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Biogenic Gold Nano-Triangles: Cargos for anticancer drug delivery

Roopa Dharmatti, Chinmay Phadke, Ashmi Mewada, Mukeshchand Thakur,Sunil Pandey, Madhuri Sharon

PII: S0928-4931(14)00491-3DOI: doi: 10.1016/j.msec.2014.08.006Reference: MSC 4822

To appear in: Materials Science & Engineering C

Received date: 6 December 2013Revised date: 16 April 2014Accepted date: 1 August 2014

Please cite this article as: Roopa Dharmatti, Chinmay Phadke, Ashmi Mewada,Mukeshchand Thakur, Sunil Pandey, Madhuri Sharon, Biogenic Gold Nano-Triangles:Cargos for anticancer drug delivery, Materials Science & Engineering C (2014), doi:10.1016/j.msec.2014.08.006

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Biogenic Gold Nano-Triangles: Cargos for

anticancer drug delivery

Roopa Dharmatti

¶, Chinmay Phadke

¶, Ashmi Mewada, Mukeshchand Thakur, Sunil Pandey and

Madhuri Sharon*

N. S. N. Research Center for Nanotechnology and Bio-Nanotechnology, Ambernath, MS, India

¶ Authors have equal contribution

*Author for correspondence: sharonmadhuri@gmail.com

Phone: +91 9552599207

Abstract

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.

Keywords: Gold Nano Triangles, Cytotoxicity, Biogenic-synthesis, HeLa Cells, MDCK cells,

Drug release kinetics

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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 physical, chemical and optical

properties of nanoparticles [9, 10]. Among these, shape plays significant role in tuning the

properties and is the exigent task to manipulate methodically [10]. In 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, eco-friendly and results in formation of

thermodynamically stable nanoparticles which extrudes the disadvantages of chemical methods

requiring expensive instruments and results in 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 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 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 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 tumours [34]. Extremely flat

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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 best option in comparison Gold nanorods and Gold

nanospheres for cancer treatment [34].

In present paper we report use of A. 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, need of GNTs is envisaged, hence efforts were directed

towards standardizing suitable separation technique. Since Sucrose Density Gradient

Centrifugation (SDGC) technique, takes the advantage of 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, 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 600nm and 800nm. 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].

2. Materials and methods

2.1. Materials:

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.

2.2. Preparation of plant extract:

Leaves of Azadirachta indica were washed with nanopure water to remove dust particles. 5 g of

leaves were crushed in 20 mL nanopure water and then filtered through 0.22µ 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.

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2.3. Synthesis of GNT:

A stock solution of 50,000 ppm gold aurochlorate was prepared in nanopure water. In order to

use 100ppm 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.

2.4. Separation of GNTs by SDGC:

Separation of GNTs from polydispersed and anisotropic GNPs was achieved with 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 minutes.

Fractions were collected separately using micropipette (Eppendorf Research Pipettes, Germany)

and characterized spectrophotometrically. After SDGC, for further purification dialysis method

was employed using pre-activated dialysis bag (12-14kD) against nanopure water for 3hrs 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 path length 1 cm was used to record the spectra.

Morphology of GNTs was studied using Field Emission Scanning Electron Microscopy (FE-

SEM) 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 was 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 NaCl. Salt solution was added to cuvette containing

GNT solution and UV-Vis spectrum was recorded. Procedure was repeated with increasing

concentrations (0.017-3.4M) of NaCl to observe the red shift in peak as compared to the original

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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:

800

600

)( dxIP Abs

Where, P – Flocculation Parameter, IAbs – Intensity of absorbance, λ – wavelength.

2.7. Attachment of DOX to biologically synthesized GNT:

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

hours and stirred continuously using a magnetic stirrer. After 4 hours, both inlet and outlet valves

were closed. The reaction was allowed to take place for 12 hours. 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)

Drug loading efficiency (DLE) of GNPs was calculated using following equation:

2.8. In Vitro release of DOX:

Dialyzed GNT-DOX solution was taken in two different pre-activated 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

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spectrophotometer at 485 nm and calculated using 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 (5x105

/mL) in 96-well plates and incubated at 37ºC and 5% CO2 for 24 h. 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±2ºC to

initiate formation of formazan. After completion of the reaction, medium was replaced with 200

µl of DMSO. The Microtitre plate containing complex was agitated slowly to dissolve formazan

crystals. Finally, the dissolved formazan in Dimethyl Sulfoxide (DMSO) was transferred to fresh

96-well plates and read on microplate reader (Thermo, USA) at 570 nm.

3. Results and Discussions:

3.1. Synthesis of GNT:

Boiling of mixture after HAuCl4 acid addition resulted in wine red solution indicating 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 optical phenomenon called Surface Plasmon resonance

(SPR) in GNPs, as a consequence of 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. Wide area under peak

indicates presence of polydispersed GNPs. This is further confirmed by FE-SEM (Inset of Fig.

1) which shows the maximum concentrations of GNT. However, nano-hexagons, nano-spheres,

nano-rods were also found in minute quantities.

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3.2. Separation of GNTs by SDGC & Characterization:

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

presence of spherical nanoparticles of varying size (Fig 2a). On the other hand, spectra of layer B

displayed a peak at 521 nm and a slight hump at 633 nm which corresponds to presence of non-

spherical GNPs which are massive than spherical ones due to hydrodynamic constraints (Fig 3a).

FE-SEM image of A fraction revealed existence of mixture of spherical, hexagons as well as

GNTs having nano-prism morphology (Fig 2b). On the other hand, FE-SEM image of fraction B

demonstrated occurrence of maximum GNTs with about 121.7 nm triangle arms and

approximately 61.1º 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 gets 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. Band at 993 cm-1

is attributed to amine N-H

bends presumably from amino acid peptide bonds. 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.

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3.3.2. GNT

Peak enhancement was observed at 588 and 691 cm-1

possibly due to increase in percentage of

C-H vibrations on the surface of GNTs. Strong IR band at 1622 cm-1

(Originally shifted

from1603 cm-1

) was due to weak interactions between GNT surface and plant extract via

hydrogen bonding involving N-H bends. Since GNTs were dispersed in aqueous solution 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. 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 3000-3668 is due to high amount of

hydroxyl groups from the drug molecule as well as aqueous solution.

3.3.4. GNT-DOX Complex

New band at 1014 cm-1

represents either C-O-C stretching vibrations may be arising from drug

molecule C=O bond and bio-functionalized GNT surface. New band at 1425 cm-1

is carboxylic

C-O stretches and 1622 cm-1

(originally 1603 cm-1

from DOX) is due 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 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):

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 resists their agglomeration. The effect of NaCl on spectral behavior of biologically capped

GNPs is shown in Fig. 5a. The initial peak of biogenic GNPs was at 521 nm, which showed 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 slight decrease in intensity

due change in volume. This red shift implies the presence of dipole interaction between

plasmons of adjacent nanoparticles in solution induced by the NaCl solution [38]. It must be

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mentioned here that, if improper folding of capping proteins and weak electrostatic attraction

between NH4 and COOH groups occurs 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].

Stability of these GNTs was assayed by recording 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 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 presence of natural linkers for attachment of drug .The drug loading

efficiency of GNTs is thus comparatively higher than previously assessed drug loading capacity

of spheres [7] and rods [40] 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 regression coefficients (R2) of above mentioned statistical

models.

In a solid tumor because of interstitial fluid pressure, which is consequence of highly acidic

environment it becomes arduous task to deliver drugs [41, 42]. 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 hours, 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 distribution from the blood vessels, tumor cells may have acidic or

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basic pH. The complex prepared in this experiment (GNT-DOX), capable of releasing drug in

acidic as well as basic environment plays 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 were 98.1% and 97.0% at their highest concentrations

respectively (Fig. 7a and 7b). GNTs showed identical impact on percentage viability of both the

cell types (MDCK and HeLa) indicating their biocompatibility. IC50 value of Complex of GNT-

DOX on HeLa cells was found to be 0.1 mM which was 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 adequate amount of drug release at both

acidic and physiological, which is suitable for tumor microenvironment.

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Acknowledgements

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.

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List of captions for Figures:

Fig. 1. UV- Vis spectra of GNPs using A. indica extract. Inset: FE-SEM image of the same

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

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.

Fig. 4. FTIR spectra of various samples used for the conjugation of final complex

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

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

Fig. 7. Cytotoxic effects of A. indica extract, GNT, GNT-DOX and Free DOX on (a) MDCK

cells and (b) HeLa cells

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4

Fig. 5

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Fig. 6

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Fig. 7

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Graphical abstract

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Highlights

Biosynthesis of Gold nanoparticles (GNPs) using A. indica leaf extract.

Separation of stable Gold Nano Triangles (GNTs) by Sucrose Density Gradient

Centrifugation (SDGC).

Attachment of anti-cancer drug Doxorubicin (DOX) on GNTs

High biocompatibility of GNTs against MDCK cells

GNT-DOX followed follow First order DOX release kinetics under both acidic and

physiological pH.