Highly Porous Hybrid Metal–Organic Nanoparticles Loaded with Gemcitabine Monophosphate: a Multimodal Approach to Improve Chemo- and Radiotherapy

Article abstract of DOI:10.1002/cmdc.201900596

Nanomedicine recently emerged as a novel strategy to improve the performance of radiotherapy. Herein we report the first application of radioenhancers made of nanoscale metal-organic frameworks (nanoMOFs), loaded with gemcitabine monophosphate (Gem-MP), a radiosensitizing anticancer drug. Iron trimesate nanoMOFs possess a regular porous structure with oxocentered Fe trimers separated by around 5 ? (trimesate linkers). This porosity is favorable to diffuse the electrons emitted from nanoMOFs due to activation by γ radiation, leading to water radiolysis and generation of hydroxyl radicals which create nanoscale damages in cancer cells. Moreover, nanoMOFs act as “Trojan horses”, carrying their Gem-MP cargo inside cancer cells to interfere with DNA repair. By displaying different mechanisms of action, both nanoMOFs and incorporated Gem-MP contribute to improve radiation efficacy. The radiation enhancement factor of Gem-MP loaded nanoMOFs reaches 1.8, one of the highest values ever reported. These results pave the way toward the design of engineered nanoparticles in which each component plays a role in cancer treatment by radiotherapy.

Full text of DOI:10.1002/cmdc.201900596

Accepted Article
Title: Highly porous hybrid metal-organic nanoparticles loaded with
gemcitabine-monophosphate: a multimodal approach to improve
chemo and radiotherapy
Authors: Xue LI, Christian Serre, Erika Porcel, Mario Menendez-
Miranda, Jingwen Qiu, Xiaomin Yang, Alexandra Pastor,
Didier Desmaële, Sandrine Lacombe, and Ruxandra Gref
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To be cited as: ChemMedChem 10.1002/cmdc.201900596
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900596
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10.1002/cmdc.201900596
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Highly porous hybrid metal-organic nanoparticles loaded with
gemcitabine-monophosphate: a multimodal approach to improve
chemo and radiotherapy
Xue Li, [a] Erika Porcel, [a] Mario Menendez-Miranda, [a] Jingwen Qiu, [a] Xiaomin Yang, [a] Christian
Serre, [b] Alexandra Pastor, [c] Didier Desmaële, [c] Sandrine Lacombe*,[a] Ruxandra Gref*[a]
[a]
Dr. X. Li (0000-0003-0254-643X), Dr. E. Porcel (0000-0001-8048-4276), Dr. M. Menendez-Miranda (0000-0002-2992-1351), J. Qiu (0000-0003-4344-
3728), X. Yang (0000-0003-0372-148X), A. Pastor, Dr. S. Lacombe (0000-0003-3334-9201), Dr. R. Gref (0000-0002-7869-0908)
Institut de Sciences Moléculaires d'Orsay,
UMR CNRS 8214, Université Paris-Sud, Université Paris-Saclay
Rue André Rivière, 91405 Orsay Cedex, France.
Emails : ruxandra.gref@u-psud.fr ; sandrine.lacombe@u-psud.fr
Dr. C. Serre (0000-0003-3040-2564)
Institut des Matériaux Poreux de Paris, FRE 2000, Ecole Normale Supérieure de Paris,
Ecole Supérieure de Physique et de Chimie Industrielles de Paris, PSL Research University
24 rue Lhomond, 75005 Paris, France
[b]
[c]
Dr. D. Desmaële (0000-0003-4013-4655)
Institut Galien,
UMR CNRS 8612, Université Paris-Sud, Université Paris-Saclay,
5 Rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France.
Supporting information for this article is given via a link at the end of the document
Abstract: Nanomedicine recently emerged as a novel strategy to
improve the performance of radiotherapy. Here we report the first
application of radioenhancers made of nanoscale metal-organic
frameworks (nanoMOFs), loaded with Gemcitabine-monophosphate
Gem is a prodrug which relies on the intracellular enzyme
deoxycytidine kinase to form its active intermediates: mono-, di-,
and triphosphate derivatives.[6] It has been shown both in vitro and
in vivo that after phosphorylation this drug exerts its cytotoxic
effects and induces radiosensitization mainly through inhibition of
DNA synthesis.[7] However, the first intracellular phosphorylation
of Gem is the rate-limiting step and especially difficult for resistant
cancer cells.[6,8] The administration of Gem monophosphate
(Gem-MP) is hampered by its poor stability in biological media
and poor cellular uptake.[6] Moreover, because of lack of tumor
targeting and specificity, healthy-tissue toxicity of this drug is the
major drawback.[5,9] In this challenging context, some of us have
shown that Gem-MP could be protected against degradation with
increased cellular uptake by encapsulation in nanoparticles
(NPs).[10] Nanotechnology has proven the effectiveness to target
tumors, strategy of particular interest for drug delivery by
achieving drug transcytosis, drug targeting and theranostics.[11,12]
In addition to the benefits of NPs for drug delivery, the potential
value of metal based NPs as radioenhancers has been
discovered in 2000.[13,14] Currently, there are several types of
high-Z NPs in clinical development, including Au NPs, Gd NPs,
and crystalline HfO2 NPs endowed with a targeting coating.[15]
More recently, porous Hf based nanoscale metal organic
frameworks (nanoMOF) outperformed dense HfO2 NPs.[16–18] The
combination of nanoMOFs mediated radiotherapy with checkpoint
blockade has been shown as a promising strategy to broaden the
application of immunotherapy.
(Gem-MP),
a radiosensitizing anticancer drug. Iron trimesate
nanoMOFs possess a regular porous structure with oxocentered Fe
trimers separated by around 5 Å (trimesate linkers). This porosity is
favorable to diffuse the electrons emitted from nanoMOFs due to
activation by g radiation, leading to water radiolysis and generation of
hydroxyl radicals which create nanoscale damages in cancer
cells. Moreover, nanoMOFs act as “Trojan horses”, carrying their
Gem-MP cargo inside cancer cells to interfere with DNA repair. By
displaying different mechanisms of action, both nanoMOFs and
incorporated Gem-MP contribute to improve radiation efficacy. The
radiation enhancement factor of Gem-MP loaded nanoMOFs reaches
1.8, one of the highest values ever reported. These results pave the
way towards the design of engineered nanoparticles in which each
component plays a role in cancer treatment by radiotherapy.
Introduction
Despite advances and refinements in cancer early detection and
treatment, the vast majority of human malignancies are not
effectively eradicated and there is a clear need to develop more
efficient
treatments
based
on combined modalities.
Chemoradiation, the combination of chemotherapy and
radiotherapy, is now the standard of care for many of solid tumors,
including lung, esophageal, head and neck cancers.[1–3]
Advantageously, synergistic effects were reported in
chemoradiation, since some anticancer drugs not only interfere
with cell metabolism, but they also make cancer cells more
sensitive to radiotherapy.[4] In clinical studies, Gemcitabine (Gem)
showed improved therapeutic effects in combination with
irradiation even at very low dosages (less than 50 mg/m2 per
week).[5]
However, there are still no examples of NPs engineered to act
both as radioenhancers and nanocarriers to target anticancer
drugs to the tumors. We developed here this concept using
porous nanoMOF loaded with Gem-MP. Iron (III) trimesate
nanoMOFs MIL-100(Fe) (MIL stands for Materials from Institut
Lavoisier) were shown to be biodegradable and devoid of toxicity
in vivo.[19–21] Furthermore, they act as efficient contrast agents for
Magnetic Resonance Imaging (MRI) constituting promising
devices for theragnostic applications.[22] NanoMOFs’ surfaces can
1
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also be engineered for cancer cell targeting and for long blood
circulation purposes.[22–26] Due to their hydrophilic/hydrophobic
character and high surface areas up to 1700 m2/g,[27] iron(III)
trimesate nanoMOFs were successfully loaded with
unprecedented amounts (up to 20-70 wt%) of a large variety of
drugs able to penetrate within their open porous MOF
structures.[27–29] For instance, phosphorylated drugs such as
Gem-MP[10], AZT-MP[30], and AZT-TP[30,31] were loaded with
efficiencies close to 100%.
MIL-100(Fe) nanoMOFs are assembled up from Fe(III)
oxocentered trimers of octahedra and trimesate linkers (1,3,5-
benzene tricarboxylate) that self-assemble to build a 3D porous
architecture delimiting large (29 Å) and small (24 Å) mesoporous
cages (Figure 1A). These cages are accessible for drug
adsorption in the 3D-porosity through pentagonal (5.6 Å) and
hexagonal windows (8.6 Å).[27] Iron trimesate nanoMOFs were
also shown to be non-toxic and to generate in biological media
reactive oxygen species (ROS).[29,32] In summary, Gem-MP
loaded nanoMOFs exhibit potentially favorable characteristics for
multimodal cancer treatment: i) they efficiently load and protect
Gem-MP; ii) their surface can be functionalized in a versatile
manner to control the interactions with biological systems and iii)
they are non-toxic, biodegradable, and potentially allow
monitoring by MRI accumulation at the tumor and disease
evolution (theranostics).
Figure 1. Schematic representation of the “green” hydrothermal synthesis of
nanoMOFs and Gem-MP encapsulation (A); Fast processes (t < 10−12 s)
involved in nanoMOF excited by  radiations (B), including the secondary
electron generation and ROS production. The porous 3D structure of nanoMOF
is prone to facilitate ROS diffusion. Morphology of as-synthesized nanoMOFs
(C), Gem-MP loaded nanoMOF before (D) and after (E) 24 h incubation in water;
and irradiated nanoMOFs (F); scale bar in C-F: 50µm.
Gem-MP and nanoMOFs due to coordination of the phosphate
moiety of Gem with the Fe trimers which possesses
two accessible unsaturated metal sites per trimer.[10,30]
Encapsulation of Gem-MP did not affect the integrity of the 3D
crystalline nanoMOF structures as demonstrated by (i)
morphology and size unaffected (Figure 1D) (ii) similar PXRD
(Figure S1) and DLS mean diameters (221 ± 18 nm versus 217 ±
25 nm, Figure S2) as well as (iii) less than 1% release of their
constitutive ligand, trimesate. NanoMOF suspensions loaded with
10 wt% Gem-MP were also stable in water during storage, with
preserved morphology (Figure 1E) and neither detectable
(<1%) Gem-MP release nor ligand detachment (<1%) over one
day, in agreement with our previous study.[10] Gem-MP release
studies were also conducted in DMEM cell culture media.
Hydrodynamic diameter didn’t change after 6 h incubation (Figure
S2). As expected, nanoMOFs progressively degraded, which
resulted in around 15% of Gem-MP release in the suspension
media after 6 h incubation. This degradation is triggered by the
phosphates from the media that coordinate to the iron sites
leading to ligand and drug release.[42] In our case, Gem-MP
release remained lower than 20% after 24 h incubation in cell
culture medium. (Figure S3). The efficient drug encapsulation
and the stable formulation built the foundation for biological
evaluation.
Here we demonstrate in vitro that nanoMOFs amplify the effect of
-rays on tumor cells killing, and for the first time, we combine
radiotherapy and chemotherapy using Gem-MP loaded
nanoMOFs. As a whole, this confers to these new type of
nanoagents the unique synergistic effect to enhance by a factor
of 1.8 the radiotoxicity of the treatment.
Results and Discussion
NanoMOFs synthesis and drug incorporation
Several methods have been described for the synthesis of MIL-
100(Fe) nanoMOFs [33–38]among which microwave-assisted
solvent-free “green” hydrothermal techniques are the most
effective to obtain high yields of nanoMOFs with controlled
sizes.[34] The nanoMOFs are crystalline (Figure S1) with
diffraction patterns in agreement with previous studies.[39,40] They
possessed
a facetted morphology (Figure 1C) and their
hydrodynamic diameters, characterized by dynamic light
scattering (DLS) and NP tracking analysis (NTA), were 217 ± 25
nm and 200 ± 64 nm, respectively (Figure S2). NTA tracks all the
individual NPs in their Brownian motion and thus gives the size
distribution based on number, whereas DLS measures the
fluctuations of the scattered light and is more sensitive to largest
Toxicity of nanoMOF, Gem-MP, and Gem-MP loaded
nanoMOF
These studies were completed by toxicity investigations of
nanoMOFs loaded or not with Gem-MP on HeLa cells used as
cellular probes. The cells were incubated with the products in the
same conditions as the ones used for irradiation studies (Figure
S4). As expected, free Gem-MP induced a cytotoxic effect killing
50% of cells after 6 h incubation at concentrations of 1.7 µg/mL.
In contrast, no significant toxicity (>95% cell viability) was
observed with unloaded nanoMOF at concentrations of 17 µg/mL.
At equivalent concentrations, the toxicity of Gem-MP was similar
whether the drug was free or incorporated in the nanoMOFs (cell
viability of 536.4% for Gem-MP & 528.1% for Gem-MP loaded
particles.
A
good agreement was found between the
complementary DLS and NTA methods (less than 10%
differences) as previously reported with other types of NPs.[41]
NanoMOFs displayed a BET (Brunauer–Emmett–Teller) specific
surface of 1740 ± 100 m2.g-1, in agreement with previously
reported data.[39,40]
Due to the strong coordination between phosphate groups and
the unsaturated iron(III) Lewis acid sites from their framework,
nanoMOFs acted as efficient “molecular nanosponges”, soaking
up Gem-MP from its aqueous solution within the large
mesoporous cages (29 Å). Almost a perfect loading efficiency
(>98%) was obtained, confirming the strong interaction between
2
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10.1002/cmdc.201900596
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nanoMOFs). The lack of toxicity of nanoMOFs in the irradiation
conditions set up the basis for the further biological investigation.
reached 7.1±0.1 wt% and a blue shift of 2-3 nm was observed for
Rhod-Ada emission after adsorption on the surface of nanoMOFs
(Figure S6B). In contrast with parent Rhod which detached readily
from nanoMOFs (Figure 2B), Rhod-Ada showed a low release
from the nanoMOFs in cell culture media, i.e. less than 10% after
Localization and quantification of nanoMOF in HeLa cells
Prior to irradiation experiments, the internalization of nanoMOF in
HeLa cells was investigated using confocal microscopy. This tool
allows detecting optical sections within cells, thus unravelling the
location of the NPs. However, confocal microscopy requires
labelling of the NPs with fluorescent dyes. Iron based nanoMOFs
are challenging to be labelled because of dye fluorescence
quenching in the pores or desorption from the surface once in
contact with biological media. Figure 2A illustrates these issues:
fluorescein [24], one of the most widely employed dyes, can
penetrate inside the porous 3D nanoMOF structure but its
fluorescence was quenched. In contrast, Rhodamine (Rhod) with
bulkier structure, unable to enter in the pores of the MOF, was
efficiently associated to the coating of the outer surface of the
nanoMOFs up to 8.2±0.2 wt%, thus allowing a successful labelling.
However, around 43 wt% of Rhod was immediately released
within 5 minutes (“burst release”) upon incubation in cell culture
medium (Figure 2B).
6
h incubation (Figure 2C). As a conclusion, the newly
synthesized Rhod-Ada ensures stable coatings for biological
evaluations such as the study of the interactions between
nanoMOFs and HeLa cells using confocal microscopy.
In this context, to achieve an efficient Rhod labelling, stable in
biological media, a new Rhod derivative functionalized with a
hydrophobic anchor was synthesized. Adamantane (Ada) was
chosen as lipophilic anchoring agent as its molecular size is well
adapted to insert as a guest into the cages at the nanoMOFs
surface, whereas the bulky hydrophilic Rhod would deploy in the
suspension medium, avoiding quenching. 1-Adamantane acetic
acid was successfully attached to Rhod via a short covalent linker
as depicted in Figure S5. Briefly, an excess of 2,2'-
(ethylendioxy)bis(ethylamine) (1) was reacted with 1-adamantane
acetyl chloride to give the corresponding mono amide (2), which
was further acylated with succinic anhydride to provide the
carboxylic acid (3). Rhod-Ada (5) was obtained by reaction with
rhodamine piperazine (4) using EDCI as coupling agent by
adapting a published procedure.[43] The purified Rhod-Ada had
Figure 3. Confocal images of nanoMOF internalization in HeLa cells.
Confocal images of HeLa cells after 6 h incubation with Rhod-Ada labeled
nanoMOFs (100µg/mL) followed by intensive washing with PBS. (scale bar
25µm).
As shown in Figure 3 and the video in the supporting information,
the confocal images showed high intensity inside the cytoplasm,
despite extensive washing to remove loosely adhered nanoMOFs.
This demonstrates that nanoMOFs localized in the cytoplasm but
not in the nucleus. The surface modification of nanoMOFs with
Rhod-Ada could affect cellular uptake and distribution, therefore
the quantification of intracellular nanoMOFs with or without Rhod-
Ada was performed.
The quantity of nanoMOFs internalized in the cells was
determined using a procedure based on iron staining with
potassium ferrocyanide.[25] This quantification of iron content in
cells was performed for concentrations ranging from 25 to 200
µg/mL nanoMOFs added to HeLa cells for 6 h incubation. Prior to
the iron dosage, the cells were washed to remove non adhered
particles and then digested. As shown in Figure 4, the amount of
nanoMOFs internalized in HeLa cells increased almost linearly as
a function of the nanoMOF concentration. This calibration curve
was used to determine the amount of internalized nanoMOFs at
the concentration of 17 µg/mL where there was no toxicity
observed. It was found that around 8 pg nanoMOFs were
associated per cell, corresponding to 1.5 pg Fe/cell, either
internalized or firmly attached on the cell membrane.
similar
fluorescence
spectrum
as
Rhod,
showing
Excitation/Emission = 566 nm/589 nm (Figure S6A).
Figure 2. NanoMOF labeling with fluorescent dyes. A. Schematic
representation of two strategies for nanoMOF labeling: i) fluorescent dyes which
penetrate inside the porous structure of nanoMOFs, such as fluorescein,
eventually quenched; ii) nanoMOFs labeled by adsorption of fluorescent dyes
(Rhod, Rhod-Ada) on the surface maintain their fluorescence (red star). Rhod
(B) and Rhod-Ada (C) release kinetics in cell culture medium.
Rhod-Ada was efficiently adsorbed on the surface of nanoMOFs
by a simple solvent-free method, consisting in 12 h incubation
under gentle stirring of nanoMOF aqueous suspensions
containing Rhod-Ada. The amount of associated Rhod-Ada
3
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where D10 nanoMOFs and D10 control correspond to the radiation doses
used to reach 10% of cell survival fraction in the samples treated
with nanoMOFs and in the controls, respectively.
Figure 4 Quantification of nanoMOFs internalized in HeLa cells. Gray: as-
synthesized nanoMOFs; red: Rhod-Ada labelled nanoMOFs. NanoMOFs with
different concentrations (25, 50, 100, 150 and 200 µg/mL) were incubated with
2×105 HeLa cells in 24 well plates for 6 h, and the mass of internalized
nanoMOFs was determined respectively.
As reported in Table 1, REF value of nanoMOF alone reached 1.2.
The DEF (2Gy) and DEF (4 Gy) of nanoMOF alone were found
close to 1.3 and 1.5, respectively. This is comparable to
the effects observed by Mazur et al. with iron oxide NPs, showing
DEF (1-4 Gy) values in the range of 1.1-1.6.[49,50] This clearly
indicates that nanoMOFs enhance radiation effects. Remarkably,
the equivalent iron concentration in the nanoMOFs used in this
study was much lower than the one reported in previous studies
with other iron oxide NPs (nanoMOFs contain 56 µM Fe
compared to iron oxide NPs containing 3.6 mM ~17.9 mM Fe[49,50]).
Moreover, the incubation time was also much shorter (6 h for
nanoMOFs in comparison with 72 h for iron oxide NPs[49,50]). The
amount of Fe internalized in cells in the case of iron oxide NPs
reached up to 40 pg/cell, whereas in this study, it was only 1.5
pg/cell. As a conclusion, the porous iron trimesate nanoMOFs are
much more efficient to amplify radiation effects of -rays than solid
iron oxide NPs. To the best of our knowledge, this is the first time
that a radioenhancing effect of Fe-based porous material is
reported.
The radioenhancement effect is due to the succession of physico-
chemical processes as described elsewhere for high-Z
compounds and dense NPs.[15] Of note we present here a
radioenhancement effect with porous low density NPs containing
iron.[15] Briefly, the radioenhancement observed with the
nanoMOFs may be explained by the activation of Fe atoms
interacting with gamma rays, followed by de-excitation and
emission of electrons. The interaction of emitted electrons with
surrounding water molecules is responsible for the production of
hydroxyl radicals, which are toxic for the cells.[15] Thus, the toxicity
of a radiation treatment increases with the number of electrons
interacting with water molecules. Therefore it is expected that the
surface area of the NPs plays an important role[51] because the
production of radicals increases with the porosity of the agent.
Inversely, the number of electrons is expected to decrease when
electrons do not travel to the surface, which is the case for
increasing diameter of the solid particle. Advantageously, MIL-
100(Fe) nanoMOFs possess a high specific BET surface area up
to 1740 m2.g-1. Moreover, their regular 3D structure with well
dispersed Fe centers separated by distances of around 5 Å
(trimesate linkers), is favorable for a better electron traveling
through the interconnected network to enhance hydroxyl radical
generation and facilitate their fast diffusion in the surrounding
biological medium. As a consequence, nanoMOFs act efficiently
as radioenhancers despite the relatively low electron density of
the emitting atoms (Fe with Z of 26), as compared to Gd (64), Au
(79), Pt (78) or Hf (72).
Impact of nanoMOFs, Gem-MP, and Gem-MP loaded
nanoMOFs on radiation effects
Prior to irradiation studies, the stability of nanoMOFs, loaded or
not with Gem-MP and Gem-MP alone was investigated upon
radiation using doses ranging from 4 to 6.5 Gy.
Noteworthy, even at the highest dose, nanoMOFs kept similar
facetted morphology as non-irradiated samples (Figures 1A and
1F) with neither significant size variation after irradiation, as
shown by NTA and DLS methods (Figure S2), nor degradation as
indicated by the negligible loss of their constitutive ligand,
trimesate (<1%, Table S1). Gem-MP was not degraded either, at
the highest irradiation dose. Remarkably, nanoMOFs did not lose
their Gem-MP content upon irradiation (Table S1), as indicated by
negligible release of Gem-MP. These data further confirm the
strong interaction between Gem-MP and nanoMOFs.
Clonogenic assay was then used to investigate the impact of
nanoMOF, Gem-MP, and Gem-MP loaded nanoMOFs on cell
killing induced by radiation. The colony-forming potential of cells
were assessed after treatment with nanoMOFs, Gem-MP, and
Gem-MP loaded nanoMOFs using a standardized procedure,
where the radiation dose was fixed in the range of 1~6.5 Gy,
comparable to the clinical dosage. [44,45]
Figure 5 The effect of nanoMOFs and Gem-MP on irradiation enhancement.
Survival fractions of HeLa cells irradiated by  rays, in the presence of nanoMOF
(red), Gem-MP (blue), Gem-MP loaded nanoMOF (pink), and in the control
(black).
The survival curves of HeLa cells in the presence or not of Gem-
MP, nanoMOFs and Gem-MP loaded nanoMOFs, irradiated with
-rays, are presented in Figure 5. The cell survival fractions (SF)
decreased exponentially with the increase of the radiation dose,
Table 1. Summary of REF (radiation enhancement factor), DEF(2Gy) (dose
enhancement factor at 2Gy), and DEF(4Gy) obtained with HeLa cells irradiated
in agreement with previous reports.[45–48] Interestingly, the bare
nanoMOFs activated by -rays decreased significantly the cell
survival. The efficiency of nanoMOFs to amplify radiation-induced
cell death was evaluated by calculating the dose enhancement
factor (DEF) and the radiation enhancement factor (REF)[15]
defined by the equations (1) and (2).
by
 source, in the presence of nanoMOFs, Gem-MP, Gem-MP loaded
nanoMOFs. Control sample was HeLa cells treated by only  irradiation.
Sample
control
REF
DEF(2Gy) DEF(4Gy)
DEF=SFcontrol/SFnanoMOFs
(1)
1
1
1
where SFcontrol and SFnanoMOFs correspond to SF of the control and
SF of treated cells at a certain dose, respectively.
nanoMOF
1.19
1.35
1.83
1.30
1.58
2.68
1.53
2.23
6.47
REF=D10control/D10
(2)
Gem-MP
nanoMOFs
Gem-MP loaded nanoMOF
4
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To characterize the type of lesions amplified by nanoMOFs, SF
sensitive to irradiation.[52] Upon irradiation, nanoMOFs amplified
the radiation effect by generating high amounts of ROS, which
generated DNA damages. Gem-MP hindered reparation of DNA
double-strand breaks in particular. Moreover, as previously
shown, when Gem-MP loaded nanoMOFs were incubated with
HeLa cells, they penetrated inside the cells, thus promoting
interaction between the drug and the cells as compared with free
drug unable to bypass cell membranes. This could be another
reason for the improved anticancer activity after irradiation of
encapsulated drug that is of high interest for clinical applications.
curves were simulated using the linear quadratic law (Eq. (3))[45]
:
SF(D)=exp‐(D+D2)
where D is the dose of irradiation. The coefficient  corresponds
to the contribution of directly-lethal lesions, whereas is
(3)

attributed to the contribution of additive sub-lethal lesions. The
values of  and  determined by a fitting procedure are reported
in Table 2. The presence of nanoMOFs alone induces an increase
in a (0.30 for nanoMOFs & 0.22 for the control). This indicates an
enhancement of the direct lethality of the radiation treatment in
the presence of nanoMOFs.
In
conclusion,
radiosensitization
of
Gem-MP
and
radioenhancement of nanoMOFs complement each other to
improve irradiation performance, synergistically contributing to
eradicate cancer cells upon irradiation.
Table 2 Calculated coefficients a and b for HeLa cells irradiated by  source, in
the presence of nanoMOF, Gem-MP, Gem-MP loaded nanoMOF, and in the
control (HeLa cells treated by only  irradiation). The dependency R2 values are
reported in the last column.
Nanoscale impact of nanoMOFs
To gain further insights onto the physico-chemical effects induced
on biological molecules by nanoMOFs activated by radiation, we
used plasmids as molecular nanoprobes to quantify the induction
of nanosize (complex) damages.[44,54] In particular, we quantified
double strand breaks (DSBs), which are bigger than 2 nm
(distance between two strands), to probe the effect of nanoMOFs
on the induction of nanosize damages (Figure 1B). The addition
of nanoMOFs alone (without irradiation) did not induce any
damage. Upon irradiation, the number of single strand breaks
(SSB) kept constant (less than 5% variation) in comparison to
control, whereas DSBs increased with the dose, indicating that
nanosize damages were induced. The effect of nanoMOFs on
DSB is illustrated in Figure 6, where the nanoprobe was mixed
with nanoMOFs and irradiated at a dose ranging from 0 up to 115
Gy. The yield of DSB, defined as the number of breaks induced
per plasmid and per gray, corresponds to the slope of the dose
response curves.[45] Remarkably, when the ratio of nanoprobe
over nanoMOFs was close to 200:1, the induction of DSBs was
dramatically enhanced (Figure 6). In the dose range of 0~70 Gy,
DSB increased linearly with the dose, in agreement with
previously reported studies with Gd-based NPs.[45] The yield of
DSB increased 2.33 times (0.003 for the control and 0.007 with
the addition of nanoMOFs). In the dose range 70~115 Gy, DSB
increased in an exponential manner (Fitting: DSB= 4×10-5x2 -
0.0045x + 0.1831, x=dose). DSB was approximately 5 times
higher than the control when irradiated at 115 Gy.
Sample
control
a (Gy-1)
b (Gy-2)
R2
0.006±
0.002
0.22 ± 0.01
0.9995
nanoMOF
Gem-MP
0.30 ± 0.01
0.36 ± 0.01
0
0
0.9979
0.9933
Gem-MP
nanoMOF
loaded
0.44 ± 0.01
0
0.9989
The same data analysis was performed for cells treated with
Gem-MP. We found that Gem-MP amplified cell death by DEF
(4Gy) of 2.23 and REF of 1.35. The increase of a (from 0.22 for
the control to 0.36) shows that direct lethal damages are
increased. This corresponds to the well-known effect of the drug,
which agrees with former studies[10]. Thus, upon irradiation, the
agent induces radiosensitization of the cells, namely an
amplification of radiation effect due to perturbation of a metabolic
pathway. Gem-MP is responsible for: i) induction of early S phase
block[52] via DNA inhibition at the moment of irradiation,
contributing to more pronounced radiosensitizing effect; ii)
inhibition of RAD51-dependent repair for DNA breaks induced by
irradiation.[53] This effect is distinct from the “radioenhancement”
presented above (which is related to early stage electronic
process).
These results suggest the activation of physico-chemical
processes including the emission of electrons and consecutive
production of water radicals, responsible for the induction of
nanosize damages as explained before. In particular, the
emission of electrons in water and therefore the production of
reactive radicals are facilitated by the porous structure of the
nanoMOFs. It thus confirms that nanoMOFs amplify the induction
of nanosize damage due to physico-chemical effects and to the
porous character of this agent.
For Gem-MP loaded nanoMOF, an unprecedented effect was
observed. In this case, cell killing drastically increased as
witnessed by the REF of 1.83 (as compared to 1.19 for nanoMOF,
and 1.35 for Gem-MP alone) and by the DEF (4 Gy) up to 6.47
(as compared to 1.53 for nanoMOF and 2.23 for Gem-MP). To the
best of our knowledge, the REF obtained with high-Z NPs is in the
range of 1.1 to 1.6.[15,56] Thus, the amplification effect obtained by
Gem-MP loaded nanoMOF is among the highest ever reported.
This is associated with a doubling of the directly lethal damage as
illustrated by a increase from 0.22 (for the control) up to 0.44,
much higher than that of nanoMOFs (0.30) and Gem-MP (0.36)
alone. This demonstrates for the first time that the use of drug
carried nanocages (Gem-MP loaded nanoMOFs) are able to
combine radioenhancement and radiosensitization, which results
in a much higher radio-enhancement effect superior to each
element (nanoMOF and Gem-MP) considered separately. This
may be explained as follows. When Gem-MP loaded nanoMOFs
were incubated with HeLa cells, Gem-MP probably inhibited DNA
synthesis and induced cell block in early S phase, which is more
5
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10.1002/cmdc.201900596
ChemMedChem
FULL PAPER
carbodiimide (EDCI), hydroxybenzotriazole (HOBt), ethyl ether (Et2O),
sodium hydroxide (NaOH) and 2,2’-(ethylendioxy)bis(ethylamine) were
obtained from Sigma-Aldrich (France). Rhodamine piperazine was
obtained according to T. Nguyen et al.[43] Chemicals obtained from
commercial suppliers were used without further purification.
Cell culture. HeLa cells were grown in DMEM medium (Life Technologies,
France) supplemented with 10% v/v decomplemented fetal bovine serum
(FBS) (Life Technologies, France) at 37°C in humidified air containing 5%
CO2.
Synthesis and characterization of MIL-100(Fe) nanoMOFs
MIL-100(Fe) nanoMOFs were synthesized as previously described by
microwave assisted hydrothermal reaction.[34] Briefly, 30 mL of aqueous
mixture containing 6.0 mM of iron chloride hexahydrate and 4.02 mM of
trimesic acid was heated by microwave at 130 °C for 6 min under stirring
Figure 6 Yields of nanosize lesions in nanoprobes (plasmids) upon  rays
irradiation in the presence of nanoMOFs for different plasmid:nanoMOFs ratio
(red: plasmids control; green: plasmid:nanoMOFs (number ratio) = 2000:1;
purple: plasmid:nanoMOFs (number ratio) = 200:1).
with power of 1600
W (Mars-5, CEM, USA). The as-synthesized
nanoMOFs were recovered by centrifugation at 10,000 g for 15 min and
washed six times with absolute ethanol to remove unreacted trimesate.
The dynamic diameter of nanoMOFs was characterized by dynamic light
scattering (DLS; Malvern Nano-ZS, Zetasizer Nano series, France). The
porous surface area was measured by nitrogen sorption experiments at -
196 °C on an ASAP 2020 (Micromeritics, USA) after sample degassing at
100 °C for 15 h under vacuum. NanoMOFs were stored in ethanol at room
temperature until further usage.
Conclusions
Here we report the first proof of concept of nanoMOFs loaded with
an anticancer drug to eradicate tumor cells upon irradiation.
Whereas free Gem-MP is a powerful radiosensitizer, iron
trimesate nanoMOFs were shown to possess intrinsic
radioenhancement properties taking advantage of the ordered
porous 3D structure. This approach could be extended to other
drugs, provided that they are not subject to degradation upon
irradiation. Non-toxic nanoMOFs acted as “Trojan horses”
significantly penetrating inside cancer cells, thus promoting the
cytotoxic action of entrapped Gem-MP. By displaying different
mechanisms of action, both nanoMOFs activated by radiation and
their Gem-MP cargo synergistically contributed to kill cancer cells.
Given that nanoMOFs could act as contrast agent for MRI imaging,
Drug loading and release studies
NanoMOFs suspensions in ethanol (1.0 mg) were centrifuged at 10,000 g
for 10 min, supernatants were discarded and the pellets were re-dispersed
in 1.0 mL of Gem-MP aqueous solution (100 µg/ml). After gently stirring
overnight, the drug-loaded nanoMOFs were recovered by centrifugation
(10,000 g, 10 min) and the supernatant was analyzed by HPLC to
determine the amount of non-encapsulated Gem-MP. For the release
studies, the Gem-MP loaded nanoMOFs (1.0 mg/mL) were centrifuged
(10,000 g, 10 min) and redispersed in cell culture medium Dulbecco
Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine
Serum (FBS), 1% penicillin/streptomycin (100 mg/mL), and 1% L-
Glutamine. Drug release studies were performed at 37°C with nanoMOFs
concentrations of 2.5 mg/mL. After different incubation times (30 min, 2 h,
4 h, 6 h, and 8 h), the samples were centrifuged (10,000 g, 10 min). The
pellets were redispersed in 10 mM phosphate buffered saline (PBS) by
vortex to totally degrade the nanoMOFs. Extracted Gem-MP was
assessed by HPLC to determine the nanoMOF drug loadings.
this opens
a
new perspective for imaging guided
chemoradiotherapy, paving the way towards a new paradigm of
NPs aided radiotherapy using drug loaded radioenhancing
carriers to synergistically play on different modes of action and
improve cancer treatments.
HPLC methods. Detection of Gem-MP and TA was carried out by HPLC
(Agilent 1100, USA) using a Phenomenex C18 column (4.6 × 250 mm, 5
μm) at a flow rate of 1.0 mL/min. A mobile phase consisting of 84% buffer
(0.2 M Triethylamine Acetate): 16% methanol was used. Compounds were
detected at 254 nm with an injection volume of 20 μl. Retention times of
trimesate and Gem-MP were 2.67 and 3.08 min, respectively.
Experimental Section
Materials
Chemical materials. Iron (III) chloride hexahydrate (98%, Alfa Aesar,
France), and 1,3,5-benzenetricarboxylic acid (trimesate, 95%, Sigma-
Aldrich, France) were used for the synthesis of nanoMOFs. Absolute
ethanol (99%, Carlo Erba, France) was used to purify the nanoMOFs. 2’,2’-
difluorodeoxycytidine monophosphate (Gem-MP) was purchased from
Toronto Research Chemicals (Canada). Triethylamine acetate and
methanol (HPLC grade) were purchased from Sigma-Aldrich (France) as
mobile phase for detection of Gem-MP.
Stability of nanoMOF, Gem-MP, and Gem-MP loaded nanoMOF upon
irradiation
NanoMOF, Gem-MP, and Gem-MP loaded nanoMOF were prepared at
the concentration of 17 µg/mL, 1.7 µg/mL, and 17µg/mL for nanoMOF,
Gem-MP and Gem-MP loaded nanoMOFs with 10 wt% drug loading,
respectively. All the samples were incubated in water at 37°C for 6 h,
followed by irradiation at 4, and 6.5 doses using a Cs-137 gamma source
(energy = 0.6617 MeV ()) generated by GSR-D1 gamma irradiator at
Institut Curie in Orsay, France. The supernatants of all samples after
irradiation were recovered after centrifugation at 17,000 g for 20 min and
analyzed by HPLC to quantify the amount of released TA and Gem-MP.
For Rhod-Ada synthesis, diethyl ether and tetrahydrofuran (THF) were
distilled from sodium/benzophenone ketyl. N,N-Dimethylformamide (DMF),
and dichloromethane (CH2Cl2) were distilled from calcium hydride, under
nitrogen atmosphere. All reactions involving air- or water-sensitive
compounds were routinely conducted in glassware which was flame-dried
under a positive pressure of nitrogen or argon. 1-Adamantane acetic acid,
oxalyl chloride, succinic anhydride, 1-ethyl-3-(3-dimethylaminopropyl)-
Fluorescent labelling of nanoMOFs.
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900596
ChemMedChem
FULL PAPER
Rhod-Ada conjugate was synthesized in order to efficiently label the
nanoMOFs.
diethyl‐3H‐xanthen‐3‐iminium chloride (Rhod-Ada, 5). To a
mixture of rhodamine piperazine (120 mg, 0.22 mmol) and acid 3 (113 mg,
0.26 mmol) in DMF (3 mL) was sequentially added HOBt (10 mg, 0.07
mmol), EDCI (56 mg, 0.29 mmol) and Hünig’s base (148 mg, 1.15 mmol).
The reaction mixture was stirred at 20 °C for 24 h and concentrated under
reduce pressure. The residue was taken up into HCl 0.1 N (5 mL) and
extracted with CH2Cl2 (3 X 10 mL). The combined organic phases were
dried over MgSO4 and concentrated to leave a dark red oil which was
chromatographed over silica gel eluting with CH2Cl2/MeOH, 20:1 and then
10:1 to give the rhodamine adamantane conjugate 5 as a dark red solid
(79 mg, 47%). 1H NMR (300 MHz, CDCl3) δ 7.90-7.67 (m, 5H, 2 NH, H-
3’Rhod, H-5’Rhod, H-6’Rhod), 7.55-7.50 (m, 1H, H-4’Rhod), 7.29 (d, J = 9.3 Hz,
2H, H-1Rhod, H-8Rhod), 7.07 (dd, J = 9.3 Hz, J = 2.4 Hz, 2H, H-2Rhod, H-7Rhod),
6.97 (d, 1H, J = 2.4 Hz, 2H, H-4Rhod, H-5Rhod), 3.69 (q, J = 7.0 Hz, 8H,
N(CH2CH3)2), 3.60 (s, 4H, OCH2CH2O), 3.57-3.30 (m, 16H,
CH2CH2OCH2CH2OCH2CH2, CON(CH2CH2)2NCO), 2.65-2.55 (m, 2H,
CH2CON), 2.50-2.42 (m, 2H, CH2CON), 2.00-1.90 (m, 5H, CH2CON, H-
3Ada, H-5Ada, H-7Ada), 1.80-1.60 (m, 12H, H-2Ada, H-4Ada, H-6Ada, H-8Ada, H-
9Ada, H-10Ada), 1.31 (t, J = 7.0 Hz, 12H, N(CH2CH3)2) ppm; 13C NMR (75
MHz, CDCl3) δ 174.7 (C, CO), 173.9 (C, CO), 172.7 (C, CO), 169.6 (C,
CORhod), 159.3 (2C, C-3Rhod, C-6Rhod), 157.2 (2C, C-4aRhod, C-4bRhod),
157.0 (C, C-1’Rhod), 136.5 (CH, C-5’Rhod), 133.2 (2CH, C-1Rhod, C-8Rhod),
132.7 (C, C-9Rhod), 131.8 (CH, C-6’Rhod), 131.3 (C and CH, C-2’Rhod, C-
32’Rhod), 128.9 (CH, C-4’Rhod), 115.4 (2CH, C-2Rhod, C-7Rhod), 114.8 (2C, C-
8aRhod, C-8bRhod), 97.3 (2CH, C-4Rhod, C-5Rhod), 71.3 (CH2, CH2O), 71.2
(CH2, CH2O), 70.7 (CH2, CH2O), 70.6 (CH2, CH2O), 51.8 (CH2,
AdaCH2CON), 46.9 (4CH2, N(CH2CH3)2), 43.7 (3CH2, C-2Ada, C-8Ada, C-
9Ada), 43.2-42.6 (m, 4CH2, N(CH2CH2)2N), 40.3 (CH2, CH2NCO), 40.1 (CH2,
CONCH2), 37.9 (3CH2, C-4Ada, C-6Ada, C-10Ada), 33.8 (C, C-1Ada), 31.5
(CH2, NCOCH2CH2CON), 30.2 (3CH, C-3Ada, C-5Ada, C-7Ada), 29.0 (CH2,
NCOCH2CH2CON), 12.8 (4CH3, N(CH2CH3)2) ppm; MS (ESI+): m/z (%) =
General
IR spectra were obtained as solid or neat liquid on a Fourier Transform
Bruker Vector 22 spectrometer. Only significant absorptions are listed. The
1H and 13C NMR spectra were recorded on Bruker Avance 300 (300 MHz
and 75 MHz, for 1H and 13C, respectively) or Bruker Avance 400 (400 MHz
and 100 MHz, for 1H and 13C, respectively) spectrometers. Recognition of
methyl, methylene, methine, and quaternary carbon nuclei in 13C NMR
spectra rests on the J-modulated spin-echo sequence. Mass spectra were
recorded on a Bruker Esquire-LC. Analytical thin-layer chromatography
was performed on Merck silica gel 60F254 glass precoated plates (0.25
mm layer). Column chromatography was performed on Merck silica gel 60
(230-400 mesh ASTM). These methods were used for all the following
compounds.
Synthesis
of
2‐(Adamantan‐1‐yl)‐N‐{2‐[2‐(2‐
solution of 1-
aminoethoxy)ethoxy]ethyl}acetamide (2). To
a
adamantane acetic acid (280 mg, 1.44 mM) in CH2Cl2 (3 mL) was added
a drop of DMF and oxalyl chloride (202 mg, 1.58 mmol). The reaction
mixture was stirred 2 h at 20 °C and then concentrated in vacuo. The
residue was taken up into CH2Cl2 (10 mL) and slowly added over three
hours to 2,2’(ethylendioxy)bis(ethylamine) (2.13 g, 14.4 mmol) in CH2Cl2
(20 mL). The reaction mixture was stirred at room temperature for 12 h and
concentrated under reduced pressure. The residue was treated with 0.5 N
NaOH (10 mL) and extracted with Et2O (2  5 mL). The organic layer was
discarded, and the aqueous phase was extracted with CH2Cl2 (5  10 mL).
The combined organic phases were dried over MgSO4 and concentrated
to leave a pale yellow oil which used directly in the next step (262 mg,
56%).
917.6 (100) [M-Cl]+, 553.3 (15), 539.3 (14), 470 (80) [M-Cl+Na]2+
.
NanoMOFs labeling with Rhod-Ada
IR (neat, cm-1) n 3330 (br), 2929, 2899, 2846, 1640, 1510, 1480, 1347,
1336, 1315, 1293, 1275, 1243, 1203, 1156, 1139, 1103, 1096, 989, 925,
906, 883, 725; 1H NMR (300 MHz, CDCl3) δ 6.10 (br s, 1H, CONH), 3.58
(s, 4H, OCH2CH2O), 3.55-3.45 (m, 4H, CH2OCH2CH2OCH2), 3.45-3.35 (m,
2H, HNCH2CH2O), 2.87 (br s, 2H), 2.19 (br s, 2H, NH2), 1.95-1.88 (m, 5H,
CH2CON, H-3, H-5, H-7), 1.75-1.60 (m, 12H, H-2, H-4, H-6, H-8, H-9, H-
10) ppm; 13C NMR (75 MHz, CDCl3) δ 171.1 (C, CON), 73.3 (CH2, CH2O),
70.3 (CH2, CH2O), 70.2 (CH2, CH2O), 70.1 (CH2, CH2O), 51.7 (CH2,
AdaCH2CON), 42.7 (3CH2, C-2, C-8, C-9), 41.8 (CH2, CH2N), 39.1 (CH2,
CONCH2), 36.9 (3CH2, C-4, C-6, C-10), 32.8 (C, C-1), 28.7 (3CH, C-3, C-
5, C-7) ppm; MS (ESI+): m/z (%) = 325.3 (100) [M-].
Rhod-Ada was dissolved in water at a concentration of 1mM and incubated
overnight with nanoMOFs at a weight ratio of 1:10 (Rhod-Ad: nanoMOFs).
Rhod-Ad labelled nanoMOFs were washed with water until no free Rhod-
Ad was found in the washing solution (concentration <0.05 µg/mL).
Because of quenching effect of nanoMOFs, Rhod adsorption on the
surface of nanoMOFs could not be directly quantified. Indirect methods
were applied consisting in quantifying the initial Rhod amount added to
nanoMOFs and the free Rhod in the supernatants after extensive
washings of the nanoMOFs using a fluorescence spectrophotometer
(VARIAN, Cary Eclipse). Rhod-Ad adsorption was calculated as the
weight ratio (%) between the adsorbed Rhod-Ad and the nanoMOFs.
Rhod-Ad release was measured in cell culture medium. After incubation at
37°C for 1, 2, 4, and 6 h, the samples were collected by centrifugation and
the detached Rhod-Ad was measured using a fluorimeter. Rhod labelling
was performed in the same way as control samples.
3‐{[2‐(2‐{2‐[2‐(Adamantan‐1‐
yl)acetamido]ethoxy}ethoxy)ethyl]carbamoyl}propanoic acid (3)
synthesis. A mixture of amine 2 (124 mg, 0.38 mmol) and succinic
anhydride (50 mg, 0.51 mmol) in THF (3 mL) was heated at 40 °C for 16
h. The mixture was concentrated in vaccuo and the residue was
chromatographed on silica gel eluting with CH2Cl2/MeOH, 20:1 to leave
the acid 3 as a colorless oil (113 mg, 70%). IR (neat, cm-1) n 3300 (br),
3080 (br), 2930, 2901, 2846, 1720, 1645, 1619, 1540, 1454, 1347, 1335,
1242, 1203, 1173, 1157, 1140, 1103, 1095, 1078, 1045, 990, 882; 1H NMR
(300 MHz, MeOH-d4) δ 3.61 (s, 4H, OCH2CH2O), 3.54 (t, J = 5.4 Hz, 4H,
CH2OCH2CH2OCH2), 3.36 (t, J = 5.4 Hz, 4H, CONHCH2CH2O), 2.61-2.55
(m, 2H, CH2CO2H), 2.51-2.45 (m, 2H, NCOCH2), 1.95 (s, 5H, CH2CON, H-
Visualization by confocal microscopy. 3.0×104 HeLa cells were seed
on sterile glass slides. After incubating overnight for the cells to get
attached, nanoMOFs labelled with Rhod or Rhod-Ad were incubated with
living cells for 6 h. After washing three times with PBS to remove free
nanoMOFs, living cells were visualized using
a
LEICA SP5
confocal system, equipped with a thermostatically controlled and CO2
regulated chamber (University Paris Sud, France). Rhod and Rhod-Ad
were excited at 514 nm and the emission from 560-600 nm were collected.
The images were processed with the Image J software.
3, H-5, H-7), 1.80-1.60 (m, 12H, H-2, H-4, H-6, H-8, H-9, H-10) ppm; 13
C
NMR (75 MHz, MeOH-d4) δ 176.0 (C, CO2H), 174.5 (C, CON), 173.8 (C,
CON), 71.3 (CH2, CH2O), 71.2 (CH2, CH2O), 70.6 (CH2, CH2O), 70.5 (CH2,
CH2O), 51.8 (CH2, AdaCH2CON), 43.7 (3CH2, C-2, C-8, C-9), 40.3 (CH2,
CH2NCO), 40.1 (CH2, CONCH2), 37.9 (3CH2, C-4, C-6, C-10), 33.7 (C, C-
1), 31.5 (CH2, CH2CH2CO2H), 30.3 (CH2, CH2CH2CO2H), 30.17 (3CH, C-
3, C-5, C-7) ppm; MS (ESI-): m/z (%) = 423.3 (100) [M-].
Fe quantification was performed by adjusting a previous reported iron
staining protocol.[25] Briefly, 2.0×105 cells were plated in 24 wells plates
and incubated for 24 h for cell adherence. Then the cells were incubated
for 6 h with 1 mL of DMEM 10 % FBS or 1 mL of culture media containing
nanoMOFs labelled or not with Rhod-Ad (nanoMOF concentration = 0~200
µg/mL). Cells were prepared in four wells for each condition. At the end of
the incubation, the cells were washed three times with PBS to eliminate
9 ‐ {2 ‐ [4 ‐ (3 ‐ {[2 ‐ (2 ‐ {2 ‐ [2 ‐ (adamantan ‐ 1 ‐
yl)acetamido]ethoxy}ethoxy)ethyl]
carbamoyl}
propanoyl)
piperazine ‐ 1 ‐ carbonyl]phenyl} ‐ 6 ‐ (diethylamino) ‐ N,N ‐
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900596
ChemMedChem
FULL PAPER
non interacting MOFs. Cells were detached using trypsin and then counted
with Luna counting (LUNA™, Logos Biosystem, Korea). Cells in four other
wells cultured in the same conditions were digested with 100 µL HCl (5M)
at 60°C for 2 h. At the end of the incubation, 100 µL of 4% potassium
ferrocyanide was added in each well. The plates were further incubated at
room temperature in the dark for 30 min. The absorbance of the treated
samples was measured at 690 nm. Calibration curves were obtained with
nanoMOFs suspensions with known concentrations (0~250 µg/mL). The
internalized nanoMOFs was quantified using HeLa cells without
nanoMOFs as control.
We acknowledge support from the Université Paris Saclay for the
« Inititative de Recherche Stratégique" IRS NanoTheRad project.
This research was funded by European Research Council
through Cyclon Hit project (People-2013-ITN Grant Agreement
608407), by the French National Research Agency (ANR) grant
ANR-14-CE08-0017 and the ARGENT project, Grant Agreement
608163. This work is supported by a public grant overseen by the
French ANR as part of the “Investissements d’Avenir” program
(Labex NanoSaclay, reference: ANR-10-LABX-0035). We
acknowledge help from Ludivine Houel Renault, responsible of
the platform CPBM/CLUPS/LUMAT FR2764 for cell culture and
confocal microscopy.
Effect of Gem-MP and nanoMOFs on SF of HeLa cells upon irradiation
HeLa cells were cultured using DMEM media supplemented with 10% FBS,
1% penicillin/streptomycin (100 mg/mL), and 1% L-Glutamine. Cells were
maintained at 37 °C in a 5% CO2 incubator. Growing cells at a density of
2×105 were seeded in flasks (25 cm3) 12 h before irradiation. NanoMOFs
loaded or not with Gem-MP were added to the cell culture medium at an
equivalent Fe concentration of 56 µM. After 6 h incubation, the compounds
(nanoMOFs, Gem-MP, and Gem-MP loaded nanoMOFs) were removed
and the cells were irradiated using the same Cs-137 gamma source as for
the stability investigation. The cells were irradiated at ambient conditions
with doses of 1, 2, 3.5, 5, and 6.5 Gy. The effect of radiation, nanoMOFs
and Gem-MP on cells was quantified by clonogenic assay. Briefly, cells
were detached with trypsin (0.05%) and plated onto Petri dishes (60 mm)
at a density of 100 surviving cells/dish. The plating efficiency was close to
39% for HeLa cells, whatever they interacted or not with nanoMOFs. When
Gem-MP was used, the plating efficiency was close to 28%. After 2 weeks
of incubation, the formed cell colonies were washed twice with PBS and
stained with 5% methylene blue in methanol. The colonies were finally
counted to determine the surviving fractions.
Keywords: metal organic frameworks • nanoparticles •
radioenhancer • radiosensitization • synergistic effect
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Total = 1.47 × S + R + L
R' = R/Total
S' = 1.47 × S/Total
L' = L/Total
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DSB yield (breaks per plasmid) = L'/(1- L')
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Entry for the Table of Contents
Insert graphic for Table of Contents here.
Gemcitabine-monophosphate (Gem-MP), a radiosensitizing anticancer drug, was successfully loaded in iron trimesate nanoMOFs, which
possess a porous structure with oxocentered Fe trimers separated by trimesate linkers. The radiation enhancement factor of Gem-MP loaded
nanoMOFs reaches 1.8, one of the highest values ever reported.
10
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