Method for recovering microglia and astroglia and application thereof

Abstract

Methods for treating Alzheimer's disease and other neurodegenerative disorders in which the capacity of microglia and / or astrocytes has been altered and impaired are described. The method comprises the step of carrying out brain targeting on PD-L1 by using a brain targeting nanogel which is loaded with a PDL1 antibody and is functionalized by two specific targeting ligands. The nanogel can successfully cross the blood brain barrier to release the antibody, reduce the expression of PD-L1 in the brain, and maintain the integrity of BBB at the same time.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 512,934, entitled “Method for the Revival of Microglia and Astrocytes and its Application,” with a filing date of July 11, 2023, and U.S. Provisional Patent Application Serial No. 63 / 589,727, entitled “Method for the Revival of Microglia and Astrocytes and Applications Thereof,” with a filing date of October 12, 2023, both of which are incorporated herein by reference for all purposes.

[0003] Federal Research Statement

[0004] This invention was made with government support under Grant No. 1R01AG054839-01A1 awarded by the National Institutes of Health. The government has certain rights in this invention. background

[0006] Alzheimer's disease (AD) is the sixth leading cause of death in the United States. The occurrence and progression of AD are the result of a toxic and inflammatory microenvironment induced by β-amyloid (Aβ) plaques, neurofibrillary tangles formed by tau protein, and microglial dysfunction. To date, most of the methods explored for the treatment of AD have focused on reducing the amount of toxic substances and alleviating the inflammatory state of the brain to protect neurons from damage. However, due to the limitations of these methods in both their targets and efficacy, the methods currently available for AD therapy have only shown limited efficacy.

[0007] Although targeting Aβ for AD therapy has failed many times, recently, several antibodies targeting Aβ, including lecanemab and aducanumab, have produced reasonable results in clinical trials, such as reducing Aβ load and slowing the progression of the disease. Unfortunately, none of them has successfully prevented AD progression due to the presence of the blood-brain barrier (BBB), which prevents 98% of small molecules and almost all macromolecules from entering the brain.

[0008] Microglia (resident macrophages of the brain) constantly survey the incidence of pathogens and cell debris in the brain and provide maintenance for brain tissue. In the early stages of AD, activated microglia engulf Aβ fibrils through phagocytosis. With the progression of the disease combined with aging, the phagocytic ability of microglia is altered and impaired, causing the accumulation of Aβ fibrils in the brain, leading to excessive activation of microglia, and promoting inflammatory responses, producing neurotoxic reactive oxygen species and proinflammatory cytokines. Therefore, damaged neurons send neuronal damage signals to microglia to further activate microglia and reproduce cyclically. The continuation of this progressive cycle causes neuronal loss and AD progression. In addition, studies have revealed that the activation of astrocytes is closely related to the progression of AD, including failure to clear Aβ fibrils and microglial debris, release of Aβ effects in pathological tau phosphorylation, abnormal production of γ-aminobutyric acid to inhibit the activity of adjacent neurons, and cause AD neuronal dysfunction.

[0009] The dysregulation of programmed death ligand 1 (PD-L1), expressed in both microglia and astrocytes, plays a key role in regulating the immune response in Alzheimer's disease (AD) and other central nervous system (CNS) diseases. This dysregulation hinders the effective clearance of amyloid β (Aβ) plaques and leads to harmful microglial activation. It has been previously reported that the expression of PD-L1 in microglia and astrocytes is upregulated in both AD patients and AD mouse models. It has also been reported that PD-L1 knockout mice have enhanced M2 microglia, which are characterized by anti-inflammatory and neuroprotective effects.

[0010] PD-L1 antibody (aPDL1) is widely used in cancer immunotherapy by blocking the interaction between PD-1 and PD-L1. Studies have explored the efficacy of aPDL1 in mouse AD models, revealing that aPDL1 can alleviate AD-related symptoms, including reducing Aβ fibril load and tau aggregation in 5XFAD and DM-hTAU models, respectively, as well as reducing inflammatory cytokines and cognitive deficits in both models. Unfortunately, due to the presence of the blood-brain barrier (BBB), only a limited fraction of aPDL1 can reach the brain region after iv administration, making its clinical application impractical.

[0011] The BBB, formed by the adhesions and tight junctions of endothelial cells, protects the brain from damage and bacterial infection by limiting the passage of high molecular weight molecules and pathogens. However, due to the presence of the BBB, more than 98% of existing therapeutic molecules cannot enter the brain. Therefore, therapeutic agents for central nervous system (CNS) diseases cannot effectively reach their targets and thus fail to show therapeutic effects. Various carrier systems have been developed to attempt to deliver therapeutic agents across the BBB, including polymers, dendrimers, nanogels, mesoporous silica particles, and gold nanoparticles.

[0012] While such developments provide improvements in the art, there is room for further improvements. What is needed in the art are methods for treating degenerative disorders such as AD that provide for successful delivery of aPD-L1 across the BBB and thereby restore the beneficial activities of microglia and astrocytes.

[0013] Overview

[0014] According to one embodiment, a method for restoring the function of damaged microglia and / or astrocytes is disclosed. For example, the method may include delivering the nanogel to microglia and / or astrocytes. The nanogel includes a cross-linked network, which in turn includes a first copolymer and a second copolymer. The first copolymer includes a main chain, a first group comprising poly(ethylene glycol) flanked by the first main chain, and a second group flanked by the main chain and conjugated to a PD-L1 antibody via a disulfide bond. The second copolymer also includes a second main chain, having a group comprising polyethylene glycol flanked by the second main chain, and another group flanked by the main chain of the second copolymer and conjugated to a nicotinic acetylcholine receptor (nAChR) targeting ligand via a disulfide bond. The nanogel also includes a receptor for advanced glycation end products (RAGE) targeting ligand, which is conjugated to the surface of the nanogel via a disulfide bond. According to the method, after the nanogel is delivered to damaged microglia and / or astrocytes, various disulfide bonds are degraded, thereby releasing the PD-L1 antibody. After the release of the PD-L1 antibody, PD-L1 expressed by microglia and / or astrocytes is degraded, and the functions of damaged microglia and / or astrocytes are restored.

[0015] Also disclosed is a method for treating neurodegenerative disorders. The method of treatment may generally include delivering a pharmaceutical composition to a subject suffering from a neurodegenerative disorder, the pharmaceutical composition comprising a nanogel as described and a pharmaceutically effective carrier. After the pharmaceutical composition is delivered to the subject, the nanogel can cross the blood-brain barrier, and then various disulfide bonds are degraded, thereby releasing the PD-L1 antibody, resulting in degradation of PD-L1 expressed by damaged microglia and / or astrocytes in the brain and functional recovery of damaged microglia and / or astrocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] A full and enabling disclosure of the subject matter, including the best mode thereof, as would occur to one of ordinary skill in the art, is more particularly set forth in the remainder of this specification, including with reference to the accompanying drawings, in which:

[0018] Figure 1 A scheme for restoring microglia and astrocytes by using disclosed materials for AD therapy is shown.

[0019] Figure 2 A reaction scheme for the formation of poly[(2-(pyridin-2-yldisulfonyl)ethyl acrylate)-co-[poly(ethylene glycol)methoxy]] (PDA-PEG-OCH3) is presented.

[0020] Figure 3 A reaction scheme for forming poly[(2-(pyridin-2-yldisulfonyl)ethyl acrylate)-co-[poly(ethylene glycol)hydroxy]] (PDA-PEG-OH) is presented.

[0021] Figure 4 A reaction scheme for forming a poly[(2-(pyridin-2-yldisulfonyl)ethyl acrylate)-co-[poly(ethylene glycol)[-co-[poly(2-mercaptoethanol)]] (PDA-PEG-BME) intermediate copolymer is presented.

[0022] Figure 5 A reaction scheme for forming a poly[(2-(pyridin-2-yldisulfonyl)ethyl acrylate)-co-[poly(ethylene glycol)[-co-[poly(4-nitrophenyl chloroformate)]] (PDA-PEG-NPC) precursor copolymer is presented.

[0023] Figure 6 Reaction schemes for forming poly[(2-(pyridin-2-yldisulfonyl)ethyl acrylate)-co-[poly(ethylene glycol)]-co-[poly(glutathione-2-methacryloyloxyethylphosphorylcholine)]] (PDA-PEG-MPC) copolymers are presented.

[0024] Figure 7Reaction schemes for forming PDA-PEG-antibody conjugate copolymers are presented.

[0025] Figure 8 A reaction scheme for forming PDA-PEG-MPC-antibody nanogels by cross-linking of PDA-PEG-MPC copolymers with PDA-PEG-antibody conjugate copolymers is presented.

[0026] Fig. 9 A reaction scheme for forming dual-targeted nanogels by surface modification of the nanogels with ligands for RAGE is presented.

[0027] Fig.10 The formation method used to form the brain-targeted nanogel as described in the Examples section is shown.

[0028] Fig.11 Provided is an image of a PAGE gel electrophoresis comprising a nanogel (BTN-PLD1) as described herein.

[0029] Fig.12 TEM images of nanogels as described herein are provided.

[0030] Fig.13 The size distribution of exemplary nanogels as described herein is presented.

[0031] Fig.14 Shown are flow cytometric profiles of cellular uptake and lysosomal escape of the nanogel (BTN-PDL1) as described herein after 4 hours of co-culture with hCMEC / d3 cells.

[0032] Fig.15 Shown is the quantitative analysis of cellular uptake and lysosomal escape of the nanogel (BTN-PDL1) as described herein after 4 hours of co-culture with hCMEC / d3 cells.

[0033] Fig.16 Shown are flow cytometric profiles of cellular uptake and lysosomal escape of the nanogel (BTN-PDL1) as described herein after 4 hours of co-culture with BV2 cells.

[0034] Fig.17 Shown is the quantitative analysis of cellular uptake and lysosomal escape of the nanogel (BTN-PDL1) as described herein after 4 hours of co-culture with BV2 cells.

[0035] Fig.18 Shown are flow cytometric profiles of cellular uptake and lysosomal escape of the nanogel (BTN-PDL1) as described herein after 4 h of co-culture with mouse astrocyte (MA) cells.

[0036] Fig.19 Shown is the quantitative analysis of cellular uptake and lysosomal escape of the nanogel (BTN-PDL1) as described herein after 4 hours of co-culture with MA cells.

[0037] Fig. 20 The BBB transwell model used in the Examples section is schematically shown.

[0038] Fig.21 Presented in Fig. 20 The penetration efficiency of BSA-Cy3 and BTN-BSACy3 was evaluated in the transwell model under various conditions.

[0039] Fig. 22 Immunofluorescence images of the in vivo biodistribution of nanogel (BTN-PDL1) in 5×FAD mice are presented.

[0040] Fig.23 The ex vivo biodistribution of the nanogel (BTN-PDL1) in the brain of 5×FAD mice is presented.

[0041] Fig.24 Shown is the expression of TRIM21 in BV2 and MA cells.

[0042] Fig.25 The expression of PD-L1 in BV2 cells was shown by gel electrophoresis.

[0043] Fig.26 The expression of PD-L1 in BV2 cells was quantitatively shown.

[0044] Fig. 27 The expression of PD-L1 in MA cells was shown by gel electrophoresis.

[0045] Fig.28 The expression of PDL-1 in BV2 cells was quantitatively shown.

[0046] Fig.29 The expression of ZO-1 and Occludin in hCME / D3 cells was shown by gel electrophoresis.

[0047] Fig.30 The expression of ZO-1 in hCMEC / D3 cells was quantitatively shown.

[0048] Fig.31 The expression of occludin in hCMEC / D3 cells was quantitatively shown.

[0049] Fig.32 Shown is the cell viability of BV2 cells after 100 ng / mL LPS challenge.

[0050] Fig.33 Shown is the cell viability of N2a-converted neurons after 48 h incubation with CMBV-2+LPS.

[0051] Fig.34 The remaining Aβ-FITC in the system including the nanogel (BTN-PDL1) or aPDL1 co-cultured with BV2 cells and 5 μM Aβ-FITC is quantitatively shown.

[0052] Fig.35 The transwell model used in the examples described herein is schematically shown.

[0053] Fig.36 Quantitatively showing the expression of neurons transformed with nanogels, Cy5-labeled BV2 cells, Aβ-FITC and N2a as described after 24 h of treatment. Fig.35 of the remaining Aβ in the system.

[0054] Fig.37 Quantification shows the expression of neurons transformed with nanogels, Cy5-labeled BV2 cells, Aβ-FITC and N2a as described after 24 h of treatment. Fig.35 BV2 cells infiltrated in the system.

[0055] Fig.38 Quantitatively showing the expression of neurons transformed with nanogels, Cy5-labeled BV2 cells, Aβ-FITC and N2a as described after 24 h of treatment. Fig.35 of the remaining neurons in the system.

[0056] Fig.39 The amount of BV2 cell debris remaining after MA cells fed with Cy5-labeled BV2 cell debris were co-cultured with Aβ-FITC for 96 hours is quantitatively shown.

[0057] Fig.40 The amount of Aβ remaining after MA cells fed with Cy5-labeled BV2 cell fragments were co-cultured with Aβ-FITC for 96 hours is quantitatively shown.

[0058] Fig.41 Representative photographs of nest-building in the mouse model during the in vivo treatment protocol are shown.

[0059] Fig.42 The nesting scores in different groups during the in vivo treatment regimen in the mouse model are shown quantitatively.

[0060] Fig.43 Representative swimming paths during the in vivo treatment protocol of the mouse model are presented.

[0061] Fig.44 Escape latency of different groups in the in vivo treatment regimen of the mouse model is quantitatively shown.

[0062] Fig.45 The number of times the escape plateau was crossed during the in vivo treatment regimen in the mouse model is quantified.

[0063] Fig.46 Shown is PD-L1 expression assessed using whole-brain immunoblotting.

[0064] Fig.47 The whole brain PD-L1 expression values ​​are quantitatively shown.

[0065] Details

[0066] The various embodiments of the disclosed subject matter will now be referred to in detail, and one or more examples of the embodiment are set forth below. Each embodiment is provided by explaining the subject matter rather than by limiting it. In fact, it will be apparent to those skilled in the art that multiple modifications and variations may be made in the present disclosure without departing from the scope or spirit of the subject matter. For example, the features illustrated or described as a part of an embodiment may be used in another embodiment to produce yet other embodiments.

[0067] Disclosed herein is a method for treating AD and other neurodegenerative disorders in which the ability of microglia and / or astrocytes has been altered and impaired. More particularly, a method of treatment is disclosed, including using a brain-targeted nanogel (BTN) loaded with aPDL1 and functionalized with two specific targeting ligands to therapeutically target PD-L1 to the brain. According to the disclosed method, when aPDL1 is delivered across the BBB, the antibody can be released from the nanogel, and the expression of PD-L1 in the brain is reduced while maintaining the integrity of the BBB, and thereby promoting the recovery of the desired activity and synergistic interaction between microglia and astrocytes, promoting the degradation of Aβ plaques, and enhancing neuronal survival and cognitive function. As disclosed herein, therapeutic targeting of PD-L1 with aPDL1 can present a promising approach to restore microglia and astrocytes, improve neuronal health and cognitive function in AD and other central nervous system (CNS) diseases.

[0068] More particularly, disclosed are therapeutic applications using brain-targeted nanogels (also referred to in the present disclosure as BTN-PDL1) that integrate the functionality of ligands targeting two specific receptor types, nAChR and RAGE, with aPDL1 to deliver the nanogel across the BBB and subsequently degrade the disulfide bonds of the nanogel in the brain environment, thereby releasing the antibody, which in turn downregulates PD-L1 in microglia and astrocytes and restores their function in clearing Aβ and alleviating neuroinflammation.

[0069] Without wishing to be bound by any particular theory, the disclosed methods are understood to work via endogenous protein degradation mechanisms that can selectively degrade proteins in mammalian cells by intracellular delivery of antibodies. The endogenous method utilizes intrinsic cellular self-defense mechanisms that include intracellular supply of PDL1 antibodies, binding of the antibodies to their target protein PDL1, and formation of an antibody / target protein complex having a tripartite motif (TRIM21)-21, followed by ubiquitination and proteasome-mediated degradation of the complex.

[0070] The first of the two receptor types targeted by the nanogel is the nicotinic acetylcholine receptor (nAchR). nAchRs are ligand-gated cation channels located in the presynaptic compartment where they regulate the release of acetylcholine or other neurotransmitters, as well as cellular excitability and neuronal integration. nAchR expression is widespread throughout the brain.

[0071] The second of the two receptor types targeted by the nanogel is the receptor for advanced glycation end products (RAGE). During neuroinflammation, RAGE is overexpressed on the BBB. In addition, RAGE is highly expressed in the brain of AD patients. Therefore, the delivery material for the disclosed method beneficially includes a ligand targeting RAGE.

[0072] By incorporating two different types of targeting ligands in conjunction with aPDL1 with nanogels designed to degrade in the relatively highly acidic and high redox potential environment of the brain, the disclosed materials can be used in therapeutic methods to deliver materials across the BBB and to targeted cell types in order to release the payload in the targeted area and thereby help prevent the progression of disease states such as AD and provide a way to improve the retention of learning and cognitive abilities in subjects treated according to the disclosed methods.

[0073] Figure 1 Methods for forming and utilizing the disclosed materials are schematically described. Figure 1 A shows the fabrication scheme of brain-targeting materials. Figure 1B schematically illustrates a method of action that includes material across the BBB via RAGE and nAChR receptor-mediated endocytosis, beyond which the delivery vehicle can enter microglia and astrocytes, and then release the aPDL1 payload upon degradation of the disulfide bonds of the nanogel, and subsequently degrade intracellular PD-L1. Through degradation of intracellular PD-L1, both microglia and astrocytes can restore normal function. For example, the restored cells can clear Aβ plaques and protect neurons and the BBB from Aβ-induced toxicity.

[0074] The brain-targeted system can be used to treat any of a variety of neurodegenerative diseases, including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, multiple sclerosis, neonatal hypoxic-ischemic encephalopathy, stroke, spinal cord injury, brain injury, retinal injury, post-traumatic stress disorder, brain tumors, glioblastoma, chemobrain, long COVID and frontotemporal dementia, among others.

[0075] The delivery system is in the form of a nanogel particle that includes a nanoparticle structure that provides a delivery system and a cross-linked copolymer that carries an aPD-LI antibody and a targeting ligand by using disulfide-containing side ligands of the nanogel. For example, the cross-linked copolymer of the nanogel can be conjugated with one or more of an antibody, a ligand targeting a receptor, and one or more additional desired components such as additional ligands for targeting specific cellular components.

[0076] As used herein, the term "nanogel" refers to particles having a nanometer average cross-sectional size, for example, less than 1000 nanometers, such as about 500 nanometers or less, about 300 nanometers or less, about 100 nanometers or less, about 50 nanometers or less, about 30 nanometers or less, about 20 nanometers, or about 10 nanometers or less, such as from about 5 nanometers to about 50 nanometers in some embodiments. The nanogel particles can also be highly hydrophilic and can have a high water content.

[0077] The copolymer of the cross-linked nanogel can be produced from the initial reaction product of poly(ethylene glycol) and a monomer containing pyridine-2-thiol. The copolymer can be a block copolymer, a random copolymer, or any combination thereof. Although illustrated as a block copolymer in the present disclosure, it should be understood that this representation is simply a shorthand for any type of copolymer (e.g., random copolymer, block copolymer, etc.) including repeating units (including side groups as described herein).

[0078] The copolymer formed after the reaction of poly(ethylene glycol) and pyridine-2-thiol-containing monomers can include a first group comprising hydrophilic poly(ethylene glycol) pendant to the polymer backbone, and a second group pendant to the polymer backbone that is terminated by pyridine and also includes a disulfide bond between the polymer backbone and the terminal pyridine group. The pyridine groups of the copolymer thus formed can be further reacted in one or more intermediate formation steps to conjugate aPDL1 and two BBB targeting ligands and any other desired components of the delivery system to the copolymer and form a copolymer that can also be cross-linked to form a nanogel.

[0079] For example, a first copolymer may include aPDL1 conjugated to a side group and a second copolymer may include a phosphorylcholine group as a component of the side group (e.g., a terminal component of the side group), and both copolymers may include a hydrophilic poly(ethylene glycol) side group as well as an unreacted group for further conjugation.

[0080] The antibody and targeting ligand can be bonded to the nanoparticle via a disulfide-containing bond that can be degraded in environments including acidic pH and / or high redox potential. Thus, due to the sensitivity of the disulfide ligand attachment bond to the blood-brain barrier lysosomal and / or nervous system environment, the nanoparticle can circulate in the subject's system, cross the BBB via targeting provided by the BBB receptor targeting ligand, and then release the aPDL1 payload.

[0081] Nanoparticles can be prepared from polymeric materials, which can be biocompatible, provide long cycle life in vivo, and can be successfully connected to at least two different parts via acidic response and / or redox potential response bond formation. As utilized herein, acid-sensitive bonds can generally refer to about pH 6.8 or lower, such as about pH 4 to about pH 6.8 environments will degrade or otherwise break and in a higher pH (such as about 7 or higher) environment with a more stable bond. Redox potential sensitive bonds can generally refer to the bond that will degrade in the following environment: the redox potential that the environment has is equal to the redox potential that glutathione concentration is about 0.1mM to about 10mM.

[0082] After cross-linking between two (or more) copolymers and between two (or more) copolymers, for example, the disulfide bonds of the unreacted side groups of the copolymers are cleaved, followed by the cross-linking reaction of air oxidation, resulting nanogel particles can be formed, which can encapsulate antibodies and hydrophilic poly (ethylene glycol) and other desirable groups displayed on the surface and / or encapsulated in the nanogel. The part of the targeting receptor can be put together with the copolymer before or after cross-linking, so as to be present on the outer surface of the cross-linked nanogel, and promote the nanogel to transport across the BBB, and target microglia and / or astrocytes in the brain. For example, one or both parts can be put together with the remaining reactive groups of one or more copolymers after cross-linking and nanogel formation. Alternatively, one or both parts can be put together with the reactive groups of one or more copolymers before cross-linking and nanogel formation.

[0083] The nanogel particles thus formed may include a hydrophilic poly(ethylene glycol) component, with a targeting ligand and aPDL1 conjugated to the nanogel copolymer via an acid-sensitive disulfide bond for therapeutic delivery. The targeting ligand may be present at a high concentration at the outer surface of the nanogel particles. The formation of nanogel particles can give advantages to central nervous system therapy. For example, due to the presence of hydrophilic poly(ethylene glycol) side groups, the circulation time of the nanogel in the systemic delivery method can be greatly extended. In addition, aPDL1 can be encapsulated in a cross-linked nanogel, and thus prevented from degrading before delivery from the nanogel after crossing the BBB. Since the copolymers that form the nanogel and conjugate the antibody to the nanogel can include environmentally sensitive bonds common on the brain side of the BBB barrier, aPDL1 can be released from the nanogel after the nanogel enters an environment that is conducive to bond degradation, such as after the nanogel crosses the BBB and in some embodiments, after being taken up by cells on the brain side of the BBB.

[0084] The poly(ethylene glycol) used to form the delivery system can include reactivity, such as methacrylate functional groups, for reaction with the pyridine-2-thiol monomer. In a specific embodiment, the initial copolymer formation scheme can include the reaction of the pyridine-2-thiol monomer with a poly(ethylene glycol) methacrylate having the general structure:

[0085]

[0086] Wherein in some embodiments, n is from about 4 to about 1,000, from about 5 to about 100, or from about 6 to about 20.

[0087] However, poly(ethylene glycol) methacrylate is not limited to the above, and may include modifications at the terminal hydroxyl group. For example, the poly(ethylene glycol) starting material may include, but is not limited to, poly(ethylene glycol) methacrylate, poly(ethylene glycol) methyl ether methacrylate, and the like.

[0088] Pyridine-2-thiol containing monomers can be copolymerized with poly(ethylene glycol) to form a copolymer including pyridine-2-thiol groups pendant to the copolymer backbone. By way of example, but not limitation, the pyridine-2-thiol monomers can include one or more of the following:

[0089] (Pyridine-2-thiol)ethyl acrylate (PDA):

[0090]

[0091] (Pyridine-2-thiol)ethyl methacrylate (PDMA):

[0092]

[0093] N-(2-(pyridin-2-yldisulfonyl)ethyl)acrylamide:

[0094]

[0095] N-(2-(pyridin-2-yldisulfonyl)ethyl)methacrylamide:

[0096]

[0097] Ethyl (2-(pyridin-2-yldisulfonyl)ethyl) carbonate

[0098]

[0099] The reaction can be promoted by any suitable catalyst. For example, the catalyst can include but is not limited to azobisisobutyronitrile (AIBN), benzoyl peroxide, potassium persulfate or a combination thereof. The polymerization can be free radical polymerization or active radical polymerization, including stable radical mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization and iodine transfer polymerization. The last monomer ((2-(pyridin-2-yldisulfonyl)ethyl)ethyl carbonate) of the above example can be polymerized by ring-opening polymerization using isopropanol as initiator and Sn(Oct)2 as catalyst.

[0100] By way of example, Figure 2 The reaction scheme of 2-(pyridin-2-yldisulfonyl)ethyl acrylate (PDSA) monomer and poly(ethylene glycol) methyl ether methacrylate (PEGMMA) is shown, and Figure 3 A reaction scheme for PDSA and poly(ethylene glycol) methacrylate (PGMA) is shown, both using azobisisobutyronitrile (AIBN) as a catalyst. As can be seen, the poly(ethylene glycol) component of the copolymer thus formed can be in the form of groups pendant to the copolymer backbone, wherein the secondary pendant groups include disulfide bonds and ester bonds and terminate in a pyridine group.

[0101] Although shown with specific chain lengths, it should be understood that the figures show only representative embodiments, and that the length of the poly(ethylene glycol) pendant units and the molar ratio of the pyridine-2-thiol containing repeating units of the polymer to the poly(ethylene glycol) pendant units are not particularly limited. For example, the molar ratio of the pyridine-2-thiol containing repeating units of the polymer to the poly(ethylene glycol) pendant units can be from about 100:1 to about 1:100 ( Figure 1 and Figure 2 In some embodiments, the ratio of x:y is from about 20:1 to about 1:20, in some embodiments, from about 10:1 to about 1:10, or in some embodiments, about 1:1.

[0102] In some embodiments, the copolymers formed may generally have a weight average molecular weight of from about 1,000 to about 100,000 or from about 5,000 to about 35,000. In one embodiment, the copolymers may have a polydispersity index (PDI) of from about 1.05 to about 3 or in some embodiments, from about 1.15 to about 1.30.

[0103] PDI is a measure of the molecular mass distribution in a given polymer sample. The calculated PDI is the weight average molecular weight divided by the number average molecular weight. It indicates the distribution of individual molecular masses in a batch of polymers. PDI has a value equal to or greater than 1, but as the polymer chains approach uniform chain length, the PDI approaches unity (i.e., 1).

[0104] Number average molecular weight (M n ) is easily calculated by those of ordinary skill in the art and generally refers to the common arithmetic mean or average of the molecular weights of individual macromolecules. It is determined by measuring the molecular weight of n polymer molecules, summing the weights, and dividing by n, such as represented by the following formula:

[0105]

[0106] Where N i is the molecular weight M i The number-average molecular weight of a polymer can be determined by gel permeation chromatography and all colligative methods, such as vapor pressure osmometry or end group determination.

[0107] Weight average molecular weight (Mw ) is easily calculated by a person skilled in the art and generally refers to:

[0108]

[0109] Where N i is the molecular weight M as above i The weight average molecular weight can be determined by light scattering, small angle neutron scattering (SANS), X-ray scattering, gel permeation chromatography and sedimentation velocity.

[0110] The copolymer formed after the reaction of poly(ethylene glycol) with a pyridine-2-thiol-containing monomer, such as poly[(2-(pyridin-2-yldisulfonyl)ethyl acrylate-co-[poly(ethylene glycol)]] (PDA-PEG), can be a precursor copolymer that can be further directly reacted with a compound or monomer containing a functional group used in the final delivery system (e.g., aPDL1 or a BBB receptor ligand) to form a copolymer that is then cross-linked to form a nanogel. In some embodiments, the copolymer formed after the initial reaction of poly(ethylene glycol) with a pyridine-2-thiol-containing monomer can be an intermediate copolymer and / or a precursor copolymer that can then be further reacted to form one or more additional intermediates, which can then be directly reacted with a material for a final delivery system and thereby form a copolymer that is then cross-linked to form a nanogel. In general, further functionalization of the precursor copolymer or intermediate copolymer can be performed via reaction of the pyridine groups (or reaction products of the pyridine groups) of the copolymer.

[0111] In order to conjugate the aPD-L1 antibody to the precursor copolymer, the pyridine group of the intermediate copolymer can be activated by a functional group that is conducive to antibody conjugation. For example, the intermediate copolymer can be functionalized by reacting with an activator having a general structure of YQX. X and Y can independently be leaving groups, one of which (Y) is capable of reacting with a pyridine group or an intermediate reaction product group provided after the monomer reacts with the pyridine group, and the other (X) is capable of conjugating with the antibody.

[0112] In such Figure 4 In one embodiment illustrated in FIG. 1 , a first intermediate copolymer formed by the reaction of poly(ethylene glycol) with a pyridine-2-thiol-containing monomer can be modified by reaction of a portion of the pyridine-terminated groups to form a second intermediate copolymer including hydroxyl-terminated groups. In further reaction of the second intermediate copolymer (e.g., Figure 5 ), the pendant hydroxyl-terminated group can be attached to the Y functional group of the activator (e.g., Figure 5The activated side groups of the resulting precursor copolymer can be activated via the remaining X functional groups (e.g., Figure 5 In this embodiment, the poly(ethylene glycol) components of the first intermediate copolymer and the second intermediate copolymer will generally not terminate in hydroxyl groups in order to avoid activation of the poly(ethylene glycol) components by reaction with the activator groups and to ensure that the activated pendant groups are generated from the pyridine-terminated groups of the initially formed copolymer.

[0113] Examples of materials that can react with pyridine groups to form hydroxyl-terminated side groups can include mercapto alcohols such as 2-mercaptoethanol (BME), 3-mercapto-1-butanol, etc., or other suitable reagents that can provide terminal hydroxyl groups via a thiol-disulfide exchange reaction (see, e.g., Figure 3 ).

[0114] The terminal hydroxyl groups of the intermediate copolymer thus formed can be used to react with an activator to provide a precursor copolymer comprising a side-linked leaving group X suitable for conjugation with an antibody. By way of example, X and Y can include, but are not limited to, a halide group, a mesyl group, a tosyl group, an aryloxy group such as a phenoxy group, and a substituted aryloxy group such as a substituted phenoxy group. Examples of activators include, but are not limited to, chloroformates, such as 4-nitrophenyl chloroformate, pentafluorophenyl chloroformate, succinimidyl chloroformate, or a combination thereof.

[0115] After activation, the precursor copolymer can be conjugated to aPDL1 through reaction of the activated side groups with antibodies under conditions as known in the art (eg, pH 8.5, etc.).

[0116] In addition to the aPDL1 conjugated thereto, the nanogel can also include a ligand for targeting nAchR. In a specific embodiment, the targeting ligand can include a phosphorylcholine group that can be used as a ligand for any neuronal subtype nAchR present on the BBB itself or on either side of the BBB, and the neuronal subtype nAchR includes twelve different nicotinic receptor subunits: homomeric combinations and heteromeric combinations of α2-α10 and β2-β4. Examples of nAchR neuronal subtypes that can be targeted by the phosphorylcholine ligands of the nanogel can include, but are not limited to, (α4)3(β2)2, (α4)2(β2)3, (α3)2(β4)3, α4α6β3(β2)2 and (α7)5, and combinations thereof. In a specific embodiment, the phosphorylcholine ligand can target α7nAchR that includes only α7 subunits.

[0117] In one embodiment, a phosphorylcholine ligand can be incorporated to target nAchRs expressed by microglia or astrocytes when aPDL1 is delivered therapeutically to the interior of the cell. The phosphorylcholine group can also be combined with a RAGE targeting ligand to promote crossing the BBB.

[0118] In the formation of the copolymer, a precursor copolymer (e.g., a PDA-PEG copolymer as initially formed) can be reacted with a monomer comprising a phosphorylcholine group. Alternatively, the PDA-PEG copolymer can be an intermediate copolymer that can be further modified to form a precursor copolymer comprising a suitable functional group for reacting with a monomer comprising a phosphorylcholine group.

[0119] In one embodiment, the phosphorylcholine group-containing monomer may include the following general structure:

[0120]

[0121] The phosphorylcholine head of the monomer includes only a single carbon between the ammonium group and the phosphate group, rather than the more common two-carbon ethyl group in phosphorylcholine monomers.

[0122] Of course, the monomer containing phosphorylcholine is not limited to such embodiment. The example of the monomer comprising phosphorylcholine group can include but is not limited to 2-acryloyloxyethyl phosphorylcholine, 2-methacryloyloxyethyl phosphorylcholine (MPC), 2-(meth)acryloyloxyethoxyethyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxyethoxynonyl phosphorylcholine, allyl phosphorylcholine, butenyl phosphorylcholine, hexenyl phosphorylcholine, octenyl phosphorylcholine, decenyl phosphorylcholine or its combination.

[0123] exist Figure 1 and Fig. 9 In the illustrated embodiment, the RAGE targeting ligand can be conjugated to the outer surface of the cross-linked nanogel. The RAGE targeting peptides or small molecules included herein include peptides or small molecules that target one or more domains (e.g., V, C1 or C2 domains), receptor isoforms or polymorphisms. By way of example, but not limitation, RAGE targeting ligands can include relatively low molecular weight advanced glycation end products such as Nε-carboxy-methyl-lysine (CML), Nε-carboxy-ethyl-lysine (CEL), quinolinic acid and methylglyoxal derived hydroimidazoles (MG-H), as well as larger peptide-based structures, including Aβ (e.g., Aβ 1-40 and Aβ 1-42 ), HMGB1, S100 / calgranulin, Aβ fibrils and cross-linked / modified Aβ.

[0124] The nanogel can be administered as a component of a pharmaceutical composition in a pharmaceutically effective amount to a subject in need thereof.

[0125] The term "pharmaceutically effective amount" refers to the amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human being that is being sought by a researcher or clinician. The amount may be a therapeutically effective amount.

[0126] The term "pharmaceutically acceptable carrier" is used herein to refer to a carrier that is generally safe, non-toxic, and neither biologically undesirable nor otherwise undesirable and is acceptable for veterinary use as well as human pharmaceutical use that can be used to prepare a pharmaceutical composition. "Pharmaceutically acceptable carrier" as used in this specification and claims may include both one such carrier and more than one such carrier. The term "pharmaceutically acceptable" refers to a carrier that is compatible with the other ingredients of the formulation and is not harmful to the recipient thereof.

[0127] The term "administration of" or "administering" a pharmaceutical composition should be understood to mean providing the pharmaceutical composition to an individual in need of treatment in a form that can be introduced into the body of that individual in a therapeutically useful form and in a therapeutically useful amount, including but not limited to: oral dosage forms such as tablets, capsules, syrups, suspensions, etc.; and injectable dosage forms such as IV, IM or IP, etc.

[0128] Pharmaceutical compositions can be prepared by any method known in the pharmaceutical field. Pharmaceutical compositions include any composition made by mixing nanogels and pharmaceutically acceptable carriers, optionally together with one or more other components as known in the art. The carrier can take a variety of forms, depending on the form of the product required for administration, such as oral or parenteral (including intravenous). Therefore, the pharmaceutical composition can be presented as discrete units suitable for oral administration, such as capsules, cachets or tablets, each containing a predetermined amount of active ingredient. In addition, the composition can be presented as a powder, a granule, a solution, a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. In addition to the common dosage forms described above, the composition can also be administered by controlled release means and / or a delivery device. The aforementioned list is only illustrative and is not intended to be limited in any way.

[0129] Pharmaceutical compositions intended for oral use may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, colorants, and preservatives to provide pharmaceutically excellent (elegant) and palatable products. Tablets may contain a composition having at least one of the compounds described herein mixed with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture. These excipients may be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating agents and disintegrants such as corn starch or alginic acid; adhesives such as starch, gelatin or gum arabic, and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, and thereby provide a sustained effect for a longer period of time. Tablets may be prepared by compression or molding, optionally with one or more auxiliary ingredients or excipients. Compressed tablets can be prepared by compressing in a suitable machine at least one disclosed compound in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant. Molded tablets can be prepared by molding in a suitable machine a mixture of the moistened powdered compound and an inert liquid diluent.

[0130] Pharmaceutical compositions for oral use may also be presented as hard gelatin capsules in which one or more of the disclosed compounds are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which one or more of the disclosed compounds are mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0131] Pharmaceutical compositions can also include aqueous suspensions containing nanogels mixed with excipients suitable for making aqueous suspensions. In addition, oily suspensions can be prepared by suspending the nanogels in vegetable oils, for example, peanut oil, olive oil, sesame oil or coconut oil or in mineral oils such as liquid paraffin. Oily suspensions can also contain various excipients. Pharmaceutical compositions can also be in the form of oil-in-water emulsions, which can also contain excipients, such as sweeteners and flavorings.

[0132] The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension, or in the form of a sterile powder for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be an effective liquid for easy injection. The pharmaceutical composition must be stable under the conditions of manufacture and storage, and should prevent the contaminating effects of microorganisms such as bacteria and fungi.

[0133] Beneficially, the disclosed aPDL1-loaded brain-targeted nanogels can effectively cross the BBB, deliver aPDL1 to microglia and astrocytes, release aPDL1 intracellularly, subsequently deplete PD-L1 in microglia and astrocytes, and restore the function of the cells in clearing Aβ and alleviating neuroinflammation in AD brains. Therefore, the disclosed materials can be beneficially used to prevent the progression of AD and maintain learning and cognitive abilities.

[0134] The disclosed materials and methods provide promising strategies for modulating the brain's immune response and addressing the underlying pathology of neurodegenerative disorders.

[0135] The present invention may be better understood with reference to the examples set forth below. Example

[0136] Chemicals

[0137] Cyanine 5 NHS ester (Cy5-NHS) was purchased from Lumiprobe Co. (Cockeysville, MD, USA). A 6.5 mm (Costar, 3413) was purchased from Fisher Scientific. Amyloid-β42 peptide was purchased from GL Biochem (Shanghai, catalog number 052487). 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Tokyo Chemical Industry Co., Ltd (Portland, OR, USA). Fluorescein isothiocyanate (FITC) and thioflavin S (ThS) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

[0138] Cell culture

[0139] BV2 (ATCC; HTB-26) was cultured in Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% v / v fetal bovine serum (FBS) (Cytiva, catalog number SH30396.03) and 1% v / v penicillin-streptomycin (PS) (P0781, Sigma) with 10,000 units of penicillin. Primary human astrocytes (Sciencell, catalog number 1800) and primary mouse astrocytes (Sciencell, catalog number 1800-57) were maintained with 1% v / v FBS and 1% v / v penicillin-streptomycin (PS) supplemented with 10,000 units of penicillin in astrocyte culture medium (Sciencell, catalog number 1801), astrocyte growth supplement (Sciencell, catalog number 1852). All cells were incubated at 37°C in a 5% CO2 incubator.

[0140] Fabrication and characterization of nanogels

[0141] As described above and as Fig.10 Synthesis of PDA-PEG-IgG (poly IgG) and PDA-PEG-PDL1 (poly PDL1) shown in . In general, the scheme includes: (a) reaction of poly (ethylene glycol) methacrylate (PEG360), 2'2-azobisisobutyronitrile (AIBN), anisole, 65 ° C. (b) further reaction with MPC-SH, rt. (c) reaction of poly (ethylene glycol) methyl ether methacrylate (PEG500), 2'2-azobisisobutyronitrile (AIBN), anisole, 65 ° C. (d) further reaction with 2-mercaptoethanol (BME), dichloromethane, rt. (e) followed by reaction with 4-nitrophenyl chloroformate, pyridine, dichloromethane, 4 ° C transferred to rt. (f) followed by conjugation with aPDL1, PBS buffer (pH = 8.4), dimethyl sulfoxide (DMSO), 4 ° C. (g) Cross-linking of functionalized copolymers in the presence of tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dimethyl sulfoxide (DMSO), rt. (h) Surface functionalization of cross-linked nanogels with RAGE peptide-Mal, dd H2O, 4°C.

[0142] In step (g), PDA-PEG-MPC was mixed with polyantibody, followed by addition of 5% tris(2-carboxyethyl)phosphine (TCEP) and crosslinked in DMSO under stirring at room temperature for 15 min. Next, the reaction mixture was added dropwise to dd H2O under stirring on ice to form nanogel. The nanogel solution was further dialyzed against dd H2O for 3 times at 4°C. The solution was then dialyzed by The dialysis tube (regenerated cellulose, MWCO: 100 kDa) was concentrated. The RAGE targeting peptide (w / w 2% polymer dissolved in DMSO) was added to the nanogel solution and stirred at 4°C for 24 h. Next, the nanogel solution was dialyzed against dd H2O at least 3 times at 4°C to obtain a MWCO of 100 kDa. 8000 kDa dialysis bag to remove free peptide and DMSO. Finally, the solution was passed through The dialysis tubes were concentrated again and stored at 4 °C.

[0143] The nanogel suspension was loaded onto a 300 mesh copper grid and air-dried. The morphology of the nanogel particles was captured using a transmission electron microscope (Hitachi HT7800 TEM, Hitachi High Technologies, Tokyo, Japan). The hydrodynamic size and zeta potential of the nanogel were measured by a Zetasizer (Nano-ZS).

[0144] Polyacrylamide gel electrophoresis of PDDA-PEG-aPDL1

[0145] To confirm that the antibody was successfully conjugated to the polymer, polyacrylamide gel electrophoresis was performed. To simulate the intracellular microenvironment, 2 mM GSH was added to release the antibody from the polymer-antibody conjugate. After electrophoresis for 90 min at rt, the gel was stained with Coomassie Brilliant Blue for 1 h and washed with water until the bands were clear. The migration of the bands on the gel was analyzed by ChemiDoc TM System (BIO-RAD) imaging.

[0146] Cytotoxicity assay

[0147] The effect of nanogel on protecting cells from LPS damage was determined using MTT assay. BV2 cells were seeded in 96-well plates (8,000 cells / well) at 37°C, 5% CO2 and cultured overnight to allow cell attachment. Cells were incubated for 3 hours with complete medium supplemented with 100ng / mL LPS. Afterwards, aPDL1 or nanogel was added and incubated for another 48 hours. The viability of the treated cells was assessed using MTT assay by measuring the absorbance at 570nm with a Molecular Devices SpectraMax i3x microplate reader. Data were analyzed and plotted using GraphPad Prism 9.4.1.

[0148] Aβ oligomer-FITC labeling and the formation of Aβ aggregates

[0149] First, freeze-dried Aβ 42The peptide was dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) to a final concentration of 5 mg / mL. Afterwards, the solution was sonicated for 20 min and then shaken at room temperature for 2 h. HFIP was removed under a gentle stream of nitrogen and stored at -20 °C. Aβ 42 Peptide samples were freshly reconstituted before each use to avoid self-assembly. After dissolving the sample in DMSO, it was sonicated at room temperature for 10 min and then diluted in PBS for use as a stock solution. For FITC labeling, Aβ oligomers (1 mg) and FITC (0.1 mg) were reacted in DMSO and stirred at room temperature. After 24 h, the solution was dialyzed 3 times against DMSO (dialysis bag MW1,000 kDa) to remove free FITC. During this period, Aβ oligomers should assemble to form Aβ aggregates.

[0150] Uptake of Aβ-FITC by BV2 cells

[0151] BV2 cells were seeded in glass bottom dishes and cultured overnight. The Aβ-FITC aggregates prepared above were added to the cells and incubated for 3 hours. Afterwards, different treatments were added to the cells without removing the old culture medium. Cell images were taken at different time points of 3, 8 and 24 hours using a confocal microscope.

[0152] Flow cytometry

[0153] HCMEC / d3, BV2 and MA cells are cultured in 6-well plates. After adding blank, aPDL1-Cy5 and BTN-PDL1-Cy5 to the cells, they are incubated at 37 ° C for 3 hours in a cell culture incubator. 0.25% trypsin is used to harvest cells for 3 minutes, and 4% paraformaldehyde (PFA) is used to fix 20 minutes at room temperature. Cells are washed three times with PBS, resuspended in 200 μL of PBS, and analyzed using BD LSRFortessa flow cytometer (BD Biosciences). Flow cytometry data analysis is performed using FACSDiva software (Becton, Dickinson and Company).

[0154] Permeation of BTN-IgG-Cy3 into Transwell Endothelial Cell HCMEC / d3 Simulated BBB Model

[0155] Transwell inserts (0.4 cm 2 ) were placed in a 24-well matching plate and incubated with collagen at 37°C for 1 hour. The inserts were then washed once with PBS. Next, 1×10 5HCMEC / d3 cells were seeded in 200 μL of complete endothelial culture medium, and 500 μL of complete endothelial culture medium was added to the lower layer. Transendothelial resistance (TEER) was measured every day until TEER readings were stable, indicating that BBB integrity was established. For the treatment group including blank, BSA-Cy3 and BTN-PDL1-BSA-Cy3, the sample was treated with complete culture medium, complete culture medium supplemented with BV2 cell supernatant culture medium concentrate or complete culture medium supplemented with LPS activated BV2 cell supernatant culture medium concentrate. At different time points, samples were collected, and fluorescence intensity was measured at an excitation wavelength of 550 nm and an emission wavelength of 562 nm to calculate penetration efficiency.

[0156] Immunoblotting

[0157] BV2 cells were cultured in 6-well plates. Cells were pretreated with LPS for 3 hours and then subjected to different treatments. Cells were collected and proteins were extracted with extraction reagent (Thermo Scientific, catalog number 78501). The BCA quantification kit was determined and bromophenol blue was added to adjust the final protein concentration to 1 mg / mL. The samples were boiled at 95°C for 15 min and then returned to room temperature. Proteins (20 μg / well) were loaded in 10% PAGE-GEL and electrophoresed under voltage. The proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% BSA at rt for 1 hour, and incubated with the primary antibody at 4°C overnight, followed by incubation with HRP-conjugated secondary antibodies at rt for 1 hour. The blots were exposed to ChemiDoc TM Imaging system (BIO-RAD).

[0158] ELISA assay

[0159] ELISA test is followed by the protocol of mouse TNFα uncoated ELISA kit (Invitrogen, catalog number 88-7324-88) and mouse IL-1β uncoated ELISA kit (Invitrogen, catalog number 88-7013-88). BV2 cells are seeded in 6-well plates at 20,000 cells / well until the cells attach. 100μg / mL LPS 100μL / well pretreatment for 3 hours, then different treatments are added. After 48 hours, the culture medium is collected separately and centrifuged at 3,000rpm for 10min to remove the flowing cells. The production of nitrate / nitrite in the culture medium is quantified.

[0160] Therapeutic effect of nanogel on AD mouse model

[0161] To study the therapeutic effect of nanogel on AD, 5×FAD mice (8 months old) were randomly divided into 4 groups (n=7) and injected with saline, aPDL1, BTN-IgG and nanogel (4 mg / kg) iv once a week for two weeks. Age-matched C57BL / 6 mice in the normal group and AD mice in the control group were administered with saline.

[0162] Biodistribution of aPDL1-Cy5 and nanogel-Cy5

[0163] The test was performed on 8 month old 5×FAD mice. The mice were depilated and then injected with nanogel-Cy5 nanoparticles iv through the tail vein. Images were taken under the IVIS system at different time points. The biodistribution of aPDL1 and BTN-PDL1 was determined in vivo by the IVIS spectral system. Four hours after injection, the mice were sacrificed and perfused with PBS and 4% paraformaldehyde. The brain, heart, liver, spleen, lungs and kidneys were collected and imaged ex vivo using the IVIS spectral system.

[0164] Therapeutic effect of nanogel on AD mouse model

[0165] To study the therapeutic effect of nanogel on AD, 5×FAD mice (8 months old) were randomly divided into 4 groups (n=7) and injected iv with saline, aPDL1, nanogel functionalized with IgG instead of PDL1, and nanogel described herein (4 mg / kg) once a week for two weeks. Age-matched C57BL / 6 mice in the normal group and AD mice in the control group were administered with saline.

[0166] Nesting assay

[0167] Nesting ability was assessed once a week. Mice were individually housed in cages containing pressed cotton cubes at noon to test their nesting behavior. The nesting results were photographed and scored the next morning. A 5-point nest rating scale was used, for example, 1, no nest; 2, incomplete nest; 3, flat nest; 4, cup-shaped nest; 5, dome nest, to characterize the constructed nests.

[0168] Morris water maze assay

[0169] The test was conducted on 5×FAD mice aged 8 months. The swimming pool was made of blue polyethylene. On the southeast side of the pool, there was a transparent plastic escape platform. The pool was filled to 10 cm above the platform, and the water was made opaque by milk. The water temperature was maintained between 22°C and 24°C. In order to eliminate competitive environmental cues, the lights in the pool room were turned off, and the lights on the four floors were set diagonally. A camera installed on the central ceiling of the test room recorded the behavior of the mice. These cameras were connected to the EthoVision3.1 computerized video tracking system. Before the final test, the mice needed 5 days of non-spatial training to adapt to the pool and underwater platform. The mice were placed in the pool in four diagonal directions and four 60-second swimming sessions were performed with an interval of more than 30 minutes. After removing the platform on the sixth day, a random position was selected, and the mice were placed in the swimming pool alone for testing. Data were collected and analyzed by ANOVA.

[0170] Immunocytochemistry

[0171] The cells were treated with 4% paraformaldehyde for 30 min, then incubated with 0.1% Triton-X 100 (TX-100) for 3 min, and blocked with 5% BSA buffer at room temperature for 1 hour. Afterwards, the samples were treated with the primary antibody overnight at 4°C. The next day, the samples were washed 3 times / 5 min with PBS-T. The samples were incubated with fluorescently labeled secondary antibodies at room temperature for 1 h and washed 3 times with PBS-T. Then, the cell nuclei of the samples were labeled with Hoechst 33342. Finally, the slides were mounted with Prolong TM The cells were mounted with Gold antifade reagent (Invitrogen, catalog, P36934) and sealed with a cover glass. TM FL microscopy imaging.

[0172] Immunohistochemistry

[0173] The tissue was treated with 4% paraformaldehyde at rt for 24 hours, followed by gradual dehydration in 15% sucrose PBS solution and 30% sucrose PBS solution for another 24 hours. The dehydrated tissue was embedded in optimal cutting temperature (OCT, Fisher Healthcare, catalog number 4587) and frozen overnight at -80°C. All organs were cut into 12 μm slices with a freezing microtome and stored at -20°C. The resulting tissue sections were incubated with 0.1% TX-100 for 3 minutes and blocked at room temperature for 1 hour under 5% BSA buffer. Afterwards, the samples were treated with the primary antibody at 4°C overnight. The next day, the samples were washed 3 times / 5 minutes with PBS-T. The samples were incubated with fluorescently labeled secondary antibodies at room temperature for 1 hour and washed 3 times with PBS-T. Then, the cell nuclei of the samples were labeled with Hoechst 33342. Finally, the slides were removed by staining with Prolong TM Gold antifade reagent (Invitrogen, catalog, P36934) was mounted and covered, and sealed with a cover glass, and then EVOS TM FL microscopy imaging.

[0174] Hematoxylin and eosin (H&E) staining

[0175] Freshly sectioned tissue sections were allowed to return to room temperature and OCT was removed by washing 3 times / 15 min with PBS. The tissue was fixed for 5 min using cooled acetone. The sections were then stained with an H&E staining kit. Afterwards, the sections were stained with Cytoseal TM XYL (Epredia, catalog number 8312-4) was mounted and imaged using an Amscope microscope.

[0176] Statistical analysis

[0177] All data are presented as mean ± standard deviation (SD) (n ≥ 3), and statistical significance was analyzed by GraphPad Prism 9.0 (GraphPad Prism Software Inc., San Diego, California) using Student's t-test or ANOVA with Tukey's significant difference. Differences were considered significant when p value was less than 0.05.

[0178] result

[0179] Characterization of nanogels

[0180] To confirm the successful encapsulation of aPDL1 into the nanogels and its release under a reducing environment simulating intracellular conditions, agarose gel electrophoresis was performed. Fig.11 Shown in. Fig.11 Lane 4 is a protein marker. The samples in lanes 5-7 (aPDL1, BTN-PDL1 and polymer) were incubated at 1 mM GSH and 37°C for 1 hour. The size distribution of the nanogels was determined to be 98.43 ± 41.43 nm (PDI: 0.16, Fig.13 ), and its morphology was spherical as determined by transmission electron microscopy ( Fig.12 ). The surface charge of BTN-PDL1 was determined by zeta sizer to be -14±9.32 mV.

[0181] Cellular uptake of TN-PDL1 by brain endothelial cells, microglia, and astrocytes

[0182] To detect the cellular uptake of nanogels by HCMEC / d3, BV2, and mouse astrocytes (MA), flow cytometry analysis was performed. Fig.14 and Fig.15 Results for HCMEC / d3 cells are shown. Fig.16 and Fig.17 Results for BV2 cells are shown, and Fig.18 and Fig.19 Results for MA cells are shown. As shown, the results revealed that BTN-PDL1 exhibited significantly higher efficacy in entering hCMEC / D3, BV2, and MA cells compared to aPDL1. The separate red signals in the confocal images further confirmed that BTN-PDL1 could effectively escape from lysosomes after entering microglia, which may be due to the proton sponge effect induced by the protonated PDA groups in the acidic lysosomal environment.

[0183] Evaluation of the penetration efficiency of BTN-BSA-Cy3 nanoparticles

[0184] To investigate the ability of nanogels to penetrate the BBB, the penetration efficiency of TN-BSA-Cy3 nanoparticles was evaluated in an in vitro BBB Transwell model ( Fig. 20 ). Nanoparticle delivery was evaluated in vitro using a transwell model, in which brain microvascular endothelial cells (HCMEC / d3) were cultured on a porous filter membrane in the upper chamber. The integrity of the BBB in vitro was determined by measuring transendothelial electrical resistance (TEER), while permeability was assessed by detecting the fluorescent signal of Cy3-labeled nanoparticles. Fig.21As indicated in , it was found that TN-BSA exhibited much higher BBB penetration efficacy than free BSA. Interestingly, after stimulation of conditioned medium from activated BV2 medium, which can induce upregulation of RAGE receptors on the BBB, an improved penetration of TN-BSA was observed. In contrast, only a slight increase was observed for the penetration of free BSA. Therefore, as Fig.21 As shown in Figure 3, it was demonstrated that TN-based nanocarriers can effectively utilize overexpressed RAGE to penetrate the BBB in an inflammatory setting.

[0185] Evaluation of RAGE expression in cerebral vessels of 5×FAD mice

[0186] To confirm the overexpression of RAGE expression in the BBB of AD mice, immunofluorescence co-localization was performed in brain sections of 8-month-old 5×FAD mice. Blood vessels and RAGE were detected with CD31 and RAGE antibodies, respectively. The results revealed that RAGE expression in the BBB was not obvious in normal mice. In contrast, strong red signals overlapping with CD31-labeled blood vessels were observed in brain sections of AD mice, indicating that RAGE is a viable target for nanoparticles to effectively deliver aPDL1 to the brain.

[0187] In vivo biodistribution analysis of BTN-PDL1-Cy5

[0188] To further evaluate the biodistribution of aPDL1 in mice, aPDL1 was labeled with the fluorescent dye Cy5 and subsequently conjugated with PDA-PEG polymers to fabricate nanogels. The biodistribution of the resulting BTN-PDL1-Cy5 was investigated in 5×FAD mice using the Spectral In Vivo Imaging System (PerkinElmer, Waltham, MA, USA). The excitation / emission wavelength pair of 610 / 650 nm provided the best signal-to-noise ratio and was therefore used for whole-body and anatomical organ imaging. Whole-body images were obtained at 0.5, 1, 2, and 4 hours after iv injection ( Fig. 22 ) (n=3 for each treatment). Afterwards, major organs were harvested and ex vivo fluorescence signal transduction was obtained ( Fig.23, brain images are shown). 0.5 hours after injection, the fluorescence intensity of aPDL1-Cy5 was mainly accumulated in the mouse bladder and was almost undetectable in the brain, indicating its poor BBB penetration ability and rapid elimination. In contrast, strong fluorescence signals of BTN-PDL1-Cy5 were observed in the animal brain both in vivo and in vitro, indicating the significant brain targeting effect of BTN-PDL1. In order to further explore whether BTN-PDL1 can penetrate the BBB, the collected brain slices were stained with CD31 and DAPI. Compared with the weak red signal in the mouse brain slices treated with aPDL1-Cy5, a large number of red dots far away from the green signal were observed in the mouse brain slices treated with BTN-PDL1-Cy5, verifying its outstanding BBB penetration ability in the AD mouse model.

[0189] TRIM21 expression in microglia and astrocytes

[0190] Since the presence of TRIM21 is considered desirable for the success of improving the disclosed methods, the expression of TRIM21 was assessed in both microglia and astrocytes. Fig.24 As shown in , it was confirmed that TRIM21 is highly expressed in both microglia and astrocytes.

[0191] BTN-PDL1 downregulates PD-L1 in microglia

[0192] To investigate whether BTN-PDL1 exhibits PD-L1-lowering effects in microglia, LPS was used to trigger the activation of BV2 cells. Fig.25 and Fig.26 As shown in , it was revealed that LPS induced upregulation of PD-L1 in microglia, confirming the PD-L1-promoting role of inflammatory conditions. Fig.25 and Fig.26 As shown in , under the stimulation of LPS, 1 μg / ml of BTN-PDL1 caused the PD-L1 expression in microglia to decrease to normal levels, indicating that PD-L1 was successfully degraded in microglia via the disclosed method.

[0193] BTN-PDL1 downregulates PD-L1 in astrocytes

[0194] To investigate whether BTN-PDL1 exhibits PD-L1-lowering effects in astrocytes, conditioned medium (CM) from BV2 cells stimulated with LPS was used to express BV-2+LPS ), which is known to contain cytokines that can activate astrocytes and upregulate PDL1 expression. Fig. 27 and Fig.28 As shown in the figure, it reveals that CM BV-2+LPSInduced upregulation of PD-L1 in astrocytes. Similar to its effect on microglia, 1 μg / ml BTN-PDL1 also led to BV-2+LPS The expression of PD-L1 in astrocytes was reduced to normal levels under the stimulation of BTN-PDL1, indicating that PD-L1 was successfully degraded in astrocytes via BTN-PDL1-based Trim-Away.

[0195] BTN-PDL1 protects BBB integrity

[0196] To further determine the safety of BTN-PDL1 without compromising the blood-brain barrier (BBB), the expression of tight junction proteins ZO-1 and occludin in endothelial cells was examined. Fig.29 , Fig.30 and Fig.31 As indicated, CM BV-2+LPS induced downregulation of ZO-1 and occludin, which are associated with the integrity of the BBB. Surprisingly, BTN-PDL1 treatment restored the expression of ZO-1 and occludin, suggesting a BBB-protective role of BTN-PDL1.

[0197] BTN-PDL1 inhibits microglial activation

[0198] Immunofluorescence imaging indicated that BTN-PDL1 also downregulated the increase of PD-L1 induced by LPS stimulation. In addition, BTN-PDL1 attenuated the activation of microglia, as confirmed by the reduction of IBA-1 expression. Fig.32 As indicated in , the viability of BV2 cells was significantly improved.

[0199] Neuroprotective effects of BTN-PDL1

[0200] CM BV-2+LPS Induces the death of N2a-differentiated neurons ( Fig.33 ), attributed to the release of toxic cytokines by activated microglia. Surprisingly, BTN-PDL1 was found to BV-2+LPS The treated neurons showed significant protective effects.

[0201] BTN-PDL1 improves the ability of microglia to degrade Aβ

[0202] Further evaluation of microglial function after BTN-PDL1 indicated downregulation of microglial PD-L1. The results revealed that BTN-PDL1-treated microglia effectively engulfed Aβ within 3 h of incubation, as confirmed by colocalization of Aβ and lysosomes in fluorescence imaging. In addition, most of the engulfed Aβ signal disappeared within 24 h, indicating Aβ degradation. In contrast, there was little or no change in Aβ in BV2 cells incubated with aPDL1-treated ( Fig.34 ).

[0203] BTN-PDL1-restored microglia protect neurons from Aβ injury

[0204] To determine whether BTN-PDL1-restored microglia can migrate and clear Aβ, a Transwell model was used ( Fig.35 The appearance of Cy5-labeled BV2 and decreased Aβ-FITC in the bottom chamber confirmed the restored microglial activity after BTN-PDL1 treatment ( Fig.36 , Fig.37 It is also worth mentioning that the number and morphology of neurons in the BTN-PDL1 treated group were significantly improved ( Fig.38 ).

[0205] BTN-PDL1 improves the ability of astrocytes to engulf and degrade Aβ and microglial cell debris

[0206] As is known, astrocytes can clear Aβ and microglial cell debris. Fig.39 and Fig.40 As shown in Figure 3, reduced BV2 fragmentation and Aβ signals after 4 days of BTN-PDL1 treatment confirm that the restored astrocytes are able to engulf and degrade Aβ and microglial debris.

[0207] Therapeutic effect of BTN-PDL1 on AD mouse model

[0208] a) Nesting assay

[0209] Nesting behavior is a widely used indicator of overall health. In this study, a previously established method for assessing the nesting ability of mice was adopted. In particular, 8-month-old 5×FAD mice were selected for the experiment. Each mouse was housed individually in a nest cage and provided with a 5×5 cm pressed cotton square overnight during the dark phase. Drug administration included a first iv injection at 8.25 months and a second injection at 8.5 months. Nest formation was assessed before and 3 weeks after drug administration. As Fig.41 and Fig.42As indicated in , the nesting skills of mice in the control, aPDL1 and TN-IgG treated groups remained unchanged. However, BTN-PDL1 treated animals significantly improved their nesting skills.

[0210] b) Morris water maze test

[0211] To evaluate the effect of BTN-PDL1 on cognitive skills and memory in 5×FAD mice, Morris water maze (MWM) was used. The performance of mice was recorded by video and XT15 software was used for analysis to determine the latency to reach the platform and the number of target platform crossings for each mouse during the test period. Interestingly, after two weeks of treatment, BTN-PDL1-treated AD mice spent most of their time in the target quadrant ( Fig.43 ), which was different from that shown in control, aPDL1, and BTN-IgG treated AD mice, but similar to that shown in normal mice. In addition, BTN-PDL1 treated AD mice showed learning ability similar to that of normal mice ( Fig.44 ), as evidenced by decreased escape latency during the training session. In addition, BTN-PDL1-treated AD mice crossed the target platform more often than controls, indicating improved memory ability ( Fig.45 ).

[0212] Expression of PD-L1 in brain tissue

[0213] The complex relationship between programmed death ligand 1 (PD-L1) expression, neuroinflammation, and clearance of Aβ plaques in the brain has attracted great interest in recent studies. In this study, the expression of PD-L1 in different groups of mice was investigated to elucidate its potential role in these processes. The study revealed that the expression of PD-L1 in the brain of AD mice was significantly increased compared with age-matched normal mice. Interestingly, a slightly attenuated increase trend was observed in the AD group treated with aPDL1. However, BTN-PDL1 treatment effectively counteracted this effect by reducing PD-L1 expression and restoring it to normal levels ( Fig.46 , Fig.47 ).

[0214] To further understand the distribution of PD-L1, PD-L1 / IBA1 immunobiological analysis was performed on brain sections from the mouse hippocampus. The analysis revealed that microglia in the control group exhibited upregulated PD-L1, implying their involvement in the inflammatory response. This finding was supported by the colocalization of PD-L1 and activated microglia. Interestingly, the aPDL1-treated group did not show a decrease in PD-L1 expression, while BTN-PDL1 treatment led to the disappearance of aggregated microglia along with a significant decrease in PD-L1 expression. A similar quenching effect on astrocyte activation was also noted in BTN-PDL1-treated mice, confirmed by a reduction in GFAP signals. At the same time, the reduction in Aβ plaques stained by ThS confirmed the recovery of microglia and astrocytes after BTN-PDL1 treatment.

[0215] While certain embodiments of the disclosed subject matter have been described using specific terms, such description is for illustrative purposes only, and it is to be understood that changes and variations may be made without departing from the spirit or scope of the subject matter.

Claims

1. A method for restoring the function of damaged microglia and / or astrocytes, comprising delivering a nanogel to the microglia and / or astrocytes, the nanogel comprising a cross-linked network comprising a first copolymer and a second copolymer, the first copolymer comprising a first main chain, a first group flanked by the first main chain, and a second group flanked by the first main chain and conjugated to a PD-L1 antibody via a first disulfide bond, the first group comprising a first poly(ethylene glycol), the second copolymer comprising a second main chain, a third group flanked by the second main chain, and a fourth group flanked by the second main chain and conjugated to a nicotinic acetylcholine receptor (nAChR) targeting ligand via a second disulfide bond, the third group comprising a second poly(ethylene glycol), the nanogel further comprising a receptor for advanced glycation end products (RAGE) targeting ligand conjugated to the surface of the nanogel via a third disulfide bond; wherein After delivering the nanogel to the damaged microglia and / or astrocytes, the first disulfide bond, the second disulfide bond, and the third disulfide bond are degraded, thereby releasing the PD-L1 antibody, and wherein After the release of the PD-L1 antibody, PD-L1 expressed by the microglia and / or astrocytes is degraded, and the functions of the damaged microglia and / or astrocytes are restored. 2 . The method according to claim 1 , wherein the PD-L1 antibody is released in the microglia and / or astrocytes.

3. The method of claim 1 or claim 2, wherein the nanogel is delivered to the microglia and / or astrocytes in an environment having a pH of about 6.8 or less.

4. The method of any one of the preceding claims, wherein the first copolymer comprises the reaction product of a third poly(ethylene glycol) and a first pyridine-2-thiol-containing monomer, and the second copolymer comprises the reaction product of a fourth poly(ethylene glycol) and a second pyridine-2-thiol-containing monomer.

5. The method of claim 4, wherein the first pyridine-2-thiol-containing monomer and the second pyridine-2-thiol-containing monomer are independently selected from (pyridine-2-thiol)ethyl acrylate, (pyridine-2-thiol)ethyl methacrylate, N-(2-(pyridin-2-yldisulfonyl)ethyl)acrylamide, N-(2-(pyridin-2-yldisulfonyl)ethyl)methacrylamide, and (2-(pyridin-2-yldisulfonyl)ethyl)ethyl carbonate.

6. The method of any one of the preceding claims, wherein the nAChR targeting ligand comprises a phosphorylcholine group.

7. The method of claim 6, wherein the fourth side group comprises the following structure:

8. The method of any one of the preceding claims, wherein the RAGE targeting ligand comprises Nε-carboxy-methyl-lysine, Nε-carboxy-ethyl-lysine, quinolinic acid, methylglyoxal-derivatized hydroimidazolide, Aβ peptide, HMGB1, or S100 / calgranulin.

9. The method according to any one of the preceding claims, wherein the function of the microglia and / or astrocytes comprises degradation of Aβ.

10. The method of any one of the preceding claims, wherein the function of the astrocytes comprises degradation of microglial debris.

11. The method according to any one of the preceding claims, wherein the function of the microglia and / or astrocytes comprises a reduction in activation of the microglia and / or astrocytes.

12. A method for treating a neurodegenerative disorder, the method comprising: A pharmaceutical composition is delivered to a subject, the composition comprising a nanogel and a pharmaceutically effective carrier, the nanogel comprising a cross-linked network comprising a first copolymer and a second copolymer, the first copolymer comprising a first backbone, a first group pendant to the first backbone, and a second group pendant to the first backbone conjugated to a PD-L1 antibody via a first disulfide bond, the first group comprising a first poly(ethylene glycol), the second copolymer comprising a second backbone, a third group pendant to the second backbone, and a fourth group pendant to the second backbone conjugated to a nicotinic acetylcholine receptor (nAChR) targeting ligand via a second disulfide bond, the third group comprising a second poly(ethylene glycol), the nanogel further comprising a receptor for advanced glycation end products (RAGE) targeting ligand conjugated at the surface of the nanogel via a third disulfide bond; After delivering the pharmaceutical composition to the subject, the nanogel crosses the blood-brain barrier; and wherein The first disulfide bond, the second disulfide bond and the third disulfide bond are degraded after crossing the blood-brain barrier, thereby releasing the PD-L1 antibody, and wherein After the release of the PD-L1 antibody, PD-L1 expressed by microglia and / or astrocytes is degraded, and the functions of damaged microglia and / or astrocytes of the brain are restored. 13 . The method according to claim 12 , wherein the nanogel enters microglia and / or astrocytes in the brain, so that the PD-L1 antibody is released in the microglia and / or astrocytes.

14. The method of claim 12 or claim 13, wherein the neurodegenerative disorder is Alzheimer's disease.

15. The method of claim 12 or claim 13, wherein the neurodegenerative disorder comprises amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, spinal muscular atrophy, multiple sclerosis, neonatal hypoxic-ischemic encephalopathy, stroke, spinal cord injury, brain injury, retinal injury, post-traumatic stress disorder, brain tumor, glioblastoma, chemo brain, long COVID, or frontotemporal dementia.

16. The method of any one of claims 12-15, wherein the first copolymer comprises the reaction product of a third poly(ethylene glycol) and a first pyridine-2-thiol-containing monomer, and the second copolymer comprises the reaction product of a fourth poly(ethylene glycol) and a second pyridine-2-thiol-containing monomer.

17. The method of any one of claims 12-16, wherein the nAChR targeting ligand comprises a phosphorylcholine group.

18. The method of claim 17, wherein the fourth side group comprises the following structure:

19. The method of any one of claims 12-18, wherein the RAGE targeting ligand comprises Nε-carboxy-methyl-lysine, Nε-carboxy-ethyl-lysine, quinolinic acid, methylglyoxal-derivatized hydroimidazolide, Aβ peptide, HMGB1, or S100 / calgranulin.

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