US20140328759A1 - Limit size lipid nanoparticles and related methods - Google Patents

Limit size lipid nanoparticles and related methods Download PDF

Info

Publication number
US20140328759A1
US20140328759A1 US14/353,460 US201214353460A US2014328759A1 US 20140328759 A1 US20140328759 A1 US 20140328759A1 US 201214353460 A US201214353460 A US 201214353460A US 2014328759 A1 US2014328759 A1 US 2014328759A1
Authority
US
United States
Prior art keywords
lipid
nanoparticle
popc
streams
lnp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US14/353,460
Other languages
English (en)
Inventor
Pieter R. Cullis
Igor V. Jigaltsev
James R. Taylor
Timothy Leaver
Andre Wild
Nathan Maurice Belliveau
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of British Columbia
Original Assignee
University of British Columbia
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of British Columbia filed Critical University of British Columbia
Priority to US14/353,460 priority Critical patent/US20140328759A1/en
Assigned to THE UNIVERSITY OF BRITISH COLUMBIA reassignment THE UNIVERSITY OF BRITISH COLUMBIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TAYLOR, JAMES R., JIGALTSEV, Igor V., CULLIS, PIETER R., BELLIVEAU, Nathan Maurice, LEAVER, Timothy, WILD, ANDRE
Publication of US20140328759A1 publication Critical patent/US20140328759A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/50273—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means or forces applied to move the fluids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61J—CONTAINERS SPECIALLY ADAPTED FOR MEDICAL OR PHARMACEUTICAL PURPOSES; DEVICES OR METHODS SPECIALLY ADAPTED FOR BRINGING PHARMACEUTICAL PRODUCTS INTO PARTICULAR PHYSICAL OR ADMINISTERING FORMS; DEVICES FOR ADMINISTERING FOOD OR MEDICINES ORALLY; BABY COMFORTERS; DEVICES FOR RECEIVING SPITTLE
    • A61J3/00—Devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/08—Drugs for disorders of the alimentary tract or the digestive system for nausea, cinetosis or vertigo; Antiemetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P23/00—Anaesthetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/431—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor
    • B01F25/4317—Profiled elements, e.g. profiled blades, bars, pillars, columns or chevrons
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/433—Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
    • B01F25/4331—Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/431—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor
    • B01F25/43197—Straight mixing tubes with baffles or obstructions that do not cause substantial pressure drop; Baffles therefor characterised by the mounting of the baffles or obstructions
    • B01F25/431971—Mounted on the wall
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0605—Metering of fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

  • the present invention is directed to limit size nanoparticles for delivery of therapeutic and/or diagnostic agents, methods for using the lipid nanoparticles, and methods and systems for making the lipid nanoparticles.
  • LNP lipid nanoparticles
  • LNP lipid nanoparticles
  • i.v. intravenous
  • LNP smaller than approximately 50 nm diameter can permeate through the lymphatics and accumulate in tissues such as bone marrow whereas particles of 30 nm or smaller can access progressively more tissues in the body.
  • Particles smaller than approximately 8 nm diameter are cleared by the kidney. It is therefore particularly important to be able to generate particles in the size range 10-50 nm as these particles are most likely to be able to access extravascular target tissue.
  • limit size LNP Methods of making limit size LNP have not progressed substantially for nearly 30 years. All of the methods employ “top down” approaches where larger structures are formed by dispersion of lipid in water, followed by mechanical disruption to produce smaller systems.
  • the preferred method for making bilayer vesicles in the 100 nm size range involves extrusion of preformed multilamellar vesicles (micron size range) through polycarbonate filters with a pore size of 100 nm or smaller and is not useful for producing systems smaller than approximately 50 nm.
  • the predominant method for making limit size systems has usually involved sonication of multilamellar vesicles, usually tip sonication, which has limitations of sample contamination, sample degradation and, most importantly, lack of scalability.
  • lipid systems containing bilayer-forming lipids such as phosphatidylcholine (PC)
  • PC phosphatidylcholine
  • Chol PC/cholesterol
  • nanoemulsions consisting of PC and non-polar lipids such as triglycerides
  • sonication or other emulsification techniques have been applied.
  • size ranges less than 50 nm has proven elusive.
  • LNPs of useful size can be prepared by conventional top down methods, a need exists for improved methods that facilitate the scalable preparation of these LNPs. The present seeks to fulfill this need and provides further related advantages.
  • the invention provides limit size lipid nanoparticles useful for delivery of therapeutic and/or diagnostic agents.
  • the limit size lipid nanoparticle has a diameter from about 10 to about 100 nm.
  • the lipid nanoparticle has a lipid bilayer surrounding an aqueous core.
  • the lipid bilayer includes a phospholipid.
  • the lipid nanoparticle has a lipid monolayer surrounding a hydrophobic core.
  • the lipid monolayer includes a phospholipid.
  • the nanoparticle includes a lipid bilayer surrounding an aqueous core, wherein the bilayer includes a phospholipid, a sterol, and a polyethylene glycol-lipid, and the core comprises a therapeutic or diagnostic agent.
  • the nanoparticle includes a lipid monolayer surrounding a hydrophobic core, wherein the monolayer comprises a phospholipid, and the core comprises a fatty acid triglyceride and a therapeutic and/or diagnostic agent.
  • the invention provides a method for administering a therapeutic agent to a subject, comprising administering a nanoparticle of the invention to a subject in need thereof.
  • the invention provides a method for administering a diagnostic agent to a subject, comprising administering a nanoparticle of the invention to a subject in need thereof.
  • the invention provides a method for treating a disease or condition treatable by administering a therapeutic agent, comprising administering a therapeutically effective amount of a nanoparticle of the invention to a subject in need thereof.
  • the invention provides a method for diagnosing a disease or condition diagnosable by administering a diagnostic agent, comprising administering a nanoparticle of the invention to a subject in need thereof.
  • the method includes making limit size lipid nanoparticles in a device having a first region adapted for flow of first and second adjacent streams and a second region for mixing the streams, comprising:
  • the invention provides devices for making limit size lipid nanoparticles.
  • the device includes:
  • a third microchannel for receiving the first and second streams, wherein the third microchannel has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a third stream comprising limit size lipid nanoparticles.
  • the device includes:
  • a plurality of microchannels for receiving the first and second streams, wherein each has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a plurality of streams compromising lipid nanoparticles;
  • each of the plurality of microchannels for receiving the first and second streams may include:
  • a third microchannel for receiving the first and second streams, wherein each has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a plurality of streams compromising lipid nanoparticles.
  • FIG. 1 is a schematic illustration of a representative system of the invention, a continuous-flow staggered herringbone micromixer.
  • the mixing of two separate streams occurs in the patterned central channel which grooved walls drive alternating secondary flows that chaotically stir the fluids injected.
  • the chaotic mixing leads to exponential increase of the interfacial area thus reducing the diffusion distances between two fluids. Rapid interdiffusion of the two phases (aqueous and ethanolic containing fully solvated lipids) results in the self-assembly of LNPs, whose size depends primarily on their lipid composition and aqueous/ethanolic flow rate ratio.
  • FIGS. 2A and 2B illustrate limit size LNP vesicles (FIG. A) and phospholipid-stabilized solid-core nanospheres (FIG. B) produced by increasing the aqueous/ethanolic flow rate ratio (FRR).
  • FRRs were varied by maintaining a constant flow rate in the ethanolic channel (0.5 ml/min) and increasing the flow rates of the aqueous channel from 0.5 to 4.5 ml/min. Size measurements were obtained using DLS (number weighting).
  • FIG. 5 illustrates the effect of the POPC/TO molar ratio on the size of LNPs.
  • the data points for the DLS measured LNP sizes represent means ⁇ SD of 3 experiments. Theoretical values were calculated as described, calculated values were used to plot a curve fit (second order exponential decay).
  • FIGS. 6A and 6B are cryo-TEM micrographs of POPC LNPs loaded with doxorubicin at 0.1 mol/mol ( FIG. 6A ) and 0.2 mol/mol ( FIG. 6B ) D/L ratios.
  • FIG. 7A is a three-dimensional view of a representative parallel fluidic structure of the invention useful for making limit size lipid nanoparticles.
  • FIG. 7B shows a top view and a side view of the representative parallel fluidic structure shown in FIG. 7A .
  • the top view shows two planar herringbone structures in parallel.
  • the side view shows that the fluidic parallel fluidic structure has three layers to give a total of six herringbone structures.
  • FIG. 7C is a three-dimensional view of a second representative parallel fluidic structure of the invention useful for making limit size lipid nanoparticles.
  • FIG. 8 is an image of the simulated pressure drop between the inlet port and the first section of each layer of the representative parallel fluidic structure shown in FIG. 7A . Due to higher downstream resistance, downstream resistance in each layer is essentially identical.
  • FIG. 9 is an image of a microfluidic scale-up device (chip) loaded into a holder. The device is pressed against the rear surface interface plate and the ports sealed with O-rings.
  • FIG. 10 compares mean vesicle diameter of representative POPC/Cholesterol vesicles (final lipid concentration was 8 mg/mL) prepared by the scale-up system (parallel fluidic device) and a single mixer device.
  • FIG. 11 compares mean vesicle diameter of representative DSPC/Cholesterol vesicles (final lipid concentration was 3 mg/mL) prepared by the scale-up system (parallel fluidic device) and a single mixer device.
  • FIG. 12 is a representative temperature-controlled fluid device of the invention having heating chambers.
  • FIG. 13 is a representative temperature-controlled fluid device of the invention having heating chambers.
  • FIG. 14 is a representative temperature-controlled fluid device of the invention having heating chambers.
  • FIG. 15 compares particle size (nm) as a function of mole percent cholesterol (Chol) in a representative lipid bilayer nanoparticle of the invention.
  • FIG. 16 compares the results of an in vivo pharmacokinetic (PK) study evaluating of retention properties of POPC and POPC/Chol (7:3) DOX loaded systems both containing 6.5% DSPE-PEG2000 (PEG) in plasma of CD-1 mice.
  • PK in vivo pharmacokinetic
  • FIG. 17 compares the results of an in vitro release study performed in presence of 50% FBS: POPC/PEG (D/L 0.1), POPC/Chol/PEG 70/30/6.5 (D/L 0.1), POPC/Chol/PEG 70/30/6.5 (D/L 0.15), POPC/Chol/PEG 65/35/6.5 (D/L 0.1), and POPC/Chol/PEG 65/35/6.5 (D/L 0.15).
  • the present invention provides limit size lipid nanoparticles, methods for using the nanoparticles, and methods and systems for making the nanoparticles.
  • limit size lipid nanoparticles are provided.
  • limit size refers to the lowest size limit possible for a particle. The limit size of a particle will depend on the particle's composition, both the particle's components and their amounts in the particle. Limit size lipid nanoparticles are defined as the smallest, energetically stable lipid nanoparticles that can be prepared based on the packing characteristics of the molecular constituents.
  • limit size lipid nanoparticles are provided in which the lipid nanoparticle has a diameter from about 10 to about 100 nm.
  • the limit size lipid nanoparticles of the invention include a core and a shell comprising a phospholipid surrounding the core.
  • the core includes a lipid (e.g., a fatty acid triglyceride) and is semi-solid, or solid.
  • the core is liquid (e.g., aqueous).
  • the shell surrounding the core is a monolayer. In another embodiment, the shell surrounding the core is a bilayer.
  • the limit size nanoparticle includes a lipid bilayer surrounding an aqueous core.
  • the nanoparticles can be advantageously loaded with water-soluble agents such as water-soluble therapeutic and diagnostic agents, and serve as drug delivery vehicles.
  • the lipid bilayer (or shell) nanoparticle includes a phospholipid.
  • Suitable phospholipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.
  • the phospholipid is a C8-C20 fatty acid diacylphosphatidylcholine.
  • a representative phospholipid is 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).
  • the nanoparticles include from about 50 to about 99 mole percent phospholipid.
  • the nanoparticle further comprises a sterol.
  • the nanoparticles include from about 10 to about 35 mole percent sterol.
  • Representative sterols include cholesterol.
  • the ratio of phospholipid to sterol e.g., cholesterol
  • the ratio of phospholipid to sterol is 55:45 (mol:mol).
  • the ratio of phospholipid to sterol is 60:40 (mol:mol).
  • the ratio of phospholipid to sterol is 65:35 (mol:mol).
  • the ratio of phospholipid to cholesterol is 70:30 (mol:mol).
  • the nanoparticle of invention can further include a polyethylene glycol-lipid (PEG-lipid).
  • PEG-lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, and PEG-modified dialkylglycerols.
  • Representative polyethylene glycol-lipids include DLPE-PEGs, DMPE-PEGs, DPPC-PEGs, and DSPE-PEGs.
  • the polyethylene glycol-lipid is DSPE-PEG (e.g., DSPE-PEG2000).
  • the nanoparticle includes from about 1 to about 10 mole percent polyethylene glycol-lipid.
  • the nanoparticle includes from 55-100% POPC and up to 10 mol % PEG-lipid (aqueous core LNPs).
  • the lipid nanoparticles of the invention may include one or more other lipids including phosphoglycerides, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lyosphosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.
  • Other compounds lacking in phosphorus, such as sphingolipid and glycosphingolipid families are useful. Triacylglycerols are also useful.
  • Representative particles of the invention have a diameter from about 10 to about 50 nm.
  • the lower diameter limit is from about 10 to about 15 nm.
  • the limit size nanoparticle includes a lipid monolayer surrounding a hydrophobic core.
  • These nanoparticles can be advantageously loaded with hydrophobic agents such as hydrophobic or difficultly, water-soluble therapeutic and diagnostic agents.
  • the hydrophobic core is a lipid core.
  • Representative lipid cores include fatty acid triglycerides.
  • the nanoparticle includes from about 30 to about 90 mole percent fatty acid triglyceride.
  • Suitable fatty acid triglycerides include C8-C20 fatty acid triglycerides.
  • the fatty acid triglyceride is an oleic acid triglyceride (triglyceride triolein).
  • the lipid monolayer includes a phospholipid. Suitable phospholipids include those described above.
  • the nanoparticle includes from about 10 to about 70 mole percent phospholipid.
  • the ratio of phospholipid to fatty acid triglyceride is from 20:80 (mol:mol) to 60:40 (mol:mol).
  • the triglyceride is present in a ratio less than about 40% and not greater than about 80%.
  • the limit size lipid nanoparticles of the invention can include one or more therapeutic and/or diagnostic agents. These agents are typically contained within the particle core.
  • the particles of the invention can include a wide variety of therapeutic and/or diagnostic agents.
  • Suitable therapeutic agents include chemotherapeutic agents (i.e., anti-neoplastic agents), anesthetic agents, beta-adrenaergic blockers, anti-hypertensive agents, anti-depressant agents, anti-convulsant agents, anti-emetic agents, antihistamine agents, anti-arrhytmic agents, and anti-malarial agents.
  • chemotherapeutic agents i.e., anti-neoplastic agents
  • anesthetic agents i.e., beta-adrenaergic blockers, anti-hypertensive agents, anti-depressant agents, anti-convulsant agents, anti-emetic agents, antihistamine agents, anti-arrhytmic agents, and anti-malarial agents.
  • anti-neoplastic agents include doxorubicin, daunorubicin, mitomycin, bleomycin, streptozocin, vinblastine, vincristine, mechlorethamine, hydrochloride, melphalan, cyclophosphamide, triethylenethiophosphoramide, carmaustine, lomustine, semustine, fluorouracil, hydroxyurea, thioguanine, cytarabine, floxuridine, decarbazine, cisplatin, procarbazine, vinorelbine, ciprofloxacion, norfloxacin, paclitaxel, docetaxel, etoposide, bexarotene, teniposide, tretinoin, isotretinoin, sirolimus, fulvestrant, vairubicin, vindesine, leucovorin, irinotecan, capecitabine, gemcitabine, mitoxantrone hydroch
  • the therapeutic agent is an anti-neoplastic agent.
  • the anti-neoplastic agent is doxorubicin.
  • the invention provides a nanoparticle that includes a lipid bilayer surrounding an aqueous core in which the bilayer includes a phospholipid, a sterol, and a polyethylene glycol-lipid, wherein the core comprises a therapeutic or diagnostic agent.
  • the nanoparticle is a limit size nanoparticle.
  • the nanoparticle has a diameter from about 10 to about 50 nm.
  • the invention provides a lipid monolayer surrounding a hydrophobic core in which the monolayer comprises a phospholipid, and the core includes a fatty acid triglyceride and/or a therapeutic or diagnostic agent.
  • the nanoparticle is a limit size nanoparticle. In certain embodiments, the nanoparticle has a diameter from about 10 to about 80 nm.
  • the lipid nanoparticles of the invention are useful for delivering therapeutic and/or diagnostic agents.
  • the invention provides a method for administering a therapeutic and/or diagnostic agent to a subject.
  • a nanoparticle of the invention comprising a therapeutic and/or diagnostic agent is administered to the subject.
  • the invention provides a method for treating a disease or condition treatable by administering a therapeutic agent effective to treat the disease or condition.
  • a nanoparticle of the invention comprising the therapeutic agent is administered to the subject in need thereof.
  • the invention provides methods for making limit size lipid nanoparticle.
  • the invention provides a method for making lipid nanoparticles in a device having a first region adapted for flow of first and second adjacent streams and a second region for mixing the streams, comprising:
  • the device is a microfluidic device.
  • the flow pre-mixing is laminar flow.
  • the flow during mixing is laminar flow.
  • the lipid nanoparticles are limit size lipid nanoparticles.
  • limit size lipid nanoparticles are prepared by rapid mixing of the first and second streams.
  • the formation of limit size nanoparticles depends on, among other factors, the rate of changing the polarity of the solution containing the lipid particle-forming materials (e.g., rapid mixing of two streams with different polarities).
  • the rapid mixing is achieved by flow control; control of the ratio of the first flow rate to the second flow rate.
  • the ratio of the first flow rate to the second flow rate is greater than 1:1 (e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, including intermediate ratios).
  • the rapid mixing is achieved by controlling the composition of the streams.
  • Rapid change in solvent polarity past a critical point results in limit size nanoparticle formation.
  • reducing the ethanol content in the second stream below 100% allows for rapid mixing of the streams at flow rate ratios near 1:1.
  • mixing the first and second streams comprises chaotic advection. In other embodiments, mixing the first and second streams comprises mixing with a micromixer. In certain embodiments, mixing of the first and second streams is prevented in the first region by a barrier (e.g., a channel wall, sheath fluid, or concentric tubing). In certain embodiments, the method further includes diluting the third stream with an aqueous buffer (e.g., flowing the third stream and an aqueous buffer into a second mixing structure or dialyzing the aqueous buffer comprising lipid particles to reduce the amount of the second solvent).
  • an aqueous buffer e.g., flowing the third stream and an aqueous buffer into a second mixing structure or dialyzing the aqueous buffer comprising lipid particles to reduce the amount of the second solvent.
  • first solvent is an aqueous buffer and the second solvent is a water-miscible solvent (e.g., an alcohol, such as ethanol).
  • the second solvent is an aqueous alcohol.
  • the second stream include lipid particle-forming materials (e.g., lipids as described above).
  • the second stream comprises a fatty acid triglyceride.
  • the fatty acid triglyceride can be present in the second stream in an amount from about 30 to about 90 mole percent.
  • Suitable fatty acid triglycerides include C8-C20 fatty acid triglycerides.
  • a representative fatty acid triglyceride is an oleic acid triglyceride (triglyceride triolein).
  • the second stream comprises a phospholipid.
  • the phospholipid can be present in the second stream in an amount from about 10 to about 99 mole percent.
  • the phospholipid is a diacylphosphatidylcholine. Suitable phospholipids include those described above, such as C8-C20 fatty acid diacylphosphatidylcholines.
  • a representative phospholipid is 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).
  • the ratio of phospholipid to fatty acid triglyceride is from 20:80 (mol:mol) to 60:40 (mol:mol).
  • the second stream comprises a sterol (e.g., cholesterol).
  • the sterol can be present in the second stream in an amount from about 10 to about 35 mole percent.
  • the sterol is cholesterol.
  • the ratio of phospholipid to cholesterol is 55:45 (mol:mol).
  • the second stream comprises a polyethylene glycol-lipid.
  • Suitable polyethylene glycol-lipids include those described above.
  • the polyethylene glycol-lipid can be present in the second stream in an amount from about 1 to about 10 mole percent.
  • a representative polyethylene glycol-lipid is DSPE-PEG (e.g., DSPE-PEG2000).
  • the method further comprises loading the lipid nanoparticle with a therapeutic and/or diagnostic agent to provide a lipid nanoparticle comprising the therapeutic and/or diagnostic agent.
  • the first or second streams can include the therapeutic and/or diagnostic agent depending on the agent's solubility.
  • the present invention provides microfluidic mixing approaches that at high fluid rate ratios can produce LNP systems of limit size for both aqueous core vesicular systems as well as solid core systems containing a hydrophobic fat such as TO.
  • the present invention provides for the formation of LNP with sizes as small as 20 nm using the staggered herringbone micromixer.
  • LNP self-assembly is driven by interdiffusion of two miscible phases.
  • the presence of the herringbone mixer results in an exponential increase in surface area between the two fluids with distance traveled, resulting in much faster interdiffusion. This allows formation of limit size vesicular and solid core LNP at aqueous buffer-to-alcohol flow rate ratios as low as 3.
  • Limit size vesicles in the size ranges 20-40 nm diameter have previously only been achieved by employing extensive sonication of large multilamellar systems. Sonication has numerous disadvantages including sample degradation and contamination.
  • the microfluidic approach which does not involve appreciable input of energy to disrupt previously formed structures, is considerably gentler and is unlikely to lead to such effects.
  • the production of limit size vesicular LNP can be readily scaled using the microfluidic approach by assembling a number of mixers in parallel.
  • LNP in the size range 10-50 nm offer particular advantages in drug delivery applications, as they are much more able to penetrate to extravascular target tissues than larger systems.
  • a major disadvantage of LNP systems (in the 80-100 nm diameter size range) containing anticancer drugs is that while they are able to extravasate in regions of tumors, there is little penetration into tumor tissue itself.
  • presently available LNP systems can penetrate tissues exhibiting “fenestrated” endothelia, such as the liver, spleen or bone marrow, but have very limited ability to penetrate into other tissues.
  • the limit size systems available through the microfluidic mixing techniques of the present invention have considerable utility for extending the applicability of LNP delivery technology.
  • the present invention provides rapid microfluidic mixing techniques to generate limit size LNP systems using a “bottom up” approach.
  • bottom up refers to methods in which the particles are generated by condensation from solution in response to rapidly increasing polarity of the surrounding medium.
  • LNP were formed using a herringbone continuous flow microfluidic mixing device that achieves chaotic advection to rapidly mix an organic (ethanol) stream which contains the lipids, with an aqueous stream.
  • Representative lipid systems included 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), POPC/cholesterol (Chol) and mixtures of POPC with the triglyceride triolein (TO).
  • Microfluidic Mixing can Produce Limit Size LNP Systems at High Flow Rate Ratios.
  • the present invention provides “limit size” systems that constitute the smallest stable LNP systems that can be made consistent with the physical properties and proportions of the lipid components.
  • LNP were formed by mixing an ethanol stream containing dissolved lipid with an aqueous stream in a microfluidic mixer. It was reasoned that the more rapidly the polarity of the medium experienced by the lipids was increased, the smaller the resulting LNP should become until some limit size was reached. Two factors can influence the rate of increase in polarity: (1) the rate of mixing and (2) the ratio of aqueous to ethanol volumes that are being mixed. The rate of mixing in the herringbone micromixer increases with total flow rate.
  • the first set of experiments was designed to determine whether limit size LNP systems could be formed by increasing the FRR using a herringbone microfluidic mixing device.
  • LNPs were formed by mixing ethanol (containing lipids) and aqueous (154 mM saline) streams where the flow rate of the ethanol was held constant (0.5 ml/min) and the flow rate of the aqueous phase was increased over the range 0.5 ml/min to 4.5 ml/min, corresponding to FRR ranging from 1 to 9. The total flow rate was therefore varied over the range 1 ml/min to 5 ml/min.
  • Three representative lipid systems were investigated.
  • the first two, POPC and POPC/Chol are known to form bilayer vesicles on hydration, whereas the third, mixtures of POPC and triolein (TO), can form “solid core” emulsions with the POPC forming an outer monolayer surrounding a core of the hydrophobic TO.
  • the third, mixtures of POPC and triolein (TO) can form “solid core” emulsions with the POPC forming an outer monolayer surrounding a core of the hydrophobic TO.
  • limit size LNP with a diameter of about 20 nm as assayed by dynamic light scattering (DLS; number mode) are observed for FRR of 3 and higher. These systems were optically clear, consistent with the small size indicated by DLS.
  • DLS dynamic light scattering
  • the limit size would be expected to be sensitive to the POPC/TO ratio, assuming that the POPC lipids form a monolayer around a solid core of TO. Assuming a POPC area per molecule of 0.7 nm 2 , a monolayer thickness of 2 nm and a TO molecular weight of 885.4 and density of 0.91 g/ml, a limit particle size of about 20 nm diameter would require a POPC/TO ratio of 60/40 (mol/mol). As shown in FIG. 2B , for FRR of 5 or greater, LNP systems were obtained with a mean particle size of 20 nm for POPC/TO (60/40; mol/mol) mixtures. These small systems were optically clear.
  • LNP size was highly reproducible (within ⁇ 2 nm) between different experiments. No particle size growth for the POPC/TO nanoemulsions incubated at 20° C. in presence of 25% ethanol for at least 24 h was observed (data not shown). Once dialyzed to remove residual ethanol, the POPC/TO 20 nm systems remained stable for at least several months.
  • 31 P-NMR techniques were used to determine whether some of the phospholipid is sequestered away from the bulk aqueous buffer, which would be consistent with bilayer vesicle structure, or whether all the POPC is in the outer monolayer, which would be consistent with a solid core surrounded by a POPC monolayer. This was straightforward to accomplish because the 31 P-NMR signal arising from the phospholipid in the outer monolayer can be removed by adding Mn 2+ . Mn 2+ acts as a broadening agent that effectively eliminates the 31 P-NMR signal of phospholipid to which it has access.
  • FIGS. 3A-3C illustrate the 31 P NMR spectra obtained for POPC ( FIG. 3A ), POPC/Chol, 55/45 mol/mol ( FIG. 3B ) and POPC/TO, 60/40 mol/mol ( FIG. 3C ) LNP systems in the absence and presence of 2 mM Mn 2+ .
  • the buffer contains no Mn 2+ , a sharp “isotropic” peak is observed in all three preparations (upper panels), consistent with rapid isotropic motional averaging effects due to vesicle tumbling and lipid lateral diffusion effects.
  • the addition of Mn 2+ reduces the signal intensity to levels 50% of the initial signal for the POPC and POPC/Chol systems ( FIGS.
  • the ratio of the lipid on the outside of the vesicle to the lipid on the inside can be used to determine the vesicle size if the bilayer thickness and area per lipid molecule is known.
  • the Ro/i for the POPC and POPC/Chol system was calculated from FIGS. 3A and 3B and found to be 1.7 and 1.35, respectively, corresponding to sizes of approximately 30 nm and 50 nm diameter, respectively, assuming a bilayer thickness of 3.5 nm. These values are larger than determined by DLS, which could arise due to increased packing density in the inner monolayer and/or the presence of a small proportion of multi-lamellar vesicles.
  • the limit size of LNP produced from POPC/TO mixtures should be dependent on the molar ratios of phospholipid to triglyceride.
  • the molar ratios required to form LNP of diameter 20, 40, 60, and 80 nm were calculated and used to produce LNP systems whose size was measured by DLS.
  • FIG. 5 shows the decrease of the mean diameter of the POPC/TO LNPs as a function of POPC/TO molar ratio, compared with the curve that represents the theoretical values.
  • Table 1 provides a direct comparison between predicted and DLS-estimated sizes of POPC/TO LNP. Good correspondence is seen between the predicted size based on the POPC/TO ratio and the actual size.
  • Doxorubicin can be Loaded and Retained in Limit Size Vesicular LNP.
  • therapeutic drugs and diagnostic agents can be loaded and retained in limit size vesicular LNP systems.
  • the low trapped volumes of such systems would be expected to limit encapsulation of solutes (such as ammonium sulphate) that can be used to drive the pH gradients (inside acidic) that lead to accumulation of weak base drugs such as doxorubicin.
  • solutes such as ammonium sulphate
  • doxorubicin a widely used anti-neoplastic agent
  • LNP systems composed of POPC containing ammonium sulfate were prepared as below. No significant change in size compared to POPC systems prepared in saline was observed.
  • the ammonium sulfate-containing LNP were incubated at 60° C. in presence of the varying amounts of doxorubicin (initial drug/lipid (D/L) ratios were set at 0.05, 0.1 and 0.2 mol/mol). In all cases, drug loading efficacies approaching 100% were achieved within 30 min (data not shown). DLS analyses of the loaded samples showed no particle size increase compared to the empty precursors (about 20 nm).
  • FIGS. 6A and 6B Representative images from the cryo-TEM studies are shown in FIGS. 6A and 6B .
  • Previous cryo-TEM studies of liposomal doxorubicin formulations have demonstrated the existence of linear precipitates of encapsulated drug resulting in a “coffee bean” shaped liposomal morphology.
  • LNPs loaded at D/L 0.1 exhibit a similar appearance, indicating the drug precipitation pattern similar to that observed in the 100 nm systems ( FIG. 6A ).
  • the ability of the loaded LNP to stably retain the encapsulated drug may be a concern.
  • the stability of the doxorubicin-loaded LNP stored at 4° C. was monitored for the period of 8 weeks. No detectable drug release/particle size change was observed.
  • 90% (0.1 mol/mol systems) and 75% (0.2 mol/mol systems) of the loaded drug remained encapsulated at 24 h time point (data not shown).
  • the presence of a sterol and a polyethylene glycol-lipid in the lipid nanoparticle improved the size characteristics of the nanoparticle (e.g., maintained advantageous particle size of from about 15 to about 35 nm.
  • phosphate buffered saline pH 7.4
  • lipid DSPC/Chol with or without DSPE-PEG2000
  • the total flow rate was 4 mL/min, the FRR was 5:1 (3.33 mL/min aqueous, 0.66 mL/min ethanol), and the initial concentration of lipid in ethanol was 20 mM.
  • the product was dialyzed overnight in phosphate buffered saline at pH 7.4 and the concentrated by Amicon Ultra Centrifugation units (10K MWCO). The results are presented in Tables 2 and 3.
  • FIG. 15 compares particle size (nm) as a function of mole percent cholesterol (Chol) in a representative lipid bilayer nanoparticle of the invention.
  • the presence of 3% mol DSPE-PEG2000 allows the size of POPC/Chol/PEG systems to be maintained up to 35% mol Chol). Size was measured by DLS, number weighting.
  • the lipid nanoparticles of the invention can be advantageously loaded with therapeutic agents.
  • doxorubicin DOX
  • POPC/PEG systems Chol-free
  • Drug loading efficacies approaching 100% were achieved within 30 min.
  • Chol-containing systems POPC/Chol/PEG
  • loading of doxorubicin into POPC/Chol/PEG systems was performed at 37° C. (3 h, D/L 0.1 mol/mol).
  • 3% PEG were included into formulation at the formation stage, additional 3.5% were post-inserted prior to loading.
  • the presence of cholesterol in the system resulted in improvement of doxorubicin retention (both in vitro and in vivo).
  • FIG. 16 compares the results of an in vivo pharmacokinetic (PK) study evaluating of retention properties of POPC and POPC/Chol (7:3) DOX loaded systems both containing 6.5% DSPE-PEG2000 (PEG) in plasma of CD-1 mice. The results shows that the system including the polyethylene glycol lipid demonstrates enhanced retention.
  • PK in vivo pharmacokinetic
  • FIG. 17 compares the results of an in vitro release study performed in presence of 50% FBS.
  • the systems evaluated were POPC/PEG (D/L 0.1), POPC/Chol/PEG 70/30/6.5 (D/L 0.1), POPC/Chol/PEG 70/30/6.5 (D/L 0.15), POPC/Chol/PEG 65/35/6.5 (D/L 0.1), and POPC/Chol/PEG 65/35/6.5 (D/L 0.15).
  • the results demonstrate that increasing of the Chol content from 30% to 35% provides increased DOX retention and that increasing D/L ratio to 0.15 mol/mol did not lead to any improvement of drug retention.
  • the invention provides devices and systems for making limit size nanoparticles.
  • the device includes:
  • a third microchannel ( 110 ) for receiving the first and second streams wherein the third microchannel has a first region ( 112 ) adapted for flowing the first and second streams and a second region ( 114 ) adapted for mixing the contents of the first and second streams to provide a third stream comprising limit size lipid nanoparticles.
  • the lipid nanoparticles so formed are conducted from the second (mixing) region by microchannel 116 to outlet 118 .
  • the second region of the microchannel comprises bas-relief structures.
  • the second region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction.
  • the second region includes a micromixer.
  • means for varying the flow rates of the first and second streams are used to rapidly mix the streams thereby providing the limit size nanoparticles.
  • one or more of the microchannels have a hydraulic diameter from about 20 to about 300 ⁇ m.
  • the devices of the invention provide complete mixing occurs in less than 10 ms.
  • the device is a parallel microfluidic structure.
  • one or more regions of the device are heated.
  • the invention provides devices that include more than one fluidic mixing structures (i.e., an array of fluidic structures).
  • the invention provides a single device (i.e., an array) that includes from 2 to about 40 parallel fluidic mixing structures capable of producing lipid nanoparticles at a rate of about 2 to about 1600 mL/min.
  • the devices produce from 2 to about 20,000 mL without a change in lipid nanoparticle properties.
  • the device for producing lipid nanoparticles includes:
  • a plurality of microchannels for receiving the first and second streams, wherein each has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a plurality of streams compromising lipid nanoparticles;
  • each of the plurality of microchannels for receiving the first and second streams includes:
  • a third microchannel for receiving the first and second streams, wherein each has a first region adapted for flowing the first and second streams and a second region adapted for mixing the contents of the first and second streams to provide a plurality of streams compromising lipid nanoparticles.
  • the device includes from 2 to about 40 microchannels for receiving the first and second streams. In these embodiments, the device has a total flow rate from 2 to about 1600 mL/min.
  • the second regions each have a hydraulic diameter of from about 20 to about 300 ⁇ m. In certain embodiments, the second regions each have a fluid flow rate of from 1 to about 40 mL/min.
  • the heating element is effective to increase the temperature of the first and second streams in the first and second microchannels to a pre-determined temperature prior to their entering the third microchannel.
  • the inlet fluids are heated to a desired temperature and mixing occurs sufficiently rapidly such that the fluid temperature does not change appreciably prior to lipid nanoparticle formation.
  • the invention provides a system for making limit size nanoparticles that includes a parallel microfluidic structure.
  • N single mixers are arrayed such that a total flow rate of N ⁇ F is achieved, where F is the flow rate used in the non-parallelized implementation.
  • Representative parallel microfluidic structures of the invention are illustrated schematically in FIGS. 7A-7C .
  • FIG. 7A A perspective view of a representative parallel microfluidic structure is illustrated in FIG. 7A and a plan view is illustrated in FIG. 7B .
  • device 200 includes three fluidic systems ( 100 a , 100 b , and 100 c ) arranged vertically with each system including one first solvent inlet ( 202 ), two second solvent inlets ( 206 and 206 ′), two mixing regions ( 110 and 110 ′), and a single outlet ( 208 ).
  • Each system includes microchannels for receiving the first and second streams ( 202 and 206 and 206 ,′ respectively).
  • each fluidic system includes:
  • microchannel 116 a conducts one of the plurality of streams from the mixing region to fourth microchannel 208 via outlet 118 a that conducts the lipid nanoparticles from the device.
  • fluidic system 100 a includes a second second solvent inlet ( 206 ′) and mixing region ( 110 a ′) with components denoted by reference numerals 102 a ′, 104 a ′, 106 a ′, 108 a ′, 112 a ′, 114 a ′, 116 a ′ and 118 a ′.
  • reference numerals 102 a ′, 104 a ′, 106 a ′, 108 a ′, 112 a ′, 114 a ′, 116 a ′ and 118 a ′ correspond to their non-primed counterparts ( 102 , 104 , 106 , 108 , 112 , 114 , 116 , and 118 ) in FIG. 7B .
  • This structure produces vesicles at higher flow rates compared to the single mixer chips and produces vesicles identical to those produced by single mixer chips.
  • six mixers are integrated using three reagent inlets. This is achieved using both planar parallelization and vertical parallelization as shown in FIGS. 7A and 7B .
  • Planar parallelization refers to placing one or more mixers on the same horizontal plane. These mixers may or may not be connected by a fluidic bus channel. Equal flow through each mixer is assured by creating identical fluidic paths between the inlets and outlets, or effectively equal flow is achieved by connecting inlets and outlets using a low impedance bus channel as shown in FIG. 7C (a channel having a fluidic impedance significantly lower than that of the mixers).
  • FIG. 7C illustrates device 300 includes five fluidic systems ( 100 a , 100 b , 100 c , 100 d , and 100 e ) arranged horizontally with each system including one first solvent inlet, one second solvent inlet, one mixing region, and a single outlet ( 208 ).
  • Device 300 includes microchannels for receiving the first and second streams ( 202 and 206 ) and a microchannel ( 208 ) for conducting lipid nanoparticles produced in the device from the device.
  • fluidic system 100 a includes:
  • a first microchannel ( 202 ) (with inlet 203 ) in fluidic communication via first inlet ( 102 a ) with a first inlet microchannel ( 104 a ) to receive the first stream comprising the first solvent;
  • microchannel 116 a conducts the third stream from the mixing region to fourth microchannel 208 via outlet 118 a .
  • Microchannel 208 conducts the lipid nanoparticles from the device via outlet 209 .
  • fluidic system 100 a includes a second second solvent inlet ( 206 ′) and mixing region ( 110 a ′) with components denoted by reference numerals 102 a ′, 104 a ′, 106 a ′, 108 a ′, 112 a ′, 114 a ′, 116 a ′ and 118 a ′.
  • reference numerals 102 a ′, 104 a ′, 106 a ′, 108 a ′, 112 a ′, 114 a ′, 116 a ′ and 118 a ′ correspond to their non-primed counterparts ( 102 , 104 , 106 , 108 , 112 , 114 , 116 , and 118 ) in FIG. 7B .
  • the invention provides a device for producing limit size lipid nanoparticles, comprising n fluidic devices, each fluidic device comprising:
  • a third microchannel ( 110 a ) for receiving the first and second streams wherein the third microchannel has a first region ( 112 a ) adapted for flowing the first and second streams and a second region ( 114 a ) adapted for mixing the contents of the first and second streams to provide a third stream comprising limit size lipid nanoparticles conducted from the mixing region by microchannel 116 a,
  • first inlets ( 102 a - 102 n ) of each fluidic device ( 100 a - 100 n ) are in liquid communication through a first bus channel ( 202 ) that provides the first solution to each of the first inlets,
  • each fluidic device ( 100 a - 100 n ) are in liquid communication through a second bus channel ( 206 ) that provides the second solution to each of the second inlets, and
  • each fluidic device ( 100 a - 100 n ) are in liquid communication through a third bus channel ( 208 ) that conducts the third stream from the device.
  • the reference numerals refer to representative device 300 in FIG. 7C .
  • n is an integer from 2 to 40.
  • Parallelized devices are formed by first creating positive molds of planar parallelized mixers that have one or more microfluidic mixers connected in parallel by a planar bus channel. These molds are then used to cast, emboss or otherwise form layers of planar parallelized mixers, one of more layers of which can then be stacked, bonded and connected using a vertical bus channels. In certain implementations, planar mixers and buses may be formed from two separate molds prior to stacking vertically (if desired). In one embodiment positive molds of the 2 ⁇ planar structure on a silicon wafer are created using standard lithography. A thick layer of on-ratio PDMS is then poured over the mold, degassed, and cured at 80 C for 25 minutes.
  • the cured PDMS is then peeled off, and then a second layer of 10:1 PDMS is spun on the wafer at 500 rpm for 60 seconds and then baked at 80° C. for 25 minutes. After baking, both layers are exposed to oxygen plasma and then aligned. The aligned chips are then baked at 80° C. for 15 minutes. This process is then repeated to form the desired number of layers. Alignment can be facilitated by dicing the chips and aligning each individually and also by making individual wafers for each layer which account for the shrinkage of the polymer during curing.
  • this chip has been interfaced to pumps using standard threaded connectors (see FIG. 9 ). This has allowed flow rates as high as 72 ml/min to be achieved. Previously, in single element mixers, flows about 10 ml/min were unreliable as often pins would leak eject from the chip.
  • chips are sealed to on the back side to glass, and the top side to a custom cut piece of polycarbonate or glass with the interface holes pre-drilled.
  • the PC to PDMS bond is achieved using a silane treatment. The hard surface is required to form a reliable seal with the o-rings. A glass backing is maintained for sealing the mixers as the silane chemistry has been shown to affect the formation of the nanoparticles.
  • the devices and systems of the invention provide for the scalable production of limit size nanoparticles.
  • the following results demonstrate the ability to produce identical vesicles, as suggested by identical mean diameter, using the microfluidic mixer illustrated in FIGS. 7A and 7B .
  • Mean vesicle diameter (nm) for scale-up formulation of representative limit size nanoparticles (DSPC/Cholesterol vesicles) produced using the parallel microfluidic structure is compared to those produced using a single mixer microfluidic device in FIG. 11 (final lipid concentration was 3 mg/mL).
  • Formulation of DSPC/Cholesterol vesicles is made using a 130 ⁇ 300 ⁇ m mixer (channel cross-section) by mixing at a buffer: lipid-ethanol volumetric flow rate ratio of 3:1, with a total flow rate of 12 ml/min in a single microfluidic mixer.
  • the scale-up mixer which enables throughput of 72 ml/min (6 ⁇ scaling), consists of 6 original mixers where three sets of two mixers are stacked vertically and placed next to each other horizontally.
  • Mean vesicle diameter (nm) for scale-up formulation of representative limit size nanoparticles (DSPC/Cholesterol vesicles) produced using the parallel microfluidic structure is compared to those produced using a single mixer microfluidic device in FIG. 11 (final lipid concentration was 3 mg/mL).
  • Formulation of DSPC/Cholesterol vesicles is made using a 130 ⁇ 300 ⁇ m mixer (channel cross-section) by mixing at a buffer: lipid-ethanol volumetric flow rate ratio of 3:1, with a total flow rate of 12 ml/min in a single microfluidic mixer.
  • the scale-up mixer which enables throughput of 72 ml/min (6 ⁇ scaling), consists of 6 original mixers where two sets of three mixers are stacked vertically and placed next to each other horizontally.
  • the invention provides temperature-controlled fluidic structures for making limit size lipid nanoparticle.
  • the solution can be rapidly heated when the streams are flowed through a chamber with a high surface area (heater area) to volume ratio.
  • COMSOL simulations showed that the solution can be heated by flowing through 10 mm ⁇ 10 mm ⁇ 100 um chamber at a flow rate of 1 mL/min. The simulation showed that the solution heats up in the first fifth of the chamber so the flow rate could probably increased to 5 mL/min.
  • FIGS. 12-14 Representative temperature-controlled fluidic structures are illustrated in FIGS. 12-14 .
  • 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine was obtained from Avanti Polar Lipids (Alabaster, Ala.). 1,2,3-Tri(cis-9-octadecenoyl) glycerol (glyceryl trioleate, TO), cholesterol (Chol), sodium chloride, ammonium sulfate, and doxorubicin hydrochloride were from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada).
  • the micromixer was a chaotic mixer for continuous flow systems with the layout based on patterns of asymmetric grooves on the floor of the channel (staggered herringbone design) that induce a repeated sequence of rotational and extensional local flows thus inducing rapid mixing of the injected streams.
  • the device was produced by soft lithography, the replica molding of microfabricated masters in elastomer.
  • the device features a 200 ⁇ m wide and 79 ⁇ m high mixing channel with herringbone structures formed by 31 ⁇ m high and 50 ⁇ m thick features on the roof of the channel (see FIG. 1 ).
  • Fluidic connections were made with 1/32′′ I.D., 3/32′′ O.D. tubing that was attached to 21G1 needles for connection with syringes. 1 ml, 3 ml, and 5 ml syringes were generally used for inlet streams.
  • a dual syringe pump (KD200, KD Scientific) was used to control the flow rate through the device.
  • Lipids POPC or POPC/Chol (55/45 molar ratio) for preparations of liposomal systems, POPC/TO at different ratios for preparations of nanoemulsions were dissolved in ethanol at 10 mg/ml of total lipid.
  • the LNP were prepared by injecting an ethanolic lipid mixture into the first inlet and an aqueous hydration solution (saline, 154 mM NaCl) into the second inlet of the mixing channel of the micromixer (see FIG. 1 ).
  • FRR flow rate ratio of aqueous stream volumetric flow rate to ethanolic volumetric flow rate
  • Limit size vesicular POPC LNP containing ammonium sulfate were formed as described above except that saline was replaced with 300 mM ammonium sulfate solution (FRR 3, 10 mg/ml POPC in ethanolic solution). After formation, the LNP were dialyzed against 300 mM ammonium sulfate and concentrated to 10 mg/ml with the use of the Amicon Ultra-15 centrifugal filter units (Millipore). An ammonium sulfate gradient was generated by exchanging the extravesicular solution with 154 mM NaCl, pH 7.4 on Sephadex G-50 spin columns.
  • Doxorubicin hydrochloride was dissolved in saline at 5 mg/ml and added to the ammonium sulfate-containing LNP to give molar drug-to-lipid ratios of 0.05, 0.1, and 0.2. The samples were then incubated at 60° C. for 30 min to provide optimal loading conditions. Unentrapped doxorubicin was removed by running the samples over Sephadex G-50 spin columns prior to detection of entrapped drug.
  • Doxorubicin was assayed by fluorescence intensity (excitation and emission wavelengths 480 and 590 nm, respectively) with a Perkin-Elmer LS50 fluorimeter (Perkin-Elmer, Norwalk, Conn.), the value for 100% release was obtained by addition of 10% Triton X-100 to a final concentration of 0.5%.
  • Phospholipid concentrations were determined by an enzymatic colorimetric method employing a standard assay kit (Wako Chemicals, Richmond, Va.). Loading efficiencies were determined by quantitating both drug and lipid levels before and after separation of external drug from LNP encapsulated drug by size exclusion chromatography using Sephadex G-50 spin columns and comparing the respective drug/lipid ratios.
  • LNP were diluted to appropriate concentration with saline and mean particle size (number-weighted) was determined by dynamic light scattering (DLS) using a NICOMP model 370 submicron particle sizer (Particle Sizing Systems, Santa Barbara, Calif.). The sizer was operating in the vesicle and solid particle modes to determine the size of the liposomes (POPC and POPC/Chol systems) and lipid core nanospheres (POPC/TO systems), respectively.
  • DLS dynamic light scattering
  • Proton decoupled 31 P-NMR spectra were obtained using a Bruker AVII 400 spectrometer operating at 162 MHz. Free induction decays (FID) corresponding to about 10,000 scans were obtained with a 15 ⁇ s, 55-degree pulse with a 1 s interpulse delay and a spectral width of 64 kHz. An exponential multiplication corresponding to 50 Hz of line broadening was applied to the FID prior to Fourier transformation. The sample temperature was regulated using a Bruker BVT 3200 temperature unit. Measurements were performed at 25° C.
  • Samples were prepared by applying 3 ⁇ L of PBS containing LNP at 20-40 mg/ml total lipid to a standard electron microscopy grid with a perforated carbon film. Excess liquid was removed by blotting with a Vitrobot system (FEI, Hillsboro, Oreg.) and then plunge-freezing the LNP suspension in liquid ethane to rapidly freeze the vesicles in a thin film of amorphous ice. Images were taken under cryogenic conditions at a magnification of 29K with an AMT HR CCD side mount camera. Samples were loaded with a Gatan 70 degree cryo-transfer holder in an FEI G20 Lab6 200 kV TEM under low dose conditions with an underfocus of 5-8 ⁇ m to enhance image contrast.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Clinical Laboratory Science (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pain & Pain Management (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cardiology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Otolaryngology (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychiatry (AREA)
  • Anesthesiology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)
  • Micromachines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
US14/353,460 2011-10-25 2012-10-25 Limit size lipid nanoparticles and related methods Abandoned US20140328759A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/353,460 US20140328759A1 (en) 2011-10-25 2012-10-25 Limit size lipid nanoparticles and related methods

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201161551366P 2011-10-25 2011-10-25
PCT/CA2012/000991 WO2013059922A1 (en) 2011-10-25 2012-10-25 Limit size lipid nanoparticles and related methods
US14/353,460 US20140328759A1 (en) 2011-10-25 2012-10-25 Limit size lipid nanoparticles and related methods

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2012/000991 A-371-Of-International WO2013059922A1 (en) 2011-10-25 2012-10-25 Limit size lipid nanoparticles and related methods

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US15/087,721 Continuation US9943846B2 (en) 2011-10-25 2016-03-31 Limit size lipid nanoparticles and related methods

Publications (1)

Publication Number Publication Date
US20140328759A1 true US20140328759A1 (en) 2014-11-06

Family

ID=48166990

Family Applications (5)

Application Number Title Priority Date Filing Date
US14/353,460 Abandoned US20140328759A1 (en) 2011-10-25 2012-10-25 Limit size lipid nanoparticles and related methods
US15/087,721 Active US9943846B2 (en) 2011-10-25 2016-03-31 Limit size lipid nanoparticles and related methods
US15/927,925 Active 2033-03-24 US10843194B2 (en) 2011-10-25 2018-03-21 Microfluidic mixing devices and systems
US17/061,247 Active 2033-01-22 US11648556B2 (en) 2011-10-25 2020-10-01 Limit size lipid nanoparticles and related methods
US18/296,954 Pending US20230256436A1 (en) 2011-10-25 2023-04-06 Limit size lipid nanoparticles and related methods

Family Applications After (4)

Application Number Title Priority Date Filing Date
US15/087,721 Active US9943846B2 (en) 2011-10-25 2016-03-31 Limit size lipid nanoparticles and related methods
US15/927,925 Active 2033-03-24 US10843194B2 (en) 2011-10-25 2018-03-21 Microfluidic mixing devices and systems
US17/061,247 Active 2033-01-22 US11648556B2 (en) 2011-10-25 2020-10-01 Limit size lipid nanoparticles and related methods
US18/296,954 Pending US20230256436A1 (en) 2011-10-25 2023-04-06 Limit size lipid nanoparticles and related methods

Country Status (5)

Country Link
US (5) US20140328759A1 (ja)
EP (3) EP3915545A1 (ja)
JP (6) JP2015502337A (ja)
CA (1) CA2853316C (ja)
WO (1) WO2013059922A1 (ja)

Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2017184786A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Cpf1 complexes with reduced indel activity
WO2017184768A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017189308A1 (en) 2016-04-19 2017-11-02 The Broad Institute Inc. Novel crispr enzymes and systems
WO2018006166A1 (en) 2016-07-06 2018-01-11 Precision Nanosystems Inc Smart microfluidic mixing instrument and cartridges
WO2018035387A1 (en) 2016-08-17 2018-02-22 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2018035388A1 (en) 2016-08-17 2018-02-22 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2018071549A1 (en) * 2016-10-11 2018-04-19 New York University Nanoparticles and uses thereof
WO2018191750A2 (en) 2017-04-14 2018-10-18 The Broad Institute Inc. Novel delivery of large payloads
WO2019094983A1 (en) 2017-11-13 2019-05-16 The Broad Institute, Inc. Methods and compositions for treating cancer by targeting the clec2d-klrb1 pathway
WO2020186213A1 (en) 2019-03-14 2020-09-17 The Broad Institute, Inc. Novel nucleic acid modifiers
WO2020191102A1 (en) 2019-03-18 2020-09-24 The Broad Institute, Inc. Type vii crispr proteins and systems
WO2020236972A2 (en) 2019-05-20 2020-11-26 The Broad Institute, Inc. Non-class i multi-component nucleic acid targeting systems
CN114247398A (zh) * 2021-12-17 2022-03-29 沈磊 一种脂质纳米颗粒制备系统及设备
US11447527B2 (en) 2018-09-18 2022-09-20 Vnv Newco Inc. Endogenous Gag-based capsids and uses thereof
WO2022224595A1 (en) * 2021-04-22 2022-10-27 Kabushiki Kaisha Toshiba Flow channel structure, method for agitating fluid and method for manufacturing lipid particles
US11572575B2 (en) 2016-10-03 2023-02-07 Precision NanoSystems ULC Compositions for transfecting resistant cell types
US11707436B2 (en) 2014-12-15 2023-07-25 Nanosphere Health Sciences Inc. Methods of treating inflammatory disorders and global inflammation with compositions comprising phospholipid nanoparticle encapsulations of NSAIDS
WO2023196818A1 (en) 2022-04-04 2023-10-12 The Regents Of The University Of California Genetic complementation compositions and methods

Families Citing this family (64)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012019168A2 (en) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
CA2821992A1 (en) 2010-10-01 2012-04-05 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
DE12722942T1 (de) 2011-03-31 2021-09-30 Modernatx, Inc. Freisetzung und formulierung von manipulierten nukleinsäuren
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
EP3492109B1 (en) 2011-10-03 2020-03-04 ModernaTX, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
WO2013059922A1 (en) * 2011-10-25 2013-05-02 The University Of British Columbia Limit size lipid nanoparticles and related methods
JP2015501844A (ja) 2011-12-16 2015-01-19 モデルナ セラピューティクス インコーポレイテッドＭｏｄｅｒｎａ Ｔｈｅｒａｐｅｕｔｉｃｓ，Ｉｎｃ． 修飾ヌクレオシド、ヌクレオチドおよび核酸組成物
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
EP2833923A4 (en) 2012-04-02 2016-02-24 Moderna Therapeutics Inc MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF PROTEINS
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
LT2922554T (lt) 2012-11-26 2022-06-27 Modernatx, Inc. Terminaliai modifikuota rnr
EP2971010B1 (en) 2013-03-14 2020-06-10 ModernaTX, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2015013596A2 (en) * 2013-07-26 2015-01-29 The University Of British Columbia Method and device for manufacturing polymer particles containing a therapeutic material
WO2015034925A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Circular polynucleotides
EP3041934A1 (en) 2013-09-03 2016-07-13 Moderna Therapeutics, Inc. Chimeric polynucleotides
EP3043781A1 (en) * 2013-09-13 2016-07-20 Arbor Therapeutics, LLC Nanoparticulate compositions for targeted delivery of lipophilic drugs and acid labile, lipophilic prodrugs of cancer chemotherapeutics and their preparation
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
EP3052521A1 (en) 2013-10-03 2016-08-10 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
WO2016011226A1 (en) 2014-07-16 2016-01-21 Moderna Therapeutics, Inc. Chimeric polynucleotides
EP3171895A1 (en) 2014-07-23 2017-05-31 Modernatx, Inc. Modified polynucleotides for the production of intrabodies
CN107533076A (zh) * 2015-02-24 2018-01-02 不列颠哥伦比亚大学 连续流微流体系统
US10632072B2 (en) * 2015-03-19 2020-04-28 University Of Connecticut Systems and methods for continuous manufacturing of liposomal drug formulations
WO2016166771A1 (en) * 2015-04-13 2016-10-20 Council Of Scientific & Industrial Research Continuous micro mixer
HUE057613T2 (hu) 2015-09-17 2022-05-28 Modernatx Inc Vegyületek és készítmények terápiás szerek intracelluláris bejuttatására
JP6661319B2 (ja) * 2015-09-28 2020-03-11 小林製薬株式会社 リポソーム
HUE059127T2 (hu) 2015-10-22 2022-10-28 Modernatx Inc Légúti vírusok elleni vakcinák
PL3386484T3 (pl) 2015-12-10 2022-07-25 Modernatx, Inc. Kompozycje i sposoby dostarczania środków terapeutycznych
DE102015226018A1 (de) * 2015-12-18 2017-06-22 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Kontinuierliches Verfahren zur Herstellung von vesikulären oder scheibenförmigen, supramolekularen Nanopartikeln, und Verwendungen hiervon
DK3394030T3 (da) 2015-12-22 2022-03-28 Modernatx Inc Forbindelser og sammensætninger til intracellulær afgivelse af midler
DK3394093T3 (da) 2015-12-23 2022-04-19 Modernatx Inc Fremgangsmåder til anvendelse af ox40-ligand-kodende polynukleotider
EP3400097B1 (en) 2016-01-06 2021-01-27 The University Of British Columbia Bifurcating mixers and methods of their use and manufacture
EP3538067A1 (en) 2016-11-08 2019-09-18 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
FI3596041T3 (fi) 2017-03-15 2023-01-31 Yhdiste ja koostumuksia terapeuttisten aineiden antamiseen solun sisään
US11969506B2 (en) 2017-03-15 2024-04-30 Modernatx, Inc. Lipid nanoparticle formulation
CA3056133A1 (en) 2017-03-15 2018-09-20 Modernatx, Inc. Crystal forms of amino lipids
AU2018246024B2 (en) 2017-03-31 2020-08-06 Fujifilm Corporation Liposome composition and pharmaceutical composition
EP3610943B1 (en) * 2017-04-13 2023-05-31 National University Corporation Hokkaido University Flow channel structure and lipid particle or micelle formation method using same
US12016919B2 (en) 2017-05-30 2024-06-25 Glaxosmithkline Biologicals Sa Methods for manufacturing an adjuvant
WO2018232120A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
US11786607B2 (en) 2017-06-15 2023-10-17 Modernatx, Inc. RNA formulations
EP3417931A1 (en) * 2017-06-22 2018-12-26 Leibniz - Institut für Analytische Wissenschaften - ISAS - E.V. Microfluidic gradient generator
WO2019046809A1 (en) 2017-08-31 2019-03-07 Modernatx, Inc. METHODS OF MANUFACTURING LIPID NANOPARTICLES
JP7379776B2 (ja) 2018-04-29 2023-11-15 プレシジョン ナノシステムズ ユーエルシー 耐性細胞型をトランスフェクトするための組成物
CN112312895B (zh) * 2018-06-20 2023-05-09 富士胶片株式会社 包含内含药物的脂质体组合物及免疫检查点抑制剂的组合医药
EP3852728B1 (en) 2018-09-20 2024-09-18 ModernaTX, Inc. Preparation of lipid nanoparticles and methods of administration thereof
US11066355B2 (en) 2019-09-19 2021-07-20 Modernatx, Inc. Branched tail lipid compounds and compositions for intracellular delivery of therapeutic agents
AU2020355000A1 (en) 2019-09-23 2022-03-17 Omega Therapeutics, Inc. Compositions and methods for modulating apolipoprotein B (APOB) gene expression
AU2020352552A1 (en) 2019-09-23 2022-03-17 Omega Therapeutics, Inc. Compositions and methods for modulating hepatocyte nuclear factor 4-alpha (HNF4α) gene expression
JP2023517326A (ja) 2020-03-11 2023-04-25 オメガ セラピューティクス， インコーポレイテッド フォークヘッドボックスｐ３（ｆｏｘｐ３）遺伝子発現をモジュレートするための組成物および方法
FI20215508A1 (en) 2020-04-09 2021-10-10 Niemelae Erik Johan Mimetic nanoparticles to prevent the spread of new coronaviruses and reduce the rate of infection
EP3915673A1 (de) * 2020-05-25 2021-12-01 Leon-Nanodrugs GmbH Verfahren zur überprüfung der funktionsfähigkeit einer anlage zur herstellung von nanopartikeln durch gezielte fällung aus übersättigten lösungen
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
WO2023283359A2 (en) 2021-07-07 2023-01-12 Omega Therapeutics, Inc. Compositions and methods for modulating secreted frizzled receptor protein 1 (sfrp1) gene expression
JPWO2023153404A1 (ja) * 2022-02-08 2023-08-17
CN114534652B (zh) 2022-02-08 2024-07-19 上海天泽云泰生物医药有限公司 波形微结构混合单元及其用途
WO2023161350A1 (en) 2022-02-24 2023-08-31 Io Biotech Aps Nucleotide delivery of cancer therapy
WO2023183369A2 (en) * 2022-03-22 2023-09-28 Respirerx Pharmaceuticals, Inc. Lipid nanoparticle compositions and methods for formulating insoluble drugs
WO2024006863A1 (en) 2022-06-30 2024-01-04 Precision NanoSystems ULC Lipid nanoparticle formulations for vaccines
GB202216449D0 (en) 2022-11-04 2022-12-21 Io Biotech Aps TGF-BETA1 vaccine
WO2024126423A1 (en) 2022-12-12 2024-06-20 Precision NanoSystems ULC Lipid nanoparticles lyophilization methods and compositions
CN116374973B (zh) * 2023-02-08 2024-09-20 浙江大学 一种双层油酸法制备水相均匀分散的羟基磷灰石的方法
CN116098985A (zh) * 2023-02-27 2023-05-12 广西大学 纳米级热带水果淀粉-内源性蛋白质-内源性脂质三元复合物的制备方法及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2123260A1 (en) * 2006-12-29 2009-11-25 Shijiazhuang Pharma. Group Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. Liposome formulation and process for preparation thereof

Family Cites Families (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981501A (en) 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
EP0907412B1 (en) 1996-06-28 2008-08-27 Caliper Life Sciences, Inc. High-throughput screening assay systems in microscale fluidic devices
WO1998033585A1 (en) 1997-02-05 1998-08-06 California Institute Of Technology Microfluidic sub-millisecond mixers
EP1203614A1 (de) * 2000-11-03 2002-05-08 Polymun Scientific Immunbiologische Forschung GmbH Verfahren und Vorrichtung zur Herstellung von Lipidvesikeln
US8137699B2 (en) 2002-03-29 2012-03-20 Trustees Of Princeton University Process and apparatuses for preparing nanoparticle compositions with amphiphilic copolymers and their use
EP1412065A2 (en) 2001-07-27 2004-04-28 President And Fellows Of Harvard College Laminar mixing apparatus and methods
US7252928B1 (en) 2002-03-12 2007-08-07 Caliper Life Sciences, Inc. Methods for prevention of surface adsorption of biological materials to capillary walls in microchannels
US7901939B2 (en) 2002-05-09 2011-03-08 University Of Chicago Method for performing crystallization and reactions in pressure-driven fluid plugs
EP3100719A3 (en) 2002-05-15 2017-02-22 California Pacific Medical Center Delivery of nucleic acid-like compounds
JP2006507921A (ja) 2002-06-28 2006-03-09 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ 流体分散のための方法および装置
DK1519714T3 (da) 2002-06-28 2011-01-31 Protiva Biotherapeutics Inc Fremgangsmåde og apparat til fremstilling af liposomer
US7214348B2 (en) 2002-07-26 2007-05-08 Applera Corporation Microfluidic size-exclusion devices, systems, and methods
GB2395196B (en) * 2002-11-14 2006-12-27 Univ Cardiff Microfluidic device and methods for construction and application
CA2520864A1 (en) 2003-03-31 2004-10-21 Alza Corporation Lipid particles having asymmetric lipid coating and method of preparing same
US7160025B2 (en) 2003-06-11 2007-01-09 Agency For Science, Technology And Research Micromixer apparatus and methods of using same
JP4533382B2 (ja) 2003-08-28 2010-09-01 セルラ・インコーポレイテッド マイクロ流体分析およびソーティング用の一体化された構造物
US20050175683A1 (en) 2003-10-24 2005-08-11 Yuanpeng Zhang Preparation of lipid particles
EP1537858A1 (en) 2003-12-04 2005-06-08 Vectron Therapeutics AG Drug delivery vehicles and uses thereof
US7507380B2 (en) 2004-03-19 2009-03-24 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Microchemical nanofactories
EP1750673B1 (en) 2004-05-17 2009-12-02 Tekmira Pharmaceuticals Corporation Liposomal formulations comprising dihydrosphingomyelin and methods of use thereof
ATE537263T1 (de) 2004-06-07 2011-12-15 Protiva Biotherapeutics Inc Kationische lipide und verwendungsverfahren
US7871632B2 (en) * 2004-07-12 2011-01-18 Adventrx Pharmaceuticals, Inc. Compositions for delivering highly water soluble drugs
JP4882307B2 (ja) 2004-08-20 2012-02-22 東ソー株式会社 複合粒子の製造方法
EP1793804A2 (en) * 2004-09-09 2007-06-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem Use of liposomal glucocorticoids for treating inflammatory states
JP5643474B2 (ja) 2004-10-01 2014-12-17 ヴェロシス，インク． マイクロチャネルプロセス技術を用いる多相混合プロセス
BRPI0606335A2 (pt) 2005-03-23 2009-09-29 Velocys Inc caracterìsticas em superfìcie na tecnologia de microprocesso
US20060219307A1 (en) 2005-03-31 2006-10-05 National Taiwan University Micromixer apparatus and method therefor
US9005654B2 (en) 2005-07-27 2015-04-14 Protiva Biotherapeutics, Inc. Systems and methods for manufacturing liposomes
EP1934246B8 (en) 2005-10-07 2012-02-08 Istituto di Ricerche di Biologia Molecolare P. Angeletti S.R.L. Matrix metalloproteinase 11 vaccine
US7794136B2 (en) * 2006-05-09 2010-09-14 National Tsing Hua University Twin-vortex micromixer for enforced mass exchange
WO2007150030A2 (en) * 2006-06-23 2007-12-27 Massachusetts Institute Of Technology Microfluidic synthesis of organic nanoparticles
WO2008053988A1 (fr) 2006-11-02 2008-05-08 National University Corporation Nagoya University Procédé de production de microcapsules
WO2008151022A2 (en) 2007-05-31 2008-12-11 Anterios, Inc. Nucleic acid nanoparticles and uses therefor
EP2167233B1 (en) 2007-06-26 2013-01-23 Micronit Microfluidics B.V. Device and method for fluidic coupling of fluidic conduits to a microfluidic chip, and uncoupling thereof
WO2009086558A1 (en) 2008-01-02 2009-07-09 Tekmira Pharmaceuticals Corporation Improved compositions and methods for the delivery of nucleic acids
US8414182B2 (en) 2008-03-28 2013-04-09 State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University Micromixers for nanomaterial production
HUE034483T2 (en) 2008-04-15 2018-02-28 Protiva Biotherapeutics Inc New lipid preparations for introducing a nucleic acid
US8187554B2 (en) 2008-04-23 2012-05-29 Microfluidics International Corporation Apparatus and methods for nanoparticle generation and process intensification of transport and reaction systems
WO2010012005A2 (en) 2008-07-25 2010-01-28 S.K. Pharmaceuticals, Inc. Methods and systems for production of nanoparticles
CN102216243B (zh) 2008-11-14 2014-09-03 Dsm精细化学奥地利Nfg两合公司 制备环丙烷衍生物的方法
JP2010180353A (ja) 2009-02-06 2010-08-19 Kyoto Univ ブロック共重合体の製造方法
EP2480208A1 (en) * 2009-09-23 2012-08-01 Indu Javeri Methods for the preparation of liposomes
CN107028886A (zh) * 2009-11-04 2017-08-11 不列颠哥伦比亚大学 含有核酸的脂质粒子及相关的方法
WO2012000104A1 (en) 2010-06-30 2012-01-05 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
US20130323269A1 (en) 2010-07-30 2013-12-05 Muthiah Manoharan Methods and compositions for delivery of active agents
US8361415B2 (en) 2010-09-13 2013-01-29 The Regents Of The University Of California Inertial particle focusing system
WO2013059922A1 (en) * 2011-10-25 2013-05-02 The University Of British Columbia Limit size lipid nanoparticles and related methods

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2123260A1 (en) * 2006-12-29 2009-11-25 Shijiazhuang Pharma. Group Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. Liposome formulation and process for preparation thereof

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11707436B2 (en) 2014-12-15 2023-07-25 Nanosphere Health Sciences Inc. Methods of treating inflammatory disorders and global inflammation with compositions comprising phospholipid nanoparticle encapsulations of NSAIDS
WO2017120612A1 (en) 2016-01-10 2017-07-13 Modernatx, Inc. Therapeutic mrnas encoding anti ctla-4 antibodies
WO2017184786A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Cpf1 complexes with reduced indel activity
WO2017184768A1 (en) 2016-04-19 2017-10-26 The Broad Institute Inc. Novel crispr enzymes and systems
WO2017189308A1 (en) 2016-04-19 2017-11-02 The Broad Institute Inc. Novel crispr enzymes and systems
WO2018006166A1 (en) 2016-07-06 2018-01-11 Precision Nanosystems Inc Smart microfluidic mixing instrument and cartridges
US11059039B2 (en) 2016-07-06 2021-07-13 Precision Nanosystems Inc. Smart microfluidic mixing instrument and cartridges
WO2018035388A1 (en) 2016-08-17 2018-02-22 The Broad Institute, Inc. Novel crispr enzymes and systems
WO2018035387A1 (en) 2016-08-17 2018-02-22 The Broad Institute, Inc. Novel crispr enzymes and systems
US11572575B2 (en) 2016-10-03 2023-02-07 Precision NanoSystems ULC Compositions for transfecting resistant cell types
WO2018071549A1 (en) * 2016-10-11 2018-04-19 New York University Nanoparticles and uses thereof
WO2018191750A2 (en) 2017-04-14 2018-10-18 The Broad Institute Inc. Novel delivery of large payloads
WO2019094983A1 (en) 2017-11-13 2019-05-16 The Broad Institute, Inc. Methods and compositions for treating cancer by targeting the clec2d-klrb1 pathway
US11447527B2 (en) 2018-09-18 2022-09-20 Vnv Newco Inc. Endogenous Gag-based capsids and uses thereof
US11505578B2 (en) 2018-09-18 2022-11-22 Vnv Newco Inc. Endogenous Gag-based capsids and uses thereof
WO2020186213A1 (en) 2019-03-14 2020-09-17 The Broad Institute, Inc. Novel nucleic acid modifiers
WO2020191102A1 (en) 2019-03-18 2020-09-24 The Broad Institute, Inc. Type vii crispr proteins and systems
WO2020236972A2 (en) 2019-05-20 2020-11-26 The Broad Institute, Inc. Non-class i multi-component nucleic acid targeting systems
WO2022224595A1 (en) * 2021-04-22 2022-10-27 Kabushiki Kaisha Toshiba Flow channel structure, method for agitating fluid and method for manufacturing lipid particles
CN114247398A (zh) * 2021-12-17 2022-03-29 沈磊 一种脂质纳米颗粒制备系统及设备
WO2023196818A1 (en) 2022-04-04 2023-10-12 The Regents Of The University Of California Genetic complementation compositions and methods

Also Published As

Publication number Publication date
US20230256436A1 (en) 2023-08-17
JP2022141709A (ja) 2022-09-29
US20210023556A1 (en) 2021-01-28
US10843194B2 (en) 2020-11-24
EP2770980A1 (en) 2014-09-03
WO2013059922A1 (en) 2013-05-02
JP2017081954A (ja) 2017-05-18
JP2024147535A (ja) 2024-10-16
CA2853316A1 (en) 2013-05-02
EP3915545A1 (en) 2021-12-01
US9943846B2 (en) 2018-04-17
US20160214103A1 (en) 2016-07-28
EP2770980A4 (en) 2015-11-04
EP3069785A1 (en) 2016-09-21
US20180280970A1 (en) 2018-10-04
JP2020121986A (ja) 2020-08-13
JP2015502337A (ja) 2015-01-22
JP2019116478A (ja) 2019-07-18
CA2853316C (en) 2018-11-27
US11648556B2 (en) 2023-05-16

Similar Documents

Publication Publication Date Title
US11648556B2 (en) Limit size lipid nanoparticles and related methods
Zhigaltsev et al. Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing
Andra et al. A comprehensive review on novel liposomal methodologies, commercial formulations, clinical trials and patents
Zhang et al. Lipid in chips: a brief review of liposomes formation by microfluidics
CA3059714C (en) Flow channel structure and lipid particle or micelle formation method using same
Bonacucina et al. Colloidal soft matter as drug delivery system
Hood et al. Microfluidic synthesis of PEG-and folate-conjugated liposomes for one-step formation of targeted stealth nanocarriers
Ilhan-Ayisigi et al. Advances in microfluidic synthesis and coupling with synchrotron SAXS for continuous production and real-time structural characterization of nano-self-assemblies
JP3131923B2 (ja) 指状突起―融合リポソームおよびゲル
JP2009132629A (ja) リポソーム製剤の製造方法
Cheung Preparation of multifunctional nanoparticles using microfluidics
Zhang Liposomes in drug delivery
KR101402794B1 (ko) 지질 이중막 구체 및 지질 이중막 구체를 포집하고 있는 다중 지질 이중막 구체의 제작법
US20240216278A1 (en) Methods for producing nanoparticle dispersions
Lim Microfluidics (NanoAssemblr©) as a potential platform for nanocarrier formation
Reddy et al. Nanoliposomes-A Review: https://doi. org/10.54037/WJPS. 2022.100308
Martin Microfluidic-assisted self-assembly of biocompatible polymersomes: size control, drug-loading and self-assembly mechanism
Akar et al. High throughput microfluidics-based synthesis of PEGylated liposomes for precise size control and efficient drug encapsulation
Yanar Liposomes encapsulating model drugs and silver nanoparticles for illumination based drug release
Hood Pharmacy-on-a-chip: Microfluidic synthesis and preparation of tumor-targeted liposomes
Kardula Exosome and liposomes as drug delivery vesicles for treatment of cancer

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE UNIVERSITY OF BRITISH COLUMBIA, CANADA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CULLIS, PIETER R.;JIGALTSEV, IGOR V.;TAYLOR, JAMES R.;AND OTHERS;SIGNING DATES FROM 20140501 TO 20140704;REEL/FRAME:033503/0250

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION