Chitosan nanoparticles for inhalation administration
By combining CXCR4 antagonist with chitosan, a modified hydroxychloroquine chitosan nanodelivery vehicle was formed, which solved the problem of insufficient drug deposition and retention time in the lung by inhaled administration, and achieved efficient pulmonary drug delivery and tumor suppression effects.
Patent Information
- Application Number
- CN202510099930.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-22
- Publication Date
- 2025-05-13
AI Technical Summary
The existing inhalation administration technology has insufficient pulmonary drug deposition and retention time, resulting in poor efficacy and increased systemic toxic side effects.
A chitosan nanoparticles were designed to form a modified hydroxychloroquine chitosan nanodelivery vector by combining CXCR4 antagonists such as hydroxychloroquine with chitosan, which utilizes the mucosal adhesion properties of chitosan and collagen interactions to increase the deposition rate and retention time of the drug in the lungs, and inhibit the invasion and metastasis of tumor cells by antagonizing the CXCR4/CXCL12 pathway.
It significantly improves the deposition rate and retention time of drugs in the lungs, enhances the local concentration of drugs in the lungs, improves bioavailability, and effectively inhibits the invasion and metastasis of tumor cells, reducing systemic toxic side effects.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biomedicine, and in particular relates to chitosan nanoparticles for inhalation administration. Background Art
[0002] Lung cancer is one of the most common malignant tumors in the world, and non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and the risk of metastasis is 55% when diagnosed. Compared with traditional drug administration methods such as oral administration and intravenous injection, the special physiological structure of the lungs determines its advantages in tumor treatment. Due to the large lung absorption area, thin alveolar epithelial cells and rich blood vessels, pulmonary inhalation administration can deliver drugs directly to the lungs without the first-pass effect in the liver, which can reduce systemic drug distribution and reduce systemic toxic side effects, increase the local concentration of drugs in the lungs, and improve drug bioavailability. It has the advantages of rapid onset, good efficacy, and reduced systemic toxicity.
[0003] However, due to the self-protection mechanism of the lungs, drugs may be cleared from the lungs through the mucociliary mechanism or cellular phagocytosis before reaching the lungs to exert their effects. Particles with aerodynamic diameters greater than 5 μm are easily cleared by the mucociliary mechanism in the upper respiratory tract; particles with a diameter of about 1 to 5 μm are easy to enter the lungs, but seem to be cleared out of the body through phagocytosis by lung phagocytes; and nanoparticles with a diameter of about 200 nm to 300 nm are not easy to be cleared, which can significantly increase the residence time of drugs in the lungs. However, after inhalation administration, the particles enter the respiratory tract with the airflow and are deposited in the lungs. Their deposition rate and retention can directly affect the efficacy and pharmacokinetic characteristics of inhaled preparations.
[0004] Chitosan is a naturally occurring cationic polysaccharide derived from chitin. It contains a large number of amino groups in its structure and is positively charged under acidic conditions. Therefore, it can not only increase the deposition rate of drugs in the lungs by interacting with negatively charged tumor cell membranes and anionic substances in the mucus layer, but also instantly open the tight junctions in epithelial cells to increase the permeability of drugs. Chitosan has low immunogenicity, good safety, biodegradability and biocompatibility, so it is widely used in the lung and nasal mucosal delivery system of chemotherapy drugs. In addition, chitosan can interact with collagen in the fibrotic lung tumor microenvironment through hydrogen bonds, so it can increase the retention rate of drugs in the lungs by taking advantage of the characteristics of fibrotic collagen deposition in the lung cancer microenvironment.
[0005] Lung cancer metastases highly express CXCR4 and have a high risk of metastasis. After binding to CXCL12, CXCR4 can activate multiple downstream signaling pathways and trigger cell migration. Antagonizing this pathway with CXCR4 antagonists can effectively resist tumor invasion and metastasis. For example, hydroxychloroquine (HCQ) is structurally similar to the CXCR4 antagonist (NSC56612) and has the effect of antagonizing the CXCR4 / CXCL12 pathway and inhibiting tumor invasion and metastasis.
[0006] Based on this, the present invention designs to modify chitosan using a CXCR4 antagonist to construct a nano delivery carrier for inhalation administration. Summary of the invention
[0007] One of the purposes of the present invention is to provide a chitosan nanoparticle, comprising chitosan, on which a CXCR4 antagonist is connected.
[0008] Furthermore, the CXCR4 antagonist is hydroxychloroquine, AMD3100 or Cyclam (1,4,8,11-tetraazacyclotetradecane).
[0009] Furthermore, the chitosan is low molecular weight chitosan, medium molecular weight chitosan or high molecular weight chitosan.
[0010] In a specific embodiment of the present invention, the CXCR4 antagonist hydroxychloroquine is connected to low molecular weight chitosan using N'N-carbonyldiimidazole (CDI) as a connecting chain to obtain chitosan modified with hydroxychloroquine, the structural formula of which is as follows:
[0011]
[0012] Wherein n is the number of repeating units of the chitosan derivative.
[0013] Specifically, the chitosan modified with hydroxychloroquine is prepared by the following reaction formula:
[0014]
[0015] In the reaction formula, 1 is hydroxychloroquine (HCQ), 2 is HCQ-CDI, and 3 is polymer CSQ;
[0016] The preparation process is as follows: first, hydroxychloroquine sulfate is desalted to obtain hydroxychloroquine (HCQ), HCQ-CDI is synthesized using N,N-carbonyldiimidazole (CDI), and then it is connected with chitosan to obtain the polymer carrier CSQ.
[0017] Preferably, the molecular weight of the chitosan is 50-100 kDa, and the mass ratio of N,N-carbonyldiimidazole, hydroxychloroquine and chitosan is 1.5-2:1-1.5:0.8-1.5.
[0018] Specifically, the steps include:
[0019] (1) Preparation of HCQ
[0020] Take hydroxychloroquine sulfate and add water to dissolve it, add 30% ammonia water under stirring until a milky white precipitate is produced, add dichloromethane to extract, collect the organic phase and remove the solvent to obtain HCQ;
[0021] (2) Preparation of HCQ-CDI
[0022] HCQ and CDI are dissolved in N,N-dimethylformamide (DMF) respectively, and CDI solution is added dropwise to the HCQ solution under nitrogen protection, and the reaction is stirred at room temperature. After the reaction is completed, water is added to quench and dichloromethane is used for extraction. The organic phase is collected and the solvent is removed to obtain HCQ-CDI;
[0023] 3) Preparation of CSQ
[0024] Chitosan was weighed and dissolved in a 1% glacial acetic acid solution, HCQ-CDI was dissolved in DMSO and added to the chitosan solution, stirred for reaction, and after the reaction was completed, dialyzed and freeze-dried to obtain the polymer carrier CSQ.
[0025] In the present invention, chitosan nanoparticles can be prepared by conventional methods in the art, such as covalent crosslinking, ionic crosslinking, macromolecular composite, precipitation, chemical modification self-assembly, spray drying, reverse micelle, filtration and emulsion droplet coagulation, etc. In a specific embodiment of the present invention, an ionic crosslinking method is used for preparation, in which a polyanionic crosslinking agent is crosslinked with a polycationic chitosan carrying a positive charge through electrostatic adsorption to prepare nanoparticles, and the crosslinking agent is preferably sodium tripolyphosphate TPP.
[0026] Specifically, the preparation process of the nanoparticles is as follows: weigh CSQ and dissolve it in 1% glacial acetic acid solution, adjust the solution pH to 4-5.5; add TPP dropwise to the CSQ solution at 0-4°C, stir and react, and obtain CSQ NPs. Preferably, the mass ratio of TPP to CSQ is 1:6-1:3.
[0027] The second purpose of the present invention is to provide the use of the chitosan nanoparticles in the preparation of drugs for treating lung cancer.
[0028] Furthermore, the drug is administered by inhalation.
[0029] Cancer metastases, such as lung cancer, highly express CXCR4 and have a high risk of metastasis. After binding to CXCL12, CXCR4 can activate multiple downstream signal transduction pathways, triggering cell migration. Antagonizing this pathway can effectively resist tumor invasion and metastasis. Hydroxychloroquine (HCQ) is structurally similar to the CXCR4 antagonist (NSC56612) and has the effect of antagonizing the CXCR4 / CXCL12 pathway and inhibiting tumor invasion and metastasis.
[0030] The present invention uses N'N-carbonyldiimidazole (CDI) and hydroxychloroquine to modify the amino group in the chitosan structure to prepare a hydroxychloroquine-based chitosan (CSQ) nano delivery carrier for pulmonary inhalation. On the one hand, the chitosan mucosal adhesion characteristics and the characteristics of the interaction with collagen are used to increase the deposition rate and retention time of the drug in the lungs after the drug is inhaled into the lungs; on the other hand, the characteristics of hydroxychloroquine antagonizing the CXCR4 / CXCL12 pathway are used to antagonize tumor invasion and metastasis. Nanoparticles that can be used for pulmonary inhalation are prepared based on the above-mentioned hydroxychloroquine-based chitosan delivery carrier.
[0031] The chitosan carrier prepared by the present invention can interact with collagen through hydrogen bonds and has mucosal adhesion properties. After atomization inhalation, the local concentration of the drug in the lungs can be increased, and the invasion and metastasis of tumor cells can be inhibited by antagonizing the CXCR4 / CXCL12 pathway. In one embodiment of the present invention, the prepared hydroxychloroquine-based chitosan nanoparticles can antagonize tumor cell metastasis and invasion, and the tumor cell healing rate is only 16.5%. BRIEF DESCRIPTION OF THE DRAWINGS
[0032] Figure 1 is the particle size distribution of CSQ NPs in Example 1.
[0033] Figure 2 is the Zeta potential of CSQ NPs in Example 1.
[0034] Figure 3 Transmission electron microscopy characterization of CSQ NPs of Example 1.
[0035] Figure 4 This is the infrared spectrum of CSQ of Example 3.
[0036] Figure 5 This is the infrared spectrum of the interaction between CSQ and collagen in Example 3.
[0037] Figure 6 The particle size change results of CSQ NPs adhering to mucin in Example 3.
[0038] Figure 7 The changes in particle size and PDI index of CSQ NPs in Example 3 within 72 hours.
[0039] Figure 8 These are the lung imaging results after nanoparticle aerosol inhalation.
[0040] Fig. 9 The results show that CSQ NPs can antagonize tumor cell metastasis.
[0041] Fig.10 The results show that CSQ NPs antagonize tumor cell invasion. DETAILED DESCRIPTION
[0042] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.
[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0045] In the following examples, the chitosan used is low molecular weight chitosan with a molecular weight range of 50-100 kDa.
[0046] Example 1
[0047] (1) Preparation of HCQ
[0048] Weigh 434 mg of hydroxychloroquine sulfate and dissolve it in 1 mL of pure water. Add 2 mL of 30% ammonia water under constant stirring until a milky white precipitate is produced. Add 10 mL of dichloromethane for extraction. Collect the organic phase, add anhydrous sodium sulfate to remove water, and evaporate the solvent to obtain the desalted product HCQ.
[0049] (2) Preparation of HCQ-CDI
[0050] The obtained 390 mg HCQ and 648 mg CDI were dissolved in 2 mL DMF, and the CDI solution was added dropwise to the HCQ solution under nitrogen protection, and the reaction was continued for 5 hours. After the reaction was completed, 4 mL of water was added to terminate the reaction, and the organic phase was extracted with dichloromethane and collected. After the organic phase was washed with water, anhydrous sodium sulfate was added to remove water, and the solvent was removed by vacuum rotary evaporation to obtain HCQ-CDI.
[0051] (3) Preparation of CSQ
[0052] 339 mg of chitosan was weighed and dissolved in a 1% glacial acetic acid solution to make the concentration of the chitosan solution 5 mg / mL. The solution was stirred magnetically overnight. The above-obtained product HCQ-CDI was dissolved in 3-4 mL DMSO and added to the chitosan solution and continued to react with stirring for 48 hours. After the reaction was completed, the product was collected and dialyzed. The dialysate was changed every 8 hours. After 48 hours, the product was collected and freeze-dried in vacuum to obtain CSQ.
[0053] (4) Preparation of CSQ NPs
[0054] CSQ was dissolved in 1% glacial acetic acid solution and the pH was adjusted to 5 using 1 M NaOH to make the CSQ concentration of 1 mg / mL. The CSQ solution was preheated at 60°C for 10 to 15 minutes and filtered using a 0.45 μm filter membrane. 1 mg / mL TPP was slowly added dropwise to the CSQ solution under an ice bath condition of 0 to 4°C. The volume ratio of CSQ to TPP was 3. The reaction solution was reacted under magnetic stirring until the Tyndall effect occurred to obtain CSQ NPs.
[0055] The particle size distribution, PDI index and Zete potential were characterized using a Malvern particle size meter. The results are as follows: Figure 1 and Figure 2 The results of characterizing the nanoparticle structure using transmission electron microscopy are shown in Figure 3 As shown, the results show that the prepared CSQ NPs are uniform spherical nanoparticles with a particle size of about 232 nm, a potential of about +16.3 mV, and a PDI of about 0.244.
[0056] Example 2
[0057] (1) Preparation of HCQ
[0058] Weigh 868 mg of hydroxychloroquine sulfate and dissolve it in 1.5 mL of pure water. Add 3 mL of 30% ammonia water under constant stirring until a milky white precipitate is produced. Add 15 mL of dichloromethane for extraction. Collect the organic phase, add anhydrous sodium sulfate to remove water, and evaporate the solvent to obtain the desalted product HCQ.
[0059] (2) Preparation of HCQ-CDI
[0060] The obtained 782 mg HCQ and 1296 mg CDI were dissolved in 2.5 mL DMF, and the CDI solution was added dropwise to the HCQ solution under nitrogen protection, and the reaction was continued for 6 hours. After the reaction was completed, 5 mL of water was added to terminate the reaction, and the organic phase was extracted with dichloromethane and collected. After the organic phase was washed with water, anhydrous sodium sulfate was added to remove water, and the solvent was removed by vacuum rotary evaporation to obtain HCQ-CDI.
[0061] (3) Preparation of CSQ
[0062] 678 mg of chitosan was weighed and dissolved in 1% acetic acid solution to make the concentration of chitosan solution 5 mg / mL. The solution was stirred magnetically overnight. The above product HCQ-CDI was dissolved in 5-6 mL DMSO and added to the chitosan solution and continued to react with stirring for 48 hours. After the reaction was completed, the product was collected and dialyzed. The dialysate was changed every 8 hours. After 48 hours, the product was collected and freeze-dried in vacuum to obtain CSQ.
[0063] (4) Preparation of CSQ NPs
[0064] CSQ was dissolved in 1% acetic acid solution and the pH was adjusted to 5.5 using 1 M NaOH to make the CSQ concentration of 1 mg / mL. The CSQ solution was preheated at 60°C for 10 to 15 minutes and filtered using a 0.45 μm filter membrane. 1 mg / mL TPP was slowly added dropwise to the CSQ solution under an ice bath condition of 0 to 4°C. The volume ratio of CSQ to TPP was 3. The reaction solution was reacted under magnetic stirring until the Tyndall effect occurred to obtain CSQ NPs.
[0065] The particle size distribution, PDI index and Zete potential were characterized using a Malvern particle size meter. The results showed that the prepared CSQNPs were uniform spherical nanoparticles with a particle size of about 234 nm, a potential of about +13.0 mV, and a PDI of about 0.268.
[0066] Example 3
[0067] (1) Preparation of HCQ
[0068] Weigh 1302 mg of hydroxychloroquine sulfate and dissolve it in 2.5 mL of pure water. Add 4 mL of 30% ammonia water under constant stirring until a milky white precipitate is produced. Add 20 mL of dichloromethane for extraction. Collect the organic phase, add anhydrous sodium sulfate to remove water, and evaporate the solvent to obtain the desalted product HCQ.
[0069] (2) Preparation of HCQ-CDI
[0070] The obtained 1122 mg HCQ and 1944 mg CDI were dissolved in 3.5 mL DMF respectively, and the CDI solution was added dropwise to the HCQ solution under nitrogen protection, and the reaction was continued with stirring for 8 hours. After the reaction was completed, 7 mL of water was added to terminate the reaction, and a large amount of dichloromethane was used to extract and collect the organic phase. After the organic phase was washed with water, anhydrous sodium sulfate was added to remove water, and the solvent was removed by vacuum rotary evaporation to obtain HCQ-CDI.
[0071] (3) Preparation of CSQ
[0072] 1017 mg of chitosan was weighed and dissolved in 1% acetic acid solution to make the concentration of chitosan solution 5 mg / mL. The solution was stirred magnetically overnight. The above product HCQ-CDI was dissolved in 7-8 mL DMSO and added to the chitosan solution and continued to react with stirring for 48 hours. After the reaction was completed, the product was collected and dialyzed. The dialysate was changed every 8 hours. After 48 hours, the product was collected and freeze-dried in vacuum to obtain CSQ.
[0073] (4) Preparation of CSQ NPs
[0074] CSQ was dissolved in 1% acetic acid solution and the pH was adjusted to 4.5 using 1 M NaOH to make the CSQ concentration of 1 mg / mL. The CSQ solution was preheated at 60°C for 10 to 15 minutes and filtered using a 0.45 μm filter membrane. 1 mg / mL TPP was slowly added dropwise to the CSQ solution under an ice bath condition of 0 to 4°C. The volume ratio of CSQ to TPP was 3. The reaction solution was reacted under magnetic stirring until the Tyndall effect occurred to obtain CSQ NPs.
[0075] The particle size distribution, PDI index and Zete potential were characterized using a Malvern particle size meter. The results showed that the prepared CSQNPs were uniform spherical nanoparticles with a particle size of approximately 249.5 nm, a potential of approximately +22.0 mV, and a PDI of approximately 0.248.
[0076] Example 4
[0077] (1) Preparation of AMD3100-CDI
[0078] Weigh 201 mg AMD3100 and 324 mg CDI and dissolve them in 5 mL amide reaction solvent such as DMSO or DMF respectively. Slowly add the CDI solution to the AMD3100 solution under nitrogen protection and stir at room temperature for 24 hours. After the reaction, separate and purify using a chromatography column with the mobile phase of ethyl acetate: methanol = 10:1. After removing the solvent, the compound AMD3100-CDI is obtained.
[0079] (2) Preparation of CS-AMD3100
[0080] Weigh 169 mg of chitosan and dissolve it in 1% glacial acetic acid solution to make the concentration of chitosan solution 5 mg / mL. Stir it magnetically overnight, and dissolve the above product in 7-8 mL DMSO and drip it into the chitosan solution and continue to stir and react for 48 hours. After the reaction is completed, collect the product and dialyze it. Change the dialysate every 8 hours. After 48 hours, collect the product and vacuum freeze-dry it to obtain CS-AMD3100.
[0081] Example 5
[0082] The synthetic route of this embodiment is shown below:
[0083]
[0084] (1) Preparation of Compound 2
[0085] 67 mg of 1,4,8,11-tetraazacyclotetradecane (Cyclam, i.e., compound 1) was weighed and dissolved in 13 mL of dichloromethane, and 218 mg of di-tert-butyl dicarbonate (Boc2O) was dissolved in 5 mL of dichloromethane and added dropwise to the above solution under ice bath conditions and stirred for reaction for 2 hours. The resulting mixture was concentrated and separated and purified by a chromatography column. The mobile phase system used was ethyl acetate: methanol = 10:1. After removing the solvent, the compound tri-tert-butyl-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate was obtained and recorded as compound 2.
[0086] (2) Preparation of Compound 3
[0087] 468 mg of α, α′-dichloro-p-xylene and 96 mg of K2CO3 were weighed and dissolved in 7 mL of acetonitrile, and compound 2 was added thereto. The reaction was refluxed at 60°C for 6 hours. The obtained compound was concentrated and separated and purified by a chromatography column. The mobile phase used was ethyl acetate: methanol = 10:1. After removing the solvent, the compound tri-tert-butyl-11-(4-(chloromethyl)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8-tricarboxylate was obtained and recorded as compound 3.
[0088] (3) Preparation of Compound 4
[0089] 687 mg of chitosan was weighed and dissolved in 1% glacial acetic acid solution to make the concentration of chitosan solution 5 mg / mL, and magnetic stirring was carried out overnight. The above compound 3 was dissolved in 5 mL of acetonitrile and added to the chitosan solution and the stirring reaction was continued for 48 hours to obtain compound 4.
[0090] (4) Preparation of Compound 5
[0091] 5 mL of trifluoroacetic acid was mixed into the above reaction solution, and dialyzed against a mixture of ethanol and water in a volume ratio of 1:1 for 48 hours. After the reaction was completed, the product was collected for dialysis treatment, and the dialysate was changed every 12 hours. After 48 hours, the product was collected and freeze-dried in vacuum to obtain the final product, compound 5.
[0092] Test Example 1
[0093] The polymer CSQ prepared in Example 3 was characterized by Fourier transform infrared spectroscopy. Figure 4 The infrared spectrum shows that the amino group in chitosan is at 1657 cm -1 、1591cm-1 There are FITR spectrum values at , and the displacement and intensity of the peak changed after reaction with HCQ-CDI. The surface HCQ-CDI reacted with chitosan to successfully prepare CSQ.
[0094] Test Example 2
[0095] The interaction between CSQ and collagen was characterized by Fourier transform infrared spectroscopy. The polymer CSQ prepared in Example 3 was reacted with collagen at a mass ratio of 3:2. The results are as follows: Figure 5 As shown. After chitosan is grafted with HCQ, the amino group (-NH2) is at 1640cm- 1 、1546cm -1 There is a peak at 1689cm-1, and the carboxyl group (-COOH) in collagen is at 1689cm-1. -1 、1541cm -1 There is a peak at , and the peak and displacement intensity change after the two are mixed (CSQ / Col). The results show that chitosan can still interact with collagen through hydrogen bonds after grafting HCQ.
[0096] Test Example 3
[0097] The CSQ NPs interacted with mucin to explore its mucosal adhesion properties. 0.5 mg / mL mucin solution was added dropwise to the CSQ NPs prepared in Example 3 and the pH of the mixed solution was adjusted to 5.5, so that the final concentration of the nanoparticles in the mixed solution was adjusted to 80 μg / mL. After the obtained mixed solution was mixed, the particle size distribution of the nanoparticles was analyzed by dynamic light scattering technology in a Malvern particle size analyzer, which was used as an important parameter for the mucosal adhesion properties of the nanoparticles. The results are shown in Figure 2. Figure 6 As shown, when mucin was added to the nanoparticles, the particle size distribution of the nanoparticles shifted toward larger sizes, indicating that CSQNPs can interact with mucin and have good mucosal adhesion properties.
[0098] Test Example 4
[0099] The CSQ NPs prepared in Example 3 were placed at 4°C and taken out at regular intervals to measure the changes in particle size and PDI index to characterize their stability. Figure 7 As shown in the figure, there was no significant change in the particle size and PDI of CSQ NPs within 72 h, indicating that the nanoparticles prepared by this method have a certain stability.
[0100] Test Example 5
[0101] The CSQ prepared in Example 3 was dissolved in 1% acetic acid solution and the pH was adjusted to 4.5 using 1M NaOH to make the CSQ concentration 1 mg / mL. The CSQ solution was preheated at 60°C for 10 to 15 minutes and filtered using a 0.45 μm filter membrane. 100 uL of 5 mg / mL dihydrochlorin e6 was dripped into the CSQ solution, and 1 mg / mL TPP was slowly dripped into the CSQ solution under ice bath conditions. The volume ratio of CSQ to TPP was 3. The reaction solution was reacted under magnetic stirring until the Tyndall effect was produced, thereby obtaining fluorescent nanoparticles Ce6@CSQ that can be used for in vivo imaging, and a nebulizer was used to administer the drug to mice. The mouse lungs were imaged at 4h, 8h, and 12h to verify their mucosal adhesion properties. The results are shown in Figure 8 As shown, Ce6@CSQ was more likely to accumulate in the lungs compared with the free group, indicating that the carrier had mucosal adhesion properties.
[0102] Test Example 6
[0103] The CSQ NPs prepared in Example 3 were used to evaluate the inhibitory effect of the preparation on tumor cell metastasis by cell scratching. The cell metastasis was photographed at 12h and 24h. The results are as follows: Fig. 9 As shown in the figure, the lowest tumor healing rate in the CSQ group was 16.5%. The inhibitory effect of the preparation on tumor cell invasion was evaluated by transwell experiment. The high concentration matrix gel was placed at 4°C for overnight freeze-thaw. The frozen and thawed matrix gel was diluted with culture medium according to the instructions. 100 μL of culture medium containing matrix gel was spread on the upper chamber of the traswell. After incubation at 37°C for 3 hours, the culture medium was aspirated and 100 μL of PBS was added for hydration. If there was no liquid leakage, cells could be inoculated in the upper chamber, and 600 μL of complete culture medium containing medicine was added to the lower chamber. After 24 hours, the well plate was taken out, washed twice with PBS, the cells on the inner side of the upper chamber were scraped off and fixed with 4% paraformaldehyde solution, and then the cells were stained with 1% crystal violet and photographed under a microscope. The results are shown in the figure. Fig.10 As shown, the number of cell invasion was the lowest in the CSQ group.
[0104] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.
Claims
1. A chitosan nanoparticle, characterized in that: The invention comprises chitosan, and a CXCR4 antagonist is connected to the chitosan.
2. The chitosan nanoparticles according to claim 1, characterized in that: The chitosan is low molecular weight chitosan, medium molecular weight chitosan or high molecular weight chitosan.
3. The chitosan nanoparticles according to claim 1, characterized in that: The CXCR4 antagonist is hydroxychloroquine, AMD3100 or Cyclam.
4. The chitosan nanoparticles according to claim 3, characterized in that: The chitosan is connected with hydroxychloroquine / AMD3100 via N'N-carbonyldiimidazole.
5. The chitosan nanoparticles according to claim 1, characterized in that: The chitosan is low molecular weight chitosan, and the CXCR4 antagonist is hydroxychloroquine.
6. Use of the chitosan nanoparticles according to any one of claims 1 to 5 in the preparation of tumor therapeutic drugs.
7. The use according to claim 6, characterized in that: The tumor is lung cancer.
8. The use according to claim 6, characterized in that: The therapeutic drug is administered by inhalation.