Synthesis and characterization of amphiphilic functional polyesters by ring-opening polymerization and click reaction

Reactive & Functional Polymers 72 (2012) 36–44

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Synthesis and characterization of amphiphilic functional polyesters by ring-opening polymerization and click reaction Rong-Jia Su a, Hong-Wei Yang b, Yann-Lii Leu a, Mu-Yi Hua b, Ren-Shen Lee c,⇑ a

Graduate Institute of Natural Products, Chang Gung University, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, Taiwan Department of Chemical and Materials Engineering, Chang Gung University, Tao-Yuan, Taiwan c Center of General Education, Chang Gung University, Tao-Yuan, Taiwan b

a r t i c l e

i n f o

Article history: Received 18 May 2011 Received in revised form 11 August 2011 Accepted 28 August 2011 Available online 1 September 2011 Keywords: Amphiphilic Graft-block functional polyester Click chemistry Micelles

a b s t r a c t During this work we have prepared novel amphiphilic graft-block (PaN3CL-g-alkyne)-b-PCL functional polyesters, comprising poly(a-azido-e-caprolactone-graft-alkyne) (PaN3CL-g-alkyne) as the hydrophilic segment and poly(e-caprolactone) (PCL) as the hydrophobic segment, by ring-opening polymerization of e-caprolactone (e-CL) with hydroxyl-terminated macroinitiator PaClCL, substituting pendent chloride with sodium azide. The copolymers were subsequently used for grafting of 2-propynyl-terminal alkyne moieties by the Cu(I)-catalyzed Huisgen’s 1,3-dipolar cycloaddition, thus producing a ‘‘click’’ reaction. 1 H NMR, FT-IR, GPC, and differential scanning calorimetry (DSC) examined the characteristics of the copolymers. Grafting of PMEs or PMPEGs onto the PaN3CL-b-PCL caused these amphiphilic copolymers to self-assemble into micelles in the aqueous phase. Fluorescence, dynamic light scattering (DLS) and transmission electron microscopy (TEM) then examined these micelles. The critical micelle concentration (CMC) ranged from 8.2 mg L1 to 39.8 mg L1 at 25 °C and the average micelle size ranged from 140 to 230 nm. The hydrophilicity and length of the hydrophilic segment influenced micelle stability. The current study describes the drug entrapment efficiency and drug loading content of the micelles, dependent on the composition of graft-block polymers. The results from in vitro cell viability assays indicated that (PaN3CL-g-alkyne)-b-PCL shows low cytotoxicity. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.

1. Introduction Aliphatic polyester have deeply impacted biomedical and engineering field, such as tissue scaffolding and therapeutic delivery. Poly(lactide) (PLA), poly(glucolide) (PGA), poly(e-caprolactone) (PCL), and their respective copolymers combine biocompatibility and biodegradability which make them leading candidates for resorbable implant materials, prosthetics, surgical sutures, vascular grafts, bone screws, and erodible polymers for drug delivery systems [1]. An apparent drawback of standard aliphatic polyesters is the lack of functionalities on the polymer backbone which allow the tailoring of polyester properties for the specific applications. The introduction of pendent functional groups along these polyester chains is highly desirable to tailor and modulate physicochemical properties, such as hydrophilicity, biodegradation rate, bioadhesion, crystallinity and biological activity [2–4]. Amphiphilic block copolymers are well known for self-assembly into micelles or larger aggregates in solvents selective for one block. In aqueous solutions, a core–shell structure commonly forms, with a hydrophobic core surrounded by a hydrated hydro⇑ Corresponding author. Tel.: +886 3 2118800x5054; fax: +886 3 2118700. E-mail address: [email protected] (R.-S. Lee).

philic shell. Previous researchers extensively studied [5–9] the properties of block copolymer micelles in biomedical applications, namely the efficacious delivery of hydrophobic drugs sequestered within micellar cores. The parameter which principally governs self-organization of amphiphilic block copolymers is hydrophilic–hydrophobic balance, even though other experimental factors such as concentration, pH, ionic strength, temperature, and sample preparation may also influence aggregation mechanisms [10]. Hence, the macromolecular structure of block copolymer aggregates somewhat predetermines their nanoscale morphology [11,12]. Modifying the hydrophilic–hydrophobic balance of diblock copolymers via coupling with additional hydrophilic or hydrophobic moieties is a possibility [13,14]. The copper-catalyzed 1,3-dipolar cycloaddition of azides and terminal alkynes is potentially a very interesting reaction for modifying block copolymer aggregates [15,16]. This particular reaction is efficient, highly selective, and generally produces very high yields in organic or aqueous media, requiring only mild experimental conditions [17]. Chemists lately describe this versatile reaction as the archetype of ‘‘click’’ chemistry [18]. Due to the simplicity and selectivity of these reactions, recent research works extensively studied copper-catalyzed azide–alkyne cycloadditions (CuAAC) in biological and materials science [19–26].

1381-5148/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.reactfunctpolym.2011.08.008

R.-J. Su et al. / Reactive & Functional Polymers 72 (2012) 36–44

The present study investigates CuAAC as a tool for varying the hydrophilic-hydrophobic balance of diblock copolymers in aqueous medium. This approach ‘‘clicks’’ a model AB amphiphilic block copolymer composed of a poly(e-caprolactone) (PCL) hydrophobic segment and a poly(a-azo-e-caprolactone-graft-2-propynyl benzoate) (PaN3CL-g-PBA) hydrophilic segment with additional hydrophilic alkyne (Scheme 1). The current work studies the influence of hydrophilic/hydrophobic chain length of the block copolymer on micelle size, drug entrapment efficiency, and drug loading content. Fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM) then evaluated the micellar characteristics of these graft-block copolymers in an aqueous phase.

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WI). m-Chloroperoxybenzoic acid (m-CPBA) was purchased from Fluka Chemical Co. (Buchs SG1, Switzerland). Stannous octoate (SnOct2) was purchased from Strem Chemical Co. (Newburyport, MA). e-CL was dried and vacuum-distilled over calcium hydride. aClCL was prepared according to a previously reported method [27]. 2-Propynyl-2-methoxyethanol (PME) and 2-propynyl-methoxypoly(ethylene glycol) (PMPEG) were prepared as previously reported [28]. Organic solvents such as tetrahydrofuran (THF), methanol, chloroform, toluene, N,N-dimethylformamide (DMF) and n-hexane were of high-pressure liquid chromatography (HPLC) grade purchased from Merck Chemical (Darmstade, Germany). Ultrapure water was obtained by purification with a Milli-Q Plus (Waters, Milford, MA).

2. Experimental 2.2. Preparation of PaClCL macroinitiators 2.1. Materials Benzyl N-(2-hydroxyethyl)carbamate, 2-chlorocyclohexanone, pyrene, 2-propynyl benzoate (PBA), indomethacin (IMC), and sodium azide were purchased from Aldrich Chemical Co. (Milwaukee,

Hydroxyl-terminated functional PaClCL was prepared with benzyl N-(2-hydroxyethyl)carbamate initiating the ring-opening polymerization of aClCL. A total of 0.765 mmol of benzyl N-(2hydroxyethyl)carbamate, 7.65 mmol of aClCL and SnOct2

Scheme 1. Synthesis of (PaN3CL-g-alkyne)-b-PCL graft-block copolymers.

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(1.5 wt.% based on the weight of aClCL) were introduced into a flask and dissolved into the toluene (30 mL) under a dry nitrogen stream. The flask was purged with nitrogen and the reaction solution was refluxed for 24 h. the solution was concentrated using a vacuum rotary evaporator and the resulting product was dissolved in CHCl3, microfiltered, and then precipitated into excess CH3OH with stirring. After purification, the PaClCL macroinitiators were dried in vacuo at 50 °C for 24 h and then analyzed. 1H NMR (CDCl3): d 7.38 (m, C6H5–), 5.22 (s, C6H5CH2–), 4.29 (m, –CH2O– and – CaClH–), 4.20 (m, –CeH2O–), 3.49 (m, –NCH2–), 2.02 (m, CbH2–), 1.73 (m, CdH2–), 1.57 (m, CcH2–). 2.3. Synthesis of PaClCL-b-PCL diblock copolymers All glassware was dried in the oven and handled under dry nitrogen stream. The typical polymerization process to provide PaClCL13-b-PCL30 was as follows. PaClCL13 (Mn = 1678 g mol1, 0.17 mmol) and e-CL (0.58 g, 5.1 mmol) were added to a flask under a dry nitrogen stream. 22 mg of SnOct2 (1.5 wt.% based on the weight of PaClCL and e-CL) and toluene (30 mL) were then added to the flask. The flask was purged with nitrogen and the reaction solution was refluxed for 24 h. The solution was concentrated using a vacuum rotary evaporator. The resulting product was dissolved in CHCl3 and then precipitated into excess n-hexane/ethyl ether (v/v 5:1) with stirring. The purified polymer was dried in vacuo at 50 °C for 24 h and analyzed. The yields ranged between 52% and 93%. Fig. 1A displays the representative IR spectrum of PaClCL14b-PCL24. 1H NMR (CDCl3): d 7.37 (m, C6H5–), 5.23 (s, C6H5CH2–), 4.24 (m, –CeH2O– and –CaClH– of PaClCL), 4.08 (t, J = 6.8 Hz, – CeH2O– of PCL), 3.50 (m, –NCH2–), 2.33 (t, J = 7.2 Hz, –CaH2– of PCL), 2.13 (m, –CbH2– of PaClCL), 1.64 (m, –CdH2– of PaClCL, and – CbH2– and –CdH2– of PCL), 1.37 –CcH2– of PaClCL and PCL). 2.4. Synthesis of PaN3CL-b-PCL diblock copolymers PaClCL14-b-PCL24 (0.22 mmol, 14 equiv of aClCL) was dissolved in 10 mL of DMF in a glass reactor followed by the addition of NaN3 (3.70 mmol). The mixture was stirred overnight at room temperature. The insoluble salt was removed by filtration and elimination of DMF in vacuo. The resulting product was dissolved in CHCl3 and

then precipitated into excess n-hexane/ethyl ether (v/v 4:1) with stirring. The purified polymer was dried in vacuo at 50 °C for 24 h and then analyzed. The yields ranged between 90% and 98%. Fig. 1B displays the representative IR spectrum of PaN3CL14-bPCL28. 1H NMR (CDCl3): d 7.39 (m, C6H5–), 5.24 (s, C6H5CH2–), 4.28 (m, –CeH2O– of PaN3CL), 4.10 (t, J = 6.8 Hz, –CeH2O– of PCL), 3.87 (m, –CaH– of PaN3CL), 3.50 (m, –NCH2–), 2.33 (t, J = 7.6 Hz, CaH2– of PCL), 1.86 (m, –CbH2– and –CdH2– of PaN3CL and PCL), 1.37 (m, –CcH2– of PaN3CL and PCL). 2.5. Typical click chemistry reaction PaN3CL61-b-PCL32 (27.9 mmol, 1.70 mol equiv of azide) was transferred into a glass reactor containing THF. 2-Propynyl benzoate (1.70 mol), CuI (2.8 mmol), and triethyl amine (2.8 mmol) were then added to the reactor. The solution was stirred at 35 °C for 4 h. The cycloaddition copolymer was precipitated in hexane with stirring. The purified polymer was dried in vacuo at 50 °C for 24 h and then analyzed. Figs. 1C and 2A and B display the representative IR, 1 H NMR and 13C NMR spectra of (PaN3CL61-g-PBA56)-b-PCL32. 2.6. Characterization 1 H NMR spectra were recorded at 500 MHz (with a Bruker WB/ DMX-500 spectrometer, Ettlingen, Germany) using chloroform (d 7.24 ppm) as an internal standard in chloroform-d (CDCl3). A thermal analysis of the polymer was performed on a DuPont 9900 system that consisted of DSC (Newcaste, DE). The heating rate was 20 °C min1. Glass-transition temperatures (Tgs) were recorded at the middle of heat capacity change and taken from the second heating scan after quick cooling. Number- and weight-average molecular weights (Mn and Mw, respectively) of the polymer were determined by a GPC system, carried out on a Jasco HPLC system equipped with a model PU-2031 refractive-index detector (Tokyo, Japan) and Jordi Gel DVB columns with pore sizes of 100, 500, and 1000 Å. Chloroform was used as an eluent at a flow rate of 0.5 mL min1. PEG standards with a low dispersion (Polymer Sciences) were used to generate a calibration curve. Data were recorded and manipulated with a Windows-based software package (Scientific Information Service). UV–vis spectra were

Fig. 1. Representative FT-IR spectra (A) PaClCL14-b-PCL24, (B) PaN3CL14-b-PCL28, and (C) (PaN3CL61-g-PBA56)-b-PCL32.

R.-J. Su et al. / Reactive & Functional Polymers 72 (2012) 36–44

Fig. 2. Representative (A) 1H NMR and (B)

obtained using a Jasco V-550 spectrophotometer (Tokyo, Japan). The pyrene fluorescence spectra were recorded on a Hitachi F4500 spectrofluorometer (Japan). Square quartz cells of 1.0  1.0 cm were used. For fluorescence excitation spectra, the detection wavelength (kem) was set at 390 nm.

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13

C NMR spectra of (PaN3CL61-g-PBA56)-b-PCL32.

pyrene concentration in the final solutions was 6.1  107 M. The flasks were allowed to stand overnight at room temperature to equilibrate the pyrene and the micelles. The emission wavelength was 390 nm for excitation spectra. 2.9. Dynamic light scattering measurements

2.7. Preparation of polymeric micelles Polymeric micelles of (PaN3CL-g-alkyne)-b-PCL copolymers were prepared using the oil-in-water (o/w) emulsion technique. 30 mg of each polymer was dissolved in dichloromethane (DCM, 5 mL). The solution was added dropwise into DI water (100 mL) with vigorous stirring at ambient temperature. The emulsion solution was then ultrasonicated for 1 h and stirred overnight at ambient temperature. After the DCM completely evaporated, the experiment yielded a polymeric micelle solution.

Size distributions of the micelles were estimated by DLS using a particle-size analyzer (Zetasizer nano ZS, Malvern, UK) at 20 °C. Scattered light intensity was detected at 90° to an incident beam. Measurements were made after the aqueous micellar solution (C = 300 mg L1) was passed through a microfilter with an average pore size of 0.2 lm (Advantec MFS, USA). An average size distribution of aqueous micellar solution was determined based on CONTIN programs of Provencher and Hendrix [30]. 2.10. Transmission electron microscopy (TEM) measurements

2.8. Fluorescence measurements To confirm micelle formation, fluorescence measurements were carried out using pyrene as a probe [29]. Fluorescence spectra of pyrene in aqueous solution were recorded at room temperature using a fluorescence spectrophotometer. The sample solutions were prepared by first adding known amounts of pyrene in acetone to a series of flasks. After the acetone had evaporated completely, measured amounts of micelle solutions with various concentrations of (PaN3CL-g-alkyne)-b-PCL ranging from 0.293 to 300 mg L1 were added to each flask and mixed by vortex. The

The morphology of the micelles was observed using TEM (JEM 1200-EXII, Tokyo, Japan). Drops of micelle solution (C = 300 mg L1, no containing stain agent) were placed on a carbon film coated on a copper grid, and were then dried at room temperature. The observation was conducted at an accelerating voltage of 100 kV. 2.11. Drug loading content and drug entrapment efficiency Using oil-in-water solvent evaporation, (PaN3CL99-g-PME90)-bPCL44 (67 mg, 50-fold CMC) was dissolved in 6 mL methylene

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chloride followed by adding IMC, serving as a model drug at various weight ratios to the polymer (1/10–1/1). The solution was added dropwise to 150 mL of distilled water containing 1 wt.% poly(vinyl alcohol) under vigorous stirring. Poly(vinyl alcohol) was used as a surfactant to reduce micelle aggregation. Sonication at ambient temperature for 60 min reduced droplet size. The emulsion was stirred overnight at ambient temperature to evaporate the methylene chloride. The unloaded residue of IMC was removed by filtration using a Teflon filter (Whatman) with an average pore size of 0.45 lm. The micelles were obtained by vacuum drying. The addition of a ten-fold excess volume of DMF disrupted a weighed amount of micelle. Drug content was assayed spectrophotometrically at 320 nm using a Diode Array UV–vis spectrophotometer. Eqs. (1) and (2) calculate the drug loading content and drug entrapment efficiency, respectively:

supplemented with 10% fetal bovine serum (FBS) in a humid atmosphere of 5% CO2 at 37 °C, for 24 h. The growth medium was replaced with a medium that contained the desired amount of polymers. Cells were incubated for 24 h, and cell viability was assayed by adding 100 lL of medium that contained 10 lL of MTT PBS solution (5 mg mL1). After incubation for 4 h, the formazan crystals were dissolved in 100 lL of DMSO. The absorbance of each well was measured using a microplate reader (Stat Fax 2100, Awareness) at a test wavelength of 570 nm and a reference wavelength of 630 nm.

Drug loading content ð%Þ

Ring-opening polymerization of e-CL with hydroxyl-terminated macroinitiator PaClCL produced various PaClCL-b-PCL diblock copolymers. Scheme 1 illustrates the synthesis of PaClCL-b-PCL diblock copolymers. Ring-opening homopolymerization of aClCL with initiator benzyl N-(2-hydroxyethyl)carbamate (at molar ratios 10/1, 30/1, and 60/1) in the presence of SnOct2 at reflux with toluene for 24 h prepared the hydroxyl-terminated PaClCL. Table 1 presents the results of the polymerization. The number-average molecular weight (Mn) of the macroinitiators with narrow polydispersities corresponded with Mn,th and Mn,NMR. The current study used the hydroxyl group of the macroinitiator as the initiation site for the ring-opening polymerization of e-CL in the presence of SnOct2 (1.5 wt.%) as the catalyst at reflux with toluene for 24 h. Table 2 presents the results of polymerization. The yields were moderate to high, ranging between 52% and 93%. The Mn of block copolymers obtained from copolymerization of the e-CL and PaClCL macroinitiator increased with increasing molar ratio of the e-CL to the PaClCL macroinitiator in the feed. The number-average molecular weight (Mn) of the block copolymers corresponded with Mn,th. Using PaClCL14 as the macroinitiator, the molar ratio of e-CL to PaClCL14 in the feed increased from 30 to 90, and the Mn of the copolymers increased from 8300 to 14,920 g mol1 with Mw/Mn between 1.45 and 1.73. Using PaClCL38 or PaClCL62 as the macroinitiator, the molar ratio of e-CL to PaClCL in the feed was fixed at 30, and the obtained Mn values of the copolymers were 12,530, and 12,760 g mol1, respectively. 1H NMR analyzed the molar compositions of the block copolymers. Comparing the integral area of the resonance peak d = 2.22– 2.39 ppm of the second carbon (C2) methylene protons of PCL with the resonance peaks d = 1.89–2.11 ppm of the third carbon (C3) methylene protons of PaClCL determined the amounts of comonomer incorporated into the copolymer. The resonance peaks are assigned to the corresponding hydrogen atoms of the copolymers.

¼ ðweight of drug in micelles=weight of micellesÞ  100

ð1Þ

Drug entrapment efficiency ð%Þ ¼ ðweight of drug in micelles=weight of drug fed initiallyÞ  100 ð2Þ 2.12. In vitro drug release The appropriate amounts of the IMC-loaded micelles (110.2 mg) were precisely weighed and suspended in 10 mL of PBS (0.1 M, pH 7.4). The micellar solution was introduced into a dialysis membrane bag (molecular weight cutoff = 3500), and the bag was placed in 50 mL of PBS release media. The media was then shaken (30 rpm) at 37 °C. At predetermined time intervals, 3 mL aliquots of the aqueous solution were withdrawn from the release media, and the same volume of a fresh buffer solution was added. The concentration of released IMC was monitored with a UV–vis spectrophotometer at a wavelength of 320 nm. The rate of controlled drug release was measured by the accumulatively released weight of IMC according to the calibration curve of IMC. 2.13. In vitro degradation In vitro degradation of about 30 mg of copolymer thin film was performed in 5 mL PBS (0.1 M, pH 7.4) at 37 °C, and the buffer solution was replaced every 2 days. At specific time intervals, the specimen was removed, washed with distilled water, lyophilized, and weighed. Weight loss (%) = 100(D0  D)/D0 where D0 is the copolymer weight before degradation and D is the copolymer weight after degradation for a specified period.

3. Results and discussion 3.1. Synthesis of PaClCL-b-PCL diblock copolymers

3.2. Substitution of the pendent Cl atoms of PaClCL-b-PCL 2.14. In vitro cell viability Human bladder cancer cells (MGH-U1) were cultured onto a plate with 96 wells (1  104 cells/well) in DMEM media

According to Scheme 1, the pendent chlorides of PaClCL-b-PCL must be converted into azides by reaction with sodium azide. This study, thus, included reaction of PaClCL-b-PCL with one equivalent

Table 1 Results of macroinitiator prepared by ring-opening polymerization of a-chloro-e-caprolactone (aClCL) with initiatora.

a

Macroinitiator

[aClCL]/[I] molar ratio in feed

[aClCL]/[I] molar ratiob

Mn,NMRb (g mol1)

Mn,thc (g mol1)

Mn,GPCd (g mol1)

Mw/Mnd

PaClCL13 PaClCL38 PaClCL62

10/1 30/1 60/1

13/1 38/1 62/1

2126 5838 9402

1680 4650 9105

1680 7210 11,480

1.57 1.48 1.30

The benzyl N-(2-hydroxy ethyl) carbamate (BHEC) was used as the initiator. Determined from 1H NMR spectroscopy of PaClCL. c Mn,th = MBHEC + MaClCL  [aClCL]/[I] (where MBHEC is the molecular weight of BHEC, MaClCL is the molecular weight of aClCL, [aClCL] is the monomer molar concentration, and [I] is the initiator molar concentration. d Number-average molecular weight (Mn) and weight-average molecular weight (Mw) are determined from GPC. b

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R.-J. Su et al. / Reactive & Functional Polymers 72 (2012) 36–44 Table 2 Results of block copolymerization of e-CL initiated with hydroxyl-terminated PaClCL and substitution of chloride of PaClCL-b-PCL by sodium azide. [e-CL]/[PaCl(N3)CL] molar ratio in feed

[e-CL]/[PaCl(N3)CL] molar ratioa

Yield (%)

Mn,NMRa (g mol1)

Mn,thb (g mol1)

Mn.GPCc (g mol1)

Mw/Mnc

PaClCL-b-PCL PaClCL14-b-PCL24 PaClCL14-b-PCL54 PaClCL14-b-PCL73 PaClCL38-b-PCL28 PaClCL62-b-PCL31 PaClCL62-b-PCL112 MPEG9-b- PaClCL14-b-PCL26

30/1 60/1 90/1 30/1 30/1 90/1 30/1

24/1 54/1 73/1 28/1 31/1 112/1 26/1

54 77 52 93 75 70 70

5012 8436 10,605 8735 12,943 22,190 8361

5548 9121 12,545 9857 12,943 19,677 8817

8300 12,630 14,920 12,530 12,760 15,560 15,090

1.45 1.54 1.73 1.48 1.28 1.18 1.19

PaN3CL-b-PCL PaN3CL14-b-PCL28 PaN3CL38-b-PCL28 PaN3CL61-b-PCL31 PaN3CL61-b-PCL112 MPEG9-b-PaN3CL14-b-PCL26

24/1 28/1 31/1 112/1 26/1

28/1 28/1 31/1 112/1 26/1

97 98 97 92 90

6700 9278 13,186 22,431 9050

7235 10,104 13,227 19,961 9064

11,220 11,890 12,120 18,080 15,540

1.51 1.50 1.25 1.12 1.22

Copolymer

a

Determined from 1H NMR spectroscopy of PaClCL-b-PCL and PaN3CL-b-PCL. Mn,th = Mn,PaClCL(or PaN3CL) + Me-CL  [M]/[I] (where Mn,PaClCL is the number-average molecular weight of PaClCL, Mn,PaN3CL is the number-average molecular weight of PaN3CL, Me-CL is the molecular weight of e-CL, [M] is the monomer molar concentration, and [I] is the macro-initiator molar concentration). c Determined from GPC. b

of sodium azide in DMF overnight at room temperature. Table 2 presents the substitution results. As expected, the IR spectrum demonstrates a new absorption peak at 2104 cm1, characteristic of the azide (Fig. 1B). 1H NMR confirmed that the conversion of the pendent chlorides into azides is almost quantitative (data not shown). Indeed, the resonance peak at 4.29 ppm for the CHCl protons completely disappears to be replaced by a new peak at 3.87 ppm, typical of the CHN3 protons. 1H NMR and integration of the resonance peaks at d = 3.87 ppm of the CHN3 protons of PaN3CL with the resonance peaks d = 2.33 ppm of the second carbon (C2) methylene protons of PCL determined the molar ratio [aN3CL]/[PCL] in the block copolymers. Table 2 demonstrates that the molecular weights of the copolymers are similar to that of the chloride substituent polymer. This observation is consistent with a small change in the hydrodynamic volume of the copolymer. The resonance peaks are assigned to the corresponding hydrogen atoms of the copolymers. 3.3. Click reaction of PaN3CL-b-PCL with alkyne To functionalize the aliphatic polyesters, chemical transformation by click reaction between alkynes and azides is favorable due to the mild conditions and quantitative yields. Copper iodide and Et3N in THF catalyzed the click reaction at a low temperature (35 °C) for 4 h. Table 3 presents the cycloaddition results. Mn,GPC was smaller than Mn,NMR or Mn,th. This is because graft copolymers are more compact structures than the linear standards used to calibrate the GPC [31]. The grafting efficiency was between 83% and 100%. Observation of the disappearance of the azide signal at 2104 cm1 and a new absorption peak at 1608 cm1 characteristic of the vibration of the triazole unsaturated group in the FTIR spectrum confirmed a successful click reaction yielding an alkyne-grafted polyester (Fig. 1C). The GPC trace showed a unimodal distribution (data not shown). According to 1H NMR analysis, the cycloaddition confirmed that the peak for CHN3 proton at 3.87 ppm disappeared, and that a new peak for the triazole proton appeared at 7.85 ppm, indicating triazole formation. Integration of 1H NMR spectral signals at d = 5.45 (Is) against the signal at d = 2.29 (Ik) determined the extent of PBA incorporation into the copolymer. As shown in Table 3, the spectroscopically calculated incorporations of PBA are similar to the feed ratios. The graft-block (PaN3CL-g-alkyne)-b-PCL copolymers are crystalline when the PCL segment is longer than the PaN3CL-g-

alkyne segment, with Tm in the range of 36–53 °C. As the PCL segment is shorter than the PaN3CL-g-alkyne segment, the graftblock copolymers are amorphous, exhibiting only a Tg. This is due to the incorporated PaN3CL-g-alkyne destroying the crystalline area of PCL. 3.4. Micelles of graft-block copolymers The amphiphilic nature of the graft-block copolymers, consisting of hydrophilic PaN3CL-g-PME or PaN3CL-g-PMPEG block and hydrophobic PCL, provides an opportunity to form micelles in water. Fluorescence techniques investigated the characteristics of the graft-block copolymer micelles in aqueous phase, including the critical micelle concentrations (CMCs) using pyrene as a probe. Fig. 3 displays the excitation spectra of pyrene in the (PaN3CL50g-PMPEG48)-b-PCL94 solution at various concentrations. Findings demonstrate that fluorescence intensity increases with increasing (PaN3CL50-g-PMPEG48)-b-PCL94 concentration. The characteristic feature of pyrene excitation spectra, a red shift of the (0, 0) band from 334 to 338 nm upon pyrene partition into micellar hydrophobic core, determined the CMC values of (PaN3CL50-g-PMPEG48)-bPCL94 block-graft copolymers. Fig. 4 presents the intensity ratios (I338/I334) of pyrene excitation spectra versus the logarithm of concentration of (PaN3CL50-g-PMPEG48)-b-PCL94 block-graft copolymers. Intersecting straight-line segments drawn through the points at the lowest polymer concentrations, which lay on a nearly horizontal line, passing through the points on the rapidly rising part of the plot, determined the CMC. Table 4 presents CMC values of the graft-block copolymers dependent on block composition. The CMC of the graft-block copolymers ranged from 8.2 to 39.8 mg L1. This is larger than for the PCL-b-(PaN3CL-g-PBA) polymeric micelles [the CMC of PCL-b-(PaN3CL-g-PBA), for example, was 2.4–7.6 mg L1] [26]. The CMC of the (PaN3CL-g-PBA)-b-PCL series of copolymers remained undetermined because the PaN3CL-g-PBA segment did not demonstrate enough hydrophilicity. Generally, if the hydrophilic block remains constant for a series of copolymers, an increase in the molecular weight of the hydrophobic block will decrease the CMC. To a lesser extent, if constant length of the hydrophobic block is maintained, then an increase in the length of the hydrophilic block will cause an increase in the value of the CMC [32]. This study observed contrasting results. At a similar the hydrophobic block length, the length of the hydrophilic segment increased and CMC values decreased. The micelle became more stable due to the increase in hydrophilic segment length.

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Table 3 Results of graft of PaN3CL-b-PCL with alkyne by click reaction. Graft copolymera

[RAC„CH]/ [PaN3CL-b-PCL] molar ratio in feed

[RAC„CH]/ [PaN3CL-b-PCL] molar ratiob

Grafting efficiency (%)

Mn,NMRb (g mol1)

Mn,thc (g mol1)

Mn,GPCd (g mol1)

Mw/Mnd

(PaN3CL14-g-PBA14)-b-PCL28 (PaN3CL38-g-PBA33)-b-PCL23 (PaN3CL61-g-PBA56)-b-PCL32 (PaN3CL61-g-PBA61)-b-PCL112 (PaN3CL62-g-PME52)-b-PCL32 (PaN3CL28-g-PMPEG25)-b-PCL96 MPEG12-b-(PaN3CL38-g-PBA37)-b-PCL26

14/1 38/1 61/1 61/1 62/1 28/1 38/1

14/1 33/1 56/1 61/1 52/1 25/1 37/1

100 86 92 100 83 87 97

8940 14,788 23,060 32,191 20,523 32,850 15,488

8940 15,589 23,861 32,191 21,664 33,192 15,648

6470 7350 10,260 10,490 12,020 27,150 21,010

1.87 1.54 1.20 1.39 1.88 1.08 1.01

Tg (°C)e

Tm (°C)e

53.1 30.3 51.1 35.7 32.8 40.9 43.8

a

Abbreiviations: PBA = 2-propynyl benzoate; PME = 2-propynyl-2-methoxyethanol; PMPEG = 2-propynyl methyl poly(ethylene glycol). Determined from 1H NMR spectroscopy of (PaN3CL-g-alkyne)-b-PCL. c Mn,th = Mn,PaN3CL-b-PCL + Malkyne  [alkyne]/[PaN3CL-b-PCL] (where Mn,PaN3CL-b-PCL is the number-average molecular weight of PaN3CL-b-PCL, Malkyne is the molecular weight of alkyne, [alkyne] is the alkyne molar concentration, and [PaN3CL-b-PCL] is the polymer PaN3CL-b-PCL molar concentration). d Determined from GPC. e Determined from DSC thermograms. b

Fig. 3. Excitation spectra of (PaN3CL50-g-PMPEG48)-b-PCL94 copolymer monitored at kem = 390 nm.

Fig. 4. Plot of I338/I334 intensity ratio (from pyrene excitation spectra; pyrene concentration = 6.1  107 mol L1) versus logarithm of concentration (log C): (PaN3CL62-gPME52)-b-PCL32 (d); (PaN3CL99-g-PME90)-b-PCL44 (j); (PaN3CL141-g-PME130)-b-PCL140 (N); (PaN3CL230-g-PME230)-b-PCL147 (.).

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R.-J. Su et al. / Reactive & Functional Polymers 72 (2012) 36–44 Table 4 Properties of IMC-loaded (PaN3CL-g-PBA)-b-PCL copolymer micelles. Entries

CMC (mg L1)

Copolymer

Micelle size (nm)

1 (PaN3CL99-g-PME90)-b-PCL44

8.9

200.8 ± 3.7

2 3 4 5 6

(PaN3CL62-g-PME52)-b-PCL32 (PaN3CL141-g-PME130)-b-PCL140 (PaN3CL230-g-PME230)-b-PCL147 (PaN3CL28-g-PMPEG25)-b-PCL96 (PaN3CL50-g-PMPEG48)-b-PCL94

16 39.8 8.2 12.6 10.4

161.3 ± 17.9 – 230.3 ± 4.6 140.5 ± 1.1 149.4 ± 1.3

Feed weight ratio IMC/polymer

Drug entrapment efficiency (%)

Drug loading content (%)

1/2 1/1 2/1 1/1 1/1 1/1 1/1 1/1

53.5 39.1 34.6 16.2 10.2 33.7 20.1 22.3

17.9 19.6 23.0 8.1 5.1 16.9 10.1 11.2

Fig. 5. TEM images of micelles formed by (PaN3CL99-g-PME90)-b-PCL44: (a) without drug; (b) with drug.

The mean hydrodynamic diameters of micelles without IMC from DLS ranged from 140 to 230 nm. Fixing the concentration at 50-fold of the CMC value (50  CMC), the mean diameter of micelles increased with increasing ratio of the lengths of the hydrophilic segment to the hydrophobic segment. When the drug was incorporated, the size of micelle increased. Fig. 5 shows a similar trend in micelle morphology. 3.5. Evaluation of drug loading content and drug entrapment efficiency This work calculated the amount of IMC incorporated into graftblock (PaN3CL-g-PME)-b-PCL micelles by the ratio of the weight of IMC in the nanosphere to the pre-weighted IMC-loaded micelles, calculated by absorbance measurement after removing free IMC by filtration. Table 4 shows the amount of IMC introduced into the micelle by controlling the weight ratio between the drug and the polymer. Drug entrapment efficiency and drug loading content decreased with the weight ratio of drug to polymer and were dependent on the polymer composition described. At a constant feed weight ratio (1/1), drug entrapment efficiency and drug

Fig. 6. IMC released from the micelles of (PaN3CL230-g-PME230)-b-PCL147 (j), PaN3CL141-g-PME130)-b-PCL140 (N), (PaN3CL99-g-PME90)-b-PCL44 (d), (PaN3CL50-gPMPEG48)-b-PCL94 (x), and (PaN3CL28-g-PMPEG25)-b-PCL96 ().

loading content increased with the ratio of hydrophilic/hydrophobic segments. Increasing micelle stability increased the amounts of drug entrapped in the micelles. 3.6. In vitro release of IMC Drug release tests, performed at 37 °C with different IMCloaded (PaN3CL-g-PME)-b-PCL and (PaN3CL-g-PMPEG)-b-PCL nanoparticles, investigated the effects of polymer composition on drug release behavior. Fig. 6 shows that the IMC-loaded (PaN3CLg-PME)-b-PCL and (PaN3CL-g-PMPEG)-b-PCL nanoparticles exhibit well-developed sustained drug release patterns with a trend for faster drug release with increasing the length of hydrophilic block or decreasing length of hydrophobic block. Increasing the length of hydrophilic block or decreasing the length of hydrophobic block caused the hydrophilicity of micelles to increase. These findings

Fig. 7. Weight loss of (PaN3CL31-g-PME31)-b-PCL48 (⁄), (PaN3CL61-g-PBA61)-bPCL112 (N), (PaN3CL14-g-PBA14)-b-PCL38 (), (PaN3CL38-g-PBA33)-b-PCL23 (j), and hydrogenolysis of (PaN3CL14-g-PBA14)-b-PCL38 (x) treated in PBS (0.1 M, pH = 7.4) at 37 °C.

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R.-J. Su et al. / Reactive & Functional Polymers 72 (2012) 36–44

ties. The average micelle size is approximately 140–230 nm. Varying the graft-block copolymer composition alters micelle sizes. These copolymers degrade under physiological conditions. Results also demonstrated that the (PaN3CL-g-alkyne)-b-PCL had low cytotoxicity. The graft-block copolymers demonstrate high potential for application in drug delivery. Acknowledgements This research was supported by Grants from the National Science Council (NSC 97-2221-E-182-009) and Chang Gung University (BMRP 123). References

Fig. 8. The cell viability of bladder cancer cell (MGH-U1) at various polymer concentrations (n = 6).

indicate that varying the length of hydrophilic or hydrophobic block modulates drug release from the micelles. 3.7. Preliminary in vitro degradation and cell viability study As a biodegradable model, this study evaluated the in vitro degradation of graft-block (PaN3CL-g-alkyne)-b-PCL from the weight loss of the sample in a thin film at 37 °C under physiological conditions (pH 7.4). Fig. 7 presents the findings. After immersion for 30 days, copolymer weight loss was minimal. Increasing the molecular weight and the length of the hydrophobic segment in the copolymers decreased their hydrophilicity, resulting in lower molecular weight losses. MTT assay using MGH-U1 human bladder cancer cells quantified the in vitro cytotoxicities of the (PaN3CL62-g-PME52)-b-PCL32 polymer at various concentrations. The wells containing only media without polymer were treated as positive controls, with a cell viability of 100%. The equation [Abs]sample/[Abs]control  100 calculated relative cell viability. Fig. 8 compares the cell viability of MGH-U1 cells at various polymer concentrations of (PaN3CL62-gPME52)-b-PCL32. At a concentration lower than 250 lg mL1, with cell survival up to 90%, the polymer demonstrated no significant cytotoxicity. 4. Conclusions The present study investigated a click strategy for modifying the hydrophilic-hydrophobic balance of polymers and successfully prepared novel graft-block amphiphilic copolymers consisting of a hydrophilic segment, PaN3CL-g-PME or PaN3CL-g-PMPEG, and a hydrophobic segment (PCL). This work conducted structural analyses, including 1H NMR and FT-IR, to confirm the conjugation of these polymers. Thermal analysis revealed that (PaN3CL-g -alkyne)-b-PCL copolymers have lower melting points than their corresponding homopolymers. Varying the amounts of PaN3CL -g-alkyne or PCL in copolymers modifies their hydrophilic proper-

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