Gold-Embedded Hollow Silica Nanogolf Balls for Imaging and

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Gold embedded hollow silica nano golf balls for imaging and photothermal therapy Woraphong Janetanakit, Liping Wang, Karla Santacruz-Gomez, Preston Boone Landon, Paul L. Sud, Nirav Patel, Grace Jang, Malvika Jain, Alice Yepremyan, Sami A Kazmi, Deependra Kumar Ban, Feng Zhang, and Ratnesh Lal ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08398 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017

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ACS Applied Materials & Interfaces

Gold Embedded Hollow Silica Nano Golf Balls for Imaging and Photothermal Therapy Woraphong Janetanakit†,‡, Liping Wang∥,¦,‡, Karla Santacruz-Gomez∇,‡, Preston B. Landon§, Paul L. Sud∥, Nirav Patel∥, Grace Jang§, Malvika Jain§, Alice Yepremyan∥, Sami A. Kazmi†, Deependra K. Ban§, Feng Zhang#,Φ, and Ratnesh Lal*,§,⊥,! †

§

Department of Nanoengineering, ∥Department of Bioengineering, Department of Mechanical and Aerospace En!

gineering, ⊥Materials Science and Engineering Program, and Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093 USA. ¦

School of Biomedical Engineering, Shanghai Jiaotong University, Shanghai, P.R.China.

∇Department #

of Physics, University of Sonora, Hermosillo, México

Agricultural Nanocenter, School of Life Sciences, Inner Mongolia Agricultural University, Inner Mongolia,

P.R.China. Φ

Department of Biomedical Engineering, School of Basic Medical Sciences, Guangzhou Medical University, Guang-

zhou 511436, P.R.China KEYWORDS Hollow nano golf balls, NIR laser, localized surface plasmon resonance, photothermal therapy (PTT), cellular uptake

ABSTRACT: Hybrid nanocarriers with multi-functional properties have wide therapeutic and diagnostic applications. We have constructed hollow silica nano golf balls (HGBs) and gold embedded hollow silica nano golf balls (Au@SiO2 HGBs) using the layer-by-layer approach on a symmetric polystyrene Janus template; the template consists of smaller polystyrene spheres attached to an oppositely charged large polystyrene core. Zeta potential measurement supports the electric force based template-assisted synthesis mechanism. Electron microscopy, UV-vis, and Near Infrared (NIR) spectroscopy show that HGBs or Au@SiO2 HGBs composed of a porous silica shell with an optional dense layer of gold nanoparticles embedded in the silica shell. In order to visualize their cellular uptake and imaging potential, Au@SiO2HGBs was loaded with quantum dots (QDs). Confocal fluorescent microscopy and atomic force microscopy imaging show reliable endocytosis of QD-loaded Au@SiO2 HGBs in HeLa cells and red blood cells (RBCs). Surface enhanced Raman spectroscopy (SERS) of Au@SiO2 HGBs in 4-MBA and RBCs cells show enhanced intensity of Raman signal specific to the RBCs membrane specific spectral markers. Au@SiO2 HGBs show localized surface plasmon resonance (LSPR) and heat-induced cell death in NIR range. These hybrid golf ball nanocarriers would have broad applications in personalized nanomedicine ranging from in vivo imaging to photothermal therapy.

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1. Introduction

shell silica nanoparticles,14 porous shell silica nano-

Gold nanoparticles (AuNPs) have wide applications in

carriers15 and multi-shell structures.16 Previous studies17

catalysis,1-3

biosens-

indicate that 70 nm solid silica nanoparticles increased

ing/bioimaging,7 photonics,8 and toxin scavenging.9 Gold

the release of lactate dehydrogenase (LDH) by human

nanoparticles are an excellent photothermal transducer

cervical carcinoma cells (HeLa), suggesting cell mem-

and have tunable localized surface plasmon resonance

brane damage and decreased metabolic activity of HeLa

(LSPR) that can convert absorbed tissue-penetrable near

cells. However, unlike 70 nm particles, 200 nm silica na-

infrared (NIR) light to heat. Hence, hollow gold nanocar-

noparticles triggered no such cytotoxicity in Hela cells.17

riers with defined pores would allow the heat-induced

Many cell types including HeLa cells can internalize larg-

release of imaging contrast molecules and therapeutics

er (100 - 200 nm diameter) silica particles via

(theranostics) as well as non-invasive photothermal ther-

endocytosis.18 Thus, larger silica nanocarriers can be engi-

apy (PTT). Such broad theranostics applications can be

neered for theranostics delivery as well as for photother-

accomplished remotely and in a highly controlled man-

mal ablation therapy (PTT).

ner. Gold nanoparticle clusters show strong particle-

Layer-by-layer (LBL) assembly method is a commonly

particle plasmonic coupling effect that would considera-

used nanocarrier fabrication technique.19 The method

bly enhance surface enhanced Raman scattering (SERS)

includes three steps: 1) synthesis and activation of a stable

and photothermal conversion efficiency. In principle,

expendable symmetrical/asymmetrical hierarchal tem-

well-assembled nano gold carriers would have maximum

plate made of, for example, polystyrene;20 2) formation of

LSPR absorbance to be red shifted towards the NIR re-

the desired shell by coating this homogeneous or hetero-

gion. As a result, the photothermal conversion efficiency

geneous template;21 and 3) removal of the template by

could be optimized for in vivo PTT.

thermal or wet chemical methods.22 Additional deposition

Gold nanocarriers, such as vesicles and clusters for light-

of gold nanoparticles in silica layers has been achieved by

triggered theranostics have been designed using a variety

modifying the shell growth step of the LBL assembly us-

of polymerization techniques.10 An efficient strategy to

ing an asymmetric polystyrene (PS) template.

achieve enhanced LSPR effect with stable plasmon is to

In this study, we leveraged the benefits of both silica and

deposit a uniform layer of gold nanoparticles within a

gold and using a homogeneous symmetric PS template,

homogeneous shell of silica. Due to its intrinsic biocom-

we have synthesized another type of silica nanocarriers -

patibility and biochemical stability, silica is often used for

hollow nano silica golf ball with gold nanoparticles em-

coating inorganic theranostic materials and for control-

bedded inside the silica shell (Au@SiO2 HGBs). The di-

ling drug release. Due to tunable carrier size, narrow pore

ameter of inner void space in the golf balls depends on

size distribution, ordered pore shape, augmented loading

the size of the template. Deposition of the gold particles

capacity, and distinctively functionalized exterior and

within the shell and porosity of the structure were con-

interior surfaces, a family of silica nanocarriers termed

firmed by TEM imaging while the open “dimple hole”

hollow/porous or porous core-shell Janus silica nano-

structure and the nanocarrier size were confirmed by

cosmetics,4-5

drug

delivery,6

11-12

Re-

SEM imaging. Further, we also confirmed loading of the

cently, more sophisticated 100 - 200 nm diameter Janus

quantum dots in Au@SiO2 HGBs. We performed experi-

silica nanoparticles have been designed and character-

ments to analyze the SERS based sensing, quantum dot

carriers have been explored as theranostic carriers.

13

based fluorescent imaging, atomic force microscopy im-

ized, including solid core silica nanoparticles, core/yolk-

2


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aging, and cellular uptake of Au@SiO2 HGBs by the HeLa

three times in DI H2O and then twice in EtOH. After the

cells and RBCs followed by photo-thermal heating using

final rinse, the polystyrene template was re-dispersed in 5

difference wavelength of lasers (488 nm, 532 nm, and 808

mL of 80% EtOH solution. Next, 50 nm carboxylate-

nm) in HeLa cells.

modified polystyrene spheres were electrostatically at-

2. Experimental Section

tached to the 200 nm PDDA-functionalized polystyrene surface as follows: 150 µL of aqueous 50 nm carboxylate-

2.1. Materials

modified colloidal polystyrene was diluted in 850 µL of DI

Carboxylate-modified polystyrene (PS-COOH) spheres

H2O. The mixture was then added to 5 mL of the PDDA-

(50 and 200 nm diameter (Cat #15913 and #08216, respec-

functionalized polystyrene solution and tumbled for 30

tively) 2.5% in water and poly-(diallyldimethylammonium

min.

chloride) (PDDA, MW ~8,500, 28% in water; Cat #24828)

2.2.2. UV irradiation

were purchased from PolySciences. Sodium citrate tribasic dihydrate, ACS reagent, ≥99.0% (Cat #S4641), chloro-

The sample was irradiated using a UV box (UV

auric acid trihydrate (HAuCl4·3H2O, 99.9%; Cat #520918),

Stratalinker 2400) with the 254 nm UV irradiation (Pow-

Tetrakis(hydroxymethyl)-phosphonium chloride (THPC,

er, 15 W) for 30 min.24 After irradiation, the solution was

80% solution in water; Cat #404861), Tetraethyl orthosili-

centrifuged at 3200 rcf for 45 min and then re-dispersed

cate (TEOS; Cat #131903) and 6 nm CdSeS/ZnS alloyed

in 5 mL of 80% EtOH solution.

quantum dots (carboxylated, ߣ௘௠ 540 nm; Cat #753777)

2.2.3. Synthesis of AuNPs

were purchased from Sigma Aldrich. Sodium hydroxide

AuNPs was synthesized by published method.23 AuNPs

(NaOH; Cat #38304) was purchased from Acros Organics.

were prepared by adding 54 mL of DI H2O to 50 µL of 10

Isopropanol (IPA; HPLC grade; Cat #A451-4) and Ammo-

M sodium hydroxide followed by stirring. 12 µL of 80%

nium hydroxide (NH4OH, 29%; Cat #A669S) were pur-

THPC was diluted in 1 mL of DI H2O and aged for 5 min.

chased from Fisher Scientific. Dimethylformamide (DMF;

The THPC solution was then added to the aqueous sodi-

Cat #4929-08) and anhydrous ethyl alcohol (EtOH; Cat

um hydroxide solution. The mixture was stirred for an

#9401-06) was purchased from Macron Chemicals and JT

additional 5 min before the addition of 2 mL of 1 wt.%

Baker, respectively. Deionized (DI) water was obtained by

HAuCl4. The solution quickly turned reddish brown in

a Millipore Advantage A10 system (18.2 MΩ resistance).

color and was stirred for 30 min prior to storage at 4 °C

2.2. Synthesis of HGBs or Au@SiO2 HGBs

for at least 24 h before use.

2.2.1. Polystyrene template synthesis Carboxylated modified polystyrene has a net negative surface charge at normal pH. A coating of polyelectrolyte such as PDDA can reverse the net charge.23 This was achieved by adding 2.5 mL of 1 wt.% aqueous PDDA solution to the solution of 0.5 mL of 2.5 wt.% 200 nm Carboxylate-modified polystyrene in 3.0 mL DI H2O while stirring. The solution was stirred for 20 minutes and then centrifuged at 10,000 rcf for 45 min to remove excessive polymers. Centrifugation and re-dispersion were repeated

3



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2.25 mL of diluted TEOS solution was added. The solution was kept stirred and incubated for an addition of 12 hr at room temperature. 2.2.6. Etching the PS template away The solution was centrifuged at 3200 rcf for 45 min to remove excessive silica precursors and to terminate the

Figure 1. Schematic illustration of the formation of hollow silica

silanization process. The mixture was then rinsed at least

nano golf balls (HGBs) and Gold embedded hollow silica nano golf balls (Au@SiO2 HGBs). The template nanoparticle was as-

twice in anhydrous EtOH at 3200 rcf for 15 min, redis-

sembled using PDDA coating over 200 nm carboxylate function-

persed with 5.7 mL DMF to dissolve the PS template25,

alized polystyrene with 50 nm carboxylate functionalized polysty-

and incubated at 60 °C while stirring for at least 2 days.

rene. Then the template was either coated with silica directly or

The solution was then centrifuged at 8000 rcf for 30 min

attached to gold nanoparticles prior to coating with silica. Later

and redispersed in anhydrous EtOH three times. After the

the silica coated template was etched with DMF to remove the

last wash, the product was redispersed in 500 µL of anhy-

polystyrene template.

drous EtOH (3.6 mg mL-1).

2.2.4. The attachment of AuNPs

2.2.7. Loading of the quantum dot particles

2.5 mL of AuNPs solution was added to the 5 mL UV

Loading the carboxylate-modified quantum dots in

treated pollen solution. The sample was incubated and

Au@SiO2 HGBs was performed by redispersing 50 µL of

stirred at 60 °C overnight. The solution was then washed

Au@SiO2 HGBs in 900 µL of HCl solution of pH 4.0. Then

with DI H2O twice at 3200 rcf for 30 min to get rid of ex-

50 µL of quantum dots (QDs) was added. The solution

cessive AuNPs and was then redispersed in 5 mL of 80%

was tumbled overnight at ambient temperature. During

EtOH solution.

this time, the tube was covered with aluminum foil to

2.2.5. Growth of silica shell

prevent photo-bleaching. The solution was then rinsed and centrifuged at 10000 rcf for 45 min to remove exces-

5 mL of the above solution was mixed with a solution of

sive quantum dot particles. Centrifugation and redisper-

17.5 mL IPA, 4.5 mL DI H2O and 150 µL NH4OH. The

sion were repeated five times in H2O. The final product

mixed solution was stirred and sonicated such that all the

was redispersed in 1 mL of DI H2O.

template particles were well-dispersed. 10 µL of TEOS was diluted in 5 mL of anhydrous EtOH. 4.5 mL of the diluted

2.3. Characterization of Golf Ball

TEOS was then slowly added to the mixed solution at the

2.3.1. UV-vis Spectroscopy

rate of 0.2 mL per min by the syringe pump while stirring

The LSPR of metal nanoparticle is very sensitive and var-

vigorously. The solution was stirred and incubated for

ies with size, shape, surface functionality, and interaction

additional 20-24 hr at room temperature. Then the solu-

with molecules. Therefore, we analyzed the LSPR of

tion was rinsed by centrifuging at 3200 rcf for 45 min and

AuNPs, HGBs, and Au@SiO2 HGBs using TECAN infinite®

redispersed in 5 mL of 80% EtOH solution. A second layer

m200 pro in the 400-850 nm scanning range. For the

coating with silica shell was then performed. 5 mL of cen-

analysis, 150 µl of samples was put in 96 wells plate, and

trifuged solution was again mixed with 17.5 mL IPA, 4.5

absorption data was collected in the scanning range.

mL DI H2O and 150 µL NH4OH and then sonicated and

2.3.2. Scanning electron microscopy (SEM)

mixed on a vortex mixer to ensure redispersion. Lastly,

4


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The surface morphology of nanocarriers and the assembly

information about the stability of template. Here, the

of the template were examined by SEM. SEM analysis was

samples were diluted in DI H2O, and ζ potential meas-

performed by deposition of the 5 µl sample on the

urement was performed with a Zeta analyzer (Malvern

aluminum holder and drying of the sample in an inert

Instruments Zetasizer Nano ZS90.)

environment. Images were obtained using a FEI XL30

DLS analysis was also performed to analyze the hydrody-

SPEG UHR SEM.

namic size analysis of SiO2-HGB, Au@SiO2HGB, and QD

2.3.3. Transmission Electron Microscopy (TEM)

loaded HGB using DI water. The measurement was per-

We also observed the deposition of AuNPs on the silica

formed using a DLS analyzer (Malvern Instruments

surface by TEM. Samples were prepared by deposition of

Zetasizer Nano ZS90.).

20 µL of highly diluted samples in DI H2O on a TED

2.3.5. Cell culture and Particles incubation

PELLA Formvar/Carbon 200 mesh copper grid. The sam-

HeLa (ATCC® CCL-2™) were cultured in Dublecco’s Modi-

ples were air dried, and images were obtained using JEOL

fied Eagle Medium (DMEM; Gibco® Cat #11965-092) sup-

1200 EX II TEM.

plement with 10% fetal bovine serum (FPS; Gib co® Cat

2.3.4. Zeta (ζ) potential and DLS measurement

#10437-028) and 1% penicillin-streptomycin solution at 37

The higher surface zeta (ζ) potential colloidal solution

°C using a 5% CO2 incubator. The cells were grown on the

indicates, the higher stability. Therefore, analyzing ζ po-

35 mm glass bottom microwell dish (MatTek Corp.) in 2

tential during different steps of synthesis provides the

mL DMEM. Cells at 80% of confluence were incubated with Au@SiO2 HGBs or QD-attached Au@SiO2 HGBs

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Figure 2. SEM images of HGBs during the synthesis process: (A,B) The attachment of 50 nm carboxylated polystyrene and the PDDA coated 200 nm carboxylated polystyrene; (C,D) the coating of silica on the surface of template; (E,F) HGBs; (G,H) TEM images of HGBs. SEM images of Au@SiO2 HGBs during the synthesis process: (I) The attachment of AuNPs to pollen particles; (J) the coating of silica on the surface of Au attached template; (K) Au@SiO2 HGBs after the removal of the sacrificial core (See Figure S1A for overall view of HGB); (L) TEM images of Au@SiO2 HGBs (See Figure S1B for overall view of AuNP distribution on HGB surface). to a final concentration of 12.5 µg mL-1 for 2 hrs. After in-

was visualized using spectral 32-channel detectors in a

cubation, cells were washed twice with phosphate buffer

virtual filter mode (quantum dot: 500- 580 nm; Mito-

saline solution (PBS) to remove extra nanoparticles and

Tracker Deep Red FM: 620-700 nm). Confocal z-stack

resuspended in fresh DMEM.

images (1024 x 1024 pixels, 0.12 resolution, 0.375 um step

2.3.6. Fluorescent imaging

size) were processed using the NIS-Elements software (Nikon) and ImageJ.

Confocal fluorescence microscopy images were obtained with a Nikon A1R laser scanning confocal microscope sys-

2.3.7. NIR irradiation

tem (Nikon Instruments; Melville, NY) attached to an

The solution was spotted by dropping on the glass slide.

ECLIPSE Ti-E microscope equipped with an oil immersion

The glass slide was then irradiated using a 808 nm laser at

objective lens (40x, 1.3 NA, CFI Plan Fluor; Nikon). Quan-

500 mW for 5 min. Then the images of glass slides were

tum dots and MitoTracker Deep Red FM were excited at

taken with Seek™ Thermal Compact thermal imaging

404 nm and 641 nm, respectively. Fluorescence emission

cameras (Seek Thermal, Inc.) to record the heating area.

6


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ACS Applied Materials & Interfaces spectra were conducted by using a 50x water immersion objective and by applying 1 s of acquisition time and were recorded between 600 to 1800 cm-1 after 1 s of exposure. The 4-MBA was used as probe molecule of SERS capability due to its non-resonant electronic distinctive, and RBCs as an example of the biological importance of SERS. 2.3.10. AFM study of RBCs RBCs were incubated with Au@SiO2 HGBs for 30 min, then RBCs were collected by centrifugation at 10000 rpm for 20 min. The collected RCBs were fixed in 4% formaldehyde solution for 30 min and then were collected by centrifugation at 10000 rpm for 20 min. They were then

Figure 3. Hydrodynamic size analysis of SiO2-HGB,

dispersed in PBS and dropped onto glass slide pre-coated

Au@HGB, and QD-Au@SiO2HGB.

with poly-L-lysine. After 20 min the slide was washed

2.3.8. Photothermal therapy tested in vitro

with DI H2O and then dried under nitrogen flow. After air

Cells were incubated with the particles (final concentra-

drying, the samples were rinsed with DI H2O to remove

tion of 12.5 µg mL-1). Cells were washed with PBS twice and

incubated

with

fresh

LIVE/DEAD®

salt crystals and then air dried again before analysis. The

Viabil-

images of RBCs were obtained by using AFM probe with a

ity/Cytotoxicity Kit, for mammalian cells (ThermoFisher;

spring constant of 0.02 N m−1 (TR400PSA from Asylum

Cat #L3224) for 30 min in the dark. Cells were then irradi-

Research) in contact mode using a Dimension Hybrid

ated with 488 nm laser, 534 nm laser, or 808 nm laser.

XYZ scanner from Bruker. The images were obtained in

The images were acquired using fluorescence microscopy

air for all samples.

with live cells without fixing. Images were obtained using

3. Result and Discussion

Olympus IX71 with a Hamamatsu EM-CCD digital camera

3.1. Characterization of hollow nano golf ball nanocarriers

2.3.9. The surface-enhanced Raman scattering (SERS)

Hollow silica nano golf balls (HGBs) and gold embedded

Blood specimens were collected from healthy volunteers

hollow silica nano golf balls (Au@SiO2 HGBs) were syn-

by phlebotomy. RBCs were isolated from whole blood by

thesized by the method described in the “experimental”

dilution with 5 mL of PBS and centrifugation at 3500 rpm

section (Figure 1), and was verified by electron microsco-

and 4° C for 10 min. The precipitate was collected for fur-

py (SEM and TEM) imaging (Figure 2). The process initi-

ther analysis. For SERS experiments, RBCs were adhered

ated with the deposition of cationic polymer PDDA on

to poly-L-lysine-coated Silicon wafer immerse in PBS in a

the colloidal carboxylated polystyrene. The deposition of

petri dish. RBCs were incubated with Au@SiO2 HGBs (1.7

the polymer on the polystyrene is a well-defined

x10-11 M) after 30 min of incubations; RBCs were washed

process.21, 26 In order to create a positive charge surface,

with PBS and resuspended in PBS for SERS measure-

adsorption of the PDDA onto the negative surface of 200

ments. Raman and SERS spectra of RBCs and 4-

nm modified carboxylate PS templates was performed.

Mercaptobenzoic acid (4-MBA) were obtained using a

The PDDA was self-assembled onto carboxylated surface

LabRaman HR Micro-spectrometer (Horiba, Jobin-Ybon)

due to the electrostatic attraction as shown by the alter-

equipped with a CCD detector and a 633 nm laser. All

nation of ζ potentials after the polymer deposition (Table

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1). The smaller negatively charged carboxylated polystyrene spheres (50 nm) were then adsorbed to the polymerdeposited spheres (200 nm) to create a pollen structure. The negative charged 50 nm satellite repelled each other as shown in Figure 2A. Hence the space between the satellites was formed. The template was then treated under the ultraviolet lamp (254 nm wavelength; 15 W) for 30 min. The UV-treated PDDA-coated carboxylic cores showed lower positive Figure 4. (A) EDX spectrum of Au@SiO2 HGBs. (B) SEM

charge compared to the untreated PDDA-coated carbox-

image of Au@SiO2 HGBs and the corresponding elemental

ylic cores. The ζ measurement confirms the degradation

EDX mappings for (C) Si, (D) Au, and (E) Both elements

of the PDDA on the surface of the nanocarriers. As de-

overlay on the SEM image.

scribed in the previous study,24 the UV induced partial degradation of PDDA layer, and made silica shell formation possible. At higher ζ potential (after PDDA partial degradation), the rapid interaction between newly formed silica colloidal particles and the positively charged PDDA area could be delayed. This delay allowed the thin layer of silane polymerization on top of the

PDDA as shown in

Table 1. ζ potential of samples performed in DI H2O

Figure 5. UV-vis spectra of HGBs (solid line), AuNPs pre-

at 25 ⁰C

pared by THPC method (dashed line), and Au@SiO2 HGBs (dotted line) Sample

ζ Potential (mV)

Carboxylated Polystyrene (PSC)

-64.63 ± 2.41

Carboxylated Polystyrene coated

38.15 ± 1.95

Figure 2C. As shown in the SEM image (Figure 2F), the electron beam was able to pass through the pores in the particle shell after the PS was etched away. The inner void

with PDDA (PSC+PDDA) UV-Treated Carboxylated Polysty-

space of the hollow golf balls was confirmed by examining 20.25 ± 1.95

the TEM images. As shown in Figure 2E, G, the nano-

rene coated with PDDA

carriers are hollow and porous. This dual porosity proper-

(PSC+PDDA+UV)

ty can be used for encapsulating theranostic agents, such

Hollowed Silica Nanoparticle

-32.53 ± 4.18

as small molecules or antibodies that can diffuse in and

Au@SiO2 Hollowed Silica Nanopar-

-26.70 ± 2.68

out through the pores of the nanocarriers freely15 or in a gated fashion.27

ticle Solid Silica Nanoparticle

Next, the template process was modified by attaching

-51.86 ± 4.21

AuNPs on the surface of the template prior to TEOS polymerization. Results of this upgraded LBL process are shown in Figure 2. AuNPs prepared by THPC reducing agent exhibit a negative ζ potential.28 The colloidal AuNPs

8


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were then attached to the surface of the PDDA-coated

nanoparticles were observed to be embedded in the silica

200 nm PS, but not on the 50 nm carboxylated PS satel-

coating layer.

lites as observed in Figure 2I. Specifically, AuNPs were

DLS analysis of Hollow silica nano golf ball (SiO2-HGB),

found only on the free space on PDDA-coated cores,

gold embedded Hollowed Silica (Au@HGB), and QD

while sparing the PS satellites. The attachment of AuNPs

loaded Au@HGB was performed (Figure 3). Hydrodynam-

onto the PDDA-coated layer was achieved via electrostat-

ic size analysis showed average sizes of 250 nm, 341 nm,

ic attraction

20

between AuNPs and PDDA-coated layer of

and 358 nm for SiO2-HGB, Au@HGB, and QD loaded

the PS core. Due to their negative surface charge, the 50

Au@HGB, respectively, with negligible amount of aggre-

nm satellites did not show any adsorption of the (negative

gates. The larger hydrodynamic size, compared to the size

surface charged) AuNPs. Thus, selective adsorption of

estimated from SEM and TEM analyses, clearly indicates the hydrophilic nature of the particle that leads to the formation of a static aqueous layer around the particle, the Brownian movement, and shows larger hydrodynamic size. 3.2. Characterization of the deposited gold Energy-dispersive X-ray spectroscopy (EDX) was used to verify the nature of the gold and silica within the sample. As shown in Figure 4A, the majority of the spectrum is

Figure 6. Optical microscopy (bright field) images of RBCs

dominated by the aluminum. This is due to the fact that

cultured with QDs-loaded Au@SiO2 HGBs (A) with objective

the sample was placed on top of aluminum foil protected

20×; (C) with objective 40×; and the fluorescence images of RBCs (B) with objective 20×; (D) with objective 40×; QDs

SEM pins. A detailed analysis of the EDX spectra in Figure

sample was excited at 480 nm, and the emitted light was

4A shows both the Si and Au peaks. The elemental map-

collected at 520 nm. AFM images of RBCs (control) not treat-

ping results for both Si and Au supported the similar dis-

ed with Au@SiO2 HGBs (E, F, G); and the AFM images of

tribution of the two elements within the nanocarriers

RBCs treated with (H, I, J).

(Figure 4E).

AuNPs to the PS cores was accomplished. Silica coating of

The UV-vis spectra were recorded for the AuNPs, HGBs,

the template was then performed, and only the PDDA

and Au@SiO2 HGBs for comparison. As shown in Figure

core part was coated with polymerized silane (Figure 2J.

5, the analysis of the UV absorbance curve show the peak

After etching away the PS template, the gold embedded

absorbance of Au@SiO2 at ~540 nm, similar to normal

hollow silica golf ball (Au@SiO2 HGBs) nanocarriers were

spherical gold nanoparticles.7, 29-30 The UV-vis absorbance

obtained. The modified LBL process produced monodis-

of Au@SiO2 HGBs is enhanced at the wavelength ~700

persed nanocarriers as shown in Figures 2K, 2L. The

nm - 850 nm compared to the wavelengths of HGBs and

bright spots inside the silica shells as shown in Figure 2

AuNPs. Compared with the AuNPs prepared by the cit-

are AuNPs. The void spaces inside the Au@SiO2 HGBs

rate method,31 AuNPs prepared by THPC method were of

were further confirmed by TEM imaging as shown in Fig-

smaller size. It is worth mentioning that the peak absorb-

ures 2K (See Supporting Information Figure S1A for over-

ance of the particles may vary according to the pH of the

all view of HGB). In Figure 2L (See Supporting Infor-

solution. Specifically, the plasmon resonance was sup-

mation Figure S1B for overall view of Au@SiO). The gold

9



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Page 10 of 17

caused by the finite-size effect of metal dimensions.28

pressed with a decrease of the solution pH, which was

Figure 7. (A) Raman spectra of 4-MBA alone (black line) and in the presence on Au@SiO2 HGBs (green line) resulting in an enhanced intensity demonstrating SERS capability on non-resonant molecules. (B) Raman spectra of RBCs alone (black line) and in the presence of Au@SiO2 HGBs (red line). Both spectra show typical Raman peaks of RBCs components, but SERS allow us to analyze membrane components. All Raman spectra were acquired using a 633 nm laser and a 50x objective.

On the contrary, when the pH of the solution was in-

constraints. Second, the interaction of nanoparticles with

creased, the AuNPs prepared by using THPC showed ab-

red blood cells (RBCs) has been of major concern in terms

sorption peak similar to one of spherical gold nanoparti-

of cytotoxicity and biocompatibility.1 When nanoparticles

cles in their UV spectrum.

enter into the biological fluid, the first cell they would

However, even at pH 6.5 in DI H2O, Au@SiO2 HGBs

interact with is most likely red blood cells (RBCs). RBC-

shows only an absorbance peak at 540 nm and does not

loaded nanoshuttles would be less likely to be affected by

demonstrate any plasmon resonance suppression as

the immune system, will be less toxic and more biocom-

shown by the THPC-method AuNPs at the same pH con-

patible. Previous works have demonstrated that mesopo-

dition. Tentatively, this phenomenon could be explained

rous silica nanoparticles with sizes around 100 nm are

by 1) a lack of solution mediated effects since AuNPs were

taken by RBCs without disturbing their biophysical prop-

encapsulated in the silica shells and/or 2) the hollow

erties.2 And finally, we explored the possibility that the

holes are confined so that the distance between encapsu-

metallic nature (gold embedded) of the Au@SiO2 HGBs

lated AuNPs could be reduced; thus causing the red shift

present the surface enhancement Raman spectroscopy

and the enhanced absorption in the NIR region.

(SERS) effect and hence these nanoshuttle-loaded RBCs could be used for bioanalytical sensing. To better under-

3.3. Red blood cell uptake and SERS capability

stand the bio-distribution of nanocarrier materials within

The reasons for selecting nanoshuttle encapsulation with-

a cell, Au@SiO2 HGBs was loaded with QDs and then co-

in RBC are many folds: first to test if they these nanoshut-

cultured with RBCs. The RBCs were then imaged in mul-

tles would be endocytosed by circulating cells. If they

tiple ways to ascertain the distribution of Au@SiO2 HGBs.

could be loaded in RBCs, their transport in the circulatory

We observed the uptake of Au@SiO2 HGBs into RBCs

system would be less likely to be impeded by viscoelastic

using fluorescence microscopy. From the microscopy re-

10


Page 11 of 17

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sults (Figure 6A, B, C & D), fluorescence signal was visible

agent while they could also achieve targeted delivery

in the RBCs after co-culture for 30 minutes, which indi-

though blood vessels.

cated high uptake of Au@SiO2 HGBs by RBCs. Some RBCs

One interesting application of multifunctional nanostruc-

was brighter than others, which might be due to the

tures is the molecular sensing based on SERS. The SERS

Au@SiO2 HGBs uptake in different RBCs, and Au@SiO2

activity of Au@SiO2 HGBs nanocarriers was tested using

HGBs distributed in a different part of RCBs.

4-MBA and RBCs as examples of non-resonant excitation

It is important to know the distribution of Au@SiO2 HGBs

and biological probe molecules, respectively. Figure 7

on the surface as well as inside of RBCs. For this purpose,

shows both the Raman and SERS spectra of 4-MBA and

we captured AFM images of control RBCs and RBCs with

RBCs. Both 4-MBA spectra exhibit typical Raman peaks

QDs-loaded Au@SiO2 HGBs. AFM enables study of the

located at 633, 801, 810, 1100, 1183, 1293 and 1595 cm-1 (dot-

uptake of the Au@SiO2 HGBs inside of RBCs and the de-

ted lines). When tested in the presence of Au@SiO2

tailed morphology changes of RBCs. The high resolution

HGBs, 4-MBA and RBCs display an increased intensity of

AFM imaging allowed observing the nanoparticles inside

the Raman signal and the presence of new peaks at 1137,

RBCs. The typical AFM images of the untreated RBCs are

1624 and 1654 cm-1 (black arrows) relative to the SERS

presented in Figure 6E, F, G. The biconcave shape and

effect. SERS signal can be discriminated from Raman

relatively smooth surface are observed. There are no par-

(Figure 7A), where EFs have shown to be on the order of

ticles inside the cells, and the diameter of the original

104/105. Raman and SERS analysis of RBCs show several

RBCs is ~6.6 µm. After treatment with Au@SiO2 HGBs,

characteristic porphyrin bands (660 cm-1 and 1446 cm-1),

the morphology of RBCs changed as shown in the AFM

amino acids (715 cm-1 and 753 cm-1) and a number of other

images (Figure 6H, I, J). Morphology of the treated RBCs

typical peaks34 (Figure 7B, typical peaks marked with dot-

was flatter than the morphology of the original RBCs. The

ted lines). Excitation of SERS in RBCs in the presence of

biconcave shape nearly disappeared and the RBCs were

Au@SiO2 HGBs reveals a significant enhancement of the

flatter.

Additionally, the diameter of the RBCs after

Raman Effect. In RBCs SERS, we observed the membrane

treatment increased to about 8.1 µm. Furthermore, small

specific spectral markers (e.g., 1576 cm-1 with EF of ~104

nanoparticles in the RBCs, under the membrane and in-

and 1072 cm-1), which are normally masked by hemoglo-

side of the RBCs are present. The Au@SiO2 HGBs uptake

bin signals. This finding indicates that SERS could be very

in RBCs and subsequent changes in their morphology

useful for RBCs membrane analysis.35

were observed. We did not observe any rupturing of RBCs

3.4. NIR irradiation

and possible potential cytotoxicity of Au@SiO2 HGBs after

Even though the spherical AuNPs, unlike gold nanorods

30 mins of co-culturing of Au@SiO2 HGB with RBCs. We

do not share the plasmon resonance characteristic in

did not examine the long-term toxicity of Au@SiO2 HGBs

NIR,29-30 when spherical gold nanoparticles are close to-

in RBC or in other type of cells. The AFM images are con-

gether to form nanoaggregates, they enhanced NIR ab-

sistent with the fluorescence images of RBCs, supporting

sorbance.10 Thus, the gold clusters with an inter-particle

the conclusion that Au@SiO2 HGBs could get into the

distance of 11.8 nm can be heated up 7°C with NIR laser.36

RBCs as well as distribute inside the RBCs membrane.

TEM image (Figure 3E) show that the Au@SiO2 HGBs

Through this mechanism, Au@SiO2 HGBs could serve as a

were deposited with many aggregated AuNPs. Occasion-

shuttle for drug delivery or as a diagnosing or imaging

ally, AuNPs can be seeded close together within the SiO2 shell. As shown in Figures 8C & 8D, when irradiated with

11



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Page 12 of 17

an NIR laser (808 nm, spot size 5 mm) at 2 W/cm2, both

Deep Red FM; and (C) Orthogonal views of single HeLa cell

dried AuNPs and Au@SiO2 HGBs nanocarriers can be

with QDs-loaded Au@SiO2 HGBs.

heated up from 21.7°C (71°F) to 25 °C (77°F).

previously reported

3.5. Cellular Uptake of Au@SiO2 HGBs into HeLa cells

particles in RBCs begins within 30 min and reaches the

Au@SiO2 HGBs were incubated with HeLa cell and cul-

alloyed carboxylated quantum dots (ߣ௘௠ , 540 nm) were

tured for 2 hr. Unlike sub 100 nm nanoparticles, which

attached to the Au@SiO2 HGBs; the quantum dots emit-

can be internalized via energy independent endocytosis

ted green fluorescence when excited. This allowed trackthe

Au@SiO2

HGBs

in

HeLa

cells.

It

that the uptake of the silica nano-

uptake saturation after 2 hr. Thus, the QD-attached

In order to obatain fluorescence signals, 6 nm CdSeS/ZnS

ing

37

pathways, the 200 nm Au@SiO2 HGBs can be internalized

was

via energy dependent endocytosis18 such as the clathrin38 or caveolin39 dependent pathways. A detailed analysis of the fluorescent images supports the internalization of the nano-carriers with green fluorescent QD by the cells stained with a red-fluorescent dye (Figure 9). ~72.96±4.19 % of HeLa cells had QD-attached Au@SiO2 HGBs. The efficiency of the cellular uptake of ~72.96±4.19 % was obtained by the number of cells with QD/ all cells.

®

Figure 8. Infrared Images taken with Thermal Seeker devic-

Confocal imaging was then performed to collect the z-

es: (A) coverslip; (B) AuNPs; (C) HGBs; (D) Au@SiO2 HGBs.

stack data for individual cells to confirm the internaliza-

The setting allows the device to capture the highest and low-

tion of the QDs-loaded Au@SiO2 HGBs in HeLa cell as

est temperature region in the image. (E) The photographic

shown in the Figure 9C.

image of the equipment set up, the blue dotted box represent

3.6. Photothermal Treatment

the location of the cover slide in the image, while the red dotted box is the actual location of the cover slide mounted

The UV-vis spectra absorption of the Au@SiO2 HGBs dis-

on the platform.

played peak absorbance similar to normal spherical AuNPs (540 nm) with the enhanced absorption in the NIR region. Based on the experimental results of the NIR irradiation, as a proof-of-concept, we then evaluated the feasibility of using these nanoparticles as photothermal tumor-ablation agents. Three kinds of lasers, 488 nm (blue laser), 534 nm (green laser), and 808 nm (IR laser), were used to heat the nanocarriers in vitro. The cell viability was evaluated by a fluorescent assay, with live cells labelled with green fluorophores and dead cells labelled with red fluorophores. Figure 10B shows that the cell viability was unaffected by the internalization of Au@SiO2 HGBs. The cells alone were also irradiated by different

Figure 9. (A) HeLa cells stained with MitoTracker® Deep Red

lasers (Figure 10C, E, G), which also did not affect the via-

FM (Thermofisher); (B) HeLa cells incubated 2 hours with

bility of the cells compared with the control.

QDs-loaded Au@SiO2 HGBs then stained with MitoTracker®

12


Page 13 of 17

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Figure 10. Fluorescent images of HeLa cells stained with LIVE/DEAD® Viability/Cytotoxicity Kit (Green = live; Red = dead). (A) -1

Cells alone, (B) Cells incubated Au@SiO2 HGBs (final concentration at 12.5 µg mL ) without any laser irradiation, (C) Cells alone irradiated with a blue laser (488 nm), (D) Cells incubated with Au@SiO2 HGBs and irradiated with a blue laser, (E) Cells alone irradiated with a green laser (534 nm), (F) Cells incubated with Au@SiO2 HGBs and irradiated with a green laser, (G) Cells alone irradiated with an IR laser (808 nm), and (H) Cells incubated with Au@SiO2 HGBs and irradiated with an IR laser. The cells significantly killed by the different lasers in the presence of nanoparticles. The scale bar is 100 µm all images.

When we combined both the irradiation and the incuba-

In this study, two variant of hollow golf ball like silica

tion of Au@SiO2 HGBs, a significant decrease in cell via-

nanostructure (HGB) with spatially oriented nanopore

bility was observed. This is shown as an increase of red

were synthesized. The homogeneous hierarchical tem-

stained cells in fluorescent images (Figure 10D, F and H).

plate particles were prepared via physically absorbing

Using a temperature sensor probe, we measured the

smaller negatively charged PS spheres onto the surface of

change in temperature of each solution for lasers of dif-

larger

ferent wavelengths. The green laser exhibited slight heat-

spheres. In order to acquire the photo-thermal potential,

ing to 43 °C in both samples with and without Au@SiO2

negatively charged gold nanoparticles attached to the

HGBs; the blue laser exhibited heating to 45 °C in both

positive charge surface of PS template. For silica hollow

samples with and without Au@SiO2 HGBs, and IR laser

golf ball synthesis, this optional step was skipped. The

exhibited heating to 43 °C only with the Au@SiO2 HGBs.

TEOS was used to grow a silica shell over a template con-

The sample without Au@SiO2 HGBs exhibited no change

taining AuNPs. Porous hybrid/silica hollow golf balls were

in temperature when irradiated with the IR laser. The

made by dissolving the PS template using DMF. The elec-

increase of temperature of solution without nanocarriers

tron microscopic (SEM and TEM) analysis clearly showed

can be attributed to the fact that at lower spectrum, the

the absorption of gold nanoparticle on the surface with

cells themselves would absorb some laser energy.

spatially oriented nanopores on the silica surface. The

4. Conclusion

surface plasmon resonance of Au@SiO2 HGB in different

PDDA

functionalized

positively

charged

PS

pH levels showed no shift while free AuNP showed SPR

13



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Page 14 of 17

shift in similar condition; clearly showed the stability of

Author Contributions

AuNP on the silica surface. In addition, ζ potential meas-

W.J., L.W., K.S.G., and N.P. designed the experiment. W.J.,

urements of the intermediates were performed to study

L.W., K.S.G., P.B.L., N.P., M.J., A.Y. and S.K. performed the

the mechanism of particle adsorption within the synthesis

experiments. W.J., L.W., K.S.G., P.L.S., and G.J. analyzed the

process. Further, the potential of Au@SiO2 HGB in photo-

data. W.J., L.W., K.S.G., D.K.B, F.Z. and R.L. wrote the paper.

thermal therapy was analyzed using HeLa cells. The fluo-

Funding Sources

rescent quantum dot was loaded in the Au@SiO2 HGB for

This project was supported in part by the National Institute

imaging distribution of nanostructure in HeLa cells. Us-

on Aging of National Institutes of Health (Grant AG028709).

ing preloaded quantum dot nanocarriers as imaging

K.S.G thanks the FUMEC and AMC for funds to support the

agents, Au@SiO2 HGBs was shown to enter HeLa cells, as

2016 summer research yield at the University of California in

well as to carry payloads during the process. The endocy-

San Diego.

tosis of those novel nanocarriers has not been specified

ACKNOWLEDGMENT

experimentally. Further investigation of the scalability of

The authors acknowledge and appreciate Dr. Preston P. Landon

the nano golf balls from different PS template size is cur-

and Dr. Chen Zhang for scientific discussion and guidance.

rently underway. We also investigated the QDs-loaded nanocarriers uptake by red blood cells; fluorescence and

SUPPORTING INFORMATION

AFM images indicate that Au@SiO2 HGBs enter into the

Transmission electron microscopy images of silica nano

red blood cells. We also observed the nanocarriers under

golf balls and AnNP embedded silica nano golf balls.

the red blood cells membrane. We have reported the SERS spectra of 4-MBA and RBCs separately in the presence of the Au@SiO2 HGBs. The results support the

REFERENCES

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AUTHOR INFORMATION Corresponding Author *Correspondence and requests for materials should be addressed to R.L. ([email protected] ), Tel +1 (858) 822-0384. Fax: +1 (858) 534-5722.

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Gold-Embedded Hollow Silica Nanogolf Balls for Imaging and

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