Maintaining Hydrophobic Drug Supersaturation in a Micelle Corona

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

Maintaining Hydrophobic Drug Supersaturation in a Micelle Corona Reservoir Ziang Li,† Theodore I. Lenk,† Letitia J. Yao,‡ Frank S. Bates,*,† and Timothy P. Lodge*,†,‡ †

Department of Chemical Engineering and Materials Science and ‡Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: Two poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PND) statistical copolymers and a series of three poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-polystyrene (PND-b-PS-C12) diblock polymers were synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization, in which the molecular weight of the thermoresponsive PND corona block was held constant while the polystyrene core block length was varied. The corona thickness and density of the micelles in phosphate-buffered saline (PBS, pH = 6.5) were quantified by a combination of dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). Two hydrophobic model drugs, phenytoin and nilutamide, were used to examine the drug−polymer interactions in aqueous solution. Intermolecular interactions between the diblock polymer micelle corona and both drugs were revealed by 2D 1H nuclear Overhauser effect spectroscopy (NOESY). The drug−polymer “binding” strength, quantified by diffusion ordered NMR spectroscopy (DOSY), increased as corona density of the diblock polymer micelle increased for both drugs. The in vitro dissolution of the amorphous solid dispersions was systematically investigated as a function of drug type, drug loading, and the solution-state assembly of the polymers by using either a selective or nonselective spray drying solvent. Forming micelles prior to spray drying significantly enhanced phenytoin dissolution and supersaturation maintenance for the diblock polymers by storing the drug molecules in the corona.

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INTRODUCTION A major challenge in the development of orally administered pharmaceuticals is the poor aqueous solubility of hydrophobic drugs, which limits bioavailability and therapeutic benefit.1,2 This challenge has inspired the research and development of drug formulation approaches for improving drug solubility and therefore bioavailability.3 One particularly effective strategy is the use of amorphous solid dispersions (ASDs), a widely used therapeutic formulation composed of an active pharmaceutical ingredient and a matrix of polymer excipients.4 In the solid state, these pharmaceutically inactive excipients stabilize the drug in a metastable amorphous state rather than the thermodynamically stable crystalline form.5,6 Upon dissolution in the gastrointestinal (GI) fluid, ASDs may greatly enhance bioavailability because the amorphous drug is significantly more soluble than its crystalline counterpart.5,7 However, drug nucleation and subsequent crystal growth would naturally occur from the supersaturated solution due to the strong thermodynamic driving force.8 Thus, the polymer excipients must inhibit drug nucleation and/or crystal growth to sustain the high drug supersaturation over the pharmaceutically relevant time scale.9 The choice of the polymer excipient in an ASD determines the ultimate dissolution behavior of the drug in aqueous solution. A variety of polymer vehicles have been used to © XXXX American Chemical Society

achieve rapid dissolution and supersaturation maintenance, yet there is not a single mechanism that can be universally applied to all drug candidates.10 For example, a widely used excipient, hydroxypropyl methylcellulose acetate succinate (HPMCAS), is particularly effective in maintaining the supersaturation of various drugs.11 However, for some rapidly crystallizing hydrophobic drugs, HPMCAS cannot sustain the high drug supersaturation due to an inability to inhibit nucleation and crystal growth.11−13 Over recent decades, block polymers with core−corona micellar structures in aqueous solution have proven to be effective carriers for the delivery of hydrophobic drugs.14 The amphiphilic character of the block polymer micelles enables a high drug uptake in the hydrophobic inner core, surrounded and stabilized by the hydrophilic corona.15 A major challenge for such a core-loading strategy is the low permeability of the entrapped drug molecules through the gastrointestinal membrane,16 which is just as important as the aqueous solubility for enhanced bioavailability of orally administered drugs.17 Although numerous external stimuliresponsive mechanisms have been developed to trigger the controlled release of the entrapped drugs, these approaches Received: October 28, 2017 Revised: December 18, 2017

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DOI: 10.1021/acs.macromol.7b02297 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules

Scheme 1. Synthesis of Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PND-C12) and Poly(N-isopropylacrylamideco-N,N-dimethylacrylamide)-b-polystyrene (PND-b-PS-C12) via RAFT Polymerization

as a function of drug type, drug loading, and solution-state assembly of the polymers by using either a selective or nonselective solvent prior to spray drying. The important influence of micelle assembly on drug−polymer interactions and subsequent in vitro dissolution performance in the corona loading strategy revealed by this study can contribute to the rational design of polymer micelles as effective therapeutic formulations.

cannot be easily realized in the human GI tract for oral drug delivery applications.18,19 To resolve this problem, recent work has shown that hydrophobic drug molecules can be sequestered in the micelle corona, rather than the core, for enhanced solubility and supersaturation maintenance.20−22 Analogous to the mechanism of reverse-phase liquid chromatography, the micelle corona serves as a “reservoir” to stabilize a high drug supersaturation by partitioning these hydrophobic drug molecules into a less hydrophilic polymerrich phase.20,21 In this study, we systematically investigate the effect of the solution state assembly on drug−polymer interactions and the subsequent in vitro dissolution of the ASDs. Three poly(Nisopropylacrylamide-co-N,N-dimethylacrylamide)-b-polystyrene (PND-b-PS-C12) diblock polymers with an identical thermoresponsive PND corona block and a hydrophobic polystyrene core block of different molecular weights were synthesized by reversible addition−fragmentation chain transfer (RAFT) polymerization along with two PND statistical copolymer controls with either dodecyl (C12) or ethyl (C2) chain ends (Scheme 1 and Figure S1). It has been shown that N-isopropylacrylamide (NIPAm)-based polymers, synthesized by RAFT polymerization using an alkyl chain transfer agent (CTA), self-assembled into micelles in aqueous solution with a C12 core but maintained their random coil conformation with a C2 chain end.21,23,24 Thus, in this study, the properties of the micelles (i.e., size, corona density, and thickness) were systematically investigated by varying the length of the hydrophobic polystyrene block. The solution-state properties of the statistical copolymers and diblock polymers in phosphate-buffered saline (PBS, pH = 6.5) were characterized by dynamic light scattering, static light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy. Phenytoin (PTN), an anticonvulsant, and nilutamide (NID), a nonsteroidal antiandrogen, were selected as the model drugs for this study. Although both drugs suffer low aqueous solubility, PTN is not only more hydrophobic but also a more rapid crystallizer than NID.25,26 Drug−polymer intermolecular interactions in aqueous solution, with the polymers as either free chains or micelles, were investigated by 2D 1H nuclear Overhauser effect spectroscopy (NOESY), and their corresponding “binding” strength was further quantified by 2D 1H diffusion ordered spectroscopy (DOSY, also known as pulsed-field-gradient NMR). The polymer free chains or micelles were then spray dried with the two model drugs, and the in vitro dissolution of the ASDs was systematically studied

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EXPERIMENTAL SECTION

Materials. All chemicals were purchased from Sigma-Aldrich and used as received unless otherwise noted: phenytoin (PTN, ≥99%), nilutamide (NID), N-isopropylacrylamide (NIPAm, ≥99%), N,Ndimethylacrylamide (DMA, 99%), styrene (S, ≥99%), 2,2-azobis(2methylpropionitrile) (AIBN, 98%), 1,4-dioxane (anhydrous, 99.8%), tetrahydrofuran (THF, ≥99.9%), hexanes (99.9%, Fisher), methanol (HPLC grade, 99.9%), acetonitrile (HPLC grade, 99.9%), water (HPLC grade), dimethyl sulfoxide-d6 (DMSO-d6, 99.9 atom % D, Cambridge Isotope Laboratories, Inc.), deuterium oxide (D2O, 99.9 atom % D, Cambridge Isotope Laboratories, Inc.), chloroform-d (CDCl3, 99.8 atom % D + 0.05% v/v TMS, Cambridge Isotope Laboratories, Inc.), and 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP, 98 atom % D). Hydroxypropyl methylcellulose acetate succinate (HPMCAS) was provided by the Dow Chemical Company (AFFINISOL, Mn = 60 kg/mol, Đ = 2.4, 10 wt % acetate, 11 wt % succinate). The RAFT chain-transfer agent (CTA), S-1dodecyl-S-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (C12), was synthesized according to procedures adapted from the literature.27 Another CTA, 4-cyano-4-[(ethylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (C2), was purchased from Strem Chemicals, Inc., and used as received. Phosphate buffered saline (PBS, pH = 6.5) was prepared using Milli-Q water with 82 mM sodium chloride (≥99.9%, Fisher), 20 mM sodium phosphate dibasic (99.5%), and 47 mM potassium phosphate monobasic (≥99%, J.T. Baker). Fasted simulated intestinal fluid powder (FaSSIF) was purchased from Biorelevant. Polymer Synthesis and Characterization. A series of three poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-polystyrene (PND-b-PS-C12) diblock polymers were synthesized by a twostep RAFT polymerization using the same macro-CTA (i.e., PNDC12), according to procedures adapted from the literature,28−30 as shown in Scheme 1. A second statistical copolymer, PND-C2, targeting the same molecular weight and NIPAm mole fraction of the PND-C12, was synthesized using a different CTA (C2) according to the protocol shown in Figure S1. The number-average molecular weight (Mn) and dispersity (Đ) of the synthesized polymers are summarized in Table 1. These polymers are designated as PND34-C2, PND34-C12, PND34-b-PS2-C12, PND34-b-PS9-C12, and PND34-b-PS14C12, with the subscripts denoting the Mn of the corresponding block in kg/mol and the suffix denoting the type of chain end based on the CTA used. B


Article

Macromolecules Table 1. Polymer Characteristicsa sample PND34-C2 PND34-C12 PND34-b-PS2-C12 PND34-b-PS9-C12 PND34-b-PS14-C12

Mn of PNDb (kg/mol) Mn of PSc (kg/mol) 34 34 34 34 34

2.0 8.8 14

became transparent. Finally, the solution was lyophilized to produce micelles with frozen polystyrene cores in the solid state. Dynamic Light Scattering (DLS). DLS measurements were performed using a Brookhaven BI-200SM system with a 637 nm laser at various temperatures. PND statistical copolymers and lyophilized PND-b-PS-C12 micelles were dissolved in PBS solution (pH = 6.5). The solutions were passed through 0.2 μm filters into clean glass tubes and placed in a temperature-controlled index-matching bath for measurements. Intensity correlation functions were acquired at five scattering angles ranging from 60° to 120° in 15° increments, with an acquisition time of 10 min per angle. The hydrodynamic radius (Rh) distributions of the samples were obtained by fitting the correlation functions to the Laplace inversion routine REPES.33 The correlation functions were also fit to a second-order cumulant model, and the mutual diffusion coefficient Dm was extracted from a linear fit of the mean decay rates (Γ ) vs the square of the scattering vector (q2) (see Supporting Information for details). The mean hydrodynamic radius (Rh) was then obtained via the Stokes−Einstein relationship. The dispersity (μ2/Γ 2 ) is reported as the average value from the secondorder cumulant model fits at the five scattering angles. Small-Angle X-ray Scattering (SAXS). SAXS experiments were carried out on the DND-CAT 5-ID-D beamline at the Argonne National Laboratory Advanced Photon Source, with an X-ray wavelength of 0.7293 Å. Sample solutions were transferred into 1.5 mm quartz capillary tubes and sealed with parafilm to prevent solvent evaporation. 2D scattering patterns were collected at 25 °C on a Rayonix CCD area detector with an exposure time of 3 s and integrated to obtain 1D data of intensity (arbitrary units) vs scattering vector q (Å−1). The PBS solvent background was subtracted from the 1D data of the polymer solution, and the resulting intensity data were analyzed and fit to models built in the SasView software (version 4.0.1). 2D 1H NMR Sample Preparation. A stock solution of deuterated PBS solution consisting of 82 mM sodium chloride, 20 mM sodium phosphate dibasic, and 47 mM potassium phosphate monobasic in D2O was prepared. TSP was added directly to the solution at a concentration of 50 or 100 μg/mL for study of PTN or NID, respectively. PND statistical polymers were dissolved directly in the solution at various concentrations. Lyophilized PND-b-PS-C12 micelles, prepared by the cosolvent method, were dissolved in the solution to achieve equal PND concentrations as the PND statistical polymers. PTN or NID was then dissolved in DMSO-d6 at a concentration of 5 or 10 mg/mL, respectively. The DMSO-d6 solution was pipetted into the prepared polymer/D2O buffer mixture at 1% DMSO-d6/D2O (v/v) to achieve a final PTN or NID concentration in the deuterated PBS solution of 50 or 100 μg/mL for each study, respectively. Nuclear Overhauser Effect Spectroscopy (NOESY). NOESY experiments were performed on a Bruker Avance III HD (500 MHz) spectrometer with a 5 mm Prodigy TCI cryoprobe at 25 °C, using the pulse sequence “noesyesgpph” that employed excitation sculpting with gradients for water suppression.34 The data were collected with 2048 data points × 256 increments × 8 scans. Unless otherwise noted, the relaxation delay and mixing time were set to be 3 and 1.5 s, respectively. The spectra were analyzed with the Topspin 3.5 software package (aligned with TSP protons at 0 ppm). Diffusion Ordered Spectroscopy (DOSY). DOSY experiments were performed on a Bruker Avance III (500 MHz) spectrometer with a 5 mm Broadband Fluorine Observe (BBFO) probe at 27 °C. For PTN, the “ledbpgp2s” pulse sequence (longitudinal eddy current delay experiment using bipolar gradients acquired in 2D) was applied with a relaxation delay of 3 and 32 scans in total. For NID, the “stebpgp1s19” pulse sequence (stimulated echoes using bipolar gradient with 1 spoil gradient for diffusion and using 3−9−19 pulse sequence with gradients for water suppression) was applied with a relaxation delay of 3 and 128 scans in total. The experimental data were analyzed with the Topspin 3.5 software package. The diffusion coefficients of the model drug (i.e., PTN or NID), TSP, and polymer in the solution were obtained from fits to eq 1:

Đ 1.04 1.04 1.09 1.16 1.21

a

All PNDs have 67 mol % NIPAm (based on 1H NMR spectroscopy). Based on SEC-MALS data (using weight-averaged dn/dc = 0.0953 mL/g). cBased on 1H NMR spectroscopy. b

PND34-C 12 macro-CTA, a statistical copolymer, was first synthesized via RAFT polymerization by using NIPAm and DMA as the comonomers. NIPAm (20.0 g, 0.177 mol), DMA (7.50 g, 0.0757 mol), CTA S-1-dodecyl-S-(α,α′-dimethyl-α″-acetic acid) trithiocarbonate (C12) (0.307 g, 0.842 mmol), and recrystallized AIBN (0.0138 g, 0.0840 mmol) were combined with 126 mL of 1,4dioxane in a Schlenk flask. Three freeze−pump−thaw cycles were performed, after which the flask was filled with argon and removed from the vacuum line. The reactants were stirred at 72 °C for 4 h, and then the reaction was quenched by placing the flask into an ice bath. The reaction product was dissolved with extra THF and precipitated by dropwise addition to ice-cold hexanes, followed by filtration. The polymer was redissolved in THF and precipitated a second time into hexanes, filtered, and dried in a vacuum oven at approximately 50 °C for 24 h. PND-b-PS-C12 diblock polymers were synthesized following a similar procedure, using the PND34-C12 macro-CTA. PND34-C12 (3.50 g, 0.104 mmol), styrene (2.69 g, 0.0259 mol), and recrystallized AIBN (1.7 mg, 0.010 mmol) were combined with 10 mL of THF in a Schlenk flask. After three freeze−pump−thaw cycles, the flask was filled with argon. The reaction was allowed to run at 80 °C for 18 h and then quenched by placing the flask in an ice bath. The polymer was dissolved in extra THF and precipitated dropwise in ice-cold hexanes, followed by filtration; this procedure was repeated. The final product was dried in a vacuum oven at approximately 50 °C for 24 h and stored in a desiccator under reduced pressure until use. Chemical composition was assessed by 1H nuclear magnetic resonance (NMR) spectroscopy on a Bruker Avance III HD (500 MHz) spectrometer (equipped with a 5 mm Prodigy TCI cryoprobe) in CDCl3 at 25 °C. The compositions of NIPAm for PND34-C2 and PND34-C12 were both 67 mol % based on 1H NMR analysis shown in the Supporting Information (Figure S2). The polystyrene compositions for PND-b-PS-C12 diblock polymers were also calculated from 1 H NMR spectra (Figure S2). The molecular weight distributions of the polymers were determined by size-exclusion chromatography (SEC) on an Agilent 1260 Infinity series system with a Wyatt Dawn Heleos II multiangle laser light scattering (MALS) detector and a Wyatt Optilab T-rEX refractive index detector with THF as the eluent at 25 °C. A weight-averaged refractive index increment (dn/dc) of 0.0953 mL/g was used for PND statistical copolymers in THF based on the composition obtained from the 1H NMR spectra and literature dn/dc values of PNIPAm (0.107 mL/g) and PDMA (0.068 mL/g) in THF.31,32 SEC traces of the polymers are shown in Figure S3. Polystyrene molecular weight for the PND-b-PS-C12 diblock polymers was determined based on the PND molecular weight from SEC and the polystyrene composition from 1H NMR. Micelle Formation. PND-b-PS-C12 diblock polymer micelles were prepared using the cosolvent method. PND-b-PS-C12 was dissolved in THF at 10 mg/mL. An equal volume of Milli-Q water was titrated dropwise into the THF solution while stirring. Typically, the transparent solution became translucent after addition of approximately 15% of the total volume of Milli-Q water, indicating the formation of micelles. The solution was transferred into a dialysis bag and dialyzed against a large amount of DI water for 3 days to remove THF. The solution was then passed through a 0.45 μm filter to remove dust and residual large aggregates, after which the solution C


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Macromolecules ⎛ I(g ) ⎞ δ⎞ 2 ⎛ ln⎜ ⎟ = −(γgδ ) D⎜Δ − 3 ⎟ ⎝ ⎠ ⎝ I(0) ⎠

(1)

where g is the applied gradient strength, varying from 2 to 95% of the maximum strength, I(g) is the recorded echo intensity, γ is the gyromagnetic ratio of 1H, δ is the length of the gradient pulse (set to 2 ms), and Δ is the diffusion time (set to 100 ms). Spray Drying. The model drug (i.e., PTN or NID) and polymer were codissolved at 10 or 25 wt % drug loading in either THF or MeOH to reach a total solids concentration of 2 wt %. The solution was transferred to a 20 mL syringe and injected into a mini spray dryer (Bend Research) with the following processing parameters: solution flow rate of 0.65 mL/min, nitrogen gas flow rate of 12.8 standard liters per minute (SLPM), and inlet temperature of 72 °C. The outlet temperature was approximately 25 °C. The spray-dried dispersions (SDDs) were collected on a filter paper, transferred into a scintillation vial, and dried in a vacuum oven at ambient temperature overnight for at least 12 h. Prepared SDDs were stored in a desiccator at reduced pressure until use. In Vitro Dissolution Assay. The in vitro dissolution tests were performed in duplicate at 37 °C using the microcentrifuge method under nonsink conditions.11,35 SDDs were weighed into 2.0 mL conical microcentrifuge tubes. PBS solution (pH = 6.5) with 0.5 wt % of FaSSIF was added to the SDDs to reach a desired drug concentration of 1000 μg/mL (i.e., either 7.2 mg of SDD with 25 wt % drug loading or 18.0 mg of SDD with 10 wt % drug loading were dissolved in 1.8 mL of PBS/FaSSIF solution). The microcentrifuge tubes were vortexed for 1 min and placed in a VWR Analogue dry heating block at 37 °C. At 4, 10, 20, 40, 90, 180, and 360 min, the dissolution medium was centrifuged for 1 min at 13 000 rpm, and then a 50 μL aliquot of the supernatant was collected in a HPLC vial and diluted with 250 μL of MeOH. The microcentrifuge tubes were then vortexed for an additional 30 s, returned to the heating block, and held at 37 °C until the next time point. The samples were outside the heating block during the aliquot collection process for approximately 2 min. Solubilized drug concentration (i.e., PTN or NID) in the supernatant was analyzed by reverse-phase high-performance liquid chromatography (HPLC) using a 1260 Infinity Quaternary liquid chromatograph system with a reverse-phase EC-C18 column (Poroshell 120, 4.6 × 50 mm, 2.7 μm, Agilent) and an Infinity multiple wavelength UV−vis detector. The mobile phase was 60:40 and 50:50 (v/v) of water/acetonitrile solution at a flow rate of 1 mL/ min for SDDs containing PTN and NID, respectively. For both drugs, calibration curves were developed at a wavelength of 240 nm to determine the sample concentration. Equilibrium solubility of the drug was determined by dissolving an excess amount of crystalline PTN or NID in the PBS/FaSSIF solution and equilibrating at 37 °C for 48 h. The sample was centrifuged for 1 min at 13 000 rpm to remove undissolved drug, and the supernatant concentration was analyzed by HPLC using the same conditions as described above.

Figure 1. Hydrodynamic radius distributions at 10 mg/mL in PBS solution (pH = 6.5, T = 25 °C) at 90° scattering angle. The diblock solutions were prepared by the cosolvent method and lyophilized before dissolution.

Table 2. Mean Hydrodynamic Radius and Dispersity of Polymers in PBS Solution (pH = 6.5)a T = 25 °C PND34-C2 PND34-C12 PND34-b-PS2-C12 PND34-b-PS9-C12 PND34-b-PS14-C12

T = 37 °C

Rh (nm)

μ2/Γ2

Rh (nm)

μ2/Γ2

4.9 13.0 20.2 24.4 29.0

0.19 0.09 0.09 0.02 0.01

5.7 15.9 19.7 22.7 27.0

0.12 0.03 0.03 ≈0 ≈0

a

The diblock polymer solutions were prepared by the cosolvent method and lyophilized before dissolution.

indicating that PND34-C2 and PND34-C12 exhibited free chain and micelle conformations in PBS solution, respectively. This result is consistent with previous studies in our group and by others that the dodecyl RAFT end group is sufficiently hydrophobic to facilitate micellar self-assembly of NIPAmbased homopolymers in aqueous solution.21,23,24 The diblock polymer micelles with frozen polystyrene cores (i.e., prepared by the cosolvent method) exhibited narrowly distributed hydrodynamic radii upon dissolution in PBS solution. At the same temperature, the mean Rh value increased with increasing PS chain length for the diblock micelles, mostly due to the increased core size. As the temperature increased from 25 to 37 °C, values of both Rh and dispersity (μ2/Γ2) of the diblock micelles decreased. This is attributed to the contraction of the PND corona chains as the temperature approached the lower critical solution temperatures (LCSTs, 40−41 °C) of the corona blocks. SAXS measurements were conducted to determine the core radius (Rc) and aggregation number (Nagg) of the polymer micelles (summarized in Table 3). Figure 2 shows the SAXS

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RESULTS Micellization of Polymer. The polymers investigated in this work all exhibited cloud points above body temperature at 40−41 °C due to the copolymerization of the more hydrophilic monomer DMA (33 mol %) with NIPAm (67 mol %) (see Figure S4 and Table S1 in the Supporting Information). Hydrodynamic radius distributions of the polymers in PBS solution (pH = 6.5, T = 25 °C) are shown in Figure 1. The corresponding mean hydrodynamic radius (Rh) and dispersity (μ2/Γ2) at both 25 and 37 °C were calculated by fitting to a second-order cumulant model and are summarized in Table 2. Representative autocorrelation functions and plots of decay rate Γ versus q2 are shown in Figure S5. The two PND statistical copolymers with the same molecular weight but different RAFT end groups showed very different Rh values upon direct dissolution in PBS solution,

Table 3. Micelle Characteristics Based on Fitting SAXS Results at 25 °C sample

Rc (nm)

PND34-C12 PND34-b-PS2-C12 PND34-b-PS9-C12 PND34-b-PS14-C12

2.0 ± 0.1 5.3 ± 0.1 9.1 ± 0.2

Nagga,b

Lcoronac (nm)

± ± ± ±

13.0 18.2 19.1 19.9

12 11 45 139

1 2 5 14

ρcorona (mg/mL) 74 18 42 79

± ± ± ±

13 4 9 16

Nagg = number of arms (f) of the Gaussian star polymer model fit for PND34-C12. bNagg of the diblock polymers is estimated by assuming a dry core. cLcorona = Rh − Rc. a

D


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as estimated by the number of arms (f) from the star polymer model fit. For the three diblock polymer micelles, the SAXS patterns were fit to the Pedersen model (eq S7), which describes the form factor of a micelle with a spherical core and Gaussian polymer chains attached to the surface.38 Based on the fitting results shown in Table 3, the core radius of the micelle increased with increasing molecular weight of the polystyrene block. This trend, and the spherical shape of the polystyrene core, were further confirmed by cryogenic transmission electron microscopy (cryo-TEM) images of the diblock polymer micelles in PBS solution (Figure S14). Because the polystyrene core was kinetically frozen in aqueous solution at the experimental temperature, the micelle aggregation number was estimated by assuming a dry core. The micelle aggregation number (Nagg, upper bound) and corona density (ρcorona) were calculated according to eqs 2 and 3, respectively:

Figure 2. Small-angle X-ray scattering (SAXS) data of the polymer samples at 10 mg/mL in PBS solution (pH = 6.5, T = 25 °C), with PBS solution scattering subtracted. The solid lines are best fits to the Pedersen model, except for PND34-C2 (fit to the Debye model) and PND34-C12 (fit to the Gaussian star polymer model). The profiles are shifted vertically by factors of 10 for clarity. The diblock polymer solutions were prepared by the cosolvent method and lyophilized before dissolution.

4 πR c 3ρNav = NaggM n,PS 3

ρcorona =

patterns of all five polymers in 10 mg/mL PBS solution at 25 °C (solvent background was subtracted, see Figures S6 and S7). Static light scattering (SLS) confirmed the free chain conformation of PND34-C2 in PBS solution (Nagg = 1; see Table S2 and Figures S8 and S9). Therefore, the SAXS pattern of PND34-C2 in PBS solution was fit to the Debye function (eqs S3 and S4),36 resulting in a radius of gyration (Rg) value of 6.0 ± 0.4 nm. For the self-assembled PND34-C12 micelles, the SAXS pattern was fit to a Gaussian star polymer model (eqs S5 and S6; the C12 core size was negligible due to the enormous molecular weight difference between the core and corona).37 The aggregation number of the micelles was 12 ± 1,

(2)

NaggM n,PND 4 Nav 3 πR h 3

(

− 3 πR c 3 4

)

(3)

where Rc is the core radius, ρ is the polystyrene core density, Nav is Avogadro’s number, Mn,PS is the molecular weight of polystyrene, Mn,PND is the molecular weight of PND, and Rh is hydrodynamic radius of the micelle. The aggregation numbers of the diblock polymer micelles increased with increasing molecular weight of the polystyrene block, indicating that a larger number of chains can be incorporated into a core with a larger core size. The micelle core radius estimated from SAXS was consistent with the results obtained from both SLS and cryo-TEM within experimental uncertainty (Table S2 and

Figure 3. (a) 2D 1H NOESY spectra of (a) 50 μg/mL PTN and 72 μg/mL PND34-b-PS14-C12 and (b) 100 μg/mL NID and 144 μg/mL PND34-bPS14-C12 in deuterated PBS solution. The diblock polymer solutions were prepared by the cosolvent method and lyophilized before dissolution. Circled cross-peaks indicate intermolecular polymer−drug interactions in solution for both model drugs. Chemical structures of PTN, NID, and diblock polymer with labeled protons are inserted. E


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Figure 4. Reduced diffusion coefficients (D/D0) of (a) PTN and (b) NID measured by DOSY in deuterated PBS solution as a function of the normalized PND concentration. PTN and NID concentrations are held at 50 and 100 μg/mL, respectively. The diblock polymer solutions were prepared by the cosolvent method and lyophilized before dissolution. Solid lines represent a two-state model that accounts for the diffusion of free and bound drug molecules, from which the indicated binding constants Kb were determined.

b-PS14-C12 were observed (Figure 3b). Both the isopropyl and methyl peaks of PND34-b-PS14-C12 were observed in the 1D spectrum sliced at the NID methyl peak (i.e., 1.57 ppm) from the 2D 1H NOESY spectrum (Figure S17), indicating NID molecules were in close spatial proximity with both NIPAm and DMA repeat units in the PND34-b-PS14-C12 micelle corona. In contrast, only cross-peaks between the methyl groups of NID and the isopropyl groups of the self-assembled PND34-C12 micelles were observed, while no cross-peaks were observed between nilutamide and individual PND34-C2 chains (Figure S18). Based on these results, both model drugs interacted most strongly with the diblock polymer PND34-bPS14-C12 micelles, followed by the self-assembled PND34-C12 micelles, and to the least extent with the individual PND34-C2 chains. The diffusion coefficients of both model drugs and a small molecule tracer (i.e., TSP) in the presence of the polymers at various concentrations were measured to further assess the drug−polymer interactions. PTN and NID concentrations were held at 50 and 100 μg/mL, respectively (with equal concentrations of TSP). The corresponding reduced diffusion coefficients (D/D0, D/D0 = 1 in the absence of polymer) are plotted as a function of PND concentration in Figure 4. The diffusion coefficients of TSP were independent of the polymer type and concentration with both model drugs in all DOSY experiments (Figure S19), indicating that the polymer concentrations in this study were low enough such that any changes in diffusion of the small molecules were caused by specific interactions between the small molecules and polymers rather than by changes in the macroscopic solution viscosity.39 Both model drugs by themselves diffused at least 1 order of magnitude faster than the polymers (Figure S20), and the measured time-weighted diffusion coefficient (D) decreased when a fraction of the drug molecules were “bound” to the polymers due to favorable drug−polymer interactions. The molecular motions of both PTN and NID can be described by a single-exponential decay (i.e., a single time-weighted D value was obtained by fitting to eq 1, Figure S20), indicating that the exchange time between the free and “bound” drug molecules was much shorter than the diffusion time of the DOSY experiments (i.e., Δ = 100 ms in eq 1).40,41 A two-state model, described in detail previously, was used to account for the partitioning of the drug into two species, as free and “bound” molecules in solution.39 By assuming the “bound” drug

Figures S10−S14). Based on the DLS and SAXS results, the corona thickness (Lcorona = Rh − Rc) and corona density (ρcorona) of the micelles were calculated and are summarized in Table 3. For the diblock micelles, the corona thickness increased with increasing molecular weight of the polystyrene block, suggesting that the corona chains were more stretched with a higher aggregation number. A more crowded corona environment (i.e., higher ρcorona) was therefore achieved by increasing the length of the polystyrene block for these diblock micelles. The self-assembled PND34-C12 micelle showed a smaller corona thickness compared to the diblock micelles, and therefore its corona density was comparable to the diblock micelle with the longest polystyrene block (i.e., PND34-b-PS14C12). The correlation between the drug−polymer interaction and micelle corona density is discussed later. Drug−Polymer Interactions. NOESY experiments were employed to explore drug−polymer interactions in deuterated PBS solution. PTN and NID were dissolved in deuterated PBS solution at 50 and 100 μg/mL, respectively. Cross-peak correlations of the aromatic group of PTN with both the isopropyl and methyl groups of PND34-b-PS14-C12 were observed (Figure 3a), indicating that PTN molecules were in close spatial proximity to both NIPAm and DMA repeat units of the PND34-b-PS14-C12 micelle corona. The 1D spectrum sliced at the PTN aromatic group peak (i.e., 7.48 ppm) from the 2D 1H NOESY spectrum of PTN and PND34-b-PS14-C12 confirmed these correlations, since both the isopropyl group from the NIPAm repeat unit and methyl groups from the DMA repeat unit were clearly observed in the 1D spectrum (Figure S15). This indicates that PTN molecules interacted with the micelle corona rather than the core. At equal PTN and PND concentrations, no drug−polymer cross-peaks were observed between PTN and PND34-C2 (Figure S16a). This suggests that PTN molecules were more likely to interact with the crowded coronas of PND34-b-PS14-C12 micelles than with individual PND34-C2 chains in solution. In addition, only crosspeaks between the aromatic group of PTN and the isopropyl group of the NIPAm repeat unit of the self-assembled PND34C12 micelles were observed (Figure S16b), implying that interactions between PTN and diblock polymer PND34-b-PS14C12 micelles were stronger than the ones between PTN and self-assembled PND34-C12 micelles. Similarly, cross-peaks between the aromatic and methyl groups of NID and the isopropyl and methyl groups of PND34F


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Figure 5. PTN dissolution concentration−time curves for SDDs with drug loadings of 10 wt % at 37 °C when polymers were (a) directly dissolved in THF or (b) dissolved in MeOH before spray drying (the diblock polymers were prepared by the cosolvent method and lyophilized before dissolution in MeOH). Dashed lines indicate the measured equilibrium solubility of the crystalline PTN in PBS/FaSSIF solution.

binding through favorable hydrophobic interactions. This is coincidently in agreement with the fact that the binding of these diblock polymer micelles was stronger with the more hydrophobic model drug PTN (i.e., PTN has lower aqueous solubility than NID in crystalline form). The impact of these drug−polymer interactions on the in vitro dissolution performance of both model drugs is discussed in the following section. In Vitro Dissolution Assay Results. To evaluate the efficacy of the polymers as excipients, polymers were spray dried with either PTN or NID and subjected to in vitro dissolution tests under nonsink conditions in PBS solution with 0.5 wt % of fasted simulated intestinal fluid powder (FaSSIF) at 37 °C for 6 h, simulating conditions in the intestinal lumen. 11,42 All dissolution experiments were prepared with a total drug concentration of 1000 μg/mL. The influence of polymer solution-state assembly prior to spray drying on the dissolution and supersaturation maintenance of SDDs was investigated at 10 wt % loading of PTN. Selective and nonselective solvents for the PND corona block were used to form micelles and free chains prior to the spray drying process, respectively. The target PTN concentration was approximately 21 times higher than its equilibrium solubility in its crystalline form in PBS/FaSSIF solution at 37 °C (indicated as a dashed line in Figure 5).20 The enhancement factor (EF), defined in eq 6 as the ratio of the area under the dissolution concentration−time curve (AUC) of the SDD to the AUC of the crystalline drug over a 6 h experimental period, was used to quantify dissolution performance for comparison purposes:

molecule has the same diffusion coefficient as the polymer (Dpolymer), the fractions of the free drug (pfree) and “bound” drug (pbound) can be estimated by the following equation (i.e., pfree + pbound = 1): D = pfree Ddrug,0 + pbound Dpolymer

(4)

where Ddrug,0 is the diffusion coefficient of free drug molecules. A binding constant (Kb), defined in eq 5, was used to quantify and compare the drug−polymer interactions across all polymers: Kb =

[drug]bound [drug]free [repeat unit]

(5)

where [drug]bound is the molar concentration of drug molecules bound to the polymer, [drug]free is the molar concentration of free drug molecules, and [repeat unit] is the molar concentration of the PND repeat unit (by assuming the frozen polystyrene core is an inert component in the case of diblock polymer micelles). The Kb values between both model drugs and the investigated polymers were averaged over all concentrations and are listed in Figure 4. The diffusion coefficients of both PTN and NID were nearly unaffected by the addition of PND34-C2, as indicated by the low Kb values (Kb = 3 ± 14 and 8 ± 4 for PTN and NID, respectively). This suggests that both model drugs have rather weak interactions with individual PND34-C2 chains, which is consistent with the absence of drug−polymer cross-peaks in their corresponding NOESY spectra (Figures S16 and S18). For the diblock polymer micelles with the same corona block, the Kb values of both PTN and NID increased systematically with increasing polystyrene core block length. In fact, the Kb value between PTN and PND34-b-PS14-C12 (Kb = 288 ± 24) was at least 3 times higher than the values of any NIPAm-based polymers reported in previous studies.20,42 The increase of Kb values correlates with the increase of the corona density values for these diblock polymer micelles (ρcorona in Table 3). Within experimental error, the Kb values of PND34-C12 were equal between PTN (Kb = 25 ± 11) and NID (Kb = 29 ± 7). These values were surprisingly lower than those of the diblock polymer micelles, although the self-assembled PND34-C12 micelle has a comparable corona density to the PND34-bPS14-C12 micelle (Table 3). We speculate that the polystyrene at the core−corona interface strengthens the drug−polymer

EF =

AUC(360 min)SDD AUC(360 min)crystalline drug

(6)

THF was chosen as a nonselective solvent for the diblock polymers when spray drying with PTN. Upon direct dissolution in THF, all polymers showed a narrowly distributed single peak with a hydrodynamic radius of approximately 4 nm, indicating free chain conformations (Figure S21 and Table S3). The dissolution concentration− time curves for SDDs of the polymers spray dried from THF, along with a leading commercial excipient control reported in our previous work (i.e., HPMCAS),20 are shown in Figure 5a. Ricarte et al. have recently shown that maintenance of high PTN supersaturation directly correlated with the existence of amorphous nanoparticles containing PTN and HPMCAS, as G


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Figure 6. NID dissolution concentration−time curves for SDDs with drug loadings of (a) 25 wt % and (b) 10 wt % at 37 °C. All polymers were dissolved in MeOH before spray drying. The diblock polymer solutions were prepared by the cosolvent method and lyophilized before dissolution in MeOH. Dashed lines indicate the measured equilibrium solubility of the crystalline NID in PBS/FaSSIF solution.

visualized by cryo-TEM and quantified by SAXS.43 However, the PTN supersaturation level dropped quickly after the initial rapid dissolution as HPMCAS failed to inhibit drug nucleation and crystal growth.20 In this study, PND34-C12 and PND34-bPS2-C12 showed superior dissolution performance than other polymers, as both polymers achieved a rapid PTN release and maintained the supersaturation near the highest achievable concentration throughout the 6 h experimental period (C360 min = 965 ± 27 and 979 ± 2 μg/mL for PND34-C12 and PND34-bPS2-C12, respectively). This is consistent with our previous results that polymeric excipients with self-micellizing capability often had higher enhancement factors,20,21 since both PND34C12 and PND34-b-PS2-C12 can self-assemble into micelles after being directly dissolved in PBS solution (Figure 1 and Figures S22 and S23). At 10 wt % PTN loading, PND34-C2 free chains achieved a dissolution profile comparable to PND34-C12 and PND34-b-PS2-C12 micelles within experimental error. In contrast, PND34-b-PS9-C12 and PND34-b-PS14-C12 did not dissolve well in PBS solution due to the longer hydrophobic polystyrene blocks, and therefore they failed to achieve a high PTN supersaturation. The supersaturation level was lower for the diblock polymer with the longer hydrophobic polystyrene block (i.e., PND34-b-PS14-C12), possibly because of its lower solubility in PBS solution as free chains. To maintain the micelle morphology of the diblock polymers with frozen polystyrene cores before spray drying, MeOH was chosen as a selective solvent for the PND corona block. Since PND34-C2 and PND34-C12 were both free chains in either MeOH (Figure S24) or THF (Figure S21), their corresponding dissolution profiles were the same within experimental error (Figure 5a,b), regardless of the spraydrying solvent. With a rather short polystyrene block, PND34b-PS2-C12 showed two populations in MeOH, mostly free chains with a small fraction of larger aggregates (Figures S24 and S25). Upon dissolution in the PBS solution, PND34-b-PS2C12 also achieved nearly full release of PTN by spray drying with MeOH (Figure 5b). This was attributed to its selfmicellizing capability in PBS solution (Figures S22 and S23). Unlike in THF, lyophilized PND34-b-PS9-C12 and PND34-bPS 14 -C 12 micelles prepared by the cosolvent method maintained their well-defined micelle morphology upon dissolution in MeOH (Figure S24). After spray drying, PTN molecules were presumably loaded in the micelle corona, rather than the frozen polystyrene core, for PND34-b-PS9-C12 and PND34-b-PS14-C12. As a result, the dissolution performance

of the two diblock polymers was improved significantly by preforming the micelles before spray drying (Figure 5b) compared to spray drying as free chains in THF (Figure 5a). For the diblock polymer with the longest polystyrene chain (PND34-b-PS14-C12), full release of PTN was achieved initially (C40 min = 1004 ± 27 μg/mL); however, the PTN supersaturation level dropped slowly afterward due to crystallization. This might be due to the induced nucleation and growth of the sequestered PTN molecules as a result of their high local concentration in the micelle corona. In fact, a higher drug− polymer binding constant from the DOSY experiments (Figure 4) indicated a higher fraction of PTN was stored in the micelle corona. Induced nucleation and growth of the sequestered PTN would be most likely to occur in the micelles with the highest binding constant (i.e., PND34-b-PS14-C12). Nevertheless, by forming the micelles prior to spray drying in MeOH, PND34-b-PS9-C12 (EF = 19.2 ± 0.6) and PND34-bPS14-C12 (EF = 16.2 ± 0.3) still significantly outperformed the commercial excipient HPMCAS (EF = 5.0 ± 0.3) in supersaturating PTN. At a higher loading of 25 wt %, the initial supersaturation level was lower for all the polymers (Figure S26a), presumably due to the lower polymer-to-drug ratio. With 25 wt % PTN, the micelle-forming PND34-C12 delivered a considerably higher supersaturation than free chains of PND34-C2 (Figure S26a). The difference between PND34-C12 and PND34-C2 was also observed at a longer time point after dissolution (i.e., C24 h = 879 ± 32 and 287 ± 1 μg/ mL for PND34-C12 and PND34-C2, respectively) even with only 10 wt % of PTN (Figure S26b). Both observations suggest that the PND34-C12 micelle is a better crystallization inhibitor than PND34-C2 free chains at equal polymer molecular weight and concentration. The polymer efficacy as an excipient in SDDs was also investigated by using NID as a model drug at both 10 and 25 wt % loading (Figure 6). All the diblock polymer micelles were formed by the cosolvent method and lyophilized before dissolution in MeOH for spray drying. The total NID concentration (i.e., 1000 μg/mL) was approximately 7.9 times higher than its measured equilibrium solubility in PBS/FaSSIF solution at 37 °C, indicated by the dashed line in Figure 6. With 25 wt % NID, PND34-C12 achieved its highest supersaturation level at 40 min (C40 min = 845 ± 32 μg/ mL), which then started to decrease after 90 min due to drug crystallization. At equal NID loading of 25 wt %, the PND34-C2 free chains had lower supersaturation at all time points H


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by hydrophobic drugs can be solubilized in the crowded corona region of the micelle, in the form of drug-rich amorphous nanoparticles.20,43 PTN may have a stronger affinity for the slightly hydrophobic micelle corona region compared to NID due to its higher hydrophobicity. Such affinity is consistent with the cross-peaks observed in the NOESY spectra (Figure 3), most likely due to hydrophobic interactions between the drug and the micelle corona. Concurrently, the diblock polymer micelles had higher binding constants with PTN than with NID based on the DOSY results (Figure 4), resulting in a better dissolution performance (i.e., higher EF in Figure 7) with PTN than with NID. In contrast, HPMCAS (as the control) had the lowest enhancement factor with PTN among all the polymers, while outperforming most of the polymers with NID (Figure 7). The polymer micelles may be more effective in stabilizing the more hydrophobic and rapidly crystallizing drugs (e.g., PTN) through the corona sequestration mechanism, and therefore the micelle corona loading strategy can be used as a replacement and/or complement to HPMCAS to improve the dissolution performance of these drugs. In addition, the enhancement factors were much lower at a higher drug loading of 25 wt % for all the polymers, presumably due to the inefficient inhibition of drug nucleation and crystal growth at a lower polymer-to-drug ratio. The predicted amorphous solubilities of both PTN (∼2000 μg/mL) and NID (∼1400 μg/mL) are above the target drug concentrations (i.e., 1000 μg/mL) during the dissolution experiments (see Supporting Information for amorphous solubility calculations). Taylor and Zhang have comprehensively discussed the “reservoir” effect of liquid−liquid phase separation in a recent review, whereby a supersaturated aqueous solution can be achieved in equilibrium with drugrich nanodroplets.16 These drug-rich nanodroplets can maintain the drug supersaturation level by replenishing the absorbed drug.16 Recent work from our group has also shown that high drug supersaturation is directly correlated with the formation of presumably drug-rich nanoparticles.20,43 Although liquid−liquid phase separation is unlikely to occur when the supersaturated concentrations of PTN and NID are below their amorphous solubility,16 a similar “reservoir” concept was demonstrated in this study by using the crowded corona region of the micelle to stabilize the high drug supersaturation level through favorable drug−polymer intermolecular interactions. A good correlation between the strength of the drug−polymer interactions (i.e., Kb in Figure 4) and the corona crowdedness (ρcorona in Table 3) was observed for the three diblock polymer micelles, suggesting that a more crowded micelle corona environment increases the drug affinity to the polymer. When the corona density of the diblock polymer micelles increased, a higher fraction of the free drug molecules in aqueous solution was “bound” to the polymers (e.g., the highest values of pbound were 0.42 and 0.38 for PTN and NID, respectively, with PND34-b-PS14-C12), reflected by a higher Kb value (Figure 4). However, this study demonstrated that a higher drug−polymer binding strength does not necessarily translate into a better dissolution performance, although it could be beneficial to inhibition of drug nucleation and crystal growth.45 We speculate that drug nucleation and crystal growth could occur not only in the aqueous solution but also in the micelle corona (e.g., induced crystallization due to its high concentration), and therefore it is important to optimize the partitioning of free and “bound” drugs by tuning the corona density of the micelle. Thus, the maintenance of a high drug

compared to the self-assembled PND34-C12 micelles. PND34C2 and PND34-C12 had either comparable or slightly better dissolution performance (EF = 5.1 ± 0.6 for PND34-C2 and 6.0 ± 0.2 for PND34-C12) compared to HPCMAS (EF = 5.3 ± 0.1). All three diblock polymer micelles had inferior dissolution performance compared to HPMCAS with 25 wt % NID. For these diblock polymer micelles, the supersaturation level of NID decreased due to drug nucleation and crystal growth after reaching an initially high level, and the drug desupersaturation was more rapid for the micelles with a larger polystyrene core. A similar trend was also observed with 10 wt % NID loading (Figure 6b), although each polymer had a slightly better supersaturation maintenance due to the higher polymer to drug ratio. At 10 wt % loading, both PND34-C2 and PND34C12, along with HPCMAS, were effective in maintaining the supersaturation of NID over the entire 6 h experimental period.

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DISCUSSION To compare the dissolution performance of the excipients for the two different model drugs, the enhancement factors for the SDDs with either PTN or NID at both drug loadings are summarized in Figure 7. PTN is not only a more hydrophobic

Figure 7. Enhancement factor (EF) as calculated at 360 min from the dissolution data of SDDs with 10 and 25 wt % loadings of (a) PTN and (b) NID. All polymers were dissolved in MeOH before spray drying (the diblock polymers were prepared by the cosolvent method and lyophilized before dissolution in MeOH). Maximum possible enhancement factors, indicated by the dashed lines, are 21 and 7.9 for PTN and NID, respectively.

compound than NID (i.e., higher octanol−water partition coefficient, log P), but it is also a stronger crystallizer, as evidenced by its higher free energy of crystallization (Table S5). Therefore, the maximum possible enhancement factor of PTN was higher than that of NID at equal target drug concentration (i.e., 1000 μg/mL). Surprisingly, the diblock polymers achieved higher enhancement factors relative to the maximum possible value with PTN than NID at 10 wt % drug loading, despite the fact that PTN has a much higher thermodynamic driving force to crystallize than NID at equal supersaturated concentration, as predicted by the Hoffman equation (eq S11).44 This result is attributed to the micelle corona sequestration mechanism proposed previously, whereI


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platform for the design of efficient oral drug delivery formulations.

supersaturation level can be achieved when a delicate balance between the free drug in the supersaturated aqueous phase and the “bound” drug in the micelle corona phase is reached. Forming the micelle structure in a selective spray-drying solvent (i.e., MeOH in this study) was important in order to load the drug molecules into the corona of the diblock polymer micelles rather than into the core. The in vitro dissolution performance of the diblock polymers with longer hydrophobic polystyrene blocks (i.e., PND34-b-PS9-C12 and PND34-b-PS14C12) was significantly improved by spray drying with the preformed micelles in MeOH rather than as free chains in THF (e.g., compare Figures 5a and 5b). Spray drying is a kinetically limited process, whereby the drug molecules are presumed to be kinetically trapped in the polymer micelle corona due to the rapid evaporation of solvent.46 The drug molecules are more likely to interact with the micelle corona when the solute concentration in MeOH is increased, which may be beneficial for drug encapsulation. However, there are practical limitations to the solute concentration achievable by the spray-drying process. First, both the drug and polymer need to be molecularly dissolved prior to spray drying. Second, while a higher solute concentration provides high initial solute saturation, it will increase the particle size of the solid dispersion,47 resulting in slower solid dispersion dissolution.48 The role of the polymer LCST is critical when designing polymer micelles as excipients for oral drug delivery applications.49 In this study, the cloud point of the micelle corona block (i.e., 40−41 °C) was tuned to be slightly above body temperature (i.e., 37 °C) by incorporating sufficient hydrophilic monomer (i.e., 33 mol % of DMA), based on the high throughput screening work performed by Ting and coworkers.42 The drug−corona interaction is speculated to be stronger (i.e., higher Kb value) with increasing mole fraction of the more hydrophobic monomer (i.e., NIPAm). Therefore, there exists a delicate balance between hydrophobic and hydrophilic monomers wherein the micelles can provide a slightly hydrophobic corona region to sequester the hydrophobic drug molecules while still remaining soluble in aqueous solution at 37 °C. In addition, the high Tg and nonpolar polystyrene core was chosen such that the dry polystyrene core was kinetically frozen in aqueous solution in order to focus on the drug−polymer interaction mechanism by avoiding micelle chain exchange.50 Results presented in this study and previous work indicate that the micelle corona sequestration mechanism can be broadly applied to a variety of hydrophobic drugs (i.e., PTN, NID, and probucol) to improve their dissolution and supersaturation maintenance.20−22 In contrast, the release of trapped drug molecules in a typical core-loading method often requires an external stimulus-responsive mechanism (e.g., change of pH and/or temperature);51,52 otherwise, the bioavailability of the trapped drug would be compromised due to its low permeability through the gastrointestinal membrane.16 Good in vitro−in vivo correlation and no liver toxicity were observed in an animal study of P(NIPAm-coDMA) polymers.42 Additionally, it has been shown that an FDA-approved styrene-containing triblock polymer (i.e., polystyrene-b-polyisobutylene-b-polystyrene) and polystyrene nanoparticles demonstrated good biocompatibility.53,54 Therefore, there is good reason to expect that biocompatibility will not be a concern in oral delivery applications for the polymers used in this work. The corona loading strategy showcased in this study, in parallel with other methods that successfully utilize drug-rich nanostructures,20,43,55 offers a promising

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CONCLUSION We demonstrate a simple yet powerful strategy to sequester hydrophobic drug molecules in the crowded corona region of well-defined micelles. The micelle corona serves as a reservoir to maintain high drug supersaturation, and such a corona loading strategy proves to be effective for two different hydrophobic drugs, PTN and NID. By forming the micelle structures with a selective solvent (i.e., MeOH) rather than dissolving the diblock polymer as free chains in a nonselective solvent (i.e., THF) prior to spray drying, in vitro dissolution performance of the SDDs was improved with PTN. For these PND-b-PS-C12 diblock polymer micelles, a more crowded corona environment was achieved by incorporating an inert polystyrene block with higher molecular weight, which resulted in a stronger drug−polymer interaction (i.e., higher Kb value) for both PTN and NID. Nevertheless, higher PTN and NID supersaturation and maintenance during in vitro dissolution tests were observed for diblock polymer micelles with shorter polystyrene blocks, possibly due to induced nucleation and growth of the highly concentrated drug molecules that were sequestered in the micelle corona. Micelles with selfassembling capability in aqueous solution (i.e., PND34-C12 and PND34-b-PS2-C12) showed superior in vitro dissolution performance (i.e., higher enhancement factor) with both drugs despite their relatively lower drug−polymer “binding” strength. Therefore, the optimization of the micelle corona density plays an important role in balancing the fraction of free and “bound” drug molecules for better drug supersaturation maintenance. This study reveals the importance of micelle solution-state assembly on both the drug−polymer interactions and the subsequent drug dissolution and supersaturation maintenance for oral drug delivery applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.7b02297.

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Polymer characterization, optical transmittance measurements, details of dynamic light scattering experiments and analysis, small-angle X-ray scattering measurements, static light scattering experiments and analysis, cryogenic transmission electron microscopy images of the micelles, additional NOESY spectra, additional DOSY experiments and details, additional in vitro dissolution tests, and amorphous solubility calculation (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Frank S. Bates: 0000-0003-3977-1278 Timothy P. Lodge: 0000-0001-5916-8834 Notes

The authors declare no competing financial interest. J


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ACKNOWLEDGMENTS This study was funded by The Dow Chemical Company through Agreement 224249AT with the University of Minnesota. The authors gratefully thank Professor Theresa M. Reineke, Professor Marc A. Hillmyer, Dr. Jodi Mecca, Dr. Jin Zhao, Dr. Robert L. Schmitt, Dr. William Porter, Dr. Jeffrey M. Ting, Peter W. Schmidt, Lindsay M. Johnson, Anatolii A. Purchel, and Dr. Ralm G. Ricarte for helpful discussions. Parts of this work were performed at the DuPont−Northwestern− Dow Collaborative Access Team (DND-CAT) located at Sector 5 of the Advanced Photon Source (APS). DND-CAT is supported by E.I. DuPont de Nemours & Co., The Dow Chemical Company, and Northwestern University. Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract DE-AC02-06CH11357. This work benefited from the use of the SasView application, originally developed under NSF Award DMR-0520547. SasView contains code developed with funding from the European Union’s Horizon 2020 research and innovation programme under the SINE2020 project, Grant Agreement No. 654000. Parts of this work were carried out in the College of Science & Engineering Characterization Facility, University of Minnesota, which has received capital equipment funding from the NSF through the UMN MRSEC program under Award DMR-1420013. NMR instrumentation was supported by the Office of the Vice President of Research, College of Science and Engineering, and the Department of Chemistry at the University of Minnesota; the Bruker HD NMR was supported by the Office of the Director, National Institutes of Health under Award S10OD011952. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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