Conjugation of the PEG Amines to the NIR Dyes

Supplementary Information

Supplementary Material and Methods

Conjugation of the PEG amines to the NIR dyes

Number average molecular weights (Mn) and weight average molecular weights (Mw) of the starting polymers PEG amine 20 kDa (P20, Fluka, St Louis, MO) or 40 kDa (P40, JenKem Technology USA Inc., Allen, TX) were determined by size exclusion chromatography (SEC) with a Viscotek GPCmax system (Malvern, UK, equipped with differential refractive index and light scattering detectors). A phosphate buffer 0.1 mmol/L (pH 7.4) was prepared and filtered immediately prior to use. Freshly prepared standard solution of PEG was used for the multidetector calibration, which was then checked with a freshly prepared dextran solution (PEG and dextran: PolyCAL ® TDS-PEO-NB, Viscotek). Standards and samples were dissolved in buffer (5 mg/mL), filtered on polyamide filters (0.2 m) and transferred in a HPLC vial closed with silicon septa. Adequate molecular weight separation was achieved using two ViscoGEL columns (GMPWXL, hydroxylated polymethacrylate) in series at a flow rate of 0.7 mL/min and a temperature of 35 °C . The molecular weight and polydispersity values obtained through SEC (P20: Mn 18.0 kDa, Mw/Mn 1.11; P40: Mn 33.6, Mw/Mn 1.05) were compatible with the values obtained by means of 1H NMR (Bruker Av400 spectrometer, Bruker BioSpin,

Fällanden, Switzerland, samples dissolved in D2O) (Table 1). The radius of gyration of P20 and P40 was calculated starting from the SEC results, using an empirical formula based on static light- scattering measurements . The correspondent hydrodynamic radius (RH) of was calculated according to Kirkwood and Riseman , and it was found to be 4.2 nm for P20 and 5.8 nm for P40.

Five hundred L reaction volume of anhydrous DMSO containing 75 nmol methoxypoly(ethylene glycol) amine and 75 nmol IRDye® 680LT NHS Ester or IRDye® 800CW NHS Ester (D680 or D800, respectively) was incubated at room temperature under stirring for 6 h in dark. The crude reaction was then diluted with 4 mL of ultrapure water and freeze-dried overnight. The lyophilizate was then reconstituted with 250 L of HEPES buffered saline (20 mmol/L HEPES, 145 mmol/L NaCl, pH 7.4). The coupled product (P20-D680, P40-680, P20-D800, or P40-D800) was purified using a Dye- removal column (Thermo Fisher Scientific, Rockford, IL). Briefly, 400 L of resin were transferred in an empty tube and centrifuged at 1000 x g for 30 s to remove the storage solution, as recommended by the supplier. The crude reaction was then added to the column and the tube was centrifuged again at 1000 x g for 30 s. The eluate was then passed for a second time on a newly packed resin to ensure complete removal of the unlabelled dye. The effectiveness of the purification was checked by means of high-performance liquid chromatography. The purified solution was diluted and analyzed by HPLC (LaChrom Elite series, Hitachi High Technologies, Mannheim, Germany). The chromatographic column used was a YMC Pack Pro C18 150 mm × 4.6 mm column with 3 μm particles (YMC Co., Kyoto, Japan). A gradient HPLC method was used to ensure a good resolution between the conjugated PEG-dye and the eventual correspondent free dye: solvent A consisted of 10 mmol/L tetrabutylammonium bromide (TBAB, Fluka) aqueous solution and solvent B contained 10 mmol/L TBAB in acetonitrile/water (90/10 v/v). Mobile phase A was filtered through a 0.45-μm polyamide filter (Sartorius, Göttingen, Germany) prior to use. The flow rate of the mobile phase was 1.0 mL/min. The HPLC gradient was kept as following (% A/B): 90/10, 80/20, 70/30, 100/0 for 25 min, with a post run time of 5 min. The detector wavelength was set at 680 and 780 nm according to the dye to be investigated. The injection volume was 10 μL. In a preliminary phase of this study, the purification procedure was set up by means of a semi-preparative HPLC procedure. Each eluted chromatographic peak was collected, freeze-dried 1 overnight, resuspended in D2O, and analysed via H-NMR in order to check the presence of the PEG chain.

The retention times (tR) for D800, D680, P20-D800, P40-D800, P20-D680, and P40-D680 were tR=20.8, 24.3, 14.4, 13.7, 18.9, and 18.3 min, respectively. The efficiency of the coupling procedure was then assessed spectrophotometrically.

1 / 8 Evaluation of the coupling efficiency

Five μL of each sample before and after the purification were diluted to 200 L with methanol HPLC grade. The absorbance of the samples was measured via visible-NIR spectroscopy (Cary 300 Bio UV- spectrophotometer, Agilent Technologies) using reduced volume cuvettes (quartz, 20-mm path length, Agilent Technologies). The coupling efficiency was calculated using equation S1:

CE = Cp/Ci * 100 [S1] where Cp is the concentration of the purified coupled dye and Ci is the concentration of the dye in the -1 -1 crude reaction. The molar extinction coefficients were from Licor (εMeOH = 250000 M cm for D680 -1 -1 and εMeOH = 300000 M cm for D800). The CE for P20-D800 was 61 %, for P40-D800 38 %, for P20-680 53 %, for P40-D680 31 %.

Pharmacokinetic (PK) study

Following tail vein injections of 0.05 nmol/g mouse weight (expressed in terms of D800 content) of P20-D800 or P40-D800 into wildtype 8 week-old mice (n=3), serial blood sampling from the saphenous vein (~30 L) was performed at t= 2 min, 15 min, 1 h and 3 h for P20-D800 and t=2 min, 15 min, 1 h, 3 h and 6h for P40-D800. A final blood sample was collected under terminal anaesthesia by cardiac puncture 6 h and 12 h post-injection for P20-D800 and P40-D800, respectively. Urine was also collected at the final time point. Blood samples were left to stand for 30 min and then were centrifuged at 3000 x g for 10 min. Serum was collected and frozen at -20°C before analysis.

PK parameters were determined with a serum concentration time profile. Serum was deproteinized following an optimized method . A volume of 10 L of serum was added to 15 L of acetonitrile/methanol (47/3 v/v). The tube was vortexed and centrifuged for 10 min at 1500 x g. Part of the supernatant (10 L) was withdrawn and loaded in a 384-wells plate from Greiner (Frickenhausen,

Germany). Fluorescence emission spectrum of each solution was acquired (λex: 750 nm, λem: 780-850 nm) immediately after the loading procedure in order to minimize evaporation of the solvent. Data were normalized to the fluorescence emission at 799 nm of the t=0 h time point. D800 concentration was calculated by analysing the data through a calibration curve created spiking mouse serum with the correspondent dye (R2=0.9775 for P20-D800 and R2=0.9974 for P40-D800). The serum concentration decay curve of P20-D800 and P40-D800 were analysed by non- compartmental analysis . Apparent first-order terminal elimination rate (Kel) was determined with a linear regression of the semilogarithmic plot of the serum concentration vs time curve, using the last 3 points of each curve. Terminal half-life t1/2β was calculated as ln 2/Kel. The areas under the curve at time t (AUC0-6h for P20-D800 and AUC0-12h for P40-D800, respectively) were computed using the trapezoidal method. Areas under the curve from time zero to infinity were calculated by adding AUC 0-t to the ratio of the last measurable concentration over Kel. Total blood clearance (CL) was calculated dividing the injected dose (in g) by AUC0-. The volume of distribution was obtained by dividing CL by Kel. In order to rule out the presence of unbound dye at the end of the kinetics, samples (t=6 h) from animals injected with P20-D800 or P40-D800 were analysed also via HPLC. First serum was deproteinized, and afterwards eluted in an analytical HPLC following the gradient protocol described above. P20-D800 and pure D800 dye were also eluted (0.9 mol/L D800 concentration) as controls.

Measurements of clearance and tissue distribution of vascular tracers

After intravenous injection of P20-D800 and P40-D800 tracers in normal mice, a series of in vivo images were acquired with the IVIS Spectrum to evaluate clearance of the tracers from the animals

(n=3 mice per tracer). Images (exposure, 2 s, λex: 745 nm, λem: 800 nm, binning of 4) were acquired with the animal in the ventral position at 1, 3, 6, and 24 h. Images of the ears were acquired

(exposure, 8 s, λex: 745 nm, λem: 800 nm, binning of 4) at 3, 6, 24, 48, and 72 h to monitor clearance from the skin. At one week post injection of tracer, the mice were sacrificed and ex vivo images of 2 / 8 blood, heart, kidney, liver, lung, spleen, and inguinal lymph nodes were acquired (exposure, 10 s, λex: 745 nm, λem: 800 nm, binning of 4). Signals were compared to mice (n=3) fed alfalfa-free chow that were not injected with tracers.

Quantifications of blood volume fraction, tissue leakage rate and vascular leakage rate

After exporting the data from the Living Image software into Microsoft Excel, signal enhancement values at each time point (t expressed in min) were calculated for the saphenous vein and each ear by subtracting the pre-injection background fluorescence signal intensities as shown in equation S2:

Signal Enhancement (t) = Fluorescence Intensity (t) – Background Fluorescence Intensity [S2]

Next, the ear signal enhancement (ESE) values at each time point were divided by the saphenous vein signal enhancement (SVSE) values for normalization to the injected dose and intravascular dynamics of the conjugates. This normalized set of values was used to calculate two quantifications based on the assumptions of the two-compartment model for ear tissue. The first, blood volume fraction (BVF), is defined as an estimation of detected signal in the tissue that is intravascular. As shown in equation

S3, it was calculated as ESE divided by SVSE at the time point t1 at which the vascular signals reach equilibrium after tail vein injection (typically around 1-2 min post-injection, a time point at which it is assumed no significant leakage has yet occurred):

At t1 at which SVSE reaches equilibrium:

BVF = ESE (t1) / SVSE (t1) [S3]

The second quantification, tissue leakage rate (TLR, expressed in min-1), is the linear fit of the plot of normalized enhancement values (ESE/SVSE) over time as shown in equation S4. A positive slope indicates an increase in extravascular signal or leakage of the tracer into the tissue.

ESE (t) / SVSE (t) = (TLR * t) + ((ESE (t0) / SVSE (t0)) [S4]

As this tissue leakage rate is dependent on the surface area of blood vessels in the tissue, an additional normalization was necessary before calculation of a vascular leakage rate (VLR). First, the SVSE values at each time point were adjusted to the SVSE at the time of peak saphenous vein enhancement t1, as shown in equation S5:

NSVSE (t) = SVSE (t) / SVSE (t1) [S5]

Second, this same normalization was performed for the ESE values, as shown in equation S6:

NESE (t) = ESE (t) / ESE (t1) [S6]

Next, the NESE divided by the NSVSE were determined for each time point and the VLR (expressed in min-1) was calculated as the linear fit of the plot of these normalized values over time.

NESE (t) / NSVSE (t) = (VLR * t) + ((NESE (t0) / NSVSE (t0)) [S7]

For most experiments, the TLR and VLR values were determined for a total of 10 min immediately after the time point t1 at which equilibrium was reached in the saphenous vein. However, in cases of measuring acute permeability after injections of proteins into tissue, the TLR and VLR values were determined over the course of the first 8 min. Analysis time for each mouse was approximately 5 min.

Intraobserver variability of BVF, TLR, and VLR was assessed by analysis of one set of data by two independent observers from wildtype mouse ears (n=6). Interclass correlation coefficients (ICC) were estimated in SPSS using a mixed-effects analysis of variation analysis. We determined single measure ICC values of 0.915, 0.855, and 0.963 for BVF, TLR, and VLR, respectively. For average measures we determined ICC values of 0.955, 0.922, and 0.981, respectively.

3 / 8 Dynamic in vivo stereomicroscopic visualization of blood and lymphatic vessels

A Zeiss StereoLumar.V12 (Carl Zeiss, Oberkochen, Germany) stereomicroscope with AxioVision (Zeiss) software was adapted for far-red and near infrared wavelength visualization. To accomplish this, we installed a cooled EMCCD camera (Evolve eXcelon, Photometrics, Tuscon, AZ) with enhanced sensitivity to the near infrared spectrum, a high-powered light emitting diode (LED) system with four illumination wavelengths at 470 nm, 550 nm, 635 nm and 770 nm (CoolLED, Andover, UK) and specific filters for FITC, Texas Red, IRDye680 (Zeiss) and IRDye800 (Semrock, Rochester, NY). Mice were anesthetized via i.p. injection of 0.2 mg/kg medetomidine and 80 mg/kg ketamine. A heating stage set at 37 °C was used to maintain body temperature.

For imaging of vascular perfusion and leakage under basal conditions or in response to systemic injection of recombinant proteins, the mice were positioned with the ear taped flat on a custom imaging platform. The autofluorescence of the FITC channel was used to focus the stereomicroscope onto a region of blood vessels in the ear. Then an imaging sequence was initiated on the far-red channel (635 nm λex, 690 nm λem, exposure:100 ms, 1 image per s), and approximately 30 s into the 16 min scan an injection of 10 to 25 mol/L P20-D680 with or without 1 g hVEGF-A165 was performed into the tail vein. Stereomicroscopic imaging of 4T1 tumors after an intravenous injection of 25 mol/L P20-D680 was also performed on a region encompassing the tumor.

For stereomicroscopy imaging of lymphatic vessels in the ear, we initiated the imaging directly after injection of 1 L of 25 mol/L P40-D680 with a 30 g syringe. Images (635 nm λex, 690 nm λem, exposures: 100-200ms) were acquired at select time points after injection to characterize the spread of dye through the lymphatic vessel network of the ear.

Quantification of lymphatic clearance after acute leakage of tracers

Dynamic NIR imaging of vascular leakage was performed at select time points after ear injection of 30 ng human VEGF-A165 in wildtype mice to determine the time point at which the leakage of P20- D800 was attenuated. Based on this data (Figure S8), we assumed that after 40 min continued leakage into tissue would be negligible in comparison to the tracer that had already leaked. Therefore, the decrease in signal intensity from this point on is an estimate of lymphatic clearance since P20-D800 tracer does not enter blood vessels after tissue injection. Wildtype (n=5) and K14-VEGF-C mice (n=4) were injected into one ear with 30 ng human VEGF-A165 and imaged as described in the methods “Acute permeability studies”. We acquired images of the ears at 40 min and then at 2 h, 4 h, 6 h, and 24 h after this time. Mice were allowed to wake up and move freely between imaging time points. Signal intensities were adjusted to baseline ear signals before injection of tracers to calculate ESE values. We then normalized the ESE data at all time points enhancement values to the 40 min value. Data for each mouse was fit to a one-phase exponential decay model with lymphatic clearance expressed as decay constant K (expressed in h-1) or as half life (expressed in h) as shown in equations S8 and S9.

Normalized Tissue Enhancement = e-Kt [S8]

Half Life = ln 2 / K [S9]

Measurement of depth penetration through a tissue phantom

4 / 8 Tubes containing 1 mL of P20-D800 (10 mol/L), ICG (10 mol/L) or BSA-Rhodamine (2 mg/mL) were prepared. IVIS images were acquired at exposure, 0.1 s, λ ex: 745 nm, λem: 800 nm, binning of 2 for P20-D800 and ICG and at exposure, 0.1 s, λex: 570 nm, λem: 620 nm, binning of 2 for BSA- Rhodamine. Immediately after each image acquisition, a 1-cm thick section of chicken breast was placed on top of the tubes and another image with the same acquisition settings was then acquired. ROI analysis of the average signal intensity in counts was performed using Living Imaging Software, with the covered signal intensity normalized to the uncovered signal intensity to estimate the tissue penetration efficiency.

Supplementary References

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Supplementary Figure Legends

5 / 8 Supplementary Fig. 1 PK profiles and clearance of vascular tracers from injected mice (a) Serum circulation profile of P20-D800. Mean ± SD (n=3). Fluorescence emission intensity was normalized to the t=0 h intensity (λex: 750 nm, λem: 799 nm). (b) Serum circulation profile of P40-D800. Mean ± SD (n=3). Fluorescence emission intensity was normalized to the t=0 h intensity (λ ex: 750 nm, λem: 799 nm)

Supplementary Fig. 2 Clearance and biodistribution of vascular tracers (a) Chromatograms of: purified P20-D800, P20-D800 extracted from mouse serum 6 h after i.v. injection, P20-D800 extracted from mouse urine 6 h after i.v. injection, and free dye D800 (top to bottom). (b) Chromatograms of purified P40-D800, P40-D800 extracted from mouse serum 6 h after i.v. injection, P40-D800 extracted from mouse urine 6 h after i.v. injection, and free dye D800 (top to bottom). (c) Signal in bladder indicating kidney clearance at 1 and 6 h after i.v. injections of P20-D800 and P40-D800. (d) Fluorescence of the plasma and organs of the mice 1 week after injections of P20-D800 and P40-D800 compared to uninjected control mouse autofluorescence signals. Mean ± SD (n=3)

Supplementary Fig. 3 Animal positioning and region of interest (ROI) selection for analysis of vascular leakage in mouse ears. (a) Surgical tape is affixed to the top edge of the ears to position the ears flat using the anesthesia nose cone. (b) Shaved saphenous vein region of the mouse is positioned using surgical tape and a custom platform to raise the leg to the same imaging plane as the ears. (c) ROI1 and ROI2 are drawn on the ears excluding the taped regions using the manual segmentation tool in Living Image software. (d) ROI3 is drawn at the brightest region of the saphenous vein of the mouse

Supplementary Fig. 4 Initial validation of quantifications by comparison of leakage of P20 and P40 dye conjugates in normal ears. (a) Quantification of BVF, TLR, and VLR values from IVIS imaging after injection of either P20-D800 or P40-D800. (b) Select images from stereomicroscope visualization of normal ears at 4 min, 15 min, and 30 min after tail vein injection of P20-D680 showing steady increase in signal outside blood vessels or of P40-D680 showing no increase in signal outside blood vessels. Far right images shows larger field of view image taken at 30 min after injection showing the imaged region in yellow box

Supplementary Fig. 5 Variability in the severity of inflammation and vascular leakage in K14- VEGFA+/+ mice. Three 10-week old K14-VEGFA+/+ mice are shown with varying degrees of vascularity and redness of the ears (left panels). NIR images and quantifications of BVF (middle panels) and TLR (right panels) also show high degree of variability that correlate well with the visual features

Supplementary Fig. 6 Correlation of ear thickness measurements with imaging quantifications during the course of inflammation. (a) Correlation of ear thickness with BVF. (b) Correlation of ear thickness with TLR. (c) Correlation of ear thickness with VLR

Supplementary Fig. 7 Assessment of acute permeability via Miles assay. (a) Representative image demonstrating Evans Blue dye extravasation into either PBS (left) or 30 ng human VEGF-A165 (right) injected ears. Picture was acquired 30 min after ear injections. (b) Evan’s Blue concentration in PBS or VEGF-A injected ears after 24 h of elution of the tissue in formamide

Supplementary Fig. 8 Rapid loss of permeability effect after ear injection of 30 ng human VEGF- A165. (a) Tissue leakage rates in the ears of a representative animal approximately 3 min after injection of VEGF-A or PBS. (b) TLR 30 min after injections. (c) TLR 60 min after injections

Supplementary Fig. 9 Increased depth penetration of P20D800 tracer compared to ICG and BSA- Rhodamine (a) BSA-Rhodamine (2 mg/mL) uncovered signal. (b) BSA-Rhodamine signal through 1- cm thick chicken breast tissue. (c) P20D800 (10 mol/L) uncovered signal. (d) P20D800 signal through 1-cm thick chicken breast tissue. (e) ICG (10 mol/L) uncovered signal. (f) ICG signal through 1-cm thick chicken breast tissue. For comparison purposes, each uncovered image was adjusted to the display maximum of 100% average signal intensity of the fluorescent tracer, while each 6 / 8 tissue phantom image was adjusted to the display maximum of 25% of the respective uncovered average signal intensity.

Legends For Movie Files

7 / 8 Supplementary Movie 1

Injection of P20-D680 while the ear of a wild type mouse is positioned under a stereomicroscope at 35X magnification. Video is 60 times normal speed. Arterioles exhibit strong vasomotion throughout sequence. Blurring of tissue outside blood vessels over time is apparent indicating leakage of tracer.

Supplementary Movie 2

Injection of P40-D680 while the ear of a wild type mouse is positioned under a stereomicroscope at 35X magnification. Video is 60 times normal speed. Arterioles exhibit strong vasomotion throughout sequence. No blurring of image over time is apparent indicating no leakage of tracer.

Co-injection of 1 g human VEGF-A with P20-D680 while the ear of a wild type mouse is positioned under a stereomicroscope at 15X magnification. Video is 50 times normal speed. Injection is performed approximately 1 min after initiation of sequence.

Co-injection of 1 g human VEGF-A with P20-D680 while the ear of a K14-VEGF-C +/- mouse is positioned under a stereomicroscope at 15X magnification. Video is 50 times normal speed. Injection is performed approximately 1 min after initiation of sequence.

Injection of P20-D680 while a subcutaneous 4T1 tumor of a Balb/c mouse is positioned under a stereomicroscope at 6X magnification. Video is 50 times normal speed. Blurring of tissue outside blood vessels over time is apparent, indicating leakage of tracer.