Supporting Information. Labelling the Structural Integrity of Nanoparticles. for Advanced in situ Tracking in Bionanotechnology

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1 Supporting Information Labelling the Structural Integrity of Nanoparticles for Advanced in situ Tracking in Bionanotechnology Fabian Meder*[a], Steffi S. Thomas[a], Laurence W. Fitzpatrick[a], Amirah Alahmari[a], Suxiao Wang[b], Jason G. Beirne[b], Gizela Vaz[a], Gareth Redmond[b] and Kenneth A. Dawson*[a] [a] Centre for BioNano Interactions, University College Dublin, School of Chemistry and Chemical Biology, Belfield 4, Dublin, Ireland [b] Functional Nanomaterials, University College Dublin, School of Chemistry and Chemical Biology, Belfield 4, Dublin, Ireland S1

2 Table of Contents 1. Additional experimental details Quantification of dyes in SiNPs Fluorescence lifetime measurements Supporting data...5 S1. Differential centrifugal sedimentation (DCS) of particles with and without shell...5 S2. Further fluorescent analysis of ThT, FITC, and RITC doped SiNPs...6 S3. Further characterization of SiNPs synthesized with varying methanol/ethanol ratios...8 S4 and S5. Increased detection limits of SiNPs in DCS when doped with ThT...9 S6. Fluorescence and light scattering of particles doped with ThT and RITC in 1 M KOH...12 S7. Relative fluorescence reduction of SiNPs exposed to 1M KOH and H 2 O 13 S8. Influence of 1M KOH on ThT, FITC, and RITC fluorescence...14 S9. Blue shift of ThT fluorescence during degradation...16 S10. ThT fluorescence and light scattering of SiNPs no shell 0.1M KOH S11. H 2 O, ALF, cmem, FBS, and HS influence on free ThT and ThT&FITC in SiNPs S12. Comparing SiNPs with and without shell in H 2 O, ALF, cmem, FBS, and HS S13. Fluorescence of bare PVK and PLGA particles and doped with ThT S14. Influence of Fe-doping on SiNPs degradation kinetics S15. Additional characterization of ThT-doped PLGA, PVK, and FeSiNPs by TEM and DLS Literature S2

3 1. Additional experimental details 1.1 Quantification of dyes in SiNPs Two mg ml -1 FITC stock solution was prepared by dissolving 4 mg of FITC in 2 ml of anhydrous ethanol, which was followed by immediate addition of 20 μl of ethanolamine (11x molar excess). The conjugate mixture was stirred at room temperature for 4 hours in darkness to covalently link FITC to a primary amine to obtain a reference conjugate similar as used in the particles (APTMS-FITC conjugate). 9.6 mg ml -1 ThT stock solution was prepared by dissolving ThT in ethanol. Then, a standard curve was prepared using a mixture of both dyes with final concentration of 1.6 µg ml -1. This was further diluted with H 2 O to obtain 5 more points on the standard curve with final point being water without any dyes. Both the standard curve and the nanoparticles were incubated with 1 M KOH at 60 C with stirring and left overnight. To determine if all the nanoparticles were degraded after reaction, DLS of the samples was recorded and degradation confirmed by observing reduced count rates of scattered light. Absorbance and fluorescence of dissolved samples were recorded after bringing the solutions to the optimum ph for analysis (ph at which highest absorbance respectively fluorescence obtained) using HCl which have been ph 3 5 for ThT and ph 7 9 for FITC as confirmed by measuring absorbance spectra (data not shown). Thus, after dissolution overnight, samples (both standard curve and the nanoparticles) were split into two equal halves. For the one half of the samples, the ph was adjusted between 3 5 to detect ThT and thus masking the effect to FITC as it has low fluorescence at this ph. The ph for the other half was adjusted between 7 9, to detect the fluorescence of FITC and thus removing any effects from ThT which has lower fluorescence S3

4 at this ph. After the ph correction, the volume of each halves were adjusted using H 2 O to the highest volume so as to keep the concentration of the samples same. 200 µl of the samples were transferred on to a 96 well plates and were scanned for ThT absorbance and FITC fluorescence and recalculated for ThT and FITC content by comparing standards and three measurements per sample. 1.2 Fluorescence lifetime measurements Lifetime measurements were performed on a home-built setup which incorporated an inverted microscope (IX71, Olympus Corp., UK), a pulsed laser diode (399 nm, LDH-D-C-405, PicoQuant GmbH, Germany) as excitation source and single-photon avalanche photodiode detector (SPCM-AQRH-14-FC, Perkin Elmer Inc., Ireland) Excitation filter (LD01-405, Semrock, Inc., UK), dichroic mirror (Di02-R405, Semrock, Inc., UK) and emission filter (BLP01-405R, Semrock, Inc., UK) were used to route excitation and emission signals. An additional 450 nm short wave pass filter (03SWP404, Melles Griot ) was used in the emission channel. A pulsed probe signal (5 MHz) at low pulse energy (< 1 pj) was used for all experiments. All experiments were performed with a 60x lens (UPLSAPO 60XW, Olympus Corp., UK) focused ~20 μm into sample droplets (> 20 μl) deposited onto glass coverslips which were sealed in a chamber to prevent evaporation. Colloidal silica (Ludox HS-40; , Sigma Aldrich, Ireland) was used to record an instrument response function (IRF) at μm concentration. Acquisition was via a time-correlated single photon counting card and software package (TimeHarp 200, PicoQuant GmbH, Germany) while data fitting was performed using analysis software (Fluofit 4.6; PicoQuant GmbH, Germany). S4

5 2. Supporting data Figure S1: Figure S1. Differential centrifugal sedimentation (DCS) of particles with and without shell. The figure shows size difference of SiNPs with and without shell which have peaks at 69 and 61 nm, respectively suggesting a shell thickness of about 4 nm. S5

6 Figure S2: Figure S2. Further fluorescent analysis of ThT, FITC, and RITC doped SiNPs. a) Red shift of ThT in presence of FITC and RITC in SiNPs. The maxima of the red shifted excitation spectra of ThT are not influenced by FITC or RITC dual labelling (emission at 475 nm), neither the red shift of the ThT emission spectra is influenced (data not shown) b) Fluorescence spectra as S6

7 function of ThT concentration in the SiNPs. ThT fluorescence (left) increases as expected with increasing ThT doping levels. FITC fluorescence (right) varies to lower extend (within about one decimal power) and is highest at medium ThT concentrations suggesting the existence of an optimum balance between ThT and FITC incorporation and for FITC fluorescence yields. Further influences next to dye loading may have particle size, particle internal quenching mechanisms, and interactions with the surroundings. c) Fluorescence lifetime measurements conducted on SiNPs containing ThT and different levels of FITC. The amplitude weighted lifetime of SiNPs doped with ThT was found to be 1.21 ns, which agrees with literature. 1 Measurements do not show any significant changes in ThT fluorescence lifetime suggesting no significant energy transfer between ThT and FITC which could be expected as emission maxima of ThT and excitation of FITC overlap. d) and e) influence of external ph on ThT and FITC in SiNPs with and without shell, respectively. In the given range of ph 1-9 (and about 10 minutes exposure time) no particle degradation is expected so that signals presented here reflect only the influence of the particle media on ThT and FITC fluorescence assuming no particle degradation. Although included into particles (either with shell or without shell), FITC fluorescence is sensitive towards acidic ph resulting in lower fluorescence and suggesting that the silica matrix might be permeable for protons leading to FITC s well-known ph sensitivity. However, ThT signal is stable over the ph range and not affected within this ph range suggesting that a variation of ThT fluorescence requires particle degradation (compare Figure 3). S7

8 Figure S3: Figure S3. Additional characterization of SiNPs synthesized with varying methanol/ethanol ratios. a) ThT and FITC loading of SiNPs synthesized with different amounts of methanol and ethanol and constant ThT (0.7%) and FITC (0.03%) addition. The results show that ThT incorporation is slightly higher when higher methanol concentrations are used while final FITC levels are relatively constant. b) UV-Vis spectra of SiNPs doped with ThT and FITC and synthesized with different amounts of methanol. Strong ThT absorption peaks can be observed in particular for small particles obtained from high methanol concentrations. c) Fluorescence emission spectra of SiNPs doped with ThT and FITC and synthesized with different amounts of methanol showing relative emission of ThT and FITC. S8

9 Figure S4 and S5: We observed another technological advantage of ThT doped SiNPs arising from their increased absorbance between 400 and 450 nm due to ThTs excitation red shift. DCS is an increasingly important technique to investigate interactions of NP with biological surroundings. It enables the investigation of binding events on NP surfaces, e.g., mapping of biomolecular coronas 2, and measurements of colloidal stability of NPs in different biological media. 3 During measurement, particles pass a 405 nm laser to record sedimentation times in a density gradient. The laser intensity is recorded by a linear extinction detector and depends on light scattering and absorbance of the NPs. Silica, however, is almost transparent for light of 405 nm wavelength and the signal mainly relies on scattering. This results in poor detectability of small or ultrasmall SiNPs particles. Figure S4a and S4b show the concentration dependency of DCS signal of SiNP of 27 nm. Particles that contain ThT and FITC give 18 times higher signals than non-doped, commercial Ludox TMA SiNPs and 4 times higher signals when doped solely with FITC. The effect is in particular remarkable for very small particles. Figure S4c shows DCS signals of 9 nm SiNPs with and without ThT. Particles without ThT were applied in 4.8 times higher concentration. Light scattering is accordingly ~4.8 times higher (table in Figure S4c). Nevertheless, despite higher concentration and higher light scattering, 9 nm SiNPs that do not contain ThT are not detectable. ThT doped SiNPs, however, give a strong signal (see also Figure S5). S9

10 Figure S4. a) Dependency of the DCS signal on SiNP concentration (27 nm, ThT doped SiNPs). b) DCS signal plotted as linear function of particle concentration and different labels (FITC+ThT and FITC alone). Commercial SiNPs Ludox TMA without FITC or ThT were analyzed as reference. c) Detectability of sub 10 nm SiNPs in DCS. ThT labeled SiNPs are detectable while unlabelled particles are not detectable although applied in 4.8 times higher concentration. Light scattering of both samples (measured by DLS, table on the right) and TEM images confirm particle sizes and concentrations. S10

11 Figure S5. Signal increase in DCS of ThT doped particles with decreasing particle size. Particle diameter measured by DCS plotted against the calculated sample weight D*Wd (D = diameter in µm, Wd = sample weight in µg µm -1 diameter). Calculations of sample weight are done by the instrument s software (CPS Control System, V 9.5c) and are based on extend of light absorption when particles pass the detector. Among the ThT doped SiNPs of different sizes, a trend towards higher signals of smaller particles was observed although same concentrations of the particles were injected. This is in contrast to the fact that smaller particles scatter less light which would reduce the signal measured by the detector but correlates with slightly higher levels of ThT doping found in smaller particles and their higher absorbance (Figure S3). Furthermore smaller particles pass the detector with lower velocities which might affect the signals. In any case, small SiNPs without ThT were not measureable (Figure S4) and ThT doping strongly increases the detectability of SiNPs. S11

12 Figure S6: Figure S6. Changes in relative fluorescence and light scattering of particles doped with ThT and RITC during exposure to 1 M KOH. Compared to ThT fluorescence, RITC fluorescence reduces only slightly upon degradation, similar as observed for particles doped with FITC and ThT. It suggests that changes in particle structure and microenvironment of the fluorophores can be sensitively detected by ThT. S12

13 Figure S7: Figure S7. Relative fluorescence reduction of SiNPs doped with FITC and ThT (left) and RITC and ThT (right) without shell exposed to 1M KOH and H 2 O. In H 2 O no significant changes of the fluorescence intensities for all fluorophores are observed (data points for ThT in H 2 O and FITC/RITC in H 2 O exactly overlap). SiNPs are expectedly stable in H 2 O and do not undergo degradation as observed in KOH. The measurements exclude photo bleaching during duration of experiment on ThT fluorescence as ThT fluorescence decrease would have been expected to occur in H 2 O in case of photo bleaching. S13

14 Figure S8: Figure S8. Influence of KOH on ThT, FITC, and RITC fluorescence in absence of SiNPs to test if KOH used to degrade SiNPs has an influence on the ThT, FITC, and RITC fluorescence signal. ThT, FITC, and RITC have been exposed to different aqueous KOH solutions of indicated concentrations for 2 hours followed by recording fluorescence spectra. ThT shows only a slight drop of fluorescence intensities with higher KOH concentrations (about maximal 2 times for ex/em 347/440 nm). Maximal 5 times decrease for ThT in solution at the same excitation/emission wavelengths as when incorporated in particles (ex/em 425/475 nm) is observed. The drop is minimal compared to the up to 2500 times fluorescence decrease that is observed upon degradation of SiNPs (Figure 3). This suggests that the main influence on ThT fluorescence at 475 nm comes from changes in particle integrity when ThT is incorporated into SiNPs and exposed to KOH. Also for RITC only a slight reduction of about 5 times is observed in 1 M KOH. FITC fluorescence instead decreases about 100 times in 1 M KOH and 2 M KOH, emission maxima were not shifted (data not shown). Fluorescence loss of FITC in this extend are not observed during the degradation experiments of SiNPs. Here, FITC signal is only slightly reduced, ~2 times after 2 hours. Why FITC maintains its fluorescence after particle degradation is S14

15 unclear. This could be due to quenching of the fluorophore when incorporated in the particle as also reported by Ow et al 4. Upon release from the particles, FITC may gain back its initial fluorescence intensities and compensating thus for the losses due to KOH which may also explain why degradation experiments in biological media (Figure S11) show increased FITC signals upon degradation. In any case, the changes of ThT fluorescence upon SiNP degradation are much larger than any observed influences of KOH suggesting that ThT fluorescence is solely influenced by the changes of SiNP integrity and not affected by the solvent. S15

16 Figure S9: Figure S9. (a) and (b) show the fluorescence decrease of 61 nm SiNPs doped with ThT (no FITC or RITC dual labelling) during exposure to 0.1 M KOH for up to 180 min. Normalizing the data by the maximum of each curve reveals a blue shift in the emission spectra recorded at 347 nm excitation (c) and the emission spectra recorded at 425 nm excitation is not shifted (d). This suggests that during degradation, ThT regains its fluorescence emission at ex/em 347/440 nm representing the rotational free state of the molecule. Due to overlaying of the 475 nm emission peak and the weaker nature of the 440 nm emission, the regained, blue shifted emission peak is relatively weak. The slightly lower emission of ThT in 0.1 M KOH additionally reduces the signal and the measured profiles do not allow direct correlation between 475 nm emission S16

17 decrease and 440 nm emission increase. We thus suggest to focus on the reduction of 475 nm emission to monitor nanoparticle integrity. Figure S10: Figure S10. Decrease of ThT fluorescence and light scattering for 61 nm ThT-doped SiNPs in 0.1 M KOH. Decrease of both signals occurs almost simultaneously indicating that the lag between the DLS and ThT signal reported in Figure 3 (b and d) is due to the shell which delays the degradation of the dye-labelled core. The fact that light scattering (which reflects the particle size and integrity) and ThT fluorescence decays simultaneously, suggests that ThT can be used as sensor for nanoparticle integrity. S17

18 Figure S11: Figure S11. a) and b) show the influence of H 2 O, ALF, cmem, FBS, and HS on fluorescence of free ThT in solution during exposure at 37 C for up to 52 hours at ex/em 425/475 nm and ex/em S18

19 347/440 nm, respectively. At both excitation wavelengths, ThT fluorescence either remains stable or increases depending on the media. This suggests that the fluorescence drop observed for ThTdoped nanoparticles at ex/em 425/475 nm is expectedly due to particle degradation. c) shows ThT and FITC signals of dual labelled SiNPs upon exposure to H 2 O, ALF, cmem, FBS, and HS. Results for ThT are already discussed in the main manuscript. FITC fluorescence increases when particles are degrading in cmem, FBS, and HS and remains almost unchanged in non-degrading H 2 O and ALF. Thus, also the FITC signal confirms degradation and as discussed in Figure S8, the increase upon degradation may be due to quenching of FITC molecules when in the particles and upon release they regain full fluorescence. Nevertheless, only 3-5 times maximal increase is observed for FITC, while ThT fluorescence decreases up to 30 times suggesting higher sensitivity for detecting degradation. S19

20 Figure S12: Figure S11. Replotted data of Figure 4 to compare degradation of SiNPs with and without shell in a) H 2 O, b) ALF, c) cmem, d) FBS, and e) HS during exposure at 37 C for up to 52 hours by analysing decay of ThT fluorescence at ex/em 425/475 nm. In H 2 O and ALF, no degradation is observed for both particle types. In cmem, FBS, and HS similar degradation kinetics are observed for either SiNPs with or without shell. For all three media, at time point 6 hours, SiNPs with shell do not show a significant signal reduction and SiNPs without shell show a higher fluorescence reduction than SiNPs with shell. This suggests that SiNPs degradation is delayed by applying the shell as also observed for degradation in KOH (Figure 2). After 24 hours, the fluorescence reduction of particles with and without shell is similar, while a larger drop of the fluorescence is observed for SiNPS with shell after 52 hours. The protective shell may thus initially delay degradation independent of the degrading media while at a later time points and S20

21 proceeded degradation (52 hours), the dye labelled core is affected more strongly resulting in higher fluorescence losses. The degradation kinetics do not show saturation, neither for SiNPs with shell nor for particles without shell, which may indicate incomplete degradation. This is also confirmed by gel electrophoresis in which still some particles do not enter the pores of the gels (first band, Figure 4). ThT-doping of SiNPs thus suggests different degradation kinetics as function of structural features such as protective shell and provides a direct measure to distinguish structural features during degradation in situ. S21

22 Figure S13: Figure S13. Fluorescence emission and excitation spectra of PLGA (a) and PVK (b) nanoparticles that are ThT-doped and non-doped are given. Non-doped PLGA particles do expectedly not show any fluorescence emission at 475 nm. ThT doped PLGA particles show the characteristic emission peak at about 475 nm and two excitation peaks at 425 nm and at about 350 nm due to ThT entrapment. The presence of two excitation peaks may result from some ThT which is entrapped in the particles but can rotate. This may be determined by the density of the polymer network and its interactions with ThT. Non-doped PVK particles show their intrinsic excitation peaks. ThT-doped PVK particles show a 450 nm and a 475 nm emission peak as well S22

23 as a broad excitation peak ranging from 360 to 425nm in addition to the polymers intrinsic excitation signals. The emission peak at 450 nm might originate from either free ThT, present after synthesis or ThT included in the particles which can rotate. The data suggests ThT incorporation in both, ThT-doped PLGA and PVK particles, by showing shifted ThT fluorescence peaks at 475 nm emission and confirm that the polymers do not have overlaying signals in this region. Figure S14: Figure S14. Relative light scattering obtained from DLS and ThT fluorescence of Fe-doped and SiNPs and SiNPs synthesized under same conditions but without Fe doping. Both methods suggest, Fe-doped SiNPs degrade faster than nondoped SiNPs. Nevertheless, it needs to be considered that also size, agglomeration, particle porosity, surface roughness etc. may influence degradation. As seen from Figure S15 Fe doped SiNPs have an intensity mean of about 200 nm and show a relatively rough surface. Intensity mean, poly dispersity index (PDI) and the TEM of S23

24 the FeSiNPs may suggest slight aggregation. In contrast, the SiNPs without Fe doping have an intensity mean of about 110 nm and show a smoother surface and are not aggregated (Figure 2 a in main document, sample with 0.7% ThT in 0% MeOH, poly dispersity index is 0.08, data not shown). While aggregation may protect particles from degradation by reducing the exposed surface area, a rougher surface or porosity increases surface area and may enhance degradation. In any case, Fe-doping varies the SiNPs in a way that they degrade faster and degradation can be sensed by ThT incorporation and is furthermore confirmed by DLS. Figure S15: Figure S15. Additional characterization of ThT-doped PLGA, PVK, and FeSiNPs by TEM and DLS. S24

25 4. Literature 1. Hutter, T.; Amdursky, N.; Gepshtein, R.; Elliott, S. R.; Huppert, D., Study of Thioflavin- T Immobilized in Porous Silicon and the Effect of Different Organic Vapors on the Fluorescence Lifetime. Langmuir 2011, 27, Kelly, P. M.; Åberg, C.; Polo, E.; O'Connell, A.; Cookman, J.; Fallon, J.; Krpetić, Ž.; Dawson, K. A., Mapping Protein Binding Sites on the Biomolecular Corona of Nanoparticles. Nat. Nanotechnol. 2015, 10, Wohlleben, W., Validity Range of Centrifuges for the Regulation of Nanomaterials: From Classification to as-tested Coronas. J. Nanopart. Res. 2012, 14, Ow, H.; Larson, D. R.; Srivastava, M.; Baird, B. A.; Webb, W. W.; Wiesner, U., Bright and Stable Core Shell Fluorescent Silica Nanoparticles. Nano Lett. 2004, 5, S25