WO2024026841A1 - 一种载药红细胞膜纳米粒及其制备方法和应用 - Google Patents

一种载药红细胞膜纳米粒及其制备方法和应用 Download PDF

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WO2024026841A1
WO2024026841A1 PCT/CN2022/110594 CN2022110594W WO2024026841A1 WO 2024026841 A1 WO2024026841 A1 WO 2024026841A1 CN 2022110594 W CN2022110594 W CN 2022110594W WO 2024026841 A1 WO2024026841 A1 WO 2024026841A1
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red blood
tumor
blood cell
cell membrane
drug
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French (fr)
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陈华兵
杨红
李明
柯亨特
翟艳华
汪媛
林雪花
杨一帆
赵振铎
王璐
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苏州大学
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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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • 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
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention discloses a red blood cell membrane nanosystem for tumor-targeted drug delivery, and specifically relates to a red blood cell membrane nanoparticle loaded with ICG-DOX and its preparation method and application.
  • Nanomedicines play an important role in enhancing drug efficacy and reducing drug side effects because of their tumor-specific targeted drug delivery characteristics.
  • nanomedicines are synthesized through biomimetic methods and biogenic natural materials such as albumin, cells, bacteria, and viruses are used for nanodrug delivery.
  • This type of nanomaterials combines natural substances with With their respective advantages, nanotechnology has increasingly become a new technology with good application prospects in tumor treatment.
  • Natural material biomimetic synthetic nanomaterials have unique biological characteristics of the material itself, such as long-lasting circulation, tumor-specific targeting, immune regulation, etc.
  • nanodrug delivery systems based on albumin and cell membranes have been widely used in precision tumor treatment (such as J Control Release, 2021, 329, 997-1022; Chem Soc Rev, 2021,50, 945-985).
  • red blood cells are widely used in nanomedicine research because of their rich sources, simple structure and no complex organelles, easy extraction of cell membranes, and strong modification of the cell membrane surface.
  • red blood cells There are currently two different strategies for drug delivery based on red blood cells: using intact red blood cells as carriers for drug delivery and using red blood cell membranes to treat existing nanoparticles such as PLGA nanoparticles, iron oxide magnetic nanoparticles, and mesoporous silica nanoparticles. , liposomes, polymer vesicles, etc. are wrapped in cell membranes for the synthesis of nanomaterials.
  • PEG ⁇ Fe 3 O 4 prepared by the existing hydrothermal method is extruded and wrapped with targeted modified red blood cell membrane fragments to prepare nanoparticle red blood cell membrane ⁇ PEG ⁇ Fe 3 O with immune evasion and sensitive magnetic responsiveness. 4 ;
  • the prior art also discloses a red blood cell membrane-coated MOFs nanomedicine carrier and its preparation method and application.
  • the MOFs nanomedicine carrier includes MOFs nanoparticles and red blood cell membranes, and the pores of the MOFs nanoparticles are loaded with boric acid. , the MOFs nanoparticles are externally wrapped with red blood cell membranes, and the MOFs nanomedicine carriers are synthesized using a hydrothermal method.
  • the purpose of the present invention is to provide a nanovesicle based on red blood cell membrane, which realizes drug reaction and preparation inside it and is used for encapsulating anti-tumor drugs. It has the characteristics of precise and controllable size, simple preparation, and drug originality. The characteristics of site synthesis can achieve higher targeting, increase drug loading efficiency, and have good biological safety, tumor targeting, deep tumor penetration and retention, and the prepared cell membrane nanoparticles retain the cell membrane itself It is soft and can deform to achieve deep tissue penetration in the tumor site, improving the efficient deep delivery of drugs in tumor tissue. At the same time, ICG and DOX form complex precipitation to reduce the unnecessary release of DOX in normal tissues, and the toxic and side effects are effective. reduce.
  • ID-RBCages can responsively release the chemotherapy drug DOX through laser irradiation.
  • light-activated photodynamic therapy and chemotherapy induce the immunogenic death of tumor cells, causing the activation of anti-tumor acquired immune effects, and achieving tumor-enhanced photochemotherapy-immune synergistic therapy.
  • a drug-loaded red blood cell membrane nanoparticle and its preparation method is as follows: mixing the red blood cell membrane and anionic reaction precursor and then extruding to obtain anionic reaction precursor red blood cell membrane vesicles; and then mixing the anionic reaction precursor Red blood cell membrane vesicles and cationic reaction precursors are mixed and reacted to obtain drug-loaded red blood cell membrane nanoparticles.
  • the invention discloses drug-loaded red blood cell membrane nanoparticles with precisely controllable size for targeted delivery of anti-tumor drugs.
  • the particle size of the drug-loaded red blood cell membrane nanoparticles is 20 to 240 nm.
  • the delivery effect of the nanodrug delivery system is closely related to its size, and size control is crucial to the regulation of tumor drug delivery.
  • Nanoparticles of different sizes prepared through size control can be used for size-regulated screening of anti-tumor drugs. The smaller the particle size, the higher the tumor drug delivery efficiency of red blood cell membrane nanoparticles. Therefore, studying nano-drug delivery methods with controllable size of biogenic materials will help to build a safer platform for multi-modal imaging and tumor treatment.
  • the red blood cell membrane serves as the skeleton of the nanoparticles;
  • the cationic reaction precursor is a metal compound or a small molecule drug;
  • the anionic reaction precursor source is a sulfide, a citric acid compound or a dye drug.
  • Cationic reaction precursors, anionic reaction precursors such as doxorubicin and indocyanine green.
  • the red blood cell membrane is obtained by hypotonic treatment of red blood cells; preferably, the red blood cells are suspended in a hypotonic buffer, left to stand and then centrifuged to obtain the red blood cell membrane; further preferably, the red blood cells are left to stand in an ice bath for 20 to 40 minutes, centrifuge at 3000-4000g for 10-20 minutes.
  • the concentration of hypotonic buffer is 20 to 30% of the concentration of its corresponding isotonic buffer.
  • the red blood cell membrane and the anionic reaction precursor are incubated in a hypotonic buffer, and then extruded with a liposome extruder to obtain the anionic reaction precursor red blood cell membrane vesicles; then the anionic reaction precursor red blood cell membrane is The vesicles and the cationic reaction precursor are stirred and reacted in a hypotonic buffer, followed by ultrafiltration to obtain drug-loaded red blood cell membrane nanoparticles.
  • the incubation time is 20 to 40 minutes, and after incubation, ultrasonic treatment is performed for 3 to 6 minutes, and then the liposome extruder is used for gradient extrusion; preferably, the pore size for gradient extrusion is 800 nm, 400 nm, 200 nm, 100 nm, 50 nm.
  • the reaction temperature of the stirring reaction is 25 to 55°C, and the reaction time is 3 to 8 hours, preferably 37°C for 4 to 5 hours.
  • the molar ratio of anionic reaction precursor and cationic reaction precursor is 1: (0.5-8), preferably 1: (0.5-2).
  • the present invention proposes the application of the above-mentioned drug-loaded red blood cell membrane nanoparticles in preparing tumor treatment drugs.
  • the drug-loaded red blood cell membrane nanoparticles of the present invention have: (1) Good size uniformity, with a particle diameter of 60 to 240 Within the nm size range, the size of the extruder polycarbonate membrane can achieve precise control of the nanometer size of the red blood cell membrane; (2) As a natural biogenic material, the red blood cell membrane has good biocompatibility and can be used as a drug delivery The carrier has good safety, and the drug-loaded red blood cell membrane nanoparticles have extremely low cytotoxicity and good blood compatibility, and they have good tumor targeting in tumor-bearing mouse models; (3) Drug-loaded red blood cell membrane nanoparticles have good softness.
  • red blood cell membrane nanoparticles Compared with cell membrane nanoparticles prepared by conventional cell membrane wrapping technology, they have significantly enhanced deformability, which can achieve efficient targeting, deep penetration and effective retention of tumor tissues; (4) Drug-loaded red blood cell membrane nanoparticles can be efficiently taken up by tumor cells and localized in acidic lysosomes. After illumination, they can destroy the lysosomal membrane and enhance the permeability of the nuclear membrane, achieving rapid cytoplasmic transport of DOX and the nucleus. deliver. In addition, ID-RBCages can induce significant cell immunogenic death and efficiently activate acquired anti-tumor immune effects; (5) As a natural membrane material, the red blood cell membrane can react under photothermal conditions and other light stimulation conditions such as photodynamic conditions.
  • the drug-loaded red blood cell membrane nanoparticles have good tumor targeting and can efficiently penetrate tissues and deliver in low-permeability Panc02 mouse pancreatic cancer tumors.
  • the drugs can reach deep into the tumor; in addition, light can also promote the rapid transport of chemotherapy drugs into the nucleus, achieving a synergistic anti-tumor effect of efficient photochemotherapy.
  • Figure 1 shows the transmission electron microscope picture of ID-RBCages (A), the dynamic light scattering particle size of ID-RBCages (B), the absorption spectrum of ID-RBCages (C), and the fluorescence spectrum of ID-RBCages (D).
  • Figure 2 shows the investigation of the photothermal heating ability of ID-RBCages (A), the transmission electron microscope image after illumination under different conditions (B), and the release under illumination conditions (C).
  • Figure 3 shows DHE and AO staining of ID-RBCages.
  • Figure 4 shows the light-induced nuclear damage experiment of ID-RBCages.
  • Figure 5 shows the cellular uptake of ID-RBCages and free drugs at different time points.
  • Figure 6 shows the intracellular transport of light-activated drugs into the nucleus by ID-RBCages.
  • Figure 7 shows the cell survival rate of (A) 4T1 cells after incubation with ID-RBCages for 24 h under light and no light conditions; (B) 4T1 cells after incubation with ID-RBCages for 24 h under light and no light conditions (785 nm, 0.5 W cm -2 , 3 min) live-dead cell staining diagram.
  • Figure 8 shows pictures of EdU staining of cells under non-illumination and 785 nm laser irradiation.
  • Figure 9 shows the immunofluorescence staining of (A) CRT protein and (B) HMGB1 protein in 4T1 cells under non-illumination and 785 nm laser irradiation; (C) flow cytometry detection of cell CRT levels and (D) fluorescence intensity statistics; (E) Detection of ATP secretion levels in 4T1 cells under non-illumination and 785 nm laser irradiation.
  • Figure 10 shows the tissue distribution of ID-RBCages.
  • A DOX distribution in each tissue 24 hours after tail vein injection of ICG and ID-RBCages (ICG, 7.5 mg kg -1 );
  • B Tail vein injection of ICG and ID- Near-infrared fluorescence imaging of isolated tissues after RBCages (ICG, 7.5 mg kg -1 ) for 24 hours and
  • C average fluorescence intensity statistics of each tissue.
  • Figure 11 shows the examination of the penetration ability of ID-RBCages into deep tumor tissue.
  • A CLSM pictures of the penetration behavior of ID-RBCages and 70 nm-mPLGA in Panc02 subcutaneous tumors and
  • B fluorescence intensity statistics in selected areas. Analysis;
  • C Distribution of DOX in tumor tissues under light and non-light conditions.
  • Figure 12 shows (A) thermal imaging of the tumor site under light conditions (785 nm, 0.5 W cm -2 ) and (B) thermal imaging of the tumor site 24 hours after injection of different concentrations of ID-RBCages into the tail vein of mice with 4T1 subcutaneous tumors. Temperature rise curve.
  • Figure 13 shows the tumor growth curve of mice with 4T1 subcutaneous tumors (A) 24 hours after tail vein injection of Free DOX, ID-RBCages or PBS and 25 days of illuminating the tumor, and (B) tumor weight statistics of each group at the end of the tumor inhibition experiment .
  • Figure 14 shows the tumor growth curve of mice with 4T1 orthotopic mammary tumors (A) 24 hours after tail vein injection of Free DOX, ID-RBCages or PBS, and 25 days of illuminating the tumor; (B) Lung metastasis at the end of the tumor inhibition experiment Bioluminescence pictures of lesions and (E) corresponding bioluminescence intensity statistics of lung metastases.
  • Figure 15 shows the photoresponsive drug entry into the nucleus of tumor tissue by ID-RBCages.
  • Figure 16 shows the synergistic use of ID-RBCages photochemotherapy and immunotherapy in the treatment of 4T1-Luc orthotopic breast cancer.
  • A After tail vein injection of Free DOX, ID-RBCages, and PBS, the tumor was illuminated for 24 hours, and aPD-L1 was injected for combined treatment. Mouse tumor growth curve within 25 days;
  • B Tumor weight statistics of mice in each group at the end of the tumor inhibition experiment;
  • C Bioluminescence pictures of lung metastases at the end of the tumor inhibition experiment and
  • D Corresponding lung metastasis Statistics of bioluminescence intensity of lesions;
  • E H&E staining pictures of lung metastases at the end of the tumor inhibition experiment.
  • Figure 17 shows the synergistic use of ID-RBCages photochemotherapy and immunity in the treatment of Panc02 pancreatic cancer subcutaneous tumors.
  • A Schematic diagram of the anti-tumor dosing regimen in mice with Panc02 subcutaneous tumors;
  • B Tumor growth curves of mice in different dosing treatment groups,
  • C Survival curves of mice and
  • D tumor growth curves of individual mice in each group.
  • Figure 18 shows (A) the tumor growth curves of blank control mice and treatment group mice after re-inoculation with tumor cells (1 ⁇ 10 5 ) on day 60 and (B) the growth curves of long-term effector T cells and central memory T cells. Flow cytometry and (C), (D) corresponding statistical analysis.
  • Figure 19 shows the synergistic use of ID-RBCages photochemotherapy and immunity in the treatment of KPC orthotopic pancreatic cancer.
  • A Schematic diagram of the dosage regimen for KPC orthotopic pancreatic cancer tumor treatment;
  • B The results of each treatment group of KPC orthotopic pancreatic cancer mice Bioluminescence photos of mouse tumors at different time points;
  • C Tumor growth curves of KPC orthotopic pancreatic cancer mouse tumor suppressor groups and
  • D Survival curves of mice in each group.
  • Figure 20 is a diagram of the products obtained under different hypotonic treatment conditions.
  • Red blood cells are existing products.
  • the red blood cells in the embodiment of the present invention are from healthy female Balb/c mice of 6 to 8 weeks old, and are separated and collected after intravenous blood collection.
  • the drug-loaded red blood cell membrane nanoparticles disclosed in the present invention have the effect of targeted delivery and anti-tumor, and their preparation method is as follows: (1) Resuspend the red blood cells collected by centrifugation in hypotonic PBS buffer (0.25 ⁇ ), and place in an ice bath Let stand for 20 to 40 minutes; then centrifuge at high speed (3500 g) for 15 minutes, discard the supernatant to collect the bottom sediment, and then wash it with hypotonic PBS buffer (0.25 ⁇ ) 3-5 times to obtain the red blood cell membrane.
  • step (2) Resuspend the red blood cell membrane prepared in step (1) into hypotonic PBS buffer (0.25 ⁇ ), place it in a 30 ml glass reaction bottle, add ICG solution, incubate for 20 to 40 minutes and then sonicate for 3 to For 6 minutes, use a liposome extruder to inject the ultrasonic cell membrane solution at 800 nm and 400 nm respectively. Extrusion is carried out under the conditions of nm, 200 nm, 100 nm and 50 nm, and the number of extrusions for each size is 11 to 25 times.
  • the red blood cell nanovesicles prepared by the above-mentioned extrusion are the anionic reaction precursor red blood cell membrane vesicles.
  • the cationic reaction precursor solution to the above-mentioned anionic reaction precursor red blood cell membrane vesicle solution to react; after the reaction is completed, take out the reaction solution and use an ultrafiltration tube with a molecular weight cutoff of 30 to 200kD to perform ultrafiltration purification of the reaction solution.
  • the reacted drug-loaded red blood cell membrane nanoparticles are obtained.
  • the ultrafiltration speed is 1200 ⁇ 5000 rpm/min, the number of ultrafiltration times is 10 to 20 times.
  • Subcutaneous tumor model In order to examine the anti-tumor biological activity of ID-RBCages, subcutaneous tumor models of 4T1 mouse breast cancer, Panc02 mouse pancreatic cancer, and KPC mouse pancreatic cancer were constructed. Among them, mouse-derived Cell 4T1 was inoculated into Balb/c female mice, and Panc02 and KPC were inoculated into C57 mice.
  • the inoculation method was to add 100 ⁇ L of cell suspension (4T1 cell density was 5 ⁇ 10 6 cells/mL, and Panc02 and KPC cell density was 5 ⁇ 10 7 /mL) was injected subcutaneously in the right leg of the mouse, and the tumor length and width were measured using a vernier caliper until the tumor grew to 70-100 mm3 .
  • KPC orthotopic pancreatic cancer model C57 mice were anesthetized after abdominal hair removal, and the left upper abdomen was surgically incised to expose the spleen and pancreas. Use surgical forceps to carefully pull the spleen and pancreatic tail out of the mouse abdominal cavity, and add matrix 50 ⁇ L of gel-coated KPC-Luc cell suspension (cell density: 1 ⁇ 10 8 cells/mL) was carefully injected into the tail end of the mouse pancreas. After the injection, the spleen and pancreas were carefully pushed back into the abdominal cavity of the mouse, and the wound was sutured. Tumor growth was monitored using bioluminescent images taken using a small animal in vivo imaging system (IVIS, Lumia II).
  • Example 1 The clinically approved photosensitizer molecule indocyanine green (ICG) and the chemotherapy drug doxorubicin (Doxorubicin, DOX) were selected for encapsulation of red blood cell membrane nanomedicines to prepare ICG/DOX red blood cell membrane nanoparticles. (ID-RBCages).
  • ICG indocyanine green
  • DOX chemotherapy drug doxorubicin
  • ID-RBCages The sulfonate group of the ICG molecule can form a complex precipitation with the protonated amino group of the DOX molecule through coordination, and co-precipitate in the cavity of the red nanocell membrane to prepare ID-RBCages.
  • the red blood cells in the embodiments of the present invention come from healthy female Balb/c mice of 6-8 weeks old. They are separated and collected after venous blood collection. Specifically, female Babl/c mice of 6-8 weeks old are taken, and the whole blood of the mice is taken after anesthesia. (1 mL) and collected into a centrifuge tube containing anticoagulant heparin sodium, centrifuged at 800 g for 10 min, discarded the supernatant and retained the lower red blood cell pellet, washed the resulting red blood cells three times with 1 ⁇ PBS buffer, and centrifuged to collect the resulting red blood cells.
  • the preparation method of ID-RBCages is as follows: (1) Resuspend the red blood cells obtained by centrifugation in 50 mL of hypotonic PBS buffer (0.25 ⁇ ), let stand in an ice bath for 30 min; then centrifuge at high speed (3500 g) for 15 min , discard the supernatant, collect the bottom pellet, and wash it three times with hypotonic PBS buffer (0.25 ⁇ ) to obtain the red blood cell membrane.
  • step (2) Resuspend the red blood cell membrane prepared in step (1) into 10 mL hypotonic PBS buffer (0.25 ⁇ ), place it in a 30 ml glass reaction bottle, add ICG solution (1.0 mg mL -1 ), Incubate for 30 minutes and then ultrasonic for 5 minutes.
  • ICG solution 1.0 mg mL -1
  • liposome extruder to extrude the ultrasonic cell membrane solution sequentially through 800 nm, 400 nm, 200 nm, 100 nm, and 50 nm microporous polycarbonate filters. Each size is extruded.
  • the number of outtakes is 25 times, the receiver is the above-mentioned 30 ml glass reaction bottle, and the 50 nm extruded red blood cell nanovesicles are ICG red blood cell membrane vesicles. Then add 2 mL of 1.0 mg mL -1 DOX solution to the above ICG red blood cell membrane vesicle solution (30 ml glass reaction bottle) (the molar ratio of ICG to DOX is 1:1), stir the reaction at 600 rpm in a 37°C water bath for 4 h, after the reaction is completed, use a 100 kd ultrafiltration tube to perform ultrafiltration to obtain drug-loaded red blood cell membrane nanoparticles; the ultrafiltration speed is 3000 rpm/min, and the number of ultrafiltration times is 15 times.
  • ID-RBCages have the characteristic absorption peak of ICG and the characteristic fluorescence emission peak of doxorubicin, indicating that ID-RBCages can successfully contain ICG and DOX; conventional calculations show that the drug loading capacity is ICG (10.5%), DOX (7.8%) ).
  • the Young's modulus of ID-RBCages was measured using AFM and averaged 18 kPa.
  • the Young's modulus of the Se-NPs previously disclosed by this research group was 68 kPa on average, using organic polymer PEG-PUSe-PEG containing monoselenium as the carrier and ICG and DOX as model drugs.
  • DOX and ICG were encapsulated into human serum albumin (HSA) through a co-precipitation reaction to prepare ICG/DOX@HSA nanoparticles with an average Young's modulus of 46 kPa.
  • HSA human serum albumin
  • Example 2 Photothermal heating ability test: Take 500 mL of ID-RBCages and free ICG solutions with concentrations (ICG) of 0.2 mM, 0.5 mM, and 1.0 mM respectively, and place them in 2 mL EP tubes. Irradiate the solution with a 785 nm laser (0.5 W cm -2 , 5 min), monitor the solution temperature using a digital display thermometer, record the solution temperature every 30 s, and draw the temperature rise curve of the solution. The photothermal warming ability of a series of ID-RBCages solutions with different concentrations was measured (Figure 2A). The results showed that ID-RBCages can effectively produce photothermal warming under 785 nm laser irradiation, and its warming effect is concentration-dependent. , as the sample concentration increases, its photothermal warming effect can be significantly enhanced.
  • ICG concentrations
  • Example 3 Investigation of the drug release behavior of light-activated ID-RBCages. Dialysis method was used to examine the in vitro drug release behavior of ID-RBCages. Take 1 mL each of ID-RBCages, ID-RBCages-Laser, ID-RBCages-Laser/Vc (785 nm, 0.5W cm -2 , 5 min, Vc concentration is 2mM) and Free DOX solution of the same concentration, and encapsulate them in In the dialysis bag (molecular weight cutoff of 3500), place the dialysis bag in a 15 mL centrifuge tube filled with dialysis medium (acetate buffer at pH 5.0 or phosphate buffer at pH 7.4). Each group has three replicates.
  • dialysis medium acetate buffer at pH 5.0 or phosphate buffer at pH 7.4
  • ID-RBCages can trigger the release of intelligent responsive chemotherapy drug DOX through the photodynamic effect under laser irradiation, laying a solid foundation for reducing drug toxic and side effects and further enhancing multi-drug synergistic tumor killing.
  • Example 4 In order to further evaluate the ability of ID-RBCages to induce singlet oxygen production in cells under light conditions, a DHE staining experiment was used to verify it. 4T1 cells were seeded in 24-well plates (1 ⁇ 10 cells per well). After the cells adhere to the wall, discard the culture medium and add pre-diluted ID-RBCages and Free I/D solution (a mixture of ICG solution and DOX solution). The concentrations are set to 0, 0.1, 0.2, 0.5, 1.0, 2.0 ⁇ g mL -1 , 3 parallel holes. Continue culturing for 6 h, remove the culture medium, and wash three times with PBS.
  • the cells in the illumination group were illuminated with a 785 nm laser (0.5 W cm -2 , 3 min) and continued to be cultured for 3 h.
  • Remove the culture medium add 1 mL of freshly prepared DHE staining solution (50 ⁇ M) to each well, and incubate in the dark for 0.5 h.
  • Remove the staining solution wash three times with PBS, observe and take photos with a laser confocal microscope, and avoid light throughout the entire process.
  • 4T1 cells were seeded in a 24-well plate (1 ⁇ 10 5 cells per well), and 1 mL of culture medium was added to each well. After cells adhere to the wall. Discard the culture medium and add pre-diluted ID-RBCages and Free I/D solutions.
  • the concentrations are set to 0, 0.1, 0.2, 0.5, 1.0, 2.0 ⁇ g mL -1 , and three parallel wells are prepared.
  • the cells in the illumination group were illuminated with a 785 nm laser (0.5 W cm -2 , 3 min) and continued to be cultured for 3 h.
  • Remove the culture medium add 1 mL of freshly prepared AO staining solution (10.0 ⁇ g mL -1 ) to each well, incubate in the dark for 0.5 h, remove the staining solution, wash three times with PBS, observe and take photos with a laser confocal microscope.
  • DHE is a superoxide anion fluorescent probe that can freely enter living cells.
  • the DHE molecule itself has no fluorescence, but the ethidium oxide produced by it under the action of ROS can produce strong red fluorescence after being incorporated into DNA, which can be used for intracellular ROS detection.
  • ID-RBCages after ID-RBCages are taken up into cells, no ROS is produced under non-light conditions, but under 785 nm laser illumination, ID-RBCages can be effective at extremely low (0.05 ⁇ g mL -1 ) drug concentrations. Induces significant ROS generation.
  • ID-RBCages taken up in cells can effectively generate ROS, which lays a good foundation for ID-RBCages to exert the photodynamic therapy effect.
  • AO staining experiments were used to analyze cells before and after light exposure.
  • the AO dye used is a membrane-permeable fluorescent dye molecule. It is weakly alkaline and exhibits green fluorescence in the cytoplasmic environment. It will be protonated in the acidic environment of the lysosome and exhibit red fluorescence. It can be used as an indicator to characterize the structure of cell lysosomes. It shows that ID-RBCages can destroy the lysosomal structure of cells under laser irradiation after being absorbed into cells, providing a strong guarantee for the drug's lysosomal escape and cytoplasmic transport.
  • Example 5 In order to further evaluate whether ROS generated by light-induced ID-RBCages will affect the structure of the cell nuclear membrane, FITC fluorescently labeled Dextran was used for verification.
  • the molecular weight of Dextran selected is 70kD. Compared with the size of the nuclear pore of the cell nucleus, Dextran with this molecular weight cannot penetrate the nuclear pores of normal cell nuclei and enter the interior of the nucleus. It can be used as an indicator of the structural integrity of the nuclear membrane.
  • 4T1 cells were seeded in a 24-well plate (1 ⁇ 10 5 cells per well), and 1 mL of culture medium was added to each well.
  • Example 6 Cellular uptake and uptake pathways. Take 4T1 cells that are growing well, plate them in a 6-well plate at a cell density of 1.0 ⁇ 10 6 cells per well, and culture them statically. After the cells adhered, ID-RBCages and free DOX (1.0 ⁇ g mL -1 ) were added respectively, and 3 duplicate wells were set up. Incubate for 2 h, 12 h, and 24 h respectively, wash the cells three times with PBS, digest, collect and count the cells, and break the cells with an ultrasonic grinder (ultrasonic power: 400 W, ultrasonic time: 5 min).
  • an ultrasonic grinder ultrasonic power: 400 W, ultrasonic time: 5 min.
  • ID-RBCages 1.0 ⁇ g mL -1
  • Efficient cellular uptake is the guarantee for the therapeutic effect of the drug.
  • the cell uptake experiment results show ( Figure 5) that the cellular uptake behavior of ID-RBCages is consistent with that of free drugs. Furthermore, the cellular uptake of ID-RBCages was related to the uptake time.
  • ID-RBCages gradually increased with time. And at the same time, ID-RBCages can cause more cellular drug uptake.
  • the uptake of ID-RBCages is 2.5 times that of free drug uptake, indicating that the use of red blood cell membrane nanoreactors for drug delivery can Effectively increases the cellular drug uptake of anti-tumor drugs, laying the foundation for further enhancing the effect of drug treatment.
  • Example 7 Use laser confocal technology to observe the dynamic process of cytoplasmic transport and nuclear delivery after intracellular DOX uptake after illumination.
  • the cells pre-incubated with ID-RBCages for 6 hours were first stained and labeled for nuclei and lysosomes, and the cell culture dish was fixed on the laser confocal imager. The focus was adjusted to find the cells on the imaging software and observe and take pictures as non-illuminated cells. image. Next, use a 785 nm laser for illumination (0.5 W cm -2 , 5 min), and use a laser confocal microscope to observe the cells at 5 min, 15 min, 30 min, and 60 min after illumination and take pictures to record the DOX cells. Internal transfer situation.
  • Example 8 Cytotoxicity. Take 4T1 cells that are in good growth status, digest and collect them, dilute them with culture medium to a cell density of 5 ⁇ 10 4 cells/mL, and inoculate them into a 96-well cell culture plate. Inoculate 100 ⁇ L cell suspension in each well and place it in a 37°C cell culture incubator. Cultivate overnight, add different concentrations of ID-RBCages nanoparticle solutions (calculated based on the concentration of DOX) into each well, and the dosage concentrations are 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, and 4.0 ⁇ g mL -1 respectively. Each concentration setting Four duplicate wells were set, and a PBS control group without drugs was set at the same time.
  • the cells were cultured for 24 h, and MTT was used to measure cell survival. Detect and calculate cell viability.
  • cells were stained using Calcein-AM/PI live cell/dead cell double staining kit. The specific steps are as follows. Take the 4T1 cells in good growth status, digest and collect them, dilute them with culture medium to a cell density of 5 ⁇ 10 4 cells/mL, and inoculate them into a 96-well cell culture plate. Inoculate 100 ⁇ L cell suspension in each well, 37 Cultivation in cell culture incubator overnight. After the cells are completely attached, ID-RBCages and Free DOX are added to each well.
  • the dosage concentration calculated as DOX is 1.0 ⁇ g mL -1 .
  • Three duplicate wells are set for each concentration, and a PBS control group without drugs is set.
  • ID-RBCages In order to study the effect of ID-RBCages on the viability of 4T1 mouse breast cancer cells, the MTT method was used to examine the cytotoxicity of ID-RBCages. As shown in Figure 7A, under non-light conditions, ID-RBCages had little effect on the growth of 4T1 cells, indicating that ID-RBCages themselves have low cytotoxicity, which is beneficial to reducing the toxic side effects of chemotherapy drugs in non-tumor sites. Under 785 nm laser illumination conditions, ID-RBCages can cause strong cell killing of 4T1 cells at a lower concentration, with an IC 50 value of 0.61 ⁇ g mL -1 . It is suggested that ID-RBCages can be used as good photochemotherapy drugs. With good photochemotherapy killing and low dark toxicity, they can selectively kill tumors through laser regulation and reduce damage to normal tissues, indicating that ID-RBCages can be used as A nanomedicine for photochemotherapy with good selectivity.
  • Example 9 In order to examine the effect of ID-RBCages on the proliferation activity of cells under light conditions, EdU staining experiment was used to characterize the cell proliferation.
  • the EdU staining experiment can specifically label cells in the S phase of active proliferation, and can indirectly examine the proliferation ability through fluorescence analysis of Azide 488. As shown in Figure 8, the fluorescence picture results can visually show the strength of the cell's active proliferation ability, in which the cell nucleus appears blue and cells in the S phase appear green.
  • ID-RBCsges have no obvious inhibitory effect on the active proliferation of tumor cells under non-light conditions, while ID-RBCages can significantly inhibit cell proliferation under light conditions, and cells with active proliferation are almost invisible in fluorescent pictures. exist. It shows that ID-RBCages can effectively inhibit the growth of tumor cells by down-regulating cell proliferation activity under light conditions, thereby damaging tumor cells.
  • Example 10 Cell immunogenic death (ICD effect) experiment.
  • ID-RBCages induce the expression of calreticulin (CRT) on the surface of tumor cells.
  • CRT calreticulin
  • 4T1 cells were seeded in a 24-well plate (1 ⁇ 10 5 cells per well), and 1 mL of culture medium was added to each well. Culture until cells are completely adherent. Discard the culture medium, add ID-RBCages and free drug control Free I/D solution (DOX, 1.0 ⁇ g mL -1 ) pre-diluted in the culture medium, and incubate for 12 hours before discarding the culture medium.
  • DOX free drug control Free I/D solution
  • the cells in the illumination group were illuminated with a 785 nm laser (0.5 W cm -2 , 3 min), and the cells were continued to be cultured for 3 h. After the cells were fixed with 4% paraformaldehyde, they were incubated with primary antibodies (2 h) and fluorescent secondary antibodies (APC). mark, 1 h). CRT expression was detected using flow cytometry. CRT immunofluorescence staining. 4T1 cells were seeded in a glass-bottomed cell culture dish (1 ⁇ 10 5 cells per well), 2 mL of culture medium was added to each well, and cultured until the cells adhered. Add ID-RBCages and Free I/D (DOX, 1.0 ⁇ g mL -1 ) respectively.
  • the illumination group was illuminated with a 785 nm laser (0.5 W cm -2 , 3 min), continued to be cultured for 6 h, fixed with 4% paraformaldehyde, and incubated with primary antibodies (12 h) and fluorescent secondary antibodies (Cy3.5 labeled , 6 h), after labeling the cell nuclei with DAPI solution (5.0 ⁇ g mL -1 ), photo analysis was performed using a laser confocal microscope.
  • ID-RBCages induce the efflux of high mobility group box 1 (HMGB1) in tumor cells.
  • 4T1 cells were seeded in a glass-bottomed cell culture dish (1 ⁇ 10 5 cells per well), 2 mL of culture medium was added to each well, and cultured until the cells were completely attached. Add ID-RBCages and Free I/D (DOX, 1.0 ⁇ g mL -1 ) respectively. Continue to incubate for 12 h, discard the culture medium, and wash three times with PBS. The illumination group was illuminated with a 785 nm laser (0.5 W cm -2 , 3 min), and the cells were continued to be cultured for 24 h. The culture medium was discarded and the cells were washed. The cells were fixed with 4% paraformaldehyde for 15 min. The fixative was removed and 0.3 was added.
  • %TritonX-100 breaks the membrane (incubate for 30 minutes). After washing with PBS, the primary antibody (12 h) and fluorescent secondary antibody (Cy3.5-labeled, 6 h) were incubated respectively. After the cell nuclei were labeled with DAPI solution (5.0 ⁇ g mL -1 ), photos were taken and analyzed using a laser confocal microscope. ID-RBCages induce the secretion of adenosine triphosphate (ATP) in tumor cells. 4T1 cells were seeded in a 24-well plate (1 ⁇ 10 5 cells per well), and 1 mL of culture medium was added to each well. Culture until cells are completely adherent.
  • ATP adenosine triphosphate
  • Example 11 In order to analyze the tumor targeting ability of ID-RBCages and its distribution in various tissues and organs of mice, a tissue distribution experiment was conducted. Panc02 subcutaneous tumor mice with tumor sizes of 70-100 mm 3 were taken, ID-RBCages and the free drug Free I/D (ICG, 7.5 mg kg -1 ) were injected into the tail vein of the tumor-bearing mice. After 24 hours, the mice were The mice were sacrificed by dislocation and their hearts, livers, spleens, lungs, kidneys and tumors were removed, and the ICG distribution was imaged and analyzed using a small animal in vivo imaging device. Strictly protect from light, then weigh each tissue and use a homogenizer to crush the tissue.
  • ICG Free drug Free I/D
  • the homogenate is extracted with a mixed solution of chloroform and methanol (1:1) to remove the organic solvent. After DMSO is reconstituted, a multifunctional microplate reader is used to analyze the tissue. The distribution of DOX in each tissue was quantitatively analyzed.
  • the tumor tissue targeting of ID-RBCages was significantly improved compared to the free drug group, and the tumor tissue accumulation amount of DOX was 15.2 ID% g -1 , which was 7.17 times that of the free drug group.
  • the reason for the enhanced targeting of ID-RBCages is that the prepared nanoparticles have a suitable size and can effectively achieve tumor accumulation through the EPR effect.
  • the deformability of ID-RBCages allows it to efficiently penetrate deep tumors, providing a basis for further exerting anti-tumor effects.
  • the application of tumor bioactivity has laid the foundation.
  • ICG can be directly applied to near-infrared fluorescence imaging, so taking small animal imaging fluorescence photos of isolated tissues can directly observe the distribution of ID-RBCages in various tissues and organs.
  • ID-RBCages has significantly improved tumor tissue targeting compared with the free drug group, and its tumor tissue fluorescence intensity is 5.25 times that of the free drug group ( Figure 10C), which is consistent with the results of quantitative tissue distribution of DOX.
  • Example 12 In order to evaluate the effective penetration ability of ID-RBCages in deep tumors, the drug delivery of ID-RBCages in deep tissues of low-permeability Panc02 mouse pancreatic cancer tumors was investigated using tumor tissue section vascular staining experiments. After the mice bearing Panc02 subcutaneous tumors were randomly divided into groups, ID-RBCages and Free-I/D (ICG, 7.5 mg kg -1 ) were injected into the tumor-bearing mice through the tail vein. 24 hours after administration, a small sample of The mouse tumors were fixed with 4% paraformaldehyde solution, and then sliced with a microtome (the thickness of the slices was 10 ⁇ m). After immunofluorescence staining of CD31, the slices were observed and photographed using a laser confocal microscope, and Image J was used to Images were subjected to statistical analysis of fluorescence intensity.
  • ID-RBCages could achieve good deep tumor penetration in the pancreatic cancer Panc02 subcutaneous tumor model with low permeability and dense tumor extracellular matrix.
  • the ID-RBCages drug still has relatively uniform and efficient deep penetration capabilities, and has a uniform ID-RBCages fluorescence signal distribution in the tissue area around the blood vessels ( Figure 11B) .
  • This is related to the appropriate size of ID-RBCages and their good softness, which can achieve deep penetration through deformation in tumor tissues to achieve efficient drug delivery.
  • ID-RBCages could be effectively taken up by tumor cells at the tumor site 24 hours after tail vein administration.
  • ID-RBCages After illumination, ID-RBCages responsively release the DOX contained therein, and the ROS generated by the ID-RBCages damages the cell nuclear membrane, accelerating the effective transport of DOX to the nucleus of tumor cells, which provides a basis for photochemotherapy of tumor cells.
  • the killing effect provides a favorable guarantee.
  • Example 13 Near-infrared thermal imaging.
  • a thermal imager was used to monitor the tumor temperature of mice under laser irradiation.
  • ID-RBCages and Free I/D ICG, 7.5 mg kg -1
  • ICG Free I/D
  • the mouse tumor site was illuminated with a 785 nm laser (0.5 W cm -2 , 5 min), and use a thermal imager to record the tumor temperature.
  • the temperature of the tumor site in the ID-RBCages group with a dose of 10.0 mg kg -1 could reach 19.2°C, and within the 5-minute observation time, the tumor site could continue to maintain the highest temperature without falling after it reached the highest temperature. This relies on the efficient targeted accumulation of ID-RBCages at the tumor site. ID-RBCages effectively enhance the photostability of ICG.
  • Example 14 Photochemotherapy is used to treat breast cancer in mice. Investigation of the anti-tumor effect of ID-RBCages using the 4T1 subcutaneous tumor model. The experiment started when the mouse tumor volume was 70-100 mm 3. ID-RBCages and Free I/D (ICG, 7.5 mg kg -1 ) were injected into the mice through the tail vein. After 24 hours, the mice in the illumination group were irradiated with laser (0.5 W cm -2 , 5 min) on the tumor site using a 785 nm laser, and the tumor size of the mice was measured using a vernier caliper within 25 days. On the 25th day, after measuring the tumors, the mice in each group were sacrificed, and the tumors of the mice were removed, photographed and weighed.
  • ICG Free I/D
  • the dosing regimen for the 4T1 orthotopic breast cancer experiment is the same as that for subcutaneous tumors. The difference is that the tumor observation time for the orthotopic breast cancer experiment is 21 days. At the 21st day, the lung tissue of each group of mice is removed for in vitro bioluminescence imaging. Photos were taken, and then the isolated lung tissue was fixed in 4% paraformaldehyde solution, sectioned and stained with H&E. At the same time, the tumors of the 21-day-old mice were photographed and weighed.
  • the tumors of the mice in the ID-RBCages administration group grew faster under non-light conditions, and their growth trend was basically the same as that of the mice in the PBS group. On the 25th day, the tumor growth multiple of the mice in this group was 42.2 times. .
  • the tumor growth of mice in the ID-RBCages administration group was significantly inhibited under near-infrared laser irradiation. During the first 9 days after light treatment, the average tumor volume gradually decreased, indicating that the tumors of mice in this group After being effectively controlled, the tumors of the mice in this group began to gradually relapse and grow, but their tumors grew slowly. On the 25th day, their average tumor growth was 5.1 times the original tumor volume.
  • ID-RBCages can effectively inhibit tumor growth under light conditions, but this inhibitory effect cannot achieve complete tumor ablation, and the drug administration strategy needs to be further changed or combined with other anti-tumor drugs to effectively treat tumors.
  • ID-RBCages can be used as an efficient photoresponsive anti-tumor drug delivery platform, which can achieve highly selective and effective tumor inhibition by near-infrared light excitation at the tumor site. It is expected to be further combined with other anti-tumor treatment strategies to achieve effective treatment of tumors.
  • the growth multiple is 4.6 times.
  • the mice in the ID-RBCages-Laser/Vc treatment group were pre-injected into the tumor with Vc to shield the photodynamic effect.
  • the tumors in this group of mice gradually receded in the first 6 days after illumination, and then the tumors recurred and grew faster.
  • the final tumor growth fold was 11.0 times. There was no significant difference in tumor growth of free drugs compared with the PBS group under light and non-light conditions.
  • Bioluminescence imaging results of isolated lung tissue showed that the lung tumor metastasis of mice in the ID-RBCages light group was significantly reduced compared with the PBS group, while the lung metastasis inhibition effect of mice in the ID-RBCages-Laser/Vc group became worse, indicating that light Dynamic therapy is not only beneficial to the suppression of in situ tumors, but also plays an important regulatory role in the suppression of metastases.
  • Example 15 In order to further examine the nuclear transport ability of ID-RBCages after DOX-responsive release at the tissue level, tumor tissue sections before and after illumination were observed. As shown in Figure 15, ID-RBCages can be effectively taken up by tumor cells at the tumor site 24 hours after tail vein administration. In the absence of light, ID-RBCages are mainly distributed in the cytoplasm of cells, and are less distributed in the nucleus.
  • the tumor sections also showed that under non-illumination conditions, the tumor tissue morphology was relatively complete, and the cells remained relatively dense, indicating that under non-illumination conditions, ID-RBCages had no obvious damaging effect on the tumor tissue; further, after illumination, Slice analysis of the tumor tissue showed that the ID-RBCages in the tumor tissue cells after illumination had a high co-localization with the nucleus, indicating that the ID-RBCages responsively released their contained DOX after illumination, and their ID-RBCages The generated ROS damages the cell nuclear membrane, accelerates the effective transport of DOX to the nucleus of tumor cells, and provides a favorable guarantee for the killing effect of photochemotherapy on tumor cells.
  • Example 16 In order to further examine the potential application value of ID-RBCages inducing tumor immunotherapy, aPD-L1 and ID-RBCages were used in combination to investigate the therapeutic effect of orthotopic breast cancer.
  • the experimental group design is PBS, aPD-L1, Free I/D, Free I/D/aPD-L1, ID-RBCages, ID-RBCages/Laser, ID-RBCages/Laser/Vc, ID-RBCages/Laser/aPD-
  • ID-RBCages and Free I/D ICG, 7.5 mg kg -1 ) were injected into the mice through the tail vein for administration.
  • mice in the light group The mice were irradiated with laser (0.5 W cm -2 , 5 min) on the tumor site using a 785 nm laser.
  • the dose of aPD-L1 was 5 mg/kg, which was administered intraperitoneally on days 2, 5, and 8 respectively.
  • the mice will be sacrificed and the mouse lung tissue will be removed for bioluminescence imaging. Then the isolated lung tissue will be fixed in 4% paraformaldehyde solution and sectioned. Afterwards, H&E staining was performed, and the tumors of the mice were photographed and weighed.
  • aPD-L1 has limited effect on the treatment of orthotopic breast cancer.
  • the free drug light group was combined with aPD-L1
  • there was no significant difference in tumor growth compared to the non-antibody group indicating that the combination of free drugs and aPD-L1 antibodies could not effectively enhance the therapeutic effect.
  • the ID-RBCages illumination group combined with aPD-L1 antibody can completely eliminate the original tumor and effectively inhibit tumor recurrence.
  • the possible reason is that ID-RBCages can induce a strong immunotherapy effect under illumination conditions and can promote the tumor site. Recruiting abundant CTLs and other anti-tumor immune cells, therefore when combined with aPD-L1 antibody, it can significantly improve the anti-tumor activity and achieve efficient synergy between photochemotherapy and immunotherapy, thereby achieving complete ablation of the in situ tumor and effectively inhibiting tumor lung The occurrence of partial transfer.
  • Example 17 In order to examine the anti-tumor biological activity of ID-RBCages in Panc02 tumor-bearing mice, the Panc02 subcutaneous tumor model was used to evaluate the anti-tumor biological effect of ID-RBCages combined with photochemotherapy and immunotherapy. The mouse Panc02 pancreatic cancer subcutaneous tumor model was used to conduct tumor inhibition experiments to examine the therapeutic effect of ID-RBCages on low-penetration tumors.
  • a total of 8 different groups were set up, namely: PBS, aPD-L1, Free I/D-Laser, Free I /D-Laser/aPD-L1, ID-RBCages, ID-RBCages/aPD-L1, ID-RBCages-Laser, ID-RBCages-Laser/aPD-L1, GEM+Abraxane.
  • ID-RBCages and Free I/D (7.5 mg kg -1 ) were injected into the tail vein on day 0.
  • the tumors in the light group were irradiated with 785 nm laser (0.5 W cm -2 , 4 min) 24 hours after administration.
  • mice 35 mg kg -1
  • Abraxane 8 mg kg -1
  • aPD-L1 antibody 5.0 mg kg -1
  • the mouse tumor growth was recorded from 0 to 35 days. When the tumor volume reached 1500 mm3 , the mouse was considered dead, and the survival of the mice was observed until 120 days.
  • mice with tumor volume of 70 mm3 were treated according to the ID-RBCages-Laser/aPD-L1 treatment plan in the tumor suppression group, and mice with complete tumor ablation were selected for tumor re-challenge experiments on the 60th day after treatment. . That is, Panc02 cells were subcutaneously inoculated on the contralateral side of the mouse where the tumor was first received (the number of cells inoculated was 1 ⁇ 10 6 /mouse), and Panc02 was also inoculated into the blank mice as a control. After inoculation, the tumor growth of the two groups of mice was recorded. At the same time, on the 60th day, six mice each with complete tumor ablation and six mice without blank were selected. After taking the tumor tissue to prepare a single cell suspension, immunostaining of long-term memory T cells was performed and analyzed using flow cytometry. analyze.
  • the tumors of the Panc02 subcutaneous tumors in the ID-RBCages illumination group were ablated on the 13th day after illumination, and then the tumors recurred, and the tumors grew rapidly after recurrence, and on the 35th day, the tumors averaged The volume can reach 945 mm 3 .
  • the tumors in this group The average mouse tumor volume was 113 mm 3 .
  • the survival curve of mice 120 days after administration was statistically analyzed ( Figure 17D).
  • mice in the treatment group were inoculated with tumor cells again, they would not grow subcutaneous tumors, indicating that the mice had a memory immune effect that could effectively inhibit the growth of tumors inoculated with tumor cells again.
  • Flow cytometry was further used to analyze T cells in the spleens of mice ( Figure 18B-D).
  • the results showed that the number of long-term memory T cells and central memory T cells in the mice in the treatment group significantly increased, among which The number of TEM was 3.1 times that of the blank control mice, and the number of TCM was 1.9 times that of the blank control mice.
  • ID-RBCages photochemotherapy and immune synergistic treatment can effectively induce long-term immune memory effects in mice and play an important role in preventing tumor recurrence or metastasis.
  • Example 18 More than 90% of human pancreatic cancer tumors will have KRAS mutations.
  • mouse KPC tumor cells with both KRAS and TP53 mutations were selected to study the anti-tumor effect.
  • the combined use of the innate immune STING agonist SR717 and aPD-L1 antibody further enhanced the effect of immunotherapy.
  • KPC-Luc cells were used to construct an orthotopic tumor model of mouse pancreatic cancer, and tumor growth was monitored using small animal in vivo imaging.
  • the tumor inhibition experiment of mouse KPC orthotopic pancreatic cancer has a total of 8 different groups, namely: PBS, SR717+aPD-L1, GEM+Abraxane, GEM+Abraxane+SR717+aPD-L1, Doxil, ID -RBCages-Laser, ID-RBCages-Laser/SR717+aPD-L1.
  • ID-RBCages and Free I/D ICG, 7.5 mg kg -1
  • mice in the light group were first anesthetized and then surgically opened their abdominal cavities. , use a 785 nm laser to illuminate the tumor site (0.5 W cm -2 , 5 min), and surgically suture the wound after the illumination is completed.
  • the SR717+aPD-L1 combined treatment group received intraperitoneal injection of aPD-L1 antibody (5.0 mg kg -1 ) and SR717 (35 mg kg -1 ) on days 2, 5, and 8.
  • GEM+Abraxane was administered by tail vein injection on days 0, 3, and 6, with the dose of GEM being 35 mg kg -1 and Abraxane being 8 mg kg -1 .
  • a small animal in vivo imaging system was used to conduct bioluminescence imaging of abdominal pancreatic tumors to monitor the growth of in situ pancreatic cancer tumors. And the survival status of mice in each group was recorded within 120 days.
  • the pancreatic cancer of the mice in the PBS group grew rapidly. On the 24th day, the bioluminescence intensity of the tumor site of the mice in this group increased by 59.7 times.
  • Other treatment groups such as SR717+aPD-L1, GEM +Abraxane and SR717+aPD-L1+GEM+Abraxane treatment groups have poor therapeutic effects on KPC orthotopic pancreatic cancer.
  • Doxil in the clinical drug control group also had no significant inhibitory effect on the treatment of KPC orthotopic pancreatic cancer, and its tumor growth multiple was 42.8 times at 24 days. Illumination of ID-RBCages can effectively inhibit the growth of pancreatic cancer in mice.
  • mice treated with ID-RBCages light were die.
  • the half survival period of mice treated with ID-RBCages light was 45 days, and 60% of mice in the ID-RBCages light combined with immunotherapy group were still alive on the 120th day after treatment. It shows that ID-RBCages photochemotherapy and immune synergy can effectively prolong the survival time of KPC orthotopic tumor mice, bringing hope for the effective treatment of orthotopic pancreatic cancer.
  • Example 19 On the basis of Example 1, the volume of the added ICG solution was adjusted to obtain drug-loaded red blood cell membrane nanoparticles with a molar ratio of ICG and DOX of 1:0.5 or 1:2.
  • step (1) On the basis of Example 1, step (1) was adjusted as follows: (1) Resuspend the red blood cells obtained by centrifugation in 50 mL of hypotonic PBS buffer (0.25 ⁇ ), and let stand in an ice bath for 30 min; then wash 3 times with hypotonic PBS buffer (0.25 ⁇ ) to obtain red blood cell membranes. The remaining steps are the same as in Example 1. The solution remains clear during stirring. Use a transmission electron microscope to observe the morphology of the solution after the reaction is completed. The results in Figure 20A show that there are irregularly shaped cell membrane fragments in the nanoparticle solution, and no morphology was observed. Nano-production, it is impossible to prepare red blood cell membrane nanoparticles with uniform size.
  • step (1) was adjusted as follows: (1) Resuspend the red blood cells obtained by centrifugation in 50 mL of hypotonic PBS buffer (0.25 ⁇ ), stir in an ice bath at 3500 rpm for 30 min; then, discard the supernatant to collect the bottom precipitate, and then wash it 5 times with hypotonic PBS buffer (0.25 ⁇ ) to obtain the red blood cell membrane; use the same method as Example 1, and when obvious turbidity appears during the stirring reaction, nanoparticles are obtained There is only a small amount of drug layer on its surface, which is in the shape of a hollow ring and cannot effectively contain drugs, see Figure 20B. In contrast, the entire reaction process in Example 1 was clarified, and electron microscopy showed that ID-RBCages nanoparticles could effectively encapsulate drugs.
  • the present invention successfully realizes the biomimetic synthesis of red blood cell membrane nanoparticles (ID-RBCages) loaded with ICG and DOX; the main technical progress achieved is as follows: (1) Using nano red blood cell membranes to encapsulate clinical anti-tumor drugs ID-RBCages were successfully prepared through the reaction of photosensitizer (ICG) and chemotherapy drug (DOX).
  • ICG photosensitizer
  • DOX chemotherapy drug
  • the average particle size of the product is 65.8 ⁇ 5.4 nm
  • the size of ICG/DOX nanoparticles is 5.3 ⁇ 3.8 nm, with regular morphology and uniform particle size.
  • ID-RBCages have good photothermal and photodynamic effects and can achieve light-activated DOX responsive release. After ID-RBCages are activated by light, they can synergistically enhance the ICD effect of tumor cells through the photodynamic effect and the chemotherapeutic effect of DOX, and have the potential to enhance the effect of immunotherapy.
  • ID-RBCages can be efficiently taken up by tumor cells and distributed in the acidic lysosomes of cells; tumor targeting is good.
  • the tumor site drug distribution of DOX is 15.2 ID% g -1 .
  • ID-RBCages can be activated by light in cells to release DOX, and are rapidly transported from lysosomes to the cytoplasm, and then rapidly enter the nucleus to exert synergistic anti-tumor effects with photochemotherapy.
  • ID-RBCages have strong tissue penetration ability in low-permeability Panc02 mouse pancreatic cancer tumors, can efficiently deliver drugs to deep tumors, and can achieve intelligent response release of chemotherapeutic drugs and cell nuclear transport, and coexistence of DOX and cell nuclei after illumination.
  • ID-RBCages have good photochemotherapy effect in 4T1 breast cancer subcutaneous and in situ tumors, can significantly inhibit tumor growth, and can effectively inhibit lung metastasis.
  • the ID-RBCages photochemotherapy-immune synergistic therapy strategy can achieve the transformation from an immune "cold tumor” to an immune "hot tumor”, reverse the immunosuppressive microenvironment, completely ablate 4T1 in situ breast cancer, and effectively inhibit lung metastasis.
  • ID-RBCages combined with aPD-L1 antibody can effectively inhibit the growth of Panc02 pancreatic cancer subcutaneous tumors.
  • ID-RBCages photochemotherapy-immune synergistic therapy strategy can degrade the extracellular matrix, reverse the immunosuppressive microenvironment, enhance tumor infiltration of macromolecule drug aPD-L1 and CTLs, and achieve effective treatment of refractory and low-penetration pancreatic cancer.
  • ID-RBCages combined with aPD-L1 and SR717 can effectively inhibit the tumor growth of orthotopic pancreatic cancer in double-mutated KPC-Luc mice.
  • the ID-RBCages photochemotherapy-immune synergistic therapy strategy relieves the inhibition of STING activation in KPC cells by TP53 mutations, dually activates acquired immunity and innate immunity, and achieves efficient treatment of double-mutated pancreatic cancer by regulating the extracellular matrix and immunosuppressive microenvironment. .

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Abstract

一种载药红细胞膜纳米粒及其制备方法和应用，载有阴离子反应前体和阳离子反应前体。以红细胞膜为反应器，在水相中制备具有光响应性药物释放功能的红细胞膜仿生纳米粒，该纳米粒具有良好的光热效果、物理化学稳定性、光稳定性、肿瘤靶向性、肿瘤深部穿透和响应性药物释放，准确定位肿瘤，在近红外光激发下产生高效光热效应，热消融杀死肿瘤细胞。

Description

一种载药红细胞膜纳米粒及其制备方法和应用 技术领域
本发明公开了一种用于肿瘤靶向药物递送的红细胞膜纳米体系，具体涉及一种载ICG-DOX红细胞膜纳米粒及其制备方法和应用。
 背景技术
恶性肿瘤是严重威胁人类健康的重大恶性疾病之一，其发病率和死亡率在国内外均呈明显的上升趋势。如何实现肿瘤的精确诊断与高效治疗是当前研究的重点及难点。纳米技术的发展为肿瘤的治疗提供了一种新的策略，纳米药物因其具有肿瘤特异性靶向药物递送的特点，在增强药物疗效、降低药物毒副作用上发挥着重要作用。
在目前文献报道的不同纳米生物平台中，通过仿生方法合成纳米药物采用生物源性的天然材料例如白蛋白、细胞、细菌以及病毒等材料进行纳米药物递送，该类型的纳米材料结合了天然物质与纳米技术的各自优势，越来越成为肿瘤治疗具有良好应用前景的新型技术。天然材料仿生合成纳米材料具有材料自身独特的生物学特征，如长效循环、肿瘤特异性靶向、免疫调节等。目前基于白蛋白以及细胞膜的纳米药物递送体系已经被广泛应用于肿瘤精确治疗当中（如J Control Release, 2021, 329, 997-1022；Chem Soc Rev, 2021,50, 945-985）。
目前基于细胞膜的纳米药物研究中，红细胞因其自身来源丰富，结构简单无复杂的细胞器，易于提取细胞膜，且其细胞膜表面可修饰性强，被广泛应用于纳米药物研究。目前基于红细胞药物递送的研究重要有两种不同的策略，以完整红细胞作为载体进行药物递送以及使用红细胞膜对现有纳米颗粒如PLGA纳米粒、氧化铁磁性纳米粒子、介孔二氧化硅纳米粒子、脂质体、聚合物囊泡等进行细胞膜包裹进行纳米材料的合成。比如现有技术水热法制备的PEG‑Fe 3O 4用经靶向修饰的红细胞膜碎片挤出包裹，制备出有免疫逃避性和灵敏磁响应性的纳米颗粒红细胞膜‑PEG‑Fe 3O 4；现有技术还公开了一种红细胞膜包覆的MOFs纳米药物载体及其制备方法与应用，该MOFs纳米药物载体包括MOFs纳米粒子、红细胞膜，所述MOFs纳米粒子的孔隙上装载有硼酸，所述MOFs纳米粒子外部包裹红细胞膜，该MOFs纳米药物载体采用水热法合成。现有技术都是采用先制备药物粒子再被红细胞膜包裹的技术方案，该药物递送存在载药量较低、靶向性较差等局限，而且合成工艺复杂、纳米材料尺寸控制依赖于内核纳米材料而难以精确调控纳米材料的尺寸大小等问题。
技术问题
为解决上述技术问题，本发明的目的是提供一种基于红细胞膜纳米囊泡，在其内部实现药物反应与制备，用于抗肿瘤药物的包载，具有尺寸精确可调控、制备简单、药物原位合成的特点，可以实现更高的靶向性，增加药物包载效率，并且具有良好的生物安全性、肿瘤靶向性、肿瘤深部穿透和滞留，并且所制备的细胞膜纳米粒保留细胞膜自身的柔软性，可以发生形变实现在肿瘤部位的深部组织穿透，提高药物在肿瘤组织的高效深部递送，同时ICG与DOX形成复合物沉淀降低了DOX在正常组织中的非必要释放，毒副作用有效降低。而在肿瘤组织中，通过激光照射，ID-RBCages可以响应性释放化疗药物DOX。同时，光激活的光动力治疗、化疗通过诱导肿瘤细胞免疫原性死亡，引起抗肿瘤获得性免疫效应激活，实现肿瘤增强的光化疗-免疫协同治疗。
 技术解决方案
本发明采用如下技术方案：一种载药红细胞膜纳米粒，其制备方法为，将红细胞膜与阴离子反应前体混合后挤出，得到阴离子反应前体红细胞膜囊泡；再将阴离子反应前体红细胞膜囊泡与阳离子反应前体混合反应，得到载药红细胞膜纳米粒。
本发明公开了具有靶向递送抗肿瘤药物的尺寸精确可控的载药红细胞膜纳米粒，所述载药红细胞膜纳米粒的粒径为20～240 nm。纳米药物递送体系的递送效果与其自身的尺寸大小有着密切的关系，尺寸调控对于肿瘤药物递送的调控至关重要。通过尺寸调控制备不同粒径大小的纳米粒可用于抗肿瘤药物的尺寸调控性筛选。粒径越小的红细胞膜纳米粒肿瘤药物递送效率越高。因此，研究制备生物源性材料尺寸可调控性的纳米药物递送方法，有助于构建多模态成像和治疗肿瘤的更加安全的平台。
上述技术方案中，红细胞膜作为纳米粒的骨架；所述阳离子反应前体为金属化合物或者小分子药物；所述阴离子反应前体源为硫化物、柠檬酸化合物或者染料药物。阳离子反应前体、阴离子反应前体比如阿霉素和吲哚菁绿。
上述技术方案中，由红细胞低渗处理得到红细胞膜；优选的，将红细胞悬于低渗缓冲液中，静置后离心处理，得到红细胞膜；进一步优选的，静置为冰浴静置20～40分钟，离心处理为3000～4000g处理10～20分钟。低渗缓冲液的浓度为其对应的等渗缓冲液浓度的20～30%。
上述技术方案中，将红细胞膜与阴离子反应前体在低渗缓冲液中孵育，然后用脂质体挤出器挤出，得到阴离子反应前体红细胞膜囊泡；然后将阴离子反应前体红细胞膜囊泡与阳离子反应前体在低渗缓冲液中搅拌反应，然后超滤，得到载药红细胞膜纳米粒。优选的，孵育的时间为20～40分钟，孵育后超声处理3～6分钟，再用脂质体挤出器梯度挤出；优选的，梯度挤出的孔径为800 nm、400 nm、200 nm、100 nm、50 nm。搅拌反应的反应温度为25～55℃，反应时间为3～8h，优选为37℃下搅拌反应4～5小时。
上述技术方案中，阴离子反应前体、阳离子反应前体的摩尔比为1∶（0.5～8），优选1∶（0.5～2）。
本发明提出上述载药红细胞膜纳米粒在制备肿瘤治疗药物中的应用。
 有益效果
本发明的载药红细胞膜纳米粒具有：（1）良好的尺寸均一性，其粒径在60~240 nm尺寸范围内，通过挤出器聚碳酯膜的尺寸可实现红细胞膜纳米尺寸的精确调控；（2）红细胞膜作为天然的生物源性材料，其生物相容性较好，其作为药物递送载体具有较好的安全性，并且载药红细胞膜纳米粒具有极低的细胞暗毒性和较好的血液相容性，其在荷瘤小鼠模型中具有良好的肿瘤靶向性；（3）载药红细胞膜纳米粒具有较好的柔软度，其相比较于常规的细胞膜包裹技术制备的细胞膜纳米粒子具有显著增强的变形性，可实现肿瘤组织的高效靶向、深部穿透以及有效滞留；（4）载药红细胞膜纳米粒可被肿瘤细胞高效摄取，并定位于酸性溶酶体中，光照后能够破坏溶酶体膜并增强细胞核膜通透性，实现DOX的快速胞浆转运及细胞核递送。此外，ID-RBCages能诱导显著的细胞免疫原性死亡，高效激活获得性抗肿瘤免疫效应；（5）红细胞膜作为天然膜性材料，在光热条件下以及其他光刺激条件如光动力条件下可进行光响应性的细胞膜破裂，为响应性药物释放奠定基础；（6）载药红细胞膜纳米粒肿瘤靶向性良好，在低渗透性Panc02小鼠胰腺癌肿瘤中能高效穿透组织，递送药物到肿瘤深部；另外，光照还能促进化疗药物快速转运入核，实现高效光化疗协同抗肿瘤效果。
 附图说明
图1为ID-RBCages的透射电镜图片（A）、ID-RBCages的动态光散射粒径（B）、ID-RBCages的吸收图谱（C）、ID-RBCages的荧光光谱（D）。
图2为ID-RBCages的光热升温能力考察（A）、不同条件下的光照后的透射电镜图（B）、光照条件下的释放（C）。
图3为ID-RBCages的DHE以及AO染色。
图4为ID-RBCages的光照细胞核损伤实验。
图5为ID-RBCages以及游离药物不同时间点的细胞摄取。
图6为ID-RBCages的光激活药物胞内转运入核。
图7为（A）4T1细胞与ID-RBCages孵育24 h后光照及未光照条件下细胞存活率；（B）4T1细胞与ID-RBCages孵育24 h后光照及未光照条件下（785 nm，0.5 W cm -2，3 min）活-死细胞染色图。
图8为非光照以及785 nm激光照射下细胞EdU染色图片。
图9为非光照以及785 nm激光照射下4T1细胞（A）CRT蛋白、（B）HMGB1蛋白的免疫荧光染色图;（C）流式细胞仪检测细胞CRT水平及其（D）荧光强度统计;（E）非光照以及785 nm激光照射下4T1细胞ATP分泌水平检测。
图10为 ID-RBCages的组织分布，（A）尾静脉注射ICG和ID-RBCages（ICG，7.5 mg kg -1）24 h后各组织中DOX分布情况；（B）尾静脉注射ICG和ID-RBCages（ICG，7.5 mg kg -1）24 h后离体组织的近红外荧光成像图以及（C）各个组织的平均荧光强度统计。
图11为ID-RBCages的深部肿瘤组织穿透能力考查，（A）ID-RBCages及70 nm-mPLGA在Panc02皮下瘤中穿透行为的CLSM图片及其在选定区域的（B）荧光强度统计分析；（C）光照以及非光照条件下肿瘤组织中DOX的分布。
图12为4T1皮下瘤小鼠尾静脉注射不同浓度的ID-RBCages 24 h后（A）肿瘤部位在光照条件下（785 nm，0.5 W cm -2）的热成像图片及（B）肿瘤部位的温度升高曲线。
图13为4T1皮下肿瘤小鼠（A）尾静脉注射Free DOX、ID-RBCages或者PBS后24 h光照肿瘤，25天内的小鼠肿瘤生长曲线以及（B）抑瘤实验结束时各组瘤重统计。
图14为4T1原位乳腺肿瘤小鼠（A）尾静脉注射Free DOX、ID-RBCages或者PBS后24 h光照肿瘤，25天内的小鼠肿瘤生长曲线；（B）抑瘤实验结束时肺部转移灶的生物发光图片及（E）相应的肺转移灶生物发光强度统计。
图15为ID-RBCages的肿瘤组织光响应药物入核。
图16为ID-RBCages光化疗免疫协同用于4T1-Luc原位乳腺癌治疗，（A）尾静脉注射Free DOX、ID-RBCages、PBS后24 h光照肿瘤，治疗注射aPD-L1进行联合治疗，25天内的小鼠肿瘤生长曲线；（B）抑瘤实验结束时各组小鼠的瘤重统计；（C）抑瘤实验结束时肺部转移灶的生物发光图片及（D）相应的肺转移灶生物发光强度统计；（E）抑瘤实验结束时肺部转移灶的H&E染色图片。
图17为ID-RBCages光化疗免疫协同用于Panc02胰腺癌皮下瘤治疗，（A）Panc02皮下瘤小鼠抑瘤给药方案示意图；（B）不同给药治疗组的小鼠肿瘤生长曲线、（C）小鼠的生存曲线以及（D）每组小鼠中单只小鼠的肿瘤生长曲线。
图18为（A）空白对照小鼠以及治疗组小鼠在第60天进行肿瘤细胞（1×10 5）再接种后的肿瘤生长曲线及其（B）长期效应T细胞以及中央记忆T细胞的流式细胞图与（C）、（D）相应统计分析。
图19为ID-RBCages光化疗免疫协同用于KPC原位胰腺癌治疗，(A) KPC原位胰腺癌肿瘤治疗的给药方案示意图；（B）KPC原位胰腺癌小鼠各治疗组的小鼠不同时间点肿瘤的生物发光照片；（C）KPC原位胰腺癌小鼠抑瘤各组小鼠的肿瘤生长曲线以及（D）各组小鼠的生存曲线。
图20为不同低渗处理条件得到的产物图。
 本发明的实施方式
脂质体挤出器（Avanti，美国），吲哚菁绿、阿霉素采购于国药集团化学试剂有限公司；超滤离心管（Millipore，美国），碳支持膜铜网、碳支持膜镍网（北京新兴百瑞科技有限公司）。红细胞为现有产品，本发明实施例的红细胞来自6-8周的雌性Balb/c健康小鼠，静脉取血后分离收集得到。
本发明公开的载药红细胞膜纳米粒具有靶向递送抗肿瘤的作用，其制备方法如下：（1）将离心收集所得红细胞重悬于低渗的PBS缓冲液中（0.25×），于冰浴中静置20～40 min；然后高速离心（3500 g）15 min，弃上清收集底层沉淀，再使用低渗PBS缓冲液（0.25×）洗涤3-5次，得到红细胞膜。
（2）将步骤（1）所制备的红细胞膜重悬至低渗PBS缓冲液（0.25×）中，并置于30 ml玻璃反应瓶中，加入ICG溶液，孵育20～40分钟后超声3～6min，将超声后细胞膜溶液使用脂质体挤出器分别在800 nm，400 nm，200 nm，100 nm以及50 nm的条件下进行挤出，每种尺寸挤出次数为11～25次。上述挤出所制备的红细胞纳米囊泡即为阴离子反应前体红细胞膜囊泡。
（3）往上述阴离子反应前体红细胞膜囊泡溶液中加入阳离子反应前体溶液进行反应；反应结束后取出反应溶液，使用截留分子量为30~200kD的超滤管对反应溶液进行超滤纯化，得到反应后的载药红细胞膜纳米粒。优选的，超滤的转速为1200~5000 rpm/min，超滤的次数为10~20次。
肿瘤模型的建立：（1）皮下瘤模型：为考察ID-RBCages抗肿瘤生物活性，构建了4T1小鼠乳腺癌、Panc02小鼠胰腺癌、KPC小鼠胰腺癌的皮下瘤模型，其中鼠源性细胞4T1接种于Balb/c雌性小鼠，Panc02、KPC接种于C57小鼠，接种方法为将100 µL细胞悬液（4T1细胞密度为5×10 6个/mL，Panc02、KPC细胞密度为5×10 7个/mL）注射在小鼠右腿皮下，使用游标卡尺测量肿瘤长和宽，待肿瘤长至70-100 mm 3时使用。肿瘤体积计算方法为：肿瘤体积＝1/2×（长径×短径×短径）。
（2）4T1原位乳腺癌模型：将6-8周的Balb/c雌性小鼠麻醉后手术切开其第二对乳腺附近皮肤，将预先稀释好的4T1-Luc细胞悬液（细胞密度为1×10 7个/mL）50 µL注射至小鼠乳腺下方脂肪垫中，缝合伤口。使用游标卡尺测量肿瘤长和宽，待肿瘤长至70-100 mm 3时使用。
（3）KPC原位胰腺癌模型：将C57小鼠腹部脱毛后麻醉，手术切开其左上腹部，暴露脾脏以及胰腺，使用手术镊子小心将脾脏及胰腺尾部拉出小鼠腹腔，将加有基质胶的KPC-Luc细胞混悬液50 µL（细胞密度为1×10 8个/mL）小心注射到小鼠胰腺尾端，注射完毕将脾脏以及胰腺小心推回小鼠腹腔后，缝合伤口。使用小动物活体成像系统（IVIS，Lumia II）拍摄生物发光图片监控肿瘤生长情况。
下面结合附图和实施例，对本发明的具体实施方式进一步详细描述。其中，实施例用于说明本发明，但不局限于本发明。本发明的具体实验操作以及测试方法为常规技术。所有数据的统计学分析使用GraphPad Prism 8.3软件，数值以mean ± SD表示，统计分析采用独立样本 t检验， P值＜0.05被认为有统计学意义。NS无显著性，* P<0.05，** P<0.01，*** P<0.001，**** P<0.0001。
实施例一：选用了临床批准的光敏剂分子吲哚菁绿（Indocyanine green，ICG）和化疗药物阿霉素（Doxorubicin，DOX）用于红细胞膜纳米药物包载，制备ICG/DOX红细胞膜纳米粒（ID-RBCages）。ICG分子的磺酸根可以与DOX分子质子化的氨基通过配位作用形成复合物沉淀，在红纳米细胞膜空腔中进行共沉淀制备ID-RBCages。
本发明实施例的红细胞来自6-8周的雌性Balb/c健康小鼠，静脉取血后分离收集得到，具体的，取6-8周雌性Babl/c小鼠，麻醉后取小鼠全血（1 mL）并收集到含有抗凝剂肝素钠的离心管中，800 g离心力下离心10 min弃上清保留下层红细胞沉淀，将所得红细胞用1×PBS缓冲液洗涤三次，离心收集所得红细胞。
ID-RBCages的制备方法如下：（1）将离心所得红细胞重悬于50 mL低渗的PBS缓冲液中（0.25×），于冰浴中静置30 min；然后高速离心（3500 g）15 min，弃上清收集底层沉淀，再使用低渗PBS缓冲液（0.25×）洗涤3次，得到红细胞膜。
（2）将步骤（1）所制备的红细胞膜重悬至10 mL低渗PBS缓冲液（0.25×）中，并置于30 ml玻璃反应瓶中，加入ICG溶液（1.0 mg mL -1），孵育30分钟后超声5min，将超声后细胞膜溶液使用脂质体挤出器依次用800 nm、400 nm、200 nm、100 nm、50 nm微孔聚碳酯滤膜进行挤出，每种尺寸挤出次数为25次，接收器为上述30 ml玻璃反应瓶，50 nm挤出的红细胞纳米囊泡即为ICG红细胞膜囊泡。然后往上述ICG红细胞膜囊泡溶液中（30 ml玻璃反应瓶）加入1.0 mg mL -1的DOX溶液2 mL（ICG、DOX摩尔比例为1∶1），于37℃水浴中600 rpm搅拌反应4 h，反应结束后，使用100 kd超滤管超滤，得到载药红细胞膜纳米粒；超滤的转速为3000 rpm/min，超滤的次数为15次。
如图1所示，透射电镜（Transmission electron microscopy, TEM）图像显示所制备的ID-RBCages纳米粒为大小均匀的圆形结构纳米颗粒，其尺寸为65.8 ± 5.4 nm，内部的ID纳米簇粒径大小为5.3 ± 3.8 nm。动态光散射（Dynamic light scattering, DLS）测定结果表明，ID-RBCages的水合粒径为79.5 ± 7.5 nm，其粒径分布峰形为单一峰，具有较好的粒径均一性。ID-RBCages具有ICG的吸收特征峰以及阿霉素的特征荧光发射峰，提示了ID-RBCages对ICG以及DOX的成功包载；常规计算得到载药量为ICG（10.5%）、DOX（7.8%）。
使用AFM对ID-RBCages的杨氏模量进行测定，平均为18kPa。而本课题组之前公开的以含有单硒的有机高分子PEG-PUSe-PEG为载体，ICG和DOX 为模型药物得到的Se-NPs的杨氏模量平均为68kPa；本课题组之前制备的将DOX和ICG通过共沉淀反应包裹进人血清白蛋白（HSA），制备出ICG/DOX@HSA纳米粒，杨氏模量平均为46kPa。
实施例二：光热升温能力考查：分别取500 mL浓度（ICG）为0.2 mM、0.5 mM、1.0 mM的ID-RBCages以及游离ICG溶液，置于2 mL的EP管中。用785 nm激光器（0.5 W cm -2，5 min）照射溶液，使用数显示温度仪监测溶液温度，每30 s记录溶液温度，绘制溶液的升温曲线。对一系列不同浓度的ID-RBCages溶液的光热升温能力的测定（图2A），结果表明在785 nm的激光照射下，ID-RBCages可以有效地产生光热升温，其升温效应具有浓度依赖性，随着样品浓度的提升，其光热升温效应可显著增强。
实施例三：光激活的ID-RBCages药物释放行为进行考察。采用透析法考察ID-RBCages体外药物释放行为。分别取相同浓度的ID-RBCages、ID-RBCages-Laser、ID-RBCages-Laser/Vc（785 nm，0.5W cm -2，5 min，Vc浓度为2mM）以及Free DOX溶液各1 mL，封装在透析袋中（截留分子量为3500），将透析袋放于装有透析介质（pH 5.0的醋酸盐缓冲液或pH 7.4的磷酸盐缓冲液）的15 mL离心管，每组三个重复，置于恒温摇床（120 rpm，37°C），分别在0.5、1、2、4、8、24、48 h更换透析缓冲液，并对DOX进行定量，并计算其累计释放量。对单次激光照射后的ID-RBCages电镜图片进行拍摄，结果显示（图2B），光照产生的单线态氧可以有效地破坏纳米粒的结构，有利于促进ID-RBCages所包载的药物的快速释放。此外分别使用0℃条件屏蔽光热效应以及加入Vc屏蔽光动力效果，结果表明光激活的药物释放主要是由光照射下所产生的ROS显著破坏纳米粒的膜结构所引发的，而单纯的光热效应对纳米粒的结构影响不大。进一步使用透析法对DOX的药物释放进行定量分析（图2C），结果显示在非光照时ID-RBCages的药物释放较少（<10%），而使用近红外激光照射溶液后，其药物释放能够显著增加到34.8%，使用Vc进行光动力效果进行屏蔽，DOX的释放量则显著降低，累计释放量减少为15.3%。综上表明，ID-RBCages在激光照射下能够通过光动力效应引发智能响应性化疗药物DOX释放，为降低药物毒副作用，并进一步增强多药协同肿瘤杀伤上奠定了坚实基础。
实施例四：为了进一步评价ID-RBCages光照条件下在细胞内诱导单线态氧产生的能力，使用DHE染色实验对其进行验证。将4T1细胞接种于24孔板中（每孔1×10 5个细胞）。细胞贴壁后，弃去培养基，加入预先稀释好的ID-RBCages、Free I/D溶液（ICG溶液与DOX溶液的混合），浓度设置为0、0.1、0.2、0.5、1.0、2.0 μg mL -1，平行3个复孔。继续培养6 h，去除培养基，PBS洗3次。光照组细胞用785 nm激光器进行光照（0.5 W cm -2，3 min），继续培养3 h。除去培养基，每孔加入新鲜配置的DHE染色液1 mL（50 μM），避光培养0.5 h，除去染色液，PBS洗三次，激光共聚焦显微镜观察并拍照，全程避光操作。将4T1细胞接种于24孔板中（每孔1×10 5个细胞），每孔加入1 mL培养基。细胞贴壁后。弃去培养基，加入预先稀释好的ID-RBCages、Free I/D溶液，按照DOX计算，浓度设置为0、0.1、0.2、0.5、1.0、2.0 μg mL -1，平行3个复孔。继续培养6 h，去除培养基，PBS洗3次。光照组细胞用785 nm激光器进行光照（0.5 W cm -2，3 min），继续培养3 h。除去培养基，每孔加入新鲜配置的AO染色液1 mL（10.0 μg mL -1），避光培养0.5 h，除去染色液，PBS洗三次，激光共聚焦显微镜观察并拍照。DHE为超氧化物阴离子荧光探针，其自身能够自由进入活细胞内部。DHE分子自身无荧光，而其在ROS作用下所产生的氧化乙啶在掺入DNA中后，可产生较强的红色荧光，可用于细胞内的ROS检测。如图3所示，ID-RBCages摄取进入细胞后，非光照条件下无ROS产生，而在785 nm激光光照下，在极低的（0.05 μg mL -1）ID-RBCages药物浓度下就可以有效诱导显著ROS生成。表明激光照射条件下，细胞中摄取的ID-RBCages可有效生成ROS，为ID-RBCages发挥光动力治疗效果奠定了良好的基础。为了进一步验证光照诱导ID-RBCages在溶酶体中所产生的ROS可以有效地破坏溶酶体的膜结构，使用AO染色实验对光照前后的细胞进行分析。所使用的AO染料是一种膜通透性的荧光染料分子，其自身为弱碱性，在细胞质环境下呈现绿色荧光，在溶酶体的酸性环境中会发生质子化而表现为红色荧光，可用做细胞溶酶体结构表征的指示剂。表明ID-RBCages摄取进入细胞后可在激光照射的条件下破坏细胞的溶酶体结构，为药物的溶酶体逃逸并进行胞浆转运提供了有力保障。
实施例五：为了进一步评价光照诱导ID-RBCages所产生的ROS是否会细胞核膜的结构产生影响，采用FITC荧光标记的Dextran进行验证。所选用的Dextran分子量为70kD，相对于细胞核核孔的尺寸，此分子量的Dextran无法穿透正常细胞核的核孔进入细胞核内部，可用作细胞核膜结构完整性的指示剂。将4T1细胞接种于24孔板中（每孔1×10 5个细胞），每孔加入1 mL培养基。细胞贴壁后，弃去培养基，加入预先稀释好的ID-RBCages、Free I/D溶液（DOX，1.0 µg mL -1），平行3个复孔。继续培养6 h，去除培养基，PBS洗3次。光照组细胞用785 nm激光器进行光照（0.5 W cm -2，3 min），光照后加入培养基稀释好的FITC荧光标记的右旋糖酐溶液（FITC-Dextran，MW：70 kD，1.0 mg mL -1），室温避光孵育3 h，除去染色液，PBS洗三次，加入1 mL Hoechst 33342溶液（5.0 μg mL -1），37℃避光染色5 min，弃去染料，PBS洗三次，激光共聚焦显微镜观察并拍照。分别对光照以及非光照条件下的细胞进行FITC-Dextran孵育，如图4所示，非光照以及PBS组的细胞在Dextran孵育后，摄取到细胞内部的Dextran只分布在胞浆中，而不进入细胞核中。相反地，ID-RBCages光照组细胞在其胞浆和细胞核内部均可以观察到较强的绿色荧光信号，表明FITC标记的Dextran在光照后的细胞内可以有效的转移到细胞核中去，证实了光照诱导ID-RBCages产生的ROS可以对细胞的核膜产生损伤，破坏了细胞核膜的完整性，利于细胞核靶向的化疗药物的有效递送。
实施例六：细胞摄取和摄取途径。取生长状态良好的4T1细胞，在6孔板中以细胞密度为每孔1.0×10 6个细胞进行铺板，静置培养。细胞贴壁后，分别加入ID-RBCages、游离DOX（1.0 µg mL -1），同时设置3个复孔。分别孵育2 h、12 h、24 h，PBS洗涤细胞三次，消化收集细胞并计数，超声粉碎仪破碎细胞（超声功率：400 W，超声时间：5 min）。随后，向细胞裂解液中分别加入1 mL的DMSO萃取药物，使用全波长酶标仪进行定量，计算药物摄取量。取生长状态良好的4T1细胞，按照每孔10 6个细胞的数量接种到6孔细胞培养板中，每孔加入2 mL培养基，培养箱静置培养至细胞贴壁。随后分别加入不同的内吞途径抑制剂，空白对照组加入等体积的PBS，单独取一块板置于4℃冰箱中低温孵育，设置三个复孔。2 h后，弃去培养基，每孔分别加入稀释好的ID-RBCages（1.0 µg mL -1），6 h后弃培养基，消化、收集细胞并计数，使用超声粉碎仪破碎细胞（超声功率：400 W，超声时间：5 min），随后加入1 mL的DMSO对药物进行萃取，使用全波长酶标仪定量，计算药物摄取量。高效的细胞摄取是药物发挥治疗效果的保障，细胞摄取实验结果显示（图5），ID-RBCages与游离药物的细胞摄取行为一致。此外，ID-RBCages细胞摄取量与摄取时间相关。在24 h的观察时间内，随着时间的延长，ID-RBCages的细胞摄取量逐渐增加。并且在相同的时间下，ID-RBCages可引起更多的细胞药物摄取，在24 h时，ID-RBCages的摄取量为游离药物摄取量的2.5倍，表明使用红细胞膜纳米反应器进行药物递送可以有效增加抗肿瘤药物的细胞药物摄取量，为进一步增强药物治疗效果奠定了基础。
实施例七：使用激光共聚焦技术对光照后的细胞内DOX摄取后的胞浆转运以及细胞核递送的动态过程进行观察。将ID-RBCages预孵育6 h的细胞首先进行细胞核以及溶酶体染色标记，并在激光共聚焦成像仪上固定好细胞培养皿，调好焦距在成像软件上找到细胞并观察拍照作为非光照细胞图像。接下来，使用785 nm激光器进行光照（0.5 W cm -2，5 min），并在光照后5 min、15 min、30 min、60 min使用激光共聚焦显微镜对细胞进行观察并拍照记录DOX的细胞内转运情况。结果显示（图6），光照后DOX可以实现快速的溶酶体逃逸，并在光照后15 min内实现均匀的胞浆分布，并且在细胞核内出现少量的DOX蓄积，随着时间延长至60 min时，DOX的细胞核转运可增加到88%。表明ID-RBCages能够实现光响应性的药物胞浆转运并实现快速的细胞核递送，为DOX发挥抗肿瘤化疗作用提供了保障。
实施例八：细胞毒性。取生长状态良好的4T1细胞，消化收集后用培养基稀释至细胞密度为5×10 4个/mL，接种于96孔细胞培养板，每孔接种100 µL细胞悬液，37℃细胞培养箱中培养过夜，每孔加入不同浓度ID-RBCages纳米粒溶液（以DOX的浓度来计算），给药浓度分别为0.05、0.1、0.2、0.5、1.0、2.0、4.0 μg mL -1，每个浓度设置4个复孔，同时设置不加药物的PBS对照组。在37℃培养箱中继续培养24 h，去除培养基，用PBS洗三遍，非光照组加入含有MTT（0.5 mg mL -1）的细胞培养基100 µL，继续培养4 h，轻轻吸出培养液，每孔加入100 µL二甲亚砜（DMSO）溶液，摇床上摇动5 min，用多功能酶标仪测定每孔在492 nm处的吸光度，并计算细胞存活率。光照组的细胞每孔加入新鲜培养基100 µL，并使用785 nm的激光器对每孔细胞光照3 min（0.5 W cm -2），光照后继续培养细胞24 h，并使用MTT对细胞存活情况进行检测，计算细胞存活率。为了直观地考察纳米粒的细胞杀伤的作用，使用Calcein-AM/PI活细胞/死细胞双染试剂盒对细胞进行染色。其具体步骤如下，取生长状态良好的4T1细胞，消化收集后用培养基稀释至细胞密度为5×10 4个/mL，接种于96孔细胞培养板，每孔接种100 µL细胞悬液，37℃细胞培养箱中培养过夜。细胞完全贴壁后，每孔加入ID-RBCages、Free DOX，给药浓度以DOX计算为1.0 µg mL -1，每个浓度设置3个复孔，同时设置不加药物的PBS对照组。在37℃培养箱中继续培养24 h，去除培养基，用PBS洗三遍，加入新鲜的细胞培养基，每孔100 µL，非光照组的细胞放回细胞培养箱中继续培养，光照组细胞使用785 nm的激光器对每孔细胞进行光照（0.5 W cm -2，3 min），光照后继续培养细胞6 h，对细胞进行染色后使用激光共聚焦对细胞进行拍照。
为了研究ID-RBCages对4T1小鼠乳腺癌细胞活力的影响，使用MTT法对ID-RBCages的细胞毒性进行了考察。如图7A所示，在非光照条件下，ID-RBCages对4T1细胞的生长影响较小，表明ID-RBCages自身的细胞毒性较低，这有利于降低化疗药物在非肿瘤部位的毒副作用。而在785 nm激光光照条件下，ID-RBCages在较低给药的浓度即可对4T1细胞造成强烈的细胞杀伤，其IC 50值为0.61 μg mL -1。提示ID-RBCages可以作为良好的光化疗治疗药物，其良好的光化疗杀伤以及较低的暗毒性，能够通过激光调控作用选择性杀伤肿瘤，并减少对于正常组织的损伤，表明ID-RBCages可以作为一种具有良好选择性的光化疗治疗纳米药物。
通过活-死细胞染色实验，分别对ID-RBCages在非光照条件下的细胞暗毒性以及激光照射条件下的光毒性进行了考察。如图7B所示，相比较于游离药物组，ID-RBCages对细胞的杀伤作用无显著性变化，其细胞杀伤能力与游离药物组基本保持一致，而在光照条件下，ID-RBCages的细胞杀伤能力则显著提升。而对于游离药物组，光照之后其细胞杀伤较非光照条件下无明显变化，仍有较多的细胞存活。综上表明，红细胞膜纳米反应器递送药物可以显著增强细胞的杀伤能力，并且ID-RBCages肿瘤细胞杀伤是光选择性，安全性良好。
实施例九：为了考察ID-RBCages光照条件下对于细胞的增殖活性产生的影响，采用EdU染色实验对细胞增殖情况进行表征。EdU染色实验可以特异性的对处于活性增殖的S期的细胞进行标记，并可以通过对Azide 488的荧光分析，来间接的进行增殖能力的进行考察。如图8所示，荧光图片结果可以直观地显示细胞活性增殖能力的强弱，其中细胞核呈蓝色，处于S期的细胞呈现绿色。ID-RBCsges在非光条件下对肿瘤细胞的活性增殖能力无明显的抑制作用，而ID-RBCages在光照条件下会显著抑制细胞的增殖，在荧光图片上几乎看不到具有活性增殖能力的细胞存在。表明ID-RBCages在光照条件下能有通过下调细胞增殖活性，实现对肿瘤细胞生长的有效抑制，从而达到损伤肿瘤细胞的作用。
实施例十：细胞免疫原性死亡（ICD效应）实验。ID-RBCages诱导肿瘤细胞表面钙网蛋白（calreticulin，CRT）表达。分别使用流式细胞术以及免疫荧光染色的方法，对肿瘤细胞表面CRT表达进行检测。将4T1细胞接种于24孔板中（每孔1×10 5个细胞），每孔加入1 mL培养基。培养至细胞完全贴壁。弃去培养基，并加入培养基预先稀释好的ID-RBCages和游离药物对照Free I/D溶液（DOX，1.0 μg mL -1），孵育12 h后弃去培养基。光照组细胞使用785 nm激光器进行光照（0.5 W cm -2，3 min），继续培养细胞3 h，4%的多聚甲醛固定细胞后，分别孵育一抗（2 h）以及荧光二抗（APC标记，1 h）。使用流式细胞仪对CRT表达量进行检测。CRT免疫荧光染色。将4T1细胞接种于玻底细胞培养皿中（每孔1×10 5个细胞），每孔加入2 mL培养基，培养至细胞贴壁。分别加入ID-RBCages、Free I/D（DOX，1.0 µg mL -1）。继续孵育12 h。光照组用785 nm激光器进行光照（0.5 W cm -2，3 min），继续培养6 h，4%的多聚甲醛固定后，分别孵育一抗（12 h）以及荧光二抗（Cy3.5标记，6 h），DAPI溶液（5.0 μg mL -1）标记细胞核后，使用激光共聚焦显微镜进行拍照分析。ID-RBCages诱导肿瘤细胞胞内高迁移率族蛋白B1（High mobility group box 1，HMGB1）外排。将4T1细胞接种于玻底细胞培养皿中（每孔1×10 5个细胞），每孔加入2 mL培养基，培养至细胞完全贴壁。分别加入ID-RBCages、Free I/D（DOX，1.0 µg mL -1）。继续孵育12 h，弃去培养基，PBS洗三遍。光照组用785 nm激光器进行光照（0.5 W cm -2，3 min），继续培养细胞24 h，弃去培养基并洗涤细胞，4%的多聚甲醛固定细胞15 min，去除固定液，加入0.3%TritonX-100破膜（孵育30 min）。PBS洗涤后，分别孵育一抗（12 h）以及荧光二抗（Cy3.5标记，6 h），DAPI溶液（5.0 μg mL -1）标记细胞核后，使用激光共聚焦显微镜进行拍照分析。ID-RBCages诱导肿瘤细胞三磷酸腺苷（Adenosine triphosphate，ATP）分泌。将4T1细胞接种于24孔板中（每孔1×10 5个细胞），每孔加入1 mL培养基。培养至细胞完全贴壁。弃去培养基，分别加入ID-RBCages、Free I/D（DOX，1.0 µg mL -1）。继续孵育12 h，弃去培养基，PBS洗三遍。光照组用785 nm激光器进行光照（0.5 W cm -2，3 min），继续培养12 h，收集细胞培养基，使用ATP检测试剂盒对ATP进行定量。化疗药物DOX以及光动力效应均可以有效地诱导肿瘤细胞发生免疫原性死亡。因此，对ID-RBCages诱导肿瘤细胞免疫原性死亡的能力进行了考察。分别对免疫原性死亡的重要指标钙网蛋白（Calreticulin，CRT）的细胞膜表面暴露、高迁移率族蛋白B1（High mobility group box 1，HMGB1）的释放、三磷酸腺苷（Adenosine triphosphate，ATP）的胞外分泌这三个ICD效应指标进行考察。结果显示，ID-RBCages光照后可有效地增强细胞膜表面CRT蛋白的暴露（图9A，C-D），为免疫细胞识别肿瘤细胞提供了更强的“吃我”信号，ID-RBCages诱导的肿瘤细胞ATP的胞外释放的增加（图9E），也使肿瘤细胞向巨噬细胞以及DC前体细胞释放更强的“发现我”信号，同时，HMGB1胞外释放的增加也进一步验证了ID-RBCages可以有效地引起ICD效应的产生（图9B）。
实施例十一：为了对ID-RBCages肿瘤靶向能力以及其在小鼠各组织脏器的分布情况进行分析，开展了组织分布实验进行研究。取肿瘤大小为70-100 mm 3的Panc02皮下瘤小鼠，尾静脉注射ID-RBCages以及游离药物Free I/D（ICG，7.5 mg kg -1）到荷瘤小鼠体内，24 h后将小鼠脱臼处死并取下其心、肝、脾、肺、肾以及肿瘤，使用小动物活体成像仪对ICG分布情况进行成像分析。严格避光，之后对各组织进行称重后使用匀浆机粉碎组织，匀浆液使用氯仿和甲醇（1:1）混合溶液萃取，去除有机溶剂，DMSO复溶后，使用多功能酶标仪对各组织中的DOX分布进行定量分析。
如图10A所示，ID-RBCages肿瘤组织的靶向性相比较于游离药物组显著提升，其DOX的肿瘤组织蓄积量为15.2 ID% g -1，是游离药物组的7.17倍。ID-RBCages靶向性增强原因在于，所制备的纳米粒尺寸合适，能够有效通过EPR效应实现肿瘤的蓄积，尤其是，ID-RBCages的变形性使其可以高效穿透深部肿瘤，为进一步发挥抗肿瘤生物活性的应用奠定了基础。ICG可以直接应用于近红外荧光成像，因此对离体组织进行小动物成像荧光照片拍摄，可直接观察ID-RBCages在各组织脏器中的分布情况。如图10B所示，ID-RBCages较游离药物组肿瘤组织靶向性有着显著的提升，其肿瘤组织荧光强度是游离药物组的5.25倍（图10C），这与DOX组织定量分布的结果一致。
实施例十二：为了评价ID-RBCages深部肿瘤中的有效穿透能力，使用肿瘤组织切片血管染色实验对ID-RBCages在低渗透性的Panc02小鼠胰腺癌肿瘤深部组织中的药物递送进行考察。将荷Panc02皮下瘤的小鼠，随机分组后，尾静脉注ID-RBCages、Free-I/D（ICG，7.5 mg kg -1）到荷瘤小鼠体内，在给药后24 h，取小鼠肿瘤并用4%的多聚甲醛溶液固定，随后切片机进行切片（切片厚度为10 µm），切片进行CD31免疫荧光染色后，使用激光共聚焦显微镜对切片进行观察并拍照，并使用Image J对图片进行荧光强度统计分析。
结果显示（图11A-B），在低渗透性的具有致密的肿瘤胞外基质的胰腺癌Panc02皮下瘤肿瘤模型中，ID-RBCages可以实现良好的肿瘤深部穿透。在血管周围组织渗透深度为80 μm的区域范围内，ID-RBCages药物仍然具有较为均匀的高效的深部穿透能力，在血管周围的组织区域中具有均匀的ID-RBCages荧光信号分布（图11B）。这与ID-RBCages合适尺寸大小以及其自身良好的柔软性，可以在肿瘤组织中通过变形进行深部穿透实现高效药物递送有关。如图16C所示，尾静脉给药24 h后，ID-RBCages在肿瘤部位可有效的被肿瘤细胞所摄取。光照后ID-RBCages响应性释放了其包载的DOX，并且其ID-RBCages所产生的ROS对细胞核膜产生了损伤，加快了DOX向肿瘤细胞核部位的有效转运，为发挥对肿瘤细胞的光化疗杀伤作用提供了有利的保障。
实施例十三：近红外热成像。为了考察ID-RBCages在体内肿瘤光热治疗能力，使用热成像仪对激光照射条件下小鼠肿瘤温度进行监测。尾静脉注射ID-RBCages、Free I/D（ICG，7.5 mg kg -1）到荷瘤小鼠体内，给药24 h后，785 nm激光器对小鼠肿瘤部位进行光照（0.5 W cm -2，5 min），并使用热成像仪对肿瘤温度进行记录。
结果表明（图12），直接激光照射PBS组小鼠，其肿瘤部位无明显升温，在5 min的持续光照下升温为4℃，表明激光照射本身不产生明显光热升温效果。ID-RBCages给药组的小鼠在激光照射下，其肿瘤部位均可以产生明显的光热升温效果，且其光热升温具有明显的浓度依赖性，给药浓度越高，肿瘤热升温能力也越强。其中，10.0 mg kg -1剂量的ID-RBCages组肿瘤部位升温可达到19.2℃，且在5 min的观察时间内，肿瘤部位升温到最高后可保持其持续维持在最高的温度不下降。这依赖于ID-RBCages在肿瘤部位的高效靶向蓄积、ID-RBCages有效增强了ICG的光稳定。
实施例十四：光化疗用于小鼠乳腺癌治疗。使用4T1皮下瘤肿瘤模型对ID-RBCages抑瘤效果的考察。小鼠肿瘤体积为70-100 mm 3时开始实验，尾静脉注射ID-RBCages、Free I/D（ICG，7.5 mg kg -1）进入小鼠体内。24 h后，光照组小鼠使用785 nm激光器对肿瘤部位进行激光照射（0.5 W cm -2，5 min），在25天内使用游标卡尺测量小鼠肿瘤大小。第25天时，测量肿瘤后将各组小鼠处死，取下小鼠肿瘤拍照并称重。4T1原位乳腺癌实验给药方案与皮下瘤相同，区别在于原位乳腺癌的肿瘤实验的肿瘤观察时间为21天，并在21天时将各组小鼠肺组织取出，进行离体生物发光成像照片拍摄，然后将离体肺组织固定在4%多聚甲醛溶液中，切片后进行H&E染色。同时对21天小鼠的肿瘤进行拍照并称重。
首先在4T1皮下瘤肿瘤模型中，对ID-RBCages进行抗肿瘤抑瘤生物学效应进行考察。结果显示（图13），在PBS对照实验组中，不管是否对肿瘤部位进行激光照射，肿瘤生长均没有明显区别，在25天的观察时间内，PBS组以及PBS光照组的肿瘤体积增长倍数分别为45.5倍以及44.4倍。Free DOX治疗组肿瘤生长相比于PBS组稍有降低，而光照对游离药物组无影响，光照非光照条件下游离的DOX治疗组的小鼠肿瘤生长倍数分别为35.5和33.2倍。ID-RBCages给药组的小鼠，非光照条件下小鼠的肿瘤生长较快，其生长趋势与PBS组小鼠基本一致，并且在第25天时，该组小鼠的肿瘤生长倍数为42.2倍。而ID-RBCages给药组的小鼠肿瘤在近红外激光照射下，肿瘤生长受到明显的抑制，在光照治疗后的前9天时间内，其平均肿瘤体积逐渐减小表明该组小鼠的肿瘤受到了有效地控制，在之后的时间该组小鼠肿瘤开始逐渐复发生长，但其肿瘤生长较为缓慢，在第25天时，其平均肿瘤增长为原始肿瘤体积的5.1倍。表明ID-RBCages在光照条件下可以有效抑制肿瘤生长，但该抑制作用不能实现肿瘤的完全消融，需要进一步改变给药策略或是联合其他抗肿瘤药物进行有效治疗肿瘤。ID-RBCages可以作为一种高效光响应性的抗肿瘤药物递送平台，能够实现在肿瘤部位实现近红外光激发的高选择性肿瘤有效抑制，有望进一步联合其他抗肿瘤治疗策略实现肿瘤的有效治疗。
接下来，在4T1-Luc小鼠原位乳腺癌模型上对ID-RBCages在原位肿瘤的抗肿瘤生物学效应进行了评价。同时通过对小鼠抑瘤实验结束时肺组织进行离体生物发光成像图片进行拍摄，对ID-RBCages抑制原位乳腺癌的肺部转移的能力进行了研究。结果表明（图14），ID-RBCages在近红外光光照条件下，可以有效抑制小鼠原位乳腺癌的生长。光照后，ID-RBCages治疗组的小鼠肿瘤体积逐渐减小，并在给药后的第9天肿瘤完全消失，但该治疗组小鼠的肿瘤随后出现复发现象，在第21天时其肿瘤最终的生长倍数为4.6倍。ID-RBCages-Laser/Vc治疗组小鼠通过将Vc瘤内预注射来屏蔽光动力效应，该组小鼠肿瘤在光照后的前6天肿瘤在逐渐消退，而后肿瘤出现较快的复发生长，最终的肿瘤生长倍数为11.0倍。游离药物在光照以及非光照条件下，其肿瘤生长较PBS组没有明显差异。离体肺组织生物发光成像结果显示，ID-RBCages光照组的小鼠肺部肿瘤转移情况较PBS组显著降低，而ID-RBCages-Laser/Vc组的小鼠肺转移抑制效果变差，表明光动力治疗不仅有利于原位肿瘤的抑制，对转移瘤的抑制也起着重要调控作用。
实施例十五：为了进一步考察ID-RBCages在组织水平上的DOX响应性释放后的细胞核转运能力，分别对光照前后的肿瘤组织切片进行观察。如图15所示，尾静脉给药24 h后，ID-RBCages在肿瘤部位可有效的被肿瘤细胞所摄取。在未光照的情况下，ID-RBCages主要分布在细胞的胞浆中，而在细胞核内的分布量较少。并且肿瘤切片也显示非光照条件下，肿瘤组织形态较为完整，其细胞之间仍保持较为致密的状态，表明在非光照条件下，ID-RBCages对肿瘤组织的无明显损伤作用；进一步对光照后的肿瘤组织进行切片分析，结果显示光照后的肿瘤其组织细胞中ID-RBCages与细胞核具有较高的共定位，表明光照后ID-RBCages响应性释放了其包载的DOX，并且其ID-RBCages所产生的ROS对细胞核膜产生了损伤，加快了DOX向肿瘤细胞核部位的有效转运，为发挥对肿瘤细胞的光化疗杀伤作用提供了有利的保障。
实施例十六：为了进一步考察ID-RBCages诱导肿瘤免疫治疗的潜在应用价值，选用aPD-L1与ID-RBCages进行联用对原位乳腺癌治疗效果进行考察。实验分组设计为PBS、aPD-L1、Free I/D、Free I/D/aPD-L1、ID-RBCages、ID-RBCages/Laser、ID-RBCages/Laser/Vc、ID-RBCages/Laser/aPD-L1共八个实验组。在小鼠肿瘤体积在70-100 mm 3时开始实验，尾静脉注射ID-RBCages、Free I/D（ICG，7.5 mg kg -1）进入小鼠体内进行给药，24 h后，光照组小鼠使用785 nm激光器对肿瘤部位进行激光照射（0.5 W cm -2，5 min），aPD-L1的给药剂量为5 mg/kg，分别在第2、5、8天进行腹腔给药。观察小鼠肿瘤到21天，使用游标卡尺测量小鼠肿瘤大小，第21天时处死小鼠并取出小鼠肺组织进行生物发光成像，然后将离体肺组织固定在4%多聚甲醛溶液中，切片后进行H&E染色，同时对小鼠的肿瘤进行拍照并称重。
结果显示（图16），ID-RBCages-Laser+aPD-L1可以实现4T1-Luc原位乳腺癌的完全消融，并在21天的观察期内无肿瘤复发的产生。此外，ID-RBCages-Laser+aPD-L1在乳腺癌的肺转移抑制上显示出较强的治疗效果，可以完全抑制乳腺癌肺部转移的发生。而单独给与aPD-L1治疗的小鼠治疗效果存在较大的差异性，该组小鼠中有两只小鼠的生长得到了较好的控制，而其余小鼠的肿瘤生长较快，整组中小鼠的平均肿瘤生长速度较快。其与PBS对照组对照无统计学差异，表明单独使用aPD-L1时存在小鼠个体响应性差异较大的缺点，总体上来看，aPD-L1对原位乳腺癌治疗效果有限。游离药物光照组联合使用aPD-L1时，肿瘤生长相比于非联用抗体组无明显差别，表明游离药物和aPD-L1抗体进行联用不能取得治疗效果上的有效增强。这可能与游离药物自身不能有效引激活小鼠的抗肿瘤免疫效应有关，因此联用aPD-L1抗体时无法实现协同增效，联用效果不佳。ID-RBCages光照组联合使用aPD-L1抗体可以实现对原位肿瘤的彻底清除并有效抑制肿瘤复发，可能的原因为ID-RBCages在光照条件下可以引起较强的免疫治疗效应，能够促使肿瘤部位招募丰富的CTLs等抗肿瘤免疫细胞，因此同时联用aPD-L1抗体时，可以显著提升抗肿瘤活性，实现光化疗与免疫治疗的高效协同，进而实现原位肿瘤的完全消融并有效抑制肿瘤肺部转移的发生。
实施例十七：为考察ID-RBCages在Panc02荷瘤小鼠的体内抗肿瘤生物活性，使用Panc02皮下瘤模型对ID-RBCages光化疗协同免疫治疗的抗肿瘤生物学效果进行评估。小鼠Panc02胰腺癌皮下瘤模型进行抑瘤实验考察ID-RBCages对低渗透性肿瘤的治疗效果，共设置8个不同分组，分别为：PBS、aPD-L1、Free I/D-Laser、Free I/D-Laser/aPD-L1、ID-RBCages、ID-RBCages/aPD-L1、ID-RBCages-Laser、ID-RBCages-Laser/aPD-L1、GEM+Abraxane。在第0天尾静脉注射ID-RBCages以及Free I/D（7.5 mg kg -1），光照组在给药24 h后使用785 nm激光照射肿瘤（0.5 W cm -2，4 min）。在第0、3、6天尾静脉注射GEM（35 mg kg -1）、Abraxane（8 mg kg -1），在第2、5、8天腹腔注射aPD-L1抗体（5.0 mg kg -1）。在0-35天记录小鼠肿瘤生长情况，肿瘤体积达到1500 mm 3时算为小鼠死亡，并观察小鼠的生存情况至120天。另取两组小鼠，各20只，一组接种皮下Panc02肿瘤，另一组作为空白鼠不接瘤。接瘤小鼠在其肿瘤体积为70 mm 3按照抑瘤分组中ID-RBCages-Laser/aPD-L1治疗方案进行治疗，并在治疗后第60天挑选肿瘤完全消融的小鼠进行肿瘤再挑战实验。即在小鼠首次接瘤的对侧皮下接种Panc02细胞（接种细胞数量为1×10 6个/只），并对空白小鼠同时接种Panc02作为对照。接种后，分别记录两组小鼠的肿瘤生长情况。同时，在第60天时，选取肿瘤完全消融的小鼠以及未空白小鼠各六只，取肿瘤组织制备单细胞悬液后，对长期记忆性T细胞进行免疫染色，并使用流式细胞仪进行分析。
如图17A-C结果显示，ID-RBCages光照组的Panc02皮下瘤在光照后，肿瘤在第13天肿瘤消融，而之后肿瘤均出现复发，且复发后肿瘤快速生长，并在第35天时肿瘤平均体积可以达到945 mm 3。而ID-RBCages光照联合aPD-L1抗体治疗组肿瘤在消融之后，在第21天有两只小鼠发生肿瘤复发，而其余三只小鼠肿瘤则一直没有复发产生，在第35天时该组小鼠肿瘤平均体积为113 mm 3。对给药后120天的小鼠生存曲线进行统计（图17D），结果显示ID-RBCages光照组的半数生存期为45天，相对于PBS组的27天明显延长了小鼠的生存期，而对于ID-RBCages光照组联用aPD-L1抗体治疗组的小鼠在120天时仍有60%的小鼠存活。表明ID-RBCages光化疗联合免疫治疗在Panc02皮下瘤的肿瘤模型的治疗中能够展现出良好的抗肿瘤效果。如图18A所示，在治疗后的第60天选取完全消融的小鼠，接种肿瘤细胞进行再挑战实验。结果显示，治疗组小鼠再次接种肿瘤细胞时，其不会生长皮下肿瘤，表明小鼠体内具有记忆性的免疫效应产生，能够有效抑制再次接种肿瘤细胞的瘤体生长。进一步使用流式细胞术对小鼠脾脏中的T细胞进行分析（图18B-D），结果表明，治疗组的小鼠体内长期记忆性的T细胞以及中央记忆T细胞的数量均明显增多，其中TEM的数量为空白对照小鼠体内的3.1倍，TCM的数量则为空白对照小鼠体内的1.9倍。综上表明，ID-RBCages光化疗免疫协同治疗可以有效引起小鼠体内的长期免疫记忆效应，为防止肿瘤的复发或者转移发挥着重要作用。
实施例十八：人源性胰腺癌肿瘤90%以上会发生KRAS突变。为了进一步评价ID-RBCages在KRAS突变的肿瘤中光化疗免疫协同治疗的能力，选用同时具有KRAS以及TP53突变的小鼠KPC肿瘤细胞进行抗肿瘤效应研究。在KPC胰腺癌模型的抑瘤实验中，联合使用先天免疫的STING激动剂SR717与aPD-L1抗体进一步增强免疫治疗的效果。使用KPC-Luc细胞构建小鼠胰腺癌原位肿瘤模型，并使用小动物活体成像对肿瘤的生长情况进行监测。并对第0、6、12、18、24天的肿瘤活体成像的生物发光强度进行统计。具体的，小鼠KPC原位胰腺癌的抑瘤实验，共设置8个不同分组，分别为：PBS、SR717+aPD-L1、GEM+Abraxane、GEM+Abraxane+SR717+aPD-L1、Doxil、ID-RBCages-Laser、ID-RBCages-Laser/SR717+aPD-L1。在第0天尾静脉注射ID-RBCages以及Free I/D（ICG，7.5 mg kg -1）进入荷瘤小鼠体内，在给药24 h后，光照组的小鼠首先麻醉后手术打开其腹腔，使用785 nm激光器对肿瘤部位进行光照（0.5 W cm -2，5 min），光照结束后手术缝合伤口。SR717+aPD-L1联合治疗组在第2、5、8天腹腔注射aPD-L1抗体（5.0 mg kg -1）、SR717（35 mg kg -1）。GEM+Abraxane给药则是在第0、3、6天时尾静脉注射给药，其中GEM给药剂量为35 mg kg -1，Abraxane为8 mg kg -1。并在第0、6、12、18、24天，使用小动物活体成像系统对其腹部胰腺肿瘤进行生物发光成像，监测其原位胰腺癌肿瘤的生长。并在120天内记录各组小鼠的生存情况。
如图19A-C所示，PBS组小鼠的胰腺癌生长迅速，第24天时，该组小鼠的肿瘤部位的生物发光强度增长倍数为59.7倍，其他治疗组如SR717+aPD-L1、GEM+Abraxane以及SR717+aPD-L1+GEM+Abraxane治疗组对KPC原位胰腺癌的治疗效果均较差。临床药物对照组的Doxil对KPC原位胰腺癌的治疗也没有显著的抑制效果，其肿瘤生长倍数在24天时为42.8倍。ID-RBCages光照可以有效的抑制小鼠胰腺癌的生长，其在第24天时肿瘤的荧光强度增长倍数为13.4倍，而使用SR717和aPD-L1对ID-RBCages光照治疗进行联合治疗时，原位胰腺癌的生长可以得到有效控制，其在第24天时，肿瘤生长倍数为3.6倍，并且五只小鼠中有两只小鼠的肿瘤被完全抑制。表明ID-RBCages光化疗联合免疫治疗可以在KRAS/TP53双突变的KPC胰腺癌治疗上发挥显著的治疗效果。对各实验组小鼠生存曲线进行统计分析（图19D），结果表明除了ID-RBCages光照治疗组以及ID-RBCages光照联合免疫治疗组的两组以外，其余各组的小鼠在45天内均完全死亡。而ID-RBCages光照治疗的小鼠其半数生存期为45天，ID-RBCages光照联合免疫治疗组的小鼠在治疗后的第120天仍然有60%的小鼠存活。表明ID-RBCages光化疗免疫协同可以有效延长KPC原位肿瘤小鼠的生存时间，为原位胰腺癌的有效治疗带来了希望。
实施例十九：在实施例一的基础上，调节加入ICG溶液的体积，得到ICG、DOX摩尔比例为1∶0.5或者1∶2的载药红细胞膜纳米粒。
对照例一：在实施例一的基础上，第（1）步调整为：（1）将离心所得红细胞重悬于50mL低渗的PBS缓冲液中（0.25×），于冰浴中静置30 min；然后使用低渗PBS缓冲液（0.25×）洗涤3次，得到红细胞膜。其余步骤与实施例一一样，在搅拌时溶液保持澄清，使用透射电镜对反应完成后的溶液进行形貌观察，图20A结果显示纳米粒溶液中为形貌不规则的细胞膜碎片，未观察到纳米产生，无法制备出尺寸均一的红细胞膜纳米粒。
对照例二：在实施例一的基础上，第（1）步调整为：（1）将离心所得红细胞重悬于50mL低渗的PBS缓冲液中（0.25×），于冰浴中3500rpm搅拌30 min；然后，弃上清收集底层沉淀，再使用低渗PBS缓冲液（0.25×）洗涤5次，得到红细胞膜；采用实施例一一样的方法，在搅拌反应时出现明显浑浊，得到纳米粒仅在其表面有少量药物层，为中空的圆环状形态，无法有效包载药物，参见图20B。与之对比，实施例一整个反应过程澄清，电镜显示ID-RBCages纳米粒可有效包载药物。
本发明基于红细胞膜纳米反应器，成功实现了载ICG和DOX的红细胞膜纳米粒（ID-RBCages）的仿生合成；取得的主要技术进步如下：（1）利用纳米红细胞膜包载临床用抗肿瘤光敏剂（ICG）和化疗药物（DOX），经过反应成功制备了ID-RBCages。产物平均粒径为65.8 ± 5.4 nm，ICG/DOX纳米粒大小为5.3 ± 3.8 nm，形貌规整，粒径均一。
（2）ID-RBCages具有良好的光热、光动力效应，并能够实现光激活的DOX响应性释放。ID-RBCages被光激活后，可通过光动力效应以及DOX的化疗作用，协同增强肿瘤细胞的ICD效应，具有增强免疫治疗效果的潜力。
（3）ID-RBCages可被肿瘤细胞高效摄取，并分布于细胞的酸性溶酶体中；肿瘤靶向良好，在4T1肿瘤皮下瘤模型中，DOX的肿瘤部位药物分布为15.2 ID% g -1。ID-RBCages在细胞内可被光激活释放DOX，并快速从溶酶体转运至胞浆，然后迅速入核发挥光化疗协同的抗肿瘤效果。ID-RBCages在低渗透性Panc02小鼠胰腺癌肿瘤中具有较强的组织穿透能力，可高效递送药物到达深部肿瘤，并能够实现化疗药物智能响应释放以及细胞核转运，光照后DOX与细胞核的共定位率可达到78.6%。ID-RBCages在4T1乳腺癌皮下以及原位瘤中均具有良好的光化疗效果，可显著抑制肿瘤生长，并能够有效抑制肺转移。ID-RBCages光化疗-免疫协同治疗策略能实现免疫“冷肿瘤”到免疫“热肿瘤”的转变，逆转免疫抑制性微环境，完全消融4T1原位乳腺癌，并有效抑制肺转移。ID-RBCages联用aPD-L1抗体能够有效抑制Panc02胰腺癌皮下瘤生长。ID-RBCages光化疗-免疫协同治疗策略可降解细胞外基质、逆转免疫抑制性微环境、增强大分子药物aPD-L1以及CTLs的肿瘤浸润，实现对难治性、低渗透性胰腺癌的有效治疗。ID-RBCages联合aPD-L1和SR717可有效抑制双突变的KPC-Luc小鼠原位胰腺癌肿瘤生长。ID-RBCages光化疗-免疫协同治疗策略解除TP53突变对KPC细胞STING激活的抑制，双重激活获得性免疫和先天免疫，并通过调控细胞外基质以及免疫抑制性微环境实现双突变胰腺癌的高效治疗。

Claims (10)

  1. 一种载药红细胞膜纳米粒的制备方法，其特征在于，将红细胞膜与阴离子反应前体混合后挤出，得到阴离子反应前体红细胞膜囊泡；再将阴离子反应前体红细胞膜囊泡与阳离子反应前体混合反应，得到载药红细胞膜纳米粒。
  2. 根据权利要求1所述载药红细胞膜纳米粒的制备方法，其特征在于，由红细胞低渗处理得到红细胞膜。
  3. 根据权利要求2所述载药红细胞膜纳米粒的制备方法，其特征在于，将红细胞悬于低渗缓冲液中，静置后离心处理，得到红细胞膜。
  4. 根据权利要求3所述载药红细胞膜纳米粒的制备方法，其特征在于，静置为冰浴静置20～40分钟，离心处理为3000～4000g处理10～20分钟。
  5. 根据权利要求1所述载药红细胞膜纳米粒的制备方法，其特征在于，所述反应的温度为25～55℃，反应时间为3～8h。
  6. 根据权利要求1所述载药红细胞膜纳米粒的制备方法，其特征在于，将红细胞膜与阴离子反应前体在低渗缓冲液中孵育，然后用脂质体挤出器挤出，得到阴离子反应前体红细胞膜囊泡；然后将阴离子反应前体红细胞膜囊泡与阳离子反应前体在低渗缓冲液中搅拌反应，然后超滤，得到载药红细胞膜纳米粒。
  7. 根据权利要求6所述载药红细胞膜纳米粒的制备方法，其特征在于，孵育的时间为20～40分钟，孵育后超声处理3～6分钟，再用脂质体挤出器梯度挤出；所述超滤时截留分子量为30～200kD，转速为1200～5000 r/min，超滤的次数为10～20次。
  8. 根据权利要求1所述载药红细胞膜纳米粒的制备方法，其特征在于，所述阳离子反应前体为金属化合物或者小分子药物；所述阴离子反应前体源为硫化物、柠檬酸化合物或者染料药物。
  9. 根据权利要求1所述载药红细胞膜纳米粒的制备方法制备的载药红细胞膜纳米粒，其特征在于，载药红细胞膜纳米粒的粒径为20～240 nm。
  10. 权利要求9所述载药红细胞膜纳米粒在制备具有近红外光热效应和多模态成像功能的试剂、具有近红外光响应性药物、细胞核靶向输送多功能试剂、肿瘤诊疗一体化纳米制剂或者肿瘤治疗药物中的应用。
PCT/CN2022/110594 2022-08-05 2022-08-05 一种载药红细胞膜纳米粒及其制备方法和应用 WO2024026841A1 (zh)

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