CN103908676A - Folic-acid-modified laponite nanometer particle, preparation thereof and applications thereof - Google Patents
Folic-acid-modified laponite nanometer particle, preparation thereof and applications thereof Download PDFInfo
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Abstract
The invention relates to a folic-acid-modified laponite nanometer particle, preparation thereof and applications thereof. Laponite is modified by utilization of a silane coupling agent. The weight ratio of the laponite (LAP) to the silane coupling agent is 5:1-5:4. The weight percentage of the silane coupling agent and folic acid is 3.01-3.34%. The preparation includes: a step of, under stirring, adding dropwise an aqueous solution of the silane coupling agent into an aqueous solution of the laponite (LAP), reacting at 45-60 DEG C for 12-16 h and dialyzing to obtain aminated laponite LM-NH2; a step of dissolving the folic acid into a solvent, adding N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride EDC, and uniformly stirring to obtain a mixed solution; and a step of adding dropwise the aqueous solution of the aminated laponite LM-NH2 into the mixed solution, stirring for 2-3 d, and dialyzing. The laponite nanometer particle is used for loading medicines. The preparation has characteristics of simple method, mild reaction conditions and easy operation and has good prospect of industrial implementation.
Description
Technical field
The invention belongs to nano drug-carrying material and preparation thereof and application, particularly a kind of hectorite nano-particle of modified with folic acid and preparation and application.
Background technology
Chemotherapy, as one of important means for the treatment of of cancer, is shown great attention to for a long time always.But the existing clinical efficacy of many antitumor drug is low, toxic and side effects is large etc., and problem has become the bottleneck in Medication for Cancer.Because uncontrollable antitumor drug concentrates on tumor locus, the normal structure and the organ that cause patient are subject to unnecessary injury, even threaten patient's life.In recent years, utilize the method for nano-carrier load antitumor drug, not only can improve drug solubility, increase medicine stability and realize medicament slow release, can also change significantly medicine distribution and metabolism status in vivo by its special size range and surface nature, strengthen the infiltration of medicine to tumor vessel wall and in tumor locus enrichment (EPR effect), prolong drug is in the action time of tumor locus.In addition, the nano-particle of modifying through targeted molecular can be by the initiatively picked-up of targeting raising tumor cell to medicine, realize drug specificity and be transported to tumor locus, in improving therapeutic effect, alleviate the injury to other organs, become focus and the emphasis of research.
Folic acid (Folic Acid, FA) is a kind of micromolecular water soluble vitamin.Tumor cell can maintain by Active transport and the folic acid in picked-up microenvironment the metabolism of overacfivity, therefore all have the folacin receptor of overexpression at most of tumor cell surface, and normal cell surface folacin receptor expression is little.Research shows folate molecule to be connected to nano-carrier surface, and the tumor cell that can improve folacin receptor high expressed is engulfed nanometer medicine-carried system, the targeted of the antitumor drug of realizing the load of nano-carrier institute to tumor cell.At present existing multinomial patent discloses the preparation method of the targeted nano pharmaceutical carrier take folic acid as targeted molecular, if publication number is the patent of CN102579353A, CN102276813A, CN101411877, CN101254309.
Consulting pertinent literature can find, the LAP of synthetic modified with folic acid carries with the research of neoplasm targeted therapy and there is not yet report for antitumor drug as pharmaceutical carrier.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of hectorite nano-particle and preparation and application of modified with folic acid, LM-FA/DOX medicament-carried nano granule of the present invention has good sustained drug release effect and pH response release characteristics, the tumor cell of homofolic acid expression of receptor is had to targeting and obvious inhibition, can be used for the targeted therapy of cancerous cell.
The hectorite nano-particle of a kind of modified with folic acid of the present invention, in the hectorite nano-particle LM-FA of described modified with folic acid, hectorite is modified with silane coupler, and the mass ratio of hectorite LAP and silane coupler is 5:1-5:4; In the hectorite clay nano granule LM-FA of modified with folic acid, the mass fraction of silane coupler and folic acid is at 3.01 – 3.34%.
Described silane coupler is (3-aminopropyl) dimethylethoxysilane APMES.
The preparation method of the hectorite nano-particle of a kind of modified with folic acid of the present invention, comprising:
(1), under stirring condition, the aqueous solution of silane coupler is dropwise added in hectorite LAP aqueous solution, under 45-60 ℃ of condition, reaction 12-16h, dialysis, obtains amidized hectorite LM-NH
2; Wherein the mass ratio of hectorite LAP and silane coupler is 5:1-5:4; Under the preferred water bath condition of reaction condition, 50 ℃, reaction 16h;
(2) folic acid is dissolved in solvent, adds 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC, stirring and evenly mixing, obtains mixed solution, then dropwise adds amidized hectorite LM-NH
2aqueous solution, stirs 2-3d, dialyses, and obtains the hectorite nano-particle LM-FA of modified with folic acid; Wherein the ratio of the molal quantity of EDC and folic acid is 10:1-15:1; The mass ratio of lithium amide saponite and folic acid is 20:1-10:1.
In described step (1), hectorite LAP concentration of aqueous solution is 5-10mg/mL.
The lithium amide saponite LM-NH obtaining in described step (1)
2in, the mass fraction of silane coupler is at 1.76-2.64%.
In described step (2), solvent is dimethyl sulfoxide DMSO.
Amidized hectorite LM-NH in described step (2)
2the concentration of aqueous solution is 6-8mg/mL.
In described step (1), (2), dialysis is pure water dialysis, and bag filter molecular cut off size is 8000-14000, and dialysis time is 2-4d.
The application of the hectorite nano-particle of a kind of modified with folic acid of the present invention, amycin aqueous solution is added to the hectorite nano-particle LM-FA aqueous solution of modified with folic acid, stir 12-24h, centrifugal, washing, disperse, obtain the hectorite nano-particle LM-FA/DOX of load amycin modified with folic acid, wherein the hectorite nano-particle LM-FA of modified with folic acid and the mass ratio of amycin DOX are 1:1-4:1.
Described stirring is magnetic agitation, and mixing speed is 100-150r/min.
The hectorite nano-particle LM-FA concentration of aqueous solution of described modified with folic acid is 2-5mg/mL; The concentration of amycin aqueous solution is 1-2mg/mL.
Described centrifugal speed is 8000-10000r/min, and the centrifugal time is 5min.
Hectorite (Laponite, LAP) is a kind of inorganic sheet stratiform clay nano material, has good colloidal stability.It is the about 1nm disk-like structure of 25nm thickness that LAP has diameter, and a large amount of negative charges are carried on surface, can be by the positively charged small-molecule drug of ion exchange load, and be a kind of good pharmaceutical carrier.There is great amount of hydroxy group on the surface of LAP, can retain the disc-shaped structure of hectorite by the modification of silane coupler, and effectively reduces being cross-linked between LAP lamellar structure, keeps the stability of material in aqueous solution.Meanwhile, silane coupler can be introduced active group at nano grain surface, for further modifying and drug loading.Obtain the amidized hectorite LM-NH of surperficial ammonia by the modification of (3-aminopropyl) dimethylethoxysilane APMES exactly in the present invention
2, and by modified with folic acid on nanometer hectorite surface, the hectorite nano-particle of preparing modified with folic acid is a kind of nano-carrier with cancerous cell target function, is expected to realize load and the targeted of medicine, gene or diagnosis molecular probe.
The hectorite LM-FA/DOX of the modified with folic acid of the load amycin that the present invention obtains can effectively control the release of amycin, and the inhibitory action that can significantly improve by the active targeting of folic acid the tumor cell to homofolic acid expression of receptor, has a extensive future.
The present invention uses the method such as ultraviolet-visible spectrophotometer, thermogravimetric analysis, transmission electron microscope, Zeta electric potential measurement to characterize the hectorite nano-particle of targeting modification prepared by the present invention, check toxicity and the targeting of the cervical cancer cell (Hela cell) of the hectorite nano-particle effects on surface folacin receptor high expressed of medicine carrying with "diazoresorcinol" method, flow cytometry and laser confocal microscope, concrete test result is as follows simultaneously:
(1) surface potential and hydration grain diameter measurement
Before and after modifying, particle diameter and the surface potential of particle are all measured by dynamic light scattering, and result is as shown in table 1.After silane coupler is modified, the surface potential of hectorite is from-change to-2.4mV of 37.9mV, and this is because APMES is positively charged, modifies the part negative charge that can shield LAP surface behind LAP surface, has caused the variation of material surface electromotive force.Further modified after electronegative FA be reduced to-5.4mV of the surface potential of LM-FA on surface.Therefore, also can prove that by the potential change before and after modifying hectorite surface successfully modified APMES and folic acid.In addition, although the hydration diameter of hectorite all increases to some extent after finishing, the size of material is less than 200nm and has good dispersibility, therefore, is still a kind of good carrier material.
Table 1LAP, LM-NH
2, LM-FA hydration diameter and surface potential
(2) thermogravimetric analysis test result
In the thermogravimetric analysis test result shown in accompanying drawing 1, compared with LAP, LM-NH
2with LM-FA having lost respectively 1.76% and 3.34% of gross mass from the temperature-rise period of 200 ℃ to 600 ℃.This result proves that silane coupling A PMES and FA are all successfully modified at LAP surface.
(3) ultraviolet-visible spectrum test result
In the ultraviolet-visible spectrum test result shown in accompanying drawing 2, before medicine carrying, LAP and LM-NH
2in 800nm wave-length coverage, all there is no obvious absworption peak at 250nm.In the ultraviolet spectra of LM-FA, occurred a stronger absworption peak at 280nm place, this is the ultraviolet characteristic absorption peak of FA.This result also confirms that FA is successfully modified hectorite surface.After load DOX, three kinds of materials all demonstrate an obvious absworption peak near 480nm.According to bibliographical information, the ultraviolet characteristic absorption peak that this absworption peak is DOX.Therefore, the hectorite nano-particle of finishing still can load antitumor drug DOX.By UV-quantitative analysis, LM-NH
2be respectively 87.6 ± 0.65% and 92.1 ± 2.2% with the medicine carrying efficiency of LM-FA.
(4) transmission electron microscope (TEM) observed result
For observe intuitively modify after and the metamorphosis of hectorite nano-particle after medicine carrying, we have adopted transmission electron microscope observation material.In the TEM picture shown in accompanying drawing 3, (a) be LAP, be (b) LM-FA, be (c) amplify the part of (b), (d) be LM-FA/DOX.Compared with LAP, the LM-FA form after modification does not have significant change, has retained distinctive disc structure, and can find out from (c) figure, and disk perimeter has adorned vestige.After load DOX, material shape does not have significant change.
(5) extracorporeal releasing experiment result
Accompanying drawing 4 is (a) LM-NH
2/ DOX and (b) the cumulative in vitro release profiles of LM-FA/DOX.In 192 hours, DOX in the weak acid environment of pH=5.0 from LM-FA/DOX rate of release compared with fast in the neutral environment of pH=7.4.This illustrates that this medicine-carried system all has pH response, with the similar weak acid environment of tumor tissues in rate of release fast, preparation is high, and is that in the similar neutral environment of normal structure, rate of release is slow, preparation is low.Accompanying drawing 4 illustrates, LM-FA/DOX can effectively reduce the release of DOX at normal structure place, and can discharge faster at tumor tissues position, reduces the toxicity of DOX normal tissue, improves the inhibition to tumor cell malignant proliferation.
(6) "diazoresorcinol" experiment
Selecting the Hela cell of homofolic acid expression of receptor and the l cell L929 cell of low folacin receptor expression is model, investigates the inhibitory action of prepared medicine-carried system to two kinds of cell proliferation.Specific practice is, by the Hela cell of adherent growth or L929 cell with add the culture medium of material to be placed in 5%CO
2under 37 ℃ of conditions, cultivate altogether 24 hours, outwelling original culture medium also cleans 3 times with aseptic PBS, adding the culture medium that contains 0.1mg/mL "diazoresorcinol" to be placed under the same terms continued to cultivate after 4 hours, sucking-off upper strata culture medium is measured it at excitation wavelength lambda=530nm, the fluorescent value at emission wavelength lambda=590nm place, the size of fluorescent value can reflect the quantity of living cells.Accompanying drawing 5 is process drug carrier material LM-NH
2(a) Hela cell and (b) the "diazoresorcinol" result of the test of L929 cell processed with LM-FA.Result shows that two kinds of carrier materials all do not show obvious cytotoxicity to Hela cell and L929 cell.
Accompanying drawing 6 is (a) Hela and (b) test result of L929 cell.Figure (a) is presented in given concentration range, and the experimental group of processing through LM-FA/DOX is than process LM-NH
2the experimental group Hela cell survival rate that/DOX processes is low, and figure (b) is presented in given concentration range, the experimental group of processing through LM-FA/DOX and process LM-NH
2the experimental group L929 cell survival rate that/DOX processes is more or less the same.The result shows that LM-FA/DOX can strengthen by the targeting of FA the inhibitory action of the tumor cell of DOX to homofolic acid expression of receptor.
(7) cytophagy experiment
The fluorescent effect producing after utilizing flow cytometer to Hela cell and L929 cytophagy medicine detects.Accompanying drawing 7 for DOX concentration be 6ppm, medicine and the co-culture of cells latter two cell of 4 hours produces the result of fluorescence owing to engulfing medicine.The Hela cell of processing through LM-FA/DOX is than process LM-NH
2the Hela cell that/DOX processes shows stronger fluorescence (p < 0.001), and the L929 cell of processing through LM-FA/DOX and process LM-NH
2the fluorescence intensity that the L929 cell that/DOX processes shows does not have notable difference.The result shows that it is that the FA receptor of FA by linking on carrier and tumor cell surface overexpression combines and realizes to the effect of engulfing of DOX that LM-FA/DOX increases tumor cell.Therefore, LM-FA/DOX of the present invention can be by receptor-ligand in conjunction with increasing tumor cell to the engulfing of DOX, thereby strengthen the inhibition to tumor cell of DOX, and effectively reduce the picked-up of normal cell to DOX, reduced side effects of pharmaceutical drugs.
(8) in cell, distribute and test
In order to observe more intuitively medicine in intracellular distribution, employing laser scanning confocal micro-scope has investigated cell and medicine is cultivated 4 hours interior fluorescence distribution situations of cell afterwards altogether.Accompanying drawing 8 is the interior fluorescence distribution result of Hela cell under DOX concentration 6ppm condition, and accompanying drawing 9 is the interior fluorescence distribution result of L929 cell under identical DOX concentration.Can find out that from accompanying drawing 8 LM-FA/DOX that the present invention reports not only can obviously strengthen the picked-up of Hela cell to DOX, the DOX being simultaneously ingested shows certain core distribution character that becomes in Hela cell.And in accompanying drawing 9, through LM-NH
2the L929 cell that/DOX processes, in cell, fluorescence intensity is lower, and does not show special distribution character.
Comprehensively above experimental result can be thought, the LM-FA/DOX that the present invention reports can obviously improve the picked-up of tumor cell to DOX, has improved the anticancer effect of DOX, reduces the toxic and side effects of DOX normal tissue cell simultaneously, has a good application prospect.
beneficial effect
(1) LM-FA/DOX medicament-carried nano granule of the present invention has good sustained drug release effect and pH response release characteristics, and the tumor cell of homofolic acid expression of receptor is had to targeting and obvious inhibition, can be used for the targeted therapy of cancerous cell;
(2) preparation method of the present invention is simple, reaction condition gentleness, and easy operating, has the prospect of industrialized implementation.
Accompanying drawing explanation
Fig. 1 is LAP, LM-NH
2thermogravimetric analysis result figure with LM-FA;
Fig. 2 is LAP, LM-NH
2, LM-FA, LM-NH
2the ultra-violet analysis result of/DOX and LM-FA/DOX;
Fig. 3 is the TEM photo of associated materials, and wherein (a) is LAP, is (b) LM-FA, is (c) that amplify the part of (b), (d) is LM-FA/DOX;
Fig. 4 be under external condition of different pH DOX from LM-NH
2the cumulative release curve discharging in/DOX and LM-FA/DOX, wherein (a) is that DOX is from LM-NH
2/ DOX discharges, and (b) discharges from LM-FA/DOX for DOX;
Fig. 5 is that "diazoresorcinol" fluorimetry is tested the different cells that obtain through PBS, DOX, LM-NH
2processing the cell viability after 24h with LM-FA, (a) be Hela cell, is (b) L929 cell;
Fig. 6 is that "diazoresorcinol" fluorimetry is tested the different cells that obtain through PBS, DOX, LM-NH
2/ DOX and LM-FA/DOX process the cell viability after 24h, (a) are Hela cell, are (b) L929 cell;
Fig. 7 is that flow cytometry test obtains through PBS, DOX, LM-NH
2/ DOX and LM-FA/DOX process after 4h, the engulf situation of different cells (Hela cell, L929 cell) to DOX (vertical coordinate is the fluorescence intensity after cell endocytic DOX, is directly proportional to the amount of engulfing, and * * * p<0.001);
Fig. 8 is that Hela cell is through PBS, DOX, LM-NH
2fluorescence distribution in cell after/DOX and LM-FA/DOX processing 4h, wherein in material, DOX concentration is 6ppm, and nucleus is dyed blueness with Hoechst33342;
Fig. 9 is that L929 cell is through PBS, DOX, LM-NH
2fluorescence distribution in cell after/DOX and LM-FA/DOX processing 4h, wherein in material, DOX concentration is 6ppm, and nucleus is dyed blueness with Hoechst33342.
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read the content of the present invention's instruction, these equivalent form of values fall within the application's appended claims limited range equally.
Embodiment 1
50mg LAP powder is dissolved in 5mL ultra-pure water, 50 ℃ of water bath condition lower magnetic forces stirrings, LAP is dissolved completely and obtain clear solution, get the aqueous solution that 1mL concentration is the APMES of 20mg/mL, be slowly added dropwise in above-mentioned LAP aqueous solution, and keep 50 ℃ of conditioned responses of bath temperature 16 hours.After reaction finishes, the solution obtaining is all transferred to and held back in the bag filter that amount of analysis size is 8000 – 14000.Bag filter is put into the beaker that 2L deionized water is housed and dialyse, dialysis continues 2-4 days, and change water 3-4 every day.After dialysis finishes, transferred product in bag filter is managed to interior 4 ℃ of preservations to 50mL EP, obtain the LM-NH that silane coupler is modified
2.
Get 2.4mg FA and be dissolved in 4mL DMSO, and add 5.2mg EDC, magnetic agitation 4 hours.Get the LM-NH that 4mL concentration is 6.95mg/mL
2aqueous solution, slowly adds in the DMSO solution of the FA that contains EDC, magnetic agitation reaction 3 days.After reaction finishes, the LM-FA solution obtaining is all transferred to and held back in the bag filter that amount of analysis size is 8000 – 14000.Bag filter is put into the beaker that 2L deionized water is housed and dialyse, dialysis continues 2-4 days, and change water 3-4 every day.After dialysis finishes, transferred product in bag filter is managed to interior 4 ℃ of preservations to 50mL EP, obtain the LM-FA of modified with folic acid.
Embodiment 2
Get the LM-NH that 3mL concentration is 6.95mg/mL
2aqueous solution, and the 3mL concentration LM-FA aqueous solution that is 2.4mg/mL, adding respectively concentration is aqueous solution 6.95mL and the 2.4mL of the DOX of 1mg/mL, magnetic agitation reaction 24 hours.After reaction finishes, solution being transferred to respectively in 15mL centrifuge tube, is centrifugal under 8000rpm condition (5min) at rotating speed, and the precipitate with deionized water obtaining washing 3 times, can obtain medicament-carried nano granule LM-NH
2/ DOX and LM-FA/DOX.
Embodiment 3
By LM-NH
2it is 1mg/mL(LM-NH that/DOX and LM-FA/DOX are dissolved into concentration with the buffer of pH=7.4 and pH=5.4 respectively
2the concentration of/DOX or LM-FA/DOX) solution, getting 1mL, to put into bag filter fixing, is placed in the container of the buffer that contains the different pH of 9mL, is placed in 37 ℃ of shaking tables and vibrates.In the 12h starting, get sample one time every 2h, get sample one time every 24h later.Get the outer liquid 1mL of bag filter at every turn, measure its 480nm place absorbance, then outside bag filter, add corresponding buffer solution 1mL.Obtain under external condition of different pH DOX from LM-NH by the method
2the release profiles discharging in/DOX and LM-FA/DOX.
Embodiment 4
Collect exponential phase Hela and L929 cell, be seeded on 96 porocyte culture plates according to the density in the 10000 every holes of cell, be placed in 5%CO
2, under 37 ℃ of conditions, hatch 24h.Discard after culture medium, 180 μ L culture medium are changed in every hole, and add 20 μ L containing LM-NH
2the PBS of/DOX, LM-FA/DOX or pure DOX or pure PBS(matched group).Tissue Culture Plate is continued to be placed on 5%CO
2, 37 ℃ are continued to hatch 24h.Add "diazoresorcinol" solution (1mg/mL) according to the concentration in the 20 every holes of μ L, the lower 37 ℃ of constant temperature culture 4h of lucifuge environment.Every hole sucks upper strata culture fluid 100 μ L in black 96 orifice plates in order, detects each hole at excitation wavelength lambda=530nm in Multifunction fluorescent microplate reader, the fluorescent value at emission wavelength lambda=590nm place, and the size of fluorescent value can reflect the quantity of living cells.
Collect logarithmic (log) phase Hela and L929 cell, be seeded on 24 porocyte culture plates according to the density in 200,000 every holes of cell, be placed in 5%CO
2, under 37 ℃ of conditions, hatch 24h.Discard after culture medium, 900 μ L culture medium are changed in every hole, and add 100 μ L containing LM-NH
2the concentration of/DOX(DOX is 6ppm), the concentration of LM-FA/DOX(DOX is 6ppm) or pure DOX(concentration be 6ppm) PBS or PBS(matched group).Continue to be placed in 5%CO
2, under 37 ℃ of conditions, hatch 4h.Go culture medium, with PBS washing 3 times, add after the about 3min of 100 μ L trypsinization, add immediately 1mL left and right PBS piping and druming collecting cell, transfer in 10mL centrifuge tube 1000rpm centrifugal 5 minutes, continue with 1mL PBS resuspended.Using fluorescence resolution as parameter, join in flow cytometer and detect Cell sap resuspended strainer filtering as sample, obtain the engulf result of cell to medicine.
Embodiment 5
The circular microscope slide of sizeable processing is put into 24 orifice plates with the density in 1, every hole, and every hole adds 0.5mL culture medium to soak 24 hours.Discard and soak after culture medium, collect exponential phase Hela and L929 cell, be seeded on circular microscope slide according to the density in the 20000 every holes of cell, be placed in 5%CO
2, under 37 ℃ of conditions, hatch 24h.Discard after culture medium, 450 μ L culture medium are changed in every hole, and add 50 μ L containing LM-NH
2the concentration of/DOX(DOX is 6ppm), the concentration of LM-FA/DOX(DOX is 6ppm) or pure DOX(concentration be 6ppm) PBS or PBS(matched group).Continue to be placed in 5%CO
2, under 37 ℃ of conditions, hatch 4h.Discard culture medium, wash 1-2 time with aseptic PBS, every hole adds the PBS solution 0.5mL of 2.5% glutaraldehyde, is statically placed under 4 ℃ of conditions and fixes 30min.Discard glutaraldehyde solution, wash 1-2 time with aseptic PBS, add Hoechst33342(1ug/ml), cover cell, be statically placed in 37 ℃ of 15min that dye.By the sucking-off of Hoechst33342 liquid, wash 3 times with aseptic PBS, on microscope slide, drip a fluorescence sealer, coverslip is ticked from 24 orifice plates, the one side that has cell is pressed onto on microscope slide, by fluorescence distribution in Laser scanning confocal microscope cellular morphology and cell.
Claims (11)
1. a hectorite nano-particle for modified with folic acid, is characterized in that: described hectorite silane coupler is modified, and the mass ratio of hectorite LAP and silane coupler is 5:1-5:4; The mass fraction of silane coupler and folic acid is at 3.01 – 3.34%.
2. the hectorite nano-particle of a kind of modified with folic acid according to claim 1, is characterized in that: described silane coupler is (3-aminopropyl) dimethylethoxysilane APMES.
3. a preparation method for the hectorite nano-particle of the modified with folic acid as described in as arbitrary in claim 1-2, comprising:
(1), under stirring condition, the aqueous solution of silane coupler is dropwise added in hectorite LAP aqueous solution, under 45-60 ℃ of condition, reaction 12-16h, dialysis, obtains amidized hectorite LM-NH
2; Wherein the mass ratio of hectorite LAP and silane coupler is 5:1-5:4;
(2) folic acid is dissolved in solvent, adds 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride EDC, stirring and evenly mixing, obtains mixed solution, then dropwise adds amidized hectorite LM-NH
2aqueous solution, stirs 2-3d, dialyses, and obtains the hectorite nano-particle LM-FA of modified with folic acid; Wherein the ratio of the molal quantity of EDC and folic acid is 10:1-15:1; The mass ratio of lithium amide saponite and folic acid is 20:1-10:1.
4. the preparation method of the hectorite nano-particle of a kind of modified with folic acid according to claim 3, is characterized in that: in described step (1), hectorite LAP concentration of aqueous solution is 5-10mg/mL.
5. the preparation method of the hectorite nano-particle of a kind of modified with folic acid according to claim 3, is characterized in that: the lithium amide saponite LM-NH obtaining in described step (1)
2in, the mass fraction of silane coupler is at 1.76-2.64%.
6. the preparation method of the hectorite nano-particle of a kind of modified with folic acid according to claim 3, is characterized in that: in described step (2), solvent is dimethyl sulfoxide DMSO.
7. the preparation method of the hectorite nano-particle of a kind of modified with folic acid according to claim 3, is characterized in that: amidized hectorite LM-NH in described step (2)
2the concentration of aqueous solution is 6-8mg/mL.
8. the preparation method of the hectorite nano-particle of a kind of modified with folic acid according to claim 3, is characterized in that: in described step (1), (2), dialysis is pure water dialysis, and bag filter molecular cut off size is 8000-14000, and dialysis time is 2-4d.
9. the application of the hectorite nano-particle of the modified with folic acid as described in as arbitrary in claim 1-2, it is characterized in that: the hectorite nano-particle LM-FA aqueous solution that amycin aqueous solution is added to modified with folic acid, stir 12-24h, centrifugal, washing, disperse, obtain the hectorite nano-particle LM-FA/DOX of load amycin modified with folic acid, wherein the hectorite nano-particle LM-FA of modified with folic acid and the mass ratio of amycin are 1:1-4:1.
10. the application of the hectorite nano-particle of a kind of modified with folic acid according to claim 9, is characterized in that: the hectorite nano-particle LM-FA concentration of aqueous solution of described modified with folic acid is 2-5mg/mL; The concentration of amycin aqueous solution is 1-2mg/mL.
The application of the hectorite nano-particle of 11. a kind of modified with folic acid according to claim 9, is characterized in that: described stirring is magnetic agitation, and mixing speed is 100-150r/min; Centrifugal speed is 8000-10000r/min, and the centrifugal time is 5min.
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| CN104208703A (en) * | 2014-08-29 | 2014-12-17 | 东华大学 | Polyethylene glycol-lactobionic acid modified aminated hectorite nano particle as well as preparation method and application thereof |
| CN104644560A (en) * | 2015-03-05 | 2015-05-27 | 东华大学 | Hyaluronic acid modified hectorite amide nanoparticle and preparation and application of hyaluronic acid modified hectorite amide nanoparticle |
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| CN104147608A (en) * | 2014-07-16 | 2014-11-19 | 东华大学 | Lithium amide soapstone nano particles modified by polyethylene glycol-folic acid as well as preparation and application of lithium amide soapstone nano particles |
| CN104147608B (en) * | 2014-07-16 | 2017-02-15 | 东华大学 | Lithium amide soapstone nano particles modified by polyethylene glycol-folic acid as well as preparation and application of lithium amide soapstone nano particles |
| CN104208703A (en) * | 2014-08-29 | 2014-12-17 | 东华大学 | Polyethylene glycol-lactobionic acid modified aminated hectorite nano particle as well as preparation method and application thereof |
| CN104644560A (en) * | 2015-03-05 | 2015-05-27 | 东华大学 | Hyaluronic acid modified hectorite amide nanoparticle and preparation and application of hyaluronic acid modified hectorite amide nanoparticle |
| CN107162011A (en) * | 2017-05-04 | 2017-09-15 | 东华大学 | A kind of preparation method for the hectorite nano particle ICG/LAP for loading indocyanine green |
| CN109045305A (en) * | 2018-07-31 | 2018-12-21 | 东华大学 | A kind of preparation method of the hectorite nano particle of TPGS modification |
| CN109045305B (en) * | 2018-07-31 | 2021-12-10 | 东华大学 | Preparation method of TPGS-modified hectorite nanoparticles |
| CN112137987A (en) * | 2020-11-02 | 2020-12-29 | 南京师范大学 | Preparation method of small-size targeted layered aluminosilicate drug-loaded platform, drug-loaded platform and application thereof |
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