Materials Science and Engineering C 44 (2014) 92–98
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Biogenic gold nano-triangles: Cargos for anticancer drug delivery Roopa Dharmatti 1, Chinmay Phadke 1, Ashmi Mewada, Mukeshchand Thakur, Sunil Pandey, Madhuri Sharon ⁎ N.S.N. Research Center for Nanotechnology and Bio-Nanotechnology, Ambernath, MS, India
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 16 April 2014 Accepted 1 August 2014 Available online 7 August 2014 Keywords: Gold nano triangles Cytotoxicity Biogenic-synthesis HeLa cells MDCK cells Drug release kinetics
a b s t r a c t We present synthesis of biogenic gold nano triangles (GNTs) using Azadirachta indica leaf extract at inherent pH (5.89) and its application in efficient drug delivery of doxorubicin (DOX) (anticancer drug). The main idea was to take advantage of large surface area of GNTs which has 3 dimensions and use the plant peptides coated on these triangles as natural linkers for the attachment of DOX. Sucrose density gradient centrifugation (SDGC) and dialysis methods were used for separation of the GNT from mixture of GNPs. Flocculation parameter (FP) was used to check stability of GNT which was found to be exceptionally high (0–0.75) due to the biological capping agents. DOX attachment to GNT was verified using Fourier transformed infra-red (FTIR) spectroscopy. The complex thus formed was found to be less toxic to normal cells (MDCK cells) and significantly toxic for the cancerous cells (HeLa cells). Drug loading efficiency was found to be 99.81% and DOX release followed first order release kinetics. Percentage drug release was found to be more than 4.5% in both acidic (5.8) as well as physiological pH (7.2) which is suitable for tumor targeting. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology is getting flourished as a consequence of developing nanomaterials and their enormous applications in various fields e.g. drug delivery [1], improvement of cancer diagnostics [2] and other diseases as well as in the field of catalysis [3], fuel cell [4], heavy metal detection [5] and therapeutics [6]. Prevailing among nanoparticles, gold nanoparticles (GNPs) have gathered more attention not only due to their higher efficiency of light absorption at their longitudinal plasmon resonance but also greater efficacy of conjugated drug delivery [7] and less toxicity [8]. The shape, morphology and size of nanoparticles govern the physical, chemical and optical properties of nanoparticles [9,10]. Among these, shape plays a significant role in tuning the properties and is the exigent task to manipulate methodically [10]. In the last few decades, several chemical and physical methods for synthesis of gold nano rods [11], discs [12], multipods [13], triangular prisms [14], cubes [15] and nano-shells [16] have been reported. However, biological methods are more simplistic and eco-friendly and result in the formation of thermodynamically stable nanoparticles which extrudes the disadvantages of chemical methods requiring expensive instruments and result in the release of toxic chemicals [17, 18]. Biological methods also produce nanoparticles having natural linkers onto which drugs can be loaded [7] directly. To this date, metal
⁎ Corresponding author. Tel.: +91 9552599207. E-mail address: [email protected] (M. Sharon). 1 Authors have equal contribution.
http://dx.doi.org/10.1016/j.msec.2014.08.006 0928-4931/© 2014 Elsevier B.V. All rights reserved.
nanoparticles are synthesized with the aid of algae [19,20], bacteria [21,22], fungi [23,24] and plant [25–27] systems. The application of GNPs as drug delivery vehicles resulted in a more efficient drug delivery system because of their controlled release of chemotherapeutic agents to diseased site and minimum use of drug [28,29]. This property of GNPs is explored to anchor drugs and transport them to specific site evading immune mechanism and avoiding damage to healthy tissues. Additionally, gold nano triangles (GNTs) also get internalized inside the cell cytoplasm [30]. Biologically synthesized GNTs are biocompatible [31] and have a large surface area which provides covalent binding of various chemical compounds like drugs [32], proteins [33], genes [30] and other molecules. The unique optical properties of GNTs make them a promising candidate for photothermal treatment and hyperthermia of tumors [34]. Extremely flat morphology is the key feature of GNTs which provides high thermal contact with tumor cells, thereby reducing exposure time. This is not possible with gold nanospheres and rods [35]. Hence, GNTs are considered to be the best option in comparison of gold nanorods and gold nanospheres for cancer treatment [34]. In the present paper we report use of Azadirachta indica leaf extract for synthesis of GNPs in which uniquely shaped triangles were found to be dominant among all other shapes such as cubes, hexagons and spherical structures. For drug delivery, the need of GNTs is envisaged; hence, efforts were directed toward standardizing suitable separation technique. Sucrose density gradient centrifugation (SDGC) technique takes the advantage of the difference in the densities of anisotropic GNPs thus forming different layers of GNPs from which the required nanoparticles can be easily separated. Therefore, GNTs were separated using SDGC based on their different sedimentation rates. Moreover,
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stability of biogenic GNT was assessed by flocculation study to figure out the efficiency of the surface proteins to resist flocculation under physiological pH (7.2) with addition of increasing concentration of NaCl and calculating integrated absorbance between 600 nm and 800 nm. An anticancer drug doxorubicin (DOX) was attached for treatment of tumor cells and drug release kinetics was studied using zero order, first order, Higuchi and Hixson–Crowell model [27,36].
NaCl. Salt solution was added to cuvette containing GNT solution and UV–vis spectrum was recorded. The procedure was repeated with increasing concentrations (0.017–3.4 M) of NaCl to observe the red shift in the peak as compared to the original peak of GNT. FP is an empirical term used for measurement of integrated absorbance between longer wavelengths (600–800 nm in this case). The equation used to calculate the integrated absorbance is as follows: 8Z00
2. Materials and methods P¼ 2.1. Materials
93
IAbs ðλÞdx 600
Gold aurochlorate (HAuCl4), doxorubicin (DOX) and triethylamine (TEA) were purchased from Sigma–Aldrich, USA. All the experiments were carried out in nanopure water. In order to remove the traces of metal contaminants glasswares were washed with Aqua regia.
where, P—flocculation parameter, IAbs—Intensity of absorbance, and λ— wavelength.
2.2. Preparation of plant extract
0.4 mM stock solution of DOX was prepared by dissolving 0.29 g in 10 mL nanopure water. In order to use 0.25 mM, 6.2 mL of stock solution was added in 3.8 mL of GNTs and allowed to react with 70 μL of TEA. The solution was subjected to purging under argon atmosphere for 4 h and stirred continuously using a magnetic stirrer. After 4 h, both inlet and outlet valves were closed. The reaction was allowed to take place for 12 h. Attachment of DOX to GNT was analyzed spectrophotometrically. The resultant GNT–DOX conjugate was dialyzed overnight against nano-pure water to remove unbound DOX. Unbound drug concentration was calculated using standard calibration curve of DOX (straight line equation y = 7.428x). The drug loading efficiency (DLE) of GNPs was calculated using following equation:
Leaves of A. indica were washed with nanopure water to remove dust particles. 5 g of leaves was crushed in 20 mL nanopure water and then filtered through 0.22 μ filter to remove cellular debris. Extract was diluted 100 times using double distilled water for experimental use. This extract was stored at 4 °C until further use. 2.3. Synthesis of GNT A stock solution of 50,000 ppm gold aurochlorate was prepared in nanopure water. In order to use 100 ppm gold aurochlorate, 0.04 mL stock solution was added in 20 mL boiling solution of reaction vessel containing diluted plant extract (inherent pH 5.89). The extract was boiled till the appearance of wine red color.
2.7. Attachment of DOX to biologically synthesized GNT
DLE ¼
Theoretical amount of drug loaded−Free drug 100 Theoretical amount of drug loaded
2.4. Separation of GNTs by SDGC Separation of GNTs from polydispersed and anisotropic GNPs was achieved with the help of sucrose density gradient centrifugation in which 2 mL solutions of each of 60%, 50%, 40%, 30%, 20% and 10% sucrose (w/v) were layered one above other in same order in a centrifuge tube. At the end i.e. above 10% sucrose layer, 2 mL of biogenic GNPs solution was carefully poured. Tube was spun at 5000 rpm in centrifuge (Remi Industries, India) for 40 min. Fractions were collected separately using a micropipette (Eppendorf Research Pipettes, Germany) and characterized spectrophotometrically. After SDGC, for further purification dialysis method was employed using pre-activated dialysis bag (12–14 kD) against nanopure water for 3 h under mild stirring. 2.5. Characterization UV–vis spectroscopy (Lambda 25 PerkinElmer, USA) was carried out using plant extract as reference. Clean quartz cuvette having a path length of 1 cm was used to record the spectra. Morphology of GNTs was studied using field emission scanning electron microscopy (FESEM) on a Carl Zeiss Microimaging, GmbH, Germany. 2–3 drops of the colloidal gold solution were dispensed onto a silicon wafer and dried under ambient condition before examination. Involvement of diverse functional groups and molecular interactions as well as molecular orientation of the complexes were verified using Fourier transformed infra red spectroscopy (FTIR) on a MAGNA-550, Nicolet instruments, USA. The sample was prepared by loading 0.1 mL of GNTs in aqueous form onto the source. 2.6. Stability testing of GNTs using flocculation parameter (FP) The stability of GNTs was checked by analyzing the changes in the optical properties of GNTs in response to the varying concentrations of
2.8. In vitro release of DOX Dialyzed GNT–DOX solution was taken in two different preactivated dialysis bags (2 mL each) and transferred to beakers containing 80 mL of phosphate buffer solution at pH 5.8 and 7.2. The drug release study was conducted at 37 °C with continuous stirring at 100 rpm. To measure the drug release content, samples (3 mL) were periodically removed and replaced with an equivalent volume of the phosphate buffer solution. The amount of released DOX was analyzed with a spectrophotometer at 485 nm and calculated using the standard calibration curve of DOX (straight line equation y = 7.428x). The experiments were performed in triplicate for each sample. With precise control of the GNT–DOX complex, the release of the drug can be tuned to achieve a desired kinetic profile. Four of the most common kinetic profiles are zero order, first order, Higuchi and Hixson–Crowell. These drug release kinetics was calculated using the standard equations as per our previous studies [27,36]. 2.9. Cytotoxicity studies Cytotoxicity effects of Neem extract, GNT, GNT–DOX and free DOX were studied on MDCK and HeLa cells using MTT assay which is based on the conversion of pale yellow MTT to violet colored formazan crystals by mitochondrial enzyme succinate dehydrogenase. Cells were seeded (5 × 105/mL) in 96-well plates and incubated at 37 °C and 5% CO2 for 24 h. The culture medium was then replaced with test solutions and incubated further for 48 h. These solutions were later replaced with MTT (200 μg/mL) and cells were incubated for 2.5 h at 28 ± 2 °C to initiate formation of formazan. After completion of the reaction, the medium was replaced with 200 μL of DMSO. The microtitre plate containing complex was agitated slowly to dissolve formazan crystals. Finally, the
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3.2. Separation of GNTs by SDGC & characterization
Fig. 1. UV–vis spectra of GNPs using A. indica extract. Inset: FE-SEM image of the same.
dissolved formazan in dimethyl sulfoxide (DMSO) was transferred to fresh 96-well plates and read on a microplate reader (Thermo, USA) at 570 nm. 3. Results and discussions 3.1. Synthesis of GNT Boiling of the mixture after HAuCl4 acid addition resulted in a wine red solution indicating the formation of GNPs [10]. There is one prominent peak at 526 nm and a slight hump at 721 nm observed in the UV– visible spectra (Fig. 1). This is an optical phenomenon called surface plasmon resonance (SPR) in GNPs, as a consequence of the peculiar behavior of electrons entrapped in nano-cages leading to quantum confinement effect [37]. Transverse surface plasmon resonance (TSPR) (526 nm) and longitudinal surface plasmon resonance (LSPR) (721 nm) depict the presence of anisotropic gold nanoparticles and/or their agglomeration in the solution. The wide area under peak indicates the presence of polydispersed GNPs. This is further confirmed by FESEM (inset of Fig. 1) which shows the maximum concentrations of GNT. However, nano-hexagons, nano-spheres, nano-rods were also found in minute quantities.
In order to use in drug delivery application, GNTs needed to be separated from the mixture of nanoparticles. Considering the high buoyant density nature of SDGC over conventional equilibrium isopycnic centrifugal strategies, GNTs were separated using the same. The differential sedimentation rates of the nanoparticles were exploited to separate them using centrifugal force [11]. After SDGC, the tubes displayed two distinct layers. Spectrophotometric observation of layer A exhibited a medium intensity peak at 505 nm showing TSPR due to the presence of spherical nanoparticles of varying size (Fig. 2a). On the other hand, the spectra of layer B displayed a peak at 521 nm and a slight hump at 633 nm which corresponds to the presence of non-spherical GNPs which are massive than the spherical ones due to hydrodynamic constraints (Fig. 3a). The FE-SEM image of A fraction revealed the existence of a mixture of spherical, hexagons as well as GNTs having nano-prism morphology (Fig. 2b). On the other hand, the FE-SEM image of fraction B demonstrated the occurrence of maximum GNTs with about 121.7 nm triangle arms and approximately 61.1° angle (Fig. 3b). Fig. 3c shows GNTs obtained after dialysis of fraction B, which was envisaged for its use in attaching drug [30]. It has been reported earlier that nanoparticles of size more than 100 nm get phagocytozed in animal cells [30]. Preliminary drug attachment studies were carried out using UV–vis spectroscopy. Fig. 3a represents red shift from 521 nm to 477 nm due to dielectric constraints also dampening of hump which was initially at 633 nm. The possible reason for dampening is due to interaction between biofabricated GNTs and drug which was further confirmed by FTIR.
3.3. FTIR studies Fig. 4 displays FTIR data of plant extract, GNT and further conjugates.
3.3.1. A. indica extract Bands at 569 and 691 cm−1 represent alkene and acetylenic C\H bending vibrations from backbones of macromolecules in the plant extract. The band at 993 cm−1 is attributed to amine N\H bends presumably from amino acid peptide bonds. The strong band at 1380 and 1603 cm−1 is again due to amine C\N stretches from peptide bonds. Feeble wide band at 3446 cm− 1 is due to hydroxyl and alcoholic vibrations due to aqueous solution.
Fig. 2. (a) UV–vis spectra of Fraction A after separation using SDGC. (b) FE-SEM image of same fraction showing presence of nano-spheres.
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Fig. 4. FTIR spectra of various samples used for the conjugation of the final complex.
Fig. 3. (a) UV–vis spectra of Fraction B. (b) FE-SEM image of same showing majority of GNT. (c) FE-SEM image of fraction B after dialysis.
3.3.4. GNT–DOX complex The new band at 1014 cm− 1 represents either C\O\C stretching vibrations may be arising from drug molecule C_O bond and biofunctionalized GNT surface. The new band at 1425 cm− 1 is carboxylic C\O stretches and 1622 cm− 1 (originally 1603 cm− 1 from DOX) is due to amide C_O bond formation. New bands at 2817 and 2939 cm − 1 are attributed to aldehyde and alkane C\H vibrations prominent after interaction of DOX with the GNT surface. Multiple IR vibrations at 3466 and 3790 cm − 1 are due to hydroxyl groups arising out of aqueous solution. 3.4. Stability testing of GNTs using flocculation parameter (FP)
3.3.2. GNT Peak enhancement was observed at 588 and 691 cm−1 possibly due to an increase in percentage of C\H vibrations on the surface of GNTs. The strong IR band at 1622 cm−1 (originally shifted from 1603 cm−1) was due to weak interactions between the GNT surface and the plant extract via hydrogen bonding involving N\H bends. Since GNTs were dispersed in an aqueous solution the strong peak at 3446 cm− 1 was observed arising from \OH vibrations.
3.3.3. DOX Bands at 609 and 752 cm− 1 arise from alkane CH2 bends, alkene C\H bends and aromatic C\H bends. The prominent peak at 1603 cm− 1 is due to aromatic C_C stretch and amine N\H bending vibrations in the molecule. Wide bands ranging from 3000 to 3668 are due to the high amount of hydroxyl groups from the drug molecule as well as aqueous solution.
The most important quality of GNPs synthesized using A. indica leaf extract was its outstanding stability under physiological conditions (pH 7.2) by virtue of ions and peptides which resist their agglomeration. The effect of NaCl on the spectral behavior of biologically capped GNPs is shown in Fig. 5a. The initial peak of biogenic GNPs was at 521 nm, which showed a red shift to 666 nm after the addition of NaCl solution of increasing concentration. The peak was found to be stable after addition of any further amount of salt with a slight decrease in intensity due change in volume. This red shift implies the presence of dipole interaction between plasmons of adjacent nanoparticles in the solution induced by the NaCl solution [38]. It must be mentioned here that, if improper folding of capping proteins and weak electrostatic attraction between NH4 and COOH groups occur it leads to aggregation of GNPs [39]. Whereas no aggregation will occur if hydrophilic charged groups are exposed to interact with water and hydrophobic groups are hindered [39].
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Fig. 5. (a) UV–vis spectra of GNT displaying high stability with addition of NaCl at pH 7.2. (b) Flocculation parameter of GNT indicating exceptional stability due to capping proteins.
The stability of these GNTs was assayed by recording the UV–vis spectrum as mentioned earlier. Fig. 5b demonstrates that FP increases with increasing concentrations of salt. Thus at particular pH the efficacy of capping proteins to prevent agglomeration can be considered as directly proportional to the FP. Smaller FP in this case points to exceptional stability of GNTs. This stability imparted by the capping proteins makes it excellent cargos for the delivery of drugs and these surface proteins are used as linkers instead of additional chemical linkers thus avoiding unnecessary toxic effects.
3.5. Drug release kinetics The idea of architecting biogenic GNTs with DOX is for sustained release of DOX in an ideal tumor microenvironment. DOX showed that exceptional drug loading efficiency was calculated to be 99.81% (calculations shown in Supporting Information) with GNTs which provided larger surface area as well as the presence of natural linkers for attachment of drug .The drug loading efficiency of GNTs is thus comparatively higher than the previously assessed drug loading capacity of spheres [7] and rods [36] in our lab. The drug release kinetics was studied using zero order, first order, Higuchi and Hixson–Crowell models at pH 5.8 and 7.2. The deciding factor for the type of drug release profile was the regression coefficients (R2) of above mentioned statistical models.
In a solid tumor because of interstitial fluid pressure, which is a consequence of the highly acidic environment it becomes an arduous task to deliver drugs [40, 41]. To tackle this crucial problem a drug delivery system which can withstand tumor conditions and carry large amounts of drug to the targeted tumor cells is needed. GNTs are ideal cargos as they have represented good drug release profiles in both basic (pH 7.2) and acidic (pH 5.8) conditions. Initially after an hour, the drug release at pH 5.8 (1.04%) was found to be greater than release at pH 7.2 (0.3%) as shown in (Fig. 6a). At the end of 72 h, the drug release was more than 4.5% in both acidic and basic conditions (Fig. 6a). Thus in solid tumor chemotherapy GNTs can be used as smart delivery vehicles. Depending upon the distribution from the blood vessels, tumor cells may have acidic or basic pH. The complex prepared in this experiment (GNT–DOX), capable of releasing drug in acidic as well as basic environment, plays an important role for tackling any type of solid tumor cell. DOX follows first order release kinetics (Fig. 6b). Most of the drugs which are administered by injection follow first order release kinetics and it was interesting to note that DOX + GNT complex also followed first order release kinetics.
3.6. Cytotoxicity studies The cytotoxicity studies revealed that A. indica extract was biocompatible, as parentage viability values of both MDCK and HeLa cells
Fig. 6. (a) Percentage drug release in in-vitro conditions with respect to time. (b) Drug release profile of DOX following 1st order release kinetics.
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Fig. 7. Cytotoxic effects of A. indica extract, GNT, GNT-DOX and Free DOX on (a) MDCK cells and (b) HeLa cells.
were 98.1% and 97.0% at their highest concentrations respectively (Fig. 7a and b). GNTs showed an identical impact on the percentage viability of both the cell types (MDCK and HeLa) indicating their biocompatibility. The IC50 value of the complex of GNT–DOX on HeLa cells was found to be 0.1 mM which was a much smaller value as compared with that of free DOX i.e. 0.2 mM, whereas the complex shows less cytoxicity on MDCK cells which was calculated to be 65.3% at its highest concentration (0.25 mM). The calculated IC50 value of free DOX on MDCK cells was 0.25 mM, which is higher as compared to that of the final complex. So, the complex of GNT–DOX is biocompatible as well as excellent armadas for tumor chemotherapy. 4. Conclusion Biogenic GNTs obtained from A. indica leaf extract are promising contenders for the delivery of anticancer drug DOX and are boon for drug delivery. GNTs have got the edge over the other drug delivery systems as a consequence of their following affirmatives: 1. High drug loading capacity because of their large surface area. 2. Extreme stability under high salt concentrations because of proper folding of capping proteins thus preventing agglomeration. 3. Importantly mild toxicity to normal cells and considerable toxicity for cancer cells (HeLa cells). 4. Also it follows first order kinetics with an adequate amount of drug release at both acidic and physiological conditions, which is suitable for the tumor microenvironment. Acknowledgments Authors are indebted to funding authorities of N.S.N. Research Center for carrying out the projects. We are also thankful to TIFR, Mumbai and IIT Bombay, SAIF department-Mumbai for their support in FTIR and FE-SEM characterization. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.08.006. References [1] Wim H. De Jong, Paul J.A. Borm, Drug delivery and nanoparticles: applications and hazards, Int. J. Nanomedicine 3 (2008) 133–149. [2] A. Aliosmanoglu, I. Basaran, Nanotechnology in cancer treatment, J. Nanomed. Biotherapeut. Discov. 2 (2012), http://dx.doi.org/10.4172/2155-983X.1000107. [3] Bing Zhou, Scott Han, Robert Raja, Gabor A. Somorja, Nanotechnology in Catalysis, vol. 3, Springer, New York, 2007.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Biogenic gold nano-triangles: Cargos for anticancer drug delivery Roopa Dharmatti 1, Chinmay Phadke 1, Ashmi Mewada, Mukeshchand Thakur, Sunil Pandey, Madhuri Sharon ⁎ N.S.N. Research Center for Nanotechnology and Bio-Nanotechnology, Ambernath, MS, India
a r t i c l e
i n f o
Article history: Received 6 December 2013 Received in revised form 16 April 2014 Accepted 1 August 2014 Available online 7 August 2014 Keywords: Gold nano triangles Cytotoxicity Biogenic-synthesis HeLa cells MDCK cells Drug release kinetics
a b s t r a c t We present synthesis of biogenic gold nano triangles (GNTs) using Azadirachta indica leaf extract at inherent pH (5.89) and its application in efficient drug delivery of doxorubicin (DOX) (anticancer drug). The main idea was to take advantage of large surface area of GNTs which has 3 dimensions and use the plant peptides coated on these triangles as natural linkers for the attachment of DOX. Sucrose density gradient centrifugation (SDGC) and dialysis methods were used for separation of the GNT from mixture of GNPs. Flocculation parameter (FP) was used to check stability of GNT which was found to be exceptionally high (0–0.75) due to the biological capping agents. DOX attachment to GNT was verified using Fourier transformed infra-red (FTIR) spectroscopy. The complex thus formed was found to be less toxic to normal cells (MDCK cells) and significantly toxic for the cancerous cells (HeLa cells). Drug loading efficiency was found to be 99.81% and DOX release followed first order release kinetics. Percentage drug release was found to be more than 4.5% in both acidic (5.8) as well as physiological pH (7.2) which is suitable for tumor targeting. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nanotechnology is getting flourished as a consequence of developing nanomaterials and their enormous applications in various fields e.g. drug delivery [1], improvement of cancer diagnostics [2] and other diseases as well as in the field of catalysis [3], fuel cell [4], heavy metal detection [5] and therapeutics [6]. Prevailing among nanoparticles, gold nanoparticles (GNPs) have gathered more attention not only due to their higher efficiency of light absorption at their longitudinal plasmon resonance but also greater efficacy of conjugated drug delivery [7] and less toxicity [8]. The shape, morphology and size of nanoparticles govern the physical, chemical and optical properties of nanoparticles [9,10]. Among these, shape plays a significant role in tuning the properties and is the exigent task to manipulate methodically [10]. In the last few decades, several chemical and physical methods for synthesis of gold nano rods [11], discs [12], multipods [13], triangular prisms [14], cubes [15] and nano-shells [16] have been reported. However, biological methods are more simplistic and eco-friendly and result in the formation of thermodynamically stable nanoparticles which extrudes the disadvantages of chemical methods requiring expensive instruments and result in the release of toxic chemicals [17, 18]. Biological methods also produce nanoparticles having natural linkers onto which drugs can be loaded [7] directly. To this date, metal
⁎ Corresponding author. Tel.: +91 9552599207. E-mail address: [email protected] (M. Sharon). 1 Authors have equal contribution.
http://dx.doi.org/10.1016/j.msec.2014.08.006 0928-4931/© 2014 Elsevier B.V. All rights reserved.
nanoparticles are synthesized with the aid of algae [19,20], bacteria [21,22], fungi [23,24] and plant [25–27] systems. The application of GNPs as drug delivery vehicles resulted in a more efficient drug delivery system because of their controlled release of chemotherapeutic agents to diseased site and minimum use of drug [28,29]. This property of GNPs is explored to anchor drugs and transport them to specific site evading immune mechanism and avoiding damage to healthy tissues. Additionally, gold nano triangles (GNTs) also get internalized inside the cell cytoplasm [30]. Biologically synthesized GNTs are biocompatible [31] and have a large surface area which provides covalent binding of various chemical compounds like drugs [32], proteins [33], genes [30] and other molecules. The unique optical properties of GNTs make them a promising candidate for photothermal treatment and hyperthermia of tumors [34]. Extremely flat morphology is the key feature of GNTs which provides high thermal contact with tumor cells, thereby reducing exposure time. This is not possible with gold nanospheres and rods [35]. Hence, GNTs are considered to be the best option in comparison of gold nanorods and gold nanospheres for cancer treatment [34]. In the present paper we report use of Azadirachta indica leaf extract for synthesis of GNPs in which uniquely shaped triangles were found to be dominant among all other shapes such as cubes, hexagons and spherical structures. For drug delivery, the need of GNTs is envisaged; hence, efforts were directed toward standardizing suitable separation technique. Sucrose density gradient centrifugation (SDGC) technique takes the advantage of the difference in the densities of anisotropic GNPs thus forming different layers of GNPs from which the required nanoparticles can be easily separated. Therefore, GNTs were separated using SDGC based on their different sedimentation rates. Moreover,
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stability of biogenic GNT was assessed by flocculation study to figure out the efficiency of the surface proteins to resist flocculation under physiological pH (7.2) with addition of increasing concentration of NaCl and calculating integrated absorbance between 600 nm and 800 nm. An anticancer drug doxorubicin (DOX) was attached for treatment of tumor cells and drug release kinetics was studied using zero order, first order, Higuchi and Hixson–Crowell model [27,36].
NaCl. Salt solution was added to cuvette containing GNT solution and UV–vis spectrum was recorded. The procedure was repeated with increasing concentrations (0.017–3.4 M) of NaCl to observe the red shift in the peak as compared to the original peak of GNT. FP is an empirical term used for measurement of integrated absorbance between longer wavelengths (600–800 nm in this case). The equation used to calculate the integrated absorbance is as follows: 8Z00
2. Materials and methods P¼ 2.1. Materials
93
IAbs ðλÞdx 600
Gold aurochlorate (HAuCl4), doxorubicin (DOX) and triethylamine (TEA) were purchased from Sigma–Aldrich, USA. All the experiments were carried out in nanopure water. In order to remove the traces of metal contaminants glasswares were washed with Aqua regia.
where, P—flocculation parameter, IAbs—Intensity of absorbance, and λ— wavelength.
2.2. Preparation of plant extract
0.4 mM stock solution of DOX was prepared by dissolving 0.29 g in 10 mL nanopure water. In order to use 0.25 mM, 6.2 mL of stock solution was added in 3.8 mL of GNTs and allowed to react with 70 μL of TEA. The solution was subjected to purging under argon atmosphere for 4 h and stirred continuously using a magnetic stirrer. After 4 h, both inlet and outlet valves were closed. The reaction was allowed to take place for 12 h. Attachment of DOX to GNT was analyzed spectrophotometrically. The resultant GNT–DOX conjugate was dialyzed overnight against nano-pure water to remove unbound DOX. Unbound drug concentration was calculated using standard calibration curve of DOX (straight line equation y = 7.428x). The drug loading efficiency (DLE) of GNPs was calculated using following equation:
Leaves of A. indica were washed with nanopure water to remove dust particles. 5 g of leaves was crushed in 20 mL nanopure water and then filtered through 0.22 μ filter to remove cellular debris. Extract was diluted 100 times using double distilled water for experimental use. This extract was stored at 4 °C until further use. 2.3. Synthesis of GNT A stock solution of 50,000 ppm gold aurochlorate was prepared in nanopure water. In order to use 100 ppm gold aurochlorate, 0.04 mL stock solution was added in 20 mL boiling solution of reaction vessel containing diluted plant extract (inherent pH 5.89). The extract was boiled till the appearance of wine red color.
2.7. Attachment of DOX to biologically synthesized GNT
DLE ¼
Theoretical amount of drug loaded−Free drug 100 Theoretical amount of drug loaded
2.4. Separation of GNTs by SDGC Separation of GNTs from polydispersed and anisotropic GNPs was achieved with the help of sucrose density gradient centrifugation in which 2 mL solutions of each of 60%, 50%, 40%, 30%, 20% and 10% sucrose (w/v) were layered one above other in same order in a centrifuge tube. At the end i.e. above 10% sucrose layer, 2 mL of biogenic GNPs solution was carefully poured. Tube was spun at 5000 rpm in centrifuge (Remi Industries, India) for 40 min. Fractions were collected separately using a micropipette (Eppendorf Research Pipettes, Germany) and characterized spectrophotometrically. After SDGC, for further purification dialysis method was employed using pre-activated dialysis bag (12–14 kD) against nanopure water for 3 h under mild stirring. 2.5. Characterization UV–vis spectroscopy (Lambda 25 PerkinElmer, USA) was carried out using plant extract as reference. Clean quartz cuvette having a path length of 1 cm was used to record the spectra. Morphology of GNTs was studied using field emission scanning electron microscopy (FESEM) on a Carl Zeiss Microimaging, GmbH, Germany. 2–3 drops of the colloidal gold solution were dispensed onto a silicon wafer and dried under ambient condition before examination. Involvement of diverse functional groups and molecular interactions as well as molecular orientation of the complexes were verified using Fourier transformed infra red spectroscopy (FTIR) on a MAGNA-550, Nicolet instruments, USA. The sample was prepared by loading 0.1 mL of GNTs in aqueous form onto the source. 2.6. Stability testing of GNTs using flocculation parameter (FP) The stability of GNTs was checked by analyzing the changes in the optical properties of GNTs in response to the varying concentrations of
2.8. In vitro release of DOX Dialyzed GNT–DOX solution was taken in two different preactivated dialysis bags (2 mL each) and transferred to beakers containing 80 mL of phosphate buffer solution at pH 5.8 and 7.2. The drug release study was conducted at 37 °C with continuous stirring at 100 rpm. To measure the drug release content, samples (3 mL) were periodically removed and replaced with an equivalent volume of the phosphate buffer solution. The amount of released DOX was analyzed with a spectrophotometer at 485 nm and calculated using the standard calibration curve of DOX (straight line equation y = 7.428x). The experiments were performed in triplicate for each sample. With precise control of the GNT–DOX complex, the release of the drug can be tuned to achieve a desired kinetic profile. Four of the most common kinetic profiles are zero order, first order, Higuchi and Hixson–Crowell. These drug release kinetics was calculated using the standard equations as per our previous studies [27,36]. 2.9. Cytotoxicity studies Cytotoxicity effects of Neem extract, GNT, GNT–DOX and free DOX were studied on MDCK and HeLa cells using MTT assay which is based on the conversion of pale yellow MTT to violet colored formazan crystals by mitochondrial enzyme succinate dehydrogenase. Cells were seeded (5 × 105/mL) in 96-well plates and incubated at 37 °C and 5% CO2 for 24 h. The culture medium was then replaced with test solutions and incubated further for 48 h. These solutions were later replaced with MTT (200 μg/mL) and cells were incubated for 2.5 h at 28 ± 2 °C to initiate formation of formazan. After completion of the reaction, the medium was replaced with 200 μL of DMSO. The microtitre plate containing complex was agitated slowly to dissolve formazan crystals. Finally, the
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3.2. Separation of GNTs by SDGC & characterization
Fig. 1. UV–vis spectra of GNPs using A. indica extract. Inset: FE-SEM image of the same.
dissolved formazan in dimethyl sulfoxide (DMSO) was transferred to fresh 96-well plates and read on a microplate reader (Thermo, USA) at 570 nm. 3. Results and discussions 3.1. Synthesis of GNT Boiling of the mixture after HAuCl4 acid addition resulted in a wine red solution indicating the formation of GNPs [10]. There is one prominent peak at 526 nm and a slight hump at 721 nm observed in the UV– visible spectra (Fig. 1). This is an optical phenomenon called surface plasmon resonance (SPR) in GNPs, as a consequence of the peculiar behavior of electrons entrapped in nano-cages leading to quantum confinement effect [37]. Transverse surface plasmon resonance (TSPR) (526 nm) and longitudinal surface plasmon resonance (LSPR) (721 nm) depict the presence of anisotropic gold nanoparticles and/or their agglomeration in the solution. The wide area under peak indicates the presence of polydispersed GNPs. This is further confirmed by FESEM (inset of Fig. 1) which shows the maximum concentrations of GNT. However, nano-hexagons, nano-spheres, nano-rods were also found in minute quantities.
In order to use in drug delivery application, GNTs needed to be separated from the mixture of nanoparticles. Considering the high buoyant density nature of SDGC over conventional equilibrium isopycnic centrifugal strategies, GNTs were separated using the same. The differential sedimentation rates of the nanoparticles were exploited to separate them using centrifugal force [11]. After SDGC, the tubes displayed two distinct layers. Spectrophotometric observation of layer A exhibited a medium intensity peak at 505 nm showing TSPR due to the presence of spherical nanoparticles of varying size (Fig. 2a). On the other hand, the spectra of layer B displayed a peak at 521 nm and a slight hump at 633 nm which corresponds to the presence of non-spherical GNPs which are massive than the spherical ones due to hydrodynamic constraints (Fig. 3a). The FE-SEM image of A fraction revealed the existence of a mixture of spherical, hexagons as well as GNTs having nano-prism morphology (Fig. 2b). On the other hand, the FE-SEM image of fraction B demonstrated the occurrence of maximum GNTs with about 121.7 nm triangle arms and approximately 61.1° angle (Fig. 3b). Fig. 3c shows GNTs obtained after dialysis of fraction B, which was envisaged for its use in attaching drug [30]. It has been reported earlier that nanoparticles of size more than 100 nm get phagocytozed in animal cells [30]. Preliminary drug attachment studies were carried out using UV–vis spectroscopy. Fig. 3a represents red shift from 521 nm to 477 nm due to dielectric constraints also dampening of hump which was initially at 633 nm. The possible reason for dampening is due to interaction between biofabricated GNTs and drug which was further confirmed by FTIR.
3.3. FTIR studies Fig. 4 displays FTIR data of plant extract, GNT and further conjugates.
3.3.1. A. indica extract Bands at 569 and 691 cm−1 represent alkene and acetylenic C\H bending vibrations from backbones of macromolecules in the plant extract. The band at 993 cm−1 is attributed to amine N\H bends presumably from amino acid peptide bonds. The strong band at 1380 and 1603 cm−1 is again due to amine C\N stretches from peptide bonds. Feeble wide band at 3446 cm− 1 is due to hydroxyl and alcoholic vibrations due to aqueous solution.
Fig. 2. (a) UV–vis spectra of Fraction A after separation using SDGC. (b) FE-SEM image of same fraction showing presence of nano-spheres.
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Fig. 4. FTIR spectra of various samples used for the conjugation of the final complex.
Fig. 3. (a) UV–vis spectra of Fraction B. (b) FE-SEM image of same showing majority of GNT. (c) FE-SEM image of fraction B after dialysis.
3.3.4. GNT–DOX complex The new band at 1014 cm− 1 represents either C\O\C stretching vibrations may be arising from drug molecule C_O bond and biofunctionalized GNT surface. The new band at 1425 cm− 1 is carboxylic C\O stretches and 1622 cm− 1 (originally 1603 cm− 1 from DOX) is due to amide C_O bond formation. New bands at 2817 and 2939 cm − 1 are attributed to aldehyde and alkane C\H vibrations prominent after interaction of DOX with the GNT surface. Multiple IR vibrations at 3466 and 3790 cm − 1 are due to hydroxyl groups arising out of aqueous solution. 3.4. Stability testing of GNTs using flocculation parameter (FP)
3.3.2. GNT Peak enhancement was observed at 588 and 691 cm−1 possibly due to an increase in percentage of C\H vibrations on the surface of GNTs. The strong IR band at 1622 cm−1 (originally shifted from 1603 cm−1) was due to weak interactions between the GNT surface and the plant extract via hydrogen bonding involving N\H bends. Since GNTs were dispersed in an aqueous solution the strong peak at 3446 cm− 1 was observed arising from \OH vibrations.
3.3.3. DOX Bands at 609 and 752 cm− 1 arise from alkane CH2 bends, alkene C\H bends and aromatic C\H bends. The prominent peak at 1603 cm− 1 is due to aromatic C_C stretch and amine N\H bending vibrations in the molecule. Wide bands ranging from 3000 to 3668 are due to the high amount of hydroxyl groups from the drug molecule as well as aqueous solution.
The most important quality of GNPs synthesized using A. indica leaf extract was its outstanding stability under physiological conditions (pH 7.2) by virtue of ions and peptides which resist their agglomeration. The effect of NaCl on the spectral behavior of biologically capped GNPs is shown in Fig. 5a. The initial peak of biogenic GNPs was at 521 nm, which showed a red shift to 666 nm after the addition of NaCl solution of increasing concentration. The peak was found to be stable after addition of any further amount of salt with a slight decrease in intensity due change in volume. This red shift implies the presence of dipole interaction between plasmons of adjacent nanoparticles in the solution induced by the NaCl solution [38]. It must be mentioned here that, if improper folding of capping proteins and weak electrostatic attraction between NH4 and COOH groups occur it leads to aggregation of GNPs [39]. Whereas no aggregation will occur if hydrophilic charged groups are exposed to interact with water and hydrophobic groups are hindered [39].
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Fig. 5. (a) UV–vis spectra of GNT displaying high stability with addition of NaCl at pH 7.2. (b) Flocculation parameter of GNT indicating exceptional stability due to capping proteins.
The stability of these GNTs was assayed by recording the UV–vis spectrum as mentioned earlier. Fig. 5b demonstrates that FP increases with increasing concentrations of salt. Thus at particular pH the efficacy of capping proteins to prevent agglomeration can be considered as directly proportional to the FP. Smaller FP in this case points to exceptional stability of GNTs. This stability imparted by the capping proteins makes it excellent cargos for the delivery of drugs and these surface proteins are used as linkers instead of additional chemical linkers thus avoiding unnecessary toxic effects.
3.5. Drug release kinetics The idea of architecting biogenic GNTs with DOX is for sustained release of DOX in an ideal tumor microenvironment. DOX showed that exceptional drug loading efficiency was calculated to be 99.81% (calculations shown in Supporting Information) with GNTs which provided larger surface area as well as the presence of natural linkers for attachment of drug .The drug loading efficiency of GNTs is thus comparatively higher than the previously assessed drug loading capacity of spheres [7] and rods [36] in our lab. The drug release kinetics was studied using zero order, first order, Higuchi and Hixson–Crowell models at pH 5.8 and 7.2. The deciding factor for the type of drug release profile was the regression coefficients (R2) of above mentioned statistical models.
In a solid tumor because of interstitial fluid pressure, which is a consequence of the highly acidic environment it becomes an arduous task to deliver drugs [40, 41]. To tackle this crucial problem a drug delivery system which can withstand tumor conditions and carry large amounts of drug to the targeted tumor cells is needed. GNTs are ideal cargos as they have represented good drug release profiles in both basic (pH 7.2) and acidic (pH 5.8) conditions. Initially after an hour, the drug release at pH 5.8 (1.04%) was found to be greater than release at pH 7.2 (0.3%) as shown in (Fig. 6a). At the end of 72 h, the drug release was more than 4.5% in both acidic and basic conditions (Fig. 6a). Thus in solid tumor chemotherapy GNTs can be used as smart delivery vehicles. Depending upon the distribution from the blood vessels, tumor cells may have acidic or basic pH. The complex prepared in this experiment (GNT–DOX), capable of releasing drug in acidic as well as basic environment, plays an important role for tackling any type of solid tumor cell. DOX follows first order release kinetics (Fig. 6b). Most of the drugs which are administered by injection follow first order release kinetics and it was interesting to note that DOX + GNT complex also followed first order release kinetics.
3.6. Cytotoxicity studies The cytotoxicity studies revealed that A. indica extract was biocompatible, as parentage viability values of both MDCK and HeLa cells
Fig. 6. (a) Percentage drug release in in-vitro conditions with respect to time. (b) Drug release profile of DOX following 1st order release kinetics.
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Fig. 7. Cytotoxic effects of A. indica extract, GNT, GNT-DOX and Free DOX on (a) MDCK cells and (b) HeLa cells.
were 98.1% and 97.0% at their highest concentrations respectively (Fig. 7a and b). GNTs showed an identical impact on the percentage viability of both the cell types (MDCK and HeLa) indicating their biocompatibility. The IC50 value of the complex of GNT–DOX on HeLa cells was found to be 0.1 mM which was a much smaller value as compared with that of free DOX i.e. 0.2 mM, whereas the complex shows less cytoxicity on MDCK cells which was calculated to be 65.3% at its highest concentration (0.25 mM). The calculated IC50 value of free DOX on MDCK cells was 0.25 mM, which is higher as compared to that of the final complex. So, the complex of GNT–DOX is biocompatible as well as excellent armadas for tumor chemotherapy. 4. Conclusion Biogenic GNTs obtained from A. indica leaf extract are promising contenders for the delivery of anticancer drug DOX and are boon for drug delivery. GNTs have got the edge over the other drug delivery systems as a consequence of their following affirmatives: 1. High drug loading capacity because of their large surface area. 2. Extreme stability under high salt concentrations because of proper folding of capping proteins thus preventing agglomeration. 3. Importantly mild toxicity to normal cells and considerable toxicity for cancer cells (HeLa cells). 4. Also it follows first order kinetics with an adequate amount of drug release at both acidic and physiological conditions, which is suitable for the tumor microenvironment. Acknowledgments Authors are indebted to funding authorities of N.S.N. Research Center for carrying out the projects. We are also thankful to TIFR, Mumbai and IIT Bombay, SAIF department-Mumbai for their support in FTIR and FE-SEM characterization. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.08.006. References [1] Wim H. De Jong, Paul J.A. Borm, Drug delivery and nanoparticles: applications and hazards, Int. J. Nanomedicine 3 (2008) 133–149. [2] A. Aliosmanoglu, I. Basaran, Nanotechnology in cancer treatment, J. Nanomed. Biotherapeut. Discov. 2 (2012), http://dx.doi.org/10.4172/2155-983X.1000107. [3] Bing Zhou, Scott Han, Robert Raja, Gabor A. Somorja, Nanotechnology in Catalysis, vol. 3, Springer, New York, 2007.
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