testing in-vitro biocompatibility of sinp chips

8/10/2019 Testing in-Vitro Biocompatibility of SiNP Chips

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Testing in-vitro Biocompatibility Of SiNP

chips

Siddharth Chadaram, Binata Joddar, Yoshihiro ItoNano Medical Engineering Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama, Japan.

[email protected],[email protected]

1

I

NTRODUCTION

Nanotechnology has received an increased attention in the biological research field. The important

examples are: the usage of nanoparticles in optical and magnetic resonance imaging, the demonstration

of potential application of metal nanoshells and carbon nanotubes for the treatment of tumor and

cancer cells, and the application of nanowire-based transistors to electrically detect specificbiomolecules. In all of these cases, the nano-materials function either inside the cells or at the vicinity of

the surface of biomolecules. Direct interconnection of the cells to the external world by interfacing

nano-materials may afford great opportunities to probe and manipulate biological processes occurring

inside the cells, across the membranes, and between neighboring cells. A nanoscale material with high

aspect ratio is good candidate for this application. For instance, silicon nanowires (SiNWs, d = 1-100 nm)

are a few orders of magnitude smaller in diameter than mammalian cells (dcell~ on the order of 10 m)

yet comparable to the sizes of various intracellular biomolecules. The nanowires have high aspect ratio

( < 103) and yet are sufficiently rigid to be mechanically manipulated. The nanometer scale diameter and

the high aspect ratio of SiNWs make them readily accesible to the interiors of living cells, which may

facilitate the study of the complex regulatory and signaling patterns at the molecular level[1].

In this study, we used SiNWs as a surface for HeLa cell culture. We coated the SiNWs with a biomaterial

dopamine, to further facilitate the immobilization of HeLa cells. Biomaterials are intended to respond

to and interact with biological systems in many ways. Whether used for tissue engineering or drug

delivery, biomaterials should support a specific function of the target tissue to help regenerate and

replace damaged and diseased tissue. To achieve these functions, it is often necessary to modify the

surfaces of existing materials to control cell adhesion and growth. Inspired by the mussel plaque protein,

dopamine, which can be easily deposited on to metals, is now commonly used for surface modification

of other biomaterials. Dopamine adsorption not only provides an organic layer, but also facilitates

further immobilization of biological molecules on the surface of various materials. It is also relatively

non-toxic in vivo[2].

The aim of this experiment was to test cell adhesion on dopamine-coated or uncoated Si and SiNP

surfaces and to identify the best conditions for cell adhesion on these substrates respectively.
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M

ATERIALS AND METHODS

2 1 DOPAMINE COATING

The surfaces of the pre-cleaned silicon discs (962mm2) were subjected to dopamine treatment. The discs

were placed in a flask containing 2 mg ml-1 of dopamine solution in water (acidic, pH 4.5) for 24h at room

temperature. The dopamine-treated Si discs were sonicated in Milli-Q water (3 x 10 min) at room

temperature. After three cycles of washing, these discs were left to dry under vacuum conditions for

24h before further use.

2 2 P

REPARATION OF CELL CULTURE MEDIA

The cell culture medium was prepared by mixing 500 mL of pre-warmed D-MEM Solution (High Glucose;

with L-Glutamine and Phenol Red), 50mL 10%FBS and 500L of 1%Penicillin-Streptomycin Solution.

2 3 H

E

L

A CELL CULTURE

A medium for the culture of HeLa cells was prepared as described earlier[2.2].Appropriate volume of

the cell suspension and media was added to each well containing the silicon discs. The plates were

covered with aluminium foil and placed in the incubator at 37C, 5% CO2 for 24h. The cells were stained

with a live cell stain - Propidium Iodide, after which the cells appeared green under fluorescence.


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Fig. 1.

Labeled picture of the setup used

where

1. Plain (coat.) = Plain silicon

disc, coated with dopami

2. SiNP (coat.) = Silicon disc

(with nano-projections on

surface), coated with

dopamine.

3. Plain (unc.) = Plain silicon

disc, uncoated.

4. SiNP (unc.) = Silicon disc

(with nano-projections on

surface), uncoated.


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Fig. 2.

Visualization of HeLa cells on various types of Si surfaces throughfluorescent microscopy:

A. HeLa cells on dopamine-coated Si surface.

B. HeLa cells on dopamine-coated SiNP surface.

C. HeLa cells on uncoated Si surface.

D. HeLa cells on uncoated SiNP surface.

E. HeLa cells in the control well.


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3

R

ESULTS

It was found out using fluorescent microscopy that only few viable cells were retained on to the silicon

surfaces.

The following observations were made:

More cells were retained on Plain (coat)surface than on SiNP (coat.) surface.

More cells were retained on SiNP (unc.)surface than on Plain (unc.)surface.

We inferred that cell adhesion was more effective on uncoated SiNP surfaces than on dopamine-

coated SiNP surfaces.

4

D

ISCUSSION

In this study, we utilized the dopamine-coated Si substrate with a vertically aligned SiNW array on it, as a

platform for growing HeLa cells. Dopamine was deposited on to the surfaces as a monolayer (pH 4.5),

following established procedures.

According to the above chart, it can be seen that more number of cells have been retained on uncoated

SiNP surface than on the dopamine-coated SiNP surface. In case of uncoated SiNP surface, the cells may

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Plain coated Si Plain uncoated Si SiNP coated SiNP uncoated Control Wells

Cell Adhesion

Samples

Numberof

cells(x105)


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have chosen to attach between the deep spaces or crevices between the nano-pillars (Fig. 3.). In case of

the coated SiNP surface, although dopamine is a good source for cell immobilization, it may have filled in

the spaces between the nano-pillars, rendering the top surface too rough for cell adhesion (Fig. 4.).

5

C

ONCLUSION

A

ND

F

UTURE

W

ORK

In conclusion, we have shown that uncoated SiNP chips are more susceptible for cell-adhesion than the

dopamine-coated SiNP chips.

We are yet to perform further studies on

The alignment of nano-particles on Si surface as there might be a chance of better cell adhesion

on SiNP with varied pillar lengths or spacing.

Decreasing the time for cell adhesion for better insight (as it is possible that the time providedfor cell adhesion (24h) was too long).

Fig. 3.

Fig. 4


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R

EFERENCES

[1] Kim W, Ng JK, Kunitake ME, Conklin BR, Yang P.Interfacing Silicon Nanowires with Mammalian Cells.

J Am ChemSoc.2007Jun 13;129(23):7228-9.

[2] Joddar B, Albayrak A, Kang J, Nishihara M, Abe H, Ito Y. Sustained delivery of siRNA from dopamine-coated stainless steel surfaces.Acta Biomater.2013 May;9(5):6753-61.

testing in-vitro biocompatibility of sinp chips

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