SOLID DISPERSIONS AND PARTICLES AND METHODS FOR CONTROLLED-RELEASE OF LIPID-SOLUBLE OR DISPESIBLE ACTIVES

Abstract

The invention provides a particle comprising a lipid core and surface-decorated (nano)particles of at least one stabilizing material, for delivery of active agents.

Claims

1-54. (canceled)

55. A particle comprising a solid lipid core, the circumference of the core being surface-decorated with a plurality of (nano)particles of at least one stabilizing material, the solid lipid core comprising or consisting at least one lipid-soluble or lipid-dispersible active agent, and wherein at least a portion of the (nano)particles comprising or associating to at least one active or non-active material, wherein the at least one lipid-soluble or lipid-dispersible active agent is same or different from said at least one active material.

56. The particle according to claim 55, wherein the solid lipid core having a size ranging from 0.3 to 200 microns.

57. The particle according to claim 55, wherein the lipid-core is free of nanoparticles.

58. The particle according to claim 55, wherein the density of the (nano)particles decorating the surface of the lipid core does not exceed 15%.

59. The particle according to claim 55, wherein the lipid-core comprises a solid lipid or water-insoluble solid polymer that is solid at a temperature between 25 and 100° C.

60. The particle according to claim 55, wherein the lipid core is of a material selected from a solid triglyceride, a wax fatty ester, a solid paraffin, a polymer, or a solid material capable of solubilizing or dispersing the lipid-soluble or water insoluble active agent.

61. The particle according to claim 55, wherein the lipid core consists the lipid-soluble or insoluble active agent.

62. The particle according to claim 55, wherein the lipid core is of a material selected from a wax, a beeswax, a paraffin wax, a hydrogenated vegetable oil, a long chain solid fatty alcohol and acid.

63. The particle according to claim 55, wherein the (nano)particles are particulate materials having a size (diameter) typically in the nanometer regime.

64. The particle according to claim 55, wherein the at least one lipid-soluble or lipid-dispersible active agent and said at least one active material, being same or different, are each independently selected pharmaceutical agents, veterinary agents, food components or additives, coloring agents and dyes, cosmetic agents, and agricultural agents.

65. The particle according to claim 64, wherein the at least one lipid-soluble or lipid-dispersible active agent and said at least one active material, being same or different, are each independently selected amongst pharmaceutical or veterinary agents.

66. The particle according to claim 65, wherein the agents are pharmaceutically acceptable agents.

67. The particle according to claim 66, wherein the pharmaceutically acceptable agent is oxybenzone, lidocaine, ibuprofen, ketoprofen or escitalopram.

68. The particle according to claim 55, wherein the active agent contained in at least a portion of the (nano)particles is a water soluble active agent, or an amphipathic active agent or a non-lipophilic agent.

69. A particle comprising a lipid core, the circumference of the core being surface-decorated with a plurality of (nano)particles of at least one stabilizing material, the lipid core being an active material or comprising at least one lipid-soluble or lipid-dispersible active material and wherein the (nano)particles optionally comprise or are associated to at least one active material.

70. A particle comprising a lipid core, the circumference of the core being surface-decorated with a plurality of (nano)particles of at least one stabilizing material, the lipid core being an active material or comprising at least one lipid-soluble or lipid-dispersible active material, wherein the lipid core is free or essentially free of nanoparticles, and wherein the plurality of (nano)particles optionally comprise or are associated to at least one active material.

71. An aqueous dispersion comprising a plurality of particles according to claim 55.

72. A product comprising at least one particle according to claim 55.

73. A method of preparing a dispersion according to claim 18, the method comprising mixing (i) a homogenous solution or a dispersion of at least one active agent in a melted lipid core material, and (ii) a dispersion of (nano)particles of a dispersing agent in an aqueous medium; under conditions permitting formation of the dispersion.

74. The method according to claim 73, wherein the homogenous solution or the dispersion is obtained by melting the lipid core material and dissolving (or dispersing) therein the at least one active agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0085] FIGS. 1A-C are SEM photos depicting analysis of the Pickering dispersion, at different resolutions. The paraffin wax forms the solid core coated with silica-NH.sub.2 nanoparticles, as shown in FIG. 1C.

[0086] FIG. 2 depicts fluorescence microscopy (x10) of solid wax particles decorated with amine modified silica nanoparticles after staining with fluorescamine. The excitation wavelength was set to 365 nm.

[0087] FIGS. 3A-C are SEM photos, at different resolutions, depicting analysis of a Pickering dispersion, according to certain embodiments of the invention. The Witepsol® H32 forms the solid core which is coated with silica-NH.sub.2 nanoparticles, as shown in FIG. 3C.

[0088] FIGS. 4A-C are SEM photos, at different resolutions, depicting an analysis of the Pickering dispersion, according to certain embodiments of the invention. The hydrogenated vegetable oil forms the solid core which is coated with silica-NH.sub.2 nanoparticles, as shown in FIG. 4C.

[0089] FIGS. 5A-C show SEM photos, at different resolutions, depicting analysis of the paraffin particles with 1% ibuprofen, according to certain embodiments of the invention. The paraffin wax forms the solid core coated with silica-NH.sub.2 nanoparticles, as shown in FIG. 5C.

[0090] FIGS. 6A-B demonstrate drug release from microparticles in aqueous media, at 25° C., according to certain embodiments of the invention.

[0091] FIG. 7 shows a confocal microscopy of hydrogenated vegetable oil Pickering particle with Nile red in the core and fluorescamine at the particle’s surface.

[0092] FIG. 8 shows a cross section SEM analysis of the hydrogenated vegetable oil decorated with TiO.sub.2—NH.sub.2. The wax forms the solid core coated with TiO.sub.2-NH.sub.2 nanoparticles.

[0093] FIGS. 9A-F present photos of particles prepared from lipid core and chitosan nanoparticles. FIGS. 9E and 9F are the surface and cross-section of a particle.

[0094] FIGS. 10A-D show fluorescamine marking of chitosan nanoparticles embedded onto the surface of a lipid microsphere.

[0095] FIGS. 11A-D show fluorescence marking of microparticles with two markers to distinguish between the core lipid and surface nanoparticles.

[0096] FIG. 12 depicts release of lidocaine from 20% loaded microparticles of Beeswax decorated with chitosan nanoparticles. Release conducted in buffer phosphate 37° C., lidocaine release was determined by HPLC.

[0097] FIG. 13 depict release of Ibuprofen from 20% loaded microparticles of Beeswax decorated with starch nanoparticles. Release conducted in water or buffer phosphate, ibuprofen release was determined by UV.

[0098] FIG. 14 shows Fluazinam loaded in hydrogenated vegetable oil decorated with modified silica nanoparticles, about 20 microns in diameter, constantly releasing fluazinam when immersed in water at room temperature.

[0099] FIG. 15 demonstrates microsphere formation with various ratios of phenylalanine-silica and hydrogenated oil using optical microscopy at X10 resolution.

DETAILED DESCRIPTION OF EMBODIMENTS

Example 1: Solid Wax Particles With Surface Colloidal Silica

[0100] Materials: Paraffin wax (m.p. 36-38° C.), Silica ~14 nm (Aerosil 200, Lot. 157010913) was obtained from Evonik, fluorescamine was obtained from Sigma Aldrich, acetone (dry), (3-minopropyl)-triethoxysilane (APTES) was obtained from Sigma Aldrich.

Methods

[0101] 1. Functionalization of silica: The method was obtained from Yakoov et al. (ACS Omega 2018, 3, 14294-14301). Silica nanoparticles were functionalized by (3-minopropyl)-triethoxysilane (APTES) to introduce amine-functionalized groups onto the particles (silica-NH.sub.2). Silica (1 g) was added to 40 mL of methanol and stirred for complete dispersion. Then, APTES (99% Sigma-Aldrich) was added slowly to the solution for a final concentration of 0.5 M. The reaction was carried out at ambient temperature for 45 min. After silanization, the particles were collected by centrifugation (9000 rpm, 10 min) and rinsed four times with excess methanol. Afterward, the silica-NH2 particles were dried at room temperature under vacuum overnight.

[0102] 2. Preparation of particles: 0.5 % w/v silica-NH.sub.2 was added to 2 mL deionized water and heated at 90° C. The mixture was homogenized at 22,000 rpm for 30 sec to obtain uniform silica dispersion. 100 .Math.L of melted wax was added and homogenization continued for 30 sec to obtain a uniform O/W emulsion. The formed hot emulsion was cooled down to room temperature by immersing in iced water while continue mixing. The dispersion was stable at room temperature. Particles were isolated by either centrifugation and decanting the water, filtration or lyophilization to form a solid thaw.

[0103] 3. Instrumentation: Homogenization: POLYTRON homogenizer, Model No. PT 2100 was used for preparing O/W Pickering emulsion. Scanning Electron Microscopy (SEM): They Pickering particles were analyzed using scanning electron microscope Magellan TM 400 L Field Emission Scanning Electron Microscope. Fluorescence microscopy: The microparticles were analyzed in the presence of fluorescamine dye using Olympus FluoView FV10i – Confocal laser scanning microscope.

Results

[0104] 1. The formed dispersion can remain as dispersion or the formed particles can be isolated by precipitation and filtration, centrifugation and decantation of the water phase or lyophilized. The dry powder was obtained after air drying which was further characterized using SEM.

[0105] 2. The SEM analysis revealed well dispersed particles of size ranging between 30-50 .Math.m. Further, as represented in FIGS. 1, a close view of the particle revealed silica nanoparticle at the surface of the particles.

[0106] 3. In order to confirm that the wax solid core is supported by silica-NH.sub.2, a fluorescamine dye was used. Fluorescamine interacts with the amines and becomes fluorescent. The dried particles were dispersed in fluorescamine aqueous medium for 10 minutes and analyzed. The excitation wavelength of the dye was set to 365 nm, considering instrument specifications.

[0107] The particles glow green in fluorescence, as shown in FIG. 2. This indicates that the silica-NH.sub.2 is at the surface of the wax particles. If the NH.sub.2 groups were not available at the surface, the particles would not have been visible in fluorescence microscopy as confirmed by analyzing particles without the dye.

[0108] Uniform, dispersed, and spherical solid particles were prepared using wax and modified silica-NH.sub.2 nanoparticles. The modification of silica using APTES did not disturb the dispersing ability or size of the nanoparticles. SEM analysis revealed the formation of ~50 .Math.m wax particles as solid core with surface embedded silica-NH.sub.2 nanoparticles. The surface silica was further confirmed by fluorescamine binding to silica particles under confocal microscopy.

Example 2: Preparation of Witepsol® H32 (m.p. 37° C.) and Hydrogenated Vegetable Oil (60-70° C.) Silica Nanoparticles Coated Particles

[0109] The method of preparation described in Example 1 was used, replacing the wax with Witepsol® H32 as solid core. The SEM analysis revealed well dispersed particles of sizes ranging in between 30-50 .Math.m (FIGS. 3). Similarly, particles with hydrogenated vegetable oil solid cores and surfaces embedded NH.sub.2-silica nanoparticles were prepared. SEM analysis revealed well dispersed particles of size ranging in between 5-40 .Math.m (FIGS. 4).

[0110] The developed method was functional and could be applied to varieties of solid lipids and waxes. By adjusting the ratio between the solid lipid and silica-NH.sub.2 size distribution of the particles could be tuned.

Example 3: Drug Loaded Nano-Shell Solid Particles

[0111] Ibuprofen was selected for incorporation in particles. The appropriate amount of drug was solubilized in molten oil at 90° C. and thereafter followed the same procedure as detailed in Example 1.

[0112] Paraffin wax particles with 1% Ibuprofen: A dispersion was formed and dried to form a powder that was visualized by SEM. The SEM analysis revealed similar particles to those without the drug, with well dispersed sizes ranging between 30-50 .Math.m (FIGS. 5).

[0113] Further preparations: Ibuprofen (1 and 5%), ketoprofen (1 and 5%) and escitalopram (5%) in wax, Witepsol® H 32 and hydrogenated vegetable oil.

[0114] Release of drugs from particles: Standard curve of drug was prepared in 10 mM phosphate buffer at pH 7.2. 10 mg of drug loaded lyophilized Pickering particles were added to 2 mL of buffer and the periodic isolated solution was analyzed by UV. The release was examined at room temperature. This was done in purpose to examine the drug release properties from the Pickering particles. The analysis of initial 30 min of release is considered as the burst release of drugs from the particles (FIGS. 6).

[0115] Release of ibuprofen from 1% (w/w) drug loaded Pickering dispersion. The release of ibuprofen was monitored by UV at the wavelength of 223 nm. It was observed that almost all the drug was released within 30 mins, in an aqueous solution (FIG. 6A).

[0116] Release of ketoprofen from 1% (w/w) drug loaded Pickering dispersion. Standard curve of ketoprofen was prepared in 10 mM phosphate buffer at pH 7.2 at wavelength of 260 nm. It was observed that almost all the drug was released within 30 mins, in aqueous solution.

[0117] The 5% loading of ibuprofen, ketoprofen and escitalopram in the dispersion were similarly prepared. Table 1 summarizes the remaining drug in the particles, after 30 min.

TABLE-US-00001 Encapsulation efficiencies of drugs in particles according to the invention Pickering dispersion Solid lipid % ibuprofen encapsulation (223 nm in UV) % ketoprofen encapsulation (260 nm in UV) % escitalopram encapsulation (238 nm in UV) Paraffin wax 46 53 92% Witepsol® H 32 57 51 64% Hydrogenated Veg oil 56.5 57 90%

[0118] The particles were checked for the drug release with time. FIGS. 6 provides the release of the drug loaded Pickering particles in a buffer medium. The drug was released in a sustained way. Further, the encapsulation efficiency seemed to be dependent on the compatibility between the drug and the solid lipid.

Example 4: Characteristics of the Particles in the Presence of the Fluorescence Dyes-Nile Red and Fluorescamine

[0119] Nile red, 200 .Math.g was dissolved in the melted wax to prepare the solid core. The dispersion was prepared as described in Example 1. The dispersion was lyophilized to obtain a complete dispersed dry powder.

[0120] Fluorescamine, 200 .Math.g/mL of the Fluorescamine aqueous solution was prepared using a stock solution of Fluorescamine in acetone. To 1 mL of Fluorescamine diluted sample, 30 mg of the Nile red containing dispersion was added and kept on shaking for 5 mins and lyophilized. Fluorescamine is water soluble and has the property to bind with primary amines; hence, it interacts only with the silica-NH.sub.2 in the outer-surface of the Pickering particle.

[0121] Fluorescence Imaging Analysis: The particles were analyzed under Olympus IX83 inverted microscope. The fluorescent dyes of the same particles were excited separately using default settings in the microscope. Nile red excitation revealed red colored particle and fluorescamine excitation revealed green color particle. The obtained images were then processed using Image J software, that merge both the Nile red and fluorescamine image into one.

[0122] Hydrogenated vegetable oil particles were analyzed under fluorescence microscopy. The particles were dispersed in water and then a drop was added over the glass slides and covered with cover slip. FIG. 7 demonstrates the fluorescence imaging.

[0123] FIG. 7 provides information about the selectivity of the dyes over the particles. The black background in the images indicates that there are no free aggregates of the dyes in the aqueous medium. The preference of both dyes can easily be seen in the figure. As such, the Nile red prefers the wax core and the fluorescamine prefers binding to the Silica-NH.sub.2. Being a solid core, it is difficult to differentiate between the red and green fluorescence; however, upon composition, it is seen that the region where there are no Slica-NH.sub.2 the red color is stronger.

[0124] The absence of the Nile red dye in the aqueous dispersion indicates the encapsulation efficiencies of the particles to hold hydrophobic molecules. This was observed in FIGS. 6 by observing a sustained release of ibuprofen and escitalopram from the Pickering particles in the release medium.

[0125] The distribution of nanoparticles within the core and surface of the microparticles were determined by detecting nanoparticles in the cross-section of particles prepared with silica, chitosan and iron oxide. SEM analysis with EDX did not detect nanoparticles in the cross-section but only on the surface of the particle as shown in FIG. 8.

Example 5: All Lipid (Nano)Particle Decorating Particles of the Invention

[0126] The formation of lipid-based particles where the surface particles lipidic as well as the core, was obtained as follows: In the first step, solid lipid nanoparticles of high melting point lipids such as stearic acid, tristearin or carnauba wax was prepared by either melt method or pre-concentrate method, followed by a second step of the preparation of the microparticle by melt method, using a lipid core that melts at temperatures that are below the melting point of the lipid of the surface. This is demonstrated below:

[0127] Step 1: Tristearin was mixed with sterylamine and/or stearol and/or stearic acid and/or phospholipid or a surface-active agent such as Tween and or Span. The mixture was melted at temperature of about 90° C. and mixed well. To the melt, hot water >90° C. containing a minute amount of a surface active agent was added and homogenized to form a homogeneous emulsion. The emulsion was cooled down while homogenizing until a solid dispersion was obtained. The average particle size of this dispersion should be below 2 microns, controlled by the preparation conditions, the ratio of aqueous media to the lipid media, the ratio of surface active agents to lipid and other additives that may be incorporated. These particles contained a lipid active agent that was dissolved or dispersed in the lipid core of this dispersion.

[0128] Alternatively, the dispersion could be prepared by dissolving the selected solid lipids in a mixture of surfactants such as Tween, Span, LipoPEG, and phospholipids, Cremophor and a water miscible solvent such as ethanol, propylene glycol, isopropanol, ethyl lactate and N-methyl pyrrolidone (NMP). This homogeneous solution was added to water at room temperature to form a nano-dispersion where the particle size was controlled by the pre-concentrate composition and the properties and amount of aqueous media where the preconcentrate is mixed in.

[0129] Step 2: A lipid that melt at a temperature about 10° C. below the melting point of the particles of Step 1 was selected for the core material. Examples are Witepsol, Wax, trilaurin, tricaprin and other lipids that melt at temperature below 50° C. The selected lipid was melted along with the selected active agents to be incorporated in the core. To this melt, the dispersion of Step 1, at an amount of solids that was 10% w/w or less, of the melted lipid, that was diluted in hot aqueous solution and added to the melt while thoroughly mixing or homogenizing to form a homogeneous emulsified dispersion. The mixing continued while the dispersion was cooled down to form the microspheres where the core was the lipid of this step and the surface particles were from Step 1.

[0130] The ability to form the particles decorated with lipid particles was dependent on the surface properties of the particles of the first step. The surface should allow interaction with the lipid melt but not incorporation within the melt. The surface properties of the particles of Step 1 can be modified by the addition on lipids with hydrophilic head such as carboxylic acid, amine or PEG chain, for example: fatty acid, fatty amine or fatty alcohol, PEG-fatty acid or a phospholipid of different hydrophilic structure. The size of the final particle can be designed based on the composition of the lipids, the surface active agents, the properties of the aqueous media, the ratio of lipid to particles, and the preparation conditions.

Example 6: Lipid Microparticles Decorated With Nanoparticles for Controlled Release of Agents

[0131] This work utilizes natural materials composed of natural lipid (beeswax) and natural polysaccharides, chitosan and starch, as nanoparticles for the encapsulation of lidocaine (L) and ibuprofen (I) as model drugs. These active agents were chosen as poorly water-soluble drugs with basic (lidocaine) or acidic (ibuprofen) nature. Lidocaine-loaded chitosan-NP-stabilized beeswax microspheres and Ibuprofen-loaded-starch-NP-stabilized beeswax microspheres, prepared by melt dispersion method without use of any surfactant, organic solvents or harsh conditions. These microspheres were fully characterized and the drug release profile was obtained.

Materials and Methods

[0132] White Beeswax was obtained from Nature’s Oil Company, Ohio. Chitosan was obtained from Primex Company. Soluble potato starch was obtained from S.D. Fine-Chem., Mumbai. Lidocaine, Fluorescein isothiocyanate and Fluorescamine were purchased from Sigma Aldrich, Rehovot, Israel. Ibuprofen was obtained from Ethyl Corporation, Orangeburg. Acetic acid and Ethanol were purchased from GADOT Group, Netanya, Israel. Sodium Hydroxide pellets were purchased from J.T. Baker. All the reagents were of analytical grade.

Preparation of Chitosan Nanoparticles

[0133] Chitosan colloidal nanoparticles were successfully synthesized by adjusting the pH value. A typical preparation procedure was as follows: 0.6 g acetic acid was added to 100 mL deionized water to form 0.1 M solution. 0.2 g chitosan was added to 20 mL acetic acid solution and a mixture was continuously stirred for 4 h at room temperature until a clear solution was obtained. The pH value was modified by adding NaOH (1 M) solution, until the transparent chitosan solution was changed to be opalescent (for example, 180 .Math.L of NaOH solution was added for each 2 mL of Chitosan solution). At this moment, the pH of the dispersion was 6.5 with chitosan nanoparticles dispersed in this solution. This nanoparticle dispersion was used immediately for preparation of solid lipid microspheres.

Preparation of Starch Nanoparticles

[0134] Starch nanoparticles were prepared by nanoprecipitation method. Soluble potato starch (2.5 gr) was added to 50 mL of deionized water and the resulting mixture was continually stirred at 90° C. for 1 h. The heating was turned off and 50 mL of Ethanol was added dropwise using additional funnel. During an addition of Ethanol white precipitate started to form. After the dispersion was reached room temperature another portion of 50 mL ethanol was added dropwise. The resulting mixture was centrifuged at 9000 rpm for 5 min. After supernatants were removed 20 mL ethanol was added and the mixture was centrifuged. These washing cycles with ethanol performed two more times in order to remove the water. The remaining white solid was dried under reduced pressure.

Preparation of Lidocaine-Loaded Solid Lipid Microspheres (SLM) Stabilized With Chitosan NP

[0135] Lidocaine loaded particles were prepared using a melting dispersion method. Beeswax was heated and melted in oil-bath at 90° C. and solid Lidocaine (base form) added whilst stirring to form a beeswax-lidocaine clear mixture (Lidocaine 10% w/w). Separately, Chitosan-NP dispersion in water was heated in the same oil-bath with stirring at 1000 rpm. The melted mixture of Lidocaine in Beeswax was subsequently added in one portion to the Chitosan dispersion with constant stirring and stirring was continued for 3 min more (70 .Math.L of melted mixture were added to each 2 mL of 1% Chitosan dispersion). The resultant mixture was cooling in a water bath with stirring till reached the room temperature. During the cooling microspheres were formed. Blank-SLM were prepared using the same method but without an addition of the drug.

Preparation of Ibuprofen-Loaded Solid Lipid Microspheres (SLM) Stabilized With Starch NP

[0136] Ibuprofen loaded particles were prepared using the same method. Beeswax was melted in oil-bath at 90° C. and solid Ibuprofen added whilst stirring to form a beeswax-ibuprofen clear mixture (Ibuprofen 22% w/w). Separately, starch-NPs were accurately weighted and the deionized water was added. The resulting mixture was sonicated for 5 min in order to make uniform dispersion. This dispersion was heated in the same oil-bath with stirring at 1000 rpm for 5 min. The melted mixture of Ibuprofen in Beeswax was subsequently added in one portion to the Starch-NPs dispersion and stirring was continued for 8 min more (70 .Math.L of melted mixture were added to each 4 mL of Starch dispersion containing 80 mg of dry Starch-NP). The resultant mixture was cooled in an ice-water bath with stirring. During the cooling microspheres were formed. Blank particles were prepared using the same method but without an addition of the drug.

Particles Characterization

Mean Particle Size Measurement

[0137] The average particle size of chitosan and starch nanoparticles was determined by using ZetaSizer NANO (Malvern Instruments, UK). Chitosan NP and Starch NP dispersions were diluted with deionized water and were equilibrated to room temperature for 2 min. A 1 cm path length Brand® disposable UV-transparent cuvettes were used for the particle size measurement. All measurements were performed at a constant temperature of 25° C.

Scanning Electron Microscopy

[0138] SEM photographs were taken using a scanning electron microscope FEI Quanta 200 SEM (Brno, Czech Republic) at suitable magnification at room temperature. The photographs were observed for morphological and structural characteristics and to confirm the spherical nature of microparticles. The samples were mounted on aluminum stubs, using double sided adhesive tape and Pd/Au coated under vacuum for 60 s using a sputter coater (SC7620, Spatter coater, UK). The average particle size of the microspheres was noted from the SEM studies.

Transmission Electron Microscopy

[0139] The morphological observations of microparticles were obtained by using JEM-1400 PLUS, Japan, the 120 kV model in JEOL’s new PC controlled TEM range. A drop of NP dispersion was placed onto a carbon-coated copper grid followed by the removal of excess dispersion using a filter paper. It was then negatively stained using NanoVan (Nanoprobes, USA) or Uranyl acetate (2%) and air-dried at room temperature for 30 min. The grid was then ready to be examined under TEM.

Fluorescent Microscopy

[0140] Fluorescent microscopy imaging was performed on Nikon Spinning Disk Microscope. The system includes the Yokogawa W1 Spinning Disk with wide field of view. Two discs are integrated with 25 .Math.m and 50 .Math.m pinholes, for optimal imaging with different objectives. The system is mounted on the new Nikon motorized fluorescent microscope Ti2E and equipped with 405/488/561/638 nm lasers. The system includes the NIS Elements software package for multi-dimensional experiments, with JOBS for high content acquisition and analysis, deconvolution, tracking, 3D automatic measurements.

Preparation of Fluorescent Solid Lipid Microspheres for Imaging

[0141] Dried Beeswax-Chitosan and Beeswax-Starch microspheres (10-20 mg) were added to 2 mL of ddH.sub.2O, followed by addition of 50 .Math.l of Fluorescein isothiocyanate/Fluorescamine solution (1 mg/mL, in DMSO). The mixture was shaken for 1 h in the dark at room temperature. The liquid was removed and the remaining particles were washed 5 times with water in order to remove all unreacted dye. The obtained microspheres were imaged using Nikon Spinning Disk Microscope.

Assay of Lidocaine and Ibuprofen by UV

[0142] The amounts of Lidocaine and Ibuprofen were measured by UV absorbance using an ultraviolet spectrophotometer (UV-Visible Spectrophotometer, Ultrospec 2100 pro, Amersham Biosciences). Lidocaine was measured at a wavelength of 215 nm in 10 mM PBS, and a wavelength of 242 nm in chloroform. The standard curves were constructed in a concentration range of 5-25 .Math.g/mL in 10 mM PBS with R.sup.2 value of 0.99 and in a concentration range of 7-100 .Math.g/mL in chloroform with R.sup.2 value of 0.995. Ibuprofen was measured at a wavelength of 223 nm in 10 mM PBS or water and a wavelength of 264 nm in chloroform. The standard curves were constructed in a concentration range of 3-70 .Math.g/mL in 10 mM PBS with R.sup.2 value of 0.9993 and in a concentration range of 15-800 .Math.g/mL in chloroform with R.sup.2 value of 0.9935.

Determination of Encapsulation Efficiency and Loading Capacity

[0143] Encapsulation efficiency is percentage of drug encapsulated in the microspheres relating to initial quantity used. 10 mg of microspheres were dissolved in 4 mL of chloroform. The absorbance of the drugs was measured using a UV-VIS spectrophotometer after appropriate dilution in order to reach calibration curve concentrations. The measured absorbance was then converted to the amount of drug by using standard calibration curve.

In Vitro Drug Release

[0144] In vitro release of Lidocaine and Ibuprofen from microspheres was studied using 10 mM PBS or ddH.sub.2O as dissolution medium at 37° C., 150 rpm (using shaking incubator). 10 mg of microspheres were mixed with 5 mL of H.sub.2O or buffer and placed in the incubator. Three samples of each formulation were run at same conditions. A 3 mL samples were withdrawn at predetermined time intervals and an equal volume of fresh dissolution medium was replaced. Collected samples were suitably diluted and then analyzed for drugs contents by measuring the absorbance at 215 nm for Lidocaine samples in 10 mM PBS and at 223 nm for Ibuprofen samples. The concentration of lidocaine and ibuprofen in test samples was calculated using calibration curves for each drug in H.sub.2O or 10 mM PBS.

Results and Discussion

Lidocaine Loaded Beeswax-Chitosan Microspheres

[0145] The transmittance of the aqueous chitosan solutions decreased with increasing pH values. At high pH (near 6.6, pKa of chitosan), chitosan’s amines becoming deprotonated and chitosan could undergo interpolymer associations that lead to nanoparticle formation. TEM images of these nanoparticles revealed spherical particles with diameter rage between 200 and 800 nm.

[0146] This nanoparticles dispersion was used as stabilizing material for the formation of solid lipid microspheres. Several parameters were studied in order to achieve spherical form with maximum yield. These p arameters included: amount of wax added to aqueous dispersion of chitosan NP, amount of base (NaOH) added that influence the pH which by itself influence the concentration of precipitated chitosan NP, temperature of oil bath where all ingredients were mixed, the stirring speed and the stirring time. The right ratio between the amount of beeswax and the concentration of chitosan NP enables the maximum yield of particles formed without any residual of unreacted wax. For comparison, without the addition of chitosan nanoparticles, the process failed and resulted in formation of aggregate cake of solid wax. It may be due to the repulsion resulting from high interfacial tension between the hydrophobic waxy material and external aqueous phase.

[0147] The formed spherical microparticles were collected and dried in an open air. The same procedure was used for the preparation of lidocaine-loaded microspheres. For this purpose, Lidocaine (mp 68.5° C.) was mixed with beeswax at 90° C., temperature which allows both ingredients to appear at their melted form, and thus, to accelerate mixing. This melted mixture was added to the chitosan nanoparticles dispersion in the same matter as pure wax was added. Important to notice, that the amount of Lidocaine drug mixed with wax has huge influence on yield of microspheres formed. Thus, while the percentage of the Lidocaine increases, the yield of formed particles decreases. For this reason, the melted mixture contained 10% of Lidocaine which allowed the reasonable formation yield. The morphological structure of the synthesized microparticles with or without Lidocaine drug was examined using SEM. It was found that there was no visible difference in shape, size and their surface morphology. SEM photographs showed that the wax microspheres were spherical in nature and had a smooth surface with size distribution of 300-600 .Math.m.

[0148] The enlarged images revealed unique “wave”-shaped surface. One of the spherical particles was successfully cut in order to understand difference between the inner and outer phases. The SEM images indicated that the core has more homogenic nature while the outer phase is very porous with rough texture which is characteristic to polysaccharides. The in vitro release of Lidocaine from particles is given in FIG. 12.

Ibuprofen Loaded Beeswax-Starch Microspheres

[0149] Commercially available soluble potato starch consists of smooth oval microparticles. In order to serve as a stabilizing material in the lipid microspheres preparations there is a need for starch nanoparticles. These starch nanoparticles were prepared by nanoprecipitation method from commercial microparticles. The formed particles were spherical in shape and had an average diameter of 306 nm, according to DLS measurements.

[0150] These nanospheres were used in further lipid microspheres preparation. Mixture of starch NP in water was sonicated prior to use in order to get homogeneous dispersion. Several factors that can influence the particles formation have been studied -amount of starch NP, amount of beeswax, stirring rate, temperature and the stirring time. It was recognized that the last parameter has of greatest importance on particle yield. Thus, only when enough time was given for the mixing of wax with starch (10 min instead of usual 3 min), nice spherical particles were formed upon cooling. The same procedure was used for the preparation of ibuprofen-loaded microspheres. For this purpose, Ibuprofen (mp 75-78° C.) was mixed with beeswax at 90° C., temperature which allows both ingredients to appear at their melted form, and thus, to accelerate mixing. In this case, it was possible to prepare rather concentrated mixture which contained 20% of Ibuprofen and yet to get good yield of microparticles without any residue wax. These microspheres were characterized by SEM imaging. SEM photographs showed that the wax microspheres were spherical in nature and had a smooth surface with size distribution of 300-600 .Math.m.

Fluorescent Labeling of Solid Lipid Microspheres

[0151] The fluorescent labeling methods for polysaccharide labeling have been widely researched in recent years. Fluorescent marker, fluorescein isothiocyanate (FITC), was conjugated to the chitosan molecule and was used to study chitosan-mucin interactions and the biodegradation and distribution of chitosan in mice. Through covalent bonding, the primary amino group of chitosan combines with isothiocyanate group (N═C═S) of fluorescein isothiocyanate to label the chitosan. The chitosan labeled by fluorescein isothiocyanate has a high fluorescence intensity, light stability and solid combination. In this work this fluorescent labeling compound was used in order to visualize the polysaccharide coatings of prepared Solid Lipid Microspheres. Thus, the microparticles were allowed to mix with the dye solution, intensively washed with water to remove the unreacted dye and visualized immediately by fluorescent microscope (FIGS. 10)

[0152] In order to distinguish between the wax core and the polysaccharide coatings the microspheres were fluorescently labeled using two different fluorescent dyes. The Nile red dye was incorporated in Beeswax prior to the formation of microspheres to mark the core. After these particles have been formed using ChitosanNP as stabilizers, they were colored with FITC dye. When the particles treated with FITC were excited at two wavelengths (561 nm and 488 nm) fluorescent images revealed red core and green coatings (FIGS. 11A-B). In order to confirm the presence of chitosan coating, the obtained microspheres were treated with different dye, Fluorescamine. Fluorescamine is a coloring reagent developed by Weigele et al. for amino acid analysis. This molecule is not fluorescent by itself but upon reaction with primary amines forms a fluorescent product. Thus, if the nanoparticles of chitosan are present on the surface of beeswax, the Fluorescamine should react with NH.sub.2 group, present in Chitosan, forming a fluorescent product, which has an excitation maximum at 390 nm. These particles were imaged using 405 nm laser to visualize the surface coating labeled with Fluorescamine (blue, FIG. 11C) and using two lasers (405 nm and 561 nm) to visualize the wax core and chitosan coating (red and blue, FIG. 11D).

Drug Encapsulation and Loading Capacity of Solid Lipid Microspheres

[0153] The loading capacity is usually expressed as the ratio of the weight of entrapped drug and the total weight of the lipid and coating particles in percentage. Some of the factors which affect the loading capacity of a drug in a lipid are the solubility of the drug in the lipid melt, the miscibility of the drug and the lipid melt, the chemical and physical properties of the drug. Thus, if the melting point of drug is similar to the lipid melting point, it is possible to melt them together, which highly increase the loading capacity. In this work we chose to use drugs which have similar or close melting point as beeswax. Thus, under the procedure conditions, 90° C., both wax and drug materials are melting (beeswax melting point is 62-64° C., Lidocaine melting point is 68° C., Ibuprofen melting point is 75-77° C.), allowing good mixing. The loading capacity of Lidocaine in Beeswax-Chitosan MSs was found to be 4.9 % and of Ibuprofen in Beeswax-Starch MSs was found to be 15.7% (average of three independent experiments for each formulation).

Drug Entrapment Efficiency

[0154] Drug entrapment efficiency is calculated in order to determine the amount of drug that is actually retained in the microparticles related to the amount of drug that was mixed with lipid before particles formation, and usually expressed as a percentage. The chemical nature of drug, like water solubility or existence of basic or acidic groups in molecule can influence the entrapment efficacy. Thus, in the case of solid lipid particles formation using chitosan nanoparticles, the pH of the dispersion is 6.5, therefore, drugs containing acidic group, like Ibuprofen, will become deprotonated, leading to their salt formation and increased water solubility. This way some portion of the drug will diffuse from lipid to the aqueous medium during particles preparation, leading to decreasing in entrapment efficiency. In addition, as a water solubility of drug is increasing, the efficacy of encapsulation using hot melting method is decreasing. Therefore, Ibuprofen-loaded microspheres obtained from beeswax and starch have been formed with good entrapment efficiency (71%), as Ibuprofen is only very slightly soluble in water (21 mg/L, at 25° C.) and the drug remains in its neutral form during particle formation. Lidocaine molecule contains a basic group (tertiary amine) which remains neutral during the particles formation using Beeswax-Chitosan (pH 6.5) but has higher water solubility (410 mg/L at 30° C.) leading to lower entrapment efficacy (49%).

Studies of Drugs Release From Solid Lipid Microspheres

[0155] In vitro drug release for the obtained microspheres was studied in 5 ml of 10 mM buffer phosphate solution, pH 7.4 or in ddH.sub.2O at 37° C. The release profile of Lidocaine from Beeswax-Chitosan

[0156] The release profile was fitted logarithmic function, with initial burst release at first 24 h, where about 70% of drug was released and the remaining amount of drug was released during at least one week. FIG. 13 illustrates the average of three different experiments.

[0157] The release profile of Ibuprofen from Beeswax-Starch MSs was studied at two dissolution media, buffer PBS and ddH.sub.2O at 37° C. As Ibuprofen possesses the acidic group — COOH, the pH of the solution has a very meaningful influence on its release rate. Thus, this statement can be proven by observing two release profiles depicted in FIG. 13. Both graphs followed the logarithmic behavior, with boost release of the drug during the first day. But it can be recognized that in case of the particles’ dissolution in PBS pH 7.4, most of the drug was released during 4 days, while in case of using water instead of buffer, the release was continued for 7 days more. Thus, by changing the dissolution medium (or formulation) it is possible to control the rate of drug release from these microparticles, leading to the desired one, depending on application.

[0158] Summary: Natural particle-stabilized solid-lipid microspheres were prepared by melt dispersion technique. Using a natural lipid melt and nanoparticles from natural source spherical microparticles were produced. These microspheres were prepared also using drug-lipid melt for the production of drug-loaded microparticles. Beeswax, chitosan and starch, due to their lower toxicity, absence of solvents, surfactant or emulsifiers in the production process can be suitable for coating of drugs which need to be released in controlled rate.

Example 7: Oxybenzone-Loaded Solid Lipid Microparticles Stabilized by TiO.SUB.2 Nano-Shell

[0159] Microparticles were prepared using hot melt homogenization technique from three different types of solid lipids, soy wax, cetyl alcohol and trilaurin, approved for cosmetic applications, using TiO.sub.2 nanoparticles (nano-shell) as stabilizing material.

[0160] In this method no surfactant was applied, but instead, nanoparticles of TiO.sub.2 were used as stabilization material, leading to the formation of solid spherical particles of micrometer size (10-50 microns). Microparticles were obtained using the three lipids: Soy wax, Cetyl alcohol and Trilaurin loaded with four different Oxybenzone concentrations: 20%, 25% 33.3% and 50%. The total yield is >90%, the isolated powder yield was in the range of 70-85% due to loss of materials during isolation of particle. The loadings ware similar to the entry loading and the average particle size of all particles was in the range of 23 to 35 microns.

[0161] Cream formulations containing 10% were prepared and sent for SPF (Sunscreen Protection Factor) evaluation. Cream was prepared using Water, Cocoa Butter, Soy lecithin, HPMC, PG and Propyl Paraben.

[0162] Earlier, another Oxybenzone-loaded microparticles were prepared as well, using Trimyristin and modified TiO.sub.2, were characterized as well and their SPF values were measured. The SPF for the free oxybenzone in cream or the titanium oxide-lipid microparticles without oxybenzone was in the range of 2.9-3.4 while all cream samples containing particles loaded with oxybenzone and titanium oxide nano-shell, demonstrated SPF in the range of 7-8.5.

Example 8: Lipid Particles Decorated With Metal Oxides

[0163] Microparticles were prepared using Titanium dioxide (TiO.sub.2). Microparticles of paraffin wax and Hydrogenated Vegetable Oil were prepared and examined. A fine free flowing dry powder was obtained after lyophilization which was further characterized using SEM. The SEM analysis revealed well dispersed particles of size ranging in between 10-50 .Math.m.

[0164] Drug encapsulation in wax microparticles: The encapsulation and release of 5% of ibuprofen, ketoprofen and escitalopram was studies. The encapsulation efficiency of the drugs ranged from 50 to 95%.

[0165] The cross section of the TiO.sub.2—NH.sub.2 onto hydrogenated vegetable oil particles was examined using SEM. This analysis proved that the decorating TiO.sub.2 nanoparticle remains only at the surface and nothing in the core.

[0166] Particles decorated with Iron oxide: Microparticles were prepared using iron oxide nanoparticles. The surface of the iron oxide was modified using silane. The silane modified metal oxide is abbreviated as Fe.sub.2O.sub.3—NH.sub.2. The preparation method is same as described above. Microparticles with paraffin wax and hydrogenated vegetable oil decorated with iron oxide were obtained as fine dry powder after lyophilization which was further characterized using SEM. The SEM analysis revealed well dispersed particles of size ranging in between 10-50 .Math.m.

Example 9: Magnetic Microparticles

[0167] Magnetic nanoparticles of different sizes, shapes and compositions are commercially available or synthesized in large scale iron oxide or iron metal nanoparticles. [0168] Antimicrobial microparticles [0169] Colored Microparticles [0170] Conductive Microparticles

Example 10: Formulation of Microparticles in Carriers

[0171] The microparticles of this invention were granulated into large granules by mixing the particles with poly(vinyl pyrrolidone) or polyvinylalcohol aqueous solution which after water evaporation large granules were obtained. The size of the granules can be tailored by the process or by grinding the large granules. The microparticles may be a mixture of microparticles with different active agents or/and, mixed with inert particulate solids such as clay, microcrystalline cellulose, starch and the like, prior to granulation. In a typical preparation,

[0172] Drug loading: DEET insect repellent 30% (w/w): 30% w/w of DEET was loaded into the microparticles. The selection of the 30% drug content was based on the dissolving the DEET into the molten oil and then cooled down. The DEET loading was examined for 1:1, 1:2, 1:10 and 1:20 ratio SiO.sub.2-NH.sub.2:hydrogenated vegetable oil. The DEET loaded particles were tested by weight loss in the DEET loaded 1:10 ratio was examined. Accelerated temperature of 50° C. was used. The weight sample was heated to 500° C. and monitored the weight loss for 1 h. 2% decrease in the weight loss of the DEET loaded microparticles was observed.

[0173] Fluazinam 30% (w/w): 30% w/w of fluazinam was loaded into particles. The selection of the 30% drug content was based on the dissolving the drug into the molten oil and then cooled down. The drug loading was examined for 1:1, 1:2, 1:10 and 1:20 ratio SiO.sub.2—NH.sub.2—hydrogenated vegetable oil by taking ~10 mg of the drug loaded particle material and vortexing strong for 15 minutes in 5% Tween 20 aqueous solution. Fluazinam release is shown in FIG. 13.

Example 11: Modification of Silica Nanoparticles by Physical Adsorption and Identifying the Formation of Solid Lipid Microparticle

[0174] The aim was to investigate the solid lipid formation using physically modified nanoparticles (NP). Surface modification of NP should be done by absorption of cationic or anionic polysaccharides, amino acids, fat, PEG, etc. Silica nanoparticles was used and surface modified by physical adsorption of materials such as, chitosan, alginate, cholesterol and amino acid.

[0175] Materials: Silica ~14 nm (Aerosil 200, Lot. 157010913) was obtained from Evonik; Cholesterol; low mol. wt. Chitosan; medium viscosity sodium alginate; DL-phenylalanine.

[0176] Choice of material: The coating material was chosen such that they are practically insoluble in aqueous conditions. This will diminish the chances of the coating that could erase from the silica nanoparticles while pickering preparation.

Method

[0177] Surface modification of the Silica NPs: 5% w/w coating agent was used in the total material unless specifically stated.

[0178] Cholesterol coating over NPs: 50 mg of cholesterol was dissolved in 50 mL ethanol. Thereafter, 950 mg of the silica NP was dispersed by vortex mixing for 30 mins. The mixture was then vacuum dried using rotaevaporator.

[0179] DL phenylalanine coating over NPs: 50 mg of phenylalanine was dissolved in 50 mL ethanol. Thereafter, 950 mg of the silica NP was dispersed by vortex mixing for 30 mins. The mixture was then vacuum dried using rotaevaporator.

[0180] Chitosan coating over NPs: 50 mg of low mol wt. chitosan was dissolved in 50 mL acidic water at pH 4. Thereafter, 950 mg of the silica NP was dispersed by vortex mixing for 30 mins. The mixture was then freeze dried using a lyophilizer.

[0181] Sodium alginate coating over NPs: 25 mg of medium viscosity sodium salt of alginic acid was dissolved in 50 mL water. 25 mg of the coating material was used because of the high viscosity of the sodium alginate. Thereafter, 975 mg of the silica NP was dispersed by vortex mixing for 30 mins. The mixture was then freeze dried using a lyophilizer.

Preparation of Microparticle Dispersion

[0182] Step 1. Various w/w ratios of surface modified silica were added to 2 mL water and heated at 90° C. The mixture was homogenized at 22,000 rpm for 35 sec to obtain a uniform NP dispersion.

[0183] Step 2. In a separate vial, solid lipid was heated at 90° C. 100 .Math.L of melted wax was added to 2 mL of hot dispersion and homogenized at 22,000 rpm for 35 sec to obtain a uniform O/w emulsion.

[0184] Step 3. The O/w emulsion from Step 2 is cooled down at room temperature and then air dried for further analysis.

[0185] Results: The obtained surface modified silica NPs are powdered material, easily dispersible in water upon vortexing or under shear force such as under homogenizer.

[0186] CHOLESTEROL-SILICA + PARAFFIN WAX: Various ratios of NP to Lipid were selected. No microparticles were formed

[0187] CHOLESTEROL-SILICA + CETYL ALCOHOL: Various ratios of NP to Lipid were selected. Good microparticles were obtained for formulations prepared from cholesterol-silica and cetyl alcohol in the ratio, 1:50, 1:20 and 1:10.

[0188] CHOLESTEROL-SILICA + Hydrogenated Vegetable oil: Various ratios of NP to Lipid were selected. Good microparticles formation is observed between cholesterol-silica and hydrogenated vegetable oil in the ratio, 1:50, 1:20; 1:10 and 1:5.

[0189] PHENYLALANINE-SILICA + PARAFFIN WAX: Various ratios of NP to Lipid were selected. Good microparticle formation was observed between phenylalanine-silica and paraffin wax in the ratio, 1:50.

[0190] PHENYLALANINE-SILICA + CETYLALCOHOL: Various ratios of NP to Lipid were selected. Good microparticle formation is observed between phenylalanine-silica and cetyl alcohol in the ratio, 1:50, 1:20, 1:10 and 1:5.

[0191] PHENYLALANINE-SILICA + Hydrogenated Vegetable oil: Various ratios of NP to Lipid were selected. Good microparticles formation was observed between phenylalanine-silica and h veg oil in the ratio, 1:50, 1:20, and 1:10.

TABLE-US-00002 Non-limiting examples of particles prepared according to the invention. ++ successful microparticles formation; + good microparticles formation; – no formulation obtained Nanoparticle (NP) Lipid (L) Nanoparticles: Lipid ratio 1:50 1:20 1:10 1:5 1:2 Cetyl alcohol Silica ++ ++ ++ ++ + Cetyl alcohol Cholesterol-Silica ++ + + - - Cetyl alcohol Phenylalanine-Silica ++ ++ ++ + + Paraffin wax Silica - - - - - Paraffin wax Cholesterol-Silica - - - - - Paraffin wax Phenylalanine-Silica + - - - - H. Veg oil Silica - - - - - H. Veg oil Cholesterol-Silica ++ ++ ++ - - H. Veg oil Phenylalanine-Silica ++ ++ ++ - -

[0192] This study indicates that surface coating of nanoparticle improve their ability to form nano-shell microparticles.

Example 12: Nano-Shell Coated Microparticles From Different Nanoparticles and Lipid Inner Core. Effect of Composition

[0193] Aim of the proposed research: Literature reveal the Pickering emulsion with oils and solid particles. It means, in either case, Oil in water or Water in oil emulsions, the inner core is liquid supported by a surface layer of solid particle.

[0194] Herein, we propose to use a solid wax, fats or lipids with combination with colloidal silica or solid particles to create Pickering dispersions with a solid core and with controlled dimensions. The solid lipid particles find wide application is food industry, cosmetics, and medical fields.

[0195] Method: Various amount of nanoparticle (2, 5, 10, 20 50 mg) was added to 2 mL water and heated at 90° C. The mixture was homogenized at 22,000 rpm for 35 sec to obtain a uniform dispersion. In a separate vial, the solid lipid was heated at 90° C. 100 .Math.L of melted wax was added to 2 mL of hot dispersion and homogenized at 22,000 rpm for 35 sec to obtain a uniform O/w emulsion. The O/w emulsion is cooled down at room temperature and then dried for further analysis. The ratios of NP to lipids is considered as, 1:50, 1:20. 1:10, 1:5 and 1:2.

Instrumentation

[0196] Homogenization: POLYTRON homogenizer, Model No. PT 2100 was used for preparing O/w Pickering emulsion.

[0197] Scanning Electron Microscopy (SEM): They Pickering particles were analyzed using scanning electron microscope Magellan TM 400 L Field Emission Scanning Electron Microscope.

[0198] Chemical modification of silica-nanoparticle surface: The method was obtained from Yakoov et al. (ACS Omega 2018, 3, 14294-14301). Silica nanoparticles were functionalized by (3-minopropyl)-triethoxysilane (APTES) to introduce amine-functionalized groups onto the particles (silica-NH2). Silica (1 g) was added to 40 mL of methanol and stirred for complete dispersion. Then, APTES (99% Sigma-Aldrich) was added slowly to the solution for a final concentration of 0.5 M. The reaction was carried out at ambient temperature for 45 min. The particles were collected by centrifugation (9000 rpm, 10 min) and rinsed four times with excess methanol. Afterward, the silica-NH.sub.2 particles were dried at room temperature under vacuum overnight.

TABLE-US-00003 List of lipids tested Lipids Melting point (°C), literature PARAFFIN WAX 47 - 65 CETYL ALCOHOL 49 STEARIC ACID 69-72 DYNASAN® 114 55-60 DYNASAN® 116 63-68 DYNASAN® 118 72 IMWITOR® 372 P 62 IMWITOR® 491 66-77 IMWITOR® 900 K 61 SOFTISAN® 378 39 WITEPSOL® E 85 43 WITEPSOL® H32 32 WITEPSOL® H16 34 COMPRITOL® 888 ATO 70 COMPRITOL® HD5 ATO 65-77 PRECIROL® ATO 5 50-60 H. VEG OIL 55-65 GV60 60-63 VGB4 68-71 VGB6 68-74 VGB22 61-66 VGB5ST 69-73 LAURIC ACID 44 BEES WAX 60-64 CARNAUBA WAX 82 COCONUT WAX 30-39 TRILAURIN 47 TRICAPRIN 31-32 POLYCAPROLACTONE 60 PALMITIC ACID 63

[0199] List of nanoparticles that were tested: Silica, zinc oxide, clay, Magnesium hydroxide, Iron oxide, aluminum oxide, titanium dioxide, copper oxide, cellulose, starch, chitosan, etc. On the other hand, surface modified nanoparticles, by both physical and chemically modification ere tested and reported in the Example above

SUMMARY OF THE FINDINGS

[0200] The formation of nano-shell microparticles was confirmed using scanning electron microscopy (SEM). The ability to form microparticles is dependent on the nanoparticle surface activity, the lipid core, the nanoparticle: lipid ratio; the preparation conditions (not shown).

TABLE-US-00004 particles according to the invention Nanoparticle (NP) Lipid (L) NP:L 1:50 1:20 1:10 1:5 1:2 Cetyl alcohol Silica ++ ++ ++ ++ + Cetyl alcohol Silica-NH.sub.2 ++ ++ ++ + + Cetyl alcohol ZnO - - - ++ ++ Cetyl alcohol CuO + - - ++ - Paraffin wax Silica-NH.sub.2 ++ ++ ++ ++ ++ Paraffin wax Silica - - - - - Paraffin wax TiO.sub.2—NH.sub.2 ++ ++ ++ ++ + Paraffin wax Hydroxyapatite - - ++ ++ ++ Paraffin wax ZnO + ++ ++ + ++ Paraffin wax CuO ++ - - - - H. Veg oil ZnO - + ++ ++ ++ H. Veg oil CuO - - - - ++ H. Veg oil Silica - - - - - H. Veg oil Silica-NH.sub.2 ++ ++ ++ ++ ++ Stearic acid Silica - - - - - Witepsol H32 Silica - - - - -

Example 13: Microparticles With Multifunctional Surface Properties

[0201] The ability to embed nanoparticles of different properties allows obtaining microparticles of designed and desired properties. Nanoparticles of different properties, sizes and shapes are commercially available from different sources or can be synthesized using procedures published in common academic and technical reports have been used for making microparticles with different surface functionalities. Thus, for example, FePt magnetic nanoparticles (J. Nano Research Vol. 1 (2008) pp. 23-29) and Zn/Cu blend nanoparticles are used to prepare microparticles with a range of lipid cores, including: hydrogenated vegetable oil, cetyl alcohol, cetyl palmitate and solid paraffin. The particles are magnetic, antimicrobial and catalytic.

[0202] Nanoparticles containing surface polymerizable residues such as vinyl groups, amino and carboxylate groups, isocyanate groups are prepared by chemical bonding to particles surface groups such as hydroxyl group reacted with for example, diisocyanates, acryloyl or methacryloyl chloride, reactive silyl molecules and the like. These surface reactive groups can be polarized by light, heat or by radical polymerization. One application is in 3D printing inks where the printed ink is polymerized by light. Another application is polymerization after coating of a surface to improve adhesion and durability. Moreover, microparticles with metallic surfaces can be sintered or fused by heat or melt and the removal of the inner core by organic extraction, evaporation or degradation.

Example 14: Microparticles With Nanoparticles of Different Sizes

[0203] Silica nanoparticles of the size: 20, 50, 100, and 600 nanometers were used to prepare microparticles with hydrogenated vegetable oil as core lipid. The particles were prepared using the method described in Examples 1-3. All particles were coated with phenylalanine prior to application. In all preparations, the weight ratio of nanoparticles to lipid cores remains constant as 1:10. The silica particles of 100 nm or below resulted in particles in the range of 20-50 microns in diameter while the larger microparticles yielded larger particles (>50 microns).