# Nanomaterials Enhanced Gene Expression in Yeast Cells

Authors: Su-Fang Chien; Shi-Hui Chen; Chhiu-Tsu Lin
Journal: Journal of Nanomaterials
 (2008-01-22)

---

## Abstract

Metal nanomaterials are shown to enhance gene expression for riceα-galactosidase gene (α-Gal) in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica-Au and silica-Ag, were prepared and characterized by UV-vis spectroscopy and TEM technique. The rice α-galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p-nitrophenyl-α-D-galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in α-galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS-gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice α-galactosidase gene in yeast cells are discussed.


---

## Body

## 1. Introduction

The size and surface area of the nanoparticles
together with their available functional groups and charges are crucial factors
in targeting and the attachment of cell-specific ligands that can lead to an
increased selectivity in delivery and expression of genes. Gold and silica
nanoparticles have been employed to investigate gene expression from the
unamplified total human RNA [1]
and in vivo in the brain [2]. It has been known for a long time that the Indian eats silver powder and
the Chinese mixes gold thin films in food. Gold nanoparticles were loaded and modified with oligonucleotide and employed as the intracellular gene regulation agents for controlling protein expression in cells [3]. With the aid of gold nanoparticles, these
intracellular gene regulating agents exhibit more than 99% cellular uptake and
can introduce oligonucleotides at a higher effective concentration than the
conventional transfection agents [3]. By employing surface plasmon resonance
spectra of Ag nanoparticles as the nanometer-size index probes, the real-time
probing and imaging of membrane transport in living microbial cells have been
demonstrated [4]. Moreover, the application of organically modified silica nanoparticles
as a nonviral vector for efficient in
vivo gene delivery has been communicated [2]. The in vivo gene delivery is an area of
current research, where genetic materials (e.g., DNA, RNA, and
oligonucleotides) could be used to inhibit undesirable gene expression or to
synthesize therapeutic proteins [5, 6].The riceα-galactosidase gene was isolated from
the stem portion of taro (Colocasia
esculenta) that has been demonstrated to have the capability of converting
group B into group O red blood cells [7]. In this paper, we describe the use of
oligonucleotide-loaded nanoparticles to enhance the expression of rice α-galactosidase
gene in yeast cells. Au, Ag, silica-Au, and silica-Ag nanoparticles were synthesized
and charaterized. The TEM images of nanomaterials transfected inside the cell
membrane will be illustrated. The results will be used to address the following
questions. Will the nanomaterials be toxic and kill micro-organisms, or be
activated to enhance gene expression? Will these α-galactosidase activities be highly selective
and specific, and also depend on the nature and type of nanomaterials? If the
gene expression is enhanced, will the protein expression be limited only to the
intracellular activity or included also the secreted activity into medium? If
the secreted activity is highly specific, will the protein expressed be a pure
form and no further protein purification should be required?


## 2. Experimental

### 2.1. Materials

Tetraethyl orthosilicate (TEOS), hydrogen tetrachloroaurate
(III) trihydrate (HAuCl4·3H2O),
silver nitrate (AgNO3), sodium citrate, and ammonium hydroxide
solution (NH4OH) were purchased from Sigma-Aldrich (St. Louis, MO 63195, USA) and used as received. Ethyl alcohol, 200
proof, was obtained from Pharmco (Shelbyville, KY 40065, USA).
The water employed in all preparations was purified by a Milli-Q system (Millipore) (Bedford, MA, USA). Rice
α-galactosidase gene was cloned from cDNA library. The yeast strain, Pichia pastoris SMD1168, and pPIC-9k
plasmid were obtained from Invitrogen (Carlsbad, Calif, USA). The synthetic pNP-substrate,
p-nitrophenyl-α-D-galactopyranoside were obtained from Sigma-Aldrich. Other
materials for cell cultivation and cloning, such as agar plate, YPD plate,
yeast extract, lactose, sodium carbonate, LB medium, SMGY medium, YNB medium, Eppendorf
tube, and Pyrex Petri dishes were used from a conventional laboratory stock.


### 2.2. Preparation and Characterization of Nanomaterials

Silica colloidal particles were processed by following the method of Stöber et al. [8]. Briefly,
it involved hydrolysis and successive condensation of tetraethylorthosilicate
(TEOS) in ethanol/water mixture with ammonium hydroxide as a catalysis. In a
typical formulation, a mixture of 0.6 mL of TEOS and 10 mL ethanol was
sonicated for 30 minutes. While stirring the sol-gel precursor solution, a 0.1 mL of 3% NH4OH solution was added dropwise. The silica colloidal
solution turned turbidly white and allowed to stir for 3-4 hours for the reaction
to be completed. The silica product solution was centrifuged at 15000 rpm for
30 minutes. The SiO2 precipitates were collected, washed with water twice,
and dried at 37°C. The silica nanoparticles were redispersed in
water to form a 1 g/L colloidal solution for further use.The aqueous colloidal solution of Au and
Ag, individually, was prepared following a chemical reduction method employed by
Turkevich et al. [9] and Fu et al. [10]. First, an aqueous solution of 2 mM for
each metal salt, HAuCl4, and AgNO3 was prepared. Second,
to synthesize Au colloidal solution, a volume of 25 mL HAuCl4 solution was heated to boil and stirred vigorously, and 2.5 mL of freshly
prepared sodium citrate solution (38.8 mM in ethanol) were added dropwise until
a wine-red color was observed. A similar procedure was used also for preparing
Ag colloidal solution.The composite nanomaterials, silica-Au and
silica-Ag, were prepared by the procedure described previously [10, 11]. For a nanoparticle,
silica-Ag as an example, a volume of 25 mL of 2 mM AgNO3 solution
and 0.2 mL of 1 g/L SiO2 colloidal solution (as prepared above) was heated
to boil and stirred vigorously, and 2.5 mL of freshly prepared sodium citrate
solution (38.8 mM, in ethanol) were slowly added. Initially, a white/turbid
solution of silica particles with silver nitrate turned pale yellow and after reacting
for 30 minutes, the color of the solution turned golden yellow indicating the
formation of silica-Ag nanocomposites. It is important to ensure that the
silver and gold ions have been reduced completely by adding an excess amount of
reducing agents. Moreover, the nanoparticles were normally centrifuged, washed,
and resuspended in solution before use, in order to minimize the alcohol cosolvent
and a possible existence of the trace amount of metal ions in solution. The
size, shape, and distribution of the synthesized nanomaterials and also the α-Gal/nanomaterial
complexes were characterized by a TEM (Jeol JSM-1200 Ex II, operated at 80 kV) and
a UV-vis spectrophotometer (Hitachi U-2000).


### 2.3. Gene Cloning and Gene Expression in Yeast Cells

Riceα-galactosidase
gene was cloned in pPIC-9k plasmid (Invitrogen, Calif, USA) and transformed
into SMD 1168 yeast strain chromosomal DNA by electroporation according to the
Invitrogen protocol described in EasySelect (http://www.Invitrogen.com)
and given by Higgins and Cregg [12]. The gene expression was done also by
following the Invitrogen EasySelect procedure. The cloned SMD 1168-α-Gal
yeast cells were cultured overnight from a single colony on YPD plate in 2 mL
SMGY medium. The cells were harvested and the induction was carried out in 2 mL
of a YNB medium containing amino acids, 2% of glycerol as carbon source, and
30% of the individual testing nanomaterials. For the preparation of control
sample, the solution of nanomaterials dispersed in water was replaced by the
same volume amount of pure water that was then used to carry out the control
experiment for each type of nanomaterials tested [7, 13]. Finally, the
expressed cells were induced by adding 1% of methanol everyday. The cell mass
was measured spectrophotometrically by recording its optical density at 600 nm (OD600).


### 2.4. Determination of Enzyme Activity and  Enzyme Assay

After each period of induction in the gene expression (24 hours, 48 hours, and up to
8 days), the enzyme activity was determined (in duplicate) by a syntheticpNP-substrate, p-nitrophenyl-α-D-galactopyranoside.
Fifty μL of the induced yeast cells and 30 μL of 4 mM pNP-substrate were pipetted into an Eppendorf tube and incubated at
37°C for 10 minutes. After incubation, a 1.0 mL of 0.2 M Na2CO3,
pH 9.8, was added to terminate the reaction. The final reaction mixture was
then centrifuged at 8000 rpm for 15 minutes (as shown in the right test tube of
Figure 1(a); the left test tube is the control), and the resulting clear yellow
supernatant (i.e., the released p-nitrophenol, pNP, representing the secreted
activity) was read in a spectrophotometer at 400 nm (as shown in Figure 1(b), a
UV-visible spectrum from 300 nm to 600 nm) [7]. The intracellular enzyme
activity was obtained by subtracting the secreted activity from the total
activity. One unit of enzyme activity is defined as the amount of enzyme that
can produce 1 μmol of pNP/min at 37°C.(a) Secreted recombinantα-galactosidase activity; see text for the details, and (b) OD at 400 nm of the secreted α-galactosidase
supernatant, representing the production of p-nitrophenol.
(a)(b)The secreted proteins  were analyzed by SDS-Polyacrylamide Gel Electrophoresis (PAGE) [14]. The SDS gel, containing
13.5% polyacrylamide with a 0.75 mm thickness, was prepared. After loading the samples,
electrophoresis was carried out with 110 V and 20 mA in a continuousbuffer
system for 2 hours. The gel was stained with Coomassie
Brilliant Blue R-250 and
destained until the background was clear. The question of a possible cell death
during induction period, as may be caused by the nanomaterials toxicity, was
monitored by cells staining with Trypan Blue [15] (4% solution from Sigma
Adrich).


## 2.1. Materials

Tetraethyl orthosilicate (TEOS), hydrogen tetrachloroaurate
(III) trihydrate (HAuCl4·3H2O),
silver nitrate (AgNO3), sodium citrate, and ammonium hydroxide
solution (NH4OH) were purchased from Sigma-Aldrich (St. Louis, MO 63195, USA) and used as received. Ethyl alcohol, 200
proof, was obtained from Pharmco (Shelbyville, KY 40065, USA).
The water employed in all preparations was purified by a Milli-Q system (Millipore) (Bedford, MA, USA). Rice
α-galactosidase gene was cloned from cDNA library. The yeast strain, Pichia pastoris SMD1168, and pPIC-9k
plasmid were obtained from Invitrogen (Carlsbad, Calif, USA). The synthetic pNP-substrate,
p-nitrophenyl-α-D-galactopyranoside were obtained from Sigma-Aldrich. Other
materials for cell cultivation and cloning, such as agar plate, YPD plate,
yeast extract, lactose, sodium carbonate, LB medium, SMGY medium, YNB medium, Eppendorf
tube, and Pyrex Petri dishes were used from a conventional laboratory stock.


## 2.2. Preparation and Characterization of Nanomaterials

Silica colloidal particles were processed by following the method of Stöber et al. [8]. Briefly,
it involved hydrolysis and successive condensation of tetraethylorthosilicate
(TEOS) in ethanol/water mixture with ammonium hydroxide as a catalysis. In a
typical formulation, a mixture of 0.6 mL of TEOS and 10 mL ethanol was
sonicated for 30 minutes. While stirring the sol-gel precursor solution, a 0.1 mL of 3% NH4OH solution was added dropwise. The silica colloidal
solution turned turbidly white and allowed to stir for 3-4 hours for the reaction
to be completed. The silica product solution was centrifuged at 15000 rpm for
30 minutes. The SiO2 precipitates were collected, washed with water twice,
and dried at 37°C. The silica nanoparticles were redispersed in
water to form a 1 g/L colloidal solution for further use.The aqueous colloidal solution of Au and
Ag, individually, was prepared following a chemical reduction method employed by
Turkevich et al. [9] and Fu et al. [10]. First, an aqueous solution of 2 mM for
each metal salt, HAuCl4, and AgNO3 was prepared. Second,
to synthesize Au colloidal solution, a volume of 25 mL HAuCl4 solution was heated to boil and stirred vigorously, and 2.5 mL of freshly
prepared sodium citrate solution (38.8 mM in ethanol) were added dropwise until
a wine-red color was observed. A similar procedure was used also for preparing
Ag colloidal solution.The composite nanomaterials, silica-Au and
silica-Ag, were prepared by the procedure described previously [10, 11]. For a nanoparticle,
silica-Ag as an example, a volume of 25 mL of 2 mM AgNO3 solution
and 0.2 mL of 1 g/L SiO2 colloidal solution (as prepared above) was heated
to boil and stirred vigorously, and 2.5 mL of freshly prepared sodium citrate
solution (38.8 mM, in ethanol) were slowly added. Initially, a white/turbid
solution of silica particles with silver nitrate turned pale yellow and after reacting
for 30 minutes, the color of the solution turned golden yellow indicating the
formation of silica-Ag nanocomposites. It is important to ensure that the
silver and gold ions have been reduced completely by adding an excess amount of
reducing agents. Moreover, the nanoparticles were normally centrifuged, washed,
and resuspended in solution before use, in order to minimize the alcohol cosolvent
and a possible existence of the trace amount of metal ions in solution. The
size, shape, and distribution of the synthesized nanomaterials and also the α-Gal/nanomaterial
complexes were characterized by a TEM (Jeol JSM-1200 Ex II, operated at 80 kV) and
a UV-vis spectrophotometer (Hitachi U-2000).


## 2.3. Gene Cloning and Gene Expression in Yeast Cells

Riceα-galactosidase
gene was cloned in pPIC-9k plasmid (Invitrogen, Calif, USA) and transformed
into SMD 1168 yeast strain chromosomal DNA by electroporation according to the
Invitrogen protocol described in EasySelect (http://www.Invitrogen.com)
and given by Higgins and Cregg [12]. The gene expression was done also by
following the Invitrogen EasySelect procedure. The cloned SMD 1168-α-Gal
yeast cells were cultured overnight from a single colony on YPD plate in 2 mL
SMGY medium. The cells were harvested and the induction was carried out in 2 mL
of a YNB medium containing amino acids, 2% of glycerol as carbon source, and
30% of the individual testing nanomaterials. For the preparation of control
sample, the solution of nanomaterials dispersed in water was replaced by the
same volume amount of pure water that was then used to carry out the control
experiment for each type of nanomaterials tested [7, 13]. Finally, the
expressed cells were induced by adding 1% of methanol everyday. The cell mass
was measured spectrophotometrically by recording its optical density at 600 nm (OD600).


## 2.4. Determination of Enzyme Activity and  Enzyme Assay

After each period of induction in the gene expression (24 hours, 48 hours, and up to
8 days), the enzyme activity was determined (in duplicate) by a syntheticpNP-substrate, p-nitrophenyl-α-D-galactopyranoside.
Fifty μL of the induced yeast cells and 30 μL of 4 mM pNP-substrate were pipetted into an Eppendorf tube and incubated at
37°C for 10 minutes. After incubation, a 1.0 mL of 0.2 M Na2CO3,
pH 9.8, was added to terminate the reaction. The final reaction mixture was
then centrifuged at 8000 rpm for 15 minutes (as shown in the right test tube of
Figure 1(a); the left test tube is the control), and the resulting clear yellow
supernatant (i.e., the released p-nitrophenol, pNP, representing the secreted
activity) was read in a spectrophotometer at 400 nm (as shown in Figure 1(b), a
UV-visible spectrum from 300 nm to 600 nm) [7]. The intracellular enzyme
activity was obtained by subtracting the secreted activity from the total
activity. One unit of enzyme activity is defined as the amount of enzyme that
can produce 1 μmol of pNP/min at 37°C.(a) Secreted recombinantα-galactosidase activity; see text for the details, and (b) OD at 400 nm of the secreted α-galactosidase
supernatant, representing the production of p-nitrophenol.
(a)(b)The secreted proteins  were analyzed by SDS-Polyacrylamide Gel Electrophoresis (PAGE) [14]. The SDS gel, containing
13.5% polyacrylamide with a 0.75 mm thickness, was prepared. After loading the samples,
electrophoresis was carried out with 110 V and 20 mA in a continuousbuffer
system for 2 hours. The gel was stained with Coomassie
Brilliant Blue R-250 and
destained until the background was clear. The question of a possible cell death
during induction period, as may be caused by the nanomaterials toxicity, was
monitored by cells staining with Trypan Blue [15] (4% solution from Sigma
Adrich).


## 3. Results and Discussion

### 3.1. Size and Size Distribution of Nanomaterials

The surface plasmon absorption bands observed in the UV-visible spectra are commonly used
for characterizing the size and shape of metallic nanoparticles. In particular,
the gold and silver nanomateterials have been well documented. Normally, the
UV-visible spectra of the synthesized nanomaterials in solution are recorded
from 200 nm to 800 nm. Small gold nanoparticles of <5 nm diameter do not
show any plasmon absorption, but gold nanoparticles of 5–50 nm show a sharp
absorption band in the 520–530 nm region [16]. As the particles grow bigger,
the absorption band broadens and covers the visible spectral range [17]. The
absorption spectrum of the silver colloids of facetted silver nanocrystals
(with a particle diameter of 40–60 nm) shows a surface plasmon absorption band
with a maximum around 420 nm. For silver nanoparticles, a shift in the plasmon
absorption band from 400 nm to 670 nm was reported as the particle shape
changed from spherical to triangular prisms during visible light irradiation
[18]. Under the experimental conditions described in Section 2. Figure 2 shows
the individual plasmon resonance absorption band for each species of the
prepared nanomaterials, recorded from 200 nm to 800 nm. The surface plasmon
absorption band maximum is observed at Figure 2(a) to be 475 nm for Ag nanoparticle,
Figure 2(b) 425 nm for silica-Ag nanocomposite, Figure 2(c) 525 nm for Au
nanoparticle, and Figure 2(d) 550 nm for silica-Au nanocomposite. The plasmon
bands are broad in all spectra suggesting a wide range of particle size
distribution. Based on the observed spectral wavelength of surface plasmon
bands, the particle size of Ag nanoparticle in Figure 2(a) should be ∼60 nm
whereas silica-Ag nanocomposite in Figure 2(b) should be in the range of 40–60 nm.
For Au nanoparticle in Figure 2(c), the size should be within 5–50 nm. On
the other hand, the
size of the silica-Au nanocomposite in Figure 2(d) should be ∼50 nm.UV-visible spectra of surface plasmon band:
(a) Ag at 475 nm, (b) silica-Ag at 425 nm, (c) Au at 525 nm, and (d) silica-Au
at 550 nm.
(a)(b)(c)(d)The particle size and size distribution of
nanomaterials assigned from the observed surface plasmon bands in Figure2 can also
be verified by the TEM images as shown in Figure 3. In Figure 3(a), the size of Au nanoparticles is not very
uniform but they are all in the ranges between 12 nm and 20 nm, as suggested by
the observed surface plasmon band at 525 nm in the UV-visible spectrum as displayed
in Figure 2(c). The size of nanocomposite of SiO2 and Au, that is,
silica-Au in TEM, Figure 3(b), is observed to be in the ranges between 40 nm and 60 nm in agreement
with the assignment of UV-visible spectrum of Figure 2(d). The sizes of Ag
particles in TEM, Figure 3(c), are also not very uniform ranging from 25 nm to 30 nm, and they
tend to form some larger agglomerates. This may explain why the surface plasmon
band in Figure 2(a) is observed at a longer wavelength of 475 nm. These
agglomerates seem to disappear in TEM, Figure 3(d), when Ag nanoparticles form composite structure
with SiO2 particles, that is, the particle size of silica-Ag is
observed to be in the ranges between 40 nm and 60 nm, as assigned in the UV-visible
spectrum of Figure 2(b). It is worthwhile to mention that the size and size
distribution of nanomaterials are critically important for gene delivery to
effectively transport into cells interior and to enhance gene expression in
cells, which will be demonstrated below.TEM images of the synthesized
nanomaterials: (a) Au, (b) silica-Au, (c) Ag, and (d) silica-Ag.
(a)(b)(c)(d)


### 3.2.α-Galactosidase Activity in Cells Assisted by Nanomaterials

When gold nanoparticles were loaded and modified with oligonucleotide, the effectiveness
of using gold nanoparticle-oligonucleotide complexes (via surface binding) as
intracellular gene regulating agents for the control of protein expression in
cells has been illustrated [3]. By chemically tailoring the density of DNA
bound to the surface of gold nanparticles, it was possible to introduce
oligonucleotides at a higher effective concentration than conventional
transfection agents, and remain nontoxic to the cells. In the present study,
Rice α-galactosidase gene is expected to load
on the surface of nanomaterials via electrostatic interactions. The
nanomaterial modified α-galactosidase complexes can then transport
effectively into yeast cells interior to enhance gene expression.The TEM imaging technique was used to trace
the location ofα-Gal/nanomaterial complexes, either
inside or outside the yeast cells to give the enhanced α-galactosidase
activity. If the nanomaterials are located in the solution medium (or outside
the yeast cells) as the unbounded or free nanomaterials, they will be easily
washed off by the double deionized water. On the other hand, if α-Gal/nanomaterial
complexes are incorporated into the yeast cells, then they will be remained
inside the cells upon water washing. However, the encapsulated nanomaterials
inside the yeast cells may be dissolved away if the “royal water”—aqua regia was used for washing. The TEM
images of α-Gal/nanomaterials incubated in yeast
cells, after 5-day induction, are shown in Figure 4, Figure 4(a) Au
nanparticles, and Figure 4(b) Ag nanoparticles. It is observed that α-Gal/nanomaterials
have a compatible size as those of free nanomaterials shown in the TEM images
of Figure 3, except that α-Gal/nanomaterials are shown to
encapsulate in a unique shape of a cell (Figure 4(a) and 4(c)). When these
incubated samples were washed with double deionized water, the nanomaterials
remained as shown in Figure 4(d) for α-Gal/Ag complexes (the same is also
obtained for α-Gal/Au complexes). This result
indicates that α-Gal/nanomaterials are not simply
dispersed in the solution medium as free nanoparticles, in fact they are
encapsulated inside the yeast cells. Furthermore, when an aqua regia solution
was used to wash the incubated samples, the α-Gal/Au complexes were shown to dissolve
away as seen in the TEM images of Figure 4(b) (the same is also obtained for α-Gal/Ag
complexes). The TEM images in Figure 4 have provided the illustrations that the
transfection of α-Gal/nanomaterials were indeed inside
the yeast cells to give the observed enhancement in gene expression.TEM images ofα-Gal/nanomaterials incubated in yeast
cells, after 5 days of induction: (a) Au nanparticles, (b) sample in (a) washed with royal water, (c) Ag nanoparticles, and (d) sample in (c) washed with double deionized water.
(a)(b)(c)(d)The nonviral transfer of DNA into cells (or
transfection) is a routine procedure in modern biochemistry [19]. Following the
protocol of the transfection mechanism, nanoparticles with DNA are given to the
cell culture medium and incorporated into the cells by endocytosis. Inside the
cytoplasm, the nanoparticles finally reach the nucleus and insert the DNA into
the nucleus. This is the location in the cell where DNA molecules are copied
for translation into proteins or are multiplied for cell division. Xu et al.
[4] studied the real-time probing of membrane transport in living microbial
cells (Pseudomonas aeruginosa) using
single nanoparticle (silver nanoparticle) optics. They showed that Ag
nanoparticles with sizes ranging up to 80 nm were accumulated in living
microbial cells.  This observation demonstrated that the Ag nanoparticles with
size of 80 nm or smaller can transport through the inner or outer membrane of
the P. aeruginosa cells. This particle
size, 80 nm or less as shown in TEM        images of Figure 3 for the nanomaterials
synthesized, would be used as the particle size reference for our works on
nanomaterials enhanced gene expression in yeast cells.In this study, the gene expression
experiments in yeast (SMD1168/α-Gal) are carried out by adding nanomaterials
(30% of the total volume of the medium) to 2 mL of the cell cultures for every
24 hours of induction. Figure 5 shows the enhanced gene expression,
α-galactosidase activity in yeast cells by adding Au (12 nm–20 nm) and
silica-Au (40 nm–60 nm) nanomaterials. The intracellular and secreted enzyme
activities resulting from day 1 to day 4 inductions are plotted in Figures 5(a)
and 5(b), respectively. The day 1 activity was measured for a 24-hour induction
after each nanomaterial was added. The
α-galactosidase activity is plotted in yellow line (-▲-), pink line (-■-), and blue line (-♦-), separately for the addition of Au, silica-Au,
and the control samples (no nanomaterial added). It shows that both Au and
silica-Au nanomaterials are not toxic to yeast cells; rather, they act to
increase the transfection activity. The enhancement of gene expression is
higher for Au nanoparticle than for silica-Au nanocomposite. It is noted that,
one unit of enzyme activity is defined as the amount of enzyme that can produce
1 μmol ofpNP/min at 37°C.
From Figure 5, the intracellular α-galactosidase activity is shown to be about
5–7 times higher than the secreted (extracellular) activity in the medium, that
is, about 20% of enzyme activity was secreted in the meduim [13]. It should be
noted that, in our experiment, the testing samples and control sample have the
same amount of cells. After induction (day 1 to day 5), the control sample was
shown to express a basal level of α-galactosidase both in the medium and inside
the cells. With the addition of nanomaterials, the secreted level of enzyme
displayed a 3-4 fold enhancement.Gene expression enhanced by Au(-▲-) and silica-Au (-■-), and the control sample (-♦-). (a) intracellular activity and (b)
extracellular (secreted) activity.
(a)(b)In Figure5, the intracelluar activity
increases from day 1 to day 5 induction, and then it starts to decrease after
the day 5 induction (including the control sample without the nanomaterials
added), while the extracellular activity increases continuously from day 1 to
day 8 induction (not shown in Figure 5). One of the possible explanations for
this observation is the cell death, where the continuously increased in
extracellular activity after day 5 induction might come from the decreasing
intracellular activity, that is, partially, due to the enzyme leakage from
inside. In order to verify that cell death was not the case, a 4% trypan blue solution
was used to stain the cells. Figure 6 shows an optical micrograph of the
stained yeast cells: Figure 6(a) the cells after induction for 5 days
containing α-Gal/nanomaterial complexes, and Figure 7(b) the cells treated with 100% methanol (i.e., dead cell). The dead cells
turned blue color, whereas no cell death was observed for the yeast cells after
induction for 5 days with α-Gal/nanomaterials.Optical micrographs of the stained yeast cells: (a) the cells after induction for 5 days containingα-Gal/nanomaterial
complexes, and (b) the cells treated with 100% methanol (i.e., dead cell).
(a)(b)Gene expression enhanced by (a) and (b) different doses of silica-Ag nanocomposites, and by (c) different types of
nanomaterials. (a) and (c) intracellular activity and (b) extracellular
(secreted) activity. See text for details.
(a)(b)(c)Figure7 shows (a) the intracellualr and (b) secreted
α-galactosidase activity assisted by adding
different amount of silica-Ag, 450 μL (yellow line, -▲-), 225 μL (pink
line, -■-), and control (blue line, -♦-, no nanomaterial added). The induction period
was carried out from day 1 to day 8. The activity was labeled in unit/mL, where
one unit of enzyme activity was defined as the amount of enzyme that can
release 1 μmol ofpNP/min at 37°C.
For intracellular activity, the activity at day 5 induction was measured to be
0.595, 0.66, and 0.74 unit/mL for the controlled sample, 225 μL added silica-Ag
sample, and 450 μL added silica-Ag sample, respectively. This represents 11%
and 24% enhancements in enzyme activity for the 225 μL and 450 μL added silica-Ag
samples, respectively, as compared to the control sample. The results indicate
that cells incubated with silica-Ag enhance the intracellular activity; while a
double dose of silica-Ag gives a double enzyme activity. After day 5 induction,
the enzyme activity went down, where the control sample lost the
α-galactosidase activity faster (i.e., the nanomaterial-α-galactosidase
complexes are less susceptible to degradation). At day 8 induction, the
intracellular activity was changed to 0.18, 0.55, and 0.68 unit/mL for the
controlled sample, 225 μL added silica-Ag sample, and 450 μL added silica-Ag
sample, respectively. This corresponds to 206% and 278% enhancements in enzyme activity
for the 225 μL and 450 μL added silica-Ag samples, respectively, as compared to
the control sample. It is noted that the amount of enzyme activity in unit/mL
is about 4-5 times smaller in the secreted medium (Figure 7(b)) as compared to
that inside the yeast cells (Figure 7(a)). This is again in agreement with ∼20% of enzyme activity secreted in the medium
reported previously [13]. However,  the  percent enhancement of extracellular
activity assisted by silica-Ag is much larger than that shown in the
intracellular activity. Unlike the intracellular activity, the secreted
activity is shown to increase from day 1 to day 8 inductions. Again, this
observation is not due to the cell death during the induction period as shown
in Figure 6. At the day 8 induction, 233% and 423% enhancements in
extracellular activity were observed for the 225 μL and 450 μL added silica-Ag
samples, respectively, as compared to the control sample. In short, a double
enzyme activity has been expressed when a double dose of silica-Ag  was given to
the yeast cells.Figure7(c) compares the intracellular enzyme
activity assisted by the same amount of different nanomaterials, Au (purple
line, -*-), Ag (yellow line, -▲-), silica-Au (light blue,
-x-), silica-Ag (pink line, -■-), and control (blue line, -♦-, no nanomaterial added). The Au (12–20 nm) and
Ag (25–30 nm) nanoparticles give a higher enhancement in enzyme activity than
the corresponding silica-Au and silica-Ag nanocomposites (with a particle size
of 40–60 nm) that presumably are due to the difference in their particle
sizes. The results indicate that the highest enhancement in intracellular
enzyme activity goes to Au nanoparticles assisted gene expression, followed by
Ag nanoparticles, silica-Au nanocomposites, and then silica-Ag nanocomposites. It
is also shown that the α-galactosidase activity is a function of the type and
proportional to the amount of nanomaterials incorporated.The use of nanomaterials to enhance
transfection by physical concentration of DNA at the cell surface is essential
for increasing gene expression and protein production. The DNA/nanomaterial
complex cannot only be brought into the cytoplasm, but can also make its way to
the cell nucleus where DNA molecules are copied for translation into protein.
The ability of synthesizing therapeutic proteins via nanomaterials-assisted
gene expression in cells is a great challenge, in particular, if the
enhancement in protein yields is selective, specific, and obtainable in a
commercial scale. The recombinant enzyme proteins in yeast cells and in the
secreted medium, resulting from the nanomaterials-assisted gene expression, that
is,α-galactosidase activity are subjected to the SDS-gel electrophoresis
analysis. In order to show that the α-galactosidase was really produced by the
cells, SDS-PAGE analysis [14] was carried out for each testing sample, dried
medium, and cells. For cell sample, about 10 μL of the 150 μL
suspension cells were used. For medium sample, the dried sample was dissolved
in 15 μL of water before use. The results are
shown in Figure 8, where α-galactosidase shows a single protein band at 46 kDa
[13, 20]. Based on having 363 amino acid residues, the enzyme could exist as a
monomer [13]. The S't and BSA represents Bio Rad low protein
standard and bovine serum albumin, respectively. In Figure 8(a), the experiments
were done with Au and silica-Au, whereas those carried out with Ag and silica-Ag
are listed in Figure 8(b). The α-galactosidase was found in the secreted
medium in Figures 8(a) lane 4, and 8(b) lane 7 and Lane 8, as well as in the
cellular materials in Figure 8(b) lane 1 through lane 4. The secreted protein
was shown to have a pure form, and it should be easier for any further protein
purification if needed. When lane 4 was compared with lane 1 in Figure 8(a),
Au nanoparticles were shown to enhance about four times of the protein
expression in the medium. When lane 7 and lane 8 in Figure 8(b) were compared,
both Ag and silica-Ag were shown to increase about 2-3 times of the protein expression
under the same experimental conditions.The SDS-polyacrylamide gel electrophoresis for the recombinant enzyme proteins obtained from SMD1168/α-Gal cells and
its medium concentrates. See text for details.
(a)(b)The nanomaterials-mediated transfer mechanism ofα-galactosidase gene into yeast cells
should be function of the characteristic of nanomaterials
used (type, size,
size distribution, interaction, etc.). The results in Figures 5
and 7 have
demonstrated that Ag and Au nanoparticles gave a higher  enzyme activity than
that assisted by silica-Ag and silica-Au nanocomposites. The synthesized Au and
Ag nanoparticles are smaller in size (12–30 nm) as compared to that of silica-Ag
and silica-Au nanocomposites (40–60 nm). It is suggested that the smaller
nanoparticles are more effective in reaching the cell surface, penetrating into
the cytoplasm via endocytosis, and making their ways to the cell nucleus. When
Au and Ag nanoparticles were added to and incubated in the yeast culture, a 3–5
fold enhancement in α-galactosidase activity was observed for
the intracellular activity as well as in the secreted medium as compared to
those of the control group. The secreted protein in the medium was shown to
have a pure form and displayed as a single protein band in the SDS-gel
electrophoresis.


## 3.1. Size and Size Distribution of Nanomaterials

The surface plasmon absorption bands observed in the UV-visible spectra are commonly used
for characterizing the size and shape of metallic nanoparticles. In particular,
the gold and silver nanomateterials have been well documented. Normally, the
UV-visible spectra of the synthesized nanomaterials in solution are recorded
from 200 nm to 800 nm. Small gold nanoparticles of <5 nm diameter do not
show any plasmon absorption, but gold nanoparticles of 5–50 nm show a sharp
absorption band in the 520–530 nm region [16]. As the particles grow bigger,
the absorption band broadens and covers the visible spectral range [17]. The
absorption spectrum of the silver colloids of facetted silver nanocrystals
(with a particle diameter of 40–60 nm) shows a surface plasmon absorption band
with a maximum around 420 nm. For silver nanoparticles, a shift in the plasmon
absorption band from 400 nm to 670 nm was reported as the particle shape
changed from spherical to triangular prisms during visible light irradiation
[18]. Under the experimental conditions described in Section 2. Figure 2 shows
the individual plasmon resonance absorption band for each species of the
prepared nanomaterials, recorded from 200 nm to 800 nm. The surface plasmon
absorption band maximum is observed at Figure 2(a) to be 475 nm for Ag nanoparticle,
Figure 2(b) 425 nm for silica-Ag nanocomposite, Figure 2(c) 525 nm for Au
nanoparticle, and Figure 2(d) 550 nm for silica-Au nanocomposite. The plasmon
bands are broad in all spectra suggesting a wide range of particle size
distribution. Based on the observed spectral wavelength of surface plasmon
bands, the particle size of Ag nanoparticle in Figure 2(a) should be ∼60 nm
whereas silica-Ag nanocomposite in Figure 2(b) should be in the range of 40–60 nm.
For Au nanoparticle in Figure 2(c), the size should be within 5–50 nm. On
the other hand, the
size of the silica-Au nanocomposite in Figure 2(d) should be ∼50 nm.UV-visible spectra of surface plasmon band:
(a) Ag at 475 nm, (b) silica-Ag at 425 nm, (c) Au at 525 nm, and (d) silica-Au
at 550 nm.
(a)(b)(c)(d)The particle size and size distribution of
nanomaterials assigned from the observed surface plasmon bands in Figure2 can also
be verified by the TEM images as shown in Figure 3. In Figure 3(a), the size of Au nanoparticles is not very
uniform but they are all in the ranges between 12 nm and 20 nm, as suggested by
the observed surface plasmon band at 525 nm in the UV-visible spectrum as displayed
in Figure 2(c). The size of nanocomposite of SiO2 and Au, that is,
silica-Au in TEM, Figure 3(b), is observed to be in the ranges between 40 nm and 60 nm in agreement
with the assignment of UV-visible spectrum of Figure 2(d). The sizes of Ag
particles in TEM, Figure 3(c), are also not very uniform ranging from 25 nm to 30 nm, and they
tend to form some larger agglomerates. This may explain why the surface plasmon
band in Figure 2(a) is observed at a longer wavelength of 475 nm. These
agglomerates seem to disappear in TEM, Figure 3(d), when Ag nanoparticles form composite structure
with SiO2 particles, that is, the particle size of silica-Ag is
observed to be in the ranges between 40 nm and 60 nm, as assigned in the UV-visible
spectrum of Figure 2(b). It is worthwhile to mention that the size and size
distribution of nanomaterials are critically important for gene delivery to
effectively transport into cells interior and to enhance gene expression in
cells, which will be demonstrated below.TEM images of the synthesized
nanomaterials: (a) Au, (b) silica-Au, (c) Ag, and (d) silica-Ag.
(a)(b)(c)(d)


## 3.2.α-Galactosidase Activity in Cells Assisted by Nanomaterials

When gold nanoparticles were loaded and modified with oligonucleotide, the effectiveness
of using gold nanoparticle-oligonucleotide complexes (via surface binding) as
intracellular gene regulating agents for the control of protein expression in
cells has been illustrated [3]. By chemically tailoring the density of DNA
bound to the surface of gold nanparticles, it was possible to introduce
oligonucleotides at a higher effective concentration than conventional
transfection agents, and remain nontoxic to the cells. In the present study,
Rice α-galactosidase gene is expected to load
on the surface of nanomaterials via electrostatic interactions. The
nanomaterial modified α-galactosidase complexes can then transport
effectively into yeast cells interior to enhance gene expression.The TEM imaging technique was used to trace
the location ofα-Gal/nanomaterial complexes, either
inside or outside the yeast cells to give the enhanced α-galactosidase
activity. If the nanomaterials are located in the solution medium (or outside
the yeast cells) as the unbounded or free nanomaterials, they will be easily
washed off by the double deionized water. On the other hand, if α-Gal/nanomaterial
complexes are incorporated into the yeast cells, then they will be remained
inside the cells upon water washing. However, the encapsulated nanomaterials
inside the yeast cells may be dissolved away if the “royal water”—aqua regia was used for washing. The TEM
images of α-Gal/nanomaterials incubated in yeast
cells, after 5-day induction, are shown in Figure 4, Figure 4(a) Au
nanparticles, and Figure 4(b) Ag nanoparticles. It is observed that α-Gal/nanomaterials
have a compatible size as those of free nanomaterials shown in the TEM images
of Figure 3, except that α-Gal/nanomaterials are shown to
encapsulate in a unique shape of a cell (Figure 4(a) and 4(c)). When these
incubated samples were washed with double deionized water, the nanomaterials
remained as shown in Figure 4(d) for α-Gal/Ag complexes (the same is also
obtained for α-Gal/Au complexes). This result
indicates that α-Gal/nanomaterials are not simply
dispersed in the solution medium as free nanoparticles, in fact they are
encapsulated inside the yeast cells. Furthermore, when an aqua regia solution
was used to wash the incubated samples, the α-Gal/Au complexes were shown to dissolve
away as seen in the TEM images of Figure 4(b) (the same is also obtained for α-Gal/Ag
complexes). The TEM images in Figure 4 have provided the illustrations that the
transfection of α-Gal/nanomaterials were indeed inside
the yeast cells to give the observed enhancement in gene expression.TEM images ofα-Gal/nanomaterials incubated in yeast
cells, after 5 days of induction: (a) Au nanparticles, (b) sample in (a) washed with royal water, (c) Ag nanoparticles, and (d) sample in (c) washed with double deionized water.
(a)(b)(c)(d)The nonviral transfer of DNA into cells (or
transfection) is a routine procedure in modern biochemistry [19]. Following the
protocol of the transfection mechanism, nanoparticles with DNA are given to the
cell culture medium and incorporated into the cells by endocytosis. Inside the
cytoplasm, the nanoparticles finally reach the nucleus and insert the DNA into
the nucleus. This is the location in the cell where DNA molecules are copied
for translation into proteins or are multiplied for cell division. Xu et al.
[4] studied the real-time probing of membrane transport in living microbial
cells (Pseudomonas aeruginosa) using
single nanoparticle (silver nanoparticle) optics. They showed that Ag
nanoparticles with sizes ranging up to 80 nm were accumulated in living
microbial cells.  This observation demonstrated that the Ag nanoparticles with
size of 80 nm or smaller can transport through the inner or outer membrane of
the P. aeruginosa cells. This particle
size, 80 nm or less as shown in TEM        images of Figure 3 for the nanomaterials
synthesized, would be used as the particle size reference for our works on
nanomaterials enhanced gene expression in yeast cells.In this study, the gene expression
experiments in yeast (SMD1168/α-Gal) are carried out by adding nanomaterials
(30% of the total volume of the medium) to 2 mL of the cell cultures for every
24 hours of induction. Figure 5 shows the enhanced gene expression,
α-galactosidase activity in yeast cells by adding Au (12 nm–20 nm) and
silica-Au (40 nm–60 nm) nanomaterials. The intracellular and secreted enzyme
activities resulting from day 1 to day 4 inductions are plotted in Figures 5(a)
and 5(b), respectively. The day 1 activity was measured for a 24-hour induction
after each nanomaterial was added. The
α-galactosidase activity is plotted in yellow line (-▲-), pink line (-■-), and blue line (-♦-), separately for the addition of Au, silica-Au,
and the control samples (no nanomaterial added). It shows that both Au and
silica-Au nanomaterials are not toxic to yeast cells; rather, they act to
increase the transfection activity. The enhancement of gene expression is
higher for Au nanoparticle than for silica-Au nanocomposite. It is noted that,
one unit of enzyme activity is defined as the amount of enzyme that can produce
1 μmol ofpNP/min at 37°C.
From Figure 5, the intracellular α-galactosidase activity is shown to be about
5–7 times higher than the secreted (extracellular) activity in the medium, that
is, about 20% of enzyme activity was secreted in the meduim [13]. It should be
noted that, in our experiment, the testing samples and control sample have the
same amount of cells. After induction (day 1 to day 5), the control sample was
shown to express a basal level of α-galactosidase both in the medium and inside
the cells. With the addition of nanomaterials, the secreted level of enzyme
displayed a 3-4 fold enhancement.Gene expression enhanced by Au(-▲-) and silica-Au (-■-), and the control sample (-♦-). (a) intracellular activity and (b)
extracellular (secreted) activity.
(a)(b)In Figure5, the intracelluar activity
increases from day 1 to day 5 induction, and then it starts to decrease after
the day 5 induction (including the control sample without the nanomaterials
added), while the extracellular activity increases continuously from day 1 to
day 8 induction (not shown in Figure 5). One of the possible explanations for
this observation is the cell death, where the continuously increased in
extracellular activity after day 5 induction might come from the decreasing
intracellular activity, that is, partially, due to the enzyme leakage from
inside. In order to verify that cell death was not the case, a 4% trypan blue solution
was used to stain the cells. Figure 6 shows an optical micrograph of the
stained yeast cells: Figure 6(a) the cells after induction for 5 days
containing α-Gal/nanomaterial complexes, and Figure 7(b) the cells treated with 100% methanol (i.e., dead cell). The dead cells
turned blue color, whereas no cell death was observed for the yeast cells after
induction for 5 days with α-Gal/nanomaterials.Optical micrographs of the stained yeast cells: (a) the cells after induction for 5 days containingα-Gal/nanomaterial
complexes, and (b) the cells treated with 100% methanol (i.e., dead cell).
(a)(b)Gene expression enhanced by (a) and (b) different doses of silica-Ag nanocomposites, and by (c) different types of
nanomaterials. (a) and (c) intracellular activity and (b) extracellular
(secreted) activity. See text for details.
(a)(b)(c)Figure7 shows (a) the intracellualr and (b) secreted
α-galactosidase activity assisted by adding
different amount of silica-Ag, 450 μL (yellow line, -▲-), 225 μL (pink
line, -■-), and control (blue line, -♦-, no nanomaterial added). The induction period
was carried out from day 1 to day 8. The activity was labeled in unit/mL, where
one unit of enzyme activity was defined as the amount of enzyme that can
release 1 μmol ofpNP/min at 37°C.
For intracellular activity, the activity at day 5 induction was measured to be
0.595, 0.66, and 0.74 unit/mL for the controlled sample, 225 μL added silica-Ag
sample, and 450 μL added silica-Ag sample, respectively. This represents 11%
and 24% enhancements in enzyme activity for the 225 μL and 450 μL added silica-Ag
samples, respectively, as compared to the control sample. The results indicate
that cells incubated with silica-Ag enhance the intracellular activity; while a
double dose of silica-Ag gives a double enzyme activity. After day 5 induction,
the enzyme activity went down, where the control sample lost the
α-galactosidase activity faster (i.e., the nanomaterial-α-galactosidase
complexes are less susceptible to degradation). At day 8 induction, the
intracellular activity was changed to 0.18, 0.55, and 0.68 unit/mL for the
controlled sample, 225 μL added silica-Ag sample, and 450 μL added silica-Ag
sample, respectively. This corresponds to 206% and 278% enhancements in enzyme activity
for the 225 μL and 450 μL added silica-Ag samples, respectively, as compared to
the control sample. It is noted that the amount of enzyme activity in unit/mL
is about 4-5 times smaller in the secreted medium (Figure 7(b)) as compared to
that inside the yeast cells (Figure 7(a)). This is again in agreement with ∼20% of enzyme activity secreted in the medium
reported previously [13]. However,  the  percent enhancement of extracellular
activity assisted by silica-Ag is much larger than that shown in the
intracellular activity. Unlike the intracellular activity, the secreted
activity is shown to increase from day 1 to day 8 inductions. Again, this
observation is not due to the cell death during the induction period as shown
in Figure 6. At the day 8 induction, 233% and 423% enhancements in
extracellular activity were observed for the 225 μL and 450 μL added silica-Ag
samples, respectively, as compared to the control sample. In short, a double
enzyme activity has been expressed when a double dose of silica-Ag  was given to
the yeast cells.Figure7(c) compares the intracellular enzyme
activity assisted by the same amount of different nanomaterials, Au (purple
line, -*-), Ag (yellow line, -▲-), silica-Au (light blue,
-x-), silica-Ag (pink line, -■-), and control (blue line, -♦-, no nanomaterial added). The Au (12–20 nm) and
Ag (25–30 nm) nanoparticles give a higher enhancement in enzyme activity than
the corresponding silica-Au and silica-Ag nanocomposites (with a particle size
of 40–60 nm) that presumably are due to the difference in their particle
sizes. The results indicate that the highest enhancement in intracellular
enzyme activity goes to Au nanoparticles assisted gene expression, followed by
Ag nanoparticles, silica-Au nanocomposites, and then silica-Ag nanocomposites. It
is also shown that the α-galactosidase activity is a function of the type and
proportional to the amount of nanomaterials incorporated.The use of nanomaterials to enhance
transfection by physical concentration of DNA at the cell surface is essential
for increasing gene expression and protein production. The DNA/nanomaterial
complex cannot only be brought into the cytoplasm, but can also make its way to
the cell nucleus where DNA molecules are copied for translation into protein.
The ability of synthesizing therapeutic proteins via nanomaterials-assisted
gene expression in cells is a great challenge, in particular, if the
enhancement in protein yields is selective, specific, and obtainable in a
commercial scale. The recombinant enzyme proteins in yeast cells and in the
secreted medium, resulting from the nanomaterials-assisted gene expression, that
is,α-galactosidase activity are subjected to the SDS-gel electrophoresis
analysis. In order to show that the α-galactosidase was really produced by the
cells, SDS-PAGE analysis [14] was carried out for each testing sample, dried
medium, and cells. For cell sample, about 10 μL of the 150 μL
suspension cells were used. For medium sample, the dried sample was dissolved
in 15 μL of water before use. The results are
shown in Figure 8, where α-galactosidase shows a single protein band at 46 kDa
[13, 20]. Based on having 363 amino acid residues, the enzyme could exist as a
monomer [13]. The S't and BSA represents Bio Rad low protein
standard and bovine serum albumin, respectively. In Figure 8(a), the experiments
were done with Au and silica-Au, whereas those carried out with Ag and silica-Ag
are listed in Figure 8(b). The α-galactosidase was found in the secreted
medium in Figures 8(a) lane 4, and 8(b) lane 7 and Lane 8, as well as in the
cellular materials in Figure 8(b) lane 1 through lane 4. The secreted protein
was shown to have a pure form, and it should be easier for any further protein
purification if needed. When lane 4 was compared with lane 1 in Figure 8(a),
Au nanoparticles were shown to enhance about four times of the protein
expression in the medium. When lane 7 and lane 8 in Figure 8(b) were compared,
both Ag and silica-Ag were shown to increase about 2-3 times of the protein expression
under the same experimental conditions.The SDS-polyacrylamide gel electrophoresis for the recombinant enzyme proteins obtained from SMD1168/α-Gal cells and
its medium concentrates. See text for details.
(a)(b)The nanomaterials-mediated transfer mechanism ofα-galactosidase gene into yeast cells
should be function of the characteristic of nanomaterials
used (type, size,
size distribution, interaction, etc.). The results in Figures 5
and 7 have
demonstrated that Ag and Au nanoparticles gave a higher  enzyme activity than
that assisted by silica-Ag and silica-Au nanocomposites. The synthesized Au and
Ag nanoparticles are smaller in size (12–30 nm) as compared to that of silica-Ag
and silica-Au nanocomposites (40–60 nm). It is suggested that the smaller
nanoparticles are more effective in reaching the cell surface, penetrating into
the cytoplasm via endocytosis, and making their ways to the cell nucleus. When
Au and Ag nanoparticles were added to and incubated in the yeast culture, a 3–5
fold enhancement in α-galactosidase activity was observed for
the intracellular activity as well as in the secreted medium as compared to
those of the control group. The secreted protein in the medium was shown to
have a pure form and displayed as a single protein band in the SDS-gel
electrophoresis.


## 4. Conclusion

We have cloned riceα-galactosidase gene into yeast chromosome cells. Using
different nanomaterials, the gene expression of α-galactosidase activity as the
parameter for monitoring the effectiveness of particle's catalytic activity has
been investigated. Au and Ag nanoparticles have shown to enhance gene
expression in yeast cells with the cloned rice α-galactosidase gene. Under our
experimental conditions, Ag, Au, silica-Ag, and silica-Au nanomaterials are not
toxic, and they are not shown to kill the yeast cells. On the contrary, they
act to enhance gene expression inside the yeast cells and also secrete into the
cultural medium. The Ag and Au nanoparticles have a smaller size ranging from
12 nm to 30 nm that may be responsible for the effectiveness of making their
ways to the cell nucleus. It is suggested that SMD 1168-α-Gal yeast cells
may be a choice of model system for studying other nonviral transfer of gene or
drug into cells assisted by nanomaterials. It is further indicated that the
size and uniform distribution of nanoparticle are important for a controllable
transfection study.



---

*Source: 391497-2008-03-17.xml*