Timed-Release Polymer NanoparticlesNguyen T. D. Tran,† Nghia P. Truong,† Wenyi Gu,† Zhongfan Jia,† Matthew A Cooper,‡

and Michael J. Monteiro*,†

†Australian Institute for Bioengineering and Nanotechnology and ‡Institute for Molecular Biosciences, The University of Queensland,Brisbane QLD 4072, Australia

*S Supporting Information

ABSTRACT: Triggered-release of encapsulated therapeutics from nanoparticleswithout remote or environmental triggers was demonstrated in this work.Disassembly of the polymer nanoparticles to unimers at precise times allowed thecontrolled release of oligo DNA. The polymers used in this study consisted of ahydrophilic block for stabilization and second thermoresponsive block for self-assembly and disassembly. At temperatures below the second block’s LCST (i.e.,below 37 °C for in vitro assays), the diblock copolymer was fully water-soluble, andwhen heated to 37 °C, the polymer self-assembled into a narrow size distribution ofnanoparticles with an average diameter of approximately 25 nm. The thermores-ponsive nature of the second block could be manipulated in situ by the self-catalyzeddegradation of cationic 2-(dimethylamino)ethyl acrylate (DMAEA) units tonegatively charged acrylic acid groups and when the amount of acid groups wassufficiently high to increase the LCST of the second block above 37 °C. The disassembly of the nanoparticles could be controlledfrom 10 to 70 h. The use of these nanoparticles as a combined therapy, in which one or more agents can be released in apredetermined way, has the potential to improve the personal point of care treatment of patients.

■ INTRODUCTIONTriggered-release of encapsulated materials from nanoparticleshas attracted considerable attention for the on-demand releaseof compounds.1 The potential applications range from drugdelivery, to fragrance release, to self-healing materials.2

Degradation or disassembly of nanoparticles can be activatedeither remotely through external light,3 electric or magneticsources,4 or through environmental triggers such as in vivobiological changes (e.g., pH changes5 or localized enzymeactivity6). While these triggers represent elegant methods forselective release, there are still many applications whereremotely activated triggers cannot be used. Environmentaltriggers (such as pH, enzymatic degradation, temperature) haveother issues due to their variability within cell lines and withinthe same tissue.7 In such cases, new nontriggered releasedelivery materials are required that would act in an environ-ment independent manner. We believe that nanoparticles,which release their payload at specific times in the absence ofan external trigger, would be of great interest for the controlledrelease of small molecules and biological therapeutic agents.Therefore, nanoparticles must be designed to encapsulate andrelease the therapeutic agent on-demand to impart a rapideffect. These nanoparticles after release should be nontoxic,allowing the application of multiple doses for high effectivetherapeutic effects. Thermoresponsive polymer (e.g., poly(N-isopropylacrylamide); PNIPAM) nanoparticles have been usedfor such a purpose,8 in which polymeric micelles degrade tounimers through an acid or base hydrolysis process. However,even a small change in pH (from 7.5 to 7.2) could slow thedegradation rate by a factor of 2.9 If such polymer nanoparticles

were used in vivo, the variability of pH within cells and at, forexample, tumors will cause the noncontrolled release of thetherapeutic. To overcome this significant hurdle, we willincorporate a self-catalyzed polymer (poly(2-(dimethylamino)-ethyl acrylate); PDMAEA) into the second hydrophobic blockwith PNIPAM. The degradation rate in water of PDMAEA topoly(acrylic acid) is independent of the physiological pHranging from 5.5 to 10.1,10 making such timed-release micellesideal for many biological applications where precise release ofthe payload is required within any physiological environment.In this work, we synthesized thermoresponsive (PNIPAM)

and cationic (PDMAEA) diblock copolymers that are water-soluble below their lower critical solution temperature (LCST,< 37 °C) and, when heated above its LCST to 37 °C, self-assemble to form small nanoparticles of approximately 20 nm.Through a self-catalyzed hydrolysis mechanism,10 the polymernanoparticles rapidly disassemble after desired times tobiologically nontoxic negatively charged diblock unimers (seeScheme 1A). The disassembly mechanism occurs when there issufficient degradation of the cationic side groups, whichprovides the mechanism to increase the LCST of the polymerabove 37 °C. We designed the polymer nanoparticles todisassemble and thus release their payload at a desired timeindependent of the local microenvironment. The disassemblyprofile for nanoparticles observed in this work, to ourknowledge, has not been previously reported and represents a

Received: November 6, 2012Revised: December 16, 2012Published: January 8, 2013

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significant step toward controlled release in the absence of anexternal trigger. Such nanoparticles also have the potential to beused in combination therapy to deliver therapeutic agents thatcan be released at desired times in one dose depending uponthe required treatment.

■ METHODS SECTIONMaterials. Dioxane (Aldrich, 99%), carbon disulfide (Aldrich,

99%), 1-butanethiol (Aldrich, 99%), methyl bromopropionate(Aldrich, 98%), dimethyl sulfoxide (DMSO, Aldrich >99.9%), N,N-dimethylformamide (DMF: Labscan, AR grade), dichloromethane(DCM: Labscan, AR grade), N-(t-BOC-aminopropyl)methacrylamide(Polysciences, 100%), trifluoroacetic acid (TFA: Merck, AR grade),triethylamine (TEA: Fluka, 98%), tri(2-carboxyethyl) phosphinehydrochloride solution (TCEP: Aldrich, 98%), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl: Al-drich, premium), N-hydroxysuccinimide (NHS: Aldrich, 98%),hexylamine (Aldrich, 99%), and folic acid (Aldrich, ≥97%) wereused as received. Styrene (STY, Aldrich, 99%), dimethylacrylamide(DMA, Aldrich, 99%), 2-(dimethylamino) ethyl acrylate (DMAEA,Sigma-Aldrich, 98%), and butyl acrylate (BA, Aldrich, 99%) werepassed through a column of basic alumina (activity I) to removeinhibitor. N-Isopropylacrylamide (NIPAM, Aldrich, 97%) wasrecrystallized from hexane. Azobisisobutyronitrile (AIBN) was alsorecrystallized twice from methanol prior to use. 9−27 oligo DNA wassynthesized by Invitrogen, 9−27F+R−MW = 14998 (23bp), Sense: 5′-GTCAGAAATAGAAACTGGTCATC-3′ Antisense: 5′-GATGAC-CAGTTTCTATTTCTGAC3′. Milli-Q water (18.2 MΩ cm−1) wasgenerated using a Millipore Milli-Q academic water purificationsystem. All other chemicals and solvents used were of at least analyticalgrade and used as received.Instruments. 1H, 1D DOSY, and 2D DOSY NMR. All NMR spectra

were recorded on Bruker DRX 500 MHz using external locks (CDCl3or D2O or DMSO-d6) and referenced to the residual nondeuteratedsolvent (CHCl3 or H2O or DMSO).Size Exclusion Chromatography (SEC) and Triple Detection−Size

Exclusion Chromatography (TD-SEC). Analysis of the molecular

weight distributions of the polymers were determined using a PolymerLaboratories GPC50 Plus equipped with differential refractive indexdetector. Absolute molecular weights of polymers were determinedusing a Polymer Laboratories GPC50 Plus equipped with dual anglelaser light scattering detector, viscometer, and differential refractiveindex detector. HPLC grade N,N-dimethylacetamide (DMAc,containing 0.03 wt % LiCl) was used as the eluent at a flow rate of1.0 mL/min. Separations were achieved using two PLGel Mixed B (7.8× 300 mm) SEC columns connected in series and held at a constanttemperature of 50 °C. The triple detection system was calibrated usinga 2 mg/mL PSTY standard (Polymer Laboratories: Mwt = 110 K, dn/dc = 0.16 mL/g, and IV = 0.5809). Samples of known concentrationwere freshly prepared in DMAc + 0.03 wt % LiCl and passed through a0.45 μm PTFE syringe filter prior to injection. The absolute molecularweights and dn/dc values were determined using Polymer LaboratoriesMulti Cirrus software based on the quantitative mass recoverytechnique.

Dynamic Light Scattering (DLS). Dynamic light scatteringmeasurements were performed using a Malvern Zetasizer Nano Seriesrunning DTS software and operating a 4 mW He−Ne laser at 633 nm.Analysis was performed at an angle of 173° and a constanttemperature of 25 °C. The sample refractive index (RI) was set at1.59 for polystyrene. The dispersant viscosity and RI were set to 0.89Ns·m−2 and 1.33, respectively. The number-average hydrodynamicparticle size and polydispersity index are reported. The polydispersityindex (PDI) was used to describe the width of the particle sizedistribution. It was calculated from a Cumulants analysis of the DLSmeasured intensity autocorrelation function and is related to thestandard deviation of the hypothetical Gaussian distribution (i.e.,PDIPSD = σ2/ZD

2, where σ is the standard deviation and ZD is the Zaverage mean size).

Syntheses and Characterization. Synthesis of the ChainTransfer Agent (CTA), Methyl 2-(Butylthiocarbonothioylthio)-propanoate (MCEBTTC). The synthesized MCEBTTC was carriedout according to the literature procedure.23 Carbondisulfide (3.1 mL,0.051 mol) in dichloromethane (50 mL) was added dropwise to astirred solution of 1-butanethiol (5 mL, 0.047 mol) and triethylamine(7.2 mL, 0.051 mol) in dichloromethane (25 mL) over 30 min at 0 °C

Scheme 1. (A) Method of Nanoparticle Formation and Degradation of DMAEA to Acrylic Acid (AA) to Trigger UnimerFormation; (B) Synthetic Methodology for Thermoresponsive/Degradable Polymers

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under an argon atmosphere. The solution gradually turned yellowduring the addition. After complete addition, the solution was stirredat room temperature for 30 min. Methyl bromopropionate (5.7 mL,0.051 mol) in dichloromethane (25 mL) was then added dropwiseover 30 min and the solution was stirred for 2 h. The dichloromethanewas removed under nitrogen and the residue was dissolved in diethylether. The solution was then washed with cold 10% HCl solution (3 ×50 mL) and Milli-Q water (3 × 50 mL) and dried over anhydrousMgSO4. The ether was removed under vacuum, and the residualyellow oil was purified by column chromatography (19:1 petroleumether/ethyl acetate on silica, second band; yield = 76%). 1H NMR(CDCl3) δ 0.90 (t, J = 7.5 Hz, 3H, CH3), 1.40 (m, J = 7.5 Hz, 2H,CH2), 1.57 (d, J = 7.5 Hz, 3H, CH3), 1.66 (q, J = 7.5 Hz, 2H, CH2),3.34 (t, J = 7.5 Hz, 2H, CH2), 3.73 (s, 3H, CH3), 4.80 (q, J = 7.5 Hz,1H, CH).Synthesis of Poly(N,N-diethylacrylamide) Macro-Chain Transfer

Agent (PDMA macro-CTA). DMA (10.40 mL, 0.10 mol), MCEBTTC(25.42 mg, 1.01 × 10−3 mol), and AIBN (14.10 mg, 8.57 × 10−5 mol)were dissolved in DMSO in a 50 mL dry Schlenk flask equipped with amagnetic stirrer bar. The mixture was deoxygenated by purging withargon for 30 min and then heated to 60 °C for 2 h. The reaction wasstopped by cooling to 0 °C in an ice bath and exposed to the air. Thesolution was then diluted with dichloromethane (500 mL) and washedwith brine (3 × 100 mL). The DCM was then dried over anhydrousMgSO4, filtered, and reduced in volume by rotary evaporation. Thepolymer was recovered by precipitation into a large excess of diethylether (1 L) and isolated by filtration. The polymer was redissolved inacetone and precipitated in diethyl ether. The redissolving andprecipitation process was repeated two times. The polymer was filteredand then dried under high vacuum for 24 h at room temperature togive a yellow powder product (yield =71%). Mn = 8200, PDI = 1.14(SEC-RI calibrated using PSTY standards in DMAc solutioncontaining 0.03 wt % of LiCl), Mn = 10000 (SEC-triple detection,dn/dc = 0.081); Mn = 9769 (1H NMR). 1H NMR (500 MHz, CDCl3):δ 0.87 (CH3CH2CH2-), 1.09 (CH3-(CH-COO)-), 2.84−3.05((CH3)2-N-), 3.29 (-CH2-S-(CS)-S-), 3.60 (CH3O-(CO)-),5.14 (-(CS)-S-CH-).Synthesis of Block Copolymers of NIPAM and DMAEA from

PDMA Macro-CTA (A). NIPAM (1.00 g, 8.85 × 10−3 mol), DMAEA(0.34 mL, 2.21 × 10−3 mol), PDMA macro-CTA (725.66 mg, 8.85 ×10−5 mol) and AIBN (1.45 mg, 8.85 × 10−6 mol) were dissolved in 20mL of dioxane in a 50 mL dry Schlenk flask equipped with a magneticstirrer bar. The mixture was deoxygenated by purging with argon for30 min and then heated to 60 °C for 7 h under an argon. The reactionwas stopped by cooling to 0 °C in an ice bath and exposed to air. Thesolution was precipitated in diethyl ether (500 mL) and filtered. Thepolymer was redissolved in acetone and precipitated in diethyl ether.The redissolving and precipitating process were repeated twice. Theyellow powder product was dried under high vacuum at roomtemperature for 48 h (yield = 78%)Synthesis of Block Copolymers of NIPAM, DMAEA, and BA from

PDMA Macro-CTA (B1, B2). For a polymerization with 5.04% of BA inthe second block, NIPAM (1.00 g, 8.85 × 10−3 mol), DMAEA (0.34mL, 2.21 × 10−3 mol), BA (0.083 mL, 5.75 × 10−4 mol), PDMAmacro-CTA (725.66 mg, 8.85 × 10−5 mol), and AIBN (1.45 mg, 8.85× 10−6 mol) were dissolved in 20 mL of dioxane in a 50 mL drySchlenk flask equipped with a magnetic stirrer bar. The mixture wasdeoxygenated by purging with argon for 30 min and then heated to 60°C for 8 h under argon. The reaction was stopped by cooling to 0 °Cin an ice bath and exposed to air. The solution was precipitated indiethyl ether (500 mL) and filtered. The polymer was redissolved inacetone and precipitated in diethyl ether. Redissolving andprecipitating were repeated twice. The yellow powder product wasdried under high vacuum at room temperature for 48 h (yield = 75%).For a polymerization with 9.38% of BA in the second block, the sameprocedure was performed as above but with double the amount of BA(0.18 mL, 1.24 × 10−3 mol; yield = 72%).Synthesis of Block Copolymers of NIPAM, DMAEA, and STY from

PDMA Macro-CTA (C1, C2). For a polymerization with 4.50% of STYin the second block, NIPAM (1.00 g, 8.85 × 10−3 mol), DMAEA (0.34

mL, 2.21 × 10−3 mol), STY (0.067 mL, 5.75 × 10−4 mol), PDMAmacro-CTA (725.66 mg, 8.85 × 10−5 mol), and AIBN (1.45 mg, 8.85× 10−6 mol) were dissolved in 20 mL of dioxane in a 50 mL drySchlenk flask equipped with a magnetic stirrer bar. The mixture wasdeoxygenated by purging with argon for 30 min and then heated to 60°C for 45 h under argon. The reaction was stopped by cooling to 0 °Cin an ice bath and exposed to air. The solution was precipitated indiethyl ether (500 mL) and filtered. The polymer was redissolved inacetone and precipitated in diethyl ether. Redissolving andprecipitating were repeated twice. The yellow powder product wasdried under high vacuum at room temperature for 48 h (yield = 76%).For a polymerization with 19.11% of STY in the second block, thesame procedure was performed as above, but with double the amountof STY (0.14 mL, 1.24 × 10−3 mol) and for a reaction time of 60 h(yield = 78%).

1H, 1D DOSY, and 2D DOSY NMR. After samples were welldissolved in CDCl3 or D2O or DMSO-d6, sample solutions were thentransferred to NMR tubes. With samples in CDCl3, the spectrometerwas set at 25 °C for determination of polymer structure. With samplesin D2O, the spectrometer was set at different temperatures fordetermination of the polymer structure before and after degradation.With samples in DMSO-d6, a DOSY experiment was run at 25 °C toacquire spectra to suppress any small molecule or solvent signals byincreasing the pulse gradient and increasing d (p30) from 1 to 3 ms.

Lower Critical Solution Temperature (LCST) of the BlockCopolymer, as Determined by DLS. Polymer samples were weighedin vials and dissolved in cold Milli-Q water at the concentration 10mg/mL. These solutions were immediately kept in an ice bath, andthen filtered directly into DLS curvets using 0.45 μm cellulose syringefilter. For measurement of the LCST, the polymer solutions werecooled to 5 °C by DLS machine, and the measurements were carriedout by slowly increasing the temperature of DLS machine from 5 to 60°C using SOP software.

Disassembly Kinetics of Block Copolymer Nanoparticles at 37 °Cby DLS. The number-average particle diameter was measured for eachsample to determine the disassembly time of the nanoparticles.Polymer samples were weighed in vials and dissolved in cold Milli-Qwater at the concentration 5 mg/mL. These solutions wereimmediately kept in ice bath, and then filtered directly into DLScurvets using 0.45 μm cellulose syringe filter. The samples were kept at37 °C water bath and the particle size at different time intervals wasmeasured. The particles size and polydispersity index (PDI) werecalculated based on five measurements. With polymer sample coded A,the sample was heated and kept at 45 °C for measurement.

Lower Critical Solution Temperature (LCST) of the BlockCopolymer after the Polymers Particles Fully Degraded by DLS.Polymers B1, B2, C1, and C2 were weighed in vials and dissolved incold Milli-Q water at the concentration 10 mg/mL. These solutionswere then cooled in an ice bath and filtered directly into DLS curvetsusing 0.45 μm cellulose syringe filter. These polymer solutions B1, B2,C1, and C2 were then kept in water bath at 37 °C for 27, 73, 26, and53 h, respectively, before being measured polymer particle sizes byDLS. The measurements of LCST were carried out by slowlyincreasing the temperature of DLS machine from 5 to 70 °C usingSOP software.

Studying of Binding Ability of Oligo DNA 9−27 andThermoresponsive Block Copolymers at Different Nitrogen-to-Phosphorus (N/P) Ratios. A total of 1.0 μg of oligo DNA 9−27(0.5 μg/μL) was complexed with each block copolymers at differentnitrogen-to-phosphorus (N/P) ratios 0.5, 1, 2, 5, and 10 in a totalamount volume of 100 μL of Milli-Q water. After shaking with avorterxer, the mixtures were allowed to complex without stirring for 30min in an ice bath. The polymers/oligo DNA complexes were thenkept at 37 °C in a water bath for another 15 min and run at the sametime on one gel. In preparation for the gel, the complexes (20 μL)were quickly mixed with 5 μL of DNA loading dye, and immediatelyloaded into a 2% agarose gel containing TAE buffer and ethidiumbromide. The gels were immersed in 1× TAE buffer (heated to 50°C). Oligo DNA 9−27 (1.0 μg) without polymer as a control. The gelswere set to run in this preheat buffer for 12 min at 80 V before being

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visualized using a UV transilluminator. The other complexes atdifferent nitrogen-to-phosphorus (N/P) ratios 0.5, 1, 2, 5, and 10 in atotal amount volume of 100 μL of Milli-Q water were prepared withthe same protocol and polymer particle sizes measured by DLS.Studying of Binding and Release of Oligo DNA 9−27/

Thermoresponsive Block Copolymer Complexes. A total of 1.0 μgof oligo DNA 9−27 (0.5 μg/μL) was complexed with polymer at anitrogen-to-phosphorus (N/P) ratio of 10 in a total amount volume of100 μL of Milli-Q water. After shaking with a vorterxer, the mixtureswere allowed to complex without stirring for 30 min in an ice bath.The polymers/oligo DNA complexes were kept at 37 °C in a waterbath for different times and run at the same time on one gel. Thecomplexes (20 μL) were quickly mixed with 5 μL of DNA loading dyeand immediately loaded into a 2% agarose gel containing TAE bufferand ethidium bromide (Biorad). The gels were immersed in 1× TAEbuffer (preheated to 50 °C). Oligo DNA 9−27 (1.0 μg) withoutpolymer as a control. The gels were run in 1× TAE buffer (heated to50 °C) for 12 min at 80 V before being visualized using a UVtransilluminator.Synthesis of Folic Acid Conjugated to a Thermoresponsive

Polymer (H); See Scheme S2. Synthesis of Random CopolymerP(NIPAM-co-BA) (D) by RAFT Polymerization. NIPAM (2.00 g, 1.77× 10−2 mol), BA (0.38 mL, 2.65 × 10−3 mol), MCEBTTC (44.68 mg,1.77 × 10−4 mol), and AIBN (2.90 mg, 1.77 × 10−5 mol) weredissolved in 12 mL of dioxane in a 50 mL dry Schlenk flask equippedwith a magnetic stirrer bar. The mixture was deoxygenated by purgingwith Argon for 30 min and then heated to 60 °C for 17.5 h underargon. The reaction was stopped by cooling to 0 °C in an ice bath andexposed to air. The solution was precipitated in diethyl ether (500mL) and filtered. The polymer was redissolved in acetone andprecipitated in diethyl ether. The redissolving and precipitating processwere repeated twice. The yellow powder product was dried under highvacuum at room temperature for 24 h (yield = 72%).Synthesis of Block Copolymer RAFT-PDMA-b-P(NIPAM-co-BA) (E)

from P(NIPAM-co-BA) Macro-CTA. DMA (0.63 mL, 6.09 × 10−3

mol), P(NIPAM-co-BA) macro-CTA (D) (0.80 g, 6.09 × 10−5 mol),and AIBN (1.00 mg, 6.09 × 10−6 mol) were dissolved in 8 mL ofDMSO in a dry Schlenk tube equipped with a magnetic stirrer bar.The mixture was deoxygenated by purging with argon for 30 min andthen heated to 60 °C for 16.5 h under argon. The reaction wasstopped by cooling to 0 °C in an ice bath and exposed to air. Thesolution was then diluted with dichloromethane (500 mL) and washedwith brine (3 × 100 mL). The dichloromethane was then dried overanhydrous MgSO4, filtered, and reduced in volume by rotaryevaporation. The polymer was recovered by precipitation in diethylether (500 mL) and filtered. The polymer was redissolved in acetoneand precipitated in diethyl ether. The redissolving and precipitatingprocess were repeated twice. The yellow powder product was driedunder high vacuum at room temperature for 48 h (yield = 72%).Synthesis of Boc-Protected Amine Functional Copolymer (F) by

Michael Addition of Copolymer (E) with N-(t-BOC-aminopropyl)Methacrylamide. Copolymer E (0.5 g, 2.20 × 10−5 mol) and TCEP(63.15 mg, 2.20 × 10−4 mol) were dissolved in 1.5 mL of DMF in adry Schlenk tube equipped with a magnetic stirrer bar. The mixturewas deoxygenated by purging with argon for 30 min. At the same time,TEA (31.3 μL, 2.20 × 10−4 mol), hexamine (31.4 μL, 2.20 × 10−4

mol), and 1.5 mL of DMF were added in another dry Schlenk tubeand purged by argon for 30 min. The mixture of TEA, hexamine, andDMF in the second tube was then transferred to the first tube by adeoxygenated syringe needle. The combined mixture was then keptstirring at room temperature under an argon flow overnight and thendialyzed against acetone for 1 day. The dialyzed solution wasconcentrated to 1 mL and precipitated three times in diethyl ether.The white powder product was then dried under high vacuum at roomtemperature for 24 h (yield = 85%).Synthesis of Amine Functional Copolymer (G) by Deprotection of

Copolymer (F) with TFA. Copolymer F (0.35 g, 1.54 × 10−5 mol) andTFA (0.82 mL, 1.08 × 10−2 mol) were dissolved in 2 mL of DCM in adry Schlenk tube equipped with a magnetic stirrer bar. The mixturewas then stirred at room temperature for overnight. The DCM in the

solution was then removed blown by under a nitrogen flow for 1 h.The viscous solution was redissolved in 1.5 mL of DCM. The residueTFA in the solution was neutralized by 1 mL of TEA. The solutionwas then passed through a 0.45 μm PTFE syringe filter, concentratedto 1 mL by nitrogen flow. The concentrated solution was thenprecipitated in diethyl ether three times. The white powder productwas then dried under high vacuum at room temperature for 24 h (yield= 80%).

Synthesis of Folic Acid Functional Copolymer H. Copolymer G(0.20 g, 8.81 × 10−6 mol), folate (11.66 mg, 2.64 × 10−5 mol),EDC·HCl (10.13 mg, 5.29 × 10−5 mol), and NHS (3.04 mg, 2.64 ×10−5 mol) were dissolved in 3.3 mL of DMSO/H2O (10/1 V/V) in adry Schlenk tube equipped with a magnetic stirrer bar. The mixturewas then stirred at room temperature for 24 h and dialyzed againstcold Milli-Q water for 24 h. The yellow solid, folate, was removed byfiltration. The filtrate was frozen and freeze-dried for 2.5 days to givethe pale yellow fluffy powder (yield = 60%).

Cell Uptaken Assay. The osteosarcoma U-2OS cells were culturedin 24-well plate (1 × 105/well) in completed DMEM medium. Folicacid functionalized copolymer H was mixed with copolymer B1, B2,C1, and C2, respectively, and dissolved in cold (ice-bath) nuclease-freewater to give a copolymer solution with 15 mol % of folic acid(Scheme S3). A model siRNA, a 21nt oligo DNA conjugated with Cy3(DNA-Cy3), was diluted and added to the polymers. The N/P ratio ofpolymer to siRNA was 50/1. The mixtures were incubated in an ice-bath for 30 min to allow the complexation between positive chargedpolymer and negative charged siRNA, followed by incubation at 37 °C(above the LCST for all the copolymers) for 10 min to allow theformation of polymer/siRNA nanoparticles. They were then added tothe cells to reach the final concentration of the siRNA 50 nM for celluptake. The cells were then incubated for 10 h before washing withPBS buffer and fixation with 4% paraffin formaldehyde. The cell nucleiwere stained with Hoechst 33341 and cell uptake was viewed underfluorescent microscope.

■ RESULTS AND DISCUSSION

The diblock copolymer was designed (see Scheme 1B) to havea first block that provides steric stabilization to the nano-particles in water and a second block to be boththermoresponsive and able to undergo a self-catalyzedhydrolysis reaction in water. First, the stabilizing hydrophilicpoly(dimethyl acrylamide),11 PDMA, was prepared using thereversible addition−fragmentation chain transfer (RAFT)polymerization technique with a number-average molecularweight (Mn) of 8200 and polydispersity index (PDI) of 1.14.This polymer was then blocked with PNIPAM and PDMAEAto form the random second block. PNIPAM has an LCST closeto 32 °C;12,13 at temperatures below its LCST, the PNIPAM isfully water-soluble, but when heated above its LCST, itbecomes water insoluble.14 We have studied the self-catalyticdegradation behavior of the cationic PDMAEA, which trans-forms into the negatively charged poly(acrylic acid) (PAA) inwater over time (see Scheme S1 in Supporting Information).10

This polymer was shown to degrade at the same rate regardlessof its molecular weight or pH (ranging from pH 5.5 to 10.1)and could bind and release negatively charged oligo DNA (amodel for siRNA).10,15 The significant advantages of thispolymer include its high binding to negative biomolecules, hightransfection (or uptake) into cells, and full release of negativelycharged biomolecules through ionic repulsion after degrada-tion.15 The resulting nontoxic PAA eliminates the accumulationof highly toxic cationic polymers, especially when repeat dosesare required. The polymer, P(DMA96-b-(NIPAM87-co-DMAEA25)), with an Mn of 34500 and PDI of 1.23, wasshown to have an LCST starting at 39 °C (see Table 1 andFigure S19), and when heated to 45 °C in water, it formed

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nanoparticles of 25 nm in diameter with a very narrow sizedistribution (0.095, where values <0.1 represents distributionsthat are narrow). These polymer particles changed from 25 to19 nm in water over the first 5 h due to the intrabinding ofnegatively charged side groups with the positively charged ones(see Figure S20 in Supporting Information). After 5 h, therewas a sharp decrease in size over 1 h due to the disassembly todiblock unimers. Unfortunately, this polymer had an LCST wellabove 37 °C and would not be suitable as a drug delivery devicefor in vivo systems. We therefore modified the polymer toreduce the LCST well below 37 °C.One method for decreasing the LCST of polymers involves

incorporating a hydrophobic monomer. The greater the weightfraction of a hydrophobic monomer in the copolymer the lowerthe LCST,16 conversely, the greater the amount of ahydrophilic monomer in the copolymer the higher theLCST.17 In this work, we incorporated either styrene (STY)or butyl acrylate (BA) with increasing mol % into the diblockcopolymer (see Table S1 in Supporting Information for Mn andPDI values) to lower the LCST to below 37 °C. Polymer B1(5% mol fraction of BA in the second block, P(DMA96-b-(NIPAM88-co-DMAEA25-co-BA6))) gave an LCST between 25and 29 °C, and when the soluble polymer in water was heatedto 37 °C, nanoparticles of approximately 27 nm formed with anarrow size distribution (Table 1). Increasing the weightfraction of BA in the second block to 9.4 mol % (B2;P(DMA96-b-(NIPAM91-co-DMAEA25-co-BA12))) resulted in amarked decrease in the LCST, ranging from 17 to 21 °C. Whenthis polymer was heated in water to 37 °C, we observed anarrow particle size distribution with an average size close to 25nm. The incorporation of 4.5 mol % STY (C1; P(DMA96-b-(NIPAM84-co-DMAEA22-co-STY5))) resulted in the lowering ofthe LCST similar to that of 5 mol % BA (B1), in which theLCST ranged between 26 and 30 °C. This polymer produced anarrow size distribution of nanoparticles with an average sizeclose to 27 nm when heated to 37 °C, which was again similarto the size produced from polymer B1. Increasing the amountof STY to 19 mol % (C2, P(DMA96-b-(NIPAM40-co-DMAEA15-co-BA13))) decreased the LCST down to a range between 15and 19 °C, which was only 2° lower than for B2. The size ofthese polymer nanoparticles at 37 °C was 20 nm. The

difference between B2 and C2 was due to the difference in themolecular weights and, in particular, the differences betweenthe number of the comonomer units (see Table S1). TheSupporting Information provides all the LCST data for the fivepolymers synthesized in this work.The four polymers, B1 to C2, were then solubilized in water

and heated to 37 °C. The resulting polymer nanoparticles werestored at this temperature and the change in size measured overtime by dynamic light scattering (DLS). Figure 1 showed the

change in size for the four polymers over 100 h. Nanoparticlesfrom polymer B1 decreased in size from 27 to 18 nm after 8 h,leveling off at this size until 21 h, after which time there was arapid decrease in size to that consistent of diblock unimers (∼5nm) after 26.5 h. The pH of the polymer solution wasmeasured to be 7.6 before and after full degradation of thePDMAEA side groups. This pH is below the pKa for PDMAEA(8.3 for the monomer)18 and above the pKa of PAA (4.3),suggesting that both cationic and anionic species will be ionizedin water. These data are consistent with a three stagedegradation process leading to disassembly: first, the smallnumber of initially degraded side chains (now negativelycharged) will bind with the positively charged side groups todecrease the size of the nanoparticles; second, a plateau regionwhere no change in size is observed; and third, thenanoparticles disassemble to unimers over ∼5.5 h. A similarthree stage size change was also observed for the other threepolymers. Polymer B2 showed that the plateau time could beextended until 66 h, after which disassembly to unimersoccurred over a 5.8 h period. When 4.5% mol fraction of STYwas incorporated in the copolymer (i.e., C1), a similar threestage behavior similar to that of B1 was observed. Disassemblystarted after 17 h, and the polymer fully disassembled intounimers after a further 5.4 h. With a similar trend, polymer C2(19 mol % STY in the second block) started to disassembleafter 47 h, and was fully disassembled to unimers after a further6.8 h. The data in Figure 1 clearly showed that the disassemblyprocess for all polymers could be controlled, suggesting that wecould control the time at which disassembly occurs by simplymanipulating the hydrophobic monomer content in the secondblock. In addition, the time for disassembly for all polymers wasapproximately 6 h, suggesting the same release profile of thepayload from the nanoparticles regardless of the time at thestart of disassembly.

Table 1. Lower Critical Solution Temperature (LCST),Hydrodynamic Diameter (Dh), Polydispersities (PDI), andDegradation Times for Thermoresponsive BlockCopolymers Determined by Dynamic Light Scattering(DLS)

polymerLCST(°C)a Dh (nm; PDI)

btstart

(tdegrade)d

A: P(DMA96-b-(NIPAM87-co-DMAEA25))

39.0−41.0

25.12 (0.095)c 5.5 (1)c

B1: P(DMA96-b-(NIPAM88-co-DMAEA25-co-BA6))

25.0−29.0

27.02 (0.028) 21 (5.5)

B2: P(DMA96-b-(NIPAM91-co-DMAEA25-co-BA12))

17.0−21.0

25.31 (0.047) 66 (5.75)

C1: P(DMA96-b-(NIPAM84-co-DMAEA22-co-STY5))

26.0−30.0

27.46 (0.024) 17 (5.44)

C2: P(DMA96-b-(NIPAM40-co-DMAEA15-co-STY13))

15.0−19.0

19.99 (0.063) 47 (6.8)

aLCST determined by DLS (10 mg/mL). bHydrodynamic diameter(Dh) determined by DLS (5 mg/mL) at 37 °C. cDh determined byDLS (5 mg/mL) at 45 °C. dDisassembly time at 37 °C: tstart = timewhen the size starts to decrease; tdegrade = time from tstart to formationof unimers.

Figure 1. Degradation kinetics profiles for B1, B2, C1, and C2. Thedata were averaged from five measurements by DLS at polymersolution concentration of 5 mg/mL at 37 °C.

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The sharp disassembly profile observed in Figure 1 supportsa change in the thermoresponsive nature of the polymers as theDMAEA side groups undergo a self-catalyzed hydrolysis. TheLCST of the four polymers (B1 to C2) measuredapproximately 30 min after full disassembly to unimers (seeTable 2 and Supporting Information) showed that the LCST of

polymers B1 to C2 were above 37 °C (ranging between 39 and41 °C for B1 and C1, and 37−39 °C for B2 and C2). The datademonstrated that with the increase in the degradation ofDMAEA side groups to carboxylic acids, the diblockcopolymer’s LCST increased, and when the LCST becamegreater than 37 °C, disassembly to water-soluble unimersoccurred. This mechanism resulted in a sharp disassemblytransition from spherical nanoparticles to water-soluble diblockcopolymer unimers. When the polymers were allowed to bestored for one month at 37 °C, no LCST was observed even attemperatures as high as 70 °C.Polymer/DNA Binding, Release, and Cell Uptake. The

efficient delivery of siRNA holds great promise in the cure forcancers and infectious diseases.19 For siRNA-based deliverycarriers to reach their full potential, they must be protectedfrom enzymatic degradation, efficiently be taken up by cells,and the siRNA released with controlled and reproduciblepharmacokinetics.20 The main problems with positively chargedpolymers for siRNA delivery are (i) the difficulty to fully releasethe siRNA21 and (ii) their high toxicity, which becomes

problematic after repeat doses due to accumulation intissues.12,22 We previously showed that PDMAEA can readilybind to oligo DNA (a 9−27 oligo DNA that is a close analog tosiRNA) and once the side groups have degraded to negativelycharged carboxylic acid groups release all the negatively chargedoligo DNA.10,15 However, the homopolymer PDMAEA gave nocontrol over the release profile. In this current work, we will usethe well-proven model compound oligo DNA to determinewhether the release profiles of the oligo DNA from fourpolymers (B1 to C2) were the same as the disassembly profiles,as shown in Figure 1.The oligo DNA and polymer were mixed in an ice bath at an

NP ratio of 10 (i.e., the ratio of nitrogen on the polymer tophosphorus on the oligo DNA) and allowed to stand for 30min without stirring and then heated to 37 °C. This NP ratiowas chosen as it showed complete binding with the oligo DNA(see Figure S25 in Supporting Information). It was found thatthere was no change in the size of the nanoparticles (between20 to 30 nm in diameter) when measured at 37 °C (i.e., abovethe LCST of the polymers) in the absence or presence of oligoDNA, as shown in Table S2. To determine the ability of theoligo DNA to bind with the polymer nanoparticles, weperformed agarose gel retardation assays. The oligo DNAcannot enter the gel when bound or complexed to the polymernanoparticles. All four polymer nanoparticles strongly boundwith oligo DNA at an NP (nitrogen to phosphorus) ratio of 10after incubation for 1 h (Figure 2). This assay also providedinsight into the leakage of oligo DNA over time and the releaseof oligo DNA over time. There was no evidence of leakage ofany oligo DNA from polymer B after 20 h, and release of alloligo DNA was observed after 26 h. This release profile wasconsistent with the disassembly profile shown in Figure 1.Disassembly occurred after 21 h and full disassembly after afurther 5.5 h, supporting the release of all oligo DNA after 26 h.Polymers B2, C1, and C2 also showed the same trends, withthe release time of all oligo DNA consistent with the fulldisassembly time of the polymer nanoparticles (see Table 1).Importantly, there was no leakage of oligo DNA untildisassembly commenced. These findings support the timedand controlled release of the oligo DNA by manipulating the

Table 2. Lower Critical Solution Temperature (LCST),Hydrodynamic Diameter (Dh), Polydispersities (PDI) forthe Block Copolymers after Being Fully Degraded (i.e., FullConversion of DMAEA to Acrylic Acid (AA))

polymer time (h)a LCST (°C)b Dh (nm)c

B1 27 39.0−41.0 16.67 ± 1.32B2 73 37.0−39.0 15.66 ± 1.42C1 26 39.0−41.0 15.39 ± 1.08C2 53 37.0−39.0 12.56 ± 0.15

aTime after which the LCST of the polymer was determined. bLCSTdetermined by DLS (10 mg/mL). cHydrodynamic diameter (Dh)determined by DLS (5 mg/mL) at a temperature just above the LCST.

Figure 2. Agarose gel assay for binding and release of oligo DNA 9−27 from the polymer/DNA complex nanoparticles at different times in Milli-Qwater at N/P ratio 10. Soluble copolymers in Milli-Q water were incubated with DNA (N/P ratio 10) at below their LCST for 30 min and thenheated and kept at 37 °C for agarose gel retardation assay over different times.

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LCST of the polymer above 37 °C as a function of the DMAEAdegradation to PAA.To test whether these timed-release nanoparticles could be

taken up by cells prior to degradation to unimers, we mixedCy3 oligo DNA with our polymers and added these complexesto osteosarcoma U2OS cells at 37 °C and were then incubatedfor 10 h. All polymers showed little or no uptake. Folic acid wasthen coupled to the end of a thermoresponsive polymer G(PDMA99-b-P(NIPAM97-co-BA13)) to form polymer H (Figure3A, and see Supporting Information) as a binding agent to

cancer cells. Polymer H (15 mol %) was coassembled with eachof the polymers B1−C2 and complexed with Cy3-DNA in onepot. This represents a simple but effective method to surfacefunctionalize such nanoparticles without changing the disasso-ciation properties of polymer B1 to C2. The uptake (Figure3B) of Cy3 oligo DNA with a representative polymers C2 andH was high (Figure 3B(i)), while C2 and H polymers only(Figure 3B (ii)) and Cy3 oligo DNA (Figure 3B(iii)) showedno uptake.

■ CONCLUSIONIn conclusion, we have demonstrated the use of designerdiblock copolymer nanoparticles to release, as a proof ofconcept, oligo DNA (a proven model compound for biologicaltherapeutics) on-demand using a timed disassembly process.The release mechanism does not require activation fromremote sources or biological or pH triggers. The polymers usedin this study consisted of a hydrophilic block for stabilizationand a second thermoresponsive block for self-assembly anddisassembly. At temperatures below the second block’s LCST(i.e., below 37 °C for in vitro assays), the diblock copolymerwas fully water-soluble, and when heated to 37 °C, the polymerself-assembled into a narrow size distribution of nanoparticleswith an average diameter of approximately 25 nm. Thethermoresponsive nature of the second block could be

manipulated in situ by the self-catalyzed degradation of cationicDMAEA units to negatively charged acrylic acid groups, andwhen the amount of acids groups was sufficiently high toincrease the LCST of the second block above 37 °C, thenanoparticles disassembled to unimers over approximately 5−6h. The disassembly time of 5−6 h was similar regardless of thetime when the nanoparticles started to disassemble, and thepolymers showed excellent binding to oligo DNA without anyleakage until full disassembly to unimers. These nakednanoparticles could only be taken up by osteosarcoma cellswhen coated with a transfection agent, folic acid. We used acoassembly method using the polymers B1−C2 and a folic acidfunctionalized thermoresponsive polymer to produce nano-particles with folic acid on the surface and without changing thedisassembly profiles of the B1−C2 polymers. The well-definedand controlled release profiles observed in this work represent asignificant step toward controlled release in the absence of anexternal trigger and, with our coassembly method, allows forother functional molecules to be decorated on the nanoparticlesurface.This new nanoparticle technology could enable sophisticated

combination therapies. For example, one or more therapeuticagents encapsulated in different timed-release nanoparticles, inwhich this particle mixture could be used in a one dosingregimen for tailored release of the agents when required. Theycould be programmed to release certain theraputics at the timeafter administration when optimal biodistribution of theparticles has occurred and when the agents will have maximalpharmacodynamic effect, and then slowly and consistently overtime. One key application we are exploring is in improvedmethods for delivery of antitumor agents, leading to anoptimized therapeutic index for cancer therapy.

■ ASSOCIATED CONTENT*S Supporting InformationSynthesis and characterizations of all polymers, including 1HNMR spectra, DLS, SEC traces, and data from biological assays.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: m.monteiro@uq.edu.au.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSM.J.M acknowledges financial support from the ARC DiscoveryGrant (DP120100973).

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Figure 3. (A) Synthesis of folic acid functionalized timed-releasenanoparticles. (B) Fluorescent microscopy photos of the osteosarcomaU2OS cells dosed with 50 nM Cy3 oligo DNA and copolymerpolyplexes in completed DMEM medium. N/P ratio (i.e polymer tosiRNA) was 50:1 in water at 37 °C. They were then added to the cellsand were incubated for 10 h before washing with PBS buffer andfixation with 4% paraffinformaldehyde. The cell nuclei were stainedwith Hoechst 33341 and cell uptake viewed under fluorescentmicroscope. Photos (i) copolymers C2 + H + Cy3-DNA, (ii) C2 +H, and (iii) Cy3-DNA.

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