prior to np[xl184 ] synthesis , the biodegradable copolymer plga 50:50 ( 17 kda ; 0.18 dlg ; lake shore biomaterials ) was modified to incorporate a peg moiety ( cooh - peg - nh2 ; 3.5 kda ; jenkem technology ) , which enhances both nanoparticle stability and circulation time .
nanoparticles were synthesized with various xl184 ( > 99.0% purity ; selleck chemicals ) and peg - plga drug / polymer ratios ranging from 110% ( w / w ) . of the tested ratios ,
the xl184 molar loading efficiency was maximal at a drug / polymer ( w / w ) ratio of 1% ( supplemental figure 2 ) .
in addition , a range of solvent : water ratios ( 1:21:10 ) were tested and the optimal ratio for np[xl184 ] formation was 1:3 acetone : water .
for the optimized synthesis protocol , xl184 was co - solubilized in 1 ml of acetone with plga - peg at a 1% ( w / w ) drug to polymer concentration .
nanoprecipitation was achieved by adding this mixture drop - wise using a 27-gauge needle to 3 ml of h2o stirred magnetically at 400 rpm .
the reaction mixture was then stirred uncovered for 6 h to allow acetone evaporation , passed through a 0.2 m filter and purified by ultrafiltration ( amicon - millipore ; 30 kda cut - off ) at 2500 rpm for 10 min with intermittent washing ( 4 cycles ; 4 ml phosphate - buffered saline per wash ) .
the xl184plga nanoparticle loading efficiency was determined by optical absorption measurements following solvation of the nanoparticles in dimethyl sulfoxide , using the formula : 100(no .
all np[xl184 ] size and charge measurements were made by dynamic light scattering ( malvern , zetasizer nano zs ) .
pmils co - encapsulating bpd ( verteporfin ; vwr international ) and np[xl184 ] were prepared by modification of existing synthesis methods .
the lipids ( dppc , dotap , cholesterol , and dspe - peg ; avanti polar lipids ) were each dissolved separately in chloroform , and then mixed together in a molar ratio of 2:0.2:1:0.2 ( dppc : dotap : cholesterol : dspe - peg ) with 100 nmoles of bpd .
this lipid composition was selected based on the previously reported pharmacological success of similar compositions .
inclusion of the cationic lipid , dotap , resulted in a zeta potential ( surface charge ) of + 3 mv .
this slightly cationic surface charge promotes cellular uptake without significant cytotoxicity to maintain biocompatibility . to form thin lipid films containing bpd ,
chloroform was removed by roto - evaporation and by placing the sample under vacuum overnight .
next , lipid film hydration was achieved by adding np[xl184 ] ( 50 nmoles of xl184 ) in 1 ml of phosphate - buffered saline . to ensure adequate encapsulation of np[xl184 ] , the thin film was subjected to 10 freeze - thaw cycles ( 6 min per cycle ) at 0c and 45c , below and above the highest transition temperature of the lipid mixture ( dppc ; tm = 41c ) .
the resulting dispersion of multilamellar liposomes was extruded through a 200-nm - diameter polycarbonate membrane using a mini - extruder system ( avanti polar lipids ) to form unilamellar liposomes .
bpd and xl184 not loaded into the pmil were removed by dialysis ( spectra / por ; 300 kda cut - off ; 1 ml sample in 4 l of phosphate - buffered saline at 4c for 18 h ) . during the dialysis period
initially , these measurements indicated a bimodal distribution with peaks at 80 nm ( np[xl184 ] ) and 150 nm ( pmil ) , which gradually became a single monodispersive peak ( pdi < 0.2 ) as purification of the pmils completed . however , electron microscopy revealed the presence of residual np[xl184 ] not incorporated within liposomes within the pmil samples ( supplementary note 3 ) .
the pmil bpd and xl184 concentrations and loading efficiencies were determined by fluorescence and absorbance spectroscopy or by high - performance liquid chromatography ( hydrosil c18 ods ; 2.0 cm  14.0 cm ; 50% acetonitrile in h2o  100% acetonitrile ; 0.5 h ) , respectively , following solvation of the pmils in dimethyl sulfoxide . all pmil size and charge measurements
pmil and np[xl184 ] size stability during storage at 4c was investigated by repeated dynamic light scattering measurements over a period of 40 d ( supplementary fig .
dark release and photoinduced drug release were measured using dialysis membranes in phosphate - buffered saline at 37c with 10% fetal bovine serum added to each dialysis tube ( spectra / por ; np[xl184 ] , 100 kda cut - off ; pmils , 300 kda cutoff ; fig .
2f ) . a 690 nm diode laser ( high power devices , inc . ) was used for all nir irradiation experiments . during dialysis samples
were collected periodically and placed immediately in acetonitrile containing 1% of the internal standard n-(1-naphthyl)ethylenediamine .
separation and quantification of drug components was achieved by liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) using standard curves for each drug normalized to the internal standard .
briefly , 1.0 l of dialysis sample was injected into a zorbaxc18 ( 2.1  50 mm ) column eluted at 0.400 ml / min with acetonitrile and 10 mm ammonium formate ( 80%  20% over 4 min ) .
detection of drug components was made using triple quadrupole ms / ms detection with an ion source esi+ in mrm scan mode to identify the product ions for bpd ( ret .
time= 2.68 min , 232 m / z ) . quantitative analysis of chromatograms allowed for area under curve integrations of each product ion normalized to the internal standard ( ret .
total moles were determined using standard curves and percent loss calculated for each time point after correcting for sample volume changes .
the resulting bpd and xl184 release profiles were fit individually to a simple one- or two - phase exponential model : a0 + a1  e + a2  e , where a0 is an offset , a1 and a2 are the maxima release plateaus ( at equilibrium ) of phases 1 and 2 , k1 and k2 are the release rate constants of phases 1 and 2 , and t is time from placing the sample in serum media within the dialysis tube .
note that xl184 release from np[xl184 ] is sufficiently described by a single - phase model ( p=0.067 , two - phase alternative hypothesis ) , whereas xl184 release from the pmil with or without laser irradiation is best described by the multi - phase model ( p = 0.00030.0079 ) .
dynamic light scattering and transmission electron microscopy were also performed before and after photoirradiation ( fig . 3 and supplementary fig .
cryo - em was performed using a fei technai g2 polara microscope equipped with an energy filter ( gatan ) and a k2 summit direct detection device ( gatan ) .
briefly , 5 l of nanomaterial sample ( ~60 m bpd for the pmil or l[bpd ] ; ~30 or 125 m xl184 for the pmil or np[xl184 ] , respectively ) mixed with 2 l of bsa gold tracer ( em - grade 6 nm ; electron microscopy sciences , 25484 ) re - suspended in phosphate - buffered saline were deposited onto glow - discharged holey carbon grids ( quantifoil r 2/1 200 mesh , copper ; electron microscopy sciences ) , blotted and rapidly vitrified in a liquid ethane and propane mixture ( 50:50 ) using a custom - built plunger ( max planck institute of biochemistry , germany ) .
kv under low - dose conditions with 4.98  or 6.12  sampling and a defocus of 3 or 6 m for 2d or 3d images , respectively .
2d images were obtained using the dose - fractionation mode of the detector ( ~2040 e /  cumulative dose ) .
tilt series ( 60 ) for tomography were collected around a single axis with a 2 sampling increment using serialem software ( ~100 e /  cumulative dose ) .
renders of 3d pmil and np[xl184 ] objects were created by manual segmentation in imod and rendered using vmd .
5 . only unambiguous np[xl184 ] objects were counted ( ~20 nm in diameter or greater ) .
mean lamellarity was calculated as : l(nl  l)/lnl , where nl is the number of objects with lamellarity l ( e.g. ,
tem ( philips cm10 ) was performed using negative staining either on untreated ( 200 mesh nickel pelco support film with a formvar / carbon coating , ted pella inc . ) or ionized carbon coated grids ( to promote sample adhesion ) .
briefly , 10 l of sample was added to the grid , air - dried and stained ( 2 l , 1.0% phosphotungstic acid ) .
imaging was performed at 100.0 kv and magnifications of ~10,00050,000. monolayer cultures of aspc1 cells ( american type culture collection , crl-1682 ; low passage number , < 20)recently tested ( july 2015 ) and found to be negative for mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza)were maintained in media ( rpmi 1640 , mediatech ) supplemented with 10% fetal bovine serum ( invitrogen ) , 100 units / ml penicillin and 100 g / ml streptomycin . the aspc1 cell line has not been listed in the database of cross - contaminated or misidentified cell lines maintained by the international cell line authentication committee .
cellular uptake of pmils and l[bpd ] was tested in multi - well plates with coverslip bottoms ( greiner bio - one ) plated with aspc1 cells allowed to attach and grow overnight .
nanoconstructs were added to the wells at staggered time points to reach a concentration of 100 nm bpd and to achieve varying incubation times at 37c ( 1590 min ) .
imaging was performed with an olympus fv1000 confocal microscope with a 20  0.75 na ( numerical aperture ) objective .
bpd excitation was performed using a 405 nm diode laser with an emission spectrograph centred on the 696 nm bpd fluorescence emission peak .
the laser , photomultiplier tube detector and pinhole settings , as well as brightness - contrast adjustment settings for display , were kept constant for all images .
in addition , phase contrast images were collected during microscopy in order to focus on a high - density field of cells ( not shown ) .
images were also collected for untreated cells ( 0 min ) to quantify the autofluorescence background and to define a fluorescence intensity threshold that rejects 99.5% of the background signal .
this intensity threshold was applied to all images to select pixels above the autofluorescence background ( true bpd signal ) for analysis .
the resulting cellular uptake data was fit to a simple biexponential pharmacokinetic model : a  ( e  e ) , where k and j are the elimination and absorption rate constants , a is a coefficient dependent on the administered bpd dose as well as its bioavailability and t is time post - administration . for in vitro pdt , 0.2510 aspc1 cells were grown on a 35-mm culture dish for 24 h and incubated with nanoconstructs containing bpd ( 250 nm equivalent ) and/or xl184 ( 100125 nm equivalent ) in 1 ml complete medium for 1 h. the incubation media was then replaced with 2 ml of fresh , complete media prior to photoirradiation .
this removal of nanoconstructs not uptaken by cells prior to irradiation limits the release of xl184 and the generation of photocytotoxic species to intracellular and cell - associated nanoconstructs .
singlet oxygen sensor green ( sosg ; molecular probes ) and d - mannitol ( sigma - aldrich ) were used to probe reactive oxygen species involvement in bpd - pdt - induce met activation .
tyrphostin ag1478 ( sigma - aldrich ) and bacterial toxin b ( toxin b , clostridium difficile - calbiochem , millipore ) were used to test for involvement of enzymes known to participate in met transactivation .
sosg and mannitol were added to cells immediately prior to laser irradiation in fresh media and then removed immediately after pdt by a second media replacement step .
ag1478 and toxin b were incubated with cells in fresh media for 30 min and 2 h , respectively , prior to laser irradiation and then removed immediately after pdt by a second media replacement .
all animal experiments were conducted with approval and according to guidelines established by the massachusetts general hospital institutional animal care and use committee .
experiments were carried out on 6-week - old male swiss nude mice weighing 2025 grams ( cox breeding laboratories ) . for tumour implantations and photoirradiation ,
tumours were implanted by injection of a 50 l volume containing 10 aspc1 cells in a 1:1 mixture of matrigel ( bd biosciences ) and culture media .
subcutaneous tumours were implanted above the hind leg and tumour volumes were estimated longitudinally by measuring the three tumour dimensions using a calliper and the hemi - ellipsoid formula : volume =   l  w  h/6 , where l , w and h , are the tumour length , width and height .
note that here , h , represents the measured height of the hemi - elliptical tumour , which is half the height of a full ellipsoid .
eighteen days following cancer cell implantation , subcutaneous tumours reached volumes of ~50 mm prior to the start of treatment . for orthotopic tumour implantation
, animals were laid supine , a small left abdominal flank incision was made to exteriorize the pancreas and the cell suspension was injected into the pancreas . a small amount of 10% povidone / iodine was applied topically to the injection site .
then the incision was closed with 4 - 0 sutures and 10% povidone / iodine was then applied to the incision site to prevent infection .
ten days after cancer cell implantation , orthotopic pancreatic tumours reached volumes of ~25 mm prior to the start of treatment .
all injections for treatment were done intravenously ( tail vein ) in 200 l sterile phosphate - buffered saline .
mice were randomized into the various treatment groups , and the tumours of mice receiving bpd were irradiated with nir light ( using the 690 nm diode laser listed above ) 1 h post - injection , delivered at an irradiance of 100 mwcm .
subcutaneous tumours were irradiated transcutaneously while orthotopic tumours were surgically exposed as for tumour implantation and irradiated .
fourteen days after treatment , orthotopic tumours were excised to estimate their volumes using callipers and the ellipsoid formula : volume =   l  w  h/6 .
briefly , orthotopic pancreatic tumours were excised 2 weeks post - treatment , embedded in optimal cutting temperature compound and frozen at 80c .
sections were ( 1 ) fixed in 1:1 acetone : methanol for 15 minutes at 20c , ( 2 ) air dried for 30 minutes , and ( 3 ) washed three times in phosphate - buffered saline .
a blocking solution ( dako protein block reagent ) was applied for 30 minutes , followed by application of the immunostains , at ~5 g / ml monoclonal antibody ( mab ) each diluted in background reducing dako antibody diluent for 2 h at room temperature in a humidifying chamber . finally , the slides were washed again three times , mounted ( invitrogen slowfade gold with 4,6-diamidino-2-phenylindole , dapi ) with a coverslip and sealed with nail polish .
confocal fluorescence imaging was performed using an olympus fluoview 1000 confocal microscope with a 10  0.4 numerical aperture ( na ) or a 20 0.75 na objective .
excitation of dapi , anti - mouse pecam-1 ( cd31 ; clone 390 ; cbl1337 , millipore ) mab - alexa fluor 568 conjugates and anti - human cytokeratin 8 ( clone lp3k ; mab3156 , r&d systems ) mab - alexa fluor 647 conjugates was carried out using 405- , 559- and 635-nm lasers , respectively .
mosaic images of entire tumour cross - sections were collected and stitched together using the olympus fluoview software .
the anti - human cytokeratin 8 stain ( a cytoskeletal protein highly expressed by aspc1 cells ) has dual selectivity for the epithelial cancer cells because it does not react with mouse proteins .
all analyses were performed using custom matlab ( mathworks ) routines for batch image processing .
microvessel density values were calculated from whole tumour sections , within viable tumour tissue only , and averaged over slices from the entire tumour rather than a more complex  hot spot  identification and calculation , which is difficult to define objectively .
intratumoural microvessel volume is calculated by multiplying microvessel density with the viable tumour volume in each slice and then summing over the whole tumour by interpolation . here
, we used the minimum tumour subsampling necessary  based on a mathematical model  to resolve a statistically significant change in intratumoural microvessel volume , as validated previously using the orthotopic aspc1 tumour model ( supplementary note 9 ) .
a quantitative reverse transcription  polymerase chain reaction ( qrt - pcr ) assay was performed on excised liver and iliac lymph nodes to estimate the number of human cancer cells in excised organs as described and validated previously .
briefly , qrt - pcr is used to measure the total number of human cancer cells from the level of human and mouse glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) housekeeping genes .
at least 300 mg of freshly excised liver and retroperitoneal lymph nodes were collected at the treatment endpoint and snap frozen in liquid nitrogen .
the frozen samples were then pulverized and homogenized , followed by rna extraction ( rnaeasy plus mini kit ; qiagen ) .
human and mouse gapdh gene were measured using custom synthesized primers ( invitrogen ) . for each specimen ,
the cycle threshold ( ct ) from human gapdh gene was normalized by ct from mouse gapdh gene .
the normalized ct was quantified into number of cancer cells using a standard curve generated with a set of organ lysates from no - tumour control mice mixed with different numbers of human cancer cells .
specific statistical tests are indicated in the figure captions and were carried out using graphpad prism ( graphpad software ) .
parametric tests ( one - way anova with tukey s post - test ) were used for in vitro drug release ( fig .
6 ) studies ; and , the dagostino & pearson omnibus normality test (  = 0.05 ; requires n  8 replicates per group ) did not identify significant deviations from normality within these data sets ( testing could only performed for groups with n  8 replicates ) . note that all groups within the drug release data were analysed together ( some groups appear only in supplementary fig .
1 ) were analysed using nonparametric tests ( the mann - whitney u test or kruskal 
the brown  forsythe test (  = 0.05 ) was applied to all data sets with n  3 replicates to test for equal variance ( regardless of whether parametric or nonparametric analysis was used ) and identified significant deviations from equal variance ( figs .
in these cases , the data were analysed following a logarithmic transform of the data to pass the brown  forsythe test .
5d ) following a natural logarithm transform of the data to pass the dagostino & pearson omnibus normality test .
no exclusion criteria were used , and no data points or animals were excluded from analysis .
animal sample sizes were selected to ensure adequate power ( 80% ) to detect a 20% difference using a maximum of 16 animals per group assuming a standard deviation of 15% .
prior to np[xl184 ] synthesis , the biodegradable copolymer plga 50:50 ( 17 kda ; 0.18 dlg ; lake shore biomaterials ) was modified to incorporate a peg moiety ( cooh - peg - nh2 ; 3.5 kda ; jenkem technology ) , which enhances both nanoparticle stability and circulation time .
nanoparticles were synthesized with various xl184 ( > 99.0% purity ; selleck chemicals ) and peg - plga drug / polymer ratios ranging from 110% ( w / w ) . of the tested ratios ,
the xl184 molar loading efficiency was maximal at a drug / polymer ( w / w ) ratio of 1% ( supplemental figure 2 ) .
in addition , a range of solvent : water ratios ( 1:21:10 ) were tested and the optimal ratio for np[xl184 ] formation was 1:3 acetone : water .
for the optimized synthesis protocol , xl184 was co - solubilized in 1 ml of acetone with plga - peg at a 1% ( w / w ) drug to polymer concentration .
nanoprecipitation was achieved by adding this mixture drop - wise using a 27-gauge needle to 3 ml of h2o stirred magnetically at 400 rpm .
the reaction mixture was then stirred uncovered for 6 h to allow acetone evaporation , passed through a 0.2 m filter and purified by ultrafiltration ( amicon - millipore ; 30 kda cut - off ) at 2500 rpm for 10 min with intermittent washing ( 4 cycles ; 4 ml phosphate - buffered saline per wash ) .
the xl184plga nanoparticle loading efficiency was determined by optical absorption measurements following solvation of the nanoparticles in dimethyl sulfoxide , using the formula : 100(no .
all np[xl184 ] size and charge measurements were made by dynamic light scattering ( malvern , zetasizer nano zs ) .
pmils co - encapsulating bpd ( verteporfin ; vwr international ) and np[xl184 ] were prepared by modification of existing synthesis methods .
the lipids ( dppc , dotap , cholesterol , and dspe - peg ; avanti polar lipids ) were each dissolved separately in chloroform , and then mixed together in a molar ratio of 2:0.2:1:0.2 ( dppc : dotap : cholesterol : dspe - peg ) with 100 nmoles of bpd .
this lipid composition was selected based on the previously reported pharmacological success of similar compositions .
inclusion of the cationic lipid , dotap , resulted in a zeta potential ( surface charge ) of + 3 mv .
this slightly cationic surface charge promotes cellular uptake without significant cytotoxicity to maintain biocompatibility . to form thin lipid films containing bpd ,
chloroform was removed by roto - evaporation and by placing the sample under vacuum overnight .
next , lipid film hydration was achieved by adding np[xl184 ] ( 50 nmoles of xl184 ) in 1 ml of phosphate - buffered saline . to ensure adequate encapsulation of np[xl184 ] , the thin film was subjected to 10 freeze - thaw cycles ( 6 min per cycle ) at 0c and 45c , below and above the highest transition temperature of the lipid mixture ( dppc ; tm = 41c ) .
the resulting dispersion of multilamellar liposomes was extruded through a 200-nm - diameter polycarbonate membrane using a mini - extruder system ( avanti polar lipids ) to form unilamellar liposomes .
bpd and xl184 not loaded into the pmil were removed by dialysis ( spectra / por ; 300 kda cut - off ; 1 ml sample in 4 l of phosphate - buffered saline at 4c for 18 h ) . during the dialysis period
initially , these measurements indicated a bimodal distribution with peaks at 80 nm ( np[xl184 ] ) and 150 nm ( pmil ) , which gradually became a single monodispersive peak ( pdi < 0.2 ) as purification of the pmils completed
. however , electron microscopy revealed the presence of residual np[xl184 ] not incorporated within liposomes within the pmil samples ( supplementary note 3 ) .
the pmil bpd and xl184 concentrations and loading efficiencies were determined by fluorescence and absorbance spectroscopy or by high - performance liquid chromatography ( hydrosil c18 ods ; 2.0 cm  14.0 cm ; 50% acetonitrile in h2o  100% acetonitrile ; 0.5 h ) , respectively , following solvation of the pmils in dimethyl sulfoxide . all pmil size and charge measurements
pmil and np[xl184 ] size stability during storage at 4c was investigated by repeated dynamic light scattering measurements over a period of 40 d ( supplementary fig .
release and photoinduced drug release were measured using dialysis membranes in phosphate - buffered saline at 37c with 10% fetal bovine serum added to each dialysis tube ( spectra / por ; np[xl184 ] , 100 kda cut - off ; pmils , 300 kda cutoff ; fig .
2f ) . a 690 nm diode laser ( high power devices , inc . ) was used for all nir irradiation experiments . during dialysis samples
were collected periodically and placed immediately in acetonitrile containing 1% of the internal standard n-(1-naphthyl)ethylenediamine .
separation and quantification of drug components was achieved by liquid chromatography - tandem mass spectrometry ( lc - ms / ms ) using standard curves for each drug normalized to the internal standard .
briefly , 1.0 l of dialysis sample was injected into a zorbaxc18 ( 2.1  50 mm ) column eluted at 0.400 ml / min with acetonitrile and 10 mm ammonium formate ( 80%  20% over 4 min ) .
detection of drug components was made using triple quadrupole ms / ms detection with an ion source esi+ in mrm scan mode to identify the product ions for bpd ( ret .
time= 2.68 min , 232 m / z ) . quantitative analysis of chromatograms allowed for area under curve integrations of each product ion normalized to the internal standard ( ret .
total moles were determined using standard curves and percent loss calculated for each time point after correcting for sample volume changes .
the resulting bpd and xl184 release profiles were fit individually to a simple one- or two - phase exponential model : a0 + a1  e + a2  e , where a0 is an offset , a1 and a2 are the maxima release plateaus ( at equilibrium ) of phases 1 and 2 , k1 and k2 are the release rate constants of phases 1 and 2 , and t is time from placing the sample in serum media within the dialysis tube .
note that xl184 release from np[xl184 ] is sufficiently described by a single - phase model ( p=0.067 , two - phase alternative hypothesis ) , whereas xl184 release from the pmil with or without laser irradiation is best described by the multi - phase model ( p = 0.00030.0079 ) .
dynamic light scattering and transmission electron microscopy were also performed before and after photoirradiation ( fig . 3 and supplementary fig .
cryo - em was performed using a fei technai g2 polara microscope equipped with an energy filter ( gatan ) and a k2 summit direct detection device ( gatan ) .
briefly , 5 l of nanomaterial sample ( ~60 m bpd for the pmil or l[bpd ] ; ~30 or 125 m xl184 for the pmil or np[xl184 ] , respectively ) mixed with 2 l of bsa gold tracer ( em - grade 6 nm ; electron microscopy sciences , 25484 ) re - suspended in phosphate - buffered saline were deposited onto glow - discharged holey carbon grids ( quantifoil r 2/1 200 mesh , copper ; electron microscopy sciences ) , blotted and rapidly vitrified in a liquid ethane and propane mixture ( 50:50 ) using a custom - built plunger ( max planck institute of biochemistry , germany ) .
imaging was performed at 300 kv under low - dose conditions with 4.98  or 6.12  sampling and a defocus of 3 or 6 m for 2d or 3d images , respectively .
2d images were obtained using the dose - fractionation mode of the detector ( ~2040 e /  cumulative dose ) .
tilt series ( 60 ) for tomography were collected around a single axis with a 2 sampling increment using serialem software ( ~100 e /  cumulative dose ) .
renders of 3d pmil and np[xl184 ] objects were created by manual segmentation in imod and rendered using vmd .
5 . only unambiguous np[xl184 ] objects were counted ( ~20 nm in diameter or greater ) .
mean lamellarity was calculated as : l(nl  l)/lnl , where nl is the number of objects with lamellarity l ( e.g. , l = 1 for unilamellar liposomes ) .
tem ( philips cm10 ) was performed using negative staining either on untreated ( 200 mesh nickel pelco support film with a formvar / carbon coating , ted pella inc . ) or ionized carbon coated grids ( to promote sample adhesion ) .
briefly , 10 l of sample was added to the grid , air - dried and stained ( 2 l , 1.0% phosphotungstic acid ) .
monolayer cultures of aspc1 cells ( american type culture collection , crl-1682 ; low passage number , < 20)recently tested ( july 2015 ) and found to be negative for mycoplasma contamination ( mycoalert mycoplasma detection kit , lonza)were maintained in media ( rpmi 1640 , mediatech ) supplemented with 10% fetal bovine serum ( invitrogen ) , 100 units / ml penicillin and 100 g / ml streptomycin .
the aspc1 cell line has not been listed in the database of cross - contaminated or misidentified cell lines maintained by the international cell line authentication committee .
cellular uptake of pmils and l[bpd ] was tested in multi - well plates with coverslip bottoms ( greiner bio - one ) plated with aspc1 cells allowed to attach and grow overnight .
nanoconstructs were added to the wells at staggered time points to reach a concentration of 100 nm bpd and to achieve varying incubation times at 37c ( 1590 min ) .
imaging was performed with an olympus fv1000 confocal microscope with a 20  0.75 na ( numerical aperture ) objective .
bpd excitation was performed using a 405 nm diode laser with an emission spectrograph centred on the 696 nm bpd fluorescence emission peak .
the laser , photomultiplier tube detector and pinhole settings , as well as brightness - contrast adjustment settings for display , were kept constant for all images .
in addition , phase contrast images were collected during microscopy in order to focus on a high - density field of cells ( not shown ) .
images were also collected for untreated cells ( 0 min ) to quantify the autofluorescence background and to define a fluorescence intensity threshold that rejects 99.5% of the background signal .
this intensity threshold was applied to all images to select pixels above the autofluorescence background ( true bpd signal ) for analysis .
the resulting cellular uptake data was fit to a simple biexponential pharmacokinetic model : a  ( e  e ) , where k and j are the elimination and absorption rate constants , a is a coefficient dependent on the administered bpd dose as well as its bioavailability and t is time post - administration . for in vitro pdt , 0.2510 aspc1 cells were grown on a 35-mm culture dish for 24 h and incubated with nanoconstructs containing bpd ( 250 nm equivalent ) and/or xl184 ( 100125 nm equivalent ) in 1 ml complete medium for 1 h. the incubation media was then replaced with 2 ml of fresh , complete media prior to photoirradiation .
this removal of nanoconstructs not uptaken by cells prior to irradiation limits the release of xl184 and the generation of photocytotoxic species to intracellular and cell - associated nanoconstructs .
singlet oxygen sensor green ( sosg ; molecular probes ) and d - mannitol ( sigma - aldrich ) were used to probe reactive oxygen species involvement in bpd - pdt - induce met activation .
tyrphostin ag1478 ( sigma - aldrich ) and bacterial toxin b ( toxin b , clostridium difficile - calbiochem , millipore ) were used to test for involvement of enzymes known to participate in met transactivation .
sosg and mannitol were added to cells immediately prior to laser irradiation in fresh media and then removed immediately after pdt by a second media replacement step .
ag1478 and toxin b were incubated with cells in fresh media for 30 min and 2 h , respectively , prior to laser irradiation and then removed immediately after pdt by a second media replacement .
all animal experiments were conducted with approval and according to guidelines established by the massachusetts general hospital institutional animal care and use committee .
experiments were carried out on 6-week - old male swiss nude mice weighing 2025 grams ( cox breeding laboratories ) . for tumour implantations and photoirradiation ,
tumours were implanted by injection of a 50 l volume containing 10 aspc1 cells in a 1:1 mixture of matrigel ( bd biosciences ) and culture media .
subcutaneous tumours were implanted above the hind leg and tumour volumes were estimated longitudinally by measuring the three tumour dimensions using a calliper and the hemi - ellipsoid formula : volume =   l  w  h/6 , where l , w and h , are the tumour length , width and height . note that here , h , represents the measured height of the hemi - elliptical tumour , which is half the height of a full ellipsoid .
eighteen days following cancer cell implantation , subcutaneous tumours reached volumes of ~50 mm prior to the start of treatment . for orthotopic tumour implantation
, animals were laid supine , a small left abdominal flank incision was made to exteriorize the pancreas and the cell suspension was injected into the pancreas . a small amount of 10% povidone / iodine was applied topically to the injection site .
then the incision was closed with 4 - 0 sutures and 10% povidone / iodine was then applied to the incision site to prevent infection .
ten days after cancer cell implantation , orthotopic pancreatic tumours reached volumes of ~25 mm prior to the start of treatment .
all injections for treatment were done intravenously ( tail vein ) in 200 l sterile phosphate - buffered saline .
mice were randomized into the various treatment groups , and the tumours of mice receiving bpd were irradiated with nir light ( using the 690 nm diode laser listed above ) 1 h post - injection , delivered at an irradiance of 100 mwcm .
subcutaneous tumours were irradiated transcutaneously while orthotopic tumours were surgically exposed as for tumour implantation and irradiated .
fourteen days after treatment , orthotopic tumours were excised to estimate their volumes using callipers and the ellipsoid formula : volume =   l  w  h/6 .
briefly , orthotopic pancreatic tumours were excised 2 weeks post - treatment , embedded in optimal cutting temperature compound and frozen at 80c .
sections were ( 1 ) fixed in 1:1 acetone : methanol for 15 minutes at 20c , ( 2 ) air dried for 30 minutes , and ( 3 ) washed three times in phosphate - buffered saline .
a blocking solution ( dako protein block reagent ) was applied for 30 minutes , followed by application of the immunostains , at ~5 g / ml monoclonal antibody ( mab ) each diluted in background reducing dako antibody diluent for 2 h at room temperature in a humidifying chamber . finally , the slides were washed again three times , mounted ( invitrogen slowfade gold with 4,6-diamidino-2-phenylindole , dapi ) with a coverslip and sealed with nail polish .
confocal fluorescence imaging was performed using an olympus fluoview 1000 confocal microscope with a 10  0.4 numerical aperture ( na ) or a 20 0.75 na objective .
excitation of dapi , anti - mouse pecam-1 ( cd31 ; clone 390 ; cbl1337 , millipore ) mab - alexa fluor 568 conjugates and anti - human cytokeratin 8 ( clone lp3k ; mab3156 , r&d systems ) mab - alexa fluor 647 conjugates was carried out using 405- , 559- and 635-nm lasers , respectively .
mosaic images of entire tumour cross - sections were collected and stitched together using the olympus fluoview software .
the anti - human cytokeratin 8 stain ( a cytoskeletal protein highly expressed by aspc1 cells ) has dual selectivity for the epithelial cancer cells because it does not react with mouse proteins .
all analyses were performed using custom matlab ( mathworks ) routines for batch image processing .
microvessel density values were calculated from whole tumour sections , within viable tumour tissue only , and averaged over slices from the entire tumour rather than a more complex  hot spot  identification and calculation , which is difficult to define objectively .
intratumoural microvessel volume is calculated by multiplying microvessel density with the viable tumour volume in each slice and then summing over the whole tumour by interpolation . here
, we used the minimum tumour subsampling necessary  based on a mathematical model  to resolve a statistically significant change in intratumoural microvessel volume , as validated previously using the orthotopic aspc1 tumour model ( supplementary note 9 ) .
a quantitative reverse transcription  polymerase chain reaction ( qrt - pcr ) assay was performed on excised liver and iliac lymph nodes to estimate the number of human cancer cells in excised organs as described and validated previously .
briefly , qrt - pcr is used to measure the total number of human cancer cells from the level of human and mouse glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) housekeeping genes .
at least 300 mg of freshly excised liver and retroperitoneal lymph nodes were collected at the treatment endpoint and snap frozen in liquid nitrogen .
the frozen samples were then pulverized and homogenized , followed by rna extraction ( rnaeasy plus mini kit ; qiagen ) .
human and mouse gapdh gene were measured using custom synthesized primers ( invitrogen ) . for each specimen ,
the cycle threshold ( ct ) from human gapdh gene was normalized by ct from mouse gapdh gene .
the normalized ct was quantified into number of cancer cells using a standard curve generated with a set of organ lysates from no - tumour control mice mixed with different numbers of human cancer cells .
specific statistical tests are indicated in the figure captions and were carried out using graphpad prism ( graphpad software ) . all reported p values are two - tailed .
parametric tests ( one - way anova with tukey s post - test ) were used for in vitro drug release ( fig .
6 ) studies ; and , the dagostino & pearson omnibus normality test (  = 0.05 ; requires n  8 replicates per group ) did not identify significant deviations from normality within these data sets ( testing could only performed for groups with n  8 replicates ) .
note that all groups within the drug release data were analysed together ( some groups appear only in supplementary fig .
1 ) were analysed using nonparametric tests ( the mann - whitney u test or kruskal 
wallis one - way anova ) . the brown  forsythe test (  = 0.05 )
was applied to all data sets with n  3 replicates to test for equal variance ( regardless of whether parametric or nonparametric analysis was used ) and identified significant deviations from equal variance ( figs .
in these cases , the data were analysed following a logarithmic transform of the data to pass the brown  forsythe test .
5d ) following a natural logarithm transform of the data to pass the dagostino & pearson omnibus normality test .
no exclusion criteria were used , and no data points or animals were excluded from analysis .
animal sample sizes were selected to ensure adequate power ( 80% ) to detect a 20% difference using a maximum of 16 animals per group assuming a standard deviation of 15% . for the subcutaneous model , significance
 nanoscale drug delivery vehicles can facilitate multimodal therapies of cancer by promoting tumour - selective drug release . 
 however , few are effective because cancer cells develop ways to resist and evade treatment . here 
 , we introduce a photoactivatable multi - inhibitor nanoliposome ( pmil ) that imparts light - induced cytotoxicity in synchrony with photo - initiated and sustained release of inhibitors that suppress tumour regrowth and treatment escape signalling pathways . 
 the pmil consists of a nanoliposome doped with a photoactivatable chromophore ( benzoporphyrin derivative , bpd ) in the lipid bilayer , and a nanoparticle containing cabozantinib ( xl184)a multikinase inhibitor  encapsulated inside . near infrared tumour irradiation , 
 following intravenous pmil administration , triggers photodynamic damage of tumour cells and microvessels , and simultaneously initiates release of xl184 inside the tumour . 
 a single pmil treatment achieves prolonged tumour reduction in two mouse models and suppresses metastatic escape in an orthotopic pancreatic tumour model . 
 the pmil offers new prospects for cancer therapy by enabling spatiotemporal control of drug release whilst reducing systemic drug exposure and associated toxicities . 
METHODS
NP[XL184] synthesis and characterization
PMIL synthesis and characterization
PMIL stability and drug photo-release kinetics
Cryo-electron microscopy
Transmission electron microscopy
Cell culture studies
None
Microvessel immunofluorescence imaging
Measurement of metastatic burden
Statistical analyses
Supplementary Material