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Reagent B: Sodium thiosulfate pentahydrate (Na2S2O3 : 5H2O, Sigma, CAS# ... Step i: NA-OPSS 0.5 mL 2.8 mg/mL 0.1X PBS with 2 mM NaN3, was added to 25 .... to the other group, then blood was collected from all mice at +60 min (N=2).
SUPPLEMENTARY INFORMATION DOI: 10.1038/NMAT3982

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization Supplementary Information Figures S1-S7, Movie S8 Etchant solution. 20X Etchant stock for in vitro use: Reagent A: Tripotassium hexacyanoferrate (III) (K3Fe(CN)6, Sigma, CAS# 13746-66-2) was typically dissolved in DPBS (Hyclone Cat#SH30028.02) at 0.20 M and stored in the dark. Warning: hexacyanoferrate salt should NOT be dissolved in strong acid (e.g. HCl, HNO3, H2SO4) due to potential of toxic HCN gas formation. Hexacyanoferrate (III), HCF, is also known commonly as ferricyanide. Reagent B: Sodium thiosulfate pentahydrate (Na2S2O3 : 5H2O, Sigma, CAS# 10102-17-7) was typically dissolved in DPBS at 0.2 M. These stocks were stored at room temperature in 50 mL polypropylene tubes in the dark for at least a month without issue. A modified etchant was used for injection experiments, see ‘in vivo etchant’ section below. In vitro etchant dilution: Typically the two solutions were combined with PBS or cell culture media to form a 2X solution, stored at room temperature in normal lighting, and used within the same day of preparation. E.g. 800 μL PBS, 100 μL Reagent A, 100 μL Reagent B. Equal volume was added to the media containing the cells and AgNPs. Etching power was verified using a stock of concentrated silver particles with observation of color change culminating at that given by the HCF solution itself (yellow). See Fig. S5 for typical colors. Preparation of silver nanoparticles. 70 nm silver nanoparticles AgNPs with PVP coating (AgNP-PVP) were prepared using a modification of a method previously reported.1 First, 0.375 g of AgNO3 (Sigma Cat#209139-25G >99%) and 1.5 g PVP (polyvinylpyrrolidone, Sigma Cat#856568-500G MW ~55000) were dissolved in 150 mL ethylene glycol (BDH Cat#BDH1125-1LP >99.99%). This mixture was then heated to 160 C and the reaction proceeded for 1 h. The silver was cooled and precipitated in a large amount of acetone followed by centrifugation at 1000 RCF for 10 min. The solution was decanted and the solids were redispersed in water with bath sonication. Note: residual PVP is necessary to stabilize the particles, but excess PVP should be minimized, wherein the optimum is observed to form a silvery sheen at the air-liquid surface that disappears on sonication, which reforms on standing. An aliquot may be dissolved in dilute PVP in water for UV-Vis characterization. We used an extinction coefficient of 1 x 1011 M-1 cm-1, for 70 nm diameter solid spherical silver nanoparticles (450 nm plasmon peak, Nanocomposix Inc, sample sheets). Typical UV-Vis spectrum showed an intense plasmon peak at 425 to 455 nm (55 nm to 80 nm diameter) depending on the particular batch.

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DOI: 10.1038/NMAT3982

NeutrAvidin (NA, Thermo Scientific) was modified for silver nanoparticle coupling by appending 5 kDa NHS-PEG-OPSS (Jenkem), where OPSS is ortho-pyridyl disulfide. NA (25 mg) was dissolved in 5 mL 10% glycerol water solution, dissolved for an hour, then brought to 1X PBS using 10X PBS. NHS-PEGOPSS, 6 mg, was dissolved in 0.5 mL glycerol/water for 15 sec then added to the NA solution. After 4-5 h the NA-OPSS was dialyzed against 0.1X PBS with 2 mM NaN3 using a 20 kDa Slide-a-Lyzer, Pierce, 4 L buffer with one change after several hours followed by overnight dialysis. The product was removed, filtered 0.22 µm (Millipore syringe filter) then assayed for OPSS using reduction by TCEP (tris(2-carboxyethyl)phosphine, 0.5M solution pH 7.0, Sigma). 55 µL NA-OPSS + 500 µL 0.1XPBS, without and then with 1 µL 0.5 M TCEP added to quantify the absorbance change at 343 nm. On average 2.2 OPSS were bound per NA, using extinction from pyridyl leaving group of 8.08x103 M-1 cm1 at 343 nm and 99.6x103 M-1 cm-1 at 280 nm for NA (and assuming 60 kDa MW for NA). Stock concentration of NA was ~2.8 mg/mL, reduced from the initial 5 mg/mL due to swelling of the casette contents during dialysis. 20 nm AgNPs were synthesized similarly as above, but using 10 kDa PVP instead of 55 kDa as given for the 70 nm AgNP procedure.1 20 g PVP 10 kDa (Sigma) was slowly dissolved in 150 mL ethylene glycol at room temperature with stirring, and to this solution the 400 mg AgNO3 was added. The suspension was stirred at room temperature until complete dissolution of the AgNO3 was achieved. Then the flask was heated up to 120 C using an oil bath, and the reaction was allowed to proceed for 1 h at this temperature. At the end of the reaction period the brown-black colloidal dispersion was cooled until the system reached room temperature. Acetone precipitation was performed as for the larger AgNPs, spun and decanted, with solids dissolved in water. Typical UV-Vis absorption showed a plasmon peak between 400 nm to 410 nm. Synthetic Scheme.

Preparation of Ag NeutrAvidin nanoparticles (Ag-NA). Step i: NA-OPSS 0.5 mL 2.8 mg/mL 0.1X PBS with 2 mM NaN3, was added to 25 mL of 70 nm AgNPPVP in water (~ pH 6, optical density at 1 cm (O.D.) 200 for 70 nm AgNP, or 2 mL NA-OPSS was added to 7 mL of O.D. 670 for 20 nm AgNP) and incubated overnight, then washed by centrifugation at 3300 RCF for 70 nm AgNP or 14000 RCF for 20 nm AgNP, decanting supernatant and sonicating into

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DOI: 10.1038/NMAT3982

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PBST, made from PBS (Hyclone) with 0.005% Tween 20 (Sigma). Optionally, MES hemisodium salt in water pH 6.2 was added to 20 mM (for 70 nm AgNP, or 5 mM for 20 nm AgNP) directly after NA, and TCEP was added the next day, 10 µM final concentration. Step ii: Lipoic PEG amine (MW 3400 g/mol, Nanocs Inc. for 70 nm AgNP, or smaller MW 1000 g/mol for 20 nm AgNP and Ag-citrate) was dissolved in 70% ethanol and added to the Ag-NA at a final concentration of 20 μM (for 70 nm AgNP, 100 µM for 20 nm AgNP) and incubated for 3 days at room temperature. After washing the particles in PBST (diluting to 100 or 700 O.D. at plasmon peak for the 70 nm and 20 nm AgNPs, respectively, 1 cm pathlength) at 3300 RCF (or 14000 RCF for 20 nm AgNP) and sonicating, NA content was determined through biotin-4-fluorescein assay (Life Technologies, Cat#B-10570) to be 102-103 NA per NP. Step iii: NHS-dyes were added to portions of the Ag-NA at 20-50 μM final concentration (see below), incubated overnight at 4 C followed by extensive centrifuge washing with PBST until supernatant was not fluorescent. These products were filtered and could be stored at 4 C for at least a year. Prior to usage the material was bath sonicated to disperse. See Supplementary Fig. S7 for addition dye details. Step iv: Biotin peptides were added at 40 μM for >1 h, then nanoparticles were centrifuge washed with PBST. Experimental details for live imaging of NP internalization (Fig. 1c) Single slice confocal images of PPC-1 cells with RPARPAR Ag-NA488 before and after etching. PPC-1 cells were maintained in growth media (DMEM + 10 % fetal bovine serum and Penicillin/ Streptomycin) on glass coverslips for two days (8 chamber, Nunc)). The cells were then incubated with 5 µg/ml CellMask Orange (Invitrogen) for 5 min in cell growth media, to visualize the membrane as well as any endosomes that later formed. The cells were washed three times in Hank’s Balanced Salt Solution (HBSS, w/ CaCl2 and MgCl2, Gibco Cat#14025-092) and ~10 pM RPARPAR Ag-NA488 in growth media was added then incubated for 45 min at 37C, 5% CO2. Unbound particles were washed off with HBSS. An equal volume of 2X etchant solution in PBS was then injected into the culture chamber while on the microscope stage (Fluoview 500, Olympus). A time-lapse series of images were collected, frames were extracted using the Fluoview software and then processed in ImageJ. Cell viability assay in vitro (Fig. 2a) PPC-1 cell viability 24 and 48 h post-etch was assayed using PrestoBlue (Invitrogen). A series of conditions compared etch solution consisting of 10 mM HCF and 10 mM thiosulfate in DMEM supplemented with 10% fetal bovine serum (FBS). Untreated cells were used for normalization. RPARPAR Ag-NA (R-Ag) or Ag-NA without a peptide (x-Ag) was added to media, incubated for 1 h at 37 C, removed to room temperature and aspirated and washed with HBSS, etched for 1 or 10 min, aspirated and washed with HBSS, then new media was added and incubation proceeded at 37 C. For the assay, media was replaced with fresh media containing PrestoBlue with 40 min incubation at 37 C. Fluorescence plate reader (TECAN) analysis at 560/590 nm excitation/emission followed as per PrestoBlue manufacturer instructions. Cells tested at 24 h were separate from those used for 48 h time points. Three concentrations of etchant were tested (1 mM, 10 mM, 30 mM each of reagent A and B, final concentration) with 10 min etch time for 1 mM, and 1 min for 10 and 30 mM. The etch times of 1 and 10 min are considered a normal exposure – more than enough time for complete etching (see Fig. 2b). Etchant was removed and replaced with fresh media and incubated at 37 C until assay time. Longer exposure to etch solution was tested by leaving the etchant for the 24 or 48 h, until directly prior to viability assay. Toxicity was seen in the long exposures only. (One possible reason for AgNP-RPAR at 48 h having greater viability than AgNP or no AgNP is the consumption of the oxidative species

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DOI: 10.1038/NMAT3982

HCF(III) during etching of cell-membrane bound Ag, forming a new composition consisting of HCF(II).) The number of replicates per condition was 6. Each condition data was averaged, and the background signal from PrestoBlue in media was subtracted prior to normalization to untreated cells. Etching kinetics (Fig. 2b) Data collected by Fluorimeter (Horiba Jobin Yvon Fluoromax 4) monitoring the 90o scattering signal from 70 nm Ag-NA. Excitation and emission were set at 470 nm and 520 nm with 5 nm slit widths. Signals were stable from Ag-NA in PBST (0.005% Tween 20) and dropped upon adding the etchant. The steepest portion with negative slope was fit using a linear function and then converted to an etch rate in units of percent of initial signal per second. The time at which signal was 10% of initial intensity was plotted by manual examination of the raw data. For the lowest concentration, stoichiometry variation was performed. Na2S2O3 at 0.067, 0.13, or 0.20 mM and fixed concentration of HCF=0.067 mM. In vivo etchant (also see Fig. S4 for in vivo toxicity) The sodium salt of HCF was prepared by sodium ion exchange resin treatment of 0.2 M K3Fe(CN)6 solution. Sodium-HCF was combined with 0.2 M Na2S2O3 and 1 M NaCl to have a nominal concentration of ~150 mM Na+, 17 mM Fe(CN)63-, and 50 mM S2O32-, and was sterile filtered. 150 µL was injected via tail vein per 20-25 g mouse. The stoichiometry chosen for the etchant was based on the etching kinetics result. Blood chemistry panel (also see Fig. S4 for in vivo toxicity) (Fig. 2b) Injected etchant should have isotonic Na+, and minimal K+ to minimize cardiotoxicity. Nonpeptide Ag-NA in PBS or a solution of ‘in vivo etchant’ was injected by tail vein into 20-25 g Balb/c female mice aged 6-8 weeks (Harlan Laboratories) and blood was collected from the orbital plexus the following day into lithium heparin collection tubes. Blood was centrifuged at 1000xg and plasma was collected and frozen at -80 ºC. On the day of analysis 100 µL of thawed plasma was pipetted into Comprehensive Diagnostic Profile Rotor #500-0038 and analyzed using the VetScan VS2. Values were evaluated by Prism using two-way ANOVA, N=3-6. Differences versus Dubelco-PBS (Hyclone) were not significant with a criterion of P