Preparation method and application of ultrasonic response nanocomposite hydrogel

Abstract

The application belongs to the technical field of novel sustained-release drug delivery system, and particularly relates to a preparation method and application of a high-molecular targeted drug-loaded nanodroplet and injectable hydrogel. The high-molecular material is modified by acetone, and is used as a carrier of drugs and phase-contrast contrast agents to construct drug-loaded nanodroplets. The chitosan-based injectable hydrogel is prepared by a freeze-salting method, and the obtained drug-loaded nanodroplets are combined in the hydrogel matrix to construct a system. The nanocomposite injectable hydrogel has the characteristics of high targeting and low toxicity, and under the action of ultrasound, the drug release can be promoted, and the macrophage phenotype can be effectively reversed, thereby providing a means for the treatment of arthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials and drug delivery technology, specifically relating to a composite hydrogel loaded with drug nanoparticles, its preparation method and application. Background Technology

[0002] Rheumatoid arthritis (RA) is a common chronic autoimmune disease that can lead to functional impairment and even disability, causing immense suffering and inconvenience to patients. To halt the progression of RA, patients should begin antirheumatic drug treatment as early as possible. Antirheumatic drugs for RA treatment include traditional synthetic antirheumatic drugs, targeted synthetic antirheumatic drugs, and biologics, such as methotrexate, leflunomide, tofacitinib, and adalimumab. Currently, methotrexate (MTX) is a first-line treatment; however, the remission rate with traditional methotrexate monotherapy is approximately 30%, while novel biologics such as TNF inhibitors and small-molecule targeted drugs combined with MTX only achieve a 70-80% remission rate. Exploring new targets and drugs for RA treatment is the future direction of research.

[0003] Macrophages play a crucial role in the development and progression of rheumatoid arthritis (RA). As an innate immune cell, macrophages can polarize into pro-inflammatory M1 and anti-inflammatory M2 phenotypes depending on the RA synovial microenvironment. As a core component of the innate immune system, macrophages play a dual role in the pathogenesis of RA, acting as both drivers of inflammation and regulators of tissue repair. Their polarization morphology and dynamic interaction with the synovial microenvironment profoundly influence the progression of RA and treatment response. Recent studies have shown that macrophages, as the main group of synovial infiltrating immune cells, play a pivotal role in the triggering and maintenance of synovitis through imbalances in polarization phenotypes and disruptions in programmed cell death patterns (such as pyroptosis and necroptosis). Macrophages play a crucial role in inducing synovial inflammation: on the one hand, imbalance in M0 macrophage polarization leads to increased pro-inflammatory M1 polarization and decreased anti-inflammatory M2 polarization, subsequently releasing various inflammatory cytokines such as TNF-α, IL-1β, and IL-6; on the other hand, increased macrophage pyroptosis can also promote the release of inflammatory factors such as IL-1β and IL-18. Maintaining macrophage polarization balance is one of the important strategies for controlling the progression of rheumatoid arthritis (RA).

[0004] Tripterygium wilfordii root bark (CEL) is the first naturally active product isolated from the root bark of Tripterygium wilfordii. It possesses anti-inflammatory and other pharmacological activities and can be used to treat rheumatoid arthritis (RA). Numerous studies have shown that CEL can affect macrophage polarization, reducing the number of inflammatory macrophages and thus alleviating the progression of RA. However, CEL has drawbacks such as low water solubility, insufficient targeting, and organ toxicity, hindering its clinical translation.

[0005] In summary, developing a drug delivery system that specifically targets inflammatory macrophages, reverses macrophage polarization, and has high drug utilization and low drug toxicity is of great significance for alleviating rheumatoid arthritis and bacterial infectious arthritis. Summary of the Invention

[0006] Based on the above problems, the primary objective of this invention is to provide a nanocomposite injectable hydrogel capable of loading triptolide, thereby addressing the issues of triptolide's high toxicity, low targeting, and poor in-situ retention. This nanohydrogel exhibits long-lasting and controlled-release effects, resulting in good biocompatibility for the ultrasound-responsive nanocomposite injectable hydrogel. Furthermore, the presence of cRGD (clinically generated RGD) enhances the targeting of this nanocomposite hydrogel. cRGD can selectively target αvβ3 integrin, which studies have shown to be highly expressed in macrophages. The calcium-phosphorus cross-linked chitosan cryogel demonstrates excellent biocompatibility, and the gel is injectable and provides porous, sustained-release drug-loaded nanoparticles. Therefore, the nanocomposite hydrogel provided by this invention also alleviates, to some extent, the organ toxicity of triptolide.

[0007] The second objective of this invention is to provide a nanodroplet composite injectable hydrogel. Under the influence of ultrasound, due to its acoustic droplet vaporization and cavitation effects, the nanodroplets can promote endocytosis, enhance therapeutic efficacy, and achieve an integrated diagnostic and therapeutic effect.

[0008] A third objective of this invention is to provide the application of the above-mentioned targeted drug-loaded nanodroplet composite injectable hydrogel.

[0009] One of the objectives of this invention is achieved through the following technical solution: The carrier and target molecules of this targeted drug-loaded nanodroplet composite injectable hydrogel are both cRGD-polycaprolactone-polyethylene glycol; the therapeutic drug is triptolide, dissolved in dimethyl sulfoxide (DMSO); and the solvent for the cRGD-polycaprolactone-polyethylene glycol modification is acetone. In the cRGD-polycaprolactone-polyethylene glycol targeted nanodroplets, the average molecular weight of polycaprolactone is 5000 g / mol, and the average molecular weight of polyethylene glycol is 2000 g / mol. The injectable hydrogel is a calcium-phosphate ion-crosslinked chitosan cryohydrogel, with the ion-crosslinking components being calcium salts and phosphates.

[0010] In the method described in this invention, the loaded drug is one of triptolide and vancomycin; Preferably, the drug is triptolide.

[0011] The specific synthesis steps are as follows: (1) Weigh 5 mg of cRGD-polycaprolactone-polyethylene glycol, add acetone and stir for 15 min to modify it; (2) Use a rotary evaporator to evaporate the acetone completely; (3) Add acetone again and stir for 15 min. During the stirring process, add 3 ml of DD water dropwise. (4) After stirring, acetone is removed by rotary evaporation again to obtain cRGD-polycaprolactone-polyethylene glycol carrier; (5) Weigh 5 mg of triptolide, add 200 ul DMSO to dissolve it completely, and then add DD water to make up to 1 ml.

[0012] (6) During the stirring of the cRGD-polycaprolactone-polyethylene glycol carrier solution, fully dissolved triptolide was added dropwise and stirred for 15 min to obtain triptolide drug-loaded nanoparticles, namely cRGD-CEL NP.

[0013] (7) Weigh out chitosan powder and dissolve it in 1% (v / v) glacial acetic acid solution, and freeze it overnight with a thickness of 0.5 cm.

[0014] (8) Add a large amount of 1.5 mol / L CaCl2 solution, let stand for 2 hours and then remove it; add a large amount of 0.9 mol / L NaH2PO4 solution, let stand for 2 hours and then remove it.

[0015] (9) Add a large amount of 0.2 mol / L NaOH solution and let stand for 5 min. Rinse thoroughly with deionized water to remove residual salts and obtain injectable hydrogel, namely CH.

[0016] (10) CH and cRGD-CEL NP were mixed in a ratio of 2:1 and frozen at -20℃ overnight. The lyophilized product was dissolved in water to obtain an ultrasonic drug-loaded nanodroplet composite injectable hydrogel, namely CND@CH.

[0017] Preferably, the volume of acetone is 0.5 to 2 ml.

[0018] Preferably, the temperature of the rotary evaporation is 40~45℃.

[0019] Preferably, the mass of the drug administered is 5-10 mg.

[0020] The second objective of this invention is achieved by the following technical solution: Perfluorohexane (PFH) was added to the nanoparticles prepared for one of the objectives, and emulsified using an ultrasonic cell disruptor to obtain nanodroplets, namely cRGD-CEL ND (CND). The ultrasonic nanocomposite hydrogel maintained its shape and initial diameter during injection, while undergoing a liquid-gas transition upon ultrasonic exposure, generating micron-sized bubbles at the site of interest—a process known as acoustic droplet vaporization. The backscattered signal generated by the microbubbles was the strongest, significantly improving the resolution, sensitivity, and specificity of ultrasound diagnosis. Furthermore, the microbubbles in the liquid underwent cavitation under ultrasonic action; this ultrasonic cavitation can widen the gaps between surrounding target cells (including vascular endothelial cells and tissue cells), promoting endocytosis.

[0021] Preferably, the phase change contrast agent is mixed with the cRGD-CEL NP prepared by the target institute at a ratio of 1 ml: 20 μl.

[0022] Preferably, the ultrasonic emulsification time is 0.5~2s; the power of the ultrasonic waves used in ultrasonic emulsification is 80~100W.

[0023] The third objective of this invention is achieved by the following technical solution: The aforementioned ultrasound-responsive nanocomposite hydrogels were applied to targeted therapy and integrated diagnosis and treatment of rheumatoid arthritis and bacterial infectious arthritis caused by Escherichia coli, methicillin-resistant Staphylococcus aureus, and other bacteria. The CND@CH composite hydrogel prepared in this study possesses ultrasound-responsive controlled release and long-lasting drug release characteristics; it can be administered via injection, reducing the frequency of punctures and avoiding mechanical damage to the joint. Simultaneously, it can exert the effects of triptolide in inhibiting synovial inflammation, clearing MRSA, and disrupting bacterial biofilms, thus balancing antibacterial and anti-inflammatory efficacy with drug safety, providing an ideal novel drug delivery system for MRSA-related suppurative arthritis. Attached Figure Description

[0024] Figure 1 This is the macroscopic form of CND@CH in Embodiment 1 of the present invention; Figure 2 These are fluorescence microscopy images of CND@CH in Embodiment 2 of the present invention; Figure 3 This is a scanning electron microscope (SEM) image of CND@CH in Embodiment 3 of the present invention; Figure 4 This refers to the cumulative release rate of CND@CH in Experimental Example 4 of this invention; Figure 5 This refers to the hemolysis of CND@CH in Experimental Example 5 of this invention; Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.

[0026] Example 1 This embodiment provides an ultrasound-responsive nanodroplet composite injectable hydrogel, which includes: a nanocarrier, a target molecule, a therapeutic drug, a novel phase change contrast agent, and an injectable hydrogel / calcium phosphate ion crosslinked chitosan cryohydrogel.

[0027] The preparation method of the nanodroplet composite injectable hydrogel in this embodiment includes the following steps: Weigh 5 mg of cRGD-polycaprolactone-polyethylene glycol, add acetone and stir for 15 min to modify it. After stirring, use a rotary evaporator to remove the acetone completely, add acetone again, and stir for 15 min. During stirring, add 3 ml of DD water dropwise. After stirring, evaporate the acetone again to obtain the cRGD-polycaprolactone-polyethylene glycol carrier, i.e., cRGDNP. Weigh 5 mg of triptolide, first add 200 μL of DMSO to dissolve it completely, and then add DD water to make up to 1 ml. While stirring the cRGD-polycaprolactone-polyethylene glycol carrier solution, add the fully dissolved triptolide dropwise and stir for 15 min to obtain triptolide drug-loaded nanoparticles, i.e., CNP. Take 1 mL of CNP, add 20 μL of PFH, and emulsify for 1 s using an ultrasonic cell disruptor at an ultrasonic power of 100 W to obtain CND. Chitosan (CS) powder was weighed and dissolved in a 1% (v / v) glacial acetic acid solution to obtain a 3% (w / v) CS solution with a thickness of approximately 0.5 cm. This solution was then frozen overnight at -20°C. A large amount of 1.5 mol / L CaCl2 solution was added sequentially, and the solution was allowed to stand for 2 hours before being aspirated. A large amount of 0.9 mol / L NaH2PO4 solution was added, and the solution was allowed to stand for 2 hours before being aspirated. A large amount of 0.2 mol / L NaOH solution was added, and the solution was allowed to stand for 5 minutes. The solution was then thoroughly rinsed with deionized water to remove residual salts, yielding an injectable hydrogel, CH. CH and CND were mixed in a 2:1 ratio and frozen overnight at -20°C. The lyophilized product was dissolved in water to obtain an ultrasonically loaded drug-eluting nanodroplet composite injectable hydrogel, CND@CH.

[0028] The macroscopic morphology of the ultrasonically responsive nanohydrogel is as follows: Figure 1As shown in the figure. Initially, it was in a liquid state. After freezing and salting out, it gradually became a solid state. When the vial was inverted, the solid in the vial did not fall out, indicating that CND@CH was successfully prepared.

[0029] Experiment Example 2: Fluorescence microscopy images of ultrasonically responsive nanocomposite hydrogels An ultrasonically responsive nanocomposite hydrogel was constructed according to the method described in Example 1. Subsequently, FITC-labeled CND@CH was prepared: 1 mg of FITC was weighed and combined with 1 mL of CND, and the mixture was thoroughly mixed using an ultrasonic cleaner. The FITC-labeled CND and CH were then mixed in a 1:2 ratio to obtain FITC-labeled CND@CH. FITC is a green fluorescein derivative; after binding FITC to CND@CH, the binding was analyzed using fluorescence microscopy to determine if the binding was successful. The results are as follows... Figure 2 As shown, CND@CH exhibits distinct green fluorescence, and the fluorescence image of CND@CH reveals that CND is successfully encapsulated within CH. Furthermore, during the preparation of CND@CH, some FITC is transferred from CND to CH, causing CH to also exhibit weak fluorescence.

[0030] Experiment Example 3: Observation of the microstructure of CND@CH using scanning electron microscopy (SEM) The microstructure of CND@CH was observed by SEM, and the results are as follows: Figure 3 As shown, CND@CH exhibits a three-dimensional network structure, with a large number of spherical particles and plate-like crystals adsorbed within the three-dimensional network structure. The spherical particles are CND, indicating that CND and CH have successfully combined, and CND@CH has been successfully prepared.

[0031] This case demonstrates the successful preparation of CND@CH, where the loose, porous network structure endows the hydrogel with drug loading capacity, facilitating controlled drug release.

[0032] Example 4: Cumulative release rate of ultrasonically responsive nanocomposite hydrogels The controlled release performance of CND@CH was investigated. The drug release patterns of CND@CH within 5 h under US treatment and untreated conditions are as follows: Figure 4 As shown, the results indicate that the drug release rate of CND@CH without US treatment was only about 10% within 5 h, while the cumulative drug release rate of the US-treated group was close to 80% at 5 h; and the drug release rate showed a significant instantaneous increase after each US stimulation.

[0033] This case demonstrates that CND@CH possesses excellent ultrasound-responsive drug release performance. This performance is attributed to two main factors: firstly, the cavitation effect generated by CND under ultrasound (US) effectively promotes drug release; and secondly, the precise control of the hydrogel network structure by the US, thereby driving rapid drug release. This fully validates the application potential of this drug-loaded hydrogel in US-mediated controlled drug release.

[0034] Example 5: Biosafety Study of Ultrasonic Response Nanocomposite Hydrogels In this case, the biocompatibility of the ultrasound-responsive nanocomposite hydrogel was verified through a hemolysis experiment. Fresh blood from healthy mice was used for the hemolysis experiment to obtain mouse erythrocyte dispersions. Nanodroplet solutions of different concentrations (1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL) were prepared using PBS (pH 7.4). Physiological saline was used as a negative control, and Triton X-100 was used as a positive control. Triton X-100 is a nonionic surfactant whose structure involves its hydrophobic end inserted into the erythrocyte membrane while its hydrophilic end is exposed outside the membrane. This structure disrupts the lipid bilayer, leading to erythrocyte membrane rupture and hemoglobin release, with a hemolysis rate typically approaching 100%. However, in the biocompatibility evaluation of biomedical materials, a hemolysis rate below 5% is considered a safety threshold. Each ultrasound-responsive nanocomposite hydrogel group, along with positive and negative controls, was mixed with erythrocyte dispersion. After standing at room temperature for 4 hours, the dispersions were centrifuged, and the centrifuge tubes were photographed at the same horizontal level. The absorbance of the supernatant at 540 nm was then measured using an ELISA reader, and the hemolysis rate was calculated. Figure 5 As shown. Figure 5 The results showed that the hemolysis rate of CND@CH was less than 5% within the concentration range of 0 ~ 100 μg / mL.

[0035] This case demonstrates that ultrasound-responsive nanocomposite hydrogels have good biocompatibility and can be used for subsequent animal experiments.

Claims

1. A nanocomposite injectable hydrogel, characterized in that, It was prepared by combining a polymer carrier, a drug, a phase change contrast agent, and an injectable hydrogel raw material. The polymeric carrier of the drug-loaded nanodroplets is cRGD-targeted cyclic peptide-polycaprolactone-polyethylene glycol (cRGD-PEG-PEI). The drug is one or a combination of two of triptolide and vancomycin; The phase change contrast agent is perfluorohexane (PFH). The injectable hydrogel is based on chitosan, with calcium salt and phosphate as ionic crosslinking components.

2. The method for preparing the nanocomposite injectable hydrogel as described in claim 1, characterized in that, Includes the following steps: (1) After cRGD-polycaprolactone-polyethylene glycol was modified by stirring with acetone and rotary evaporation, it was stirred with tripterygium nitrate solution; (2) Adding PFH emulsification to prepare nanodroplets; (3) Chitosan was prepared into an injectable hydrogel using the calcium phosphate ion crosslinking-freeze salting-out method. / Chitosan was dissolved in glacial acetic acid, and calcium chloride and sodium dihydrogen phosphate were added sequentially. After pH adjustment, the mixture was frozen and molded. An injectable hydrogel was prepared using the calcium phosphate ion crosslinking-freeze salting-out method. (4) Nanocomposite injectable hydrogels are prepared by mixing nanodroplets and injectable hydrogels in a certain proportion, freeze-drying and then re-dissolving.

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