pH-responsive destabilization and facile bioconjugation of new hydroxyl-terminated block copolymer micelles

pH-Responsive Destabilization and Facile Bioconjugation of New

Hydroxyl-Terminated Block Copolymer Micelles

Behnoush Khorsand, Jung Kwon Oh

Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada H4B 1R6

Correspondence to: J. K. Oh (E-mail: [email protected])

Received 13 October 2012; accepted 10 December 2012; published online 9 January 2013

DOI: 10.1002/pola.26533

ABSTRACT: New hydroxyl-terminated amphiphilic block copoly-

mers (HO-ABPs) having pendant t-butyl groups for pH-respon-

siveness and terminal OH groups for bioconjugation are

reported. These HO-ABPs consist of hydrophilic poly(oligo

(ethylene oxide) monomethyl ether methacrylate) and hydro-

phobic poly(t-butyl methacrylate) blocks and were synthesized

by a consecutive atom transfer radical polymerization in the

presence of an OH-terminated bromine initiator. Aqueous self-

assembly of HO-ABPs resulted in colloidally stable micellar

aggregates being capable of encapsulating hydrophobic guest

molecules. They were nontoxic to cells and destabilized in

response to low pH. A facile bioconjugation of HO-ABP

micelles for active targeting is demonstrated by conjugation

with biotin (vitamin H) and competitive assay exhibiting >93%

ABP chains conjugated with biotin in each micelle. VC 2013

Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem.

2013, 51, 1620–1629

KEYWORDS: amphiphilic block copolymer; atom transfer radical

polymerization (ATRP); bioconjugation; controlled release;

diblock copolymers; drug delivery systems; micelles;

pH-responsive degradation; POEMA; PtBMA

INTRODUCTION In recent years, polymer-based drug deliv-ery systems (DDS) have gained significant attention in phar-maceutical science and nanotechnology.1,2 In particular,micelles based on amphiphilic block copolymers (ABPs) ena-ble the physical encapsulation of hydrophobic drugs.3–5

These micelles offer benefit as drug delivery carriers. Spe-cific features include colloidal stability with low critical mi-cellar concentration (CMC); tunable sizes with narrow sizedistribution; and protection of drugs from possible deactiva-tion.6,7 However, control over several properties is stillrequired for the design and development of block copolymermicelles as effective DDS.

Besides biocompatibility (i.e., noncytotoxicity to healthycells), bioconjugation with cell targeting biomolecules canpromote active targeting to specific diseased cells through,for example, specific ligand–receptor interactions.8,9 Forfacile bioconjugation, functional groups such as hydroxyl(AOH), amino (ANH2), and sulfhydryl (ASH) groups areincorporated into ABP micelles, and further react withligands such as antibodies, peptides, proteins, and folic acids.Another desirable is stimuli-responsive release of encapsu-lated drugs as results of degradation or destabilization ofABP-based micelles in response to external triggers.10–12

Here, the pH-sensitive (or acid-sensitive) micelles, while sta-ble at physiological pH, are destabilized in acidic conditions,facilitating the release of encapsulated drugs in a controlled

manner.13,14 Recently, the pH-responsive nanoparticles asoral delivery carriers of hydrophobic curcumin having antitu-mor properties are reported.15,16 One convenient method toprepare pH-responsive micelles involves the incorporation ofpendant pH-responsive cleavable groups into the hydropho-bic block. The cleavage of these groups in acidic pHincreases the polarity of the hydrophobic block, causing themicelles to destabilize or disrupt. For example, acid-sensitivemicelles have been prepared by self-assembly of blockcopolymers containing poly(aspartic acid) (PAp)17 and poly-lysine dendron (PLL).18 These PAp and PLL were synthesizedby ring-opening polymerization of cyclic monomers function-alized with orthoesters. pH-sensitive poly(methacrylamides)(PMAm)-based micelles designed with pendant cyclic acetalmoieties are also reported.19–21 These PMAm block copoly-mers contain a hydrophilic poly(ethylene oxide) (PEO) blockand are prepared by controlled radical polymerization meth-ods.22,23 Despite these advances, there are still needs todesign new types of degradable micelles for better under-standing of structure–property relationship between mor-phological variance and stimuli-responsive degradation.

Herein, we report new hydroxyl (OH)-terminated ABP(HO-ABPs) micelles having pendant pH-responsive elementsas well as bioconjugation ability and biocompatibility. TheHO-ABPs consist of hydrophilic poly(oligo(ethylene oxide)monomethyl ether methacrylate) (POEOMA) and

Additional Supporting Information may be found in the online version of this article.

VC 2013 Wiley Periodicals, Inc.

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hydrophobic poly(t-butyl methacrylate) (PtBMA) blocks.They were prepared by consecutive atom transfer radical po-lymerization (ATRP)24,25 of OEOMA and tBMA in the pres-ence of an OH-functionalized bromine initiator. ATRP hasbeen extensively used to synthesize well-controlled homopol-ymers and copolymers of either POEOMA26–30 or PtBMA31,32

as well as random copolymers of P(OEOMA-co-tBMA).33 Toour knowledge, however, the synthesis of block copolymerscontaining POEOMA and PtBMA in the presence of functionalATRP initiators using ATRP is not yet reported.

Scheme 1 illustrates our synthetic route and design space ofOH-terminated POEOMA-b-PtBMA ABPs as pH-responsivedelivery carriers. Frist, POEOMA having pendant oligo(ethy-lene oxide) chains is an analog of liner PEO which is biocom-patible and approved by FDA for clinical use.34,35 Cell viabil-ity results indicate noncytotoxicity of HO-ABPs. Second,PtBMA formed hydrophobic cores enabling the encapsulationof hydrophobic guest molecules including model drugs.t-Butyl groups of PtBMA blocks are hydrolyzed to hydro-philic poly(methacrylic acid) (PMAA) in acidic conditions;thus, low pH-responsiveness of PtBMA micellar cores cancause micelles to destabilize to form large aggregates, whichmay lead to enhanced release of encapsulated drugs. Third,to demonstrate facile bioconjugation of HO-ABPs with celltargeting biomolecules, as a proof-of-concept approach here,terminal OH groups were conjugated with biotin (vitamin H)for the synthesis of biotin-terminated ABPs.

EXPERIMENTAL

Materials and InstrumentationEthylene glycol (EG), 2-bromoisobutyric acid (Br-iBuA), N,N0-dicyclohexyl carbodiimide (DCC), N,N-dimethylaminopyridine(DMAP), N-(3-dimethylaminopropyl)-N0-ethylcarbodiimidehydrochloride (EDC), N,N,N0,N00,N00-pentamethyldiethylenetri-amine (PMDETA, >98%), 2,20-bipyridyl (bpy), copper(I) bro-mide (CuBr, >99.99%), copper(I) chloride (CuCl, >99.99%),

trifluoroacetic acid (CF3COOH), avidin from egg white, biotin(>99%, lyophilized powder), 2-(4-hydroxyphenylazo)-ben-zoic acid (HABA), and potassium hydrogen phthalate (KHP)were purchased from Aldrich Canada. Dulbecco’s modifiedeagle medium (DMEM, 100 lL) and fetal bovine serum (FBS)from Wisent and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT, a yellow tetrazole) from Promegawere purchased for cell viability assay. OEOMA with MW ¼300 g mol�1 and pendant ethylene oxide (EO) units � 5 andtBMA from Aldrich were purified by passing it through a col-umn filled with basic alumina to remove inhibitors.

2-Hydroxyethyl 20-bromoisobutyrate (OH-iBuBr) was synthe-sized as described elsewhere.36 Briefly, Br-iBuA (11.4 g, 68.7mmol) was mixed with EG (6.4 g, 103.1 mmol, 1.5 equiva-lents to hydroxyl groups) in the presence of DCC (15.1 g,72.7 mmol) and a catalytic amount of DMAP (0.7 g) in THF(250 mL) at room temperature overnight. After the removalof dicyclohexylurea formed as a by-product, the product wascollected as the third of the total four bands off a silica gelcolumn by column chromatography using 1/4–2/3 v/v mix-ture of diethyl ether/hexane. Yield ¼ 5.6 g (54.7%). Rf ¼ 0.3on silica (1/4 diethyl ether/hexane).1H NMR (CDCl3, ppm): 1.92 (s, 6H, AC(CH3)2Br), 3.82 (t, 2H,ACH2OH), 4.28 (t, 2H, AOCH2CH2OH).

13C NMR (CDCl3,ppm) 30.7, 55.8, 60.8, 67.4, 171.9.

1H NMR spectra were recorded using a 500-MHz Varianspectrometer. The CDCl3 singlet at 7.26 ppm and DMSO-d6multiplet at 2.5 ppm were selected as the reference stand-ard. Spectral features are tabulated in the following order:chemical shift (ppm); multiplicity (s, singlet; d, doublet; t, tri-plet; m, complex multiple); number of protons; position ofprotons. Molecular weight and molecular weight distributionwere determined by gel permeation chromatography (GPC)with a Viscotek VE1122 pump and a refractive index detec-tor. Three PolyAnalytik columns (PAS-103L, 105L, 106L,designed to determine molecular weight up to 2,000,000

SCHEME 1 Synthesis of HO-terminated POEOMA-b-PtBMA ABP by consecutive ATRP of OEOMA and tBMA in the presence of a

HO-terminated ATRP initiator, aqueous micellization and pH-responsive properties.

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g mol�1) were used with THF as an eluent at 30 �C at a flowrate of 1 mL min�1. Linear poly(methyl methacrylate)(PMMA) standards were used for calibration. Aliquots ofpolymer samples were dissolved in THF and the clear solu-tions were filtered using a 0.25-lm PTFE filter to removeany THF-insoluble species. A drop of anisole was added as aflow rate marker. Monomer conversion was determinedusing GPC for ATRP of OEOMA.30 The micellar sizes inhydrodynamic diameters by volume were measured bydynamic light scattering (DLS) at a fixed scattering angle of175� at 25 �C with a Malvern Instruments Nano S ZEN1600equipped with a 633-nm He–Ne gas laser. All micellar disper-sions without dilution were filtered by 0.45-lm PES filter toremove large aggregates. Fluorescence spectra were recordedon Varian Cary Eclipse fluorescence spectrometer. Thermalproperties including glass transition temperature (Tg) ofpolymers were measured with TA Instruments DSC Q10 calo-rimeter over a temperature range of �70 to 150 �C at aheating rate of 10 �C min�1.

ATRP of OEOMA for Synthesis of HO-POEOMA-BrMacroinitiatorsA series of ATRP of OEOMA was conducted in the presenceof HO-iBuBr under various conditions. As an example, thedetailed procedure for HO-macroinitiator (MI)-3 with[OEOMA]0/[OH-iBuBr]0/[CuBr]0/[bpy]0 ¼ 50/1/0.5/1 isdescribed as follows; OH-iBuBr (0.42 g, 2.0 mmol), OEOMA(30.0 g, 100.0 mmol), bpy (0.32 g, 2.0 mmol), acetone(25 mL), and anisole (0.3 mL) were added to a 100-mLSchlenk flask. The resulting mixture was deoxygenated bythree freeze-pump-thaw cycles. The reaction flask was filledwith nitrogen and then CuBr (0.14 g, 1.0 mmol) was addedto the frozen solution. The flask was sealed, evacuated withvacuum and backfilled with nitrogen once. The mixture wasthawed and the flask was then immersed in an oil bath pre-heated to 47 �C to start the polymerization. Aliquots werewithdrawn at different time intervals during the polymeriza-tion to monitor conversion and molecular weight by GPC.The polymerization was stopped by exposing the reactionmixture to air.

In order to remove residual copper species and unreactedmonomers, as-prepared polymer solutions of HO-POEOMA-Br in acetone were added dropwise into hexane under stir-ring. The precipitated green residues were passed through acolumn filled with basic aluminum oxide with THF as an elu-ent to remove copper species twice. Solvent was removed byrotary evaporation and residual solvents were furtherremoved in a vacuum oven at 50 �C overnight to isolatepurified polymers.

ATPR of tBMA for Synthesis of HO-POEOMA-b-PtBMABlock CopolymerThe purified, dried HO-POEOMA-Br (1.9 g, 0.49 mmol), tBMA(14 g, 98.4 mmol), bpy (76.9 mg, 0.49 mmol), acetone(14 mL), and anisole (0.1 mL) as an internal standard wereadded to a 50-mL Schlenk flask. The resulting mixture wasdeoxygenated by three freeze-pump-thaw cycles. The reac-tion flask was filled with nitrogen and then CuBr (35.3 mg,

0.25 mmol) was added to the frozen solution. The flask wasclosed, evacuated with vacuum and backfilled with nitrogenonce. The mixture was thawed, and the flask was thenimmersed in an oil bath preheated to 47 �C to start the poly-merization. The polymerization was stopped by exposing thereaction mixture to air. The resulting HO-ABP was purifiedas described above.

Aqueous MicellizationWater was added dropwise into an organic solution consist-ing of the purified, dried HO-ABP dissolved in THF. Theresulting dispersion was stirred for >12 h to remove THF,yielding colloidally stable micellar dispersions at variousconcentrations. For a typical example, a micellar dispersionat 0.5 mg mL�1 was prepared with the use of HO-ABP(10 mg), THF (2 mL), and water (20 mL).

Determination of Critical Micellar ConcentrationUsing a NR ProbeA stock solution of NR in THF at 5 mg mL�1 and a stock solu-tion of HO-ABP in THF at 1 mg mL�1 were prepared. Water(12 mL) was then dropwise added into mixtures consisting ofthe same amount of the stock solution of NR (20 lL, 0.1-mgNR) and various amounts of the stock solution of HO-ABP.The resulting dispersions were stirred for 12 h to removeTHF, and then were subjected to filtration using 0.45-lm PESfilters to remove excess NR, forming a series of NR-loadedmicelles at various concentrations of HO-ABP ranging from10�5 to 0.1 mg mL�1. Their fluorescence spectra wererecorded at kex ¼ 480 nm, and the fluorescence intensity wasrecorded at maximum kem ¼ 620 nm.

Transmission Electron MicroscopyTransmission electron microscopy (TEM) images were takenusing a Philips CM200 HR-TEM, operated at 200-kV electronsand equipped with thermionic LaB6 cathode filament, anti-contamination cold finger, Genesis EDAX system, and AMTV600 2k 2K� CCD camera. The point-to-point resolution andthe line resolution of the machine are 0.24 and 0.17 nm,respectively. To prepare specimens, samples were droppedonto the TEM copper grids (400 mesh, carbon coated). Thegrids were dried in air.

MTT Cell Viability AssayHEK293T and HeLa cells were plated at 5 � 105 cells/wellinto a 96-well plate and incubated for 24 h in DMEM (100lL) containing 10% FBS. They were then treated with vari-ous amounts of HO-ABP micelles for another 48 h. Blankcontrol without micelles was run simultaneously. Cell viabil-ity was measured using CellTiter 96 non-radioactive cell pro-liferation assay kit (MTT, Promega) according to manufac-turer’s instruction. Briefly, a solution of MTT supplied byPromega (20 lL) was added into each well, and then cellmedia were carefully removed after 4 h. DMSO (100 lL) wasadded into each well to dissolve MTT crystals, and then theabsorbance was recorded at k ¼ 570 nm using PowerwaveHT Microplate Reader (Bio-Tek). Each concentration was 12-replicated. Cell viability was calculated as the percent ratioof absorbance of mixtures with micelles to control.


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Hydrolytic Cleavage of t-Butyl Groups of PtBMA Blocksin Acidic ConditionsPtBMA blocks in HO-ABP were hydrolyzed in the presence ofCF3COOH in CH2Cl2 at room temperature. Typically, HO-ABP(100 mg) dissolved in CH2Cl2 (1.6 mL) was mixed withCF3COOH (321.6 lL, 4.3 mmol) under stirring for 15 h. Thedegraded product was precipitated from hexane (10 mL).The white solids were isolated and further dried in a vac-uum oven at 50 �C.

pH-Responsive Destabilization of OH-ABP Micelles atLow pHThe similar procedure described above was used to preparea micellar dispersion at 5 mg mL�1, which was divided intothree aliquots of equivalent volume in a 20-mL vial. Two ali-quots were adjusted to pH ¼ 5 and 3.2 using an aqueousKHP buffer solution at pH ¼ 3 and another one at pH ¼ 7was used as a control. They were then characterized withsize and size distribution using DLS over time.

Loading of NR into HO-ABP MicellesNR-loaded micellar dispersions were prepared as follows.Typically, a stock solution of NR in THF (5 mg mL�1, 1 mL)and HO-ABP (50 mg) were mixed with water (100 mL). Theresulting mixture was stirred overnight to remove THF andsubjected to centrifugation (8000 rpm � 15 min � 4 �C) toprecipitate nondissolved NR. The supernatant was further fil-tered using a 0.45 lm PES filter to remove residual NR. Thefinal concentration of NR-loaded micelles was 0.5 mg mL�1.To determine the loading level of NR in micelles, an aliquotof NR-loaded micellar dispersion (30 mL) was taken, andwater was removed by a rotary evaporation. The remainingresidues were further dried in a vacuum oven at 50 �C for2 h, and then dissolved in THF (10 mL). The UV–Vis spec-trum was recorded to measure the absorbance at a maxi-mum kabs ¼ 527 nm. Similar procedure was repeated threetimes with freshly prepared NR-loaded micelles to obtain re-producible data. A loading level of NR (%) was determinedby the weight ratio of NR encapsulated in micelles to driedpolymers. A loading efficiency (LE, %) was calculated by theweight ratio of NR encapsulated in micelles to NR initiallyadded.

Bioconjugation of HO-ABP with BiotinBiotin (1.9 mg, 8 � 10�3 mmol), HO-ABP (80 mg, 8 � 10�3

mol), and EDC (1.5 mg, 8 � 10�3 mmol) was dissolved inDMF (3 mL) at room temperature. The mixture was allowedto stir for 48 h at the same temperature. The mixture was

then dialyzed against aqueous NaHCO3 solution for 3 days toremove free biotins. The biotinylated ABP was isolated bythe removal of water using rotary evaporator and was fur-ther dried in vacuum oven.

Avidin-HABA Binding Assay to DetermineAvailability of Biotin in ABPsThe dried biotinylated ABP (200 mg) was dissolved in THF(2.5 mL) and added dropwise into deionized water (41 mL).The resulting dispersion was kept under stirring overnightto remove THF, yielding an aqueous dispersion of micelles ofbiotinylated ABP at 5.0 mg mL�1 concentration. Avidin-HABAcomplex solution was prepared as follows; HABA (5.0 mg)was dissolved in water (8 mL) and undissolved specieswere removed using a 0.2-lm PES filter. Avidin (5.0 mg)was added to an aliquot of the HABA solution (5 mL). Theresulting avidin-HABA solution was equilibrated at roomtemperature for 2 h. In a vial, an aliquot of avidin-HABA so-lution (1.0 g) was then mixed with an aqueous biotinylatedABP micellar dispersion (1.0 g). In another vial, the sameamount of avidin-HABA solution (1.0 g) was mixed with thesame amount of water (1.0 g) as a control. After a week,the precipitates (corresponding to complexes of biotin-micelles with avidin) were removed by filtration with0.2-lm PES filter. The UV–Vis spectrum of the supernatantwas recorded.

RESULTS AND DISCUSSION

Synthesis of HO-POEOMA-b-PtBMA ABPsOur experiments began with the synthesis of HO-iBuBr ini-tiator by reacting BriBuA with excess EG using a carbodii-mide coupling reaction. The initiator was purified byextensive column chromatography and its structure wasconfirmed by 1H and 13C NMR. After the successful synthe-sis of HO-iBuBr initiator, a series of ATRP of OEOMA in thepresence of HO-iBuBr was conducted under various condi-tions. ATRP of OEOMA initiated with ethyl 2-bromoisobuty-rate in toluene is reported elsewhere.37 In our experi-ments, the parameters that influence the kinetics andcontrol of ATRP of OEOMA initiated with HO-iBuBr in ace-tone were investigated to synthesize well-controlled HO-POEOMA-Br macroinitiators (MIs). Table 1 summarizes theresults.

With the ratio of [OEOMA]0/[HO-iBuBr]0 ¼ 50/1, defined asthe targeting number average degree of polymerization, DP ¼50, the ATRP of OEOMA in the presence of CuBr/PMDETA

TABLE 1 Characteristics for ATRP of OEOMA in the Presence of OH-iBuBr at 47 8C in Acetonea

Recipe Catalyst Ligand Time (h) Conversionb Mn,theoc (g mol�1) Mn

b (g mol�1) Mw/Mnb

HO-MI-1 CuBr PMDETA 2.0 0.81 12,150 15,400 1.22

HO-MI-2 CuCl PMDETA 2.2 0.80 12,000 15,600 1.39

HO-MI-3 CuBr bpy 4.0 0.78 11,700 14,300 1.40

a [OEOMA]0/[HO-iBuBr]0/[catalyst]0/[ligand]0 ¼ 50/1/0.5/0.5 for PMDETA

and 1/0.5/1 for bpy; OEOMA/acetone ¼ 1.5/1 v/v.b Determined by GPC with PMMA standards.

c Theoretically calculated molecular weight: Mn,theo ¼ [OEOMA]0/[HO-

iBuBr]0 � MW(OEOMA) � conversion.



(HO-MI-1) and CuCl/PMDETA (HO-MI-2) was fast, reaching80% conversion in 2 h. This result suggests no significant dif-ference in kinetics for catalysts complexed with PMDETA.However, CuBr/PMDETA complex gave better control withMw/Mn < 1.25, compared with CuCl/PMDETA active complexin this system. CuBr/bpy (HO-MI-3) slowed down polymeriza-tion, with first-order kinetics (Supporting Information Fig.S1a) and increasing molecular weight over conversion withMw/Mn < 1.4 (Supporting Information Fig. S1b).

ATRP of OEOMA was carried out with the ratio [OEOMA]0/[HO-iBuBr]0/[CuBr]0/[bpy]0 ¼ 50/1/0.5/1 for 30 min tosynthesize HO-POEOMA-Br MIs. As seen in Figure 1(a), 1HNMR spectrum of HO-POEOMA-Br exhibits a peak (a)appeared at 1.4 ppm corresponding to two methyl protonsin the initiating species and a singlet (b) at 3.4 ppm corre-sponding to methoxy protons. From the integral ratio of [(b/3)/(a/6)], the DP of HO-POEOMA-Br was determined to be19. GPC results indicate molecular weight Mn ¼ 9,500 gmol�1 with monomodal and narrow molecular weight distri-bution as low as Mw/Mn ¼1.2 (Fig. 2).

In the presence of the purified HO-POEOMA19-Br as an ATRPMI, ATRP of tBMA was carried out in acetone at 47 �C underthe conditions of [tBMA]0/[HO-POEOMA-Br]0/[CuBr]0/[bpy]0¼ 200/1/0.5/1. The GPC trace evolved to a high molecularweight region, with Mn ¼ 20,800 g mol�1 and Mw/Mn ¼1.24 (Fig. 2). DSC results indicate two distinct glass transi-tion temperatures (Tg

0s) at �55.2 and 98.2 �C which corre-

spond to immiscible POEOMA and PtBMA blocks (SupportingInformation Fig. S2). From the integral ratio of [(c/9)/(b/3)]with the DP ¼ 19 for HO-POEOMA-Br, 1H NMR results allowfor determining the DP of PtBMA block to be 67 [Fig. 1(b)].

These results confirm the synthesis of well-controlled HO-POEOMA19-b-PtBMA67 (HO-ABP).

Aqueous Micellization of HO-ABPOwing to its amphiphilic nature, the purified HO-ABP formedcolloidally stable HO-functionalized micelles through self-assembly in water. Critical micellar concentration (CMC) wasdetermined using fluorescence spectroscopy with a Nile Red(NR) probe. This method follows the change in fluorescence

FIGURE 1 1H NMR spectra of HO-MI (a), HO-ABP before (b), and after (c) hydrolytic cleavage of PtBMA block in the presence of tri-

fluoroacetic acid.

FIGURE 2 GPC traces HO-POEOMA-Br macroinitiator and HO-

ABP.


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of NR over the concentration of polymers; the fluorescenceof NR is significantly low in water, but it is intense when NRis entrapped in hydrophobic micellar cores.38,39 In theexperiments, a series of mixtures consisting of the sameamount of NR and various amounts of HO-ABP ranging from10�5 to 0.1 mg mL�1 in aqueous solutions was prepared bya solvent evaporation method. After the removal of THF byevaporation and free NR by filtration, their fluorescencespectra (kex ¼ 480 nm) were measured [Fig. 3(a)]. At lowerconcentrations of HO-ABP, fluorescence intensity is signifi-cantly low and does not change. However, it increased withan increasing concentration of HO-ABP [Fig. 3(b)]. From twoequations obtained by fitting each dataset to linear relation-ship, the CMC of HO-ABP was determined to be 6.3 lg mL�1.

Next, the size and morphology of micelles of HO-ABP wereinvestigated using DLS technique and TEM. Solvent evapora-tion method40 was used to prepare colloidally stable micellesat concentrations ranging from 0.05 to 5 mg mL�1, aboveCMC. As seen in Figure 4(a), an example of CONTIN plot for

micellar aggregates at 0.05 mg mL�1 shows monomodal sizedistribution with a diameter ¼ 28.4 6 0.1 nm (triplicates forreproducibility). Interestingly, the micelle sizes remained notsignificantly changed, although the concentration of HO-ABPsin the same volume of THF varied [Fig. 4(b)]. TEM imageindicates spherical micelles with average diameter ¼ 12.5 6

2.5 nm [inset in Fig. 4(a)]. Micellar size was determined tobe larger by DLS than by TEM. The difference in micellarsizes between DLS and TEM can be attributed to the dehy-drated state of the micelles.41

MTT NoncytotoxicityCell viability of the resulting HO-ABP micelles was examinedusing MTT assay (a standard colorimetric method to mea-sure cytotoxicity). Human embryonic kidney HEK293T andHeLa cancer cell line were cultured with different concentra-tions of HO-ABP. Cells without polymers were also includedas a control. After 48 h incubation, absorbance was meas-ured using an absorbance-based plate reader to determine

FIGURE 3 Evolution of fluorescence spectra (a) and fluores-

cence intensity at kem,max ¼ 620 nm (b) of Nile Red in a series

of aqueous mixtures having various amounts of HO-ABP to

determine CMC.

FIGURE 4 DLS diagram and TEM image (inset) with scale bar

¼ 100 nm (a) and diameter over concentration (b) of HO-ABP

micelles.



viability. Figure 5 suggests >95% viability of cells, with nosignificant difference compared to control cells, indicatingthat HO-ABP is nontoxic to cells up to 1 mg mL�1.

Low pH-Responsive Cleavage of HO-ABP andDestabilization of Micellar AggregatesThe hydrolysis of t-butyl groups of PtBMA under acidic con-ditions in the presence of either trifluoroacetic acid42 orhydrogen chloride33,43 has been previously reported. The hy-drolysis resulted in hydrophobic PtBMA being converted tohydrophilic PMAA. In this experiment, 1H NMR was used todetermine the cleavage of t-butyl groups in the PtBMA blockof HO-ABP in the presence of trifluoroacetic acid. Figure 1(c)shows 1H NMR spectrum of the hydrolyzed HO-ABP inDMSO-d6. A peak at 1.5 ppm, which corresponds to t-butylprotons disappeared; this suggests significant hydrolysis of t-butyl groups of PtBMA, to yield doubly hydrophilic HO-POEOMA-b-PMAA block copolymers. In CDCl3, HO-ABP wasdissolved before hydrolysis, but precipitated after hydrolysis.The solubility change also supports the complete hydrolysisof the PtBMA block.

Next, pH-responsive destabilization of PtBMA micellar coreswas further examined. Aliquots of HO-ABP micellar disper-sion at 5 mg mL�1 were adjusted to pH ¼ 5 and 3 using anaqueous KHP buffer solution at pH ¼ 3. DLS was followed tomeasure the change in particle size distribution after 4 days(Fig. 6). At pH ¼ 7 as a control, the micellar size and its dis-tribution did not change. However, at pH ¼ 5, the sizeslightly increased from 26.9 to 29.1 nm, with a new popula-tion of large aggregates (>2 lm). The digital picture alsoevidenced the formation of larger aggregates. When pH wasfurther decreased to 3, the size significantly increased from26.9 to 35.6 nm with more amounts of large aggregates.These results suggest that PtBMA core-containing micellesare destabilized in acidic conditions.

Our several attempts (including the use of 1H NMR andFTIR) to determine the extent of cleavage of t-butyl groupsto the corresponding methacrylic acid groups in aqueous mi-cellar cores was not straightforward. However, PtBMA blockscould be partially hydrolyzed to hydrophilic PMAA blocks inmicellar cores in response to acids, yielding POEOMA-b-P(tBMA-co-MAA) block copolymers. Although these copoly-mers form aggregates with larger sizes at pH <5.0, it can beanticipated that such conversion of t-butyl groups to metha-crylic acid groups changes the polarity of micellar cores tobe hydrophilic, thus leading to enhanced release of encapsu-lated drugs. Similar results for acid-responsive micelles withacid-labile groups exhibiting the increase in particle size andenhanced release in response to acidic pH are reported.19,21

Physical Entrapment of Model DrugsTo examine applicability of self-assembled HO-ABP as poten-tial delivery carriers, the encapsulation of model drugs wasexamined. There are several methods to encapsulate hydro-phobic drugs in micellar cores. Micellar drug conjugatedesign involves the covalent conjugation of drugs into micel-lar cores through relatively stable or cleavable linkers (calledpro-drugs).44,45 This method has been explored to reducepremature drug release; however, a drawback includes the

FIGURE 5 Viability of HEK293T and HeLa cells cultured with

OH-ABP micelles for 48 h.

FIGURE 6 DLS diagrams of HO-ABP (a) of HO-ABP micelles at pH ¼ 7, 5, and 3 after 4 days (b) and digital images.


POLYMER SCIENCE


limitation in the design and synthesis of hydrophobic blocksthat should bear pendant functional groups. Physical entrap-ment is a facile method to encapsulate small drugs in micel-lar cores.46–48 For initial assessment here, Nile Red (NR), afluorescent dye as a hydrophobic model drug, was used toinvestigate the effect of the ratio of NR/HO-ABP on physicalloading. Micellization of HO-ABP with NR in water throughsolvent evaporation method yielded NR-loaded micelles. Af-ter removal of excess NR by centrifugation and filtration andwater by a rotary evaporation, UV–Vis spectroscopy wasused to determine the loading level and loading efficiency(LE) of NR in micelles. All samples were performed in tripli-cate. The extinction coefficient of NR in THF was also deter-mined to be e ¼ 37,300 M�1 cm�1 at kabs ¼ 527 nm (Sup-porting Information Fig. S3). An example of UV–Vis spectrumof NR-loaded micelles dissolved in THF exhibits a strongabsorption of NR at kmax ¼ 527 nm (Supporting InformationFig. S4). As presented in Table 2, with the weight ratio ofNR/HO-ABP ¼ 1/10 at 0.5 mg mL�1 concentration of HO-ABP micelles, loading level was 0.4% with LE ¼ 4.1%. Witha decreasing ratio of NR/HO-ABP to 0.2/10 wt/wt at higherconcentration of HO-ABP micelles (3.3 mg mL�1), the loadinglevel increased to 1.1% and LE to 56%. Supporting Informa-

tion Figure S5 shows a typical example of DLS diagram ofNR-loaded micelles in aqueous solution. Their hydrodynamicdiameters remained similar to that of micelles without NR,suggesting no significant effect of the presence of NR onmicellization of HO-ABP in aqueous solutions.

Bioconjugation with Biotin and Avidin-HABA AssayActive targeting to specific cells through bioconjugation ofdelivery vehicles with cell targeting agents is a desired prop-erty for polymer-based DDS. Specific targeting could reducethe serious side effects of drugs as well as enhance drug effi-ciency. To assess the applicability of HO-ABP toward targeteddelivery, the conjugation of terminal hydroxyl groups (AOH)in HO-ABP with biotin was examined. Unreacted biotin wasremoved by extensive dialysis against aqueous base solution.The purified biotin-labeled HO-ABP (Biotin-ABP) was dis-solved in water to form biotinylated micelles at concentra-tion of 5.0 mg mL�1 [Fig. 7(a)].

The availability of biotin presented from Biotin-ABP wasevaluated by a competitive avidin-HABA binding assay usingUV–Vis spectroscopy.49,50 Avidin is a protein having fourbinding pockets. Because of the stronger affinity of biotin toavidin as opposed to HABA, biotin molecules replace HABA

TABLE 2 Encapsulation of NR in HO-ABP Micelles at Different Weight Ratios of NR/HO-ABP

HO-ABP (mg mL�1) NR/HO-ABP (wt/wt) Loadinga (%) LEb (%) d (DLS) (nm)

0.5 – – – 28.2

0.5 1.0/10 0.4 6 0.04 4.1 6 0.4 28.3

3.3 0.2/10 1.1 6 0.1 55.9 6 0.1 27.3

a Determined by the weight ratio of NR encapsulated in micelles to

dried polymers.

b Calculated by the weight ratio of NR encapsulated in micelles to NR

initially added.

FIGURE 7 Schematic illustration of bioconjugation of OH-ABP with biotin to form biotin-conjugated ABP (a) and UV–Vis spectra of

avidin–HABA complex before and after addition of biotinylated micelles in aqueous solution (b).



molecules in an avidin-HABA complex. An aliquot of biotinyl-ated micellar dispersion was mixed with aqueous avidin-HABA complex. As seen in Figure 7(b), the absorption isattributed to HABA at 350 nm and avidin-HABA complex at500 nm. Upon the addition of biotinylated micelles, the ab-sorbance at 500 nm decreased by DA ¼ 0.79; this indicatesavidin–biotin complexation. Using the calibration curve,50 theavailability of biotin was calculated to be 0.93 mg biotin/gpolymer. Thus, terminal biotin molecules should be able toenhance the active targeting ability of biotinylated micellestoward cancer cells in vivo.51

CONCLUSIONS

Well-defined OH-terminated HO-POEOMA-b-PtBMA blockcopolymers were synthesized by a consecutive ATRP ofOEOMA and tBMA in the presence of a HO-terminated Br ini-tiator in acetone. 1H NMR and GPC results suggest that poly-merizations preceded in living fashion, yielding both HO-MIand HO-ABP with monomodal and narrow molecular weightdistributions as low as Mw/Mn < 1.25. Owing to the amphi-philic nature, these HO-ABPs self-assembled, through aque-ous micellization, to form spherical micellar aggregates,confirmed by DLS and TEM measurements, at above CMC of6.8 lg mL�1 determined by NR fluorescence probe method.These micelles enabled the encapsulation of hydrophobicmodel drugs. Hydrolytic cleavage of t-butyl group of PtBMAresulted in doubly hydrophilic HO-POEOMA-b-PMAA blockcopolymers. In acidic pH, HO-ABP micelles were destabilizedto form large aggregates. The terminal OH groups reactedwith biotin by a facile carbodiimide coupling reaction. Avi-din-HABA assay results indicate that the availability of biotinwas 0.93 biotin per HO-ABP chain. These results, combinedwith noncytotoxicity and facile bioconjugation of HO-ABPs,suggest that new HO-terminated ABP micelles can find theirapplications as controlled delivery carriers in response tolow pH.

ACKNOWLEDGMENTS

Financial supports from Concordia University Start-Up, CanadaResearch Chair (CRC) Award, and FQRNT Equipment Grant aregratefully acknowledged. The authors thank S. Aleksanian forhelpful discussions and M. Sacher for our access to cell culturefacilities at Concordia University. JKO is Tier II Canada ResearchChair (CRC) in Nanobioscience as well as a member of CentreQu�eb�ecois sur les Mat�eriaux Fonctionnels (CQMF) funded byFQRNT and of Concordia Composite (CONCOM) Center at Con-cordia University.

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