CN108815521B - Photosensitive cell membrane bionic targeted nano-drug for tumor combined therapy and preparation thereof - Google Patents
Photosensitive cell membrane bionic targeted nano-drug for tumor combined therapy and preparation thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of nano-medicines, and discloses a photosensitive cell membrane bionic targeted nano-medicine for tumor combination therapy and a preparation method thereof. The invention provides a bionic drug delivery system with bionic characteristics, high drug loading capacity and specificity targeting tumor cells and synergistic anticancer sensitization, which is prepared by using a human O-type erythrocyte membrane as a delivery platform, albumin as a drug carrier, DACHPt as a chemotherapeutic drug, ICG as a photosensitive reagent and RGD as a targeting molecule. Realizes the purpose of tumor chemotherapy and photothermal therapy multi-mechanism combined tumor treatment, and provides a new idea and platform for tumor treatment.
Description
Technical Field
The invention belongs to the technical field of nano-medicines, and particularly relates to a photosensitive cell membrane bionic targeted nano-medicine for tumor combination therapy and a preparation method thereof.
Background
RBCs have many unique characteristics, such as wide circulation range, automatic clearance of aged and damaged RBCs by the reticuloendothelial system, long half-life cycle, optimal surface area to volume ratio, making them candidates for in vivo delivery of many natural or synthetic payloads, red cell membrane carriers have gained wide attention over the past decade, as the most commonly used active agent carrier in blood, commonly referred to as "red cell carrier" or "RBCs carrier", which have good biocompatibility of primary cells, retained a number of membrane proteins that are important factors for their long circulation in vivo, early in 1973, GARRET M et al (G.M.IHLER, H.GLAO R, W.SCHNUREF.Proc.Natl.Acad.Sci. S A, RBC 3,70, prepared red blood cell membrane- α -red cell membrane- α -A. the inventors have also discovered that the stability of red cell membrane- α -red cell membranes is increased after the preparation of red cell membrane- α -red cell membrane carrier, which has been a little disclosed as a rapid-red cell membrane- α -red cell membrane carrier, which has been a conventional red cell membrane carrier with no-red membrane-red cell membrane-red membrane carrier, no longer than the conventional red cell membrane carrier, no-red cell membrane carrier has been prepared by conventional procedures, no-red cell membrane carrier, no-red cell membrane carrier, no-red membrane carrier, no longer, No. 3, No. 3, No. 7, No. 3, No. 3, No. 3, No. 3, No. 3, No. no.
The erythrocyte membrane as a transport vehicle can provide long-circulation characteristics for the drug carrier, but whether the drug carrier has high drug loading capacity, tumor microenvironment responsiveness and biocompatibility is also one of important factors to be considered in drug carrier design. Albumin, the most abundant protein in plasma. It plays an important role in maintaining plasma osmotic pressure and transporting endogenous substances. Many hydrophobic molecules, such as fat-soluble vitamins, hormones, etc., are carried by proteins to achieve blood circulation. The abundant functional groups on the surface of albumin, such as amino, carboxyl and sulfydryl, enable albumin to be compounded with various drugs through covalent bond action, electrostatic action and hydrophobic action. The most successful drug-loading model is that researchers combine paclitaxel, the first anticancer drug, with human serum albumin through hydrophobic interaction to prepare "albumin-bound paclitaxel", which has been clinically used for treating various types of cancer patients and has been approved by the U.S. food and drug administration.
Tumor therapy has now developed into a variety of fields including chemotherapy, radiotherapy, photothermal therapy and magnetic hyperthermia. The chemotherapy has wide application and instant effect, and the photothermal therapy has the advantages of low corrosivity, no repetition and the like. However, monotherapy is often characterized by long treatment cycle and easy induction of tumor drug resistance. Therefore, a treatment mode of chemotherapy/photodynamic therapy combined medication is selected to construct a safe drug carrier capable of carrying a plurality of different action mechanisms, so that the aim of dual-tube treatment is achieved, and the effect of tumor treatment can be further improved.
Divalent platinum compounds are the most widely applied antitumor drugs at present, and the inhibition effect of the cisplatin complexes on cell propagation is discovered since Rosenberg and the like in the sixties of the last century, so that the divalent platinum compounds become common clinical antitumor drugs and are widely applied to treatment of head and neck cancer, lung cancer, lymphoma and other diseases. Bivalent platinum compounds become a conventional means for systematically removing tumor cells after operations and radiotherapy at present, and are even known as penicillin in the field of antitumor drugs. Bivalent platinum compounds inhibit tumor cell proliferation and initiate apoptosis primarily by targeting DNA replication.
Indocyanine green (ICG) was an amphiphilic dye developed by kodak research laboratory in 1955. As a small molecular organic near-infrared dye, the dye is widely applied to diagnosis and treatment of tumors. Photothermal therapy, as a promising method for tumor therapy, has a mechanism that after photosensitizers in tumors are concentrated in lesions, cytotoxic singlet oxygen (singlet oxygen) is generated under the action of laser light with matched absorption wavelength,1O2) Killing tumor cells, destroying the vascular system of the tumor and causing the body to generate certain immune response to the tumor. The damage degree of the photothermal therapy to the tissues is smaller compared with the conventional treatment means such as operation, chemotherapy, radiotherapy and the like. However, indocyanine green has short half-life, is easy to be specifically bound with in vivo lipoprotein, is easy to aggregate in a solution, has poor light stability, causes self-quenching of fluorescence, and limits the application of indocyanine green in the medical field.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a photosensitive cell membrane biomimetic targeted nano-drug for tumor combination therapy.
The invention also aims to provide a preparation method of the photosensitive cell membrane bionic targeted nano-drug for tumor combination therapy.
The purpose of the invention is realized by the following scheme:
a cell membrane bionic light-sensitive targeting nano-drug for tumor combined therapy comprises a human O-type erythrocyte membrane, albumin, (1, 2-diaminocyclohexane) platinum dichloride, indocyanine green (ICG) and arginine-glycine-aspartic acid pentapeptide (RGD), wherein the human O-type erythrocyte membrane is used as a carrying platform, the albumin is used as a drug carrier, (1, 2-diaminocyclohexane) platinum dichloride is used as a chemotherapeutic drug, the indocyanine green (ICG) is used as a light-sensitive reagent, and the arginine-glycine-aspartic acid pentapeptide (RGD) is used as a targeting molecule.
The albumin is preferably Bovine Serum Albumin (BSA) or human serum albumin (FBS), which are all available from sigma company;
the preparation method of the cell membrane bionic targeting nano-drug for tumor combination therapy mainly comprises the following steps:
(1) preparation of carboxyl modified albumin: at room temperature, dissolving albumin in a buffer solution, then adding a 1, 4-dioxane solution of succinic anhydride, carrying out surface modification reaction on albumin, filtering the obtained reaction solution after the reaction is finished, then carrying out centrifugal ultrafiltration on the filtrate, and freeze-drying the liquid in an ultrafiltration tube to obtain carboxyl-modified albumin;
(2) preparation of albumin-indocyanine green nanoparticles: adding Dithiothreitol (DTT), indocyanine green (ICG) and water into the carboxyl modified albumin obtained in the step (1), reacting under an anaerobic condition, carrying out centrifugal ultrafiltration on the obtained reaction liquid after the reaction is finished, and taking the liquid in an ultrafiltration tube, namely albumin-indocyanine green nano particles;
(3) preparation of albumin-loaded DACHPt/indocyanine green nanoparticles: adding (1, 2-diaminocyclohexane) platinum dichloride (DACHPt) water solution into the albumin-indocyanine green nanoparticles obtained in the step (2) for reaction, carrying out centrifugal ultrafiltration on the obtained reaction solution after the reaction is finished, and taking the liquid in an ultrafiltration tube to obtain albumin-loaded DACHPt/indocyanine green nanoparticles which are named as BPtI;
(4) preparation of human O-type erythrocyte membrane: taking human O-type blood whole blood, centrifuging to remove supernatant and leukocyte layer, performing hypotonic treatment on lower layer cells to remove intracellular matrix, and extruding in a polycarbonate porous membrane extruder to obtain erythrocyte membrane vesicles;
(5) preparation of targeting ligand: dissolving arginine-glycine-aspartic acid pentapeptide (RGD) in PBS (phosphate buffer solution) containing tris (2-carboxyethyl) phosphine (TCEP), adding the PBS solution of maleimide-polyethylene glycol-phospholipid (DSPE-PEG-MAL), stirring at room temperature for reaction, centrifugally ultrafiltering the obtained reaction liquid after the reaction is finished, and freeze-drying the liquid in an ultrafiltration tube to obtain the target ligand (RGD-PEG-DSPE) for modifying the erythrocyte membrane;
(6) preparation of targeting ligand modified erythrocyte membranes: mixing the targeting ligand RGD-PEG-DSPE for modifying the erythrocyte membrane with the erythrocyte membrane vesicle in the step (4) at room temperature, vortexing at 800rpm for 20-120s, and standing at 0-25 ℃ for 10-50min to obtain the targeting ligand modified erythrocyte membrane;
(7) preparing a cell membrane bionic targeting nano-drug: uniformly mixing the albumin-loaded DACHPt/indocyanine green nanoparticles prepared in the step (3) and the target ligand-modified erythrocyte membrane obtained in the step (6) in PBS, extruding and coating the mixture in a polycarbonate porous membrane extruder with the thickness of 200nm, dialyzing the mixture in a dialysis bag for 24 hours, and purifying to obtain the photosensitive erythrocyte membrane bionic target nanoparticles named as BPtI @ RBC-R.
The buffer solution in the step (1) is PBS buffer solution with the pH value of 6-9; the concentration of the 1, 4-dioxane solution of succinic anhydride in the step (1) is 2-5 mg/mL; the using amount of the albumin and the succinic anhydride in the step (1) meets the requirement that the mass ratio of the albumin to the succinic anhydride is 1:2-2: 1;
the reaction in the step (1) is carried out at room temperature for 1-4 h;
the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration separation in the step (1) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min;
the mass ratio of the modified albumin and the indocyanine green in the step (2) is 5-20: 1 to 6; the dosage of Dithiothreitol (DTT) in the step (2) is that 4-20mg of DTT is correspondingly added into each 1mg of indocyanine green; the amount of the water in the step (2) is such that 1-10mL of water is correspondingly added to each 1mg of indocyanine green;
the reaction in the step (2) is carried out at 37 ℃ for 1-2h under the anaerobic condition;
the molecular weight cut-off of the ultrafiltration membrane in the centrifugal ultrafiltration in the step (2) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min;
the (1, 2-diaminocyclohexane) platinum dichloride (DACHPt) aqueous solution in the step (3) refers to (1, 2-diaminocyclohexane) platinum dichloride (DACHPt) aqueous solution with the mass concentration of 0.5-1 mg/ml;
the using amount of the albumin-indocyanine green nanoparticles and the (1, 2-diaminocyclohexane) platinum dichloride aqueous solution in the step (3) meets the requirement that the mass ratio of the albumin-indocyanine green nanoparticles to the solute (1, 2-diaminocyclohexane) platinum dichloride (DACHPt) is 2-10: 1;
the reaction in the step (3) is carried out at 25 ℃ for 48-150h;
the molecular weight cut-off of the ultrafiltration membrane in the centrifugal ultrafiltration in the step (3) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min;
the preparation of the human O-type erythrocyte membrane in the step (4) specifically comprises the following steps: centrifuging human O-type blood whole blood at 4 ℃ and 2500rpm for 5min, removing upper layer liquid, adding physiological saline into the lower layer, pumping, centrifuging at 4 ℃ and 2500rpm for 5min, removing supernatant, repeating the steps of adding physiological saline into the lower layer, pumping, centrifuging and removing supernatant twice, and collecting lower layer cell membranes of a centrifuge tube; performing hypotonic treatment on the collected lower cell membrane, namely suspending the cell membrane in 1/4 XPBS, placing the cell membrane on ice for 20min, centrifuging the cell membrane for 5min at 800rcf, removing the upper layer, washing the cell membrane at the bottom layer for 2 times by using 1 XPBS, performing ultrasonic treatment for 5-10min at 53kHz and 100W, and extruding the cell membrane in a polycarbonate porous membrane extruder with the wavelength of 400nm and 200nm in sequence to prepare the erythrocyte membrane vesicle;
the mol ratio of arginine-glycine-aspartic acid pentapeptide (RGD), tris (2-carboxyethyl) phosphine (TCEP) and maleimide-polyethylene glycol-phospholipid (DSPE-PEG-MAL) in the step (5) is 4-7:0.2-0.8: 1-3; the concentration of the PBS solution containing the tris (2-carboxyethyl) phosphine (TCEP) in the step (5) is 0.5-1 mg/ml; the concentration of the PBS solution of maleimide-polyethylene glycol-phospholipid (DSPE-PEG-MAL) in the step (5) is 30-50 mg/ml;
the stirring reaction in the step (5) is a reaction at a speed of 200 and 500rmp for 1 to 5 hours at room temperature.
The molecular weight cutoff of the ultrafiltration membrane in the ultrafiltration separation in the step (5) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min;
the mass ratio of the targeting ligand RGD-PEG-DSPE for modifying the erythrocyte membrane to the erythrocyte membrane vesicle in the step (6) is 0.005: 1;
the mass ratio of the albumin-loaded DACHPt/indocyanine green nanoparticles to the targeting ligand-modified erythrocyte membrane in the step (7) is 1-5: 1-5; the PBS in the step (7) is used in an amount that 5mL of PBS is used for every 1-5mg of albumin-loaded DACHPt/indocyanine green nanoparticles; the cut-off molecular weight of the dialysis bag in the step (7) is 7000-10000 Da;
the room temperature in the present invention means 18 to 28 ℃.
The mechanism of the invention is as follows:
the invention provides a bionic drug delivery system with bionic characteristics, high drug loading capacity and specificity targeting tumor cells and combined anticancer sensitization, which is prepared by using a human O-type erythrocyte membrane as a delivery platform, albumin as a drug carrier, DACHPt as a chemotherapeutic drug, ICG as a photosensitive reagent and RGD as a targeting molecule. Realizes the purpose of tumor chemotherapy and photothermal therapy multi-mechanism combined tumor treatment, and provides a new idea and platform for tumor treatment.
Compared with the prior art, the invention has the following advantages and beneficial effects:
erythrocytes are a natural organism with a long circulation time in vivo, up to 120 days, excellent biocompatibility and low immunogenicity. The erythrocyte is treated by centrifugation, washing, hypotonic treatment and the like to prepare a hollow erythrocyte membrane, and the modified erythrocyte membrane is used as a drug delivery platform, so that a large amount of protein on the surface of the erythrocyte is reserved, and the erythrocyte membrane has the traditional advantages of high biocompatibility, low immunogenicity and long circulation, and also has the characteristics of small size and high entrapment rate.
The research of albumin as a drug carrier is very extensive and common, the invention provides that albumin is functionalized, DACHPt is used as a chemotherapeutic drug, ICG is used as a photosensitive reagent to be loaded on the functionalized albumin, a protein nano drug-loaded inner core with a combined treatment effect of chemical drugs and photothermal therapy is prepared, and then an erythrocyte membrane wraps the albumin drug-loaded inner core, so that the problems of short circulation time, drug leakage and multi-drug resistance in the albumin drug-loaded inner core are effectively solved.
The erythrocyte membrane modified by the targeting agent enables an erythrocyte membrane drug-loading system to have the purpose of specifically targeting tumors, effectively controls the distribution of antitumor drugs in tissues and cells, and has the effects of efficient and accurate drug delivery and site-specific release.
Drawings
FIG. 1 is a schematic diagram of the preparation of BPtI @ RBC-R, wherein (A) the preparation of BIPt; (B) preparation process of BIPt @ RBC-R.
FIG. 2 is a transmission electron micrograph of the nanoparticles of example 1. Wherein A is a transmission electron micrograph of BSA supported DACHPt/indocyanine green nanoparticles (BPtI); b is a transmission electron microscope image of the photosensitive erythrocyte membrane bionic targeted nano-drug BPtI @ RBC-R; c is a transmission electron microscope picture of a photosensitive erythrocyte membrane bionic targeted nano-drug BPtI @ RBC-R after being irradiated by 808nm laser, and picture scales are 500 nm.
FIG. 3 is a graph of the amount of internalization of BPtI, BPtI @ RBC, and BPtI @ RBC-R in macrophages at various drug concentrations in example 1.
FIG. 4 is a graph of cell viability under different operating conditions at different drug concentrations in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
The reagents used in the examples are commercially available without specific reference. Human blood type O in the examples was purchased from blood supply from a hospital blood bank or from blood provided by the patient himself.
Example 1
(1) Preparation of protein drug carrier: dissolving 50mg of bovine serum albumin (BSA, purchased from sigma company) in a phosphate buffer solution with pH of 9 at 25 ℃, then dropwise adding 10ml of 1, 4-dioxane containing 45mg of succinic anhydride to modify the surface of the bovine serum albumin so as to graft carboxyl groups on the surface of the bovine serum albumin as much as possible, reacting for 2 hours at room temperature, filtering the obtained reaction solution after the reaction is finished, placing the obtained filtrate in an ultrafiltration centrifugal tube at 4 ℃, and carrying out centrifugal ultrafiltration, wherein the cut-off molecular weight of the ultrafiltration membrane is 10000KDa, the centrifugation speed is 4000rpm, and the centrifugation time is 20 min; and (3) freeze-drying the liquid in the ultrafiltration tube to obtain the carboxyl modified albumin.
(2) Taking 15mg of the freeze-dried carboxyl modified albumin prepared in the step (1), adding 28mg of DTT and 6mg of indocyanine green (purchased from Tokyo, Japan) into the freeze-dried carboxyl modified albumin, adding 20 ml of pure water, reacting for 1h at 37 ℃ under an anaerobic condition, placing the mixture in an ultrafiltration centrifugal tube at 4 ℃ after the reaction is finished, and carrying out centrifugal ultrafiltration, wherein the cut-off molecular weight of the ultrafiltration membrane is 10000KDa, the centrifugation speed is 4000rpm, and the centrifugation time is 20 min; and taking the liquid in the ultrafiltration tube to obtain bovine serum albumin-indocyanine green nanoparticles (BSA-ICG).
(3) Mixing the bovine serum albumin-indocyanine green nanoparticles obtained in the step (2) with 0.8mg/ml of (1, 2-diaminocyclohexane) platinum dichloride (DACHPt) (purchased from sigma company) water solution according to the mass ratio of the bovine serum albumin-indocyanine green nanoparticles to the (1, 2-diaminocyclohexane) platinum dichloride (DACHPt) of 5:1, reacting for 5 days at 25 ℃, then carrying out centrifugal ultrafiltration on the obtained reaction liquid, wherein the molecular weight cut-off of the ultrafiltration membrane is 10000KDa, the centrifugal speed is 4000rpm, the centrifugal time is 20min, and taking the liquid in the ultrafiltration tube to obtain the BSA-loaded DACHPt/indocyanine green nanoparticles, namely BPtI;
(4) preparation of human O-type erythrocyte membrane: taking 5mL of human O-type blood whole blood, centrifuging at 4 ℃ and 2500rpm for 5min, and removing upper-layer liquid; adding 5mL of normal saline into the lower layer, repeatedly beating, centrifuging at 4 deg.C and 2500rpm for 5min, removing supernatant, repeatedly washing the lower layer cells with normal saline, beating, centrifuging twice, and collecting the lower layer cell membrane. The collected cell membranes were then hypotonic, first suspended in 1/4 × PBS and placed on ice for 20min and centrifuged at 800rcf for 5 min. Removing the upper layer, washing the cell membrane at the bottom layer with 1 × PBS for 2 times, performing ultrasonic treatment on the washed cell membrane for 8min under the condition of 53kHz and 100W, and sequentially extruding the cell membrane in a polycarbonate porous membrane extruder (Avanti miniature extruder) with the particle size of 400nm and 200nm to obtain the erythrocyte membrane vesicle;
(5) preparation of targeting ligand modified erythrocyte membranes: 20mg of arginine-glycine-aspartic pentapeptide (RGD) (purchased from Yaoqian Biotechnology Ltd.) was dissolved in 0.5mL of PBS solution containing 0.5mg of tris (2-carboxyethyl) phosphine (TCEP). Then, 2mL of a PBS solution containing 80mg of maleimide-polyethylene glycol-phospholipid (DSPE-PEG-MAL, molecular weight 5000) was added to the above mixed solution and stirred at room temperature for 4 hours. Placing the obtained reaction solution in an ultrafiltration centrifugal tube at 4 ℃ for centrifugal ultrafiltration, wherein the cut-off molecular weight of the ultrafiltration membrane is 10000KDa, the centrifugation speed is 4000rpm, the centrifugation time is 20min, collecting a product in the ultrafiltration tube, and freeze-drying to obtain the target ligand for modifying the erythrocyte membrane;
(6) and (3) mixing 5mg of targeting ligand for modifying the erythrocyte membrane with 1g of the erythrocyte membrane vesicle prepared in the step (4) at room temperature, vortexing at 800rpm for 80s, and standing at 15 ℃ for 30min to obtain the erythrocyte membrane modified by the targeting ligand.
(7) Preparing a photosensitive cell membrane bionic targeting nano-drug: mixing 5mg of the BSA loaded DACHPt/indocyanine green nanoparticles prepared in the step (3) and 5mg of the targeting ligand modified erythrocyte membrane prepared in the step (6) in 5mL of PBS, extruding and coating the mixture in a polycarbonate porous membrane extruder with the thickness of 200nm, dialyzing the mixture in a dialysis bag (7000Da) for 24 hours for purification, and finally preparing the bionic targeting BSA double drug-loaded system with the erythrocyte membrane, wherein the system is named as BPtI @ RBC-R.
(8) Preparing a photosensitive cell membrane bionic nano-drug: mixing 5mg of the BSA loaded DACHPt/indocyanine green nanoparticles prepared in the step (3) and 5m g of the erythrocyte membrane vesicles prepared in the step (4) in 5mL of PBS, extruding and coating the mixture in a polycarbonate porous membrane extruder with the thickness of 200nm, dialyzing the mixture in a dialysis bag for 24 hours for purification, and finally successfully preparing the photosensitive erythrocyte membrane biomimetic nano-drug, wherein the nano-drug is named as BPtI @ RBC.
The preparation process of BPtI @ RBC-R is schematically shown in FIG. 1, wherein (A) is the preparation process of BIPt; (B) is the preparation process of BIPt @ RBC-R.
The transmission electron microscope image of the nanoparticles prepared in example 1 is shown in fig. 2, wherein a is the transmission electron microscope image of BSA-loaded DACHPt/indocyanine green nanoparticles BPtI; b is a transmission electron microscope image of the photosensitive erythrocyte membrane bionic targeted nano-drug BPtI @ RBC-R; c is a transmission electron microscope picture of a membrane rupture of a photosensitive erythrocyte membrane bionic targeted nano-drug BPtI @ RBC-R after being irradiated by 808nm laser for 5min, and picture scales are all 500 nm. Fig. 2 (a) is a graph of BSA-loaded DACHPt/indocyanine green nanoparticle BPtI, that is, a non-coated BSA dual carrier, with a particle size of about 70nm, and fig. 2 (B) is a transmission electron microscope graph of a photosensitive erythrocyte membrane biomimetic targeted nano-drug, that is, a BSA dual drug-loaded system coated with a nano erythrocyte membrane, with a particle size of about 167nm, which indicates that a photosensitive erythrocyte membrane biomimetic targeted nano-drug with a particle size of about 200nm is successfully prepared. Fig. 2C is a transmission electron microscope image of the light-sensitive erythrocyte membrane biomimetic targeting nano-drug after illumination, wherein the erythrocyte membrane is ruptured after illumination, which shows that the BSA double-drug-loading system coated with nano-erythrocytes of the present invention is helpful to release the coated DACHPt drug and photosensitizer ICG under the effect of laser matching the absorption wavelength.
Adding DACHPt/indocyanine green nanoparticles (BPtI) loaded with BSA (with water as a solvent) with different concentrations, a BSA double-drug-loaded system (BPtI @ RBC) with a bionic erythrocyte membrane and a target BSA double-drug-loaded system (BPtI @ RBC-R) with a bionic erythrocyte membrane into a pore plate (1 ten thousand cells/pore) cultured with macrophage RW264.7 (American ATCC cell bank), cracking the macrophage RW264.7 after 6 hours, and testing the fluorescence intensity in the pore plate to obtain the endocytosis of the BPtI, BPtI @ RBC and BPtI @ RBC-R in the macrophage. As shown in FIG. 3, it can be seen from FIG. 3 that the amount of internalization of BPtI @ RBC and BPtI @ RBC-R encapsulating the erythrocyte membrane in macrophages is much less than that of BPtI without the erythrocyte membrane. The nanoparticles are usually regarded as foreign substances and removed by macrophages after acting in vivo, but the nanoparticles can effectively relieve the removal of the macrophages after wrapping erythrocyte membranes, wherein the wrapping groups have obvious escape phagocytosis capacity, which shows that the nanoparticles have excellent immune escape function after wrapping erythrocyte membranes.
In addition, cytotoxicity tests were also performed. Adding different concentrations (water as solvent) of BSA loaded DACHPt/indocyanine green nanoparticles (BPtI), photosensitive erythrocyte membrane bionic nano-drug (BPtI @ RBC) and photosensitive erythrocyte membrane bionic targeted nano-drug (BPtI @ RBC-R) into a pore plate (1 ten thousand cells/pore) cultured with B16F10 cells (American ATCC cell bank), and treating under illumination conditions (1 w/cm)2808nm) and simultaneously setting photosensitive erythrocyte membrane bionic nano-drugs (BPtI @ RBC) and photosensitive red with different concentrationsCell membrane biomimetic targeted nanoparticle drug (BPtI @ RBC-R) was added to the well plate (1 ten thousand cells/well) in which B16F10 cells (american ATCC bank) were cultured, respectively, for comparative test in dark, after 24 hours, the liquid in the well plate was aspirated, 10 microliters of cytotoxicity test reagent (CCK8) was added for toxicity test, and the graph of fig. 4 was obtained.
As can be seen from fig. 4, under the non-illumination condition, the cell survival rate of the BPtI @ RBC-R group is lower than that of the BPtI @ RBC group, which indicates that the erythrocyte membrane modified by the targeting agent enables the erythrocyte membrane drug-loading system to have the purpose of specifically targeting tumors, effectively controls the distribution of the antitumor drug in tissues and cells, and has the effects of efficient and accurate drug delivery and site-specific release.
As can be seen from FIG. 4, the BPtI @ RBC-R group had the lowest cell survival rate under the illumination condition, indicating that the cytotoxicity of the combination of the chemical drug and the photothermal therapy is significantly improved, and the targeted envelope dual-vector BPtI @ RBC-R has the capability of killing tumor cells more effectively.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A cell membrane bionic light-sensitive targeted nano-drug for tumor combined therapy is characterized by comprising a human O-type erythrocyte membrane, albumin, (1, 2-diaminocyclohexane) platinum dichloride, indocyanine green and arginine-glycine-aspartic acid pentapeptide, wherein the human O-type erythrocyte membrane is used as a carrying platform, the albumin is used as a drug carrier, (1, 2-diaminocyclohexane) platinum dichloride is used as a chemotherapeutic drug, the indocyanine green is used as a light-sensitive agent, and the arginine-glycine-aspartic acid pentapeptide is used as a targeted molecule;
the cell membrane bionic photosensitive targeted nano-drug for tumor combined therapy is prepared by the following method:
(1) preparation of carboxyl modified albumin: at room temperature, dissolving albumin in a buffer solution, then adding a 1, 4-dioxane solution of succinic anhydride, carrying out surface modification reaction on albumin, filtering the obtained reaction solution after the reaction is finished, then carrying out centrifugal ultrafiltration on the filtrate, and freeze-drying the liquid in an ultrafiltration tube to obtain carboxyl-modified albumin;
(2) preparation of albumin-indocyanine green nanoparticles: adding dithiothreitol DTT, indocyanine green ICG and water into the carboxyl modified albumin obtained in the step (1), reacting under an anaerobic condition, carrying out centrifugal ultrafiltration on the obtained reaction liquid after the reaction is finished, and taking the liquid in an ultrafiltration tube, namely albumin-indocyanine green nano particles;
(3) preparation of albumin-loaded DACHPt/indocyanine green nanoparticles: adding a (1, 2-diaminocyclohexane) platinum dichloride aqueous solution into the albumin-indocyanine green nanoparticles obtained in the step (2) for reaction, carrying out centrifugal ultrafiltration on the obtained reaction solution after the reaction is finished, and taking the liquid in an ultrafiltration tube to obtain albumin-loaded DACHPt/indocyanine green nanoparticles;
(4) preparation of human O-type erythrocyte membrane: taking human O-type blood whole blood, centrifuging to remove supernatant and leukocyte layer, performing hypotonic treatment on lower layer cells to remove intracellular matrix, and extruding in a polycarbonate porous membrane extruder to obtain erythrocyte membrane vesicles;
(5) preparation of targeting ligand modified erythrocyte membranes: dissolving arginine-glycine-aspartic acid pentapeptide in a PBS (phosphate buffer solution) containing tris (2-carboxyethyl) phosphine, adding a PBS solution of maleimide-polyethylene glycol-phospholipid, stirring for reaction at room temperature, performing centrifugal ultrafiltration on the obtained reaction solution after the reaction is finished, and freeze-drying the liquid in an ultrafiltration tube to obtain the target ligand for modifying the erythrocyte membrane;
(6) mixing the targeting ligand for modifying the erythrocyte membrane with the erythrocyte membrane vesicle in the step (4) at room temperature, vortexing at 800rpm for 20-120s, and standing at 0-25 ℃ for 10-50min to obtain the erythrocyte membrane modified by the targeting ligand;
(7) preparing a cell membrane bionic targeting nano-drug: uniformly mixing the albumin-loaded DACHPt/indocyanine green nanoparticles prepared in the step (3) and the target ligand-modified erythrocyte membrane obtained in the step (6) in PBS, extruding and coating in a polycarbonate porous membrane extruder with the thickness of 200nm, and dialyzing in a dialysis bag for 24h for purification to obtain the light-sensitive erythrocyte membrane bionic target nanoparticles.
2. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the albumin is bovine serum albumin or human serum albumin.
3. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the buffer solution in the step (1) is PBS buffer solution with the pH value of 6-9; the concentration of the 1, 4-dioxane solution of succinic anhydride in the step (1) is 2-5 mg/mL; the using amount of the albumin and the succinic anhydride in the step (1) meets the requirement that the mass ratio of the albumin to the succinic anhydride is 1:2-2: 1;
the reaction in the step (1) is carried out at room temperature for 1-4 h;
the molecular weight cut-off of the ultrafiltration membrane in the ultrafiltration separation in the step (1) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min.
4. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the mass ratio of the modified albumin and the indocyanine green in the step (2) is 5-20: 1 to 6; the dosage of the DTT in the step (2) meets the condition that 4-20mg of DTT is correspondingly added into each 1mg of indocyanine green; the amount of the water in the step (2) is such that 1-10mL of water is correspondingly added to each 1mg of indocyanine green;
the reaction in the step (2) is carried out at 37 ℃ for 1-2h under the anaerobic condition;
the molecular weight cut-off of the ultrafiltration membrane in the centrifugal ultrafiltration in the step (2) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min.
5. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the (1, 2-diaminocyclohexane) platinum dichloride aqueous solution in the step (3) refers to (1, 2-diaminocyclohexane) platinum dichloride aqueous solution with the mass concentration of 0.5-1 mg/ml;
the using amounts of the albumin-indocyanine green nanoparticles and the (1, 2-diaminocyclohexane) platinum dichloride aqueous solution in the step (3) meet the requirement that the mass ratio of the albumin-indocyanine green nanoparticles to the solute (1, 2-diaminocyclohexane) platinum dichloride is 2-10: 1;
the reaction in the step (3) is carried out at 25 ℃ for 48-150h;
the molecular weight cut-off of the ultrafiltration membrane in the centrifugal ultrafiltration in the step (3) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min.
6. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the preparation of the human O-type erythrocyte membrane in the step (4) specifically comprises the following steps: centrifuging human O-type blood whole blood at 4 ℃ and 2500rpm for 5min, removing upper layer liquid, adding physiological saline into the lower layer, pumping, centrifuging at 4 ℃ and 2500rpm for 5min, removing supernatant, repeating the steps of adding physiological saline into the lower layer, pumping, centrifuging and removing supernatant twice, and collecting lower layer cell membranes of a centrifuge tube; and (3) performing hypotonic treatment on the collected lower cell membrane, namely suspending the cell membrane in 1/4 XPBS (poly (butylene succinate)), placing the cell membrane on ice for 20min, centrifuging the cell membrane for 5min at 800rcf, removing the upper layer, washing the cell membrane at the bottom layer for 2 times by using 1 XPBS, performing ultrasonic treatment for 5 to 10min at 53kHz and 100W, and extruding the cell membrane in a polycarbonate porous membrane extruder with the wavelength of 400nm and the wavelength of 200nm in sequence to prepare the erythrocyte membrane vesicle.
7. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the mol ratio of the arginine-glycine-aspartic acid pentapeptide, the tri (2-carboxyethyl) phosphine and the maleimide-polyethylene glycol-phospholipid in the step (5) is 4-7:0.2-0.8: 1-3; the concentration of the PBS solution containing the tris (2-carboxyethyl) phosphine in the step (5) is 0.5-1 mg/ml; the concentration of the maleimide-polyethylene glycol-phospholipid PBS solution in the step (5) is 30-50 mg/ml;
the stirring reaction in the step (5) refers to a reaction at a speed of 200 and 500rmp for 1-5h at room temperature;
the molecular weight cutoff of the ultrafiltration membrane in the ultrafiltration separation in the step (5) is 3500-20000 KDa; the centrifugation speed is 2000-5000 rpm, and the centrifugation time is 5-30 min;
the mass ratio of the targeting ligand for modifying the erythrocyte membrane to the erythrocyte membrane vesicle in the step (6) is 0.005: 1.
8. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to claim 1, characterized in that:
the mass ratio of the albumin-loaded DACHPt/indocyanine green nanoparticles to the targeting ligand-modified erythrocyte membrane in the step (7) is 1-5: 1-5; the PBS in the step (7) is used in an amount that 5mL of PBS is used for every 1-5mg of albumin-loaded DACHPt/indocyanine green nanoparticles; the cut-off molecular weight of the dialysis bag described in the step (7) is 7000-10000 Da.
9. The cell membrane biomimetic light-sensitive targeted nano-drug for tumor combination therapy according to any one of claims 1-8, characterized in that:
the room temperature stated in steps (1) to (7) is 18 to 28 ℃.
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