A drug delivery carrier complex targeting the central nervous system and its preparation method and application

Abstract

The present invention discloses a drug delivery carrier complex targeting the central nervous system and a preparation method and application thereof, and relates to the field of biomedical technology. The preparation method comprises the following steps: azide-modifying a carrier cell to obtain an azide-modified carrier cell; modifying thiol and cholesterol to both ends of a therapeutic miRNA respectively, and then connecting the modified therapeutic miRNA with an propylene complex to form a miRNA complex; incubating the azide-modified carrier cell and the miRNA complex together to form the drug delivery carrier complex through a click chemistry reaction. The drug delivery carrier complex prepared by the present invention has good targeting to the central nervous system and can be used to prepare drugs for treating central nervous system diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a drug delivery carrier complex targeting the central nervous system, and a preparation method and application thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​is a barrier between plasma and brain cells formed by the walls of brain capillaries and glial cells, and a barrier between plasma and cerebrospinal fluid formed by the choroid plexus. It can strictly control the entry of specific nutrients (such as amino acids, glucose, nucleosides and fatty acids) from the vascular system into the extracellular fluid of the central nervous system, and limit the passage of harmful exogenous molecules (such as neurotoxins) to maintain the homeostasis of the central nervous system. At the same time, the blood-brain barrier also prevents certain drugs and large molecule therapeutic drugs such as biopharmaceuticals from entering the brain, and low levels of drug concentrations will affect the therapeutic efficiency of the drugs. Therefore, there is an urgent need for an effective non-invasive technology to efficiently deliver biological drugs to the central nervous system.

[0003] Currently, the technologies for targeting the central nervous system can be roughly divided into the following categories: (1) receptor-mediated endocytosis (RMT); (2) application of neurotropic viruses; (3) nanoparticle delivery systems; (4) exosome-mediated delivery systems; and (5) other delivery systems.

[0004] For the development of RMT delivery technology in the CNS, the ideal receptor should be expressed only on blood-brain barrier cells and have a high transport capacity mediated by selective ligands that do not interfere with its natural function and do not trigger normal lysosomal degradation. Although such an "ideal" receptor has not yet been found, clinical trials are ongoing for drugs targeting transferrin receptor (TfR), insulin receptor, and LDL receptor-related protein 1 (LRP1), and these results can confirm whether RMT-based technology can safely and effectively deliver therapeutically relevant drug doses to the CNS while minimizing peripheral and / or off-target toxic effects.

[0005] Neurotropic viruses, a class of viruses with an affinity for neural tissue, have also been tried as vectors to enter the central nervous system, the most prominent of which include adeno-associated virus (AAV) and lentiviruses, others include herpes simplex virus (HSV) and virus-like particles (VLPs). The application of these viral vectors is currently limited to gene therapy and often relies on invasive administration methods that require additional patient monitoring, such as intrathecal or intrarenal injection. The development of a new type of viral vector that crosses the BBB, such as certain AAV variants, could reduce the reliance on invasive treatment methods during neurosurgical treatment. Other issues that should be considered when using neurotropic viruses as vectors include vector purity, regulatory elements and transgene sequences, the use of non-integrating or self-inactivating vectors, the nature of the packaged DNA, the route of administration, formulation, and infusion volume and rate. More research is needed to conduct robust safety assessments and select the best viral vector.

[0006] Nanoparticle systems are widely used in the exploration of carriers, such as liposomes, polymeric nanoparticles, and solid lipid nanoparticles (SLNPs). Studies have shown that non-targeted liposomes are more passively taken up, which may be due to the damage of blood-brain barrier integrity rather than active blood-brain barrier transport mechanism. The application of targeted liposomes, such as glutathione (GSH)-polyethylene glycol liposomes, has been confirmed in some preclinical studies for its efficient brain targeting. Nanoparticle polymers represented by polylactic acid-polyethylene glycol copolymer (PLGA) have also been used in neurodegenerative diseases and brain tumors. In general, although studies have shown that drug delivery using nanoparticles as carriers has certain clinical value in the treatment of central nervous system diseases, it has to be admitted that the success of most cases is achieved in the context of brain cancer, or more precisely, under the premise that the integrity of the BBB is damaged. The inherent properties of the nanoparticles themselves (such as particle size, surface charge, etc.) will also affect drug delivery. The intrinsic stability of nanoparticles is also important, and the carrier needs to remain intact until it reaches the brain to produce therapeutic effects. Degradation is a problem that many nanoparticle carriers need to face. In short, there is currently no effective way to target nanoparticles to the brain and increase blood drug concentrations in the brain, and further research is needed.

[0007] Exosomes are small cell-derived vesicles with a diameter of 40-100nm, which can be secreted by most types of cells. Exosomes can contain a variety of substances, including proteins and nucleic acids. In recent years, more and more studies have shown that exosomes have the ability to be used as a carrier to achieve drug delivery to the central nervous system, but there are still some problems to be solved to make them suitable for the treatment of central nervous system diseases. The first is the acquisition of exosomes. Their content depends on the cells in which they are produced. Exosomes from different cells can cause different effects. Identifying the appropriate source of exosomes can help avoid potential side effects. Secondly, if there is no brain-targeted modification, only a small part of the injected exosomes can successfully reach the brain. The mechanism by which exosomes cross the BBB is still unclear. The main mechanism of interaction between exosomes and cells is membrane fusion, followed by the delivery of their contents into cells. Another important issue is the loading efficiency of exosomes. The existing exosome loading strategies are all inefficient, which will limit the maximum applicable dose of the load and make it reach a therapeutically inefficient level. In order to make exosomes a delivery tool for central nervous system therapeutic drugs, these problems must be solved.

[0008] Cell-mediated brain delivery is a relatively new approach that can be viewed as a specific application within the broader context of cell therapy. These cells can also be engineered to produce therapeutically relevant peptides, proteins, or RNA molecules, or to carry drug-encapsulated liposomes, for either autologous, patient-specific therapies or as allogeneic, universal cell therapies infused into the systemic circulation. Immune cells such as macrophages and monocytes are suitable candidates for brain delivery in neuroinflammatory diseases or neurodegenerative diseases with a neuroinflammatory component, as these cells are recruited to cross the blood-brain barrier during brain inflammation. These immune cells rely on their own “infiltration” process to change their shape in order to cross the blood-brain barrier.

[0009] Currently, the exploration of cell-mediated delivery vectors to the central nervous system is still in its early stages, and there are many unanswered questions. For example, it is not clear whether the number of cells that can cross the blood-brain barrier is sufficient for effective treatment, or whether the cells reach the brain areas relevant for treatment. Since only a small number of vector cells may enter the brain through peripheral intravenous administration, while most cells remain in other organs; therefore, the distribution of vector cells in other major organs in the periphery is also a major issue that must be considered. Summary of the invention

[0010] The purpose of the present invention is to provide a drug delivery carrier complex targeting the central nervous system and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The drug delivery carrier complex has good targeting to the central nervous system and can be used to prepare drugs for treating central nervous system diseases.

[0011] To achieve the above object, the present invention provides the following solutions:

[0012] The present invention provides a method for preparing a drug delivery carrier complex targeting the central nervous system, comprising the following steps:

[0013] The carrier cell is subjected to azide modification to obtain an azide-modified carrier cell;

[0014] The two ends of the therapeutic miRNA are modified with thiol and cholesterol respectively, and then the modified therapeutic miRNA is connected with the acryl complex to form a miRNA complex;

[0015] The azide-modified carrier cell and the miRNA complex are co-incubated to form the drug delivery carrier complex through a click chemistry reaction.

[0016] Furthermore, the carrier cells include microglia or neutrophils.

[0017] Furthermore, the carrier cells are microglia.

[0018] Furthermore, the therapeutic miRNA is miR-138-5pmimic.

[0019] Furthermore, the acrylic compound is diphenylcyclooctyne-polyethylene glycol-acrylate.

[0020] Furthermore, the method for azide-modifying a vector cell comprises co-culturing the vector cell with azide-modified mannose.

[0021] The present invention also provides a drug delivery carrier complex targeted to the central nervous system prepared according to the above preparation method.

[0022] The present invention also provides the use of the above-mentioned drug delivery carrier complex in improving the targeting of the central nervous system in the pharmaceutical process.

[0023] The present invention also provides a drug for treating central nervous system diseases, comprising the above-mentioned drug delivery carrier complex.

[0024] The present invention discloses the following technical effects:

[0025] The present invention constructs a novel and original drug delivery carrier complex targeting the central nervous system through a bioorthogonal labeling strategy based on metabolic sugar engineering and bioorthogonal click chemistry. The drug delivery carrier complex can smoothly cross the blood-brain barrier and has good targeting to the central nervous system. The present invention develops a new drug delivery system for preparing drugs for treating central nervous system diseases, so that the treatment of central nervous system diseases can be achieved through peripheral intravenous administration, avoiding the infection risk caused by in situ injection administration in the lateral ventricle. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 Schematic diagram of the construction of the carrier complex of the present invention;

[0028] Figure 2 The results of in vitro and in vivo effect verification experiments of carrier complexes carrying therapeutic miRNA; A is the fluorescence verification of miR-138-5pmimic attached to the cell surface in vitro; red represents CY5, and blue represents the cell nucleus; B is the quantitative statistical result of the fluorescence value of CY5 in vitro; C is the distribution of two carrier complexes (mimic-NE and mimic-HAPI) in the main organs of MCAO rats; D is the carrier complex carrying miR-138-5pmimic to the central nervous system of rats; red represents CY5, blue represents the cell nucleus, and the arrow represents the representative miR-138-5pmimic labeled with CY5; E is the quantitative analysis result of CY5 in vivo; F is the observation of rat neurons responding to miR-138-5pmi by confocal microscopy mic uptake; red indicates CY5, green indicates neurons, blue indicates cell nuclei, and arrows indicate representative miR-138-5p mimics labeled with CY5; G is the result of quantitative analysis of CY5 in neurons; H is the Western blot analysis result of CES2 expression in the injured area of ​​MCAO rats; I is the gray value analysis result of Western blot of CES2; J is the Western blot analysis result of the downstream target genes of miR-138-5p; K is the gray value analysis result of Western blot of the downstream target genes of miR-138-5p; all data represent the mean ± SEM of three independent experiments; P values ​​were determined using two-way ANOVA or Student t tests; nsP ≥ 0.05,* P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Terminology Note:

[0035] The miR-138-5pmimic referred to in the present invention is a chemically synthesized miRNA mimic that can simulate endogenous miRNA and upregulate miRNA activity.

[0036] The DBCO-PEG-AC complex referred to in the present invention refers to diphenylcyclooctyne-polyethylene glycol-acrylate, which was purchased from Shanghai Tuoyang Biological Company.

[0037] Example 1

[0038] 1. Methods

[0039] 1.1 Construction of carrier complex

[0040] like Figure 1 The build strategy shown does the following:

[0041] (1) Preparation and pretreatment of carrier cells

[0042] Primary neutrophils (NE) were prepared from the tibia and femur of SD rats aged 8-12 weeks and cultured in RPMI1640 medium (Gibco) containing 10% fetal bovine serum. HAPI cells (rat microglia) were purchased from the Shanghai ATCC cell bank. HAPI cells were cultured in DMEM medium (Gibco) supplemented with 10% FBS and 100U / mL penicillin-streptomycin.

[0043] (2) Pre-modification of therapeutic miRNA

[0044] The HAPI cells and NE cells cultured in step (1) were co-cultured with azide-modified mannose (CAS No.: 361154-30-5) for 1.5 hours, and co-incubated for pretreatment before bioorthogonal labeling to form cell complexes, namely azide-modified HAPI cells and azide-modified NE cells, respectively.

[0045] Both ends of miR-138-5pmimic were chemically modified in advance. The 5' end of the antisense strand of the mimic was modified with thiol, and the 3' end was modified with cholesterol. To facilitate fluorescence observation, the 3' end was labeled with the fluorescent dye Cyanine5 (CY5). The modified miR-138-5pmimic was incubated with the diphenylcyclooctyne-polyethylene glycol-acryl (DBCO-PEG-AC) complex at 25°C for 0.5h. The two mimic compounds were connected by an ester bond between the thiol and the acryl to form a mimic complex.

[0046] (3) Connect the vector cell and mimic complex through click chemistry reaction

[0047] Azide-modified HAPI cells and azide-modified NE cells were co-incubated with the mimic complex at 25°C, and HAPI cell-carrier complex (mimic-HAPI) and NE cell-carrier complex (mimic-NE) were formed respectively through click chemistry reaction.

[0048] 1.2 The vector complex is loaded with therapeutic miRNA, and the experimental effect is verified by in vitro fluorescence observation, in vivo living imaging, fluorescence observation, and changes in downstream protein levels.

[0049] 2. Results

[0050] After the construction of the carrier complex was completed, fluorescence microscopy observed that miR-138-5pmimic was loaded onto the cell surface ( Figure 2 A and B). Taking advantage of the property of inflammatory cells to target damaged inflammatory areas, the present invention selected different inflammatory cells (neutrophils NE and microglia HAPI) as carriers, and explored their efficiency in targeting and delivering miRNA to damaged areas. The present invention loaded miR-138-5pmimic onto the surface of NE cells or HAPI cells. By observing the distribution of miR-138-5pmimic labeled with Cyanine5 (CY5) in the organs of MCAO rats, it was found that both mimic-NE and mimic-HAPI could reach the ischemic injury area, and the delivery ability of HAPI cells was better than that of NE cells, while the distribution of miRNA complexes in other organs was mainly concentrated in the lungs and kidneys ( Figure 2 The staining results also showed that CY5-labeled miR-138-5pmimic could reach the ischemic damaged area ( Figure 2 D and E) and taken up by neurons ( Figure 2 F and G). Therefore, the present invention found that both HAPI cells and NE cells can be used as carriers for central nervous system targeting, and experiments confirmed that HAPI cells have better targeting, therefore, the present invention used HAPI cells as carriers in subsequent experiments.

[0051] After miRNA is transported to the damaged area by HAPI cells, it must be separated from the loaded cells in order to function. To this end, the present invention modifies miRNA with thiol groups and forms ester bonds by esterifying the propylene group on the cell surface. Because the cells in the damaged area are broken and release esterases, such as carboxylesterases, these esterases can cut the ester bonds and separate the miRNA from the cell surface. Over the past few decades, the molecular characteristics of carboxylesterases (including CES1 and CES2) and their biological effects in vivo and in exogenous metabolism have been widely studied. Recent studies have shown that CES is involved in the hydrolysis of a large number of endogenous esters. In vitro experiments using CES2 cells co-cultured with mimic-HAPI for 8 hours showed that mimic was separated from HAPI ( Figure 2 In vivo experiments confirmed that the expression of CES2 was significantly increased in the ischemic injury area of ​​MCAO rats ( Figure 2 H and I), indicating that there is an internal environment in the body where ester bonds dissociate.

[0052] Finally, the expression of DNMT3A, Rheb, and Rhebl1 in the injured area of ​​MCAO rats after treatment was detected. The results showed that miR-138-5pmimic can play a similar function after reaching the damaged area, reducing the expression of DNMT3A and thus increasing the level of Rhebl1. Similar to the previous results, HAPI cells have a stronger targeting ability than NE cells ( Figure 2 J and K).

[0053] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a drug delivery carrier complex targeting the central nervous system, characterized in that: The following steps are involved: The carrier cell is subjected to azide modification to obtain an azide-modified carrier cell; The two ends of the therapeutic miRNA are modified with thiol and cholesterol respectively, and then the modified therapeutic miRNA is connected with the acryl complex to form a miRNA complex; incubating the azide-modified carrier cell and the miRNA complex together to form the drug delivery carrier complex through a click chemistry reaction; The carrier cells are microglia or neutrophils; The acryl complex is diphenylcyclooctyne-polyethylene glycol-acrylate; The method for azide-modifying a vector cell comprises co-culturing the vector cell with azide-modified mannose; the CAS number of the azide-modified mannose is 361154-30-5; The therapeutic miRNA is miR-138-5p mimic.

2. A drug delivery carrier complex targeting the central nervous system prepared according to the preparation method according to claim 1.

3. A drug for treating central nervous system diseases, characterized in that: Comprising the drug delivery carrier complex according to claim 2.

Images