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 . such materials are peer reviewed and may be reorganized for online delivery , but are not copyedited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors .