CN113402630A

CN113402630A - Chitosan derivative drug delivery carrier and preparation method and application thereof

Info

Publication number: CN113402630A
Application number: CN202110658164.3A
Authority: CN
Inventors: 喻樊
Original assignee: Yancheng Teachers University
Current assignee: Yancheng Teachers University
Priority date: 2021-06-11
Filing date: 2021-06-11
Publication date: 2021-09-17
Anticipated expiration: 2041-06-11
Also published as: CN113402630B

Abstract

The invention discloses an arginine and ursolic acid modified chitosan nano drug delivery carrier, a preparation method thereof and application thereof as an anti-tumor drug carrier, wherein the chemical structure of the chitosan nano drug delivery carrier is shown as a general formula I. Compared with the prior art, the preparation method of the inventionThe preparation method has the advantages that the process is simple, the chitosan is modified by ursolic acid and arginine, the prepared nano micelle carrier can effectively improve the delivery efficiency of the antitumor drug, the arginine and hydroxyl on the chitosan 6-C generate ester, the water solubility is higher, the drug loading capacity is high, the drug can be more effectively transferred to tumor cells, and the nano micelle carrier also has good functions of penetrating through a membrane and deeply penetrating, inhibiting P-glycoprotein from reversing tumor drug resistance and preventing tumor cell metastasis. The drug delivery carrier can increase the sensitivity of the tumor to drugs, inhibit the discharge of P-glycoprotein to anticancer drugs, reverse tumor drug resistance, improve anticancer effect, reduce the dosage of the anticancer drugs and improve treatment efficiency.

Description

Chitosan derivative drug delivery carrier and preparation method and application thereof

Technical Field

The invention belongs to the technical field of nano drug delivery carriers, and particularly relates to an arginine and ursolic acid modified chitosan nano drug delivery carrier as well as a preparation method and application thereof.

Background

At present, chemotherapy, surgery and radiotherapy are indispensable important treatment methods for malignant tumors.

During clinical chemotherapy, the failure of malignant tumor chemotherapy is frequently seen, and the main reason is the phenomenon of multidrug resistance (MDR) of tumors formed after long-term application of anti-tumor drugs. The term "Multidrug Resistance (MDR)" refers to the development of cross-Resistance in tumor cells to untouched, structurally unrelated, mechanistically distinct antineoplastic drugs after long-term exposure to a chemotherapeutic drug. Chemotherapeutic drugs currently known to be associated with MDR include doxorubicin, epirubicin, daunorubicin, bleomycin, mitomycin, vinblastine, etoposide, taxanes, as well as cisplatin and melphalan, and the like; with the frequent use of chemotherapy drugs, the problem of drug resistance in tumor therapy is more and more prominent, and the drug resistance is one of the most serious obstacles in tumor chemotherapy at present. The american cancer society estimates that death in 90% of cancer patients is associated with the development of drug resistance to varying degrees.

To date, various approaches have been attempted to overcome tumor MDR, including targeted therapies using chemosensitizers and anti-tumor drugs, among others. However, the existing tumor MDR reversal agent generally has the problems of single action target, large self-toxic and side effects and the like, so that the clinical application of the tumor MDR reversal agent is greatly influenced. In addition, the liposome is adopted as a carrier to simultaneously encapsulate the antitumor drug and other drugs for treating tumors, but further research shows that the problem of multidrug resistance cannot be well solved after the drugs are combined with the antitumor drug. In addition, most chemotherapy drugs in clinical application are hydrophobic drugs and can be used after the carrier is solubilized, so that the development of a safe and effective drug-loaded delivery system has important value for realizing clinical application of the chemotherapy drugs. But drug delivery systems are under investigation: 1. toxicity problems of the carrier itself, which is a material that can be used as a high-efficiency delivery carrier, often show poor metabolism and excretion, and thus have potential carrier toxicity problems; 2. the carrier can not help the drug to achieve the purpose of treatment, and the carrier is inert and has no function, so that the carrier can not achieve the purpose of synergistic treatment of tumors; 3. the tumor has a defense mechanism, the disc root of the surrounding tissue is staggered, the medicine is difficult to deeply permeate into the periphery of the tumor and enter into tumor cells, and the problem is also troublesome for the design and constructors of the carrier to face frequently; 4. the targeting problem can reduce the toxic and side effects on normal organs and improve the anti-tumor curative effect if the tumor microenvironment is sensitive; 5. biocompatibility, toxic and side effects caused by the injection of a large amount of synthesized high molecular polymers into blood, and is not suitable for intravenous injection administration; 6. the problem of tumor metastasis, which can occur during the process of tumor generation, growth and treatment, makes the cancer not easy to be cured radically, reduces the treatment effect and brings pain to patients. 7. The synthesis of the anti-cancer drug is not easy, the price is high, and a smaller amount of the anti-cancer drug is urgently needed to achieve a better treatment effect, so that the menstrual flow smoothness burden of a patient is relieved. 8. The problem of drug resistance, how to reverse the drug resistance of drug-resistant tumor cells and enhance the sensitivity of the drug-resistant tumor cells to chemotherapeutic drugs, and the method has important value for improving the chemotherapeutic effect. Therefore, how to reasonably select from a plurality of alternative materials and skillfully construct an ideal drug-carrying system, particularly a targeted drug-carrying system capable of resisting drug-resistant tumors is not easy, but has important value and significance for the effective treatment of the drug-resistant tumors.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the technical problems, the invention utilizes ursolic acid and arginine to modify chitosan, prepares a nano drug delivery carrier which has the functions of penetrating membrane and deeply penetrating, inhibiting P-glycoprotein from reversing tumor drug resistance and resisting tumor metastasis, can be used as a carrier of an anti-tumor drug, reduces the dosage of the anti-cancer drug and improves the treatment efficiency.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

an arginine and ursolic acid modified chitosan nano drug delivery carrier has a chemical structure shown as a general formula I:

the general formula I represents chitosan molecules which can participate in reaction, n is the mole number of deacetylated chitosan monomers, m represents the mole number of arginine directly grafted on chitosan, n-k represents the mole number of ursolic acid on chitosan or the substitution degree of ursolic acid, and k-m represents the mole number of deacetylated chitosan monomers which do not participate in reaction. In general, m, k-m, n-k represent different values depending on the molecular weight and the reaction conditions. n is 10 to 3105, k is 8 to 776, and m is 3 to 3074.

Preferably, in the nano drug delivery carrier, the molecular weight of chitosan is 1-500kDa, the deacetylation degree is 70-95%, the modification proportion of ursolic acid is 1-25%, and the modification proportion of arginine is 10-200%.

The preparation method of the arginine and ursolic acid modified chitosan nano drug delivery carrier comprises the following steps:

(1) preparing an ursolic acid solution, adding NHS and EDC, stirring uniformly, dropwise adding into a chitosan aqueous solution, adjusting the pH to be in a range of 4-7, and reacting at room temperature to obtain N-ursolic acyl-chitosan shown in formula II;

(2) preparing a suspension aqueous solution of N-ursolic acyl-chitosan, adding NHS, EDC and DMAP, stirring uniformly, dropwise adding an arginine aqueous solution, adjusting the pH value to be 4-7, and reacting at room temperature to obtain O-arginyl-N-ursolic acyl-chitosan shown in formula I, namely the nano drug delivery carrier.

Preferably, in the step (1), the ursolic acid solution is prepared by dissolving ursolic acid in DMSO with the concentration of 1-30 mg/ml; the molar ratio of the ursolic acid, the NHS, the EDC and the chitosan is 0.5-2: 1-2: 3-6: 1-3; the molecular weight of the chitosan is 1-500kDa, and the concentration of the chitosan aqueous solution is 1-30 mg/ml; the reaction time at room temperature is 40-56 h.

Preferably, in the step (1), after the room temperature reaction is finished, precipitating with absolute ethanol, filtering, sufficiently washing the precipitate with dichloromethane, ethanol and water, and freeze-drying to obtain the N-ursolic acyl-chitosan shown in the formula II.

Preferably, in the step (2), the concentration of the suspension aqueous solution of the N-ursolic acyl-chitosan is 1-30 mg/ml; the molar ratio of the arginine, the NHS, the DMAP and the EDC is 1: 0.1: 3-5; the concentration of the arginine aqueous solution is 1-300 mg/ml, and the molar ratio of arginine to N-ursolic acyl-chitosan is 1-10: 1; the reaction time at room temperature is 40-56 h.

Preferably, in the step (2), after the reaction at room temperature is finished, the O-arginyl-N-ursolic acyl-chitosan shown in formula I is prepared by dialysis, suction filtration and freeze drying of the filtrate.

Preferably, the preparation method of the blank nano-micelle comprises the following steps: dissolving 0-arginyl-N-ursolic acyl-chitosan in water, wherein the concentration of the solution is 0.1-50 mg/ml, performing ultrasonic treatment in a water bath, and freeze-drying to obtain a target product, namely O-arginyl-N-ursolic acyl-chitosan nano micelle.

The preparation process has the advantages of simple operation, simple process, low manufacturing cost and the like, and can adjust the substitution degree of the amphiphilic copolymer by changing the feeding ratio and the reaction time so as to change the particle size of the nano micelle, wherein the formed O-arginyl-N-ursolic acid yl-chitosan nano micelle is regular spherical, and the average particle size is 50-500 nm.

The invention also provides application of the arginine and ursolic acid modified chitosan nano drug delivery carrier as an anti-tumor drug carrier.

Preferably, the antitumor drug is selected from paclitaxel, docetaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, capecitabine, vincristine, hydroxycamptothecin, doxorubicin, adriamycin or mitomycin, etc.

Preferably, the application method comprises the steps of dissolving the nano drug delivery carrier, preparing a micelle solution, mixing the micelle solution with a solution containing the anti-tumor drug, incubating, and removing free drug to obtain the O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle.

The research of the invention shows that the O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle has the capability of inhibiting P-gp from reversing the multidrug resistance of the tumor, and the O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle has obvious anti-tumor activity.

The invention firstly takes arginine, ursolic acid and chitosan as materials to synthesize an amphiphilic micelle material, and prepares the paclitaxel nano micelle which has the functions of penetrating the membrane and deeply permeating and inhibiting P-glycoprotein from reversing tumor drug resistance, so that the drug reaches the action part with minimum loss, the drug effect of paclitaxel can be improved, the dosage is reduced, and the paclitaxel nano micelle is a novel dosage form and is the result of technical innovation. In the prior art, ursolic acid and paclitaxel are directly combined for use, however, paclitaxel is a hydrophobic compound, has poor solubility in water, is a P-gp substrate, is easily discharged by P-gp, is easy to generate drug resistance, has a defense mechanism for tumors, is ragged in the disc root nodes of surrounding tissues, and is difficult to deeply permeate into the periphery of the tumors and enter tumor cells. In the invention, a novel amphiphilic chitosan derivative, namely O-arginin-N-ursalic-chitosan micelle (OAUCS), which has membrane penetration and P-gp reversal multidrug resistance inhibition functions and simultaneously has an anti-tumor metastasis effect is synthesized, wherein arginine can imitate membrane penetration peptide to promote drugs to better enter tumors, and ursolic acid can play a role in P-gp inhibition efflux and reversal of tumor resistance. The nanometer preparation has better tissue penetration effect, and hydrophilic arginine is used for modifying chitosan, and the hydrophilic arginine and the hydrophobic ursolic acid are respectively positioned on 6-and 2-C, so that the nanometer preparation is beneficial to the extension of high-molecular chitosan molecules in water, and has better water solubility and more stability. The arginine-enriched chitosan not only can improve the hydrophilicity of the chitosan, but also can carry medicaments and deeply permeate into tumor cells. Arginine can help hydrolyzed ursolic acid to form salt, and the solubility of the ursolic acid in tumor cells is increased, so that a better effect is achieved. In addition, arginine with a certain concentration is beneficial to preventing tumor metastasis, ursolic acid can be used as a hydrophobic part to improve the carrying capacity of a hydrophobic drug, and can also play a role in inhibiting the efflux of P-gp to the drug, reversing tumor drug resistance and reducing the dosage of the anti-tumor drug. Therefore, the modification of arginine and ursolic acid can achieve synergistic effect and increase the treatment effect.

The technical effects are as follows: compared with the prior art, the preparation method has simple process, and as the chitosan is modified by adopting the ursolic acid and the arginine, the prepared nano micelle carrier not only has good functions of penetrating deeply into a membrane and inhibiting P-glycoprotein from reversing tumor resistance and tumor metastasis, but also can effectively improve the delivery efficiency of the anti-tumor drug, has higher drug-loading rate and better water solubility, so that the drug can be more effectively transferred to tumor cells.

Drawings

FIG. 1 is an infrared spectrum of O-arginyl-N-ursolic acyl-chitosan.

FIG. 2 is a nuclear magnetic spectrum of O-arginyl-N-ursolic acyl-chitosan.

FIG. 3 is a transmission electron micrograph of O-arginyl-N-ursolic acid acyl-chitosan nano micelle.

FIG. 4 shows the results of toxicity experiments of OAUCS on MCF-7/PTX cells.

FIG. 5 shows the results of toxicity experiments of PTX-OAUCS on MCF-7/PTX cells.

FIG. 6 is a graph of the effect of groups on the level of apoptosis of MCF-7/PTX cells.

FIG. 7 is a graph of the effect of groups on tumors in mice; wherein, the graph A is a tumor body growth curve; panel B post-tumor-ablation volume; panel C shows the tumor mass after detachment.

FIG. 8 shows the results of the rhodamine efflux assay.

FIG. 9 shows that OAUCS down-regulates P-gp expression.

Detailed Description

The present invention will be further described with reference to the following examples in order to provide a thorough understanding of the present invention.

Example 1

Weighing 500mg of ursolic acid, dissolving in 50ml of dimethyl sulfoxide, adding a proper amount of 0.125g of NHS and 0.58g of EDC, stirring uniformly, dropwise adding 1mg/ml of chitosan solution (1% of acetic acid water: 1 of dimethyl sulfoxide by volume), adjusting the pH value to be within 5.0 range, reacting at room temperature for 48 hours, precipitating with 1000ml of absolute ethanol, performing suction filtration, fully washing the precipitate with 30ml of dichloromethane, 30ml of ethanol and 30ml of water for 3 times, and freeze-drying to obtain the N-ursolic acyl-chitosan shown in formula II; weighing 500mg of N-ursolic acyl-chitosan shown as II, suspending in water, adding 1g of NHS, 0.11g of DMAP and 4.7g of EDC, stirring uniformly, adding 1.5g of arginine, adjusting the pH value to 5.0, reacting at room temperature for 48h, dialyzing, filtering, and freeze-drying the filtrate to obtain O-arginyl-N-ursolic acyl-chitosan shown as formula I; dissolving 100mg O-arginyl-N-ursolic acyl-chitosan in 20ml water, and performing ultrasonic treatment in ice bath to obtain O-arginyl-N-ursolic acyl-chitosan nanometer micelle (OAUCS).

Preparation of O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle: weighing O-arginyl-N-ursolic acyl-chitosan, dissolving in deionized water, performing ultrasonic treatment for 30 times by using a probe, controlling the power to be 200w, stopping working for 2s and 3s, and preparing a 2mg/mL micelle solution. And adding dimethyl sulfoxide into paclitaxel to obtain solution with concentration of 2 mg/mL. According to the weight percentage of paclitaxel: adding dimethyl sulfoxide solution containing paclitaxel of 2mg/mL into the dosage of O-arginyl-N-ursolic acyl-chitosan with mass ratio of 25%, stirring at room temperature in dark place for 2 hours, transferring into a dialysis bag with molecular weight cutoff of 3500 after the stirring is finished, dialyzing with pure water for 24 hours, collecting the dialyzed product, centrifuging at low temperature of 8000r for 10min to remove paclitaxel which is not encapsulated by micelle, and collecting the supernatant to obtain O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle (PTX-OAUCS).

And (3) measuring the drug loading rate and the encapsulation efficiency of the paclitaxel in the drug-loaded micelle by using HPLC. Taking paclitaxel-loaded micelle, diluting with mobile phase, and subjecting to Supersil ODS 2C chromatography₁₈(4.6X 250mm, 5 μm), methanol/water (70: 30, v/v) as mobile phase, flow rate of 1ml/min, detection wavelength of 227nm, and sample amount of 20 μ l. And calculating the drug concentration in the drug-loaded micelle according to the standard curve.

Entrapment rate ═ paclitaxel administration mass-unencapsulated free paclitaxel mass)/paclitaxel administration mass × 100%

The drug loading is the mass of paclitaxel in the micelle/mass of the micelle x 100%.

The drug-loading rate of the drug-loaded micelle is 22.7% and the encapsulation rate is 84.6% through calculation.

Preparing blank micelle and drug-loaded micelle solution, and respectively measuring the particle size and surface potential of the drug-loaded micelle by using a particle size and surface potential measuring instrument. The particle size of the drug-loaded micelle is 127.5 +/-3.47 nm and the Zeta potential is 23.2 +/-3.21 mV.

1.1 Fourier Infrared Spectroscopy (FTIR)

The infrared spectrum was measured by means of a Nicolet 2000 type Fourier transform infrared spectrometer (Nicolet, Inc., USA). The determination method comprises the following steps: adding about 1-2 mg of the prepared O-arginyl-N-ursolic acyl-chitosan freeze-dried sample into about 200mg of ground and dried KBr powder, fully mixing, tabletting, and scanning in an infrared spectrometer, wherein the scanning range is as follows: 4000cm^-1～400cm^-1The IR spectrum was recorded as shown in FIG. 1.

1.2 Nuclear magnetic resonance 1H-NMR

The freeze-dried sample of O-arginyl-N-ursolic acyl-chitosan prepared above is dissolved in D₂O or deuterated DMSO, irradiated at a frequency of 150MHz and recorded at 20 ℃ on a nuclear magnetic resonance apparatus of the Bruker (AVACE) AV-500 type (Bruker, Germany), as shown in FIG. 2.

1.3 measurement of degrees of substitution of Arbuterol and arginyl groups

1.3.1 measurement of degree of substitution of Arbuterol with Ursolic acyl group

C, N, H element mass percentages of the chitosan and ursolic acid chitosan were measured respectively by an Elementar Vario EL III element analyzer (Elementar, germany).

The substitution degree of ursolic acid by elemental analysis was 20.91%.

1.3.2 determination of the degree of arginyl substitution

The C, N, H element mass percentages of the chitosan, ursolic acid-based chitosan, and O-arginyl-N-ursolic acid-based chitosan were determined by an Elementar Vario EL III element analyzer (Elementar, Germany).

The degree of substitution of arginine by elemental analysis was 178%.

1.4 Transmission Electron microscope

And taking a proper amount of prepared drug-loaded nanoparticles, diluting the drug-loaded nanoparticles by 100 times with distilled water, dripping the drug-loaded nanoparticles on a copper net, standing for a period of time, sucking the drug-loaded nanoparticles by using filter paper, dripping 2% phosphotungstic acid solution for negative dyeing for 5-10 min, naturally volatilizing, and observing the shape by using a transmission electron microscope. The results are shown in FIG. 3, where the under-mirror particle size is < 200nm, which is a relatively round particle.

Example 2

Weighing 250mg of ursolic acid, dissolving the ursolic acid in 25ml of dimethyl sulfoxide, adding a proper amount of 0.125g of NHS and 0.58g of EDC, stirring uniformly, dropwise adding 1mg/ml of chitosan solution (1% of acetic acid water to the volume ratio of the dimethyl sulfoxide is 1: 1), adjusting the pH value to be within the range of 5.0, reacting at room temperature for 48 hours, precipitating with 1000ml of absolute ethyl alcohol, performing suction filtration, fully washing and precipitating with 30ml of dichloromethane, 30ml of ethanol and 30ml of water for 3 times, and freeze-drying to obtain the N-ursolic acyl-chitosan shown in the formula II; weighing 250mg of N-ursolic acyl-chitosan shown in II, suspending in water, adding 1g of NHS, 0.11g of DMAP and 4.5g of EDC, stirring uniformly, adding 1.5g of arginine, adjusting the pH value to 5.0, reacting at room temperature for 48h, dialyzing, performing suction filtration, and freeze-drying the filtrate to obtain O-arginyl-N-ursolic acyl-chitosan shown in formula I; dissolving 100mg of O-arginyl-N-ursolic acyl-chitosan in 20ml of water, and carrying out ice bath ultrasonic treatment to obtain the O-arginyl-N-ursolic acyl-chitosan nano micelle.

Preparation of O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle: weighing O-arginyl-N-ursolic acyl-chitosan, dissolving in deionized water, performing ultrasonic treatment for 30 times by using a probe, controlling the power to be 100w, stopping working for 2s to 1s, and preparing a 2mg/mL micelle solution. And adding dimethyl sulfoxide into paclitaxel to obtain solution with concentration of 2 mg/mL. According to the weight percentage of paclitaxel: adding dimethyl sulfoxide solution containing paclitaxel of 2mg/mL into the dosage of O-arginyl-N-ursolic acyl-chitosan with the mass ratio of 25%, stirring at room temperature in dark place for 2 hours, transferring into a dialysis bag with the molecular weight cutoff of 3500 after the stirring is finished, dialyzing with pure water for 24 hours, collecting the dialyzed product, centrifuging at low temperature of 8000r for 10min to remove paclitaxel which is not encapsulated by micelle, and collecting the supernatant to obtain the O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle.

The drug-loading rate of the drug-loaded micelle is 21.8% and the encapsulation rate is 83.5% through calculation.

Preparing blank micelle and drug-loaded micelle solution, and respectively measuring the particle size and surface potential of the drug-loaded micelle by using a particle size and surface potential measuring instrument. The particle size of the drug-loaded micelle is 129.2 +/-3.20 nm and the Zeta potential is 24.6 +/-1.73 mV.

Example 3:

MTT method for detecting influence of OAUCS on MCF-7/PTX tumor cell proliferation capacity

MTT method detection cultured cells are inoculated into a 96-well culture plate and placed in saturated humidity CO₂Continuously culturing in incubator for 24h (37 deg.C, 5% CO)₂) Discarding the culture solution, adding a proper amount of DMEM culture solution into each well, adding Blank OAUCS micelles without PTX into the control group and Blank OAUCS group, adding PTX-OAUCS containing 3 mu M, PTX-OAUCS containing 6 mu M and PTX-OAUCS containing 9 mu M into the positive medicine group, and adding calf serum respectively. And continuously culturing for 24h to prepare a test cell liquid. Adding MTT solution into each hole, incubating for 4h at 37 ℃, absorbing the culture solution in each hole, adding dimethyl sulfoxide (DMSO) into each hole, measuring the OD value of each hole by adopting an enzyme-linked immunosorbent assay detector after oscillation, measuring at a certain wavelength, and only containing DMSO in the zeroing hole. The mean OD value of each well was calculated as the tumor cell inhibition rate according to the following formula:

the inhibition ratio (%) (average OD value of control group-average OD value of administration group)/average OD value of control group × 100%.

Data processing: and (3) inputting the OD value into an Excel table, calculating the cell inhibition rate according to a formula, wherein data statistics and processing are performed through software SPSS, Student's t-test is adopted for comparison among measurement data groups, one-wayANOVA is adopted for comparison among multiple groups, p is less than 0.05 to indicate that the statistical difference exists, and p is less than 0.01 to indicate that the statistical difference exists.

The results show that: after the OAUCS acts at different concentrations (1mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 30mg/ml and 60mg/ml), 1-30mg/ml has no statistical difference on the proliferation inhibition rate of MCF-7/PTX cells; after 30mg/ml OAUCS, the proliferation inhibition rate of MCF-7/PTX cells is obviously higher than that of a control group, and the difference has statistical significance (P is less than 0.05). The OAUCS has low proliferation inhibition rate on MCF-7/PTX cells in the range of 0-20mg/ml and is nontoxic. In the later examination of the tumor-killing effect of PTX-OAUCS, vehicle concentrations below 20mg/ml were selected to exclude the tumor-killing effect of the drug vehicle (see fig. 4).

Reversal of MCF-7/PTX resistance by OAUCS

Taking human breast cancer paclitaxel-resistant cell line (MCF-7/PTX) cells growing in logarithmic phase, adjusting cell density according to 8 × 10 per well³Inoculating each cell in a 96-well plate, adding 180 mu L of cell suspension into each well, culturing for 24h, sucking the culture medium for changing the liquid, replacing a PTX group and a PTX-OAUCS group with 100 mu L of DMEM culture medium, adding 100 mu L of OAUCS into the OAUCS group for culturing for 24h, and then adding PTX with different concentrations, wherein the final concentrations are respectively 0, 1, 5, 25, 125 and 250 mu g/mL, and the total volume of each well is 200 mu L; the control wells were left without drug; the zero setting hole is culture solution without cells, and each group is provided with 3 multiple holes. Placing at saturated humidity, 37 deg.C and 5% CO₂Culturing in incubator for 48 hr, adding MTT (5mg/mL)10 μ L into each well, culturing for 4 hr, discarding culture supernatant, adding DMSO into each well, shaking on shaker for 10min at 150 μ L, measuring OD570 value of each well, and calculating inhibition rate of paclitaxel on MCF-7/PTX cell proliferation and IC₅₀Value, resistance index. Separately taking PTX-OAUCS, calculating inhibition rate and IC by adopting the same PTX concentration and experimental method as those of the PTX group₅₀Value, resistance index.

The calculation formula is as follows: cell growth inhibition (%) [1- (experimental OD value-blank OD value)/(control OD value-blank OD value)]X is 100%; by using SigMaPlot 10.0 software computing IC₅₀A value; drug resistance reversal multiple (RI) ═ drug pre-effect cell IC₅₀Post-operative cellular IC₅₀。

As shown in Table 1, OAUCS enhances the proliferation inhibitory effect of PTX on MCF-7/PTX cells and increases the sensitivity of cells to PTX. This result indicates that OAUCS can reduce resistance of MCF-7/PTX cells to PTX.

TABLE 1 reversion of MCF-7/PTX resistance in each sample

Sample (I)	IC₅₀(μM)	Reversal multiple of drug resistance
			Paclitaxel PTX	30.89	/
OAUCS	10.44	2.95
			PTX-OAUCS	1.95	15.84

In conclusion, OAUCS and PTX-OAUCS can be developed into drugs for reversing the drug resistance of breast cancer to paclitaxel.

Example 4: effect of OAUCS on reversing drug resistance of MCF-7/PTX cells to paclitaxel

MTT method for detecting MCF-7/PTX cell proliferation inhibition by PTX-OAUCS

The experimental procedure was the same as in example 3. Taking human breast cancer paclitaxel-resistant cell line (MCF-7/PTX) cells growing in logarithmic phase, adjusting cell density according to 8 × 10 per well³Inoculating each cell in a 96-well plate, adding 180 mu L of cell suspension into each well, culturing for 24h, and adding PTX-OAUCS and PTX with different concentrations, wherein the total volume of each well is 200 mu L; the control wells were left without drug; the zero setting hole is culture solution without cells, and each group is provided with 3 multiple holes. Placing at saturated humidity, 37 deg.C and 5% CO₂And (3) continuously culturing for 48h in the incubator, adding 10 mu L of MTT (5mg/mL) into each well, continuously culturing for 4h, discarding culture supernatant, adding 150 mu L of DMSO into each well, shaking on a shaking table for 10min, measuring the OD570 value of each well, and calculating the tumor cell inhibition rate.

The results show that: compared with the single PTX 3 mu M, the 3 mu M PTX-OAUCS has the advantages that the proliferation inhibition rate of MCF-7/PTX cells is obviously increased, and the difference has obvious statistical significance (P is less than 0.01); compared with 3 mu M PTX-OAUCS, the proliferation inhibition rate of MCF-7/PTX cells is obviously increased, and the difference has obvious statistical significance (P is less than 0.01). It was shown that PTX-OAUCS significantly inhibited the proliferation of MCF-7/PTX cells (see FIG. 5).

2. Flow cytometry detection of PTX-OAUCS promotion of MCF-7/PTX cell apoptosis

MCF-7 and MCF-7/PTX cells were divided into 6 groups: control group, 3. mu.M PTX group, 6. mu.M PTX group, 3. mu.M PTX-OAUCS group, 6. mu.M PTX-OAUCS group. Taking cells growing in logarithmic phase, adjusting the number of the cells to a proper number according to the requirement, inoculating the cells to a culture plate for culture, observing the cells the next day, adjusting the concentration of the medicine to a proper concentration, and applying the medicine to a subsequent experiment after 48 hours of action; adjusting the cells to 2.5X 10⁵One per ml, inoculated in six-well plates, i.e. 5X 10⁵Per well; discarding the old culture medium after the cells adhere to the wall the next day, diluting the PTX or PTX-OAUCS to the required concentration by using a DMEM complete culture medium, adding the diluted PTX or PTX-OAUCS into a six-hole plate, and continuously culturing for 48 hours by using an incubator; preparing PBS phosphate buffer solution, a straw, pancreatin without EDTA, a flow tube, a BD apoptosis kit and the like for later use; marking flow tubes, respectively sucking old culture medium in six-hole plate into corresponding flow tubes, washing with PBS phosphate buffer solution for 3 times, adding 0.5mL pancreatin without EDTA, digesting completely, and neutralizing with old culture mediumPlacing the cells into a flow tube labeled accordingly; centrifuging at 1000rmp for 5min, discarding the supernatant, adding 1mL PBS buffer solution into each tube, centrifuging after slight shaking on a vortex shaker, repeating for 3 times at 2000rmp for 10min, and discarding the supernatant; diluting 10 times of Annexin V Binding Buffer to 1 time, adding 100 mu L of Buffer into each tube, continuously adding 7-AAD and PE Annexin V, keeping each tube at 2.5 mu L, keeping out of the sun for 30min, adding 1 time of Annexin V Binding Buffer, and keeping at 200 mu L/hole; and (4) performing analysis on the machine, and recording the apoptosis rate of each group of cells by using an experimental book.

The results show that: compared with the single use of 3 mu M PTX, the apoptosis rate of MCF-7/PTX cells of 3 mu M PTX-OAUCS is obviously increased, and the difference has obvious statistical significance (P is less than 0.01); compared with the 3 mu M PTX-OAUCS, the apoptosis rate of MCF-7/PTX cells is obviously increased, and the difference has obvious statistical significance (P is less than 0.01). It was shown that PTX-OAUCS significantly promoted the level of apoptosis in MCF-7/PTX cells (see FIG. 6).

Example 5: effect of ursolic acid on reversing drug resistance of mouse subcutaneous transplantation tumor to paclitaxel

MCF-7/PTX cells grown in log phase were taken, resuspended in PBS and adjusted to 3X 10⁷one/mL, 3X 10 injection per mouse from the underarm⁶One cell, 100. mu.L/cell. 10 days after cell injection, mice were randomly grouped and administered individually, and animals were divided into 6 groups: a control group (PBS 100. mu.L is injected every 3 days), a blank vector OAUCS group (OAUCS 10mg/kg is injected every 3 days), an LPTX group (PTX 10mg/kg is injected every 3 days), a low-dose LPTX-OAUCS group (PTX-OAUCS 10mg/kg is injected every 3 days), a medium-dose MPTX-OAUCS group (PTX-OAUCS 25mg/kg is injected every 3 days), a high-dose HPTX-OAUCS group (PTX-OAUCS 50mg/kg is injected every 3 days), and LPTX, LPTX-OAUCS, MPTX-OAUCS and HPTX-OAUCS groups are injected with doses calculated by drug PTX for 4 weeks to strip tumor bodies.

The length and width of the tumor were measured by a vernier caliper every 3 days, and the tumor volume was calculated (V ═ L × W)²X 0.5, L represents the length of the tumor; w represents the width of the tumor), data were recorded, and a curve of tumor growth in mice was plotted. After the final drug is applied for 3 days, the tumor is taken out, the stripped tumor body weight is weighed by a balance, the stripped tumor body length and width are measured by a vernier caliper, data are recorded, and a graph is drawn.

The results show that: the low dose L-PTX-OAUCS group has slower tumor growth under the skin of mice compared with the L-PTX group, and the difference has statistical significance (P is less than 0.05); the high dose H-PTX-OAUCS group has slower tumor growth under the skin of the mice and the difference has statistical significance (P is less than 0.05) compared with the low dose L-PTX-OAUCS group; after tumor body exfoliation, compared with the L-PTX group, the low-dose L-PTX-OAUCS group has smaller tumor body volume and obvious statistical significance of difference (P is less than 0.05); the tumor volume of the high-dose H-PTX-OAUCS group is reduced compared with that of the low-dose L-PTX-OAUCS group, and the difference has statistical significance (P is less than 0.05); after tumor exfoliation, the high dose H-PTX-OAUCS group had a lighter tumor weight and the difference was statistically significant (P < 0.05) compared to the low dose L-PTX-OAUCS group. It was shown that the effect of L-PTX-OAUCS significantly inhibited the growth of tumors in mice compared to the effect of L-PTX alone (see FIG. 7).

Example 6: determination of P-gp function by rhodamine efflux test

The function of P-gp was performed using the classical method: the fluorescent dye rhodamine 123(R123) that stains cells is used to identify, and rhodamine is also a recognized, classical P-gp substrate. There are many reports that the function of P-gp is significantly associated with rhodamine efflux, and inhibition of P-gp leads to dye retention. Rhodamine can be almost completely encapsulated in nanomicelle systems. Accumulation of rhodamine in cells was detected by flow cytometry.

Preparation of R123-OAUCS: weighing O-arginyl-N-ursolic acyl-chitosan, dissolving in deionized water, performing ultrasonic treatment for 30 times by using a probe, controlling the power to be 100w, stopping working for 2s to 1s, and preparing a 2mg/mL micelle solution. And adding dimethyl sulfoxide into R123 to prepare a solution with the concentration of 2 mg/mL. According to the weight percentage of paclitaxel: adding dimethyl sulfoxide solution containing 2mg/mL R123 into 25% of O-arginyl-N-ursolic acyl-chitosan, stirring at room temperature in dark place for 2 hours, transferring into a dialysis bag with molecular weight cutoff of 3500 in dark place, dialyzing with pure water for 24 hours, collecting the dialyzed product, centrifuging at 8000R for 10min at low temperature to remove R123 which is not encapsulated by micelle, and collecting supernatant to obtain R123-OAUCS.

Taking MCF-7/PTX cells grown in logarithmic phaseAdjusting the number of cells to a proper number as required, inoculating the cells to a culture plate for culture, observing the cells the next day, adjusting the concentration of the medicine to a proper concentration, and performing subsequent experiments after 48 hours of action; adjusting the cells to 2.5X 10⁵One per ml, inoculated in six-well plates, i.e. 5X 10⁵Per well; the old culture medium is discarded after the cells are attached to the wall the next day, the rhodamine solution and R123-OAUCS are added into the culture medium by using a DMEM complete culture medium, the culture medium contains 5 mu g/mL of rhodamine, and the rhodamine solution and the R123-OAUCS are incubated for 2 hours at 37 ℃, and the control group is a DMEM culture medium. Discarding the supernatant, adding 200 mu L of PBS for washing for 3 times, digesting with pancreatin, adding 500 mu L of precooled PBS for resuspending cells, detecting in a flow cytometer, wherein the excitation wavelength is 480nm, the emission wavelength is 540-660 nm, and the intracellular rhodamine concentration is expressed by the average value of fluorescence intensity.

Rhodamine 123(R123) is a classical P-gp substrate, is subjected to P-gp efflux, has fluorescence, and can reflect the strength of the P-gp efflux through the strength of intracellular fluorescence. As shown in figure 8, the OAUCS has the influence on rhodamine that is discharged by P-gp, and the graph shows that R123 singly incubates with cells for 2h, and the R123 entering the cells is very little, while the R123 is made into R123-OAUCS micelles, the R123 entering the cells is greatly increased, the fluorescence intensity of the R123 in the cells is increased, and the difference has obvious statistical significance (P is less than 0.01); it is shown that OAUCS can indeed inhibit the efflux of R123 by P-gp.

Western blot detection of expression of P-gp

Taking cells growing in logarithmic phase, adjusting the number of the cells to a proper number according to the requirement, inoculating the cells to a culture plate for culture, observing the cells the next day, adjusting the concentration of the medicine to a proper concentration, and applying the medicine to a subsequent experiment after 48 hours of action; adjusting the cells to 2.5X 10⁵One per ml, inoculated in six-well plates, i.e. 5X 10⁵Per well; the next day, the old medium was discarded after the cells were attached to the wall, and the control group, the low dose OAUCS group (L-OAUCS, 0.1mg/ml), the medium dose OAUCS group (M-OAUCS, 1mg/ml), and the high dose OAUCS group (H-OAUCS, 10mg/ml) were diluted to the required concentration with DMEM complete medium and added to six well plates at 200. mu.L, and the incubator was continued for 48 hours. Collecting MCF-7/PTX cells incubated with OAUCS of different concentrations, extracting total protein, quantifying protein amount by BCA method, performing sample electrophoresis, transferring protein on gel toSealing the PVDF membrane at room temperature for 1h, adding primary antibodies (P-gp primary antibody and beta-actin primary antibody), incubating overnight at 4 ℃, placing the PVDF membrane in GST rabbit secondary antibody for incubation, washing for 3 times, developing, detecting and recording the expression of P-gp.

The OAUCS and MCF-7/PTX cells are incubated for 48h, Western blot is used for detecting the expression of P-gp, the influence result of the OAUCS on the expression of the P-gp is shown in figure 9, the expression of the P-gp is reduced remarkably in a low-dose L-OAUCS group compared with a control group, and the difference has remarkable statistical significance (P is less than 0.05); compared with the low-dose L-OAUCS group, the high-dose H-OAUCS group has the advantages that the expression of P-gp is reduced remarkably, and the difference has extremely remarkable statistical significance (P is less than 0.001). The MCF-7/PTX cells showed a gradual decrease in P-gp expression with increasing OAUCS concentration, indicating that OAUCS can down-regulate P-gp expression.

Example 7:

MTT method for measuring cell adhesion

10g/L BSA; a96-well plate was added to each well of 50. mu.l of 25mg/L Matrigel, and air-dried overnight with BSA as a control substrate. 50 μ l of serum-free medium containing 0.1% BSA was added to each well to hydrate the basement membrane at 37 ℃ for 1 h. Taking MCF-7 cells cultured by conventional subculture or co-incubating with OAUCS (1mg/ml, 10mg/ml) of different concentrations for 48h, digesting and washing, and adjusting cell density to 1 × 10 with RPMI1640 of 0.1% BSA-1% newborn calf serum⁵One per ml, 100. mu.l of cell suspension was added to a 96-well plate, 4 samples in parallel per group. Culturing for 1h, removing culture medium by aspiration, washing with PBS to remove non-adherent cells, adding 20 μ l MTT solution and CO per well₂The incubator was incubated for 4 hours, MTT solution was discarded, 150. mu.l of DMSO was added to each well, and absorbance (OD value) of each well was measured at a wavelength of 490nm in an enzyme-labeled photometer. The adhesion rate and adhesion inhibition rate of the cells on the artificial basement membrane Matrigel were calculated.

The calculation formula is as follows: the adhesion rate (%) was [ (Matrigel-based cell OD/BSA-based cell OD) -1] × 100%. The sticking inhibition ratio (%) [1- (applied cell adhesion ratio/non-applied cell adhesion ratio) ] × 100%.

TABLE 2 adhesion of OAUCS-treated MCF-7/PTX cells to Matrigel

OAUCS(mg/ml)	Adhesion Rate (%)	Adhesion inhibition ratio (%)
			0	66.42±5.42	/
1	43.95±5.11*	33.83
			10	29.17±4.79**	56.08

Comparison of OAUCS-treated groups with control groups for t-test (P < 0.05; P < 0.01)

Adhesion is the initiation step of cancer cell invasion and metastasis, and can inhibit tumor cell adhesion and prevent tumor cell metastasis. The experimental results show that: the adhesion inhibition effect of OAUCS on MCF-7/PTX cells is obviously improved at different concentrations (1mg/ml and 10mg/ml), and the difference has obvious statistical significance (P is less than 0.05). Indicating that 1mg/ml and 10mg/ml OAUCS can inhibit the adhesion of MCF-7/PTX tumor cells and prevent the metastasis of the tumor cells.

In summary, the following steps: the invention relates to a novel anti-tumor metastasis nano drug delivery carrier with functions of penetrating membrane deeply and inhibiting P-glycoprotein reversal tumor resistance, which has high encapsulation efficiency and drug loading capacity.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

1. An arginine and ursolic acid modified chitosan nano drug delivery carrier has a chemical structure shown as a general formula I:

2. the arginine-and-ursolic acid-modified chitosan nano drug delivery carrier according to claim 1, wherein in the nano drug delivery carrier, the molecular weight of chitosan is 1-500kDa, the degree of deacetylation is 70-95%, the modification proportion of ursolic acid is 1-25%, and the modification proportion of arginine is 10-200%.

3. The method for preparing arginine-and ursolic acid-modified chitosan nano drug delivery carrier according to claim 1 or 2, comprising the following steps:

4. The method for preparing an arginine-and ursolic-modified chitosan nano drug delivery carrier according to claim 3, wherein in the step (1), the concentration of the ursolic acid solution is 1-30 mg/ml; the molar ratio of the ursolic acid to the NHS to the EDC to the chitosan is 0.25-3: 1-3: 2-6: 1-5; the molecular weight of the chitosan is 1-500kDa, and the concentration of the chitosan aqueous solution is 1-30 mg/ml; the reaction time at room temperature is 40-56 h.

5. The method for preparing an arginine-and-ursolic acid-modified chitosan nano drug delivery carrier according to claim 3, wherein in the step (1), after the room temperature reaction is finished, absolute ethanol is precipitated, the filtration is carried out, dichloromethane, ethanol and water are used for fully washing the precipitate, and the N-ursolic acid-chitosan shown in the formula II is prepared by freeze-drying.

6. The preparation method of an arginine-and-ursolic-modified chitosan nano drug delivery carrier according to claim 3, wherein in the step (2), the concentration of the N-ursolic acyl-chitosan suspension aqueous solution is 1-30 mg/ml; the molar ratio of arginine, NHS, DMAP and EDC is 0.25-2: 0.5-3: 0.05-0.3: 1-5; the concentration of the arginine aqueous solution is 1-300 mg/ml, and the molar ratio of arginine to N-ursolic acyl-chitosan is 1-10: 1; the reaction time at room temperature is 40-56 h.

7. The method for preparing an arginine-and-ursolic acid-modified chitosan nano drug delivery carrier according to claim 3, wherein in the step (2), after the reaction at room temperature is finished, the O-arginyl-N-ursolic acid-based chitosan shown in formula I is prepared by dialysis, suction filtration and filtrate freeze-drying.

8. The arginine-and ursolic acid-modified chitosan nanopharmaceutical delivery vehicle of claim 1 or 2 for use as an anti-tumor drug carrier.

9. The use according to claim 8, wherein said antineoplastic agent is selected from paclitaxel, docetaxel, carboplatin, cisplatin, oxaliplatin, gemcitabine, capecitabine, vincristine, hydroxycamptothecin, doxorubicin or mitomycin, and the like.

10. The use of claim 8, comprising dissolving the nano drug delivery carrier to prepare a micelle solution, mixing the micelle solution with a solution containing an anti-tumor drug, incubating, and removing free drug to obtain the O-arginyl-N-ursolic acyl-chitosan drug-loaded micelle.