Solubilization of poorly water soluble drugs in micelles of hydrophobically modified hydroxypropylcellulose copolymers

Journal of Controlled Release 93 (2003) 59 – 68 www.elsevier.com/locate/jconrel

Solubilization of poorly water soluble drugs in micelles of hydrophobically modified hydroxypropylcellulose copolymers Mira F. Francis a, Mariella Piredda a, Francßoise M. Winnik a,b,* b

a Faculty of Pharmacy, University of Montreal, C.P. 6128 Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7 Department of Chemistry, University of Montreal, C.P. 6128 Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7

Received 11 July 2003; received in revised form 8 August 2003; accepted 8 August 2003

Abstract The main objective of this study is to exploit the solubilizing potential of hydroxypropylcellulose-g-polyoxyethylene alkyl ether (HPC-g-(POE)y-Cn) polymeric micelles towards poorly water soluble drugs in order to improve their oral bioavailability. Hydrophobically modified HPC graft copolymers of various compositions were synthesized by attaching hexadecyl or octadecyl residues to the hydrophilic HPC backbone via short POE linkers of different lengths. The onset of micellization was estimated by fluorescence spectroscopy. The hydrodynamic diameter of different HPC-g-(POE)y-Cn micelles was evaluated by dynamic light scattering (DLS). Cyclosporin A (CsA), a poorly water soluble immunosuppressant, was selected as model drug. CsA-loaded HPC-g-(POE)y-Cn micelles were prepared by a dialysis procedure and the amount of CsA incorporated in the micelles was assayed by high-performance liquid chromatography. Following 24-h incubation with human colon adenocarcinoma, Caco-2 cells, the cytotoxicity of various HPC-g-(POE)y-Cn copolymers was evaluated using the MTT colorimetric assay and compared to those of unmodified HPC and free (POE)y-Cn. In aqueous solution, different HPC-g(POE)y-Cn copolymers formed polymeric micelles of low critical association concentrations (CAC) and micelle mean diameters ranging from 78 to 90 nm. CsA loading into HPC-g-(POE)y-Cn polymeric micelles was significantly larger than in unmodified HPC. It increased with increasing number of (POE)y-Cn units grafted per HPC chain. On the cellular level, unmodified HPC showed no cytotoxicity, while free (POE)y-Cn molecules inhibited cell growth. Most importantly, the study revealed that HPCg-(POE)y-Cn exhibited no significant cytotoxic effect. D 2003 Elsevier B.V. All rights reserved. Keywords: Polymeric micelles; Cyclosporin A; Hydroxypropylcellulose (HPC); Solubilization; Cytotoxicity

1. Introduction Oral drug delivery continues to be the preferred gateway of a drug into the bloodstream, especially * Corresponding author. Faculty of Pharmacy, University of Montreal, C.P. 6128 Succ. Centre-ville, Montreal, Quebec, Canada H3C 3J7. Tel.: +1-514-343-6123; fax: +1-514-343-2362. E-mail address: [email protected] (F.M. Winnik). 0168-3659/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2003.08.001

when repeated or routine administration is necessary [1]. However, for effective delivery via the oral route, a therapeutic agent must first dissolve in the gastrointestinal lumen [2,3]. This represents a major challenge in the field of pharmaceutical drug formulation, namely the design of an oral dosage form for highly lipophilic drugs, that would enhance their notoriously poor bioavailability [4]. Among various strategies investigated for oral delivery of poorly water soluble

60

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

drugs, such as nanosuspensions [5], microemulsions [6], lipids [7] and liposomes [8], the use of polymers has gained much attention because of their high diversity, biocompatibility, biodegradability and the multiple functional groups they display for the conjugation of various pilot molecules [9]. Among the different polymer-based drug delivery systems, ‘‘polymeric micelles’’ represent a promising delivery vehicle for poorly water soluble pharmaceutical active ingredients [10,11]. Polymeric micelles form spontaneously when amphiphilic polymers, containing both hydrophilic and hydrophobic fragments, are dissolved in water. They consist of a hydrophobic core created upon assembly of the hydrophobic residues stabilized by a corona of highly hydrated hydrophilic polymeric chains [12]. A review of past achievements readily convinces one that, in the case of injectable drug formulations, polymeric micelles are highly effective drug delivery vehicles [13,14]. They have been largely ignored, however, in oral drug delivery formulations. We present here a study of polymeric micelles as drug carriers capable of solubilizing high levels of lipophilic drugs, so as to improve their absorption from the gastrointestinal tract and, consequently, their bioavailability following oral administration. Cyclosporin A (CsA), a highly lipophilic undecapeptide [15], was selected as model drug. Its water solubility, 23 Ag/ml at 20 jC, is extremely

low. CsA is administered as immunosuppressant to prevent allograft rejection following various organ transplantations [16]. Its oral administration has always been complicated due to the presence of the metabolizing enzyme cytochrome P-450 and the multidrug transporter P-glycoprotein (P-gp) in the small intestine [17]. We set out to investigate the suitability of polymeric micelles as carriers in the oral delivery of CsA. Hydroxypropylcellulose (HPC), a non-ionic watersoluble polymer, was selected to form the hydrophilic shell of the micelles. HPC is widely used as an excipient in oral solid dosage forms, in which it acts as a disintegrant [18], and as a binder in granulation [19]. It is essentially a non-toxic and non irritant polysaccharide [20]. Our strategy is (1) to prepare hydrophobically modified hydroxypropylcelluloses, (HPC-g-(POE)yCn) (see Fig. 1) by attachment of hydrophobic cetyl (C16) or octadecyl (C18) groups to hydrophilic HPC via a short polyoxyethylene (POE) linker of varying length; (2) to monitor the formation and characteristics of HPC-g-(POE)y-Cn micelles in aqueous environment; (3) to exploit the solubilizing power of HPC-g-(POE)y-Cn polymeric micelles towards CsA; and (4) to evaluate the cytotoxicity of the polymeric micelles towards epithelial intestinal cells. This approach will allow us to create nanosized entities, which entrap CsA in their hydrophobic core

Fig. 1. Chemical structure of HPC-g-(POE)y-Cn copolymer where n = 16 and 18 for (POE)y-C16 and (POE)y-C18, respectively, and y = 10 and 20 for (POE)10-Cn and (POE)20-Cn, respectively.

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

while forming a stable aqueous suspension via the steric stabilization promoted by hydrated HPC chains.

61

1

H-NMR spectroscopy in DMSO-d6 using a Bruker ARX-400 400 MHz spectrometer. 2.3. Critical association concentration of the HPC-g(POE)y-Cn polymeric micelles

2. Materials and methods 2.1. Materials CsA, polyoxyethylene (20) cetyl ether [(POE)20C16; C16H33(OCH2CH2)20OH; Brij 58R], polyoxyethylene (20) stearyl ether [(POE)20-C18; C18H37 (OCH2CH2)20OH; Brij 78R], polyoxyethylene (10) cetyl ether [(POE)10-C16; C16H33(OCH2CH2)10OH; Brij 56R], sodium chloride (NaCl), monobasic sodium phosphate (NaH2PO4), dibasic sodium phosphate (Na2HPO4), sodium dodecyl sulphate (SDS), HPC (MW 80,000 Da, molar substitution level (MS): 3.7, where MS is defined as the average number of alkylene oxide per anhydroglucose unit [21,22]) and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Deuterated dimethyl sulfoxide (DMSO-d6), triethylamine (Et3N) and trimethylamine hydrochloride (Me3N
HCl) were purchased from Aldrich Chemicals. HPLC-grade acetonitrile (ACN) and water were obtained from Anachemia Science. Ethanol (95%) was obtained from Commercial Alcohols (Brampton, ON, Canada). The Caco-2 cell line was purchased from American Type Culture Collection (ATCC) at passage 18. Dulbecco’s modified Eagle medium (DMEM), penicillin – streptomycin (10,000 U/ml penicillin G and 10,000 Ag/ml streptomycin), fetal bovine serum (FBS), 0.25% (w/v) trypsin –1 mM EDTA
4Na and non-essential amino acids (NEAA) were supplied from Invitrogen. 2.2. Synthesis of HPC-g-(POE)y-Cn copolymers Various hydrophobically modified HPC-g-(POE)yCn graft copolymers were synthesized as described elsewhere [23]. Briefly, the terminal hydroxyl group of POE was activated via tosylation. Subsequently, the tosylated (POE)y-Cn was coupled to HPC by a Williamson ether synthesis. The resulting HPC-g-(POE)yCn copolymers were purified by a soxhlet extraction with n-hexane to remove all free (POE)y-Cn residues. The level of (POE)y-Cn grafting was determined by

The critical association concentration (CAC) of the HPC-g-(POE)y-Cn copolymers was estimated by fluorescence spectroscopy using pyrene, a hydrophobic fluorescence probe that preferentially partitions into the hydrophobic core of the micelle. It undergoes changes in its photophysical properties as a result of the change in the micropolarity it experiences upon diffusion from bulk water (hydrophilic environment) into the micelle core (hydrophobic environment) [24,25]. An estimation of the CAC value was obtained by monitoring the changes in the ratio of the pyrene excitation spectra intensities [26] at k = 333 nm (I333) for pyrene in water and k = 336 nm (I336) for pyrene in the hydrophobic medium within the micelle core. The method used is exemplified in Fig. 2 where we present (A) a series of excitation spectra of pyrene in water and in the presence of increasing concentrations of the non ionic surfactant (POE)20-C18 and (B) traces of the concentration dependence of the ratio I336/I333 in solutions of (POE)10-C16, (POE)20-C16 and (POE)20C18. The critical micelle concentration (CMC) values are taken as the surfactant concentration corresponding to the onset of the increase in the ratio I336/I333. The CAC value was determined for each polymer solution from the intersection of two straight lines (the horizontal line with an almost constant value of the ratio I336/I333 and a line approximating the steep upward section of the sigmoidal curve (see Fig. 2B)). Samples for spectroscopic analysis were prepared as follows: A pyrene-saturated solution in deionized water was prepared by stirring overnight a suspension of pyrene in water, followed by filtration to remove excess undissolved pyrene microcrystals. Polymer or surfactant stock solutions (10 g/l) were prepared in pyrene-saturated water. They were left to equilibrate under agitation over 24 h away from light. Subsequently, the stock solutions were diluted with pyrenesaturated water to obtain solutions of varying polymer concentrations (10 3 – 10 g/l), which were further equilibrated under agitation for 24 h. Excitation spectra were monitored at kem = 390 nm (excitation and emission slits were set at 0.5 and 1.0 mm, respective-

62

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

two solutions to a final volume of 4 ml, keeping the polymer concentration constant (2.5 mg/l). The different mixtures were stirred 2 h at room temperature. To remove free CsA and form CsAloaded micelles, the mixtures were transferred into pre-swollen semi-permeable Spectra/PorR 1 dialysis membranes (molecular weight cutoff 6000 –8000 g/ mol, Spectrum Laboratories) and were dialysed against distilled water for 48 h. During the first 2 h, the water was exchanged two times (every hour) and then six times during the following 46 h. After a total of 48 h of dialysis, each solution was filtered through a 0.22-Am pore-size nylon filter and the filtrate was freeze-dried. 2.5. Micelle size measurement The hydrodynamic diameter of freeze-dried CsAfree and CsA-loaded HPC-based polymeric micelles in aqueous solution (5 mg/ml) was evaluated by dynamic laser light scattering (DLS) at 25 jC using a Brookhaven system with an Uniphase ABlue laser at wavelength of 532 nm and a scattering angle of 90j. All measurements were performed in triplicate; the data presented are the mean F S.D. Fig. 2. (A) Excitation spectra of pyrene-saturated aqueous solution monitored at kem 390 nm in absence (.) or presence of increasing concentrations of (o) 10 mg/l, (n) 20 mg/l and (x) 40 mg/l (POE)20-C18. (B) Changes in the I336 nm/I333 nm ratio of pyrene fluorescence intensity as a function of concentration (0.3 – 2.5  103 mg/l) for (.) (POE)10-C16, (x) (POE)20-C16 and ( w ) (POE)20-C18.

ly). Steady-state fluorescence spectra were measured at 25 jC with a Fluorolog Tau-3 spectrometer (JobinYvon Horiba) equipped with a GRAMS/32 (Galactic Ind.) data analysis system. 2.4. Physical loading of CsA in HPC-g-(POE)y-Cn polymeric micelles A dialysis method was employed to prepare CsAloaded polymeric micelles. A HPC-g-(POE)y-Cn solution (5 mg/ml) in deionized water (Milli-Q water purification system) and a CsA solution (5 mg/ml) in ethanol were prepared separately. Each solution was stirred 1 h at room temperature. Subsequently, different mixtures of polymer with varying CsA concentrations (2.5 – 40% w/w) were prepared by mixing the

2.6. HPLC analysis The micelle-loaded CsA was extracted from freezedried micelles using acetonitrile (ACN). The resulting suspensions were sonicated in an ultrasonic bath for 10 min, then agitated for 8 h. They were then filtered through 0.45-Am pore-size Gelman GHP Acrodisc filters and assayed by high performance liquid chromatography (HPLC) [27] using an Agilent Technologies HP 1100 chromatography system with a quaternary pump, a UV-visible detector, a column thermostat and a HP Vectra computer equipped with HP-Chemstation software. A symmetryR octadecylsilane C18 (5 Am, 250  4.6 mm I.D.) column and the corresponding guard column of similar characteristics (20  3.9 mm I.D.) were used. The mobile phase consisted of ACN/water (80:20) with a flow rate of 1.2 ml/min. The column was thermostated at 70 jC. In all cases, the injection volume was 50 Al and the run time was 10 min. The CsA peak, monitored at 210 nm, appeared at a retention time of 6.5 min. A CsA calibration curve was prepared using standard solutions of concentrations ranging from 3.125 to 400 mg/

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

l, with a first order correlation coefficient (r2) greater than 0.99. Finally, the extent of drug loading (DL) was calculated using Eq. (1): DL ð%Þ ¼ 100ðWC =WM Þ

ð1Þ

where WC is the weight of CsA loaded in micelles calculated using the calibration curve and WM is the weight of CsA-loaded micelles before extraction with ACN. It should be noted that the minimum amount of CsA detectable by this assay is 0.05% w/w.

phosphate-buffered saline (PBS; 75 mM NaCl, 53 mM Na2HPO4, 13 mM NaH2PO4, pH 7.2) were added to each well. After 4 h of additional incubation at 37 jC, 100 Al of 10% SDS in 0.01 N HCl solution were added to each well to stop the reaction and to ensure solubilization of formazan crystals. The plates were incubated overnight at 37 jC, after which the optical density values were measured at 570 nm using a multiwell-scanning spectrophotometer (PowerWave; Biotek Instruments).

2.7. Cell culture

3. Results and discussion

The human colon adenocarcinoma cells, Caco-2, were grown as described previously [28]. Briefly, Caco-2 cells were routinely maintained in DMEM with 4.5 g/l D-glucose, supplemented with 10% (v/v) heat-inactivated FBS, 1% (v/v) NEAA and 1% (v/v) penicillin – streptomycin antibiotics solution (100 U/ ml penicillin G and 100 Ag/ml streptomycin). Cells were allowed to grow in a monolayer culture in 75 cm2 T-flasks in an incubator at 37 jC with controlled atmosphere containing 5% CO2 and 90% relative humidity. Culture medium was changed every 48 h and cells were passaged at 80 –90% confluency at a split ratio of 1:3 using 0.25% trypsin –1 mM EDTA.

3.1. Characterization of the modified HPC copolymers

2.8. Colorimetric MTT cytotoxicity assay The cell viability in presence of unmodified HPC, free (POE)20-C16, (POE)20-C18 and (POE)10-C16 surfactants, or various HPC-g-(POE)y-Cn copolymers was evaluated using the MTT colorimetric assay. Caco-2 cells were seeded in triplicate in 96-well culture plates at a density of approximately 5  104 cells in 100 Al of cell culture medium per well. The cells were cultured at 37 jC in a humidified atmosphere of 5% CO2 in air for 48 h. Subsequently, increasing concentrations of unmodified HPC, free (POE)y-Cn residues or various HPC-g-(POE)y-Cn copolymers (0 – 10 g/l in culture medium) were added to the cells. Caco-2 cells were further incubated at 37 jC for 24 h. Thereafter, cell viability was determined by a MTT test according to the procedure described by Mosmann [29]. The test is based on mitochondrial dehydrogenase cell activity as an indicator of cell viability. Ten microliters of 5 mg/ml MTT solution in

63

Various hydrophobically modified HPC copolymers were synthesized (Fig. 1 and Table 1). They differed in three molecular aspects: (i) the level of grafting, i.e. the number of (POE)y-Cn substituents linked to the HPC backbone; (ii) the size of the (POE)y moiety; and (iii) the size of the hydrophobic alkyl group (Cn; hexadecyl group or octadecyl group). They were purified thoroughly to ensure complete removal of unreacted (POE)y-Cn and residual solvent. Analysis by GPC confirmed that the molecular weight of HPC was not affected significantly by the chemical modification (data not shown). The degree of grafting was determined by 1H-NMR spectroscopy, comparing the relative intensity of signals due to the POE and Cn protons to the intensity of signals due to protons linked to the polysaccharide backbone [23]. The association properties of the amphiphilic polymers in water, summarized in Table 2, were gathered from fluorescence probe experiments, which yield the CAC, and from DLS measurements, which give the average size of the polymeric micelles, as discussed in detail in the following sections. 3.1.1. Critical association concentration of HPC-g(POE)y-Cn micelles The polymer concentration corresponding to the onset of micellization was deduced from a fluorescence assay, based on the changes in the photophysics of pyrene, a hydrophobic probe added in minute amounts to a polymer solution. This probe partitions preferably in hydrophobic microenvironments, as de-

64

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

Table 1 Molecular and physicochemical characteristics of (POE)y-Cn surfactants and HPC-g-(POE)y-Cn copolymers Polymer sample

HPC (POE)10-C16 (POE)20-C16 (POE)20-C18 HPC-g-(POE)10-C16 HPC-g-(POE)10-C16 HPC-g-(POE)20-C16 HPC-g-(POE)20-C16 HPC-g-(POE)20-C18 HPC-g-(POE)20-C18

Grafted (POE)y-Cna (mol%)

Average number of (POE)y-Cn units per HPC chain

CAC Polymer concentrationb (mg/l)

(POE)y-Cn concentration (  10 6 mol/l)

0 – – – 0.9 F 0.1 4.7 F 0.1 1.1 F 0.1 3.9 F 0.1 1.1 F 0.1 3.1 F 0.1

0 – – – 5 19 5 18 5 16

– 4.3 F 1.0c 4.6 F 0.6c 3.7 F 0.5c 75 F 14 17 F 3 65 F 12 15 F 5 135 F 10 22 F 6

– 6.3 F 1.4c 4.1 F 0.5c 3.2 F 0.4c 1.8 F 0.3 2.1 F 0.4 1.9 F 0.3 1.5 F 0.5 3.9 F 0.3 1.8 F 0.4

a

Determined by 1H-NMR measurement in DMSO-d6 [23]. Determined by change in I336 nm/I333 nm ratio of pyrene fluorescence with log polymer concentration at 25 jC. c These values refer to the critical micelle concentrations (CMC) of the (POE)y-Cn surfactants. b

scribed in detail in Section 2 where we present the approach to determine the CMC values of (POE)y-Cn. Aqueous solutions of different HPC-g-(POE)y-Cn copolymers exhibited a shift in the pyrene excitation spectra, similar to that observed with solutions of (POE)y-Cn. Plots of the I336/I333 ratios versus the logarithm of the concentration of the aqueous solutions of HPC-g-(POE)y-Cn of varying compositions are shown in Fig. 3. The CAC values of the copolymers in water are given in Table 1, in units of mg/l of polymer and 10 7 mol/l of (POE)y-Cn units. We notice that, for a given type of hydrophobic group grafted on HPC, the CAC (mg/l) decreases with increasing molar content of (POE)y-Cn residues, reflecting the increase in hydrophobicity of the polymer and consequent enhanced stability of the poly-

meric micelle. The major driving force for the assembly of amphiphilic copolymers in water is the removal of hydrophobic fragments from the aqueous surroundings resulting in the formation of micelles consisting of a hydrophobic core stabilized by hydrated hydrophilic chains exposed into water [30]. The CAC values are reported also in terms of (POE)y-Cn molar concentrations (Table 1). We notice that, as anticipated, the CMC of the free surfactants with longer (POE) segments is lower than the CMC of the corresponding surfactant with a shorter (POE) head group (see for example (POE) 20 -C 16 vs. (POE)10-C16). Conversely, for the same POE segment length (20 units), the surfactant bearing a C18 group has lower CMC than the surfactant having C16 group. The CAC values of the polymeric micelles, reported

Table 2 Size of CsA-free and CsA-loaded HPC-g-(POE)y-Cn polymeric micelles and maximum CsA loading capacity (% w/w) of the micelles Polymer sample

HPC-g-(POE)10-C16 HPC-g-(POE)10-C16 HPC-g-(POE)20-C16 HPC-g-(POE)20-C16 HPC-g-(POE)20-C18 HPC-g-(POE)20-C18 a b

Grafted (POE)y-Cn (mol%)

Maximum a CsA loading (% w/w)

0.9 F 0.1 4.7 F 0.1 1.1 F 0.1 3.9 F 0.1 1.1 F 0.1 3.1 F 0.1

2.3 F 0.2 5.3 F 0.3 2.5 F 0.3 6.7 F 0.4 1.7 F 0.3 4.8 F 0.3

Mean diameterb F S.D. (nm) CsA-free polymeric micelles

CsA-loaded polymeric micelles

85 F 2 80 F 1 90 F 1 78 F 1 85 F 1 83 F 2

65 F 2 50 F 3 66 F 2 44 F 4 75 F 4 74 F 3

Determined by HPLC analysis with UV detection at 210 nm. Determined by DLS measurements of 5 mg/ml aqueous solution at 25 jC with a scattering angle of 90j.

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

65

3.2. Characterization of the CsA-loaded polymeric micelles

Fig. 3. Changes in the I336 nm/I333 nm ratio of pyrene fluorescence intensity as a function of concentration (1 – 1  104 mg/l) for the (o) HPC-g-(POE)10-C16 (1 mol%), (.) HPC-g-(POE)10-C16 (4.7 mol%), (5) HPC-g-(POE)20-C16 (1 mol%), (n) HPC-g(POE)20-C16 (4 mol%), (D) HPC-g-(POE)20-C18 (1 mol%) and (E) HPC-g-(POE)20-C18 (3.1 mol%) copolymers.

in units of alkyl group concentration, are nearly identical, within experimental errors, independently of the (POE)y-Cn grafting level. Overall, though, the onset of micellization of polymeric micelles occurs at lower alkyl group concentrations than the CMC of the corresponding surfactants. 3.1.2. Size of the HPC-g-(POE)y-Cn polymeric micelles The hydrodynamic diameters of polymeric micelles, determined by DLS, ranged from 40 to 90 nm (Table 2). In all cases, the size distributions were unimodal, indicative of the absence of free polymer chains and of large polymeric aggregates. We note that, for each (POE)y-Cn substituent, the size of micelles decreases with increasing level of grafting on the HPC backbone. Moreover, CsA-free HPC-g(POE)y-Cn micelles are generally larger than the corresponding CsA-loaded micelles independently of the size of the hydrophobic substituent and of the level of modification. It should be pointed out that the uptake of particles within the intestine and the extent of drug absorption increase with decreasing particle size and increasing specific surface area [31]. Thus, the small size exhibited by all the polymeric micelles studied here shows a favorable trend towards oral drug delivery.

Cyclosporin A was incorporated into the polymeric micelles by a dialysis method which involved treatment of an aqueous polymer solution with a solution of CsA in ethanol, followed by extensive dialysis of the mixed solution against water. The amount of CsA released upon dissolution of the CsA-loaded polymeric micelles was then determined by an HPLC assay and plotted against the CsA concentration added initially for each preparation. The amount of free CsA is expected to be low since the undissolved fraction of the drug was removed by filtration after the dialysis procedure. We evaluated first the ability to solubilize CsA in (POE)y-Cn micelles and by unmodified HPC (Fig. 4). (POE)10-C16, (POE)20-C16 and (POE)20-C18 micelles were able to incorporate relatively high levels of CsA; 17.5%, 21% and 15.5% w/ w, respectively. Interestingly, CsA has some affinity for unmodified HPC, with a level of CsA incorporation of 1.4% w/w. This effect may reflect the presence of some ‘‘hydrophobic pockets’’ within the HPC structure. The structure of HPC is known to be heterogenous with some longer isopropoxyl side chains [32]. Next, we assessed the incorporation of CsA within polymeric micelles (Table 2 and Fig. 5). For all copolymer samples, the amount of CsA incorporated in the micelles was larger than in the case of unmodified HPC (Fig. 4). The maximum amount of CsA loaded within the different micelles

Fig. 4. CsA loading (% w/w) in micelles of (POE)10-C16 (.), (POE)20-C16 (n) and (POE)20-C18 (E) surfactants as well as unmodified HPC polymer (x) at 2.5 – 40 (% w/w) CsA initial loading in 2.5 mg/ml micelles. Mean F S.D. (n = 3).

66

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

CsA concentration < 15%, then increases rapidly as the initial CsA concentration exceeds 15%. 3.3. In vitro cytotoxicity study

Fig. 5. CsA loading (% w/w) in micelles of ( w ) HPC-g-(POE)10-C16 (1 mol%), (x) HPC-g-(POE)10-C16 (4.7 mol%), (5) HPC-g(POE)20-C16 (1 mol%), (n) HPC-g-(POE)20-C16 (4 mol%), (D) HPC-g-(POE)20-C18 (1 mol%) and (E) HPC-g-(POE)20-C18 (3.1 mol%) copolymers at 2.5 – 40 (% w/w) CsA initial loading in 2.5 mg/ml micelles. Mean F S.D. (n = 3).

ranged from 4.8% to 6.7% (w/w) depending on the level of grafting and on the type of grafted hydrophobic modifier. Several trends are apparent if one compares the highest CsA loading percentages (at 40% w/w CsA added initially) determined for the various polymers (Table 2). The percentage of CsA loading increases with increasing grafting level of (POE)y-Cn on HPC backbone. For a constant number of (POE)y-Cn units ( f 5 units per HPC chain), the CsA loading achieved with HPC-g-(POE)20-C18 (1.7% w/w) is lower than those determined for HPC-g-(POE)10-C16 (2.3% w/w) and HPC-g-(POE)20-C16 (2.5% w/w). These results are consistent with the fact that (POE)10-C16 and (POE)20-C16 micelles are more effective in solubilizing CsA than (POE)20-C18 micelles, 17.5%, 21% and 15.5% (w/w), respectively (Fig. 4). This may be taken as an indication that the micropolarity of the hexadecyl-POE chains core might present a better solubilizing environment for CsA than that offered by the octadecyl-POE moieties. It is interesting to note also that, at low CsA initial concentrations ( < 15% w/w), HPC-g-(POE)y-Cn polymeric micelles exhibit a solubilizing/loading trend different from that displayed by (POE)y-Cn micelles (Figs. 4 and 5). While the degree of CsA incorporation within (POE)y-Cn micelles increases steadily with increasing initial CsA concentration, the level of CsA incorporation in polymeric micelles remains low and nearly constant with initial

The cytotoxicity of HPC, (POE)20-C16, (POE)20C18, (POE)10-C16 and various HPC-g-(POE) y-Cn copolymers was examined using a MTT assay performed with Caco-2 cells. This study is of particular importance in view of the known toxicity of the (POE)y-Cn surfactants when they are not linked to polymers. The dose-dependent viability of Caco-2 cells treated with unmodified HPC, free (POE)20-C16 surfactant and HPC-g-(POE)20-C16 (4 mol%) copolymer over 24 h is presented in Fig. 6. Similar cell viability patterns were observed in the case of free and HPC-grafted (POE)10-C16 as well as (POE)20-C18 (results not shown). As anticipated, free POE-Cn surfactants inhibited cell growth even at concentrations below 1 g/l. Such deleterious effect is consistent with the propensity of polyoxyethylene alkyl ether surfactants to affect membrane integrity [33]. The polysaccharide HPC showed no toxicity up to a concentration of 10 g/l, confirming the inertness of HPC [34]. It may also be pointed out that the World Health Organization (WHO) has not specified an acceptable daily intake for HPC since the levels consumed were not considered to represent any hazard to health [35]. Of interest to our study is the fact that none of the HPC-g-(POE) y -C n copolymers exhibited any significant toxicity towards Caco-2

Fig. 6. Effect of (.) unmodified HPC, (D) free (POE)20-C16 surfactant and (o) HPC-g-(POE)20-C16 (4 mol%) copolymer concentration (0 – 10 g/l) on Caco-2 cell viability measured by MTT assay following 24-h incubation at 37 jC/5% CO2. Mean F S.D. (n = 3).

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

cells, up to concentrations of 10 g/l. This important result indicates that by grafting the (POE)y-Cn residues on HPC, we succeeded in eliminating their toxic effect on cells. The non-toxic hydrophilic HPC chains forming the polymeric micelle outer shell are in contact with the cells and effectively insulate them from the surfactant residues assembled in the inner core.

4. Conclusion In summary, we have demonstrated that aqueous solutions of HPC-g-(POE)y-Cn copolymers of various compositions increase the solubility of the lipophilic drug, cyclosporin A, through solubilization of the drug in the hydrophobic core of polymeric micelles, while the hydrophilic, non-toxic HPC outer shell stabilizes the system in the aqueous milieu. The molar contents, as well as the length of the hydrophobic substituent, have an important effect on the solubilizing power of HPC-g-(POE)y-Cn polymeric micelles towards hydrophobic drugs. The small size of the polymeric micelles, the very low concentration of micellization onset and the absence of cytotoxic effects towards intestinal cells represent promising characteristics for the development of a novel polymeric drug carrier for the oral delivery of poorly water soluble drugs.

Acknowledgements This work was supported by the Natural Sciences and Engineering Research Council of Canada under its strategic grants program. M.F. Francis acknowledges a scholarship from the Rx&D Health Research Foundation. We would like to thank Dr. Patrice Hildgen and Dr. Albert Adam for providing some useful pieces of equipment.

References [1] E.C. Lavelle, S. Sharif, N.W. Thomas, J. Holland, S.S. Davis, The importance of gastrointestinal uptake of particles in the design of oral delivery systems, Adv. Drug Deliv. Rev. 18 (1995) 5 – 22.

67

[2] D. Horter, J.B. Dressman, Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract, Adv. Drug Deliv. Rev. 46 (2001) 75 – 87. [3] W.N. Charman, V.J. Stella, Transport of lipophilic molecules by the intestinal lymphatic system, Adv. Drug Deliv. Rev. 7 (1991) 1 – 14. [4] A.T. Florence, P.U. Jani, Particulate delivery: the challenge of the oral route, in: A. Rolland (Ed.), Pharmaceutical Particulate Carriers: Therapeutic Applications, Marcel Dekker, New York, NY, 1993, pp. 65 – 107. [5] R.H. Muller, C. Jacobs, O. Kayser, Nanosuspensions as particulate drug formulations in therapy. Rationale for development and what we can expect for the future, Adv. Drug Deliv. Rev. 47 (2001) 3 – 19. [6] K. Itoh, S. Matsui, Y. Tozuka, T. Oguchi, K. Yamamoto, Improvement of physicochemical properties of N-4472: Part II. Characterization of N-4472 microemulsion and the enhanced oral absorption, Int. J. Pharm. 246 (2002) 75 – 83. [7] T. Gershanik, S. Benita, Self-dispersing lipid formulations for improving oral absorption of lipophilic drugs, Eur. J. Pharm. Biopharm. 50 (2000) 179 – 188. [8] S. Minato, K. Iwanaga, M. Kakemi, S. Yamashita, N. Oku, Application of polyethyleneglycol (PEG)-modified liposomes for oral vaccine: effect of lipid dose on systemic and mucosal immunity, J. Control. Release 89 (2003) 189 – 197. [9] A.K. Andrianov, L.G. Payne, Polymeric carriers for oral uptake of microparticulates, Adv. Drug Deliv. Rev. 34 (1998) 155 – 170. [10] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev. 47 (2001) 113 – 131. [11] V.S. Trubetskoy, Polymeric micelles as carriers of diagnostic agents, Adv. Drug Deliv. Rev. 37 (1999) 81 – 88. [12] M.C. Jones, J. Leroux, Polymeric micelles—a new generation of colloidal drug carriers, Eur. J. Pharm. Biopharm. 48 (1999) 101 – 111. [13] X. Zhang, H.M. Burt, G. Mangold, D. Dexter, D. Von Hoff, L. Mayer, W.L. Hunter, Anti-tumor efficacy and biodistribution of intravenous polymeric micellar paclitaxel, Anticancer Drugs 8 (1997) 696 – 701. [14] Y. Mizumura, Y. Matsumura, M. Yokoyama, T. Okano, T. Kawaguchi, F. Moriyasu, T. Kakizoe, Incorporation of the anticancer agent KRN5500 into polymeric micelles diminishes the pulmonary toxicity, Jpn. J. Cancer Res. 93 (2002) 1237 – 1243. [15] W. Vine, L. Bowers, Cyclosporine: structure, pharmacokinetics, and therapeutic drug monitoring, Crit. Rev. Clin. Lab. Sci. 25 (1987) 275 – 311. [16] A. Hamwi, A. Salomon, R. Steinbrugger, M. Fritzer-Szekeres, W. Jager, T. Szekeres, Cyclosporine metabolism in patients after kidney, bone marrow, heart-lung, and liver transplantation in the early and late posttransplant periods, Am. J. Clin. Pathol. 114 (2000) 536 – 543. [17] V.J. Wacher, J.A. Silverman, Y. Zhang, L.Z. Benet, Role of Pglycoprotein and cytochrome P450 3A in limiting oral absorption of peptides and peptidomimetics, J. Pharm. Sci. 87 (1998) 1322 – 1330.

68

M.F. Francis et al. / Journal of Controlled Release 93 (2003) 59–68

[18] Y. Machida, T. Nagai, Directly compressed tablets containing hydroxypropyl cellulose in addition to starch or lactose, Chem. Pharm. Bull. 22 (1974) 2346 – 2351. [19] G.W. Skinner, W.W. Harcum, P.E. Barnum, J.H. Guo, The evaluation of fine particle hydroxypropylcellulose as a roller compaction binder in pharmaceutical applications, Drug Dev. Ind. Pharm. 25 (1999) 1121 – 1128. [20] Final report on the safety assessment of hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, hydroxypropyl methylcellulose and cellulose gum, J. Am. Coll. Toxicol. 5 (1986) 1 – 60. [21] Y. Tezuka, K. Imai, M. Oshima, T. Chiba, Determination of substituent distribution in cellulose ethers by 13C- and 1H-NMR studies of their acetylated derivatives: O-(2-hydroxypropyl)cellulose, Carbohydr. Res. 196 (1990) 1 – 10. [22] M.G. Wirick, M.H. Waldman, Some solution properties of fractionated water-soluble hydroxypropylcellulose, J. Appl. Polym. Sci. 14 (1970) 579 – 597. [23] M. Piredda, M.F. Francis, F.M. Winnik, Macromolecules (2003) (submitted for publication). [24] C.L. Zhao, M.A. Winnik, G. Riess, M.D. Croucher, Fluorescence probe techniques used to study micelle formation in water-soluble block copolymers, Langmuir 6 (1990) 514 – 516. [25] D.C. Dong, M.A. Winnik, The Py scale of solvent polarities, Can. J. Chem. 62 (1984) 2560 – 2565. [26] M. Wilhelm, C.L. Zhao, Y. Wang, R. Xu, M.A. Winnik, J.L. Mura, G. Riess, M.D. Croucher, Poly(styrene-ethylene oxide) block copolymer micelle formation in water: a fluorescence probe study, Macromolecules 24 (1991) 1033 – 1040.

[27] E. Ugazio, R. Cavalli, M.R. Gasco, Incorporation of cyclosporin A in solid lipid nanoparticles (SLN), Int. J. Pharm. 241 (2002) 341 – 344. [28] I.J. Hidalgo, T.J. Raub, R.T. Borchardt, Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability, Gastroenterology 96 (1989) 736 – 749. [29] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55 – 63. [30] Z. Gao, A. Eisenberg, A model of micellization for block copolymers in solution, Macromolecules 26 (1993) 7353 – 7360. [31] A.T. Florence, N. Hussain, Trancytosis of nanoparticle and dendrimer delivery systems: evolving vistas, Adv. Drug Deliv. Rev. 50 (2001) S69 – S89. [32] E.D. Klug, Some properties of water-soluble hydroxyalkyl celluloses and their derivatives, J. Polymer Sci., Part C 36 (1971) 491 – 508. [33] D. Dimitrijevic, A.J. Shaw, A.T. Florence, Effects of some non-ionic surfactants on transepithelial permeability in Caco2 cells, J. Pharm. Pharmacol. 52 (2000) 157 – 162. [34] S. Obara, H. Muto, H. Kokubo, N. Ichikawa, M. Kawanabe, O. Tanaka, Primary dermal and eye irritability tests of hydrophobically modified hydroxypropyl methylcellulose in rabbits, J. Toxicol. Sci. 17 (1992) 21 – 29. [35] FAO/WHO, Evaluation of certain food additives and contaminants: thirty-fifth report of the joint FAO/WHO expert committee on food additives, Tech. Rep. Ser. Wld. Hlth. Org. No. 789 (1990).