CN106511271A - KGM (Konjac Glucomannan)-g (grafted)-AH (Alicyclic Amine) drug loaded nano micelle and preparation method - Google Patents
KGM (Konjac Glucomannan)-g (grafted)-AH (Alicyclic Amine) drug loaded nano micelle and preparation method Download PDFInfo
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Abstract
The invention relates to synthesis of a KGM (Konjac Glucomannan)-g(grafted)-AH (Alicyclic Amine) material and preparation of drug loaded nano micelle thereof, and belongs to the technical field of biological materials and slow release. The synthesis of the KGM-g-AH material comprises the following steps: firstly carrying out oxidization ring opening on KGM by using periodate, thus obtaining DAK (Dialdehyde KGM); then enabling the DAK to react with alicyclic amine, thus obtaining amphipathic KGM-g-AH. A grafted copolymer can be assembled to form nano micelle through an ethanol injection method; the nano micelle has pH (Potential of Hydrogen) sensitivity; in a neutral environment of normal tissues and blood, the nano micelle is relatively stable; in a weak acid environment of tumor tissues, imine bonds of molecules of the nano micelle are in reversible breakage, loaded drugs can be rapidly released, and the loaded drugs can be gathered in tumor cells, so that site-specific delivery of the drugs on the tumor tissues is realized. The prepared KGM-g-AH drug-loaded nano micelle is stable in structure, simple to synthesise, high in drug loading capacity, good in biocompatibility and low in cytotoxicity, has pH sensitivity and has a wide application prospect in the field of drug controlled release.
Description
Technical field
The present invention relates to a kind of synthesis of the grafting of the fatty amine with pH sensitiveness Konjac Glucomannan and its load medicine
The preparation of nano-micelle, belongs to biomaterial and slow release method field.
Background technology
With expanding economy and the increasingly destruction of ecological environment, the disease of cancer is sent out rate also more and more higher, threatens people
Health.Chemotherapy is the treatment method of most widely used cancer, very fast for the killing and suppression of cancer cell
Speed effectively, and has the advantages that globality, comprehensive therapy, but there is also many problems, such as medicine poorly water-soluble, administration
Poor selectivity in vivo afterwards, with multi-drug resistant, circulation time is short, and bioavailability is low（Expert opinion on
drug delivery,2012,9:687-700）.In order to solve these problems, micron and nanoscale drug delivery system such as
Vesica, micella and microcapsules etc. are widely studied in recent years and are developed rapidly, to improve drug effect and reduce toxicity.Especially
Nano level micella, has many advantages and is widely paid close attention to, such as can be with direct injection without the risk of blocking blood vessel
The time of drug cycles can be extended with relatively low blood clearance, strengthen infiltration and stick effect（EPR）Effect, is allowed in tumour
Tissue retention is simultaneously enriched with, and medicine is limited in tumor locus as far as possible, is reduced while the lethality that improve drug on tumor
The infringement of medicine normal tissue（Nanomedicine : nanotechnology, biology and medicine,
2010,6:714-729;European journal of pharmaceutics and biopharmaceutics, 2007,
65:259-269）.But people are systematically expected far from staying in this for ideal carrier.
The research of the carriers of response type such as environmental response type delivery system such as pH, enzyme, temperature is opened for the administration of chemicals
New approaches are opened up, whole carrier system can be made to become the participant of an active during chemotherapy.Tumor locus are with nothing
Oxygen glycolysis provides energy, with the gradually increase of lactic acid, makes tumor locus extracellular fluid pH to value between 5-6, compared to
Liquid outside normal histocyte（pH=7.4）Slant acidity.Therefore, pH is conventional to prepare antineoplastic environmental response for designing
One of factor of property carrier.
Natural polysaccharide has high safety, nontoxic, and the features such as biodegradability and good biocompatibility, and it is wide to originate
General, processing cost is low, is the drug carrier material that a class has preferable application prospect.Containing a large amount of on the strand of natural polysaccharide
Active group, such as hydroxyl, carbonyl, amido etc. are conducive to its chemistry and bio-modification to give its amphipathic and pH sensitiveness
（Carbohydrte polymers, 2015, 123:53-66）.The C=N double bonds contained due to schiff bases are in neutral and alkaline bar
It is stable under part, and selective fracture in acid condition, it is widely used in the sensitive pharmaceutical carriers of pH in recent years
（Polymer chemistry, 2012, 3:3045-3055 ）.For example with the alginic acid of the doubly linked glycosyl galactoses of C=N
Sodium-curcumin pH sensitivity micellas（International journal of biological macromolecules,2016,
86:1-9）, aoxidize the gel carrier that KGM is connected with C=N double bonds with shitosan（Reactive & functional
polymers, 200666.10:1055-1061）, aoxidize the C=N double bonds that KGM is formed as bridge with oneself two hydrazines with aminosalicylic acid
PH sensitive carriers（Polymer bulletin, 2014, 15.6:183-193）Deng.
Konjaku glucomannan（KGM）In being a kind of a kind of hydrophilic natural degradable extracted from konjac tuber
Property polysaccharide, is widely used in pharmaceutical carrier the features such as with nontoxic, biodegradable, good biocompatibility
（Carbohydrate polymers, 2005, 60:27-31）.For example, treat can dissolving in stomach for chronic gastropathy
KGM capsules（Patent CN 1275619）, the pharmaceutical carrier of site specific DDS for colon（Polymer, 2005, 46.16:6274-6281）,
KGM- polymethylacrylic acid gel carriers（Carbohydrate polymers, 2013,97.2:565-70）Deng.But KGM
Strongly hydrophilic limits its application in pharmaceutical carrier, and the KGM of hydrophobically modified is remarkably improved which to lipophilic drugs
Ability is contained, so as to increase its range of application.Such as the Carboxymethyl Konjac Glucomannan graft containing cholesterol and its micella
Prepare（Patent CN 102399301）, aoxidize the balloon borne body of micro emulsion of photoresponse prepared by KGM（Biomacromoecules,
2014, 15.6:2166-71）, the preparation of Carboxymethyl Konjac Glucomannan nano drug-carrying microsphere（Patent CN 105055372A）.
Modified K GM can extend curative effect of medication with Drug controlled release, reduce poisonous side effect of medicine, improve hydrophobic medicine as pharmaceutical carrier
Thing is to membrane passage and the stability and change method of administration of medicine.At present, contain C=N with KGM as prepared by base material
The report of the pH sensitivity micellas of double bond is less.
The content of the invention
It is an object of the invention to provide one kind is with dialdehyde-based konjaku glucomannan（DAK）With containing that fatty amine reaction is obtained
The konjaku glucomannan of the hydrophobic grafts of C=N double bonds（KGM-g-AH）, further make the load curcumin with pH sensitiveness and receive
The preparation of rice glue beam and detection method.
The present invention carries out hydrophobically modified to hydrophilic KGM so as to amphipathic by the synthesis of two steps, and can be from group
Dress is formed with hydrophobic aliphatic chain as core, nanosized micelles systems of the hydrophilic KGM as shell, is remarkably improved which to hydrophobic drug
Contain and solubilising power.In addition, the micellar system is stable in neutral environment, and may be selected in weakly acidic tumor microenvironment
Property rupture, and quick release goes out the medicine of package-contained so as in tumor cell enrichment, so as to realize the control release of medicine, and
The toxic and side effect of medicine is effectively reduced, drug effect is improved.
The purpose of the present invention is achieved through the following technical solutions, and a kind of fatty amine is grafted konjaku glucomannan medicament-carried nano glue
The preparation method of beam, comprises the steps：
（1）Dialdehyde-based konjaku glucomannan（DAK）Preparation：Weigh KGM to be scattered in distilled water, in the bar of room temperature mechanical stirring
Under part, swelling 8-24 h, are then dissolved in sodium metaperiodate in distilled water, and are added drop-wise in KGM dispersion liquids, KGM and sodium metaperiodate
Mass ratio be 1:0.4-1:1.5（KGM is 1 with the mass ratio of sodium metaperiodate:When 0.4, the oxidizability of DAK is 25%, Weight-average molecular
Measure as 20000-400000 g/molKGM, KGM is 1 with the mass ratio of sodium metaperiodate:When 0.8, the oxidizability of DAK is 45%, and weight is equal
Molecular weight is 8000-10000 g/mol）, room temperature lucifuge stirring reaction 12-48 h, 40-60 DEG C of reduced pressure concentration are filtered, and are turned
Dialysis in moving on to bag filter removes salt and small molecule product, and freeze-drying had both obtained the DAK of white.
（2）The preparation of the konjaku glucomannan of fatty amine grafting（KGM-g-AH）：By step（1）The DAK dissolvings of middle preparation
In a small amount of water, by fatty amine（The chain length of fatty amine is shorter, KGM-gMicella particle diameter prepared by-AH is less, more stable）Dissolving
In ethanol or hexamethylene, and it is added in DAK solution, flow back at the boiling point of ethanol or hexamethylene 6-18 h, removes reaction bulb,
30-40 DEG C is removed under reduced pressure ethanol or hexamethylene, with the organic reagent immiscible with water（Ethyl acetate or chloroform）Wash three times,
Organic phase reduced pressure concentration is taken, then dialysis removes unreacted amine, and freeze-drying had both obtained the KGM- of browng- AH samples；
Described dialdehyde-based konjaku glucomannan with the rate of charge of fatty amine is：The aldehyde radical of dialdehyde-based konjaku glucomannan and fat
The ratio of the amount of the material of the amido of amine is 1:1；
Described fatty amine is straight-chain fatty amine, short chain amine in being more preferably dissolvable in water ethanol or methyl alcohol, be can dissolve
Long-chain amine in hexamethylene；
Described short chain amine is preferably octylame, lauryl amine, and long-chain amine is preferably octadecylamine.
（3）The preparation of the konjaku glucomannan micella/carrier micelle of fatty amine grafting：
1. alcohol injection：By KGM-g- AH is dissolved in ethanol, in the condition injection distilled water of magnetic agitation, is stirred at room temperature
Certain hour, dialyses in distilled water in proceeding to bag filter and removes ethanol, and every 4 h changes a water, takes dislysate 200-400
W ultrasound 3-10 min, cross 0.45 μm and 0.22 μm of miillpore filter obtains micellar solution.
2. dialysis：The KGM- for being dissolved in ethanol or chloroform of measured amountsg- AH, proceeds to certain molecular cut off
In bag filter, room temperature is dialysed two days, takes 200-400 W ultrasound 3-10 min of dialyzed solution, crosses 0.22 μm and 0.45 μm of micropore
Filter membrane obtains micellar solution.
3. the preparation of carrier micelle：By curcumin ethanol solution and KGM-g-AH ethanol or chloroformic solution are well mixed, ginger
Flavine is 1 with the mass ratio of KGM-g-AH:20～2:5, inject under conditions of magnetic agitation in the distilled water of 10 mL, room temperature
30 min are stirred, is dialysed 2 days in distilled water in proceeding to bag filter, every 4 h changes a water, takes 200 W of dislysate ultrasound 3-
10 min, cross 0.45 μm and 0.22 μm of miillpore filter obtains carrier micelle.
（4）Tablets in vitro is tested:Polypeptide drug-loaded micelle solution is transferred in bag filter, is then immersed in containing 0.5-
2.0%（w/v）Tween 80 pH=5.0 and pH=7.4 phosphate buffer in, and under the conditions of being placed on 37 DEG C a period of time,
The buffer solution for taking out certain volume under the time for setting determines its light absorption value at 424 nm, while supplementing same volume phase
Synthermal fresh buffer solution.
（5）Cytotoxicity experiment:By cryodesiccated KGM-g-AH8Micella and 20 μ L are dissolved in the MTT of PBS
（5 μg/mL）With cultured 4 h of HepG2 cell culture, KGM-g-AH8The concentration of micella is 2-125 μ g/mL, then will be thin
Born of the same parents are rinsed well with the fresh cell culture medium of 200 μ L, remove MTT, then dissolve first with 200 μ L DMSO, 570
Its optical density is determined at nm.
（6）Cell phagocytosis experiment:HepG2 cells are dispersed in the cell culture fluid containing 10% hyclone and 1% antibiotic
In, then with 1 × 105The culture of cells/well density is in 6 orifice plates.Then at 37 DEG C, 5% CO2Under conditions of cultivate 24 h, then
Its culture medium is rinsed well, respectively with the KGM-g-AH marked containing 10% hyclone, 1% antibiotic, FITC8Micella 1
ML cell culture mediums and 10% hyclone, 1% antibiotic, the KGM-g-AH of FITC marks81 mL cell culture mediums of carrier micelle
4 h are cultivated, culture medium is then removed, then with dye 15 min of nucleus of Hoechst 33342 of 50 μ L, culture is removed and is situated between
Matter, is rinsed 3 times with 1 mL PBS cushioning liquid, then cell is put under laser co-focusing and is observed.
For prior art, beneficial effects of the present invention are as follows：
1st, the present invention carries out hydrophobically modified with hydrophilic konjaku glucomannan as sill to which so as to amphipathic, be modified
The preparation of the konjaku glucomannan of fatty amine grafting afterwards（KGM-g-AH）Can be self-assembly of with hydrophobic aliphatic chain as core, it is hydrophilic
KGM is the nanosized micelles system of shell, is remarkably improved konjaku glucomannan containing and solubilising power to hydrophobic drug；Separately
Outward, the micella also contains C=N double bonds, and alternative ruptures in sour environment, with pH sensitiveness, when carrier micelle reaches cancer
When becoming tissue, weakly acidic tumor environment makes the degraded of carrier micella shell and quickly removes, so as to discharge the medicine of package-contained,
It is allowed to be enriched in tumor tissues, realizes passive target.
2nd, the size of nano-particle can be adjusted by the length of regulation aliphatic chain, and the chain length of fatty amine is shorter, KGM-
Micella particle diameter prepared by g-AH is less, more stable（Fig. 3）.
3rd, nano-micelle particle prepared by the present invention, can be used for pharmaceuticals industry, with preferable biocompatibility and biology
Degradability, the application for konjaku glucomannan as pharmaceutical carrier in pharmaceuticals industry provide new approaches.
The polymer of the surface carbohydrates modification that the 4th, prepared by the present invention can be self-assembly of nano level spherical micelle particle, have
There are preferably dispersiveness and stability.
Description of the drawings
Fig. 1 fatty amines are grafted the infrared spectrogram of KGM.A, b, c, d, e in figure refers to KGM, DAK respectively, octylame grafting
KGM, the KGM of lauryl amine grafting and the KGM, R=- (CH of octadecylamine grafting2)nCH3。
Fig. 2 fatty amines are grafted the nuclear magnetic spectrum of KGM.A, b, c, d in figure refers to DAK, the KGM of octylame grafting, 12 respectively
The KGM of the amine grafting and KGM of octadecylamine grafting.
The transmission electron microscope picture of the nano-micelle of Fig. 3 fatty amines grafting KGM.A, b in figure refers to prepared by alcohol injection respectively
Octylame grafting KGM unentrappeds curcumin and contain curcumin micella pattern, c refer to dialysis prepare octylame grafting
The pattern of KGM micellas, d, e refer to the pattern of the KGM micellas of the lauryl amine of alcohol injection preparation and octadecylamine grafting respectively.
The control release figure of the nano-micelle of Fig. 4 fatty amines grafting KGM.A, b in figure refers to octylame grafting KGM bags respectively
Carry curcumin after pH=5.0 and pH=7.4 elution profiles.
The cytotoxicity figure of the nano-micelle of Fig. 5 octylames grafting KGM.
The cellular uptake figure of the nano-micelle of Fig. 6 octylames grafting KGM.
Specific embodiment
Further detailed description is made with reference to embodiment and description of the drawings, but the embodiment invented is not limited to
This.
Embodiment 1：Dialdehyde-based konjaku glucomannan（DAK）Preparation
The KGM of 3.00 g is weighed, is scattered under conditions of mechanical agitation in 500 mL distilled waters, then swelling 12 under room temperature
H, is obtained KGM dispersion liquids；Weigh 2.40 g sodium metaperiodates to be dissolved in 100 mL distilled waters, be added dropwise in KGM dispersion liquids, lucifuge
24 h are reacted under room temperature condition, reaction bulb is removed, and be evaporated to 300 mL or so, transfer them under the conditions of 55 DEG C
In the bag filter of 3500 molecular cut offs, dialyse 7 days in double steamings, by dialyzed solution freeze-drying both white dialdehyde-based is magic
Taro Glucomannan（DAK）.
As shown in the infared spectrum b of Fig. 1, in 3429 cm-1There is-OH a stretching vibration peak at place, in 2938 cm-1Place has
One C-H stretching vibration peak, in 1733 cm-1There is a carbonylic stretching vibration peak at place, after illustrating KGM Jing sodium periodate oxidations
Generate C=O.As shown in the nuclear magnetic spectrum of Fig. 2, it can be seen that on the peak of 9.20-9.28 ppm is DAK from Fig. 2 a
The proton peak of aldehyde radical, 3.81-4.97 ppm are the anomeric proton peak on sugared ring.
Embodiment 2：Octylame is grafted the preparation of KGM（KGM-g-AH8）
The DAK of 0.3g is weighed in the distilled water of 30 mL, 10 min under the conditions of 50 DEG C, are dissolved；Measure 0.19 mL of octylame dissolvings
In 100 mL ethanol, the ethanol solution of octylame is mixed with the DAK aqueous solution, there aring 8 h of backflow at the boiling point of ethanol, removing anti-
Bottle is answered, 30 DEG C are removed under reduced pressure ethanol, washed three times with ethyl acetate or chloroform, take organic phase, be removed under reduced pressure at 30 DEG C or so
Machine reagent, finally, is dissolved with the ethanol of 2 mL, is proceeded in bag filter, dialysed 2 days in distilled water, and every 4 h changes a water, takes
Go out dialyzed solution, freeze-drying had both obtained the konjaku glucomannan KGM- of the grafting of browng-AH8。
As shown in the infared spectrum of Fig. 1 c, in 3317 cm-1There is-OH a stretching vibration peak at place, in 2969 cm-1With
2924 cm-1There are the C-H stretching vibration peaks of a methyl and methylene at place, in 1665cm-1There are a sharp C=N peak, table in place
The amido of the C=O and octylame of bright DAK there occurs that reaction generates KGM-g-AH8。
As shown in the nuclear magnetic spectrum of Fig. 2 b, have the proton peak of a methyl at 0.87 ppm, 1.2-1.99 ppm it
Between have the proton peak of methylene, the peak between 3.28 ppm-3.90 ppm is the anomeric proton peak on DAK sugar rings, shows octylame
The aldehyde radical of amido and DAK there occurs reaction.
Embodiment 3：Lauryl amine is grafted the preparation of KGM（KGM-g-AH12）
KGM-g-AH12Preparation process：The DAK of 0.3g is weighed in the distilled water of 30 mL, under the conditions of 50 DEG C, 10 is dissolved
min；Weigh 0.22 g lauryl amines to be dissolved in 100 mL ethanol, the ethanol solution of lauryl amine is mixed with the DAK aqueous solution, is being had
Flow back at the boiling point of ethanol 10 h, removes reaction bulb, and 30 DEG C are removed under reduced pressure ethanol, are washed three times with ethyl acetate, take organic phase,
Organic reagent is removed under reduced pressure at 30 DEG C or so, finally, is dissolved with the ethanol of 2 mL, is proceeded in bag filter, dialysed in distilled water
2 days, every 4 h changed a water, took out dialyzed solution, and freeze-drying had both obtained the konjaku glucomannan KGM- of the grafting of browng-A
H12。
As shown in the infared spectrum of Fig. 1 d, in 3356 cm-1There is-OH a stretching vibration peak at place, in 2973 cm-1With
2925 cm-1There are the C-H stretching vibration peaks of a methyl and methylene, 1656 cm in place-1There is a sharp C=N peak at place, shows
The amido of the C=O and lauryl amine of DAK there occurs that reaction generates KGM-g-A H12.As shown in the nuclear magnetic spectrum of Fig. 2 c, in 1.2-
There is the proton peak of methylene between 1.99 ppm, and have the proton peak of methyl between 0-0.9 ppm, 3.21 ppm-3.71
Peak between ppm is the anomeric proton peak on DAK sugar rings, shows that the amido of lauryl amine there occurs reaction with the aldehyde radical of DAK.
Embodiment 4：Octadecylamine is grafted the preparation of KGM（KGM-g-AH18）
KGM-g-AH18Preparation process：The DAK of 0.3 g is weighed in the distilled water of 30 mL, under the conditions of 50 DEG C, 10 is dissolved
min；Weigh 0.32 g octadecylamines to be dissolved in 100 mL hexamethylenes, the cyclohexane solution of octadecylamine mixed with the DAK aqueous solution,
There aring 12 h of backflow at the boiling point of hexamethylene, removing reaction bulb, reactant liquor is proceeded to and stand in separatory funnel 30 min, take organic
Phase, 30 DEG C are removed under reduced pressure hexamethylene, are washed three times with chloroform, take organic phase, and 30 DEG C are removed under reduced pressure organic reagent, finally, use 2
The ethanol and ether of mL（v/v=1:1）Mixed solvent dissolving, proceed in bag filter, in distilled water dialyse 2 days, every 4 h is changed
Water, takes out dialyzed solution, and freeze-drying had both obtained the konjaku glucomannan KGM- of the grafting of browng-A H18。
As shown in the infared spectrum of Fig. 1 e, in 3412 cm-1There is-OH a stretching vibration peak at place, in 2920 cm-1With
2852 cm-1There are the C-H stretching vibration peaks of a methyl and methylene, 1661 cm in place-1There is a sharp C=N peak at place, shows
The amido of the C=O and octadecylamine of DAK there occurs that reaction generates KGM-g-A H18.As shown in the nuclear magnetic spectrum of Fig. 2 d, in 1.23-
There is the proton peak of methylene between 1.69 ppm, and have the proton peak of methyl between 0.03-0.86 ppm, 3.2 ppm-4.28
Peak between ppm is the anomeric proton peak on DAK sugar rings, shows that the amido of lauryl amine there occurs reaction with the aldehyde radical of DAK.
Embodiment 5：KGM-g-AH8The preparation of micella
（1）Alcohol injection：Measure the KGM-g-AH of 0.5 mL 10mg/mL8Ethanol solution, injects in the condition of magnetic agitation
In the distilled water of 10 mL, 30 min are stirred at room temperature, are dialysed 2 days in distilled water in proceeding to bag filter, every 4 h changes a water, takes
200 W of dislysate, 5 min of ultrasound, cross 0.45 μm and 0.22 μm of miillpore filter obtains micellar solution.
Fig. 3 a, b are KGM-g-AH8Alcohol injection prepare nano-micelle transmission electron microscope picture.Can from figure
Go out micella spherical in shape, and it is dispersed.
（2）Dialysis：Measure 10 mL 1mg/mL KGM-g-AH8Ethanol solution in molecular cut off be 1000 it is saturating
In analysis bag, room temperature is dialysed 2 days, and every 4 h changes a water, takes 200 W of dialyzed solution, 5 min of ultrasound, crosses 0.22 μm and 0.45 μm
Miillpore filter obtains micellar solution.
（3）Carry curcumin KGM-g-AH8The preparation of micella：By the curcumin ethanol solution and 0.5 mL of 1 mL, 2 mg/mL
The KGM- of 10 mg/mLg-AH8Ethanol solution is well mixed, and injects in the distilled water of 10 mL, room under conditions of magnetic agitation
Temperature 30 min of stirring, dialyse 2 days in proceeding to bag filter in distilled water, and every 4 h changes a water, takes 200 W ultrasounds 5 of dislysate
Min, crosses 0.45 μm and 0.22 μm of miillpore filter obtains carrier micelle.
Embodiment 6：KGM-g-AH8The control release experiment of carrier micelle
5 mL carrier micelles are transferred in bag filter, be then immersed in 25 mL contains 0.5%（w/v）Tween 80 pH
In=5.0 and pH=7.4 phosphate buffer, and 48 h under the conditions of being placed on 37 DEG C, 3 mL are taken out under the time for setting
Buffer solution determines its light absorption value at 424 nm, while supplementing the synthermal fresh buffer solution of 3 mL phases.
Fig. 4 is KGM-g-AH8The control release figure of nano-micelle.Solid line and dotted line in figure refers to octylame grafting respectively
KGM contains the elution profiles after curcumin in pH=5.0 and pH=7.4.It can be seen that medicine is released when pH=5.0
What is put is fast, and discharged before 24 h it is fast, tend towards stability after 24 h, substantially discharge in 48 h and finish, explanation
KGM-g- AH micellas have pH sensitiveness.
Embodiment 7：KGM-g-AH8The cytotoxicity experiment of micella
By cryodesiccated KGM-g-AH8Micella and 20 μ L are dissolved in the MTT of PBS（5μg/mL）With it is cultured
4 h of HepG2 cell culture, KGM-g-AH8The concentration of micella is 2-125 μ g/mL, then that cell is fresh with 200 μ L
Cell culture medium is rinsed well, removes MTT, then dissolves first with 200 μ L DMSO, at 570 nm determines its optical density.
As shown in Figure 5 it can be seen that KGM-g-AH8It is relatively low to cytotoxicity, when 125 μ g, the survival rate of cell
Also as many as 75%.
Embodiment 8：KGM-g-AH8The cellular uptake experiment of carrier micelle
HepG2 cells are dispersed in the cell culture fluid containing 10% hyclone and 1% antibiotic, then with 1 × 105Cell/
Hole density culture is in 6 orifice plates.Then at 37 DEG C, 5% CO2Under conditions of cultivate 24 h, its culture medium is rinsed into dry then
Only, respectively with the KGM-g-AH marked containing 10% hyclone, 1% antibiotic, FITC81 mL cell culture mediums of micella and 10%
Hyclone, 1% antibiotic, the KGM-g-AH of FITC marks81 mL cell culture mediums culture of carrier micelle, 4 h, training of then going out
Foster medium, then with dye 15 min of nucleus of Hoechst 33342 of 50 μ L, removes culture medium, is buffered with 1 mL PBS molten
Liquid is rinsed 3 times, then cell is put under laser co-focusing and is observed.
B, e as shown in Fig. 6 is the nuclear picture of the blueness of the marks of Hoechst 33342, in a, c around nucleus
Part is for marking KGM-g-AH8The green fluorescences that send of FITC, it is as can be seen from the figure when 4 h, intracellular
There is very strong fluorescence, illustrate that carrier enters cell, perinuclear part is KGM- in d, fg-AH8The ginger that carrier is contained
The green fluorescence that flavine sends, it is as can be seen from the figure when 4 h, intracellular to have very strong fluorescence, illustrate KGM-g-AH8
Curcumin has successfully been loaded into carrier cell.
Embodiment 9：KGM-g-AH12The preparation of micella
（1）Alcohol injection：Measure 10 mg/mL of 0.5mL（It is higher that concentration can be prepared）KGM-g-AH12Ethanol solution,
The condition of magnetic agitation is injected in the distilled water of 10 mL, and 30 min are stirred at room temperature, and dialyses 2 in proceeding to bag filter in distilled water
My god, every 4 h changes a water, takes 400 W of dislysate, 3 min of ultrasound, crosses 0.45 μm and 0.22 μm of miillpore filter obtains glue
Beam solution.
（2）Dialysis：Measure 10 mL 1mg/mL KGM-g-AH12Ethanol solution in molecular cut off be 3500 it is saturating
In analysis bag, room temperature is dialysed two days, and every 4 h changes a water, takes 200 W of dialyzed solution, 3 min of ultrasound, crosses 0.22 μm and 0.45 μm
Miillpore filter obtains micellar solution.
（3）Carry curcumin KGM-g-AH12The preparation of micella：By the curcumin ethanol solution and 0.5 of 0.5 mL, 2 mg/mL
The KGM- of 10 mg/mL of mLg-AH12Ethanol solution is well mixed, and is injected in the distilled water of 10 mL under conditions of magnetic agitation,
30 min are stirred at room temperature, are dialysed 2 days in distilled water in proceeding to bag filter, every 4 h changes a water, taken 400 W of dislysate and surpass
3 min of sound, crosses 0.45 μm and 0.22 μm of miillpore filter obtains carrier micelle.
Fig. 1 d are KGM- prepared by dialysisg-AH12The transmission electron microscope picture of micella, it can be seen that micella is rounded
And be uniformly dispersed.
Embodiment 10：KGM-g-AH18The preparation of micella
（1）Alcohol injection：Measure 10 mg/mL of 0.5mL（It is higher that concentration can be prepared）KGM-g-AH18Ethanol ether is molten
Liquid, injects in the distilled water of 10 mL in the condition of magnetic agitation, 30 min is stirred at room temperature, and proceeds in bag filter in distilled water
Dialysis 2 days, every 2 h changes a water, takes 200 W of dislysate, 10 min of ultrasound, crosses 0.45 μm and 0.22 μm of miillpore filter is
Obtain micellar solution.
（2）Dialysis：Measure 5 mL, 2 mg/mL KGM-g-AH18Chloroformic solution in molecular cut off be 3500 it is saturating
In analysis bag, room temperature is dialysed two days, and every 2 h changes a water, takes 200 W of dialyzed solution, 10 min of ultrasound, crosses 0.22 μm and 0.45 μ
M miillpore filters obtain micellar solution.
（3）Carry curcumin KGM-g-AH18The preparation of micella：By the curcumin ethanol solution and 1 mL of 0.5 mL, 2 mg/mL
The KGM- of 20 mg/mLg-AH18Chloroformic solution is well mixed, and injects in the distilled water of 10 mL, room under conditions of magnetic agitation
Temperature 30 min of stirring, dialyse 2 days in proceeding to bag filter in distilled water, and every 4 h changes a water, takes 200 W of dislysate ultrasonic
10 min, cross 0.45 μm and 0.22 μm of miillpore filter obtains carrier micelle.
Fig. 1 e are KGM- prepared by dialysisg-AH18The transmission electron microscope picture of micella, it can be seen that micella is rounded
And be uniformly dispersed.
Claims (2)
1. a kind of fatty amine is grafted the preparation method of konjaku glucomannan medicament-carried nano micelle, it is characterised in that：The method includes
Following steps：
（1）The preparation of dialdehyde-based konjaku glucomannan：Weigh konjaku glucomannan to be scattered in distilled water, stir in room temperature mechanical
Mix, swelling 8-24 h, konjaku glucomannan dispersion liquid is obtained；Then sodium metaperiodate is dissolved in distilled water, and is added drop-wise to evil spirit
In taro Glucomannan dispersion liquid, room temperature lucifuge stirring reaction 12-48 h, 40-60 DEG C of reduced pressure concentration are filtered, are finally transferred to
In analysis bag, dialysis removes salt and small molecule product, and freeze-drying had both obtained the dialdehyde-based konjaku glucomannan of white;
Described konjaku glucomannan is 1 with the mass ratio of sodium metaperiodate:0.4-1:1.5；
（2）Fatty amine is grafted the synthesis of konjaku glucomannan：By step（1）The dialdehyde-based konjaku glucomannan of middle preparation is dissolved in
In water；During fatty amine is dissolved in ethanol or hexamethylene, and it is added in the aqueous solution of dialdehyde-based konjaku glucomannan, in ethanol
Or the 6-18 h that flow back at the boiling point of hexamethylene, reaction bulb is removed, 30-40 DEG C is removed under reduced pressure ethanol or hexamethylene, with water mutually not
The organic reagent for mixing is washed three times, takes organic phase reduced pressure concentration, and then dialysis removes unreacted fatty amine, and freeze-drying was both obtained
Konjaku glucomannan is grafted to fatty amine；
Described dialdehyde-based konjaku glucomannan with the rate of charge of fatty amine is：The aldehyde radical of dialdehyde-based konjaku glucomannan and fat
The ratio of the amount of the material of the amido of amine is 1:1；
Described fatty amine is straight-chain fatty amine, short chain amine in being more preferably dissolvable in water ethanol or methyl alcohol, be can dissolve
Long-chain amine in hexamethylene, short chain amine are preferably octylame, lauryl amine, and long-chain amine is preferably octadecylamine；
（3）Fatty amine is grafted the preparation of konjaku glucomannan carrier micelle：The ethanol solution of dewatering medicament and fatty amine grafting evil spirit
The ethanol or chloroformic solution of taro Glucomannan is well mixed, and the mass ratio that dewatering medicament is grafted konjaku glucomannan with fatty amine is
1:20～2:5, above-mentioned mixed liquor is injected in distilled water under conditions of magnetic agitation, 30min is stirred at room temperature, is proceeded to
Dialysis in analysis bag removes ethanol and/or chloroform, takes dislysate 200-400 W ultrasound 3-10 min, crosses 0.45 μm and 0.22 μ
M miillpore filters obtain polypeptide drug-loaded micelle solution.
2. a kind of fatty amine as claimed in claim 1 is grafted the preparation method of konjaku glucomannan medicament-carried nano micelle, and which is special
Levy and be：Described dewatering medicament is curcumin.
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| CN108186572A (en) * | 2018-02-11 | 2018-06-22 | 湖北工业大学 | The konjaku glucomannan of injection tool pH sensibility-liposome composite Nano pharmaceutical carrier and its preparation and application |
| CN108576779A (en) * | 2018-02-11 | 2018-09-28 | 湖北工业大学 | Konjaku glucomannan-liposome composite Nano food delivering system and its preparation method and application |
| CN109998987A (en) * | 2019-04-08 | 2019-07-12 | 上海理工大学 | A kind of composite hydrogel and its preparation method and application based on natural konjaku flour |
| CN110833141A (en) * | 2019-11-23 | 2020-02-25 | 福建农林大学 | Boiling-resistant konjac glucomannan noodles and preparation method thereof |
| CN111096950A (en) * | 2019-12-31 | 2020-05-05 | 青岛农业大学 | Curcumin double-layer emulsion with colon-targeted delivery function and preparation method and application thereof |
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| CN1687195A (en) * | 2005-04-07 | 2005-10-26 | 武汉大学 | Glucomannan microgel particles of konnjaku and preparation method |
| CN102432760A (en) * | 2011-09-16 | 2012-05-02 | 马东晖 | Quaternary amine konjac glucomannan adduct, its preparation method and application |
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| CN1687195A (en) * | 2005-04-07 | 2005-10-26 | 武汉大学 | Glucomannan microgel particles of konnjaku and preparation method |
| CN102432760A (en) * | 2011-09-16 | 2012-05-02 | 马东晖 | Quaternary amine konjac glucomannan adduct, its preparation method and application |
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| CN108186572A (en) * | 2018-02-11 | 2018-06-22 | 湖北工业大学 | The konjaku glucomannan of injection tool pH sensibility-liposome composite Nano pharmaceutical carrier and its preparation and application |
| CN108576779A (en) * | 2018-02-11 | 2018-09-28 | 湖北工业大学 | Konjaku glucomannan-liposome composite Nano food delivering system and its preparation method and application |
| CN108576779B (en) * | 2018-02-11 | 2021-11-12 | 湖北工业大学 | Konjac glucomannan-liposome composite nano food delivery system and preparation method and application thereof |
| CN109998987A (en) * | 2019-04-08 | 2019-07-12 | 上海理工大学 | A kind of composite hydrogel and its preparation method and application based on natural konjaku flour |
| CN110833141A (en) * | 2019-11-23 | 2020-02-25 | 福建农林大学 | Boiling-resistant konjac glucomannan noodles and preparation method thereof |
| CN111096950A (en) * | 2019-12-31 | 2020-05-05 | 青岛农业大学 | Curcumin double-layer emulsion with colon-targeted delivery function and preparation method and application thereof |
| CN112618515A (en) * | 2020-12-29 | 2021-04-09 | 江南大学 | Preparation method of exosome-loaded oral colon-targeted drug delivery polymer |
| CN112618515B (en) * | 2020-12-29 | 2021-09-24 | 江南大学 | Preparation method of exosome-loaded oral colon-targeted drug delivery polymer |
| CN112830979A (en) * | 2021-01-15 | 2021-05-25 | 江南大学 | Modified xanthan gum and preparation method and application thereof |
| CN113105651A (en) * | 2021-03-25 | 2021-07-13 | 暨南大学 | Sprayable hydrogel and preparation method and application thereof |
| CN117100869A (en) * | 2023-08-14 | 2023-11-24 | 常熟耐素生物材料科技有限公司 | Biological-based pH-sensitive nano material and preparation method thereof |
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