Multi-responsive albumin-lonidamine conjugated hybridized gold nanoparticle as a combined photothermal-chemotherapy for synergistic tumor ablation

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Multi-responsive albumin-lonidamine conjugated hybridized gold nanoparticle as a combined photothermal-chemotherapy for synergistic tumor ablation Hima Bindu Ruttala , Thiruganesh Ramasamy , Bijay Kumar Poudel , Raghu Ram Teja Ruttala , Sung Giu Jin , Han-Gon Choi , Sae-Kwang Ku , Chul Soon Yong , Jong Oh Kim PII: DOI: Reference:

S1742-7061(19)30743-3 https://doi.org/10.1016/j.actbio.2019.11.003 ACTBIO 6434

To appear in:

Acta Biomaterialia

Received date: Revised date: Accepted date:

9 July 2019 28 October 2019 1 November 2019

Please cite this article as: Hima Bindu Ruttala , Thiruganesh Ramasamy , Bijay Kumar Poudel , Raghu Ram Teja Ruttala , Sung Giu Jin , Han-Gon Choi , Sae-Kwang Ku , Chul Soon Yong , Jong Oh Kim , Multi-responsive albumin-lonidamine conjugated hybridized gold nanoparticle as a combined photothermal-chemotherapy for synergistic tumor ablation, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.11.003

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Multi-responsive albumin-lonidamine conjugated hybridized gold nanoparticle as a combined photothermal-chemotherapy for synergistic tumor ablation

Hima Bindu Ruttala1,2, Thiruganesh Ramasamy1,3, Bijay Kumar Poudel1, Raghu Ram Teja Ruttala4, Sung Giu Jin5, Han-Gon Choi6, Sae-Kwang Ku7*, Chul Soon Yong1*, Jong Oh Kim1* 1

College of Pharmacy, Yeungnam University, 214-1, Dae-dong, Gyeongsan 712-749, Republic

of Korea 2

Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261,

USA 3

Center for Ultrasound Molecular Imaging and Therapeutics, Department of Medicine,

University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA 4

Sri Vasavi Institute of Pharmaceutical sciences, Tadepalligudem, West Godavari, Andhra

Pradesh, India. 5

Department of Pharmaceutical Engineering, Dankook University, 119 Dandae-ro, Dongnam-gu,

Cheonan, 31116, Republic of Korea 6

College of Pharmacy, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-

791, Republic of Korea 7

Department of Anatomy and Histology, College of Korean Medicine, Daegu Haany University,

Gyeongsan 712-715, Republic of Korea *

Corresponding authors:

E-mail: [email protected] (Jong Oh Kim) Tel: +82-53-810-2813, Fax: +82-53-810-4654 E-mail: [email protected] (Chul Soon Yong) Tel: +82-53-810-2812, Fax: +82-53-810-4654 E-mail: [email protected] (Sae Kwang Ku) Tel: +82-53-819-1549, Fax: +82-53-819-1860

Abstract Herein, we developed a multifunctional nanoplatform based on the nanoassembly of gold nanoparticles (GNP) conjugated with lonidamine (LND) and aptamer AS1411 (AS-LAGN) as an effective cancer treatment. Conjugating AS1411 aptamer on the surface of the nanoparticle significantly improved particle accumulation in cancer cells via specific affinity toward the nucleolin receptors. In vitro study clearly revealed that laser irradiation-based hyperthermia effect enhanced the chemotherapeutic effects of LND. Combinational treatment modalities revealed significant apoptosis with higher cell killing effect due to increased ROS production and inhibition of cell migration. GNP’s ability to convert the excited state photon energy into thermal heat enabled synergistic photothermal/chemotherapy with improved therapeutic efficacy in animal models. Moreover, immunohistochemistry staining assays confirmed the ability of ASLAGN to induce cellular apoptosis/necrosis and ablation in tumor tissues, without causing evident damages to the surrounding healthy tissues. Altogether, this AS-LAGN nanoplatform could be a promising strategy for mitochondria-based cancer treatment. Keywords Gold nanoparticles, lonidamine, photothermal, chemotherapy, mitochondria

Statement of Significance We have designed a facile biodegradable multifunctional nanocarrier system to target the mitochondria, the major “power house” of the cancer cells. We have constructed a multifunctional nanoassembly of protein coronated gold nanoparticles (GNP) conjugated with

lonidamine (LND) and aptamer AS1411 (AS-LAGN) as an effective combination of phototherapy with chemotherapy for cancer treatment. The LND was conjugated with albumin which was in turn conjugated to GNP via redox-liable disulfide linkage to generate oxidative stress and ROS to kill cancer cells. GNP’s ability to convert the excited state photon energy into thermal heat enabled synergistic photothermal/chemotherapy with improved therapeutic efficacy in animal models. Consistently, AS-LAGN showed enhanced antitumor efficacy in xenograft tumor model with remarkable tumor regression property.

1. Introduction The mitochondrion has emerged as a major target in cancer therapeutics owing to its diverse regulatory functions, including ATP synthesis, synaptic transmission, maintenance of danger signaling pathways, and regulation of mitochondrial DNA [1,2]. As the mitochondrion is also intricately associated with tumor progression and multidrug resistance (MDR) in cancer cells [3,4], mitochondria-targeting interventions would serve as a promising strategy in cancer therapy. Accumulation of mitochondria-targeting drugs would trigger mitochondrial apoptosis pathway, thereby leading to programmed cell death as well as cancer cell suicide [5]. Several mitochondrial targeting drugs are used as effective cell suicide weapons to initiate the intrinsic apoptotic pathway for cancer cell destruction through mitochondrial dysfunction and apoptosis [6,7]. Lonidamine (LND) is an anticancer drug that initiates its cancer killing effect by inhibiting hexokinase and acting directly on adenine nucleotide translocase (ANT). LND opens the mitochondrial permeability transition pore complex and induces apoptosis of cancer cells [8,9].

Due to the complex tumor microenvironment, therapy based on single molecular target strategy has resulted in limited therapeutic efficacy. In addition, the limited penetration ability of anticancer drugs and their therapeutic efficacy have become an obstacle for the clinical translation of nanomedicine [10,11]. Therefore, strategies should be focused on an integrated tumor targeting approach to improve the transport and accumulation of drugs in tumor tissues. In this perspective, combination of photothermal therapy with chemotherapy is emerging as an interesting approach for effective cancer therapy. Hyperthermia is expected to increase the flow of blood to deeper tissues and enhance microvascular permeability in tumors [12,13]. Gold nanoparticles (GNP) have been reported to exhibit hyperthermia effect owing to their optical properties and surface plasmon resonance (SPR) effect. GNP effectively converts extrinsic light energy into localized heat at a plasmonic resonant wavelength [14]. The thermal energy induced by GNP triggers a series of intracellular biochemical reactions and induces reactive oxygen species (ROS) and cell death [15]. However, GNP is highly unstable in normal conditions, which makes it important to functionalize the particles to accomplish the photothermal property [16]. Surface modification of GNP allows the conversion of plasmonic nanoparticles into versatile bio-nanoparticles. The surface modification of GNP allows the incorporation of multiple anticancer drugs which otherwise would be difficult to accumulate in tumor tissue [17]. Several GNP-based drugs have been developed by CytImmune with their lead drug, Aurimune (TNF bearing PEGylated GNPs), in clinical trials. In this study, we opted to use albumin to modify the surface of GNP. Albumin is one of the important biocompatible proteins present in human plasma with crucial physiological roles, including transport of drugs, free radical scavenging, and maintenance of osmotic pressure [18]. From a drug delivery perspective, albumin functionalization of NP confers the long circulation property, avoids rapid

clearance, and increases systemic bioavailability. The presence of protein on the outer surface of GNP leads to the formation of a core-corona structure, which has a great impact on colloidal stability, cellular targeting, and distribution in photo-thermal ablation applications [19]. A multifunctional nanostructure was fabricated by applying the knowledge of chemistry and integrating multiple components, including GNP, albumin (BSA), and LND into a single system with the tumor-specific targeting moiety, Aptamer AS1411 (AS-LAGN). We hypothesized that laser irradiation of AS-LAGN would elevate the local temperature, leading to the induction of a ROS-mediated chain reaction and accumulation of LND in the mitochondria of tumor cells, ultimately contributing to the creation of an effective chemo-photothermal therapy. In vitro studies have been performed with several cancer cell lines (MCF-7, MDA-MB-231, and DU145) and the mechanism involved in mitochondria-mediated apoptosis and synergistic potential of dual therapy have been demonstrated in detail. Herein, we sought to determine the in vivo antitumor efficacy of a nanosystem in DU145-bearing xenograft nude mice.

2. Materials and Methods 2.1. Materials LND was purchased from Abcam (Biotech, Life sciences, Cambridge, United Kingdom). Albumin, Gold(III) chloride trihydrate, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and Nhydroxysuccinimide (NHS) were obtained from Sigma-Aldrich Co. (St Louis, MO, USA). The 26-mer

AS1411

DNA

aptamer

(sequence:

50-amino-C6

linker;

NH2-5′-

(GGTGGTGGTGGTTGTGGTGGTGGTGG)-3′, molecular weight: 8272.3 g/mole) was custom

synthesized and purchased from BIONEER (South Korea). Human breast adenocarcinoma (MCF-7 and MDA-MB-231), human prostate carcinoma (DU145), and human lung carcinoma (A549) cells were obtained from the Korean Cell Line Bank (Seoul, South Korea). All cell lines were cultured in DMEM 1640 containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin (Life Technologies). Cells were incubated at 37 °C in a 5% CO2 atmosphere. All other chemicals were of reagent grade and were used as supplied.

2.2. Preparation of the denatured albumin (dBSA) and cationic albumin (cBSA) Briefly, denatured albumin (dBSA) was prepared by NaBH4 reduction; the aqueous solution concentration of 500 µM BSA was treated with 28 mg NaBH4. The reaction was continually stirred for 1 h at room temperature and incubated for 20 min in a water bath at 70 °C. The sample was then dialyzed (Mw 10,000 cutoff) to remove free NaBH4. Thiol groups were quantified by DTNB assay. On the other hand, cationic BSA was prepared by the following process: briefly, 500 mg of BSA was treated with 32 ml (0.9 M) ethylediamine by adjusting pH 4.75. Subsequently, 332.4 µL EDC was added and continually stirred for 2 h at room temperature. The reaction was terminated by adding 1.3 ml of 4 M acetate buffer and then centrifuged at 5000 rpm for 15 min using a protein concentrator (Mw: 10,000 Da). The sample was dialyzed against water and filled to 250 ml before freezing.

2.3. Preparation of aptamer and lonidamine-conjugated albumin-decorated gold nanoparticles (AS-LAGN)

First, the citrate-stabilized GNP was prepared by the method below. Briefly, 6 ml of HAuCl4 was dissolve in 50 ml distilled water and allow to boil for 10 min. Subsequently, 8.5 ml trisodium citrate was introduced under vigorous stirring. After 10 min, the reaction mixture was cooled to room temperature and a clear change from colorless to blue was observed, which eventually turned wine red. Wine red depicted the clear formation of gold nanoparticles and later, these nanoparticles were filtered through a 0.45-µM membrane. Albumin-decorated GNPs (AGN) were prepared by the following method; 1 mg/1ml of BSA (2:1 proportion of denatured cationic albumin and native denatured albumin) was added to 1 ml of GNP and 1 ml of HBS buffer, stirred for 6 h at room temperature, and then centrifuged for 8 min at 12,000 rpm. Samples were washed thrice with distilled water and filled to a final volume of 1 ml to form AGN. To conjugate the carboxylic groups of LND (321.16 µg) to albumin, the amine groups of albumin decorated on the surface of the GNP was treated with 1.9 mg EDC and 1.3 mg NHS (LAGN). After overnight stirring of the solution, LAGN was obtained after centrifugation at 12,000 rpm for 8 min and the process was repeated twice to remove excess EDC/NHS. LAGN in DNase/RNase free water was then incubated with 400 mM EDC and 100 mM NHS for 1 h at room temperature with gentle stirring. The resultant NHS-activated particles were washed twice with DNase/RNase free water to remove the free residue of EDC/NHS by centrifugation for 15 min at 12000 rpm. Further, 1 ml of activated NPs was covalently incubated with 1 µM of 5’NH2-modified AS1411 aptamer (1µg/mL) at room temperature for 16 h with gentle stirring. Finally, the resultant AS1411 aptamer conjugated LAGN (AS-LAGN) was washed twice with DNase/RNase free water to remove excess aptamer by centrifugation for 15 min at 12000 rpm before storage at 4 °C.

2.4. Physicochemical characterization 2.4.1. Dynamic light scattering (DLS) The hydrodynamic particle size (nm), PDI, and zeta potential (mV) of the NPs were determined using a NanoZS light-scattering particle size analyzer (Malvern Instruments, UK) with DTS software (version 5.0). Data are expressed as mean ± standard deviation (S.D; n=3). 2.4.2. Morphology Morphological analysis of the nanoparticles was performed using transmission electron microscopy (TEM) and atomic force microscopy (AFM). TEM was performed by placing a drop of NP dispersion onto a copper grid with a carbon film stained with phosphotungstic acid (2% w/v), which was viewed under the TEM. Similarly, the NPs were deposited on an ultra-flat mica sheet and used to perform AFM analysis using Nanoscope IIIa scanning probe microscope (Digital Instruments, Murray Hill, NJ, USA). 2.4.3. Solid state characterization, EDX spectra, and UV analysis A differential scanning calorimeter (DSC-Q200, TA Instruments, New Castle, DE, USA) was used to study the thermal behavior of free LND, albumin, LAGN, and AS-LAGN. Differential scanning calorimetry (DSC) scans were recorded at a heating rate of 10 °C/min from 40 °C to 250 °C. Fourier transform infrared spectroscopy (FTIR) analysis was performed using a Thermo Scientific Nicolet Nexus 670 FTIR spectrometer over the range, 550−4000 cm-1. Elemental analyses of freeze-dried AS-LAGN were performed using a liquid N2-cooled EDX detector (EDAX Inc., Mahwah, NJ, USA) attached to scanning electron microscope (SEM, S-4100, Hitachi, Japan). GNPs and AS-LAGN were monitored between 400 and 700 nm by UV–vis

spectroscopy (Perkin Elmer U-2800, Hitachi, Tokyo, Japan) using a quartz cuvette, with distilled water as reference. 2.4.4. Photothermal analysis by NIR irradiation Photothermal effect of nanoformulations was determined using a NIR laser source with 808-nm fiber-coupled infrared diode laser module (FC-W-808 nm-30W, Changchun New Industries Optoelectronics Technology, China) in a continuous wave operating mode at 1W/cm2 for 5 min. The laser was passed via a silica optical fiber onto samples and thermal imaging and temperature recording were performed with an infrared thermal camera (Therm-App TH, Opgal Optronic Industries Ltd, Israel). 2.4.5. Agarose gel electrophoresis To demonstrate the amide-coupling chemistry of the DNA aptamer to the surface of the ASLAGN, Agarose (2%) gel electrophoresis was performed. The DNA aptamers, LAGN and ASLAGN (2 and 1µM), were loaded on the gel (containing 0.2 mg/mL ethidium bromide). The gel was run in a tris-borate-ethylenediaminetetraacetic acid (EDTA) buffer at 80 V for 20 min followed by imaging and analysis using a standard transilluminator. 2.4.6. Drug release study The release of LND was investigated in the release medium (phosphate buffered saline, PBS, pH 7.4) using a dialysis bag (molecular weight cutoff of 3500 Da). Briefly, 1 mL of AS-LAGN formulation was treated with laser irradiation (5 min, 2 W/cm2, 808 nm). Later, these formulations were placed in a dialysis bag, sealed on both ends, placed in 10 mL PBS, and incubated at 37 °C in shaking water bath at 100 rpm. One ml of sample was withdrawn at desired time intervals and replenished with fresh buffer of equal volume. Further, LND was quantified

using an Agilent 1200 series high performance liquid chromatography (HPLC) with an Inertsil ODS-3 reverse-phase C18 column (Inertsil® ODS3: 0.5 µm, 15 cm 0.46 cm, GL Sciences Inc., Tokyo, Japan) maintained at 30 °C. The mobile phase consisted of acetonitrile and water with 0.1% formic acid (55:45 v/v). It was eluted at a flowrate of 1.0 mL/min and monitored using UV-Vis detector at 230 nm to validate LND. The release experiments were conducted in triplicate and the results are presented as average data with standard deviations. 2.5. Biological assays 2.5.1. Cytotoxicity assay In vitro cytotoxicity of the GNP, AGN, AGN (NIR+), LND, LAGN, AS-LAGN, and AS-LAGN (NIR+) formulations was evaluated in MCF 7, MDA MB 231, and DU145 cells by MTT assay. Cells (1 x 104) were seeded in 96-well plates and exposed to the respective formulations for 48 h at various concentrations (0.001-10 µg/mL). After a 48-h incubation time, the cells were treated with MTT reagent (100 µL; 1.25 mg/mL) and incubated for 3 h at 37 °C in the dark. The cells were then lysed and formazan crystals were dissolved by the addition of 100 µL of DMSO. Absorbance was measured at 570 nm using a microplate reader (Multiskan EX, Thermo Scientific, Waltham, MA, USA). 2.5.2. Cellular uptake MCF 7, MDA MB 231, and DU145 cells (3 x 105) were seeded in a 6-well plate and incubated for 24 h. Cells were pretreated with excess free Aptamer before treatment to observe the competitive uptake of FAM labelled-AS-LAGN into the cancer cell for a 1-h incubation time at 37 °C. Cells were washed, trypsinized, and centrifuged twice at 1500 rpm for 3 min and the pellet was resuspended in 1 mL PBS. The suspended cells were directly introduced into a

fluorescence activated cell sorting (FACS (flow cytometer)) from BD FACS Calibur

TM

(USA)

and analyzed with FL 1 channels. For confocal imaging (CLSM), FAM labelled-AS-LAGN were incubated in cancer cells for 1 h, washed twice with ice cold PBS, later stained with Lysotracker red, and washed twice with PBS. Samples were further fixed with 4% paraformaldehyde (5 min) and washed twice with PBS. Finally, coverslips that contained cells were mounted onto slides and observed using confocal laser scanning microscopy (CLSM, Nikon A1+, Nikon, Tokyo, Japan). 2.5.3. Cell Apoptosis Quantitative apoptosis of individual formulations were measured by using annexin V/propidium iodide (PI) apoptosis assay. The cells were seeded at a density of 2 x 105 in a 6-well plate and incubated for 24 h. The cells were treated with various formulations for 24 h. Subsequently, the cells were washed, collected, and stained with annexin V-FITC and PI (BD Biosciences, CA, USA) for 15 min in the dark. Cells were then enumerated via flow cytometry using a FACS Calibur instrument (BD Biosciences). The qualitative apoptosis is demonstrated by nuclear morphology staining by Hoechst. The cells were treated with LND, LAGN, AS-LAGN, and ASLAGN (+) at a concentration of 10 µg/mL and incubated for 24 h. Thereafter, the cells were washed twice with PBS and stained with (1 µL/mL) Hoechst 33342 for 10 min at room temperature in the dark. Finally, cells were rinsed with PBS and images were observed using a fluorescence microscope (Nikon Eclipse Ti). 2.5.4. Cellular reactive oxygen species analysis (ROS) detection during irradiation To evaluate the generation of ROS, 2’,7’-dichlorodihydrofluorescein diacetate (H2DCF-DA) was used as a fluorescent probe. Briefly, cells (3 x 105) were incubated with different formulations

for 6 h and stained with H2DCF-DA (10 mM) for 30 min at 37 °C in the dark. Subsequently, the cells were washed and exposed to NIR (808 nm, 1 W/cm2, 5 min) using SDL-660-LM-1000T laser (Shanghai Dream Lasers Technology Co., Ltd., China). Cells were then trypsinized and rinsed twice with PBS. The cellular fluorescence of the oxidized product, DCF, was analyzed by flow cytometry. At least 10,000 data events were collected for each sample. 2.5.5. Photothermal ablation of cancer cells upon NIR irradiation Briefly, 3 x 105 cells were seeded in a 6-well plate and incubated overnight. The following day, AS-LAGN formulation was administered to cells and incubated for 3 h. Subsequently, cells were washed and exposed to 808 nm laser (2 W/cm2) beam and irradiated with a spot diameter of ~3mm for 5 min. The cells were then washed and replenished with fresh media and incubated for 3 h. Eventually, cells were stained with calcein-acetoxymethyl (calcein-AM) and ethidium homodimer-1 (EthD-1) in PBS at final concentrations of 2 µM. Cells were observed using an inverted fluorescence microscope (Nikon Eclipse Ti, Tokyo, Japan). 2.6. Biodistribution and in vivo photothermal imaging The in vivo distribution and tumor accumulation of LAGN and AS-LAGN were investigated in the DU145 bearing xenograft mice using FOBITM (Fluorescence In Vivo Imaging System, Neoscience, South Korea). Cy 5.5-conjugated formulations were administered intravenously to tumor-bearing mice via the tail vein. After 24 h, mice were scanned with FOBI using the red channel. The mice were then anaesthetized with chloroform before bioimaging. Tumors and major organs were excised, washed with PBS, and imaged under FOBI. The fluorescence intensities were determined using NEOimage software with operator-defined regions of interest (ROI) measurements in tumors and other organs. In vivo photothermal imaging of AS-LAGN in

Balb/c nude mice was performed with digital thermal camera photographs (Therm-App TH, Israel). Mice were intravenously injected with saline and AS-LAGN formulation and after 24 h, tumor area were exposed to NIR irradiation (808 nm, 1 W/cm2, and 5 min). Digital images were taken to determine the photothermal effects. 2.7. In vivo antitumor study The in vivo antitumor effect of LND, LAGN, AS-LAGN, and AS-LAGN (NIR+) was evaluated in DU-145 tumor bearing Balb/c nude mice. Briefly, 1 x 107 cells/100 µL was injected subcutaneously into rear flanks of 5-week old male Balb/c mice. The tumor was allowed to grow around 100 mm3 to further carryout the study. Two weeks after initial implantation, animals were randomly divided into six groups (n = 5 per group). Mice were intravenously administered samples via tail vein at a fixed dose of 5 mg/kg on days 1, 4, 7, and 10. Tumors were measured twice weekly using a digital caliper and tumor volume (V) was calculated as V = ((width) 2 X length)/2. The animals were sacrificed after four weeks of treatment and primary tumor was excised, weighed, and subjected to further analysis. All experiments were approved and strictly conducted in accordance with the rules of the Institutional Animal Ethical Committee of Yeungnam University (IACUC No. 2018-031, Republic of Korea).

2.8. Histopathology analysis Xenograft tumor masses were cut, immersed in 10% formalin, embedded in paraffin, serially sectioned (3–4 µm), and stained with hematoxylin and eosin (H&E) prior to histopathological examination under a light microscope (Nikon, Tokyo, Japan). Tumor cell volumes and intact

tumor cell-occupied regions (%/mm2 of tumor mass) were calculated using a computer-based automated image analyzer (iSolution FL ver 9.1, IMT i-solution Inc., Quebec, Canada). The apoptotic markers, caspase-3, poly (ADP-ribose) polymerase (PARP), CD31, and Ki67, were investigated immunohistochemically using purified primary antibodies and biotinylated secondary antibodies with avidin-biotin-peroxidase complex (ABC) and a peroxidase substrate kit (Vector Labs, Burlingame, CA, USA). Briefly, endogenous peroxidase activity was blocked with methanol and 0.3% H2O2 for 30 min and non-specific immunoglobulin binding was blocked with normal horse serum blocking solution in 10 mM citrate buffer (pH 6.0). Tissue sections were incubated with primary antisera overnight at 4 °C in a humidified chamber, and then incubated with biotinylated universal secondary antibodies and ABC reagents for 1 h at room temperature. Finally, sections were reacted with the peroxidase substrate kit for 3 min at room temperature. Cells were counted as immunopositive if they had >20% immunoreactivity to the apoptotic markers (caspase-3 or PARP). The percentage of the region occupied by caspase- 3and PARP-positive cells within the tumor mass (%/mm2 of tumor mass) was measured using an automated image analyzer.

2.9. Statistical analysis To determine whether differences between test groups were statistically significant, data were analyzed by analysis of variance (ANOVA) or the Student’s t-test using IBM SPSS Statistics for Windows (Release 14.0K, SPSS Inc., Chicago, IL, USA). Data are presented as mean ± standard deviation of four independent experiments. A P-value < 0.05 was considered statistically significant.

3. Results and Discussion 3.1. Fabrication and physicochemical characterization of the GNP-based nanoconstruct A schematic presentation of the GNP-based nanoconstruct is shown in Figure 1. Overall scheme involves the synthesis of the thiol-group liberated cationic BSA (cBSA), which was capped on the GNP surface; this was followed by the conjugation of LND and the targeting agent (Aptamer). Abundant carboxylic groups and amine groups are present in albumin. Albumin contains 35 cysteine residues which are involved in stabilizing the disulfide bonds and structure of molecules [21]. First, albumin was denatured by reducing the 17 disulfide bonds to thiol groups for interaction with GNP based on metal-thiol interactions. The 4 µmol of denatured albumin converts 4 molecules of S-S bonds into 8 SH groups as confirmed by the DTNB assay. Albumin is reported to possess approximately 57 amine groups by employing ethylenediamine. The number of amine groups was quantified by the Ninhydrin assay [22]. We have employed 1:1 weight fraction of cationic BSA (containing >150 NH2 group) and native BSA (containing ~57 NH2 and ~100 COOH group). During the first step, amine group was employed for the LND conjugation using EDC/NHS reaction and thoroughly washed. The free –COOH group was employed for AS1411 conjugation and we have meticulously calculated the each part of conjugation chemistry. The citrate-stabilized GNP was prepared by the seed-mediated method with an average size of 32.6 ± 2.5 nm and a surface charge of -14.6 ± 1.7 mV. Upon mixing albumin with GNP, an immediate layer of protein around the particle was formed with an increase in size (54.3 ± 4.4 nm). The successful protein capping of GNP (AGN) was confirmed by a change in surface charge from -14.6 ± 1.7 mV of single GNP to -3.3 ± 1.9 mV. The strong

binding of albumin to GNP was attributed to the strong thiol-metal interaction. The presence of an outer protein corona stabilized the GNP particles under various pH conditions and ionic strengths where GNP would flocculate under similar conditions. Interestingly, a densely packed protein layer will confer maximum stability than a monolayer formation around the plasmonic particles [23]. As a next step, LND was chemically conjugated to albumin via CDI chemistry where the amine group of LND formed an amide linkage with the carboxylic group of LND (92.5 ± 5.1 nm). In addition, drug binding association to BSA usually occurs in subdomain IIA due to the presence of large hydrophobic cavity in site I that can accommodate several drugs [21]. Finally, Aptamer AS1411 was conjugated to the carboxylic group of albumin via CDI chemistry with a final particle size of 134.4 ± 2.7 nm for the nanoconstruct (Figure S1). We have used 1 ml of GNP solution (4.7 mg) and coated with BSA (1 mg/ml). The surface coated BSA was then conjugated with LND (321.16 µg) equivalent to approximately 30% w/w of BSA and observed 85% of loading/conjugation efficiency. We have confirmed the thiolated BSA interaction with a GNP via DTNB assay (4 µmol of BSA contains 8 –SH groups) and GNP was maximally coated with BSA. The morphology of the nanoparticle was examined by TEM as shown in Figure 2A. The nanoparticle construct remained well-separated without any signs of aggregation, indicating successful protein capping. In addition, surface topography and height profile were evaluated by AFM imaging. As shown in Figure 2B, GNP particles were circular and nanosized with a flat object on the mica surface; particle size increased after albumin capping on the particles. The 3D image clearly revealed the difference between particles before and after albumin assembly with a notable difference in the height profile. Elemental microanalysis via energy-dispersive X-ray spectroscopy (EDS) was acquired to identify the component elements of the composite

nanoparticles. The EDS of AS-LAGN revealed a strong signal with 33.35 wt% of gold at 2.1 keV and a high presence of C and O atoms; this further verified the successful construction of the composite nanoparticles (Figure 2C). Successful encapsulation of drug in the nanoparticle system was confirmed by DSC. Figure 2D shows that LND had a sharp endothermic peak at 210 °C corresponding to its melting point; however, such peaks were not observed after conjugation with albumin and aptamer. These results clearly indicate the presence of drug in the amorphous form after its conjugation to albumin. SPR peaks of bare GNP and AS-LAGN were examined by UV-Vis spectroscopy (Figure 3A). Results showed that surface modification of GNP slightly changed the refractive index around GNP. A slight red shift was observed (~8 nm) after albumin assembly on the plasmonic particles. The successful binding of albumin to GNP was confirmed by FTIR spectroscopy (Figure S2). GNP possesses symmetric and antisymmetric COO stretching bands at ~1390 cm -1 and ~1560 cm-1 [24]. AGN showed two characteristics bands corresponding to amide I (1650 cm1

) and amide II (1540 cm-1) [25]. The amide I band was due to carbonyl stretching vibrations in

albumin while amide II band resulted from the coupling of C-N stretching and N-H bending in BSA. Furthermore, the conjugation of aptamer with size 26 nucleotides, to nanoparticle was determined by agarose gel electrophoresis. As shown in Figure 3B, free aptamer could migrate on the gel, resulting in a bright band. Lane 4 sample (AS-LAGN, 1 µM aptamer) could not travel on the gel, confirming the successful conjugation of aptamer to solid particle; the light band that appeared in lane 3 represents the unconjugated aptamer. The thermal behavior of GNP, AGN, and AS-LAGN was studied under the influence of NIR light. As expected, temperature increased steadily for GNP and AS-LAGN as a function of time with the exposure of NIR. Temperature increased from 20 °C to 45 °C within 300 s whereas a ~5 °C change in temperature was observed

for the control during the time-period. Of note, surface modification of GNP did not change the plasmonic property of gold (Figure 3C). The stability of AS-LAGN nanosystem in physiological conditions (pH 7.4) was observed to be stable throughout the study period (Figure S3). LND release from nanoconstruct was tested in the presence and absence of NIR irradiation. As seen in Figure 3D, ~50% of drug was released from the nanoparticle under the influence of NIR light within 4 h in the release study whereas less than 10% of drug was released in the absence of NIR light during the time period. Similar pattern of drug release continued until the end of 24 h with an eventual release of ~70% from the nanoparticles under the influence of NIR. Since the BSA is conjugated with GNP using the –s-s disulfide linkage, presence of GSH at acidic tumor environment will break the assembly of nanosystem paving way for the release of LND. The results clearly reveal the influence of NIR irradiation on the nanoparticles. The higher drug release was mainly attributed to the hyperthermia effect of GNP that disrupted the drug conjugation and resulted in eventual release.

3.2. In vitro cellular assessment We assessed the tumor targeting potential of AS-LAGN and LAGN in MCF-7, MDA-MB-231, and DU-145 cancer cells using flow cytometry. These cells are known to have a higher surface expression of nucleolin receptors than other cancer cells. Across all cell lines, AS-LAGN showed remarkably higher fluorescence intensity than GNP. Mean fluorescence intensity (MFI) of AS-LAGN was 2.1-, 1.6-, and 1.9-fold higher than that of GNP in MCF-7, MDA-MB-231, and DU-145 cells, respectively (Figure 4A, Figure S4). The results indicate that AS-LAGN was actively internalized by the cancer cells via a specific-receptor based internalization mechanism.

This is due to the targeting ability of AS1411 toward the nucleolin receptors of cancer cells [26]. This result is consistent with our hypothesis that surface conjugation of AS1411 could more effectively and efficiently target cells. Consistently, we studied the subcellular distribution of AS-LAGN in these cancer cells using confocal laser scanning microscopy (CLSM) (Figure 4B). Lysosome was stained with LysoTracker Red and FAM labeled aptamer to guide particle distribution in the cells. A bright green fluorescence was observed in the cellular cytoplasm with no obvious nucleus accumulation after a 2-h incubation, implying a typical receptor-mediated cellular internalization. Green fluorescence was mainly located in the cytoplasmic region; the merged well with the red fluorescence denoted lysosomes. Based on these findings, AS-LAGN was initially located in the acidic organelles (late endosome-lysosome), and drug release occurred after nanoparticle destabilization. Intracellular drug release in lysosomes will act on mitochondria and interfere with the energy metabolism of cancer cells by acting on mitochondrially-bound hexokinase, which will greatly enhance the efficacy of cancer treatment [27]. In vitro anticancer effect of different formulations was tested in MCF-7, MDA-MB-231, and DU149 cells (Figure 4C). Without NIR, neither GNP nor AGN caused any damage to cancer cell viability, indicating its good compatibility as a drug delivery carrier system. LND decreased the cellular viability at higher concentration, but showed a high IC50 value in the range, 20-50 µg/ml. Data showed that monotherapy relied on either LND or photothermal effect, which had limited therapeutic effects with more than 65-70% of cells being viable. Continuous laser irradiation was performed at 808 nm with power density of 2 W/cm2 for 5 min. Ligand targeting and NIR-based photothermal effects significantly enhanced LND-based mitochondrial damage. For example, AS-LAGN (+NIR) resulted in 75% of cell death compared to 30% for

LND and 40% for GNP (+NIR) in MDA-MB-231 cells. Similar results were observed in MCF-7 and DU-145 cancer cells. We speculated that NIR induction triggered the release of LND from GNP surface after the disassembly of GNP due to hyperthermia effect. The aptamer present on the particle surface will interact favorably with the receptor overexpressed on cancer cells and result in higher accumulation of particles compared to non-targeted particles [28,29]. Across all cell lines, combination of photothermal and chemotherapy killed more than 80% of cancer cells, indicating its superior anticancer effect. Combination therapy reduced the IC50 value by 4-fold (~5 µg/ml vs ~22 µg/ml) for all cancer cells compared to monotherapy. Overall, conjugation of aptamer (AS1411) might enhance the receptor-mediated cellular uptake and laser irradiationbased hyperthermia effect of GNP could potentially enhance the antitumor effect of LND in cancer therapy.

3.3. Apoptosis and Reactive oxygen species analysis The combination effect of chemotherapy and phototherapy was determined by Annexin V/PI-based apoptosis assay using flow cytometer. Early apoptosis is characterized by Annexin V+/PI- while late apoptosis is denoted by Annexin V+/PI+. As shown in Figure 5A, GNP could not induce apoptosis alone, whereas LND induced definite apoptosis of cancer cells. The nucleolin targeted AS-LAGN (-NIR) relatively increased the apoptotic ratio with higher percentage of cells in early and late apoptosis. Upon laser irradiation at 808 nm, cells remarkable underwent the early and late apoptosis phases. For example, total apoptosis (early and late apoptosis) of AS-LAGN (+NIR) was ~90%, which is significantly higher than that of AS-LAGN (-NIR). In contrast, free LND had 20% apoptotic cells in MDA-MB-231 cancer cells, indicating

the potential of a photothermal effect and the significance of combination therapy. Overall, apoptosis induction was in the order, control < LND < LAGN < AS-LAGN (-NIR) < AS-LAGN (+NIR). FACS data clearly revealed that the mitochondrial-targeting effect of LND along with laser irradiation-based phototherapy had a synergistic effect on the induction of higher apoptosis in cancer cells. The apoptotic effect was further confirmed by Hoechst 33342 staining (Figure 5B). The untreated cells maintained its morphology and showed normal nuclei. The cells treated with LND and LAGN showed altered morphology and visible spots. The cells treated with ASLAGN (under the influence of NIR) had distorted nuclei with highly condensed chromatin granules. Most cells died because of remarkable cell shrinkage, fragmentation of nucleus and DNA, and chromatin condensation which are typical signs of cells undergoing apoptosis. The destructive effect of phototherapy was demonstrated by a live/dead assay (Figure 6A). The monolayer of cells was incubated with AS-LAGN and a section of monolayer cells was irradiated with 2 W/cm2 for 5 min. Calcein AM binds to live cells while ethidium bromide binds to dead cells. Irradiated areas were evidently stained with red color, indicating dead cells, while non-irradiated cells were mainly green, indicating their viability. This result clearly explains the site-specific action of lasers, which exhibited marked cell death when incubated with AS-LAGN. A slight presence of dead cells in the non-irradiated region might be due to the drug which is released during irradiation and is likely diffused to surrounding cells [30]. The influence of photothermal action was further probed by a reactive oxygen species (ROS) study for evaluating the cause of apoptosis [31]. As shown in Figure 6B, ROS levels were definitively higher after laser irradiation of AS-LAGN (+NIR) than either GNP or LND or AS-LAGN (-NIR) alone. Fluorescence change caused by AS-LAGN (+NIR) was 1.8-fold higher than that of AS-LAGN (-NIR), indicating the potential of laser irradiation in inducing ROS

production. Of note, LND or other formulations also exhibited DCFH fluorescence, indicating ROS production that might be due to various intracellular actions of therapeutic agents. However, excessive ROS production in AS-LAGN (+NIR) stems from the GNP core. The data clearly reveal that excessive ROS generation in cancer cells after NIR light exposure might enhance oxidative damage to mitochondria which could be the main reason for enhanced apoptosis [32]. Cell migration is considered to be an important parameter in tumor proliferation and tumor angiogenesis; therefore, we evaluated the inhibitory potential of individual formulations. Figure S5 shows that untreated control cells could retain their migration capacity, and scratch was completely healed after 18 h in all cancer cells. LND and LAGN inhibited the migration of cells to an extent; however, they were not effective. Most remarkably, however, AS-LAGN (NIR+) showed the maximum anti-migratory effect (~80%), with most cells either apoptotic or dead. This indicated that a combination of photothermal therapy could potentially synergize the anticancer effect of anticancer agents. Metastasis is the result of multiple changes in cancer cells and the surrounding microenvironment that allows cells to migrate into healthy tissues. The superior inhibitory effect of AS-LAGN (NIR+) could potentially influence cell-cell interactions and decrease the rate of healing. Therefore, a higher cell migratory capacity of formulations could be a potential benefit in cancer therapy.

3.4. In vivo biodistribution and antitumor efficacy In vivo biodistribution of AS-LAGN is presented in Figure 7A. AS-LAGN showed remarkable accumulation in tumor compared to that of non-targeted LAGN after 24 h of administration. The

fluorescence signal from LAGN was identified all over the body, while AS-LAGN showed strong signal intensity in tumor tissue. The strong affinity of AS1411 toward the surface expressing nucleolin receptors could be the main reason for enhanced accumulation of particles in the tumor tissue. To quantitatively evaluate the distribution of particles in various organs, animals were sacrificed and their organs were collected (liver, lung, spleen, kidney and heart) for ex vivo analysis (Figure S6). Notably, ex vivo fluorescence imaging further confirmed the superior tumor accumulation capacity of AS-LAGN with a significantly higher fluorescence signal at tumor sites than in other organs. Consistently, week fluorescent signals were observed in the heart, kidney, and spleen, suggesting a superior biodistribution pattern. The high accumulation of AS-LAGN in tumors could be attributed to multiple reasons: 1) high affinity of AS1411 toward the nucleolin receptor which will enhance its accumulation via specific ligandreceptor binding; 2) surface of the carrier was modified with albumin which could extend circulation time, thereby increasing the accumulation in tumor; 3) particle size of AS-LAGN was within the desired range of 120 nm which could potentially avoid rapid systemic clearance through extravasation and leakage from blood vessels for accumulation in cancerous tissues [33,34]. Furthermore, the photothermal effect of AS-LAGN in xenograft mice was validated using a thermal camera. Upon laser irradiation, local temperatures immediately rose to 45 °C - a temperature that sufficiently ablated tumor cells (Figure 7B). It is worth noting that the surrounding healthy tissues did not show any increase in temperature, indicating the site-specific phototherapy effect of GNP. Moreover, the temperature was achieved within a short span of 5 min. In PBS-administered mice, however, upon NIR irradiation temperature did not increase.

3.5. In vivo anticancer efficacy analysis

Motivated by the encouraging tumor targeting potential of AS-LAGN, we further explored the antitumor efficacy profile of AS-LAGN (+NIR) in the DU145-bearing xenograft model (Figure 8A). Mice were intravenously administered the respective formulations and NIR lasers were exposed to tumors 6 h post-injection at 2 W/cm2 for 5 min (808 nm). LND and ASLAGN were minimally effective in controlling tumor growth in mice. As expected, AS-LAGN irradiated at 808 nm showed remarkable tumor inhibitory effect in animals and significantly controlled tumor burden compared to any treated group. The high regression rate in AS-LAGN (+NIR) treated group clearly revealed the combinational potential of NIR irradiation and chemotherapy. We believe that external NIR exposure increased the local temperature, allowing the release of conjugated drug and acting in a synergistic manner. In vitro studies clearly highlighted the ROS inducing effect of GNP that leads to cell apoptosis and cell death. Of note, a high dose of LND is required to initiate an antitumor action, but in the present study, we observed complete regression of tumor upon combination with laser irradiation. The superior antitumor efficacy might be attributed to multiple reasons, including higher drug accumulation in tumors, surface modification with albumin, small particle size, and synergistic therapeutic activity [35-38]. The safety profiles of AS-LAGN were analyzed by measuring body weights (Figure 8B). In this work, we did not find any signs of toxicity or death due to any of the formulations. This indicated the good safety profile of the administered agents. Organs were subsequently removed and stained with H&E to observe the pathological profile. No signs of organ damage or toxicity was observed after treatment with AS-LAGN (±NIR irradiation), suggesting that the drug was effectively encapsulated in the nanocarrier. Its controlled release behavior could allow its long-term administration. Furthermore, NIR irradiation did not cause

any adverse effects in normal tissues. As the nanoconstruct did not cause any organ damage without compromising its therapeutic efficacy, it is of great clinical potential (Figure S7) [39]. The potential mechanism of action of AS-LAGN was investigated by immunostaining primary tumors (Figure 8C). Through H&E staining, most tumor cells were found to undergo notable tumor necrosis with loss of their membrane integrity. This finding indicates the notable inhibition of tumor growth and cellular necrosis in sections treated with AS-LAGN (+NIR). Consistently, the AS-LAGN (+NIR) treated group had the lowest tumor cell volume (~23%) compared to either AS-LAGN (-NIR) (~35%) or free LND (~70%), which had significantly higher volumes. These data clearly suggest that the NIR-laser irradiation-mediated hyperthermia effect could be the main reason for the higher cellular necrosis. TUNEL staining was performed to evaluate apoptosis. As seen, AS-LAGN (+NIR) treated group showed a remarkably higher TUNEL positive cells which is evidence of marked apoptosis of cancer cells. Higher TUNEL positive cell is an indicator of higher cell death relative to other groups that had lower TUNEL staining. Apoptosis was further confirmed by caspase-3 and PARP immunostaining. Mice treated with AS-LAGN (+NIR) showed 2-fold and 3-fold higher caspase-3-immunolabeled cells than control LAGN and LND, respectively. Similarly, 2- and 3-fold higher PARP-immunolabeled cells were observed for these groups, respectively. The expression levels of caspase-3 and PARP are hallmarks of apoptosis. ROS generated by GNP and LND might result in mitochondrial pores that lead to downstream activation and release of caspase-3; this will in turn activate the cleavage of PARP [40-42]. Ki67 staining was carried out to investigate the proliferative activity of tumor cells and CD31+ to investigate the angiogenesis. As seen, 80% of control cells were CD31+ and Ki67+, indicating uninterrupted tumor growth. LND treatment slightly reduced the number of Ki67 positive cells, however, combining AS-LAGN + laser resulted in a 10-fold reduction in

Ki67 and CD31+, indicating a major suppression of tumor cells as illustrated by TUNEL staining (higher apoptosis). The number of CD31+ and Ki-67+cancer cells was significantly lower in the AS-LAGN + laser treated group than in the other groups. Overall, the data suggest that the better therapeutic outcomes from the AS-LAGN (+NIR) treated group could arise from the synergistic combination of chemotherapeutic drug and laser irradiation that target tumors. In this study, we clearly demonstrated that local hyperthermia resulted from laser-induced GNP destruction in cancer cells. The primary mechanism of action of cancer cell death was identified as cellular apoptosis/necrosis, resulting from thermal ablation and mitochondria-mediated ROS generation [43,44].

4. Conclusion In summary, we developed a facile approach to synthesize a multifunctional AS-LAGN for targeted cancer therapeutic applications. This robust nanoplatform was constructed with a unique conjugation chemistry where Au with a core size of 10 nm was entangled with albumin and LND was conjugated at specific pockets on the protein surface. AS1411 surface conjugation retained its nucleolin binding activity, which led to an enhanced accumulation of the nanoconstruct in nucleolin-positive cancer cells via active targeting. By conjugating BSA to GNP via redox-liable disulfide linkage and LND to the albumin layer via disulfide bridges, AS-LAGN showed remarkable therapeutic efficacy in cancer cells, which was attributed to photothermal ablation and chemotherapeutic effect. The ability of GNP to convert excited state photon energy into thermal heat enabled synergistic photothermal/chemotherapy with improved efficacy. Overall, the developed AS-LAGN showed the following characteristics; (i) nanochemistry on the surface

of plasmonic nanoparticles; (ii) enhanced delivery of therapeutics to cancer cells; (iii) synergistic activity of LND and GNP in a single nanosystem; and (iv) higher accumulation in tumors and superior antitumor efficacy, with good safety profile. The present study highlighted the possibility of creating mitochondria-based cancer therapeutics by meticulously engineering a multifunctional nanoplatform. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements This research was supported by a grant from the National Research Foundation of Korea (NRF), funded

by

the

Korea

government

(MSIP)

(No.

2018R1A2A2A05021143,

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Figure 1. Schematic illustration of aptamer and lonidamine-conjugated albumin-decorated gold nanoparticles (AS-LAGN).

Figure 2. (A) TEM images of GNP and AS-LAGN. (B) Surface topography and height profile analysis of GNP and AS-LAGN by AFM. (C) Elemental analysis of Au metal in AS-LAGN by Energy dispersive X-ray spectroscopy (EDX). (D) Differential scanning calorimetry of free LND, BSA, LAGN, and AS-LAGN.

Figure 3. Characterization of AS-LAGN. A) UV-vis absorbance spectra of GNP and AS-LAGN. B) Agarose gel electrophoresis (2% gel) of samples: lane 1 (free AS1411), lane 2 (LAGN), lane 3 and lane 4 AS-LAGN (2 µM and 1 µM of AS1411 was conjugated to 1mg LAGN, respectively). C) In vitro temperature elevation of PBS, GNP, AGN, and AS-LAGN was performed using NIR thermal imaging. D) LND release profile at pH 7.4 PBS in the absence and presence of laser irradiation (808 nm, 2 W/cm2, 5 min).

Figure 4. A) Cellular uptake analysis of LAGN and AS-LAGN was investigated by FACS after 1 h of incubation (yellow: LAGN; pink: AS-LAGN). B) CLSM images of intracellular trafficking of AS-LAGN in MCF 7, MDA MB 231, and DU145 cells after 15 min of incubation (In AS-LAGN, FAM labelled AS1411 was used as the fluorescence dye.). C) Cytotoxicity assessment of different formulations by MTT assay after 48 h of incubation. * P<0.05 indicates statistical difference between AS-LAGN (-NIR) and AS-LAGN (+NIR).

Figure 5. A) Annexin V/PI staining of LND, LAGN, AS-LAGN (-NIR), and AS-LAGN (+NIR) upon NIR irradiation (808 nm, 5 min, 2 W/cm2) by flow cytometry analysis. B) Nuclear morphology was observed after a 10-min staining with Hoechst in the presence and absence of laser irradiation in DU-145 and MDA-MB-231 cell lines by fluorescence microscopy.

Figure 6. A) Photothermal ablation of AS-LAGN with and without NIR irradiation after staining with Calcein-AM (green) and EthD-1 (red). B) Cellular reactive oxygen species (ROS) analysis of various formulations after treatment with DCF-DA in MCF-7, MDA-MB-231, and DU-145 cancer cells.

Figure 7. A) Biodistribution of LAGN and AS-LAGN in the xenograft tumor mice model following intravenous injection and ex-vivo images of different organs excised at 24 h. B) In vivo photothermal effect of an increase in temperature post-NIR irradiation following treatment with PBS and AS-LAGN for 5 min, 808 nm, and 2 W/cm2.

Figure 8. In vivo antitumor efficacy. A) Changes in tumor volume and B) bodyweight comparison in DU145 tumor xenograft nude mice after treatment with different samples: (a) control, (b) LND, (c) LAGN, (d) AS-LAGN (-NIR), and (e) AS-LAGN (+NIR) with and without NIR laser irradiation. The formulation was administered via tail vein at a fixed dose of 5 mg/kg on days 1, 4, 7, and 10. Data are presented as mean ± SD (n=4) (*p<0.05, **p<0.01, ***p<0.0001). C) Immunohistopathology and immunohistochemical analyses of different formulations were performed with caspase 3 and PARP apoptotic markers, and CD31 and Ki67, angiogenesis and anti-proliferative markers. (a) control, (b) LND, (c) LAGN, (d) AS-LAGN (NIR) and (e) AS-LAGN (+NIR). Scale bar = 120 µm.

Graphical abstract