CN109381428A - Nanometer formulation for the treatment of photodynamic therapy combined immunization - Google Patents
Nanometer formulation for the treatment of photodynamic therapy combined immunization Download PDFInfo
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- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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Abstract
The invention belongs to field of pharmaceutical preparations, it is related to the nanometer formulation treated for photodynamic therapy combined immunization, which orders blocking agent by photosensitizer, immunologic test and auxiliary material constitutes size in the pharmaceutical dosage form of nanometer range.The nanometer formulation makes photosensitizer and immunologic test order blocking agent and is enriched in tumor locus, play optical dynamic therapy and Immunotherapy after intravenously administrable.The results showed, compared with conventional dosage systems and medication, drug is loaded into the nanometer formulation, it can improve photosensitizer and/or immunologic test orders blocking agent and reaches the amount of tumor locus, and/or realize the positioning release of drug in tumour, while enhancing therapeutic effect, systemic toxic side effect is reduced.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, and in particular to a kind of novel treats for photodynamic therapy combined immunization
Nanometer formulation, it by photosensitizer, immunologic test order partial size that blocking agent and auxiliary material form nanometer range pharmaceutical dosage form.
More specifically, the present invention modifies drug by galenic pharmacy means, can reach following purpose: with conventional dosage systems and administration
Method is compared, and can be improved photosensitizer and/or immunologic test orders blocking agent and reaches the amount of tumor locus, and/or realize in tumour
The positioning of drug discharges, and while enhancing therapeutic effect, reduces systemic toxic side effect.
Background technique
Blocking prior art discloses immunologic test point be after operation, radiotherapy, chemotherapy, molecular targeted therapy another
Novel treating malignant tumor method, achieves significant clinical therapeutic efficacy in recent years, it has also become the research heat of oncotherapy
Point.Immunologic test point refers to the signaling molecule access for playing in immune system and adjusting immunologic cellular activity, it is for maintaining certainly
The duration and intensity of body tolerance and regulatory T-cell response play a significant role.Studies have shown that malignant tumour can be by immune
Checkpoint inhibits the activation of T cell, to escape immunologic cytotoxicity.
Studies have reported that inhibition immune signal can be blocked using monoclonal antibodies block immunologic test point, T is activated
Cell makes patient generate effective and lasting antitumor response；Anti-immunity checkpoint antibody, such as anti-cell toxic T lymphocyte
Antigen -4 (CTLA-4) Antybody therapy can inhibit regulatory T cells (Treg), increase cd8 t cell/Treg ratio；Anti- journey
(programmed cell death protein 1, the PD-1) antibody of programmed cell death albumen -1 and anti-PD-1 ligand
(Programmed death-ligand 1, PD-L1) Antybody therapy, can block PD-1/PD-L1 access, improve exempting from for tumour
Epidemic disease inhibition microenvironment；Practice display, although immunologic test point Blocking therapy achieves treatment effect well in clinical studies
Fruit, but the global response rate of patient is not high, it is invalid to some patients' treatment.
Photodynamic therapy is a kind of joint nontoxicity/hypotoxicity photosensitizer and corresponding light source, anti-by photodynamics
Reactive oxygen species should be generated, the treatment technology of tumor tissues is destroyed；Photosensitizer due to entering tissue only reaches a certain concentration simultaneously
It is irradiated by sufficient light, can just cause photodynamics reaction and kill target cell, therefore it is a kind of high specific and safety
Local therapeutic approaches；Damage caused by reactive oxygen species causes part while killing tumour cell, release tumor related antigen
Acute inflammation induces host immune response, however photodynamic therapy can not generate lasting, strong immune response, this is main
It is the inhibitive ability of immunity microenvironment due to tumour, it cannot effective inducing T cell mediated immunity response.
Experiment and clinical study results in recent years confirms, the blocking treatment of immunologic test point is mutually tied with photodynamic therapy
It closes, synergistic therapeutic effect can be generated, and be able to suppress metastatic tumo(u)r；Wherein, on the one hand, optical dynamic therapy directly kills,
Killing tumor cell is released tumour specific antigen, increases immunocyte and invades profit to tumor locus, another aspect is led to
The microenvironment of the blocking reversing tumor inhibitive ability of immunity of immunologic test point is crossed, collaboration photodynamic therapy activates immune cell,
It induces host to generate strong lasting immune response, kills residual tumor cells.
Researcher thinks, realizes that the key of above-mentioned combination therapy is that design provides a kind of pharmaceutical preparation, it can take simultaneously
It carries immunologic test and orders blocking agent and photosensitizer, realize that both drugs are delivered in the joint of tumor locus.Currently, there is not yet related
The research of preparation is reported.
Summary of the invention
The purpose of the present invention is in view of the drawbacks of the prior art, providing, one kind is novel to combine for photodynamic therapy
The nanometer formulation of immunization therapy.
The present invention uses galenic pharmacy technology, and preparation can carry photosensitizer simultaneously and immunologic test orders blocking agent, and size exists
The pharmaceutical dosage form of nanometer range.The nanometer formulation intravenous administration, can convey photosensitizer simultaneously and immunologic test orders blocking agent
To tumor locus, optical dynamic therapy and Immunotherapy are played.The nano-drug preparation, can compared with conventional dosage systems
It improves photosensitizer and/or immunologic test and orders blocking agent and reach the amount of tumor locus, and/or realize that the positioning of drug in tumour is released
It puts, while enhancing therapeutic effect, reduces systemic toxic side effect.
More specifically, nanometer formulation of the invention orders the partial size that blocking agent and auxiliary material form by photosensitizer, immunologic test
In the pharmaceutical dosage form of nanometer range；
The nanometer formulation can be mixed by the way that photosensitizer and immunologic test are ordered blocking agent with auxiliary material solution, in the method for stirring
It is made.Photosensitizer in the nanometer formulation is the photosensitizer containing porphyrin ring structure, can be m- four (hydroxy phenyl) porphyrins,
5,10,15,20- tetra- (4- benzene sulfonic acid sodium salt) -21H, 23H- porphyrin, protoporphyrin IX, 5,10,15,20- tetra- (1-4- picolyl)
Porphyrin toluene fulfonate, chlorin e 6, Benzoporphyrin derivative monoacid ring A, chlorophyll, pheophorbide salt A and these
The form of the salt of compound；
In the present invention, which is made liposome, and the blocking of immunologic test point is wherein contained in liposome interior water phase
Agent contains photosensitizer in lipid bilayer.
In the present invention, it is monoclonal antibody that the immunologic test in the nanometer formulation, which orders blocking agent, which is to belong to
In immunoglobulin G (IgG) molecule, which can be the IgG in different genera source, can be source of people, source of mouse
Etc.；
Specifically, the described monoclonal antibody for ordering blocking agent can be anti-cell toxic T lymphocyte antigen -4
(CTLA-4) antibody, anti-apoptosis albumen -1 (programmed cell death protein 1, PD-1) are anti-
Body, anti-programmed death ligand 1 (Programmed death-ligand 1, PD-L1) antibody, 9 antibody of AntiCD3 McAb, anti-CD73 are anti-
One of body is several；
The monoclonal antibody for making an inventory of blocking agent is also possible to other IgG molecules.
In the present invention, the auxiliary material in the nanometer formulation is polyvinylpyrrolidone, the molecular weight model of polyvinylpyrrolidone
5000 are trapped among to 50000Da.
In the present invention, studies have shown that has the photosensitizer molecule of porphyrin ring structure can be with by intermolecular hydrophobic effect
Monoclonal antibody (IgG) stable bond, under solution state, photosensitizer, IgG, polyvinylpyrrolidone three are self-assembled into and receive
Rice compound；The affinity of photosensitizer molecule and IgG with porphyrin ring structure be significantly higher than its with it is sero-abluminous affine
Power, therefore, immunologic test, which orders blocking agent IgG, can significantly improve stability of such photosensitizer in blood, improve photosensitizer and exist
Concentration in blood enhances optical dynamic therapy effect to increase it in the enrichment of tumor tissues.
In the present invention, in the photosensitizer, antibody, polyvinylpyrrolidone self-assembled nanometer compound, photosensitizer
With the quality of antibody than range between 1:0.1 to 1:5；The quality of photosensitizer and polyvinylpyrrolidone is than range in 1:0.01
To between 1:100.
The photosensitizer, antibody, polyvinylpyrrolidone self-assembled nanometer compound partial size in 10nm to 200nm
Between.
Nanometer formulation according to the present invention is also possible to light-operated temperature sensitive liposome, which, which contains, has
The photosensitizer and dipalmitoylphosphatidylcholine thermo-sensitive material of porphyrin ring structure, utilize the light of the photosensitizer with porphyrin ring structure
Thermal transition characteristic is irradiated by 808nm laser in tumor locus, and the sensitiser absorption luminous energy in liposome is converted into thermal energy, when
When its local temperature is higher than phase transition temperature (41 DEG C) of dipalmitoylphosphatidylcholine, the antibody of package-contained is released in liposome
It puts, realizes the blocking of immunologic test point；After photosensitizer is absorbed by tumour cell, realize through 660nm laser irradiation to tumour cell
Optical dynamic therapy, the liposome have in tumour can it is light-operated release drug characteristic.
In the present invention, immunologic test will be contained by the preparation method of liposome in liposome interior water phase orders blocking agent and resist
Body (IgG) contains photosensitizer in lipid bilayer, using long circulating action of the liposome in blood, and its in tumor tissues
In increase penetrate be detained (Enhanced Permeability and Retention, EPR) effect, improve photosensitizer and
Enrichment of the antibody in tumour.
In the present invention, the photosensitizer of liposome package-contained is the photosensitizer containing porphyrin ring structure, can be m- four (hydroxyl
Base phenyl) porphyrin, 5,10,15,20- tetra- (4- benzene sulfonic acid sodium salt) -21H, 23H- porphyrin, protoporphyrin IX, tetra- (1- of 5,10,15,20-
4- picolyl) porphyrin toluene fulfonate, chlorin e 6, Benzoporphyrin derivative monoacid ring A, chlorophyll, de-magging leaf be green
The form of the salt of hydrochlorate A and these compounds；
The immunologic test of liposome package-contained orders blocking agent antibody (IgG) and can be anti-cell toxic T lymphocyte antigen-
4 (CTLA-4) antibody, anti-apoptosis albumen -1 (programmed cell death protein 1, PD-1) are anti-
Body, anti-programmed death ligand 1 (Programmed death-ligand 1, PD-L1) antibody, 9 antibody of AntiCD3 McAb, anti-CD73 are anti-
One of body is several；
The immunologic test of liposome package-contained orders blocking agent antibody (IgG) and can be other IgG molecule
In the present invention, liposome is prepared using emulsification film dispersion method, reverse phase evaporation, second emulsifying method；Wherein,
Film dispersion method: phosphatide, cholesterol, pegylated phospholipids and photosensitizer are sufficiently dissolved to mixing organic molten
In agent (generally chloroform or chloroform: methanol mixed solution), decompression step by step is evaporated up to organic solvent and thoroughly volatilizes, at bottom of bottle
One layer of dry immobilized artificial membrane is obtained, buffer salt antibody-containing (generally phosphate buffer) aquation of appropriate volume is added, i.e.,
Liposome is obtained, the methods of ultrasound, microjet, high-pressure homogeneous or film extrusion are taken to obtained liposome, preparation meets grain
The liposome that diameter requires；
Reverse phase evaporation: phosphatide, cholesterol, pegylated phospholipids and photosensitizer are substantially dissolved in unmixing with water
Organic solvent (usually ether, chloroform) in, it is abundant with buffer salt water phase (usually phosphate buffer) antibody-containing
Mixing forms it into water-in-oil emulsion using the methods of ultrasound and homogeneous, sets decompression step by step evaporation in a round bottom flask and has flung to
Solvent obtains gel state substance, and addition buffer, which fullys shake, can be obtained liposome；
Second emulsifying method: the first water phase and oil are mixed to prepare water in oil emulsion using method similar with reverse phase evaporation
Agent, by this emulsion be added the second water phase in, that is, form the emulsion of water-in-oil-in water, be evaporated under reduced pressure, remove organic solvent to get
Liposome；
Cholesterol and/or other phospholipid compositions can also be added in above-mentioned liposome, can be distearoylphosphatidyl
The one or several kinds of choline, hydrogenated soya phosphatide, lecithin, 1- myristoyl -2- stearyl lecithin；
Pegylated phospholipids in above-mentioned liposome are Pegylation Distearoyl Phosphatidylethanolamine.Wherein, gather
Molecular weight glycol range is between 1000 to 6000.The effect of pegylated phospholipids is hydrophilic in the offer of the surface of liposome
Property macromolecular chain polyethylene glycol (PEG).The polyethylene glycol is usually mono methoxy polyethylene glycol, by the rouge of PEG surface modification
Plastid, it is possible to reduce plasma protein absorption avoids the phagocytosis of mononuclear phagocyte system, plays the role of " stealth ", significantly extends its blood
Half-life period is starched, to increase its chance for reaching tumor locus, improves a possibility that passing medicine in tumour.
The present invention provides a kind of novel nanometer formulations for the treatment of photodynamic therapy combined immunization, by photosensitive
Agent, immunologic test order blocking agent and auxiliary material composition partial size nanometer range pharmaceutical dosage form.With conventional dosage systems and administration
Method is compared, and the nanometer formulation of preparation can improve photosensitizer and/or immunologic test orders blocking agent and reaches the amount of tumor locus, and/
Or realize the positioning release of drug in tumour, while enhancing therapeutic effect, reduce systemic toxic side effect.
Detailed description of the invention
The Ce6- time is bent in blood after PD-L1-Ce6-PVP nanometers of Fig. 1 mouse tail vein injection α or Ce6-PVP solution
Line, %ID/mL account for the percentage of injection dosage, * p < 0.05, n=3 for Ce6 in every milliliter of blood.
After PD-L1-Ce6-PVP nanometers of Fig. 2 lotus GL261 glioma mouse tail vein injection α or Ce6-PVP solution 1h
Fluorescence distribution in each tissue, %ID account for the percentage of injection dosage for Ce6 in each tissue internal organs of unit area, and * p <
0.05, n=3.
Specific embodiment
Embodiment 1
With rat anti-mouse PD-L1 monoclonal antibody (α PD-L1), chlorin e 6 (Ce6), polyvinylpyrrolidone
(PVP) for, self-assembled nanometer compound is prepared, illustrates that formulation and technology, preparation process and its tumour of the nano-complex are passed
The characteristic of medicine；
1) prescription and preparation process
Prepare the phosphate buffer (0.01M, pH=7.4) for being 6.7mg/mL containing PVP (MW=10000Da) concentration.It measures
15 μ L (0.1mg containing PVP) of this solution is added dropwise to the phosphorus that 60 μ L contain 0.2mg α PD-L1 under 500rpm magnetic agitation
In acid buffer (0.01M, pH=7.4), continue to stir 5min.4mg Ce6 is weighed, the 0.1%NaOH for being dissolved in 1mL is molten
It in liquid, draws the 25 μ L Ce6 solution and is added dropwise in the mixed solution of above-mentioned α PD-L1 and PVP, continue to stir 5min, mixing is equal
Up to α PD-L1-Ce6-PVP nano-solution after even.Substitute the above-mentioned PD-L1's containing α with phosphate buffer (0.01M, pH=7.4)
Ce6-PVP solution is made as experimental comparison group in phosphate buffer；
Particle diameter distribution: by transmission electron microscope observation, the partial size of α PD-L1-Ce6-PVP nanoparticle is in 30nm or so；
2) the power credit of Ce6, Ce6+PVP and α PD-L1, human serum albumins (HSA), mice serum albumin (MSA)
Analysis
By the coupling of albumen and CM5 chip, chemical combination is carried out using interaction of biomacromolecules instrument (Biacore T200)
The dynamic analysis of object Ce6, Ce6 and PVP (1:1, w/w) mixture and α PD-L1, HSA, MSA albumen.Solution system is HBS-
EP+ buffer, PH=7.4；
The results show that the affine force value (KD) of Ce6 and α PD-L1 is 6.096 × 10-8The affine force value of M, Ce6 and MSA
It (KD) is 1.617 × 10-6M.The affine force value (KD) of Ce6 and HSA is 2.118 × 10-6The affinity of M, Ce6 and α PD-L1 is
Its 26.5 times with MSA；It is its 34.7 times with HSA, in the case where Ce6 and PVP (1:1 w/w) mixing, Ce6 and α PD-L1
Affine force value (KD) be 7.002 × 10-8M, it was demonstrated that the PVP in preparation does not significantly affect the combination of Ce6 and α PD-L1；Above-mentioned parent
With power the experiment results show that Ce6 and α PD-L1 has natural strong affinity, nano-solution can be self-assembled into.Between them
Affinity be higher than Ce6 and sero-abluminous affinity, α PD-L1 can play the role of stablizing Ce6 in blood；
3) α PD-L1-Ce6-PVP nanometers with Ce6-PVP solution intravenous injection after in mouse Internal pharmacokinetics compared with
PD-L1-Ce6-PVP nanometers of C57BL/6 mouse tail vein injection α (α containing 0.2mg PD-L1 and 0.1mg Ce6) or
Ce6-PVP solution (0.1mg Ce6), respectively after administration 0.5h, 1h, 2h, 4h, 8h, 12h, tail vein takes 20 μ L of blood for 24 hours, set
In plastic centrifuge tube, near-infrared spectroscopy is carried out, fluorescence intensity in quantitative analysis blood is control so that preceding blank blood is administered
Group calculates the drug concentration in blood；
The results show that PD-L1-Ce6-PVP nanometers of α can be improved stability of the Ce6 in blood, increase blood level, Fig. 1
It shows after PD-L1-Ce6-PVP nanometers of C57BL/6 mouse tail vein injection α between 0.5h to 4h, fluorescence intensity is significant in blood
Higher than Ce6-PVP solution group, for example, Ce6 concentration is Ce6- in the blood of PD-L1-Ce6-PVP nanometers of 1h after injection, α groups
1.64 times of PVP solution group；
4) raw in lotus glioma Mice Body after α PD-L1-Ce6-PVP nanometers and Ce6-PVP solution intravenous injection 1h
The comparison of object distribution
C57BL/6 mouse intracranial injection 5 × 105A GL261 brain glioblastoma cell (5 μ L), encephalic original position brain glue after 13 days
Matter tumor model foundation, PD-L1-Ce6-PVP nanometers of tail vein injection α (α containing 0.2mg PD-L1 and 0.1mg Ce6) or Ce6-PVP
Solution (0.1mg Ce6), 1h after administration, tail vein take 20 μ L of blood, rear to put to death, and tissue internal organs are given in separation, carry out near-infrared
The fluorescence intensity in internal organs is respectively organized in optical imagery, quantitative analysis, calculates its fluorescence intensity in each tissue internal organs；
As a result as shown in Fig. 2, 1h after injection, Ce6 fluorescence intensity in the brain tumors of PD-L1-Ce6-PVP nanometers of α groups
It is 1.85 times of Ce6-PVP solution group；
Experimental result confirm, α PD-L1, Ce6, PVP three after being self-assembly of nano-complex, with Ce6, PVP
The two physical composition compares, and the stability that can improve Ce6 in blood increases its effective concentration in blood, improves it in tumour
In concentration.
Embodiment 2
By taking serum IgG, Ce6, PVP as an example, self-assembled nanometer compound is prepared, illustrates the prescription of the nano-complex
Technique, preparation process and its tumour pass the characteristic of medicine.
α PD-L1 is replaced with serum IgG, using 1 preparation process of embodiment, IgG-Ce6-PVP nano-solution is made,
Transmission electron microscope observation, partial size test the affinity for measuring Ce6 and serum IgG in 30nm or so, binding kinetics
Being worth (KD) is 7.394 × 10-8M, the 1h after mouse tail vein injection, Ce6 concentration is Ce6- in the blood of IgG-Ce6-PVP nanometers of groups
1.68 times of PVP solution group；Ce6 fluorescence intensity is 1.71 times of Ce6-PVP solution group in the brain tumor of IgG-Ce6-PVP nanometers of groups；
Experimental result confirms that after being self-assembly of nano-complex, Ce6 and IgG have the three of rat IgG, Ce6, PVP
There is its high-affinity similar with α PD-L1, and to increase it effective dense in blood for the stability that equally can be improved Ce6 in blood
Degree, improves its concentration in tumour.
Embodiment 3
By taking the anti-CTLA-4 monoclonal antibody IgG of humanization (α CTLA-4), pheophorbide salt A (PheoA), PVP as an example,
Self-assembled nanometer compound is prepared, illustrates that formulation and technology, preparation process and its tumour of the nano-complex pass the characteristic of medicine.
Prepare the phosphate buffer (0.01M, pH=7.4) for being 6.7mg/mL containing PVP (MW=10000Da) concentration.It measures
15 μ L (0.1mg containing PVP) of this solution is added dropwise to the phosphorus that 60 μ L contain 0.2mg α CTLA-4 under 500rpm magnetic agitation
In acid buffer (0.01M, pH=7.4), continue to stir 5min.4mg pheophorbide salt A is weighed, is dissolved in 1mL's
In 0.05%NaOH solution, it is molten to draw the mixing that above-mentioned α CTLA-4 and PVP is added dropwise in the 50 μ L pheophorbide salt solution A
In liquid, continue to stir 5min, after mixing up to α CTLA-4-PheoA-PVP nano-solution, with phosphate buffer (0.01M,
PH=7.4 PheoA-PVP solution is made as experimental comparison group in the phosphate buffer for) substituting the above-mentioned CTLA-4 containing α；
By transmission electron microscope observation, the partial size of α CTLA-4-PheoA-PVP nanoparticle is in 45nm or so, mouse tail
PheoA concentration is 2.34 times of PheoA-PVP solution group in the blood of CTLA-4-PheoA-PVP nanometers of 1h after intravenous injection, α groups；
CTLA-4-PheoA-PVP nanometers of groups of α PheoA fluorescence intensity in BALB/c mouse lotus 4T1 breast cancer orthotopic tumour is PheoA-
2.51 times of PVP solution group；
Experimental result confirms that the three of anti-CTLA-4 monoclonal antibody IgG, PheoA, the PVP of humanization is through being self-assembly of
After nano-complex, the stability that the anti-CTLA-4 monoclonal antibody IgG of humanization can be improved PheoA in blood increases it in blood
Effective concentration, improve its concentration in tumour；
Above-described embodiment 1,2 and 3 as a result, it was confirmed that with IgG molecular structure monoclonal antibody immunologic test point resistance
Disconnected agent has strong affinity to the photosensitizer molecule containing porphyrin ring structure, can be improved photosensitive in the stability and tumour in blood
The concentration of agent.
Embodiment 4
With rat anti-mouse PD-L1 monoclonal antibody IgG (α PDL-1), Hamster anti-mouse CTLA-4 monoclonal antibody IgG
For (α CTLA-4), pheophorbide salt A (PheoA), preparation is loaded with liposome (the α PD- of α PDL-1, α CTLA-4 and PheoA
L1/ α CTLA-4-PheoA), illustrate that formulation and technology, preparation process and its tumour of the liposome pass the characteristic of medicine.
Each material in following basic prescription is weighed according to molar ratio: dipalmitoylphosphatidylcholine (DPPC)/cholesterol/
PheoA/ Pegylation Distearoyl Phosphatidylethanolamine (DSPE-PEG2000) (55:35:5:5), using film aquation knot
It closes extrusion molding and prepares α PD-L1/ α CTLA-4-PheoA liposome, steps are as follows: keeping its molten above-mentioned nano material addition chloroform
Solution is placed in eggplant-shape bottle and depressurizes rotary evaporation removing chloroform, α PD-L1 and α CTLA-4 are concentrated by ultrafiltration to containing both monoclonal antibodies point
Not Wei 10mg/mL PBS solution, by this solution 1mL be added eggplant-shape bottle in, 4 DEG C aquation 2 days, during which vortex oscillation disperse, will carry
Medicine lipid hydrating fluid is repeatedly extruded through 100nm nucleopore membranes, then elutes past what the removal of Sepharose CL-4B gel column dissociated
Antibody is to get drug-loaded liposome；
α PD-L1/ α CTLA-4-PheoA liposome is prepared using film aquation combination extrusion molding, observes and receives under transmission electron microscope
The grain of rice is more blister-shape structures, average grain diameter 140nm.The encapsulation rate of α PD-L1 and α CTLA-4 are respectively 18.4 ± 3.4% Hes
19.1 ± 1.6%；
Under 808nm laser action, through 0.5W/cm2After irradiating 5min, solution temperature reaches temperature-sensitive nano material DPPC's
Phase transition temperature, the antibody α PD-L1 contained is released 95% or more, without 808nm laser irradiation, the antibody α PD-L1 that contains
It is released less than 5%, which generates singlet oxygen, 808nm laser (0.5W/cm under 660nm laser action2) irradiation
The ability that 5min generates singlet oxygen to it has little effect；
After being injected intravenously α PD-L1/ α CTLA-4-PheoA liposome 6h, BALB/c mouse lotus 4T1 breast cancer orthotopic tumour
In PheoA fluorescence intensity be 2.54 times of simple PheoA solution group；
After being injected intravenously IRDye800CW label α PD-L1/ α CTLA-4-PheoA liposome 6h, BALB/c mouse lotus 4T1
IRDye800CW label α PD-L1 fluorescence intensity in breast cancer orthotopic tumour is simple IRDye800CW label α PD-L1 solution
2.03 times of agent group；
Mice with tumor vein is given drug-carrying nanometer particle 6h and is followed by by 808nm laser (0.5W/cm2) 3min is irradiated, tumour is flat
Equal temperature reaches~42 DEG C, realizes the temperature requirement of light-operated release antibody.
Embodiment 5
By taking Humanized anti-human PD-1 monoclonal antibody IgG (α PD-1), protoporphyrin IX (PpIX) as an example, preparation is loaded with α PD-1
With the liposome (α PD-1-PpIX) of protoporphyrin IX, illustrate that formulation and technology, preparation process and its tumour of the liposome pass medicine
Characteristic；
Using reverse phase evaporation, by DPPC 2.8mg, cholesterol 0.93mg, pegylated phospholipids (DSPE-PEG2000,
0.77mg) and photosensitizer PpIX 0.2mg is substantially dissolved in 1mL chloroform, with the phosphate for containing α PD-1 antibody (0.5mg)
Buffer 0.33mL is sufficiently mixed, and forms it into water-in-oil emulsion using ultrasonic method, sets decompression step by step steaming in a round bottom flask
Organic solvent is removed in performance, obtains gel state substance, and buffer is added and fullys shake；Medicine lipid hydrating fluid will be carried and repeatedly extruded and passed through
100nm nucleopore membranes, then the free antibody of Sepharose CL-4B gel column removal is eluted past to get α PD-1-PpIX lipid
Body；
It is more blister-shape structures that nanoparticle is observed under transmission electron microscope, and the encapsulation rate of average grain diameter 130nm, α PD-1 are
55.3 ± 6.7%, under 808nm laser action, through 0.5W/cm2After irradiating 5min, the antibody α PD-1 contained is released 95%
More than, which generates singlet oxygen, 808nm laser (0.5W/cm under 660nm laser action2) irradiation 5min it is produced
The ability of raw singlet oxygen has little effect；
After being injected intravenously α PD-1-PpIX liposome 6h, the PpIX in BALB/c mouse lotus 4T1 breast cancer orthotopic tumour is glimmering
Luminous intensity is 3.12 times of simple PpIX solution group；
After being injected intravenously IRDye800CW label α PD-1-PpIX liposome 6h, BALB/c mouse lotus 4T1 breast cancer orthotopic
IRDye800CW label α PD-1 fluorescence intensity in tumour is 2.34 times of simple IRDye800CW label α PD-1 solution group；
Mice with tumor vein is given drug-carrying nanometer particle 6h and is followed by by 808nm laser (0.5W/cm2) 3min is irradiated, tumour is flat
Equal temperature reaches~42 DEG C, realizes the temperature requirement of light-operated release antibody.
The present invention is illustrated by above description and examples, that has been described as unrestricted, is not intended to limit this hair
Bright scope of the claims.
Claims (14)
1. it is a kind of for photodynamic therapy combined immunization treatment nanometer formulation, it is characterised in that said preparation by photosensitizer,
Immunologic test order blocking agent and auxiliary material composition partial size nanometer range pharmaceutical dosage form；
The nanometer formulation by photosensitizer, immunologic test order blocking agent and auxiliary material mixing be made；
Or,
Liposome is made in the nanometer formulation, and immunologic test is wherein contained in liposome interior water phase and orders blocking agent, and lipid is double
Photosensitizer is contained in molecular layer.
2. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
The photosensitizer stated contains porphyrin ring structure, is selected from m- four (hydroxy phenyl) porphyrins, 5,10,15,20- tetra- (4- benzene sulfonic acid sodium salts)-
21H, 23H- porphyrin, protoporphyrin IX, 5,10,15,20- tetra- (1-4- picolyl) porphyrin toluene fulfonate, chlorin e 6,
Benzoporphyrin derivative monoacid ring A, chlorophyll, pheophorbide salt A and the compound salt form.
3. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
It is monoclonal antibody that the immunologic test stated, which orders blocking agent,.
4. the nanometer formulation according to claim 3 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
The monoclonal antibody stated is immunoglobulin G molecule.
5. the nanometer formulation according to claim 3 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
The monoclonal antibody stated is selected from anti-cell toxic T lymphocyte antigen -4 (CTLA-4) antibody, anti-apoptosis albumen -
1 (programmed cell death protein 1, PD-1) antibody, 1 (Programmed of anti-programmed death ligand
Death-ligand 1, PD-L1) antibody, 9 antibody of AntiCD3 McAb, one of anti-CD73 antibody or several.
6. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
It states nanometer formulation blocking agent and pharmaceutic adjuvant is ordered by photosensitizer, immunologic test and nanometer formulation is made using the method for solution stirring.
7. the nanometer formulation according to claim 6 for the treatment of photodynamic therapy combined immunization, which is characterized in that adopt
It is prepared in nanometer formulation with the method that solution stirs, the pharmaceutic adjuvant is polyvinylpyrrolidone.
8. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
It states nanometer formulation and liposome is made, wherein the auxiliary material of use is made of phosphatide, cholesterol, pegylated phospholipids.
9. the nanometer formulation according to claim 8 for the treatment of photodynamic therapy combined immunization, which is characterized in that institute
The pegylated phospholipids stated are Pegylation Distearoyl Phosphatidylethanolamine.
10. the nanometer formulation according to claim 8 for the treatment of photodynamic therapy combined immunization, which is characterized in that
The phosphatide contains dipalmitoylphosphatidylcholine.
11. pressing claim, the nanometer formulation described in 6 for the treatment of photodynamic therapy combined immunization, which is characterized in that
The phosphatide also contains or not contain Distearoyl Phosphatidylcholine, hydrogenated soya phosphatide, lecithin, 1- myristoyl-
2- stearyl lecithin.
12. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that
The liposome is temperature sensitive liposome, the characteristic with light-operated release drug.
13. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that
Freeze drying protectant mannitol is added in the preparation, is prepared into lyophilized preparation.
14. the nanometer formulation according to claim 1 for the treatment of photodynamic therapy combined immunization, which is characterized in that
The partial size of the nanometer formulation is 10-1000nm.
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