Biomaterials 280 (2022) 121258 Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials A multivalent Ara-C-prodrug nanoconjugate achieves selective ablation of leukemic cells in an acute myeloid leukemia mouse model Victor Pallarès a, b, c, 1, Ugutz Unzueta a, b, c, f, 1, Aïda Falgàs a, b, c, 1, Anna Aviñó c, d, Yáiza Núñez a, b, Annabel García-León a, b, Laura Sánchez-García c, e, f, Naroa Serna c, e, f, Alberto Gallardo a, g, Lorena Alba-Castellón a, b, Patricia Álamo a, b, c, Jorge Sierra b, h, Lídia Cedó a, i, Ramon Eritja c, d, Antonio Villaverde c, e, f, Esther Vázquez c, e, f, ***, Isolda Casanova a, b, c, **, Ramon Mangues a, b, c, * a Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona, 08041, Spain b Josep Carreras Leukaemia Research Institute, Barcelona, 08916, Spain c CIBER en Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Madrid, 28029, Spain d Institute for Advanced Chemistry of Catalonia (IQAC), CSIC, Barcelona, 08034, Spain e Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain f Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, 08193, Spain g Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain h Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain i CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, 28029, Spain A R T I C L E I N F O A B S T R A C T Keywords: Current therapy in acute myeloid leukemia (AML) is based on chemotherapeutic drugs administered at high Targeted protein nanoparticle doses, lacking targeting selectivity and displaying poor therapeutic index because of severe adverse effects. Here, Multivalency we develop a novel nanoconjugate that combines a self-assembled, multivalent protein nanoparticle, targeting Targeted drug delivery the CXCR4 receptor, with an Oligo-Ara-C prodrug, a pentameric form of Ara-C, to highly increase the delivered Ara-C prodrug CXCR4 payload to target cells. This 13.4 nm T22-GFP-H6-Ara-C nanoconjugate selectively eliminates CXCR4+ AML cells, Acute myeloid leukemia which are protected by its anchoring to the bone marrow (BM) niche, being involved in AML progression and chemotherapy resistance. This nanoconjugate shows CXCR4-dependent internalization and antineoplastic ac­ tivity in CXCR4+ AML cells in vitro. Moreover, repeated T22-GFP-H6-Ara-C administration selectively eliminates CXCR4+ leukemic cells in BM, spleen and liver. The leukemic dissemination blockage induced by T22-GFP-H6- Ara-C is significantly more potent than buffer or Oligo-Ara-C-treated mice, showing no associated on-target or off-target toxicity and, therefore, reaching a highly therapeutic window. In conclusion, T22-GFP-H6-Ara-C ex­ ploits its 11 ligands-multivalency to enhance target selectivity, while the Oligo-Ara-C prodrug multimeric form increases 5-fold its payload. This feature combination offers an alternative nanomedicine with higher activity and greater tolerability than current intensive or non-intensive chemotherapy for AML patients. 1. Introduction Ara-C (cytosine arabinoside; cytarabine) combined with anthracyclines, usually daunorubicin or idarubicin [1,2]. In most patients, this therapy Over the last four decades, the 7 + 3 remission induction therapy has is able to eliminate leukemic cells achieving a complete remission, but been the standard of care for acute myeloid leukemia (AML). This fatally killing normal hematopoietic cells, which, in turn, may put the intensive chemotherapy treatment is based on the use of high doses of patient at risk of suffering complications such as infections, * Corresponding author. Biomedical Research Institute Sant Pau (IIB-Sant Pau), Josep Carreras Leukaemia Research Institute (IJC) and CIBER-BBN, Spain. ** Corresponding author. Biomedical Research Institute Sant Pau (IIB-Sant Pau), Josep Carreras Leukaemia Research Institute (IJC) and CIBER-BBN, Spain. *** Corresponding author. Institute of Biotechnology and Biomedicine (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona and CIBER-BBN, Spain. E-mail addresses: esther.vazquez@uab.cat (E. Vázquez), icasanova@santpau.cat (I. Casanova), rmangues@santpau.cat (R. Mangues). 1 VP, UU and AF contributed equally to this work. https://doi.org/10.1016/j.biomaterials.2021.121258 Received 26 May 2021; Received in revised form 22 October 2021; Accepted 13 November 2021 Available online 24 November 2021 0142-9612/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). V. Pallarès et al. Biomaterials 280 (2022) 121258 inflammatory processes, cardiotoxicity or even death [3–5]. In addition, min at 20,000 g) and charged in a HiTrap Chelating HP 1 mL column (GE other patients, specially the elderly or those with unfavorable profiles, Healthcare) for IMAC protein purification with an ÄKTA pure (GE cannot be considered candidates for intensive chemotherapy [1]. Healthcare). Protein elution was performed by a lineal gradient of In recent years, novel therapies have been approved by the FDA to elution buffer (20 mM Tris, 500 mM NaCl, 500 mM Imidazole) and treat patients with specific molecular alterations, for instance, FLT3 or purified protein fraction was then dialyzed against sodium carbonate IDH inhibitors, antibody drug conjugates targeting overexpressed CD33 with salt buffer (166 mM NaCO3H, 333 mM NaCl pH = 8). Protein purity or CD123 surface receptors, or inhibitors of proteins that regulate was determined by SDS-PAGE electrophoresis followed by western-blot leukemic cell survival or proliferation pathways, such as BCL or immunodetection with an anti-His monoclonal antibody (Santa Cruz Hedgehog [6–9]. However, most of these drugs are small molecules that Biotechnology) and protein integrity was determined by MALDI-TOF use passive diffusion, have a short half-life in blood, undergo renal mass spectrometry. Purified protein nanoparticles amount was finally clearance and do not efficiently accumulate in tumors [10–12]. There­ determined by Bradford assay. fore, new specific targeted drugs that overcome these limitations, and consequently increase antineoplastic efficacy while reducing systemic 2.3. T22-GFP-H6-Ara-C conjugation toxicity, are still needed. To this purpose, nanomedicines can become a new tool to improve the pharmacokinetic properties and the bio­ Oligonucleotides containing Ara-C (Oligo-Ara-C) were prepared distribution of classical drugs by selectively delivering potent cytotoxic using the commercial protected N4-Acetyl-2′ -O-acetyl-5′ -O-DMT-arabi­ drugs directly to leukemic cells exploiting the receptor overexpression nosyl cytosine 3′ -CE phosphoramidite unit (Carbosynth) following observed in malignant cells [13–15]. In fact, we have designed a standard methodologies (Supporting Information and Fig. S1). T22-GFP- multivalent nanoparticle capable of selective internalization in leukemic H6-Ara-C nanoconjugates (NCs) were generated by covalent binding of cells that overexpress the receptor CXCR4 [16]. CXCR4 is a chemokine T22-GFP-H6 nanoparticles and Oligo-Ara-C (Ara-C pentameric form) receptor that is overexpressed in leukemic cells in a large number of through protein lysine amines in a two-step reaction. For that, thiol AML patients, conferring a poor prognosis or higher relapse rate to them functionalized Oligo-Ara-C molecules were first separated from DTT by [17–22]. In addition, CXCR4 and its SDF1 ligand are determinants of NAP-10 sephadex desalting column (GE Healthcare) and then reacted minimal residual disease (MRD) and limit chemotherapy effectiveness in with an EMCS bi-functional cross-linker (6-Maleimidohexanoic acid N- AML patients [23,24]. T22-GFP-H6 is a self-assembled protein nano­ hydroxysuccinimide ester) by thiol-Maleimide reaction in a 1:1 (Oligo- particle that incorporates around 11 T22 peptidic ligands, acquiring Ara-C: linker) molar ratio for 10 min at R.T. This reaction efficiently multivalency for CXCR4 binding. This property confers the nanoparticle incorporates active NHS-ester groups to Oligo-Ara-C. NHS-ester func­ the capacity to selectively internalize in CXCR4-overexpressing tionalized Oligo-Ara-C molecules were then immediately reacted with (CXCR4+) leukemic cells. In fact, we have previously demonstrated T22-GFP-H6 external lysine-amines at a 1:5 (protein:Oligo-Ara-C) molar the ability of this nanoparticle, once conjugated to a microtubule in­ ratio O/N at R.T. Generated NCs were finally dialyzed against sodium hibitor, to internalize and eliminate target leukemic cells [25]. Here, we carbonate with salt buffer in a high permeability 12–14 MWCO mem­ demonstrate that the conjugation of the multivalent T22-GFP-H6 brane (Spectrum labs) in order to remove non-reacted free Oligo-Ara-C nanoparticle to a new prodrug, a pentameric form of Ara-C, accommo­ molecules. Generation of NCs was determined by MALDI-TOF mass dates a high payload of this drug, which is used in standard care of spectrometry, and conjugated Oligo-Ara-C amount was finally quanti­ several hematological cancers. Thus, the use of T22-GFP-H6-Ara-C could fied by UV/visible light spectrophotometry at 260 nm. be more effective than equimolar doses of Oligo-Ara-C in a disseminated CXCR4+ AML mouse model. 2.4. Light scattering analysis 2. Material and methods Volume size distribution and zeta potential of T22-GFP-H6 nano­ particles and T22-GFP-H6-Ara-C NCs were determined by Dynamic 2.1. Cell lines Light Scattering (DLS) and Electrophoretic Light Scattering (ELS) respectively at 633 nm in a Zetasizer Nano ZS (Malvern instruments). THP-1 and SKM-1 AML cell lines were purchased from DSMZ Samples were measured in triplicate and average peak size (nm), zeta (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell (mV) and polydispersion index (pdi) were calculated. Cultures, Braunschweig, Germany). Cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 10 mmol/L L-glutamine, 100 2.5. Electron microscopy U/mL penicillin, 10 mg/mL streptomycin and 0.45 μg/mL fungizone (Gibco, Thermo Fisher Scientific (TFS), Waltham, MA, US) and kept at Ultrastructural morphology (size and shape) of T22-GFP-H6-Ara-C 37 ◦ C in a humidified atmosphere of 5% CO2. Furthermore, THP-1 cells nanoconstructs was visualized with two rapid high-resolution imaging were transfected with a plasmid encoding the luciferase gene using techniques. Drops of 5 μL of sample diluted at 0.2 μg/mL in its buffer Lipofectamine LTX and PLUS reagents (A12621, Invitrogen, TFS) ac­ were deposited both in silicon wafers (Ted Pella) for field emission cording to the manufacturer’s instructions which conferred biolumi­ scanning electron microscopy (FESEM) and negatively stained with 2% nescence (BLI) to the cells (hereinafter referred to as THP-1-Luci). uranyl acetate (Merck) in 400 mesh carbon-coated copper grids (Elec­ tron Microscopy Sciences) for transmission electron microscopy (TEM). 2.2. Protein nanoparticles production and purification Images of general fields and nanoparticle details were obtained at three magnifications with a FESEM Merlin (Zeiss) operating at 1 kV and Synthetic gene encoding for T22-GFP-H6 protein was designed in equipped with an in-lens secondary electron detector and a TEM JEM house and provided by Geneart (Thermo Fisher) into pET22b plasmid 1400 (Jeol) operating at 80 kV and equipped with an Orius SC200 CCD (Novagen). T22-GFP-H6 nanoparticles were produced in E.coli Origami camera (Gatan), respectively. B (Novagen) O/N at 20 ◦ C upon induction with 0.1 mM IPTG (isopropyl- β-D-thiogalactopyronaside). Cells were then harvested by centrifugation 2.6. Fluorescence determination (15 min at 5000 g) and disrupted in a Tris buffer (20 mM Tris, 500 mM NaCl, 10 mM Imidazole) supplemented with EDTA-Free protease in­ Fluorescence of T22-GFP-H6 nanoparticles and T22-GFP-H6-Ara-C hibitor cocktail (cOmplete™ EDTA-Free, Roche Diagnostics, Basel, NCs was measured in a Varian Cary Eclipse fluorescence spectropho­ Switzerland) by 3 rounds of French press (Thermo) at 1200 psi. Protein tometer (Agilent Technologies) upon excitation at 450 nm and emission containing soluble fraction was then separated by centrifugation (45 scan from 500 to 525 nm. Samples were measured in triplicate. 2 V. Pallarès et al. Biomaterials 280 (2022) 121258 2.7. Confocal laser microscopy subcutaneously in a single dorsal flank of each mice. Tumor growth was monitored twice a week with a caliper (tumor volume = width2 x THP-1 cells were cultured in 24 well plates and incubated with 1 μM length/2). When tumors reached a volume of 300–500 mm3, control T22-GFP-H6-Ara-C or buffer for 2 h to evaluate NC internalization by mice (“Buffer”) received a single intravenous dose of NC buffer (NaCO3H confocal microscopy. Cell nuclei and membranes were then labeled with NaCl pH = 8, n = 3) whereas experimental mice (“T22-GFP-Ara-C”) an 0.2 μg/mL Hoechst 33 342 (Molecular Probes) and 2.5 μg/mL CellMask intravenous dose of 200 μg T22-GFP-H6-Ara-C, which contains the GFP Deep Red (Molecular Probes) respectively for 10 min and subsequently fluorescent protein that allows the in vivo NC tracking. Experimental washed in DPBS (Gibco) twice before adding fresh medium. Cells were mice were euthanized at different time points (10 min, 30 min, 2 h, 5 h, finally transferred to a slide, covered by a glass cover and recorded in a 24 h and 48 h, n = 3/group) after the NC administration. The ex vivo TCS-SP5 confocal laser scanning microscope (Leica Microsystems) using fluorescence intensity (FLI) was quantified in SC tumors, spleen, liver, a Plan Apo 63 x/1.4 (oil HC x PL APO lambda blue) objective and a blue heart, lungs, backbone, brain and plasma. Plasma was obtained by diode (405 nm), an Ar laser (488 nm) and a HeNe laser (633 nm) for intracardiac puncture (25G) and blood centrifugation at 600g for 10 min Hoechst, GFP and CellMask excitation respectively. Z stacks of different at 4 ◦ C. Moreover, we also collected urine from control and experimental sections were also acquired every 0.5 μm and 3D images analyzed using mice. Imaris 7.2.1 software (Bitplane). The emitted FLI was registered with the IVIS Spectrum (124 262, PerkinElmer, Waltham, MA, USA). FLI correlates to the amount of 2.8. Internalization and antineoplastic capacities of T22-GFP-H6-Ara-C accumulated protein in each tissue and is expressed as average radiant in CXCR4+ AML cell lines efficiency. FLI from experimental mice was calculated subtracting the FLI auto-fluorescence of control mice. Finally, the T22-GFP-H6-Ara-C First, the internalization capacity of the T22-GFP-H6-Ara-C was biodistribution in all organs from 2 to 48 h was measured by cumula­ evaluated in AML cells. THP-1 and SKM-1 were cultured at 20 × 104 tive FLI using the area under the curve (AUC). cells/mL and were treated with the NC at 50, 125 and 250 nM for 1 or 24 h. After that, cells were washed with PBS and were treated with Trypsin- 2.10. Detection of T22-GFP-H6-Ara-C in plasma and urine by western EDTA (0.5%, no phenol red, Gibco, TFS) at 1 mg/mL for 15 min to blot remove the membrane-bound NCs that were not internalized. After 2 washes with PBS, GFP fluorescence was measured by flow cytometry The same volume of plasma (2 μL) or urine (10 μL) from mice treated using FACS Calibur cytometer. Two technical and biological replicates with buffer or T22-GFP-H6-Ara-C at different time points were used for were performed for each condition. Data acquisition was analyzed by the NC detection. The rest of volume until 20 μL was added with Milli-Q Cell Quest Pro software and results were expressed as mean fluorescence water and 4 μL of LB6X-DTT 3 M was mixed to each sample. Then, intensity (MFI) relative to the MFI of the buffer (MFIR) ± standard error samples were separated using 15% SDS-PAGE and transferred to a (SE). nitrocellulose blotting membrane. Membranes were blocked with 5% Secondly, a receptor competition assay was performed pre-treating skim milk in TBST for 2 h at room temperature, and then incubated with or not THP-1 cells with AMD3100, an antagonist of CXCR4, at 7500 the 1:500 GFP primary antibody (sc-9996, Santa Cruz Biotechnology) nM for 1 h prior to the exposure to 250 nM of T22-GFP-H6-Ara-C (ratio O/N. Membranes were washed with TBST and then incubated with the 30:1) for 1, 24 or 48 h. Trypsin-EDTA incubation and FACS analyses mouse secondary antibody (1:10 000, Jackson Immune Research, West were performed as described above and results were presented as per­ Grove, Pennsylvania, USA) for 1 h. The positive control for GFP detec­ centage of cells with T22-GFP-H6-Ara-C internalization after exposed tion was 1 ng of T22-GFP-H6-Ara-C. Western blot visualization was them with AMD3100 compared with the group of cells exposed to NC performed using the SuperSignal West Pico Chemiluminescent Substrate without AMD3100. (Thermo Fisher Scientific) and the Universal Hood II Gel Doc Imaging Finally, T22-GFP-H6-Ara-C antineoplastic activity was evaluated in System (BioRad). CXCR4+ AML cell lines and compared with the Oligo-Ara-C anticancer activity at equimolar doses. THP-1 and SKM-1 were cultured at 30 × 104 2.11. Evaluation of the in vivo antineoplastic effect of T22-GFP-H6-Ara- cells/mL and treated with T22-GFP-H6-Ara-C or Oligo-Ara-C at different C in a CXCR4+ AML mouse model concentrations between 25 and 500 nM (equimolar doses for Oligo-Ara- C between 91.5 and 1830 nM) for 48 h. Then, cells were incubated for 4 To assess the antineoplastic activity of T22-GFP-H6-Ara-C in vivo, a h with the XTT reagents (Cell Proliferation Kit II, Roche Diagnostics) and CXCR4+ AML disseminated mouse model was used [25]. the absorbance was measured at 492 nm in a FLUOstar OPTIMA spec­ NSG (NOD-scid IL2Rgammanull) female mice (4 weeks old) were trophotometer (BMG Labtech, Ortenberg, Germany). Cell viability per­ obtained from Charles River Laboratories and housed in microisolator centage was obtained by subtracting the absorbance of the blanks and units with sterile food and water ad libitum. In a preliminary assay, we expressing each condition as percentage of cell growth (±standard error, run an experiment to identify the most effective anticancer dosage in SE), as compared with untreated controls (buffer treated cells). Five vivo that showed no toxicity. After one week of quarantine, twenty NSG technical and three biological replicates were performed for each mice were intravenously (i.v.) injected with THP-1-Luci cells (1 × 106 condition. cells/200 μL) and randomized into a control group and four different experimental groups (n = 4/group). Two days later, mice were i.v. daily 2.9. T22-GFP-H6-Ara-C pharmacokinetics and biodistribution in a injected with NC buffer (NaCO3H NaCl pH = 8) in the control group, and subcutaneous (SC) AML mouse model the other four groups with daily doses of 5, 25, 100 or 200 μg of T22- GFP-H6-Ara-C for 10 days. The AML progression in the mice was Four-week old female Swiss Nude mice were obtained from Charles monitored every 2 or 3 days in IVIS Spectrum by detecting in vivo BLI in River Laboratories (Wilmington, MA, US). Mice were maintained in mice. Weight of mice was controlled during the experiment and all of specific pathogen-free (SPF) conditions with sterile food and water ad them were euthanized the first day at which any mouse presented libitum. All in vivo procedures were conducted in accordance with the relevant signs of disease such as lack of mobility or 10% weight loss. guidelines approved by the institutional animal Ethics Committee of Afterwards, a more exhaustive study with a higher number of mice Hospital Sant Pau (project number 10108 by the Government of Cata­ was performed with the most effective T22-GFP-H6-Ara-C dosage that lonia) and performed following the European Union Directive 2010–63- lacks off-target toxicity (200 μg, n = 8) compared to mice treated with EU for welfare of the laboratory animals. the Oligo-Ara-C at an equimolar dose (23.85 nmol, n = 8) and the NC After 1 week of quarantine, 10 million THP-1 cells were injected buffer (NaCO3H NaCl pH = 8, n = 8) (Fig. S2A). Following the 3 V. Pallarès et al. Biomaterials 280 (2022) 121258 experimental design from the previous experiment, each treatment was oligonucleotides of Ara-C (Oligo-Ara-C) (Supporting Information and administered for 10 daily doses and the AML mouse progression as well Fig. S1) to the T22-GFP-H6 targeting vector (Fig. 1A). First, a 6-Maleimi­ as mouse weight were also registered every 2 or 3 days. BLI of relevant dohexanoic acid N-hydroxysuccinimide ester (EMCS) bifunctional cross- organs and tissues such as spleen, liver, hindlimbs, cranium and back­ linker was covalently bound to a thiol functionalized Oligo-Ara-C bone were analyzed ex vivo after euthanasia. Then, tissues were fixed in (Oligo-Ara-C-SH) by maleimide-thiol reaction, generating a thioester formaldehyde 3.7% and paraffin-embedded for further histological and bond. Then, the generated Oligo-Ara-C-Linker complexes were subse­ immunohistochemistry analyses. Results were expressed as Total flux of quently reacted with T22-GFP-H6 protein through exposed lysine- BLI (photons/second; Radiance photons) ± SE for the in vivo studies and amines by NHS ester-amino reaction generating an amide bond. as Average Radiance of BLI (photons/second/cm2/sr) ± SE for the ex This process resulted in the efficient binding of an average of 3–4 vivo studies. pentameric Oligo-Ara-C molecules per protein, that was calculated by UV light spectrophotometry and further corroborated by MALDI-TOF 2.12. Hepatic and renal function for toxicity assessment mass spectrometry. In concordance with the spectrophotometry data, T22-GFP-H6-Ara-C NCs showed up to four additional peaks with Blood samples were collected from the in vivo preliminary assay sequential molecular weight (MW) increase of around 2172 Da (range: (mice treated with buffer or 5, 25, 100 or 200 μg of T22-GFP-H6-Ara-C 2138–2242 Da) over the MW of T22-GFP-H6 (30.6 kDa) in its MALDI- for a total of 10 doses, n = 4/group) at the last day (day 14) after TOF spectrum (Fig. 1B). Each of those peaks correspond to the addi­ intracardiac puncture (25G). Plasma of each mouse was obtained by tion of an increasing number of Oligo-Ara-C (calculated MW:2271 Da) centrifugation of total blood at 600 g for 10 min (min) at 4 ◦ C. Aspartate molecules per proteins. transaminase (AST) and alanine transaminase (ALT) enzyme activities Volume size distribution analysis showed low but significant differ­ for the hepatic toxicity and creatinine levels for renal toxicity were ences between T22-GFP-H6 nanoparticles and T22-GFP-H6-Ara-C NCs determined using commercial kits (ASTL ref. 20764949 322; ALTL ref. size. DLS assays determined that the size of T22-GFP-H6 was 12.2 ± 0.2 20764957 322 and CREJ2 ref. 04810716 190, Roche Diagnostics) and nm, whereas the NC size was 13.4 ± 0.2 nm. In addition, the incorpo­ adapted for a COBAS 6000 autoanalyzer (Roche Diagnostics). ration of negatively charged Oligo-Ara-C nucleotides to the protein resulted in a significant increase in the negative charge of T22-GFP-H6- 2.13. Histological and immunohistochemical staining Ara-C NCs compared to the unconjugated T22-GFP-H6 (zeta potential: − 12.1 ± 0.6 vs − 9.7 ± 0.5 mV, respectively) (Fig. 1C). Transmission Detection of leukemic cells in the disseminated AML mice, treated Electron Microscopy (TEM) and Field Emission Scanning Electron Mi­ with T22-GFP-H6-Ara-C, Oligo-Ara-C or buffer, was performed by croscopy (FESEM) images revealed pseudoespherical NCs that validated immunohistochemistry (IHC) using the anti-human CD45 antibody the size determined by DLS (Fig. 1D). Furthermore, the fluorimetry (IR75161-2, Agilent, Dako) to stain only the leukemic THP-1-Luci cells assay showed no significant differences in the specific fluorescence in tissues such as liver, spleen, backbone and hindlimbs. Dako Autos­ levels between the conjugated and unconjugated nanoparticles (Fig. 1E). tainer Link 48 was used to perform this IHC staining following the Finally, the incubation of T22-GFP-H6-Ara-C with CXCR4+ THP-1 cells manufacturer’s instructions. Leukemic cell infiltration was evaluated resulted in the intracellular localization of the NCs as determined by and quantified using an Olympus BX53 microscope (Olympus, Tokyo, confocal laser microscopy (Fig. 1F). Japan) and the cellSens Dimension software (Olympus) using the Counter and Measurement tool choosing 4 fields randomly for each 3.2. Internalization capacity of T22-GFP-H6-Ara-C and competition with tissue. Results were presented as percentage of stained area ±SE. Images AMD3100 in CXCR4+ AML cell lines were acquired using an Olympus DP73 digital camera and processed with cellSens Dimension 1.9 software (Olympus). All samples were First, in order to evaluate the capacity of T22-GFP-H6-Ara-C NCs to evaluated by two independent observers with an inter-observer agree­ internalize into leukemic cells through the CXCR4 receptor, we used two ment of 95%. AML cell lines, THP-1 and SKM-1, that present high CXCR4 levels in To analyze the potential toxicity of treatments in tissues, hematox­ their surface, which we have reported in a previous work [25]. Flow ylin and eosin staining was performed in lung, kidney, liver, heart, cytometry assays showed that T22-GFP-H6-Ara-C internalized in both backbone, hindlimbs and spleen. Two independent observers and an CXCR4+ leukemic cell lines (THP-1 and SKM-1) in a dose-dependent expert pathologist evaluated the possible toxicity in non-leukemic cells manner after 1 h exposure to the NC. Twenty-four hours later, the NC of these tissues using the Olympus BX53 microscope (Olympus). internalization increased significantly at 125 and 250 nM in both cell lines (Fig. 2A and B). Moreover, we analyzed if the internalization was 2.14. Statistical analysis dependent on the CXCR4 receptor by performing competition assays with the antagonist of CXCR4 AMD3100. T22-GFP-H6-Ara-C internali­ Statistical analyses were performed in the IBM SPSS Statistics zation was reduced significantly, in the range of 60–80%, at 1, 24 and (Release 25.0, New York, NY, USA). Mann-Whitney U test was used in 48 h after NC exposure and 1 h pre-treatment with AMD3100 (Fig. 2C). both in vitro and in vivo assays, and any differences were considered statistically significant when the p-value was lower than 0.05. p < 0.05 3.3. Antineoplastic effect of T22-GFP-H6-Ara-C in CXCR4+ AML cell and p < 0.01 were indicated in figures and graphs by * or **, respec­ lines tively. AUC analyses were performed with GraphPad Prism software (Version 6.01, GraphPad Software, Inc., San Diego, CA, USA). IC50 was Afterwards, we analyzed the antineoplastic activity of T22-GFP-H6- calculated with SigmaPlot (Release 12.0.0.182; Systat Software, Inc., Ara-C in THP-1 and SKM-1 cell lines. Exposure to T22-GFP-H6-Ara-C San Jose, CA, USA) using non-linear regression test with Hill-4 param­ showed a dose-dependent antineoplastic effect in THP-1 and SKM-1 eter adjustment. cells (Fig. 2D and E). IC50 of T22-GFP-H6-Ara-C was calculated for both cell lines. In THP-1, the IC50 was 108 ± 35 nM, whereas in SKM-1 was 3. Results 78 ± 7 nM. No significant differences in anticancer activity were found between both lines, either when considering specific concentrations or 3.1. Characterization and synthesis of T22-GFP-H6-Ara-C NCs when comparing THP-1 and SKM-1 IC50 values. In order to generate T22-GFP-H6-Ara-C NCs, a two-step reaction was used for the covalent binding of prior synthetized pentameric 4 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 1. Physicochemical characterization of T22-GFP-H6-Ara-C NCs. (A) Schematic representation of the two-step chemical Oligo-Ara-C conjugation to T22-GFP-H6 nanoparticles through protein lysine-amines by a bifunctional EMCS crosslinker. (B) MALDI-TOF mass spectrometry of T22-GFP-H6 nanoparticles (top) and T22-GFP- H6-Ara-C NCs (bottom). Each peak over 30.6 kDa in T22-GFP-H6-Ara-C indicates the incorporation of an additional Oligo-Ara-C molecule. (C) Volume size dis­ tribution (Size) and Z-potential (Zeta) of T22-GFP-H6 nanoparticles (top) and T22-GFP-H6-Ara-C NCs (bottom) measured by light scattering. Pdi indicates poly­ dispersion index. Data is presented as mean ± SE. (D) Representative high-resolution images of electron microscopy (TEM and FESEM) of polydisperse well-formed nanoconstructs of T22-GFP-H6-Ara-C. Bars size: 20 nm. (E) Relative specific fluorescence of T22-GFP-H6 nanoparticles and T22-GFP-H6-Ara-C NCs measured at 510 nm upon excitation at 488 nm. (F) Confocal laser microscopy images of THP-1 cells upon exposure to 2 μM of T22-GFP-H6-Ara-C NCs for 24 h. Cell nuclei are labeled in blue, cell membranes are labeled in red and green signal correspond to T22-GFP-H6-Ara-C NCs fluorescence. Scale bars indicate 10 μm. SE, standard error. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3.4. Pharmacokinetics and biodistribution of T22-GFP-H6-Ara-C in a sacrificing the mice bearing SC tumors with THP-1 cells at different CXCR4+ AML mouse model time points (10 min, 30 min, 2 h, 5 h, 24 h and 48 h) to measure the fluorescence emitted by the NC or its detection by WB with an anti-GFP We evaluated the NC pharmacokinetics and biodistribution, after a antibody, in plasma, urine and different tissues. single intravenous administration of 200 μg of T22-GFP-H6-Ara-C The highest peak of T22-GFP-H6-Ara-C in plasma appeared 10 min 5 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 2. Internalization and antineoplastic capacities of the T22-GFP-H6-Ara-C nanoparticle in CXCR4+ AML cell lines. (A–B) Internalization capacity of T22- GFP-H6-Ara-C in THP-1 and SKM-1 at 1 and 24 h after exposure to different concentrations of the NC measured by flow cytometry. Results are presented by the mean fluorescence intensity (MFI) normalized by the MFI of buffer treated cells (MFI ratio or MFIR) ± SE. (C) Competition assay was performed by cytom­ etry in THP-1 cells adding or not 7500 nM AMD3100 1 h prior to the administration of 250 nM T22-GFP- H6-Ara-C exposed for 1, 24 and 48 h (ratio 30:1). Results are presented by the mean percentage of internalization ± SE. (D–E) The antineoplastic activ­ ity was measured by XTT exposing THP-1 and SKM-1 to different concentrations of the T22-GFP-H6-Ara-C or the Oligo-Ara-C at equimolar doses incubated for 48 h. Results are presented by the mean percentage of viability ± SE. Mann-Whitney U test was used to test differences between groups indicated by * when p- value was <0.05. AML, Acute myeloid leukemia; MFIR, Mean fluorescence intensity ratio; SE, standard error. after the intravenous injection of the NC (1.82 × 108 ± 0.40 × 108) and located to SC tumor. decreased 7.3-times (0.25 × 108 ± 0.07 × 108) at 30 min. Afterwards, the presence of NC in circulation was considerably reduced, 20-times at 2 h, 38.2-times at 5 h, 83.5-times at 24 h and, finally, almost unde­ 3.5. T22-GFP-H6-Ara-C dose response study in a CXCR4+ AML mouse tectable levels were observed at 48 h (Fig. 3A and B). The levels of NC in model plasma were very similar to the sum of the FLI detected in liver and kidneys at short times (0.40 × 108 ± 0.09 × 108 and 1.56 × 108 ± 0.28 To evaluate the anticancer activity of T22-GFP-H6-Ara-C in vivo, a × 108 at 10 min and 0.09 × 108 ± 0.04 × 108 and 0.38 × 108 ± 0.08 × disseminated AML mouse model was generated by intravenous injection 108 at 30 min in liver and kidneys, respectively) (Fig. 3C and D). of CXCR4+ THP-1-Luci cells, as reported [25]. Two days after the i.v. Moreover, by western blot, we observed the presence of the nanoparticle injection of THP-1-Luci cells, 10 daily doses of buffer (NaCO3H NaCl pH (anti-GFP staining) in plasma samples obtained from mice treated after = 8) or T22-GFP-H6-Ara-C (5, 25, 100 and 200 μg) were i.v. adminis­ 10 min with T22-GFP-H6-Ara-C and much lower NC expression at 30 tered in different groups of mice in order to choose the NC dose with the min. In contrast, we did not observe any presence of T22-GFP-H6-Ara-C highest antineoplastic effect and the lowest systemic toxicity. The results in urine samples at any time of the biodistribution study (Fig. 3E). showed that mice administered with 5 or 25 μg of T22-GFP-H6-Ara-C Finally, the cumulative FLI quantification of T22-GFP-H6-Ara-C at did not reduce the leukemia dissemination compared to buffer-treated longer times (2–48 h) showed a high amount of NC accumulation in SC mice. In contrast, we observed significant differences between mice tumor tissue (2.67 × 108 ± 0.50 × 108), whereas much lower levels of treated with buffer and mice treated with 100 μg or 200 μg of NC were present at the non-tumor organs, such as liver (0.54 × 108± T22-GFP-H6-Ara-C at day 13 (2.3-fold and 5.6-fold lower BLI, respec­ 0.16 × 108), kidneys (0.51 × 108± 0.16 × 108) or lungs (0.55 × 108± tively). Indeed, 200 μg-T22-GFP-Ara-C-treated mice group was the only 0.09 × 108) (Fig. 3F). one which showed a significantly lower leukemic dissemination (lower Thus, all these results suggest that the NC transiently accesses the BLI signal) already on day 8 and day 10 compared to buffer-treated mice fenestrated vessels in the liver and kidneys during a short time period, to (Fig. 4A and B). return to the bloodstream, without penetrating the parenchyma or being Furthermore, none of the mice treated with T22-GFP-H6-Ara-C excreted through the urine, since at longer times the NC is mainly showed significant mouse weight reduction at the end of the AML dissemination follow-up compared to control mice. Exceptionally, at day 6 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 3. Pharmacokinetics and biodistribution of T22- GFP-H6-Ara-C in a SC CXCR4+ AML mouse model. (A) FLI images of 100 μL of plasma, acquired by IVIS Spectrum, from mice treated with a single dose of buffer (“Buffer”, n = 3/group) or a single dose of 200 μg T22-GFP-H6-Ara-C (“T22-GFP-Ara-C”) at 10 min, 30 min, 2 h, 5 h, 24 h and 48 h. (B) Plasma FLI quantification from buffer-treated mice (n = 3/ group) and 200 μg NC-treated mice at different time points (10 min, 30 min, 2 h, 5 h, 24 h and 48 h, n = 3/ group). (C–D) FLI quantification emitted by liver and kidneys ex vivo at different time points after the NC administration. (E) Western Blot analysis detecting the NC (anti-GFP staining) in plasma and urine sam­ ples collected from mice treated with buffer or NC at different time points. C+ is a positive control for GFP staining. (F) Cumulative FLI registered from mice treated with NC from 2 to 48 h in SC tumors and normal organs. Cumulative FLI were quantified using the area under the curve (AUC). FLI quantifications were performed subtracting the auto-fluorescence emitted from buffer samples or organs. B, buffer. 3 after cell injection, mice treated with 200 μg of T22-GFP-H6-Ara-C mice treated with Oligo-Ara-C was significantly lower than buffer- showed a significant loss of mouse weight, which was immediately treated mice from the third day and until the eighth day after the in­ restored (Fig. 4C). Moreover, the biochemical parameters determining jection of cells (that is, during the first 7 doses). After that period, mouse hepatic (ALT and AST) and renal (creatinine) toxicity did not exhibit weight between both groups was similar. In contrast, the weight of NC- significant differences between control mice and mice treated with any treated mice was significantly lower than this in buffer-treated group concentration of T22-GFP-H6-Ara-C (Fig. 4D and E). Thus, the mice only at the third day after cell injection, exactly the same event that group treated with 200 μg of T22-GFP-H6-Ara-C demonstrated to have occurred in the preliminary experiment. No differences in body weight the highest antineoplastic effect without inducing neither loss of mouse were found between the Oligo-Ara-C and the T22-GFP-Ara-C-treated weight nor hepatic or renal functional toxicity at the end of the groups (Fig. S2B). experiment. Disease progression was monitored, along the experiment, by analyzing the bioluminescence emitted (BLI) by each mouse with the IVIS spectrum system. Treatment with T22-GFP-H6-Ara-C decreased 3.6. Comparison of the antineoplastic effect of T22-GFP-H6-Ara-C and mice BLI compared to the buffer or Oligo-Ara-C-treated mice after the free Oligo-Ara-C in a CXCR4+ AML mouse model sixth dose (day 8) (Fig. 5A and B). In contrast, treatment with Oligo-Ara- C needed two additional doses to decrease disease progression (day 10). Here, using the same design as the previous experiment, we evalu­ However, in a more global study of BLI along time, performed by ated the antineoplastic effect of the most effective T22-GFP-H6-Ara-C analyzing the AUC of the different treatments, T22-GFP-H6-Ara-C dosage (200 μg T22-GFP-H6-Ara-C for a total of 10 doses) and this ef­ treatment induced a significant blockade of AML progression by fect was compared to that observed in mice treated with free Oligo-Ara- reducing the emitted luminescence a 75 ± 2% (9.8E+07 ± 2.6E+06 BLI C at equimolar doses or administered with NC buffer. The weight of the 7 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 4. T22-GFP-H6-Ara-C dose response study in a disseminated CXCR4þ AML mouse model. (A) BLI registered in mice treated with ten doses of NC buffer (“Buffer”, n = 4) or ten doses of different T22- GFP-H6-Ara-C concentrations (5, 25, 100 and 200 μg, “T22-GFP-Ara-C”, n = 4/group) at day 13 after the intravenous administration of THP1-Luci cells. (B) BLI quantification of mice treated with buffer or different T22-GFP-H6-Ara-C concentrations (5, 25, 100 and 200 μg) throughout the leukemia dissemi­ nation follow-up. Results are presented in total flux units [p/s]. (C) Mouse weight of buffer- or NC- treated-mice during the experiment. (D) Functional analysis measuring the levels of hepatic trans­ aminases (AST and ALT) and (E) creatinine in mice treated with buffer or mice groups treated with different concentrations of T22-GFP-H6-Ara-C at the end of the experiment. * denotes statistical difference (p < 0.05) between buffer-treated mice and 200 μg- NC-treated mice. # denotes statistical difference (p < 0.05) between buffer-treated mice and 100 μg-NC- treated mice. Mann-Whitney U test was used to analyze differences between groups. All results are expressed as mean ± SE. BLI, bioluminescence; ALT, alanine aminotransferase; AST, aspartate amino­ transferase; SE, standard error. Total Flux reduction) compared to the buffer-treated group, whereas the reduction of CD45 positive leukemic cells compared to buffer treatment Oligo-Ara-C treatment reached only a 21 ± 10% reduction (2.7E+07 ± (Fig. 7A and B). Moreover, the NC treatment reduced significantly 1.3E+07 BLI Total Flux) (Fig. 5C). leukemic dissemination in all tissues compared to the Oligo-Ara-C When the animals had signs of advanced disease, mice of all groups treatment, including the spleen. were euthanized and AML dissemination was studied locally in tissues by analyzing BLI ex vivo using the IVIS spectrum. In contrast to treat­ 3.7. Evaluation of toxicity in NC on-target and off-target mouse tissues ment with T22-GFP-H6-Ara-C that decreased significantly BLI levels in bone marrow tissues, including backbone, hindlimbs and cranium, Finally, hematoxylin-eosin staining was performed to assess the Oligo-Ara-C treatment only significantly reduced BLI in cranium possible toxicity of the different treatments in tissues. All analyzed (Fig. 6A, B and C); however, this reduction was significantly lower than samples showed a lack of relevant toxicity for the T22-GFP-H6-Ara-C or the exerted by the NC. In addition, BLI in spleen and liver was decreased the Oligo-Ara-C treatment, compared to the buffer treatment, after a significantly by both treatments, as compared to buffer-treated mice, careful inspection of the possible occurrence of inflammatory processes, however the reduction of AML cell dissemination in these organs was steatosis, hemorrhages or morphological changes in normal tissues (on- more pronounced in the NC-treated mice (Fig. 6D and E). target NC toxicity) such as liver, spleen, backbone, or hindlimbs. Finally, AML target tissues were analyzed by IHC to detect and Furthermore, the off-target tissues of experimental mice, such as lungs, quantify the area occupied by leukemic cells using the human CD45 kidneys or heart, did not show congestion, edema or intraalveolar marker. T22-GFP-H6-Ara-C treatment decreased significantly the hemorrhage, neither the glomerulus and surrounding renal tubules dissemination of leukemic cells in liver, spleen, backbone and hindlimbs showed cytoplasmic vacuolation or eosinophilic protein accumulation compared to that of the buffer-treated mice in a 71 ± 1, 99 ± 0, or 76 ± 3 nor the heart tissue had morphological alterations (Fig. 8). and 61 ± 5%, respectively. In contrast, Oligo-Ara-C treatment only decreased significantly AML dissemination in spleen reaching a 74 ± 1% 8 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 5. Antineoplastic effect of T22-GFP-H6-Ara-C and free Oligo-Ara-C in a CXCR4+ AML mouse model. (A) Follow-up of AML dissemination in NSG mice treated with buffer (NaCO3H + NaCl pH = 8) (“Buffer”, n = 8), T22-GFP-H6-Ara-C (“T22-GFP-Ara-C”, n = 8) or free Oligo-Ara-C (Oligo-Ara-C, n = 8) during treatment monitoring mice BLI in IVIS spectrum. (B) Evolution of AML dissemination of mice treated with buffer, T22-GFP-H6-Ara-C or Oligo-Ara-C measuring mice BLI by IVIS Spectrum. Results are presented by mean of total flux [p/s] ± SE. (C) Cumulative BLI of the different treated mouse groups during the treatment quantifying area under the curve of AML evolution for each mouse. Results are presented as mean of area under the curve in total flux units [p/s] ± SE. Mann-Whitney U test was used to analyze significant differences between groups, and they are indicated by ** when p-value was <0.01. AML, Acute myeloid leukemia; BLI, bioluminescence; i.v., intravenous; SE, standard error. 4. Discussion T22-GFP-H6-Ara-C NC, as compared to free-Ara-C, since it avoids he­ patic metabolism and renal excretion (cut-off 7 nm), leading to a high We demonstrated that the novel T22-GFP-H6-Ara-C NC reaches a accumulation in CXCR4+ cancer tissues, while evading its accumulation high therapeutic activity in an AML mouse model through selective in normal tissues. This biodistribution change leads, in turn, to a potent blockade of CXCR4+ leukemic cell dissemination, as compared to the antineoplastic effect of T22-GFP-H6-Ara-C specifically to CXCR4 over­ free Oligo-Ara-C, in the absence of on-target or off-target toxicities. This expressing leukemic cells in the absence of toxicity that could signifi­ high therapeutic window is triggered by the multivalency of the protein- cantly reduce the adverse effects of classical Ara-C-based AML therapy. nanocarrier together with the 5-fold increased drug payload delivered to To support this argument, we report that T22-GFP-H6-Ara-C displays target leukemic cells, that is achieved by the conjugation of a pentameric comparable physicochemical characteristics to the T22-GFP-H6 nano­ form of Ara-C. We believe that the conjugation strategy applied in our carrier, which was our first protein nanoparticle displaying a well- NC can be widely exploited because an ADC that uses the same chemical characterized multivalent targeting approach. Here, we have demon­ strategy (e.g.trastuzumab emtansine, “Kadcyla”) was already approved strated that the conjugation of the Oligo-Ara-C prodrug to T22-GFP-H6 by the FDA [26]. does not affect the tertiary structure of the protein, its stability or its Furthermore, this novel therapy displays several advantages as functionality neither its capacity to internalize in CXCR4+ cells in vitro compared to classical chemotherapy or to previously developed nano­ and in vivo [16,30]. Moreover, regarding its molecular mechanism of medicines. First, as Ara-C (Cytarabine) has a short plasma half-life, anticancer activity, once the NC specifically internalizes in target cancer extense hepatic metabolism and rapid renal elimination (within 24 h cells, the molecules of Ara-C are sequentially released in the cytosol by about 80% of the drug is excreted in the urine) [27], it must be the 3′ exonucleases that cleave the phosphodiester bond between sugars administered as a continuous intravenous infusion to reach sustained of the Oligo-Ara-C in the 3′ end to 5’ direction [31]. Next, the released and effective antineoplastic concentrations. Nevertheless, the prolonged Ara-C is metabolized intracellularly into its active triphosphate form exposure to high Ara-C concentrations triggers severe adverse effects that acts as an antimetabolite by competing with cytidine for incorpo­ that include bone marrow suppression, hepatic dysfunction or infectious ration into DNA [27]. complications [28,29]. In contrast, our results support a dramatic Because of its high therapeutic index in our disseminated AML mouse change in the pharmacokinetics and pharmacodynamics of the 13.4 nm model, our NC could be indicated in more than a half of AML patients 9 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 6. Antineoplastic effect in CXCR4+ AML affected organs ex vivo after mice treatment with T22-GFP-H6-Ara-C or Oligo-Ara-C. (A–E) Ex vivo BLI analysis of backbone, hindlimbs, cranium, spleen and liver of mice after treatment with buffer, T22-GFP-H6-Ara-C or Oligo-Ara-C. Graphs represent the quantitation of BLI presented as mean of average radiance ± SE for each tissue and representative images of these analyses show the BLI levels measured in IVIS Spectrum. * or ** indicate p-value <0.05 or <0.01, respectively, using Mann-Whitney U test to analyze significant differences between groups. AML, Acute myeloid leukemia; Avg, average; BLI, bioluminescence; SE, standard error. that display CXCR4 overexpression in their leukemic cells, who the development of AML as a consequence of the reduction of LSCs currently lack clear therapeutic options. The AML patient subsets homing and trafficking in the bone marrow [24,38–46]. In this work, bearing CXCR4+ leukemic cells, candidates to be treated with our NC, T22-GFP-H6-Ara-C has a high cytotoxic effect against the THP-1 include patients with AML de novo (50–55%), which associate with poor leukemic cell line that contains a large proportion of cells with high prognosis and shorter disease-free survival; elderly (≥65 years) or co­ ALDH activity, a property directly linked to the LSC phenotype [47]. morbid AML patients in whom intensive chemotherapy is not indicated, Thus, our NC displays a clear advantage over the current therapy and AML patients scheduled for a second round of intensive chemo­ because of its capacity of selectively targeting and ablating CXCR4+ therapy or an invasive allogenic transplant, involving 50–70% of these AML cells and consequently the LSCs. that relapse after Ara-C based-therapy [1,2,17,18,18–22,32–35]. Besides that, most nanoparticles being tested in clinical trials are Most importantly, it is known that leukemic stem cells (LSCs) pro­ untargeted [15,48,49]. For example, the CPX-351, a marketed liposome tection in the bone marrow niche is dependent on CXCR4 and its that encapsulates Ara-C and daunorubicin, changes only the drug interaction with CXCL12 [24,36,37]. Accordingly, eliminating CXCR4+ pharmacokinetics, rather than proving preferential uptake by leukemia leukemic cells is considered a sound and effective strategy to eradicate cells, as it is claimed. Thus, CPX-351 achieves low rate of clinical 10 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 7. Detection and quantitation of disseminated leukemic cells in tissues of T22-GFP-Ara-C or Oligo- Ara-C-treated mice. (A) Quantitation of leukemic cells in liver, spleen, backbone and hindlimbs per­ forming human CD45 detection (marker of leukemic cells) by IHC in mice treated with buffer, T22-GFP- H6-Ara-C or Oligo-Ara-C. Results are presented as mean of surface percentage with CD45 positive cells ± SE. ** indicates significant differences between groups with a p-value <0.01 using Mann-Whitney U test. (B) Representative images of the CD45 detection by IHC in liver, spleen, backbone and hindlimbs ac­ cording to the different treated mouse groups. IHC, immunohistochemistry; SE, standard error. response in AML at high risk or relapsed-patients, showing similar capacity and stimulates their phagocytosis by the reticuloendothelial toxicity to intensive chemotherapy, which has prompted its use in system [52,53]. In sharp contrast, our protein-based nanoparticles combination [50]. Similarly, additional untargeted nanomedicines maintain their targeting capacity because their protein nature avoids include PLA/PLGA polymeric nanoparticles containing Aurora kinase B corona formation [30,54]. inhibitors, or liposomes encapsulating Annamycin or Grb2 Mainly, two T22-GFP-H6-Ara-C features determine the advantage of antisense-encapsulated that need also to be used in combination with our nanotechnological approach as compared to other nanoparticle targeted drugs [51]. Thus, the preclinical trend is now to develop lipo­ drugs. On the one hand, the multivalency of the NC, based on the somes or polymeric nanoparticles that target relevant markers for AML incorporation of around 11 ligands that target the CXCR4 receptor in a (e.g., anti-CD33, anti-CD45 mAbs) [14]; however, once injected in the single self-assembled nanoparticle, confers super-selectivity. This bloodstream they acquire a “protein corona” that limits their targeting feature permits the interaction of the target receptor only when its 11 V. Pallarès et al. Biomaterials 280 (2022) 121258 Fig. 8. Toxicity study in H&E stained tissue sections of mice treated with T22-GFP-H6-Ara-C or Oligo-Ara-C. Hematoxylin and eosin staining of target AML tissues such as liver, spleen, backbone and hindlimbs, and non-target AML tissues such as lung, kidney and heart of mice treated with buffer, T22-GFP-H6-Ara-C or Oligo- Ara-C. density in the cell surface is higher than a specific threshold, showing an this is the first time that an oligomeric form of Ara-C is synthetized to be ON/OFF effect [55]. Therefore, T22-GFP-H6-Ara-C could only inter­ used as a prodrug. nalize in leukemic cells with high CXCR4 expression (e.g. LSCs) while Finally, as our study represents a prove-of-concept assay that dem­ avoiding internalization in normal cells, which display low CXCR4 onstrates the high potential of T22-GFP-H6-Ara-C, we expect, in the near expression [56]. This argument is supported by the fact that the future, to replace the GFP scaffold protein in our NC by the recently T22-GFP-H6 nanocarrier achieves 86.0% accumulation in cancer tis­ engineered and structurally identical human counterpart (HSNBT) that sues, as compared to untargeted nanoparticles that reach only an is derived from the G2 domain of the human nidogen and, thus, avoids average of 0.7% of the administered dose accumulated in cancer tissues the immunogenicity that could be associated to the exogenous fluores­ or the antibody-drug conjugates that reach only 0.1% [57,58]. On the cent protein [61]. other hand, our NC is able to incorporate a multimeric Oligo-Ara-C prodrug that allows to deliver a huge drug payload, which conse­ 5. Conclusion quently achieves a high antineoplastic effect in target cells. Although we conjugated previously oligomeric drugs to protein nanoparticle [59,60], In conclusion, we have generated a novel T22-GFP-H6-Ara-C 12 V. Pallarès et al. Biomaterials 280 (2022) 121258 protein-based NC that highly enhances the therapeutic window of cur­ covering the therapeutic use of T22. All other authors report no conflicts rent drugs by combining nanoparticle multivalency, to achieve selective of interest in this work. targeting CXCR4+ cancer cells, with a multimeric prodrug to increase the conjugated payload drug. In addition, the T22-GFP-H6-Ara-C in­ Appendix A. Supplementary data duction of negligible toxicity and higher antineoplastic effect observed in the disseminated CXCR4+ AML mouse model, suggest their use as an Supplementary data to this article can be found online at https://doi. alternative approach to non-intensive therapies in AML patients that org/10.1016/j.biomaterials.2021.121258. show a low rate of complete remissions and still severe adverse effects. References Funding [1] H. Döhner, E. Estey, D. Grimwade, S. Amadori, F.R. Appelbaum, T. Büchner, H. Dombret, B.L. Ebert, P. Fenaux, R.A. Larson, R.L. Levine, F. Lo-Coco, T. Naoe, This work was supported by Instituto de Salud Carlos III (ISCIII, Co- D. Niederwieser, G.J. Ossenkoppele, M. Sanz, J. Sierra, M.S. Tallman, H.-F. Tien, A. funding from FEDER) [PI18/00650, PIE15/00028, PI15/00378 and EU H. Wei, B. Löwenberg, C.D. 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