Intelligent polymer-lipid-based nanodrug delivery system for improving BBB permeability through a dual active brain targeting strategy

Abstract

The blood-brain barrier (BBB) ​​and the blood-cerebrospinal fluid (CSF) barrier (BCSFB) impede effective systemic drug delivery to the CNS. This application provides compounds and nanoparticles for increasing drug permeability across the BBB. Specifically, this application provides nanoparticles for the diagnosis and treatment of central nervous system (CNS) diseases, as well as methods for preparing them. These nanoparticles are polymer-lipid-based nanoparticles (PLNPs) functionalized to promote permeability across the blood-brain barrier (BBB) ​​and accumulation within CNS disease areas. In particular, the nanoparticles target LDL receptors and / or glucose transporters. In various embodiments, the nanoparticles include terpolymers containing polysorbates (such as polysorbate 80), polyacrylic acids (such as polymethacrylic acid (PMAA)), and various polysaccharides (including maltodextrin), and the nanoparticles also contain cholesterol and lipids. The nanoparticles encapsulate payloads, which are therapeutic molecules, biomolecules, contrast agents, or nucleotides.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 508,505, filed on June 15, 2023, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Systemic drug treatment of neurological diseases such as brain tumors, congenital metabolic disorders (e.g., lysosomal storage diseases), infectious diseases, and neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), stroke, amyotrophic lateral sclerosis (ALS), Friedreich's ataxia (FRDA), and multiple sclerosis (MS)) is a difficult challenge, which is due to the unique protective barriers of the central nervous system (CNS). Such innate barriers, mainly the blood - brain barrier (BBB) and the blood - cerebrospinal fluid (CSF) barrier (BCSFB), not only play an important role in maintaining the ionic and volumetric environment while protecting the CNS from toxic and infectious substances, but also cause obstacles to effective systemic drug delivery to the CNS. Although many drugs have the potential for treatment of CNS diseases, most of these drugs are not clinically used because of the brain barriers. Different strategies for efficient CNS delivery have been studied to facilitate passage through these barriers to enhance drug delivery to the CNS.

[0003] Among these strategies, nanotechnology has emerged as an exciting and promising new platform for treating CNS diseases, demonstrating great potential to overcome problems associated with conventional therapeutic approaches. Molecules can be nanoengineered to perform multiple specific functions, such as crossing the blood-brain barrier (BBB), targeting specific cells or signaling pathways, responding to endogenous stimuli, functioning as a vehicle for gene delivery, and promoting nerve regeneration and cell survival. Various types of nanoparticles (NPs), namely liposomes, lipid nanoparticles, polymer nanoparticles, dendrimers, cyclodextrins, silica nanoparticles, magnetic nanoparticles, gold nanoparticles, quantum dots, and carbon nanotubes, have been cited as attractive candidates for enhancing drug permeability across the BBB. The suitability of nanosystems for delivery to the brain depends on properties such as nanometer size, surface charge, morphology, and, in particular, molecular recognition and interaction (active targeting) between specific ligands conjugated on the nanoparticle surface and molecules overexpressed at target sites in the brain.

[0004] Active targeting is particularly important when designing solutions to achieve this goal because this strategy allows nanoparticles to be directed to desired locations, thereby enabling the transport and delivery of drugs to sites of action in the brain. In fact, these nanosystems have a large surface area-to-volume ratio, which increases the chemical reactivity of the nanoparticles and allows for surface modification by molecules that can be recognized by receptors / transporters overexpressed in the brain bone ducts (BBB) ​​and cell-specific receptors overexpressed in brain tissue. Essentially, there are three different strategies to achieve this objective: adsorption-mediated transcytosis, transporter-mediated transcytosis, and receptor-mediated transcytosis.

[0005] Adsorption-mediated transcytosis (AMT) provides a pathway for delivering nanoparticles to the brain across the blood-brain barrier (BBB). BBB endothelial cells constitute a phospholipid-rich membrane covered by a polysaccharide coat of heparan sulfate proteoglycans (HSPGs), i.e., glypican and syndecane. Furthermore, numerous carboxyl groups of sialycoproteins and sialycolipids are present on this side of the BBB. Collectively, these contribute to the highly negatively charged luminal side of the BBB. Therefore, AMT can be facilitated by electrostatic interactions between the negatively charged portion exposed on the luminal surface of brain endothelial cells and the cationic groups of ligands conjugated on the nanoparticle surface. However, cell-specific targeting is not guaranteed by AMT alone, as positively charged molecules can be rapidly and indiscriminately adsorbed by all negatively charged cell membranes and penetrate various cells.

[0006] An alternative strategy for drug delivery to the brain is the use of BBB-specific transporters for the efficient delivery of low molecular weight nutrients from the bloodstream to the CNS. More than 20 different transporters are known in the BBB, and it is possible to synthesize nanoparticles with surface-conjugated molecules that are well recognized by transporters overexpressed in brain endothelial cells. Transporter-mediated transcytosis (TMT) is an important approach for designing nanocarriers for delivery to the brain.

[0007] Another way to reach brain tissue is through receptor-mediated transcytosis (RMT), utilizing receptors overexpressed in the blood-brain barrier (BBB). Nanoparticles can be modified with specific ligands for these receptors, and therefore can be taken up by brain endothelial cells. Once inside the brain, the nanoparticles should reach their correct cellular targets. Active targeting is particularly important when designing solutions to achieve this goal, as this strategy allows nanoparticles to be directed to the desired site of action by modifying the surface of the NPs with molecules that can be specifically recognized by receptors or transporters overexpressed in the brain.

[0008] Over the past several decades, various strategies have been applied to nanoparticles to improve the efficiency of therapeutic payload delivery to the brain. Unfortunately, however, nanomedicine has yet to yield results in clinical trials for the treatment of neurological disorders. Therefore, there is a continuing and urgent medical need to find novel strategies to enhance drug permeability across the blood-brain barrier (BBB). [Overview of the project]

[0009] In one embodiment, the present disclosure relates to a multifunctional nanoparticle composition including a pharmaceutical composition for diagnosing and treating central nervous system (CNS) diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and stroke.

[0010] In another embodiment, the present invention provides polymer lipid-based nanoparticles (PLNPs) functionalized by a dual active brain targeting strategy, namely transporter-mediated transcytosis by glucose transporter proteins (e.g., GLUT-1) and receptor-mediated transcytosis by low-density lipoprotein (LDL) receptors, these multifunctional nanoparticles are configured to facilitate permeability and accumulation across the blood-brain barrier in disease areas of the central nervous system. The present invention is considered to be the first embodiment of a compound for simultaneously targeting both LDL receptors and glucose transporters to transport cargo across the BBB.

[0011] In a third aspect, the present invention provides nanoparticles having a surface covered with a layer of novel terpolymer. The novel terpolymer is selected from a library of novel terpolymers consisting of varying amounts of polysorbate (e.g., polysorbate 80) and polyacrylic acid (e.g., polymethacrylic acid [PMAA]) grafted onto maltodextrins (dextrose equivalents: 3-20) of different molecular weights.

[0012] In its fourth embodiment, the novel terpolymer coats the surface of polymer lipid nanoparticles, and "Polysorbate 80," one of the endogenous components derived from the terpolymer, adsorbs ApoE from plasma and promotes the passage of multifunctional nanoparticles through the blood-brain barrier (BBB) ​​via receptor-mediated transcytosis through cerebral vascular endothelial cells.

[0013] In a fifth embodiment, another endogenous component, "maltodextrin," forms the backbone of the novel terpolymer, which is highly functionalized with glucose units at both ends and enhances the passage of multifunctional nanoparticles through the blood-brain barrier via the facilitative glucose transporter protein 1 (GLUT1) pathway.

[0014] In its sixth embodiment, EDC chemistry is used to covalently functionalize the polymer with glucose in order to further enhance the permeability of the BBB via the GLUT1 pathway.

[0015] The present invention provides a method for synthesizing payload-encapsulated polymer lipid nanoparticles (PLNPs), the method comprising the steps of: dissolving a lipid in a solvent; dissolving a payload in a solvent; dissolving a terpolymer in a solvent; mixing the lipid and payload to obtain a lipid / payload complex; adding an aqueous solution of the terpolymer to the lipid / payload complex to form a terpolymer / lipid payload emulsion; and homogenizing the terpolymer / lipid payload emulsion to obtain payload-supported PLNPs, in which case the lipid is selected based on its ability to form a complex with the payload, and the terpolymer is a graft polymer obtained by grafting polysorbate and acrylic acid onto maltodextrin having a dextrose equivalent of 1 to 30. In one aspect of the present invention, the polymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the maltodextrin is a low molecular weight maltodextrin with a molecular weight of less than 10 kDa. In yet another aspect, the maltodextrin is a low molecular weight maltodextrin with a dextrose equivalent in the range of about 15 to about 17. In yet another embodiment, the polysorbate is polysorbate 80. In yet another embodiment, the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacrylic-(C1-C25)-OH methacrylic-(C1-C25)-NH2.

[0016] The present invention provides a method for synthesizing payload-encapsulated polymer lipid nanoparticles (PLNPs), the method comprising the steps of: dissolving a lipid in a solvent; dissolving a payload in a solvent; dissolving a terpolymer in a solvent; mixing the lipid and payload to obtain a lipid / payload complex; adding an aqueous solution of the terpolymer to the lipid / payload complex to form a terpolymer / lipid payload emulsion; and homogenizing the terpolymer / lipid payload emulsion to obtain payload-supported PLNPs, in which case the lipid is selected based on its ability to form a complex with the payload, and the terpolymer is a graft polymer in which polysorbate 80 and methacrylic acid are grafted onto a polysaccharide, with a molecular weight in the range of about 3 to about 800 kDa. In one aspect of the present invention, the terpolymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the polysaccharide is a low molecular weight polysaccharide in the range of about 3 to about 10 kDa. In yet another aspect, the polysaccharide is a low molecular weight polysaccharide with a molecular weight of less than about 40 kDa. In yet another embodiment, the polysaccharide is a low molecular weight polysaccharide with a molecular weight in the range of about 20 to about 40 kDa. In yet another embodiment, the polysaccharide is a low molecular weight polysaccharide with a dextrose equivalent in the range of about 15 to about 17. In yet another embodiment, the polysorbate is polysorbate 80. In yet another embodiment, the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacrylic-(C1-C25)-OH methacrylic-(C1-C25)-NH2.

[0017] The present invention provides a method for synthesizing payload-encapsulated polymer lipid nanoparticles (PLNPs), the method comprising the steps of: dissolving a lipid in a solvent; dissolving a payload in a solvent; dissolving a terpolymer in a solvent; mixing the lipid and payload to obtain a lipid / payload complex; adding an aqueous solution of the terpolymer to the lipid / payload complex to form a terpolymer / lipid payload emulsion; and homogenizing the terpolymer / lipid payload emulsion to obtain payload-supported PLNPs, wherein the lipid is selected based on its ability to form a complex with the payload, and the terpolymer is polysorbate 80 The present invention provides a graft polymer obtained by grafting molecules having a carboxyl group onto a polymer selected from the list consisting of polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(methacrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate, stearyl methacrylate, and methacrylic-(C1-C25)-OH methacrylic-(C1-C25)-NH2, wherein the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the molecule having a carboxyl group includes monomers having a carboxyl group. In another embodiment, the molecule having a carboxyl group is methacrylic acid. In yet another embodiment, the molecule having a carboxyl group is acrylic acid. In yet another embodiment, the polymer further comprises glucose monomers conjugated to the terpolymer.

[0018] The present invention provides a method for synthesizing payload-encapsulated polymer lipid nanoparticles (PLNPs), the method comprising the steps of: dissolving lipids in a solvent; dissolving payloads in a solvent; dissolving terpolymers in a solvent; mixing lipids and payloads to obtain a lipid / payload complex; adding an aqueous solution of terpolymers to the lipid / payload complex to form a terpolymer / lipid payload emulsion; and homogenizing the terpolymer / lipid payload emulsion to obtain payload-supported PLNPs, wherein the lipids are selected based on their ability to form complexes with payloads, and the terpolymers are bound to apolipoprotein E. A graft polymer is obtained by grafting molecules and molecules having a carboxyl group onto a polymer selected from the list consisting of polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(methacrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate, stearyl methacrylate, and methacrylic-(C1-C25)-OH methacrylic-(C1-C25)-NH2, wherein the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the molecule that binds to apolipoprotein E is polysorbate 80. In another aspect, the molecule that binds to apolipoprotein E is a fatty acid having an unsaturated chain, or a phospholipid having an unsaturated fatty acid chain. In yet another aspect, polyethylene glycol is incorporated into the molecule that binds to apolipoprotein E.

[0019] The present invention provides a method for synthesizing payload-encapsulated polymer lipid nanoparticles (PLNPs), the method comprising the steps of: dissolving a lipid in a solvent; dissolving a payload in a solvent; dissolving a terpolymer in a solvent; mixing the lipid and payload to obtain a lipid / payload complex; adding an aqueous solution of the terpolymer to the lipid / payload complex to form a terpolymer / lipid payload emulsion; and homogenizing the terpolymer / lipid payload emulsion to obtain payload-supported PLNPs, in which case the lipid is selected based on its ability to form a complex with the payload, and the terpolymer is a graft polymer in which molecules that bind to apolipoprotein E and molecules having carboxyl groups are grafted onto a polymer containing glucose chains, and the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the polymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the payload comprises a nucleotide-based payload, and the lipid comprises at least one of ionic lipids or cationic phospholipids.

[0020] The present invention provides a compound for transporting a payload across the blood-brain barrier, the compound targeting both LDL receptors and glucose transporters. In one embodiment of the present invention, the compound has a diameter in the range of about 50 nm to about 250 nm, and the compound has a negative surface charge. In another embodiment, the glucose transporter is GLUT-1. In another embodiment, the payload comprises a nucleotide. In another embodiment, the payload comprises a therapeutic molecule. In another embodiment, the payload comprises a biomolecule. In another embodiment, the payload comprises a contrast agent.

[0021] The present invention provides a method for synthesizing a compound for transporting a payload across the blood-brain barrier, wherein the compound targets both LDL receptors and glucose transporters, and the method comprises the steps of: dissolving a lipid in a solvent; dissolving a payload in a solvent; dissolving a terpolymer in a solvent; mixing the lipid and payload to obtain a lipid / payload complex; adding an aqueous solution of the terpolymer to the lipid / payload complex to form a terpolymer / lipid payload emulsion; and homogenizing the terpolymer / lipid payload emulsion to obtain a PLNP carrying the payload, in which case the lipid is selected based on its ability to form a complex with the payload, and the terpolymer is a graft polymer in which molecules having molecules that bind to apolipoprotein E and molecules having carboxyl groups are grafted onto a polymer containing glucose chains, and the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the polymer further comprises glucose monomers conjugated to the terpolymer.

[0022] The present invention provides a compound for transporting a payload across the blood-brain barrier, the compound using apolipoprotein E to target the LDL receptor and glucose for transport via GLUT-1. In one embodiment of the present invention, the glucose transporter is GLUT-1. In another embodiment, the payload comprises a nucleotide. In another embodiment, the payload comprises a therapeutic molecule. In another embodiment, the payload comprises a biomolecule. In another embodiment, the payload comprises a contrast agent.

[0023] The present invention provides nanoparticles for transporting a payload across the blood-brain barrier, the nanoparticles targeting both LDL receptors and glucose transporters. In one embodiment of the present invention, the glucose transporter is GLUT-1. In another embodiment, the payload comprises a nucleotide. In yet another embodiment, the payload comprises a therapeutic molecule. In yet another embodiment, the payload comprises a biomolecule. In yet another embodiment, the payload comprises a contrast agent.

[0024] The present invention provides nanoparticles that target both LDL receptors and glucose transporters, have a diameter in the range of approximately 50 nm to approximately 250 nm, and have a negative surface charge. In one aspect of the present invention, the nanoparticles do not have lipids exposed on their surface. In another aspect, the glucose transporter is GLUT-1. In another aspect, the payload contains nucleotides. In another aspect, the payload contains a therapeutic molecule. In another aspect, the payload contains a biomolecule. In another aspect, the payload contains a contrast agent.

[0025] The present invention provides nanoparticles comprising a terpolymer containing a polysaccharide, a hydrophobic monomer, and a hydrophilic or amphiphilic monomer, which transport a payload across the blood-brain barrier by targeting both the LDL receptor and the glucose transporter. In one aspect of the present invention, targeting of the LDL receptor is performed by nanoparticles that recruit ApoE in the bloodstream before encountering the endothelium. In another aspect, the free glucose terminus of the terpolymer is conjugated to the glucose transporter. In another aspect, the hydrophilic or amphiphilic monomer can conjugate to ApoE in the bloodstream. In another aspect, the nanoparticles further comprise glucose conjugated with the terpolymer. In another aspect, the polysaccharide has a DE of about 20 or less. In another aspect, the polysaccharide has a molecular weight in the range of about 1 to about 800 kDa.

[0026] According to the present invention, there is provided a polymeric lipid nanoparticle (PLNP) comprising a terpolymer, a lipid, cholesterol, and a payload, wherein the lipid forms a complex with the payload, the terpolymer forms a shell surrounding the complex, and the terpolymer is a graft polymer obtained by grafting polysorbate 80 and methacrylic acid onto maltodextrin having a dextrose equivalent of 1 to 30. In one aspect of the present invention, the PLNP further comprises a helper lipid. In one aspect, the polymer further comprises a glucose monomer conjugated to the terpolymer. In another aspect, the maltodextrin is a low molecular weight maltodextrin having a molecular weight of less than 10 kDa. In another aspect, the maltodextrin is a low molecular weight maltodextrin having a dextrose equivalent in the range of about 15 to about 17.

[0027] According to the present invention, there is provided a polymeric lipid nanoparticle (PLNP) comprising a terpolymer, a lipid, cholesterol, and a payload, wherein the lipid forms a complex with the payload, the terpolymer is a graft polymer obtained by grafting polysorbate 80 and methacrylic acid onto a polysaccharide, and the molecular weight is in the range of about 3 to about 800 kDa. In one aspect of the present invention, the PNLP further comprises a helper lipid. In another aspect, the terpolymer further comprises a glucose monomer conjugated to the terpolymer. In another aspect, the polysaccharide is a low molecular weight polysaccharide having a molecular weight in the range of about 3 to about 10 kDa. In another aspect, the polysaccharide is a low molecular weight polysaccharide having a molecular weight of less than about 40 kDa. In one aspect of the present invention, the polysaccharide is a low molecular weight polysaccharide having a molecular weight in the range of about 20 to about 40 kDa. In another aspect, the polysaccharide is a low molecular weight polysaccharide having a dextrose equivalent in the range of about 15 to about 17.

[0028] The present invention provides polymer lipid nanoparticles (PLNPs) comprising a terpolymer, lipids, cholesterol, and a payload, wherein the lipids form a complex with the payload, and the terpolymer is a graft polymer in which polysorbate 80 and molecules having a carboxyl group are grafted onto a polymer selected from the list consisting of polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), and poly(methacrylamide), and the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the PNLP further comprises a helper lipid. In another aspect, the molecule having a carboxyl group comprises a monomer having a carboxyl group. In another aspect, the molecule having a carboxyl group is methacrylic acid. In another aspect, the molecule having a carboxyl group is acrylic acid. In another aspect, the polymer further comprises a glucose monomer conjugated to the terpolymer.

[0029] The present invention provides polymer lipid nanoparticles (PLNPs) comprising a terpolymer, lipids, cholesterol, and a payload, wherein the lipids form a complex with the payload, and the terpolymer is a graft polymer in which molecules that bind to apolipoprotein E and molecules having a carboxyl group are grafted onto a polymer selected from the list consisting of polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), and poly(methacrylamide), the polymer having a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the PNLP further comprises a helper lipid. In another aspect, the molecule that binds to apolipoprotein E is polysorbate 80. In another aspect, the molecule that binds to apolipoprotein E is a fatty acid having an unsaturated chain, or a phospholipid having an unsaturated fatty acid chain. In another aspect, polyethylene glycol is incorporated into the molecule that binds to apolipoprotein E.

[0030] According to the present invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising a terpolymer, a lipid, cholesterol, and a payload, wherein the lipid forms a complex with the payload, and the terpolymer is a graft polymer obtained by grafting a molecule that binds to apolipoprotein E and a molecule having a carboxy group onto a polymer containing a glucose chain, and the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the PNLP further comprises a helper lipid. In another aspect, the polymer further comprises a glucose monomer conjugated to the terpolymer. In another aspect, the payload comprises a nucleotide-based payload, and the lipid comprises at least one of an ionic lipid or a cationic phospholipid.

[0031] According to the present invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising a terpolymer, a lipid, cholesterol, and a payload, wherein the lipid forms a complex with the payload, and the terpolymer is a graft polymer obtained by grafting a molecule that binds to apolipoprotein E and a molecule having a carboxy group onto a polymer containing a glucose chain, and the polymer has a molecular weight in the range of about 1 to about 800 kDa. In one aspect of the present invention, the PLNP further comprises a helper lipid. In another aspect, the polymer further comprises a glucose monomer conjugated to the terpolymer.

[0032] Based on the above and other advantages and features of the present invention described hereinafter, the essence of the present invention will be more clearly understood by referring to the following detailed description of the present invention and the appended claims.

Brief Description of the Drawings

[0033] [Figure 1] A schematic diagram showing a synthesis process of a PLNP designed to cross the blood-brain barrier (BBB) according to the present invention is shown. [Figure 2] A schematic diagram of the PLNP structure according to the present invention is shown. [Figure 3] PNLP crossing the BBB is shown. [Figure 4A]This paper presents the results of a CNS permeability study comparing bispecific TERP chemistry with a single-receptor approach using MR imaging. [Figure 4B] The results of a CNS permeability test comparing bispecific TERP chemistry (square) and a single-receptor approach using ICP technology (circle) are shown. [Figure 5] The results of tests on the brain localization of TERP nanoparticles are shown, with mRNA-related staining regions displayed and quantified in the presence of neurons, astrocytes, and microglia. [Figure 6] This paper compares the blood-brain barrier (BBB) ​​permeability between polymer lipid nanoparticles containing polysorbate 80 (TERP) (TERP-PS80), TERP containing polysorbate 80 (PS80) and endogenous glucose units (TERP-PS80-iGLU), and TERP containing polysorbate 80 (PS80), endogenous glucose units, and covalent glucose (TERP-PS80-iGLU-cGUL). [Modes for carrying out the invention]

[0034] Legend for the figure

[0035] 6. Polysaccharide components of terpolymers

[0036] 7. Lipid components of terpolymers

[0037] 8. Polysorbate component of terpolymer

[0038] 9. Glucose component of terpolymer

[0039] 10. Lipid components (e.g., ionic lipids, phospholipids, helper lipids)

[0040] 11 Cholesterol

[0041] 12 payloads

[0042] 14. Droplets of lipid / payload complexes

[0043] 16 Terpolymer

[0044] 18. Emulsions of terpolymer / lipid-payload complexes

[0045] 19 Polymer Lipid Nanoparticles (PLNPs)

[0046] 20. Lipid-cargo complexes, lipid-payload complexes

[0047] 21 Cargo, RNA

[0048] 22 Terpolymer

[0049] 23 Anionic active sites of terpolymers (e.g., carboxylic acids)

[0050] 24 Lipids

[0051] 26 Cholesterol

[0052] 28 Helper lipids

[0053] 29 Negative surface charge of PLNP

[0054] 30 PLNP

[0055] 32 Apolipoprotein E (ApoE)

[0056] 34 Nanoparticles having free glucose ends and exposed ApoE

[0057] 35 Exposure of Nanoparticles ApoE

[0058] 36 Free glucose terminus of nanoparticles

[0059] 38 GLUT-1 transporter

[0060] 40. Lipoprotein (LDL) receptor

[0061] 41 Blood

[0062] 42 Brain

[0063] 43 Endothelium

[0064] 44. Tight junctions of the endothelium

[0065] 50 staining mRNA

[0066] 52 Neurons stained with Map2

[0067] Astrocytes stained with 54 GFAP

[0068] 56 Microglia stained with Iba1

[0069] As used herein, the term "alkyl" means a linear or branched hydrocarbon containing 1 to 25 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1, 2, 3, 4, 5, or 6 carbon atoms. Typical examples of alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0070] As used herein, the term "amino" refers to the -NH2 group.

[0071] As used herein, the term "carboxy" refers to a -COOH group, which may be protected as an ester group: -COO-alkyl.

[0072] As used herein, the term "hydroxy" refers to the -OH group.

[0073] The term "nucleic acid" is a technical term referring to a set of at least two base-sugar-phosphate combinations. For naked DNA delivery, polynucleotides contain more than 120 monomer units, which is necessary to distinguish them from oligonucleotides. However, for the purpose of delivering RNA, RNAi, and siRNA, which are either single-stranded or double-stranded, polynucleotides contain two or more monomer units. A nucleotide is a monomer unit of a nucleic acid polymer. This term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of messenger RNA, antisense, plasmid DNA, parts of plasmid DNA, or viral genetic material. Antisense is a polynucleotide that interferes with the function of DNA and / or RNA. The term "nucleic acid" refers to a set of at least two base-sugar-phosphate combinations. Natural nucleic acids have a phosphate backbone, while artificial nucleic acids may contain other types of backbones but the same bases. A nucleotide is a monomer unit of a nucleic acid polymer. This term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). RNA can take the forms of tRNA (transfer RNA), snRNA (small RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, RNAi, siRNA, and ribozymes. This term also includes PNA (peptide nucleic acid), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.

[0074] The term "siRNA" refers to small inhibitory ribonucleic acid. siRNAs are generally less than 30 nucleotides in length and can be single-stranded or double-stranded. Ribonucleotides can be natural or artificial and can be chemically modified. Longer siRNAs may contain cleavage sites that can be enzymatically or chemically cleaved to produce siRNAs less than 30 nucleotides in length, generally 21-23 nucleotides. siRNAs share sequence homology with their corresponding target mRNA. Sequence homology may be less than 100 percent, but is sufficient to cause sequence-specific association between the siRNA and the targeted mRNA.

[0075] In this specification, Cx means an acyclic straight-chain or branched hydrocarbon of the longest length x, and therefore C1-C5 includes methyl, ethyl, propyl, butyl, pentyl, isopropyl, and the like.

[0076] This invention relates to the development of novel polymer-lipid nanoparticles for improving BBB permeability. Passive diffusion across the blood-brain barrier is often ineffective because the payload or cargo is too large to pass through the blood-brain barrier passively at concentrations sufficient to exert an effect. This invention shows the design and development of novel polymer-lipid nanoparticles having a surface or shell covered with a layer of a novel polymer, which has polysorbate and glucose units, which are endogenous dual active brain-targeting components.

[0077] The novel polymer-lipid nanoparticles have a polymer layer functionalized by a dual active brain targeting strategy: transcytosis by glucose transporter protein 1 and receptor-mediated transcytosis by the low-density lipoprotein (LDL) receptor.

[0078] Endogenous glucose units originate from the ends of the polymer backbone, such as maltodextrin, which contains molecular weights with 3 to 20 dextrose equivalents.

[0079] Using low molecular weight polysaccharides is an improvement compared to terpolymers with higher molecular weight polysaccharide backings, because low molecular weight polysaccharides provide significantly additional glucose terminal units on the surface of nanoparticles, better facilitating transport via glucose transporters. In preferred embodiments, the low molecular weight polysaccharides have a dextrose equivalent (DE) of about 20 or less. Such terpolymers can then be improved by conjugating glucose to the terpolymer. Such terpolymers can also be improved by conjugation with polysorbate (e.g., PS80) to access a second transport mechanism through the BBB. Furthermore, this terpolymer can be improved by both conjugation of glucose onto the terpolymer and conjugation with PS80 to access a second transport mechanism through the BBB. Other materials exist that can be used instead of PS80 to create a second transport mechanism through the BBB, as described below.

[0080] To further facilitate the glucose transporter protein 1 (GLUT1) pathway, polymers can be covalently functionalized with glucose using well-known EDC chemistry. "EDC" refers to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or other suitable aqueous carboxylic acid crosslinking agents.

[0081] Figure 1 shows a schematic diagram illustrating the manufacturing process of PLNPs as a carrier system for delivery to the CNS, particularly across the blood-brain barrier. PLNPs are partially synthesized by utilizing the three core materials and methods used in the production of LNPs, namely cationic lipids, cholesterol, and helper lipids. However, this invention hereby indicates that instead of using PEG-lipids, a fourth material in the synthesis of LNPs, a terpolymer designed to further improve the chemical properties and stability of the nanoparticle surface and to cross the BBB is used. (Note that PLNPs also cross the intranasal and intrathecal barriers, but are not specifically designed for this purpose). As shown in Figure 1, the synthesis of the nanocomplex begins with rapidly mixing the lipid component 10 in alcohol or an organic solvent (preferably ethanol) with cholesterol 11 and payload(s) 12 in the solvent (the choice of solvent depends on the payload(s)). In Figure 1, the payload depicted is RNA, but this approach can work with a wide range of payloads, including nucleotides, therapeutic molecules, biomolecules, and contrast agents. The choice of lipids depends on the payload(s) (note that a given PLNP may have multiple payloads), and the solvent used should reflect the specific lipids. The lipids interact with the payload, and through electrostatic and hydrophobic / hydrophilic interactions, the lipids self-assemble to surround the payload and form a lipid / payload complex. Further changes in the polarity of the lipid solution (including charged lipids, cholesterol, and helper lipids, as is well known to those skilled in the art) generate large lipid / payload complex droplets 14 through electrostatic, hydrophobic, and van der Waals interactions. The amount of lipid required to form a complete complex around the payload will depend on the specific payload. In preferred embodiments, the lipid-to-payload weight ratio is in the range of 2.5:1 to 15:1, and in other embodiments, the lipid-to-payload weight ratio is in the range of 2.5:1 to 50:1.

[0082] Continuing from Figure 1, as the manufacturing process progresses, aqueous terpolymer 16 is added, which further interacts with the droplet 14 through electrostatic and hydrophobic interactions to coat the surface of the lipid payload complex 14 and form a polymer / lipid payload emulsion 18. The polymer-lipid-payload complex is then treated through sonication, homogenization, or microfluidic techniques to produce miniature PLNPs 19.

[0083] The process shown in Figure 1 can accommodate a wide range of useful payloads. These include both positively and negatively charged payloads, and encompass nucleotides, therapeutic molecules, biomolecules, and contrast agents.

[0084] As is known to those skilled in the art, further purification, buffer exchange to physiological pH, and nanoparticle concentration can be used as desired, as long as the PLNP particles retain a negative charge due to the presence of anionic polymers on their surface.

[0085] When the cargo is nucleic acid or nucleotide, the weight ratio of terpolymer to total lipids can be increased to 10:1, but the preferred weight ratio of terpolymer to total lipids is 0.01:1 or 0.5:1 to 6:1, more preferably about 3:1.

[0086] The diameter of the PNLP resulting from this synthesis can be controlled during the homogenization and sonarization stages, as is known to those skilled in the art. For the purpose of optimally designing the PNLP to deliver the payload to the brain, a preferred diameter is about 5 nm to about 500 nm, preferably 100 nm to about 150 nm, and another preferred diameter is about 100 nm.

[0087] Unlike typical lipid nanoparticles, it should be noted that PLNPs produced by the above method do not contain lipids exposed on the shell or surface of the PLNPs. As a result, the PNLPs produced by this synthesis have improved stability and reduced immunogenicity due to the absence of lipids exposed to external interactions.

[0088] Lipid 10 can be positively charged, loaded, or neutrally charged, but should be selected to reflect the payload and produce a lipid / payload droplet with an outer lipid layer.

[0089] The helper lipids may include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin, such as DSPC and DPPC, as well as any zwitterionic phospholipid.

[0090] The polymer (which forms the main chain of the terpolymer) is preferably a maltodextrin with a molecular weight in the range of about 3 to about 20 dextrose equivalents. In another embodiment, the polymer is a maltodextrin having a molecular weight in the range of about 3 to about 30, or about 40 dextrose equivalents. However, any polymer that produces glucose chains in the backbone when PLNP is synthesized can be used. For a suitable polymer, in a preferred embodiment, the polymer is characterized by short glucose chains having hydroxyl-active groups on the backbone.

[0091] The efficiency of crossing the blood-branch barrier (BBB) ​​is influenced by the glucose density on the surface of the PNLP (Particle Network Polymer). Using short-chain glucose polymers increases the number of glucose terminals, thereby promoting transport.

[0092] The polymer monomers in the polymer side chains can be selected from a range of materials including, but not limited to, PS80, fatty acids having unsaturated chains, and phospholipids having unsaturated fatty acid chains (preferably having carboxylic acid groups).

[0093] In a preferred embodiment, the polymer is a maltodextrin having a molecular weight in the range of about 3 to about 20 dextrose equivalents, with a side chain of PS80.

[0094] In a more specific example, the payload is manganese dioxide, the lipids are DSPC and cholesterol, and the polymer is maltodextrin with a molecular weight of approximately 3 to 20 dextrose equivalents and a side chain of PS80. In this specific example, there are no helper lipids.

[0095] The above synthesis can be carried out using clinically approved lipids, cholesterol, and helper lipids used in conventional clinically approved LNPs. (Those skilled in the art will recognize that some of the materials described in the above synthesis description are not clinically approved.)

[0096] Importantly, in the present invention, the fourth material commonly used in the synthesis of LNPs, PEG lipids (such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) or other PEG lipid complexes), is replaced with a specific class of terpolymer, further improving the surface chemistry and stability of the nanoparticles and facilitating transport across the blood-brain barrier (BBB). Compared with conventional LNPs that have a net neutral charge under physiological conditions, the PLNPs described in the present invention have a net negative surface charge under physiological conditions (i.e., conditions in the blood), thereby being more stable during blood circulation.

[0097] In modified versions of the above synthesis method, a step of conjugating glucose into the polymer can be included to increase the glucose concentration on the surface or shell of the PNLP. However, introducing glucose onto a general (polymer) backbone is relatively difficult. In some embodiments, it is advantageous from the viewpoint of synthesis and scale-up to rely on the use of short-chain glucose polymers as the backbone.

[0098] Figure 2 shows a schematic diagram of the BBB transport structure. Referring to Figure 2, a BBB transport PLNP is shown, comprising a coating of a lipid payload complex 20 along with a terpolymer 22 having a carboxylic acid active site 23. The polymer interacts with the lipid-payload complex due to electrostatic and hydrophobic / hydrophilic interactions. The PLNP is partially synthesized as described above with reference to Figure 1, using three core materials used in the production of LNPs, such as lipids 24, cholesterol 26, and helper lipids 28 surrounding the cargo 21. A fourth component introduced to generate the BBB transport PLNP is a terpolymer 22 having a carboxylic acid group. Unlike LNPs, which are neutral under physiological conditions, the proposed system has a negative surface charge 29 at physiological pH and has better stability due to electrostatic repulsion between particles. In a preferred embodiment, the terpolymer 22 has glucose terminal units on the polymer backbone that facilitate BBB transport.

[0099] In a preferred embodiment, the BBB transport PLNP has a terpolymer, which is a maltodextrin having a molecular weight in the range of about 3 to about 20 dextrose equivalents, with a side chain of PS80.

[0100] In a more specific example, BBB transport PLNP has a manganese dioxide payload, the lipid components are DSPC and cholesterol, and the terpolymer contains maltodextrin and PS80 side chains with a molecular weight of approximately 3 to 20 dextrose equivalents.

[0101] In preferred embodiments, BBB transporter PLNPs utilize D-glucose transporter protein (GLUT), one of the important nutrient transporters. GLUT is present in particularly high concentrations in the brain's microvessels and is approximately 100 times more abundant than transferrin receptors, which are widely used as brain-specific targets for active drug delivery. The brain's massive and uninterrupted energy demands are met almost entirely by bD-glucose, which can penetrate the brain very efficiently via facilitative GLUT.

[0102] Specifically, in some embodiments, in the case of BBB-transporting PNLP, the terpolymer backbone is made from glucose units, and the terminal units of the backbone are recognized by the GLUT-1 transporter, thereby facilitating the passage of PNLP through the BBB.

[0103] In another embodiment, the novel polymer-lipid nanoparticles have a layer of the polymer described above that is functionalized by a dual active brain targeting strategy, namely transcytosis by glucose transporter protein 1 and receptor-mediated transcytosis by a low-density lipoprotein (LDL) receptor. This dual-functional structure enables transcytosis by glucose transporter protein 1, as well as receptor-mediated transcytosis by a low-density lipoprotein (LDL) receptor, such as the 2-LDLR receptor. In a preferred embodiment, PS80 is used on the side chain that binds to the 2-LDLR receptor in the target for receptor-mediated transcytosis to the brain.

[0104] Figure 3 shows PNLP traversing the BBB via migration from blood 41 through endothelium 42 to brain tissue 43 (characteristic cell types shown in the bottom layer of the figure: neurons, astrocytes, microglia). The PNLP 30 absorbs apolipoprotein E (ApoE) 32 from the blood to create nanoparticles 34 having both a free glucose terminus 36 and exposed ApoE 35. The nanoparticles 34 traverse from the blood through the endothelium into the brain using two transport mechanisms: one is transporter-mediated transcytosis, where the glucose unit 36 ​​is recognized by the GLUT-1 transporter 38, facilitating the nanoparticles 34 to cross the endothelium (BBB); the other is receptor-mediated transcytosis, where ApoE 35 interacts with the lipoprotein (LDL) receptor 40, which also facilitating the nanoparticles 36 to cross the endothelium (BBB).

[0105] Generally, synthesizing nanoparticles using bifunctional BBB crossovers is difficult. By using a short-chain glucose polymer as the backbone, the synthesis disclosed above facilitates the addition of a second transport mechanism (in this case, by using PS80 in the side chain and a 2-LDLR receptor). As a result, this approach to synthesizing nanoparticles using bifunctional BBB crossovers is easily scaled up.

[0106] In some embodiments, the terpolymer is a graft terpolymer of poly(methacrylic acid)-polysorbate 80-maltodextrin. Briefly, polysorbate 80, maltodextrin, and methacrylic acid are mixed by emulsion polymerization at 70°C in the presence of potassium persulfate (KPS), sodium thiosulfate (STS), and water. [ka]

[0107] It should be noted that the terpolymers obtained above contain a skeletal carboxylic acid group for each MAA unit of the polymer, while the polysorbate contains a side-chain carboxylic acid group. The effect of this is to provide several carboxylic acid groups per unit of the terpolymer. In some embodiments, the amount of maltodextrin or polysorbate can be varied in a manner understandable to those skilled in the art, thereby changing the average number of suchly modified terpolymer units. In some embodiments, the terpolymer structure can be varied by using polysorbate having different values ​​of w, x, y, or z (or combinations thereof).

[0108] In some embodiments, C1-C25 acrylate is used instead of MAA in the above synthesis. In some embodiments, ethyl acrylate is used. In some embodiments, C1-C25-disubstituted aminoC1-C25 acrylate is used (wherein Cx may be a different x in each case). In some embodiments, 2-(dimethylamino)ethyl methacrylate is used. In some embodiments, 2-(diisopropylamino)ethyl methacrylate is used. In some embodiments, C1-C25 methacrylate is used. In some embodiments, stearyl methacrylate is used. In some embodiments, methacrylic-(C1-C25)-OH is used. In some embodiments, methacrylic-(C1-C25)-NH2 (including the hydrochloride as a complex ion, if applicable) is used. In some embodiments, any polymerizable monomer containing an acrylic group or a methacrylic group is used.

[0109] Furthermore, such terpolymers can also be synthesized by generally following the approach given in U.S. Patent No. 10,233,277 (which is incorporated herein by reference in its entirety).

[0110] In some embodiments, the PLNP described herein has a therapeutically effective payload for diagnosing or treating CNS diseases. In some embodiments, the CNS disease is Alzheimer's disease. In some embodiments, the CNS disease is Parkinson's disease. In some embodiments, the CNS disease is Huntington's disease. In some embodiments, the CNS disease is seizures. In some embodiments, the payload comprises nucleotides. In some embodiments, the payload comprises therapeutic molecules. In some embodiments, the payload comprises biomolecules. In some embodiments, the payload comprises contrast agents.

[0111] The present invention discloses a pharmaceutical composition comprising a PLNP having at least one cargo useful for the treatment or diagnosis of at least one CNS disease, and a pharmaceutical component enabling parenteral dosage forms for human or veterinary use. Parenteral dosage forms can be administered to patients via various routes, including subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Because these administrations typically bypass the patient's natural defenses against contaminants, parenteral dosage forms may be particularly sterile or sterilized before administration to the patient. Examples of parenteral dosage forms include immediate injection solutions, dried products dissolved or suspended in a pharmaceutically acceptable vehicle for injection, immediate injection suspensions, and emulsions. Pharmaceutical compositions for parenteral injection include pharmaceutically acceptable aqueous or non-aqueous sterile solutions, dispersions, suspensions, or emulsions, and sterile powders that can be reconstituted into sterile injection solutions or dispersions. Suitable fluidity of the composition may be maintained, for example, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial action may be ensured by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. The inclusion of isotonic agents, such as sugars and sodium chloride, may also be desirable. Sustained absorption of the injectable pharmaceutical form can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin.

[0112] In some cases, it is often desirable to slow down the absorption of a drug from subcutaneous or intramuscular injection in order to prolong its effects. This may be achieved by using a liquid suspension of a crystalline or amorphous material that is poorly water-soluble. The rate of drug absorption then depends on its dissolution rate, which in turn depends on the crystal size and morphology.

[0113] Injectable depot formulations are prepared by forming a microencapsulated matrix of the drug in a biodegradable polymer such as polylactide-polyglycolide. The drug release rate can be adjusted depending on the ratio of the drug to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoester) and poly(anhydrous). Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a filter that retains bacteria, or by incorporating a sterilizer in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media immediately before use.

[0114] Injectable preparations, such as sterile injectable suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspension agents. Sterile injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenterally acceptable diluents or solvents. Acceptable vehicles and solvents that may be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. [Examples]

[0115] Example 1: To investigate the degree of BBB penetration of a payload using a bispecific TERP system containing both LDL-r and glucose transporters (polysorbate 80 (PS80), a TERP short-chain maltodextrin containing endogenous glucose units, and covalently bound glucose) compared with a single-receptor-mediated TERP composition targeting only LDL-r (a long-chain polysaccharide containing PS80), gadolinium (Gd) was encapsulated as the payload in both formulations as described above. TERP-Gd nanoparticles were intravenously injected into healthy naive animals. CNS permeability of Gd was confirmed in vivo using MR imaging 2 hours after treatment. It was observed that the bispecific TERP chemistry increased the MR contrast signal (R1 value) 2.6 times compared to treatment with the single-specific receptor approach. The results are shown in Figure 4A as the percentage increase relative to the initial (pre-treatment) signal.

[0116] To investigate the brain pharmacokinetics of TERP-enabled Gd nanoparticles, animals were euthanized 0.5, 1, 2, and 4 hours after injection. Brain samples were collected, and the amount of Gd in brain tissue was measured using the ICP method. The results are shown in Figure 4B. The results shown in Figure 4B represent the percentage increase compared to treatment with physiological saline. The data confirmed that these bispecific TERP nanoparticles resulted in more than 2.7 times superior brain permeability of the brain payload compared to treatment with the LDL-r monotherapy pathway.

[0117] Example 2: To investigate the brain localization of TERP nanoparticles, TERP nanoparticles carrying Cy5-mRNA (TERP short-chain maltodextrin containing polysorbate 80 (PS80) and endogenous glucose units, and covalently bound glucose) were intravenously injected into healthy, untreated animals. The animals were euthanized two hours after treatment. Brain tissue was collected, and tissue slides were prepared for IHC testing. Neurons (Map2), astrocytes (Gfap), and microglia (Iba1) were stained and imaged using fluorescence imaging.

[0118] The results are shown in Figure 5. mRNA is stained red 50 in all three slides. Intracortical: The neuron slide Map2 is green 52. Intracortical: The astrocyte slide Gfap is green 54. Intracortical: The microglia slide Iba1 is green 56. The data are reported as the number of colocalization signals from cells and mRNA. The results indicate that these TERP nanoparticles are taken up mainly by neurons, and less by astrocytes and microglia.

[0119] Example 3: Chemical substances and reagents

[0120] The amphiphilic polymer, poly(methacrylic acid)-polysorbate 80, was grafted onto maltodextrin (DE=17) to synthesize PLNP as described above. The phospholipid DPPC was obtained from NOF America. Cholesterol was obtained from Spectrum Chemical, USA. Anhydrous ethanol was obtained from Greenfield Canada. Polyvinyl alcohol (PVA) and potassium permanganate (KMnO4) were purchased from Sigma-Aldrich (Oakille, ON, Canada). All chemicals were analytical grade and used without further purification unless otherwise indicated.

[0121] Laboratory animals

[0122] For in vivo brain delivery efficiency, 8-week-old female Balb / c mice (Jackson Laboratory, Maine, USA) were used. Throughout the study, the animals were given free access to food and water.

[0123] Preparation of MnO2-NP-supported TERP

[0124] To verify the BBB permeability of polymer-lipid nanoparticles, MnO2-NPs were supported on PNLP and covered with a polymer layer or a polymer layer pre-conjugated with glucosamine.

[0125] In short, crude inorganic MnO2 NP-lipid PNLP was first synthesized by reducing KMnO4 to MnO2 in an aqueous medium in the presence of PVA, followed by the addition of ethanol solutions of phospholipid (DPPC) and cholesterol. The emulsion was then mixed with an amphiphilic polymer, and hydrophobic interactions between the polymer and the lipid domains of the nanoparticles led to self-assembly and the formation of MnO2-TERP. The emulsion was then passed through a high-pressure homogenizer at a pressure exceeding 25 kpsi to produce small NPs. The final sample was collected in ice-cold water, filtered, and purified using tangent flow filtration (TFF) to remove unreacted reagents. The purified solution was freeze-dried in the presence of sucrose as a cryoprotectant.

[0126] In vivo brain delivery efficiency using MnO2-TERP

[0127] To investigate the blood-brain barrier (BBB) ​​permeability of TERP via dual active brain targeting strategies (GLUT1 and low-density lipoprotein (LDL) receptor pathways), BALB / c mice were intravenously injected with 100 μmol Mn / kg of MnO2-TERP via the tail vein. Two groups of animals (n=5 / group) were treated with MnO2-TERP using an amphiphilic polymer, and one group was fasted for 12 hours. After fasting, glucose solution (20 wt%) was injected to raise blood glucose levels, and 30 minutes later, a single dose of MnO2-TERP was administered. One hour later, the mice were perfused, brain tissue was collected, and homogenized for Mn quantification using inductively coupled plasma atomic emission spectroscopy (ICP-OES). In another experiment, MnO2-TERP, prepared using a polymer conjugated with glucosamine, was administered to fasted mice (n=5 / group), and brain tissue was then collected using the same procedure to quantify Mn under ICP-OES.

[0128] Figure 6 shows a comparison of BBB permeability for polymer lipid nanoparticles (TERPs) containing endogenous glucose units and polysorbate 80 (without glucose pre-injection to activate GLUT transporters) (indicated as TERP-PS80), TERP containing endogenous glucose units and polysorbate 80 (PS80) with GLUT transporter activation by glucose injection (indicated as TERP-PS80-iGLU), and TERP containing polysorbate 80 (PS80), endogenous glucose units, and covalent glucose with GLUT transporter activation by glucose injection (indicated as TERP-PS80-iGLU-cGUL). GLUT transport data normalized to inactivated GLUT conditions (left) shows that the PMAA-PS80-maltodextrin terpolymer described herein, having endogenous glucose units (center) and especially covalent glucose units (right), has a significantly higher ability to transport across the BBB. This finding indicates that such PLNPs can be actively transported via GLUT.

[0129] Predicted Example 4: To further evaluate the BBB entry of PLNPs via transporters, modified versions of the above experiments can also be performed. One possible experiment would evaluate the PLNPs described herein in a similar experiment, but with the expectation of elimination of BBB entry in the presence of a selective GLUT1 transporter antagonist well known in the art (with or without pre-injection of glucose). Another possible experiment would evaluate the PLNPs described herein in a similar experiment for elimination of BBB entry in an animal (whose genome includes a transgenic transmutation, deletion, knockdown, knockout, or any other suitable means known in the art that causes at least partial loss of GLUT1 function for BBB transport in the animal). In some experiments, the mutation may be located within the Slc2a1 gene.

[0130] Predicted Example 5: Cell-based screening can also be performed to evaluate the blood-brain barrier (BBB) ​​entry of PLNPs via transporters. To assess GLUT transport efficiency, commercially available human SLC2A1 (glucose transporter GLUT1) knockout A549 cell lines and wild-type A549 cells are incubated with PLNPs containing a fluorescent dye payload. Following incubation at predetermined time points selected based on calibration, literature precedents, or both, the uptake rate of PLNPs is examined using confocal microscopy.

Claims

1. A method for synthesizing polymer-lipid nanoparticles (PLNPs) for encapsulating a payload, Solubilize lipids in a solvent, The payload is solubilized in a solvent, Soluble the terpolymer in the solvent, The lipid and payload are mixed to obtain a lipid / payload complex. An aqueous solution of the terpolymer is added to the lipid / payload complex to form a terpolymer / lipid-payload emulsion. The step includes homogenizing the terpolymer / lipid-payload emulsion to obtain a PLNP on which the payload is supported, The synthesis method wherein the lipid is selected with respect to its ability to form a complex with the payload, and the terpolymer is a graft polymer obtained by grafting polysorbate and acrylic acid onto maltodextrin in an amount of 1 to 30 dextrose equivalents.

2. The method according to claim 1, wherein the terpolymer further comprises a glucose monomer conjugated to the terpolymer.

3. The method according to claim 1, wherein the maltodextrin is a low molecular weight maltodextrin having a molecular weight of less than 10 kDa.

4. The method according to claim 1, wherein the maltodextrin is a low molecular weight maltodextrin having a dextrose equivalent in the range of about 15 to about 17.

5. The method according to claim 1, wherein the polysorbate is polysorbate 80.

6. The method according to claim 1, wherein the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacrylic-(C1-C25)-OH methacrylic-(C1-C25)-NH2.

7. A method for synthesizing polymer-lipid nanoparticles (PLNPs) for encapsulating a payload, Solubilize lipids in a solvent, The payload is solubilized in a solvent, Soluble the terpolymer in the solvent, The lipid and payload are mixed to obtain a lipid / payload complex. An aqueous solution of the terpolymer is added to the lipid / payload complex to form a terpolymer / lipid-payload emulsion. The step includes homogenizing the terpolymer / lipid-payload emulsion to obtain a PLNP on which the payload is supported, The synthesis method wherein the lipid is selected with respect to its ability to form a complex with the payload, and the terpolymer is a graft polymer obtained by grafting polysorbate and acrylic acid onto a polysaccharide, and has a molecular weight in the range of about 3 to about 800 kDa.

8. The method according to claim 7, wherein the terpolymer further comprises a glucose monomer conjugated to the terpolymer.

9. The method according to claim 7, wherein the polysaccharide is a low molecular weight polysaccharide having a molecular weight in the range of about 3 to about 10 kDa.

10. The method according to claim 7, wherein the polysaccharide is a low molecular weight polysaccharide having a molecular weight of less than approximately 40 kDa.

11. The method according to claim 7, wherein the polysaccharide is a low molecular weight polysaccharide having a molecular weight in the range of about 20 to about 40 kDa.

12. The method according to claim 7, wherein the polysaccharide is a low molecular weight polysaccharide having a dextrose equivalent in the range of about 15 to about 17.

13. The method according to claim 7, wherein the polysorbate is polysorbate 80.

14. The method according to claim 7, wherein the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacrylic-(C1-C25)-OH methacrylic-(C1-C25)-NH2.

15. A method for synthesizing polymer-lipid nanoparticles (PLNPs) for encapsulating a payload, Solubilize lipids in a solvent, The payload is solubilized in a solvent, Soluble the terpolymer in the solvent, The lipid and payload are mixed to obtain a lipid / payload complex. An aqueous solution of the terpolymer is added to the lipid / payload complex to form a terpolymer / lipid-payload emulsion. The step includes homogenizing the terpolymer / lipid-payload emulsion to obtain a PLNP on which the payload is supported, The lipids are selected based on their ability to form a complex with the payload, and the terpolymer includes polysorbate 80 and molecules having carboxyl groups, polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(methacrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate, stearyl methacrylate, and methacrylic-(C1-C25)-OH The synthesis method is a graft polymer obtained by grafting onto a polymer selected from a list consisting of methacrylic-(C1-C25)-NH2, wherein the polymer has a molecular weight in the range of about 1 to about 800 kDa.

16. The method according to claim 15, wherein the molecule having a carboxyl group comprises a monomer, and the monomer has a carboxyl group.

17. The method according to claim 15, wherein the molecule having a carboxyl group is methacrylic acid.

18. The method according to claim 15, wherein the molecule having a carboxyl group is acrylic acid.

19. The method according to claim 15, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

20. A method for synthesizing polymer-lipid nanoparticles (PLNPs) for encapsulating a payload, Solubilize lipids in a solvent, The payload is solubilized in a solvent, Soluble the terpolymer in the solvent, The lipid and payload are mixed to obtain a lipid / payload complex. An aqueous solution of the terpolymer is added to the lipid / payload complex to form a terpolymer / lipid-payload emulsion. The step includes homogenizing the terpolymer / lipid-payload emulsion to obtain a PLNP on which the payload is supported, The lipids are selected based on their ability to form a complex with the payload, and the terpolymer includes molecules that bind to apolipoprotein E and molecules having a carboxyl group, such as polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(methacrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25-N,N'-disubstituted amino-(C1-C25)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate, stearyl methacrylate, and methacrylic-(C1-C25)-OH The synthesis method is a graft polymer obtained by grafting onto a polymer selected from a list consisting of methacrylic-(C1-C25)-NH2, wherein the polymer has a molecular weight in the range of about 1 to about 800 kDa.

21. The method according to claim 20, wherein the molecule that binds to apolipoprotein E is polysorbate 80.

22. The method according to claim 20, wherein the molecule that binds to apolipoprotein E is a fatty acid having an unsaturated chain or a phospholipid having an unsaturated fatty acid chain.

23. The method according to claim 20, wherein polyethylene glycol is incorporated into the molecule that binds to apolipoprotein E.

24. A method for synthesizing polymer-lipid nanoparticles (PLNPs) for encapsulating a payload, Solubilize lipids in a solvent, The payload is solubilized in a solvent, Soluble the terpolymer in the solvent, The lipid and payload are mixed to obtain a lipid / payload complex. An aqueous solution of the terpolymer is added to the lipid / payload complex to form a terpolymer / lipid-payload emulsion. The step includes homogenizing the terpolymer / lipid-payload emulsion to obtain a PLNP on which the payload is supported, The synthesis method wherein the lipid is selected based on its ability to form a complex with the payload, the terpolymer is a graft polymer in which molecules that bind to apolipoprotein E and molecules having a carboxyl group are grafted onto a polymer containing glucose chains, and the polymer has a molecular weight in the range of about 1 to about 800 kDa.

25. The method according to claim 24, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

26. The method according to claim 24, wherein the payload comprises a nucleotide-based payload and the lipid comprises at least one of an ionic lipid or a cationic phospholipid.

27. A compound for transporting a payload across the blood-brain barrier, wherein the compound targets both an LDL receptor and a glucose transporter.

28. The compound according to claim 27, wherein the compound has a diameter in the range of about 50 nm to about 250 nm, and the compound has a negative surface charge.

29. The compound according to claim 27, wherein the glucose transporter is GLUT-1.

30. The compound according to claim 27, wherein the payload comprises a nucleotide.

31. The compound according to claim 27, wherein the payload comprises a therapeutic molecule.

32. The compound according to claim 27, wherein the payload includes a biomolecule.

33. The compound according to claim 27, wherein the payload comprises a contrast agent.

34. A method for synthesizing a compound for transporting a payload across the blood-brain barrier, wherein the compound targets both an LDL receptor and a glucose transporter, and the method is Solubilize lipids in a solvent, The payload is solubilized in a solvent, Soluble the terpolymer in the solvent, The lipid and payload are mixed to obtain a lipid / payload complex. An aqueous solution of the terpolymer is added to the lipid / payload complex to form a terpolymer / lipid-payload emulsion. The step includes homogenizing the terpolymer / lipid-payload emulsion to obtain a PLNP on which the payload is supported, The synthesis method wherein the lipid is selected based on its ability to form a complex with the payload, the terpolymer is a graft polymer in which molecules that bind to apolipoprotein E and molecules having a carboxyl group are grafted onto a polymer containing glucose chains, and the polymer has a molecular weight in the range of about 1 to about 800 kDa.

35. The method according to claim 34, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

36. A compound for transporting a payload across the blood-brain barrier, wherein the compound uses apolipoprotein E to target the LDL receptor and glucose for transport via GLUT-1.

37. The compound according to claim 36, wherein the glucose transporter is GLUT-1.

38. The compound according to claim 36, wherein the payload comprises a nucleotide.

39. The compound according to claim 36, wherein the payload comprises a therapeutic molecule.

40. The compound according to claim 36, wherein the payload includes a biomolecule.

41. The compound according to claim 36, wherein the payload includes a contrast agent.

42. Nanoparticles for transporting a payload across the blood-brain barrier, wherein the nanoparticles target both LDL receptors and glucose transporters.

43. The nanoparticle according to claim 42, wherein the glucose transporter is GLUT-1.

44. The nanoparticle according to claim 42, wherein the payload comprises a nucleotide.

45. The nanoparticle according to claim 42, wherein the payload comprises a therapeutic molecule.

46. The nanoparticles according to claim 42, wherein the payload includes a biomolecule.

47. The nanoparticles according to claim 42, wherein the payload comprises a contrast agent.

48. Nanoparticles that target both LDL receptors and glucose transporters, have a diameter in the range of approximately 50 nm to 250 nm, and have a negative surface charge.

49. The nanoparticles according to claim 48, wherein the nanoparticles do not have lipids exposed on their surface.

50. The nanoparticle according to claim 48, wherein the glucose transporter is GLUT-1.

51. The nanoparticle according to claim 48, wherein the payload comprises a nucleotide.

52. The nanoparticles according to claim 48, wherein the payload comprises a therapeutic molecule.

53. The nanoparticles according to claim 48, wherein the payload includes a biomolecule.

54. The nanoparticles according to claim 48, wherein the payload comprises a contrast agent.

55. Nanoparticles comprising a terpolymer containing polysaccharides, hydrophobic monomers, and hydrophilic or amphiphilic monomers, The nanoparticles transport a payload by targeting both LDL receptors and glucose transporters, thereby crossing the blood-brain barrier.

56. The nanoparticles according to claim 55, wherein the targeting of the LDL receptor is carried out by the nanoparticles that recruit ApoE in the bloodstream before encountering the endothelium.

57. The nanoparticle according to claim 55, wherein the free glucose terminus of the terpolymer is bound to a glucose transporter.

58. The nanoparticle according to claim 55, further comprising the hydrophilic or amphiphilic monomer capable of binding to ApoE in the blood circulation.

59. The nanoparticle according to claim 55, further comprising glucose conjugated with the terpolymer.

60. The nanoparticle according to claim 55, wherein the polysaccharide has a DE of about 20 or less.

61. The nanoparticles according to claim 55, wherein the polysaccharide has a molecular weight in the range of about 1 to about 800 kDa.

62. Terpolymer, Lipids, cholesterol, payload Polymer lipid nanoparticles (PLNPs) containing, The PLNP is a graft polymer in which polysorbate 80 and methacrylic acid are grafted onto maltodextrin in an amount of 1 to 30 dextrose equivalents.

63. The PLNP according to claim 62, further comprising a helper lipid.

64. The PLNP according to claim 62, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

65. The PLNP according to claim 62, wherein the maltodextrin is a low molecular weight maltodextrin having a molecular weight of less than 10 kDa.

66. The PLNP according to claim 62, wherein the maltodextrin is a low molecular weight maltodextrin having a dextrose equivalent in the range of about 15 to about 17.

67. Terpolymer, Lipids, cholesterol, payload Polymer lipid nanoparticles (PLNPs) containing, The PLNP is a graft polymer in which the lipid forms a complex with the payload, and the terpolymer is a graft polymer in which polysorbate 80 and methacrylic acid are grafted onto a polysaccharide, and the molecular weight is in the range of about 3 to about 800 kDa.

68. The PNLP according to claim 67, further comprising a helper lipid.

69. The PNLP according to claim 67, wherein the terpolymer further comprises a glucose monomer conjugated to the terpolymer.

70. The PNLP according to claim 67, wherein the polysaccharide is a low molecular weight polysaccharide having a molecular weight in the range of about 3 to about 10 kDa.

71. The PNLP according to claim 67, wherein the polysaccharide is a low molecular weight polysaccharide having a molecular weight of less than approximately 40 kDa.

72. The PNLP according to claim 67, wherein the polysaccharide is a low molecular weight polysaccharide having a molecular weight in the range of about 20 to about 40 kDa.

73. The PNLP according to claim 67, wherein the polysaccharide is a low molecular weight polysaccharide having a dextrose equivalent in the range of about 15 to about 17.

74. Terpolymer, Lipids, cholesterol, payload Polymer lipid nanoparticles (PLNPs) containing, The terpolymer is a graft polymer obtained by grafting polysorbate 80 and molecules having a carboxyl group onto a polymer selected from the list consisting of polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), and poly(methacrylamide), wherein the polymer has a molecular weight in the range of about 1 to about 800 kDa.

75. The PNLP according to claim 74, further comprising a helper lipid.

76. The PNLP according to claim 74, wherein the molecule having a carboxyl group comprises a monomer, and the monomer has a carboxyl group.

77. The PNLP according to claim 74, wherein the molecule having a carboxyl group is methacrylic acid.

78. The PNLP according to claim 74, wherein the molecule having a carboxyl group is acrylic acid.

79. The PNLP according to claim 74, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

80. Terpolymer, Lipids, cholesterol, payload Polymer lipid nanoparticles (PLNPs) containing, The terpolymer is a graft polymer obtained by grafting molecules that bind to apolipoprotein E and molecules having a carboxyl group onto a polymer selected from the list consisting of polysaccharides, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), and poly(methacrylamide), wherein the polymer has a molecular weight in the range of about 1 to about 800 kDa.

81. The PNLP according to claim 80, further comprising a helper lipid.

82. The PNLP according to claim 80, wherein the molecule that binds to apolipoprotein E is polysorbate 80.

83. The PNLP according to claim 80, wherein the molecule that binds to apolipoprotein E is a fatty acid having an unsaturated chain or a phospholipid having an unsaturated fatty acid chain.

84. The PNLP according to claim 80, wherein polyethylene glycol is incorporated into the molecule that binds to apolipoprotein E.

85. Terpolymer, Lipids, cholesterol, payload Polymer lipid nanoparticles (PLNPs) containing, The PLNP wherein the lipid forms a complex with the payload, and the terpolymer is a graft polymer in which molecules that bind to apolipoprotein E and molecules having a carboxyl group are grafted onto a polymer containing glucose chains, and the polymer has a molecular weight in the range of about 1 to about 800 kDa.

86. The PNLP according to claim 85, further comprising a helper lipid.

87. The PNLP according to claim 85, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

88. The PNLP according to claim 85, wherein the payload comprises a nucleotide-based payload, and the lipid comprises at least one of an ionic lipid or a cationic phospholipid.

89. Terpolymer, Lipids, cholesterol, payload Polymer lipid nanoparticles (PLNPs) containing, The PLNP wherein the lipid forms a complex with the payload, and the terpolymer is a graft polymer in which molecules that bind to apolipoprotein E and molecules having a carboxyl group are grafted onto a polymer containing glucose chains, and the polymer has a molecular weight in the range of about 1 to about 800 kDa.

90. PLNP according to claim 89, further comprising a helper lipid.

91. The PLNP according to claim 89, wherein the polymer further comprises a glucose monomer conjugated to the terpolymer.

Images