Supplementary Materials for Colour-Tunable Fluorescent Multiblock Micelles Zachary M. Hudson, David J. Lunn, Mitchell A. Winnik,* Ian Manners* *To whom correspondence should be addressed. E-mail: mwinnik@chem.utoronto.ca (M. A. W.); ian.manners@bristol.ac.uk (I. M.) This file includes:. Supplementary Figures 2. Supplementary Tables 3. Supplementary Methods
List of Acronyms and Abbreviations BCP = block copolymer BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene CDSA = crystallization-driven self-assembly δ = chemical shift DSC = differential scanning calorimetry EtOAc = ethyl acetate GPC = gel permeation chromatography LSCM = laser scanning confocal microscopy L n = number average contour length L w = weighted average contour length MALDI-TOF = matrix-assisted laser desorption-isonization time-of-flight N = sample size NMR = nuclear magnetic resonance PDI = polydispersity index (ratio of L w /L n for micelle contour lengths and M w /M n for polymer chain lengths) PDMS = poly(dimethylsiloxane) PMVS = poly(methylvinylsiloxane) PFS = poly(ferrocenyldimethylsilane) TEM = transmission electron microscopy T g = glass transition temperature THF = tetrahydrofuran T m = melting temperature σ = standard deviation WLE = white-light emitting XL = crosslinked 2
. Supplementary Figures A Normalized Frequency.35.3.25.2.5..5 2 3 4 Contour Length / nm B Normalized Frequency.35.3.25.2.5..5 5 3 45 6 Contour Length / nm C Normalized Frequency.35.3.25.2.5..5 25 25 375 Contour Length / nm Supplementary Figure Fluorescent cylindrical micelles. TEM micrographs of (A) M(), (B) M(2) and (C) M(3) cylindrical micelles with corresponding contour length statistics. The PDMS and PMVS micelle coronas are not visible in TEM micrographs due to insufficient electron density contrast. 3
Supplementary Figure 2 Tunable-Colour Fluorescent Micelles. Top: Raw digital photographs of solutions of fluorescent micelles under UV irradiation at 365 nm. Bottom: LSCM images, TEM micrographs and fluorescence emission spectra (λ ex = 365 nm) of cylindrical micelles of tunable colour. Normalized emission >7 nm was obtained separately with λ ex = 58 nm to eliminate interference from the second-order diffraction of the 365 nm excitation beam. A B C D E F G H I J K L M N O P WLE D65 A.2.8.6.4.2 5 nm 4 5 6 7 B.2.8.6.4.2 5 nm 4 5 6 7 C.2.8.6.4.2 5 nm 4 5 6 7 4
D.2.8.6.4.2 nm 4 5 6 7 E.2.8.6.4.2 5 nm 4 5 6 7 F.2.8.6.4.2 5 nm 4 5 6 7 G.2.8.6.4.2 5 nm 4 5 6 7 H.2.8.6.4.2 5 nm 4 5 6 7 5
Emission Intensity / a.u. I.2.8.6.4.2 4 5 6 Wavelength / nm 7 4 5 6 Wavelength / nm 7 4 5 6 Wavelength / nm 7 4 5 6 Wavelength / nm 7 4 5 6 Wavelength / nm 7 5 nm Emission Intensity / a.u. J.2.8.6.4.2 5 nm Emission Intensity / a.u. K.2.8.6.4.2 5 nm Emission Intensity / a.u. L.2.8.6.4.2 5 nm Emission Intensity / a.u. M.2.8.6.4.2 5 nm 6
N.2.8.6.4.2 nm 4 5 6 7 O.2.8.6.4.2 nm 4 5 6 7 P.2.8.6.4.2 nm 4 5 6 7 WLE.2.8.6.4.2 5 nm 4 5 6 7 D65.2.8.6.4.2 nm 4 5 6 7 7
3 µm Supplementary Figure 3 Micelle colour distribution. Sequential LSCM scan of white light-emitting fluorescent micelles (Supplementary Table, entry M) showing uniform distribution of each primary colour along the length of the micelle. Emission was sequentially detected between 45-47 nm (top left, λ ex = 45 nm), 5-57 nm (top right, λ ex = 488 nm) and 65-72 nm (λ ex = 594 nm), as well as all three channels simultaneously (bottom right). 8
Normalized Frequency.3.25.2.5..5 2 3 4 5 Contour Length / nm Supplementary Figure 4 Fluorescent -block co-micelles. TEM micrograph of -block M(3)-b- M(PFS 6 -b-pdms 66 )-b-m(2)-b-m(pfs 6 -b-pdms 66 )-b-m()-b-m(pfs 6 -b-pdms 66 )-b-m()-b- M(PFS 6 -b-pdms 66 )-b-m(2)-b-m(pfs 6 -b-pdms 66 )-b-m(3) block co-micelles. 9
5 µm Supplementary Figure 5 Independent colour addressing. Sequential LSCM scan of -block M(3)- b-m(pfs 6 -b-pdms 66 )-b-m(2)-b-m(pfs 6 -b-pdms 66 )-b-m()-b-m(pfs 6 -b-pdms 66 )-b-m()-b- M(PFS 6 -b-pdms 66 )-b-m(2)-b-m(pfs 6 -b-pdms 66 )-b-m(3) block co-micelles showing the independent addressing of each primary colour. Shown is the individual illumination of the M(2) blocks only (top left, λ ex = 488 nm), the M() blocks only (top right, λ ex = 594 nm), the M(3) blocks only (bottom left, λ ex = 45 nm), as well as all three fluorescent block types simultaneously (bottom right).
5 nm 5 nm Normalized Frequency.35.3.25.2.5..5 C Normalized Frequency.25.2.5..5 D Contour Length / nm Contour Length / nm Supplementary Figure 6 Triblock co-micelles. (A) TEM micrograph of M(PFS 6 -b-pdms 66 ) micelles. (B) TEM micrograph of M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 )-b-m(pfs 53 -b-pmvs 6 ) triblock co-micelles. Histograms (C) and (D) of the contour length distributions of (A) and (B) respectively.
nm Supplementary Figure 7 Crosslinked triblock co-micelles. Representative TEM micrograph of XL M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 )-b- XL M(PFS 53 -b-pmvs 6 ) triblock co-micelles. 2
Supplementary Figure 8 Non-centrosymmetric micelles. Representative TEM micrograph of noncentrosymmetric XL M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 ) A-B micelles after addition of PFS 6 -b- PDMS 66 unimer. 3
Normalized Frequency.2.5..5 3 6 9 2 5 Contour Length / nm Supplementary Figure 9 Non-centrosymmetric 7-block co-micelles. TEM micrograph and corresponding contour length histogram of non-centrosymmetric 7-block XL M(PFS 53 -b-pmvs 6 )-b- M(PFS 6 -b-pdms 66 )-b-m()-b-m(pfs 6 -b-pdms 66 )-b-m(2)-b-m(pfs 6 -b-pdms 66 )-b-m(3) block comicelles. Scale bars =. 4
µm Supplementary Figure Non-centrosymmetric 7-block micelles by LSCM. LSCM images of noncentrosymmetric 7-block XL M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 )-b-m()-b-m(pfs 6 -b- PDMS 66 )-b-m(2)-b-m(pfs 6 -b-pdms 66 )-b-m(3) block co-micelles. 5
4 µm 5 µm 5 µm Supplementary Figure Scarf-like micelles. TEM micrograph and LSCM images of fluorescent scarf-like micelles. 6
Normalized Refractive Index / a.u...9.7.5.3. PFS 62 homopolymer PFS 62 -b-(pdms 65 -r-pmvs 2 ) 2 3 4 5 9.5.5 3.5 5.5 7.5 -. Retention Time / min Supplementary Figure 2 GPC data. Overlaid refractive index GPC traces for luminescent polymers -3 and their precursors. The retention time and PDI of 2-mercaptoethylamine-functionalized 5 are affected by interactions with the GPC column. 7
2. Supplementary Tables Supplementary Table : Preparation of Cylindrical Micelles of Tunable Colour Sample Vol. (µl) Vol. (µl) 2 Vol. (µl) 3 CIE (x, y) A.69,.3 B.28,.66 C.9,.2 D 9.6,.37 E 7.5 2.5.52,.45 F 5 5.42,.54 G 2.5 7.5.34,.6 H 7.5 2.5.45,.25 I 5 5.3,.23 J 2.5 7.5.24,.2 K 7.5 2.5.25,.5 L 5 5.22,.39 M 2.5 7.5.2,.3 N 2 6 2.3,.49 O 2 2 6.25,.3 P 8.52,.34 WLE 3.7 2.7 3.6.33,.33 D65 3. 3. 3.9.3,.33 Supplementary Table 2: Polymer Characterization Data Sample M n First Block a Block Ratio b M n Diblock b PDI (g/mol) (g/mol) PFS 28 -b-pdms 56 6,7 :2 48,2.2 PFS 6 -b-pdms 66 4,8 : 63,7.6 PFS 63 -b-pdms 53 5,2 :8 53,4.6 PFS 53 -b-pvms 6 2,8 : 64,5.3 PFS 62 -b-(pdms 65 -r-pmvs 2 ) 5, : 6,7.6 PI 76 -b-pfs 76 5,2 : 23,6. a Determined by MALDI-TOF mass spectrometry. b Determined by relative integration of H NMR signals from each block. 8
3. Supplementary Methods Preparation of M(PFS 63 -b-pdms 53 ) Crystallite Seed Micelles: A sample of long M(PFS 63 -b-pdms 53 ) cylinders (> µm) were prepared by heating 2 mg of PFS 63 -b- PDMS 53 in 2 ml EtOAc for h at 75 C, then allowing the sample to cool to room temperature and stand for 4 days. M(PFS 63 -b-pdms 53 ) crystallite seeds were then prepared by sonication of 4 ml of this solution for 3 h at C using a 5 W sonication processor equipped with a titanium sonotrode. Preparation of Cylindrical Micelles of Tunable Fluorescent Colour: To a 7 ml screw-cap vial was added 2. ml EtOAc and 5 µl of pre-sonicated M(PFS 63 -b- PDMS 53 ) crystallite seeds in EtOAc (. mg/ml,.5 mg). To this mixture was then added varying amounts of, 2, and/or 3 with gentle stirring as mg/ml THF solutions, such that µl of fluorescent polymer solution was added in total. These solutions were then left to stand for 2 days. The precise composition of each sample of fluorescent micelles is given in Supplementary Table, with the CIE coordinates of the emission colour of that sample. TEM micrographs of drop-cast aliquots of these solutions showed the formation of cylindrical micelles. Three representative samples were then measured to obtain contour length statistics (given below), and indeed were found to give micelles of equal length (σ < 3%) (Supplementary Figure 4). From µl of : L n = 98 nm, PDI =.2, σ/l n =.5, N = 35. From µl of 2: L n = 986 nm, PDI =.7, σ/l n =.27, N = 347. From µl of 3: L n = 97 nm, PDI =.3, σ/l n =.8, N = 38. LSCM images and representative TEM micrographs were then obtained for all samples. LSCM images are plotted using the CIE 93 chromaticity values for each sample (Supplementary Figure 5). 9
Preparation of Centrosymmetric Red-Green-Blue Fluorescent -Block Co-Micelles: To ml EtOAc was added a solution of pre-formed M(PFS 6 -b-pdms 66 ) cylindrical micelles (.2 mg,. mg/ml in EtOAc). (L n = 244 nm, vide supra) To this solution was added 4 µl of a solution of in THF (.4 mg, mg/ml) with stirring. The solution was allowed to age for 24 h, then 2 µl of PFS 6 -b-pdms 66 solution in THF (.4 mg, 2 mg/ml) was added with stirring. The solution was allowed to age for 24 h, then 4 µl of a solution of 2 in THF (.4 mg, mg/ml) was added with stirring. The solution was allowed to age for 24 h, then 2 µl of PFS 6 -b-pdms 66 solution in THF (.4 mg, 2 mg/ml) was added with stirring. The solution was allowed to age for 24 h, then 4 µl of a solution of 3 in THF (.4 mg, mg/ml) was added with stirring. The solution was once again aged for 24 h. TEM micrographs of a drop-cast aliquot showed the formation of cylindrical micelles with an average contour length of 259 nm (PDI =.2, σ/l n =.6, N = 37) (Supplementary Figure 6). Preparation of M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 )-b-m(pfs 53 -b-pmvs 6 ) Triblock Co- Micelles: To 95 ml EtOAc was added 4 ml of pre-formed M(PFS 63 -b-pdms 53 ) crystallite seeds ( mg/ml in EtOAc) and.8 ml of a THF solution of PFS 6 -b-pdms 66 (2 mg/ml, 6 mg) was added with stirring. This solution was allowed to age for 2 days. TEM micrographs of a drop-cast aliquot showed M(PFS 6 -b-pdms 66 ) micelles of average length 244 nm (PDI =.6, σ/l n =.25, N = 47) (Supplementary Figure 7A). Next, 2 µl of a THF solution of PFS 53 -b-pmvs 6 (2 mg/ml, 4 mg) was added to a gently stirred 2 ml suspension of the prepared M(PFS 6 -b-pdms 66 ) micelles (. mg/ml, 2 mg). This solution was allowed to age for 2 days. TEM micrographs of a drop-cast aliquot showed the formation of cylindrical micelles with an average contour length of 57 nm (PDI =.5, σ/l n =.22, N = 33) (Supplementary Figure 7B). 2
Crosslinking of M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 )-b- M(PFS 53 -b-pmvs 6 ) Triblock Co- Micelles: To a 2 ml screw-cap vial was added 2 ml of the prepared solution of M(PFS 53 -b-pmvs 6 )-b- M(PFS 6 -b-pdms 66 )-b-m(pfs 53 -b-pmvs 6 ) triblock co-micelles (.3 mg/ml,.6 mg), 96 µl of an EtOAc solution of 2,2-dimethoxy-2-phenylacetophenone (2 mg/ml,.92 mg, 2 equiv./vinyl group) and ml EtOAc. The solution was bubbled with N 2, sealed and irradiated 3 cm from a mercury lamp for 3 min at 25 C. TEM micrographs of a drop-cast aliquot showed the darkening of the PMVS corona region, consistent with highly crosslinked PMVS chains (Fig S8). Preparation of Non-Centrosymmetric XL M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b-pdms 66 ) A-B Micelles: To a 2 ml screw-cap vial was added 6.5 ml of the prepared solution of XL M(PFS 53 -b- PMVS 6 )-b-m(pfs 6 -b-pdms 66 )-b- XL M(PFS 53 -b-pmvs 6 ) triblock co-micelles and.6 ml dry THF (2% v/v). The mixture was placed in an ultrasonic bath operating at 35 khz and 6 W for min, then left to stir at 3 rpm on an orbital shaker for 3 h. At this point, the suspension was transferred to a length of regenerated cellulose tubing for dialysis into % EtOAc for 6 h. To examine the growth of these XL M(PFS 53 -b-pmvs 6 ) micelles to further CDSA, 2 µl of a THF solution of PFS 6 -b-pdms 66 (2 mg/ml, 2.4 mg) was added to 2 ml of the above solution with gentle stirring, and the mixture was aged for 24 h. TEM micrographs of a drop-cast aliquot showed that 77% of the of the micelles remain active to CDSA in one direction only, giving non-centrosymmetric micelles. Approximately 5% of the micelles remain active to CDSA at both micelle termini, while 8% of the micelles appear inactive to CDSA (N = 362) (Supplementary Figure 8). Preparation of Non-Centrosymmetric Red-Green-Blue Fluorescent Micelles: To a 7 ml screw-cap vial was added ml of a solution of XL M(PFS 53 -b-pmvs 6 )-b-m(pfs 6 -b- PDMS 66 ) micelles (.5 mg,.5 mg/ml). To this solution was added 2 µl of a solution of in THF 2
(.2 mg, mg/ml) with stirring. The solution was allowed to age for 48 h, then 3 µl of PFS 6 -b- PDMS 66 solution in THF (.6 mg, 2 mg/ml) was added with stirring. The solution was allowed to age for 24h, then 2 µl of a solution of 2 in THF (.2 mg, mg/ml) was added with stirring. The solution was allowed to age for 48 h, then 3 µl of PFS 6 -b-pdms 66 solution in THF (.6 mg, 2 mg/ml) was added with stirring. The solution was allowed to age for 24h, then 2 µl of a solution of 3 in THF (.2 mg, mg/ml) was added with stirring. The solution was once again aged for 48 h. TEM micrographs of a drop-cast aliquot showed the formation of cylindrical micelles with an average contour length of 595 nm (PDI =.5, σ/l n =.22, N = 328) (Supplementary Figure ). Preparation of Encoded Pentablock Micelles: Colours of Canada, the United Kingdom and Ireland To 4.2 ml EtOAc was added µl of a solution of pre-formed M(PFS 63 -b-pdms 53 ) crystallite seeds (. mg,. mg/ml in EtOAc). To this solution was added 2 µl of a THF solution of unimer (.2 mg, mg/ml, see compositions below) with stirring. The solution was allowed to age for 48 h, then 4 µl of a second THF solution of unimer (.4 mg, mg/ml) was added with stirring. After aging for a further 48 h, 4 µl of a third THF solution of unimer (.4 mg, mg/ml) was added with stirring. After standing for 48 h, pentablock micelles with encoded fluorescent colours could be obtained. st Addition 2 nd Addition 3 rd Addition Canadian Micelles (2 µl) (4.8 µl), 2 (.8 µl), (4 µl) 3 (4.4 µl) British Micelles (2 µl) (4.8 µl), 2 (.8 µl), 3 (4 µl) 3 (4.4 µl) Irish Micelles (5 µl), 2 (5 µl) (4.8 µl), 2 (.8 µl), 3 (4.4 µl) 2 (4 µl) Formation of Fluorescent Scarf-Like Micelles: To a mixture of 3.5 ml decane and.5 ml xylenes was added.5 mg PI 76 -b-pfs 76. The mixture was heated to C for h with stirring, then cooled to 8 C, removed from stirring and left to stand overnight. The mixture was then placed in an ultrasonic bath operating at 35 khz and 6 W for min. 22
To ml of this solution was then added µl of a solution of in THF (. mg, mg/ml) with stirring. The solution was allowed to age for 48 h, then µl of PFS 6 -b-pdms 66 solution in THF (.5 mg, 5 mg/ml) was added with stirring. The solution was allowed to age for 24h, then µl of a solution of 2 in THF (. mg, mg/ml) was added with stirring. The solution was allowed to age for 48 h, then µl of PFS 6 -b-pdms 66 solution in THF (.5 mg, 5 mg/ml) was added with stirring. The solution was allowed to age for 24h, then µl of a solution of 3 in THF (.2 mg, mg/ml) was added with stirring. The solution was once again aged for 48 h. TEM micrographs of a drop-cast aliquot showed the formation of scarf-like micelles (Supplementary Figure ). 23