Correction notice: Pair correlation microscopy …...escape from early endosomes occurred on two...

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In the version of the Supplementary Information originally published, Supplementary Schemes 1 and 2 were missing. This has now been corrected in this file 4 October 2016.

Correction notice: Pair correlation microscopy reveals the role of nanoparticle shape in intracellular transport and site of drug releaseElizabeth Hinde, Kitiphume Thammasiraphop, Hien T. T. Duong, Jonathan Yeow, Bunyamin Karagoz, Cyrille Boyer, J. Justin Gooding and Katharina Gaus

Nature Nanotechnology http://dx.doi.org/10.1038/nnano.2016.160 (2016)

http://dx.doi.org/10.1038/nnano.2016.160


SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2016.160

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

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SUPPLEMENTARY MATERIAL

The shape of polymeric nanoparticles impacts on intracellular translocation and the site of drug release

Elizabeth Hinde, Kitiphume Thammasiraphop, Hien T. T. Duong, Jonathan Yeow, Bunyamin Karagoz, Cyrille Boyer, J. Justin Gooding, Katharina Gaus.

Pair correlation microscopy reveals the role ofnanoparticle shape in intracellular transport and

site of drug release



2 NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology

SUPPLEMENTARY INFORMATION DOI: 10.1038/NNANO.2016.160

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Characteristics of nanoparticles

Supplementary Figure 1: Characteristics of nanoparticles of four different shapes to target doxorubicin to the nucleus. a. Transmission electron micrographs of the four types of polymeric nanoparticles with different shapes. Scale bar = 100 nm. b. Physical properties of the four differently shaped polymeric nanoparticles: nanoparticle dimensions (end diameter and length), polymerization time (the reaction of the block polymer mixture to create nanoparticles of different shape), IC50 values of nanoparticle cytotoxicity when loaded with doxorubicin (concentration of doxorubicin that was required to induce cytotoxicity in 50% of a population of MCF7 cells). The chemical properties of the four different nanoparticle shapes are identical.





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Autocorrelation analysis of the mobile nanoparticle concentration gradient

Supplementary Figure 2: The total nanoparticle concentration gradient compared to the mobile nanoparticle population that is undergoing translocation to a target destination. a-d. The total (intensity based analysis, top row) versus mobile (autocorrelation analysis, bottom row) nanoparticle concentration gradient of micelles (a), vesicles (b), rods (c) and worms (d), recorded along the confocal line scans presented in Fig. 3. a-d. In b-d the stationary population of nanoparticle in the cytoplasm was not detected in the autocorrelation analysis of mobile particles and this sub-population may reduce the number of particles that could reach the nucleus.





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ATP-dependent and –independent diffusion of nanoparticles across the plasma membrane

To assess the energy dependence of nanoparticle uptake, we quantified the degree of Cy-5 labelled nanoparticle accumulation in the cytoplasm of MCF7 cells in the presence and absence of ATP (Fig. S3a-b). As a control, we co-incubated the cells with Alexa-555 tagged transferrin, a glycoprotein that can only enter the cells via endocytosis. In the presence of ATP, both Cy-5 labelled rods (red, Fig. S3a) and Alexa-555 tagged transferrin (green, Fig. S3a) entered the MCF7 cells. In the absence of ATP only the Cy-5 labelled rods (red, Fig. 4b) entered the cells while transferrin (green, Fig. S3b)remained in the extracellular space. From auto- and pair-correlation analysis of the intensity fluctuations acquired along line scans that extended from the extracellular matrix into the cytoplasm (insert in Fig. S3a-b), we learnt that a large proportion of the nanoparticles, independently of shape, crossed the plasma membrane by passive diffusion (Fig. S3c-d). That is, although the number of nanoparticles detected in the cytoplasm was reduced for each shape upon ATP depletion (Fig. S3c), the majority of nanoparticles still gained intracellular access by an energy independent mechanism. Interestingly, the small reduction in cytoplasmic nanoparticle accumulation observed in the absence of ATP was accompanied by an increase in the average delay time for each shape to cross the plasma membrane under these conditions (Fig. S3d). Given that pair correlation analysis of ATP-mediated transport of transferrin-Alexa555 into the cytoplasm occurred on a timescale of 1-10 ms (Fig. S3e), and this fast time scale transit was inhibited for all four nanoparticle shapes when ATP was depleted (Fig. S3f-i), the increase in the average delay time suggests that in addition to the majority of nanoparticles entering the cell by passive diffusion, a fraction of nanoparticles entered the cell by a facilitated endocytosis.

Supplementary Figure 3: ATP-dependent and ATP-independent uptake of nanoparticles. a-b.Merged fluorescence image of a MCF7 cell incubated with Cy5-labelled rods (red) and Alexa555-transferrin (green) in the presence (a) and absence (b) of ATP. Inserts show where line scans wereacquired. Scale bar = 3 µm. c. Concentration of mobile nanoparticles in the cytoplasm in the presence (solid bars) and absence (chequered bars) of ATP, obtained from autocorrelation analysis. d. Transit times of mobile nanoparticles across the plasma membrane in the presence (solid bars) and absence (chequered bars) of ATP, obtained from pair correlation analysis (δr = 8-10 pixels). In c-d, three cells were analysed for each condition and three line scans were acquired per cell. This resulted in n = 9measurements for statistical analysis by one-way Anova with no significant difference found between nanoparticles and ± ATP. e. Pair correlation analysis of the distribution of transit times for transferrin mobility across the plasma membrane in the presence and absence of ATP demonstrates ATP driven





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transport to occur on a timescale of approximately 1-10 ms. f-i. Pair correlation analysis of the distribution of transit times for micelles (f), vesicles (g), rods (h) and worms (i) crossing the plasma membrane in the presence (blue curves) and absence (red curves) of ATP. In e-i solid lines in the plots represent average pair correlation profiles and shaded areas are standard deviations. For each condition 2-4 cells were analysed and two line scans were acquired per cell. This resulted in n = 4-8measurements.





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Endosomal accumulation and escape

As illustrated in Fig. 4c in the main manuscript, the escape time from both early and late endosomes was rather broad and no shape dependency on the average time for endosomal escape was found (Fig. 4e). When we examined the distributions of escape times in more detail (Fig. S4), it was found thatescape from early endosomes occurred on two timescales (fast and slow) and this resulted in two characteristic escape times (arrows in Fig. S4b). In contrast, the distribution profiles of escape times from the late endosomes was shape dependent (Fig. S4c). Spherical nanoparticles (micelles and vesicles) predominately escaped the late endosomes on a slow timescale (arrow in Fig. S4c, red and dark blue curve) while high-aspect ratio nanoparticles mainly adopted fast escapes from the late endosomes (arrow in Fig. S4c, green and light blue curve). Thus the differences in late endosomal accumulation (Fig. 4c) may be caused by the differences in the number of nanoparticles that escaped the late endosomes on a slow versus fast time scale.

Supplementary Figure 4: Escape times from early and late endosomes. a. Merged fluorescence image of MCF7 cells expressing Rab7-GFP (green) incubated with Cy-5-modified nanoparticles (red). Rods and worms show the highest nanoparticle accumulation as found from autocorrelation analysis in Fig. 4d. b. Pair correlation analysis of the distribution of escape times for the spherical (micelles (dark blue) and vesicles (red)) and high aspect ratio nanoparticles (rods (light blue) and worms (green)) from Rab5-positive endosomes (early endosomes). c. Pair correlation analysis of the distribution of escape times for the spherical (micelles (dark blue) and vesicles (red)) and high aspect ratio nanoparticles (rods (light blue) and worms (green)) from Rab7-positive endosomes (lateendosomes). In b-c solid lines in the plots represent average pair correlation profiles and shaded areas are standard deviations. For each condition 2-4 cells were analysed and four line scans were acquired across four different endosomes per cell. This resulted in n = 8-16 measurements. Dashed vertical lines separate fast and slow timescale nanoparticle populations and arrows show the peak time of transit for each population.





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Crossing the nuclear envelope

Supplementary Figure 5: Nuclear localization signal peptide selectively enhanced nuclear import of high aspect ratio shapes. a-c. Pair correlation analysis of micelle (a), rod (b) and worm (c) shaped nanoparticles across the nuclear envelope without an NLS (top row) and with an NLS (bottom row) in the cytoplasm-to-nucleus (blue curves) and nucleus-to-cytoplasm (red curves) direction (δr = 10). Comparison of micelles (a) with rods and worms (b-c), reveals the NLS modification to selectively enhance import (blue curves) of high aspect ratio shaped nanoparticles on a fast timescale. Solid lines in the plots represent average pair correlation profiles and shaded areas are standard deviations. For each condition 3-6 cells were tested and two line scans were acquired per cell. This resulted in n = 6-12 measurements.





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Doxorubicin delivery to the nucleus

Supplementary Figure 6: Cross pair correlation analysis of nanoparticle doxorubicin release.a. Merged fluorescence intensity images of MCF7 cells incubated with rhodamine-labelled micelles, vesicles, rods and worms (red) loaded with doxorubicin (green) after a total incubation of 0.25 h, 6 and 24 h. b-c. Fraction of doxorubicin bound to the differently shaped nanoparticles in the cytoplasm (b) and nucleus (c) at 0.25 h, 6 and 24 h. For each condition 6 cells were tested and two line scans were acquired per cell. This resulted in n = 12 measurements for statistical analysis by one-way Anova (no significance was found for the data in b-c, P > 0.05).





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Supplementary Figure 7. 1H-NMR spectra of P(OEGMA32-co-FMA1) and P(OEGMA32)-co-FMA1)-b-P(ST-co-VBA) block copolymer in d-chloroform where FMA refers to fluorescein O-methacrylate.

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Supplementary Figure 8. Size exclusion chromatography (SEC): size evolution of the M-CTA and dispersion polymerization of styrene and 3-vinylbenzaldhyde at different polymerization times: A: 8h (spherical micelles); (B): 12 h (rod-like); (C): 18 h (worm-like) and D: 48 h (vesicles).

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M-CTA 8600 - 1.2 A 22900 - 1.5 B 23800 - 1.6 C 25100 - 1.7 D 37400 - 1.7

Mn PDI





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Supplementary Figure 9. UV-Vis absorption spectra of POEGMA-b-P(ST-co-VBA) nanoparticles, showing the successful attachment of Rhodamine and DOX.

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Supplementary Scheme 1. Schematic illustration of fluorescein labelled macro-CTA synthesis. The structures of the resulting polymers in each step were identified by 1H-NMR spectra (Figure S1). In the FMA containing macro-CTA spectrum all the aromatic protons belonging to FMA were clearly observed at around 7 ppm as assigned in the Figure. On the other hand, when we investigated the block copolymer spectrum, typical aromatic protons of PST and PVBA were observed at 7.3-6.7 and 7.5 ppm, respectively. Aldehyde proton belonging to PVBA was also clearly observed at 9.9 ppm. 1H-NMR results clearly showed that both FMA containing macro-CTA and chain extension were successfully achieved.





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Supplementary Scheme 2. P(OEGMA32-co-FMA1)-b-PST-co-PVBA block copolymer synthesis by RAFT dispersion polymerization





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Supplementary Scheme 3. Schematic representation of functionalization of POEGMA-b-PST-co-PVBA nanoparticles with Rhodamine followed by conjugation of doxorubicin via the formation of Schiff base. Size exclusion chromatography traces of the block copolymers (Figure S8), show that the number molecular weight (Mn) increases with the polymerization time. Small shoulders on the size exclusion chromatography traces were acceptable due to the PST coupling character. The results obtained from the dispersion polymerization were collected and are shown in Table S1.





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Supplementary Table 1. Summary of the RAFT dispersion block copolymerization of styrene and vinyl benzaldehyde in the presence of FMA based macro-CTA.

morphology Reaction Time (h)

VBA Conv. (%) a

ST Conv. (%) b

Total Conv. (%) c

Mn(NMR)

gmol-1 d

Mn(SEC)

gmol-1 e

PDI

Micelle (A) 8 8.76 4.06 4.29 33800 22900 1.5rod-like (B) 12 10.57 4.37 4.68 35900 23800 1.6worm-like (C) 18 16.95 4.89 5.49 40600 25100 1.7vesicle(D) 48 19.24 9.06 9.57 62000 37400 1.7

Polymerization conditions are explained in the experimental section.a． VBA conversion was calculated from 1H-NMR spectrum of the reaction mixture by dividing of aldehyde protons of the poly(vinylbenzaldehyde) peak (9.75 ppm) integral with the sum of aldehyde protons of vinyl benzaldehyde (10.03 ppm) and PVBA peak integrals.b． ST conversion was calculated from 1H-NMR spectrum of the reaction mixture by dividing of the polystyrene, poly(vinyl benzaldehyde) aromatic protons and one vinyl proton of the both monomer peaks integral between 7.2 ppm to 6.3 ppm with the sum of vinyl peaks (5.8 and 5.3 ppm) of both ST and VBA and the peak integrals that appeared at 7.2 ppm to 6.3 ppm. c． Total conversion was calculated using; ConvT = 0.95*Conv(ST) + 0.05* Conv(VBA)d． NMR molecular weight was calculated according to Mn= ( [MST]o/ [RAFT]o)* xST* MwST +( [MVBA]o/ [RAFT]o)* xVBA* MwVBA + MwRAFT , where [MST]o , [MVBA]o [RAFT]o , xST, xVBA, MwST , MwVBA and MwRAFT are monomers and RAFT agent concentration, monomers conversion, molecular weight of monomer and RAFT agent, respectively.e． The experimental Mn and PDI were determined by SEC using dimethyl acetamide as eluent solvent with polystyrene standards (the molecular weight ranging from 168 to 106 gmol-1).


Correction notice: Pair correlation microscopy …...escape from early endosomes occurred on two...

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