Understanding Plasmonic Heat-triggered drug release from gold based nanostructure

Accepted Manuscript Understanding Plasmonic Heat-triggered drug release from gold based nanostructure Nebu John, Sony George

PII:

S1773-2247(17)30737-2

DOI:

10.1016/j.jddst.2018.05.036

Reference:

JDDST 679

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 2 September 2017 Revised Date:

4 April 2018

Accepted Date: 23 May 2018

Please cite this article as: N. John, S. George, Understanding Plasmonic Heat-triggered drug release from gold based nanostructure, Journal of Drug Delivery Science and Technology (2018), doi: 10.1016/ j.jddst.2018.05.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Understanding Plasmonic Heat-Triggered Drug Release from Gold based Nanostructure Nebu Johna, Sony Georgea,* a

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Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom, Trivandrum 695581, India. *

Corresponding author- Refereeing, publication and Post-publication. Department of Chemistry, School of Physical and Mathematical Sciences, University of Kerala, Kariavattom, Trivandrum -695581, Kerala, India.

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E-mail address: [email protected] Mobile: +91 9446462933

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Abstract

Targeted drug delivery in a spatiotemporal fashion with high specificity could aid numerous therapeutic applications having reduced nonspecific cytotoxicity. In a typical nanoconstruct, gold nanoparticle synthesized using folic acid as reducing agent is conjugated with stearic acid coupled Pluronic F-127. The pluronic F-127 can hold 5-FU on its hydrophilic corona of

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the micelle, acting as a drug reservoir and the folic acid reduced Au NPs on its outer surface ensures the targeting ability as well as provides surface plasmon resonance induced plasmonic heating required for triggered drug release. Thus, engineered nanostructure has high stability and ability to load 73.07% of 5-FU. In-vitro drug release profile with and

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without laser irradiation at plasmonic resonance wavelength of 532 nm (50 mW commercial laser) was studied and found the nanoconstruct was effective for controlled drug release via

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laser induced plasmonic heating from Au NPs. In-vitro biocompatible studies proves the nanocarrier was nontoxic and the drug loaded nanocarrier was effective towards A549 lung cancer cells. The overall results of the study reveals that the multifunctional nanoconstruct based on gold nanoparticle can find promising therapeutic application in targeted triggered drug release and has the potential application during oncosurgical pocedures via (i) laser triggered targeted drug release, (ii) Photothermal therapy, (iii) SERS based onsite detection and imaging of cancer cells.

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Graphical Abstract

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Key words

Gold nanoparticle; Folic acid; Pluronic F-127; 5-Fuorouracil; Surface Plasmon resonance;

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Laser triggered drug release

Introduction

Chemotherapy is a widely accepted method of cancer treatment that uses drugs to destroy cancer cells, which divide and grow quickly [1]. The chemotherapy drugs like 5-fluorouracil (5-FU) can inhibit the action of an enzyme, thymidylate synthase, present in the nucleotide of

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DNA, that leads to the destruction of DNA of cancer cells [2]. However, due to the lack of specificity of 5-FU, it leads to gastrointestinal, cardiovascular and dermatologic side effects that may last even after the treatment [3-4]. In order to overcome these drawbacks of conventional chemotherapy, multifunctional nanomaterials for targeted drug delivery have

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attained much attraction [5-8]. Advances in the cancer therapy prove that the human cancer cell surface over expresses the folate receptor, which throws light on the targeting ability of

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folic acid (FA). Many researchers employed FA to use it as a ligand for synthesizing gold nanoparticle (Au NPs) [9-10]. By making use of this targeting ability of FA, along with surface Plasmon resonance (SPR) effect of Au NPs and by conjugating it with modified pluronic F-127 (PF-127), a multifunctional drug delivery system with reduced side effect on normal cells can be synthesized. Researchers have explored the targeting ability of folic acid, which can respond to the increased affinity of the over expressed folate receptors in cancer cells [11-12]. Folate receptors are folate-binding proteins, which are of four isoforms, α, β, γ/ γ΄ and δ. The α and β isoforms are distinguishable by high affinities for folic acid, and its corresponding reduced

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ACCEPTED MANUSCRIPT folates. Folic acid is vitamin-B, which plays a major role in the synthesis and repair of DNA. Folic acid is made up of three residues including pteridine, p-aminobenzoate and glutamic acid, that is poorly soluble in water, although it is categorized as a water soluble vitamin [13]. Previously, Wang et al. synthesized Au NPs using folic acid as reducing as well as stabilizing agent[14]. Sun et al. studied the microwave assisted synthesis of Au NPs using folic acid as

PF-127, which may give extra stability to Au NPs [15].

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reducing agent [9]. The folic acid reduced Au NPs can be allowed to adsorb on the surface of

Pluronic F-127 is a triblock copolymer composed of poly(ethylene oxide)poly(propylene oxide)- poly(ethylene oxide) blocks, PEO100-PPO65-PEO100, (70% PEO

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segment) with amphiphilic nature. It can form self assembled micelle with hydrophobic PPO segment core above its critical micelle concentration (CMC) in aqueous solution, which has

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been extensively explored as a controlled drug delivery vehicle [16]. Sun et al. modified PF127 with stearic acid (SA) in order to lower the CMC of the copolymer and to overcome the rapid release of drug before it reaches the targeted site[17]. The amphipilic nature of the PF127 in aqueous environment has been shown to be suitable for the encapsulation of both hydrophilic and hydrophobic drugs [18-19]. In the previous studies, the drugs like ibuprofen, aspirin, erythromycin and 5-fluorouracil were loaded in the micelle of the PF-127 [19].

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Miyazaki et al. studied the effects of concentration of PF-127, temperature, and the drug concentration on the drug release study by means of in-vitro release method [20]. Astilean et al. synthesized pluronic- nanogold hybrid nanoparticles in a single step reaction using PF-127 as both reducing and stabilizing agent [21]. PF-127 was reported as the effective stabilizing

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agent for Au NPs among pluronic block copolymers due to the increased hydrophilic chain lengths (PEO 70%). Thus the unique thermo reversible nature, excellent stabilizing property

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towards Au NPs, biocompatibility, and promising drug release property enables it to be used as a potential drug delivery vehicle. In photo-thermal therapy (PTT), the photon energy is converted into heat, sufficient to

destroy cancer cells, in which the heating sources includes infra red, or visible light, radiofrequency waves, microwaves, and ultra sound waves [22]. Among, a variety of trigger for drug release, low power laser light has been considered as a highly promising heating source in the controlled release of drugs. The laser light (λ=532 nm) can be used to induce SPR in Au NPs, which generate heat to the local area around it, and local temperature can rise to tens or hundreds of degrees above physiological temperatures [23]. The temperature of Au NPs due to plasmonic heating depends on laser frequency and increasing when 3

ACCEPTED MANUSCRIPT wavelength of the laser is approaching to the SPR wavelength [24]. El-Sayed et al. advanced the optical properties and applications of gold nanoparticles in cancer diagnosis and photothermal therapy [25]. Yelin et al. studied the laser irradiated triggered release of Rituximab, an anti-CD20 monoclonal antibody-based drug conjugated with gold nanospheres [26]. This implies that photothermal heating could be utilized to destroy cancer cells at the

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targeted site as well as it heats the drug carrier, PF-127, which enables the easy release of drugs.

Diagnosing cancer at the primary stage is important and considerable attention should be given for diagnosing the depth of the tumour affected tissues in the specific organs. The

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major concerns in surgical treatment of cancer are the challenges in detecting the depth of proliferation of cancer tissues in organs. Numerous methods are available for the detection

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and diagnosis of cancer cells. Recently, SERS has also been explored as a tool for cancer diagnosis, as it can provide detailed information on the chemical composition of cells [27]. SERS manifests by enhanced polarizability of the analyte molecule at the proximity of surface plasmons occurring in metal nanoparticle, where the molecule adsorbed on its surface. This results in increase in the Raman scattering intensities by (i) Electromagnetic enhancement by increasing the intensity of the electric field of the incident radiation and (ii)

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chemical enhancement due to (a) interaction between the molecule and the nanostructure in the ground state (b) resonance between excitation wavelength and molecular transition (c) resonance between molecule-nanostructure charge-transfer transition and excitation wavelength [28-29]. When conjugated to tumour targeting molecules like folic acid this gold

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nanoparticle may serve as diagnostic signatures for cancer cells during intraoperative surgical procedures. The SERS imaging can enables precise positioning of proliferation of cancer

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sites, thus minimizing the devastating side effects of surgery. In this work, a dual-functionalized folic acid having both protecting and targeting

ability, is used as the reducing moiety for the synthesis of Au NPs, which is conjugated with stearic acid coupled PF-127 (SA-PF-127) that serve as a drug carrier for a model drug, 5-FU. The as-synthesized Au NPs can assemble around the SA-PF-127, which also serve as a protecting agent to prevent aggregation of Au NPs. The nanosystem with modified pluronic micelle having Au NPs around it is loaded with 5-FU. The pluronic undergo reversible swelling and de-swelling behaviour when the temperature was cycled between 20 and 37 °C. The developed nanostructure is subjected to laser irradiation to evaluate the plasmonic heat induced triggered drug release. Herein, we report the targetability and biocompatibility of the 4

ACCEPTED MANUSCRIPT modified drug carrier, PF-127, possibility of photothermal therapy, via plasmonic heat generation through laser irradiation and subsequent triggered drug release, and potentiality of

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multifunctional nanosystem for SERS imaging of cancer cells (Scheme 1).

Scheme 1. Schematic illustration representing the potential applications of gold based nanostructure.

2. Material and Methods

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2.1. Materials

The following materials were purchased from Sigma-Aldrich: Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4.3H2O) and pluronic F-127 (PF-127). Analytical grade chemicals such as folic acid, stearic acid, 5-fluorouracil, ethyl acetate and chloroform were purchased

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from Merck Millipore (Mumbai, India). All chemicals were used as received without further purification. A549 (Lung carcinoma) cell lines was initially procured from National centre

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for cell sciences (NCCS), Pune, India and maintained Dulbecos modified Eagles medium (Gibco, Invitrogen). Millipore water was used in all experiments. 2.2. Synthesis of gold nanoparticles All glass wares were cleaned with aqua regia solution (1:3 mixture of con. HNO3 and con. HCl) to prevent artificial nucleation. Synthesis of Au NPs was done following the previously reported method with slight modification [9]. To 10 mL of aqueous HAuCl4 (0.8 mM), 0.4 mL of folic acid solution (0.6 mM) was added, and the mixture was ultrasonicated for 5 minutes (min). To improve the solubility of FA in solution, 1M NaOH solution was added dropwise and the pH of the solution was maintained at 10.6. The mixture was stirred for 30 5

ACCEPTED MANUSCRIPT min, and transferred into a sealed vessel. Immediately the solution was heated in a domestic microwave oven (Power Solo 17 D, ONIDA) for 10 min with a microwave output power of 700 Watts. Upon reaction, the colour of the solution changed to wine red which indicates the formation of Au NPs and it was further characterized by UV-visible spectrometer (Shimadzu,UV-2450). The size of the Au NPs was characterized by the TEM analysis The ATR-FTIR spectra of folic acid and Au NPs were recorded

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(JEOL, JEM-2100).

(Shimadzu, IR Affinity-1S). The solution was incubated for 8 days at room temperature, and UV-visible spectrum of the sample was taken at regular intervals to investigate the stability of

2.3. Synthesis of stearic acid coupled pluronic F-127

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the synthesized Au NPs solution.

SA-PF-127 were synthesized according to a literature method [17]. In a typical procedure, 5

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g of PF-127 and 5 g of stearic acid were mixed and heated to 150 °C for 5 h. The unreacted stearic acid was removed by dropping the solution into the petroleum ether/ethyl acetate (v/v) 1: 1 mixture and filtering the insoluble substance. The SA-PF-127 was dried by rotary evaporator and kept at room temperature under vacuum. The products were characterized by ATR-FTIR.

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2.4. Synthesis of Au NPs conjugated SA-PF-127

The nanostructures were synthesized by adding 0.5 g SA-PF-127 in 5 mL Au NPs solution under ultrasonication for about 2 h. After cooling to room temperature, the solution was

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lyophilized to obtain a dried sample.

2.5. Synthesis of 5-FU loaded Au NPs conjugated SA-PF-127

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About 100 mg of 5 –FU was added to 100 mg nanostructure and allowed to dissolve in 10 mL chloroform in an RB flask. The mixture was stirred continuously for 2 h, and then kept in vacuum overnight at room temperature. The resultant thin film was washed with 10 mL water, and then filtered, lyophilized, and sonicated for about 30 min. The remaining solution was used for determining the encapsulation efficiency of the nanostructure. 2.6. Determination of encapsulation efficiency The percentage of drug encapsulated was determined spectrophotometrically at 266 nm (λmax of 5-FU) using UV-visible Spectrophotometer. The encapsulation efficiency was calculated with the following relationship 6

ACCEPTED MANUSCRIPT Encapsulation efficiency % =

Total drug − Free drug × 100 Total drug

2.7. In vitro drug release studies The in vitro drug release studies were done on a period of 5 days at pH 7.4 phosphate

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buffered saline (PBS) solution. Dialysis bag (Membra-cel M C 18 ×100 CLR, product of USA) was activated using EDTA/ sodium bicarbonate solution, followed by rinsing thoroughly with distilled water.

Drug loaded nanostructure (equivalent to 1 mg 5-FU)

dispersed in 1 mL PBS was taken in a previously activated dialysis bag of 12 cm initial length and 2.5 cm diameter. The bag was closed at both ends with cotton thread and tested

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for diffusion. The dialysis bag was immersed in a beaker containing 250 mL PBS as release medium. The temperature was set at 37 °C and the rotation speed was at 600 rpm. At

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appropriate time intervals, 0.5 ml of release medium was withdrawn and subjected to UV spectrophotometric analysis at a wave length of 266 nm (λmax of 5-FU) with a blank solution of PBS.

2.8. Laser irradiated drug release studies

The laser irradiated study was done at physiological condition (37 ºC and PBS with pH 7.4).

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The drug loaded nanostructures in the dialysis bag was irradiated with laser (50 mW power) for about 50 min and UV-visible spectra were recorded at regular interval of time. 2.9. Live dead cell assay

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The cell line was cultured in 25 cm2 tissue culture flask with DMEM supplemented with 10% fetal bovine serum (FBS), L-glutamine, sodium bicarbonate and antibiotic solution

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containing: Penicillin (100 u/mL), streptomycin (100 µg/mL), and amphotericin B (2.5 µg/mL). Cultured cell lines were kept at 37 °C in a humidified 5% CO2 incubator (NBS Eppendorf, Germany).

The viability of cells was evaluated by direct observation of cells by inverted phase

contrast microscope. Two days old confluent monolayer of cells were trypsinized and the cells were suspended in 10% growth medium, 500 µL cell suspension (25×104 cells/well) was seeded in 96 well tissue culture plate and incubated at 37 °C in a humidified 5% CO2 incubator.

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ACCEPTED MANUSCRIPT The nanostructure with and without 5-FU was added to cells at concentration of 25, 50 and 100 µg/mL. The cells were incubated for 24 h and subjected to fluorescent microscopy. Live dead staining was performed using mixture of acridine orange (100 µg/mL) and ethidium bromide (100 µg/mL) at room temperature for 10 min. The stained cells were

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washed twice with 1×PBS and observed by a fluorescence microscope in blue filter for acridine orange and a green filter for ethidium bromide (Olympus CK×41 with Optika Pro 5 camera).

To evaluate the efficacy of laser irradiation the same procedure was repeated with 100

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µL cell suspension (5×104 cells/well) and the samples was irradiated with laser beam (commercial green laser source, λ= 532 nm, 50 mW) for 1 h and was added to cells at

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concentration of 25 and 50 µg/mL. Simultaneously the samples without laser irradiation was also added to cells at the same concentration. The cells were incubated for 24 h and subjected to fluorescent microscopy.

3. Results and discussion

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3.1. Characterization of folic acid reduced gold nanoparticle based nanostructure The formation and stability of Au NPs was investigated by comparing the UV-visible spectrum (Figure. S1) of the synthesized solution in regular intervals of time. The absorption maximum is found to be at 532 nm immediately after the synthesis of the Au NPs (Figure 1).

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However, the absorption peak of the Au NPs is red shifted from 532 nm to 560 nm after 6 days. The red shifting of the absorption maxima of the Au NPs confirms the aggregation

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tendency of Au NPs on prolonged storage. The decrease in intensity and broadening of peaks also reveals the aggregation of Au NPs after few days. This may be due to Ostwald ripening process [30]. The presence of un-reacted gold ions at the reaction sites may also leads to the aggregation phenomena.

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0.3 0.2 0.1 0.0 600 700 Wavelength (nm)

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Figure 1. UV-visible absorption spectra of Au NPs (a) and Au NPs based nanostructure loaded with 5Fluorouracil (b). Inset: From left to right are the photographs showing colorimetric response of HAuCl4.3H2O, Au NPs and Au NPs based nanostructure.

TEM analyses were performed and images indicate the average size of the Au NPs spans within 15.05 ± 1.25 nm in diameter (Figure. 2). In the HRTEM image of Au NPs, the lattice fringes observed corresponds to the inter planar separation (111) plane of face-

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centered cubic structure of Au NPs. The selected area electron diffraction pattern (SAED) of Au NPs as shown in Figure 2(c) consists of several diffraction rings indexed to (111), (220),

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(200), (311) and (222) indicative of the polycrystalline nature of Au NPs.

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Figure 2. TEM image (a,b), HRTEM image (c) and Selected area electron diffraction (SAED) pattern (d) of folic acid reduced Au NPs.

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Figure 3. TEM image (a, b), HRTEM image (c) and Selected area electron diffraction (SAED) pattern (d) of 5-

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FU loaded gold nanoparticle based nanostructure.

To examine the conjugation of stearic acid with PF-127, ATR-FTIR measurements

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were employed (Figure S2). To understand the interaction between SA and PF-127, we compared the ATR-FTIR spectra of bare SA, bare PF-127 and SA-PF-127. The sharp peak at 1740 cm-1 due to the stretching of C=O bond arising from the ester group attests the successful coupling of SA with PF-127[17]. The synthesized Au NPs is conjugated with stearic acid coupled pluronic F-127 (SAPF-127) at its corona. Figure S3 displays the ATR-FTIR spectra of the FA stabilized Au NPs compared with bare FA. The peaks of the folic acid appeared at 1600 cm-1 for the C=O bond stretching vibration of the -CONH2 group, 1700 cm-1 for the C=O bond stretching of the carbonyl group, and between 3000 cm-1 and 3600 cm-1 for the –NH stretching and the –OH 11

ACCEPTED MANUSCRIPT stretching vibration. The ATR-FTIR spectrum of Au NP showed the peaks at 1699 cm-1 and 1604 cm-1 for the C=O of the –CONH2 group, owing to the successful linkage of the folate ions on the surface of the Au NPs [14]. However the sharp peaks between 3000 and 3600 cm-1 gets broadened. The broad peaks of the Au-FA-SA-PF-127 nanostructure appeared at 1700 cm-1 and 1500 cm-1 are due to the interaction of SA-PF-127 with folic acid reduced gold 1

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nanoparticle (Au-FA). Moreover, the disappearance of the peak of SA-PF-127 at 2908 cmand 2845 cm-1 in the ATR-FTIR spectrum of gold nanoparticle conjugated with stearic acid

pluronic F-127 (Au-FA-SA-PF-127) indicates the formation of the intermolecular hydrogen bonding between –NH group of FA and C=O group of SA-PF127 [31]. TEM and HRTEM

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analysis (Figure 3) also revealed that the Au NPs get conjugated around PF-127 micelles. The HRTEM image of Au NPs in Au-FA-SA-PF-127 nanostructure clearly indicates the interaction of Au NPs with SA-PF-127and in the SAED pattern the diffraction rings indexed

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to (220), (200), (311) and (222) are clearly observed.

After successfully attaching Au NPs on the surface of SA-PF-127, 5-Fluorouracil is loaded on the hydrophilic end of micelle. The UV-visible spectrum of the nanostructure loaded with 5-FU revealed the persistence of plasmonic peak at 532 nm of Au NPs is retained without any shift of absorption maxima although its intensity of absorbance decreases slightly

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(Figure 1). In the ATR-FTIR spectrum (Figure S4) of bare 5-FU, the peak assigned to conjugated (1655-1670 cm-1) and non-conjugated (1690 cm-1) C=O stretch, which disappeared in the spectrum of 5-fluorouracil (5-FU) loaded nanostructure (Au-FA-SA-PF127-5-FU). These results suggested that the 5-FU was physically adsorbed in the micelle and

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no strong interaction has occurred.

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3.2. Drug release study

The drug encapsulation efficiency of the nanostructure was calculated by measuring the absorption maximum of UV-visible spectrum and it was found to be 73.07%. The release of 5-FU from the drug carrier followed a biphasic process at pH 7.4 (37 ± 0.1 °C) with an initial rapid drug release (up to ~20%) in 2 h and about 59 % drug released in a sustained manner over a period of 120 h (Fig. S5). The initial fast release of the drug into the incubation medium is due to the leakage of poorly adsorbed/entrapped drug on the surface of the drug carrier. The sustained release of 5-FU in 120 h suggests that the majority of the drug was entrapped in the nanostructure.

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indicates that hot electrons absorb further incoming photons only after they relax to the

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ground state [32]. This may also occurs when the laser irradiation leads to the simultaneous size growth and size reduction of Au NPs [33]. The slight Plasmon band broadening reveals the plasmonic heating of Au NPs, which has a resonant character [24]. The laser light absorbed by the plasmonic electronic system creates nascent nonthermal electrons, which is

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in thermal equilibrium with cold conduction-band electrons after a few femtoseconds. Then the electron energy is transferred to the nanoparticle lattice via electron-phonon coupling. Finally the lattice energy is transferred to the surrounding medium through phonon-medium

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interaction leading to heat diffusion within the medium [34]. The origin of slight blue shift of the Au NPs is due to the thermal expansion of the nanoparticle. The nanoparticle volume is temperature dependent as

V T = Vₒ 1 + β ΔT

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Where Vₒ is the volume of the nanoparticle at room temperature, ∆T = T-Tₒ is the temperature variation from the room temperature and β is the volume thermal expansion coefficient. The SPR blue shift take place owing to the fact that the permittivity of the water

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decreases with increase of temperature [24].

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0 min 5 min 10 min 20 min 30 min 40 min 50 min

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Wave length (nm)

Figure 4. UV-visible spectra of Au NPs (1µM) in PBS buffer solution (pH=7.4) under laser irradiation at 532 nm for different interval of time.

3.3. Laser induced plasmonic heating and triggered drug release The drug release profile of the nanostructure irradiated with laser was studied. Figure 5(a) shows percent release of 5-FU versus time upon laser irradiation. The 5-FU was detected at

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levels of 10.11%, 19.68%, 28.83%, 45.5%, 70.45% and 77.56% after irradiation for 5, 10, 20, 30, 40 and 50 min, respectively. Longer exposure of laser light gave faster release of 5-FU, owing to the fact that plasmonic heating decreases the viscosity of the extramicellar water

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passage which enhances the release of drugs from the hydrophilic end of the micelle. Here it is found that the drug release attained for laser irradiated 5-FU loaded SA-PF127 micelle is 12.52%, which indicates the inability of the material for laser triggered fast drug release in

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the absence of Au NPs (Figure 5(b)). Correspondingly the release of the drug was only 11.75% without laser irradiated sample (Figure 5(c)). This results show that the nanostructure is suitable for both sustainable release and laser triggered fast release of the drug. The irradiation time of the laser for the fast release of drug can be reduced by increasing the power of the laser source within the biological limits as in photodynamic therapy.

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ACCEPTED MANUSCRIPT 90 Laser irradiated 5-FU loaded nanocomposite Laser irradiated 5-FU loaded SA-PF-127 Without laser irradiation

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Drug release (%)

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Figure 5. In vitro drug release profiles of 5-FU from the Au NPs based nanostructure in PBS buffer (pH = 7.4) with laser irradiation (a), stearic acid coupled pluronic F-127 (without Au NPs) with laser irradiation (b) and Au NPs based nanostructure without laser irradiation (c) at 37 °C.

3.4. Cytotoxic assay on A549 lung cancer cells

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The in-vitro cytotoxicity of 5-FU loaded nanostructure was studied in A549 lung cancer cells. As can be seen in Figure 6, at low nanostructure concentration, no significant toxicity related to nanostructure was observed owing to the excellent biocompatibility of the drug carrier. However, when the concentration of the drug carrier increases to 100 µg/mL, cell viability

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decreases, which may be due to the masking of cellular surface, hence reduce the accessibility of oxygen to cells and cause unexpected cell death.

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The in-vitro imaging studies were done on A549 lung cancer cells that express folate

receptors on its surface. The results revealed that 5-FU loaded nanostructure exhibit a dosedependent cytotoxic effect. At low concentration, the 5-FU loaded nanostructure exhibit higher cytotoxicity, which further increases to 95% at 100 µg/mL of 5-FU loaded nanostructure. This enhancement in cytotoxicity is due to targeted delivery of 5-FU on the surface of cancer cells. First the folic acid on the surface of Au NPs were attracted by the folate receptors on the surface of A549 lung cancer cells leading to the accumulation and internalization of 5-FU into the cell via endocytosis and leads to cell death. The results support the evidence of targeting ability of folic acid thereby increasing concentration of drug in cells. 15

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Figure 6. Fluorescence image of A549 lung cancer cells alone (control) (a), gold based nanostructure (drug carrier) alone (25, 50 & 100 µg/mL) (b), (c), (d), and drug loaded gold based nanostructure (25, 50,100 µg/mL) (e), (f), (g)

Gold-based nanostructure exposed to laser light has a stronger cytotoxicity compared

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to its unexposed state (Figure 7). These results further ensure that laser irradiation on Au nanostructures probably triggers plasmonic heating of the Au NPs. The Au NPs are able to absorb light radiation and convert into heat energy, which decreases the viscosity of the water passage suitable for easy drug release. In this study, the maximum cytotoxicity is found for laser irradiated nanostructure. Further the cytotoxicity is dose dependent of the drug. Thus

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towards the targeted site.

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Au-FA-SA-PF-127 is suitable for targeted and controlled delivery of anticancer drugs

Figure 7. Fluorescence image of A549 Lung cancer cells alone (control) (a), drug loaded gold based nanostructure without laser irradiation (25 & 50 µg/mL) (b), (c), and drug loaded gold based nanostructure with laser irradiation (25 & 50 µg/mL) (d), (e).

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ACCEPTED MANUSCRIPT In this work, the developed nanostructure has the ability to target cancer cells, where the conjugated SA-PF-127 gives extra stability to the Au NPs. Here SA-PF-127 acts as a hydrophilic drug carrier and thus 5-FU gets loaded on it.

The laser irradiation on

nanostructure results in plasmonic heating of Au NPs via SPR effect which can directly destroy cancer cells. Moreover the heat energy generated on Au NPs due to SPR effect is

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imparted to the drug carrier. Since the drug is released by diffusion through extramicellar water channels, the drug release depends on (i) the microviscocity of the extramicellar fluid, (ii) the size of the water channels (iii) and the drug equilibrium between the micellar phase and the aqueous phase [35]. Thus the increase in temperature decreases the microviscocity

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of the water channels and increases the size of it. This result in fast release of drug through plasmonic heat generated via laser irradiation. In addition to it, the gold nanoparticle having sharp edges and tips has been known to exhibit a very high sensitivity towards the changes in

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the dielectric environment as well as the enhancements of the electric field around it [36]. Thus the sharp edged Au NPs has the potential to act as SERS agent for cancer detection and imaging. It is expected that the nanostructure can find promising application in targeted triggered drug release and effective especially during intraoperative surgical treatment of cancer via (i) laser triggered drug release (ii) cancer cell death via plasmonic heating (iii)

Conclusion

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SERS based detection and imaging of cancer cells.

Targeted therapy using multi-functional Au-FA-SA-PF-127-5-FU nanostructure holds

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promising potential for minimizing non-specific toxicity of drugs. We have demonstrated the laser light triggered target specific delivery of 5-FU using multi-functional nanostructure

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system. Upon laser irradiation, the heat generated due to SPR effect enhances the rupture of the micelle, thus resulting in drug release. Laser irradiation induced plasmonic heat generation, triggers a rapid drug release from the nanostructure and the presence of folic acid ensuring the targeting ability. The cytotoxicity study reveals the biocompatibility of the drug carrier, and the efficacy of the nature of drug loaded nanostructure. The targeting ability and the ease of drug entrapment coupled with the controlled release of drug ensure the ability of the nanostructure as a valuable tool for delivering drug at the targeted site. Thus developed nanostructure can find potential application in plasmonic heat induced triggered drug release at targeted cancer region and subsequent cell death via heat generation at the site of activity. This dual plasmonic phenomenon can be employed during surgical procedures.

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ACCEPTED MANUSCRIPT Notes The authors declare no competing financial interest.

Acknowledgement

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The authors thank the Head, Department of Chemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram for pursuing the platform to conduct the research. The authors also

thank

the

Director,

SICC,

University

of

Kerala,

Kariavattom

campus,

Thiruvananthapuram; Director, SAIF-STIC-CUSAT, Kochi; RGCB, Thiruvananthapuram,

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DST-SAIF, M.G. University, Kottayam. The author N.J. acknowledge support for this work by University Grants Commission, Bangalore, India through the teacher fellowship

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(F.No.FIP/12th plan/KLMG035, TF: 03) under faculty development programme during XIIth plan period.

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Figure S1. UV-visible absorption spectra of folic acid reduced Au NPs in different days (1 to 8 days in definite intervals of time).

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Figure S2. ATR FTIR spectra of PF-127 (a), stearic acid (b) and PF-127 coupled with stearic acid (c).

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Figure S4. ATR FTIR spectra of 5-Fluorouracil (a), Au NPs conjugated with SA-PF127 (b) and 5-Fluorouracil loaded Au NPs based nanostructure (5-FU-Au-FA-SA-PF-127).

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Figure S5. In vitro drug release profile of 5-FU from the gold based nanostructure in PBS (pH= 7.4) at 37 °C, over a long period of time.

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