CN111888475B

CN111888475B - Sustained-release preparation, preparation method and application thereof in preparing in-situ tumor immune combined treatment medicine

Info

Publication number: CN111888475B
Application number: CN202010725637.2A
Authority: CN
Inventors: 张志平; 胡梅; 张娇
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2022-01-07
Anticipated expiration: 2040-07-24
Also published as: CN111888475A

Abstract

The invention discloses a sustained and controlled release preparation, a preparation method thereof and application thereof in preparing in-situ tumor immune combination treatment medicines, relating to the technical field of biomedical nano materials. The sustained and controlled release preparation takes liquid crystal gel as a carrier, and the liquid crystal gel is loaded with an immunogenicity inducer and an immunomodulator. The sustained and controlled release preparation can be prepared into a liquid crystal precursor by adopting a simple stirring and mixing method, and then an immunogenicity inducer and an immunomodulator are loaded. The invention co-loads the immunogenicity inducer and the immunomodulator in the in-situ slow-release liquid crystal gel, can form a semisolid gel in situ after intratumoral, peritumoral or postoperative intratumoral administration, slowly and slowly release the medicine, effectively regulates the tumor microenvironment, plays a role in inhibiting the growth, recurrence and metastasis of the tumor, simultaneously reduces the systemic toxic and side effects caused by the traditional administration way, and has potential application prospect in the in-situ local treatment of the tumor.

Description

Sustained-release preparation, preparation method and application thereof in preparing in-situ tumor immune combined treatment medicine

Technical Field

The invention relates to the technical field of biomedical nano materials, in particular to a sustained-release preparation, a preparation method and application thereof in preparing in-situ tumor immune combination treatment medicines.

Background

Cancer has long been one of the global public health threatening diseases and is also one of the public problems that seriously threaten the health of the people in China. Common cancer therapies include chemotherapy, radiation therapy, surgical resection and immunotherapy, with surgical resection being the predominant treatment for solid tumors at present. However, tumor recurrence and metastasis from post-operative residual tumors and circulating tumor cells or from an immunosuppressive microenvironment remains a major cause of patient death.

Cancer immunotherapy has proven to be a promising therapeutic approach that can activate the host's immune system, selectively eliminate residual tumors and circulating tumor cells, and achieve a long-lasting anti-tumor immune response. Although immunotherapy with chimeric antigen receptor-T (CAR-T), immune checkpoint Inhibitors (ICB), tumor vaccines, and cytokines have met with some clinical success, only a small fraction of patients can develop a sustained immune response with a single immunotherapy. In addition, treatment-related side effects such as cytokine storm, potentially immunosuppressive microenvironment and ineffective activation of the immune system still prevent widespread use of immunotherapy.

Recently, many studies have shown that cancer therapies such as chemotherapy, radiation, photodynamic therapy, photothermal therapy, etc. can cause immunogenic death (ICD) of tumor cells, and this immunogenic apoptotic effect promotes the release of tumor-associated antigens, risk-associated molecular patterns, and pro-inflammatory cytokines, which contribute to the processing and presentation of tumor-associated antigens by antigen-presenting cells, thereby eliciting innate and adaptive immune responses. Wherein, the immunogenic apoptosis inducing cells generate the tumor neoantigen as an in situ vaccine without complicated and complicated identification and separation steps. Nevertheless, during wound healing, the body induces the release of Vascular Endothelial Growth Factor (VEGF), PDGF, prostaglandin, blood coagulation factors and complement in large quantities, which in turn leads to the accumulation of inflammatory cells such as tumor-associated macrophages of type M2 (TAM), myeloid-derived suppressor cells (MDSCs), regulatory T cells (tregs) and like immunosuppressive microenvironments that form an immunosuppressive microenvironment by releasing immunomodulatory cytokines such as transforming growth factor β (TGF- β), IL-10, cyclooxygenase-2 (COX-2) and like in large quantities or promoting the expression of checkpoint inhibitory molecules. This immunosuppressive microenvironment not only promotes tumor growth, accelerating escape of tumor cells also leads to immune tolerance, but also immune responses elicited solely by immunogenic inducers are generally too weak to reverse the tumor-mediated immunosuppressive microenvironment. Therefore, to achieve better clinical results, it is very important to regulate the postoperative tumor microenvironment.

Combining an immunogenicity inducing agent with an immunomodulator is thought to generate a stronger anti-tumor immune response and reverse the tumor immunosuppressive microenvironment. However, the safety of the combination therapy is still the focus of clinical trials, and some combination therapy schemes may further increase the toxic and side effects of the drugs on the body. The traditional infusion route often generates huge toxic and side effects due to poor drug targeting, so that the drug is difficult to play a role in local tumor.

Disclosure of Invention

The invention solves the technical problems of tumor recurrence and metastasis caused by postoperative residual tumor and circulating tumor cells or immunosuppressive microenvironment in the prior art, provides a sustained-release preparation, a preparation method and application for preparing an in-situ postoperative tumor immune medicament, can be used for sustained-release administration of in-situ postoperative tumor immune combination treatment, can slowly and continuously release the medicament in vivo, effectively regulates postoperative tumor microenvironment and inhibits tumor recurrence and metastasis.

According to a first aspect of the present invention, there is provided a sustained-release preparation, which uses a liquid crystal gel as a carrier, wherein the liquid crystal gel is loaded with an immunogenicity inducer and an immunomodulator.

Preferably, the liquid crystal gel includes 30-60 parts by mass of phospholipid, 30-60 parts by mass of glyceride, and 10-20 parts by mass of organic solvent.

Preferably, the phospholipid is soybean phospholipid and/or egg yolk lecithin; the glyceride is at least one of dehydrated sorbitol monooleate, glyceryl dioleate, glyceryl oleate ether, phytic glyceride and plant triol.

Preferably, the liquid crystal gel further comprises at least one of tocopherol, tocopherol acetate and tricaprylate.

Preferably, the immunogenicity-inducing agent is a chemotherapeutic drug, or a mixture of a chemotherapeutic drug and a photosensitizer; the immunomodulator is Toll-like receptor agonist, STING pathway agonist, immunodetection point inhibitor or cytokine.

Preferably, the chemotherapeutic drug is doxorubicin, paclitaxel, oxaliplatin or gemcitabine; the photosensitizer is indocyanine green, chlorin, gold nanoparticles or black phosphorus nanoparticles; the Toll-like receptor agonist is TLR3, TLR4, TLR7/8 or TLR 9; the STING pathway agonist is C-di-GMP; the immune checkpoint inhibitor is a CTLA4 antibody, a PD1 antibody, or a PD-L1 antibody; the cytokine is IL-10 or GM-CSF.

According to another aspect of the present invention, there is provided a method for preparing any one of the sustained-release preparations, comprising the steps of:

(1) mixing phospholipid, glyceride and an organic solvent, and fully dissolving to obtain a liquid crystal precursor;

(2) and (2) adding an immunogenicity inducer and an immunomodulator into the liquid crystal precursor obtained in the step (1), and fully dissolving to obtain a clear, transparent and uniform drug-loaded sustained-release preparation.

Preferably, the mass parts of the phospholipid, the glyceride and the organic solvent are respectively 30-60 parts, 30-60 parts and 10-20 parts;

preferably, the phospholipid is soybean phospholipid and/or egg yolk lecithin; the glyceride is at least one of dehydrated sorbitol monooleate, glyceryl dioleate, glyceryl oleate ether, phytic glyceride and plant triol; the liquid crystal gel precursor also comprises at least one of tocopherol, tocopherol acetate and tricaprylate.

Preferably, the mixing in step (1) is heating mixing, or stirring mixing on a magnetic stirrer; in the step (2), the immunogenicity inducer and the immunomodulator are firstly prepared into stock solution in a solvent and then added into the liquid crystal precursor.

According to another aspect of the invention, there is provided the use of any one of the sustained or controlled release formulations for the manufacture of a medicament for the in situ tumour immunotherapy combination.

Generally, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:

(1) the invention creatively loads the immunogenicity inducer and the immunomodulator in the in-situ slow-release liquid crystal gel, can form the semisolid gel in situ after the administration in tumor, tumor periphery or postoperative tumor cavity, slowly controls the release of the medicine, effectively regulates the tumor microenvironment, plays a role in inhibiting the growth, recurrence and metastasis of the tumor, simultaneously reduces the systemic toxic and side effects caused by the traditional administration way, and has potential application prospect in the in-situ local treatment of the tumor.

(2) The invention has high maximum drug loading, good drug loading for drug molecules with different physicochemical properties, stable sustained release effect, easy large-scale production, simple preparation, good injectability and high patient compliance.

(3) The in-situ gel in the invention is a preparation which can generate phase transition immediately at the application site after being administrated in a solution state and can be transformed from a liquid state to a semisolid gel. The sustained and controlled release preparation has injectability, and can immediately generate phase transition to form semisolid gel after meeting water solution. The gel has good histocompatibility, and long retention time at administration position, and can form drug reservoir for controllably releasing drug. Therefore, the immune drug and the immunogenicity inducer are co-loaded in the in situ gel, so that the tumor immune microenvironment is effectively adjusted to enhance the cancer immunotherapy.

(4) The sustained-release liquid crystal gel medicinal preparation can be used for administration around tumor, in tumor or in postoperative tumor cavity, immediately forms gel in vivo after in situ administration, slowly releases the medicament, is used for improving tumor microenvironment, inhibiting tumor recurrence and metastasis, simultaneously reduces toxic and side effects on normal tissues, not only improves the local medicinal effect, but also reduces the toxic and side effects on other tissues and organs.

(5) The phospholipid material selected by the slow-release liquid crystal gel is preferably soybean phospholipid and yolk lecithin, and in order to ensure that clear, uniform and transparent solution can be formed, the soybean phospholipid and the yolk lecithin are phospholipids with lower phase transition temperature and have better stability.

(6) After the medicine is administrated, the liquid crystal precursor is immediately converted into semisolid gel and continuously releases the medicine in a controllable manner, wherein the released chemotherapeutic medicine or photosensitizer can convert residual tumor or recurrent tumor into tumor antigen to form in-situ vaccine, and further combines the immune stimulation effect of the immunomodulator, so that the tumor microenvironment is improved, antigen specific immune reaction is generated, the growth, recurrence and metastasis of the tumor are effectively inhibited, and the effect of in-situ tumor immune combined treatment is achieved. The preparation can effectively entrap drug molecules (small-molecule drugs to large-molecule protein drugs) with different physicochemical properties, has biodegradability and biocompatibility, simple and convenient preparation steps, is suitable for large-scale production, and has obvious sustained and controlled release effects and low toxicity.

(7) The present invention preferably depends on the phospholipid type, cost considerations, viscosity requirements prior to injection, sustained release period and desired release behavior, generally speaking, the higher the amount of phospholipid used, the greater the viscosity prior to injection and the longer the release period. Therefore, the ratio of the phospholipid, the amphiphilic liquid crystal forming material and the solvent can be adjusted to an optimum state according to specific requirements, wherein the mass parts of the phospholipid is 30-60 parts, and the mass parts of the glycerol ester is 30-60 parts.

(8) Preferably, the sustained-release preparation further comprises tocopherol, tocopherol acetate or tricaprylate, which can increase the curvature of a bicontinuous layer inside the liquid crystal, facilitate the formation of cubic phase or hexagonal phase liquid crystal gel, and prevent the oxidation of phospholipid.

(9) According to the invention, preferably, after the sustained-release preparation is added with the photosensitizer, the liquid crystal gel has heat sensitivity, and the conversion from a gel state to a sol state can be realized through near-infrared light irradiation, so that the local light-controlled-release immune medicine is realized. Specifically, the gel state is obtained at a temperature of 40 ℃ or lower, and the gel state is converted into a sol state at 40 to 50 ℃.

Drawings

FIGS. 1-2 are photographs showing the behavior of the gel formulation of the present invention, wherein FIG. 1 is a liquid crystal precursor solution (left), and the liquid crystal precursor forms a non-flowable gel after contacting with PBS buffer (right). FIG. 2 shows the formation of a spherical gel after injection of a liquid crystal precursor into PBS via a syringe.

Fig. 3 and fig. 4 are the rheological property and the polarization optical property of the sustained-release liquid crystal gel before and after gelation, respectively.

FIG. 5 shows the in vitro photothermal conversion efficiency of the sustained release liquid crystal gel of the present invention.

FIG. 6 shows the drug release of the sustained-release liquid crystal gel in tumor-bearing mice.

Fig. 7-9 are B16F10 postoperative mouse survival analysis and weight change graphs, wherein fig. 7 is mouse postoperative tumor recurrence rate, fig. 8 is tumor postoperative mouse survival, and fig. 9 is mouse postoperative weight change.

FIGS. 10-12 show the regulation of the post-operative tumor microenvironment by the sustained-release liquid crystal gel of the present invention, wherein FIG. 10 shows the number of CD3+ CD8+ T cells in the tumor tissue, FIG. 11 shows the ratio of macrophage type 1 cells to macrophage type 2 cells in the mouse tumor tissue, and FIG. 12 shows the ratio of MDSCs in the mouse tumor tissue.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1

Weighing the formula according to the table 1, putting soybean lecithin (SPC100), Sorbitan Monooleate (SMO), Tocopherol Acetate (TA) and absolute ethyl alcohol with different mass ratios into a 10mL EP tube (wherein the mass part of the TA is controlled to be 10), sealing with a sealing glue, then placing on a multipoint magnetic stirrer, stirring at 1000rpm/min overnight, and then placing in an ultrasonic instrument to remove bubbles by ultrasound to obtain a clear and transparent pale yellow liquid crystal precursor solution. And taking off the needle part of the 1ml syringe, sucking 0.1ml of the liquid crystal precursor, then returning the needle of the syringe to inject the liquid crystal precursor into the PBS buffer solution, and observing whether the liquid crystal precursor can be injected successfully or not and simultaneously observing whether semisolid gel can be formed or not and recording the gel time of the semisolid gel.

As shown in Table 1, it was found that clear and transparent liquid crystal precursors are more easily formed and the fluidity is better as the amount of absolute ethyl alcohol increases, but the amount of organic solvents increases to easily stimulate the body, and thus 10% absolute ethyl alcohol is preferable as the solvent. In addition, when the ethanol content is 10%, the higher the content of the phospholipid is, the more difficult the formation of a clear and transparent liquid crystal precursor is, and on the contrary, the lower the content of the phospholipid is, the longer the gelling time of the liquid crystal precursor is, so that the liquid crystal precursor is not favorable for forming a semisolid gel in vivo immediately, and therefore, the mass parts of the phospholipid, the mass parts of the glyceride and the solvent are adjusted to be 30-60 parts and 10-20 parts respectively.

TABLE 1 examination of the viscosity behavior and the gelling behavior of liquid crystal precursors of different compositions

Example 2

On the basis of example 1, a blank liquid crystal precursor was prepared by weighing a recipe of 45 parts by mass of SPC100, 35 parts by mass of SMO, 10 parts by mass of TA, and 10 parts by mass of Ethanol, and after a clear liquid crystal precursor solution was formed, a portion of the solution was taken and added to a blank ampoule, and the mobility of the liquid crystal precursor was observed by tilting the ampoule. Then, a certain amount of PBS buffer was added, and after 10min, the bottom of the ampoule was again observed by tilting the ampoule to observe the fluidity of the liquid crystal precursor, as shown in FIG. 1, and after 10min, a semi-solid gel was formed. In addition, the 1ml syringe needle was removed, 0.1ml of the liquid crystal precursor was aspirated, and the syringe needle was returned to inject the liquid crystal precursor into the PBS buffer, resulting in the formation of a gel immediately after the gel precursor meets the aqueous medium as shown in FIG. 2.

Example 3

And further weighing 45 parts by mass of SPC100, 45 parts by mass of GDO and 10 parts by mass of ethanol in a 10mL EP tube, sealing with a sealing compound, placing the tube on a multipoint magnetic stirrer, stirring at 1000rpm/min overnight, and placing the tube in an ultrasonic instrument for removing bubbles by ultrasonic waves to obtain a clear and transparent pale yellow liquid crystal precursor solution. And taking down the needle part of the 1ml syringe, sucking 0.1ml of the liquid crystal precursor, returning the needle of the syringe, and injecting the liquid crystal precursor into PBS buffer solution, wherein the gel precursor forms gel immediately when meeting water.

Example 4

And further weighing 45 parts by mass of SPC100, 45 parts by mass of GMO and 10 parts by mass of ethanol in a 10mL EP tube, sealing with sealing glue, placing the tube on a multi-point magnetic stirrer, stirring at 1000rpm/min overnight, and placing the tube in an ultrasonic instrument for removing bubbles by ultrasonic waves to obtain a clear and transparent pale yellow liquid crystal precursor solution. And taking down the needle part of the 1ml syringe, sucking 0.1ml of the liquid crystal precursor, returning the needle of the syringe, and injecting the liquid crystal precursor into PBS buffer solution, wherein the gel precursor forms gel immediately when meeting water.

Example 5

A blank liquid crystal gel was prepared according to the method of example 2, and 1ml of a lyotropic liquid crystal precursor was injected into a PBS buffer solution, and after 5 hours of equilibration, the rheological behavior was tested using a variable pitch rheometer. Rheological conditions: the temperature was 25 ℃, the plate spacing 0.5mm, the rotor 40mm, and the test was started 5min after the sample was stabilized. The frequency was 1Hz and the shear strain was from 0.1% to 100%, and the region where the corresponding storage modulus G ', G' did not change with shear strain was recorded as the Linear Viscoelastic Region (LVR). Then, selecting proper strain and stress in the linear viscoelastic region, measuring the viscosity and vibration modulus of the fluid, and measuring the frequency sweep range from 0.1Hz to 10Hz, and the result is shown in FIG. 3. FIG. 3 shows that the storage modulus and the loss modulus of the liquid crystal precursor have no obvious difference along with the change of the frequency, which indicates that the lyotropic liquid crystal precursor is still in a fluid state. After the liquid crystal precursor is added into the aqueous medium PBS, the difference between the storage modulus and the loss modulus of the liquid crystal precursor is rapidly increased along with the change of the frequency, and the storage modulus is always remarkably higher than the loss modulus, which indicates that the lyotropic liquid crystal exists in a gel state. The liquid crystal precursor is evenly laid on a glass slide, PBS buffer solution is dripped to completely cover the lyotropic liquid crystal precursor, the mixture is kept stand for 5min, a cover glass is covered, and the birefringence phenomenon is observed by a polarized light microscope, so that as shown in figure 4, the mesophase of the lyotropic liquid crystal has an obvious polygonal structure which is a specific structure of the reversed hexagonal phase lyotropic liquid crystal, and the liquid crystal gel is the reversed hexagonal phase liquid crystal gel.

Example 6

A blank liquid crystal gel was prepared according to the method of example 2, R848 or R837 or STING agonist stock solutions were prepared in DMSO, and 1mg of each was added to the blank liquid crystal precursor, and the blank liquid crystal precursor was stirred at room temperature on a multi-point magnetic stirrer at 1000rpm/min and then ultrasonically debubbled, R848@ LCFS, R837@ LCFS, C-di-GMP @ LCFS, respectively.

Example 7

A blank liquid crystal gel was prepared according to the method of example 2, and DOX HCl and indocyanine green (ICG) were prepared in DMSO at concentrations of 0.2 mg/. mu.L, respectively, and then 1mg amounts of DOX HCl and indocyanine green (ICG) were added to the blank liquid crystal precursor, respectively, and then the blank liquid crystal precursor was stirred at room temperature on a multi-point magnetic stirrer at 1000rpm/min for a period of time, followed by ultrasonic debubbling, to obtain DOX @ LCFS, ICG @ LCFS, and D/I @ LCFS, respectively. Then, 1mL of blank LCFS, DOX @ LCFS, ICG @ LCFS and D/I @ LCFS are respectively taken and placed in a 10mL of EP tube, laser excitation is carried out in a near infrared band of 808nm, the power is set to be 2W, and photo-thermal conversion efficiency of different groups within 0-10 min is recorded, as shown in fig. 5, the temperature of free ICG or liquid crystal gel carrying the ICG gradually rises along with the increase of infrared irradiation time, and the ICG @ LCFS and the D/I @ LCFS have obvious photo-thermal conversion efficiency.

Example 8

According to the examples4 preparing DOX and DiO (instead of small molecule immunoregulation medicament) loaded liquid crystal precursor (DOX/DiO @ LCFS), selecting about 20g C57BL/6 mouse, and implanting 2 × 10 subcutaneous implant under right side armpit⁵Melanoma cells, to a tumor size of about 200mm³Injecting 0.1mL of medicine-carrying lyotropic liquid crystal around the tumor, killing the mice at a preset time point, stripping the tumor, freezing at-80 ℃, preparing a frozen section, observing the fluorescence intensity of the tumor tissue by laser confocal imaging, and continuously enhancing the fluorescence intensity of DOX and DiO in the tumor tissue within one week as shown in figure 6, which indicates that LCFS can continuously and slowly release small-molecule medicines into the tumor tissue.

Example 9

A blank liquid crystal gel was prepared according to the method of example 2, and DOX HCl and R848 were prepared in DMSO at concentrations of 0.2 mg/. mu.L, respectively, and then 1mg DOX HCl and R848 were added to the blank liquid crystal precursor, respectively, and the mixture was stirred at room temperature on a multi-point magnetic stirrer at 1000rpm/min for a period of time, and then ultrasonically debubbled to obtain DOX @ LCFS, R848@ LCFS, and D/R @ LCFS, respectively. Selecting a C57BL/6 mouse (6-8 weeks old) with the weight of about 20g, carrying out trypsinization when B16F10 tumor cells are in a logarithmic phase, adjusting the concentration to be 2 x 106, wiping the abdomen of the mouse with 75% alcohol, and inoculating 100 mu L of cell suspension subcutaneously. The size of the tumor to be detected is about 500mm³Mice were anesthetized with 1% pentobarbital (10mg/kg) intraperitoneally. The tissue surrounding the tumor was wiped with 75% alcohol, the tumor was carefully stripped off, and 1% of the tumor was left in the wound, which was closed with surgical suture. Mice after surgical resection were randomly divided into the following 5 groups of 10 mice each:

1) PBS group

2) Free DOX/R848(DOX:5 mg/kg; r848:5mg/kg)

3)DOX@LCFS(DOX:5mg/kg)

4)R848@LCFS(R848:5mg/kg)

5)D/R@LCFS(DOX:5mg/kg；R848:5mg/kg)

After surgical excision for 12h, 100. mu.L PBS, DOX/R848, DOX @ LCFS, R848@ LCFS, and D/R @ LCFS were injected subcutaneously at the wound site. When the tumor volume is about 5X 5mm³Determining the tumor recurrence, and measuring every 2 or 3 daysDetermining the volume of the tumor, when the tumor volume reaches 2000 mm³Or determining the mouse to die if the unilateral diameter reaches 20mm, recording the growth curve of the recurrence condition of the mouse tumor, and recording the result as shown in figure 7, figure 8 and figure 9, wherein D/R @ LCFS can effectively inhibit the recurrence of the postoperative tumor and obviously prolong the survival time of the mouse, and the weight of the mouse does not change obviously.

Example 10

A model for melanoma excision in mice was established as in example 6, and the total tumor recurrence volume in mice was about 100mm³Randomized into 4 groups of 4, each administered subcutaneously at the site of tumor recurrence: PBS, DOX @ LCFS, R848@ LCFS, and D/R @ LCFS. After 2 weeks, the mice were sacrificed by cervical dislocation, and tumors were detached and flow-tested as follows:

1) cutting the tumor tissue into small pieces, soaking in blank culture medium containing 1mg/mL collagenase IV, and incubating in a 37 deg.C gas bath constant temperature oscillator for 70min to obtain single cell suspension.

2) After filtration of the single cell suspension through a 40 μm mesh screen, the single cell suspension was centrifuged at 2000rpm for 5min and then resuspended in 3mL of lymph isolate, 500 μ L of blank medium was carefully added to the surface of the isolate, and centrifuged at 800g for 30 min.

3) After centrifugation, the intermediate buffy coat cell layer was carefully collected in 10mL of blank medium and centrifuged by 250g for 10min by inversion and mixing.

4) Collecting the cell sediment to obtain the lymphocyte.

5) Each tube of cells was divided into three equal portions as required for the experiment, and CD16/32 was added and incubated at 4 ℃ for 10min to block specific staining.

6) Different antibodies were added to stain the single cell suspensions with different fluorescent antibodies: MDSC (CD11b, Ly-6G/Ly-6C antibody), T cells (CD3, CD4, CD8 antibody), tumor-associated macrophages (CD11b, F4/80, CD206, CD86 antibody), incubated at 4 ℃ for 30 min.

7) After the incubation was completed, 1mL of PBS was added to each tube, and the mixture was centrifuged at 2000rpm for 5min to discard the supernatant.

8) After one PBS wash, 300. mu.L PBS was resuspended in the cells and tested on the machine. The results are shown in FIG. 10, FIG. 11 and FIG. 12, and D/R @ LCFS contributes to the immune effector CD3⁺CD8⁺Infiltration of T cells and M1 type macrophages reduces the proportion of immunosuppressive M2 type macrophages and MDSCs, so that the D/R @ LCFS can effectively improve the tumor immunosuppressive microenvironment.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The sustained and controlled release preparation is characterized in that liquid crystal gel is used as a carrier, and the liquid crystal gel is reversed-phase hexagonal liquid crystal gel; the liquid crystal gel is loaded with an immunogenicity inducer and an immunomodulator; the sustained and controlled release preparation is prepared by the following steps:

(1) mixing 45 parts by mass of soybean phospholipid, 35 parts by mass of sorbitan monooleate, 10 parts by mass of tocopherol acetate and 10 parts by mass of ethanol, and fully dissolving to obtain a liquid crystal precursor;

2. The sustained-release formulation of claim 1, wherein the immunogenicity inducing agent is a chemotherapeutic drug, or a mixture of a chemotherapeutic drug and a photosensitizer; the immune modulator is a Toll-like receptor agonist, a STING pathway agonist, an immune checkpoint inhibitor or a cytokine.

3. The sustained or controlled release formulation of claim 2, wherein the chemotherapeutic agent is doxorubicin, paclitaxel, oxaliplatin or gemcitabine; the photosensitizer is indocyanine green, chlorin, gold nanoparticles or black phosphorus nanoparticles; the Toll-like receptor agonist is TLR3, TLR4, TLR7/8 or TLR 9; the STING pathway agonist is C-di-GMP; the immune checkpoint inhibitor is a CTLA4 antibody, a PD1 antibody, or a PD-L1 antibody; the cytokine is IL-10 or GM-CSF.

4. The sustained-release preparation according to claim 1, wherein the mixing in step (1) is heating mixing or stirring mixing on a magnetic stirrer; in the step (2), the immunogenicity inducer and the immunomodulator are firstly prepared into stock solution in a solvent and then added into the liquid crystal precursor.

5. Use of the sustained or controlled release formulation according to any one of claims 1 to 4 for the manufacture of a medicament for the immunological combination therapy of tumors in situ.