- Email: [email protected]
Synthesis, characterization and biocompatibility studies of oligosiloxane modified polythiophenes
European Polymer Journal 39 (2003) 1405–1412 www.elsevier.com/locate/europolj
Synthesis, characterization and biocompatibility studies of oligosiloxane modified polythiophenes Marlene Waugaman, Biswajit Sannigrahi, Paul McGeady, Ishrat M. Khan
*
Department of Chemistry, Clark Atlanta University, Atlanta, GA 30314, USA Received 12 September 2002; received in revised form 10 January 2003; accepted 25 January 2003
Abstract This paper describes the preparation and characterization of homopolymers of 3-oligo(dimethylsiloxane)thiophene macromonomers, V–VIII, and copolymers with 3-methylthiophene. The thiophene macromonomers were prepared by hydrosilylation reaction between x-(Si–H)-oligo(dimethylsiloxane), I–IV, and 3-propenylthiophene using a platinumdivinyltetramethyldisiloxane complex as the catalyst. The products were characterized by 1 H, 13 C, 29 Si NMR and IR spectroscopy; DSC (differential scanning calorimetry) and GPC studies. Two distinct glass transition temperatures are observed for poly[VIII], a Tg at )79 °C corresponds to the soft oligo(dimethylsiloxane) phase and the Tg at 190 °C corresponds to the hard thiophene backbone. Homopolymers of V and VI, and copolymers may be doped with I2 to generate electronic conductive material, a copolymer of poly[V]-co-poly[3-methylthiophene] (50/50, w/w) has an electronic conductivity value of 5 105 S/cm at 25 °C. The polymers are tractable and may be molded into thin films; a number of the polymers are soluble in organic solvents. Polythiophene modified with oligosilioxanes are biocompatibile; the polymers minimally interfere with the growth of HeLa cells. Ó 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction There is considerable interest in electronic conductive polymers for application in biological and therapeutic systems [1]. Electronic conductive polymers have been utilized in tissue engineering and drug delivery systems [2–5]. Polypyrrole, with good electronic conductive properties and biocompatibility, has been utilized in a number of biomedical applications including biosensors and controlled drug delivery [5]. Biofunctional activity (secretory function of adrenal chromaffin cells) of polypyrrole (PPy) on an indium–tin oxide (ITO) electrode has been reported [6]. The processability of polypyrrole is easily improved by modification to obtain materials with requisite properties necessary for fabrication into device components [7,8]. Additionally, the relationship between the polypyrrole structure with cell attachment and proliferation has been studied. The
study suggests that factors such as hydrophobicity, hydrophilicity and charge density play important role in interaction with cells [1]. Polythiophenes are an excellent class of electronic conductive polymers and may be tailored (synthetically) to provide processable electronic conductive materials [9–11]. To the best of our knowledge, correlation between the structure of polythiophene based polymers with cell attachment and growth has not studied. In this paper, we report the preparation and characterization of soluble and biocompatible oligosiloxane modified polythiophenes. Oligosiloxanes were used as the modifying agent because polysiloxanes are physiologically inert and are attractive for biomedical applications [12].
2. Experimental procedures 2.1. General
*
Corresponding author. Fax: +1-404-880-6849. E-mail address: [email protected] (I.M. Khan).
Unless otherwise noted all glassware were flame dried. Chromatographic silica gel (230–425 mesh, Fisher),
0014-3057/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0014-3057(03)00035-1
1406
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
ranged between 40% and 50%. The products were characterized by 1 H, 13 C and 29 Si NMR and IR spectroscopy. IR (NaCl, 2130 cm1 ).
n-butyl lithium (1.6 M and 2.4 M solutions in hexanes, Aldrich), chlorodimethylsilane (CDMS, Acros), hexamethylcyclotrisiloxane (D3 , Acros) and platinumdivinylhexamethyldisiloxane complex (Huls) were used as received. Hexanes (Fisher) were distilled from calcium hydride. Tetrahydrofuran (THF, Fisher) and diethyl ether (Fisher) were distilled under nitrogen gas from sodium/benzophenone. 3-bromothiophene (3BT, Acros) was distilled from calcium hydride. Allyl bromide was distilled from magnesium sulfate. 3-Methylthiophene (Aldrich, 98%) was purified by stirring with CaH2 overnight followed by distillation.
2.4. Preparation of 3-[oligo(dimethylsiloxane)]thiophene macromonomers [V–VIII] The macromonomers were prepared by the hydrosilylation of I–IV with 3-PT using platinum-divinyltetramethyldisiloxane complex in THF (Scheme 2). The macromonomers were purified by first removing the THF on a rotary evaporator, ammonium hydroxide was added to the resulting black liquid, and the liquid was extracted with three 100 ml portions of hexanes. The organic layer was extracted with 100 ml portions of water until the waterÕs pH reached 6.5. The macromonomers were obtained in 35–50% yield. The products were characterized by 1 H, 13 C and 29 Si NMR and IR spectroscopy.
2.2. Preparation of 3-propenylthiophene (3-PT) 3-PT was prepared utilizing RiekeÕs method [13]. 4.9 g (30 mmol) of 3-bromothiophene was added to 24.4 ml (39 mmol) of 1.6 M n-butyl lithium in 30 ml of hexanes at )60 °C. Following the addition of 3 ml of THF and another 10 ml of hexanes, 4.1 g (34 mmol) of allyl bromide was added dropwise over a 15 min period at )60 °C. The reaction was allowed to proceed for 1 h. 3PT was purified by vacuum distillation, followed by silica gel chromatography using hexanes as the eluant. 3PT was obtained in 60% yield as a clear liquid. 1 H NMR (CDCl3 , d relative to TMS): 3.44 (2H), 5.12 (1H), 5.17 (1H), 6.03 (1H), 6.94 (1H), 6.97 (1H) and 7.29 (1H). GCMS: m/e (Mþ 124).
2.5. Homopolymerization and copolymerization Homopolymerization of the macromonomers [V– VIII] and copolymerization with 3-methylthiophene were carried out by oxidative coupling with FeCl3 in CHCl3 for 24 h. at room temperature by the methods reported earlier [14]. Poly[3-methylthiophene] was prepared by the same method.
2.3. Preparation of x-(Si–H)-oligo(dimethylsiloxane) [I–IV]
2.6. Measurements
The functional PDMS oligomers were prepared by dissolving 5.0 g (22.4 mmol) of D3 in 50 ml of THF. Upon cooling the flask to )78 °C, appropriate amount of 1.6 M n-BuLi (amount dependent on the desired repeat unit for the oligomers) was added to the solution (Scheme 1). After 30 min, the reaction was terminated by the addition of chlorodimethylsilane. After removal of the solvent, the salt was separated from the oligomer by precipitation into chloromethane. The oligomeric product was purified by precipitation into methanol. The oligomers were a clear viscous liquid and yields
Routine 1 H NMR spectra were recorded on a Bruker ARX 400 NMR spectrometer in CDCl3 . Tetramethylsilane was used as the internal standard. Routine 13 C CP-MAS NMR were recorded on a Bruker MSL 200 solid state NMR spectrometer. DSC studies were carried out on a Perkin–Elmer DSC-4 under dry nitrogen. The DSC was calibrated using indium as standard. The thermograms were obtained at a heating rate of 20 °C per minute and the reported values were obtained from the second heating after quench cooling at a rate of 320 °C per minute. The Tg Õs were taken at the midpoints of
CH 3 1.
CH3
BuLi/-78 o
2. Me2SiHCl Si
CH3
C
O
Bu
Si CH 3
3 CH3
I - IV n = 3 [I],10 [II], 25 [III], 50 [IV]
Scheme 1.
O
Si n
CH3
H
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
CH3 Bu
Si
1407
CH3 O
+
H
Si n
S
CH3
CH3
Pt catalyst/25 O C
CH3 Si
CH2CH2CH2
CH3
CH3 O
Si
Bu
n
CH3
S V -VIII n = 3 [V], 10 [VI], 25 [ VII], 50 [VIII ]
Scheme 2.
the heat capacity changes, the Tm Õs were taken at the maximum of the enthalpy endothermic peaks. GPC studies were carried out on a Perkin–Elmer 250 liquid chromatography system using toluene as solvent. Polystyrene standards were used for calibration. IR spectra were obtained on a Nicolet Impact 400 spectrometer. Doping and conductivity measurements: Iodide was used in vapor phase doping of the polymers under vacuum for 72 h. The doped polymers were pressed into pellets under three metric tons of pressure. Electroactive characterization of the polymers was performed using the four point probe as published earlier [15]. A Fluke 79 Series II multimeter was used to measure the potential difference between the probes. The outer two probes tips were connected to a Peschel Instrument Inc. power generator that supplied a constant current. 2.7. Cell culture HeLa Cells (human ovarian cancer cells)(ATTC) were cultured in RPMI-1640 medium (Cellgro, USA), supplemented with 5% fetal bovine serum (FBS) and 1% antibiotic solution. Five mg of oligo(siloxane) modified polythiophenes in fine powdery form was placed into the bottom of a 24-well polystyrene tissue culture plate (Corning Incorporated, USA). Polymers in the well were sterilized by 70% alcohol. 1 ml of cell suspension with a cell concentration of about 2.0 104 /ml were added to each
well. Cells were incubated under 5% CO2 /95% air at 37 °C for 7 days. In this study, unmodified wells were used as control. The cells attachment and growth observed by microscopy (Nikon-TMS, Japan) and digital photomicrographs were recorded (Sony-CCD-IRIS, Japan). 3. Results and discussion 3.1. Structural characterization of I–VIII The functional oligosiloxanes (I–IV) were synthesized by the ring opening polymerization of D3 using n-BuLi as initiator (Scheme 1). The repeat units of the functional oligomers ranged from 4 to 50. The 400 MHz 1 H NMR spectrum for the functional oligomer I is shown in Fig. 1. The NMR spectrum is consistent with the structure of the oligomer; the peak at 0.05 ppm is the resonance of the protons of the repeat unit, –[SiO(CH3 )2 ]n –; the peak at 0.17 ppm correspond to the protons of the terminal dimethylsiloxane, –Si(CH3 )2 H; the triplet at 0.53 ppm (protons of the methylene of the butyl group attached to a silicon atom, CH3 CH2 CH2 CH2 [SiO(CH3 )2 ]n ); a triplet at 0.87 ppm (protons of the terminal methyl of the butyl group, CH3 CH2 CH2 CH2 –); a peak at 1.31 ppm (the internal methylenes of the butyl group, CH3 CH2 CH2 CH2 –) and the multiplet at 4.69 ppm is the resonance of the terminal x-(Si–H). The 13 C and 29 Si NMR spectra are consistent with the structure of the functional oligomers.
1408
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
Fig. 1. 400 MHz 1 H NMR spectrum of I.
The 79.5 MHz 29 Si NMR spectrum of I is shown in Fig. 2. The peak at )21.5 ppm corresponds to the c-silicon and the one at )20.0 ppm to the b-silicon (BuSi(CH3 )2 OSi(CH3 )2 OSi(CH3 )2 –); the )6.87 ppm peak is assigned to the (OSi(CH3 )2 H) silicon nuclei and the peak at 7.73 is due to the a-silicon nuclei (BuSi(CH3 )2 OSi(CH3 )2 Si(CH3 )2 –). The experimental Mn , as determined by 1 H NMR end group analysis, of the functional oligomers agreed with the theoretical values. The degree of functionalization of the purified x-(Si– H)-oligo(dimethylsiloxane)s [I–IV] was determined from 1 H NMR spectra by comparing the ratio of the methyl protons attached to the x-terminal silicon with the amethylene of the butyl group. For lower molecular weight oligomers [I–II], the degree of functionalization were close to 100%, and for III it was 85%. IR spectra of I–IVshow strong absorption band at 2130 cm1 for the Si–H stretching [16]. The thiophene macromonomers [V–VIII] were prepared by the hydrosilylation reaction between the x-(Si–H)-oligo(dimethylsiloxane) oligomers and 3-pro-
Fig. 2. 79 MHz
29
penylthiophene (Scheme 2). The addition reaction was catalyzed by a platinum-divinyltetramethyldisiloxane complex. The 400 1 H NMR spectrum for the macromonomer V is shown in Fig. 3. The spectrum show the following resonances: a the peak at 0.08 ppm (–[SiO(CH3 )2 ]n –); a peak at 0.59 ppm (CH2 [SiO(CH3 )2 ]n ); a resonance at 0.90 ppm (CH3 CH2 CH2 CH2 –); a peak at 1.33 ppm (CH3 CH2 CH2 CH2 –); a multiplet at 1.68 ppm (–CH2 CH2 CH2 –[SiO(CH3 )2 ]n ); the peak at 2.67 ppm (–CH2 CH2 CH2 –[SiO(CH3 )2 ]n ) is the methylene group attached to the thiophene and the aromatic proton resonances are between 6.9 and 7.4 ppm. The resonances are consistent with the structure of the macromonomer. The 1 H NMR spectrum is complemented by 29 Si NMR. The silicon resonances are as follows: )21.59 to )20.00 ppm (silicons of the repeat units –[SiO(CH3 )2 ]); 6.87 ppm (Si(CH3 )2 H); and 7.73 ppm (a-silicon of the repeat unit, BuSiO(CH3 )2 –). The hydrosilylation reaction was followed by IR and NMR spectroscopy. The reaction was run until no residual Si–H bonds could be detected by IR.
Si NMR spectrum of I.
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
1409
Fig. 3. 400 MHz 1 H NMR spectrum of V.
Table 1 Molecular weights of V, VI and VIII
V VI VIII
Mn (Theory)
Mw (GPC)
Mn (GPC)
450 900 3900
– – 6000
300 980 4500
Mw =Mn
1.3
The molecular weights of the various macromonomers are listed in Table 1. The observed Mn values were close to the theoretical values. The highest molecular weight macromonomer VIII shows a polydispersity of 1.3. Because of the rather low molecular weights of macromonomers V–VI, the molecular weight distributions were not determined. The functional oligomers I– IV did not elute through the GPC column, most likely, because the Si–H may have reacted with the column packing material. The degree of functionality of the purified macromonomers, as determined by integrating 1 H NMR spectra, was close to 100%. Therefore, following purification, via this approach pure 3(oligodimethylsiloxane)thiophene macromonomers may be obtained.
Table 2 Glass transition and melting temperature (°C) of functional oligomers and macromonomers Tg
Tm
Oligomers I II III IV
)144 )139 )135 )132
– – – )45
Macromonomers V VI VII VIII
)122 )129 )118 )120
– – – )46
3.2. Thermal studies Differential scanning calorimetry (DSC) data for the functional oligomers and the macromonomers are listed in Table 2. The DSC thermograms of three of the oligomers are shown in Fig. 4. The glass transition (Tg ) of the x-(Si–H)-oligo(dimethylsiloxane)s [I–IV] varied from )144 to )132 °C, the Tg value increasing with increasing molecular weight. Segmental motion, a second order transition is even observed for the oligomer with four siloxane repeat units. At such low temperatures, translational motion may not be possible and thus segmental motion is all that is present. The oligomer IV
Fig. 4. DSC thermograms of V, VII and VIII.
with 50 repeat units shows endothermic melting of crystalline forms at )45 °C. With the addition of the thiophene group, the Tg values of the macromonomers [V–VIII] are shifted to higher temperatures, with
1410
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
observed Tg values around )120 °C. Also, for VIII, a crystalline melting temperature is observed at )46 °C. 3.3. Characterization of the homopolymers and the copolymers Homopolymers and copolymers were prepared by oxidative coupling with FeCl3 in CHCl3 . The homopolymers and copolymers form aggregates in NMR solvents and hence solution NMR cannot be obtained. Apparently, the aggregates do no have sufficient motion in the NMR timescale and thus the polymers are characterized by CP-MAS NMR. The 13 C CP-MAS of a poly(V)-co-poly(3-methylthiophene) (75/25, w/w) is shown in Fig. 5. The carbons of the methyl groups attached to silicon atoms are observed at zero ppm. The alkyl carbons appear between 10 and 40 ppm and the aromatic thiophene carbons are between 120 and 150 ppm. The glass transition temperatures of the homopolymers are observed at around 190 °C and are listed in Table 3. Poly(VIII) also shows a low Tg at )79 °C, indicating that the polymer forms a microphase separated system. The formation of a microphase separated system has been observed in comb homopolymers of 3-(2,5,8,11,14,17,20,23-octaoxyhexadodecyl)thiophene, abbreviated as P[3-(OHDT)], by the observation of two glass transition temperatures [15]. The lower Tg corresponded to the oligo(oxyethylene) side chain and the higher Tg was attributed to the hard polythiophene phase of P[3-(OHDT)]. A 40°C increase in the low Tg between the macromonomer VIII and poly(VIII) may
Table 3 Glass transition temperature (°C) of homopolymers Poly(V) Poly(VI) Poly(VII) Poly(VIII)
Tg1
Tg2
– – – )79
196 190 195 187
reflect a decrease in the free volume in the densely packed oligo(dimethylsiloxane) side chains in the comb polymer. This is similar to the trend observed for the P[3-(OHDT)] system. The poly[VI]-co-poly[3-methylthiophene] (50/50, w/w) and poly[VII]-co-poly[3-methylthiophene] (50/50, w/w) copolymers showed a single glass transition temperature between 170 and 190 °C. The homopolymers and copolymers are dark red to black powdery materials and may be molded into thin films. The materials are significantly more tractable than the parent poly(3-alkylthiophene)s. The homopolymer poly[VIII] is soluble in THF; copolymer poly[VII]co-poly[3-methylthiophene] (50/50, w/w) is soluble in THF and CHCl3 . At 25 °C, the iodine doped homopolymers of V and VI show conductivity values of 1.7 106 and 9.3 108 S/cm, respectively. P[3-(OHDT)] has been shown to have an electronic conductivity value of 3.6 105 S/cm [15]. The lower conductivities for V and VI are most likely due the difference in the nature of the side chain (siloxane versus ethylene oxide). Oligo(oxyethylene) phase is more dopant counter ion friendly compared to the hydrophobic oligo(dimethylsiloxane). The homopolymers of VII and VIII are non-conductive. Electronic conductive values may be increased (or tailored) by copolymerization with 3-methylthiophene. The iodine doped copolymer of poly[V]-co-poly[3-methylthiophene] (50/ 50, w/w) has an electronic conductivity of 5 105 S/cm at room temperature and this value is in the range of electronic conductivities observed for the 3-methylthiophene/3-(2,5,8,11,14,17,20,23-octaoxyhexadodecyl)thiophene comb copolymers [14]. 3.4. Biocompatibility studies
Fig. 5. 50 MHz 13 C CP-MAS of poly[V]-co-poly[3-methylthiophene] (75/25, w/w).
In Fig. 6, the inverted micrographs of HeLa cells grown in the absence and presence of the oligosiloxane modified thiophenes powders are shown. Poly[3-methylthiophene] were used as the polymer control, the cells did not attach to the thiophene homopolymers. Most likely, factors such as hydrophobicity, hydrophilicity and charge density are not favorable for protein adsorption and hence cell attachment on poly[3-methylthiophene] homopolymers is not observed. Cell attachment and proliferation studies were carried out on two copolymers. Panels A and D, are TCPS (tissue culture polystyrene)
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
1411
Fig. 6. Inverted micrographs of HeLa cells grown in the absence and presence of the oligosiloxane modified thiophenes. Panels A and D, are control after 18 h and 7 days, respectively. Panels B and E, are poly[VI]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively. Panels C and F, are poly[VII]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively.
control after 18 h and 7 days, respectively. Panels B and E, are poly[VI]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively. Panels C and F, are poly[VII]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively. The polymers appear to minimally interfere with the growth of HeLa cells. The only effect appears to be the somewhat slower growth in the presence of the polymer. The inverted micrographs show that the cells are of normal morphology and are growing over the polymer (appear underneath in the inverted microscope). The attachment and growth of cells on the siloxane modified poly[thiophene] polymers
suggest that modification with oligosiloxanes results in balancing the factors (e.g. hydrophobicity, hydrophilicity and charge density) required for interaction with cells. Cells were not counted because of difficulties encountered due to the presence of the polymer powder.
4. Conclusions Electronic conductive polymers that are biocompatible may be prepared by the homopolymerization of V– VIII and copolymerization with 3-methylthiophene. The
1412
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
polymers and copolymers are tractable materials and may be easily fabricated into thin films. The polymers appear to minimally interfere with the growth of HeLa cells. The only effect appears to be the somewhat slower growth in the presence of the polymer. It is possible to tailor the electronic properties and processability by preparing copolymers of V–VIII. The polymers are potentially important in tissue engineering applications.
Acknowledgements The authors thank National Institute of Health for research support through NIH/NIGMS/MBRS/SCORE Grant #SO6GM08247 and RCMI Grant #G12RR0306.
References [1] Shastri VR, Pishko MV. Biomedical applications of electroactive polymers. In: Wise DL, Wnek GE, Trantolo DJ, Cooper TM, Gresser JD, editors. Electrical and optical polymer systems: fundamentals, methods and application. New York: Marcel Dekker; 1998. p. 1031.
[2] Valentini R, Vargo T, Gardella J, Aebischer P. Biomaterials 1992;13:183. [3] Langer R, Vacanti JP. Science 1993;260:920. [4] Smith AB, Knowles CJ. Biotech Appl Biochem 1990; 12:661. [5] Miller LL, Zhou QX. Macromolecules 1987;20:1594. [6] Aoki T, Tanono M, Sanui K, Ogata N, Kumakura K. Biomaterials 1996;17:1971. [7] Ruckenstein E, Yang S. Polymer 1993;34:4655. [8] Ruckenstein E, Hng L. Synth Met 1994;66:249. [9] Ahn SH, Czae MZ, Kim ER, Lee H, Han SH, Noh J, Hara M. Macromolecules 2001;34:2522. [10] Cik G, Krajcovic J, Veis P, Vegh D, Sersen F. Synth Met 2001;118:111. [11] deSouza JM, Pereira EC. Synth Met 2001;118:167. [12] Stevens M. Polymer chemistry, an introduction. 3rd ed. NY: Oxford University Press; 1999. p. 427. [13] Wu X, Chen T, Zhu L, Rieke RD. Tetrahedron Lett 1994;35:3673. [14] Burke Jr LM, Khan IM. Macromol Chem Phys 2000;201: 2228. [15] Arnold S, Pratt L, Khan I. Smart materials. SPIE Proc 1995;2441:29. [16] Silverstein RM, Webster FX. Spectrometric identification of organic compounds. 6th ed. NY: Wiley & Sons; 1998. p. 108.
Synthesis, characterization and biocompatibility studies of oligosiloxane modified polythiophenes Marlene Waugaman, Biswajit Sannigrahi, Paul McGeady, Ishrat M. Khan
*
Department of Chemistry, Clark Atlanta University, Atlanta, GA 30314, USA Received 12 September 2002; received in revised form 10 January 2003; accepted 25 January 2003
Abstract This paper describes the preparation and characterization of homopolymers of 3-oligo(dimethylsiloxane)thiophene macromonomers, V–VIII, and copolymers with 3-methylthiophene. The thiophene macromonomers were prepared by hydrosilylation reaction between x-(Si–H)-oligo(dimethylsiloxane), I–IV, and 3-propenylthiophene using a platinumdivinyltetramethyldisiloxane complex as the catalyst. The products were characterized by 1 H, 13 C, 29 Si NMR and IR spectroscopy; DSC (differential scanning calorimetry) and GPC studies. Two distinct glass transition temperatures are observed for poly[VIII], a Tg at )79 °C corresponds to the soft oligo(dimethylsiloxane) phase and the Tg at 190 °C corresponds to the hard thiophene backbone. Homopolymers of V and VI, and copolymers may be doped with I2 to generate electronic conductive material, a copolymer of poly[V]-co-poly[3-methylthiophene] (50/50, w/w) has an electronic conductivity value of 5 105 S/cm at 25 °C. The polymers are tractable and may be molded into thin films; a number of the polymers are soluble in organic solvents. Polythiophene modified with oligosilioxanes are biocompatibile; the polymers minimally interfere with the growth of HeLa cells. Ó 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction There is considerable interest in electronic conductive polymers for application in biological and therapeutic systems [1]. Electronic conductive polymers have been utilized in tissue engineering and drug delivery systems [2–5]. Polypyrrole, with good electronic conductive properties and biocompatibility, has been utilized in a number of biomedical applications including biosensors and controlled drug delivery [5]. Biofunctional activity (secretory function of adrenal chromaffin cells) of polypyrrole (PPy) on an indium–tin oxide (ITO) electrode has been reported [6]. The processability of polypyrrole is easily improved by modification to obtain materials with requisite properties necessary for fabrication into device components [7,8]. Additionally, the relationship between the polypyrrole structure with cell attachment and proliferation has been studied. The
study suggests that factors such as hydrophobicity, hydrophilicity and charge density play important role in interaction with cells [1]. Polythiophenes are an excellent class of electronic conductive polymers and may be tailored (synthetically) to provide processable electronic conductive materials [9–11]. To the best of our knowledge, correlation between the structure of polythiophene based polymers with cell attachment and growth has not studied. In this paper, we report the preparation and characterization of soluble and biocompatible oligosiloxane modified polythiophenes. Oligosiloxanes were used as the modifying agent because polysiloxanes are physiologically inert and are attractive for biomedical applications [12].
2. Experimental procedures 2.1. General
*
Corresponding author. Fax: +1-404-880-6849. E-mail address: [email protected] (I.M. Khan).
Unless otherwise noted all glassware were flame dried. Chromatographic silica gel (230–425 mesh, Fisher),
0014-3057/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0014-3057(03)00035-1
1406
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
ranged between 40% and 50%. The products were characterized by 1 H, 13 C and 29 Si NMR and IR spectroscopy. IR (NaCl, 2130 cm1 ).
n-butyl lithium (1.6 M and 2.4 M solutions in hexanes, Aldrich), chlorodimethylsilane (CDMS, Acros), hexamethylcyclotrisiloxane (D3 , Acros) and platinumdivinylhexamethyldisiloxane complex (Huls) were used as received. Hexanes (Fisher) were distilled from calcium hydride. Tetrahydrofuran (THF, Fisher) and diethyl ether (Fisher) were distilled under nitrogen gas from sodium/benzophenone. 3-bromothiophene (3BT, Acros) was distilled from calcium hydride. Allyl bromide was distilled from magnesium sulfate. 3-Methylthiophene (Aldrich, 98%) was purified by stirring with CaH2 overnight followed by distillation.
2.4. Preparation of 3-[oligo(dimethylsiloxane)]thiophene macromonomers [V–VIII] The macromonomers were prepared by the hydrosilylation of I–IV with 3-PT using platinum-divinyltetramethyldisiloxane complex in THF (Scheme 2). The macromonomers were purified by first removing the THF on a rotary evaporator, ammonium hydroxide was added to the resulting black liquid, and the liquid was extracted with three 100 ml portions of hexanes. The organic layer was extracted with 100 ml portions of water until the waterÕs pH reached 6.5. The macromonomers were obtained in 35–50% yield. The products were characterized by 1 H, 13 C and 29 Si NMR and IR spectroscopy.
2.2. Preparation of 3-propenylthiophene (3-PT) 3-PT was prepared utilizing RiekeÕs method [13]. 4.9 g (30 mmol) of 3-bromothiophene was added to 24.4 ml (39 mmol) of 1.6 M n-butyl lithium in 30 ml of hexanes at )60 °C. Following the addition of 3 ml of THF and another 10 ml of hexanes, 4.1 g (34 mmol) of allyl bromide was added dropwise over a 15 min period at )60 °C. The reaction was allowed to proceed for 1 h. 3PT was purified by vacuum distillation, followed by silica gel chromatography using hexanes as the eluant. 3PT was obtained in 60% yield as a clear liquid. 1 H NMR (CDCl3 , d relative to TMS): 3.44 (2H), 5.12 (1H), 5.17 (1H), 6.03 (1H), 6.94 (1H), 6.97 (1H) and 7.29 (1H). GCMS: m/e (Mþ 124).
2.5. Homopolymerization and copolymerization Homopolymerization of the macromonomers [V– VIII] and copolymerization with 3-methylthiophene were carried out by oxidative coupling with FeCl3 in CHCl3 for 24 h. at room temperature by the methods reported earlier [14]. Poly[3-methylthiophene] was prepared by the same method.
2.3. Preparation of x-(Si–H)-oligo(dimethylsiloxane) [I–IV]
2.6. Measurements
The functional PDMS oligomers were prepared by dissolving 5.0 g (22.4 mmol) of D3 in 50 ml of THF. Upon cooling the flask to )78 °C, appropriate amount of 1.6 M n-BuLi (amount dependent on the desired repeat unit for the oligomers) was added to the solution (Scheme 1). After 30 min, the reaction was terminated by the addition of chlorodimethylsilane. After removal of the solvent, the salt was separated from the oligomer by precipitation into chloromethane. The oligomeric product was purified by precipitation into methanol. The oligomers were a clear viscous liquid and yields
Routine 1 H NMR spectra were recorded on a Bruker ARX 400 NMR spectrometer in CDCl3 . Tetramethylsilane was used as the internal standard. Routine 13 C CP-MAS NMR were recorded on a Bruker MSL 200 solid state NMR spectrometer. DSC studies were carried out on a Perkin–Elmer DSC-4 under dry nitrogen. The DSC was calibrated using indium as standard. The thermograms were obtained at a heating rate of 20 °C per minute and the reported values were obtained from the second heating after quench cooling at a rate of 320 °C per minute. The Tg Õs were taken at the midpoints of
CH 3 1.
CH3
BuLi/-78 o
2. Me2SiHCl Si
CH3
C
O
Bu
Si CH 3
3 CH3
I - IV n = 3 [I],10 [II], 25 [III], 50 [IV]
Scheme 1.
O
Si n
CH3
H
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
CH3 Bu
Si
1407
CH3 O
+
H
Si n
S
CH3
CH3
Pt catalyst/25 O C
CH3 Si
CH2CH2CH2
CH3
CH3 O
Si
Bu
n
CH3
S V -VIII n = 3 [V], 10 [VI], 25 [ VII], 50 [VIII ]
Scheme 2.
the heat capacity changes, the Tm Õs were taken at the maximum of the enthalpy endothermic peaks. GPC studies were carried out on a Perkin–Elmer 250 liquid chromatography system using toluene as solvent. Polystyrene standards were used for calibration. IR spectra were obtained on a Nicolet Impact 400 spectrometer. Doping and conductivity measurements: Iodide was used in vapor phase doping of the polymers under vacuum for 72 h. The doped polymers were pressed into pellets under three metric tons of pressure. Electroactive characterization of the polymers was performed using the four point probe as published earlier [15]. A Fluke 79 Series II multimeter was used to measure the potential difference between the probes. The outer two probes tips were connected to a Peschel Instrument Inc. power generator that supplied a constant current. 2.7. Cell culture HeLa Cells (human ovarian cancer cells)(ATTC) were cultured in RPMI-1640 medium (Cellgro, USA), supplemented with 5% fetal bovine serum (FBS) and 1% antibiotic solution. Five mg of oligo(siloxane) modified polythiophenes in fine powdery form was placed into the bottom of a 24-well polystyrene tissue culture plate (Corning Incorporated, USA). Polymers in the well were sterilized by 70% alcohol. 1 ml of cell suspension with a cell concentration of about 2.0 104 /ml were added to each
well. Cells were incubated under 5% CO2 /95% air at 37 °C for 7 days. In this study, unmodified wells were used as control. The cells attachment and growth observed by microscopy (Nikon-TMS, Japan) and digital photomicrographs were recorded (Sony-CCD-IRIS, Japan). 3. Results and discussion 3.1. Structural characterization of I–VIII The functional oligosiloxanes (I–IV) were synthesized by the ring opening polymerization of D3 using n-BuLi as initiator (Scheme 1). The repeat units of the functional oligomers ranged from 4 to 50. The 400 MHz 1 H NMR spectrum for the functional oligomer I is shown in Fig. 1. The NMR spectrum is consistent with the structure of the oligomer; the peak at 0.05 ppm is the resonance of the protons of the repeat unit, –[SiO(CH3 )2 ]n –; the peak at 0.17 ppm correspond to the protons of the terminal dimethylsiloxane, –Si(CH3 )2 H; the triplet at 0.53 ppm (protons of the methylene of the butyl group attached to a silicon atom, CH3 CH2 CH2 CH2 [SiO(CH3 )2 ]n ); a triplet at 0.87 ppm (protons of the terminal methyl of the butyl group, CH3 CH2 CH2 CH2 –); a peak at 1.31 ppm (the internal methylenes of the butyl group, CH3 CH2 CH2 CH2 –) and the multiplet at 4.69 ppm is the resonance of the terminal x-(Si–H). The 13 C and 29 Si NMR spectra are consistent with the structure of the functional oligomers.
1408
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
Fig. 1. 400 MHz 1 H NMR spectrum of I.
The 79.5 MHz 29 Si NMR spectrum of I is shown in Fig. 2. The peak at )21.5 ppm corresponds to the c-silicon and the one at )20.0 ppm to the b-silicon (BuSi(CH3 )2 OSi(CH3 )2 OSi(CH3 )2 –); the )6.87 ppm peak is assigned to the (OSi(CH3 )2 H) silicon nuclei and the peak at 7.73 is due to the a-silicon nuclei (BuSi(CH3 )2 OSi(CH3 )2 Si(CH3 )2 –). The experimental Mn , as determined by 1 H NMR end group analysis, of the functional oligomers agreed with the theoretical values. The degree of functionalization of the purified x-(Si– H)-oligo(dimethylsiloxane)s [I–IV] was determined from 1 H NMR spectra by comparing the ratio of the methyl protons attached to the x-terminal silicon with the amethylene of the butyl group. For lower molecular weight oligomers [I–II], the degree of functionalization were close to 100%, and for III it was 85%. IR spectra of I–IVshow strong absorption band at 2130 cm1 for the Si–H stretching [16]. The thiophene macromonomers [V–VIII] were prepared by the hydrosilylation reaction between the x-(Si–H)-oligo(dimethylsiloxane) oligomers and 3-pro-
Fig. 2. 79 MHz
29
penylthiophene (Scheme 2). The addition reaction was catalyzed by a platinum-divinyltetramethyldisiloxane complex. The 400 1 H NMR spectrum for the macromonomer V is shown in Fig. 3. The spectrum show the following resonances: a the peak at 0.08 ppm (–[SiO(CH3 )2 ]n –); a peak at 0.59 ppm (CH2 [SiO(CH3 )2 ]n ); a resonance at 0.90 ppm (CH3 CH2 CH2 CH2 –); a peak at 1.33 ppm (CH3 CH2 CH2 CH2 –); a multiplet at 1.68 ppm (–CH2 CH2 CH2 –[SiO(CH3 )2 ]n ); the peak at 2.67 ppm (–CH2 CH2 CH2 –[SiO(CH3 )2 ]n ) is the methylene group attached to the thiophene and the aromatic proton resonances are between 6.9 and 7.4 ppm. The resonances are consistent with the structure of the macromonomer. The 1 H NMR spectrum is complemented by 29 Si NMR. The silicon resonances are as follows: )21.59 to )20.00 ppm (silicons of the repeat units –[SiO(CH3 )2 ]); 6.87 ppm (Si(CH3 )2 H); and 7.73 ppm (a-silicon of the repeat unit, BuSiO(CH3 )2 –). The hydrosilylation reaction was followed by IR and NMR spectroscopy. The reaction was run until no residual Si–H bonds could be detected by IR.
Si NMR spectrum of I.
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
1409
Fig. 3. 400 MHz 1 H NMR spectrum of V.
Table 1 Molecular weights of V, VI and VIII
V VI VIII
Mn (Theory)
Mw (GPC)
Mn (GPC)
450 900 3900
– – 6000
300 980 4500
Mw =Mn
1.3
The molecular weights of the various macromonomers are listed in Table 1. The observed Mn values were close to the theoretical values. The highest molecular weight macromonomer VIII shows a polydispersity of 1.3. Because of the rather low molecular weights of macromonomers V–VI, the molecular weight distributions were not determined. The functional oligomers I– IV did not elute through the GPC column, most likely, because the Si–H may have reacted with the column packing material. The degree of functionality of the purified macromonomers, as determined by integrating 1 H NMR spectra, was close to 100%. Therefore, following purification, via this approach pure 3(oligodimethylsiloxane)thiophene macromonomers may be obtained.
Table 2 Glass transition and melting temperature (°C) of functional oligomers and macromonomers Tg
Tm
Oligomers I II III IV
)144 )139 )135 )132
– – – )45
Macromonomers V VI VII VIII
)122 )129 )118 )120
– – – )46
3.2. Thermal studies Differential scanning calorimetry (DSC) data for the functional oligomers and the macromonomers are listed in Table 2. The DSC thermograms of three of the oligomers are shown in Fig. 4. The glass transition (Tg ) of the x-(Si–H)-oligo(dimethylsiloxane)s [I–IV] varied from )144 to )132 °C, the Tg value increasing with increasing molecular weight. Segmental motion, a second order transition is even observed for the oligomer with four siloxane repeat units. At such low temperatures, translational motion may not be possible and thus segmental motion is all that is present. The oligomer IV
Fig. 4. DSC thermograms of V, VII and VIII.
with 50 repeat units shows endothermic melting of crystalline forms at )45 °C. With the addition of the thiophene group, the Tg values of the macromonomers [V–VIII] are shifted to higher temperatures, with
1410
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
observed Tg values around )120 °C. Also, for VIII, a crystalline melting temperature is observed at )46 °C. 3.3. Characterization of the homopolymers and the copolymers Homopolymers and copolymers were prepared by oxidative coupling with FeCl3 in CHCl3 . The homopolymers and copolymers form aggregates in NMR solvents and hence solution NMR cannot be obtained. Apparently, the aggregates do no have sufficient motion in the NMR timescale and thus the polymers are characterized by CP-MAS NMR. The 13 C CP-MAS of a poly(V)-co-poly(3-methylthiophene) (75/25, w/w) is shown in Fig. 5. The carbons of the methyl groups attached to silicon atoms are observed at zero ppm. The alkyl carbons appear between 10 and 40 ppm and the aromatic thiophene carbons are between 120 and 150 ppm. The glass transition temperatures of the homopolymers are observed at around 190 °C and are listed in Table 3. Poly(VIII) also shows a low Tg at )79 °C, indicating that the polymer forms a microphase separated system. The formation of a microphase separated system has been observed in comb homopolymers of 3-(2,5,8,11,14,17,20,23-octaoxyhexadodecyl)thiophene, abbreviated as P[3-(OHDT)], by the observation of two glass transition temperatures [15]. The lower Tg corresponded to the oligo(oxyethylene) side chain and the higher Tg was attributed to the hard polythiophene phase of P[3-(OHDT)]. A 40°C increase in the low Tg between the macromonomer VIII and poly(VIII) may
Table 3 Glass transition temperature (°C) of homopolymers Poly(V) Poly(VI) Poly(VII) Poly(VIII)
Tg1
Tg2
– – – )79
196 190 195 187
reflect a decrease in the free volume in the densely packed oligo(dimethylsiloxane) side chains in the comb polymer. This is similar to the trend observed for the P[3-(OHDT)] system. The poly[VI]-co-poly[3-methylthiophene] (50/50, w/w) and poly[VII]-co-poly[3-methylthiophene] (50/50, w/w) copolymers showed a single glass transition temperature between 170 and 190 °C. The homopolymers and copolymers are dark red to black powdery materials and may be molded into thin films. The materials are significantly more tractable than the parent poly(3-alkylthiophene)s. The homopolymer poly[VIII] is soluble in THF; copolymer poly[VII]co-poly[3-methylthiophene] (50/50, w/w) is soluble in THF and CHCl3 . At 25 °C, the iodine doped homopolymers of V and VI show conductivity values of 1.7 106 and 9.3 108 S/cm, respectively. P[3-(OHDT)] has been shown to have an electronic conductivity value of 3.6 105 S/cm [15]. The lower conductivities for V and VI are most likely due the difference in the nature of the side chain (siloxane versus ethylene oxide). Oligo(oxyethylene) phase is more dopant counter ion friendly compared to the hydrophobic oligo(dimethylsiloxane). The homopolymers of VII and VIII are non-conductive. Electronic conductive values may be increased (or tailored) by copolymerization with 3-methylthiophene. The iodine doped copolymer of poly[V]-co-poly[3-methylthiophene] (50/ 50, w/w) has an electronic conductivity of 5 105 S/cm at room temperature and this value is in the range of electronic conductivities observed for the 3-methylthiophene/3-(2,5,8,11,14,17,20,23-octaoxyhexadodecyl)thiophene comb copolymers [14]. 3.4. Biocompatibility studies
Fig. 5. 50 MHz 13 C CP-MAS of poly[V]-co-poly[3-methylthiophene] (75/25, w/w).
In Fig. 6, the inverted micrographs of HeLa cells grown in the absence and presence of the oligosiloxane modified thiophenes powders are shown. Poly[3-methylthiophene] were used as the polymer control, the cells did not attach to the thiophene homopolymers. Most likely, factors such as hydrophobicity, hydrophilicity and charge density are not favorable for protein adsorption and hence cell attachment on poly[3-methylthiophene] homopolymers is not observed. Cell attachment and proliferation studies were carried out on two copolymers. Panels A and D, are TCPS (tissue culture polystyrene)
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
1411
Fig. 6. Inverted micrographs of HeLa cells grown in the absence and presence of the oligosiloxane modified thiophenes. Panels A and D, are control after 18 h and 7 days, respectively. Panels B and E, are poly[VI]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively. Panels C and F, are poly[VII]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively.
control after 18 h and 7 days, respectively. Panels B and E, are poly[VI]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively. Panels C and F, are poly[VII]-co-poly[3-methylthiophene] (50/50, w/w), after 18 h and 7 days, respectively. The polymers appear to minimally interfere with the growth of HeLa cells. The only effect appears to be the somewhat slower growth in the presence of the polymer. The inverted micrographs show that the cells are of normal morphology and are growing over the polymer (appear underneath in the inverted microscope). The attachment and growth of cells on the siloxane modified poly[thiophene] polymers
suggest that modification with oligosiloxanes results in balancing the factors (e.g. hydrophobicity, hydrophilicity and charge density) required for interaction with cells. Cells were not counted because of difficulties encountered due to the presence of the polymer powder.
4. Conclusions Electronic conductive polymers that are biocompatible may be prepared by the homopolymerization of V– VIII and copolymerization with 3-methylthiophene. The
1412
M. Waugaman et al. / European Polymer Journal 39 (2003) 1405–1412
polymers and copolymers are tractable materials and may be easily fabricated into thin films. The polymers appear to minimally interfere with the growth of HeLa cells. The only effect appears to be the somewhat slower growth in the presence of the polymer. It is possible to tailor the electronic properties and processability by preparing copolymers of V–VIII. The polymers are potentially important in tissue engineering applications.
Acknowledgements The authors thank National Institute of Health for research support through NIH/NIGMS/MBRS/SCORE Grant #SO6GM08247 and RCMI Grant #G12RR0306.
References [1] Shastri VR, Pishko MV. Biomedical applications of electroactive polymers. In: Wise DL, Wnek GE, Trantolo DJ, Cooper TM, Gresser JD, editors. Electrical and optical polymer systems: fundamentals, methods and application. New York: Marcel Dekker; 1998. p. 1031.
[2] Valentini R, Vargo T, Gardella J, Aebischer P. Biomaterials 1992;13:183. [3] Langer R, Vacanti JP. Science 1993;260:920. [4] Smith AB, Knowles CJ. Biotech Appl Biochem 1990; 12:661. [5] Miller LL, Zhou QX. Macromolecules 1987;20:1594. [6] Aoki T, Tanono M, Sanui K, Ogata N, Kumakura K. Biomaterials 1996;17:1971. [7] Ruckenstein E, Yang S. Polymer 1993;34:4655. [8] Ruckenstein E, Hng L. Synth Met 1994;66:249. [9] Ahn SH, Czae MZ, Kim ER, Lee H, Han SH, Noh J, Hara M. Macromolecules 2001;34:2522. [10] Cik G, Krajcovic J, Veis P, Vegh D, Sersen F. Synth Met 2001;118:111. [11] deSouza JM, Pereira EC. Synth Met 2001;118:167. [12] Stevens M. Polymer chemistry, an introduction. 3rd ed. NY: Oxford University Press; 1999. p. 427. [13] Wu X, Chen T, Zhu L, Rieke RD. Tetrahedron Lett 1994;35:3673. [14] Burke Jr LM, Khan IM. Macromol Chem Phys 2000;201: 2228. [15] Arnold S, Pratt L, Khan I. Smart materials. SPIE Proc 1995;2441:29. [16] Silverstein RM, Webster FX. Spectrometric identification of organic compounds. 6th ed. NY: Wiley & Sons; 1998. p. 108.