Synthesis and Functionalization of Dextran – Based Single – Chain Nanoparticles in Aqueous Media

Dextran from Leuconostoc spp. (DXT-40, Mr ~40 kDa), glycidyl methacrylate (GMA) (97%), dimethyl sulfoxide (DMSO) (98%), 3-mercaptopropionic acid (≥99%), 4-(4,6dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM·HCl) (96%) and 2,2′-(ethylenedioxy)diethanethiol [3,6-dioxa-1,8-octane-dithiol (DODT)] (95%) were purchased from Aldrich. Phosphate-buffered saline (PBS) was purchased from Scharlau. 4-(Dimethylamino)pyridine (DMAP) was purchased from Acros-Organics. 2,2'-(7-(4-((2-Aminoethyl)amino)-1-carboxy-4-oxobutyl)-1,4,7-triazonane-1,4diyl)diaceticacid (NH2-NODA-GA) (98%) was purchased from CheMatech. Water (H2O) used in the syntheses, unless otherwise stated, was deionized water from a MilliQ A10 Gradient equipment (Millipore).

OH, Guard-Aquagel-OH columns and a differential refractive index (RI) detector.0.3 M NaNO 3 , 0.01 M NaH 2 PO 4 , pH 7 was used as eluent at a flow rate of 1 mL/min.The system was calibrated using polyethylene oxide (PEO) standards.
Transmission electron microscopy (TEM): TEM analyses were performed in a TECNAI G2 20 TWIN microscope (FEI, Eindhoven, The Netherlands), operating at an accelerating voltage of 200 KeV in a bright-field image mode.One drop of the sample dispersion in water (~3 μL, 0.035 mg/mL) was deposited on a carbon film supported on a copper grid (300 mesh), hydrophilized by a glow discharge process just prior to use.After staining for 20 seconds with a uranyl acetate aqueous solution (1% w/v), the sample was rotated at high speed in order to dry at room temperature quickly by spinning process.Numberaverage diameter was calculated by ImageJ platform analysis using a Gaussian curve fitting after counting about 300 nanoparticles.
Nuclear magnetic resonance ( 1 H NMR and DOSY NMR): NMR spectra were recorded on a Bruker AVANCE III spectrometer at 500 MHz and 25 ºC.Chemical shifts (δ) are given in ppm relative to the residual signal of the solvent.Splitting patterns: b, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet.
Diffusion Coefficient calculations: Taylor dispersion analysis (TDA) studies were performed on a Viscosizer-TD using fused silica capillaries.The mobile phase was water and the solutes were monitored by UV absorbance (UV wavelength filter 214 nm) at two fixed windows.Diffusion coefficient experiments were run at 25 ºC and 140 mbar.
Fourier transform infrared (FTIR) spectra were registered at room temperature in a Jasco FT/IR 4100 spectrophotometer, using a Gladi ATR accessory.

Synthesis of the dextran methacrylated precursor polymer [DXT-MA (DS ~52%)]
Dextran methacrylated polymer (DXT-MA) was synthesized following a slightly modified published procedure [1S].Dextran (DXT-40, 1g, 6.2 mmol) was dissolved in 30 mL of dimethyl sulfoxide (DMSO) under a nitrogen atmosphere, to this solution 200 mg of 4-(N,N-dimethylamino)pyridine (DMAP, 1.6 mmol) was added.Then, 1 mL of 1S van Dijk-Wolthuis, W. N. E.; Kettenes-van den Bosch, J. J.; van der Kerk-van Hoof, A.;Hennink, W. E. Macromolecules 1997, 30, 3411−3413.glycidyl methacrylate (GMA, 7.5 mmol) was incorporated and the mixture was stirred at room temperature during 4 days.The reaction was stopped by adding an equimolar amount of concentrated HCl solution (37% v/v, 1.6 mmol, 0.132 mL) to neutralize DMAP.The modified dextran solution was purified by dialysis against distilled water (MWCO 3500 Da) at room temperature until reaching deionized water conductivity values < 1µS (9 days, refreshing with 4 L of deionized water twice per day).Yield: 75%, DS 52%.The degree of substitution (DS, percent of modified hydroxyl groups per repeating unit) was calculated by 1 H NMR through integration of the MA proton signal (integration reference 1.0) with respect to the signals at 3.3-4.2ppm corresponding to the protons of the glucose (Glc) moiety (6H for unsubstituted Glc and 5H for substituted Glc) except the anomeric protons and the substituted positions (mainly position 3).

Functionalization of the
percentage) was calculated as the ratio between the amount of radioactivity in the filter and the total amount of radioactivity in all fractions.Finally, the nanoparticles were suspended in 0.2 M sodium acetate buffer solution (pH 4.2).Radiochemical yield was calculated as the ratio between the amount of radioactivity in the resuspended fraction and the starting amount of radioactivity.In vivo imaging studies: Immediately after administration of the radiolabeled SCPNs, and without recovering from sedation, animals were positioned in an eXplore speCZT CT preclinical imaging system (GE Healthcare, USA) to perform in vivo studies.Body temperature was maintained with a homeothermic blanket control unit (Bruker BioSpin GmbH, Karlsruhe, Germany) to prevent hypothermia, and SPECT scans were acquired for 30 min.After the SPECT scan, a CT acquisition was performed to provide anatomical information of each animal.The SPECT images were reconstructed using a orderedsubset expectation maximization (OSEM) iterative algorithm (3 iterations/3 subsets, 128 x 128 x 32 array with a voxel size of 0.55 x 0.55 x 2.46 mm 3 ), whereas for the CT a cone beam filtered back-projection a Feldkamp algorithm (437 x 437 x 800 array with a voxel size of 0.2 x 0.2 x 0.2 mm 3 ) was used.After reconstruction, images were quantified using πMOD analysis software (version 3.4, PMOD Technologies Ltd.).Volumes of interest (VOIs) were manually drawn in the lungs on the CT images and translated to the SPECT images.The relative concentration of radioactivity in the different VOIs was finally determined.

Figure S5 :
Figure S5: TEM (uranyl staining) and distribution analysis (number-average diameter calculated by ImageJ platform using a Gaussian curve fitting after counting about 300 nanoparticles) of DXT-SCPN-F.
SCPNs: Radiolabelled nanoparticles prepared as described above were incubated in sodium acetate buffered solution at 37 °C using a digital block heater.At different time points(1, 3, 24, 48, 72 and 144 h)samples were withdrawn and the amount of radioactivity was measured.The 67 Ga-radiolabelled SCPNs were filtered, washed twice with ultrapure water, and the amount of radioactivity in the filter and the filtrate/washings was measured.The radiochemical stability was calculated as the percentage of radioactivity in the pellet with respect to the total amount of radioactivity (pellet + filtrate + washings).Animals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals (European Convention for the Protection of using spacers in the syringe plunger) was administered (amount of radioactivity around 1.85 MBq).Immediately after, rats were submitted to in vivo imaging studies.
Century.Inc.) was used for correct visualization of the epiglottis, ensuring a correct positioning of the tip just above the carina.A pre-defined volume of radiolabelled SCPNs (50 µL, established by