group with
The synchronous rise of control group blood glucose.
In addition, compared with other oral insulins transport the research of system, in vivo in terms of effect, this research synthesis
LBL4 has the characteristics that significant rapid-action, significant effect.Blood glucose is significantly reduced to initial blood glucose value by the 1h after medication
40%, compared with control group at this time, the blood glucose level of LBL4 group is the 50% of control group.What this result and intestinal villus absorbed
As a result consistent, the infiltration rate for all showing LBL4 particle is fast, this rapid-action feature.
It is successfully synthesized layer by layer in conclusion insulin nano ball, polygalacturonic acid, chitosan is applied successfully in the present invention
The oral insulin of self assembly transports system, and self-assembling nanoparticles thermal stability is higher than free insulin.Wrap up 4 layer materials
LBL system has more independent structure, lesser partial size (being less than 200nm), surface positively charged.Encapsulation rate be 55.21 ±
4%, drug-loading efficiency is 60.73 ± 6%, Drug loading capacity with higher.Extracorporeal releasing experiment shows the hydrochloric acid in pH=1.1
In solution, LBL system has obvious controlled-release function, and in the PBS buffer solution of pH=6.8, LBL system shows burst release mould
Formula, it is believed that LBL system has pH response characteristic, and insulin releasing rate at low ph conditions is slightly slower than under the conditions of high pH
Rate of release.It finds in vivo experiment, this transports the control of hyperglycemia that system can be significant and long-acting, after the 1h that takes medicine i.e.
There is blood glucose decline, touches the bottom in 3h, have the ability of regulating and controlling blood sugar in 10h.
Claims (8)
1. the preparation method that a kind of oral insulin of LBL self-assembly structure transports system, which is characterized in that first in low pH
Under the conditions of, positively charged insulin nano ball is precipitated using salting out method, according to the electrical property and electrostatic force of material, application layer
Layer self-assembly method uses electronegative polygalacturonic acid and electropositive chitosan successively Islets element nanosphere respectively；
The preparation method, includes the following steps:
S1. under 10~20 DEG C of environment, insulin is dissolved in dilute hydrochloric acid solution, NaCl is added and is allowed to final concentration and reach
0.5~0.7M, solution stir 0.4~0.6h, are centrifuged to obtain precipitating；
S2. polygalacturonic acid solution is added in the precipitating of step S1, is stirred for 0.4~0.6h, 0.8~1.5min of ultrasound, continues
0.4~0.6h is stirred, precipitating is centrifuged to obtain；
S3. chitosan solution is added in the precipitating of step S2, stirs 0.4~0.6h, is centrifuged to obtain precipitating；
S4. it is repeated in step S2 and step S3 repeatedly, that is, is alternately added polygalacturonic acid solution and chitosan solution carries out
Package, the oral insulin for obtaining different layers of LBL self-assembly structures transport system；
The pH of dilute hydrochloric acid solution described in step S1 is 1.0~1.4.
2. preparation method according to claim 1, which is characterized in that the mass body of step S1 insulin and dilute hydrochloric acid solution
Product is than being 4~6mg/ml.
3. preparation method according to claim 1, which is characterized in that the pH of polygalacturonic acid solution described in step S2 is
3~5.
4. preparation method according to claim 1, which is characterized in that the preparation method of step S2 polygalacturonic acid solution
Are as follows: polygalacturonic acid is dissolved in dilute hydrochloric acid solution, NaCl is added, NaCl final concentration is made to reach 0.5~0.7M, pH is
3~5.
5. preparation method according to claim 1, which is characterized in that the preparation method of chitosan solution described in step S3
Are as follows: it dissolves the chitosan in dilute hydrochloric acid solution, adds NaCl solid, NaCl final concentration is made to reach 2~4M, pH is 3~5.
6. preparation method according to claim 1, which is characterized in that precipitating and polygalacturonic acid solution described in step S2
Mass volume ratio be 0.8~1.2g:100ml；The mass volume ratio of precipitating and chitosan solution described in step S3 is 4~6g:
100ml。
7. the oral insulin for the LBL self-assembly structure that any the method is prepared according to claim 1~6 transports body
System.
8. the oral insulin of LBL self-assembly structure described in claim 7 transports system in terms of preparing oral insulin medicament
Application.
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