| Section 1:Anti-av?3 antibody guided three-step pretargeting approach using magnetoliposomes for molecular magnetic resonance imaging of breast cancer angiogenesisObjectivePretargeting of biomarkers with nanoparticles in molecular imaging is promising to improve diagnostic specificity and realize signal amplification,but data regarding its targeting potential in magnetic resonance(MR)imaging are limited.The purpose of this study was to evaluate the tumor angiogenesis targeting efficacy of the anti-av?3 antibody guided three-step pretargeting approach with magnetoliposomes.Materials and methods1.1 Materials1,2-Distearoyl-sn-glycero-3-phospho-rac-glycerol,sodium salt(DSPG)was purchased from Lipoid GmbH(Ludwigshafen,Germany).1,2-distearoyl--sn-glycero-3-phosphoethanol-amine-N-[biotinyl(polyethylene glycol)2000](Biotin-PEG2000-DSPE)and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy[polyethylene glycol]-2000)(DSPE-PEG-2000)were obtained from Avanti Polar Lipids(Alabaster,AL,USA).LissamineTM rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine,triethylammonium salt(Rho-DHPE)was from Invitrogen corporation(Carlsbad,CA,USA).2-(Tris(hydroxymethyl)methylamino)ethane-1-sulphonic acid(TES),methyl thiazdyl tetrazolium(MTT),dimethyl sulfoxide(DMSO),paraformaldehyde were from Sigma(Saint Louis,SA,USA).Ferric chloride(FeCl3·6H2O),ferrous sulfate(FeSO4·7H2O),concentrated hydrochloric acid(28%NH4·OH in water solution)and laurate were purchased from Guangzhou chemical reagent Co.Ltd.All other chemicals were of analytical grade.1.2 Preparation of SPIOUsing a modification of a previously described method[1],appropriate amounts of FeSO4·7aq,FeCl3-6aq were mixed at a 1:2 molar ratio and transferred to a 3-necked flask,to which 15ml ammonia water was added gradually with agitation by magnetic stirring apparatus.The reaction was maintained at 60 ? for 15 min to obtain Fe3O4 nanoparticles.The black precipitates were isolated by applying a permanent magnet,washed with 50ml ammonia/water(5/95)3 times in sequence,the particles were combined with 1g laurate[dissolved in 50ml ammonia/water(5/95)],reacted at 90 ? for several minutes,and finally obtained laurate-stabilized SPIO.1.3 Preparation of magnetoliposomes Liposomes were prepared by a thin lipid film hydration method.DSPG,cholesterol,and biotin-PEG2000-DSPE with a molar ratio of 3:2:0.15 were dissolved in chloroform.Nonbiotinylated control liposomes contained DSPG,cholesterol,and PEG2000-DSPE at a molar ratio of 3:2:0.15.As a fluorescent marker,0.2 mol%of Rho-DHPE was added.The solvent was removed by rotary evaporation,followed by additional drying under nitrogen.The dry lipid film was hydrated in 10ml TES(5mM,PH 7.0).Subsequently,the mixture was dispered by ultrasonic cleaner(200W,10min)and then sonicated by ultrasonic disintegrator(150W,30min).Phospholipid vesicles was obtained by centrifuged at 37? for 20 min at 2500g/min.Magnetoliposomes(ML)and biotinylated magnetoliposomes(Bt-ML)were prepared from laurate-stabilized SPIO(0.5ml;Fe concentration 22mg/ml),which were dialyzed(molecular weight cut-off:10,000)at 37? for 3 days in the presence of phospholipid vesicles(10ml containing 0.15mmol phospholipid)with regular changes of the buffer(5mM TES,PH 7.0)by self-assembly procedure to form lipid-coated SPIO.1.4 Biotinylation of monoclonal antibodiesAnti-avP3 monoclonal antibodies(Biolegend,San Diego,USA)were biotinylated with Sulfo-NHS-LC-Biotin following the manufacturer's(Pierce,Rockford,USA)protocol.After the purification by ultrafiltration with an Amicon Ultra-15 Centrifugal Filter Unit with a 10 kDa membrane from Millipore(Billerica,MA,USA),the final biotin/antibody ratio was =4 as determined by the HABA method(Pierce).1.5 Physico-chemical properties of ML Particle size and morphology determinationA droplet of the SPIO,ML,and Bt-ML suspension were deposited on a Formvar-coated grid,respectively.After 5 minutes,excess fluid was withdrawn,and the preparations were stained with 1%phosphotungstic acid and allowed to sit for 30 seconds for negative staining to distinguish their shell from the core.Liquid was then removed from the carbon grid,the grid samples were examined using a transmission electron microscope(TEM,H-7650,HITACHI,Japan)operating at 200 kV.The mean size distribution of SPIO,ML,and Bt-ML were determined using a Malvern Zeta sizer 3000HS(Malvern,UK)at a scattering angle of 90° at 25 ?operating at 632.0 nm Phosphate analysis&iron concentrationsPhosphate analysis was done according to the method of Vaskovsky et al.(1975)and iron concentrations were measured by atomic absorption at 372.2nm(AAS,Z-5000,HITACHI,Japan).The loading content of Fe in SPIO,ML,Bt-ML was determined using an atomic absorption spectrophotometer(AAS,Z-5000,HITACHI,Japan).Briefly,3.0 ml of the sample was digested in a medium containing of 1 ml of HN03(65%)and 2 ml H202(30%)to decompose the iron oxide.The clear solution gained and some distilled water was added to make sure the metered volume.The assay was linear between 0 and 25 mg/ml with a correlation coefficient of 1.0.X-ray powder diffraction(XRPD)Sample crystallization was analysed using XRPD with a X-ray diffraction system(D-MAX2200 VPC,RIGAKU,Japan)using Cu Ka radiation with = 1.542 °A and a divergence slit of 1°.The samples were gently consolidated in flat aluminium sample holders and scanned at 40kV and 30mA from 10° to 70° to collect 2 data using a scanning speed of 0.1285° min-1 and a step size of 0.0084°.The identification of SPIO,ML,and Bt-ML were carried out by comparing the diffraction pattern of the sample with library data in the powder diffraction files using Diffract-plus software.1.6 Magnetic property measurementsThe nanoparticles were prepared in 0.01,0.02,0.03,0.04,and 0.05mM of Fe concentrations.T2 of these solutions were determined on a 3.0T MR system(Signa Excite,General Electric,USA).A T2 mapping sequence(TR:3000ms,TE:20,40,60,80ms,matrix 512×160,field of view[FOV]200 mm)was used to measure transverse relaxation times.Images of the various.solutions were analyzed by defining regions of interest(ROI)in each test tube.Relaxivity(r2)was calculated through the curve fitting of T2(s-1)vs the Fe concentration(mM).A vibration sample magnetometer(VSM,MPMS XL-7 magnetometer,Quantum design,USA)was used to characterize the magnetic properties of SPIO,ML and Bt-ML.The hysteresis of the magnetization was recorded at 300 K obtained under circulate magnetic field ranged between +1 and-1Tesla.1.7 Cell cultureAll the cell lines were gifts from research center of clinical medicine of Nanfang Hospital(Guangzhou,China).Both breast cancer cell lines of MDA-MB-435S(avp3-integrin positive)and MCF-7(av?3 negative)cells were grown in Dulbecco's modified Eagle's medium(DMEM),and RAW264.7 in RPMI-1640 supplemented with 10%fetal bovine serum(FBS),penicillin(100 U/ml),and streptomycin(100 U/ml)in a humidified incubator at 37? with 5%C02 in the air.1.8 Fluorocytometry and immunofluorescenceCells(MDA-MB-435S,MDA-MB-231,and MCF-7)were detached with trypsin/EDTA,washed once in complete medium and allowed to remain at 37? for 30 min.Cells were then washed in ice-cold wash buffer phosphate buffered saline/2%bovine serum albumin were incubated with a FITC conjugated primary monoclonal antibody for 30 min at 4?.Cells were washed and resuspended in wash buffer and analysed by fluorocytometry on a FACScan analyzer.Sterile 13-mm coverslips were placed in the wells of a 24-well plate;500 gl of cell suspension(2 x 105 cells ml-1)was added to the wells and incubated at 37?.Morphology of the cells on the different substrates was examined by precoating the coverslips with various ECM proteins and adding the cells in a serum-free medium.At certain time intervals thereafter,the coverslips were washed with 0.1%BSA in phosphate-suffered saline(PBS)and fixed in 1:10 formalin for 10 min.Cells were then incubated incubated with the FITC-labeled primary antibody at 4? overnight.After further washing,the coverslips were mounted onto glass slides and examined with a fluorescence microscope.1.9 Cytotoxictiy assayIn vitro cytotoxicity of the nanoparticles were assessed using the MTT assay of MDA-MB-435S and MCF-7.Briefly,the cells were seeded in a 96-well plate at the density of 4×104 cells/well for 48h,then culture medium was replaced with 200?l medium containing ML or Bt-ML(at 200,400,800,1200,1600 and 2000 ?M Fe)and kept incubated for 20h.Subsequently,50?l MTT solution(5 mg/ml)was added to the wells and incubated for 4 h in a humidified atmosphere prior to the addition of 150?l of DMSO into each well to dissolve the formazan crystals,gentle shaking for 10 min so that complete dissolution was achieved.The reaction mixture on each well of the 96-well culture plate was measured using the ELISA reader BIOTEK ELX800 at wavelength 570 nm.The control group contained cells and cell culture medium without nanoparticles.1.10 Visualization of the cellular uptake by Prussian blue stainingRAW264.7 cells were seeded onto the 6-well plate with 5×105 cells/well and incubated for 24h.Then the supernatant was removed and 2ml of the full RPMI-1640 culture medium containing SPIO,ML,and Bt-ML(Fe concentration of 50 ?g/ml)was added to RAW264.7 cells,respectively.After incubation at 37? for 2 h,the supernatant was removed and cells were washed three times with phosphate-buffered saline(PBS).Then the cells were fixed by 2ml 4%paraformaldehyde solution for 20 min.After fixation,the cells were stained with a filtrated and fresh prepared potassium ferrocyanate solution(mixture of equal volume of 2%potassium ferrocyanate with 2%hydrochloric acid)for 30 min at 37 ? causing a deep blue complex,rewashed and then counterstained with nuclear fast red for 5 min.The wells without nanoparticles were used as blank.1.11 In vivo biodistributionAll nanoparticles were administered intravenously(IV,ear marginal vein)in New Zealand rabbits(n=2/groups)at 100 ?mol Fe/kg.The animals were sacrificed by air embolism.Tissue samples of the liver,1ung,spleen,heart,kidney,and cervical lymph nodes were removed for histochemistry.Following formalin fixation and embedding in paraffin,deparaffinated tissue sections(10 ?m thick)were stained for ferric iron using Perls' reaction(Prussian Blue stain,counterstained with Nuclear Fast Red).1.12 In vivo MR imagingAll procedures were approved by the animal center of Southern Medical University(Guangzhou,China).Tumor xenograft with 1×107 MDA-MB-435S cells were implanted into the right caudal mammary fat pad of female mice(NOD/SCID,Huafukang company,Beijing,China).6-8 weeks after implantation when the tumor reached 0.8-1.2 cm in diameter,the animals were randomized into two groups:targeted Bt-ML and non-targeted ML group for MR scan(n=5/group).The mice were subjected to injections via the tail vein with 150 ?g biotinylated anti-avP3 MoAb over 2 min(first step).After 36 h,1 mg of cold avidin(Santa Cruz,California,USA)was injected over 2 min,followed by an additional 0.5mg of cold streptavidin 10 min later(second step).2 h later,ML or Bt-ML was injected via the tail vein at a dose of 80 ?mol Fe/kg(third step).Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium(60mg/kg)before the MR studies.MR imaging was performed at before and 2h postinjection with a 3.0T MR system(Signa Excite,General Electric,USA)equipped with a mouse-imaging coil.Coronal T2-weighted SE(TR/TE= 4000/85ms,FOV = 12 cm,matrix = 320x224,NEX =4,thickness/interval 2.5/0.2 mm).The pixel intensities from T2-weighted images were analyzed with MATLAB software(The Math-Works,Natick,MA,USA).17,18 In each baseline and 2h postinjection T2-weighted image slice,a region of interest(ROI)was manually placed around the tumor edge on each baseline slice,and the standard deviation(SD)of the average tumor signal at baseline was calculated.Subsequent,serial images were spatially coregistered using a cross-correlation routine,and the tumor ROI mask was copied to each time point.At 2h postinjection,pixels with an MR signal intensity decrease?3 SD below the baseline tumor signal were considered significant.The enhanced fraction within the whole tumor for each individual mouse was determined by the number of enhanced pixels divided by the number of total pixels within the tumor.1.13 Fluorescence Immunohistochemistry and Prussian blue stainingAfter MR imaging,animals were euthanized,and tumors were resected,and quickly frozen in optimal cutting temperature compound for immunohistochemistry.Frozen tumor tissue slices(5 ?m thick)were fixed in acetone for 10 min,dried in the air for 30 min.Nonspecific binding of antibodies was blocked by incubation with donkey serum for 30 min at room temperature.The sections were incubated overnight with rat anti-mouse CD31 monoclonal antibody(BD Pharmingen,San Jose,CA,USA),then washed and incubated with fluorescein isothiocyanate(FITC)goat anti-rat secondary antibody(Jackson ImmunoResearch,West Grove,PA,USA).4,6'-diamidino-2-phenylindole(DAPI)(Molecular Probes,Eugene,Oregon,USA)was used for cell nuclei staining.Sections were also stained with Prussian blue according to standard clinical pathology protocols.1.14 Statistical analysisStatistical analysis was performed using two-tailed unpaired student t-test.at the p<0.05 level.All data were expressed in the form of the mean±standard deviation.All tests were performed using SPSS version 13.0.Results2.1 Synthesis and characterizationTEM micrographs of magnetite nanoparticles showed that all the synthesized nanoparticles(NPs)were relatively uniform in size and almost spherical(Fig.l).ML and Bt-ML demonstrated core-shell structure loaded with SPIO.The particle sizes,hydrodynamic diameters,and their polydispersity index are summerized in Table 1.The difference of hydrodynamic diameters from TEM can be explained by the flexibility of the PEG chain.The crystallography of the three SPIO colloids was verified by XRD(Fig.2).The identical peaks for Fe3O4,which were located at 30.1°,35.5 °,43.1 °,53.4 °,57.0 °,and 62.6 °,corresponding to their indices(220),(311),(400),(422),(511),and(400)were observed in all of the samples.The results indicated that liposomal encapsulation of the magnetic cores did not make significant changes in the crystal phase of Fe3O4.2.2 Magnetic property measurementsThe T2 relaxivity of NPs at 3.0T MR was examined(Fig.3).The obtained results showed that with the increase of Fe concentration,the MRI signal intensity of NPs decreased(Fig.3a).As shown in Fig.3b,nanoparticles were well-fitted by a line within the analyzed range of iron concentration(0.01-0.05 mM),thus exhibiting the typical property of SPIO in shortening T2 relaxation time.The specific relaxivity values of SPIO,ML,and Bt-ML were found to be 0.722×103,0.611×103,0.675×103 mM-1 s-1,(Fig.3b),respectively.The magnetic property of SPIO,ML,and Bt-ML was evaluated by VSM toconfirm the feasibility and sensitivity as MR imaging nanoprobes(Fig.4).Typical hysteresis curves regarding superparamagnetic behaviors were observed.The magnetization decreased from plateau value to zero when magnetic field intensity decreased,which implied the NPs would respond well to magnetic fields without any permanent magnetization.The specific saturation magnetism ?s at IT for ML,and Bt-ML were 11.7,and 8.05 emu/g,which is much smaller than the value of SPIO(41.2 emu/g).The decrease of the magnetization of such coated particles might be due to the coating of non-magnetic liposomes on the magnetic nanoparticles or the decrease in their iron oxide content.2.3 Fluorocytometry and immunofluorescenceThe integrin av?3 was strongly expressed in MDA-MB-435S cells,and in MDA-MB-231 cells only at a relatively low level,but only weakly in MCF-7 cells.The fluorescence of integrin av?3 was observed mainly on the surface of MDA-MB-435S cells.2.4 Cytotoxicity assayIn order to investigate the cytotoxicity of the novel carrier nanoparticles,the MTT assay of viability of MDA-MB-435S and MCF-7 was used.As shown in Fig.5,no significant toxicity in cell viability was observed up to 2000 ?M Fe concentrations in both cell lines.This resullt indicate that ML and Bt-ML are biocompatible and low-toxic at the given Fe concentration range(200-2000 ?M).2.5 Nanoparticles uptake by macrophagesThe presence of intracellular SPIO allowed direct visualization of their uptake by Prussian blue staining.As shown in Fig.6A,our results showed RAW264.7 cells incubated with SPIO were stained in intensive blue color under 50 ?g/ml of the Fe concentration in culture medium.Blue areas or spots could be seen in almost every cell.In comparison,the RAW264.7 cells incubated with ML or Bt-ML(Fig.6B-C)showed a little weak blue color appearance.The result indicated that SPIO entrapment into liposomes and modification by PEG could dramatically decrease the internalization of the NPs by mouse macrophage cells.2.6 BiodistributionPrussian Blue staining of tissue specimens revealed marked uptake of SPIO,while little uptake of ML and Bt-ML in the liver.For the SPIO preparation,iron pigment accumulated primarily within the Kupffer cells.In the spleen,there was a high uptake of SPIO by macrophages in the red pulp,while hardly no uptake of ML and Bt-ML.For the lung,no uptake in alveolar macrophages was observed.A remarkably complete lack of uptake was seen lymph nodes,kidney and heart for all nanoparticles.2.7 In vivo MR imagingThe in vivo MR imaging was performed before injection and 2h postinjection(Figure 7A).The precontrast images also contained some "enhanced" pixels,as their signal intensities were below the threshold.For mice receiving Bt-ML,the spatial distribution of enhanced pixels was heterogeneous and mainly in the periphery of the tumor.For mice receiving ML,pixel signal enhancement was much less pronounced and scattered within the tumor rim.The enhanced fraction in tumors was 7.00±1.8%(t=5.226,P<0.05)at 2 h postinjection of targeted Bt-ML,while only 2.O±11.1%decrease was observed in the non-targeted group(Figure 7B).Additionally,we found high signal reductions in the liver at 2 h postinjection of Bt-ML and ML,respectively.2.8 Fluorescence immunohistochemistry and Prussian blue stainingEx vivo fluorescence microscopy was used to investigate the binding location of NPs inside tumor tissues.Rhodamine fluorescence from av?3-targeted Bt-ML was colocalized with the angiogenic endothelial cells within tumor tissues and mainly distributed to the periphery of tumor(Fig.8A-C).Little fluorescence was also observed outside the vessel wall or lumen.In contrast,fluorescence from ML was only found in the interstitial space of the tumor but not colocalized with endothelial cells(Fig.8D-F).To further verify the accumulation of the SPIO-encapsulated nanoparticles in the tumor tissue,Prussian blue staining was carried out,as shown in Fig.9.In good agreement with fluorescence immunohistochemistry,blue spots of Bt-ML showed vascular localization.In nontargeting group,only a little ML was observed with an unspecific distribution.The neovascular spatial distribution seen microscopically,was consistent with the contrast enhancement patterns of angiogenesis observed in MR imaging.ConclusionsIn conclusion,we have developed SPIO-based magnetoliposomes with superparamagnetism,biocompatibility,and low cytotoxicity.Our results demonstrated that with an antibody-guided three step pretargeting approach,magnetoliposomes were sensitive enough to allow for MRI detection of tumor angiogenesis.Additionally,the MR enhancement revealed the predominantly peripheral distribution of angiogenesis,which was highly consistent with the histological distribution of av?3-targeted rhodamine nanoparticles.Thus,our strategy with liposomal carriers may provide a versatile platform for assessment of angiogenic activity at the molecular level and can be used as a target-specific anticancer drug delivery system.Section 2:Use of intravoxel incoherent motion diffusion-weightedMR imaging for assessment of treatment response to invasive fungal infection in the lungObjectivesThe purpose of this study was to determine whether intravoxel incoherent motion(IVIM)-derived parameters and apparent diffusion coefficient(ADC)could act as imaging biomarkers for predicting antifungal treatment response.Materials and methodsThis study was approved by the local ethics committee and informed consent was obtained from all participants.Forty-six consecutive patients(mean age,33.9 ± 13.0 y)with newly diagnosed IFI in the lung according to ORTC/MSG criteria were prospectively enrolled.All patients underwent diffusion-weighted magnetic resonance(MR)imaging at 3.0 T using 11 b values(0-1000 sec/mm2).ADCtotai,pseudodiffusion coffiecient D*,perfusion fraction f,and the diffusion coefficient D were calculated using the IVIM model.Interobserver agreement were evaluated using the intraclass correlation coefficient(ICC).Patients were stratified into favorable(n=32)and unfavorable response(n=14)groups based on follow-up for determination of the predictive powers of IVIM parameters using Student t test and receiver operating characteristic(ROC)curve analyses.ResultsDWI signal decay curves in favorable response group were found to be biexponential fit in comparison with the monoexponential fit for the unfavorable response group.D was consistently lower than ADC in all tissues.Interobserver agreement on the measurements between the two observers were excellent(ICC=0.954-0.988;narrow width of 95%limits of agreement).f values were significantly lower in the unfavorable response group(12.6%±4.4%)than in the favorable response group(30.2%± 8.6%)(Z=4.989,P<0.001).However,the ADCtotal,D,and D*were not significantly different between the two groups(P>0.05).Receiver operating characteristic curve analyses showed f to be a significant predictor for differentiation(AUC=0.967),with a sensitivity of 93.8%and a specificity of 92.9%.ConclusionsIVIM-MRI is potentially useful in the prediction of antifungal treatment response to patients with IFI in the lung.Our results indicate that a low perfusion fraction f may be a noninvasive imaging biomarker for unfavorable response. |
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