CN105669857B - A kind of quaternized chitosan oligosaccharide modifier of ES-2 peptide and the preparation method and application thereof - Google Patents
A kind of quaternized chitosan oligosaccharide modifier of ES-2 peptide and the preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention discloses a kind of quaternized chitosan oligosaccharide modifier of ES-2 peptide, by carboxyl and the amino of quaternized chitosan oligosaccharide molecule on ES-2 peptide molecule by amido bond in conjunction with, structural formula are as follows: (ES-2) n-CO-NH-O-HTCC；In formula, n=1~10；The molecular weight of ES-2 is 1223Da, isoelectric point 10.42；The molecular weight of O-HTCC is 1000Da or more, and free amino molar content is not less than 20%.The invention also discloses the preparation methods of the quaternized chitosan oligosaccharide modifier of ES-2 peptide.The quaternized chitosan oligosaccharide modifier of ES-2 peptide of the invention is compared with ES-2, remain ES-2 molecule Antiangiogenic activity and anti-tumor activity, the superperformance of O-HTCC is incorporated, makes it have stability height, the characteristic that long half time, cellular affinity is strong, targeting is strong etc.；Compared with TMC, O-HTCC modifies the more efficient of ES-2, therefore has better using effect and application range.
Description
Technical field
The present invention relates to quaternized chitosan oligosaccharide modifiers of a kind of ES-2 peptide and the preparation method and application thereof, belong to biological doctor
Medicine technical field.
Background technique
Endostatin (Endostatin, ES) is a kind of endogenic angiogenesis inhibitor factor, can be effectively
Inhibit the formation of new vessels, to cut off the nutrition supply approach and wastes metabolism approach of tumour, plays the life for inhibiting tumour
Long and transfer effect.ES-2 peptide (IVRRADRAAVP) be in ES structure one there is obvious Cancer therapy, antitumor
Active small peptide section, it is easier to enter cell interior, and be easier to obtain and transformation, but it is the same with ES, ES-2 peptide there is also
The disadvantages of Half-life in vivo is short and stability is poor, these disadvantages greatly limit ES-2 peptide and further apply.
Chitosan oligosaccharide is unique a kind of cationic polysaccharide existing for presently found nature, and structure is clear and has targeting,
It can be further improved its positively charged ability after quaternized.
In N, N, N- trimethyl quaternary ammonium chitosan (TMC) preparation process, the 2-NH of chitosan oligosaccharide2It is raw first after being methylated
At 2-NH (CH3) even 2-N (CH3)2, then can just be further formed 2-N (CH3)3 +Quaternary ammonium group, it is therefore, water-soluble good
Free amino amount in TMC is few (generally < 10%), leads to its, conjugate active holding lower to the joint efficiency of ES-2
Rate is poor, constrains quaternized chitosan oligosaccharide and is chemically modified to ES-2 peptide molecule.
Moreover, because ES-2 short peptide molecules amount is smaller, and isoelectric point position 10.42, belong to basic protein, so just with same band
The difficulty that the quaternized chitosan oligosaccharide of charge modifies it wants greatly more compared to the protein modified difficulty of other non-alkaline.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of quaternized chitosan oligosaccharide modifier of ES-2 peptide and its
Preparation method.
Since during tumour occurrence and development, the generation of new vessels can be tumor tissue with nutrients and transport useless
Object, so if inhibiting the generation of new vessels caused by tumour, so that it may play the role of " tumour hungry to death "；In diabetes etc.
In lysis, the generation of new vessels will lead to the severe reactions such as bleeding, can be bright for the inhibition of the generation of new vessels
The complication such as aobvious treatment patient's retina lesion.Therefore, it is a further object to provide the quaternized shells of the ES-2 peptide
Oligosaccharides modifier is preparing the application in Cancer therapy and/or anti-tumor drug.
To achieve the above object, the present invention adopts the following technical solutions:
A kind of quaternized chitosan oligosaccharide modifier of ES-2 peptide, by the carboxyl and quaternized chitosan oligosaccharide (O- on ES-2 peptide molecule
HTCC) amino of molecule is combined by amido bond, and structural formula is as follows:
(ES-2)n-CO-NH-O-HTCC；
In formula, n=1~10；The molecular weight of ES-2 is 1223Da, isoelectric point 10.42；The molecular weight of O-HTCC is
1000Da or more, free amino molar content are not less than 20%.
Preferably, n=1~5, i.e. a molecule O-HTCC and 1~5 molecule ES-2 covalent bond form modifier；O-
The molecular weight of HTCC is 1000Da~10000Da, and free amino molar content is not less than 50%.
The molecular weight of n-trimethyl chitosan chloride will affect the activity of modified ES-2 peptide, if the molecular weight of n-trimethyl chitosan chloride
It is excessive, it will cause serious steric hindrance and conformation change, significantly reduce the activity of the ES-2 peptide of small molecule；But quaternized shell
The molecular weight of glycan is also unsuitable too low, need to guarantee a certain amount of free amino amount.The present invention comprehensively considers modified ES-2
The activity of peptide and with n-trimethyl chitosan chloride Percentage bound, preferably the molecular weight of O-HTCC be 1000Da~10000Da, preferably protecting
While holding ES-2 peptide activity, the Percentage bound with n-trimethyl chitosan chloride is also improved.
Unless otherwise noted, the free amino of term-": primary amine group (i.e.-NH2), also known as level-one amido.
Term " quaternized chitosan oligosaccharide ": the C of chitosan oligosaccharide6It is grafted obtained from a group containing quaternary ammonium salt and spreads out on-OH
Biology.
The preparation method of the quaternized chitosan oligosaccharide modifier of above-mentioned ES-2 peptide, step are as follows:
(1) carbodiimide (EDC) is added into ES-2 peptide solution and n-hydroxysuccinimide (NHS) carries out carboxyl and lives
Change, the molar ratio of ES-2 peptide and EDC are 1:(1-100), the molar ratio of EDC and NHS are 3:(0.1-5), reaction temperature is 20 DEG C
It~30 DEG C, is slowly stirred and is protected from light 15~45min；
(2) solution after step (1) reaction is added in n-trimethyl chitosan chloride solution, keeps ES-2 peptide and quaternized shell few
The mass ratio of sugar is 1:(0.1~30), 4~8h is reacted, crude product is obtained, through isolating and purifying the quaternized chitosan oligosaccharide to get ES-2 peptide
Modifier.
In step (1), the ES-2 peptide solution is that ES-2 peptide is dissolved in solution obtained from PBS buffer solution；ES-2 peptide is molten
The pH of liquid is 4.0-6.0, and wherein the concentration of ES-2 peptide is 0.5~5mg/ml.
In step (2), the n-trimethyl chitosan chloride solution is molten obtained from PBS buffer solution for n-trimethyl chitosan chloride to be dissolved in
Liquid；The pH of n-trimethyl chitosan chloride solution is preferably 7.0-9.0, and wherein the concentration of n-trimethyl chitosan chloride is 1~5mg/ml.Due in
Property slight alkali environment be conducive to the carboxyl of ES-2 peptide molecule and the amino of quaternized chitosan oligosaccharide molecule amido bond formation, therefore this
The pH of n-trimethyl chitosan chloride solution is selected as 7.0-9.0 by invention.
It is described to isolate and purify in step (2) method particularly includes: to use SephadexG-25 or Sephadex30 gel layer
Analysis column is isolated and purified；It is 6.0 with pH, concentration 25mmol/L phosphate buffer carries out linear elution, flow velocity 1.0ml/
Min, Detection wavelength 254nm collect eluting peak component；It uses molecular cut off for the 48h that dialyses in 500~1000D bag filter, goes
Except salinity, freeze-drying.Due to SephadexG-25 and Sephadex30 gel chromatography column isolate and purify range be 0~
10000, ES-2 and the molecular weight of season ammonification chitosan oligosaccharide and modifier be exactly in the molecular ranges of both gel chromatography columns
Among, so the present invention selects SephadexG-25 or Sephadex30 to isolate and purify as gel chromatography column, separation
Purification effect is preferable.
Preferably, in step (1), the molar ratio of ES-2 peptide and EDC are 1: the molar ratio of (10~50), EDC and NHS are 3:
(0.2~2).
Preferably, in step (2), the mass ratio of ES-2 peptide and quaternized chitosan oligosaccharide is 1:(0.5~5).
Preferably, in step (2), the n-trimethyl chitosan chloride the preparation method comprises the following steps: chitosan is dissolved in aqueous acetic acid
In, ethyl alcohol is added, is stirred at room temperature, benzaldehyde sufficiently is added after swelling, milky glue reaction solution is made；By above-mentioned milky white coloring agent
Ground after shape reaction solution is dry, isopropanol be added, 2,3- epoxypropyltrimethylchloride chloride is reacted, reaction solution through filtering,
It after washing, drying, adds ethanol solution hydrochloride and is reacted, n-trimethyl chitosan chloride crude product is obtained after being precipitated, being dried；Above-mentioned season
Ammonium chitosan crude product is soluble in water, uses molecular cut off for the 48h that dialyses in 100~500D bag filter, and removal small molecule is miscellaneous
Matter, dialyzate freeze-drying, is made n-trimethyl chitosan chloride (O-HTCC).
2,3- epoxypropyltrimethylchloride chloride (GTMAC) is grafted to shell widow by n-trimethyl chitosan chloride prepared by the present invention
O- (hydroxyl) propyl -3- trimethyl ammonium chloride chitosan oligosaccharide (O-HTCC) derivative is obtained on sugared C6-OH, it is water-soluble more preferable, freely
Amino rate is 90% or more.
Carboxyl is activated using EDC-NHS, is the common method in organic synthesis, but is also difficult point place,
The factors such as the additional amount of EDC and NHS, the pH of reaction system, reaction temperature and reaction time can all influence the efficiency of activation；And
There is also larger differences for the Percentage bound of carboxyl and amino after different condition activation.The present invention is basic protein for ES-2 peptide,
Modification difficulty is relatively carried out to other non-alkaline albumen using the difficulty modified with positively charged n-trimethyl chitosan chloride it
Big more problems are wanted, the additional amount, reaction temperature and means of purification of EDC and NHS are optimized, the collaboration for passing through above-mentioned condition is made
With successfully preparing that stability is higher, half-life period is longer, cellular affinity is higher, the stronger n-trimethyl chitosan chloride-of bioactivity
ES-2 peptide modifier.
The quaternized chitosan oligosaccharide modifier of above-mentioned ES-2 peptide prepare with new vessels generate disease therapeutic agent and/
Or the application in anti-tumor drug is also protection scope of the present invention.Specifically, the adjoint new vessels generation disease includes
Diabetic retinopathy, treating senile maculopathy and arthritis etc..
Beneficial effects of the present invention:
(1) present invention can make by adjusting conditions such as EDC and NHS molar ratio, reaction temperature and the reaction time being added
It is standby to obtain the modifier of ES-2 Yu quaternized chitosan oligosaccharide difference Percentage bound.
(2) the quaternized chitosan oligosaccharide modifier of ES-2 peptide prepared by the present invention is compared with ES-2 peptide, and stability is higher, half
Declining, the phase is longer, cellular affinity is higher, bioactivity is stronger.
Detailed description of the invention
Fig. 1: the MALDI-TOF-MS mass spectrum mirror of the quaternized chitosan oligosaccharide modifier of ES-2 peptide prepared by the embodiment of the present invention 2
Determine result.
Fig. 2: the result of suppressing cell reproduction test.
Fig. 3: inhibiting endothelial cell migration test result, and in figure, a is blank, and b is mixture, c ES-2, d O-
HTCC-ES-2。
Fig. 4: heat stabilization test result.
Fig. 5: cellular affinity test result.
Specific embodiment
The present invention is further illustrated in conjunction with the embodiments, it should which explanation, following the description is merely to explain this
Invention, is not defined its content.
Raw material sources:
Chitosan oligosaccharide, ordinary commercial products, viscosity average molecular weigh 3000Da；
ES-2 peptide, ordinary commercial products, molecular weight 1223Da.
Embodiment 1: the preparation of n-trimethyl chitosan chloride
The preparation method of n-trimethyl chitosan chloride, steps are as follows:
1. 3g chitosan oligosaccharide is taken to be dissolved in the aqueous acetic acid that 120ml mass concentration is 10%, ethyl alcohol 60ml is added, room temperature is stirred
30min is mixed, is sufficiently swollen, benzaldehyde 15.8g (15.105ml) is added dropwise, continues to be stirred to react 1h, it is gluey that milky is made
Reaction solution adjusts pH to neutrality in 60 DEG C of oven drying 20h, and filtering is collected precipitating, washed with methanol, dries, and reaction is made
Object A；
2. setting in a round bottom flask after step 1. 2.75g reactant A grinding obtained, 50ml isopropanol, stirring being added
9g 2 is added in 30min, and 3- epoxypropyltrimethylchloride chloride, 70 DEG C of oil bath return stirring 16h, reaction solution filtering, what is obtained consolidates
Body successively uses methanol, acetone washing, and reactant B is made in drying；
3. setting the salt for adding 50ml 0.25mol/L in a round bottom flask after step 2. 2.7g reactant B grinding obtained
Sour ethanol solution after being stirred to react for 24 hours at room temperature, boils off most of solvent, adds 15ml water, after completely dissolution, was added
Acetone to be measured, is precipitated, precipitating is collected in filtering, and it is dry, n-trimethyl chitosan chloride (O-HTCC) crude product is made.By step 3. 1g obtained
O-HTCC crude product is then dissolved in water, uses molecular cut off for the 48h that dialyses in 100~500D bag filter, and removal small molecule is miscellaneous
Matter, dialyzate freeze-drying, is made n-trimethyl chitosan chloride (O-HTCC).
Through detecting, the molecular weight of n-trimethyl chitosan chloride manufactured in the present embodiment is 3000Da, and free amino content is
91.4%.
The preparation of the quaternized chitosan oligosaccharide modifier of embodiment 2:ES-2 peptide
Preparation step is as follows:
(1) ES-2 peptide 50mg is dissolved in the PBS buffer solution that 25ml concentration is 10mM, pH 5.0, ES-2 peptide solution is made,
The concentration of ES-2 peptide is 2.0mg/ml；80mg EDC and 20mg NHS, concussion reaction 30min under greenhouse are added, reaction is made
Liquid A；
(2) weighing quaternized chitosan oligosaccharide (O-HTCC) 25mg made from embodiment 1 to be dissolved in 25ml concentration is 10mM, pH 8.0
PBS buffer solution in, be made quaternized chitosan oligosaccharide solution, the concentration of O-HTCC is 1mg/ml, and it is obtained that step (1) is then added
Reaction solution A, concussion reaction 6h, the SephadexG-25 gel through 1.6 × 100cm are isolated and purified, with pH 6.0, concentration
25mmol/L phosphate buffer carries out linear elution, and flow velocity 1.0ml/min, Detection wavelength 254nm collect each elution fraction,
Quaternized chitosan oligosaccharide-ES-2 peptide modifier is made.
Using MALDI-TOF-MS Mass Spectrometric Identification as a result, result is as shown in Figure 1, the results showed that most ES-2 are logical
It crosses-CO-NH- to be modified by O-HTCC, the modification rate of ES-2 molecule about 85%.
The preparation of the quaternized chitosan oligosaccharide modifier of embodiment 3:ES-2 peptide
Preparation step is as follows:
(1) ES-2 peptide is dissolved in the PBS buffer solution of 50ml concentration 10mM, pH 5.0, ES-2 peptide solution, ES-2 peptide is made
Concentration be 2.0mg/ml (1.635 μm of ol/ml)；80mg EDC and 20mg NHS, concussion reaction 30min under greenhouse are added,
Reaction solution A is made；
(2) weighing quaternized chitosan oligosaccharide (O-HTCC) 50mg made from embodiment 1 to be dissolved in 25ml concentration is 10mM, pH 8.0
PBS buffer solution in, chitosan oligosaccharide solution is made, the concentration of O-HTCC is 2.0mg/ml, and step (1) reaction obtained is then added
Liquid A, concussion reaction 6h, the SephadexG-25 gel through 1.6 × 100cm are isolated and purified, with pH 6.0, concentration
25mmol/L phosphate buffer carries out linear elution, and flow velocity 1.0ml/min, Detection wavelength 254nm collect each elution fraction,
Quaternized chitosan oligosaccharide-ES-2 peptide modifier is made.
Using MALDI-TOF-MS Mass Spectrometric Identification as a result, showing that most ES-2 pass through amido bond and repaired by O-HTCC
The modification rate of decorations, ES-2 molecule is greater than 70%.It should be noted that the value of modification rate is not to be the bigger the better, but it will basis
Actual needs is adjusted, but too low modification rate is unobvious for the raising effect of modified outcome compatibility and stability,
Also can be limited for the prolongation effect of half-life period, general recommendations modification rate is higher than 70%.
The preparation of the quaternized chitosan oligosaccharide modifier of embodiment 4:ES-2 peptide
Preparation step is as follows:
(1) ES-2 peptide (ES-2) is dissolved in the PBS buffer solution of 25ml concentration 10mM, pH 6.0, ES-2 peptide solution is made,
The concentration of ES-2 peptide is 2.0mg/ml；40mg EDC and 10mg NHS, concussion reaction 30min under greenhouse are added, reaction is made
Liquid A；
(2) weighing quaternized chitosan oligosaccharide (O-HTCC) 50mg made from embodiment 1 to be dissolved in 25ml concentration is 10mM, pH 8.0
PBS buffer solution in, chitosan oligosaccharide solution is made, the concentration of O-HTCC is 2.0mg/ml, and step (1) reaction obtained is then added
Liquid A, concussion reaction 6h, the SephadexG-25 gel through 1.6 × 100cm are isolated and purified, with pH 6.0, concentration
25mmol/L phosphate buffer carries out linear elution, and flow velocity 1.0ml/min, Detection wavelength 254nm collect each elution fraction,
Quaternized chitosan oligosaccharide-ES-2 peptide modifier is made.
In this experimental example, the amount of EDC and NHS, which are compared, have been carried out halving processing in other examples, through MALDI-TOF-MS mass spectrum
Identification, most of ES-2 are modified yet by amido bond by O-HTCC, and modification rate is greater than 50%.But due to the input amount of EDC and NHS
It is of great significance for modification, it is lower that this example compares other embodiments modification rate.
Comparative example 1:
N, N, N- trimethyl quaternary ammonium chitosan oligosaccharide (TMC)-ES-2 peptide modifier are prepared using existing EDC condensation method.
TMC 200mg, ES-2 40mg that free amino rate is 23.2% are weighed, is dissolved in the 0.1mol/L's of pH7.4 respectively
Na2HPO4-NaH2PO4In buffer 10ml, TMC solution is slowly added into ES-2 solution, is slowly stirred at 25 DEG C, in
30min is separately added into EDCHCl 100mg, and overnight, 4 DEG C of dialysis, SephadexG-25 gel is separated for 4 DEG C of dark place reactions
Purifying.
Using MALDI-TOF-MS Mass Spectrometric Identification as a result, showing that the modification rate of ES-2 molecule is only 20%.
Comparative example 2:
ES-2 peptide and the EDC molar ratio being added are adjusted to 1: 5, are 3:0.1 by the molar ratio of EDC and NHS, ES-2 peptide with
The mass ratio of quaternized chitosan oligosaccharide is 1:0.2；Quaternized chitosan oligosaccharide-ES-2 peptide modifier is made with embodiment 2 in remaining operation.
Using MALDI-TOF-MS Mass Spectrometric Identification as a result, showing that the modification rate of ES-2 molecule is only 35%.
Comparative example 3:
It is 1:1, ES-2 peptide and season by the molar ratio that ES-2 peptide and the EDC molar ratio being added are adjusted to 1: 60, EDC and NHS
The mass ratio of ammonium chitosan oligosaccharide is 1:6；Quaternized chitosan oligosaccharide-ES-2 peptide modifier is made with embodiment 2 in remaining operation.Using
MALDI-TOF-MS Mass Spectrometric Identification is as a result, show that the modification rate of ES-2 molecule is only 45%.
The conditions such as molar ratio, reaction temperature and the reaction time that EDC and NHS is added it can be seen from above-mentioned comparative example, can
To directly affect the Percentage bound of ES-2 Yu quaternized chitosan oligosaccharide.
Test example:
1. suppressing cell reproduction is tested
(1) trial drug: the quaternized chitosan oligosaccharide modifier of ES-2 peptide prepared by embodiment 2, ES-2 and ES-2 peptide
Quaternized chitosan oligosaccharide modifier is the mixture that 1:1 is mixed with ES-2 in mass ratio.
(2) test method:
Logarithmic phase Human umbilical vein endothelial cells EAhy926 is collected, adjustment concentration of cell suspension is 10000/ hole, is divided in 96 holes
Plate, every 200 μ L of hole (the DMEM culture medium containing 10%FBS), is placed in 37 DEG C, 5%CO2Constant incubator in culture paste cell
Wall；The trial drug of various concentration gradient is added, 5 multiple holes of every kind of drug continue to cultivate 48h；CCK-8 solution 10 is added in every hole
μ L, 37 DEG C are continued termination culture after 2~4h of culture, and enzyme-linked immunosorbent assay instrument measures each hole absorbance (OD) at wavelength 450nm
Value, and calculate its inhibiting rate: [(experimental group-blank group)/(control-blank group) of inhibiting rate=1-.It repeats experiment three times, makes even
Mean value.
Suppressing cell reproduction test result see Fig. 2, as seen from Figure 2, the ES-2 peptide of various concentration gradient it is quaternized
After acting on 48h, the anti-cell of O-HTCC-ES-2 modifier increases by chitosan oligosaccharide modifier (O-HTCC-ES-2 modifier) and ES-2
Function and effect are grown obviously to be eager to excel much than the ES-2 of homogenous quantities concentration and the two mixture.
2. inhibiting endothelial cell migration test
(1) trial drug: the quaternized chitosan oligosaccharide modifier of ES-2 peptide prepared by embodiment 2, ES-2 and ES-2 peptide
Quaternized chitosan oligosaccharide modifier is the mixture that 1:1 is mixed with ES-2 in mass ratio.
(2) test method:
Logarithmic phase Human umbilical vein endothelial cells EAhy926 is collected, adjustment concentration of cell suspension is 5 × 104/ hole, is inoculated in 6
In orifice plate, every hole 2ml is placed in 37 DEG C, 5%CO2Incubator in overnight incubation；Ruler is put into ultraviolet photograph in super-clean bench in advance
After penetrating 30min, 6 orifice plates cell is taken, supernatant is abandoned, is cleaned 2 times with PBS, is gently drawn while sterile ruler compares with yellow pipette tips
Cell surface is crossed, a cell-free straight line is formed, 3 straight lines are drawn in each hole, and PBS cleans 2 times to remove the cell crossed out.Point
Not Jia Ru the O-HTCC-ES-2 modifier and ES-2 that contain of 2ml DMEM culture medium (containing 10%FBS), and using PBS as negative
Control, drug concentration is adjusted to 125 μ g/mL, and bFGF is added makes its final concentration of 5ng/ml, every kind of drug be arranged 3 it is parallel
Hole is placed in CO2It is cultivated respectively in constant incubator for 24 hours and 48h.6 orifice plates are taken out, supernatant is abandoned, is placed under inverted fluorescence microscope
It takes pictures, observes the width of scratch.Test 5 times is repeated, is averaged.
The result of endothelial cell migration test is inhibited to see Fig. 3, as seen from Figure 3, under identical drug concentration effect, O-
HTCC-ES-2 inhibits the effect of endothelial cell migration most strong.
3. heat stabilization test
Suitable ES-2 and O-HTCC-ES-2 (preparation of embodiment 2) is taken to be dissolved in the PBS buffering of isometric pH7.4 respectively
It in liquid, is incubated for respectively at 37 DEG C of water-baths, is sampled respectively at different time points, measure it according to CCK-8 method and endothelial cell is increased
The inhibitory activity grown.Retain hundred for activity is calculated compared with inhibiting rate of the inhibiting rate for the different time points surveyed when the 0min
Divide ratio, as ordinate, using the time as abscissa, draws stability curve.
As a result see Fig. 4, it can be seen that after O-HTCC-ES-2 modifier and ES-2 are incubated for same time in 37 DEG C of environment,
The retentive activity percentage of O-HTCC-ES-2 modifier is higher, that is, its thermal stability is better than ES-2.
4. cellular affinity is tested
(1) trial drug: the quaternized chitosan oligosaccharide modifier of ES-2 peptide prepared by embodiment 2, ES-2 and ES-2 peptide
Quaternized chitosan oligosaccharide modifier is the mixture that 1:1 is mixed with ES-2 in mass ratio.
(2) test method:
1) FITC marks Endostatin and its conjugate
O-HTCC-ES-2 modifier, ES-2 and each 3mg of the two mixture are weighed, after being dissolved respectively with distilled water l mL,
To 9~9.5 carbonate buffer solution dialysed overnight of pH, solution liquid is moved into small beaker after dialysis: being weighed FITC 1mg, is added two
First sulfoxide (DMSO) 1mL, makes final concentration of 1mg FITC/1mL DMSO, is about 50 μ gFITC/1mg albumen according to FITC/ES-2
The ratio of matter FITC-DMSO solution is added dropwise in the ES-2 and its modifier, mixture solution after dialysis, by marker
It with PBS plus sets to 2.5mL, magnetic stirring apparatus is protected from light stirring 2h at room temperature, removes free fluorescein with Scphadex G10 column, first
PBS is collected with 25mLPBS elution G10 column and elutes first fluorescein binding protein peak, measures F/P ratio.It calculates: F/P=
2.87 × A495/ [A280-0.35 × A495], which should be between 2.5~4.5.
2) the FITC marker pair of flow cytomery O-HTCC-ES-2 modifier, ES-2 and the two mixture
The binding ability of EAhy926
Endothelial cell is inoculated in (30 × 10 in 12 orifice plates4A/hole), serum free medium is added (after including FITC label
O-HTCC-ES-2 modifier, ES-2 and the two mixture, each medication group is according to 125 μ g/mL of protein concentration) l mL.Respectively
In 37 DEG C, 5%CO22h is incubated in incubator, the PBS of frost is rinsed 3 times, is transferred in streaming pipe, and flow cytometer measurement is each
Group positive rate.As a result see Fig. 5, ES-2 is obviously increased after O-HTCC is modified with endothelial cell compatibility, and drug is easier to enter
Cell plays a role.
Compared with natural ES-2, ES-2 is after O-HTCC is modified by amido bond, the O- of identical mass concentration gradient
HTCC-ES-2, inhibition of endothelial cell proliferation and migration etc. effect in terms of, than ES-2 itself have it is apparent enhancing and
It improves；The thermal stability of O-HTCC-ES-2 has compared with ES-2 to be significantly increased, and furthermore the cellular affinity of O-HTCC-ES-2 also obtains
Very big improvement is arrived, drug is more easier to carry out cell, plays drug effect.
Claims (8)
1. a kind of preparation method of the quaternized chitosan oligosaccharide modifier of ES-2 peptide, which is characterized in that step are as follows:
(1) molar ratio of the addition EDC and NHS progress activated carboxylic into ES-2 peptide solution, ES-2 peptide and EDC are 1: (10~
50), the molar ratio of EDC and NHS is 3:(0.2~2), 30min is stirred and is protected from light at room temperature, and the ES-2 peptide sequence is
IVRRADRAAVP；
(2) solution after step (1) reaction is added in quaternized chitosan oligosaccharide solution, makes ES-2 peptide and quaternized chitosan oligosaccharide
Mass ratio is 1:(0.5~5), concussion reaction 6h obtains crude product, through isolating and purifying the quaternized chitosan oligosaccharide modification to get ES-2 peptide
Object.
2. preparation method as described in claim 1, which is characterized in that in step (1), the pH of the ES-2 peptide solution is 5.0,
Wherein the concentration of ES-2 peptide is 0.5~5mg/ml.
3. preparation method as described in claim 1, which is characterized in that in step (2), the pH of the quaternized chitosan oligosaccharide solution
It is 8.0, wherein the concentration of quaternized chitosan oligosaccharide is 1~5mg/ml.
4. preparation method as described in claim 1, which is characterized in that in step (2), the specific method isolated and purified
Are as follows: it is isolated and purified using SephadexG-25 or Sephadex30 gel chromatography column；It is 6.0 with pH, concentration 25mmol/L
Phosphate buffer carries out linear elution, and flow velocity 1.0ml/min, Detection wavelength 254nm collect eluting peak component.
5. preparation method as described in claim 1, which is characterized in that in step (2), the preparation side of the quaternized chitosan oligosaccharide
Method are as follows: chitosan oligosaccharide is dissolved in aqueous acetic acid, ethyl alcohol is added, is stirred at room temperature, benzaldehyde sufficiently is added after swelling, is made milky white
Coloring agent shape reaction solution；It will be ground after the drying of above-mentioned milky glue reaction solution, isopropanol, 2,3- epoxy chlorine be added
Change ammonium to be reacted, reaction solution after filtration, washing and drying, adds ethanol solution hydrochloride and reacted, and is precipitated, is dried
Afterwards quaternized chitosan oligosaccharide crude product；Above-mentioned quaternized chitosan oligosaccharide crude product is soluble in water, uses molecular cut off saturating for 100~500D
Dialyse 48h in analysis bag, removes small molecular weight impurity, and quaternized chitosan oligosaccharide is made in dialyzate freeze-drying.
6. the ES-2 peptide that the preparation method of the quaternized chitosan oligosaccharide modifier of any one of the claim 1-5 ES-2 peptide obtains
Quaternized chitosan oligosaccharide modifier.
7. the quaternized chitosan oligosaccharide modifier of ES-2 peptide as claimed in claim 6 is preparing controlling with new vessels generation disease
Treat the application in drug and/or anti-tumor drug.
8. the use as claimed in claim 7, which is characterized in that it includes diabetes view that the adjoint new vessels, which generate disease,
Film lesion, treating senile maculopathy or arthritis.
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| CN107261124B (en) * | 2017-06-09 | 2020-05-22 | 山东大学 | Preparation method and application of angiogenesis inhibiting peptide and hyaluronic acid modifier thereof |
| CN113499447B (en) * | 2021-07-07 | 2024-04-30 | 山东大学 | CRGD-quaternized chitosan oligosaccharide modified ES2 peptide-methotrexate conjugate, and preparation method and application thereof |
| CN114306340B (en) * | 2021-12-14 | 2023-03-07 | 山东大学 | Preparation method and application of cholic acid-quaternized chitosan oligosaccharide-ES 2 peptide/camptothecin conjugate |
| CN114163549B (en) * | 2022-01-06 | 2022-10-04 | 苏州乙水茉生物科技有限公司 | Chitosan oligosaccharide and bivalent plant vaccine covalent conjugate and preparation method and application thereof |
| CN115353555B (en) * | 2022-08-22 | 2024-04-30 | 山东大学 | Quaternized chitosan oligosaccharide-mu-conotoxin conjugate as well as preparation method and application thereof |
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