Development of polyion complex micelles for encapsulating and delivering amphotericin B

Biomaterials 30 (2009) 3352–3358

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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Development of polyion complex micelles for encapsulating and delivering amphotericin B Chau-Hui Wang a, Wei-Ting Wang b, Ging-Ho Hsiue b, * a b

Polymer Technology Division, Material and Chemical Research Laboratories, Industrial Technology Research Institute, Hsinchu 300, Taiwan Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 300, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 January 2009 Accepted 28 February 2009 Available online 19 March 2009

A block copolymer poly(2-ethyl-2-oxazoline)-block-poly(aspartic acid) (PEOz-b-PAsp) was synthesized and investigated as the carrier of antifungal drug amphotericin B (AmB). Polyion complex (PIC) micelles with clear core–shell structures were identified by TEM, which revealed that the PAsp segment became hydrophobic after it interacted with AmB. PEOz-b-PAsp increased not only the solubility of AmB but also simultaneously the drug potency. The prolonged release of AmB from micelles effectively inhibited the growth of Candida albicans even after three days of administration. Moreover, the in vitro cytotoxicity of AmB-loaded micelles was less than that of FungizoneÒ, which is a powerful antifungal antibiotic that is adopted to treat various fungal infections. The PEOz-b-PAsp PIC micelles with lower cytotoxicity and higher potency than FungizoneÒ represent a potential means of encapsulating basic/amphoteric drugs. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Amphotericin B Poly(2-ethyl-2-oxazoline) Poly(aspartic acid) Polyion complex micelles

1. Introduction Amphotericin B (AmB) is an antifungal drug, which is commonly used intravenously to treat systemic fungal infections. AmB has a wide range of antifungal activities against most opportunistic and endemic fungi [1]. However, it is insoluble in water and most organic solvents. In order to make it biologically active, dissolution in an aqueous milieu is necessary. Two drug formulations of AmB are commercially available. The use of first type of AmB with deoxycholate is limited due to dose-dependent adverse effects such as nausea, fever and cytotoxicity. For example, the formulation sodium deoxycholate (FungizoneÒ) is clinically used but nephrotoxicity is a frequent complication [2,3]. Unlike the deoxycholate formulation, the second type is a lipid-based formulation with low nephrotoxicity [4,5]. However, cost and other factors limit its widespread use [6–8]. Much effort has been made to develop costeffective delivery systems with reduced AmB toxicity. Polymeric micelles for drug delivery have been demonstrated in the previous studies [9–14]. The core–shell structure of such selfassembling systems presents advantages such as lower cytotoxicty, higher solubility and higher bioavailability than free drugs [15–18]. We have developed a series of block copolymers to encapsulate water-insoluble drugs in the form of nano-scale micelles [19,20]. Hydrophobic drugs are incorporated into AB block copolymer mostly by chemical bonding or physical entrapment. Few works * Corresponding author. Tel.: þ886 3 571 9956; fax: þ886 3 572 6825. E-mail address: [email protected] (G.-H. Hsiue). 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.02.041

have focused on such non-covalent interactions as electrostatic forces between polymers and drugs. AmB is a zwitterion compound and has been reported to form a complex with various phospholipids [21,22]. The lipid formulations of AmB showed a lower toxicity than AmB deoxycholate. To reduce the cytotoxicity and enhance efficacy, the block copolymer-based on poly(2-ethyl-2-oxazoline) (PEOz) and poly (aspartic acid) (PAsp) was synthesized for the encapsulation of AmB. PEOz is a water-soluble polymer that has been approved by FDA (US Food and Drug Administration) for use as an indirect food additive. Liposomes on which is grafted PEOz have been shown to circulate for a long time in blood and to be associated with a reduction of the clearance rates in spleen and liver [23]. On the other hand, PAsp was designed as the core domain of micelles because of its biocompatibility and its ability to interact with basic drugs [24–26]. This work investigates the synthesis and characterization of PEOz-b-PAsp diblock copolymer. The in vitro drug release, cytotoxicity and antifungal activity of PEOz-b-PAsp PIC micelles were also studied. 2. Materials and methods 2.1. Materials 2-Ethyl-2-oxazoline (Aldrich) and methyl p-toluenesulfonate (MeOTs, Aldrich) were purified by vacuum distillation over calcium hydride. Tetrahydrofuran (THF, Tedia), chloroform (Tedia), and acetonitrile (ACN, Fisher) were dried over calcium hydride and distilled under nitrogen. b-Benzyl L-aspartate (Alfa Aesar), triphosgene (Fluka), amphotericin B, L-aspartic acid 4-benzyl ester (Alfa Aesar), trifluoromethanesulfonic acid (TFMSA, Lancaster), trifluoroacetic acid (TFA, Alfa Aesar) and thioanisole (TCI) were used as-received.

C.-H. Wang et al. / Biomaterials 30 (2009) 3352–3358 2.2. Synthesis of N-carboxy-b-benzyl L-aspartate anhydride (Asp(OBzl)–NCA)

b-Benzyl L-aspartate (10.0 g) was dissolved in THF (100 mL) under nitrogen. Triphosgene (13.3 g) was then introduced and the mixture was reacted with gentle stirring at 40  C. After the mixture became transparent (about 3 h), the solution was precipitated in n-hexane (300 mL) and then stored at 20  C overnight. The resulting product was filtered and washed with n-hexane (30 mL). The obtained monomer N-carboxy-b-benzyl L-aspartate anhydride (Asp(OBzl)–NCA) was dried under vacuum and characterized by 1H NMR and FT-IR spectroscopy. 1H NMR spectra were obtained using an AMX-500 Bruker spectrometer. FT-IR spectra were recorded from KBr pellets on a Perkin–Elmer Spectrum 824 spectrometer. Asp(OBzl)–NCA 1H NMR (500 MHz, CDCl3): d 2.9, 3.1 (CHCH2COOCH2C6H5); 4.6 (CHCH2COOCH2C6H5); 5.2 (CHCH2COOCH2C6H5); 7.3 (CHCH2COOCH2C6H5). Asp(OBzl)–NCA FT-IR (KBr): 1735 cm1 (C]O of ester, stretching); 1160 cm1 (C–O of ester, stretching); 2400–3400 cm1 (OH of carboxyl acid, stretching); 1720 cm1 (C]O of carboxyl acid, stretching); 1560–1640 cm1 (NH2, bending); 1475 and 1600 cm1 (C]C of benzyl, stretching). 2.3. Synthesis of macroinitiator amino-poly(2-ethyl-2-oxazline) (PEOz–NH2) Amino-poly(2-ethyl-2-oxazline) (PEOz–NH2) was synthesized by the cationic ring-opening polymerization of 2-ethyl-2-oxazoline (10 mL) in ACN (20 mL) at 100  C for 12 h, using MeOTs (0.075 mL) as the initiator. The living polymerization of PEOz was terminated using 0.1 N NH3 acetonitrile solution at 0  C for 24 h [27]. The polymer solution was passed flow through silica gels in a flush column and then precipitated in cooled diethyl ether (400 mL). After vacuum drying, a white powdery product was obtained. Gel permeation chromatography (GPC) measurements were made using a Phenomenex Phenogel apparatus equipped with a refractive index detector. THF was adopted as the mobile phase at a flow rate of 1.0 mL/min. The columns were calibrated using poly(methyl methacrylate) standards (Polymer Laboratories). PEOz–NH2 1H NMR (500 MHz, CDCl3): d 1.1 (N(COCH2CH3)CH2CH2); 2.1–2.4 (N(COCH2CH3)CH2CH2); 3.4 (N(COCH2CH3)CH2CH2). PEOz–NH2 FT-IR (KBr): 1640 cm1(C]O of amide). 2.4. Synthesis of poly(2-ethyl-2-oxazoline)-block-poly(aspartic acid) (PEOz-b-PAsp) Asp(OBzl)–NCA (6.3 g) was polymerized in chloroform (40 mL) at 40  C for 48 h using PEOz–NH2 (5.0 g) as the macroinitiator. The reaction mixture was then poured into cooled diethyl ether (800 mL), precipitating out a white solid. The degree of polymerization of each polymer segment was estimated using 1H NMR spectra. PEOz-b-PAsp(OBzl) 1H NMR (500 MHz, CDCl3): d 1.1 (N(COCH2CH3)CH2CH2); 2.1–2.4 (N(COCH2CH3)CH2CH2); 2.6, 3.1 (COCH(CH2COOCH2C6H5)NH); 3.4 (N(COCH2CH3) CH2CH2); 4.2 (COCH(CH2COOCH2C6H5)NH); 4.9, 5.1 (COCH(CH2COOCH2C6H5)NH); 7.2 (COCH(CH2COOCH2 C6H5)NH). PEOz-b-PAsp(OBzl) FT-IR (KBr): 3300 cm1 (NH of amide, stretching); 1735 cm1 (C]O of ester, stretching); 1160 cm1 (C–O of ester, stretching); 1640 cm1 (C]O of amide); 1530 (NH of amide, bending); 1475, 1600 cm1 (C]C of benzyl, stretching); 690, 750 cm1 (monosubstrate of benzyl, stretching).

O S O CH3 O

H3C

N +

Et

PEOz-b-PAsp(OBzl) (9 g) was deprotected with 1.5 M TFMSA–thioanisole (molar ratio 1:1) in TFA (9.9 mL) [28]. This mixture was gently stirred at room temperature for 30 min and then precipitated in cooled diethyl ether (200 mL). The resulting PEOz-bPAsp was then purified by dialysis (MWCO 1000, Spectrum). The removal of the benzyl group on PAsp(OBzl) was confirmed by 1H NMR and FT-IR spectra. PEOz-b-PAsp 1H NMR (500 MHz, DMSO-d6): d 1.1 (N(COCH2CH3)CH2CH2); 2.1–2.4 (N(COCH2CH3)CH2CH2); 2.7 (COCH(CH2COOH)NH); 3.4 (N(COCH2CH3)CH2CH2); 4.5 (COCH(CH2COOH)NH); 8.0 (COCH(CH2COOH)NH); 12.3 (COCH(CH2COOH)NH). FT-IR (KBr): 3300 cm1 (NH of amide, stretching); 2400–3400 cm1 (OH of carboxyl acid, stretching); 1720 cm1 (C]O of carboxyl acid, stretching); 1630, 1640 cm1 (C]O of amide, stretching), 1530 cm1 (NH of amide, bending).

2.5. Characterization of PEOz-b-PAsp and PIC micelles The optical transmittance of aqueous PEOz-b-PAsp copolymer solutions with various pH values (I ¼ 0.01) was monitored at 500 nm by means of a UV/Vis spectrophotometer (Lambda 2S, Perkin Elmer) [29]. The samples (1 mg/mL) were placed in Teflon-stopped quartz cuvettes. The transmittance of distilled water at 25  C was set to 100% and the pH range was 2–11. The PIC micelles were prepared by thin film hydration [30,31]. PEOz-b-PAsp (10 mg) and certain amounts of AmB were dissolved in 10 mL of N,N-dimethylformamide (DMF). After 3 h of vacuum drying in a rotary evaporator at 40  C, the thin film was formed around the bottom of the flask. Ten milliliter of pH 7.4 phosphate buffer solution (PBS) was added for hydration and the solution was then sonicated for 10 min. The transparent yellow solution was filtered through the 0.45 mm syringe filter to remove the unloaded AmB. To determine the drug content, 1 mg/mL of PIC micelles at pH 7.4 PBS was mixed with an equal volume of DMSO. The UV absorbance was measured at 405 nm; the AmB concentration was estimated according to the standard curves of free AmB at DMSO/pH 7.4 PBS (1/1 vol.). The drug content is calculated using the formula, drug content (wt%) ¼ (the weight of AmB in micelles)/(total weight of PIC micelles)  100. The encapsulation efficiency is defined as: encapsulation efficiency (%) ¼ (the weight of AmB after filtration)/(the weight of AmB before filtration)  100. The hydrodynamic diameter of PIC micelles was measured by photon correlation spectroscopy using a Malvern Zetasizer Nano ZS at 25  C. The measurements were made at a scattering angle of 90 . To observe the micellar core–shell structure, transmission electron microscopy (TEM, Hitachi H-600 microscope) was utilized to record the images. A drop of the sample solution was allowed to settle on a copper grid for 1 min. Excess sample was wiped away with filter paper and the negative staining of sample by 1% phosphotungstic acid (PTA) was conducted for 1 min [32]. The in vitro release of AmB from PIC micelles was determined using the membrane diffusion technique at 37  C. Five milliliter of micelles (1 mg/mL) was placed in a dialysis tube (MWCO 3500, SPECTRUM), which was immersed in 50 mL of pH 7.4 PBS. The amount of AmB released from the dialysis tube was measured using a UV/Vis spectrometer at a wavelength of 405 nm. UV/Vis spectra were recorded on a Perkin Elmer Lamda 2S spectrometer.

0.1 N NH3 ACN sol'n

2-ethyl-2-oxazoline

H2N

R = CH2COOBzl R' = CH2COOH

O

Et

O R OH

Triphosgene 50 °C, 3 h

40 °C, 48 h CHCl3

O

HN

O

β-Benzyl L-aspartate

Asp(OBzl)-NCA

R'

H N O

N H

H

y

poly(2-ethyl-2-oxazoline)

R

y

NH2 N

O

O

N

H3C O

methyl p-toluenesulfonate

H3C

3353

1.5 M TFMSA–thioanisole TFA

Et PEOz-b-PAsp

H3C O

R

H N N

y

O

N H

Et PEOz-b-PAsp(OBzl)

Scheme 1. Synthetic routes for PEOz-b-PAsp.

H

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C.-H. Wang et al. / Biomaterials 30 (2009) 3352–3358

Fig. 1. 1H NMR spectrum of PEOz-b-PAsp in DMSO-d6. 2.6. In vitro cytotoxicity test Human HT-1080 fibroblasts were incubated in Dulbecco’s modified Eagle’s medium (DMEM, Biosource, Rockville, MD) that was supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 mg/mL streptomycin at 37  C in a humidified atmosphere of 5% CO2. The mitochondrial activity of PEOz-b-PAsp, PIC micelles and FungizoneÒ treated cells was measured using the CellTiter 96Ò AQueous Assays (an MTS assay, Promega) following the manufacturer’s protocol. HT1080 cells were seeded on a 24-well plate at a concentration of 1 105 cells/well. After 12 h of incubation, the growth medium was replaced with 1500 mL of DMEM that contained the desired amount of PEOz-b-PAsp, PIC micelles and FungizoneÒ. The viable cells reduced the methoxyphenyl-tetrazolium salt (MTS) compound into a colored formazan product, which was quantified colorimetrically at 490 nm using a spectrofluorometer (Sunrise/Tedan, Austria). Untreated HT-1080 cells incubated only with growth medium were used as a control. 2.7. Antifungal activity The minimal inhibitory concentration (MIC), which is defined as the concentration at which fungi in wells are fully inhibited, was determined by a dilution method in the YPD medium [33–35]. Candida albicans were incubated in YPD medium at 37  C in a humidified atmosphere of 5% CO2. A suspension of C. albicans (1 106/well) was seeded on a 24-well plate and samples with different concentrations were added. The MICs of Fungizone and PIC micelles were determined by the naked eye as the concentrations at which visible growth was completely inhibited. The viability of C. albicans was quantified colorimetrically at 600 nm using a spectrofluorometer (Sunrise/Tedan, Austria). Untreated C. albicans incubated with only YPD medium were used as a control.

conducted using an initiator with amino/hydroxyl functional groups. Additionally, strong nucleophiles such as tertiary amine and metal oxide reportedly initiate NCA and yield a poly(amino acid) of high molecular weight [36]. In this work, PEOz-b-PAsp block copolymer was designed to encapsulate antifungal drug AmB. The carboxylic acid on PAsp was expected to interact with AmB and thereby form a hydrophobic core within micelle. Scheme 1 presents the synthetic route of PEOz-b-PAsp. 2-Ethyl-2-oxazoline was polymerized by MeOTs and the living end was then terminated by NH3 acetonitrile solution. The molecular weight of PEOz–NH2 was determined by GPC. PEOz-b-PAsp(OBzl) was then synthesized in an SN2 reaction with Asp(OBzl)–NCA using PEOz–NH2 as the macroinitiator. The benzyl group of PAsp(OBzl) was then removed using TFMSA–thioanisole in TFA. The molecular weight of PEOz-b-PAsp was calculated from the integral ratios of signals between two blocks in the 1H NMR spectra. As presented in Fig. 1, the chemical shifts of the peaks at 2.6, 4.2, and 12.3 ppm were assigned to the protons of the methylene, methyne and carboxylic acid on the PAsp unit, respectively. Amphiphilic block copolymers that comprise hydrophilic and hydrophobic segments are commonly used as carriers of water-

120

3. Results and discussion

N-Carboxyanhydride (NCA) ring-opening polymerization is one of the methods for preparing poly(amino acid). It is commonly Table 1 Molecular weights and compositions of PEOz-b-PAsp block copolymers. Code

In feed (mmol) PEOz–NH2

Asp(OBzl)–NCA

X57A09 X57A20 X92A13 X92A36

0.89 0.89 0.54 0.54

10.08 20.16 10.08 20.16

a b

Determined by GPC. Determined by 1H NMR.

PEOz (Mn)a

PAsp (Mn)b

Yield (wt%)

5700 5700 9200 9200

900 2000 1300 3600

50 69 70 42

Transmittance (%)

100

3.1. Synthesis and characterization of PEOz-b-PAsp

80 60 X92A36 X92A13 X57A20 X57A09

40 20 0

0

2

4

6 pH

8

10

12

Fig. 2. The optical transmittance of PEOz-b-PAsp in different pH solutions (I ¼ 0.01).

C.-H. Wang et al. / Biomaterials 30 (2009) 3352–3358

Absorbance

1.3

3355

Fungizone Micelles AmB

0.8

0.3

Fig. 5. TEM images of PIC micelles with core–shell structure.

300

400 Wavelength (nm)

500

Fig. 3. UV/Vis spectra of FungizoneÒ, PIC micelles and AmB in mixed solution of pH 7.4 PBS and DMSO (1:1 v/v).

insoluble drugs. It is worthy to note that PEOz and PAsp herein are both hydrophilic under neutral and alkaline conditions. Four samples of PEOz-b-PAsp with different polymer compositions were synthesized. Table 1 presents their feed ratios and characteristics. Deprotection of the benzyl group on PAsp(OBzl) reduced the yield of PEOz-b-PAsp. Fig. 2 shows the optical transmittance of PEOz-bPAsp in aqueous solutions with different pH values. The solution became turbid when the pH was below the pKa of PAsp (pH 5.2) [37]. Furthermore, the copolymers with longer PAsp segments exhibited a great drop in transmittance as the pH value decreased. This behavior may be attributed to the hydrogen bonding between PEOz and the carboxylic acid on PAsp, which results in the aggregation of copolymers [38]. X92A13 was selected for use in the following experiments because of its good solubility at low pH and suitable PAsp chain length. Notably, all samples were completely soluble at a pH of over 7. The dissociation of PAsp increased the solubility of copolymer in aqueous solution and enabled the functional groups to associate with AmB. In our previous works, PEOz copolymers also exhibited temperature-sensitive properties in the aqueous solution at around 35  C [39]. The transmittance of polymer solution decreased markedly above characteristic temperatures. Actually, the lower critical solution temperature (LCST) of PEOz homopolymer ranged from 62 to 82  C, depending on the molecular weight and polymer concentration [40]. The introduction of hydrophobic segment to

Drug content (wt%)

80

180 Drug content Average diameter

160

60

140

40

120

20

100

0

0.09

0.19 0.28 Drug/Polymer (mol/mol)

0.47

80

Average diameter (nm)

100

Fig. 4. Drug content and mean diameter of PIC micelles at various drug/polymer feed ratios (w/w) (n ¼ 3).

PEOz is considered to decrease its LCST. In contrast, the phase separation temperature was raised by introducing hydrophilic PAsp into PEOz. No precipitated or phase separated phenomena were observed in the following experiments including in vitro release, cytotoxicity and antifungal activity. 3.2. Characterization of PIC micelles As mentioned above, electrostatic force was adopted to encapsulate AmB into micelles. The UV/Vis spectra were measured to determine whether AmB was associated with PEOzb-PAsp. As shown in Fig. 3, free AmB absorbs at wavelength of 365, 385 and 405 nm, whereas PIC micelles exhibit a broad peak with high intensity at 335 nm. The spectrum of PIC micelles is similar to that of FungizoneÒ, suggesting that AmB was associated with PAsp and formed complexes as like deoxycholate salt. Fig. 4 plots the drug content and average diameter of PIC micelles as a function of the drug/polymer mol ratio in the feed.

100

80

Release of AmB (%)

-0.2 200

60

40

20 Fungizone Micelles 0 0

50

100

150

Time (h) Fig. 6. In vitro release of AmB from PIC micelles and FungizoneÒ in pH ¼ 7.4 buffer solutions at 37  C (n ¼ 2).

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C.-H. Wang et al. / Biomaterials 30 (2009) 3352–3358

Cell viability (%)

A 100 80 60 40 20 0

1 day 3 day 10

100 1000 Concentration (ug/mL)

10000

Cell viability (%)

B 100 80 60 40 20 0

Fungizone Micelles 10

100 1000 AmB Concentration (ug/mL)

10000

C 100 Fig. 8. Photographs of broth dilution method applied to model C. albicans after (A) one day and (B) three days.

Cell viability (%)

80

the control group. According to Fig. 6, the micelles continuously released AmB and the amount of which reached a plateau after 48 h. The release profile followed first order-like kinetics in the first 7 h and an accumulated release of about 60% was observed thereafter. The micelle solution was transparent at 37  C, suggesting that hydrophilic PEOzs surrounding PAsp–AmB complexes made micelles well dispersed. In contrast, FungizoneÒ was precipitated after 12 h because of its intrinsically hydrophobic nature.

60 40 20 0

Fungizone Micelles 10

100 1000 AmB Concentration (ug/mL)

10000

Fig. 7. In vitro cytotoxicity of (A) PEOz-b-PAsp (B) PIC micelles and FungizoneÒ on day one (C) PIC micelles and FungizoneÒ on day three on HT-1080 cells (n ¼ 4).

The results indicate that the size of micelle decreased as the drug/polymer mol ratio increased. The encapsulation efficiency of AmB was 97% when the drug/polymer ratio was 0.47. The mean diameter determined by DLS was 108 nm in PBS at pH 7.4. Fig. 5 presents the TEM image of PIC micelles and the core–shell structure was clearly identified. 3.3. In vitro release The in vitro release of PIC micelles was monitored in the phosphate buffer solution at pH 7.4 and 37  C. FungizoneÒ, which utilizes sodium desoxycholate as the excipient of AmB, was used as

3.4. In vitro cytotoxicity test Fig. 7 shows the cell viability of PEOz-b-PAsp, PIC micelles and FungizoneÒ at concentrations from 10 to 1000 mg/mL. The wells with only medium were regarded as a positive control with a cell viability of 100%. The relative cell viability was determined as [Abs]sample/[Abs]control  100%. As presented in Fig. 7A, the cell viability exceeded 80% after one and three days of incubation, suggesting that PEOz-b-PAsp is a new material with low cytotoxicity. Besides the PEOz mentioned above, the incorporation of PAsp may be helpful because it is supposed to reduce the cytotoxicity of polymer-based drug vector [24,25]. On the other hand, the cell viability of micelles was over 70% at 100 mg/mL of AmB concentration, which is the minimum dose of FungizoneÒ for clinical use (Fig. 7B and C) [2,3]. The result indicates PIC micelles successfully reduce the cytotoxicity of AmB. However, it was noted that the cell viabilities of micelles and FungizoneÒ were similar when AmB concentration was relatively high (1000 mg/mL). The solubility of

C.-H. Wang et al. / Biomaterials 30 (2009) 3352–3358

Viability of C. albicans (%)

A

120 100 80 60 40 20 0

Viability of C. albicans (%)

B

Micelles Fungizone 1

10 100 1000 AmB Concentration (ug/mL)

10000

120

3357

drug AmB. PIC micelles with a clear core–shell structure were identified by TEM, and the results indicated that the PAsp segment became hydrophobic when it interacted with AmB. The PEOz-b-PAsp increased not only the solubility of AmB but also simultaneously its efficacy. The sustained release of micelles effectively suppressed the growth of C. albicans even after three days of administration. Moreover, the cytotoxicity of micelles was less than that of FungizoneÒ. This behavior may be attributed to the release of the AmB in a monomeric (non-aggregated) state. Monomeric AmB is less toxic to mammalian cells than to fungal cells because it selectively interacts with ergosterol. Aggregated AmB is nonselective, thus resulting in high cytotoxicity. The water-soluble block copolymer PEOz-b-PAsp described here provides another means of encapsulating basic/amphoteric drugs. The PIC micelles with low cytotoxicity, high drug content and high efficacy represent a potential carrier for delivering basic/ amphoteric drugs. Appendix

100

Figures with essential colour discrimination. Certain figures in this article, in particular Figs. 1, 3 and 8 may be difficult to interpret in black and white. The full colour images can be found in on-line version at doi:10.1016/j.biomaterials.2009.02.041.

80 60

References

40 Micelles Fungizone

20 0

1

10 100 1000 AmB Concentration (ug/mL)

10000

Fig. 9. Antifungal activity of PIC micelles and FungizoneÒ at various AmB concentrations after (A) one day and (B) three days (n ¼ 4).

FungizoneÒ may reach its upper limit, preventing more AmB from dissolving or being activated in the medium. 3.5. Antifungal activity of PIC micelles and FungizoneÒ In microbiology, MIC is the lowest concentration of an antimicrobial that inhibits the visible growth of a microorganism after overnight incubation. The lowest concentration (highest dilution) of antibiotic that prevents the appearance of turbidity is regarded as the MIC. At this dilution, the antibiotic is bacteriostatic. Fig. 8 shows a photograph of the application of the broth dilution method to the model C. albicans. The MIC of micelles was at 20 mg/mL on days one and three of incubation. In contrast, medium applied with FungizoneÒ was associated with turbidity at all concentrations after three days, suggesting that its potency was lost (Fig. 8B). Fig. 9 presents the antifungal activity of PIC micelles and FungizoneÒ at various AmB concentrations. The growth inhibition rate of micelles was more effective than that of FungizoneÒ on days one and three of incubation. In contrast, FungizoneÒ could not inhibit fungal growth on the third day. The maintenance of, or even improvement of antifungal activity may reflect the sustained release of the drug. 4. Conclusions In this work, a novel block copolymer PEOz-b-PAsp was successfully synthesized and was used as the carrier of antifungal

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