Preparation method of gold nanoparticle-chitosan-hydroxy phospholime nano drug-loading system loaded with camptothecin and derivatives of camptothecin

Abstract

The invention provides a novel nano drug-loading system loaded with camptothecin and derivatives thereof, which is based on hydroxyl phospholime (HA) nanoparticles and chitosan-gold nanoparticles, and realizes efficient loading and accurate release of camptothecin through excellent specific surface area, biocompatibility and photothermal conversion capability, so that the drug-loading system has good application prospects. Under near-infrared light irradiation, heat energy can be effectively converted, tumor tissues can be precisely targeted, the bioavailability and the treatment effect of drugs are improved, a more efficient and safer strategy is provided for precise treatment of cancers, and important innovative significance and wide application prospects are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of preparation of anti-tumor drug delivery systems, and in particular relates to a method for preparing a nano drug delivery system loaded with camptothecin and its derivatives. Background Art

[0002] In recent years, camptothecin and its derivatives have shown great potential in the field of tumor treatment, especially in the treatment of various tumor types, and have achieved significant anti-tumor effects. Camptothecin compounds, as an important class of anti-tumor drugs, have a mechanism of preventing the proliferation and spread of cancer cells by inhibiting the activity of DNA topoisomerase I. A large number of studies have shown that camptothecin and its derivatives not only have strong anti-tumor activity, but also show good prospects in the treatment of different types of tumors.

[0003] Although camptothecins have shown excellent anti-tumor effects in in vitro and in vivo studies, their clinical application still faces some important challenges. Among them, the most prominent problem is the poor water solubility of camptothecins, which limits their effective use in clinical treatment. In addition, the lack of tumor targeting is also a serious problem. Traditional camptothecins are difficult to accurately target tumor sites in vivo, resulting in large distribution and toxic side effects of the drugs in normal tissues. Therefore, how to improve the bioavailability, targeting and therapeutic effects of camptothecins has become a key issue that researchers need to solve urgently.

[0004] Photothermal therapy (PTT) is an emerging anti-tumor treatment method that can achieve thermal ablation of tumor cells by local heating. However, after PTT, tumor cells may develop heat resistance, which affects the treatment effect. To solve this problem, combining PTT with chemotherapy can effectively make up for the shortcomings of a single treatment method and enhance the overall treatment effect. Gold nanoparticles (AuNPs) play an important role in this combined treatment due to their excellent photothermal conversion ability and low cytotoxicity. Gold nanoparticles can efficiently absorb and convert light energy into heat energy under near-infrared light irradiation, generate high temperatures locally, thereby enhancing the photothermal effect and accurately killing tumor cells. At the same time, the surface of gold nanoparticles is easy to functionalize and can be combined with drugs or targeting molecules to improve the targeting of treatment and the bioavailability of drugs. Due to their low cytotoxicity and good biocompatibility, gold nanoparticles cause minimal damage to normal tissues during treatment, which helps to reduce side effects. In addition, gold nanoparticles also have controllable size and morphology, which can adjust their distribution and drug release characteristics in the body according to needs, further improving the treatment effect. By combining gold nanoparticles with photothermal therapy and chemotherapy, it is possible to enhance the anti-tumor effect while reducing the drug resistance of tumor cells, thereby improving the accuracy and efficacy of treatment.

[0005] Metal nanoparticles (AuNPs) can be prepared using specific polymers as stabilizers, and their surfaces can be modified to control particle size. Currently, the synthesis of gold nanoparticles using natural biomass materials has attracted great attention based on the green synthesis or biosynthesis of AuNPs. Chitosan can act as a reducing agent and stabilizer in the process of synthesizing gold nanoparticles because of the presence of a large number of amino and hydroxyl groups. Chitosan can be used to reduce chloroauric acid to generate chitosan-gold nanoparticle complexes.

[0006] Hydroxyapatite (HA) nanoflowers have many significant advantages as drug carriers. First, due to their good biocompatibility and degradability, HA nanoflowers can be safely decomposed in the body, reducing the risk of toxicity caused by long-term accumulation. Secondly, the nanoscale and high specific surface area of ​​HA nanoflowers enable them to effectively load a large amount of drugs, and improve the targeting and stability of drugs through surface modification. In addition, HA nanoflowers can achieve precise drug release by adjusting their morphology, size and surface properties, and have a good controlled release effect. Because HA has a natural affinity with calcium-phosphorus compounds in the body, it can also promote the enrichment of drugs in the lesion area, especially in the treatment of bone-related diseases and tumors. It shows good prospects. In short, as a drug carrier, HA nanoflowers not only improve the bioavailability of drugs, but also enhance the therapeutic effect through precise targeting and controlled release characteristics.

[0007] The combination of hydroxyapatite (HA) nanoflowers and gold nanoparticles has a significant synergistic effect in photothermal therapy. As a drug carrier, HA nanoflowers can effectively load drugs and improve the bioavailability of drugs in targeted therapy, while gold nanoparticles have excellent photothermal conversion capabilities and can be quickly converted into thermal energy under near-infrared light irradiation, killing tumor cells by local temperature increase. When the two are combined, gold nanoparticles can enhance the photothermal effect of HA nanoflowers in the tumor area, making the effect of photothermal therapy more accurate and efficient. At the same time, the biocompatibility and degradability of HA combined with the thermal effect of gold nanoparticles can reduce damage to normal tissues and enhance drug accumulation in the tumor site, thereby achieving better therapeutic effects. In addition, the complex of HA nanoflowers and gold nanoparticles can achieve stronger targeting and controlled release functions through surface modification, so that drugs can be accurately released during photothermal therapy, thereby improving treatment efficiency and reducing side effects. This composite material provides an innovative solution for photothermal therapy of tumors. Summary of the invention

[0008] In order to solve the problems of the prior art, the purpose of the present invention is to provide a method for preparing a NIR-responsive nano-drug delivery system of camptothecin and its derivatives. The NIR-responsive nano-drug delivery system of camptothecin and its derivatives prepared by the present invention has a high drug loading rate, encapsulation rate and good stability, and can achieve NIR-responsive release in the tumor microenvironment.

[0009] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0010] The present invention provides a method for preparing a nano drug delivery system loaded with camptothecin and its derivatives, comprising the following steps:

[0011] (1) Dissolve camptothecin in methanol at a concentration of 100 ug / mL and sonicate for 30 min at 25°C to 35°C to allow camptothecin to fully dissolve in methanol.

[0012] (2) 1 g of solid chloroauric acid was dissolved in 50 mL of deionized water to prepare a 2% chloroauric acid solution; 0.1 g of chitosan was dissolved in 100 mL of 1% acetic acid aqueous solution, stirred overnight to prepare a 0.1% chitosan solution, and filtered through a 0.22 μm filter membrane.

[0013] (3) Take 30 ml of the chitosan solution after membrane treatment, adjust the pH to 5.5 with ammonia water, add 0.6 ml of 2% chloroauric acid solution, stir and react in a water bath at 80° C. for 3 h, and set reflux to prevent solvent evaporation.

[0014] (4) Calcium chloride dihydrate and ammonium dihydrogen phosphate were prepared into 1 mol / L solutions respectively. To ensure the preparation of the optimal calcium hydroxyphosphate, the Ca / P molar ratio was set to 1.67. The pH value of the calcium chloride dihydrate solution was adjusted to 11.5, and ammonium dihydrogen phosphate was added. Urea and L-glutamic acid were added, stirred for 20 minutes, and then placed in a reactor at 160° C. for 10 hours to obtain calcium hydroxyphosphate microspheres.

[0015] (5) Adding the calcium hydroxyphosphate prepared in step (4) above to the chitosan-gold nanocomposite solution prepared in step (3) above, stirring evenly, magnetically stirring at 45° C. for 3 h, centrifuging at 10,000 rpm for ten minutes, washing three times with deionized water, and freeze-drying to obtain a camptothecin derivative and its derivative bio-nano drug delivery system with near-infrared response.

[0016] (6) Add the chitosan-gold nanoparticles prepared in step (5) to the camptothecin methanol solution prepared in step (1), stir evenly, shake at 37°C for 24 hours on a shaking oscillator at 150 rpm, centrifuge and freeze-dry to obtain a nano drug delivery system of camptothecin and its derivatives.

[0017] In the above technical solution, the camptothecin derivative in step (1) is a camptothecin derivative.

[0018] In the above technical solution, the volume of chloroauric acid in step (2) is 0.1 ml to 1 ml, and the concentration of chitosan is 0.1% to 0.2%.

[0019] In the above technical solution, the pH of the chitosan-chloroauric acid mixed solution in step (3) is 3.5-6.5.

[0020] In the above technical solution, the pH value of the calcium chloride dihydrate solution in step (4) is 7-11.

[0021] In the above technical solution, in step (5), the mass ratio of hydroxy calcium phosphate to chitosan is 7:3.

[0022] A nano drug-carrying system loaded with camptothecin and its derivatives prepared by the preparation method, wherein the particle size of chitosan-gold nano-hydroxyapatite is 5-10um.

[0023] A nano drug-carrying system loaded with camptothecin and its derivatives prepared by the preparation method has a drug-carrying rate of 25% to 29%.

[0024] The present invention provides a method for preparing a nano drug delivery system loaded with camptothecin and its derivatives. Hydroxyapatite nanoparticles can effectively load camptothecin derivatives and gold nanoparticle photosensitizers, effectively convert near-infrared light into heat energy, and enhance the therapeutic effect of tumors; the drug delivery system can realize NIR-responsive drug release in the tumor microenvironment, can be applied to the targeted treatment of anti-tumor drugs, and has good application prospects.

[0025] Compared with the prior art, the beneficial effects of the present invention are: the composite material of hydroxyapatite (HA) and gold nanoparticles as a carrier for loading camptothecin (CPT) has multiple advantages, especially showing unique potential in cancer treatment. First, HA nanoflowers can provide a large specific surface area and good biocompatibility, effectively load camptothecins, and enhance their targeting and stability through surface modification. Gold nanoparticles have excellent photothermal conversion capabilities, which can be efficiently converted into thermal energy under near-infrared light irradiation, locally heat tumor tissue, and enhance the effect of photothermal therapy. After the two are combined, the degradability of HA can ensure the gradual release of drugs in the body, while gold nanoparticles provide additional thermal effects under light irradiation, promote the release of drugs and enhance the thermal sensitivity of tumor cells, further improving the therapeutic effect.

[0026] In addition, during photothermal therapy, the composite material of HA and gold nanoparticles can not only accurately target tumor tissues and avoid damage to normal tissues, but also improve the bioavailability of camptothecins by enhancing drug accumulation and controlled release at the tumor site. The thermal effect of gold nanoparticles helps to break down the blood vessel walls of tumor tissues, promote drug penetration, and further enhance its therapeutic effect. Therefore, the composite material of HA and gold nanoparticles loaded with camptothecins not only optimizes the delivery and release of drugs, but also enhances the accuracy and effect of treatment through photothermal effect, providing a more efficient and safe strategy for cancer treatment. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods:

[0028] Embodiment 1:

[0029] A method for preparing a nano drug delivery system loaded with camptothecin and its derivatives, comprising the following steps:

[0030] (1) Disperse 5 mg of camptothecin in 50 mL of methanol and ultrasonicate at 25°C for 30 min to obtain a methanol solution of camptothecin.

[0031] (2) Dissolve 0.1 g of chitosan powder in 100 mL of 1% (v:v) acetic acid water, stir overnight and filter, record as solution A, dissolve 1 g of chloroauric acid powder in 50 mL of deionized water, record as solution B, mix well, then slowly add 0.6 ml of solution B to solution A, stir magnetically at 80°C for 3 h. After the reaction is completed, place in a 4°C refrigerator away from light for later use.

[0032] (3) Dissolve 0.295 g of calcium chloride dihydrate in 20 mL of deionized water, referred to as solution A, dissolve 0.138 g of ammonium dihydrogen phosphate in 20 mL of deionized water, referred to as solution B, and dissolve 0.6 g of urea in 10 ml of deionized water, referred to as solution C. Slowly add solution B to solution A, stir magnetically at 25°C for 5 min, then add solution C, stir for 5 min, then add 2.2 g of L-glutamic acid and stir for 20 min. Transfer the mixed solution into a reactor, react at 160°C for 10 h, centrifuge at 10,000 r / min for 10 min after the reaction, wash with deionized water 3 times, discard the supernatant, and freeze-dry to obtain hydroxy calcium phosphate particles.

[0033] (4) Dissolve 70 mg of the hollow nanoparticles in 30 ml of the solution of step (1), stir magnetically at 45° C. for 3 h, centrifuge at 10,000 r / min for 10 min after the reaction, wash three times with deionized water, and freeze-dry the precipitate after centrifugation to obtain chitosan-gold nano-hydroxycalcium phosphate particles.

[0034] (5) Dissolve 25 mg of chitosan-gold nano-hydroxycalcium phosphate particles in the methanol solution of camptothecin in step (1), shake at 150 rpm at 37°C for 24 h. After the reaction is completed, centrifuge at 10,000 rpm for 10 min, wash with methanol three times, and freeze-dry the precipitate after centrifugation to obtain chitosan-gold nano-hydroxycalcium phosphate loaded camptothecin nanoparticles.

[0035] (6) The prepared drug-loaded nanoparticles were prepared into a 1 mg / mL PBS buffer solution (pH 5.5) and placed in a constant temperature oscillator (37°C, 100 RPM). After (1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h), the centrifuge tubes were taken out and centrifuged to obtain 2mL of the supernatant. 2mL of PBS buffer solution was added and ultrasonically dispersed. The nanoparticles were then placed in a constant temperature oscillator and waited for the next time point. These experiments were performed under 808nm laser irradiation (2.1W / cm 2 ) or without irradiation with 808 nm laser. Take out the sample, measure the concentration of camptothecin and its derivatives in the release medium by high performance liquid chromatography, and calculate the cumulative release rate.

[0036] The release of camptothecin was 54.77%, and when irradiated with near-infrared light, the release of camptothecin was 79.5%. With the irradiation of near-infrared light, chitosan-gold nano-hydroxycalcium phosphate particles can quickly release camptothecin and its derivatives, indicating that the nano drug delivery system has NIR-responsive release performance.

[0037] Embodiment 2:

[0038] A method for preparing a nano drug delivery system loaded with 10-hydroxycamptothecin (HCPT) and its derivatives comprises the following steps:

[0039] (1) Disperse 5 mg of 10-hydroxycamptothecin in 50 mL of methanol and ultrasonicate for 30 min at 25° C. to obtain a methanol solution of 10-hydroxycamptothecin.

[0040] (2) Dissolve 0.1 g of chitosan powder in 100 mL of 1% (v:v) acetic acid water, stir overnight and filter, record as solution A, dissolve 1 g of chloroauric acid powder in 50 mL of deionized water, record as solution B, mix well, then slowly add 0.6 ml of solution B to solution A, stir magnetically at 80°C for 3 h. After the reaction is completed, place in a 4°C refrigerator away from light for later use.

[0041] (3) Dissolve 0.295 g of calcium chloride dihydrate in 20 mL of deionized water, referred to as solution A, dissolve 0.138 g of ammonium dihydrogen phosphate in 20 mL of deionized water, referred to as solution B, and dissolve 0.6 g of urea in 10 ml of deionized water, referred to as solution C. Slowly add solution B to solution A, stir magnetically at 25°C for 5 min, then add solution C, stir for 5 min, then add 2.2 g of L-glutamic acid and stir for 20 min. Transfer the mixed solution into a reactor, react at 160°C for 10 h, centrifuge at 10,000 r / min for 10 min after the reaction, wash with deionized water 3 times, discard the supernatant, and freeze-dry to obtain hydroxy calcium phosphate particles.

[0042] (4) Dissolve 70 mg of the hollow nanoparticles in 30 ml of the solution of step (1), stir magnetically at 45° C. for 3 h, centrifuge at 10,000 r / min for 10 min after the reaction, wash three times with deionized water, and freeze-dry the precipitate after centrifugation to obtain chitosan-gold nano-hydroxycalcium phosphate particles.

[0043] (5) Dissolve 25 mg of chitosan-gold nano-hydroxycalcium phosphate particles in the methanol solution of 10-hydroxycamptothecin in step (1), shake at 150 rpm at 37°C for 24 h, centrifuge at 10,000 rpm for 10 min after the reaction, wash three times with methanol, and freeze-dry the precipitate after centrifugation to obtain chitosan-gold nano-hydroxycalcium phosphate loaded with 10-hydroxycamptothecin nanoparticles.

[0044] (6) The prepared drug-loaded nanoparticles were prepared into a 1 mg / mL PBS buffer solution (pH 5.5) and placed in a constant temperature oscillator (37°C, 100 RPM). After (1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h), the centrifuge tubes were taken out and centrifuged to obtain 2mL of the supernatant. 2mL of PBS buffer solution was added and ultrasonically dispersed. The nanoparticles were then placed in a constant temperature oscillator and waited for the next time point. These experiments were performed under 808nm laser irradiation (2.1W / cm 2 ) or without irradiation with 808 nm laser. The samples were taken out and the concentration of 10-hydroxycamptothecin and its derivatives in the release medium was measured by high performance liquid chromatography, and the cumulative release rate was calculated.

[0045] The release of 10-hydroxycamptothecin was 47.5%, and when irradiated with near-infrared light, the release of 10-hydroxycamptothecin was 75.5%. With the irradiation of near-infrared light, chitosan-gold nano-hydroxycalcium phosphate particles can quickly release 10-hydroxycamptothecin and its derivatives, indicating that the nanodrug delivery system has NIR-responsive release performance.

[0046] Embodiment 3:

[0047] A method for preparing a nano drug delivery system loaded with irinotecan (CPT-11) and its derivatives comprises the following steps:

[0048] (1) Disperse 5 mg of irinotecan in 50 mL of methanol and ultrasonicate at 25° C. for 30 min to obtain a methanol solution of irinotecan.

[0049] (2) Dissolve 0.1 g of chitosan powder in 100 mL of 1% (v:v) acetic acid water, stir overnight and filter, record as solution A, dissolve 1 g of chloroauric acid powder in 50 mL of deionized water, record as solution B, mix well, then slowly add 0.6 ml of solution B to solution A, stir magnetically at 80°C for 3 h. After the reaction is completed, place in a 4°C refrigerator away from light for later use.

[0050] (3) Dissolve 0.295 g of calcium chloride dihydrate in 20 mL of deionized water, referred to as solution A, dissolve 0.138 g of ammonium dihydrogen phosphate in 20 mL of deionized water, referred to as solution B, and dissolve 0.6 g of urea in 10 ml of deionized water, referred to as solution C. Slowly add solution B to solution A, stir magnetically at 25°C for 5 min, then add solution C, stir for 5 min, then add 2.2 g of L-glutamic acid and stir for 20 min. Transfer the mixed solution into a reactor, react at 160°C for 10 h, centrifuge at 10,000 r / min for 10 min after the reaction, wash with deionized water 3 times, discard the supernatant, and freeze-dry to obtain hydroxy calcium phosphate particles.

[0051] (4) Dissolve 70 mg of the hollow nanoparticles in 30 ml of the solution of step (1), stir magnetically at 45° C. for 3 h, centrifuge at 10,000 r / min for 10 min after the reaction, wash three times with deionized water, and freeze-dry the precipitate after centrifugation to obtain chitosan-gold nano-hydroxycalcium phosphate particles.

[0052] (5) Dissolve 25 mg of chitosan-gold nano-hydroxycalcium phosphate particles in the methanol solution of irinotecan in step (1), shake at 150 rpm at 37° C. for 24 h, and after the reaction, centrifuge at 10,000 rpm for 10 min, wash with methanol three times, and freeze-dry the precipitate after centrifugation to obtain chitosan-gold nano-hydroxycalcium phosphate loaded irinotecan nanoparticles.

[0053] (6) The prepared drug-loaded nanoparticles were prepared into a 1 mg / mL PBS buffer solution (pH 5.5) and placed in a constant temperature oscillator (37°C, 100 RPM). After (1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h), the centrifuge tubes were taken out and centrifuged to obtain 2mL of the supernatant. 2mL of PBS buffer solution was added and ultrasonically dispersed. The nanoparticles were then placed in a constant temperature oscillator and waited for the next time point. These experiments were performed under 808nm laser irradiation (2.1W / cm 2 ) or without irradiation with 808 nm laser. Take out the sample, measure the concentration of irinotecan and its derivatives in the release medium by high performance liquid chromatography, and calculate the cumulative release rate.

[0054] The release of irinotecan was 50.5%, and when irradiated with near-infrared light, the release of irinotecan was 76.5%. With the irradiation of near-infrared light, chitosan-gold nano-hydroxycalcium phosphate particles can quickly release irinotecan and its derivatives, indicating that the nano drug delivery system has NIR-responsive release performance.

Claims

1. A method for preparing a nano drug delivery system loaded with camptothecin and its derivatives, characterized in that: The preparation method comprises the following steps: (1) dissolving camptothecin (or hydroxycamptothecin and irinotecan) in methanol at a concentration ratio of 100ug / mL:(50-100ug / mL), and ultrasonicating for 30 minutes at 25°C-35°C so that the alkali can be fully dissolved in methanol. (2) 1 g of solid chloroauric acid was dissolved in 50 ml of deionized water to prepare a 2% chloroauric acid solution; 0.1 g of chitosan was dissolved in 100 ml of 1% acetic acid aqueous solution, stirred overnight to prepare a 0.1% chitosan solution, and filtered through a 0.22 μm filter membrane. (3) Take 30 ml of the chitosan solution after membrane treatment, adjust the pH to 5.5 with ammonia water, add 0.6 ml of 2% chloroauric acid solution, stir and react in a water bath at 80° C. for 3 h, and set reflux to prevent solvent evaporation. (4) Calcium chloride dihydrate and ammonium dihydrogen phosphate were prepared into 1 mol / L solutions respectively. To ensure the preparation of the optimal calcium hydroxyphosphate, the Ca / P molar ratio was set to 1.

67. The pH value of the calcium chloride dihydrate solution was adjusted to 11.5, and ammonium dihydrogen phosphate was added. Urea and L-glutamic acid were added, stirred for 20 minutes, and then placed in a reactor at 160° C. for 10 hours to obtain calcium hydroxyphosphate microspheres. (5) Adding the calcium hydroxyphosphate prepared in step (4) above to the chitosan-gold nanocomposite solution prepared in step (3) above, stirring evenly, magnetically stirring at 45° C. for 3 h, centrifuging at 10,000 rpm for ten minutes, washing three times with deionized water, and freeze-drying to obtain a camptothecin derivative and its derivative bio-nano drug delivery system with near-infrared response. (6) Add the chitosan-gold nanoparticles prepared in step (5) to the camptothecin methanol solution prepared in step (1), stir evenly, shake at 37°C for 24 hours on a shaking oscillator at 150 rpm, centrifuge and freeze-dry to obtain a nano drug delivery system of camptothecin and its derivatives.

2. The method for preparing a nano drug delivery system loaded with camptothecin and its derivatives according to claim 1, characterized in that: The drug loaded in step (1) is camptothecin.

3. The method for preparing a nano drug delivery system loaded with camptothecin and its derivatives according to claim 1, characterized in that: In step (2), a 2% chloroauric acid solution and a 0.1% chitosan solution are prepared.

4. The method for preparing a nano drug delivery system loaded with camptothecin and its derivatives according to claim 1, characterized in that: The pH value of the chitosan solution in step (3) is 5.

5.

5. The method for preparing a nano drug delivery system loaded with camptothecin and its derivatives according to claim 1, characterized in that: In step (3), the volume ratio of the chitosan solution to the chloroauric acid solution is 50:

1.

6. The method for preparing a nano drug delivery system loaded with camptothecin and its derivatives according to claim 1, characterized in that: In step (4), Ca 2+ With PO4 - The molar ratio was 1.67 and the pH was 11.

7. The method for preparing a nano drug delivery system loaded with camptothecin and its derivatives according to claim 1, characterized in that: In step (5), the mass ratio of chitosan to hydroxyapatite is 30:

70.

8. The nano drug delivery system loaded with camptothecin and its derivatives prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The prepared chitosan-gold nano-hydroxy calcium phosphate has a particle size of 5-10 um.

9. The nano drug delivery system loaded with camptothecin and its derivatives prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The drug loading rate is 25% to 29%.