Supporting Information for:
To: Journal of Nanoparticle Research
An integrated approach for the systematic evaluation of polymeric nanoparticles in healthy
and diseased organisms
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Leopoldo Sitia Dr , Katia Paolella Dr , Michela Romano Dr , Martina Bruna Violatto Dr , Raffaele
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Ferrari PhD2, Stefano Fumagalli PhD1,3, Laura Colombo Dr1, Ezia Bello Dr , Maria-Grazia De
Simoni PhD1, Maurizio D’Incalci MD1, Massimo Morbidelli PhD2-4, Eugenio Erba Dr1, Mario
Salmona PhD1 ,Davide Moscatelli PhD2 and Paolo Bigini PhD1*
* Corresponding author Dr. Paolo Bigini: IRCSS - Mario Negri Institute for Pharmacological
Research, Via La Masa 19, 20156Milan, Italy. Phone number +39 02 39014221; Fax: +39 02
39014744.
E-mail address:paolo.bigini@marionegri.it
1. Materials and Methods
1.1. Materials
Rhodamine B (RhB, Sb sensitivity <0.1 µg/mL, Carlo Erba reagents), dicyclohexylcarbodiimide
(DCC, 99% purity, Sigma-Aldrich), 2-hydroxyethyl methacrylate (HEMA, >99% purity, SigmaAldrich), 4-(dimethylamino)-pyridine (DMAP; Sigma-Aldrich), methyl methacrylate (MMA, 99%
purity, Sigma-Aldrich), potassium persulfate (KPS, >99% purity, ACS reagent), Azobis(2methylpropionamidine) dihydrochloride (>99% purity, Fluka), polyoxyethylenesorbitan monooleate (Tween80, Sigma-Aldrich), acetonitrile (99% purity, Sigma-Aldrich), trifluoroacetic acid
(Sigma-Aldrich), DMSO and DMSO-d6 (both Sigma Aldrich) D12731 1,1′-dioctadecyl-3,3,3′,3′tetramethylindotricarbocyanine iodide (‘DiR′; DiIC18(7)) (Molecular Probes)were all used as
received.
Ham’s F12 (Biowest, Nuaille – France) supplemented with supplemented with 10% FBS and 1% Lglutamine (200 mM) (Biowest). MDA-MB231.1833 cells were maintained in DMEM-High
Glucose (Dulbecco's Modified Eagle Medium High Glucose) supplemented with 10% FBS and 1%
L-glutamine (200 mM) (Biowest).
Both cell lines were maintained at 37°C in a humidified atmosphere at 5% CO2 in T25 or T75 cm2
flasks (IwakyBibbySterilin, Staffordshire, UK). (Fluoromont, Sigma Aldrich).
1.2. Fluorescent macromonomer synthesis
Briefly, RhB and HEMA were dissolved in acetonitrile and loaded together in a beaker, then DCC
and DMAP, both dissolved in acetonitrile, were added dropwise. The reaction was run for 24h at
40°C under magnetic stirring. After that, the reaction medium was quenched in an ice bath, and the
dicyclohexylurea (DCU) from the DCC hydration was removed by filtration. Dried macromonomer
(HEMA-RhB) was obtained after acetonitrile removal by solvent evaporation under vacuum. The
raw product was chromatographically purified through HPLC, The HEMA-RhB macromonomer
was purified on an Agilent 1100 Series HPLC, equipped with an auto-sampler, a diode array
detector, an online degasser and an isocratic pump (buffer: 40% acetonitrile, 0.1 % trifluoroacetic
acid). A Gilson FC 203B fraction collector was connected at the outlet of the HPLC to collect
fractions during elution. A Kromasil 100-10C18 preparative column was used (250x4.6mm).
1.3. Fluorescent macromonomer characterization
ElectroSpray Ionization (ESI)
The purified HEMA-RhB macromonomer was analyzed using a maXis ESI-Q-TOF
(BrukerDaltonics) mass spectrometer (MS) equipped with an automatic syringe pump for sample
injection from KD Scientific. The ESI-Q-TOF mass spectrometer was running at 4500V, with a
desolvation temperature of 200 °C. The mass spectrometer was operating in the positive ion mode.
Nitrogen was used as the nebulizer and drying gas. The standard electrospray ion source was used
to generate the ions. The solvent was chloroform. Sixty shots from each spot were averaged to
obtain one mass spectrum. The ESI-Q-TOF MS instrument was calibrated in the m/z range 50–1300
with the use of an external calibration standard (Tunemix solution) supplied by Agilent.
In order to confirm the structure and the molecular weight of the obtained purified macromonomers,
ESI analyses were performed and the obtained results are reported in Fig. S1.
Fig. S1 ESI spectra performed on the purified macromonomer
Fig. S1-A confirms the structure of the purified HEMA-RhB. The main peak obtained is relative to
a molecular weight equal to 555.28 Da that corresponds to the macromonomer with added a sodium
atom (+23 Da) and in which chlorine coming from the RhB salt is not present (-35 Da).
Additionally, the spectrum shown in Fig. S1b confirms the high purity of the HEMA-RhB
macromonomer, wherein, despite some low molecular weight peak, due to ESI ionization process,
the peaks of pure RhB, HEMA and by-products of the esterification reaction are not present.
Nuclear Magnetic Resonance (NMR) Spectroscopy
The structure of macromonomer has been determined by 1H-NMR using a 500 MHz Ultrashield
NMR spectrometer (Bruker). The sample was dissolved in DMSO-d6.
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H-NMR analysis has been conducted to improve the characterization and to validate the chemical
structure of the purified macromonomer. In Fig. S2a the spectrum of pure RhB in DMSO-d6 is
reported and it can be compared with that of HEMA-RhB macromonomer (Fig. S2b).
(a)
(b)
Fig. S2 H-NMR spectra of: (a) RhB and (b) HEMA-RhB macromonomer
By a close inspection of Fig. S2 it is possible to detect the peaks in the region comprised between
5.7 to 6.1 ppm (labeled as P in Fig. S2b) relative to the vinyl hydrogens of HEMA which are not
detectable in Fig. S2a. In addition, the ratio between the area of the peak P and the others, labeled
as A, B, C and D (which correspond to the hydrogens in the benzyl group bonded to the HEMA
molecule) is in the correct ratio of 1:1 for each peak. The presence of the HEMA molecule
determines a change in the shift of the closest hydrogen atoms and these differences between pure
RhB and HEMA-RhB macromonomer spectra can be detected from the peaks labeled as B, C, E
and F in Fig. S2b where their chemical shift is increased in respect of the ones reported in Fig. S2a.
1.4. Poly(methyl-methacrylate)(PMMA) nanoparticles synthesis
Poly(methyl-methacrylate) nanoparticles(PMMA-NPs) were synthesized through batch emulsion
polymerization (BEP) and monomer-starved semi-batch emulsion polymerization (MSSEP) using
KPS or 2,2'-Azobis(2-methylpropionamidine) dihydrochloride as initiators, and Tween80 as
surfactant. The reaction was carried out in batch and semi-batch conditions using a 100 mL threenecked glass flaskwith a reflux condenser. Temperature was controlled with an external oil bath set
at 80±1 °C. A different mass of Tween80 was added to 100 mL of deionized water and thereactor
atmosphere was kept inert through vacuum-nitrogen cycles. After that, for the BEP,MMA with a
proper amount of HEMA-RhB macromonomer previously dissolved in it (0.1% wHEMARhB/wMMA+HEMA-RhB),was
injected into the purged system with a syringe. The system was maintained
under magnetic stirring at 350 rpm. Finally, 0.08 g of the proper initiator were added to initiate the
reaction. For MSSEP the initiator was injected into the purged solution and the solution of the two
monomers was injected into the system at a flow rate of 3 mL/h using a syringe pump (Model NE300, New Era Pump System, Farmingdale, US). The reaction was run for 3h. Final monomer
conversion was measuredgravimetrically and calculated as higher than 99.5%. After the synthesis
the pH was adjusted to 7 using NaOH 0.1 M. Different particle sizes were obtained by varying the
emulsifier-to-monomer ratio, the monomer concentration in the aqueous phase, and the feeding
condition.
1.5 PMMA NPs characterization
Dynamic Light Scattering
The average size of PMMA-NPs was measured in dH2O using dynamic light scattering (DLS,
Zetanano ZS, Malvern). Micro UV-Cuvettes with dimension 12.5×12.5×45 mm (70 μL) and light
path 1 cm (Brand GmbH) were used for size measurements and Zeta dip cell (Malvern) for ζ-
potential measurement. Reported data are the average of three measurements of the same sample.
The incorporation of RhB in the polymer matrix has been validated from fluorescence intensity
analysis. PMMA-NPs obtained from the polymerization process have been precipitated using a 5 M
solution of CaCl2and centrifuged at 13000 rpm for 30 minutes. After the complete aggregation of
the PMMA-NPs, a transparent supernatant is obtained, as shown in Fig. S3b (reported data for 100PMMA-NPs).
a)
b)
Fig. S3 PMMA-NP (a) as obtained after polymerization reaction and (b) after precipitation with a 5
M solution of CaCl2 and followed by centrifugation
The qualitative results shown in Fig. S3 can be quantitatively proved through FI analysis. As it is
reported in Fig. S4, no fluorescence can be detected in the clear solution; thus, it can be concluded
that all the RhB added is present as HEMA-RhB macromonomer covalently linked to the PMMA
chains and then localized into the PMMA-NPs.
Fig. S4 Fluorescence analysis of the PMMA-NPsuspension and the correspondent supernatant
obtained after aggregation of the NPs latex
1.6. Cell segmentation and quantification of fluorescence in-vitro and in-vivo
In upper panels three representative images from the same field of view are shown to explain how
this analysis was performed. The blue spots (nuclei - left) enabled us to count the number of cells,
the interference phase contrast (Nomarsky - middle) was used to measure the cell surface and the
signal associated with NPs (RhB red, right panel).
a
CELL #
CELL SURFACE (µm2)
Rh-B SPREAD (µm2)
b
Fig. S5 (a) Representative confocal analysis showing cell nuclei (left), the shape of cells (middle)
and the spread of RhB (right) in 4T1 cells after 24 hours of incubation with PMMA-100+. The
analysis of the three channels for each single field of view with the dedicated software TissueQuest
(TissueGnostics GmbH, Austria), described in the lower panel, indicated : i) the mean number of
nuclei for each single condition and average cell surface; ii) the intracellular surface of RhB signal
(the overlap among the two images automatically excluded all red pixels falling outside the cell
borders).
(b) Representative scatterograms showing the profile of nuclear segmentation (left) and the pattern
associated with RhB (right). For both channels the fluorescence intensityand the area of each single
eventare reported
1.7. Biparametric BrdU/DNA analysis
Exponentially growing MDA-MB231.1833 cells were incubated with PMMA-100+ at the three
different reported concentrations. After 24, 48 and 72 hours of incubation 30 µM 5-bromo-2-
deoxyuridine (BrdU) was added for 15’, then the cells were collected and fixed in 70% ethanol.
For detection of BrdU incorporation into DNA, the fixed cells were washed with cold PBS and the
DNA was denatured with 1 mL of 3N HCl for 30’ at room temperature (RT) to allow the anti-BrdU
monoclonal antibody (MAb) reaction with the BrdU incorporated in the DNA chain. DNA
denaturation was stopped by adding 3 mL 0.1 M sodium tetraborate pH 8.5. After centrifugation,
the pellet was incubated with 1 mL 0.5% Tween-20 (Sigma Chemicals Co., St Louis, MO, USA) in
PBS and 1% (v/v) bovine serum albumin (BSA) (Sigma) for 15’ at RT. The incorporated BrdU was
visualized by incubating the cells with the monoclonal antibody anti-BrdU (Becton Dickinson)
diluted 1:10 in 0.5% (v/v) Tween-20 in PBS for 1 h at RT in the dark. After centrifugation the pellet
was incubated with 1 mL of 0.5% (v/v) Tween-20 and 1% (v/v) BSA for 15’ at RT. Then, cells
were incubated with the secondary antibody Alexa Fluor 488 (Molecular Probe) diluted 1:500 in
0.5% (v/v) Tween-20 in PBS for 1 h at RT in the dark. The cells were resuspended in 2 mL of a
solution containing 2.5 μg/mL PI in PBS and 25 μLRNAse 1 mg/mL in water, and stained
overnight at 4°C in dark. Biparametric BrdU/DNA analysis was done on at least 20000 cells for
each sample using a FACS Calibur instrument (Becton Dickinson). The data are the average of
three replications and were analyzed using Cell Quest software. The cell cycle phase distribution
percentages were calculated by the method of Krishan and Frei.
1.8. Immunohistochemistry
At the end of each incubation medium was removed, cells were washed with PBS, fixed with 4%
solution of paraformaldehyde, and staining was done for each marker, as follows:
a. Nuclei: Hoechst33258 (Sigma-Aldrich; 1:1000); b. Mitochondria: Mitotracker Green® (1:2000)
incomplete medium pre-heated at 37 °C, 5% CO2; c. Lysosomes: Polyclonal rat antibody
(GeneTex®, Inc.) anti- Lamp II (1:400) +Triton X-100 10% + FBS 10% in PBS 0.1M pH 7.4
without Ca2+/Mg2+, O/N at 4°C; d. Endosomes: Primary Polyclonal rabbit antibody anti-M6PR
1:400 + FBS 10% in PBS 0.1M pH 7.4 without Ca2+/Mg2+, O/N at 4°C; e. Golgi Apparatus:
Primary monoclonal mouse antibody anti-GM130 (1:400) (BD Transduction LaboratoriesTM) +
Triton X-100 10% + FBS 10% in PBS 0.1M pH 7.4 without Ca2+/Mg2+, O/N at 4°C.
Cells were then examined with a confocal microscope and quantitative evaluation of fluorescent
markers was performed with the image cytometry software TissueQuest (TissueGnostics GmbH,
Austria) as previously reported.
1.9. Two-photon confocal microscopy
Animals were allowed overnight recovery before imaging. Two hundred μL of PMMA-100+ [2
mg/mL] were injected into the tail vein and the mice were transferred to the two-photon microscope
stage. In-vivo imaging was done keeping the animals under gaseous anaesthesia. Images were
acquired with a BX51WI microscope coupled to a FV300 scanner head (Olympus Corporation,
Tokyo, Japan), equipped with a multiphoton laser, Chamaleon ultra II (Coherent, Santa Clara,
USA). A 20x magnification water immersion objective (XLUMPFL20X W/IR Objective with NA
0.95 WD 2.0mm, Olympus, Tokyo) was used to obtain images 30’ and 24h after NP injection.
Cortical volumes measuring sized 350x350x100μm were imaged and RhB was excited using
844nm wavelength. To measure RBC speedrepetitive line scans were acquired at 830Hz and
positioned at the center of the vessels pertinent to the imaged area. RBC speed was calculated from
the slopes of non-fluorescent red blood cell tracks [16]and expressed as mm/second. Two-photon
images were managed by Imaris (Bitplane, Switzerland).
2. Results
2.1. PMMA-NPs characterization
NP (name)
Diameter [nm]
Polydispersity
Index (PDI)
ζ-potential
[mV]
[NPs]
(#/ml W)
[PMMA]
(mg/ml)
PMMA-50+
50.2
0.127
34.5
2.01 E+14
15.90
PMMA-50-
53.6
0.076
-26.2
1.73 E+14
16.60
PMMA-100+
95.7
0.095
34.9
4.40 E+13
24.00
PMMA-100-
94.8
0.064
-26.9
3.29 E+13
17.40
PMMA-200+
188.6
0.026
8.6
4.62 E+12
19.30
PMMA-200-
196.0
0.107
-42.5
5.70 E+12
26.70
Fig. S6 Characterization of the main physico-chemical parameters of the different types of PMMANP synthesized for this project. PMMA: poly(methylmethacrylate); RhB: Rhodamine-B
2.2. In-vitro platform results
6 hours
24 hours
72 hours
Serum
CTR
a
b
Fig. S7 (a) Representative pictures showing the kinetics of internalization in 4T1 cells of PMMA100+ diluted in distilled water (upper panels) or after 48 hours of pre-incubation in human serum at
37°C.
(b) Histograms reporting the fluorescence intensity of RhB in 4T1 cells after 6, 24 and 72 hours
PMMA-100+. The fluorescence intensity at 6h in 4T1 cells incubated with PMMA-100+ diluted in
distilled water (grey bars) was normalized to 1, and the other data are reported in proportion to this
value. Analysis was done by unpaired Student’s t-test, matching the values at the same times.
***p<000.1
Fig. S8 Bi-parametric analysis of YO-PRO-1/propidium iodide (PI) after PMMA-100+ in MDAMB 231.1833 cells. The scatterogram is representative of cell distribution depending on the
expression of the two markers (YO-PRO to define early apoptosis) and propidium iodide to reveal
the membrane leakage and therefore the necrosis. The presence of both markers is associated with a
late apoptosis, the lack of both is typical of living cells.
Statistical analyses by five different experiments did not reveal any significant pattern of cell
distribution (as percentage) among the different groups. In particular the low percentages of cells in
R2 (apoptosis) upon exposure to NPs at the first time-point, did not significantly differ from those
observed in control condition
a
MDA-MB231.1833 cells, PMMA-100 +
CTRL
[0,5 *1011]
[1*1011]
[2*1011]
Number of cells / channel
24h
48h
72h
DNA CONTENT
b
Ratio BrdU +/BrdU BASAL CONDITION
PMMA-100+ [0.5*1011 ]
PMMA-100+ [1*1011 ]
PMMA-100+ [2*1011 ]
MDA-MB231.1833 Cell Line
24 h
13.46
13.15
12.31
11
48 h
8.84
8.03
7.77
7.51
Fig. S9 (a) Cell cycle analysis in MDA-MB231.1833 cells at different times after PMMA-100+
incubation.
(b) Percentage of BrdU positive and BrdU negative cells MDA-MB231.1833 at three different
PMMA-100+ concentrations and at two different times.