A pH-responsive nanodiamond drug delivery system and a preparation method thereof

Abstract

This invention discloses a pH-responsive nanodiamond drug delivery system and its preparation method, belonging to the field of nanomedicine carrier technology. The delivery system uses polyglycerol-modified nanodiamond (NDPG) as the base carrier, covalently loading doxorubicin (DOX) via pH-sensitive hydrazone bonds, and combining it with hyaluronic acid (HA) to achieve targeted modification and surface-stabilized coating, ultimately forming an HA-DOX / NDPG composite system or an NDPG-DOX@HA core-shell structure. The preparation method includes: in-situ ring-opening polymerization grafting of polyglycerol onto the nanodiamond surface to obtain NDPG; activation of hyaluronic acid followed by coupling with adipic acid dihydrazide, then forming hydrazone bonds with doxorubicin to obtain HA-DOX; activation of NDPG followed by grafting of connecting arms, then coupling with doxorubicin to obtain NDPG-DOX, and finally coating with hyaluronic acid to obtain the core-shell product. The carrier of this invention exhibits good water dispersibility, stable drug loading rate, low drug leakage under physiological conditions, rapid drug release in the weakly acidic environment of tumors, and also possesses active targeting capability and good biocompatibility. It can be used to prepare highly effective and low-toxicity antitumor drugs, and has high clinical application and translational value.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine carrier technology, specifically relating to a pH-responsive nanodiamond drug delivery system and its preparation method, and particularly to an antitumor nanodrug delivery system based on polyglycerol-modified nanodiamond, hyaluronic acid targeting, and acid-sensitive hydrazone bond cleavage. Background Technology

[0002] Doxorubicin (DOX) is a widely used anthracycline broad-spectrum antitumor drug in clinical practice, showing good therapeutic effects on various solid tumors. However, it suffers from problems such as a short half-life in vivo, lack of targeted distribution throughout the body, severe cardiotoxicity and bone marrow suppression, and a high risk of drug burst release. Traditional nanodelivery systems mostly rely on physical adsorption for drug loading, which has drawbacks such as high leakage rate in the bloodstream, insufficient stability, poor biocompatibility, and lack of responsiveness to the tumor microenvironment.

[0003] Nanodiamonds (ND) possess advantages such as high biocompatibility, high specific surface area, easy surface modification, and low cytotoxicity, making them ideal drug carrier materials. However, pristine nanodiamonds exhibit poor water dispersibility and are prone to aggregation, limiting their application in the biopharmaceutical field. Surface grafting modification with polyglycerol (PG) can significantly improve their hydrophilicity and colloidal stability, while also providing a large number of modifiable hydroxyl sites.

[0004] Hyaluronic acid (HA) can specifically bind to the CD44 receptor, which is highly expressed on the surface of tumor cells, enabling active targeted delivery. The hydrazone bond is stable in a neutral physiological environment (pH 7.4) and rapidly hydrolyzes and breaks down in the weakly acidic extracellular environment of tumors (pH 6.0) and the acidic environment of lysosomes (pH 5.0), achieving precise drug release.

[0005] Currently, there is no integrated drug delivery system that combines polyglycerol-modified nanodiamonds, pH-sensitive hydrazone bond drug delivery, and hyaluronic acid targeting, nor is there a matching industrially feasible preparation route. Therefore, developing a highly dispersed, highly stable, pH-responsive, actively targeted, low-toxicity, and highly efficient nanodiamond drug delivery system is of great significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a pH-responsive nanodiamond drug delivery system and its preparation method, which integrates hydrophilic modification of nanodiamond carriers, covalent drug loading, acid-responsive controlled release, and active targeting, thereby improving drug safety and therapeutic efficacy.

[0007] The technical solution of the present invention is as follows: A pH-responsive nanodiamond drug delivery system includes two types of structures: (1) HA-DOX / NDPG complex: formed by the adsorption of HA-DOX and NDPG through electrostatic and hydrogen bonding; (2) NDPG-DOX@HA core-shell structure: formed by the encapsulation of NDPG-DOX as the core and hyaluronic acid as the shell through electrostatic and hydrogen bonding.

[0008] Among them, NDPG is obtained by in-situ ring-opening polymerization of nanodiamond and glycidyl ether under heating and nitrogen protection; HA-DOX is obtained by activating HA, coupling ADH, and forming hydrazone bonds with DOX; NDPG-DOX is obtained by activating NDPG with CDI, grafting ADH, and forming hydrazone bonds with DOX.

[0009] This invention achieves the following by adjusting the structural composition and preparation parameters: low leakage in the physiological environment, rapid drug release in the acidic environment, high colloidal stability, and active targeting of tumor cells, significantly reducing toxic side effects and improving anti-tumor effects.

[0010] The beneficial effects of this invention are as follows: 1. Polyglycerol grafting significantly improves the water dispersibility and stability of nanodiamonds, making them less prone to aggregation; 2. Using hydrazone covalent drug delivery avoids burst release and improves blood circulation stability; 3. It exhibits significant pH-responsive release characteristics, enabling precise drug release at the tumor site; 4. Hyaluronic acid provides active targeting capabilities, enhancing drug accumulation at tumor sites; 5. The preparation process is mild, the purification is simple, and the reproducibility is good, making it suitable for large-scale production. Attached Figure Description

[0011] Figure 1. Schematic diagram of NDPG synthesis route; Figure 2. Schematic diagram of the HA-DOX synthesis route; Figure 3. Schematic diagram of the synthesis of the NDPG-DOX@HA core-shell structure; Figure 4. In vitro drug release curves of the delivery system under different pH conditions. Detailed Implementation

[0012] The present invention will be further described in detail below with reference to specific embodiments.

[0013] Example 1: Preparation of NDPG 500 mg of nanodiamond powder was dispersed in 10 mL of ethylene glycol and sonicated until homogeneous and stable. Under nitrogen protection, the mixture was heated in an oil bath at 100 °C, with 4 mL of glycidyl ether added dropwise every 30 min, for a total of 19 mL, and the reaction was continued for 16 h. The reaction was terminated by adding 50 mL of distilled water. The reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da, dialyzed with ultrapure water for 3 days, and freeze-dried to obtain NDPG with a polyglycerol grafting amount of approximately 36 wt%.

[0014] Example 2 Preparation of HA-DOX Weigh 200 mg of sodium hyaluronate and dissolve it in 100 mL of MES buffer. Stir to dissolve. Add 48 mg of EDCI and activate at room temperature for 20 min. Add 436 mg of adipic acid dihydrazide solution and react at room temperature for 2 h. Adjust the pH to 7.0, transfer to a 3500 Da dialysis bag, dialyze with deionized water for 2 days, filter and freeze dry to obtain HA-ADH.

[0015] Dissolve 100 mg HA-ADH in sodium acetate buffer, add 2 mL of doxorubicin solution (2 mg / mL), adjust the pH to 5.0, and react in the dark for 2 h; adjust the pH to 7.0, dialyze until the external solution is colorless, and freeze dry to obtain HA-DOX with a drug loading of about 5.5 wt%.

[0016] Example 3 Preparation of HA-DOX / NDPG complex Dissolve 20 mg of HA-DOX in 10 mL of deionized water; separately disperse 20 mg of NDPG in 10 mL of deionized water and sonicate until homogeneous; mix the two and stir overnight at room temperature in the dark to obtain the HA-DOX / NDPG complex.

[0017] Example 4: Preparation of NDPG-DOX 40 mg of NDPG was dispersed in anhydrous dimethyl sulfoxide, and 80 mg of CDI was added under nitrogen protection. The mixture was stirred at 45 °C for 4 h. 200 mg of adipic acid dihydrazide and 100 μL of triethylamine were added, and the mixture was stirred under nitrogen protection for 12 h. The reaction was terminated by adding 80 mL of deionized water, and the mixture was transferred to a 4000 Da dialysis bag for dialyzing for 3 days. NDPG-ADH was obtained by lyophilization with 5% trehalose.

[0018] 20 mg of NDPG-ADH was dispersed in deionized water, doxorubicin solution was added dropwise, the pH was adjusted to 5.0, and the reaction was carried out in the dark for 2 h. The mixture was then transferred to a 14000 Da dialysis bag and dialyzed for 2 days to obtain NDPG-DOX with a drug loading of approximately 2.8 wt%.

[0019] Example 5: Preparation of NDPG-DOX@HA core-shell structure 20 mg of NDPG-DOX was ultrasonically dispersed in 10 mL of deionized water; 25 mg of sodium hyaluronate was dissolved in 10 mL of deionized water; the HA solution was slowly added dropwise to the NDPG-DOX dispersion and stirred continuously for 1 h; the solution was then transferred to a 14000 Da dialysis bag and dialyzed for 24 h, and then lyophilized to obtain NDPG-DOX@HA.

[0020] Example 6 In vitro release experiment Release tests were conducted using dialysis at 37°C in PBS buffer solutions at pH 7.4, pH 6.0, and pH 5.0. The results showed that at pH 7.4, the cumulative release after 72 h was ≤35%; at pH 6.0, the cumulative release after 72 h was approximately 55%; and at pH 5.0, the cumulative release after 72 h was ≥80%, exhibiting typical pH-responsive drug release characteristics.

[0021] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention. All modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pH-responsive nanodiamond drug delivery system, characterized in that, It includes polyglycerol-modified nanodiamond (NDPG), doxorubicin (DOX) linked by hydrazone bonds, and hyaluronic acid (HA); the delivery system is an HA-DOX / NDPG complex or an NDPG-DOX@HA core-shell structure, which rapidly releases the drug under acidic conditions of pH 5.0–6.0, is stable under physiological conditions of pH 7.4, and has a low leakage rate.

2. The delivery system according to claim 1, characterized in that, The NDPG is prepared by ring-opening polymerization of nanodiamond (ND) and glycidyl ether under nitrogen protection at 100°C, with a polyglycerol grafting amount of 30–38 wt%.

3. The delivery system according to claim 1, characterized in that, The HA-DOX is prepared by reacting sodium hyaluronate with adipic dihydrazide (ADH) under EDCI activation to generate the HA-ADH intermediate, which is then reacted with doxorubicin at pH=5 to form a hydrazone bond, with a drug loading rate of 5.0–6.0 wt%.

4. The delivery system according to claim 1, characterized in that, The NDPG-DOX is prepared by activating NDPG with carbonyl diimidazole (CDI), grafting adipate dihydrazide, and then coupling it with doxorubicin via a hydrazone bond, with a drug loading rate of 2.5–3.5 wt%.

5. The delivery system according to claim 1, characterized in that, The NDPG-DOX@HA has a core-shell structure, with the core being NDPG-DOX and the outer shell being hyaluronic acid, which is coated by electrostatic interactions and hydrogen bonds; the average hydrated particle size is 80–135 nm, and the zeta potential is -20 mV to -7 mV.

6. The method for preparing the pH-responsive nanodiamond drug delivery system according to any one of claims 1–5, characterized in that, The process includes the following steps: (1) Preparation of NDPG: Disperse nanodiamonds in ethylene glycol, add glycidyl ether dropwise under nitrogen protection, react at 100°C for 16 h, and after the reaction, dialyze and purify, freeze dry to obtain NDPG; (2) Preparation of HA-DOX: Dissolve sodium hyaluronate in buffer, add EDCI to activate the carboxyl group, and then add adipic acid dihydrazide to react to obtain HA-ADH intermediate, dialyze and freeze dry, and react with doxorubicin under pH=5 conditions in the dark to form hydrazone bonds, dialyze and purify, freeze dry to obtain HA-DOX; (3) Preparation of delivery system: Mix HA-DOX solution with NDPG suspension and stir to obtain HA-DOX / NDPG complex; or activate NDPG, graft connecting arms and couple with doxorubicin to obtain NDPG-DOX, and then add hyaluronic acid solution for coating to obtain NDPG-DOX@HA core-shell structure.

7. The preparation method according to claim 6, characterized in that: Step (1) Dialysis was performed for 3 days using a dialysis bag with a molecular weight cutoff of 8000 Da; Step (2) Dialysis was performed for 2 days using a dialysis bag with a molecular weight cutoff of 3500 Da.

8. The preparation method according to claim 6, characterized in that: The hydrazone reaction conditions are pH 4.5–5.5, room temperature, and reaction in the dark for 2–12 h.

9. The use of the delivery system according to any one of claims 1–5 in the preparation of a pH-responsive antitumor drug, characterized in that, The delivery system targets and releases doxorubicin in the weakly acidic tumor microenvironment, reducing cardiotoxicity and improving drug accumulation efficiency at the tumor site.

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