WO2003079990A2 - Microencapsulation a l'aide d'atomiseurs ultrasoniques - Google Patents

Microencapsulation a l'aide d'atomiseurs ultrasoniques Download PDF

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WO2003079990A2
WO2003079990A2 PCT/US2003/008559 US0308559W WO03079990A2 WO 2003079990 A2 WO2003079990 A2 WO 2003079990A2 US 0308559 W US0308559 W US 0308559W WO 03079990 A2 WO03079990 A2 WO 03079990A2
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polymer
microcapsules
solvent
aqueous
solution
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PCT/US2003/008559
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WO2003079990A3 (fr
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Kinam Park
Yoon Yeo
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Purdue Research Foundation
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Publication of WO2003079990A3 publication Critical patent/WO2003079990A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • 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
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/20—After-treatment of capsule walls, e.g. hardening
    • B01J13/203—Exchange of core-forming material by diffusion through the capsule wall

Definitions

  • the present invention relates to pharmaceutical compositions and methods of drug delivery.
  • the invention especially relates to methods and compositions for controlled release of easily denatured drugs utilizing microcapsules.
  • Microencapsulation technologies have advanced significantly during the last few decades and the current technologies are at such a level that drugs can be delivered at predetermined rates for days and years depending on applications. These advances, however, usually apply only to low molecular weight drugs.
  • Microencapsulation of high molecular weight drugs, such as peptides and proteins is still complicated due to the intricate nature of their physical and chemical properties.
  • protein-based pharmaceuticals have become especially important since mass production of such drugs has been enabled by recombinant DNA technology.
  • completion of the human genome project is expected to bring about an improved understanding of the therapeutic roles of specific proteins, which should lead to numerous new protein drugs. Since the early promise of sustained delivery of proteinaceous drugs [1], studies on protein microencapsulation have increased exponentially.
  • the emulsion (solvent evaporation or extraction) methods utilize volatile organic solvents for dissolving water-insoluble polymers, such as poly (lactic acid-co-glycolic acid) (PLGA).
  • PLGA poly (lactic acid-co-glycolic acid)
  • a double emulsion process is commonly used for encapsulation of water-soluble drugs such as protein or peptide. Both solid/oil/water (s/o/w) and water/oil/water (w/o/w) systems are used depending on the type of protein drug.
  • the organic solvent is removed by evaporation at a raised temperature and/or under vacuum. See, for example, U.S. Patent No. 3,523,906 (issued to Vrancken, et al. [4]).
  • the organic solvent is extracted into a large volume of continuous phase, thereby turning the emulsion drops into solid polymer microspheres. See, for example, U.S. Patent No. 4,389,330 (issued to Tice, et al. [5]).
  • the organic solvents conventionally employed in this method are typically chlorinated hydrocarbons, such as methylene chloride. In both methods, the use of chlorinated solvents often becomes a substantial drawback for environmental and human safety reasons.
  • microspheres prepared by this method limits the permitted daily exposure to 6 mg per day for methylene chloride.
  • loading capacity of microspheres prepared by this method is in general very low.
  • the way emulsions are created increases not only the total interfacial area the bioactive substances are subjected to, but also extensive shear and cavitation stress, which may be destructive to bioactive substances [6].
  • Biodegradable polymer is dissolved in an organic solvent, such as methylene chloride, together with a protein powder and then atomized over a bed of frozen ethanol overlaid with liquid nitrogen.
  • the microdrops freeze upon contacting the liquid nitrogen and then sink onto the frozen ethanol layer.
  • the frozen microspheres sink into the ethanol.
  • the methylene chloride solvent in the microspheres then thaws and is slowly extracted into eth.anol, resulting in hardened microspheres containing proteins .and polymer matrix.
  • this method can be costly and laborious, especially for scaled-up production.
  • the coacervation method is based on salting out (or phase separation) of polymers from a homogeneous solution into small drops of a polymer-rich phase upon addition of extra substances.
  • aqueous polymer solution e.g., gelatin or carboxymethylcellulose
  • a strongly hydrophilic substance e.g., sodium sulfate
  • a water-miscible non-solvent e.g., ethanol, acetone, dioxane, isopropanol, or propanol
  • the water-soluble polymer is concentrated to form the polymer-rich phase. This is known as simple coacervation.
  • the polymer-rich phase is formed on the drug particle surface to afford a capsule.
  • the polymer-rich complex (coacervate) phase is induced by interaction between two dispersed hydrophilic polymers (colloids) of opposite electric charges. Since electrostatic interactions are involved, it is important to control the pH of the medium in order to control the charges of the polymers.
  • a drug is mixed with a polymer, which is melted at high temperatures.
  • the mixture is then suspended in a non- miscible solvent with continuous stirring at a temperature several degrees above the melting point of the polymer.
  • the system is cooled until the polymer p.articles are solidified. This method requires the drug to be stable at the polymer melting temperature.
  • Interfacial cross-linking and interfacial polymerization employ two reactive phases that can form a solid boundary at their interface, which becomes the surface of microparticles.
  • the polymer For interfacial cross-linking, the polymer must possess functional groups that can be cross-linked by ions or multi-functional molecules contained in a continuous phase.
  • Interfacial polymerization requires two reactive monomers dissolved in immiscible solvents that can be polymerized at the interfaces. Capsules are collected after quenching the polymerization reaction with a third phase.
  • Spray-drying involves spraying a mixture of a drug and a polymer and evaporating the solvent in a drying chamber to solidify the atomized drops.
  • This seemingly simple process has not been widely used in the pharmaceutical industry due at least in part to difficulties in the scale-up process.
  • the parameters optimized in the laboratory scale spray dryer do not usually work for the industrial scale spray dryer.
  • the temperature of inlet gas that is used for drying the microdrops can reach 90-l"50°C [9, TOjj " which may not ' be tolerable for encapsulation of heat-sensitive biomaterials.
  • Ultrasonic atomizers have reportedly been used for microparticle formation in connection with the spray-drying or spray-congealing techniques discussed hereinabove.
  • the ultrasound energy was used to break up drug and carrier mixture into microparticles [11].
  • a lipid excipient and a drug were mixed at a temperature higher than the melting point of the excipient.
  • the resulting fluid was poured onto the oscillating surface, and the liquid was atomized into small drops upon hitting the surface.
  • the microdrops were collected in a cold chamber in which the liquid drops were solidified (spray-congealing).
  • a water/oil (w/o) emulsion consisting of an aqueous bovine serum albumin (BSA) solution and PLGA solution in methylene chloride was atomized using an ultrasonic atomizer adapted to a conventional spray dryer [12].
  • BSA bovine serum albumin
  • PLGA PLGA solution in methylene chloride
  • ultrasonic atomizer adapted to a conventional spray dryer [12].
  • protein loaded microspheres were produced using an ultrasonic atomizer (U.S. Patent No. 5,389,379, issued to Dirix et al. [13]). They collected the particles in a non-solvent bath to remove the organic solvent. The hardened drops were subsequently tansferred into the second non-solvent to harden the microspheres.
  • Supercritical fluids have recently been utilized for their unique characteristics: high compressibility and liquid-like density. Microparticles have been prepared by either rapid expansion of supercritical solutions (RESS) or supercritical antisolvent crystallization (SAS) [8].
  • RESS exploits the liquid-like solvent power of the supercritical fluids whereas SAS uses supercritical fluid as an antisolvent.
  • Carbon dioxide is most commonly used because it is environmentally benign, relatively non-toxic, non-inflammable, and inexpensive, and the condition for the critical fluid is easily attainable.
  • RESS is limited by the requirement that all solutes should be soluble in the supercritical fluid. For this reason, RESS may not be used to make protein loaded polymeric microcapsules.
  • the SAS method utilizes the supercritical fluid as an anti-solvent that causes precipitation of the solids. Therefore, the SAS method is suitable for solids that are difficult to solubilize in supercritical fluids, such as peptides and proteins.
  • the supercritical fluid approach is in its infancy and it is as yet hard to anticipate mass-production of microparticles using this method.
  • Microparticles can be categorized as “microspheres” and “microcapsules”.
  • microspheres usually refers to monolithic type formulations in which the drug molecules are dispersed throughout the polymeric matrix [14].
  • microcapsules' * refers * to ⁇ eservo' ⁇ r devices in which a drug-containing core is surrounded by a continuous polymeric layer, shell, or membrane.
  • microcapsules can be multinuclear microcapsules, where multiple drug cores are embedded throughout the polymer matrix, or mononuclear microcapsules, for which a single drug core is surrounded by the polymer membrane [15].
  • microspheres or multinuclear microcapsules One of the disadvantages of microspheres or multinuclear microcapsules is that degradation products of the polymer can easily build up to generate acidic microenvironments within the microparticles, which can be undesirable for acid-labile drugs [16]. Furthermore, the presence of abundant polymer in proximity to a large amount of drug can cause unfavorable interaction between two substances. It can be a significant problem when it comes to protein drugs, which are highly susceptible to denaturation due to hydrophobic interactions with the polymer [17].
  • microcapsules can provide a number of advantages.
  • microcapsules provide much more drug reservoir space than microspheres.
  • the reservoir space can accommodate protective excipients as well as drugs.
  • drugs located in the single core are not in extensive contact with the polymer, but only those on the surface are exposed to the degrading polymer.
  • the degradation products of the polymer would not build up within the microcapsules because they are more likely to diffuse out to the release medium rather than to the core structured by the constituent excipients.
  • the release profile can be further modified by building heterogeneous layers of membranes.
  • Mononuclear microcapsules can be produced by the interfacial cross-linking and interfacial polymerization methods. However, the complexity of the procedures and handling of the unreacted monomers can be issues that make them impractical. Gustavsson et al. [18] describe a method to coat starch microparticles with a polymer shell for control of the drug release profile. The polymer coating is obtained by suspending the core microparticles in an air-suspension coater providing a polymer solution [19]. However, the time and the labor required to finish the multi-step procedures may become obstacles when modifying the production scale.
  • the present invention achieves the objectives identified above by employing atomization technology, particularly ultrasonic atomization, in conjunction with 'solvent exchange' to produce microencapsulated particles.
  • a microencapsulation method based on solvent exchange which employs ink-jet technology, has been described previously, see, e.g., U.S. Patent Publication 2002/0160109 Al.
  • the solvent exchange process can also be achieved by simultaneously producing a large number of drops of aqueous and polymer solutions, and spatially concentrating them to facilitate collision among the drops.
  • a method for preparing a microencapsulated composition comprises: (1) providing an aqueous solution containing a composition to be encapsulated dissolved therein; (2) providing a polymer solution containing a water-insoluble polymer dissolved in a hydrophilic solvent; (3) generating a plurality of first microdroplets from the aqueous solution; (4) generating a plurality of second microdroplets from the polymer solution, wherein the first and second microdroplets are generated by at least one atomizing device; and (5) contacting the first and second microdroplets to form a plurality of pre-encapsulant particles.
  • the pre- encapsulant particles have a core domain containing the aforementioned composition and an outer layer containing the polymer, such that solvent exchange occurs between the core domain and the outer layer thereby forming a polymer shell around the encapsulated composition.
  • Atomizers for use in conducting a production metho of the present mven o ⁇ include those that employ centrifugal, pressure, kinetic, and sonic energy. Preferred atomizers are ultrasonic ones, as illustrated by examples hereinafter.
  • Substances that can be encapsulated include any water-soluble compound, but particularly interesting are bioactive substances, such as low molecular weight drugs, proteins, oligonucleotides, gene, and polysaccharides.
  • a particularly advantageous property of microcapsules produced according to the present invention is that they can exhibit controlled release properties.
  • a number of atomizer configurations for use in generating microcapsules according to the present invention are contemplated.
  • a particularly surprising and useful configuration is one in which a coaxial ultrasonic atomizer is submerged into an aqueous collection bath. In this configuration, immediate solvent exchange can occur between the microparticles and the bath in addition to the solvent exchange that can occur between core .and shell domains.
  • Pre-encapsulant particles formed by a method of the present invention can be hardened, i.e., the polymer-containing shell can be solidified, through solvent exchange.
  • Such hardening can be due to solvent exchange between the aqueous core and polymer-containing shell of the particles and/ or between the shell and surrounding bath.
  • solvent removal from the polymer-containing shell can be effected by spray drying or freeze drying techniques.
  • microbubbles can be produced using an atomizer.
  • hydrophobic drugs can be loaded alongside polymer in a single phase.
  • Microcapsules containing hydrophobic compounds within a non-aqueous core can also be made.
  • FIG. 1 schematically illustrates a solvent exchange method of the present invention as implemented with a coaxial ultrasonic atomizer.
  • a plurality of microdrops of each solution is produced by the atomizer (A). Collision occurs among the drops resulting in coalescence (B). Solvent exchange begins as soon as two different microdrops come in contact (C).
  • Fig. 2 shows several views of microcapsules typically produced by a method of the invention.
  • Panels A and B show bright field microscopic pictures and Panel C shows scanning electron microscopic pictures of mononuclear microcapsules produced by an ultrasonic atomizer according to principles of the present invention. Scale bars: 100 ⁇ m.
  • Fig. 3 depicts four different configurations of ultrasonic atomizer assemblies contemplated for generation of microcapsules according to principles of the present invention.
  • Fig. 4 schematically illustrates particle size reduction using an extra piezo device interposed between the ultrasonic atomizer and a collection bath.
  • Fig. 5 depicts a plot of various solvents as a function of the fractions of their dispersion force (fd), polmty (f p ), and hydrogen bonding (f h ) for use in hydrophilic solvent selection.
  • fd dispersion force
  • f p polmty
  • f h hydrogen bonding
  • Fig. 6 illustrates selection of hydrophilic organic solvents by the hydrogel method with preferred solvents marked by a shadow.
  • Fig. 7 illustrates selection of hydrophilic solvents using optical density as a measure of membrane quality (Panel A) and film diameter reflecting the degree of spreading (Panel B).
  • Fig. 8 depicts a schematic representation of the use of a spray dryer apparatus according to principles of the present invention.
  • Fig. 9 illustrates bright field microscopic images of microcapsules produced with an ultrasonic atomizer using 2% PLGA solution in ethyl acetate and several combinations of aqueous solution and collection bath as set forth in Example 1.
  • the solvent exchange method for forming microcapsules is based on a phenomenon that a solid polymer film forms at the interface between an aqueous solution and a solution containing a water-insoluble polymer upon contact of the two solutions as a result of phase separation of the polymer.
  • This method permits production of mononuclear drug-containing microcapsules (consisting of a single aqueous core surrounded by a thin biodegradable polymer membrane) whenever solvent exchange occurs between a polymer-containing solvent shell surrounding an aqueous core microdrop containing the drug.
  • a solvent exchange method of microcapsule formation has been previously described by Y. Yeo et al. in U.S. Serial No. 10/017,338, the pertinent disclosure of which is incorporated herein by reference.
  • Formation of a polymer film enveloping the surface of an aqueous drop depends on spreading of the polymer solution on the aqueous surface and subsequent phase-separation of the water-insoluble polymer. It is observed that spreading of the polymer solution is mainly dictated by physical properties of the hydrophilic or water-miscible polymer solvent. On the hydrogel surface, the polymer solution is exposed to two Ends of interracesran interface "with a hydrogel (which consists of >90% of water, so it is basically an interface with water) and an interface with air. For favorable spreading of the polymer solution over the aqueous surface, the solvent is required to have a low interfacial tension against both water and air (i.e., surface tension).
  • Phase-separation of the polymer film is a result of mass transfer between the polymer (e.g., organic) solvent and water (i.e., solvent exchange) leading to a decrease in the solubility of the polymer in the solvent.
  • Organic solvents should be miscible with water to a certain degree in order to cause instant phase-separation of the polymer film.
  • One way to achieve this objective is to produce a plurality of drops of the aqueous and the polymer solutions and induce collision among the drops. The collision is followed by coalescence of the drops.
  • the hydrodynamic fates of the two liquid drops are determined by the relative surface tensions of the liquids.
  • the organic solvent preferentially deforms and spreads on and around the aqueous drop upon contact, while the aqueous drop having a higher surface tension relative to that of the organic solvent tends to maintain its spherical shape.
  • a coaxial ultrasonic atomizer can be employed to produce a plurality of drops of each solution (Fig. 1, Panel A).
  • Two liquids comprising aqueous drug solution and the biodegradable polymer dissolved in (an) org.anic solvent(s) flow through the ultrasonic atomizer.
  • both liquids form a double layered film on the surface of the atomizer tip and are simultaneously fragmented into a large number of drops. Collision occurs among drops in proximity, which is followed by coalescence of the drops (Fig. 1, Panel B).
  • a coaxial atomizer is preferably used because it efficiently generates a space where the drops are highly populated, and thus collision of heterogeneous drops is very likely.
  • the solvent exchange process begins as soon as the two microdrops come in contact (Fig. 1, Panel C). Hardening of the microcapsules can be completed in a water bath as t ⁇ e solvent exchange process is accelerated in the presence of an abundance of water. The air and the water bath can thus be called primary and secondary continuous phases, respectively.
  • One of the unique aspects of this approach is that solvent exchange occurs very fast and microcapsule formation is complete in a matter of seconds. Exemplary microcapsules are shown in Fig. 2 under different levels of magnification.
  • microcapsules appear transparent when observed by a bright field microscope, since the surrounding polymer layer is only a membrane, as shown in Panels A and B.
  • the main spherical bodies of the microcapsules appear blue when stained with Coomassie Blue, indicating that the aqueous cores consist of the aqueous encapsulation solution.
  • the scanning electron microscopic image shows that the surface of the microcapsules appears smooth and free of major defects, indicating that solvent exchange occurring at the interface of the aqueous core and the polymer-containing organic solvent shell produces a continuous polymer membrane.
  • Typical particles sizes for microcapsules produced according to the present invention are in the range of about 0.1 to about 500 ⁇ m.
  • the liquid film When a liquid film is introduced onto a vibrating surface, such that the direction of vibration is perpendicular to the surface, the liquid film absorbs the vibration energy and creates unique capillary waves, which form regularly alternating crests and troughs in the liquid film [22]. Beyond the critical amplitude, the capillary waves cannot maintain their stability, .and the waves collapse and tiny drops of liquid emerge from the top of the waves.
  • An ultrasonic atomizer is a device used to generate such vibrations leading to atomization of a liquid [23].
  • the atomizer body consists of three principal sections: front horn, the atomizing section; rear horn, the rear section, and a section consisting of a pair of disc-shaped piezoelectric transducers. Working in unison, these three elements provide means for creating the vibration required to atomize liquids delivered to the atomizing surface. Liquid enters through a fitting on the rear, passes through the tube and then the central axis of the front hom. Finally, the liquid reaches the atomizing surface where atomization takes place.
  • Piezoelectric transducers convert electrical energy provided by an external power source into high-frequency mechanical motion [22].
  • the vibration energy applied to the atomizing surface lets the liquid overcome the surface tension and spread on the surface forming a liquid film.
  • the liquid film absorbs the underlying vibration energy and generates capillary waves. When the amplitude of the capillary waves exceed-ra critical value, the waves collapse ejecting small drops of the liquid.
  • Ultrasonic spray technology has been employed in industrial and research applications related to the electronics and biomedical areas, mainly for surface coating and liquid dispensing. [22].
  • the ultrasonic atomizers have been used for coating the interior of blood-collecting tubes with anti-coagulants, applying adhesives to sutures, or dispensing reagents into well-plates for diagnostic testing [22].
  • the popularity of the ultrasonic atomizer in such areas is mainly attributed to its ability to produce drops of small size and low inertia.
  • the velocity of the drops produced from an ultrasonic atomizer is 1 — 10% that of a hydraulic or air-atomizing nozzle, and this virtually eliminates overspray problems.
  • Another advantage of ultrasonic atomization is that the mechanical stress caused by the vibration is relatively minor so that it does not render bioactive substances inactive [22].
  • Ultrasonic atomizers operate at low energy levels, which are unlikely to compromise the viability of biological materials, such as blood, antibodies, and bacteria.
  • the configuration of the atomizers can be varied without limit as long as it guarantees collision among liquid microdrops.
  • a coaxial atomizer is preferably used because it efficiently generates a space where the drops are highly populated, and thus collision of heterogeneous drops is most likely (Fig. 3-A).
  • two liquids flow under the influence of a single ultrasonic generator. One liquid flows through the inner nozzle, and the other flows through the outer one. Both liquids delivered to the same atomizing surface are broken into microdrops as the vibration energy is applied on the surface. Coalescence occurs among the liquid drops in close proximity. Microencapsulation is not affected by the pathway each liquid is allowed to follow.
  • the procedure can be performed without a coaxial cable.
  • the liquid inlets . are not limited to two, but can be expanded as many as desired.
  • Two separate atomizers can be arranged in such a way that two sprays of microdrops can collide in air (Fig. 3-B).
  • the distance from each atomizer to the space where the collision takes place is important, since it determines the population density of drops of two liquids in the space and thereby the yield of microencapsulation.
  • Each spray of microdrops can be produced in a thin sheet form and arranged to cross each other so that the probability of collision can be maximized.
  • the number of atomizers is not limited to two, but can be expanded to as many as desired. See Fig. 3-C.
  • a closed receptacle can be used to prevent airflow around atomizers, which can cause difficulty in controlling the trajectory of the liquid spray (Fig. 3-C). Furthermore, vacuum or heat can be applied into the closed receptacle to facilitate solvent removal.
  • microcapsules can also be produced by submerging the atomizer in a collection bath to induce the solvent exchange directly by contact between polymer drops and the collection bath (Fig. 3-D).
  • formed microparticles are mononuclear microcapsules containing bath materials in the core.
  • Lipophilic/hydrophobic drugs can be loaded into the microcapsules by co-dissolving the drugs with the polymer.
  • Hydrophilic drugs can be loaded into the microcapsules by generating them in an aqueous bath containing the drug(s) to be encapsulated.
  • microcapsules can be reduced by fragmenting the embryonic pre-encapsulant particles before microcapsule formation is complete. As shown in Fig. 4, embryonic particles emerging from the atomizer are broken into smaller particles upon hitting an extra piezoelectric device.
  • the piezoelectric surface can assume any form including sheet, cylinder, sphere, and combinations thereof.
  • An exemplary piezoelectric device is commercially available from Sono Tek Corp. (Milton, NY).
  • biodegradable polymers are preferable, especially when their degradation products are known to be innocuous or biocompatible. They need not be surgically removed at the end of a treatment.
  • biodegradable polymers which have been investigated for the controlled delivery of protein drugs, include homopolymers of poly(lactic acid) (PLA) or poly(glycolic acid)
  • PGA poly(lactic acid)-co-(glycolic acid)
  • PLA poly(ortho esters)
  • polyanhydrides Due to the biocompatibility and the long history of clinical applications, PLGA and PLA are most preferably used.
  • Other biodegradable polymers that can be used include polycaprolactone, polycarbonates, polyesteramides, poly(amino acids), poly(dioxanones), ⁇ oly(alkylene alkylate)s, polyacetals, polycyanoacrylates, biodegradable polyurethanes, blends and copolymers thereof.
  • non-biodegradable polymers can be used.
  • Examples include polyacrylates, polymethacrylates, polymers of ethylene- vinyl acetates and other acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chiorosulphonaie po-yoiefms, polyethylene oxide, blends .and copolymers thereof.
  • the solvent should be able to spread out favorably on the aqueous surface.
  • the solvent should be miscible with water to a certain degree, thus allowing fast phase separation of the polymer.
  • Solvent screening can begin with the Hildebrand solubility parameters of the organic solvents.
  • 60 solvents having the Hildebrand solubility parameter of 16 - 24 MPa 1 2 are screened, however, only half of them are able to produce clear PLGA solutions, and the rest are poor solvents or marginally able to swell the PLGA.
  • the results are summarized on a triangular graph according to the Teas method (Fig. 5).
  • ⁇ d , ⁇ p , and ⁇ h are Hansen's multicomponent solubility parameters which stand for the contributions from dispersion forces, polar interactions, and hydrogen bonding, respectively.
  • Hildebrand solubility parameter in the range of 16 to 26 MPa 1 2 .
  • Selected good solvents for PLGA can be refined further by a screening method using a hydrogel (the hydrogel method). Solutions of polymer in different solvents are placed on a layer of hydrogel, and thus formed polymer films are evaluated with respect to the diameter and the optical density (Fig. 6). The diameter of the film reflects the degree of spreading of each solvent, and the turbidity allows for evaluation of the quality of the polymer membrane.
  • Solvents that form membr-anes of relatively large diameters are preferable, since it means that the polymer solution spreads easily.
  • Polymer membranes displaying relatively low optical densities are preferred since high turbidity reflects the formation of a rough and discontinuous precipitate, which is likely to fail in control of the drug release across the membrane.
  • the quality of the polymer membrane represented by the optical density is linked to the solubility of the solvent in water (Fig. 7-A).
  • Solvents that have low to medium solubility in water (0 ⁇ 60% w/w) result in a transparent and dense membrane, whereas water-miscible solvents (-100% w/w) make a turbid and discontinuous membrane.
  • Preferred hydrophilic solvents have a water solubility of about 5 to about 100%.
  • the spreading capability of the polymer solution represented by the film diameter is related to the surface tension of the solvent (Fig. 7-B). Solvents having relatively low surface tension tend to spread favorably. Surface tension of 30 dynes/cm appeared to be a rough threshold. Therefore, it is also possible to make a quick judgment in solvent selection by examining the surface tension and the water-solubility of the solvent.
  • a preferred hydrophilic solvent has a surface tension of less than about 45 mN/m.
  • Relatively hydrophilic organic solvents such as ethyl acetate
  • ethyl acetate have been used with increasing regularity as an alternative to methylene chloride [19, 27-29].
  • its relatively high solubility in water often makes it difficult to produce dense and regular microspheres using conventional emulsion methods [27] and sometimes requires doping the continuous phase with additional ethyl acetate in order to delay diffusion of the solvent out of the discontinuous phase [30].
  • the morphology of microspheres is fair more dependent on the phase ratio (the volume ratio of the organic to aqueous phase) than methylene chloride when ethyl acetate is used in the emulsion method [27].
  • the hydrophilicity of methylethyl ketone caused the same problem [31].
  • the solvent exchange method takes advantage of the hydrophilicity of the organic solvent.
  • utilizing hydrophilic organic solvents is the major feature of the solvent exchange method as previously described.
  • the solvent exchange method what makes the microcapsule a sphere is the surface tension of the aqueous solution. Therefore, facile formation of a w/o emulsion is not a requirement for capsule formation. Elimination of such a requirement affords more flexible selection of the organic solvents, which is in turn beneficial to toxicological and environmental safety.
  • the aqueous core initiates the phase separation of the polymer, and serves as a reservoir for drug molecules and protective excipients (if necessary). Moreover, it plays an important role in maintaining the mechanical strength of the microcapsules.
  • a hydrophilic polymer that is capable of undergoing the sol-to-gel transition, as the aqueous solution. This unique property, present in many polysaccharides and hydrophilic polymers, makes it possible to process the drug formulation in a liquid state until particle formation and to solidify the particles later by providing appropriate conditions.
  • sodium alginate which forms an ionotropic hydrogel in the presence of divalent cations such as calcium, can be used as a base for the aqueous solution.
  • the alginate solution containing a drug substance is ejected from the atomizer and is fragmented into microdrops. Microcapsules formed in air are collected in the aqueous bath containing calcium ions. It appears that calcium ions are able to diffuse into the aqueous core before the polymer membrane is completely sealed by precipitation of the polymer.
  • a variety of aqueous polymer solutions can serve the same function. See Table 2.
  • Poloxamer 407 (20 - 25%) > 20 - 30 °C (temperature) [34]
  • the materials that can be used for the aqueous core are not limited to the polymers capable of sol-to-gel transition.
  • a variety of hydrophilic polymers can be included along with drugs in the aqueous solution. The presence of the hydrophilic polymer can increase the viscosity of the aqueous solution, and thus contribute to preventing diffusion of the drug substances into the bath during collection.
  • the hydrophilic polymer is a polyelectrolyte or has functional groups that can interact with the drug substances, it can play an assistant role in control of the drug release.
  • hydrophilic polymers examples include polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), polyoxazoline, polyacrylic acid, polyacrylamide, polymethacrylic acid, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).
  • PEG polyethylene glycol
  • PVP polyvinylpyrrolidone
  • PVA polyvinyl alcohol
  • polyoxazoline polyacrylic acid
  • polyacrylamide polyacrylamide
  • polymethacrylic acid polymethacrylic acid
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Protective excipients can be included in the aqueous solution to increase the stability of the encapsulated drugs.
  • examples of such protective excipients include carbohydrates, e.g., sucrose, lactose, mannitol, trehalose, cyclodextrins, surface active agents such as Tweens, stearate salts, Poloxamers, polyvinyl alcohol, hydrophilic polymers such as carboxymethyl cellulose, gelatin, albumin, dextran, polyethylene glycol, or buffer salts such as calcium carbonate, calcium orthophosphate, sodium acetate, magnesium hydroxide, calcium hydroxide, and zinc carbonate.
  • carbohydrates e.g., sucrose, lactose, mannitol, trehalose, cyclodextrins, surface active agents such as Tweens, stearate salts, Poloxamers, polyvinyl alcohol, hydrophilic polymers such as carboxymethyl cellulose, gelatin, albumin, dextran,
  • Microcapsules can be collected in an aqueous bath. Conditions that can induce a sol- to-gel transition of the aqueous core can be provided via the collection bath. When polyelectrolytes such as alginate are included in the aqueous solution, microcapsules are collected in an aqueous bath containing counter-ions such as calciums that can complex with the polyelectrolytes. When the transition is temperature-sensitive, the temperature of the bath can be adjusted appropriately to solidify the aqueous core. Emulsifying agents can be included in the bath to prevent aggregation of the embryonic microcapsules. Polyvinylalcohols, Poloxamers, and Tweens are representative emulsifying agents.
  • the materials that can be used for the collection bath are not limited to water. Any kind of liquid miscible with the solvent for the polymer but which does not dissolve the polymer can be used for this purpose. Examples are alcohols, ketones, and oils.
  • microcapsules When microcapsules are collected in the water bath, it is important to disturb the surface of the water bath efficiently, otherwise, it is possible to accumulate films of polymer solution on the surface, which form a solid layer that obstructs entr-ance of the microcapsules into the bath.
  • simple magnetic stirring can easily break the stability of the water surface and allow introduction of the microcapsules into the bath. Vibrating the bath can serve the same purpose and the vibration energy can be provided using an ultrasonic bath or sonication probes submerged in the bath.
  • the polymer solvent can be removed by evaporation. Extending the path that microcapsules fly in air and/or applying mild heat to the flying microcapsules can dry the solvent before the microcapsules reach the collection bath. The microcapsules can be directly collected as dried particles or can be collected in the bath.
  • the advantages of combining the solvent exchange method with spray-drying are that further hardening procedures are not required and microencapsulation can be performed in a single step. Moreover, the entire procedure is continuous .and is, therefore, convenient for aseptic processing and scale-up.
  • ultrasonic atomizers can be employed with a conventional spray- dryer or drying chamber to evaporate the solvent.
  • the polymer solution and the aqueous solution loaded with drugs are atomized using ultrasonic atomizers into a drying chamber of the spray-dryer. Collision among the drops results in microcapsules in air.
  • a stre,am of warm gas introduced from a separate inlet evaporates the liquids and solidifies the microcapsules.
  • a drying gas air, nitrogen, or reducing gas
  • the resulting outlet temperature will be lower, e.g., 30 °C.
  • the unheated drying gas flow can be maintained for a predefined period of time (e.g., 30 min, 2 hours, etc.)
  • the formed microcapsules are separated from the carrier gas in a cyclone attached to the drying chamber and collected in a receiver connected to the cyclone.
  • Suitable commercial spray dryers are available from Buchi Analytical, Inc. (New Castle, DE), Niro A/S (Soeborg, Denmark), and L.P. Technology, Ltd. (Leeds, England).
  • Microcapsules formed in air as a result of collision among the drops are directly frozen in the liquefied gas.
  • the frozen microcapsules are then transferred to a pre-cooled chamber connected to a freeze-dryer.
  • the solvent is removed by lyophilization. This method is especially advantageous when the active ingredient is sensitive to a high temperature.
  • the ratio of flow rates of two solutions plays a significant role in determining the probability of collision between microdrops of two liquids.
  • Qp 0 ⁇ the flow rate of the polymer solution
  • Q A q the flow rate of the aqueous solution
  • the drops of polymer solution preferentially deformed and surrounded the aqueous drops, while the aqueous drops that had a relatively higher surface tension as compared to that of the organic solvent resisted deformation and were encapsulated within the polymer drops.
  • the ratio of Qpoi QAq is in the range of about 1 to about 10.
  • Qp 0 ⁇ is lowered to 1/3 of the original flow rate, reversed capsules emerge.
  • Qp 0 ⁇ was reduced, the polymer drops were exposed to a relatively larger number of aqueous drops. Aqueous drops, which had considerably high surface tension, tended to coalesce with other aqueous drops to form bigger ones. Polymer drops present within the aqueous drops are believed to be those entrapped during the coalescence between aqueous drops.
  • Optimum microcapsules are obtained using a coaxial atomizer when Qpoi/QAq ranges from 1.5/0.25 to 3/0.25.
  • Qp 0 ⁇ /Q A q ratio need not be so constrained and ratios of 1/1 to 10/1 are acceptable.
  • particle size is influenced by the higher flow rate (i.e., major flow) between the two liquids.
  • major flow i.e., major flow
  • Microcapsules of reasonable sizes are obtained when the major flow, which is typically Qp 0 ⁇ , is 1.5 - 3.0 ml/min with the ultrasonic atomizer working at 60kHz.
  • the solvent exchange method is based on an instantaneous mass transfer (solvent exchange) between the solvent and water, which decreases the ability of the solvent to dissolve polymer causing precipitation of the polymer.
  • solvent exchange an instantaneous mass transfer
  • the concentration of organic solvent in the proximity of the microcapsules in the bath should be maintained as low as possible: at least lower than the saturation solubility of the solvent in water. Therefore, it is preferable to use a large volume of collection bath to make a sink condition around the solvent exchange area.
  • the saturation solubility of ethyl acetate in water is 8 %w/w.
  • the bath size is small to such an extent that the solvent concentration exceeds the saturation solubility, diffusion of the solvent and subsequent solidification of the polymer membrane is significantly delayed.
  • the polymer drops associated with the aqueous core are gradually detached to form a separate phase instead of leaving a polymer membrane on the core.
  • Microbubbles Polymeric microcapsules filled with gas (i.e. microbubbles) can be produced using ultrasonic atomizers immersed in the aqueous bath.
  • Microbubbles are in clinical use as ultrasound contrast agents for sonographic applications [41].
  • Microbubbles create an acoustic impedance mismatch from biological tissues and fluids, and thus efficiently reflect ultrasound.
  • microbubbles have been used for local drug delivery and especially for targeted gene delivery [42].
  • Drugs can be incorporated into the membrane of the microbubbles or internalized into the gas-filled interior of the microbubbles and are released when ultrasound energy ruptures the microbubbles.
  • Cavitation refers to the formation and subsequent dynamic life of bubbles in liquids. It can be hydrodynamic, thermal, or acoustic in origin and can occur in a variety of liquids under a wide range of conditions [43].
  • acoustic cavitation caused by ultrasonic irradiation is an important source of a number of sonochemical phenomena [44] . Sound is transmitted through liquids as a wave consisting of alternating compression and rarefaction cycles. When the rarefaction wave is sufficiently powerful, it can develop a negative pressure large enough to overcome the intermolecular forces present in the liquid. In this situation the molecules can be separated from each other to form tiny bubbles in the medium.
  • the bubbles can originate from density fluctuations of a pure liquid at a given pressure and temperature (i.e., homogeneous nucleation) or foreign substances that stabilize pockets of gas, which become 'nuclei' for the bubble growth (i.e., heterogeneous nucleation).
  • Microbubbles can be produced by stabilizing the transient gas bubbles, which otherwise will be collapsed by the compression cycle following the rarefaction cycle.
  • ultrasonic energy has been used to stabilize the gas-liquid interface by cross- linked proteins [45].
  • a protein solution was ultrasonically irradiated in the presence of oxygen to cause acoustic cavitation.
  • the acoustic cavitation played dual functions: dispersion of gas into the protein solution and generation of oxidative radicals such as H- and OH- in water, which oxidized free cysteine residues present in the protein.
  • the gas bubbles dispersed in the protein solution were fixed by cross-linking of the cysteine residues of the surrounding proteins and forming proteinaceous microbubbles.
  • polymeric microbubbles are produced by dispersing a solution of water-insoluble polymer, such as PLGA, in an aqueous bath using an ultrasonic atomizer.
  • the ultrasonic atomizer is immersed within the aqueous bath.
  • the function of the ultrasonic atomizer is not only to deliver and atomize the polymer solution to the liquid bath but also to provide ultrasonic irradiation into the bath.
  • Gas bubbles occur in the aqueous bath as the liquid is ultrasonically irradiated. Simultaneously, the bubbles are stabilized by polymer precipitate formed as a result of solvent exchange between the polymer solution and the aqueous bath.
  • the end product is a suspension of hollow polymeric microbubbles less than about 10 ⁇ m in diameter. Confocal microscopic images clearly visualize that the microbubbles have a polymeric membrane surrounding an air-filled core.
  • the dark appearance of the bubble interior indicates that the core does not contain bath materials.
  • the Nile Red signals show that the polymer phase is a single layer surrounding the gas core.
  • PNA emulsifying agent
  • the major drawbacks of commercial ultrasound contrast agents are short plasma half-life and its acoustic instability relative to pressure changes [45].
  • the polymeric microbubbles described above are able to withstand acoustic pressure.
  • the present method does not require specific properties of the encapsulating materials other than solubility in water.
  • the polymers can be utilized to make microbubbles.
  • the use of a separate polymer solution can become another advantage since the drugs can be easily loaded onto the polymer membrane simply by dissolving or suspending them in the polymer solution.
  • non-aqueous microcapsules are produced by extruding a solution of water-insoluble polymer, such as PLGA, and water-insoluble liquids, such as n-dodecane, n-decane, n-hexane, cyclohexane, and toluene, into an aqueous bath using a coaxial ultrasonic atomizer.
  • Hydrophobic drugs can be loaded as a solution in the non-aqueous liquid. Unlike previous examples that produce microcapsules in air, this method can be used to encapsulate hydrophobic drugs.
  • the present solvent exchange method is a single-step process and thus is much simpler than any other existing microencapsulation techniques.
  • the production scale can be easily modified without affecting the quality of the final products.
  • the simplicity of the procedure will significantly bring down the overall cost of microcapsule production.
  • conventional methods often include a harsh condition such as an emulsification step, which can exert unfavorable influences on the stability of encapsulated proteins by exposing them to the w/o interface and intensive physical'stress.
  • ultrasonic atomization is mild enough to preserve the bioactivity of the drugs.
  • ultrasonic atomizers utilize very low energy. Even if the mild stress would become a problem, although unlikely, the time the drug is exposed to the ultrasonic vibration is only a fraction of a second. Second, in the mononuclear microcapsules produced by the solvent exchange method, undesirable interactions between drug and the organic solvent or the polymer matrix are limited only to the surface of the core, if any, and do not affect the majority of the drugs.
  • the solvent for polymers can be chosen with more flexibility.
  • the double emulsion-solvent extraction/evaporation method requires the organic solvent to be hydrophobic so that it can form an emulsion in water.
  • the solvent cannot be too hydrophobic because if the emulsion drops do not stay too long in a liquid state, a significant loss of drugs will be lost into the continuous phase [36]. Therefore, the solvents that can be successfully used in the double emulsion methods are limited to only a few solvents: practically to methylene chloride. This is one of the disadvantages of this method, since methylene chloride is a possible carcinogen and its residual amount should be tightly controlled to meet the regulation.
  • the solvent exchange method can easily overcome these limitations.
  • the high water solubility of solvents is not a problem since the solvent exchange method does not depend on formation of an emulsion.
  • the low water solubility of solvents is also not a limiting condition, because the absolute amount of the organic solvent to be removed from individual microcapsules is less than for those produced by the emulsion method.
  • solidification of the polymer membrane occurs quickly before the drug is lost.
  • encapsulation efficiency can be significantly improved in the solvent exchange method. Drug loss across the dispersed drop interfaces occurs only during the first minutes before polymer precipitates [36]. When the microparticle is sealed as the polymer solidifies, diffusion of the drug into the continuous phase is limited.
  • the precipitation occurs quickly, not only because the preferred solvent is hydrophilic, but also because the absolute amount of solvent to be removed is small.
  • the encapsulation efficiency of this encapsulation method is as high as 80% on average. Depending on the formulation variables, the encapsulation efficiency can reach near 100%.
  • the solvent exchange method can be used not only as a microencapsulation technique, but also as a means to coat stents with a mixture of a polymer and a water-soluble drug.
  • Drug coated stents are becoming an increasing popular approach fbrthe controlled delivery of drugs.
  • Microcapsules produced in air can be captured on the stent surface to form one or multiple layers of coating.
  • the use of ultrasonic atomizers is particularly advantageous for this application because of the unique capability of the device to produce a low-velocity spray that eliminates overspray problems.
  • microcapsules were collected in a water bath containing 0.15 M calcium chloride for stabilization of the microcapsules through formation of calcium-alginate gel.
  • the size distribution of the microcapsules was determined using a Microrrac Full Range Particle Size Analyzer 9200.
  • microcapsules were imaged using a bright field microscope (Panels A,B) and a confocal or scanning electron microscope (Panel C) as shown in Fig. 2.
  • microspheres were also produced using a double emulsion-solvent evaporation method described in the literature [37] with a slight modification.
  • 50 ⁇ l of aqueous FITC-dextran solution (20%) was poured into 1 ml of methylene chloride containing 33% PLGA and 0.003% Nile Red. The solution was mixed for 1 min using a vortex mixer.
  • w/o emulsion was poured under magnetic stirring into 2 ml of aqueous 1% PVA solution saturated with methylene chloride to form a w/o/w emulsion.
  • the w/o/w double emulsion was poured into 200 ml of water containing 0.1% PVA and continuously stirred for 3 hours at room temperature until most of methylene chloride evaporated, leaving solid microspheres.
  • the formed microcapsules were imaged using a confocal microscope.
  • Fig. 9 illustrates bright field microscopic pictures of microcapsules produced wi h an ultrasonic atomizer using 2% PLGA solution in ethyl acetate and various combinations of aqueous solution and collection bath following the procedure set forth above.
  • Scale bar 100 ⁇ m (A); 50 ⁇ m (B, C, and D) Panel Aqueous solution Collection bath
  • a solution of 2% PLGA in ethyl acetate and an aqueous solution containing 0.2% sodium alginate were delivered into a coaxial ultrasonic atomizer using syringe pumps at controlled flow rates as shown in Fig 3 (Panel A).
  • the polymer solution flowed through the inner nozzle at 1.5 ml/min and the aqueous solution flowed through the outer nozzle at 0.25 ml/min.
  • 0.02% Coomassie Blue was added to the aqueous solution for visualization by light microscopy.
  • both liquids were fragmented into microdrops. The collision of multiple drops in air produced microcapsules.
  • Thus formed microcapsules were collected as described in Example 1.
  • Example 3 Microencapsulation using an ultrasonic atomizer without a coaxial cable.
  • a solution of 2% PLGA in ethyl acetate and an aqueous solution containing 0.2% sodium alginate were delivered into an ultrasonic atomizer through separate inlets.
  • the polymer solution flowed at 1.5 ml/min and the aqueous solution flowed at 0.25 ml/min.
  • both liquids were fragmented into microdrops.
  • the collision of multiple drops in air produced microcapsules.
  • Thus formed microcapsules were collected as described in Example 1.
  • Example 4 Microencapsulation using separate ultrasonic atomizers.
  • a solution of 2% PLGA in ethyl acetate and an aqueous solution containing 0.2% sodium alginate were delivered into two ultrasonic atomizers respectively using syringe pumps at controlled flow rates.
  • the polymer solution flowed through one atomizer at 1.5 ml/min and the aqueous solution flowed through the other atomizer at 0.25 ml/min.
  • 0.02% Coomassie Blue was added to the aqueous sol tion for visualization by light microscopy.
  • both liquids were fragmented into microdrops.
  • Two atomizers were aligned so that two liquid sprays could be coincided as shown in Fig. 3 (Panel B). The collision of multiple drops in air pro- quiz scored microcapsules.
  • the microcapsules formed were collected as described in Example 1.
  • Example 5 Microencapsulation using coaxial ultrasonic atomizer submerged in collection bath.
  • Microcapsules can be produced by submerging the atomizer in a collection bath and inducing solvent exchange by contact of the polymer solution and the collection bath as shown in Fig. 3 (Panel D).
  • 0.4 mg/ml Nile Red was added to the polymer solution.
  • the atomizer was submerged in the collection bath consisting of water.
  • the PLGA solution delivered via the ultrasonic atomizer was fragmented into microcapsules ranging 5 - 100 ⁇ m in diameter.
  • the microparticles were visualized by confocal microscope, they were observed to be hollow microcapsules.
  • Example 6 Particle size reduction using extra piezoelectric device.
  • the particle size can be reduced after microencapsulation by secondary fragmentation of the embryonic microcapsules.
  • a piezoelectric device vibrating at a high frequency was placed underneath the atomizer (Fig. 4).
  • the emerging microcapsules were broken into smaller particles upon hitting the vibrating piezoelectric device.
  • Thus formed microcapsules were collected as described in Example 1.
  • the solvent exchange method can be used as a means to deposit polymer coating containing water-soluble drugs on stents.
  • the microcapsules produced by the method described in Example 1 are deposited on the stents.
  • the solvent can be removed by evaporation.
  • the path that microcapsules fly in air can be extended and/or mild heat can be applied to the flying microcapsules.
  • the layer of microcapsules thus formed on the stent contains aqueous cores including drug substances, and the polymer that controls the drug release rate.
  • the microcapsules were captured on a glass plate an observed by microscope.
  • a solution of 2% PLGA in ethyl acetate labeled with 0.4 mg/ml Nile Red and an aqueous solution containing 0.2% sodium alginate and 2.8 mg/ml FITC-dextran were delivered into a coaxial ultrasonic atomizer using syringe pumps at controlled flow rates.
  • the aqueous solution flowed through the inner nozzle at 0.25 ml/min and the polymer solution flowed through the outer nozzle at 1.5 ml/min.
  • both liquids were fragmented into microdrops.
  • the microdrops were captured on the glass plate by quickly passing the plate under the atomizer so that only one layer of spray could be deposited. Captured microdrops were observed using a fluorescence microscope with polymer drops appearing red due to the presence of fluorescence dye Nile Red and the aqueous drops appearing green due to FITC-dextran.
  • microcapsules are products of coalescence of two drops in air
  • the microcapsules were captured on a glass plate and observed by microscope.
  • a solution of 2% PLGA in ethyl acetate labeled with 0.4 mg/ml Nile Red and an aqueous solution containing 0.2% sodium alginate and 2.8 mg/ml FITC-dextran were delivered into a coaxial ultrasonic atomizer using syringe pumps at controlled flow rates.
  • the aqueous solution flowed through the inner nozzle at 0.25 ml/min and the polymer solution flowed through the outer nozzle at 1.5 or 0.5 ml/min.
  • both liquids were fragmented into microdrops.
  • the microdrops were captured on the glass plate by quickly passing the plate under the atomizer so that only one layer of spray could be deposited. Captured microdrops were observed using a fluorescence microscope.
  • Organic solvents having the Hildebrand solubility parameter of 16 - 24 MPa 1 2 were screened for polymer solvency. 125 mg of PLGA was added to glass vials containing 5 ml of solvents. The vials were agitated overnight at room temperature. Solubility of PLGA in a particular solvent was judged by visual examination. Solvents were classified into four groups: good solvents (forming clear polymer solution); intermediately good solvents (forming turbid polymer solution upon heating); intermediately poor solvents (marginally able to swell the PLGA); and poor solvents. The results are summarized on a triangular graph developed by Teas [38], according to Hansen's solubility parameterS"[3'9] of the solvents (Fig. 5).
  • the second and third qualities of the solvent were examined by a simple screening tool called the hydrogel method.
  • a library of PLGA solutions (5% w/v) was constructed using the good solvents selected above.
  • a 10 ⁇ l drop of each solution was placed on a layer of hydrogel containing 0.5% agarose.
  • the diameter of the polymer film that formed on the gel was measured 10 seconds after the placement.
  • a microplate containing 200 ⁇ l of agarose gel in each well was prepared to which 5 ⁇ l of each PLGA solution was simultaneously applied.
  • the turbidity of the film developed after 1 minute and was measured at 620 nm using a microplate reader. See Figs. 6 and 7.
  • TLE (%) 100 x (Protein used for encapsulation / Microparticle weight)
  • Microcapsules accurately weighed ( ⁇ 10mg), were put into a microcentrifuge tube, to which 0.2 ml of dimethyl sulfoxide (DMSO) was added. The microcapsules were dissolved by vortexing. 0.8 ml of NaOH/SDS/Citric acid trisodium salt solution (0.05N/0.5%/0.075M) was then added to the tube and mixed. After sonicating for 90 min at 25 °C, samples were centrifuged at 10,000 rpm for 5 min. Aliquots of the clear DMSO/NaOH/SDS/Citrate solution were pipetted into the wells of a microplate. Samples were analyzed using the bicinchoninic acid (BCA) assay method.
  • BCA bicinchoninic acid
  • Microparticles were put into a microcentrifuge tube, to which 1.0 ml of NaOH/SDS/Citric acid trisodium salt solution (0.05N/0.5%/0.075M) was added. The microparticles were dissolved by vortexing. After sonicating for 90 min at 25 °C, samples were centrifuged at 10,000 rpm for 5 min. Aliquots of the clear NaOH/SDS/Citrate solution were pipetted into the wells of a microplate. Samples were analyzed using the BCA assay method. The results are shown in Table 3.
  • Remaining microcapsules were collected by centrifugation at 10000 rpm for 3 min and washed with DW three times to remove BSA present in the outside of the microcapsules.
  • Microcapsules were frozen at -20°C and freeze-dried overnight, lml of DMSO was added to the dried microcapsules to dissolve PLGA selectively. DMSO supernatant containing PLGA was discarded and the protein debris was washed with fresh DMSO three times to remove polymer remnant. Protein debris was collected by centrifugation (6000 rpm, 1 min), and was dissolved in 1 ml of PBS. The protein solution was subjected to non-reducing PAGE. The gel was stained by the Coomassie Blue method. BSA powder was treated with the same procedure in order to make sure that the above procedure did not bring about any conformational change to the BSA.
  • microcapsules were collected by centrifugation at 10000 rpm for 3 min and washed with DW three times to remove lysozyme present in the outside of the microcapsules.
  • Microcapsules were frozen at -20 °C and freeze-dried for overnight.
  • 300 ⁇ l of DMSO was added to the dried microparticles to dissolve both lysozyme and PLGA.
  • 700 ⁇ l of DW was added to the solution.
  • Resulting suspension was centrifuged at 6000 rpm for 1 min. Proteins in the supernatant were subjected to non-reducing PAGE. The gel was stained by the Coomassie Blue method. Lysozyme powder was subjected to the same procedure in order to make sure that the above procedure did not bring about any conformational change to the lysozyme.
  • Example 17 Formation of microbubbles using ultrasonic atomizer submerged in bath.
  • Microbubbles can be produced by submerging the atomizer in a water bath and inducing solvent exchange from contact of the polymer solution and the collection bath.
  • 0.4 mg/ml Nile Red was added to the polymer solution.
  • the atomizer was submerged in the collection bath consisting of water and 0.5% PVA. In order to visualize the air entrapped within the microbubble, the bath was labeled with FITC.
  • the PLGA solution delivered via the ultrasonic atomizer was fragmented into microbubbles in the range of about 5 to 100 ⁇ m in diameter. Confocal microscopic images of the microbubbles show the cores appearing dark, indicating that they did not contain either bath materials or polymer but were filled with air.
  • Example 18 Formation of microcapsules filled with non-aqueous liquids using a coaxial ultrasonic atomizer submerged in the bath.
  • Microcapsules filled with non-aqueous liquids can be produced by submerging a coaxial ultrasonic atomizer in the bath and extruding a solution of water-insoluble polymer such as PLGA and water-insoluble liquids.
  • a solution of 2% PLGA m ethyl acetate and n decane were delivered into a coaxial ultrasonic atomizer using syringe pumps at controlled flow rates. The n-decane flowed through the inner nozzle at 0.25 ml/min and the polymer solution flowed through the outer nozzle at 1.5 ml/min.
  • the polymer solution and the non-aqueous liquid were labeled with two lipophilic dyes, Nile Red and DiO (3,3'-dioctadecyloxacarbocyanine perchlorate), respectively, which are commercially available from Sigma Corp. (St. Louis, MO).
  • Nile Red and DiO 3,3'-dioctadecyloxacarbocyanine perchlorate
  • both liquids were fragmented into microdrops in the collection bath.
  • Mononuclear microcapsules formed as drops of the two solutions coalesced. It is likely that since the non-aqueous liquids are also non-solvents for the polymer the contact between two drops induced immediate precipitation of polymer at their interfaces through solvent exchange.
  • the microcapsules looked mononuclear, of which a non- aqueous core was surrounded by polymer membrane. Later the microcapsules appeared homogeneously in orange when observed with confocal microscopy, a combination of red (Nile Red) and green (DiO), most likely because the two lipophilic dyes partitioned into the other phases with time.
  • atomizers other than ultrasonic ones can be used to generate microdroplets that undergo solvent exchange to afford microcapsules according to the principles of the present invention. Even though the above examples are implemented using ultrasonic atomizers as an exemplary atomizer, a variety of atomization techniques can be employed for the same purpose. Different commercial atomizers are categorized in terms of the different types of energy used to break up bulk liquid into drops. Possible examples include rotary atomizers that utilize centrifugal energy, centrifugal atomizers using pressure energy, and pneumatic atomizers utilizing kinetic energy, in addition to the sonic atomizers using sonic energy.

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Abstract

La présente invention concerne un procédé permettant de générer une pluralité de microgelules contenant un médicament qui utilise un ou plusieurs atomiseurs de façon à former ces microgelules par le phénomène d'échange de solvant. Une pluralité de microgouttelettes d'une solution aqueuse sont mises en contact avec une pluralité de microgouttelettes contenant un polymère dissout dans un solvant hydrophile dans des conditions dans lesquelles cette solution polymère enveloppe la microgouttelette aqueuse. L'échange de molécules de solvant entre le noyau aqueux et sa coque contenant le polymère dépose le polymère sous la forme d'une membrane autour du noyau aqueux. Un atomiseur préféré est un atomiseur coaxial ultrasonique. On peut générer des microgelules dans l'air de même qu'immergées dans un bain de recueil. On peut obtenir des propriétés recherchées de ces microgelules, par exemple une libération commandée, en apportant des excipients de protection à l'intérieur du noyau aqueux, fournissant un polymère hydrophile capable d'entreprendre une transition sol-gel à l'intérieur de ce noyau aqueux, optimisant la sélection du solvant de polymère, réglant les débits de flux relatifs et offrant d'autres avantages similaires.
PCT/US2003/008559 2002-03-19 2003-03-19 Microencapsulation a l'aide d'atomiseurs ultrasoniques WO2003079990A2 (fr)

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US8313676B2 (en) 2007-10-23 2012-11-20 Koninklijke Philips Electronics N.V. Methods for preparing polymer microparticles
US8846035B2 (en) 2007-10-23 2014-09-30 Koninklijke Philips N.V. Methods for preparing polymer microparticles
EP2098220A3 (fr) * 2008-03-03 2016-10-26 Hitachi, Ltd. Dispositif de fluide et ensemble de dispositif de fluide
CZ306334B6 (cs) * 2013-12-19 2016-12-07 C2P S.R.O. Přípravek pro administraci nikotinamidu a způsob jeho výroby
CZ306333B6 (cs) * 2013-12-19 2016-12-07 C2P S. R. O. Přípravek pro administraci kyseliny nikotinové a způsob jeho výroby
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US8313676B2 (en) 2007-10-23 2012-11-20 Koninklijke Philips Electronics N.V. Methods for preparing polymer microparticles
US8846035B2 (en) 2007-10-23 2014-09-30 Koninklijke Philips N.V. Methods for preparing polymer microparticles
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CZ306334B6 (cs) * 2013-12-19 2016-12-07 C2P S.R.O. Přípravek pro administraci nikotinamidu a způsob jeho výroby
CZ306333B6 (cs) * 2013-12-19 2016-12-07 C2P S. R. O. Přípravek pro administraci kyseliny nikotinové a způsob jeho výroby
CN115041074A (zh) * 2022-06-20 2022-09-13 安徽中科大禹科技有限公司 一种超声波雾化等离子体处理装置
CN115041074B (zh) * 2022-06-20 2023-09-29 安徽中科大禹科技有限公司 一种超声波雾化等离子体处理装置

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