CN109381428B - Nanometer medicinal preparation for treating tumor by photodynamic therapy and immunotherapy - Google Patents
Nanometer medicinal preparation for treating tumor by photodynamic therapy and immunotherapy Download PDFInfo
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Abstract
The invention belongs to the field of medicinal preparations, and relates to a nano preparation for tumor photodynamic therapy combined immunotherapy, which is a medicinal preparation with the size in a nano range and consists of a photosensitizer, an immune checkpoint blocker and auxiliary materials. After intravenous administration, the nanometer preparation enriches photosensitizer and immune checkpoint blocker at tumor part, and plays roles of photodynamic therapy and immunotherapy. Experiments show that compared with the conventional administration system and administration method, the nano preparation loaded with the medicine can improve the amount of the photosensitizer and/or the immune checkpoint blocker reaching the tumor part, and/or realize the positioning release of the medicine in the tumor, and reduce the systemic toxic and side effects while enhancing the treatment effect.
Description
Technical Field
The invention belongs to the field of medicinal preparations, and particularly relates to a novel nano preparation for combined tumor photodynamic therapy and immunotherapy, which is a medicinal preparation with a nano-scale particle size and consists of a photosensitizer, an immune check point blocker and auxiliary materials. More specifically, the invention modifies the medicine by a pharmaceutical means, and can achieve the following purposes: compared with the conventional administration system and the conventional administration method, the method can improve the amount of the photosensitizer and/or the immune checkpoint blocker reaching the tumor part, and/or realize the positioning release of the medicament in the tumor, and reduce the systemic toxic and side effects while enhancing the treatment effect.
Background
The prior art discloses that the blocking of immune check points is a novel malignant tumor treatment method after surgery, radiotherapy, chemotherapy and molecular targeted therapy, and the method has remarkable clinical treatment effect in recent years and becomes a research hotspot of tumor treatment. Immune checkpoints refer to signaling molecule pathways in the immune system that function to modulate immune cell activity, which play an important role in maintaining self-tolerance and modulating the persistence and intensity of T cell responses. Studies have shown that malignant tumors can evade immune killing by inhibiting activation of T cells through immune checkpoints.
Research reports that the monoclonal antibody is adopted to block an immune check point, so that inhibitory immune signals can be blocked, T cells are activated, and effective and lasting anti-tumor response is generated for patients; treatment with anti-immune checkpoint antibodies, such as anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibodies, can suppress regulatory T cells (tregs), increasing the ratio of CD8T cells/tregs; the treatment of an anti-Programmed cell death protein-1 (PD-1) antibody and an anti-PD-1 ligand (PD-1) antibody can block a PD-1/PD-L1 channel and improve the immunosuppressive microenvironment of tumors; practice has shown that although immune checkpoint blockade therapy works well in clinical studies, the overall response rate of patients is not high, and it is ineffective for partial patient treatment.
Photodynamic therapy is a treatment technology which combines a non-toxic/low-toxicity photosensitizer and a corresponding light source, generates active oxygen clusters through photodynamic reaction and destroys tumor tissues; the photosensitizer entering the tissue can trigger photodynamic reaction to kill target cells only when reaching a certain concentration and being irradiated by enough light, so that the photosensitizer is a high-specificity and safe local treatment method; the injury caused by the active oxygen cluster can kill tumor cells, release tumor-related antigens, simultaneously cause local acute inflammation and induce host immune response, however, the photodynamic therapy can not generate continuous and strong immune response, which is mainly due to the immunosuppressive microenvironment of the tumor and can not effectively induce T cell mediated immune response.
The results of recent experiments and clinical researches prove that the combination of blocking treatment of immune check points and photodynamic therapy can generate a synergistic treatment effect and inhibit metastatic tumors; on one hand, photodynamic therapy directly kills and kills tumor cells, so that tumor specific antigens are released, immune cells are increased to infiltrate into tumor parts, and on the other hand, the blocking of an immune check point reverses a tumor immunosuppressive microenvironment, and the photodynamic therapy is cooperated to activate immune cells of an organism, so that a host is induced to generate strong and lasting immune response, and residual tumor cells are killed.
Researchers believe that the key to achieving the above combination therapy is the design of a pharmaceutical formulation that can carry both an immune checkpoint blocker and a photosensitizer, enabling the combined delivery of these two drugs at the tumor site. At present, no research report of related preparations is found.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a novel nano preparation for combined tumor photodynamic therapy and immunotherapy.
The invention uses the pharmaceutical technology to prepare the drug form which can carry the photosensitizer and the immune checkpoint blocker at the same time and has the size in the nanometer range. The nanometer preparation can be injected intravenously to deliver photosensitizer and immune checkpoint blocker to tumor part simultaneously, and has photodynamic therapy and immunotherapy effects. Compared with the conventional drug delivery system, the nano-drug preparation can improve the amount of the photosensitizer and/or the immune checkpoint blocker reaching the tumor part, and/or realize the positioning release of the drugs in the tumor, thereby enhancing the treatment effect and reducing the systemic toxic and side effects.
More specifically, the nano preparation is a medicine preparation with the particle size in a nano range, which is composed of a photosensitizer, an immune checkpoint blocker and auxiliary materials;
the nanometer preparation can be prepared by mixing photosensitizer and immune checkpoint blocker with adjuvant solution, and stirring. The photosensitizer in the nano preparation is a photosensitizer containing a porphyrin ring structure, and can be m-tetra (hydroxyphenyl) porphyrin, 5,10,15,20-tetra (4-sodium benzenesulfonate) -21H, 23H-porphyrin, protoporphyrin IX, 5,10,15,20-tetra (1-4-methylpyridyl) porphyrin tosylate, dihydroporphin e6, benzoporphyrin derivative monocyclic acid A, chlorophyll, pheophorbide A, and the salt forms of the compounds;
in the invention, the nano preparation is prepared into liposome, wherein an immune checkpoint blocker is encapsulated in the internal water phase of the liposome, and a photosensitizer is encapsulated in a lipid bilayer.
In the invention, the immune checkpoint blocker in the nano preparation is a monoclonal antibody, the monoclonal antibody belongs to immunoglobulin G (IgG) molecules, and the monoclonal antibody can be IgG from different species, human source, mouse source and the like;
specifically, the monoclonal antibody of the point blocker can be one or more of an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody, an anti-Programmed cell death protein-1 (PD-1) antibody, an anti-Programmed death ligand-1 (PD-L1) antibody, an anti-CD 39 antibody and an anti-CD 73 antibody;
the monoclonal antibody of the checkpoint blockade agent may also be another IgG molecule.
In the invention, the auxiliary material in the nano preparation is polyvinylpyrrolidone, and the molecular weight range of the polyvinylpyrrolidone is 5000-50000 Da.
According to the invention, researches show that photosensitizer molecules with porphyrin ring structures can be stably combined with monoclonal antibodies (IgG) through intermolecular hydrophobic interaction, and the photosensitizer, the IgG and polyvinylpyrrolidone are self-assembled into a nano compound in a solution state; the affinity of the photosensitizer molecule with the porphyrin ring structure and IgG is obviously higher than that of the photosensitizer molecule and serum albumin, so that the immune checkpoint blocker IgG can obviously improve the stability of the photosensitizer in blood and the concentration of the photosensitizer in blood, thereby increasing the enrichment of the photosensitizer in tumor tissues and enhancing the photodynamic therapy effect.
In the photosensitizer, the antibody and the polyvinylpyrrolidone self-assembly nano compound, the mass ratio of the photosensitizer to the antibody is in the range of 1:0.1 to 1:5 or more; the mass ratio of the photosensitizer to the polyvinylpyrrolidone is in the range of 1:0.01 to 1:100, respectively.
The particle size of the photosensitizer, the antibody and the polyvinylpyrrolidone self-assembly nano compound is between 10nm and 200 nm.
The nano preparation related to the invention can also be a light-operated temperature-sensitive liposome, the light-operated temperature-sensitive liposome contains a photosensitizer with a porphyrin ring structure and a dipalmitoyl phosphatidylcholine thermosensitive material, the photosensitizer in the liposome absorbs light energy and converts the light energy into heat energy by utilizing the photothermal conversion characteristic of the photosensitizer with the porphyrin ring structure and irradiating the tumor part by 808nm laser, and when the local temperature of the photosensitizer is higher than the phase transition temperature (41 ℃) of dipalmitoyl phosphatidylcholine, an antibody encapsulated in the liposome is released, so that the blocking of an immune check point is realized; after the photosensitizer is taken up by tumor cells, the photodynamic therapy of the tumor cells is realized through 660nm laser irradiation, and the liposome has the characteristic of optically controlled release of drugs in tumors.
In the invention, an immune checkpoint blocker antibody (IgG) is entrapped in an internal water phase of the liposome through a preparation method of the liposome, a photosensitizer is entrapped in a lipid bilayer, and the enrichment of the photosensitizer and the antibody in a tumor is improved by utilizing the long circulation effect of the liposome in blood and the Enhanced Penetration and Retention (EPR) effect of the liposome in a tumor tissue.
In the invention, the photosensitizer carried by the liposome is a photosensitizer containing a porphyrin ring structure, and can be m-tetra (hydroxyphenyl) porphyrin, 5,10,15,20-tetra (4-sodium benzenesulfonate) -21H, 23H-porphyrin, protoporphyrin IX, 5,10,15,20-tetra (1-4-methylpyridyl) porphyrin tosylate, dihydroporphine 6, benzoporphyrin derivative monocyclic acid A, chlorophyll, pheophorbide A and the salt forms of the compounds;
the immune checkpoint blocker antibody (IgG) carried by the liposome can be one or more of an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody, an anti-Programmed cell death protein-1 (PD-1) antibody, an anti-Programmed death ligand 1 (PD-L1) antibody, an anti-CD 39 antibody and an anti-CD 73 antibody;
the liposome-entrapped immune checkpoint blocker antibody (IgG) may be other IgG molecules.
In the invention, an emulsion film dispersion method, a reverse phase evaporation method and a secondary emulsification method are adopted to prepare liposome; wherein the content of the first and second substances,
film dispersion method: fully dissolving and uniformly mixing phospholipid, cholesterol, pegylated phospholipid and photosensitizer in an organic solvent (generally chloroform or a chloroform-methanol mixed solution), gradually decompressing and evaporating until the organic solvent is completely volatilized, obtaining a layer of dried phospholipid membrane at the bottom of a bottle, adding a proper volume of buffer salt (generally a phosphate buffer solution) containing an antibody for hydration to obtain liposome, and preparing the liposome meeting the particle size requirement by adopting methods such as ultrasound, microjet, high-pressure homogenization or film extrusion and the like;
reverse phase evaporation method: fully dissolving phospholipid, cholesterol, polyethylene glycol phospholipid and photosensitizer in an organic solvent (usually diethyl ether and chloroform) immiscible with water, fully mixing with a buffer salt water phase (usually phosphate buffer solution) containing an antibody, forming a water-in-oil emulsion by using methods such as ultrasound, homogenization and the like, placing in a round-bottomed flask, gradually reducing pressure, steaming to remove the organic solvent to obtain a gel-state substance, adding a buffer solution, and fully shaking to obtain a liposome;
a secondary emulsification method: mixing the first water phase and oil by a method similar to reverse phase evaporation method to obtain water-in-oil emulsion, adding the emulsion into the second water phase to form water-in-oil-in-water type multiple emulsion, evaporating under reduced pressure, and removing organic solvent to obtain liposome;
the liposome may also contain cholesterol and/or other phospholipid components, such as one or more of distearoyl phosphatidylcholine, hydrogenated soybean phospholipid, lecithin, and 1-myristoyl-2-stearoyl lecithin;
the pegylated phospholipid in the liposome is pegylated distearoyl phosphatidyl ethanolamine. Wherein the molecular weight of the polyethylene glycol ranges from 1000 to 6000. The function of the pegylated phospholipid is to provide hydrophilic polymer chains polyethylene glycol (PEG) at the surface of the liposomes. The polyethylene glycol is usually monomethoxy polyethylene glycol, and the liposome modified on the surface of the PEG can reduce the adsorption of plasma protein, avoid the phagocytosis of a mononuclear phagocyte system, play a role in invisibility, obviously prolong the half-life period of the plasma, thereby increasing the chance of reaching a tumor part and improving the possibility of drug delivery in tumors.
The invention provides a novel nano preparation for combined tumor photodynamic therapy and immunotherapy, which is a medicinal preparation with the particle size in a nano range and consists of a photosensitizer, an immune checkpoint blocker and auxiliary materials. Compared with the conventional drug delivery system and the drug delivery method, the prepared nano preparation can improve the amount of the photosensitizer and/or the immune checkpoint blocker reaching the tumor part, and/or realize the positioning release of the drug in the tumor, and reduce the systemic toxic and side effects while enhancing the treatment effect.
Drawings
Figure 1. Ce 6-time curve in blood after intravenous injection of α PD-L1-Ce6-PVP nano or Ce6-PVP solution at the tail of mice,% ID/mL, which is the percentage of Ce6 per mL of blood in the injected dose,% p <0.05, n =3.
Fig. 2 shows the fluorescence distribution in each tissue after GL261 brain glioma-bearing mouse tail vein injection of alpha PD-L1-Ce6-PVP nano or Ce6-PVP solution for 1h,% ID, which is the percentage of Ce6 in each tissue organ per unit area in the injection dose,% p <0.05, n =3.
Detailed Description
Example 1
Taking rat anti-mouse PD-L1 monoclonal antibody (alpha PD-L1), chlorin e6 (Ce 6) and polyvinylpyrrolidone (PVP) as examples, preparing a self-assembled nano compound, and describing the prescription process, the preparation process and the tumor drug delivery characteristics of the nano compound;
1) Prescription and preparation process
A phosphate buffer (0.01m, ph = 7.4) containing PVP (MW =10000 Da) at a concentration of 6.7mg/mL was prepared. This solution was measured out in 15. Mu.L (containing PVP 0.1 mg), added dropwise to 60. Mu.L of phosphate buffer (0.01M, pH = 7.4) containing 0.2mg of. Alpha.PD-L1 under magnetic stirring at 500rpm, and stirring was continued for 5min. Weighing 4mg Ce6, dissolving in 1mL of 0.1% NaOH solution, sucking 25 μ L of Ce6 solution, dropwise adding into the mixed solution of alpha PD-L1 and PVP, continuously stirring for 5min, and uniformly mixing to obtain alpha PD-L1-Ce6-PVP nano solution. A phosphate buffer (0.01M, pH = 7.4) is used for replacing the phosphate buffer containing alpha PD-L1 to prepare a Ce6-PVP solution as an experiment control group;
particle size distribution: the particle size of the alpha PD-L1-Ce6-PVP nano-particle is about 30nm through observation of a transmission electron microscope;
2) Kinetic analysis of Ce6, ce6+ PVP with α PD-L1, human Serum Albumin (HSA), mouse Serum Albumin (MSA)
Coupling of proteins to CM5 chips, kinetic analysis of mixtures of compounds Ce6, ce6 and PVP (1, w/w) with α PD-L1, HSA, MSA proteins was performed using a biomacromolecule interaction apparatus (Biacore T200). The solution system is HBS-EP + buffer solution, and the pH is =7.4;
the results show that Ce6The affinity value (KD) with alpha PD-L1 is 6.096X 10 -8 The affinity value (KD) of M, ce6 and MSA is 1.617X 10 -6 And M. The affinity value (KD) of Ce6 for HSA was 2.118X 10 -6 M, ce6 has 26.5 times greater affinity for α PD-L1 than for MSA; 34.7 times that of HSA, and the affinity value (KD) of Ce6 to α PD-L1 was 7.002 × 10 in the case of Ce6 mixed with PVP (1, w/w) -8 M, demonstrating that PVP in the formulation does not significantly affect the binding of Ce6 to α PD-L1; the affinity experiment result shows that Ce6 and alpha PD-L1 have natural strong affinity and can be self-assembled into a nano solution. The affinity between the two is higher than that between Ce6 and serum albumin, and alpha PD-L1 can play a role in stabilizing Ce6 in blood;
3) Comparison of pharmacokinetics in mice after intravenous injection of alpha PD-L1-Ce6-PVP nano and Ce6-PVP solution
Injecting alpha PD-L1-Ce6-PVP nano (containing 0.2mg of alpha PD-L1 and 0.1mg of Ce6) or Ce6-PVP solution (0.1mg of Ce6) into the tail vein of a C57BL/6 mouse, taking 20 mu L of blood from the tail vein after 0.5h, 1h, 2h, 4h, 8h, 12h and 24h after administration, placing the blood in a plastic centrifuge tube, performing near infrared optical imaging, quantitatively analyzing the fluorescence intensity in the blood, and calculating the drug concentration in the blood by taking blank blood before administration as a control group;
the results show that the alpha PD-L1-Ce6-PVP nanometer can improve the stability of Ce6 in blood and increase the concentration in blood, and fig. 1 shows that the fluorescence intensity in blood is significantly higher than that of the Ce6-PVP solution group between 0.5h and 4h after the alpha PD-L1-Ce6-PVP nanometer is injected into the tail vein of a C57BL/6 mouse, for example, the Ce6 concentration in blood of the alpha PD-L1-Ce6-PVP nanometer group is 1.64 times that of the Ce6-PVP solution group at 1h after injection;
4) Comparison of biodistribution of alpha PD-L1-Ce6-PVP nano and Ce6-PVP solution in vivo in brain-bearing glioma mice after intravenous injection for 1h
Intracranial injection of 5X 10C 57BL/6 mice 5 GL261 brain glioma cell (5 muL), intracranial in-situ brain glioma model establishment 13 days later, tail vein injection alpha PD-L1-Ce6-PVP nanometer (containing 0.2mg alpha PD-L1 and 0.1mg Ce6) or Ce6-PVP solution (0.1mg Ce6), tail vein blood sampling 20 muL after 1h after administration, then sacrifice, separation to tissue organs, near infrared optical imaging, quantitative analysis of tissue organs in each tissue organFluorescence intensity, which is calculated for each tissue organ;
the results are shown in fig. 2, and the fluorescence intensity of Ce6 in the brain tumor of alpha PD-L1-Ce6-PVP nano group is 1.85 times higher than that of the Ce6-PVP solution group at 1h after injection;
the experimental result proves that after the three components of the alpha PD-L1, the Ce6 and the PVP are self-assembled to form the nano composite, compared with the physical composition of the two components of the Ce6 and the PVP, the stability of the Ce6 in blood can be improved, the effective concentration of the Ce6 in blood can be increased, and the concentration of the Ce6 in tumors can be improved.
Example 2
Rat serum IgG, ce6 and PVP are taken as examples to prepare the self-assembly nano compound, and the prescription process, the preparation process and the tumor drug delivery characteristics of the nano compound are explained.
Rat serum IgG replaces alpha PD-L1, the preparation process of the embodiment 1 is adopted to prepare IgG-Ce6-PVP nano solution, the particle size is about 30nm through observation of a transmission electron microscope, and the affinity value (KD) of Ce6 and rat serum IgG is 7.394 multiplied by 10 measured by a binding kinetics experiment -8 M, 1h after intravenous injection of the tail of the mouse, the Ce6 concentration in blood of the IgG-Ce6-PVP nano group is 1.68 times that of the Ce6-PVP solution group; the Ce6 fluorescence intensity in the brain tumor of the IgG-Ce6-PVP nano group is 1.71 times that of the Ce6-PVP solution group;
experimental results prove that after rat IgG, ce6 and PVP are self-assembled to form a nano-composite, ce6 and IgG have high affinity similar to alpha PD-L1, the stability of Ce6 in blood can be improved, the effective concentration of Ce6 in blood can be increased, and the concentration of Ce6 in tumors can be increased.
Example 3
Taking humanized anti-CTLA-4 monoclonal antibody IgG (alpha CTLA-4), pheoA and PVP as examples, a self-assembly nano-composite is prepared, and the prescription process, the preparation process and the tumor drug delivery characteristics of the nano-composite are described.
A phosphate buffer (0.01m, ph = 7.4) containing PVP (MW =10000 Da) at a concentration of 6.7mg/mL was prepared. This solution was measured out in 15. Mu.L (containing PVP 0.1 mg), added dropwise to 60. Mu.L of phosphate buffer (0.01M, pH = 7.4) containing 0.2mg of α CTLA-4 under magnetic stirring at 500rpm, and stirred for 5min. Weighing 4mg of pheophorbide A, dissolving the pheophorbide A in 1mL of 0.05-percent NaOH solution, sucking 50 μ L of the pheophorbide A solution, dropwise adding the pheophorbide A solution into the mixed solution of the alpha CTLA-4 and PVP, continuously stirring for 5min, uniformly mixing to obtain an alpha CTLA-4-PheoA-PVP nano solution, and replacing the phosphate buffer solution containing the alpha CTLA-4 with a phosphate buffer solution (0.01M, pH = 7.4) to prepare a PheoA-PVP solution as an experimental control group;
the particle size of the alpha CTLA-4-PheoA-PVP nano-particles is about 45nm through observation of a transmission electron microscope, the blood PheoA concentration of the alpha CTLA-4-PheoA-PVP nano group is 2.34 times that of the PheoA-PVP solution group after 1 hour of mouse tail vein injection; the PhoA fluorescence intensity of the alpha CTLA-4-PhoA-PVP nano group in BALB/c mouse 4T 1-charged breast cancer in situ tumor is 2.51 times that of the PhoA-PVP solution group;
the experimental result proves that after the humanized anti-CTLA-4 monoclonal antibody IgG, the PheoA and the PVP form a nano compound through self-assembly, the humanized anti-CTLA-4 monoclonal antibody IgG can improve the stability of the PheoA in blood, increase the effective concentration of the PheoA in blood and improve the concentration of the PheoA in tumor;
the results of examples 1,2 and 3 above demonstrate that an immune checkpoint blocker of a monoclonal antibody having an IgG molecular structure has a strong affinity for a photosensitizer molecule having a porphyrin ring structure, and can improve the stability in blood and the concentration of the photosensitizer in a tumor.
Example 4
Rat anti-mouse PD-L1 monoclonal antibody IgG (alpha PDL-1), hamster anti-mouse CTLA-4 monoclonal antibody IgG (alpha CTLA-4) and pheoA are taken as examples to prepare liposome (alpha PD-L1/alpha CTLA-4-pheoA) loaded with the alpha PDL-1, the alpha CTLA-4 and the pheoA, and the prescription process, the preparation process and the tumor drug delivery characteristics of the liposome are explained.
Weighing the following materials in the basic formula according to the molar ratio: dipalmitoylphosphatidylcholine (DPPC)/cholesterol/PheoA/pegylated distearoylphosphatidylethanolamine (DSPE-PEG 2000) (55: dissolving the nano material by adding chloroform, placing the nano material in a solanaceous bottle, reducing pressure, rotating and evaporating to remove the chloroform, ultrafiltering and concentrating alpha PD-L1 and alpha CTLA-4 to PBS (phosphate buffer solution) containing 10mg/mL of the two monoclonal antibodies respectively, adding 1mL of the solution into the solanaceous bottle, hydrating for 2 days at 4 ℃, carrying out vortex oscillation and dispersion, repeatedly extruding a drug-loaded lipid hydration solution to pass through a 100nm nuclear pore membrane, eluting and removing free antibodies through a Sepharose CL-4B gel column to obtain a drug-loaded liposome;
the alpha PD-L1/alpha CTLA-4-PheoA liposome is prepared by adopting a film hydration combined extrusion method, and the nano particles are observed to be of a multi-vesicular structure under a transmission electron microscope, and the average particle size is 140nm. The encapsulation efficiency of the alpha PD-L1 and the alpha CTLA-4 is 18.4 +/-3.4 percent and 19.1 +/-1.6 percent respectively;
under the action of 808nm laser, passing through a 0.5W/cm 2 After irradiating for 5min, the solution temperature reaches the phase transition temperature of the thermosensitive nano material DPPC, the coated antibody alpha PD-L1 is released by more than 95%, the coated antibody alpha PD-L1 is released by less than 5% without 808nm laser irradiation, the nanoparticle generates singlet oxygen under the action of 660nm laser, and 808nm laser (0.5W/cm) 2 ) Irradiation for 5min has little effect on its ability to generate singlet oxygen;
after intravenous injection of the alpha PD-L1/alpha CTLA-4-PheoA liposome for 6 hours, the PheoA fluorescence intensity of BALB/c mouse with 4T1 breast cancer in situ tumors is 2.54 times that of the pure PheoA solution;
after the IRDye800CW marked alpha PD-L1/alpha CTLA-4-PheoA liposome is injected for 6 hours intravenously, the fluorescence intensity of IRDye800CW marked alpha PD-L1 in BALB/c mouse 4T 1-bearing breast cancer in situ tumor is 2.03 times of that of a simple IRDye800CW marked alpha PD-L1 solution group;
the tumor-bearing mice receive 808nm laser (0.5W/cm) after intravenous administration of the drug-loaded nanoparticles for 6h 2 ) Irradiating for 3min, wherein the average tumor temperature reaches 42 ℃, and the temperature requirement of optically controlling the release of the antibody is realized.
Example 5
Taking humanized anti-human PD-1 monoclonal antibody IgG (alpha PD-1) and protoporphyrin IX (PpIX) as examples, preparing liposome (alpha PD-1-PpIX) loaded with the alpha PD-1 and the protoporphyrin IX, and describing the prescription process, the preparation process and the tumor drug delivery characteristics of the liposome;
adopting a reverse phase evaporation method, fully dissolving 2.8mg of DPPC, 0.93mg of cholesterol, 0.77mg of polyethylene glycol phospholipid (DSPE-PEG 2000,0.77 mg) and 0.2mg of photosensitizer PpIX in 1mL of chloroform, fully mixing with 0.33mL of phosphate buffer solution containing alpha PD-1 antibody (0.5 mg), forming a water-in-oil emulsion by using an ultrasonic method, placing in a round bottom flask, gradually reducing pressure and steaming to remove an organic solvent to obtain a gel state substance, adding the buffer solution, and fully shaking; repeatedly squeezing the drug-loaded lipid hydration solution to pass through a 100nm nuclear pore membrane, eluting and removing free antibodies through a Sepharose CL-4B gel column to obtain the alpha PD-1-PpIX liposome;
the nanoparticles are observed to be of a multi-vesicular structure under a transmission electron microscope, the average particle diameter is 130nm, the encapsulation rate of alpha PD-1 is 55.3 +/-6.7%, and the nanoparticles are encapsulated by 0.5W/cm under the action of 808nm laser 2 After 5min of irradiation, the entrapped antibody alpha PD-1 is released by more than 95%, the nanoparticles generate singlet oxygen under the action of 660nm laser, and 808nm laser (0.5W/cm) 2 ) Irradiation for 5min has little effect on its ability to generate singlet oxygen;
after the alpha PD-1-PpIX liposome is injected intravenously for 6h, the PpIX fluorescence intensity in the breast cancer in situ of the lotus 4T1 live tumor of a BALB/c mouse is 3.12 times that of a pure PpIX solution group;
after 6h of intravenous injection of IRDye800CW marked alpha PD-1-PpIX liposome, the fluorescence intensity of IRDye800CW marked alpha PD-1 in BALB/c mouse 4T 1-bearing breast cancer orthotopic tumor is 2.34 times of that of a simple IRDye800CW marked alpha PD-1 solution group;
the tumor-bearing mice receive 808nm laser (0.5W/cm) after intravenous administration of the drug-loaded nanoparticles for 6h 2 ) Irradiating for 3min, wherein the average tumor temperature reaches 42 ℃, and the temperature requirement of optically controlling the release of the antibody is realized.
The present invention is illustrated by the above description and examples, which are not limitative and do not limit the scope of the claims of the present invention.
Claims (6)
1. A nanometer preparation for photodynamic therapy and combined immune checkpoint blockade therapy of tumors is characterized in that the nanometer preparation is prepared by mixing photosensitizer containing porphyrin ring structure, monoclonal antibody of immune checkpoint blockade agent and polyvinylpyrrolidone as adjuvant, wherein the photosensitizer containing porphyrin ring structure is chlorin e6 and/or pheophorbide A;
the monoclonal antibody is an immunoglobulin G molecule, the monoclonal antibody is selected from one or more of an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody, an anti-Programmed cell death protein-1 (PD-1) antibody and an anti-Programmed death ligand-1 (PD-L1) antibody, or,
the nanometer preparation is a light-controlled temperature-sensitive liposome prepared from a photosensitizer with a porphyrin ring structure, an immune checkpoint blocker monoclonal antibody and auxiliary materials of phospholipid, cholesterol and pegylated phospholipid, wherein the immune checkpoint blocker monoclonal antibody is encapsulated in an internal water phase of the liposome, the photosensitizer with a porphyrin ring structure is encapsulated in a lipid bilayer of the liposome, the phospholipid contains dipalmitoyl phosphatidylcholine, the photosensitizer with a porphyrin ring structure is demagnetised chlorophyllin A and/or protoporphyrin IX, the light-controlled temperature-sensitive liposome is irradiated by laser of 808nm, the photosensitizer in the liposome absorbs light energy and converts the light energy into heat energy, and when the local temperature of the photosensitizer is higher than the phase transition temperature of the dipalmitoyl phosphatidylcholine, the immune checkpoint blocker monoclonal antibody encapsulated in the liposome is released; the monoclonal antibody is an immunoglobulin G molecule, and is selected from one or more of an anti-cytotoxic T lymphocyte antigen-4 (CTLA-4) antibody, an anti-Programmed cell death protein-1 (PD-1) antibody and an anti-Programmed death ligand-1 (PD-L1) antibody.
2. The nano-preparation for photodynamic therapy in combination with immune checkpoint blockade therapy according to claim 1, wherein the nano-preparation is prepared from the photosensitizer containing a porphyrin ring structure, the monoclonal antibody of the immune checkpoint blockade agent and polyvinylpyrrolidone as a pharmaceutic adjuvant by a solution stirring method.
3. The Nanopropreparate for photodynamic therapy of tumor in combination with immune checkpoint blockade therapy according to claim 1, wherein said pegylated phospholipid is pegylated distearoylphosphatidylethanolamine.
4. The Nanopropreparate for the photodynamic therapy of cancer in combination with immune checkpoint blockade therapy according to claim 1, wherein said phospholipid additionally or alternatively does not comprise distearoylphosphatidylcholine, hydrogenated soya phospholipid, lecithin, 1-myristoyl-2-stearoyl lecithin.
5. The NanoPremulation for photodynamic therapy of tumor in combination with immune checkpoint blockade therapy according to claim 1, wherein mannitol, a lyoprotectant, is added to the preparation to prepare a lyophilized preparation.
6. The nano-formulation for use in the photodynamic therapy of tumor in combination with immune checkpoint blockade therapy as claimed in claim 1, wherein the particle size of said nano-formulation is 10 to 1000 nm.
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