Organelle-Targeting Gold Nanorods for Macromolecular Profiling of

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Organelle-Targeting Gold Nanorods for Macromolecular Profiling of Subcellular Organelles and Enhanced Cancer Cell Killing Yanting Shen, Lijia Liang, Shuqin Zhang, Dianshuai Huang, Rong Deng, Jing Zhang, Huixin Qu, Shuping Xu, Chongyang Liang, and Weiqing Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01320 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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ACS Applied Materials & Interfaces

Organelle-Targeting Gold Nanorods for Macromolecular Profiling of Subcellular Organelles and Enhanced Cancer Cell Killing Yanting Shen a, Lijia Liang a, Shuqin Zhang b, Dianshuai Huang b, Rong Deng a, Jing Zhang a, Huixin Qu b, Shuping Xu a*, Chongyang Liang a*, Weiqing Xua a

State Key Laboratory of Supramolecular Structure and Materials, Institute of

Theoretical Chemistry, Jilin University, Changchun 130012 China b

Institute of Frontier Medical Science, Jilin University, Changchun 130021, People’s

Republic of China KEYWORDS:

in

situ

analysis,

organelle-targeting,

SERS,

PTT,

nucleus,

mitochondria, lysosome

ABSTRACT Subcellular organelles, e.g., nucleus, mitochondria and lysosome, are the vital organelles with responsibilities that maintain cell operation and metabolism. Owing to their roles in energy production and programmed cell death, these organelles have become prime therapeutic targets in different diseases and states. In this study, biocompatible, organelle-targeting nanoprobes were developed by modifying gold nanorods (AuNRs) with specific targeting peptides. These nanoprobes were employed to directly profile subcellular biomolecules and vital organelles by surface-enhanced Raman scattering (SERS) spectroscopy. Macromolecular spectral profiles of subcellular organelles were achieved and compared. Further, these 1 ACS Paragon Plus Environment

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organelle-targeting AuNRs were used for the photothermal treatment of cancer cells (HepG2, Hela and MCF-7 cell lines). The ell viability assays show that nucleus- and mitochondria-targeting AuNRs provide higher photothermal efficiencies under an 808 nm laser relative to the lysosome-targeting ones. This study makes critical insight into the spectral profiles of subcellular organelles, and also inspires people in the development

of

high-efficacy cancer

therapeutic

strategies

by subcellular

organelle-targeting drugs.

INTRODUCTION

As the vital organelles of cells, nucleus, mitochondria and lysosome can participate in many important physiological and pathological processes including cell proliferation, organism metabolism and intracellular transportation and play essential roles in regulating cellular biological functions.1, 2 The dysfunction of these organelles would lead to a variety of aberrant regulations and multiple diseases. For example, any turbulence to nucleus may lead to the abnormal regulation of cell activity and even causes the programmed death of the cell. 3 Inflammation, tumor, silicosis, and various lysosomal storage diseases are related to lysosomal dysfunction strictly. Besides, deficiency in mitochondrial function would accompany with multifarious pathological symptoms, such as the cardiovascular, neurodegenerative diseases and cancers as well.1, 4 Therefore, the concept of organelle and subcellular targeting has gained more and more attention in the near future. To explore the molecular mechanism and specific functions of organelles, a variety of organelle-targeting molecules and ligands were developed.5-7 For example, 2 ACS Paragon Plus Environment

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importing exotic materials to lysosome seems more accessible due to the superior accumulation effect of lysosomes.8 Small molecules (such as N, N-dimethyl ethylenediamine) can also help nanoparticles to achieve a precisely targeting feature based on the permeation and accumulation of the monobasic amine.9 For nucleus targeting, small molecules can enter and escape from nuclear pore complex (NPC) freely with a passive way because NPCs equipped with 9 nm channels. However, macromolecules with a diameter of over 9 nm or with a weight of above 45 kDa require the assistance of nuclear location sequence (NLS) to target nucleus, which is an active transport way.10 Similar to nucleus targeting, transporting nanoparticles to mitochondria is not easy. The most common-used ligands for targeting mitochondria are the lipophilic cations and the mitochondrial targeting sequence peptides.11-13 Based on the targeting strategies, more and more organelle-targeting nanoplatforms were developed to detect subcellular analytes and monitor intracellular environments. As a powerful analytical tool, surface-enhanced Raman scattering (SERS) spectroscopy can not only provide detailed fingerprint spectral information, but also combine the advantages of high sensitivity, showing notable superiority in the analysis of bio-samples, such as cells and tissues, as well as living bodies.14,

15

Recently, efforts have been made for achieving subcellular organelles information at molecular level.9,

16

For example, Lim and co-workers designed three SERS-tag

nanoprobes with nucleus-targeting, mitochondria-targeting and cytoplasm-targeting functions for subcellular imaging.17 Hu et al. also presented a single SERS probes (membrane or nucleus targeting nanoprobes) with either labelling or label-free 3 ACS Paragon Plus Environment


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strategies to achieve targeted SERS images (nucleus and cell membrane) by a confocal Raman system. However, owing to an indirect exploration of their method, very little information of cell organelles can be achieved. Also, these studies care less about the precise locations of these targeting nanoprobes. Thus, the obtained label-free SERS spectra may lack accuracy in interested detection positions. Therefore, the direct and precise detections and analysis of the biomolecular information from specific organelles are still challenging, which is of great significance in expounding molecular mechanisms during critical physiological and pathological processes. Photothermal therapy (PTT) is a widely used therapeutic approach by converting light energy into heat to generate hyperthermia and further cause cell death. As one of the critical treatments, metal nanoparticles were widely adopted as a PTT-sensitive agent to improve the light collection efficiency due to the plasmonic property of metal nanomaterials.18-21 Recently, PTT nanomaterials combined with organelle targeting agents, e.g., nucleus, have been extensively exploited to obtain maximum therapeutic efficacy.22-26 However, no one compares the PTT nanotherapy effect among different organelle-targeting strategies. In this paper, with the aim of obtaining precise bimolecular information of subcellular organelles and exploring the different efficacy among different organelle-targeting platforms in PTT treatment toward cancer cell, we designed biocompatible and organelle-targeting nanoprobes based on the gold nanorods (AuNRs) modified with specific targeting peptides to target nucleus, mitochondria 4 ACS Paragon Plus Environment

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①

ANT: AuNR-based Nucleus-Targeting probe PEG

RGD

ALT: AuNR-based Lysosome-Targeting probe

AuNRs

AMT: AuNR-based Mitochondria-Targeting probe

②

Cell culture

633 nm 808 nm

Organelle-selective SERS detection

③

Targeting organelles

④

Organelle-targeting photothermal therapy

Scheme 1 The schematic illustration of the SERS detections and the NIR photothermal

therapy

of

cancer

cells

with

the

AuNR-based

subcellular

1 -○ 4 ; the preparation of the organelle-targeting organelle-targeting nanoprobes. ○

nanoprobes,

cell

culture

with

the

organelle-targeting

nanoprobes,

the

organelle-selective SERS detection, and organelle-targeting PTT. and lysosome. The profound plasmonic effects of these targeting nanoprobes are found in two aspects: profiling the molecular information of specific organelles and performing PTT to induce cell death. Valuable insights of our work can be outlined as (1) coincident experimental evidences on the successful targeting of our designed nanoprobes toward their organelles were achieved by the super-resolution fluorescence microscopy and the Bio-transmission electron microscopy (Bio-TEM), which is of great importance in accurately obtaining the molecular information of organelles. (2) The inherent macromolecular Raman profiles of nucleus, mitochondria and lysosome were obtained simultaneously and compared for the first time, which 5 ACS Paragon Plus Environment


are helpful in learning cells from molecular level and creating comprehensive reference maps of cancer cells. (3) The PTT efficacies of different organelle-targeting platforms were investigated under the same condition. Nuclear-targeting and mitochondria-targeting probes possess relatively higher killing ability, which plays a guidance role in the design of targeting antitumor drugs.

EXPERIMENT SECTION Preparation of AuNR-based nanoprobes.

AuNRs were synthesized using a seed growth

method 27 and the achieved AuNR has a length of 66 nm and a width of 30 nm. The detailed procedures are described in supporting information. Transmission electron microscope (TEM), ultraviolet-visible (UV-Vis) and dynamic light scattering (DLS) spectroscopies were used to measure the size, morphology, plasmonic property and zeta potential of the obtained AuNRs. Then, the AuNRs were modified with methoxy poly(ethylene glycol)-thiol (mPEG-SH) with MW=5000 to inhibit aggregation and improve biocompatibility. 28 The number of the mPEG-SH on each particle is about 1000. Then, the lysosome, mitochondria and nucleus targeting nanoprobes (ALT, AMT and ANT) were obtained by modifying RGD, RGD/MLS, and RGD/NLS on the 1 in Scheme 1) via the covalent linking between gold surface of PEGylated AuNRs (○

and thiol group of cysteine (bold in peptide sequence of MLS, NLS and RGD). The mixture was incubated overnight at room temperature. And the molar ratio of MLS (or NLS) with AuNRs is 104:1, while RGD is 103:1 according to literatures.29, 30 Following by a step of centrifugation (4000 rpm, 8 min), the cleaned AuNRs were 6 ACS Paragon Plus Environment

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redispersed in water for use. The AuNRs concentration was eventually evaluated by the absorbance value by using Uv-vis spectroscopy. Observation of intracellular distribution of targeting nanoprobes by super-resolution confocal fluorescence microscopy.

To take the confocal fluorescent images of nanoprobes, the

FITC-labelled targeting nanoprobes were prepared first as we established a method.28 The FITC-labelled nanoprobes (0.1 nM) were cultured with cancer cells for 24 hrs, and then the mitochondria, nucleus and lysosome of the cells were stained with commercial organelle dyes for 15 min, respectively. Then we used a super-resolution fluorescence microscopy and an analysis software to record and deal with the fluorescent images of an individual cell as the method we developed in the published paper.28 Nucleus (blue, Hoechst 33342, excited by a 405 nm laser), mitochondria (red, Mito Tracker red CMXRos, excited by a 543 nm laser) and lysosome (red, LysoTracker red DND-99, excited by a 543 nm laser) will give colors on the fluorescent images. Bio-TEM imaging for cells.

After HepG2 cells were cultured with 0.1 nM of AMT,

ANT and ALT nanoprobes for 24 h incubation. The cells were harvested and fixed with 0.25 % trypsin and glutaraldehyde at 4°C. Afterwards, cells were washed with PBS for two times and then dehydrated with increasing ethanol gradients. Then, they were treated with propylene oxide and embedded in Epon. Finally, the cells were cut into thick sections about 80 nm and placed on a carbon film supported by the copper grids, followed by a staining procedure with uranyl acetate and lead citrate for Bio-TEM observation. 7 ACS Paragon Plus Environment


SERS detections. The

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confocal Raman system (LabRAM Aramis, Horiba Jobin Yvon)

was used for SERS detections of mitochondria, nucleus and lysosome, respectively. The laser is a He-Ne laser (λex =632.8 nm) and the power is about 7.1 mW. After the cells were cultured with nanoprobes for 24 hrs, they were fixed on coverslips for SERS detections. Experimental conditions, t=10 s and accumulations=2 times, were set. Photothermal performance of AMT, ANT and ALT.

PTT efficacies of the AuNRs, AMT,

ANT and ALT were investigated by irradiating a 2 mL quartz plate containing 1.0 mL of AuNRs (or AMT, ANT, ALT nanoprobes) with a concentration of 2.0 nM under a laser (808 nm, 4.0 W/cm2) for irradiation 10 min. The temperature of the irradiated aqueous dispersion was recorded on a thermocouple combined with a digital temperature controller. Then cells cultured in glass-bottom dishes (φ30 mm, NEST) were incubated with AMT, ANT and ALT nanoprobes for 24 hrs and the concentration is 0.2 nM. After irradiation for 5 min, the culture medium was removed and 1.0 mL of PBS mixed with 2.0 µM of PI and 3.0 µM of Calcein-AM were added. After incubation for 30 min, the cells were washed with PBS for two times, and further imaged by confocal fluorescence microscope (Olympus). Calcein-AM (green) and PI (red) were used to stain living and dead cells, respectively.

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Absorbance (a.u.)

b)

631

0.8

632

637

629

20

AuNRs ALT ANT AMT

Zeta potential (mV)

1.0

a)

50nm

100

10

0.4

0.0 400

d) d)

600

700

Wavelength (nm)

800

Temperature (℃)

60 40 20

ALT AMT ANT

45

AMT

ANT

ALT

f) f) H2O

50

AuNRs

900

e)e)

55

5

0 500

60

80

c)

15

0.6

0.2

Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 35 30

Absorbance (a.u.)

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ALT

ANT

25

0 Control

AMT

ANT

ALT

AMT

20

0

100 200 300 400 500 600 Time (s)

400

500

600

700

Wavelength (nm)

800

900

Figure 1. a) TEM image of the synthesized AuNRs. b) and c) UV−vis spectra and zeta potentials of the AuNRs, AMT, ANT and ALT nanoprobes, respectively. d) Cell viabilities of HepG2 cells after they were incubated with different nanoprobes with a concentration of 0.1 nM for 24 hrs. e) Temperature elevation profiles of water and AMT, ANT and ALT contained solutions (2.0 nM) after they were irradiated with a 808 nm laser under a power density of 4.0 W/cm2 for 10 min. f) Uv-vis spectra of the AMT, ANT and ALT nanoprobes before (solid) and after 6-month storage (dash)

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For the WST-1 assay, 104 cells were seeded in 96 well plates and respectively cultured with AMT, ANT and ALT with a concentration of 0.2 nM for 24 hrs. Finally, the cell viabilities without and with irradiation were measured by a standard WST-1 assay. Statistical analysis.

According to standard deviation, a two-paired t-test was conducted

for statistical analysis. *** means significantly different with the p-value

Organelle-Targeting Gold Nanorods for Macromolecular Profiling of

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