KR102313988B1 - Reversible gel for Parenteral drug delivery comprising non-concentric multi-nanodomain vesicles and nanoliposome, and Composition for drug delivery comprising the same - Google Patents

Abstract

The present invention relates to a non-concentric multi-nanodomain vesicle comprising two or more liposomes in contact with and connected to each other, and an outer wall surrounding the two or more liposomes; and a nanoliposome, wherein the non-concentric multi-nanodomain vesicle is composed of an organic phase containing a hydrophobic drug and an aqueous phase containing a hydrophilic drug and has a negative charge, wherein the nanoliposome has a positive charge, A reversible gel for parenteral drug delivery is provided. The reversible gel for parenteral drug delivery according to the present invention can increase the encapsulation efficiency of various hydrophobic and hydrophilic drugs, and can be easily injected using a single syringe. The present invention that overcomes the limitations of vesicle or liposome-based drug delivery systems that are difficult to induce sustained release behavior by moving to surrounding tissues, can be used in surgical sites, etc., and is loaded with drugs that control the functions of cancer cells and various immune cells When used in combination with an immune checkpoint inhibitor, the reversible gel for parenteral drug delivery according to

Description

Reversible gel for parenteral drug delivery comprising non-concentric multi-nanodomain vesicles and nanoliposomes, and composition for drug delivery comprising the same drug delivery comprising the same}

The present invention relates to a reversible gel for parenteral drug delivery comprising non-concentric multi-nanodomain vesicles and nanoliposomes, and a drug delivery composition comprising the same.

In order to solve the problems of toxicity and side effects caused by excessive drug injection, sustained-release drug delivery systems having various characteristics have been developed and utilized.

Most hydrogels developed as such sustained-release drug delivery systems have been developed limited to specific polymer groups because they utilize the phenomenon of sol-gel transition by in vivo temperature or pH.

Among them, most hydrogels prepared using biocompatible materials or natural polymers have the disadvantage of being easily decomposed in the body within 1 or 2 days.

In addition, although hydrogels made of synthetic polymers have the property of releasing drugs in a sustained-release form, they have the disadvantage that they remain in the body for a long time and may cause side effects and toxicity problems in the body.

In addition, most of the hydrogels were irreversible gels that did not show flow again after the viscosity increased after gel formation. Such an irreversible gel has a disadvantage in that there is a limitation in its use in a system in which two or more drugs having different physicochemical properties and functions must be loaded.

For example, in a system in which two different substances are mixed to form an irreversible gel, it was impossible to use a single syringe. A system for allowing the formation of a gel has been developed and used. However, there was a problem in that the tip of the syringe was clogged or gelation occurred quickly, so there was a difficulty in its utilization.

And, the general polymer-based hydrogel system had a problem in that the loaded hydrophilic or lipophilic drug was initially released in excess through burst release.

On the other hand, cancer cells are recognized and destroyed by T cells, which are immune cells, unlike normal normal cells. However, these cancer cells suppress the function of T cells through immune checkpoint inhibitory target proteins that suppress immune function by changing the tumor microenvironment to avoid immune attack, and these proteins include PD-1, PD-L1, CTLA- 4 and so on.

Recently, immune checkpoint inhibitors are being used to solve this immune evasion function. Immune checkpoint inhibitors (anti-PD-1, anti-PD-L1, anti-CTLA-4, etc.) do not directly attack cancer cells, but bind to immune checkpoint inhibitory proteins and neutralize their functions, thereby reducing the immune evasion function of cancer cells. By inhibiting it, the strength of immune cells is increased, and it plays a role in helping T cells destroy cancer cells. Inhibitors of the "immune checkpoint" of immune cells that act as a switch to inactivate immune cells through specific signal transduction are representative immunotherapeutic agents that have led the advent of the era of immunotherapy.

These immune checkpoint inhibitors can increase the survival rate compared to existing anticancer drugs, but have a limited therapeutic effect in monotherapy and no longer effective when resistance to the therapeutic agent develops. This is because, in addition to immune checkpoint inhibitors, various immunosuppressive cells and immunosuppressive factors exist.

Therefore, if an immune checkpoint inhibitor is combined with a drug delivery system to control various immunosuppressive cells and immunosuppressive factors around tumor cells, it is expected that the efficiency of the existing immune checkpoint inhibitor-based anticancer immunotherapy will be dramatically improved. , In order to increase the efficacy of combination treatment with an immune checkpoint inhibitor, when these drugs are administered by vascular injection, many side effects such as lowering the function of T cells for treatment have been reported.

Therefore, it is very important to develop a drug delivery system that allows the loaded drug to be slowly released in a sustained-release form for a certain period of time at the injection site.

Accordingly, the present inventors have developed a drug delivery system capable of forming a gel regardless of physiological factors such as temperature and pH in vivo, using non-concentric multi-nanodomain vesicles and nanoliposomes that are currently approved for use in the human body. A novel gel with reversible properties was developed using

US Patent 4,761,288

The present invention provides a reversible gel for parenteral drug delivery comprising a non-concentric multi-nanodomain vesicle and nanoliposome, and a drug delivery composition comprising the same.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description. There will be.

The present invention relates to a non-concentric multi-nanodomain vesicle comprising two or more liposomes in contact with and connected to each other, and an outer wall surrounding the two or more liposomes; and nanoliposomes, wherein the non-concentric multi-nanodomain vesicle has a negative charge and consists of an organic phase and an aqueous phase, and the nanoliposome provides a reversible gel for parenteral drug delivery, characterized in that it has a positive charge.

In one embodiment of the present invention, the non-concentric multi-nanodomain vesicle having a negative charge and the nanoliposome having a positive charge are formed by electrostatic attraction to each other, and the structure is temporarily collapsed according to the shear stress, such Since the phenomenon can be repeated, the form of a reversible gel can be stably maintained.

In another embodiment of the present invention, the ratio of the non-concentric multi-nanodomain vesicle to the nanoliposome is preferably 10:1 (w/w) to 10:9 (w/w) for normal gel formation. and more preferably 10:3 (w/w).

In another embodiment of the present invention, the diameter of the non-concentric multi-nanodomain vesicle may be preferably 1 to 100 μm, more preferably 10 to 50 μm, and the diameter of the nanoliposome is preferably Preferably, it may be 20 nm to 900 nm, and more preferably 50 to 500 nm.

In another embodiment of the present invention, the organic phase, the liposome membrane, and/or the outer wall of the non-concentric multi-nanodomain vesicle may include a hydrophobic drug generally used in the art, preferably lipophilic ( lipophilic) may include an immunoactivating substance, an immunosuppressive factor controlling substance, an immuno-oncology agent, etc., more preferably imiquimod, MPLA (Monophosphoryl lipid A), resiquimod, flagellin, CPG (CpG). ), poly(I:C), etc., if the drug has lipophilic properties, it is not limited thereto.

In another embodiment of the present invention, the aqueous phase of the non-concentric multi-nanodomain vesicle, that is, the inside of the liposome may include a hydrophilic drug generally used in the art, preferably hydrophilic It may include an immune activating substance, an immunosuppressive factor controlling substance, an immune anticancer agent, etc., more preferably gemcitabine, clodronate, mitomycin, cisplatin, adriamycin, etc., but limited to drugs with hydrophilic properties doesn't happen

In another embodiment of the present invention, the inside of the nanoliposome may include a hydrophilic drug generally used in the art, preferably a hydrophilic immune activation material, an immunosuppressive factor controlling material, an immuno-cancer agent and the like, and more preferably gemcitabine, clodronate, mitomycin, cisplatin, adriamycin, etc., but is not limited thereto as long as it is a drug having hydrophilic properties.

In another embodiment of the present invention, the membrane of the nanoliposome may include a hydrophobic drug generally used in the art, and preferably a lipophilic immune activating substance, an immunosuppressive factor controlling substance, immunity It may include an anticancer agent, etc., more preferably imiquimod, MPLA (Monophosphoryl lipid A), resiquimod, flagellin, PG (CpG), poly icy (poly(I:C)) It is not limited thereto, as long as it is a drug having lipophilic properties.

In another embodiment of the present invention, the non-concentric multi-nanodomain vesicle includes various immuno-activating substances having hydrophilic properties inside the liposome, the membrane of the liposome and/or the lipophilic immuno-activating substance on the outer wall of the capsule. By simultaneous loading, it is possible to increase the effective duration of immune cell activation function.

In another embodiment of the present invention, the non-concentric multi-nanodomain vesicle is various immunosuppressive factor control substances having hydrophilic properties inside the liposome, the membrane of the liposome and/or the outer wall of the capsule for lipophilic immunosuppression By simultaneously loading the factor-controlling substance, the effective duration of the immunosuppressive factor-controlling substance capable of controlling the functions of the immunosuppressive cells and the immunosuppressive substance can be increased.

In another embodiment of the present invention, the reversible gel for parenteral drug delivery is stably maintained without spreading from the injected site to the surroundings or the structure is not collapsed, thereby allowing the drug to be released in a sustained release form.

In addition, the present invention is a reversible gel for parenteral drug delivery; And it provides a composition for drug delivery, characterized in that it comprises at least one selected from the group consisting of a hydrophobic drug and a hydrophilic drug.

In one embodiment of the present invention, the hydrophobic drug comprises an organic phase, a liposome membrane, and/or an outer wall of a non-concentric multi-nanodomain vesicle contained in a reversible gel for drug delivery; And/or it may be included in the membrane of the nanoliposome, and the hydrophilic drug may be included in the liposome and/or the nanoliposome of the non-concentric multi-nanodomain vesicle contained in the reversible gel for drug delivery.

In another embodiment of the present invention, the composition for drug delivery may be used in the form of an injection.

In another embodiment of the present invention, the composition for drug delivery may further include immune checkpoint inhibitors, and the immune checkpoint inhibitors are preferably anti-PD-1, anti-PD-L1. , anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40 , anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti TIGIT, etc., but there is no limitation as long as it is an immune checkpoint inhibitor used in the art.

In addition, the present invention is a reversible gel for parenteral drug delivery of claim 1; And it provides a pharmaceutical composition for preventing or treating cancer, characterized in that it comprises at least one selected from the group consisting of hydrophobic drugs and hydrophilic drugs.

In one embodiment of the present invention, the pharmaceutical composition may induce immunogenic cell death, or induce activation of dendritic cells, natural killer cells, and/or T cells, or regulatory T Cells, myeloid derived suppressor cells, and M2 macrophage may exhibit anticancer effects by inhibiting the functions.

In another embodiment of the present invention, the pharmaceutical composition is preferably characterized in that it induces a systemic antitumor immune response.

In another embodiment of the present invention, the pharmaceutical composition is characterized in that it inhibits cancer proliferation, metastasis, recurrence, etc., or inhibits resistance to anticancer therapy, but is a part of a commonly used cancer treatment method If so, it is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition may be injected into a local injection site, such as a tumor, but is not limited thereto as long as it is a method generally used as a treatment method for cancer.

In addition, the present invention provides a method for preventing or treating cancer comprising administering the composition for drug delivery to an individual.

In addition, the present invention provides a use for preventing or treating cancer of the composition for drug delivery.

In addition, the present invention provides the use of the composition for drug delivery to produce a medicament used for the prevention or treatment of cancer.

The reversible gel for parenteral drug delivery according to the present invention can independently load two or more drugs having various physicochemical properties and functions, and has a reversible property that can be injected using a single syringe, non-concentric multiple The drug loaded into the nanodomain vesicle has an effect that the encapsulation efficiency is increased by more than 15 times compared to the drug loaded on the existing liposome, and the loaded drug is also released in a sustained release form for a period of 7 to 30 days or more.

And the reversible gel for parenteral drug delivery according to the present invention can increase the encapsulation efficiency of various hydrophobic and hydrophilic drugs, and can be easily injected using a single syringe. It overcomes the limitations of vesicle or liposome-based drug delivery systems, which are difficult to induce sustained release behavior by moving to surrounding tissues during use, and can be used in surgical sites, etc.

In addition, the reversible gel for parenteral drug delivery according to the present invention loaded with drugs that control the functions of cancer cells and various immune cells can dramatically improve the efficiency of anticancer immunotherapy when used in combination with an immune checkpoint inhibitor.

In addition, since the reversible gel for parenteral drug delivery according to the present invention can contain both hydrophobic and/or hydrophilic drugs, it is expected to be widely applied to the treatment of various diseases in addition to cancer treatment.

1 shows a structure in which a non-concentric multi-nanodomain vesicle having a negative charge and a nano-sized liposome having a positive charge are formed by electrostatic attraction, the structure is temporarily collapsed according to a shear stress, and this phenomenon can be repeated A schematic diagram of a reversible gel is shown.
2 shows the structure of a non-concentric multi-nanodomain vesicle containing imiquimod and gemcitabine.
3 is a graph comparing the drug encapsulation efficiency and durability of non-concentric multi-nanodomain vesicles and general liposomes.
4 is a result of confirming the stability of a non-concentric multi-nanodomain vesicle constituting an injection-type gel.
5 is a result of observing the structure according to the ratio difference between the non-concentric multi-nanodomain vesicle and the nanoliposome constituting the injection-type gel.
6 is a result of observing nanoliposomes that serve as a bridge between non-concentric multi-nanodomain vesicles.
7 is a graph comparing the viscosity and shear stress of an injection-type gel composed of a non-concentric multi-nanodomain vesicle and a nanoliposome.
8 is a result of confirming the stability of an injection-type gel composed of a non-concentric multi-nanodomain vesicle and nanoliposome.
9 is a graph comparing the drug sustained-release properties of non-concentric multi-nanodomain vesicles and injection-type gels.
10A is a result of confirming the persistence of the injection-type gel in an actual mouse body.
10b shows a control group injected with only a fluorescent substance (G1), a group injected with only a non-concentric multi-nanodomain vesicle (G2), an injection-type gel composed of a non-concentric multi-nanodomain vesicle and a positively charged nanoliposome (G3) , and fluorescence signal measurement results of the group (G4) injected with a non-concentric multi-nanodomain vesicle and a negatively charged nanoliposome.
Figure 10c is the result of confirming the injection site in real mice after 2 weeks.
11a is a result of comparing the concentration of cytokine (IL-6) in mouse serum and the mouse body weight for confirming the biostability of the injection-type gel.
11B is a graph showing comparative data of alanine aminotransferase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) for confirming the biostability of the injection-type gel.
12 is a schematic diagram related to injection-type gel anticancer immunotherapy composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
13 is a measurement of immunogenic cell death induced by gemcitabine.
14 is a graph confirming the immune response increased by gemcitabine and imiquimod.
15 is a graph showing apoptosis of the 4T1 cell line and bone marrow-derived immunosuppressive cells induced by gemcitabine.
16 is a graph confirming the efficacy of positively charged clodronate-nanoliposomes using macrophages.
17 shows a surgical method using an injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
18 is a graph confirming the inhibition of tumor growth and the increase in survival rate of an injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
Figure 19 confirms the tumor metastasis inhibitory efficacy of the injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
20 is a graph showing the change in the ratio of immune cells in the tumor by the injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
Figure 21 is a graph confirming the cytokine change in the tumor by the injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
22 is a graph confirming the change in the ratio of immune cells in the lymphatic organ by the injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
23 is a result of measuring an antigen-specific immune response by an injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
24 is a magnetic resonance imaging image of tumor metastasis inhibited by an implantable gel.
25 is a result confirming the effect according to the type of immune cells in the anticancer immunotherapy.
26 is a result confirming the anticancer immunotherapy efficacy of the injection-type gel using the secondary tumor.
27 is a confirmation of the immune response by memory T cells (memory T cells) induced by the injection-type gel.
28 is a schematic diagram of a combination therapy with an immune checkpoint inhibitor using a reversible injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes.
29 is a graph comparing the activation of the immune checkpoint system of an injection-type gel composed of a non-concentric multi-nanodomain vesicle and a nanoliposome in an animal experiment using a 4T1 tumor cell line.
30 is an injection-type gel composed of a non-concentric multi-nanodomain vesicle and nanoliposomes containing chlordronate in an animal experiment using a 4T1 tumor cell line, and an immune checkpoint inhibitor combination therapy efficacy evaluation result.
FIG. 31 is a graph comparing the ratio of immune cells in an injection-type gel composed of a non-concentric multi-nanodomain vesicle and a nanoliposome containing chlordronate in an animal experiment using a 4T1 tumor cell line and an immune checkpoint inhibitor combination therapy.
Figure 32 is a graph comparing the antigen-specific immune response of an injection-type gel composed of a non-concentric multi-nanodomain vesicle and nanoliposomes containing chlordronate in an animal experiment using a 4T1 tumor cell line and an immune checkpoint inhibitor combination therapy; am.
33 is a graph comparing the activation of the immune checkpoint system of an injection-type gel composed of a non-concentric multi-nanodomain vesicle and a nanoliposome in an animal experimental model using the TC1 tumor cell line.
34 is a graph evaluating the efficacy of an injection-type gel composed of a non-concentric multi-nanodomain vesicle and a nanoliposome containing chlordronate and an immune checkpoint inhibitor combination therapy in an animal experimental model using the TC1 tumor cell line.
35 is a graph comparing immune cell ratios of an injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes and immune checkpoint inhibitor combination therapy in an animal experiment using the TC1 tumor cell line.
36 is a graph comparing antigen-specific immune responses of an injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes and an immune checkpoint inhibitor combination therapy in an animal experiment using the TC1 tumor cell line.

Hereinafter, a reversible gel for parenteral drug delivery and a drug delivery composition comprising the same according to specific embodiments of the present invention will be described in more detail.

Conventional hydrogels were mostly irreversible gels that did not show flow again after the viscosity increased after gel formation. Such an irreversible gel has a disadvantage in that there is a limitation in its use in a system in which two or more drugs having different physicochemical properties and functions must be loaded. In addition, if an immune checkpoint inhibitor is used in combination with a drug delivery system to control various immunosuppressive cells and immunosuppressive factors around tumor cells, it is expected that the efficiency of the existing immune checkpoint inhibitor-based anticancer immunotherapy can be dramatically improved. , In order to increase the efficacy of combination treatment with an immune checkpoint inhibitor, when these drugs are administered by vascular injection, many side effects such as lowering the function of T cells for treatment have been reported.

Accordingly, the present inventors used non-concentric multi-nanodomain vesicles and liposomes, which are currently approved for use in the human body, in order to develop a drug delivery system that can form a gel regardless of physiological factors such as temperature and pH in the body. Thus, a novel gel with reversible properties was developed.

The present invention relates to a non-concentric multi-nanodomain vesicle comprising two or more liposomes in contact with and connected to each other, and an outer wall surrounding the two or more liposomes; and nanoliposomes, wherein the non-concentric multi-nanodomain vesicle has a negative charge and consists of an organic phase and an aqueous phase, and the nanoliposome provides a reversible gel for parenteral drug delivery, characterized in that it has a positive charge.

In the present invention, a structure is formed by electrostatic attraction between a micrometer-sized non-concentric multi-nanodomain vesicle having a negative charge and a nano-sized nanoliposome having a positive charge, and the structure is temporarily collapsed according to the shear stress, such A reversible gel in which the development can be repeated and a drug delivery system loaded with a drug can be provided.

In the present invention, a non-concentric multi-nanodomain vesicle with a size of several tens of micrometers that can increase the encapsulation efficiency of various water-soluble and poorly-soluble drugs was used, and electrostatic attraction with the non-concentric multi-nanodomain vesicle bearing a negative charge A new type of gel was prepared by combining positively charged nanoliposomes as a crosslinking agent that helps to form a gel by

In addition, the structure is easily collapsed temporarily by the shear force acting during the injection process using a single syringe, and as the shear stress is removed at the injected site, the gel is formed again by electrostatic attraction. , Reversible gel formation was induced by controlling the composition ratio of non-concentric multi-nanodomain vesicles and cationic nanoliposomes.

The ratio of the non-concentric multi-nanodomain vesicle to the nanoliposome may be 10:1 (w/w) to 10:9 (w/w), preferably 10:3 (w/w).

On the other hand, in the gel containing the non-concentric multi-nanodomain vesicle and nanoliposome according to the present invention, the gel is formed again by electrostatic attraction at the injection site, and the storage modulus (G') and loss modulus (G") values It can exhibit the properties of a reversible gel that restores the properties of this gel.

In addition, it is possible to prevent a cytokine storm or side effects caused by drugs due to a sudden influx of loaded drugs into blood vessels.

In addition, the reversible gel for parenteral drug delivery loaded with various drugs according to the present invention enhances the anticancer treatment effect by inducing apoptosis of cancer cells in cancer tissues, activating immune cells for treatment and inhibiting the functions of immunosuppressive cells. It can help to kill cancer cells and effectively recognize cancer antigens formed by the killed cancer cells by antigen-presenting cells such as dendritic cells and macrophages.

According to another aspect of the present invention, the reversible gel for drug delivery; And it provides a composition for drug delivery, characterized in that it comprises at least one selected from the group consisting of a hydrophobic drug and a hydrophilic drug.

In one embodiment of the present invention, the drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes induces immunogenic cell death in cancer cells, thereby inducing expression of carleticulin or HMGB1.

In one embodiment of the present invention, a drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce the death of MDSCs, which are immunosuppressive cells.

In one embodiment of the present invention, a drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce M2-->M1 polarization of macrophages, which are immunosuppressive cells.

In one embodiment of the present invention, an injection-type gel composed of a drug-loaded non-concentric multi-nanodomain vesicle-nanoliposome can induce the death of regulatory T cells, which are immunosuppressive cells.

In one embodiment of the present invention, the injection-type gel composed of drug-loaded non-concentric multi-nanodomain vesicle-nanoliposomes induces the activation of antigen-presenting cells, dendritic cells and macrophages, thereby promoting the expression of proinflammatory cytokines. can

In one embodiment of the present invention, the drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce Type I interferon (IFN-alpha, IFN-beta) through the activation of dendritic cells.

In one embodiment of the present invention, a drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce the activity of CD4+ T cells.

In one embodiment of the present invention, the drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce the activity of CD8+ T cells.

In one embodiment of the present invention, a drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce the activity of natural killer cells (NK).

In one embodiment of the present invention, the drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can function to prevent the growth or recurrence of cancer cells at the treatment site.

In one embodiment of the present invention, a drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes can induce a systemic immune response.

In one embodiment of the present invention, the drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes releases the drug at a local site, thereby inducing immune activation in lymph nodes.

In one embodiment of the present invention, the drug-loaded non-concentric multi-nanodomain vesicle-gel composed of nanoliposomes releases the drug at a local site, thereby inducing immune activation in spleen.

In one embodiment of the present invention, a drug-loaded non-concentric multi-nanodomain vesicle-a gel composed of nanoliposomes may have a function of preventing metastasis of primary cancer to the lung or liver.

In one embodiment of the present invention, a gel composed of a drug-loaded non-concentric multi-nanodomain vesicle-nanoliposome not only prevents the recurrence of primary cancer, but also prevents the formation of secondary cancer in other tissues or sites. can have

In one embodiment of the present invention, in a group in which cancer cells are completely regressed by a gel composed of a drug-loaded non-concentric multi-nanodomain vesicle-nanoliposome, memory T cells are generated, and cancer recurrence may have a function to prevent

Technology to additionally control various immunosuppressive cells and immunosuppressive factors other than immune checkpoint in order to maximize the effects of checkpoint inhibitors in the non-responding group and resistance group to the existing immune checkpoint inhibitors This is very important.

Drugs that control these immunosuppressive cells and immunosuppressive factors should not inhibit the function of therapeutic T cells activated by existing immune checkpoint inhibitors, and should not cause toxicity and side effects, so the vascular drug delivery system (systemic drug) delivery), a local treatment drug delivery system is advantageous.

Although sustained-release drug delivery systems with various properties have been developed and utilized, most hydrogels prepared using biocompatible materials have the disadvantage of being easily decomposed in the body within 1 or 2 days, and hydrogels made of synthetic polymers. Although gels remain in the body for a long period of time and release the drug in a sustained-release form, they have the disadvantage that they may cause side effects and toxicity problems in the body.

However, the reversible gel for drug delivery and the drug delivery composition comprising the drug according to the present invention do not cause the above problems, and when combined with an immune checkpoint inhibitor, the anticancer immunotherapeutic effect increases, and antigen-specific It may have effects such as an increase in immune response.

In the present invention, the composition for drug delivery may further include immune checkpoint inhibitors, and the immune checkpoint inhibitors are anti-PD-1, anti-PD-L1, and anti-CTLA-4, anti- KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, It may be selected from the group consisting of anti-CD40L, anti-BTLA, anti-TCR, anti TIGIT, and the like.

In the present invention, the multi-nanodomain vesicle is composed of an organic phase and an aqueous phase, the organic phase includes a first immunomodulatory substance and a fluid oil, and the organic phase is the membrane of the liposome, and the outer wall of the multi-nanodomain vesicle The aqueous phase comprises a second immunomodulatory substance, the aqueous phase is an inner aqueous phase of the liposome membrane and an external aqueous phase of the liposome membrane, and the first immunomodulatory substance is a fat-soluble immunoactive substance, and the first 2 The immunomodulatory substance is a water-soluble immunoactive substance, and the fluid oil is characterized in that it improves the structural stability of two or more liposomes that are in contact with each other and are linked, a multi-nanodomain vesicle can be provided.

In the present invention, the multi-nanodomain vesicle comprising two or more liposomes has a longer duration of immune cell activating material, immune cell activation efficacy, encapsulation efficiency, or physiological stability compared to conventional single liposomes and single emulsions. may be improved.

In the present invention, the immunoactivating substance is a toll-like receptor agonist, saponin, antiviral peptide, inflammasome inducer, NOD ligand (NOD ligand), CDS ligand ( It may include one or more substances selected from the group consisting of a cytosolic DNA sensor ligand), a stimulator of interferon genes (STING) ligand, and combinations thereof, but may not be limited thereto.

According to another aspect of the present invention, as a multi-nanodomain vesicle comprising two or more liposomes in contact with and connected to each other, and an outer wall of a multi-nanodomain vesicle surrounding the two or more liposomes, the multi-nanodomain vesicle comprises an organic phase and an aqueous phase, wherein the organic phase includes a first immunomodulatory substance and a fluid oil, the organic phase forms the membrane of the liposome and the outer wall of the multi-nanodomain vesicle, and the aqueous phase includes a second immunomodulatory substance wherein the aqueous phase is an inner aqueous phase of the liposome membrane and an external aqueous phase of the liposome membrane, the first immunomodulatory substance and the second immunomodulatory substance are immunosuppressive factor control substances, and the fluid oil is in contact with each other And characterized in that to improve the structural stability of the two or more liposomes that are linked, a multi-nanodomain vesicle can be provided.

In the present invention, the multi-nanodomain vesicle-based solid cancer microenvironment control composition is a new type of immunomodulatory composition for regulating the microenvironment of cancer. It is characterized in that it contains a drug that can control the functions of suppressor cells and immunosuppressive substances (immunosuppressive factor control substances).

According to one embodiment of the present invention, a plurality of liposomes are linked to each other while forming respective domains based on an immunosuppressive factor, that is, an immunosuppressive factor controlling substance capable of controlling the functions of immunosuppressive cells and immunosuppressive substances. , it is possible to prepare a multi-nanodomain vesicle for immunomodulation having a micro-sized capsule form with improved structural stability of multiple liposomes linked by the introduced fluid oil component. In addition, according to one embodiment of the present invention, a new multi-layer that can overcome the disadvantages of low encapsulation efficiency and short effective duration of a single liposome material used as various pharmaceutical compositions, and increase the effective duration of the immune function modulating effect A nanodomain vesicle-based anticancer immunotherapeutic composition can be prepared.

The multi-nanodomain vesicle according to an embodiment of the present invention can control the functions of immunosuppressive cells and immunosuppressive substances loaded on the outer wall and inside of the capsule while slowly collapsing from the outer wall to the inner membrane of the capsule. Since the immunosuppressive factor controlling substance is released, it has the advantage that the effective duration of the immune mechanism modulating substance can be increased.

In addition, the multi-nanodomain vesicle according to an embodiment of the present invention can control the functions of various immunosuppressive cells and immunosuppressive substances having lipophilic properties on the membrane of liposomes and/or the outer wall of the multi-nanodomain vesicle. By loading a possible immunosuppressive factor control substance, the effective duration of the immunoactive substance can be increased.

The multi-nanodomain vesicle according to an embodiment of the present invention is an immunosuppressive factor by loading various immunosuppressive cells having hydrophilic properties and an immunosuppressive factor controlling material capable of controlling the functions of the immunosuppressive material in the liposome. It can increase the effective duration of the control substance.

In the multi-nanodomain vesicle according to an embodiment of the present invention, various immunosuppressive factor controlling substances having hydrophilic properties inside liposomes, lipophilic immunosuppressive factor controlling substances on the membrane of the liposome and/or the outer wall of the capsule are simultaneously added. By loading, it is possible to increase the effective duration of the immunosuppressive factor controlling substance capable of controlling the functions of the immunosuppressive cells and the immunosuppressive substance.

The nanoliposome according to an embodiment of the present invention may include various immunosuppressive factor controlling substances having hydrophilic properties inside the liposome, and lipophilic immunosuppressing factor controlling substances in the membrane of the liposome.

In the present invention, the drug that can be loaded into the multi-nanodomain vesicle and the nanoliposome is not limited to the specific embodiment of the present invention, and may include other drugs that can be loaded by analogy conventionally in the industry.

In one example of the present invention, as a drug capable of controlling the function of Myeoloid-Derived Suppressor Cell (MDSC), that is, an immunosuppressor control substance, Tadalafil, Sildenafil, L-AME, Nitroaspirin, Celecoxib, NOHA, Bardoxolone methyl, D ,L-1-methyl-tryptophan, 5-Fluorouracil, Gemcitabine, 17-DMAG, Peptide-Fc fusion proteins, ATRA, Vitamin A, Vitamin D3, Vitamin E, GR1 antibodies, Zoledronic acid, Sunitinib, Axitinib, Decetaxel, Sorafenib, Cucurbitacin B, JSI-124, Anti IL-17 antibodies, Anti-glycan antibodies, Anti-VEGF antibodies, Bevacizumab, Antracycline, Tasquinimod, Imatinib, cyclophosphamide, but are not limited thereto.

In one embodiment of the present invention, the PI3K inhibitors are PX-866, Wortmannin, PI-103, Pictilisib, GDC-0980, PF-04691502, BEZ235, XL765, XL147, BAY80-6946, GSK-2126458, Buparlisib, BYL719, AZD8186, It is characterized in that it is GSK-2636771, CH5132799, INK-1117, etc.

In an example of the present invention, the PI3Kdelta inhibitors are AMG-319, Idelalisib, TRG-1202, INCB050465, IPI-145, Duvelisib, Acalisib, TG-1202, RV1729, RP-6530, GDC-0032, etc. .

In one example of the present invention, the PI3Kgamma inhibitors are IPI-549, IPI-145, and the like.

In an example of the present invention, as a drug that can control the function of Treg (Regulatory T cell), that is, an immunosuppressive factor control substance, Anti-CD25 antibodies (daclizumab), Basiliximab, LMB-2, Denileukin diftitox (Ontak), Bivalent IL-2 fusion toxin, Anti-TGF-beta antibodies, fresolimumab, TGF-betaR kinase inhibitors, LY2157299, Soluble TGF-betaR I/II, Ipilimumab, Tremelimumab, Pembrolizumab, Nivolumab, TIM-3 antibodies, LAG-3 antibodies, Anti -CD39 antibodies, Anti-73 antibodies, A(2A)R inhibitors, Celecoxib, Indomethacin, Diclofenac, Ibuprofen, TNFR2 antibodies, Anti-GITR antibodies, Bevacizumab, Anti-OX40(CD134) antibodies, soluble GITR ligand, Blockades for chemokine receptors (CCR4, 5, 6,10), cyclophosphamide, Sunitinib, Fludarabine, PI3K p110 (delta) inhibitors, CliniMACs, Mogamulizumab, Fingolimod, Regulators for miRNA (miR-155, miR-146a, miR-181a), 5-aza- 2-deoxycytidine, paclitaxel, Imatinib, Sorafenib, Cyclosporin A, Tacrolimus, Dasatinib, Poly-G-oligonucleotide, TLR8 ligands, gemcitabine, and 5-fluorouracil.

An example of the present invention is a drug that can tune the function of TAM (tumor associated macrophage), that is, a drug that can inhibit the recruitment of macrophage as an immunosuppressor control substance, CCL2 / CCR2 inhibitors (Yondeli, RS102895), It is characterized by being M-CSF or M-CSFR inhibitors (anti-M-CSF antibodies, JNJ-28312141, GW2580), chemoattractants (CCL5, CXCL-12, VEGF) and their receptors, such as inhibitors, HIFs inhibitors, etc. However, the present invention is not limited thereto.

In addition, it is a drug that can inhibit the survival of TAM, that is, as an immunosuppressive factor control substance, it is a drug that can induce the expression of Bisphosphonates, Clodronate, Dasatinib, anti-FRbeta antibodies, Shigella flexneri, Legumain and CD1d. , but is not limited thereto.

And, as a drug that can improve the properties of M1 macrophage, that is, as an immunosuppressive factor control substance, NF-kB agonist TLR agonist, Anti-CD40 antibodies, Thiazolidinediones, Tasquinimod, Anti-IL-10R antibodies, Anti-IL -10 antibodies, oligonucleotides (Anti-IL-10R Anti-IL-10), STAT1 agonist interferon, SHIP capable of inducing M1 pathway, GM-CSF, IL-12, Thymosin alpha1, etc. characteristic, but is not limited thereto.

In addition, drugs that can inhibit the M2 macrophage-based cancer cell growth mechanism, i.e., immunosuppressive factor control substances, include STAT3 inhibitors sunitinib, sorafenib, WP1066, corosolic acid, oleanolic acid, STAT6 inhibitors and the M2 pathway (c- Myc, PPAR-alpha/gamma, PI3K, KLF4, HIFs, Ets2, DcR3, mTOR) inhibitors and HRG, CuNG, MDXAA, Silibinin, PPZ, etc., but are not limited thereto.

And, it is characterized in that the target miRNA that can control the function of macrophage under the tumor microenvironment is miR-155, miR-511-3p, miR-26a, and the like.

And, by targeting Macrophage under the tumor microenvironment, target drugs that can increase anticancer efficacy include Paclitaxel, Docetaxel, 5-Flurouracil, Alendronate, Doxorubicin, Simvastatin, Hydrazinocurcumin, Amphotericin B, Ciprofloxacin, Rifabutin, Rifampicin, Cisplatinfloxacin, Rifabutin, Rifampicin, Efavirenz, Theophyline, Pseudomonas exotoxin A, Zoledronic acid, Trabectedin, Siltuximab (Anti-IL-6 antibodies), Dasatinib, CpG-ODN, Interferon-alpha, -beta, -gamma, GM-CSF, IL-12, Thymosin alpha-1, Sunitinib, 5,6-Dimethylxanthenone-4-acetic acid, Silibinin, CCL2-CCR2 inhibitors (PF-04136309, Trabectedin, Carlumab), CSF1-CSF1R signaling blocker (BLZ945, PLX3397, Emactuzumab (RG7155), AMG-820, IMC -CS4, GW3580, PLX6134) and toll-like receptor 7 ligands (imiquimod, 852A), NF-kB inhibitors (N-acetyl-l-cystein, Vitamin C, bortezomib, aspirin, salicylates, indolecarboxamide derivatives, quinazoline analogues, Thalidomide, prostaglandin metabolites), HIF-1 inhibitors (2ME2, 17-AAG, Camptothecin, Topotecan, Pleurotin, 1-methylpropyl, 2-imidazolyl dissulphide, YC-1), CXCR4 agonists (AMD3100, AMD1498, ALX40-4C, T 22, T140, CGP64222, KRH-1636) and the like), but is not limited thereto.

One example of the present invention is immunosuppressive environmental factor inhibitors (Transforming growth factor beta (TGF-beta) inhibitors, Nitro aspirin, Cycloxygenase-2 (COX2) inhibitors, Indoleamine 2,3-dioxygenase (IDO) inhibitors, Phosphodiesterase-5 ( It is possible to provide a multi-nanodomain vesicle-based composition containing PDE-5) inhibitors, Anti-Interleukin 10 (IL-10)) drugs.

In one example of the present invention, TGF-beta inhibitors include, but are not limited to, SB-505124, LY-364974, and the like.

Nitro aspirin in one example of the present invention includes, but is not limited to, NCX 4040 and the like.

In one example of the present invention, the COX-2 inhibitor includes, but is not limited to, Celecoxib.

In one example of the present invention, IDO inhibitors include, but are not limited to, Indoximod, NLG919, and the like.

In one example of the present invention, the PDE-5 inhibitor includes, but is not limited to, Tadalafil (Cialis) and the like.

In one embodiment of the present invention, the solid cancer microenvironmental immunosuppressor control material contained in the multi-nanodomain vesicle may be composed of a combination of two or more of the above drugs.

In one embodiment of the present invention, natural killer cells and T cells having therapeutic ability to find and directly kill cancer cells in the body effectively survive in the body, and multi-nanodomain vesicles that can improve the therapeutic efficacy are prepared. It may be an immunomodulatory substance comprising.

An example of the present invention provides a multi-nanodomain vesicle-based composition comprising co-activators (OX40, CD137, CD27, CD40), etc. as a method of activating T cells through direct binding in a solid cancer microenvironment.

Anti-OX40 in one example of the present invention includes, but is not limited to, RG7888 and the like.

In one example of the present invention, Anti-CD137 includes, but is not limited to, Urelumab.

Anti-CD27 in one example of the present invention includes, but is not limited to, Varlilumab and the like.

Anti-CD40 in one example of the present invention includes, but is not limited to, BMS-986090 and the like.

An example of the present invention is a multi-nanodomain vesicle containing a drug capable of inhibiting immunosuppressive factors (Treg, MDSC, TAM, IDO, PD-L1) as a method of T cell activation through indirect binding in a solid cancer microenvironment A base composition is provided.

An example of the present invention may provide a multi-nanodomain vesicle-based composition comprising an anticancer agent that increases the efficacy of immune cells through induction of immunogenic cell death through chemotherapy.

An example of the present invention provides a multi-nanodomain vesicle-based composition including a drug capable of killing cancer cells or controlling the tumor microenvironment through epigenetic machinery.

As an example of an epigenetic mechanism in the present invention, as a DNA methyltransferase inhibitor (DNMTi) substance, 5-Azacytidine, 5-Aza-2-deoxycytidine, Decitabine, SGI-110, Zebularine, CP-4200, Cladribine, Fludarabine, Clofarabine, Procainamide, Procaine, Hydralazine, Disulfiram, RG108, Nanaomycin A, Genistein, Equol, Curcumin, EGCG, Resveratrol, Parthenolide and the like are selected from, but not limited to.

As an example of an epigenetic mechanism in the present invention, histone deacetylase inhibitor (HDACi) substances include Vorinostat, Abexinostat, Suberoylanilide, Hydroxamic acid, Belinostat, Panobinostat, Romidepsin, Valproic acid, Entinostat, Givinostat, Resminostat, Quisinostat, Pracinostat, Pracinostat, Dacinostat, Pyroxamide, CHR-3996, CBHA, Trichostatin A, Oxamflatin, MC1568, Tubacin, PCI-30451, Tacedinaline, Mocetinostat, Chidamide, BML-210, M344, Butyrate, Sodium butyrate, Trapoxin A, Apicidin, Nicotinamide, Splitomicin, Nicotinamide -527, Dihydrocoumarin, Tenovin-D3, AGK2, AEM1, AEM2, Cambinol, Sirtinol, Salermide, Tenovin-6, TMP-269, Psammaplin A, Nexturastat A, RGFP966 and the like.

As an example of the present invention, immunotherapy refers to drugs that prevent cancer cells from evading the body's immune system or allow immune cells to better recognize and attack cancer cells, for example, immune checkpoint inhibitors ipilimumab, pemb There are rolizumab, nivolumab, atezolizumab, and the like, and blinatumomab and the like as an agent for enhancing the action of human immune cells, but is not limited thereto.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example One : Imiquimod (R837) and Gemcitabine containing non-concentric Injection-type gel synthesis composed of multi-nanodomain vesicles and nanoliposomes containing clodronate

Multi-domain vesicles containing imiquimod and gemcitabine were prepared through a w-o-w emulsification process. Dissolve imiquimod in DOPC (16mg), DPPG (2mg), triolein (4mg), cholesterol (8mg), squalene (6mg) and oleic acid 6mg in 1mL chloroform to dissolve the solution. This solution was emulsified using a microtip sonicator like a 5% sucrose and 7.5% glucose mixed solution mixed with gemcitabine 10mg (water in oil).

Next, a solution of 40 mM lysine and 7.5% glucose was prepared using a homogenizer to prepare a wow emulsion, chloroform was removed using a rotary evaporator, and the unencapsulated drug was non-concentric. It was removed in the multi-nano domain vesicle washing process.

Nanoliposomes were prepared by dissolving DOPE (0.006 mmole) and DOTMA (0.006 mmole) in 1 mL chloroform and then forming a thin film using an evaporator. The lipid mixture formed in the form of a thin film is soluble in deionized water containing 1 mg Chlodronate. The solution prepared in this way is subjected to sonication for 1 minute and reacted for 2 hours to form a stable form. Unencapsulated clodronate was removed using a centrifuge filter. The non-concentric multi-nanodomain vesicles and nanoliposomes made through this process were mixed in a ratio of 10:3 (w/w), respectively, to form an injection-type gel.

In Figure 1, the structure of a micrometer-sized non-concentric multi-nanodomain vesicle having a negative charge and a nano-sized liposome having a positive charge are formed by electrostatic attraction, and the structure is temporarily collapsed according to the shear stress, and this phenomenon is A schematic diagram of a reversible gel that can be repeated is shown.

In FIG. 2, the structure of a non-concentric multi-nanodomain vesicle divided into hydrophilic and hydrophobic regions is shown through fluorescence images.

In Figure 3, the drug encapsulation efficiency and sustained release, compared to general liposomes, non-concentric multi-nanodomain vesicles show a more excellent effect as a graph.

Example 2: Confirmation of stability of injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes

In order to confirm the stability of the non-concentric multi-nano domain vesicles, the non-concentric multi-nano domain vesicles were prepared and stored at 4° C. and 37° C. at the biological temperature, respectively, to check whether there was a change in the structure. Significant structural changes of the multi-nanodomain vesicles were not observed at both temperatures for 4 weeks under an optical microscope. In addition, the size of the non-concentric multi-nanodomain vesicles also did not show a significant difference with time at 4°C and 37°C (FIG. 4).

Next, the difference in the structure of the injection-type gel due to the difference in the ratio of non-concentric multi-nanodomain vesicles and nanoliposomes was confirmed. When the ratio of the nanoliposomes is small, the gel form is not formed, but it was confirmed that a gel was formed at an appropriate ratio of 10:3, and when the ratio of the nanoliposomes was increased, it was confirmed that the gel structure collapsed (FIG. 5).

In FIG. 6 , the fluorescence images of the non-concentric multi-nanodomain vesicles and the injection-type gel were compared to identify nanoliposomes that act as a bridge between the non-concentric multi-nanodomain vesicles. When only non-concentric multi-nano-domain vesicles were present, it was confirmed that the non-concentric multi-nano-domain vesicles were separated from each other.

7 shows the results of comparing the viscosity and shear stress of non-concentric multi-nanodomain vesicles and injection-type gels. It was confirmed that the viscosity of the injection-type gel was higher than that of the non-concentric multi-nanodomain vesicle, and that it was a reversible gel in which the temporary collapse and recovery of the structure were repeated according to the shear stress.

In Figure 8, the stability of the injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes was confirmed at 4°C and 37°C, respectively. As a result of observation for 4 weeks, it was not significantly different from the initial state at 4°C, but at 37°C, a structural change of the injection-type gel was confirmed from the 14th day. From the 14th day, the decomposition of the injection-type gel began to be gradually observed, and the decomposition proceeded with time, and it was confirmed that the decomposition was almost completely decomposed on the 28th day. Through the above results, it was confirmed that the injection-type gel sufficiently delivered drugs such as imiquimod, gemcitabine, and clodronate in vivo and was decomposed in the body.

In Figure 9, the drug sustained release of the non-concentric multi-nanodomain vesicle and the injection-type gel was compared. In the drug sustained-release comparison of imiquimod and gemcitabine, no significant differences were observed between non-concentric multi-nanodomain vesicles and injectable gels. Therefore, it can be said that there will be no difference in drug delivery efficacy even after the gel is formed.

10a, 10b, and 10c, the persistence of such an injection-type gel was confirmed in the actual mouse body. After injecting a sample containing a fluorescent material into the body of the mouse, it was confirmed whether the structure was maintained over time (FIG. 10a). In the control group injected with only the fluorescent substance (G1), all fluorescence signals disappeared within 1 day, whereas the group injected with only non-concentric multi-nanodomain vesicles (G2) and the multi-domain vesicle and negatively charged nanoliposomes were injected together. In the group (G4), it was confirmed that the fluorescence signal was rapidly weakened after about 3 days. In contrast, it was confirmed that the injection-type gel (G3) composed of a non-concentric multi-nanodomain vesicle and positively charged nanoliposomes maintained the fluorescence signal for more than 2 weeks (Fig. 10b). After 2 weeks, as a result of checking the injection site in real mice, the injection-type gel maintained its shape, but it was difficult to find the shape of other samples ( FIG. 10c ).

In order to confirm the biostability of the injection-type gel, as a result of comparing the cytokine (IL-6) concentration in mouse serum and the mouse body weight, no significant change was confirmed by the injection-type gel compared to the control group (FIG. 11a). In addition, it was found that no significant difference was observed in the injection-type gel compared to other controls in the comparative data of alanine aminotransferase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) (FIG. 11b).

Example 3: Imiquimod (R837) and Gemcitabine inclusive non-concentric multi-nano domain vesicle and Clodronate inclusive with nanoliposomes Constructed injection type gel Efficacy evaluation of anticancer treatment

12 shows a schematic diagram related to anticancer immunotherapy using an injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes and a surgical method. Gemcitabine contained in non-concentric multi-nanodomain vesicles removes bone marrow-derived immunosuppressive cells (mdsc) and simultaneously attacks cancer cells to generate cancer cell antigens through immunogenic cell death. Imiquimod enhances the immune response, and nanoliposomes containing clodronate reduce tumor associated macrophages, leading to activation of the immune response.

In FIG. 13, the immunogenic cell death induced by gemcitabine was confirmed by measuring calreticulin and HMGB1.

In FIG. 14, it was confirmed that the immune response was increased by the supernatant obtained by treating the 4T1 cell line with gemcitabine and imiquimod. That is, dendritic cell activation markers CD40 and CD80 were measured, and inflammatory cytokines TNF-α and IL-6 were measured. The supernatant contained the antigen produced by gemcitabine, and imiquimod improved the immune response, so it was confirmed that the immune response increased the most in the group treated with both.

In FIG. 15, apoptosis induced by gemcitabine was confirmed. That is, it was confirmed that the rate of induced apoptosis increased according to the concentration of gemcitabine treatment.

In Figure 16, clodronate, positively charged liposomes, and positively charged clodronate-liposomes were treated in macrophages to compare their efficacy. It was confirmed that positively charged clodronate-nanoliposomes effectively reduced macrophages even at low concentrations compared to clodronate and positively charged liposomes.

17 shows a surgical method using an injection-type gel. In animal experiments, BALB/c (female 5-6 weeks old, orient) mice without specific pathogens were used. First, anesthetized by injecting 0.01 ml of a 2.5% avertin (2,2,2-tribromoethanol-tert-amylacohol, Sigma-Aldrich) solution into the abdominal cavity of a mouse per body weight (g), 1x10 ⁶ breast cancer cells in the right flank Cells (4T1) were injected subcutaneously. After 14 days, when the tumor size reached about 300 mm³, surgery was performed to remove about 90% of the tumor. At this time, samples were injected into 6 groups. The anticancer effect of the injection-type gel was confirmed by comparing the ratio of various immune cells (CD4 ⁺ T cells, CD8 ⁺ T cells, NK cells, dendritic cells, bone marrow-derived immunosuppressive cells) on the 10th day after surgery. Immune cells were separated from the spleen and lymph nodes and analyzed. First, the spleen was isolated from the mouse, and then red blood cells were removed using a red blood cell lysis buffer. Only splenocytes were left and used. Lymph nodes were isolated from mice and then treated with collagenase for 1 hour to separate cells from tissues. After the cells are separated, they go through a filtering process to prevent the inclusion of tissues other than lymphocytes. The cells thus obtained were labeled with various immune cells using various antibodies (anti-CD4, anti-CD8, anti-CD3, anti-CD335, anti-CD11b, anti-CD11c, anti-Gr1), followed by flow cytometry (flow cytometry). cytometer).

In FIG. 18, through the measurement of the survival rate after surgery, it was confirmed that the survival rate was significantly increased compared to the control group by the injection-type gel. The group containing only imiquimod and gemcitabine also increased the survival rate compared to the control group, but it was confirmed that the effect of the injection-type gel containing clodronate was greater.

In FIG. 19 , it was confirmed that tumor metastasis was most effectively inhibited when the injection-type gel of the present invention was used as a result of confirming whether or not tumor metastasis in the lung using the property of easily metastasizing 4T1 breast cancer cells.

Example 4 : non-concentric multi-nano domain vesicle and with nanoliposomes Constructed injection type on the gel Immune response induced by

In FIG. 20, it was confirmed that the ratio of various immune cells in the tumor was changed on the 10th day after tumor surgery and gel injection. In the case of CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells, it was confirmed that the ratio increased in the gel-injected group. Conversely, it was confirmed that the ratio of cells that suppress the immune response, such as bone marrow-derived immunosuppressive cells and M2 macrophages, decreased.

In Figure 21, as a result of measuring various cytokines (TNF-α, IL-6, INF-γ, IFN-α), it was confirmed that the highest cytokine was measured in the group treated with the injection-type gel.

In Figure 22, it was confirmed the change in the ratio of immune cells inside the lymphatic organ after surgery. When the injection-type gel was used ^{, the ratio of CD4 +} T cells and CD8 ⁺ T cells in the spleen increased, and the ratio of bone marrow-derived immunosuppressive cells decreased. The ratio of dendritic cells and NK cells also increased, and the same pattern was confirmed not only in the spleen but also in the lymph nodes. As a result of confirming the ratio of regulatory T cells in the lymph node, it was confirmed that the ratio was significantly reduced when the injection-type gel was used.

In FIG. 23, cytokines (INF-γ, IL-2, TNF-α) secreted from lymphocytes isolated from the spleen and lymph nodes were measured. When the injection-type gel was used, proinflammatory cytokines increased in the tumor, and cytokines secreted from lymphocytes through this were stimulated by the tumor lysate. It was confirmed that an antigen-specific immune response was induced. Overall, it was confirmed that the highest immune enhancing effect was observed when the injection-type gel was used in the tumor, spleen, and lymph nodes.

In FIG. 24 , it was confirmed that tumor metastasis was suppressed when the injection-type gel was used through magnetic resonance imaging (MRI) images. In the case of the control group, it was confirmed that the tumor had metastasized to the internal organs of the mouse and displayed white.

In FIG. 25, CD4 ⁺ T cells, CD8 ⁺ T cells, and NK cells were respectively reduced in the mouse body using an antibody, and then the effect was confirmed. Although all three types of immune cells had an effect on the anticancer immunotherapy effect, it was confirmed that ^{CD8 +} T cells and NK cells had a ^{greater effect than CD4 + T cells.} It can be seen that both tumor weight and tumor metastasis were ^{less affected by CD4 + T cells.}

In FIG. 26, a systemic antitumor immune response was confirmed using a secondary tumor. The experiment was carried out in the same manner as in Example 1, but the secondary tumor was injected into the contralateral side of the mouse at the same time as a part of the primary tumor was surgically removed on the 14th day after the primary tumor injection. After observing the size and weight of the tumor ^{and checking the ratio of immune cells (CD4 +} T cells, CD8 ⁺ T cells) within the tumor, both the primary and secondary tumors in the group treated with the injection-type gel significantly decreased in size and weight. , it was confirmed that the ratio of immune cells increased.

In Figure 27, the immune response by memory T cells (memory T cells) was confirmed. Splenocytes isolated from the spleen were labeled with memory T cell-related antibodies anti-CD44 and anti-CD62L, and the ratio of memory T cells was compared using flow cytometry. It was confirmed that the highest ratio was measured when the injection-type gel was used for both ^{CD4 +} memory T cells and CD8 ^{+ memory T cells.} In the course of the tumor re-injection experiment, mice (experimental group) in which the tumors generated during the survival rate measurement process in Example 2 completely disappeared were used. After 45 days from the surgical procedure, the tumor was injected into the contralateral flank. At this time, a mouse (control group) that was not treated with anything was also injected with the tumor. After tumor re-injection, as a result of observing the size of the tumor, it was confirmed that the immune response by the injection-type gel greatly inhibited the growth of the tumor. In the control group, the tumor continued to grow within a few days after tumor injection, whereas in the experimental group, tumor growth was suppressed for about 3 weeks. In addition, when tumor metastasis was confirmed in the lung, tumor metastasis was suppressed in the experimental group compared to the control group, so that no tumor was identified in the lung.

Example 5: Imiquimod (R837) and Gemcitabine inclusive non-concentric multi-nano domain vesicle and Clodronate inclusive with nanoliposomes Constructed injection type gel and Immune checkpoint inhibitor combination therapy

28 shows a schematic diagram of a combination therapy using a reversible injection-type gel composed of non-concentric multi-nanodomain vesicles and nanoliposomes and an immune checkpoint inhibitor. When the injection-type gel is used for non-immunogenic tumors, myeloid-derived immunosuppressive cells (MDSCs) and tumor-associated macrophages (Tumor- associated macrophage), making immune checkpoint inhibitors work well. Here, an immune checkpoint inhibitor (anti-PD-1, anti-PD-L1) can be treated to provide an even better anticancer immunotherapeutic effect.

In FIG. 29, the activation of the immune checkpoint system induced by the injection-type gel composed of a non-concentric multi-nanodomain vesicle and nanoliposome was confirmed. In animal experiments, BALB/c (female 5-6 weeks old, orient) mice without specific pathogens were used. First, anesthetized by injecting 0.01 ml of a 2.5% avertin (2,2,2-tribromoethanol-tert-amylacohol, Sigma-Aldrich) solution into the abdominal cavity of a mouse per body weight (g), 1x10 ⁶ breast cancer cells in the right flank Cells (4T1) were injected subcutaneously. After 14 days, when the tumor size reached about 300 mm³, surgery was performed to remove about 90% of the tumor. The remaining tumors were isolated on the 10th day after surgery, and then the cells were separated and analyzed using a flow cytometer. When the injection-type gel was used, it was confirmed that the expression level ^{of PD-1 in the T cells (CD3 +} cells) in the tumor was increased, and the expression level of ^{PD-L1 in the tumor cells (CD45 - cells) was increased.} Through this, it was confirmed that when the immune checkpoint inhibitor was used in combination, an improved immune response was exhibited than when only the injection-type gel was used.

In Figure 30, the efficacy of the injection-type gel and immune checkpoint inhibitor combination therapy was confirmed. The procedure was performed in the same manner as in FIG. 29 until the operation, and from the third day after the operation, immune checkpoint inhibitors (anti-PD-1, anti-PD-L1) were injected into mice 5 times at 200 μg each time for 2 days. . When measuring the survival rate, it was confirmed that the tumor completely disappeared in 7 out of 10 mice in the mice treated with anti-PD-L1 compared to 4 out of 10 mice using the injection gel alone, and thus the survival rate has increased significantly.

In FIG. 31, the experiment was conducted in the same manner as in FIG. 30, and the ratio of ^{immune cells (CD8 + T cells) in the tumor and spleen was compared on the 3rd day after the last immune checkpoint inhibitor injection.} Even when only the injection-type gel was used alone, the immune cell ratio was significantly increased, but it was confirmed that it was further increased when combined with anti-PD-L1. No significant change was observed with the administration of the immune checkpoint inhibitor alone without the injection-type gel.

In Figure 32, when the lymphocytes isolated from the spleen were stimulated with the tumor lysate, the degree of cytokine secretion was confirmed. As a result, it was confirmed that the secretion of cytokine (IFN-γ) was most remarkably increased when the injection gel and anti-PD-L1 were used in combination. Through this, it was confirmed that the antigen-specific immune response was increased by the combination of the immune checkpoint inhibitor.

33, it was confirmed whether the same immune response as the breast cancer model was activated through the lung cancer model (TC1). In the lung cancer model, C57/BL6 (female 5-6 weeks old, orient) mice were used, and the other experimental methods were the same as in FIG. 29 . In the lung cancer model, it was confirmed that the expression level ^{of PD-1 in the T cells (CD3 +} cells) in the tumor was increased, and the expression level of ^{PD-L1 in the tumor cells (CD45 -} cells) was increased in the same way as in the breast cancer model.

In FIG. 34 , the survival rate was measured in the same manner as in FIG. 30 . Although the survival rate was greatly increased even when only the injection-type gel was used alone, it was confirmed that the survival rate was significantly increased when used in combination with anti-PD-1. Again, no significant change was observed with the administration of the immune checkpoint inhibitor alone.

^{In FIG. 35, the ratio of immune cells (CD8 +} T cells) in the tumor and the spleen was compared in the same manner as in FIG. 31 . Similar to the breast cancer model, even when only the injection-type gel was used alone, the immune cell ratio was significantly increased, but it was confirmed that the ratio was further increased when combined with anti-PD-1 in the lung cancer model. As in the breast cancer model, no significant change was confirmed by administration of an immune checkpoint inhibitor alone without an injection-type gel.

Next, the lymphocytes isolated from the spleen were stimulated with a TC1 tumor lysate to confirm an antigen-specific immune response. Even in this case, it was confirmed that the antigen-specific immune response was increased only by the injection-type gel alone treatment, but further increased when it was used in combination with anti-PD-1 (FIG. 36).

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (23)

a non-concentric multi-nanodomain vesicle comprising two or more liposomes in contact with each other, and an outer wall that surrounds the two or more liposomes and is composed of the same component as the liposome; And as a reversible gel for parenteral drug delivery comprising a nanoliposome,
The non-concentric multi-nanodomain vesicle has a negative charge on the surface,
The nanoliposome has a positive charge on the surface,
The interior of the liposome, the interior of the nanoliposome, and the space between the outer wall and the liposome are in an aqueous phase,
The outer wall, the liposome membrane, the nanoliposome membrane, and the contact portion between the outer wall and the liposome are made of an organic phase,
The reversible gel for parenteral drug delivery is a drug in any one or more selected from the group consisting of an organic phase of a non-concentric multi-nanodomain vesicle, an aqueous phase of a non-concentric multi-nanodomain vesicle, an organic phase of nanoliposomes, and an aqueous phase of nanoliposomes. A reversible gel for parenteral drug delivery comprising a. According to claim 1,
Reversible gel for parenteral drug delivery, characterized in that the ratio of the non-concentric multi-nanodomain vesicle and the nanoliposome is 10:1 (w/w) to 10:9 (w/w). 3. The method of claim 2,
Reversible gel for parenteral drug delivery, characterized in that the ratio of the non-concentric multi-nanodomain vesicle and the nanoliposome is 10:3 (w/w). According to claim 1,
Reversible gel for parenteral drug delivery, characterized in that the non-concentric multi-nanodomain vesicle has a diameter of 1 μm to 100 μm. According to claim 1,
The reversible gel for parenteral drug delivery, characterized in that the nanoliposome has a diameter of 20 nm to 900 nm. According to claim 1,
A reversible gel for parenteral drug delivery, characterized in that the organic phase, the liposome membrane, and/or the outer wall of the non-concentric multi-nanodomain vesicle contains a hydrophobic drug. According to claim 1,
A reversible gel for parenteral drug delivery, characterized in that the aqueous phase of the non-concentric multi-nanodomain vesicle contains a hydrophilic drug. According to claim 1,
Reversible gel for parenteral drug delivery, characterized in that it contains a hydrophilic drug inside the nanoliposome. According to claim 1,
Reversible gel for parenteral drug delivery, characterized in that the membrane of the nanoliposome contains a hydrophobic drug. 10. The method of claim 6 or 9,
The hydrophobic drug is a reversible gel for parenteral drug delivery, characterized in that it is an immunoactivating substance or an immunosuppressive factor controlling substance. 9. The method of claim 7 or 8,
The hydrophilic drug is a reversible gel for parenteral drug delivery, characterized in that it is an immunoactivating substance or an immunosuppressive factor controlling substance. According to claim 1,
The reversible gel for parenteral drug delivery is a non-concentric multi-nanodomain vesicle having a negative charge on the surface and a nanoliposome having a positive charge on the surface to form a structure by electrostatic attraction. A reversible gel for parenteral drug delivery, characterized in that it does not disintegrate and is stably maintained, thereby allowing the drug to be released in a sustained release form. The reversible gel of claim 1 for parenteral drug delivery; and
A composition for drug delivery, comprising at least one selected from the group consisting of a hydrophobic drug and a hydrophilic drug. 14. The method of claim 13,
The composition is characterized in that it is used in the form of an injection, a composition for drug delivery. 14. The method of claim 13,
The composition is characterized in that it further comprises immune checkpoint inhibitors, drug delivery composition. 16. The method of claim 15,
The immune checkpoint inhibitor is anti-PD-1, anti-PD-L1, and anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti- 1 or more selected from the group consisting of GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti TIGIT Characterized in, a composition for drug delivery. The reversible gel of claim 1 for parenteral drug delivery; and
A pharmaceutical composition for preventing or treating cancer, comprising at least one selected from the group consisting of hydrophobic drugs and hydrophilic drugs. 18. The method of claim 17,
The pharmaceutical composition is characterized in that inducing immunogenic cell death (immunogenic cell death), the pharmaceutical composition. 18. The method of claim 17,
The pharmaceutical composition is characterized in that inducing the activation of dendritic cells, natural killer cells, and / or T cells, the pharmaceutical composition. 18. The method of claim 17,
The pharmaceutical composition is a pharmaceutical composition, characterized in that it inhibits the functions of regulatory T cells, myeloid derived suppressor cells, M2 macrophage. 18. The method of claim 17,
The pharmaceutical composition is characterized in that inducing a systemic antitumor immune response (systemic antitumor immune response), the pharmaceutical composition. 18. The method of claim 17,
The pharmaceutical composition is characterized in that for inhibiting cancer recurrence (recurrence), metastasis (metastasis) or resistance to anticancer therapy, the pharmaceutical composition. 18. The method of claim 17,
The pharmaceutical composition is characterized in that the injection into a local site (local injection), such as a tumor, the pharmaceutical composition.

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