Drug delivery carrier complex targeting central nervous system, preparation method for drug delivery carrier complex, and use thereof

Abstract

The present invention relates to the technical field of biomedicine. Disclosed are a drug delivery carrier complex targeting a central nervous system, a preparation method for the drug delivery carrier complex, and a use thereof. The preparation method comprises the following steps: carrying out azide modification on carrier cells to obtain azide-modified carrier cells; respectively modifying sulfydryl and cholesterol to two ends of a therapeutic miRNA, and then linking the modified therapeutic miRNA to a propylene-based complex for reaction to form an miRNA complex; and co-incubating the azide-modified carrier cells and the miRNA complex, and forming the drug delivery carrier complex by means of a click chemistry reaction. The drug delivery carrier complex prepared in the present invention has good targeting performance for the central nervous system, and can be used in the preparation of a drug for treating central nervous system diseases.

Description

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

[0001] The present invention relates to the field of biomedicine 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 the plasma and brain cells formed by the walls of brain capillaries and glial cells, and a barrier between the plasma and cerebrospinal fluid formed by the choroid plexus. It strictly controls 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 limits the passage of harmful exogenous molecules (such as neurotoxins) to maintain central nervous system homeostasis. 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 central nervous system, 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 underway on drugs targeting transferrin receptor (TfR), insulin receptor, and LDL receptor-related protein 1 (LRP1). These results can confirm whether RMT-based technology can safely and effectively deliver therapeutically relevant drug doses to the central nervous system 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 explored as vectors for CNS entry. The most prominent include adeno-associated virus (AAV) and lentiviruses, with others including 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 novel BBB-crossing viral vectors, such as certain AAV variants, could reduce the reliance on invasive treatment modalities during neurosurgery. 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, route of administration, formulation, and infusion volume and rate. More research is needed to conduct robust safety assessments and select the optimal viral vector.

[0006] Nanoparticle systems, such as liposomes, polymeric nanoparticles, and solid lipid nanoparticles (SLNPs), have been widely used in drug delivery vehicle exploration. Studies have shown that non-targeted liposomes are more likely to be passively taken up, likely due to impaired blood-brain barrier integrity rather than active transport mechanisms across the barrier. Targeted liposomes, such as glutathione (GSH)-polyethylene glycol liposomes, have demonstrated effective brain targeting in preclinical studies. Nanoparticle polymers, such as poly(lactic-co-glycolic acid) copolymers (PLGA), have also been used in neurodegenerative diseases and brain tumors. Overall, while research has demonstrated the clinical value of nanoparticle-based drug delivery in the treatment of central nervous system diseases, it is undeniable that most successful cases have been achieved in the context of brain cancer, specifically under conditions where BBB integrity is compromised. Furthermore, the intrinsic properties of the nanoparticles (such as particle size and surface charge) can also influence drug delivery. The intrinsic stability of the nanoparticles is also crucial; the carrier must remain intact until reaching the brain to achieve therapeutic efficacy. Degradation is a challenge faced by many nanoparticle carriers. 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 measuring 40-100 nm in diameter, secreted by most cell types. Exosomes can contain a variety of substances, including proteins and nucleic acids. In recent years, increasing research has demonstrated the potential of exosomes as a vehicle for drug delivery to the central nervous system (CNS). However, several challenges remain before they can be applied to the treatment of CNS disorders. First, the availability of exosomes: their content depends on the cells from which they originate, and exosomes from different cell types can have varying effects. Identifying the appropriate exosome source can help avoid potential side effects. Second, without brain-targeting modifications, only a small fraction of injected exosomes successfully reach the brain. The mechanism by which exosomes cross the brain barrier (BBB) ​​remains unclear; the primary mechanism of interaction between exosomes and cells is membrane fusion, followed by delivery of their contents into the cell. Another key issue is exosome loading efficiency. Existing exosome loading strategies are inefficient, limiting the maximum dose of the cargo and resulting in therapeutically ineffective levels. Addressing these issues is crucial for the successful application of exosomes as a delivery vehicle for CNS therapeutics.

[0008] Cell-mediated brain delivery is a relatively new approach that can be considered a specialized 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, allowing for either autologous, patient-specific therapies or 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 because 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 many unanswered questions remain. For example, it is unclear whether the number of cells that can cross the blood-brain barrier is sufficient for effective treatment or whether the cells reach the brain regions relevant for treatment. Because only a small fraction of vector cells may enter the brain via peripheral intravenous administration, while the majority remain in other organs, the distribution of vector cells to other major peripheral organs is also a major issue that must be considered.

[0010] Summary of the Invention

[0011] The present invention aims to provide a drug delivery carrier complex targeting the central nervous system, and its preparation method and application, in order 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.

[0012] To achieve the above object, the present invention provides the following solutions:

[0013] The present invention provides a method for preparing a drug delivery carrier complex targeting the central nervous system, comprising the following steps:

[0014] performing azide modification on the carrier cell to obtain an azide-modified carrier cell;

[0015] The two ends of the therapeutic miRNA are modified with sulfhydryl and cholesterol, respectively, and the modified therapeutic miRNA is then ligated with the propylene complex to form a miRNA complex;

[0016] The azide-modified carrier cell and the miRNA complex are co-incubated to form the drug delivery carrier complex through a click chemistry reaction.

[0017] Furthermore, the carrier cells include microglia or neutrophils.

[0018] Furthermore, the carrier cells are microglial cells.

[0019] Furthermore, the therapeutic miRNA is miR-138-5p mimic.

[0020] Furthermore, the propenyl complex is diphenylcyclooctyne-polyethylene glycol-acrylate.

[0021] Furthermore, the method for azide-modifying the vector cell comprises co-culturing the vector cell with azide-modified mannose.

[0022] The present invention also provides a drug delivery carrier complex targeting the central nervous system prepared according to the above preparation method.

[0023] 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.

[0024] The present invention also provides a drug for treating central nervous system diseases, comprising the above-mentioned drug delivery carrier complex.

[0025] The present invention discloses the following technical effects:

[0026] This invention constructs a novel, original drug delivery vehicle complex targeting the central nervous system (CNS) through a bioorthogonal labeling strategy based on metabolic glycoengineering and bioorthogonal click chemistry. This drug delivery vehicle complex can successfully cross the blood-brain barrier and exhibits excellent CNS targeting. This invention develops a novel drug delivery system for the preparation of drugs for treating CNS diseases, enabling the treatment of CNS diseases via peripheral intravenous administration, avoiding the infection risks associated with in situ intracerebroventricular injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0028] FIG1 is a schematic diagram of the construction of the carrier complex of the present invention;

[0029] Figure 2 shows the results of in vitro and in vivo validation experiments of the carrier complex carrying therapeutic miRNA; A is the fluorescence verification of miR-138-5p mimic 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-138-5p mimic to the central nervous system of rats; red represents CY5, blue represents the cell nucleus, and the arrow represents a representative miR-138-5p mimic labeled with CY5; E is the quantitative analysis result of CY5 in vivo; F is the uptake of miR-138-5p mimic by rat neurons observed by confocal microscopy; red represents CY5, green represents neurons, blue represents the cell nucleus, and the arrow represents a representative miR-138-5p labeled with CY5 mimic; G is the result of quantitative analysis of CY5 in neurons; H is the result of Western blot analysis of CES2 expression in the injured area of ​​MCAO rats; I is the result of grayscale value analysis of CES2 by Western blot; J is the result of Western blot analysis of downstream target genes of miR-138-5p; K is the result of grayscale value analysis of downstream target genes of miR-138-5p by Western blot; 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

[0030] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Terminology Notes:

[0036] The miR-138-5p mimic referred to in the present invention is a chemically synthesized miRNA mimic that can simulate endogenous miRNA and upregulate miRNA activity. It was constructed and modified by Suzhou Genetron Health Co., Ltd.

[0037] 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.

[0038] Example 1

[0039] 1. Methods

[0040] 1.1 Construction of carrier complex

[0041] The build strategy shown in Figure 1 performs the following operations:

[0042] (1) Preparation and pretreatment of carrier cells

[0043] Primary neutrophils (NE) were prepared from the tibia and femur of 8-12-week-old Sprague-Dawley rats and cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum. HAPI cells (rat microglia) were purchased from the Shanghai ATCC Cell Bank. HAPI cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco) supplemented with 10% FBS and 100 U / mL penicillin-streptomycin.

[0044] (2) Pre-modification of therapeutic miRNA

[0045] 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 pre-treated by co-incubation before bioorthogonal labeling to form cell complexes, namely azide-modified HAPI cells and azide-modified NE cells, respectively.

[0046] The miR-138-5p mimic was chemically modified at both ends. The antisense strand of the mimic was modified with a thiol at the 5' end and cholesterol at the 3' end. To facilitate fluorescence observation, the 3' end was labeled with the fluorescent dye Cyanine 5 (CY5). The modified miR-138-5p mimic was incubated with a diphenylcyclooctyne-polyethylene glycol-acryl (DBCO-PEG-AC) complex at 25°C for 0.5 hours. The two mimic compounds were linked by an ester bond between the thiol and acryl groups, forming the mimic complex.

[0047] (3) Connect the carrier cell and mimic complex through click chemistry reaction

[0048] 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.

[0049] 1.2 The carrier complex is loaded with therapeutic miRNA, and the experimental effect is verified by in vitro fluorescence observation, in vivo live imaging, fluorescence observation, and changes in downstream protein levels.

[0050] 2. Results

[0051] After the construction of the carrier complex was completed, fluorescence microscopy observed that miR-138-5p mimic was loaded onto the cell surface (A and B in Figure 2). Utilizing the characteristics of inflammatory cells targeting damaged inflammatory areas, the present invention selected different inflammatory cells (neutrophils NE and microglia HAPI) as carriers and explored their efficiency in targeting the delivery of miRNA to damaged areas. The present invention loaded miR-138-5p mimic onto the surface of NE cells or HAPI cells. By observing the distribution of miR-138-5p mimic labeled with Cyanine5 (CY5) in the organs of MCAO rats, it was found that both mimic-NE and mimic-HAPI could reach the ischemic damage 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 (C in Figure 2). The staining results also showed that CY5-labeled miR-138-5p mimic could reach the ischemic damaged area (D and E in Figure 2) and be taken up by neurons (F and G in Figure 2). 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.

[0052] After miRNA is transported to the damaged area by HAPI cells, it must be separated from the loaded cells to function. To this end, the present invention modifies miRNA with sulfhydryl groups by esterifying the propylene group on the cell surface to form an ester bond. Because the damaged area cells are broken and release esterases, such as carboxylesterases, these esterases can cut the ester bond and then 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 and HAPI separated (Figure 2 A and B). In vivo experiments confirmed that CES2 expression 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.

[0053] Finally, the expression of DNMT3A, Rheb, and Rhebl1 in the injured area of ​​treated MCAO rats was examined. The results showed that the miR-138-5p mimic exerted a similar function upon reaching the injured area, reducing DNMT3A expression and thereby increasing Rhebl1 levels. Similar to previous results, HAPI cells showed a stronger targeting ability than NE cells (Figure 2, J and K).

[0054] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined 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 the modified therapeutic miRNA is then linked with the propylene complex to form a miRNA complex; The azide-modified carrier cell and the miRNA complex are co-incubated to form the drug delivery carrier complex through a click chemistry reaction.

2. The preparation method according to claim 1, characterized in that: The carrier cells include microglia or neutrophils.

3. The preparation method according to claim 2, characterized in that: The carrier cells are microglial cells.

4. The preparation method according to claim 1, characterized in that: The therapeutic miRNA is miR-138-5p mimic.

5. The preparation method according to claim 1, characterized in that: The acryl compound is diphenylcyclooctyne-polyethylene glycol-acrylate.

6. The preparation method according to claim 1, characterized in that: The method for azide-modifying a vector cell comprises co-culturing the vector cell with azide-modified mannose.

7. A drug delivery carrier complex targeting the central nervous system prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the drug delivery carrier complex according to claim 7 in the preparation of drugs for treating central nervous system diseases.

9. A drug for treating central nervous system diseases, characterized in that: Comprising the drug delivery carrier complex according to claim 7.

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