WO2016089309A1

WO2016089309A1 - Method of preparing hollow microparticles and hollow microparticles prepared thereof

Info

Publication number: WO2016089309A1
Application number: PCT/SG2015/050483
Authority: WO
Inventors: Say Chye Joachim Loo; Sharad KHAREL; Wei Li Lee
Original assignee: Nanyang Technological University
Priority date: 2014-12-02
Filing date: 2015-12-02
Publication date: 2016-06-09

Abstract

A method of preparing hollow microparticles is provided. The method comprises dissolving a first hydrophobic polymer, preferably poly-lactic-co-glycolic acid (PLGA), in an organic solvent, such as dichloromethane (DCM), comprising an osmolyle, preferably sodium chloride, to form a polymer solution. Optionally, a second hydrophobic polymer, preferably poly-L-lactide (PLLA), is further added to the polymer solution. The polymer solution is then dispersed into an aqueous medium comprising a surfactant, preferably polyvinyl alcohol (PVA), to form an emulsion. When the organic solvent is evaporated from the emulsion, the aqueous medium penetrates through the polymer solution to form aqueous droplets and the hydrophobic polymer(s) precipitates to form a shell around one or more of the aqueous droplets which can be removed by lyophilisation to obtain hollow microparticles. Hollow microparticles prepared by the method are also provided.

Description

METHOD OF PREPARING HOLLOW MICROPARTICLES AND HOLLOW

MICROPARTICLES PREPARED THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore patent application No. 10201408019R filed on 2 December 2014, the content of which is incorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] Various embodiments relate to methods of preparing hollow microparticles, and hollow microparticles prepared thereof.

BACKGROUND

[0003] Controlled drug delivery systems to achieve controlled release of therapeutic drugs have been developed. These systems are believed to reduce institutional healthcare load for long term disease management, and increase patient compliance for diseases requiring extended treatment and monitoring regimes.

[0004] Hollow microparticles may be used in micro-encapsulation for such controlled drug deliveries. State of the art methods to prepare hollow microparticles include (a) template based fabrication techniques, whereby a surface of colloidal template of ionic particles is coated with a thin layer of desired material or its precursor, followed by selective removal of the templates by chemical etching or dissolution, (b) microfluidic fabrication techniques, involving use of three coaxial nozzles to produce a smooth coaxial jet comprising a carrier, annular shell and core streams, and which are acoustically excited to break the streams up into core-shell droplets, and (c) coaxial electrohydrodynamic micro -bubbling technique, whereby solution of a water-insoluble polymer, such as polymethylsilsesquioxane, is perfused through an outer needle portion of a co- axial needle arrangement, while an air flow is simultaneously passed through an inner needle portion, with both needles placed in an electric field, eventually collecting the microbubbles in distilled water in a hollow core- shell morphology.

[0005] Due to use of the inorganic particles as colloidal templates in template based fabrication techniques, removal by dissolution is required. As such, to coat polymers over such colloidal templates, the polymer shell coating has to be cross-linked in order to withstand this dissolution process to retain the morphology. Consequently, the process becomes relatively complicated, laborious and time consuming.

[0006] Likewise, microfluidic fabrication techniques require additional, selective dissolution of the core material to form a hollow cavity.

[0007] For coaxial electrohydrodynamic microbubbling techniques, many parameters affect the electrohydrodynamic process, such as viscosity, density, electrical conductivity, and relative permittivity, along with process parameters such as flow rates, and applied voltage. As such, successful fabrication of hollow microparticles becomes rather complicated.

[0008] In view of the above, there exists a need for an improved method to prepare hollow microparticles that overcomes or at least alleviates one or more of the above-mentioned problems.

SUMMARY

[0009] In a first aspect, a method of preparing hollow microparticles is provided. The method comprises

a) dissolving a first hydrophobic polymer in an organic solvent comprising an osmolyte to form a polymer solution,

b) dispersing the polymer solution into an aqueous medium comprising a surfactant to form an emulsion,

c) extracting the organic solvent from the emulsion, wherein the aqueous medium penetrates through the polymer solution to form aqueous droplets, and the first hydrophobic polymer precipitates to form a shell around one or more of the aqueous droplets, and

d) removing the aqueous droplets to obtain the hollow microparticles.

[0010] In a second aspect, hollow microparticles prepared by a method according to the first aspect are provided.

BRIEF DESCRIPTION OF THE DRAWINGS [0011] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0012] FIG. 1 shows schematic diagrams depicting general process flow according to embodiments for (A) single layer hollow microparticle; and (B) double layer hollow microparticle.

[0013] In the embodiment depicted in FIG. 1(A), a polymer solution comprising a first hydrophobic polymer dissolved in an organic solvent comprising an osmogen, otherwise termed herein as an osmolyte, is shown. The polymer solution is dispersed into an aqueous medium comprising a surfactant to form an oil in water (O/W) emulsion, wherein the polymer solution constitutes the oil phase and the aqueous medium comprising the surfactant constitutes the continuous phase. In the embodiment shown, the aqueous medium is water. The polymer solution that is dispersed in the aqueous medium form polymer solution droplets in the aqueous medium continuous phase. The aqueous medium, being driven by osmotic drive of the osmogen, penetrates through the polymer solution to form aqueous droplets in the polymer solution. The aqueous droplets may be scattered throughout the polymer solution. At the same time, or subsequently, the organic solvent is extracted from the emulsion which may take place by evaporation, and the first hydrophobic polymer precipitates to form a shell around an aqueous droplet. The aqueous droplets may migrate and coalesce in the polymer solution of the emulsion to grow in size. Similarly, the first hydrophobic polymer may migrate and coalesce on the coalesced aqueous droplets, to form microparticles comprising a shell of first hydrophobic polymer surrounding or encapsulating a core of aqueous droplet. The aqueous droplet is removed such as by lyophilization to obtain hollow microparticles.

[0014] In the embodiment depicted in FIG. 1(B), a polymer solution comprising a first hydrophobic polymer ("Polymer B") and a second hydrophobic polymer ("Polymer A") dissolved in an organic solvent comprising an osmolyte is shown. The polymer solution is dispersed into an aqueous medium comprising a surfactant to form an oil-in-water (O/W) emulsion, wherein the polymer solution constitutes the oil phase and the aqueous medium comprising the surfactant constitutes the continuous phase. In the embodiment shown, the aqueous medium is water. The polymer solution that is dispersed in the aqueous medium form polymer solution droplets in the aqueous medium continuous phase. The aqueous droplets may be scattered throughout the polymer solution. The organic solvent is extracted from the emulsion, wherein the aqueous medium, being driven by osmotic drive of the osmogen, penetrates through the polymer solution to form aqueous droplets, and the first hydrophobic polymer and the second hydrophobic polymer precipitates to form a first polymer layer and a second polymer layer around an aqueous droplet to form a core-shell structure. The aqueous droplets may migrate and coalesce in the polymer solution of the emulsion to grow in size. Similarly, the first hydrophobic polymer and the second hydrophobic polymer may migrate and coalesce on the coalesced aqueous droplets. By subsequently removing the aqueous droplet from the microparticle, for example, by freeze drying, a double-layered hollow microparticle is obtained.

[0015] FIG. 2 shows scanning electron micrograph (SEM) of (A) overall particle distribution, and (B) cross section of a microparticle. Scale bar in the figures denote 200 μιη and 20 μιη, respectively.

[0016] FIG. 3 shows scanning electron micrographs of a cross section of hollow microspheres fabricated with (A) 20 % w/w osmolyte, (B) 10 % w/w osmolyte, (C) 5 % w/w osmolyte, and (D) 1.25 % w/w osmolyte. Scale bar in the figures represent (A) 100 μιη, (B) 50 μιη, (C) 50 μιη, and (D) 20 μιη.

[0017] FIG. 4 shows scanning electron micrographs of cross section of hollow/porous microparticles fabricated using (A) sodium chloride (NaCl) as osmolyte, (B) potassium chloride (KC1) as osmolyte, and (C) sucrose as osmolyte. Scale bar in the figures represent 20 μιη.

[0018] FIG. 5 shows scanning electron micrograph of hollow microspheres fabricated using polymer solution concentration of (A) 6.67 % w/v, and (B) 3 % w/v. Scale bar in the figures represent 20 μιη and 50 μιη, respectively.

[0019] FIG. 6 (A) to (D) shows scanning electron micrograph of double layered hollow microparticles fabricated according to embodiments. Scale bar in the figures represent: (A) and (B) 50 μιη; and (C) and (D) 20 μιη.

[0020] FIG. 7 shows scanning electron micrograph of cross section of (A) microparticles distribution, and (B) single microparticles. Scale bar in the figures represent: (A) 100 μιη; and (B) 20 μιη. [0021] FIG. 8 shows scanning electron micrograph of cross-sectioned hollow microparticles showing the bovine insulin microcrystals embedded in the shell. Scale bar in the figure represents 10 μιη.

[0022] FIG. 9 shows Energy Dispersive X-Ray (EDX) spectrum of (A) blank carbon tape; (B) insulin microcrystals on carbon tape; (C) insulin microcrystals devoid region of hollow poly(lactic-co-glycolic acid) (PLGA) microparticle, and (D) insulin microcrystals embedded in shell of microparticle.

[0023] FIG. 10 depicts development of a high-performance liquid chromatography (HPLC) method where (A) a graph showing peaks for insulin at different concentrations for calibration curve, and (B) calibration curve and equation.

[0024] FIG. 11 is a table showing encapsulation efficiency obtained by extraction method, where extraction efficiency of hollow microparticles was calculated to be 83.96 % ± 2.56.

[0025] FIG. 12 shows hollow microparticles fabricated with varying shell thickness and insulin loaded in solid and solution form, where (A) to (F) are scanning electron micrographs of (A) standard hollow microparticle; (B) solid microparticle; (C) thick shell microparticle; (D) thin shell microparticle; (E) microparticle loaded with insulin solution; and (F) microparticle loaded with insulin solution and salt. Scale bar in the figures (A) to (C): 20 μιη; (D) and (E): 50 μιη; and (F): 100 μιη. FIG. 12(G) and (H) are graphs showing comparative release analyzed in different release medium, where (G) shows release in phosphate buffer saline (PBS); and (H) shows release in simulated gastric fluid (SGF).

[0026] FIG. 13 is (A) a graph depicting results of cell work for bioactivity assessment, and (B) flow chart of protocol followed for cell work. From the results obtained, it may be seen that level of bioactivity of model peptide insulin loaded in the hollow particles in different batches are on par with the positive control, and there is minimum loss of bioactivity in the peptide released from the hollow microparticles fabricated by a method disclosed herein.

[0027] FIG. 14 depicts morphologies of the paclitaxel and docetaxel-loaded microparticles with different size of hollow cavity, where scanning electron micrographs of (A) Sample Al; (B) Sample A2; (C) Sample A3; (D) Sample A4; (E) Sample A5; (F) Sample A6; (G) Sample A7; and (H) Sample A8 listed in TABLE 1 are shown.

[0028] FIG. 15 shows graphs depicting the encapsulation efficiency (E.E) (%) of (A) paclitaxel and (B) doxorubicin in different formulations (sample size n, which represents number of times experiment was repeated, is 3). The E.E (%) of paclitaxel and doxorubicin in PLLA microparticles was higher than those of PLGA microparticles regardless of amount of salt used. This is due to hydrophobicity of PLLA. Poly-L-lactic acid (PLLA) microparticle exhibited increased E.E of both drugs regardless of the amount of salt, as higher lactide content generally increased the yield and E.E. Further formulations including cyclodextrins were conducted using 5 mg of salt due to good E.E (%) of paclitaxel and doxorubicin in both polymers.

[0029] FIG. 16 shows scanning electron micrographs of internal morphologies of (A) Sample B l, (B) Sample B3, and (C) Sample B5 listed in TABLE 2. Scale bar in the figures represents 20 μιη.

[0030] FIG. 17 shows graphs depicting encapsulation efficiency (E.E) (%) of (A) paclitaxel and (B) docetaxel in different formulations (n = 3). In the presence of HPCD, a progressive increase of E.E (%) of PTX from about 60 % to 90 % (w/w) was observed. PLLA or PLGA microspheres were able to encapsulate only a small amount of hydroxypropyl-β- cyclodextrin (HPCD) in polymer layer, probably as a result of the high water solubility of the HPCD, which reduces its affinity for the PLLA and PLGA matrices and was located in hollow cavity during water influx due to salt.

[0031] FIG. 18 is a graph showing release profiles of paclitaxel (PTX) from A3, B l, B3 and B5 for 480 h in PBS with 0.1% polyoxyethylene sorbitan monooleate (Tween 80) (n = 3).

[0032] FIG. 19 is a graph showing release profiles of PTX from A3, B l, B3 and B5 for 480 h in PBS with 0.1% Tween 80 (n = 3).

[0033] FIG. 20 shows a MCF-7 cell proliferation assay using CCK-8 assay, depicting evaluation of effectiveness of hollow microparticles fabricated by a method disclosed herein in preserving bioactivity of the loaded peptide.

[0034] FIG. 21 shows a graph summarizing results of a circular dichroism spectroscopy study.

DETAILED DESCRIPTION

[0035] Methods according to embodiments disclosed herein provide a single-step method to fabricate hollow microparticles having a shell comprising a hydrophobic polymer surrounding a void to assume a core-shell structure. By involving use of osmolyte as a water attractant to draw water into a polymer solution through osmotic pressure, and precipitation of polymer through solvent evaporation, hollow microparticles may be easily, economically, and efficiently prepared.

[0036] Advantageously, methods to fabricate hollow microparticles according to methods disclosed herein are simple and fast, yet fully controllable. Morphology such as hollow core volume, shell thickness, porosity, and particle size of the hollow microparticles may be kinetically controlled by varying process parameters, such as concentration of polymer in solution, osmolyte type and concentration, stirring speed of solution, and oil to water ratio. By adjusting the process parameters, polymer precipitation rate and osmotic pressure may be varied, which may in turn be used to tailor morphology of the hollow microparticles formed.

[0037] With the above in mind, various embodiments refer in a first aspect to a method of preparing hollow microparticles. As used herein, the term "microparticle" refers to a microscopic particle having a size measured in micrometers (μιη). The term "hollow microparticle" refers to a microparticle having a void or a cavity. One or more than one void or cavity may be present in the microparticle, such as one, two, three, four or five voids or cavities. For example, the hollow microparticle may have a single void which forms a core of the microparticle, with a shell surrounding the single void. When more than one void is present in the microparticle, the microparticle may have structure of a shell surrounding the one or more voids to form the microparticle, such that the microparticle assumes a porous structure within an interior of the microparticle. The voids or cavities may have the same size or a different size. The one or more voids may be contained entirely within an interior of the microparticle, for example, contained within a shell and is/are not present on an exterior surface of the microparticle.

[0038] In various embodiments, the hollow microparticle has a shell surrounding a single void to assume a core-shell structure.

[0039] The method comprises dissolving a first hydrophobic polymer in an organic solvent comprising an osmolyte to form a polymer solution.

[0040] The term "hydrophobic" is generally used to describe a substance that repels water. In line with this definition, the term "hydrophobic polymer" refers to a polymer having a low affinity for aqueous solutions including water. For example, hydrophobic polymers may include polymers that do not dissolve in, be mixed with, or be wetted by water. As another example, hydrophobic polymers may also include polymers that do not absorb an appreciable amount of water.

[0041] In embodiments disclosed herein, a suitable hydrophobic polymer may be one that is able to dissolve in an organic solvent, and be precipitated using a solvent extraction process.

[0042] In various embodiments, the hydrophobic polymer may be a natural polymer or a synthetic polymer. The term "natural polymer" as used herein refers generally to a polymeric material that may be found in nature. Examples of a natural polymer include, but are not limited to, natural rubber and cellulose such as ethyl cellulose. Examples of synthetic polymers include, but are not limited to, polyolefin, polystyrene, polyester, polyamide, polyether, polysulfone, polycarbonate, polyurea, polyurethane, polysiloxane, copolymers thereof, and mixtures thereof.

[0043] The hydrophobic polymer may be biodegradable or non-biodegradable, which may depend on the intended application. For drug delivery applications, biodegradable polymers are generally used. In various embodiments, the hydrophobic polymer is a biodegradable polymer or a biocompatible material.

[0044] Biodegradable polymers refer generally to natural or synthetic polymers that gradually degrade in vivo to produce biocompatible or non-toxic byproducts over a period of time (e.g., within days, or months, or years). Disintegration may for instance occur via hydrolysis, may be catalyzed by an enzyme and may be assisted by conditions to which the microparticles are exposed in the cell. The term "biocompatible" refers to a material that is capable of interacting with a biological system without causing cytotoxicity, undesired protein or nucleic acid modification or activation of an undesired immune response.

[0045] Examples of biodegradable polymers include, but are not limited to, polymers and oligomers of glycolide, lactide, polylactic acid (PLA), polyesters of a-hydroxy acids, including lactic acid and glycolic acid, such as the poly(a-hydroxy) acids including polyglycolic acid (PGA), poly(DL-lactic-co-glycolic acid) (PLGA), poly-L-lactic acid (PLLA), and terpolymers of DL-lactide and glycolide; ε-caprolactone and ε-caprolactone copolymerized with polyesters; polylactones and polycaprolactones including poly(caprolactone) (PCL), poly(8-caprolactone), poly(valerolactone) and poly(gamma- butyrolactone); polyanhydrides; polyorthoesters; polydioxanones; and other biologically degradable polymers that are non-toxic or are present as metabolites in the body. [0046] Non-exhaustive examples of biocompatible polymers include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose such as methyl cellulose and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, and hydroxybutyl methyl cellulose, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly (ethylmethacry late), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(laurylmethacrylate), poly(phenylmethacrylate), poly(methacrylate), poly (isopropacrylate), poly(isobutacrylate), poly(octadecacrylate), polyethylene, polypropylene, poly (ethylene glycol), poly (ethylene oxide), poly (ethylene terephthalate), poly (vinyl alcohols), poly(vinyl acetate) such as ethylene vinyl acetate (EVA), poly(vinyl chloride), polystyrene, polyhyaluronic acids, casein, gelatin, gluten, polyanhydrides, polyacrylic acid, alginate, chitosan, any copolymers thereof, and mixtures thereof.

[0047] Choice of hydrophobic polymer to be used in a method disclosed herein may depend on the end application. For example, for application in biomedical areas, polymers such as PCL, PGA, PLA and PLGA are of interest because of their biocompatibility and biodegradability properties. In particular, PLGA has been approved by US Food and Drug Administration (FDA) for human therapy. When degrading, PLA and PLGA chains are cleaved in the body to monomeric acids, i.e. lactic and glycolic acids that are eliminated from the organism through Kreb's cycle as C0₂ and in urine as water. A person skilled in the art is able to choose and determine the appropriate polymer type based on the area of specific application.

[0048] In various embodiments, the first hydrophobic polymer is selected from the group consisting of poly-lactic-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), poly- caprolactone (PCL), polyglycolide (PGA), derivatives thereof, copolymers thereof, or mixtures thereof.

[0049] The first hydrophobic polymer is dissolved in an organic solvent comprising an osmolyte to form a polymer solution. As used herein, the term "organic solvent" refers to a solvent comprised of a carbon-containing chemical. The organic solvent may be immiscible with an aqueous medium. By dissolving the first hydrophobic polymer in an organic solvent, a homogeneous solution may be formed. Therefore, a suitable solvent may be one that is able to dissolve the first hydrophobic polymer.

[0050] Amount of hydrophobic polymer in the polymer solution may be any suitable amount that is able to at least substantially dissolve in the organic solvent. This may also depend on, for example, thickness of the hydrophobic polymer shell to be formed. Generally, a higher concentration of hydrophobic polymer in the polymer solution results in formation of a thicker polymer shell on the microparticle.

[0051] Concentration of the hydrophobic polymer in the polymer solution may be calculated by dividing the weight of polymer present by the volume of organic solvent (expressed as % w/v). In various embodiments, concentration of the first hydrophobic polymer in the polymer solution is in the range of about 1 % (w/v) to about 30 % (w/v), such as between about 10 % (w/v) to about 30 % (w/v), about 20 % (w/v) to about 30 % (w/v), about 1 % (w/v) to about 20 % (w/v), about 3 % (w/v) to about 15 % (w/v), about 3 % (w/v) to about 10 % (w/v), about 8 % (w/v) to about 12 % (w/v), about 8 % (w/v), about 10 % (w/v), or about 12 % (w/v). In various embodiments, concentration of the first hydrophobic polymer in the polymer solution is about 10 % (w/v).

[0052] Suitable organic solvents that may be used include, but are not limited to, methylene chloride (dichloromethane or DCM), methanol, dimethylformamide (DMF), tetrahydrofuran (THF), methyl ethyl ketone (MEK), chloroform, pentane, benzene, benzyl alcohol, carbon tetrachloride, ethyl acetate (EAc), acetone, acetonitrile, dimethyl sulfoxide, propylene carbonate (PC), and mixtures thereof. In various embodiments, the organic solvent comprises or consists essentially of dichloromethane.

[0053] In various embodiments, the organic solvent is a volatile organic solvent such as chloroform or dichloromethane. The term "volatile" as used herein refers to a compound that may be readily vaporized at ambient temperature. One measure of the volatility of a substance is its boiling point at one atmosphere. For example, the volatile organic solvent may have a boiling point at one atmosphere of less than about 100 °C, such as about 75 °C or about 50 °C or about 40 °C or about 30 °C. [0054] To enhance the rate at which the first hydrophobic polymer dissolves in the organic solvent, agitation, for example, by stirring or sonication may be used. Heat energy may also be applied to increase the dissolve rate of polymer in the solvent.

[0055] An osmolyte is comprised in the organic solvent. The term "osmolyte", otherwise referred to herein as "osmogen", refers generally to compounds or substances that affect osmosis. In embodiments disclosed herein, an osmolyte may be added to provide increased osmotic pressure to drive water from the aqueous medium into the polymer solution to form aqueous droplets within the polymer solution.

[0056] Examples of osmolyte that may be used include, but are not limited to, sodium chloride, potassium chloride, sucrose, sodium bromide, sodium citrate, sodium lactate, sodium hydroxide, sodium iodide, sodium carbonate, sodium hydrogen carbonate, sodium nitrate, sodium fluoride, sodium sulfate, potassium carbonate, potassium citrate, potassium lactate, potassium hydrogen carbonate, potassium bromide, potassium hydroxide, potassium iodide, potassium nitrate, potassium sulfate, cesium chloride, rubidium chloride, lithium chloride, and mixtures thereof. In various embodiments, the osmolyte comprises or consists essentially of sodium chloride.

[0057] Amount of osmolyte in the polymer solution may be 20 % (w/w) or less. For example, amount of osmolyte in the polymer solution may be in the range of about 1 % (w/w) to about 18 % (w/w), about 1 % (w/w) to about 15 % (w/w), about 1 % (w/w) to about 13 % (w/w), about 1 % (w/w) to about 10 % (w/w), about 5 % (w/w) to about 20 % (w/w), about 8 % (w/w) to about 20 % (w/w), about 10 % (w/w) to about 20 % (w/w), about 12 % (w/w) to about 14 % (w/w), about 3 % (w/w) to about 15 % (w/w), about 8 % (w/w) to about 16 % (w/w), or about 3 % (w/w) to about 8 % (w/w). In specific embodiments, amount of osmolyte in the polymer solution is in the range of about 1.25 % (w/w) to about 10 % (w/w).

[0058] The osmolyte may remain substantially undissolved, or undissolved in the polymer solution. In various embodiments, the osmolyte may be at least substantially uniformly distributed within the polymer solution.

[0059] The method comprises dispersing the polymer solution into an aqueous medium comprising a surfactant to form an emulsion.

[0060] The term "aqueous medium" as used herein refers to water or a solution based primarily on water such as phosphate buffered saline (PBS), or water containing a salt dissolved therein. [0061] The aqueous medium comprises a surfactant, which may be added to stabilize the emulsion, thereby functioning as a stabilizer.

[0062] The surfactant may be an amphoteric surfactant, an anionic surfactant, a cationic surfactant, a nonionic surfactant, or mixtures thereof.

[0063] Examples of amphoteric surfactants include, but are not limited to, dodecyl betaine, sodium 2,3-dimercaptopropanesulfonate monohydrate, dodecyl dimethylamine oxide, cocamidopropyl betaine, 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]- propanesulfonate, coco ampho glycinate, and mixtures thereof.

[0064] Examples of an anionic surfactant include, but are not limited to, sodium dodecyl sulfate (SDS), sodium pentane sulfonate, dehydrocholic acid, glycolithocholic acid ethyl ester, ammonium lauryl sulfate and other alkyl sulfate salts, sodium laureth sulfate, alkyl benzene sulfonate, soaps, fatty acid salts, and mixtures thereof.

[0065] Examples of a cationic surfactant include, but are not limited to, cetyl trimethylammonium bromide (CTAB), dodecylethyldimethylammonium bromide (D12EDMAB), didodecyl ammonium bromide (DMAB), cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), hexadecyltrimethylammonium p-toluenesulfonate, benzalkonium chloride (BAC), benzethonium chloride (BZT), and mixtures thereof.

[0066] In various embodiments, the surfactant is a non-ionic surfactant. Examples of non- ionic surfactants include, but are not limited to, poloaxamers, alkyl poly(ethylene oxide), diethylene glycol monohexyl ether, polyvinyl alcohol (PVA), copolymers of poly(ethylene oxide) and poly(propylene oxide), hexaethylene glycol monohexadecyl ether, alkyl polyglucosides, digitonin, ethylene glycol monodecyl ether, cocamide monoethanolamine (cocamide MEA), cocamide diethanolamine (cocamide DEA), cocamide triethanolamine (cocamide TEA), fatty alcohols, and mixtures thereof.

[0067] In specific embodiments, the surfactant comprises or consists of polyvinyl alcohol (PVA).

[0068] Concentration of surfactant in the aqueous medium may be in the range of about 0.1 % (w/v) to about 5 % (w/v). For example, the surfactant may be present in the aqueous medium in the range of about 0.1 % (w/v) to about 3 % (w/v), about 0.1 % (w/v) to about 2 % (w/v), about 0.1 % (w/v) to about 1 % (w/v), about 0.5 % (w/v) to about 5 % (w/v), about 1 % (w/v) to about 5 % (w/v), about 3 % (w/v) to about 5 % (w/v), about 1 % (w/v) to about 4 % (w/v), about 2 % (w/v) to about 3.5 % (w/v), or about 1 % (w/v) to about 3 % (w/v). [0069] The polymer solution is dispersed into the aqueous medium comprising a surfactant to form an emulsion. The term "emulsion" as used herein refers to a disperse system of two or more immiscible liquids. Therefore, emulsifying of one liquid in the other can result in formation of two different phases, in which small droplets of one liquid may be dispersed, i.e. separated and distributed throughout the space, in the other liquid. The small droplets of liquid is called the dispersed phase, while the other liquid, within which the small droplets of liquid is dispersed, is called the continuous phase.

[0070] Most emulsions consist of water and oil or fat as immiscible phases. Depending on the composition and ratio of the phases two distribution options exist. In case the aqueous phase, such as water "W" is the continuous phase and the oil "O" is the dispersed phase, the result is an "O/W emulsion" or oil-in-water emulsion, whose basic character is determined by the aqueous phase. If oil "O" is the continuous phase and water "W" the dispersed phase, the result is a "W/O emulsion" or water-in-oil emulsion, wherein the basic character is determined by the oil.

[0071] In embodiments disclosed herein, the organic solvent-based polymer solution is dispersed into the aqueous medium to form an emulsion, which is an oil-in-water (O/W) emulsion. The surfactant that is present in the aqueous medium may function to stabilize the polymer solution dispersed therein to form the oil-in-water emulsion.

[0072] The term "oil-to-water ratio" as used herein may refer to the volumetric ratio of polymer solution to aqueous medium (expressed as v/v). It may be calculated by dividing the volume of polymer solution by the volume of the aqueous medium. Generally, the oil-to- water ratio may be controlled to affect the time required for evaporation of the solvent as well as mobility of the polymer in solution, which may in turn affect the precipitation rate of the polymers.

[0073] In various embodiments, ratio of the polymer solution to the aqueous medium may be in the range of about 0.01 to about 0.1 v/v, such as about 0.01 to about 0.08 v/v, about 0.02 to about 0.09 v/v, about 0.03 to about 0.08 v/v, or about 0.05 to about 0.07 v/v.

[0074] Dispersing the polymer solution into the aqueous medium may be carried out under continuous stirring, or any form of dispersing method that is able to emulsify two different immiscible phases. Examples of dispersing methods include, but are not limited to, continuous stirring, ultrasonic emulsification and homogenization using a homogenizer. [0075] Advantageously, use of continuous stirring allows size of hollow microparticles formed to be controlled simply by varying the speed of stirring. For example, a lower stirring speed may generally result in a larger emulsion droplet size, which may in turn result in an increase in size of the hollow microparticles formed. On the other hand, a higher stirring speed may result in a smaller emulsion droplet size, which may translate into formation of smaller sized hollow microparticles. Accordingly, speed of stirring may be used to affect the size of microparticles formed.

[0076] Stirring speed as used herein may have a range of between about 150 rpm to about 2000 rpm, such as between about 500 rpm to about 2000 rpm, about 1000 rpm to about 2000 rpm, about 150 rpm to about 1500 rpm, about 150 rpm to about 1000 rpm, about 200 rpm to about 800 rpm, about 300 rpm to about 500 rpm, about 300 rpm, about 400 rpm, or about 500 rpm. In various embodiments, continuous stirring is carried out at a speed of about 300 rpm to about 2000 rpm.

[0077] Continuous stirring may be carried out for any suitable amount of time. For example, the continuous stirring may be carried out for a time period in the range of about 1 hour to about 12 hours, such as about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 6 hours to about 12 hours, about 3 hours to about 6 hours, about 5 hours, about 4 hours or about 3 hours. In various embodiments, the continuous stirring is carried out for a time period in the range of about 3 hours to about 5 hours.

[0078] The method comprises extracting the organic solvent from the emulsion. Extracting the organic solvent from the emulsion may be carried out in such a way that the aqueous medium penetrates through the polymer solution to form aqueous droplets, and the first hydrophobic polymer precipitates to form a shell around one or more of the aqueous droplets.

[0079] In various embodiments, extracting the organic solvent from the emulsion is carried out by solvent evaporation. When the organic solvent is removed from the emulsion droplets dispersed in the aqueous medium, concentration of hydrophobic polymer in the emulsion droplets may increase until a point at which the hydrophobic polymer and organic solvent phase separate, i.e. coacervate forming a first coacervate phase within the emulsion droplet. The first coacervate phase comprising the first hydrophobic polymer may coacervate to form a layer on one or more of the aqueous droplets. The polymer precipitation rate may take place at an appropriate speed that is sufficient to allow time for the coacervate droplets to move and to coalesce. The process may continue until residual solvent is removed, after which microparticles comprising a polymer shell and core of aqueous droplet(s) are formed.

[0080] In various embodiments, the hydrophobic polymer precipitates to form a shell around a single aqueous droplet to obtain a microparticle having a core-shell structure.

[0081] The mean diameter of the aqueous droplets formed, which constitute a void or core of the resulting hollow microparticles, may be in the range of about 40.5 + 13.20 μιη to about 84 + 24.5 μιη.

[0082] Formation of the aqueous droplets and precipitation of the hydrophobic polymer shell around one or more of the aqueous droplets may take place concurrently. For example, while the aqueous medium penetrates through the polymer solution of the emulsion and coalesces to form one or more aqueous droplets in the polymer solution, the hydrophobic polymer that is contained in the polymer solution may precipitate to form a shell around one or more of the aqueous droplets. The rate at which the aqueous droplets and the polymer shell are formed may be the same or different. In some embodiments, formation of the aqueous droplets takes place at a faster rate than precipitation of the hydrophobic polymer shell around one or more of the aqueous droplets.

[0083] The polymer shell may be defined by a single wall with an internal and an external surface (i.e., balloon-like). Thickness of the hydrophobic polymer shell formed may be in the range of about 1 μιη to about 100 μιη, such as about 1 μιη to about 80 μιη, about 1 μιη to about 60 μιη, about 1 μιη to about 40 μιη, about 1 μιη to about 20 μιη, about 1 μιη to about 5 μιη, about 10 μιη to about 100 μιη, about 30 μιη to about 100 μιη, about 50 μιη to about 100 μιη, about 25 μιη to about 75 μιη, or about 30 μιη to about 60 μιη.

[0084] Upon forming microparticles having a polymer shell and core of aqueous droplet(s), the aqueous droplets are removed to obtain the hollow microparticles. For example, the hydrophobic polymer may precipitate to form a shell around a single aqueous droplet to obtain a microparticle having a core-shell structure. With removal of the single aqueous droplet, a hollow microparticle having a core-shell structure may be obtained.

[0085] Removing the aqueous droplets may be carried out by lyophilization.

Lyophilization, otherwise termed herein as freeze drying, refers to freezing of a material at temperatures sufficiently low to dehydrate the material by sublimation. The lyophilization process is usually carried out under vacuum. [0086] Lyophilizing the microparticles may be carried out at any suitable temperature which is sufficient to sublime water present in the microparticles. In various embodiments, lyophilizing the microparticles is carried out at a temperature in the range of about -50 °C to about 0 °C, such as about -30 °C to about 0 °C, about -10 °C to about 0 °C, about -50 °C to about -10 °C, about -50 °C to about -20 °C, about -50 °C to about -30 °C, about -40 °C to about -10 °C, about -30 °C to about -20 °C. In some embodiments, freeze drying the microparticles is carried out at a temperature of about -45 °C.

[0087] The method of the first aspect may further comprise at least one centrifugation and at least one washing step after removing the aqueous droplets.

[0088] Besides forming hollow microparticles comprising a single layer of polymer shell surrounding a void or cavity, methods disclosed herein may be used to form hollow microparticles having two or more layers surrounding a void or cavity. Accordingly, the method may further comprise dissolving a second hydrophobic polymer in the organic solvent comprising an osmolyte. In so doing, the hollow microparticles formed may have a two-layered structure of a first layer comprising the first hydrophobic polymer, and a second layer comprising the second hydrophobic polymer. The first layer and the second layer of hydrophobic polymers may surround one or more voids to form a multilayered hollow microparticle. Examples of suitable hydrophobic polymers that may be used for the second hydrophobic polymer have already been discussed above.

[0089] In various embodiments, the first hydrophobic polymer and the second hydrophobic polymer are independently selected from the group consisting of poly-lactic-co- glycolic acid (PLGA), poly-l-lactic acid (PLLA), poly-caprolactone (PCL), polyglycolide (PGA), derivatives thereof, copolymers thereof, and mixtures thereof.

[0090] In specific embodiments, the first hydrophobic polymer is poly-lactic-co-glycolic acid and the second hydrophobic polymer is poly-l-lactic acid.

[0091] As in the case where one hydrophobic polymer is used to form the shell of the hollow microparticles, total concentration of the first hydrophobic polymer and the second hydrophobic polymer in the polymer solution may be in the range of about 1 % (w/v) to about 30 % (w/v).

[0092] Examples of applications in which the hollow microparticles may be used include, but are not limited to, a drug delivery system (DDS) for delivery of drugs, proteins, peptides, DNA, cells, dyes and other biomedical-applied systems, and therapeutic agents. [0093] Accordingly, the method disclosed herein may further comprise adding a hydrophobic active compound to be encapsulated to the polymer solution. The term "hydrophobic active compound" as used herein refers to a hydrophobic compound which is intended to be delivered or released. In various embodiments, the hydrophobic active compound is dissolved in the organic solvent and is thereby encapsulated within the shell of the hollow microparticles. Advantageously, this allows incorporating hydrophobic active compounds into the hollow microparticles in a single step process, which translates into processing simplicity and efficiency.

[0094] One or more different types of hydrophobic active compound may be added. The type of interaction between the hydrophobic active compound and the first hydrophobic polymer may be physical or chemical in nature. In various embodiments, one or more hydrophobic active compounds may be loaded in the first hydrophobic polymer via physical bonding, for example, by any one of hydrophobic forces, hydrogen bonding, van der Waals interaction, or electrostatic forces.

[0095] In various embodiments, one or more hydrophobic active compounds may be loaded in the first hydrophobic polymer via chemical bonding, for example, by covalent bonding, ionic bonding, or affinity interactions (e.g. ligand/receptor interactions, antibody/antigen interactions, etc.). Therefore, one or more types of hydrophobic active compound may be loaded and localized in the shell of the microparticle by choosing the appropriate type of interaction between the hydrophobic active compound and the first hydrophobic polymer. This may in turn be exploited for controlled release of the hydrophobic active compound in application.

[0096] Examples of hydrophobic active compound that may be encapsulated include, but are not limited to, a drug, a protein, an enzyme, an antibody, a peptide, a growth factor, an organic molecule, a nucleic acid, a cell, a pesticide, a dye, a chemical indicator and a fertilizer.

[0097] In some embodiments, the hydrophobic active compound comprises or consists of a drug. The term "drug" refers to a substance useful for the treatment of or the prevention of a human or an animal disorder or in the regulation of a human or animal physiological condition or metabolic state. Examples of drug include, but are not limited to, antihistamines, e.g. diphenhydramine and chlorphenirmine, and drugs affecting the cardiovascular, renal, hepatic and immune systems, such as antihypertensives, beta blockers, and cholesterol lowering agents; sympathomimetic drugs, such as the catecholamines, e.g. epinephrines; noncatecholamines, e.g. phenylephrine and pseudoephedrine; anti-infective agents, including antibacterial, antiviral and antifungal agents, such as the aminoglycosides, e.g., streptomycin, gentamicin, kanamycin; anti-arthritis drugs, such as narcotic pain relievers; anti- inflammatory agents, e.g. indomethacin, dexamethasone and triamcinolone; antitumor agents, e.g. 5-fluorouracil and methotrexate; and tranquilizers, such as diazepam.

[0098] In specific embodiments, the drug is a hydrophobic drug selected from the group consisting of ibuprofen, paclitaxel, doxorubicin, docetaxel, and combinations thereof.

[0099] In some embodiments, the hydrophobic active compound comprises or consists of a peptide. In specific embodiments, the peptide comprises or consists of bovine insulin. During the fabrication process, the hydrophobic nature of bovine insulin allows localization of the peptide in the oil phase of the emulsion, and which forms the shell of the hollow microparticle.

[00100] As freeze drying may subject the hollow particles and active compounds contained therein to destabilizing stresses, suitable excipients and stabilizing additives may be included in formulation for stability during freeze drying. Lyoprotectants such as dextran, glycols, glycerol and cyclodextrins may be used to minimize instability in some freeze-dried formulations. In various embodiments, a cyclodextrin is added to the polymer solution during hollow microparticles preparation for stabilizing active compounds during freeze drying. Further, inclusion of cyclodextrin may also help in better encapsulation of the active compounds and tuning the release profile of the system.

[00101] Various embodiments refer in a second aspect to hollow microparticles prepared by a method according to the first aspect.

[00102] Mean diameter of the hollow microparticles formed may be in the range of about 100 μπι to about 800 μιη.

[00103] In various embodiments, the hollow microparticles formed are essentially monodispersed.

[00104] As mentioned above, the hollow microparticles may be used in a drug delivery system (DDS) for delivery of drugs and therapeutic agents. For a hollow microparticle comprising a hydrophobic active compound, the hollow microparticle may be degraded at a site of intended usage to release the hydrophobic active compound contained therein. The term "degrade" as used herein refers to breaking down of the microparticle to smaller molecules. The hollow microparticle may degrade at different rates depending on the environment the microparticles are placed in, and the conditions at which the microparticles are subjected to.

[00105] Degradation may take place under certain conditions, such as temperature, abrasion, pH, ionic strength, electrical voltage, current effects, radiation and biological means. In some embodiments, degradation of the hollow microparticles takes place over a time period ranging from a few seconds to a few days or months. The time period required for the microparticle to degrade may be dependent on a few parameters, for example, constituent of the microparticles, such as type of polymer and hydrophobic active compound used, size of the microparticles, temperature, pH and pressure. In various embodiments, the hollow microparticles formed using a method disclosed herein are used for sustained release of a hydrophobic active compound encapsulated therein.

[00106] In various embodiments, hollow microparticles containing the hydrophobic active compounds are comprised in a pharmaceutical composition. The composition may be poured or injected into a mold having a desired shape, and then hardened to form a matrix having hollow microparticles dispersed therein. The shell comprising the hydrophobic polymer may degrade, leaving only the active compound. Therefore, a sustained release of the target substance such as fertilizer or pesticide may be achieved along with degradation of the microparticles. In some embodiments, the composition is adapted to be deliverable to a site, such as a defect site, in an animal or a human body. The composition may be injected directly into a site, such as a defect site, in a patient, where the polymer may harden into a matrix having microparticles dispersed therein. The polymer may be biodegradable. Therefore, a sustained release of the active compound, such as drugs, may be achieved along with degradation of the polymer.

[00107] Hereinafter, the present invention will be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, lengths and sizes of layers and regions may be exaggerated for clarity. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. [00108] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00109] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00110] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

EXPERIMENTAL SECTION

[00111] As disclosed herein, various embodiments relate to a facile single-step fabrication technique to form polymeric microparticles with well-defined hollow core-shell structures. Applications may range from micro -encapsulation to drug delivery, and other applications that require hollow core- shell structures.

[00112] Compared to state of the art methods, methods of preparing hollow microparticles disclosed herein is simpler, faster and more robust, as it avoids multiple steps necessarily used in the earlier techniques. A simple but effective single-step fabrication technique to develop hollow microparticles, and the key parameters that are able to control the morphological properties of these hollow microparticles, has been demonstrated. [00113] A fabrication technique for preparation of hollow microparticles, which may be used for drug delivery, is described as follow:

[00114] The hollow microparticles were prepared using O/W emulsion solvent evaporation method. Briefly, the polymer and osmolyte (NaCl) were added to a volatile organic solvent and stirred until polymer dissolved completely and the osmolyte particles were suspended uniformly. The resultant polymer solution was added into the water bath containing surfactant and emulsified using an overhead stirrer allowing for the extraction and evaporation of solvent which gave rise to final hollow microspheres. Finally, the microspheres produced were filtered, rinsed with de-ionized water, lyophilized and stored in a desiccator.

[00115] Example 1; Manipulation of process parameters

[00116] Selection of polymer: Any water-insoluble polymers may be used regardless of their miscibility or immiscibility in the organic solvent. For example, dichloromethane was used in the fabrication of the particles because of the miscibility of the polymers (i.e. PLGA) in dichloromethane.

[00117] Polymer solution concentration: Lowering the polymer solution concentration decreased the size of the hollow microspheres significantly.

[00118] Osmolyte content: Amount of osmolyte may be carefully tuned. With decreasing amount of osmolyte, thickness of shell increased while the hollow core volume diminishes. Also, increasing the amount of osmolyte beyond a certain concentration (such as 20 % w/w of NaCl) may result in loss of hollow core-shell morphology in certain embodiments. For example, a hollow core shell morphology may be formed for as low as 1.5 % w/w of osmolyte to as much as 20% w/w osmolyte. In some embodiments, increasing amount of osmolyte to more than 20% w/w (such as 25% w/w) may result in difficulty in controlling morphology of the microparticles, and extremely porous random sized particles may be obtained instead.

[00119] Osmolyte type: Different osmolytes with their own unique osmotic property may be used to control the morphology of the hollow microspheres.

[00120] Overhead stirring speed: It may be manipulated to control the size of the hollow microspheres.

[00121] Precipitation rate of polymer: Precipitation rate of polymer during fabrication affects the internal morphology of the particle, i.e. well-defined hollow core-shell or scattered multiple pores. The process parameters that affect precipitation rate such as a) the amount of osmolyte, b) polymer solution concentration, c) oil to water ratio, and d) stirring speed, therefore, should be carefully adjusted to achieve suitable precipitation rate of the polymer. The precipitation rate should be slow enough just to allow the coalescence of the inner water droplets into the core of the polymer emulsion droplets.

[00122] Example 2: Fabrication of hollow microparticles (Embodiment 1)

[00123] 0.2 g of poly(lactic-co-glycolic acid) (PLGA) (6.67 % w/v) and 5 mg of sodium chloride (NaCl) (2.5 % w/w) were added to 3 ml of dichloromethane (DCM). The content was put under magnetic stirring for 3.5 hours and vortexed for 20 minutes to ensure that the polymer was completely dissolved and finer salt particles were uniformly distributed within the polymer solution. Subsequently, the polymer/salt solution was poured into 50 ml of polyvinyl alcohol (PVA) aqueous solution (3 % w/v) and emulsified under overhead stirring for 3.5 hours at 500 rpm. This amount of PVA aqueous solution was carefully chosen based on solubility limit of DCM in water (2 % v/v). At this oil to water ratio (o/w: 0.06), the slow diffusion of DCM into the external aqueous phase led to slower precipitation of the polymers. FIG. 2(B) is a scanning electron micrograph showing a cross- sectional view of a hollow microparticle fabricated.

[00124] When the polymer solution containing uniformly dispersed osmolyte was emulsified with the external aqueous phase, o/w emulsion droplets were first formed. As the polymer droplets contained dispersed osmolyte, the osmotic pressure drove water from the external aqueous phase into the polymer droplet, giving rise to spontaneous formation of inner water droplets within the polymer droplets.

[00125] Since oil to water ratio and amount of osmolyte were key factors in altering precipitation rate of the polymer, they were carefully controlled to maintain the precipitation rate such that it was slow enough to allow penetrating water droplets to coalesce into the core to reduce the interfacial area between oil and inner water phases. After freeze drying, the water was removed and finally the microspheres with hollow core were formed. The microparticles were homogenous when the internal hollow structure was observed (FIG. 2(A)).

[00126] Example 3: Effect of amount of osmolyte (Embodiment 2)

[00127] In a similar experiment, changing osmolyte (NaCl) amount to vary the osmotic pressure resulted in microspheres with varied volume of the hollow core and thickness of the shell. The results were in agreement to the different magnitude of osmotic pressure by corresponding amount of osmolyte. FIG. 3 shows cross-sectional views of the microspheres fabricated with different percentage weights of same osmolyte in the polymer solution, keeping all other process parameters the same.

[00128] The osmotic pressure in the emulsion droplet increased with increasing the amount of osmolyte. High osmolyte content drove tremendous amount of water into the emulsion droplet causing the precipitation rate to increase significantly. Hence, the polymer started to precipitate faster which did not provide enough time for the inner water droplets to coalesce into the core, and resulted in the formation of multiple large pores dispersed within the particles (FIG. 3A).

[00129] Decreasing the osmolyte amount to obtain desirable slower precipitation rate, however, subsequently facilitated coalescence of inner water droplets into the core resulting in hollow core-shell morphology (FIG. 3B). With further decrease of osmolyte amount, area of the hollow core decreased, while shell thickness increased as less water penetrated into the o/w emulsion droplet (FIG. 3(C) and FIG. 3(D)).

[00130] Example 4: Effect of osmolyte type (Embodiment 3)

[00131] In a similar experiment as Embodiment 1, when the osmolyte (a) NaCl was replaced with (b) potassium chloride (KC1) or (c) sucrose, the relationship between osmotic pressure of osmolyte to the precipitation rate of polymer and the final morphology of the hollow core-shell microparticles was elucidated.

[00132] The osmotic pressure (OP) of 5 mg of NaCl, 5 mg KC1 and 10 mg sucrose in the polymer emulsion droplet, assuming same initial conditions for each osmolyte-containing emulsion droplet, given by the relation, OP = iMRT, where i = Van't Hoff Factor, M= Molar Concentration, R= Universal Gas Constant, and T= Absolute temperature, is in the decreasing order as OP of NaCl > OP of KC1 > OP of sucrose.

[00133] Morphology of the hollow microsphere varies in the same order, i.e. bigger hollow core and thinner shell structure for NaCl, smaller hollow core and thicker shell for KC1 and multiple scattered pores for sucrose. FIG. 4 shows the cross sectional view of microspheres fabricated using each osmolyte keeping all other process parameters same.

[00134] When NaCl was replaced by KC1 which has lower osmotic pressure, lesser water was driven in, resulting in comparatively smaller hollow core and thicker shell as shown in FIG. 4(B). Likewise sucrose, having almost 6- fold lower osmotic pressure than NaCl, was not able to provide enough osmotic force to drive in appreciable amount of water that was able to coalesce into single core at the center as shown in FIG. 4(C).

[00135] Example 5: Effect of polymer solution concentration (Embodiment 4)

[00136] In a similar experiment, when the polymer solution concentration was decreased to 3 % w/v from 6.67 % w/v, size of the hollow microspheres was decreased dramatically to 40.5 + 13.20 μηι from 84 + 24.5 μιη respectively. Lowering the polymer solution concentration decreased the viscosity of the solution, forming finer emulsion droplets under mechanical stirring. FIG. 5 shows the cross-section of the microparticles fabricated with different polymer solution concentrations while keeping all other process parameters same.

[00137] Example 6: Fabrication of multilayered hollow microparticles (Embodiment 5}

[00138] With careful optimization of the fabrication parameters, it has been demonstrated herein that with the aid of osmolyte, double layered and potentially multilayered hollow microparticles may be fabricated without altering the layer configuration.

[00139] The process is similar to single layered hollow microparticle fabrication. The only major difference is instead of one polymer dissolved in DCM (oil phase), two different polymers are dissolved in DCM. In the exemplified formulation, PLGA and PLLA were used as the different polymers.

[00140] Firstly, 0.1 g of PLGA and 0.1 g of poly-L-lactide (PLLA) (6.5% w/v) and 10 mg of NaCl (5 % w/w) were added to 3.07 ml of DCM. The content was put under magnetic stirring for 3.5 hours and vortexed for 20 minutes. Subsequently, the polymer/salt solution was poured into 100 ml of PVA aqueous solution (3 % w/v) and emulsified under overhead stirring for 3.5 hours at 500 rpm. At this oil to water ratio (o/w: 0.06), the slow diffusion of DCM into the external aqueous phase led to slower precipitation of the polymers allowing the two polymers and water phase to separate and coalesce into respective regions. FIG. 6 shows the cross-sectional view of the double layered hollow microparticles fabricated.

[00141] Example 7: Loading of model drug ibuprofen

[00142] In a similar experiment, a hydrophobic drug, ibuprofen, was used as model drug to show the potential of various embodiments disclosed herein for drug loading. 40 mg of ibuprofen (20 % w/w) was added in the polymer solution along with 5 mg NaCl (2.5 % w/w). FIG. 7 shows a cross-section image of the hollow microparticles loaded with ibuprofen. [00143] Example 8: Loading of model peptide Bovine Insulin

[00144] Bovine insulin (a very hydrophobic peptide) was chosen as model peptide to be loaded in the hollow microparticles developed to evaluate the efficiency of the fabrication technique.

[00145] Example 8.1: Fabrication of PLGA microparticles loaded with bovine insulin microcrystals

[00146] Briefly, 10 mg of insulin microcrystals was added along with 5 mg of NaCl in the fabrication process explained earlier. The insulin microcrystals, despite insulin being a very hydrophobic peptide, were insoluble in DCM, and were used in their solid form (instead of a solution form) so as to further reduce interaction of the peptide with the solvent, so as to preserve bioactivity of the peptide. The insulin microcrystals were uniformly distributed in the polymer solution and after emulsifying the resultant polymer solution with external aqueous phase containing PVA, the hollow microparticles with insulin microcrystals embedded throughout the shell of the microparticles were formed. FIG. 8 shows a cross- sectional image of microparticles, where the embedded insulin microcrystals are highlighted by the squares or rectangles.

[00147] Example 8.2: Confirmation of the observed embedded microcrystals to be insulin microcrystals

[00148] EDX spectrum analysis was used to identify the embedded microcrystals as insulin microcrystals. EDX spectrum of the blank carbon tape (FIG. 9A) and insulin microcrystals devoid region of microparticle showed the peaks of two elements carbon and oxygen (FIG. 9C). Whereas, EDX spectrum of pure microcrystals of bovine insulin showed a peak for element sulphur (FIG. 9B). The strong signal at 2.3 keV was assigned to the sulphur atoms present in the insulin molecule. Six sulphur atoms per insulin monomer contributed to this peak at 2.3 keV. The sulphur atoms were present as disulphide bridges between three pairs of cysteinyl residues in Insulin.

[00149] Finally, when the embedded microcrystals in the shell of cross-sectioned hollow microcparticles were targeted for EDX the spectrum shows peak for sulphur at 2.3 keV as well (FIG. 9D). Comparing the peaks in FIG. 9D and FIG. 9B, it became apparent that the microcrystals seen embedded in the shell were bovine insulin microcrystals. Hence, the peptide was loaded successfully in the microparticles fabricated with the hollow core-shell configuration remaining unaffected. [00150] Example 9: Commercial Application

[00151] By optimizing certain process parameters, its density may be reduced to be less than gastric fluids. This would allow these microspheres to be used for oral drug delivery, with the potential for gastric retention, thus prolonging and sustaining drug release in the gastric region.

[00152] These hollow core microparticles may be optimized to have different bulk densities by altering the hollow core-shell thickness, making these particles suitable for pulmonary drug delivery.

[00153] Tunable drug release kinetics may be achieved by encapsulating the drugs either in the hollow core or in the solid shell. Through careful selection of the polymers and thickness of shell layer, a zero-order controlled release may be achieved.

[00154] Hollow or porous structure may potentially reduce the possibility of creating an acidic microenvironment in biodegradable microparticles, which may lead to the destabilization and noncovalent aggregation of encapsulated acid-labile biomacromolecules. The hollow microparticles disclosed herein are able to avoid this issue, and may thus be a promising platform for peptide or protein delivery, as these agents are generally more susceptible to environmental changes.

[00155] To the best of the inventors' knowledge, no literatures were found whereby hollow microspheres were fabricated in a single-step fabrication process based on the osmotic properties of an osmolyte. Developing the technique by capitalizing the relationship between the osmotic pressure of the osmolyte and its corresponding effect on the precipitation of polymer to produce such hollow particles by single oil-in-water (O/W) emulsion has yet to be reported. Also, there is now sufficient knowledge by tweaking specific process variables to:

[00156] 1. Control the size of the hollow microparticles

[00157] 2. Control the thickness of the shell of hollow microparticles

[00158] 3. Control the formation of hollow core or scattered pores.

[00159] Achieving these would provide a robust, yet facile technique of fabricating hollow microparticles with fully controllable particle parameters.

[00160] Example 10: HPLC method

[00161] A Reverse-phase high performance liquid chromatography (RP-HPLC) was used to quantify the encapsulation efficiency and the concentration of insulin. BC-Zorbax SB-C18 Analytical (4.6 x 250 mm, 5 μιη) chromatographic column was used for the chromatographic separation. The mobile phase comprised of acetonitrile and water (30:70) containing 0.1 % TFA. Sample injection volume was 100 μΐ and the mobile phase was eluted at a flow rate of 1.5 ml/min and effluent was monitored at 214 nm. The mobile phase was freshly prepared, filtered and degassed by ultrasonication before use every time

[00162] Example 11; Encapsulation efficiency obtained by extraction method

[00163] 10 mg (n = 3) of insulin loaded hollow microparticles were dissolved in 0.5 ml dichloromethane and 1 ml of 0.01 N HC1 was added for extraction of the insulin. The solution was vortexed vigorously, allowed to settle and then centrifuged (5000 rpm, 5 mins). The insulin amount in the upper aqueous phase was determined by using a reversed-phase HPLC Agilent separations module equipped with a UV-visible detector by the method mentioned above. The encapsulation efficiency of the hollow microparticles was determined to be 83.96 + 2.56 % compared to 84.13 + 2.55 % of the solid microparticles.

[00164] Example 12; Release profiles comparison and optimization

[00165] 20 mg of insulin loaded microparticles (n = 3) were suspended in 2 ml of Phosphate Buffer Saline (20 mM pH 7.4) in Eppendorf tubes and incubated at 37 °C incubator. After every 24 hours 0.5 ml of the supernatant was accurately withdrawn and replenished with 0.5 ml of fresh buffer. The supernatant were then analyzed using the same reversed-phase HPLC system under the same operating conditions as described earlier for the HPLC method development. The cumulative amount of insulin released at time t was calculated from the equation below and was plotted over time.

[00166] Cumulative amount of insulin release at time t (%) = (amount of insulin released at time t)/( total amount of insulin released at time infinity)

[00167] The release profiles of insulin loaded solid particles and hollow particles with different shell thickness as a result of different osmolyte amounts and also microparticles loaded with bovine insulin in solution form with and without osmolyte (FIG. 12A to 12F) were analyzed. The release study was carried out in two different buffer systems viz: Phosphate Buffer Saline (PBS) (FIG. 12G) and Simulated Gastric Fluid (FIG. 12H). In both buffer systems, it was observed that the higher the amount of osmolyte, the higher was the amount of released insulin at each time point due to smaller shell thickness and consequently shorter diffusion distance. Osmolyte content in the formulation of microparticles hence is established as an effective way of altering the release profile of the microparticles. [00168] Example 13: Evaluating effectiveness of hollow microparticles in preserving bioactivity of loaded peptide

[00169] Example 13.1; MCF-7 Cell proliferation assay using CCK-8 Assay

[00170] Insulin sensitive human breast cancer cell line was used to access the bioactivity of the bovine insulin. Briefly, after 21 days of incubation in PBS, the remaining insulin extracted from the particles were quantified using reversed phase HPLC as described earlier.

Cells were seeded at 10000 cells per well in 96 well plates in Dulbecco's Modified Eagle's

Medium (DMEM) medium supplemented with 10 % FBS, 1 % penicillin and 1 % L- glutamine. When the cells had reached 70 % confluency, the cells were serum starved for 12 hours and cultivated for 4 days in serum free DMEM medium and 10 μg/ml of extracted insulin from the hollow microparticles (n = 10) and solid microparticles (n = 10) respectively.

Medium supplemented with 10 μg/ml of freshly prepared bovine insulin served as a positive control and medium only (devoid of insulin or FBS) served as a negative control (n = 10).

Serum free condition was practiced so that the proliferative effect could be attributed to the insulin which then could be correlated to the bioactivity. Finally, cell number was quantified using Cell Counting Kit-8 following the protocol supplied by the manufacturer (Dojindo

Molecular Technologies).

[00171] The level of bioactivity shown by the peptide loaded in the hollow microparticles fabricated by a method disclosed herein was approximately equal to the positive control. Meaning, the microparticles were able to preserve the optimum level of bioactivity of the peptide for 21 days and possibly can preserve for even longer period of time.

[00172] Example 13.2: Circular dichroism (CD) spectroscopy study

[00173] The insulin remaining in the hollow microparticles and the solid microparticles were extracted after 21 days. Briefly, 100 mg (n = 3) of hollow and solid microparticles were incubated in PBS at 37 °C for 21 days. The samples were centrifuged (5000 rpm, 5 minutes) and washed three times to wash away the insulin released in the PBS during the period. Finally, the insulin trapped in the particles were extracted with exactly the same method as described earlier. The extracted insulin were then analyzed using circular dichroism (CD) spectroscopy. Measurements were performed using a 1 cm path cuvette at 100 nm min^"1 scanning speed, with a step size of 0.5 nm at 25 °C and wavelength range of 190 nm to 260 nm (far UV). CD spectrum of freshly prepared insulin solution and heat denatured (24 hours) insulin were also obtained for comparative analysis. Each spectrum is the average of 10 runs. [00174] The CD spectroscopy result of the peptide loaded in the system disclosed herein showed two minima around 208 nm and 222 nm, which is typical of predominant a-helix structure of peptides such as insulin. The profile of the insulin extracted from hollow particles (after 21 days) superimposed almost entirely to that of standard insulin profile. This indicates that the structural integrity of insulin loaded in microparticles disclosed herein was preserved well over the period of 21 days. This result also further supports the obtained results in MCF- 7 cell proliferation assay discussed above.

[00175] Example 14: Effects of addition of cyclodextrin to hollow particles

[00176] An oil/water emulsion solvent evaporation method was used to prepare the PLGA or PLLA microparticles. Briefly, DOX (20 mg) was dissolved in MeOH (1 ml) under magnetic stirring. PTX (10 mg) and different amount of salt (5 mg, 10 mg or 20 mg) were dissolved in DCM (3 ml) under magnetic stirring. For fabrication of B and C-series, different amount of HPCD was added in DCM or MeOH to form inclusion complex with PTX or DTX, respectively. Where appropriate, a certain amount of HPCD was also added in DCM for fabrication of HPCD-containing microparticles. MeOH solution was added into DCM solution under stirring for 1 h to mix together. And then, the homogenized polymer solution was poured into 50 ml of 3 % (w/v) PVA aqueous solution using overhead stirrer at 500 rpm for 4 h. During this step, the different osmotic pressure of salt resulted in different size of hollow cavity. Lastly, the particles were centrifuged, washed with deionized water, lyophilized and stored in a dessicator.

[00177] TABLE 1: Various parameters to figure out release patterns and drug loading capacity of the dual-drug loaded microparticles (DOX: doxorubicin; PTX: paclitaxel)

Organic Phase

Samples MeOH 1 mL DCM 3 mL

Al DOX 20 mg PTX 10 mg

PLGA 200 mg

A2 DOX 20 mg PTX 10 mg

PLLA 200 mg

A3 DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 5 mg A4 DOX 20 mg PTX 10 mg

PLLA 200 mg

Salt 5 mg

A5 DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 10 mg

A6 DOX 20 mg PTX 10 mg

PLLA 200 mg

Salt 10 mg

A7 DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 20 mg

A8 DOX 20 mg PTX 10 mg

PLLA 200 mg

Salt 20 mg

[00178] TABLE 2: Various parameters to figure out release patterns and drug loading capacity of the dual-drug loaded microparticles (5 mg salt).

Organic Phase

Samples MeOH 1 mL DCM 3 mL

Bl DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 5 mg

HPCD 20mg

B2 DOX 20 mg PTX 10 mg

PLLA 200 mg

Salt 5 mg

HPCD 20mg

B3 DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 5 mg HPCD 40mg

B4 DOX 20 mg PTX 10 mg

PLLA 200 mg

Salt 5 mg HPCD 40mg

B5 DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 5 mg HPCD 80mg

B6 DOX 20 mg PTX 10 mg

PLLA 200 mg

Salt 5 mg HPCD 80mg : Comparison of samples A3, Bl, B3 and B5

Organic Phase

Samples MeOH 1 mL DCM 3 mL

A3 DOX 20 mg PTX 10 mg

PLGA 200 mg Salt 5 mg

Bl DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 5 mg HPCD 20mg

B3 DOX 20 mg PTX 10 mg

PLGA 200 mg

Salt 5 mg HPCD 40mg

B5 DOX 20 mg PTX 10 mg

PLGA 200 mg Salt 5 mg HPCD 80mg

[00180] TABLE 4: Release rate constant of different formulations (c is concentration and t is time) (Values in the table has unit of %/hr).

[00181] To evaluate the release rate of different formulations, a rate constant (k) was determined as changing speed factor. Ko-24 and K24-480 as initial burst release and second sustained release exhibited increasing tendency according to the amount of HPCD used.

[00182] TABLE 5: The released amount (%) of different formulations (Values in the table has unit of %/hr).

[00183] The released amount of PTX from B5 showed highest among tested formulations. Not only did B5 exhibit the highest burst release, it also exhibited the highest amount of following sustained release of PTX. With different amounts of HPCD, the initial burst release and second sustained release may be modulated.

[00184] While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

A method of preparing hollow microparticles, the method comprising

d) removing the aqueous droplets to obtain the hollow microparticles.

The method according to claim 1, wherein the first hydrophobic polymer is selected from the group consisting of poly-lactic-co-glycolic acid (PLGA), poly-l-lactic acid (PLLA), poly-caprolactone (PCL), polyglycolide (PGA), derivatives thereof, copolymers thereof, and mixtures thereof.

The method according to claim 1 or 2, wherein concentration of the first hydrophobic polymer in the polymer solution is in the range of about 1 % (w/v) to about 30 % (w/v).

The method according to any one of claims 1 to 3, wherein the organic solvent is selected from the group consisting of dichloromethane (DCM), methanol, dimethylformamide (DMF), tetrahydrofuran (THF), methyl ethyl ketone (MEK), chloroform, pentane, benzene, benzyl alcohol, carbon tetrachloride, ethyl acetate, acetone, acetonitrile, dimethyl sulfoxide, propylene carbonate, and mixtures thereof.

The method according to any one of claims 1 to 4, wherein the organic solvent comprises or consists of dichloromethane.

6. The method according to any one of claims 1 to 5, wherein the osmolyte is selected from the group consisting of sodium chloride, potassium chloride, sucrose, sodium bromide, sodium citrate, sodium lactate, sodium hydroxide, sodium iodide, sodium carbonate, sodium hydrogen carbonate, sodium nitrate, sodium fluoride, sodium sulfate, potassium carbonate, potassium citrate, potassium lactate, potassium hydrogen carbonate, potassium bromide, potassium hydroxide, potassium iodide, potassium nitrate, potassium sulfate, cesium chloride, rubidium chloride, lithium chloride, and mixtures thereof.

7. The method according to any one of claims 1 to 6, wherein the osmolyte comprises or consists of sodium chloride.

8. The method according to any one of claims 1 to 7, wherein amount of osmolyte in the polymer solution is 20 % (w/w) or less.

9. The method according to any one of claims 1 to 8, wherein amount of osmolyte in the polymer solution is in the range of about 1.25 % (w/w) to about 10 % (w/w).

10. The method according to any one of claims 1 to 9, wherein the osmolyte is at least substantially uniformly distributed within the polymer solution.

11. The method according to any one of claims 1 to 10, wherein the surfactant is a non- ionic surfactant.

12. The method according to any one of claims 1 to 11, wherein the surfactant comprises or consists of polyvinyl alcohol.

13. The method according to any one of claims 1 to 12, wherein concentration of surfactant in the aqueous medium is in the range of about 0.1 % (w/v) to about 5 % (w/v).

14. The method according to any one of claims 1 to 13, wherein dispersing the polymer solution into the aqueous medium is carried out under continuous stirring.

15. The method according to claim 14, wherein the continuous stirring is carried out at a speed in the range of about 300 rpm to about 2000 rpm.

16. The method according to any one of claims 1 to 15, wherein ratio of the polymer solution to the aqueous medium is in the range of about 0.01 to about 0.1.

17. The method according to any one of claims 1 to 16, wherein the hydrophobic polymer precipitates to form a shell around a single aqueous droplet to obtain a hollow microparticle having a core-shell structure.

18. The method according to any one of claims 1 to 17, wherein removing the aqueous droplets is carried out by lyophilization.

19. The method according to any one of claims 1 to 18, further comprising dissolving a second hydrophobic polymer in the organic solvent comprising an osmolyte.

20. The method according to claim 19, wherein the first hydrophobic polymer and the second hydrophobic polymer are independently selected from the group consisting of poly-lactic-co-glycolic acid (PLGA), poly-l-lactide (PLLA), poly-caprolactone (PCL), polyglycolide (PGA), derivatives thereof, copolymers thereof, and mixtures thereof.

21. The method according to claim 19 or 20, wherein the first hydrophobic polymer is poly-lactic-co-glycolic acid and the second hydrophobic polymer is poly-l-lactide.

22. The method according to any one of claims 19 to 21, wherein total concentration of the first hydrophobic polymer and the second hydrophobic polymer in the polymer solution is in the range of about 1 % (w/v) to about 30 % (w/v).

23. The method according to any one of claims 19 to 22, wherein the hollow microparticles formed have a two-layered structure of a first layer comprising the first hydrophobic polymer, and a second layer comprising the second hydrophobic polymer.

24. The method according to any one of claims 1 to 23, wherein the hollow microparticles formed are essentially monodispersed.

25. The method according to any one of claims 1 to 24, wherein the mean diameter of the hollow microparticles formed is in the range of about 100 μιη to about 800 μιη.

26. The method according to any one of claims 1 to 25, further comprising adding a hydrophobic active compound to be encapsulated to the polymer solution.

27. The method according to claim 26, wherein the hydrophobic active compound is selected from the group consisting of a drug, a protein, an enzyme, an antibody, a peptide, a growth factor, an organic molecule, a nucleic acid, a cell, a pesticide, a dye, a chemical indicator, and a fertilizer.

28. The method according to claim 26 or 27, wherein the hydrophobic active compound comprises or consists of a drug.

29. The method according to claim 28, wherein the drug is selected from the group consisting of ibuprofen, paclitaxel, doxorubicin, docetaxel, and combinations thereof.

30. The method according to claim 26 or 27, wherein the hydrophobic active compound comprises or consists of a peptide.

31. The method according to claim 30, wherein the peptide comprises or consists of bovine insulin.

The method according to any one of claims 26 to 31, further comprising adding cyclodextrin to the polymer solution.

33. Hollow microparticles prepared by a method according to any one of claims 1 to 32.