Supporting Information

Specific drug delivery to cancer cells with double-imprinted

nanoparticles against EGFR membrane receptor

Francesco Canfarotta #†, Larissa Lezina#‡⊥, António Guerreiro †, Joanna Czulak† Alexey

Petukhov ⊥˚, Alexandra Daks ⊥, Katarzyna Smolinska-Kempisty ∥, Alessandro Poma §, Sergey

Piletsky*∥, Nickolai A. Barlev *⊥@

#
These authors contributed equally to this work.
†
MIP Diagnostics Ltd., Fielding Johnson Building, University of Leicester, LE1 7RH, UK.
‡
Department of Cancer Studies, University of Leicester, LE1 7RH. ∥ Department of Chemistry,

University of Leicester, LE1 7RH, UK. ⊥ Laboratory of Gene Expression and Regulation,

Institute of Cytology, 194064 Saint-Petersburg, Russia, Almazov National Medical Research

Centre, 197341, St Petersburg, Russia,. § Chemistry Department, University College London,

WC1H 0AJ, UK, ˚Institute of Hematology, @Moscow Institute of Physics and Technology,

Dolgoprudny, Moscow Region 141700, Russia.

Corresponding Authors

*To whom correspondence should be addressed, email: nick.a.barlev@gmail.com and

sp523@le.ac.uk

S1
Methods

Preparation of EGFR epitope-modified glass beads (solid-phase)

Glass beads (Spheriglass® 2429 CP00, 53-106 µm diameter, from Blagden Chemicals) were first

activated by boiling in a 2 M NaOH for 15 min, washed with deionized water (until a pH of 7.5

was measured on the wash solution) then rinsed with acetone, dried at 80 °C and subsequently

incubated overnight in 2 % v/v 3-aminopropyltriethyloxysilane (Sigma Aldrich) in dry toluene

solution (0.4 mL solution/g glass beads). Then 60 g of glass beads were placed in a solution of

succinimidyl iodoacetate (Thermo Fisher), at 0.2 mg/mL in acetonitrile for 2 h in the dark (0.4

mL solution/g glass beads). Afterwards, the beads were washed with 400 mL of acetonitrile in a

sintered glass funnel and incubated with 7 mg of cysteine-modified peptide epitope (primary

template) in 40 mL of deoxygenated 1× phosphate buffered saline (PBS) containing 5 mM

EDTA, pH 8.2. For preparation of biotin-derivatized glass beads (for synthesis of biotin-

imprinted control nanoMIP), 60 g of amine-derivatized glass beads were incubated with a

solution of biotin (Sigma Aldrich) containing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride (EDC) and N-hydroxysuccinimide (NHS) (both from Sigma Aldrich) at 0.5, 10

and 15 mg/mL respectively, in 1× PBS, pH 7.4. After overnight incubation, the beads were

washed with 500 mL of deionized water in a sintered glass funnel and used for the synthesis of

nanoMIPs.

Synthesis of fluorescent and doxorubicin-loaded nanoMIPs

The procedure has been adapted from Poma et al. and Canfarotta et al.1, 2
The following

monomers (from Sigma Aldrich) were dissolved in PBS 5 mM, pH 7.4 (100 mL): N-

isopropylacrylamide (39 mg), N,N'-methylenebisacrylamide (2 mg), N-tert-butylacrylamide (33

mg), N-(3-aminopropyl)methacrylamide hydrochloride (5.8 mg), acrylic acid (2.2 µL) and N-

S2
fluoresceinylacrylamide (2.6 mg). N-tert-butylacrylamide was previously dissolved in ethanol (1

mL) and then added to the aqueous solution. The fluorescent monomer N-

fluoresceinylacrylamide was prepared according to Tan et al.3 When producing EGFR-

nanoMIPs double imprinted against doxorubicin, 2.9 mg of doxorubicin HCl (Sigma Aldrich)

was added to the polymerization mixture as described above. For synthesis of control nanoMIPs

(non-EGFR-imprinted), biotin-derivatized glass beads were used, and all remaining conditions

were kept identical.

The solution containing the monomers was degassed under vacuum and sonication for 5 min,

and then purged with N2 for 20 min. Then, 60 g of EGFR-derivatized glass beads added to the

solution. Polymerization was initiated by adding ammonium persulfate aqueous solution (800

µL, 60 mg/mL) and N,N,N',N'-tetramethylethylenediamine (24 µL), both from Sigma Aldrich.

The headspace was flushed with N2 and the bottle sealed with a screw cap. Polymerization was

carried-out at room temperature for 1 h. Subsequently, the content of the polymerization vessel

was poured into a solid-phase extraction (SPE) cartridge (60 mL) equipped with a frit (20 µm

porosity). A total of 9 washes with 20 mL of distilled water at 20 ºC were carried out to remove

low affinity nanoMIPs, polymer and unreacted monomer. Afterwards, the SPE cartridge

containing the solid-phase was placed in a water bath at 70 °C for 15 min. An aliquot of 20 mL

of distilled water pre-warmed at 65 °C was poured into the SPE to collect the high-affinity

nanoMIPs. This action was repeated 7 times, until about 140 mL of a solution of high-affinity

nanoMIPs in water were collected. To ensure complete removal of all unreacted monomer from

the bulk of the nanoparticles (which might otherwise interfere with the subsequent cell assays)

the collected solution was concentrated down to 20 mL using a centrifugal dialysis cartridge

fitted with a membrane with 30 kDa molecular weight cutoff (Amicon Ultra, Merck Millipore).

S3
This was followed by seven washes (14 mL) with deionized water on the same dialysis cartridge,

after which the NPs were re-suspended in 50 mL deionized water. The whole procedure was

repeated using biotin-derivatized glass beads to prepare control nanoMIPs imprinted against

biotin.

Before using the nanoMIPs on cytotoxicity assays, nanoMIPs (at a concentration of 300

µg/mL) were incubated in a solution of doxorubicin in water (final concentration 50 µM)

overnight. After incubation, the nanoMIPs loaded with doxorubicin were washed in a centrifugal

dialysis cartridge fitted with a membrane with 30 kDa molecular weight cutoff (Amicon Ultra,

Merck Millipore) 9 times (14 mL water used for each wash), until no free doxorubicin could be

detected by fluorescence in the filtrate. Both types of doxorubicin-loaded nanoMIPs (EGFR and

biotin MIPs) at the same concentration (300 µg/mL) exhibited a similar fluorescence intensity

emission at 588 nm (excitation 473 nm), which can be ascribed to an equal amount of

doxorubicin loaded into the nanoMIPs. In the MIP suspension, this was the equivalent to 1.43

µM. Before addition to the cells, the doxorubicin-loaded nanoMIP solution was diluted from 300

µg mL-1 to 20 µg mL-1, equivalent to 95 nM doxorubicin.

Size and concentration analysis of nanoMIPs

Particle size was measured with a Zetasizer Nano (Nano-S) particle-size analyzer from Malvern

Instruments Ltd (UK). An aliquot of the dispersion of nanoMIPs in distilled water was sonicated

for 1 min and then analyzed by DLS at 25 °C in a 3 mL disposable polystyrene cuvette.

Attenuator position, measurement duration and number of runs were automatically chosen by the

instrument. The values are reported as an average of 4 measurements. TEM images of nanoMIPs

were taken using a JEOL JEM 1010, 100 kV high contrast TEM equipped with a Gatan SC1000

Orius CCD camera (Gatan, Abingdon Oxon, UK). Samples for the analysis have been prepared

S4
by depositing a drop of the nanoMIPs dispersion, previously filtered through a 0.45 µm PES

syringe filter, on a carbon-coated TEM copper grid (400 mesh, from E.M. Resolutions Ltd., UK),

and leaving them to dry at room temperature. The nanoMIP mass concentration was assessed by

freeze-drying an aliquot (20 mL) of the particle solution and then weighing the solid.

Fluorescence analysis

Measurements of fluorescence intensity were performed using a Cary Eclipse spectrofluorometer

(Varian Australia Pty Ltd) at 25 °C. Excitation and emission wavelengths were set at 492 nm and

515 nm. The fluorescence measurements were performed using 1 mL quartz cuvettes of 10 mm

path length.

SPR analysis

Analysis was performed on SIA Au SPR gold chips (GE Healthcare, UK) modified with lipoic

acid (Sigma Aldrich). Bare gold chips were first cleaned by hydrogen plasma at 50 W during

five minutes on an Emitech K1050X Plasma Cleaner (Emitech, UK) and then placed in ethanol

containing 0.3 mg/mL lipoic acid and 5 % (v/v) acetic acid (Fisher Scientific), overnight in a

sealed vial. After surface modification, chips were rinsed with ethanol and dried under a stream

of N2, assembled on the holder following the manufacturer instructions and docked onto the SPR

instrument (Biacore 3000, GE Healthcare, UK). For polymer coupling, the chips were activated

by injection of 100 µl EDC 0.2 M and NHS 0.05 M (both from Sigma Aldrich) in water at 10

µl/min, followed by 3-5 injections of nanoMIPs (0.1 mg mL-1) in water at 15 µl/min until 90-100

% surface capacity was reached. For EGFR immobilization, the same activation procedure was

used, then 80µL EGFR (), or BSA for the control channels, was injected (0.2 mg mL-1) in 1×

PBS, pH 7.4 at 15 µl/min. Remaining NHS esters were deactivated by injection of 100 µl of 0.1

M ethanolamine hydrochloride (Sigma Aldrich) at 10 µl min-1. The EGFR peptide was then

S5
injected onto the nanoMIP-modified chip in concentrations ranging from 0.01 to 50 nM, and

analysis was performed in 1× PBS at pH 7.4. For binding assays with the EGFR receptor,

nanoMIPs were injected onto the chip with immobilized protein. Kinetic analysis of the

sensorgrams was performed with the BiaEvaluation software v4.1 assuming a 1:1 Langmuir

binding model. Chi2 values for data reported are below 10 % of the respective instrument

response.

Cell culture conditions

MDA-MB 231, MDA-MB-468 and SKBR-3 cells (obtained from ATCC) were cultured in

DMEM (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO). In SKBR-3 cells

media insulin was added to a final concentration of 1 mg/mL. Cells were incubated in the

presence of 100 U mL-1 penicillin/100 µg mL-1 streptomycin (P/S) in humidified atmosphere of

5% CO2 and 95% air at 37 °C. All cell lines used in this study were checked for mycoplasma

contamination by PCR using specific primers to mycoplasma 16S rRNA.

Binding analysis of fluorescent nanoMIPs to the cells surface by FACS

In the experiments with breast cancer cell lines, cells were collected by gentle scraping, washed

with PBS and resuspended in blocking buffer (1xPBS, 0.5% BSA, 0.1% NaN3) in concentration

1x106 cells in 100 µl in three replicates per experimental condition. Fluorescent nanoMIPs were

briefly sonicated for dispersion and added to the cells suspension to achieve the final

concentration of 10 and 40 µg mL-1. Mixtures were incubated for 2 h in the dark at 4°C.

Following their incubation, the samples were centrifuged, washed and resuspended in blocking

buffer. Binding of fluorescent nanoMIPs to the cells surface were analyzed by BD FACSCalibur

(BD Biosciences) and FlowJo software (from Tree Star).

Peptide Binding Competition assay

S6
For competition experiments, MDA-MB-468 cells were prepared essentially the same way as for

nanoMIPs binding assay. Briefly, 1x106 of cells were resuspended in 100 µl of the blocking

buffer, which contained 10 nM of the same EGFR peptide that was used for synthesis of EGFR-

MIPs to reduce non-specific binding. Fluorescent nanoMIPs (EGFR-nanoMIPs and biotin-

nanoMIPs) were briefly sonicated before adding to the cells suspension to achieve the final

concentration of 20 µg mL-1. Following resuspension in PBS, the EGFR peptide was then added

to the binding mixture to achieve the concentrations indicated in the text. Incubation, washing

and flow cytometry analysis were performed as described above in the “Binding analysis of

fluorescent nanoMIPs to the cells surface by FACS” section.

Cell cycle analysis and MTS tests

Cells were collected by trypsinization, resuspended in RPMI medium in concentration 2×106

cells in 2 mL per experimental condition, and treated with doxorubicin-loaded nanoMIPs, briefly

sonicated prior to the experiment, at final concentration of 20 µg/mL for 2 h at 37°C under

rotation. After incubation, the samples were centrifuged, washed with PBS and plated in RPMI

in three replicas for subsequent staining with propidium iodine (PI) or MTS test. For PI staining,

48 h later all cells, both attached and floating, were collected, washed with PBS, fixed with 70%

ethanol, stained with propidium iodide and analyzed as described previously.31 For MTS Cell

proliferation assay treated cells were plated in 96-well plates at a density of 500 cells per well in

5 replicas. After five days, MTS tetrazolium compound (from Abcam) was added to the cell

culture and the absorbance was recorded at 492 nm using a 96-well plate reader (Tecan Spectra

Mini Microplate Reader, Switzerland).

Confocal microscopy analysis

S7
MIP binding was assessed using two different breast cancer cell lines (MDA-468 and SKBR-3

with high and low EGFR expression, respectively) at two different time points (2 and 24 h). One

day prior the treatment cell suspensions were seeded on gelatin-covered coverslips to achieve 40

– 70% confluency. NanoMIPs were washed with 70% ethanol and twice with PBS before adding

to the cells, using centrifugation cartridges as mentioned before. The following day the medium

was replaced with the nanoparticle suspension (10 µg/mL) in full media and the plates were

incubated for 2 h or 24 h. Following the incubation, the cover slips were washed 4 times with

fresh pre-warmed PBS to eliminate the excess of nanoMIPs that were not bound. Cells were then

fixed with a solution of 4% PFA in PBS for 10 min at room temperature. PFA fixed coverslips

were placed in blocking buffer containing 5% BSA and 0.3% Triton-X100 in PBS for 1 h. The

coverslips were then incubated with primary anti γ-catenin antibodies in blocking buffer in a

moist chamber for 1 h (dilution 1:500). After washing with PBS for 5 times coverslips were

incubated with secondary Alexa-fluor 546 donkey anti-mouse (1 µg/mL) antibodies in blocking

buffer in a moist chamber for 1 hour. After washing with PBS for 5 times coverslips were

mounted with DAPI-antifade mountant. γ-tubulin staining was employed for membrane staining.

Images were taken on a TCS SP5 (Leica) confocal microscope and High Content Imaging

System Operetta CLS (PerkinElmer, UK).

Measurements of intracellular levels of doxorubicin by FACS

Since doxorubicin is a fluorescent anthracycline with excitation wavelength at 480 nm, we

decided to measure the intracellular levels of doxorubicin in MDA-MB-468 and SKBR-3 cells

treated either with free doxorubicin, or as part of EGFR-nanoMIPs. Specifically, MDA-MB-468

and SKBR-3 cells were cultivated as described in previous figure legends. The night before the

experiment cells were seeded into 12-well plates (1x105 cells per well) to ensure exponential

S8
growth. EGFR-nanoMIPs loaded with doxorubicin were added to cells in concentration of 35

µg/ml in PBS solution supplemented with 100nM doxorubicin. After two hours incubation with

nano-MIPs cells were washed with fresh medium and either subjected to FACS analysis at 480

nm using Guava® easyCyte 8 flow cytometer (Merck, Millipore). The data were obtained using

FCS Express 6 (Denovosoftware).

Treatment of MDA-MB-468 cells with MIPs and IF staining of phospho-H2A.X foci

MDA-MB-468 cells were cultured in DMEM supplemented with 10% FBS, L-glutamine and

penicillin/streptomycin on coverslips in 24-well plates. MIP incubation with doxorubicin was

performed during 3 h at room temperature (0,1 µM doxorubicin in a solution of MIPs at 350

µg/ml). Next, MIPs solutions were sonicated to avoid clumps and added to 100 000 cells per well

in the 24-well plate.

After 2 h incubation the media was replaced with fresh full DMEM (except positive control that

included 16 h incubation with 1 µM doxorubicin). 1 h after change of medium the cells were

fixed with 4% PFA for 10 minutes at room temperature. After fixation, the cells were washed

with PBS and incubated with blocking buffer containing 5% BSA and 0.3% Triton X-100 in PBS

for 1 h at room temperature. Then the cells were incubated with a solution of primary anti-

phospho-H2A.X antibodies in blocking buffer (Cell Signalling, 20E3, 1:300) at +4°C overnight.

The next day, the cells were washed with PBS and stained with a solution of secondary Alexa

Fluor 488 conjugated antibodies (Invitrogen, A-11034, 1:1000) for 1 h at room temperature.

After washing with PBS, the preparations were mounted with mounting medium including

antifade and DAPI (Invitrogen). The images were obtained with a confocal microscope TCS SP5

(Leica) as described above.

S9
Analysis of EGFR internalization after MIPs treatment

The MDA-MB-468 cells were incubated with 35 µg/ml MIPs solution, or with 20nM EGF

(Sigma-Aldrich, E9644) solution in full media (used as positive control). In addition, the EGFR

internalization was analyzed after treatment of cells with 35 µg/ml MIPs+20nM EGF mixture.

The treatment was performed during 6 h.

After 6 h of incubation, the cells were fixed with 4% PFA for 10 minutes at room temperature.

After fixation, the cells were washed with PBS and incubated with a blocking buffer containing

5% BSA and 0.3% Triton X-100 in PBS for 1 h at room temperature. Then the cells were

incubated with a solution of primary anti-EGFR antibodies in blocking buffer (Santa Cruz

Biotechnology, 1005, 1:100) at +4°C overnight. The next day, the cells were washed with PBS

and stained with a solution of secondary Alexa Fluor 488 conjugated antibodies (Invitrogen, A-

11034, 1:1000) for 1 h at room temperature. After washing with PBS, the preparations were

mounted with mounting medium including antifade and DAPI (Invitrogen). The images were

obtained with a confocal microscope TCS SP5 (Leica).

Supplementary results

S10
Figure S1. Excitation (---) and emission (―) spectra of N-fluoresceinyl-acrylamide (a) and

EGFR-MIPs prepared with addition of N-fluoresceinyl-acrylamide monomer (b).

Figure S2. DLS analysis performed on biotin nanoMIPs (a) and EGFR nanoMIPs (b) in distilled

water, showing an average hydrodynamic diameter of ca. 223 ± 5 nm (a) and 242 ± 13 nm (b),

with a polydispersity index of 0.255 and 0.203 respectively.

S11
Figure S3. SPR sensorgrams for binding of the EGFR epitope peptide or the extracellular

protein domain (aa 1-641, used as template) in a range of concentrations (from 0.01 to 50 nM) to

immobilized EGFR-nanoMIP (a), to immobilized control nanoMIPs (biotin-nanoMIP) (b). Part

(c) shows the binding response for EGFR-nanoMIP binding to immobilized extracellular EGFR

(aa 1-641), the response was subtracted from a control channel containing immobilized BSA, or

EGFR-nanoMIP injected in a range of concentrations from 0.01 to 1 nM.

S12
Figure S4. Confocal microscopy images of fluorescent nanoMIPs (green) in MDA-468 and

SKBR-3 cells after 2 and 24 h incubation. DAPI was employed to stain the nucleus (blue), and γ-

tubulin (red) for the membrane.

S13
Figure S5. FACS analysis on fluorescent EGFR nanoMIPs in breast cancer cells. (a) Binding of

EGFR nanoMIPs to MDA-MB-231 expressing low levels of EGFR on their surface, as

demonstrated by Western blot (Fig. 2a, main manuscript). (b) Confocal microscopy on MDA-

MB-231 cells, showing the poor binding of EGFR nanoMIPs (green) to the target protein, as

detected by FACS (Fig. 2a, main manuscript). DAPI was used to stain nuclei, gamma-catenin for

the cytoplasm.

Figure S6. Binding competition assay between EGFR-MIPs and free EGFR epitope peptide for

MDA-MB-468 or SKBR-3 cells (left or right panel, respectively), monitored by FACS.

Statistical analysis was done by one-way ANOVA. All treated samples are compared with the

untreated control, ** P < 0.01. Note, that due to a very low binding of EGFR-MIPs to SKBR-3

cells, only relative binding of EGFR-MIPs was calculated. Binding of the MIPs to SKBR-3 cells

in the absence of the corresponding peptide was arbitrary set as 1, and the binding efficacy of

EGFR-MIPs in the presence of 1000 nM of the peptide was calculated as fold-difference

compared to the value obtained for binding at 0 nM of the peptide.

S14
Figure S7. Assessment of the cytotoxicity effects of EGFR and biotin nanoMIPs loaded and

unloaded with doxorubicin in the presence of cells expressing high levels of EGFR (MDA-468)

and with cells expressing low levels of EGFR (SKBR-3). Prior to incubation with cells, the

amount of doxorubicin loaded into both types of nanoMIPs (EGFR and biotin) and added to the

cells was adjusted and assessed to be equal by fluorescence, at 95 nM. The control represents

cells incubated in the absence of nanoMIPs. Statistical analysis was done by one-way ANOVA.

All treated samples are compared with the untreated control, ** P < 0.01. Survival of cells after

treatment with EGFR MIPs was set as 100%, and EGFR (doxo) is the percentage ratio of

survival to EGFR MIPs.

S15
Figure S8. Concentration-dependent effects of EGFR-nanoMIPs loaded with different amounts

of doxorubicin on survival of MDA-MB-468 cells. Cells were incubated with increasing

amounts of EGFR-nanoMIPs loaded or unloaded with the indicated amounts of doxorubicin for

2 hours. Following washes, cells were analyzed by MTS test as described for Figure 3. Lanes 1-

3: Increasing amounts (0, 10, and 30 µg/ml, respectively) of empty EGFR-nanoMIPs were

incubated with MDA-MB-468 cells; lanes 4-6: 30 µg/ml of EGFR-nanoMIPs loaded with 0, 50,

and 100 nM of doxorubicin; lanes 7-9: note, that free doxorubicin (0, 50, and 100 nM,

respectively) affects MDA-MB-468 cells only at the highest concentration. Statistical analysis

was done by two-ways t-test. Samples 5 and 8, and 6 and 9 were compared pairwise, * P< 0,05,

** P < 0.01.

S16
Figure S9. Representative images of doxorubicin absorbance in SKBR-3 (A) and MDA-MB-468

(B) cells. SKBR-3 and MDA-MB-468 cells were treated with 0.1 µM doxorubicin for 2 hours in

the presence or absence of EGFR-nano MIPs. Following washes, cells after incubation with

EGFR-nanoMIPs loaded with doxorubicin were harvested either immediately (black lane), or 24

S17
hours later (red lane). Cells pulsed with free doxorubicin for 2 hours and then chased for 24

hours were used as control (blue lane). The quantification of doxorubicin retained in cells as

described in (A) and (B (lower panel). 1- Cells with EGFR-nanoMIPs loaded with doxorubicin

were harvested immediately after2 hours incubation;, 2- Cells with EGFR-nanoMIPs loaded with

doxorubicin were harvested 24 hours later after the 2 hours incubation; 3- Cells pulsed with free

doxorubicin for 2 hours were then chased for 24 hours. (C) Statistical analysis was performed

using two-ways t-test as shown, * P< 0,05, ** P < 0.01.

S18
Figure S10. EGFR-MIPs with doxorubicin induce the formation of phospho-H2A.X foci.

(A) MDA-MB-468 cells were incubated with unloaded or loaded with doxorubicin (0,1 µM)

EGFR-MIPs or biotin-MIPs. Cells were also incubated with free doxorubicin at 0,1 µM for 2 hrs

or 1 µM for 16 hrs as reference controls. Subsequently, cells were stained with anti-phospho-

H2A.X antibodies (Cell Signalling, 20E3, 1:300) followed by confocal microscopy analysis. (B)

Statistical analysis of the results shown in panel A. Each dot represents at least 50 nuclei in the

view field of microscope. Thus, ~500 different nuclei were scored for ɣ-H2Ax positive staining

for each cell treatment variant. Statistical analysis was performed using two-ways t-test as shown

with asterics, * P< 0,05, ** P < 0.01.

S19
 pERK
0,1 0,6 0,1 2,0 0,11 0,5

Actin

pAKT
1,7 1,5 1,0 0,6 0,9 0,

Actin
1 2 3 4 5 6

Figure S11. Treatment of cells with EGFR nano-MIPs does not cause significant activation

of EGFR signaling. A representative Western blot analysis (the experiment was repeated three

times with essentially similar results) of SKBR-3 (lanes 1, 3, and 5) and MDA-MB-468 cells

(lanes 2, 4, and 6). Both types of cells were treated with 0.1 µM doxorubicin for 2 hours in the

absence (lanes 1, and 2), or presence of EGFR nano-MIPs (lanes 3 and 4), or biotin nano-MIPs

(lanes 5 and 6). Following washes, cells were harvested 24 hours later and analyzed by western

blotting for levels of pERK and pAKT. Actin was used as loading control. Relative strengths of

signal for Erk and Akt were calculated as fold-difference over the actin signal.

S20
Figure S12. Internalization of EGFR upon treatment with EGFR-MIPs with or without

EGF. (A) MDA-MB-468 cells were incubated with MIPs against EGFR or biotin for 6 hrs. After

incubation, cells were fixed and stained with anti-EGFR antibodies. Nuclei were stained with

DAPI. The images were obtained with a confocal microscope TCS SP5 (Leica). (B) Statistical

analysis was performed using two-ways t-test as shown, * P< 0,05, ** P < 0.01.

Supplementary References

S21
(1) Poma, A.; Guerreiro, A.; Whitcombe, M. J.; Piletska, E. V.; Turner, A. P. F.; Piletsky, S. A.,

Solid-Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles with a Reusable

Template – “Plastic Antibodies”. Advanced Functional Materials 2013, 23 (22), 2821-2827.

(2) Canfarotta, F.; Waters, A.; Sadler, R.; McGill, P.; Guerreiro, A.; Papkovsky, D.; Haupt, A.;

Piletsky S., Biocompatibility and internalization of molecularly imprinted nanoparticles. Nano

Research, In Press, doi: DOI 10.1007/s12274‐016‐1222‐7.

(3) Tan, W.; Shi, Z. Y.; Kopelman, R., Development of submicron chemical fiber optic sensors.

Analytical Chemistry 1992, 64 (23), 2985-2990.

(4) Lezina, L.; Aksenova, V.; Fedorova, O.; Malikova, D.; Shuvalov, O.; Antonov, A. V.;

Tentler, D.; Garabadgiu, A. V.; Melino, G.; Barlev, N. A., KMT Set7/9 affects genotoxic stress

response via the Mdm2 axis. Oncotarget 2015, 6 (28), 25843-25855.

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