Supporting Information to

Cargo Shuttling by Electrochemical Switching of Core-Shell

Microgels Obtained by a Facile One-Shot Polymerization

Olga Mergel,a,b Sabine Schneider,a Rahul Tiwari,c Philipp T. Kühn,b Damla Keskin,b Marc C. A. Stuart,d Sebastian Schöttner,e Martinus de Kanter,f Michael Noyong,g Tobias Caumanns,h Joachim Mayer,h Christoph Janzen,i Ulrich Simon,g Markus Gallei,e Dominik Wöll,a Patrick van Rijnb and Felix A. Plampera,j*

a Institute of Physical Chemistry, RWTH Aachen University, Landoltweg 2, 52056 Aachen, Germany b Department of Biomedical Engineering-FB40, University of Groningen, University Medical Center Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands c DWI - Leibniz Institute for Interactive Materials, RWTH Aachen University, Forckenbeckstraße 50, 52056 Aa-chen, Germany d Groningen Biomolecular Sciences and Biotechnology Institute and Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands e Ernst-Berl-Institute for Chemical Engineering and Macromolecular Chemistry, Technische Universität Darm-stadt, Alarich-Weiss-Straße 4, D-64287 Darmstadt, Germany f Chair for Laser Technology LLT, RWTH Aachen University, Steinbachstr. 15, 52074 Aachen, Germany g Institute of Inorganic Chemistry and JARA-SOFT, RWTH Aachen University, Landoltweg 1, 52056 Aachen, Germany h GFE Central Facility for Electron Microscopy, RWTH Aachen University, Ahornstraße 55, D-52074 Aachen, Germany

i Fraunhofer Institute for Laser Technology (ILT), Steinbachstr. 15, 52074 Aachen, Germany j Institute of Physical Chemistry, Freiberg University of Mining and Technology, Leipziger Straße 29, 09599 Freiberg, Germany

EXPERIMENTAL SECTION

REAGENTS AND CHEMICALS . Iron(III) choride hexahydrate (98%, FeCl3 . 6H2O) and L(+) ascorbic acid (99%) were pur-

chased from Sigma-Aldrich. Pluronics F68 was obtained from Applichem, Darmstadt, Germany.

SMALL SCALE SYNTHESIS – TUNING THE MICROGEL STRUCTURE . 0.855 g NIPAM (7.56 mmol), 0.069 g BIS (0.45 mmol),

0.0025 g CTAB (12.5 % of the cmc = 0.92 mM) and 0.034 g V50 (0.13 mmol) were dissolved in 60 mL bidestilled water. 10

mL of this solution was added to a 20 mL vial equipped with a septum and degassed with nitrogen over 45 minutes. 0.432 g of

CD were dissolved in 15 mL bidestilled water to solubilize 0.099 g (0.47 mmol) of the hydrophobic VFc. The solution was

degassed with nitrogen over 45 minutes. 2 mL of this solution was added to the 10 mL of the NIPAM solution. The solution

was heated up to 70 °C (oil bath temperature). 15 minutes after turbidity was observed the reaction was stopped by exposing

the solution to air and letting it cool down to room temperature. The microgel dispersion was purified by ultracentrifugation (3

times at 30.000 rpm) and redispersed in water.

CHEMICAL OXIDATION OF P(NIPAM- CO-VFC). 100 mg of P(NIPAM-co-VFc) microgel (µG) were dissolved in 10 mL of

H2O and pH=4 was adjusted with 0.1 M HCl. After addition of 27 mg FeCl3 . 6H2O (molar ratio 1:3) the reaction mixture was

stirred for 1 h at room temperature.1 Thereby the color changed from yellow to green. Subsequently the mixture was either

dialyzed against acidic water, or centrifuged 2x at 40.000 rpm and freeze dried for further use.

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2018

CHEMICAL REDUCTION OF P(NIPAM- CO-VFC+). 45 mg of P(NIPAM-co-VFc) µG (in its oxidized Fc+ state) were dissolved

in 5 mL of H2O 24 mg of L(+) ascorbic acid (molar ratio 1:3) were added. The reaction mixture was stirred for 1 h at room

temperature, the color changed from green to yellow within 10 min. The mixture was centrifuged 2x at 40.000 rpm and freeze

dried for further use.

ELEMENTARY ANALYSIS was performed by Mikroanalytisches Labor Kolbe, Mülheim, Germany.

CYCLIC VOLTAMMETRY . Voltammetry experiments of an aqueous solution of P(NIPAM-co-VFc) (9.5 g / L; 1 wt%; yielding

a Fc concentration of 2.1 mM) containing 0.01 M KCl were performed. Cyclic voltammogramms were recorded by sweeping

the potential in the range of 0 V - 0.5 V vs. Ag/AgCl at a scan rate of 5 mV s-1.

Voltammetry experiments in organic media of P(NIPAM-co-VFc) µG (3 g/L; 0.3 wt%; yielding a Fc concentration of 1 mM)

in 0.1 M tetrabutylammonium perchlorate TBAP in CH3CN were conducted by sweeping the potential between 0.4 V- (-0.3) V.

Also measurements in DMSO were conducted in a potential range of 0.12 V - (-0.4V), with microgel concentration of 2 g/L

(0.2 wt%; yielding a Fc concentration of 0.7 mM) in 0.1 M TBAP in DMSO. An Ag/AgNO3 reference electrode stored in 1 M

TBAP in CH3CN served as non-aqueous reference electrode in organic media.

HYDRODYNAMIC VOLTAMMETRY . Hydrodynamic voltammograms were recorded of 0.13 mM [Fe(CN)6]3- in 0.1 M KCl as

reference system using a platinum rotating disk electrode (rotation at 100 rpm). Further samples containing a solution of 0.13

mM [Fe(CN)6]3- in presence of 1 g/L (0.1 wt%) P(NIPAM-co-VFc) microgel in its neutral reduced (pristine) Fc state and in its

positively charged, oxidized Fc+ state were prepared. Thereby an initial charge ratio of icr = 1.2 between negatively charged

counterion and positively charged microgel was adjusted, assuming full chemical oxidation with FeCl3. After centrifugation at

40°C and 40.000 rpm the supernatant was analyzed. The potential was ranged between 0.5 V and 0 V vs. Ag/AgCl at a constant

scan rate of 5 mV s-1 and rotation rate of 100 rpm.

SPECTROELECTROCHEMISTRY . Where stated, some spectroelectrochemical experiments were performed with a SEC2000-

UV/VIS spectrometer from ALS (this setup includes a thermostatable cuvette holder qpod from Quantum Northwest, USA;

excitation at 470 nm was achieved with a LLS-470 High-Power LED Light Source from Ocean Optics, connected by fiber

optics to the cuvette holder and eventually to the spectrometer; 90° optics: optical path length for excitation: 0.5 mm; optical

path length for fluorescence light, which is partly obstructed by the electrode: ~ 4 mm). As cuvette, a SEC-C05 Thin Layer

Quartz Glass Spectroelectrochemical Cell Kit with Pt grid electrodes from ALS, Japan, was employed. The Pt grid working

electrode was slightly bent to allow maximum passage of fluorescence light from the vicinity of the electrode to the detector.

An Ag/AgCl electrode (stored in 1 M KCl) served as aqueous reference electrode and a Pt wire as counter electrode, all

connected to the potentiostat (held either at 0.5 V or 0.0 V against Ag/AgCl; Metrohm Autolab PGSTAT128N, Filderstadt,

Germany). Fluorescence spectra were subtracted with background signal obtained at 20°C (reduced state, where hydrophobic

4HP fluorescence dye is hardly entrapped; the weak fluorescence band of 4HP at 620 nm in the hydrophilic state was neglected;

during redox action, 10 spectra - each measured for 6 min - were averaged and smoothed, i.e. the fluorescence data of one hour

is represented in each curve).

DETERMINATION OF FLUORESCENCE QUANTUM YIELD . Absorption spectra of 4HP in aqueous 0.1 M KCl solution and in n-

propanol, respectively, were measured with a Jasco V-550 UV/VIS spectrophotometer. Fluorescence spectra of the same

solutions were recorded on a Jasco FP-8200 spectrofluorometer. The corresponding spectra are presented in Fig. S21. The

fluorescence quantum yield φfl,aq of 4HP in 0.1 M aqueous KCl solution was determined using the following equation

with the known fluorescence quantum yield ffl,nPr of 4HP in n-propanol of 0.57,2 the absorbances (A) of 0.0198 and 0.0027 of

4HP in the n-propanol and aqueous medium used for measurements, the refractive indices (n) of 1.379 and 1.333 for n-propanol

and water, and the relative fluorescence intensities F at 602 nm. The resulting fluorescence quantum yield for 4HP in 0.1 M

aqueous KCl solution is 1.4%.

1H-NMR SPECTROSCOPY. Spectra were measured with a 400-MHz Bruker DRX 400 NMR spectrometer at room

temperature. For the kinetic study, 2 mL samples were taken from the polymerization mixture before and after polymerization

initiation (within the first hour in 1-10 min intervals, every 20 min during the second hour of polymerization and in 30 min

intervals during the third hour). Polymerization was terminated by cooling of the taken samples, which were freeze dried for

further use. DMSO-d6 was used as solvent and polymer concentration was 4 mg.mL-1. The chemical shifts are presented in

parts per million (ppm) downfield from the TMS standard. The proton signal of residual DMSO-d6 was used as reference. The

conversion was calculated by use of methyl-β-cyclodextrin (CD) as internal standard.

UV-VIS SPECTROSCOPY. Spectra were recorded from a stock solution containing 0.2 g/L (0.02 wt%) 0.055 mM PSS in the

range of λ 500 nm-200 nm using a QS high precision cell, made of quartz SUPRASIL (10 mm, Hellma Analytics), in a PG

Instruments T92+ UV Spectrophotometer. Further 3 g/L (0.3 wt%) of the reduced (pristine) P(NIPAM-co-VFc) and the

chemically oxidized microgel (icr = 1) were dissolved in the stock solution and centrifuged at 40°C at 40.000 rpm. The

supernatant was analyzed by UV-VIS spectroscopy.

ANALYSIS OF SUPERNATANT . The content of fluorescence probe 4HP in supernatant could be followed by the fluorescence

intensity, giving indication of the 4HP content in the sedimented microgels. For this reason, the dispersions of a 1 mL sample

containing a 3 g/L CS1 dispersion (in 0.1 M KCl and 5.10-7 M 4HP) were kept at 34°C (equilibrated overnight). After decanting,

the solvent of the supernatant was exchanged to n-propanol (due to the high quantum yield) by freeze drying and by dissolving

the lyophilisate in an aliquot of n-propanol. Then, the remaining fluorescence in the supernatant was evaluated (with help of a

FP-8200 spectrofluorometer; excitation at 470 nm; actual fluorescence measurements conducted at room temperature).

SCANNING TRANSMISSION ELECTRON M ICROSCOPY (STEM) experiments (Figure S24, S25) were carried out using a JEOL

JEM-2100F microscope (JEOL, Tokyo, Japan) equipped with a field emission gun operating at a nominal acceleration voltage

of 200 kV. The samples were investigated using a JEOL single tilt holder. The Energy Dispersive X-Ray Spectroscopy EDX

data were collected with an Oxford X-Max 80 TEM Si-drift detector (Oxford Instruments GmbH, Wiesbaden, Germany).

STEM experiments (Figure S5) were performed on a Carl Zeiss Libra 200FE microscope operating at a voltage of 200 kV.

Approx. 1 µL of the microgel dispersion were added onto a carbon coated copper grid that was pretreated in Argon plasma.

After drying measurements were performed using a high-angle annular dark field detector. EDX line scans were performed

using an EDS-detector from Bruker.

ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY (EIS) measurements were performed on the CH Instruments Electrochemical

Workstation Potentiostat CHI760D (Austin, Texas, USA) Thereby, the experiments were carried out temperature-dependently

from 20-50°C in a conventional three-electrode setup in a water jacketed cell connected to a thermostat (thermo scientific

Haake A28) of a 10 mM Fc of P(NIPAM-co-VFc) microgel as redox probe in a supporting electrolyte solution of 0.1 M KCl.

A platinum gauze electrode served as counter electrode, while the platinum rotating disk electrode, 4 mm disk diameter

(without rotation) was connected as working electrode. An Ag/AgCl electrode stored in 1 M KCl served as reference

electrode in all three cases. All potentials in the text and figures are referenced to the Ag/AgCl couple. The (dc) potential

was held at the open circuit potential measured at each temperature, while a small oscillating voltage of 5 mV amplitude was

applied (leading to an alternating current – ac – readout). The measuring frequency f used for EIS measurements ranged from

0.02 Hz to 100 kHz. Impedance data analysis was performed according to proper transfer function derivation and identification

procedures, which involved complex nonlinear last-squares (CNLS) fitting based on the Marquardt-Levenberg algorithm using

the CH Instruments Beta software.

Atomic Force Microscopy. Surface morphology of the microgel coated glass slides was determined with AFM (Dimension

3100 Nanoscope V, Veeco, Plainview, NY, USA) in contact mode using DNP cantilevers (spring constant k=0.06 N/m and

resonant frequency f0=18 kHz) made from silicon nitride. For investigation of single adsorbed microgels a 20 µL of a 0.025

wt% microgel solution were spin coated (60 s at 81 ps) onto a plasma activated silicon wafer.

Quantitative analysis of the mechanical properties was performed on a Catalyst Nanoscoop V instrument (Bruker, Billerica,

MA, USA) using the PeakForce QNM mode of Bruker with a large amplitude in liquid (water). Bruker SCANASYST-FLUID

silicon nitride cantilevers (k=0.7 N/m, f0=120-180 kHz,) with nominal tip (20 nm) were used. The system was calibrated before

each measurement by determining the exact spring constant of the tip for the Young’s modulus assessment. NanoScope®

Analysis software was used for data evaluation.

M ICROGEL COATING . A glass slide (Menzel GmbH, Braunschweig, Germany, 76 × 26 × 1 mm) was rinsed with ethanol (70

%) and water and subsequently dried with pressurized air. Plasma oxidation was performed for 10 min (at 100 mTorr and 0.2

mbar, on Plasma Active Flecto 10 USB). The glass slides were hydrophobized with 4 drops of trichloro(octyl)silane in vacuo

(desiccator) overnight, afterwards sonicated, rinsed three times with water. After drying at room temperature, a microgel

suspension (5 mg/mL, 0.5 wt%) was sprayed onto the hydrophobic glass slide (tilted 45°) until the whole surface is wetted (8-

12 times) to coat the surface. After drying at room temperature the surface was dried overnight in the oven at 50 °C. The slides

with the dried microgel (multi) layer were immersed in water for at least 6 h while the water was replaced three times. The

washing step assures that only microgels that are physically bound to the surface remain attached and create a homogeneous

monolayer.

ASSESSMENT OF BACTERIAL ADHESION . Bacterial adhesion on microgel coated glass surfaces was performed using a custom-

built parallel plate flow chamber by flowing bacterial suspension 3 × 108 mL-1 for 2 h at room temperature at a set shear rate

of 12 s− 1, as stated by a protocol previously described.3, 4 Before starting each experiment, PBS was circulated through the flow

chamber to remove air bubbles. After 2h, the bacteria flow was stopped and switched to PBS buffer solution at the same flow

rate for 30 min in order to remove non-adhering bacteria from the system. Bacterial adhesion was monitored using a microscope

(OlympusBH-2) equipped with a 40x long working distance objective (Olympus ULWD-CD Plan 40 PL). Each live image

(1392 x 1040 pixels with 8-bit resolution) was acquired after summation of 15 consecutive images (time interval 1 s) to increase

the signal to noise ratio and to eradicate moving bacteria from the analysis.

Four images were taken after 2h flow and 30 min PBS wash from different spots of the coated substrate. The total number of

bacteria still adhering to the surface were counted from these images. Differences were considered significant if p < 0.05. All

values given in this paper are the averages of experiments on three separately microgel coated surfaces and were performed

with separately cultured bacteria.

Scheme S1: Schematic illustration of the P(NIPAM-co-VFc) microgel polymerization.

RESULTS

Results of the elementary analysis are in good agreement with the theoretically calculated values adjusted during the synthesis

(Scheme S1 and Table S1). Further, the two batches CS1 and CS2 with the same polymerization parameters demonstrate the

reproducibility as indicated by the similar amount of Fe incorporated into the microgel (Table S1) as well as the final

hydrodynamic radius Rh and the temperature and redox response (Figure S7 and Figure 3, main part). In Figure S1, the 1H-

NMR spectra, which were used for the kinetic study are depicted before polymerization initiation t0 and after 2.5 h t2.5 h

indicating a faster conversion of VFc. The spectrum of the purified final microgel contains hardly any signals of the

cyclodextrin anymore (4.7 – 5.2 ppm). Hence, the repeated washing with excess water helps to strip off the cyclodextrin

moieties during workup (taking the equilibrium constant for the association between ferrocene and β-cyclodextrin, 17 . 10-3

L/mol,5 already at synthesis conditions 10 % of the ferrocene units are without any cyclodextrin cover; in reality, due to steric

influence of the close vinyl group / polymer backbone, the association constant for polymer-bound ferrocene and cyclodextrin

is even less, providing microgels with largely undecorated ferrocene units after workup). The reversible temperature responsive

behavior of the P(NIPAM-co-VFc) microgel is given in Figure S2 (/Figure S11) and the redox response in organic and aqueous

medium in Figure S6. A facile adjustment of the internal structure (a VFc-rich microgel without dominant gradient) is

demonstrated in Figure S3 – S5 (see discussion below). Chemical and electrochemical oxidation of the ferrocene unit Fc reveal

very similar final hydrodynamic radii Rh (Figure S8). The transferred charge during one cycle of electrolysis process of

P(NIPAM-co-VFc) microgel is shown in Figure S9. Thereby 15% of the Fc units are switched electrochemically, and the time

scale of the reduction process is increased probably due to better solubility and swelling of the oxidized Fc+ species, increasing

the average distance between redox-active groups (reducing the charge transfer/electron hopping). However, full

electrochemical Fc oxidation (Figure S10), does not lead to a significant increase in microgel swelling, which can be explained

by the swelling-restriction by the surrounding PNIPAM shell.

8 7 6 5 4 3 2 1 0

Chemical Shift (ppm)

Isopropyl-H +

Ferrocenring-H9

Amide-H

11.61.60

DMSOH2O

1.00

CD

Figure S1: 1H-NMR spectra of P(NIPAM-co-VFc) polymerization before initiation t0 (left), at t2.5 h (right) and after workup (pure microgel CS1; bottom) in DMSO-d6.

Table S1. Elementary analysis of P(NIPAM-co-VFc).

Element P(NIPAM- co-VFc)a P(NIPAM- co-VFc)b

H 9.7 wt% 9.4 wt% N 10.7 wt% 11.1 wt% O 12.3 wt% n.d. Fe 1.7 wt% 1.85 wt%

a theoretically calculated values assuming full conversion, b obtained from elementary analysis exemplary shown for CS2 (Fe content in CS1 1.23wt%)

5 10 15 20 25 30 35 40 45 50 55

200

250

300

350

400

450

Heating Cooling

Rh

[nm

]

T [°C]

Figure S2: Hydrodynamic radius against temperature of P(NIPAM-co-VFc) microgel in H2O showing the reversibility of the temperature responsive behavior. Premature abortion of the microgel synthesis (15 min after turbidity) was seen to alter the resulting microgel structure. Due to the early stop of the reaction only the “core region” of the otherwise synthesized core-shell microgels results. Thus, smaller microgels (~52 nm at room temperature) with a higher VFc content are obtained (Figure S3, Figure S4). DLS measurements show microgels in the size of 52 ± 0.5 nm in hydrodynamic radius showing hardly any temperature-dependent behavior due to the predominance of the VFc. Moreover, STEM(-EDX) measurements show a homogeneous and comparable Fe and C distribution in a line scan across a single microgel (Figure S5).

8 7 6 5 4 3 2 1 0

19.34.64

Chemical Shift [ppm]

1.00

Amide-H

Isopropyl-H +Ferrocenring-H

9

Figure S3: 1H-NMR spectrum of P(NIPAM-co-VFc) microgel – reaction aborted after 15 min.

10 15 20 25 30 35 40 45 50 550

100

200

300

400 Heating Cooling

RH [n

m]

T [°C] Figure S4: Hydrodynamic radius against temperature of P(NIPAM-co-VFc) for synthesis time of 4 hours (black) and of 15

minutes (red) in H2O showing the lack of temperature responsive behavior for the aborted reaction.

Figure S5: High-angle annular dark-Field Scanning Transmission Electron Microscopy (STEM) (left) and energy dispersive

X-ray spectroscopy (EDX) analysis (right, line-scan across a single microgel mapping carbon (green) and iron (red)).

0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4

-15

-10

-5

0

5

10

i [µ

A]

E [V] vs. Ag/AgNO3(org.)

0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4

-1

0

1

i [µ

A]

E [V] vs. Ag/AgNO3(org.)0.5 0.4 0.3 0.2 0.1 0.0

-0.2

0.0

0.2

0.4

i [µ

A]

E [V] vs. Ag/AgCl(aq.)

Figure S6: Cyclic voltammogram of 1 mM Fc as monomer (dark red curve) or incorporated into the microgel P(NIPAM-co-VFc) (red curve; 3 g/L, 0.3 wt%; please note, viscosity has hardly changed upon microgel addition at these low concentrations),6 both in organic medium 0.1 M tetrabutylammonium perchlorate (TBAP) in CH3CN and in aqueous medium 0.1 M KCl; at room temperature and with scan rate v = 5 mV/s.

Analogous the previous derivations, we try to assess the influence of incorporating several ferrocene units into one particle on the CV properties (see scenario 2 in Ref.6). We abbreviate the Randles-Sevcik equation with help of the Stokes-Einstein equation in following way (K is a constant, dependent on scan rate, electrode area, viscosity, which are all basically unchanged; n equals the number of transferable electrons per electroactive species; C is the concentration of the electroactive species and Rh its hydrodynamic radius):

= /

It reads for ferrocene monomer (n=1)

, = ,

When uniting n ferrocene units at constant ferrocene concentration, the concentration of electroactive species will be reduced by 1/n. The hydrodynamic radius will increase. We can assess the increase of hydrodynamic volume VMG of the “agglomerate”

in following way (for monomer, the volume VFc equals , ), taking into account the major NIPAM content of the microgels

(for each Fc unit, we assume – due to synthesis stoichiometry – 25 NIPAM units coincorporated, which provide a 20 times dry volume increase for each ferrocene due to the molar mass and density differences: ferrocene density is 1.5 g/mL; NIPAM 1.1 g/mL):

, = 20 ∙ ∙ = 20 ∙ ∙ 43 ,

Assessing a volume related swelling ratio of approximately 30 from the temperature dependent DLS data and 50 % residual volume occupied by water in the collapse microgels (Figure S4), the swollen volume of the ferrocene microgels can be postulated:

, = 30 ∙ 20 ∙ ∙ 43 ,

This is consistent with , = !30 ∙ 20 ∙ ∙ ," ≈ 13 ∙ %/ ∙ ,

Now we can assess the peak currents, if all the ferrocene units would be addressable at once (at constant Fc concentration):

, = / ,

≈ /&%

!13 ∙ %/ ∙ ,≈ ,

%/4

One can assess the order of magnitude of n for our microgels by again taking the collapsed radius and assuming 50 % volume occupied by residual entrapped water, which is in the order of millions (by taking 1 nm3 for VFc comparing it to 1/20th of the dry microgel volume). Hence, if all ferrocene units would be electrochemically addressable at once, the peak current would even increase when going from monomer to microgel (at constant ferrocene concentration). However, the opposite is observed, indicating the hampered electron hopping into the microgel.

10 20 30 40 50

200

250

300

350

400

450

500

T [°C]

Rh

[nm

]

CS 2 unswitched CS 1 unswitched

5 10 15 20 25 30 35 40 45 50 55150

200

250

300

350

400

450

500Fc+ P(NIPAM-co-VF)Fc P(NIPAM-co-VF)

Rh

[nm

]

T [°C]

Figure S7: Hydrodynamic radius against temperature of pristine P(NIPAM-co-VFc), CS1 and CS2 assign different batches, but the same polymerization parameters (left) and of the oxidized state Fc+ and the reduced (pristine) state Fc of P(NIPAM-co-VFc) microgel in H2O (right).

10 20 30 40 50200

250

300

350

400

450

500 el chem ox chem ox with FeCl

3

Rh

[nm

]

T [°C] Figure S8: Hydrodynamic radius against temperature of the fully electrochemically oxidized (black) and chemically oxidized (FeCl3) state Fc+ (green) of P(NIPAM-co-VFc) microgel in H2O.

0 50 100 150 200-350

-300

-250

-200

-150

-100

-50

0

t [min]

Oxidation

Cha

rge

[mC

]

0 2000 4000 6000 8000 10000

-4.8

-4.6

-4.4

-4.2

-4.0

log(

-I/[A

])

t [s]

Figure S9: Transferred charge during oxidation process at 0.5 V (left) and reduction at 0.1 V (right) of an initial solution containing 2.1 mM Fc, (9.5 g / L; ~1 wt%) of P(NIPAM-co-VFc) microgel (1.23 wt% of Fe, CS1) at 23 °C, indicating faster oxidation than reduction; 15% of the Fc oxidized; center: first order kinetic plot of the oxidation process, demonstrating non-linear behavior and therefore different kinetic regimes.

-2 0 2 4 6 8 10 12 14 16 18

0

50

100

150

200

250

300

350

Reduction

Cha

rge

[mC

]

t [h]

0 -400 -800 -2000240

260

280

300

320

340

Oxidation

Rh

[nm

]

Q [mC]0 1 2 3 4 5 6 7

240

260

280

300

320

340

Oxidation

Rh

[nm

]

t [d] Figure S10: Hydrodynamic radius at 34 °C against the transferred charge (left) and against time (right) during stepwise oxidation of an initial solution of 1 mM Fc 0.3 wt%, 45 mg/15 mL of P(NIPAM-co-VFc) in 0.1 M KCl; applied potential: 0.5 V at 30 °C.

0 5 10 15 20 25 30 35

650

700

750

800

850

900

950

Particle Diameter

Par

ticle

Siz

e (n

m)

Measurement (a.u.)

100 1000 10000-5

0

5

10

15

20

25

Mean distributionM

ittel

wer

t (%

)

Particle Size (nm)

Figure S11: Hydrodynamic diameter and particle size distribution obtained by backscattering probe for measurement of a 3 g/L solution of reduced (pristine) P(NIPAM-co-VFc), Fc state, at 23 °C.

UPTAKE OF GUEST MOLECULES

After structural investigation and insight into redox properties of the microgels, we are interested in changes in polarity of these

constructs for targeted uptake/release. In order to demonstrate the change in polarity of the microgel upon switching, uptake

experiments were performed with negatively charged, low molecular weight counterions and linear polyelectrolytes, namely

poly(styrene sulfonate) PSS (Mw=3610 Da, Figure S12).

UPTAKE OF HEXACYANOFERRATES

Hereby, the uptake and release of the components was studied by analysis of the supernatant after centrifugation by

hydrodynamic voltammetry in case of redox responsive ferricyanide counterions and UV-VIS spectroscopy in case of PSS

polyelectrolyte. Uptake occurs due to electrostatic attraction of the guest molecules to the microgel in its oxidized Fc+ state.

The uptake of the ferricyanide counterions can be verified by the diminished limiting current arising from the redox responsive

counterions remained in the supernatant after centrifugation (Figure S12). Thereby the decrease in limiting current iL can be

directly correlated to a decrease in counterion concentration, according to the Levich equation.7

0,5 0,4 0,3 0,2 0,1 0,0-0,5

0,0

0,5

1,0

1,5

2,0

2,5

3,0 [Fe(CN)6]3-

SN of [Fe(CN)6]3- + Fc µG

SN of [Fe(CN)6]3- + Fc+ µG

i [µ

A]

E [V vs. Ag/AgCl]250 300 350 400 450

0,0

0,1

0,2

0,3

0,4

Abs

orba

nce

[a.u

.]

λ [nm]

PSS SN of PSS / Fc µG

SN of PSS / Fc+ µG

Figure S12: Demonstration of uptake of multivalent anions: Hydrodynamic voltammogramms of 0.13 mM [Fe(CN)6]3- in 0.1 M KCl at Pt RDE at a rotation rate of 100 rpm (black, left) and the supernatant of an initial dispersion containing 1 g/L (0.1 wt%) of P(NIPAM-co-VFc) in the reduced (pristine) Fc state (orange) and the oxidized state Fc+ (full chemical oxidation with FeCl3, icr=1.2, green) and demonstration of uptake of linear polyelectrolte (right): UV-VIS spectra of PSS reference solution 0.2 g/L (0.02 wt%, 0.055 mM PSS, black, right) and the supernatant of an initial dispersion containing 3 g/L (0.3 wt%) of P(NIPAM-co-VFc) in the reduced (pristine) Fc state (orange) and the oxidized state Fc+ (chemically oxidized with FeCl3, icr=1, green); dispersions centrifuged at 40 °C and 40.000 rpm.

UPTAKE OF LINEAR POLYELECTROLYTE (PSS)

The uptake of PSS18 into the positively charged microgel was verified by the decreasing PSS absorption band at λ= 262 nm in

case of Fc+ containing dispersion after centrifugation. Additionally, the hydrodynamic radius of microgel in the oxidized state

decreases remarkably in presence of the linear polyelectrolyte PSS, based on entropically favored interpolyelectrolyte-

complexation (IPEC formation) and a concomitant release of monovalent counterions (Figure S12).8, 9 Thereby the volume

phase transition of the microgel-based interpolyelectrolyte complex is strongly decreased (26 °C) in comparison with the pure

positively charged microgel (35 °C; Figure S13).

5 10 15 20 25 30 35 40 45 50

200

300

400

500 PSS / Fc P(NIPAM-co-VF) PSS / Fc+ P(NIPAM-co-VF) el. chem.

Rh

[nm

]

T [°C]

Figure S13: Hydrodynamic radius against temperature of interpolyelectrolyte complexes (IPEC) in reduced (pristine) P(NIPAM-co-VFc), Fc state (black), and after electrochemical oxidation (E=0.5V) in presence of linear PSS, Fc+ state (blue) of the P(NIPAM-co-VFc) µG, icr=1.

UPTAKE OF NONPOLAR SUBSTANCES

550 600 650 700

0

20

40

60

80

Flu

ores

cenc

e In

tens

ity [a

.u.]

Wavelength [nm]

Temperature Rise (from 20 °C to 57 °C)

Figure S14: Change in 4HP fluorescence intensity upon heating the sample in the spectroelectrochemical setup (SEC2000-UV/VIS Spectrometer, ALS, Japan, including a thermostatable cuvette holder qpod from Quantum Northwest, USA; without electrochemical action; 1 mL of 3 g/L dispersion of pristine P(NIPAM-co-VFc) CS1 in 0.1 M KCl and 1.10-5 M 4HP; acquisition time for each curve 1 min; heating rate 1K/min; all spectra subtracted with background signal at 20 °C)

The uptake of the hydrophobic fluorescence dye 4HP is shown in Figure S14. With increasing temperature, the microgels

collapse and expel water, thus allowing the uptake of hydrophobic substances. The fluorescence band of 4HP with its maximum

at around 600 nm appears due to the incorporation of the dye into the hydrophobic domains of the microgels (Figure S17). This

increase can be mainly attributed to the uptake of dyes. In line with these observations, a significant decrease in the fluorescence

of the separated supernatant was observed in presence of microgel, mainly in its reduced (hydrophobic) state (Figure S15;

indicating an uptake of up to 75% of dye).

550 600 6500

50

100

150

Cor

r. F

luor

esce

nce

Inte

nsity

[a.u

.]

Wavelength λ [nm]

Figure S15: Demonstration of uptake of fluorescence probe 4HP into microgels by analyzing supernatant equilibrated over night at 34°C: 4HP solution without microgels (black), supernatant of suspensions of reduced pristine microgels (blue) and chemically oxidized microgels (red) of a ~ 1 mL sample containing a 3 g/L P(NIPAM-co-VFc) CS1 dispersion (after sedimentation of microgel; in 0.1 M KCl and 5.10-7 M 4HP; a volume was freeze-dried and redispersed in the same volume of n-propanol prior fluorescence analysis of supernatant; most likely, peak at 540 nm can be assigned as a Raman signal)

Further, the nonreactivity of 4HP under the electrochemical conditions was demonstrated again for 1.10-5 M 4HP solution

containing 10 g/L Pluronics F68, being treated under the same voltages as done for the microgel samples. Neither applying 0

V nor applying 0.5 V for several hours each did change the fluorescence signal. Hence, the changes in fluorescence response

of 4HP do not originate from any redox reaction of 4HP, but are directly linked to the uptake and release of the dye (Figure

S16).

Figure S16: Change of 4HP fluorescence upon electrochemical oxidation at 0.5 V (left), reduction at 0 V (center; all against Ag/AgCl) and the subsequent repetitive switching (right) of 1 mL of a 3 g/L CS1 dispersion (in 0.1 M KCl and 1.10-5 M 4HP; each colored curve represents 1 h; all measured at 34°C and subtracted with background signal at 20°C; excitation wavelength: 470 nm; measured with SEC2000-UV/VIS Spectrometer, ALS, Japan, including a thermostatable cuvette holder qpod from Quantum Northwest, USA).

300 350 400 450 500 550 6000.000

0.005

0.010

0.015

0.020

Abs

orba

nce

Wavelength λ [nm]

4HP in 0.1M KCl 4HP in n-propanol

500 550 600 650

10

100

1000

10000F

luor

esce

nce

Inte

nsity

[a.u

.]

Wavelength λ [nm]

4HP in 0.1M KCl 4HP in n-propanol

Figure S17: UV-Vis spectra (left) and fluorescence spectra of 5 . 10-7 M 4HP (right) in different solvents for determination of the fluorescence quantum yield in in aqueous 0.1 M KCl solution.

Staphylococcal Killing by Triclosan-Loaded Microgels and Released Drug upon Oxidation in Planktonic Mode of Growth (Figure S18)

Figure S18: Representative image of the oxidation agent FeCl3 control on a TSB agar plate for determination of the MBC (Minimal Bactericidal Concentration) of S. aureus ATCC12600. FeCl3 control shows no bactericidal killing at the used concentration range for chemical oxidation of the P(NIPAM-co-VFc) micogel, (section 1) =1.1 mg/mL.

Influence of Microgel Stiffness on Bacterial Adhesion on Microgel Coated Substrates.

Microgel coated surfaces show anti-fouling behaviour for mammalian cells and prevent protein adsorption.10, 11 In order to

investigate the bacterial adhesion on a microgel coated surface the non-motile model microbe S. Aureus ATCC 12600 was

used. Thereby the mechanical properties can be varied by switching between two oxidation states of the P(NIPAM-co-VFc)

microgel. Here we investigate the influence of stiffness on the bacterial adhesion. Atomic force microscopy images of the

coatings at two different oxidation states are shown in Figure S19, 1a) and b). The surface absorbed microgel turns from a

compact and stiff P(NIPAM-co-VFc) structure to a more extended, swollen and more flexible structure upon oxidation to

P(NIPAM-co-VFc+), see Figure S19, 1c). Quantitative analysis of the mechanical properties was performed by atomic force

microscopy in the force mapping mode. Thereby the unswitched state of the microgel, shows with a Young´s Modulus of

400 kPa a significantly stiffer particle center compared to the outer layer and is in line with the core shell structure as indicated

by the TEM images. Upon oxidation and swelling this particle turns with a Youngs Modulus of 50 kPa into a significantly

softer gel (see Figure S19 2c). The bacterial adhesion was evaluated on both types of surfaces and on an uncoated glass substrate

as control experiment. Although the microgel coatings shows remarkable anti-fouling performance compared to bare glass, the

difference in stiffness seems not to be sufficient to alter the fouling behavior (see Figure S19, 3c). This could be connected to

the more positively charged state upon oxidation, which induces some additional attraction to the bacteria. An increase in anti-

fouling performance was previously reported for softer gels, with detaching L929 mouse fibroblasts upon temperature induced

swelling.10

Figure S19: Atomic force microscopy images of P(NIPAM-co-VFc) microgel coated glass substrates before (1a) and after oxidation (1b) in dry state and corresponding height profile (1c) in wet state, changes in Young´s modulus upon oxidation (2c) and optical micrographs illustrating the bacterial adhesion of S. Aureus ATCC 12600 on P(NIPAM-co-VFc) and P(NIPAM-co-VFc+) coated glass substrates and the corresponding number of adhering bacteria after 2h in comparison to a uncoated glass substrate, *** assigns p=0.0001.

CYCLIC VOLTAMMETRY

The electrochemical accessibility of the redox-active moiety located in the microgel strongly depends on the solvent quality,

not only for the ferrocene unit, but also for the surrounding NIPAM shell. In aqueous media, the low solubility of ferrocene

and in addition the collapsed NIPAM shell causes an encapsulation and insulation of the ferrocence units at temperatures above

the VPTT (Figure S20). Consequently, the redox active groups in microgel core are not addressable. In contrast ferrocene units

in acetonitrile are better soluble and are addressable even at high temperatures (Figure S21). However, a non-linear behaviour

(linear dependence shown for the VFc monomer in CH3CN in Figure S21) of peak currents with increasing temperature

indicates a probable structural change even in organic media. In DMSO, which is considered to be a good solvent for both

ferrocene and PNIPAM over the whole investigated temperature range, the linear increase of peak currents with temperature

can be observed, probably due to changing viscosity and kinetic effects (Figure S22).

0.6 0.4 0.2 0.0

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

20°C 24°C 28°C 32°C 34°C 36°C 40°C 44°C 48°C 50°C

i [µ

A]

E [V] vs. Ag/AgCl

∆ T

Figure S20: 2.1 mM Fc (9.5 g/L; ~ 1wt%) of P(NIPAM-co-VFc) µG (CS1) in 0.01 M KCl at different temperatures, scan rate v = 5 mV/s (left) and extracted peak currents (right).

0.5 0.4 0.3 0.2 0.1 0.0 -0.1 -0.2 -0.3 -0.4

-3

-2

-1

0

1

2

20°C 24°C 30°C 34°C 36°C 40°C 44°C 50°C

i [µA

]

E [V] vs. Ag/AgNO3

∆ T

20 25 30 35 40 45 50-3

-2

-1

0

1

2

3

ipa ipc

i [µ

A]

T [°C]

Figure S21: Cyclic voltammograms of 1mM Fc (3 g/L, 0.3 wt%) of P(NIPAM-co-VFc) µG in 0.1 M TBAP in CH3CN at different temperatures, scan rate v = 5 mV/s (left) and extracted peak currents from cyclic voltammograms (right).

0,2 0,1 0,0 -0,1 -0,2 -0,3 -0,4-2

-1

0

1

2

20°C 24°C 28°C 32°C 36°C 40°C 44°C 48°C 50°C

i [µ

A]

E [V] vs. Ag/AgNO3

∆ Τ

20 25 30 35 40 45 50-1,6

-1,2

-0,8

-0,4

0,0

0,4

0,8

1,2

1,6

ipa ipc

i p[µ

A]

T [°C]

Figure S22: Cyclic voltammograms of 0.7 mM Fc+, P(NIPAM-co-VFc) µG after full electrochemical oxidation (2 g/L, 0.2 wt%) in 0.1 M TBAP in DMSO at different temperatures, scan rate v = 5 mV/s (left) and extracted peak currents (right).

20 25 30 35 40 45 50 55

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

ip ia

ip [m

A]

T [°C]

ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY

Electrochemical impedance spectroscopy can help to assess the temperature dependent electrochemical behavior. Therefore,

we applied oscillations around the open circuit potential using the pristine microgel suspension. The resulting spectra (Nyquist

plots) are shown in Figure S23. We applied one general equivalent circuit for fitting the impedance data (see Figure S23): a

parallel combination of a constant phase element CPEDL,12 which describes the capacitive properties of the diffuse double layer

more realistically, and a charge transfer resistance RCT. This arrangement was supplemented in series with the electrolyte

resistance RElect. This equivalence circuit gives a reasonable description of the impedance data at high temperatures, when the

charge transfer is restricted and hence any diffusional impedance is negligible. At low temperatures, a second constant phase

element was introduced phenomenologically in series to RCT accounting for the diffusional properties. Hereby, we offered more

freedom to the equivalent circuit instead of a classical Warburg-type diffusional impedance (which would give a modified

Randles circuit).13 This freedom includes also open and closed boundary diffusion as one might expect in such complex

colloidal systems.12 Open boundary diffusion might contribute when convection due to heating sets in. Closed boundary

diffusion could describe the scenario in the time scale of microgel impact on the electrode, where the charge transport (by

redox-site exchange/electron hopping) can only occur to a certain distance into the microgel (the maximum of this distance is

limited to the diameter of the actual microgel).

The parameters n and q describing the CPEDL hardly change with temperature (blue symbols in Figure S23). As expected, n is

close to unity. However, q scatters more for CPEDiff , but n is also near unity, which illustrates the dominance of a closed

boundary diffusion. Most prominent is the behavior of RCT. RCT shows a non-monotonic behavior, which starts on lower level,

but decreases close to the VPTT of the microgel. Above the VPTT, RCT increases one order of magnitude, illustrating the

difficulty of any electrochemical addressability in the collapsed/glassy state. This is in line with the previous CV data. The

minimum close to the VPTT can be explained with the enhanced electroaddressability due to the increased proximity of the

redox-active centers in the (thin) shell network in consequence of the collapsing microgel yet without the obstructions of the

more and more glassy network.

0 40000 80000 120000 160000 2000000

-40000

-80000

-120000

-160000

0 40000 80000 120000 160000 2000000

-40000

-80000

-120000

-160000

20°C 24°C 28°C 32°C 36°C 40°C 44°C 48°C 50°C

Z``

(O

hm)

T

T

Z´ (Ohm)

20 25 30 35 40 45 500.0

0.2

0.4

0.6

0.8

1.0

n

T [°C] 20 25 30 35 40 45 50

0.0

5.0x10-6

1.0x10-5

1.5x10-5

q [S

iem

ens

. sec

n ]

T [°C]

20 25 30 35 40 45 50

100000

1000000

RC

T [O

hm]

T [°C]

Figure S23: Temperature-dependent Nyquist plots of P(NIPAM-co-VFc) microgel and extracted parameters from the equivalent circuit shown, including up to two constant phase elements CPE (the red data points refer to the constant phase element referring to diffusion and the blue ones refer to the double layer; the squares refer to the q parameter, while the circles define the dimensionality n of the CPE; the green hexagons assign the charge transfer resistance – the line is a guide to the eye only; the bulk electrolyte resistance is always in the range of 40-100 Ω).

SCANNING TRANSMISSION ELECTRON M ICROSCOPY STEM WITH ENERGY DISPERSIVE X-RAY SPECTROSCOPY EDX ANALYSIS

The ferrocene distribution within a microgel can be assessed by help of electron microscopy using elemental mapping. Hence

Energy Dispersive X-Ray Spectroscopy EDX was used to investigate the structure of pristine microgels (Figure S24) and

oxidized microgels (Figure S25). The pristine microgels indicate a higher iron content close to the center of the microgels,

while the surface of the shell indicates much lower content of iron, which is also seen for the oxidized state. However, the core

shows weaker signals in the oxidized state, which might be due to a partial redistribution of ferrocenium units upon swelling

of the formerly collapsed core, which is partly retained even after drying.

Figure S24: Dark-Field Scanning Transmission Electron Microscopy (STEM) of pristine P(NIPAM-co-VFc) microgel CS1 with energy dispersive X-ray spectroscopy (EDX) analysis

Figure S25: Dark-Field Scanning Transmission Electron Microscopy (STEM) of chemically oxidized P(NIPAM-co-VFc) microgel CS1 with energy dispersive X-ray spectroscopy (EDX) analysis

- Shell

- Shell

Figure S26: Cryo-TEM images of the electrochemically reduced state Fc after chemical oxidation (dark green; CS1) of P(NIPAM-co-VFc) microgel in H2O vitrified from a dispersion at 34 °C; enlargement of marked microgel including a comparison of the radii from Figure 5 (main part).

0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

******* **

**

Control0 wt%

0.0001 wt%0.001 wt%0.01 wt%

Nor

mal

ized

Abs

orba

nce

Incubation Condition

Control Fc µG Fc+ µG

0.1 wt%

#*

Figure S27: Normalized absorbance (cell viability of L929 mouse fibroblasts) at varying concentrations of P(NIPAM-co-VFc) µG, reduced (pristine) Fc and electrochemically oxidized Fc+ state at 37 °C after 5 d, * assigns statistical significant difference p<0.05 and ** p<0.0005. # assigns the control group.

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