CN115252580A - Drug-loaded erythrocyte membrane nanoparticle and preparation method and application thereof - Google Patents
Drug-loaded erythrocyte membrane nanoparticle and preparation method and application thereof Download PDFInfo
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- CN115252580A CN115252580A CN202210938574.8A CN202210938574A CN115252580A CN 115252580 A CN115252580 A CN 115252580A CN 202210938574 A CN202210938574 A CN 202210938574A CN 115252580 A CN115252580 A CN 115252580A
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Abstract
The invention discloses a drug-loaded erythrocyte membrane nanoparticle and a preparation method and application thereof. The erythrocyte membrane is used as a reactor, the erythrocyte membrane bionic nanoparticles with the photoresponsive drug release function are prepared in a water phase, the nanoparticles have good photo-thermal effect, physical and chemical stability, light stability, tumor targeting property, tumor deep penetration and responsive drug release, tumors are accurately positioned, a high-efficiency photo-thermal effect is generated under the excitation of near infrared light, tumor cells are killed by thermal ablation, high-efficiency, safe, visual and accurate tumor treatment is realized, and the potential of further development and application in clinic is realized.
Description
Technical Field
The invention discloses an erythrocyte membrane nano system for tumor targeted drug delivery, and particularly relates to ICG-DOX loaded erythrocyte membrane nanoparticles and a preparation method and application thereof.
Background
Malignant tumor is one of the major malignant diseases seriously threatening human health, and the morbidity and the mortality of the malignant tumor show obvious rising trends at home and abroad. How to realize accurate diagnosis and efficient treatment of tumors is the key and difficult point of current research. The development of the nanotechnology provides a new strategy for the treatment of tumors, and the nano-drug plays an important role in enhancing the curative effect of the drug and reducing the toxic and side effects of the drug due to the characteristic of tumor-specific targeted drug delivery.
In different nano biological platforms reported in the literature at present, the synthesis of nano drugs by a biomimetic method adopts natural materials of biological origin, such as albumin, cells, bacteria, viruses and the like, to carry out nano drug delivery, and the nano materials of the type combine respective advantages of natural substances and nano technology, and are increasingly becoming novel technologies with good application prospects in tumor treatment. The natural material biomimetic synthesis nano material has unique biological characteristics of the material, such as long-acting circulation, tumor specific targeting, immune regulation and the like. Currently, albumin and cell membrane based nano drug delivery systems have been widely used in precise tumor therapy (e.g., J Control Release, 2021, 329, 997-1022, chem Soc Rev, 2021,50, 945-985.
In the current research of nano-drugs based on cell membranes, erythrocytes are easy to extract cell membranes due to abundant sources, simple structure and no complex organelles, and the cell membrane surfaces have strong modifiability, so that the erythrocytes are widely applied to the research of nano-drugs. At present, two different strategies are important for research based on erythrocyte drug delivery, and complete erythrocytes are used as carriers for drug delivery, and erythrocyte membranes are used for wrapping cell membranes of existing nanoparticles such as PLGA nanoparticles, iron oxide magnetic nanoparticles, mesoporous silica nanoparticles, liposomes, polymer vesicles and the like to synthesize nanomaterials. Such as PEG-Fe prepared by hydrothermal method of the prior art 3 O 4 Extruding and wrapping with target-modified erythrocyte membrane fragments to prepare nanoparticle erythrocyte membrane-PEG-Fe with immune evasion and sensitive magnetic response 3 O 4 (ii) a The prior art also discloses an MOFs nano-drug carrier coated by erythrocyte membranes, and a preparation method and application thereof, wherein the MOFs nano-drug carrier comprises MOFs nano-particles and erythrocyte membranes, boric acid is loaded on pores of the MOFs nano-particles, the erythrocyte membranes are coated outside the MOFs nano-particles, and the MOFs nano-drug carrier is synthesized by a hydrothermal method. The prior art adopts the technical scheme that the drug particles are prepared firstly and then are wrapped by the erythrocyte membrane, the drug delivery has the limitations of low drug loading, poor targeting property and the like, the synthesis process is complex, the size control of the nano material depends on the core nano material, and the size of the nano material is difficult to accurately regulate and control, and the like.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a nano vesicle based on erythrocyte membranes, which realizes drug reaction and preparation in the nano vesicle, is used for encapsulating antitumor drugs, has the characteristics of accurate and controllable size, simple preparation and in-situ synthesis of the drugs, can realize higher targeting property, increases the drug encapsulation efficiency, has good biological safety, tumor targeting property and deep penetration and detention of tumors, maintains the flexibility of the cell membranes, can deform to realize deep tissue penetration at tumor parts, improves the efficient deep delivery of the drugs in tumor tissues, and simultaneously reduces the unnecessary release of DOX in normal tissues and effectively reduces toxic and side effects by forming compound precipitation of ICG and DOX. In tumor tissue, however, ID-RBCages can responsively release the chemotherapeutic drug DOX by laser irradiation. Meanwhile, the light-activated photodynamic therapy and chemotherapy induce the immunogenic death of tumor cells to cause the activation of the acquired anti-tumor immune effect, thereby realizing the enhanced photochemical therapy-immune synergistic therapy of the tumor.
The invention adopts the following technical scheme:
a medicine carrying erythrocyte membrane nanoparticle is prepared by mixing erythrocyte membrane with anion reaction precursor, and extruding to obtain anion reaction precursor erythrocyte membrane vesicle; then mixing the anion reaction precursor erythrocyte membrane vesicles with the cation reaction precursor for reaction to obtain the drug-loaded erythrocyte membrane nanoparticles.
The invention discloses a drug-loaded erythrocyte membrane nanoparticle with precisely controllable size and capable of delivering an anti-tumor drug in a targeted manner, wherein the particle size of the drug-loaded erythrocyte membrane nanoparticle is 20-240 nm. The delivery effect of the nano-drug delivery system has close relation with the size of the nano-drug delivery system, and size regulation is important for regulation of tumor drug delivery. The nanoparticles with different particle sizes prepared by size regulation can be used for size regulation and control screening of antitumor drugs. The smaller the particle size, the higher the tumor drug delivery efficiency of the erythrocyte membrane nanoparticle. Therefore, the research on the preparation of the nano-drug delivery method with the size controllability of the biological source material is helpful for constructing a safer platform for multi-modal imaging and tumor treatment.
In the technical scheme, the erythrocyte membrane is used as a skeleton of the nanoparticle; the cation reaction precursor is a metal compound or a micromolecular drug; the anion reactive precursor source is a sulfide, a citrate compound, or a dye drug. Cationic reactive precursors, anionic reactive precursors such as doxorubicin and indocyanine green.
In the technical scheme, the erythrocyte membrane is obtained by hypotonic treatment of the erythrocyte; preferably, the erythrocyte is suspended in hypotonic buffer solution, and centrifugal treatment is carried out after standing to obtain erythrocyte membrane; more preferably, the standing is performed in an ice bath for 20 to 40 minutes, and the centrifugation treatment is performed at 3000 to 4000g for 10 to 20 minutes. The concentration of the hypotonic buffer solution is 20-30% of the concentration of the corresponding isotonic buffer solution.
In the technical scheme, an erythrocyte membrane and an anion reaction precursor are incubated in a hypotonic buffer solution, and then a liposome extruder is used for extruding to obtain an anion reaction precursor erythrocyte membrane vesicle; then stirring the anion reaction precursor erythrocyte membrane vesicles and the cation reaction precursor in a hypotonic buffer solution for reaction, and then performing ultrafiltration to obtain the drug-loaded erythrocyte membrane nanoparticles. Preferably, the incubation time is 20-40 minutes, the ultrasonic treatment is carried out for 3-6 minutes after the incubation, and then the liposome extruder is used for gradient extrusion; preferably, the pore diameter of the gradient extrusion is 800 nm,400 nm,200 nm,100 nm and 50 nm. The reaction temperature of the stirring reaction is 25-55 ℃, the reaction time is 3-8 h, and the stirring reaction is preferably carried out for 4-5 h at 37 ℃.
In the above technical scheme, the molar ratio of the anion reaction precursor to the cation reaction precursor is 1: 0.5-8, preferably 1: 0.5-2.
The invention provides application of the drug-loaded erythrocyte membrane nanoparticles in preparing a tumor treatment drug.
The drug-loaded erythrocyte membrane nanoparticle of the invention has the following characteristics: (1) The size uniformity is good, the particle size is in the size range of 60 to 240 nm, and the size of the polycarbonate film of the extruder can realize the accurate regulation and control of the nanometer size of the erythrocyte film; (2) The erythrocyte membrane is used as a natural biological source material, has good biocompatibility, has good safety when being used as a drug delivery carrier, and the drug-loaded erythrocyte membrane nanoparticle has extremely low cell dark toxicity and good blood compatibility, and has good tumor targeting property in a tumor-bearing mouse model; (3) The drug-loaded erythrocyte membrane nanoparticles have better softness, have obviously enhanced deformability compared with the cell membrane nanoparticles prepared by the conventional cell membrane wrapping technology, and can realize efficient targeting, deep penetration and effective detention of tumor tissues; (4) The drug-loaded erythrocyte membrane nanoparticles can be efficiently absorbed by tumor cells, are positioned in acid lysosomes, can damage lysosome membranes and enhance the permeability of cell nucleus membranes after illumination, and realize the rapid cytoplasmic transport and cell nucleus delivery of DOX. In addition, the ID-RBCages can induce obvious cell immunogenic death and efficiently activate acquired anti-tumor immune effect; (5) The erythrocyte membrane is used as a natural membranous material, can perform photoresponsive cell membrane rupture under the photo-thermal condition and other photo-stimulation conditions such as the photodynamic condition, and lays a foundation for the release of responsive drugs; (6) The drug-loaded erythrocyte membrane nanoparticle tumor has good targeting property, can efficiently penetrate tissues in pancreatic cancer tumors of low-permeability Panc02 mice, and delivers drugs to deep parts of the tumors; in addition, the illumination can also promote the rapid transport of chemotherapeutic drugs into the nucleus, and the high-efficiency photochemical therapy synergistic anti-tumor effect is realized.
Drawings
FIG. 1 shows a transmission electron micrograph of ID-RBCages (A), a dynamic light scattering particle size of ID-RBCages (B), an absorption spectrum of ID-RBCages (C), and a fluorescence spectrum of ID-RBCages (D).
FIG. 2 is a transmission electron microscope image (B) of ID-RBCages under different conditions after photo-thermal temperature-raising capability examination (A) and under different conditions, and a release (C) under the illumination conditions;
FIG. 3 shows DHE and AO staining of ID-RBCages.
FIG. 4 shows the light-induced nuclear damage assay of ID-RBCages.
FIG. 5 shows cellular uptake of ID-RBCages and free drug at various time points.
FIG. 6 shows intracellular transport of ID-RBCages light-activated drugs into the nucleus.
FIG. 7 is a chemical formula (A)A) After the 4T1 cells and the ID-RBCages are incubated for 24 hours, the cell survival rate is ensured under the conditions of illumination and no illumination; (B) 4T1 cells were incubated with ID-RBCages for 24 h and then irradiated (785 nm,0.5W cm, g) in the absence of light -2 3 min) live-dead cell staining pattern.
FIG. 8 is a photograph of EdU staining of cells under non-illuminated and 785 nm laser illumination.
FIG. 9 shows immunofluorescence staining patterns of 4T1 cells (A) CRT protein, (B) HMGB1 protein under non-illumination and 785 nm laser irradiation, (C) CRT level and fluorescence intensity statistics of cells detected by a flow cytometer, (E) ATP secretion level detection of 4T1 cells under non-illumination and 785 nm laser irradiation.
FIG. 10 shows the tissue distribution of ID-RBCages, (A) Tail vein injection of ICG and ID-RBCages (ICG, 7.5 mg kg) -1 ) After 24 h, the distribution condition of DOX in each tissue is determined; (B) Tail vein injection of ICG and ID-RBCages (ICG, 7.5 mg kg) -1 ) Near infrared fluorescence imaging of ex vivo tissues after 24 h and (C) mean fluorescence intensity statistics for each tissue.
FIG. 11 is an examination of the deep tumor tissue penetration of ID-RBCages, (A) CLSM pictures of the penetration behavior of ID-RBCages and 70 nm-mPLGA in Panc02 subcutaneous tumors and (B) statistical analysis of fluorescence intensity in selected regions; (C) Distribution of DOX in tumor tissue under light as well as non-light conditions.
FIG. 12 shows the results of the tail vein injection of different concentrations of ID-RBCages 24 h after 4T1 subcutaneous tumor mice (A) tumor sites were under light conditions (785 nm,0.5W cm) -2 ) And (B) a temperature increase curve of the tumor site.
FIG. 13 shows 4T1 subcutaneous tumor mice (A) irradiated 24 h after tail vein injection of Free DOX, ID-RBCages or PBS, tumor growth curves of the mice within 25 days, and (B) tumor weight statistics for each group at the end of the tumor suppression experiment.
FIG. 14 is a graph of mouse tumor growth over 25 days after 4T1 in situ breast tumor mice (A) irradiated with Free DOX, ID-RBCages or PBS 24 h after tail vein injection; (B) And (E) carrying out bioluminescence picture of lung metastasis at the end of tumor inhibition experiment and carrying out corresponding lung metastasis bioluminescence intensity statistics.
FIG. 15 shows the light response of ID-RBCages tumor tissue to drugs into the nucleus.
FIG. 16 is a mouse tumor growth curve within 25 days of ID-RBCages photopathy and immunotherapy synergy for 4T1-Luc in situ breast cancer treatment, (A) 24 h tumor illumination after tail vein injection of Free DOX, ID-RBCages, PBS, treatment injection of aPD-L1 for combination therapy; (B) tumor weight statistics of each group of mice at the end of the tumor inhibition experiment; (C) Bioluminescence pictures of lung metastases and (D) corresponding bioluminescence intensity statistics of the lung metastases at the end of the tumor inhibition experiment; (E) H & E staining of lung metastases at the end of tumor suppression experiments.
FIG. 17 is a schematic representation of the immuno-synergy of ID-RBCages photochemotherapy for Panc02 pancreatic carcinoma subcutaneous tumor treatment, (A) a tumor suppression dosing regimen for Panc02 subcutaneous tumor mice; (B) Tumor growth curves of mice of different dosing treatment groups, (C) survival curves of mice, and (D) tumor growth curves of a single mouse in each group of mice.
FIG. 18 shows (A) tumor cells (1X 10) in the blank control mice and the treated mice on day 60 5 ) And (C) performing corresponding statistical analysis on a tumor growth curve after re-inoculation and flow cytograms of (B) long-term effector T cells and central memory T cells.
FIG. 19 is a schematic of ID-RBCages photochemotherapy for immune synergy for KPC orthotopic pancreatic cancer treatment, (A) dosing regimens for KPC orthotopic pancreatic cancer tumor treatment; (B) Bioluminescent photographs of tumors at different time points in mice of each treatment group of KPC orthotopic pancreatic cancer mice; (C) KPC orthotopic pancreatic cancer mice tumor growth curves for each group of mice and (D) survival curves for each group of mice.
FIG. 20 is a graph of the products obtained under different hypotonic treatment conditions.
Detailed Description
Liposome extruder (Avanti, usa), indocyanine green, doxorubicin purchased from national pharmaceutical group chemicals ltd; ultrafiltration tubes (Millipore, usa), carbon-supported membrane copper mesh, carbon-supported membrane nickel mesh (new bairi technologies ltd, beijing). The red blood cells are the existing products, and the red blood cells of the embodiment of the invention are obtained by taking blood from a female Balb/c healthy mouse in 6-8 weeks, separating and collecting the blood after venous blood taking.
The invention discloses a drug-loaded erythrocyte membrane nanoparticle with the function of targeted delivery and anti-tumor, which is prepared by the following steps:
(1) Resuspending the collected red blood cells in hypotonic PBS buffer solution (0.25 x), and standing in ice bath for 20-40 min; then, the mixture was centrifuged at high speed (3500 g) for 15 min, the supernatant was discarded, and the bottom layer precipitate was collected, followed by washing 3 to 5 times with hypotonic PBS buffer (0.25X) to obtain erythrocyte membranes.
(2) Resuspending the red cell membrane prepared in the step (1) into a hypotonic PBS buffer solution (0.25 x), placing the red cell membrane into a 30 ml glass reaction bottle, adding an ICG solution, incubating for 20-40 minutes, then performing ultrasound for 3-6 min, and extruding the cell membrane solution after ultrasound under the conditions of 800 nm,400 nm,200 nm,100 nm and 50 nm respectively by using a liposome extruder, wherein the extrusion times of each size are 11-25 times. The erythrocyte nano vesicle prepared by extrusion is an anion reaction precursor erythrocyte membrane vesicle.
(3) Adding a cation reaction precursor solution into the anion reaction precursor erythrocyte membrane vesicle solution for reaction; and (3) taking out the reaction solution after the reaction is finished, and carrying out ultrafiltration purification on the reaction solution by using an ultrafiltration tube with the molecular weight cutoff of 30 to 200kD to obtain the drug-loaded erythrocyte membrane nanoparticles after the reaction. Preferably, the rotating speed of the ultrafiltration is 1200 to 5000 rpm/min, and the ultrafiltration frequency is 10 to 20 times.
Establishment of tumor model
(1) Subcutaneous tumor model: in order to investigate the anti-tumor biological activity of the ID-RBCages, subcutaneous tumor models of breast cancer of a 4T1 mouse, pancreatic cancer of a Panc02 mouse and pancreatic cancer of a KPC mouse are constructed, wherein murine cells 4T1 are inoculated to a Balb/C female mouse, and the Panc02 and KPC are inoculated to a C57 mouse, and the inoculation method is to inoculate 100 mu L cell suspension (the density of 4T1 cells is 5 multiplied by 10) 6 Cell density of Panc02 and KPC 5 × 10/mL 7 One per mL) was injected subcutaneously into the right leg of the mouse, and the length and width of the tumor were measured using a vernier caliper until the tumor length reached 70-100 mm 3 It is used when in use. The tumor volume calculation method comprises the following steps: tumor bodyProduct =1/2 × (long diameter × short diameter).
(2) 4T1 in situ breast cancer model: after 6-8 weeks Balb/c female mice are anesthetized, the skin near the second pair of mammary glands is incised, and 4T1-Luc cell suspension (with the cell density of 1X 10) diluted in advance is added 7 individual/mL) 50 µ L was injected into the fat pad under the mammary gland of the mouse and the wound was sutured. Measuring the length and width of the tumor with vernier caliper until the length of the tumor reaches 70-100 mm 3 It is used when in use.
(3) KPC in situ pancreatic cancer model: c57 mice were anesthetized after abdominal hair removal, the left upper abdomen was incised by surgery to expose the spleen and pancreas, the spleen and pancreas tail were carefully pulled out of the abdominal cavity of the mice using surgical forceps, and the KPC-Luc cell suspension added with matrigel was 50 μ L (cell density 1X 10) 8 individual/mL) was carefully injected into the tail end of the mouse pancreas, and after completion of the injection, the spleen and pancreas were carefully pushed back into the mouse abdominal cavity, and the wound was closed. Tumor growth was monitored by taking bioluminescent images using a small animal in vivo imaging system (IVIS, lumia II).
The following detailed description of the present invention is provided in connection with the accompanying drawings and examples. The examples are provided to illustrate the present invention, but are not intended to limit the present invention. The specific experimental procedures and test methods of the present invention are conventional techniques. Statistical analysis of all data GraphPad Prism 8.3 software was used, values are expressed as mean + -SD, and statistical analysis was performed using independent samplestAnd (4) checking the test result,Pvalues < 0.05 were considered statistically significant. NS is not significantP<0.05，**P<0.01，***P<0.001，****P<0.0001。
Example one
Clinically approved photosensitizer molecules of Indocyanine green (ICG) and chemotherapeutic drug Doxorubicin (DOX) are selected for red cell membrane nano drug entrapment, and ICG/DOX red cell membrane nanoparticles (ID-RBCages) are prepared. The sulfonic acid group of ICG molecule and the protonated amino group of DOX molecule can form compound precipitate through coordination, and the compound precipitate is coprecipitated in the cavity of red nanometer cell membrane to prepare ID-RBCages.
The red blood cells of the embodiment of the invention are from female Balb/c healthy mice in 6-8 weeks, are obtained by separation and collection after intravenous blood collection, and specifically, the female Balb/c mice in 6-8 weeks are taken, the whole blood (1 mL) of the mice is taken after anesthesia and is collected into a centrifuge tube containing anticoagulant heparin sodium, the mice are centrifuged for 10 min under the centrifugal force of 800 g, the supernatant is discarded, the lower layer red blood cell precipitate is reserved, the obtained red blood cells are washed three times by 1 XPBS buffer solution, and the obtained red blood cells are collected by centrifugation.
The preparation method of the ID-RBCages comprises the following steps:
(1) Resuspending the centrifuged erythrocytes in 50mL of hypotonic PBS buffer (0.25X), and standing in ice bath for 30 min; then, the mixture was centrifuged at high speed (3500 g) for 15 min, the supernatant was discarded to collect the bottom layer precipitate, and the precipitate was washed with hypotonic PBS buffer (0.25X) 3 times to obtain erythrocyte membranes.
(2) The red cell membrane prepared in step (1) was resuspended in 10 mL of hypotonic PBS buffer (0.25X) and placed in a 30 mL glass reaction flask, and ICG solution (1.0 mg mL of ICG solution) was added -1 ) And performing ultrasonic treatment for 5min after incubation for 30 min, sequentially extruding the cell membrane solution subjected to ultrasonic treatment by using 800 nm,400 nm,200 nm,100 nm and 50 nm microporous polycarbonate filter membranes by using a liposome extruder, wherein the extrusion frequency of each size is 25 times, the receiver is the 30 ml glass reaction bottle, and the erythrocyte nano vesicles extruded at 50 nm are ICG erythrocyte membrane vesicles. Then, 1.0 mg mL of the above ICG erythrocyte membrane vesicle solution (30 mL of glass reaction flask) was added -1 Stirring and reacting 2 mL of DOX solution (the molar ratio of ICG to DOX is 1: 1) in water bath at 37 ℃ at 600 rpm for 4 hours, and performing ultrafiltration by using a 100 kd ultrafiltration tube after the reaction is finished to obtain drug-loaded erythrocyte membrane nanoparticles; the rotational speed of ultrafiltration was 3000 rpm/min, and the number of ultrafiltration was 15.
As shown in fig. 1, transmission Electron Microscopy (TEM) images show that the prepared ID-RBCages nanoparticles are round-structured nanoparticles with uniform size, the size of which is 65.8 ± 5.4 nm, and the size of the inner ID nanocluster is 5.3 ± 3.8 nm. Dynamic Light Scattering (DLS) measurement results show that the hydrated particle size of the ID-RBCages is 79.5 +/-7.5 nm, the particle size distribution peak is a single peak, and the particle size uniformity is better. The ID-RBCages have an ICG absorption characteristic peak and a doxorubicin fluorescence emission peak, and successful entrapment of the ID-RBCages on the ICG and the DOX is prompted; drug loadings of ICG (10.5%), DOX (7.8%) were routinely calculated.
The Young's modulus of ID-RBCages was measured by AFM, and the average value was 18kPa. The Young modulus of Se-NPs obtained by using organic polymer PEG-PUSE-PEG containing single selenium as a carrier and ICG and DOX as model drugs, which is disclosed in the subject group before, is 68kPa on average; previously prepared in this subject group, human Serum Albumin (HSA) was wrapped in DOX and ICG by coprecipitation reaction to prepare ICG/DOX @ HSA nanoparticles with Young's modulus of 46kPa on average.
Example two
Examination of photothermal heating capacity: 500 mL of solutions of ID-RBCages and free ICG at concentrations (ICG) of 0.2 mM, 0.5 mM and 1.0 mM, respectively, were placed in 2 mL EP tubes. With a 785 nm laser (0.5W cm) -2 5 min), monitoring the solution temperature using a digital display thermometer, recording the solution temperature every 30 s, and drawing a temperature rise curve of the solution. The result of the measurement of the photothermal heating capacity of a series of ID-RBCages solutions with different concentrations (figure 2A) shows that under 785 nm laser irradiation, the ID-RBCages can effectively generate photothermal heating, the heating effect has concentration dependence, and the photothermal heating effect can be obviously enhanced along with the increase of the sample concentration.
EXAMPLE III
The drug release behavior of light activated ID-RBCages was examined. And (4) observing the in-vitro drug release behavior of the ID-RBCages by adopting a dialysis method. Respectively taking ID-RBCages, ID-RBCages-Laser and ID-RBCages-Laser/Vc (785 nm,0.5W cm) -2 5min, vc concentration 2 mM) and Free DOX solution, each 1 mL, in dialysis bags (molecular weight cut-off 3500), the dialysis bags were placed in 15 mL centrifuge tubes containing dialysis media (acetate buffer at pH 5.0 or phosphate buffer at pH 7.4), each set of three replicates, placed in a constant temperature shaker (120 rpm,37 ℃), dialysis buffer was changed at 0.5, 1, 2, 4, 8, 24, 48 h, and the amount of DOX released was quantified and calculated. ID-RBCages electron microscope picture after single laser irradiationThe result of shooting shows (fig. 2B) that singlet oxygen generated by illumination can effectively destroy the structure of the nanoparticle, which is beneficial to promoting the rapid release of the drug encapsulated by ID-RBCages. In addition, the photothermal effect is shielded under the condition of 0 ℃ and the photodynamic effect is shielded by adding Vc, and the result shows that the light-activated drug release is mainly caused by that ROS generated under light irradiation obviously damages the membrane structure of the nanoparticles, and the simple photothermal effect has little influence on the structure of the nanoparticles. Further quantitative analysis of DOX drug release by dialysis (FIG. 2C) showed that ID-RBCages released less drug when not illuminated (<10%), whereas the drug release can be significantly increased to 34.8% after the solution is irradiated with near-infrared laser, the photodynamic effect is shielded by Vc, the release of DOX is significantly reduced, and the cumulative release is reduced to 15.3%. In conclusion, ID-RBCages can trigger the release of intelligent response chemotherapeutic drugs DOX through photodynamic effect under laser irradiation, and a solid foundation is laid for reducing the toxic and side effects of the drugs and further enhancing the multi-drug synergistic tumor killing.
Example four
To further evaluate the ability of ID-RBCages to induce singlet oxygen production in cells under light conditions, they were validated using DHE staining experiments. 4T1 cells were seeded in 24-well plates (1X 10 per well) 5 Individual cells). After the cells were attached to the wall, the medium was discarded, and previously diluted ID-RBCages and Free I/D solutions (mixture of ICG solution and DOX solution) were added at concentrations of 0, 0.1, 0.2, 0.5, 1.0, and 2.0. Mu.g mL -1 Parallel to 3 multiple wells. The culture was continued for 6 h, the medium was removed and washed 3 times with PBS. The cells in the light-irradiation group were irradiated with light (0.5W cm) using a 785 nm laser -2 3 min), continue culturing for 3 h. Removing the culture medium, adding 1 mL (50 μ M) of freshly prepared DHE staining solution into each well, culturing for 0.5 h in a dark place, removing the staining solution, washing with PBS for three times, observing and photographing by a laser confocal microscope, and performing dark operation in the whole process. 4T1 cells were seeded in 24-well plates (1X 10 per well) 5 One cell), 1 mL of medium was added per well. After the cells are attached to the wall. Discarding the culture medium, adding pre-diluted ID-RBCages and Free I/D solution, and calculating according to DOXConcentrations were set at 0, 0.1, 0.2, 0.5, 1.0, 2.0. Mu.g mL -1 Parallel to 3 multiple wells. The culture was continued for 6 h, the medium was removed and washed 3 times with PBS. The cells in the light-irradiation group were irradiated with light (0.5W cm) using a 785 nm laser -2 3 min), continue culturing for 3 h. The medium was removed and 1 mL (10.0. Mu.g mL) of freshly prepared AO staining solution was added to each well -1 ) Culturing in dark for 0.5 h, removing staining solution, washing with PBS for three times, observing with laser confocal microscope, and taking pictures. DHE is a superoxide anion fluorescent probe that is itself free to enter the interior of living cells. The DHE molecule has no fluorescence, and after the oxidopyridine generated by the DHE molecule under the action of ROS is doped into DNA, strong red fluorescence can be generated, so that the DHE molecule can be used for detecting ROS in cells. As shown in FIG. 3, there was no ROS production under non-light conditions after uptake of ID-RBCages into cells, but very low (0.05. Mu.g mL) under 785 nm laser light -1 ) Significant ROS production can be induced effectively under the drug concentration of ID-RBCages. The result shows that the ID-RBCages taken in the cells can effectively generate ROS under the laser irradiation condition, and a good foundation is laid for the ID-RBCages to exert the photodynamic treatment effect. To further verify that ROS generated in lysosomes by light-induced ID-RBCages can effectively destroy the membrane structure of the lysosome, cells before and after irradiation were analyzed using AO staining experiments. The AO dye is a membrane-permeable fluorescent dye molecule, is alkalescent, presents green fluorescence in a cytoplasmic environment, can be protonated in an acidic environment of a lysosome to present red fluorescence, and can be used as an indicator for structural characterization of the cell lysosome. The ID-RBCages can damage the lysosome structure of the cell under the condition of laser irradiation after being taken into the cell, and powerful guarantee is provided for lysosome escape of the drug and cytoplasmic transportation.
EXAMPLE five
To further evaluate whether the ROS produced by the light-induced ID-RBCages influence the structure of the nuclear membrane, a FITC-labeled Dextran was used for validation. The molecular weight of the selected Dextran is 70kD, relative to the size of nuclear pore of nucleus, the molecular weight of Dextran can not penetrate the nuclear pore of normal nucleus into the inside of nucleus, and can be used as nucleusAn indicator of the structural integrity of the film. 4T1 cells were seeded in 24-well plates (1X 10 per well) 5 One cell), 1 mL of medium was added per well. After the cells are attached to the wall, the culture medium is discarded, and pre-diluted ID-RBCages and Free I/D solution (DOX, 1.0 mu g mL) is added -1 ) Parallel to 3 multiple wells. The culture was continued for 6 h, the medium was removed and washed 3 times with PBS. The cells were irradiated with 785 nm laser (0.5W cm) -2 3 min), after light irradiation a diluted FITC fluorescently labeled Dextran solution of the culture medium (FITC-Dextran, MW:70 kD,1.0 mg mL -1 ) Incubated at room temperature in the dark for 3 hours, the staining solution was removed, washed three times with PBS, and 1 mL of Hoechst 33342 solution (5.0. Mu.g mL) was added -1 ) Dyeing at 37 ℃ in dark for 5min, discarding the dye, washing with PBS three times, observing by a laser confocal microscope and taking a picture. The cells were incubated with FITC-Dextran in the illuminated and non-illuminated conditions, respectively, as shown in FIG. 4, after incubation with Dextran, the Dextran taken up inside the cells was distributed only in the cytoplasm and not into the nucleus of the cell. On the contrary, the ID-RBCages light-irradiated cells can observe stronger green fluorescence signals in the cytoplasm and the inside of the cell nucleus, which indicates that FITC-labeled Dextran can be effectively transferred to the cell nucleus in the irradiated cells, and confirms that ROS generated by the ID-RBCages induced by light can damage the nuclear membrane of the cells, the integrity of the nuclear membrane of the cells is damaged, and the effective delivery of the nuclear-targeted chemotherapeutic drugs is facilitated.
EXAMPLE six
Cellular uptake and uptake pathways. 4T1 cells in good growth state were sampled in 6-well plates at a cell density of 1.0X 10 per well 6 The individual cells were plated and cultured by static culture. After the cells are attached to the wall, ID-RBCages and free DOX (1.0 mu g mL) are added respectively -1 ) And 3 multiple holes are simultaneously arranged. Respectively incubating for 2 h, 12 h and 24 h, washing the cells for three times by PBS, digesting and collecting the cells, counting the cells, and crushing the cells by an ultrasonic crusher (ultrasonic power: 400W, ultrasonic time: 5 min). Subsequently, 1 mL of DMSO-extracted drug was added to each cell lysate, and the amount of drug uptake was calculated by quantifying using a full-wavelength microplate reader. Taking 4T1 cells in good growth state, according toEach hole 10 6 The number of each cell is inoculated into a 6-hole cell culture plate, 2 mL of culture medium is added into each hole, and the incubator stands and cultures until the cells adhere to the wall. Then adding different endocytosis pathway inhibitors, adding PBS with the same volume in a blank control group, independently taking a plate, placing the plate in a refrigerator at 4 ℃ for low-temperature incubation, and setting three multiple holes. After 2 h, the medium was discarded, and diluted ID-RBCages (1.0 μ g mL) was added to each well -1 ) After 6 h, the medium was discarded, the cells were digested, collected and counted, and the cells were disrupted using an ultrasonic disrupter (ultrasonic power: 400 W, ultrasonic time: 5 min), followed by 1 mL of DMSO to extract the drug, quantitated using a full-wavelength plate reader, and drug uptake was calculated. Efficient cellular uptake guarantees therapeutic effects of the drugs, and cellular uptake experimental results show that the ID-RBCages and free drugs have consistent cellular uptake behaviors (figure 5). In addition, the amount of ID-RBCages taken up by the cells correlates with the time of uptake. The cell uptake of ID-RBCages increased gradually over the 24 h observation period. And under the same time, the ID-RBCages can cause more cell drugs to be taken in, and the taking amount of the ID-RBCages is 2.5 times of that of free drugs in 24 hours, which shows that the cell drug taking amount of the antitumor drugs can be effectively increased by using the erythrocyte membrane nano-reactor for drug delivery, and lays a foundation for further enhancing the drug treatment effect.
EXAMPLE seven
The dynamic processes of cytoplasmic transport and nuclear delivery following illuminated uptake of intracellular DOX were observed using confocal laser techniques. Cells pre-incubated for 6 h by ID-RBCages are firstly subjected to nucleus and lysosome staining and marking, a cell culture dish is fixed on a laser confocal imaging instrument, the focus is adjusted to find the cells on imaging software, and the cells are observed and photographed to be used as non-illumination cell images. Next, light irradiation (0.5W cm) was performed using a 785 nm laser -2 5 min) and observing the cells by using a laser confocal microscope at 5min, 15 min, 30 min and 60 min after illumination and photographing to record the intracellular transport condition of DOX. The results show (fig. 6) that DOX after illumination can achieve rapid lysosomal escape and within 15 min after illuminationHomogeneous cytoplasmic distribution and a small accumulation of DOX in the nucleus, with up to 88% increase in nuclear transport of DOX over time up to 60 min. The ID-RBCages can realize the photoresponsive drug cytoplasmic transport and the rapid cell nucleus delivery, and provides guarantee for the DOX to play the role of anti-tumor chemotherapy.
Example eight
Cytotoxicity. Collecting 4T1 cells with good growth state, digesting, collecting, and diluting with culture medium to cell density of 5 × 10 4 Inoculating each cell/mL into a 96-well cell culture plate, inoculating 100 mu L of cell suspension into each well, culturing overnight in a cell culture box at 37 ℃, adding ID-RBCages nanoparticle solutions with different concentrations (calculated by the concentration of DOX) into each well, wherein the administration concentrations are 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 4.0 mu g mL respectively -1 Duplicate wells were set for each concentration, and a PBS control without drug was set. The incubation was continued for 24 h at 37 ℃ in an incubator, the medium was removed, washed three times with PBS and MTT (0.5 mg mL) was added to the non-illuminated group -1 ) The cell culture medium is cultured for 4 hours continuously, the culture solution is sucked out gently, 100 muL of dimethyl sulfoxide (DMSO) solution is added into each hole, the shaking table is shaken for 5min, the absorbance of each hole at 492 nm is measured by a multifunctional microplate reader, and the cell survival rate is calculated. Adding fresh culture medium to each well of the cells in the illumination group at 100 μ L, and illuminating each well of the cells for 3 min (0.5W cm) by using a 785 nm laser -2 ) And continuously culturing the cells for 24 hours after illumination, detecting the survival condition of the cells by using MTT, and calculating the survival rate of the cells. In order to visually examine the cell killing effect of the nanoparticles, cells were stained using a Calcein-AM/PI live cell/dead cell double staining kit. The method comprises collecting 4T1 cells with good growth state, digesting, collecting, and diluting with culture medium to cell density of 5 × 10 4 And inoculating each cell/mL into a 96-well cell culture plate, inoculating 100 mu L of cell suspension into each well, and culturing overnight in a 37 ℃ cell culture box. After the cells are completely attached to the wall, adding ID-RBCages and Free DOX into each hole, and calculating the administration concentration by DOX to be 1.0 mu g mL -1 3 duplicate wells were set for each concentration, and a PBS control without drug was set. Continuously culturing at 37 deg.C for 24 hrRemoving the culture medium, washing with PBS for three times, adding fresh cell culture medium, culturing in a cell culture box with each well of 100 μ L, and culturing the cells in the non-illumination group by irradiating the cells in each well with 785 nm laser (0.5W cm) -2 3 min), continuously culturing the cells for 6 h after illumination, staining the cells, and then photographing the cells by using laser confocal light.
To investigate the effect of ID-RBCages on the viability of breast cancer cells in 4T1 mice, the cytotoxicity of ID-RBCages was examined using the MTT method. As shown in FIG. 7A, the growth of 4T1 cells was less affected by ID-RBCages under non-light conditions, indicating that ID-RBCages themselves are less cytotoxic, which is beneficial for reducing the toxic side effects of chemotherapeutic drugs on non-tumor sites. Under 785 nm laser light, the ID-RBCages can cause strong cell killing to 4T1 cells at lower administration concentration, and the IC thereof 50 The value was 0.61. Mu.g mL -1 . The ID-RBCages can be used as a good photo-chemotherapy treatment drug, has good photo-chemotherapy killing and low dark toxicity, can selectively kill the tumor through the laser regulation and control effect, and reduces the damage to normal tissues, and the ID-RBCages can be used as a photo-chemotherapy treatment nano-drug with good selectivity.
The dark cytotoxicity of ID-RBCages under non-illumination conditions and the phototoxicity under laser illumination conditions are respectively examined through live-dead cell staining experiments. As shown in FIG. 7B, compared to the free drug group, the killing effect of ID-RBCages on cells did not change significantly, the cell killing ability of ID-RBCages was substantially consistent with that of the free drug group, and the cell killing ability of ID-RBCages was significantly improved under the light condition. In the free drug group, the cell killing after illumination has no obvious change compared with the cell killing under the non-illumination condition, and more cells still survive. In conclusion, the drug delivered by the erythrocyte membrane nano reactor can obviously enhance the killing capability of cells, and the ID-RBCages tumor cell killing is photo-selectivity and has good safety.
Example nine
In order to examine the influence of the light condition of the ID-RBCages on the proliferation activity of the cells, the cell proliferation condition is characterized by adopting an EdU staining experiment. The EdU staining assay allows for the specific labeling of cells in the actively proliferating S phase and allows for indirect investigation of proliferative capacity by fluorescence analysis of Azide 488. As shown in fig. 8, the fluorescence image results can visually indicate the intensity of the active proliferation capacity of the cells, wherein the cell nuclei are blue, and the cells in S phase are green. The ID-RBCsges has no obvious inhibition effect on the active proliferation capacity of tumor cells under the non-light condition, while the ID-RBCages can obviously inhibit the proliferation of the cells under the light condition, and the cells with the active proliferation capacity can hardly be seen on a fluorescent picture. The ID-RBCages can effectively inhibit the growth of the tumor cells by reducing the cell proliferation activity under the illumination condition, thereby achieving the effect of damaging the tumor cells.
Example ten
Experiment of cell immunogenic death (ICD effect). ID-RBCages induce the expression of Calreticulin (CRT) on the surface of tumor cells. CRT expression on the surface of tumor cells was detected using flow cytometry and immunofluorescence staining, respectively. 4T1 cells were seeded in 24-well plates (1X 10 per well) 5 Cells), add 1 mL of medium per well. Culturing until the cells are completely attached to the wall. The medium was discarded and pre-diluted ID-RBCages of medium and Free drug control Free I/D solution (DOX, 1.0. Mu.g mL) were added -1 ) After incubation for 12 h the medium was discarded. Illumination of cells in the tissue group was performed using a 785 nm laser (0.5W cm) -2 3 min), cells were cultured for 3 h, and after 4% paraformaldehyde fixing the cells, primary antibody (2 h) and fluorescent secondary antibody (APC labeling, 1 h) were incubated, respectively. The CRT expression level was measured by flow cytometry. CRT immunofluorescence staining. 4T1 cells were seeded in glass-bottom cell culture dishes (1X 10 per well) 5 Cells), 2 mL of medium is added to each well and cultured until the cells adhere to the wall. Respectively adding ID-RBCages and Free I/D (DOX, 1.0 mu g mL) -1 ). Incubation was continued for 12 h. The illumination group was illuminated with a 785 nm laser (0.5W cm) -2 3 min), continuously culturing for 6 h, fixing 4% paraformaldehyde, and respectively incubating primary antibodies (1)2 h) And fluorescent secondary antibody (Cy3.5-labeled, 6 h), DAPI solution (5.0. Mu.g mL) -1 ) After labeling the nuclei, photographic analysis was performed using a laser confocal microscope. ID-RBCages induce the efflux of High mobility group box 1 (HMGB1) protein from tumor cells. 4T1 cells were seeded in glass-bottom cell culture dishes (1X 10 cells per well) 5 Cells), 2 mL of culture medium is added into each well, and the cells are cultured until the cells are completely attached. Respectively adding ID-RBCages and Free I/D (DOX, 1.0 mu g mL) -1 ). Incubation was continued for 12 h, medium was discarded and washed three times with PBS. The illumination group was illuminated with a 785 nm laser (0.5W cm) -2 3 min), continuing to culture the cells for 24 h, discarding the medium and washing the cells, fixing the cells with 4% paraformaldehyde for 15 min, removing the fixing solution, adding 0.3% TritonX-100, and rupturing the membranes (incubating for 30 min). After PBS wash, primary antibody (12 h) and fluorescent secondary antibody (Cy3.5-labeled, 6 h), DAPI solution (5.0. Mu.g mL) were incubated -1 ) After labeling the nuclei, photographic analysis was performed using a laser confocal microscope. ID-RBCages induce the secretion of Adenosine Triphosphate (ATP) by tumor cells. 4T1 cells were seeded in 24-well plates (1X 10 per well) 5 Cells), add 1 mL of medium per well. Culturing until the cells are completely attached to the wall. The medium was discarded, and ID-RBCages and Free I/D (DOX, 1.0 μ g mL) were added -1 ). Incubation was continued for 12 h, medium was discarded and washed three times with PBS. The illumination group was illuminated with a 785 nm laser (0.5W cm) -2 And 3 min), continuously culturing for 12 h, collecting cell culture medium, and quantifying ATP by using an ATP detection kit. The chemotherapy drug DOX and the photodynamic effect can effectively induce the tumor cells to generate immunogenic death. Thus, the ability of ID-RBCages to induce immunogenic death of tumor cells was examined. Three ICD effect indexes, namely, the cell membrane surface exposure of Calreticulin (CRT), the release of High mobility group box 1 (HMGB1) and the extracellular secretion of Adenosine Triphosphate (ATP), which are important indexes of immunogenic death, are examined respectively. The results show that ID-RBCages can effectively enhance the exposure of CRT proteins on the surface of cell membranes after illumination (figure)9A, C-D), which provides a stronger "eat-me" signal for immune cells to recognize tumor cells, the ID-RBCages-induced increase in extracellular release of tumor cell ATP (FIG. 9E) also provides a stronger "find-me" signal for tumor cells to macrophages and DC precursor cells, and the increase in extracellular release of HMGB1 further verifies that ID-RBCages can effectively elicit the ICD effect (FIG. 9B).
EXAMPLE eleven
In order to analyze the ID-RBCages tumor targeting capability and the distribution condition of the ID-RBCages tumor targeting capability in each tissue organ of a mouse, a tissue distribution experiment is carried out for research. Taking tumor with size of 70-100 mm 3 Panc02 subcutaneous tumor mice, tail vein injection of ID-RBCages and Free drug Free I/D (ICG, 7.5 mg kg) -1 ) And (3) in a tumor-bearing mouse, dislocating the mouse after 24 h, taking down the heart, the liver, the spleen, the lung, the kidney and the tumor, and carrying out imaging analysis on the ICG distribution condition by using a small animal living body imaging instrument. Strictly protected from light, after each tissue was weighed, the tissue was pulverized using a homogenizer, the homogenate was extracted using a mixed solution of chloroform and methanol (1).
As shown in FIG. 10A, the targeting property of ID-RBCages tumor tissue is significantly improved compared to the free drug group, and the tumor tissue accumulation amount of DOX is 15.2 ID% g -1 7.17 times that of the free drug group. The reason for enhancing the targeting property of the ID-RBCages is that the prepared nanoparticles have proper sizes, can effectively realize the accumulation of tumors through an EPR effect, and particularly, the deformability of the ID-RBCages enables the nanoparticles to efficiently penetrate deep tumors, thereby laying a foundation for further exerting the anti-tumor bioactivity. ICG can be directly applied to near infrared fluorescence imaging, so that the in vitro tissue is subjected to small animal imaging fluorescence photo shooting, and the distribution condition of ID-RBCages in each tissue organ can be directly observed. As shown in fig. 10B, the targeting of ID-RBCages was significantly improved compared to the tumor tissue of the free drug group, and the fluorescence intensity of the tumor tissue was 5.25 times that of the free drug group (fig. 10C), which is consistent with the result of DOX tissue quantitative distribution.
Example twelve
To evaluate the effective penetration capacity in ID-RBCages deep tumors, drug delivery of ID-RBCages in low permeability Panc02 mouse pancreatic cancer tumor deep tissues was investigated using tumor tissue section vascular staining experiments. Mice bearing Panc02 subcutaneous tumors were randomized into groups and injected tail vein with ID-RBCages, free-I/D (ICG, 7.5 mg kg) -1 ) In tumor-bearing mice, 24 h after administration, the mice tumors were taken and fixed with 4% paraformaldehyde solution, then sectioned by a microtome (the section thickness is 10 μm), and after CD31 immunofluorescent staining of the sections, the sections were observed and photographed by using a laser confocal microscope, and the images were subjected to fluorescence intensity statistical analysis by using Image J.
The results show (fig. 11A-B) that ID-RBCages can achieve good deep tumor penetration in a low permeability Panc02 subcutaneous tumor model of pancreatic cancer with dense tumor extracellular matrix. The ID-RBCages drugs still had relatively uniform and highly efficient deep penetration in the region of 80 μm depth of penetration of the perivascular tissue, and a uniform distribution of the ID-RBCages fluorescence signals in the perivascular tissue region (FIG. 11B). This is associated with the appropriate size of ID-RBCages and their own good flexibility to allow deep penetration through the tumor tissue by deformation for efficient drug delivery. As shown in FIG. 16C, ID-RBCages were efficiently taken up by tumor cells at the tumor site 24 h after tail vein administration. After illumination, the ID-RBCages responsively releases the DOX encapsulated therein, and ROS generated by the ID-RBCages damages a cell nucleus membrane, so that the effective transportation of the DOX to a tumor cell nucleus part is accelerated, and a favorable guarantee is provided for exerting the effect of killing tumor cells by photochemical therapy.
EXAMPLE thirteen
Near infrared thermal imaging. In order to examine the tumor photothermal treatment capacity of the ID-RBCages in vivo, the tumor temperature of the mice under the laser irradiation condition was monitored by using a thermal imaging instrument. Tail vein injection of ID-RBCages, free I/D (ICG, 7.5 mg kg) -1 ) After the drug is administered for 24 h in the tumor-bearing mice, a 785 nm laser enters the tumor part of the miceLine illumination (0.5W cm) -2 5 min) and tumor temperature was recorded using a thermal imager.
The results show (fig. 12) that the tumor site of the PBS mice was not significantly heated by direct laser irradiation, and the temperature was 4 ℃ under 5min of continuous illumination, indicating that the laser irradiation itself did not produce significant photothermal heating effect. Under the laser irradiation of the mice of the ID-RBCages administration group, the tumor parts can generate obvious photothermal warming effects, the photothermal warming has obvious concentration dependence, and the higher the administration concentration is, the stronger the tumor hyperthermic warming capability is. Wherein, 10.0 mg kg -1 The tumor site temperature of the ID-RBCages group of the doses can reach 19.2 ℃, and the tumor site can be kept to be continuously maintained at the highest temperature without reduction after the tumor site temperature is raised to the highest within 5min of observation time. This relies on the highly targeted accumulation of ID-RBCages at the tumor site, which effectively enhances the photostability of ICGs.
Example fourteen
Phototherapy is used for the treatment of breast cancer in mice. And (4) using a 4T1 subcutaneous tumor model to investigate the tumor inhibition effect of the ID-RBCages. The tumor volume of the mouse is 70-100 mm 3 The experiment was started by tail vein injection of ID-RBCages, free I/D (ICG, 7.5 mg kg) -1 ) Into the body of the mouse. 24 After h, the mice in the light group were irradiated with laser light (0.5W cm) to the tumor site using a 785 nm laser -2 5 min), mouse tumor size was measured using a vernier caliper over 25 days. On day 25, groups of mice were sacrificed after tumor measurement, and mice were removed and tumors were photographed and weighed. The administration scheme of the 4T1 in-situ breast cancer experiment is the same as that of subcutaneous tumor, the difference is that the tumor observation time of the tumor experiment of the in-situ breast cancer is 21 days, lung tissues of each group of mice are taken out at 21 days, in-vitro bioluminescence imaging photos are taken, then in-vitro lung tissues are fixed in 4 percent paraformaldehyde solution, and H is carried out after slicing&And E, dyeing. Tumors of 21-day mice were also photographed and weighed.
Firstly, in a 4T1 subcutaneous tumor model, the anti-tumor and anti-tumor biological effect of ID-RBCages is investigated. The results show (fig. 13) that in the PBS control experimental group, no difference was observed in tumor growth regardless of whether the tumor site was irradiated with laser light, and the tumor volume was increased by 45.5-fold and 44.4-fold in the PBS group and the PBS illumination group, respectively, within the observation period of 25 days. Tumor growth in Free DOX treated group was slightly reduced compared to PBS group, while illumination had no effect on Free drug group, and the fold growth of tumor in mice in Free DOX treated group under non-illumination condition was 35.5 and 33.2 fold, respectively. The tumor growth of the mice of the ID-RBCages administration group is faster under non-light conditions, the growth trend of the mice is basically consistent with that of the mice of the PBS group, and the tumor growth multiple of the mice of the group is 42.2 times at the 25 th day. The tumor growth of the mice of the ID-RBCages administration group is obviously inhibited under the irradiation of near-infrared laser, the average tumor volume of the mice is gradually reduced within the first 9 days after the light treatment, which indicates that the tumors of the mice are effectively controlled, the tumors of the mice begin to gradually recur and grow at the later time, but the tumor growth is slower, and the average tumor growth is 5.1 times of the original tumor volume at the 25 th day. The ID-RBCages can effectively inhibit the growth of the tumor under the illumination condition, but the inhibition effect cannot realize the complete ablation of the tumor, and the tumor needs to be effectively treated by further changing the administration strategy or combining other antitumor drugs. The ID-RBCages can be used as a high-efficiency photoresponsive anti-tumor drug delivery platform, can realize high-selectivity tumor effective inhibition excited by near infrared light at a tumor part, and is expected to be further combined with other anti-tumor treatment strategies to realize effective treatment of tumors.
Next, the anti-tumor biological effect of ID-RBCages on in situ tumors was evaluated in a 4T1-Luc mouse in situ breast cancer model. Meanwhile, the lung tissue is subjected to in vitro bioluminescence imaging picture shooting when the mouse tumor suppression experiment is finished, and the lung metastasis capability of the ID-RBCages in situ breast cancer is researched. The results show (FIG. 14) that ID-RBCages can effectively inhibit the growth of mouse in-situ breast cancer under the near infrared light illumination condition. After illumination, the tumor volume of the mice in the ID-RBCages treatment group gradually decreased, and the tumors completely disappeared at the 9 th day after administration, but the tumors in the mice in the treatment group subsequently recurred, and the final growth multiple of the tumors was 4.6 times at the 21 st day. The ID-RBCages-Laser/Vc treatment group of mice is used for shielding the photodynamic effect by carrying out intratumor pre-injection on Vc, tumors of the group of mice gradually regress in the first 6 days after the illumination, then the tumors have faster recurrent growth, and the final tumor growth multiple is 11.0 times. The tumor growth of the free drug under illumination and non-illumination conditions is not obviously different from that of the PBS group. The isolated lung tissue bioluminescence imaging result shows that the lung tumor metastasis condition of the mouse in the ID-RBCages illumination group is obviously reduced compared with that of the PBS group, and the lung metastasis inhibition effect of the mouse in the ID-RBCages-Laser/Vc group is poor, so that the photodynamic therapy is favorable for inhibiting the in-situ tumor and plays an important regulation and control role in inhibiting the metastatic tumor.
Example fifteen
To further examine the cell nucleus transport capacity of ID-RBCages after DOX-responsive release at the tissue level, tumor tissue sections before and after illumination were observed separately. As shown in FIG. 15, ID-RBCages were efficiently taken up by tumor cells at the tumor site 24 h after tail vein administration. In the absence of light, the ID-RBCages are distributed predominantly in the cytoplasm of the cell and in lesser amounts in the nucleus. The tumor section also shows that the tumor tissue is relatively complete in shape under the non-illumination condition, and the cells of the tumor section still keep a relatively compact state, which shows that the ID-RBCages has no obvious damage effect on the tumor tissue under the non-illumination condition; the tissue of the tumor after illumination is further subjected to section analysis, and the result shows that the ID-RBCages and cell nuclei in the tissue cells of the tumor after illumination have higher co-localization, which indicates that the ID-RBCages responsively release DOX carried by the tissue cells after illumination, and ROS generated by the ID-RBCages damage the cell nuclear membrane, accelerates the effective transportation of the DOX to the nuclear part of the tumor, and provides favorable guarantee for exerting the effect of photochemical therapy and killing on the tumor cells.
Example sixteen
In order to further investigate the potential application value of ID-RBCages induced tumor immunotherapy, aPD-L1 and ID-RBCages are selected to be used together to investigate the treatment effect of the in-situ breast cancer. The experimental grouping is designed to be PBS, aPD-L1, free I/D/aPD-L1, ID-RBCages/Laser/Vc, ID-RBCages/Laser/aPD-L1 total eight experimental groups. The tumor volume in mice is 70-100 mm 3 The experiment was started by tail vein injection of ID-RBCages, free I/D (ICG, 7.5 mg kg) -1 ) The preparation is administered in mice, and 24 h later, the mice in the light irradiation group are irradiated with laser (0.5W cm) to tumor part with 785 nm laser -2 5 min), aPD-L1 was administered at a dose of 5 mg/kg intraperitoneally on days 2, 5, and 8, respectively. Observing the tumor of the mouse to 21 days, measuring the tumor size of the mouse by using a vernier caliper, killing the mouse at the 21 st day, taking out the lung tissue of the mouse for bioluminescence imaging, fixing the isolated lung tissue in 4% paraformaldehyde solution, slicing and carrying out H&E staining, while tumors of the mice were photographed and weighed.
The results show (FIG. 16), that ID-RBCages-Laser + aPD-L1 can achieve complete ablation of 4T1-Luc breast cancer in situ, and no tumor recurrence occurs within the 21 day observation period. In addition, ID-RBCages-Laser + aPD-L1 shows a strong therapeutic effect on the inhibition of the lung metastasis of the breast cancer, and can completely inhibit the occurrence of the lung metastasis of the breast cancer. The treatment effect of the mice treated by the aPD-L1 is different greatly, the growth of two mice in the group of mice is controlled well, the tumor growth of the rest mice is faster, and the average tumor growth speed of the mice in the whole group is faster. The recombinant human aPD-L1 has no statistical difference with a PBS control group, which indicates that the individual responsiveness of mice has the defect of large difference when the aPD-L1 is used alone, and the treatment effect of the aPD-L1 on the in-situ breast cancer is limited on the whole. When the free drug light irradiation group is used together with the aPD-L1, the tumor growth has no obvious difference compared with the non-combined antibody group, which indicates that the combination of the free drug and the aPD-L1 antibody can not obtain effective enhancement on the treatment effect. This may be related to the fact that the free drug itself cannot effectively induce and activate the anti-tumor immune effect of the mice, so that no synergistic effect can be achieved when the aPD-L1 antibody is used in combination, and the combination effect is not good. The ID-RBCages light group can be used for combining with the aPD-L1 antibody to completely remove the in-situ tumor and effectively inhibit the tumor recurrence, and the probable reason is that the ID-RBCages can cause stronger immunotherapy effect under the light condition and can promote tumor parts to recruit abundant anti-tumor immune cells such as CTLs and the like, so when the aPD-L1 antibody is used simultaneously, the anti-tumor activity can be obviously improved, the high-efficiency cooperation of the light chemotherapy and the immunotherapy is realized, the complete ablation of the in-situ tumor is realized, and the occurrence of tumor lung metastasis is effectively inhibited.
Example seventeen
To investigate the in vivo anti-tumor biological activity of ID-RBCages in Panc02 tumor-bearing mice, the anti-tumor biological effect of ID-RBCages phototherapy-immunotherapy was evaluated using the Panc02 subcutaneous tumor model. A mouse Panc02 pancreatic cancer subcutaneous tumor model is used for tumor inhibition experiment investigation of the treatment effect of ID-RBCages on low-permeability tumors, and 8 different groups are set in total, wherein the groups are respectively as follows: PBS, aPD-L1, free I/D-Laser/aPD-L1, ID-RBCages/aPD-L1, ID-RBCages-Laser/aPD-L1, and GEM + Abraxane. On day 0, tail vein injections of ID-RBCages and Free I/D (7.5 mg kg) -1 ) The light group irradiated the tumor with 785 nm laser (0.5W cm) 24 h after administration -2 4 min). Gem (35 mg kg) was injected into tail vein on days 0, 3, and 6 -1 ）、Abraxane（8 mg kg -1 ) The aPD-L1 antibody (5.0 mg kg) was intraperitoneally injected on days 2, 5, and 8 -1 ). The growth of the mouse tumor is recorded at 0-35 days, and the tumor volume reaches 1500 mm 3 Mice were counted as dead and observed for survival up to 120 days. Two groups of mice were also taken, 20 mice each, one group was inoculated with subcutaneous Panc02 tumor, and the other group was used as empty mice without tumor. The tumor volume of the tumor-inoculated mice is 70 mm 3 Treatment was performed according to the ID-RBCages-Laser/aPD-L1 treatment protocol in the tumor suppressor cohort, and mice with completely ablated tumors were selected on day 60 post-treatment for tumor re-challenge experiments. Namely, the Panc02 cells are inoculated subcutaneously on the opposite side of the first tumor inoculation of the mice (the inoculation cell number is 1 multiplied by 10) 6 One/mouse) and air mice were inoculated with Panc02 as a control. After inoculation, tumor growth was recorded for each of the two groups of mice. Meanwhile, on day 60, six mice with completely ablated tumors and six mice without blanks are selected, and after tumor tissues are taken to prepare single cell suspensions, long-term memory T cells are exemptedThe cells were stained and analyzed using flow cytometry.
As shown in the results of FIGS. 17A-C, after the Panc02 subcutaneous tumors of the ID-RBCages lighting group were lighted, the tumors were ablated on day 13, and then all tumors had recurrences, and the tumors grew rapidly after the recurrence, and the average tumor volume reached 945 mm on day 35 3 . After the tumors of the ID-RBCages light combined aPD-L1 antibody treatment group are ablated, two mice have tumor recurrence on the 21 st day, the tumors of the other three mice have no recurrence, and the average tumor volume of the mice in the group is 113 mm on the 35 th day 3 . Statistics of the mouse survival curves at 120 days post-dose (FIG. 17D) showed that half of the survival time for the ID-RBCages light group was 45 days, significantly extending the survival time of the mice over 27 days for the PBS group, while 60% of the mice survived at 120 days for the ID-RBCages light group in combination with the aPD-L1 antibody treatment group. The combination of ID-RBCages phototherapy and immunotherapy can show good antitumor effect in the treatment of a tumor model of Panc02 subcutaneous tumors. As shown in fig. 18A, fully ablated mice were picked at day 60 post-treatment and inoculated with tumor cells for re-challenge experiments. The result shows that when the mice in the treatment group are inoculated with the tumor cells again, subcutaneous tumors cannot grow, and the result shows that the mice have memory immune effect in vivo and can effectively inhibit the tumor body growth of the tumor cells inoculated again. Further analysis of T cells in the mouse spleen using flow cytometry (fig. 18B-D) showed that mice in the treated group had significantly increased numbers of long term memory T cells as well as central memory T cells, with a TEM count of 3.1-fold in the placebo mice and a TCM count of 1.9-fold in the placebo mice. In conclusion, the ID-RBCages photochemical therapy and immune synergistic treatment can effectively cause long-term immune memory effect in mice and play an important role in preventing the recurrence or metastasis of tumors.
EXAMPLE eighteen
KRAS mutation occurs in more than 90% of human pancreatic cancer tumors. To further evaluate the ability of ID-RBCages to perform a chemo-immune synergistic treatment in KRAS-mutated tumors, a chemotherapeutic agent with bothKRAS and TP53 mutant mouse KPC tumor cells were studied for anti-tumor effects. In tumor inhibition experiments of KPC pancreatic cancer model, combination of the innate immunity STING agonist SR717 and aPD-L1 antibody further enhances the effect of immunotherapy. KPC-Luc cells are used for constructing an in-situ tumor model of the pancreatic cancer of the mouse, and the growth condition of the tumor is monitored by using in-vivo imaging of small animals. And counting the bioluminescence intensity of the live tumor images at days 0, 6, 12, 18 and 24. Specifically, the tumor inhibition experiment of mouse KPC in-situ pancreatic cancer sets up a total of 8 different groups, which are: PBS, SR717+ aPD-L1, GEM + Abraxane + SR717+ aPD-L1, doxil, ID-RBCages-Laser/SR717+ aPD-L1. On day 0, tail vein injections of ID-RBCages and Free I/D (ICG, 7.5 mg kg) -1 ) Entering tumor-bearing mice, after 24 h administration, the mice in the light group were anesthetized and the abdominal cavity was opened by surgery, and the tumor site was irradiated with light (0.5W cm) using a 785 nm laser -2 5 min), and closing the wound after the illumination is finished. SR717+ aPD-L1 combination treatment group was intraperitoneally injected with aPD-L1 antibody (5.0 mg kg) on days 2, 5, and 8 -1 ）、SR717（35 mg kg -1 ). GEM + Abraxane was administered by tail vein injection on days 0, 3, and 6, wherein GEM was administered at a dose of 35 mg kg -1 Abraxane 8 mg kg -1 . And on days 0, 6, 12, 18, 24, their abdominal pancreatic tumors were bioluminescent imaged using a small animal in vivo imaging system to monitor their growth of in situ pancreatic cancer tumors. And survival was recorded for each group of mice over 120 days.
As shown in FIGS. 19A-C, the pancreatic cancer of the PBS group mice grew rapidly, the tumor site bioluminescence intensity of the group mice increased 59.7-fold on day 24, and the other treatment groups, such as SR717+ aPD-L1, GEM + Abraxane and SR717+ aPD-L1+ GEM + Abraxane, had poor treatment effect on KPC in situ pancreatic cancer. Doxil in the clinical drug control group also has no obvious inhibition effect on the treatment of KPC in-situ pancreatic cancer, and the tumor growth multiple of the clinical drug control group is 42.8 times at 24 days. The ID-RBCages light effectively inhibited the growth of pancreatic cancer in mice with a tumor fluorescence intensity increase by a factor of 13.4 at day 24, whereas the combined treatment of ID-RBCages light with SR717 and aPD-L1 resulted in an effective control of pancreatic cancer growth in situ with a tumor growth factor of 3.6 at day 24 and complete inhibition of tumors in two out of five mice. The ID-RBCages photochemical treatment and immunotherapy can play a significant treatment effect on KRAS/TP53 double-mutation KPC pancreatic cancer treatment. Statistical analysis of mouse survival curves for each experimental group (fig. 19D) revealed that mice in each group died completely within 45 days, except for the two groups, the ID-RBCages light-treated group and the ID-RBCages light-combined immunotherapy group. While half of the survival time of the ID-RBCages light-treated mice was 45 days, 60% of the mice in the ID-RBCages light-combined immunotherapy group survived at day 120 after the treatment. The ID-RBCages photochemical therapy immune synergy is shown to be capable of effectively prolonging the survival time of KPC tumor-in-situ mice, and hopes are brought for effective treatment of pancreatic cancer in situ.
Example nineteenth
On the basis of the first embodiment, the volume of the ICG solution is adjusted to obtain the drug-loaded erythrocyte membrane nanoparticles with the molar ratio of ICG to DOX of 1: 0.5 or 1: 2.
Comparative example 1
On the basis of the first embodiment, the step (1) is adjusted as follows:
(1) Resuspending the centrifuged erythrocytes in 50mL of hypotonic PBS buffer (0.25X), and standing in ice bath for 30 min; then, the membrane was washed 3 times with hypotonic PBS buffer (0.25X) to obtain red cell membrane. The rest steps are similar to the embodiment, the solution is kept clear during stirring, the appearance of the solution after the reaction is finished is observed by using a transmission electron microscope, and the result shown in fig. 20A shows that the nanoparticle solution is cell membrane fragments with irregular appearance, no nanometer generation is observed, and the erythrocyte membrane nanoparticles with uniform size cannot be prepared.
Comparative example II
On the basis of the first embodiment, the step (1) is adjusted as follows:
(1) Resuspending the centrifuged erythrocytes in 50mL of hypotonic PBS buffer (0.25X), and stirring at 3500rpm in ice bath for 30 min; then, discarding the supernatant, collecting the bottom layer precipitate, and washing with hypotonic PBS buffer solution (0.25X) for 5 times to obtain erythrocyte membrane; by the method of one example, obvious turbidity appears during the stirring reaction, and the obtained nanoparticles have a small amount of drug layer on the surface, are in a hollow circular ring shape, and cannot effectively encapsulate the drug, as shown in fig. 20B. In contrast, the whole reaction process of the example is clear, and an electron microscope shows that the ID-RBCages nanoparticles can effectively entrap the drug.
The invention successfully realizes the biomimetic synthesis of the erythrocyte membrane nanoparticles (ID-RBCages) carrying ICG and DOX based on the erythrocyte membrane nano-reactor; the main technical progress is as follows:
(1) The ID-RBCages is successfully prepared by carrying clinical antitumor photosensitizer (ICG) and chemotherapeutic Drug (DOX) through a nano erythrocyte membrane and reacting. The average grain diameter of the product is 65.8 +/-5.4 nm, the size of ICG/DOX nano-particles is 5.3 +/-3.8 nm, the appearance is regular, and the grain diameter is uniform.
(2) The ID-RBCages has good photo-thermal and photo-dynamic effects and can realize photo-activated DOX responsive release. After the ID-RBCages are activated by light, the ICD effect of tumor cells can be synergistically enhanced through the photodynamic effect and the chemotherapy effect of DOX, and the potential of enhancing the immunotherapy effect is realized.
(3) The ID-RBCages can be efficiently absorbed by tumor cells and distributed in acidic lysosomes of the cells; the tumor targeting is good, and in a 4T1 tumor subcutaneous tumor model, the tumor part drug distribution of DOX is 15.2 ID% g -1 . The ID-RBCages can be activated by light to release DOX in cells, and the DOX is rapidly transported from lysosomes to cytoplasm and then rapidly enters nuclei to exert the synergistic anti-tumor effect of the light chemotherapy. The ID-RBCages has stronger tissue penetration capability in pancreatic cancer tumors of low-permeability Panc02 mice, can efficiently deliver drugs to deep tumors, can realize intelligent response release of chemotherapeutic drugs and nuclear transport, and can achieve 78.6% of co-localization rate of DOX and nuclei after illumination. The ID-RBCages has good phototherapeutic effect in 4T1 breast cancer subcutaneous and in-situ tumor, can obviously inhibit tumor growth, and can effectively inhibit lung metastasis. The ID-RBCages light chemotherapy-immune synergistic treatment strategy can realize immune cold swellingThe transformation from tumor to immune heat tumor reverses the immunosuppressive microenvironment, completely ablates 4T1 in situ breast cancer, and effectively inhibits lung metastasis. The ID-RBCages combined with the aPD-L1 antibody can effectively inhibit the growth of Panc02 pancreatic cancer subcutaneous tumors. The ID-RBCages light chemotherapy-immune synergistic treatment strategy can degrade extracellular matrix, reverse immunosuppressive microenvironment, enhance tumor infiltration of macromolecular drugs aPD-L1 and CTLs, and realize effective treatment on refractory and low-permeability pancreatic cancer. The ID-RBCages can effectively inhibit the growth of the in-situ pancreatic cancer tumor of the KPC-Luc mouse with double mutations by combining the aPD-L1 and the SR 717. The ID-RBCages light chemotherapy-immune synergistic treatment strategy relieves the inhibition of TP53 mutation on the STING activation of KPC cells, double activates acquired immunity and innate immunity, and realizes the high-efficiency treatment of double-mutation pancreatic cancer by regulating and controlling extracellular matrix and immunosuppressive microenvironment.
Claims (10)
1. A preparation method of drug-loaded erythrocyte membrane nanoparticles is characterized in that erythrocyte membranes and anion reaction precursors are mixed and then extruded out to obtain erythrocyte membrane vesicles of the anion reaction precursors; then mixing the anion reaction precursor erythrocyte membrane vesicles with the cation reaction precursor for reaction to obtain the drug-loaded erythrocyte membrane nanoparticles.
2. The method for preparing the drug-loaded erythrocyte membrane nanoparticle according to claim 1, wherein the erythrocyte membrane is obtained by hypotonic treatment of erythrocytes.
3. The preparation method of the drug-loaded erythrocyte membrane nanoparticle according to claim 2, wherein the erythrocyte is suspended in a hypotonic buffer solution, and the erythrocyte membrane is obtained by centrifugation after standing.
4. The preparation method of the drug-loaded erythrocyte membrane nanoparticle of claim 3, wherein the standing is ice bath standing for 20-40 minutes, and the centrifugal treatment is 3000-4000 g treatment for 10-20 minutes.
5. The preparation method of the drug-loaded erythrocyte membrane nanoparticle according to claim 1, wherein the reaction temperature is 25-55 ℃ and the reaction time is 3-8 h.
6. The preparation method of the drug-loaded erythrocyte membrane nanoparticle according to claim 1, wherein erythrocyte membranes and anion reaction precursors are incubated in hypotonic buffer solution and then extruded by a liposome extruder to obtain anion reaction precursor erythrocyte membrane vesicles; then stirring the anion reaction precursor erythrocyte membrane vesicles and the cation reaction precursor in a hypotonic buffer solution for reaction, and then performing ultrafiltration to obtain the drug-loaded erythrocyte membrane nanoparticles.
7. The preparation method of the drug-loaded erythrocyte membrane nanoparticle of claim 6, wherein the incubation time is 20-40 minutes, the ultrasound treatment is carried out for 3-6 minutes after the incubation, and then the gradient extrusion is carried out by a liposome extruder; the molecular weight cut-off during ultrafiltration is 30-200 kD, the rotating speed is 1200-5000 r/min, and the ultrafiltration frequency is 10-20 times.
8. The preparation method of the drug-loaded erythrocyte membrane nanoparticle of claim 1, wherein the cation reaction precursor is a metal compound or a small molecule drug; the anion reactive precursor source is a sulfide, a citrate compound, or a dye drug.
9. The drug-loaded erythrocyte membrane nanoparticles prepared by the preparation method of the drug-loaded erythrocyte membrane nanoparticles according to claim 1 are characterized in that the particle size of the drug-loaded erythrocyte membrane nanoparticles is 20-240 nm.
10. The application of the drug-loaded erythrocyte membrane nanoparticles of claim 9 in preparing reagents with near-infrared thermal effect and multi-modal imaging function, near-infrared photoresponsive drugs, cell nucleus targeted delivery multifunctional reagents, tumor diagnosis and treatment integrated nano preparations or tumor treatment drugs.
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