CN115518152B - Polypeptide micelle for enhancing light immunotherapy based on tumor vascular normalization and immune checkpoint blocking and preparation and application thereof - Google Patents
Polypeptide micelle for enhancing light immunotherapy based on tumor vascular normalization and immune checkpoint blocking and preparation and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- 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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
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- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Inorganic Chemistry (AREA)
- Biophysics (AREA)
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Abstract
The invention belongs to the technical field of biological medicines, and in particular relates to a polypeptide micelle for enhancing photo-immune therapy based on tumor vascular normalization synergic immune check point blocking, and preparation and application thereof.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a polypeptide micelle for enhancing photo-immunotherapy based on tumor vascular normalization and immune checkpoint blocking, and preparation and application thereof.
Background
Solid tumors, in order to obtain a sufficient nutritional supply, will meet their need for rapid proliferation by the newly created large blood vessels. The pathological blood vessel has the characteristics of high leakage, loose endothelial cell connection, low pericyte adhesion degree and the like, so that the efficiency of oxygen transmission and blood perfusion is weakened; meanwhile, infiltration of immune cells can be reduced by down regulating expression of vascular adhesion factors, and conversion of macrophages to M2 type is promoted, so that an immunosuppressive microenvironment is formed, and tumor cell escape is assisted. Thus, anti-angiogenic therapies have great research significance and clinical value for inhibiting tumor growth and metastasis.
At present, the traditional cancer treatment means mainly comprising operation, chemotherapy and radiotherapy is to eliminate cancer cells by means of external force, and the toxic and side effects brought by the cancer cells can generally reduce the survival quality of patients and exacerbate the risks of metastasis and recurrence of malignant tumors. Therefore, it is urgent to find new cancer treatments that specifically recognize, track and destroy distant metastases while eliminating the primary tumor. Among them, immune checkpoint blocking therapy (ICB) can promote the killing effect of the immune system of the organism on cancer cells by weakening the immune evasion ability of the cancer cells, however, for some cold tumors with extremely low immunogenicity (such as microenvironment with high expression of immunosuppressive factors and tumor blood vessels with abnormal growth, which are not favorable for T cell infiltration and the exertion of the cell killing effect thereof), the single drug treatment effective rate of ICB therapy is only 10-30%. Although down-regulation of PD-L1 expression by gene silencing is also a means of ICB therapy, currently commonly used gene delivery vectors (e.g., liposomes) are not readily available to assist siRNA in lysosomal escape. Photodynamic therapy (PDT) can trigger immunogenic death (ICD) of tumor cells, promote immunogenicity of cold tumor, and promote specific recognition and killing effect of immune system on cancer cells, so that the therapeutic effect of ICB therapy is promoted, but the curative effect of oxygen-consuming PDT is usually limited by hypoxia in tumor areas. Therefore, there is a need to develop new photo-immune therapies to overcome the shortcomings of low response rate of immune checkpoint monotherapy and weak specific immune response of existing anti-tumor strategies.
Disclosure of Invention
In order to overcome the above-mentioned shortcomings of the prior art, a primary object of the present invention is to provide a polypeptide micelle.
The second object of the present invention is to provide a method for producing the above-mentioned polypeptide micelle. The polypeptide micelle is formed by self-assembling a polypeptide and a hydrophobic compound and then further adsorbing the polypeptide and the hydrophobic compound with SiPD-L1 through electrostatic attraction.
A third object of the present invention is to provide an antitumor application of the above polypeptide micelle. The polypeptide micelle can be blocked based on tumor vascular normalization and cooperative immune check points to enhance the effect of photo-immune treatment, so that the anti-tumor effect is improved.
The first object of the present invention is achieved by the following technical solutions:
the invention provides a polypeptide micelle, which is formed by self-assembling a polypeptide and a hydrophobic compound and then further adsorbing the polypeptide and SiPD-L1 through electrostatic attraction, wherein the polypeptide comprises a cationic sequence peptide segment R9, an MMP-2 (matrix metalloproteinase-2) substrate peptide segment GPLGVRG, an anti-angiogenesis peptide segment ATWLPPR, a photosensitizer Ce6 and polyethylene glycol (PEG), and the sequence of the polypeptide is shown as SEQ ID NO:1 (PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR).
Preferably, the hydrophobic compound comprises lecithin, polylactic-co-glycolic acid (PLGA).
In the polypeptide, a cationic sequence peptide segment RRRRRRRRR (R9) is used for loading small interfering RNA, an MMP-2 substrate peptide segment GPLGVRG is used for realizing MMP-2 responsive degradation of a polypeptide micelle, an anti-angiogenesis peptide ATWLPPR is used for realizing tumor vascular normalization, a photosensitizer Ce6 is used for realizing photodynamic therapy, and PEG is used for increasing the biocompatibility of the micelle.
When the polypeptide micelle enters a tumor microenvironment, the polypeptide micelle can be degraded into two parts under the action of MMP-2, wherein the part coupled with Ce6 and carrying the siPD-L1 can enter tumor cells under the action of a transmembrane peptide R9, the Ce6 plays a role of PDT to trigger ICD, specific anti-tumor immune reaction is stimulated, active oxygen (ROS) generated by PDT damages a lysosome membrane, and the escape of the siPD-L1 adsorbed on R9 by electrostatic attraction is promoted, so that the expression of the PD-L1 is regulated down. Meanwhile, the other section of anti-angiogenic peptide A7R degraded in the tumor microenvironment can exert the vascular normalization effect of the Vascular Endothelial Growth Factor Receptor (VEGFR) and the neuropilin (NRP-1) of the tumor endothelial cells through targeting, so that the hypoxia state of the tumor area is relieved, the curative effect of oxygen consumption PDT is promoted, on the other hand, the normalized blood vessel can promote the infiltration of immune cells, and the PD-L1 is cooperated to down regulate and relieve the immunosuppression microenvironment, so that the immune response of specific anti-tumor immune response is facilitated, and the effect of light immunotherapy can be enhanced through the tumor vascular normalization cooperated with immune checkpoint blocking.
The second object of the present invention is achieved by the following technical solutions:
the invention also provides a preparation method of the polypeptide micelle, which comprises the following steps:
s1, synthesizing SEQ ID NO by adopting Fmoc solid-phase polypeptide synthesis method: 1;
s2, mixing the polypeptide and the hydrophobic compound in the step S1 in an organic solvent, then dripping the obtained mixture into water, uniformly mixing, and standing for more than 2 hours;
s3, adding the SiPD-L1 into the solution in the step S2, and incubating to obtain the polypeptide micelle.
Preferably, the molar ratio of the polypeptide to the hydrophobic compound is 1: (2-8). Further, the molar ratio of the polypeptide to the hydrophobic compound is 1:2.
preferably, the polypeptide has a nitrogen to phosphorus ratio (N/P) of not less than 5 to siPD-L1. P (P) A7R When the N/P ratio with the SiPD-L1 is more than 5, the carrier can completely load siRNA.
According to the invention, a polypeptide which simultaneously comprises a cationic sequence peptide fragment, a matrix metalloproteinase-2 substrate peptide fragment, an anti-angiogenesis peptide fragment and a photosensitizer is synthesized through an Fmoc solid-phase polypeptide synthesis method, then self-assembled with a hydrophobic compound, and then incubated with small interfering RNA (siPD-L1) at room temperature to obtain a stable and uniform polypeptide micelle. The polypeptide micelle overcomes the defects of low response rate of single drug treatment of immune checkpoints and weak specific immune response of the existing anti-tumor strategy, and reverses the immune inhibition microenvironment of a tumor region through tumor vascular normalization and immune checkpoint blocking therapy, thereby providing the treatment effect of the photo-immunotherapy.
Preferably, the organic solvent comprises dimethyl sulfoxide (DMSO).
Preferably, the mixing is vortex and ultrasonic mixing.
Further, the swirling time is 10-20s; the frequency of the ultrasonic wave is 40kHZ, and the power is 100-150W. The ultrasonic power and frequency should not be as great as possible so as not to destroy the stability of the siRNA.
The third object of the present invention is achieved by the following technical means:
the invention also provides application of the polypeptide micelle in preparing an anti-tumor medicament.
Preferably, the anti-tumor is one that promotes tumor vascular normalization and/or attenuates immune evasion ability of cancer cells by means of immune checkpoint blockade.
The invention also provides an anti-tumor drug, which comprises the polypeptide micelle.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a polypeptide micelle, which is formed by self-assembling a polypeptide (PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR) and a hydrophobic compound and then further adsorbing the polypeptide with a siPD-L1 through electrostatic attraction, wherein the polypeptide comprises a cationic sequence peptide segment R9, an MMP-2 (matrix metalloproteinase-2) substrate peptide segment GPLGVRG, an anti-angiogenesis peptide segment ATWLPPR, a photosensitizer Ce6 and polyethylene glycol (PEG), can realize gene loading at the same time, has the functions of MMP-2 responsive degradation, vascular normalization and photo-immunotherapy in a tumor area, and can play an ICB function with the siPD-L1 combined with the cationic sequence peptide segment through electrostatic attraction.
The polypeptide micelle prepared by the invention has excellent photodynamic treatment effect and gene transfection capability, also has good biocompatibility and biodegradability, can be degraded into two parts in the tumor microenvironment in a responsive way, one part enters tumor cells to play a role in promoting ICD effect through a photosensitizer Ce6 to play a PDT effect, meanwhile, the siPD-L1 down regulates the expression of the tumor cells PD-L1, and the A7R in the other part plays a role in normalizing blood vessels by blocking the combination of vascular endothelial cell factor (VEGF) and VEGFR and NRP-1 of tumor vascular endothelial cells, relieves the hypoxia state of the tumor microenvironment and promotes infiltration of immune cells, thereby synergistically enhancing the specific anti-tumor immunity effect. The polypeptide micelle overcomes the defects of low response rate of single drug treatment of immune checkpoints and weak specific immune response of the existing anti-tumor strategy, and reverses the immune inhibition microenvironment of a tumor region through tumor vascular normalization and immune checkpoint blocking therapy, thereby providing the treatment effect of the photo-immunotherapy.
Drawings
FIGS. 1a and 1b are, respectively, polypeptide micelles P A7R Transmission Electron Microscopy (TEM) of @ sIPD-L1 after degradation by MMP-2;
FIGS. 2a and 2b are polypeptide micelles P, respectively A7R Particle size distribution diagram of @ SiPD-L1 and Zeta potential diagram before and after loading the SiPD-L1;
FIG. 3 is a polypeptide micelle P A7R Ultraviolet-visible light absorption spectrum of @ SiPD-L1 (P in the figure A7R Represents P A7R @siPD-L1)；
FIG. 4 is a polypeptide micelle P A7R A critical micelle concentration profile of @ siPD-L1;
FIG. 5 shows a polypeptide micelle P A7R In vitro singlet oxygen production Capacity test (P in the figure) A7R Represents P A7R @siPD-L1)；
FIG. 6 is a polypeptide micelle P A7R Gel blocking test results of @ siPD-L1 (lanes 1-8, lanes 1-7 are P, respectively) A7R N/P ratio (P A7R siRNA) 0.5, 1, 2.5, 5, 10, 20, 25; lane 8 is: free siRNA; p in the figure A7R Represents P A7R @siPD-L1)；
FIG. 7 shows (a) a micrograph and (b) an Image J statistical plot of HUVEC tube results (P in the figure) A7R Represents P A7R @siPD-L1)；
FIG. 8 is a view of a laser confocal microscope of the ROS-producing capability of 4T1 cells (658 nm laser excitation, 5min irradiation; P in the figure) A7R Represents P A7R @siPD-L1)。
Detailed Description
The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.
EXAMPLE 1 Synthesis of polypeptide PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR
The Fmoc solid-phase polypeptide synthesis method is adopted to synthesize polypeptide PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR, wherein the polypeptide simultaneously comprises a cationic sequence peptide segment R9, an MMP-2 substrate peptide segment GPLGVRG, an anti-angiogenesis peptide segment ATWLPPR, a photosensitizer Ce6 and PEG. The method comprises the following specific steps:
(1) Fmoc-Arg (Pbf) -Wang Resin with Loading of 0.32mmol/g is selected, and Fmoc protecting groups are removed after swelling;
(2) Coupling is carried out sequentially from the C end to the N end according to a polypeptide sequence PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR until the 1 st amino acid Fmoc-Arg (Pbf) -OH at the N end, wherein the side chain protecting groups of Arg, lys, thr, trp are Arg, dde, tBu, boc respectively; all amino acids were protected with Fmoc at amino position alpha; taking a small sample, removing Fmoc protecting groups and Dde protecting groups, then cracking, and judging the correctness of the polypeptide through mass spectrometry after cracking;
(3) After determining that the fragment polypeptide Fmoc-RRRRRRRRRK (Dde) -LLGPLGVRG-ATWLPPR mass spectrum is correct, removing Fmoc protecting groups, adding PEG with 3 times of the molar multiple of the polypeptide for reaction, and ending the reaction after ninhydrin detection is negative; taking a small sample, cracking, verifying the completion of PEG reaction through mass spectrum and HPLC, hydrazinolysis of Dde protecting group of a Lys side chain, washing three times by using DMF and DCM respectively after ninhydrin detection is negative, pumping, adding Ce6 with the molar multiple of 2 times of polypeptide for reaction, and ending the reaction until ninhydrin detection is negative to obtain linear peptide resin;
(4) Reacting the cleavage solution (TFA: triisopropylsilane: water=95:2.5:2.5) with a linear peptide resin to obtain a linear peptide with all side chain protecting groups removed;
(5) Dissolving the linear peptide in water, purifying by using semi-preparative chromatography, separating out qualified liquid, collecting, evaporating and freeze-drying to obtain the target peptide PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR.
Example 2 preparation of polypeptide micelles
1mg of the polypeptide prepared in example 1 was dissolved in 10. Mu.L of dimethyl sulfoxide at room temperature, then mixed with 14. Mu.L of a DMSO solution of lecithin (concentration: 6 mg/mL) (molar ratio of polypeptide to lecithin: 1:2), and the mixture was added dropwise to 1mL of ultrapure water, vortexed for 15s, sonicated in a water bath at 40kHZ under 100-150W for 5-10min, allowed to stand for 2 hours or more, and then reacted with siPD-L1 [ silenced small interfering RNA (siRNA) expressed by programmed death receptor-ligand 1 (PD-L1), P A7R siRNA (N/P) =10, purchased from Guangzhou Ruibo Biotechnology Co., ltd.) was incubated at room temperature for 30min to obtain polypeptide micelle P A7R @siPD-L1。
And observing the morphology of the polypeptide micelle by using a transmission electron microscope. As shown in fig. 1a, the polypeptide micelle has an obvious spherical structure and uniform particle size. Meanwhile, hydrodynamic particle size of the polypeptide micelle was measured to be about 30-50nm using a bruck Lin Lidu instrument (fig. 2 a). In addition, by measuring the potential of the polypeptide micelle, the potential of the polypeptide micelle was changed from positive to negative after incubation with siPD-L1 (fig. 2 b), indicating that it was successfully loaded with siPD-L1.
Experimental example 1 performance test
(1) Matrix metalloproteinase degradability assay of polypeptide micelles
Under the water bath condition of 37 ℃, a proper amount of polypeptide micelle prepared in the embodiment 2 is incubated with MMP-2 (matrix metalloproteinase-2) (the concentration of MMP-2 is 2.5 mug/mL) for 1-2 hours, and then a sample is treated by adopting a negative dyeing technology and is observed by using a transmission electron microscope.
As shown in figure 1b, after the polypeptide micelle is treated by MMP-2, the spherical structure is not existed, which indicates that the polypeptide micelle has good MMP-2 response degradation property.
(2) Ultraviolet-visible light absorption spectrum of polypeptide micelle
The prepared polypeptide micelle P A7R After dissolving @ siPD-L1 in water, its uv-vis absorption spectrum was examined.
As shown in fig. 3, P A7R At 400nm and 66 @ sPD-L1There are two characteristic absorption peaks at 0nm, demonstrating successful coupling of Ce6 to the polypeptide.
(3) Critical micelle concentration determination
P at different concentrations A7R Aqueous solution @ siPD-L1 (0-200. Mu.M) and pyrene fluorescent probe (10) -6 M) is incubated at 37℃for 2h, and its emission at the 300-550 band is then detected with a fluorescence spectrophotometer at 334nm of excitation light. The Critical Micelle Concentration (CMC) value is the fluorescence intensity ratio (I384/I373) of the emitted light at 384nm and 373 nm.
As shown in FIG. 4, the polypeptide micelle has a low critical micelle concentration (6.04. Mu.M).
(4) In vitro singlet oxygen production capability assessment
Will P A7R Either @ sPD-L1 or MMP-2 pretreated P A7R Aqueous solution @ sPD-L1 (P is added A7R Incubation of @ sPD-L1 with MMP-2 at 2.5. Mu.g/mL for 1-2h, removal and dissolution in water at 1. Mu.g/mL) with a singlet oxygen detection probe SOSG (2X 10) - 6 M) and then at 658nm (100 mW/cm) 2 ) Irradiation was performed and its emission wavelength intensity at 525nm was detected with a fluorescence spectrophotometer at an excitation wavelength of 488 nm.
As can be seen from FIG. 5, P A7R P pretreated with MMP-2, having excellent singlet oxygen generating capability @ SiPD-L1 A7R The production rate of singlet oxygen by @ siPD-L1 is faster than that of P without MMP-2 pretreatment A7R @siPD-L1。
(5) Gel retardation experiment
0.27 μg of siPD-L1 was taken with different concentrations of P A7R Mixing @ SiPD-L1 polypeptide micelle, and incubating at room temperature for 30min to obtain P with different N/P ratios (0.5, 1, 2.5, 5, 10, 20, 25) A7R The @ siPD-L1 was then mixed with loading buffer and then added to a 1% agarose gel for electrophoresis experiments, which were completed after 80V for 30 minutes on Tris-Acetate-EDTA (TAE) buffer.
As shown in FIG. 6, P A7R When the N/P ratio with the SiPD-L1 is more than 5, the carrier can completely load siRNA.
(6) HUVEC tube forming experiment
HUVEC cells were seeded into 96-well plates covered with matrigel (Corning; cat# 356234) and plated with P A7R Either @ SiPD-L1 was pretreated with MMP-2 (P A7R Incubation of @ sPD-L1 with MMP-2 at 2.5. Mu.g/mL for 1-2 h) P A7R Incubation at siPD-L1 and using HUVEC cells without drug treatment (PBS treatment) as control. After incubation for 4h in a carbon dioxide incubator at 37 ℃, the formation of the tubules was observed under a microscope and the tubule length was counted with Image J.
As shown in FIG. 7, MMP-2 pretreated P A7R The @ siPD-L1 significantly reduces the tubule forming ability of HUVEC cells, because the polypeptide micelle structure collapses after MMP-2 pretreatment, and the anti-angiogenic peptide A7R is dissociated, so that the HUVEC cells can play a better role in resisting angiogenesis.
(8) Intracellular ROS production assay
Inoculating 4T1 cells into a laser confocal dish, culturing for 24h, replacing fresh complete culture solution (DMEM culture medium), and adding free Ce6, P into the culture solution A7R P pretreated with @ SiPD-L1 and MMP-2 A7R Culturing @ sPD-L1 (Ce 6 content 5. Mu.g/mL) in a carbon dioxide incubator for 12h, removing the medium containing the drug, washing 1 time with PBS, adding 1mL of 10. Mu.M 2, 7-dichlorofluorescein diacetate (DCFH-DA) working solution, and irradiating 5min (100 mW/cm) with 658nm laser 2 ) Incubation was then continued in a 37 ℃ cell incubator for 30min under light protection, and finally observation was performed by laser confocal microscopy.
As can be seen from FIG. 8, P A7R ROS generated by the @ siPD-L1 group after illumination are stronger than those of the free Ce6 group, and P after MMP-2 treatment A7R ROS production levels were higher in the @ SiPD-L1 experimental group than in P without MMP-2 treatment A7R Group @ siPD-L1.
In summary, the polypeptide in the polypeptide micelle prepared by the invention comprises a cationic sequence peptide segment R9, an MMP-2 substrate peptide segment GPLGVRG, an anti-angiogenesis peptide segment A7R, a photosensitizer Ce6 and PEG. Wherein the cationic motif peptide segment RRRRRRRRR (R9) is used for loading small interfering RNA, the MMP-2 substrate peptide segment GPLGVRG is used for realizing MMP-2 responsive degradation of polypeptide micelle, the antiangiogenic peptide ATWLPPR is used for realizing tumor vascular normalization, the photosensitizer Ce6 is used for realizing photodynamic therapy, and the PEG is used for increasing the biocompatibility of the micelle.
Meanwhile, the experiment shows that the polypeptide micelle has excellent photodynamic treatment effect and gene transfection capability, has good biocompatibility and biodegradability, can be degraded into two parts in the tumor microenvironment in a responsive way, one part enters tumor cells to play a PDT role through a photosensitizer Ce6 to promote ICD effect, meanwhile, the sPD-L1 down regulates the expression of the tumor cells PD-L1, and the A7R in the other part plays a vascular normalization role by blocking the combination of vascular endothelial cell factor (VEGF) and VEGFR and NRP-1 of tumor vascular endothelial cells, relieves the hypoxia state of the tumor microenvironment and promotes infiltration of immune cells, so that the specific anti-tumor immunity effect is synergistically enhanced.
The polypeptide micelle overcomes the defects of low response rate of single drug treatment of immune checkpoints and weak specific immune response of the existing anti-tumor strategy, and reverses the immune inhibition microenvironment of a tumor region through tumor vascular normalization and immune checkpoint blocking therapy, thereby providing the treatment effect of the photo-immunotherapy.
The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.
Sequence listing
<110> university of Zhongshan
<120> polypeptide micelle for enhancing photo-immune therapy based on tumor vascular normalization synergistic immune checkpoint blocking, preparation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> PRT
<213> polypeptide (Artificial Sequence)
<400> 1
Arg Arg Arg Arg Arg Arg Arg Arg Arg Lys Leu Leu Gly Pro Leu Gly
1 5 10 15
Val Arg Gly Ala Thr Trp Leu Pro Pro Arg
20 25
Claims (9)
1. The polypeptide micelle is formed by self-assembling a polypeptide and a hydrophobic compound, and then further adsorbing the polypeptide and siPD-L1 through electrostatic attraction, wherein the polypeptide comprises a cationic sequence peptide segment R9, an MMP-2 substrate peptide segment GPLGVRG, an anti-angiogenesis peptide segment ATWLPPR, a photosensitizer Ce6 and polyethylene glycol, the polypeptide is shown as PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR, and the hydrophobic compound comprises lecithin and a polylactic acid-glycolic acid copolymer.
2. The method for preparing the polypeptide micelle as claimed in claim 1, which comprises the following steps:
s1, synthesizing a polypeptide shown as PEG-RRRRRRRRRK (Ce 6) -LLGPLGVRG-ATWLPPR by adopting an Fmoc solid-phase polypeptide synthesis method;
s2, mixing the polypeptide and the hydrophobic compound in the step S1 in an organic solvent, then dripping the obtained mixture into water, uniformly mixing, and standing for more than 2 hours;
s3, adding the SiPD-L1 into the solution in the step S2, and incubating to obtain the polypeptide micelle.
3. The method for preparing a polypeptide micelle according to claim 2, wherein the molar ratio of the polypeptide to the hydrophobic compound is 1: (2-8).
4. The method for preparing a polypeptide micelle according to claim 2, wherein the nitrogen to phosphorus ratio of the polypeptide to siPD-L1 is not less than 5.
5. The method for preparing a polypeptide micelle according to claim 2, wherein the organic solvent comprises dimethyl sulfoxide.
6. The method for preparing polypeptide micelles of claim 2, wherein the mixing is vortex+ultrasonic mixing.
7. The use of the polypeptide micelle of claim 1 in the preparation of an antitumor drug.
8. The use according to claim 7, wherein the anti-tumor is promoting tumor vascular normalization and/or impairing the immune evasion ability of cancer cells by means of immune checkpoint blockade.
9. An antitumor drug comprising the polypeptide micelle of claim 1.
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| CN111956609A (en) * | 2020-08-31 | 2020-11-20 | 中国医学科学院生物医学工程研究所 | Immune checkpoint blockade polypeptide prodrug nano micelle and preparation method and application thereof |
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| CN107157928A (en) * | 2017-05-18 | 2017-09-15 | 中国科学技术大学 | A kind of matrix metalloproteinase responsive polymer pharmaceutical carrier and its preparation method and application |
| CN109985238A (en) * | 2019-04-16 | 2019-07-09 | 中山大学 | A kind of antineoplastic pharmaceutical compositions and its application based on the blocking of immunologic test point |
| CN110804086A (en) * | 2019-10-10 | 2020-02-18 | 上海维洱生物医药科技有限公司 | Polypeptide nano-carrier and preparation method and application thereof |
| CN111956609A (en) * | 2020-08-31 | 2020-11-20 | 中国医学科学院生物医学工程研究所 | Immune checkpoint blockade polypeptide prodrug nano micelle and preparation method and application thereof |
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