An engineered stem cell membrane biomimetic formulation, and methods of making and using the same

Abstract

This invention discloses an engineered stem cell membrane biomimetic formulation, its preparation method, and its applications. The biomimetic formulation of this invention consists of core-shell structured drug-loaded nanoparticles. The core is a lipid polymer hybrid nanoparticle loaded with a drug for reprogramming microglia metabolism; the outer shell is an engineered stem cell membrane on which ligands that specifically target microglia are highly expressed. The engineered stem cell membrane-coated biomimetic formulation provided by this invention has a simple preparation process and features high drug loading capacity, controlled release, high blood-brain barrier penetration, and precise microglia targeting capability. It can achieve synergistic therapeutic effects through microglia energy metabolism reprogramming and has high clinical research value in the treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention belongs to the field of nanobiopharmaceuticals, specifically relating to an engineered stem cell membrane biomimetic preparation, its preparation method, and its application. Background Technology

[0002] Neurodegenerative diseases are a group of diseases caused by the progressive degeneration or death of neurons in terms of structure and function, resulting in dysfunction of the nervous system. These include Alzheimer's disease, Parkinson's disease, stroke, and multiple cherry blossom syndrome. Neurodegenerative diseases are characterized by the aggregation of abnormally processed and misfolded proteins, as well as neuroinflammation. Microglia, as multifunctional executors of abnormal protein clearance and the release of inflammatory factors, are closely related to these pathological features. Therefore, microglia can serve as an important target for the treatment of neurodegenerative diseases.

[0003] Microglia are resident immune cells in the central nervous system (CNS) of the brain, maintaining brain homeostasis by monitoring the microenvironment. In the event of injury or disease, microglia rapidly recognize and migrate to the damaged site, exerting an immune protective function by clearing pathogens, cell debris, and misfolded proteins. This process requires a large amount of energy. Adenosine triphosphate (ATP), produced from glucose metabolism, is the main energy source for microglia, while glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the main metabolic pathways for glucose. Under normal physiological conditions, microglia primarily exert their immune surveillance function by continuously providing energy through highly efficient OXPHOS. However, in neurodegenerative diseases, pathogens, cell debris, and misfolded proteins accumulate in large quantities. To efficiently clear these foreign substances, microglia need to proliferate, migrate, and phagocytose rapidly. This requires a rapid energy supply, causing the energy supply mode to shift from OXPHOS to the faster-producing glycolysis (10-100 times faster than OXPHOS), enabling them to complete the energy-intensive foreign substance clearance process. However, while continuous glycolysis produces energy rapidly, it suffers from low glucose utilization and insufficient ATP production (approximately 1 / 16th of OXPHOS), leading to impaired chemotaxis and phagocytosis in microglia and weakened foreign body clearance. Furthermore, lactate, a glycolytic metabolite, can directly promote the release of inflammatory factors from microglia, further enhancing glycolysis and creating a vicious cycle of abnormal energy metabolism. Therefore, targeted reprogramming of microglia's energy metabolism can improve microglia dysfunction, increase ATP production to enhance their ability to clear foreign bodies, and reduce inflammatory factor production to alleviate neuroinflammation, thus effectively treating neurodegenerative diseases.

[0004] While targeting and reprogramming microglia's energy metabolism could be beneficial in treating neurodegenerative diseases, developing drugs targeting this site requires overcoming challenges related to the blood-brain barrier (BBB) ​​and microglia-specific delivery. Therefore, developing a formulation that simultaneously possesses BBB penetration capability, microglia targeting, and microglia energy metabolism reprogramming capabilities would be an effective strategy for treating neurodegenerative diseases; however, no such formulations have yet been publicly reported. Summary of the Invention

[0005] This invention addresses the technical problems of poor blood-brain barrier penetration and poor cell targeting in existing drugs for the treatment of neurodegenerative diseases. It develops a class of engineered stem cell membrane-coated biomimetic preparations, their preparation methods, and their application in reprogramming energy metabolism in microglia.

[0006] The first objective of this invention is to provide an engineered stem cell membrane-coated biomimetic formulation comprising a lipid polymer hybrid nanoparticle core, a drug encapsulated in the core, and an engineered stem cell membrane encapsulating the outside of the core.

[0007] As a preferred embodiment of the present invention, the drug is a drug for reprogramming microglia metabolism.

[0008] The drug for reprogramming microglia metabolism described in this invention refers to a drug that can convert microglia energy metabolism from glycolysis to mitochondrial oxidative phosphorylation.

[0009] As a further preferred embodiment of the present invention, the drug for reprogramming microglia metabolism is selected from one or any combination of resveratrol, quercetin, scutellarin, tea polyphenols, emodin, rutin, ferulic acid, and magnolol.

[0010] As a preferred embodiment of the present invention, the lipid polymer hybrid nanoparticles comprise lipids and biocompatible polymers.

[0011] As a preferred embodiment of the present invention, the lipid component is selected from one or any combination of phosphatidylcholine, dipalmitoylphosphatidylcholine, dilauroylphosphatidylcholine, trimethyl-2,3-dioleoyloxypropylammonium bromide, phosphatidylethanolamine, and polyethylene glycol succinate of vitamin E.

[0012] As a preferred embodiment of the present invention, the biocompatible polymer is selected from one or any combination of polylactic acid-glycolic acid copolymer, polyetherimide, polylactic acid, polystyrene, maltodextrin, polyglycolic acid, polyurethane and polycaprolactone.

[0013] The engineered stem cell membrane described in this invention refers to the cell membrane extracted from engineered stem cells prepared through methods such as genetic engineering, ligand coupling, and pretreatment.

[0014] As a preferred embodiment of the present invention, the stem cells are selected from one or any combination of embryonic stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, neural stem cells, adipose stem cells and hematopoietic stem cells.

[0015] As a preferred embodiment of the present invention, the engineered stem cell membrane is extracted from engineered stem cells, and the membrane surface of the engineered stem cells highly expresses key proteins that target microglia.

[0016] As a preferred embodiment of the present invention, the stem cells include one or any combination of embryonic stem cells, mesenchymal stem cells, neural stem cells, adipose stem cells and hematopoietic stem cells.

[0017] As a preferred embodiment of the present invention, the engineered stem cells include one or any combination of stem cells that highly express chemokine receptor 4 on their cell membrane, stem cells that highly express vascular endothelial cell adhesion molecule-1 on their cell membrane, stem cells that highly express intercellular adhesion molecule-1 on their cell membrane, and stem cells that highly express immunoadhesins on their cell membrane.

[0018] As a preferred embodiment of the present invention, the method for stem cell engineering includes one or any combination of lentiviral transfection, hypoxia induction, lipopolysaccharide stimulation, and surface grafting of targeting peptides.

[0019] Preferably, the engineered stem cell membrane is prepared by gradient centrifugation after cell disruption. The cell disruption method includes one or any combination of hypotonic lysis, mechanical grinding, repeated freeze-thaw cycles, and ultrasonic disruption. The gradient centrifugation is performed at 4°C, 3000–8000g for 10–20 min, and the supernatant is collected. Then, the supernatant is centrifuged at 4°C, 100000–250000g for 1–2 h, and the precipitate is collected.

[0020] In this invention, the engineered stem cell membrane-coated biomimetic nanoparticles have a particle size between 50 and 250 nm, which is 5 to 30 nm larger than that of uncoated lipid-polymer hybrid nanoparticles. Their potential ranges from -5 to -20 mV, drug loading is between 10% and 30%, and encapsulation efficiency is between 75% and 98%.

[0021] The second objective of this invention is to provide a method for preparing an engineered stem cell membrane-coated biomimetic formulation, comprising the following steps:

[0022] (1) Dissolve the polymer and drug in an organic reagent that is miscible with water to obtain an organic phase;

[0023] (2) The lipid was dissolved in ethanol, and a certain mass of the lipid solution was added to water to obtain an aqueous phase.

[0024] (3) The organic phase is added dropwise to the aqueous phase and ultrasonically treated to obtain lipid polymer hybrid nanoparticles.

[0025] (4) The engineered stem cell membrane described in claim 4 is mixed with the lipid polymer hybrid nanoparticle core obtained in step (3), and the engineered stem cell membrane is coated on the surface of the lipid polymer hybrid nanoparticle by ultrasonication or extrusion.

[0026] Furthermore, in step (1), the feed ratio (drug:polymer) is 1:20 to 1:5 (mass ratio).

[0027] Furthermore, in step (2), the lipid ratio (lipid:polymer) is 1:20 to 1:3 (mass ratio).

[0028] Furthermore, in step (3), the ratio of organic phase to aqueous phase is 1:20 to 1:5 (volume ratio).

[0029] Furthermore, in step (4), the feed ratio (stem cell membrane: polymer) is 1:15 to 1:1 (mass ratio).

[0030] Furthermore, in step (4), the ultrasonic power of the ultrasonic method is 10-80 Hz and the ultrasonic time is 1-5 min.

[0031] Further, in step (4), the extruder of the extrusion method is an extruder containing a polycarbonate film with a pore size of 200 nm, and the number of extrusions is 3 to 20.

[0032] Furthermore, in step (5), the centrifugation speed is 3000-15000 rpm and the centrifugation time is 10-30 min.

[0033] Furthermore, the preparation method of the present invention also includes step (5) centrifuging and purifying the mixture obtained in step (4) to remove excess stem cell membrane, thereby obtaining a biomimetic preparation coated with engineered stem cell membrane.

[0034] The third objective of this invention is to provide the application of an engineered stem cell membrane-coated biomimetic formulation in the preparation of a drug, preferably in the preparation of a drug for the treatment of neurodegenerative diseases by reprogramming microglia energy metabolism.

[0035] The energy metabolism reprogramming is defined as inhibiting microglia glycolysis while promoting mitochondrial oxidative phosphorylation metabolism.

[0036] Furthermore, the biomimetic formulation coated with engineered stem cell membranes can cross the BBB and precisely target microglia, inhibiting glycolytic metabolism in microglia, promoting mitochondrial oxidative phosphorylation metabolism, and playing a role in metabolic reprogramming.

[0037] The beneficial effects of this invention are as follows:

[0038] The lipid polymer hybrid nanoparticles used in this invention have the advantages of high drug loading capacity and controlled release.

[0039] The engineered stem cell membrane-coated biomimetic formulation prepared in this invention has brain-targeting capabilities and can precisely target microglia.

[0040] The engineered stem cell membrane-coated biomimetic preparation prepared in this invention can reprogram microglia metabolism, increase energy production, thereby promoting foreign body clearance, relieving neuroinflammation, and treating neurodegenerative diseases. Attached Figure Description

[0041] Figure 1 This is a flow cytometry analysis of the expression levels of immunoadhesins in mesenchymal stem cells after hypoxic culture.

[0042] Figure 2 This is a flow cytometry result analysis of the expression level of chemokine receptor 4 after lipopolysaccharide treatment of neural stem cells.

[0043] Figure 3 This is a flow cytometry analysis of the expression level of adhesion molecule-1 in vascular endothelial cells after hypoxic culture of embryonic stem cells.

[0044] Figure 4 Transmission electron microscopy (TEM) images of the uncoated lipid polymer hybrid nanoparticles and the engineered stem cell membrane-coated biomimetic formulations constructed in this invention.

[0045] Figure 5 This is a particle size distribution diagram of the uncoated lipid polymer hybrid nanoparticles and the engineered stem cell membrane-coated biomimetic formulation constructed in this invention.

[0046] Figure 6 This is an in vitro release diagram of the engineered stem cell membrane-coated biomimetic formulation constructed according to the present invention.

[0047] Figure 7 This is a potential characterization diagram of the uncoated lipid polymer hybrid nanoparticles and the engineered stem cell membrane-coated biomimetic formulation constructed in this invention.

[0048] Figure 8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis images of the uncoated lipid polymer hybrid nanoparticles and the engineered stem cell membrane-coated biomimetic formulations constructed in this invention.

[0049] Figure 9 This is a serum stability diagram of the uncoated lipid polymer hybrid nanoparticles and the engineered stem cell membrane-coated biomimetic formulation constructed in this invention.

[0050] Figure 10This is a graph showing the cytotoxicity of the engineered stem cell membrane-coated biomimetic formulation constructed in this invention.

[0051] Figure 11 This is a graph showing the BBB penetration ability of the engineered stem cell membrane-coated biomimetic formulation constructed in this invention.

[0052] Figure 12 This diagram illustrates the microglia affinity of the engineered stem cell membrane-coated biomimetic formulation constructed in this invention.

[0053] Figure 13 This figure shows the ability of the engineered stem cell membrane-coated biomimetic formulation constructed in this invention to be taken up by microglia. Detailed Implementation

[0054] The following detailed embodiments are further descriptions of the present invention. These embodiments are for illustrative purposes only and should not be considered as limiting the present invention in any way.

[0055] I. Preparation process of biomimetic formulations coated with engineered stem cell membranes

[0056] 1. Extraction of stem cell membranes

[0057] Example 1

[0058] This embodiment provides a process for extracting stem cell membranes, including the following steps:

[0059] (1) After washing the expanded mesenchymal stem cells three times with PBS, the cells were scraped off and collected using a cell scraper.

[0060] (2) Add hypotonic lysis buffer to the cells collected in step (1) and grind them at 70 Hz for 5 min in a low-temperature grinder to obtain a cell-broken mixture.

[0061] (3) Centrifuge the mixture obtained in step (2) at 5000g for 10min, collect the supernatant, and continue centrifuging at 214000g for 2h to obtain cell membrane precipitate, which is stored in a -80℃ refrigerator for later use.

[0062] Example 2

[0063] In this embodiment, the engineered stem cells are mesenchymal stem cells that highly express immunoadhesins, the engineering method is hypoxia induction, and the cell membrane extraction method is a combination of hypotonic lysis and mechanical destruction.

[0064] A method for preparing engineered stem cells that highly express immunoadhesins includes the following steps:

[0065] (1) The purified mesenchymal stem cells were cultured normally in an incubator to allow them to adhere to the wall.

[0066] (2) After adhesion, mesenchymal stem cells were cultured in a three-gas incubator for 24 hours under the following conditions: oxygen concentration of 5%, carbon dioxide concentration of 5%, and nitrogen concentration of 90%.

[0067] (3) After 24 hours, remove the cell culture medium, wash three times with PBS, and digest with trypsin to obtain cells.

[0068] Flow cytometry analysis of the immunoadhesin expression levels in the obtained cells showed that the expression level of immunoadhesins in mesenchymal stem cells was significantly increased after hypoxic culture. Figure 1 ).

[0069] Example 3

[0070] A process for preparing and extracting an engineered stem cell membrane that highly expresses immunoadhesins includes the following steps:

[0071] (1) The purified mesenchymal stem cells were cultured normally in an incubator to allow them to adhere to the wall.

[0072] (2) After adhesion, mesenchymal stem cells were cultured in a three-gas incubator for 24 hours under the following conditions: oxygen concentration of 5%, carbon dioxide concentration of 5%, and nitrogen concentration of 90%.

[0073] (3) After 24 hours, remove the culture medium, wash three times with PBS, and scrape off the cells with a cell scraper to collect the cells.

[0074] (4) Add hypotonic lysis buffer to the cells collected in step (3) and grind them at 70 Hz for 5 min in a low-temperature grinder to obtain a cell-broken mixture.

[0075] (5) Centrifuge the mixture obtained in step (4) at 5000g for 10min, collect the supernatant, and continue centrifuging at 214000g for 2h to obtain cell membrane precipitate, which is stored in a -80℃ refrigerator for later use.

[0076] Example 4

[0077] This embodiment provides a method for preparing engineered stem cells that highly express chemokine receptor 4, comprising the following steps:

[0078] (1) The purified neural stem cells were cultured normally in an incubator to allow them to adhere to the wall.

[0079] (2) Remove the culture medium and replace it with fresh culture medium containing 100 ng / mL lipopolysaccharide, and continue culturing for 12 h.

[0080] (3) After 12 hours, remove the cell culture medium, wash three times with PBS, and digest with trypsin to obtain cells.

[0081] Flow cytometry analysis of the expression levels of chemokine receptor 4 in the obtained cells showed that the expression level of chemokine receptor 4 in neural stem cells was significantly increased in lipopolysaccharide (LPS). Figure 2 ).

[0082] Example 5

[0083] A process for preparing and extracting an engineered stem cell membrane that highly expresses chemokine receptor 4 includes the following steps:

[0084] (1) The purified neural stem cells were cultured normally in an incubator to allow them to adhere to the wall.

[0085] (2) Remove the culture medium and replace it with fresh culture medium containing 100 ng / mL lipopolysaccharide, and continue culturing for 12 h.

[0086] (3) After 12 hours, remove the culture medium, wash three times with PBS, and scrape off the cells with a cell scraper to collect the cells.

[0087] (4) Add hypotonic lysis buffer to the cells collected in step (3) and grind them at 70 Hz for 5 min in a low-temperature grinder to obtain a cell-broken mixture.

[0088] (5) Centrifuge the mixture obtained in step (4) at 8000g for 15min, collect the supernatant, and continue centrifuging at 250000g for 2h to obtain cell membrane precipitate, which is stored in a -80℃ refrigerator for later use.

[0089] Example 6

[0090] In this embodiment, the engineered embryonic stem cells highly expressing vascular endothelial cell adhesion molecule-1 were engineered using lentiviral transfection, and the cell membrane was extracted using a combination of repeated freeze-thaw cycles and mechanical disruption. This embodiment provides a method for preparing an embryonic stem cell membrane highly expressing vascular endothelial cell adhesion molecule-1, comprising the following steps:

[0091] (1) Neural stem cells were transfected with lentiviruses to enable them to express vascular endothelial cell adhesion molecule-1 efficiently and stably. Then, they were expanded and cultured to obtain engineered stem cells that highly expressed vascular endothelial cell adhesion molecule-1.

[0092] (2) The expression level of vascular endothelial cell adhesion molecule-1 in the engineered stem cells obtained in step (1) was analyzed by flow cytometry. The results showed that the expression level of vascular endothelial cell adhesion molecule-1 in lentivirus-transfected embryonic stem cells was significantly increased. Figure 3 ).

[0093] (3) After lentivirus transfection, remove the culture medium, wash three times with PBS, and scrape off the cells with a cell scraper to collect the cells.

[0094] (4) Place the cells collected in step (3) in a -20°C freezer for 4 hours, then thaw them at 25°C for 3 hours. Repeat this process 10 times to obtain cell fragments.

[0095] (5) The cell fragments obtained in step (4) were sonicated at 80 Hz for 3 min using a probe sonicator. Centrifuged at 3000 g for 20 min and the supernatant was collected. Centrifuged at 210000 g for 2 h to obtain cell membrane precipitate, which was stored at -80℃ for later use.

[0096] 2. Preparation of lipid polymer hybrid nanoparticle cores for loading reprogrammed microglial cell metabolic drugs

[0097] Table 1. Formulation of lipid polymer hybrid nanoparticle cores for loading reprogrammed microglial cell metabolic drugs

[0098]

[0099]

[0100] The polymer and the reprogrammed metabolite were dissolved in a water-miscible organic solvent at a certain mass ratio to form the organic phase. The phospholipid solution was added to water to form the aqueous phase. The organic phase was then added dropwise to the aqueous phase. The mixed solution was ultrasonicated in a water bath for 20 minutes to obtain lipid polymer hybrid nanoparticles.

[0101] 3. Preparation of engineered stem cell membrane biomimetic formulations for loading reprogrammed microglia metabolic drugs

[0102] Table 2. Process conditions for preparing engineered stem cell membrane biomimetic formulations loaded with reprogrammed microglia metabolic drugs using the ultrasonic method.

[0103]

[0104]

[0105] The stem cell membranes or engineered stem cell membranes prepared in Examples 1-6 were mixed with lipid polymer hybrid nanoparticles prepared in Examples 7-14 and sonicated. The mixture was then centrifuged at 8000 rpm for 20 min to remove excess stem cell membranes, resulting in a biomimetic formulation coated with an engineered stem cell membrane loaded with reprogrammed microglia metabolic drugs.

[0106] Table 3. Process conditions for the extrusion method to prepare engineered stem cell membrane biomimetic formulations loaded with reprogrammed microglia metabolic drugs.

[0107]

[0108] The engineered stem cell membranes prepared in Examples 1-6 were mixed with the lipid polymer hybrid nanoparticles prepared in Examples 7-14. The mixture was repeatedly extruded using an extruder, and excess stem cell membrane was removed by centrifugation at 8000 rpm for 20 min, yielding a biomimetic formulation coated with an engineered stem cell membrane loaded with a drug for microglia metabolism. II. Characterization of the Biomimetic Formulation Coated with Engineered Stem Cell Membrane Further studies were conducted using the biomimetic formulation coated with an engineered stem cell membrane loaded with a drug for microglia metabolism constructed in Example 25.

[0109] 1. Morphological and particle size characterization of biomimetic formulations coated with engineered stem cell membranes

[0110] The morphology of nanoparticles was observed using transmission electron microscopy, such as... Figure 4 As shown, the engineered stem cell membrane-coated biomimetic formulation is a perfectly round sphere with a typical "shell-core" structure.

[0111] The particle size of nanoparticles was determined using a laser particle size analyzer, such as... Figure 5 As shown, the engineered stem cell membrane-coated biomimetic formulation has a particle size of 96 nm, which is 18 nm larger than the uncoated lipid hybrid nanoparticles prepared in Example 7.

[0112] 2. Investigation on the encapsulation efficiency and drug loading of biomimetic formulations coated with engineered stem cell membranes

[0113] 500 μL of the engineered stem cell membrane-coated biomimetic formulation was precisely measured and placed in the inner tube of an ultrafiltration centrifuge tube. The tube was centrifuged at 3000 g for 10 min, and 200 μL of the supernatant was collected. This supernatant was diluted to 1 mL with methanol and injected under specific chromatographic conditions to determine the resveratrol content. The encapsulation efficiency and drug loading were calculated using the following formulas: Encapsulation efficiency (%) = ((drug dosage - free drug dosage) / drug dosage) * 100%, Drug loading (%) = ((drug dosage - free drug dosage) / nanoparticle mass) * 100%. The encapsulation efficiency of the resveratrol-loaded biomimetic nanoparticles was found to be 89%, and the drug loading was 21%.

[0114] 3. In vitro release of biomimetic agents coated with engineered stem cell membranes

[0115] The in vitro release characteristics of the formulation were investigated using dialysis. Using PBS (pH 7.4) as the release medium, 1 mL of the biomimetic formulation coated with an engineered stem cell membrane was placed in 50 mL of PBS. 1 mL of dialysate was collected at 1, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 hours, and an equal volume of fresh membrane was added. The mixture was then placed in a shaker for further analysis. The resveratrol content was determined by injection under specific chromatographic conditions, and the cumulative resveratrol release was calculated. Results are as follows: Figure 6As shown, the biomimetic formulation coated with engineered stem cell membrane can be continuously released within 72 hours, indicating that the biomimetic formulation coated with engineered stem cell membrane plays a sustained-release role in the drug.

[0116] 4. Potential characterization of biomimetic formulations coated with engineered stem cell membranes

[0117] The potential of nanoparticles was measured using a laser particle size analyzer, such as... Figure 7 As shown, the biomimetic agent coated with the engineered stem cell membrane has a potential of -13.5 mV, which is closer to the engineered stem cell membrane's -14.1 mV than the -0.2 mV of the lipid polymer hybrid nanoparticles.

[0118] 5. Protein characterization of biomimetic formulations coated with engineered stem cell membranes

[0119] Nanoparticle proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, such as Figure 8 As shown, the proteins on the biomimetic formulation coated with the engineered stem cell membrane are basically the same as those on the engineered stem cell membrane, indicating that the stem cell membrane was successfully coated with lipid polymer hybrid nanoparticles.

[0120] 6. Serum stability of biomimetic formulations coated with engineered stem cell membranes

[0121] The lipid polymer hybrid nanoparticles prepared in Example 7 and the biomimetic formulation coated with engineered stem cell membrane prepared in Example 25 were respectively added to PBS containing 10% (v / v) fetal bovine serum and incubated at room temperature for one week. The particle size and particle size distribution index (PDI) were then measured using a laser particle size analyzer at pre-arranged time intervals. Figure 9 As shown, within one week, the particle size and PDI of the biomimetic formulation coated with engineered stem cell membrane did not change significantly, while the lipid polymer hybrid nanoparticles aggregated on the fourth day, indicating that the biomimetic formulation coated with engineered stem cell membrane has good serum stability.

[0122] 7. In vitro safety of biomimetic formulations coated with engineered stem cell membranes

[0123] BV-2 microglia were seeded into 96-well plates and cultured for 24 h. The culture medium was then replaced with fresh medium containing different concentrations of free resveratrol and a biomimetic formulation coated with engineered stem cell membranes (prepared in Example 25). The resveratrol concentration ranged from 1 to 100 μg / mL. Incubation continued for another 24 h. The culture medium was then discarded, and the cells were washed twice with PBS. 10 μL of 5 mg / mL MTT phosphate solution was added, and the cells were incubated at 37°C for 4 h. The supernatant was discarded, and 150 μL of DMSO was added. The absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as follows.

[0124] Cell viability = (OD) sample -OD blank ) / (OD control -OD blank )×100%

[0125] Among them, OD sample It is the absorbance (OD) of the test solution well after the test solution treatment. control This is the absorbance, OD of the control wells treated with only blank culture medium. blank The absorbance of the zeroing well with complete culture medium as blank.

[0126] like Figure 10 As shown, the cell viability of the biomimetic agents coated with engineered stem cell membranes at different concentrations was close to 100%, indicating that the biomimetic agents coated with engineered stem cell membranes have good cell safety. III. Targeting Ability Assessment of Biomimetic Agents Coated with Engineered Stem Cell Membranes

[0127] 1. Investigation of the brain-targeting ability of biomimetic agents coated with engineered stem cell membranes.

[0128] An in vitro BBB model was constructed using mouse brain microvascular endothelial cells bEnd.3. The method is as follows: bEnd.3 cells were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 mcg in the upper chamber of a Transwell plate and cultured at 37°C for 7 days. The trans-epithelial electrical resistance (TEER) was measured using a transmembrane resistance meter. When the TEER reached and stabilized at 180 Ω, the BBB model was considered successfully constructed. Coumarin-6-labeled lipid polymer hybrid nanoparticles prepared in Example 7, the biomimetic formulation coated with a stem cell membrane prepared in Example 24, and the biomimetic formulation coated with an engineered stem cell membrane prepared in Example 25 were added to the upper chamber and incubated for 4 h. The culture medium from both the upper and lower chambers was collected, and the absorbance of coumarin-6 was measured using a microplate reader to calculate the BBB penetration rate.

[0129] like Figure 11 As shown, the biomimetic formulation coated with engineered stem cell membranes labeled with coumarin-6 exhibits the highest BBB penetration rate, indicating that this biomimetic formulation can enhance BBB penetration ability.

[0130] 2. Microglial cell affinity of biomimetic agents coated with engineered stem cell membranes

[0131] BV-2 cells were planted at a density of 3 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 mcg / well in 96-well plates and cultured at 37°C for 12 hours. Once the cells had grown to approximately 80%, the medium was replaced with fresh serum-free medium and Aβ was added. 1-42After incubating the solution for 24 hours, the lipid polymer hybrid nanoparticles prepared in Example 7, the biomimetic stem cell membrane-coated formulation prepared in Example 24, and the biomimetic engineered stem cell membrane-coated formulation prepared in Example 25 were added, and incubation continued for another 24 hours. BV-2 cells were then introduced at a rate of 1 × 10⁻⁶ cells per well. 3 Cells were seeded at a density of [number] cells per cell in the lower chamber of a Transwell plate. 600 μL of culture medium containing lipid polymer nanoparticles, a biomimetic preparation coated with a stem cell membrane, and a biomimetic preparation coated with an engineered stem cell membrane was added to the lower chamber. The plates were cultured for 24 h. After removing unmigrated cells from the upper membrane layer, the lower membrane layer was fixed with 4% paraformaldehyde for 15 min, followed by staining with 0.2% crystal violet for 30 min. The number of migrating cells was observed under an inverted fluorescence microscope.

[0132] like Figure 12 As shown, the number of cells that migrated in the group treated with the biomimetic agent coated with engineered stem cell membrane was significantly higher than that in other groups, indicating that the biomimetic agent coated with engineered stem cell membrane has a good affinity for microglia and is beneficial for the agent to target microglia.

[0133] 3. Microglial cell-targeted uptake capability of biomimetic agents coated with engineered stem cell membranes

[0134] Using the microglial cell line BV-2 as the experimental subject, BV-2 cells were spaced at 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 24-well plates and cultured at 37°C for 12 hours. Then, the lipid polymer hybrid nanoparticles prepared in Example 7, labeled with coumarin-6, the biomimetic preparation with stem cell membrane coating prepared in Example 24, and the biomimetic preparation with engineered stem cell membrane coating prepared in Example 25 were added and incubated for 4 hours. Cell uptake was observed under an inverted microscope.

[0135] like Figure 13 As shown, the biomimetic formulation coated with an engineered stem cell membrane labeled with coumarin-6 exhibits the strongest fluorescence intensity, indicating that the biomimetic formulation has microglia targeting capabilities.

[0136] IV. Application of biomimetic agents coated with engineered stem cell membranes in energy metabolism of reprogrammed microglia

[0137] 1. Construct an in vitro energy metabolism disorder model

[0138] Using the microglial cell line BV-2 as the experimental subject, BV-2 cells were spaced at 3 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mcg / well in 24-well plates and cultured at 37°C for 12 hours. Once the cells reached approximately 80% confluence, the medium was replaced with fresh serum-free medium and Aβ was added. 1-42 Incubate the solution for 24 hours.

[0139] 2. Investigation on the ability of engineered stem cell membrane-coated biomimetic nanoparticles to regulate microglial cell metabolism

[0140] BV-2 cells were planted at a density of 3 × 10⁶ cells per well. 3 Cells were seeded at a density of 1000 mcg / well in 96-well plates and cultured at 37°C for 12 hours. Once the cells had grown to approximately 80%, the medium was replaced with fresh serum-free medium and Aβ was added. 1-42 After incubating the solution for 24 hours, lipid polymer hybrid nanoparticles, biomimetic agents coated with stem cell membranes, and biomimetic agents coated with engineered stem cell membranes were added and incubated for another 24 hours. A separate experiment was conducted without Aβ. 1-42 The pre-stimulation group served as a control, and glycolytic stress and mitochondrial stress were tested using Seahorse XFe96.

[0141] Table 4. Effects of Examples 7-32 on microglia glycolysis, mitochondrial oxidative phosphorylation, and ATP levels.

[0142]

[0143]

[0144]

[0145] Compared to not using Aβ 1-42 Pre-stimulation group, Aβ 1-42 Pre-stimulation significantly increased basal, maximal, and reserve glycolytic capacity in microglia, while significantly decreasing basal, maximal, and reserve respiration. This indicates that Aβ-induced enhanced glycolytic metabolism and weakened mitochondrial oxidative phosphorylation in microglia, along with decreased ATP levels. In other words, microglia metabolism underwent a shift from mitochondrial oxidative phosphorylation to glycolytic metabolism, leading to reduced ATP production. Treatment with a biomimetic agent coated with engineered stem cell membranes significantly reduced basal, maximal, and reserve glycolytic capacity, while significantly increasing basal, maximal, and reserve respiration. ATP levels also increased, approaching those of the blank control group. This demonstrates that the biomimetic agent coated with engineered stem cell membranes can effectively reverse the Aβ-induced shift from glycolysis to mitochondrial oxidative phosphorylation, re-enhancing mitochondrial phosphorylation levels and promoting ATP production.

Claims

1. A biomimetic formulation with engineered stem cell membrane coating, characterized in that... The invention comprises a lipid polymer hybrid nanoparticle core, a drug encapsulated within the core, and an engineered stem cell membrane surrounding the core. The engineered stem cell membrane is extracted from engineered stem cells, and the membrane surface of these stem cells highly expresses a key protein targeting microglia. The engineered stem cells are selected from neural stem cells with high expression of chemokine receptor 4, embryonic stem cells with high expression of vascular endothelial cell adhesion molecule-1, and mesenchymal stem cells with high expression of immunoadhesins. The mesenchymal stem cells with high expression of immunoadhesins are engineered using hypoxia-induced methods. The drug is a microglia reprogramming drug, selected from one or more combinations of resveratrol, quercetin, scutellarin, tea polyphenols, emodin, rutin, ferulic acid, and magnolol.

2. The biomimetic formulation according to claim 1, characterized in that... The lipid polymer hybrid nanoparticles comprise lipids and a biocompatible polymer. The lipid component is selected from one or any combination of phosphatidylcholine, dipalmitoyl phosphatidylcholine, dilauroyl phosphatidylcholine, trimethyl-2,3-dioleoyloxypropylammonium bromide, phosphatidylethanolamine, and polyethylene glycol succinate of vitamin E. The biocompatible polymer is selected from one or any combination of polylactic acid-glycolic acid copolymer, polyetherimide, polylactic acid, polystyrene, maltodextrin, polyglycolic acid, polyurethane, and polycaprolactone.

3. The engineered stem cell membrane-coated biomimetic formulation according to any one of claims 1-2, characterized in that, The method for preparing the engineered stem cell membrane-coated biomimetic formulation includes: (1) Dissolve the polymer and drug in an organic reagent that is miscible with water to obtain an organic phase; (2) The organic phase is added dropwise to the lipid-containing aqueous phase under stirring; (3) The mixture obtained in step (2) is ultrasonically treated to obtain lipid polymer hybrid nanoparticles; (4) The engineered stem cell membrane is mixed with the lipid polymer hybrid nanoparticle core obtained in step (3), and the engineered stem cell membrane is coated on the surface of the lipid polymer hybrid nanoparticle by ultrasonication or extrusion.

4. The method for preparing engineered stem cell membranes according to claim 3, characterized in that, In step (1), the mass ratio of drug to polymer is 1:20 to 1:5; in step (2), the mass ratio of lipid to polymer is 1:20 to 1:3; in step (3), the volume ratio of organic phase to aqueous phase is 1:20 to 1:5; in step (4), the mass ratio of stem cell membrane to lipid polymer hybrid nanoparticle core is 1:15 to 1:

1.

5. The preparation method according to claim 4, characterized in that, The ultrasonic method described in step (4) has an ultrasonic power of 10~80 Hz and an ultrasonic time of 1~5 min; the extrusion method described in step (4) uses an extruder containing a polycarbonate film with a pore size of 200 nm and the number of extrusions is 3~20.

6. The preparation method according to claim 3, characterized in that, It also includes step (5) centrifuging and purifying the mixture obtained in step (4) to remove excess stem cell membrane, thereby obtaining a biomimetic preparation coated with engineered stem cell membrane.

7. The use of the engineered stem cell membrane-coated biomimetic formulation according to any one of claims 1 to 2 in the preparation of Alzheimer's disease treatment drugs.

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