Editing Silica Nanoparticles

== Chemicals<br>  ==

Sigma:<br> 

Surfactant: Aerosol OT (98%)<br> 

cosurfactants, 1-butanol (99.8%)<br> 

triethoxyvinylsilane: VTES (97%)<br> 

[[Image:Triethoxyvinylsilane.png]] 

99% (Aldrich) <br> 

175560-100ML: $37.40 <br> 

175560-500ML: $115.00<br> 

<br>≥98% (Sigma-Aldrich) <br> 

A3648-100ML $61.70<br> 

A3648-500ML: $212.50<br> 

<br>≥98.0%<br> 

440140-100ML: $64.40<br> 

440140-500ML: $221.50<br> 

<br> 

(3-Aminopropyl)triethoxysilane , APTES (99%)<br> 

[[Image:(3-Aminopropyl)triethoxysilane.png]] 

A3648-100ML: $61.70<br> A3648-500ML: $212.50<br> 

<br> 

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.<br> 

<br> 

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.<br> 

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== Synthesis<br>  ==

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{| width="200" cellspacing="1" cellpadding="1" border="1"
|-
| Name<br> 
| 
Aerosol-OT 

g<br> 

| 
nBuOH<br> 

µl 

| 
Water 

ml<br> 

| 
DMSO<br> 

µl 

| 
VTES 

µl<br> 

| 
APTES<br> 

µl 

| Diameter nm<br>
|-
| ORMA40<br> 
| 0.44<br> 
| 800<br> 
| 20<br> 
| 100<br> 
| 200<br> 
| 40<br> 
| 15-20<br>
|-
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br> 
| <br>
|}

<br> 

<br>dissolve&nbsp; Aerosol-OT and 1-butanol in 20 ml of double-distilled water by vigorous magnetic stirring. <br> 

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

Add 100 µl dye in DMSO or just DMSO<br>Add 200 µl of neat VTES&nbsp; to the micellar system<br> 

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

Precipitate ORMOSIL nanoparticles by adding aqueous <br> 

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

Stirring for ~20 h at room temperature. <br> 

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. <br> 

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

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

<br> 

<br> 

=== Use commercial nanoparticles  ===

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

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

Incubate at room temper- ature for at leas&nbsp; 2 h <br> 

&nbsp;All modifications used constant molar concentration of silane with each modification having differing amounts of APTES and MTES; <br> 

A100M0 was modified in a solution of 100% APTES, <br> 

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

Recover nanoparticles by spin.<br> 

Wash repeatedly in ethanol followed by water. <br> 

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

<br> 

== Transfection<br>  ==

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

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

Mix two solutions together gently 

Incubated at room temperature for 30 min.<br>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<br> 

<br> 

== Silicalite nanoparticles<br>  ==

=== 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, <br> 

TPAOH: tetrapropylammonium hydroxide.&nbsp; <br> 

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

The resulting solution is filtered to separate the functionalized nanoparticles<br> 

<br> 

Transfection 

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

Plasmid DNA&nbsp; is&nbsp; 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<br>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<br>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<br>serum containing medium for 44 h.<br> 

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=== 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&nbsp;: 1&nbsp;: 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&nbsp;: 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,<br>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<br>water-in-oil microemulsion without rigorous experimental conditions, such as high temperature, high pressure, etc. The size of the nanoparticles can be controlled through<br>varying the molar ratio of water to surfactant (R w ) and the molar ratio of TEOS to the ammonia (R N 19 ,20 J• 

<br> 

amino-modified silica nanoparticles are directly produced by the synchronous hydrolysis of tetraethoxysilane and N-(beta-amimoethyl)-gamma-aminopropyltri-ethoxysilane in<br>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<br>modify with aminosilanes, which provide a new, simple and safe method for the application of nanoparticles in the nanoparticles in the biomedicine research.<br>age of the amino-modified nanoparticles nanoparticles are (50 ± 4) urn in size I mg nanoparticles can bind 97.2 flg plasmid DNA<br> 

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=== Protocol 3<br>  ===

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

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

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

<br> 

<br> 

== Study on the Effect of Synthesis Parameters of SilicaNanoparticles Entrapped with Rifampicin<br>  ==

DOI: 10.3303/ACOS1311044<br> 

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

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

<br> 

[[Image:Silica nanoparticle size.png]] 

<br>

== Engineering of Hollow Mesoporous Silica Nanoparticles for Remarkably Enhanced Tumor Active Targeting Efficacy<br> ==

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 &amp; Weibo Cai 1<br>

SCIENTIFIC REPORTS | 4 : 5080 | DOI: 10.1038/srep05080<br>

<br>

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

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

[[Category:Transfection]] [[Category:Paul]]