Immune System

Evaluation of cellular uptake mechanisms for AuNP-collagen-Avemar

nanocarrier on transformed and non-transformed cell lines

1. Introduction

Nowadays, development of metallic nanoparticles such as gold and

silver nanoparticles has successfully trapped the attentions and promised its effects and benefits on biosensing, bioimaging, and biomedical applications such as diseases therapeutics, diagnostics and drugs

delivery [1–3]. Gold nanoparticles (AuNPs) is one of most investigated

and popular metallic nanoparticles and have been well applied in

imaging and carriers of drugs and/or biomolecules for diseases and

cancers therapeutics in which are resulted from their easy fabrication

process, tunable sizes, charges and unique physicochemical properties

as well as easy functionalized with biomolecules, native nontoxic and

biocompatibility [4,5]. Although the versatile benefits and advantages

of AuNPs, several limitations such as penetration and/or permeability,

drug retention, and vascular barriers are major problems in nanoparticles-based therapeutics and/or theranostics. Therefore, in order to

improve the above defects and increase drug utilization, several ameliorating methods on nanoparticles such as surface chemistry modification with biopolymers and penetrated molecules to increase the

cellular uptake ability, intracellular targeting and stability have been

development [6,7].

1. Introduction

Nowadays, development of metallic nanoparticles such as gold and

silver nanoparticles has successfully trapped the attentions and promised its effects and benefits on biosensing, bioimaging, and biomedical applications such as diseases therapeutics, diagnostics and drugs

delivery [1–3]. Gold nanoparticles (AuNPs) is one of most investigated

and popular metallic nanoparticles and have been well applied in

imaging and carriers of drugs and/or biomolecules for diseases and

cancers therapeutics in which are resulted from their easy fabrication

process, tunable sizes, charges and unique physicochemical properties

as well as easy functionalized with biomolecules, native nontoxic and

biocompatibility [4,5]. Although the versatile benefits and advantages

of AuNPs, several limitations such as penetration and/or permeability,

drug retention, and vascular barriers are major problems in nanoparticles-based therapeutics and/or theranostics. Therefore, in order to

improve the above defects and increase drug utilization, several ameliorating methods on nanoparticles such as surface chemistry modification with biopolymers and penetrated molecules to increase the

cellular uptake ability, intracellular targeting and stability have been

development [6,7].

Evaluation of cellular uptake mechanisms for AuNP-collagen-Avemar

2. Materials and methods

2.1. Preparation of gold-collagen-Avemar nanocomposites (AuNP-Col-Ave)

and FITC conjugation

The gold nanoparticle-collagen (AuNP-Col) has generated and described as the previous study [21,22]. No reference was found before

published the novel method for preparation this AuNP-Col-Ave nanocarrier. Briefly, to prepare the AuNP-collagen-Avemar nanocarrier

(AuNP-Col-Ave), the AuNP (Gold Nanotech Inc, Taiwan) was sonicated

for 15 min and next thoroughly mixed with 100 μl of collagen (0.5 mg/

ml, BD Bioscience, USA) in a 1:1 vol ratio at RT for 30 min and obtained

the AuNP-Col nanocomposites with a volume of 200 μl (20 ppm). The

AuNP-Col nanocomposites were further interacted with Avemar

(0.5 mg/ml) in a 3:2 vol ratio at 4 °C for 2 h and obtained the AuNP-ColAve nanocomposites. For preparing a FITC conjugation, the AuNP-ColAve nanocomposites were generally interacted with FITC (0.5 mg/ml,

Sigma) in a 50:1 vol ratio at 4 °C for 8 h. The generated AuNP-Col-AveFITC nanocomposites were further washed with deionized water for

twice and kept in a dark at 4 °C.

2.2. Characterization

The fabricated AuNP-Col and AuNP-Col-Ave were further characterized their physical and chemical properties which have described

as previously study [21]. Briefly, the UV–vis absorption spectrum was

analyzed using UV–vis spectrophotometry (Helios Zeta, Thermo fisher

Scientific Inc, USA) and the infrared (IR) spectra was obtained using a

Fourier transform IR spectrometer (Shimadzu Pretige-21, Japan). Further, the hydrodynamic sizes were analyzed using Dynamic light


2.2. Characterization

The fabricated AuNP-Col and AuNP-Col-Ave were further characterized their physical and chemical properties which have described

as previously study [21]. Briefly, the UV–vis absorption spectrum was

analyzed using UV–vis spectrophotometry (Helios Zeta, Thermo fisher

Scientific Inc, USA) and the infrared (IR) spectra was obtained using a

Fourier transform IR spectrometer (Shimadzu Pretige-21, Japan). Further, the hydrodynamic sizes were analyzed using Dynamic light 2.2. Characterization

The fabricated AuNP-Col and AuNP-Col-Ave were further characterized their physical and chemical properties which have described

as previously study [21]. Briefly, the UV–vis absorption spectrum was

analyzed using UV–vis spectrophotometry (Helios Zeta, Thermo fisher

Scientific Inc, USA) and the infrared (IR) spectra was obtained using a

Fourier transform IR spectrometer (Shimadzu Pretige-21, Japan). Further, the hydrodynamic sizes were analyzed using Dynamic light.

2.3. Cell culture

The transformed cells of oral squamous cell carcinoma SCC-4 cells

were kindly provided by Prof. Da-Tian Bao [23] and were maintained in

DMEM contained with 10% FBS, 2 mM glutamine, and 1% penicillin/

streptomycin/neomycin. Non-transformed cells such as human skin fibroblasts (HSF) and bovine aortic endothelial cells (BAEC) were obtained from the American Type Cell Culture (ATCC) and maintained in

low glucose Dulbecco’s modified Eagle medium supplemented with

10% FBS and 1% (v/v) antibiotics (10,000 U/ml penicillin G and

10 mg/ml streptomycin). All the cells were maintained in a humidified

atmosphere incubator with 5% CO2 and 37 °C.

2.4. Cell viability assay

The cytotoxic effects of AuNP-Col-Ave were detected using a colorimetric MTT assay and Calcein AM Assay. Cells (1 × 104 cells/well)

were seeded in 96 wells culture plate and then incubated with various

doses of AuNP-Col-Ave (0.02, 0.04, 0.08, 0.16, 0.24, 0.48 and 0.96 mg/

ml) for 48 h. Cells were then incubated with MTT agent (0.5 mg/ml) for

2 h at 37 °C and the further dissolved colorimetric formazan complex was analyzed at the absorption wavelength of 570 nm using an ELISA

reader (SpectraMax M2, Molecular Devices, USA). The Calcein AM cell

viability assay was executed according to the manufacturer’s instructions. Briefly, after cells were treatment of AuNP-Col-Ave, cells were

then incubated with Calcein AM solution (2 μM) in PBS for 30 min at

37 °C. the fluorescence images and fluorescence intensity were measured and recorded using a fluorescent microscopy (ZEISS AXIO Z1,

USA) and Flow cytometer combined with FACS software (Becton

Dickinson, USA), respectively.

2.5. Cellular uptake assay

Cells (1 × 105

) were treated with AuNP-Col-Ave-FITC (0.5 mg/ml)

for 2 h and washed away the excess nanoparticles with PBS. The cells

were incubated for another 48 h at 37 °C. For fluorescence analysis,

cells were washed with PBS and then subjected to 4% paraformaldehyde fixation, 0.5% Triton X-100 permeability and F-actin phalloidin

staining (Rhodamine phalloidin, Sigma-Aldrich, USA) as well as DAPI

nuclear staining (Invitrogen). The fluorescence images were obtained

and carried out using a fluorescent microscopy (ZEISS AXIO Z1, USA).

Moreover, the cellular uptake ability was further validated by detected

the fluorescein-positive cells using flow cytometer and quantified with

the FACS software (Becton Dickinson, USA). Next, to verify the cellular

uptake mechanisms, cells (1 × 105

) were pre-treated with several endocytosis inhibitors included Cytochalasin D (Cyto, 5 μM),

Chlorpromazine (CPZ, 2 μM), Bafilomycin (Baf, 100 nM) and Methyl-βcyclodextrin (β-MCD, 2.5 mM) for 1 h and then treated with AuNP-Col Ave-FITC (0.5 mg/ml) for 2 h. After cells were washed away the excess

nanoparticles with PBS and incubated in culture medium for another

48 h at 37 °C, cells were subjected to fluorescence image assay and flow

cytometry and the fluorescein positive cells were further quantified

using the FACS software (Becton Dickinson, USA), respectively.

2.6. Statistical analysis

Data from multiple samples (n = 3–6) were collected for a given

experiment and expressed as mean ± standard deviation. All experiments were independently repeated at least three times. Student’s t-test

and the single-factor analysis of variance (ANOVA) method were used

to examine the difference between groups. For ANOVA, Bonferroni was

chosen for post hoc analysis. The p values less than 0.05 (p < 0.05)

were considered statistically significant.

section_15

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