Block copolymer micelles for drug delivery: loading and release of doxorubicin

Journal of Controlled Release 48 (1997) 195–201

Block copolymer micelles for drug delivery: loading and release of doxorubicin a b c c c b, G. Kwon , M. Naito , M. Yokoyama , T. Okano , Y. Sakurai , K. Kataoka * a

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6 G 2 N8 b Department of Materials Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda, Japan c Institute for Biomedical Engineering, Tokyo Women’ s Medical College, Kawada-cho, Shinjuku-ku, Tokyo 162, Japan Received 16 April 1996; revised 10 September 1996; accepted 7 October 1996

Abstract Micelles of poly(ethylene oxide)-block-poly(b-benzyl-L-aspartate) (PEO-PBLA) were loaded with doxorubicin (DOX) and were characterized in relation to their use as drug vehicles. First, an oil-in-water emulsion method was developed to load DOX in PEO-PBLA micelles. The level of DOX in PEO-PBLA micelles was 5–12% w / w. Whereas the mean diameter of unloaded, PEO-PBLA micelles was ca., 19 nm, the mean diameter of PEO-PBLA micelles loaded with DOX was ¯37 nm. Minimal chemical degradation of DOX occurred as a result of loading in PEO-PBLA micelles. In addition, DOX in PEO-PBLA micelles was less susceptible to chemical degradation than free DOX in aqueous solution. There was evidence for retention of DOX in PEO-PBLA micelles even after freeze-drying and reconstitution in water. Lastly, PEO-PBLA micelles served as drug depots, slowly releasing DOX (days), even in the presence of 10% w / v serum albumin. The results suggest a number of pharmaceutical advantages of PEO-PBLA micelles for the delivery of DOX.  1997 Elsevier Science B.V. Keywords: Copolymer micelles; Doxorubicin; Drug delivery

1. Introduction Block copolymers self-assemble into micelles showing promise as long circulating vehicles for drug delivery [1–6]. The micelles have core / shell structures of AB or ABA block copolymers, where their shells consist of hydrophilic blocks, e.g. poly(ethylene oxide) (PEO), and their cores consist of hydrophobic blocks, e.g. poly(b-benzyl-L-aspartate) (PBLA). The cores serve as nonaqueous reservoirs for hydrophobic drugs [2,3], and the shells interact with the biological milieu (brush of PEO), affecting *Corresponding author. Tel.: 181 471 241501, ext. 4310.

their pharmacokinetics and disposition [1]. Block copolymers having PEO blocks form micelles that are small relative to other drug vehicles (except viruses), having diameters of 20–30 nm. For micelle-forming, poly(ethylene oxide-blockaspartate)-doxorubicin (DOX) conjugates, there is evidence for prolonged circulation times, low uptake by the mononuclear phagocyte system (MPS), and passive accumulation at solid tumors of mice [1,5]. Furthermore, there is evidence for the complete regression of several tumors, e.g. colon 26 carcinoma, as a result of treatment by micelle-forming, poly(ethylene oxide-block-aspartate)-DOX conjugates [1].

0168-3659 / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0168-3659( 97 )00039-4

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Herein, we report on a physical method of loading DOX into PEO-PBLA micelles (chemical methods of loading drugs into block copolymer micelles are given elsewhere [1]). Notably, there are negligible levels of DOX loaded into PEO-PBLA micelles by simply equilibrating PEO-PBLA and DOX in water. The loading of hydrophobic drugs into PEO-PBLA micelles ideally occurs at high levels, with high efficiency, and without chemical degradation of drug. Thus, an oil-in-water (o / w) emulsion method was proposed to fulfill these requirements. It is known that block copolymers stabilize emulsions [7]. The loading method of PEO-PBLA micelles may be suitable for a host of other hydrophobic drugs. We also report on the properties of PEO-PBLA micelles loaded with DOX. First, evidence for the loading of DOX in PEO-PBLA micelles was obtained by dynamic light scattering (DLS) measurements and by size exclusion chromatography (SEC)HPLC. Second, evidence for the chemical stability of DOX loaded in PEO-PBLA micelles was obtained by reverse-phase HPLC and by visible spectroscopy, and comparisons were made with free drug. Lastly, evidence for the release of DOX from PEO-PBLA micelles was obtained by fluorescence spectroscopy and by SEC-HPLC.

2. Materials and methods DOX–HCl was purchased from Sanraku Co., Japan. The purity of DOX was checked by reversephase HPLC. Other chemicals were of reagent grade. PEO-PBLA was synthesized and characterized as described previously [6]. In brief, b-benzyl-L-aspartate N-carboxyanhydride was polymerized by addition of a-methyl-v-amino-poly(ethylene oxide) (12 000 g / mol). The reaction was done in a solution of chloroform and DMF (10:1) at 358C. PEO-PBLA was precipitated in diethyl ether at 08C and dried under vacuum. The PBLA block had a degree of polymerization of 20 by 1 H-NMR. DOX–HCl (0.50–3.0 mg) was added to CHCl 3 (1.0 ml) and solubilized by the addition of 2.0 equivalents of triethylamine with sonication (30 s). PEO-PBLA (5.0 mg) was dissolved in 10.0 ml of distilled water with sonication (30 s). The CHCl 3 solution of DOX was added to the stirred aqueous

solution, forming the o / w emulsion. The o / w emulsion was kept overnight in the dark at 258C and in an open atmosphere, allowing evaporation of CHCl 3 . Free DOX was removed from PEO-PBLA micelles by ultrafiltration (Amicon YM-30). DOX loaded in the micelles was quantitated by measuring its UV absorbance at 485 nm after addition of DMF (4-fold volume). Samples were diluted with 0.10 M PBS, pH 7.4, to 10 mg / ml DOX and stored in a liquid state at 258C, in a frozen state, or in a freeze-dried state (Eyela, FD-5N, Japan). The mean, hydrodynamic diameters of PEOPBLA micelles were obtained by DLS at 258C (Photal, DLS-700, Otsuka Electronics, Japan) [8]. The instrument was equipped with an argon laser at a wavelength of 488 nm. A scattering angle of 908 was used. The level of DOX was 10 mg / ml. The estimation of the size distribution was carried out from correlation function profile by the histogram method. SEC of PEO-PBLA micelles loaded with DOX was done with an Asahipak GS-520H column (molecules with molecular weights in excess of 300 000 g / mol based on pullulan standards eluted at the void volume). Samples of 100 ml (0.10 M PBS, pH 7.4) were injected, and separations were done at 408C using flow rates of 1.0 ml / min (JASCO, Intel. Hyper LC, Japan). DOX was quantitated by measurement of its UV absorbance at 485 nm (JASCO, 870-UV/ VIS detector, Japan) and by measurement of its fluorescence (JASCO, 820-FP, Japan) (l ex 5471 nm, l em 5595 nm). The level of DOX was 10 mg / ml. Reverse-phase chromatography of PEO-PBLA micelles loaded with DOX was done with a column from Waters (mBondsphere 5m C4-300, USA). Samples of 20 ml diluted to 10 mg / ml DOX with 0.10 M sodium phosphate buffer, pH 7.4, were separated at 408C at a flow rate of 1.0 ml / min. The mobile phase was a linear gradient mixture of an aqueous solution of 1% acetic acid and acetonitrile (15% v / v to 85% v / v). Detection of DOX was done by measuring its UV absorbance at 485 nm. The following samples were studied: (i) free DOX, (ii) solution of DOX and PEO-PBLA prepared by simple equilibration in buffered solution (PEO-PBLA1DOX), and (iii) PEO-PBLA micelles loaded with DOX. The absorbance of DOX at 485 nm was followed

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micelles, ¯19 nm. In the SEC study, DOX eluted at the void volume consistent with its loading in PEOPBLA micelles. Free DOX, on the other hand, adsorbed to gel of the column and did not elute off the column. The maximum yield of DOX loaded into PEOPBLA micelles was 65% based on the initial level of DOX, with the DOX content of the micelles being 12% (w / w). The yield attained by the dialysis method was 8% [4]. Analysis of DOX in PEO-PBLA micelles by reverse-phase HPLC gave evidence that most of the DOX was loaded without degradation (Fig. 1). DOX eluted primarily in its monomeric form at ¯9 min, with a small portion of degradation product eluting at ¯13 min, area ratio52.2:1. Free DOX also eluted as a single peak at ¯9 min, as was the case where DOX was simply equilibrated in an aqueous solution of PEO-PBLA, i.e. DOX1PEO-PBLA. Although degradation products of DOX in alkaline, aqueous solution have not been completely elucidated [9], degradation of DOX does occur. The loss of the amino sugar along with the aromatization of the aglycone moiety was suggested to be the major degradation pathway of DOX [9]. Efforts to prevent degradation of drug during loading of PEO-PBLA micelles are ongoing, focusing on the levels of the components, e.g. level of triethylamine, and on other anthracyclines, e.g. daunorubicin, having better stability at elevated pH 1 . Evidence for the chemical stability of DOX loaded in PEO-PBLA micelles was revealed by visible spectroscopy (Fig. 2). The absorbance of free DOX

over time (JASCO, Ubest 50, Japan). The level of DOX was 10 mg / ml. All samples were in 0.10 M PBS, pH 7.4, and were stored in the dark at 258C. Samples were free DOX, PEO-PBLA1DOX, and PEO-PBLA micelles loaded with DOX. A fluorescence study of DOX was carried out (JASCO, 770F, Japan). Excitation and emission bandwidths were both 10 nm with a l ex of 471 nm. The level of DOX was 5.0 mg / ml. Samples were free DOX, PEO-PBLA1DOX, and PEO-PBLA micelles loaded with DOX. A water-soluble, quenching agent, I 2 , was used for the fluorescence quenching study. The level of I 2 (KI) ranged from 0.0 to 0.60 M, with the ionic strength of the solution kept constant by the addition of NaCl. Also, Na 2 S 2 O 4 (10 25 M) was added to prevent oxidation of iodide.

3. Results and discussion Block copolymer micelles, like low molecular weight surfactant micelles, solubilize hydrophobic drugs in aqueous solutions. But little is known about the loading of drugs into block copolymer micelles and further about the properties of the drug-loaded, block copolymer micelles. As mentioned, there are negligible levels of DOX loaded into PEO-PBLA micelles by simply equilibrating PEO-PBLA and DOX in water. This is the second method developed for physically loading DOX in PEO-PBLA micelles, the goal being improved loading efficiency [4]. At low levels of DOX (0.50–1.0 mg), DOX loading of PEO-PBLA micelles proceeded smoothly without precipitation and with formation of micelles (Table 1). The o / w emulsion had a milky appearance, which disappeared upon evaporation of CHCl 3 . PEO-PBLA micelles loaded with DOX had hydrodynamic diameters of ¯37 nm, larger than unloaded

1

Daunorubicin is berift of an a-ketol group present in DOX, which leads to degradation of DOX through an enolization pathway at pH.4. Accordingly, daunorubicin has greater stability than DOX at elevated pH [9].

Table 1 DOX loading of PEO-PBLA micelles by o / w emulsion method PEO-PBLA (mg) / H 2 O (ml)

DOX (mg)

Yield (%)

load (w / w)

Micelle formation a

5 / 10

0.5 1.0 1.5 2.5 3.0

43 65 43 43 39

5 12 12 18 22

o o — — —

a

Determined by DLS (weight average) and SEC.

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Fig. 1. Chromatogram of DOX loaded in PEO-PBLA micelles by o / w emulsion method. Gradient conditions are shown in the inset.

at 485 nm decreased from 0.20 to 0.050 over 250 h due to the chemical degradation of the drug [10]. In contrast, the absorbance of DOX loaded in PEOPBLA micelles was largely constant over the same period, ¯0.20. The cores of PEO-PBLA micelles were largely devoid of water, preventing hydrolytic reactions of DOX. The sample of DOX1PEO-PBLA

had a decrease in absorbance from 0.20 to 0.13 over 250 h but more gradually than free DOX, perhaps as a result of interaction of DOX with PEO in the shell regions of PEO-PBLA micelles. Besides indicating the chemical stability of DOX inside PEO-PBLA micelles, visible spectroscopy results also suggested that DOX was released slowly

G. Kwon et al. / Journal of Controlled Release 48 (1997) 195 – 201

Fig. 2. Time dependency of DOX absorbance at 485 nm: (i) DOX, (ii) sample of DOX1PEO-PBLA, (iii) PEO-PBLA micelles loaded with DOX (PEO-PBLA / DOX).

from the micelles since a decrease in UV absorbance at 485 nm due to released DOX was not evident. Furthermore, there was only a slight increase in total fluorescence intensity of PEO-PBLA micelles loaded with DOX over the same time period, whereas free DOX lost its ability for fluorescence (Fig. 3). Self-association of DOX occurred in the PEO-PBLA micelles (self-quenched fluorescence), contributing to the slow release of the drug from the micelles. Upon release, DOX dissociated and regained its ability for fluorescence. The fluorescence of DOX loaded in PEO-PBLA micelles was regained by addition of

Fig. 3. Time dependency of DOX fluorescence: (i) DOX, (ii) PEO-PBLA micelles loaded with DOX (PEO-PBLA / DOX).

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sodium dodecyl sulfate, which likely disrupted the PEO-PBLA micelles and caused the release of DOX [4]. PEO-PBLA micelles loaded with DOX were incubated in 0.10 M PBS, pH 7.4, containing 10% w / v serum albumin, and again slow release of DOX from PEO-PBLA micelles was revealed by SECHPLC. Over 100 h, a gradual decrease in the absorbance of DOX (485 nm), eluting with PEOPBLA micelles at the void volume, was notable. But most of DOX, ¯80%, was still loaded in the PEOPBLA micelles after 100 h. The slow release of DOX from PEO-PBLA micelles is unique among micellar systems, where rates of release usually occur on the order of msec [11]. The gradual release of DOX may be due to the solid-like cores of PEO-PBLA micelles [2]. In contrast, a majority of micelles have liquid-like cores and quickly release hydrophobic molecules. Thus, dose dumping of DOX after intravenous injection of PEO-PBLA micelles loaded with DOX is not expected, but rather a gradual release of DOX from the micelles, i.e. depot effect. The slow release of DOX from PEO-PBLA micelles allows for use in parenteral delivery, where long circulation of the PEO-PBLA micelles may lead to extravasation at sites of solid tumors and localized DOX release [5]. Table 2 summarizes the sizes of PEO-PBLA micelles loaded with DOX as determined by DLS. The mean diameters of PEO-PBLA micelles are largely unchanged by freezing or by freeze-drying; the latter process is advantageous since residual solvent such as CHCl 3 is removable under such conditions. Weight-averaged size distribution for PEO-PBLA micelles with DOX before and after freeze-drying are shown on Fig. 4. Although there were a small fraction of PEO-PBLA micelles that had undergone secondary association, observed around 120 nm, size distributions were unchanged after freeze drying. The results indicate that it is possible to reconstitute PEO-PBLA micelles loaded with DOX after freeze-drying and obtain a micellar solution. For the freeze-dried samples, it was also important to determine whether or not DOX was present inside the cores of the PEO-PBLA micelles after reconstitution in distilled water. To this end, collisional quenching constants, KSV , were estimated from

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Table 2 Weight-average, diameters of PEO-PBLA micelles loaded with DOX and PEO-PBLA micelles a Sample

Non-treated sample (nm)

Frozen sample (nm)

Freeze-dried sample (nm)

PEO-PBLA micelles loaded with DOX PEO-PBLA micelles

37 (139) 19 (83)b

38 (147) 19 (68)

28 (116) 16 (72)

a

Samples at 258C, freeze-dried samples reconstituted with distilled water. Values in parenthesis are the diameters of particles due to the secondary association of micelles.

b

Fig. 4. Size distributions of PEO-PBLA micelles loaded with DOX: (i) sample without freeze-drying (ii) reconstituted sample after freeze-drying.

Stern-Volmer plots of the fluorescence quenching data (Fig. 5), using: F ]0 5 1 1 KSV f I 2 g F where F and F0 are the total fluorescence intensities of DOX with and without I 2 , respectively, and KSV is the collisional quenching constant. The equation describes a dynamic quenching process [12]. DOX had a KSV of 22 M 21 , whereas PEO-PBLA micelles loaded with DOX had a KSV of 5.8 M 21 . In the former case, DOX was in an aqueous milieu and was readily quenched by I 2 . In the latter case, DOX was only slightly quenched by I 2 and was not readily accessible to I 2 , consistent with DOX located inside the cores of PEO-PBLA micelles. KSV of PEO-PBLA micelles loaded with DOX was largely unaffected by freeze-drying and reconstitution, ¯6.7 M 21 , suggesting that PEO-PBLA micelles were not disrupted and kept DOX in their cores. The sample of DOX1PEO-PBLA had a KSV of 21 M 21 . Lastly, SEC-HPLC of PEO-PBLA micelles loaded with DOX after freezing and freeze-drying also evidenced retention of DOX inside PEO-PBLA

Fig. 5. Stern-Volmer plots of (i) DOX, (ii) sample of DOX1PEOPBLA, (iii) PEO-PBLA micelles loaded with DOX (PEO-PBLA / DOX) (before and after freeze drying).

G. Kwon et al. / Journal of Controlled Release 48 (1997) 195 – 201

micelles, with DOX eluting with the PEO-PBLA fraction (data not shown).

4. Conclusions DOX was loaded into PEO-PBLA micelles by an o / w emulsion method. DOX loaded in PEO-PBLA micelles was stable towards chemical degradation, in contrast to free drug. It was possible to freeze-dry PEO-PBLA micelles loaded with DOX and obtain micelles loaded with drug upon reconstitution in water. PEO-PBLA micelles released DOX slowly (depot effect), in contrast to other micellar systems. Finally, PEO-PBLA micelles may be sterilizable by simple filtration due to their small size. The pharmaceutical properties of PEO-PBLA micelles hopefully will be exploited in their use as vehicles for DOX delivery in cancer chemotherapy.

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