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Almaraz, R. Gref, V. Agostoni, C. kreuz, P. Clayette , C. Serre, P. Couvreur and P. Horcajada, J. Mater.
Chem. B, 2017, DOI: 10.1039/C7TB01933E.

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 Page 1 of 18 Journal of Materials Chemistry B
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DOI: 10.1039/C7TB01933E

Towards an improved HIV-microbicide activity through the co-encapsulation of NRTI drugs in

biocompatible Metal Organic Frameworks nanocarriers

Journal of Materials Chemistry B Accepted Manuscript

M.T. Marcos-Almaraz1, R. Gref2,3, V. Agostoni2, C. Kreuz4, P. Clayette4, C. Serre1,5, P. Couvreur2,
P. Horcajada1,6*
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1
Institut Lavoisier, Université de Versailles St-Quentin, UMR CNRS 8180, 45 avenue des Etats-
Unis, 78035 Versailles Cedex, France.
2
Institut Galien Paris-Sud, UMR 8612, CNRS, Université Paris-Sud, Université Paris Saclay,
Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry Cedex, France.
3
Institut de Sciences Moléculaires, UMR 8214, CNRS, Université Paris-Sud, Université Paris
Saclay, Orsay, France.
4
Laboratoire de Neurovirologie, Bertin-Pharma, CEA, 18 route du Panorama, B.P. 6, 92265
Fontenay aux Roses Cedex, France.
5
Institut des Matériaux Poreux de Paris, FRE 2000 CNRS, Ecole Normale Supérieure, Ecole
Supérieure de Physique et de Chimie Industrielles, PSL Research University, 75005, Paris,
France.
6
IMDEA Energy, Avda. Ramón de la Sagra 3, 28935 Móstoles-Madrid, Spain.

ABSTRACT

The efficacy of the routinely used anti-HIV (Human Immunodeficiency Virus) therapy based on
nucleoside reverse transcriptase inhibitors (NRTIs) is limited by the poor cellular uptake of their
active triphosphorylated metabolites and the low efficiency of intracellular phosphorylation of
their prodrugs. Nanoparticles of iron(III) polycarboxylate Metal-Organic Frameworks
(nanoMOFs) are promising drug nanocarriers. In this study, two active triphosphorylated NRTIs,
 Journal of Materials Chemistry B Page 2 of 18
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the azidothymidine triphosphate (AZT-Tp) and lamivudine triphosphate (3TC-Tp), were

successfully co-encapsulated into the biocompatible mesoporous iron(III) trimesate MIL-100(Fe)

Journal of Materials Chemistry B Accepted Manuscript

nanoMOF in order to improve anti-HIV therapies. Drug loaded nanoMOFs could be stored up to
2-months and reconstituted after freeze drying, keeping similar physicochemical properties.
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Their antiretroviral activity was evidenced in vitro on monocyte-derived macrophages

experimentally infected with HIV, making these co-encapsulated nanosystems excellent HIV-
microbicide candidates.

INTRODUCTION

In recent years, new human immunodeficiency virus (HIV) infections and acquired immune
deficiency syndrome (AIDS)-related deaths have decreased but the viral latency, the persistence
of viral reservoirs and the still insufficient efficacy of the current antiretroviral treatments do
not allow to eradicate HIV in an efficient way yet. Nucleoside reverse transcriptase inhibitors
(NRTIs) are of major importance, as they are being considered among the most effective
molecules routinely used in anti-HIV therapy. Azidothymidine (AZT) and lamivudine (3TC) are
elements of this family of compounds and, as the rest of NRTI, protect cells from HIV infection
by inhibiting both retrotranscription and synthesis of the proviral DNA. 1,2 In order to exert their
antiretroviral activity, these prodrugs must be triphosphorylated by intracellular kinases into
their active derivatives. These kinases are among the main parameters which limit the clinical
efficacy of NRTIs, leading to the emergence of drug resistance and unwanted side-effects due
to the necessity of using high therapeutic doses. Two options are possible to increase the
efficiency of the current anti-HIV therapy, either to identify new compounds or to by-pass the
limits of the current molecules. Connected with this last option, a promising strategy is the
direct delivery of active triphosphorylated NRTI such as azidothymidine triphosphate (AZT-Tp).
Unfortunately, the cellular uptake of these derivatives is hampered by their high hydrophilicity,
which also decreases their stability in biological fluids. This justifies the necessity to develop
effective carriers able to encapsulate these active triphosphorylated NRTIs and release them
3-7
inside cells susceptible to be infected by HIV. Moreover, in absence of an efficient HIV
 Page 3 of 18 Journal of Materials Chemistry B
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vaccine, the development of effective microbicides is an interesting alternative for the HIV
prophylaxis. Cells of the macrophage lineage are present in every organ and are the second

Journal of Materials Chemistry B Accepted Manuscript

major HIV cell target. Because of the very long life span of HIV-1-infected macrophages, they
act as a source of virus for prolonged period of time in HIV-infected patients and, consequently,
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can disseminate HIV throughout the organism. Since macrophages are one of the cell
populations in the vaginal and anal mucosas involved in the entry of HIV to the body,
microbicide candidates should prevent infection of macrophages to be effective. Despite the
efficiency of some surfactants (e.g. quaternary ammoniums), they are associated with mucosal
irritation, facilitating the virus infection and/or replication. In this context, we have identified
the newly developed nanoparticles of biocompatible iron(III) polycarboxylate metal-organic
frameworks (nanoMOFs) as ideal candidates to convey triphosphorylated NRTI up to major HIV
8 9
target cells such as macrophages. These highly porous crystalline coordination polymers,
based on cations connected to polydentate organic linkers, have been shown previously to
successfully incorporate high loadings of phosphorylated nucleoside analogues10-12 as well as
other drugs13-15 into their porosity, but not yet in combination. In particular, the benchmarked
biocompatible MIL-100(Fe) nanoparticles (NPs), based on oxocentered trimers of iron(III)
octahedra and trimesate anions, exhibit an exceptional porosity (Brunauer-Emmett-Teller
surface SBET ∼ 2000 m2g-1; pore volume Vp ∼ 1.0 cm3g-1; cages size ∼ 25 and 29 Å accessible via
microporous pentagonal or hexagonal windows of free apertures of 4.7*5.5 and 8.5 Å,
respectively; Figure 1),16 able to host important drug cargoes and deliver it on a controlled
manner. 8,10-12 Therefore, in order to mimic routinely used anti-HIV multitherapies and to limit
the number of taking drugs for treated patients, MIL-100(Fe) nanoMOF was here concomitantly
loaded with the triphosphorylated NRTIs, AZT-Tp and 3TC-Tp (Figure 1), and further assessed in
vitro for their anti-HIV activity.
 Journal of Materials Chemistry B Page 4 of 18
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Journal of Materials Chemistry B Accepted Manuscript

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Figure 1. Chemical structure of 3TC-Tp and AZT-Tp. Schematic view of a large cage of the MIL-100(Fe) NPs. Iron
polyhedra, carbon and oxygen are represented in orange, black and red, respectively (hydrogen atoms are omitted
for clarity).

EXPERIMENTAL

Materials: Iron(III) chloride hexahydrate (FeCl3-6H2O; Alfa Aesar, 98%), 1,3,5-

benzenetricarboxylic acid (BTC or trimesic acid; Aldrich, 95%), azido 3´-deoxythimidine 5´-
triphosphate, tetratriethylammonium salt (AZT-Tp, 3.8 Ci.mmol-1, Moravek Biochemicals), (-)- (-
)-ß-L-2',3'-dideoxy 3'-thiacytidine triphosphate, tetraammonium salt (3TC-Tp; 2 μmol, Moravek
Biochemicals), phosphate buffer solution (PBS, pH = 7.4, Sigma) with 10% of heat-inactivated
fetal bovine serum (FBS; Fischer), ammonium dihydrogenphosphate (NH4H2PO4; Alfa Aesar,
98%), tetrabutylammonium perchlorate (TBAP, C16H36CINO4; TCI, 98%) and acetonitrile (AcN ;
for HPLC Super Gradient Reagent, VWR Chemicals) were purchased and used without any
further purification.

Synthesis of MIL-100(Fe) nanoparticles: MIL-100(Fe) NPs were synthesized by microwave-

assisted hydrothermal procedure, according to the previously reported method. 16 Activation or
purification of 2.5 g of MIL-100(Fe) consisted of the re-dispersion and centrifugation (10,500
rpm, 20 min) of the NPs in 20 mL of distilled water and five successive times in 20 mL absolute
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ethanol. Further activation was carried out by re-dispersing the solid for 1 h 40 min in 20 mL of
a 0.1 M KF solution under stirring. NPs were collected by centrifugation (10500 rpm, 20 min)

Journal of Materials Chemistry B Accepted Manuscript

and washed twice with 20 mL of distilled water and once with 20 mL of absolute ethanol.
Activated MIL-100(Fe) NPs were isolated by centrifugation and stored wet with few droplets of
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fresh absolute ethanol to avoid complete drying of the product. Prior to the experiments, NPs
were exchanged into MilliQ water (H2Omq).

Drug encapsulation: 25 mg of wet MIL-100(Fe) NPs (Note well: weighted wet, considering the
wet : dry ratio previously determined from NPs dry at 100 ºC overnight) were incubated with 2
mg of AZT-Tp and 1 mg of 3TC-Tp in 5 mL of aqueous solution, under magnetic stirring at room
temperature for 24 h (n = 4). In order to avoid the evaporation leak during the encapsulation
time, the container was covered with a lid. The encapsulation rate was estimated by HPLC from
the supernatant obtained after the first centrifugation of the just encapsulated product.

Drug release and chemical stability studies: 1 mg of the co-encapsulated product was
recovered by centrifugation at 10,000 rpm for 10 min. The supernatant was isolated to estimate
the encapsulation efficiency by HPLC. The remaining solid was resuspended in 400 µL of PBS
(pH = 7.4) with 10% of FBS and dispersed in this simulated physiological medium by ultrasound
tip (Digital Sonifer 450, Branson) during 10 s at 10% of amplitude. The resulting colloidal
solutions were incubated under bidimensional shaking (100 rpm) at 37 °C for different times
(from 30 min to 6 days). At each time point, the sample was centrifuged (10,000 rpm, 10 min)
and the half volume of the supernatant was isolated to quantify the drug release by HPLC. This
same volume was replaced in the sample with fresh physiological medium at 37°C.
When the co-encapsulated sample was lyophilized, the procedure to analyze its colloidal
stability in physiological media was similar to this one, adding the corresponding volume of PBS
(pH = 7.4) with 10% of FBS to reach a concentration of approx. 0.1 mg.mL-1, and dispersing
immediately after that by ultrasound tip. The resulting colloidal solution was incubated as
previously to simulate physiological conditions. The same as before, for the different time
points these colloidal samples were vortexed.
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HPLC analysis was performed in all the cases using a HPLC system (Waters E2695) connected to
a C18 column (4 x 250 mm, 5 μm, PurospherR) coupled with a WatersTM 2998 photodiode array

Journal of Materials Chemistry B Accepted Manuscript

detector and Waters EmpowerTM2 acquisitions software. The mobile phase consisted in a
mixture of AcN:H2O (10:90), NH4H2PO4 (50 mM) and TBAP (5 mM), at pH = 2.5. The flow rate
was 1 mL min-1 and the injected volume 50 μL. Retention times were: 7, 9, 17 and 24 min for
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3TC-Tp, AZT-Tp, azido 3´-deoxythimidine 5´-monophosphate AZT-Mp and BTC, respectively.

Standard calibration curves of the different tested compounds were performed in the 50-1.5,
175-6.2, 125-175 and 500-2.5 μg mL-1 ranges for 3TC-Tp, AZT-Tp, AZT-Mp and BTC, respectively.

Physicochemical characterization: powder X ray powder diffraction (PXRD) was collected using
a D8 Advance Bruker diffractometer with Cu Kα1 radiation (lambda = 1.54056 angstroms) from
3 to 30˚ (2θ) using a step size of 0.02˚ and 2.5 s per step in continuous mode. N2 adsorption
measurements were carried out at 77K by using a BELsorp Max (Bel, Japan). Prior to the
analysis, about 15 mg of the coencapsulated sample were evacuated at 130 °C under secondary
vacuum for 3 h. Size and ƺ-potential of the NPs were measured by dynamic light scattering
(DLS; Zetasizer nano ZS, Malvern Instruments), previous dispersion by ultrasound tip during 1-2
min at 10% of amplitude. Note here that to monitor the aggregation/degradation pattern of the
NPs as a function of the time (hours/days) under physiological conditions (PBS-10% FBS, 37 °C,
under shaking), the samples were not dispersed again in each time point. Transmission electron
microscopy (TEM) was performed on a Zeiss EM902 microscope, in the « Plateforme de
microscopie et d’imagerie » of INRA (Jouy en Josas, France). Carbon-coated TEM grids, carbon
type-B, 200 mesh copper grids were purchased from Ted Pella, Inc. Samples were prepared by
dispersing the dried NPs at 0.1 mg·mL-1 in absolute ethanol by means of ultrasounds, drop
casting the solution on a carbon-coated TEM grid and allowing the solvent evaporate under air.

Freeze-drying: 17 mg of the co-encapsulated sample were freeze-dried by using a traditional

glass desiccator under primary vacuum. The system was also connected to a cool container
(refrigerated by liquid N2) which was receiving the just evaporated solvent.
 Page 7 of 18 Journal of Materials Chemistry B
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Statistical analysis: Data were expressed as mean of 5 experiments (± S.E.M) in case of drug
release analysis and 3 experiments (± S.E.M) for the evolution of colloidal stability under

Journal of Materials Chemistry B Accepted Manuscript

physiological conditions.

In vitro anti-HIV activity: The anti-HIV activity of the co-loaded, 3TC-Tp-loaded and AZT-Tp-
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loaded NPs was tested in primary cultures of human monocyte-derived macrophages (MDM)
17
experimentally infected with the reference macrophage-tropic HIV-1/Ba-L strain (see SI). As
previously described, MDM were obtained after 7-day cell differentiation of monocytes isolated
from human peripheral blood mononuclear cells (PBMC; magnetic CD14+ cell sorting). MDM
were treated and infected, and HIV-1/Ba-L replication was quantified throughout the cell
cultures until day 28 using the RetroSys RT Activity Kit (Innovagen AB, Lund, Sweden). In
parallel, cell viability of co-loaded NPs was assessed on uninfected macrophages by a typical
methyltetrazolium (MTT) salt assay. Experiments were performed in triplicates and repeated
with cells isolated from 3 other donors (four independent experiments; n = 4). The
18
antiretroviral effects of compounds were calculated as previously described by determining
the cumulative RT activity, the percentage of the control RT activity, and the 50, 70 and 90%
effective doses (ED50, ED70, ED90).

RESULTS & DISCUSSION

Drug co-encapsulation and physicochemical characterization

The co-encapsulation of the challenging antiretroviral drugs, AZT-Tp and 3TC-Tp, was
successfully achieved following an one-step, biocompatible, fast and efficient impregnation
method, using an aqueous solution of both drugs (see Experimental section). Although similar
impregnation approaches have been widely applied for the adsorption of different active
19,20
molecules within porous MOFs , this is the first time that this protocol succeeds to
incorporate two complementary drugs within an unique nanoMOF with, in addition, very high
loading efficiencies (up to 78 and 84 % of the initial drug concentration were incorporated
within the nanoMOF for AZT-Tp and 3TC-Tp, respectively, as quantified by HPLC). These high
efficiency values evidence an important affinity of the drugs for the MIL-100(Fe) matrix. This is
 Journal of Materials Chemistry B Page 8 of 18
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in good agreement with the formation of specific interactions between the phosphate groups
of the NRTIs and the coordinatively unsaturated iron(III) metal sites (CUS) with a Lewis acid

Journal of Materials Chemistry B Accepted Manuscript

character of the MIL-100(Fe) NPs, as previously reported for the AZT-Tp drug. 10,11 Remarkably,
important drug loadings up to 24 wt% (expressed as the weight percentage with respect to the
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empty dry MIL-100(Fe) NPs) were adsorbed within the nanoMOF depending on the drug :
nanoMOF weight ratio. For instance, drug loadings of around 7.0 ± 0.5 wt% were reached after
11
an unique AZT-Tp impregnation in diluted drug aqueous solutions. Despite the dilution, the
incorporation yields were high, suggesting the remarkable affinity of AZT-Tp for the nanoMOFs.
In this study, AZT-TP loadings similar to that one observed in the unique AZT-Tp encapsulation
were achieved (6.2 ± 0.5 wt%), suggesting that the co-encapsulation of the 3TC-Tp does not
interfere with the AZT-Tp adsorption. Remarkably, starting from an AZT-Tp:3TC-Tp weight ratio
2:1, similar to that one of the commercialized current tritherapy formulation (Combivir®,
21
GlaxoSmithKline), we were able to reach similar drug content in the final nanoMOF
formulation (i.e. 1.8:1), corresponding to a 3TC-Tp loading of 3.4 ± 0.5 wt%. This indicates a
similar affinity of both drugs for the MIL-100(Fe) NPs. In fact, the two studied NRTIs exhibited
comparable molecular dimensions and chemical structures (Figure 1), so they might similarly
interact with the porous hybrid framework. Finally, considering the previously reported higher
loadings of AZT-Tp (up to 24 ± 2 wt%) upon successive impregnations with more concentrated
drug solutions, 8 one could rationally expect to increase the loadings of both AZT-Tp and 3TC-Tp,
if needed.

600

400
Va (cm3(STP)g-1)

MIL-100_AZT-Tp_3TC-Tp

MIL-100_AZT-Tp
200

MIL-100
0

3 10 20 30 0.0 0.2 0.4 0.6 0.8 1.0

P/P0
2-Theta - Scale
 Page 9 of 18 Journal of Materials Chemistry B
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Figure 2. XRPD patterns (on the left) and N2 sorption isotherms at 77K (on the right) of MIL-100(Fe) NPs containing
AZT-Tp (blue) and/or 3TC-Tp (red) in comparison with the empty NPs (black).

Journal of Materials Chemistry B Accepted Manuscript

After the NRTIs entrapment, the NPs were fully characterized by a set of complementary
techniques. PXRD confirmed the preservation of the crystalline structure of the MIL-100(Fe)
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NPs (Figure 2) upon co-encapsulation. Note here the different intensity of the first Bragg
reflections between the patterns of MIL-100_AZT-Tp and MIL-100_AZT-Tp_3TC-Tp; which is
probably due to a different hydration state (after encapsulation, non-controlled solvation state
since samples, considered as cytotoxic, are introduced in a closed capillary for PXRD analysis).
The particle size and morphology remained unchanged, disclosing well-faceted cubic
nanocrystals of around 150 nm, as observed by Transmission Electron Microscopy (TEM; Figure
S3). Similarly, the particle mean diameter, determined by dynamic light scattering (DLS) in
aqueous solution just before and after the encapsulation procedure did not show any
significant difference (141 ± 43 vs. 167 ± 50 nm, respectively; Table 1), although a higher
polydispersity was observed (PdI = 0.41 vs. 0.14, respectively). Note here that the negative
surface of the NPs became even more negative upon co-encapsulation (ƺ-potential values
shifted from -21 to -46 mV), in agreement with the presence of NRTI molecules exhibiting
deprotonated phosphates, adsorbed on the external surface of the NPs. As reported before for
other phosphate biomolecules, this highly-negative surface improves the colloidal stability of
these nanoMOFs by creating enough electrostatic repulsion between NPs, 22 in agreement with
the absence of aggregation after encapsulation. The colloidal stability of the co-encapsulated
MIL-100(Fe) NPs was also evaluated in simulated physiological media (see below, co-delivery
section).

Table 1. Physicochemical properties of MIL-100(Fe) NPs in aqueous solution before and after encapsulation of AZT-
Tp and 3TC-Tp, as well as after 2 months of freeze-drying of the co-loaded nanoMOF, in terms of estimated
hydrodynamic size, surface charge (ƺ-potential), polydispersity index (PdI) and porosity.

Before encapsulation After encapsulation After freeze-drying

Size (nm) 141 ± 43 167 ± 50 147 ± 49
ƺ-potential (mV) -21 ± 4 -46 ± 5 -34 ± 3.5
PdI 0.14 0.41 0.20
BET surface (m2.g-1) 1450 870 -
Vmicropore (cm3.g-1) 0.60 0.42 -
 Journal of Materials Chemistry B Page 10 of 18
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Both the high total drug loading (around 9.6 wt%) and the low external surface when compared

Journal of Materials Chemistry B Accepted Manuscript

with the inner pores surface area (180 vs. 1450 m2.g-1; estimated by the t-plot and Brunauer-
Emmett-Teller (BET) methods, respectively), suggest that the drugs are mostly adsorbed within
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the MOF porosity and not at the outer surface. In addition, this is further supported by N2
porosimetry whereas lower specific surface area and pore volume are obtained after
encapsulation (Table 1 and Figure 2). When compared with the single AZT-Tp encapsulation
with a 6 wt% drug loading, a slightly lower porosity is, as expected, present for the 9.6 wt% co-
encapsulated sample (SBET = 1020 vs. 870 m2.g-1).

Our previous data based on molecular simulations suggested that AZT-Tp molecules were
preferentially adsorbed within the large mesoporous cages (Ø ∼ 29 Å) of the MIL-100(Fe) via
their hexagonal microporous windows (∼8.6 Å) due to a size exclusion effect (AZT-Tp
dimensions ∼12x9x7 Å; Figure 1) in the smaller cages only accessible by pentagonal windows
10,23
(∼5.5 Å-diameter). Similarly, considering the dimensions of the 3TC-Tp (13x8x4 Å3; Figure
1), this NRTI-Tp would be exclusively located within these larger cages. Thus, one could roughly
estimate the presence of almost four AZT-Tp and around two 3TC-Tp molecules within each
large cage of MIL-100(Fe). Considering that AZT-Tp and 3TC-Tp molecules occupy a volume of
ca. 356 and 308 Å3 (according to molecular simulation calculations), 10
one can conclude that
the large MOF cages (∼12700 Å3) would be only partially occupied (∼ 16%). This suggests that
even higher drug cargoes could be achieved, in agreement with the improved loadings
previously reached when using pure AZT-Tp (24 wt%). 8

Co-delivery of the NRTI-TP

The release profile of the active triphosphorylated NRTIs from MIL-100(Fe) NPs was evaluated
in phosphate buffer solution (PBS) supplemented with 10% (w/v) fetal bovine serum (FBS) at
37°C and under bidimensional shaking. The amount of each released drug was monitored by
HPLC in a time course up to 6 days (see Experimental section). In parallel, the effect of the drug
loading on the nanoMOFs degradation was monitored by the estimation of the release of the
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constitutive organic linker of the MIL-100(Fe) NPs, i.e. the trimesic acid (BTC). Finally, the
hydrodynamic size of the particles was determined along with the release experiment,

Journal of Materials Chemistry B Accepted Manuscript

evidencing an initial increase of the particle size of around 80 nm (from ~170 to 250 nm) after
30 min. Although one cannot rule out a partial aggregation, this could be explained by the
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formation of a protein blanket, due to the rapid adsorption on the outer nanoMOF surface of
serum proteins present in the environmental medium, as also suggested by the fact that the
particle size remained afterwards unchanged over the whole release experiment (6 days; Figure
3).

Remarkably, different release kinetics were observed for each NRTI-Tp. 3TC-Tp exhibits the
fastest release with almost 80% of the loaded drug released within the first 8 h and with a
complete release after 24 h (Figure 3, pink line). On the contrary, AZT-Tp is released in a more
progressive manner during 3 days, possibly because of its higher affinity for the nanoMOFs and
larger dimensions as compared to 3TC-Tp at least in two directions (12x9x7 vs. 13x8x4 Å3,
respectively). The larger size of AZT-Tp might thus hinder its rapid diffusion through the pores.
The slow AZT-Tp release kinetics exhibited three different steps (Figure 3A, green line): i) an
induction time of 4 h, in which a very few amount of drug was released (< 0.5%), ii) then,
around 90% of the encapsulated AZT-Tp was released to the medium within 3 days, and can be
empirically fitted with a regression factor >0.99 to a zero order kinetics ([AZT-Tp] = Kt in which K
is the kinetic constant and t the time; K = 1.32±0.03 with an intercept of -4.83±0.12, in
agreement with the previous mentioned induction time); note here that this second step is
predictable independent of the drug-concentration ; and finally, iii) the remaining 10% of the
drug content was very slowly released in the following 3 days, with a complete leakage of the
cargo after 6 days.

Considering the previously reported hydrolysis of the AZT-Tp in aqueous solution into their
24,25
inactive mono- and/or di-phosphorylated metabolites (AZT-Mp or AZT-Dp), we have also
evaluated the degradation of this NRTI-Tp during their release process from MOFs. Note that
another very weak retention peak was seen in the HPLC chromatogram of the release medium,
with a similar absorption spectrum than AZT-Tp (Figure S1). We confirmed that a few amount
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(0.6% after 6 days) of the initial AZT-Tp was progressively dephosphorylated to their
monophosphated metabolite (AZT-Mp) by analyzing the HPLC retention time and UV-Vis

Journal of Materials Chemistry B Accepted Manuscript

spectrum of the available purified AZT-Mp product under the same experimental conditions
(Figure S2). It was concluded that the progressive AZT-Tp release resulted in a partial protection
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of the drug from degradation.

100
3TC-Tp
Drug release (%)

BTC AZT-Tp
50

AZT-Mp
0

500 0 1 2 3 4 5 6
Particle size (nm)

200

0
0 1 2 3 4 5 6
time (days)

Figure 3. Release of 3TC-Tp, AZT-Tp, AZT-Mp and the constitutive organic linker BTC from MIL-100(Fe) nanoMOFs
(on the top), together with their colloidal stability (determined by DLS; on the bottom) in PBS supplemented with
FBS at 10% (w/v) at 37°C under stirring (n = 5).

The release of different NRTI-Tp from porous MOFs is a consequence of three different
parameters: drug diffusion thought the pores, drug-matrix interactions and MOF chemical
disintegration. Indeed, concomitantly to the leakage of both anti-HIV NRTI-Tp, the hybrid
network of the nanoMOF suffered also a continuous degradation, as evidenced by the HPLC
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detection of the leaching of the constitutive BTC linker into the nanoMOFs suspension medium
(Figure 3A, blue line).

Journal of Materials Chemistry B Accepted Manuscript

Stability during the storage of NRTI-Tp-loaded MOFs by freeze drying
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The biocompatible and biodegradable MIL-100(Fe) nanocarriers 26 were shown to efficiently co-
encapsulate and co-release the challenging antiviral compounds AZT-Tp and 3TC-Tp. In view of
further developments and for eventual industrial translation, their storage by freeze drying was
investigated for the first time.

The MIL-100(Fe) NPs co-loaded or not were freeze dried just after their preparation. Then, the
dried NPs were kept for 2 months at room temperature. Remarkably, after reconstitution in
water and redispersion by sonication, they perfectly preserved their structure and
physicochemical properties, as shown by their morphology, size and charge (estimated by TEM
and DLS; Figure S3 and Table 1) together with the PXRD patterns (Figure 2). For instance, the
mean diameter before freeze-drying was 167 ± 50 and 147 ± 49 after, even if the polydispersity
index was observed to increase after freeze-drying (0.41 vs. 0.20). Moreover, the colloidal
stability of the redispersed freeze-dried particles was similar to the untreated ones (Figure S3).
This study shows the excellent preservation of nanoMOFs loaded drugs by freeze-drying.

In vitro anti-HIV activity

Triphosphorylated NRTI-loaded nanoMOFs previously demonstrated significant anti-HIV

activity, especially in macrophages, the second major HIV cell target; it is probably related with
their high phagocytose capabilities. 8,11 Therefore, the comparison of the anti-HIV effects of the
3TC-Tp and AZT-Tp co-loaded nanoMOFs with those of the individual 3TC-Tp and AZT-Tp-loaded
MIL-100(Fe) NPs was investigated using primary cultures of human monocyte-derived
macrophages (MDM) experimentally infected with the reference macrophage-tropic HIV-1/Ba-L
17
strain (see Experimental section). The retroviral replication was measured all along the
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cultures in cell culture supernatants by quantifying the enzymatic reverse transcriptase (RT)
activity 18 (Table 2) and 50, 70 and 90% effective doses (ED50, ED70, ED90) were calculated. In

Journal of Materials Chemistry B Accepted Manuscript

parallel, cell viability of co-loaded nanoMOFs was assessed on uninfected macrophages using a
methyltetrazolium salt (MTT) assay (see Experimental section).
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27
As expected, the bare nanoMOFs did not show any antiretroviral activity and cytotoxicity
(Table 2 and Figure S4). In our experimental conditions, 3TC-Tp and AZT-Tp-co-loaded
nanoMOFs dose-dependently decreased the HIV replication in human MDM, in the absence of
any cytotoxicity (Table 2, Figure S4). Similar anti-HIV effects were observed with AZT-Tp-loaded
nanoMOF and AZT (Table 2; 8 ± 9 nM for AZT-loaded nanoMOF ED50 vs. 4 ± 3 nM for AZT
ED50). No significant differences were also observed for 3TC-TP-loaded nanoMOF vs. 3TC and
for co-loaded nanoMOF vs. AZT-Tp- or 3TC-Tp-loaded nanoMOF (Table 2). Despite the observed
similar antiviral activity, this is of great interest. First, because co-encapsulating drugs could
modify the pharmacokinetic and biodistribution of parental NRTI to reach HIV reservoirs and
could help to eradicate the virus from the reservoirs in HIV-infected patients. Secondly, because
intracellularly delivering the active already triphosphorylated forms of NRTI using nanovectors12
may overcome the limitation associated with their intracellular phosphorylation by cell kinases.
This is particularly interesting when considering the microbicide approach, because nanoMOFs
can be easily phagocytized by macrophages, releasing their active NRTI-Tp cargo directly inside
these cells and targeting the major HIV entry in the body (i.e. at the level of mucous barriers).11
Therefore, NRTI-Tp loaded nanoMOFs seem excellent candidates as HIV microbicides.

In addition, the efficiency of this coencapsulated nanoparticulate system was compared with
the commercialized microbicide Tenofovir (PMPA) using our experimentally HIV-1/Ba-L-infected
MDM model and obtaining similar results. Indeed, ED50, ED70 and ED90 were respectively 1.3,
5.0 and 12.5 nM for PMPA and 11, 21 and 41 nM for MIL-100(Fe)_AZT-Tp_3TC-Tp, PMPA
exhibiting a slightly higher efficacy.
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Table 2. Comparison of anti-HIV effects of AZT-Tp/3TC-Tp, AZT-Tp- and 3TC-Tp-loaded nanoMOF in HIV-1/Ba-L-

Journal of Materials Chemistry B Accepted Manuscript

infected MDM. Results were obtained from 3 independent experiments, each one made in triplicate and expressed
as nM.
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ED50 ED70 ED90

(nM)
MIL-100(Fe) ≥1000 ≥1000 ≥1000
AZT 4±3 8±9 29 ± 26
MIL-100(Fe)_AZT-Tp 8±9 19 ± 14 65 ± 41
3TC 22 ± 26 61 ± 70 214 ± 172
MIL-100(Fe)_3TC-Tp 6±5 33 ± 29 181 ± 176
MIL-100(Fe)_AZT-Tp_3TC-Tp 11 ± 5 21 ± 13 41 ± 25

CONCLUSIONS

Biocompatible iron carboxylate nanoMOFs of the MIL-100(Fe) architecture were successfully

loaded with the AZT-Tp/3TC-Tp drug combination, reaching a total loading of around 9.6 wt%
with an equivalent AZT/3TC ratio than in the currently commercialized HIV prodrug based
tritherapy (Combivir®, GlaxoSmithKline). A convenient method was developed to store the drug
loaded particles by freeze-drying and reconstituted nanoMOF showed similar physico-chemical
properties, even two months after storage. Moreover, as determined in vitro, the incorporated
triphosphorylated NRTIs maintained their antiretroviral activity. These nanoparticles might find
potential future applications for combination therapy in the treatment of HIV patients,
particularly by decreasing NRTI medications, targeting HIV reservoirs or acting as protecting
microbicides (e.g. at mucous barriers after rectal or vaginal administration routes).

Conflict of interest

The authors declare no competing conflict of interests.

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Acknowledgments

Journal of Materials Chemistry B Accepted Manuscript

This work was partially supported by the French Research Agency (ANR) through a VirMIL
MatePro ANR-project. We thank Karen Storck for excellent technical assistance. PH
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acknowledges the Spanish Ramon y Cajal Programme (grant agreement n° 2014-16823) and the
People Programme (Marie Curie Actions) of the European Union's Seventh Framework
Programme (FP7/2007-2013) under REA grant agreement n° 291803.

References

1. PN. Kumar, A. Rodriguez-French, MA. Thompson, KT. Tashima, D. Averitt, PG. Wannamaker,
VC. Williams, MS. Shaefer, GE. Pakes, KA. Pappa and ESS40002 Study Team, HIV Med, 2006,
7(2), 85–98.

2. M. Stürmer, S. Staszewski and HW. Doerr, Antivir Ther, 2007, 12(5), 695–703.

3. SV. Vinogradov, E. Kohli and AD. Zeman, Mol Pharm, 2005, 2(6), 449–461.

4. T. Mamo, EA. Moseman, N. Kolishetti, C. Salvador-Morales, J. Shi, DR. Kuritzkes, R. Langer, U.

von Andrian and OC. Farokhzad, Nanomedicine, 2010, 5(2), 269–285.

5. D. Desmaële, R. Gref and P. Couvreur, J Control Release, 2012, 161(2), 609–618.

6. G. Giacalone, H. Hillaireau and E. Fattal, Eur J Pharm Sci, 2015, 75, 40–53.

7. EV. Batrakova, HE. Gendelman and AV. Kabanov, Expert Opin Drug Deliv, 2011, 8(4), 415-433.

8. P. Horcajada, T. Chalati, C. Serre, B. Gillet, C. Sebrie, T. Baati, J.F. Eubank, D. Heurtaux, P.

Clayette, C. Kreuz, J.S. Chang, Y.K. Hwang, V. Marsaud, PN. Bories, L. Cynober, S. Gil, G. Férey, P.
Couvreur and R. Gref, Nat Mater, 2010, 9, 172-178.

9. See special issue: Chem Soc Rev. 2014; 43(16): 5403-6176.

 Page 17 of 18 Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/C7TB01933E

10. V. Agostoni, R. Anand, S. Monti, S. Hall, G. Maurin, P. Horcajada, C. Serre, K. Bouchemal and R.
Gref, J. Mater. Chem B, 2013, 1, 4231-4242.

Journal of Materials Chemistry B Accepted Manuscript

11. V. Agostoni, T. Chalati, P. Horcajada, H. Willaime, R. Anand, N, Semiramoth, T. Baati, S. Hall,
G. Maurin, H. Chacun, K. Bouchemal, C. Martineau, F. Taulelle, P. Couvreur, C. Rogez-Kreuz, P.
Published on 29 September 2017. Downloaded by University of Newcastle on 30/09/2017 01:04:33.

Clayette, S. Monti, C. Serre and R. Gref, Adv Healthc Mater, 2013, 2, 1630-1637.

12. V. Rodriguez-Ruiz, A. Maksimenko, R. Anand, S. Monti, V. Agostoni, P. Couvreur, M.

Lampropoulou, K. Yannakopoulou and R. Gref, J Drug Target, 2015, 23 (7-8), 759-767.

13. M. Giménez-Marqués, T. Hidalgo, C. Serre and P. Horcajada, Coord. Chem. Rev., 2016,
307(2), 342-360.

14. Z. Tian, X. Yao,K. Ma, X. Niu, J. Grothe, Q. Xu, L. Liu, S. Kaskel and Y. Zhu, ACS Omega, 2017,
2(3), 1249−1258

15. W. Liu, Y.M. Wang, Y.H. Li, X.B. Yin, X.W. He and Y.K. Zhang, Small, 2017, DOI:
10.1002/smll.201603459

16. A. García Márquez, A. Demessence, AE. Platero-Prats, D. Heurtaux, P. Horcajada, C. Serre,

JS. Chang, G. Férey, VA. de la Peña-O’Shea, C. Boissière, D. Grosso and C. Sanchez, Eur J Inorg
Chem, 2012, 32, 5165–5174.

17. S. Gartner, P. Markovits, DM. Markovitz, MH. Kaplanm, RC. Galloand M. Popovic, Science,
1986, 233, 215-219.

18. M. Martin, N. Serradji, N. Dereuddre-Bosquet, G. Le Pavec, G. Fichet G, A. Lamouri, F.

Heymans, JJ. Godfroid, P. Clayette and D. Dormont, Antimicrob Agents Chemother, 2000,
44(11), 3150–3154.

19. P. Horcajada, C. Serre, M. Vallet-Regí M, M. Sebban, F. Taulelle and G. Férey, Angew. Chem.
Int. Ed Engl, 2006, 45 (36), 5974-5978.

20. P. Horcajada, R. Gref, T. Baati, PK. Allan, G. Maurin, P. Couvreur, G. Férey, RE. Morris and C.
Serre, Chem Rev, 2012, 112(2), 1232-1268.
 Journal of Materials Chemistry B Page 18 of 18
View Article Online
DOI: 10.1039/C7TB01933E

21. SD. Portsmouth and CJ. Scott, Ther Clin Risk Manag, 2007, 3(4), 579-583.

22. RJ. Hunter, Zeta Potential in Colloid Science, Academic Press, UK, 1988.

Journal of Materials Chemistry B Accepted Manuscript

23. P. Horcajada, S. Surblé, C. Serre, D.Y. Hong, YK. Seo, J.S. Chang, J.M. Grenèche, I. Margiolaki
and G. Férey, Chem Commun (Camb), 2007, 27, 2820-2822.
Published on 29 September 2017. Downloaded by University of Newcastle on 30/09/2017 01:04:33.

24. WH. Pitcher 3rd, TW. Kirby, EF. DeRose and RE. London, Antiviral Res, 2003, 58(3), 227-233.

25. S. Dharmasena, Z. Pongracz, E. Arnold, SG. Sarafianos and MA. Parniak, Biochemistry, 2007,
23, 46(3), 828-836.

26. T. Baati, F. Bourasset, N. Gharbi, L. Njim, M. Abderrabba, A. Kerkeni, H, Szwarc and F.

Moussa, Biomaterials, 2012, 33(19), 4936-4946.

27. R. Grall, T. Hidalgo, J. Delic, A. García-Márquez, S. Chevillard and P. Horcajada, J Mater Chem
B, 2015, 3, 8279—8292.