WO2024026039A2 - Têtes d'impression à commande interne (magic) à canaux multiples et guidées - Google Patents

Têtes d'impression à commande interne (magic) à canaux multiples et guidées Download PDF

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Publication number
WO2024026039A2
WO2024026039A2 PCT/US2023/028884 US2023028884W WO2024026039A2 WO 2024026039 A2 WO2024026039 A2 WO 2024026039A2 US 2023028884 W US2023028884 W US 2023028884W WO 2024026039 A2 WO2024026039 A2 WO 2024026039A2
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WO
WIPO (PCT)
Prior art keywords
printhead
mixture
fluid mixer
fluid
linkers
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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.)
Ceased
Application number
PCT/US2023/028884
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English (en)
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WO2024026039A3 (fr
Inventor
Mohammed MANIRUZZAMAN
Yu Zhang
Faez ALKADI
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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Publication of WO2024026039A2 publication Critical patent/WO2024026039A2/fr
Publication of WO2024026039A3 publication Critical patent/WO2024026039A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/20—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • B29C64/205—Means for applying layers
    • B29C64/209—Heads; Nozzles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/10—Processes of additive manufacturing
    • B29C64/106—Processes of additive manufacturing using only liquids or viscous materials, e.g. depositing a continuous bead of viscous material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/307—Handling of material to be used in additive manufacturing
    • B29C64/314—Preparation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/386—Data acquisition or data processing for additive manufacturing
    • B29C64/393—Data acquisition or data processing for additive manufacturing for controlling or regulating additive manufacturing processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y10/00—Processes of additive manufacturing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y40/00—Auxiliary operations or equipment, e.g. for material handling
    • B33Y40/10—Pre-treatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y50/00—Data acquisition or data processing for additive manufacturing
    • B33Y50/02—Data acquisition or data processing for additive manufacturing for controlling or regulating additive manufacturing processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y70/00—Materials specially adapted for additive manufacturing
    • B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C64/00—Additive manufacturing, i.e. manufacturing of three-dimensional [3D] objects by additive deposition, additive agglomeration or additive layering, e.g. by 3D printing, stereolithography or selective laser sintering
    • B29C64/30—Auxiliary operations or equipment
    • B29C64/307—Handling of material to be used in additive manufacturing
    • B29C64/321—Feeding
    • B29C64/336—Feeding of two or more materials

Definitions

  • This invention is in the field of printing material, such as particles (e.g., microparticles and nanoparticles) or hydrogels (gelations), and delivery of live cells, biologies, or active pharmaceutical ingredients (drugs) by microparticles or nanoparticles.
  • This invention relates generally to devices, systems, and methods for extrusion-based printing of microparticles, nanoparticles, microparticle formulations, nanoparticle formulations, hydrogels, or the like.
  • Microparticles and nanoparticles have attracted worldwide research interests and have emerged as a powerful tool for the delivery of pharmaceutical reagents.
  • the effectiveness and function of biodegradable microparticles depend on several physiochemical properties, including size, surface charge, shape, as well as hydrophobicity, and hydrophilicity.
  • Formulated microparticles allow the encapsulation of a variety of agents, including proteins, plasmid DNA, lipophilic and hydrophilic drugs.
  • fabricated microparticles are suitable for many administration routes, such as inhalation, injection, and oral delivery. Different ligands and antibodies can also be attached to the microparticle surface for targeted drug delivery.
  • PEGylated microparticles with stealth properties are developed to increase the circulation time in vivo for improved therapeutic efficiency.
  • multi-channel controlled pneumatic printheads which contain linkers (e.g., Luer locks), designed functional systems (such as microfluid systems, coaxial systems, biaxial systems, reactors, mixers, stirrers, etc.), and output ports (e.g., multi-nozzles, multi-channels) are provided.
  • linkers e.g., Luer locks
  • designed functional systems such as microfluid systems, coaxial systems, biaxial systems, reactors, mixers, stirrers, etc.
  • output ports e.g., multi-nozzles, multi-channels
  • the linker parts are useful to connect a printhead to a printer, such as a bioprinter including one or multiple pneumatically controlled fluid reservoirs.
  • the linkers can include various linking configurations to link with different printer systems and printheads, such as Luer locks, Luer slips, or the like.
  • the designed functional system can comprise a microfluidic system that includes a structure having a controlled fluid path length, controlled fluid path size, and/or controlled fluid path cross-sectional shape, which may vary in two dimensions or three dimensions.
  • the designed functional system can be coupled with multiple fluid pneumatic injectors via the linkers and can include multiple mixing chambers.
  • the designed functional system is not limited to microfluidics systems, but can include coaxial systems, reactors, mixing/stirring systems, or the like.
  • the designed functional system is referred to as a fluid mixer, which may be configured to or used to achieve complete or partial mixing of components or may be configured to or used to achieve coaxial, biaxial, triaxial, or higher order axial flow of components.
  • various output ports may be used.
  • one or more than one nozzles can be coupled to the designed functional system for outputting fluid from the nozzle(s).
  • Various sizes, cross-sections, shapes, lengths, or the like of the nozzles can be configured so as to achieve output of material from the nozzle according to desired parameters.
  • the printhead is customized and designed using computer-aided design (CAD) software, which can advantageously allow the printheads to be adapted and used in a variety of different commercial printers.
  • CAD computer-aided design
  • the various portions of the printhead can be designed using CAD software and manufactured using various additive manufacture technology, such as 3D laser sintering, stereolithography (SLA) printing, or the like.
  • the disclosed printheads have advantages for printing nanoparticles, microparticles, and/or hydrogels, such as, but not limited to, proteins, polymers, mRNA, lipids, liposomes, etc.
  • nanoparticles and/or microparticles can be prepared using printheads that are designed and/or configured by adjusting the above parameters (e.g., using CAD software) and printed using additive manufacturing.
  • flow simulation software e.g., SOLIDWORKS® Flow Simulation
  • SOLIDWORKS® Flow Simulation can be used to computationally model fluid dynamics and simulate liquid and gas flows for customizing designs for the printheads.
  • suitable 3D models of printheads can be generated and flow simulations performed to optimize the printhead, with the printheads prepared quickly (e.g., comparing other manufacturing processes) using additive manufacturing.
  • microparticles and nanoparticles can be prepared using the printheads described herein.
  • the printheads can be used for extrusion-based printing and emulsion/mixture evaporation techniques to fabricate novel polymeric microparticles or nanoparticles, such as comprising polymeric poly(lactide-co-glycolide) (PLGA) or other materials, which may be optionally biocompatible or biodegradable.
  • PLGA polymeric poly(lactide-co-glycolide)
  • PLGA poly(lactide-co-glycolide)
  • PLGA poly(lactide-co-glycolide)
  • PLGA is an example biocompatible and biodegradable FDA-approved copolymer, which can be hydrolyzed into lactic and glycolic acid monomers.
  • a method of this aspect comprises providing a first component to a first linker of a plurality of linkers of a printhead; providing a second component to a second linker of the plurality of linkers of the printhead; contacting the first component and the second component in a fluid mixer of the printhead; forming a mixture of the first component and the second component in the printhead; and printing or flowing the mixture from the printhead through one or more outlets.
  • the first component comprises a polymer, a non-polymeric excipient or carrier, and/or an active pharmaceutical ingredient.
  • the second component comprises a solvent.
  • additional components can be provided to additional linkers of the plurality of linkers, such that the additional components are mixed with the first and second component in the fluid mixer. Printing may subject the mixture to shear forces that separate the mixture into a plurality of droplets including particles.
  • Contacting the first component and the second component may comprise providing the first component to an inlet of a fluid mixer in the printhead and providing the second component to the same or a different inlet of the fluid mixer in the printhead.
  • the fluid mixer may comprise a coaxial or other arrangement of a first fluidic channel in fluid communication with a first linker and a second fluid channel in fluid communication with a second linker, for example.
  • the first component is provided to the first linker
  • the second component is provided to the second linker, which can direct the components to the fluid mixer.
  • the fluid mixer may comprise a stirrer.
  • an electric field or a magnetic field may be used for activating the stirrer.
  • the stirrer may optionally rotate or be rotatable about an axis parallel to a flow of the mixture (e.g., through one or more of the outlets) or one or more components, or about an axis perpendicular to a flow of the mixture or one or more of the components, or about an axis with any other orientation relative to the flow of the mixture or one or more of the components.
  • the fluid mixer may optionally comprise a mixing architecture characterized by an “S” shape, a “Y” shape, or a helix shape. Again, one or more additional linkers can be included in the printhead to allow for mixing of 3 or more components in the fluid mixer.
  • the particles and/or droplets prepared according to this aspect may be subjected to further processing.
  • methods of this aspect may further comprise subjecting the droplets to evaporation conditions to evaporate the solvent from the droplets and leave the particles.
  • Methods of this aspect may further comprise washing the plurality of particles, for example.
  • Methods of this aspect may further comprise lyophilizing the plurality of droplets or the particles.
  • the mixture may comprise or further comprise one or more of a cosolvent, a surfactant, a preservative, live cells, cellular components, an additional active ingredient, a salt, a preservative, a protein, a peptide, an amino acid, or a nucleic acid component.
  • the mixture may be a homogeneous mixture or a non- homogeneous mixture.
  • a non-homogeneous mixture may be desirable so as to prevent complete mixing of the first component and a second component, such as to form a coaxial feed of the first component around or adjacent to the second component.
  • example active ingredients may comprise a protein, an antibody, a nucleic acid, messenger ribonucleic acid (mRNA) molecules, a lipid nanoparticle, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), homing endonucleases or mega-nucleases, a growth factor, a plasmid, a hydrophilic pharmaceutical, a lipophilic pharmaceutical, a viral particle, a virus-like particle, a live yeast cell, a live recombinant yeast cell, a live fungus, a live bacterial cell, a live recombinant bacterial cell, a live insect cell, a live mammalian cell, or a live mesenchymal stem cell.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALENs transcription activator-
  • a weight ratio of the active pharmaceutical ingredient to the polymer or the non-polymeric excipient in the mixture is from 1 :8 to 1 : 15, such as from 1 :8 to 1 :9, from 1 :9 to 1 : 10, from 1 : 10 to 1 : 11, from 1 : 11 to 1 : 12, from 1 : 12 to 1 : 13, from 1 : 13 to 1 : 14, or from 1 : 14 to 1 : 15.
  • the polymer may be a biodegradable polymer.
  • Example biodegradable polymers include, but are not limited to, poly(lactide-co-glycolide), polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), Pluronic F127, sodium alginate, hyaluronic acid, chitosan, cyclodextrin, dextran, agarose, gelatin, albumin, collagen, fibroin, lipids, a polyethylene glycol (PEG) derivative, a pharmaceutical grade polymer, poly(hydroxy butyrate), poly(P-malic acid), or poly(L-lysine).
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • the non-polymeric excipient may be a hydrophilic substance or a hydrophobic substance.
  • Example non-polymeric excipients include, but are not limited to, a non-reducing sugar, such as trehalose or sucrose, a polyol, such as mannitol, sorbitol, xylitol, or an amino acid, such as leucine or L-arginine.
  • the particles can be prepared using any suitable printing parameters and any suitable environmental parameters.
  • the printing may occur at ambient conditions (e.g., at atmospheric pressure and at room temperature), though control of the temperature of the mixture may be achieved by including a heat exchanger or other temperature controller (e.g., a coolant jacket, a Peltier cooler, etc.) in the printhead (e.g., at the fluid mixer).
  • Temperatures for collecting the plurality of droplets may correspond to ambient temperature or cryogenic temperatures.
  • collecting the plurality of droplets optionally comprises receiving the plurality of droplets on a surface having a temperature of from about -200 °C to about -78 °C or at room temperature or from about 4 °C to about 50 °C.
  • the extrusion-based printing method subjects the mixture to a pressure of from 1 kPa to 700 kPa, such as from IkPa to 600 kPA, from 1 kPa to 500 kPa, from 1 kPa to 400 kPa, from 1 kPa to 300 kPa, from 1 kPa to 200 kPa, from 1 kPa to 100 kPa, from 1 kPa to 50 kPa, from 1 kPa to 40 kPa, from 1 kPa to 30 kPa, from 1 kPa to 20 kPa, from 1 kPa to 10 kPa, from 10 kPa to 600 kPa, from 10 kPa to 500 kPa, from 10 kPa to 400 kPa, from 10 kPa to 300 kPa, from 10 kPa to 200 kPa, from 10 kPa to 100 kPa, from
  • an extrusion pressure of the extrusion-based printing method greater than or about 700 kPa.
  • the extrusion-based printing method uses a nozzle having a diameter of from 1 pm to 1000 pm, such as from 1 pm to 10 pm, from 10 pm to 100 pm, from 100 pm to 700 pm, from 300 pm to 700 pm, from 100 pm to 200 pm, from 200 m to 300 pm, from 300 pm to 400 pm, from 400 pm to 500 pm, from 500 pm to 600 pm, from 600 pm to 700 pm, from 700 pm to 800 pm, from 800 pm to 900 pm, or from 900 pm to 1000 pm.
  • a temperature of the mixture during the printing is from about 4 °C to about 50 °C, such as from 4 °C to 10 °C, from 10 °C to 20 °C, from 20 °C to 30 °C, from 30 °C to 40 °C, or from 40 °C to 50 °C.
  • printing the mixture comprises receiving the particles or droplets on a surface, wherein the surface has a temperature of about room temperature or less than or about -78 °C or less than or about -180 °C.
  • a system of this aspect comprises a printer, such as a bioprinter, which may include one or more pneumatically controlled fluid extruders.
  • systems of this aspect comprise a plurality of supply containers for preparing or storing respective components, such as but not limited to components comprising a polymer or a non-polymeric excipient, a solvent, or an active pharmaceutical ingredient; a printhead in fluid communication with the plurality of supply containers, the printhead comprising a plurality of linkers, such as where each linker is in fluid communication with an outlet of one of the plurality of supply containers; a fluid mixer for mixing components from the plurality of linkers into a mixture; and one or more outlets in fluid communication with the fluid mixer.
  • the system may optionally comprise a collection surface for receiving a plurality of droplets of the mixture from the one or more outlets.
  • the mixture may comprise water, a polymer or a non-polymeric excipient, a solvent, and an active pharmaceutical ingredient.
  • the collection surface comprises a sterile vial.
  • Systems of this aspect can include various components or adjustable parameters to allow for preparing particles, such as according to the methods described herein.
  • the collection surface may optionally be cooled to a temperature of from about -200 °C to about -75 °C.
  • a system of this aspect may further comprise a cooling or refrigeration system coupled to the collection surface for cooling the collection surface to a temperature of from about -200 °C to about -75 °C.
  • a system of this aspect may comprise one or more temperature sensors or temperature controllers for monitoring or controlling a temperature of the collection surface.
  • the collection surface is a moving or movable or translating or translatable collection surface.
  • a system of this aspect may further comprise a translation stage for generating a relative translation between one or more printheads and/or nozzles and the collection surface.
  • a system of this aspect may further comprise one or more pressure sensors or pressure controllers for monitoring or controlling an extrusion pressure associated with the printheads and/or nozzles.
  • a system of this aspect may further comprise one or more actuators for monitoring or controlling an extrusion speed associated with one or more printheads and/or nozzles.
  • a system of this aspect may further comprise a housing for maintaining at least one or more printheads and/or nozzles and the collection surface in a sterile environment.
  • a system of this aspect may further comprise sterilization equipment positioned to sterilize one or more of the plurality of supply containers, the one or more printheads and/or nozzles, or the collection surface.
  • compositions are provided herein, such as microparticle-based therapeutic compositions.
  • a composition may comprise particles having diameters of from 10 nm to 1100 pm; and one or more live cells.
  • the particles are attached to surfaces of the one or more live cells.
  • the one or more live cells are at least partially encapsulated into the particles.
  • Example live cells include, but are not limited to, live yeast cells, live recombinant yeast cells, live fungal cells, live bacterial cells, live recombinant bacterial cells, live insect cells, live mammalian cells, or live mesenchymal stem cells.
  • the particles may be in a lyophilized condition.
  • Example particles include particles comprising a polymer or a non-polymeric excipient, such as prepared according to various methods described herein or prepared using various systems described herein.
  • the polymer is a biodegradable polymer selected from the group consisting of poly(lactide-co-glycolide), polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), pluronic F127, sodium alginate, hyaluronic acid, chitosan, cyclodextrin, dextran, agarose, gelatin, albumin, collagen, lipids, fibroin, a polyethylene glycol (PEG) derivative, a pharmaceutical grade polymer, poly(hydroxy butyrate), poly(P-malic acid), or poly(L-lysine).
  • PEG polyethylene glycol
  • the non- polymeric excipient is a hydrophilic substance, a hydrophobic substance, a non-reducing sugar, trehalose, sucrose, a polyol, mannitol, sorbitol, xylitol, an amino acid, leucine, or L-arginine.
  • the particles further comprise an active ingredient embedded within or adsorbed to the particles.
  • the active pharmaceutical ingredient may include one or more of a protein, an antibody, a nucleic acid, messenger ribonucleic acid (mRNA) molecules, a lipid nanoparticle, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), homing endonucleases or meganucleases, a growth factor, a plasmid, a hydrophilic pharmaceutical, a lipophilic pharmaceutical, a viral particle, a virus-like particle, a live yeast cell, a live recombinant yeast cell, a live fungus, a live bacterial cell, a live recombinant bacterial cell, a live insect cell, a live mammalian cell, or a live mesenchymal stem
  • printheads are described herein, such as printheads for preparing particles, optionally according to the methods described herein.
  • the printheads may comprise or correspond to various printheads described herein.
  • the printheads may include a plurality of linkers defining inlets of the printhead; a fluid mixer in fluid communication with the plurality of linkers for mixing components from the inlets into a mixture; and one or more outlets in fluid communication with the fluid mixer.
  • one or more of the plurality of linkers comprises a Luer lock, a Luer slip, or a slip tip.
  • the plurality of linkers may be characterized by a diameter of less than or about 5.0 mm.
  • the linkers may be used and/or configured for establishing sealed fluid communication with other devices, such as a bioprinter, pneumatically controlled fluid extruders, supply containers, other printheads or fluid mixers, etc.
  • the fluid mixer may comprise a coaxial or other arrangement of a first fluidic channel in fluid communication with a first linker and a second fluid channel in fluid communication with a second linker.
  • One or more additional fluidic channels in fluid communication with one or more additional linkers may optionally be used.
  • the fluid mixer may comprise a stirrer, which may or may not include an electric field or a magnetic field for activating the stirrer.
  • the stirrer may optionally rotate or be rotatable about an axis parallel to a flow of the mixture (e.g., through one or more of the outlets) or one or more components, or about an axis perpendicular to a flow of the mixture or one or more of the components, or about an axis with any other orientation relative to the flow of the mixture or one or more of the components.
  • the fluid mixer may be formed from a simple 2D structure to a complex 2D structure to a 3D structure.
  • the fluid mixer may be any shape with any angle size/channel cross-section and, in examples, may comprise a mixing architecture characterized by a “Y” shape, an “S” shape, or a helix shape.
  • mixing architecture can be configured according to any suitable shape, configuration, or scheme for a particular application, such as to achieve a specific amount of mixing and/or to achieve complete and/or partial mixing of two or three or more different components, for example.
  • the one or more outlets may be characterized by a diameter of less than or about 5.0 mm.
  • the one or more outlets may comprise one or more extrusion-based printing nozzles for generating a plurality of droplets of the mixture including particles having diameters of from 10 nm to 1100 pm.
  • the one or more outlets may comprise additional linkers, such as for establishing fluid communication with a linker of an additional printhead, such as to provide a mixture of two or more components as an input for the additional printhead.
  • any of the printheads described herein can be prepared or manufactured.
  • the printhead comprises a plurality of linkers defining inlets of the printhead; a fluid mixer in fluid communication with the plurality of linkers for mixing components from the inlets into a mixture; and one or more outlets in fluid communication with the fluid mixer.
  • multiple printheads may be linked together, such as where a linker of one printhead is coupled to an outlet of another printhead.
  • Such a configuration may be useful for achieving complex mixing configurations, such as where a coaxial or multi -axial mixture flow of components is achieved by a first printhead and where the mixture flow from the first printhead is further mixed with additional components, such as in a further coaxial or multi -axial mixture flow.
  • manufacturing the printhead may comprise an additive manufacturing process (e.g., 3D printing using 3D laser sintering, SLA printing, or the like).
  • the methods may include 3D modelling the printhead prior to manufacturing the printhead.
  • the methods may include designating a 3D model for the printhead.
  • the method may include modeling flow and mixing within the fluid mixer prior to manufacturing the printhead.
  • FIG. 1 provides a schematic illustration of selected operations for preparing a mixture for microparticle generation.
  • FIG. 2A, FIG. 2B, and FIG. 2C provide schematic illustrations of printhead elements for component mixing and microparticle and/or nanoparticle generation.
  • FIG. 3 provides a schematic illustration showing a system for microparticle and or nanoparticle generation including a printhead for component mixing.
  • FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12, and FIG. 13 provide schematic illustrations of printheads for component mixing and microparticle and/or nanoparticle generation.
  • FIG. 14 and FIG. 15 show flow simulations for MAGIC printheads.
  • FIG. 16, FIG. 17, FIG. 18, and FIG. 19 show dynamic light scattering (DLS) analysis results of particles generated from printheads according to examples described herein.
  • DLS dynamic light scattering
  • FIG. 20 provides data showing Circular Dichroism spectra obtained for fibroin particles fabricated according to examples described herein.
  • FIG. 21 provides data showing Fourier Transform-Infrared spectra for fibroin particles fabricated according to examples described herein.
  • FIG. 22, FIG. 23, FIG. 24, FIG. 25, FIG. 26, FIG. 27, and FIG. 28 show dynamic light scattering (DLS) analysis results of fibroin particles generated from printheads according to examples described herein.
  • DLS dynamic light scattering
  • FIG. 29 show scanning electron micrograph images of fibroin particles generated from printheads according to examples described herein.
  • FIG. 30, FIG. 31, and FIG. 32 provide schematic illustrations of printhead elements for component mixing and microparticle and/or nanoparticle generation.
  • Extrusion-based 3D printing is often performed using a single ink or switching between two or more inks.
  • the ability to create compositional variation in-situ by rapid mixing or biaxial or coaxial printing of two different inks inside a printhead with dynamically changing ratios can produce a broad palette of material compositions that are useful in making functionally graded materials for applications in, for example, soft robotics, tissue engineering, or integrated sensors.
  • rapid combination of high viscosity bio-inks in low Reynolds number flows has been a challenge.
  • multi-channeled and guided inner controlling (MAGIC) printheads are capable of coaxial, biaxial, and mixing dynamically controllable ratios of different bio-inks to print graded materials.
  • Bio-hydrogels have emerged as promising platforms for drug release systems due to inherent biocompatibility, controllable degradability, and tunable physical properties.
  • Single contingent gel systems contain drug molecules that may lead to burst drug release.
  • a platform for drug delivery to targets organs (tissues) may be required.
  • the coaxial hydrogel structures were developed that could simultaneously exert both affinity and diffusion control over the release of chemotherapeutic drugs.
  • a range of coaxial hydrogel structures were developed through MAGIC 3D-printing techniques, with the purpose of building suitable therapeutic platforms for sustained and local release of drugs (e.g. anti-cancer drugs, anti-bacterial drugs, painkiller, etc.) to tissues/organs.
  • the printing systems described herein can be used to make a bi/tri cylinder that can load multiple drugs and release them at the same time.
  • a printed cylinder can permit one side to load lightweight and/or floating materials and the other side to load a drug, such that the resultant scaffold can float (e.g., in the stomach) with a particular orientation.
  • Such aspects allow extension to new medical application areas such as floating tablets.
  • the present disclosure provides multi-channel controlled pneumatic printheads which contain a linker (e.g. Luer lock), a designed functional system (e.g., a microfluidic system, coaxial system, or a mixing system) and one or more outlets (e.g., nozzle part or output linker).
  • a linker e.g. Luer lock
  • a designed functional system e.g., a microfluidic system, coaxial system, or a mixing system
  • outlets e.g., nozzle part or output linker.
  • Customized designs for the printheads can be created, for example, using computer aided design software and flow simulation software can be used to identify the extent and range of mixing within the printheads.
  • Air pressure controlled pneumatic printheads are capable of extruding a wide range of high and low viscosity materials.
  • the connection of printheads with state-of-the-art 3D bioprinters allows for printing with a wide range of materials.
  • printers allow for delicate control of the temperature of the print bed and printhead, enabling a high level of printing quality, regardless of the bio-ink’s viscosity.
  • Mixing is a process by which uniformity of concentration is achieved. Depending on the context, mixing may be in reference to the concentration of a particular component or set of components in a fluid.
  • the design and implementation of mixers in microfluidics differs considerably from that on the macroscale. The small length scale leads to different physical phenomena being dominant at the microscale. First, inertial effects that typically result in turbulence and good mixing on the macroscale are weak in microfluidics, while methods of actuation based on electro kinetics, surface tension or other phenomena that are not relevant on the macroscale become feasible on the microscale. Secondly, many mechanical designs such as stirrers that can be easily implemented on the macroscale are very difficult to implement on the microscale.
  • MVC mixing variance coefficient
  • N is the number of sub-domains
  • Ci is the concentration in the 1 th sub-domain
  • c av g is the average concentration for the entire domain. MVC approaches zero when mixing is complete.
  • methods for producing multi-channeled and guided inner controlling (MAGIC) printheads described herein can be performed by using a resin composition for stereolithography, for example, using liquid resin as a raw material.
  • printheads can be fabricated using SLA resin compositions using a variety of stereolithography methods including LCD (stereolithography liquid display method: Liquid Crystal Display), DLP (stereolithography projector (surface exposure) method: Digital Light Processing), and SLA (stereolithography laser method: Stereolithography Apparatus).
  • LCD stereolithography liquid display method: Liquid Crystal Display
  • DLP stereolithography projector (surface exposure) method: Digital Light Processing
  • SLA stereolithography laser method: Stereolithography Apparatus
  • printheads can be fabricated by using direct metal laser sintering (DMLS).
  • DMLS is an industrial metal 3D printing process that builds fully functional metal prototypes and production parts in 7 days or less. A range of metals produce final parts that can be used for end-use applications.
  • the DMLS machine begins sintering each layer — first the support structures to the base plate, then the part itself with a laser aimed onto a bed of metallic powder. After a cross-section layer of powder is micro-welded, the build platform shifts down, and a recoating blade moves across the platform to deposit the next layer of powder into an inert build chamber. The process is repeated layer by layer until the build is complete.
  • Techniques described herein include those employing pneumatic, pressure assisted, extrusion-based 3D printing and emulsion/mixture evaporation for fabricating microparticles or nanoparticles, such as useful for particle-based drug delivery systems encapsulating an active pharmaceutical ingredient for the treatment of different diseases.
  • the techniques provide for encapsulation of a variety of substances including proteins, plasmid DNA, lipophilic pharmaceutical compositions, hydrophilic pharmaceutical compositions, live cells, and/or cellular components into polymeric particles.
  • a variety of biocompatible polymers can be used to formulate the particles, such as, but not limited to, poly (lactide-co-glycolide) (PLGA), polylactide (PLA), polycaprolactone (PCL), etc.
  • non-polymeric excipients such as nonreducing sugars, such as trehalose, sucrose, or polyols (e.g., mannitol, sorbitol, xylitol), or amino acids (e.g., leucine) can be used as suitable carrier matrices to form the particles.
  • an active pharmaceutical ingredient (if hydrophilic) can be dissolved in a poly(vinyl alcohol) (PVA) or other suitable polymeric aqueous solution including but not limited to, polyethylene glycol (PEG) or polyvinyl pyrrolidone (PVP), or lipidic solutions, optionally with a cosolvent to aid in dissolution in the case of hydrophobic active pharmaceutical ingredients.
  • PVA poly(vinyl alcohol)
  • PEG polyethylene glycol
  • PVP polyvinyl pyrrolidone
  • lipidic solutions optionally with a cosolvent to aid in dissolution in the case of hydrophobic active pharmaceutical ingredients.
  • the active pharmaceutical ingredient dissolved in the aqueous PVA solution can be further added to a polymer dissolved in an organic solvent, such as chloroform, followed by mixing completely to form a primary mixture.
  • the primary mixture can be further added to another PVA aqueous solution, such as with a higher PVA concentration, followed by mixing completely to generate a secondary mixture.
  • the secondary mixture can be transferred to a pneumatic syringe with a fine gauge needle, then printed by a bioprinter employing an extrusion-based printing step.
  • the resultant particles can optionally be washed (e.g., by ultracentrifugation) one or more times, and then collected.
  • FIG. 1 provides a schematic overview of a process 100 of preparing mixtures according to various techniques described herein.
  • Process 100 may include providing a first component at block 105 and providing a second component at block 110.
  • the first component may include a polymer, a non-polymeric excipient, or an active pharmaceutical ingredient.
  • the active pharmaceutical ingredient may comprise a protein, an antibody, a nucleic acid, messenger ribonucleic acid (mRNA) molecules, a lipid nanoparticle, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), homing endonucleases or mega-nucleases, a growth factor, a plasmid, a hydrophilic pharmaceutical, a lipophilic pharmaceutical, a viral particle, a virus-like particle, a live yeast cell, a live recombinant yeast cell, a live fungus, a live bacterial cell, a live recombinant bacterial cell, a live insect cell, a live mammalian cell, or a live mesenchymal stem cell.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALENs transcription activator
  • the polymer may be a biodegradable polymer, such as selected from the group consisting of poly(lactide-co-glycolide), polylactide (PLA), polyglycolide (PGA), poly caprolactone (PCL), Pluronic Fl 27, sodium alginate, hyaluronic acid, chitosan, cyclodextrin, dextran, agarose, gelatin, albumin, collagen, fibroin lipids, a polyethylene glycol (PEG) derivative, a pharmaceutical grade polymer, poly(hydroxy butyrate), poly(P-malic acid), or poly(L-lysine).
  • PEG polyethylene glycol
  • PEG polyethylene glycol
  • the non-polymeric excipient may be a hydrophilic substance, a hydrophobic substance, a nonreducing sugar, trehalose, sucrose, a polyol, mannitol, sorbitol, xylitol, an amino acid, leucine, or L-arginine.
  • the second component may be a solvent.
  • the first component and the second component are contacted.
  • Contacting the first component and the second component may include providing the first component to an inlet of a fluid mixer in a printhead and providing the second component to the inlet of the fluid mixer in the printhead to form a mixture, such as at block 120.
  • the fluid mixer may be a coaxial arrangement of a first fluidic channel in fluid communication with a first linker and a second fluid channel in fluid communication with a second linker.
  • the first component may be provided to the first linker, and the second component may be provided to the second linker, in order to feed the components to a mixing chamber or region (fluid mixer).
  • the fluid mixer may be a stirrer. If present in the fluid mixer, a stirrer may be activated, such as by providing an electric field or a magnetic field.
  • the fluid mixer may include a mixing architecture (e.g., flow pathway within the fluid mixer) characterized by an “S” shape or a helix shape.
  • the mixture may be suitable for generating particles of active ingredients embedded in the materials using extrusion-based printing techniques described herein.
  • the mixture can be prepared without the use of sonication or other ultrasonic mixing techniques that can result in raising the mixture temperature, allowing for temperature sensitive active ingredients to be incorporated into particles without being subjected to excessive temperatures.
  • process 100 may include printing the mixture from the printhead through one or more outlets (e.g., printing nozzles).
  • the printing may subject the mixture to shear forces that separate the mixture into a plurality of droplets including particles.
  • process 100 optionally includes subjecting the droplets to evaporation conditions to evaporate the solvent from the droplets and leave the particles.
  • process 100 optionally includes washing the particles.
  • process 100 optionally includes lyophilizing the plurality of droplets or the particles.
  • FIG. 2A provides a schematic depiction of a printhead 200 for generating particles.
  • the printhead 200 may include a plurality of linkers 202A, 202B, 202C defining inlets of the printhead 200.
  • the printhead 200 may include a fluid mixer 204 in fluid communication with the plurality of linkers 202A, 202B, 202C for mixing components from the inlets into a mixture.
  • the fluid mixer 204 may be in fluid communication with the plurality of linkers 202 A, 202B, 202C via a plurality of primary fluid lumens.
  • the printhead 200 may include one or more outlets in fluid communication with the fluid mixer 204.
  • the one or more outlets may be in fluid communication with the fluid mixer 204 via a one or more secondary fluid lumens.
  • the outlet of component 200 includes an outlet linker 206 A and a nozzle 206B.
  • outlet linker 206 A and a nozzle 206B are shown as two separate components, the outlet linker 206A and a nozzle 206B may be a unitary structure with fluid mixer 204 such that no outlet linker 206A is present and the nozzle 206B is joined to the fluid mixer 204.
  • FIG. 2A also shows other nozzle configurations on opposite sides of nozzle 206B, which may be substituted for nozzle 206B.
  • the other nozzle configurations may include a plurality of nozzle ports, while nozzle 206B may include a single nozzle port.
  • Printhead 200 is also shown including sub-printhead 210, which includes linkers 212A and 212B, fluid mixer 214 and outlet 216.
  • Outlet 216 is depicted as an outlet linker, which may be coupled to the central linker 202B, for example.
  • Such sub-printhead 210 is depicted to illustrate a configuration where multiple printheads are coupled to one another, so as to provide additional opportunities and configurations for mixing additional components before they are introduced into a fluid mixer.
  • One or more of the plurality of linkers 202 A, 202B, 202C, 212A, 212B may include a Luer lock, a Luer slip, a slip tip, or other couplers for establishing sealable fluid communication.
  • the plurality of linkers 202A, 202B, 202C, 212A, 212B may be various types of couplings or connectors.
  • each of the plurality of linkers 202A, 202B, 202C, 212A, 212B may be the same type of coupling or connector.
  • the linkers 202A, 202B, 202C, 212A, 212B may be characterized by a diameter of less than or about 5.0 mm, such as less than or about 4.5 mm, less than or about 4.0 mm, less than or about 3.5 mm, less than or about 3.0 mm, less than or about 2.5 mm, less than or about 2.0 mm, less than or about 1.5 mm, less than or about 1.4 mm, less than or about 1.3 mm, less than or about 1.2, mm, less than or about 1.1 mm, less than or about 1.0 mm, less than or about 0.9 mm, less than or about 0.8 mm, less than or about 0.7 mm, less than or about 0.6 mm, less than or about 0.5 mm, or less.
  • the fluid mixer 204 may be a coaxial arrangement of a first fluidic channel in fluid communication with a first linker and a second fluid channel in fluid communication with a second linker.
  • the fluid mixer may include a stirrer as shown in FIG. 2B.
  • the mixer may be activated using any means, including an electric field or a magnetic field for activating the stirrer. While the stirrer is shown at an upstream location of the fluid mixer, it is contemplated the stirrer may be positioned at any location along the fluid mixer. Further, it is contemplated that a plurality of fluid mixers, such as stirrers, may be used to form the mixture.
  • the fluid mixer may include a mixing architecture characterized by a “Y” shape, an “S” shape, a helix shape, or any other desirable shape to establish a desired amount of mixing of components introduced from various linkers.
  • the one or more outlets or nozzles 206A, 206B, 216 of the printhead 200 or subprinthead 216 may be characterized by a diameter of less than or about 5.0 mm, such as less than or about 4.5 mm, less than or about 4.0 mm, less than or about 3.5 mm, less than or about 3.0 mm, less than or about 2.5 mm, less than or about 2.0 mm, less than or about 1.5 mm, less than or about 1.4 mm, less than or about 1.3 mm, less than or about 1.2, mm, less than or about 1.1 mm, less than or about 1.0 mm, less than or about 0.9 mm, less than or about 0.8 mm, less than or about 0.7 mm, less than or about 0.6 mm, less than or about 0.5 mm, or less.
  • the one or more outlets may include one or more extrusion-based printing nozzles, such as nozzle 206B, for generating a plurality of droplets of the mixture including particles having diameters of from 10 nm to 1100 pm.
  • FIG. 2C shows photographs of various nozzles including nozzle linkers for joining to an output linker of a printhead.
  • the outlet of the printhead 200 may include one or more outlets, such as in the form of one or more nozzle ports.
  • a system 300 for generating particles is schematically depicted.
  • the system may include a plurality of supply containers 302 for preparing or storing a respective component, such as comprising a polymer or a non-polymeric excipient, a solvent, or an active pharmaceutical ingredient.
  • the system 300 may also include a printhead 304, which may include any of the features or characteristics previously discussed with regard to printhead 200 or other printheads described herein.
  • the system 300 may include a collection surface 306 for receiving a plurality of droplets 308 of the mixture from the one or more outlets of the printhead, such as droplets 308 that contain particles.
  • the collection surface 306 may be, for example, a sterile vial.
  • the collection surface 306 of system 300 may be cooled to a temperature of from about -200 °C to about -75 °C during operation.
  • a cooling or refrigeration system coupled to the collection surface 306 may cool the collection surface to a temperature of from about -200 °C to about -75 °C.
  • One or more temperature sensors or temperature controllers may monitor or control a temperature of the collection surface 306.
  • the collection surface 306 may be a moving or translating collection surface.
  • System 300 may optionally include one or more pressure sensors or pressure controllers, temperature sensors or temperature controller, position sensors, translation stages, or other sensors or controllers.
  • the temperature may be controlled over a wide range, such as from about -120 °C to about 250 °C
  • the printhead 304 may be compatible with use of a wide range of FDA approved thermoplastics or for establishing low temperature, useful to maintain viability of cells or other biomaterial in the mixture to be printed using the printhead.
  • the printhead can be used to extrude thermoplastic materials, such as for making pharmaceutical tablets with crystal drugs (e.g., biopharmaceutics classification system class 3).
  • the printhead is compatible with temperature sensitive biomaterials.
  • the system 300 may include a Peltier cooler, a heat exchange jacket, or other heat exchanger in contact with the printhead 304 or fluid mixer region, such as adjacent to or surrounding the S-shaped flow path of the fluid mixer depicted, to add or remove heat and/or control temperature of the mixture within the printhead 304.
  • water from a bath can be circulated in a heat exchange apparatus which surrounds the fluid mixer or fabricated as flow paths within the periphery of the fluid mixer.
  • the present disclosure also encompasses methods of creating, designing, forming, and/or fabricating printheads, such as printhead 200 previously described.
  • the methods may include manufacturing a printhead, the printhead including a plurality of linkers defining inlets of the printhead, a fluid mixer in fluid communication with the plurality of linkers for mixing components from the inlets into a mixture, and one or more outlets in fluid communication with the fluid mixer.
  • Manufacturing the printhead may include additive manufacturing such as 3D laser sintering, SLA printing, or the like.
  • the methods may include 3D modeling the printhead prior to manufacturing the printhead.
  • the methods may include designating a 3D model for the printhead.
  • the methods may also include studying properties of the printhead prior to manufacturing, such as modeling flow and mixing within the fluid mixer, and droplet formation via one or more nozzles or nozzle ports prior to manufacturing the printhead.
  • the processes of modeling and designating a 3D model for the printhead and modeling flow, mixing, and printing may be performed iteratively, such as to optimize the printhead for printing droplets or particles of particular mixtures or mixtures of particular properties.
  • the printheads, methods, and systems described herein are useful for preparing microparticles and/or nanoparticles, such as using sprayed multi adsorbed-droplet reposing technology.
  • Example techniques and particle components and compositions are described in U.S. Provisional Application No. 63/219,258, filed on July 7, 2021, and PCT International Application No. PCT/US2022/036336, filed on July 7, 2022, which are hereby incorporated by reference in their entireties.
  • the invention may be further understood by the following non-limiting examples.
  • FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, FIG. 11, FIG. 12, and FIG. 13 provide numerous views of printheads, including various fluid mixer types and architectures, as well as various dimensions.
  • the top rows in FIGS. 4-7 show depictions of 3D models of the printheads, while the bottom rows in FIGS. 4-7 show photographs of fabricated printheads, created using SLA printing techniques using a standard clear SLA resin. These different printheads were used to establish the manufacturing limits according to the particular SLA printing system used, to identify the smallest flow path and/or nozzle sizes.
  • FIG. 8 shows fabricated printheads with various nozzle sizes.
  • the flow rate may be controlled by the input pressure.
  • the flow rate is one parameter that is related to the size of the final particles.
  • FIG. 9 shows 3D helix shaped MAGIC printheads with various length (e.g., the number of coils). In FIG. 9, the number of coils are as follows: A) 5 coils, (B) 10 coils, (C) 15 coils, and (d) 20 coils. All inner tubes in FIG. 9 have a fixed 2 mm diameter.
  • FIG. 10 shows fabricated coaxial printheads with various inner length. In FIG. 10, the inner lengths are as follows: A) 10 mm, (B) 15 mm, (C) 20 mm, and (d) 30 mm.
  • FIG. 11 shows printheads with multiple inputs (e.g., three or more).
  • the printhead may include three inputs or four inputs, or any number of inputs.
  • the diameter for the input is 1 mm.
  • FIGS. 4-11 3D flow simulations were performed for each of the printheads depicted in FIGS. 4-11 to evaluate flow conditions (e.g., flow rate, flow direction, pressure, shear force, density, viscosity, etc.) in the printhead system and the extent of mixing.
  • FIG. 12 provides a schematic of the mixing in a printhead with two or three inputs.
  • FIG. 13 provides example stirrers that may be used in the printhead to encourage mixing.
  • FIG. 14 provides data showing the extent of mixing determined by the flow simulations for several printhead designs, including a colinear fluid mixer, a fluid mixer characterized by an “S” shape, and a fluid mixer characterized by a 3D helix. The flow simulation mimics the changing of the flow density via the microfluidic system.
  • FIG. 15 shows additional details from the flow simulation for printheads having a fluid mixer characterized by an “S” shape for different pressure conditions at the fluid inlets, identifying flow trajectories and pressures within the different regions of the printhead, showing how the flow simulation mimics the flow direction under different input pressure.
  • a first component includes a regenerated bombyx mori silk fibroin aqueous solution.
  • a second component includes an organic solvent, including, but not limited to, one or more of ethanol, methanol, acetone, isopropanol, etc. Then, the first and second components are transferred to two pneumatic syringes and connected to a printhead by the top linkers, separately. Both components print by a bioprinter employing an extrusion-based printing step. For some examples, 0.1 mg/ml regenerated silk fibroin solution was used as the first component.
  • FIG. 16 shows differential light scattering (DLS) analysis of the particles made from vertexing, dropping, and coaxial printing. These three methods of making particles were used for comparing to methods of printing using the printheads described herein.
  • DLS differential light scattering
  • FIG. 17 shows DLS analysis of the particles made using a “Y” shaped printhead described herein.
  • Printheads with nozzle size 1.5 mm and 1.0 mm diameter were used.
  • 20 kPa and 50 kPa extraction pressures were tested separately with both “Y” shaped printheads.
  • Silk fibroin and various organic solvents methanol, isopropanol, acetone, and ethanol were selected and analysis of the particles using various different nozzle sizes, pressures, and solvents and are displayed in FIG. 17.
  • FIG. 18 shows DLS analysis of the particles made from 3D-helix shaped printhead fluid mixers described herein and compared with those with “Y” shaped printheads.
  • Printheads with nozzle sizes 1.5 mm and 0.8 mm diameter were used for the “Y” shaped printheads, with 3 mm diameter nozzle size used for the 3D helical printhead.
  • PLGA in acetone and PVA in deionized water were used for the components.
  • FIG. 19 show DLS analysis results of particles generated from printheads according to examples described herein.
  • Protein-based particles may be useful as drug delivery vehicles (DDVs) due to their biocompatibility.
  • DDVs drug delivery vehicles
  • fibroin-protein-based DDVs have gained attention due to their biodegradability, excellent mechanical properties, processing flexibility, and ‘green’ regeneration properties.
  • SFPs silk fibroin particles
  • This Example describes use of the Multi-Channeled and Guided Inner Controlling (MAGIC) techniques described herein to synthesize the SFPs.
  • MAGIC Multi-Channeled and Guided Inner Controlling
  • this Example describes a quality-by-design (QbD) study to identify correlations between the particle size and organic solvents (ethanol, methanol, isopropanol, acetone, etc.), design of the MAGIC system (simple ‘ Y’ shape, 2D zigzag shape, 3D spiral shape, etc.), or printing parameters (pressure or nozzle size), respectively.
  • the size of the generated particles was analyzed by dynamic light scattering (DLS), and scanning electron microscopy (SEM).
  • the physico-chemical characterization of the SFPs was analyzed by circular dichroism (CD) Spectroscopy and Fourier Transform-Infrared (FTIR) Spectroscopy.
  • a fluid simulation model (Solidworks) has been used to study the designed system's mixing process in a 1 mm, 2 mm, or 3 mm-pm-wide channel integrating a MAGIC printing system by a standard derivation of color index at individual pixels.
  • the concentration distribution was obtained by successfully solving the Navier-Stokes equation and the diffusion-convection equation in the steady-state form. Because of the large range of Reynolds numbers studied (100 ⁇ Re ⁇ 600), the diffusion-convection simulations are carried out with high diffusion coefficients. Accordingly, mixing indexes in the channel flow are compared at different cross sections and the relationship between the mixing quality and size of the SFPs are calculated. The results illustrated the effects of both pressure and channel geometry on hydrodynamics and mixing efficiency.
  • APIs active pharmaceutical ingredients
  • PTC proximal tubule cells
  • Circular Dichroism (CD) Spectroscopy.
  • the fabricated fibroin particles were suspended in acetone, ethanol, methanol, or isopropanol solvent with a final concentration of 0.1 mg/mL.
  • Circular Dichroism (CD) spectroscopy was used to investigate the secondary structure of the encapsulated trypsin.
  • a JASCO-810 Spectrometer (Japan) was equipped with RTE bath/circulator (NESLAB RTE-111) and purged with N2 gas at a flow-rate of 3-5 mL/min.
  • the CD spectrometer was used for the scanning, which was carried out for 190-260 nm wavelengths with a resolution of 0.2 nm at 25 °C and accumulation of six scans. The scan speed was 100 nm/min and the response time was 0.25 s. Six measurements were carried out to obtain each spectrum. Samples of the 0.05 mg/mL and 0.01 mg/mL solutions were stored in 0.1-cm and 1-cm path length cells, respectively. The mean residue ellipticity values [0] were expressed in degree cm 2 - dm ol' 1 and were calculated using the equation:
  • FIG. 20 provides a plot showing Circular Dichroism spectra obtained for the fibroin particles fabricated using the MAGIC system.
  • FTIR Fourier Transform-Infrared
  • the infrared (IR) spectral region between 1750 cm' 1 and 1450 cm' 1 was classified as absorption by the peptide backbones of amide I (1700-1600 cm' 1 ) and amide II (1600-1500 cm' 1 ), which were mostly used for the analysis of different secondary structures of RSF.
  • the FTIR spectra are shown in FIG. 21, where the peaks at 1661-1663 cm' 1 , 1575-1777 cm' 1 , and 1525-1522 cm' 1 are characteristic of silk II secondary structure, whereas the absorptions at 1672-1669 cm' 1 and 1531- 1529 cm' 1 are indicative of silk I conformation.
  • Silk particles cargos A variety of particles comprising silk fibroin were prepared using a MAGIC system employing ethanol as a solvent, with varying amounts of another component, dimethylsulfoxide (DMSO). The various formulations tested are listed in Table 1. The particles were analyzed using dynamic light scattering (DLS), and scanning electron microscopy (SEM). Results from the dynamic light scattering measurements are shown in FIG. 22, FIG. 23, FIG. 24, FIG. 25, FIG. 26, FIG. 27, and FIG. 28. FIG. 29 shows scanning electron microscopy images of the particles, with an individual particle circled in the image as an example.
  • DLS dynamic light scattering
  • SEM scanning electron microscopy
  • Encapsulating mRNA in lipid nanoparticles is a valuable technique used in mRNA-based vaccines and therapies. Lipid nanoparticles protect the fragile mRNA molecules and facilitate their delivery into cells, where they can be translated into proteins or used for gene therapy. Encapsulation of mRNA in lipid nanoparticles may include one or more of the following: a. Selection of Lipids: Suitable lipids may be chosen for nanoparticle formation. Example lipids may include a mixture of cationic lipids (positively charged) and helper lipids.
  • Cationic lipids help to bind and condense the negatively charged mRNA, while helper lipids stabilize the nanoparticle structure and improve delivery efficiency.
  • b. Preparation of Lipid Solution The selected lipids may be dissolved in an organic solvent, such as ethanol or chloroform, to create a lipid solution.
  • c. Formation of Lipid-MRNA Complex The mRNA molecule may be combined with the lipid solution. The positively charged cationic lipids will electrostatically interact with the negatively charged mRNA, forming a lipid-mRNA complex.
  • An aqueous solution (buffer) may be added to the lipid-mRNA complex and subject the mixture to controlled agitation or sonication.
  • lipid nanoparticles can lead to the spontaneous formation of lipid nanoparticles, with the mRNA encapsulated within their core.
  • This may include using a MAGIC system as a new nanoparticle formation system for lipid-mRNA system.
  • e. Purification The lipid nanoparticles may be purified to remove any excess lipids, free mRNA, or other impurities. Example purification methods include ultrafiltration or dialysis.
  • Characterization The lipid nanoparticles may be analyzed to ensure their size, stability, and encapsulation efficiency. Techniques such as dynamic light scattering and transmission electron microscopy can be used for characterization.
  • Storage The encapsulated mRNA in lipid nanoparticles may be stored at appropriate conditions (e.g., low or subzero temperatures) to maintain stability until they are ready for use.
  • Encapsulating mRNA in liposome particles is a technique used for mRNA delivery in various applications, including gene therapy and vaccine development.
  • Liposomes are lipid-based vesicles that can encapsulate the mRNA and protect it from degradation, facilitating its delivery into cells.
  • Encapsulation of mRNA in liposomes nanoparticles may include one or more of the following: a. Selection of Lipids: Appropriate lipids may be chosen for liposome formation. Lipids with varying properties, such as cationic lipids or neutral lipids, can be used to tailor the characteristics of the liposomes for specific applications. b.
  • Preparation of Lipid Solution The selected lipids may be dissolved in an organic solvent, such as chloroform or ethanol, to create a lipid solution.
  • c. Formation of Liposomes The lipid solution may be combined with the mRNA solution. The lipids may spontaneously self-assemble into liposomes in the presence of water and the mRNA.
  • Extrusion or Sonication To further refine the liposome size and improve homogeneity, the liposome mixture can be extruded through porous membranes or subjected to sonication. Instead of using extrusion or sonication method, this Example includes using a MAGIC printing system to form mRNA-liposome.
  • the liposomes may be purified to remove any excess lipids, free mRNA, or other impurities.
  • Example purification methods include gel filtration or dialysis.
  • Characterization The liposomes may be analyzed to determine their size, stability, and encapsulation efficiency. Techniques like dynamic light scattering and transmission electron microscopy can be used for characterization.
  • Storage The encapsulated mRNA in liposomes may be stored under appropriate conditions (e.g., low or subzero temperatures) to maintain stability until ready for use.
  • FIG. 30, FIG. 31, and FIG. 32 provide numerous views of example printheads and components.
  • FIG. 30 depicts 3D models of several example linker systems, which may be used to provide for inlets for combining the multiple components in a printhead.
  • FIG. 31 depicts 3D models of an example linker system coupled to a helical mixing system (left) as well as additional example helical mixing systems.
  • FIG. 31 depicts 3D models of example linker systems (top) as well as additional example helical mixing systems.

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  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne des techniques, des systèmes et des procédés comprenant ceux qui utilisent des têtes d'impression pneumatiques commandées par canaux multiples qui contiennent un élément de liaison (par exemple, un verrou Luer), un système fonctionnel conçu (par exemple, un système microfluidique, un système coaxial, bi-axial/tri-axial, ou un système de mélange), et une ou plusieurs sorties (par exemple, une partie buse ou un élément de liaison de sortie). Les têtes d'impression sont utiles pour l'impression 3D pneumatique, assistée par pression, basée sur l'extrusion et l'évaporation d'émulsion/mélange pour fabriquer des particules qui peuvent encapsuler un ingrédient pharmaceutique actif ou des cellules vivantes dans un polymère biocompatible.
PCT/US2023/028884 2022-07-27 2023-07-27 Têtes d'impression à commande interne (magic) à canaux multiples et guidées Ceased WO2024026039A2 (fr)

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US20220118416A1 (en) * 2020-10-18 2022-04-21 Pathak Holdings Llc Crosslinked multilayered material compositions, methods for their preparation and applications thereof
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US10926454B2 (en) * 2016-05-20 2021-02-23 Sartorius Stedim Biotech Gmbh Dispensing device and system for biological products
US12157260B2 (en) * 2019-09-09 2024-12-03 Xerox Corporation Melt emulsion extrusion methods for producing thermoplastic polymer particles
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