WO2009132206A1 - Compositions et procédés pour administration et libération intracellulaire de chargement - Google Patents

Compositions et procédés pour administration et libération intracellulaire de chargement Download PDF

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Publication number
WO2009132206A1
WO2009132206A1 PCT/US2009/041559 US2009041559W WO2009132206A1 WO 2009132206 A1 WO2009132206 A1 WO 2009132206A1 US 2009041559 W US2009041559 W US 2009041559W WO 2009132206 A1 WO2009132206 A1 WO 2009132206A1
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Prior art keywords
reaction product
mold
less
particles
crosslinker
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PCT/US2009/041559
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English (en)
Inventor
Andrew Murphy
Ashley Galloway
Jason Rolland
Patrick Pohlhaus
Derek Schorzman
Michael Hunter
Joseph Desimone
Chris Clark
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Liquidia Technologies, Inc.
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Publication of WO2009132206A1 publication Critical patent/WO2009132206A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles

Definitions

  • the present invention relates to compositions for nanoparticles for intracellular delivery and release of cargo. More particularly, nanoparticles formed from the compositions package oligonucleotides and resist extracellular degradation.
  • the aim of encapsulating or including the active agent in a delivery vehicle is to enable the active agent to be transported to a site of therapeutic action while at the same time protecting it from degradation such as hydrolysis, enzymatic digestion, etc.
  • Another aim of creating a delivery vehicle is to control release of the active agent at a site of action so that the amount available to the organism is maintained at a desired level.
  • the present invention includes a reaction product of a polymer, a crosslinker, a nucleic acid, and between about 2 wt% and about 75 wt% of a charged monomer wherein the reaction results in a particle having a substantially predetermined three dimensional shape and a largest cross-sectional dimension of less than about 5 micron.
  • the reaction occurs in a mold comprising perfluoropolyether and the charged monomer make up between about 10 wt % and about 60 wt% of the reaction product components.
  • the charged monomer include between about 20 wt% and about 60 wt% of the reaction product components.
  • the nucleic acid comprises less than about 30 nucleotides and the polymer comprises poly( vinyl pyrrolidinone).
  • the polymer comprises a volatile monomer prepolymerized into an oligomer.
  • the crosslinker comprises a disulfide or a ketal and the particle can have an overall positive charge.
  • the mold is washed before conducting the reaction.
  • the particles are washed after being molded.
  • the mold comprises less than about 1 wt% initiator, less than about 0.5 wt% initiator, or less than about 0.1 wt% initiator.
  • the present invention includes a reaction product of a polymer, between about 10 wt% and about 25 wt% of a crosslinker, a nucleic acid, and a charged monomer wherein the reaction results in a particle having a substantially predetermined three dimensional shape and a largest cross-sectional dimension of less than about 5 micron.
  • the present invention also includes a reaction product of a polymer, a crosslinker, between about 0.25 wt% and about 20 wt% of a nucleic acid, and a charged monomer wherein the reaction results in a particle having a substantially predetermined three dimensional shape and a largest cross-sectional dimension of less than about 5 micron.
  • the present invention further includes a method of making drug delivery nanoparticles that includes prepolymerizing a monomer having a vapor pressure of greater than about 5MPa at about 25 degrees C into an oligomer having a vapor pressure of less than about 10 mm Hg at about 25 degrees C.
  • the method also includes forming a mixture of the oligomer and an active agent, introducing the mixture into cavities of a mold, wherein the cavities have a cross- sectional dimension of less than about 10 micrometers, and treating the mixture in the cavities of the mold to form nanoparticles.
  • the present invention includes a method for isolated particles fabrication, including introducing a mixture into cavities of a mold, wherein the cavities are less than about 10 micron in diameter and wherein the mold comprises a first surface energy, laminating a cover sheet onto the cavity side of the mold wherein the cover sheet includes a second surface energy that is greater than the first surface energy of the mold, and peeling the cover sheet away from the mold such that the mixture remains in the cavities of the mold and on the cover sheet but not on the mold outside the cavities.
  • the cover sheet is peeled away from the mold by wrapping the laminate around a freely rotatable roller.
  • a harvest sheet is laminated onto the cavity side of the mold.
  • the mixture is treated while in the cavities of the mold such that the mixture is hardened into particles.
  • a harvest sheet is laminated onto the cavity side of the mold after hardening the mixture.
  • the harvest sheet is peeled from the mold wherein the particles remain on the harvest sheet.
  • Figure 1 shows dose dependent knockdown of the lucif erase gene in HeLa cells with intracellular delivery nanoparticles, according to some embodiments of the present inventions
  • Figure 2 shows dose dependent knockdown of the lucif erase gene in HeLa cells with intracellular delivery nanoparticles, according to another embodiment of the present inventions
  • Figure 3 shows dose dependent knockdown of the luciferase gene in HeLa cells with intracellular delivery nanoparticles, according to still further embodiments of the present inventions
  • Figure 4 shows dose dependent knockdown of the luciferase gene in HeLa cells with intracellular delivery nanoparticles, according to yet another embodiment of the present inventions
  • Figure 5 shows dose dependent knockdown of the luciferase gene in HeLa cells with intracellular delivery nanoparticles, according to certain embodiments of the present inventions
  • Figure 6 shows dose dependent knockdown of the luciferase gene in HeLa cells with intracellular delivery nanoparticles, according to further embodiments of the present inventions
  • Figures 7-11 show dose dependent knockdown of the luciferase gene in HeLa cells with various formulations of PEGiooo dimethacrylate based intracellular delivery nanoparticles, according to alternative embodiments of the present invention.
  • Figure 12 shows dose dependent knockdown of the luciferase gene in HeLa cells with intracellular delivery naonparticles, according to a further embodiment of the present invention.
  • the present invention generally includes materials and methods for packaging, delivering, and releasing cargo, such as drug or biological materials into cells or tissues.
  • the drug or biological materials e.g., cargo or active agent
  • the nanoparticles have engineered shapes, sizes, and compositions and are fabricated from techniques that are compatible with sensitive biological and/or pharmaceutical cargo materials.
  • the nanoparticles in some embodiments can be engineered for applications such as delivering a cargo to tissues or organs, delivering a cargo to cells, delivering a cargo into cells, delivering a cargo to targeted cells and/or tissues, and the like by altering parameters of the nanoparticle such as, for example, matrix composition, particle size, particle shape, cargo loading concentration, particle charge or charge distribution, targeting agents, particle degradation rate and/or response, crosslinking, combinations thereof, and the like.
  • the nanoparticle materials in some embodiments of the present invention can be constructed to undergo tailored breakdown, for example, in response to a predetermined stimuli or upon encountering an environmental condition to trigger release of cargo, illicit a response, or treat a condition.
  • the components of the broken down nanoparticle can be cleared from the cell or tissue.
  • the nanoparticle of the present invention can also be targeted with, for example, ligands to target specific cells or tissues needing treatment.
  • the cargo is housed within the matrix of the nanoparticle such as crosslinked polymer networks.
  • the nanoparticles are also fabricated into a selected or predetermined size, shape, and volume.
  • the nanoparticle and/or its constituent components are, in some embodiments, biocompatible, non-toxic, and may be water soluble.
  • a nanoparticle in accordance with the present invention is less than about 500 ⁇ m in a broadest dimension (e.g., largest cross-sectional dimension). In some embodiments, the nanoparticle is less than about 450 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 400 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 350 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 300 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 250 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 200 ⁇ m in a broadest dimension.
  • a broadest dimension e.g., largest cross-sectional dimension.
  • the nanoparticle is less than about 150 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 100 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 75 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 50 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 40 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 30 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 20 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 10 ⁇ m in a broadest dimension.
  • the nanoparticle is less than about 5 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 1 ⁇ m in a broadest dimension. In some embodiments, the nanoparticle is less than about 900 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 800 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 700 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 600 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 500 nm in a broadest dimension.
  • the nanoparticle is less than about 400 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 300 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 200 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 100 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 80 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 75 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 70 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 65 nm in a broadest dimension.
  • the nanoparticle is less than about 60 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 55 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 50 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 45 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 40 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 35 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 30 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 25 nm in a broadest dimension.
  • the nanoparticle is less than about 20 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 15 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 10 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 7 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 5 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 2 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 0.5 nm in a broadest dimension. In some embodiments, the nanoparticle is less than about 0.1 nm in a broadest dimension.
  • the nanoparticle of the present invention has an aspect ratio of substantially 1 :1. According to other embodiments of the present invention the nanoparticle has an aspect ratio of about 1.5:1. In yet further embodiments of the present invention the nanoparticle has an aspect ratio of about 2:1. In still further embodiments of the present invention the nanoparticle has an aspect ratio of about 2.5:1. In some embodiments of the present invention the nanoparticle has an aspect ratio of about 3:1. In other embodiments of the present invention the nanoparticle has an aspect ratio of about 3.5:1. According to other embodiments of the present invention the nanoparticle has an aspect ratio of about 4 : 1. In yet further embodiments of the present invention the nanoparticle has an aspect ratio of about 4.5:1.
  • the nanoparticle has an aspect ratio of about 5:1. In some embodiments of the present invention the nanoparticle has an aspect ratio of about 5.5:1. In other embodiments of the present invention the nanoparticle has an aspect ratio greater than about 6:1. In other embodiments of the present invention the nanoparticle has an aspect ratio of at least about 10:1. As used herein, aspect ratio refers to the ratio of the longest axis to the shortest axis of the nanoparticle.
  • the nanoparticles of the present invention are fabricated from a composition or matrix of materials that include a polymer, a charged monomer, a crosslinker, and an active cargo.
  • the nanoparticles generally are biodegradable crosslinked oligomeric polymer nanoparticles that form intracellular delivery devices.
  • the intracellular delivery devices include, for example, oligomeric vinyl pyrrolidinone crosslinked with a disulfide crosslinker.
  • the matrix materials of the intracellular delivery nanoparticles of the present invention include a biodegradable crosslinked polymer, such as oligomeric vinyl pyrrolidinone crosslinked with a ketal crosslinker.
  • Animal means humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians, and fish.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig).
  • An animal may be a transgenic animal.
  • Biocompatible refers to having the property of being biologically compatible by not producing a toxic, injurious, or immunological response in living tissue.
  • Biodegradable means compounds that, when introduced to a biological fluid, are broken down by cellular machinery, proteins, enzymes, hydrolyzing chemicals, intracellular constituents, and the like into components that the cells can either reuse or dispose of without significant toxic effect on the cells (i.e., fewer than about 20% of the cells are killed).
  • biodegradable refers to both enzymatic and non-enzymatic breakdown or degradation of the polymeric structure. Bio degradation can take place intracellularly or intercellularly. In certain preferred embodiments, the chemical reactions relied upon to break down the biodegradable compounds are uncatalyzed.
  • Biodegradable nanoparticle refers to a nanoparticle of the present invention that is selectively formulated to break down under selected conditions, such as for example, the oxidation/reduction of crosslink components in a nanoparticle system.
  • the biodegradable nanoparticle can be formulated to break down in response to pH, an enzyme, ionic strength gradient, water, selected biologic fluids, light, temperature change, combinations thereof, or the like.
  • Cargo refers to any biologically active agent such as, for example, RNA, siRNA, dsRNA, ssRNA, shRNA, miRNA, rRNA, tRNA, snRNA, DNA, ssDNA, dsDNA, plasmid DNA, antisense DNA, antisense RNA, vaccine, protein, amino acid, biological molecule, pharmaceutically active agent, drug, virus, bacteria, tag, diagnostic agent or other substance that can treat or otherwise result in an effect on biological tissue or an organism and the like.
  • Crosslinker or “crosslinking agent” refers to a molecule with two or more functional groups that can join adjacent chains of a polymer through covalent bonds and/or form a three- dimensional network when reacted with the appropriate co-monomers.
  • Degradable refers to having the property of breaking down or degrading under certain conditions, e.g., at neutral or basic pH, in a biological solution, and can include biodegradable.
  • Degradable crosslink or “degradable crosslinker” means a linkage formed by the crosslinker or crosslinking agent is capable of being severed by a chemical reaction which may or may not be accelerated by a certain local environment, i.e., inside a cell, outside a tumor, etc.), an enzymatic process, or by reaction with reducing agents such as glutathione as well hydrolysis in an acidic environment.
  • An example of degradation of such a degradable crosslink is hydrolysis of a ketal group bridging two polymer chains at low pH which is generated from using a crosslinker with a ketal between two polymerizable groups, the cleavage of a disulfide bond, by a reducing agent, or the like.
  • Effective amount means an amount necessary to elicit a desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of particles may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the encapsulating matrix, the target tissue, etc.
  • “Inhibit” or “down-regulate” or “knock-down” means that the expression of a gene, level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits, such as pathogenic protein, viral protein or cancer related protein subunit(s), is reduced below that observed in the absence of the compounds or combination of compounds of the invention.
  • Initiator refers to any compound that initiates polymerization, or produces a reactive species that initiates polymerization.
  • “Monomer” refers to a molecule that can combine with another to form a polymer; it is the repeating unit of a polymer.
  • Modulate means that the expression of a gene, level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunit(s) of a protein is up-regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of the nanoparticles of the invention.
  • Nucleotide means a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar. Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non- natural nucleotides, non-standard nucleotides and other; see for example, Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al, International PCT Publication No. WO 93/15187; all of which are hereby incorporated by reference herein.
  • modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
  • Polymer refers to a natural or synthetic compound of at least two repeating monomeric units.
  • Polymerization refers to the bonding of two or more monomers to form a polymer.
  • Polymerizable group refers to monomers which polymerize upon introduction of an initiator or radical source.
  • Polynucleotide or “oligonucleotide” means a polymer of nucleotides. Typically, a polynucleotide comprises at least three nucleotides.
  • RNA means ribonucleic acid that synthesizes protein within a cell, transferring information from DNA to the protein- forming system of the cell. RNA is also involved in expression and repression of hereditary information and its four main types include heterogeneous nuclear RNA (hRNA); messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).
  • hRNA heterogeneous nuclear RNA
  • mRNA messenger RNA
  • tRNA transfer RNA
  • rRNA ribosomal RNA
  • mRNA means messenger RNA and these represent the products of the majority of genes.
  • rRNA means ribosomal RNA and forms the structural component of the ribosome, the machine that translates mRNA into protein.
  • tRNA means types of RNA that form a “t” shape. Each of these RNAs can recognize 1-3 codons (the 3 nucleotide code present in DNA and RNA) on one end via its anti-codon loop and is attached to an amino acid via its other end. As the ribosome "translates" mRNA into protein, tRNAs enter the ribosome and match amino acids to the mRNA's successive codons.
  • snRNA means small nuclear RNA or RNAs and are catalytic RNAs that perform mRNA splicing.
  • siRNA means small interfering RNAs that play a broad role in gene expression. These small RNAs are used to destroy mRNAs with complementary sequences and can be used to inhibit gene expression. This process is called RNAi (for RNA interference).
  • Up-regulate means that the expression of a gene, level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits is greater than that observed in the absence of the nanoparticles of the invention.
  • the expression of a gene such as a viral or cancer related gene can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.
  • Zeta potential means the electrical potential that exists at the shear plane of a particle, which is some small distance from the surface. Zeta potential is commonly calculated as a function of the surface charge of a particle, any adsorbed layer at the interface, and the nature and composition of the surrounding medium in which the particle is suspended.
  • the mold materials and processes for fabricating the intracellular delivery nanoparticles of the present invention utilize a low-surface energy, liquid curable, polymeric material, such as for example, FLUOROCURTM materials (Liquidia Technologies, Inc., North Carolina) disclosed in the above-referenced patent applications.
  • the molds include a plurality of cavities of precisely defined shape and size in which the nanoparticles of the present invention are molded or fabricated.
  • the mold materials described and incorporated herein including PFPE and FLUOROCURTM include properties such as low surface energy (less than about 20 mN/m and in some embodiments, less than about 18 mN/m, and in further embodiments, less than 15 mN/m, 12 mN/m, or 10 mN/m), high resistance to swelling in the presence of a substantial majority of organic solvents, low change of volume between the liquid and solid state, highly non-reactive, and the like result in a superior molding material.
  • PFPE based mold materials are substantially chemically resistant, thereby limiting effecting chemical and/or biochemical activity of the cargo, including but not limited to biological cargo.
  • PFPE based mold materials are PFPE based mold materials that the low surface energy yields a non- wetting surface such that material to be molded does not remain on the mold areas between adjacent cavities and, therefore, little or no residual layer is formed between or connecting the intracellular delivery nanoparticles.
  • the molding process and materials of the present invention include a gentle treatment such that delicate cargo, such as for example, nucleic acids, biologic materials, proteins, enzymes, genetic materials, and the like can be processed without degradation, denaturing, loss of function, or other damage.
  • the cavities of the mold have sizes and shapes that substantially match the desired predetermined sizes and shapes of the nanoparticles to be produced.
  • the mold cavity is less than about 500 ⁇ m in a broadest dimension (e.g., largest cross-sectional dimension or diameter).
  • the mold cavity is less than about 450 ⁇ m in a broadest dimension.
  • the mold cavity is less than about 400 ⁇ m in a broadest dimension.
  • the mold cavity is less than about 350 ⁇ m in a broadest dimension.
  • the mold cavity is less than about 300 ⁇ m in a broadest dimension.
  • the mold cavity is less than about 250 ⁇ m in a broadest dimension.
  • the mold cavity is less than about 200 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 150 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 100 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 75 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 50 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 40 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 30 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 20 ⁇ m in a broadest dimension.
  • the mold cavity is less than about 10 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 5 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 1 ⁇ m in a broadest dimension. In some embodiments, the mold cavity is less than about 900 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 800 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 700 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 600 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 500 nm in a broadest dimension.
  • the mold cavity is less than about 400 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 300 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 200 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 100 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 80 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 75 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 70 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 65 nm in a broadest dimension.
  • the mold cavity is less than about 60 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 55 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 50 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 45 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 40 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 35 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 30 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 25 nm in a broadest dimension.
  • the mold cavity is less than about 20 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 15 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 10 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 7 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 5 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 2 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 0.5 nm in a broadest dimension. In some embodiments, the mold cavity is less than about 0.1 nm in a broadest dimension.
  • particle matrix composition is introduced into cavities of the mold, hardened or cured to form particles, and removed from the cavities.
  • the hardening or curing can result from a treatment, such as but not limited to, actinic radiation, thermal energy, evaporation, crosslinking, chemical reaction, temperature change, combinations thereof, or the like.
  • a treatment such as but not limited to, actinic radiation, thermal energy, evaporation, crosslinking, chemical reaction, temperature change, combinations thereof, or the like.
  • the mold materials are highly non- wetting, substances molded in the cavities do not bind or adhere to the molds and can be released as discrete particles substantially mimicking the shape and size of the cavity from which it was fabricated and yielding a plurality of monodisperse nanoparticles.
  • the mold includes a plurality of the same size and shaped cavities, that mold will yield a plurality of discrete particles having substantially the same size and shape.
  • the components of the matrix materials can be pre- mixed before being introduced into the cavities of the molds such that the compositions within each cavity is substantially equivalent or homogeneous.
  • the matrix materials can be added sequentially or in stages to the cavities of the molds such that certain components can be processed in different steps and/or under different conditions.
  • the matrix materials can be cured, partially cured, or processed prior to being introduced into the cavities of the molds.
  • a sandwich sheet or layer (e.g., a cover sheet) is introduced onto the molds to sandwich the matrix composition between the low surface energy polymeric mold and the sandwich sheet or layer.
  • the sandwich sheet is a polymer or polyester sheet.
  • the sandwich sheet or layer has a surface energy that is greater than the surface energy of the mold.
  • removal of the sandwich sheet or layer includes peeling the the sandwich sheet or layer such that the matrix composition remains in the cavities of the mold and on the sandwich sheet or layer, but not on the mold outside the cavities.
  • the sandwhich sheet or layer is removed from the mold by wrapping the laminate around a freely rotatable roller.
  • the molds include low surface energy perfluoropoly ether materials having low concentrations of initiator.
  • the molds used in the present invention can include between about 0.05 wt% and about 10 wt% initiator, such as photoinitiator.
  • the mold includes between about 0.1 wt% and about 5 wt% initiator.
  • the mold includes between about 0.5 wt% and about 2.5 wt% initiator.
  • the mold includes between about 0.1 wt% and about 1 wt% initiator.
  • the mold includes between about 0.1 wt% and about 0.5 wt% initiator.
  • the initiator can be a photoinitiator, thermal initiator, combinations thereof, or the like.
  • the initiator is a photoinitiator, such as DEAP, DPT, or the like.
  • the molds can be washed prior to being used to fabricate the nanoparticles of the present invention.
  • the molds can be washed with a solvent, subjected to a second cure, combinations thereof, or the like to remove or inactivate components of the mold, such as for example un-reacted or excess photoinitiator.
  • the filled mold is brought into contact with a second polymer or polyester sheet, such as a harvest sheet.
  • the harvest sheet is an untreated polyethylene terephthalate sheet.
  • the harvest sheet can be a corona treated sheet of polyethylene terephthalate.
  • the harvest sheet can been treated with a coating of arabinogalactan, a corona treatment, a poly( vinyl pyrrolidone) layer, or a substance that has a greater affinity for the particles than the affinity between the particles and the mold materials.
  • the harvest sheet treatment is a soluble and non-toxic substance.
  • the curing station can include exposure to actinic radiation, fusion treatment, an LED, a UV lamp, thermal exposure, or the like.
  • the curing acts to solidify or harden the matrix composition materials in the cavities of the mold and thereby form the intracellular delivery nanoparticles according to some embodiments of the present invention.
  • the mold sheet is separated from the second polymer sheet, or harvest sheet, to remove the intracellular delivery nanoparticles from the cavities of the mold and leave the particles exposed on the harvest sheet.
  • the mold sheet is separated from the harvest sheet at an angle that imparts a greater work of adhesion between the particles and the harvest sheet than the particles and the mold materials.
  • the particles can be collected or harvested from the harvest sheet by, for example, scraping, dissolving the coating, treating the harvest sheet, or the like.
  • water or another solvent can be applied to dissolve the coating from the harvest sheet and release the particles.
  • the particles can be washed or purified.
  • the particles are washed to lessen or alleviate a toxic or otherwise unwanted component associated with the particle.
  • the particles can be washed to remove excipients used for harvesting or residual components from the polymerization process.
  • the particles can be washed or purified by washing in water and/or an organic solvent, and concentrated via filtration or centrifugation.
  • particles can also be washed to lessen or remove a coating layer used to remove particles from the mold.
  • the particles can also be purified after harvesting.
  • the particles can be purified by known methods in the art, such as for example filtering, collection on a filter, centrifuging, dialysis, combinations thereof, or the like.
  • the intracellular delivery nanoparticles of the present invention are composed of monomer materials that are polymerized and/or crosslinked into the shape and size specific intracellular delivery nanoparticles.
  • the monomer can be a water soluble monomer that is made water insoluble when polymerized and/or crosslinked into the intracellular delivery nanoparticle.
  • the water insoluble intracellular delivery nanoparticle is, however, typically degradable under selected intracellular or biologic conditions into water soluble non-toxic components that can be excreted from cells, tissues, organs, and/or an organism according to some embodiments.
  • the particle can have different properties such as varying bio degradation conditions, varying hydrophobic/hydrophilic conditions, water solubility, non-water solubility, and the like.
  • the polymer includes natural or synthetic polymers.
  • the nanoparticle matrix materials of the present invention can include synthetic polyelectrolytes and polar polymers, such as poly(acrylic acid), poly(styrene sulfonate), carboxymethylcellulose (CMC), poly( vinyl alcohol), poly(ethylene oxide) (PEO), poly( vinyl pyrrolidone) (PVP), dextran, and the like.
  • water insoluble polymers are made water soluble by ionization or protonation of a pendant group.
  • water insoluble polymers containing pendent anhydride or ester groups can be solubilized when the anhydride or esters hydro lyze to form ionized acids on the polymer chain.
  • water soluble polymers are preferred polymers for the polymer component of the intracellular delivery nanoparticle because the polymers can be solublized in cellular and body fluids and excreted therefrom.
  • the polymers of the matrix are selected or tuned to degrade upon encountering a dissolution condition, which in some embodiments, can be a condition selected from a cellular or biologic environment, such as for example pH.
  • the water soluble polymer useful in the intracellular delivery particles can include poly( vinyl pyrrolidinone), reactive oligomeric poly(vinyl pyrrolidinone), poly(ethylene glycol), protected polyvinyl alcohol, poly(DMAEMA), HEA, HEMA, branched PEGs, combinations thereof, and the like.
  • the polymer is a non-water soluble polymer such as, for example poly(beta-amino esters), PLGA, PLA, poly(caprolactone),
  • the synthesis of well-defined polymers having controlled molecular structures can be essential to the preparation of certain intracellular delivery nanoparticles.
  • the intracellular delivery nanoparticle can be fabricated from prepolymers having well-defined pre-determined molecular weight, low volatility, high volatility, narrow molecular weight distribution, combinations thereof, and the like.
  • polymers for forming the intracellular delivery nanoparticle can be prepolymerized from volatile or otherwise unstable monomers.
  • the present invention includes prepolymerization techniques to reduce evaporation, reactivity, or other loss of the volatile component by initially forming a prepolymer or oligomer of the volatile or unstable monomer.
  • a prepolymer or oligomer of the volatile monomer can be produced by, but is not limited to, living polymerization reactions, anionic polymerization reactions, free radical living polymerization, catalytic chain transfer agent (CCT), iniferter mediated polymerization, stable free radical mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), reversible addition- fragmentation chain transfer (RAFT) polymerization, step-growth polymerization, combinations thereof, and the like.
  • CCT catalytic chain transfer agent
  • SFRP iniferter mediated polymerization
  • SFRP stable free radical mediated polymerization
  • ATRP atom transfer radical polymerization
  • RAFT reversible addition- fragmentation chain transfer
  • the monomer can be, but is not limited to, butadienes, styrenes, propene, acrylates, methacrylates, vinyl ketones, vinyl esters, vinyl acetates, vinyl chlorides, vinyl fluorides, vinyl ethers, vinyl pyrrolidone, acrylonitrile, methacrylnitrile, acrylamide, methacrylamide allyl acetates, fumarates, maleates, ethylenes, propylenes, tetrafluoroethylene, ethers, isobutylene, fumaronitrile, vinyl alcohols, acrylic acids, amides, carbohydrates, esters, urethanes, siloxanes, formaldehyde, phenol, urea, melamine, isoprene, isocyanates, expoxides, bisphenol A, chlorsianes, dihalides, dienes, alkyl olefins, ketones, aldehydes, vinylidene chloride, anhydrides,
  • the prepolymer can include, but is not limited to polyamides, proteins, polyesters, polystyrene, polyethers, polyketones, polysulfones, polyurethanes, polysiloxanes, polysilanes, cellulose, amylase, polyacetals, polyethylene, glycols, poly(acrylate)s, poly(methacrylate)s, poly(vinyl alcohol), poly(vinyl pyrrolidone), poly(vinylidene chloride), poly( vinyl acetate), poly(ethylene glycol), polystyrene, polyisoprene, polyisobutylenes, poly(vinyl chloride), poly(propylene), poly(lactic acid), polyisocyanates, polycarbonates, alkyds, phenolics, epoxy resins, polysulf ⁇ des, polyimides, liquid crystal polymers, heterocyclic polymers, polypeptides, conducting polymers including polyacetylene, polyquinoline, polyaniline
  • the reactive prepolymer is generally capable of undergoing further polymerization, post-prepolymerization, and in some embodiments can be made by living polymerization.
  • Living polymerizations are chain polymerizations from which chain transfer and chain termination are absent. In many cases the rate of chain initiation is fast compared with the rate of chain propagation so that the number of kinetic-chain carriers is essentially constant throughout the polymerization, leading to controlled polymer architecture.
  • reactive prepolymers for particle compositions can be made by anionic living polymerizations. In other embodiments, reactive prepolymers for particle compositions can be made by free radical living polymerization.
  • the free radical living polymerization includes one or more of the following: catalytic chain transfer agent (CCT), the iniferter mediated polymerization, stable free radical mediated polymerization (SFRP), atom transfer radical polymerization (ATRP), and reversible addition-fragmentation chain transfer (RAFT) polymerization.
  • CCT catalytic chain transfer agent
  • SFRP stable free radical mediated polymerization
  • ATRP atom transfer radical polymerization
  • RAFT reversible addition-fragmentation chain transfer
  • Reactive prepolymers can also be made through a variety of other polymerization techniques that allow for controlled chain length.
  • Techniques include catalytic chain transfer polymerization, which is a very efficient and versatile free-radical polymerization technique for the synthesis of functional macromonomers. This process is based on the ability of certain transition metal complexes, most notably of low-spin Co complexes such as cobaloximes, to catalyze the chain transfer to monomer reaction, as described in Australian Journal of Chemistry 55(7) 381-398, which is incorporated herein by reference in its entirety.
  • NMP Nitroxide mediated polymerization
  • TEMPO a radical scavenger
  • ARP Atom transfer radical polymerization
  • the ATRP technique includes an easy experimental setup, use of readily accessible and inexpensive catalysts (usually copper complexes formed with aliphatic amines or imines, or pyridines, many of which are commercially available), and simple initiators, such as alkyl halides.
  • RAFT is a form of free radical polymerization that shows living characteristics the presence of RAFT agents by a reversible addition and fragmentation chain transfer process.
  • polymers made by step growth methods increase in molecular weight at a very slow rate at lower conversions and only reach moderately high molecular weights at very high conversion.
  • Step growth polymers are defined as polymers formed by the stepwise reaction between functional groups of monomers. Most step growth polymers are also classified as condensation polymers, but not all step growth polymers release condensates. Further related disclosure and compositions are found in the following: U.S. Patent Nos.
  • an intracellular delivery nanoparticle can be fabricated from a monomer having a high vapor pressure at about room temperature, such as for example above about 5 MPa, by first performing a prepolymerization such that the corresponding reactive prepolymer has a vapor pressure less than about 0.1 mm Hg (25 0 C).
  • a monomer having a vapor pressure at room temperature of about, for example, 8.0 MPa can be employed into the polymer component of the intracellular delivery nanoparticle of the present invention by first prepolymerizing the monomer into an oligomer having a vapor pressure less than about 0.01 mm Hg (25 0 C).
  • poly(vinylpyrolidone-co-vinyl acetate-co-vinyl alcohol) can be used as the polymer component of the matrix for the nanoparticles of the present invention.
  • the poly(vinylpyrolidone-co-vinyl acetate-co-vinyl alcohol) can be formed, in one embodiment for example, by treating a solution of poly(vinylpyrolidone-co-vinyl acetate) (BASF Luvitec-
  • poly(vinylpyrollidone-co-vinyl acetate-co-vinyl methacrylate) can be used as the polymer component of the matrix materials for the nanoparticles of the present invention.
  • the poly(vinylpyrollidone-co-vinyl acetate-co-vinyl methacrylate) can be formed, in one embodiment for example, by treating a suspension of poly(vinylpyrollidone-co- vinyl acetate-co-vinyl alcohol) in toluene equipped with a mechanical stirrer and Dean-Stark trap and heating under nitrogen atmosphere at reflux to azeotropically remove residual water.
  • the particles of the present invention further include, in some embodiments, a biologically compatible and/or degradable crosslinker.
  • the crosslinker preferably is selected or tuned to degrade under specific conditions, such as for example, in response to a selected pH, in response to a selected enzyme, after a selected time in an aqueous environment, combinations thereof, or the like.
  • Degradable crosslinkers of the present invention can degrade, in some embodiments, through hydrolysis, enzymatic cleavage, a change of temperature, pH, or other environments such as oxidation or reduction.
  • Crosslinking groups can include hydrolytically labile carbonate, ester, ketal, acetal, orthoester, hydrazone, silicon based hydrolyzable crosslinkers, and phosphazene linkers, lactide or glycolide, and succinic acid and alpha hydroxy acids such as glycolic, or lactic acid.
  • crosslinkers of the present invention may also include a degradable region containing one or more groups such as anhydride, a ketal, an acetal, an orthoester and/or a phosphoester.
  • the biodegradable region may contain at least one amide functionality.
  • the crosslinker can also include an ethylene glycol oligomer, oligo(ethylene glycol), poly(ethylene oxide), poly( vinyl pyrolidone), poly(propylene oxide), poly(ethyloxazoline), or combinations of these substances.
  • crosslinkers of the present invention include reduction/oxidation cleavable cross linkers, such as a disulfide bridges, azo linkages, combinations thereof, or the like.
  • Crosslinkers susceptible to pH changes are also included in some embodiments; these systems can be stable under acidic or basic conditions and start to degrade at blood pH or can be base or acid catalyzed.
  • Hydrolytically degradable crosslinking agents that may be utilized as the degradable crosslinker of the present invention include, but are not limited to, poly(epsilon -caprolactone)- b- tetraethylene glycol-b- poly(epsilon -capro lactone) dimethacrylate, poly(epsilon- caprolactone)-b-poly(ethylene glycol)-b-poly(epsilon-capro lactone) dimethacrylate, poly(lactic acid)-b-tetraethylene glycol-b-poly(lactic acid)dimethacrylate, poly(lactic acid)-b-poly(ethylene glycol)-b-poly(lactic acid)dimethacrylate, poly(glycolic acid)-b-tetraethylene glycol-b- poly(glycolic acid)dimethacrylate, poly(gly colic acid)-b-poly(ethylene glycol)-b-poly(gly colic acid)dimeth
  • silicon based crosslinkers useful in the nanoparticles of some embodiments of the present invention can be found in Patrickios C. S., Themistou E., Synthesis and Characterization of Star Polymers and Cross-Linked Star Polymer Model Networks Containing a Novel, Silicon-Based, Hydrolyzable Cross-Linker, Macromolecules 2004, 37, 6734-6743; Roh, Y.S., Kim, M.S., Chung, CM., Photopolymerization and Curing Shrinkage of Silicon-Containing Multifunctional Methacrylates, J. of Materials Science Letters 21, 2002, 1093-1095; Davidson, R. S., Ellis, R., Very, S., and Wilkinson, S. A., Polymer 33 (1992) 3031; Japanese patent Kokai-H- 10-245247 (1997); Timmer, M.S., et al, Biomacromolecules 2003, 4, 1026-1033;
  • Enzymatically degradable crosslinking agents that may be useful as the degradable crosslinker according to some embodiments of the present invention include, but are not limited to, crosslinking agents in which a short sequence of amino acids (for example, from 3-5 amino acids) are linked to two methacrylate or acrylate groups.
  • Examples of enzymatically degradable crosslinking agents include, but are not limited to, alanine-proline-glycineleucine- poly( ethylene glycol )-alanine-proline-glycine-Ieucine )-diacrylate, alanineproline-glycine- leucine-diacrylate, alanine- proline-glycine-leucine -poly(ethylene glycol)-alanine-proline- glycine-leucine)-dimethylacrylate, and alanine-proline-glycineleucine-dimethylacrylate, combinations thereof, and the like.
  • enzymatically degradable crosslinking agents are disclosed in West & Hubbell (1999) Macromolecules 32(l):241-4, which is incorporated herein by reference in its entirety. Still other enzymatically cleaved crosslinkers contain azobonds.
  • a hydrolytically labile crosslinker can be fabricated for use in the particles of the present invention.
  • An example of a hydrolytically labile crosslinker includes poly(epsilon-caprolactone)-b- tetraethylene glycol -b-poly(epsilon- caprolactone)dimethacrylate.
  • disulfide and ketal based crosslinkers are utilized as the degradable crosslinker of the present invention.
  • Disulfide crosslinkers are preferable, in some embodiments, due to their reversibility and relative stability in blood plasma.
  • degradable crosslinking monomers useful in the intracellular delivery particle of the present invention include acid sensitive crosslinkers such as, but not limited to, orthoester and hydrazide crosslinkers.
  • alternative crosslinkers include ester crosslinkers, HEMA and HEA based crosslinkers, acrylamide based crosslinkers, proteins such as but not limited to azo reagents, orthocarbonates, combinations thereof, and the like.
  • light or sonic activated therapeutic treatments can be formed from the intracellular delivery particles of the present invention by utilizing a light or sonic sensitive crosslinker that will breakdown when subjected to a selected stimuli, such as light or ultrasound, respectively, such that a cargo is thereby released to treat a tissue following the application of such stimuli.
  • a selected stimuli such as light or ultrasound
  • the end groups of the crosslinker monomers can include an acrylate, methacrylate, allyl, acrylamide, epoxy, vinyl ether, vinyl ester, vinyl amides, no endgroups, combinations thereof, and the like.
  • the crosslink monomers having preselected dissolution or degradation characteristics include the 2,2'-bis(methacryloylethoxy)-propane (also referred to as ketal dimethacryate) of the following structure:
  • the crosslink monomers include the 2,2' -bis(2-allyloxylethoxy)- propane having the following structure:
  • the crosslink monomers include the 2,2'- bis(monoacryloxy(polyethylenegylcol)ethoxy)-propane having the following structure:
  • the crosslink monomers include the bis(acryloxyethyl)disulf ⁇ de (also referred to as disulfide diacrylate) having the following structure:
  • the crosslink monomers include the 3,3' -dithiopropionic acid, bis(polyethylgylcolmonomethacrylate) (also referred to as long chain (LC) disulfide diacrylate) ester having the following structure:
  • the crosslink monomers have preselected dissolution or degradation characteristics and include the 2,2'-bis(acryloylethoxy)-propane (also referred to as ketal diacrylate) of the following structure:
  • the nanoparticle matrix material of the present invention includes less than about 10 wt% crosslinker. In some embodiments, the matrix material includes at least about 10 wt% crosslinker. According to some embodiments, the matrix materials of the present invention include between about 10 wt% and about 25 wt% crosslinker. In other embodiments, the matrix material includes between about 10 wt% and about 50 wt% crosslinker. In other embodiments, the matrix material includes between about 10 wt% and about 75 wt% crosslinker. In some embodiments, the matrix material includes at least about 75 wt% crosslinker. In other embodiments, the matrix material includes about 0 wt% crosslinker.
  • the matrix material includes about 5 wt% crosslinker. In other embodiments, the matrix material includes about 10 wt% crosslinker. In other embodiments, the matrix material includes about 15 wt% crosslinker. In other embodiments, the matrix material includes about 20 wt% crosslinker. In other embodiments, the matrix material includes about 25 wt% crosslinker. In other embodiments, the matrix material includes about 30 wt% crosslinker. In other embodiments, the matrix material includes about 35 wt% crosslinker. In other embodiments, the matrix material includes about 40 wt% crosslinker. In other embodiments, the matrix material includes about 45 wt% crosslinker. In other embodiments, the matrix material includes about 50 wt% crosslinker.
  • the matrix material includes about 55 wt% crosslinker. In other embodiments, the matrix material includes about 60 wt% crosslinker. In other embodiments, the matrix material includes about 65 wt% crosslinker. In other embodiments, the matrix material includes about 70 wt% crosslinker. In other embodiments, the matrix material includes about 75 wt% crosslinker. In other embodiments, the matrix material includes about 80 wt% crosslinker. In other embodiments, the matrix material includes about 85 wt% crosslinker. In other embodiments, the matrix material includes about 90 wt% crosslinker. In other embodiments, the matrix material includes about 95 wt% crosslinker.
  • the cargo or active agent of the intracellular delivery particle can include a pharmaceutically active agent, chemically active agent, biologically active agent, combinations thereof, or the like.
  • the cargo can include RNA, siRNA, dsRNA, ssRNA, shRNA, miRNA, rRNA, hRNA, mRNA, tRNA, snRNA, DNA, ssDNA, dsDNA, plasmid DNA, antisense DNA, antisense RNA, vaccine, combinations thereof, and the like.
  • the active agent includes an oligonucleotide having at least 5 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 10 base pair.
  • the active agent includes an oligonucleotide having at least 15 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 20 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 25 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 30 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 35 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 40 base pair. In some embodiments, the active agent includes an oligonucleotide having at least 45 base pair.
  • the active agent includes an oligonucleotide having at least 50 base pair. In some embodiments, the active agent includes an oligonucleotide having between 5 and 50 base pair. In some embodiments, the active agent includes an oligonucleotide having between 10 and 40 base pair. In some embodiments, the active agent includes an oligonucleotide having between 15 and 35 base pair. In some embodiments, the active agent includes an oligonucleotide having between 15 and 30 base pair. In some embodiments, the active agent includes an oligonucleotide having between 15 and 25 base pair.
  • the active agent includes an oligonucleotide having a formal charge between neutral and negative sixty. In other embodiments, the active agent includes an oligonucleotide having a negative charge with a value of twice the number of base pair plus two. In some embodiments, the active agent includes an oligonucleotide having a molecular weight of at least about 6,000 g/mol. In some embodiments, the active agent includes an oligonucleotide having a molecular weight at of least about 10,000 g/mol. In some embodiments, the active agent includes an oligonucleotide having a molecular weight between about 6,000 g/mol and about 20,000 g/mol. In some embodiments, the active agent includes an oligonucleotide having a molecular weight of at least about 20,000 g/mol.
  • the cargo can introduce a parameter to the intracellular delivery particle such as, for example, a negative charge, a positive charge, hydrophobic or hydrophilic properties or the like, that can require offsetting by addition of further monomers or additives to the particle.
  • a parameter to the intracellular delivery particle such as, for example, a negative charge, a positive charge, hydrophobic or hydrophilic properties or the like, that can require offsetting by addition of further monomers or additives to the particle.
  • the positive or negative charge of a cargo can be offset by including one or more oppositely charged monomers or additives to the particle.
  • the nucleotide sequence may introduce a negative charge to the particle that can be offset by the inclusion of a charged (e.g., positively charged) monomer.
  • the cargo can include polynucleotides, such as oligonucleotides and the like used to modulate splicing of pre-mRNA; these polynucleotides are often called splice switching oligonucleotides. These splice switching oligonucleotides hybridize to splicing elements in pre-mRNA and redirect splicing from one splice variant to another. As a result a disease causing splice variant may be reduced and a therapeutic splice variant may be increased.
  • polynucleotides such as oligonucleotides and the like used to modulate splicing of pre-mRNA; these polynucleotides are often called splice switching oligonucleotides. These splice switching oligonucleotides hybridize to splicing elements in pre-mRNA and redirect splicing from one splice variant to another. As a result a disease causing splice
  • the chemical composition of these oligonucleotides may include, in some embodiments, one or more nucleotides or nucleosides independently selected from the group consisting of 2- deoxyribonucleotides, 2'O-methyl (2'-methoxy) ribonucleotides, 2'O- MOE (-O-ethyl-0-methyl) ribonucleotides, hexitol (HNA) nucleotides or nucleosides, 2'0-4'C- linked bicyclic ribofuranosyl (LNA) nucleotides or nucleosides, phosphorothioate analogs of any of the foregoing, methylphosphonate analogs of any of the foregoing, N3'-P5' phosphoramidate analogs of any of the foregoing and combinations thereof.
  • nucleotides or nucleosides independently selected from the group consisting of 2- deoxyribonucleotides, 2'O-methyl (2'
  • the splice switching oligonucleotides, or oligomers may include phosphorodiamidate morpholino nucleotide analogs and peptide nucleic acid (PNA) nucleotide analogs.
  • PNA peptide nucleic acid
  • the cargo of the present invention can include derivatives of polynucleotides.
  • Such derivatives can include, but are not limited to, modifications in the bases, sugars, and/or phosphate linkages of the polynucleotide.
  • Modified bases can include, but are not limited to, those found in the following nucleoside analogs: 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3- methyl adenosine, 5-methylcytidine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2- thiocytidine.
  • Modified sugars include, but are not limited to, T- fluororibose, ribose, T- deoxyribose, 3 '-azido-2', 3'-dideoxyribose, T, 3'-dideoxyribose, arabinose (the 2'-epimer of ribose), acyclic sugars, hexoses, combinations thereof, and the like.
  • the nucleosides may be strung together by linkages other than the phosphodiester linkage found in naturally occurring DNA and/or RNA.
  • Modified linkages can include, but are not limited to, phosphorothioate and 5'-N-phosphorarnidite linkages.
  • combinations of the various modifications described herein may be used in a single polynucleotide. These modified polynucleotides, in some embodiments, can be fabricated by using synthetic chemistry in vitro or by other techniques known in the art.
  • the cargo of the particles is not necessarily restricted to any physical form of polynucleotide cargo.
  • the cargo can be, for example, a circular plasmid, a linearized plasmid, a cosmid, a viral genome, a modified viral genome, an artificial chromosome, combinations thereof, or the like.
  • the polynucleotide may also be of any sequence or encode for a protein or peptide.
  • the encoded proteins may be enzymes, structural proteins, receptors, soluble receptors, ion channels, pharmaceutically active proteins, cytokines, interleukins, antibodies, antibody fragments, antigens, coagulation factors, albumin, growth factors, hormones, insulin, combinations thereof, and the like.
  • the polynucleotide can also include regulatory regions to control gene expression. These regulatory regions may include, but are not limited to, promoters, enhancer elements, repressor elements, TATA box, ribosomal binding sites, stop sites for transcription, combinations thereof, and the like. In other embodiments, the polynucleotide is not intended to encode a protein, for example, the polynucleotide may be used to fix an error in the genome of the cell being transfected.
  • the cargo can include a polynucleotide such as an antisense agent.
  • Antisense therapy is described to mean, for example, administration or in situ provision of single- or double-stranded oligonucleotides or their derivatives which specifically hybridize, e.g., bind, under cellular conditions, with cellular mRNA and/or genomic DNA, or mutants thereof, so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation.
  • Antisense therapy is further described in Crooke, Molecular Mechanisms of Action of Antisense Drugs, Biochem. Biophys.
  • the binding can be conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix as described in Chan et al. J. MoI. Med. 75(4):267-282, 1997; which is incorporated herein by reference in its entirety.
  • the cargo of the particle of the present invention can be siRNA or microRNAs.
  • siRNA's are small RNAs that play a broad role in eukaryotic gene expression. These RNA's typically function to destroy mRNAs with complementary sequences.
  • the cargo includes tRNA, which can recognize 1-3 codons (3 nucleotide code present in DNA and RNA) on one end via its anti-codon loop and is attached to an amino acid via its other end. As the ribosome "translates" mRNA into protein, tRNAs enter the ribosome to match amino acids to the mRNA's successive codons.
  • the cargo is small nuclear RNA, which catalyzes RNAs that perform mRNA splicing.
  • the polynucleotide cargo can be associated with other agents in the particles such as poly-amines, or the like that can neutralize the negative charge in the phosphate backbone of the polynucleotide.
  • Neutralizing or charged agents may allow for the passage of the neutral complex through cellular and nuclear membranes. These agents can also protect the polynucleotide from degradation once the polynucleotide is in the cell.
  • the polynucleotide cargo includes a sequence encoding an antigenic peptide or protein.
  • particles containing such cargo can be delivered to a patient as a vaccine to induce an immunologic response sufficient to decrease the chance of a subsequent infection and/or lessen the symptoms associated with such an infection.
  • the polynucleotide of these vaccines may be combined with interleukins, interferon, cytokines, and adjuvants such as cholera toxin, alum, Freund's adjuvant, combinations thereof, or the like.
  • the active cargo can be pharmaceutical drugs, metal complexes, combinations thereof, or the like.
  • the nanoparticle matrix material of the present nanoparticles includes at least about 0.25 wt% oligonucleotide active agent. In some embodiments the matrix material of the present nanoparticles includes between about 0.25 wt% and about 25 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 0.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 1 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 1.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 2 wt % oligonucleotide active agent.
  • the matrix material includes about 2.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 3 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 3.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 4 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 4.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 5.5 wt % oligonucleotide active agent.
  • the matrix material includes about 6 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 6.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 7 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 7.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 8 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 8.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 9 wt % oligonucleotide active agent.
  • the matrix material includes about 9.5 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 10 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 11 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 12 wt % oligonucleotide active agent.
  • the matrix material includes about 13 wt % oligonucleotide active agent.
  • the matrix material includes about 14 wt % oligonucleotide active agent.
  • the matrix material includes about 15 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 16 wt % oligonucleotide active agent.
  • the matrix material includes about 17 wt % oligonucleotide active agent.
  • the matrix material includes about 18 wt % oligonucleotide active agent.
  • the matrix material includes about 19 wt % oligonucleotide active agent.
  • the matrix material includes about 20 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 21 wt % oligonucleotide active agent.
  • the matrix material includes about 22 wt % oligonucleotide active agent.
  • the matrix material includes about 23 wt % oligonucleotide active agent.
  • the matrix material includes about 24 wt % oligonucleotide active agent.
  • the matrix material includes about 25 wt % oligonucleotide active agent. In other embodiments, the matrix material includes about 30 wt % oligonucleotide active agent.
  • the matrix material includes about 35 wt % oligonucleotide active agent.
  • the matrix material includes about 40 wt % oligonucleotide active agent.
  • the matrix material includes about 45 wt % oligonucleotide active agent.
  • the matrix material includes about 50 wt % oligonucleotide active agent. In some embodiments the matrix material of the present nanoparticles includes at least about 25 wt% oligonucleotide active agent. In some embodiments the matrix material of the present nanoparticles includes at least about 50 wt% oligonucleotide active agent.
  • the intracellular delivery nanoparticle of the present invention also can include charged monomers.
  • the charged monomers can include cationically charged monomers such as, for example [2-(acryloyloxy)ethyl]trimethyl ammonium chloride (AETMAC), 2-aminoethyl methacrylate hydrochloride (AEM-HCl), anionically charged monomers, combinations thereof, or the like.
  • AETMAC [2-(acryloyloxy)ethyl]trimethyl ammonium chloride
  • AEM-HCl 2-aminoethyl methacrylate hydrochloride
  • anionically charged monomers combinations thereof, or the like.
  • the charged monomer contributes one positive charge for each molecule of the charged monomer added to the composition.
  • the charged monomers can be included to offset the charge of the cargo or other matrix components and configure the intracellular delivery particle with a localized or overall charge that can assist the particle in crossing a cell membrane and entering a cell or otherwise delivering or maintaining a charged cargo within the particle through ionic interactions.
  • the measurement or effect of including the charged monomer with the particle matrix is to create a zeta potential of the particle that will assist the particle in crossing a cellular membrane.
  • the charged monomer can be included in an N/P (nitrogen of the charged monomer to phosphate of the oligonucleotide cargo ratio) of between about 1 and about 65.
  • the N/P ratio can be between about 1 and about 30.
  • the N/P ratio can be between about 1 and about 15.
  • the N/P ratio can be between about 2 and about 10.
  • the N/P ratio can be between about 3 and about 6.
  • the intracellular delivery nanoparticles are configured to have a positive zeta potential. In other embodiments, the nanoparticles are configured to have a zeta potential of between about negative 50 mV to about positive 50 mV. In some embodiments, the nanoparticles are configured to have a zeta potential between about negative 25 mV and about positive 50 mV. In other embodiments, the nanoparticles have a zeta potential of between about negative 10 mV and about negative 25 mV. In yet further embodiments, the nanoparticles have a zeta potential of between about positive 20 mV and positive 50 mV.
  • the nanoparticles can be configured to have a positive zeta potential of between about 5 mV and about 150 mV. In other embodiments, the nanoparticles are configured with a zeta potential of between about 15 mV and about 100 mV. In other embodiments, the nanoparticles are configured with a zeta potential of between about 20 mV and about 75 mV. In other embodiments, the nanoparticles are configured with a zeta potential of between about 25 mV and about 50 mV.
  • the nanoparticle matrix material is composed of between 2 wt% and 75 wt% charged monomer. In other embodiments, the matrix material includes at least about 2 wt % charged monomer. In other embodiments, the matrix material includes about 5 wt % charged monomer. In other embodiments, the matrix material includes about 10 wt % charged monomer. In other embodiments, the matrix material includes about 15 wt % charged monomer. In other embodiments, the matrix material includes about 20 wt % charged monomer. In other embodiments, the matrix material includes about 25 wt % charged monomer. In other embodiments, the matrix material includes about 30 wt % charged monomer.
  • the matrix material includes about 35 wt % charged monomer. In other embodiments, the matrix material includes about 40 wt % charged monomer. In other embodiments, the matrix material includes about 45 wt % charged monomer. In other embodiments, the matrix material includes about 50 wt % charged monomer. In other embodiments, the matrix material includes about 55 wt % charged monomer. In other embodiments, the matrix material includes about 60 wt % charged monomer. In other embodiments, the matrix material includes about 65 wt % charged monomer. In other embodiments, the matrix material includes about 70 wt % charged monomer. In other embodiments, the matrix material includes about 75 wt % charged monomer. In other embodiments, the matrix material includes at least about 75 wt % charged monomer.
  • the intracellular delivery nanoparticle can also include an initiator.
  • the initiator can include a photoinitiator, such as for example HCPK or the like. Ideally, the initiator should not be toxic and is otherwise safe to a cell, tissue, organ, or organism.
  • the initiator is included in the matrix materials to begin the reactions in the cavity of the molds to form or solidify the matrix compositions into the intracellular delivery nanoparticles of the present invention.
  • the matrix material includes less than about 10 wt% initiator. In some embodiments, the matrix material includes less than about 5 wt% initiator. In some embodiments, the matrix material includes less than about 2.5 wt% initiator. In some embodiments, the matrix material includes less than about 1 wt% initiator.
  • the matrix material includes less than about 0.5 wt% initiator. In some embodiments, the matrix material includes less than about 0.1 wt% initiator. In some embodiments, the nanoparticle matrix material includes between about 0.05 wt% and about 10 wt% initiator. In some embodiments, the matrix material includes between about 0.5 wt% and about 5 wt% initiator. In some embodiments, the matrix material includes between about 0.5 wt% and about 2.5 wt% initiator In some embodiments, the matrix material includes between about 0.1 wt% and about 1 wt% initiator. In some embodiments, the matrix material includes between about 0.1 wt% and about 0.5 wt% initiator.
  • the intracellular delivery nanoparticles of the present invention can also include lysosomotropic compounds, endosomo lytic compounds, combinations thereof, and the like.
  • the lysosomotropic or endosomolytic compounds can be encapsulated, packaged, or coupled with or within the biodegradable crosslinked polymer matrix of the nanoparticle and be configured to assist release of the cargo released from the nanoparticle but retained in a cellular body, such as an endosome or the like.
  • lysosomotropic and endosomolytic compounds can lyse endosomal membranes.
  • lysosomotropic compounds useful for combining with the intracellular delivery particles of the present invention include, but are not limited to, cholesterol, chloroquine, ammonium chloride, fluoxitine, amitriptyline, diphenhydramine, chlorphentermine, propranolol, chlorpromazine, perhexaline, amantadine, gentamicin, quinacrine, glycylphenylalanine 2-naphthyl amide (GPN), combinations thereof, or the like.
  • endosomolytic agents useful for combining with the intracellular delivery particles of the present invention include, but are not limited to chloroquine, fusogenic peptides, inactivated adenoviruses, polyethyleneimine, cholesterol, cholesterol derivatives, cationic lipids, ethanol, combinations thereof, and the like.
  • Further lysosomotropic and/or endosomolytic agents and their function(s), useful for combining with the intracellular delivery particles of the present invention can be found in International Patent Application Publication No. WO 2000/063409; U.S Patent No. 6,849,272; and U.S. Published Application No. 2007- 0036865, each of which is incorporated herein by reference in its entirety.
  • the intracellular delivery particles includes a charged monomer such as for example, AETMAC or AEM, a degradable crosslinker such as for example, bis(acryloxyethyl) disulfide (also referred to as disulfide diacrylate) or 2,2' di(2- methacryloxy ethyl) propane (also referred to as ketal dimethacrylate), a polymer such as for example, reactive oligomeric PVP, and a cargo such as, for example, an oligonucleotide.
  • a charged monomer such as for example, AETMAC or AEM
  • a degradable crosslinker such as for example, bis(acryloxyethyl) disulfide (also referred to as disulfide diacrylate) or 2,2' di(2- methacryloxy ethyl) propane (also referred to as ketal dimethacrylate)
  • a polymer such as for example, reactive oligomeric PVP
  • a cargo such as, for example
  • the matrix composition from which the intracellular delivery nanoparticles are formed includes between about 2 wt% to about 60 wt% charged monomer, between about 1 wt% and about 25 wt% degradable crosslinker, and between about 0.25 wt% and about 5 wt% cargo with substantially the remainder of the particle being composed of the polymer component.
  • the matrix composition from which the intracellular delivery nanoparticles are formed includes between about 10 wt% to about 50 wt% charged monomer, between about 5 wt% and about 25 wt% degradable crosslinker, and between about 0.25 wt% and about 20 wt% cargo with substantially the remainder of the particle being composed of the polymer component.
  • the matrix composition includes between about 2 wt% to about 10 wt% charged monomer, between about 10 wt% and about 25 wt% disulfide diacrylate crosslinker, and between about 0.5 wt% and about 5 wt% active oligonucleotide with substantially the remainder of the particle being composed of a water soluble polymer component such as for example, oligomeric polyvinyl pyrrolidone.
  • the matrix composition includes between about 6 wt% to about 20 wt% charged monomer, between about 15 wt% and about 25 wt% ketal dimethacrylate degradable crosslinker, and between about 0.25 wt% and about 5 wt% active oligonucleotide with substantially the remainder of the particle being composed of a water soluble polymer component such as for example, oligomeric polyvinyl pyrrolidone.
  • the matrix composition can also include 4,4'Bis(diethylamino)-benzophenone, a curing agent such as a photoinitiator (e.x., 1- hydroxycyclohexylphenyl ketone), cholesterol, dimethyl sulfoxide (DMSO), combinations thereof, and the like.
  • a curing agent such as a photoinitiator (e.x., 1- hydroxycyclohexylphenyl ketone), cholesterol, dimethyl sulfoxide (DMSO), combinations thereof, and the like.
  • Table 1 shows matrix composition from which intracellular delivery nanoparticles were formed that produced in vitro dose dependent knock-down of a targeted gene. The table also shows percent photoinitiator used in the molds to make the nanoparticles of the selected compositions, the type of curing applied to the nanoparticles, the harvest layer used, the purification technique utilized, and the resulting zeta potential for the respective nanoparticles made from the listed matrix compositions. Table 1:
  • the intracellular delivery particles can include a charged monomer such as for example, AEM, a degradable crosslinker such as for example, N,N'-bis(acryloyl)cystamine, a polymer such as for example, poly(vinylpyrolidone-co-vinyl acetate-co-vinyl methacrylate), an initiator such as for example, 1 -hydroxy cyclohexyl phenyl ketone, and a cargo such as, for example, an oligonucleotide.
  • AEM charged monomer
  • a degradable crosslinker such as for example, N,N'-bis(acryloyl)cystamine
  • a polymer such as for example, poly(vinylpyrolidone-co-vinyl acetate-co-vinyl methacrylate)
  • an initiator such as for example, 1 -hydroxy cyclohexyl phenyl ketone
  • a cargo such as, for example,
  • the matrix composition from which the intracellular delivery nanoparticles can be formed can include between about 20 wt% to about 50 wt% AEM, between about 0 wt% and about 25 wt% N 5 N'- bis(acryloyl)cystamine, between about 0.1 wt% and about 2 wt% 1 -hydroxy cyclohexyl phenyl ketone, and between about 0.5 wt% and about 15 wt% oligonucleotide with substantially the remainder of the particle being composed of poly(vinylpyrollidone-co-vinyl acetate-co-vinyl methacrylate).
  • the matrix composition from which the intracellular delivery nanoparticles can be formed can include between about 30 wt% to about 45 wt% AEM, between about 0 wt% and about 15 wt% N,N'-bis(acryloyl)cystamine, between about 0.1 wt% and about 2 wt% 1 -hydroxy cyclohexyl phenyl ketone, and between about 1 wt% and about 10 wt% oligonucleotide with substantially the remainder of the particle being composed of poly(vinylpyrolidone-co-vinyl acetate-co-vinyl methacrylate).
  • the matrix composition from which the intracellular delivery nanoparticles can be formed can include between about 30 wt% to about 40 wt% AEM, between about 0 wt% and about 10 wt% N,N'-bis(acryloyl)cystamine, between about 0.1 wt% and about 1 wt% 1 -hydroxy cyclohexyl phenyl ketone, and between about 0.5 wt% and about 7.5 wt% oligonucleotide with substantially the remainder of the particle being composed of poly(vinylpyrolidone-co-vinyl acetate-co-vinyl methacrylate).
  • intracellular delivery nanoparticles of the present invention were fabricated from AETMAC, PEGiooo dimethacrylate or mPEGsooo monoacrylate, ketal diacrylate or butanediol diacrylate, and a 21mer siRNA.
  • the intracellular delivery nanoparticles include about 20 wt% AETMAC, about 57 wt% PEGiooo dimethacrylate, about 20 wt% ketal diacrylate, and about 1 wt% of a 21mer siRNA (anti-luciferase RNA), with about 1 wt% each of a fluorescent tag and a photoinitiator, such as HCPK.
  • This formulation showed dose dependent knockdown of the luciferace gene in HeLa cells as shown in Figure 7.
  • the intracellular delivery nanoparticles include about 40 wt% AETMAC, about 52 wt% PEGiooo dimethacrylate, about 5 wt% ketal diacrylate, and about 1 wt% of a 21mer siRNA (anti-luciferase RNA), with about 1 wt% each of a fluorescent tag and a photoinitiator, such as HCPK.
  • This formulation showed dose dependent knockdown of the luciferace gene in HeLa cells as shown in Figure 8.
  • the intracellular delivery nanoparticles include about 60 wt% AETMAC, about 33 wt% PEGiooo dimethacrylate, and about 5 wt% of a 21mer siRNA (anti-luciferase RNA), with about 1 wt% each of a fluorescent tag and a photoinitiator, such as HCPK.
  • This formulation showed dose dependent knockdown of the luciferace gene in HeLa cells as shown in Figure 9.
  • the intracellular delivery nanoparticles include about 20 wt% AETMAC, about 57 wt% PEGiooo dimethacrylate, about 20 wt% butanediol diacrylate, and about 1 wt% of a 21mer siRNA (anti-luciferase RNA), with about 1 wt% each of a fluorescent tag and a photoinitiator, such as HCPK.
  • This formulation showed dose dependent knockdown of the luciferace gene in HeLa cells as shown in Figure 10.
  • the intracellular delivery nanoparticles include about 50 wt% AETMAC, about 23 wt% PEGiooo dimethacrylate, about 20 wt% mPEG 5O oo monoacrylate, and about 5 wt% of a 21mer siRNA (anti-luciferase RNA), with about 1 wt% each of a fluorescent tag and a photoinitiator, such as HCPK.
  • This formulation showed dose dependent knockdown of the luciferace gene in HeLa cells, as shown in Figure 11.
  • intracellular delivery nanoparticles that show in vitro gene knockdown of a specific target gene by siRNA interference include nanoparticles fabricated from the methods disclosed and incorporated herein and from the mixture of about 50 wt%
  • intracellular delivery nanoparticles that produce in vitro gene knockdown of a specific target gene by siRNA interference include nanoparticles fabricated from the methods disclosed and incorporated herein and from the mixture of between about 10 wt% and about 50 wt% AEM, about 23 wt% PEGiooo DMA, and about 5 wt% siRNA.
  • intracellular delivery nanoparticles producing in vitro gene knockdown of a specific target gene by siRNA interference include nanoparticles fabricated from the methods disclosed and incorporated herein and from the mixture of about 20 wt% AEM, about 20 wt% DEAEMA, between about 28 wt% and about 33 wt% PEGiooo DMA, about 20 wt% mPEGsooo monoacrylate, and between about 5 wt% and about 10 wt% siRNA.
  • a second film consisting of a 6" wide by 2 mil thick sheet (D-316 from DuPont Teijin films) having a patterned fluoropolymer mold consisting of 200 x 200 nm holes adhered to one side (see U.S. patent application no. 11/633,763, filed December 4, 2006, which is incorporated herein by reference in its entirety) is brought into mold side contact with the liquid of film 1 at the nip point whereby the two films are laminated together. After traveling about 2 feet at about 7 feet/minute the two films of the laminate are passed around a freely rotating 3 inch diameter steel peel roller and the films are peeled apart from each other. After passing around this roller, the two films are separated.
  • the isolated film mold consists of liquid matrix solution in the nanocavities of the mold and is absent liquid on the land areas.
  • the isolated PET film (film 1) carries with it any excess matrix material.
  • the film mold (film 2) is then fed into another nip point where it is laminated with a third film (film 3) having a thickness of 2 mils.
  • the third film includes a PET sheet with a corona treated side that is brought into contact with the with the filled mold cavities.
  • the third film includes PET sheet having untreated surfaces.
  • the third film is a polyester sheet that is coated with a layer of arabinogalactan (between 1 and 10 microns thick) and the arabinogalactan is brought into contact with the filled mold cavities.
  • the nip consists of two rollers in contact with one another, a top rubber roller with a Shore A hardness of 90 having a 4" diameter and approximately 8" width and a bottom stainless steel roller approximately 4" in diameter and 8" in width.
  • Example 1 The liquid matrix solution in the cavities is then cured with the laminate traveling at about 7 feet per minute with film 2 side up.
  • the matrix material is cured by an LED band 8 inches in length and 2 inches in width emitting light at 395 nm (UV Process
  • the matrix material in the cavities is cured by a UV lamp (Fusion Model D bulb) with a power of about 3 W positioned about 2.5 inches from the film.
  • the UV exposure acts to cure the liquid confined to the mold cavities into solid particles.
  • the laminate is separated X no. inches after curing into its constituent two films.
  • Particles are removed from film three and collected into water using a polyethelene scraper in Examples 1 and 2. Approximately 1 mL of water is used to collect a 3 foot length of particles.
  • particles are removed from film 3 by running film 3 with particles through a closed nip point of a 1 inch diameter steel roller having a width of 8" and being coated with a Teflon® FEP (DuPont) sleeve and a second %" diameter roller having a width of 8" and being coated with rubber having a Shore A hardness of 60.
  • the film is fed such that the side consisting of the arabinogalactan coating having particles of 200nm on the surface is facing the Teflon FEP sleeve.
  • a bead of water having a volume of 1.8 mL is placed and held in the nip.
  • the film is now fed through the nip at a speed of ⁇ 4 feet / minute such that the bead of water dissolves the arabinogalactan layer and releasing the particles into the bead of water.
  • the suspension of particles in water is removed using a pipette and placed into an eppendorff tube for further purification.
  • the solids composition of this Liquidia PRINTTM Particle formulation is approximately 8% AETMAC, 25% disulfide diacrylate, 2.5% BDAB, 1% fluorescein-o-acrylate, 1.5% anti-luciferase siRNA, and the remainder PVP-oil.
  • Particles were gently removed from the corona treated PET sheet using a cell scraper and a bead of nuclease free water. Particles were then washed by performing an initial spin (1,000 rpm for 2 min to remove any debris that may have been introduced in the manufacturing process. The particle solution was then transferred into a 0.1 um PVDF Centricon Tube (Millipore). The solution was spun at 4,000 rpm until the filtrate passed through the membrane leaving particles suspended on the membrane.
  • Particles were then resuspended in 1 mL of nuclease free water followed by a second centrifugal filtration at 4,000 rpm. This washing was repeated with a second 1 mL of nuclease free water. Particles were then resuspended in 200 ⁇ L of nuclease free water and stored at 4 0 C prior to cell studies.
  • Cells were infected with particles as outlined in the cell assay section. Particles were determined to have a zeta potential in pure water of 40 mV and an effective diameter of 452 nm in an aqueous solution. These particles were shown to induce luciferase knockdown in HeLa cells in a dose dependent fashion as shown in Figure 1.
  • the solid composition of this Liquidia PRINTTM Particle formulation is approximately 6% AETMAC, 20% ketal dimethacrylate, 1% HCPK, 1% fluorescein-o-acrylate, 0.5% anti- luciferase siRNA, and the remainder PVP-oil.
  • Particles were gently removed from the PET sheet using a cell scraper and a bead of nuclease free water. Particles were then washed by performing an initial spin (1,000 rpm for 2 min to remove any debris that may have been introduced in the manufacturing process. The particle solution was then transferred into a 0.1 um PVDF Centricon Tube (Millipore). The solution was spun at 4,000 rpm until the filtrate passed through the membrane leaving particles suspended on the membrane. Particles were then resuspended in 1 mL of nuclease free water followed by a second centrifugal filtration at 4,000 rpm. This washing was repeated with a second 1 mL of nuclease free water. Particles were then resuspended in 200 ⁇ L of nuclease free water and stored at 4 0 C prior to cell studies. Cells were infected with particles as outlined in the cell assay section.
  • Particles were determined to have a zeta potential in pure water of 100 mV while the dynamic light scattering shows particles in the 250 nm size range. These particles were also shown to induce luciferase knockdown in HeLa cells in a dose dependent fashion as shown in Figure 2.
  • Example 3 In this formulation, 100 ⁇ L of 80% AETMAC, 100 ⁇ L ketal dimethacrylate, 280 ⁇ L of reactive oligomeric PVP, 4.7 mg of 1-hydroxycyclohexylphenyl ketone, 4.8 mg of fluorescein- o-acrylate, and 20 ⁇ L of water were added to a nuclease free eppendorf tube. After mixing well, 20 ⁇ L of this solution was added to 100 ⁇ g of anti-luciferase siRNA in water immediately before particle fabrication.
  • the solids composition of this Liquidia PRINTTM Particle formulation is approximately 20% AETMAC, 20% ketal dimethacrylate, 1% HCPK, 1% fluorescein-o-acrylate, 0.5% anti-luciferase siRNA, and the remainder PVP-oil.
  • Particles were gently removed from the AG harvest layer into a stream of nuclease free water. Particles were then washed by performing an initial spin (1,000 rpm for 2 min to remove any debris that may have been introduced in the manufacturing process. The particle solution was then transferred into a fresh nuclease free eppendorf tube. The solution was spun at 13,000 rpm for 10 minutes to pellet the particles. The supernatant was removed and the particles were resuspended in 1 mL of nuclease free water with the aid of vortexing and sonication. The process was repeated IX for a total of 2 x 1 mL washings.
  • the particles were resuspended in 200 ⁇ L of nuclease free water, again with the aid of vortexing and sonication. Particles were then stored at 4 0 C prior to cell studies. Cells were infected with particles as outlined in the cell assay section.
  • Particles were determined to have a zeta potential in pure water of 18 mV and an effective diameter of 411 nm in an aqueous solution. These particles were shown to induce lucif erase knockdown in HeLa cells in a dose dependent fashion as shown in Figure 3.
  • the solids composition of this Liquidia PRINTTM Particle formulation is approximately 20% AETMAC, 20% ketal dimethacrylate, 1% HCPK, 1% fluorescein-o- acrylate, 0.25% anti-luciferase siRNA, and the remainder PVP-oil.
  • Particles were gently removed from the AG harvest layer into a stream of nuclease free water. Particles were then washed by performing an initial spin (1,000 rpm for 2 min to remove any debris that may have been introduced in the manufacturing process. The particle solution was then transferred into a fresh nuclease free eppendorf tube. The solution was spun at 13,000 rpm for 10 minutes to pellet the particles.
  • the supernatant was removed and the particles were resuspended in 1 mL of nuclease free water with the aid of vortexing and sonication. The process was repeated IX for a total of 2 x 1 mL washings. After the final pelleting, the particles were resuspended in 200 ⁇ L of nuclease free water, again with the aid of vortexing and sonication. Particles were then stored at 4 0 C prior to cell studies. Cells were infected with particles as outlined in the cell assay section.
  • Particles were determined to have a zeta potential in pure water of 25 mV and while the dynamic light scattering shows particles in the 200 nm size range in an aqueous solution. These particles were shown to induce luciferase knockdown in HeLa cells in a dose dependent fashion as shown in Figure 4.
  • the solids composition of this Liquidia PRINTTM Particle formulation is approximately 20% AETMAC, 20% ketal dimethacrylate, 1% HCPK, 1% fluorescein-o-acrylate, 0.5% cholesterol, 0.5% anti- luciferase siRNA, and the remainder PVP -oil.
  • Particles were gently removed from the AG harvest layer into a stream of nuclease free water. Particles were then washed by performing an initial spin (1,000 rpm for 2 min to remove any debris that may have been introduced in the manufacturing process. The particle solution was then transferred into a fresh nuclease free eppendorf tube. The solution was spun at 13,000 rpm for 10 minutes to pellet the particles. The supernatant was removed and the particles were resuspended in 1 mL of nuclease free water with the aid of vortexing and sonication. The process was repeated IX for a total of 2 x 1 mL washings.
  • the particles were resuspended in 200 ⁇ L of nuclease free water, again with the aid of vortexing and sonication. Particles were then stored at 4 0 C prior to cell studies. Particles were determined to have a zeta potential in pure water of 24 mV and an effective diameter of 444 nm in an aqueous solution. These particles were shown to induce luciferase knockdown in HeLa cells in a dose dependent fashion as shown in Figure 5.
  • Example 6 In this formulation, 50 ⁇ L of 80% AETMAC, 50 ⁇ L disulfide diacrylate, 395 ⁇ L of reactive oligomeric PVP, 5.2 mg of fluorescein-o-acrylate, 20 ⁇ L of water, and 125 ⁇ L of DMSO were added to a nuclease free eppendorf tube. After mixing well, 100 ⁇ L of this solution was added to 1.0 mg of 1 -hydroxy cyclohexylphenyl ketone in a second nuclease free eppendorf tube. 20 ⁇ L of this solution and 15 ⁇ L of DMSO were added to 200 ⁇ g of anti- luciferase siRNA in water immediately before particle fabrication.
  • the solids composition of this Liquidia PRINTTM Particle formulation is approximately 10% AETMAC, 10% disulfide diacrylate, 1% HCPK, 1% fluorescein-o-acrylate, 1% anti- luciferase siRNA, and the remainder PVP-oil.
  • Particles were gently removed from the AG harvest layer into a stream of nuclease free water. Particles were then washed by performing an initial spin (1,000 rpm for 2 min to remove any debris that may have been introduced in the manufacturing process. The particle solution was then transferred into a fresh nuclease free eppendorf tube. The solution was spun at 13,000 rpm for 10 minutes to pellet the particles. The supernatant was removed and the particles were resuspended in 1 mL of nuclease free water with the aid of vortexing and sonication. The process was repeated IX for a total of 2 x 1 mL washings.
  • the particles were resuspended in 200 ⁇ L of nuclease free water, again with the aid of vortexing and sonication. Particles were then stored at 4 0 C prior to cell studies. Cells were infected with particles as outlined in the cell assay section. Particles were determined to have a zeta potential of 15 mV and an effective diameter of 373 nm in water. These particles were shown to induce luciferase knockdown in HeLa cells in a dose dependent fashion as shown in Figure 6.
  • the film was then brought into contact with FLUOROCUR® mold containing 200 nm diameter and 200 nm depth nanocavities (produced as reported previously) at the nip point of a hot laminator (Chemlnstruments HL- 100, Fairfield, OH) set at roll temperature ofl50 0 F and speed setpoint of 2, or approximately 3.5 ft/min) and separated immediately after the nip point such that both sheets remain in contact with the rolls immediately after delamination.
  • a hot laminator Chemlnstruments HL- 100, Fairfield, OH
  • the majority of the nanocavities of the FLUOROCUR® mold are filled with polymer within 0-50 nm of the top of the cavity.
  • each stock solution to prepare 200 ⁇ L total volume as a 5 wt% solution was added to a 1.5 mL polyethylene tube in the following order: 2-aminoethyl methacrylate hydrochloride, PEGiooo-dimethacrylate, mPEGsooo-monoacrylate, ketal diacrylate or 1,4-butanediol diacrylate, fluorescein-o-acrylate, hydroxycylohexylphenyl ketone, and anti- luciferase RNA. After addition of all the components, the mixture was vortex for 30 s.
  • Table II below, outlines relative wt% of solid components in different casting solutions that made up five different particle matrix materials for particles Pl, P2, P3, P4, and P5. Data of gene expression/knock-down are shown in Figures 7-11, which correspond to particles P1-P5, respectively.
  • the solution was then coated onto a 5 Mil thick PET substrate using a #2 Mayer rod (RD Specialties, Webster, NY), and the solvent was evaporated with a flow of warm air.
  • the thin, transparent film was then laminated to a FLUOROCUR® mold containing 200 nm diameter and 200 nm depth nanocavities using a rubber roller. The two sheets were then separated. Using this processing method, the majority of the nanocavities of the FLUOROCUR® mold are filled with polymer within 0-50 nm of the top of the cavity. Filled mold was then hand laminated with a 2 Mil PET film using a rubber roller, and the laminate was cured by irradiation at 50-60 mW/cm 2 for 5 minutes.
  • the mold layer was peeled away from the PET resulting primarily in transfer of solid nanoparticles from mold cups to the PET film.
  • the particles were removed from the PET surface using a polyethylene blade in a small volume of water. The particles were then pelleted via centrifugation (13,000 x g, 10 minutes) and resuspend into water twice. After a final pelleting, the particles were resuspended to the desired volume.
  • Nanoparticles Pl having the composition shown in Table II, resulted in luciferase gene expression knockdown as shown in Figure 7.
  • a solution containing the following was prepared at 5% solids in dimethyl formamide: 23 wt% mg PEG 5000 mono-acrylate; 35 wt% mg N,N'-Bis(acryloyl)cystamine;
  • the solution was coated onto the raw side of a polyester film (MELINEX® 453, DuPont Teijin Films) using a number 2 mayer rod and the DMF was evaporated at ambient temperature using an air dryer.
  • the film was laminated at a speed of 6 feet/minute to a FLUOROCUR® mold having 200 x 200 nm cavities (Liquidia Technologies, Inc., North Carolina) in a nip point consisting of a 6" rubber roller (durometer 85) and a 6" steel roller driven together at a pressure of 60 psi.
  • the mold and the polyester sheet remained laminated for 3 feet until the polyester sheet was separated from the mold. After separation from the polyester sheet, the mold cavities remained filled with the monomer mixture.
  • the mold was then laminated at a speed of 6 feet/minute to the raw side of a second polyester sheet (MELINEX® 453, DuPont Teijin Films) at a second nip point consisting of a 6" rubber roller (durometer 85) and a 6" steel roller driven together at a pressure of 60 psi.
  • the laminate was then taken through a UV fusion lamp (Fusion UV 1250 Systems, Inc.) using a Type D bulb positioned about 5" from the laminate at a rate of 6.8 ft/min during which the monomer mixture cured into solid particles within the cavities of the mold.
  • the laminate was then separated resulting in the fully cured particles in an array on the polyester sheet.
  • the particles on polyester sheet were collected into a volume of water using a polyethylene cell scraper (NUNCTM Cell Scrapers, distributed through Thermo Fisher Scientific, Rochester Site). The resulting suspension was collected and placed into a 1.5 mL eppendorf tube. The particles were purified by centrifugation at a speed of 13.5k rpm for 5 minutes and removing the supernatant and replacing with nuclease-free water. Particles were then resuspended by sonication for 1 minute and vortexing for 10 seconds and the process was repeated 3 times. The final concentration of the particles was 5 mg/mL.
  • NUNCTM Cell Scrapers distributed through Thermo Fisher Scientific, Rochester Site
  • Particles were dosed onto luciferase-expressing HeLa and diluted in a 2.5 fold serial dilution in several wells. The particles decreased luciferase expression in a dose-dependent manner as seen in

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Abstract

Une nanoparticule est fabriquée à partir d’une réaction d’un polymère, d’un agent de réticulation, d’un acide nucléique et entre environ 2 % en poids et environ 75 % en poids d’un monomère chargé dans une cavité d’un moule conduisant à une particule comportant une forme tridimensionnelle sensiblement prédéterminée et une dimension en coupe transversale la plus importante inférieure à environ 5 microns. Le moule peut comprendre du perfluoropolyéther et l’acide nucléique peut comprendre moins d’environ 30 nucléotides et le polymère peut être de la poly(vinylpyrrolidinone). Les particules peuvent être lavées après avoir été moulées et le moule peut comprendre moins d’environ 1 % en poids d’initiateur, moins d’environ 0,5 % en poids d’initiateur ou moins d’environ 0,1 % d’initiateur.
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