Bottlebrush block copolymer-lipid for the formation of lipid nanoparticles with rough surface topography

Abstract

Described are amphiphilic bottlebrush block copolymers (BBCPs) to precisely tailor the surface topography of lipid nanoparticles. The amphiphilic BBCPs are diblock and contain a hydrophilic block containing poly(ethylene glycol) side chains, and a hydrophobic block containing lipid side chains. The lipid nanoparticles have a monolayer membrane containing a blend of one or more of the amphiphilic BBCPs and lipid molecules. The hydrophilic block protrudes outward on the external surface of the membrane, and imparts roughness to the surface of the lipid nanoparticles. The data show that amphiphilic BBCPs produce stable lipid nanoparticles, optionally encapsulating agents to be delivered. These lipid nanoparticles can be used in the efficient delivery of agents (e.g., mRNA) to cells, tissues, and / or organs with longer circulation time after systemic injection.

Description

[0001] BOTTLEBRUSH BLOCK COPOLYMER-LIPID FOR THE FORMATION OF LIPID NANOPARTICLES WITH ROUGH SURFACE TOPOGRAPHY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 507,503 filed June 12, 2023, which is hereby incorporated by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under AU 45965 and HL147352 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE INVENTION

[0007] This invention is in the field of amphiphilic bottlebrush block copolymers (BBCPs) and nanoparticles containing these polymers, particularly hybrid polymer-lipid BBCPs composed of polyethylene glycol (PEG) and a phospholipid (e.g., l,2-distearoyl-sn-glycero-3- phosphoethanolamine lipid (DSPE)) to form lipid nanoparticles with a rough membrane containing a PEG-block protruding on the exterior of the membrane, for the efficient delivery of nucleic acids, such as mRNA.

[0008] BACKGROUND OF THE INVENTION

[0009] Nanoparticle-based therapeutics and diagnostics offer immense potential in a wide range of biomedical applications. For example, drug- loaded nanoparticles have garnered great interest in drug delivery as a strategy to improve the pharmacokinetics of drugs (Banik, et al. WIREs Nanomed Nanobiotechnol 2016, 8 , 271-299) However, the impact of nanoparticle-based drug delivery in clinical medicine has been limited with only a small number of FDA-approved formulations to date. (Anselmo, et al. Bioeng. Transl. Med. 2016, 7, 10-29; An Update. Bioeng. Transl. Med. 2019, 4, 1-16)' Most formulations tested in clinical trials ultimately fail to show beneficial effects due to their rapid blood clearance and low drug delivery efficiency (Cho et al. Biomaterials 2012, 33, 1190-1200; Qian, et al. Polym. Chem. 2016, 7, 3300-3310).

[0010] A widely used method to enhance the blood circulation time of nanoparticles is based on coating the surface with a dense layer of poly (ethylene glycol) (PEG), which reduces opsonization and subsequent clearance by the mononuclear phagocytosis system (Du, et al. Biomaterials 2015, <59, 1—11). While this stealthy PEG-coating effectively inhibits the adsorption of serum proteins on the nanoparticle surface via steric repulsion, the same characteristic inherently reduces their uptake into cells (Cao, et al. ACS Nano 2020, 14, 3563-3575). Several physicochemical parameters of PEG-coated nanoparticles such as size (Walkey, et al. J. Am. Chem. Soc. 2012, 134, 2139-2147), shape (Kinnear, et al. Chem. Rev. 2017, 117, 11476- 11521), rigidity (Hui, et al. ACS Nano 2019, 13, 7410-7424; Mullner, et al. ACS Nano 2015, 9, 1294-1304 ), and surface functionality (Sutton, et al. Pharm. Res. 2007, 24, 1029-1046, Deng, et al Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 11453-11458) have been investigated as methods to modulate cell uptake and protein adsorption. However, only a small number of studies have focused on surface topography as a strategy to optimize nanoparticle design.

[0011] Tuning the surface topography of inorganic nanoparticles has resulted in improvements in nanoparticle pharmacokinetics, including cell uptake (Niu et al. J. Mater. Chem. B 2015, 4, 212-219; Wang, et al. Nat. Nanotechnol. 2018, 13, 1078-1086; Song, et al. J. Am. Chem. Soc. 2017, 139, 18247-18254; Chen, et al. ACS Cent. Sci. 2019, 5, 960-969; Lord, et al. Nano Today 2010, 5, 66-78 ; Roach, et al. J. Am. Chem. Soc. 2006, 128, 3939-3945). For example, Wang et al. ACS Cent. Sci. 2017, 3, 839-846 observed greatly enhanced circulation half-lives and cellular internalization of mesoporous silica nanoparticles with virus-mimicking spiky surface topography compared to a smooth-surfaced control.

[0012] Although surface properties are critical for nanoparticle performance, it is difficult to control the surface topography of polymer materials. Control of the surface topography of polymeric nanoparticles is a formidable challenge due to the limited conformational control of linear polymers that form the nanoparticle surface. Despite their frequent use in biomedical applications, the effect of surface topography is poorly understood in the context of PEGylated polymeric nanoparticles. This may be in part due to the difficulty of controlling the surface topography and morphology of polymeric nanoparticles. The flexible nature of linear polymer building blocks that readily undergo conformational changes if exposed on the nanoparticle surface makes it challenging to modulate the surface without affecting their conformation. In particular, PEG chains transition from mushroom to brush conformation as their surface density increases, leading to drastically reduced serum protein adsorption and macrophage uptake (Du, et al. Biomaterials 2015, 69, 1-11). Zhou et al. ACS Nano 2018, 12, 10130— 10141 demonstrated that a second PEG layer can be conjugated onto PEGylated nanoparticles to create a dynamic topographical structure that prolongs circulation times in vivo. However, their results were attributed to the combined effect of conformational fluctuations of the low-density outer PEG layer and the steric repulsion of the dense inner PEG layer rather than surface topography.

[0013] To independently evaluate the effect of surface topography on the nanoparticle’ s biological fate, it is critical to alter surface topography without changing the density and conformation of PEG chains on the nanoparticles surface. Although surface properties are critical for nanoparticle performance, it is difficult to control the surface topography of lipid nanoparticles.

[0014] Therefore, it is an object to provide lipid nanoparticles with controlled rough surface topography, which was previously not possible using conventional linear hydrophilic polymer- lipids.

[0015] It is another object to provide lipid nanoparticles with controlled rough surface topography, which are useful for enhanced, controlled drug delivery.

[0016] It is a further object provide PEGylated lipid nanoparticles with controlled rough surface topography, which was previously not possible using conventional linear PEG-lipids. SUMMARY OF THE INVENTION

[0017] Described are amphiphilic bottlebrush block copolymers (BBCPs) with a backbone containing unsaturated polyolefin (e.g., a polynorbornylene) and: (i) a hydrophilic block containing poly(ethylene glycol) side chains covalently bonded directly or indirectly to the backbone, and (ii) a hydrophobic block containing l,2-distearoyl-sn-glycero-3- phosphoethanolamine side chains covalently bonded directly or indirectly to the backbone. Preferably, the amphiphilic BBCPs are diblock.

[0018] The amphiphilic BBCPs can be used to precisely tailor the surface topography of lipid nanoparticles due to their shape-persistent properties. The lipid nanoparticles contain a membrane with the amphiphilic BBCPs anchored in the membrane via a hydrophobic block containing lipid side chains, while the hydrophilic block of the amphiphilic BBCPs protrudes outward on the external surface of the membrane. The protruding amphiphilic BBCPs impart roughness to the surface of the lipid nanoparticles. Accordingly, also described are lipid nanoparticles with a rough surface, and methods to precisely tailor the surface topography of the lipid nanoparticles, to allow better controlled interactions with proteins, cells, and tissues; and / or for enhanced delivery of agents.

[0019] In preferred forms, the lipid nanoparticles have a monolayer membrane containing a blend of one or more of the amphiphilic BBCPs described herein and lipid molecules. The lipid molecules include ionizable lipids (e.g., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate), helper lipids (e.g., distearoylphosphatidylcholine), steroids (e.g., cholesterol), or combinations thereof. Preferably, the helper lipids increase the lipid nanoparticles’ stability and fluidity.

[0020] In other forms, the lipid nanoparticles also encapsulate a second set of particles containing a lipid membrane. This second set of particles can also encapsulate one or more of the agents to be delivered. In these forms, the lipid membrane of the second set of particles preferably contains (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate, distearoylphosphatidylcholine, or a combination thereof. The data show that amphiphilic BBCPs (e.g. , PEG-DSPE BBCPs) produce stable lipid nanoparticles, optionally encapsulating agents (e.g., mRNA). The lipid nanoparticles have a mean hydrodynamic diameter between about 100 nm and about 175 nm, as measured using dynamic light scattering, and can find application in the efficient delivery of agents (e.g. , mRNA) to cells, tissues, and / or organs with longer circulation time after systemic injection.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a schematic showing the synthesis of a norbomene-PEG (PEG-MM), prepared by attaching methoxy-PEG-amine to a norbornene-A- hydroxysuccinimidyl-ester following Liu, et al. J. Am. Chem. Soc. 2012, 134, 16337-16344.

[0023] FIGs. 2A and 2B are schematics showing the synthesis via ROMP of an amphiphilic BBCP containing PEG-MM and DSPE lipid. In FIG. 2A, G3 was prepared from second-generation Grubbs catalyst by stirring in toluene following Love, et al. Angew. Chemie Int. Ed. 2002, 41, 4035-4037. In FIG. 2B, the degree of polymerization of the PEG block backbone (n) is 20, and the A-hydroxysuccinirnidyl block backbone (m) is 10.

[0024] FIG. 3 shows characterization of bottle brush polymers synthesized herein, based on gel permeation chromatography.

[0025] FIG. 4 shows characterization of bottle brush polymers synthesized herein, based on nuclear magnetic resonance spectroscopy.

[0026] FIG. 5 is a schematic of the encapsulation of an agent in a lipid nanoparticle via self-assembly of amphiphilic BBCPs and various lipid molecules.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] I. Definitions

[0029] “Biocompatible” and “biologically compatible,” as used herein, generally refer to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient. “Monodisperse” describes a plurality of nanoparticles where the nanoparticles have the same or nearly the same diameter or aerodynamic diameter. As used herein, a monodisperse distribution refers to particle distributions in which 80, 81, 82, 83, 84, 85, 86, 86, 88, 89, 90, 91, 92, 93, 94, 95% or greater of the distribution lies within 5% of the mass median diameter or aerodynamic diameter.

[0030] “Hydrophilic,” as used herein, refers to the property of having affinity for water. For example, hydrophilic polymers (or hydrophilic polymer segments) are polymers (or polymer segments) which are primarily soluble in aqueous solutions and / or have a tendency to absorb water. In general, the more hydrophilic a polymer is, the more that polymer tends to dissolve in, mix with, or be wetted by water.

[0031] “Lipophilic” refers to compounds having an affinity for lipids.

[0032] “Amphiphilic” refers to a molecule combining hydrophilic and lipophilic (hydrophobic) properties. As used herein, an amphiphilic bottlebrush block copolymer is one which has one end formed of a hydrophilic polymer and one end formed of a lipid. As a result, when dispersed into a mixture of water and low water-solubility solvent such as many of the organic solvents, the hydrophilic end orients into the water and the hydrophobic end orients into the low water-solubility end.

[0033] “Self-assembling,” “self-assembly,” and related terms, as used herein, refer to the use of an amphiphilic bottlebrush block copolymers, alone or in mixture with lipids and / or hydrophobic polymers, which orient in a mixture of aqueous and non-aqueous solvents to form lipid nanoparticles, wherein the hydrophilic ends orient with the other hydrophilic ends and the hydrophobic ends orient with the other hydrophobic ends.

[0034] “Hydrophobic” as used herein refers to substances that lack an affinity for water; tending to repel and not absorb water as well as not dissolve in or mix with water.

[0035] The term "therapeutic agent" refers to an agent that can be administered to prevent or treat a disease or disorder. Therapeutic agents can be a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof. “Pharmaceutically acceptable,” as used herein, refers to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio, in accordance with the guidelines of agencies such as the Food and Drug Administration.

[0036] “Biocompatible” and “biologically compatible,” as used herein, generally refer to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient. Generally speaking, biocompatible materials are materials which do not elicit a significant inflammatory or immune response when administered to a patient.

[0037] “Molecular weight,” as used herein, generally refers to the relative average chain length of the bulk polymer, unless otherwise specified. In practice, molecular weight can be estimated or characterized using various methods including gel permeation chromatography (GPC) or capillary viscometry. GPC molecular weights are reported as the weight- average molecular weight (Mw) as opposed to the number- average molecular weight (Mn). Capillary viscometry provides estimates of molecular weight as the inherent viscosity determined from a dilute polymer solution using a particular set of concentration, temperature, and solvent conditions.

[0038] As used herein, bottlebrush polymers, also known as molecular brushes, are a special class of polymers characterized by having a “sidechain” polymer grafted to every repeating unit along the main chain polymer “backbone”. The physical size and structure of bottlebrush polymers is governed by the length (DP, or number-average molecular weight, Mn) of the side-chain and backbone polymers. The chemical properties (such as solubility, responsive behavior, etc) are governed primarily by side-chain polymer type or monomer selection. Together, the side-chain and backbone polymers determine bottlebrush molecular structure properties such as size, shape and stiffness. The type of side-chain polymer dictates bottlebrush functional properties, such as interaction with environment, hydrophobicity, and self-assembled structures. Polyethylene glycol (PEG) is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is commonly expressed as H-iO-CH^-CHA -OH. PEG, PEO, and POE refer to an oligomer or polymer of ethylene oxide. The three names are chemically synonymous, but historically PEG is preferred in the biomedical field, whereas PEO is more prevalent in the field of polymer chemistry. Because different applications require different polymer chain lengths, PEG has tended to refer to oligomers and polymers with a molecular mass below 20,000 g / mol, PEO to polymers with a molecular mass above 20,000 g / mol, and POE to a polymer of any molecular mass. PEGs are prepared by polymerization of ethylene oxide and are commercially available over a wide range of molecular weights from 300 g / mol to 10,000,000 g / mol. PEG and PEO are liquids or low-melting solids, depending on their molecular weights. While PEG and PEO with different molecular weights find use in different applications, and have different physical properties (e.g., viscosity) due to chain length effects, their chemical properties are nearly identical. Different forms of PEG are also available, depending on the initiator used for the polymerization process. The most common initiator is a monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated mPEG. Lower-molecular- weight PEGs are also available as purer oligomers, referred to as monodisperse, uniform, or discrete.

[0039] II. Compositions

[0040] More efficient delivery of therapeutic, diagnostic, and / or prophylactic agents, collectively “agents,” to cells, tissues, and / or organs, could lead to enhanced treatment outcomes in a variety of diseases or disorders. For example, more efficient methods of mRNA delivery to cells and tissues could lead to new treatments for diseases such as cancer. Delivery vehicles (such as nanoparticles) have been used for the controlled and / or sustained delivery of these agents. Lipid nanoparticles (LNPs) containing a large amount of ionizable lipids have been widely used in delivering one or more of these agents. Although, these lipid nanoparticles show high mRNA transfection efficiency, they suffer from high toxicity and rapid clearance from circulation, thereby preventing their accumulation at the target site despite their incorporation of polymeric systems, such as polyethylene glycol-lipids, taught to enhance circulation. It is believed that the PEG bottlebrush helps with toxicity and circulation.

[0041] The instant project involves modifying the surface topography of lipid nanoparticles with shape-persistent amphiphilic BBCPs (e.g., PEG- DSPE BBCPs) to allow better controlled interactions with proteins, cells, and tissues, and the improved delivery of these agents (e.g., mRNA) in many applications. Shape-persistent, refers to the persistent cylindrical / rod-like shape of bottlebrush polymers due to their semirigid extended backbone with large persistence length. Because of the fluidity of membranes containing lipid molecules, it can be challenging to develop LNPs with amphiphilic BBCPs inserted into these membranes, such as a population of monodispersed LNPs with amphiphilic BBCPs incorporated, due to their large size especially since the amphiphilic BBCPs form stable micelles by themselves. Surface topography can be controlled by systematically changing structural parameters of the amphiphilic BBCPs’ architecture. The data show that amphiphilic BBCPs e.g., PEG-DSPE BBCPs) produce stable LNPs, optionally encapsulating agents (e.g., mRNA). The formed LNPs contain a membrane with the amphiphilic BBCPs anchored in the membrane via a hydrophobic block containing lipid side chains, while the hydrophilic block of the amphiphilic BBCPs protrudes outward on the external surface of the membrane. The protruding amphiphilic BBCPs impart roughness to the surface of the LNPs. These LNPs find application in the efficient delivery of agents (e.g., mRNA) to cells, tissues, and / or organs with longer circulation time after systemic injection as well as enhanced uptake into solid tumors.

[0042] A. Amphiphilic Bottlebrush Block Copolymers

[0043] The amphiphilic BBCPs described herein, contain a backbone and: (i) a hydrophilic block containing hydrophilic polymer side chains bonded directly or indirectly to the backbone, and (ii) a hydrophobic block containing lipid side chains bonded directly or indirectly to the backbone. In some forms of the amphiphilic BBCPs, (i) the hydrophilic polymer side chains contain polyethylene glycol, and / or (ii) the lipid side chains contain a phospholipid (such as DSPE). BBCPs (including the amphiphilic BBCPs) were characterized via size exclusion chromatography (SEC), preferably gel permeation chromatography (GPC), and NMR. SEC analysis revealed successful chain extension with monomodal molecular weight distribution and a negligible amount of unreacted intermediate bottlebrush polymer after the addition of the hydrophobic block. The amphiphilic BBCPs described herein, can be diblock or triblock. Preferably, the amphiphilic BBCPs are diblock.

[0044] The weight-average molecular weight (Mw) or the number- average molecular weight (Mn) can vary for a given amphiphilic BBCP but is generally from about 1000 Daltons to 1,000,000 Daltons, 1000 Daltons to 500,000 Dalton, 1000 Daltons to 250,000 Daltons, 1000 Daltons to 100,000 Daltons, 5,000 Daltons to 100,000 Daltons, or 5,000 Daltons to 75,000 Daltons. These values can be determined via GPC.

[0045] Further details of the amphiphilic BBCPs are described below. i. Backbone

[0046] Preferably, the backbone of the amphiphilic BBCPs can contain, or can be formed by, one or more polymeric units. The backbone can be an unsaturated polyolefin (e.g. , a poly norbornylene), a saturated polyolefin, a polyester, a polyamide, a polyamine, a polyanhydride, or a combination thereof. Preferably, the backbone is an unsaturated polyolefin (<?.g., a poly norbornylene) . ii. Hydrophilic polymer side chains

[0047] Preferably, the hydrophilic polymer side chains are covalently bonded, directly or indirectly, to the backbone. The hydrophilic polymer side chains of the amphiphilic BBCPs include neutral hydrophilic polymers, charged hydrophilic polymers, or a combination thereof. Preferably, the hydrophilic polymer side chains include neutral hydrophilic polymers. Suitable examples of hydrophilic polymer side chains include, but are not limited to, polyalkylene oxides such as polyethylene glycol (PEG) and polyethylene oxide) (PEG); poly(oxyethylated polyol), poly(olefinic alcohol) such as poly(vinyl alcohol); polyvinylpyrrolidone; polysaccharides; hydrophilic polypeptides such as poly-L-glutamic acid (PGS), gammapolyglutamic acid, poly-L-aspartic acid, poly-L-serine, and poly-L-lysine; poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), or combinations thereof.

[0048] Any number of biocompatible hydrophilic polymers can be used to prepare hydrophilic polymer side chains of the amphiphilic BBCPs. In some forms, the biocompatible hydrophilic polymer(s) are biodegradable. In other forms, the biocompatible hydrophilic polymer(s) are non-biodegradable. In other forms, the biocompatible hydrophilic polymer(s) are a mixture of degradable and non -degradable polymers. In preferred forms, the hydrophilic polymer side chains contain polyalkylene oxides such as polyethylene glycol (PEG) and poly(ethylene oxide) (PEO).

[0049] The Mw or Mn of a hydrophilic polymer side chain can be between about 1 kDa and about 10 kDa, between about 1 kDa and about 7.5 kDa, or between about 1 kDa and about 5 kDa, as determined via GPC. iii. Lipid side chains

[0050] The lipid side chains of the amphiphilic BBCPs contain phospholipids, sphingolipids, saturated fatty acid, unsaturated fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), or combinations thereof. In preferred forms, the lipid side chains contain phospholipids.

[0051] Preferably, the lipid side chains are covalently bonded to the backbone via a hydrophilic chemical moiety of the lipid. In these forms, the headgroup of the lipid side chain is covalently bonded, directly or indirectly, to the backbone, while the hydrophobic tail group or segment of the lipid side chain orients away from the backbone.

[0052] Typically, a lipid side chain is non-polymeric, and has a molecular weight between 50 Da and 2.5 kDa.

[0053] B. Lipid Nanoparticles

[0054] Also described are LNPs containing a membrane that contains one or more of the amphiphilic BBCPs disclosed herein. The membrane can be a monolayer or a bilayer, and is preferably a monolayer. In these LNPs, some or all of the hydrophilic blocks of the amphiphilic BBCPs protrude to the exterior surface of the LNPs. In preferred forms, the membrane contains a blend of the amphiphilic BBCPs and lipid molecules. The lipid molecules in the membrane can include ionizable lipids, helper lipids, or combinations thereof. Preferably, the helper lipids increase the LNPs’ stability and fluidity. Suitable lipid molecules include, but are not limited to, lipid molecules comprise phospholipids, sphingolipids, saturated fatty acid, unsaturated fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), or combinations thereof. In some forms, the lipid molecules in the membrane include (i) (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (MC3), (ii) distearoylphosphatidylcholine (DSPC), and / or (iii) cholesterol. i. Agents to be delivered

[0055] In some forms, the LNPs have encapsulated therein, dispersed therein, and / or covalently or non-covalently associated with the surface one or more agents, such as therapeutic, prophylactic and / or diagnostic agents. The therapeutic agent can be a small molecule (molecular weight under 2500 Da, more preferably under 2000 Da or under 1500 Da, most preferably under 1000 Da); protein or peptide; polysaccharide or saccharide; nucleic acid molecule; and / or a therapeutic or prophylactic lipid.

[0056] In some forms, the LPNs have a higher proportion of the agents encapsulated within the LNPs than on the surface of the LNPs. In some forms, the LPNs have the agents encapsulated within the LNPs and not present on the surface of the LNPs.

[0057] The agents can be encapsulated directly in the core of the LNPs. In other forms, the LNPs contain a second set of particles encapsulated within the LNPs, optionally wherein the second set of particles encapsulates one or more of the agents. In some forms, the agents are encapsulated directly in the LNPs and also in the second set of particles.

[0058] Preferably, this second set of particles contains a lipid membrane. The lipid membrane of this second set of particles can be a monolayer or bilayer, preferably a monolayer. In some forms, the lipid membrane of this second set of particles contains ionizable lipids, helper lipids, or combinations thereof, optionally wherein the helper lipids increase the

[0059] LNPs’s stability and fluidity. In some forms, the lipid membrane of this second set of particles contains phospholipids, sphingolipids, saturated fatty acid, unsaturated fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), or combinations thereof. In some forms, the lipid membrane of this second set of particles contains phospholipids. In some forms, the lipid membrane of this second set of particles contains (i) (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31 -tetraen- 19-yl 4-(dimethylamino)butanoate (MC3), and / or (ii) distearoylphosphatidylcholine (DSPC).

[0060] Exemplary classes of therapeutic and / or prophylactic agents include, but are not limited to, analgesics, anti-inflammatory drugs, hormones, cardiovascular drugs, anti-proliferatives, such as anti-cancer agent, anti- infectious agents, such as antibacterial agents and antifungal agents, gastrointestinal drugs, nutritional agents, vitamins and nutraceuticals. These may be vitamins, supplements such as calcium or biotin, or natural ingredients such as plant extracts or phytohormones.

[0061] In some forms, the agents are one or more nucleic acids (such as mRNA). The nucleic acid can alter, correct, or replace an endogenous nucleic acid sequence. Nucleic acid therapeutics can be used in vaccines; to treat cancers; to correct defects in genes, such as in pulmonary diseases and metabolic diseases affecting lung function; or to mitigate symptoms of neurodegeneration.

[0062] In this work, LNPs with a rough surface were generated either encapsulating nucleic acids (e.g., mRNA) or not encapsulating mRNA, as evidenced from cryo-transmission electron microscopy (cryo-TEM). ii. Lipid nanoparticle size

[0063] The LNPs have a particle diameter between 50 nm and less than 1 ,000 nm, as measured using dynamic light scattering. The diameter can be the mean hydrodynamic diameter of the LNPs. In some forms, the LNPs have a mean hydrodynamic diameter between about 50 nm and about 200 nm, between about 50 nm and about 175 nm, or between about 100 nm and about 175 nm, as measured using dynamic light scattering. III. Methods of Making and Reagents therefor

[0064] A. The amphiphilic bottlebrush block copolymers

[0065] Non-limiting examples of synthesizing amphiphilic BBCPs via ringopening metathesis polymerization (ROMP) are collectively shown in the schematics in FIG. 1, FIG. 2A, and FIG. 2B.

[0066] FIG. 1 describes an example of making PEG-macromonomer (PEG- MM). PEG-MM and A-(hydroxyethyl)-cE-5-norbornene-cxo-2,3- dicarboximide were prepared according to Matson, et al., J. Am. Chem. Soc. 2008, 130, 6731-6733.

[0067] Briefly, cE-5-norbornene-exo-2,3-dicarboxylic anhydride can be refluxed in an organic solvent (such as toluene) in the presence of 1.5 equiv. of 4-aminobutanoic acid, to form A-(hydroxyethyl)-<?z5-5-norbomene-exo- 2,3-dicarboximide. Reaction with this product and A-hydroxy succinimide under conditions shown in FIG. 1 leads to the formation of NHS-Nb. The PEG-MM can be formed following the steps further outlined in FIG. 1, via routine modification of reaction conditions.

[0068] FIG. 2A shows the synthesis of PEG-DSPE BBCP. The G3 can be prepared from a second-generation Grubbs catalyst by stirring in toluene following Love, et al., Angew. Chemie Int. Ed. 2002, 41, 4035-4037. Polymerization of PEG-MM can be performed via ROMP catalyzed by a Grubbs catalyst, such as G3. ROMP is a type of olefin metathesis chaingrowth polymerization. The driving force of the reaction is relief of ring strain in cyclic olefins (e.g., norbornene or cyclopentene). The NHS-Nb synthesized as shown in FIG. 1, can then be reacted with the polymerized PEG-block to form PEG-NHS BBCP. A lipid (e.g., DSPE) can be reacted with this intermediate product to form PEG-DSPE BBCP under conditions shown in FIG. 2B.

[0069] In general, the amphiphilic BBCPs can be synthesized by ROMP using (i) the hydrophilic polymer side chain-cyclic olefin and cyclic olefin- jV-hydroxysuccinimidyl-ester followed by reaction of the cyclic olefin-A'- hydroxysuccinimidyl-ester with a lipid, (ii) lipid side chain-cyclic olefin and cyclic olelin- / V-hydroxysuccinimidyl-ester followed by reaction of the cyclic olefin- / V-hydroxysucci nimidyl-ester with a hydrophilic polymer, or (iii) the hydrophilic polymer side chain-cyclic olefin and lipid side chain-cyclic olefin, each initiated by a Grubbs catalyst.

[0070] In more specific forms, the amphiphilic BBCPs can be synthesized by ROMP using (i) the hydrophilic polymer side chain-norbornene and norbomene-N-hydroxysuccini midyl-ester followed by reaction of the norbomene-A-hydroxysuccini midyl-ester with a lipid, (ii) lipid side chain- norbornene and norbomene-iV-hydroxysuccinimidyl-ester followed by reaction of the norbomene- -hydroxysuccinimidyl-ester with a hydrophilic polymer, or (iii) the hydrophilic polymer side chain-norbomene and lipid side chain-norbomene, each initiated by a Grubbs catalyst.

[0071] Methods of forming the backbones of BBCPs and BBCPs are generally described in Ree, et al., NPG Asia Materials 2017, 9, e453, the contents of which are hereby incorporated by reference.

[0072] B. The Lipid Nanoparticles

[0073] The preferred method for making the LNPs microfluidics, using a microfluidics device (<?.g. , NanoAssemblr). Briefly, the amphiphilic BBCPs, optionally in the presence of lipid molecules are dissolved in an organic solvent. Suitable organic solvents for dissolving the BBCPs and the optionally lipid molecules include, but are not limited to, polar protic organic solvents (<?.g., alcohols (such as anhydrous ethanol), etc. ,) and polar aprotic organic solvents (<?.g., dimethyl formamide, dimethyl sulfoxide, etc.) The total lipid concentration can be between about 1 mg / mL and about 10 mg / mL; about 1 mg / mL and about 7.5 mg / mL, about 2.5 mg / mL and about 7.5 mg / mL, such as about 5.0 mg / mL. The agent(s) to be delivered can be dissolved in solvents suitable for dissolving the agents, based on the lipophilic properties of the agent(s). Separate solutions containing (i) the amphiphilic BBCPs, optionally in the presence of lipid molecules, on the one hand, and (ii) the agent(s) to be delivered, on the other hand, can next be introduced into a microfluidic mixing device at a (i): (ii) volume ratio of between about 1:1 and about 1 :5, such as about 1:3.

[0074] Non-limiting details of making the LNPs using a microfluidics device are provided in the Examples section. The LNPs can also be produced via a thin film hydration method followed by ultrasonication. Formation of these types of LNPs has not been demonstrated before. In some forms, the feed mole ratios of components to make the LNPs, not including the agents to be delivered, can be as follows: amphiphilic BBCPs, between 1 mol% and 15 mol%, such as 1.5 mol%; lipid molecules, 85 mol% and 99 mol%, such as 90 mol%.

[0075] In other forms, the feed mole ratios of components to make the LNPs, not including the agents to be delivered, can be as follows: amphiphilic BBCP 1 mol% and 15 mol%, such as 1.5 mol%; phospholipids, between 40 mol% and 70 mol%, such as 60 mol%; and steroids (e.g., cholesterol), between 30 mol% and 45 mol%, such as 38.5 mol%, with the proviso that the total feed mole ratio is 100 mol%.

[0076] In other forms, the feed mole ratios of components to make the LNPs, not including the agents to be delivered, can be as follows: amphiphilic BBCP 1 mol% and 15 mol%, such as 1.5 mol%; ionizable lipids, between 40 mol% and 60 mol%, such as 50 mol%; helper lipids, between 5 mol% and 15 mol%, such as 10 mol%; and cholesterol, between 30 mol% and 45 mol%, such as 38.5 mol%, with the proviso that the total feed mole ratio is 100 mol%.

[0077] Quantitative characterization of LNPs surface topography (i.e., surface roughness and surface area) is a challenge due to the small characteristic length scale of the surface. Standard surface characterization methods such as atomic force microscopy and nitrogen adsorption isotherms that have been used for nanoparticles in the past are inadequate to analyze soft LNPs in their native hydrated state as their membrane can deform and collapse upon dehydration. A model can be used to approximate the surface topography by depicting the LNP membrane as a number of individual cylinders with an overlapping portion close to a solid core.

[0078] In this model, the mean-line surface radius (RMLS) represents the radius where the brush volume above equals the valley volume below the MLS. The inner shell sphere radius (Riss) corresponds to the radius of the overlapping portion of the bottlebrush cylinder shell.

[0079] The radius of cylinders representing the outer segment of the PEG bottlebrush polymer (Rbrusii) can be determined from cryo-TEM images. The radius of the inner shell sphere (Riss) containing the overlapping cylinder portion can be estimated based on the fraction of fast-relaxing PEG end group protons from NMR Ti relaxation. The mean line sphere radius ’MI.S ) representing a sphere of equal volume can be calculated. The arithmetic mean roughness can be determined based on the sum of height deviations from the mean line sphere (MLS) divided by the MLS surface area (A) and the MLS radius (r) to account for the nanoparticle curvature according to previously reported Eq 1, where |Z(x, y) | represents the integral of the cylinder volume above the MLS and valley volume below (Chang et al RSC Adv. 2017, 7, 40255-40261).

[0080] The LNP surface area can be calculated from the surface area of the non-overlapping portion of bottlebrush cylinders as follows: IV. Methods of Using

[0081] The LNPs are useful for the delivery of agents, such as therapeutic, diagnostic, and / or prophylactic agents. The LNPs can be injected intravenously, subcutaneously, or intramuscularly, encapsulated and administered orally, formulated as a suspension, gel, spray, or other formulation for administration to a mucosal surface (nasal, pulmonary, vaginal, rectal, buccal, sublingual), or formulated for topical delivery. The dosage is determined using standard techniques based on the agent(s) to be delivered and the method and form of administration. The LNPs may be administered as a dry powder, as an aqueous suspension (in water, saline, buffered saline, etc.), in a hydrogel, organogel, or liposome, in capsules, tablets, troches, or other standard pharmaceutical excipient.

[0082] These formulations are typically administered by injection, preferably intravenously.

[0083] The present invention will be further understood by reference to the following non- limiting examples. Examples

[0084] Example 1: Synthesis of lipid nanoparticles containing an amphiphilic BBCP and an agent

[0085] Materials and methods

[0086] (i) Synthesis ofPEG-NHS BBCP

[0087] Stock solutions of PEG-MM (0.05 M), NHS-Nb (0.05 M), and G3 (0.01 g / mL) in anhydrous dichloromethane (DCM) were prepared inside an argon-filled glovebox. ROMP of PEG-MM (2 kDa, 21 .3 pmol) was initiated via the addition of G3 (1.1 pmol) followed by continued stirring for 30 min. After taking an aliquot of the reaction mixture for GPC analysis, NHS-Nb (10.7 pmol) was added dropwise and the solution stirred for additional 60 min before quenching with ethyl vinyl ether.

[0088] (ii) Synthesis of PEG-DSPE BBCP

[0089] PEG-NHS BBCP (1 equiv.) and DSPE (0.7 equiv.) were dissolved in anhydrous chloroform and triethylamine (5 equiv.) was added. The solution was stirred at 40 °C for 2 h. After evaporating the solvent using a rotary evaporator, the crude was redissolved in ultrapure water and filtered through a syringe filter. The solution was then dialyzed against nanopure water for 24 h using regenerated cellulose dialysis tubing (10 kDa MWCO, Spectrum Labs) followed by lyophilization to obtain a colorless viscous liquid.

[0090] (Hi) Formation of lipid nanoparticles (LNPs)

[0091] PEG-DSPE BBCP (1.5 mol%), MC3 ionizable lipid (50 mol%), DPSC helper lipid (10 mol%), and cholesterol (38.5 mol%) were dissolved in anhydrous ethanol at a total lipid concentration of 5 mg / mL and FLuc mRNA (0.18 mg / mL) in 100 mM sodium acetate buffer (pH 4.0) for mRNA loaded LNPs. A microfluidic mixing device (NanoAssemblr Ignite, Precision NanoSystems) was used at a 1:3 ratio of ethanol to sodium acetate buffer solution to form LNPs with instrument settings according to the manufacturer’s recommendations. The collected LNPs were then purified using Amicon centrifuge filter tubes (100 kDa MWCO) and redispersed in PBS before DLS analysis and storage at -80 °C.

[0092] Results

[0093] BBCPs composed of PEG-MM and NHS-Nb were synthesized via sequential graft-through ROMP with a targeted backbone degree of polymerization of 20 and 10, respectively (FIGs. 2A and 2B). GPC- multiangle light scattering (MALS) analysis in DMF (FIG. 3) reveals the successful formation of the PEG block with a backbone DP of 21 (48.7 kDa) as well as chain extension with NHS-Nb (DP = 12). Incorporation of NHS- Nb at the desired ratio was also confirmed via1H-NMR. DSPE lipids were conjugated to the PEG-NHS BBCP via amine coupling in chloroform in the presence of triethylamine at 40 °C followed by purification via dialysis against water (FIG. 2B). 'H-NMR analysis confirmed the complete displacement of NHS with DSPE as indicated by the lipid carbohydrate tail proton peak at 1.2 ppm and the absence of the NHS proton peak at 2.8 ppm (FIG. 4).

[0094] LNPs were formed via microfluidic mixing of PEG-DSPE BBCP, MC3, DSPC, and cholesterol dissolved in ethanol and FLuc mRNA in sodium acetate buffer (pH 4). After LNP formation and buffer exchange with PBS, LNPs were analyzed via DLS and cryo-TEM (FIG. 5). DLS analysis shows the formation of LNPs with low polydispersity (<0.07) around 145 nm in size compared to a LNP control containing a conventional linear PEG-DMG lipid with a diameter of 135 nm. Without wishing to be bound by theory, it is believed that the size difference is due to the increased length of the PEG-DSPE BBCP compared to linear PEG-DMG on the LNP surface. In fact, cryo-TEM images confirm the incorporation of PEG-DSPE BBCP in the LNP membrane as evident by protruding structures present on the nanoparticle surface (approximately 5-10 nm in length) resulting in a rough LNP surface.

[0095] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. An amphiphilic bottlebrush block copolymer (BBCP) comprising a backbone and:(i) a hydrophilic block comprising hydrophilic polymer side chains bonded directly or indirectly to the backbone, and(ii) a hydrophobic block comprising lipid side chains bonded directly or indirectly to the backbone.

2. The amphiphilic BBCPs of claim 1, wherein the hydrophilic polymer side chains comprise neutral hydrophilic polymers, charged hydrophilic polymers, or a combination thereof.

3. The amphiphilic BBCPs of claim 1 or 2, wherein the hydrophilic polymer side chains comprise polyalkylene oxides such as polyethylene glycol (PEG) and poly(ethylene oxide) (PEO); poly(oxy ethylated polyol), poly(olefinic alcohol) such as poly(vinyl alcohol); polyvinylpyrrolidone; polysaccharides; hydrophilic polypeptides such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, and poly-L-lysine; poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), or combinations thereof.

4. The amphiphilic BBCPs of any one of claims 1 to 3, wherein the hydrophilic polymer side chains comprise polyalkylene oxides such as polyethylene glycol (PEG) and poly(ethylene oxide) (PEO).

5. The amphiphilic BBCPs of any one of claims 1 to 4, wherein the lipid side chains comprise phospholipids, sphingolipids, saturated fatty acid, unsaturated fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), or combinations thereof.

6. The amphiphilic BBCPs of any one of claims 1 to 5, wherein the lipid side chains comprise phospholipids.

7. The amphiphilic BBCPs of any one of claims 1 to 6, wherein the lipid side chains are covalently bonded to the backbone via a hydrophilic chemical moiety of the lipid.

8. The amphiphilic BBCPs of any one of claims 1 to 7, wherein the polymer backbone is an unsaturated polyolefin (e.g., a polynorbornylene), asaturated polyolefin, a polyester, a polyamide, a polyamine, a polyanhydride, or a combination thereof.

9. The amphiphilic BBCPs of any one of claims 1 to 8, synthesized by ring-opening metathesis polymerization (ROMP) using (i) the hydrophilic polymer side chain-cyclic olefin and cyclic olefin-jV-hydroxysuccinimidyl- ester followed by reaction of the cyclic olefin-7V-hydroxysuccinimidyl-ester with a lipid, (ii) lipid side chain-cyclic olefin and cyclic olefin-A'- hydroxysuccinimidyl-ester followed by reaction of the cyclic olefin-7V- hydroxysuccinimidyl-ester with a hydrophilic polymer, or (iii) the hydrophilic polymer side chain-cyclic olefin and lipid side chain-cyclic olefin, each initiated by a Grubbs catalyst.

10. The amphiphilic BBCPs of any one of claims 1 to 9, synthesized by ROMP using (i) the hydrophilic polymer side chain-norbomene and norbornene-Mhydroxysuccinimidy 1-ester followed by reaction of the norbornene- / V-hydroxysuccinimidyl-ester with a lipid, (ii) lipid side chain- norbornene and norbomene-7V-hydroxysuccinimidy l-ester followed by reaction of the norbornene- / V-hydroxysuccinimidyl-ester with a hydrophilic polymer, or (iii) the hydrophilic polymer side chain-norbornene and lipid side chain-norbomene, each initiated by a Grubbs catalyst.

11. The amphiphilic BBCPs of any one of claims 1 to 10, wherein:(i) the hydrophilic polymer side chains comprise polyethylene glycol, and / or(ii) the lipid side chains comprise l,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE).

12. Lipid nanoparticles (LNPs) comprising a membrane that comprises the amphiphilic BBCPs of any one of claims 1 to 11, wherein some or all of the hydrophilic blocks protrude to the exterior surface of the LNPs.

13. The LNPs of claim 12, wherein the membrane comprises a blend of the amphiphilic BBCPs and lipid molecules.

14. The LNPs of claim 13, wherein the lipid molecules comprise ionizable lipids, helper lipids, or combinations thereof, optionally wherein the helper lipids increase the LNPs’ stability and fluidity.

15. The LNPs of claim 13 or 14, wherein the lipid molecules comprise phospholipids, sphingolipids, saturated fatty acid, unsaturated fatty acids,glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), or combinations thereof.

16. The LNPs of any one of claims 13 to 15, wherein the lipid molecules comprise:(i) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (MC3),(ii) distearoylphosphatidylcholine (DSPC), and / or(iii) cholesterol.

17. The LNPs of any one of claims 12 to 16, wherein the membrane is a monolayer.

18. The LNPs of any one of claims 12 to 17, having encapsulated therein agents selected from therapeutic, prophylactic, and / or diagnostic agents.

19. The LNPs of claim 18, wherein the agents are one or more nucleic acids (such as mRNA).

20. The LNPs of claim 18 or 19, (i) having a higher proportion of the agents encapsulated within the LNPs than on the surface of the LNPs, or (ii) having the agents encapsulated within the LNPs and not present on the surface of the LNPs.

21. The LNPs of any one of claims 18 to 20, further comprising a second set of particles encapsulated within the LNPs, optionally wherein the second set of particles encapsulates one or more of the agents.

22. The LNPs of claim 21, wherein the second set of particles comprises a lipid membrane.

23. The LNPs of claim 22, wherein the lipid membrane is a monolayer or bilayer.

24. The LNPs of claim 22 or 23, wherein the lipid membrane comprises ionizable lipids, helper lipids, or combinations thereof, optionally wherein the helper lipids increase the LNPs’s stability and fluidity.

25. The LNPs of any one of claims 22 to 24, wherein the lipid membrane comprises phospholipids, sphingolipids, saturated fatty acid, unsaturated fatty acids, glycerides, monoglycerides, diglycerides, triglycerides, hormones, steroids (e.g., cholesterol, bile acids), vitamins (e.g., vitamin E), or combinations thereof.

26. The LNPs of any one of claims 22 to 25, wherein the lipid membrane comprises phospholipids.

27. The LNPs of any one of claims 22 to 26, wherein the lipid membrane comprises:(i) (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen- 19-yl 4- (dimethylamino)butanoate (MC3), and / or(ii) distearoylphosphatidylcholine (DSPC).

28. The LNPs of any one of claims 12 to 27, having a mean hydrodynamic diameter between about 50 nm and about 200 nm, between about 50 nm and about 175 nm, or between about 100 nm and about 175 nm, as measured using dynamic light scattering.

29. A method for delivering therapeutic, prophylactic or diagnostic agents to a subject in need thereof, the method comprising administering the LNPs of any one of claims 12 to 28.

30. The method of claim 29, wherein the nanoparticles are administered systemically.

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