EP1385488A2 - Procedes et compositions utiles pour encapsuler des agents actifs - Google Patents

Procedes et compositions utiles pour encapsuler des agents actifs

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Publication number
EP1385488A2
EP1385488A2 EP01989162A EP01989162A EP1385488A2 EP 1385488 A2 EP1385488 A2 EP 1385488A2 EP 01989162 A EP01989162 A EP 01989162A EP 01989162 A EP01989162 A EP 01989162A EP 1385488 A2 EP1385488 A2 EP 1385488A2
Authority
EP
European Patent Office
Prior art keywords
solvent
particles
couoidosomes
colloidosome
agent
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.)
Withdrawn
Application number
EP01989162A
Other languages
German (de)
English (en)
Inventor
Andreas Bausch
Anthony Dinsmore
Ming Hsu
Michael Nikolaides
David Weitz
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.)
Harvard College
Original Assignee
Harvard College
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 Harvard College filed Critical Harvard College
Publication of EP1385488A2 publication Critical patent/EP1385488A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • A61K9/5089—Processes

Definitions

  • the present invention relates generally to methods for making self- assembled, selectively permeable elastic microscopic structures that have controlled pore-size, porosity and superior mechanical properties, as well as the structures formed and various uses thereof.
  • encapsulation of living cells in alginate has been accomplished. Additionally, electrostatic deposition of alternating layers of particles on the surfaces of living cells provides a flexible approach. However, these approaches are not readily generalized for encapsulation of other materials. Additionally, the alginate capsules may not provide a sufficiently narrow distribution of pore sizes to prevent isolation of the encapsulated cell from various immune system components, such as antibodies. Other approaches, such as use of microfabrication technology, require demanding lithographic capabilities, yield only one capsule at a time and are not easily applicable to polymeric or other inorganic molecules. Thus, alternative, general approaches for preparation of elastic, micron-to-millimeter sized capsules that exhibit size-selective permeability are needed. The present invention addresses this need.
  • coUoidosomes Methods for making self-assembled, selectively permeable elastic microscopic structures, referred to herein as coUoidosomes, that have controlled pore-size, porosity and desired mechanical properties have been discovered. Accordingly, methods of forming coUoidosomes are provided.
  • a method of forming coUoidosomes includes providing particles formed from a biocompatible material in a first solvent and forming an emulsion by adding a first fluid to the first solvent wherein the emulsion is defined by droplets of the first fluid surrounded by the first solvent.
  • the method includes coating the surface of the droplets with the particles and then stabilizing the particles on the surface of the droplets to form stable coUoidosomes.
  • the coUoidosomes produced typically have a yield strength of at least about 20 Pascals.
  • the method may be performed with an oil-in-water system or a water-in- oil system.
  • the particles are spherical and are formed from a biocompatible polymer.
  • a method includes providing particles formed from a biocompatible material in a first solvent and forming an emulsion by adding a second solvent containing the active agent to the first solvent.
  • the emulsion is defined by droplets of the second solvent surrounded by the first solvent.
  • the method includes coating the surface of the droplets with the particles and stabilizing the particles on the surface of the droplets to form stable coUoidosomes.
  • the coUoidosomes typically have a yield strength of at least about 20 Pascals.
  • the particles are substantially spherical and are formed from a biocompatible polymer.
  • a colloidosome in at least some embodiments, includes a shell formed of biocompatible, substantially spherical particles wherein each of the particles are linked to neighboring particles.
  • the shell defines an inner chamber and has a plurality of pores extending therethrough.
  • the chamber in certain embodiments is sized for housing an active agent.
  • the coUoidosomes typically have a yield strength of at least about 20 Pascals.
  • the particles that form the colloidosome may be linked by a variety of methods to stabilize the colloidosome, including use of van der Waals forces, polyelectrolytes, by a swelling method or by a sintering process.
  • a colloidosome suspension in other aspects of the invention, includes a colloidosome suspended in a first solvent wherein the colloidosome has a shell formed of biocompatible, substantially spherical particles. Each of the particles are linked to neighboring particles.
  • the shell defines an inner chamber and has a plurality of pores extending therethrough.
  • the chamber is sized for housing an active agent and filled with a second solvent that is substantially identical to the first solvent.
  • FIG. 1 depicts a drawing of the steps in a method of forming a colloidosome in a water-in-oil system described herein, fexample 5
  • FIG. 2 depicts a drawing showing a cross-sectional view of a colloidosome in decalin formed according to the methods described herein.
  • PMMA polymethylmethacrylate.
  • FIG. 3 depicts a side view of self-assembled particles forming the shell of a colloidosome according to at least some embodiments of the invention.
  • FIG. 4 depicts brightfield optical micrographs of coUoidosomes formed with polystyrene particles and stabilized with poly-L-lysine according to the method described in example 1.
  • (a) shows coUoidosomes formed from 1.3 ⁇ m diameter particles and
  • (b) shows coUoidosomes formed from 0.5 ⁇ m diameter particles.
  • the coUoidosomes in this figure have been transferred into water from a toluene/octanol solution as described in example 1.
  • FIG. 5 depicts brightfield optical micrographs of coUoidosomes formed with polystyrene particles without stabilization in a toluene/octanol solvent as more fully described in example 1.
  • (a) shows coUoidosomes formed from 0.5 ⁇ m diameter particles and
  • (b) shows coUoidosomes fomed from 1.0 ⁇ m diameter particles.
  • FIG. 6 depicts 3-dimensional confocal fluorescence images of coUoidosomes formed with 0.7 ⁇ m polymethylmethacrylate beads in a water-in-oil system without stabilization as more fully described in example 2.
  • Top a 3- dimensional projection;
  • Bottom a 3-dimensional reconstruction.
  • FIG. 7 depicts scanning electron micrographs of a 10 ⁇ m diameter colloidosome formed from 0.9 ⁇ m diameter polystyrene spheres in 50 volume % vegetable oil and 50 volume % toluene.
  • the coUoidosomes have been dried after sintering at 105°C for 5 minutes and interface removal as more fully described in example 4;
  • (b) shows a 10 ⁇ m diameter colloidosome and
  • (a) shows a close-up view of (b).
  • the arrow in (a) points to one of the 0.15 ⁇ m pores that define the permeability.
  • FIG. 8 depicts micrographs of coUoidosomes demonstrating their selective permeability.
  • CoUoidosomes formed from 0.9 ⁇ m diameter particles in 50 volume % vegetable oil and 50 volume % toluene in an aqueous solvent were subject to interface removal and were exposed to 0.5 ⁇ m and 0.1 ⁇ m probe particles in the exterior phase for 8 hours prior to recording the images in (a) and (b); (a) brightfield microscope image, the arrows point to larger probe particles that are excluded from the interior of the colloidosome; (b) a fluorescence micrograph, arrow points to smaller probe particles that can pass through the pores of the colloidosome and enter the chamber therein.
  • FIG. 9 depicts scanning electron micrographs of coUoidosomes prepared with 0.9 ⁇ m polystyrene beads modified with aldehyde sulfate groups after sintering for various periods of time [0 minutes (upper left); 5 minutes (upper right); 20 minutes (lower left); and 2 hours (lower right)] as more fully described in example 4.
  • FIG. 10 depicts confocal fluorescence images of colloidsomes formed with polymethylmethacrylate in decalin and stabilized by swelling as described in example 5.
  • Top a top view of a colloidosome;
  • Middle an oblique view of a colloidosome;
  • Bottom a view of a broken colloidosome.
  • FIG. 11 is a brightfield optical micrograph of a multi-layered colloidosome formed with polystyrene (latex) beads functionalized with sulfate in dodecane/ethanol according to the method described in example 6.
  • FIG. 12 depicts a brightfield optical micrograph of coUoidosomes formed with amidine-modified polystyrene beads as described in example 7. (a) top view of a colloidosome; (b) bottom view of the colloidosome in (a).
  • FIG. 13 depicts a colloidosome encapsulating a fibroblast cell.
  • the colloidosome was formed from polymethylmethacrylate beads in decalin as more fully described in example 8.
  • FIG. 14 is a drawing that depicts a cross-section of a colloidosome having encapsulated therein a pancreatic cell that secretes insulin. As seen in the figure, antibodies are prevented from entering through the pores of the colloidosome whereas insulin can exit the colloidosome through the pores.
  • a method includes providing particles formed from a biocompatible material in a first solvent and forming an emulsion by adding a first fluid to the first solvent, wherein the emulsion is defined by droplets of the first fluid surrounded by the first solvent.
  • the emulsion may be an oil-in-water or a water-in-oil emulsion.
  • the method includes coating the surface of the droplets with the particles and stabilizing the particles on the surface of the droplets to form a stable colloidosome that has a yield strength of at least about 20 Pascals.
  • the coUoidosomes formed include an outer layer, or shell, of the particles that define an internal enclosure, such as a chamber or cavity, and will be more fully described herein.
  • the method may include transferring the colloidosome into a second fluid and isolating or otherwise recovering substantially intact coUoidosomes, wherein the second fluid is substantially identical to the first fluid, or alternatively, the second fluid is substantially different from the first solvent.
  • substantially intact it is meant herein that at least about 80%, or at least about 90%, or at least about 95%, and even at least about 99% of the coUoidosomes remain intact after removing the oil-water interface by transferring the coUoidosomes from the, for example, first solvent into a second fluid substantially the same as the first fluid as described herein.
  • substantially identical it is meant herein that the fluids involved are chemically similar to each other and/or have similar solubility properties. Additionally, “substantially identical" fluids include fluids in which one can not observe separate phases if the fluids are mixed together and/or the fluids are 5 otherwise miscible.
  • the second fluid and the first fluid can be aqueous solvents.
  • the second fluid and the first fluid can be organic solvents.
  • substantially different it is meant herein that the fluids involved are not chemically similar to each other and/or do not have similar solubility properties.
  • substantially different fluids include fluids in which one can observe separate phases if the fluids are mixed together and/or the fluids are otherwise immiscible.
  • the second fluid can be an organic solvent and the first fluid can be an aqueous solvent.
  • the coUoidosomes are surprisingly able to withstand a large amount of yield stress.
  • the formed structures may be advantageously used, for example, for encapsulating a desired active agent. Accordingly, in another aspect of the invention, methods for encapsulating desired active agents are also provided.
  • a method for encapsulating a desired active agent includes providing particles formed from a biocompatible material in a first solvent and forming an emulsion by adding a first fluid, such as a solvent, containing an active agent to the first solvent wherein the emulsion is defined by droplets of the first fluid surrounded by the first solvent.
  • the method includes
  • the 30 includes a shell formed of biocompatible, substantially spherical particles wherein each of the particles are linked to its neighboring particles.
  • the outer shell defines an inner chamber and has a plurality of pores.
  • the chamber is sized for housing an active agent.
  • the colloidosome is non-biodegradeable, but may be biodegradeable upon selection of appropriate starting materials in selected circumstances as desired. Additionally, in at least some embodiments, the colloidosome has a yield strength of at least about 20 Pascals.
  • the particles are linked to neighboring particles by van der Waals forces, or other electrostatic forces; chemical cross-linking of the particles, from coalescence of the particles in one or more regions of the particles or from a combination thereof.
  • a method includes providing particles formed from a biocompatible material in a first solvent and forming an emulsion by adding a first fluid to the first solvent, wherein the emulsion is defined by droplets of the first fluid surrounded by the first solvent.
  • the method includes coating the surface of the droplets with the particles and stabilizing the particles on the surface of the droplets to form stable coUoidosomes having a yield strength of at least about 20 Pascals.
  • the method in certain embodiments, includes transferring the coUoidosomes from the first solvent into a second fluid substantially identical to the first fluid and recovering substantially intact coUoidosomes as described herein.
  • FIG. 1 a fabrication method used to form coUoidosomes in at least some embodiments of the invention is described.
  • the method is described for a water-in-oil system, but may readily be used to obtain oil-in-water emulsions.
  • colloidal particles are first suspended in oil.
  • aqueous solution For clarity, only a single droplet of aqueous solution is shown being added to form an emulsion.
  • the solution may be swirled or otherwise mixed slightly, if desired. However, high shear is not required to self-assemble the colloidosome. Beads are locked together as indicated in the figure by a swelling process or with use of a polyelectrolyte, such as a polycationic agent as more fully described herein.
  • coUoidosomes are then isolated and subject to interface removal by a centrifugation process. It has been determined herein that at least about 100, or in other embodiments at least 1000, coUoidosomes can be produced in a single test tube according to the methods described herein and it is expected that the process can be scaled to larger quantities.
  • the fluids such as the solvents, utilized in the methods described herein are, in certain embodiments, liquids, such as organic solvents and aqueous solvents, although use of gaseous fluids is also envisioned as more fully described herein.
  • the fluids are selected such that the fluid used to form the droplet and the fluid in which the droplet is placed to form the emulsion are immiscible.
  • the choice of fluids selected will depend on the nature of the particles used to make the colloidosome, and the nature of the internal liquid phase of the colloidosome.
  • the particles may be suspended in an organic solvent as the first solvent and the emulsion can be formed with water or other aqueous solution as the first fluid. If a colloidosome with a cavity filled with an organic phase is desired, then the particles may be suspended in an aqueous solvent as the first solvent and the emulsion may be formed with an organic solvent as the first fluid.
  • aqueous solvents include water, and liquids highly soluble in water, such as glycerol, ethylene glycol, formamide or similar solvents and combinations thereof.
  • the solvent includes water.
  • organic solvents may be utilized. Such organic solvents are generally water- immiscible fluids, or fluids that that, when combined, can form discrete interfaces. Organic solvents typically will dissolve only trace quantities of an aqueous solution, such as no more than about 0.0001 g to 0.001 g aqueous solution/g of solvent. As described herein, such organic solvents include various oils.
  • Suitable organic solvents include hydrocarbons, including alkanes such as dodecane and hexadecane; aromatic hydrocarbons, including toluene and benzene; decalin, selected alcohols, such as octanol; silicon oil, vegetable oil or other natural oil or similar solvents, and combinations thereof.
  • the particles utilized in the methods are typically formed of biocompatible materials that can self-assemble at an oil-water interface.
  • Use of the term “oil” herein includes organic solvents as described herein.
  • the particles are, in certain forms of the invention, formed of hydrophilic or hydrophobic components or other materials, or combinations thereof.
  • hydrophilic and hydrophobic are used herein and are defined in the art to mean “water-loving' and “water-hating”, respectively.
  • hydrophilic component denotes a material that has functional or other chemical groups which have a strong affinity for water compared to a hydrophobic group whereas the term “hydrophobic component” denotes a material that has functional or other chemical groups which have little or no affinity for water compared to a hydrophilic group as known in the art.
  • the components may be monomeric, but are polymeric in other embodiments.
  • Exemplary hydrophobic materials used to form the particles include polystyrene, polyalkylmethacrylat.es, such as polymethylmethacrylate, polyethylmethyacrylate, polybutylmethacrylate; polyalkylenes, including polyethylene and polypropylene; and inorganic materials such as ceramics and including silica, alumina, titania that are surface-functionalized to make them hydrophobic.
  • Suitable hydrophilic materials used to form the particles include organic polymers that can be functionalized with hydrophilic groups; clay particles, such as disk-shaped particles; biological materials, including pollen grains, seeds, and virus particles that have been treated so as to be non-infective or to otherwise to not cause disease; and particles, including nanoparticles, composed of metallic, electrically semiconducting or insulating materials, including gold, cadmium sulfide, cadmium selenide, zinc sulfate and combinations thereof.
  • nanoparticles refers to particles with diameters less than about 20 nm
  • the materials used to form the particles are derivatized or otherwise modified with selected functional groups in order to, for example, decrease aggregation of the particles.
  • selected functional groups for example, hydrophobic polystyrene particles are suspended in an aqueous solvent
  • introduction of ionic groups leads to sufficient repulsion of particles so that they will not associate to the point of forming agglomerates.
  • the functional groups may be anionic or cationic.
  • Suitable anionic groups include, for example, carboxylate, sulfate, aldehyde sulfate, aldehyde amidine, aliphatic amines and other groups and combinations thereof.
  • Suitable cationic groups include amine, amidine and combinations thereof.
  • the particles are substantially spherical or some similar shape. Thus, at least about 90% of the particles, in other embodiments at least about 95%, and in yet other embodiments at least about 100% of the particles are spherical or otherwise in the form of a bead.
  • the emulsion is formed by adding or otherwise suspending a first fluid in the first solvent.
  • the first fluid is in the form of small drops, or droplets, in certain forms of the invention and is substantially immiscible in the first solvent.
  • the droplets may be formed by adding the first fluid to the first solvent and gently agitating the container in which the first solvent is contained. Such a process also accelerates the self-assembly process. As this may generate some shear stress on the system, in some embodiments the droplets are formed with little or no shear stress during the self-assembly process by use of a pipet or by injecting the droplets into the solution with conventional droplet-forming machines known to the art.
  • the size of the gas droplets may be similarly controlled by appropriate modification of the convention machine described herein.
  • shear stress may be determined by measuring the solvent velocity gradient and the solvent viscosity as known in the art and multiplying these values together.
  • the size of the coUoidosomes formed in the method depends primarily on the size of the template emulsion droplet and the diameter of the particles utilized. In at least some embodiments, the droplet may range from, for example, about 50 nm to about 1000 ⁇ m, or about 10 ⁇ m to about 300 ⁇ m.
  • the coUoidosomes can similarly range in size from about 50 nm to about 1000 ⁇ m, and about 10 ⁇ m to about 300 ⁇ m, depending on the thickness of the colloidosome shell. Jt is realized that the structural integrity of the colloidosome decreases as a function of increasing diameter and should be taken into account when forming such structures.
  • the particles self-assemble at the interface between the two fluids.
  • self-assembly of the particles is driven by the minimization of total interfacial energy and whether they self-assemble is determined by the three interfacial energies (i.e., oil/water, oil/particle and water/particle) as discussed in, for example, Pieranski (1980) Physics Rev. Lett. 45:569-572.
  • at least about 90%, or at least about 95%, or even at least about 99% of the surface area of the droplets are covered with the particles.
  • the coUoidosomes may then be stabilized in a variety of ways.
  • the particles may be linked to each other by van der Waals forces or other electrostatic interactions, with use of chemical cross-linking agents for inter- particle cross-linking, by a swelling process or by a sintering process. The latter processes can lead to physical linking or attachment of the particles to each other.
  • the particles are linked by cross-linking between reactive surfaces of adjacent beads.
  • the cross-linking agent is added to the first solvent after the colloidosome is formed.
  • a wide variety of cross-linking agents may be used, including dicyclohexylcarbodiimide (DCC), or other similar cross-linking agents known to the art, and combinations thereof.
  • DCC dicyclohexylcarbodiimide
  • the particles are linked by mechanically locking adjacent beads. This is accomplished by forming bridge, or necks, between beads.
  • the solvent-suspended coUoidosomes are incubated in an oven at the glass transition temperature (T g ) of the polymer that the particle is formed of for a period of time sufficient to at least partly coalesce the particles or otherwise merge or join the particles to increase the structural integrity of the coUoidosomes. If T g is higher than the boiling point of the solvent, the boiling point of the solvent may be increased by addition of solutes known to the art to increase the boiling point.
  • glycerol for an aqueous solution, glycerol, ethylene glycol, or other known solution or composition that increases the boiling temperature of an aqueous solution, or a combination thereof, may be utilized to increase the boiling point of the solution.
  • organic solvent other organic solvents having a higher boiling point may be added to the organic solvent utilized to increase the boiling point of the solution.
  • the particles may at least partly coalesce and linkages or "necks" between neighboring particles may be formed.
  • part coalesce it is meant herein that a region of one particle and an opposing region of a neighboring particle will melt and mix together such that a continuous linkage or other bridge between the particles is formed and remains after the sintering process is completed and the particles have cooled to their initial termperature prior to the process, such as room temperature.
  • deformation of the beads can increase the bead-bead contact area, making the attractive force between the beads stronger without coalescence.
  • the time period for the sintering process should be selected such that complete coalescence does not occur whereby a non-porous shell is formed, unless such complete coalescence, and coUoidosomes without pores, are desired. Although this time period may vary depending on the nature of the colloidosome and components and solvents utilized to form the coUoidosomes, in some embodiments of the invention the coUoidosomes are heated for a period of about 2 minutes to about 120 minutes, or no more than about 5 minutes.
  • the structure of the coUoidosomes is stabilized using a swelling method.
  • the colloidal particles can be at least partially coalesced to form a structurally stronger shell, or may otherwise exhibit increased interparticle attraction.
  • the first solvent is organic
  • one or more organic solvents in which the colloidal particles are soluble in are added to the first solvent to otherwise contact the particles for a period of time sufficient for a region of the particles to at least partially solubilize and thereby at least partially coalesce with a region of its neighboring colloidal particles.
  • an appropriate organic solvent includes toluene.
  • an appropriate organic solvent includes a combination of chlorobenzene and decalin in a volume ratio of about 35:65.
  • suitable organic solvents may be determined by the skilled artisan taking into account the nature of the colloidal particle.
  • the time required to stabilize the particles with the use of organic solvents described herein will vary with the nature of the solvents and the particles utilized. Generally, the amount of time the solvents contact the particles is about 1 minute to about 10 minutes.
  • the coUoidosomes are stabilized by utilizing one or more polyelectrolytes.
  • the polyelectrolyte can be added to the solvent which is emulsified or may be added to the first solvent that includes the, for example, beaded particles.
  • the nature of the polyelectrolyte will depend on the nature of the charge on the surface of the colloidal particles.
  • polycationic agents can be utilized when the net charge on the surface of the colloidal particle is predominantly negative or when only negatively charged functional groups are on the surface
  • polyanionic agents can be utilized when the net charge on the surface of the colloidal particle is predominantly positive or when the surface of the colloidal particles includes only positively charged functional groups.
  • Exemplary polycationic agents include polyamino acids, including poly-L-lysine; poly(diallyldimethylammonium chloride)(PDMAC), poly(allylamine hydrochloride) or other similar polycationic agents or combinations thereof.
  • Suitable polyanionic agents include, for example, poly(styrene sulfonate), including poly(sodium 4-styrenesulfonate); or other suitable agents or combinations thereof.
  • the surface of the particles can be modified utilizing various ionic functional groups as previously described. Additionally, the surface can be modified with other agents that will bind to another agent that may be added to the first fluid. For example, the particles can be modified with biotin and avidin can be added to the system. Other such combinations of agents include, for example, biotin and streptavidin.
  • the coUoidosomes are isolated in a variety of ways.
  • the interface is removed and the coUoidosomes are isolated by use of centrifugal force, such as by transferring coUoidosomes that are suspended in an organic solvent into an aqueous solvent wherein the chamber of the coUoidosomes is filled with an aqueous solvent, or vice versa.
  • the coUoidosomes are being transferred from an organic solvent into an aqueous solvent, for example, aliquots of the coUoidosomes are placed on the top of the desired aqueous solution, which can include a non-ionic surfactant such as, for example, Tween, SPAN, Triton or other suitable non-ionic surfactant.
  • a non-ionic surfactant such as, for example, Tween, SPAN, Triton or other suitable non-ionic surfactant.
  • the coUoidosomes are being transferred from an aqueous solvent into an organic solvent, such as where the internal chamber of the colloidosome is filled with an organic solvent, the coUoidosomes are placed on the top of the desired organic solvent which has a density greater than the density of the aqueous solvent, but less than the density of organic solvent in the chamber of the colloidosome.
  • the coUoidosomes are then centrifuged at a centrifugal force and for a period of time sufficient for isolation. Although this time period may vary depending on the circumstances, the coUoidosomes can be centrifuged at about 2000 g to about 14000 g for about 5 minutes to about 30 minutes. Typically, the coUoidosomes can be centrifuged at about 9300 g for about 10 minutes in order to remove the coUoidosomes from the oil-water interface.
  • Other methods of isolating the coUoidosomes herein include drying. The drying process is performed by soaking the coUoidosomes in ethanol to remove the interface and then allowing the ethanol to evaporate. This is an effective drying process when both solvents used are miscible in ethanol.
  • the coUoidosomes formed have advantageous mechanical properties. For example, not only are the coUoidosomes elastic, they have a yield strength of about 20 Pascals to about 100 Pascals, or about 20 Pascals to about 500 Pascals, or about 20 Pascals to about 1000 Pascals and even at least about 20 Pascals to about 2000 Pascals. Additionally, the coUoidosomes have a yield strength of at least about 20 Pascals, or at least about 50 Pascals, or at least about 100 Pascals, or at least about 200 Pascals, or at least about 500 Pascals, or at least about 750 Pascals and even at least about 1000 Pascals. The yield strength can be determined by using a cantilever to mechanically deform the colloidosome with a known stress and observing the response.
  • coUoidosomes which release their contents upon exposure to an applied force.
  • the yield strength of the coUoidosomes is selected to allow capsule destruction during chewing to be no more than about 10 MegaPascals.
  • the yield strength of the coUoidosomes can be more than about 1 MegaPascals
  • the coUoidosomes formed are structurally stable or otherwise have sufficient structural integrity so that at least about 80%, or at least about 90%, or at least about 95%, and even at least about 99% of the coUoidosomes remain intact after removing the oil-water interface as described herein.
  • the interface can be removed as noted herein by, for example, transferring the coUoidosomes from the first solvent into a second solvent substantially identical to the first fluid or by other methods described herein that do not substantially affect the structural stability of the coUoidosomes, or otherwise damage the coUoidosomes.
  • the number of coUoidosomes that remain intact after the transfer is typically determined by inspection utilizing an optical microscope.
  • FIG. 2 a drawing of a cross-section of a colloidosome formed with polymethylmethacrylate (PMMA) colloidal particles utilizing an aqueous solution in decalin oil according to the methods described herein is shown.
  • colloidosome 10 includes shell 20 formed of a monolayer of particles 30 that defines inner chamber 40.
  • FIG. 3 depicts a drawing showing a cross-section of a colloidosome 50 that includes close-packed spheres 60 and interstitial pores 65.
  • the pore size may be controlled by, for example, the size of the particles utilized to form the colloidosome. For example, use of beaded particles of larger diameter lead to larger pore sizes whereas use of beads of smaller diameter lead to smaller pore sizes. Additionally, a mix of both smaller particles and larger particles can be used in forming a colloidosome to achieve a smaller pore size while retaining the advantageous properties of coUoidosomes made with only larger particles.
  • a method includes providing particles formed from biocompatible materials in a first solvent and forming an emulsion by adding a first fluid, such as a solvent, containing the active agent to the first solvent wherein the emulsion is defined by droplets of the first fluid surrounded by the first solvent.
  • the method includes coating the surface of the droplets with the particles and stabilizing the particles on the surface of the droplet to form a colloidosome having encapsulated therein the desired active agent.
  • the coUoidosomes formed have a yield strength of at least about 20 Pascals or otherwise as described above.
  • the method includes transferring the coUoidosomes from the first solvent into a second fluid substantially identical to the first fluid and recovering substantially intact coUoidosomes as described herein.
  • the particles utilized in the methods of encapsulating an active agent are also substantially spherical and can be formed of biocompatible polymers as described herein.
  • one form of the method of encapsulation is similar to the methods of forming the coUoidosomes described herein with the exception that the first fluid is a solvent that includes or otherwise contains the desired active agent.
  • the first fluid can be selected based on the nature of the active agent, and may be a liquid or a gas. Therefore, the first fluid can be chosen such that it can solubilize the active agent or will otherwise be compatible with the active agent.
  • the active agent is a hydrophobic material, such as certain drugs
  • methods would include utilizing an oil-in-water emulsion such that the first solvent is aqueous and the first fluid is an organic solvent or oil.
  • the active agent is a hydrophilic material, such as some biological macromolecules, or is a material that is otherwise compatible with aqueous solutions, such as biological cells, then a water-in-oil system is used wherein the first solvent is an organic solvent or oil and the first fluid is an aqueous solution.
  • active agents may be encapsulated according to the methods described herein.
  • Active agent refers to an agent that has a beneficial effect in a biological system, such as in or on the body of a patient, or otherwise provides advantages when added or otherwise applied to a system as described herein.
  • the active agent can be, for example, a biological agent or a chemical agent.
  • Chemical agents include, for example, drugs or other pharmaceutical agents, flavoring agents or chemicals that give rise to fragrances.
  • the biological agents include, for example, biological macromolecules, such as proteins, nucleic acids, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that can encode, for example, a desired protein; vitamins, fats or other lipids; carbohydrates, and combinations thereof to form various food products.
  • the active agent can also be a gas, such that when the coUoidosomes are added to, or are formed in, such a system in which foams are formed, the foams are stabilized.
  • the foams formed in this manner are opaque from the scattering of light by the particles that are present on the gas droplet surfaces. This can be advantageous when applied to foods, as opaque foams are more aesthetically pleasing.
  • the active agents can diffuse out of the colloidosome through the pores if the active agent is sized to fit through the pores. If it is desired that such active agents whose size is larger than the pore size be released from the capsule in use, a wide variety of methods are available. For example, it has been determined herein that the contents can be released by rupture if sufficient shear is applied, or by application of compressive stresses. Additionally, because the fabrication process depends only on the surface properties of the colloidal particles, there is substantial freedom to choose the material in the core of the particles to add functionality. In at least some embodiments, a portion of the particles may be made of a material that increases its volume, for example, upon increasing the pH as in alkali-swellable microgel particles.
  • some of the particles could be made from a material easily dissolved in situ (chemically or photochemically), thus creating large holes in the capsule and releasing the contents.
  • a colloidosome in one form, includes a shell formed of biocompatible, substantially spherical particles wherein each of the particles are linked to a neighboring particle, typically each of its neighboring particles.
  • the shell is an outer layer that defines an inner chamber or cavity and has a plurality of pores extending therethrough.
  • the chamber is sized to house or otherwise contain an active agent as described herein.
  • the shell is formed of a monolayer of the spherical particles, although multi-layer shells are also envisioned as more fully described below.
  • the coUoidosomes are quite strong, having the preferred yield strengths as described above.
  • the coUoidosomes can withstand relatively high yield shear rates.
  • the coUoidosomes such as those having a diameter of about 10 ⁇ m to about 50 ⁇ m in water, have a yield shear rate of at least about 10s- 1 , or at least about 25 s " ⁇ or at least about 50 s "1 , even at least about 75 s "1 and even at least about 100 s "1 .
  • Such yield strengths may further be greater than about 100 s "1 in certain forms of the invention.
  • the nature of the spherical particles or other components of the coUoidosomes has already been described above.
  • the coUoidosomes can be substantially spherical, elliptical or other rounded shape.
  • the aspect ratio of the colloidosome can be about 2:1.
  • the thickness of the outer shell, or layer is dependent on the diameters of the particles utilized to form the colloidosome and the number of layers present.
  • the shell is an outer layer that is a monolayer of the particles and thus the thickness of the shell can range from about 20 nm to about 20 ⁇ m, or about 100 nm to about 10 ⁇ m, or about 0.5 ⁇ m to about 1 ⁇ m.
  • the shell may be formed from multiple layers of the particles, including two, three, four or more layers, and thus the diameter of the outer layer can be two, three, four or more times the diameters mentioned above. Such multiple layers can be formed, for example, by allowing aggregation of the particles when suspended in the first solvent as described above.
  • the outer shell defines an enclosure, such as a chamber or cavity that may advantageously be utilized to house or otherwise contain an active agent as described herein.
  • the size of the chamber is dependent on the size of the emulsion droplet template, and can thus be varied accordingly as described herein. As described above, the droplet, and thus the diameter of the chamber, can range in size from, for example, 50 nm to about 1000 ⁇ m, or about 10 ⁇ m to about 300 ⁇ m. The size of the chamber is chosen depending on the application.
  • the chamber is sized to accommodate the cell and is, for example, at least about 10 ⁇ m in diameter.
  • the diameter of the colloidosome can be about 50 nm to about 1000 ⁇ m or about 10 ⁇ m to about 300 ⁇ m,.
  • At least about 50% of the coUoidosomes, further at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% and even at least about 95% of the coUoidosomes have a diameter of about 50 ⁇ m to about 200 ⁇ m or can be.greater than at least about 50 ⁇ m.
  • the coUoidosomes have well-defined pores whose size can be varied depending on the application. For example, if a colloidosome has encapsulated therein a biological cell, the pores are large enough to allow any desirable substance produced by the cell to diffuse out of the chamber through the pores and external to the colloidosome, as well as allow desirable substances necessary to sustain the cell, such as glucose or other nutrients, to enter the chamber. It is realized that the pores for such an application are sufficiently small or otherwise sized to prevent entry into the chamber by immune system cells or immune system components, such as various antibodies, as well as to prevent the encapsulated cell from exiting the chamber through the pores. As previously described herein, the pore size can be adjusted by the size of the particles utilized.
  • pore size can vary depending on the application, pore sizes can range from about 3 nm to about 3 ⁇ m, about 10 nm to about 1000 nm, or about 75 nm to about 200 nm. When encapsulating a biological cell, pore sizes are typically no more than about 1 ⁇ m to about 3 ⁇ m.
  • the pore sizes in a colloidosome are substantially uniform. That is, at least about 90%, or about 95%, or even about 100% of the pores of the colloidosome are of the same size and may, for example, have the same radius and thus the same diameter.
  • the radius of the pores may differ by about 50% to about 300%, resulting in pores differing in diameter by up to a factor of about 1.5, or even by a factor up to about 4.
  • the pores may differ in radius by up to about 50%.
  • the coUoidosomes described herein are be included in a suspension.
  • the colloidosome suspension includes, in at least one embodiment, a colloidosome suspended in a first solvent wherein the colloidosome has a shell formed of biocompatible, substantially spherical particles. Each of the particles are linked to neighboring particles as previously described.
  • the shell defines an inner chamber and has a plurality of pores extending therethrough.
  • the chamber is sized for housing an active agent and filled with a second solvent that is substantially identical to the first solvent.
  • the chamber is filled with an aqueous solution and the solvent that the colloidosome is suspended in (i.e., the exterior solvent) may be the same or a similar aqueous solution.
  • solvents, as well as other solvents, have been previously described herein.
  • the suspensions of polystyrene beads were obtained from Interfacial Dynamics Corporation (IDC).
  • IDC Interfacial Dynamics Corporation
  • Divinylbenzene crosslinked beads 1.3 and 0.5 ⁇ m in diameter with carboxyl surface charge groups (DVB carboxyl beads) were used, along with biotin-coated 0.9 ⁇ m-diameter beads with aldehyde sulfate surface charge groups (aldehyde sulfate beads).
  • VVB carboxyl beads carboxyl surface charge groups
  • aldehyde sulfate beads biotin-coated 0.9 ⁇ m-diameter beads with aldehyde sulfate surface charge groups
  • the carboxyl-modified fluorescent polystyrene probe particles (1.0 ⁇ m-diameter, excitation/emission wavelengths of 580/605nm; 0.5 ⁇ m, 580/605; 0.1 ⁇ m, 505/515nm) were provided by Molecular Probes.
  • the 1-octanol, toluene, dodecane, glycerol (all 99% pure), dimethyldichlorosilane, TWEEN20, and SPAN80 were purchased from Aldrich and not subject to further purification before use.
  • the silicone oil (Fluka), ethanol (200 proof, Pharmco), acetone (Baker) and poly-L-lysine 0.1 % w/v aqueous solution (Sigma, P8920) were also used as obtained from the manufacturers.
  • the deionized water (Dl) used for the experiments was purified by a Millipore Milli-Q system. Wesson vegetable oil was filtered with a 0.45 ⁇ m-pore hydrophobic syringe filter prior to use.
  • Polystyrene beads cross-linked with divinyl-benzene (DVB-PS; 0.5 ⁇ m and 1.3 ⁇ m diameter) and carboxylate-modified were obtained from Interfacial Dynamics, Portland, OR.
  • the internal cross-linking with divinyl benzene prevents dissolution of the particles in toluene.
  • the beads were suspended in a solution of 90 volume % toluene and 10 volume % octanol at a volume fraction of about 10 "3 .
  • the beads were then locked together to form a strong shell that remains intact after the water-oil interface is removed.
  • the above procedure was repeated, except that the aqueous phase was a 1 mg/ml polycationic poly-L-lysine (150-300 kD) solution.
  • the exterior phase was then replaced with water.
  • the capsules were washed in octanol and approximately 0.1 ml of the octanol-capsule solution was added to the top of 1 ml of an aqueous solution of non-ionic surfactant (10 mg/ml of Tween 20).
  • the capsules were centrifuged at 9300 g for 10 minutes.
  • the permeability of the coUoidosomes to probe colloidal particles of various sizes was then quantified.
  • the coUoidosomes were suspended in water containing polystyrene spheres of various sizes. Individual coUoidosomes were inspected in an optical microscope and the number and sizes of particles within the coUoidosomes was determined. Analysis
  • FIG. 5 shows brightfield optical micrographs of colloidosome formed as described herein with 1.3 ⁇ m polystyrene beads as described herein.
  • the image in (a) depicts coUoidosomes formed utilzing 1.3 ⁇ m diameter beads whereas the image in (b) depicts coUoidosomes formed from 0.5 ⁇ m diameter particles.
  • the image was taken by optical microscopy as described herein and known to the art.
  • coUoidosomes were formed as in example 1 , with the exception that, instead of polystyrene beads in toluene/octanol, polymethylmethacrylate (PMMA) beads (0.7 ⁇ m diameter) were suspended in decahydronapthalene (decalin) and no poly-L-lysine, or other stabilizing agent, was used for stabilization.
  • PMMA polymethylmethacrylate
  • decalin decahydronapthalene
  • the coUoidosomes were not transferred into water in this example. In this system an ordered, complete monolayer of colloidal spheres was also observed as seen in the fluorescence confocal microscope image seen in FIG. 6.
  • polystyrene spheres (0.9 ⁇ m diameter functionalized with biotin, from Interfacial Dynamics, Portland, OR) were suspended in water at a volume fraction of 10 "3 .
  • the polystyrene beads assembled at the surface of the oil droplets as discussed in the preceding examples, except that the particles surprisingly adhered to one another at the surface.
  • the beads were unexpectedly stable in the aqueous solution and it was therefore not necessary to add any stabilization agent to the oil phase to lock the beads together at the surface.
  • the external water phase was replaced with oil by placing a 0.1 ml aliquot of the aqueous solution with coated droplets in a vial on top of 1 ml of dense silicone oil and the coUoidosomes were centrifuged at 9300 g for 10 minutes.
  • the stabilization arises from the enhanced interparticle attraction provided by the silicon oil due to diminished electrostatic repulsion.
  • polystyrene spheres that were biotinylated and functionalized with aldehyde sulfate were added to water at a volume fraction of about 10 "3 .
  • a sintering process was utilized.
  • 50 volume % glycerol was added to the exterior aqueous phase to increase the boiling point of the solution prior to exposing the solution to a temperature of 105°C for about five minutes.
  • the polystyrene particles coalesced slightly, creating 150 nm necks between them.
  • the shell therefore contained a continuous polystyrene shell with a regular array of holes.
  • the coUoidosomes were washed with ethanol and dried in a vacuum so they could be viewed under an electron microscope. Scanning electron micrographs of a dried collidosome prepared by this method are shown in FIG 7. It was found that sintering the particles for longer times had an effect on the pore size and, it is believed, the strength of the capsule. For example, after sintering the particles for 20 minutes, the particles coalesced completely and the holes were completely filled.
  • FIG. 8 depicts microscope images of coUoidosomes used in determining the permeability of the coUoidosomes. The colloidosome were formed as described above for the colloidosome in FIG. 7 but were not dried prior to analysis.
  • the image in (a) is a brightfield microscope image showing that the larger probe particles (i.e., 0.5 ⁇ m diameter) denoted by the arrow are excluded from the interior of the colloidosome. Note that diffraction from the particles that form the colloidosome shell is faintly visible.
  • the image in (b) is a fluorescence image showing that smaller probe particles (i.e., 0.1 ⁇ m diameter) as indicated by the arrow are able to pass through the pores and into the interior of the colloidosome. It was determined by the method described in example 1 that coUoidosomes prepared by this method after sintering for 5 minutes were impermeable to 0.5 ⁇ m diameter probe particles but were permeable to 0.1 ⁇ m diameter probe particles.
  • the effect of sintering time on coUoidosomes formed with 0.9 ⁇ m biotin-coated polystyrene beads with aldehyde sulfate surface charge groups after drying is shown in FIG. 9.
  • the coUoidosomes prepared with polystyrene functionalized with aldehyde sulfate and biotinylated were sintered for a period of 5 minutes, 20 minutes and 2 hours and scanning electron micrographs were taken. After sintering, the coUoidosomes were soaked in ethanol for 24 hours and allowed to dry in air for 24 hours. As seen in FIG.
  • PMMA particles (0.03 ml of 10-20 volume % PMMA in decalin) (provided by Andrew Schofield, University of Edinburgh), were suspended in 0.3 ml decalin.
  • the volume fraction of PMMA particles in this mixture was about 1 -2%.
  • About 1 to 10 microliters of water (first fluid) was then added (the water can include 0.1 M NaCI and/or fluorescein dye. The dye is for aiding visualization of the particles).
  • the sample was then shaken to produce small (20-300 micron) water drops. Alternatively, the water phase can be added as one large droplet (about 400 microns). Chlorobenzene was then added to the decalin contaning the coated droplets in an amount of 0.2 ml.
  • this step swells the particles, since chlorobenzene is a good solvent for PMMA.
  • chlorobenzene is a good solvent for PMMA.
  • about 0.1 mL of the above chlorobenzene/decalin/PMMA/water solution was added to 3mL of a mixture of 50 volume% toluene and 50 volume % decalin.
  • the coated droplets were transferred to 4 ml of decalin by withdrawing the droplets in a pipette and injecting them into a vial with decalin in order to wash away toluene.
  • the resulting coated droplets are stable against coalescence and it is believed they can be stored in decalin indefinitely.
  • Figure 6 depicts a coUoidosomes that is representative of this particlar stage in colloidosome formation as it has not yet been stabilized.
  • FiG. 10 depicts confocal fluorescence images taken of the coUoidosomes formed according to the procedure outlined in this example. As seen in FIG. 10, bottom, a coUoidosomes wherein the droplet has ruptured is shown. The two- dimensional rafts of PMMA particles are stuck to one another as seen in the FIG. 10, bottom, thus providing evidence that the stabilization process worked.
  • coUoidosomes were formed as in example 1 , with the exception that, instead of carboxylate-modified (DVB-PS) in toluene/octanol, 1.0 micron beads with sulfate groups were suspended in dodecane (90 volume %) and ethanol (10 volume %), also at a volume fraction of 10 "3 . About 10 microliters of water were added per ml of dodecane/ethanol solution and the solution was vortexed to break the water droplets (mean droplet diameter ranged from 50 ⁇ m to 500 ⁇ m) and to accelerate particle adsorption. Results
  • An emulsion of silicon oil droplets was prepared in water by mixing equal volumes of silicon oil and water with 2g/L SDS. The mixture was pushed through a syringe filter with 1.2-micron diameter holes. The filtration was performed a total of 5 times with the aim of making a fairly uniform distribution of oil droplet sizes
  • a volume of 0.01 ml of the above oil-droplet solution was added to 1 ml of de-ionized water.
  • the SDS concentration at this point was about 0.02g/L.
  • Polystyrene beads (1 -micron-diameter and amidine-functionalized) were added to the solution (bead volume fraction about 1%). Within a few minutes, the sample was observed in an optical microscope (see attached figures). Oil droplets that were about 5 microns in diameter were observed, some of the oil droplets were fully coated with amidine beads. Not all droplets were fully coated, however, possibly due to an insufficient quantity of SDS on the droplets. Brightfield optical micrographs were taken of the resulting coUoidosomes that were formed and are seen in FIG. 12.
  • amidine beads were drawn to and/or held at the oil-water interface by electrostatic attraction.
  • the beads are cationic and the interface is anionic due to the SDS.
  • This example shows how a rat fibroblast may be encapsulated in a colloidosome as described herein.
  • the procedure is identical to example 2, except that the aqueous phase contain rat 3T3 fibroblasts (supplied by Justin Jiang, Harvard University). Cells were cultured in Hank's buffered saline (cat #14025-092, Life Technologies, Rockville, MD). About 0.1 mL of cell/buffer solution was injected into the decalin solution which contained PMMA beads, 0.7 micron diameter, about 1-2 volume%.
  • Colloidosome solution and control were stored in an incubator with controlled temperature and atmosphere.
  • Control cells and droplet-encapsulated cells were compared visually using an optical microscope after 30 minutes and 90 minutes. In both cases, cells appeared round in shape, a general indicator of good health (dying cells are distinguishable by shape).
  • the cells were seen to adhere and spread on the surface of the petri dish (an indicator of good health) and were seen to adhere on the inner (aqueous) surface of the coated droplets, as seen in FIG 13.
  • FIG. 14 is a drawing that depicts a cross-section of a colloidosome with an encapsulated pancreatic cell that secretes insulin. As seen in the figure, antibodies are prevented from entering the colloidosome whereas insulin can exit the colloidosome. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety.

Abstract

La présente invention concerne des procédés de production de structures microscopiques, élastiques, sélectivement perméables, auto-assemblées, appelées colloïdosomes, qui présentent une dimension des pores et une porosité régulées ainsi que des propriétés mécaniques intéressantes. Dans une forme de réalisation de l'invention, un procédé de formation de colloïdosomes consiste à utiliser des particules formées à partir d'un matériau biocompatible dans un premier solvant, puis à former une émulsion par ajout d'un premier fluide au premier solvant, ladite émulsion étant définie par des gouttelettes du premier fluide entourées par le premier solvant. Le procédé consiste à recouvrir la surface des gouttelettes avec les particules et à stabiliser les particules sur la surface des gouttelettes. Les colloïdosomes produits présentent une limite d'élasticité spécifique qui est au moins égale à 20 Pascals. Dans certaines formes de la présente invention, les particules sont sphériques et sont formées d'un polymère biocompatible. Des colloïdosomes formés selon les procédés de cette invention sont également présentés. Dans une forme de réalisation, un colloïdosome comprend une enveloppe formée de particules biocompatibles sensiblement sphériques qui sont chacune liées aux particules voisines. L'enveloppe définit une chambre interne dimensionnée pour loger un agent actif souhaité et comprend une pluralité de pores qui la traversent. Les colloïdosomes sont stables du point de vue de leur structure et présentent une limite d'élasticité propre qui est au moins égale à 20 Pascals. La présente invention concerne également une suspension colloïdale et des procédés d'encapsulation d'un agent actif voulu.
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