Silica Nanoparticles

Chemicals

Sigma:

Surfactant: Aerosol OT (98%)

cosurfactants, 1-butanol (99.8%)

triethoxyvinylsilane: VTES (97%)

99% (Aldrich)

175560-100ML: $37.40

175560-500ML: $115.00

≥98% (Sigma-Aldrich)

A3648-100ML $61.70

A3648-500ML: $212.50

≥98.0%

440140-100ML: $64.40

440140-500ML: $221.50

(3-Aminopropyl)triethoxysilane , APTES (99%)

A3648-100ML: $61.70
A3648-500ML: $212.50

he main advantage of ORMOSIL nanoparticles over other nanoparticles is that the presence of both hydrophobic and hydrophilic groups on the precursor alkoxy-organosilane helps them to self-assemble both as normal micelles and reverse micelles under appropriate conditions. The resulting micellar (and reverse micellar) cores can be used for entrapping biomolecules like drugs, proteins, etc. Such a system has a number of advantages including: (a) they can be loaded with both hydrophilic as well as hydrophobic dyes, (b) they can be precipitated in oil-in-water microemulsions so that corrosive solvents like cyclohexane and complex purification steps like solvent evaporation, ultra-centrifugation etc., can be avoided (c) their organic group can be further modified for the attachment of targeting molecules, and (d) they may be can be possibly bio-degraded through the biochemical decomposition of the Si—C bond . The presence of the organic group also reduces the overall rigidity and density of the particle, which is expected to enhance the stability of such particles in aqueous systems against precipitation.

Spherical, monodisperse particles of colloidal silica can be conveniently prepared by hydrolysis of tetraalkyl silane. This method, commonly referred to as the ‘sol-gel’ method, can be further extended in the synthesis of organically modified silica (ORMOSIL) particles, where the precursor alkoxysilane molecules also include one or two organic groups. The incorporation of organic groups modify the final structure of the silica network, e.g. leading to the formation of mesoporous matrices, characterized by a network structure of ordered and uniform porosity. Such porous matrices can host a number of optically as well as biologically active molecules like fluorescent dyes, proteins, anticancer drugs, image contrast agents, etc.

Synthesis

Name	Aerosol-OT g	nBuOH µl	Water ml	DMSO µl	VTES µl	APTES µl	Diameter nm
ORMA40	0.44	800	20	100	200	40	15-20

dissolve  Aerosol-OT and 1-butanol in 20 ml of double-distilled water by vigorous magnetic stirring.

optional: dissolve dye in DMSO to make 10 mM solution (for visual or florescent tracking)

Add 100 µl dye in DMSO or just DMSO
Add 200 µl of neat VTES  to the micellar system

Stire the resulting solution was stirred for ~ 30 min, or for about one hour, or till the solution becomes clear

Precipitate ORMOSIL nanoparticles by adding aqueous

• ammonia solution (for no amine)
  
• or amine containing compounds such as 3-aminopropyltriethoxysilane, APTES (for amine functionalized, i.e. transfection reagent),  resulting in the incorporation of cationic amino groups on the surface of the ORMOSIL nanoparticles.
  

Stirring for ~20 h at room temperature.

Remove surfactant Aerosol-OT and cosurfactant 1-butanol by dialyzing the solution against water in a 12- to 14-kDa cutoff cellulose membrane (Spectrum Laboratories) for 50 h.

buffer: phosphate-buffered saline (PBS), or HEPES

Filter through the dialyzed solution 0.2-µm cutoff membrane filter (Nalgen).

Use commercial nanoparticles

Spin down Silica nanoparticles (Poly-sciences, Inc., Warrington, PA)  at 3500 rpm for 5 min in a glass conical centrifuge tube

Wash with 100% ethanol several times
Add Silane solutions (United Chemical Technologies, Bristol, PA) of aminopropyl triethoxy silane (APTES) and methyl triethoxy silane (MTES)  to the nanoparticles

Incubate at room temper- ature for at leas  2 h

 All modifications used constant molar concentration of silane with each modification having differing amounts of APTES and MTES;

A100M0 was modified in a solution of 100% APTES,

A50M50 was modified by 50% APTES and 50% MTES on a molar basis.

Recover nanoparticles by spin.

Wash repeatedly in ethanol followed by water.

This results in nanoparticles with varying surface amino and methyl group content.

Transfection

Mix   DNA stock solution 6 ug  with 0.25 ml of OPTI-MEM medium in one microcentrifuge tube.

Add 50 ul of the ORMA40 nanoparticles aqueous suspension  to 0.25 ml of OPTI-MEM medium in another microcentrifuge tube.

Mix two solutions together gently

Incubated at room temperature for 30 min.
Add the resulting DNA–ORMOSIL complex suspension to a 60-mm culture plate of COS-1 cells containing 5 ml of the medium

Incubate for 24 h (37°C, 5% CO 2 ).

After incubation, replace medium

Silicalite nanoparticles

protocol 1

Silicalite-1 nanocrystals with a mean approximate size of 55 nm can be synthesized from a clear solution that is composed of 9 TPAOH: 0.16 NaOH: 25 Si: 495 H2O: 100 EtOH,

TPAOH: tetrapropylammonium hydroxide. 

The clear solution is refluxed at 60 ◦ C for 240 h, washed and centrifuged.
To functionalize the silicalite nanoparticles, samples (55 nm silicalite calcined at 500 ◦ C in air)  are mixed with 3-aminopropyltriethoxysilane (APTES) in 60 ml of toluene in a round-bottom flask.
The sample is heated in an oil bath for 4 h at 120 ◦ C.

The resulting solution is filtered to separate the functionalized nanoparticles

Transfection

Amine functionalized silicalite nanoparticles and unfunctionalized silicalite nanoparticles were prepared in individual batches of
0 . 01 mg ml − 1 in purified and 0 . 2 μ m filtered water.

Plasmid DNA  is  first mixed with PEI (high molecular weight, water free; Sigma Aldrich, St Louis, MO) at an N:P ratio of 5:1, briefly vortexed and allowed to sit for 25 min at
room temperature. After 25 min, the PEI–pDNA polyplexes (1 μ g pDNA) or pDNA (1 μ g) is incubated with 20 μ l of the silicalite nanoparticle solution for another 8 min. Finally, the
silicalite nanoparticles and/or polyplexes/pDNA are added to the cells in Opti-MEM transfection medium (serum-free) and incubated for 4 h at 37 ◦ C. This is followed by incubation in
serum containing medium for 44 h.

Protocol 2

Amino-modified silica nanoparticles were prepared using the synchronous hydrolysis of tetraethoxysilane and N-(beta-amimoethyl)-gamma­-aminopropyltyiethoxysilane in water-in-oil microemulsion. First, a stable and transparent microemulsion was prepared by using the method as previously reported. A solution containing cyclohexane, TritonX-100, n-hexanol (with the ratio of volume: 4 : 1 : 1) was mixed, followed by the addition of adequate water and was stirred for 5 min. Then well-distributed mixture of tetraethoxysilane and N -(beta-amimoethy l)-gamma-aminopropyltyiethoxysilane (with the ratio of volume: 1 : 1) was added to the microemulsion. With the initiation of ammonia, tetraethoxysilane and N-(beta-amimoethyl)-gamma-aminopropyltyiethox­ysilane were synchronously hydrolyzed in the microemulsion to produce amino-modified silica nanoparticles. The whole reaction lasted for 24 h. Pure silica nanoparticles were prepared simultaneously by the controlled hydrolysis of tetraethoxysilane in microemulsion with the initiation of ammonia. At the same time, fluorescent nanoparticles have been prepared for studying the biocompatibility of the silica nanoparticles with cells, employing a highly luminescent dye complex of ruthenium ion and bipyridyl,
Tris(2,2 '-bipyridyl) dichlororuthenium (II) hexahydrate as the core of the nanoparticles silica nanoparticles could be easily prepared by using the hydrolysis of tetraethoxysilane in the
water-in-oil microemulsion without rigorous experimental conditions, such as high temperature, high pressure, etc. The size of the nanoparticles can be controlled through
varying the molar ratio of water to surfactant (R w ) and the molar ratio of TEOS to the ammonia (R N 19 ,20 J•

amino-modified silica nanoparticles are directly produced by the synchronous hydrolysis of tetraethoxysilane and N-(beta-amimoethyl)-gamma-aminopropyltri-ethoxysilane in
water-in-oil microemulsion with the initiation of ammonia. Nanoparticles produced by this method are uniform in size and well dispersed. There is no need to further
modify with aminosilanes, which provide a new, simple and safe method for the application of nanoparticles in the nanoparticles in the biomedicine research.
age of the amino-modified nanoparticles nanoparticles are (50 ± 4) urn in size I mg nanoparticles can bind 97.2 flg plasmid DNA

Protocol 3

from Synthesis of cationic mesoporous silica nanoparticles as a carrier for the delivery of nucleic acids (Iranian)

Synthesis and surface modification of MSNPs was
carried out through three major steps. At the first step,
silica nanoparticles were synthesized by template
removing method in which CTAB, templating surfactant,
was mixed with TEOS, the silica precursor, in a basic
aqueous solution (pH 11). 100 mg of CTAB was added to
the solution of 48 mL deionized water and 350 μL of
sodium hydroxide (2M) and heated to 80°C. Stirrer’s
rotational rate was a variable in this study (Table 1).
When the temperature stabilized, TEOS was added drop
wise. The TEOS concentration was the other variable
(Table 1). After 2 h, the solution was centrifuged and the
particles were refluxed in a solution of 30 mL of ethanol
and 3 mL hydrochloric acid (12 M). After 6 h, the

mixture was centrifuged and the precipitate was washed
with water and ethanol, 3 times each. The surfactants
were completely removed by calcination at 600 °C for 6
h.
The second step of the fabrication was expanding the
pores of MSNPs through the calcination method in the
presence of complex salts [7]. In this step, MSNP-2 with
an average size of 100 nm were dispersed with ultrasonic
in a solution containing NaCl, LiCl and KNO3. The
mixture was dried at 70 °C, and then calcinated at 350,
400, or 450 °C for 2 h.
The final step in the fabrication was the surface
functionalization. During this step the MSNPs (MSNP-2-
1 to MSNP-2-4) were functionalized with N-(2-
aminoethyl)-3-aminopropyltrimethoxy-silane
(EDS),
which causes a relatively positive charge on the MSNPs
surface.

Study on the Effect of Synthesis Parameters of SilicaNanoparticles Entrapped with Rifampicin

DOI: 10.3303/ACOS1311044

Nor Ain Zainal a , Syamsul Rizal Abd Shukor* a , Hajaratul Azwana Ab. Wab b ,Khairunisak Abdul Razak

The materials required for the synthesis of silica nanoparticles are described as follows. Tween 80 - viscous
liquid, trimethoxyvinylsilane (TMVS 98% pure), and rifampicin were obtained from Sigma-Aldrich Co. (MO,
USA). 2-butanol (99% pure), and ammonium hydroxide solution 10 M (31.5% NH 3 pure) were obtained from
Fischer Scientific (Fairlawn, NJ). All these chemicals were of analytical grade and used without any
purification. De-ionized water from a Millipore filtration system (operating at 18.2 MΩ cm) was used in this
study.
2.2 Preparation of Silica Nanoparticles
Silica nanoparticles were prepared by micelles formation approach. First, 5.5 mL Tween 80 was dissolved in
200 mL of de-ionized water. The mixture was stirred for 15 minutes before 200 μL of prepared NH 3 ( 1 mL NH 3
solution was dissolved in 1 mL de-ionized water) was added to ensure the pH is maintain in the range 9 - 11.
Then, 2-butanol was poured into the mixture and continuously stirred for five minutes. The mixture was then
transferred into a preheated reactor at the set temperature and continuously stirred at 320 rpm for one hour.
After that, the prepared rifampicin drug solution ( 0.0839 g of rifampicin dissolved in 1.5 mL methanol) was
added into the above mixture and continuously stirs under the same condition. After an hour,
trimethoxyvinylsilane (TMVS) as silica precursor was added. The mixture was left overnight with continuously
stirred at 320 rpm and maintain with the set temperature, which yield a total volume of 250 mL. The effect of
synthesis temperature, butanol and TMVS on the particle size of silica was investigated. The produced silica
nanocolloids were then subjected to dialysis process for five days to remove the excess surfactant. Finally, the
sample will then be collected in the bottle and stored in the refrigerator until further test.

Engineering of Hollow Mesoporous Silica Nanoparticles for Remarkably Enhanced Tumor Active Targeting Efficacy

Feng Chen 1 *, Hao Hong 1 *, Sixiang Shi 2 , Shreya Goel 2 , Hector F. Valdovinos 3 , Reinier Hernandez 3 , Charles P. Theuer 4 , Todd E. Barnhart 3 & Weibo Cai 1

SCIENTIFIC REPORTS | 4 : 5080 | DOI: 10.1038/srep05080

Synthesis of uniform HMSN. Uniform HMSN with an average particle size of
150 nm was synthesized following the literature procedure with improved Na 2 CO 3 -
etching process 16 . Three major steps were involved in the synthesis of HMSN. Firstly,
synthesis of uniform ,100 nm sized dSiO 2 using a modified Sto ̈ber method 17 . In a
typical synthesis, 35.7 mL of absolute ethanol was mixed with 5 mL water and 0.8 mL
of ammonia and stirred for 5–10 minutes at room temperature. Then 1 mL of TEOS
was added and the mixture was allowed to react at room temperature for 1 h.
Afterward, dSiO 2 nanoparticles was washed with water and ethanol and suspended in
20 mL of water.
Secondly, synthesis of dSiO 2 @MSN. CTAC (2 g) and TEA (20 mg) were dissolved
in 20 mL of high Q water and stirred at room temperature for 1 h. Then, 10 mL of

dSiO 2 water solution was added and stirred at room temperature for 1 h before
addition of 0.15 mL of TEOS. The mixture was stirred for 1 h at 80uC in a water bath
to form dSiO 2 @MSN.
Thirdly, etching of dSiO 2 @MSN to form HMSN. Both mixture and water bath were
cooled down to 50uC followed by addition of 636 mg of sodium carbonate (Na 2 CO 3 ),
which was under constant stirring for 30 min to form HMSN. To remove the CTAC,
the product was extracted for 24 h with a 1 wt% solution of NaCl in methanol at room
temperature. This process was carried out for at least 3 times to ensure complete
removal of CTAC.
Synthesis of HMSN-NH 2 . To functionalize the HMSN surface with –NH 2 groups, as-
synthesized HMSN was first dispersed in 20 mL of absolute ethanol, followed by
addition of 1 mL of APS. The system was sealed and kept at 86–90uC in a water bath
for 24 h. Afterward, the mixture was centrifuged and washed with ethanol for several
times to remove the residual APS. The HMSN-NH 2 could be well-dispersed in water,
and the concentration of –NH 2 groups (nmol/mL) was measured using a Kaiser test kit.
Synthesis of NOTA-HMSN-ZW800-PEG-TRC105. To conjugated HMSN with
ZW800, 2 nmol of ZW800 was mixed with HMSN-NH 2 (with ,100 nmol of -NH 2
groups), and reacted for 2 h at room temperature (pH 8.5–9.0) to form HMSN-
ZW800. Then, p-SCN-Bn-NOTA (,53 nmol) in dimethyl sulfoxide was allowed to
react with HMSN-NH 2 at pH 8.5 to obtain NOTA-HMSN-ZW800. Afterward, 2 mg
(400 nmol) of SCM-PEG 5k -Mal was added and reacted for another 1 h, resulting in
NOTA-HMSN-ZW800-PEG-Mal. NOTA-HMSN-ZW800-PEG-TRC105 could be
obtained by reacting TRC105-SH (2.5 nmol, 50 nmol/mL) with NOTA-HMSN-
ZW800-PEG-Mal (0.5 nmol, 2 nmol/mL).
Cu labeling and serum stability studies. 64 CuCl 2 (,148 MBq) was diluted in
300 mL of 0.1 M sodium acetate buffer (pH 6.5) and added to NOTA-HMSN-
ZW800-PEG-TRC105 or NOTA-HMSN-ZW800-PEG. The reaction was allowed to
proceed at 37uC for 30 min with constant shaking. 64 Cu-NOTA-HMSN-ZW800-
PEG-TRC105 and 64 Cu-NOTA-HMSN-ZW800-PEG were purified using PD-10
columns with PBS as the mobile phase. The radioactivity fractions (typically between
3.0 and 4.0 mL, total of 1 mL) were collected for further in vivo PET imaging
experiments. After 6 mL of PBS, the unreacted 64 Cu may start to elute from the
column. The whole procedure of 64 Cu labeling and purification of the HMSN nano-
conjugates could be completed within 60 min.
For serum stability studies, 64 Cu-HMSN-ZW800-TRC105 was incubated in whole
mouse serum at 37uC for up to 24 h (the time period investigated for serial PET
imaging, which is about two half-lives of 64 Cu). Portions of the mixture were sampled at
different time points and filtered through 10 kDa cutoff filters. The filtrates were col-
lected, and the radioactivity was measured. The percentages of retained (i.e. intact) 64 Cu
on the HMSN-PEG conjugates ( 64 Cu-HMSN-ZW800-TRC105) were calculated using
the equation [(total radioactivity - radioactivity in filtrate)/total radioactivity] 3 100%