Intelligent polymer-lipid based NANO drug delivery system to improve the bbb-penetration by dual active brain targeting strategies

Abstract

The blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB) create an obstacle for effective systemic drug delivery to the CNS. This application provides compounds and nanoparticles for increasing the penetration of drugs through the BBB. Specifically, this application provides nanoparticles for the diagnosis and treatment of central nervous system (CNS) diseases and preparation methods therefor. These nanoparticles are polymer-lipid based nanoparticles (PLNPs) functionalized to facilitate blood brain barrier (BBB) penetration and accumulation in a disease area of the CNS. Notably, said nanoparticles target an LDL receptor and / or glucose transporter. In various embodiments, the nanoparticles comprise terpolymers which comprise polysorbates (such as polysorbate 80), poly acrylic acids (such as poly methacrylic acid (PMAA)) and various polysaccharides (including maltodextrin) and the nanoparticles also comprise cholesterol and lipids. The nanoparticles encapsulate a pay load which is a therapeutic drug molecule, biomolecule, contrast agent or nucleotide.

Description

Intelligent Polymer-Lipid based Nano Drug Delivery System to Improve the BBB- penetration by Dual Active Brain Targeting StrategiesCROSS-REFERENCE TO RELATED APPLICATIONThis application claims the benefit of U.S. Provisional Patent Application Ser. No. 63 / 508,505, filed June 15, 2023, which is hereby incorporated by reference in its entirety.BACKGROUND

[0001] Systemic drug treatment of neurological diseases, such as brain tumors, inborn metabolic errors (e.g., lysosomal storage diseases), and infectious and neurodegenerative diseases (e.g., Alzheimer disease (AD), Parkinson disease (PD), Huntington’s disease (HD), stroke, Amyotrophic Lateral Sclerosis (ALS), Friedreich's ataxia (FRDA) and Multiple sclerosis (MS)), is a daunting challenge 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 a critical role in protecting the CNS against toxic and infectious agents while maintaining the ionic and volumetric environments, but also create an obstacle for effective systemic drug delivery to the CNS. Although many agents have therapeutic potential for CNS diseases, few of these agents have been clinically used because of brain barriers. To facilitate penetration across these barriers for enhanced drug delivery to the CNS, different strategies for efficient CNS delivery have been studied.

[0002] Among these strategies, nanotechnology has come out as an exciting and promising new platform for treating CNS diseases and has shown great potential to overcome problems related to the conventional treatment approaches. Molecules can be nanoengineered to carry out multiple specific functions such as to cross the BBB, target specific cell or signaling pathway, respond to endogenous stimuli, and act as a vehicle for gene delivery, support nerve regeneration and cell survival. There are various kinds of nanoparticles (NPs), namely, liposomes, lipid nanoparticles, polymeric nanoparticles, dendrimers, cyclodextrins, silica nanoparticles, magnetic nanoparticles, gold nanoparticles, quantum dots, and carbon nanotubes, have been described as appealing candidates for increasing the penetration of drugs through the BBB. The suitability of nano systems for brain delivery depends on properties such as nanometric size, surface charge, morphology, and, especially, the molecular recognition and interaction between a specific ligand conjugated on the nanoparticle surface and the molecule overexpressed on the brain target place (active targeting).

[0003] Active targeting is particularly important when designing solutions to achieve this goal, since this strategy allows directing the nanoparticles to the desired place and, consequently, transporting and delivering drugs to the site of action into the brain. In fact, these nano systems display a great surfacearea to volume ratio, which enables the nanoparticles to be highly chemically reactive, allowing surface modification with molecules that may be recognized by the receptors / transporters overexpressed in the BBB and cell-specific receptors in the brain tissue. There are, essentially, three different strategies for achieving this purpose: adsorptive-mediated transcytosis, transporter-mediated transcytosis, and receptor-mediated transcytosis.

[0004] The adsorptive-mediated transcytosis (AMT) provides a route for the brain delivery of nanoparticles across the BBB. The endothelial cells of BBB constitute a phospholipid rich membrane covered by a glycocalyx composed of heparan sulfate proteoglycans (HSPGs), namely glypican and syndecan. Moreover, there are many carboxyl groups of sialoglycoproteins and sialoglycolipids on this side of the BBB. Together, these make the luminal side of the BBB highly negatively charged. Therefore, AMT can be promoted using the electrostatic interactions between the negative moieties exposed at the luminal surface of cerebral endothelial cells and the cationic groups of ligands conjugated on the nanoparticles surface. However, by itself, AMT does not ensure cell-specific targeting, since positively charged molecules can be rapidly and indiscriminately adsorbed by all negatively charged cell membranes and, thus, penetrate a variety of different cells.

[0005] An alternative strategy for the brain delivery of drugs is the use of BBB-specific transporters, for efficient supply of nutrients of low molecular weight from the bloodstream to the CNS. There are more than twenty different transporters are well known in the BBB and it is possible to synthesize nanoparticles with molecules conjugated on their surface that are well recognized by the transporters overexpressed in brain endothelial cells. Transporter-mediated transcytosis (TMT) is an important approach for the design of nanocarriers for brain delivery.

[0006] Another way of reaching the brain tissue is taking advantage of receptors overexpressed in the BBB; the so-called receptor-mediated transcytosis (RMT). Nanoparticles can be modified with specific ligands of receptors and, hence, can be taken up by brain endothelial cells. Once in the brain, nanoparticles must reach the right cell target. Active targeting is particularly important when designing solutions to achieve this goal, since this strategy allows directing the nanoparticles to the desired site of action, by modifying the surface of NPs with molecules that may be recognized specifically by receptors or transporters overexpressed in the brain.

[0007] During the past decades, different strategies have been applied in nanoparticles for improving the brain delivery efficiency of therapeutic payloads; but unfortunately, nanomedicine has yet to make its mark in clinical studies for the treatment of neurological disorders. Hence, there is a continued and urgent medical need for finding new strategies to increase the penetration of drugs through BBB.SUMMARY

[0008] In one embodiment, the present disclosure relates to multifunctional nanoparticles compositions, including pharmaceutical compositions, for the diagnosis and treatment of a central nervous system (CNS) disease such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington’s Disease, stroke and so forth.

[0009] In another embodiment, the present invention provides polymer-lipids based nanoparticles (PLNPs) functionalized with dual active brain targeting strategies: transporter-mediated transcytosis by glucose transporter protein (for example, GLUT-1), and receptor-mediated transcytosis by low-density lipoprotein (LDL) receptor; the multifunctional nanoparticles being configured to facilitate blood brain barrier penetration and accumulation in a disease area of the central nervous system. It is believed that the present invention is the first working example of a compound to transport cargo across the BBB that simultaneously targets both an LDL receptor and a glucose transporter.

[0010] In its third aspect, the present invention provides nanoparticles with a surface covered with a layer of novel terpolymers. The novel terpolymers are selected from a library of novel terpolymers composed of various quantities of polysorbates (such as polysorbate 80) and poly acrylic acids (such as poly methacrylic acid [PMAA]), grafting onto different molecular weights of maltodextrins (dextrose equivalent: 3-20).

[0011] In its fourth aspect, the novel terpolymers cover the surface of polymer-lipid nanoparticles, in which one intrinsic component “polysorbate 80” from the terpolymer adsorbs ApoE from plasma and enhances multifunctional nanoparticle crossing of the BBB by receptor-mediated transcytosis through brain vascular endothelial cells.

[0012] In its fifth aspect, another intrinsic component “maltodextrin” forms the backbone of a novel terpolymer, the backbone being heavily functionalized by glucose units at both ends, and enhances multifunctional nanoparticle crossing of the BBB by facilitative glucose transporter protein 1 (GLUT1) pathway.

[0013] In its sixth aspect, to further improve the penetration of BBB by GLUT1 pathway, the polymers are covalently functionalized with glucose using EDC chemistry.

[0014] In accord with the invention, there is provided a method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid-payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a polysorbate and an acrylicacid grafted onto maltodextrin with a dextrose equivalent of 1-30. In an aspect of the 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 below 10 kDa. In still 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 aspect, the polysorbate is polysorbate 80. In still yet another aspect, the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, C1-C25- N,N’ -disubstituted amino-(Ci-C2s)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci- C2S)-OH methacryl-(Ci-C25)-NH2.

[0015] In accord with the invention, there is provided a method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid-payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and methacrylic acid grafted onto a polysaccharide with a molecular weight within the range of about 3 to about 800 kDa. In an aspect of the invention, the terpolymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the polysaccharide is a low molecular weight polysaccharide with a molecular weight is within the range of about 3 to about 10 kDa. In another aspect, the polysaccharide is a low molecular weight polysaccharide with a molecular weight is less than about 40 kDa. In still another aspect, the polysaccharide is a low molecular weight polysaccharide with a molecular weight in the range of about 20 to about 40 kDa. In still another aspect, the polysaccharide is a low molecular weight polysaccharide with a dextrose equivalent in the range of about 15 to about 17. In yet another aspect, the polysorbate is polysorbate 80. In still yet another aspect, the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, Ci-Cbs-N.N’ -disubstituted amino-(Ci- C25) -acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-OH methacryl-(Ci-C2s)-NH2.

[0016] In accord with the invention, there is provided a method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid-payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to formcomplexes with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly (meth acrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, CI-C25-N,N’ -disubstituted amino-(Ci-C2s)-acrylate, 2- (dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-OH methacryl-(Ci-C2s)-NH2, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the molecule with carboxylic groups comprises a monomer where the monomer has carboxylic groups. In another aspect, the molecule with carboxylic groups is methacrylic acid. In another aspect, the molecule with carboxylic groups is acrylic acid. In still another aspect, the polymer further comprises glucose monomers conjugated to the terpolymer.

[0017] In accord with the invention, there is provided a method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid-payload emulsion; and homogenizing of the terpolymer / lipid- payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(meth acrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, Ci-C25-N,N’-disubstituted amino-(Ci-C2s)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-OH methacryl-(Ci- C2S)-NH2, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the molecule that binds with apolipoprotein E is polysorbate 80. In another aspect, the molecule that binds with apolipoprotein E is a fatty acid with unsaturated chains or a phospholipid with unsaturated fatty chains. In another aspect, the molecule that binds with apolipoprotein E incorporates a polyethylene glycol.

[0018] In accord with the invention, there is provided a method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous solution to the lipid / payloadcomplex to form a terpolymer / lipid-payload emulsion; and homogenizing of the terpolymer / lipid- payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the polymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the payload comprises a nucleotide-based payload and the lipids comprise at least one of ionizable lipids or cationic phospholipids.

[0019] In accord with the invention, there is provided a compound for transporting a payload across the blood brain barrier, where the compound targets both the LDL receptor and the glucose transporter. In an aspect of the invention, the compound has a diameter in the range of around 50 nm to around 250 nm and the compound has a negative surface charge. In another aspect, the glucose transporter is GLUT- 1. In another aspect, the payload comprises a nucleotide. In another aspect, the payload comprises a therapeutic drug molecule. In another aspect, the payload comprises a biomolecule. In another aspect, the payload comprises a contrast agent.

[0020] In accord with the invention, there is provided a method for synthesizing a compound for transporting a payload across the blood brain barrier, where the compound targets both an LDL receptor and a glucose transporter, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid-payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the polymer further comprises glucose monomers conjugated to the terpolymer.

[0021] In accord with the i n vention , there is provided a compound for transporting a payload across the blood brain barrier, where the compound uses apolipoprotein E to target the LDL receptor and glucose to transport through GLUT-1. In an aspect of the invention, the glucose transporter is GLUT-1. In another aspect, the payload comprises a nucleotide. In another aspect, the payload comprises a therapeutic drug molecule. In another aspect, the payload comprises a biomolecule. In another aspect, the payload comprises a contrast agent.

[0022] In accord with the invention, there is provided a nanoparticle for transporting a payload across the blood brain barrier, where the nanoparticle targets both the LDL receptor and the glucosetransporter. In an aspect of the invention, the glucose transporter is GLUT-1. In another aspect, the payload comprises a nucleotide. In another aspect, the payload comprises a therapeutic drug molecule. In another aspect, the payload comprises a biomolecule. In another aspect, the payload comprises a contrast agent.

[0023] In accord with the invention, there is provided a nanoparticle, where the nanoparticle targets both the LDL receptor and a glucose transporter and the nanoparticle has a diameter in the range of around 50 nm to around 250 nm and the nanoparticle has a negative surface charge. In an aspect of the invention, the nanoparticle has no exposed lipids on the surface. In another aspect, the glucose transporter is GLUT-1. In another aspect, the payload comprises a nucleotide. In another aspect, the payload comprises a therapeutic drug molecule. In another aspect, the payload comprises a biomolecule. In another aspect, the payload comprises a contrast agent.

[0024] In accord with the invention, there is provided a nanoparticle comprising: a terpolymer comprising a polysaccharide, and a hydrophobic monomer, and a hydrophilic or amphiphilic monomer; where the nanoparticle transports a payload across the blood brain barrier by targeting both the LDL receptor and the glucose transporter. In an aspect of the invention, the targeting of the LDL receptor is by the nanoparticle recruiting ApoE in the blood circulation before encountering the endothelium. In another aspect, the free glucose ends of the terpolymer bind with a glucose transporter. In another aspect, the hydrophilic or amphiphilic monomer being capable of binding with ApoE in the blood circulation. In another aspect, the nanoparticle further comprises glucose conjugated with the terpolymer. In another aspect, the polysaccharide has a DE of around 20 or less. In another aspect, the polysaccharide has a molecular weight with the range of about 1 to about 800 kDa.

[0025] In accord with the invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising: a terpolymer; lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer forms a shell surrounding the complex, and the terpolymer is a grafted polymer with polysorbate 80 and methacrylic acid grafted onto maltodextrin with a dextrose equivalent of 1-30. In an aspect of the invention, the PLNP, further comprises a helper lipid. In an aspect, 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 below 10 kDa. In another aspect, the maltodextrin is a low molecular weight maltodextrin with a dextrose equivalent in the range of about 15 to about 17.

[0026] In accord with the invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising: a terpolymer; lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and methacrylic acid grafted onto a polysaccharide with a molecular weight within the range of about 3 to about 800 kDa. In an aspect ofthe invention, the PNLP further comprises a helper lipid. In another aspect, the terpolymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the polysaccharide is a low molecular weight polysaccharide with a molecular weight is within the range of about 3 to about 10 kDa. In another aspect, the polysaccharide is a low molecular weight polysaccharide with a molecular weight is less than about 40 kDa. In an aspect of the invention, the polysaccharide is a low molecular weight polysaccharide with a molecular weight in the range of about 20 to about 40 kDa. In another aspect, the polysaccharide is a low molecular weight polysaccharide with a dextrose equivalent in the range of about 15 to about 17.

[0027] In accord with the invention, there is provided a polymer dipid nanoparticle (PLNP) comprising: a terpolymer; lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly (methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(meth acrylamide, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the PNLP further comprises a helper lipid. In another aspect, the molecule with carboxylic groups comprises a monomer where the monomer has carboxylic groups. In another aspect, the molecule with carboxylic groups is methacrylic acid. In another aspect, the molecule with carboxylic groups is acrylic acid. In another aspect, the polymer further comprises glucose monomers conjugated to the terpolymer.

[0028] In accord with the invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising: a terpolymer; lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly (methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(meth acrylamide, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the PNLP further comprises a helper lipid. In another aspect, the molecule that binds with apolipoprotein E is polysorbate 80. In another aspect, the molecule that binds with apolipoprotein E is a fatty acid with unsaturated chains or a phospholipid with unsaturated fatty chains. In another aspect, the molecule that binds with apolipoprotein E incorporates a polyethylene glycol.

[0029] In accord with the invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising: a terpolymer; lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymerhas a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the PNLP further comprises a helper lipid. In another aspect, the polymer further comprises glucose monomers conjugated to the terpolymer. In another aspect, the payload comprises a nucleotide-based payload and the lipids comprise at least one of ionizable lipids or cationic phospholipids.

[0030] In accord with the invention, there is provided a polymer-lipid nanoparticle (PLNP) comprising: a terpolymer; lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa. In an aspect of the invention, the PLNP further comprises a helper lipid. In another aspect, the polymer further comprises glucose monomers conjugated to the terpolymer.

[0031] With the foregoing and other advantages and features of the invention that will become hereafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the invention and the appended claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows a schematic diagram depicting the process of synthesis of PLNPs designed to cross the blood brain barrier (BBB) in accord with the invention.

[0033] Figure 2 shows a schematic diagram of PLNP structure in accord with the invention.

[0034] Figure 3 illustrates the PNLP crossing the BBB.

[0035] Figure 4A shows the results of CNS penetration tests comparing dual specific TERP chemistry versus a single-receptor approach using MR imaging.

[0036] Figure 4B shows the results of CNS penetration tests comparing dual specific TERP chemistry (squares) versus a single-receptor approach using ICP technique (circles).

[0037] Figure 5 shows the results of a study of brain localization of the TERP nanoparticles, displaying and quantifying stained regions associated with mRNA in the presence of neurons, astrocytes, and microglia.

[0038] Figure 6 shows the comparison of BBB penetration efficiency among polymer-lipid nanoparticles (TERP) with polysorbate 80 (TERP-PS80), TERP with polysorbate 80 (PS80) and intrinsic glucose units (TERP-PS80-iGLU), and TERP with polysorbate 80 (PS80) and intrinsic glucose units, and covalently linked glucose (TERP-PS80-iGLU-cGUL).DETAILED DESCRIPTION

[0039] Figure Legend

[0040] 6 Polysaccharide component of the terpolymer

[0041] 7 Lipid component of the terpolymer

[0042] 8 Polysorbate component of the terpolymer

[0043] 9 Glucose component of the terpolymer

[0044] 10 Lipid components (e.g., ionizable lipid, phospholipid, helper lipid)

[0045] 11 Cholesterol

[0046] 12 Payload

[0047] 14 Lipid / payload complex droplet

[0048] 16 Terpolymer

[0049] 18 Terpolymer / lipid-payload complex emulsion

[0050] 19 Polymer dipid nanoparticle (PLNP)

[0051] 20 Lipid-cargo complex, lipid-payload complex

[0052] 21 Cargo, RNAs

[0053] 22 Terpolymer

[0054] 23 Anionic active sites (e.g., carboxylic acid) of the terpolymer

[0055] 24 Lipid

[0056] 26 Cholesterol

[0057] 28 Helper lipids

[0058] 29 Negative surface charge of the PLNP

[0059] 30 PLNP

[0060] 32 Apolipoprotein E (ApoE)

[0061] 34 Nanoparticle having free glucose ends and exposed ApoE

[0062] 35 Exposed ApoE of the Nanoparticle

[0063] 36 Free glucose ends of the Nanoparticle

[0064] 38 GLUT-1 transporter

[0065] 40 Lipoprotein (LDL) receptor

[0066] 41 Blood

[0067] 42 Brain

[0068] 43 Endothelium

[0069] 44 Tight junction of the endothelium

[0070] 50 Stained mRNA

[0071] 52 Neuron stained with Map2

[0072] 54 Astrocyte stained with Gfap

[0073] 56 Microglia stained with Ibal

[0074] The term “alkyl” as used herein means a straight or branched chain hydrocarbon containing from 1 to 25 carbon atoms, preferably from 1 to 10 carbon atoms, more preferably 1, 2, 3, 4, 5, or 6carbons. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso-butyl, 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.

[0075] The term “amino” as used herein means a — NH2 group.

[0076] The term “carboxy” as used herein means a — COOH group, which may be protected as an ester group: — COO-alkyl.

[0077] The term “hydroxy” as used herein means an — OH group.

[0078] The term "nucleic acid" is a term of art that refers to a string of at least two base-sugar- phosphate combinations. For naked DNA delivery, a polynucleotide contains more than 120 monomeric units since it must be distinguished from an oligonucleotide. However, for purposes of delivering RNA, RNAi and siRNA, either single or double stranded, a polynucleotide contains 2 or more monomeric units. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of a messenger RNA, anti-sense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus. Anti-sense is a polynucleotide that interferes with the function of DNA and / or RNA. The term nucleic acids — refers to a string of at least two base-sugar-phosphate combinations. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). RNA may be in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi, siRNA, and ribozymes. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.

[0079] The term "siRNA" means a small inhibitory ribonucleic acid. The siRNA are typically less than 30 nucleotides in length and can be single or double stranded. The ribonucleotides can be natural or artificial and can be chemically modified. Longer siRNAs can comprise cleavage sites that can be enzymatically or chemically cleaved to produce siRNAs having lengths less than 30 nucleotides, typically 21 to 23 nucleotides. siRNAs share sequence homology with corresponding target mRNAs. The sequence homology can be 100 percent or less but sufficient to result in sequence specific association between the siRNA and the targeted mRNA.

[0080] By Cx, we mean herein an acyclic straight or branched hydrocarbon of longest length x; thus,C1-C5 includes methyl, ethyl, propyl, butyl, pentyl, isopropyl, etc.

[0081] The present invention relates to the development of novel polymer-lipid nanoparticles for improving the BBB penetration. In many cases, passive diffusion across the BBB is ineffective as the payload or cargo is too large to passively pass the blood brain barrier in sufficient concentrations to beeffective. This invention teaches the design and development of novel polymer-lipid nanoparticles with a surface or shell covered with a layer of novel polymers, in which the polymers have the intrinsic dual active brain targeting components, polysorbate and glucose units.

[0082] The novel polymer-lipid nanoparticles have a layer of polymer, functionalized with dual active brain targeting strategies: transporter-mediated transcytosis by glucose transporter protein 1, and receptor-mediated transcytosis by low-density lipoprotein (LDL) receptor.

[0083] The intrinsic glucose units are from the ends of the backbone of polymer, in one example maltodextrin, which includes the molecular weight with dextrose equivalent from 3 to 20.

[0084] The use of low molecular weight polysaccharides is an improvement over terpolymers with higher weight polysaccharide backbones because low molecular polysaccharides will provide significant additional glucose end units on the surface of the nanoparticle, better facilitating transportation through glucose transporter. In a preferred embodiment, the low molecular weight polysaccharides have a dextrose equivalent (DE) of around 20 or less. Such terpolymers can then be improved by conjugating glucose onto the terpolymer. Such terpolymers can also be improved by conjugation with polysorbate (e.g., PS80) to access a second transport mechanism through the BBB. Also, the terpolymers can be improved by both conjugating glucose onto the terpolymer and conjugation with PS80 to access a second transport mechanism through the BBB. As noted below, there are other materials that can be used instead of PS80 to create a second transport mechanism through the BBB.

[0085] To further facilitate glucose transporter protein 1 (GLUT1) pathway, the polymers can be covalently functionalized with glucose using the well-known EDC chemistry. By “EDC”, we mean 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or other suitable aqueous carboxylic acid crosslinking agents.

[0086] Figure 1 shows a schematic diagram depicting the process of manufacturing of PLNPs as a carrier system for delivery to the CNS, specifically across the blood brain barrier. The PLNPs are synthesized in part by use of the three-core material and methods used for manufacturing of LNPs; that is, utilizing a cationic lipid, cholesterol, and a helper lipid. However, in the present invention, rather than use the fourth material in the synthesis of LNPs, PEG-lipid, it is herein taught to instead use a terpolymer that further improves the nanoparticle surface chemistry and stability and is designed to cross the BBB. (Note that the PLNPs will also cross internasal and intrathecal barriers but were not designed specifically for this purpose). Turning to Figure 1, the nanocomplex synthesis starts with the rapid mixing of lipid components 10 in an alcohol or an organic solvent (preferably ethanol) and cholesterol 11 and the payload(s) 12 in a solvent (the choice of the solvent depends on the payload(s)). In Figure 1, the depicted payload is RNA, but this approach can work with a wide range of payloads, including nucleotides, therapeutic drug molecules, biomolecules, and contrast agents. The choice of lipid dependsupon the payload(s) (note that there can be more than one payload in a given PLNP), and the solvent used should reflect the specific lipid. The lipid interacts with the payloads and due to electrostatic and hydrophobic / hydrophilic interactions the lipids self-assemble, covering the payload to form a lipid / payload complex. Further change in the polarity of the solution of the lipids (including charged lipid, and cholesterol, and helper lipids, as will be known to a person skilled in the art) creates a shell around multiple lipid-payload droplets due to electrostatic, hydrophobic and Van der Waal interactions creating a large lipid / payload complex droplet 14. The amount of lipid necessary to form a complete complex around the payload will depend upon the specific payload; in a preferred embodiment, the weight ratio of lipid to payload is in the range of 2.5:1 to 15:1, in another embodiment, the weight ratio of lipid to payload is in the range of 2.5 : 1 to 50: 1.

[0087] Continuing with Figure 1, as the manufacturing process proceeds the addition of aqueous terpolymer 16 which through further interactions with the droplets 14 due to electrostatic and hydrophobic interactions, covers the surface of lipid-payload complex 14 to form a polymer / lipid- payloads emulsion 18. The polymer-lipid-payloads complex is then processed through sonication or homogenization or microfluidic technique to produce the small PLNPs 19.

[0088] The process as illustrated in Figure 1 can accommodate a wide range of useful payloads. Including both positively charged and negatively charged payloads, and including nucleotides, therapeutic drug molecules, biomolecules, and contrast agents.

[0089] As known to a person skilled in the art, further purification, buffer exchange to physiological pH and concentration of nanoparticles can be used as desired, as long as the PLNP particles retain a negative charge due to presence of anionic polymer on the surface.

[0090] In the case of the cargo being a nucleic acid or nucleotide, the weight ratio of terpolymer to total lipid can go as high as 10: 1 , but a preferred weight ratio of terpolymer to total lipid is between 0.01 :1 or 0.5: 1 to 6:1, and more preferably is around 3:1.

[0091] The diameter of the PNLP resulting from this synthesis can be controlled at the homonization and sonification stage, as known to persons skilled in the art. For the purposes of the PNLP being best designed to deliver a payload to the brain, a preferred diameter is around 5 nm to around 500 nm, and preferably 100 nm to around 150 nm, and another preferred diameter is around 100 nm.

[0092] It is notable that, unlike typical lipid nanoparticles, the PLNPs produced by the above method do not have any exposed lipids on the shell or surface of the PLNP. As a result, the PNLPs produced by this synthesis have increased stability and reduced immunogenic reactions, since there are no exposed lipids for external interactions.

[0093] The lipid 10 can be positively, negatively, or neutrally charged, but should be selected to reflect the payloads and result in lipid / payloads droplets with an outside layer of lipid.

[0094] The helper lipid can be any zwitterionic phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine, phosphatidyl serine, and sphingomyelin such as DSPC, DPPC.

[0095] The polymer (which is the backbone of the terpolymer) is preferably maltodextrin with a molecular weight ranging between around 3 to around 20 dextrose equivalents. In another embodiment, the polymer is maltodextrin with a molecular weight ranging between around 3 to around 30 or to around 40 dextrose equivalents. However, any polymer can be used that results in glucose chains on the backbone once the PLNP is synthesized. To be a suitable polymer, in preferred embodiments, the polymer features short glucose chains with hydroxyl-active groups on the backbone.

[0096] The efficiency of crossing the BBB is affected by the density of glucose on the surface of the PNLP. By using shorter-chain glucose-based polymers, there are more glucose ends to facilitate transport.

[0097] The monomers of polymer in the sidechains of the polymer can be selected from a range of materials including but not limited to PS80, fatty acids with unsaturated chains, and phospholipids with unsaturated fatty chains, preferably with carboxylic groups.

[0098] In the preferred embodiment, the polymer is maltodextrin with a molecular weight ranging between around 3 to around 20 dextrose equivalents and the sidechains are PS80.

[0099] In a more specific example, the payload is manganese dioxide, and the lipid is DSPC and cholesterol, and the polymer is maltodextrin with a molecular weight ranging between around 3 to around 20 dextrose equivalents and the sidechains are PS80. There is no helper lipid in this specific example.

[0100] The synthesis described above can be implemented using clinically approved lipids, cholesterol and helper lipids that are used for clinically approved conventional LNPs. (A person skilled in the art should be aware that some of the materials described in the description of the synthesis above are not clinically approved.)

[0101] Importantly, in the present invention the fourth material usually employed in the synthesis of LNPs, PEG-lipid (such as l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG) or other PEG-lipid conjugates) has been replaced with a specific class of terpolymers to further improve the nanoparticle surface chemistry and stability and facilitate transport through the BBB. Compared to conventional LNPs that have a net neutral charge under physiological condition, the present PLNPs described in this invention have net negative surface charge under physiological conditions (i.e., the condition in the blood) making them more stable during blood circulation.

[0102] In a variation of the above method of synthesis, there can be a step of conjugating glucose onto the polymer in order to increase the concentration of glucose on the surface or shell of the PNLP.However, it is relatively difficult to introduce the glucose onto a general (polymer) backbone. It is advantageous from a synthesis and scale-up point of view to rely on the use of short-chain glucose polymers as a backbone, in some embodiments.

[0103] Figure 2 shows a schematic diagram of the BBB-transport structure. Turning to Figure 2, a BBB-transport PLNP is illustrated, including a coating of lipid-payloads complex 20 with terpolymer 22 having carboxylic acid active sites 23. The polymer interacts with the lipid-payloads complex due to electrostatic and hydrophobic / hydrophilic interactions. The PLNPs are synthesized in part as described above in reference to Figure 1 using the three-core material used for manufacturing of LNPs such as a lipid 24, cholesterol 26 and helper lipid 28 to surround the cargo 21. The fourth component introduced to produce the BBB-transport PLNP is a terpolymer 22 with carboxylic acid groups. Unlike LNPs that are neutral at physiological condition, the proposed system has negative surface charge 29 under physiological pH and has a better stability due to electrostatic repulsion between particles. In preferred embodiments, the terpolymer 22 has glucose end-units on the polymer backbone that facilitate the BBB transport.

[0104] In a preferred embodiment, the BBB-transport PLNP has an terpolymer is maltodextrin with a molecular weight ranging between around 3 to around 20 dextrose equivalents and the sidechains are PS80.

[0105] In a more specific example, a BBB-transport PLNP has a payload of manganese dioxide, the lipid components are DSPC and cholesterol, and the terpolymer comprises maltodextrin with a molecular weight ranging between around 3 to around 20 dextrose equivalents and sidechains of PS80.

[0106] In a preferred embodiment, a BBB-transport PLNP uses D-Glucose transporter protein (GLUT), one of the important nutrient transporters. GLUT has a particularly high concentration in brain micro vessels; approximately 100-fold more abundant than the transferrin receptor which has been widely used as a brain-special target for active drug delivery. The large and uninterrupted energy demand of the brain is provided almost exclusively by b-D-glucose, which can highly efficiently penetrate into the brain via faci I itati ve GLUT.

[0107] Specifically, in some embodiments, for a BBB-transport PNLP the terpolymer backbone is made of glucose units, and the end-units of the backbone are recognized by the GLUT-1 transporter, which facilitates the PNLP crossing the BBB.

[0108] In another embodiment, the novel polymer-lipid nanoparticles have a layer of polymer mentioned above, functionalized with dual active brain targeting strategies, transporter-mediated transcytosis by glucose transporter protein 1, and receptor-mediated transcytosis by low-density lipoprotein (LDL) receptor. This dual-function structure allows for transporter-mediated transcytosis by glucose transporter protein 1, and also receptor-mediated transcytosis by low-density lipoprotein (LDL)receptor, such as the 2-LDLR receptor. In a preferred embodiment, the PS80 is used on the side chains which engages the 2-LDLR receptors in a subject for receptor-mediated transcytosis into the brain.

[0109] Figure 3 illustrates the PNLP crossing the BBB by way of travel from the blood 41 through the endothelium 42 and into tissue of the brain 43 (characteristic cell types therein being shown in the bottom layer of the Figure: neurons, astrocytes, microglia). A PNLP as described above 30 absorbs apolipoprotein E (ApoE) 32 from the blood to create a nanoparticle 34 which has both free glucose ends 36 and exposed ApoE 35. The nanoparticle 34 moves from the blood through the endothelium and into the brain using two transport mechanisms: transporter-mediated transcytosis where the glucose units 36 are recognized by the GLUT-1 transporter 38, which facilitates the nanoparticle 34 crossing through the endothelium (BBB), and receptor-mediated transcytosis where the ApoE 35 interacts with the lipoprotein (LDL) receptor 40, which also facilitates the nanoparticle 36 crossing through the endothelium (BBB)

[0110] It is generally difficult to synthesize nanoparticles with bi-functional BBB crossing systems. By using short-chain glucose polymers as the backbone, the synthesis disclosed above makes it easier to accommodate adding a second transport mechanism (in this case by using PS80 on the side chains and using the 2-LDLR receptors). As a result, this approach to synthesizing nanoparticles with bifunctional BBB crossing systems will more readily scaled-up.

[0111] In some embodiments, the terpolymer is a graft terpolymer of poly(methacrylic acid)- polysorbate 80-maltodextrin. Briefly, polysorbate 80, maltodextrin, and methacrylic acid are combined in an emulsion polymerization in the presence of potassium persulfate (KPS), sodium thiosulfate (STS), and water at 70 degrees Celsius:Maltodextrin

[0112] It should be noted that the illustrated resultant terpolymer above includes a backbone carboxylic acid group for each MAA unit in the polymer and the polysorbate includes a side chaincarboxylic acid group; the effect of which is to provide several carboxylic acid groups per unit of terpolymer. In some embodiments, the quantity of maltodextrin or polysorbate can be varied in a manner understood by those skilled in the art to change the average number of terpolymer units so decorated. In some embodiments, a polysorbate with a different value of w, x, y, or z (or combinations thereof) can be used to vary the terpolymer structure.

[0113] In some embodiments, in lieu of MAA, a C1-C25 acrylate is used in the above synthesis. In some embodiments, ethyl acrylate is used. In some embodiments, a Ci-C25-disubstituted amino C1-C25 acrylate is used (where Cxmay 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, a C1-C25 methacrylate is used. In some embodiments, stearyl methacrylate is used. In some embodiments, methacryl-(Ci-C2s)-OH is used. In some embodiments, methacryl-(Ci-C2s)-NH2 (with hydrochloride as complex ion, as applicable) is used. In some embodiments, any polymerizable monomer comprising an acryl- or methacryl group is used.

[0114] It is also possible to synthesize such a terpolymer by generally following the approach given in U. S. Patent 10,233,277 (hereby incorporated by reference in its entirety).

[0115] In some embodiments, a PLNP as described herein has a payload that is therapeutically effective to diagnose or treat a CNS disease. 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 stroke. In some embodiments, the payload comprises a nucleotide. In some embodiments, the payload comprises a therapeutic drug molecule. In some embodiments, the payload comprises a biomolecule. In some embodiments, the payload comprises a contrast agent.

[0116] A pharmaceutical composition is hereby disclosed, the composition comprising PLNPs having at least one cargo that is therapeutically or diagnostically useful for at least one CNS disease, and pharmaceutic ingredients that enable a parenteral dosage form for human or veterinary use. Parenteral dosage forms can be administered to patients by various routes including subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are specifically sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. Pharmaceutical compositions for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous soludons, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable fluidity of the composition maybe maintained, for example, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservative agents, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by various antibacterial and antif ungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0117] In some cases, in order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.

[0118] Injectable depot forms are made by forming microencapsulated matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly (anhydrides). Depot injectable formulations also are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

[0119] Injectable preparations, for example, sterile injectable suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic, parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.Example 1:

[0120] To explore the degree of BBB-penetration of payloads using the dual-specific TERP system (TERP low-chain maltodextrin with polysorbate 80 (PS80) and intrinsic glucose units, and covalently linked glucose) with both LDL-r and glucose transporters compared to the single-receptor mediated TERP composition (long-chain polysaccharides with PS80) targeting LDL-r alone, gadolinium (Gd) was encapsulated as the payload in to these two formulations as described above. TERP-Gd nanoparticles were injected intravenously in healthy naive animals. The CNS penetration of Gd wasconfirmed in-vivo using MR imaging at 2 hours post treatment. It was observed that the dual-specific TERP chemistry increases the MR contrast signal (R1 value) by 2.6-fold compared to that after treatment with the single-specific receptor approach. The results are shown in Figure 4A as a percent increase over the initial (pre-treatment) signal.

[0121] To explore the brain PK of the TERP-enabled Gd nanoparticles, the animals were sacrificed at 0.5, 1, 2, and 4 hours post injection. Brain samples were collected and the amount of Gd in the brain tissues was measured using ICP technique. The results are shown in Figure 4B. The results as shown in Figure 4B are a percent increase over a treatment with saline. The data confirmed that these dualspecific TERP nanoparticles offer superior brain-penetration of the payload in the brain compared to that after treatment with the LDL-r alone pathway, by more than 2.7-fold.Example 2:

[0122] To study the brain localization of the TERP nanoparticles, Cy5-mRNA-loaded TERP nanoparticles (TERP low-chain maltodextrin with polysorbate 80 (PS80) and intrinsic glucose units, and covalently linked glucose) were injected intravenously in healthy naive animals. Animals were sacrificed 2 hours post treatment. The brain tissues were collected, and tissue slides were prepared for the IHC studies. The slides were stained for neurons (Map2), astrocytes (Gfap), and microglia (Ibal) imaged using fluorescent imaging.

[0123] The results are shown in Figure 5. In all three slides, the mRNA is stained red 50. In the Cortex: Neuron slide Map2 is green 52; in the Cortex: Astrocyte slide Gfap is green 54; and in the Cortex: Microglia slide Ibal is green 56. The data has been reported as the number co-localized signals from cells and the mRNA. Results showed that these TERP nanoparticles are mainly taken up by neurons, and less by astrocytes and microglia, respectively.Example 3:

[0124] Chemical and Reagents

[0125] The amphiphilic polymer, poly (methacrylic acid)-polysorbate 80 was grafted onto maltodextrin (DE=17) and the PLNPs were synthesized as previously described. 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) purchased from Sigma-Aldrich (Oakville, ON, Canada). All chemicals were of analytical grade and used without further purification if not indicated otherwise.

[0126] Experimental Animals

[0127] For in vivo brain delivery efficiency, eight weeks old female Balb / c mice (Jackson laboratory, Maine, USA) were used. The animals had free access to food and water throughout the study.

[0128] Preparation of MnO2-NP loaded TERP

[0129] To verify the BBB penetration of polymer-lipid nanoparticles, MnO2-NP was loaded into the PNLP, covered with a layer of polymer or polymer pre-conjugated with glucosamine.

[0130] Briefly, the crude inorganic Mn02 NP-lipid PNLP were first synthesized via reduction of KMnO4 to Mn02 in presence of PVA in an aqueous medium, followed by the addition of ethanolic solution of phospholipid (DPPC) and cholesterol. The emulsion was then mixed with amphiphilic polymer, during which the hydrophobic interaction between the polymer and the lipid domain of the nanoparticles led to the self-assembly and formation of MnO2-TERP. The emulsion was then passed through a high-pressure homogenizer at a pressure above 25 Kpsi to produce small NPs. The final sample was collected in ice cold water, filtered, and purified using tangential flow filtration (TFF) to remove the unreacted reagents. The purified solution was lyophilized in the presence of sucrose as a cryo-protectant agent.

[0131] In Vivo brain delivery efficiency using MnO2-TERP

[0132] To investigate the BBB-penetration of TERP by dual active brain targeting strategies (GLUT1 and low-density lipoprotein (LDL) receptor pathways), the BALB / c mice were injected IV through the tail vein at a dose of 100 pmole Mn / kg of MnO2-TERP. Two groups of animals (n=5 / group) were treated with MnO2-TERP using the amphiphilic polymer, in which one group of animals were fasting for 12 hours. After fasting, glucose solution (20 wt %) was injected to elevate the blood glucose concentration, and 30 min later, a single dose of MnO2-TERP was administrated. After 1 hour, the mice were perfused and brain tissues were collected, homogenized for the quantification of Mn using Inductively Coupled Plasma Optical Emission spectroscopy (ICP-OES). In another experiment, Mn02- TERP prepared using polymer conjugated with glucosamine were administrated into the fasting mice (n=5 / group), and then followed the same procedure to collect the brain tissues for Mn quantification under ICP-OES.

[0133] Figure 6 shows the comparison of BBB penetration efficiency among polymer-lipid nanoparticles (TERP) with intrinsic glucose units with polysorbate 80 (without glucose pre-injection to activate GLUT transporters) (labelled TERP-PS80), TERP with polysorbate 80 (PS80) and intrinsic glucose units with glucose injection to activate GLUT transporters (labelled TERP-PS80-iGLU), and TERP with polysorbate 80 (PS 80) and intrinsic glucose units, and covalently linked glucose with glucose injection to activate GLUT transporters (labelled TERP-PS80-iGLU-cGUL). The data for GLUT transport, normalized to the non-activated GLUT condition (left), shows that a PMAA-PS80- maltodextrin terpolymer as described herein having intrinsic glucose units (middle) and especially having covalently linked glucose units (right) are significantly more capable of transport across the BBB. This finding demonstrates that such PLNPs can be actively transported via GLUT.Prophetic Example 4:

[0134] It is also possible to perform modified versions of the experiment given above to further evaluate transporter-mediated BBB entry of PLNPs. In one possible experiment, PLNPs described herein will be evaluated in a similar experiment, with the expected observation of ablation of BBB entry in the presence of such selective GLUT1 transporter antagonists (with or without glucose pre-injection) 5 as are known in the art. In another possible experiment, PLNPs described herein will be evaluated in a similar experiment for ablation of BBB entry in an animal whose genome has a transgenically introduced mutation, a deletion, a knock-down, a knock-out, or any other appropriate means known to the art that results in at least partial loss of function of GLUT1 for BBB transport in that animal. In some experiments, the mutation may be in the Slc2al gene. 0 Prophetic Example 5:

[0135] It is also possible to perform cell-based screens to evaluate transporter-mediated BBB entry of PLNPs. To evaluate GLUT transport efficiency, the commercially available Human SLC2A1 (Glucose Transporter GLUT1) knockout A549 cell line and wild type A549 cell will each be incubated with a PLNP having a fluorescent dye payload. Following the incubation at predetermined time points, 5 selected based on calibration or on literature precedent or both, the uptake kinetics of the PLNP will be studied using a confocal microscope.

Claims

CLAIMS:1.What is claimed is:

1. A method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous soludon to the lipid / payload complex to form a terpolymer / lipid- payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a polysorbate and an acrylic acid grafted onto maltodextrin with a dextrose equivalent of 1-30.

2. The method of claim 1 where the terpolymer further comprises glucose monomers conjugated to the terpolymer.

3. The method of claim 1 where the maltodextrin is a low molecular weight maltodextrin with a molecular weight below 10 kDa.

4. The method of claim 1 where the maltodextrin is a low molecular weight maltodextrin with a dextrose equivalent in the range of about 15 to about 17.

5. The method of claim 1 where the polysorbate is polysorbate 80.

6. The method of claim 1 where the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, CI-C25-N,N’ -disubstituted amino-(Ci-C2s)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-OH methacryl-(Ci-C2s)-NH2.

7. A method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex;adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid- payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a polysorbate and an acrylic acid grafted onto a polysaccharide with a molecular weight within the range of about 3 to about 800 kDa.

8. The method of claim 7 where the terpolymer further comprises glucose monomers conjugated to the terpolymer.

9. The method of claim 7 where the polysaccharide is a low molecular weight polysaccharide with a molecular weight is within the range of about 3 to about 10 kDa.

10. The method of claim 7 where the polysaccharide is a low molecular weight polysaccharide with a molecular weight is less than about 40 kDa.

11. The method of claim 7 where the polysaccharide is a low molecular weight polysaccharide with a molecular weight in the range of about 20 to about 40 kDa.

12. The method of claim 7 where the polysaccharide is a low molecular weight polysaccharide with a dextrose equivalent in the range of about 15 to about 17.

13. The method of claim 7 where the polysorbate is polysorbate 80.

14. The method of claim 7 where the acrylic acid is selected from methacrylic acid, C1-C25 acrylate, ethyl acrylate, CI-C25-N,N’ -disubstituted amino-(Ci-C2s)-acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-OH methacryl-(Ci-C2s)-NH2.

15. A method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous soludon to the lipid / payload complex to form a terpolymer / lipid- payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly (methyl methacrylate / methacrylic acid),poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(meth acrylamide), methacrylic acid, C1-C25 acrylate, ethyl acrylate, CI-C25-N,N’ -disubstituted amino-(Ci-C2s)- acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-0H methacryl-(Ci-C2s)-NH2, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

16. The method of claim 15 where the molecule with carboxylic groups comprises a monomer where the monomer has carboxylic groups.

17. The method of claim 15 where the molecule with carboxylic groups is methacrylic acid.

18. The method of claim 15 where the molecule with carboxylic groups is acrylic acid.

19. The method of claim 15 where the polymer further comprises glucose monomers conjugated to the terpolymer.

20. A method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous soludon to the lipid / payload complex to form a terpolymer / lipid- payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly (methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(meth acrylamide, methacrylic acid, C1-C25 acrylate, ethyl acrylate, CI-C25-N,N’ -disubstituted amino-(Ci-C2s)- acrylate, 2-(dimethylamino)ethyl methacrylate, 2-(diisopropylamino)ethyl methacrylate, C1-C25 methacrylate stearyl methacrylate, and methacryl-(Ci-C2s)-OH methacryl-(Ci-C2s)-NH2, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

21. The method of claim 20, where the molecule that binds with apolipoprotein E is polysorbate 80.

22. The method of claim 20, where the molecule that binds with apolipoprotein E is a fatty acid with unsaturated chains or a phospholipid with unsaturated fatty chains.

23. The method of claim 20, where the molecule that binds with apolipoprotein E incorporates a polyethylene glycol.

24. A method of synthesizing polymer-lipid nanoparticles (PLNPs) encapsulating a payload, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex; adding the terpolymer aqueous soludon to the lipid / payload complex to form a terpolymer / lipid- payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain payload loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

25. The method of claim 24 where the polymer further comprises glucose monomers conjugated to the terpolymer.

26. The method of claim 24 where the payload comprises a nucleotide-based payload and the lipids comprise at least one of ionizable lipids or cationic phospholipids.

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

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

29. The compound of claim 27, where the glucose transporter is GLUT- 1.

30. The compound of claim 27, where the payload comprises a nucleotide.

31. The compound of claim 27, where the payload comprises a therapeutic drug molecule.

32. The compound of claim 27, where the payload comprises a biomolecule.

33. The compound of claim 27, where the payload comprises a contrast agent.

34. A method for synthesizing a compound for transporting a payload across the blood brain barrier, where the compound targets both an LDL receptor and a glucose transporter, comprising the steps of: solubilizing lipids in a solvent; solubilizing the payload in a solvent; solubilizing a terpolymer in a solvent; mixing the lipids and payload to obtain a lipid / payload complex;adding the terpolymer aqueous solution to the lipid / payload complex to form a terpolymer / lipid- payload emulsion; and homogenizing the terpolymer / lipid-payload emulsion to obtain pay load loaded PLNPs; where the lipids are chosen for their ability to form complexes with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

35. The method of claim 34 where the polymer further comprises glucose monomers conjugated to the terpolymer.

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

37. The compound of claim 36, where the glucose transporter is GLUT- 1.

38. The compound of claim 36, where the payload comprises a nucleotide.

39. The compound of claim 36, where the payload comprises a therapeutic drug molecule.

40. The compound of claim 36, where the payload comprises a biomolecule.

41. The compound of claim 36 where the payload comprises a contrast agent.

42. A nanoparticle for transporting a payload across the blood brain barrier, where the nanoparticle targets both the LDL receptor and the glucose transporter.

43. The nanoparticle of claim 41, where the glucose transporter is GLUT-1.

44. The nanoparticle of claim 41 , where the payload comprises a nucleotide.

45. The nanoparticle of claim 41, where the payload comprises a therapeutic drug molecule.

46. The nanoparticle of claim 41 , where the payload comprises a biomolecule.

47. The nanoparticle of claim 41 , where the payload comprises a contrast agent.

48. A nanoparticle, where the nanoparticle targets both the LDL receptor and a glucose transporter and the nanoparticle has a diameter in the range of around 50 nm to around 250 nm and the nanoparticle has a negative surface charge.

49. A nanoparticle of claim 48, where the nanoparticle has no exposed lipids on the surface.

50. The nanoparticle of claim 48, where the glucose transporter is GLUT-1.

51. The nanoparticle of claim 48, where the payload comprises a nucleotide.

52. The nanoparticle of claim 48, where the payload comprises a therapeutic drug molecule.

53. The nanoparticle of claim 48, where the payload comprises a biomolecule.

54. The nanoparticle of claim 48, where the payload comprises a contrast agent.

55. A nanoparticle comprising :a terpolymer comprising a polysaccharide, and a hydrophobic monomer, and a hydrophilic or amphiphilic monomer; where the nanoparticle transports a payload across the blood brain barrier by targeting both the LDL receptor and the glucose transporter.

56. The nanoparticle of claim 55, where the targeting of the LDL receptor is by the nanoparticle recruiting ApoE in the blood circulation before encountering the endothelium.

57. The nanoparticle of claim 55, where the free glucose ends of the terpolymer bind with a glucose transporter.

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

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

60. The nanoparticle of claim 55, where the polysaccharide has a DE of around 20 or less.

61. The nanoparticle of claim 55, where the polysaccharide has a molecular weight with the range of about 1 to about 800 kDa.

62. A polymer-lipid nanoparticle (PLNP) comprising: a terpolymer, lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer forms a shell surrounding the complex, and the terpolymer is a grafted polymer with polysorbate 80 and methacrylic acid grafted onto maltodextrin with a dextrose equivalent of 1-30.

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

64. The PLNP of claim 62 where the polymer further comprises glucose monomers conjugated to the terpolymer.

65. The PLNP of claim 62 where the maltodextrin is a low molecular weight maltodextrin with a molecular weight below 10 kDa.

66. The PLNP of claim 62 where the maltodextrin is a low molecular weight maltodextrin with a dextrose equivalent in the range of about 15 to about 17.

67. A polymer-lipid nanoparticle (PLNP) comprising: a terpolymer, lipids; cholesterol;a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and methacrylic acid grafted onto a polysaccharide with a molecular weight within the range of about 3 to about 800 kDa.

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

69. The PNLP of claim 67 where the terpolymer further comprises glucose monomers conjugated to the terpolymer.

70. The PNLP of claim 67 where the polysaccharide is a low molecular weight polysaccharide with a molecular weight is within the range of about 3 to about 10 kDa.

71. The PNLP of claim 67 where the polysaccharide is a low molecular weight polysaccharide with a molecular weight is less than about 40 kDa.

72. The PNLP of claim 67 where the polysaccharide is a low molecular weight polysaccharide with a molecular weight in the range of about 20 to about 40 kDa.

73. The PNLP of claim 67 where the polysaccharide is a low molecular weight polysaccharide with a dextrose equivalent in the range of about 15 to about 17.

74. A polymer-lipid nanoparticle (PLNP) comprising: a terpolymer, lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with polysorbate 80 and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly (ethylene-alt-maleic anhydride), poly(meth acrylamide, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

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

76. The PNLP of claim 74 where the molecule with carboxylic groups comprises a monomer where the monomer has carboxylic groups.

77. The PNLP of claim 74 where the molecule with carboxylic groups is methacrylic acid.

78. The PNLP of claim 74 where the molecule with carboxylic groups is acrylic acid.

79. The PNLP of claim 74 where the polymer further comprises glucose monomers conjugated to the terpolymer.

80. A polymer-lipid nanoparticle (PLNP) comprising:a terpolymer, lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer chosen from the list consisting of polysaccharide, chitosan, chitosan derivatives, polyacrylic acid, poly(methyl methacrylate / methacrylic acid), poly(butadiene / maleic acid), poly(ethylene-alt-maleic anhydride), poly(meth acrylamide, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

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

82. The PNLP of claim 80, where the molecule that binds with apolipoprotein E is polysorbate 80.

83. The PNLP of claim 80, where the molecule that binds with apolipoprotein E is a fatty acid with unsaturated chains or a phospholipid with unsaturated fatty chains.

84. The PNLP of claim 80, where the molecule that binds with apolipoprotein E incorporates a polyethylene glycol.

85. A polymer-lipid nanoparticle (PLNP) comprising: a terpolymer, lipids; cholesterol; a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

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

87. The PNLP of claim 85 where the polymer further comprises glucose monomers conjugated to the terpolymer.

88. The PNLP of claim 85 where the payload comprises a nucleotide-based payload and the lipids comprise at least one of ionizable lipids or cationic phospholipids.

89. A polymer-lipid nanoparticle (PLNP) comprising: a terpolymer, lipids; cholesterol;a payload; and where the lipids form a complex with the payload, and the terpolymer is a grafted polymer with a molecule that binds with apolipoprotein E and a molecule with carboxylic groups grafted onto a polymer comprising a glucose chain, and the polymer has a molecular weight within the range of about 1 to about 800 kDa.

90. The PLNP of claim 89, further comprising a helper lipid.

91. The PLNP of claim 89 where the polymer further comprises glucose monomers conjugated to the terpolymer.

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