This is the submitted version of the article: Solórzano R., Tort O., García-Pardo J., Escribà T., Lorenzo J., Arnedo M., Ruiz-Molina D., Alibés R., Busqué F., Novio F.. Versatile iron-catechol-based nanoscale coordination polymers with antiretroviral ligand functionalization and their use as efficient carriers in HIV/AIDS therapy. Biomaterials Science, (2019). 7. : 178 - . 10.1039/c8bm01221k. Available at: https://dx.doi.org/10.1039/c8bm01221k Please do not adjust margins Journal Name ARTICLE Versatile Iron-Catechol based Nanoscale Coordination Polymers. Received 00th January 20xx, Antiretroviral Ligand Functionalization and their use as Efficient Accepted 00th January 20xx Carriers in HIV/AIDS Therapy DOI: 10.1039/x0xx00000x a,b c a c d Rubén Solórzano ,Olivia Tort , Javier García-Pardo , Tuixent Escribà , Julia Lorenzo , Mireia c a b b a Arnedo , Daniel Ruiz-Molina , Ramon Alibés , Félix Busqué* Fernando Novio* A novel chemical approach integrating the benefits of nanoparticles with the versatility of coordination chemistry is reported here to increase the effectiveness of well-known HIV antiretroviral drugs. The novelty of our approach is illustrated using a catechol ligand tethered to the known antiretroviral AZT as a consitutive building block of the nanoparticles. The resulting nanoscale coordination polymers ensure good encapsulation yields and equivalent antiretroviral activity while significantly diminishing its cytotoxicity. Moreover, this novel family of nanoparticles also offer: i) long lasting drug release dissimilar inside and outside the cells depending on pH, ii) triggering in the presence of esterases, activating in an on-off manner the antiviral activity, thanks to a proper chemical design of the ligand and iii) improved colloidal stabilities and cellular uptakes (up to 50 fold increase). The presence of the iron nodes also adds multifunctionality as possible contrast agents. The present study demonstrates the suitability of NCPs bearing pharmacologically active ligands as an alternative to conventional antiretroviral treatments. Introduction toxicity and the development of new treatment strategies that do not rely on daily medication through the development of 8-13 The lifelong treatment of Human Immunodeficiency Virus new nanoscopic platforms for drug delivery. Nanocarriers (HIV) infection nowadays still faces several drawbacks: 1) daily can also facilitate drug transport and entrance to all infected + dosing can be cumbersome for the patient; 2) the existence of CD4 cells reservoirs throughout the body, present even in cellular reservoirs of latent HIV viruses that escape the patients who have had undetectable HIV RNA plasma levels for 1-4 14 treatment; 3) limited capacity of antiretroviral (ARV) drugs years, facilitating its elimination. to access tissue reservoirs and sanctuaries i.e. central nervous So far, different combinations of nanostructured ARV drugs 5 15 system, lymph nodes or lungs; 4) low solubility and poor have been reported using protein-based lactoferrin, 16-18 19,20 bioavailability of the antiviral drugs; and 5) drug resistance polymeric nanoparticles or lipids. In most of these development after continued treatment and the examples modest to low drug loading contents, usually 6,7 corresponding side effects. With no definitive cure and oscillating around 5 wt%, are achieved. More effective forced to suffer these long-life side effects, the target for HIV encapsulation processes have been shown on nanoscale 21-24 drug administration has concentrated on decreasing long-term metal-organic frameworks (nano-MOFs) with antiretroviral 23,25 drug loadings up to 42 wt% or in drug solid nanoparticles ( a. up 70 wt%), obtained using emulsion-templated freeze- Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, 26,27 Campus UAB, Bellaterra, 08193 Barcelona, Spain. drying. However, and in spite of these pioneering results, E-mail: fernando.novio@icn2.cat questions such as minimization of side effects, biodistribution b. Departament de Química, Universitat Autònoma de Barcelona (UAB), Campus improvement or the development of novel formulation UAB. Cerdanyola del Vallès 08193, Barcelona, Spain. c. Laboratory of Retrovirology and Viral Immunopathogenesis, Institut allowing for the metabolization into a pharmacologically active d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), 08036 Barcelona, drug, mainly intracellularly, still represents a real challenge. Spain. d. Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Therefore, the development of novel nanoformulations has Biologia Molecular. Universitat Autónoma de Barcelona, 08193 Bellaterra, become a foremost objective in HIV therapy. Barcelona, Spain. Nanoscale coordination polymer particles (NCPs) embody a Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See novel family of hybrid nanoparticles combining metal ions and DOI: 10.1039/x0xx00000x organic ligands that can use drugs as constitutive building J. Name., 2013, 00, 1-3 | 1 Please do not adjust margins Please do not adjust margins ARTICLE Journal Name 28-31 blocks (chemical entrapment), though physical nucleoside analogue AZT was tethered to this compound using 32-34 encapsulation has also been reported. Chemical 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b] entrapment allows for a better fine-tuning of the release pyridinium 3-oxid hexafluorophosphate (HATU) as a coupling kinetics (up to many hours) as well as better formulation with reagent and N,N-Diisopropylethylamine (DIPEA) as base in THF 35 increased encapsulation yields. The use of active metal drugs, such as Pt(IV), as polymeric nodes of coordination polymers is the most representative and successful example of 29,36-38 chemical entrapment. Less explored has been the chemical entrapment through tethering of active drugs as chelating ligands, in spite of the fact that its efficiency has 39 already been reported. Moreover, the chemical flexibility of organic synthesis may allow for the design of drugs (ligands) 40 cleaved under physiological conditions. As a model compound for these studies, we have selected the broadly studied ARV drug azidothymidine (AZT) that selectively inhibits the HIV-1 reverse transcriptase (Fig. 1a). AZT was long used in the past as an effective antiretroviral drug that rapidly metabolized in the liver to the inactive glucoronide form resulting in poor bioavailability with high residual toxic effects 41 when administered orally. Therefore, this antiretroviral drug represents an adequate example to validate our approach. Specifically we proposed the functionalization of AZT at the 5´- OH position with a chelating catechol group (catAZT, see Fig. 1a). Functionalization at this position has already been shown to be successful for the synthesis of AZT prodrugs with 42,43 chemical and/or enzymatic hydrolysis. Afterwards, this ligand, in combination with an iron salt and a bisdentate ligand is used to form the nanoparticles (see Fig 1a). Finally, the release of the drug is expected to take part following a two- step mechanism: I) liberation of the catAZT from the nanoparticle in a pH controlled process and second, liberation of the free AZT drug from the catechol unit in the presence of the corresponding enzymes (see Fig. 1b). For comparison purposes, to test possible effects of the material without Fig. 1 (a) Schematic representation of a catechol ligand linked to the antiviral drug AZT by an enzymatically-cleavable bond via the carboxylic containing an antiretroviral drug, the use of the analogous but group. Posterior incorporation of catAZT or catTHY into a mixture inactive thymidine (THY) is also proposed (see Fig. 1a). containing iron and a bridging bis-imidazol ligand results in nanoparticle formation induced by fast precipitation of the coordination polymer formed. (b) Schematic representation of the antiviral release process, from Results and discussion the nanoparticles constructs to the AZT drug. It involves two main steps: I) release of the catAZT ligand most likely due to particle degradation and II) Synthesis and characterization of catAZT and catTHY enzymatic cleavage of the free catechol derivative containing AZT. The synthesis of catAZT was achieved through a 5-step synthetic sequence in 32% overall yield starting from leading to derivative 5 in 86% yield. Final removal of the commercially available 3,4-dibenzyloxybenzaldehyde (see Fig. protecting silyl groups of 5 was achieved by using 2a and ESI S1† for details) which reacted with 4- triethylamine trihydrofluoride affording the target compound carboxybutyltriphenylphosphonium bromide and sodium CatAZT in 68% yield. Synthesis of catTHY, changing hydride in dry toluene to afford the olefin 2 in 88% yield as a azidothymidine by thymidine, was also faced from the 5:1 mixture of the Z and E isomers. The use of other bases and common intermediate 4. However, initial attempts to carry out solvents, such as potassium tert-butoxide in dry THF proved to the coupling reaction of 4 with thymidine using conventional be less efficient for this process. Simultaneous hydrogenation coupling reagents (HATU, EDCl/HOBt or CDI) under standard of the alkene moiety and removal of the benzyl protecting conditions were unsuccessful. After some experimentation, it group, at high pressure of H2 under Pd/C catalyst in EtOAc, was found that the reaction of thymidine with 4 under leaded to catechol 3 in almost quantitative yield. Protection of Mitsunobu conditions (Ph3P and DBAB) delivered the 5’- the hydroxyl groups of 3 as their tert-butyldiphenylsilyl substituted compound 6 in 56% yield. After coupling, (TBDPS) derivatives was accomplished by using TBDPSCl and treatment with triethylamine trihydrofluoride removed the 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in dry acetonitrile, silyl protecting group to afford CatTHY in 68% yield (see Fig. 2a affording common intermediate 4 in 64% yield. Next, the and ESI S1† for details). 2 | J. Name., 2012, 00, 1-3 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE Fig. 2 (a) Schematic of catAZT and catTHY synthesis. (b) Preparation of catAZT and catTHY-containing nanoparticles. Next experiments aimed to determine the kinetics of the shown in Fig. 3, in the absence of pig liver esterase (PLE), and enzymatic hydrolysis of catAZT in PBS/BSA 0.5 mM, 37 °C. As independently of the pH used, there was almost no hydrolysis of the ligand (ca. 35% in 72 h, pH 7.4). On the contrary, addition of PLE under the same experimental conditions resulted in a fast release of the antiviral drug (ca. 90% in 1 h). The release profile for both pH 5.1 and 7.4 offered similar results, indicating a weak dependence of the enzymatic activity to the pH. Synthesis and characterization of the nanoparticles Ligand catAZT was used to prepare the corresponding antiretroviral nanoparticles (ARV-NCP) catAZT-NCP following a 32 well stablished methodology consisting in the direct reaction of the catechol ligand and 1,4-bis(imidazol-1-ylmethyl)benzene (bix) with iron(II) acetate using polyvinylpyrrolidone (PVP) as stabilizing agent (Fig. 2b). After vigorous stirring at room temperature, a dark-purple precipitate was collected by centrifugation, washed several times with ethanol, and dried under vacuum. The synthetic procedure is detailed in the experimental section and complete nanoparticle characterization is summarised in Supplementary Information (see ESI S2†). SEM images revealed the formation of spherical Fig. 3 Hydrolysis kinetics of catAZT in the absence (no esterases) or catAZT-NCPs nanoparticles with an average diameter of presence (esterases) of pig liver esterases (PLE) at the indicated pH 147±33 nm (Fig. 4a) while dynamic light scattering (DLS) values. In all cases, experiments were performed in a PBS/BSA 0.5 studies showed an average hydrodynamic radius of 202 ± 10 mM buffer at 37 °C. At the top, schematic representation of the nm (PDI = 0.07) in ethanol (see ESI S2†, Fig. S1). enzymatic cleavage of catAZT by pig liver esterases (PLE). J. Name., 2013, 00, 1-3 | 3 Please do not adjust margins Please do not adjust margins ARTICLE Journal Name The X-ray powder diffraction (XRD) pattern was characteristic of an amorphous material (see ESI S2†, Fig. S2). The Fourier- transform infrared (FTIR) spectra confirmed the coordination of the bix and prodrug ligands to the metal (see Fig. 4b). Characteristic bands for the asymmetric azido stretching band -1 are observed at both catAZT (2108 cm ) and catAZT-NCPs -1 (2106 cm ). Characteristic bands for Bix (1508, 1228 and 1103 -1 cm ) are also observed at catAZT-NCPs FTIR spectrum (1519, -1 1263 and 1097 cm , respectively). Finally, elemental analysis, NMR and ICP measurements data agreed with the presence of the different elements with a general chemical formula [Fe(catAZT)1.5(bix)0.6(AcO)(H2O)2.4] (see ESI S2†, Table S1 and Fig. S3). The stoichiometric deviation from the theoretical results, quite common for NCPs, arises from the out-of- equilibrium conditions used for the synthesis of the nanoparticles. In any case, the reaction is really reproducible as shown by elemental analysis and a complete chemical characterization of at least three different and independent synthetic batches. HPLC quantification of the AZT prodrug showed a loading content of 25% in weight (44% of catAZT), in agreement with the expected value from the previous chemical formula (for detailed characterization see ESI S2†, Fig. S4-S5). ICP measurements for the iron metal ion (5.46%), were also in agreement with that deduced from the formula of elemental analysis (5.50%). Although the original source of 2+ 3+ iron is a Fe salt, the complex was stabilized as high-spin Fe as shown by Mössbauer spectroscopy (see ESI S2†, Fig. S6). This electronic modification results from a redox interplay between the metal ion and the electroactive catechol ligands 44 in air as previously reported. Complementarily, model catTHY-NCPs nanoparticles were also synthesized and tested for comparison purposes. catTHY-NCPs nanoparticles showed comparable physicochemical features to those found for catAZT-NCPs , for example, an average diameter of 87± 26 nm was measured by SEM micrographs and an average hydrodynamic radius of 170±2 nm (PDI = 0.14) in ethanol by DLS. (for complete characterization see ESI S3†). Colloidal stability. Zeta-potential values close to -8.0 mV were obtained for catAZT-NCPs dispersed in 20 mM PBS buffer at pH 7.4. In agreement with such low values, a time dependent aggregation process was observed that eventually ended up with the precipitation of the sample, even at low-moderate concentrations. The addition of Bovine Serum Albumin (BSA) has been extensively used for dispersing inorganic and 45,46 polymeric nanoparticles. In our case, fluorescence quenching studies of BSA in the presence of NCPs (see ESI S2†, Fig. S7) suggested an interaction of the NCPs with the surface of this model protein. Accordingly, in the presence of such protein nanoparticles turn out to be stable except at very high Fig. 4 (a) Representative SEM image of catAZT-NCPs. Inset: histogram concentrations (i.e concentrations >2 mg/ml) where the of particle size extracted from SEM micrographs (200 particles, mean 47 size 147 ± 33 nm. (b) FTIR spectra of i) Bix, ii) catAZT and iii) catAZT- addition of additional sucrose is needed (see ESI S2†, Fig. S8). NCPs. (c) Effect of catAZT-NCPs and free AZT on the cell viability of Cell viability. Toxicity to lymphocytic function is one of the human primary CD4+ T lymphocytes. CD4+T cells were incubated major considerations in the clinical applicability of novel during 24h in the presence of the indicated concentrations of catAZT- antiviral compounds. Moreover, it has been extensively NCPs or AZT. Cell viability is expressed as percentage compared to an 48 untreated control. Values are mean ± standard error of the mean demonstrated that AZT is highly toxic to human lymphocytes . (SEM) (n = 3). (d) 1H MRI T1 and T2 phantoms maps of catAZT-NCPs in Therefore, we have examined the cytotoxic effect of the a PBS/agarose 1% solution at pH 7.4 at different concentrations (0, 1, catAZT-NCPs nanoparticles against endogenous human CD4+ T 5, 10, 25 mM, referred to the Fe concentration). 4 | J. Name., 2012, 00, 1-3 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE lymphocytes. Primary CD4+ T cells were prepared by the rapid expansion method (REM) and treated for 24 h with catAZT- NCPs and free AZT, used as the reference compound (Fig. 4c). When the CD4+ T cells were treated with AZT, a clear cytotoxic effect was observed already at concentrations of 10 µM (or higher), with an IC50 value of 64 µM. To our surprise, when the same cells were treated with catAZT-NCPs, irrelevant cytotoxic effects were detected at least up to the highest concentration tested (500 µM). At this concentration, the nanoparticles maintained a cell viability of 70%, while for free AZT was <10% (IC50 > 500µM). These results confirmed that + AZT remarkably reduced the toxicity on primary CD4 T lymphocytes upon nanostructuration. MRI experiments. To confirm that the functionalization with AZT does not disrupt the multifunctional character of these nanoparticles, MR relaxometry experiments at different concentrations under an external magnetic field of 7 Teslas and in two phantom sequences, were done (Fig. 4d). The nanoprobes were dispersed in a PBS/agarose 1% to ensure a good colloidal stability. The obtained relaxation rate values were plotted versus the iron concentration reporting good linear correlations (see ESI S2†, Fig. S9). The iron-based NCPs Fig. 5 (a) Schematic representation of the different release steps for exhibit a signal enhancement in a concentration-dependent the catAZT prodrug (step 1) and subsequent release of AZT (step 2). (b) -1 -1 manner and a T1 positive contrast of r1 = 0.15 mM s , and T2 AZT release kinetics from catAZT-NCPs in the absence (no esterases) or -1 -1 negative contrast of r2 = 117.5 mM s . Curiously, these in the presence (esterases) of pig liver esterase (PLE) at pH 5.1 or 7.4. nanoparticles exhibit a low r1 in comparison to the commonly In all the cases, the experiments were performed in PBS/BSA buffer at 37 ºC (see experimental section for details). used gadolinium contrast agents (i.e. Gd-DTPA; r1= 3.3 −1 −1 mM s ) or other related Iron-based NCPs (i.e. Fe-NCPs; −1 −1 49 (r1=4.4 mM s ). However, they present a high T2 negative circulate throughout the body at physiological pH enhancing contrast, which make these nanoparticles a very promising T2 its biodistribution. contrast agent with r2 relaxivities nearly 25 times higher than Completely different results were obtained in the presence of Gd-DTPA. the model esterase PLE (180 U/L). In this case, AZT release appeared to be faster, reaching almost up to 100% after 50 Drug release profile hours and with estimated half-lives of ∼4.5 h (pH 5.1) and ∼10 h (pH 7.4). These results confirmed that the enzymatic The release profile of AZT from the catAZT-NCP nanoparticles hydrolysis was fast, as previously described for the free catAZT involves two main steps: 1) release of the catAZT ligand from ligand, so the limiting step was the release of the catAZT from the nanoparticles and 2) its transformation onto the active AZT the nanoparticles. prodrug upon enzymatic cleavage by esterases (see Fig. 5a). First experiments were done to determine the kinetics of HIV-Antiviral activity of catAZT-NCPs catAZT release (step 1) by using HPLC for the quantification, upon incubation at 37 °C of the nanoparticles in a PBS/BSA 0.5 The antiviral activity of the whole set of synthesized mM buffer solution at two different pHs, 5.1 and 7.4. First nanoparticles and control molecules was tested on a model experiments in the absence of esterases at pH 7.4 showed no MT-2 human lymphocytic cell line by means of the MTT assay detectable presence of catAZT outside the dialysis bag, in a biohazard P3 laboratory specially prepared for it (for more indicating the high stability of the coordination polymer at this info see Experimental Section). MT-2 cells is an established cell pH (for more details see Experimental Section). Nevertheless, line of CD4+ T cells, easier to manipulate and obtain than significant amounts were found at the lowest pH of 5.1, even primary human cells, thus suitable for first line studies of anti- at early stages of the release, due to the lower stability of the HIV effect and, therefore broadly used to test the efficacy coordinative bond between the iron and the catechol at low experimental antiviral agents thanks to its high 50-52 pH values. This pH-triggered liberation of the catAZT ligand reproducibility. HIV-1 exerts a profound cytopathic (CPE) could favour the release inside the cells (i.e. acidic pH present effect against CD4+ T lymphocytes. Once infected, the in lysosomes), decreasing toxicity-associated side effects. lymphocytes accumulate viral DNA and actively produce HIV Independently of the pH, and in the absence of esterases the proteins, which results in the concomitant lysis of such presence of free AZT was low (≈ 15% at 72 h, see Figure 5b). infected cells by apoptosis. MT-2 cells are profoundly sensitive Overall, the release rates are sufficient for the NCPs to to the CPE effect of HIV-1. J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins Please do not adjust margins ARTICLE Journal Name Uptake experiments. The therapeutic efficacy of an HIV-1 report, in which the nanoformulation of the antiviral drug inhibitor depends on its intracellular concentration, which in dolutegravir resulted in higher uptake by human monocyte- 61 turn is directly related with its uptake kinetics (in addition to derived macrophages in comparison with the free molecule. 53, 54 other factors such as its metabolism and/or cellular efflux). It is worth to mention that cellular dolutegravir levels reached To assess this question, MT-2 cells were incubated for 4 hours a maximum at 4 h after treatment with 100 µM of the in the presence of different concentrations of catAZT-NCPs nanoformulated drug. (AZT and catAZT were also used as model compounds). After HIV-Antiviral activity. The cytotoxicity on non-infected human this time, the amount of intracellular AZT was determined by MT-2 lymphocytes was at first determined. No appreciable HPLC-UV using an optimized procedure (see ESI S4† and cytotoxicity was observed for any of the antiretroviral agents Experimental Section for more details). The results shown in at concentrations of 20 μM or lower after 3 or 7 days Fig. 6 indicate intracellular levels of AZT after direct exposure incubation except for the highest concentrations of the of the MT-2 cells to AZT, catAZT and catAZT-NCPs. The prodrug/ligand catAZT (100 μM), which showed reduced cell intracellular concentration of AZT up to concentrations of 500 viability of ~70 % (for more info see ESI S5†, Fig. S18). When 6 µM (<0.070 nmols/10 cells) was undetectable after AZT or the same experiments were now repeated using MT-2 cells catAZT incubation. Only at extremely and unrealistic high infected with the virus, a decrease of the cytopathic effect of concentration of such compounds of 1000 µM (see Fig. 6), HIV on human MT-2 cells was observed in the cultures treated detectable intracellular levels of AZT (0.81 ± 0.15 and 0.62 ± with AZT, catAZT and catAZT-NCPs for 3 and 7 days treatment 6 0.11 nmols/10 for AZT and catAZT, respectively), were (see Fig. 7). In particular, a substantial increase in the cell measured. Interestingly, concentrations of the catAZT-NCPs as viability was observed after three days following the order AZT low as 100 µM (normalized versus the AZT concentration) > catAZT-NCPs > catAZT. Certainly, the most efficient antiviral already resulted in detectable intracellular levels of AZT, with a significant concentration-dependent increase up to 500 µM 6 (3.48 ± 0.33 nmols of AZT/10 cells). Fig. 6 Intracellular levels of AZT in MT-2 cells after 4h of incubation in the presence of different concentrations (100 µM, 200 µM, 500 µM, referred to the AZT equivalent concentrations) of AZT, catAZT or catAZT-NCPs. The asterisk (*) indicates those conditions with levels of AZT in the samples under the limit of detection of the method (<0.070 nmols/106 cells). For AZT and catAZT, an additional concentration of 1000 µM was assayed. Note that Fig. 7 HIV-antiviral activity. Cell viability of different concentrations of AZT, this concentration was not assayed for catAZT-NCPs (**) because catAZT, catAZT-NCPs, and catTHY-NCPs on HIV-infected MT-2 cell culture nanoparticles were not stable at concentrations higher than 10 mg/ml. after 3 days (a,b), and 7 days incubation (c,d) . The cytopathic effect of HIV (decrease in cell viability) is observed after incubating HIV-infected MT-2 Considering the detection limit of the HPLC method used for cells with the compounds with no antiviral effect (catTHY-NCPs and 6 the quantification as a reference (<0.070 nmols/10 cells), we saccharose). The antiviral effect of the compounds was indirectly measured can easily estimate that treatment with the nanostructured as an increase in cell viability (increase in absorbance at 620 nm), as shown when incubating HIV-infected MT-2 cells in the presence of AZT, catAZT- material resulted in a >50 fold increase at 500 µM in NCPs or catAZT (a,c). (b,d) Relative Anti-HIV activity expressed as percentage comparison with free AZT. This could potentially be due to the compared to the absence of activity (saccharose) and maximal anti-viral mechanism of uptake. Nanoparticles are generally taken up by activity in the assay determined for the most effective concentration of AZT 55-58 (4 μM) after b) 3 days or d) 7 days incubation. An unpaired t-test was endocytosis mechanisms, as opposed to small molecules, performed to compare anti-HIV efficacies of the compounds at 4 μM which are taken up by other different pathways. In the case of compared to the reference compound AZT. Note that the anti-viral efficacy AZT, it has been described that enters inside the cell through of catAZT-NCPs was similar to that of AZT, while the effect of the prodrug membranes via passive diffusion and/or by uptake catAZT and the control catTHY-NCPs were significantly lower. ns, non- 59,60 transporters. Similar results were observed in a recent significant; * p< 0.05; ** p< 0.01; *** p<0.0001. 6 | J. Name., 2012, 00, 1-3 Please do not adjust margins Please do not adjust margins Journal Name ARTICLE response was obtained for the free AZT (followed closed by Acknowledgements with the catAZT-NCPs), which recovered the cell viability This work was supported by project MAT2015-70615-R, within the 0.16-40 μM concentration range and with a MAT2012-35324, CTQ2013-44161-R, CTQ2016-75363-R and maximum antiviral activity centered at 4 μM. At this BIO2016-78057-R from the Spanish Government and FEDER concentration, the efficiency of catAZT-NCPs nanoparticles was funds. Authors also thank Instituto de Salud Carlos III (ISCIII), slightly -but non-significantly- inferior than AZT, while the Madrid, Spain - Red de Investigación en SIDA (RIS), ISCIII-RETIC ligand catAZT showed the lowest activity (Fig. 7a and 7b). (RD16/0025/0002). R.S. thanks the Ministerio de Educación, Control systems (i.e. catTHY-NCPs and saccharose) did not Cultura y Deporte for the predoctoral grant FPU14/03170. The show any significant effect. Interestingly, when the same ICN2 is funded by the CERCA programme/Generalitat de experiment was evaluated after seven days of incubation, the Catalunya and supported by the Severo Ochoa programme of relative activity of AZT and catAZT-NCPs becomes now very the Spanish Ministry of Economy, Industry and similar (Fig. 7c), fact attributed to the accumulated long lasting Competitiveness (MINECO, grant no. SEV-2013-0295). MR release effect of the nanoparticles, while the difference of studies were carried out at the joint NMR facility of UAB and activity with catAZT ligand becomes more evident (Fig. 7d). CIBER-BBN, Unit 25 of NANBIOSIS, with a 7T horizontal. Conclusions Notes and references We have designed and synthesized novel iron-catechol based ‡ Footnotes relating to the main text should appear here. These nanoscale coordination polymeric particles that incorporate a might include comments relevant to but not central to the prodrug molecule tethered to a catechol ligand. To illustrate matter under discussion, limited experimental and spectral data, the potential of this nanoplatform we have used as catechol and crystallographic data. ligand (catAZT) a catechol linked, through an enzymatically § cleavable ester bond, to the well-known antiretroviral AZT. The presence of a chelating catechol group allowed for the 1. D. A. Kulpa and N. Chomont, J. Virus Erad., 2015, 1, 59-66. reproducible incorporation of catAZT with high payloads within 2. T.-W. Chun, S. Moir and A. S. Fauci, Nat. Immunol., 2015, coordination polymer nanoparticles of 147±5 nm average size. 16, 584-589. Following this approach, we have successfully reproduced the 3. C. Schwartz, S. Bouchat, C. Marban, V. Gautier, C. Van effective antiretroviral activity of the free AZT prodrug while Lint, O. Rohr and V. Le Douce, Biochem. Pharmacol., 2017, the nanostructuration allows for the following significant 146, 10-22. advantages: I) stabilization of the drug in physiological media 4. A. Sosnik, Nanomedicine, 2010, 5, 833-837. as a colloidal suspension; II) control over the release properties 5. R. Rose, D. J. Nolan, E. Maidji, C. A. Stoddart, E. J. Singer, of the drug by the pH and the presence of enzymes, III) the S. L. Lamers and M. S. McGrath, AIDS Res. Hum. Retroviruses, 2017. nanoparticles retain the inherent multifunctionality thanks to 6. B. K. Titanji, D. Pillay and C. Jolly, J. Gen. Virol., 2017, 98, the presence of iron ions with MRI responses, IV) significant 821-834. reduction of the AZT toxicity and V) enhancement of the 7. A. F. Capetti, M. Micale, L. Carenzi, F. Niero, S. Landonio, cellular uptake (up to 50 fold increase). catAZT-NCPs S. Vimercati, G. Dedivitiis and G. 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