Reduction-Controlled Release of Organic Nanoparticles fromDisulfide Cross-linked Porous PolymerNeil Grant,† Hong Wu,‡ and Haifei Zhang*,†

†Department of Chemistry, University of Liverpool, Oxford Street, Liverpool, L69 7ZD, United Kingdom‡Department of Pharmaceutical Chemistry and Pharmaceutical Analysis, School of Pharmacy, Fourth Military Medical University,Xi’an 710032, China

*S Supporting Information

ABSTRACT: Reduction-controlled release is favored for many applications. The cleavage of disulfide bonds is known to besensitive to reducing agents. Here, a cross-linker containing a disulfide bond is prepared and then used to prepare cross-linkedporous polymer via an emulsion templating approach. Oil-in-water (O/W) emulsions are first formed where an organic dye isdissolved in the oil droplet phase and monomer/cross-linker/surfactant are added into the continuous aqueous phase. Bypolymerizing the O/W emulsion followed by freeze-drying, organic nanoparticles are formed in situ within the disulfide-cross-linked porous polymer. The release of organic nanoparticles in water is demonstrated and can be tuned by the presence ofreducing agents such as dithiothreitol and tris(2-carboxyethyl)phospine. This approach has the potential to be used for thereduction-controlled release of poorly water-soluble drug nanoparticles from porous polymers or hydrogels.

1. INTRODUCTION

Biodegradable polymers and hydrogels are used in a variety ofapplications including tissue engineering1 and drug release.2−4

Hydrogels are chemically or physically cross-linked hydrophilicpolymers, which are insoluble in water but swell. Biocompat-ibility, biodegradability, and easily tuned mechanical stabilityhave made hydrogels highly attractive in biological andbiomedical applications. For example, hydrogels have beenextensively investigated for targeted drug delivery andcontrolled release.5,6

Among the chemically cross-linked hydrogels, disulfide cross-linkers have been widely used.7−10 Disulfide matrices play animportant role in pharmaceutical and biological applicationsdue to their stability under normal conditions and degrading ina reductive environment.11−13 The disulfide bond can becleaved in aqueous media by reducing agents such asdithiothreitol (DTT), glutathione (biologically available) andtris(2-carboxyethyl)phosphine (TCEP),11−14 and others.15,16

Disulfide cross-linked hydrogels, capsules, and micelles aremostly investigated.8,17−25 Various studies have shown theenhanced release by the addition of a reducing agent or withincells.17−28

Drug solubility in water is a major issue because over 40% ofdrugs in development pipelines are classed as poorly soluble.29

This can lead to issues such as low bioavailability, erraticabsorption profiles, and reduced patient compliance.29,30 Oneapproach to addressing this issue is to form drug nano-particles.30,31 Not only can the drug dissolution rate increasesignificantly with reduced particle sizes, aqueous drug nano-dispersion may be administrated directly.30−33 We havedeveloped a new emulsion-freeze-drying approach to formorganic/drug nanoparticles in situ within porous polymers.34,35

The nanoparticles can be released simply by dissolution34,35 orvia a temperature trigger to produce stable aqueous nano-dispersion.36 Although reduction-controlled release via disulfide

cleavage has been widely used for hydrogel, capsule, and micellesystems, all the studies have been focused on the release ofsoluble molecules in aqueous systems.17−28 Considering thelarge number of poorly water-soluble drugs and the intensivestudies on drug nanoparticles, an investigation on thereduction-controlled release of poorly water-soluble drugnanoparticles would be highly appealing. Here, using anorganic dye as a model compound, we report a study on theformation of organic nanoparticles within a disulfide cross-linked emulsion-templated porous polymer and the reduction-controlled release of the organic nanoparticles into water.

2. EXPERIMENTAL SECTION2.1. Materials. Acrylamide (AM, 99%, Mw 71.08),

N,N,N′,N′-tetramethylethylenediamine (TMEDA), Triton X-405 (70% in water, density 1.096 g cm−3), oil red O (OR),cystamine dihydrochloride (96%), acryloyl chloride (97%),tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Mw286.65), DL-dithiothreitol (DTT, Mw 154.25), and all theother chemicals were purchased from Sigma-Aldrich and usedas received. Chloroform, ethyl acetate, heptane, acetonitrile,and sodium hydroxide were all of analytical grade and used asreceived. Distilled water was used in each case.

2.2. Preparation of the Bisacryloylcystamine (BAC)Cross-Linker. N,N′-bis-acrylcystamine (Mw 204.35) wassynthesized following the method reported previously:7 Briefly,8 g of cystamine dihydrochloride was dissolved in 80 cm3 of3.12 M NaOH solution, and then 9.6 g of acryloyl chloride in20 cm3 of acetonitrile was added dropwise to the solutionunder vigorous stirring at 50 °C for 15 min or until

Received: September 11, 2013Revised: November 15, 2013Accepted: December 12, 2013Published: December 12, 2013

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effervescence disappeared. The reaction product was extractedwith hot chloroform at 50 °C. The extract was washed with 0.1M HCl and saturated sodium chloride aqueous solution anddried on sodium sulfate for 1 day. After removal of the solventunder vacuum conditions, the residue was recrystallized usingethyl acetate/heptane (2:1) and identified by 1H NMR andmicroanalysis.2.3. Preparation of Emulsion-Templated Polyacryla-

mide (PAM). Preparation of oil-in-water emulsions andnanoparticles/porous polymers using BAC as a cross-linkerfollowed a similar procedure reported before.36 The molarratios of AM/BAC were fixed at 20:1 and 40:1 in a 10 wt %monomer solution. BAC was dissolved in the monomersolution by sonication until fully dissolved. A total of 10 wt%aqueous ammonium persulphate solution (0.1 cm3) was addedto the monomer/cross-linker solution (2 cm3) followed byTriton X-405 (0.3 cm3). A solution of OR in cyclohexane (0.02wt/v%, 6 cm3) with TMEDA (30 μL) was added dropwise tothe aqueous phase while stirring using a lab egg stirrer. Theformed emulsion was left to stir for 5 min before transferring toan oven at 60 °C overnight. In the polymerized samples, 44 wt% was contributed from AM (others were mainly surfactant andcross-linker). For emulsions with an internal phase volume of50%, the preformed emulsion was homogenized for 1.5 min atspeed 5 using a Fisher Brand homogenizer. The polymerizedemulsions were frozen and freeze-dried to remove the solvents,yielding porous material. All the OR in the original emulsionswas transformed into nanoparticles within the emulsion-templated macropores during freeze-drying.35,36

2.4. Release of OR Nanoparticles from DisulfideCross-Linked Porous PAM. Release using DTT as thereducing agent: Solutions of DTT were prepared at theconcentrations of 0.1, 0.2, and 1 wt % in pH 9 water andbubbled with nitrogen for 10 min. Prepared porous polymerwith OR nanoparticles (0.05 g) were cut and placed in a vialwith a septa and flushed with nitrogen for 10 min. DTTsolution (5 cm3) was added via a syringe to the vial. The vialwas placed into a water bath at 45 °C. Periodically, the aqueousmedium was agitated five times to ensure uniform mixing of thesuspensions, and 200 μL of clear red suspension was removedfor UV analysis. The removed volume was replaced by a freshvolume of DTT. The collected samples were analyzed by aUV−vis plate reader at room temperature.Release using TCEP as the reducing agent: TCEP solutions

in water at the concentrations of 0.02, 0.2, and 1 wt % wereprepared. The prepared porous polymer with OR nanoparticles(0.05 g) was soaked in the TCEP solution (5.5 cm3) at roomtemperature at pH 7. Periodically, 200 μL of the aqueousmedium was taken for UV analysis as described above. Theremoved volume was replaced with fresh TCEP solution.Monitoring of the release was performed using a UV plate

reader (Quant, Bio-Tek Instrument Inc.). A total of 200 μL ofthe released aqueous suspension was pipetted into a well of a96-well flat bottomed polypropylene plate. The absorbance wasmonitored by scanning from 200 to 800 nm in 2 nm steps (ORabsorption at 514 nm was used). The height of the peak wassubtracted from the baseline to give the absorption reading ofeach sample. The absorbance data were normalized using

=∞

MAA

t

(1)

where M is the normalized unit, At is the absorbance at time t,and A∞ is the highest final absorbance reading from the tests.The release rates were calculated using the Higuchi model forrelease of molecules from an insoluble matrix:

= =∞

MAA

ktt 1/2

(2)

where k is the rate constant and t is the release time. Theregression function on Excel was used to calculate the R2 foreach release curve.

2.5. Characterization. The dried materials were sectionedto reveal the internal porous structures. The samples wereadhered to an aluminum stub using a silver colloidal suspensionand allowed to dry. A sputter coater (EMITECH K550X) wasused to coat the samples with gold at 30 mA for 3 min. AHitachi S-4800 field emission SEM was used to reveal the porestructure at 3 kV. The pore sizes and pore volumes of the driedmaterials were examined using a Micromeritics Autopore IV9500 porosimeter. Samples were subjected to a pressure cyclestarting at approx 0.5 psi, increasing to 60000 psi in predefinedsteps.The OR nanoparticles were released into water in the

presence of a reducing agent. The OR nanoparticles werereleased and an aqueous OR nanoparticle dispersion wasformed. The OR nanodispersions were centrifuged for 15 minat 13 000 rpm using an Eppendorf Centrifuge 5415D, and thesupernatant liquid was filtered through a 1 μm syringe toremove the degraded polymer. OR nanoparticles were notprecipitated during centrifuging, as a clear red solution was stillobserved. The OR dispersions were analyzed at 25 °C bydynamic laser scattering (DLS) using a Malvern Zetasizer witha backscattering detection at 173°. The scattering intensitysignal for the detector was passed through a correlator wherethese data were analyzed by the software and gave a sizedistribution. The size of the hydrated OR nanoparticles in waterwere obtained. Each measurement was repeated at least threetimes, and the average data were used to plot the DLS curvesand obtain the particle size. A total of 10 μL of a diluted ORnanodispersion was dropped onto holey carbon filmed coppergrids (400 mesh) and allowed to dry overnight. A scanningtransmission electron microscopic (STEM) detector attachedto the Hitachi S-4800 SEM was used to observe the dry ORnanoparticles at 30 KV.Breaking of the disulfide bonds and therefore the formation

of thiols were performed using DTT or TCEP. To prove theformation of thiols, the TCEP Ellman’s stain method was used.The stain was prepared based on a reported procedure:37 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB; 0.15 g, 0.034 mmol) in1:1 ethanol and tris-HCl (150 mL total).Preparation of 1 M tris-HCl: Tris(hydroxymethyl)-

aminomethane (30.28 g) was dissolved in distilled water (150cm3) and acidified to pH 7.4 with concentrated HCl. Thesolution was made up to 250 cm3 using distilled water. Toprove the formation of the thiols, a drop of the releasedmedium was placed onto a TLC plate and submerged in thestain solution. The formation of the thiols was observed by abright yellow spot where the drop was placed.

3. RESULTS AND DISCUSSION

N,N′-bis-acrylacystamine (BAC), the disulfide cross-linker, wassynthesized following a procedure reported previously7 andconfirmed by the NMR spectrum and the microanalysis (Figure

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1). The organic dye OR was used as the model compoundbecause the release can be easily observed and monitored.36

OR-cyclohexane (CH) solution (0.02 wt/v%) was emulsifiedinto aqueous solution containing monomer acrylamide (AM,10 wt %), cross-linker BAC, and surfactant Triton X-405 toform oil-in-water emulsions. The molar ratios of AM to BACwere 40:1 and 20:1. The oil to water ratios in the emulsionswere 1:1 (50 v/v % oil phase) and 3:1 (75 v/v % oil phase).These emulsions were polymerized at 60 °C in an ovenovernight and then frozen in liquid nitrogen and freeze-dried toproduce dry porous cross-linked polyacrylamide (PAM) with insitu formed OR nanoparticles.35,36 The compositions of theformed emulsions and the porosity of the polymers(characterized by Hg porosimeter) are given in Table 1.Sample PAM_X2.5_O50 indicates the porous polymerprepared from the emulsion with 2.5% cross-linker (BAC/AM = 1:40) and 50% oil phase. Highly interconnected porousstructures are formed from the emulsions with 75% oil phase(Figure 2A and C). While for the emulsions with 50% oil phase,isolated cellular pores are still well connected by ice-templatedpores, consistent with the previous studies.34,38 Hg intrusionporosimetry measurements show the distribution of macro-pores (Figure 2E and F). Bimodal pore size distributions areobserved for the structures made from 75% oil-phase emulsion,indicating higher pore interconnectivity.When the OR/polymer composites were placed in water, a

negligible release of OR nanoparticles was observed. Reducingagent DTT or TCEP was then added to enhance the release.

DTT is not stable in water, and oxidation can occur whenexposed to O2 or metal ions such as Fe3+.39 TCEP, however, isa nonvolatile solid and can be easily handled in the air.40 Inaqueous solutions, TCEP is significantly more stable than DTTand is a stronger reductant than DTT.41

It was believed that the addition of a reducing agent such asDTT or TCEP could cleave the disulfide bonds, degrade thecross-linked polymer, and enhance the release of ORnanoparticles from the porous scaffolds. In our study, bothDTT and TCEP were dissolved in water as reducing agents(0.05 g composite sample soaked in 5 cm3 aqueous solution) totune the release of OR nanoparticles. Red color (from ORnanoparticles) was observed to diffuse out of the polymer intowater, and clear red nanodispersions were formed. As thedisulfide bonds in the polymer were cleaved, the size of thepolymeric scaffolds reduced, and in some cases the scaffoldsbroke apart before being completely dissolved.Release by the addition of DTT was conducted in a water

bath at pH 9 and 45 °C, although it was possible to perform aslow cleavage at 37 °C.19 DTT concentrations of 0.1, 0.2, and 1wt % in water were studied for all four prepared composites(Table S1). The DTT solution was kept under nitrogen toensure that the DTT did not self-oxidize. The number of molesof DTT was in excess of that of the disulfide bonds (3.24 ×10−5 mol [0.1 wt % DTT] to 7.77 × 10−6 mol [2.5% cross-linked polymer]). As a result, the rates of the disulfide cleavagereaction were not changed proportionally with the DTT

Figure 1. 1H NMR spectrum for the synthesized cross-linker BAC. 1H NMR data (400 MHz, CDCl3): δ 2.85 (m, 4H, environment a), δ 3.7 (m, 4H,environment b), δ 5.62 (m, 2H, environment f), δ 6.2−6.4 (m, 4H, trans-H on alkene, environments d and e), δ 6.7 (s, 2H, -NHc). Elementalpercentage by microanalysis: %C = 45.64 (46% calculated), %H = 6.11% (6.2% calculated), %N = 10.28% (10.8% calculated).

Table 1. Preparation Conditions and Porosity of Disulfide Crosslinked Polyacrylamide

sample AM solution (cm3) BAC (%) OR-CH (cm3) oil volume ratio (%) pore volume (cm3 g−1) peak pore size (μm)

PAM_X2.5_O50 4 2.5 4 50 7.0 ± 0.4 3.9PAM_X2.5_O75 2 2.5 6 75 8.5 ± 0.4 26.0, 2.0PAM_X5_O50 4 5 4 50 6.9 ± 0.4 3.8PAM_X5_O75 2 5 6 75 7.2 ± 0.3 25.9, 1.5

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concentrations, as demonstrated by the slowly increased releaseof OR nanoparticles (Figure 3).The intermediate in the DTT reduction is highly unstable.

The reducing capabilities of DTT are limited due to the factthat only the negatively charged S-thiolate is the reactivespecies; the reaction is often limited to pH values above pH 7.We had thus chosen pH 9 for the release with DTT. TCEP,however, is a useful reductant over a much wider pH range(1.5−8.5) than DTT is.14 The release studies with TCEP werethus performed at room temperature and pH 7 (Table S2).With TCEP as a stronger reducing agent and more stable insolution, lower starting concentrations of TCEP (0.02, 0.2, 1 wt%) were used. The release profiles for PAM_X2.5_O50 areshown in Figure 4A. All the release data in this study arecorrelated using the classical Higuchi model where a linearrelationship between the normalized absorbance and squareroot of releasing time is observed.42 The correlation parameterR2 is close to 1, indicating that the release profiles conform tothe Higuchi model (Figures 3B and 4B). As shown in Figure4B, the rate of OR release increases from 0.0181 to 0.0295 and0.0437 (min−1/2) for concentrations of 0.02, 0.2, and 1 wt %,respectively. At 0.02 wt % TCEP, the number of moles ofTCEP in solution is 3.48 × 10−6 mol, which is lower than thatof disulfide bonds. As TCEP reaction with disulfide is astoichiometric reaction,40,41 the cleavage rate of disulfide bondsmay increase linearly until the TCEP (1 wt %) is in excess.Correspondingly, this results in the largely increased release ofOR nanoparticles.The porosity of the polymer can be tuned by varying the

internal phase volume of the original emulsion, as shown inFigure 2. When the same concentration of OR solution is used,

this could result in the increased loading of OR nanoparticles inthe porous polymer. Figure 5A shows the fast release of ORnanoparticles from the porous polymer prepared from theemulsion with a 75% oil phase. Both the highly interconnectedporosity and increased OR loading contributed to thisobservation. To identify the effect of porosity, a diluted ORsolution may be used to form the emulsions and then test therelease in a further study.Another factor that affects the release is the degree of cross-

linking. Generally for hydrogels, low cross-linking degree canresult in a higher degree of swelling and often fast release.There is additional influence for disulfide cross-linked polymersin the presence of a reducing agent such as TCEP. Lower cross-linking degree (i.e., lower ratio of BCA to monomer AM)means a smaller number of disulfide bonds available forcleavage. That can lead to fast degradation of the polymer. Asshown in Figure 5B, when the cross-linking ratio was changedfrom 2.5% to 5.0% while the other conditions were kept thesame, fast release is observed for the polymer with 2.5% cross-linking ratio. The change in release rate is not to a largerdegree. However, this has been consistent for the samplestested under other conditions (Table S1, Table S2).Figure 6 shows that the sizes of the released OR

nanoparticles by the addition of DTT are around 200 nm, ascharacterized by transmission electron microscopy (TEM). Acopper grid with holey carbon film was used for the TEMimaging. Therefore, the black spots are nanoparticles, while thewhite features are the holes in the carbon film in Figure 6. Asexpected, release by DTT or TCEP (Figure S1) had noinfluence on the OR nanoparticles. All the OR nanoparticledispersions were examined by a dynamic laser scattering

Figure 2. The micrographs show the pore morphology of sample (A) PAM_X2.5_O75, (B) PAM_X2.5_O50, (C) PAM_X5_O75, and (D)PAM_X5_O50 and the relevant pore size distributions (E and F).

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technique (Table S3). The hydrated diameter of the ORnanoparticles is on average 210 nm, which is consistent withthe results from TEM imaging. The cross-linking ratio had littleeffect on particle size while higher porosity of the polymerslightly increased the particle size. However, all the measure-ments showed that the average particle sizes were below 300nm, which suggests that stable aqueous nanoparticle dispersionscould be readily formed.31,35

4. CONCLUSIONIn summary, a disulfide cross-linker is synthesized and used toprepare a reduction-responsive porous polymer. By combiningemulsion templating and freeze-drying, organic nanoparticlesare formed in situ within the porous polymer. Through thecleavage of disulfide bonds by reductants, the reduction-controlled release of organic nanoparticles (<300 nm) issuccessfully demonstrated from the porous scaffolds. The

Figure 3. The release profiles (A) and the rate determination (B) forthe release of OR nanoparticles based on the Higuchi model forsample PAM_X2.5_O50 under DTT concentrations of 0.1 wt % (▲),0.2 wt % (■), and 1 wt % (◆).

Figure 4. The release profiles with time (A) and linear correlation ofnormalized absorbance to time square root (B) based on the Higuchimodel for the sample of PAM_X2.5_O50 by the addition of TCEP atconcentrations of 0.02 wt % (◆), 0.2 wt % (■), and 1 wt % (▲).

Figure 5. (A) Comparison of the release of OR nanoparticles from50% and 75% emulsion-templated samples. (B) Comparison of therelease for samples with different cross-linking ratios.

Figure 6. TEM image of the OR nanoparticles released in the presenceof DTT.

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release rates can be tuned by varying the concentrations or thetypes of the reducing agents (DTT and TCEP). The emulsiontemplating technique is very convenient in changing theporosity of the scaffolds, which can in turn influence the releaserate of the organic nanoparticles. There is potential to extendthis approach to the preparation and reduction-responsiverelease of poorly water-soluble drug nanoparticles. Drugnanoparticles of such sizes may generally produce stableaqueous dispersions and can be highly efficient for targeteddelivery.32

■ ASSOCIATED CONTENT*S Supporting InformationThe OR nanoparticle release data and one TEM image. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel.: +44 151 7943545. Fax: +44 151 7943588. E-mail:zhanghf@liv.ac.uk.Funding

This study was funded by an EPSRC DTA studentship to N.G.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe are grateful for access to the facilities in the Centre forMaterials Discovery at the University of Liverpool.

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Industrial & Engineering Chemistry Research Article

dx.doi.org/10.1021/ie403001r | Ind. Eng. Chem. Res. 2014, 53, 246−252252