Dispersion of poloxamer-protein particles, methods of manufacturing and uses thereof

Abstract

The present invention relates to a method for preparing poloxamer-protein particles. It also relates to poloxamer-protein particles obtainable by this method, dispersion thereof, and their use in methods of encapsulation, in particular of microencapsulation.

Claims

1. A method for preparing a dispersion of poloxamer-protein particles, said method comprising the steps of: i) preparing an aqueous solution comprising a protein and a poloxamer; and ii) contacting the obtained solution with a water-miscible protein non-solvent in a sufficient amount to form a dispersion of poloxamer-protein particles, wherein the water-miscible protein non-solvent is glycofurol and wherein said poloxamer-protein particles are solid.

2. The method of claim 1, wherein the poloxamer is selected from the group consisting of poloxamer 188, 407, 338 and 237.

3. The method of claim 1, wherein the aqueous solution further contains a salt.

4. The method of claim 3, wherein the salt is present in a concentration ranging from 0.01 to 3M.

5. The method of claim 3, wherein the salt is NaCl.

6. The method of claim 1, wherein the protein is a therapeutic protein.

7. The method of claim 6, wherein the protein is an enzyme, a growth factor, a cytokine, a hormone, an antibody, an antibody fragment or a coagulation factor.

8. The method of claim 1, wherein the poloxamer-protein particles are recovered after centrifugation.

9. The method of claim 1, wherein the water-miscible protein non-solvent further contains a dissolved wall-forming polymer.

10. The method of claim 1, further comprising the step of iii) recovering the obtained poloxamer-protein particles.

11. A dispersion of poloxamer-protein particles obtainable according to claim 1.

12. The dispersion according to claim 11, wherein the water-miscible protein non-solvent, further contains a dissolved wall-forming polymer.

13. Poloxamer-protein particles obtainable according to claim 1.

14. The protein particles according to claim 13, of which median diameter ranges from 50 to 200 nm.

15. A method for encapsulating poloxamer-protein particles comprising: i) preparing a s/o/w emulsion containing: as a continuous phase, an aqueous phase, and as a discontinuous phase, an organic solvent containing dispersed poloxamer-protein particles according to claim 13 and a wall-forming polymer, said wall-forming polymer being soluble in said organic solvent, and insoluble in said continuous phase, and ii) solidifying said discontinuous phase, thereby forming encapsulated poloxamer-protein particles.

16. The method of claim 15, wherein the wall-forming polymer is a polymer of lactic acid, a copolymer of lactic acid and glycolic acid or a polyethylene glycol conjugated with a copolymer of lactic acid and glycolic acid or with a polymer of lactic acid.

17. The method of claim 16, wherein the wall-forming polymer is poly(D,L-lactide-co-glycolide) (PLGA) or PLGA-PEG-PLGA.

18. The method of claim 15, further comprising the step of iii) recovering the obtained encapsulated poloxamer-protein particles.

19. A method for encapsulating poloxamer-protein particles comprising: i) providing a dispersion of poloxamer-protein particles obtainable according to claim 1, wherein the water-miscible protein non-solvent further contains a dissolved wall-forming polymer or dispersing poloxamer-protein particles obtainable according to claim 1 in a solution of a wall-forming polymer dissolved in a solvent; ii) forming droplets of the dispersion of step i); and iii) solidifying said droplets.

20. The method of claim 19, wherein the droplets are solidified by extracting the solvent of the dispersion.

21. The method of claim 19, further comprising the step of iv) recovering the obtained encapsulated poloxamer-protein particles.

22. Encapsulated poloxamer-protein particles obtainable according to claim 15.

23. A pharmaceutical composition comprising encapsulated poloxamer-protein according to claim 22.

Description

FIGURES

(1) FIG. 1: Optimization of the lysozyme precipitation: experimental design. The table represents the values of each factor (U.sub.1, U.sub.2, U.sub.3) for the fifteen experiments.

(2) FIG. 2: Optimization of the lysozyme precipitation: spatial representation of the experimental design results.

(3) Numbers in the circles refer to the percentage of lysozyme recovered under a biologically active form, after precipitation and dissolution, for each experiment.

(4) FIG. 3: Evolution of lysozyme zeta potential when adding poloxamer 188 in solution.

(5) FIG. 4: In vitro release profile of lysozyme (meanSD) from PLGA microspheres (MS) without poloxamer 188 (2 batches twice) and with poloxamer 188 (example 7) (3 batches) and from PLGA-PEG-PLGA microspheres containing poloxamer 188 (example 9) (7 batches).

(6) FIG. 5: In vitro release profile of TGF beta 3 with poloxamer 188 from PLGA microspheres assessed by ELISA and biological activity of released TGF beta 3 assessed by a bioassay.

EXAMPLES

(7) Materials

(8) Lysozyme (chicken egg white) and its substrate: Micrococcus lysodeikticus, glycofurol (tetraglycol or -[(tetrahydro-2-furanyl)methyl]--hydroxy-poly(oxy-1,2-ethanediyl) and buffer compounds were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). TGF beta 3 was purchased from Abcys (Paris, France). TGF beta 3 ELISA kit was from R&D systems (Lille, France). Poloxamer was kindly supplied by BASF (Levallois-Perret, France). Capped 75/25 PLGA, provided by Phusis (Saint-Ismier, France), had a mean molecular weight of 27,000 Da (Polydispersity index, I=1.9) as determined by size exclusion chromatography (standard: polystyrene). PLGA50:50-PEG-PLGA50:50 (RGP t 50106, 10% PEG with 6,000 Da, i.v. 0.75) was purchased from Boehringer-Ingelheim (Ingelheim, Germany). Polyvinyl alcohol (Mowiol 4-88) was from Kuraray Specialities Europe (Frankfurt, Germany).

(9) Method for Preparing Protein Particles

(10) Firstly, the protein was dissolved in a non-buffered saline solution and then mixed with glycofurol at room temperature. 30 minutes later, the protein particles were recovered by centrifugation (10,000 g, 30 min, 4 C.). Mixing and centrifugation times of 30 min were selected in order to optimize the precipitation yield.

(11) Method for Preparing Protein-poloxamer Particles

(12) Firstly, protein and poloxamer 188 were codissolved in a saline solution. This protein-poloxamer solution was then mixed with glycofurol to prepare a protein-poloxamer dispersion. 30 minutes later, the protein-poloxamer particles were recovered by centrifugation (10,000 g, 30 min, 4 C.). Mixing and centrifugation times of 30 min were selected in order to optimize the precipitation yield.

(13) Optimization of the Precipitation Yield of a Protein Widely Available Such as Lysozyme

(14) To define the optimum conditions of precipitation of lysozyme, an experimental design was used. Preliminary studies (not shown) using the technique described revealed that three parameters influence protein precipitation yield: the ratio between the volumes of aqueous phase and of glycofurol, the ionic strength and the mass of protein. The presence of poloxamer does not affect the protein precipitation.

(15) To study these three variables, a Doehlert matrix was chosen. Fifteen experiments were carried out. Each experiment was repeated three times. The experimental domain for each factor is described as follows: ionic strength of the aqueous phase (U.sub.1): 0.01 to 0.59 M (5 levels), volume of the aqueous phase (U.sub.2): 25 to 155 l (7 levels) protein quantity (U.sub.3): 0.1 to 0.9 mg (3 levels).

(16) The volume of glycofurol was the complement for 1 ml of suspension.

(17) Experimental design is reported in FIG. 1.

(18) The measured response was the percentage of reversible lysozyme particles collected (precipitation yield) (FIG. 2). For its determination, the dispersion of protein particles in glycofurol was centrifuged, the supernatant eliminated and the pellet of protein particles dissolved in TRIS-HCl 0.01M buffer, pH 7.4 in order to determine its active mass. Biological activity of lysozyme was determined by measuring the turbidity change in a Micrococcus lysodeikticus bacterial cell suspension.

(19) Nemrod W software (2000, LPRAI, Marseille) was used for generation and exploitation of the statistical experimental design.

(20) Optimization of the Precipitation Yield of a Protein Scarcely Available (TGF-3)

(21) To define protein behaviour in the presence of salt and glycofurol, a rapid salt screening may be employed. An aqueous protein solution (50 l) containing the protein quantity wanted to be precipitated (from 20 to 1000 g) is deposited in the well of a 96-well plate. Then, saline solutions (50 l) with growing salt concentrations (from 0 to 3M) are added in each well. Finally, glycofurol (300 l) is adjoined and the absorbance is measured at 350 nm. An increase in the absorbance is related to the formation protein particles. The presence of poloxamer does not affect the formation of protein particles.

(22) Size of the Lysozyme-poloxamer 188 Particles

(23) The size of the lysozyme-poloxamer 188 particles dispersed in glycofurol was determined by light diffraction (Mastersizer 2000, Malvern Instruments, Worcestershire, UK).

(24) Zeta Potential

(25) To monitor the formation of a combination between lysozyme and poloxamer 188 in solution, lysozyme solution in acetic acid 0.1M containing increasing amount of poloxamer 188 were prepared. The protein concentration was maintained at 10 mg/ml. The corresponding zeta potential was measured as a function of protein to poloxamer ratio, using a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK).

(26) FTIR

(27) The secondary structure of lysozyme with or without poloxamer 188 inside the microspheres was determined by FTIR spectroscopy. Microspheres loaded with 5% w/w of protein (with respect to the amount of PLGA) were studied in order to detect the protein. FTIR studies were conducted with a Bucker IFS 28 equipped with a DTGS detector. 500 scans (4000-400 cm.sup.1) at 2 cm.sup.1 resolution were averaged to obtain each spectrum. Lyophilized microspheres were measured as KBr pellets (4-5 mg of microspheres per 200 mg of KBr). All spectra were analyzed in the amide I region (1700-1600 cm.sup.1) using the program OPUS version 2.0. In all cases, a linear baseline between 2000-1800 cm.sup.1 was subtracted. Infrared band position and the number of bands in the amide I region were calculated by Levenberg-Marquardt algorithm using the program OPUS. The secondary structure contents were calculated from the area of the individual assigned bands and their fraction of the total area in the amide I region.

(28) BSA-FITC Distribution in Microsphere

(29) Microspheres were loaded with BSA-FITC or with BSA-FITC/poloxamer 188 as described above. A laser scanning confocal imaging system (Olympus light microscope Fluoview FU300, Paris, France) was employed to observe BSA-FITC distribution in microspheres. Dry microspheres were dispersed on a glass slide; fluorescence images of cross-sections were taken by an optical sectioning. All the images were obtained using a single resolution.

(30) Preparation of the Microencapsuled Lysozyme-poloxamer 188 Particles

(31) PLGA microspheres loaded with lysozyme-poloxamer 188 particles were prepared using a s/o/w emulsion solvent extraction-evaporation process adapted from Pean et al. (Pean et al., 1998). Briefly, 0.9 mg of protein-poloxamer 188 particles (0.6% w/w with respect to the amount of PLGA) were prepared in the optimum conditions of precipitation and collected as described above. Then, they were carefully dispersed in an organic solution (2 ml; 3:1 methylene chloride:acetone) containing 150 mg of PLGA. The resulting organic suspension was then emulsified in a poly(vinyl alcohol) aqueous solution (90 ml, 4% w/v) maintained at 1 C. and mechanically stirred at 550 rpm for 1 min (heidolph RZR 2041, Merck Eurolab, Paris, France). After addition of 100 ml of deionized water and stirring for 10 min, the resulting s/o/w emulsion was added to deionized water (500 ml) and stirred further for 20 min to extract the organic solvent. Finally, the formed microparticles were filtered on a 5 m filter (HVLP type, Millipore S A, Guyancourt, France), washed five times with 100 ml of deionized water and freeze-dried. The average volume diameter and the size distribution of the resulting microspheres were evaluated using a Multisizer 3 Coulter Counter (Beckman Coulter, Roissy C D G, France).

(32) Lysozyme Encapsulation Efficiency

(33) Protein encapsulation yield was determined considering the biologically-active entrapped protein. Lysozyme PLGA microspheres (10 mg, 3 batches) were dissolved in 0.9 ml DMSO in silanized glass tube. After 1 hour, 3 ml of 0.01M HCl was added. The solution was left to stand for one more hour, and then incubated with Micrococcus lysodeikticus suspension for lysozyme activity determination.

(34) In Vitro Release Profile of Lysozyme from Microspheres

(35) The in vitro release profile of lysozyme from PLGA microspheres was determined by adding 500 L of TRIS-HCl 0.01M buffer, pH 7.4, containing 0.1% w/v BSA and 0.09% w/v NaCl to 10 mg of microspheres into centrifugation tubes. The tubes were closed, incubated in a water bath at 37 C. and agitated at 125 rpm. At determined time intervals, the tubes were centrifuged for 5 min at 3000 rpm. The 500 l of the supernatant were collected for analysis and replaced by fresh buffer. The percentage of biologically-active released lysozyme was measured by enzymatic assay.

(36) Results

EXAMPLE 1

Preparation of 900 g of Lysozyme Particles Coupled with Poloxamer 188

(37) 45 l of a solution containing 900 g of lysozyme and 9 mg of poloxamer 188 in NaCl 0.3 M are added to glycofurol to form a 1 ml suspension at room temperature. The complex particles are recovered by centrifugation (10,000 g, 30 min, 4 C.) and elimination of the supernatant.

EXAMPLE 2

Preparation of 300 g of Lysozyme Particles Coupled with Poloxamer 188

(38) 10 l of a solution containing 300 g of lysozyme and 3 mg of poloxamer 188 in NaCl 0.3 M are added to glycofurol to form a 1 ml suspension. The poloxamer-protein particles are recovered by centrifugation (10,000 g, 30 min, 4 C.) and elimination of the supernatant.

EXAMPLE 3

Preparation of 9 mg of Lysozyme Particles Coupled with Poloxamer 188

(39) 450 l of a solution containing 9 mg of lysozyme and 90 mg of poloxamer 188 in NaCl 0.3 M are added to glycofurol to form a 10 ml suspension. The complex particles are recovered by centrifugation (10,000 g, 30 min, 4 C.) and elimination of the supernatant.

EXAMPLE 4

Activity of Lysozyme Particles Coupled with Poloxamer 188 after Dissolution

(40) The pellet of poloxamer-protein particles described in Example 1 and 2 is dissolved in an appropriate solvent in order to determine its active mass (TRIS-HCl 0.01M buffer, pH 7.4). Biological activity of lysozyme is determined by measuring the turbidity change in a Micrococcus lysodeikticus bacterial cell suspension. More than 80% of the protein is recovered in an active form.

EXAMPLE 5

Size of the Lysozyme/Poloxamer 188 Particles

(41) The size of the poloxamer-protein particles suspended in glycofurol (described in example 1) was determined by light diffraction. The average particle size is about 100 nm with a narrow distribution.

EXAMPLE 6

Evolution of Lysozyme Zeta Potential in Presence of Poloxamer 188

(42) The interaction of lysozyme with increasing amount of poloxamer 188 in aqueous solution was controlled by zeta potential measurements. When poloxamer 188 was added, lysozyme zeta potential shifts from 15 to 5 mV (FIG. 3). This decrease of the net surface charge on lysozyme may be considered as the result of molecular combination of lysozyme with poloxamer 188.

EXAMPLE 7

Microencapsulation of the Lysozyme/Poloxamer 188 Particles in PLGA Microspheres

(43) Preparation of the PLGA Microspheres Loaded with Lysozyme/Poloxamer 188 Particles

(44) Lysozyme-loaded PLGA microspheres were prepared using a solid-in-oil-in-water (s/o/w) emulsion solvent extraction-evaporation process. Briefly, 900 g of protein particles coupled with poloxamer 188 (0.6% protein w/w with respect to the amount of PLGA) were prepared as described in example 1 and collected as described above. Then, they were carefully dispersed in an organic solution (2 ml; 3:1 methylene chloride:acetone) containing 150 mg of uncapped 75/25 PLGA (mean molecular weight of 27,000 Da, polydispersity index of 1.9). The resulting organic suspension was then emulsified in a poly(vinyl alcohol) aqueous solution (90 ml, 4% w/v) maintained at 1 C. and mechanically stirred at 550 rpm for 1 min. After addition of 100 ml of deionized water and stirring for 10 min, the resulting s/o/w emulsion was added to deionized water (500 ml) and stirred further for 20 min to extract the organic solvent. Finally, the formed microparticles were filtered on a 5 m filter washed with 500 ml of deionized water and freeze-dried. The resulting microspheres had an average volume diameter of about 60 m.

(45) In Vitro Release Study

(46) The in vitro release profile of lysozyme from PLGA microspheres was determined by adding 500 L of TRIS-HCl 0.01M buffer, pH 7.4, containing 0.1% w/v BSA and 0.09% w/v NaCl to 10 mg of microspheres into eppendorf tubes, incubated in a water bath at 37 C., agitated at 125 rpm. At determined time intervals, the tubes were centrifuged for 5 min at 3000 rpm. The 500 l of the supernatant were collected for analysis and replaced by fresh buffer. The percentage of released biologically-active lysozyme was measured by enzymatic assay. The effect of poloxamer 188 on the in vitro release profile is shown in FIG. 4.

EXAMPLE 8

(47) To characterize the encapsulated protein-poloxamer particles of example 7, the secondary structure of encapsulated lysozyme was characterized by Fourier transform infrared (FTIR) spectroscopy (Table 1). The protein amide I IR infrared spectra were analyzed for the secondary structure composition and the secondary structure was quantified. Firstly, FTIR spectroscopy demonstrated that few protein structural perturbations were induced by the encapsulation and by the presence of poloxamer. Only minor spectral changes occurred in the amide I band (1700-1600 cm.sup.1), which is sensitive to protein structure. Analysis of the spectra by Gaussian curve-fitting revealed few change in the -helical and in -sheet content; the secondary structure was within the error the same as for the powder prior to encapsulation.

(48) TABLE-US-00001 TABLE 1 Secondary structure of lysozyme under various conditions (as determined by FTIR spectroscopy) Conditions -helix -sheet Native lysozyme (powder) 24.5 37.9 Lysozyme in PLGA microspheres 24.9 34.9 Lysozyme + poloxamer188 in PLGA 23.9 20.2 microspheres

EXAMPLE 9

Microencapsulation of the Lysozyme/Poloxamer 188 Particles in PLGA-PEG-PLGA Microspheres (150 mg Microsphere Batch)

(49) PLGA-PEG-PLGA (10% PEG 6 000 Da) was employed. The same procedure as example 7 was followed to prepare the lysozyme-loaded PLGA-PEG-PLGA microspheres except that the mechanically stirring was adjusted to 850 rpm to obtain 60 m microspheres. The effect of the polymer type on the in vitro release profile is shown in FIG. 4.

EXAMPLE 10

BSA-FITC/Poloxamer 188 Distribution in PLGA-microspheres

(50) Preparation of the BSA-FITC/Poloxamer 188 Particles

(51) 45 l of a solution containing 900 g of BSA-FITC and 9 mg of poloxamer 188 in NaCl 0.3 M are added to glycofurol to form a 1 ml suspension at room temperature. The complex particles are recovered by centrifugation (10,000 g, 30 min, 4 C.) and elimination of the supernatant.

(52) Preparation of the PLGA Microspheres Loaded with Lysozyme/Poloxamer 188 Particles

(53) BSA-FITC loaded PLGA microspheres were prepared according to the procedure described in example 7. Briefly, 900 g of protein particles coupled with poloxamer 188 (0.6% protein w/w with respect to the amount of PLGA) were carefully dispersed in an organic solution (2 ml; 3:1 methylene chloride:acetone) containing 150 mg of PLGA. The resulting microspheres had an average volume diameter of about 60 m.

(54) Confocal Analysis

(55) A confocal image system was employed to observe BSA-FITC distribution in microspheres. Dry microspheres were dispersed on a glass slide; fluorescence images of cross-sections were taken by an optical sectioning. The presence of poloxamer revealed a better protein distribution inside the microspheres.

EXAMPLE 11

Preparation of 125 ng of TGF-3 (Transforming Growth Factor-beta 3) particles coupled with poloxamer 188

(56) 100 l of a solution containing 125 ng of TGF-3 and 1.25 mg of poloxamer 188 in 10 mM phosphate buffer (pH7, NaCl 2M) were added to 740 mg glycofurol. The complex particles was recovered by centrifugation (10,000 g, 30 min) and elimination of the supernatant. The presence of poloxamer 188 did not affect the precipitation yield as determined by ELISA quantification.

EXAMPLE 12

Preparation of 50 g of TGF 3 Particles Coupled with Poloxamer 188

(57) 1.220 ml of solution containing 50 g of TGF beta 3 and 3 mg of poloxamer 188 in 10 mM phosphate buffer (pH 7.4, NaCl 2M) are added to 8.68 g of glycofurol; After 30 min, the particles are recovered by centrifugation (10,000 g, 30 min, 4 C.) and elimination of the supernatant.

EXAMPLE 13

Preparation of 50 mg PLGA Microspheres Batch Loaded with Protein/Poloxamer 188 Particles

(58) Protein particles coupled with poloxamer 188 were carefully dispersed in an organic solution (670 l; 3:1 methylene chloride:acetone) containing 50 mg of capped 75/25 PLGA (mean molecular weight of 27,000 Da, polydispersity index of 1.9). The resulting organic suspension was then emulsified in a poly(vinyl alcohol) aqueous solution (30 ml, 4% w/v) maintained at 1 C. and mechanically stirred at 550 rpm for 1 min. After addition of 33 ml of deionized water and stirring for 10 min, the resulting s/o/w emulsion was added to deionized water (167 ml) and stirred further for 20 min to extract the organic solvent. Finally, the formed microparticles were filtered on a 5 m filter washed with 500 ml of deionized water and freeze-dried. The resulting microspheres had an average volume diameter of about 60 m.

EXAMPLE 14

Microencapsulation of TGF 3

(59) TGF beta 3 was precipitated as mentioned in example 12.

(60) In parallel, HAS (Human Albumin Serum) particles was prepared: 10 l of NaCl 0.3 M containing 250 g HAS are added to 1.077 g of glycofurol. After 30 min, the HAS particles were recovered by centrifugation (10 000 g, 30 min, 4 C.) and elimination of the supernatant.

(61) The HAS particles were dispersed in 3100 l polymer organic solution (composition described in example 13), particles of poloxamer-TGF beta 3 were dispersed in the same conditions. Both suspensions were mixed and used to prepare PLGA microspheres as described in example 13.

EXAMPLE 15

TGF-3 (Transforming Growth Factor-Beta3) Profile Release

(62) TGF beta 3 was encapsulated as mentioned in example 14.

(63) The in vitro release profile of TGF beta 3 from PLGA microspheres was determined by 500 l of PBS buffer, pH, containing 1% w/v BSA to 10 mg of microspheres into eppendorf tubes, incubated in a bath at 37 C., agitated at 125 rpm. The 500 l of the supernatant were collected for analysis and replaced by fresh buffer. The percentage of released TGF beta3 was determined by ELISA and the biological activity of released TGF beta 3 was evaluated by a bioassay (FIG. 5).

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