Solubilization of hydrophobic drugs by methoxy poly(ethylene glycol)‐block‐polycaprolactone diblock copolymer micelles: Theoretical and experimental data and correlations

PHARMACEUTICS, PREFORMULATION AND DRUG DELIVERY Solubilization of Hydrophobic Drugs by Methoxy Poly(Ethylene Glycol)-Block-Polycaprolactone Diblock Copolymer Micelles: Theoretical and Experimental Data and Correlations KEVIN LETCHFORD, RICHARD LIGGINS, HELEN BURT Faculty of Pharmaceutical Sciences, University of British Columbia, 2146 East Mall, Vancouver, British Columbia, Canada V6T 1Z3

Received 15 November 2006; revised 2 April 2007; accepted 9 April 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21037

ABSTRACT: The solubilization of five model hydrophobic drugs by a series of micelleforming, water-soluble methoxy poly(ethylene glycol)-block-polycaprolactone diblock copolymers (MePEG-b-PCL) with varying methoxy poly(ethylene glycol) (MePEG) and polycaprolactone (PCL) block lengths was investigated. Variation of the feed weight ratio of MePEG to caprolactone resulted in the synthesis of copolymers with predictable block lengths. The micelle diameter and pyrene partition coefficient (Kv) were directly related to the PCL block length whereas the critical micelle concentrations (CMC) were inversely related to the PCL block length. The aqueous solubilities of the model hydrophobic drugs, indomethacin, curcumin, plumbagin, paclitaxel, and etoposide were increased by encapsulation within the micelles. Drug solubilization was directly related to the compatibility between the solubilizate and PCL as determined by the Flory– Huggins interaction parameter (xsp). Furthermore, the concentration of solubilized drug was also directly related to the PCL block length. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1179–1190, 2008

Keywords: polymeric drug delivery systems; micelle; biodegradable polymers; solubility; biomaterials; copolymer; polycaprolactone; poly(ethylene glycol); Flory– Huggins interaction parameter; drug compatibility

INTRODUCTION It has been estimated that the number of poorly water soluble drug candidates has risen sharply to the order of 40% of all new chemical entities, thus presenting one of the most frequent and greatest challenges for drug development.1 The advent of combinatorial chemistry and high throughput screening has allowed for the selection of lead compounds that are excellent ligands, but poor Correspondence to: Helen Burt (Telephone: 1-604-822-2440; Fax: 1-604-822-3035; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1179–1190 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association

potential drug candidates due to their high molecular weights and increased lipophilicity.2 The poor water solubility of these compounds can hinder or even prevent the progress of the drug into clinical use. A search for improved solubilization methods for lipophilic drugs has led to the use of colloidal drug delivery systems to solubilize these agents. In particular there has been considerable interest in the use of polymeric nanoparticulate delivery systems composed of biocompatible, biodegradable amphiphilic diblock copolymers for the intravenous administration of hydrophobic compounds.3–7 These polymers are composed of a hydrophobic block, often a polyester such as poly(lactic acid), poly(lactic-co-glycolic

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acid), or poly(caprolactone) bound via an ester linkage to a hydrophilic block which is typically poly(ethylene glycol) (PEG).8–16 Depending on the diblock composition and upon addition to aqueous media, these copolymers self-assemble to form nano-sized structures termed micelles, which are characterized by a hydrophobic core surrounded by a highly water bound hydrophilic PEG corona. The corona acts as a barrier between the hydrophobic core and the surrounding aqueous media, thus preventing aggregation between micelles and restricting protein adsorption and subsequent clearance by the reticuloendothelial system. It has been shown in some cases that these nanoparticles display increased circulation time and accumulation at sites of leaky vasculature, which can be attributed to the ‘‘stealthlike’’ properties of the corona and the small particle size.17,18 The core of the micelle can be utilized as a cargo space into which hydrophobic drugs can be solubilized, often dramatically increasing the aqueous solubility of the entrapped drug.9–11,19–22 Several reports have shown that the amount of drug solubilized in micelles varies depending on the copolymer system used. For example, Burt et al.14 found that the hydrophobic anticancer agent, paclitaxel, was encapsulated up to levels of 5–10% w/w, 15–25% w/w, and 15–20% w/w in block copolymers composed of methoxypolyethylene glycol (MePEG) with hydrophobic blocks of poly(D,L lactide), poly(D,L lactide-co-caprolactone), and poly(glycolide-co-caprolactone), respectively. Similarly, Liu et al.22 found variations in the amount of ellipticine encapsulated by methoxy poly(ethylene glycol)-polycaprolactone (MePEGPCL) and MePEG-PLA with the former having a 127-fold increase in the loading efficiency over that of MePEG-PLA. Several investigators have solubilized doxorubicin with a variety of diblock copolymer systems with varying results, achieving drug loadings ranging from 0.5% w/w up to 22% w/w.8,23–28 Clearly, as illustrated by these examples, the extent to which solutes are solubilized by different core-forming blocks varies tremendously. In an attempt to explain the differences in the solubilization of solutes by block copolymer micelles, Nagarajan et al.29 investigated the solubilization of various small molecular weight hydrocarbons by copolymers composed of poly(ethylene oxide-propylene oxide) and poly(Nvinylpyrrolidone-styrene). This group found that there was an unusual selectivity for the solubilization of aromatic hydrocarbons by these block JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

copolymers. In attempts to correlate these solubility differences, it was found that the Flory– Huggins interaction parameter (xsp) described the experimental solubilization capacity of the various low molecular weight hydrocarbon solutes.29–32 The xsp is a calculated parameter which provides a measure of the degree of compatibility between the core of the micelle and the solute. The lower the xsp value, the more compatible the solute is with the micellar core and hence the higher the predicted amount of solubilization. This parameter has been used to qualitatively explain the different extents of solubilization of hydrophobic drugs by micellar drug delivery systems formed from amphipathic copolymers.6,7,22,33,34 However, to the best of our knowledge, there have been no studies that have systematically quantified the micellar solubilization of a series of hydrophobic drugs possessing a range of compatibilities with a chosen core-forming block and explored the empirical relationship between the extent of solubilization and xsp. In the current study, we investigated the solubilization of several model hydrophobic drugs by a series of MePEG-b-PCL diblock copolymers with varying block lengths. Empirical relationships between the extent of solubilization and either the xsp or the core-forming block length were developed.

MATERIALS AND METHODS Materials MePEG (Fluka, Bucks SG, Switzerland), e-caprolactone (CL; Fluka), stannous octoate (SigmaAldrich Canada Ltd., Oakville, Ont.), and pyrene (Sigma) were used as supplied without further purification. The solvents, chloroform (Fisher Scientific Co., Ottawa, Ont., HPLC grade), deuterated chloroform (Cambridge Isotope Laboratories, Andover, MA), acetone (Fisher, HPLC grade), acetonitrile (Fisher, HPLC grade), ethanol (Fisher, HPLC grade), methanol (Fisher, HPLC grade) were also used as supplied. Etoposide (Polymed Therapeutics Inc., Houston, TX), paclitaxel (Hauser, Boulder, CO), curcumin (Sigma), indomethacin (Sigma), and plumbagin (Sigma) were used as supplied. Synthesis and Characterization of MePEG-b-PCL Diblock Copolymers MePEG with a molecular weight of 750, 2000, or 5000 g/mol was combined with CL in varying DOI 10.1002/jps

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weight ratios to control the final composition of the copolymer. The reagents, totaling 50 g, were placed in a round bottom flask sealed with a ground glass stopper and immersed in a heavy mineral oil bath heated to 1408C. The reagents were mixed for 30 min to produce a homogenous liquid, at which time 0.15 mL of stannous octoate was added to the flask. The polymerization reaction was allowed to proceed for 24 h. Cooling the polymer to room temperature terminated the reaction. The copolymer molecular weight was determined by gel permeation chromatography (GPC) against polyethylene glycol standards (Polymer Laboratories, Inc., Amherst, MA) in the range of 670–11800 g/mol. Samples were injected using a Waters model 717 plus autosampler. Chloroform with a flow rate of 1 mL/min was used as the mobile phase and separation was achieved through two Waters Styragel columns (HR 3 and HR 1) connected in series. Detection was by a Waters model 2410 refractive index detector with a cell temperature of 408C. Proton NMR spectra of 10% w/v solutions of copolymer in deuterated chloroform were obtained using a 400 MHz NMR instrument (Bruker Bio Spin Corp., Billerica, MA). Peak positions and areas were analyzed to determine the degree of polymerization using MestRe-C 2.3a software and comparing integrals of peaks from the PCL block and PEG block.

Characterization of MePEG-b-PCL Micelles: CMC, Pyrene Partition Coefficient, and Size of Micelles The critical micelle concentration (CMC) and pyrene partition coefficient (Kv) were determined by a steady-state pyrene fluorescence method as previously described.25,35–37 A 1.5
105 M solution of pyrene in acetone was aliquoted into a series of amber glass vials and the acetone was evaporated under a stream of nitrogen. To the vials were added copolymer solutions in PBS (0.01 M, pH 7.4) ranging in concentration to give a final pyrene concentration of 6.0
107 M. Samples were incubated for 24 h at 378C in the dark with stirring. The excitation spectra of each sample were recorded at 378C on a Shimadzu RF 540 spectrofluorometer with an emission wavelength of 390 nm and slit widths of 2 nm. Light scattering measurements were carried out on a Malvern 3000HS Zetasizer with a He–Ne laser (532 nm) and 908 collecting optics. MeasureDOI 10.1002/jps

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ments were made at 378C on copolymer samples at a concentration of 1% w/v prepared in filtered (0.22 mm filter, Millipore, Billerica, MA) PBS (0.01 M, pH 7.4), with the exception of MePEG17-bPCL2 which was measured at a concentration of 2% w/v. Data were analyzed using CONTIN algorithms provided by Malvern Instruments (Malvern, UK).

Drug Solubilization: Aqueous and Micellar Solubilities The aqueous solubilities of the drugs after 3 h ([drug]aqueous) were determined by the incubation of excess amounts of drug in PBS at 378C. The solutions were centrifuged (Beckman Coulter, Mississauga, Ont.) at 18000g for 5 min to remove any precipitate and an aliquot of the supernatant was diluted in methanol for etoposide, ethanol for indomethacin, and a 60/40 mixture of acetonitrile and water for plumbagin. The concentrations of etoposide, indomethacin, and plumbagin in the supernatants were determined by UV VIS spectrophotometry (Hewlett Packard, Santa Clara, CA 8452A) using absorption maxima at 285 nm, 320 nm, and 420 nm, respectively. The aqueous solubility of paclitaxel was too low to be measured by UV VIS spectrophotometry. Therefore, it was determined by an HPLC assay in which an aliquot of the supernatant was dried under a nitrogen stream then reconstituted with a 60/40 mixture of acetonitrile and water. The sample was analyzed by HPLC (Waters, Milford, MA) described elsewhere.38 Curcumin was practically insoluble in water and therefore the aqueous solubility was taken from a literature value.39 The micellar solubilization of the five model drugs, etoposide, paclitaxel, plumbagin, curcumin, and indomethacin was investigated at a copolymer concentration of 1% w/v. Drugs were loaded into micelles by a solvent evaporation technique in which drug and polymer were dissolved in a common solvent (Tab. 2) and each component was aliquoted into 1 mL glass vials so that the final polymer concentration ([polymer]total) was 1% w/v and the added drug concentration ([drug]added) was incrementally increased. The solvent was removed by evaporation at 408C overnight under vacuum. Micelles were formed by the addition of 378C PBS (0.01 M, pH 7.4) followed by vortexing until the polymer/ drug matrix had dissolved. Drug solutions were incubated at 378C in a water bath and then transferred to microcentrifuge tubes and centrifuged JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

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at 18000g for 3 min to pellet any precipitate. An aliquot of the supernatant was diluted in a common solvent for both the drug and polymer as previously described. The supernatants were assayed by UV VIS spectrophotometry to determine the concentration of drug solubilized ([drug]micelle) using the wavelengths above. Paclitaxel and curcumin were assayed at 232 nm and 422 nm, respectively using a 60% solution of acetonitrile in water to dilute the supernatant. Drug loading efficiencies were calculated by % Loading efficiency ¼

½drugmicelle
100 ½drugadded

d2drug ¼ d2d þ d2p þ d2h

(5)

d2polymer ¼ d2d þ d2p þ d2h

(6)

and (1)

For each drug–polymer combination, plots of [drug]added versus [drug]micelle and % loading efficiency were generated. The highest [drug]micelle at which 100% loading efficiency was achieved (max[drug]micelle100%) was used to calculate the maximum concentration of drug contained in micelles (max[drug]micelle) by

Each individual component was calculated according to the following equations: dd ¼

¼ max½drugmicelle 100%  ½drugaqueous

(2)

To determine if there was a change in the solubilized drug concentration over time each drug was solubilized at its max[drug]micelle in MePEG17-b-PCL4 and incubated for 2, 4, and 8 h and the solubilized drug concentrations assayed. The partition coefficients of each drug in each copolymer at their max[drug]micelle was determined using the equation9: ½drugmicelle C ¼ KXPCL r ½drugaqueous

dh ¼

(7)

V 2 Fpi

1=2 (8)

V X

Ehi V

1=2 (9)

For each structural group in the molecules, Fdi and Fpi are the molar dispersion and polar attraction constants, respectively, and Ehi is the hydrogen bonding energy. Values for these parameters were found in tables published by van Krevelen.41

(3)

where K is the partition coefficient of the drug, XPCL the mole fraction of PCL in the copolymer, C the concentration of copolymer, and r the bulk density of polycaprolactone (1.146 g/mL).

Drug Solubilization Correlation Plots The max[drug]micelle was normalized to the concentration of polymer participating in the formation of micelles ([polymer]micelle) calculated by ½polymermicelle ¼ ½polymertotal  CMC

Drug–Polymer Compatibility Calculations The compatibility between the drug and the micelle core-forming block (PCL) was calculated by the Hildebrand–Scatchard equation: V RT

X Fdi

P dp ¼

max½drugmicelle

xsp ¼ ðddrug  dpolymer Þ

the molar volume of the drug as calculated by the group contributions method according to Fedors.40 Values for ddrug and dpolymer were calculated by the additive group contribution method as described by van Krevelen.41 In this method, each solubility parameter is the sum of the dispersion (dd), polar (dp), and hydrogen bonding components (dh) such that

(4)

where ddrug is the solubility parameter for the drug, dpolymer the solubility parameter for the coreforming block of the copolymer, R the gas constant, T the temperature in Kelvin, and V JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

(10)

In order to explore the relationship between drug and core-forming block compatibility and the concentration of drug micellized, plots of the ratio max[drug]micelle/[polymer]micelle versus xsp were plotted for each copolymer. Similarly, to investigate the relationship between the amount of drug micellized and the length of the coreforming block, plots of the ratio max[drug]micelle/ [polymer]micelle versus the PCL block length were generated. DOI 10.1002/jps

DOI 10.1002/jps

Feed weight ratio of MePEG: caprolactone. Degree of polymerization of MePEG: MWMePEG/44, DPn of PCL: rounded off value determined by NMR. c Theoretical MW based on feed weight ratio. d MW determined by NMR. e MW determined by GPC. f Polydispersity index determined by GPC. g Critical micelle concentration determined by the pyrene fluorescence method at 378C. h Partition coefficient of pyrene between the core-forming block and water determined by the fluorescence method at 378C. i Micelle hydrodynamic diameter determined by PCS at 378C. j Multimodal distribution with peaks at 1, 11, and 130 nm representing 11.6, 35.3, and 53.1% of total peak areas, respectively. b

a

70:30 60:40 70:30 60:40 70:30 80:20

Feed Ratio

1183

45.3 1.3 22.3 0.7 14.7 0.2 12.4 0.1 13.5 0.3 11.1 1.2j 2.80
105 1.34
105 6.81
104 4.15
104 3.88
104 6.99
103 7143 3333 2857 1250 1071 939

7166 3320 2852 1214 1073 947

7044 2825 2594 1287 1096 980

1.18 1.24 1.08 1.19 1.19 1.13

6.29
107 1.69
106 7.36
106 5.96
105 1.11
104 1.89
103

DPn

MePEG114-b-PCL19 MePEG44-b-PCL12 MePEG44-b-PCL7 MePEG17-b-PCL4 MePEG17-b-PCL3 MePEG17-b-PCL2

PDI

MW GPCe (g/mol) MW NMRd (g/mol) MW Theoc (g/mol) b a

5000 2000 2000 750 750 750

The CMCs and Kv of the series of copolymers were determined with the use the fluorescent excitation

MePEG MW (g/mol)

Characterization of Micelles

Table 1. Physicochemical Data for Synthesized MePEG-b-PCL Series

In this study we synthesized a series of watersoluble methoxy poly(ethylene glycol)-block-polycaprolactone (MePEG-b-PCL) diblock copolymers by a ring opening mechanism of CL using the hydroxyl group of MePEG as the initiator and stannous octoate as a catalyst. The molecular weight of MePEG used in the synthesis was 750 g/ mol, 2000 g/mol, or 5000 g/mol and varying the feed ratio of MePEG to CL in the reaction mixture controlled the final amount of PCL in the copolymer (Tab. 1). As previously reported, comparisons were made of the integrated peak area of the multiplet at 3.55 ppm, assigned to the methylene groups of MePEG, and the sum of the integrated peak areas of the multiplets at 1.5 ppm and 1.3 ppm, respectively, assigned to methylene protons in the caprolactone repeat unit and used to determine the degree of polymerization37,42 (Tab. 1). The degree of polymerization of MePEG was calculated by dividing the molecular weight of MePEG by 44 (the MW of the repeat unit), giving approximately 17, 44, and 114 for MePEGs with molecular weights of 750 g/mol, 2000 g/mol, and 5000 g/mol, respectively. Theoretical molecular weights were calculated from the feed ratios of MePEG and caprolactone whereas the NMR molecular weights were calculated from the degree of polymerization values determined by NMR. GPC was used to estimate the MW of the synthesized copolymers to indicate the presence of unreacted CL and to determine the polydispersity index of the final products. Neither the NMR spectra nor the GPC chromatograms showed any evidence of additional compounds such as unreacted monomer or side reaction products, therefore no further purification steps were performed. The MWs determined through GPC were close in value to those determined by NMR and the polydispersity values were between 1.08 and 1.24. The nomenclature used for the diblock copolymers displays the degree of polymerization of each component as a subscript behind each block abbreviation, rounded to the nearest whole number. For example, the copolymer MePEG114-b-PCL19 has 114 repeat units of MePEG and 19 repeat units of caprolactone.

f

Synthesis and Characterization of MePEG-b-PCL Diblock Copolymers

Kvh

RESULTS

CMCg (mol/L)

Diameteri (nm)

SOLUBILIZATION OF HYDROPHOBIC DRUGS

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spectra of pyrene in aqueous solutions of polymer with increasing concentrations as previously described.37 Plots of the fluorescent emission intensity ratio (I336/I333) as a function of copolymer concentration illustrated that as the copolymer concentration in solution increased, the ratio remained constant until a threshold polymer concentration was reached at which there was a rapid increase in I336/I333. The increase in the ratio was due to a red shift of the maximum intensity peak from 333 nm to 336 nm indicative of partitioning of the probe from an aqueous to a hydrophobic environment upon the formation of micelles. The CMC was taken as the copolymer concentration at which the rapid rise in I336/I333 occurred. In comparing the CMC values between copolymers it was apparent that there was an inverse correlation between the CMC and the PCL block length with CMC values ranging from 1.89
103 mol/L to 6.29
107 mol/L as the PCL block length increased from 2 to 19 repeat units, respectively (Tab. 1). The partition coefficient of pyrene between the core-forming block and water was found to increase from 6.99
103 to 2.80
105 as the PCL block length increased from 2 to 19 repeat units (Tab. 1). The average micelle diameter determined by dynamic light scattering increased as the molecular weight of the diblock copolymer increased (Tab. 1). Micelle size distributions were found to be monomodal with the exception of MePEG17-b-PCL2 which displayed three peaks at 1 nm, 11 nm, and 130 nm.

Figure 1. Solubilization of plumbagin by a 1% w/v solution of MePEG17-b-PCL4. (– – –) Theoretical drug solubilization, (*) experimental drug solubilization, (~) loading efficiency. Data points and error bars represent the mean SD (n ¼ 3).

some of the drugs tested. It was established that the loading efficiencies of the micellar solutions were constant at 100% in the clear solutions while the loading efficiencies fell below 100% in solutions containing precipitate (Fig. 1). Micellar solutions of MePEG17-b-PCL4 at their saturation drug concentration for each drug were incubated for 2, 4, and 8 h without a change in the solubilized drug concentration. Therefore, subsequent experiments were conducted with a 3 h incubation time (Fig. 2). The average of the max[drug]micelle 100% in the copolymer solutions was used to calculate the max[drug]micelle and this value was plotted as a function of xsp for each drug/copolymer combina-

Drug Solubilization The solubilization capacity of five model hydrophobic drugs, etoposide, paclitaxel, plumbagin, curcumin, and indomethacin in aqueous solutions of each of the synthesized copolymers at a final copolymer concentration of 1% w/v was investigated. All solutions were clear up to a threshold amount of total drug added, after which precipitation was observed upon visual inspection. The solutions were centrifuged to remove any precipitate and the supernatants assayed. It was found that the amount of drug solubilized increased up to a maximum concentration, above which the solubilized concentration decreased significantly corresponding with the formation of the precipitate (Fig. 1). Each copolymer increased the solubility of the drug over its aqueous solubility with the exception of MePEG17-b-PCL2, which only marginally increased the solubility of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

Figure 2. Time course equilibration of drugs at max[drug]micelle in 1% MePEG17-b-PCL4 in PBS at 378C. (Black) indomethacin, (white) curcumin, (diagonal stripes) plumbagin, (grey) paclitaxel, (horizontal stripes) etoposide. All bars represent the average drug concentration SD (n ¼ 3). DOI 10.1002/jps

SOLUBILIZATION OF HYDROPHOBIC DRUGS

Figure 3. Plots of maximum amount of drug solubilized in micelle as a function of xsp. (&) MePEG17-bPCL2, (&) MePEG17-b-PCL3, (~) MePEG17-b-PCL4, (!) MePEG44-b-PCL7, (*) MePEG44-b-PCL12, (^) MePEG114-b-PCL19. All points represent the mean max[drug]micelle SD (n ¼ 3).

tion (Fig. 3). These plots demonstrated an inverse correlation between the amount of drug solubilized and the xsp with those drugs with a high xsp values being solubilized to a lesser extent than those with a low xsp value. Using the max[drug]micelle values and aqueous drug concentrations, the partition coefficients of the model drugs in each copolymer were calculated. In general it was found that the partition coefficient increased with the core-forming block length particularly when comparing copolymers with the same MePEG block length (Tab. 3). However, there was not a clear trend for etoposide. Inspection of the plots in Figure 3 revealed that each drug was solubilized to a greater extent as the PCL block length increased, as indicated by increased slopes of the best-fit lines. Plots of max[drug]micelle as a function of PCL block length for each drug produced good linear correlations with high correlation coefficients indicating that more drug was solubilized by copolymers with longer PCL blocks (Fig. 4).

DISCUSSION A series of MePEG-b-PCL diblock copolymers with a range of MePEG and PCL block lengths was successfully synthesized by condensation reaction, involving the ring opening of CL using the hydroxyl terminus of MePEG as an initiator. The copolymer block compositions used in this DOI 10.1002/jps

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Figure 4. Plots of maximum amount of drug solubilized in micelles as a function of PCL block length. (&) etoposide, (~) paclitaxel, (!) plumbagin, (^) curcumin, (^) indomethacin. All points represent the mean max[drug]micelle SD (n ¼ 3).

study were chosen because they resulted in copolymers that were water soluble and therefore could be easily dispersed in aqueous solution, simplifying the drug solubilization studies and eliminating more complex procedures for loading micelles such as dialysis. In order to ensure that the copolymers were water soluble, it was necessary to increase the MePEG molecular weights while increasing the PCL block length. Characterization of the copolymers by NMR indicated that the reaction was successful due to the upfield shift of cyclic caprolactone monomer peaks from 2.5 ppm and 1.7 ppm to 2.2 ppm, 1.6 ppm, and 1.3 ppm and the appearance of the triplet at 4.15 ppm assigned to the terminal methylene in the last repeat unit of the MePEG signifying the bonding of the MePEG to polycaprolactone as described previously by our group.42 The reaction reliably produced copolymers with predictable molecular weights and degree of polymerization as suggested by the similarity in the molecular weights determined theoretically and by NMR with less than 3% deviation between the values (Tab. 1). The molecular weights determined by GPC deviated from the theoretical molecular weights more so than those determined by NMR, particularly those with longer PCL block lengths. It was speculated that the reason for this deviation was due to the fact that the standards used were composed of PEG and thus do not accurately represent those copolymers with a greater proportion of PCL. Nevertheless, the GPC traces showed that the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

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copolymer molecular weight distributions were monomodal, indicating the absence of side reaction products or unreacted caprolactone, and the polydispersity index values close to 1.0 implying a relatively narrow molecular weight distribution. It has been well documented that when amphiphilic diblock copolymers are added to an aqueous environment at a concentration termed the CMC, they self-assemble to form micelles.3,4,43–45 The CMC provides an indication of the thermodynamic stability of the micelles or the minimum concentration at which these nanoparticles will stay self-assembled. In this study, a hydrophobic fluorescent probe, pyrene, was used to detect the CMC. Pyrene displays a characteristic shift in the fluorescent excitation spectrum from 333 nm to 336 nm upon partitioning into a hydrophobic environment such as the PCL core of the micelles. Similar to previous reports, an inverse relationship was found between the PCL block length and the CMC (Tab. 1).37,42,46,47 Interestingly, even with the large increases in the MePEG block length which can cause increases in the CMC, the modest increases in the PCL block length still produced a decrease in CMC confirming previous reports that the hydrophobic block length is the primary factor determining the CMC.43 When considering the development of a micellar formulation of a hydrophobic compound it is important to consider whether the drug payload will remain in the micelle structure upon injection in the body and during subsequent dilution in the bloodstream. The CMC of the copolymer indicates at which point the micelles will disassemble upon dilution. The drug-loaded micelles used in this work were shown to be physically stable for a minimum of 8 h without precipitation (data not shown). However, it is likely that not all copolymers used in this work would make good candidates for in vivo application, as dilution would lead to disassembly and loss of the loaded drug, due to relatively high CMC values (e.g., MePEG17-b-PCL2). However, even though a formulation may possess a low CMC, this factor alone is not sufficient to predict that the drug will remain with the micelle. Our group has shown, in the case of paclitaxel-loaded MePEG-b-PDLLA micelles, that the drug rapidly dissociated from the copolymer into the blood regardless of the low CMC.14 Certainly interactions with various blood components are responsible for this dissociation; however this area still remains to be investigated. Other than providing an indication of the thermodynamic stability of JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

the micelle systems, the CMC was also used to estimate the amount of diblock copolymer taking part in the micelle formation according to Eq. 10. Several researchers have stated that one of the most important determinants of drug solubilization by micelles is the compatibility between the core-forming block and the solute.7,22,45,48,49 This compatibility has been used to justify the preferential solubilization of one compound over another or the selection of a particular copolymer for solubilization of a drug; however these comparisons are usually done in a qualitative manner. In the present study we investigated the solubilization of several compounds by a series of diblock copolymers with varying core-forming block lengths. The drugs investigated were selected due to their range of molecular weights, molar volumes, and xsp values. Additionally, we selected relatively hydrophobic drugs as these drugs would partition into the hydrophobic core of the micelles. Amphiphilic drugs such as doxorubicin were not selected as these drugs have a tendency to partition into the hydrophilic/hydrophobic interface.50 In order to quantify the compatibility between the drug and copolymer, two assumptions were made with regard to the calculation of the xsp values: (1) the micellized drug was solubilized only within the core of the micelles, not in the MePEG corona or at the core corona interface and (2) the core of the micelles could be represented by bulk PCL. It was shown that the aqueous solubility of all drugs tested could be significantly increased by solubilization within the MePEG-b-PCL diblock copolymers. This is illustrated by the increase in aqueous solubility of all drugs by MePEG114-b-PCL19 (Tab. 2). During the drug solubilization experiments, it was found that the amount of drug solubilized for a given amount of copolymer increased up to a point after which there was precipitation. Precipitation occurred once the maximum solubilization capacity of the micelles was reached and the micelles could not encapsulate any more drug. At this point the total amount of drug in solution was the sum of the amount of drug held within the micelles plus that which was contained in the surrounding aqueous media (i.e., the aqueous solubility of the drug). Addition of drug beyond this point resulted in the saturation of the drug in solution and the subsequent precipitation of the drug and presumably some of the copolymer resulting in a drop in the loading efficiency and concentration of solubilized drug (Fig. 1). During solubilization, equilibration of the DOI 10.1002/jps

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Table 2. Drug Solubility and Drug/Core-Forming Block Compatibility Data

Drug

Aqueous Solubilitya (mol/L)

dsb (MPa1/2)

Vc (cm3/mol)

xspd

Micellar Solution Solubilitye (mol/L)

Solubility Increase

Solvent Used For Micelle Loading

2.55
104 1.17
106 6.75
104 2.99
108 1.24
103

28.02 25.65 28.94 25.64 24.83

332.6 559.8 134.9 253.1 229.8

7.89 6.45 4.00 2.91 1.93

6.69
104 1.25
103 3.65
103 3.89
103 5.92
103

2.62
1068
5.41
130100
4.77


Methanol Acetonitrile Acetonitrile Acetonitrile Ethanol

Etoposide Paclitaxel Plumbagin Curcumin Indomethacin a

Aqueous solubility at 378C after 3 h equilibration in PBS (0.01 M, pH 7.4). Solubility parameter for the drug as calculated by Eq. 5. c Molar volume of the drug, calculated according to Fedors.41 d Flory–Huggins interaction parameter calculated by Eq. 4. Note dp was calculated as 20.20 MPa1/2 at T ¼ 310 K. e Total solubility of drug in a 1%w/v solution of MePEG114-b-PCL19 in PBS (0.01 M, pH 7.4) after 3 h equilibration at 378C. b

drug with the micelles occurred quickly as shown by a time course incubation of the model drugs with MePEG17-b-PCL4 at their maximum micellar solubilization (Fig. 2). During the time course it was found that there was no change in the amount of drug solubilized. Therefore, in subsequent experiments an equilibration time of 3 h was employed. In the plots of maxdrug]micelle normalized to the concentration of copolymer as a function of xsp generated for each copolymer/drug combination, an inverse correlation was found (Fig. 3). This lends support to the theory that drug and core-forming block compatibility is a contributing factor in determining the degree of drug solubilization. For each copolymer, the amount of drug encapsulated within micelles increased in the following order: etoposide, paclitaxel, plumbagin, curcumin, indomethacin, as was predicted by the calculated xsp values. To our knowledge this is the first report providing evidence that the micellar solubilization of a series of drugs is directly related to the compatibility of the drug with the core-forming block as determined by the calculated xsp value. The data presented in Table 1 illustrate that the partition coefficient for the hydrophobic probe pyrene (Kv) increased significantly as the PCL block length increased from 2 to 19 repeat units.

This indicates that as the hydrophobic block increased, the hydrophobicity of the micelles increased resulting in more pyrene present in the micelle core and hence a higher Kv. The partition coefficients for the model drugs in each diblock copolymer were calculated. It can be seen in Table 3 that, in general, for each drug, the partition coefficient increased as the PCL block length increased. The trend is clear when comparing diblocks of the same MePEG molecular weight; however, the trend does not always hold when the MePEG block length changes, for example, when comparing the solubilization of plumbagin and paclitaxel by MePEG44-b-PCL12 and MePEG114-b-PCL19. An increase in the MePEG block length without a corresponding hydrophobic block length increase can decrease the aggregation number and core volume as well as introducing water into the core, all factors which may lead to a decrease in the partition coefficient of the drug.51 There was no clear correlation between the PCL block length and the partition coefficient of etoposide. This result may be attributed to the low affinity of the drug for PCL as predicted by the high xsp value and thus an increased PCL block length did not result in increased solubilization of the drug. The magnitude of the partition coefficient was governed by

Table 3. Drug Partition Coefficients in Diblock Copolymers Copolymer MePEG114-b-PCL19 MePEG44-b-PCL12 MePEG44-b-PCL7 MePEG17-b-PCL4 MePEG17-b-PCL3 MePEG17-b-PCL2 DOI 10.1002/jps

K Indomethacin 3

1.45
10 1.45
103 1.37
103 1.09
103 4.95
102 0

K Curcumin 7

4.97
10 4.85
107 3.46
107 3.19
107 2.07
107 3.47
106

K Plumbagin 3

1.68
10 1.79
103 1.56
103 1.46
103 9.90
102 5.31
102

K Paclitaxel 5

4.07
10 6.26
105 2.64
105 3.15
105 1.63
105 1.91
104

K Etoposide 6.20
102 7.21
102 1.21
103 6.79
102 0 0

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the aqueous solubility of the drug; therefore those drugs with the lowest aqueous solubility had the greatest partition coefficient and resulted in the greatest solubility increase (Tabs. 2 and 3). However, the total amount of drug solubilized in the micellar solutions was not related to the partition coefficient but was better described by the xsp values. Inspection of Figure 3 revealed that for any one drug, more drug was solubilized as the PCL block length increased, resulting in increased slopes of the linear regression lines. Plotting the max [drug]micelle per mole of copolymer as a function of PCL block length confirmed this correlation (Fig. 4). It has been shown in previous studies that the amount of drug solubilized is strongly dependent on the length of the hydrophobic block resulting in higher drug loading in micelles with longer hydrophobic block lengths.20,52,53 One reason for this trend is the pronounced effect that the hydrophobic block length has on the CMC and core volume. As can be seen in Table 2 and as previously discussed, as the hydrophobic block length increased, the CMC decreased. It has been shown that a decrease in the CMC results in an increase in the total number of copolymer molecules participating in the formation of micelles thus increasing the number of micelles in solution available for the solubilization of the solute.54 However, other groups have noted that as the hydrophobic block length of a series of copolymers increases, the aggregation number of the micelles correspondingly increases, resulting in a larger core volume providing more space for the solubilization of greater amounts of solute.55 With the exception of MePEG17-b-PCL2, which was multimodal, all the diblocks displayed monomodal particle size distributions in which the increase in PCL block length corresponded with a general increase in the hydrodynamic diameter. This increased diameter may be attributed to the increase in the core volume and hence providing a larger cargo space for more of the drug to be solubilized. It is difficult to determine whether an increase in the core volume is indeed the cause for the increased solubilization, as the increased diameter may be due to the increase in MePEG block length. Since MePEG17-b-PCL2 has a high CMC compared to the other diblocks in the series, it is speculated that the smallest diameter peak may be due to the presence of unassociated unimers in solution, whereas loosely formed aggregates with a very limited core volume may be responsible for the larger peaks. The JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 3, MARCH 2008

observation that MePEG17-b-PCL2 marginally increased the solubilization of the model drugs (Fig. 4) and the low Kv value (Tab. 1) suggests that particles formed by this copolymer contain a very small hydrophobic core volume.

CONCLUSIONS In these studies we synthesized a series of watersoluble MePEG-b-PCL diblock copolymers with varying MePEG and PCL block lengths. The length of the PCL block was controlled by the weight ratio of caprolactone added to the reaction mixture. This series of copolymers formed micelles in which the CMC was inversely proportional to the PCL block length whereas the partition coefficient of pyrene and hydrodynamic diameter were found to be directly dependent on the PCL block length. Solubilization of a variety of hydrophobic drugs by this series of diblock copolymers led to the development of empirical relationships between the amount of micellized drug and it’s compatibility with the core-forming block (PCL) and the drug, as determined by the calculation of the xsp as well as the PCL block length. It was shown that the increase in the aqueous solubility of the drugs was related to the partition coefficient; however, the amount of drug solubilized was correlated with the xsp.

ACKNOWLEDGMENTS These studies were financially supported by grants from the Canadian Institutes of Health Research (CIHR) and Natural Sciences and Engineering Research Council (NSERC) awarded to Helen Burt and by a CIHR Clinical Research Initiative Doctoral Research Award provided to Kevin Letchford.

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DOI 10.1002/jps