METHOD OF PRODUCTION OF A COMPOSITE OF YEAST-DERIVED BETA GLUCAN PARTICLE WITH INCORPORATED POORLY-WATER-SOLUBLE LOW-MOLECULAR-WEIGHT COMPOUND, PHARMACEUTICAL PREPARATION AND USE THEREOF

Abstract

A formulation of composites having yeast-derived beta glucan particles (GPs) and water-insoluble or poorly-water-soluble low-molecular-weight compounds, such as medicaments or food supplements is disclosed. The composites can exhibit different crystallinity degrees depending on the formulation and, consequently, dissolution kinetics can be controlled. Yeast-derived beta glucan particles are used as carriers for the encapsulation and amorphization of insoluble or poorly water-soluble low-molecular-weight compounds; amorphous formulations exhibiting faster dissolution rates, and consequently, enhanced oral bioavailability. A method of preparation of the composites by spray drying is also disclosed.

Claims

1. A method of production of a composite of yeast-derived beta-glucan particle with incorporated poorly-water-soluble low-molecular-weight compound, the poorly-water-soluble low-molecular-weight compound in crystalline form having solubility in 10 mM PBS of at most 30 mg/mL, measured at 37° C. and pH 7.4, and molecular mass of at most 5,000 Da, and weight ratio of the poorly-water-soluble low-molecular-weight compound to the yeast-derived beta-glucan particle is in the range of from 0.1.Math.10−3 to 3, comprising the following steps: i) the poorly-water-soluble low-molecular low-molecular-weight compound is dissolved in an organic solvent, selected from a group comprising ethanol, methanol, acetone, isopropanol, ethylacetate, dichloromethane, trichloromethane, chloroform, hexane, cyclohexane, heptane, toluene or mixtures thereof; ii) yeast-derived beta glucan particles are added to the solution from step i) to form a suspension; iii) the suspension obtained in step ii) is spray dried under inert atmosphere to form the composite of yeast-derived beta glucan particle with poorly-water-soluble low-molecular-weight compound incorporated in its amorphous form inside the yeast-derived glucan particles.

2. The method according to claim 1, wherein the concentration of the solution of the poorly-water-soluble low-molecular-weight compound in the organic solvent in step i) is up to and including 150 mg/ml.

3. The method according to claim 1, wherein the suspension in step ii) has concentration of from 50 mg to 4 g of beta glucan particles per 100 ml of solution from step i).

4. The method according to claim 1, wherein the step iii) of spray drying is performed at volumetric gas-to-liquid flow ratio of from 50 to 10,000, and temperature in the range of from 30 to 350° C.

5. The method according to claim 1, wherein the beta glucan particles are obtained from Saccharomyces cerevisiae.

6. The method according to claim 5, wherein the beta-glucan particles are prepared by alkaline and acidic treatments of Saccharomyces cerevisiae, comprising the following steps: a) natural or dried yeast is mixed with aqueous hydroxide, preferably in 1M NaOH or KOH, forming a suspension; b) the suspension from step a) is homogenized and heated to at least 50° C. for at least 1 hour, preferably heated to 95° C. for 1 hour; c) the suspension from step b) is centrifuged and the supernate is removed; d) aqueous inorganic acid is added to the solid residue to adjust pH to about 4-5, and the suspension is heated to at least 50° C. for at least 2 hours; e) the suspension from step d) is centrifuged and the supernate is removed; f) the solid residue from step e) is washed with water and eventually water-miscible organic solvents, preferably selected from the group comprising isopropanol and acetone, and freeze-dried.

7. The method according to claim 1, wherein the poorly-water-soluble low-molecular-weight compound is selected from a group comprising ibuprofen, curcumin, atorvastatin, diplacone, artemisinin, morusin, epigallocatechin gallate, resveratrol, acetylsalicylic acid, nilotinib, ellagic acid, acetyl-boswellic acid, and amlodipine.

8. A composite of yeast-derived beta-glucan particle with one or more incorporated poorly-water-soluble low-molecular-weight compounds, the poorly-water-soluble low-molecular-weight compound having in crystalline form solubility in 10 mM PBS of at most 30 mg/mL, measured at 37° C. and pH 7.4, and molecular mass of at most 5,000 Da, obtained by the method according to claim 1, wherein the weight ratio of the poorly-water-soluble low-molecular-weight compound to the yeast-derived beta-glucan particle is in the range of from 0.1-10.sup.−3 to 3, and wherein the poorly-water-soluble low-molecular-weight compound incorporated in the yeast-derived beta-glucan particle is in its amorphous form.

9. The composite according to claim 8, wherein the poorly-water-soluble low-molecular-weight compound is selected from the group comprising ibuprofen, curcumin, atorvastatin, diplacone, artemisinin, morusin, epigallocatechin gallate, resveratrol, acetylsalicylic acid, nilotinib, ellagic acid, acetyl-boswellic acid, and amlodipine.

10. A pharmaceutical composition for gastrointestinal administration, characterized in that it comprises the composite according to claim 8 as a carrier of the poorly-water-soluble low-molecular-weight compound, wherein the poorly-water soluble low-molecular-weight compound incorporated in the beta-glucan particle is a medicament, and at least one pharmaceutically acceptable carrier, selected from the group comprising fillers, stabilizers, excipients, binders, disintegrants, wherein the medicament is selected from the group consisting of ibuprofen, curcumin, atorvastatin, diplacone, artemisinin, morusin, epigallocatechin gallate, resveratrol, acetylsalicylic acid, nilotinib, ellagic acid, acetyl-boswellic acid and amlodipine.

11. The pharmaceutical composition according to claim 10, characterized in that it further comprises a poorly-water-soluble low-molecular-weight medicament in crystalline form, not encapsulated in glucan particles, wherein the poorly-water-soluble medicament in crystalline form has solubility in 10 mM PBS of at most 30 mg/mL, measured at 37° C. and pH 7.4, and molecular mass of at most 5,000 Da.

12. The pharmaceutical composition according to claim 11, wherein the poorly-water-soluble low-molecular-weight medicament in crystalline form is selected from the group consisting of ibuprofen, curcumin, atorvastatin, diplacone, artemisinin, morusin, epigallocatechin gallate, resveratrol, acetylsalicylic acid, nilotinib, ellagic acid, acetyl-boswellic acid and amlodipine.

13. The pharmaceutical composition according to claim 11, wherein the poorly-water-soluble low-molecular-weight medicament in crystalline form is the same poorly-water-soluble low-molecular-weight compound as the one incorporated in the composite present in the pharmaceutical composition.

14. A method of treatment, comprising the step of administering the composite according to claim 8 as a carrier of the poorly-water-soluble low-molecular-weight compound in medicine to a subject in need thereof.

15. A method of treatment, comprising the step of administering the pharmaceutical composition according to claim 11 as a controlled release medicament to a subject in need thereof.

16. A method of food supplementation, comprising the step of administering the composite according to claim 8 as a food supplement to a subject in need thereof.

17. A pharmaceutical composition for gastrointestinal administration, characterized in that it comprises the composite according to claim 9, and at least one pharmaceutically acceptable carrier selected from the group consisting of fillers, stabilizers, excipients, and binders, disintegrants.

Description

BRIEF DESCRIPTION OF FIGURES

[0044] FIG. 1: The morphology of the microparticles produced in Example 2, evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope.

[0045] FIG. 2: Relative drug content of composites of Example 2.

[0046] FIG. 3: X-ray diffraction, evaluating the crystallinity of samples from Example 2, using a PANaytical X'Pert PRO with High Score Plus diffractometer.

[0047] FIG. 4: The morphology of the microparticles produced in Example 3, evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope. Arrows are showing curcumin found outside of the glucan particles.

[0048] FIG. 5: Fluorescent microscopy of composites of Example 3.

[0049] FIG. 6: Confocal microscopy of composites of Example 3.

[0050] FIG. 7: Relative drug content of composites of Example 3.

[0051] FIG. 8: X-ray diffraction, evaluating the crystallinity of the samples produced in Example 3, using a PANaytical X'Pert PRO with High Score Plus diffractometer.

[0052] FIG. 9: The morphology of the microparticles produced in Example 4, evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope. Circles and arrows are showing crystals found in the samples.

[0053] FIG. 10: X-ray diffraction patterns of ibuprofen and spray-dried glucan particles (SD-GP, IBU-GP-0.1, IBU-GP-0.2, IBU-GP-0.5, IBU-GP-1.0, IBU-GP-2.0), evaluating the crystallinity of the samples produced in Example 4, using a PANaytical X'Pert PRO with High Score Plus diffractometer.

[0054] FIG. 11: Dissolution kinetics of crude micronized ibuprofen, (IBU+ASA)/GP composites, and crude acetylsalicylic acid, produced according to Example 5.

[0055] FIG. 12: Dissolution kinetics of crude amlodipine, and AML/GP composites, produced according to Example 6.

[0056] FIG. 13: Comparison of wettability and dispersion of IBU/GP composites (left vial), produced according to Example 7, and crude ibuprofen (right vial), immediately after contact with water (a) and mildly shaken after 5 minutes (b).

[0057] FIG. 14: Dissolution kinetics of crude micronized ibuprofen, IBU/GP composites, and mixtures of them, produced according to Example 7.

[0058] FIG. 15: Powder rheology results for crude atorvastatin, ATO/GP composites produced by spray drying and by rotary evaporation, according to Example 8: (a) Crude atorvastatin; (b) ATO/GP-RE; (c) ATO/GP-SD.

[0059] FIG. 16: The morphology of the composites produced in Comparative Example 9, evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope ATO/GP composites, magnification 500× (a) and 2000× (b); ATO/PVP composites, magnification 500× (c); ATO/SLP composites, magnification 500× (d).

[0060] FIG. 17: The morphology of the composites produced in Comparative Example 10, evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope: GP-EtOH, magnification 500× (a) and 2000× (b); GP-EtOH/water, magnification 500× (c) and 2000× (d); and GP-water, magnification 500× (e) and 2000× (f).

[0061] FIG. 18: Particle size distributions of the composites produced in Comparative Example 10, evaluated by static light scattering using Horiba Partica LA 950/S2 equipment.

[0062] FIG. 19: Phagocytosis of macrophages according to Example 10, observed after 3 hours using an Olympus Fluoview FV1000 confocal system for: GP/CC-EtOH sample observed using objective 40× with zoom mode (a), and with excitation wavelength 405 nm and zoom mode (b); GP/NR-EtOH sample observed using objective 60× (c), and with excitation wavelength 550 nm (d).

EXAMPLES

[0063] The invention is further illustrated by, but not limited to, specific examples.

Example 1—General Method of Preparation of Composites of Yeast-Derived Beta-Glucan Particles and Poorly-Water-Soluble Low-Molecular-Weight Compound

[0064] The composites of yeast-derived beta glucan particles and poorly-water-soluble low-molecular-weight compounds according to the present invention were produced by spray drying using a Mini Spray Dryer B-290 from Büchi operated in inert loop under N.sub.2 atmosphere, and equipped with a 2-fluid nozzle (0.7 mm of diameter) or an ultrasonic package (ultrasonic nozzle and controller). A solution of the poorly-water-soluble low-molecular-weight compound in an organic solvent (such as ethanol, methanol, acetone, isopropanol, dichloromethane or mixtures thereof) with desired concentration (typically in the range of from 0.5 to 20 mg/mL) is prepared, and glucan particles are added to the low-molecular-weight compound solution to form a suspension, containing from 2 to 40 mg of glucan particles per 1 ml of the suspension. The resulting suspension is spray dried under inert atmosphere, typically nitrogen, and using previously defined parameters. The spray drying process promotes the rapid evaporation of the organic solvent and the subsequent precipitation of the drug within or within and outside the glucan particles. The spray-drying parameters can be changed to produce different composite formulations. The inlet temperature is selected based on the boiling point of the organic solvent and/or thermal degradation properties of the starting materials. Feeding rate and gas flow rate mainly influence droplet size. Feeding rate in the experiments varied between 1 and 20 milliliters per minute, and the gas flow rate from 100 to 600 L/h.

[0065] The beta glucan particles for the experiment were prepared from Saccharomyces cerevisiae based on the methodology described in Salon̆, et al., Suspension stability and diffusion properties of yeast glucan microparticles. Food and Bioproducts Processing, 2016. 99: p. 128-135. First, baker's yeast was subjected to alkaline treatment. For that, 600 mL of 1 M NaOH solution were added to 150 grams of yeast. The suspension was heated to 90° C. and stirred with magnetic pill for one hour; then, it was centrifuged, and the supernatant was discarded. The alkaline treatment was repeated three times. The pH of the slurry obtained alter the alkaline treatments was adjusted between 4 and 5 by adding HCl solution (35%). The acidic suspension was stirred for 2 hours at 75° C. and centrifuged to discard the supernatant. Finally, the slurry was washed with deionized water (three times), isopropanol (four times) and acetone (two times), freeze-dried for two days and stored in a refrigerator for further use.

[0066] Formulation of yeast-derived beta-glucan particles and insoluble or poorly-water soluble low-molecular-weight compounds:

[0067] Various formulations of yeast-derived beta-glucan particles and insoluble or poorly-water soluble drugs were prepared by using different low-molecular-weight compounds and/or combination of them, different solvents and/or combination of them, and varying the drug/GP mass ratios. The solvent may be, for example, ethanol, methanol, acetone, isopropanol, dichloromethane or other organic solvents, and/or mixtures of them. The scale of the experiment may vary from milligrams to hundreds of grams of the glucan particles, thus covering the industrial production. The weight of the poorly-water soluble low-molecular-weight compound is then given by the desired fraction of the drug in the composite, which can range from 0.1 to over 3.0. The ratio between the weight of the glucan particles and the volume of the solvent may range from tens of milligrams to tens of grams of particles per liter of solvent according to the desired properties of the composites. Examples of the preparations used for testing are given in the following Table 1.

TABLE-US-00001 TABLE 1 Low- Low- molecular- molecular- Model low- weight weight molecular- compound GPs compound/ Spray weight Solvent concentration concentration GPs weight drying compound used (mg/mL) (mg/mL) ratio conditions * Ibuprofen Ethanol 1.00 10.0 0.10 2FN, S and L Ibuprofen Ethanol 2.00 20.0 0.10 2FN, S and L Ibuprofen Ethanol 2.00 10.0 0.20 2FN, S and L Ibuprofen Ethanol 5.00 10.0 0.50 2FN, S and L Ibuprofen Ethanol 10.0 10.0 1.0 2FN, S and L Ibuprofen Ethanol 20.0 10.0 2.0 2FN, S and L Ibuprofen Ethanol 5.00 20.0 0.25 USN Curcumin Ethanol 0.0100 20.0 0.50 × 10.sup.−3 USN Curcumin Ethanol 0.0200 20.0  1.0 × 10.sup.−3 USN Curcumin Ethanol 0.100 20.0  5.0 × 10.sup.−3 USN Curcumin Ethanol 0.200 20.0 0.010 USN Curcumin Ethanol 1.00 20.0 0.050 2FN (S and L), USN Curcumin Ethanol 2.00 20.0 0.10 USN Curcumin Ethanol 3.60 20.0 0.18 2FN, L Curcumin Ethanol 4.00 20.0 0.20 USN Curcumin Ethanol 6.00 20.0 0.30 USN Diplacone Ethanol 2.50 × 10.sup.−3 20.0 0.13 × 10.sup.−3 USN Diplacone Ethanol 0.0130 20.0 0.63 × 10.sup.−3 USN Diplacone Ethanol 0.127 20.0  6.3 × 10.sup.−3 USN Artemisinin Ethanol 8.40 × 10.sup.−3 20.0 0.42 × 10.sup.−3 USN Epigallocatechin Ethanol 0.0140 20.0 0.68 × 10.sup.−3 USN gallate Resveratrol Ethanol 6.80 × 10.sup.−3 20.0 0.34 × 10.sup.−3 USN Ellagic acid Ethanol 9.00 × 10.sup.−3 20.0 0.45 × 10.sup.−3 USN Morusin Ethanol 0.0130 20.0 0.63 × 10.sup.−3 USN Acetyl- Ethanol 0.50 10.0 0.050 USN boswellic acid Atorvastatin Ethanol 0.0170 20.0 0.85 × 10.sup.−3 USN Atorvastatin Ethanol 0.0330 20.0  1.7 × 10.sup.−3 USN Atorvastatin Ethanol 0.167 20.0  8.4 × 10.sup.−3 USN Atorvastatin Ethanol 1.00 20.0 0.050 USN Atorvastatin Ethanol 2.00 20.0 0.10 USN Atorvastatin Ethanol 3.00 20.0 0.15 USN Atorvastatin Methanol 1.00 20.0 0.05 USN Amlodipine Ethanol 5.00 20.0 0.25 USN Amlodipine DCM 5.00 20.0 0.25 USN Amlodipine DCM/Ethanol 5.00 20.0 0.25 USN (1/1 V/V) Ibuprofen/ Ethanol 5.00 20.0 0.25 USN Acetylsalicylic acid (1/1) Nilotinib Methanol/DCM 3.0 20.0 0.15 2FN (7/3 V/V) Nilotinib Methanol/DCM 2.0 20.0 0.10 2FN (7/3 V/V) Nilotinib Methanol/DCM 1.0 20.0 0.050 2FN (7/3 V/V) Nilotinib Methanol/DCM 0.20 20.0 0.010 2FN (7/3 V/V) Nilotinib Methanol/DCM 0.0020 20.0 0.10 × 10.sup.−3 2FN (7/3 V/V) Nilotinib Methanol/DCM 0.00040 20.0 0.020 × 10.sup.−3  2FN (7/3 V/V) * 2FN: 2-fluid nozzle; S: small-droplet configuration; L: large-droplet configuration; USN: ultrasonic nozzle (with which is possible to produce extra-large droplets).

Example 2—Preparation of Composites with Different Processing Conditions

[0068] Composites of yeast-derived beta glucan particles with incorporated poorly-water-soluble low-molecular-weight compound were prepared according to the procedure of Example 1, using ibuprofen (IBU) as the poorly-water-soluble low-molecular-weight compound model, with a fixed IBU-to-GP weight ratio of 0.1. Different samples were produced by changing the processing conditions, namely initial solid content and spray-drying parameters (feeding rate and flow rate). The initial solid contents tested were 10 and 20 mg/mL, i.e. 1 or 2 grams of glucan particles were added in 100 milliliters of ibuprofen solution with concentration of 1 mg/mL or 2 mg/mL respectively. Ethanol was used as the organic solvent.

[0069] The prepared 100-mL suspensions were spray-dried using the 2-fluid nozzle. In order to evaluate the influence of droplet size in the final composites, two different set of operating conditions were tested. The first one (small droplets) consisted of 3.5 mL/min feeding rate and 600 L/h (50%) N.sub.2 flow rate; the second set (large droplets) consisted in 7.0 mL/min feeding rate and 473 L/h (40%) N.sub.2 flow rate. In both cases, the outlet temperature was kept constant at (75±2)° C., for which the inlet temperature was varied between 120 to 130° C.

[0070] The samples are labeled as: [0071] S10: Composites prepared with initial solid content 10 mg/mL and small droplets set of parameters. [0072] S20: Composites prepared with initial solid content 20 mg/mL and small droplets set of parameters. [0073] L10: Composites prepared with initial solid content 10 mg/mL and large droplets set of parameters. [0074] L20: Composites prepared with initial solid content 20 mg/mL and large droplets set of parameters.

[0075] Morphology of the Composites

[0076] The morphology of the produced microparticles (FIG. 1) was evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope. Before the SEM analysis, the samples were coated with a 5-nm gold layer using an Emitech K550X sputter coating equipment.

[0077] The glucan particles present the typical ellipsoidal morphology with 2-4 μm particle size, exhibiting a wrinkled surface that can be attributed to the hydrolysis of the yeast outer cell wall and intercellular components, product of the alkaline and acid treatments. No evidence of ibuprofen outside of the glucan particles is observed.

[0078] Encapsulation Efficiency

[0079] For the determination of the encapsulation efficiency (FIG. 2), ibuprofen was extracted from the produced IBU/GP composites by adding 10.0 mg of the microparticles to a 10.0 mL of phosphate buffer solution (pH 7.4). The dispersions were placed in an ultrasonication bath for 10 min to guarantee the complete extraction of the ibuprofen from the glucan particles. Afterwards, the glucan particles were separated by centrifugation (5 min at 7000 rpm), and 500 μL of supernatant were collected. The concentration was evaluated by high-performance liquid chromatography (HPLC) with UV detection (Agilent), coupled with C18 column (100 mm×4.6 mm, 5 μm) and mobile phase consisting of 0.01 M ammonium phosphate buffer (pH 2.0) and acetonitrile (60%). The encapsulation efficiency of the IBU/GP composite microparticles was calculated as the experimental concentration of active compound (C.sub.E), measured by HPLC, divided by the theoretical concentration (C.sub.T) of ibuprofen in the composites.

[0080] Significantly higher encapsulation efficiencies (C.sub.E/C.sub.T) were obtained for the samples produced with large-droplets settings (10 L and 20 L) when compared with the samples produced with small-droplet settings. In addition, higher encapsulation efficiencies were obtained for the samples produced from dispersions with higher solid content.

[0081] X-Ray Diffraction

[0082] Crystallinity of the samples (FIG. 3) was evaluated by recording the diffraction intensities of the produced microparticles from 5° to 50° 2θ angle using a PANaytical X'Pert PRO with High Score Plus diffractometer. Unlike the micronized crude ibuprofen, all IBU/GP composites produced are completely amorphous.

Example 3—Preparation of Composites with Different Spray-Drying Nozzles

[0083] Composites of yeast-derived beta-glucan particles with incorporated poorly-water-soluble low-molecular-weight compound were prepared according to the procedure of Example 1 using curcumin (CC) as a poorly-water-soluble low-molecular-weight compound model, with a fixed CC-to-GP weight ratio of 0.05. For that, 50-mL suspensions (20 mg/mL) were prepared by adding 1 gram of glucan particles in 50 milliliters of curcumin solution with concentration of 1 mg/mL of ethanol. Afterwards, the suspensions were spray-dried using different spray-drying nozzles, namely a 2-fluid nozzle (0.7 min of diameter) and the ultrasonic nozzle. The different nozzles can mainly influence droplet size and morphology of the samples.

[0084] For the sample spray dried using the 2-fluid nozzle (labeled 2FN), the operating conditions used consisted of 3.5 mL/min feeding rate and 473 L/h (40%) N.sub.2 flow rate. For the sample spray-dried using the ultrasonic nozzle (labeled USN), the operating conditions consisted of 3.5 mL/min feeding rate, 246 L/h (20%) N.sub.2 flow rate, and 1.8 watts (ultrasonic nozzle power). In both cases the inlet temperature was 120° C., for which the outlet temperature was (75±2)° C.

[0085] Morphology of the Composites

[0086] The morphology of the produced microparticles (FIG. 1) was evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope. Before the SEM analysis, the samples were coated with a 5-nm gold layer using an Emitech K550X sputter coating equipment. Besides the typical ellipsoidal, wrinkled morphology of the glucan particles, another type of particles with spherical morphology were observed and attributed to curcumin precipitated outside of the glucan particles. The curcumin outside of the glucan particles is much more evident in the 2FN sample than in the USN sample.

[0087] Fluorescent and Confocal Microscopy

[0088] Samples were analyzed by fluorescent (FIG. 5) and confocal microscopy (FIG. 6) using an Olympus Fluoview FV1000 confocal system (488 nm excitation wavelength). From fluorescent microscopy images is possible to observe that the loading of curcumin in the glucan particles was uniform for both samples (2FN and USN). In the confocal microscopy images, a large amount of curcumin particles outside of the glucan particles (small dots) are observed in the 2FN-sample, whereas in the USN-samples such particles are not observed, evidencing that all the curcumin was encapsulated inside the glucan particles.

[0089] Encapsulation Efficiency

[0090] The curcumin content of the CC/GP composite microparticles (FIG. 7) was calculated as the experimental concentration (C.sub.E) of curcumin, measured by UV-Vis spectrophotometry, divided by the theoretical concentration (C.sub.T) of curcumin. For the determination of the experimental concentration, curcumin was extracted from the produced CC/GP composites by adding 5.0 mg of the microparticles to 10.0 mL of methanol. The dispersions were placed in an ultrasonication bath for 10 min to guarantee the complete extraction of the curcumin from the glucan particles. Afterwards, the glucan particles were separated by centrifugation (10 min at 5000 rpm), and 3.0 mL of supernatant were filtered and placed into a spectrophotometer cuvette. Dilutions were done when necessary. Absorbance (λ=425 nm) was measured by UV-Vis spectrophotometry, using a Specord 205 BU UV-Vis spectrophotometer, and related to the concentration of curcumin using a calibration curve previously plotted.

[0091] The encapsulation efficiency was approximately 100% for the USN-sample and 61.5% for the 2FN-sample. The 40% difference is attributed to losses of curcumin that precipitated outside of the glucan particles in the case of the 2FN-sample. Such curcumin particles are very small; therefore, there is a high possibility that they were not collected in the cyclone of the spray dryer, causing the losses along the spray dryer.

[0092] X-Ray Diffraction

[0093] Crystallinity of the samples (FIG. 8) was evaluated by recording the diffraction intensities of the produced microparticles from 5° to 50° 2θ angle using a PANaytical X'Pert PRO with High Score Plus diffractometer. Unlike the pure curcumin, all CC/GP composites produced are completely amorphous.

Example 4—Preparation of the Composites with Increasing Low-Molecular-Weight Compound Loading

[0094] Composites of glucan particles and ibuprofen (IBU), as poorly-water soluble model low-molecular-weight compound, were prepared according to the procedure of Example 1, considering increasing IBU/GP mass ratios (0.1, 0.2, 0.5, 1.0 and 2.0). For that, 100-mL ibuprofen solutions were prepared with concentrations 0.1. 0.2, 0.5, 1.0 and 2.0% (w/v), using ethanol as organic solvent. Afterwards, 1.0 g of glucan particles was added to each solution and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray-drying. The dispersions were incubated overnight at room temperature before spray-drying. The samples are labeled as IBU-GP-0.1, IBU-GP-0.2, IBU-GP-0.5, IBU-GP-1.0 and IBU-GP-2.0 respectively. An analogous unloaded sample referred as “SD-GP” was also prepared and spray-dried.

[0095] The 100-mL samples were spray-dried using the Mini Spray Dryer B-290 equipped with the 2-fluid nozzle (0.7 mm of diameter) and operated in inert loop under N.sub.2 atmosphere. Two different set of operating conditions were used. The first one (small droplets) consisted of 120° C. inlet temperature, 3.5 mL/min feed rate and 600 L/h (50%) N.sub.2 flow rate. The second set of operating conditions (large droplets) was: 130° C. inlet temperature, 7.0 mL/min feed rate and 473 L/h (40%) N.sub.2 flow rate. In both cases, the outlet temperature was from 66 to 72° C.

[0096] Morphological Characterization

[0097] The morphology of the produced microparticles (FIG. 9) was evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope. Before the SEM analysis, the samples were coated with a 5-nm gold layer using an Emitech K550X sputter coating equipment. The presence of ibuprofen crystals outside of the glucan particles was observed in the samples with higher IBU content (IBU/GP mass ratio ≥0.5). The crystals appeared larger in size and quantity in the samples produced with the large-droplet spray-drying settings.

[0098] X-Ray Diffraction

[0099] Crystallinity of the samples (FIG. 10) was evaluated by recording the diffraction intensities of the produced microparticles from 5° to 50° 2θ angle using a PANaytical X'Pert PRO with High Score Plus diffractometer. A tendency of crystallinity to increase with IBU content and droplet size was observed, in accordance to the SEM observations.

Example 5—Preparation of Composites with Combination of More than One Poorly-Water Soluble Low-Molecular-Weight Compounds

[0100] Composites of glucan particles and two different poorly-water soluble model low-molecular-weight compounds, ibuprofen (IBU) and acetylsalicylic acid (ASA), were prepared according to the procedure of Example 1. The composites were prepared considering a drug/GP mass ratio of 25% (IBU/GP=12.5% wt. and ASA/GP=12.5% wt.) and using ethanol as organic solvent. For that, 50-mL of drug solution were prepared by dissolving 125 mg of IBU and 125 mg of ASA, using ethanol as common organic solvent. Afterwards, 1.0 g of glucan particles was added to the drug solution and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray drying. The sample was spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated in inert loop under N.sub.2 atmosphere. The operating conditions used consisted of 120° C. inlet temperature, 5.0 mL/min feed rate, 246 L/h (20%) N.sub.2 flow rate and 2.0 W power outlet at nozzle. The outlet temperature was 76° C.

[0101] Dissolution Kinetics

[0102] Dissolution tests (FIG. 11) were performed for crude micronized ibuprofen, crude acetylsalicylic acid and for the produced (IBU+ASA)/GP composite particles, in powder form and using 10 mM HCl (pH 2.0) as dissolution medium. For that, 20.0 mg of crude drug (IBU or ASA) or 100.0 mg of (IBU+ASA)/GP composite, were added to 200 mL of continuously stirred dissolution medium (for a maximum concentration of 0.1 mg of drug per ml of medium). The mixtures were continuously stirred at 250 rpm and room temperature in a 250-mL beaker. At pre-defined time-points (ranging from 0 to 60 min), 500 μL of sample were collected, centrifuged and filtered (200-nm pore size filtration membrane), and the concentration was evaluated by high-performance liquid chromatography (HPLC) with UV detection (Agilent), coupled with C18 column (100 mm×4.6 mm, 5 μm) and mobile phase consisting of 0.01 M ammonium phosphate buffer (pH 2.0) and acetonitrile, in gradient according to the Table 2:

TABLE-US-00002 TABLE 2 Time Flow (min) % A % B (ml/min) 0.0 80 20 1 3.0 80 20 1 3.5 40 60 1 6.5 40 60 1 7.0 80 20 1 9.0 80 20 1

[0103] After 2 minutes, both IBU and ASA encapsulated in the glucan particles were completely dissolved, whereas crude IBU and crude ASA exhibit slower dissolution rates.

Example 6—Preparation of Composites with Different Pure Organic Solvents and Combinations of Them

[0104] Composites of glucan particles and amlodipine (AML), as poorly-water soluble model low-molecular-weight compound, were prepared according to the procedure of Example 1, considering an AML/GP mass ratio of 25% and using different organic solvents and combinations of them. For that, 50-mL amlodipine solutions were prepared with concentration 5 mg/mL, using ethanol, dicloromethane (DCM) and a mixture of ethanol/DCM (50/50) as organic solvents. Afterwards, 1.0 g of glucan particles was added to each solution and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray drying.

[0105] The samples are labeled: [0106] AML/GP-EtOH: For the composites prepared using 100% ethanol as solvent. [0107] AML/GP-DCM-EtOH: For the composites prepared using 50% DCM and 50% ethanol as solvents. [0108] AML/GP-DCM: For the composites prepared using 100% DCM as solvent.

[0109] The three samples were spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated in inert loop under N.sub.2 atmosphere. The operating conditions used consisted of 120° C., 90° C. and 80° C. inlet temperature respectively for AML/GP-EtOH, AML/GP-DCM-EtOH and AML/GP-DCM samples. In all cases, 5.0 mL/min feeding rate, 246 L/h (20%) N.sub.2 flow rate and 2.4 W power outlet at nozzle were set. The outlet temperature was 76° C., 56° C. and 54° C., respectively.

[0110] Dissolution Kinetics

[0111] Dissolution tests (FIG. 12) were performed for crude amlodipine and for the produced AML/GP composite particles in powder form and using distilled water as dissolution medium. For that, 20.0 mg of crude amlodipine or 100.0 mg of composites were added to 200 mL of dissolution medium (for a maximum concentration of 0.1 mg of amlodipine per ml of medium). The mixtures were continuously stirred at 250 rpm and room temperature in a 250-mL beaker. At pre-defined time-points (ranging from 0 to 60 min), 500 μL of sample were collected, centrifuged and filtered (200-nm pore size filtration membrane) before measurements. The concentration was evaluated by UV-Vis spectrophotometry (λ=366 nm), using a Tecan Infinite M200 spectrometer. Faster dissolution was obtained for the samples AML/GP-DCM-EtOH and AML/GP-DCM. In addition, 100% of amlodipine was dissolved after 30 minutes in the case of the AML/GP composites, independently of the solvent used, while only 80% of amlodipine was dissolved after 60 minutes in the case of crude amlodipine.

Example 7—Preparation of Composites with Improved Dispersibility Properties and Controlled Release

[0112] Composites of glucan particles and ibuprofen (IBU), as poorly-water soluble model low-molecular-weight compound, were prepared according to the procedure of Example 1, considering an IBU/GP mass ratio of 25%. For that, 50-mL ibuprofen solution was prepared with concentration 5 mg/mL, using ethanol as organic solvent. Afterwards, 1.0 g of glucan particles was added to the solution and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray drying. The sample was spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated in inert loop under N.sub.2 atmosphere. The operating conditions used consisted of 120° C. inlet temperature, 5.0 mL/min feed rate, 246 L/h (20%) N.sub.2 flow rate and 2.4 W power outlet at nozzle. The outlet temperature was 76° C.

[0113] Dispersion Properties

[0114] Dispersion properties of IBU/GP composites versus micronized crude ibuprofen (FIG. 13) were analyzed by observing the behavior of the samples in suspension. For that, 20.0 mg of each sample were weighted and added to 10.0 mL of 10 mM HCl (pH 2.0). The IBU/GP composites exhibit improved dispersion properties even without the use of a surfactant. Due to their good wettability, the dispersion of the composites was fast and spontaneous.

[0115] Dissolution Kinetics

[0116] Since ibuprofen is poorly soluble under acidic conditions, it can be expected that crystalline and amorphous forms of ibuprofen will show significantly different dissolution rates in acidic medium. Therefore, dissolution tests (FIG. 14) were performed for crude micronized ibuprofen (crystalline) and for the produced IBU/GP composite particles (amorphous), as well as for physical mixtures of the crude IBU and the composite particles, in powder form and using 10 mM HCl (pH 2.0) as dissolution medium. For that, 20.0 mg of ibuprofen (crude crystalline, GP composite or physical mixtures—see Table 3) were added to 200 mL of continuously stirred dissolution medium (250 rpm at room temperature) in a 250-mL beaker. At pre-defined time-points (ranging from 0 to 60 min), 500 μL of sample were collected, centrifuged and filtered (200-nm pore size filtration membrane), and the concentration was evaluated by high-performance liquid chromatography (HPLC) with UV detection (Agilent), coupled with C18 column (100 mm×4.6 mm, 5 μm) and mobile phase consisting of 0.01 M ammonium phosphate butter (pH 2.0) and acetonitrile (60%).

TABLE-US-00003 TABLE 3 Crystalline/amorphous Mass of crude Mass of composite (mg) ibuprofen proportion ibuprofen (mg) IBU/GP mass ratio = 25% 100/0  20.0 0.0 75/25 15.0 25.0 50/50 10.0 50.0 25/75 5.0 75.0  0/100 0.0 100.0

[0117] Progressively faster dissolution profiles were obtained with increasing mass fraction of encapsulated ibuprofen, until the solubility limit was reached. For the samples with the highest mass fraction of encapsulated ibuprofen (crystalline/amorphous ibuprofen proportion=25/75 and 0/100), the fast release lead to supersaturation.

Example 8—Preparation of Composites with Improved Powder Flowability

[0118] Composites of glucan particles and atorvastatin (ATO), as poorly-water soluble model low-molecular-weight compound, were prepared according to the procedure of Example 1, considering an ATO/GP mass ratio of 25%. For that, 50-mL atorvastatin solution was prepared with concentration 5 mg/mL, using ethanol as organic solvent. Afterwards, 1.0 g of glucan particles was added to the solution and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray drying. The sample was spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated in inert loop under N.sub.2 atmosphere. The operating conditions used consisted of 120° C. inlet temperature, 5.0 mL/min feed rate, 246 L/h (20%) N.sub.2 flow rate and 2.4 W power outlet at nozzle. The outlet temperature was 76° C.

[0119] Composites of glucan particles and atorvastatin (ATO) with ATO/GP mass ratio of 25%, were also prepared by an alternative method, using rotary evaporator. For that, 200 mL of atorvastatin solution (1.25 mg/mL in ethanol) were added to 1.0 g of glucan particles in a round bottom flask. The obtained suspension was homogenized in an ultrasonication bath for 15 minutes, and the solvent was removed by evaporation using an IKA® HB10 basic rotary evaporator. The operating conditions used consisted of 60° C. water-bath temperature and 175 RPM rotation speed. The pressure was slowly decreased from atmosphere pressure to 330 mBar. When most of the ethanol was removed, the pressure was decreased to 80-90 mBar, and the sample was dried at low pressure for 20 minutes. The obtained powder was collected from the round bottom flask and freeze-dried for 48 hours.

[0120] The samples are labeled as: [0121] ATO/GP-SD: For the composites prepared by spray drying. [0122] ATO/GP-RE: For the composites prepared by rotary evaporator.

[0123] Both composite samples (ATO/GP-SD and ATO/GP-RE) and crude atorvastatin were subjected to characterization.

[0124] Powder Rheology

[0125] The moisture (water content) of the samples was firstly measure using a moisture analysis balance (simple test, 100° C., 5 mg initial mass, infrared drying). The samples, ATO/GP-SD, ATO/GP-RE and crude ATO, contained 5% wt., 3% wt. and 2.5% wt. of moisture respectively. Afterwards, the samples were exposed to laboratory humidity (21° C. and 28% relative humidity), for 24 hours and the moisture content was measured again (9% wt. for ATO/GP-SD, 9% for ATO/GP-RE, and 5% for ATO). The samples were then dried for 24 hours in the oven with very-slowly-moving fan at 30° C. Finally, a shear test (FIG. 15) was performed on the samples a Powder Rheometer FT4 (pre-sheared at 3 kPa consolidation and initial mass of 0.6-0.7 g).

[0126] Crude atorvastatin exhibits the highest cohesion and internal friction, while the composites (ATO/GP-SD and ATO/GP-RE), both show improved flowability (Table 4). For both composite samples, cohesion is the same, but they differ in internal friction. Therefore, ATO/GP-SD and ATO/GP-RE composite samples will flow similarly in high-shear and compressive specific processes, but the spray-dried sample (ATO/GP-SD) has improved flowability than ATO/GP-RE in low-stress processes.

TABLE-US-00004 TABLE 4 Sample Cohesion (kPa) UYS* (kPa) AIF* (°) Crude ATO 0.879 4.27 45.3 ATO/GP-RE 0.534 2.12 36.6 ATO/GP-SD 0.529 1.74 27.3 *UYS: Unconfmed Yield Strength; AIF: Angle of Internal Friction.

Comparative Example 9—Preparation of Composites Using Polymeric Matrices Other than Yeast Glucan Particles

[0127] Composites of glucan particles and atorvastatin (ATO), as poorly-water soluble model low-molecular-weight compound, were prepared according to the procedure of Example 1, considering an ATO/GP mass ratio of 10%. For that, 50-mL atorvastatin solution was prepared with concentration 2 mg/mL, using ethanol as organic solvent. Afterwards, 1.0 g of glucan particles was added to the solution and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray drying. The sample was spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated in inert loop under N.sub.2 atmosphere. The operating conditions used consisted of 120° C. inlet temperature, 5.0 mL/min feed rate, 246 L/h (20%) N.sub.2 flow rate and 2.4 W power outlet at nozzle. The outlet temperature was 76° C.

[0128] For comparison, composites of hydrophilic polymers and atorvastatin with ATO/polymer mass ratio of 10% were also prepared. The selected polymers were polyvinylpyrrolidone (PVP) and polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus). These polymers are commonly used to produce amorphous solid dispersions. For the preparation of the composites, 50-mL atorvastatin solutions were prepared with concentration 2 mg/mL, using ethanol as organic solvent. Afterwards, 1.0 g of polymer was added to the solution and mixed until complete dissolution. Each sample was spray-dried using the same conditions as described above for ATO/GP. The composite with PVP is labeled as “ATO/PVP”, and the composite with Soluplus is labeled as “ATO/SLP”.

[0129] Morphology of the Composites

[0130] The morphology of the composites (FIG. 16) was evaluated by Scanning Electron Microscopy (SEM) using a Jeol JCM-5700 microscope. Before the SEM analysis, the samples were coated with a 5-nm gold layer using an Emitech K550X sputter coating equipment.

[0131] The ATO/GP composites present the typical ellipsoidal morphology with 2-4 μm particle size, exhibiting a wrinkled surface that can be attributed to the hydrolysis of the yeast outer cell wall and intercellular components, product of the alkaline and acid treatments. No evidence of atorvastatin outside of the glucan particles is observed. In the case of the composites with ATO/PVP and ATO/SLP, the particles present mushroom-like morphology, with much larger particle sizes, ranging between approximately 5 to 50 μm.

[0132] Encapsulation Efficiency

[0133] For the determination of the encapsulation efficiency, atorvastatin was extracted from the produced composites by adding 10.0 mg of the particles to 10.0 mL of methanol, in which atorvastatin is freely soluble. The dispersions were placed in an ultrasonication bath for 10 min to guarantee the complete extraction of the atorvastatin from the composites. Afterwards, the samples were centrifuged (5 min at 7000 rpm), and 500 μL of supernatant were collected. The concentration was evaluated by high-performance liquid chromatography (HPLC) with UV detection (Agilent), coupled with C18 column (100 mm×4.6 mm, 5 μm) and mobile phase consisting of 0.01 M ammonium phosphate buffer (pH 2.0) and acetonitrile (60%).

[0134] The encapsulation efficiency of the composites was calculated as the experimental concentration of active compound (C.sub.E), measured by HPLC, divided by the theoretical concentration (C.sub.T) of atorvastatin in the composites. The highest encapsulation efficiency (C.sub.E/C.sub.T) was obtained for the ATO/GP composite followed by ATO/SLP sample and ATO/PVP, as shown in Table 5.

TABLE-US-00005 TABLE 5 Encapsulation efficiency Sample [%] ATO/GP 92.40 ± 0.03 ATO/SLP  89.2 ± 0 01 ATO/PVP 75.60 ± 0.02

[0135] Powder Rheology

[0136] Shear tests were performed on the samples a Powder Rheometer FT4 (pre-sheared at 3 kPa consolidation and initial mass of 0.6-0.7 g). The tests were carried out per duplicate under laboratory conditions (21.2±0.7° C. and 35.1±2.0% relative humidity).

[0137] Crude atorvastatin exhibits the highest cohesion and internal friction (see Table 6). ATO/PVP also shows high cohesion but slightly lower than crude ATO. On the other hand, ATO/GP and ATO/SLP samples are the best flowable materials, belonging to the “easy flowing” materials category.

TABLE-US-00006 TABLE 6 Sample Cohesion (kPa) UYS* (kPa) AIF* (°) FF* Category Crude ATO 0.81 ± 0.21 4.57 ± 1.07 51.24 ± 0.82 2.43 ± 0.48 Cohesive ATO/GP 0.31 ± 0.01 1.14 ± 0.04 33.52 ± 0.09 4.33 ± 0.14 Easy flowing ATO/SLP 0.30 ± 0.02 1.04 ± 0.05 29.60 ± 0.12 4.74 ± 0.22 Easy flowing ATO/PVP 0.65 ± 0.04 2.10 ± 0.12 26.35 ± 0.11 2.65 ± 0.06 Cohesive *UYS: Unconfined Yield Strength; AIF: Angle of Internal Friction; FF: Flow Function.

Comparative Example 10—Preparation of Yeast Glucan Particles Spray Dried from Pure Water, Pure Organic Solvent and Water/Organic Solvent Mixtures

[0138] Given the hydrophilic nature of beta glucans, it is of interest to evaluate the effect of the use of water as a solvent or co-solvent in the preparation of spray-dried pure yeast glucan particles. Pure glucan particles were prepared according to the procedure of Example 1, using ethanol as organic solvent. For that, 1.0 g of glucan particles was added to 50 mL of ethanol and dispersed using an IKA® T10 basic ultra-turrax for 5 minutes before spray drying. The sample was spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated in inert loop under N.sub.2 atmosphere. The operating conditions used consisted of 120° C. inlet temperature, 5.0 mL/min feed rate, 246 L/h (20%) N.sub.2 flow rate and 2.4 W power outlet at nozzle. The outlet temperature was from 60 to 70° C.

[0139] Alternatively, for comparison, pure glucan particles were prepared using water and water/ethanol mixture (50/50) as solvents. For that, each sample was spray-dried using the Mini Spray Dryer B-290 equipped with the ultrasonic nozzle and operated under air atmosphere. The inlet temperature and power outlet at nozzle were adjusted adequately for each solvent. The operating conditions used consisted of 130-140° C. inlet temperature, 5.0 mL/min feed rate, 246 L/h (20%) air flow rate and 3.0 W power outlet at nozzle. The outlet temperature was from 60 to 70° C. The samples are labeled according to the solvent used as GP-EtOH, GP-water, GP-EtOH/water respectively for ethanol, water and ethanol/water mixture.

[0140] Morphology of the Pure Glucan Particles

[0141] The morphology of the pure glucan particles (FIG. 17) was evaluated by Scanning Election Microscopy (SEM) using a Jeol JCM-5700 microscope. Before the SEM analysis, the samples were coated with a 5-nm gold layer using an Emitech K550X sputter coating equipment.

[0142] The glucan particles spray dried from organic solvent (GP-EtOH) preserve the typical ellipsoidal morphology with 2-4 μm particle size, and wrinkled surface, the same morphology as was observed for GPs prepared in Example 1 and 2, whereas the samples prepared from water and water/ethanol mixture (GP-water and GP-EtOH/water) present mushroom-like morphology, and much larger particle sizes, ranging between approximately 5 to 50 μm.

[0143] Particle Size Distributions

[0144] Particle size distributions of the samples (FIG. 18) were obtained by static light scattering using Horiba Partica LA 950/S2 equipment. Prior measurement, the samples were dispersed in distilled water at a concentration of 1.0 g/L and homogenized using an IKA® T10 basic ultra-turrax for 1 minute. Mean size, D(v, 0.1), D(v, 0.5), and D(v, 0.9) are shown in Table 7.

TABLE-US-00007 TABLE 7 Sample Mean Size (μm) D(v, 0.1) (μm) D(v, 0.5) (μm) D(v, 0.9) (μm) GP-EtOH 6.17396 ± 1.9706 3.92591 5.89407 8.78317 GP-EtOH/water 24.80679 ± 9.0110  12.81026 25.04354 36.24339 GP-water 27.94065 ± 10.2500 15.73663 27.02836 41.11664

[0145] In all cases, particle size distributions are monodispersed with sizes ranges in accordance to what was observed in the SEM images. The particle size of GPs has a fundamental influence on the phagocytosis by macrophages. It has been proven that particles in the size range of 0.1-10 μm are the most biologically active in macrophage immune response. Given that human macrophages are about 21 μm in size, the engulfment of particles that are larger than themselves is limited and can potentially cause the death of the cells. Therefore, it is expected that macrophages can phagocytize small particles, such as GP-EtOH, more efficiently and in larger amounts than bigger particles, such as GP-EtOH/water and GP-water.

[0146] In-Vitro Phagocytosis by Macrophages

[0147] Phagocytosis by macrophages was evaluated for pure yeast glucan particles prepared using ethanol as solvent (GP-EtOH). First, a cell line J774A.1 (mouse macrophages) was cultivated using the culture method recommended by ATCC: The Gobal Bioresource Centre. The cells were cultivated by resuspending approximately 75 000 cells/well in 0.5 ml of FluoroBrite™ DMEM medium/well. Separately, the glucan particles were labeled using curcumin and Nile Red (GP/CC-EtOH and GP/NR-EtOH respectively). The labeled glucan particles were suspended in a concentration of 0.8 mg/ml of FluoroBrite™ DMEM medium and homogenized using an IKA® T10 basic ultra-turrax for 1 minute. The suspensions of labeled glucan particles were added into the wells containing the macrophages in volumes of 3, 6 and 9 μL/well. Macrophages without labeled glucan particles were used as a control group. The cells were incubated at 37° C., 5% CO.sub.2 and >93% relative humidity. The interaction of macrophages with labeled glucan particles was observed after 3, 5- and 24-hours using Olympus Fluoview FV1000 confocal system (405 nm and 550 nm excitation wavelength) and the scans were analyzed by Imaris (program for analysis of confocal scans).

[0148] The phagocytosis of few composites or dyed glucan particles was observed after 3 hours (FIG. 19). The macrophages show the highest phagocytosis activity after 5 hours. After 24 hours some macrophages saturated with microparticles swelled and died, but most of macrophages revealed phagocylosed the labeled glucan particles inside the cell body.

[0149] On the other hand, due to their large size, phagocytosis of GPs prepared from water and water/ethanol mixture (GP-water and GP-EtOH/water) by macrophages is expected to be limited and even cause macrophage's death.