ORIGINAL PAPER

Synthesis and characterization of thermosensitive, star-shapedpoly(ε-caprolactone)-block-Poly(N-isopropylacrylamide)with porphyrin-core for photodynamic therapy

Xiao-Hui Dai & Hua Jin & Si-Song Yuan & Jian-Ming Pan &

Xiao-Hong Wang & Yong-Sheng Yan & Dong-Ming Liu &

Lin Sun

Received: 22 December 2013 /Accepted: 6 March 2014 /Published online: 22 May 2014# Springer Science+Business Media Dordrecht 2014

Abstract Thermosensitive, amphiphilic, star-shaped poly (ε-caprolactone)-block-poly (N-isopropylacrylamide) withprophyrin-core (SPPCL-b-PNIPAAm) was synthesized byring-opening polymerization (ROP) and reversible addition-fragmentation chain transfer polymerization (RAFT) andcharacterized by NMR, IR, GPC in this study. The opticaltransmittance results showed that the lower critical solutiontemperatures (LCST) of the copolymer solutions were attrac-tively close to normal physiologic temperature at around37 °C. Due to its amphiphilic property, the obtained SPPCL-b-PNIPAAm had a low critical micelle concentration (CMC)in an aqueous solution and self-assembled into micelles inwater. Transmission electron microscopy (TEM) and dynamiclight scattering (DLS) analysis of micelle morphology re-vealed a well-defined spherical shape with a diameter ofaround 100 nm. Moreover, SPPCL-b-PNIPAAm micelles ob-tained high singlet oxygen quantum yields and indicated asensitivity to temperature, suggesting that the SPPCL-b-PNIPAAm was a stimulus-responsive photosensitizer whichhad a potential application in targeted photodynamic therapy.

Keywords Thermosensitive . RAFT . Singlet oxygen .

Star-shaped . PDT

Introduction

Photodynamic therapy (PDT) is a clinically approved methodfor the treatment of various diseases, including psoriasis,rickets, vitiligo, and many kinds of cancer [1–4], which util-ities the reaction of photosensitizers (PS), molecular oxygen,and light irradiation of a specific wavelength. Reactive oxy-gen species (ROS) can be produced and then lead to a local-ized damage in diseased tissue [5, 6]. PDT, as a localizedtreatment, is less invasive than other procedures such assurgery, chemotherapy, or radiotherapy versus other therapies,due to being preferentially accumulated in targeted tissue andsubsequently activated through selective illumination. How-ever, as it generally has large π-conjugation domains such as aporphyrin structure, PS are unsuitable for biomedical applica-tions because of aggregation in aqueous solution, phototoxic-ity to the skin, hydrophobicity, and low-selectivity [7]. Todevelop new photosensitizing agents with a high biocompat-ibility and efficacy, the PS should have no toxicity beforeirradiation with light of appropriate energy, whereas they havelong-term medical effects on target cells under light irradia-tion. In addition, biodegradation, chemical homogeneity, andtumor selectivity must also be considered.

Previous reports show that polymer-drug conjugates [8],liposomes [9], nanoparticles [10], and polymeric micelles [7,11] have each been considered as a simple but effective meansof realizing those properties mentioned above. Polymericmicelles combining hydrophilic and hydrophobic segmentspioneered by researchers [12] are especially appealing in thatthe hydrophobic micelle core creates a microenvironment forthe incorporation of lipophilic drugs, significantly enhancing

X.<H. Dai (*) :H. Jin : S.<S. Yuan : J.<M. Pan (*) :X.<H.Wang :Y.<S. YanDepartment of Chemical Engineering, School of Chemistry andChemical Technology, Jiangsu University, Zhenjiang 212013,People’s Republic of Chinae-mail: daixiaohui@ujs.edu.cne-mail: pjm@ujs.edu.cn

Y.<S. YanState Key Laboratory of Natural and Biomimetic Drugs,Peking University, Beijing 100191, People’s Republic of China

D.<M. LiuHospital Affiliated to Jiangsu University, Zhenjiang 212013,People’s Republic of China

L. SunCSR Qingdao Sifang Co. Ltd., Qingdao, Shandong 266111, China

J Polym Res (2014) 21:412DOI 10.1007/s10965-014-0412-9

the solubility of hydrophobic drugs, biocompatibility, andstability in vitro and in vivo [13, 14]. Moreover, polymericmicelles sensitive to the environment, especially temperature-responsive materials for biomedical applications, have re-ceived much attention [12]. Poly (N-isopropylacrylamide)(PNIPAAm) and related copolymers, the most widely usedin copolymers for the preparation of temperature-sensitivepolymers currently, exhibit a lower critical solution tempera-ture (LCST) in aqueous solution [15]. When the temperatureis below the LCST, the PNIPAAm is water soluble, while onthe contrary, above the LCST it becomes insoluble and pre-cipitates from the aqueous solution. More interestingly, theLCST value can be modified by changing the hydrophilic/hydrophobic balance of the copolymer [16]. To date, numer-ous copolymers contained in PNIPAAm have been developedto achieve thermo-sensitive temporal drug delivery control. Amajor problem, however, is how to adjust the LCST value forclinical applications. Fortunately, this issue might be tackledthrough inarching poly (ε-caprolactone) (PCL), a U.S. Foodand Drug Administration-(FDA) approved biomedical poly-mer. Herein, PCL is selected as the hydrophobic segment tocombine PNIPAAm segments and PS based on its nontoxicity,superior mechanical properties, and good biodegradable prop-erties [17]. It is should be noted that although a large amountof published data exist about thermosensitive polymeric drugdelivery, the study of thermosensitive star-shaped copolymerswith a porphyrin-cored is relatively limited and more work isgreatly needed to understand the particular characteristicsinvolving singlet oxygen quantum yield and relatedapplications.

In this study, we attached the hydrophobic inner core ofstar-shaped porphyrin-cored poly (ε-caprolactone) (SPPCL)to hydrophilic poly (N-isopropylacrylamide) (PNIPAAm)blocks for preparing a photosensitizer-centered amphiphilicstar block copolymer, which can form temperature-sensitiveand biodegradable core-shell nanoparticles. As shown in

Scheme 1, the SPPCL was conveniently prepared by ring-open polymerization (ROP) of ε-caprolactone with atetrahydroxyethyl-terminated porphyrin as an initiator. Thereversible addition-fragmentation chain transfer polymeriza-tion (RAFT) approach was adopted to use trithiocarbonate-terminated star-shaped porphyrin-cored poly (ε-caprolactone)(SPPCL) as a tetrafunctional macro-RAFT agent. By thosemeans, a series of star-shaped prophyrin-cored poly (ε-caprolactone)-block-poly (N-isopropylacrylamide) (SPPCL-b-PNIPAAm) copolymers was prepared and characterizedby means of 1H NMR, GPC, FT-IR, UV–Vis. In this study,the “n” of the abbreviations SPPCL-b-PNIPAAmn means therepeat units of PNIPAAm on each arm of the copolymer.Fluorescent spectroscopy and transmission electron microsco-py (TEM), as well as the critical micelle concentration (CMC),lower critical solution temperature (LCST), and morphologyprofiles of nanoparticles were investigated. Our results showthat we have exploited a stimuli-responsive polymer system,i.e., one that is temperature-responsive, presenting a potentialmethodology to treat cancer that combines chemotherapy/photodynamic therapy strategies in one delivery system.

Experimental

Materials

S t a n n o u s o c t o a t e ( S nOc t 2 ) a n d 5 , 1 0 , 1 5 , 2 0 -Tetraphenylporphyrin (TPP) were purchased from Aldrich,China, and used as received. N, N-dimethylformamide(DMF), dimethyl sulfoxide (DMSO), Toluene (C7H8), meth-ylene chloride (CH2Cl2), and n-hexane were obtained fromShanghai Chemical Reagent Co. (China) and distilled beforeuse. ε-Caprolactone (ε-CL) was purchased from Aldrich,China, and dis t i l led from CaH2 before use. N-i s op ropy l a c r y l am ide (N IPAAm) , DCC (N ,N ’ -

Scheme 1 Synthesis of SPPCL-b-PNIPAAm copolymers

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dicyc lohexy lca rbod i imide ) , and DMAP (N,N ’ -Dimethylpyridin-4-amine) were purchased from ShanghaiChemical Reagent Co. and used after recrystallization withn-hexane. All other reagents and solvents were of analyticalgrade and used as received without further purification.

The tetrahydroxyethyl-terminated porphyrin used in thestudy was synthesized according to our previous publication[18]:1HNMR (DMSOd6, ppm): 3.89 (t, 8H, -CH2-OH), 4.28 (t,8H, -O-CH2-CH2-), 5.03 (s, 4H, -OH), 7.38 (d, 8H, o-Ar-H),8.12 (d, 8H, m-Ar-H), and 8.87 (s, 8H, β-pyrrole-H). Also, thesynthesis of the benzylsulfanylthiocarbonylsufanylpropionicacid (BSPA) used in the study was performed as documentedpreviously [19]:1H NMR (DMSOd6, ppm): 2.68 (t, 2H, CH2-C=O), 3.54 (t, 2H, CH2-S), 4.68 (s, 2H, CH2-Ph), 7.29∼7.40(m, 5H, Ph), 12.5 (b, 1H, OH). IR (KBr): 1,710, 1,495, 1,451,1,242, 1,064 cm−1.

Methods

Fourier transform infrared (FT-IR) spectra were recorded on aPerkin-Elmer Paragon 1,000 spectrometer (USA) at frequen-cies ranging from 400 to 4,000 cm−1. Before the measure-ments, the samples were thoroughly mixed with KBr andpressed into pellet form.

The 1H NMR spectra of polymers were recorded using aVarian Mercury-400 spectrometer (Varian, USA) at 400 MHzby using CDCl3 as a solvent and TMS as an internal standard.

The number- and weight-average molecular weights (Mn

and Mw, respectively) of the polymers were determined by agel permeation chromatograph (GPC, Perkin-Elmer Series200) and a refractive index detector at 30 °C, with DMF asthe eluent (1.0 mL/min), and polystyrene as the calibrationstandard.

A drop of micelle suspension was placed on a copper gridwith carbon film for TEM investigation and stained by a 0.2%(w/v) solution of phosphotungstic acid before measurement.

Observation was performed at an acceleration voltage of200KV by JEM-2010 TEM.

Fluorescent spectra were recorded at room temperatureusing a Perkin-Elmer LS 50B luminescence spectrometer.

UV–Vis spectra were recorded at room temperature using aSpectrumlab54 UV-visible spectrophotometer. Pyrene waschosen as the hydrophobic fluorescent probe to measure thecritical micelle concentration (CMC) of the polymer.

Dynamic light-scattering spectrophotometer (DLS) mea-surements was used to determine the size and size distributionof self-assembled micelles, using a Malvern 4,700 system(Malvern Ltd., Malvern, U.K.) consisting of an Autosizer4,700 spectrometer, a pump/filter unit, a model 2013 air-cooler argon ion laser (75 mW, 488 nm, equipped with amodel 2,500 remote interface controller, Uniphase), and acomputer with DLS software (PCS, version 3.15, Malvern).The measurement temperature was 37 °C, and the measure-ment angle was 90°.

Synthesis of star-shaped porphyrin-cored poly(ε-caprolactone) (SPPCL) and the macro-RAFT agentSPPCL-BSPA

Star-shaped porphyrin-cored poly (ε-caprolactone) was syn-thesized by ring-opening polymerization (ROP) using ε-caprolactone as a monomer and tetrahydroxyethyl-terminated porphyrin as an initiator, as described in our pre-vious literature [20]. The detailed synthesis route is illustratedin Scheme 1. The reaction conditions of ROP polymerizationare shown in Table 1. 1H NMR (CDCl3d6, ppm): 1.35–1.45(δHc), 1.55–1.79 (δHb), 2.28–2.43 (δHa), 4.05–4.19 (δHd).3.68 (HOCH2-, δH

e), 4.48–4.53 (PhOCH2CH2O, δHf), 4.64–

4.69 (PhOCH2CH2O, δHg).

The macro-RAFT agent SPPCL-BSPAwas synthesized byesterification between the hydroxy of the SPPCL and thecarboxylic focal point of the BSPAwith a DCC/DMAP cou-pling. SPPCL (139.8 mg, 0.01 mmol), BSPA (43.52 mg,

Table 1 Synthesis of biomimetic star-shaped porphyrin-cored SPPCL-b-PNIPAAm block copolymers via the RAFT of NIPAAm monomer in THFsolution at 50 °C

Entrya CTA (mmol) AIBN (mmol) NIPAAm (mmol) fPCL/fPNIPAAmb (%/%) MnGPC

c Mw/Mnd Mn,NMR

e

SPPCL – – – – 17,348 1.29 14,535

SPPCL-b-PNIPAAM9 0.001 0.0004 0.07 77/23 20,116 1.30 19,675

SPPCL-b-PNIPAAM22 0.001 0.0004 0.12 23/77 40,704 1.41 43,853

a The subscript numbers represent the repeating units of polymersb Denotes the weight fractions of PCL and/or PNIPAAm within block copolymers, which was calculated from 1H NMR datacWeight-average molecular weight (Mw) and number-average molecular weight (Mn) are determined by GPCdMw/Mn denotes the molecular weight distribution of polymer, where weight-average molecular weight (Mw) and number-average molecular weight(Mn) are determined by GPC in DMFeMn,NMR was determined from the integral ratio of the signal on the main chain of PCL (2.31 ppm) and the signal on the main chain of PNIPAAm(1.16 ppm) from the 1H NMR spectra

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0.16 mmol), DMAP (4.89 mg, 0.04 mmol) and DCC(16.51 mg, 0.08 mmol) were added into an over dry tubequickly with an ice-water bath. The tube was then connectedto a Schlenk line, where an exhausting-refilling process wasrepeated three times. Then, the reactive mixture were dis-solved with CH2Cl2 (3 ml). The esterification was conductedat room temperature (25 °C) for 72 h in a nitrogen atmosphere.The polymers were purified by adding a drop of acetone,followed by filtration of the deposit (DCU). The polymerswere collected by centrifugation after precipitation in diethylether. The purified SPPCL-BSPA polymer was dried in vacuoat 40 °C until a constant weight was obtained, and the polymeryield was 53.4 %.

Synthesis of star-shaped porphyrin-cored poly(ε-caprolactone)-block-Poly (N-isopropylacrylamide)(SPPCL-b-PNIPAAm)

The star-shaped porphyrin-cored poly (ε-caprolactone)-block-poly (N-isopropylacrylamide) was synthesized by the revers-ible addition-fragmentation chain transfer polymerization(RAFT) polymerization of NIPAAM (13.8 mg, 0.12 mmol),in THF (0.2 mL), using AIBN (0.07 mg, 0.0004 mmol) as aninitiator and SPPCL-BSPA as a maro-RAFT agent. All thereactants were dissolved in an overdry round-bottom flask(25 mL), which was put into an oil bath and kept at 25 °Cwith vigorous stirring. The flask was then connected to aSchlenk line, where an exhausting-refilling process was re-peated three times. The polymerization was conducted at25 °C for 12 h in a nitrogen atmosphere. The polymers werecollected by centrifugation after precipitation in diethyl ether.The polymers were purified by dissolving them in THF,followed by filtration of the deposit to move the unreactedmonomer NIPAAm. The purified SPPCL-b-PNIPAAm poly-mer was dried in vacuo at 40 °C until a constant weight(20.9 mg) was obtained, and the polymer yield was 72.8 %.

Determination of the Critical Micelle Concentrations (CMCs)

The CMC of the block copolymers was determined byemploying the hydrophobic dye solubilization method, usingpyrene as a fluorescencent probe [21]. A series of polymersolution (5 mL) ranging from 5×10−5 to 1 mg/mL was addedto5uL of pyrene dissolved in methanol (concentration, 1×10−4 mol/L) after incubated nitrogen to allow for the evapo-ration of methanol. The fluorescence excitation spectra ofpyrene were obtained as a function of the polymer concentra-tion using a Horiba Fluorolog fluorometer (at a 90° angle).The excitation spectra were recorded at 37 °C from 280 to360 nm with the emission wavelength at 668 nm. The excita-tion and emission band slits were 5.0 and 2.5 nm, respectively.

Self-assembly behavior of the SPPCL-b-PNIPAAm

The micelles of corresponding SPPCL-b-PNIPAAm copoly-mers were prepared by dialysis method. In brief, the SPPCL-b-PNIPAAm copolymers (2 mg) were dissolved in THF(2 mL) at an initial concentration of 1 mg/mL under rapidagitation at room temperature (25 °C), and then distilled waterwas added gradually at a speed of 10 μL/min using amicrosyringe. After the appearance of a blue tint (water con-tent is about 10–12wt.%) indicating the formation ofmicelles,another 20–30 wt.% of water was added to stabilize themicelles. The micelles solution can be conducted by removingTHF with the rotary evaporation apparatus. Both the meansize andmorphology ofmicelles were determined byDLS andTEM, respectively.

Transmittance measurements

The optical transmittance of the aqueous polymer solution(1 mg/mL−1, 0.1 wt.%) at various temperatures ranging from22 to 52 °C was measured at 300 nm with a UV–vis spectro-photometer. The heating rate was set to 0.1 °C/min−1 [22]. TheLCST values for the polymer solutions were determined bythe temperature at which there was 50% optical transmittance.

Detecting singlet oxygen (1O2) production

In our s tudy, the chemica l method us ing 1 ,3 -diphenylisobenzofuran (DPBF) was proposed for detecting1O2 generated by SPPCL-b-PNIPAAm photosensitizers(5,10,15,20-Tetraphenylporphyrin (TPP) as a standard regent)[23–25]. Due to DPBF, the TPP reacts irreversibly with 1O2

and causes a decrease in the intensity of the DPBF absorptionband at 456 nm. In a typical experiment, 150 μL TPP(0.147 mM) or SPPCL-b-PNIPAAm (0.147 mM) was mixedwith 3 mL of DPBF (2.5 μM) in DMF, respectively. Then, thesolutions were irradiated with a 650-nm laser source (5 mw),and their absorbencies at 456 nm were recorded every minuteby a luminescence spectrometer. The singlet oxygen quantumyield (η) of SPPCL-b-PNIPAAm in DMF was calculatedusing the following equation [26, 27]:

η ¼ φTPPtTPP

tSPPCL−b−PNIPAAmð1Þ

tTPP is the time for decrease in absorption of DPBF in thepresence of free TPP in DMF solution, adjusted to a first-orderexponential decay;

tSPPCL-b-PNIPAAm is the time for decrease in absorption ofDPBF in the presence of SPPCL-b-PNIPAAm in DMF, ad-justed to a first-order exponential decay.

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Results and discussion

Synthesis of star-shaped porphyrin-cored poly(ε-caprolactone)-block-Poly (N-isopropylacrylamide)(SPPCL-b-PNIPAAm)

The synthesis of the SPPCL-b-PNIPAAm block copolymershas three steps, as shown in Scheme 1. First, SPPCL wasprepared by the ring opening polymerization (ROP) usingtetrahydroxyethyl-terminated porphyrin as an initiator. Sec-ond, preparation of the SPPCL-BSPA macro-RAFT wasemployed by esterification between the hydroxy of the SPPCLand the carboxylic focal point of the BSPA. Third, preparationof SPPCL-b-PNIPAAm block copolymers was conducted byRAFT polymerization, using SPPCL-BSPA as macro-RAFTchain transfer reagent. The reaction conditions of RAFT po-lymerization are shown in Table 1.

1H NMR was used to characterize the actual polymermolecular weight or the degree of polymerization of resultedcopolymers. Comparing the 1H NMR of the resulting SPPCL-BSPA macro-RAFT agent with that of the SPPCL precursor,the proton signals at 3.68 ppm assignable to the primaryhydroxy methylene end-group (HOCH2-, δH

e) of the SPPCLprecursor wholly disappeared, while new signals correspond-ing to protons of the primary phenyl methylene end-group(PhCH2-, δH

j) and protons of methylene next to sulfur (-S-

CH2-, δHi) appeared at 5.73 and 3.12 ppm, respectively. These

results proved that the SPPCL with hydroxyl end-groups wasindeed converted into SPPCL-BSPA with trithiocarbonateend-groups. Those indicated that the completion of the ester-ification obtained SPPCL-BSPA macro-RAFT agent (seeFig. 1a and b).

Figure 1c shows the coexistence of characteristic peaks ofPNIPAAm and PCL blocks in the SPPCL-b-PNIPAAm22

copolymer, which has typical framework absorption peaksof SPPCL at 1.35–1.45 (δHc), 1.55–1.79 (δHb), 2.28–2.43(δHa), and 4.05–4.19 (δHd), and PNIPAAm at 1.00–1.15 ppm(-CH(CH3)2,δH

i), 1.74–1.86 ppm(-CH2-CH,δHf), 1.86–

2.14 ppm(-CH2-CH,δHe), and 4.00–4.08 ppm(-CH(-CH3)2,δH

d) are still observed. All those signals ascribed tothe block copolymer are observed distinctly, which confirmsthat the synthesis of the copolymer is successful. By calculat-ing the integration of relative intensity of the characteristicpeak at 1.16 ppm for PNIPAAm and that at 2.31 ppm for PCL,the molecular weights of SPPCL-b-PNIPAAm (Mn,NMR

d) canbe obtained (listed in Table 1).

Also, FT-IR was used to characterize the structure of theprepared polymers. As exhibited in Fig. 2, a strong absorbanceat 1,730 cm−1 was observed in all the polymers (a, b, and c)because of the stretch vibration of C=O in PCL. In the Fig. 2c,however, another three peaks at 3,300 cm−1, 1,650 cm−1 and1,550 cm−1 appear, and are attributed to the absorbance of N-

Fig. 1 1H NMR spectra (CDCL3) of an SPPCL (a), SPPCL-BSPA (b), and SPPCL-b-PNIPAAm (c) sample

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H, C=O, and C-H of the amide groups in the PNIPAAmsegment, respectively. Those confirm the successful comple-tion of the polymerization process.

GPC was used to determine the molecular weight distribu-tion (Mw/Mn) and number-averagemolecular weight (Mn, GPC)of the prepared polymers. As shown in the Fig. 3, the traces ofthe SPPCL and SPPCL-b-PNIPAAm are monomodal, sug-gesting that narrow molecular weight distribution polymerswere formed. The GPC results of the unimodal peak at differ-ent elution times denote a progression of the polymer molec-ular weight. However, the molecular weights of SPPCL-b-PNIPAAm determined by GPC (Mn, GPC) were lower thanthose determined by 1H NMR (Mn, NMR). This is partlyattributed to the fact that the star-shaped polymer has a smallerhydrodynamic volume density in comparison to the linearpolystyrene as a calibration standard for GPC measurement[28].

Self-assembly behavior of the SPPCL-b-PNIPAAm

Due to its amphiphilic property, the SPPCL-b-PNIPAAm inaqueous phase can easily form the micelle.

The fluorescence probe technique was used to study themicelle formation with a serial of different concentrationspyrene. It is known that the fluorescent properties of pyrenelargely depend on its microenvironment. Once pyrene prefer-entially partitioned from a hydrophilic into a hydrophobicmicroenvironment, an abrupt increase in fluorescence inten-sity can be observed with increasing copolymer concentra-tions. Figure 4 shows the intensity of the pyrene excitationspectra versus the logarithm of the SPPCL-b-PNIPAAm co-polymer concentrations. At the lower concentration range, thenegligible changes within the experimental error for the inten-sity of fluorescence are detected. With increasing concentra-tion of the SPPCL-b-PNIPAAm22, however, the maximumresonance excitation spectra intensity at 337 nm increaseddramatically, suggesting that pyrene was gradually partitionedfrom a hydrophilic into hydrophobic microenvironment uponmicellar aggregation. The excitation spectra of SPPCL-b-PNIPAAm9 was similar to the SPPCL-b-PNIPAAm22. CMCwas determined by intersecting straight-line segments drawnthrough the points at the lowest polymer concentrations, lyingon a nearly horizontal line, and through the points on therapidly rising part of the plot according to previous study[16]. The CMC values of SPPCL-b-PNIPAAm9 (CMC1)and SPPCL-b-PNIPAAm22 (CMC2) were 0.0166 and0.0447 mg/mL, respectively, suggesting that a micelle in theaqueous phase was easily formed in a lower concentration.More interestingly, CMC increasedwith increasing hydrophil-ic segment under the same hydrophobic association. Thisquite lower CMC value shows that the copolymers are suit-able for a potential drug delivery system, and it also helps to

Fig. 2 FT-IR spectra of SPPCL, SPPCL-BSPA, and SPPCL-b-PNIPAAm22

Fig. 3 GPC traces of the SPPCL sample and SPPCL-b-PNIPAAmsamples

Fig. 4 Intensity of the pyrene excitation spectra for different concentra-tions of SPPCL-b-PNIPAAm

412, Page 6 of 10 J Polym Res (2014) 21:412

determine increasing concentrations for suitable temperaturesin pharmaceutical applications.

Morphology of the polymeric micelles

The morphology of the self-assembled aggregates from theseSPPCL-b-PNIPAAm block copolymers was investigated byTEM and DLS. As shown in Fig. 5a, it is evident from TEMmeasurements that the micelles self-assembled from copoly-mers are well dispersed as individual nanoparticles with aregular spherical shape. A unimodal size distribution forSPPCL-b-PNIPAAm22 was observed by DLS. Figure 5bshows the mean diameter of SPPCL-b-PNIPAAm22 micelles,as measured by DLS, which is 104.7 nm. TEM observationsshowed similar results. In fact, there is a little differencebetween the particle size measured by TEM and DLS, whichwas mainly attributed to the fact that the component measuredby the DLS is the solution of the micells, while the TEMmeasures the freeze-dried micelles. As it has been reportedthat colloidal particles showed increasing blood circulationtimes with increasing hydrophil surface grafting [29, 30], we

believe that this copolymer has a favorable property for drugdelivery purposes.

LCST behavior of SPPCL-b-PNIPAAm

It is well-known that PNIPAAm exhibits a coil-to-globuletransition upon heating, which results in the precipitation ofa sol gel of PNIPAAm and related copolymers. Also, thisphase transition characteristic in aqueous solution is attributedto the critical hydrophilic/hydrophobic balance of side groupsin a polymer [12, 31]. In this study, the solubility of the blockpolymer in water can be influenced by two kinds of interac-tions. On the one hand, the strong hydrogen bonding betweenthe hydrophilic group (-CONH-) in the PNIPAAm chain andwater results in solubility. On the other hand, the hydrophobicinteraction between hydrophobic side groups (-CH(CH)3)2-)and water leads to precipitation [32]. Both interactions existsimultaneously, and the ratio between these processes dependson the temperature of environment. With an increase of tem-perature, the hydrophobic interaction will exceed the hydro-phobic interaction. The synthesized SPPCL-b-PNIPAAmconsists of the PNIPAAm chain as the hydrophilic shell,prophyrin as the core, and hydrophobic PCL as the inner blockat the arm, and thus behaves in the temperature-sensitivemanner. We characterized the LCST behaviors of SPPCL-b-PNIPAAm copolymer solutions bymeasuring the temperatureat which there was 50 % optical transmittance (transmittanceat 300 nm). As shown in Fig. 6, the LCSTs of the SPPCL-b-PNIPAAm9 and SPPCL-b-PNIPAAm22 micelles were 36 °Cand 37.4 °C, respectively. The LCST of HOOC-PNIPAAm-COOH was measured to be 42.5 °C in distilled water, asreported in previous results in literature [19]. Generally, forthe copolymers of NIPAAm polymerized with hydrophobiccomonomers, the LCST shifts to lower temperatures withincreasing hydrophobic composition, because the incorpora-tion of hydrophobic comonomers facilitates the chain aggre-gation [33]. The results obtained for the star copolymer sug-gested that the hydrophobic porphyrin core had a significanteffect on the LCST. This depression of the LCST after thePNIPAAm was chained to the star core is partly because the

Fig. 5 a TEM micropictures ofthe micelles of SPPCL-b-PNIPAAm22. b Size distributionof the micelles of SPPCL-b-PNIPAAm22 detected by DLS(the concentration of copolymerswas 1 mg/mL)

Fig. 6 Thermosensitive behavior of SPPCL-b-PNIPAAm micelles

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hydrophobic PCL with porphyrin cores facilitates chain ag-gregation. According to this regularity, it is not surprising thatwe observed the LCST of the copolymer typically shifting tolower values as the ratio of the hydrophobic core group toPNIPAAm increased,. These results are attributed to the factthat the strong interaction of the hydrophobic segmentsformed phase-separated micellar structures, showing that bothof those copolymers exhibited thermally responsive changesin polymer phase-transitions and self-assembled into micelleswith a spherical shape. This reversible thermosensitive char-acteristic provides the possibility to construct the drug deliv-ery systems in conjunction with localized hyperthermia. Thedelivery to a specific site can be enhanced by local heating/cooling procedures, inducing changes in the physical proper-ties of polymeric micelles.

Singlet oxygen measurements

It was known that under a certain wavelength, light porphyrinand its derivatives could generate singlet molecular oxygen[1O2], an initial agent in the anticancer treatment known asphotodynamic therapy (PDT). To determine the efficiency ofsinglet oxygen release and the influence of outer shell com-pounds (PNIPAAm below the LCST in our study), we used achemical method employing the DPBF molecule as a scaven-ger, which shows a decrease in the intensity of fluorescenceabsorption after it irreversibly reacts with the 1O2. In Fig. 7,there is a continuous decrease in fluorescence intensity at456 nm as a function of irradiation time, both in free porphyrin(TPP) and the copolymer (SPPCL-b-PNIPAAm).

Based on the reported estimation method of singlet oxygengeneration, using TPP solution (0.67±0.09) [27] as the stan-dard, the singlet oxygen release delivery (η) of SPPCL-b-

PNIPAAm is estimated from Eq. 1 (see Experimental Section)to be 0.117 in DMF solution. Compared with TPP, the reduc-tion of fluorescence intensity of DPBF caused by SPPCL-b-PNIPAAm is smoother. This difference in the release of 1O2

has already been reported [34], and this may be attributed toseveral trivial effects, such as the scattering of the nanoparti-cles, the local sequestration of generated 1O2 by the nanopar-ticle matrix, or an intrinsic, lower, encapsulated copolymersinglet oxygen quantum yield. It is clear, however, that thesinglet oxygen production ability of SPPCL-b-PNIPAAm canbe adequately controlled by irradiation time, which indicatesthat this structure shows an advantageous technology for PDT[35, 36].

Conclusions

We have demonstrated for the first time that thermo-sensitive,four-armed, star-shaped porphyrin-cored poly (ε-caprolactone)-b-poly (N-isopropylacrylamides) are synthe-s ized by RAFT using AIBN as an ini t ia tor, N-isopropylacrylamide as a monomer and macro-RAFT agentSPPCL-BSPA. The SPPCL-BSPA macro-RAFT agent isemployed by esterification between the hydroxy of the SPPCLand the carboxylic focal point of the BSPA. Importantly, thenumber-average molecular weight and molecular weight dis-tribution of the copolymer can be nicely controlled by theRAFT polymerization. Due to their amphiphilic property, thecore–shell micelles formed from SPPCL-b-PNIPAAm blockcopolymers have a better-defined completely phase-separatedstructure, and thus exhibit solid-like and stable core-shellstructures, which act as nanosized photosensitizing agentsfor PDT. Our results show that these spherical micelles withdiameter of approximately 100 nm have an ideal sensitivity totemperature. The LCST and CMC values of SPPCL-b-PNIPAAm block copolymers are directly proportional to therate of hydrophilic and hydrophobic components. Interesting-ly, these micelles exhibited efficient singlet oxygen genera-tion. These features, together with the controllable preparationmethod, sensing a slightly temperature signal and respondingby changing their structures, make these micelles very suitableas a potential dual delivery vehicle for the combination ofphotodynamic therapy and chemotherapy for the treatment ofcancer.

Acknowledgments The authors are grateful for the financial support ofthe National Natural Science Foundation of China (21004031), the Nat-ural Science Foundation of Jiangsu Province (BK2011459), the NationalPostdoctoral Foundation of China (20090461065), the National Postdoc-toral Foundation of Jiangsu Province (1001034B), the Open Foundationof State Key Laboratory of Natural and Biomimetic Drugs, PekingUniversity (K20110105), and the Social Development Foundation ofZhen Jiang (SH2012024).

Fig. 7 Fluorescence emission spectra of DPBF as a function of irradia-tion at 456 nm in aqueous solutions of porphyrin and SPPCL-b-PNIPAAm22

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