Sting agonist liposome-thermosensitive gel, and preparation method and application thereof
By combining liposomes with thermosensitive gels, the targeting and stability issues of Sting agonist delivery methods have been resolved, achieving controlled drug release and highly effective local treatment.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
- Filing Date
- 2022-02-26
- Publication Date
- 2026-06-09
AI Technical Summary
Existing Sting agonist delivery methods lack targeting and cannot achieve stable local concentrations, which may lead to off-target inflammation or autoimmune diseases. Furthermore, it is difficult to precisely control drug release, affecting efficacy.
A drug delivery system combining Sting agonist-loaded liposomes with a thermosensitive gel was developed. The liposomes were prepared using the lipid calcium phosphate nanoparticle method and then a thermosensitive gel, such as PLGA-PEG-PLGA gel, was added to form a gel at body temperature, thereby achieving controlled drug release.
This improved the stability and local drug concentration of Sting agonists, reduced side effects, achieved precise drug control and long-term release, and improved the efficacy of tumor treatment.
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Figure CN114469864B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to clinical formulations of Sting agonists, and more particularly to Sting agonist liposome-thermosensitive gels, their preparation methods, and their application in the treatment of tumors, belonging to the field of novel Sting agonist formulations. Background Technology
[0002] Sting agonists can activate the innate immune system and show promising application prospects against high-risk tumor cells (NBs) lacking T cells in the tumor microenvironment. However, their in vivo delivery faces multiple challenges. Most Sting agonists are negatively charged hydrophilic small molecules, limiting their penetration across the plasma membrane. Structurally, Sting agonist cGAMP contains phosphodiester bonds that are easily degraded by extracellular nucleotide pyrophosphatases / phosphodiesterases, leading to rapid degradation and inactivation in vivo or metabolic clearance. Therefore, to obtain sufficient biological activity, cGAMP is usually used at relatively high concentrations. However, excessive intratumoral cGAMP may lead to overexpression of programmed death ligand 1 (PD-L1) in tumor cells and increase tumor-infiltrating regulatory T cells (Tregs), thereby negatively impacting anti-tumor immunity and severely affecting its efficacy. Currently reported Sting agonist administration methods or systems cannot guarantee a consistently stable concentration in vivo.
[0003] Currently, most Sting agonists under research are administered systemically or intratumorally, lacking targeting and failing to achieve stable local concentrations. Furthermore, they may induce off-target inflammation or autoimmune diseases. In recent years, a few studies have begun to report the use of biomaterial-based delivery strategies to control the tissue and cellular localization of Sting agonists, thereby improving their immunomodulatory activity. For example, systemically administered PEGylated lipid nanoparticles, compared to free drugs, can increase the amount of Sting agonist uptake into cells and induce the expression of pro-inflammatory genes. Locally administered cationic silica nanoparticles, poly(β-amino ester) polymer nanoparticles, and acid-sensitive acetalized dextran microparticles can all achieve immune responses tens of times higher than free drugs, demonstrating significantly superior immunotherapeutic effects. However, none of these reported delivery systems can precisely control the stable concentration of Sting agonists, requiring further improvement. Summary of the Invention
[0004] One objective of this invention is to provide a Sting agonist liposome-thermosensitive gel;
[0005] A second objective of this invention is to provide a method for preparing the Sting agonist liposome-thermosensitive gel;
[0006] A third objective of this invention is to apply the Sting agonist liposome-thermosensitive gel to the treatment of tumors.
[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:
[0008] One aspect of the present invention provides a Sting agonist liposome-thermosensitive gel, comprising: a liposome loaded with a Sting agonist and a thermosensitive gel.
[0009] The Sting agonist mentioned above includes cyclic dinucleotide Sting agonists, preferably cGAMP.
[0010] The liposomes loaded with Sting agonist cGAMP can be prepared by using conventional liposome preparation methods to obtain liposomes loaded with Sting agonist cGAMP.
[0011] The "thermosensitive gel" described in this invention refers to a drug delivery system that, when used in a solution state, forms a gel at body temperature, effectively releasing the drug and acting on the body to exert a therapeutic effect. The gel's thermosensitivity, erosive degradation, and hydrophilicity / hydrophobicity can be controlled through block composition and proportions to meet drug loading and release requirements. The gel matrix protects the integrity of liposomes, improves their stability, and is suitable for local drug delivery, thus expanding the application range of liposomes.
[0012] In a preferred embodiment of the present invention, the thermosensitive gel comprises any one or a mixture of more than one of chitosan, PNIPAM, PEG block polymer, poloxamer, or polypeptides. Preferably, the in-situ thermosensitive gel is a PEG block polymer, and more preferably, the PEG block polymer is a PLGA-PEG-PLGA gel.
[0013] Another aspect of the present invention provides a method for preparing a Sting agonist liposome-thermosensitive gel, comprising:
[0014] (I) Preparation of liposome solutions loaded with Sting agonist;
[0015] (II) Add the prepared liposome solution loaded with Sting agonist to the in situ thermosensitive gel and stir until completely dissolved to obtain the product.
[0016] The liposome solution loaded with Sting agonist described in this invention can be prepared by any one of the following methods: lipid calcium phosphate nanoparticle method, ammonium sulfate gradient method, calcium acetate gradient method, or thin film dispersion method. Through experiments, this invention has found that the encapsulation efficiency of the liposome solution loaded with Sting agonist prepared by the lipid calcium phosphate nanoparticle method is significantly better than that of other preparation methods. Therefore, this invention preferably uses the lipid calcium phosphate nanoparticle method to prepare the liposome solution loaded with Sting agonist.
[0017] As a preferred embodiment of the present invention, the method for preparing a liposome solution loaded with Sting agonist using the lipid calcium phosphate nanoparticle method includes:
[0018] Na2HPO4 aqueous phase or aqueous phase composed of CaCl2 and Sting agonist was dispersed into cyclohexane oil phase containing nonylphenol polyoxyethylene ether (Igepal) and dispersed evenly to obtain Na2HPO4 phase microemulsion and CaCl2 phase microemulsion loaded with Sting agonist, respectively. The two were mixed and demulsified and centrifuged to obtain CaP-core. Finally, DSPE-PEG2000, cationic liposome material and CaP-core were mixed evenly to obtain liposome solution loaded with Sting agonist.
[0019] As a preferred embodiment, the present invention provides a method for preparing a liposome solution loaded with Sting agonist using the lipid calcium phosphate nanoparticle method, comprising:
[0020] (1) Add Na2HPO4 aqueous solution to an oil phase composed of cyclohexane and Igepal, sonicate, and then add DOPA to obtain Na2HPO4 phase microemulsion; (2) Add an aqueous solution composed of CaCl2 and Sting agonist to an oil phase composed of cyclohexane and Igepal, sonicate to obtain CaCl2 phase microemulsion loaded with Sting agonist; (3) Add CaCl2 phase microemulsion loaded with Sting agonist to Na2HPO4 phase microemulsion, mix and stir, demulsify, centrifuge, and obtain CaP microspheres; (4) Dissolve CaP microspheres in chloroform, centrifuge, discard the calcium phosphate precipitate not coated with DOPA, and take the supernatant to obtain CaP-core; (5) Mix DSPE-PEG2000, cationic liposome material and CaP-core evenly, evaporate chloroform, and obtain liposome solution loaded with Sting agonist.
[0021] Preferably, in step (1), the volume ratio of the Na2HPO4 aqueous solution to the oil phase composed of cyclohexane and Igepal is (0.1-5):(10-30), wherein the volume ratio of cyclohexane to Igepal is preferably 2-5:1, the concentration of Na2HPO4 is preferably 10-50 mmol, and its pH value is 6-9.
[0022] Preferably, the ultrasonic conditions described in step (1) are 50-200W ultrasound for 1-5 minutes, ultrasonic treatment for 15 minutes, and then 20 mg / ml DOPA is added.
[0023] Preferably, in step (2), the volume ratio of the aqueous solution composed of CaCl2 and Sting agonist to the oil phase composed of cyclohexane and Igepal is (0.1–5):(10–30), wherein the volume ratio of cyclohexane to Igepal is 2–5:1. The concentration of the CaCl2 solution is preferably 100–1000 mmol, and the concentration of the Sting agonist is preferably 2 mg / mL.
[0024] Preferably, the stirring in step (3) is magnetic stirring; the demulsification is performed by adding ethanol; and the centrifugation is preferably performed at 10,000 to 20,000 g for 1 to 30 minutes.
[0025] Preferably, the cationic lipid material in step (5) includes, but is not limited to, any one or more of DOTAP, DOTMA, DIMRIE, DOTIM, DOGS, DOSPA, Cholesterol, DC-Chol or BGTC in any proportion. More preferably, the cationic lipid material is composed of DOTAP and Cholesterol, and the mass ratio of the two is preferably 1 to 10:1.
[0026] In a preferred embodiment, in step (II), PLGA-PEG-PLGA gel is added to the prepared liposome solution loaded with Sting agonist, and its final concentration is controlled between 5-30% (w / w). Then, the mixture is magnetically stirred at 4°C until the particles are completely dissolved.
[0027] The preparation of the Sting agonist liposome-thermosensitive gel of this invention mainly consists of two steps:
[0028] 1. Construction of liposomes carrying the Sting agonist cGAMP: Liposomes can prevent the drug from being degraded by exonucleotide pyrophosphatases / phosphodiesterases and also prevent its rapid release from the hydrophilic pores of the gel.
[0029] 2. Liposome-In-Situ Thermosensitive Gel System: This system is constructed using a thermosensitive gel with sustained-release properties. This system combines the advantages of liposomes and in-situ thermosensitive gels, increasing drug uptake into cells while avoiding burst release, reducing side effects, achieving good sustained-release and controlled-release, and improving efficacy.
[0030] This invention first prepares a pH-sensitive calcium phosphate (CaP) core with a hollow structure for targeted delivery of water-soluble drugs. An anionic phospholipid-dioleoylphosphatidylcholine (DOPA) is used as a pre-coating agent to prepare a Sting agonist delivery carrier; that is, the Sting agonist is encapsulated within the nanoscale CaP core during its formation. The lipid coating prevents the CaP core from agglomerating during centrifugation and makes it soluble in chloroform. The DOPA layer is the inner lipid layer of the CaP surface lipid bilayer. The outer lipid material is added to the chloroform solution of the CaP core, followed by rotary evaporation, film formation, and hydration to obtain the lipid bilayer encapsulating the CaP core.
[0031] This invention relates to a Sting agonist liposome-thermosensitive gel. The liposomes have small particle size and good stability, improving the cellular entry and release behavior of Sting agonists, avoiding burst release effects, and providing continuous and stable Sting agonist stimulation in vivo. This results in better drug efficacy, increased local drug concentration, reduced side effects, and improved drug efficacy. The gel matrix protects the integrity of the liposomes, improving their stability and making them suitable for local drug delivery, thus expanding the application range of liposomes. Furthermore, the materials used have good biocompatibility and a simple preparation process. The drug release performance of the in-situ thermosensitive gel can be modulated by changing the chemical structure of the materials, thereby precisely controlling the release of Sting agonists. Especially for hydrophilic drugs, combining the advantages of liposomes and gels increases drug uptake and entry into cells while achieving excellent sustained-release performance.
[0032] This invention investigated the physicochemical properties, in vitro release, and pharmacodynamics of the constructed Sting agonist liposome-thermosensitive gel through experiments. The results demonstrated that the constructed Sting agonist liposome-thermosensitive gel can achieve long-term release and exert a good therapeutic effect on tumor control.
[0033] Abbreviations involved in this invention
[0034] DOPA: Anionic phospholipid - dioleoyl phospholipid.
[0035] DOTAP: (2,3-dioleoxypropyl)trimethylammonium chloride.
[0036] DOTMA: Trimethyl-2,3-diolenopropylammonium chloride.
[0037] DMRIE: Dimethyl-2-hydroxyethyl-2,3-bistetradecoxypropylammonium bromide.
[0038] DOTIM: 1-[2-(oleoyloxy)ethyl]-2-oleoyl-3-(2-hydroxyethyl)imidazoline ammonium chloride.
[0039] DOGS: N-(2-Spermineformyl)-N',N'-bisoctadecylglycineamide.
[0040] DOSPA: Dimethyl-2,3-dioleenooxypropyl-2-(2-sperminecarbamoylamino)ethylammonium trifluoroacetate.
[0041] DC-Chol: 3β-[N-(N',N'-dimethylaminoethyl)aminoformyl]cholesterol.
[0042] BGTC: Biguanide-ethylamine-cholesterol.
[0043] PNIPAM: Poly(N-isopropylacrylamide). Attached Figure Description
[0044] Figure 1 Electron micrograph of the Sting agonist liposome of the present invention.
[0045] Figure 2 Electron micrograph of the Sting agonist liposome of the present invention.
[0046] Figure 3 Particle size distribution of the Sting agonist liposomes of the present invention.
[0047] Figure 4 Release curve of the Sting agonist liposome-thermosensitive gel of the present invention.
[0048] Figure 5 The present invention relates to tumor volume-time changes in the treatment of tumors with Sting agonist liposome-thermosensitive gel.
[0049] Figure 6 The Sting agonist liposome-thermosensitive gel of the present invention treats tumor weight changes over time. Detailed Implementation
[0050] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.
[0051] Preliminary Example 1: Preparation of Sting Agonist Liposomes
[0052] I. Preparation of Sting agonist liposomes using the lipid calcium phosphate nanoparticle method
[0053] (I) CaP-core preparation
[0054] 1. Preparation of Na2HPO4 phase microemulsion
[0055] Oil phase: 10-30 ml cyclohexane / Igepal = 2-5:1;
[0056] Aqueous phase: 0.1–5 ml of 10–50 mmol Na₂HPO₄, pH = 6–9;
[0057] Add 100–300 μL of aqueous phase to the oil phase and sonicate at 50–200 W for 1–5 mins;
[0058] Add 100-300 μL of 20 mg / ml DOPA after 15 minutes;
[0059] 2. Preparation of CaCl2 phase microemulsion
[0060] Oil phase: 10-30 ml cyclohexane / Igepal = 2-5:1;
[0061] Aqueous phase: 0.1–5 ml 100–1000 mmol CaCl2;
[0062] 10–300 μL 2 mg / mL cGAMP;
[0063] Add 100–300 μL of aqueous phase to the oil phase and sonicate at 50–200 W for 1–5 mins;
[0064] 3. Add the CaCl2 phase microemulsion prepared in step 2 to the Na2HPO4 phase microemulsion prepared in step 1, mix for 10-40 mins, and stir at 100-800 rpm on a magnetic stirrer;
[0065] 4. Add 10-100 ml of anhydrous ethanol to break the emulsion, centrifuge (10000-20000 g, 1-30 mins), and wash with ethanol 2-3 times;
[0066] 5. Dissolve the obtained CaP microspheres in 1 ml of chloroform, centrifuge at -1000 to 5000 g for 5 mins, discard the calcium phosphate precipitate that is not coated with DOPA, and store the supernatant at 4℃.
[0067] (II) Preparation of CaP-liposome
[0068] 1. Preparation of chloroform solution for resin materials
[0069] 0.01~5ml DSPE-PEG2000 1~10mmol
[0070] 0.01–5 ml DOTAP / Cholesterol (1–10:1) 1–30 mmol (including cationic lipids including but not limited to DOTAP, DOTMA, DIMRIE and DOTIM, multivalent cationic lipids DOGS and DOSPA, and cationic cholesterol derivatives DC-Chol and BGTC)
[0071] 0.01~5ml CaP-core
[0072] 2. Evaporate chloroform
[0073] Add 0.1–5 ml of 5 mmol Tris-HCl Buffer (pH = 7.4) and sonicate (10–200 W, 1–10 mins) until pale blue or clear.
[0074] II. Preparation of Sting agonist liposomes by thin-film dispersion method
[0075] Accurately weigh the prescribed amounts (Table 1) of DOTAP, cholesterol (Chol), DSPE-MPEG2000, and DOPE into a round-bottom flask, and add an appropriate amount (approximately 30 mL) of chloroform to dissolve them completely. Remove the solvent by rotary evaporation under reduced pressure in a 37°C water bath until a uniform thin film is formed (approximately 30 min). Add 100-300 μL of an aqueous solution of cGAMP (1-3 mg cGAMP dissolved in 1-10 mL of pure water) and vortex. Add the drug to a 70°C water bath for 1 h (with the round-bottom flask cap on). Sonicate the liposome suspension using an ultrasonic homogenizer until it exhibits a pale blue opalescence. Remove free cGAMP by passing through a Sephadex G-50 gel column to obtain liposomes, which are then poured into vials and stored at 4°C.
[0076] Table 1. Component dosages for preparing Sting agonist liposomes by different methods
[0077] Preparation method DOTAP (mg) HSPC (mg) Chol (mg) <![CDATA[DSPE-MPEG 2000 (mg)]]> DOPE (mg) Thin film dispersion 10-25 1-5 1-5 1-10 Calcium acetate gradient method 10-25 10-25 1-5 1-5 Ammonium sulfate gradient method 10-25 10-25 1-5 1-5
[0078] III. Preparation of Sting agonist liposomes using the calcium acetate gradient method
[0079] 1. Accurately weigh the prescribed amounts (Table 1) of HSPC, DSPE-PEG2000, DOTAP, and cholesterol into a round-bottom flask, and add an appropriate amount (approximately 30 mL) of chloroform to dissolve them completely. Remove the solvent by rotary evaporation under reduced pressure in a 37°C water bath until a uniform thin film is formed (approximately 30 min). Add 1 mL of 130 mmol·L⁻¹ calcium acetate hydration solution (adjust pH to 6.0 with glacial acetic acid), and vortex. Hydrate in a 70°C water bath for 15 min (with the round-bottom flask cap on). Use an ultrasonic homogenizer (100%) to sonicate the liposome suspension until it exhibits a pale blue opalescent appearance.
[0080] 2. After cooling to room temperature, remove free calcium acetate by passing the solution through a Sephadex G-50 gel column to obtain blank liposomes. Add 100-300 μL of cGAMP aqueous solution (1-3 mg cGAMP dissolved in 1-10 mL of pure water); load the solution in a 70°C water bath for 1 h. After cooling to room temperature, remove free cGAMP by passing the solution through a Sephadex G-50 gel column to obtain the liposome solution.
[0081] IV. Preparation of Sting agonist liposomes using the ammonium sulfate gradient method
[0082] 1. Accurately weigh the prescribed amounts (Table 1) of HSPC, DSPE-PEG2000, DOTAP, and cholesterol into a round-bottom flask, and add an appropriate amount (approximately 30 mL) of chloroform to dissolve them completely. Remove the solvent by rotary evaporation under reduced pressure in a 37°C water bath until a uniform thin film is formed (approximately 30 min). Add 1 mL of 120 mmol·L⁻¹ ammonium sulfate aqueous solution and vortex. Hydrate in a 70°C water bath for 15 min (with the round-bottom flask cap on). Use an ultrasonic homogenizer (100%) to sonicate the liposome suspension until it exhibits a pale blue opalescent appearance.
[0083] 2. After cooling to room temperature, remove free ammonium sulfate by passing the solution through a Sephadex G-50 gel column to obtain blank liposomes. Add 100-300 μL of cGAMP aqueous solution (1-3 mg cGAMP dissolved in 1-10 mL of pure water); load the solution in a 70°C water bath for 1 h. After cooling to room temperature, remove free cGAMP by passing the solution through a Sephadex G-50 gel column to obtain the liposome solution.
[0084] Triton was added to the Sting agonist-containing liposomes prepared by the above four methods to induce membrane disruption (for liposomes prepared using the lipid calcium phosphate nanoparticle method, hydrochloric acid was added to disrupt the calcium phosphate, releasing the Sting agonist). The encapsulation efficiency was measured by HPLC.
[0085] Accurately measure 1 mL of liposomes into a centrifuge tube, add Triton vortex to rupture the membrane, and determine by high performance liquid chromatography (HPLC).
[0086] Encapsulation rate % = (Amount of drug encapsulated in the system / Amount of drug administered) × 100%
[0087] Comparison of encapsulation efficiency: lipid calcium phosphate nano-method > ammonium sulfate gradient method > calcium acetate gradient method > thin film dispersion method.
[0088] According to the encapsulation efficiency test results, among the four preparation methods mentioned above, the lipid calcium phosphate nano-method has the highest encapsulation efficiency for Sting agonist.
[0089] Example 1: Preparation of Sting agonist liposome-thermosensitive gel
[0090] Liposome thermosensitive gel was prepared using a cold-melting method:
[0091] Add an appropriate amount of PLGA-PEG-PLGA gel to the freshly prepared liposome solution (liposomes containing Sting agonist prepared by lipid calcium phosphate nanoparticle method in Preliminary Example 1), control its final concentration between 5-30% (w / w), and then magnetically stir at 4°C until the particles are completely dissolved.
[0092] Blank gels were also prepared using a similar method, namely, adding the gel to deionized water and stirring in an ice bath until a transparent solution was formed.
[0093] Example 2: Preparation of Sting agonist liposome-thermosensitive gel
[0094] Liposome thermosensitive gel was prepared by cold dissolution method: an appropriate amount of chitosan was added to freshly prepared liposome solution (liposomes containing Sting agonist prepared by ammonium sulfate gradient method in preliminary example 1), and the final concentration was controlled between 5-30% (w / w). Then, the solution was magnetically stirred at 4°C until the particles were completely dissolved.
[0095] Example 3: Preparation of Sting agonist liposome-thermosensitive gel
[0096] Liposome thermosensitive gel was prepared by cold dissolution method: an appropriate amount of poloxamer was added to freshly prepared liposome solution (liposomes containing Sting agonist prepared by calcium acetate gradient method in preliminary example 1), and the final concentration was controlled between 5-30% (w / w). Then, the solution was magnetically stirred at 4°C until the particles were completely dissolved.
[0097] Example 4: Preparation of Sting agonist liposome-thermosensitive gel
[0098] Liposome thermosensitive gel was prepared by cold dissolution method: an appropriate amount of poloxamer was added to freshly prepared liposome solution (liposomes containing Sting agonist prepared by thin film dispersion method in preliminary example 1), and its final concentration was controlled between 5-30% (w / w). Then, the solution was magnetically stirred at 4°C until the particles were completely dissolved.
[0099] Experimental Example 1: Screening Experiment for Phase Transition Temperature Control Based on Block Composition and Ratio of PEG Block Polymers
[0100] The thermosensitivity, erosion and degradation properties, and hydrophilicity / hydrophobicity of the gel can be controlled by adjusting the block composition and ratio of the PEG block polymer according to the needs of drug loading and release.
[0101] This invention screened the block composition and ratio of PEG block polymers, and the relationship between the block composition and ratio and the phase transition temperature is shown in Table 2.
[0102] Table 2. Composition and proportion of PEG block polymers and their phase transition temperatures
[0103]
[0104] Example 2: Physicochemical characterization of Sting agonist liposome-thermosensitive gel
[0105] 1. Test material: Sting agonist liposome-thermosensitive gel prepared in Example 1.
[0106] 2. Experimental methods and results
[0107] (1) Transmission electron microscopy (TEM) images of liposomes: TEM results show that the liposomes are spherical, and the particle size is similar to that measured by DLS. TEM results are shown below. Figure 1 and Figure 2 .
[0108] (2) The liposome thermosensitive gel is a flowing liquid below the phase transition temperature and a solid at 37°C.
[0109] (3) Liposome particle size distribution: The average particle size of SAL measured by dynamic light scattering (DLS) was approximately 186 nm, with a PDI of 0.188. Transmission electron microscopy results showed that the liposomes were spherical, and the particle size results were similar to those obtained by DLS. Figure 3 ).
[0110] (4) In vitro release: In vitro drug release from liposomes and liposome-thermosensitive gels was studied using the dialysis bag method. Dialysis bags with a molecular weight cutoff of 12000-14000 Da were pre-swollen by soaking in deionized water overnight. Before the release experiment, different formulations containing 40 μg cGAMP were added to the dialysis bags, sealed with dialysis clamps, and equilibrated at 37°C for 1-20 minutes. The bags were then immersed in 1-100 mL of PBS release medium and shaken at 40 rpm in an air bath shaking incubator. At preset time points, all release media were removed and replenished with an equal volume of release medium. The cGAMP concentration was determined by HPLC, and the release curves are shown in [Figure number missing]. Figure 4 .
[0111] Experimental Example 3: Pharmacodynamic Evaluation of Sting Agonist Liposome-Thermosensitive Gel
[0112] 1. Establishment of a mouse model of neuroblastoma: The mouse neuroblastoma Neuro2a cell line was cultured in DMEM (high glucose) medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. After adherence, the cells were washed with PBS during the logarithmic growth phase, then digested with trypsin containing 0.25% EDTA. After terminating digestion, the cells were centrifuged at 1000 rpm for 5 min, resuspended in basal medium, and the cell concentration was adjusted to 1.0 × 10⁶ cells / mL. 6 50 μL was injected into A / J mice, and 50 μL was injected into the axillary region of the chest.
[0113] 2. Dosage regimen: When the tumor volume reaches 50-100 mm 3 Mice were randomly divided into two groups, and injected intratumorally with either 100 μL of Sting Agonist (SA) or 100 μL of the Sting agonist liposome-thermosensitive gel (SAL-Gel) prepared in Example 1, with a dosage equivalent to 30 μg / mouse. Tumor volume was monitored, the survival status of the mice was observed, and any obvious toxic reactions at the injection site were noted. A tumor volume-time graph was plotted. Simultaneously, body weight was measured, and a curve showing body weight changes over time was plotted.
[0114] Tumor volume-time changes are shown in Figure 5 Weight changes over time are shown in the figure. Figure 6 .
[0115] The experimental results show that by combining liposomes and thermosensitive gels, the uptake of Sting agonists by cells is increased while burst release is avoided, achieving good sustained-release and significantly improving the therapeutic effect on tumors.
Claims
1. A Sting agonist liposome-thermosensitive gel, characterized in that, include: Liposomes and thermosensitive gels loaded with Sting agonist; Its preparation methods include: (I) Preparation of liposome solutions loaded with Sting agonist; (II) Add thermosensitive gel to the prepared liposome solution loaded with Sting agonist and stir until completely dissolved to obtain the product; The Sting agonist-loaded liposome solution was prepared using the lipid calcium phosphate nanoparticle method. The method for preparing the Sting agonist-loaded liposome solution using the lipid calcium phosphate nanoparticle method includes: (1) adding an aqueous solution of Na2HPO4 to an oil phase composed of cyclohexane and nonylphenol polyoxyethylene ether Igepal, ultrasonically treating the mixture, and then adding DOPA to obtain a Na2HPO4 phase microemulsion; (2) adding an aqueous solution composed of CaCl2 and the Sting agonist to an oil phase composed of cyclohexane and nonylphenol polyoxyethylene ether Igepal. (3) The CaCl2 phase microemulsion loaded with Sting agonist was obtained by ultrasonic treatment in the phase; (4) The CaCl2 phase microemulsion loaded with Sting agonist was added to the Na2HPO4 phase microemulsion, mixed and stirred, and then demulsified and centrifuged to obtain CaP microspheres; (5) The CaP microspheres were dissolved in chloroform, centrifuged, and the uncoated calcium phosphate precipitate was discarded. The supernatant was taken to obtain CaP-core; (6) DSPE-PEG2000, cationic lipid material and CaP-core were mixed evenly, and the chloroform was evaporated to obtain the liposome solution loaded with Sting agonist. The thermosensitive gel is a PLGA-PEG-PLGA gel.
2. The Sting agonist liposome-thermosensitive gel according to claim 1, characterized in that, In step (1), the volume ratio of the Na2HPO4 aqueous solution to the oil phase composed of cyclohexane and nonylphenol polyoxyethylene ether Igepal is (0.1-5):(10-30), wherein the volume ratio of cyclohexane to nonylphenol polyoxyethylene ether Igepal is 2-5:1, the concentration of Na2HPO4 is 10-50 mmol, and its pH value is 6-9. The ultrasound conditions described in step (1) are 50-200W ultrasound for 1-5 minutes, followed by 20mg / ml DOPA after 15 minutes of ultrasound treatment; In step (2), the volume ratio of the aqueous solution composed of CaCl2 and Sting agonist to the oil phase composed of cyclohexane and nonylphenol polyoxyethylene ether Igepal is (0.1-5):(10-30), wherein the volume ratio of cyclohexane to nonylphenol polyoxyethylene ether Igepal is 2-5:1; the concentration of the CaCl2 solution is 100-1000 mmol, and the concentration of the Sting agonist is 2 mg / mL. The stirring in step (3) is done by magnetic stirring; the demulsification is done by adding ethanol; the centrifugation is done at 10,000 to 20,000 g for 1 to 30 minutes. The cationic lipid material mentioned in step (5) includes, but is not limited to, any one or more of DOTAP, DOTMA, DIMRIE, DOTIM, DOGS, DOSPA, DC-Chol or BGTC in any proportion.
3. The Sting agonist liposome-thermosensitive gel according to claim 1, characterized in that, The cationic lipid material is composed of DOTAP and Cholesterol in a mass ratio of 1 to 10:
1.
4. The Sting agonist liposome-thermosensitive gel according to claim 1, characterized in that, In step (II), PLGA-PEG-PLGA gel is added to the prepared liposome solution loaded with Sting agonist, and its final concentration is controlled between 5-30 wt%.
5. Use of the Sting agonist liposome-thermosensitive gel according to any one of claims 1-4 in the preparation of a medicament for treating tumors, wherein the tumor is neuroblastoma.