CN115920081A - Nano prodrug with spontaneous directional coating of red cell membrane and ROS response and application of nano prodrug - Google Patents
Nano prodrug with spontaneous directional coating of red cell membrane and ROS response and application of nano prodrug Download PDFInfo
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- CN115920081A CN115920081A CN202211361359.2A CN202211361359A CN115920081A CN 115920081 A CN115920081 A CN 115920081A CN 202211361359 A CN202211361359 A CN 202211361359A CN 115920081 A CN115920081 A CN 115920081A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a nano prodrug with a spontaneous directional envelope of an erythrocyte membrane and ROS response and application thereof. The preparation method of the nano prodrug comprises the following steps: HO-PEG modified with oxalyl chloride 2K -Mal coupling lovastatin with polypeptide ligand P4.2 to obtain LVTNPs; and then performing co-incubation on the LVTNPs and the erythrocyte membrane to prepare the nano prodrug. The invention creatively uses the natural affinity between the mouse polypeptide ligands P4.2 and band3 as a connection mode for constructing the nano drug-carrying carrier, and provides a feasible and feasible method for directionally assembling inner leaves of the cell membrane bionic nano carrierAnd (4) innovative strategies. Meanwhile, the invention also provides a simple mixing method, and the nano prodrug with the cell membrane orientation direction can be spontaneously and efficiently prepared without using an additional co-extrusion method. The nano prodrug provided by the invention can effectively treat endothelial cell injury.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a nano prodrug with a spontaneous directional envelope of an erythrocyte membrane and ROS response and application thereof.
Background
The cell membrane bionic nano-carrier is a drug coated by utilizing the cell membrane of endogenous cells, aims to prolong the in vivo circulation time of the drug, improve the targeting property and biocompatibility of drug delivery, and endows the source cells with corresponding functions, and is one of the emerging directions in the field of drug targeted delivery in recent years. Research shows that cell membrane assembly nano-carriers generally use methods such as freeze thawing, ultrasound, extrusion and electric shock, but the assembly success rate and the loss rate are low due to the steps in the assembly process. In addition, because of the asymmetric biological characteristics of the cell membrane, how the bionic cell membrane nano-carrier realizes efficient spontaneous assembly and how to assemble in the correct direction is particularly important for further research and application of the cell membrane bionic nano-carrier.
Besides the need for further research on spontaneous and directed assembly of cell membrane biomimetic nanocarriers, accurate release of the following nanomedicine is another key challenge for effective treatment. The strong coupling effect between the cell membrane and the nano carrier is beneficial to improving the drug delivery capability of the carrier, reducing the premature release of the drug in blood circulation and obviously improving the treatment effect of pathological changes. The drug release strategy triggered by endogenous pathological stimulation can adaptively regulate and trigger local drug release so as to improve the final treatment effect and reduce toxic and side effects to the minimum. Therefore, there is a need to further optimize core nanomedicines to respond to endogenous stimuli, release the drugs "on demand" in the lesions, and develop prodrugs to increase the pharmacokinetic efficiency of the drugs based on the pathological stimuli. Endothelial cell damage and dysfunction are early events in the development of cardiovascular disease. Maintaining and restoring the integrity of endothelial cells is essential for the restoration of vascular function and disease caused by endothelial cells. In clinical treatment, anti-inflammatory and antihypertensive medicines taken orally for a long time are commonly used for restoring the functions of damaged endothelial cells, but have the problems of low bioavailability, serious toxic and side effects and the like.
Su 26104, et al, in "study of interaction of Protein4.2 with other proteins on human red blood cell membranes": protein4.2 (P4.2 or band 4.2) is an important peripheral membrane protein on the membrane of human erythrocytes, and the P4.2 gene is located in the q15 segment of human chromosome 15, is approximately 20Kb in length, contains 13 exons coding for 691 amino acids, and has a theoretical molecular weight of 76, 841Da. P4.2 can interact directly with band3, ankyrin and spectrin, and P4.2 can bind to Protein 4.1, CD47, etc., binding Rh complex into the cytoskeletal network of erythrocytes. Band3 is one of the most important proteins on the membrane of human erythrocyte, and the cytoplasmic segment (cdb 3) of the Band interacts with cytoskeletal proteins ankyrin, protein 4.1 and protein4.2 and the like to regulate the interaction between the cell membrane and the cytoskeleton. However, the paper mainly studies the mechanism and mode of interaction between P4.2 and ankyrin, band3, cdb3, and does not relate to nanomaterial or drug delivery vehicle design.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a composition for preparing a cell membrane biomimetic nanocarrier with spontaneous directional assembly.
In order to achieve the purpose, the invention adopts the following technical scheme:
the composition for preparing the cell membrane bionic nano-carrier with the spontaneous directional assembly consists of an erythrocyte-containing membrane, an internal-loaded drug and a polypeptide ligand P4.2; the erythrocyte membrane contains the transmembrane protein band 3.
The internal medicine comprises any one or more of lovastatin, simvastatin, pravastatin, atorvastatin, rosuvastatin, rapamycin and docetaxel.
The internal drug is preferably lovastatin.
The second objective of the present invention is to provide a cell membrane bionic nano-carrier which is spontaneously directionally assembled.
In order to achieve the purpose, the invention adopts the following technical scheme:
a cell membrane biomimetic nanocarrier assembled in a spontaneous orientation, the cell membrane biomimetic nanocarrier comprising the composition; the erythrocyte membrane is a shell and coats the internal drug and the polypeptide ligand P4.2; the polypeptide ligand P4.2 specifically binds to the transmembrane protein band3 in the erythrocyte membrane.
The invention provides a feasible and innovative cell membrane spontaneous orientation strategy, adopts a polypeptide ligand P4.2 with high affinity with the intracellular domain of a transmembrane protein band3 of a Red Blood Cell (RBC), and directionally assembles the polypeptide ligand on a core nano carrier by the specific combination of the intracellular fragment of the band3 and the P4.2.
Further, the amino acid sequence of the polypeptide ligand P4.2 is shown as SEQ ID NO:1 is shown.
Further, the polypeptide ligand P4.2 is of murine origin.
The invention also aims to provide a nano prodrug prepared by using the cell membrane bionic nano carrier.
In order to achieve the purpose, the invention adopts the following technical scheme:
the nano prodrug is prepared by utilizing the cell membrane bionic nano carrier, and is an ROS-responsive prodrug.
The fourth purpose of the present invention is to provide a preparation method of the nano prodrug.
In order to realize the purpose, the invention adopts the following technical scheme:
the preparation method of the nano prodrug comprises the following steps:
s1: HO-PEG modified with oxalyl chloride 2K -Mal coupling said lovastatin to said polypeptide ligand P4.2 to produce LVTNPs;
s2: and co-incubating LVTNPs prepared by S1 and the erythrocyte membrane to prepare the nano prodrug.
Further, S1 specifically is:
(1) Dissolving the lovastatin by using dichloromethane, and adding oxalyl chloride for reaction to obtain a reaction solution 1;
(2) Removing redundant oxalyl chloride in the reaction solution 1 obtained in the step (1) through rotary evaporation, and redissolving to obtain a redissolution;
(3) Adding HO-PEG into the redissolution obtained in the step (2) 2K -Mal reacting to obtain a reaction solution 2;
(4) Carrying out rotary evaporation on the reaction solution 2 obtained in the step (3), and then adding a polypeptide ligand P4.2 and DMSO for reaction to obtain a reaction solution 3;
(5) And (5) dialyzing and drying the reaction liquid 3 obtained in the step (4) to obtain LVTNPs.
Further, in the step (1), the reaction time was 4 hours.
Further, in the step (2), the redissolving agent is dichloromethane.
Further, the reaction conditions in the step (3) are as follows: the reaction was carried out at room temperature overnight.
Further, the reaction conditions in the step (4) are as follows: the reaction was carried out at 40 ℃ overnight.
Further, in the step (5), the dialysis time was 4 hours.
Further, in the step (5), dialysis was carried out using a dialysis bag having a molecular weight of 3500 Da.
Further, in S2, the incubation temperature was 37 ℃ and the incubation time was 30min.
Further, the extraction method of the erythrocyte membrane comprises the following steps: blood is taken from eyeballs after mice are anesthetized, and the erythrocyte cyst membrane is extracted by a hypotonic and repeated freeze-thawing method.
The fifth purpose of the invention is to provide the application of the combination of the polypeptide ligand P4.2 and the transmembrane protein band3 in the preparation of the cell membrane bionic nano-carrier with spontaneous directional assembly.
The invention also aims to provide the application of the combination of the polypeptide ligand P4.2 and the transmembrane protein band3 in preparing the nano prodrug capable of spontaneously directionally coating red blood cell membrane and responding to ROS.
The seventh purpose of the present invention is to provide an application of the composition, the cell membrane biomimetic nano-carrier and/or the nano-prodrug in preparation of a drug for vascular endothelial injury repair.
The eighth purpose of the present invention is to provide an application of the nano prodrug in preparation of a medicine for inhibiting uptake of macrophages.
The ninth purpose of the present invention is to provide an application of the nano prodrug in preparing drugs for inhibiting vascular endothelial cell proliferation, and reducing vascular permeability and inflammatory reaction.
The nano prodrug coated by the erythrocyte membrane and responding to the bionic ROS is used for repairing vascular endothelial injury. Specifically, the method comprises the following steps: lovastatin (LVT) has the effects of improving endothelial cell function, increasing NO production, inhibiting inflammatory cytokines, and is therefore selected as a model drug for the construction of prodrugs, functionalized in a manner of lateral directed conjugation. HO-PEG modified with Oxalyl Chloride (OC) 2K Mal couples the hydrophilic prodrug LVT to the P4.2 polypeptide. The RBC-coated LVTNPs (RBC-LVTNPs) provided by the patent spontaneously self-assemble in the inner lobe of the erythrocyte membrane in a directional way through the specific affinity between the intracellular domain of the key transmembrane protein band3 on the erythrocyte membrane and the corresponding P4.2 polypeptide modified nano-drug without using an additional co-extrusion method. The nanometer prodrug can be stimulated by high-concentration ROS at an endothelial injury part to trigger the release of the prodrug, so that the efficient repair of injured endothelial cells is promoted.
The invention has the beneficial effects that:
1. the invention provides a feasible and innovative strategy for directional assembly of inner leaves of a cell membrane bionic nano-carrier, and the invention creatively adopts a polypeptide ligand P4.2 with high affinity with the intracellular domain of the red cell membrane transmembrane protein band3, and the directional assembly is carried out on the core nano-carrier through the specific combination of the intracellular fragment of the band3 and the P4.2. Particularly, an effective solution is provided for the condition of lacking affinity between some cell membrane-core nano carriers;
2. the patent uses natural affinity between a polypeptide ligand P4.2 and band3 of a mouse source as an innovative connection mode for constructing a nano drug-carrying carrier, and connects a prodrug and an ROS sensitive chemical bond by using an erythrocyte membrane to coat the P4.2 polypeptide and a high molecular chain, so that the prodrug can be accurately released at a focus position, the drug utilization rate and the biological safety are improved, and the drug has a good curative effect of promoting damaged endothelium repair;
3. the invention can spontaneously and efficiently prepare the nano prodrug with the cell membrane orientation direction by a simple and convenient mixing method without using an additional co-extrusion method, the nano prodrug is stimulated by high-concentration ROS at the endothelial injury part to trigger the release of the prodrug, the cumulative drug release rate of the nano prodrug in PBS under the stimulation of ROS reaches 71.2 percent, and the efficient repair of the injured endothelial cells can be effectively promoted;
4. the nano prodrug provided by the invention not only has good cell compatibility and blood compatibility, but also can obviously inhibit the uptake of macrophages, and has favorable effects on prolonging the blood circulation time in the administration process, reducing undesirable clearance, increasing blood exposure and the like;
5. the nano prodrug provided by the invention can effectively treat endothelial cell injury, and remarkably reduces undesirable accumulation of nanoparticles in main organs, thereby reducing non-specific toxicity and improving in-vivo biocompatibility;
6. the nano prodrug provided by the invention has good treatment safety in vivo and is expected to become a promising and feasible nano treatment method for repairing endothelial cell injury;
7. the nano prodrug provided by the invention can effectively inhibit the proliferation of vascular endothelial cells, reduce vascular permeability and inflammatory reaction, recover the barrier and function of the endothelial cells and effectively repair damaged endothelial cell monolayers.
Drawings
FIG. 1 is a partial characterization of RBC-LVTNPs, wherein FIG. 1A is the mass ratio of LVTNPs to erythrocyte membranes; FIG. 1B is the hydrodynamic diameters of LVTNPs, RBC-LVTNPs and RBC-LVTNPs; FIG. 1C is the Zeta potential;
FIG. 2 is an electron microscope image of LVTNPs, RBC membrane, eRBC-LVTNPs and RBC-LVTNPs at different magnification;
FIG. 3 is a Western blot analysis of CD47 in RBC, RBC membrane, eRBC-LVTNPs and RBC-LVTNPs;
FIG. 4 is a flow cytometric analysis of DiD-RBC membrane and FITC-P4.2 polypeptide-modified LVTNPs;
FIG. 5 is a typical confocal image of DiD-RBC membrane with FITC-P4.2 polypeptide;
FIG. 6 is an XYZ-axis showing co-localization of erythrocyte membranes with P4.2 polypeptide in different directions;
FIG. 7 is a partial characterization of RBC-LVTNPs; wherein, FIG. 7A is the emission spectra of the probe RBC-LVTNPs and probe RBC-PLGA before and after adding ssDNA quencher to PBS under 480nm excitation, and FIG. 7B is the membrane orientation of the probe RBC-LVTNPs and probe RBC-PLGA quantified by FRET;
FIG. 8 is an electron microscope image of RBC-LVTNPs with or without hydrogen peroxide at various times;
FIG. 9 is a graph of the drug release profiles of LVTNPs and RBC-LVTNPs with and without hydrogen peroxide at different times;
FIG. 10 is a representative confocal image of RAW264.7 macrophage uptake of LVTNPs and RBC-LVTNPs;
FIG. 11 flow cytometry analysis of the uptake of LVTNPs and RBC-LVTNPs by RAW264.7 macrophages; wherein, fig. 11A is the result of the uptake of LVTNPs by RAW264.7 macrophages; FIG. 11B shows the RBC-LVTNPs uptake by RAW264.7 macrophages;
FIG. 12 is a quantitative analysis of RAW264.7 macrophage uptake of LVTNPs and RBC-LVTNPs;
FIG. 13A is a diagram of a parallel flow chamber mechanical loading apparatus model; FIG. 13B is a confocal image of endothelial cell uptake of LVTNPs and RBC-LVTNPs;
FIG. 14 is a flow cytometric analysis of endothelial cell uptake of LVTNPs and RBC-LVTNPs;
FIG. 15 is a quantitative analysis of endothelial cell uptake of LVTNPs and RBC-LVTNPs;
FIG. 16 is white sheets of culture medium, LVT, LVTNPs and RBC-LVTNPs treated for HUVEC migration at 0 and 24h, respectively, wherein FIG. 16A is white sheets of culture medium treated for HUVEC migration at 0 and 24 h; FIG. 16B is a white patch of LVT treated HUVEC migration at 0 and 24 h; FIG. 16C is a white patch of LVTNPs treated for HUVEC migration at 0 and 24 h; FIG. 16D is white disc of HUVEC migration treated with RBC-LVTNPs at 0 and 24 h;
FIG. 17 shows the migration of HUVECs treated with medium, LVT, LVTNPs and RBC-LVTNPs at 0 and 24 h; wherein, FIG. 17A is the migration of medium treated HUVEC at 0 and 24 h; FIG. 17B shows LVT treated HUVEC migration at 0 and 24 h; FIG. 17C shows the migration of LVTNPs treated HUVEC at 0 and 24 h; FIG. 17D shows the migration of HUVEC treated with RBC-LVTNPs at 0 and 24 h;
FIG. 18 shows cell viability of ECs, SMCs, and RAW264.7 cells after 24h incubation with different doses of free LVT, LVTNPs, eRBC-LVTNPs, and RBC-LVTNPs.
FIG. 19 is a hemolysis assay using LVT, LVTNPs, eRBC-LVTNPs and RBC-LVTNPs;
FIG. 20 is a graph of the quantitative analysis of absorbance at 540nm for free LVT, LVTNPs, eRBC-LVTNPs and RBC-LVTNPs;
FIG. 21 is a fluorescence image of the left and right carotid arteries of a small animal;
FIG. 22 is a quantitative analysis of LCA and RCA treated with LVTNPs and RBC-LVTNPs;
FIG. 23 is an in vitro fluorescence image of each organ;
FIG. 24 is an in vivo study, specifically a quantitative analysis, of the promotion of endothelial repair by RBC-LVTNPs;
FIG. 25 is a pharmacokinetic study of LVTNPs and RBC-LVTNPs;
FIG. 26 is H & E staining of carotid sections of mice treated for 1 and 5 days, where "I" represents intima and "M" represents media;
FIG. 27 is a graph quantifying the number of endothelial cells in the "I" region of the neointima stained with H & E;
FIG. 28 is a graph of SMC number in the "M" region of the membrane in the quantitative analysis;
FIG. 29 is an analysis of RBC, WBC, ALP, ALT, BUN and CK in blood or serum of mice after 1 and 5 days of different treatments; fig. 29A: RBC, fig. 29B: WBC, fig. 29C: ALT, fig. 29D: ALP, fig. 29E: BUN, fig. 29F: CK;
FIG. 30 shows H & E staining of mouse major organs.
Detailed Description
The technical solution of the present invention will be further clearly and completely described with reference to the following specific examples. It should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by those skilled in the art without inventive efforts shall fall within the scope of the present invention.
In the following examples, polypeptide P4.2 was purchased from Nanjing peptide industries, inc., and the amino acid sequence of polypeptide P4.2 is shown in SEQ ID NO:1 is shown in the specification; HO-PEG 2K Mal purchased from pengcheng rich biology ltd; oxalyl chloride and Lovastatin were purchased from Maxam Biotechnology Ltd; antibodies and other related reagents were purchased from domestic suppliers and used as per the instructions.
Example 1 ROS-responsive Nanopropiodrug Synthesis
40.4mg of LVT is weighed and added into a round-bottom flask, 5mL of dichloromethane is added to dissolve the medicine, 39 μ L of OC is added, stirring is started on ice, and the reaction solution is reacted for 4 hours to obtain reaction solution 1. And (4) after the reaction is finished, performing rotary evaporation until excessive OC is evaporated, and adding dichloromethane for redissolving to obtain LVT-OC (redissolution solution). Weighing 200mg of HO-PEG 2K Adding Mal into LVT-OC, reacting overnight at room temperature to obtain LVT-OC-PEG 2K Mal (reaction solution 2). Weighing 98mg of polypeptide and LVT-OC-PEG 2K -Mal, adding 5mL DMSO, reacting overnight at 40 ℃ to obtain reaction solution 3, and dialyzing with a dialysis bag with molecular weight of 3500Da for 4h. Finally, the sample is freeze-dried to obtain the LVT-PEG 2K P, abbreviated as LVTNPs.
Example 2 Synthesis of ROS-responsive Nanopropiodrugs spontaneously directed coating of Red blood cell membranes
Anesthetizing mice, taking blood from eyeballs, extracting erythrocyte membranes by using a hypotonic and repeated freeze-thawing method, and incubating the LVTNPs prepared in the embodiment 1 and erythrocyte membranes at 37 ℃ for 30min to prepare the nano prodrug (RBC-LVTNPs for short).
Comparative example 1
As a control group, edrc-LVTNPs were prepared by coextruding the LVTNPs prepared in example 1 with a red blood cell envelope.
Example 3 preparation and characterization of RBC-LVTNPs
For the synthesis of ROS sensitive polymeric prodrugs, HO-PEG was paired with OC 2K Acylation of MAL followed by coupling with LVT to give LVT-PEG 2K Mal, confirmed by 1H NMR. P4.2 is a cytoskeletal protein in erythrocytes, composed of multiple fragments, with specific affinity for the intracellular domain of band 3. To further orient the assembly of the polymeric prodrug in the correct orientation in the lobules of the erythrocyte membrane, LFVRRGQPFTIILYC was further combined with LVT-PEG 2K Mal reaction, michael reaction between maleimide and thiol, successfully confirmed the synthesis of a functional polymer prodrug by MALDI-TOF-MS spectroscopy (LVT-PEG) 2K -P). Unlike the conventional method, the present invention efficiently, simply and conveniently prepares RBC-LVTNPs by self-assembly of co-incubated erythrocyte membranes and LVTNPs without any additional treatment using co-extrusion or ultrasound. To further investigate the spontaneous assembly of red cell membranes with LVTNPs without co-extrusion. In an aqueous solution of erythrocyte membranes and LVTNPs, dh of RBC-LVTNPs decreases with increasing mass ratio of LVTNPs to erythrocyte membranes until a ratio of 3:1, and maintains a stable Dh value at a higher mass ratio. Therefore, a mass ratio of 3:1 RBC-LVTNPs and erythrocyte membranes for all subsequent studies, see FIG. 1A for details.
LVTNPs, co-extrusion of erythrocyte membranes and LVTNPs (eRBC-LVTNPs) and Dh and Zeta potentials of RBC-LVTNPs were characterized. As shown in FIGS. 1B and 1C, the mean Dh of RBC-LVTNPs was 210. + -. 2.5nm, the Zeta potential was-26.3 mV, and it is very close to that of eRBC-LVTNPs. In order to further confirm the appearance and appearance, LVTNPs, erythrocyte membranes, eRBC-LVTNPs and RBC-LVTNPs were detected by a transmission electron microscope. The LVTNPs are uniform spheres and are uniformly dispersed, the erythrocyte membrane presents irregular shapes, and the eRBC-LVTNPs and the RBC-LVTNPs present uniform nanospheres with the diameter of about 200 nm. In particular, the pronounced corona layer on the surface of RBC-LVTNPs indicates that the erythrocyte membranes successfully coat LVTNPs even without the co-extrusion method. Importantly, the corona layer thickness of RBC-LVTNPs is much thinner than that of ebbclvtnps, which is likely due to the repeated co-extrusion of the ebbclvtnps resulting in a thicker outer layer, as detailed in fig. 2. In addition, western blot analysis showed that CD47 functional protein was well retained in RBC-LVTNPs, as shown in FIG. 3.
In order to quantitatively detect the content of erythrocyte membranes and P4.2 polypeptides on RBC-LVTNPs, a double-fluorescence labeling technology is adopted to co-locate DID labeled erythrocyte membranes and FITC labeled P4.2 polypeptides. Under a confocal laser microscope, the P4.2 polypeptide and an erythrocyte membrane have better co-localization, as shown in figure 5; further processing the 3D view image with Imaris to reveal details of the co-localization of the erythrocyte membrane and the P4.2 polypeptide; as shown in fig. 6, XYZ axes show that the erythrocyte membrane is co-localized with the P4.2 polypeptide in different directions. Meanwhile, flow cytometry analysis further shows that the double positive rates of the eRBC-LVTNPs and the RBC-LVTNPs reach 70.9% and 68.4% respectively, as shown in FIG. 4, which indicates that band3 on an erythrocyte membrane is successfully combined with P4.2 peptide of the LVTNPs. In order to further determine the correct orientation of the erythrocyte membrane on the nanoparticle, a probe RBC-LVTNPs is detected by a method for marking the outer surface of the erythrocyte membrane, and the erythrocyte membrane coated PLGA nanoparticle prepared by the traditional co-extrusion method is taken as a typical bionic nanoparticle as a negative control. As shown in FIG. 7, by co-extrusion, 92.8% of the membranes in the probe RBC-LVTNPs retained the correct orientation, while only 51.3% of the membranes in the probe RBC-PLGA retained the correct orientation. This indicates that most RBC-LVTNPs can possess the correct orientation without co-extrusion. The results prove that the RBC-LVTNPs with the cell membrane orientation direction can be prepared spontaneously and efficiently by a simple mixing method.
Example 3 safety test
For the products prepared in examples 1-2 and comparative example 1, the viability of the cells was determined by MTT method, which comprises inoculating HUVEC, RAW264.7, SMCs in 96-well plates, adding LVT, LVTNPs, edrbc-LVTNPs and RBC-LVTNPs at different concentrations in serum-free medium, incubating for 24h, and determining the viability of each group by uv spectrophotometer.
Before in vivo administration treatment, the safety and good blood compatibility of nano-drugs are basic prerequisites for further application. In the cell compatibility study, this example systematically studied the cytotoxic effects of LVT, LVTNPs prepared in example 1, eRBC-LVTNPs prepared in comparative example 1, and RBC-LVTNPs prepared in example 2 on endothelial cells, smooth muscle cells, and macrophages. As shown in FIG. 18, after incubation of cells with LVT, LVTNPs, eRBC-LVTNPs and RBC-LVTNPs at concentrations of 20, 40, 60, 80 and 100. Mu.g/mL for 24h, good cell viability was detected even at the same drug concentration of 100. Mu.g/mL, suggesting that LVTNPs, eRBC-LVTNPs and RBC-LVTNPs have good cell compatibility.
Example 4 drug Release test
To study ROS-responsive prodrug release, this example measured drug release by dialysis, LVTNPs obtained in example 1 and RBC-LVTNPs obtained in example 2 were added with or without H, respectively 2 O 2 Dialysis bags (MWCO: 3500 Da) (500. Mu.M) and soaked in 50mL of release medium. 2mL of dialysis release fluid was collected at regular intervals and an equal amount of fresh medium was added. The drug release concentration was calculated by uv spectrophotometry at a wavelength of 280nm according to a standard curve.
This example is carried out with or without H 2 O 2 The release efficiency of LVTNPs and RBC-LVTNPs was tested in PBS (Amersham pharmacia Biotech). As shown in fig. 8, and without H 2 O 2 Comparison of stimulated Stable Nanomorphs, transmission Electron microscopy images showed that most RBC-LVTNPs were in H 2 O 2 Gradual destruction under stimulation, which is attributed to the breakdown of OC bonds in LVTNPs induced by ROS stimulation leading to poor thermodynamic stability. As shown in FIG. 9, the cumulative drug release rate of RBC-LVTNPs in PBS significantly increased from 35.5% to 71.2% under ROS stimulation.
Example 5 cell uptake assay
To test the ability of the RBC-LVTNPs prepared by the present invention to evade immune cell uptake in vivo, the following experiment was performed in this example:
one, RAW264.7 is added at a rate of 1X 10 per well 5 Cell density was inoculated in 10-vol% FBS-containing DMEM high-sugar medium at 37 ℃ with 5% CO 2 Culturing for 24h under the condition, and then adding FITC labeled RBC and FITC labeled RBC-LVTNPs respectively to incubate for different times. Washed 3 times with 1 × PBS and fixed in 4% paraformaldehyde at room temperature for 30min. Staining with DAPI for 15min, andthe cell morphology was observed by confocal laser microscopy.
(II) HUVEC at 1X 10 per well 5 Cell density was inoculated into 10% fetal bovine serum-containing RPMI1640 culture medium, at 37 ℃ with 5% CO 2 Culturing for 24h, adding FITC-labeled LVTNPs and FITC-labeled RBC-LVTNPs into each well, incubating for 3h, and observing cell morphology by using a confocal laser microscope.
The results confirmed that: the erythrocyte membrane-wrapped nano prodrug can inhibit the uptake of macrophages and prolong the long-term blood circulation of the nano prodrug. As shown in fig. 10, uptake of FITC-labeled LVTNPs and FITC-labeled RBC-LVTNPs was detected in RAW264.7 macrophages, and the fluorescence intensity of RBC-LVTNPs taken up by macrophages was significantly reduced compared to LVTNPs. As shown in FIGS. 11-12, the flow cytometry analysis results further confirmed that the LVTNPs had 1.2, 8.3, 2.6 and 20-fold higher fluorescence intensities at 0.5h, 1h, 2h and 4h, respectively, than the RBC-LVTNPs. The results indicate that RBC-LVTNPs can significantly inhibit macrophage uptake, which is highly advantageous for prolonging blood circulation time during dosing, reducing poor clearance and increasing blood exposure.
And (3) detecting the uptake behaviors of the endothelial cells to the LVTNPs and the RBC-LVTNPs by adopting a laser confocal microscope, and quantifying by using a flow cytometer. As shown in FIG. 13, the uptake of RBC-LVTNPs by endothelial cells was significantly higher than that of LVTNPs, and flow cytometry analysis also confirmed that the fluorescence intensities of LVTNPs and RBC-LVTNPs taken up by endothelial cells were 3 times and 3.4 times higher than those of LVTNPs and RBC-LVTNPs, respectively, in a static state. FIG. 14 is a flow cytometric analysis of endothelial cell uptake of LVTNPs and RBC-LVTNPs; FIG. 15 is a quantitative analysis of endothelial cell uptake of LVTNPs and RBC-LVTNPs.
Example 6 endothelial cell scratch test
HUVECs were seeded into cell slides and cultured to 95% confluence. Cross-scratches were made on the monolayer of cells using a 200. Mu.L pipette tip. Physiological saline, LVT, LVTNPs and RBC-LVTNPs were added separately. At 37 ℃ 5% CO 2 Incubate for various periods of time. Images of the scratched wounds were captured with a phase contrast microscope.
The wound healing rate was calculated according to the following formula: (a-b) -a -1 ×100％，
Where a is the original scratch width and b is the post-healing scratch width.
As a result: as shown in fig. 16, this example measured endothelial cell migration in scratch experiments, resulting in a scratch wound of a monolayer of endothelial cells, which was incubated with the culture medium (blank), LVT, LVTNPs prepared in example 1 and RBC-LVTNPs prepared in example 2, and the scratch was recovered when recording 0 and 24h under a microscope, the endothelial cell migration rate was significantly increased at 24h compared to 0h, i.e., LVT (55%), LVTNPs (59%) and RBC-LVTNPs (92%), as detailed in fig. 17. The results demonstrate that RBC-LVTNPs significantly promote repair of endothelial scratches.
Example 7 in vitro cytotoxicity and hemocompatibility
This example demonstrates the hemocompatibility of RBC-LVTNPs by in vitro hemolysis experiments. The method comprises the following steps: the volume ratio of the mixture is 4:5 dilution of C57BL/6 mice blood with saline for hemolysis test. 40 mu.L of blood was added to physiological saline, LVT, LVTNPs and RBC-LVTNPs, respectively, and incubated in a water bath at 37 ℃ for 1h. The supernatant was collected after centrifugation at 500 Xg for 5 min. The absorbance of each group of supernatants was measured at 545nm using an ultraviolet spectrophotometer.
Hemolytic activity calculation formula: hemolysis rate = (OD) T -OD NC )/(OD PC -OD NC )×100％
Wherein, OD T Is the OD test value, OD, obtained in the case of LVT, LVTNPs and RBC-LVTNPs NC Is a negative control, OD PC Is a positive control.
As shown in FIGS. 19 to 20, no significant hemolysis was observed in LVT, LVTNPs, eRBC-LVTNPs and RBC-LVTNPs. The hemolysis rate of all samples was less than 5%, which is widely considered as a safety standard for in vivo applications, indicating that LVT, LVTNPs, ebbclvtnps and RBC-LVTNPs have good hemolysis properties.
Example 8 pharmacokinetic experiments
In order to simulate a special blood flow environment in a pathological environment, a C57BL/6 mouse vascular guide wire injury model is established and improved, and the targeting capability of the RBC-LVTNPs prepared in example 2 on endothelial cell injury is evaluated. This process can lead to endothelial cell injury or loss of a significant endothelial cell monolayer, which can lead to diffuse vascular inflammation, new intimal formation and ultimately risk of vascular occlusion if endothelial cell repair lacks integrity.
To investigate the half-life of RBC-LVTNPs in circulation, 200 μ L FITC-labeled LVTNPs or FITC-labeled RBC-LVTNPs were injected into the tail vein of mice. Blood was collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48 and 60h post-injection, respectively. Blood samples were run in 96-well plates containing 0.2X 10 -3 M EDTA2K in 40 u L PBS dilution, using a fluorescence spectrophotometer to determine the fluorescence intensity.
As a result: FITC labeled LVTNPs and FITC labeled RBC-LVTNPs were injected via tail vein, and 24h after administration, mice were sacrificed and the major organs, right and left carotid arteries (RCA and LCA), were removed. Imaging of small animals clearly observed a significant accumulation of RBC-LVTNPs at the site of injury to endothelial cells. As shown in fig. 21 and 22, the lesion of the damaged area of LCA endothelial cells has stronger fluorescence; in contrast, the fluorescence of LCA endothelial cell injury area of LVTNPs group is relatively weak. The fluorescence intensity of the RBC-LVTNPs group was 3 times that of the LVTNPs group. This result indicates that RBC-LVTNPs can selectively accumulate in the endothelial cell lesion of LCA lesions. And at 24 hours after injection, the liver fluorescence intensity of LVTNPs is obviously higher than that of RBC-LVTNPs, and the fluorescence detection of other organs is not obvious, which is detailed in figures 23 and 24. Pharmacokinetic studies also confirmed that the blood circulation time of RBC-LVTNPs was much longer than that of LVTNPs, as shown in detail in fig. 25. The results show that the treatment of the erythrocyte membrane bionic nano prodrug can obviously reduce the undesirable accumulation of the nano particles in main organs, thereby reducing the non-specific toxicity and improving the in vivo biocompatibility.
Example 9 Experimental animals
7 weeks old C57BL/6 male mice were purchased from Jiangsu Jiejiegaokang Biotechnology GmbH, bred under SPF-level conditions with 12h alternating light and dark, bred adaptively for 1 week, and then the experiment was started. The animal experiment was approved by the welfare and ethics committee of the experimental animals of Chongqing university. After anesthesia of the mice, the Left Carotid Artery (LCA) was isolated with a sterile surgical instrument and the external carotid artery was ligated with 8-0 sutures proximal to the bifurcation. The blood flow in the internal carotid artery, external carotid artery, and common carotid artery was blocked with vascular clamps. A guide wire with a diameter of 0.38mm was inserted into the LCA incision, rotated 10 times and removed. After careful removal of the guide wire, the clamps are released to restore blood flow, but still maintain ligation of the internal and external carotid arteries to create a local low oscillation shear stress. Mice were randomized into 4 groups after injury and injected daily via the tail vein with (a) 150 μ L saline, (b) LVT, (c) LVTNPs, (d) RBC-LVTNPs. The injection dose is 20mg/kg, and the administration is continued for 5 days. After the treatment, the mice were sacrificed and the left and right carotid arteries and major organs of the mice were collected.
Example 10 in vivo biosafety and in vivo therapeutic Effect test
To assess biosafety in vivo, potential side effects of 5 days of treatment were investigated. The complete blood count showed no significant changes in Red Blood Cells (RBC) and White Blood Cells (WBC), as detailed in fig. 29. Alanine Aminotransferase (ALT) and alkaline phosphatase (ALP) were not significantly changed in liver function assays. Renal function measurements of Blood Urea Nitrogen (BUN) and Creatine Kinase (CK) also vary within the normal range. And H & E staining is carried out on the main organs (heart, liver, spleen, lung and kidney) of the mouse, and as shown in figure 30, RBC-LVTNPs have no obvious histological toxicity on the main organs. The result shows that the RBC-LVTNPs have good treatment safety in vivo and are expected to become a promising and feasible nano treatment method for repairing endothelial cell injury.
To evaluate the in vivo therapeutic effect, H & E staining was performed on carotid vessels of mice treated for 1 day and 5 days, as detailed in fig. 26-28. Compared with 1 day of treatment, after 5 days of treatment, the vascular tissues of the RBC-LVTNPs group become more regular obviously, endothelial cells in the intima recover obviously, and are distributed along the intima in a cobblestone-like manner, and the number of smooth muscles in the media area does not proliferate obviously, which indicates that the RBC-LVTNPs group has the effects of promoting the proliferation of the endothelial cells and inhibiting the proliferation of the smooth muscle cells.
Claims (10)
1. The composition for preparing the cell membrane bionic nano-carrier with spontaneous directional assembly is characterized by comprising a red cell membrane, an internal loaded drug and a polypeptide ligand P4.2; the erythrocyte membrane contains the transmembrane protein band 3.
2. The composition of claim 1, wherein the internal drug comprises any one or more of lovastatin, simvastatin, pravastatin, atorvastatin, rosuvastatin, rapamycin, docetaxel.
3. A spontaneously directionally assembled cell membrane biomimetic nanocarrier, wherein the cell membrane biomimetic nanocarrier comprises the composition of any of claims 1-2; the erythrocyte membrane is a shell and coats the internal drug and the polypeptide ligand P4.2; the polypeptide ligand P4.2 is specifically combined with transmembrane protein band3 in the erythrocyte membrane.
4. The cell membrane biomimetic nanocarrier of claim 3, wherein the amino acid sequence of the polypeptide ligand P4.2 is as set forth in SEQ ID NO:1 is shown.
5. A nano prodrug produced using the membrane biomimetic nanocarrier of any of claims 3-4, wherein the nano prodrug is a ROS-responsive prodrug.
6. The method of preparing a nano prodrug of claim 5, comprising the steps of:
s1: HO-PEG modified with oxalyl chloride 2K -Mal coupling said lovastatin to said polypeptide ligand P4.2 to produce LVTNPs;
s2: and co-incubating LVTNPs prepared by S1 and the erythrocyte membrane to prepare the nano prodrug.
7. The preparation method according to claim 6, wherein S1 is specifically:
(1) Dissolving the lovastatin by using dichloromethane, and adding oxalyl chloride for reaction to obtain a reaction solution 1;
(2) Removing redundant oxalyl chloride in the reaction liquid 1 obtained in the step (1) through rotary evaporation, and redissolving to obtain redissolved solution;
(3) Step (2) isAdding HO-PEG into the redissolved solution 2K -Mal reacting to obtain a reaction solution 2;
(4) Carrying out rotary evaporation on the reaction liquid 2 obtained in the step (3), and adding a polypeptide ligand P4.2 and DMSO for reaction to obtain a reaction liquid 3;
(5) And (5) dialyzing and drying the reaction liquid 3 obtained in the step (4) to obtain the LVTNPs.
8. The method according to claim 6, wherein the incubation temperature in S2 is 37 ℃ and the incubation time is 30min.
9. The use of the combination of the polypeptide ligand P4.2 as defined in claim 1 and the transmembrane protein band3 for the preparation of a spontaneously oriented assembled cell membrane biomimetic nanocarrier.
10. Use of the composition of claim 1, the cell membrane biomimetic nanocarrier of claim 2, and/or the nanoproberide of claim 4 in the preparation of a medicament for vascular endothelial injury repair.
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