Advanced Powder Technology xxx (2013) xxx–xxx
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Original Research Paper
Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery G. Devanand Venkatasubbu a, S. Ramasamy a,⇑, V. Ramakrishnan b, J. Kumar a a b
Crystal Growth Centre, Anna University, Chennai 600 025, Tamil Nadu, India Department of Laser Studies, School of Physics, Madurai Kamaraj University, Madurai, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 20 August 2012 Received in revised form 24 December 2012 Accepted 16 January 2013 Available online xxxx Keywords: Titanium dioxide nanoparticles PEG Folic acid Drug targeting Paclitaxel
a b s t r a c t A novel titanium dioxide nanocarrier was synthesized for targeted delivery of the anticancer drug, paclitaxel, by grafting folic acid (FA) onto the PEGylated titanium dioxide nanoparticles. Titanium dioxide is used in biomedical field for its stability and no toxicity characteristics. Titanium dioxide is one of the most promising nanoparticles (NPs) capable of a wide variety of applications in medicine and life science. Polyethylene glycol (PEG), when attached to the surface of the nanoparticles, increases the biocompatibility of the nanoparticles. PEGylated nanocarriers evade the reticuloendothelial system (RES). Folic acid (FA) is used as the ligand to target folate receptors, which are found abundant in cancer cells. FA–PEG– TiO2 nanoparticles when used as drug carriers have the ability to target cancer cells and also capable of evading the reticuloendothelial system. Titanium dioxide nanoparticles were synthesized by wet chemical method. It was annealed at 450° for 3 h. XRD analysis confirms the formation of anatase titanium dioxide. Analyses by transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed that the nanoparticles had an average size of 12 nm and uniform size distribution. The PEGylation and folic acid grafting was confirmed by UV spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and thermogravimetric analysis (TGA). The study on the loading of anticancer drug paclitaxel revealed that the titanium dioxide nanocarrier possessed a considerably higher adsorption capability. In addition, the in vitro release profile of paclitaxel from FA–PEG–TiO2 nanoparticles was characterized by an initial fast release followed by a sustained release phase. Ó 2013 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved.
1. Introduction Conventional chemotherapeutic agents were distributed nonspecifically in the body when given intravenously. They affect both cancerous and normal cells, thereby limiting the dose achievable within the tumor and also resulting in suboptimal treatment due to excess toxicity [1]. Targeted drug delivery systems promises to expand the therapeutic windows of drugs by increasing delivery to the target tissue as well as the target non-target tissue ratio. The major objective of targeted drug delivery is to reduce the non-discriminate uptake of toxic agents as well as enhancing drug accumulation at the target site. In order to target drugs to specific tissue system within the body, drug molecules can be directly attached to a targeting agent or complexed with a vehicle, that contains targeting moieties [2]. Nanostructure mediated targeted drug delivery, a key technology for the realization of nanomedicine, has ⇑ Corresponding author. Tel.: +91 044 22359207, mobile: +91 9444062269. E-mail addresses:
[email protected] (G. Devanand Venkatasubbu),
[email protected] (S. Ramasamy),
[email protected] (V. Ramakrishnan),
[email protected] (J. Kumar).
the potential to enhance drug bioavailability, improve the timed release of drug molecules and enable drug targeting [3,4]. The nanoparticles are recognized by the phagocytic cells (mainly the cells of the mononuclear phagocyte system and the polymorphonuclear leukocytes), are detected as foreign products and quickly removed from blood circulation mainly by macrophages located in the reticuloendothelial system (RES) [5]. Various attempts were made to retain the nanodrugs for long time in blood circulation by avoiding RES recognition, mainly by chemically attaching or adsorbing appropriate polymers or molecules at the particle surface [6]. PEGylated nanocarriers have the ability to evade the reticuloendothelial system and extend the circulation time of encapsulated drugs in the bloodstream. [7]. When attached to the surfaces of nanoparticles, PEG has been shown to increase their biocompatibility and reduce the adsorption of proteins and blood components on the surface of nanoparticles [8,9]. The efficiency of nanocarriers that reach the sites of interest is increased by active targeting of drugs through the incorporation of targeting moieties on the exterior of nanocarriers [10,11]. The folate receptor (FR), also known as the folate binding protein (FBP or Folbp), is a glycopolypeptide with a high affinity
0921-8831/$ - see front matter Ó 2013 The Society of Powder Technology Japan. Published by Elsevier B.V. and The Society of Powder Technology Japan. All rights reserved. http://dx.doi.org/10.1016/j.apt.2013.01.008
Please cite this article in press as: G. Devanand Venkatasubbu et al., Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery, Advanced Powder Technology (2013), http://dx.doi.org/10.1016/j.apt.2013.01.008
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(Kd 109 M) for folic acid [12]. Overexpression of FR on many cancer cells obviously identifies this receptor as a potential target for ligand directed cancer therapeutics [13]. Among the available tumor targets/markers, FR has certain distinctive advantages in that (i) it binds to an innocuous small molecule (folic acid) (ii) FR quantitatively recycles between the cell surface and intracellular compartments effectively internalizing receptor-bound folate/anti folate compounds and folate conjugates and (iii) FR expression in most proliferating normal tissues is restricted to the luminal surface of certain epithelial cells where it is inaccessible to the circulation whereas it is consistently expressed in specific types of major malignant tumors and leukemic cells where it is accessible via the circulation [14]. Titanium dioxide nanoparticles (TiO2 NPs) are bio friendly [15,16] and have good biocompatibility with weak or no toxicity in vitro and in vivo [17], which indicates its great probability of the applications in life science. It has the property of killing bacteria, viruses, fungi, and even cancer cells [18]. It is an inert material in vivo. Thus, TiO2 is one of the most promising nanoparticles capable of a wide variety of applications in medicine and life science, the unique property and high reactivity of nano-titanium dioxide makes it possible to be utilized in the fields of biomedical and bioengineering. The TiO2 is widely used in paint, pharmaceutical, and cosmetics industries. It is considered as biologically inert [19]. Jugan et al. and Yaling et al. studied the influence of TiO2 nanoparticles on human lung cells and mouse liver cells [20,21]. Zhi et al. confirmed the reduction in toxicity of TiO2 nanoparticles when functionalized with polymer [22]. However, to the best of our knowledge, no extensive studies have been carried out on TiO2 nanoparticles in the biomedical field so far. This paper reports for the first time the possibility of using TiO2 for biomedical applications, especially in targeted drug delivery for cancer therapy. The work discusses for the first time the surface modification of titanium dioxide nanoparticles for the targeted drug delivery of paclitaxel, an anticancer drug, from PEG-functionalized titanium dioxide nanoparticles attached to folic acid. 2. Experimental procedure 2.1. Synthesis of titanium dioxide nanoparticles A 7.4 ml of titanium tetra isopropoxide was added, drop by drop, to 30 ml of 1 M HNO3 aqueous solution, and then agitated for 2 h to give a transparent sol, in which 2.0 g TiO2 are contained. The pH of the colloidal solution was adjusted to pH 3, with the addition of 1 M NaOH solution after dilution of the colloid with 100 ml water, resulting in a turbid TiO2 colloid. The suspension was agitated at room temperature, centrifuged and then washed with distilled water. The isolated TiO2 was dried for 1 h at 100 °C in air. The resulting powder was then calcinated 450 °C for 3 h [23,24]. Powder X-ray Diffraction (XRD, Seifert, JSO-DE BYEFLEX 2002, Germany) was utilized to identify the crystalline phase composition. The morphology and crystal structure of the product were observed by Transmission Electron Microscopy (TEM). The instrument was JEOL 2000Fx-II operated at 200 kV, High Resolution, analytical TEM with a W-source and a point–point resolution of 2 Å. The functional groups present in the titanium dioxide were analyzed by FTIR (FTIR, Perkin Elmer Spectrum One). 2.2. Chemical synthesis of PEG-functionalised titanium dioxide nanoparticles In order to synthesize PEG functionalized TiO2 nanoparticles, the TiO2 nanoparticles were mixed with the 1% concentration
PEG 6000 solution, degree of polymerization being 96 in the mass ratio of 1:1 and stirred at 750 rpm overnight. A low PEG concentration was preferred to avoid encapsulation of nanoparticles with a thicker layer of PEG. The PEG coated nanoparticles were centrifuged at 18,000 rpm for 1 h at 10 °C, washed with distilled water and freeze dried and collected. 2.3. Folic acid modified PEG-functionalized titanium dioxide nanoparticles Folic acid is generally difficult to conjugate to the surface of the polymer because of the weak chemical reactivity of the carboxylic acid group associated with the PEG. So the carboxylic group of folic acid was first activated with dicyclohexyl carbodiimide (DCC). DCC activates the folic acid and isourea is formed. Folic acid was dissolved in dimethyl sulfoxide (DMSO) solution in different concentration. DCC was added to the solution corresponding to a folic acid: DCC ratio of 1:1 and stirred at 750 rpm for 2 h in nitrogen atmosphere. PEG functionalized titanium dioxide nanoparticles were added in the activated folic acid solution in the mass ratio 1:1 for 1 h. The PEGylated nanoparticles and conjugated with folic acid were separated by centrifugation, washed with distilled water and freeze dried. Amount of FA incorporated was determined by UV spectrophotometer method. 50.0 mg folic acid was dissolved in 50 ml DMSO solution, and then the solution was diluted to different concentrations. Folic acid has three maximum absorption peaks at a wavelength of 256, 283 and 365 nm [25]. The UV cut off wavelength of DMSO is 268 nm, in addition, at the wavelength of 283 nm, the intensity of absorption peak definitely decreases. Therefore 365 nm was used as incident wavelength and the standard curve was obtained. The amount of FA attached was determined by finding the difference in FA concentration in the solution before and after attachment. Percentage of FA attachment is calculated using the equation:
Percentage of FA attached ¼ ½ðA BÞ=A 100
ð1Þ
where A and B represent the initial and final FA concentration of the FA solution. 2.4. Conjugation of paclitaxel to functionalized titanium dioxide nanoparticles The anticancer drug paclitaxel was dissolved in dichloromethane (DCM). Folic acid modified PEG functionalized nanoparticles was added to drug solution (drug concentration = 1 mg/ml) in a mass ratio of 1:1 and stirred at room temperature for various time period. The suspension was then centrifuged (2000 rpm, 5 min) and the supernatant and precipitate were separated. The amount of drug loaded was determined by finding the difference in paclitaxel concentration in the solution before and after loading. Percentage of drug loading is calculated using the equation:
Percentage of drug loading ¼ ½ðA BÞ=A 100
ð2Þ
where A and B represent the initial and final drug concentration of the drug solution. 2.5. Drug release – in vitro study In order to determine the drug release profile, 100 mg of the paclitaxel attached folic acid modified PEG-functionalized titanium dioxide nanoparticles were introduced into a screw capped glass bottle containing 50 ml of phosphate buffered saline (PBS) medium at 37 °C and pH 7.4 under sterile condition [26,27]. The composition of PBS is 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 dissolved in 1 l of H2O. 5 ml samples were withdrawn by a pipette at a regular interval of 1 h and replaced immediately with 5 ml of
Please cite this article in press as: G. Devanand Venkatasubbu et al., Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery, Advanced Powder Technology (2013), http://dx.doi.org/10.1016/j.apt.2013.01.008
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fresh PBS medium, which was accounted for when calculating the amount released. Paclitaxel concentration in the collected samples was measured spectrophotometrically at a wavelength of 227 nm. The percentage of drug released is given by:
Percentage of drug released ¼ ½ðA BÞ=A 100
ð3Þ
where A is the weight of initial paclitaxel that was incorporated into the folic acid modified PEG-functionalized titanium dioxide nanoparticles and B represent the weight of drug released at a particular time, t. 2.6. In vitro assay for cytotoxicity activity (MTT assay) The cytotoxicity of samples on HepG2 cells was determined by the MTT assay. Cells (1 105/well) were plated in 100 ll of medium/well in 24-well plates (Hi media chemicals Ltd., Mumbai, India). After 48 h incubation the cell reaches the confluence. Then cells were incubated in the presence of various concentrations of the samples in 0.1% dimethyl sulfoxide (DMSO) for 48 h at 37 °C. After removal of the sample solution and washing with phosphate-buffered saline (pH 7.4), 200 ll/well (5 mg/ml) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide cells (MTT) phosphate-buffered saline solution was added. After 4 h incubation, 0.04 M HCl/isopropanol were added. Viable cells were determined by the absorbance at 570 nm with reference at 655 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 samples on the proliferation of Human Liver cancer cells (HepG2) was expressed as the% cell viability, using the following formula:
% Cell viability ¼ ðA570 of treated cells=A570 of control cellsÞ 100%:
3. Results and discussion Fig. 1a shows the X-ray diffraction patterns for TiO2 nanoparticles annealed at 450 °C. The diffraction peaks corresponds to anatase phase. Existence of strong crystalline peaks at 2h values of 25.20°, 37.80°, 48.04°, 53.890° and 62.68° corresponding to the crystal planes of (1 0 1), (0 0 4), (2 0 0), (1 0 5) and (2 0 4) indicates the formation of anatase titanium dioxide (JCPDS card No. 21-1272).
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The TEM image of titanium dioxide nanoparticles annealed for 3 h at 450 °C is given in Fig. 1b. TEM images confirm the formation of spherical particles. The average particle size is very small after annealing at 450 °C. This agrees well with the results of Siegel et al. [28]. Grains with much agglomeration are clearly observable from the images. The SAED analyses confirmed the formation of tetragonal structure of titanium dioxide, and are in agreement with the XRD results. The particle size distribution is shown as inset in Fig. 1b. The size is around 12 nm (81%) at 450 °C. the samples have a narrow size distribution as analyzed by DLS. UV–Vis spectra of titanium dioxide nanoparticles, TiO2 nanoparticles functionalized with PEG and TiO2 nanoparticles containing both FA and PEG are given in Fig. 2. It clearly shows a red shift in the UV spectra of TiO2 nanoparticles functionalized with PEG (kmax 342 nm) when compared with the UV spectra of pure TiO2 nanoparticles (kmax 339 nm). This shows the bonding of PEG to TiO2 nanoparticles. After the bonding of FA to PEG molecule on the surface of TiO2 nanoparticles, a further red shift is observed (kmax 348 nm). This is due to surface plasmon resonance effect. From the absorption maxima at 372 nm we can confirm the incorporation of folic acid. The FTIR spectrum of TiO2 nanoparticles is shown in Fig. 3a. The spectroscopic band is observed around the 3500 cm1, which is described to the stretching vibration of the hydroxyl group of the TiO2 nanoparticles. The broad intense band below 1139 cm1 is due to TiAOATi vibrations [29]. The band at 1628 cm1 and 1784 cm1 corresponds to the bending mode of adsorbed water and TiAOH modes. The sharp band at 1383 cm1 corresponds to the TiAO modes. The band at 490 cm1 corresponds to the TiAOATi [30,24]. The peaks over when 400–1250 cm1 are characteristic of OATiAO modes [31]. The FTIR spectrum of PEG coated titanium dioxide nanoparticles is given in Fig. 3a. The appearance of characteristic absorption peaks of PEG such as CAOAC (antisymmetric stretching, 1342 cm1) and ACH (out-of-plane bending, 955 cm1), 1110 cm1, 1229 cm1 and 2872 cm1 peak in the FTIR spectrum of PEG and PEG coated TiO2 nanoparticles suggested that PEG was grafted to the nanoparticles. The band around 2888 cm1 in the PEG spectrum corresponds to ACH2 stretching vibrations. In PEG coated TiO2 nanoparticles, it is shifted to 2872 cm1. This exhibits hydrogen bonding nature. The band at 1110 cm1 in both PEG and PEG coated TiO2 nanoparticles corresponds to ACAOACA stretching vibration. The band at 1281 cm1 corresponds to CAO stretching vibration. The band at 1242 cm1 in PEG corresponds to CH2 twisting. It gets shifted to 1229 cm1 in PEG coated TiO2 nanoparticles. This exhibits a hydrogen bonding nature. A sharp strong band at 842 cm1 is due to the CAC stretching. This peak
Fig. 1. (a) XRD pattern of titanium dioxide nanoparticles, (b) TEM images of titanium dioxide nanoparticles (inset shows particle size distributions of TiO2 nanoparticles analyzed by dynamic light scattering).
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Fig. 2. UV–Vis spectrum of folic acid modified PEG functionalized titanium dioxide nanoparticles.
is present in both pure PEG and PEG coated TiO2 nanoparticles, supporting the presence of PEG molecules on the surface of TiO2 nanoparticles by hydrogen bonding.
The FTIR spectrum of folic acid and folic acid modified PEG functionalized TiO2 nanoparticles is given in Fig. 3b. The FTIR spectrum of pure FA is characterized by a number of characteristic bands occurring at 3543, 3416, 3324, 2926, 2844, 1694, 1605, 1485 and 1411. The bands between 3600 and 3000 cm1 are due to hydroxyl stretching and NHA stretching vibration bands. The band at 1638 cm1 belongs to the C@O bond stretching of ACONH2. The bond at 1605 cm1 relates to the bending mode of NHA vibration and at 1411 cm1 corresponds to OH deformation band of phenyl skeleton. The band at 1485 cm1 was due to absorption band of phenyl ring. In the FTIR spectrum of FA modified PEG functionalized TiO2, the bands at 1107, 1229 cm1 confirms the presence of FA. The absorption band at 3435 cm1 is due to coupling of hydroxyl (OH) stretching and NH-stretching vibrations. The small shift in the peak position with respect to pure FA occurred. This demonstrated the successful FA conjugation on the surface of the PEG modified TiO2 nanoparticles. The FTIR spectrum of PAC loaded FA modified PEG functionalized TiO2 nanoparticles is given in Fig. 3c. In the FTIR spectrum of PAC the peak in the range of 3336–3436 cm1 was observed due to stretching of hydroxyl (AOH) groups. The peak observed in the range of 2356–2364 cm1 was due to amine (NH) group stretching frequency. The aromatic ring (C@C) stretching frequency was observed in the range of 1590–1735 cm1. Further,
Fig. 3. FTIR spectrum of: (A) PEG functionalized titanium dioxide nanoparticles: (a) pure TiO2; (b) pure PEG; (c) TiO2–PEG, (B) folic acid modified PEG functionalized titanium dioxide nanoparticles: (a) TiO2–PEG; (b) pure folic acid; (c) TiO2–PEG–FA and (C) FTIR spectrum of paclitaxel attached functionalized titanium dioxide nanoparticles.
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Fig. 5. Folic acid attachment profile.
Fig. 4. Thermogravimetric profiles of folic acid modified PEG functionalized titanium dioxide nanoparticles: (a) pure TiO2, (b) TiO2–PEG and (c) TiO2–PEG–FA.
the peak at 1451 cm1 represent CH2 scissoring mode of PAC [32,33]. The appearance of PAC peak in the FTIR spectrum of TiO2–PEG–FA–PAC nanoparticles confirms the loading of the anticancer drug, PAC, on the surface modified TiO2 nanoparticles. In the FTIR spectrum of TiO2–PEG–FA–PAC nanoparticles, the amide group peak of the PAC is shifted to 2325 cm1. TGA is a useful method for the determination of the presence or absence of residual components in nanoparticles. It is based on the observation of the mass loss of individual components. Fig. 4 shows the TGA thermogram of TiO2 nanoparticles, PEG coated TiO2 and folic acid modified PEG functionalized TiO2 nanoparticles. The initial, large mass loss below 100 °C due to free adsorbed water. The mass loss around 250 °C corresponds to the decomposition of the condensation of residual AOH groups and condensation of nonbonded oxygen [34]. In the case of PEG–TiO2 the sharp mass loss at 250 °C is attributed to the elimination of tightly bound hydroxyl species accompanied by crystallization of TiO2 [35]. The weight loss occurs between 300 °C and 450 °C relating to the thermal degradation of the PEG [36]. The total weight loss for pure TiO2 is 6.2%, for TiO2–PEG is 12.7% and for TiO2–PEG–FA is 16.5%. TiO2 may interact with PEG through hydrogen bond or other coordinate bonds between the TiO2 inorganic network and polymeric chains [37]. There are three stages in the decomposition of folic acid. Folic acid starts melting at 220 °C with simultaneous decomposition. The first mass loss was observed at 95 °C. From the TGA curve, it appears that the sample decomposes in three stages over the temperature range 95–800 °C. The first step occurs at 25–95 °C. The second step starts at 250 °C and ends at 721 °C. The last one within the range 721–754 °C. All the three stages overlap with each stage. It may be due to disintegration of folic acid into individual molecules with increase in temperature [38,39]. 3.1. Determination of folic acid content The percentage of folic acid attached to TiO2–PEG increases with increase in the folic acid concentration (Fig. 5). With the concentration of 1 mg/ml the folic acid attachment is 42% and reaches up to 65% with 10 mg/ml folic acid concentration. After that the percentage of attachment of folic acid becomes constant. The TiO2–PEG nanoparticles with maximum attachment of folic acid are chosen for drug attachment. Standard error on the basis of triplicate determinations (mean ± S.E., n = 3).
Table 1 Drug loading in percentage for different stirring time. S. no.
Stirring time (min)
Drug loading (%)
mg of Paclitaxel/mg of TiO2–PEG–FA nanoparticles
1 2 3 4
30 60 120 180
35 ± 2.3 47 ± 1.3 54 ± 1.6 60 ± 1.8
0.35 ± 0.023 0.47 ± 0.013 0.54 ± 0.016 0.60 ± 0.018
3.2. Drug loading capacity The drug loading percentage and the amount of drug loaded is given in Table 1. Drug loading capacity increases with increase in the stirring time. For 30 min stirring time a drug loading of 35% is achieved. It increases to a maximum of 60% for 3 h stirring time and then becomes constant. A maximum drug loading capacity of 60% was obtained with a stirring time of 3 h. Standard error is calculated on the basis of triplicate determinations (mean ± S.E., n = 3). The schematic representation of TiO2–PEG–FA–PAC is given in Fig. 6. The drug loading ability of nanocrystals is lesser because of the PEG functionalization and FA modification on the surface of the nanoparticles. The PEG coating has the advantage of reducing protein opsonization on the surface of the nanoparticles. It helps the nanoparticles to escape from reticuloendothelial system (RES). It reduces the rate of clearance [40]. 3.3. Drug release profile The drug release profile of paclitaxel from folic acid modified PEG functionalized titanium dioxide nanoparticles in PBS is given in Fig. 7. Drug release profile shows a 90% release of paclitaxel in 50 h. The drug is released gradually over a period of time. This shows that the drug is released in a sustained manner. There is an initial burst release of paclitaxel from the folic acid modified PEG functionalized TiO2 nanoparticles. By this initial burst release the drug molecule is brought close to the surface. Then the drug molecules which have interactions with folic acid are released. Then the drug release becomes sustained. Error bars are the standard error on the basis of triplicate determinations (mean ± S.E., n = 3). In the initial stage there is a rapid release of drug. It corresponds to the drug molecules present close to the surface. It is followed by a slow, steady and controlled release of drug and relates
Please cite this article in press as: G. Devanand Venkatasubbu et al., Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery, Advanced Powder Technology (2013), http://dx.doi.org/10.1016/j.apt.2013.01.008
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Fig. 6. Schematic representation of TiO2–PEG–FA–PAC.
drug release is nearly constant with time. The drug needs to diffuse through the polymer matrix and pores in the matrix. The matrix relaxation and polymer erosion are also important in the drug release through the biodegradable polymer matrix. Excluded volume, hydrodynamic interaction and specific interactions like columbic interaction and hydrogen bonding plays an important role in the drug diffusion from the polymer surface. Drug solubility, polymer degradation, polymer–drug interaction, drug-folic acid interaction are other important factors that influence the drug release [41–44]. 3.4. Cell viability (MTT assay)
Fig. 7. Drug release profiles of paclitaxel from folic acid modified PEG functionalized titanium dioxide nanoparticles error bars represent calculation of standard error on the basis of triplicate determinations (mean ± S.E., n = 3).
to drug molecules that are strongly bound to the polymer and are released from below the surface. The second stage signifies that the
In this study, the exponentially grown human hepatocellular carcinoma cells HepG2 were treated with various concentrations of TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticles ranging from 1.953 to 1000 lg ml1, and the cell viability was measured by the MTT assay. The significant inhibition (P < 0.05; P < 0.01; P < 0.001) of cell viability by nanoparticles were clearly observed in a dose dependent manner. TiO2–PEG–FA and TiO2–PEG–FA–PAC showed cytotoxicity against HepG2 cells. The cell viability for TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticles are given in Fig. 8. The cell viability decreases with increase in the concentration of the
Fig. 8. Cytotoxicity effect of TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticles at different Concentration vs control.
Please cite this article in press as: G. Devanand Venkatasubbu et al., Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery, Advanced Powder Technology (2013), http://dx.doi.org/10.1016/j.apt.2013.01.008
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Fig. 9. Cell viability on TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticle.
nanoparticles. As the concentration of TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticles is increased from 1.953 to 1000 lg ml1 the cell viability decreased. The variation in IC50 values for TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticles are given in Fig. 9. The IC50 value for TiO2–PEG–FA nanoparticles is 59.6 lg ml1, and for TiO2–PEG–FA–PAC is 22.75 lg ml1. The IC50 value for TiO2–PEG–FA–PAC is less than TiO2–PEG–FA nanoparticles. This is due to the presence of paclitaxel attached to the surface modified nanoparticles. As paclitaxel was incorporated in the nanoparticles the cell viability decreases. The IC50 value for pure titanium dioxide nanoparticles is 61.25 lg ml1 [45]. This is higher than TiO2–PEG–FA and TiO2–PEG–FA–PAC nanoparticles. The IC50 value of TiO2–PEG–FA is lower than pure TiO2. The IC50 value of TiO2–PEG–FA–PAC is lower than TiO2–PEG–FA nanoparticles. The IC50 values are in the order of TiO2 > TiO2–PEG–FA > TiO2–PEG–FA–PAC. This reveals that folate moieties in TiO2–PEG–FA–PAC NPs played an important role in enhancing cytotoxic effect by binding of TiO2–PEG–FA–PAC NPs with folate receptors on HepG2 cells, and subsequently increasing their intracellular uptake as a result of the receptor-mediated endocytosis.
4. Conclusion Titanium dioxide nanoparticles have been synthesized with a narrow size distribution. Surface modification of titanium dioxide is done with PEG and functionalized with Folic acid. Anticancer drug paclitaxel is attached to TiO2–PEG–FA molecule. The drug release profile shows an initial burst release and later sustained drug release. The in vitro anticancer studies showed that the TiO2–PEG– FA–Pac has higher anticancer activity than TiO2–PEG–FA nanoparticles.
Acknowledgements Prof. S. Ramasamy, CSIR Emeritus Scientist, acknowledge the financial support given to him to carryout this work under CSIR Emeritus Scientist Scheme number 21(0714)/08/EMR-II dated 2804-2008. Mr. G. Devanand Venkatasubbu, CSIR SRF, acknowledge CSIR for giving him SRF. The authors are greatful to Dr. Mrs. V. Meenakumari, Professor, Department of English, A.P.A College for women, Palani an autonomous college of Mother Theresa University, for making English correction in the manuscript.
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Please cite this article in press as: G. Devanand Venkatasubbu et al., Folate targeted PEGylated titanium dioxide nanoparticles as a nanocarrier for targeted paclitaxel drug delivery, Advanced Powder Technology (2013), http://dx.doi.org/10.1016/j.apt.2013.01.008
