CN112375158A - Chitosan-based nano-drug carrier and preparation method thereof - Google Patents
Chitosan-based nano-drug carrier and preparation method thereof Download PDFInfo
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- CN112375158A CN112375158A CN202011091108.8A CN202011091108A CN112375158A CN 112375158 A CN112375158 A CN 112375158A CN 202011091108 A CN202011091108 A CN 202011091108A CN 112375158 A CN112375158 A CN 112375158A
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- chitosan
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- drug
- deionized water
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Classifications
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- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention provides a chitosan-based nano-drug carrier and a preparation method thereof, belonging to the technical field of biomedical materials. The chitosan-based nano-drug carrier and the preparation method thereof provided by the invention have the advantages of long-acting circulation in vivo, high-efficiency targeting on tumors, improvement on the bioavailability of the drug and lower production cost.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a chitosan-based nano-drug carrier and a preparation method thereof.
Background
Cancer is known to become one of the major "killers" that endanger human health, and most patients generally receive conservative chemotherapy because of the disadvantages of poor selectivity of radiotherapy and surgical resection, long treatment period, and easy recurrence. However, most anticancer drugs have the defects of poor water solubility, poor stability and the like, and meanwhile, the effect of cancer treatment is greatly reduced due to the defects of poor selectivity, large toxic and side effects, easy generation of drug resistance of tumor cells and the like when small molecule administration is carried out, and sometimes serious toxic reactions and side effects force treatment interruption.
Compared with the traditional micromolecule chemotherapy drugs, the nano-drug has unique advantages in the field of tumor treatment: (1) the tumor transmission efficiency of the anti-tumor drug is remarkably improved, and the side effect of the drug is reduced; (2) the solubility of the drug is obviously increased, and the circulation time of the drug in blood is prolonged, so that the tumor treatment effect is greatly enhanced; (3) the stability of the medicine is improved, the blood half-life of the medicine is further improved, the targeting property and the tumor enrichment capacity of the medicine are enhanced, and the release rate of the medicine is controlled; (4) facilitates delivery of biomacromolecule drugs (such as DNA, siRNA, mRNA and protein, etc.) to extracellular active sites; (5) the drug transport capacity across the biological barrier is improved, and the drug resistance mechanism is overcome; (6) the leaky nature of tumor neovasculature and lack of effective lymphatic return allows systemically injected nanopharmaceuticals to accumulate and remain in tumor tissue.
The prior art discloses a method for preparing a hydrophobic drug magnetic targeting sustained and controlled release carrier by adopting chitosan and beta-cyclodextrin, such as a Chinese patent with the publication number of CN 104800169A, which comprises the following steps: (1) obtaining Fe by adopting a chemical coprecipitation method3O4Magnetic nanoparticles, adding ethyl orthosilicate into the magnetic fluid to obtain Fe3O4@SiO2Magnetic microspheres; (2) the Fe modified by epoxy group is obtained by using gamma- (2, 3-epoxypropoxy) -propyl trimethoxy silane as a coupling agent3O4@SiO2Magnetic microspheres; (3) epoxy-modified Fe3O4@SiO2Adding the magnetic microspheres into the alkalified beta-cyclodextrin solution to obtain cyclodextrin modified magnetic microspheres; (4) and (3) crosslinking the chitosan with the cyclodextrin modified magnetic microsphere to prepare the chitosan/cyclodextrin modified magnetic microsphere. The magnetic polymer microsphere has the particle size of 0.085-8.046 mu m and good magnetic responsiveness. The maximum drug loading rate of the magnetic microsphere to hydrophobic drug ibuprofen can reach 233.08mg/g, and the sustained release time can reach 24 h. The method provides a new method for preparing the magnetic targeting sustained and controlled release carrier with biocompatibility.
Disclosure of Invention
The invention aims to provide a chitosan-based nano-drug carrier which can circulate in vivo for a long time, target tumors efficiently, improve the bioavailability of drugs and has low production cost and a preparation method thereof.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the preparation method of the modified chitosan comprises the following steps:
a. reacting chitosan with phthalic anhydride to obtain an amino-protected chitosan product shown in formula I;
b. reacting the chitosan product with amino protection with 6-aminocaproic acid to obtain an esterified chitosan product shown in a formula II;
c. and reducing the esterified chitosan product to obtain the modified chitosan shown in the formula III.
The 6-aminocaproic acid can be connected with chitosan through an ester bond, an amino group and a carbon chain are introduced, the introduction of the carbon chain can enhance hydrophobicity, improve the drug loading rate and the encapsulation rate of hydrophobic drugs, and reduce the drug loss, and the introduction of the amino group can improve the modification amount of lactobionic acid, so that the prepared nanoparticles carrying the anti-tumor drugs can circulate in vivo for a long term and carry out efficient targeted delivery on tumor cells, and the toxic and side effects of the drugs on organs are weakened.
In the present invention, the specific process of step a includes: adding dimethylformamide into chitosan, stirring and swelling for 3-4h, then adding phthalic anhydride, stirring and reacting for 12-14h at the temperature of 120-150 ℃, precipitating a reaction product by using acetone, washing by using ethanol and diethyl ether in sequence, and finally drying by using phosphorus pentoxide.
Preferably, the mass ratio of the chitosan to the phthalic anhydride is 1: 0.9-1.3.
Preferably, the mass-volume ratio of the chitosan to the dimethylformamide is 1g:20-40 mL.
In the present invention, the specific process of step b includes: dissolving the chitosan protected by the amino group in deionized water, adding 6-aminocaproic acid, adding EDC and DMAP, stirring at room temperature for reaction for 12-16h, and dialyzing the reaction solution in the deionized water for 22-26h to remove impurities.
Preferably, the mass ratio of the amino-protected chitosan to the 6-aminocaproic acid is 1: 0.8-1.4.
Preferably, the EDC and DMAP are present in a molar ratio of 8-9: 1.
In the present invention, the specific process of step c includes: adding a hydrazine hydrate aqueous solution into the esterified chitosan product, reacting for 6-10h at 40-60 ℃, cooling to room temperature, dialyzing the reaction solution in deionized water for 3-5d to remove impurities, and freeze-drying the filtrate to obtain the modified chitosan.
Preferably, the mass fraction of hydrazine hydrate in the hydrazine hydrate aqueous solution is 40-50%.
Preferably, the molecular weight of the membrane used for dialysis is 2500-.
The preparation method of the chitosan-based nanoparticle specifically comprises the following steps:
s1, carrying out lactobionic acid modification on the modified chitosan to obtain lactobionic acid modified chitosan shown as a formula IV, a formula V or a formula VI;
s2, preparing modified chitosan nano particles modified by lactobionic acid.
In the present invention, the specific process of step S1 includes: firstly, dissolving lactobionic acid in deionized water, stirring and reacting with EDC and NHS for 0.5-1h at room temperature, then adding into a modified chitosan aqueous solution, reacting for 18-22h at room temperature, dialyzing the reaction solution in deionized water for 22-26h to remove impurities, and freeze-drying the filtrate to obtain the lactobionic acid modified chitosan.
Preferably, the mass ratio of the modified chitosan to the lactobionic acid is 1: 0.7-1.5.
Preferably, the EDC and NHS are present in a molar ratio of 1:1 to 1.5.
In the present invention, the specific process of step S2 includes: dissolving modified chitosan powder modified by lactobionic acid in deionized water, adding 30-35 v/v% ethanol solution, adding glutaraldehyde solution for crosslinking for 6-8h, dialyzing the mixed solution with deionized water for 22-26h to remove impurities, removing dialysate, and freeze-drying the filtrate to obtain chitosan-based nano-ions.
Preferably, the lactobionic acid modified chitosan dissolved in deionized water is 15-25 mg/mL.
Preferably, 0.17-0.26mg/mL of sodium lauryl sarcosinate and 0.02-0.07mg/mL of disodium uridylate are also dissolved in the deionized water. More preferably, the mass ratio of the sodium dodecyl sarcosinate to the disodium uridylate is 3.8-5.6: 1. The addition of the sodium dodecyl sarcosinate and the disodium uridylate according to the mass ratio of 3.8-5.6:1 is possibly beneficial to the contact winding among flexible molecular chains, the winding among the chains is gradually compact, a stable hydrophobic micro-area is formed, the entrapment of hydrophobic drugs can be facilitated, and meanwhile, the chitosan-based nanoparticles have smaller particle size, better uniformity and higher zeta potential, and are beneficial to being taken by cells, so that the long-acting circulation and the targeting property of the drug-loaded nanoparticles can be improved.
Provides a nanoparticle preparation of an antitumor drug, and the preparation method comprises the following steps: preparing the suspension of the chitosan-based nanoparticles, adding the antitumor drug, carrying out ultrasonic treatment for 5-8min under ice bath conditions, dialyzing with a dialysis membrane to remove free drug, removing dialysate, and freeze-drying filtrate to obtain the nanoparticle preparation of the antitumor drug. The 6-aminocaproic acid can be connected with chitosan through ester bonds, and the prepared chitosan-based nanoparticles load hydrophobic antitumor drugs, so that the hydrophobic antitumor drugs can be easily taken by tumor cells, the antitumor drugs can circulate in vivo for a long time and are efficiently delivered in a targeted manner to the tumor cells, and toxic and side effects on organs are weakened.
Preferably, the specific parameters of the ultrasound are: power 750W, frequency 20KHz, stop 2s per 3s of ultrasound.
Preferably, the anti-tumor drug is a hydrophobic drug.
Provides an application of chitosan-based nanoparticles in preparing an anti-tumor drug delivery system.
The invention adopts 6-aminocaproic acid to modify chitosan, thereby having the following beneficial effects: the nano particle can be connected with chitosan through an ester bond, an amino group and a carbon chain are introduced, the introduction of the carbon chain can enhance hydrophobicity, improve the drug-loading rate and the encapsulation rate of hydrophobic drugs and reduce the drug loss, and the introduction of the amino group can improve the modification amount of lactobionic acid, so that the prepared nano particle carrying the anti-tumor drug can circulate in vivo for a long term and carry out efficient targeted delivery on tumor cells, and the toxic and side effects of the drug on organs are weakened.
The invention adopts the sarcosyl and the uridylic acid disodium in the mass ratio of 3.8-5.6:1 when preparing the chitosan-based nano particles, thereby having the following beneficial effects: the chitosan-based nanoparticles are beneficial to entrapping hydrophobic drugs, have small particle size, good uniformity and high zeta potential, are beneficial to being taken by cells, and further can improve the long-acting circulation and targeting property of the drug-loaded nanoparticles.
Therefore, the chitosan-based nano-drug carrier can circulate in vivo for a long time, efficiently target tumors, improve the bioavailability of the drug and have lower production cost and the preparation method thereof.
Drawings
FIG. 1 is an infrared spectrum of chitosan before and after modification in example 1 of the present invention;
FIG. 2 is a graph showing the results of measurement of relative cell viability in test example 1 of the present invention;
FIG. 3 shows the results of the determination of the modification rate of lactobionic acid in test example 1 according to the present invention;
FIG. 4 is a graph showing the results of measurement of drug loading and encapsulation efficiency in test example 1 of the present invention;
FIG. 5 shows the results of measurement of the cellular uptake efficiency in test example 1 of the present invention;
FIG. 6 is a graph showing the results of measuring the concentration of DOX in blood and organs in test example 1 of the present invention;
FIG. 7 shows the results of measuring DOX concentration in tumor tissue in test example 1 of the present invention.
Detailed Description
The present invention is further described in detail with reference to the following examples:
example 1:
1. a preparation method of modified chitosan comprises the following steps:
experimental materials: the deacetylation degree of chitosan is 91%, and the molecular weight is 20000.
Adding 30mL of dimethylformamide into 1g of chitosan, stirring and swelling for 3h, then stirring and adding 1.2g of phthalic anhydride, stirring and reacting for 13h at 130 ℃, after the reaction product is settled by acetone, washing for 2 times by 75 v/v% ethanol, washing for 1 time by diethyl ether, drying by phosphorus pentoxide, dissolving 1g of the obtained solid in 50mL of deionized water, adding 1.2g of 6-aminocaproic acid, adding 1.72g of EDC and 0.36g of DMAP, stirring and reacting for 14h at room temperature, dialyzing the reaction solution in deionized water by a dialysis membrane with the molecular weight of 3000 for 24h to remove impurities, removing the dialysate, adding 300mL of 40 wt% hydrazine hydrate into the filtrate, reacting for 8h at 50 ℃, cooling to room temperature, dialyzing the reaction solution in deionized water by a dialysis membrane with the molecular weight of 3000 for 5d to remove impurities, removing the dialysate, and (5) taking the filtrate for freeze drying to obtain the modified chitosan. The infrared spectrogram of chitosan before and after modification is shown in figure 1.
As can be seen from FIG. 1, the modified chitosan was found to be 3423cm in length as compared with chitosan-1The peak of N-H stretching vibration is enhanced, 2866cm-1The C-H stretching vibration peak of alkyl group is enhanced, 1358cm-1Enhancement of C-H in-plane bending vibration peak of alkyl group, 1172cm-1Enhanced C-C stretching vibration peak of alkyl group, 1740cm-1C in which an ester group is presentO stretching vibration peak, 1306cm-1The peak of C-O-C stretching vibration of ester group appears, which shows that 6-aminocaproic acid and chitosan are covalently connected together by ester bond, and amino group and carbon chain are introduced.
2. A preparation method of a chitosan-based nano-drug carrier comprises the following steps:
2.1 modification of lactobionic acid: firstly, dissolving 1.2g lactobionic acid in 120mL deionized water, stirring and reacting with 0.77g EDC and 0.47g NHS at room temperature for 1h, then adding into 50mL of 20mg/mL modified chitosan aqueous solution prepared above, reacting at room temperature for 20h, dialyzing the reaction solution in deionized water for 24h to remove impurities, removing dialysate, and freeze-drying the filtrate to obtain the lactobionic acid modified chitosan.
The chemical structures of lactobionic acid, modified chitosan and lactobionic acid modified chitosan are determined by nuclear magnetic resonance hydrogen spectroscopy (1H NMR) and common characteristic peaks for lactobionic acid and lactobionic acid modified chitosan appear at chemical shifts δ 4.22 and δ 4.54, whereas chitosan does not show corresponding peaks at this shift, indicating successful modification of lactobionic acid onto chitosan.
2.2 preparation of chitosan-based nanoparticles: dissolving 1g of modified chitosan powder modified by lactobionic acid in 50mL of deionized water, adding 80mL of 32 v/v% ethanol solution, adding 10mL of 0.25 wt% glutaraldehyde solution for crosslinking for 7h, dialyzing the mixed solution with deionized water for 24h to remove impurities, removing dialysate, centrifuging the filtrate to obtain modified chitosan nanoparticle precipitate modified by lactobionic acid, and dispersing the precipitate in PBS buffer (pH7.4) to obtain chitosan-based nanoparticle suspension.
3. The preparation method of the anti-tumor nano-drug delivery system comprises the following steps:
taking 30mL of 0.2mg/mL chitosan-based nanoparticle suspension prepared above, adding 5mg DOX, performing ultrasonic treatment with an ultrasonic instrument for 5min under ice bath condition (ultrasonic interval, stopping for 2s every 3s, power 750W, frequency 20KHz), dialyzing with a dialysis membrane with molecular weight of 3000 to remove free DOX, and freeze-drying the filtrate.
Example 2:
the preparation method of the chitosan-based nano-drug carrier does not comprise the process of preparing modified chitosan, the chitosan used in the part of '2.1 lactobionic acid modification' in the preparation method of the chitosan-based nano-drug carrier is unmodified chitosan, and the rest part is completely consistent with the embodiment 1.
Example 3:
2.2 preparation of chitosan-based nanoparticles: dissolving 1g of modified chitosan powder modified by lactobionic acid, 10mg of sodium lauryl sarcosinate and 2mg of disodium uridylate in 50mL of deionized water, then adding 80mL of 32 v/v% ethanol solution, then adding 10mL of 0.25 wt% glutaraldehyde solution for crosslinking for 7h, then dialyzing the mixed solution with deionized water for 24h to remove impurities, removing dialysate, centrifuging the filtrate to obtain modified chitosan nanoparticle precipitate modified by lactobionic acid, and dispersing the precipitate in PBS buffer (pH7.4) to obtain chitosan-based nanoparticle suspension. The rest of the process was identical to example 1.
Example 4:
2.2 preparation of chitosan-based nanoparticles: dissolving 1g of modified chitosan powder modified by lactobionic acid, 10mg of sodium lauryl sarcosinate and 3.5mg of disodium uridylate in 50mL of deionized water, then adding 80mL of 32 v/v% ethanol solution, then adding 10mL of 0.25 wt% glutaraldehyde solution for crosslinking for 7h, then dialyzing the mixed solution with deionized water for 24h to remove impurities, removing dialysate, centrifuging the filtrate to obtain modified chitosan nanoparticle precipitate modified by lactobionic acid, and dispersing the precipitate in PBS buffer (pH7.4) to obtain chitosan-based nanoparticle suspension. The rest of the process was identical to example 1.
Example 5:
2.2 preparation of chitosan-based nanoparticles: dissolving 1g of modified chitosan powder modified by lactobionic acid, 10mg of sodium lauryl sarcosinate and 1.5mg of disodium uridylate in 50mL of deionized water, then adding 80mL of 32 v/v% ethanol solution, then adding 10mL of 0.25 wt% glutaraldehyde solution for crosslinking for 7h, then dialyzing the mixed solution with deionized water for 24h to remove impurities, removing dialysate, centrifuging the filtrate to obtain modified chitosan nanoparticle precipitate modified by lactobionic acid, and dispersing the precipitate in PBS buffer (pH7.4) to obtain chitosan-based nanoparticle suspension. The rest of the process was identical to example 1.
Test example 1:
1.1 in vitro cytotoxicity assay:
10mL of freshly prepared chitosan-based nanoparticle suspension is filtered by a disposable sterile filter of 0.22 mu m in an ultra-clean workbench to obtain sterile nanoparticle mother liquor. The mother liquor was diluted with DMEM to obtain 1mg/mL nanoparticle suspension.
Respectively digesting fetal mouse fibroblast MEF, mouse breast cancer cell 4T1 and human liver cancer cell MCF-7 in exponential growth phase, and inoculating on 96-well plate (200 μ l/well, 1 × 10)4One/ml), placed at 37 ℃ in 5 v/v% CO2And culturing under the condition of 95% humidity. After the cells grow into a cell monolayer by adherence, absorbing the original culture solution, adding 200 mul of sterile nanoparticle suspension, setting 5 parallel samples for each sample, and taking a DMEM culture solution as negative control; after 48h of incubation, MTT solution (2.5mg/ml, 20. mu.L/well) was added, after 4h of further incubation, the supernatant was carefully aspirated, 100. mu.l DMSO was added to each well, the mixture was shaken on a constant temperature shaker for 10min, the absorbance of each well at 490nm was measured on a microplate reader, and the relative viability of the cells was calculated:
relative survival rate (OD)Sample(s)×100％/ODBlank space
Wherein, ODSample(s)And ODBlank spaceThe absorbance values at 490nm wavelength for the sample and blank were obtained, respectively. The results of the relative viability assay are shown in FIG. 2.
As can be seen from FIG. 2, when the concentration of the chitosan-based nanoparticles prepared in examples 1 and 3 is 1mg/mL, the relative survival rates of MEF cells, 4T1 cells and MCF-7 cells are all above 90%, which indicates that the two nanoparticles have no cytotoxicity and good cell compatibility in the concentration range of less than 1 mg/mL.
1.2 determination of the modification rate of lactobionic acid:
dissolving 1.5g of lactobionic acid in 150mL of deionized water, stirring and reacting with 0.96g of EDC and 0.59g of NHS at room temperature for 1h, then adding into 50mL of 20mg/mL chitosan aqueous solution, reacting at room temperature for 20h, dialyzing the reaction solution in deionized water for 24h to remove impurities, removing dialysate, taking the filtrate, freeze-drying to obtain the lactobionic acid modified chitosan, and weighing. Group A chitosan was unmodified chitosan and group B chitosan was modified chitosan obtained in example 1 above. The modification rate was calculated according to the following formula:
modification ratio [ (M)1-M0)/M1]×100％
In the formula, M1The mass of the modified chitosan;
M0the mass of the chitosan before modification. The results of the lactobionic acid modification are shown in FIG. 3.
As can be seen from FIG. 3, the lactobionic acid modification rate of group B is significantly higher than that of group A, which indicates that 6-aminocaproic acid is connected with chitosan by an ester bond, and the lactobionic acid modification rate can be improved.
1.3 nanoparticle characterization:
the zeta potential, particle size and particle size distribution coefficient (PDI) of the chitosan-based nanoparticles were measured using a laser particle sizer. The detection wavelength was 633nm, the detection angle was 90 ℃ and the temperature was 25 ℃. The average particle size and particle size distribution of the chitosan-based nanoparticles are shown in table 1.
TABLE 1 average particle diameter and particle diameter distribution of Chitosan-based nanoparticles
As can be seen from table 1, the zeta potential of the chitosan-based nanoparticles of example 3 is significantly greater than that of examples 1, 4 and 5, and the particle size and PDI of the chitosan-based nanoparticles of example 3 are significantly less than those of examples 1, 4 and 5, which indicates that the addition of sarcosyl and disodium uridylate at a mass ratio of 3.8-5.6:1 is beneficial to the contact winding between flexible molecular chains, the winding between chains is gradually compact, and a stable hydrophobic micro-region is formed, so that the chitosan-based nanoparticles have a smaller particle size, better uniformity and a higher zeta potential.
1.4 determination of drug loading and encapsulation efficiency: respectively drawing standard curves of DOX in PBS (pH7.4 and 4.9) with different pH values, accurately weighing 10mg DOX in a brown volumetric flask, and diluting with PBS buffer solution with different pH values to constant volume to obtain the final productDOX solution at a concentration of 100. mu.g/mL. Taking certain amount, diluting in multiple proportion to obtain gradient dilution solutions with concentrations of 100 μ g/mL, 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL and 1.5625 μ g/mL respectively, determining OD of each dilution solution at 480nm wavelength of ultraviolet spectrophotometer with corresponding buffer solution as blank control480A standard curve of DOX in PBS buffer (pH7.4 and 4.9) was prepared, and the concentration (C) -absorbance (OD) of doxorubicin was measured under the environmental conditions of pH7.4 and 4.9480) The curves all show good linear relation, and the standard curve equation is OD at pH7.4480＝0.0094C-0.0015，R20.972; OD at pH4.9480＝0.0111C-0.01893，R2＝0.968。
Taking 30mL of 0.2mg/mL chitosan-based nanoparticle suspension prepared above, adding 5mg of DOX, carrying out ultrasonic treatment for 5min by an ultrasonic instrument under ice bath conditions (ultrasonic interval, stopping for 2s every 3s, power is 750W, frequency is 20KHz), centrifuging to obtain supernatant, measuring the absorbance value at 480nm on an ultraviolet spectrophotometer, calculating the mass of DOX in the nanoparticles according to a DOX standard curve, and calculating the drug loading and encapsulation rate of the nanoparticles according to the following formula:
(m) drug loading rateD/mGeneral assembly)×100％
Encapsulation efficiency (m)D/mTotal D)×100％
In the formula, mDMass of DOX in the nanoparticles;
mgeneral assemblyThe weight of the drug-loaded nanoparticles;
mtotal DThe dosage is DOX. The results of the drug loading and encapsulation efficiency measurements are shown in FIG. 4.
As can be seen from FIG. 4, the drug loading and encapsulation efficiency of example 1 are significantly higher than those of example 2, which shows that 6-aminocaproic acid and chitosan are connected by ester bond, so that the drug loading and encapsulation efficiency of chitosan-based nanoparticles on hydrophobic drugs can be improved, and the drug loss can be reduced. The drug loading rate and the encapsulation efficiency of example 3 are obviously higher than those of examples 1, 4 and 5, which shows that the addition of the sarcosyl and the uridylic acid disodium according to the mass ratio of 3.8-5.6:1 can promote the contact winding among flexible molecular chains, the winding among the chains is gradually compact, and a stable hydrophobic micro-region is formed, so that the entrapment of hydrophobic drugs can be facilitated.
1.5 in vitro cell uptake detection of chitosan-based nanoparticles:
1.5.1 Synthesis of Fluorescein Isothiocyanate (FITC) -labeled chitosan-based nanoparticles:
40mg of chitosan-based nanoparticles are weighed and ultrasonically dispersed in 20mL of deionized water, and the pH is adjusted to 7.5 by using 1mol/L NaOH. Weighing FITC, dissolving in 4mL of anhydrous methanol, dropwise adding into deionized water containing chitosan-based nanoparticles, reacting at room temperature in a dark place for 6h, adding 48mL of a methanol/ammonia water (v/v,7:3) mixed solution into the reaction system, centrifuging at 8000 Xg for 10min, removing the supernatant, and washing the obtained precipitate with methanol until the filtrate has no fluorescence absorption at 495 nm. And (5) obtaining a yellow target product after freeze drying.
1.5.2 drawing of Standard Curve for Fluorescein Isothiocyanate (FITC) in Water:
10mg FITC was weighed out and dissolved in 2mL dimethyl sulfoxide and made up to 100mL with 0.5 wt% Triton X-100 in a brown volumetric flask to give 0.1mg/mL FITC standard. The solutions were quantitatively diluted in sequence to obtain gradient solutions of 2. mu.g/mL, 1.5. mu.g/mL, 1.25. mu.g/mL, 1. mu.g/mL, 0.75. mu.g/mL, 0.5. mu.g/mL and 0.25. mu.g/mL, respectively. Each 100 μ L of the dilutions was accurately pipetted into a 96-well plate, and the fluorescence intensity of each gradient dilution was measured on a fluorescence microplate reader (λ ex ═ 488nm, λ em ═ 525nm), and a standard curve of the FITC solution was plotted. The fluorescence intensity value (FI) -sample concentration (N) shows a good linear relation, and the standard curve equation is that FI is 3871.2N-1.65, R2＝0.981。
1.5.2 taking MCF-7 cells of human breast cancer cells in exponential growth phase, digesting with pancreatin, and inoculating on 96-well cell culture plate (100 muL/well, inoculation density of 1 × 10)4One), 5 v/v% CO at 37 ℃2Culturing for 12h in a carbon dioxide incubator with 95% humidity until cells grow into a monolayer in an adherent manner, sucking out original culture solution, carrying out balanced culture in the incubator for 30min by using 5mL of D-Hank's buffer solution, sucking out the D-Hank's, adding 200 mu g/mL of fluorescent chitosan-based nanoparticle suspension prepared by the D-Hank's, respectively culturing for 1h in the carbon dioxide incubator,after 2h and 4h, the fluorescent nanoparticle suspension was aspirated, the suspension was gently rinsed three times with PBS buffer (ph7.4) to remove free nanoparticles, 100 μ L of 0.5 wt% Triton X-16.3010.70 cell lysate was added to each well to lyse the cells for 30min, and finally the fluorescence intensity of the separated cell lysate was measured with a fluorescence microplate reader (λ ex ═ 488nm, λ em ═ 525 nm). Wherein, the untreated blank cells are used as a control, the FITC content in the cells is calculated by referring to an FITC standard curve, and finally the uptake efficiency of the MCF-7 cells to the fluorescent nanoparticles is calculated according to a formula:
the intake efficiency is (W)1/W)×100％
In the formula, W1The content of FITC in cell lysate obtained by processing the fluorescent chitosan-based nanoparticles;
w is the total content of FITC in the fluorescent nanoparticles. The results of the measurement of the cellular uptake efficiency are shown in FIG. 5.
As can be seen from FIG. 5, the cellular uptake efficiency of example 1 is significantly higher than that of example 2, which indicates that 6-aminocaproic acid and chitosan are connected by an ester bond, and the modification rate of lactobionic acid can be improved, and the uptake of tumor cells can be promoted. And the bioavailability of the medicine is increased. The cellular uptake efficiency of example 3 is obviously higher than that of examples 1, 4 and 5, which shows that the chitosan-based nanoparticles are advantageously taken up by cells when the sarcosyl and the uridylic acid disodium are added according to the mass ratio of 3.8-5.6: 1.
1.6 in vivo distribution condition detection of the drug-loaded nanoparticles: taking BALN/c mouse with average weight of 25g, disinfecting the back skin of the mouse with medical alcohol, and mixing to obtain about 1 × 106Mouse mammary cancer cells 4T1 were suspended in 0.1mL of physiological saline and injected subcutaneously into the left underarm of the mouse, and two weeks later, tumors were formed. Killing tumor-bearing mice, and soaking in medical alcohol for 1 min; taking out tumor tissue in sterile operating table, and cutting into 1mm3The tissue mass was prepared into a tissue mass suspension by adding 25ml of HBSS to the tissue mass. 45 BALB/c mice were treated, the skin on the back of the mice was sterilized with medical alcohol, and the prepared tissue mass suspension was inoculated under the skin (0.2 ml/mouse) on the right back of the mice, and tumors were formed after about 8 days. Suspending the drug-loaded nanoparticle lyophilized powder in physiological saline, and injecting the drug-loaded nanoparticle lyophilized powder into mice via tail veinThe amount was 1mg (actual DOX content)/1 kg body weight. Mice were sacrificed 4h, 8h, 16h, 24h after injection, blood, tumors, organs, such as heart, liver, spleen, lung, kidney, were collected and weighed. Adding into buffer solution of pH7.4 containing 0.1M Tris-HCl, 2mM EDTA and 0.1% Triton X-100, grinding, standing for 10min to fully lyse cells, and centrifuging the extractive solution at 5000rpm for 10 min. The fluorescence intensity of DOX in the supernatant was measured with a microplate reader at an excitation wavelength of 480nm and an emission wavelength of 590nm, and the concentration of DOX in each sample was calculated from the standard curve. The results of the measurement of DOX concentration in blood and organs are shown in FIG. 6, and the results of the measurement of DOX concentration in tumor tissues are shown in FIG. 7.
As can be seen from FIGS. 6 and 7, compared with example 2, the DOX concentration in heart, liver, spleen and lung of the mice injected with the drug-loaded nanoparticles of example 1 is significantly less, the DOX concentration in tumor tissues is significantly higher, the DOX concentration in blood is reduced from 3.94% ID/g to 1.32% ID/g, the DOX concentration in blood of example 4 is reduced from 3.30% ID/g to 0.69% ID/g, and the DOX concentration in blood of example 5 is reduced from 3.23% ID/g to 0.62% ID/g, which indicates that 6-aminocaproic acid and chitosan are connected by ester bond, so that the long-acting circulation of the chitosan drug-loaded nanoparticles in vivo and the efficient targeted delivery to tumor tissues can be improved, and the toxic and side effects of drugs on organs can be reduced.
In addition, as can be seen from fig. 6 and 7, compared with examples 1, 4 and 5, the mouse injected with the drug-loaded nanoparticles of example 3 has significantly less DOX enriched in heart, liver, spleen and lung, and significantly higher DOX concentration in tumor tissue, the DOX concentration in blood is reduced from 3.22% ID/g to 0.63% ID/g, while the DOX concentration in blood of example 2 is reduced from 2.48% ID/g to 0.24% ID/g, which indicates that the addition of sarcosyl and uridine monophosphate disodium according to the mass ratio of 3.8-5.6:1 can improve the long-acting circulation and targeting of the chitosan-loaded nanoparticles in vivo, and reduce the toxic and side effects of the drug on organs.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (9)
1. The preparation method of the modified chitosan is characterized by comprising the following steps:
a. reacting chitosan with phthalic anhydride to obtain an amino-protected chitosan product;
b. reacting the chitosan product with amino protection with 6-aminocaproic acid to obtain an esterified chitosan product;
c. and reducing the esterified chitosan product to obtain the modified chitosan.
2. The modified chitosan of claim 1, wherein: the specific process of the step a comprises the following steps: adding dimethylformamide into chitosan, stirring and swelling for 3-4h, then adding phthalic anhydride, stirring and reacting for 12-14h at the temperature of 120-150 ℃, precipitating a reaction product by using acetone, washing by using ethanol and diethyl ether in sequence, and finally drying by using phosphorus pentoxide.
3. The modified chitosan of claim 1, wherein: the specific process of the step b comprises the following steps: dissolving the chitosan with the amino protection in deionized water, adding 6-aminocaproic acid, adding EDC and DMAP, stirring and reacting for 12-16h at room temperature, dialyzing the reaction solution in the deionized water for 22-26h, preferably 24h, and removing impurities.
4. The modified chitosan of claim 1, wherein: the specific process of the step c comprises the following steps: adding a hydrazine hydrate aqueous solution into the esterified chitosan product, reacting for 6-10h at 40-60 ℃, cooling to room temperature, dialyzing the reaction solution in deionized water for 3-5d to remove impurities, and freeze-drying the filtrate to obtain the modified chitosan.
5. The chitosan-based nanoparticle is characterized in that the preparation method specifically comprises the following steps:
s1, performing lactobionic acid modification on the modified chitosan of any one of claims 1-4;
s2, preparing modified chitosan nano particles modified by lactobionic acid.
6. A chitosan-based nanoparticle according to claim 5, wherein: the specific process of step S1 includes: firstly, dissolving lactobionic acid in deionized water, stirring and reacting with EDC and NHS for 0.5-1h at room temperature, then adding into a modified chitosan solution, reacting for 18-22h at room temperature, dialyzing the reaction solution in deionized water for 22-26h to remove impurities, and freeze-drying the filtrate to obtain the lactobionic acid modified chitosan.
7. A chitosan-based nanoparticle according to claim 5, wherein: the specific process of step S2 includes: dissolving modified chitosan modified by lactobionic acid in deionized water, adding 30-35 v/v% ethanol solution, adding glutaraldehyde solution for crosslinking for 6-8h, dialyzing the mixed solution with deionized water for 22-26h to remove impurities, removing dialysate, and freeze-drying the filtrate to obtain chitosan-based nano-ions.
8. The nanoparticle preparation of the antitumor drug is characterized in that the preparation method of the drug-loaded nanoparticle comprises the following steps: preparing a suspension of chitosan-based nanoparticles as described in any one of claims 5-7, adding an antitumor drug, performing ultrasonic treatment for 5-8min under ice bath condition, dialyzing with a dialysis membrane to remove free drug, removing dialysate, and freeze-drying the filtrate to obtain the nanoparticle preparation of the antitumor drug.
9. The nanoparticle formulation for antitumor drugs according to claim 8, characterized in that: the anti-tumor drug is a hydrophobic drug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113896812A (en) * | 2021-10-09 | 2022-01-07 | 华中科技大学鄂州工业技术研究院 | Chitosan derivative and preparation method and application thereof |
| CN114984238A (en) * | 2022-06-25 | 2022-09-02 | 中国海洋大学 | Multifunctional amphiphilic self-assembly nano-carrier based on chitosan and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113896812A (en) * | 2021-10-09 | 2022-01-07 | 华中科技大学鄂州工业技术研究院 | Chitosan derivative and preparation method and application thereof |
| CN114984238A (en) * | 2022-06-25 | 2022-09-02 | 中国海洋大学 | Multifunctional amphiphilic self-assembly nano-carrier based on chitosan and preparation method thereof |
| CN114984238B (en) * | 2022-06-25 | 2024-01-30 | 中国海洋大学 | Multifunctional amphiphilic self-assembled nano-carrier based on chitosan and preparation method thereof |
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