CN116212015A - In-situ ultrasonic visual controlled-release phase-change type immune hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of drug controlled release gel, in particular to in-situ ultrasonic visual controlled release phase-change immune hydrogel and a preparation method and application thereof. The preparation method of the in-situ ultrasonic visual controlled-release phase-change immune hydrogel comprises the following steps: preparing sodium alginate solution and aqueous solution of sound-induced droplet phase-change nanoparticles; the sonoliquid drop phase change nanoparticle consists of a shell formed by PLGA and capable of loading hydrophobic drugs and a liquid fluorocarbon core loaded with hydrophilic drugs; and mixing the sodium alginate solution and the acoustic drop phase change nanoparticle aqueous solution according to a certain proportion to obtain a phase change type hydrogel precursor solution. The technical scheme solves the technical problem that the nano drug delivery system is difficult to rapidly transform and apply in clinical work due to low drug loading, abrupt drug release, poor biocompatibility and the like. If the immune medicines such as tranilast and the like are used as functional medicines, the problems of insufficient immune treatment response rate and poor medicine permeability can be solved, and the application prospect is wide.

Description

A phase-change immune hydrogel with visualized and controlled release by in-situ ultrasound and its preparation method and application

technical field

The invention relates to the technical field of drug controlled-release gels, in particular to a phase-change immune hydrogel with in-situ ultrasonically visualized and controlled release, a preparation method and application thereof.

Background technique

Acoustic Droplet Vaporization (ADV) means that ultrasonic energy can excite acoustically responsive droplets from a liquid state to a bubble, increasing the ultrasonic imaging signal. The latest research shows that using acoustically induced droplet phase transitions can generate microjets that emit shock waves and eject fluids. The high-temperature and high-pressure gas produced by the droplet becoming larger and collapsing during the oscillation process can be used for sonoporation, ultrasonic penetration, ultrasonic thrombolysis, opening the blood-brain barrier, and even directly killing tumor cells. In recent years, the unique biological function of acoustically induced droplet phase transition has attracted extensive attention from researchers. As a new type of molecular probe, liquid-coated fluorocarbon nanoparticles can efficiently target tumor sites. Under the excitation of focused ultrasound, the nanoparticles can undergo a phase transition, transforming from small to large into microbubbles, thus changing the acoustic environment and And promote drug penetration. Therefore, the use of focused ultrasound and acoustically induced phase change droplets is promising for ultrasound-visualized drug delivery to tumors and facilitated drug penetration.

Chinese patent CN101780285B discloses a heat-enhanced polymer nano-ultrasonic imaging micelle loaded with liquid fluorocarbon and a preparation method thereof. This scheme uses the following method to synthesize nanoparticles loaded with liquid fluorocarbons: under the protection of argon, the hydroxyl-terminated PEG with a molecular weight of 2.0-3.0 kg/mol is dried in vacuum for several hours and then cooled to room temperature, and then injected with dry lactide and A small amount of stannous octoate; add anhydrous toluene after vacuum drying at room temperature, and then carry out reflux polymerization. After the reaction, reprecipitate in anhydrous ether, filter and dissolve with dichloromethane, carry out secondary reprecipitation in anhydrous ether, filter and vacuum dry to obtain the polymer nano-ultrasonic imaging micellar carrier material PEG -PDLLA. Co-dissolve the copolymer PEG-PDLLA and perfluoropentane in carbon tetrachloride, disperse it in the polyvinyl alcohol aqueous solution in an ice bath under the action of ultrasound, and remove the solvent to obtain the heat-enhanced load liquid Fluorocarbon-based polymer nanomicelles for ultrasound imaging. The obtained micelles are nanoscale, with a narrow particle size distribution, and have significant in vitro ultrasound imaging effects and thermal enhancement effects. Under the action of ultrasound, they can undergo a phase transition from liquid state excitation to bubbles, and generate micro jets to achieve therapeutic effects. However, the nanoparticles in the prior art have problems such as low drug loading rate, need for repeated administration, drug burst release and potential toxicity, which hinder their rapid transformation and application in clinical work. There is an urgent need to develop a new type of drug controlled release system based on acoustic droplet phase change, in order to increase drug loading, reduce drug toxicity, achieve effective control of drug release, and enhance the clinical transformation potential of drug controlled release system.

Contents of the invention

The present invention intends to provide a method for preparing a phase-change immune hydrogel with in-situ ultrasonically visualized and controlled release, so as to solve the problem of low drug loading, drug burst release, and biocompatibility in the prior art nano-drug delivery system. And other reasons make it difficult to quickly transform and apply technical problems in clinical work.

To achieve the above object, the present invention adopts the following technical solutions:

A method for preparing a phase-change immune hydrogel with visualized and controlled release by in-situ ultrasound, comprising the following steps in sequence:

S1: Preparation of sodium alginate solution and aqueous solution of sonic-induced droplet phase-change nanoparticles; the shell of sonic-induced droplet phase-change nanoparticles is composed of a polymer or lipid membrane material and a liquid fluorocarbon core loaded with hydrophilic drugs composition;

S2: Mixing the sodium alginate solution and the aqueous solution of sonic-induced droplet phase-change nanoparticles to obtain a phase-change hydrogel precursor solution for forming a hydrogel.

This scheme also provides a hydrogel obtained by the preparation method of a phase-change immune hydrogel visualized and controlled release by in-situ ultrasound.

This solution also provides the application of the hydrogel obtained by the preparation method of the in-situ ultrasonically visualized and controlled-release phase-change immune hydrogel in the preparation of a controlled-release drug system.

The principle and beneficial effect of this technical solution are:

This technical solution combines drug-loaded sonic phase change nanoparticles with sodium alginate in-situ gelation, loads various tumor treatment drugs into drug-loaded sonophase change nanoparticles, and disperses the nanoparticles into alginic acid In situ injection in sodium solution to form a phase-change hydrogel for in-situ ultrasonically visualized drug-controlled release. This technical solution solves the technical problem that the ordinary acoustic phase change nano-drug delivery system in the prior art is difficult to be quickly transformed and applied in clinical work due to low drug loading, drug burst release, biocompatibility and other reasons. . If immune drugs are used as functional drugs, this technical solution can further solve the problems of insufficient response rate and poor drug permeability in current clinical immunotherapy.

Sodium alginate is a natural biomacromolecule isolated from brown algae. Under the action of calcium and magnesium ions, sodium alginate molecules will cross-link to form a biocompatible porous hydrogel, which is widely used in drug delivery, food engineering, and tissue engineering. In this technical solution, by injecting a certain concentration of sodium alginate aqueous solution, the in situ sodium alginate hydrogel is formed by using the in situ physiological concentration of calcium and magnesium ions in the tumor, which greatly improves the drug loading concentration and reduces drug toxicity, and Long-term drug release is achieved with only a single injection. This technical solution uses the combination of drug-loaded acoustic phase change nanoparticles and sodium alginate in situ gelation, which can deliver a large amount of phase change materials to the tumor in situ, release drugs for a long time, and use ultrasonic observation to improve the safety of drug application .

In summary, on the basis of the previous research on acoustically induced phase-change nano-droplets and immune hydrogels, an in situ-generated hydrogel was used as a carrier to load phase-change nanoparticles containing functional drugs, and an in situ Ultrasonic phase-change hydrogel for drug release. Under the excitation of low-energy focused ultrasound, the nanoparticles undergo an acoustic phase transition, which visualizes the controlled release of functional drugs and eliminates tumors. If the functional drug is an immune drug (for example, TGF-β inhibitor tranilast), this technical solution can improve the immune microenvironment, normalize tumor blood vessels, enhance oxygen supply, and increase the penetration of immune checkpoint blockers in tumors It can completely eliminate residual cancer and inhibit recurrence and metastasis, providing an efficient, safe and precise tumor treatment strategy for the treatment of breast cancer.

Further, in S1, the functional drugs include tranilast, imiquimod, resiquimod, CpG oligodeoxynucleotide, lipopolysaccharide, 1-methyl tryptophan, doxorubicin, gemcitabine and At least one of paclitaxel.

Further, in S1, the raw material of the liquid fluorocarbon core includes at least one of perfluoropropane, perfluoropentane, perfluorohexane and perfluorooctyl bromide; the raw material of the membrane material includes polylactic acid- Glycolic acid copolymer and/or phospholipid material; the phospholipid material includes at least one of DPPA, DPPC, DPPG, DSPE, DSPE-PEG, and DSPG.

The drugs that can be loaded on the hydrogel of this solution include but are not limited to Tranilast (Tranilast), Imiquimod (R837), Resiquimod (R848), CpG oligodeoxynucleotide (CpG-ODN), Lipopolysaccharide (LPS), 1-methyltryptophan (1-MT) and common chemotherapy drugs (doxorubicin, gemcitabine, paclitaxel), etc. Perfluoropentane, perfluorohexane, and perfluorooctyl bromide are commonly used phase change materials in this field, and polylactic acid-glycolic acid copolymers, liposomes, etc. are commonly used membrane materials in nano drug delivery systems, and can be used In the preparation of in situ ultrasonically visualized controlled-release phase-change immunohydrogel in this protocol.

The hydrogel of the technical solution is particularly suitable for slow-release therapy of immunomedicine for tumors or other diseases. Immunotherapy refers to a treatment method that uses the autoimmune system to kill tumor cells, and has shown great potential in preventing tumor recurrence and distant metastasis. Taking immune checkpoints as an example, studies on antibodies against programmed cell death protein 1 and its ligands (anti-PD-1/PD-L1) or antibodies against toxic T lymphocyte-associated protein 4 (anti-CTLA-4) And clinical use is changing the treatment outcome of cancer patients, and is expected to expand the application to different tumor types. However, studies have shown that the clinical response rate of immune checkpoint therapy is still insufficient, and only 15% of tumor patients can benefit from immune checkpoint therapy. Therefore, it is urgent to find new therapeutic modalities to maximize the clinical efficacy of immune checkpoints. The hypoperfusion, hypoxia, and immunosuppressive microenvironment of solid tumors are important factors that make it difficult for immunotherapy to work. Due to the low perfusion of the tumor, it is difficult for immune drugs to penetrate uniformly in the tumor, which often leads to residual tumor and long-term recurrence. In addition, the hypoxic microenvironment induced by tumor hypoperfusion can induce immunosuppression, such as inhibiting the polarization of macrophages to the M1 direction, promoting the expression of tumor cell immune checkpoints, hindering the presentation of related antigens and activation of immune cells, etc., affecting the efficacy of immunotherapy. Each link. The hydrogel adopting the technical solution can promote the uniform penetration of immune drugs and other anti-tumor drugs in tumor cells, improve the effect of tumor treatment, and avoid tumor recurrence and metastasis.

Among the above drugs, tranilast is a clinically approved TGF-β inhibitor antiallergic drug. Studies have shown that tranilast can improve tumor perfusion, normalize tumor stroma, and repair tumor blood vessels Abnormalities, thereby restoring tumor perfusion and oxygenation, improving the efficacy of immune checkpoints and the effect of anti-tumor immunotherapy. As a hydrophobic drug, tranilast is still a problem how to deliver it safely and efficiently. The hydrogel adopting the technical solution can increase the delivery amount of tranilast in the affected area, and stimulate drug release according to actual needs, thereby realizing safe and efficient drug delivery.

Further, in S1, the liquid fluorocarbon is perfluoropentane.

Further, in the phase change hydrogel precursor solution of S2, the concentration of sodium alginate is 5 mg/mL-40 mg/mL, and the concentration of sonic droplet phase change nanoparticles is ≤10 mg/mL and >0 mg/mL .

Further, in S1, the sonic-induced droplet phase change nanoparticles were prepared by the following method: the functional drug was dissolved in dichloromethane to obtain solution B; perfluoropentane was added to solution B, and after sonic shock treatment , to obtain solution C; add PVA solution to solution C, and obtain solution D after acoustic shock treatment; The nanoparticles are taken from the supernatant, and after the nanoparticles are rinsed with water, the nanoparticles are resuspended in water to obtain an aqueous solution of the acoustic-induced droplet phase transition nanoparticles.

Further, in S2, the volume ratio of the sodium alginate solution and the aqueous solution of sonic droplet phase change nanoparticles is 3:2-4:1; when preparing the phase change type hydrogel precursor solution, the sonic droplet The phase-change nanoparticles are added to the sodium alginate solution, and then stirred at 200 rpm for 5-10 minutes.

Further, the hydrogel includes a gel skeleton formed by sodium alginate, and acoustically induced droplet phase-change nanoparticles attached to the skeleton; the hydrogel is injected into the body from a phase-change hydrogel precursor solution formed after solidification.

Further, the drug controlled release system is used to release functional drugs under the excitation of low-energy focused ultrasound; the hydrogel is formed in situ in vivo from a phase-change hydrogel precursor solution, and the phase-change hydrogel The precursor solution does not contain a crosslinker.

In summary, the beneficial effects of this technical solution are:

The in-situ ultrasonically visualized and controlled-release phase-change hydrogel proposed by the present invention has significant advantages such as in-situ synthetic gelation, high-efficiency loading, simple synthesis, and controlled release of ultrasonically visualized drugs. The tranilast-loaded phase-change ultrasonic controlled-release immune hydrogel prepared by this method has both the ability of space-time controlled drug release and synergistic immune checkpoint therapy. Utilizing acoustic-induced liquid droplet phase transition and hydrogel in situ gelation for local ultrasound visualization of controlled release of tranilast, improving the immunosuppressive microenvironment of tumors, promoting the normalization of tumor blood vessels in residual cancer, improving tumor perfusion and oxygenation, Promote the penetration of anti-PD-1/PD-L1 antibodies in tumor sites, enhance immune checkpoint therapy, prevent tumor recurrence and metastasis, and explore new strategies for efficient, safe and economical postoperative recurrence treatment. In addition, this system can also freely adjust various parameters such as hydrogel fluidity, drug dosage, drug type, etc., and has good universality. In a word, the in situ ultrasound visualized controlled release phase-change immune hydrogel proposed by the present invention establishes an in situ ultrasound visualized drug controlled release system to realize the dual controlled release of the hydrophobic drug tranilast in time and space, Provide new ideas for local visualization of drug delivery and potentiation of immune checkpoint therapy.

Description of drawings

Figure 1 is a physical picture of the phase-change immune hydrogel visualized and controlled release by in situ ultrasound.

Figure 2 is a cryo-scanning electron micrograph of the phase-change immunohydrogel visualized and controlled by in situ ultrasound.

Figure 3 is an image of the in vitro gelation verification results of the phase-change immunohydrogel visualized and controlled by in situ ultrasound.

Figure 4 is an image of the in vivo gelation verification results of the phase-change immunohydrogel visualized and controlled by in situ ultrasound.

Fig. 5 is the ultrasonic imaging of the phase-change immune hydrogel visualized and controlled release by in situ ultrasound.

Figure 6 is the cumulative drug release curve of the phase-change immune hydrogel visualized and controlled by in situ ultrasound.

Fig. 7 is the experimental result of the influence of the mixing speed of Comparative Example 1 on the state of solution G.

Fig. 8 is the experimental result of the effect of sodium alginate concentration on gelation effect in Comparative Example 2.

Fig. 9 is the experimental result of the influence of the concentration of phase-change nanoparticles on the state of solution G in Comparative Example 3.

Detailed ways

The present invention will be described in further detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

Example 1

A phase-change immune hydrogel visualized and controlled release by in situ ultrasound is prepared by the following method:

Stir 100 mg of sodium alginate powder in 3 mL of pure water until fully dissolved to obtain solution A (sodium alginate solution), which is stored at 4°C. Dissolve 2 mg of tranilast and 50 mg of PLGA (Jinan Daigang SJ10202, PLGA-COOH, 12,000 molecular weight) in 2 mL of dichloromethane, and use conventional ultrasonic treatment to promote dissolution during the dissolution process to obtain solution B. Add 200 μL of perfluoropentane to solution B in an ice bath, and emulsify the system for 3 minutes using a conventional acoustic shock instrument (40% intensity, 50% duty cycle, 5s on, 5soff) to obtain solution C. Add 8 mL of 4% PVA solution to solution C, and emulsify again for 3 min using a sonicator (35%, 50% duty cycle; 5s on, 5s off) to obtain solution D. 10 mL of 2% isopropanol solution was added to solution D, and magnetically stirred for 4-6 h under ice bath conditions (5 h was specifically used in this example), to obtain solution E. Centrifuge solution E at 10,000 rpm and 4°C for 8 minutes, discard the supernatant, reconstitute and rinse with pure water, repeat three times, and reconstitute with 2mL ultrapure water to obtain solution F (aqueous solution of sonic-induced droplet phase change nanoparticles), Store at 4°C (need to use as soon as possible). The concentration of nanoparticles in solution F can be determined according to the effective concentration of the drug. In this embodiment, a concentration of nanoparticles (in solution G) of about 10 mg/mL is used according to the effective concentration.

When in use, mix solution A and solution F in a volume ratio of 3:2 in an ice bath and mix them uniformly by magnetic force to obtain solution G (phase-change hydrogel precursor solution), which is stored at 4°C. injection. During the mixing process of solution A and solution F, the rotation speed of magnetic stirring was maintained at about 200 rpm, and the stirring time was 5-10 min. Before stirring and mixing, solution F needs to be added dropwise to solution A. Reverse dropwise addition will cause a phase change of nanoparticles and a large number of bubbles will appear, which will affect the subsequent in-situ drug release effect. See Figure 3-6 for the experimental results of gelation ability, ultrasonic imaging effect and in vitro drug release ability.

The inventor has tried the following method: Prepare solution A and solution F in the manner described in Example 1, and when the two solutions are mixed to form solution G, slowly add solution A (sodium alginate) to solution F (TPP NPs) , and try to ensure that all solution A is added to solution F during the addition process. Then magnetically stir at 200 rpm for 10 min to form solution G. During this process, the inventors found that the mixing order of the above materials would lead to the generation of a large number of bubbles during the stirring process of the solution G, and a large number of nanoparticles in the finally formed solution G undergo phase transitions, and there are many bubbles (similar to the one shown in Figure 7). State of solution G at 1000 rpm). After the above phenomena occurred, the inventor analyzed that the cause of excessive bubbles may be that the stirring speed of 200rpm was too high, so the stirring speed was randomly adjusted to 100rpm, and the above experiment was repeated. It was found that the phase transition of nanoparticles in solution G was still not improved, and there were still many bubbles in solution G, which may affect the in-situ gelation and drug release effects. In addition, due to the low rotation speed, the material was not fully mixed, the shape of solution G was not uniform, and after 4 hours of storage, obvious stratification appeared. The above attempts show that the mixing order of solution A and solution F is very critical to inhibit the premature phase transition of nanoparticles, and in the case of incorrect mixing order, only by reducing the rotation speed, the phase transition of nanoparticles still cannot be effectively avoided. The gel preparation of this scheme needs to solve the incompatibility problem when sodium alginate and nanoparticles are mixed, so as to realize the in situ ultrasonic visualization and controlled release of the gel. Through extensive experimental research, the inventor found that adjusting the mixing order of solution A and solution F can overcome the above problems. When preparing solution G, slowly add solution F (TPP NPs) to solution A (sodium alginate), and at a speed of 200 rpm, a phase-change hydrogel precursor solution (solution G), obtained the in situ ultrasonically visualized controlled-release phase-change immunohydrogel as shown in Example 1. It can be seen that the mixing order of solution A and solution F has played an important role in the realization of this scheme, and unexpected technical effects have been obtained.

Example 2

This example is basically the same as Example 1, except that the dosage of sodium alginate is adjusted to 60 mg, and the volume ratio of solution A to solution F is adjusted to 4:1. The in situ ultrasonically visualized controlled-release precursor solution prepared in this example showed an appearance consistent with typical results (see Figure 1), and the precursor solution showed a morphology similar to Figure 2 under a cryo-SEM after gelling. And the precursor solution of this example shows similar gelation ability, ultrasonic imaging effect and drug release ability in vitro as in Example 1.

Example 3

This embodiment is basically the same as Embodiment 1, except that the same amount of tranilast is replaced by doxorubicin. The in situ ultrasonically visualized controlled-release precursor solution prepared in this example showed an appearance consistent with typical results (see Figure 1), and the precursor solution showed a morphology similar to Figure 2 under a cryo-SEM after gelling. And the precursor solution of this example shows similar gelation ability, ultrasonic imaging effect and drug release ability in vitro as in Example 1.

Example 4

This example is basically the same as Example 1, except that the same amount of tranilast is replaced by the immunostimulant R837. The in situ ultrasonically visualized controlled-release precursor solution prepared in this example showed an appearance consistent with typical results (see Figure 1), and the precursor solution showed a morphology similar to Figure 2 under a cryo-SEM after gelling. And the precursor solution of this example shows similar gelation ability, ultrasonic imaging effect and drug release ability in vitro as in Example 1.

Example 5

This example is basically the same as Example 1, except that the same amount of tranilast is replaced by the immunostimulant R848. The in situ ultrasonically visualized controlled-release precursor solution prepared in this example showed an appearance consistent with typical results (see Figure 1), and the precursor solution showed a morphology similar to Figure 2 under a cryo-SEM after gelling. And the precursor solution of this example shows similar gelation ability, ultrasonic imaging effect and drug release ability in vitro as in Example 1.

Example 6

This example is basically the same as Example 1, except that the same amount of tranilast is replaced by the immunostimulant CpG oligodeoxynucleotide (CpG-ODN). The in situ ultrasonically visualized controlled-release precursor solution prepared in this example showed an appearance consistent with typical results (see Figure 1), and the precursor solution showed a morphology similar to Figure 2 under a cryo-SEM after gelling. And the precursor solution of this example shows similar gelation ability, ultrasonic imaging effect and drug release ability in vitro as in Example 1.

Take the in-situ ultrasonically visualized and controlled-release phase-change hydrogel precursor solution (solution G) for visual observation. The typical results are shown in Figure 1. Nanoparticle aqueous solution (TPP NPs), and the mixed solution formed by the two in a certain ratio (phase change hydrogel precursor solution, TPP ALG), the three solutions are uniform and stable in shape.

The typical results of cryo-scanning electron microscope observation of the hydrogel in this scheme can be seen in Figure 2, which is a cryo-scanning electron micrograph of a phase-change hydrogel visualized and controlled by in-situ ultrasound. It can be seen that the porous network structure inside the gel and Among them are nanoparticles. Cryo-scanning electron microscopy experiment references (10.1002/adfm.201670071): prepare physiological concentration of calcium and magnesium mixture (1.8mM Ca ²⁺ , 1.5mM Mg ²⁺ ), inject 1mL of solution G into 30mL of calcium and magnesium mixture, and then use Filter paper filters the hydrogel out for cryo-EM scanning.

Figure 3 is the in vitro gelation verification experiment results of the in situ ultrasonically visualized and controlled-release phase-change immune hydrogel, which shows that the phase-change hydrogel can be stably gelled in vitro. The upper left image of Figure 3 shows the process of injecting 1mL of acoustic droplet phase change nanoparticle aqueous solution (TPP NPs) in 30mL of calcium and magnesium solution, and the lower left image shows the gelation situation. Leaks, absorbs quickly and cannot stay in the target area for a long time. The upper picture in Figure 3 shows the process of injecting 1mL of the phase-change hydrogel precursor solution (i.e. solution G) in 30mL calcium and magnesium solution, and the middle and lower pictures show the gelation situation, using the phase-change hydrogel precursor The solution is capable of forming a gel in vitro. Figure 3 top right and bottom right show the appearance of the formed gel.

Figure 4 shows the results of the in vivo gelation verification experiment of the phase-change immune hydrogel visualized and controlled by in situ ultrasound, which shows that the phase-change hydrogel can be stably gelled in vivo. Subcutaneously inject 0.1mL sonic droplet phase change nanoparticle aqueous solution (TPP NPs) and 0.1mL phase change hydrogel precursor solution (TPP ALG) in mice, after 1h, 24h, 48h and 7 days, observe Gel formation. Acoustic-induced liquid droplet phase change nanoparticles leak and diffuse rapidly in the surrounding tissue after injection, and cannot be fixed in the target area, while the phase change hydrogel precursor solution quickly forms a hydrogel gel after injection, immobilizing the drug in the target area. In the area, the gel state remains for 7 days.

Figure 5 is an in-vivo ultrasound imaging effect diagram of in situ ultrasound visualized and controlled release. Using low-energy focused ultrasound, the ultrasound contrast signal is significantly enhanced. The specific experimental process is as follows: inject 0.1mL sodium alginate aqueous solution (ALG) and 0.1mL tranilast PLGA ALG solution (non-phase-change hydrogel precursor solution, TP ALG) subcutaneously into the mouse (using an equal amount of double distilled water instead of PFP ) and 0.1mL phase-change hydrogel precursor solution (TPP ALG), immediately observe the contrast-enhanced ultrasound signal, and then use low-energy focused ultrasound (LIFU, 4W) to irradiate for 180s. B-mode (left) and contrast-enhanced ultrasound imaging (CEUS, right) observations before and after material LIFU excitation.

Before the above-mentioned in vivo experiments were carried out, according to the conventional knowledge of the prior art, due to the low and fluctuating calcium and magnesium concentrations at physiological concentrations, the inventors predicted that it might be difficult to effectively form gels in vivo and realize controlled release of drugs. Therefore, the inventors first tried to add a certain amount of calcium and magnesium ions to the phase change hydrogel precursor solution (TPP ALG, solution G), and after the injectable hydrogel was formed in vitro, the mice were subjected to Subcutaneous injection. Forming a hydrogel and then injecting is a conventional operation mode adopted for the combination of hydrogel and nanoparticles in the prior art. However, for the material of this solution, it is very easy to make the phase transition of nanoparticles in this way, and the cross-linking of the gel is not uniform, and the uniform drug release in the body cannot be controlled. Due to the failure of the above attempts, the inventors then tried to directly inject the phase-change hydrogel precursor solution (TPP ALG) without adding any cross-linking agent (such as calcium and magnesium ions) into the mouse subcutaneously. The in situ colloidal TPP ALG prepared by calcium and magnesium ions can uniformly and stably immobilize drug-loaded phase-change nanoparticles in the target area, and can monitor drug release behavior using ultrasound visualization.

Figure 6 is the cumulative drug release curve of the phase-change immune hydrogel visualized and controlled by in situ ultrasound (the arrow is low-energy focused ultrasound excitation). The results show that low-energy focused ultrasound can significantly stimulate drug release. The specific experimental process is: in 30mL of ethanol (30%) aqueous solution containing calcium (1.8mM), magnesium (1.5mM) and Tween-80 (0.01%), inject 3mL phase change type hydrogel precursor solution (ie solution G), the solution G gels immediately, and then carries out the in vitro drug release experiment. For the experimental group (repetition), low-energy focused ultrasound excitation was performed at a specified time point, that is, low-energy focused ultrasound (LIFU, 4w) was irradiated for 180s, and then the release amount of drug tranilast was detected by ultraviolet method and the cumulative release rate was calculated; for the control group In the control group, only the ultrasound probe was placed without low-energy focused ultrasound excitation, and the release amount of the drug tranilast was directly detected at the specified time point and the release rate was calculated. Cumulative drug release curve experiments were repeated three times at each time point, and the error bars in the figure are standard deviations (SD).

Comparative example 1

Referring to Example 1, the mixing conditions of solution A and solution F were studied, and the experimental results are shown in FIG. 7 . At 1000rpm for 5min, a large number of dense bubbles can be seen in the TPP ALG. This indicates that TPP NPs are prone to phase transition at high rotational speeds. There are still a few bubbles at 600rpm for 5 minutes, and TPP NPs can be evenly dispersed at 200rpm for 5 minutes without obvious bubbles. Therefore, this technical solution uses a speed of 200rpm to mix solution A and solution F.

In addition, on the basis of Example 1, the inventor adjusted the stirring speed to 100 rpm. Because the rotating speed is too low, the material is not fully mixed, the morphology of the phase change type hydrogel precursor solution (solution G) is not uniform, and after standing for 4 hours, there is obvious layering phenomenon, which shows that the rotating speed is too low. The resulting solution G has poor stability.

Comparative example 2

Referring to Example 1, the concentration of sodium alginate in solution G (TPP ALG) was explored. 1mL of TPP ALG with a concentration of 5-20mg/mL ALG (the concentration of TPP NPs is fixed at 5mg/mL) was injected into a solution simulating 50mL of physiological calcium and magnesium concentration (Ca ²⁺ 1.8mM, Mg ²⁺ 1.5mM), with the increase of ALG concentration , the formed hydrogel has better strength, and it is easier to control the injection volume and direction (see Figure 8). In practical application, the concentration of sodium alginate in solution A can be 15mg/3mL-200mg/3mL, and the concentration of sodium alginate in solution G can be 5-40mg/mL. This technical solution preferably adopts a sodium alginate concentration (in solution G) of 16-25 mg/mL, which can fully ensure that solution G is stable and gelled.

Comparative example 3

Referring to Example 1, the concentration of nanoparticles (TPP NPs) in solution G (TPP ALG) was explored. The concentration of TPPNPs needs to be adjusted according to the actual effective dose of the drug. In the range of 0-10 mg/mL (final concentration in solution G), sodium alginate solution and nanoparticles can form a uniform mixture (see Figure 9), thereby realizing into glue. 10mg/mL is the maximum concentration of TPP NPs, beyond this concentration TPP NPs are difficult to disperse. The gel prepared according to this technical scheme can have a load of TPP NPs in the solution G of 0-10 mg/mL, which can realize effective gel formation and control of drug release. It shows that the loading capacity of TPP NPs can be changed in a relatively large range after the gel is formed, and users can adjust the loading capacity of TPP NPs within the above range according to actual needs, with a greater degree of freedom.

What is described above is only an embodiment of the present invention, and common knowledge such as specific technical solutions and/or characteristics known in the solutions will not be described here too much. It should be pointed out that for those skilled in the art, without departing from the technical solutions of the present invention, some modifications and improvements can also be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation of the present invention effect and utility of the patent. The scope of protection required by this application shall be based on the content of the claims, and the specific implementation methods and other records in the specification may be used to interpret the content of the claims.

Claims (10)

1. The preparation method of the in-situ ultrasonic visual controlled-release phase-change immune hydrogel is characterized by comprising the following steps of: the method comprises the following steps of:

s1: preparing sodium alginate solution and aqueous solution of sound-induced droplet phase-change nanoparticles; the sonoliquid drop phase change nanoparticle consists of a shell formed by a polymer and/or lipid membrane material and a liquid fluorocarbon core loaded with hydrophilic drugs;

s2: and mixing the sodium alginate solution and the acoustic droplet phase-change nanoparticle aqueous solution to obtain a phase-change hydrogel precursor solution for forming the hydrogel.

2. The method for preparing the in-situ ultrasonic visual controlled release phase-change immune hydrogel, which is characterized by comprising the following steps of: in S1, the functional drug comprises at least one of tranilast, imiquimod, resiquimod, cpG oligodeoxynucleotide, lipopolysaccharide, 1-methyltryptophan, doxorubicin, gemcitabine and paclitaxel.

3. The method for preparing the in-situ ultrasonic visual controlled release phase-change immune hydrogel, which is characterized by comprising the following steps of: in S1, the liquid fluorocarbon core material includes at least one of perfluoropropane, perfluoropentane, perfluorohexane, and perfluorobromooctane; the raw materials of the membrane material comprise polylactic acid-glycolic acid copolymer and/or phospholipid material; the phospholipid material comprises at least one of DPPA, DPPC, DPPG, DSPE, DSPE-PEG and DSPG.

4. The method for preparing the in-situ ultrasonic visual controlled release phase-change immune hydrogel according to claim 3, which is characterized in that: in S1, the sonoliquid droplet phase change nanoparticle is prepared by the following method: dissolving a functional medicine in dichloromethane to obtain a solution B; adding perfluoropentane into the solution B, and obtaining a solution C after acoustic shock treatment; adding PVA solution into the solution C, and obtaining solution D after acoustic shock treatment; adding an isopropanol solution into the solution D, and stirring to obtain a solution E; and (3) centrifuging the solution E, discarding the supernatant to obtain nanoparticles, rinsing the nanoparticles with water, and re-suspending the nanoparticles with water to obtain an aqueous solution of the sonoliquid droplet phase-change nanoparticles.

5. The method for preparing the in-situ ultrasonic visual controlled release phase-change immune hydrogel, which is disclosed in claim 4, is characterized in that: in the phase-change hydrogel precursor solution of S2, the concentration of sodium alginate is 5mg/mL-40mg/mL, the concentration of the sonoliquid drop phase-change nanoparticles is less than or equal to 10mg/mL, and is more than 0mg/mL.

6. The method for preparing the in-situ ultrasonic visual controlled release phase-change immune hydrogel, which is characterized by comprising the following steps of: in S2, the volume ratio of the sodium alginate solution to the aqueous solution of the sound-induced droplet phase-change nanoparticles is 3:2-4:1; when the phase-change hydrogel precursor solution is prepared, the sonoliquid droplet phase-change nanoparticles are added into the sodium alginate solution, and then stirred at 200rpm for 5-10min.

7. A hydrogel obtainable by the method of preparing an in situ ultrasound visualized controlled release phase-change immune hydrogel according to any one of claims 1 to 6.

8. The hydrogel obtained by the method for preparing the in-situ ultrasonic visual controlled release phase-change immune hydrogel, which is characterized in that: the hydrogel comprises a gel skeleton formed by sodium alginate, and sound-induced liquid drop phase-change nanoparticles attached to the skeleton; the hydrogel is formed by solidifying a phase-change hydrogel precursor solution after being injected into a body.

9. The use of a hydrogel obtained by the method for preparing an in situ ultrasound visualized controlled release phase-change immune hydrogel according to claim 7 or 8 in the preparation of a drug controlled release system.

10. The application of the hydrogel obtained by the preparation method of the in-situ ultrasonic visual controlled-release phase-change immune hydrogel in preparing a drug controlled-release system, which is characterized in that: the medicine controlled release system is used for releasing functional medicines under the excitation of low-energy focused ultrasound; the hydrogel is formed in situ in the living body from a phase-change hydrogel precursor solution, and the phase-change hydrogel precursor solution is free of a cross-linking agent.

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