pg ( mw 10  kda ) was synthesized by anionic , ringopening , multibranching polymerization of glycidol with slow monomer addition.37 , 38 according to our previous work , 10  kda pg has better dispersity and be synthesized more easily .
alexa fluor  488 wga and hoechst  33342 cell markers were obtained from molecular probes inc .
other reagents were obtained from beijing chemical reagent co. ( beijing , china ) . the water used throughout the study was purified with a milliq integral a10 system from millipore co. ( usa ) .
firstly , we synthesized pgnh2 with 30  % amine functionality according to the literature.23 , 24 for h  nmr spectra of pgnh2 , see figure  s11 .
pgnh2 was dissolved into methanol with a concentration of 20  mg  ml as a stock solution .
firstly , pgnh2 stock solution ( 600  l ) , methanol ( 2  ml ) , and a specific volume teos were mixed and stirred in a sealed vessel for 12  h. in this recipe , 10 , 50 , 100 , or 200  l of teos was added for parallel controls .
then , a mixture of ammonium hydroxide ( 1.5  ml ) and water ( 1.5  ml ) were quickly added into the above solution and stirred vigorously for 6  h. the products were centrifuged at a high speed ( 10  000  rpm , 5  min ) and washed with methanol and water several times .
the final obtained products were marked as pgsns10 , pgsns50 , pgsns100 , and pgsns200 , respectively , according to the different volumes of teos in the recipe .
4  mg ) were incubated in 0.5  mg  ml dox aqueous solution ( 1.33  ml ) for 24  h. the loading efficiency was calculated according to the change of characteristic absorption of dox ( 480  nm ) before and after incubation with pgsns100 . then
the samples ( denoted as pgsnsdox ) were split equally into two parts and dispersed into 1.2  ml of either phosphatebuffered saline ( pbs ) ( ph  7.2 ) or mes buffer ( ph  5.0 ) .
therefore , the concentrations of the pgsnsdox samples in different buffers were the same ( ca .
1.6  mg  ml ) . at a preset time point , the sample suspension was centrifuged , and then 0.3  ml of the supernatant was removed .
the absorbance at 480  nm of the supernatant was measured using a uv / vis spectrometer .
after that , 0.3  ml of fresh buffer solution was added to the sample suspension .
the pgsns100 sample was dispersed into pbs at a low concentration ( 0.1  mg  ml ) following shaking of the culture at 37  c . at a preset time point ,
a series of aliquots ( 5  l ) of the solution was removed and dropped on a copper grid for tem observation . at the same time , 5  ml of the dispersed solution was taken out and filtered by using millipore tubular ultrafiltration centrifugation modules ( molecular weight cut off 10  000 ) .
hela and mcf7 cells were cultured at 37  c in a dmem medium ( gibco brl , usa ) complemented with fbs ( 10  % ) , lglutamine ( 2  mm ) , penicillin ( 100  u  ml ) , and streptomycin ( 25  mg  ml ) in a humidified atmosphere with 5  % co2 . for the following experiments ,
cells were detached from culture flasks by using pbs containing edta ( 0.02  % ) and trypsin ( 0.05  % ) , and seeded to a 35  mm glassbottom petri dish for clsm observation or 96well plates for cytotoxicity measurements .
after the cells were incubated in the logarithmic growth phase , the 2  mg  ml pgsnbased dispersed sample solutions ( 50  l ) were added for 4  h of coculturing .
then , the cells were washed twice with pbs to remove the dispersed nanoparticles and dead cells in the growth media . the cell nuclei and membranes
were stained with hoechst  33342 and alexa  488 wga ( 0.025  mg  ml , 10  l ) for 15  min , respectively .
after that , the cells were washed with pbs three times , and supplemented with fresh cell culture medium .
the cytotoxicity assay was conducted with mcf7 cells in 96well plates grown to 7080  % confluency .
every 4 wells were taken as one group and incubated with different concentration of pgsn , pgsndox and dox for 48  h , respectively .
the dox loading amount was 0.18  mg per mg of pgsns . for every well in each group
, cells were cultured in 0.007 , 0.02 , 0.04 , 0.06 , and 0.08  mg  ml pgsns or pgsnsdox suspension ( in cell culture media ) as parallel experiments . at the same time
, the same amount of dox was added to the cells in another group and cultured as a positive control .
then , sterile filtered cck8 in pbs ( 10  l ) was added to each well and incubated with the cells for 1  h at 37  c , followed by measuring the absorbance at 450  nm with elisa ( tecan infinite 200 ) .
cell viability was expressed as the percentage of viable cells compared with blank control ( cells without particles ) .
fluorophore cy7 was labeled on the surface of pgsns before applying fluorescence imaging in  vivo . in detail , the cy7 nhs ester reacted with pgsns directly in ph  8.0 buffer solution for 24  h in the dark .
cy7labeled pgsns ( cy7pgsns ) were purified by repeatedly washing with saline before further application .
tumorbearing mice were prepared by inoculating 0.2  ml of pbs containing 210 mcf7 cells at the left hind leg of female balb / c nude mice ( 202  g ) , and the tumor was allowed to grow for approximately 7  days .
when the tumor volume reached 600  mm , 200  l cy7pgsn nanoparticles ( in pbs , 1  mg  ml ) was injected through the tail vein . for in  vivo imaging , mice were placed on the warmed stage and anaesthetized with 2.5  % isoflurane .
image acquisition was performed at different time intervals on an in  vivo imaging system . meanwhile ,
the fluorescence intensities of the tumor and the background were analyzed by using the corresponding software .
when the average tumor volume reached 120  mm , the balb / c nude mice ( n=15 ) were randomly and equally divided into three groups .
free dox ( 200  l , in pbs ) or pgsnsdox ( 200  l , in pbs ) was intravenously injected into the tail vein of every animal in the other two respective groups .
the concentration of free dox was 0.12  mg  ml . the dispersed solution of pgsnsdox had a concentration of 2.5  mg  ml , containing an equal amount of dox , that is , 0.12  mg  ml .
mice were treated either with the drug or saline ( control ) every 2  days with a total of 5  doses per rat .
on the seventh day of posttumor implantation , the mice were sacrificed randomly , and the tissue was extracted and fixed by 4  % formaldehyde solution overnight ; tumors were completely excised , and the tumor volume was calculated by using equation  ( 1):(1)volume(mm3)=v=0.5ab2 
  where a and b are the maximum and minimum diameters of the tumor , respectively .
the tumor growth inhibition was calculated by using equation  ( 2):(2)tumorgrowthinhibition(%)=[(volumeincontrolgroup - volumeintreatedgroup)(volumeincontrolgroup)]100 
  where  volume  always refers to the volume of the tumor at the endpoint .
statistical significance was evaluated by using the oneway analysis of variance ( anova ) and student 's ttest .
multiple comparisons were statistically analyzed by using spss software version 13.0 ( significance was established at p<0.05 ) .
clsm images were taken with an olympus fv1000 confocal system , which has a 60oilimmersion objective and a numerical aperture of 1.4 .
the tem images and selectedarea electron diffraction ( saed ) patterns were acquired by using a jem1011 and jem2011(jeol , japan ) .
the scanning electron microscopy ( sem ) images were obtained with an s4800 instrument with 10  kv accelerating voltage ( hitachi , japan ) .
the degradation of pgsns was monitored by using both tem morphology investigation and analysis of the above supernatant through icp  oes ( thermo icap 6300 ) . in  vivo fluorescence imaging
was performed at different time intervals on the in  vivo imaging system ( fx pro , carestream health ) .
the excitation and emission bandpass filters were at 740 and 760  nm , respectively .
the fluorescence intensities of the tumor and the background were analyzed by using corresponding software .
zeta potential and size distribution were documented by using a dynamic light scattering technique ( zetasizer nano , malvern ) .
pg ( mw 10  kda ) was synthesized by anionic , ringopening , multibranching polymerization of glycidol with slow monomer addition.37 , 38 according to our previous work , 10  kda pg has better dispersity and be synthesized more easily .
alexa fluor  488 wga and hoechst  33342 cell markers were obtained from molecular probes inc .
other reagents were obtained from beijing chemical reagent co. ( beijing , china ) . the water used throughout the study was purified with a milliq integral a10 system from millipore co. ( usa ) .
firstly , we synthesized pgnh2 with 30  % amine functionality according to the literature.23 , 24 for h  nmr spectra of pgnh2 , see figure  s11 .
pgnh2 was dissolved into methanol with a concentration of 20  mg  ml as a stock solution .
firstly , pgnh2 stock solution ( 600  l ) , methanol ( 2  ml ) , and a specific volume teos were mixed and stirred in a sealed vessel for 12  h. in this recipe , 10 , 50 , 100 , or 200  l of teos was added for parallel controls .
then , a mixture of ammonium hydroxide ( 1.5  ml ) and water ( 1.5  ml ) were quickly added into the above solution and stirred vigorously for 6  h. the products were centrifuged at a high speed ( 10  000  rpm , 5  min ) and washed with methanol and water several times .
the final obtained products were marked as pgsns10 , pgsns50 , pgsns100 , and pgsns200 , respectively , according to the different volumes of teos in the recipe .
4  mg ) were incubated in 0.5  mg  ml dox aqueous solution ( 1.33  ml ) for 24  h. the loading efficiency was calculated according to the change of characteristic absorption of dox ( 480  nm ) before and after incubation with pgsns100 .
the samples ( denoted as pgsnsdox ) were split equally into two parts and dispersed into 1.2  ml of either phosphatebuffered saline ( pbs ) ( ph  7.2 ) or mes buffer ( ph  5.0 ) .
therefore , the concentrations of the pgsnsdox samples in different buffers were the same ( ca .
the sample suspension was centrifuged , and then 0.3  ml of the supernatant was removed .
the absorbance at 480  nm of the supernatant was measured using a uv / vis spectrometer .
after that , 0.3  ml of fresh buffer solution was added to the sample suspension .
the pgsns100 sample was dispersed into pbs at a low concentration ( 0.1  mg  ml ) following shaking of the culture at 37  c . at a preset time point ,
a series of aliquots ( 5  l ) of the solution was removed and dropped on a copper grid for tem observation . at the same time , 5  ml of the dispersed solution was taken out and filtered by using millipore tubular ultrafiltration centrifugation modules ( molecular weight cut off 10  000 ) .
hela and mcf7 cells were cultured at 37  c in a dmem medium ( gibco brl , usa ) complemented with fbs ( 10  % ) , lglutamine ( 2  mm ) , penicillin ( 100  u  ml ) , and streptomycin ( 25  mg  ml ) in a humidified atmosphere with 5  % co2 . for the following experiments ,
cells were detached from culture flasks by using pbs containing edta ( 0.02  % ) and trypsin ( 0.05  % ) , and seeded to a 35  mm glassbottom petri dish for clsm observation or 96well plates for cytotoxicity measurements .
after the cells were incubated in the logarithmic growth phase , the 2  mg  ml pgsnbased dispersed sample solutions ( 50  l ) were added for 4  h of coculturing .
then , the cells were washed twice with pbs to remove the dispersed nanoparticles and dead cells in the growth media . the cell nuclei and membranes
were stained with hoechst  33342 and alexa  488 wga ( 0.025  mg  ml , 10  l ) for 15  min , respectively .
after that , the cells were washed with pbs three times , and supplemented with fresh cell culture medium .
the cytotoxicity assay was conducted with mcf7 cells in 96well plates grown to 7080  % confluency .
every 4 wells were taken as one group and incubated with different concentration of pgsn , pgsndox and dox for 48  h , respectively .
for every well in each group , cells were cultured in 0.007 , 0.02 , 0.04 , 0.06 , and 0.08  mg  ml pgsns or pgsnsdox suspension ( in cell culture media ) as parallel experiments . at the same time
, the same amount of dox was added to the cells in another group and cultured as a positive control .
then , sterile filtered cck8 in pbs ( 10  l ) was added to each well and incubated with the cells for 1  h at 37  c , followed by measuring the absorbance at 450  nm with elisa ( tecan infinite 200 ) .
cell viability was expressed as the percentage of viable cells compared with blank control ( cells without particles ) .
fluorophore cy7 was labeled on the surface of pgsns before applying fluorescence imaging in  vivo . in detail , the cy7 nhs ester reacted with pgsns directly in ph  8.0 buffer solution for 24  h in the dark .
cy7labeled pgsns ( cy7pgsns ) were purified by repeatedly washing with saline before further application .
tumorbearing mice were prepared by inoculating 0.2  ml of pbs containing 210 mcf7 cells at the left hind leg of female balb / c nude mice ( 202  g ) , and the tumor was allowed to grow for approximately 7  days .
when the tumor volume reached 600  mm , 200  l cy7pgsn nanoparticles ( in pbs , 1  mg  ml ) was injected through the tail vein . for in  vivo imaging , mice were placed on the warmed stage and anaesthetized with 2.5  % isoflurane .
image acquisition was performed at different time intervals on an in  vivo imaging system . meanwhile ,
the fluorescence intensities of the tumor and the background were analyzed by using the corresponding software .
when the average tumor volume reached 120  mm , the balb / c nude mice ( n=15 ) were randomly and equally divided into three groups .
free dox ( 200  l , in pbs ) or pgsnsdox ( 200  l , in pbs ) was intravenously injected into the tail vein of every animal in the other two respective groups .
the concentration of free dox was 0.12  mg  ml . the dispersed solution of pgsnsdox had a concentration of 2.5  mg  ml , containing an equal amount of dox , that is , 0.12  mg  ml .
mice were treated either with the drug or saline ( control ) every 2  days with a total of 5  doses per rat .
on the seventh day of posttumor implantation , the mice were sacrificed randomly , and the tissue was extracted and fixed by 4  % formaldehyde solution overnight ; tumors were completely excised , and the tumor volume was calculated by using equation  ( 1):(1)volume(mm3)=v=0.5ab2 
  where a and b are the maximum and minimum diameters of the tumor , respectively .
the tumor growth inhibition was calculated by using equation  ( 2):(2)tumorgrowthinhibition(%)=[(volumeincontrolgroup - volumeintreatedgroup)(volumeincontrolgroup)]100 
  where  volume  always refers to the volume of the tumor at the endpoint .
statistical significance was evaluated by using the oneway analysis of variance ( anova ) and student 's ttest .
multiple comparisons were statistically analyzed by using spss software version 13.0 ( significance was established at p<0.05 ) .
clsm images were taken with an olympus fv1000 confocal system , which has a 60oilimmersion objective and a numerical aperture of 1.4 .
the tem images and selectedarea electron diffraction ( saed ) patterns were acquired by using a jem1011 and jem2011(jeol , japan ) .
the scanning electron microscopy ( sem ) images were obtained with an s4800 instrument with 10  kv accelerating voltage ( hitachi , japan ) .
the degradation of pgsns was monitored by using both tem morphology investigation and analysis of the above supernatant through icp  oes ( thermo icap 6300 ) . in  vivo fluorescence imaging
was performed at different time intervals on the in  vivo imaging system ( fx pro , carestream health ) .
the excitation and emission bandpass filters were at 740 and 760  nm , respectively .
the fluorescence intensities of the tumor and the background were analyzed by using corresponding software .
zeta potential and size distribution were documented by using a dynamic light scattering technique ( zetasizer nano , malvern ) .
as a service to our authors and readers , this journal provides supporting information supplied by the authors .
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 abstractmesoporous silicabased nanoparticles are generally accepted as a potential platform for drug loading with a lot of advantages , except for their complex purification procedures and structures that are difficult to decompose . in this work , 
 biocompatible hyperbranched polyglycerol is introduced to synthesize mesoporous silica nanoparticles ( msns ) . 
 the materials possess good biocompatibility , controlled release , and biodegradability . 
 they also show passive targeting capability through the enhanced permeability and retention effect and can be excreted from the biological system . 
 the method avoids the needs to employ traditional surfactants and complicated purified procedures , which make these msns an efficient delivery system for cancer therapy . 
Experimental Section
Materials
Preparation of PGSNs
Guest Molecules Loaded into PGSNs and Released in Response to pH
Biodegradability of Silica Nanostructures
Cell Culture
Staining of Cells for CLSM Investigation
Cytotoxicity Assay of PGSNs and PGSNsDox InVitro
InVivo Fluorescence Imaging
Tumor Therapy InVivo
Characterization and Instrumentation
Supporting information