Supporting Information

UV-induced Vesicle to Micelle Transition: A Mechanistic StudyCraig A. Machado, Roger Tran, Taylor A. Jenkins, Amanda M. Pritzlaff, Michael B. Sims, Brent S. Sumerlin, Daniel A. Savin*

George & Josephine Butler Polymer Research Laboratory, Center for Macromolecular Science & Engineering, Department of Chemistry, University of Florida, Gainesville, FL 32611, USA. E-mail: savin@chem.ufl.edu

Figure S1. 1H NMR spectrum of Nitrobenzyl Chloroformate

Electronic Supplementary Material (ESI) for Polymer Chemistry.This journal is © The Royal Society of Chemistry 2019

Figure S2. 13C NMR spectrum of Nitrobenzyl Chloroformate

Figure S3. 13C NMR spectrum of nitrobenzyl hydroxypropyl methacrylate

Mass Spectrometry and Liquid Chromatography Characterization of Monomer NbHPMA

Methods

High Resolution Mass Spectrometry (HRMS) and High Resolution LC-MS

HRMS and high resolution LC-MS were performed on an Agilent 6220 Time-of-Flight instrument. Monomers in solution were ionized via electrospray ionization (ESI) using a heated dry gas (N2) for desolvation: flow rate – 8.0 L/min; gas temperature – 350 ºC, nebulization pressure – 30 psig. The injector was an Agilent 1100 series system (Santa Clara, CA) outfitted with a degasser (G13793), binary pump (G1312B), and auto sampler (G1367C). The mobile phase used for HRMS was methanol with 0.1% formic acid. The sample was prepared in methanol at an approximately 1 mg/mL concentration and was fully solubilized.

All LC-MS of NbHPMA was performed using a Thermo Scientific Hypersil GOLD C8 column (2.1 x 100 mm, 5 μm particle size), and mobile phases of water with 50 mM ammonium formate and 0.1% formic acid (mobile phase A) and methanol with 50 mM ammonium formate and 0.1% formic acid (mobile phase B) at a flow rate of 0.20 mL/min. Samples were separated using a gradient method starting at 10% B for 2 minutes, followed by a linear increase to 100% B at 15 minutes, and held at 100% B for 10 minutes. The sample was prepared in water with 50 mM ammonium formate and 0.1% formic acid at an approximately 1 mg/mL concentration although not all sample dissolved.

Low Resolution LC-MS/MS and Direct Infusion MS

LC-MS/MS with fragmentation by collision induced dissociation (CID) and direct infusion mass spectrometry of NbHPMA were performed on a Thermo Fisher Scientific LTQ XL Linear Ion Trap Mass Spectrometer. An electrospray ionization (ESI) ionization source was used with a heated dry gas (N2) for desolvation. ESI method parameters included: N2 sheath gas = 7 (unit less), N2 aux gas = 0 (unit less), capillary temperature = 300°C, and spray voltage of 5kV. Fragmentation (MS2) was performed using an isolation width of m/z 4.0, normalized collision energy (NCE) of 35%, an activation q of 0.250, and a detection range of m/z 100-1200. For direct infusion MS, the sample was prepared in methanol at an approximately 1 mg/mL concentration. For LC-MS/MS, the mobile phases, gradient parameters, and sample preparation matched those listed above for high resolution LC-MS.

Characterization Data

HRMS Accurate Mass Confirmation of NbHPMA

The molecular formula of NbHPMA was confirmed using HRMS with a mass error of 4.1 ppm using the [M+NH4]+ ion labeled in Figure S4 (Theoretical mass = m/z 341.1343). Peaks present at m/z 369 and 127 were reproducibly observed in replicate injections.

6x10

00.05

0.10.15

0.20.25

0.30.35

0.40.45

0.50.55

0.60.65

0.70.75

0.80.85

0.90.95

11.05

1.11.15

1.21.25

1.31.35

1.41.45

1.51.55

1.61.65

1.7

+ESI Scan (rt: 0.201-0.384 min, 23 scans) Frag=175.0V 2019-04-19_CAM-C-139-Frac33-45_01_ESI.d Subtract

341.1357

127.0756

369.1665

669.1919296.1245223.0595 414.0771

Counts vs. Mass-to-Charge (m/z)120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680

Figure S4. HRMS of NbHPMA. The indicated peak at m/z 341.1357 represents the [M+NH4]+ adduct of NbHPMA. [M+Na]+ is indicated at m/z 346.0902.

High Resolution LC-MS to Determine Purity of NbHPMA

High resolution LC-MS of NbHPMA was performed to determine purity of the sample and one peak was observed in the chromatogram suggesting a pure sample (Figure S5, top). The mass spectrum extracted from this peak is featured in Figure S6 and the predominant masses observed were m/z 341, 369, and 664. The masses m/z 341 and 664 were the sample related ions [M+NH4]+ and [2M+NH4]+, respectively. The impurity at m/z 369 was suspected to be sample-related due to co-elution with the NbHPMA [M+NH4]+ ion at m/z 341. This is demonstrated by the overlapping extracted ion chromatograms (EIC) for each species in Figure S5 (m/z 341 (middle, red) and m/z 369 (bottom, green)).

[M+NH4]+

[2M+Na]+

[M+Na]+

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0

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2

3

+ESI TIC Scan Frag=175.0V 2019-04-19_CAM-C-139-Frac33-45_01_ESI-LCMS.d

1 1

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1

+ESI EIC(341.1023-341.1963) Scan Frag=175.0V 2019-04-19_CAM-C-139-Frac33-45_01_ESI-LCMS.d

1 1

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0

0.5

1

+ESI EIC(369.1389-369.2136) Scan Frag=175.0V 2019-04-19_CAM-C-139-Frac33-45_01_ESI-LCMS.d

1 1

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

Figure S5. LC-MS chromatogram of NbHPMA (top, blue) with an extracted ion chromatogram (EIC) of m/z 341 ([M+NH4]+ for NbHPMA) (middle, red) and an extracted ion chromatogram for impurity m/z 369 (bottom, green).

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2.1

2.2+ESI Scan (rt: 13.167-14.929 min, 214 scans) Frag=175.0V 2019-04-19_CAM-C-139-Frac33-45_01_ESI-LCMS.d Subtract

341.1404

369.1722

664.2469

296.1307228.1983 523.2334439.2148 493.0896

Counts vs. Mass-to-Charge (m/z)200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680

Figure S6. Mass spectrum of NbHPMA extracted from the single peak in the LC-MS chromatogram between 13.2 and 14.9 minutes.

[2M+NN4]+

[M+NN4]+

LC-MS Chromatogram

EIC of m/z 341

EIC of m/z 369

CID Fragmentation Studies

To determine if the HRMS peak at m/z 369 was related to NbHPMA, low resolution LC-MS and CID fragmentation were performed with a Thermo Fisher Scientific LTQ XL Linear Ion Trap Mass Spectrometer (Figure S7). It was observed that the masses m/z 341 and 369 both decomposed into a fragment at m/z 127, which was also observable in the HRMS spectrum (Figure S4). The [M+Na]+ peak for NbHPMA at m/z 346 was also fragmented and produced a daughter ion of m/z 127 (Figure S7). Two common fragment masses between m/z 341 and 369 were m/z 127 and 323 which suggested these masses were related and that m/z 369 can be included in overall sample purity.

Figure S7. Parent mass spectrum of NbHPMA and fragmentation spectra (CID) of m/z 341, 346, and 369 (top to bottom).

Parent Mass Spectrum of NbHPMA

CID of NbHPMA [M+NH4]+ m/z 341

CID of NbHPMA [M+Na]+ m/z 346

CID of m/z 369

Direct Injection MS of NbHPMA

Direct-infusion mass spectrometry, in which the sample is injected by syringe pump directly into the ESI ionization source, was employed to determine if m/z 369 was an artefact of the auto-injection process. The full mass spectrum shown in Figure S8 does not include m/z 369. This result, combined with the results of the CID fragmentation study, suggested that m/z 369 was a derivative of NbHPMA formed during the auto sampler injection process prior to ionization.

Figure S8. Low-resolution direct infusion mass spectrum of NbHPMA featuring high intensity peaks m/z 341 [M+NH4]+ and m/z 346 [M+Na]+.

Figure S9. 1H NMR spectrum of polyPEGMA

0 500 1000 1500 2000 25000.0

0.2

0.4

0.6

0.8

ln([M

] o/[M

])

Time (min)Figure S10. Pseudo-first order kinetic plot for initial block polymerization of PEGMA with NbHPMA at 70 oC. Plot is linear until 560 min, then has a decrease in slope, indicating the presence of termination events.

0 10 20 30 40 50 606000

8000

10000

12000

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16000

Mn (actual) Ð Mn (theoretical)

Conversion (%)

Mn (

g/m

ol)

1.0

1.2

1.4

1.6 D

isper

sity

Figure S11. Plot of molecular weight and dispersity (determined via GPC against poly(methyl methacrylate) standards) as a function of conversion (determined via 1H NMR) for initial block polymerization of PEGMA with NbHPMA at 70 oC.

14 15 16 17 180.0

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1.0 t=0 min t=78 min t=244 min t=403 min t=592 min t=1260 min t=1746 min t=2013 min t=2367 min

RI R

espo

nse

Elution Time (min)Figure S12. GPC traces of initial block polymerization 70 oC.

0 500 1000 1500 20000.0

0.2

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0.8

1.0

ln([M

] o/[M

])

Elution Time (min)Figure S13. Linear pseudo-first order kinetic plot for poly(NbHPMA) polymerized at 70 oC.

0 10 20 30 40 50 600

5000

10000

15000

20000

25000

30000 Mn

Ð

Conversion (%)

Mn (g

/mol

)

1.2

1.6

2.0

2.4

Ð

Figure S14. Plot of molecular weight and dispersity (determined via GPC against poly(methyl methacrylate) standards) as a function of conversion (determined via 1H NMR) for poly(NbHPMA) polymerized at 70 oC.

11 12 13 14 15 16 17 180.0

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RI R

espo

nse

Elution Time (min)

t=0 min t=62 min t=130 min t=208 min t=338 min t=670 min t=1286 min t=1754 min

Figure S15. GPC traces of poly(NbHPMA) polymerized at 70 oC.

0 500 1000 1500 2000

0.0

0.5

1.0

1.5

ln([M

] o/[M

])

Time (min)Figure S16. Linear pseudo-first order kinetic plot for poly(NbHPMA) polymerized at 35 oC.

0 10 20 30 40 50 60 700

5000

10000

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20000

25000 Mn

Ð

Conversion (%)

Mn (g

/mol

)

1.1

1.2

1.3

1.4

1.5

1.6

Ð

Figure S17. Plot of molecular weight and dispersity (determined via GPC against poly(methyl methacrylate) standards) as a function of conversion (determined via 1H NMR) for poly(NbHPMA) polymerized at 35 oC.

12 13 14 15 16 17 180.0

0.2

0.4

0.6

0.8

1.0

RI R

eson

se

Elution Time (min)

t=0 min t=353 min t=1335 min t=1671 min

Figure S18. GPC traces of poly(NbHPMA) polymerized at 35 oC.

0 100 200 300 4000.0

0.2

0.4

0.6

ln([M

] o/[M

])

Time (min)Figure S19. Linear pseudo-first order kinetic plot for Nb 94 polymerized at 35 oC.

0.0 0.1 0.2 0.3 0.4 0.5 0.66000

9000

12000

15000

18000

Mn

Ð

Conversion (%)

Mn (

g/m

ol)

1.17

1.20

1.23

1.26

1.29

1.32

Ð

Figure S20. Plot of molecular weight and dispersity (determined via GPC against poly(methyl methacrylate) standards) as a function of conversion (determined via 1H NMR) for Nb 94 polymerized at 35 oC.

13 14 15 16 17 180.0

0.2

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0.6

0.8

1.0

RI R

espo

nse

Elution Time (min)

t=0 min t=49 min t=123 min t=189 min t=320 min t=379 min Final

Figure S21. GPC traces of Nb 94 polymerized at 35 oC.

Figure S22. 1H NMR of Nb 94.

14 15 16 17 18 19 200.0

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1.0

RI R

espo

nse

Elution Time (min)

t = 0 min t = 138 min t = 236 min t = 447 min Final

Figure S23. GPC traces of Nb 176 polymerized at 35 oC.

Note: Conversions at t = 138 and t=236 were too low to accurately measure via 1H NMR (negative conversion was measured at t = 138), so kinetic plots involving conversion have been omitted.

Figure S24. 1H NMR of Nb 176.

0 5 10 15 20

1x105

2x105

3x105

4x105

5x105

Inte

nsity

(a.u

.)

Time (hours)Figure S25. Scattered light intensity with crossed polarizers of Nb 94 solution as a function of time after 18 minutes of UV-irradiation. Due to the isotropic nature of the vesicles, very low light intensity reaches the detectors through the crossed polarizers, so the laser attenuation was adjusted accordingly. Spikes in intensity cause noise in the data, but the overall trend is the same as that in Figure 6.

Figure S26. Representative CONTIN size distribution plot of Nb 94 assemblies prior to irradiation.

Figure S27. Representative CONTIN size distribution plot of Nb 94 assemblies after 100 min irradiation.

0 100 200 300 4000

20

40

60

80

100

0.00.51.01.52.02.53.03.54.0

ln([P

G] o/[

PG])

Conversion ln([PG]o/[PG])

Time (min)

Conv

ersio

n (%

)

Figure S28. Kinetic plot of photolysis of preliminary block polymer. Block polymer was assembled in water and irradiated as detailed in experimental section. After given time intervals, aliquots were taken, solvent was removed, and 1H NMR was used to quantify extent of photolysis of nitrobenzyl groups. Methoxy protons from PEGMA were integrated as 3H and the integration of phenyl peaks was used to quantify the residual protecting group (PG). The conversion of photolysis was plotted as a function of irradiation time and shows a logarithmic plateau (shown in black). The pseudo-first order kinetics appear linear for the process (shown in blue). After 7 hours of irradiation, over 96% of nitrobenzyl groups were cleaved.

Figure S29. Image of a smaller Nb 176 assembly showing the fused vesicle morphology, indicating that the apparent bilayered appearance is not an artefact of focusing.

Figure S30. Additional TEM image of Nb 94 assemblies prior to irradiation, zoomed out to give a representative image of the assemblies.

Figure S31. Additional TEM image of Nb 94 assemblies after 16 minutes of irradiation, zoomed out to give a representative image of the assemblies. Due to the complex shapes of the clover-like assemblies and the size agreement between DLS and TEM, we believe these assemblies are appear as they are in solution and are not a result of drying-induced-aggregation.

Figure S32. Additional TEM image of Nb 176 assemblies prior to irradiation, zoomed out to give a representative image of the assemblies. The bridges between the assemblies are likely an artifact of drying, based on Rh determined by DLS.

Table S1. Summary of self-assembly methods’ effect on Rh for Nb 94.

Solvent Method Rh (nm)Dialysis 13THF Solvent Switch1 15

DMF Solvent Switch2 438

Table S2. Summary of self-assembly methods’ effect on Rh for Nb 176.

Solvent Method Rh (nm)Dialysis 34

Solvent Switch1 24THFSolvent Switch2 13

Dioxane Solvent Switch2 28DMF Solvent Switch2 973

1Polymer was dissolved at 0.1 mg/mL in organic solvent and water was added at 1 mL/hr. Organic solvent was allowed to evaporate under ambient conditions2Polymer was dissolved at 20 mg/mL in organic solvent and water was added at 1.8 vol% per minute until an equal volume of water and organic solvent was achieved. The resultant solution was then dialyzed against deionized water.