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Journal of Chemistry
Volume 2014 (2014), Article ID 173814, 7 pages
Research Article

Synthesis, Characterization and In Vitro Anticancer Evaluation of Itaconic Acid Based Random Copolyester

PG & Research Department of Chemistry, Pachaiyappa’s College, Chennai 600 030, India

Received 23 May 2013; Revised 4 October 2013; Accepted 22 October 2013; Published 16 January 2014

Academic Editor: Xinyong Liu

Copyright © 2014 J. Gowsika and R. Nanthini. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The present study deals with the synthesis and characterization of an aliphatic copolyester, poly [butylene fumarate-co-butylene itaconate] (PIFB) copolymer was obtained from itaconic acid, fumaric acid, and 1,4-butanediol using titanium tetraisopropoxide (TTiPO) through a two step process of transesterification and melt polycondensation. The synthesized aliphatic random copolyester was characterized with the help of FT-IR, 1H-NMR, 13C-NMR, viscosity measurements, Gel Permeation Chromatography (GPC) and X-ray diffraction (XRD) analysis. Thermal properties have been analyzed using thermogravimetric analysis (TGA) and Differential Scanning Calorimetry (DSC). Hydrolytic degradation studies were carried out in acid and alkaline regions of various pH values. The synthesized copolymer was subjected to in vitro anticancer activity studies against human breast cancer (MCF-7) cell line.

1. Introduction

The polymer synthesis was found to be an effective development on human chemotherapy. Polyesters, polyanhydrides, and so forth are mainly used in pharmaceutical, biomedical, soft tissue engineering, and drug delivery. Drug device polyester is used for plasma expanders and also tablet coating. Whenever a new drug molecule is synthesized, it is given orally or injected into the affected tissues. Nevertheless, this system of intake has disadvantage like an undesirable effect, poor drug efficiency, duration, concentration, bioavailability, and the drug that might not be controlled. To overcome this drawback, a new controlled release technology was developed. In this technology, a drug remains inside the human body for a prolonged period of time by releasing in a controlled manner [1]. The first drug delivery application is reported using hydrogel in 1960 [2]. In the beginning, biodegradable poly (glycolic acid) and poly (lactic acid) were used for tissue engineering system [35]. Later, poly (lactide-co-glycolide) was synthesized for medical application like dental implant and scaffold for bone TE [6]. In recent years, biodegradable polyesters are widely used for drug delivery, especially for anticancer drugs [7, 8]. Biodegradable polyesters have also attracted much attention as green materials and biomaterials in biodegradable fibers, nonwovens, films, sheets, bottles, injection-molded products, pharmaceutical, medical, biomedical engineering applications including drug delivery systems, and functional materials in tissue engineering [911]. Polyesters have good biocompatibility and biodegradation property which are concluded by many researchers in the past decades. Aliphatic polyesters with alkane diol like ethylene glycol, propylene glycol, butane diol, and so forth have good biodegradation some of polymers have biocompatibility nature [1215]. Biodegradable and biocompatible polyester poly (propylene fumarate) has been used for bone cement, bone tissue engineering, and drug delivery [1618]. Nowadays biodegradable polyesters are studied for drug release application; here polyesters are used only for the purpose of controlled delivery at targeted position. While releasing a drug, polyester also undergoes some activity against targeted cells. It should happen when the polymer has cytotoxic and anti-cancer activity on it. It is possible when preparation of polyester is using bioactive monomers [19]. Bioactive monomers, on polymerization enhance their bioactive nature. In the present study, it is proposed to synthesize polyester using a bioactive monomer which itself has good anti-cancer activity and drug release system. In order to enhance the biocompactibility and biodegradation, a polymer is designed from fumaric acid, 1,4-butanediol, and itaconic acid. Itaconic acid is a naturally occurring bioactive monomer and is also derived from citric acid. It can be obtained directly from fermentation of glucose [20]. Itaconic acid is used to enhance bioactivities when it undergoes polymerization and can be used in tissue engineering [21]. With all the above facts in mind, we have to synthesize an aliphatic copolyester using fumaric acid, 1,4-butanediol, and itaconic acid.

2. Experimental

2.1. Materials

Itaconic acid, fumaric acid and 1,4-butanediol were purchased from Sigma Aldrich. Titanium tetraisopropoxide, used as a catalyst, was purchased from Lancaster. All other chemicals and solvents (AR Grade) were used as such. MCF-7 cell line was utilized from King Institute, Guindy, Chennai. The cells were maintained in Minimal Essential Media (MEM) supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified atmosphere of (50 μg/mL) CO2 at 37°C. MEM was purchased from Hi Media Laboratories and Fetal bovine serum (FBS) was purchased from Cistron laboratories. Trypsin, methylthiazolyl diphenyl- tetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO) were purchased from Sisco Research Laboratory Chemicals, Mumbai. All other chemicals and reagents were purchased from Sigma Aldrich, Mumbai.

2.2. Instruments

FT-IR spectrum of copolyester was recorded using Perkin Elmer IR spectrometer in the range of 4000–400 cm−1 using KBr pellets. 1H NMR and 13C-NMR spectra were recorded on Bruker 300 MHz and 70 MHz instruments. Viscosity of polyester was performed in CHCl3 at room temperature using Ubbelohde Viscometer. Gel Permeation Chromatography (GPC) analysis was used to determine number average molecular weight () and weight average molecular weight of the polymer () in THF. The flow rate was 1 mL/min which was performed at room temperature. Bruker B8 diffractometer with Cu radiation was used for assessing the crystallinity of the polymer. The sample was scanned over the range angle (2θ), from 5° to 80°. Hydrolytic degradation study was done using phosphate buffer (PH-7.4) and gradual degradation of polymer was observed. DSC thermogram was recorded on a DSC Q200 V24.10 Build 122 differential scanning calorimeter. About 2–4 mg of the polymer sample was heated in an aluminium pan with pierced lid under nitrogen atmosphere at a scanning rate of 10°C/min between the temperature −80°C and 500°C.

2.3. Synthesis of Copolyester

The aliphatic copolyester was synthesized by a two-step melt polycondensation and transesterification method as follows. A mixture of fumaric acid (0.01 mol), itaconic acid (0.01 mol), and 1,4-butanediol (0.02 mol) with 0.1 mmol of TTiPO as catalyst taken in reaction bath was slowly heated to 160°C and stood for 2 h under dry nitrogen atmosphere and after 2 h the temperature was further increased to 190°C and kept under vacuum for 1 h to remove the traces of water. The white amorphous copolyester obtained was purified by dissolution in CHCl3 and reprecipitated in 10 fold of ice cold methanol, then dried in a vacuum at 40°C (yield: 98%) (Scheme 1).

Scheme 1: Synthetic route of copolyester.
2.4. Anticancer Evaluation

In vitro biocompatibility assessment was done using MCF-7 cell line which was maintained in Minimal Essential Media (MEM) sublimented with Fetal bovine serum (FBS), penicillin (100 μg/mL) and streptomycin (100 μg/mL) in a humidified atmosphere of 50 μg/mL CO2 at room temperature.

The cytotoxicity of polymers on MCF-7 was determined by the MTT assay [22]. Cells (1 × 105/well) were plated in 1 mL of medium/well in 24-well plates (Costar Corning, Rochester, NY). After 48 hours of incubation, the cell reaches the confluence. Then, cells were incubated in the presence of various concentrations of the samples in 0.1% DMSO for 48 h at 37°C. After removal of the sample solution and washing with phosphate-buffered saline (pH 7.4) 200 μL/well, 5 mg/mL of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) phosphate-buffered saline solution was added. After 4 h incubation, 0.04 M HCl/isopropanol was added. Viable cells were determined by the absorbance at 570 nm. Measurements were performed and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The absorbance at 570 nm was measured with a UV- Spectrophotometer using wells without sample containing cells as blanks. The effect of the polymers on the proliferation of MCF-7 was expressed as the % cell viability, using the following formula:

3. Results and Discussion

3.1. Viscosity Measurement and GPC Analysis

Inherent viscosity of the polymer was reported using chloroform at the concentration of 1 mg/mL using Ubbelohde Viscometer by calculating the values of flow time of pure solvent and polymer. The inherent viscosity of the polymer PIFB is 0.86 dL/g. GPC is used to analyze the number average molecular weight () and weight average molecular weight (). From these values, we can calculate poly dispersity () of the synthesized polymer. In the GPC technique, a single peak which differs in magnitude and elution time was received. From the above polydispersity index and value, we concluded that polymer formed in a random manner and lower molecular weight of the polymer is suitable for the biological application.

3.2. IR Spectral Studies

FT-IR spectrum of synthesized copolyester is presented in Figure 1. The copolyester showed characteristic absorption band for ester carbonyl stretching at 1723 cm−1. The polymer was observed peaks at 1040, 2900, and 1400 cm−1. These peaks are assigned to C–O–C stretching, aliphatic C–H of methylene, and C–C stretching respectively. The IR data of the polymer is shown in Table 1. A new ester bond that was formed during polycondensation can be revealed from the report.

Table 1: IR spectral data of copolyester PIFB in cm−1.
Figure 1: FT-IR spectrum of copolyester PIFB.
3.3. NMR Spectral Studies

1H-NMR spectrum of the copolyester (Figure 2) was recorded at RT in CDCl3. The peak at 1.8 ppm was attributed to central methylene protons of 1,4-butanediol while peak at 4.25 ppm is due to terminal methylene groups of 1,4-butanediol moiety [23]. In addition, the peak at 3.3–3.6 ppm is due to methylene proton of itaconic acid 7.4 ppm for CH of fumaric acid. Copolyester prepared by molten state polycondensation has generally been considered to have a random distribution of the structural units because of the almost equal reactivities of the monomers and the random transesterification reaction during the polycondensation process [24]. 13C-NMR spectrum of polymer (Figure 3), the peaks at 164, 133 & 25 ppm belongs to O–C=O, C=C of fumaric acid & methylene group of 1,4-butanediol respectively [25]. The signals at 64 and 77 ppm correspond to O–CH2 group of 1,4-butadiol. Based on these spectral data, it may be concluded that the following structural units are randomly distributed in the copolyester.

Figure 2: 1H-NMR spectrum of copolyester PIFB.
Figure 3: 13C-NMR spectrum of copolyester PIFB.

3.4. DSC & TGA Thermal Studies

The DSC thermogram of the polymer PIFB shows glass transition temperature () at −23°C and melting temperature () at 72°C [14, 15] which are shown in Figure 4 and the values are in Table 2. These values show that these temperatures of the polymer lie in the range of temperature of the polymer used for drug delivery application, which is partially matched with the temperature of the human body and from the TGA, the decomposition temperature () of the polymer is observed to be 362°C which is shown in Figure 5.

Table 2: DSC data of copolyester PIFB.
Figure 4: DSC thermogram of polymer PIFB.
Figure 5: TGA thermogram of polymer.
3.5. X-Ray Diffraction Analysis

X-ray diffractogram of the synthesized polymer is shown in Figure 6. The crystalline nature of polyester was determined from X-ray diffractogram. Gaussian curves are used to describe the amorphous phase and all crystal reflections of a diffractogram. In the X-ray diffractogram, the intensity of diffraction peaks increases with the increase in the length of the flexible spacer group. This is in accordance with the study of Chen et al. [26]. This indicates that the crystallinity of the polymer increases with the length of flexible segments. From the X-ray diffractogram, it is observed that PIFB is amorphous in nature.

Figure 6: X-ray diffractogram of PIFB.
3.6. Hydrolytic Degradation

The polymer is subjected to hydrolytic degradation in phosphate-buffered saline pH 7.4, alkaline, and acidic medium for 24 h. There is not any degradation that took place in an acidic medium. Very slight degradation takes place in alkaline medium but complete degradation takes place in phosphate buffer saline pH 7.4, which matches pH range of human body.

3.7. Cytotoxic Anticancer Evaluation of Synthesized Polymer

Viable cells were determined by the absorbance. Measurements were performed and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The absorbance was measured with a UV- Spectrophotometer using wells without sample containing cells as blanks. The effect of the polymer on the proliferation of MCF-7 was expressed as the % cell viability. The affected MCF-7 cell line at different concentration was shown in Figure 7. IC50 of the polymer was determined and was shown in Table 3. In Figure 8, a graphical representation of the polymer effect on cancer cells by % cell viability is shown.

Table 3: Anticancer effect of copolyester on MCF7 cell line.
Figure 7: Cytotoxic anticancer evaluation of polymer on MCF-7 cell line at different concentrations: (a) normal MCF-7 cell line, (b) 1000 μg/mL (c) 125 μg/mL (d) 62.5 μg/mL (IC50), and (e) 31.2 μg/mL.
Figure 8: Graphical representation of polymer on MCF-7 cancer cell line.

4. Conclusion

The polyester PIFB was successfully synthesized by transesterification and melt polycondensation method and was characterized. The probable structure of the repeating units present in the polyester was assigned on the basis of NMR spectral data. The inherent viscosity value is found to be 0.86 dL/g which predicts that the degree of polymerization is high for the polymer. TGA analysis shows the decomposition temperature of the polymer at 362°C which is the usual decomposition temperature range for many polyester with drug delivery application and good biodegradation properties. The cytotoxic assay shows that the PIFB is toxic to the MCF-7 cell and 40 to 60% of these cells were killed after incubation for two days with the extract. These lines of evidence show that the polyester may further be studied for drug delivery application.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


The authors thank the Central Leather Research Institute (CLRI), Chennai, for providing GPC technique and thermal analysis.


  1. U. A. Edlund and A. C. Albertsson, Advances in Polymer Science, Springer, New York, NY, USA, 2002.
  2. O. Wichterle and D. Lim, “Hydrophilic gels for biological use,” Nature, vol. 185, pp. 117–118, 1960. View at Google Scholar
  3. R. K. Kulkarni, K. C. Pani, C. Neuman, and F. Leonard, “Polylactic acid for surgical implants,” Archives of Surgery, vol. 93, no. 5, pp. 839–843, 1966. View at Google Scholar · View at Scopus
  4. E. E. Schneider and R. A. Polistina, “Polyglycolic acid prosthetic devices,” US Patent 3463158, 1969.
  5. A. K. Schneider, “Polylactide sutures,” US Patent 3636956, 1972.
  6. D. Wasserman and C. C. Versfelt, “Use of stannous octoate catalyst in the manufacture of l(-)lactide-glycolide copolymer sutures,” US Patent 3839297, 1974.
  7. S. Sharifi, H. Mirzadeh, M. Imani et al., “Injectable in situ forming drug delivery system based on poly(ε-caprolactone fumarate) for tamoxifen citrate delivery: gelation characteristics, in vitro drug release and anti-cancer evaluation,” Acta Biomaterialia, vol. 5, no. 6, pp. 1966–1978, 2009. View at Publisher · View at Google Scholar · View at Scopus
  8. W.-X. Guo, Z.-L. Shi, K. Liang, Y.-L. Liu, X.-H. Chen, and W. Li, “New unsaturated polyesters as injectable drug carriers,” Polymer Degradation and Stability, vol. 92, no. 3, pp. 407–413, 2007. View at Publisher · View at Google Scholar · View at Scopus
  9. J. Guillet, G. Scott, and D. Gilead, Degradable Polymers, Springer, New York, NY, USA, 2002.
  10. A. C. Albersson and O. J. Ljungquist, “Degradable polymers. II. Synthesis, characterization, and degradation of an aliphatic thermoplastic block copolyester,” Journal of Macromolecular Science A, vol. 23, pp. 411–422, 1986. View at Publisher · View at Google Scholar
  11. T. Ferro, L. Fraco, G. Rodriguez, and A. Puiggali, “Poly(ester amide)s derived from 1,4-butanediol, adipic acid and 6-aminohexanoic acid. Part II: composition changes and fillers,” Journal of Polymer, vol. 44, pp. 6139–6152, 2003. View at Publisher · View at Google Scholar
  12. M. S. Nikolic, D. Poleti, and J. Djonlagic, “Synthesis and characterization of biodegradable poly(butylene succinate-co-butylene fumarate)s,” European Polymer Journal, vol. 39, no. 11, pp. 2183–2192, 2003. View at Publisher · View at Google Scholar · View at Scopus
  13. V. Tserki, P. Matzinos, E. Pavlidou, and C. Panayiotou, “Biodegradable aliphatic polyesters. Part II. Synthesis and characterization of chain extended poly(butylene succinate-co-butylene adipate),” Polymer Degradation and Stability, vol. 91, no. 2, pp. 377–384, 2006. View at Google Scholar
  14. C. Liu, J. B. Zeng, S. L. Li, Y. S. He, and Y. Z. Wang, “Improvement of biocompatibility and biodegradability of poly(ethylene succinate) by incorporation of poly(ethylene glycol) segments,” Polymer, vol. 53, pp. 481–489, 2012. View at Publisher · View at Google Scholar
  15. J. Du, Y. Zheng, J. Chang, and L. Xu, “Synthesis, characterization and properties of high molecular weight poly(butylenes succinate) reinforced by mesogenic units,” European Polymer Journal, vol. 43, pp. 1969–1977, 2007. View at Publisher · View at Google Scholar
  16. S. Jo, P. S. Engel, and A. G. Mikos, “Synthesis of poly(ethylene glycol)-tethered poly(propylene fumarate) and its modification with GRGD peptide,” Polymer, vol. 41, no. 21, pp. 7595–7604, 2000. View at Google Scholar · View at Scopus
  17. K.-U. Lewandrowski, J. D. Gresser, D. L. Wise, R. L. White, and D. J. Trantolo, “Osteoconductivity of an injectable and bioresorbable poly(propylene glycol-co-fumaric acid) bone cement,” Biomaterials, vol. 21, no. 3, pp. 293–298, 2000. View at Publisher · View at Google Scholar · View at Scopus
  18. M. D. Timmer, C. G. Ambrose, and A. G. Milkos, “In vitro degradation of polymeric networks of poly(propylene fumarate) and the crosslinking macromer poly(propylene fumarate)-diacrylate,” Biomaterials, vol. 24, pp. 571–577, 2003. View at Publisher · View at Google Scholar
  19. D. P. Uma, S. F. Emerson, and A. C. Sarada, “In vivo tumor inhibitory and radiosensitizing effects of an Indian medicinal plant, Plumbago rosea on experimental mouse tumors,” Indian Journal of Experimental Biology, vol. 32, pp. 523–528, 1994. View at Google Scholar
  20. O. Goerz and H. Ritter, “Polymers with shape memory effect from renewable resources: crosslinking of polyesters based on isosorbide, itaconic acid and succinic acid,” Polymer International, vol. 62, pp. 709–712, 2013. View at Publisher · View at Google Scholar
  21. J. Margaret Marie, R. Puvanakrishnan, and R. Nanthini, “Design, synthesis and characterization of elastomers based on itaconic acid,” Journal of Chemical Pharmaceutical Research, vol. 4, pp. 175–179, 2012. View at Google Scholar
  22. T. Mosmann, “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays,” Journal of Immunological Methods, vol. 65, pp. 55–63, 2013. View at Publisher · View at Google Scholar
  23. W.-X. Guo, K.-X. Huang, R. Tang, and H.-B. Xu, “Synthesis, characterization of novel injectable drug carriers and the antitumor efficacy in mice bearing Sarcoma-180 tumor,” Journal of Controlled Release, vol. 107, no. 3, pp. 513–522, 2005. View at Publisher · View at Google Scholar · View at Scopus
  24. B. D. Ahn, S. H. Kim, Y. H. Kim, and J. S. Yang, “Synthesis and characterization of the biodegradable copolymers from succinic acid and adipic acid with 1,4-butanediol,” Journal of Applied Polymer Science, vol. 82, no. 11, pp. 2808–2826, 2001. View at Publisher · View at Google Scholar · View at Scopus
  25. T. Kajiyama, T. Taguchi, H. Kobayashi, K. Kataoka, and J. Tanaka, “Synthesis of high molecular weight poly(α,β-malic acid) for biomedical use by direct polycondensation,” Polymer Degradation and Stability, vol. 81, no. 3, pp. 525–530, 2003. View at Publisher · View at Google Scholar · View at Scopus
  26. B.-K. Chen, S.-Y. Tsay, and J.-Y. Chen, “Synthesis and properties of liquid crystalline polymers with low T m and broad mesophase temperature ranges,” Polymer, vol. 46, no. 20, pp. 8624–8633, 2005. View at Publisher · View at Google Scholar · View at Scopus