Comparison of Shell-Cross-Linked Micelles with Soft and Glassy

Article pubs.acs.org/Macromolecules

Comparison of Shell-Cross-Linked Micelles with Soft and Glassy Cores as a Drug Delivery Vehicle for Albendazole: Is There a Difference in Performance? Yoseop Kim,†,‡ Elviana D. Liemmawal,† Mohammad H. Pourgholami,‡ David L. Morris,‡ and Martina H. Stenzel*,† †

Centre for Advanced Macromolecular Design (CAMD) and ‡Cancer Research Laboratories, Department of Surgery, St. George Hospital, University of New South Wales, Sydney NSW 2052, Australia S Supporting Information *

ABSTRACT: The understanding of glass transition temperatures Tg in drug and polymer systems is indispensable for drug encapsulation and delivery. Amphiphilic block copolymers consisting a various ratios of poly(methyl methacrylate) (Tg = 100 °C) and poly(ethyl acrylate) (Tg = −22 °C) as the hydrophobic block have been synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization as drug delivery carrier for albendazole (ABZ). Self-assembled micelles with diameters of ∼25 nm with glassy (PMMA) and soft (PEA) core have been synthesized. Differential scanning calorimetry (DSC) has been used to evaluate crystallinity and miscibility of ABZ with the core-forming polymer. All drug− polymer systems are compatible, but they become less miscible with increasing amount of PMMA. The most noticeable difference was the suppression of the crystallinity of the drug with increasing PEA content, a prerequisite for long shelf life of the drug carrier. Since the different micelles are subject to different thermodynamic stability, shell-cross-linking was carried out. Cell experiments against OVCAR-3 cell lines show a fast and efficient uptake of these nanoparticles. Shell-cross-linked micelles were found to be 2−4 times more efficient against OVCAR-3 cells at low concentrations. In contrast, there was no significant difference in the IC50 value of drug carriers with glassy and soft cores.

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INTRODUCTION In the past, drugs were commonly administered in their crystalline state. “Disordered drug delivery”,1 a strategy that uses the drug in its amorphous form, is now an increasingly used method of delivery since it improves bioavailability, especially when the drug has been delivered orally.1−3 Since the crystalline state is the lowest energy form, drugs in the amorphous state are metastable and therefore have a strong driving force to recrystallize. Recrystallization can be suppressed by external factors such as storing the drug at low temperature, but a more permanent approach is by blending the drug with other materials such as polymers. Thermodynamic and kinetic factors determine suppression of drug crystallization, and it is often recommended that a drug be stored at least 50 K below its glass transition temperature (Tg) to prevent crystallization.1−3 Much attention has been drawn to kinetic factors focusing on ways to prevent drug mobility in the matrix. Glassy matrixes can act as barriers to drug movement, effectively freezing the drug in its current position. In contrast, use of low-Tg polymers should see faster recrystallization of the drug. However, although kinetic factors play an important role, the thermodynamic stability of the drug delivery system is vital. © 2012 American Chemical Society

Thermodynamic stability is determined by the interactions between drug and polymer.4 High compatibility between drug and polymer can lead to the permanent or long-lasting suppression of the crystallization of the drug.5 It therefore seems to be paramount to choose a polymer system with the best possible compatibility between drug and polymer. To eliminate the need to synthesize a range of unnecessary polymers for testing, solubility parameters of polymers and drug can act as a fast predictive tool by basing compatibilities on intermolecular forces.6−11 The total solubility parameter can be expressed as δ2 = δd2 + δp2 + δh2, where δd, δp, and δh are the dispersion, polar, and hydrogen-bonding partial solubility parameters, respectively.12 Similar interactive forces between drug and polymer can then lead to low enthalpy values according to ΔHm = ϕ̷ 1ϕ̷ 2[(δd1 − δd2)2 + (δp1 − δp2)2 + (δh1 − δh2)2 ] and therefore to a negative Gibbs free energy of mixing (ΔGm; ΔGm = ΔHm − TΔSm), which favors the miscibility of two substances. The enthalpic component (ΔHm) of the Gibbs Received: March 29, 2012 Revised: May 21, 2012 Published: June 22, 2012 5451

dx.doi.org/10.1021/ma300644v | Macromolecules 2012, 45, 5451−5462

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Poly(ethylene glycol) methyl ether methacrylate (PEGMEMA, Mn = 475 g mol−1) (Aldrich, reagent), methyl methacrylate (MMA) (Aldrich, reagent), ethyl acrylate (EA) (Aldrich, reagent), and methacrylic acid (MAA) were destabilized by passing them over a column of basic alumina and were stored at −7 °C. 2,2-Azobis(isobutyronitrile) (Fluka, 98%) was purified by recrystallization from methanol. The RAFT agent 4-cyanopentanoic acid dithiobenzoate (CPADB)34 and the P(PEGMEMA) macroRAFT agent35,36 were prepared according to the procedure described elsewhere. Synthesis, Micelle Preparation, Drug Loading, and Biological Evaluation. Synthesis of Statistical Copolymers Poly(methyl methacrylate-s-ethyl acrylate) P(MMA-s-EA) via RAFT Polymerization. Five different copolymers were prepared: EA (4.0 × 10−1 g, 4.0 × 10−2 mol) or EA (3.0 × 10−1 g, 3.0 × 10−2 mol)−MMA (1.0 × 10−1 g, 1.0 × 10−2 mol) or EA (2.0 × 10−1 g, 2.0 × 10−2 mol)−MMA (2.0 × 10−1 g, 2.0 × 10−2 mol) or EA (1.0 × 10−1 g, 1.0 × 10−2 mol−1)−MMA (3.0 × 10−1 g, 3.0 × 10−2 mol) or MMA (4.0 × 10−1 g, 4.0 × 10−2 mol) were mixed with CPADB (1.1 × 10−2 g, 4.0 × 10−5 mol) and AIBN (1.3 × 10−3 g, 8.0 × 10−6 mol) in 10 mL of toluene. The mixtures were degassed with nitrogen in an ice bath for 30 min. The reaction was carried out in an oil bath at 70 °C for 24 h. By introducing air and lowering the temperature with an ice bath, the reaction was terminated. The polymers were purified using a tubular dialysis membrane (MWCO 3500) against acetone. Acetone was removed under reduced pressure. Synthesis of Block Copolymer Poly(poly(ethylene glycol) methyl ether methacrylate)-block-poly(methyl methacrylate-s-ethyl acrylate) P(PEGMEMA)-b-P(MMA-s-EA) via RAFT Polymerization. P(PEGMEMA) with 85 repeating units (Mn(theo) = 40 375 g mol−1) was used as a macroRAFT agent for chain extension with MMA and EA. Five different block copolymers were prepared with the molar ratio of MMA varying from 0 to 100 mol %. EA (2.0 × 10−1 g, 2.0 × 10−2 mol) or EA (1.5 × 10−1 g, 1.5 × 10−2 mol)−MMA (5.0 × 10−2 g, 5.0 × 10−3 mol) or EA (1.0 × 10−1 g, 1.0 × 10−2 mol)−MMA (1.0 × 10−1 g, 1.0 × 10−2 mol) or EA (5.0 × 10−2 g, 5.0 × 10−3 mol−1)− MMA (1.5 × 10−1 g, 1.5 × 10−2 mol) or MMA (2.0 × 10−1 g, 2.0 × 10−2 mol) were mixed with PEGMEMA macroRAFT agent (0.78 g, 2.0 × 10−5 mol) and AIBN (6.5 × 10−4 g, 4.0 × 10−6 mol) into 10 mL of toluene. The mixtures were purged with nitrogen for 30 min in an ice bath to avoid the evaporation of the solvent and the monomers. The polymerization was carried out in an oil bath at 70 °C. The reactions were terminated by reducing the temperature in an ice bath for 5 min and introducing air. Precipitation against anhydrous diethyl ether was performed to remove the unreacted monomers. After decanting the solvent, the polymers were dried under reduced pressure overnight. Preparation of Self-Assembled Micelles from Block Copolymers. 20 mg of polymers was dissolved in 8 mL of DMF for the preparation of P(PEGMEMA)-b-P(MMA-s-EA) micelles. 2 mL of distilled water was added dropwise to the solution. The samples were dialyzed against distilled water for 24 h using a tubular dialysis membrane (MWCO 3500). The water was replaced in frequent time intervals. Synthesis of Shell-Cross-Linked Micelles via RAFT Process. Poly(poly(ethylene glycol) methyl ether methacrylates-s-methacrylic acid) P(PEGMEMA-s-MAA) was synthesized to generate a functional polymer for shell-cross-linking. PEGMEMA (3.04 g, 6.4 × 10−3 mol), MAA (1.38 g, 1.6 × 10−3 mol), CPADB RAFT agent (2.2 × 10−2 g, 8.0 × 10−5 mol), and initiator AIBN (2.6 × 10−3 g, 1.6 × 10−5 mol) were mixed in toluene (20 mL). After degassing with nitrogen, the polymerization was carried out at 70 °C. The polymers were purified following the same steps as P(PEGMEMA) polymerization. The conversion was determined by 1H NMR (CDCl3) from the PEGMEMA monomer methyl vinylic peak (−CCH2, δ = 5.7−6.0 ppm), P(PEGMEMA) −CH2−CH2 peak (δ = 3.8−4.2 ppm), and statistical polymer P(PEGMEMA-s-MAA) −CH2−CH2 peak (δ = 0.8−1.2 ppm). The composition was calculated to be P(PEGMEMA67s-MAA18). P(PEGMEMA67-s-MAA18) was used as a macroRAFT agent to chain extend with MMA and EA. The chain extension procedures were the same as above. P(PEGMEMA67-s-MAA18)-b-PMMA80 and P(PEGMEMA67-s-MAA18)-b-PEA86 were synthesized. 30 mg of

function of mixing is controlled by the relative strength of the cohesive drug and polymer intercomponent interactions. A drug has a lower chemical potential when mixed with a suitable polymer, which can then delay the devitrification of the drug. It therefore seems that a stable drug delivery system can be achieved when the interactions between polymer and drug are high creating high thermodynamic stability while a high-Tg polymer may contribute to kinetic stability.13 However, the presence of the drug in the polymer matrix can profoundly affect the glass transition temperatures Tg. Miscibility between drug and polymer is indicated by a single Tg, which is influenced by the Tg of polymer and drug depending on weight fraction of both components (Gordon−Taylor equation). Although many systems follow a linear relationship, deviations from the Gordon−Taylor equation are widespread.14−17 These effects have been studied in detail on drug delivery systems such as tablets where surface energies play only a minor role.18,19 In recent years, however, more and more nanoparticles have been found at the forefront as carriers for drugs. Among them, polymeric micelles with their core−shell structure received much attention.7,11,20−24 The small size of a micelle compared to a tablet may mean that there are added complications such as the effect of the surface energy on Tg. Recent studies suggested that Tg of polymeric materials can deviate substantially from the bulk when confining the dimensions (e.g., film thickness, when polymers form thin films, or pore diameter, when polymers are confined in porous matrices), including the decrease of the size of the drug carrier to the nano length scale. Keddie et al. showed that thin polystyrene (PS) films supported on silica exhibited a reduction in T g with decreasing thickness whereas poly(methyl methacrylate) (PMMA) films exhibited a thickness-dependent Tg that was impacted by the type of supporting substrate.25 The majority of studies have focused on the size-dependent Tg of polymer thin films, but only a handful of studies have focused on extending investigations beyond the thin film geometry to polymer nanoparticles,26 nanopores,27,28 or nanocomposites.29,30 Robertson et al. recently demonstrated that block copolymers with divergent solubility parameters for soft and hard blocks show no variation in Tg due to nanoscale domain size, and at this size range, the Tg might be affected more due to surface tension or nonequilibrium chain orientation and packing.31 However, it must be stated that there is no general experimental consensus and certainly no widely accepted physical description of how size affects Tg. In this work, we explore the use of micelles with either a glassy or a soft core as a drug delivery system for albendazole (ABZ), (methyl [5-(propylthio)-1H-benzimidazol-2yl]carbamate), which is a benzimidazole derivative compound frequently used for the treatment of several helminthiases, but also a highly efficient anticancer drug.32,33 The glass transition temperature was varied systematically with the aim to have similar compatibilities between polymer and drug throughout. In addition, the shell of the micelle will be cross-linked to eliminate the possibility of disassociation: Micelles with glassy cores show a significantly higher stability while low Tg micelles are more dynamic in nature.

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EXPERIMENTAL SECTION

Materials. Toluene (Ajax, 99.4%), N,N-dimethylacetamide (DMAc; Aldrich, 99.9%), dimethyl sulfoxide (DMSO; Ajax, 98.9%), N,N-dimethylformamide (DMF; Aldrich), diethyl ether (Univar), and Nile Red (Aldrich) were used without any further purification. 5452


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200 μg mL−1. The solutions were subsequently incubated at 37 °C/5% CO2 for 1 h. OVCAR-3 cells were washed 5 times with PBS solution. Cell uptake pictures were acquired by using a confocal fluorescence microscope with mercury lamp of λex = 475 nm and λem = 535 nm to detect fluoro micelles and λex = 535 nm and λem = 590 nm to track the release of Nile Red. Analysis. Nuclear Magnetic Resonance (NMR) Spectroscopy. The NMR spectra were recorded using a Bruker 300 MHz spectrometer. Samples were analyzed in CDCl3 at 25 °C. Size Exclusion Chromatography (SEC). Molecular weight distributions of the block copolymers were determined by size exclusion chromatography (SEC) using a Shimadzu modular system, comprising an autoinjector, a Polymer Laboratories 5.0 μm bead-size guard column (50 × 7.5 mm), followed by three linear PL columns (105, 104, and 103 Å) and a differential refractive index detector. The eluent was N,N-dimethylacetamide (DMAc; 0.05% w/v LiBr, 0.05% BHT) at 50 °C with a flow rate of 1 mL min−1. The system was calibrated using narrow polystyrene standards ranging from 500 to 106 g mol−1. The samples were prepared followed by filtration using a filter with a pore size of 0.22 μm, which eliminates higher aggregates. Dynamic Light Scattering (DLS). Hydrodynamic diameters were obtained using a Malvern particle size analyzer. Samples were filtered with microfilters of 0.45 μm size to remove any dust particles before analyzing, and run for at least three times at 25 °C. Transmission Electron Microscopy (TEM). The TEM micrographs were obtained using a JEOL 1400 transmission electron microscope. The instrument operates at an accelerating voltage of 100 kV. Samples were negative stained with phosphotungstic acid (2% w/w). A Formvar-coated grid was cast by putting it onto the surface of a polymer aqueous solution for 1 min. Excess solution was removed using filter paper. In the staining process, the cast grid was gently put onto the surface of a drop of phosphotungstic acid for 30 s. The stained grid was dried under air. Thermal Analysis. The P(MMA-s-EA) samples synthesized in the previous stage were used for this step: 7.5 mg of polymers and ABZ ranging from 0 to 2.5 mg to achieve ratios of polymer to ABZ of 3:1, 5:1, 15:1, and 0 w/w were dissolved in 4 mL of THF. The samples were left in the shaker overnight to allow enough time for the drug to interact with the polymers. THF was removed under reduced pressure over 2 days. In addition, the drug loaded micelles were obtained after loading the micelles according to the procedure described above followed by freeze-drying. The thermal events were studied using a Perkin-Elmer PE 7 Series differential scanning calorimeter (DSC). 5−10 mg of polymers and ABZ mixture were weighed in an aluminum pan and were scanned from −50 to 100 °C at a heat rate of 10 K/min. After an initial heating and cooling cycle, the glass transition temperatures Tg reported in this paper were obtained from the second heating scan. To determine crystallization event over several week only the first heating curve from −50 to 240 °C was recorded. Once ABZ reached its melting point, it started decomposing and the second heating curve does not show any transitions. The Tg of the drug was determined by heating ABZ to the onset of the melting point followed by rapid cooling at 300 K/min with a subsequent heating cycle. Calculations. Solubility Parameters Using the Group Contribution Method. Hoftyzer and van Krevelen established a method to estimate the partial solubility parameters to calculate the total solubility parameter for the polymers by using the group contribution method using the following equations:37

polymers was taken to form micelles. Micellization steps were the same as above. 5 mg of 1,8-diaminooctane and 5 mg of l-ethyl-3-(3′dimethylaminopropyl)carbodiimide hydrochloride (EDC) catalyst were added to cross-link the shell of the micelles under room temperature overnight. The cross-linked micelles were purified via dialysis. Drug Loading and Release Efficiency from Polymeric Micelles. Five different P(PEGMEMA)-b-P(MMA-s-EA) block copolymers and two shell-cross-linked micelles P(PEGMEMA67-s-MAA18)-b-PMMA80 and P(PEGMEMA67-s-MAA18)-b-PEA86 were used for both drug loading and release experiments. The dialysis method was used to load ABZ into the micelles.36 Initially, polymers and drug were dissolved in DMF and incubated overnight (2 mg/mL polymer concentration, polymer/ABZ = 5/1 w/w) followed by dialysis against distilled water using a dialysis membrane (MWCO 6000) for 8 h. Water was replaced once after 4 h. To determine the amount of loaded drug, the solution was lyophilized to remove water and CDCl3 was added to fully dissolve the polymer and remaining ABZ. The loading of ABZ was determined by 1H NMR (CDCl3) from the PPEGMEMA −CH2− CH2 peak (δ = 3.8−4.2 ppm) and ABZ −CH2−CH2 peak (δ = 2.8− 3.2 ppm). Drug loading efficiency (DLE) was calculated according to the equation

DLE (%) = (amount of ABZ in micelle) /(amount of ABZ added initially) × 100 The drug release was studied at room temperature by the dialysis method against 200 mL of distilled water. At different time intervals, 1 mL of the medium outside the dialysis membrane was taken, freezedried, and dissolved in 1 mL of DMF for UV−vis (λ = 298 nm) analysis. In Vitro Cytotoxicity Assay. Cell proliferation was measured using sulforhodamine B (SRB) assay. Briefly, OVCAR-3 (human ovarian cancer) cell lines were seeded in 96-well plates (3000 cells per well) with culture medium 10% (fetal bovine serum) RPMI-1640 [2 × 10−3 M glutamine, 1.5 g L −1 sodium bicarbonate, 0.010 M 2hydroxyethylpiperazinesulfonic acid (HEPES), 4.5 g L−1 glucose, and 10−3 M sodium pyruvate] at 37 °C in a 5% CO2 environment for 24 h. The medium was refreshed with 0.2 mL of a solution consisting of 0.1 mL cell growth medium and 0.1 mL of micelle solution of P(PEGMEMA)-b-P(MMA-s-EA) block copolymers and two shell cross-linked micelles P(PEGMEMA67-s-MAA18)-b-PMMA80 and P(PEGMEMA67-s-MAA18)-b-PEA86 micelles with and without ABZ loading to reach a final micelle concentration of 10, 50, 100, and 200 μg mL−1, respectively, followed by incubation at 37 °C in the incubator for 72 h. Subsequently, the medium was removed and washed 5 times with tap water and 5 times with 1% acetic acid. After drying overnight, 100 μg of 0.010 M Tris (pH = 10.5) solution was added to solubilize the dye. Absorbance was measured at 570 nm using S90 plate reader (Metertech, Taiwan). Nontreated cells were used as controls. The optical density (OD) was used to calculate cell viability

cell viability (%) = (OD570,sample − OD570,blank ) /(OD570,control − OD570,blank ) × 100 Cell Uptake Analysis. Fluorescein o-acrylate (1 mol % of PEGMEMA) was copolymerised during the PEGMEMA polymerization of P(PEGMEMA)-b-P(MMA-s-EA), cross-linked P(PEGMEMA 67 -s-MAA 18 )-b-PMMA 80 , and P(PEGMEMA 67 -sMAA18)-b-PEA86 in order to evaluate the interactions between prepared polymeric micelles and OVCAR-3 cells and the release of Nile Red as a replacement of ABZ. The general procedure for synthesis of Nile Red loaded fluorescein-labeled micelles is similar to the method stated above. OVCAR-3 cells were incubated in a 96-well plate (30 000 cells well−1) with culture medium 10% RPMI-1640 at 37 °C in 5% CO2 environment for 24 h. The medium was refreshed, and 0.2 mL solutions were added to each well, which were prepared from 0.05 mL of 10% RPMI-1640 and 0.15 mL of distilled water with Nile Red loaded fluorescence micelles, leading to a final micelle concentration of

δd =

∑ Fdi/V

δp = (∑ F 2 pi)0.5 /V δ h = (∑ E hi /V )0.5 where Fdi, Fpi, and Ehi refer to the functional group contributions of the intermolecular forces (dispersion, dipole−dipole interactions, and hydrogen bonding). The total molar volume (V) of drug and polymers was obtained from the Hansen method.6 δd, δp, and δh for ABZ were 5453


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the values should be used with caution.38,39 More sophisticated extensions of this approach have been developed by Stefanis and Panayiotou.12,40,41 The reader is referred to Koenig and coworkers39 and the book by Coleman et al.42 for a detailed discussion on different models and their shortcomings and advantages. This simple approach is sufficient for the current work as it has also frequently applied by others and found sufficient for simple systems.7,11,43 Prior to any compatibility studies using micelles, the interaction between ABZ and different MMA/EA copolymers was studied using DSC. Polymers with 70−80 repeating units with varying MMA and EA ratio were synthesized using RAFT polymerization. A slight preference for the incorporation of MMA was in agreement with earlier reported reactivity ratios rEA = 0.22 and rMMA= 2.04.44 Table 2 summarizes the polymers synthesized.

obtained from the method by Stefanis and Panayiotou which consists of simple linear equations based on the molecular structure of the compounds that can be used to predict the Hansen solubility parameters of organic compounds with three or more carbon atoms.12 The enthalpy of mixing (ΔHm/MPa1/2) can be predicted using

ΔHm = ϕ1ϕ2[(δd1 − δd2)2 + (δp1 − δp2)2 + (δ h1 − δ h2)2 ]

(1)

where ϕ1 and ϕ2 are the volume fractions of drug and polymer, respectively.11 The volume fraction can be expressed as ϕi = Nivi/V (Nivi is the volume of component i and V is the total volume) which is the volume occupied by a single substance over the total volume.

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RESULTS AND DISCUSSION The aim of this work is the preparation of micelles with compatibilities between polymer to the drug ABZ, but with systematically altered Tg to investigate the effect of a soft or hard core on other drug delivery parameters. In order to choose a suitable monomer pairone monomer that will lead to a high-Tg polymer while the other results in a low-Tg polymerwe employed group contribution method calculation. Our aim is to (a) select monomers with the highest compatibility with ABZ (lowest ΔHmix) and (b) to select two monomers, which have similar compatibilities with the intention to keep ΔHmix of polymer and monomer independent from Tg. We chose to investigate methacrylate/acrylate monomers since the resulting polymers have commonly high (methacrylate) and low (acrylates) Tg, respectively. When the solubility parameter of drug and polymer are similar, ΔHm reaches a minimum based on eq 1. The validity of this relationship between the miscibility and the ΔHm value has been demonstrated elsewhere,7,11,12 although from our experience this approach cannot easily be applied when polymer−drug interactions are based on very strong forces such as H-bonds are present. The monomers MMA and EA were identified as building blocks with the highest compatibility with ABZ. In addition, ΔHm values of MMA and EA against ABZ are similar; therefore, both prerequisites are fulfilled with this monomer pair (Table 1). It is thus expected that the

Table 2. Initial and Actual Molar Ratios of MMA in the Copolymerization of MMA and EA in the Presence of CPADB RAFT Agenta

methyl methacrylate (MMA) ethyl acrylate (EA) ABZ

δd/ MPa1/2

δp/ MPa1/2

δh/ MPa1/2

ΔHm/ MPa

15.8

6.5

5.4

2.6

15.8 18.9

6.3 7.4

7.2 6.3

2.6

conv (%)

MMA units polymerized

EA units polymerized

Mn(theo)/ g mol−1

MMA ratio in polymer FMMA

0 0.25 0.50 0.75 1

77 73 70 78 80

0 22 39 63 80

77 52 34 15 0

7939 7618 6567 8069 8000

0 0.30 0.55 0.84 1

Obtained by polymerization using CPADB RAFT agent (1.1 × 10−2 g, 4.0 × 10−5 mol), EA (4.0 × 10−1 g, 4.0 × 10−2 mol), EA (3.0 × 10−1 g, 3.0 × 10−2 mol)−MMA (1.0 × 10−1 g, 1.0 × 10−2 mol), EA (2.0 × 10−1 g, 2.0 × 10−2 mol)−MMA (2.0 × 10−1 g, 2.0 × 10−2 mol) and EA (1.0 × 10−1 g, 1.0 × 10−2 mol−1)−MMA (3.0 × 10−1 g, 3.0 × 10−2 mol), AIBN (1.3 × 10−3 g, 8.0 × 10−6 mol) in toluene (10 mL). The number of repeating units as well as the conversion was calculated from NMR using the ratio between monomer and polymer. a

Fully miscible binary blends show a single glass transition temperature Tg and can often be described by the Fox equation 1/Tg = ϕA/Tg,A + ϕB/Tg,B, where ϕA and ϕB are the volume fractions and Tg,A and Tg,B are the glass transition temperatures of A and B, respectively. This equation is based on the assumption that the polymers are additive without any changes in interaction taking place. However, the relationship between polymer compositions and Tg is often nonlinear. The negative or positive deviation can then be described either by the Kwei equation, the Jenckel−Heusch equation, the Couchman− Karasz equation, or the Brekner−Schneider−Cantow relationship among others.45,46 Kalogeras et al. further extended the theory and introduced a quadratic polynomial term to mathematically describe variations in the strength of interactions of the miscible binary mixture and irregular free volume modifications (BCKV equation):17,19

Table 1. Partial Solubility Parameter and Enthalpy of Mixinga for Albendazole and a High (PMMA) and Low (PEA) Tg Polymer polymer

MMA feed ratios f MMA

a

Obtained using eq 1. Volume fractions of 1:1 between polymer and drug were used.

comparability between MMA and EA with the drug will be similar leading to a similar loading capacity and retention time of the drug within the polymeric micelles. The hardness of the core can be systematically varied over a broad range by employing statistical copolymers altering the glass transition temperature between a Tg of 95 °C (PMMA) and −22 °C (PEA). This system theoretically allows the investigation of the effect of the Tg without comprising on compatibility. It is important to note here that solubility parameters calculation based on these simple equations should only act as a guide to help identifying monomers with similar polarities, but

Tg = ϕA Tg,1 + (1 − ϕA )Tg,2 + ϕA (1 − ϕA ) [a0 + a1(2ϕA − 1) + a 2(2ϕA − 1)2 ]

(2)

where a0, a1, and a2 are fitting parameters.This BCKV equation was applied to different binary mixtures and could describe copolymers as well as the interactions between drugs and polymers. Prior to investigation of the compatibility between drugs and polymer, the Tg of the different copolymers was determined. In 5454


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the fitting parameter a0 indicates an increasing plasticizing effect with increasing MMA content. These high negative values are often indicative of decreasing intercomponent interaction between drug and polymer.48 The high negative values are very common for various polymer−drug delivery system, but they are mainly prevalent for mixtures were the drug has a Tg lower than the polymer.48 However, negative derivation with drugs with a high Tg are known and have been reported using PMMA and the drug celecoxib49 and poly(N-vinylpyrrolidone) and its copolymer with sucrose.50 Although other techniques to determine crystallinity at ambient temperature such as X-ray diffraction. (XRD) are preferred, DSC analysis can give an estimation on the crystallinity of the sample. At ∼20% weight ratio of the drug, a crystalline melting point starts to appear while lower drug concentrations lead to fully amorphous polymers. The crystallinity of ABZ was evident by the visible melting endotherm in the heating curve. Although heating can potentially induce crystallization in the sample, there was no visible recrystallization exotherm visible in any of the samples prepared that might suggest so. The measured melting point of the drugs remained almost independent from the composition at around 175 ± 10 °C, which is below the melting point of ABZ of 210 °C. The melting point suppression is typical for improved polymer−drug interaction, and it can directly be related to the Flory−Huggins parameter χ.9,16 It was noticeable that with increasing amount of PMMA in the copolymer the melting endotherm increased although the amount of ABZ was similar. Although it was expected that the high-Tg systems would hamper crystallization tendencies due to the entrapment of the drug in the glassy matrix, the opposite was observed (Figure 2). A low drug ratio of 7% w/w leads to completely

addition, Tg of the drug alone was obtained by carefully heating the sample above the melting point in order to avoid decomposition of the drug at this temperature, followed by rapid cooling at 300 K min−1. The general feature of the second heating cycle in the DSC of all samples is the single glass transition of all the copolymer compositions, indicating good miscibility between each other. Figure 1 also exhibited the

Figure 1. Variation of Tg of P(MMA-s-EA) polymers at different ratios with increasing of ABZ. ★: block copolymer with PMMA core. The dotted lines represent the calculated results from BCKV equation using the values listed in the legend (eq 2).

relationship of Tg values as a function of the weight ratio of the PEA and PMMA component. The glass transition value increased with the PMMA fraction ranging from −22 °C (in agreement with the literature value of pure PEA) to 95 °C for pure PMMA. The Tg deviated negatively from the linear Fox relationship. The negative derivation is usually prevalent when the interactions between the building blocks of the stiffer polymer; here PMMA is disrupted by more flexible segments (Supporting Information, Figure S1).19 The polymers were then incubated with different ratios of ABZ in THF, followed by solvent evaporation. Care was taken to dry the polymer sufficiently under high vacuum since traces of solvent will affect the measured Tg. The ratio of polymer and ABZ is expected to have a significant effect on miscibility and crystallization suppression. As displayed in Figure 1, the presence of the drug leads to Tg values well below the linear Fox equation, which is typical for a plasticizing effect. The presence of the drug increases the degree of molecular flexibility resulting in a reduced glass transition temperature.47 When the drug concentration was increased, the Tg of the polymers decreased further (Figure 1). The nonlinear relationship between drug−polymer ratio and measured Tg can be analyzed using the empirical BCKV equation (eq 2), which requires the Tg value of the polymer with and without drugs and ABZ (342 K) for calculations. Only the blend of PMMA and ABZ was fully fitted using all parameters a0, a1, and a2. The data set for the remaining polymers is limited to drug ratios lower than polymer ratios (Figure 1). Therefore, the higher order fitting parameters a1 and a2, which may indicate an asymmetric relationship,16 were omitted for these polymers. The increasing negative number of

Figure 2. Changes in ABZ melting point and heat of fusion for the sample over time depending on polymer structure. Mass fraction of ABZ per homopolymer is 7% w/w and around 5% w/w for the micelles (see Table 5).

amorphous systems directly after preparation (Tg of ABZ loaded PMMA is above ambient temperature, PEA is below). After 3 days, however, crystallization events take place which, over the course of many days, are significantly more pronounced in the PMMA-rich matrix (Figure 2), while the changes in PEA can almost be neglected. The melting enthalpy 5455


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Scheme 1. Synthesis of Block Copolymers via the RAFT Polymerization

Table 3. Synthesis of Block Copolymers Using P(PEGMEMA)85 MacroRAFT Agent and Different Molar Feed Ratio of MMA and EA and the Number of Repeating Units N of Each Monomer in the Final Polymera MMA feed ratios f MMA

conv/%

NMMA

NEA

Mn(theo)/g mol−1

Mn(SEC)/g mol−1

PDI

MMA ratio in polymer FMMA

0 0.25 0.50 0.75 1b

80 70 86 70 80

0 20 46.3 53.7 80

80.4 48.3 40 13.3 0

47 280 46 068 47 870 45 938 43 880

48 300 47 500 48 000 46 000 46 000

1.17 1.15 1.20 1.22 1.18

0 0.29 0.54 0.80 1

a Obtained by polymerization of macroRAFT agent P(PEGMEMA)85 (0.78 g, 2.0 × 10−5 mol), EA (2.0 × 10−1 g, 2.0 × 10−2 mol), EA (1.5 × 10−1 g, 1.5 × 10−2 mol)−MMA (5.0 × 10−2 g, 5.0 × 10−3 mol), EA (1.0 × 10−1 g, 1.0 × 10−2 mol)−MMA (1.0 × 10−1 g, 1.0 × 10−2 mol) and EA (5.0 × 10−2 g, 5.0 × 10−3 mol−1)−MMA (1.5 × 10−1 g, 1.5 × 10−2 mol), AIBN (6.5 × 10−4 g, 4.0 × 10−6 mol) in toluene (10 mL). The number of repeating units as well as the conversion was calculated using the ratio between monomer and polymer by using NMR. bP(PEGMEMA)78 was employed as macroRAFT agent.

for PHEA and PHEA micelles is consistently below 10 J g−1 over many weeks. It can even be argued that the endotherm observed in PHEA may not be a crystalline melting point at all since the measured temperature is well below the melting temperature of ABZ, only complementary studies can confirm if ABZ starts crystallizing. The better stability of the low-Tg PEA system can probably be explained with a more favorable entropic contribution. The high flexibility of PEA leads to a higher conformational entropy. Although ABZ has still a plasticizer effect in PHEA, the effect is much less pronounced than in PMMA. This is in agreement with the DSC data presented in Figure 1. The increase in crystallinity of ABZ in PEA in contrast can almost be neglected (Figure 2). The differences between PMMA and PHEA in their effect on the Tg of the ABZ system highlights again that there are some shortcoming in using solubility parameters, which has also been observed by others,38 but it needs to be highlighted again that this was only a guide to identify two polymers, one with a low Tg and one with a high Tg, that can encapsulate ABZ in an approximately similar manner. After this monomer pair was selected, RAFT polymerization51−53 was used to synthesize a range of amphiphilic block copolymers for micelle preparation.54,55 The P(PEGMEMA) macroRAFT agent was first obtained by polymerization of poly(ethylene glycol) methyl ether methacrylate in toluene in the presence of CPADB at 70 °C. After 24 h of reaction time, P(PEGMEMA) with a theoretical molecular weight of 39 230 g mol−1 (Mn(SEC) = 38 500 g mol−1) and a narrow molecular weight distribution with a polydispersity index (PDI) of 1.12 was obtained. Purification was carried out via precipitation of the polymer in anhydrous diethyl ether. The resulting P(PEGMEMA)85 homopolymer was subsequently used as a macroRAFT agent to achieve a chain extension with different ratios of MMA and EA (Scheme 1). Potential formation of side products during the block copolymerization such as MMA and EA homopolymers are suppressed by the choice of a low radical flux.21 Deviations

between the theoretical molecular weight and the experimental molecular weights can be assigned to the polystyrene SEC calibrations. The molecular weight distributions were narrow, and byproducts were clearly absent (PDI < 1.3) (Supporting Information, Figure S2). Table 3 summarizes only the polymers which have been employed for the formation of micelles. Since the length of the hydrophobic block can affect drug loading,36 block copolymers with constant block lengths were chosen. The final composition of the statistical copolymer blocks shows a slight preference for the incorporation of MMA compared with EA (Table 3). This is not surprising considering that the reactivity ratios mentioned above,44 although it needs to be taken into account that the presence of a RAFT agent can alter the reactivity ratios.56 The polymer will therefore have a gradient structure with an enrichment of MMA close to the P(PEGMEMA) block (Scheme 1). Five different copolymers, P(PEGMEMA)85-b-P(EA)80, P(PEGMEMA)85-b-P(MMA20-s-EA48), P(PEGMEMA)85-b-P(MMA46-s-EA40), P(PEGMEMA)85-b-P(MMA54-s-EA13), and P(PEGMEMA)75-b-PMMA80, were generated with 75−85 repeating units in the hydrophilic block and ∼75 units in the hydrophobic block. The molar ratios of PMMA within the hydrophobic block ranged from 0 to 100%. Factors affecting the morphology of the self-assembled aggregates include the relative hydrophobic block length and the nature and presence of ions and different polydispersities (PDI).57,58,50 The polymers listed in Table 3 were dissolved in DMF and dialyzed against distilled water. The hydrodynamic diameters Dh of all PPEGMEMA-b-P(MMA-s-EA) micelles were determined by DLS in water. The micelle diameters ranged from 16 to 28 nm (Table 4).59 TEM analysis confirmed the results obtained from dynamic light scattering. Micelles with sizes of ∼20 nm could be observed (Figure 3, left). The small deviation of the DLS and TEM results is derived from the dry state of the micelles under which the micrographs were taken. 5456


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Table 4. Hydrodynamic Diameters Dh (via DLS) of Micelles in Water (1 g L−1) polymer

Dh/nm

P(PEGMEMA)85-b-P(EA)80 P(PEGMEMA)85-b-P(MMA20-s-EA48) P(PEGMEMA)85-b-P(MMA46-s-EA40) P(PEGMEMA)85-b-P(MMA54-s-EA13) P(PEGMEMA)78-b-PMMA80

16.0 18.9 28.0 22.0 25.6

The thermal properties of the block copolymer micelles were initially measured without the drug. As mentioned in the Introduction, there is evidence that the Tg of nanoparticles can deviate from the bulk material. The Tg of P(PEGMEMA)75-bPMMA80 is at 90 °C slightly lower than the one measured using PMMA80 alone. Although the reduction of the Tg from 95 °C could be caused by the confined space of the core, it could also be the incomplete phase separation with the P(PEGMEMA) shell. Indicative for the presence of P(PEGMEMA) in the core is that the Tg of P(PEGMEMA) increased from −12 to 9 °C at the same time. It seems therefore that there are no significant effects of the size on the Tg, and the deviation from the original value can be explained by mixed phase boundaries. Encapsulation of ABZ leads to a further decline to 85 °C. Considering the encapsulation efficiency, which equates to 5.5% w/w of drug per loaded PMMA weight (Table 5), the measured Tg is close to the observed values for bulk polymers. It seems therefore that the micelle structure has no influence. Unfortunately, micelles with EA content could not be analyzed since the Tg was too close to the Tg of P(PEGMEMA) to get any meaningful data. Similar to the bulk material with different ratios between MMA and EA, ABZ in the micelle slowly started crystallizing (Figure 2). Again, crystallization in PEA could be neglected while crystallization in the PMMA core proceeded almost at a similar rate to the PMMA bulk material. It seems therefore that there are no significant differences in the behavior of the drug in the bulk material and in a confined micelle space. The much smaller melting point, which can be assigned to smaller crystallite sizes, is noticeable however.60 It should be noted here that there is a possibility that the drug may interact with the water-soluble shell although the low water solubility of ABZ might suggest otherwise. Earlier calculations showed that there is indeed a possibility that various drugs can interact with poly(ethylene glycol).61 At this point no differences in terms of interactions between drug and polymer were observed between bulk material and micelles. However, there was a visible effect of the softness of the core on the ability of drug to crystallize. In contrast to expectation, which assumes the kinetic stabilization of the amorphous state in a glassy matrix, a soft core was found to lead to better stability. Thus, considerations regarding the mobility of the drug in a soft or glassy matrix are obsolete. In contrast, the soft, low-Tg matrix may even provide a higher entropic contribution. However, a significant difference between soft and glassy micelle core is the stability of the micelle. Micelles with low-Tg cores are characterized by higher CMC values and lower kinetic stability. This is even more important considering that micelles are often not stable in cell growth media.62 The presence of up to 20 amino acids, vitamins, and other ingredients lead to the fast disassociation. This could consequently mean that it is not the effect of the Tg of the softness of the core that will be observed in in vitro experiments but the ability of the micelle to disassociate or not. To eliminate this factor, micelles were cross-linked using a simple route proposed by Wooley and coworkers by incorporating carboxylic acid groups to the watersoluble block.63−65 Scheme 2 summarizes the synthesis of shellcross-linked micelles. Initially, the reactive block copolymers, P(PEGMEMA-s-MAA)-b-PMMA and P(PEGMEMA-s-MAA)b-PEA, were prepared similar to an earlier procedure with the difference that PEGMEMA was polymerized in the presence of 10 mol % of MAA to obtain P(PEGMEMA67-s-MAA16) as macroRAFT agent. Subsequent chain extension with MMA or

Figure 3. TEM images of non-cross-linked (left) and cross-linked (right) P(PEGMEMA67-s-MAA18)-b-P(MMA80). The micelles appear as white spherical objects due to the negative staining. Large dark areas: remnant stain.

According to the thermal analysis of different PMMA-s-PEA copolymers, the drug is miscible with all polymer compositions. In contrast to the blending process of the drug into the bulk material, micelles are typically loaded via incubation of the drug in a good solvent followed by addition of water, which encapsulates the drug while forming micelles. The amount of drug loaded is then determined by the feed ratio of drug and polymer, but even more by the nature of the core,36 especially by the similarity of the drug and polymer polarity based on the enthalpy of mixing.7,11 ABZ loading was carried out by incubating 10 mg of polymers with 2 mg drug in DMF, which is a solvent that can fully dissolve both the drug and the polymers. The subsequent dialysis step against water resulted in drug loaded micelles while nonencapsulated drug and solvent were removed. The drug loading efficiency was determined via 1 H NMR by collecting the samples within the dialysis membrane. Table 5 summarizes the drug loading capacities of Table 5. Drug Loading Efficiency (DLE) of Micelles Having Different MMA and EA Ratio in the Micellar Corea polymer

DLE/%

ABZ mass fraction of polymer/ % w/w

P(PEGMEMA)85-b-P(EA)80 P(PEGMEMA)85-b-P(MMA20-sEA48) P(PEGMEMA)85-b-P(MMA46-sEA40) P(PEGMEMA)85-b-P(MMA54-sEA13) P(PEGMEMA)75-b-PMMA80

24.2 27.2

4.6 5.2

25.1

4.8

25

4.8

29

5.5

a

2 mg of ABZ incubated with 10 mg of polymers.

the micelles with different ratios of MMA and EA in the core highlighting the observation that the drug loading efficiency is independent from the glass transition temperature. As predicted using solubility parameters and measured using melting point depression, compatibility and therefore loading are independent from the glass transition temperature of the core-forming polymer. 5457


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Scheme 2. P(PEGMEMA-co-MAA)-b-PMMA and P(PEGMEMA-co-MAA)-b-PEA Block Copolymer Synthesis Using PMMA or PEA MacroRAFT Agent, Followed by Self-Assembly and Shell-Cross-Linking Using 1,8- Diaminooctane

encapsulated drugs, and the immediate cross-linking step was then concluded with the purification of the ABZ loaded shellcross-linked micelles from solvent, excess drug and cross-linker via dialysis against water. Loading efficiencies of both drug carriers, one with PMMA and one with PEA core, were now both around 50%, which is double the loading efficiency of the un-cross-linked micelle. The drug release experiment was performed against distilled water to study the effect of the degree of polymer/drug interactions on drug retention time. The release profile for ABZ from P(PEGMEMA-s-MAA)-b-PMMA and P(PEGMEMA-sMAA)-b-PEA cross-linked and un-cross-linked micelles is shown in Figure 4. No significant difference was observed in the drug release between micelles with PMMA and PEA in the core. Shell-cross-linking in contrast leads to the network formation, which can act as obstacle for the diffusing drug67 leading to a reduced release rate with less than 10% release after 6 h. The slow release from the cross-linked micelle can explain the higher loading efficiency. Drug loaded micelles are typically purified via dialysis to remove free drug. This step however can also lead to some premature release of the drug.36 The crosslinked shell represents a tight network reducing the mobility of the drug;67 thus, the cross-linked micelles do not have a higher loading capacity, but fewer drugs have been lost during purification.

EA resulted in the formation of the block copolymers P(PEGMEMA67-s-MAA16)-b-PMMA80 and P(PEGMEMA67-sMAA16)-b-PEA86 (Scheme 2). It should be noted here that the length of both blocks was similar to the block copolymer listed in Table 3. Self-assembly led to micelles with a hydrodynamic diameter of around 40−50 nm. It is should be highlighted here that the micelles were significantly bigger despite a similar number of repeating units owing to the negatively charged, thus repulsive, carboxylate units in the shell. Subsequently, micelles were cross-linked by adding 1,8-diaminooctane in the presence of l-ethyl-3-(3′-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to P(PEGMEMA67-s-MAA16)-b-PMMA80 and P(PEGMEMA 67 -s-MAA 16 )-b-PEA 86 micelles, respectively (Scheme 2). The successful formation of cross-linked micelles was confirmed via DLS analysis. The particle size of crosslinked micelles (∼15 nm) was smaller than non-cross-link micelles, which is in agreement with earlier studies (Figure 3, right).66 The structural integrity of the micelle was studied by DLS. Micelles disassociate into unimers when dissolved in a good solvent for both blocks such as DMF, but once crosslinked, they retain their size (Supporting Information, Figure S3). The subsequent loading step of shell cross-linked micelles was carried out simultaneously with cross-linking. Block copolymers and ABZ were incubated with the drug in a good solvent (DMF). Addition of water leads to micelles with 5458


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To examine whether the prepared micelles were internalized by cells, the drug carrier was labeled with green fluorescence marker while ABZ was replaced by Nile Red, a red fluorescent dye being hydrophobic as ABZ. Figure 6 contains confocal

Figure 4. Drug release rate from cross-linked and un-cross-linked P(PEGMEMA 67-s-MAA 18)-b-P(MMA 80) and P(PEGMEMA67 -sMAA18)-b-P(EA86) micelles at 25 °C.

OVCAR-3 cell lines were chosen to test the toxicity of ABZ loaded micelles as in vivo and preclinical trials and pilot clinical trials showed that ABZ works efficiently as an anticancer drug against ovarian cancer.68−71 Initially, the cytotoxicities of P(PEGMEMA-s-MAA)-b-PMMA and P(PEGMEMA-s-MAA)b-PEA micelles as well as shell cross-linked micelles were tested. Figure 5 shows the percentage viability of OVCAR-3 cells

Figure 5. Cell viability percentage after incubation with cross-linked and non-cross-linked P(PEGMEMA67-s-MAA18)-b-PMMA80 and P(PEGMEMA67-s-MAA18)-b-PEA86 micelles after 72 h.

Figure 6. Fluorescence microscopy images of Nile Red loaded micelles endocytosed by OVCAR-3 cells. Green fluorescence showing the location of the micelles (left column) and the location of red fluorescence of Nile Red (right column) after various incubation times.

relative to the control sample after 72 h of incubation at 37 °C with four different micelle concentrations of 10, 50, 100, and 200 μg mL−1. Although the CPADB RAFT agent was found to be toxic,72 several studies showed the incorporation of the thiocarbonylthio groups from the RAFT agent into the polymer might reduce the toxicity substantially, which would diminish its concentration and reduce the toxicity to a nontoxic level.11,73 Similarly, cross-linked and non-cross-linked empty micelles that were used could be considered as biocompatible against OVCAR-3 cells as no significant levels of toxicity were recorded as shown in Figure 5.

fluorescence micrographs of fluorescence micelles loaded with Nile Red acting as a hydrophobic probe replacing ABZ to track the drug release and the location of the micelle independently using OVCAR-3 cell lines at different time intervals. After 3, 10, 30, and 60 min of incubation, the cells were washed 5 times with PBS buffer solution. It was observed that micelles are taken up by the cell in an efficient manner, and the Nile Red has been released from the micelles within the cells over time 5459


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(Figure 6). Overlay of both micrographs (green and red at the same time) revealed that the nanoparticles are not necessarily located at the same position as the Nile Red, indicating the efficient release of the drug inside the cell. The micelle concentrations of 200 mg L−1 are well above a potential CMC, and therefore disassociation of the micelle can be neglected. The uptake of micelles was efficient for all drug carriers, and no significant differences between soft and glassy core or un-crosslinked and shell-cross-linked micelles can be observed at the concentrations employed. Subsequently, the toxicity of the same polymeric micelles but loaded with ABZ were tested against the OVCAR-3 cells (Figure 7). Since micelles with PEA and PMMA core show

Figure 8. Microscopy images of OVCAR-3 cells incubated with uncross-linked micelles loaded with ABZ (left, drug crystals are visible) and cross-link micelles loaded with ABZ (right, no visible drug crystals).

micelles via endocytosis mainly takes place well above the CMC of the micelle. Several examples in the literature show that cell uptake declines at lower concentrations, which coincides with the disassociation of the micelles into unimers,75,76 although it seems that pluronics benefits from the disassembly into unimers enhancing drug uptake via a different mechanism.60,77,78 Although the PEA core should result in micelles with lower kinetic stability, resulting in formation of unimers and possible drug crystallization, the results are identical to the glassy PMMA core. This leads to the conclusion that both types of micelles may have low kinetic stability in cell growth media and the disassociation prevents the uptake of the drug carrier but also leads to the premature release of drugs outside the tumor, which is accompanied by fast crystallization of the free drug. The shell-cross-linked micelles in contrast is stable even at lowest concentration and under different and can efficiently be taken up together with their toxic load.

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Figure 7. Cell viability percentage after incubation of ABZ loaded cross-linked and non-cross-linked P(PEGMEMA67-s-MAA18)-bPMMA80 and P(PEGMEMA67-s-MAA18)-b-PEA86 micelles after 72 h by OVCAR-3 cells.

CONCLUSION The aim of this work was to compare two micellar systems, one with a soft low-Tg core and one with a glassy core, in relation to their performance as a drug delivery carrier for albendazole. This study has been sparked by earlier observations that, to keep the drug in an amorphous state, a glassy matrix is required to limit drug mobility. This is certainly true for a kinetically stabilized system. However, we were interested in creating a drug delivery system that is stable from a thermodynamic point of view where drug and polymer are highly compatible. To create comparable systems with similar mixing enthalpy, but using a low-Tg and a high-Tg polymer, group contribution theory was employed to calculate partial solubility parameters. Although this technique is limited in its application, it can occasionally work for simple systems. The two systems chosen, PMMA (high Tg) and PEA (low Tg), showed very good miscibility with the drug ABZ although the drug delivery system showed signs of drug crystallization over weeks indicative that the group contribution theory has some shortcomings in estimating the best possible polymer matrix for the drug. Importantly, however, it was found that crystallization was more prevalent in the high-Tg PMMA core, probably because of a better entropic contribution of the more flexible PEA. Subsequent drug loading proved that the loading capacity and the rate of release is similar in both systems, which again can be related to the compatibility between polymer and drug. Toxicity of both ABZ loaded micelles against OVCAR-3 tumor cells was also comparable. Since micelles with low Tg core tend to disassembly into unimers more easily than their glassy counterpart, shell-cross-

similar drug loading and drug release rates as well as a similar cell uptake, the observed toxicity is as expected within similar range. At high polymer vector/drug concentrations 90% cell death was observed in the un-cross-linked micelles, which is slightly reduced at lower concentrations. At first glance this may not be too surprising since cell death is drug dependent and a low micelle concentration is equivalent to a low drug concentration.36 The behavior of shell-cross-linked micelles is therefore surprising: at even the lowest concentrations a high cell death of more than 90% was observed. This observation cannot be attributed only to the shell cross-linked micelles having a higher ABZ content compensated by a much lower release rate. At such low concentrations the stability of micelles need to be considered. While there is still debate if a glassy polymer can easily disassociate at low concentrations due to their kinetic stability, the highly dynamics self-assembly of lowTg block copolymers such as Pluronics74 is well-known. In addition that there is evidence that the cell growth media may even contribute to the destabilization of block copolymer micelles.62 Disassociation at low concentrations leads to premature drug release outside the cell. In fact, crystallization of the drug could be observed under the microscope outside the cells on the incubation plates. In contrast, shell-cross-linked micelles were stable and any signs of crystallization were absent at all concentrations (Figure 8). The low stability of the drug carrier is also known to affect the cellular uptake. There is mounting evidence in the literature that the cellular uptake of 5460


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(17) Brostow, W.; Chiu, R.; Kalogeras, I. M.; Vassilikou-Dova, A. Mater. Lett. 2008, 62 (17−18), 3152−3155. (18) Nair, R.; Nyamweya, N.; Gönen, S.; Martínez-Miranda, L. J.; Hoag, S. W. Int. J. Pharm. 2001, 225 (1−2), 83−96. (19) Kalogeras, I. M.; Brostow, W. J. Polym. Sci., Part B: Polym. Phys. 2009, 47 (1), 80−95. (20) Riess, G. Prog. Polym. Sci. 2003, 28 (7), 1107−1170. (21) Stenzel, M. H. Chem. Commun. 2008, 30, 3486−503. (22) Uhrich, K. E.; Cannizzaro, S. M.; Langer, R. S.; Shakesheff, K. M. Chem. Rev. 1999, 99 (11), 3181−3198. (23) Letchford, K.; Burt, H. Eur. J. Pharm. Biopharm. 2007, 65 (3), 259−269. (24) Liu, L.; Gao, X.; Cong, Y.; Li, B.; Han, Y. Macromol. Rapid Commun. 2006, 27 (4), 260−265. (25) Keddie, J. L.; Jones, R. A. L.; Cory, R. A. Faraday Discuss. 1994, 98, 219−230. (26) Rharbi, Y. Phys. Rev. E 2008, 77 (3), 031806. (27) Schönhals, A.; Goering, H.; Schick, C. J. Non-Cryst. Solids 2002, 305, 140−149. (28) Schönhals, A.; Goering, H.; Schick, C.; Frick, B.; Zorn, R. J. NonCryst. Solids 2005, 351, 2668−2677. (29) Ash, B. J.; Siegel, R. W.; Schadler, L. S. J. Polym. Sci., Polym. Phys. 2004, 42, 4371−4383. (30) Kropka, J. M.; Pryamitsyn, V.; Ganesan, V. Phys. Rev. Lett. 2008, 101, 075702. (31) Robertson, C. G.; Hogan, T. E.; Rackaitis, M.; Puskas, J. E.; Wang, X. J. Chem. Phys. 2010, 132, 104904. (32) HORTON, J. Parasitology 2000, 121 (Suppl. S1), S113−S132. (33) Hossein Pourgholami, M.; Yan Cai, Z.; Lu, Y.; Wang, L.; Lawson Morris, D. Clin. Cancer Res. 2006, 12 (6), 1928−1935. (34) Mitsukami, Y.; Donovan, M. S.; Lowe, A. B.; McCormick, C. L. Macromolecules 2001, 34 (7), 2248−2256. (35) Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. J. Mater. Chem. 2011, 21 (34), 12777−12783. (36) Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. Macromol. Biosci. 2010, 11 (2), 219−233. (37) Hoftyzer, P. J.; Krevelen, D. W. v. Properties of Polymers, Their Estimation and Correlation with Chemical Structure, 2nd ed.; Elsevier Scientific Pub. Co.: Amsterdam, 1976. (38) Nair, R.; Nyamweya, N.; Gönen, S.; Martínez-Miranda, L. J.; Hoag, S. W. Int. J. Pharm. 2001, 225 (1−2), 83−96. (39) Miller-Chou, B. A.; Koenig, J. L. Prog. Polym. Sci. 2003, 28, 1223−1270. (40) Stefanis, E.; Tsivintzelis, I.; Panayiotou, C. Fluid Phase Equilib. 2006, 240 (2), 144−154. (41) Stefanis, E.; Panayiotou, C. Int. J. Pharm. 2012, 426 (1−2), 29− 43. (42) Coleman, M. M.; Graf, J. F.; Painter, P. C. Specific Interactions and the Miscibility of Polymer Blends: Practical Guides for Predicting & Designing Miscible Polymer Mixtures; Taylor and Francis: London, 1991. (43) Elsabahy, M.; Perron, M.-È.; Bertrand, N.; Yu, G.-e.; Leroux, J.C. Biomacromolecules 2007, 8 (7), 2250−2257. (44) Grassie, N.; Torrance, B. J. D.; Fortune, J. D.; Gemmell, J. D. Polymer 1965, 6 (12), 653−658. (45) Jayachandra Babu, R.; Brostow, W.; Kalogeras, I. M.; Sathigari, S. Mater. Lett. 2009, 63 (30), 2666−2668. (46) Yue, Y.-M.; Xu, K.; Liu, X.-G.; Chen, Q.; Sheng, X.; Wang, P.-X. J. Appl. Polym. Sci. 2008, 108 (6), 3836−3842. (47) Chi, M.-S. J. Polym. Sci., Polym. Chem. Ed. 1981, 19 (7), 1767− 1779. (48) Kalogeras, I. M. Eur. J. Pharm. Sci. 2011, 42 (5), 470−483. (49) Albers, J.; Alles, R.; Matthée, K.; Knop, K.; Nahrup, J. S.; Kleinebudde, P. Eur. J. Pharm. Biopharm. 2009, 71 (2), 387−394. (50) Shamblin, S. L.; Taylor, L. S.; Zografi, G. J. Pharm. Sci. 1998, 87 (6), 694−701. (51) Moad, G.; Rizzardo, E.; Thang, S. H. Aust. J. Chem. 2005, 58 (6), 379−410.

linking was employed to eliminate the different kinetic stabilities. It seems that shell-cross-linking again had a similar effect on both types of systems but also showed an overall improvement of the performance of the carrier leading to significantly higher toxicities at low concentrations. In summary, it seems that when thermodynamic factors were considered there were no differences between a glassy and a soft core; both carriers showed similar performances in our system, derived by similar mixing enthalpy and kinetic stability could be neglected as soon as thermodynamic stability was achieved. As disassociation results in premature release of the drug and reduced cellular uptake, it appears that stabilization of the drug carrier is a much more important parameter to enhance the performance of micelles as drug carriers.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.S. thanks the ARC (Australian Research Council) for funding in form of a Future Fellowship (FT0991273). M.S., M.P., and D.M. thank the ARC for financial support for this project (DP110102409). The authors thank the Centre for Advanced Macromolecular Design (CAMD) and UNSW Analytical Centre for support. The authors express their sincere thanks to one of the reviewers for his/her excellent input.

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Macromolecules

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