Editing Silica Nanoparticles (section)

== 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>

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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>

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