CN104138602B

CN104138602B - Type II diabetes resisting long-acing nano complex peptides and preparation method and application

Info

Publication number: CN104138602B
Application number: CN201410367708.0A
Authority: CN
Inventors: 马义; 洪岸; 陈填烽
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2014-07-29
Filing date: 2014-07-29
Publication date: 2016-09-14
Anticipated expiration: 2034-07-29
Also published as: CN104138602A

Abstract

Open a kind of type II diabetes resisting long-acing nano complex peptides of the present invention and preparation method and application.With nanometer selenium as carrier, chitosan is for connecting, it is achieved active polypeptide and the covalent bond of nanometer selenium particle.Nano combined peptide obtained by the present invention has in physiological environment and carries, protects and slow release therapeutical peptide, the effect of effective extended treatment polypeptide half-life; the effective dose playing therapeutical effect can substantially less than be used alone the dosage of therapeutical peptide, and can play the biological action of medicament slow release, long-acting treatment；Overcome when being used alone therapeutical peptide, owing to molecular weight is little, the shortcoming the most easily removed by tachymetabolism, Half-life in vivo and the bioavailability of small molecule therapy polypeptide can be effectively improved.The nano combined peptide of the present invention can be applicable to preparation treat following disease medicine: diabetes, hyperglycemia.The preparation method efficiency of the present invention is high, low cost, has a extensive future.

Description

Type II diabetes resisting long-acing nano complex peptides and preparation method and application

Technical field

The invention belongs to field of nano biotechnology, be combined particularly to a kind of type II diabetes resisting long-acing nano Peptide and preparation method and application.

Background technology

Pituitary adenylate cyclase activating peptide (PACAP) is nineteen ninety to be found having by pituitary secretion The nerve polypeptide of important biomolecule function, is the newcomer in secretin/glucagon/VIP family. PACAP has two kinds of existence form: PACAP38, is made up of 38 aminoacid；PACAP27, by PACAP38 27 aminoacid compositions of nitrogen end.PACAP, by activating its special g protein coupled receptor, improves target The concentration of the second message,second messengers such as cell cAMP, thus play extensive and important biological function.PACAP has Three class g protein coupled receptors: PAC1, VPAC1 and VPAC2；Three receptoroids are distributed not in organism With, role also differs.Wherein VPAC2 receptor is distributed in the tissues such as islets of langerhans, with energy metabolism Closely related, in terms of clinical point, the specific PACAP as VPAC2 receptor specific agonist derives Thing can be effectively reduced blood glucose levels, protection Instreptozotocin Induced and the potentiality of prevention disease development, and Risk without hypoglycemia, for the newtype drug of generally acknowledged treatment type Ⅱdiabetes mellitus.

PACAP Yu VPAC2 receptor acting is main and adenyl cyclase (AC) Rhizoma Nelumbinis joins: pass through cAMP Signal transduction pathway, activates AC, catalysis ATP and is converted into cAMP, cAMP and activates its dependency egg therewith White kinases A (PKA), PKA can make intracellular multiple proteins phosphorylation, produce physiological effect, especially It is to transcribe at promotion related gene to play an important role.PACAP passes through VPAC2 merit receptor-mediated biology Can mainly have: neurotransmitter/quenched, promotion hormone secretion, endocrine regulation balances；Regulation gonad function Generation with sexual cell；Regulation energy metabolism balance etc..

Research shows, VPAC2 receptor specific agonist can effectively facilitate the insulin secretion of dependence on the glucose, Improve body and the emergency capability of high concentration blood glucose is effectively reduced blood sugar concentration, protection Pancreatic beta cells function, Increase β cell quantity and suppression β apoptosis.But PACAP and many derivants thereof, as DBAYL, BAY55-9837 etc., due to molecular weight (< 6000Da.), in vivo due to the filtration of glomerule, Easily by kidney rapid drainage and metabolism, therefore, its Half-life in vivo is shorter, bioavailability is affected.

Nanometer selenium (SeNPs) has good biocompatibility, the most also has the function removing free radical, It is gradually explored with Preliminary Applications in medicine transmission and therapy system.Chitosan is by (1,4)-linked 2-amino-2-deoxy-β-D-glucose forms the cation polysaccharide of a kind of poisonless biological degradable.Chitosan With still there is the premium properties of high molecular polymer, such as biocompatibility, biological fall after nanoparticle effect Xie Xing, avirulence, low cost etc..Chitin nanometer is used for drug carrier and treatment, is increasingly subject to The concern of researcher.

VPAC2 receptor specific agonist class active polypeptide has good glucose dependency blood sugar lowering and makees With, but often molecular weight, its Half-life in vivo is shorter, bioavailability reduces, to long-acting performance These have the biological action of active polypeptide of good hypoglycemic activity, these active polypeptide and chitosan The nano-carrier particle coupling modified as newtype drug system, can be effectively improved polypeptide drugs water solublity and Internal stability, the internal release controlling medicine and targeting selectivity, effectively extend the half of active poly peptide medicine Decline the phase, there is important medical value.

Summary of the invention

In order to overcome the shortcoming of prior art with not enough, the primary and foremost purpose of the present invention is to provide one anti-II type The preparation method of diabetes long-acing nano complex peptides.This preparation method production efficiency is high, and production cost is low.

Another object of the present invention is to provide that the type II diabetes resisting obtained by above-mentioned preparation method is long-acting to be received Rice complex peptides.This nano combined peptide Peptide-CS-SeNPs has synergism, have more high stability, The long half-lift of more, Peptide refers to the VPAC2 receptor specific agonist class activity such as DBAYL, BAY55-9837 Polypeptide.With nanometer selenium as carrier, chitosan (Chitosan, CS) is for connecting linker, it is achieved active polypeptide Covalent bond with nanometer selenium particle (SeNPs).

It is still another object of the present invention to provide the application of described type II diabetes resisting long-acing nano complex peptides.

The purpose of the present invention is achieved through the following technical solutions: a kind of type II diabetes resisting long-acing nano complex peptides Preparation method, comprises the steps:

(1) prepared by nanometer selenium particle (SeNPs)

Under normal temperature and pressure, draw ascorbic acid (Vc) solution, be added dropwise over Na₂SeO₃Solution, limit edged Shake up, treat that color is no longer deepened, transfer to low-temp reaction, obtain nanometer selenium particle solution；

(2) polypeptide and chitosan coupling

Weighing polypeptide, dissolve with distilled water, the chitosan (Chitosan, CS) adding 0.8mg/mL is molten Liquid, stirring reaction, obtain polypeptide-chitosan conjugate solution；

(3) preparation of nano combined peptide Peptide-CS-SeNPs

Polypeptide-chitosan conjugate prepared by nanometer selenium particle solution step (1) prepared and step (2) After solution mixing, stirring reaction, after having reacted, dialysed overnight in distilled water, obtain highly purified anti- Type Ⅱdiabetes mellitus long-acing nano complex peptides Peptide-CS-SeNPs.

The concentration of the Vc solution described in step (1) is preferably 20～60mM；More preferably 20mM；

Na described in step (1)₂SeO₃The concentration of solution is preferably 5～15mM；More preferably 5mM；

Nanometer selenium particle solution described in step (1), wherein Vc and Na₂SeO₃Final concentration than preferably For 4:1；

Low temperature described in step (1) is preferably 0～4 DEG C；

The time of the reaction described in step (1) is preferably 3～5h, more preferably 4h；

Polypeptide described in step (2) is preferably DBAYL, BAY55-9837, RMBAY or RMROM Deng VPAC2 receptor specific agonist class active polypeptide；More preferably DBAYL or BAY55-9837；

The time of the reaction described in step (2) is preferably 10～16h；More preferably 12h；

Polypeptide described in step (2)-chitosan conjugate solution, wherein the final concentration of polypeptide is preferably 1mg/mL；The final concentration of polysaccharide is preferably 0.08mg/mL；

Mixing described in step (3) is preferably nanometer selenium particle solution and step (2) prepared by step (1) Polypeptide-chitosan conjugate solution the 2:(0.5 by volume～3 of preparation) mix；More preferably 2:1；

The time of the reaction described in step (3) is preferably 10～16h；More preferably 12h；

A kind of type II diabetes resisting long-acing nano complex peptides, is prepared by above-mentioned preparation method.

Described type II diabetes resisting long-acing nano complex peptides is applied to preparation and treats the medicine of following disease: sugar Urine disease, hyperglycemia.

Described type II diabetes resisting long-acing nano complex peptides is applied to preparation treatment type Ⅱdiabetes mellitus relevant disease Medicine, have a following effect: the insulin secretion of long-acting promotion dependence on the glucose, improve body to high concentration The emergency capability of blood glucose, long-acting reduction blood sugar concentration, protection Pancreatic beta cells function, increase β cell quantity Act on suppression β apoptosis and regulation function of immune system etc..

The present invention has such advantages as relative to prior art and effect:

(1) in the nano combined peptide Peptide-CS-SeNPs drug system prepared by the present invention, covalent coupling Peptide-CS-SeNPs in vivo by native enzyme or physiological environment (as the physicochemical properties such as PH become Change), can slowly discharge Peptide (DBAYL, BAY55-9837 isoreactivity polypeptide), at nano combined peptide In, the nanometer selenium particle (SeNPs) that chitosan (Chitosan, CS) is modified has therapeutical peptide Peptide Carrier carry, protect and slow releasing function, and release Peptide after SeNPs still have protection and repair β- Cell functions etc. act on；Peptide has targeting guiding function to complex peptides Peptide-CS-SeNPs, and can By the receptor-mediated biological action playing treatment type Ⅱdiabetes mellitus of VPAC2.

(2) nano combined peptide Peptide-CS-SeNPs overcomes and is used alone therapeutical peptide Peptide Time (DBAYL, BAY55-9837 isoreactivity polypeptide), due to molecular weight little (less than 6000Da.), The internal shortcoming easily removed by tachymetabolism, can be effectively improved small molecule therapy polypeptide Half-life in vivo and Bioavailability.

These differences make nano combined peptide Peptide-CS-SeNPs have in physiological environment to carry, protect With slow release therapeutical peptide, the effect of effective extended treatment polypeptide half-life, nano combined peptide plays treatment The effective dose of effect can substantially less than be used alone the dosage of therapeutical peptide, and it is slow to play medicine Release, the biological action of long-acting treatment.Under physiological condition, the therapeutical peptide of releasing of release can be with VPAC2 receptor After specific bond, promote generation and the secretion of insulin, protection Pancreatic beta cells function, increase β cell number Amount and suppression β apoptosis, and activate Insulin receptor INSR signal path, thus play treatment type Ⅱdiabetes mellitus Effect.And SeNPs has protection and repairs the effect of Instreptozotocin Induced.

(3) the nano combined peptide Peptide-CS-SeNPs prepared by the present invention is in treatment diabetes and concurrent Disease, hyperglycemia and obesity have significant curative effect.

(4) the nano combined peptide Peptide-CS-SeNPs utilizing preparation method of the present invention to obtain makes The elimination Increased Plasma Half-life 25 of therapeutical peptide Peptide (DBAYL, BAY55-9837 isoreactivity polypeptide) More than Bei.The preparation method efficiency of the present invention is high, low cost, has a extensive future.

Accompanying drawing explanation

Fig. 1 is the structural representation of nano combined peptide Peptide-CS-SeNPs.

Fig. 2 be the nano combined PEPD BAYL-CS-SeNPs (Fig. 2 A) prepared and The result figure that the transmission electron microscope (TEM) of BAY55-9837-CS-SeNPs (Fig. 2 B) is analyzed.

Fig. 3 is nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs prepared The result figure that Zeta potential is analyzed.

Fig. 4 is that nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs prepared exists The result figure of the half-life in db/db Mice Body.

Detailed description of the invention

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention It is not limited to this.

Embodiment 1

The preparation of nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs

The aminoacid sequence of DBAYL is:

MHSDAVFTDQYTRLRKQLAAKKYLQSLKQKRY；

The aminoacid sequence of BAY55-9837 is:

HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY；

Under normal temperature and pressure, weigh 8.7mg Na₂SeO₃Being dissolved in distilled water, constant volume, to 10mL, i.e. obtains 5mM Na₂SeO₃Solution；Weigh 35.2mg ascorbic acid (Vc), be dissolved in distilled water, constant volume to 10mL, I.e. obtain the Vc solution of 20mM；Weigh 8mg chitosan, be dissolved in distilled water, constant volume to 10mL, I.e. obtain the chitosan solution of 0.8mg/mL.The Vc solution that absorption 1mL prepares is in small beaker, dropwise Add 1mL Na₂SeO₃Solution, limit edged shakes up, and treats that color is no longer deepened, and transfers to low temperature (0～4 DEG C) React 4 hours, i.e. obtain rough nanometer selenium particle solution.

Weigh 1mg polypeptide Peptide (referring to DBAYL or BAY55-9837) in small beaker, add 900 μ L Distilled water dissolves, and adds the chitosan solution of 100 μ L 0.8mg/mL, stirring reaction 12 hours.Then Being joined by reactant liquor in rough nanometer selenium particle solution, constant volume, to 5mL, is stirred for reacting 12 hours. After having reacted, dialysed overnight in distilled water, i.e. obtain nano combined PEPD BAYL-CS-SeNPs or BAY55-9837-CS-SeNPs。

The structure of the nano combined peptide Peptide-CS-SeNPs of preparation is as shown in Figure 1.

Embodiment 2

The transmission electricity of nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs of preparation Mirror (TEM) detects

Nano combined peptide is observed with TECNAI-10 type transmission electron microscope (Philips) The pattern of DBAYL-CS-SeNPs and BAY55-9837-CS-SeNPs: nano combined peptide is uniformly dispersed, Dipping sample with copper mesh, to determine the size of gained sample microgranule, pattern and uniformity, and shooting has representative The electromicroscopic photograph of property.

Experimental result such as Fig. 2, the nano combined PEPD BAYL-CS-SeNPs (Fig. 2 A) of preparation and BAY55-9837-CS-SeNPs (Fig. 2 B), has good dispersibility, and its size about 100nm is micro- Particle shape is the most spherical, and uniformity is good.

Embodiment 3

The Zeta potential of nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs of preparation And Fourier transform infrared spectroscopy (FT-IR) detection

Receive with Nano-ZS (Malvern Insruments Limited) mensuration SeNPs, SeNPs-CS and two kinds Rice complex peptides DBAYL-CS-SeNPs and BAY55-9837-CS-SeNPs Zeta potential, compare them it Between difference.Experimental result as it is shown on figure 3, the zeta current potential of SeNPs is-8.5mV, the zeta of SeNPs-CS Current potential be the Zeta potential of 47.5mV, DBAYL-CS-SeNPs be 34.9mV, The Zeta potential of BAY55-9837-CS-SeNPs is 29.8mV.

Embodiment 4

Nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs of preparation is at db/db Half-life detection in Mice Body

Experiment is divided into four groups, often 6 db/db mices of group, respectively the intravenous injection 500 nano combined peptide of μ g/kg DBAYL-CS-SeNPs, BAY55-9837-CS-SeNPs, DBAYL or BAY55-9837, injection drug Thing 1～the mouse blood sample after 20 hours are collected from tail vein, and each time point blood sample collection is containing anti- In the centrifuge tube of solidifying agent sodium citrate.After sample is centrifugal, LC/MS/MS technology is utilized to detect.High-efficient liquid Phase chromatographic conditions is: mobile phase A (pure water of 100%, the formic acid containing 1%), Mobile phase B (100% Acetonitrile, the formic acid containing 1%), graded is: 0.0～0.4min, 90% mobile phase A；0.4～ 0.8min, from 90% mobile phase A to 10% mobile phase A；0.8～1.8min, 10% mobile phase A；1.8～ 2.0min, from 10% mobile phase A to 90% mobile phase A；2.0～3.0min, 90% mobile phase A；Loading Volume is 10 μ L.Each time point concentration of DBAYL or BAY55-9837 utilize the standard curve set up and Win-Nonlin version 3.1 software (Pharsight Inc. Products, the U.S.) calculates meansigma methods gained.

Experimental result as shown in Figure 4, nano combined PEPD BAYL-CS-SeNPs and The BAY55-9837-CS-SeNPs elimination half-life in db/db Mice Body is respectively 19.7 hours and 17.3 Hour；Polypeptide DBAYL and the BAY55-9837 elimination half-life in db/db Mice Body is respectively 0.77 Hour and 0.35 hour；Test result indicate that, after therapeutical peptide is compound with chitosan-modified nanometer selenium particle Internal elimination half-life of nano combined peptide, the most single therapeutical peptide.

Embodiment 5

Nano combined PEPD BAYL-CS-SeNPs and BAY55-9837-CS-SeNPs of preparation is to db/db The impact of mouse blood sugar

The type Ⅱdiabetes mellitus model db/db mice of 40 10 week old, is randomized into 5 groups according to body weight, often Organize 8.Often group mice measures blood sugar concentration before drug injection, and intravenous injection 500 μ g/kg receives the most respectively Rice complex peptides DBAYL-CS-SeNPs, BAY55-9837-CS-SeNPs, DBAYL, BAY55-9837 Or normal saline, within 2,4,6,8,10,12 hours after medicine or physiological saline, take from tail vein Blood, measures blood glucose value blood with OneTouch Ultra Meter blood glucose meter (Johnson Products, the U.S.) Sugar concentration.

Blood sugar level detection (mmol/L) of the db/db mice of nano combined peptide or polypeptide injected by table 1

Each group

2h

4h

6h

8h

10h

12h

DBAYL-CS-SeNPs

7.2±0.7^a

7.1±0.6^a,d

7.3±0.8^a,c

7.2±0.7^a,c

7.2±0.6^a,c

8.3±0.9^a,c

BAY55-9837-CS-SeNPs

7.5±0.8^a,c

7.6±0.7^a,c

8.6±0.9^a,c

9.6±1.1^a,c

14.6±1.3^b,d

15.9±1.5^b,d

DBAYL

7.7±0.8

8.9±1.0

12.5±1.2

14.0±1.1

16.7±1.5

17.1±1.3

BAY55-9837

14.2±1.1

15.5±1.2

16.3±1.3

15.7±1.2

17.2±1.3

17.5±1.4

Normal saline

16.8±1.6

15.9±1.5

17.2±1.5

16.1±1.3

17.5±1.7

17.4±1.6

Numerical value is mean+SD, n=8；4 hours, 8 hours point diet after injection medicine；

A:P < 0.01, DBAYL-CS-SeNPs group or BAY55-9837-CS-SeNPs group vs normal saline group；

B:P < 0.05, DBAYL-CS-SeNPs group or BAY55-9837-CS-SeNPs group vs normal saline group；

C:P < 0.01, DBAYL-CS-SeNPs group vs DBAYL group；Or BAY55-9837-CS-SeNPs group vs BAY55-9837 group；

D:P < 0.05, DBAYL-CS-SeNPs group vs DBAYL group；Or BAY55-9837-CS-SeNPs group vs BAY55-9837 group.

Experimental result is as shown in table 1: compared with polypeptide DBAYL and BAY55-9837, nano combined peptide DBAYL-CS-SeNPs and BAY55-9837-CS-SeNPs can more effectively reduce type Ⅱdiabetes mellitus model The blood sugar level of db/db mice, DBAYL-CS-SeNPs and BAY55-9837-CS-SeNPs can exist respectively Maintaining the blood glucose of db/db mice in 10 hours and 4 hours within normal range, its hypoglycemic activity is notable Be better than polypeptide DBAYL and BAY55-9837, show DBAYL-CS-SeNPs and BAY55-9837-CS-SeNPs can play the hypoglycemic biological action of long-acting glucose dependency.

Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-mentioned enforcement The restriction of example, the change made, modifies, replaces under other any spirit without departing from the present invention and principle In generation, combine, simplify, all should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims

1. the preparation method of a type II diabetes resisting long-acing nano complex peptides, it is characterised in that include walking as follows Rapid: with nanometer selenium as carrier, chitosan for connect, it is achieved VPAC2 receptor specific agonist class active polypeptide with The covalent bond of nanometer selenium particle；

The preparation method of described type II diabetes resisting long-acing nano complex peptides, comprises the following specific steps that:

(1) prepared by nanometer selenium particle

Under normal temperature and pressure, draw Vc solution, be added dropwise over Na₂SeO₃Solution, limit edged shakes up, and treats color No longer deepen, transfer to low-temp reaction, obtain nanometer selenium particle solution；

(2) polypeptide and chitosan coupling

Weigh polypeptide, dissolve with distilled water, add the chitosan solution of 0.8mg/mL, stirring reaction, obtain To polypeptide-chitosan conjugate solution；

(3) preparation of nano combined peptide Peptide-CS-SeNPs

Polypeptide-chitosan conjugate prepared by nanometer selenium particle solution step (1) prepared and step (2) After solution mixing, stirring reaction, after having reacted, dialysed overnight in distilled water, obtain anti-II type glycosuria Sick long-acing nano complex peptides Peptide-CS-SeNPs.

Preparation method the most according to claim 1, it is characterised in that: many described in step (2) Peptide is DBAYL, BAY55-9837, RMBAY or RMROM.

Preparation method the most according to claim 1, it is characterised in that: the Vc described in step (1) The concentration of solution is 20～60mM；

Na described in step (1)₂SeO₃The concentration of solution is 5～15mM；

Nanometer selenium particle solution described in step (1), wherein Vc and Na₂SeO₃Final concentration than for 4:1.

Preparation method the most according to claim 1, it is characterised in that: many described in step (2) Peptide-chitosan conjugate solution, the wherein final concentration of 1mg/mL of polypeptide；Chitosan final concentration of 0.08mg/mL。

Preparation method the most according to claim 1, it is characterised in that: the mixing described in step (3) Polypeptide-chitosan conjugate solution prepared by the nanometer selenium particle solution prepared for step (1) and step (2) 2:(0.5～3 by volume) mix.

Preparation method the most according to claim 1, it is characterised in that: anti-described in step (1) The time answered is 3～5h；

The time of the reaction described in step (2) is 10～16h；

The time of the reaction described in step (3) is 10～16h.

7. a type II diabetes resisting long-acing nano complex peptides, it is characterised in that by claim 1～6 One described preparation method prepares.

The application of type II diabetes resisting long-acing nano complex peptides the most according to claim 7, its feature exists In: described type II diabetes resisting long-acing nano complex peptides for preparing the medicine treating following disease: glycosuria Disease, hyperglycemia.

The application of type II diabetes resisting long-acing nano complex peptides the most according to claim 7, its feature exists In: described type II diabetes resisting long-acing nano complex peptides is applied to preparation treatment type Ⅱdiabetes mellitus relevant disease Medicine.