Nanometer vesicle delivery system based on adipose tissue as well as preparation and application of nanometer vesicle delivery system

Abstract

The invention provides a nano-vesicle delivery system based on adipose tissue. The nano-vesicle delivery system is composed of adipose tissue-derived mesenchymal stem cells and polypeptide CAQK-CP05 according to a protein concentration of 1: 1. According to the nano-vesicle delivery system, itaconic acid nano-particles or dexamethasone small molecules are loaded respectively to provide enough nutrient substances and a stable living environment for cultured cells, so that drugs can efficiently pass through a barrier to reach a disease site. The delivery system disclosed by the invention can realize penetration of different central nervous system barriers of a blood-brain barrier and a blood-spinal cord barrier by utilizing a transendothelial effect of phagocytizing the nano-vesicles, realizes drug delivery of the central nervous system, realizes local aggregation of a nerve injury part, realizes targeted therapy of the injury part, and has a good application prospect. By simply extruding and loading nano materials and small molecular drugs, delivery of different nano materials and drugs is realized. The nano-vesicle delivery system is simple in preparation steps, and raw materials are easy to obtain.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials, and specifically relates to a nanovesicle delivery system based on adipose tissue, a preparation method and an application thereof. Background Art

[0002] Spinal cord injury (SCI) is a difficult-to-treat central nervous system (CNS) injury that usually leads to motor and sensory dysfunction. Due to the lack of effective treatments, the prognosis of SCI patients is not ideal. Nanomaterials have shown superiority in SCI treatment, but crossing the blood-spinal cord barrier (BSCB) remains a challenge. After injury, the dysfunctional BSCB allows transient leakage, but it is believed that the manner and extent of entry into the injured site are insufficient to support the therapeutic effect. Because the BSCB begins to repair within 24 hours and restores function within 96 hours. On the other hand, the presence of microenvironment turbulence prevents leakage at the gap. Therefore, the development of an ideal drug delivery method across the BSCB will help nanomaterials to treat spinal cord injury.

[0003] Transcytosis is a special cell activity phenomenon, that is, after the cell swallows foreign substances, it spits them out on the other side of the cell membrane without being processed. Transcytotic nanomaterials have the potential to cross cell barriers and are used for deep tumor delivery therapy. Nanoparticles can be endowed with transcytotic ability by changing their surface charge. However, the conditions that trigger transcytosis are often elusive and often have biological toxicity, such as the positive charge on the surface of nanoparticles. Therefore, transcytotic nanomaterials have been used in tumor treatment. Specific peptides or proteins that trigger transcytosis (such as endostatin-2 and RVG29) need to be attached to the surface of nanomaterials by chemical methods, which requires complex design, process and high cost. Therefore, a simple and safe method is needed to effectively cross the BSCB barrier and deliver drugs to the central nervous system.

[0004] On the other hand, extracellular vesicles (EVs) have been reported to be able to cross the CNS barrier, but the mechanism is not clear

[13] . The biocompatibility of exosomes is thought to prolong circulation time and facilitate passage through weak parts of the barrier. In addition, EVs from breast cancer brain metastases were found to be able to cross the blood-brain barrier (BBB) ​​by transcytosis. More importantly, under inflammatory conditions, EVs were found to be able to cross the brain microvascular endothelial cell (BMEC) monolayer barrier by transport. Although the mechanism remains unclear, EV-extended nanomaterials seem to be a breakthrough in cracking the CNS. However, the low yield of EVs and complex and expensive extraction methods have become the main obstacles to EV delivery.

[0005] Inspired by the phenomenon of EVs crossing the BBB, we demonstrated a simple adipose tissue-based nanovesicle delivery system with transcytosis ability and can cross the BSCB. This is based on our discovery that EV-like nanovesicles (NVs) can cross the blood-spinal cord barrier by transcytosis. NVs are generated by extrusion from intact adipose-derived stem cells (ADSCs), a cell type that has entered the clinical trial stage, and have similar characteristics to EVs and their similar characteristic proteins. Our evidence shows that the nanovesicle delivery system can utilize endosomal trafficking and be transported to the other side through Rab11-positive recycling endosomes for exocytosis, thereby completing the cell-crossing process. Finally, the local homing ability is conferred by attaching the targeting peptide CAQK on the surface of the nanovesicles. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a nanovesicle delivery system based on adipose tissue, wherein the nanovesicle delivery system is composed of adipose tissue-derived mesenchymal stem cells and polypeptide CAQK-CP05 at a protein concentration of 1:1. The amino acid sequence of the polypeptide (CAQK-CP05) is shown in SEQ ID NO.1: CRHSQMTVTSRLCAQK

[0007] Another object of the present invention is to provide a method for preparing the adipose tissue-based nanovesicle delivery system, which is achieved by the following steps: (1) Adipose tissue of 2-week-old rats was extracted, digested with type I collagenase, and cultured in adipose stem cell culture medium (90% Dulbecco's modified Eagle's medium + 10% fetal bovine serum) to obtain adipose-derived stem cells; (2) Extruding adipose-derived stem cells into 10 μm, 5 μm, 400 nm, 200 nm, and 100 nm liposome extruders in sequence to obtain nanovesicles; (3) The nanovesicles were mixed with the polypeptide CAQK-CP05 at 4°C overnight and then purified by ultracentrifugation at 100,000 g for 1.5 hours to obtain a nanovesicle delivery system.

[0008] The concentration of the adipose-derived stem cells can be selected within a certain range. In a preferred embodiment of the present invention, in order to obtain a higher yield of nanovesicles, a cell concentration of 1 million per milliliter is used for extrusion, and the concentration of the polypeptide is the protein quantitative condition, and is mixed with the nanovesicles in a 1:1 ratio.

[0009] The operation of the extrusion process in step (2) can be operated in a manner that can be understood by those skilled in the art, for example, by comparing the extrusion process of liposome synthesis. In addition, nanomaterials and drugs can be added during the extrusion process to achieve the purpose of loading drugs. In one embodiment of the present invention, itaconic acid nano micelles are added in step (2) as loaded drugs. In another embodiment of the present invention, dexamethasone is added in step (2) as loaded drugs.

[0010] The material purification method in step (3) is to dilute to 20 ml and ultracentrifuge at 100000 g to obtain a precipitated product.

[0011] Another object of the present invention is to provide the use of the nanovesicle delivery system as a carrier in the preparation of drugs for treating central nervous system diseases.

[0012] The nanovesicle delivery system provided by the present invention provides sufficient nutrients and a stable living environment to cultured cells by loading itaconic acid nanoparticles and dexamethasone small molecules respectively, so that the drug can efficiently cross the barrier to reach the disease site. At the same time, the nanovesicle delivery system can effectively gather at the site of injury, thereby further increasing the local drug concentration. In addition, the preparation steps of the nanovesicle delivery system are simple and the raw materials are easily available.

[0013] The present invention has the following features and advantages: (1) Compared with simple nano-micelles and small molecule drugs, this delivery system utilizes the transcytosis effect of endothelial cells engulfing nanovesicles to achieve penetration of different central nervous system barriers such as the blood-brain barrier and the blood-spinal cord barrier, thereby achieving drug delivery to the central nervous system. (2) Compared with simple nano-micelles and small molecule drugs, this delivery system achieves local aggregation at the site of nerve damage and achieves targeted treatment of the damaged site. (3) This delivery system can achieve the delivery of different nano-materials and drugs by simply extruding and loading nano-materials and small molecule drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0015] Figure 1 .Synthesized nanovesicle delivery system and in vitro penetration ability test. Among them (A) Schematic diagram of nanovesicles, (B) Schematic diagram of in vitro blood-spinal cord barrier construction, (C) Measurement of the effect of different temperatures on the penetration ability of nanovesicles in the blood-spinal cord barrier in vitro, (D) Measurement of the effect of different phagocytosis inhibitor dynasore concentrations on the penetration ability of nanovesicles in the blood-spinal cord barrier in vitro, (E) Measurement of the effect of different phagocytosis inhibitors on the penetration ability of nanovesicles in the blood-spinal cord barrier in vitro, (F) Transmission electron microscopy and dynamic light scattering to show the morphology and size of CNVs@PIM, (G) FACS analysis of the surface modification of nanovesicles CAQK, (H) Effect of CNVs coating on the penetration ability of itaconic acid nanomicelles PIM in the blood in vitro Measurements in the spinal cord barrier, (I) Different concentrations of the phagocytosis inhibitor dynasore affect the ability of CNVs@PIM to cross the blood-spinal cord barrier in vitro, (J) siRab11a affects the ability of CNVs@PIM to cross the blood-spinal cord barrier in vitro, (K) CNVs coating affects the ability of small molecule drug dexamethasone (Dex) to cross the blood-spinal cord barrier in vitro, (L) Different concentrations of the phagocytosis inhibitor dynasore affect the ability of CNVs@Dex to cross the blood-spinal cord barrier in vitro, (M) siRab11a affects the ability of CNVs@Dex to cross the blood-spinal cord barrier in vitro.

[0016] Figure 2 . Application of nanovesicle delivery system to deliver nanoparticles and small molecules to the site of spinal cord injury and cerebral ischemia. (A) IVIS images of the spinal cord with nanoparticles labeled with Cy5.5 after injection of PIM, NVs, CNVs@PIM, and excessive CAQK followed by CNVs@PIM; (B) Analysis of the fluorescence intensity of the Cy5.5 signal in the center of the spinal cord injury in IVIS images; (C) Representative fluorescence images of Cy5.5-labeled nanoparticles and neurocan in the center of the spinal cord injury; (D) IVIS images of the brain with drugs labeled with Cy5 after injection of Dex, NVs@PIM, CNVs@PIM, and excessive CAQK followed by CNVs@PIM; (E) Analysis of the fluorescence intensity of the Cy5.5 signal in the center of the spinal cord injury in IVIS images; (F) Representative fluorescence images of Cy5-labeled Dex in the brain. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with the accompanying drawings and specific implementations. It should be understood that the specific implementations described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0018] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0019] The present invention will be described in detail below through examples. In the following examples, DMEM culture medium is a commercial product of ThermoFisher Scientific, liposome extruder is a commercial product of Avanti, and CAQK-CP05 polypeptide is a commercial product of Anhui Hesheng Biological Co., Ltd.

[0020] Example 1 The present invention provides a nanovesicle delivery system based on adipose tissue. The nanovesicle delivery system consists of adipose tissue-derived mesenchymal stem cells and polypeptide CAQK-CP05.

[0021] Preparation method: (1) Adipose tissue of young rats was extracted, rinsed with PBS, mechanically cut with a blade for 5 minutes, digested with type I collagenase for 30 minutes, and then cultured in DMEM low-glucose medium to obtain adipose-derived stem cells, which were then expanded to the P1 generation; (2) 1 million cells per ml were sequentially extruded using 10 μm, 5 μm, 400 nm, 200 nm and 100 nm liposome extruders, and the product was diluted with 20 ml PBS and then ultracentrifuged at 100,000 g to obtain nanovesicles; (3) The nanovesicles were mixed with the polypeptide CAQK-CP05 at a protein concentration of 1:1 at 4°C overnight, and then diluted again with 20 ml of PBS and ultracentrifuged at 100,000 g to obtain nanovesicles.

[0022] Example 2 Nanovesicle D1 was prepared according to the preparation method of Example 1, except that the polypeptide CAQK-CP05 was not used in step (3).

[0023] Example 3 The method of Example 1 was followed, except that 5 mg / mL of itaconic acid nano-micelles were added in step (2) to prepare nanovesicles D2.

[0024] Example 4 The method of Example 1 was followed, except that 5 mg / mL of dexamethasone was added in step (2) to prepare nanovesicle D3.

[0025] Example 5 Nanovesicle D4 was prepared according to the method of Example 3, except that the polypeptide CAQK-CP05 was not used in step (3).

[0026] Example 6 The method of Example 4 was followed, except that the polypeptide CAQK-CP05 was not used in step 3), to prepare nanovesicle D5.

[0027] Embodiment 7 (Application Example 1) (1) A contusion model of spinal cord injury in mice was established by the impact method. In brief, a T8-10 laminectomy was performed to expose the thoracic spinal cord. Then, the exposed spinal cord was impacted by a 5-g cylinder (2.5 mm in diameter) controlled by a gravity drop device (NYU II) that fell freely from a height of 12 mm. Subsequently, the muscles, fascia, and skin were sutured layer by layer with 4-0 nylon sutures. After surgery, mice were injected with penicillin daily, provided with easily accessible water and food for 1 week, and underwent manual bladder massage twice a day until bladder function was restored; (2) The mice were randomly divided into the itaconic acid micelle group, the D4 group, the D2 group, and another group that was injected with CAQK peptide for blocking in advance. 30 minutes after injury, each group was intravenously injected with 150 μL of itaconic acid micelles, D4, and D2 (6 mg / mL).

[0028] Example 8 (Test Example 1) First, the obtained nanovesicle delivery system was measured by transmission electron microscopy and dynamic light scattering. Figure 1 -F; One week after the model was constructed in Example 7 and the drug was injected, the mouse heart was perfused and the spinal cord was removed, and then the results were detected using an in vivo fluorescence imaging device. Figure 2 -A and Figure 2 -B; Furthermore, the obtained spinal cord was dehydrated, embedded, and frozen in sections, and then examined using a fluorescence microscope. Figure 2 -C; At the same time, after coating the 0.4 μm transwell chamber with 1 mg / ml fibronectin, the endothelial cell line HCMEC / D3 cells were cultured in the upper chamber, and 5 ug itaconic acid micelles, nanovesicles D2, 80 μmol phagocytosis inhibitor dynasore, siRNA-Rab11a and control siRNA were added to the upper chamber respectively. After 2 hours, the concentration in the lower chamber was detected. The results are as follows Figure 1 -H, Figure 1 -I. Figure 1 -J.

[0029] Embodiment 9 (Application Example 2) (1) A transient cerebral ischemia model was established in mice using the t-MCAO method. C57BL / 6 mice were anesthetized and a small incision was made in the neck to expose the carotid artery. A special coil or clamp was used to temporarily block the middle cerebral artery for 1 hour. After 1 hour, the coil or clamp was removed to restore blood flow in the middle cerebral artery. (2) The mice were randomly divided into the dexamethasone group, the D5 group, the D3 group, and another group, the D2 group, which was injected with CAQK peptides in advance for blocking. 30 minutes after injury, each group was intravenously injected with 150 μL of dexamethasone, nanovesicles D5 and D3 (6 mg / mL).

[0030] Example 10 (Test Example 2) After 2 days, the mouse hearts in Example 9 were perfused, and the mouse brains were detected using an in vivo fluorescence imaging device. Figure 2 -D and Figure 2 -E; Furthermore, the obtained brain was dehydrated, embedded, and frozen into sections, and then examined using a fluorescence microscope. Figure 2 -F; At the same time, after coating the 0.4 μm transwell chamber with 1 mg / ml fibronectin, the endothelial cell line HCMEC / D3 cells were cultured in the upper chamber, and 5 ug dexamethasone, nanovesicle D3, 80 μmol phagocytosis inhibitor dynasore, siRNA-Rab11a and control siRNA were added to the upper chamber respectively. After 2 hours, the concentration in the lower chamber was detected. The results are as follows Figure 1 -K, Figure 1 -L and Figure 1 -M.

[0031] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0032] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0033] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A nanovesicle delivery system based on adipose tissue, the nanovesicle delivery system is composed of adipose tissue-derived mesenchymal stem cells and polypeptide CAQK-CP05 at a protein concentration of 1:

1.

2. The method for preparing the adipose tissue-based nanovesicle delivery system according to claim 1 is achieved by the following steps: (1) Adipose tissue of 2-week-old rats was extracted, digested with type I collagenase, and cultured in adipose stem cell culture medium (90% Dulbecco's modified Eagle's medium + 10% fetal bovine serum) to obtain adipose-derived stem cells; (2) Extruding adipose-derived stem cells into 10 μm, 5 μm, 400 nm, 200 nm, and 100 nm liposome extruders in sequence to obtain nanovesicles; (3) The nanovesicles were mixed with the polypeptide CAQK-CP05 at 4°C overnight and then ultracentrifuged to prepare the nanovesicle delivery system.

3. The preparation method according to claim 2, characterized in that: The composition of the adipose stem cell culture medium in step (2) is: 90% Dulbecco's modified Eagle's medium + 10% fetal bovine serum.

4. The preparation method according to claim 2, characterized in that: The extrusion process of step (2) is performed in a manner that can be understood by those skilled in the art, and nanomaterials and drugs are added during the extrusion process to achieve the purpose of loading drugs.

5. The preparation method according to claim 4, characterized in that: In step (2), itaconic acid nano-micelles are added as loaded drugs or dexamethasone is added as loaded drugs.

6. The preparation method according to claim 2, characterized in that: The material purification method in step (3) is to dilute to 20 ml and ultracentrifuge at 100000 g to obtain a precipitated product.

7. Use of the nanovesicle delivery system according to claim 1 as a carrier in the preparation of drugs for treating central nervous system diseases.

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