Polymeric micellar pH-sensitive drug delivery system for doxorubicin

Journal of Controlled Release 103 (2005) 137 – 148 www.elsevier.com/locate/jconrel

Polymeric micellar pH-sensitive drug delivery system for doxorubicin Martin Hruby´*, Cˇestmı´r Konˇa´k, Karel Ulbrich Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky´ Sq. 2, 162 06 Prague 6, Czech Republic Received 13 August 2004; accepted 15 November 2004 Available online 15 December 2004

Abstract A novel polymeric micellar pH-sensitive system for delivery of doxorubicin (DOX) is described. Polymeric micelles were prepared by self-assembly of amphiphilic diblock copolymers in aqueous solutions. The copolymers consist of a biocompatible hydrophilic poly(ethylene oxide) (PEO) block and a hydrophobic block containing covalently bound anthracycline antibiotic DOX. The starting block copolymers poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PEO-PAGE) with a very narrow molecular weight distribution (M w/M n ca. 1.05) were prepared by anionic ring opening polymerization using sodium salt of poly(ethylene oxide) monomethyl ether as macroinitiator and allyl glycidyl ether as functional monomer. The copolymers were covalently modified via reactive double bonds by the addition of methyl sulfanylacetate. The resulting ester subsequently reacted with hydrazine hydrate yielding polymer hydrazide. The hydrazide was coupled with DOX yielding pH-sensitive hydrazone bonds between the drug and carrier. The resulting conjugate containing ca. 3 wt.% DOX forms micelles with R ha=104 nm in phosphate-buffered saline. After incubation in buffers at 37 8C DOX was released faster at pH 5.0 (close to pH in endosomes; 43% DOX released within 24 h) than at pH 7.4 (pH of blood plasma; 16% DOX released within 24 h). Cleavage of hydrazone bonds between DOX and carrier continues even after plateau in the DOX release from micelles incubated in aqueous solutions is reached. D 2004 Elsevier B.V. All rights reserved. Keywords: Diblock copolymers; Drug delivery systems; Micelles; Doxorubicin; pH-sensitive

1. Introduction Polymeric micelles attract an increasing interest in contemporary drug research because they could be

* Corresponding author. Tel.: +42 296 809 212; fax: +42 296 809 410. E-mail address: [email protected] (M. Hruby´). 0168-3659/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2004.11.017

used as a very efficient drug delivery system [1,2]. Polymeric micellar drug delivery systems (MDDSs) of core-shell architecture based on amphiphilic AB diblock or ABA triblock copolymers possess numerous advantages. They improve solubility and bioavailability of hydrophobic drugs that are poorly soluble or insoluble in water [1,2]. Micelles with biocompatible hydrophilic shell show low uptake by the reticuloendothelial system even if they have a

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nonbiocompatible core [3] and significantly protect the incorporated drug from fast degradation, blood clearance and elimination from the body [1,2]. Micelle formation/disintegration can be controlled by stimuli-responsive process (to pH [4], ultrasound [5–7], temperature, etc.), and degradation products of lower molecular weight could be more easily eliminated from the organism after the drug delivery system fulfilled its task. Micelles are, due to thermodynamic reasons, of very narrow size distribution, which is very important for their biodistribution, and they behave like single molecules in the cases where biodistribution is dependent on molecular weight. This is advantageous especially in the case of drug delivery systems designed for delivery of cancerostatics where high molecular weight together with biocompatibility of the system is often beneficial to achieve effective passive targeting into solid tumour tissue due to leaky vasculature and reduced or missing lymphatic drainage in these tissues (the so-called Enhanced Permeation and Retention (EPR) effect) [8,9]. Moreover, some polymeric MDDSs can avoid induced multidrug resistance of cancer cells by hypersensitizing them against cancerostatics that they carry [10]. Anthracycline antibiotics, particularly doxorubicin (DOX) [11], rank among the most used cancerostatics in current oncological chemotherapy. DOX shows high antitumour activity but also strong side effects [11]. Some MDDSs for the delivery of DOX were developed mainly using hydrophobic interactions to entrap DOX into e.g., poly(ethylene oxide) (PEO)block-poly(propylene oxide)-block-poly(ethylene oxide) [12] or poly(ethylene oxide)-block-poly(hbenzyl aspartate) micellar structures [3,13]. MDDSs with drugs noncovalently bound by hydrophobic interactions into the micelle core possess certain advantages (they are easy to prepare, can be used for all hydrophobic drugs even if they do not contain suitable functional groups for covalent attachment) but also some disadvantages. This is above all their drug release rate, which is hard to control and modify, especially in the case of bsmartQ stimuli-responsive systems. However, the micellar systems with a drug covalently bound to the polymer via stimuli-responsive cleavable bond have potential to overcome these difficulties. In particular, pH-sensitive bonds cleav-

able under mildly acidic conditions and stable under neutral pH are studied [4,14] because the pH value of the interstitial space of solid tumours as well as the interior of endosomes is usually more acidic (pH close to 5) than blood plasma (pH 7.4) [3]. DOX possesses two types of groups suitable for covalent attachment to a carrier, amino and keto groups. The primary amino group was used for, e.g., ionic-bond-mediated entrapment of DOX into poly(ethylene oxide)-block-poly(methacrylic acid) [15] micelles or for covalent attachment of DOX into poly(ethylene oxide)-block-poly(aspartic acid) [16] through amide bond. The keto function was used for the hydrazone-bond-mediated entrapment of DOX into poly(ethylene oxide)-block-poly(aspartic hydrazide-co-h-benzyl aspartate) [17] micelles or hydrazone-mediated conjugation to terminal OH-activated poly(ethylene oxide)-block-poly(lactide) [4]. In this paper, we present a novel pH-sensitive MDDS based on hydrazone-bound DOX. The starting diblock copolymer intermediate poly(ethylene oxide)block-poly(allyl glycidyl ether) (PEO-PAGE) was prepared by ring-opening polymerization, which ensures very narrow molecular weight distribution [18]. Furthermore, the copolymer could be easily purified, hence, the system is well-defined. The copolymer forms micelles of narrow size distribution and hydrodynamic radius R h~10–20 nm in aqueous solutions. This paper deals with subsequent transformation of the double bonds in the hydrophobic PAGE block to hydrazide functional groups suitable for pH-sensitive covalent attachment of DOX and conjugation of this polymer with DOX. We also focused on hydrolytic stability of the hydrazone bond and drug release from the polymer and the whole micellar system under conditions modeling blood and intracellular environment. The effect of hydrazide groups formed after DOX is released on micelle integrity, and polymer dissolution is also discussed.

2. Materials and methods 2.1. Materials Allyl glycidyl ether (AGE, Aldrich, Czech Republic) was dried with anhydrous magnesium sulfate and distilled under reduced pressure (b.p. 51 8C at 2 kPa),

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benzene (Lachema, Czech Republic) was dried with sodium and distilled, tetrahydrofuran (THF; Lachema) was kept over sodium and distilled before use, cyclohexane (Lachema) was dried with sodium metal and distilled before use. PEO monomethyl ether (I, M w=2000 and 5000), dimethyl sulfoxide (DMSO), hydrazine hydrate, DOX hydrochloride and methyl sulfanylacetate were purchased from Fluka (Czech Republic) and were used without additional purification. Other chemicals were purchased from Lachema and were used without purification. Dialysis tubing Spectra/Por 3 (molecular weight cutoff 3500 Da) was purchased from Serva Electrophoresis (Germany), Sephadex LH-60 and Sephadex G-25 gel permeation chromatography media were purchased from Amersham Pharmacia Biotech (Sweden). 2.2. Preparation of poly(allyl glycidyl ether)-blockpoly(ethylene oxide) (PAGE-PEO, II) by anionic ringopening polymerization (Scheme 1) The polymers are marked by Arabic numbers according to their functional groups in Scheme 1

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and letters according to their PEO block length. Thus, polymers a were prepared from PEO monomethyl ether M w =2000 (Ia) according to procedures described below, and polymers b were prepared by analogy from PEO monomethyl ether M w=5000 (Ib). The synthesis was performed by a modified procedure according to Ref. [18]: PEO monomethyl ether (10 g, Ia—M w=2000, 5 mmol OH) was twice azeotropically dried with benzene. Sodium hydride (after removing mineral oil by washing with dry cyclohexane; 120 mg, 5 mmol) was then added, and the mixture was heated to 100 8C under nitrogen with stirring until the hydride dissolved in the melt of Ia (ca. 30 min). AGE (20.0 g, 175.3 mmol) was added, and the mixture was heated with stirring under nitrogen at 100 8C for 6 h. Acetic acid (AcOH; 570 AL, 10 mmol) was added to stop the polymerization, and the crude IIa (30.0 g) was cooled to room temperature. The crude PAGE-PEO IIa (4.00 g) was dissolved in THF (180 mL) and added to silica gel (Kieselgel 60 Fluka, 45 g), and the solvent was removed in vacuo. The polymer-coated silica was transferred onto a column and washed with isopropyl alcohol (500 mL).

Scheme 1. Reaction scheme of the preparation of the polymeric drug carrier IV.

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The eluate, containing PAGE homopolymer and other impurities, was discharged, and IIa was washed from the column with a isopropyl alcohol–chloroform mixture (2:3 v/v, 1000 mL). Solvents were removed from the eluate in vacuo, and the residue (pure IIa, 1.91 g, 48%) was directly used in the next reaction step. The polymerization degree n (average number of AGE units per chain) of the PAGE block according to 1 H NMR [18] is 14.3 for IIa and 19.9 IIb. 2.3. Addition reaction of methyl sulfanylacetate on PAGE-PEO (Scheme 1) The synthesis was performed by a modified procedure according to Ref. [18], briefly: the purified PAGE-PEO (IIa, 1.91 g, 7.53 mmol CjC) was dissolved in THF (pH 5.0 mL) and methyl sulfanylacetate (4.00g, 3.4 mL, 37.7 mmol) and 2,2´-azobisisobutyronitrile (AIBN; 185 mg, 1.13 mmol) were added. This mixture was refluxed under nitrogen for 5 h. After that all volatiles were removed in vacuo (105 8C bath, 13 Pa) to produce a highly viscous liquid IIIa (2.72 g, 100%). Found S: 9.05%, 2.82 mmol g 1. 2.4. Conversion of methyl ester to hydrazide (Scheme 1) The ester IIIa (7.53 mmol COOCH3) was dissolved in THF (120 mL), hydrazine hydrate (37.7 g, 36.6 mL, 753 mmol) was added, and the mixture was refluxed for 7 h. After that, solvent and most hydrazine hydrate were removed in vacuo, and the residue was dissolved in water (100 mL), dialyzed against distilled water for 72 h and freeze-dried. Yield 2.23 g (79%) of IVa. Found N: 7.77% N, 5.55 mmol g 1, S: 9.35%, 2.92 mmol g 1. 2.5. Covalent attachment of DOX (Scheme 2) The polymer IVa (189 mg, 524 Amol hydrazide by N analysis) and DOX hydrochloride (21 mg, 36.2 Amol) were dissolved in dry DMSO (15 mL), and AcOH (10.5 mg, 10 Al, 0.175 mmol) and anhydrous sodium sulfate (1.00 g) were added. The mixture was stirred in the dark at room temperature for 48 h and filtered. The filtrate was separated on a Sephadex LH60 column (bed volume 120 mL) in a mixture AcOH–

DMSO (1:199 v/v). The red polymer fraction was collected, and most DMSO was removed in vacuo (13 Pa). This solution (ca 10 mL) was separated on a Sephadex G-25 column (bed volume 100 mL) in phosphate-buffered saline, pH 7.4, as a mobile phase. The red micellar fraction (45.0 mL) was collected. The solution could be stored for a limited time after freezing at 78 8C or could be freeze-dried with lactose (5 mg lactose/1 mg of polymer) after desalting on a Sephadex G-25 column using water as a mobile phase. The lyophilized powder could be redissolved in water into a micellar solution without the formation of aggregates. The DOX content in polymer V (see Scheme 2 for structure) was assayed spectrophotometrically (k max=480 nm) after quantitative release of DOX from micelles by the incubation in acid medium (500 AL AcOH/4000 AL of the solution, incubation at room temperature overnight). The calibration curve was obtained using various concentrations of DOX hydrochloride dissolved in aqueous AcOH (500 AL AcOH/ 4000 AL of the solution). Quantitative release of DOX from micelles was proved by gel permeation chromatography (GPC), no DOX was found in micellar fraction in GPC on a PD-10 column using water as a mobile phase. The polymer content in the micellar solution of V was assayed gravimetrically after desalting on a PD-10 column. Va and Vb contain 3.38 and 3.12 wt.% DOX, respectively. The total yield is 185 mg (88%) of Va and 188 mg (90%) of Vb. 2.6. Characterization of (co)polymers 1

H NMR spectra were measured in THF-d 8 on a Bruker Avance MSL 200 MHz NMR spectrometer (Bruker Daltonik, Germany). IR spectra were recorded in KBr pellets on a Perkin-Elmer Paragon 1000 PC FT-IT spectrometer (Perkin-Elmer, USA). Gel permeation chromatography (GPC) was performed in THF as mobile phase on a PL Gel Mixed-B LS (10 Am) column (Polymer Laboratories, UK) using a Delta Chrom SDS 030 chromatograph (Watrex, USA) equipped with a PL-ELS 1000 Evaporative detector (Polymer Laboratories). PEO was used for the molecular weight calibration. Mass spectra were obtained with a MALDI-TOF mass spectrometer Biflex III (Bruker Daltonik) using dithranol as a matrix and sodium trifluoroacetate as a cationizing agent.

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Scheme 2. Structure of the conjugate V.

2.7. Preparation of aqueous micellar solutions for light scattering experiments The micellar solution of polymer V was diluted with phosphate-buffered saline, pH 7.4, to the polymer content 1 mg mL 1, and then pH was adjusted to the required value with aqueous sodium hydroxide (1 mol L 1) or hydrochloric acid (1 mol L 1), respectively. The solution was filtered before measurement through a 0.22 Am PVDF membrane filter.

scattering curves in the Zimm plot were extrapolated to the zero scattering angle. For the micelles under study, the concentration dependence was neglected, which seems to be justified by low concentrations of micellar solutions (110 3 g mL 1) and by low values of the second virial coefficient for micelles [19]. Extrapolation to the zero scattering angle was carried out by linear or quadratic fits of scattering curves. The refractive index increments of the copolymers under study were taken from the previous publication [18]. The effect of DOX attachment (~3 wt.%) was neglected.

2.8. Static light scattering (SLS) 2.9. Dynamic light scattering (DLS) Static light scattering measurements were performed on a Sophica 42,000 instrument (Wippler and Schebling, Strasbourg, France) equipped with a He–Ne laser as a light source at 25 8C. The apparatus was calibrated with toluene as a standard. To obtain the weight–average molecular weight M w of micelles,

Two types of measurements were performed: (1)

The time correlation functions were measured at the scattering angle h=1738 on a Nano-ZS (Malvern, UK) zetasizer. The DTS(Nano) pro-

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(2)

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gram was used for evaluation of data. The mean positions of the size distribution peaks were taken for data representation. Angle dependences of time correlation functions were measured in the angular range h=30–1208 using a light scattering apparatus equipped with a He–Ne (632.8 nm) and an ALV 5000 multibit multitau autocorrelator covering approximately 10 decades in a delay time t. The inverse Laplace transform using the REPES [20] method of constrained regularization (which is similar in many respects to the inversion routine CONTIN [21]) was used for the analysis of autocorrelation functions. The apparent average hydrodynamic radius R ha was calculated from the average diffusion coefficient using the Stokes–Einstein equation. To obtain the hydrodynamic radius R h of micelles, the reciprocal values of R ha were plotted against sin2h/2 and extrapolated to the zero scattering angle. The experimental error of the R h determination for micelles was typically ca. 3%.

2.10. DOX release from the micelles into the aqueous buffer The amount of released DOX into aqueous buffer was assayed from the DOX fraction remaining in micelles after separation from released DOX by gel permeation chromatography. A micellar solution of Vb in phosphate-buffered saline (1 mg mL 1 polymer, 4 mL total volume) of a particular pH value was incubated at 37 8C in the dark. Twohundred-microliter samples were taken at selected incubation times, and micellar fraction was separated on a PD-10 gel permeation column in phosphatebuffered saline of the same pH as the incubated solution. The volume of the micellar solution was adjusted by the addition of phosphate-buffered saline to 4000 AL, and 500 AL AcOH was added to each sample. The samples were incubated overnight at room temperature to hydrolyze hydrazone bonds and liberate free DOX. The concentration of released DOX in solution was determined by measurement of fluorescence emission intensity at 588 nm (excitation at 480 nm). Steady-state fluorimetric assay of DOX was performed with a Hitachi Perkin-Elmer MPF-2A spectrofluorimeter (without delay) in L-format using

quartz glass cuvette (114 cm). The relative error of the assay was ca. 3%. 2.11. Chemical release of DOX from the copolymer (hydrolysis of the hydrazone bond) The hydrolysis of the hydrazone bonds was measured in a similar way as the DOX release into buffer, but polymeric DOX was separated from free DOX by gel permeation chromatography on a Sephadex LH-60 column (10 mL bed volume) in DMSO–AcOH (199:1 v/v). The volume of the polymer solution was adjusted by the addition of DMSO–AcOH (199:1 v/v) to 4000 AL, and 1000 AL water was added to each sample. The samples were incubated overnight at room temperature to release DOX, and its concentration was estimated from fluorescence emission intensity measured at 592 nm (excitation 480 nm).

3. Results and discussion 3.1. Preparation of block copolymers The starting poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO; II) block copolymers of very narrow polydispersity (M w/M n ca. 1.05) and different lengths of the blocks were prepared using two different PEO monomethyl ethers (I; M n=2000 and 5000) as macroinitiators at a macroinitiator/ monomer ratio optimized to reach optimal micellar behavior [18]. Copolymers II were purified by desorption from silica as described in Ref. [18]. In contrast to the procedure described in Ref. [18], the purification step using Amberlite XAD-4 was omitted. Results of MALDI-TOF and GPC showed no presence of free PEO in the crude PAGE-PEO copolymer. See Table 1 for molecular weights of the polymers. Radical addition reaction of the SH group of the methyl sulfanylacetate to allyl double bonds of II was used to introduce ester groups to the hydrophobic polymer chain (III, a-methyl-poly(oxirane)-blockpoly(3-{[({[methoxycarbonyl]methyl}sulfanyl)propoxy]methyl}oxirane), see Scheme 1). III could be subsequently used after conversion to hydrazide (IV, a-methyl-poly(oxirane)-block-poly(3-{[({[hydrazino-

M. Hruby´ et al. / Journal of Controlled Release 103 (2005) 137–148 Table 1 Molecular weights of the polymers according to GPC Polymer

Mn

Mw

Ia

IIa IIb IIIa IIIb IVa IVb

3632 7272 5160 9411 5315 9693

3741 7926 5418 10634 6218 11729

1.03 1.09 1.05 1.13 1.17 1.21

a

I=M w/M n.

carbonyl]methyl}sulfanyl)propoxy]methyl}oxirane)) to bind DOX by a pH-sensitive hydrazone bond forming conjugate V (see Scheme 2). The hydrazone bond is cleavable under biologically relevant conditions under slightly acidic conditions (pH ca. 5, typical of the interstitial space of most solid tumours and of the endosomal environment) and is relatively stable under neutral conditions in blood plasma (pH 7.4) [3,22]. This should minimize release of toxic free DOX during delivery into tumour tissue and thus decrease side effects and increase the range of doses exploitable in therapy. Moreover, block copolymer IV containing hydrazides is sufficiently hydrophilic to be readily soluble in water forming true solutions without the formation of multimolecular assemblies, like micelles or aggregates (a 1 mg mL 1 solution of IVb in distilled water is already below the critical association concentration as proved by DLS). This causes disruption of the micelles after release of the drug into relatively small polymer chains that could be eliminated by kidneys. The addition of methyl sulfanylacetate to II was carried out according to Ref. [18] with one exception. The reaction time was prolonged to 5 h to ensure complete conversion of the reaction, especially in the case of IIb. Conversion of ester to hydrazide was achieved by refluxing a solution of III in THF with excess hydrazine hydrate by analogy with low-molecularweight compounds [23]. One-hundred-fold molar excess of hydrazine hydrate was used to prevent cross-linking probably by the formation of diacylhydrazines [23]. The ester CjO band in infrared spectrum of III at 1732 cm 1 completely vanished in the product IV, while the hydrazide CjO band at 1667 cm 1 appeared, proving the quantitative conversion of ester

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groups to hydrazides. The elemental analysis (found 7.77% N, 5.55 mmol g 1, 9.35% S, 2.92 mmol g 1, N/S molar ratio 1.90) was in good agreement with the theoretically calculated values (7.53% N, 5.38 mmol g 1, 8.62% S, 2.69 mmol g 1, N/S molar ratio 2.00). At that, cross-linking was very low (see Table 1 for polydispersities). The excess of hydrazine hydrate cannot be fully removed by evaporation probably due to the formation of hydrogen bonds between polymer and hydrazine, so the crude polymer was dissolved in water and dialyzed against water. The freeze-dried product was then used for the next step, the attachment of DOX. DOX was bound by condensation in DMSO catalyzed by AcOH with addition of a dehydrating agent insoluble in the reaction mixture (anhydrous sodium sulfate) to shift equilibrium towards the product V. Polymer fraction was separated by gel permeation chromatography in a DMSO–AcOH mixture because all other tested mobile phases showed adsorption of the polymer on the sorbent. AcOH was added to prevent the formation of unstable free base of DOX and DOX dimer that would otherwise form in neat DMSO. 3.2. Preparation of micelles of V The micelles were formed by passing a solution of V in acidified DMSO through a Sephadex G-25 column using aqueous phosphate-buffered saline, pH 7.4, as a mobile phase. The DOX content in the resulting micelles was assayed spectrophotometrically. The DOX content was 3.38 wt.% in Va (34% yield, 2.2% hydrazide groups modified) and 3.12% wt. DOX in Vb (31% yield, 2.6% hydrazide groups modified) at the DOX to polymer weight ratio in the reaction mixture 0.10. This is the maximum DOX loading to obtain stable micelles, more DOX causes the formation of large aggregates and macroscopic precipitation as proved by the preparation of polymers using 0.05, 0.10, 0.20 or 0.40 DOX to polymer weight ratios in the reaction mixture. The DOX content 3.38% in Va corresponds to the average content 0.32 DOX moieties per chain (the value of x on Scheme 2), and the DOX content 3.12% in Vb corresponds to 0.53 DOX moieties per chain. This means that some unmodified IV in V is necessary for stabilization of the micelles or, more exactly, micelle-like nano-

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particles (see the discussion below). Although the achievable DOX content in the polymeric micellar system is only ca. 3%, our experience obtained with soluble doxorubicin conjugates show that the drug loading ca. 4–5% [24] is sufficient to achieve excellent anticancer activity of the conjugate, both in vitro and in vivo. Moreover, in contrast to the soluble carrier systems, the DOX-containing micellar system is expected to exhibit a significantly more pronounced EPR effect (more efficient accumulation of the drug in tumour), and faster elimination of the soluble hydrazide copolymer (M w~10 kDa) from body could result in higher safe achievable dosing of the drug. Evaluation of antitumour activity of the micelles is under way. 3.3. Association of PEO-PAGE copolymers with attached DOX groups Copolymer Vb forms nanoaggregates in aqueous solutions with a defined distribution, R ha=104 nm, M w=2.2106, and the width of the intensity distribution ca. 90 nm. In the case of Va, bimodal distribution was observed (see Fig. 1). The small peak at R ha=14 nm corresponds to that of micelles of IVa (9.5 nm) [18], and the higher peak at R ha=60 nm can be related to aggregates formed by DOX-modified copolymers. This means that Va is a mixture of copolymers without attached and with attached DOX moieties.

Fig. 1. R ah distribution of Va in phosphate-buffered saline, pH 7.4; c=110 3 g mL 1, h=1738.

Characteristics of aggregates are the following: R h=66.7 nm, M w=1.8106 and the width of the distribution ca. 38 nm. M w of aggregates is higher by an order of magnitude than that of IVa micelles. The aggregates could be understood as swollen micellelike nanoparticles. The swelling is due to hydrophilic free hydrazide groups. The chemically modified PAGE blocks are more hydrophilic than the original block of II, and, therefore, micelle cores are swollen. Moreover, an increase in R h can also reflect a higher M w of aggregates. The broad polydispersity of particles could be due to chemical heterogeneity of the copolymers with attached DOX moieties. To characterize particle structure, the structural density of particles (q h) of polymers Va and Vb were calculated from the hydrodynamic volumes of particles V h and from their corresponding molecular weights (in g mol 1); q h=M w/N A V h. q h=4.310 3 g mL 1 and q h=1.510 3 g mL 1 were found for aggregates of Va and Vb copolymers, respectively. Both the q h values are by an order of magnitude lower than those of regular micelles [25]. The q h of Vb nanoparticles is smaller than that of Va nanoparticles, which might be due to a lower content of DOX in Vb compared with Va. 3.4. The release of DOX from copolymer V micelles The release of DOX from micelles of Vb (Va contains some larger particles) was assayed in phosphate-buffered saline (67 mmol L 1) pH 5.0 (pH tumour interstitial space and endosome content) and 7.4 (pH of blood plasma) [3,22]. A solution of the polymer was incubated in the particular buffer at 37 8C. Aliquot samples were taken after selected incubation times, and the micellar fraction was then separated from these samples by gel permeation chromatography on Sephadex G-25 in the same buffer as used for incubation. The DOX release was evaluated from the decrease in the DOX content in the micellar fraction because free DOX is partly adsorbed on the Sephadex G-25 column unlike the micellar fraction. This approach is more accurate than dialysis which also involves the effect of stirring and diffusion of free DOX through the dialysis bag [3]. The DOX content in the polymer fraction was estimated fluorimetrically because the DOX content is

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Fig. 2. DOX release from micelles of Vb into aqueous phosphatebuffered saline; t—incubation time.

too low for UV-Vis spectrophotometry. The fluorescence intensity of a molecule in solution is generally highly dependent on the chemical environment of the molecule, and hence free DOX cannot be used for calibration. This is why calibration was made with a micellar solution of the Vb copolymer. The calibration curve shows very good linearity (R 2=0.9990) in the relevant range of concentrations (0–100% bound DOX). The DOX release from micelles into aqueous environment (see Fig. 2) is relatively fast (faster at pH 5.0 than at 7.4) in the beginning and then apparently stops at 48% at pH 5.0 or 21% at pH 7.4. This is a similar profile to release of DOX physically entrapped in micelles [3], where equilibrium between DOX physically entrapped in micelles and free DOX in solution is established. Probably, in our static system, such equilibrium between DOX released in solution and DOX released but adsorbed in hydrophobic parts of the micelle core (hydrophobic chain, DOX still bound) is established. With the aim to understand better the mechanism of drug release from the micelles, we measured the rate of chemical hydrolysis of the bond between polymer chain and the drug.

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this is due to equilibrium between free DOX molecules in solution and DOX molecules trapped in the aggregates by hydrophobic interactions. The free DOX content is higher at pH 5.0 probably due to the increased protonation of amine and hydrazide groups of copolymers. Therefore, the equilibrium is more shifted towards release of free DOX molecules into solution at pH 5.0 than at pH 7.4. The real kinetics of DOX release from copolymers should be different from the observations in Fig. 2 and should continue even after an apparent equilibrium. To confirm this hypothesis, we developed a method that makes it possible to estimate the extent of chemically cleaved DOX under similar conditions avoiding the effect of hydrophobic entrapment. Thus, a sample after incubation in buffer was separated in acidified DMSO where the polymer is soluble as nonmicellar solution and no free DOX is adsorbed on the polymer. The polymer fraction was collected, and the DOX content was estimated fluorimetrically in analogy to the latter case. The calibration curve shows very good linearity (R 2=0.9994) in the relevant range of concentrations (0–100% bound DOX). The DOX chemical cleavage (see Fig. 3) follows the first-order kinetics (R 2=0.9847 at pH 5.0 and R 2=0.9868 at pH 7.4 with the corresponding halftimes 8.62 h at pH 5.0 and 198 h at pH 7.4, see Fig. 4) in the initial period (12 h at pH 5.0 and 144 h at pH 7.4) and then slows down but does not reach a plateau as fast as the release in aqueous buffer. This is probably due to the reversible nature of hydrazone bond formation and subsequent establishment of equilibrium between free DOX in solution, DOX physically entrapped in

3.5. Chemical release of DOX from the copolymer (hydrolysis of the hydrazone bond) There is a question of why the release of DOX from micelles slows down at values which are only fractions of the total DOX loadings. We suppose that

Fig. 3. Chemical release of DOX from Vb (cleavage of hydrazone bonds) in phosphate-buffered saline; t—incubation time.

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very similar (correlation coefficient 0.9907), and the formation of DOX dimers is strongly pH-dependent in the studied pH range [3]. Because free DOX is in vivo continuously removed by renal excretion and binding on cellular structures, this system should release all DOX after some time under these conditions. 3.6. Effect of DOX release on the behavior of aggregates Fig. 4. First-order kinetics of chemical DOX release from Vb (cleavage of hydrazone bonds); t—incubation time, c—concentration of free DOX, c 0—total concentration of DOX at t=0. .—pH 5.0; n—pH 7.4.

micelles and DOX chemically bound to polymer. It is also unlikely that the formation of dimer between DOX chemically bound to polymer and already released DOX may play a significant role in the DOX release from the micelles. An argument for this is that, although the release of DOX is faster at pH 5.0, if we use a time scale not in absolute values (hours) but as (incubation time)/(release halftime at particular pH), the release curves are

The time development of the R ha distribution in Vb solutions (c=1x10 3 g mL 1) at pH 5.0 due to the DOX release from particles is shown in Fig. 5, curve a. The formation of small particles (ca. 15 nm) was already observed after 27 h of incubation. The size of the particles practically coincides with the size of IVb micelles (12.3 nm) in phosphate-buffered saline at concentration 1 mg mL 1 (the same concentration in distilled water is already below critical association concentration as proved by DLS, see above). Therefore, it is plausible to assume that the small particles are micelles of copolymer IVb after cleavage of DOX. The weight concentration of the micelles increases and becomes dominant after 190 h.

Fig. 5. R ah distribution of Vb in phosphate-buffered saline after indicated incubation times; c=110 b—pH 7.4.

3

g mL 1, h=1738. Curve a—pH 5.0; curve

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A different behavior of aggregates was observed in Vb solutions (c=110 3 g mL 1) at pH 7.4 (see Fig. 5, curve b). Only minor changes of the R ha distribution were observed after 76-h incubation. The distribution was only slightly broadened. The formation of bimodal distribution was observed after long-term incubation. In addition to large aggregates, smaller particles with a maximum of R ha at ca. 70 nm were observed. The results reflect the above finding that the release of DOX from particles is faster and more extensive at pH 5.0 than at pH 7.4, in agreement with expectations. 3.7. Interaction of micelles with albumin Serum albumin is the main blood serum protein showing hydrophobic interactions with numerous low-molecular-weight drugs. To test the interaction of Vb with serum albumin, 1 mg mL 1 of bovine serum albumin (BSA) was added to micelles of Vb (c=110 3 g mL 1) in phosphate-buffered saline, pH 7.4, and changes in apparent molecular weight M wa of particles were followed in the course of 5 h. M wa was calculated under the assumption that the concentration of aggregates is not changed by BSA addition. As no changes of M wa were observed, a negligible interaction of BSA with the micelles of Vb can be assumed.

4. Conclusions A new polymeric micellar pH-sensitive system for the drug delivery of doxorubicin was synthesized and characterized. Polymeric micelle-like nanoparticles were prepared by self-assembly of amphiphilic diblock copolymers in aqueous solutions. The copolymers consist of a biocompatible hydrophilic poly(ethylene oxide) (PEO) block and a hydrophobic block containing doxorubicin covalently bound to the carrier by a pH-sensitive hydrazone bond. The drug release kinetics was studied in aqueous buffers at pH 5.0 (close to pH in endosomes) and 7.4 (pH of blood plasma). The drug was released much faster at pH 5.0 than at 7.4. The particle behavior in aqueous solutions at pH 5.0 and 7.4 was studied by static (SLS) and dynamic light scattering (QELS).

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Aknowledgements The authors acknowledge financial support of the Grant Agency of the Academy of Sciences of the Czech Republic (grants No. A4050403, A100500501 and B4050408) and Grant Agency of the Czech Republic (grant No. 305/02/1425A). References [1] G.S. Kwon, K. Kataoka, Block-copolymer micelles as longcirculating drug vehicles, Adv. Drug Deliv. Rev. 16 (1995) 295 – 309. [2] G.S. Kwon, T. Okano, Polymeric micelles as new drug carriers, Adv. Drug Deliv. Rev. 21 (1996) 107 – 116. [3] K. Kataoka, T. Matsumoto, M. Yokoyama, T. Okano, Y. Sakurai, S. Fukushima, K. Okamoto, G.S. Kwon, Doxorubicin-loaded poly(ethylene glycol)-poly(h-benzyl-l-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance, J. Control. Release 64 (2000) 143 – 153. [4] H.S. Yoo, E.A. Lee, T.G. Park, Doxorubicin-conjugated biodegradable polymeric micelles having acid-cleavable linkages, J. Control. Release 82 (2002) 17 – 27. [5] G.A. Husseini, R.I. El Fayoumi, K.L. O’Neill, N.Y. Rapoport, W.G. Pitt, DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study, Cancer Lett. 154 (2000) 211 – 216. [6] G.A. Husseini, N.Y. Rapoport, D.A. Christensen, J.D. Pruitt, W.G. Pitt, Kinetics of ultrasonic release of doxorubicin from pluronic P105 micelles, Colloids Surf., B Biointerfaces 24 (2002) 253 – 264. [7] A. Marin, M. Muniruzzaman, N. Rapoport, Mechanism of the ultrasonic activation of micellar drug delivery, J. Control. Release 75 (2001) 69 – 81. [8] Y.J. Son, J.S. Jang, Y.W. Cho, H. Chung, R.W. Park, I.C. Kwon, I.S. Kim, J.Y. Park, S.B. Seo, C.R. Park, S.Y. Jeong, Biodistribution and anti-tumour efficacy of doxorubicin loaded glycol-chitosan nanoaggregates by EPR effect, J. Control. Release 91 (2003) 135 – 145. [9] K. Kataoka, G.S. Kwon, M. Yokoyama, T. Okano, Y. Sakurai, Block-copolymer micelles as vehicles for drug delivery, J. Control. Release 24 (1993) 119 – 132. [10] V.Y. Alakhov, E.Y. Moskaleva, E.V. Batrakova, A.V. Kabanov, Hypersensitization of multidrug resistant human ovarian carcinoma cells by pluronic P85 block copolymer, Bioconjug. Chem. 7 (1996) 209 – 216. [11] M. Wenke, Farmakologie, Avicenum, Prague, Czech Republic, 1990. [12] E.V. Batrakova, T.Y. Dorodnych, E.Y. Klinskii, E.N. Kliushnenkova, O.B. Shemchukova, O.N. Goncharova, S.A. Arjakov, V.Y. Alakhov, A.V. Kabanov, Anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity, Br. J. Cancer 74 (1996) 1545 – 1552.

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