numerous studies have 
 revealed the importance of tams on tumor 
 growth and development .
they are present in both tumor stroma and nests , accounting for up to 50% of tumor mass .
tams contribute to tumor growth by producing stromal breakdown factors 
 and by suppressing adaptive immunity .
moreover , 
 tams induce chemoresistance by inhibiting tumor cell apoptosis . recently , there is also compelling evidence 
 that tam infiltration in tumors is correlated with poor prognosis 
 of many cancers , including breast , pancreatic , ovarian , prostate , 
 cervix , bladder cancer , and certain types of glioma and lymphoma in 
 patients .
tams are thus considered a potentially viable target in designing 
 innovative imaging and therapy strategies .
in fact , there are various 
 reported methods to target tams . among them , using the membrane receptors 
 on tams is commonly explored .
for example , the endocytic cd163 protein was recently 
 proposed as a tam target for anticancer and antiangiogenesis drug 
 design .
folate receptor- , another 
 protein commonly expressed on the surface of macrophages , had also 
 been exploited to target toxins to tams .
because tams overexpress mannose receptor ( mr ) , there had been efforts to increase the delivery of oligos , 
 dna , imaging agents , and vaccines to tams by surface - modifying the 
 delivery systems with mannose derivatives , anti - mr nanobody , or galactose derivative .
unfortunately , many of the previously reported 
 delivery systems can be inevitably taken up by macrophages that are 
 not in tumors because those macrophages often express similar membrane 
 receptors as the tams do . to address 
 this issue , we recently constructed an acid - sensitive 
 sheddable pegylated , mannose - modified nanoparticle platform ( as - m - nps ) . 

the nanoparticles are prepared with plga , surface - modified with mannose , 
 and pegylated with an acid - sensitive peg amphiphile , peg - hydrazone - c18 
 ( or phc ) , which was synthesized by conjugating peg ( molecular weight , 
 2000 ) with stearic hydrazide using a hydrazone bond .
injection , 
 the long flexible peg chains shield the mannose on the surface of 
 the nanoparticles and prevent the interaction of the nanoparticles 
 with macrophages before the nanoparticles reach tumors .
once the nanoparticles 
 accumulate in tumors by the enhanced permeability and retention ( epr ) 
 effect , however , the slightly acidic 
 tumor microenvironment (  ph 6.84 ) helps catalyze the hydrolysis of the hydrazone bond and facilitate 
 the shedding of the peg chains , exposing the mannose for interaction 
 with mrs on the surface of tams . previously , we showed fluorescence 
 images of the histological sections of tumors in mice injected with 
 fluorescein - labeled as - m - nps , which indicated that the as - m - nps can 
 potentially target tams .
however , direct 
 evidence is needed to demonstrate that the as - m - nps can indeed target 
 tams . in the present report , we present ex vivo fluorescence 
 imaging data to demonstrate that surface - modification of plga nanoparticles 
 incorporated with the self - fluorescent dox with acid - sensitive sheddable 
 peg and mannose can effectively target the dox into tumors by interacting 
 with tams . moreover , by taking advantage of the cytotoxicity of dox , 
 we showed that the dox - as - m - nps were more effective than free dox 
 in inhibiting tumor growth . the dox - as - m - nps significantly decreased 
 tam population in tumors , but showed only minimum effect on macrophages 
 in mouse mps organs such as liver and spleen .
o - stearoyl mannose ( m - c18 ) , polyethylene 
 glycol 2000-hydrazone - c18 ( phc ) , and polyethylene glycol 2000-amide - c18 
 ( pac ) were synthesized following our previously published methods .
zoledronic acid , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 
 bromide ( mtt ) , plga ( 752h ) , and poly - d - lysine were from sigma - aldrich 
 ( st .
hematoxylin - eosin ( h&e ) and anti - cd31 antibody 
 were from abcam ( cambridge , ma ) .
the 5-bromo-2-deoxyuridine ( brdu ) and primary 
 brdu monoclonal antibody were from bd biosciences ( san jose , ca ) . 

anti - cd206 , rm0029 - 11h3 , and fitc - labeled anti - cd206 antibody were 
 from santa cruz biotechnology , inc .
j774a.1 macrophage cells , b16-f10 
 murine melanoma cells , bxpc-3 human pancreatic cancer cells , and tc-1 
 murine lung cancer cells were from american type culture collection 
 ( atcc , manassas , va ) and cultured in dmem ( or rpmi1640 for bxpc-3 ) 
 at 37 c and 5% co2 .
media were supplemented with 
 10% fetal bovine serum ( fbs ) , 100 u / ml of penicillin , and 100 g / ml 
 of streptomycin , all from invitrogen ( carlsbad , ca ) .
female c57bl/6 
 mice ( 68 weeks ) and male athymic nude mice were from charles 
 river laboratories ( wilmington , ma ) .
animal studies were performed 
 in accordance with the national research council guide for the care 
 and use of laboratory animals .
animal protocol was approved by the 
 institutional animal care and use committee at the university of texas 
 at austin .
briefly , 0.9 ml of tetrahydrofuran ( thf ) containing plga 752h ( 3 
 mg ) and dox ( 0.3 mg ) was added dropwise into 4.5 ml of water under 
 stirring .
the nanoparticles were collected by centrifugation ( 13  000 
  g , 10 min , 4 c ) after the evaporation 
 of thf . for the purpose of surface modification , m - c18 ( 1.2 mg ) , pac 

( 1.2 mg ) , or phc ( 3.6 mg ) were dissolved together with plga and dox 
 in thf and added into water .
particle 
 sizes and zeta potentials of the nanoparticles were determined using 
 a malvern zeta sizer nano zs ( westborough , ma ) .
the morphology of 
 the nanoparticles was analyzed using a zeiss supra 40 vp scanning 
 electron microscope ( sem ) ( zeiss smt ag , oberkochen , germany ) in the 
 icmb microscopy and imaging facility at the university of texas at 
 austin .
the entrapment efficiency ( ee ) 
 of dox was determined spectrophotometrically at 490 nm by measuring 
 the amount of free unentrapped dox in the external aqueous solution 
 after centrifugation of the nanoparticles in suspension for 5 min 
 at 13  000  g. to determine the total 
 amount of dox in the nanoparticles , 500 l of nanoparticles 
 in suspension were mixed with 4.5 ml of dimethyl sulfoxide ( dmso ) 
 for 5 min by sonication , and the concentration of dox was determined . 

the ee was calculated according to the following equation : the rate at which dox 
 was released from nanoparticles was measured 
 as a function of time when the nanoparticles were incubated in phosphate - buffered 
 saline ( pbs , ph 7.4 or 6.8 , 10 mm ) .
triplicate samples of 5 mg of 
 nanoparticles were suspended in 0.5 ml of pbs and sonicated briefly 
 in an ultrasonic water bath .
the samples were then incubated in an 
 orbital shaker at 37 c , 100 rpm . at various time points
, the 
 particles were centrifuged at 13  000  g for 5 min , and 100 l of supernatant was removed and replaced 
 with fresh pbs .
the fluorescence intensity of dox in the supernatant 
 was measured using a biotek synery ht multi - mode microplate reader 
 ( winooski , vt , usa ) at excitation 470 nm / emission 590 nm to determine 
 the dox released from nanoparticles .
j774a.1 
 cells were seeded with a density of 5  10 cells 
 per well on poly - d - lysine - precoated glass coverslips , placed 
 inside wells of a 6-well tissue culture plate , and incubated overnight . 
 on the second day , cell culture medium was replaced with 2.5 ml of 
 50 m dox in various nanoparticles ( i.e. , dox - nps , dox - m - nps , 
 dox - ai - m - nps , and dox - as - m - nps ) .
cells were incubated for 20 min , 
 followed by five additional minutes of incubation with 50 m 
 hoechst 33342 in a 37 c incubator and protected from light . 

cells were then washed with pbs three times , fixed with 4% paraformaldehyde , 
 and observed under a fluorescence microscope ( olympus bx 53 , center 
 valley , pa ) connected to the olympus cellsens dimension software . 
 to investigate the effect of ph on the cellular uptake
, nanoparticles 
 were preincubated with pbs ( ph 6.8 ) at 37 c for 6 h before further 
 incubating with cells . in order to track the intracellular fate 
 of nanoparticles ,
dox - as - m - nps were prepared with 5% ( w / w ) of plga 
 752h that was conjugated with fluorescein isothiocyanate ( fitc ) .
cells were incubated with nanoparticles for 
 15 , 30 , or 60 min , followed by five additional minutes of incubation 
 with hoechst 33342 .
cells were then washed , fixed , and observed under 
 a microscope as mentioned above .
b16-f10 
 cells ( 80% confluence ) were plated at a cell density of 5  10 cells / well into 96-well plates . after 24 h of incubation 
 at 37 c with 5% co2 ,
the growth medium was removed , 
 and the cells were incubated for another 24 h with various dox formulations , 
 with dox concentrations ranging from 0.001 to 10 g / ml .
controls 
 included cells incubated with dox - free medium or blank nanoparticles 
 ( i.e. , dox - free as - m - nps ) .
cells were washed twice with pbs and incubated 
 with 20 l of mtt ( 5 mg / ml ) solution for 4 h. formazan crystals 
 were solubilized with 150 l of dmso .
cell viability was calculated by 
 comparing the absorbance with the untreated cells . to evaluate 
 the cytotoxicity of the dox - nps in macrophages ,
j774a.1 cells were 
 seeded into 96-well plates at a cell density of 1  10 cells / well .
after 24 h of incubation , 
 cells were treated with free dox or various dox - nps , and cell viability 
 was determined 24 h later using an mtt assay as mentioned above .
similarly , 
 controls included cells incubated with dox - free medium or dox - free 
 as - m - nps .
c57bl/6 mice were subcutaneously 
 ( s.c . ) injected with b16-f10 cells ( 5  10/mouse ) 
 in the right flank .
injected with pbs , free dox solution , dox - nps , dox - m - nps , 
 dox - ai - m - nps , dox - as - nps , or dox - as - m - nps ( dox dose , 10 mg / kg ) .
mice 
 were euthanized 6 h later to collect blood , tumor , and major organs 
 ( e.g. , heart , kidneys , liver , spleen , and lung ) .
all samples were 
 then imaged using an ivis spectrum ( caliper , hopkinton , ma ) ( em / ex 
 of 465/600 nm ) . in order to evaluate the effect of tams on the 
 uptake of the dox - as - m - nps by tumors in mice ,
b16-f10 tumor - bearing 
 c57bl/6 mice were intraperitoneally ( i.p . ) injected with zoledronic 
 acid ( 5 mg / kg ) 7 days after tumor cell injection to reduce macrophages 
 in mice .
two days after the last injection 
 ( i.e. , 25 days after tumor cells injection ) , mice were i.v .
injected 
 with dox - as - m - nps ( dox , 10 mg / kg ) . as controls ,
b16-f10 tumor - bearing 
 mice that were not treated with zoledronic acid were i.v .
mice were euthanized 6 h later to 
 collect tumor and major organs for ex vivo imaging 
 using ivis spectrum .
tumor tissues were also stained with rm0029 - 11h3 , 
 a macrophage marker , to confirm the reduction of tams by treatment 
 with zoledronic acid . finally , to evaluate the kinetics of the 
 biodistribution of the 
 dox - as - m - nps in mice , b16-f10 tumor - bearing mice were i.v .
injected 
 with dox - as - m - nps ( 10 mg dox / kg ) , and three mice were euthanized 6 , 
 12 , 24 , or 48 h later to collect tumor , blood , and major organs , which 
 were then imaged using the ivis spectrum . for mice bearing bxpc-3 
 and tc-1 tumor ,
when tumor grew to around 
 7 mm in diameter , mice were grouped and i.v .
injected with dox - as - m - nps , 
 free dox , or sterile pbs .
mice were euthanized 6 h ( bxpc-3 tumor - bearing 
 mice ) or 12 h ( tc-1 tumor - bearing mice ) later to collect tumors and 
 major organs for ex vivo imaging .
mice were s.c . injected with b16-f10 cells ( 5  10/mouse ) in the right flank on day 0 . on day 6 ,
mice were randomized 
 ( n = 8) and i.v . injected with pbs , free dox solution , 
 or dox - as - m - nps .
tumor volume was calculated using the following equation : tumor volume = ( length  width)/2 . on day 16 , mice were euthanized to collect tumor , liver , and 
 spleen .
injected with brdu , 100 g / g body weight , 
 30 min prior to euthanasia .
tumor tissues were stained with 
 h&e or anti - cd206 , anti - cd31 , or anti - brdu antibodies .
when tumors reached about 9 mm , mice were randomized and i.v . 
 injected with pbs , free dox , or various dox - nanoparticles .
tumor cell suspensions were stained with fitc - labeled anti - cd206 antibodies 
 ( 1:200 dilution ) for 20 min on ice and washed 3 times with pbs .
cells 
 were then analyzed using a bd facs aria flow cytometer ( san jose , 
 ca ) .
the percent of cd206 cells that took up dox ( i.e. , 
 dox / cd206% ) was analyzed with the flow jo 
 software ( tree star inc .
dead cells and cells debris 
 were excluded based on the fsc - ssc plot .
live cells were then plotted 
 in the green ( fitc ) vs red ( dox ) dot plot quadrants .
double negative 
 control was the tumor tissue single cell suspension from mice that 
 were injected with sterile pbs .
positive control was the fitc - anti - cd206 
 stained tumor tissue single cells isolated from mice that were injected 
 with sterile pbs .
positive control for the dox was the tumor tissue 
 single cell suspension from mice that were injected with sterile pbs , 
 and the cells were coincubated with a dox solution in culture for 
 20 min .
statistical analyses were completed by performing 
 anova followed by fisher s protected least significant difference 
 procedure .
the following dox - incorporated 
 nanoparticles were prepared : dox - nps ( dox - incorporated plga nanoparticles 
 without surface modification ) , dox - m - nps ( dox - nps that were surface - modified 
 with mannose but not pegylated ) , dox - as - nps ( dox - nps that were pegylated 
 with acid - sensitive sheddable peg , but not surface - modified with mannose ) , 
 dox - as - m - nps ( dox - nps that were surface - modified with mannose and 
 pegylated with acid - sensitive sheddable peg ) , and dox - ai - m - nps ( dox - nps 
 that were surface - modified with mannose and pegylated with the acid - insensitive 
 peg2000-amide - c18 ) ( figure 1a ) .
all dox - loaded 
 nanoparticles were around 130150 nm in diameter , with a narrow 
 size distribution ( see polydispersity indices ) ( figure 1a ) .
the zeta potentials of dox - loaded nanoparticles were 24 
 to 35 mv ( figure 1a ) .
the nanoparticles were spherical ( e.g. , dox - as - m - nps , figure 1b ) .
the release of the dox from the nanoparticles 
 was biphasic ; a typical burst release phase was followed by a slower 
 release phase ( figure 1c ) .
the cytotoxicity 
 of the dox - loaded nanoparticles was tested in b16-f10 mouse melanoma 
 cells and in j774a.1 mouse macrophage cells . in b16-f10 cells , the 
 ic50 values of all dox - loaded nanoparticles
were not different 
 from one another , but they were significantly higher than that of 
 free dox ( figure 1d ) . in j774a.1 cells , except 
 the dox - m - nps , the ic50 values of all other dox - loaded 
 nanoparticles were not different from that of free dox ( figure 1d ) .
however , after the dox - as - m - nps were preincubated 
 in a ph 6.8 buffer for 6 h to facilitate the shedding of the peg chains 
 before they were added into cells , their ic50 value in 
 j774a.1 cells was significantly decreased ( by more than 50% ) ( figure 1d ) . the cytotoxicity of dox - as - m - nps after preincubation 
 at ph 6.8 was evaluated only in j774a.1 cells because there is evidence 
 that the murine macrophages cells express mannose receptor .
( b ) a representative sem graph of dox - as - m - nps .
( c ) the in 
 vitro release profile of dox from dox - as - m - nps ( inset , the 
 release profile in the initial 8 h ) .
( d ) ic50 values of 
 free dox and various dox - nanoparticles in b16-f10 cells and j774a.1 
 cells . as a control ,
the cytotoxicity of the dox - free as - m - nps was 
 also evaluated ( * the ic50 values of the dox - free as - m - nps 
 are the equivalent dox concentrations , if the as - m - nps were incorporated 
 with dox ) . data in panels a , c , and d are mean  sd ( n  3 ) . in panel
d , p < 0.05 . to verify the surface modification 
 of dox - nanoparticles ( i.e. , pegylation and/or mannose - modification ) , 
 the uptakes of various dox - loaded nanoparticles ,
dox - nps , dox - m - nps , 
 dox - ai - m - nps , and dox - as - m - nps , by j774a.1 mouse macrophages were 
 evaluated microscopically after the nanoparticles were preincubated 
 in pbs ( ph 6.8 ) for 6 h. a minimum 6 h of incubation is needed for 
 the shedding of 50% of the peg chains . without the 6 h preincubation at
ph 6.8 , only the uptake of the 
 dox - m - nps was extensive ( figure 2a ) .
however , 
 after 6 h of preincubation , the cellular uptake of the dox - as - m - nps , 
 but not the dox - ai - m - nps , was significantly increased to a level similar 
 to that of the dox - m - nps ( figure 2a ) .
( a ) uptake of different dox - nanoparticles ( red ) that were 
 preincubated or not , at ph 6.8 for 6 h. cells were incubated with 
 dox - nanoparticles for 20 min .
( b ) intracellular location of dox ( red ) 
 or dox - as - m - nps ( green ) after incubated with j774a.1 cells for 20 
 min , 30 min , or 1 h. cell nuclei were stained with hoechst ( blue ) .
the red fluorescent signals in 
 figure 2a 
 were from the dox and were an indirect indication of the uptake of 
 the dox - nanoparticles .
a further step was taken by labeling the as - m - nps 
 with fluorescein ( fitc was chemically conjugated to plga molecules ) 
 to directly observe the cellular uptake of the nanoparticles .
as shown 
 in figure 2b , dox ( red fluorescence signal ) 
 was observed in the cytoplasm of cells in 20 min , and within 1 h after 
 incubation , it was in both cytoplasm and cell nuclei ( i.e. , overlap 
 of red and blue signals ) .
fitc - labeled nanoparticles ( green signals ) 
 were observed in the cytoplasm in 20 min as well , and the fluorescence 
 intensity in the cytoplasm was increased as the incubation time was 
 increased ( to 1 h ) ( figure 2b ) . however , the 
 green fluorescence signals remained in the cytoplasm , not detectable 
 in the cell nuclei ( figure 2b ) .
the dox that 
 accumulated in the cell nuclei may be released from the dox - as - m - nps 
 before and/or after the cellular internalization of the nanoparticles . to evaluate the 
 extent to which the as - m - nps can deliver dox into tumors , while minimizing 
 its accumulation in mps organs ( e.g. , liver and spleen ) , the distribution 
 of dox in tumors , blood , and major organs in c57bl/6 mice with pre - established 
 subcutaneous ( s.c . )
injected into the mice . as shown in figure 3a ( and figure s1 , supporting 
 information ) , in mice that were injected with dox - nps or dox - m - nps , 
 especially the dox - m - nps ,
significant dox accumulation was observed 
 in mouse liver , spleen , lung , and kidneys , but not in tumors , 6 h 
 after the injection . in mice that were injected with the dox - ai - m - nps , 

dox distribution in the liver , spleen , lung , and kidneys of the mice 
 was reduced , but increased in tumor ( figures 3a and s1 , supporting information ) . in 
 mice that were injected with the dox - as - m - nps , dox distribution in 
 tumor
was further increased , as compared to in mice that were injected 
 with dox - ai - m - nps ( figures 3a and s1 , supporting information ) . shown in figure 3b
are the fluorescence intensities of dox in the 
 mouse blood 6 h after mice were injected with free dox or various 
 dox - nanoparticles .
the fluorescence intensity was higher in mice that 
 were injected with dox - ai - m - nps or dox - as - m - nps and relatively lower 
 in mice that were injected with the dox - nps or dox - m - nps ( figure 3b ) .
d 
 ( and figure s2 , supporting information ) 
 are the biodistribution of the dox - as - m - nps in s.c .
b16-f10 tumor - bearing 
 mice 6 , 12 , 24 , and 48 h after i.v . injection .
the fluorescence intensity 
 of dox - as - m - nps peaked in tumors about 24 h after i.v .
injection , 
 whereas the contents dox - as - m - nps in blood and other organs slowly 
 decreased as a function of time ( figures 3c , d 
 and s2 , supporting information ) .
representative ex vivo fluorescence images of b16-f10 tumors and other 
 major organs ( a ) and mean fluorescent intensity of dox in mouse blood 
 samples ( b ) , 6 h after mice were i.v . injected with pbs , dox , dox - nps , 
 dox - m - nps , dox - ai - m - nps , or dox - as - m - nps ( p < 0.05 ) .
( c ) representative ex vivo images of b16-f10 tumors and other major organs 6 , 12 , 24 , or 48 
 h after mice were i.v . injected with dox - as - m - nps .
( d ) mean fluorescent 
 intensity of dox in tumors , organs , and blood at various time points 
 after mice were i.v .
( e ) representative ex vivo images of b16-f10 tumors and other major organs 
 6 h after mice were injected with dox - as - m - nps or dox - as - nps .
imaging 
 was repeated in at least 3 mice , with similar trends ( t = tumor , k 
 = kidneys , h = heart , lr = liver , s = spleen , and ln = lung ) . in order to verify the function 
 of the mannose - modification on 
 the biodistribution of the nanoparticles ,
the biodistribution of the 
 dox - as - m - nps in b16-f10 tumor - bearing mice was compared with that 
 of the dox - as - nps .
the dox - as - nps was significantly lower than 
 in mice that were i.v .
injected with the dox - as - m - nps ( figures 3e and s3 , supporting information ) . finally , to confirm that the dox - as - m - nps are effective 
 in delivering 
 dox into tumors other than the b16-f10 tumors , athymic nude mice with 
 s.c .
injected tc-1 mouse lung cancer cells were used . as shown 
 in figure 4 ,
dox - as - m - nps significantly increased 
 the delivery of dox in the bxpc-3 tumors ( figure 4a ) and tc-1 tumors ( figure 4b ) , as 
 compared to free dox .
( a ) relative fluorescence intensities of dox in tumors 
 and major 
 organs of athymic nude mice 6 h after they were i.v . injected with 
 dox - as - m - nps or free dox .
( b ) relative fluorescence intensities of 
 dox in tumors and major organs of c57bl/6 mice 12 h after they were 
 i.v . injected with dox - as - m - nps or free dox . imaging was repeated 
 in 23 mice / group ( k = kidneys , h = heart , and s = spleen ) 
 ( * p < 0.05 , dox - as - m - nps , vs dox or pbs in tumors ) .
to understand the role 
 of tams in the as - m - nps ability to target dox into tumors , 
 b16-f10 tumor - bearing mice were treated with zoledronic acid to reduce 
 the macrophage population , including tams .
immunohistochemical staining 
 confirmed the reduction of cd206 staining ( an m2 macrophage 
 marker ) in tumors in mice that were treated with zoledronic acid ( figure 5a ) .
the accumulation of dox - as - m - nps in tumors in 
 mice that were treated with zoledronic acid was significantly decreased , 
 as compared to that in mice that were not treated with zoledronic 
 acid ( figures 5b and s4 , supporting information ) .
( a ) representative images of b16-f10 tumors 
 stained with rm0029 - 11h3 . 

tumor - bearing mice were treated , or not , with zoledronic acid ( za ) . 

( b ) representative ex vivo images of b16-f10 tumors 
 and other major organs 6 h after mice were injected with dox - as - m - nps . 
 prior to the injection of dox - as - m - nps
, one group of mice was treated 
 with za ( t = tumor , k = kidneys , h = heart , lr = liver , s = spleen , 
 and ln = lung ) . to test whether dox - as - m - nps 
 increase the uptake of dox by tams , 

b16-f10 tumor - bearing mice were i.v . injected with free dox or various 
 dox - nanoparticles , and the percent of cd206 cells that 
 contained dox ( i.e. , dox / cd206% ) was measured 
 using flow cytometry 6 h after injection .
as shown in figure 6a , b , the percent of dox / cd206 cells in tumors in mice that were i.v . injected with dox - as - m - nps 

was significantly higher than in mice that were injected with free 
 dox , dox - ai - m - nps , or dox - as - nps .
in fact , it was 23-fold 
 higher in tumors in mice that were injected with the dox - as - m - nps 
 than with other nanoparticles or free dox ( figure 6b ) .
uptake of dox by cd206 cells in b16-f10 tumors 6 h 
 after tumor - bearing mice were i.v . injected with free dox or various 
 dox - nanoparticles .
( a ) representative flow cytometric graphs of single 
 tumor cell suspensions after stained with fitc - labeled anti - cd206 . 

( b ) the percentage of cd206 cells in b16-f10 tumors that 
 took up dox ( i.e. , dox / cd206% ) .
data are mean 
  sem from at least 3 mice ( p < 0.05 ) . because the dox - as - m - nps 
 were shown to be more effective than other 
 nanoparticles in delivering dox into tumors , while minimizing the 
 accumulation of dox in mps organs such as liver , the antitumor activity 
 of dox - as - m - nps was evaluated in b16-f10 tumor - bearing mice and compared 
 to that of free dox .
b16-f10 tumors in mice that were i.v . injected 
 with the dox - as - m - nps grew significantly slower than in mice that 
 were injected with free dox at an identical dox dose ( figure 7a ) . at the end of the study ( i.e. , 16 days after 
 tumor cell injection ) ,
the mean weight of tumors in mice that were 
 treated with dox - as - m - nps was about 50% of that in mice that were 
 treated with free dox , less than 20% of that in mice left untreated 
 ( figure 7b ) .
shown in figure 7c are digital images of tumors at the end of the study , and 
 the mean body weights of the mice during the treatment period are 
 in figure 7d . in vivo antitumor activity 
 of dox - as - m - nps .
( a ) 
 growth curves of b16-f10 tumors in c57bl/6 mice that were treated 
 with dox - as - m - nps (  ) or free dox (  ) .
arrows indicate days on which 
 mice were treated ( * p < 0.05 , dox vs dox - as - m - nps ) . 

mean weight ( b ) and digital photographs ( c ) of tumors on day 16 ( in 
 panel b , p < 0.05 ) .
( e ) representative micrographs of tumors stained with h&e , 
 anti - cd31 antibody , or anti - brdu antibody ( necrotic areas , hemorrhagic regions , and blood vessels ) . for h&e 
 and anti - cd31 staining , bar = 100 m ; for anti - brdu staining , 
 bar = 50 m . in panels
a , b , and d , data are mean  sem 
 ( n = 8) .
tumors in mice that were injected with sterile pbs ( i.e. , 
 negative 
 control ) showed large cell nuclei and small intercellular spaces , 
 with necrosis and hemorrhage rarely observable ( h&e ) , while cd31 ( an angiogenesis maker ) and brdu staining ( a 
 cell proliferation marker ) were extensive ( figure 7e ) .
tumors in mice that were treated with free dox had some 
 necrotic areas and small hemorrhagic regions , and cd31 and brdu staining remained extensive ( figure 7e ) .
in contrast , tumors in mice that were treated 
 with dox - as - m - nps showed numerous necrotic and hemorrhagic areas , 
 with decreased cd31 staining and brdu staining 
 ( figure 7e ) . to evaluate the effect of the dox - as - m - nps 
 on tams and macrophages in mps , the tumor , liver , and spleen tissues 
 from b16-f10 tumor - bearing mice that were treated with dox - as - m - nps 
 were stained with anti - cd206 ( an m2 macrophage marker ) or rm0029 - 11h3 
 ( a pan - macrophage marker ) .
mice were treated with dox - as - m - nps or 
 free dox at 6 and 12 days after tumor cell injection , and tissues 
 were collected 4 days after the second treatment . as shown in figure 8a , the extent of cd206 staining was 
 significantly lower in tumors in mice that were treated with dox - as - m - nps 
 than in mice that were treated with free dox or left untreated .
however , 
 there was no apparent difference in the extents of rm0029 - 11h3 staining in the liver and spleen in mice that were treated 
 with dox - as - m - nps or free dox ( figure 8b ) .
representative 
 micrographs of ( a ) tumor tissues stained with anti - cd206 
 antibody ( bar = 50 m ) and ( b ) liver and spleen tissues stained 
 with rm0029 - 11h3 ( bar = 100 m ) .
traditionally , active tumor - targeting is 
 focused on exploiting 
 receptors and proteins that are overexpressed by tumor cells or molecules 
 that are overexpressed in tumor neovasculature .
recently , there is increasing interest in exploring other cellular 
 or noncellular components in tumor tissues , such as immune cells , 
 fibroblasts , and extracellular matrix materials , for active tumor 
 targeting .
in fact , there have been some efforts in utilizing tams as a target 
 for tumor imaging .
altering the tumor microenvironment 
 that is involved in tumor angiogenesis and progression could markedly 
 decrease metastasis , gaining better response to tumor resistance .
in addition , since tams exist mainly in the stroma of many tumors , targeting of tams could be an effective antitumor strategy for a 
 wide variety of tumors .
our data indicated that acid - sensitive sheddable 
 pegylation and mannose - modification of plga nanoparticles could increase 
 the distribution of the plga nanoparticles in tumor tissues via interaction 
 with tams while decreasing their accumulation in mps organs such as 
 liver .
injection , 
 free dox hardly accumulated in tumors and major organs such as liver , 
 spleen , heart , lung , and kidneys .
the un - pegylated dox - nps mainly 
 accumulated in mps organs such as liver , likely due to opsonization .
dox - m - nps were un - pegylated , but surface - modified 
 with mannose , and their accumulation in mouse liver , lung , and kidneys 
 were further increased , as compared to dox - nps , likely because of 
 the presence of mr - expressing macrophages in those organs as well .
both dox - as - m - nps and dox - ai - m - nps are pegylated , and as expected , 
 their distributions in major organs such as liver , lung , and kidneys 
 were significantly decreased , but increased in tumor tissues ( figure 3a ) , and data in figure 3b 
 indicate that pegylation increased the blood circulation time of the 
 nanoparticles .
importantly , compared to dox - ai - m - nps , the distribution 
 of dox - as - m - nps in tumor was significantly higher , demonstrating the 
 importance of acid - sensitive sheddable pegylation in increasing the 
 delivery of the nanoparticles into tumors . in order to understand 
 the significance of surface - modification with mannose on the nanoparticles 
 ability to target tumors ,
the biodistribution of dox - as - m - nps and 
 dox - as - nps were compared , and data in figure 3e showed that the tumor accumulation of dox - as - m - nps was significantly 
 higher than that of dox - as - nps , confirming that both acid - sensitive 
 sheddable pegylation and surface - modified with mannose are required 
 for successful targeting of the dox - nps into tumors .
injection 
 dox - as - m - nps increased the delivery of dox into s.c .
the observed 
 relatively higher accumulation in spleen was likely related to the 
 relatively higher background fluorescence signal in the spleen ( especially 
 in the tc-1 tumor bearing mice ( figure 4b ) ) , 
 as compared to in other organs tested .
a similar trend was also observed 
 when tested in male nude mice with orthotopic panc-1 human pancreatic 
 tumors and in female nude mice with orthotopic mda - mb-231 human breast 
 tumors ( data not shown ) .
therefore , the as - m - nps ability to 
 increase the biodistribution of molecules carried by them into tumors 
 is not limited to the b16-f10 tumors .
tams are critical for the as - m - nps 
 to target tumors , and thus , the distribution of as - m - nps in different 
 tumors will likely be affected by the population of tams in the tumors . 

therefore , the as - m - nps will 
 likely be more effective in targeting tumors that contain a higher 
 population of tams .
zoledronic acid belongs to a group of bisphosphonates , 
 which are 
 often used to reduce macrophage population in mouse models .
data in figure 5b showed 
 that the distribution of dox - as - m - nps in tumors in mice that were 
 treated with zoledronic acid was significantly reduced , as compared 
 to in similar tumor - bearing mice that were not treated with zoledronic 
 acid , demonstrating that tams are required for the as - m - nps to successfully 
 improve the distribution of dox into tumors .
in fact , dox - as - m - nps 
 increased the uptake of dox by tams in b16-f10 tumor - bearing mice 
 by 23-fold , as compared to dox - ai - m - nps or dox - as - nps ( figure 6 ) , further indicating that the interaction between 
 the mannose on the surface of the dox - as - m - nps and tams after acid - sensitive 
 shedding of the peg chains from the nanoparticles in tumor tissues 
 is critical for the enhanced delivery of dox into tumors .
data 
 in figure 7 showed that dox - as - m - nps 
 are significantly more effective than free dox in controlling tumor 
 growth .
in addition , the dox - as - m - nps were also more effective than 
 free dox in suppressing angiogenesis ( cd31 staining ) and 
 cell proliferation ( brdu staining ) in tumor tissues ( figure 7e ) . the stronger antitumor activity of the dox - as - m - nps 
 is likely in part due to the nanoparticle s ability to increase 
 the delivery of dox into tumors ( figure 3a ) . 

however , it remains unknown to what extent the stronger antitumor 
 activity of the dox - as - m - nps can be attributed to their ability to 
 reduce the tam population in tumors ( figure 8a ) . in vitro cytotoxicity data
showed that dox - as - m - nps 
 were cytotoxic not only to b16-f10 tumor cells , but also to the j774a.1 
 macrophages , and shedding of the peg chains on the dox - as - m - nps significantly 
 increased the nanoparticle s cytotoxicity to j774a.1 cells 
 ( figure 1d ) .
in b16-f10 tumor - bearing mice , i.v . 
 injection of dox - as - m - nps almost doubled the percentage of tams that 
 took up dox , as compared to i.v .
therefore , 
 the strong antitumor activity of the dox - as - m - mps is likely related 
 to their ability to reduce tam population in tumors .
it is possible 
 that b16-f10 tumors with a reduced population of tams are smaller 
 than the same b16-f10 tumors with a normal population of tams . in 
 addition , since tams promote tumor cell proliferation and induce immune 
 suppression , a reduction of tam population in tumors may also have 
 contributed to the slower tumor growth in mice that were treated with 
 dox - as - m - nps . of course ,
although dox - as - m - nps increased the delivery 
 of dox into tams ( figure 6 ) , it is unlikely 
 that all the dox - as - m - nps that reached the tumor were taken up by 
 tams .
some dox - as - m - nps that were delivered to tumor tissues were 
 likely taken up by b16-f10 tumor cells .
in addition , some dox that 
 was released from the dox - as - m - nps , before or after the nanoparticles 
 reached tumors , may also have been taken up by tumor cells .
therefore , 
 the mechanism underlying the strong antitumor activity of the dox - as - m - nps 
 is expected to be multifactorial .
for example , the dox - as - m - nps were 
 also more effective than dox alone in inhibiting angiogenesis and 
 cell proliferation in tumors ( figure 7e ) .
it 
 was noted that extensive necrosis was present in tumors in mice treated 
 with dox - as - m - nps ( figure 7e ) .
it is possible that the dox that was slowly released from the dox - as - m - nps 
 maintained a low concentration of dox in the tumor tissues and caused 
 the significant necrosis . shown in figure 8 are the micrographs of 
 tumor , liver , and spleen tissues of b16-f10 tumor - bearing mice after 
 the tissues were stained with anti - cd206 ( an m2 macrophage marker ) 
 or rm0029 - 11h3 ( a pan - macrophage marker ) .
compared to the tumor tissues 
 from mice treated with free dox or left untreated , the extent of cd206 staining was significantly decreased in tumors in mice that 
 were treated with dox - as - m - nps ( figure 8a ) , 
 demonstrating dox - as - m - nps ability to reduce tam population 
 in tumor tissues .
importantly , the extents of rm0029 - 11h3-positive 
 staining in both liver and spleen of mice that were treated with dox - as - m - nps 
 appear not different from that in mice treated with free dox or left 
 untreated ( figure 8b ) , indicating that the 
 dox - as - m - nps did not significantly affect the total macrophage population 
 in mps organs such as liver and spleen . many compounds including 
 the bisphosphonate zoledronic acid can 
 be used to deplete tams .
however , zoledronic 
 acid also significantly affects macrophages that do not reside in 
 tumors .
the as - m - nps are advantageous because they can target tumors 
 and tams but have only minimum effects on macrophages that are not 
 tumor - associated .
of course , since macrophages are white blood cells 
 differentiated from monocytes , arising from progenitor cells in the 
 bone marrow , there is the possibility 
 that 4 days after the second treatment of the b16-f10 tumor - bearing 
 mice with dox - as - m - nps any major effects that the dox - as - m - nps may 
 have had on macrophages in mps organs were restored , whereas the tam 
 population in tumors may take a longer time to replenish .
more experiments 
 will need to be carried out to study the effect of the dox - as - m - nps 
 on the dynamics of macrophages in tumor and nontumor tissues in the 
 future . nonetheless , when fully developed , the tam - targeting nanoparticle 
 platform can potentially be applied to deliver cytotoxic agents or 
 macrophage - modulating agents into tumors to decouple the interaction 
 between tams and tumor cells , making the tumor microenvironment less 
 favorable for tumor growth , but more favorable for chemotherapy .
we demonstrated that surface - modification of plga nanoparticles 
 with acid - sensitive sheddable peg molecules and mannose as a ligand 
 of mr , which is overexpressed on tams , allows the nanoparticles to 
 effectively target dox into tumors .
the targeting is dependent on 
 the presence of sufficient tams in tumors . compared to free dox , dox 
 carried by the acid - sensitive sheddable pegylated , mannose - modified 
 plga nanoparticles
more effectively inhibited tumor growth , reduced 
 tam population in tumors , but showed no or only minimum effect on 
 the macrophage population in mps .
 tumor - associated 
 macrophages ( tams ) are increasingly considered 
 a viable target for tumor imaging and therapy . 
 previously , we reported 
 that innovative surface - functionalization of nanoparticles may help 
 target them to tams . in this report , using poly(lactic - co - glycolic ) acid ( plga ) nanoparticles incorporated with doxorubicin 
 ( dox ) ( dox - nps ) , we studied the effect of surface - modification of 
 the nanoparticles with mannose and/or acid - sensitive sheddable polyethylene 
 glycol ( peg ) on the biodistribution of dox and the uptake of dox by 
 tams in tumor - bearing mice . 
 we demonstrated that surface - modification 
 of the dox - nps with both mannose and acid - sensitive sheddable peg 
 significantly increased the accumulation of dox in tumors , enhanced 
 the uptake of the dox by tams , but decreased the distribution of dox 
 in mononuclear phagocyte system ( mps ) , such as liver . 
 we also confirmed 
 that the acid - sensitive sheddable pegylated , mannose - modified dox - nanoparticles 
 ( dox - as - m - nps ) targeted tams because depletion of tams in tumor - bearing 
 mice significantly decreased the accumulation of dox in tumor tissues . 
 
 furthermore , in a b16-f10 tumor - bearing mouse model , we showed that 
 the dox - as - m - nps were significantly more effective than free dox in 
 controlling tumor growth but had only minimum effect on the macrophage 
 population in mouse liver and spleen . 
 the as - m - nps are promising in 
 targeting cytotoxic or macrophage - modulating agents into tumors to 
 improve tumor therapy . 
Introduction
Materials and Methods
Results
Discussion
Conclusions