Journal of Controlled Release 162 (2012) 591–598

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Journal of Controlled Release

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Poly(PS-b-DMA) micelles for reactive oxygen species triggered drug release

Mukesh K. Gupta, Travis A. Meyer, Christopher E. Nelson, Craig L. Duvall ⁎Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, USA

⁎ Corresponding author at: Vanderbilt University, Depaing, PMB 351631, 2301 Vanderbilt Place, Nashville, TN 3723598; fax: +1 615 343 7919.

E-mail address: craig.duvall@vanderbilt.edu (C.L. Du

0168-3659/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.jconrel.2012.07.042

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 June 2012Accepted 30 July 2012Available online 6 August 2012

Keywords:Poly(propylene sulfide) (PPS)Reversible addition fragmentation chaintransfer (RAFT) polymerizationReactive oxygen species (ROS)InflammationSmart polymer micellesTargeted drug delivery

Anewmicelle drug carrier that consists of a diblock polymer of propylene sulfide (PS) andN,N-dimethylacrylamide(poly(PS74-b-DMA310)) has been synthesized and characterized for site-specific release of hydrophobic drugs tosites of inflammation. Propylene sulfide was first polymerized using a thioacyl group transfer (TAGT) methodwith the RAFT chain transfer agent (CTA) 4-cyano-4-(ethylsulfanylthiocarbonylsulfanyl) pentanoic acid (CEP),and the resultant poly(PS74-CEP)macro-CTAwas used to polymerize a second polymer block of DMA using revers-ible addition-fragmentation chain transfer (RAFT). The formation of the poly(PS74-b-DMA310) diblock polymer wasconfirmed by 1H NMR spectra and gel permeation chromatography (GPC). Poly(PS74-b-DMA310) formed 100 nmmicelles in aqueous media as confirmed by dynamic light scattering (DLS) and transmission electron microscopy(TEM). Micelles loaded with the model drugs Nile red and DiO were used to demonstrate the ROS-dependentdrug release mechanism of these micelles following treatment with hydrogen peroxide (H2O2), 3-morpholinosydnonimine (SIN-1), and peroxynitrite. These oxidants were found to oxidize the micelle PPS core,making it more hydrophilic and triggeringmicelle disassembly and cargo release. Delivery of poly(PS74-b-DMA310)micelles dual-loaded with the Förster Resonance Energy Transfer (FRET) fluorophore pair DiI and DiO was used toprove that endogenous oxidants generated by lipopolysaccharide (LPS)-treated RAW 264.7 macrophages signifi-cantly increased release of nanocarrier contents relative to macrophages that were not activated. In vitro studiesalso demonstrated that the poly(PS74-b-DMA310) micelles were cytocompatible across a broad range of concentra-tions. These combined data suggest that the poly(PS74-b-DMA310) micelles synthesized using a combination ofTAGT andRAFThave significant potential for site-specific drug delivery to tissueswith high levels of oxidative stress.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Rheumatoid arthritis, neurodegenerative diseases, atherosclerosis,diabetes, and many cancers are among the pathologies characterizedby hyper-activation of enzymes (i.e. NADPH oxidase, iNOS, etc.) thatcreate high concentrations of reactive oxygen species (ROS) such ashydrogen peroxide, hydroxyl radicals, singlet oxygen, superoxide an-ions, nitric oxide, nitroxyl, and nitrogen dioxide [1]. Nitric oxide and su-peroxide anion can also react with each other to generate other morepotent reactive oxygen and nitrogen species such as peroxynitrite [2].Excessive levels of these ROS, known as oxidative stress, can causeDNA mutations and can alter function of proteins, leading to apoptosisor other aberrant cell activities that can cause or exacerbate a disease[1–3].

Because of the role of imbalanced ROS activity in the etiology ofnumerous diseases, delivery platforms that enable targeted releaseof antioxidants or other drugs at sites of high ROS activity have thepotential for high therapeutic impact [4–8]. Stimuli-responsive,

rtment of Biomedical Engineer-35-1631, USA. Tel.:+1 615 322

vall).

rights reserved.

“smart” polymer-based micelles have attracted considerable atten-tion for drug delivery because they can stably package their cargountil disassembly and drug release is triggered in response to changesin environmental signals such as temperature [9–11], pH [12], oxida-tion [13–15], light [16,17], or other specific molecules [18]. The aim ofthe current study was to investigate a new smart micelle for targeteddrug release in regions of high ROS activity.

Sulfur(II)-containing materials undergo a phase transition from a hy-drophobic to a hydrophilic state under oxidative environments [19], withpoly(propylene sulfide) (PPS) being an example synthetic polymerknown to exhibit oxidation responsiveness [14]. Specifically, hydropho-bic PPS has a two-stage transition to more hydrophilic poly(propylenesulphoxide) and ultimately poly(propylene sulphone) upon oxidation[14]. Block copolymers of PEG and PPS have been explored by Hubbellet al. for engineering of polymersomes for vaccine delivery and micellesfor encapsulation of hydrophobic drugs [20,21]. PEG-b-PPS micelleswere shown to enable sustained, slow release of the hydrophobic drugcyclosporin A, but, to our knowledge, these or similar polymers havenot been rigorously investigated for triggered release of hydrophobicdrug cargo in response to different ROS. Furthermore, a thioacyl grouptransfer (TAGT) method [22] was utilized here that enables pairing ofthe anionic ring opening polymerization of PPS with the RAFT polymeri-zation of the hydrophilic polymer block. RAFT is amenable to use with awide variety ofmonomers [23], andhere, DMAwas chosen for the corona

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forming polymer block due to its solubility in water and low toxicity[24,25]. The focus of the current study was on an amphiphilic diblock co-polymer of poly(PS74-b-DMA310). This diblock polymer self-assembledintomicelles, and its oxidation, disassembly, and cargo releasewere thor-oughly characterized under H2O2, 3-morpholinosydnonimine (SIN-1),peroxynitrite, and endogenous ROS generated by lipopolysaccharide(LPS)-activated macrophages.

2. Materials and methods

2.1. Materials

Propylene sulfide (PS), N,N-dimethylacrylamide (DMA),ethanethiol, carbon disulfide (CS2), sodium hydride (NaH), 4,4′-azobis(4-cyanovaleric acid) tetraphenylphosphonium chloride(TPPCl), 2,2′-azobis(isobutyronitrile) (AIBN), Nile red, DiO (3,3′-dioctadecyloxacarbocyanine perchlorate), DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate), and hydrogen peroxide(H2O2) were purchased from Aldrich Chemical Co. (Milwaukee, WI,USA). SIN-1 was purchased from Invitrogen (San Diego, CA,USA) as packages of 1 mg plastic vials. Peroxynitrite was pur-chased from EMD Millipore (Billerica, MA, USA). 4-Cyano-4-(ethylsulfanyltiocarbonyl)sulfanylpentanoic acid (CEP) wassynthesized following the previously reported procedure [26] (1H NMR(400 MHz CDCl3) δ: 1.35 (t, \S\CH2\CH3); δ 1.85 (s, \C(CN)\CH3);δ 2.4–2.67 (m,\CH2\CH2\); δ 3.42 (q,\S\CH2\CH3)). Propylene sul-fide was dried and distilled over calcium hydride (CaH2) before use, andN,N-dimethylacrylamide (DMA) was purified by distillation under re-duced pressure just before polymerization. 1-Methyl-2-pyrrolidone(NMP) was dried and distilled over CaH2.

2.2. Polymer synthesis and characterization

2.2.1. Synthesis of PPS macro CTA poly(PS74-CEP)Poly(propylene sulfide) was synthesized using the RAFT chain trans-

fer agent CEP through the TAGT polymerization method [22]. Briefly, PS(0.30 mL, 3.8 mmol), CEP (24.9 mg, 0.095 mmol), TPPCl (7.1 mg,0.019 mmol), and NMP (0.95 mL) were placed into a flame-dried am-poule equipped with a three-way stopcock and degassed for 30 minwith three freeze–pump–thaw cycles. The ampoule was immersed inan oil bath at 60 °C for 20 h, and afterwards, cooled in a liquid nitrogenbath. The crude polymerization mixture was precipitated twice fromNMP into hexane, which is nonsolvent for PPS. The resulting polymerwas dissolved into chloroform and precipitated into tenfold excessof cold methanol to better remove the TPPCl catalyst [22]. The prod-uct was dried at 60 °C under vacuum to yield a yellow viscous oil(0.15 g) (poly(PS74-CEP), Mn=5,800 g/mol, PDI=1.32).

2.2.2. Synthesis of poly(PS74-b-DMA310) diblock polymerRAFT chain extension of poly(PS74-CEP) macro-CTA was done to

prepare poly(PS74-b-DMA310). Poly(PS74-CEP) (0.58 g, 1 mmol), DMA(4.0 mL, 403 mmol), AIBN (0.8 mg, 0.05 mmol), and dioxane(10.52 mL) were placed in a dry ampoule, and the solution wasdegassed by purging with nitrogen for 30 min. The polymerizationwas conducted at 60 °C for 3 h and then quenched by cooling the re-action vessel in an ice bath and exposure to air. The final polymer(poly(PS74-b-DMA310), Mn 36,700 g/mol, PDI=1.29) was purifiedby precipitation into diethyl ether.

2.2.3. Polymer characterization1H NMR spectra were recorded in CDCl3 and D2O with a Brüker

400 MHz spectrometer. Formolecularweight determination, gel perme-ation chromatography (GPC) was performed using dimethylformamide(DMF)+0.1 M LiBr mobile phase at 60 °C through three serial TosohBiosciences TSKGel Alpha columns (Tokyo, Japan). A Shimadzu RID-10A refractive index detector and a Wyatt miniDAWN Treos multi-

angle light scattering detector was used to calculate absolute molecularweight based on dn/dc values experimentally determined throughoffline injections into the RI detector.

2.3. Micelle preparation and characterization

To prepare micelle solutions, 10 mg of purified poly(PS74-b-DMA310)was dissolved in 250 μL of THF. PBS (10 mL, pH 7.4)was added dropwiseinto the THFpolymer solutionunder vigorous stirring todilute the sampleto a final polymer concentration of 1 mg/mL. The solution was pushedthrough a 0.45 μm syringe filter, and dynamic light scatter (DLS) mea-surements were done to measure hydrodynamic diameter using aMalvern Zetasizer Nano-ZS (Malvern Instruments Ltd, Worcestershire,U.K) equipped with a 4 mW He–Ne laser operating at λ=632.8 nm.Transmission electronmicroscopy (TEM) sampleswerepreparedbyplac-ing onedropof solution (micellar dispersion at 1.0 mg/mL concentration)onto copper grids (400 mesh, carbon coated, Ted Pella Inc., Redding, CA).The grids were dried overnight in a desiccator under vacuum before im-aging with a Philips CM20 HR-TEM (Philips, EO, Netherlands).

2.4. Determination of critical micelle concentration (CMC)

The critical micelle concentration was assessed fluorescently usingNile red [27] and by identifying particle morphological changes withDLS. The Nile red dye is hydrophobic and exhibits strong fluorescencein the presence of intact micelles but is poorly soluble and minimallyfluorescent if released into an aqueous environment when micellesdestabilize. Different dilutions were prepared from a 1 mg/mL stocksolution to obtain micelle samples ranging in concentration from0.0001 to 1 mg/mL. Then, 10 μL of a 1 mg/mL Nile red stock solutionin THF was added to 1 mL of each micelle sample and incubated over-night in the dark at room temperature. The next day, samples werefiltered with a 0.45 μm syringe filter and their Nile red fluorescencewas measured in 96 well plates using a micro plate reader (Tecan In-finite 500, Tecan Group Ltd., Mannedorf, Switzerland) at an excitationwavelength of 535±20 nm and an emission wavelength of 612±25 nm. The CMC was defined, as previously described [28], as the in-tersection point on the plot of the Nile red fluorescence versus the co-polymer concentration. For DLS measurements, a range of micelleconcentrations were prepared by serial dilutions of a 1 mg/mL stocksolution to obtain samples of concentration ranging from 0.001 to1 mg/mL in PBS. DLS was then used to assess micelle hydrodynamicsize and to determine the concentration at which the micellesexhibited morphological changes [29].

2.5. Measuring micelle loading with Nile red and DiO

A fluorescence-based method was used to calculate drug loadingand encapsulation efficiency of Nile red and DiO across a range ofweight ratios from 6.25 to 200 μg dye per mg poly(PS74-b-DMA310)based on a reported method [30]. After removal of any free dyethrough centrifugation, the loaded dyes were extracted from the mi-celles by diluting the samples into 90% DMF. Nile red (excitation at535±20 nm, emission 612±25 nm) or DiO (excitation at 485±20 nm, emission 535±25 nm) content was quantified in the respec-tive samples relative to fluorescence standard curves of the moleculesmeasured in 90% DMF. Encapsulation efficiency (EE) was defined asthe weight percent of the drug loaded versus what was added tothe micelle solution, and drug loading (DL) was calculated as theweight percent of dye relative to polymer.

2.6. ROS-mediated release of Nile red and DiO

To assess ROS-dependent release from the poly(PS74-b-DMA310)micelles, Nile red and DiO were utilized as model small moleculedrugs. To prepare 1% Nile red loaded micelle solution, 50 μL of a

Scheme 1. Synthetic route for preparation of ROS-responsive poly(PS74-b-DMA310)diblock copolymer via a combination of TAGT and RAFT polymerizations.

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1 mg/mL Nile red stock solution in THF was added to 5 mL of micellesolution (1 mg/mL). The residual THF was removed through rotaryevaporation and the micelle solution was incubated overnight in thedark at room temperature. The next day, samples were filteredusing a 0.45 μm syringe filter prior to use. Nile red-loaded micelleswere exposed to a range of concentrations (0 to 3.3 vol.%) of hydro-gen peroxide. Fluorescence intensity of Nile red was monitored in a96 well plate using a micro plate reader (Tecan Infinite 500) at an ex-citation wavelength of 535±20 nm and an emission wavelength of612±25 nm. Release of the dye due to micelle oxidation and destabi-lization was assessed over time based on disappearance of Nile redfluorescence. The loss of fluorescence for each sample at each timepoint was determined by subtracting the fluorescent value fromthat of the sample prior to H2O2 addition, and the percent fluores-cence remaining was determined by normalization to the samevalue (before addition of H2O2). This value for percent fluorescenceremaining was subtracted from 100% and expressed as a percent re-lease for each sample at each time point. An analogous experimentwas completed to assess the H2O2-dependent release of DiO from mi-celles prepared using the same technique. A similar study was carriedout to assay the release of Nile red from the micelles upon treatmentwith SIN-1, a molecule known to generate superoxide, nitric oxide,and peroxynitrite [31]. Nile red loaded micelles were treated with arange of SIN-1 concentrations (1 to 100 mM), and a fresh dose ofthe respective concentration of SIN-1 was added every 24 h due tothe short half life of SIN-1 [31]. Nile red release was quantified as de-scribed for H2O2 experiments. Because SIN-1 generates nitric oxideand superoxide that then react to form peroxynitrite, micelle releasewas also studied following direct peroxynitrite treatment. A range offinal concentrations of peroxynitrite (1–100 μM) were tested by ad-dition of a basic peroxynitrite stock solution into buffered (PBS) solu-tions of Nile red-loaded micelles. The final pH of the samples was 7.4after this addition, and fluorescent intensities were measured after15 min to quantify release as described above. The full time coursewas not done with peroxynitrite because of its very short half-life atphysiologic pH.

2.7. FRET-based imaging of micelle release by activated macrophages

Micelle release of the Förster Resonance Energy Transfer (FRET)dye pair DiO and DiI was used as a readout for model drug release invitro [32]. To prepare FRET micelles, 10 μL of DiO solution (1 mg/mLsolution in THF) and 10 μL of DiI solution (1 mg/mL solution in THF)were added into 1 mL of micelle solution (1 mg/mL). The THF wasthen removed by rotary evaporation and the samples were left over-night in the dark. To remove any unloaded dye, the micelle solutionwas diluted four fold in sterile DI water and reconcentrated using acentrifuge filtration tube with 10,000 MW cutoff for 30 min. This pro-cess was repeated four times. Control micelles loaded with just DiO orDiI were prepared in the samemanner as FRETmicelles. Generation ofthe FRET effect within the dual loaded micelles was confirmed using aJobin Yvon/Horiba Fluorolog-3 FL3-111 Spectrophotofluorometer atan excitation wavelength of 484 nm.

To image micelle release mediated by endogenously-produced ROSin vitro, RAW 264.7 mouse macrophages were seeded in phenol-redfree DMEM at 5000 cells/well onto multi-chambered #1 borosilicatecover-glass slides (Fisher Scientific) and allowed to adhere overnight.After 24 h, the media was replaced with phenol-red free DMEM sup-plemented with 100 ng/mL lipopolysaccharide (LPS) or vehicle control.After 24 h, LPS-activated and control cells were treated with vehiclecontrol or with DiO/DiI FRETmicelle solutions to achieve a final concen-tration of 1.67 μg/mL for each dye. Images were acquired 24 h after mi-celle treatmentwith a Zeiss LSM 510META confocal microscope using a63× oil immersion lens equipped with a 488 nm Argon laser with505–550 band-pass filters for green emission and 560–615 band-passfilter for red (FRET) emission. The exposure time, gain, and all other

microscope settings were held constant during fluorescent imaging ofall treatment groups. Image J was used to quantitatively assess cellularfluorescence. Cells were also seeded onto 96 well plates and treated asexplained above to confirm quantitative FRET data using a SpectraMaxM5 (Molecular Devices, Sunnyvale, CA) plate reader (see additional de-tails in Fig. S-8).

2.8. Cell viability assay

RAW264.7macrophages (RAWs)were plated at 8000 cells/well ina 96-well plate and incubated at 37 °C in Alpha MEM supplementedwith 10% FBS and 1% penicillin–streptomycin. After 24 h, the mediumwas replaced with a fresh medium containing a range of polymer mi-celle concentrations (62.5–1000 μg/mL). The cells were then incubat-ed at 37 °C for 24 h or 48 h. At the respective endpoints, the cells werelysed and analyzed for intracellular LDHwith a Cytotoxicity DetectionKit (Roche Applied Science) as previously reported [33]. With thisassay, a colorimetric readout (absorbance at 492 nm with referenceat 595 nm) is acquired that represents relative cell number. Eachgroup was assayed in triplicate, and LDH quantities in the micelletreatment groups were normalized to samples receiving no treatment(NT).

3. Results and discussion

3.1. Synthesis and characterization of poly(PS74-b-DMA310)

The AB diblock copolymer poly(PS74-b-DMA310) was successfullyprepared using the TAGT and RAFT methods described (Scheme 1).The final diblock copolymer poly(PS74-b-DMA310) displayed relativelylow polydispersity (Mw/Mn=1.29) and showed the expected decreasein the elution time following chain extension from poly(PS74-CEP)(Fig. 1).

Successful diblock polymerization was also confirmed by 1H NMRspectra of the poly(PS74-CEP)macro-CTA and the poly(PS74-b-DMA310)diblock copolymer (Fig. 2). The 1H NMR spectra of the resultingpoly(PS74-b-DMA310) taken in CDCl3, a solvent in which micelles donot form, displayed characteristic signals attributed to both PPS and

Fig. 1. Confirmation of RAFT polymerization of a DMA second block from a poly(PS74-CEP)macro-CTA. TheGPC elugram showed a characteristic shift to a decreased elution time fol-lowing RAFT polymerization of the larger molecular weight poly(PS74-b-DMA310) diblockfrom the poly(PS74-CEP) macro-CTA.

Before H2O2A

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PDMA. The use of RAFT for polymerization of the corona-forming poly-mer block provides synthetic flexibility because it enables the possibil-ity of using other monomers or even random copolymer compositions(i.e., for incorporation of pendant groups that can be used to attachcell-specific targeting moieties within the micelle corona).

3.2. Micelle preparation and characterization

Formation of poly(PS74-b-DMA310) micelles with a PPS core and aPDMA corona were successfully prepared by a solvent evaporationmethod using THF. Verification that the poly(PS74-b-DMA310) existedin water as micelles stabilized by a hydrophilic poly(DMA) block wasdone by recording 1H NMR spectra in CDCl3 and D2O (Fig. S-1). The 1HNMR spectrum of the diblock copolymer in CDCl3 showed all peaksassociated with both the PPS and PDMA blocks (Fig. S-1A). Underthese conditions, both blocks are solvated and show free segmentalmotion, which is consistent with a molecularly dissolved unimeric poly-mer. In contrast, 1H NMR of poly(PS74-b-DMA310) in D2O (Fig. S1-B)showed suppression of the PPS peaks (note suppression of peak ‘a’ inFig. S-1B D2O spectrum versus S-1A CDCl3 spectrum) and resembled thespectrum for a homopolymer of PDMA (Mn=20 KDa) taken in D2O(Fig. S-1D). These combined results indicate that, in aqueous conditions,the poly(PS74-b-DMA310) assembles into micelles with the PPS polymerblock confined in the micelle core away from the D2O solvent, and thusunable to generate 1H NMR signal.

1H NMR spectra of micelles were also compared in D2O before andafter H2O2 treatment to verify that ROS could oxidize the PPS in themicelle core and increase its water solubility. For reference, 1H NMRspectra were first recorded for a PPS homopolymer in D2O before

Fig. 2. 1H NMR spectra of (A) poly(PS74-CEP) and (B) poly(PS74-b-DMA310) in CDCl3show characteristic peaks for both PS and DMA, providing additional evidence ofdiblock polymer formation.

and after treatment with H2O2 (Fig. S-1E, S-1F). The appearance of anew peak at 1.53 ppm in the 1H NMR spectra of poly(PS74-b-DMA310)in D2O following treatment with H2O2 is a characteristic shift [14] in-dicating that, in the micelle form, the H2O2 was still able to oxidizethe PPS from a sulfide into a more hydrophilic sulfone (Fig. S-1C).The shifting of the PPS peaks (CH2 and CH protons) from 2.5–3.0 ppm to 3.2–4.2 ppm (circled and labeled ‘b+c’) was also ob-served (Fig. S-1C) and further confirmed PPS conversion into a morehydrophilic sulfone, which also matched observations for PPS homo-polymer H2O2 oxidation by us (Fig. S-1E, S-1F) and others [14].

Physical characterization of the micelles was also done using DLSand TEM. The average hydrodynamic diameter of the poly(PS74-b-DMA310) micelles was measured by DLS to be 99 nm (Fig. 3A). Thissize, along with the spherical morphology of the micelles, was con-firmed using TEM (Fig. 3B). These micelles fall in a desirable sizerange for nanotherapeutics and may be well-suited for tumor targetingthrough the enhanced permeation and retention (EPR) effect [34].

DLS and TEM were also utilized to confirm the H2O2-responsivedisassembly of the micelles. Fig. 3A shows the change in hydrody-namic diameter of micelles before and after H2O2 (3.3 vol.% for24 h) treatment. The average size of micelles was found to changefrom 99 nm to 5 nm after treatment with H2O2, and this behaviorwas also supported by the absence of visible micelles in TEM imagesof H2O2 treated micelle samples (Fig. 3C). These data indicate thatthe poly(PS74-b-DMA310) micelles may be useful for cargo deliveryto sites with high ROS through environmentally-triggered micelledisassembly.

3.3. Determination of critical micelle concentration (CMC)

The critical micelle concentration (CMC) was determined usingNile red as a fluorescent probe [35]. Nile red is hydrophobic anddoes not fluoresce in an aqueous environment but is strongly fluores-cent when it partitions itself into the hydrophobic core of intact mi-celles. The fluorescence intensity of Nile red in poly(PS74-b-DMA310)aqueous solutions of various concentrations were recorded, and themaximum fluorescence intensity of the Nile red emission is shownin Fig. 4 as a function of micelle concentration. The CMC of the diblockcopolymer was determined using the crossing point of the plot andwas estimated to be 0.09 mg/mL. The CMC value of the micelles wasalso estimated using a DLS-based dilution method [29]. Table 1

0.1 1 10 100 1000 10000

Log (Dh) (nm)

After H2O2

B C

Inte

nsi

ty

Fig. 3. Poly(PS74-b-DMA310) micelles disassembled following exposure to H2O2. (A) DLSmeasurement of poly(PS74-b-DMA310) demonstrated transition from nanoparticulate mi-celles to smaller polymer unimers following H2O2 treatment. TEM imaging (B) before and(C) after H2O2 treatment visually confirmed the presence of micelles that disassembled fol-lowing oxidation. TEM scale bars=100 nm.

0

25

50

75

100

0 50 100 150 200

Per

cen

t R

elea

se

Hours

Poly(PS74-b-DMA310) Micelle H2O2-Dependent Nile Red Release

3.3%1.66%0.33%0.166%0.0033%0%

Fig. 5. H2O2 concentration-dependent release of the model drug Nile red frompoly(PS74-b-DMA310)micelles. Figure legend indicates volume % of H2O2 in PBS (n=3).

Fig. 4. Changes in the fluorescence intensity of Nile red (1.0×10−6 M) with differentconcentrations of poly(PS74-b-DMA310) indicated that the CMC was approximately0.09 mg/mL.

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shows the hydrodynamic diameter (Dh) of poly(PS74-b-DMA310) inPBS from concentrations ranging 1.0 mg/mL to 0.001 mg/mL, andthe full DLS spectra for these samples are also shown in Fig. S-2. Thehydrodynamic diameter of micelles was stable down to 0.1 mg/mL,and dilution to concentrations below this value caused the micellesto become increasingly unstable, resulting in the formation of aggre-gates. This indicated that the CMC value fell in the range from 0.05 to0.1 mg/mL, which was in agreement with the Nile red based assay.

3.4. Assessment of micelle loading with Nile red and DiO

The drug loading and encapsulation efficiency of poly(PS74-b-DMA310) micelles were determined using both Nile red andDiO. For the drug to polymer ratios tested, the maximum drug loadingcontent and encapsulation efficiency of Nile red were calculated to be1.76 wt.% (Fig. S-3A) and 63.1% ((Fig. S-3B)), respectively. The valueswere consistent with the previously reported literature [30]. DiO alsoshowed similar maximum drug loading content (1.48 wt.%, (Fig.S-3C)) and encapsulation efficiency (63%, (Fig. S-3D)).

3.5. ROS dependent release of Nile red

Nile red was chosen as a model hydrophobic drug for the investi-gation of drug release [27] in response to H2O2, which was used tomimic the presence of pathophysiologic oxidative stress [36]. Fig. 5shows in vitro release kinetics of Nile red mediated by different con-centrations of H2O2. The fluorescence intensity of Nile red-loaded mi-celles treated with H2O2 was found to decrease over time, and therate of this decrease correlated to the concentration of H2O2 present.The loss of Nile red fluorescence intensity under oxidative environ-ments can be explained by the conversion of the PPS block from a hydro-phobic sulfide to more hydrophilic sulfone. This triggers disassembly ofthe micelle into unimeric polymers and releases the Nile red into themore polar aqueous environmentwhere its fluorescence is no longer ap-parent. The fluorescence intensity of the negative control samples thatwere not treated with H2O2 remained relatively constant over thetimeframe studied, indicating that the micelles were stable in the ab-sence of ROS. This observation suggests that the poly(PS74-b-DMA310)micelles would have little nonspecific drug “leak” in vivo prior totargeted disassembly and drug release at sites of oxidative stress.

Table 1DLS measurements of poly(PS74-b-DMA310) demonstrating that the polymer CMCfalls below 0.1 mg/mL.

Polymer concentration Size (nm)

poly(PS74-b-DMA310) — 1 mg/mL 99.45poly(PS74-b-DMA310) — 0.5 mg/mL 97.55poly(PS74-b-DMA310) — 0.1 mg/mL 99.28poly(PS74-b-DMA310) — 0.05 mg/mL 506.6poly(PS74-b-DMA310) — 0.01 mg/mL 175.00poly(PS74-b-DMA310) — 0.001 mg/mL 407.2

To verify that the decrease in fluorescence intensity of Nile redduring the release experiments was due to micelle release and notH2O2 degradation of the dye itself, the free Nile red molecule wasdissolved in a mixture of THF/water (15/85) and treated with arange of concentrations of H2O2. The fluorescence intensity was foundto remain constant over the period of 170 h, confirming that loss of signalwas due to dye released from the micelles due to destabilization of themicelle structure (Fig. S-4). Furthermore, because subsequent cell exper-iments were done using the dyes DiO and DiI, an additional experimentwas done to confirm that H2O2 dose dependent micelle release of thesemodel drugswas similar to the release profile forNile red-loadedmicelles(Fig. S-5).

To assess the responsiveness of the poly(PS74-b-DMA310) micellesto other ROS, the in vitro release kinetics of Nile red were measuredover a period of 92 h following treatment with a range of concentra-tions (1–100 mM) of the peroxynitrite generator SIN-1 (Fig. S-6). Therate of release of Nile red from the micelles was found to be depen-dent on the concentration of SIN-1. To confirm that release was medi-ated by peroxynitrite, Nile red release in response to treatment withperoxynitrite alone (1–100 μM) was also tested. Use of exogenousperoxynitrite to model endogenous production is difficult to mimic invitro because it has a very short (~1 s) half-life at physiological pH. How-ever, it was found that 20% Nile Red release was triggered from micellesafter a single treatment with 100 μMperoxynitrite (Fig. S-7), further val-idating the multi-faceted response of the (PS74-b-DMA310) micelles tovarious ROS species. These results indicate that poly(PS74-b-DMA310)may be broadly applicable for in vivo delivery to pathological sites withhigh oxidative stress.

Direct comparison of hydrogen peroxide or peroxynitrite concen-trations in vivo versus those used in the cell-free experimentsreported here is difficult because cells at sites of inflammation invivo continuously generate ROS (many of which have very shorthalf-lives), whereas, for practical reasons, bolus dose delivery is uti-lized in cell free studies. It has been postulated, for example, thatmuch higher concentrations of exogenous peroxynitrite (half-lifeb1 sat physiologic pH) may be required to achieve biologic responses sim-ilar to those produced by much lower concentrations of continuouslyproduced endogenous peroxynitrite in vivo [37]. Here, we have com-pared cell-free bolus doses to the total quantity of H2O2 or peroxynitritethat would be generated within a given tissue volume during a definedtimeframe. These calculations are based on an average (spherical)macro-phage diameter of 21 μm [38] and published values of 0.63–1.258 nmol/(106 cells)/min and 0.11 nmol/(106 cells)/min for activated macrophageproduction of H2O2 and peroxynitrite, respectively [39–41]. Using thesevalues, one can estimate that, over a brief 6 hour period, a concentrationof 47–93 mMH2O2 or 8 mM peroxynitrite would be accumulated locallyin vivo if there was no diffusional loss or degradation of these species.Note that the intermediate H2O2 concentration of 0.33 vol.% tested inthe Nile red release study corresponds to 97 mM and correlates well tothe higher end values in this calculation, while the calculated value for

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peroxynitrite exceeds the highest dose (100 μM) tested in theperoxynitrite Nile red release study by 80-fold. It is estimated that theperoxynitrite generation rate of SIN-1 is 1 μM/min/(mM SIN-1), and anapproximately 1 mM dose has been used to mimic pathological condi-tions on cultured cells [42]. Over 6 h, the 1 mM SIN-1 would generate aconcentration of 360 μM peroxynitrite under the no-loss assumption,which is 3.6-fold higher than the highest dose tested in our peroxynitritebolus delivery experiment (Fig. S-7).

The preceding analysis suggests that the peroxynitrite and SIN-1 con-centrations used in our drug release experiments span physiologically-relevant ranges, while the hydrogen peroxide concentrations may havebeen higher than what would be present in inflamed tissues. Becausesites of inflammation in vivo consist of a complex milieu of differentROS and reactive nitrogen species, it is potentially advantageous thatpoly(PS74-b-DMA310) micelles were responsive to multiple reactive spe-cies, and it is not necessarily a problem if the micelles do not sufficientlyrespond to H2O2 alone to trigger rapid release. The release behavior ofthe poly(PS74-b-DMA310) carriers may in fact be ideally tuned to remainstable “in transit” and slowly respond to achieve sustained release ofcargo once a pathophysiological environment is encountered. Thoughmore switch-like release from thenanocarriersmaybe ideal for some ap-plications, it is conceivable that the sustained release profile that thesenanocarriers display may be therapeutically advantageous in some sce-narios, and in vivo testing will need to be done to better validate the be-havior of these carriers for different pathologies.

3.6. FRET-based imaging of micelle release by activated macrophages

Proof-of concept studies in LPS-activated macrophages indicatedsuccessful use of poly(PS74-b-DMA310) micelles for environmentally-targeted drug release in response to endogenously-produced ROS. Todo so, release frommicelles co-loadedwith DiO and DiI wasmeasuredusing FRET. A FRET signal (emission of DiI, a red fluorophore, upon ex-citation of DiO, a green fluorophore) occurs only when the two dyesare in close proximity, i.e., co-loaded into themicelle core. Fluorimetrymeasurements following excitation at 484 nmwas utilized to confirm

Fig. 6. Demonstration of FRET effect in poly(PS74-b-DMA310) micelles co-loaded withDiO and DiI. (A) Fluorescence emission spectra of PPS74-b-PDMA310 FRET micelles(1% DiI and 1% DiO), 1% DiO micelles, and 1% DiI micelles with 484-nm excitation.(B) Fluorescence spectra of micelles diluted tenfold into PBS or DMF. FRET signal is in-dicated by the quenching of emission at 510 nm and increase in emission at 580 nm inintact, dual-loaded micelle samples.

FRET signal in dual DiO/DiI-loaded Poly(PS74-b-DMA310) micelles(Fig. 6A). Tenfold dilution of dual DiO/DiI-loaded micelles into DMF,an organic solvent that disrupts the micelle core, disrupted the FRETsignal, but a similar tenfold dilution into PBS did not (Fig. 6B), furtherconfirming successful generation of FRET micelles.

ROS endogenously produced by LPS-activated macrophages wereshown to trigger release of the DiO/DiI dual-loaded poly(PS74-b-DMA310) micelles in vitro using this FRET-based readout. BacterialLPS was used to activate RAW macrophages since it models

Fig. 7. Cell-mediated micelle release of FRET pair DiO and DiI by LPS-activated macro-phages. (A) Representative FRET confocal microscopy images of RAW 264.7 cells deliv-ered poly(PS74-b-DMA310) micelles co-loaded with DiI and DiO with and withoutpre-activation with LPS. Upon excitation at 485 nm, decreased red emission(560–615 nm) and increased green emission (505–550 nm) were visible in cells treat-ed with LPS relative to controls, indicating that LPS-driven ROS generation triggered re-lease of micelle cargo. Scale bars=25 μm. (B) Green and red fluorescent emissionintensities integrated across the longest dimension of macrophages (n=4) showedquantitative differences, with a significant drop in the red (FRET-based) signal in thecell populations treated with LPS. Standard error is indicated by the shaded regionoverlaying each data set.

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inflammatory disease states, activates ROS-producing enzymes, andstimulates production of ROS and peroxynitrite in macrophage[43–45]. Confocal microscopy imaging of control cells showed thepresence of the FRET signal (quenched DiO/green, bright DiI/red) incontrol cells excited at 488 nm, suggesting cell internalization of theintact DiO/DiI dual-loaded micelles. In LPS-activated macrophages, adecrease in DiI/red signal and increase in DiO/green signal suggestedthat endogenously-produced ROS had triggered micelle disruptionand intracellular dye release (Fig. 7A). Quantitative analysis of themacrophage images confirmed this change in the fluorescence profilebetween LPS-treated and control cells (Fig. 7B). Macroscopic platereader data also indicated that there was a significant reduction inFRET signal for cells treated with LPS relative to vehicle controls(pb .005) (Fig. S-8). These combined FRET microscopy and plate read-er data strongly indicate that “inflamed” macrophages generate suffi-cient ROS to facilitate release of poly(PS74-b-DMA310) micelle cargo.This proof-of-principle experiment confirmed the potential feasibilityin using this system for environmentally-targeted delivery of hydro-phobic drugs to sites of inflammation.

3.7. Cell viability

The cytotoxicity of the poly(PS74-b-DMA310) micelles was evaluat-ed in RAW 264.7 cells for a range of concentrations (0–1000 μg/mL)using the LDH assay. This assay offers a simple way to measure LDH,a stable cytoplasmic enzyme present in most cells that provides anaccurate marker for cell viability [46]. Fig. 8 shows that viability ofthe RAW cells remained high in the presence of micelles across theentire range of concentrations tested after both 24 h and 48 h incuba-tion times.

4. Conclusion

This report presents novel, ROS-responsive polymeric micelles thathave the potential to be applied to preferentially release entrapped hy-drophobic drug cargo under pro-inflammatory, oxidative environ-ments. Thorough characterization of the “smart” poly(PS74-b-DMA310)micelles demonstrated responsiveness to H2O2, SIN-1, and peroxynitrite,indicating that multiple reactive species would contribute to cargorelease at sites of inflammation. The reaction of PPS-containing poly-mers with SIN-1/peroxynitrite is a novel finding to our knowledge,and it is anticipated that response to oxidants other than H2O2 isimportant for accomplishing drug release under pathophysiologically-relevant conditions. The importance of this finding is further supportedby the fact that other sulfides have been shown to react three timesfaster with peroxynitrite than to H2O2 [47]. To this end, it was foundthat LPS-activated macrophages, a model system known to robustlyproduce peroxynitrite [44], preferentially triggered cell-mediated

Fig. 8. Poly(PS74-b-DMA310) cytocompatibility in RAW 264.7 macrophages was foundto be high across the entire range of micelle concentrations tested at both 24 and48 h. The cell viability was determined by LDH assay and each treatment group wasnormalized to no treatment (NT) controls (n=3).

micelle release of the FRET pair DiO and DiI. It was also shown thatthe micellar carrier did not cause any in vitro cytotoxicity, and it isanticipated that conversion of polypropylene sulfide to more watersoluble forms (i.e., polypropylene sulfoxide) would result in safe re-moval of the micelle constituents from body after exposure to ROSrich environments. These combined data indicate that poly(PS74-b-DMA310) micelles provide a promising platform for targeted drugtherapy at sites of oxidative stress and that this carrier may be an es-pecially good candidate for delivery to pathological sites with highperoxynitrite production.

Acknowledgments

This research was supported by Vanderbilt University School ofEngineering startup funds. Dynamic light scattering and TEM wereconducted through the use of the core facilities of the Vanderbilt In-stitute of Nanoscale Sciences and Engineering (VINSE). Confocal mi-croscopy was performed in part through the use of the VUMC CellImaging Shared Resource, (supported by NIH grants CA68485, DK20593,DK58404, HD15052, DK59637, and Ey008126).

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jconrel.2012.07.042.

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