A cannabinoid heat-sensitive nano-hydrogel preparation and a preparation method thereof

Abstract

The application discloses a kind of cannabidiol heat-sensitive nano hydrogel preparation and its injection and its preparation method, the injection includes cannabidiol 5-15mg / mL, Poly (I:C) 0.5-1.5mg / mL and Pluronic F127 concentration is 20-30wt%.The injection can slow-release cannabidiol and Poly (I:C) and prolong its residence time at tumor site, reduce dosing frequency, effectively and continuously synergistic inhibition tumor, reduce side effects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to a cannabidiol thermosensitive nanohydrogel preparation and its preparation method, particularly a cannabidiol thermosensitive nanohydrogel injection and its use in anti-tumor treatment. Background Technology

[0002] Cancer is one of the most serious diseases threatening human health and a leading cause of death worldwide. In recent years, the incidence and mortality rates of cancer have been rising rapidly. According to the World Health Organization, there were 18.1 million new cancer cases and 960 cancer-related deaths globally in 2018. Traditional cancer treatments, including surgical resection, chemotherapy, and radiotherapy, cannot completely cure cancer. Therefore, exploring new cancer treatments that are highly effective, have fewer side effects, and are less costly is extremely important.

[0003] Tumor immunotherapy is a treatment method that kills tumor cells by modulating the body's immune defense mechanisms. It has attracted much attention due to its advantages such as fewer side effects and high specificity. However, single-modality immunotherapy still faces significant challenges. Various tumor-infiltrating immune cells can directly destroy tumor cells, but they can also maintain an immunosuppressive microenvironment, allowing tumor immune escape. Therefore, an ideal cancer immunotherapy must induce tumor cell death while inhibiting tumor immune escape. To this end, the inventors designed an injectable thermosensitive nanohydrogel, a nanoformation (CP) formed by combining ferroptosis-inducible nanoparticles of cannabidiol (hereinafter referred to as "CBD") with the immune adjuvant polyinosinic acid (Poly(I:C)). Compared with traditional specific immunotherapies, the synthetic CP does not involve additional chemical modifications and has low preparation costs. CBD exerts strong anti-tumor activity through direct iron-promoting effects and downregulation of Poly(I:C) or IFN-γ-induced PD-L1 expression, generating systemic anti-tumor immunity, inhibiting the growth of poorly immunogenic tumors, and prolonging the survival time of tumor-bearing mice. The nanohydrogel formulation of this invention holds promise as a safe and effective treatment option for clinically relevant cancers. Summary of the Invention

[0004] The present invention aims to provide a cannabidiol (CBD) thermosensitive nanohydrogel formulation, its injection solution, and a preparation method thereof. The CBD thermosensitive nanohydrogel formulation of the present invention prepares water-soluble nanoparticles (CBDNPs) from a poorly soluble ferroptosis inducer (CBD) using a droplet-limited deposition method combined with freeze-drying driven crystallization. Then, an immunoadjuvant (Poly(I:C)) is loaded into the addition polymer F127 of polypropylene glycol and ethylene oxide using a physicochemical method. CBDNPs and Poly(I:C) synergistically exert a strong antitumor effect, while a high concentration of F127 (25wt%) prolongs the residence time of the antitumor drug at the tumor site, reducing the frequency of administration and minimizing side effects.

[0005] To achieve the objectives of this invention, the following implementation scheme is provided.

[0006] In one embodiment, the present invention provides a cannabidiol thermosensitive nanohydrogel formulation comprising cannabidiol and Poly(I:C), and a thermosensitive gel matrix poloxamer.

[0007] Preferably, in the thermosensitive nanohydrogel formulation of the present invention, the poloxamer is Pluronic F127.

[0008] In another embodiment, a cannabidiol thermosensitive nanohydrogel injection of the present invention comprises 5-15 mg / mL cannabidiol, 0.5-1.5 mg / mL Poly(I:C) and 20-30 wt% Pluronic F127.

[0009] Preferably, the thermosensitive nanohydrogel injection solution of the present invention contains 10 mg / mL cannabidiol, 1 mg / mL Poly(I:C) and 25 wt% Pluronic F127.

[0010] On the other hand, the present invention also provides a method for preparing a cannabidiol thermosensitive nanohydrogel injection, comprising the following steps:

[0011] 1) Dissolve cannabidiol in chloroform and add Pluronic F127 to form a mixture;

[0012] 2) The mixture is converted into an O / W emulsion by stirring or ultrasonic vibration;

[0013] 3) The O / W emulsion is placed in liquid nitrogen to convert it into a solid state.

[0014] 4) The solid emulsion was freeze-dried in a freeze dryer to obtain cannabidiol nanocrystal particles;

[0015] 5) Cannabidiol nanocrystal particles are dissolved in physiological saline to obtain an aqueous solution of cannabidiol;

[0016] 6) Dissolve Poly(I:C) powder in physiological saline to obtain a Poly(I:C) aqueous solution;

[0017] 7) Mix the cannabidiol aqueous solution from step 5) and the Poly(I:C) aqueous solution from step 6) to obtain the nano-formulation solution;

[0018] 8) Mix the nano-formulation solution with Pluronic F127 (50% wt) solution in an equal volume ratio to obtain a thermosensitive nano-hydrogel injection solution (also known as "CP injectable thermosensitive nano-hydrogel formulation").

[0019] Furthermore, the present invention also provides a method for preparing a cannabidiol thermosensitive nanohydrogel injection, comprising the following steps:

[0020] 1) Dissolve 20 mg of cannabidiol powder in 200 μL of chloroform;

[0021] 2) Add 2 mL of Pluronic F127 (1.0 wt%) and mix;

[0022] 3) The mixture is converted into an O / W emulsion by stirring or ultrasonic vibration;

[0023] 4) Immerse the emulsion in liquid nitrogen and freeze it rapidly into a solid emulsion;

[0024] 5) Place the frozen solid emulsion in a freeze dryer and freeze dry it;

[0025] 6) Wash the lyophilized product with repeated centrifugation-redispersion cycle to obtain pure cannabidiol nanoparticles.

[0026] 7) The nanoparticles were added to 500 μL of physiological saline to obtain a cannabidiol aqueous solution of 40 mg / mL;

[0027] 8) Add 2 mg of Poly(I:C) powder to 500 μL of physiological saline to obtain a Poly(I:C) aqueous solution of 4 mg / mL;

[0028] 9) Mix the aqueous solutions from steps 7) and 8) in equal proportions (v / v) until homogeneous, and then add an equal volume of Pluronic F127 (50wt%) physiological saline solution to obtain the thermosensitive nanohydrogel injection solution.

[0029] Preferably, in the method of the present invention described above, the thermosensitive nanohydrogel injection solution contains cannabidiol at a concentration of 10 mg / mL, Poly(I:C) at a concentration of 1 mg / mL, and Pluronic F127 at a concentration of 25 wt%.

[0030] Preferably, in the method of the present invention described above, the cannabidiol nanoparticles have a particle size of 48.7 ± 4.7 nm.

[0031] The use of the cannabidiol thermosensitive nanohydrogel formulation or cannabidiol thermosensitive nanohydrogel injection of the present invention in the preparation of drugs for treating tumors.

[0032] In one specific embodiment, the present invention provides water-soluble cannabidiol nanoparticles, wherein cannabidiol is a compound represented by Formula I, and its English name is cannabidiol, i.e., CBD. A schematic diagram of the preparation of these nanoparticles is shown below. Figure 1 As shown.

[0033]

[0034] In another specific embodiment, the present invention provides an injectable thermosensitive nanohydrogel formulation, as shown in Formula III, which is an injectable thermosensitive nanoformulation, CP hydrogel (hereinafter abbreviated as "CP"), composed of CBDNPs and a double-stranded RNA analog, polyinosinic acid (Poly(I:C),) loaded into Pluronic F127 (25 wt%). A detailed preparation diagram is shown below. Figure 1 As shown.

[0035] The injectable thermosensitive nanohydrogel formulation of the present invention demonstrates its antitumor activity in various tumor models. Based on experimental results from tumor models, as follows... Figure 2 and 3 Mouse B16F10 tumor growth curve (data presented in...) The survival curves of B16F10 tumor mice (n=4) and B16F10 tumor survival curves (data are presented in the original text). The expression (n=4) indicates that the dosage range of the active pharmaceutical ingredient CBDNPs in the CP nanoformulation is 12.5-50 mg / kg, preferably 50 mg / kg.

[0036] Further experimental results show that, compared with CBDNPs and Poly(I:C) alone, CP can significantly inhibit tumor progression in mice, significantly improve the survival rate of tumor-bearing mice, significantly promote tumor cell necrosis and apoptosis, and inhibit tumor cell proliferation. On the other hand, the CP nanoformulation uses the amphiphilic polymer Pluronic F127 (25wt%), which is a flowable liquid at 4°C and a semi-solid gel at 37°C. This endows the CP nanoformulation with sustained-release properties at the tumor site, greatly reducing the frequency of anti-tumor drug administration and improving compliance.

[0037] Therefore, the injectable thermosensitive nanoformulation of the present invention plays a role in anti-tumor activity, particularly in inhibiting the progression of in situ tumors, providing a new treatment option for various cancer patients such as melanoma, breast cancer, and lung cancer. Attached Figure Description

[0038] Figure 1 Schematic diagram of CBD nanoparticle preparation;

[0039] Figure 2 Growth curve of mouse B16F10 tumor;

[0040] Figure 3 Survival curves of mice with B16F10 tumors;

[0041] Figure 4 A representative transmission electron microscope image of CBDNPs;

[0042] Figure 5 A statistical diagram of the particle size distribution of CBDNPs;

[0043] Figure 6 This is a representative figure from a thermosensitive experiment of CP nanohydrogel formulation;

[0044] Figure 7 A representative scanning electron microscope image of the CP nanohydrogel formulation;

[0045] Figure 8 Representative in vivo imaging image of a small animal to illustrate the sustained-release effect of CP nanohydrogel formulation at the tumor site;

[0046] Figure 9 A summary diagram of the antitumor effects of CP nanohydrogel formulations in the melanoma B16F10 tumor model;

[0047] Figure 10 A summary diagram of the antitumor effects of CP nanohydrogel formulations in a 4T1-luc breast cancer tumor model;

[0048] Figure 11 This is a summary diagram of the antitumor effects of CP nanohydrogel formulations in a lung cancer LLC tumor model; where Saline represents the Pluronic F127 alone (25wt%) group, CBD represents the CBDNPs + Pluronic F127 (25wt%) group, Poly(I:C) represents the Poly(I:C) + Pluronic F127 (25wt%) group, and CP represents the CBDNPs + Poly(I:C) + Pluronic F127 (25wt%) group. Detailed Implementation

[0049] The present invention will be further described below with reference to embodiments, but the embodiments are only used to understand the spirit of the present invention and not to limit the scope of the present invention.

[0050] Example 1: Preparation of CBDNPs and CP injectable thermosensitive nanoformulations

[0051] Preparation schematic diagram as shown Figure 1 As shown. The CP injectable thermosensitive nano-formulation is the CBD thermosensitive nano-hydrogel formulation of this invention.

[0052] The specific preparation process of CBDNPs is as follows:

[0053] 1. Preparation of CBDNPs (Formula II nanoparticles)

[0054] Ultrafine CBD nanoparticles with a particle size of 48.7 ± 4.7 nm were prepared mainly by a combination of nanodroplet-confined deposition and freeze-drying-driven crystallization. The specific implementation scheme is as follows:

[0055] 1) Using cannabidiol (CBD) powder (compound of formula I) as raw material, CBD (20 mg) and chloroform (200 μL) were added sequentially to a test tube;

[0056] 2) Add Pluronic F127 (1.0 wt%, 2 mL) to 1) and mix;

[0057] 3) Convert 2) into an O / W emulsion by ultrasonic vibration (500V, 2kHz, 20% power);

[0058] 4) Immediately immerse 3) in liquid nitrogen to rapidly freeze and transform it into a solid state;

[0059] 5) Quickly place the frozen solid emulsion from step 4) into a freeze dryer and freeze dry overnight;

[0060] The target product obtained through this preparation method is CBD nanocrystal particles;

[0061] To remove unattached Pluronic F127, the nanocrystals were washed using a repeated centrifugation-redispersion cycle (800,000 g, 20 min) to obtain pure CBD nanoparticles.

[0062] Experimental results: Transmission electron microscopy revealed that CBDNPs consisted of clusters of small nanoparticles. Figure 4 As shown. The hydrodynamic diameter of CBDNPs was determined by dynamic light scattering, and the results showed that the particle size of CBD nanoparticles was 48.7 ± 4.7 nm. Figure 5 .

[0063] 2. Preparation of CP injectable thermosensitive nanoparticles (Formula III hydrogel)

[0064] The CBDNPs and Poly(I:C) are mixed by loading them into Pluronic F127 using a physical method. The specific implementation scheme is as follows:

[0065] 1) Using the CBDNPs obtained in step 1 as raw material 1, add physiological saline (500uL) to a test tube to obtain a CBD aqueous solution (40mg / mL);

[0066] 2) Using Poly(I:C) powder as raw material 2 (2mg), add physiological saline (500uL) to a test tube to obtain Poly(I:C) aqueous solution (4mg / mL);

[0067] 3) Mix 1) and 2) in equal proportions (v / v) to obtain CP nano-formulation (where the CBD concentration is 20 mg / mL and the Poly(I:C) concentration is 2 mg / mL);

[0068] 4) Add Pluronic F127 (50 wt%) saline solution to the solution in step 3) at a moderate ratio (v / v) (on ice) to obtain the target product CP injectable thermosensitive nanoparticle formulation (where the CBD concentration is 10 mg / mL, the Poly(I:C) concentration is 1 mg / mL, and the Pluronic F127 concentration is 25 wt%).

[0069] Experimental results: The Pluronic F127 hydrogel loaded with CP nanoparticles was a flowable liquid at 4℃ and a gel at 37℃. (See figure) Figure 6 Scanning electron microscopy revealed the microstructure of the CP nanoformulation as a regular porous structure, which created regions allowing for liquid exchange between the matrix and its surrounding environment. Figure 7 As shown.

[0070] Example 2: Preparation and sustained-release test of Cy5.5CP injectable thermosensitive nanohydrogel formulation

[0071] 1) Using cannabidiol (CBD) powder as raw material, add CBD (20 mg) and chloroform (200 μL) sequentially to a test tube;

[0072] 2) Add Cy5.5 DMSO solution (1:1000 molar ratio) to 1);

[0073] 3) Subsequent steps are the same as in Example 1;

[0074] 4) Subcutaneous inoculation of B16F10 tumors into the back of C57BL / 6 mice (inoculation density 1*10⁻⁶) 6 The tumor is approximately 100mm in size. 3 At that time, Cy5.5-CP nano-formulation (0.9% NaCl / 25% F127) was injected into the tumor, with an injection volume of 100uL per animal.

[0075] Experimental Results: The fluorescence signal intensity of the tumor site at various time points was monitored using a small animal fluorescence imaging system. In vivo imaging results from small animals showed that the CP nanoparticle formulation (25% F127, hydrogel state) significantly prolonged the drug retention time in the tumor (see...). Figure 8 As shown in the figure, persistent release is achieved.

[0076] Example 3: Anti-B16F10 Tumor Effect Test of CP Nanohydrogel Formulation

[0077] First, an animal tumor model was established to observe the antitumor effects of CBDNPs, Poly(I:C), and CP nanoparticles alone.

[0078] Female C57BL / 6 mice were acclimatized and fed for one day before modeling, at a dose of 1*10 6A mouse model of tumor was established by subcutaneous injection of B16F10 tumor cells (melanoma) into the back of the tumor. The tumor volume was approximately 100 mm². 3 Mice were randomly divided into two batches of four groups (n=5 per group). The drugs were administered intratumorally every three days for a total of five times. The specific groups were: Saline group (Pluronic F127 alone (25wt%)), CBD group (CBD NPs + Pluronic F127 (25wt%), with CBD concentration of 10 mg / mL), Poly(I:C) group (Poly(I:C) + Pluronic F127 (25wt%), with Poly(I:C) concentration of 1 mg / mL), and CP group (CBD NPs + Poly(I:C) + Pluronic F127 (25wt%), with CBD concentration of 10 mg / mL and Poly(I:C) concentration of 1 mg / mL). Tumor size and volume were measured every three days during the administration period. After administration, one batch of mice was used to collect tumors, weigh the tumors, fix them with animal tissue fixative, embed them, section them, and perform HE staining, immunohistochemical staining, and immunofluorescence staining; another batch of mice was used to observe the survival rate, and the mice's condition was observed and mortality was recorded daily.

[0079] Experimental Results: Compared with the model group (Saline) alone, the drug-treated groups (CBD, Poly(I:C), and CP) all showed a certain inhibitory effect on the progression of B16F10 tumors, with the CP group showing a significant effect (P<0.0001). To test anti-tumor activity, B16F10 tumor tissues were stained with H&E, TUNEL, and Ki67 to evaluate the anti-proliferative and pro-apoptotic effects of CP on tumor cells. Compared with the CBD and Poly(I:C) treatment groups alone, the CP nanoparticle formulation significantly inhibited the proliferation of B16F10 tumor cells. Simultaneously, TUNEL immunofluorescence staining showed that the CP nanoparticle formulation significantly promoted the apoptosis of B16F10 tumor cells. Furthermore, mouse survival curves showed that compared with the CBD and Poly(I:C) treatment groups alone, the CP nanoparticle formulation significantly improved the survival rate of mice with B16F10 tumors. (See attached table). Figure 9 CP nanoparticle formulation inhibits B16F10 tumor growth. (A) Mouse tumor growth curve; (B) Mouse tumor weight; (C) Mouse survival curve; (D) Representative images of H&E, immunofluorescence (TUNEL), and immunohistochemistry (Ki67) staining. Data are expressed as x±s, n=5, where ** P<0.01, *** P<0.001, **** P<0.0001.

[0080] Example 4: Anti-tumor effect test of CP nanohydrogel formulation on 4T1-Luc tumors

[0081] First, an animal tumor model was established to observe the antitumor effects of CBDNPs, Poly(I:C), and CP nanoparticles alone.

[0082] Female BALB / c mice were acclimatized for one day before modeling, with 1*10 6 A mouse model of tumor was established by subcutaneous inoculation of 4 T1-Luc tumor cells into the left breast pad. The tumor volume was approximately 100 mm². 3 Mice were randomly divided into two batches of four groups of five mice each. The mice received intratumoral injections every three days for a total of five injections, with the specific grouping details as in Example 3. During the administration period, tumor size and volume were measured every three days. On days 14 and 20 after tumor inoculation, luciferase (200 μL / mouse) was administered intraperitoneally. After anesthesia, in vivo imaging was performed to observe changes in tumor size. After administration, one batch of mice was used to harvest tumors for photographing and weighing; the other batch was used to observe survival rates, with daily observation and recording of mortality.

[0083] Experimental Results: Compared with the model group (Saline) alone, the drug-treated groups (CBD, Poly(I:C), and CP) all showed a certain inhibitory effect on the progression of 4T1-Luc tumors (breast cancer), with the CP group showing a significant effect (P<0.0001). Furthermore, mouse survival curves showed that the CP nanoparticle formulation significantly improved the survival rate of mice with 4T1-Luc tumors compared with the CBD and Poly(I:C) treatment groups alone. (See attached figures). Figure 10 CP nanoparticles inhibited the growth of 4T1-Luc tumors. (A) Small animal imaging; (B) Mouse tumor growth curve; (C) In vitro mouse tumor image; (D) Mouse tumor weight; (E) Mouse survival curve. Data are expressed as x ± s, n = 5, where ** P<0.01, *** P<0.001, **** P<0.0001.

[0084] Example 5: Anti-LLC Tumor Effect Test of CP Nanohydrogel Formulation

[0085] First, an animal tumor model was established to observe the antitumor effects of CBDNPs, Poly(I:C), and CP nanoparticles alone.

[0086] Female C57BL / 6 mice were acclimatized and fed for one day before modeling, at a dose of 1*10 6A mouse model of tumor was established by subcutaneous inoculation of LLC tumor (lung cancer) cells into the left breast pad. The tumor volume was approximately 100 mm². 3 Mice were randomly divided into two batches, with four groups of five mice per batch. The mice received intratumoral injections every two days for a total of six injections, following the same grouping method as in Example 3. During the administration period, tumor size and volume were measured every two days. After administration, tumors were harvested from one batch of mice for photographing and weighing; the other batch was used to observe survival rates, with daily observation and recording of mortality.

[0087] Experimental Results: Compared with the model group (Saline) alone, all treatment groups (CBD, Poly(I:C), and CP) showed some inhibitory effect on LLC tumor progression, with the CP group showing a significant effect (P<0.0001). Furthermore, mouse survival curves showed that the CP nanoparticle formulation significantly improved the survival rate of mice with LLC tumors compared with the CBD and Poly(I:C) treatment groups alone. (See attached figures). Figure 11 CP nanoparticle formulations inhibit LLC tumor growth. (A) Image of mouse tumor in vitro; (B) Tumor growth curve in mice; (C) Tumor weight in mice; (D) Survival curve in mice. Data are presented in... This means that n = 5, where ** P<0.01, *** P<0.001, **** P<0.0001.

Claims

1. A cannabidiol thermosensitive nanohydrogel formulation comprising cannabidiol and Poly(I:C), and a thermosensitive gel matrix poloxamer, wherein the poloxamer is Pluronic F127, and the concentration of cannabidiol in the hydrogel formulation is 5-15 mg / mL, the concentration of Poly(I:C) is 0.5-1.5 mg / mL and the concentration of Pluronic F127 is 20-30 wt%.

2. A cannabidiol thermosensitive nanohydrogel injection, comprising cannabidiol 5-15 mg / mL, Poly(I:C) 0.5-1.5 mg / mL and Pluronic F127 at a concentration of 20-30 wt%.

3. The thermosensitive nanohydrogel injection solution as described in claim 2, comprising 10 mg / mL cannabidiol, 1 mg / mL Poly(I:C) and 25 wt% Pluronic F127.

4. A method for preparing the thermosensitive nanohydrogel injection solution of claim 2 or 3, comprising the following steps: 1) Dissolve cannabidiol in chloroform and add 1.0 wt% Pluronic F127 to form a mixture; 2) The mixture is converted into an O / W emulsion by stirring or ultrasonic vibration; 3) The O / W emulsion is placed in liquid nitrogen to convert it into a solid emulsion; 4) The solid emulsion was freeze-dried in a freeze dryer to obtain cannabidiol nanoparticles; 5) Cannabidiol nanoparticles are dissolved in physiological saline to obtain an aqueous solution of cannabidiol; 6) Dissolve Poly(I:C) powder in physiological saline to obtain a Poly(I:C) aqueous solution; 7) Mix the cannabidiol aqueous solution from step 5) and the Poly(I:C) aqueous solution from step 6) to obtain a nano-formulation solution; 8) Mix the nano-formulation solution with Pluronic F127 solution in equal volume ratio to obtain a thermosensitive nano-hydrogel injection solution.

5. A method for preparing a cannabidiol thermosensitive nanohydrogel injection, comprising the following steps: 1) Dissolve 20 mg of cannabidiol powder in 200 μL of chloroform; 2) Add 1.0 wt% Pluronic F127 (2 mL) and mix; 3) The mixture is converted into an O / W emulsion by stirring or ultrasonic vibration; 4) Immerse the emulsion in liquid nitrogen and freeze it rapidly into a solid emulsion; 5) Place the frozen solid emulsion in a freeze dryer and freeze dry it; 6) Wash the lyophilized product using a repeated centrifugation-redispersion cycle to obtain pure cannabidiol nanoparticles; 7) The nanoparticles were added to 500 μL of physiological saline to obtain a cannabidiol aqueous solution of 40 mg / mL; 8) Add 2 mg of Poly(I:C) powder to 500 μL of physiological saline to obtain a Poly(I:C) aqueous solution of 4 mg / mL; 9) Mix the aqueous solutions from steps 7) and 8) in equal proportions (v / v) until homogeneous, and then add an equal volume ratio of 50wt% of Pluronic F127 physiological saline solution to obtain the thermosensitive nanohydrogel injection solution.

6. The method of claim 4 or 5, wherein the cannabidiol nanoparticles have a particle size of 48.7 ± 4.7 nm.

7. Use of the cannabidiol thermosensitive nanohydrogel formulation of claim 1 or the cannabidiol thermosensitive nanohydrogel injection of any one of claims 2-3 in the preparation of a drug for treating tumors, wherein the tumor is melanoma, breast cancer or lung cancer.

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