Core–shell poly-methyl methacrylate nanoparticles covalently functionalized with a non-symmetric porphyrin for anticancer photodynamic therapy

Article abstract of DOI:10.1016/j.jphotobiol.2018.07.013

Photodynamic therapy (PDT) is an anticancer modality that exploits singlet oxygen and other reactive oxygen species, that are formed by selective irradiation of photosensitive molecules, to kill cancer cells. Most photosensitizers (PS) are hydrophobic and poorly soluble in water and several nanoplatforms have been established to achieve a more efficient delivery. Moreover, the covalent binding of the PS to nanoparticles could in principle reduce unwanted bleaching of the PS, while preserving its photodynamic activity. In this study we report the synthesis of a novel non-symmetrical diaryl-porphyrin suitably modified with a polymerizable pendant, that was used for the preparation of core-shell poly-methyl methacrylate nanoparticles covalently loaded with the diaryl-porphyrin (PMMA@PorVa). Particles, which were prepared with two different porphyrin loadings, are spherical in shape and with a narrow hydrodynamic diameter around 70 nm and a positive zeta potential. Their photo-toxicity was tested against the human colon carcinoma cell line HCT116 and the human ovarian adenocarcinoma cell line SKOV3. PMMA@PorVa were able to inhibit tumor cells proliferation similarly to the free porphyrin, thus confirming that the covalent attachment of the PS to PMMA nanoparticles allows to preserve PS photodynamic activity and in vitro efficacy. Flow cytometric analysis of apoptotic cells demonstrates that, especially in SKOV3 cells, the free diaryl-porphyrin is more effective in inducing apoptosis.

Full text of DOI:10.1016/j.jphotobiol.2018.07.013

Accepted Manuscript
Core–shell poly-methyl methacrylate nanoparticles covalently
functionalized with a non-symmetric porphyrin for anticancer
photodynamic therapy
Marco Ballestri, Enrico Caruso, Andrea Guerrini, Claudia
Ferroni, Stefano Banfi, Marzia Gariboldi, Elena Monti, Giovanna
Sotgiu, Greta Varchi
PII:
DOI:
Reference:
S1011-1344(18)30299-9
doi:10.1016/j.jphotobiol.2018.07.013
JPB 11299
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date:
Revised date:
Accepted date:
20 March 2018
11 June 2018
17 July 2018
Please cite this article as: Marco Ballestri, Enrico Caruso, Andrea Guerrini, Claudia
Ferroni, Stefano Banfi, Marzia Gariboldi, Elena Monti, Giovanna Sotgiu, Greta Varchi
, Core–shell poly-methyl methacrylate nanoparticles covalently functionalized with a
non-symmetric porphyrin for anticancer photodynamic therapy. Jpb (2018), doi:10.1016/
j.jphotobiol.2018.07.013
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ACCEPTED MANUSCRIPT
Core–shell poly-methyl methacrylate
nanoparticles covalently
functionalized with a non-symmetric porphyrin for anticancer
photodynamic therapy
Marco Ballestria, Enrico Carusob, Andrea Guerrinia, Claudia Ferronia, Stefano Banfib, Marzia
Gariboldib, Elena Montib, Giovanna Sotgiua and Greta Varchia*
aItalian National Research Council - Institute of Organic Synthesis and Photoreactivity (ISOF), Via
P.
Gobetti 101, 40129 Bologna, Italy.
bDepartment of Biotechnology and Life Sciences (DBSV). University of Insubria, Via J.H. Dunant 3,
21100 Varese (VA), Italy.
*Correspondence Greta Varchi, PhD greta.varchi@isof.cnr.it

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Abstract
Photodynamic therapy (PDT) is an anticancer modality that exploits singlet oxygen and other
reactive oxygen species, that are formed by selective irradiation of photosensitive molecules, to kill
cancer cells. Most photosensitizers (PS) are hydrophobic and poorly soluble in water and several
nanoplatforms have been established to achieve a more efficient delivery. Moreover, the covalent
binding of the PS to nanoparticles could in principle reduce unwanted bleaching of the PS, while
preserving its photodynamic activity. In this study we report the synthesis of a novel non-
symmetrical diaryl-porphyrin suitably modified with a polymerizable pendant, that was used for the
preparation of core-shell poly-methyl methacrylate nanoparticles covalently loaded with the diaryl-
porphyrin (PMMA@PorVa). Particles, which were prepared with two different porphyrin loadings,
are spherical in shape and with a narrow hydrodynamic diameter around 70 nm and a positive zeta
potential. Their photo-toxicity was tested against the human colon carcinoma cell line HCT116 and
the human ovarian adenocarcinoma cell line SKOV3. PMMA@PorVa were able to inhibit tumor
cells proliferation similarly to the free porphyrin, thus confirming that the covalent attachment of the
PS to PMMA nanoparticles allows to preserve PS photodynamic activity and in vitro efficacy. Flow
cytometric analysis of apoptotic cells demonstrates that, especially in SKOV3 cells, the free diaryl-
porphyrin is more effective in inducing apoptosis.
Keywords porphyrins, PMMA nanoparticles, covalent loading, photodynamic therapy, cancer

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1
Introduction
Photodynamic therapy (PDT) is a minimally invasive tumor therapeutic option that involves the
systemic administration of a photosensitizer (PS), followed by its activation with visible light. The
activated PS reacts with oxygen present in the tissue, generating reactive oxygen species (ROS) and
other free radicals by type I and II photochemical reactions and promoting the selective destruction of
1–3
target cells
.
4,5
PDT has been widely used for treating superficial tumors, such as skin and head and neck cancers,
as well as solid tumors in the esophagus, lungs, larynx, uterine cervix and colon 6–8. The first PS
granted regulatory approval was porfimer sodium (Photofrin), a purified form of hematoporphyrin
derivative consisting of a mixture of porphyrins 9,10. Despite its proven effectiveness against a variety
of cancers, Photofrin has a number of disadvantages, most notably low tissue penetration of
activating light, extended skin photosensitivity and low selectivity between tumor and healthy tissues
in the early phases of treatment. Over the past decade, researchers have actively focused on the
development of more effective PS, that would efficiently generate singlet oxygen upon activation
with longer wavelengths light (650 – 900 nm) and preferentially accumulate in tumor tissues, while
being rapidly cleared from normal tissues. However, despite the fact that some second- and third-
generation PSs have demonstrated improved activity over Photofrin, challenges, such as poor tumor
targeted accumulation and skin photosensitivity due to PS unwanted accumulation, still have to be
addressed in order to establish PDT in the clinical setting.
In the last decades, nanotechnology has emerged as a powerful tool for the design and manipulation
of matter at the nanometric scale, providing (nano)objects with augmented physicochemical
properties 11,12. Among nanotechnology applications, nanomedicine has attracted considerable
attention from researchers, clinicians and pharmaceutical companies, due to its potential to radically
13–15
change the pharmacological approach to cancer management
.
Nanoparticles (NPs) are ideal carriers to promote accumulation of therapeutic agents within tumor
16,17
tissues through the enhanced permeability and retention effect (EPR)
and currently several NPs-
based drugs have either been approved or are being tested in clinical trials with very encouraging
18
results
.
In particular, a variety of nano-sized matrices have been employed to develop nanoparticle-based
PDT agents 19,20. Indeed, besides providing more targeted delivery of the cargo, in the case of PDT,
nanoparticles protect the PS from the environment without affecting ROS production while multiple

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irradiations of the same PS-loaded nanoparticles can be performed, thus reducing the need for
21
repetitive administrations
.
In this regard, we have previously shown that positively charged biocompatible core–shell
22,23
polymethylmethacrylate nanoparticles (PMMA-NPs) are suitable vehicles for dyes and drugs
.
PMMA-NPs with specifically tailored features can be obtained by an emulsion co-polymerization
reaction, yielding particles with sizes ranging from 30 nm to 1m and characterized by a
hydrophobic PMMA inner core surrounded by a cationic hydrophilic shell made of quaternary
ammonium salts. These nanoparticles were successfully employed as delivery vehicles of anionic
PSs electrostatically loaded on the outer shell 24. We additionally demonstrated that PS-loaded
PMMA-NPs are able to efficiently kill cancer cells in vitro and in vivo upon irradiation with light
25,26
.
In order to improve the system and extend the use of our nanoparticles to non-charged
photosensitizers, we herein investigated for the first time the covalent functionalization of our
PMMA-NPs with a photosensitizer suitably modified with a polymerizable and hydrophilic pendant.
In particular, a non-symmetric di-methyl-amino-ethylacrylate diaryl-porphyrin was synthesized and
used as co-polymer in PMMA-NPs synthesis. PMMA nanoparticles covalently loaded with
porphyrin, e.g. PMMA@PorVa, were obtained with different porphyrin loading, and their in vitro
phototoxic effect was evaluated on human colon (HCT116) and ovarian (SKOV3) cancer cell lines.
Furthermore, we evaluated the ability of our newly synthesized NPs to facilitate PS internalization
and to induce apoptotic cell death and ROS production in the two cell lines.
2
Material and Methods
2.1 General Chemistry
2-(Dimethylamino)ethyl
methacrylate
(DMAEMA),
1-bromooctane,
2,2’-azobis(2-
methylpropionamidine) dihydrochloride (AIBA), methyl methacrylate (MMA, 99.0%) (distilled
1
before use) and other chemicals were purchased from Sigma-Aldrich (Italy). H NMR spectra were
recorded on a Bruker 500 MHz spectrometer in CDCl3 and on an Agilent NMR DD2 500 MHz
equipped with single ADC console and OneNMR™ probe; chemical shifts are expressed in ppm
relative to chloroform (7.28) and are reported as s (singlet), d (doublet), t (triplet) and m (multiplet).
UV-vis absorption spectra were measured on a Perkin-Elmer Lambda 10 instrument. Mass
spectrometric measurements were performed on a Bruker Esquire 3000 Plus. Merck 60 F254 silica
gel pre-coated sheets (0.2 mm thick) were used for analytical thin-layer chromatography (TLC).
Silica gel 60 (70-230 mesh, Merck) was used for column chromatography. Dichloromethane used for

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porphyrin synthesis was distilled from CaCl2 directly into the reaction flask. The di-pyrromethane
was obtained as previously reported 27. A tungsten-halogen lamp (500W) was used as source for
visible light, (and its fluence rate (irradiance) was determined with a LI-COR 1800 spectrum-
radiometer (22 mW/cm2).
Chemistry
2.1.1 5-phenyl-15-[4-(4-bromobuthoxy)phenyl]-21H,23H-porphyrin (2)
Ten drops of TFA were added to a 400 mL of CH2Cl2 solution containing 127 µL (1.25 mmol) of
28
benzaldehyde, 321 mg (1.25 mmol) of 4-(4-bromobuthoxy)benzaldehyde (1)
and 365 mg (2.5
mmol) of 2,2’-dipyrromethane (Plater et al., 2002). The mixture was kept at room temperature for 2
h. The reaction was controlled over time by TLC (SiO2; Eluent: CH2Cl2/petroleum ether = 6/4).
Afterwards, 380 mg (1.67 mmol) of 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) was added
when the aldehydes were completely reacted, and the mixture was kept at room temperature for 2 h.
The solvent was then evaporated and the crude product was purified by column chromatography
(SiO2; Eluent: CH2Cl2/petroleum ether = 6/4). Three different compounds were separated: 5,15-
diphenyl-21H,23H-porphyrin
(48
mg,
0.104
mmol,
8.3%);
5-phenyl-15-[4-(4-
bromobuthoxy)phenyl]-21H,23H-porphyrin (2) (120 mg, 0.196 mmol, 15.7%); and 5,15-di[4-(4-
bromobuthoxy)phenyl]-21H,23H-porphyrin (26 mg, 0.034 mmol, 2.7%).
Characterization of compound 2: UV-vis(CH2Cl2): 406 nm (=274000); 1H NMR (CDCl3)
, 2H), 2.20 (m, 2H, CH2-CH2Br), 2.31 (m, 2H, CH2-CH2O), 3.68 (t, 2H, CH2Br, J = 10
Hz), 4.35 (t, 2H, CH2O, J = 7.5 Hz), 7.36 (d, 2H, J = 10 Hz), 7.84 (m, 3H), 8.21 (d, 2H, J = 10 Hz),
8.30 (m, 2H), 9.10 (d, 2H, J = 5 Hz), 9.14 (d, 2H, J = 5 Hz), 9.42 (d, 4H, J = 5 Hz), 10.34 (s, 2H). MS
(ESI): M+ found: 612.98 calcd for C36H29BrN4O 613.59.
2.1.2 5-phenyl-15-[4-[4-(2-methacryloyl)ethyl-dimethyl ammonium bromide)]buthoxy) phenyl]-
21H,23H-porphyrin (3)
10 mg (0.045 mmol) of 2,6-diterbutyl-4-methylphenol (BHT) were added to a mixture of porphyrin 2
(110 mg, 0.178 mmol) and 120 µL (0.712 mmol) of 2-(dimethylamino)ethyl methacrylate
(DMAEMA) in 5 mL of THF. The mixture was kept at 60 °C for 24 h. During the reaction time a
violet solid product was formed. After filtration, the product was washed 4 times with 10 mL of
diethyl ether affording 48 mg (35%) of compound 3 as violet solid. 1H NMR (DMSO)
m3.53 (s, 6H, CH3N), 3.93 (t, 2H, CH2N), 4.16 (t, 2H, CH2N), 4.31

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(t, 2H, CH2O), 4.68 (t, 2H, CH2OCO), 5.74 (s, 1H), 6.21 (s, 1H), 7.30 (d, 2H), 7.80 (m, 3H), 8.17 (d,
2H), 8.27 (m, 2H), 9.08 (d, 4H), 9.38 (d, 4H), 10.29 (s, 2H). 13C NMR (DMSO) 166.4, 158.9,
147.2, 145.2, 140.9, 136.3, 135.9, 135.1, 133.2, 133.1, 132.9, 131.1, 128.5, 127.8, 119.1, 119.0,
113.9, 106.2, 67.5, 64.2, 58.7, 51.2, 26.3, 19.7, 18.4.
2.1.3 Nanoparticle synthesis
The positively charged nanoparticles were obtained by emulsion polymerization of methyl
methacrylate in the presence of the ionic co-monomer 2-(dimethyl-octyl) ammonium ethyl
methacrylate bromide (4, Figure 1), as emulsion stabilizer, and different amounts of compound (3)
(Table 1). The ionic co-monomer 2-(dimethyl-octyl) ammonium ethyl methacrylate bromide was
obtained as previously described 29. PMMA@PorVa were obtained as follows: 10 mL of distilled
water, 106 mg of 4 (0.29 mmol) and 3 (Table 1) were placed into a 15 mL thermostated flask
equipped with magnetic stirring, a nitrogen inlet and a cooling apparatus. The mixture was heated
under nitrogen till 80 °C and 0.2 mL of meta methyl methacrylate (1.87 mmol) were added drop
wise. After 15 min, AIBA (Table 1) was added and the reaction was stirred for 4 h at 80 °C.
Nanoparticles were initially purified by dialysis to remove residual monomer and stabilizer, followed
by subsequent washings on a stirred ultrafiltration cell Amicon system (Millipore model 8050) with
milliQ water (100 kDa filters; 5 x).
2.1.4 Nanoparticle characterization
PMMA@PorVa NPs UV spectra were determined by spectrophotometric measurement with a
Lambda 20 Perkin Elmer spectrophotometer (Waltham, MA, USA). The hydrodynamic diameter of
the nanoparticles was determined by photon correlation spectroscopy (PCS) at 25 °C using a
NanoBrook Omni Particle Size Analyzer (Brookhaven Instruments Corporation, USA) equipped with
a 35 mW red diode laser (nominal 640 nm wavelength). The ζ-potential was measured at 25 °C by
means of the same system. The loading of quaternary ammonium groups per gram of nanoparticles
was determined by potentiometric titration of the bromide ions, obtained after complete ion
exchange. The ion exchange was accomplished by dispersing 10.8 mg of the nanoparticles sample in
25 mL of 1 M KNO3 at room temperature for 30 min. The mixture was then adjusted to pH 2 with
dilute H2SO4 and bromide and chloride ions were titrated with a 0.0201 M solution of AgNO3.
Particle morphology was further studied using the EVO LS 10 LaB6 scanning electron microscopy
(SEM) (Zeiss, Italy) with an acceleration voltage of 5 kV and a working distance of 5 mm. The
samples were sputter coated under vacuum with a thin layer (10– 30 Å) of gold. Particle morphology

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was further studied through atomic force microscopy (AFM) measurements; samples were prepared
by spin-coating deposition of 1 mL of NPs (1 mg/mL) on atomic flat silicon substrate at 1500 rpm.
These experimental conditions allow to prevent the aggregation of the nanoparticles and to obtain a
uniform deposition over 1 cm2 of isolated nanoparticles, which can be clearly distinguished from the
substrate. AFM topographical images were collected using an NT- MDT (Italy) solver scanning
probe microscope in tapping mode. Colloidal particles stability in serum was performed by
suspending 500 µg of nanoparticles (PMMA@PorVa-06) in 2 ml of a FBS/H2O (1/1) mixed solution
at 37°C for several hours. NPs size during time was controlled by DLS measurements at 25°C
(Figure S7).
2.1.5 Comparative singlet oxygen generation measurements
An aerated isopropanol solution containing 50 μM of the singlet oxygen scavenger 1,3-
diphenylisobenzofuran (DPBF) (Abs = 2.3 at 410 nm) and 1 μM of photosensitizer (Abs = 0.12 at
410 nm) was prepared and kept in the dark. Afterward, 2 mL of this solution were transferred into a
cuvette and irradiated from the open top side with green LED (maximum light irradiance 7.52 × 10-5
W/cm2 at 526 nm) at room temperature up to a maximum of 20 min. At predetermined intervals, the
absorbance at 410 nm was measured. The rate of singlet oxygen production was determined from the
reduction in intensity of absorbance recorded over time. A sample of DPBF-isopropanol solution was
used as the negative control. Green LED light was specifically used for the irradiation of DPBF
because this compound is very unstable and undergoes self-photobleaching when irradiated with blue
30
LED and white halogen lamps
.
2.1.6 Photo-degradation of the porphyrin 3 and nanoparticles
PS dispersions were prepared by dissolving Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06
in water and diluted in PBS at a concentration of 10 μM. The solutions were then irradiated for 2 h at
room temperature with a halogen lamp (500 W; fluence of 158.4 J/cm2); during this period 1 mL
sample was collected every 30 min and analysed with a UV/vis spectrophotometer. The decrease in
the absorbance intensity vs time plot was used to determine the photo-degradation rate.
2.2 Biology

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2.2.1 Cytotoxicity studies.
The human colon carcinoma cell line HCT116 and the human ovarian adenocarcinoma cell line
SKOV3 were obtained from the American Type Culture Collection (Rockville, MD, USA) and
maintained in Dulbecco’s modified Eagle medium (DMEM, Mascia-Brunelli, Milano, Italy)
supplemented with 10% fetal bovine serum, 1% glutamine and 0.5% antibiotic mixture (penicillin,
streptomycin and neomycin), under standard culture conditions at 37 °C in a humidified 5% CO2
atmosphere. The antiproliferative activity of PMMA@PorVa nanoparticles at different Porphyrin 3
31
loading (PMMA@PorVa-05 and PMMA@PorVa-06) was assessed using the MTT assay
and
compared with the effect induced by free Porphyrin 3. Briefly, cells (HCT116: 2 x 103 and SKOV3:
2.5 x 103 cells/well) were seeded onto 96-well plates and allowed to grow for 48 h prior to the
treatment with the PS either in its unbound form or loaded onto NPs. The final PS concentrations
ranged between 0.1 and 100 nM and were prepared from 1 mM stock solutions of the three
formulations. After 24 h, the PS-containing medium was replaced by fresh PBS, and cells were
irradiated under visible light for 2 h using a tungsten-halogen lamp (500 W; fluence of 158.4 J/cm2).
At the end of the irradiation, cells were incubated in the dark for 24 h at 37 °C in drug-free medium,
then MTT was added to each well (final concentration 0.4 mg/mL) for 3 h at 37 °C. Formazan
crystals, formed by MTT metabolism in viable cells, were dissolved in DMSO and the optical
densities were read at 570 nm using a Universal Microplate Reader EL800 (Bio-Tek Instruments).
As described above, possible intrinsic (i.e. not photodynamic) effects on cells viability due to PSs
without irradiation, were assessed by exposing cells to different PS concentrations tenfold higher
than those used in the PDT experiments but omitting the irradiation step. IC50 values (i.e. the PS
concentration causing a 50% reduction in the cell survival fraction) were obtained by non-linear
regression analysis, using the GraphPad PRISM 4.03 software (GraphPad Software Inc., San Diego
CA).
2.2.2 Flow cytometric analysis
The induction of apoptotic cell death, following the PS irradiation, was evaluated by flow cytometric
analysis. Cells were exposed to Porphyrin 3 in its unbound form or loaded onto PMMA-NPs at the
respective IC50 values, and irradiated for 2 h in drug-free PBS, followed by 24 h incubation in the
dark and in drug-free medium. To assess the percentage of apoptotic cells, at the end of treatment
cells were harvested by trypsinization (pooling both adherent and detached cells), washed in PBS and
fixed in 70% ethanol at -20 °C for (at least) 20 min. After a further wash in PBS, DNA was stained
with a solution of propidium iodide (PI) in PBS (50 g/mL) in the presence of RNAse A (30 U/mL)

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at room temperature for 15 min before the sample analysis. Intracellular ROS production was
evaluated using 2,7-dichlorodihydrofluoresceindiacetate (DCFH-DA) as probe. Cells were detached
at the end of the irradiation period, washed and re-suspended (1 x 106 cells/mL) in PBS containing 10
μM DCFH-DA; after 45 min incubation in the dark at 37 °C, cell samples were analyzed.
Intracellular accumulation of free Porphyrin 3 and PMMA@PorVa-05 and PMMA@PorVa-06 was
measured in HCT116 and SKOV3 cells exposed to the three compounds (100 nM for 24 h); for this
set of experiments, cells were not irradiated. At the end of the exposure time, treated cells were
detached by trypsinization, washed thoroughly in ice-cold PBS, resuspended in PBS and analyzed.
All the samples were analyzed with a FACSCalibur flow cytometer (Becton Dickinson Mountain
View, CA, USA) and data were processed using CellQuest software (Becton Dickinson). Fluorescent
emission of PI was collected through a 575 nm band-pass filter acquired in log mode and the
percentage of apoptotic cells in each sample was determined based on the sub-G1 peaks detected in
mono-parametric histograms. Fluorescein fluorescence was collected through a 530-nm band-pass
filter and intracellular ROS generation was quantitated in arbitrary units based on the mean
fluorescence intensity (MFI). PS fluorescent emission was collected through a 575 nm band-pass
filter. Statistical analysis of biological data was performed by means of one- or two-way ANOVA,
with Bonferroni’s test for multiple comparisons, using GraphPad PRISM 4.03 software.
3
Results
3.1 Chemistry
The non-symmetric diaryl-porphyrin 2 (Scheme 1) was synthetized via acid-catalysed condensation
32
33
of 2,2’-dipyrromethane
with aldehyde 1
and benzaldehyde, following the general procedure
described by Lindsey and co-workers 34. The porphyrinogen intermediate was then oxidized to the
corresponding porphyrin with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (Scheme 1A).
After reaction completion and workup, three different compounds were isolated by chromatographic
purification: the two symmetrically substituted diaryl-porphyrins, e.g. 5,15-diphenyl-21H,23H-
porphyrin (2a, Scheme 1A) and 5,15-di[4-(4-bromobuthoxy)phenyl]-21H,23H-porphyrin (2b,
Scheme 1A) in 8% and 3% yield, respectively, along with the desired non-symmetric 5-phenyl-15-
[4-(4-bromobuthoxy)phenyl]-21H,23H-porphyrin (2) in 16% yield. As outlined in Scheme 1B, the
reaction of 2 with 2-(dimethylamino)ethyl methacrylate (DMAEMA), in the presence of 2,6-
diterbutyl-4-methylphenol (BHT) as antioxidant, produced Porphyrin 3 in 35% yield.

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Scheme 1. Synthesis of Porphyrin 3. A) Synthesis of 5-phenyl-15-[4-(4-bromobuthoxy)phenyl]-
21H,23H-porphyrin (2); i) TFA, CH2Cl2, rt, 2 h; DDQ, rt, 2 h. B) Synthesis of 5-phenyl-15-[4-[4-(2-
methacryloyl)ethyl-dimethyl ammonium bromide)]butoxy) phenyl]-21H,23H-porphyrin (3); i) THF,
BHT, 60 °C, 24 h.
Core-shell PMMA@PorVa nanoparticles (Figure 1) were obtained by an emulsion co-polymerization
reaction with yields ranging from 20 to 40%. During the polymerization, all the reagents concur to
form an initial micelle where hydrophobic chains locate inside the micelle (core), while the
hydrophilic ones, e.g. chains bearing the quaternary ammonium salts, remain in the external water
phase (shell). Nanoparticles were obtained with two different porphyrin loading, e.g.
PMMA@PorVa-05 and PMMA@PorVa-06, containing 6.4 g and 9.7 g of 3 per mg of
nanoparticles, respectively (Table 1 and Figure 2). We assumed that 3 completely reacted during
particles formation; indeed, during the purification steps, e.g. dialysis and ultra-filtration, no
fluorescence was observed in the waste water (data not shown), thus indicating the absence of
unreacted porphyrin.

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Figure 1. Schematic representation of PMMA@PorVa nanoparticle synthesis.
As demonstrated by SEM (Figure 2, A-B) and AFM analyses (Figures S2), PMMA@PorVa
nanoparticles are spherical in shape with an average SEM diameter of 65.3 ± 12 nm and 63 ± 11 nm
for PMMA@PorVa-05 and PMMA@PorVa-06, respectively. As reported in Table 1, the average
hydrodynamic diameter is 71.3 ± 0.92 (polydispersity index 0.09 ± 0.02) for PMMA@PorVa-05 and
of 71.0 ± 0.32 (polydispersity index 0.04 ± 0.01) for PMMA@PorVa-06 (average of five measures,
Tables S1 and S2).
The spectrophotometric analysis of PMMA@PorVa-05 and PMMA@PorVa-06 (Figure 2, C-D)
shows a significant increase in absorbance at increasing loading of 3, as well as an increase in the
fluorescence peak at 650 nm, thus confirming that despite the higher loading no aggregates are
formed.

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Figure 2. Morphological and photochemical characterization of PMMA@PorVa-05 and
PMMA@PorVa-06. SEM analysis of PMMA@PorVa-05 (A) and PMMA@PorVa-06 (B).
Absorbance (C) and emission spectra (excitation at  = 407 nm) (D) of PMMA@PorVa-05 and
PMMA@PorVa-06. (E) Photobleaching of Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06
following 120 min of irradiation with 500W tungsten halogen lamp.
The density of the cationic groups (quaternary ammonium salts) on the particle shell were found to
be 640 and 811 mol per gram of PMMA@PorVa-05 and PMMA@PorVa-06, respectively. Finally,
the -potential, measured after dialysis purification, was 42.11 mV and 60.95 mV for

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PMMA@PorVa-05 and PMMA@PorVa-06, respectively (Tables 1, S1 and S2), thus confirming that
the cationic co-monomers employed in the polymerization reaction are covalently bound to
nanoparticle surface.
Table 1. Reaction conditions for PMMA@PorVa nanoparticles synthesis.
Sample
Porphyrin 3
(mmol)
AIBA
(mmol)
Hydrodynamic
diameter (nm)
Br-
(µmol/g)
-potential (mV)
PMMA@PorVa-05
PMMA@PorVa-06
5.19 x 10-4
1.49 x 10-3
2.3 x 10-3
2.1 x 10-3
71.3 ± 0.92
71.0 ± 0.32
640
811
42.1 ± 2.76
60.9 ± 2.54
As ancillary experiment to the phototoxicity studies, the stability of a 10 µM phosphate-buffered
solution of free and loaded porphyrins was evaluated by recording their absorbance at 30 min
intervals for a total of 120 min irradiation with a 500W tungsten halogen lamp (Figure 2E). The
results show that Porphyrin 3 photostability drops of about 50% after 120 min irradiation, while
porphyrin loaded on PMMA nanoparticles retains a good photostability (80% and 85% respectively
for PMMA@PorVa-06 and PMMA@PorVa-05).
3.2 Biology
3.2.1 In vitro phototoxicity and apoptotic cell death induction.
The photodynamic efficacy of Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 was
evaluated on HCT116 human colon carcinoma and SKOV3 human ovarian adenocarcinoma cells and
expressed as IC50 values (Figure 4A). As a preliminary experiment, we determined the possible
cytotoxic effects of naked PMMA-NPs, both in the dark or under light irradiation; our results indicate
that cell viability is not affected by exposure to naked particles (data not shown). The data of
phototoxicity were obtained from dose-response curves 24 h after exposure of the two cell lines to
the three PS formulations, followed by 2 h irradiation and 24 h incubation in the dark. These results
were compared with that obtained in PS-free medium. The intrinsic cytotoxicity of Porphyrin 3 and
PMMA@PorVa nanoparticles was also assessed, using concentrations up 10-fold higher than those
used in the experiments, as described in the Materials and Methods section (max concentration 1
M), and omitting the irradiation step from the treatment protocol. No significant intrinsic
cytotoxicity was observed for any of the formulations tested (not shown), thus indicating that PS
photoactivation is an absolute requirement to trigger the cytotoxic activity of these compounds.

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All the formulations tested exerted phototoxic effects at nanomolar concentrations on both cell lines;
in particular, in HCT116 cells PMMA@PorVa-06 were as potent as Porphyrin 3 (IC50 2.68 ± 0.08
nM and 1.59 ± 0.34 nM, respectively), and both were significantly more potent than
PMMA@PorVa-05 (IC50 5.13 ± 0.59 nM). On the other hand, in SKOV3 cells no significant
differences in IC50 values were observed among the three compounds (IC50: Porphyrin 3 1.29 ± 0.06
nM; PMMA@PorVa-05 1.92 ± 0.16 nM; PMMA@PorVA-06 1.77 ± 0.22 nM).
Figure 3. Photo-toxicity and apoptosis induction of Porphyrin 3, PMMA@PorVa-05 and
PMMA@PorVa-06 in HCT116 and SKOV3 cells. IC50 values (A) were obtained by the MTT
assay in Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 treated cells 24 h after irradiation
with a tungsten-halogen lamp (500 W for 2 h). (mean ± SE of 3-5 independent experiments, **
p<0.01 vs Porphyrin 3 and PMMA@PorVa-06). For apoptosis experiments (B and C), cells were
exposed or not (control) to Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 at their
respective IC50 values for 24 h, irradiated for 2 h (light: white bars) or not (dark: black bars) and

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incubated for 24 h in drug-free medium. The percentage of apoptotic cells was determined by flow
cytometry. Mean ± SE of 4-6 independent experiments. **p < 0.0001 versus control, dark and
*p<0.001 vs control, dark and Porphyrin 3.
Apoptosis induction in HCT116 (Figure 3B) and SKOV3 (Figure 3C) cells was evaluated following
photodynamic treatment using Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 at
concentrations corresponding to their respective IC50 values. Our data confirm that all formulations
are able to induce a significant increase in apoptosis over control samples in both cell lines. In
particular, in agreement with the previously observed phototoxic effect, the fraction of HTC116
apoptotic cells is around 50% for all considered formulations, with a slight increase for cells treated
with PMMA@PorVa-06 (30% vs Porphyrin 3, 19% vs PMMA@PorVa-05). All formulations also
induced a significant increase in apoptosis in SKOV3 cells, although free Porphyrin 3 appears to
exert a stronger pro-apoptotic effect than the two nanoparticle formulations.
3.2.2 Intracellular uptake, oxidizing species and singlet oxygen levels
Following 24 h incubation in the presence of 100 nM of the three tested compounds, differences in
the intracellular uptake of Porphyrin 3 were detected by flow cytometric analysis. Figure 4 shows
that in HCT116 cells both free Porphyrin 3 and PMMA@PorVa-06, which were more potent in terms
of IC50 values, also exhibit greater intracellular accumulation. Interestingly, in SKOV3 cells
Porphyrin 3 shows a significantly higher uptake as compared to PMMA@PorVa-05 and
PMMA@PorVa-06, even though all the considered porphyrin formulations afforded similar IC50
values (cfr Figure 3A). It is likely that in the two-dimensional and static in vitro model used for this
experiment, the lower molecular weight of free Porphyrin 3 allows for faster/higher uptake; however,
this behavior could be completely reversed in in vivo settings where many other parameters and
conditions occur.

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Figure 4. Cellular uptake of Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 in
HCT116 and SKOV3 cells. Intracellular accumulation determined upon 24 h exposure to the
different PS formulations (100 nM) and measured by flow cytometric analysis; control cells were not
exposed to any PS-containing formulations (mean ± SE of 3-5 independent experiments, ***
p<0.001 vs control: § p<0.01 vs PMMA@PorVa-05 and PMMA@PorVa-06; # p< 0.05 vs
PMMA@PorVa-05).
As shown in Figure 5A, equitoxic concentrations of the PS (IC50 values) induced variable increases
in ROS generation, even though statistical significance was only attained for Porphyrin 3 and
PMMA@PorVa-06 in HCT116 cells line and for Porphyrin 3 and PMMA@PorVa-05 in SKOV3
cells. Furthermore, in both cell lines ROS levels induced by Porphyrin 3 were significantly higher
than those induced by PMMA@PorVa-05 and PMMA@PorVa-06. A significant increase in ROS
production following treatment with PMMA@PorVa-05, as compared to PMMA@PorVa-06, was
also observed in SKOV3 cells. No di-chlorofluorescein (DCF) fluorescence was detectable when
flow cytometry was performed 24 h later (data not shown), indicating that ROS generation abates
within 24 h of the end of irradiation.

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Figure 5. ROS and singlet oxygen generation induced by Porphyrin 3, PMMA@PorVa-05 and
PMMA@PorVa-06. A) ROS generation in HCT116 and SKOV3 cells following 24 h exposure to
Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 at their respective IC50 values and 2 h
irradiation in compounds-free PBS; control cells were not exposed to any PS-containing
formulations. Intracellular ROS production was determined by flow cytometry at the end of the
irradiation period. Mean ± SE of three independent experiments. *** p<0.0001 vs control, **
p<0.001 vs control, p<0.05 vs PMMA@PorVa-06. B) Singlet oxygen quantum yields for Porphyrin
3, PMMA@PorVa-05 and PMMA@PorVa-06. All values are normalized vs Porphyrin 3. Mean ± SE
of three independent experiments.
1
The production of O2 for Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 was indirectly
determined measuring the disappearance of the absorption band (at 410 nm) of 1,3-
1
diphenylisobenzofuran (DPBF), a well-known O2 scavenger. Experiments were performed following
the method described by Kochevar et al., using isopropanol as solvent, with a 50 M initial
concentration of DPBF and in the presence of 1 M of porphyrin 30. Irradiation was carried out with
a green LED device, which does not allow a high production of singlet oxygen (poor overlap of the
LED emission band with the PS absorbing band) however ensures DPBF photostability. The relative
1
rates of O2 production of the two PMMA@PorVa were obtained by comparing the kinetics of DPBF
degradation with those induced by Porphyrin 3. Results are reported in Figure 5B, showing that
Porphyrin 3 bound to PMMA has a singlet oxygen quantum yield 1.6 to 1.7 times greater than the
free compound.
4
Discussion

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In a previous work, we demonstrated that 5, 15-diaryl-porphyrins exert greater photodynamic activity
as compared to tetra-aryl-porphyrins 35; moreover, among diaryl-porphyrins, we showed that non-
symmetrical diaryl-porphyrins act as more potent photosensitizers than symmetrical ones.
Accordingly, in this paper we report the synthesis of a novel non-symmetrically substituted diaryl-
porphyrin, namely compound 3, carrying only one polymerizable pendant suitable for covalent
bonding with poly-methyl methacrylate nanoparticles.
In this regard, a number of different procedures have been reported for the synthesis of 5,15-diaryl-
porphyrins 36,37, mostly based on a (2 + 2)-type condensation, in which two dipyrrolic compounds
incorporating one type of carbon bridge are fused together and are ultimately in situ oxidized for
providing the desired porphyrin. However, for the present study, we used the 5-unsubstituted
dipyrromethane that was reacted with a mixture of two different aromatic aldehydes, following the
procedure described by Plater and co-workers 38. This procedure yielded three different compounds,
including the desired non-symmetric porphyrin 2, which was isolated by column chromatography
and then reacted with di-methyl-amino-ethylacrylate (DMAEMA) affording Porphyrin 3 in fairly
good yield.
Porphyrin 3 was then used as reagent in the polymerization reaction at different molar ratios, in order
to obtain particles with different loading of photosensitizer. In particular, two positively charged
PMMA nanoparticles covalently functionalized with Porphyrin 3, i.e. PMMA@PorVa-05 and
PMMA@PorVa-06, were obtained. Particles have comparable sizes (≈ 70 nm) and carry different
amounts of Porphyrin 3, e.g. 6.4 g and 9.7 g per mg of nanoparticles, respectively. We can assume
that, due to the presence of the quaternary ammonium salt on the porphyrin side chain, the porphyrin
itself would be mostly localized on the outer shell of the NPs rather than within the hydrophobic
PMMA core, thus being more accessible to light for photosensitization. However, photostability
studies demonstrated that once loaded onto PMMA nanoparticle, Porphyrin 3 is significantly more
photostable. One possible explanation of the behavior could be ascribed to the protective effect
exerted by nanoparticles shell on porphyrin photobleaching, possibly catching the oxidizing species
before they could reach the photosensitizer.
The anticancer photodynamic activity of PMMA@PorVa-05 and PMMA@PorVa-06 was studied in
vitro in HCT116 and SKOV3 human tumor cell lines and compared with the effect produced by
unbound Porphyrin 3. The analysis of dose/response curves obtained using the same range of
Porphyrin 3 concentrations, either in its unbound form or loaded onto PMMA NPs, shows that in
HCT116 cells, PMMA@PorVa-06 is as potent as Porphyrin 3, while in SKOV3 cells no significant
differences in IC50 values were observed among the three formulations. Similarly to what has been

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described for other photosensitizers 39–41, apoptosis appears to play a pivotal role in tumor cell death
induced by the three formulations. More specifically, no significant differences were observed in
HCT116 cells following exposure to equi-toxic concentrations of the three compounds
(corresponding to their IC50 values). On the other hand, while Porphyrin 3 was able to induce the
same percentage of apoptosis in SKOV3 and HTC116 cells, both PMMA@PorVa nanoparticles
were less effective in inducing apoptosis in this cell line (> 45% vs < 25%). Provided that comparable
intracellular concentrations of the three formulations are achieved and that IC50 values are similar for
all compounds, this result might suggest that other modalities of cell death, such as necrosis or
autophagy, could be involved when treating this cell line with PMMA@PorVa-05 and
PMMA@PorVa-06. However, the present study does not specifically address this issue; therefore,
we cannot rule out whether and to what extent these mechanisms contribute to the overall
cytotoxicity of the system.
42,43
PS uptake has been reported to correlate with activation of cell death program in PDT
.
Therefore, we performed a flow cytometric analysis of HCT116 and SKOV3 cells incubated with
Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 exploiting the intrinsic fluorescence of
Porphyrin 3. As compared to untreated controls, both HCT116 and SKOV3 cells displayed a
significant intracellular fluorescent signal 24 h after treatment with 100 nM of the PS, regardless to
its formulation. In SKOV3 cells, a higher uptake of free Porphyrin 3 was observed, which could
partially explain the differences observed in apoptosis induction. However, as discussed above, the
ability to induce photodynamic cells killing does not seem to rely exclusively on apoptosis induction,
especially in SKOV3 cells. Indeed, at the cellular level, irreparable damage to mitochondria,
endoplasmic reticulum and lysosomes caused by singlet oxygen and/or other reactive oxygen species
could lead to necrotic, apoptotic or autophagic cell death, depending on different factors, such as the
cell type, the photosensitizer structure/formulation and subcellular localization and the treatment
protocol 44.
In photodynamic therapy, light is used to elicit the photosensitizer to produce singlet oxygen and
ROS, which are considered the main effectors of tumor cells death 45,46; therefore, we considered the
ability of Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 to generate reactive oxygen
species. In particular, ROS production induced by equitoxic concentration of the three formulations
was determined by flow cytometric analysis of DCF fluorescence.
Our results indicate that
Porphyrin 3 is more effective than PMMA@PorVa in generating ROS; in particular, in HTC116 cells
a ratio (treated/control) of 9.3, 2.21 and 3.42 was observed for Porphyrin 3, PMMA@PorVa-05 and
PMMA@PorVa-06, respectively while in SKOV 3 cells this ratio was of 12.8, 3.6 and 1.52. Singlet

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oxygen quantum yields of Porphyrin 3, PMMA@PorVa-05 and PMMA@PorVa-06 were measured
through spectrophotometric analysis, indicating that Porphyrin 3 has a greater singlet oxygen
quantum yield (1.6-1.7 fold) once loaded onto PMMA-NPs. These results suggest that, depending on
its formulation, Porphyrin 3, as free form or bound to nanoparticles, exerts its photodynamic activity
preferentially through type I (ROS production) or type II reaction (singlet oxygen production),
respectively. Both type I and type II reactions occur simultaneously, and their ratio depends on
several factors, such as the type of sensitizer, the concentrations of substrate and oxygen, as well as
the binding affinity of the sensitizer for the substrate 47. Moreover, it has been shown that, when
loaded onto NPs, the carrier microenvironment may affect the photo-physical properties of PS and
48
subsequently, its biological efficacy/modality in killing cancer cells
Conclusion
.
5
In conclusion, our results demonstrate that PMMA-NPs covalently loaded with a photosensitizer can
be easily obtained with different PS loading ratios, starting from an appropriately functionalized
hydrophobic sensitizer. Our data confirm that once loaded onto nanoparticles, the PS maintains its
ability to kill cancer cells upon light irradiation. Indeed, in vitro experiments performed on two
different cancer cells lines exhibited similar cytotoxic effects, whether treated with the unbound PS
or the NP formulations. Although further studies are needed to clarify the mechanisms underpinning
the action of PMMA@PorVa, this strategy paves the way to the preparation of a novel class of NPs
covalently functionalized with PS, therefore allowing the precise tuning and targeting of PSs.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest.
Author Contributions
G.V., A.G., M.B. and E.C. conceived the study. G.V. M.G., E.M., C.F. and E.C. performed
experiments, analyzed the data and wrote the paper. M.B., E.C., A.G., C.F. and G.S. performed
compound synthesis and characterization and analyzed the data. M.G., E.M. and E.C. performed in
vitro studies. All authors reviewed the manuscript.
Funding
This work was supported by the Investigator Grant of the Italian Association for Cancer Research
(AIRC) 16740 to G.V.

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Supplementary Material
Supplementary Material includes titration curves of PMMA@PorVa nanoparticles; AFM images of
PMMA@PorVa nanoparticles; copies of Porphyrin 3 NMR spectra; DLS and zeta-potential
measurements.

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Highlights




Core-shell PMMA nanoparticles covalently loaded with a non-symmetric di-aryl porphyrin
were synthesized and characterized
Porphyrin-loaded PMMA nanoparticles displayed IC50 values in the nanomolar range on
HTC116 and SKOV3 cell lines
Porphyrin-loaded PMMA nanoparticles produce ROS and singlet oxygen upon light
irradiation
Porphyrin-loaded PMMA nanoparticles could represent a valuable system for in vivo PDT
applications