5-Fluorouracil Loaded Chitosan–PVA/Na+MMT Nanocomposite Films for Drug Release and Antimicrobial Activity
- 2.6k Downloads
In the present study, chitosan and polyvinyl alcohol (PVA) were blended with different concentrations of sodium montmorillonite (Na+MMT) clay solution by a solvent casting method. X-ray diffraction and transition electron microscope results show that the film properties are related to the co-existence of Na+MMT intercalation/exfoliation in the blend and the interaction between chitosan–PVA and Na+MMT. 5-Fluorouracil (5-FU) was loaded with chitosan–PVA/Na+MMT nanocomposite films for in vitro drug delivery study. The antimicrobial activity of the chitosan–PVA/Na+MMT films showed significant effect against Salmonella (Gram-negative) and Staphylococcus aureus (Gram-positive), whereas 5-FU encapsulated chitosan–PVA/Na+MMT bio-nanocomposite films did not show any inhibition against bacteria. Our results indicate that combination of a flexible and soft polymeric material with high drug loading ability of a hard inorganic porous material can produce improved control over degradation and drug release. It will be an economically viable method for preparation of advanced drug delivery vehicles and biodegradable implants or scaffolds.
KeywordsBiopolymer Chitosan–PVA/Na+MMT Montmorillonite 5-Fluorouracil Drug release Antimicrobial activity
In the past few decades, drug delivery systems have been of great interest and resulted in many efforts to realize the effectiveness and targeted drug delivery tendency as well as to reduce the associated side effects. Controlled drug delivery system is necessary in order to develop new nano-medicines. Thus, the carriers used for drug release are generally biodegradable polymers  and hard inorganic porous matrices . In recent past, biodegradable polymer attracted much attention owing to its potential applications as a carrier in drug delivery systems.
Chitosan is a semi-crystalline and linear polysaccharide composed of (1–4)-2-acetamido-2-deoxy-b-d-glucan (N-acetyl d-glucosamine) and (1-4)-2-amino-2-deoxy-b-d-glucan (d-glucosamine) units. It is not widely present in the environment but can be easily obtained from the partial deacetylation of a natural polymer chitin . The deacetylation degree of chitosan provides valuable information regarding to the number of amino groups (–NH2) along the chains and it can be measured as the ratio of d-glucosamine to the sum of d-glucosamine and N-acetyl d-glucosamine. For a chitosan, the deacetylated chitin must have at least 60 % of d-glucosamine residues  and controlled by chemical hydrolysis under harsh alkaline conditions or enzymatic hydrolysis in the presence of particular enzymes among of chitin deacetylase . The presence of amino groups in the chitosan structure differentiates the chitosan from chitin and allows this polymer to have several unique properties. Undoubtedly, the amino groups of the d-glucosamine residues might be protonated, when it is soluble in aqueous acidic solutions (pH < 6). Whereas the applications of chitin are tremendously limited due to its weak solubility in water or other organic solvents. Interestingly, the aqueous acidic solubility of chitosan is pH dependent, permitting its processability under warm conditions, which opens the door for many applications, particularly in the field of pharmaceutical and cosmetics . This polysaccharide has been extensively studied in the field of biomaterials because of its biodegradability, biocompatibility, and biological properties. Among various polymer development processes, polymer blending is one of the most economical and rapid ways to innovate novel materials with vital properties and it has made great scientific and commercial progresses .
Polyvinyl alcohol (PVA)-based nanocomposites are one of the familiar polymer composites that have been used in various biomedical applications (implants, artificial organs, contact lenses, drug delivery devices, wound dressings, etc.) due to its good biocompatibility behavior [7, 8, 9, 10, 11]. There are numerous methods available for crosslinking PVA chains to synthesize PVA composites, including bulk mixing with crosslinking agents such as glutaraldehyde (GA), freezing–thawing cyclic process , as well as electron beam irradiation .
Nowadays increased attention has been focused on drug intercalated smectites, particularly montmorillonite (MMT) pharmaceutical grade mineral clay [14, 15]. MMT has cation exchange capacity, good adsorption capacity, large specific surface area, and drug-carrying ability. It is hydrophilic and highly dispersible in water and can aid in the synthesis of a wide variety of hydrophilic and protonated organic molecules, which can be released in controlled fashion by replacement with other types of cations in the drug release processes [16, 17, 18]. Therefore, MMT is a good delivery carrier of hydrophilic drugs due to its high aspect ratio and can afford mucoadhesive ability for the nanoparticles to cross the gastrointestinal barrier [19, 20]. So far, MMT has been used as a controlled release system and proved to be nontoxic by hematological, biochemical and histopathological analyses in rat models . It is also utilized as a sustained release carrier for various therapeutic molecules, such as 5-fluorouracil (5-FU) , sertraline , vitamin B1 [14, 15], promethazine chloride , and buspirone hydrochloride .
5-FU is an effective chemotherapy option available for the treatment of colorectal cancer [26, 27], stomach cancer , breast cancer , brain tumor [29, 30], liver cancer , pancreatic cancers [32, 33, 34], and lung cancer [35, 36, 37, 38]. It is a pyrimidine analog that restrains the biosynthesis of deoxyribonucleotides for DNA replication through constraining thymidylate synthase activity, resulting to thymidine exhaustion, incorporation of deoxyuridine triphosphate into DNA and subsequently causing cell death [39, 40, 41]. However, 5-FU has limitations, such as short biological half-life due to rapid metabolism, non-uniform and incomplete oral absorption owing to metabolism by dihydropyrimidine dehydrogenase [42, 43, 44, 45], toxic side effects on bone marrow and gastrointestinal tract, and non-selective action against healthy cells . For successful cancer treatment, overcoming the toxic side effects on bone marrow is highly essential, which might possibly be achieved by the control release of the drug by intercalated in the clay interlayer and biopolymeric systems.
High molecular weight chitosan (viscosity of between 800 and 2000 cps), PVA (96 % hydrolysed, molecular weight 85,000–145,000, and the degree of deacetylation higher than 75 %), and 5-FU (99 %) were sourced from Sigma-Aldrich (South Africa). GA, sodium chloride (NaCl), sodium hydroxide (NaOH), silver nitrate (AgNO3), acetic acid (CH3COOH), and de-ionized water were supplied by Merck. Na+MMT was supplied as powder by Southern Clay Products, Inc. (Texas, USA). The Department of Microbiology (SRM University, India) provided the standard cultures of the organisms. All the chemicals and reagents were used without further purification. Double-distilled water was used for the preparation of all solutions.
2.2 Solutions Preparation
50 mL of chitosan solution (1 % wt/v in acetic acid) and 50 mL of PVA solution (1 % wt/v in water) (1:1) were mixed together in a 250 mL beaker and stirred at room temperature until a homogeneous solution was obtained. Then, different amounts of Na+MMT (1–5 wt%) nanoparticles were added to above mixture and stirring was continued for a further 6 h. Before casting, 1 mL of 2 % GA solution in water (a cross-linking agent) was added under stirring at room temperature. The solution was transferred immediately into Petri dishes (10 mm × 10 mm) and dried at room temperature. The formed cross-linked chitosan–PVA/Na+MMT films were washed with double-distilled water for neutralization and dried at room temperature [47, 48].
2.3 Swelling Studies
Bruker D8 advanced refractometer X-ray diffraction (XRD) was used to determine the intercalation or exfoliation (or both) of nanocomposite films. Transition electron microscope (TEM) images were recorded using a Tecnai F 12 JEOL-JEM 2100 at an accelerating voltage of 15 kV. The morphologies of the bio-nanocomposite films were observed by scan electron microscopy (SEM, JEOL FESEM JSM-7600F) equipped with energy-dispersive X-ray spectroscopy. Fourier transform infra-red (FTIR) spectroscopy measurement was carried on Perkin-Elmer UATR two using diamond Zn/Se plate. Small amount of sample was pressed on a Zn/Se plate and the spectra were recorded over a range of 550–4000 cm−1. Thermal studies of the films were carried out using TGA 7 instrument (Perkin-Elmer) at a heating rate of 10 °C min−1 under a constant nitrogen flow of 20 mL min−1.
2.5 5-Fluorouracil Loading and Encapsulation Efficiency
2.6 Release of 5-FU
For the control release studies of 5-FU from the loaded nanocomposite films, known weights were placed in a measured volume (50 mL) of 7.4 pH phosphate buffer solution at room temperature and the released amount of 5-FU was determined at different time intervals by recording the absorbance of the release medium using the UV–vis spectrophotometer . The recorded absorbance was then related to the amount of 5-FU released using a calibration plot. The absorption of the solutions of 5-FU was measured at λ max value of 266 nm.
2.7 Antimicrobial Activity
The antibacterial activity of the nanocomposites was investigated by a disk method and the standard procedure was described elsewhere . Nutrient agar medium was prepared by mixing peptone (5.0 g), beef extract (3.0 g), and sodium chloride (NaCl, 5.0 g) in 1000 mL distilled water and the pH was adjusted to 7.0. Finally, agar (15.0 g) was added to the solution. The agar medium was sterilized in an autoclave at a pressure of 6.8 kg m−2 (15 lbs) for 30 min. This medium was transferred into sterilized Petri dishes in a laminar air flow chamber (Microfilt Laminar Flow Ultra Clean Air Unit, Mumbai, India). After solidification of the media, bacteria (Salmonella, Staphylococcus aureus, Streptococcus mutants and Escherichia coli) (50 µL) culture were spread on the solid surface of the media. Over the inoculated Petri dish, one drop of gel solution (20 mg/10 mL distilled water) was added using a 10 µL tip and the plates were incubated for 48 h at 37 °C.
3 Results and Discussion
XRD data of Na+MMT and chitosan/PVA–Na+MMT nanocomposite films
1 wt% clay
2 wt% clay
3 wt% clay
4 wt% clay
5 wt% clay
The TEM image of nanocomposite (5 wt% Na+MMT) is illustrated in Fig. 3. The images show typical morphology of layered materials, in which the dark lines correspond to Na+MMT clay layers, while bright areas represent the chitosan–PVA polymer matrices. As seen in Fig. 3, the interlayer distance of clay was obviously enlarged, following the addition of the mixture of chitosan–PVA polymers. Moreover, as seen from the higher magnification image (100 nm), layered silicates were exfoliated and uniformly dispersed in chitosan–PVA polymer matrices at nano-level and it is supported from the XRD patterns.
Drug loading efficiency of chitosan/PVA–Na+MMT nanocomposite films
Weight of the film (mg)
Weight of drug in film (mg)
% Drug loading efficiency
1 wt% clay
2 wt% clay
3 wt% clay
4 wt% clay
5 wt% clay
Bio-nanocomposite films-based chitosan–PVA/Na+MMT were developed by a simple solution casting technique. It was found that the biodegradable polymer was successfully intercalated into clay galleries. Drug loading efficiency of chitosan–PVA/Na+MMT films increases with increasing clay content and higher clay content exhibited higher rate of drug release. Compared with chitosan and chitosan/Na+MMT nanocomposite films which did not show clear microbial inhibition zones, chitosan–PVA/Na+MMT nanocomposite films showed distinctive microbial inhibition zones against two test microorganisms and exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria. The present study is useful in developing novel antimicrobial agents for applications in wound burns/dressing, antimicrobial packaging, and prevention/treatment of infections due to the control release ability. In addition, the chitosan–PVA/Na+MMT nanocomposite films are a promising material for the development of new membranes for waste water treatment.
The author ABR wishes to acknowledge the Tshwane University of Technology for their financial support.
- 16.F. Bergaya, G. Lagaly (eds.), Handbook of Clay Science (Elsevier Science, Oxford, 2013)Google Scholar
- 26.E. Healey, G.e. Stillfried, S. Eckermann, J.p. Dawber, P.r. Clingan, M. Ranson, Comparative effectiveness of 5-fluorouracil with and without oxaliplatin in the treatment of colorectal cancer in clinical practice. Anticancer Res. 33(3), 1053–1060 (2013), http://ar.iiarjournals.org/content/33/3/1053.long
- 30.D. Ostertag, K.K. Amundson, F. Lopez Espinoza, B. Martin, T. Buckley et al., Brain tumor eradication and prolonged survival from intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a nonlytic retroviral replicating vector. Neurooncology 14(2), 145–159 (2012). doi: 10.1093/neuonc/nor199 Google Scholar
- 31.J.M.G.H. van Riel, C.J. van Groeningen, S.H.M. Albers, M. Cazemier, S. Meijer, R. Bleichrodt, F.G. van den Berg, H.M. Pinedo, G. Giaccone, Hepatic arterial 5-fluorouracil in patients with liver metastases of colorectal cancer: single-centre experience in 145 patients. Ann. Oncol. 11(12), 1563–1570 (2000). doi: 10.1023/A:1008369520179 CrossRefGoogle Scholar
- 32.S. Cascinu, R.R. Silva, S. Barni, R. Labianca, L. Frontini et al., A combination of gemcitabine and 5-fluorouracil in advanced pancreatic cancer, a report from the Italian Group for the Study of Digestive Tract Cancer (GISCAD). Br. J. Cancer 80(10), 1595–1598 (1999). doi: 10.1038/sj.bjc.6690568 CrossRefGoogle Scholar
- 34.W.H. Isacoff, H.A. Reber, F.M. Purcell, B.M. Clerkin, K.M. Clerkin, Low-dose continuous infusion 5-fluorouracil combined with weekly leucovorin, nab-paclitaxel, oxaliplatin, and bevacizumab for patients with advanced pancreatic cancer: a pilot study. J. Clin. Oncol. (Meet. Abstr.) 28(15), e14545 (2010), http://meeting.ascopubs.org/cgi/content/abstract/28/15_suppl/e14545
- 35.J. Nakano, C. Huang, D. Liu, D. Masuya, T. Nakashima, H. Yokomise, M. Ueno, H. Wada, M. Fukushima, Evaluations of biomarkers associated with 5-FU sensitivity for non-small-cell lung cancer patients postoperatively treated with UFT. Br. J. Cancer 95(5), 607–615 (2006). doi: 10.1038/sj.bjc.6603297 CrossRefGoogle Scholar
- 40.B. Van Triest, H.M. Pinedo, G. Giaccone, G.J. Peters, Downstream molecular determinants of response to 5-fluorouracil and antifolate thymidylate synthase inhibitors. Ann. Oncol. 11(4), 385–391 (2000), http://annonc.oxfordjournals.org/content/11/4/385
- 42.E. Aranda, E. Díaz-Rubio, A. Cervantes, A. Antón-Torres, A. Carrato et al., Randomized trial comparing monthly low-dose leucovorin and fluorouracil bolus with weekly high-dose 48-hour continuous-infusion fluorouracil for advanced colorectal cancer: a Spanish Cooperative Group for Gastrointestinal Tumor Therapy (TTD) study. Ann. Oncol. 9(7), 727–731 (1998). doi: 10.1023/A:1008282824860 CrossRefGoogle Scholar
- 43.R.B. Diasio, Z. Lu, Dihydropyrimidine dehydrogenase activity and fluorouracil chemotherapy. J. Clin. Oncol. 12(11), 2239–2242 (1994), http://jco.ascopubs.org/content/12/11/2239
- 44.E.C. Gamelin, E.M. Danquechin-Dorval, Y.F. Dumesnil, P.J. Maillart, M.J. Goudier et al., Relationship between 5-fluorouracil (5-FU) dose intensity and therapeutic response in patients with advanced colorectal cancer receiving infusional therapy containing 5-FU. Cancer 77(3), 441–451 (1996). doi: 10.1002/(sici)1097-0142(19960201)77:3<441:aid-cncr4>3.0.co;2-n CrossRefGoogle Scholar
- 45.N.J. Meropol, D. Niedzwiecki, D. Hollis, R.L. Schilsky, R.J. Mayer, Cancer and The Leukemia Group B, Phase II study of oral eniluracil, 5-fluorouracil, and leucovorin in patients with advanced colorectal carcinoma. Cancer 91(7), 1256–1263 (2001). doi: 10.1002/1097-0142(20010401)91:7<1256:aid-cncr1126>3.0.co;2-v CrossRefGoogle Scholar
- 55.M. Kouchak, A. Ameri, B. Naseri, S. Kargar Boldaji, Chitosan and polyvinyl alcohol composite films containing nitrofurazone: preparation and evaluation. Iran. J. Basic Med. Sci. 17(1), 14–20 (2014), http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3938881/pdf/ijbms-17-014
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.