CN104524594B - Medicine of nanometer diamond surface modification load methotrexate (MTX) and preparation method thereof - Google Patents
Medicine of nanometer diamond surface modification load methotrexate (MTX) and preparation method thereof Download PDFInfo
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Abstract
A kind of the invention provides medicine of nanometer diamond surface modification load methotrexate (MTX) and preparation method thereof, and application of the medicine in antineoplastic is prepared.Nanometer diamond surface with crosslinking agent polyethylene glycol diamines are modified by the present invention first so as to after acid amides reaction is coupled with glycidol, recycle the carboxyl in methotrexate (MTX) structure drug target ND PEG GLY MTX to be obtained with hydroxyl into ester.Through MTT experiment and flow cytometer test cell cycle and apoptosis experiment, show ND PEG GLY MTX can inducing apoptosis of tumour cell and curative effect is better than free MTX, which can be applied in antineoplastic is prepared.
Description
Technical field
The present invention relates to Nano medication, and in particular to a kind of nanometer diamond surface modification back loading chemotherapeutics methotrexate (MTX)
Nano medication and preparation method thereof, and application of the medicine in antineoplastic is prepared.
Background technology
With advancing by leaps and bounds for current development in science and technology, the environment that people depend on for existence is increasingly severe, causes malignant tumour
Incidence of disease more and more higher.In at present clinically traditional cancer treatment method, chemotherapy is one of method the most simple and effective, but
Being that cancer therapy drug mostly is micromolecular compound, whole body after blood dosing, being distributed in rapidly, the secondary work of larger poison is produced to patient
With drug loss amount is big, and multiple dosing can make tumour cell produce multidrug resistance, so that chemotherapy is extremely inefficient.
Therefore the new anticancer drug of research and development high-efficiency low-toxicity is particularly urgent.
Recently as the combination of the subjects such as nano science and life science, medical science, nano biological medical science into
For one of noticeable emerging cross discipline.In view of the particular advantages of nano material, research and development Nano medication is in medicine neck
Domain has huge development prospect, and wherein of greatest concern is using nano particle as pharmaceutical carrier, and medicine is inhaled by physics
The mode of attached or covalent coupling is attached on nano particle is used for drug delivery.As nanometer diamond is in all carbon nanometer materials at present
There is in material derivative the features such as toxicity is minimum, bio-compatibility is high and surface easily modifies, which is applied to give birth to as pharmaceutical carrier
Thing medical domain has very big potential value.And methotrexate (MTX) is used as clinically applying more anti-folic acid based chemotherapy medicine,
Cause drug loss after blood dosing during systemic circulation while injuring normal cell.In view of nanometer diamond carries medicine and fortune
The superiority of loading thing, we by modification after nanometer diamond Nano medication is prepared into by chemical coupling methotrexate (MTX), and grind
Study carefully its impact to activity of tumor cells, cycle, apoptosis etc..
Content of the invention
It is an object of the invention to provide a kind of medicine of nanometer diamond surface modification load methotrexate (MTX) and its preparation side
Method, and application of the medicine in antineoplastic.
A kind of preparation method of the medicine of nanometer diamond surface modification load methotrexate (MTX) that the present invention is provided, including as follows
Step：
(1) nanometer diamond of dry carboxylated is weighed, and 1-1.5mL concentration is added by the nanometer diamond of every 1mg carboxylated
In for the MES cushioning liquid of 0.1M, pH5.8, ultrasonic disperse 30min forms suspension, then by the nanometer diamond per 1mg carboxylated
Middle addition 0.2mg EDC and 0.3mg NHS, are stirred at room temperature reaction 6h, and timing is centrifuged 5min with 15000rpm after terminating, discards
Clear liquid, obtains the nanometer diamond sediment of activated carboxyl；
(2) the nanometer diamond sediment of activated carboxyl is distributed in the borate buffer solution that concentration is 0.1M, pH8.4,
Ultrasonic disperse forms suspension, adds 0.5mg H by the nanometer diamond of every 1mg activated carboxyls2N-PEG-NH2, continue to be stirred at room temperature
Reaction 12h, after question response terminates, washs 3 with the borate buffer solution that concentration is 0.1M, pH8.4 with 15000rpm centrifugation 5min
Secondary, obtain nanometer diamond-polyethylene glycol diamines (ND-PEG-NH2) sediment；
(3) by per 1mg ND-PEG-NH2Sediment is dissolved in 0.5mL absolute ethyl alcohols, ultrasonic disperse 30min, is formed suspended
Liquid, by every 1mg ND-PEG-NH2Sediment is added dropwise over the ethanol solution 0.5mL of the glycidol that volume fraction is 1%, then
Add a few drop triethylamines, maintain reaction system pH in 8-9, room temperature lucifuge stirring reaction 24h, be precipitated thing, respectively with anhydrous
Ethanol and sterilizing distilled water are washed 3 times with 15000rpm centrifugation 5min, obtain nanometer diamond-polyethylene glycol diamines-glycidol
(ND-PEG-GLY) carrier, is placed in vacuum drying chamber and keeps in dark place；
(4) add in 1mL sterilizing distilled waters by the ND-PEG-GLY dried per 1mg, lucifuge ultrasonic disperse 30min forms outstanding
Turbid liquid, then presses ND-PEG-GLY and 5 1-3 of methotrexate (MTX) (MTX) mass ratio and adds MTX, then by addition in every 1mg MTX
0.6mg EDC and 0.35mg NHS lucifuge reaction 24h in 37 DEG C of water baths, are centrifuged 5min with 15000rpm, double with sterilizing
Steaming water washing 3 times, is obtained the medicine ND-PEG-GLY-MTX that nanometer diamond surface modification loads methotrexate (MTX), and vacuum drying is cold
Hide.
Beneficial effects of the present invention compared with prior art：The nanometer diamond material that the present invention is selected has biocompatibility
Many advantages, such as good, nontoxic, stable chemical nature, surface are easily modified.Thin by nano-carrier ND-PEG-GLY and human cervical carcinoma
Born of the same parents (HeLa) act on, and show that ND-PEG-GLY has bio-compatibility；Nano-carrier ND-PEG-GLY is passed through linkage
Drug methotrexate (MTX) is treated, ND-PEG-GLY-MTX Nano medications are prepared into, thin through MTT experiment and flow cytometer test
Born of the same parents' cycle and apoptosis experiment show ND-PEG-GLY-MTX can inducing apoptosis of tumour cell and curative effect is better than free MTX, which can
Apply in antineoplastic is prepared.
Description of the drawings
Scatter diagrams of the Figure 1A with flow cytomery HeLa cells
Scatter diagrams of the Figure 1B with flow cytomery HeLa cellular uptake Nano medication ND-PEG-GLY-MTX
Impacts of Fig. 2 Nano medications ND-PEG-GLY-MTX in vitro culture HeLa cytoactive
Results of Fig. 3 A with the flow cytomery HeLa cell cycles
Fig. 3 B affect the result of HeLa cell cycles with flow cytomery methotrexate (MTX) MTX
Fig. 3 C affect the result of HeLa cell cycles with flow cytomery nano-carrier ND-PEG-GLY
Fig. 3 D affect the result of HeLa cell cycles with flow cytomery Nano medication ND-PEG-GLY-MTX
The block diagram that Fig. 3 E Fig. 3 A-D cell cycles compare when each
The block diagram that each experimental groups of Fig. 4 are affected on Apoptosis with the prolongation of process time
Fig. 5 cell endocytic Nano medication ND-PEG-GLY-MTX mechanism
Specific embodiment
It is below the material used in embodiment：
Nanometer diamond (ND, element six, diameter about 140nm, Ireland).
Methotrexate (MTX) (MTX) is anti-folic acid based chemotherapy medicine, and molecular formula is C20H22N8O5, molecular weight is 454.45, Jiangsu
C is pressed in permanent auspicious medical limited company's production, specification20H22N8O5It is calculated as 100mg.
(74.08) GLY, molecular weight produce glycidol for Sigma companies.
Polyethylene glycol diamines (H2N-PEG-NH2, molecular weight is 2000) to produce for the precious biology Co., Ltd in Shanghai west.
Embodiment 1：
(1) preparation of nanometer diamond-polyethylene glycol diamines (ND-PEG) compound
The nanometer diamond of 5mg carboxylated is accurately weighed, dried overnight in 70 DEG C of vacuum drying chambers, it is placed in 5mL MES
In (0.1M, pH5.8) cushioning liquid, ultrasonic disperse 30min forms suspension, is subsequently added into 1.0mg EDC, 1.5mg NHS, room
Warm stirring reaction 6h, is centrifuged 5min with 15000rpm, removes supernatant, obtains the nanometer diamond sediment of activated carboxyl；
The nanometer diamond of activated carboxyl is re-dispersed in boric acid (BBS, 0.1M, pH8.4) cushioning liquid, ultrasonic disperse
30min forms suspension, adds 2.5mg H2N-PEG-NH2, continue to be stirred at room temperature reaction 12h, with borate buffer solution (BBS,
0.1M, pH8.4) washed 3 times with 15000rpm centrifugation 5min, obtain nanometer diamond-polyethylene glycol diamines (ND-PEG-NH2) heavy
Starch, is vacuum dried standby.
(2) preparation of nanometer diamond-polyethylene glycol diamines-glycidol (ND-PEG-GLY) nano-carrier
The ND-PEG of 5mg dryings is accurately weighed, boric acid (BBS0.1M, the pH8.4) cushioning liquid of 2.5mL is subsequently added into, is surpassed
Sound dispersion 30min forms suspension, is added dropwise over the glycidol ethanol solution that 2.5mL volume fractions are 1%, then to reaction
A few drop triethylamines being added dropwise in liquid, maintaining reaction system pH 8 or so, room temperature lucifuge stirring reaction 24h is centrifuged with 15000rpm
5min, removes supernatant, is washed twice with sterilizing distilled water, abandoning supernatant, obtain ND-PEG-GLY nano-carrier sediments,
Vacuum drying is standby.
(3) nanometer diamond-polyethylene glycol diamines-glycidol-methotrexate (MTX) (ND-PEG-GLY-MTX) Nano medication
Prepare
The ND-PEG-GLY of 5mg drying is accurately weighed, and ultrasonic disperse is resuspended in 5mL sterilizing distilled waters, is then put
In 37 DEG C of water-baths, 2mg MTX, 1.2mg EDC, 0.7mg NHS, lucifuge stirring reaction 24h is then rapidly added.Question response is tied
Shu Hou, is centrifuged 5min in 15000rpm, cleans precipitation till supernatant is colourless with sterilizing distilled water, collects all supernatants
And its volume is recorded, obtained Nano medication ND-PEG-GLY-MTX is vacuum dried standby by unreacted MTX to be determined.
In the borate buffer solution (BBS) of pH 8.0, MTX solution of the compound concentration for 2.2M, respectively dilution prepare 2.2
×10-3M、6.6×10-3M、11×10-3M、15.4×10-3M、19.8×10-3M、33×10-3M、44×10-3M、55×10- 3M、66×10-3M, surveys its absorbance with UV-VIS spectrophotometry at 303nm.With MTX concentration as abscissa,
Absorbance at 303nm is ordinate, draws the calibration curve of MTX.According to calibration curve formula, with addition MTX before reaction
Total amount deducts the quality of MTX in supernatant after reaction, that is, obtain every milligram of ND-PEG-GLY be coupled the quality of MTX for (132 ±
1.75)μg.
Embodiment 2：
(1) preparation of～(2) is with embodiment 1
(3) nanometer diamond-polyethylene glycol diamines-glycidol-methotrexate (MTX) (ND-PEG-GLY-MTX) Nano medication
Prepare
The ND-PEG-GLY of 5mg drying is accurately weighed, and ultrasonic disperse is resuspended in 5mL sterilizing distilled waters, is then put
In 37 DEG C of water-baths, 1mg MTX, 1.2mg EDC, 0.7mg NHS, lucifuge stirring reaction 24h is then rapidly added.Question response is tied
Shu Hou, is centrifuged 5min in 15000rpm, cleans precipitation till supernatant is colourless with sterilizing distilled water, collects all supernatants
And its volume is recorded, absorbances of the wherein MTX at 303nm is determined by ultraviolet-visible spectrophotometer, is calculated with this every
It is (102 ± 0.25) μ g to be coupled MTX on milligram ND-PEG-GLY.
Embodiment 3：
(1) preparation of～(2) is with embodiment 1
(3) nanometer diamond-polyethylene glycol diamines-glycidol-methotrexate (MTX) (ND-PEG-GLY-MTX) Nano medication
Prepare
The ND-PEG-GLY of 5mg drying is accurately weighed, and ultrasonic disperse is resuspended in 5mL sterilizing distilled waters, is then put
In 37 DEG C of water-baths, 3mg MTX, 1.2mg EDC, 0.7mg NHS, lucifuge stirring reaction 24h is then rapidly added.Question response is tied
Shu Hou, is centrifuged 5min in 15000rpm, cleans precipitation till supernatant is colourless with sterilizing distilled water, collects all supernatants
And its volume is recorded, absorbances of the wherein MTX at 303nm is determined by ultraviolet-visible spectrophotometer, is calculated with this every
It is (122 ± 0.6) μ g to be coupled MTX on milligram ND-PEG-GLY.
Embodiment 4：
The experiment of flow cytomery HeLa cellular uptake Nano medications (ND-PEG-GLY-MTX)
Take the logarithm growth period HeLa cells with 2.0 × 105The density of/dish is inoculated in 35mm culture dishes, cultivates 16h
Afterwards, liquid is abandoned, PBS is washed, the ND-PEG-GLY-MTX Nano medications prepared with embodiment 1 in this experiment add ND-PEG-GLY-
MTX (containing 100 μ g/mL MTX) Nano medication is incubated 4h in 37 DEG C, and incubation is finished, and collects cell, is determined by flow cytometer
The fluorescence intensity of cell, not do the cell of any process as control.
Figure 1A and Figure 1B for HeLa cells forward scattering luminous intensity (forward scatter intensity, FSC) and
The scatter diagram of lateral scattering luminous intensity (side scatter intensity, SSC), FSC reflect the relative size of cell, SSC
The complexity of reflection intracellular granular material, particle are more, and SSC scattering strengths are bigger.Compared by Figure 1B and 1A, observed
SSC signals after ND-PEG-GLY-MTX Nano medications are with cytosis are significantly stronger than the SSC intensity of cell itself, show thin
The particulate matter of intracellular increases, this be due to Nano medication ND-PEG-GLY-MTX enter in a large number intracellular cause, illustrate ND-
It is intracellular that PEG-GLY-MTX Nano medications can enter HeLa.And the intensity of both FSC does not have notable difference, show cell
Size is not changed substantially, illustrates that Nano medication will not produce impact to cell nature.
Embodiment 5：
Impact experiment of the ND-PEG-GLY-MTX Nano medications to HeLa cytoactives
By HeLa cells (10%FBS/DMEM culture mediums, 5%CO in exponential phase2, 37 DEG C) and by per hole 5 × 103
The individual each experimental group (A for being inoculated in 96 well culture plates, after cell attachment, changing the preparation of 200 μ L 10%FBS/DMEM culture mediums：ND-
230.8 μ g/mL of PEG-GLY, ND-PEG-GLY-MTX 230.8 μ g/mL (containing 30 μ g/mL of MTX), MTX30 μ g/mL；B:ND-
384.6 μ g/mL of PEG-GLY, ND-PEG-GLY-MTX 384.6 μ g/mL (containing 50 μ g/mL of MTX), 50 μ g/mL of MTX；C：ND-
769.2 μ g/mL of PEG-GLY, ND-PEG-GLY-MTX 769.2 μ g/mL (g/mL of μ containing MTX100), 100 μ g/mL of MTX), per
With untreated HeLa cells as blank control group, 6 multiple holes of per group of setting add 20 μ L 5mg/ to group per hole after culture 48h
The PBS solution of mL MTT, continues culture 4h, then old nutrient solution in reject hole, adds 150 μ L DMSO per hole, uniformly shakes
10min, carries out machine testing on ELIASA, as a result sees Fig. 2.
Fig. 2 is the impact that each experimental group is bred to HeLa cells.Compare with blanc cell group, find nano material ND-
PEG-GLY is had little to no effect to HeLa cytoactives under the conditions of variable concentrations, shows that ND-PEG-GLY carriers have biology
Compatibility；With the increase of drug concentration, Nano medication ND-PEG-GLY-MTX and free MTX are produced to cell in various degree
Lethal effect, and the ND-PEG-GLY-MTX of big concentration to the lethality of cell more than free MTX, show ND-PEG-PEG-
MTX Nano medications can more effectively kill tumour cell.
Embodiment 6：
Impact of the ND-PEG-GLY-MTX Nano medications to the HeLa cell cycles
In order to further study activity influences of the Nano medication ND-PEG-GLY-MTX to HeLa cells, we adopt streaming
Cell instrument has carried out Cell Cycle analysis.By the HeLa cells of exponential phase with 2.0 × 105The density inoculation of/dish
In 35mm culture dishes, respectively with MTX, ND-PEG-GLY, ND-PEG-GLY-MTX process (each group medicine after cell attachment
Dosage is with above-mentioned MTT experiment), after culture 48h, old liquid is discarded, PBS is washed 3 times, cell is collected solid with 70% cold ethanol
Determine overnight, to be centrifuged before upper machine after being washed with PBS and add RNase to remove interference of the RNA to test result, finally dyeed with PI
30min, upper machine testing, not do the cell of any process as control.
Fig. 3 A, B, C, D are respectively cellular control unit and the cell with MTX, ND-PEG-GLY, ND-PEG-GLY-MTX process
PI coloration results, figure includes a high and thin G1 peak, a relatively gentle S phase platform and a short and thick G2
Peak, being fitted statistics to the area at each peak must cell proportion of the source in each cell cycle.From Fig. 3 E,
MTX and ND-PEG-GLY-MTX treatment group versus cell control groups, the S phases of cell are significantly raised, and the G2 phases significantly reduce, and medicine is described
Thing selectively acts on the S phases, that is, makes cell-cycle arrest in the S phases, blocks process of the cancer cell from the S phases to the G2 phases, resistance
The mitosis of stagnant cell, hinders the biosynthesis block of DNA and RNA, promotes Apoptosis, growth of tumour cell to be suppressed, with
The MTX toxicological mechanisms of clinical report are consistent.After this conclusion shows Nano medication ND-PEG-GLY-MTX entrance cells, ester bond breaks
Split release MTX and play anticancer therapeutic.
Embodiment 7：
ND-PEG-GLY-MTX Nano medications induce HeLa cell apoptosis assays
Learn that ND-PEG-GLY-MTX Nano medications enter cell and can actually suppress by Cell cycle influences experiment
HeLa cell divisions are bred, and induce the apoptotic situations of HeLa to carry out which so we devise three different incubation times
Quantitative determination.Equally, by the HeLa cells of exponential phase with 2.0 × 105The density of/dish is inoculated in 35mm culture dishes,
After cell attachment, with MTX, ND-PEG-GLY, ND-PEG-GLY-MTX process, (each group drug dose is with above-mentioned MTT realities respectively
Test), it be arranged three groups in parallel, respectively after culture 24h, 48h, 72h, discards old liquid, PBS is washed 3 times, will be pasted with the pancreatin without EDTA
It is collected in fresh medium after parietal cell dissociation, is washed with PBS before upper machine, adds after washing and contain Ca2+Cushioning liquid
(Binding buffer), is added thereto to examination with computer after double dyestuffs (PI+Annexin-V) dyeing 15min of new mixing.
Fig. 4 represents the apoptotic situation of each experimental group of different disposal time, thin not to be the HeLa of any process
Born of the same parents are used as control.Can be found by figure, prolongation over time, the Apoptosis of nano-carrier ND-PEG-GLY treatment groups with compare
Group is more or less the same, and illustrates that material has good biocompatibility；And Nano medication ND-PEG-GLY-MTX and free MTX with thin
After born of the same parents' effect energy inducing cell apoptosis, and 72h, Nano medication ND-PEG-GLY-MTX is significantly greater than to the lethality of cell and swims
From MTX, this phenomenon shows that the Nano medication prepared by us can improve the inhibitory action to tumour cell.
Embodiment 8：
The mechanism of flow cytomery HeLa cellular uptake ND-PEG-GLY-MTX Nano medications
In order to probe into the transporting mechanism of cell endocytic ND-PEG-GLY-MTX Nano medications, from low temperature and different inhibitor
Interaction (the ND-PEG- prepared in this experiment of ND-PEG-GLY-MTX Nano medications and cell is have studied from embodiment 1
GLY-MTX Nano medications), as a result such as Fig. 5.As seen from the figure, compared with 37 DEG C, when cultivating for 4 DEG C, HeLa cells are to ND-PEG-
The intake of GLY-MTX Nano medications is significantly reduced, and up to 60%, this is as at 4 DEG C, fluidity of erythrocyte membrane can drop to inhibiting rate
Low, exogenous material is affected with the interaction of cell membrane, so causing the uptake ratio of cell exogenous material to reduce；
The inhibitor NaN for needing energy with impact endocytosis3After (consuming intracellular ATP) is pre-processed to HeLa cells, inhibiting rate is
2.73%, illustrate the cellular uptake Nano medication with little need for energy；For further inquiring into ND-PEG-GLY-MTX with which kind of
Endocytosis mode enters cell, is methylated-β-ring paste from clathrin disrupting agent sucrose (Sucrose) and caveolin disrupting agent
Smart (M- β-CD) is pre-processed to HeLa cells respectively.As seen from Figure 5, Sucrose is to HeLa cellular uptake ND-PEG-
The inhibiting rate of GLY-MTX reaches 75% for the inhibiting rate of 16.2%, M- β-CDHeLa cellular uptake ND-PEG-GLY-MTX, thus
It is to enter cell by the endocytic pathway that caveolin is mediated to be inferred to ND-PEG-GLY-MTX.More than synthesis, as a result show ND-
PEG-GLY-MTX enters the endocytic pathway entrance HeLa cells that cell is had temperature dependency and mediated with caveolin.
In sum, effects of the ND-PEG-GLY-MTX to HeLa cells is tested by MTT, and flow cytometer is determined
The experiment of different disposal group Cellular cycle and apoptosis, is compared with free methotrexate (MTX) medicine, is fully shown with nano-carrier ND-
The ND-PEG-GLY-MTX Nano medications that PEG-GLY is prepared into by linkage chemotherapeutics methotrexate (MTX) MTX are to cancer cell
Tumour cell can more effectively be killed.Therefore Nano medication ND-PEG-GLY-MTX can be answered in high efficiency anti-tumor medicine is prepared
With.
Claims (4)
1. a kind of nanometer diamond surface modification loads the preparation method of the medicine of methotrexate (MTX), it is characterised in that including following step
Suddenly：
(1) nanometer diamond of dry carboxylated is weighed, by the nanometer diamond addition 1-1.5mL concentration of every 1mg carboxylated is
In the MES cushioning liquid of 0.1M, pH5.8, ultrasonic disperse 30min is formed in suspension, then the nanometer diamond by every 1mg carboxylated
0.2mg EDC and 0.3mg NHS are added, reaction 6h is stirred at room temperature, timing is centrifuged 5min, supernatant discarded with 15000rpm after terminating
Liquid, obtains the nanometer diamond sediment of activated carboxyl；
(2) the nanometer diamond sediment of activated carboxyl is distributed in the borate buffer solution that concentration is 0.1M, pH8.4, ultrasound
Dispersion forms suspension, adds 0.5mg H by the nanometer diamond of every 1mg activated carboxyls2N-PEG-NH2, continue reaction is stirred at room temperature
12h, after question response terminates, is washed 3 times with 15000rpm centrifugation 5min with the borate buffer solution that concentration is 0.1M, pH8.4, is obtained
Obtain nanometer diamond-polyethylene glycol diamines (ND-PEG-NH2) sediment；
(3) by per 1mg ND-PEG-NH2Sediment is dissolved in 0.5mL absolute ethyl alcohols, ultrasonic disperse 30min, is formed suspension, is pressed
Per 1mg ND-PEG-NH2Sediment is added dropwise over the ethanol solution 0.5mL of the glycidol that volume fraction is 1%, adds several
Drop triethylamine, maintains reaction system pH in 8-9, room temperature lucifuge stirring reaction 24h, is precipitated thing, respectively with absolute ethyl alcohol and
Sterilizing distilled water is washed 3 times with 15000rpm centrifugation 5min, obtains nanometer diamond-polyethylene glycol diamines-glycidol (ND-
PEG-GLY) carrier, is placed in vacuum drying chamber and keeps in dark place；
(4) add in 1mL sterilizing distilled waters by the ND-PEG-GLY dried per 1mg, lucifuge ultrasonic disperse 30min forms suspended
Liquid, then presses ND-PEG-GLY and 5 1-3 of methotrexate (MTX) (MTX) mass ratio and adds MTX, then by addition 0.6mg in every 1mgMTX
EDC and 0.35mg NHS lucifuge reaction 24h in 37 DEG C of water baths, are centrifuged 5min with 15000rpm, are washed with sterilizing distilled water
Wash 3 times, the medicine ND-PEG-GLY-MTX that nanometer diamond surface modification loads methotrexate (MTX), vacuum drying, refrigeration is obtained.
2. a kind of nanometer diamond surface modification loads the preparation method of the medicine of methotrexate (MTX) as claimed in claim 1, and which is special
Levy and be, ND-PEG-GLY is 52 with the mass ratio of methotrexate (MTX) in step (4).
3. the nanometer diamond surface modification that prepared by method as claimed in claim 1 or 2 loads the medicine of methotrexate (MTX).
4. the medicine of nanometer diamond surface modification load methotrexate (MTX) as claimed in claim 3 is in antineoplastic is prepared
Application.
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| CN104906593B (en) * | 2015-05-29 | 2017-10-17 | 山西大学 | A kind of linkage nanometer diamond adriamycin medicine and its preparation method and application |
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| US10799587B2 (en) * | 2016-05-11 | 2020-10-13 | Huan NIU | Ion implantation of neutron capture elements into nanodiamond particles to form composition for neutron capture therapy usage |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008733A (en) * | 2010-11-24 | 2011-04-13 | 首都医科大学 | Anti-tumor controlled release nanocomposite and preparation method thereof |
| CN103203024A (en) * | 2013-04-11 | 2013-07-17 | 山西大学 | Targeting nano diamond carrier and targeted drug, and preparation method and application thereof |
| CN103705944A (en) * | 2014-01-07 | 2014-04-09 | 山西大学 | Preparation method and application of folic acid-modified nano-diamond targeting medicine |
-
2015
- 2015-01-06 CN CN201510005266.XA patent/CN104524594B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008733A (en) * | 2010-11-24 | 2011-04-13 | 首都医科大学 | Anti-tumor controlled release nanocomposite and preparation method thereof |
| CN103203024A (en) * | 2013-04-11 | 2013-07-17 | 山西大学 | Targeting nano diamond carrier and targeted drug, and preparation method and application thereof |
| CN103705944A (en) * | 2014-01-07 | 2014-04-09 | 山西大学 | Preparation method and application of folic acid-modified nano-diamond targeting medicine |
Non-Patent Citations (1)
| Title |
|---|
| 纳米钻石-甲氨蝶呤可控释放体系的制备及其生物效应;王青;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20160215;B016-560 * |
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