115 (2006) 197–207www.elsevier.com/locate/jconrel

Journal of Controlled Release

Acid-cleavable polymeric core–shell particles for deliveryof hydrophobic drugs

Yannie Chan a, Volga Bulmus a,⁎, M. Hadi Zareie b, Frances L. Byrne c,Leonie Barner a, Maria Kavallaris c

a Centre for Advanced Macromolecular Design, CAMD, School of Chemical Sciences and Engineering,The University of New South Wales, Sydney 2052, NSW, Australia

b Faculty of Science (INT), University of Technology Sydney, UTS, Sydney 2007, NSW, Australiac Children's Cancer Institute Australia for Medical Research, Sydney 2031, NSW, Australia

Received 9 June 2006; accepted 25 July 2006Available online 29 July 2006

Abstract

Here we describe the combined use of acid-labile microgel approach and RAFT-mediated seeded dispersion polymerization technique toprepare an acid-cleavable core–shell like polymeric colloidal system for the delivery of hydrophobic drugs at slightly acidic sites. A newbisacrylate acetal crosslinker was copolymerized with n-butyl acrylate (BA) in the presence of a RAFT agent using a dispersion polymerizationtechnique, which yielded crosslinked spherical particles with the size ranging between 150 and 500 nm. The particles were cleaved in a pH-dependent manner similar to the acid-labile hydrolysis behaviour of the crosslinker. In order to mask the hydrophobic surface of the particles,polyethylene glycol acrylate (PEG-A) was grafted onto poly(BA) seed particles via the RAFT agent groups on the particle surface. The acidic-siteselective delivery potential of the poly(BA)-g-poly(PEG-A) particles was assessed in-vitro using a lipophilic fluorescent dye as a modelhydrophobic drug. Ca. 73% and 34% of the total dye loaded in the particles was found to be released at pH 5.0 and 7.4 in 24 h, respectively. Thegrowth of human neuroblastoma cells was not affected by the incubation with the core–shell particles and their cleavage by-products up to 3 mg/ml concentration. The physicochemical and the functional features support the potential value of the acid-cleavable poly(BA) core–poly(PEG-A)shell particles as carriers for the delivery of hydrophobic drugs at acidic sites.© 2006 Elsevier B.V. All rights reserved.

Keywords: pH-sensitive particles; Drug delivery; PEG grafting; Dispersion polymerization; Reversible addition-fragmentation chain transfer (RAFT)

1. Introduction

For controlled drug delivery applications, an ideal carriersystem should deliver its drug payload only to the site where thetherapeutic activity is required. Recent studies on the site-selective delivery of therapeutics have utilized the environmen-tal-stimuli to trigger the release of the drugs to a particular bodycompartment [1–4]]. A very elegant release strategy in thiscontext is to use pH-gradients in the body for the local deliveryof the drugs. For example, the pH of the extracellular fluid ofsome tumors is slightly lower than the pH of the normal tissueand the blood (i.e. pH 7.4) [5–7]]. Similarly, the intracellular

⁎ Corresponding author. Tel.: +61 2 9385 5745; fax: +61 2 9385 4749.E-mail address: vbulmus@unsw.edu.au (V. Bulmus).

0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jconrel.2006.07.025

vesicles of cells involved in the endocytosis mechanism, i.e.endosomes and lysosomes are also acidic [1,8,9]. Many thera-peutic strategies such as antitumor chemotherapy, gene andoligonucleotide therapies and vaccine delivery applications inwhich the relevant therapeutics reach these acidic sites as a partof their therapeutic pathway would greatly benefit from thedevelopment of new delivery systems offering low pH-triggeredrelease of therapeutics.

pH-sensitive liposomes have been investigated widely forthe controlled release of therapeutics at acidic sites, especiallyfor the intracellular delivery of drugs and genes [10–14]]. Morerecently, pH-sensitive polymeric particulate systems such asnano/microparticles and micelles have attracted attention due totheir relatively higher stability. The recent studies [4,15–26]demonstrate that polymeric particles with a low-pH triggered

198 Y. Chan et al. / Journal of Controlled Release 115 (2006) 197–207

release profile are highly likely to prove superior for the thera-peutic strategies which involve the acidic sites as a part of thetherapeutic's pathway, e.g. endosomes/lysosomes for biomacro-molecular therapeutics, or as a target site, e.g. tumor extra-cellular environment for antitumor drugs.

The studies using colloidal systems as therapeutic carriersshow that the size and the surface characteristics critically deter-mine the fate of these systems in the body. In the case of tumorchemotherapy, particles with size ranging between 70 and 200 nmare found to efficiently accumulate at the tumor tissues due to thewell-known EPR effect [27]. Regarding the surface characteristic,the particulate system needs to have a hydrophilic and neutralsurface which reduce the attack of the phagocytic cells.

The use of advanced polymerization techniques such as thereversible addition-fragmentation chain transfer (RAFT)[28,29] polymerization in the generation of polymer colloidswould potentially provide process advantages leading to theversatility in the control of physicochemical properties. Forexample, polymeric spheres bearing a surface functionalizedwith RAFT-groups could be synthesized via a RAFT mediatedprecipitation polymerization [30]. The RAFT groups on theparticle surface could be used to modify the surface easily byfurther polymerizing a co-monomer, leading to a core–shell likepolymeric particle with a polymeric shell covalently attached tothe core. Alternatively, these groups can be utilized to conjugate

Scheme 1. Scheme for the preparation of the acid-cleavable nanometer size particdispersion polymerization technique. The particles are cleaved to polymer chains an

preformed polymers, or functional agents such as cell targetingmolecules, directly to the surface. This potential motivated us toinvestigate the use of RAFT technique to produce new acid-cleavable polymer colloids with versatile properties for thedelivery of drugs at slightly acidic sites such as intracellularsites. Herein, we report the synthesis of nanometer size, acid-cleavable, oligoethylene glycol modified poly(n-butyl acrylate)particles as carriers for the hydrophobic molecules using aRAFT agent mediated seeded dispersion polymerizationtechnique. We focused on the acidic site-selective delivery ofhydrophobic molecules since a number of widely used anti-tumor drugs such as Paclitaxel and Doxorubicin are hydropho-bic. In addition, the delivery of such drugs involves slightlyacidic sites, i.e. tumor and intracellular sites as a part of theirtherapeutic pathway. Considering the hydrophobicity of thesedrugs, a hydrophobic monomer, n-butyl acrylate (BA) wascopolymerized with a new acid-cleavable bisacrylate monomerin the presence of a RAFT agent to produce crosslinked poly(BA) particles with surface RAFT groups (Scheme 1). TheRAFT agent-functionalized particles were then modified viagraft polymerization of polyethylene glycol acrylate monomer(PEG-A) to mask the hydrophobicity of the surface. The poten-tial of the particles for the acidic-site selective release of ahydrophobic drug was then assessed in vitro using a lipophilicfluorescent dye.

les with poly(BA) core and poly(PEG-A) shell using RAFT-mediated seededd by-products at acidic pH.

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2. Materials and methods

2.1. Materials

n-Butyl acrylate (BA) (Fluka), ethylene glycol dimethacry-late (EGDMA) and PEG-acrylate (PEG-A) (Aldrich) (454 g/mol, number of ethylene glycol units per acrylate: 8–9) waspurified using a basic aluminum oxide column. Azobisisobutyr-onitrile (AIBN) (DuPont) was recrystallized twice from ethanolprior to use. Cumyl dithiobenzoate (CDB) was synthesized asreported elsewhere [31]. p-Methoxybenzaldehyde (p-MBA), 2-hydroxyethyl acrylate (HEA), p-toluene sulfonic acid mono-hydrate were purchased from Sigma-Aldrich. Triethylamine(TEA) and poly(N-vinyl pyrrolidone) (PVP) (MW: 40,000 g/mol) were purchased from Riedel-de Haen and Polysciences.Inc., respectively. All other chemicals were purchased fromAjaxFinechem and used without further purification.

2.2. Synthesis and characterization of acid-cleavable cross-linker

p-MBA (0.0485 mol) and HEA (0.34 mol) were reacted inan ice-bath for 15 h using p-toluene sulfonic acid (0.0084 mol)as catalyst and 4 Å molecular sieves as drying agent. Thereaction was quenched by adding TEA (0.072 mol). Followingthe extraction with 0.1 M aqueous potassium carbonate so-lution, the reaction mixture was purified using a basic aluminacolumn and n-hexane/dichloromethane/TEA mixtures of 85/5/10 and 90/0/10. The bisacrylate acetal crosslinker, di(2-acry-loyloxy ethoxy)-[4-methoxy-phenyl]methane), was obtainedin ca. 98 mol% purity as confirmed by 1H NMR. Chemicalshifts in dimethylsulfoxide-d6: 3.6 t (4H), 3.7 s (3H), 4.2 t (4H),5.6 s (1H), 5.9 d (2H), 6.1 q (2H), 6.2 d (2H), 6.8 d (2H), 7.2 d(2H). ESI-MS (theoretical mass: 350.4 g/mol, measured mass:350.1 g/mol).

The pH-dependent hydrolysis of the acetal crosslinker wasinvestigated at pH 4.5, 5.5, 6.5, and 7.4 using a UV–vis

Table 1RAFT-mediated dispersion polymerization of BA and bisacrylate acetal crosslinker

Exp # C-linker [M]×10−2 AIBN [M]×10−3 CDB [M]×10−2 PVP [M]×10

1 17 12 3.0 142 17 12 3.0 143 17 6 3.0 144 7 6 3.0 145 7 6 1.4 146 4 6 1.4 167 4 6 1.4 228 2 6 1.4 229e 5 6 1.4 14

BA concentration was 0.5 M in all polymerizations. Polymerization temperature anddynamic light scattering (DLS). Standard deviation was calculated based on at leasta Average size is based on DLS measurement performed with dust filter ignoringb Size distribution is based on DLS measurement without dust filter. The percent

particles.c EtOH/pyridine solvent mixture was used in this polymerization.d No DLS measurement without the dust filter is available.e In this experiment, a non-cleavable crosslinker, EGDMA was used instead of af Determined by SEM.

spectrophotometer (Cary 300). 50 μl of the crosslinker solution(1.7 mg/ml anhydrous dimethylformamide (DMF)/TEA, 1%)and 450 μl of the buffer solution at varying pHs were incubatedat 37 °C. The concentration of the hydrolysis product, p-MBAwas measured at the predetermined time intervals by UV-absorbance analysis of the sample solutions at 270 nm. At theend of the experiment, the complete hydrolysis was determinedafter the incubation of the crosslinker at pH 3 for 24 h.

2.3. Synthesis of crosslinked poly(BA) particles by RAFTmediated dispersion polymerization

BA was copolymerized with the bisacrylate acetal cross-linker or EGDMA in the presence of CDB using a dispersionpolymerization technique. PVP and the solvent was mixed withthe monomer phase which contained BA, the crosslinker, AIBNas an initiator and CDB as a RAFT agent. The polymerizationrecipes are given in Table 1. Following the nitrogen purging for30 min, the polymerization mixture was placed in a shakingwater bath (Grant OLS 200) at room temperature. Polymeriza-tion was performed at 65 °C with a shaking rate of 150 rpm for36 h. After the polymerization, the crosslinked poly(BA) par-ticles was purified by dialyzing the polymerization mixture witha dialysis membrane (MWCO: 50,000 g/mol) using dichlor-omethane (DCM)/TEA and ethanol/TEA (98/2 v/v) mixturesfor 2 days. The collected mixture was centrifuged at 6000×g for20 min. Following the removal of supernatant, the collectedparticles were washed twice with DCM and final time withethanol by centrifugation. Finally, supernatant was removed andthe particles were dried under vacuum.

2.4. Grafting of poly(PEG-A) onto RAFT agent functionalizedpoly(BA) particles

Firstly, the conditions for the CDB-mediated solutionpolymerization of PEG-A in ethanol/TEA mixture (98/2 v/v)was optimized. The polymerization of PEG-A (at 65 °C) was

(Clinker)−4 MeOH/pyridine (v/v) Average sizea (nm) Size distributionb (nm)

95:5 247±28 115–496; 752–1200 (5.4%)95:5c 478±79 329–57795:5 432±67 311–502; 720–1800 (2.3%)95:5 457±55 350–564; 800–3200 (2.2%)95:5 230±30 131–257; 791–1100 (1.5%)98:2 190±10 161–224; 900–2000 (1.3%)98:2 274±36 204–307d

98:2 195±45 158–274; 750–1000 (0.2%)100:0 160±70f 43–260; 1000–3000 (1.5%)

time was 65 °C and 36 h, respectively. Average particle size was measured bythree different DLS measurements of each sample.particles larger than 1 μm.

age shows the number of particles larger than 1 μm percent of total number of

cid-cleavable crosslinker.

200 Y. Chan et al. / Journal of Controlled Release 115 (2006) 197–207

found to proceed via RAFT mechanism when the molar ratio ofPEG-A/AIBN/CDB was 1/2×10−3/8×10−3, as determined byGPC and 1H NMR. These conditions were utilized for graftingPEG-A to the CDB functionalized poly(BA) particles. In atypical grafting experiment, PEG-A, AIBN and CDB wereadded to the seed poly(BA) particle dispersion (approx. 20 mg/ml) in a mixture of ethanol/TEA (98/2, v/v). Following thenitrogen purging of the polymerization mixture for 30 min, thepolymerization was carried out at 65 °C for 6 h. The additionalRAFT agent was used in the grafting polymerization to ensurethat the concentration of the RAFT agent is sufficient to controlthe polymerization both in the solution and on the particlesurface via RAFT mechanism. After polymerization, the mix-ture was first dialyzed in ethanol/water/TEA solutions (MWCO50,000). The particles were then collected by centrifuging thedialyzed solution at 6000×g for 15 min and removing thesupernatant. The final particles were redispersed in ethanol/TEA (99/1 v/v) and the washing procedure was repeated threetimes.

2.5. Characterization of the particles

The size of the particles was measured by a BrookhavenParticle Size and Zeta Potential Analyser, with a measurementangle of 90°. Following sonication for 30 min, the dynamic lightscattering (DLS) of the particle dispersion in ethanol/TEA (98/2 v/v) was measured at least in triplicates by scanning 3 timesfor 1 min duration using a dust filter with 1 μm cutoff.

Thermal analysis was carried out with a PerkinElmer DSC 7equipped with a PerkinElmer CCA 7 controlled coolingaccessory and a TAC 7/DX thermal analysis controller. Theinstrument was calibrated using cyclohexane and indiumstandards. Samples (20 mg in a 40 μl aluminum pan) werescanned from −100 °C to 100 °C, with a heating rate of40 °C min−1 under nitrogen. Tg was calculated from the secondheating scan at the temperature which half the increase in heatcapacity had occurred.

The particle cleavage kinetic was determined via a UV–visspectrophotometer measuring the concentration of the cleavageby-product, p-MBA at 270 nm. The dialysis membrane(MWCO 35,000) containing poly(BA) particles (0.25 ml,10 mg/ml in dimethylformamide) was immersed into 5 ml ofthe buffer solution at pH 4.5. and 7.4, and incubated at 37 °Cwith reciprocal shaking at 40 rpm. At the predetermined timepoints, 0.25 ml of the dialysis medium was removed for themeasurement. The volume of the dialysis medium was retainedby adding 0.25 ml of fresh buffer at relevant pH after everysampling. After 24 h, the pH of the particle solutions wasdecreased to pH 3 and the solutions were incubated for another24 h to hydrolyze the particles completely to determine thequantity of MBA released in the case of complete cleavage.

Atomic force microscopy (AFM): The particles wereincubated in a citric acid/phosphate buffer at pH 4.5 and aphosphate buffer at pH 7.4 for 4 h (at room temperature) and10 μl was then sampled on a silicon (111) substrate for theanalysis with a Digital Instruments Multimode Nanoscope IIIscanning force microscope using tapping mode. Oxide-sharp-

ened silicon nitride tips with integrated cantilevers with anominal spring constant of 0.38 N/m were used.

Scanning electron microscope (SEM): A LEO Supra 55VPSEM (Zeiss) equipped with an in-lens secondary electrondetector was used. Images were manipulated using a Scan-ning Probe Image Processor while contrast was enhancedusing Adobe PhotoShop to enhance discrimination of the finedetail.

X-ray Photoelectron Spectrometer (XPS): A Kratos AxisULTRA XPS incorporating a 165 mm hemispherical electronenergy analyzer was used. The incident radiation wasMonochromatic A1 X-rays (1486.6 eV) at 225 W (15 kV,15 ma). Survey (wide) scans were taken at an analyzer passenergy of 160 eV and multiplex (narrow) higher resolutionscans at 20 eV. Survey scans were carried out over 1200–0 eVbinding energy range with 1.0 eV steps and a dwell time of100 ms. Narrow higher resolution scans were run with 0.05 eVsteps and 250 ms dwell time. Base pressure in the analysischamber was 1.0×10−9 Torr and during sample analysis1.0×10−8 Torr.

A modular gel permeation chromatography (GPC) system(Shimadzu) was used for the molecular weight measurements.The mobile phase was dimethylacetamide/0.05% LiBr with acolumn set consisted of a PL 5.0 μm bead size guard columnand a set of 3×5.0 μm PL linear columns (103, 104 and 105 Å)(70 °C) and a DRI detector. The linear polystyrene was used asstandards.

2.6. Loading and pH-dependent release of a hydrophobicmolecule

A lipophilic fluorescent dye, BODIPY® (4,4-difluoro-3a,4adiaza-s-indacene) (Molecular Probes) was used as amodel hydrophobic molecule. 8 mg of BODIPY® wasincubated with 50 mg of particles in 10 ml of dichloro-methane/TEA mixture (99/1 v/v) for 2 days at roomtemperature. After solvent removal, 10 ml of cold methanolwas added to the dye–particle mixture and the solution wastransferred into a dialysis membrane with MWCO 50,000. Thesolution was dialyzed using methanol/TEA (99/1 v/v) for 6 h byreplacing the medium with the fresh one every 2 h. The dialyzedparticle solution was then divided into equal volume fractionswhich were then centrifuged and dried under vacuum. Todetermine the quantity of the dye loaded in the particles, thedye-loaded particles were hydrolyzed in a citric acid buffersolution at pH 3.0 for 24 h and the dye concentration wasdetermined by a fluorescence spectrometer.

For the determination of the pH-dependent release profile ofthe particles, 4 ml of phosphate buffer saline (PBS, 0.15 M) atpH 5.0 or pH 7.4 was added to the dye-loaded dried particlefractions. These solutions were transferred into dialysismembranes, MWCO 50,000 which were then introduced into40 ml of PBS at the relevant pH and incubated at 37 °C in thedark with reciprocal shaking at 40 rpm. At predetermined timeintervals, 3 ml of each dialysis medium was removed for mea-surement and the same amount of fresh PBS was added to thesolution. After 24 h, the solutions in the dialysis bags were

Fig. 2. Cleavage of poly(BA) particles crosslinked with the acetal crosslinker atpH 4.5 and 7.4, which was monitored by the release of p-methoxybenzaldehydeusing UV–vis spectrophotometry (Exp #8, Table 1). Images are representativeAFM images of the particles (Exp #2, Table 1) after the incubation at pH 7.4 andpH 4.5 for 4 h. The scale bar is 2 μm.

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transferred to vials to completely hydrolyze the particles todetermine the total quantity of the dye loaded in the particlefraction as described above. The quantity of dye in each samplewas determined by fluorescence spectroscopy (excitation at475 nm, emission at 510 nm) after the appropriate dilutioncorrections using a calibration curve built from the knownconcentrations of BODIPY. Each experiment was performed induplicate, and the quantity of the released dye was expressed asthe percentage of the total dye loaded in the particles (w/w %).The release of BODIPY dye from the particles crosslinked witha non-cleavable crosslinker, i.e. EGDMAwas also investigatedas control. The same procedure was used for loading the dye.The total quantity of the dye loaded in these non-cleavableparticles was determined by measuring the difference in theconcentration of the dye solution before and after incubatingwith the particles.

2.7. Cell viability assay

The effect of poly(BA)-g-poly(PEG-A) particles and theircleavage products on the cell viability was assessed by AlamarBlue assay using human neuroblastoma (SH-EP) cells [32,33].Briefly, SH-EP cells were seeded a day prior to sample ex-posure at 3000 cells/well (96 well plate) in culture mediumcontaining 10% FCS/DMEM a day prior to sample exposure.Sample stocks were prepared in either DMSO Hybri-Max®(Sigma Aldrich), or DMSO/MQ-H2O solution, followedby dilution in culture medium. The final concentration ofDMSO exposed to the cells was no more than 0.5% (v/v) forthe duration of the experiment. Varying concentrations ofparticle samples or their cleavage products were added to thecells in triplicate (5–3000 μg/ml) and then incubated at 37 °C/5% CO2 for 72 h. Following the 72 h incubation, metabolicactivity was detected by addition of Alamar blue and spec-trophotometric analysis. Absorbance was determined and ex-pressed as a percentage of control, untreated cells (i.e. cellswith no sample). The cleavage products of the particles wereprepared after the complete hydrolysis of a known quantity ofthe particles in a citric acid-phosphate buffer at pH 4.0 for 3

Fig. 1. SEM image of acid-cleavable bisacrylate acetal crosslinked poly(BA)particles synthesized by the RAFT agent-mediated dispersion polymerization(Exp #8, Table 1). The scale bar shows 1 μm.

days followed by drying and resuspending in the DMSO/culture medium for the assay.

3. Results

3.1. Synthesis and characterization of RAFT agent-functiona-lized acid-cleavable poly(BA) particles

Firstly, a bisacrylate crosslinker, di(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) was synthesized in one-step withan average reaction yield of 55 mol% (by 1H NMR) using acommercially available hydroxylated acrylate monomer,hydroxyethyl acrylate (HEA) and p-methoxybenzaldehyde (p-MBA). The pH-dependent hydrolysis of the crosslinker wasinvestigated at four different pHs (i.e. pH 4.5, 5.5, 6.5 and 7.4).The hydrolysis reaction was monitored by UV–vis spectrom-eter measuring the increase in the concentration of p-MBA thatforms upon the hydrolysis of the crosslinker. At pH 4.5 and5.5, the crosslinker was found to be hydrolyzed with a half-lifeof approx. 15 and 175 min, respectively. At pH 6.5, thehydrolysis was slower but it still reached to ca. 40% in 4 hwhile at neutral pH the crosslinker remained stable for the sameperiod of time.

The new bisacrylate crosslinker and n-butyl acrylate (BA)was copolymerized via a RAFT agent mediated dispersionpolymerization technique using a biocompatible stabilizer, PVP.Cumyl dithiobenzoate (CDB) was used as a RAFTagent to havefunctional sites for the further modification of the acid-cleavable seed particles to have a hydrophilic and biocompat-ible surface. To prevent the degradation of the acid-labilestructure, a base i.e. pyridine was also included in the poly-merization recipes. The particles synthesized using CDB (which

Fig. 3. Molecular weight vs. conversion for PEG-A polymerization in ethanol/TEA mixture (98:2, v/v) at 65 °C (mol ratio of PEG-A/AIBN/CDB=1/2×10−3/8×10−3). The numbers on the graph show the PDIs.

Fig. 4. (A) SEM image of poly(PEG-A) grafted poly(BA) particles. Scale bar is1 μm. (B) XPS C1s spectra of CDB-functionalized poly(BA) particles beforeand after grafting with poly(PEG-A). Peak assignment: C1s peaks of C–C at abinding energy of 285.0 eV, C*–COO at 285.7 eV, C–O at 286.6 eV, C=O at287.7 eV, and COO at 289.0 eV.

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is pink in color) appeared to have a pink color similar to thecolor of CDB, whereas the particles synthesized by the CDB-free dispersion polymerization yielded a whitish latex solution.Table 1 shows the average size of the particles obtained from thepolymerizations. The size of the crosslinked particles was foundto be between 150 and 500 nm. Here it should be noted that theaverage particle size shown in Table 1 was measured using adust filter, eliminating the particles with size larger than 1 μm.The number of the large particles (N1 μm) was found tocompose usually less than 10% of the total number of theparticles for all polymerizations performed. The yield of theformation of the crosslinked particles was found to be between10 and 15 wt.% of the total monomer quantity in the feed for allpolymerizations probably due to the reactivity ratios of the co-monomers. The monomer conversion for all polymerizationswas found to be higher than 85 wt.%. Fig. 1 shows a repre-sentative SEM image of the poly(BA) particles crosslinked withthe acid-cleavable crosslinker.

The kinetic of the pH-dependent cleavage of particles(Ex #8, Table 1) was determined by monitoring the release ofthe cleavage by-product, i.e. p-MBA that was formed upon thehydrolysis of the acetal bonds in the particles. The particlesshowed a degradation half-life of approximately 12 h at pH 4.5,whereas the half-life of the particles at pH 7.4 appeared to belonger than 24 h (Fig. 2). The acid-sensitive degradation be-havior of the particles was also confirmed by AFM (Fig. 2). InAFM experiments, particles obtained from Exp #2 (Table 1)were used. From the AFM images, most of the particlesappeared to preserve their spherical shape after the incubation atpH 7.4 for 4 h whereas the particles incubated at pH 4.5 for thesame period of time clearly lost their spherical shape which ledto the change in the morphology and the loss of the roughness ofthe AFM substrate surface.

3.2. Surface grafting of RAFT agent-functionalized poly(BA)particles

At the next step, the seed poly(BA) particles were graftedwith poly(PEG-acrylate) (poly(PEG-A)) by RAFT polymeriza-tion of the relevant monomer in ethanol/TEA mixture (98:2 v/v). In a control experiment, RAFT polymerization of PEG-Awas first performed without using the particles to optimize theconditions. The molecular weight (Mw) versus conversion datafor the RAFT polymerization of PEG-A is given in Fig. 3. Thelinear increase of Mw with the increase in conversion, and

the low polydispersity indexes (PDI) (b1.17) indicated that thepolymerization proceeded via RAFT mechanism under theconditions utilized.

The grafting polymerization was then performed with adilute solution of CDB-functionalized particles using theconditions obtained from the control polymerization. Theparticles after grafting appeared to lose the defined sphericalshape (Fig. 4A). This might be due to the inhomogeneity of thegrafted polymer layer on the particle surface, which may becaused by the inhomogeneous localization of the macro-RAFTgroups (poly(BA) chains with RAFT end-groups) on the surfaceof the particles. The presence of PVP as stabilizer along with thepolar polymerization media may be expected to cause theinhomogeneous localization of the hydrophobic poly(BA)chains near the particle surface during the particle formationprocess. After the grafting polymerization, the particles werewashed by dialysis and subsequent centrifuging steps to removethe poly(PEG-A) physically attached to the particle surface.Following the washing steps, particles were analyzed by DSCand XPS to confirm the grafting of poly(PEG-A). From DSCdata, the Tg of the particles before and after grafting was found

Fig. 6. The percent viability of human neuroblastoma SH-EP cells after 72 hincubation with poly(BA)–co-poly(PEG) particles or their cleavage productsrelative to controls (cells with no sample). The core–shell particles used in theassay were obtained by PEG-grafting of poly(BA), Exp #8, Table 1. Thecleavage products of the particles were prepared by the complete cleavage of apredetermined quantity of the same particles in citric-acid phosphate buffer atpH 4.0 for 3 days at room temperature followed by lyophilization.

203Y. Chan et al. / Journal of Controlled Release 115 (2006) 197–207

to be −21.0±3.0 and −54.1±0.4 °C, respectively. The decreasein the Tg of the particles after the grafting reaction towards theTg of the soluble poly(PEG-A) (−59.7 °C) evidenced thepresence of poly(PEG-A) grafted to particles. The Tg of thesoluble poly(BA) was found to be −37.4 °C. Further inves-tigation by XPS revealed clearly the existence of poly(PEG-A)layer on the surface of the particles (Fig. 4B). A significantincrease in the intensity of the C1s peak at a binding energy of286.6 eV that is assigned to the C–O bond was observed in theXPS spectra of poly(PEG-A) grafted particles. The presence ofpoly(PEG-A) layer on the surface of the particles is expected toincrease the intensity of C–O specific C1s peak due to theaddition of a large number of oligoethylene glycol (CH2–CH2–O) units to the structure. From the XPS data, 92% of the surfaceatomic composition was found to be composed of poly(PEG-A).The particle size analysis by DLS revealed that the average sizeof the seed particles increased by 32±5% of the original averagesize after grafting with poly(PEG-A).

3.3. pH-dependent release of a hydrophobic dye from theparticles

The pH-dependent release profile of the crosslinked poly(BA)-g-poly(PEG-A) particles was investigated using a lipo-philic fluorescent dye as a mimic of a hydrophobic drug. PEGgrafted poly(BA) particles obtained from Exp #8 (Table 1) wereused in this experiment. The dye was loaded into the particlesby first incubating the particles and the dye in a good solvent forboth the particles and the dye, i.e. dichloromethane and thenreplacing the solvent with a poor one for the polymer, i.e.methanol. The loading content was found to be ca. 8 μg dye/mgparticle (0.8 wt.%). The dye loaded particles were incubated inthe buffer solution at two different pHs, i.e. pH 5.0 and 7.4 for24 h and the release of the drug was quantified at predeterminedtime intervals using a fluorescence spectrometer. The results ofthis experiment are shown in Fig. 5. After 24 h, at pH 5.0, ca.73% of the dye loaded in the particles was released from the

Fig. 5. Release of a lipophilic dye from the poly(BA)-g-poly(PEG-A) particlescrosslinked with bisacrylate acetal (cleavable, circles) and EGDMA (non-cleavable, squares) crosslinkers at pH 5.0 and 7.4. The filled symbols representpH 5.0 while the blank symbols are used for pH 7.4. The cleavable and non-cleavable core–shell particles used in the release experiment were obtained byPEG-grafting of poly(BA) particles, Exp #8 and 9 (Table 1), respectively.

particles whereas at pH 7.4 the release of the dye was ca. 34%.The non-cleavable particles (i.e. PEG grafted poly(BA) cross-linked with EGDMA, Exp #9, Table 1) showed a release kineticwith no pH-dependency and released ca. 41% of their dyecontent in 24 h.

3.4. Cell viability

The effect of the particles and their cleavage products on theviability of cells was determined via an Alamar Blue assay[32,33] using a drug-sensitive cancer cell line, human neuro-blastoma cells (i.e. SHEP cells). The conversion of Alamar blueto its reduced form by redox enzymes in viable cells can bequantitatively analyzed by optical density [32]. Fig. 6 shows thepercentage of viable cells relative to control cells afterincubation with the PEG-grafted particles (Exp #8, Table 1) ortheir cleavage products for 72 h. Neither the particles nor theircleavage products (Scheme 1) caused a significant inhibition inthe growth of SH-EP cells in the studied concentration range.

4. Discussions

4.1. Design, synthesis and characterization of acid-cleavablecrosslinker

For the development of an acid-labile colloidal system, weadopted the approach introduced recently by Murthy et al.[17,18]. In this approach, acid-cleavable gel-like nano/micro-particles could be produced using the crosslinkers containingacid-cleavable acetal bonds. While the crosslinkers used in theprevious studies were synthesized through multi-steps proce-dures, in this study we aimed to synthesize an acid-cleavablecrosslinker with a suitable hydrolysis kinetic utilizing a simplerprocedure and commercially available materials.

The key point in the synthesis of such crosslinkers is toachieve the desired pH-responsive behavior, i.e. rapid cleavage

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in slightly acidic pHs such as those present in tumors (pH∼6.0–7.2) and intracellular vesicles of the cells (pH∼4.5–6.8),and stable for long time in the blood, i.e. pH 7.4. The acetalsformed from p-substituted benzaldehydes have been shown bythe previous studies [17,18] to be particularly well suited to thispurpose as their acid labile behavior can be tailored by intro-ducing different substituents at the para-position. Thus the useof the crosslinkers containing p-substituted benzaldehyde ace-tals as an intrinsic acid-responsive element provides an easy andversatile way to incorporate and engineer the acid-cleavabilityto the polymeric colloidal systems for the site-selectivity of thedrug release.

In this study, we reported the synthesis of a bisacrylate acetalcrosslinker performed in one-step using commercially availablematerials, i.e. HEA monomer and p-MBA. This new cross-linker, di(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane)hydrolyzed rapidly in slightly acidic environment while itstayed stable at pH 7.4 for considerably longer time, which maybe suitable for intracellular drug delivery applications. It hasbeen well-established that the low pH causes the protonation ofthe acetal yielding a resonance stabilized carbonium ion, whichfacilitates the hydrolysis of the compound to form the relevantalcohol and the aldehyde [34].

Along with the acid-responsive character, the polarity of thecrosslinker is also an important parameter that may criticallyaffect the polymeric particle formation process which isgenerally very sensitive to the polarity of the polymerizationmedium. In the dispersion polymerization of various hydro-phobic monomers including n-butyl acrylate, the continuousphase is usually composed of polar solvents. [35,36]. The use ofa polar medium for the dispersion polymerization of the hydro-phobic monomers facilitates the particle formation. As thegrowing polymer chains reach a certain molecular weight, theytend to aggregate due to their decreased solubility in thecontinuous phase, which leads to the phase separation and theformation of spherical particles in the presence of a suitablemacromolecular stabilizer. The bisacrylate crosslinker synthe-sized in this study is soluble in non-polar solvents such asdichloromethane along with the polar aprotic solvents such asDMF. Additionally, it is soluble in the hydrophobic co-monomer, i.e. n-butyl acrylate (BA) and when it is dissolvedin BA, no phase-separation occurs upon mixing with the polarcontinuous phase, i.e. methanol/pyridine, which enables thedispersion polymerization to be performed. In addition the non-polar character of the bisacrylate crosslinker is suitable toform emulsions in an aqueous phase, and hence to derive thecrosslinked colloidal systems by an emulsion polymerizationtechnique.

4.2. Development of acid-cleavable poly(BA)-g-poly(PEG-A)particles

In general, the physicochemical properties of the colloidalsystems must be tailored carefully for a particular drug deliveryapplication. It is well-known that the size and the surfacechemistry critically determine the site of deposition of thecolloidal systems within the body [27,37]. In this study, we

intended to introduce a new approach that employs the ver-satility of the RAFT technique to produce submicron size col-loids bearing functional surface features modifiable for variousbiotechnology applications including drug delivery. CombiningRAFT technique with dispersion polymerization make it pos-sible to produce a variety of polymeric gel-like colloids with thesurface RAFT groups that allow the particle surface to bemodified with a biocompatible polymer or a bio-responsiveelement. The combination of the RAFT technique with theprecipitation polymerization and the subsequent grafting of apolymer shell have been previously reported for the synthesis ofmicron-size spheres by our group [30].

The dispersion polymerization of n-BA in the presence of abisacrylate acetal crosslinker and a RAFT agent (CDB) resultedin the formation of RAFT agent-functionalized, hydrophobic,cross-linked spheres in nanometer sizes. In general, the size ofthe particles obtained in this study was smaller than the size onewould expect from the dispersion polymerization that usuallyyields monodisperse particles above 500 nm. However, it wouldbe possible to obtain particles with the smaller size at the cost ofthe monodispersity utilizing a relatively good solvent for boththe polymer and the monomer. It was reported that a broadparticle size distribution in the dispersion polymerization ofpoly(BA) performed in 100% methanol was obtained whichappeared as a sediment comprised of large particles and acloudy supernatant phase of small particles [35]. Our resultswhich are in good accordance with this observation showedusually two populations of the particles in which the number ofthe large particles (N1 μm) usually constitutes less than 10% ofthe total number of the particles (Table 1). Despite their smallnumber in the total particle population, the large particles wouldclearly constitute a higher fraction in volume, which signifi-cantly limits the yield of the nanometer size particles that couldbe obtained by this polymerization technique. On the otherhand, it should be also noted that the formation of the largerparticle population during the polymerization process in someextent helps the formation of the smaller size particles. Duringthe polymerization process, since the capability of largerparticles to adsorb stabilizer or oligomers is poor due to theirlower surface area, the formation of larger particles facilitatesthe secondary nucleations to occur, which helps the formationof smaller size particles [36]. The large particle population canbe efficiently separated from the small one by a simplesedimentation or filtration process, which easily allows thecollection of nanometer size particles with narrower sizedistribution. As seen in Table 1, the number average particlesize for the smaller size fraction of the whole particle population(i.e. excluding the particles larger than 1 μm) was found to beless than 500 nm for all the polymerizations performed. In somecases, e.g. Exp #6 and #8 in Table 1, particles with diametersmaller than 200 nm were obtained. Several polymerizationparameters such as the stabilizer concentration and the solventpolarity might be further optimized to form particles withsmaller size and narrower distribution.

The hydrophobic bulk characteristic of the acid-cleavablepoly(BA) particles could be masked by grafting a hydrophilicpolymer to the surface using the RAFT groups available. PEG-

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A was copolymerized on CDB-functionalized particle surface,which lead to the core–shell like particles with poly(BA) coreand poly(PEG-A) surface layer (as determined by XPS). Anunexpectedly large increase in the average size after grafting(ca. 32% of the original size) was consistently observed, whichmay be explained by the interparticle crosslinking due to theaddition of the growing poly(PEG-A) chains on the surface oftwo different particles and also by the occurrence of thepolymerization inside the crosslinked particles [38]. However,these hypotheses require a more detailed investigation of thegrafting process.

The controlled character of CDB mediated polymerization ofPEG-A was confirmed by the results presented in Fig. 3. Thelinear increase in the number average molecular weight with theincreasing monomer conversion indicated that the polymeriza-tion of PEG-A under the conditions utilized occurs via RAFT-controlled mechanism. In conventional radical polymerizations,molecular weight of polymer chains formed at early stages ofpolymerization is high and reduces with conversion whereas inliving radical polymerizations molecular weight increaseslinearly with conversion [28].

4.3. pH-dependent cleavage and release profile of particles

The pH-dependent cleavage behavior of the particles can beattributed to the hydrolysis of the acetal bonds crosslinking poly(BA) chains in the particle bulk structure. The particle cleavagekinetic (Fig. 2), similar to the hydrolysis kinetic of the acetalcrosslinker is accelerated by the increase in the hydronium ionconcentration in the solution. This acceleration in the hydrolysiskinetic of the acetal bonds and the particle cleavage may beexplained briefly as follows: In an acidic solution, the acidprotonates one oxygen of the acetal, creating a weak base of therelevant neighboring group of the carbonyl carbon. This makesit easier for the water to attack the carbonyl carbon resulting inthe cleavage of the acetal bonds to the relevant aldehyde andalcohol. Since there is almost 800-fold increase in theconcentration of hydronium ions in the solution at pH 4.5compared to the solution at pH 7.4, the protonation of thecarbonyl oxygen and thus the hydrolysis of the acetal isexpected to accelerate proportionally when the pH is decreasedfrom pH 7.4 to pH 4.5. As a result of the hydrolysis of the acetalbonds that crosslink the particle network, copolymeric chainssuch as poly(BA)–co-poly(HEA) and poly(BA)-co-poly(HEA)-co-poly(PEG-A) forming the polymeric network be-come free and mobile. Dissolution of the copolymeric chains inthe aqueous solution eventually leads to the disintegration of theparticles (Fig. 2, lower AFM image).

Although the bisacrylate acetal crosslinker at pH 4.5hydrolyzes completely in less than 1 h (see Section 3.1), thecrosslinked particles at the same pH undergo cleavage slower(Fig. 2). This may be explained as follows: The microenviron-ment of the acetal bonds localized in the poly(BA) particlestructure is highly likely to be less accessible for the hydroniumions in the aqueous solution due to the hydrophobic bulk of thesurrounding poly(BA) chains with respect to the accessibility ofthe acetal bonds of the crosslinker in solution. The time

necessary for the diffusion of the aqueous solution into thehydrophobic polymer bulk would cause the delay in thehydrolysis of the acetal bonds located in the crosslinkedparticles. In addition, during the particle cleavage experiment,the time spent for the diffusion of the hydrolysis by-product, p-MBA through the polymer bulk and the dialysis membrane intothe surrounding medium from which the samples were taken forthe measurements, might also have delayed the observation ofthe particle cleavage.

The difference in the cleavage rate of the particles observedin the UV–vis spectrometer and AFM experiments (Fig. 2)might be due to the possible difference in the crosslinkingdensity of the particles since two different particle batchesproduced by different feed compositions were used, i.e. Exp #8and #2 (Table 1) in the UV–vis and AFM experiments,respectively. The particles with higher crosslinker quantity maybe expected to disintegrate and lose their spherical shape in afaster way compared to the particles with less crosslinkingdensity. Because the hydrolysis of the particles with highercrosslinking density would cause the formation of the higherquantity of the copolymeric chains containing poly(HEA). Thiswould decrease the hydrophobicity of the particle bulk andmake the diffusion of the aqueous solution into the polymerbulk and the dissolution of the freed copolymeric chains easierand faster, which may lead to the faster disintegration of theparticles.

The release kinetic of a lipophilic dye from the particles(Fig. 5) was found to be very similar to the release kinetic of p-methoxybenzaldehyde (Fig. 2). This similarity may suggest thatthe release of the dye occurs due to the pH-dependent cleavageof the polymeric matrix and the physical interactions betweenthe lipophilic dye and the polymer matrix do not exist. Thedisintegration of the particle network due to the hydrolysis ofthe acetal bonds leads to the release of the dye loaded inside thenetwork. As the hydrolysis of the particle network at acidic pHis faster than the hydrolysis at pH 7.4, the acceleration of the dyerelease at acidic pH is an expected result.

For specific applications of pH-responsive drug deliverysystems such as endosomal and tumor-site selective deliveryapplications, an ideal carrier systemwould be expected to offer aquick and efficient drug release profile in response to very smallpH changes. Considering the cleavage and the release profile,the pH-sensitivity of poly(BA)-co-poly(PEG-A) particles maybe considered moderate to provide an efficient drug release andneed to be improved particularly for these applications. It wouldbe possible to improve the pH-sensitivity of the particles byreplacing p-methoxybenzaldehyde used in the synthesis of theacid-cleavable crosslinker with a benzaldehyde bearing a lesselectron-withdrawing substituent at its para-position [17,18].Various acetal structures displaying a broad range of hydrolysisrates at mildly acidic pHs have been also reported by Frechetet al. [39]. Since the hydrolysis kinetic of the acetal basedcrosslinkers can be tuned, the pH-dependent cleavage and therelease kinetic of the crosslinked particles can be manipulatedeasily to suit for a particular drug delivery application.

The loading content of the particles (8 μg dye/mg particles,0.03 μmol dye/mg particles, efficiency 0.8 wt.%) was found to

206 Y. Chan et al. / Journal of Controlled Release 115 (2006) 197–207

be low compared to some of the nanoparticulate polymericsystems reported for the delivery of anticancer drugs [40,41]and needs to be improved for an efficient drug deliveryapplication. However, it would be sufficient in cell culture toprovide the therapeutic activity dose range of commonly usedhydrophobic anticancer drugs (10−5 M toxic dose–10−12 M for100% survival) by using the drug loaded particles at aconcentration ranging from 0.030 to 350 μg/ml.

4.4. Effect of particles and their cleavage products on cellviability

As seen in Fig. 6, neither the particles nor the by-productsformed upon the cleavage of the particles caused a significantinhibition in cell growth up to 3000 μg/ml. This concentration isalmost eight times higher than the particle concentration(350 μg/ml) required to deliver the drug at its toxicconcentration level (10−5 M). The by-products that would re-lease upon the cleavage of the core–shell particles are copo-lymeric chains of BA, PEG-A, and HEA and also by-product,p-MBA (Scheme 1). Acrylate based polymers including poly(BA) and poly(HEA) have been previously used in various invivo and in vitro applications and are known as biocompatiblematerials [42–44] The by-product, p-MBA is an FDA approveddirect food additive for human consumption [45]. Benzalde-hydes are metabolized in vivo followed by the excretion asbenzoic acid derivatives [46]. However, in vitro applications ofbenzaldehyde compounds at the cellular level may be ofconcern due to the lack of in vivo metabolic activities. Inaddition, the aldehyde group of these compounds may beexpected to interact with the amine groups of the intracellularmolecules. However, given the cell viability results (Fig. 6), theconcentrations of p-MBA released from the cleaved particles atthe concentrations needed to release the required doses of drugappear to have acceptable minimal effects on the cell viabilityof the human neuroblastoma cells. In addition to our study,previous studies by others have also employed benzaldehydecompounds for intracellular drug delivery applications andhave not indicated any significant effect of these compoundson cell viability and proliferation [18,47]. Nevertheless, inorder to rule out potential cytotoxic effects of benzaldehydecompounds and their possible interactions with amine contain-ing molecules of cells, a detailed investigation on the fate ofbenzaldehyde compounds in the cells will need to beperformed in the future as it is beyond the scope of thecurrent study.

5. Conclusions

A new approach combining the versatility of the RAFT andthe dispersion polymerization techniques with the acid-trig-gered release strategy was developed to prepare an acid-cleavable polymeric colloidal system. The size range, hydro-philic shell and the hydrophobic bulk structure along with theacid-labile character suggest the use of this system in thedelivery of hydrophobic drugs at acidic sites. Our future effortswill focus on the exploitation of the approach introduced in this

study to develop various nanoparticulate systems with im-proved pH-sensitivity and loading capacity for the intracellularand tumor-site selective delivery of hydrophobic drugs.

Acknowledgement

This work was supported by The Australian ResearchCouncil Discovery Project Grant (VB and MK; DP0664805),The University of New South Wales Early Career ResearchGrant (VB; PS06820) and The UNSW Vice Chancellor'sPostdoctoral Research Support Grant (VB; PS05036). VB, YCand MK were supported by a UNSW Vice Chancellor's Post-doctoral Research, UNSW International Postgraduate and aNational Health and Medical Research Council RD WrightCareer Development Award, respectively. The authors acknowl-edge also The Centre for Advanced Macromolecular Design(UNSW) and Microstructural Analysis Unit (UTS) for theresearch facilities, Drs. Barry Wood (Queensland University)and Bill Bong (UNSW) for XPS measurements, Mr. JohnStarling and Dr. Grainne Moran (UNSW) for the dynamiclight scattering and the fluorescence spectrometer facilities,respectively.

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