FORUMULATION OF MONODISPERSE KINETICALLY FROZEN POLYMER MICELLES VIA EQUILIBRATION-NANOPRECIPITATION

Abstract

A formulation and method of micelle production including the steps of dissolving amphiphilic block copolymers in a mixed solvent comprising water and a non-aqueous co-solvent, conducting a single-step dialysis against water or saline in order to produce monodisperse kinetically frozen polymer micelles with DLS size polydispersities less than about 0.2 in aqueous conditions or conducting an evaporation process for removal of non-aqueous solvent content in order to produce monodisperse kinetically frozen polymer micelles with DLS size polydispersities less than about 0.2 in aqueous conditions.

Claims

1. A micelle formulation made by a method comprising the steps of: dissolving amphiphilic block copolymers in a mixed solvent comprising water and a non-aqueous co-solvent, and conducting a single-step dialysis against water or saline in order to produce monodisperse kinetically frozen polymer micelles with DLS size polydispersities less than about 0.2 in aqueous conditions or conducting an evaporation process for removal of non-aqueous solvent content in order to produce monodisperse kinetically frozen polymer micelles with DLS size polydispersities less than about 0.2 in aqueous conditions.

2. The micelle formulation of claim 1 wherein the amphiphilic block copolymers include strongly hydrophobic blocks having water-polymer interfacial tensions greater than about 15 mN/m at room temperature.

3. The micelle formulation of claim 1 wherein the amphiphilic block copolymers comprise styrene monomer units.

4. The micelle formulation of claim 1 wherein the mixed solvent includes an overall non-aqueous solvent composition between about 10% and about 90% w/w.

5. The micelle formulation of claim 4 wherein the non-aqueous solvent content is between about 10% and about 90% w/w of the mixed solvent.

6. The micelle formulation of claim 1 wherein the step of dissolving includes the step of forming micelles in a molecularly uniform solvent composition and allowing time for approximate equilibration.

7. A method of forming monodisperse kinetically frozen polymer micelles in aqueous conditions, the method comprising the steps of: dissolving amphiphilic block copolymers in a mixed solvent comprising water and a non-aqueous co-solvent to create a micelle solution, and conducting a single-step dialysis against water or saline or conducting an evaporation process for removal of non-aqueous solvent content.

8. The method of claim 7, wherein the amphiphilic block copolymers comprise styrene monomer units.

9. The method of claim 7, wherein the amphiphilic block copolymers comprise a poly(styrene) block and a poly(ethylene glycol) block.

10. The method of claim 7, wherein the step of dissolving includes the step of: dissolving PS-PEG block copolymers in a mixture of acetone and water.

11. The method of claim 7, wherein the step of dissolving includes the step of: sonicating the solution at a certain point during the equilibration process.

12. The method of claim 7, wherein the step of dissolving includes the step of: mechanically agitating the solution for at least two minutes during the equilibration process.

13. The method of claim 7, wherein the step of conducting a single-step dialysis against water or saline includes the step of: dialyzing the solution using a dialysis device against a water or saline reservoir for at least 10 minutes.

14. The method of claim 7, wherein the step of conducting a single-step dialysis against water or saline includes the step of: replacing the aqueous reservoir with fresh water or saline at least once during the process.

15. The method of claim 7, wherein the step of conducting a single-step dialysis against water or saline, wherein the aqueous reservoir is at least larger in volume than the initial micelle solution being dialyzed.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The above-mentioned and other features of this disclosure, and the manner of attaining them, will become more apparent and the disclosure itself will be better understood by reference to the following description of embodiments of the disclosure taken in conjunction with the accompanying drawings, wherein:

[0013] Unless otherwise stated, a reference to a compound or component includes the compound or component by itself, as well as in combination with other compounds or components, such as mixtures of compounds.

[0014] As used herein, the singular forms “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise.

[0015] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

[0016] FIG. 1A: Schematic of conventional formulation methods to forming micelles in aqueous environment of amphiphilic BCP with strongly hydrophobic block.

[0017] FIG. 1B: Schematic of proposed mixed solvent method to forming micelles in aqueous environment of amphiphilic BCP with strongly hydrophobic block.

[0018] FIG. 2A: DLS hydrodynamic diameter size distributions for 100% acetone composition post dialysis.

[0019] FIG. 2B: DLS hydrodynamic diameter size distributions for 80% acetone and 20% water mixture composition post dialysis.

[0020] FIG. 2C: DLS hydrodynamic diameter size distributions for 70% acetone and 30% water mixture composition post dialysis.

[0021] FIG. 2D: DLS hydrodynamic diameter size distributions for 60% acetone and 40% water mixture composition post dialysis.

[0022] FIG. 2E: DLS hydrodynamic diameter size distributions for 50% acetone and 50% water mixture composition post dialysis.

[0023] FIG. 2F: DLS hydrodynamic diameter size distributions for 40% acetone and 60% water mixture composition post dialysis.

[0024] FIG. 3: Surface pressure-area isotherm for micelle systems post dialysis formed at different initial solvent conditions.

[0025] FIG. 4A: DLS hydrodynamic diameter size distributions for batch 1 using direct dialysis formulation method.

[0026] FIG. 4B: DLS hydrodynamic diameter size distributions for batch 2 using direct dialysis formulation method.

[0027] FIG. 4C: DLS hydrodynamic diameter size distributions for batch 3 using direct dialysis formulation method.

[0028] FIG. 5: Surface pressure-area isotherms for three different batches using direct dialysis method.

[0029] FIG. 6A: DLS hydrodynamic diameter size distributions for batch 1 using the mixed solvent formulation method.

[0030] FIG. 6B: DLS hydrodynamic diameter size distributions for batch 2 using the mixed solvent formulation method.

[0031] FIG. 6C: DLS hydrodynamic diameter size distributions for batch 3 using the mixed solvent formulation method.

[0032] FIG. 7: Surface pressure-area isotherms for three different batches using the mixed solvent formulation method.

[0033] Corresponding reference characters indicate corresponding parts throughout the several views. Although the drawings represent embodiments of the present disclosure, the drawings are not necessarily to scale and certain features may be exaggerated in order to better illustrate and explain the present disclosure.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

[0034] The embodiments disclosed below are not intended to be exhaustive or limit the disclosure to the precise forms disclosed in the following detailed description. Rather, the embodiments are chosen and described so that others skilled in the art may utilize their teachings.

[0035] Experimental Procedures and Materials

[0036] Equilibration-Nanoprecipitation (ENP) Micelle Formulation Method. PS-PEG (10 mg) is dissolved in 2 mL mixture of acetone (Sigma-Aldrich) and Milli-Q purified water (18 MΩ.Math.cm resistivity) under sonication. The solution is then repeatedly vortexed and sonicated until the solution appears transparent. The solution is then stored under gentle rocking at room temperature for 24 h to allow for equilibration. Acetone is then removed by dialyzing the 2 mL mixture using Slide-A-Lyzer Mini Dialysis device (20 kDa MWCO) against Milli-Q-purified water for 24 h, replacing the water reservoir at 1, 2, 4 and 6 h time points. The water reservoir is 45 mL.

[0037] Direct Dialysis Micelle Formulation Method. The procedure is the same as for Equilibration-Nanoprecipitation procedure except that the polymer is dissolved into acetone only and not an acetone/water mixture.

[0038] Polymer Materials. The experiments detailed in this research report are done using PS(5.2 kDa)-PEG(5.5 kDa) purchased from Polymer Source, Inc.

[0039] Surface Pressure-Area (SP-A) Isotherms. The surface tension-area isotherms are measured using a KSV Nima Langmuir trough (51 cm×14.5 cm) with double symmetric barriers. The total surface area of the trough is 780 cm.sup.2, and the subphase volume is 750 mL. A filter paper or platinum Wilhelmy probe is used for surface tension measurements. Micelle samples are spread onto water using a Hamilton micro syringe. The compressions are done at a rate of 3 mm/minute. The temperature of the subphase is held constant at 25° C. using a circulating water bath.

[0040] Polymer Micelle Characterizations. The hydrodynamic diameters of the block copolymer micelles are measured at 25° C. by dynamic light scattering (DLS) using a Brookhaven ZetaPALS instrument. The scattering intensities are measured using a 659 nm laser at a scattering angle of 90°. The hydrodynamic diameters were calculated from the measured diffusion coefficients using the Stokes-Einstein equation. The results were averaged over 5 runs.

[0041] Results/Discussion

[0042] The difference between the directly dialyzing PS(5.2k)-PEG(5.5k) dissolved in acetone (10 mg/mL) and dialyzing micelle systems formed in acetone/water mixtures (10 mg/mL) was demonstrated using DLS and SP-A isotherms. The DLS data in FIG. 2A-FIG. 2F show that for micelle systems formulated from dialyzing dissolved polymer in acetone (“100% Acetone”) two size populations are formed which is also reflected in the high DLS polydispersity (PD) values listed in Table 1. The maximum intensities of the two populations occur at 18.8 nm and 118.0 nm respectively. A similar result is obtained for 20% Acetone system except that the smaller population shows a greater contribution to the size distribution and is centered around a slightly larger value of 22.4 nm. The 70% Acetone, 60% Acetone, and 50% Acetone systems (FIG. 2C, FIG. 2D, and FIG. 2E, respectively) show a narrower distribution with only one size population and smaller PD values. The average hydrodynamic diameter increases with decreasing acetone content which is expected due to the increase in interfacial tension between the core and solvent mixture at higher water contents.

TABLE-US-00001 TABLE 1 DLS effective diameter and PD for micelle systems post dialysis formed at various solvent conditions Initial Acetone Composition 100% 80% 70% 60% 50% 40% Effective 40.1 ± 0.7  28.1 ± 0.5  28.8 ± 0.2  29.6 ± 0.2  32.8 ± 0.1  52.7 ± 0.2  Diameter (nm) PD 0.327 ± 0.010 0.245 ± 0.003 0.068 ± 0.018 0.052 ± 0.015 0.041 ± 0.005 0.163 ± 0.003

[0043] FIG. 3 shows the surface pressure-area (SP-A) isotherms for the various micelle systems post dialysis. The 100% Acetone system produces an isotherm curve which falls much below the other initial solvent compositions until it reaches a similar maximum surface pressure as the 80% Acetone case of around 60 mN/m. The 40% and 50% Acetone cases can achieve nearly complete lowering of the surface tension at the air-water interface as the surface pressure approaches 72 mN/m at high surface concentrations. The demands of the polymer lung surfactant application are such that being able to achieve a surface pressure of greater than about 60 mN/m under high compression is required for proper functioning of the lungs. Thus, the importance of controlling the formulation size characteristics is relevant, and the direct dialysis method leaves room for improvement for this application.

[0044] The reproducibility of the direct dialysis method was tested by forming three batches using the same polymer (PS(5.2k)-PEG(5.5k)) and formation conditions (10 mg/mL polymer concentration). The direct dialysis method implies that the polymer is initially dissolved in 100% Acetone then is directly dialyzed. Table 2 and FIG. 4A-FIG. 4C show that there are differences among each batch in the effective diameter, PD, location of the two size populations, and the relative intensities of the smaller and larger populations.

TABLE-US-00002 TABLE 2 DLS effective diameter and PD for three different batches formed using the direct dialysis method. Sample Batch 1 Batch 2 Batch 3 Effective Diameter 32.7 ± 1.2  31.6 ± 1.8  35.5 ± 1.5  (nm) PD 0.208 ± 0.020 0.213 ± 0.015 0.222 ± 0.015

[0045] As illustrated in FIG. 5, SP-A isotherm data were collected for each of the three batches, shown in FIG. 4A-FIG. 4C. The differences in DLS data are reflected in the differences in the SP-A isotherm behavior which shows the importance of controlling size characteristics via the formulation procedure. Since the SP-A behavior is directly linked to efficacy, it is relevant that the isotherm behavior is reproducible for different batches.

TABLE-US-00003 TABLE 3 DLS effective diameter and PD for three batches formed using mixed solvent formulation method. Sample Batch 1 Batch 2 Batch 3 Effective Diameter 28.0 ± 0.1  29.4 ± 0.2  28.8 ± 0.2  (nm) PD 0.098 ± 0.015 0.121 ± 0.013 0.068 ± 0.018

[0046] The reproducibility of the mixed solvent method was tested by forming three batches at the 30% acetone solvent mixture condition. The DLS size data post dialysis are shown in Table 3 and FIG. 6A-6C. All three batches show a similar effective diameter and low PD. Batch 2 does show a small contribution of larger sized micelles but the maximum intensities for all in the range of 27-29 nm. The SP-A isotherm data in FIG. 7 reflects the similarity in size distributions as all three isotherms give a very similar shape.

[0047] This disclosure is proposing a new micelle formulation method using a mixed solvent approach with a single-step dialysis against water in order to produce monodisperse kinetically frozen polymer micelles in aqueous conditions. This method is an alternative to previous methods involving initial dissolution of BCPs in a non-aqueous co-solvent followed by either direct dialysis or slow addition of water as it initially forms equilibrium micelles in a mixed solvent environment as opposed to an environment containing solvent concentration gradients.

[0048] While this disclosure has been described as having an exemplary design, the present disclosure may be further modified within the spirit and scope of this disclosure. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains.