International Journal of Nanomedicine Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH Specific Cytotoxic Effect of an Auristatin Nanoconjugate Towards CXCR4+ Diffuse Large B-Cell Lymphoma Cells This article was published in the following Dove Press journal: International Journal of Nanomedicine Aïda Falgàs, 1–3 Victor Background and Purpose: Around 40–50% of diffuse large-B cell lymphoma (DLBCL) Pallarès,1–3 Ugutz Unzueta,1–4 patients suffer from refractory disease or relapse after R-CHOP first-line treatment. Many Yáiza Núñez,1,2 Jorge Sierra,2,5 ongoing clinical trials for DLBCL patients involve microtubule targeting agents (MTAs), Alberto Gallardo,1 Lorena Alba- however, their anticancer activity is limited by severe side effects. Therefore, we chose to Castellón,1,2 Maria Antonia improve the therapeutic window of the MTA monomethyl auristatin E developing Mangues, 3,6 Patricia Álamo,1–3 a nanoconjugate, T22-AUR, that selectively targets the CXCR4 receptor, which is over­ Antonio Villaverde,3,4,7 Esther expressed in many DLBCL cells (CXCR4+) and associated with poor prognosis. Vázquez,3,4,7 Ramon Methods: The T22-AUR specificity towards CXCR4 receptor was performed by flow cytometry in different DLBCL cell lines and running biodistribution assays in Mangues, 1–3 Isolda a subcutaneous mouse model bearing CXCR4+ DLBCL cells. Moreover, we determined Casanova1–3 T22-AUR cytotoxicity using cell viability assays, cell cycle analysis, DAPI staining and 1 Biomedical Research Institute Sant Pau (IIB- immunohistochemistry. Finally, the T22-AUR antineoplastic effect was evaluated in vivo in Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 2Josep an extranodal CXCR4+ DLBCL mouse model whereas the toxicity analysis was assessed by Carreras Leukaemia Research Institute (IJC), histopathology in non-infiltrated mouse organs and by in vitro cytotoxic assays in human Barcelona, 08916, Spain; 3CIBER de PBMCs. Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid, 28029, Spain; Results: We demonstrate that the T22-AUR nanoconjugate displays CXCR4-dependent 4 Department of Genetics and Microbiology, targeting and internalization in CXCR4+ DLBCL cells in vitro as well as in Universitat Autònoma de Barcelona, Barcelona, 08193, Spain; 5Department of a subcutaneous DLBCL mouse model. Moreover, it shows high cytotoxic effect in Hematology, Hospital de la Santa Creu i Sant CXCR4+ DLBCL cells, including induction of G2/M mitotic arrest, DNA damage, mitotic Pau, Barcelona, 08025, Spain; 6Department catastrophe and apoptosis. Furthermore, the nanoconjugate shows a potent reduction in of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 7Institute of lymphoma mouse dissemination without histopathological alterations in non-DLBCL infil­ Biotechnology and Biomedicine (IBB), trated organs. Importantly, T22-AUR also exhibits lack of toxicity in human PBMCs. Universitat Autònoma de Barcelona, Conclusion: T22-AUR exerts in vitro and in vivo anticancer effect on CXCR4+ DLBCL Barcelona, 08193, Spain cells without off-target toxicity. Thus, T22-AUR promises to become an effective therapy for CXCR4+ DLBCL patients. Correspondence: Esther Vázquez Keywords: nanomedicine, targeted drug delivery, MMAE, DLBCL Institute of Biotechnology and Biomedicine (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona and CIBER-BBN, Introduction Barcelona, Spain Tel +34 935812148 Diffuse Large B-cell lymphoma (DLBCL) is the most frequent non-Hodgkin Email Esther.Vazquez@uab.es lymphoma (NHL).1 Although the addition of rituximab to the standard chemother­ Ramon Mangues apy has improved the outcome of DLBCL patients, about 40–50% of them develop Biomedical Research Institute Sant Pau relapsed/refractory (R/R) disease after treatment with rituximab plus cyclopho­ (IIB-Sant Pau), Josep Carreras Leukaemia Research Institute (IJC) and CIBER-BBN, sphamide, doxorubicin, vincristine and prednisone (R-CHOP).2,3 This is usually Barcelona, Spain managed with salvage chemotherapy and, after a complete or partial response, Tel +34 935537918 Email RMangues@santpau.cat patients undergo a hematopoietic stem cell transplant (HSCT). However, patients submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1869–1888 1869 DovePress © 2021 Falgàs et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. http://doi.org/10.2147/IJN.S289733 php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Falgàs et al Dovepress younger than 60 years old with high International Hodgkin’s lymphoma cells,19 whereas polatuzumab vedo­ Prognostic Index (IPI) and disseminated DLBCL do not tin targets the CD79b marker and was recently approved have a standard first-line therapy. Moreover, some DLBCL for the treatment of R/R DLBCL patients.20 Nevertheless, patients do not respond to the high-dose chemotherapy or all ADCs, including these two ADCs-MMAE that have they are not initially candidate for HSCT (second-line reached the clinical setting, share the issue of limited therapy), or even fail to the HSCT (needing third-line efficacy because less than 1% of the total administered therapy).4 Furthermore, current chemotherapy can induce ADC dose reaches the tumor.21,22 Here, we tested the T22- life-threatening side effects in DLBCL patients.5 Thus, AUR nanoconjugate that displays around twelve CXCR4 new treatments are urgently needed to enhance their anti­ ligands per drug-loaded nanoparticle,16 which enhances neoplastic effect in lymphoma cells without damaging tumor uptake and selective CXCR4+ DLBCL cell killing normal cells. Actively targeted nanoparticles, containing and leads to a potent blockade of lymphoma dissemination a ligand that allows their specific targeting to a receptor while reducing systemic toxicity in non-infiltrated organs. overexpressed in cancer cells, have appeared as a promising option.6,7 Here, we chose the CXCR4 receptor Materials and Methods to target lymphoma cells, since around 30–50% of DLBCL biopsies overexpress this receptor (CXCR4+)8,9 T22-AUR Nanoconjugate Production and and its expression in lymphoma cells is much higher than Characterization in normal B cells.10 Moreover, CXCR4 overexpression is T22-GFP-H6 nanocarrier was produced in E. coli Origami a poor prognostic factor in DLBCL patients.9,11 B strain as previously described in Int J Nanomedicine. Interestingly, microtubule-targeting agents (MTAs) 2012;7:4533–44. Maleimide functionalized Monomethyl promise to become potent drug-payloads in anticancer Auristain E (MC-MMAE) was acquired as custom synth­ treatments. Microtubules are tubulin polymers and essen­ esis from Levena Biopharma (Levena Biopharma, San tial components of the cytoskeleton that undergo constant Diego, CA, USA). T22-GFP-H6-MMAE (T22-AUR) assembling and disassembling within the cell. The main nanoconjugate was synthetized by the covalent binding role of microtubules is the segregation of the chromo­ of MC-MMAE to T22-GFP-H6 through protein lysine somes to the spindle poles during mitosis with the conse­ amines (generation of Alkylamine bonds) in a one-pot quent formation of daughter cells.12 However, they are reaction. For that, T22-GFP-H6 was incubated in presence involved in many other cellular processes, such as intra­ of a 1:50 molar excess of MC-MMAE for 4 h at R.T in cellular trafficking, migration and angiogenesis.13,14 sodium carbonate buffer (166 mM NaCO3H, 333 mM Therefore, their dynamic features and their implication in NaCl pH=8). T22-AUR nanoconjugate was then re- the cancer cell survival renders them good antineoplastic purified by IMAC affinity chromatography in order to targets. remove non-reacted free MC-MMAE molecules. Finally, Our group designed a T22-AUR therapeutic nanocon­ re-purified nanoconjugates were dialyzed against sodium jugate that includes the T22-GFP-H6 protein nanocarrier, carbonate buffer and filtered through 0.22 µm pore filter. previously demonstrated to selectively target CXCR4 Conjugation efficiency was checked by MALDI-TOF receptor-overexpressing DLBCL cells,15 and is conjugated mass spectrometry and nanoconjugate final concentration with the MTA monomethyl auristatin E (MMAE), aiming determined by Bradford assay. at selectively killing CXCR4+ DLBCL cells. Furthermore, Volume size distribution and zeta potential of parental this nanoconjugate has recently demonstrated efficacy in T22-GFP-H6 nanocarrier and T22-AUR nanoconjugate treating an aggressive acute myeloid leukemia dissemi­ was determined by Dynamic Light Scattering (DLS) and nated mouse model.16 Electrophoretic Light Scattering (ELS), respectively, in MMAE has been tested in different clinical trials a Zetasizer Nano ZS (Malvern instruments) at 633 nm. always conjugated with monoclonal antibodies in the Average molar mass of T22-GFP-H6 nanoparticle and form of antibody-drug conjugates (ADCs).17 Currently, T22-AUR nanoconjugate was determined by size exclu­ there are two ADCs-MMAE approved by the FDA, sion chromatography coupled to a multi angle light scat­ being both indicated for the treatment of hematological tering (SEC-MALS). For that, 200 µg of each sample was malignancies.18 Brentuximab vedotin targets CD30, which injected in a Superdex 200 increase 10/300 GL column is expressed in anaplastic large cell lymphoma and (GE Healthcare, Chicago, Illinois, USA) and run in 1870 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al a sodium carbonate buffer supplemented with zinc (166 In vitro Nanoconjugate Internalization mM NaCO3H, 333 mM NaCl, 0.1 mM ZnCl2 pH=8). The evaluation of the T22-AUR internalization in different Eluent was monitored by an in-line UV-Vis detector, DLBCL cell lines was performed measuring the GFP a Dawn Heleos MALS detector and an Optilab rEX RI levels of the nanoconjugate and using the FACS Calibur detector (Wyatt Technology Corporation, Santa Barbara, flow cytometer (BD Biosciences, San Jose, CA, USA). California, USA). All data were then analyzed by Astra Cells were incubated with 10 nM T22-AUR for 6.0.2.9 software (Wyatt Technology Corporation) using 1 h. Then, cells were washed with PBS and trypsinized dn/dc value of 0.185 (mL/g) and protein UV molar extinc­ for 15 min (1 mg/mL trypsin, Thermo Fisher Scientific) in tion coefficient value of 1.099 (mL/mg.cm). order to remove nonspecific binding of nanoconjugates to the cell membrane. The competition assays were done by DLBCL Cell Lines and Human PBMCs preincubating the cells for 1 h with 100 nM of the CXCR4 The human Toledo and U-2932 DLBCL cell lines were antagonist AMD3100 (Sigma-Aldrich, St-Louis, MO, cultured with RPMI 1640 medium whereas the human USA). Results were analyzed with Cell Quest Pro software SUDHL-2 DLBCL cell line was cultured in IMDM med­ (BD Biosciences) and expressed as mean fluorescence ium. All cell lines were supplemented with 10% fetal intensity (MFI). bovine serum (FBS), 1% glutamine and 100 U/mL peni­ cillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and were incubated at 37°C and 5% CO2 in Cytotoxicity Assays The in vitro cytotoxic effect of T22-AUR was evaluated humidified atmosphere. Toledo cell line was purchased using the colorimetric cell proliferation kit (XTT) after the from the American Type Culture Collection (ATCC, exposure of the three DLBCL cell lines (3.5·105 cells/mL) Manassas, Virginia, USA) and U-2932 cell line from the or human PBMCs (10·105 cells/mL) to buffer (166 mM German Collection of Microorganisms and Cell Cultures NaCO3H 333 mM NaCl, pH=8) or different concentrations (DSMZ, Braunschweig, Germany). Finally, SUDHL-2 was of T22-AUR for 48 h in 96 well plates. Then, XTT reagent kindly provided by Dr L. Pasqualucci (Columbia (Roche Diagnostics, Basel, Switzerland) was added and, University, NY, USA) and its use approved by IIB-Sant after 4 h incubation, cell viability was quantified by mea­ Pau research ethics committee. suring the absorbance at 492 nm wavelength using U-2932 cell line was transfected with the Luciferase a FLUOstar OPTIMA spectrophotometer (BMG Labtech, gene (pPK-CMV- F3, Promokine, TE Huissen, The Ortenberg, Germany). Moreover, the cytotoxic effect of Netherlands) by electroporation (Nucleofector TM 2b free MMAE (Levena Biopharma) or vehicle DMSO Device, Lonza, Basel, Switzerland). Then, transfected (Sigma-Aldrich) in the DLBCL cell lines was also evalu­ cells were selected with 0.4 mg/mL of geneticin (Thermo ated following the same procedure. IC50 of free MMAE Fisher Scientific) in order to achieve stable clones. for all DLBCL cell lines was calculated using the Fresh peripheral blood was obtained from healthy GraphPad Prism 6 program. Competition viability assays donors after acquiring informed consent and approval of were done by 1 h pretreatment of U-2932 cells with the Hospital de la Santa Creu i Sant Pau Ethical AMD3100 followed by the addition of the T22-AUR Committee for Clinical Research. Human PBMCs were nanoparticle (ratio 10 AMD300 – 1 T22-AUR). Data isolated by Lymphoprep gradient centrifugation (Stem were shown as percentage of cell death in relation to its Cell Technologies Vancouver, BC, Canada) followed by control (buffer, DMSO or AMD3100). red blood cell lysis (Thermo Fisher Scientific) according to the manufacturer’s instructions. Human PBMCs were maintained in culture for 48 h in Iscove’s modified CXCR4 Flow Cytometry Dulbecco’s medium (IMDM) supplemented with 3% heat CXCR4 membrane determination was done by flow cyto­ inactivated FBS, 2 mM L-glutamine (Thermo Fisher metry in human PBMCs and DLBCL cells, after washing Scientific), 20% BIT 9500 Serum Substitute (StemCell with PBS 0.5% BSA and incubating with either PE-Cy5 Technologies), 5 ng/mL IL-3 (Peprotech, Rocky Hill, NJ, mouse anti-human CXCR4 monoclonal antibody (BD USA), 5 × 10−5 M β-mercaptoethanol (Sigma-Aldrich, St Biosciences) or PE-Cy5 Mouse IgG2a isotype (BD Louis, MO, USA), 1 mM sodium pyruvate and 0.1 mM Biosciences) as negative control. Results were analyzed non-essential amino acids (Thermo Fisher Scientific). using the FACS Calibur flow cytometer with the Cell submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1871 DovePress Falgàs et al Dovepress Quest Pro software and expressed as ratio of CXCR4 MFI percentage of apoptosis (early/late) for each condition was divided by isotype MFI. evaluated using the Annexin V-CF Blue/PI apoptosis detection kit (Abcam, Cambridge, UK) and following the Cell Cycle Analysis manufacturer’s instructions. Data were analyzed by Cells were treated with buffer or 125 nM of T22-AUR for MACSQuant analyzer flow cytometry using the MACS 24 h and 48 h. Then, 1.2·106 cells were centrifuged and Quantify version 2.3 software (Miltenyi Biotec). washed with PBS. Afterwards, cells were fixed with cold 66% ethanol for 2 h at 4°C, centrifuged and washed with Western Blotting PBS. Finally, propidium iodide (Merck Millipore, DLBCL cells were seeded at 3.5·105 cells/mL and treated Burlington, Massachusetts, USA) and RNAse (Thermo with buffer or 125 nM T22-AUR for 24 h and 48 h. Then, Fisher Scientific) were added at a final concentration of cells were washed with PBS and resuspended in lysis buffer 40 µg/mL and 20 µg/mL, respectively, and cells were (1 M Tris/acetate, 1 M sucrose, 100 mM EDTA, 100 mM incubated at 37°C for 20 min. Cells were analyzed using EGTA, 10% Triton-X-100, 100 mM Naorto, 100 mM the FACS Calibur flow cytometer and data were obtained Naβglycerol, 0.5 M NaF, 100 mM Napyro, β-mercapto, using the Cell Quest Pro software (BD Biosciences). 100 mM Benzamidine, 1.74 mg/mL PMSF and 2 mg/mL leupeptin). The cell suspension was sonicated and rested for Paraffin-Embedded Cell Blocks 20 min on ice and centrifuged for 10 min at 18000g at 4°C. Toledo, U-2932 and SUDHL-2 cells were seeded at Protein concentration in supernatant was determined using 3.5·105 cells/mL in 75 cm2 cell culture flasks. Moreover, the Bradford protein assay (BioRad, Hercules, California, U-2932 cells were either treated with buffer or 125 nM of USA), according to the manufacturer’s instructions. Cell T22-AUR for 24 h and 48 h. Then, paraffin-embedded cell lysates (50 μg) were separated using 12–15% SDS-PAGE blocks were prepared from the pellet of centrifuged cell and transferred to a nitrocellulose blotting membrane. suspension by adding five drops of plasma and thrombin to Membranes were blocked with 5% skim milk in TBST for enmesh the cellular material in a clot. Then, cell clots were 2 h at room temperature, and then incubated with primary placed in a cassette, fixed in 4% paraformaldehyde and antibodies, 1:2000 PARP (556494, BD) O/N, 1:1000 cas­ paraffin-embedded in a tissue processor (Sakura, Tokyo, pase-3 (610322, BD) O/N, 1/1000 cleaved caspase-3 (9661, Japan) for future histology or immunohistochemistry Cell Signaling Technology, Danvers, Massachusetts, USA) (IHC) evaluations. O/N, 1:10000 GAPDH (MAB374, Merck Millipore) for 1 h or 1:1000 α/β tubulin (2148, Cell Signaling Technology) for DAPI Staining 1 h. Membranes were washed with TBST and then incu­ U-2932 cells (3.5·105 cells/mL) were incubated with buffer bated with the appropriate secondary antibody (1:10000, or 125 nM T22-AUR for 24 h and 48 h. After, cells were Jackson Immune Research, West Grove, Pennsylvania, washed with PBS, resuspended with 3.7% of paraformalde­ USA). Western blot visualization was performed using the hyde (Thermo Fisher Scientific) and fixed for 10 min at SuperSignal West Pico Chemiluminescent Substrate −20°C. Then, cells were resuspended with 10 μL of PBS (Thermo Fisher Scientific) and the Universal Hood II Gel and were let air dry on a slide. Finally, cells were stained Doc Imaging System (BioRad). with DAPI mounting medium (Thermo Fisher Scientific) and visualized using a fluorescence microscope (Olympus BX53, Olympus, Tokyo, Japan). Representative pictures were taken Animal Maintenance using an Olympus DP73 digital camera and processed with Four-week-old female NOD/SCID mice were obtained the cellSens Dimension 1.9 software (Olympus) at 400x. from Charles River Laboratories. Mice were maintained Apoptotic cells were visualized as chromatin condensation in specific pathogen-free (SPF) conditions with sterile and/or formation of apoptotic bodies, while mitotic cata­ food and water ad libitum. All animal experiments were strophe (MC) cells as aberrant mitosis formation. approved by the Hospital de la Santa Creu i Sant Pau Animal Ethics Committee (project number 10108 by the Annexin/PI Staining Government of Catalonia) and performed following the DLBCL cells were seeded at 3.5·105 cells/mL and treated European Union Directive 2010–63-EU for welfare of with buffer or 125 nM T22-AUR for 24 h or 48 h. The the laboratory animals. 1872 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al In vivo Nanoconjugate Biodistribution significant weight loss. At that day, the BLI of lymphoma- The nanoconjugate biodistribution was assessed in infiltrated organs was analyzed ex vivo. Finally, all organs a subcutaneous (SC) DLBCL mouse model. Firstly, were collected, fixed in 4% formaldehyde and paraffin- 10 million DLBCL Toledo cells were injected in both embedded for further histopathological or immunohisto­ dorsal flanks of NOD/SCID mice. Tumor growth was chemical evaluations. monitored twice a week with bilateral caliper measure­ ments. The mean tumor volumes were calculated using Immunofluorescence Microscopy the equation (length·width2)/2. When tumors reached Tumor paraffin sections were used for the simultaneous a volume of 600–800 mm3, mice received a single intra­ immunodetection of GFP, CXCR4 and DAPI staining in venous (IV) dose of 325 μg T22-AUR (n=3/time point) or a confocal microscopy. Tumor slides were heated 15 min 166 mM NaCO3H 333 mM NaCl pH=8 buffer (n=3). at 60°C, dewaxed and rehydrated. Antigen retrieval was Fluorescence intensity (FLI) was measured ex vivo at performed using pH=9 citrate buffer with DeCloaking two time points (5 h and 24 h) in subcutaneous tumors Chamber. For the immunodetection, sections were washed and all organs after carefully washing each organ with 3 times with TBS for 5 min and blocking buffer (TBS1x + physiological serum. The emitted FLI, expressed as aver­ 0.5% triton + 3% donkey serum) was added 1 h at RT. age radiant efficiency, measures the amount of accumu­ Slides were incubated with the GFP chicken IgY 1:250 lated T22-AUR, detected by the fluorescence emission of (AVES, Davis, CA, USA) and the CXCR4 rabbit IgG the GFP domain, in each tissue. FLI from experimental 1:250 (Abcam) primary antibodies overnight at 4°C and mice was calculated subtracting the FLI auto-fluorescence of control mice. FLI was registered in the IVIS Spectrum 2 h at RT. Then, sections were washed and the secondary 200 Imaging System (Perkin Elmer, Waltham, MA, USA). antibodies, an anti-chicken IgY-Cy2 1:50 antibody The area under the curve (AUC) of the emitted FLI during together with an anti-rabbit IgG-Cy5 1:200 antibody, the 0–24 h period was calculated using the GraphPad were added to the tissue sections for 2 h at 37°C. After Prism 6 program. three additional TBS washes, the sections were stained Finally, tumors were collected, fixed and paraffined to with DAPI 1:10000 for 10 min at RT and, finally, the perform immunofluorescence assays. mounting medium was added. Furthermore, to determine whether T22-AUR coloca­ lized with lysosomes, live U-2932 cells were incubated for In vivo Nanoconjugate Antineoplastic 5 h with 1 µM T22-AUR. Then, cells were centrifuged and Effect resuspended with 1 mL of cultured medium. NOD/SCID mice were intravenously injected with LysotrackerTM DeepRed (Thermofisher) and Hoescht 20 million luciferase-transfected U-2932 cells in 200 μL bisBenzimide H 33342 trihydrochloride (Sigma-Aldrich) of physiological serum. Three days after cell injection, were added at a final concentration of 70 nM (37°C for mice were divided randomly in the control group, admi­ 1 h) and 1 μg/mL (37°C for 30 min), respectively. nistered with buffer (166 mM NaCO3H 333 mM NaCl, All immunofluorescence pictures were taken with 4.17 pH=8), and the experimental group administered with 100 or 5.00 zoom using the 63X/1.30 objective in a Confocal μg T22-AUR. The administration of buffer or T22-AUR TCS SPE microscope (Leica, Wetzlar, Germany) using was performed three times per week for twelve doses. LAS AF software (Leica). Lymphoma dissemination was monitored, once per week, using bioluminescence imaging (BLI, total radiance photons) in the IVIS Spectrum (Perkin-Elmer). Mice Histopathology were anesthetized with 3% isoflurane in oxygen and BLI All organ sections of paraffin-embedded samples were ana­ was captured 5 min after intraperitoneal injection of firefly lyzed histopathologically (H&E staining) by two indepen­ D-luciferin (2.25 mg/mouse, Perkin Elmer). Moreover, dent observers to assess either the antineoplastic effect in mouse body weight was registered throughout the course DLBCL infiltrated organs or the possible toxicity in non- of the study. DLBCL infiltrated organs. Pictures were taken at 100x or All mice were euthanized when the first mouse pre­ 200x using an Olympus DP73 digital camera and processed sented relevant signs of disease such as poor mobility or with the cellSens Dimension 1.9 software (Olympus). submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1873 DovePress Falgàs et al Dovepress Immunohistochemistry (IHC) reaction (Figure 1A). MALDI-TOF mass spectrometry Anti-CXCR4 (1:300, Abcam) antibody was used for the spectra revealed up to 12 peaks with a sequential incre­ evaluation of CXCR4 expression in cellular blocks from ment of around 911 Da over the T22-GFP-H6 protein three different DLBCL cell lines. Anti-H2AX pSer139 molecular weight (30.6 kDa) (Figure 1B), corresponding (γH2AX, 1:800, Novus, Centennial, CO, USA) and anti- each one to the incorporation of an additional MC-MMAE cleaved Poly (ADP-ribose) polymerase (cleaved PARP, molecule (911 Da). T22-GFP-H6 conjugation did not sig­ 1:300, Promega, Madison, Wisconsin, USA) antibodies nificantly disturb nanoparticles supramolecular structure, were used in cellular blocks from U-2932 cells treated with which showed a mild increment in size (+5.5 nm) upon T22-AUR. Anti-CD20, anti-CD79a, anti-Ki67 (Dako, Santa conjugation (Figure 1C). Finally, SEC-MALS analysis Clara, CA, USA) and anti-CXCR4 (1:300, Abcam) IHC showed an average molar mass increment of 207 kDa in were performed in mouse paraffin-embedded organs. All T22-AUR nanoconjugate (564 kDa) over parental T22- IHC analyses were performed in a DAKO Autostainer GFP-H6 nanoparticles (357 kDa). This fact suggested an Link48 following the manufacturer’s instructions. The average incorporation of around 19 MMAE molecules per stained area of positive cells was quantified using the protein (or around 228 MMAE molecules per nanoparti­ Olympus CellD Imaging 3.3 software in 6-fields for cellular cle) considering that each nanoparticle is composed by blocks and 12-fields for mouse organs. Data were shown as approximately 12 monomers of T22-GFP-H6 protein positive stained area of samples treated with T22-AUR (Figure 1D). divided by the positive stained area of buffer-treated samples. The pictures were taken at 100x, 200x or 400x. Moreover, for the CXCR4 determination in cellular blocks, pictures were Specific T22-AUR Entrance Through the captured with the exact same exposure time and the CXCR4 CXCR4 Receptor in DLBCL Cell Lines intensity was measured using the Image J software Before evaluating the therapeutic effect of the T22-AUR, (1.8.0.172). We used the Colour Deconvolution Plugin with we first determined the in vitro internalization of the the H DAB vector to split the brown staining adjusting the nanoconjugate inside the DLBCL cell lines through the threshold to 30. After that, the “Analyze particles” plugin CXCR4 receptor expressed in the cell membrane. Toledo was used to detect all stained areas and the mean gray value and U-2932 DLBCL cell lines had high levels of CXCR4 was obtained combining all selected black areas and using receptor (CXCR4+ DLBCL cells), achieving intensity the ROI Manager. The intensity value of each analyzed slide mean values as high as 227.68±0.06 and 226.42±0.05, was calculated subtracting 255 to the mean gray value respectively, over a total of 255. In contrast, the obtained by the Image J analysis. SUDHL-2 cell line did not express the receptor (Figure 2A and B). Then, the T22-AUR internalization was mea­ Statistical Analysis sured, after 1 h exposure, by quantifying the fluorescence All results were expressed as mean ± standard error (SE). emission of the GFP domain incorporated to the nanocon­ Differences between groups were analyzed using either the jugate. T22-AUR internalized in CXCR4+ DLBCL cells in T-Student test or Mann–Whitney U-test after the testing of a high amount, being the MFI 27.88±3.01 and 14.25±1.64 normality (Shapiro Wilk test). Differences were consid­ in Toledo and U-2932 cells, respectively. This internaliza­ ered statistically significant at p≤0.05. Statistical calcula­ tion was CXCR4-dependent since when CXCR4+ DLBCL tions were performed using SPSS software v21 cells were pre-incubated 1 h with AMD3100, an antago­ (New York, NY, USA). nist of CXCR4 receptor, the nanoconjugate entrance was blocked and the MFI significantly decreased, reaching Results levels as low as 5.92±0.22 in Toledo cells and 4.02±0.09 Characterization of Functionalization of in U-2932 cells. Besides that, the negative-CXCR4 T22-AUR Nanoconjugate SUDHL-2 cell line showed low MFI levels (3.15±0.13) T22-AUR nanoconjugate was generated by the covalent after 1 h exposure to T22-AUR, which practically did not binding of maleimidocaproyl functionalized MMAE mole­ differ from control cells treated with buffer (2.97±0.12) or cules (MC-MMAE) through solvent exposed protein cells pre-incubated with AMD3100 before the T22-AUR lysine-amines of T22-GFP-H6 nanocarrier in a one-pot addition (2.97±0.01) (Figure 2C). 1874 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al Figure 1 T22-AUR nanoconjugate characterization. (A) T22-GFP-H6 nanoparticle conjugation with maleimide functionalized MMAE (MC-MMAE) through protein lysine amines in order to form the T22-AUR nanoconjugate. The number of conjugated MMAE molecules (purple stars) is only illustrative. (B) MALDI-TOF mass spectrometry spectra of parental T22-GFP-H6 and T22-AUR nanoconjugate. Each peak over 30.6 kDa in T22-AUR indicates the covalent incorporation of an additional MMAE molecule (+911Da). (C) Volume size distribution (size) and zeta potential (zeta) of T22-GFP-H6 nanoparticle (red) and T22-AUR nanoconjugate (purple) determined by light scattering. Pdi indicates polydispersion index. Data are presented as mean ± standard error. (D) Average molar mass distribution of parental T22-GFP-H6 nanoparticle (red) and T22-AUR nanoconjugate (purple) determined by size exclusion chromatography coupled to a multi angle light scattering (SEC-MALS). submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1875 DovePress Falgàs et al Dovepress Figure 2 Assessment of CXCR4 expression intensity and nanoconjugate internalization in different DLBCL cell lines. (A) CXCR4 IHC of Toledo, U-2932 and SUDHL-2 cellular blocks. Pictures were taken at 400x (scale bars=50 μm). (B) Quantitation of mean CXCR4 expression intensity counting six IHC-stained fields (400x) for Toledo, U-2932 and SUDHL-2 cellular blocks. (C) MFI quantification by flow cytometry in Toledo, U-2932 and SUDHL-2 cells after their incubation in buffer, 100 nM AMD3100 or 10 nM T22-AUR for 1 h, or after pretreatment for 1 h with 100 nM AMD3100 followed by the addition of 10 nM of nanoconjugate for 1 h (AMD3100 + T22-AUR). This experiment was performed in biological triplicates. All data are shown as mean ± standard error. **p≤0.01. Abbreviations: IHC, immunohistochemistry, MFI, mean fluorescence intensity. High Tumor Uptake and Internalization of dose accumulated in SC tumors (57.91 ±5.01%), followed T22-AUR in SC Tumors of CXCR4+ by the uptake in kidneys (15.10±2.32%) and liver (10.74 ±1.84%) (Figure 3B below). DLBCL Cells Furthermore, we analyzed the in vivo T22-AUR inter­ After a single intravenous dose of 325 μg of T22-AUR in nalization in the CXCR4+ Toledo cells of SC tumors. By a subcutaneous (SC) mouse model bearing CXCR4+ immunofluorescence, we could detect the localization of Toledo cells, the nanoconjugate achieved a high tumor T22-AUR, with an anti-GFP antibody, observing that it uptake in SC tumors and negligible fluorescence levels was located in the membrane as well as in the cytosol of (FLI) in the normal organs (spleen, pancreas, liver, heart, Toledo cells in the SC tumors of mice treated with a single lungs, kidneys and bone marrow) (Figure 3A). The FLI bolus of T22-AUR. In the buffer-treated animals, there peak of T22-AUR in SC tumors was at 5 h (1.36±0.51·107 was no presence of GFP in the SC tumors. Regarding the radiant efficiency) and decreased considerably at 24 CXCR4 expression, the receptor was detected predomi­ h (0.61±0.29·107 radiant efficiency). The highest FLI nantly in the membrane of SC tumor cells of animals levels in non-DLBCL infiltrated organs were observed in treated with buffer whereas the CXCR4 expression was kidneys at 5 h (0.35±0.06·107 radiant efficiency) and liver more dot-pointed in the group of animals treated with the at 24 h (0.22±0.22·107 radiant efficiency), which showed nanoconjugate, suggesting the endocytosis of the receptor 3.88-fold and 2.77-fold lower levels than SC tumors, inside the cytosol of lymphoma cells. In the merged respectively, at each corresponding time point (Figure 3B images, a co-localization of CXCR4 and T22-AUR was above). Moreover, the AUC of the emitted FLI revealed visualized in the membrane and inside of lymphoma cells that the highest percent of the total T22-AUR injected (Figure 3C). 1876 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al Figure 3 T22-AUR tissue biodistribution in a SC mouse model bearing CXCR4+ DLBCL cells. (A) Representative images of the FLI emitted in SC Toledo tumors and normal organs in animals treated either with buffer or the fluorescent nanoconjugate T22-AUR at 5 and 24 h. (B) Above: FLI quantification measured in SC Toledo tumors and normal organs in T22-AUR- treated mice 5 h (n=3) and 24 h (n=3) postinjection. FLI was calculated subtracting the autofluorescence of each tissue from buffer-treated mice (n=3) and represented as radiant efficiency. Below: Quantification of the percentage of T22-AUR injected dose that accumulated in SC tumors and non-infiltrated organs (spleen, pancreas, liver, heart, lungs, kidneys and BM). This percentage was measured using the area under the curve (AUC) of emitted FLI registered in each organ calculated in relation to the total emitted FLI during the 0–24 h period. (C) Confocal images visualizing cell nuclei (DAPI), CXCR4 receptor, GFP protein and the merged of the three staining in the SC Toledo tumors from animals treated with buffer or T22- AUR at 5 h. Scale bars=7.5 μm. All data are shown as mean ± standard error. Abbreviations: BM, bone marrow; FLI, fluorescence intensity; SC, subcutaneous. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1877 DovePress Falgàs et al Dovepress Cytotoxic Effect of Untargeted Free was shown in U-2932 cells (52.14±2.51% at 125 nM T22- MMAE and CXCR4-Targeted T22-AUR AUR) followed by Toledo cells (18.95±1.93% at 125 nM T22-AUR), and finally the SUDHL-2 cell line, which is Nanoconjugate on the DLBCL Cell Lines the CXCR4-negative DLBCL cell line, only showed Here, we assessed the sensitivity of the three DLBCL cell barely cell death (3.76±2.44% at 125 nM T22-AUR). lines to untargeted free MMAE (the drug conjugated to the Moreover, U-2932 cells underwent a significantly higher T22-AUR nanoconjugate). Our results demonstrated that level of cell death than Toledo or SUDHL-2 cells at both U-2932 cells were the most sensitive (IC50=0.33±0.14 ng/ mL) followed by SUDHL-2 (IC50=0.50±0.08 ng/mL) and low and high concentrations, whereas Toledo cell line finally Toledo (IC50=0.87±0.09 ng/mL). Thus, Toledo cells achieved significant differences with SUDHL-2 at high (with high CXCR4 expression) incubated with free T22-AUR concentrations (Figure 4C). Thus, despite MMAE showed significantly less cell death and, therefore, Toledo cells express higher levels of CXCR4 receptor significantly higher IC50 than U-2932 (with high- than U-2932 cells, they are less sensitive to T22-AUR intermediate CXCR4 expression) and SUDHL-2 cells because of their lower sensitivity to the untargeted drug (lacking CXCR4 expression) (Figure 4A and B). These itself (free MMAE) (Figure 4A and B). Moreover, data show a lack of relationship of the cytotoxicity of free SUDHL-2 cells, which are more sensitive to free MMAE MMAE with CXCR4 expression in DLBCL cell lines. than Toledo cells, but lack CXCR4 expression, show very Next step was to assess the cytotoxic effect of low levels of cell death induction after T22-AUR treat­ T22-AUR as well as its CXCR4-dependent cytotoxicity ment demonstrating that the entry of the nanoconjugate in in the three DLBCL cell lines. The higher cytotoxic effect the cells through CXCR4 is essential for the Figure 4 Cytotoxic effect of untargeted free MMAE and CXCR4-targeted T22-AUR nanoconjugate on the DLBCL cell lines. (A) Percentage of cell death induced after the incubation of free-MMAE for 48 h in CXCR4+ DLBCL cell lines (U-2932 and Toledo) and CXCR4− DLBCL cell line (SUDHL-2) measured by colorimetric cell proliferation kit (XTT). (B) Quantification of the half-maximal inhibitory concentration (IC50) of free-MMAE in each DLBCL cell line. (C) Percentage of cell death induced by different concentrations of T22-AUR for 48 h in DLBCL cell lines. (D) Competition assays (measured as percentage of cell death) done by 1 h pretreatment of U-2932 cells with 100 nM AMD3100 followed by the addition of 10 nM of nanoconjugate. All experiments were performed in biological triplicates and data are shown as mean ± standard error. *p≤0.05; **p≤0.01 and ***p≤0.005. 1878 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al nanoconjugate cytotoxic effect. Additionally, we con­ significant with respect to buffer-treated cells. The ratio of firmed the CXCR4-dependence of the T22-AUR cytotoxic apoptosis in cells treated with T22-AUR was significantly effect because after blocking the receptor with AMD3100 higher compared to buffer-treated cells, 17.35-fold and the cell death of U-2932 cells decreased significantly from 38.93-fold at 24 h and 48 h, respectively (Figure 6A and B). 32.42±2.00% to 2.83±0.40% (Figure 4D). Moreover, by performing the Annexin/PI staining in U-2932 cells, we identified a significant decrease in the percentage of viable cells treated with the nanoconju­ Mechanism of Action and Cell Death gate, from 89.62±0.97% in cells treated with buffer to Induction by T22-AUR in CXCR4+ 70.54±1.01% after 24 h exposure to T22-AUR and DLBCL Cells 44.17±1.53% after 48 h exposure. The low viability Since T22-AUR included a non-cleavable linker in its was associated with a significant increase of the percen­ conjugation with MMAE, the T22-AUR must be proteo­ tage of T22-AUR-treated cells undergoing early apopto­ lyzed within the lysosomes for the release of the MMAE sis (from 3.82±1.76% to 12.27±2.30% after 24 h and into the cytoplasm, where it can exert the drug cytotoxic 11.15±0.99% after 48 h) and late apoptosis (from 6.06 effect in DLBCL cells, as it has been described for ADCs ±0.72% to 15.68±0.72% at 24 h and 40.55±1.25% at that use non-cleavable linkers to bind and transport 48 h) (Figure 6C). MTAs.23 To check this process, we performed an immu­ Afterwards, we quantified by IHC the phosphorylation of nofluorescence assay with nuclear (Hoescht dye) and lyso­ the histone H2AX (γH2AX) in order to know whether DNA somal (lysotracker Deep Red dye) staining in live U-2932 double-strand breaks were induced by T22-AUR. U-2932 cells treated with T22-AUR. We detected the presence of cells treated with the nanoconjugate achieved a significant T22-AUR (green), by the GFP protein, into lysosomes 5.09-fold and 3.98-fold increase of γH2AX-positive area at (red), which both merged (yellow) within U-2932 cells. 24 h and 48 h, respectively, compared to cells treated with Moreover, we also observed the presence of the nanocon­ buffer. Additionally, the positive area of cleaved PARP, jugate in the cell membrane (Figure 5A). These results a marker of apoptosis, was also significantly higher in these suggested the endocytosis of T22-AUR and its subsequent cells treated with T22-AUR, 2.38-fold at 24 h and 5.27-fold destination to lysosomes, undergoing complete proteolysis at 48 h, compared to buffer group (Figure 6D and E). Finally, of the protein nanocarrier which releases the attached we confirmed the activation of the apoptotic pathway, by MMAE to the cytosol where it binds and inhibits tubulin Western blot, detecting the expression of cleaved PARP as to induce cell death. Thus, T22-AUR lysosomal entry and well as cleaved caspase-3 in U-2932 cell extracts treated with the subsequent CXCR4-dependent cell death confirm the T22-AUR for 24 h and 48 h (Figure 6F). successful conjugation of MMAE to the nanocarrier. In order to further elucidate the cell death mechanisms underlying T22-AUR cytotoxicity, we performed different In vivo Antineoplastic Effect of the experiments. We analyzed the cell cycle phase distribution T22-AUR Nanoconjugate in a CXCR4+ after 24 h and 48 h of 125 nM T22-AUR exposition in DLBCL Disseminated Mouse Model U-2932 cells. As time elapsed, the percentage of U-2932 Afterwards, we evaluated the in vivo therapeutic effect cells in G0/G1 phase decreased from 70.37±0.68% in the of T22-AUR in a disseminated mouse model of buffer-treated cells to 54.44±5.01% after 48 h with the nano­ CXCR4+ U-2932-Luci cells. The lymphoma dissemina­ conjugate incubation. This G0/G1 reduction was mainly due tion, detected by bioluminescence imaging (BLI) to the increase of the G2/M phase, which represented the emitted by U-2932-Luci cells, was exhibited on CNS 18.29±1.96% of total cells in the buffer group and the 36.58 and BM (frequently backbone and rarely cranium or ±4.80% of T22-AUR treated cells for 48 h (Figure 5B and C). jaw). Three days after the intravenous injection of lym­ After performing DAPI staining, we observed a 9.16- phoma cells, we started the administration of buffer or fold significant increase of U-2932 cells undergoing mitotic 100 μg T22-AUR (≈60 nmol of MMAE in one dose), catastrophe (MC) after 24 h of T22-AUR incubation com­ three days per week and a total of 12 doses (≈720 nmol pared to cells treated with buffer. At 48 h, cells undergoing of MMAE in the total dosage). The emitted BLI was MC diminished compared to the T22-AUR incubation for reduced in the T22-AUR-treated animals compared to 24 h, although the increase (2.42-fold) continued being buffer-treated animals (Figure 7A). Indeed, the AUC submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1879 DovePress Falgàs et al Dovepress Figure 5 T22-AUR-lysosomal colocalization and cell cycle arrest by T22-AUR in CXCR4+ DLBCL cells. (A) Hoechst (blue staining), GFP protein (green staining) and lysotracker (red staining) immunofluorescence detection in live U-2932 cells exposed to 1 μM T22-AUR for 5 h. Scale bars=5 μm. (B) Representative graphics for cell cycle analysis in U-2932 cells treated with buffer or 24 or 48 h with 125 nM T22-AUR. M1: G0/G1 phase, M2: S phase and M3: G2/M phase. (C) Quantification of the percentage of total U-2932 cells in different cell cycle phases (G0/G1, S and G2/M) after incubation with buffer or 125 nM T22-AUR (24 and 48 h). This experiment was performed in biological triplicates and data are shown as mean ± standard error. *p≤0.05. from the whole study showed a significant reduction of the infiltrated lymphoma organs of the U-2932-Luci BLI in the mice group treated with the therapeutic disseminated mouse model. The organs with highest nanoconjugate (0.53±0.06·108 total radiance photons) lymphoma-cell infiltration were bone marrow (BM, compared to the buffer-group (1.71±0.51·108 total radi­ backbone) and central nervous system (CNS, brain). ance photons) (Figure 7B). We ended the experiment Both organs were infiltrated in 7 out of 9 buffer- when the first buffer-treated mice showed a significant treated mice. The BM was not infiltrated in any mice weight loss. Nevertheless, the nanoconjugate-treated treated with T22-AUR and the CNS was infiltrated in mice did not lose weight nor did present any symptoms only 3 out of 10 T22-AUR-treated mice. Moreover, the of illness (Figure 7C). At that time, we analyzed ex vivo levels of BLI in these organs (BM and CNS) were 1880 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al Figure 6 Further mechanisms of cell death induction by T22-AUR in CXCR4+ DLBCL cells. (A and B) DAPI staining pictures and ratio quantification of MCs (yellow arrows) or apoptotic bodies (red arrows) in U-2932 cells treated with buffer or 125 nM-T22-AUR (24 and 48 h). Ratio quantification was measured dividing the number of cells undergoing MC or apoptosis in 10 fields of T22-AUR samples (24 and 48 h) by 10 fields of buffer-exposed samples. (C) Annexin/PI assay showing the total percentage of viable, early apoptotic and late apoptotic U-2932 cells treated with buffer or 125 nM T22-AUR (24 h and 48 h). These experiments were performed in biological triplicates. (D) IHC staining using γH2AX and cleaved PARP antibodies after buffer or 125 nM T22-AUR treatment (24 and 48 h) in U-2932 cells. (E) Ratio quantification of the IHC positive stained area marked by γH2AX and cleaved PARP in buffer or T22-AUR-treated samples. Ratio quantification was represented dividing the area of positive cells from 6 fields of T22-AUR-treated samples (24 and 48 h) by 6 fields of buffer-treated samples. (F) Anti-PARP, cleaved PARP, pro-caspase-3 and cleaved caspase-3 detection, by Western blotting, in U-2932 extracts treated with buffer or 125 nM T22-AUR for 24 h and 48 h. GAPDH and α/β tubulin were used as endogenous controls. All pictures were taken at 400x (scale bars= 50 μm). All data are shown as mean ± standard error. *p≤0.05; ***p≤0.005. Abbreviation: MC, mitotic catastrophe. submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1881 DovePress Falgàs et al Dovepress Figure 7 In vivo antineoplastic effect of T22-AUR in a disseminated mouse model of CXCR4+ DLBCL-Luci cells. (A) Representative images of lymphoma dissemination, registered by measuring the BLI emitted by U-2932-Luci cells, in animals treated with buffer or T22-AUR at the last days of the experiment (days 23 and 30 postinjection of lymphoma cells). (B) Area under the curve (AUC) of the lymphoma follow-up (from day 1 to day 30) registered by total BLI emission from buffer-treated mice (n=9) and T22-AUR-treated mice (n=10). (C) Body weight of mice treated with buffer (n=9) or T22-AUR (n=10) during the whole experiment. (D) Quantification of the total BLI registered (above) and representative images (below) from CNS and BM in buffer (n=9) and T22-AUR-treated mice (n=10). All data are shown as mean ± standard error. *p≤0.05; **p≤0.01. Abbreviations: BLI, bioluminescence imaging; BM, bone marrow; CNS, central nervous system. 1882 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al significantly reduced in the T22-AUR-treated compared infiltrated by DLBCL cells, such as liver, spleen, kidneys, to buffer-treated animals (Figure 7D). heart and lungs (Figure 9A). Most importantly, since human hematopoietic cells are reported to express the CXCR4 receptor, we wanted to Lymphoma-Burden Reduction by evaluate the possible hematopoietic toxicity produced by T22-AUR Nanoconjugate in CXCR4+ T22-AUR in human PBMCs. Thus, we firstly evaluated DLBCL Infiltrated Organs the levels of CXCR4 in human PBMCs of three healthy By H&E staining, CD20 and CD79a IHC, we confirmed donors, which were significantly lower (4.36±0.14 MFI) human B cell infiltration in the CNS and BM. Those data than CXCR4+ DLBCL cells (99.32±12.13 MFI) (Figure showed that buffer treated animals presented a high lymphoma 9B). Then, we evaluated the cytotoxicity of the T22-AUR infiltration in the meninges, which covers the CNS parench­ to human PBMCs observing almost negligible levels of cell yma, and, at many points, this infiltration penetrated to the death induction (0.47±0.45% at 10nM; 4.05±4.05% at 50nM brain parenchyma. Otherwise, animals treated with T22-AUR and 4.24±2.93% at 125nM) as compared to the cytotoxicity presented only residual CD20+/CD79a+ B cells in the observed in CXCR4+ DLBCL cells (24.17±7.76% at 10 nM; meninges, which did not penetrate to the parenchyma in any 56.99±5.86% at 50 nM and 63.78±5.93% at 125 nM) case. Thus, the quantification of the ratio of CD20/CD79a- (Figure 9C). Therefore, T22-AUR did not produce side positive area showed significant differences between the ani­ toxicity in mice neither in human PBMCs. mals treated with T22-AUR (ratio 0.25±0.03 CD20 and 0.15 ±0.04 CD79a) and buffer (ratio 1.00±0.14 CD20 and 1.00 Discussion ±0.18 CD79a). Furthermore, we observed that the expression We have here demonstrated the T22-AUR nanoconjugate of the CXCR4 receptor was maintained in the human B cells, capacity to potently block lymphoma dissemination in meaning that we detected also a reduction of the ratio of a CXCR4+ DLBCL mouse model, as well as its induction CXCR4-positive area in the T22-AUR group (0.21±0.04) of mitotic arrest and apoptosis in CXCR4+ DLBCL cells. compared to buffer group (1.00±0.11). Finally, we also demon­ Importantly, we have proved that MMAE conjugation strated that DLBCL cells in the CNS of buffer-treated mice to the nanocarrier, in order to generate the T22-AUR expressed Ki67 (1.00±0.22), a marker of proliferation, and the nanoconjugate, does not alter the nanocarrier targeting Ki67-positive-cells were significantly reduced in mice treated and specific internalization ability through the CXCR4 with T22-AUR (0.11±0.03) (Figure 8A). receptor in DLBCL cell lines nor the tumor and non- Similar findings were observed in the BM, animals treated tumor tissue distribution pattern in the mouse model. with buffer had a high lymphoma infiltration (CD20/CD79a- Thus, T22-AUR displayed a CXCR4-dependent targeting, positive cells) which was significantly reduced in T22-AUR- as we had previously demonstrated for the unconjugated treated mice (ratio 1.00±0.13 CD20-positive area and ratio T22-GFP-H6 nanoparticle. In fact, T22-AUR nanoconju­ 1.00±0.15 CD79a-positive area in buffer treated mice vs 0.10 gate achieved a high tumor uptake and internalization ±0.04 and 0.17±0.08, respectively, in T22-AUR-treated within CXCR4+ DLCBL tumor cells in a SC mouse mice). Moreover, the ratio of the CXCR4-positive area as model reaching its peak at 5 h post-injection, exactly the well as the ratio of Ki67-positive area in the animals treated same tumor accumulation time we have reported for the with T22-AUR was also significantly lower (0.21±0.07 nanocarrier.15 CXCR4-positive area and 0.14±0.13 Ki67-positive area) The pharmacokinetics of our nanoconjugate may be than in the animals treated with buffer (1.00±0.15 CXCR4- similar to that reported for ADCs, in which the antibody positive area and 1.00±0.13 Ki67-positive area) (Figure 8B). rather than the drug determines its half-life in blood.24 Moreover, the size of T22-AUR (17.9 nm ± 0.7 nm), Absence of Histopathological Alterations which is higher than the renal filtration cut-off (7 nm), avoids its clearance by renal filtration, as also reported by in Normal Organs of Mice and Lack of ADCs.25 in vitro Cytotoxicity in Human PBMCs The MMAE, which belongs to the vinkaloid group and After T22-AUR Treatment dolastatins family of MTAs, is increasingly used for tar­ The repeated T22-AUR administration in mice did not geted drug delivery approaches in cancer therapy. show any histopathological alteration in the organs non- Microtubules are actively involved in essential processes, submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1883 DovePress Falgàs et al Dovepress Figure 8 T22-AUR effect on lymphoma infiltration burden in the affected organs (CNS and BM). (A and B) Hematoxylin and eosin (H&E) staining, anti-CD20, anti-CD79a, anti-CXCR4 and anti-Ki67 immunohistochemical representative pictures and quantification of the ratio of the positive stained tissue area for each marker in CNS and BM organs from animals treated either with buffer or T22-AUR. Ratio quantification was obtained dividing the area of positive cells in 12 counted fields in samples T22-AUR- treated by the counted positive area in 12 fields of buffer-treated samples. Pictures were taken at 100x (scale bars= 200 μm) for CNS and 200x (scale bars= 100 μm) for BM. All data are shown as mean ± standard error. ***p≤0.005. Abbreviations: BM, bone marrow; CNS, central nervous system. 1884 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al Figure 9 T22-AUR does not induce systemic toxicity in CXCR4+ DLBCL-Luci disseminated mice or in vitro cytotoxicity on human PBMCs. (A) Hematoxylin and eosin (H&E) staining of non-DLBCL infiltrated organs (liver, spleen, kidneys, lungs and heart) from buffer and T22-AUR-treated mice. Pictures were taken at 200x (scale bars= 100 μm). (B) Membrane CXCR4 expression measured by cell cytometry in U-2932 cells and human PBMCs from three independent healthy donors. Data were shown as mean fluorescence intensity (MFI) resulted from dividing the CXCR4 MFI by its own isotype MFI. (C) Percentage of cell death measured by colorimetric cell proliferation kit (XTT) in U-2932 and human PBMCs after exposure to T22-AUR in the 10–125 nM range. All experiments were performed in biological triplicates and data are shown as mean ± standard error. *p≤0.05; ***p≤0.005. Abbreviations: MFI, mean fluorescence intensity; PBMCs, peripheral blood mononuclear cells. both in dividing and non-dividing cells, which cannot be may also be activated independently of MC in non- replaced by other cellular systems. One of the main rea­ cycling cells. Therefore, our data support the notion sons for the increasing interest in the clinical use of MTAs that MMAE may act by two different apoptotic mechan­ is because they can kill mitotic, interphase and quiescent isms, with and without previous MC trigger, in CXCR4+ cancer cells.13,14 DLBCL cells. Actually, MTAs, such as MMAE, are Regarding the mechanism of action of the MMAE mechanistically different from DNA-directed drugs delivered payload drug, our results showed that the T22- used in chemotherapy and mitosis-specific drugs that AUR cytotoxic effect in CXCR4+ DLBCL cells involves kill only cancer cells that are actively dividing.28 G2/M cell cycle arrest, MC and DNA damage (as mea­ For the evaluation of the in vivo T22-AUR antineo­ sured by γH2AX), followed by PARP cleavage and plastic effect, we used an extranodal DLBCL disseminated induction of apoptotic bodies. There is an ongoing mouse model, with CXCR4+ DLBCL infiltration in the debate on whether MC is a specific cell death program CNS and BM, which we consider a relevant model to by itself or a previous event process leading to apoptosis test novel therapies since extranodal DLBCL patients or necrosis.26,27 We observed that MC appeared earlier involving more than one site present worse overall as (ie, 24 h) than apoptosis; however, at a later time point well as progression-free survival than nodal DLBCL (ie, 48 h), the proportion of cells involved in apoptotic patients.29–31 In this DLBCL model, repeated administra­ cell death was extremely higher than the proportion of tion of the T22-AUR nanoconjugate induces a decrease of cells undergoing MC at an earlier time point (ie, 24 h). It lymphoma dissemination during the whole experimental is likely that the MMAE-induced apoptosis in our model follow-up. This cytotoxic effect was observed in the submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1885 DovePress Falgàs et al Dovepress CXCR4+ DLBCL-infiltrated CNS and especially in the tissues, whereas ADCs accumulate less than 1% of BM, where a nearly complete elimination of CXCR4+ the administered dose in cancer tissues.21,22 DLBCL cells was achieved in all nanoconjugate-treated 4. Whereas gene expression of CD79b, the target of animals. Therefore, the use of MMAE as a payload drug is this ADC, is around a median of 7.5 fold-higher in especially relevant to eliminate quiescent resistant cells in DLBCL patients than non-neoplastic controls, the R/R DLBCL patients, which remain in the hypoxic niches, CXCR4 gene expression, targeted by the T22-AUR including bulky necrotic lymphoma masses and the BM.32 nanoconjugate in DLBCL samples is on average Moreover, the hypoxic tumor BM microenvironment 140-fold higher than in normal controls.10,38 Thus, enhances cancer cell survival and reduces the sensitivity our nanoconjugate can be targeted more specifically to therapy.33–35 due to the huge CXCR4 overexpression existing in Furthermore, the absence of toxicity, either on non- DLBCL patients. DLBCL infiltrated mouse organs or human PBMCs, 5. Our nanoconjugate achieves a potent block of lym­ phoma dissemination with complete lack of associated could be explained by the “super-selectivity” reported in toxicity in normal organs. In contrast, polatuzumab multivalent systems, such as nanoparticles or nanoconju­ vedotin is effective at a dosage that induces severe side gates, which consists in the ability of interacting with their effects and life-threatening toxicities, such as grade target receptor only when its density in the cell surface is 3–4 cytopenias, peripheral neuropathy, progressive above a certain threshold.36,37 multifocal leukoencephalopathy or tumor lysis Finally, a successful preclinical development of our syndrome.38 T22-AUR nanoconjugate could add a therapeutic option for transplantation-ineligible R/R DLBCL patients. In this Conclusions sense, it must be necessarily compared with the perfor­ The association of the overexpression of CXCR4 in mance of polatuzumab vedotin ADC, which has been DLBCL cells with therapy resistance, the specific recently approved for clinical use in R/R DLBCL patients CXCR4 targeting of the nanoconjugate and the use of together with rituximab and bendamustine.38,39 Despite the payload MMAE, which is capable to kill quiescent both polatuzumab vedotin and T22-AUR incorporate the cancer cells, make the T22-AUR a promising therapeutic same payload drug (MMAE), we believe that, as compared approach in CXCR4+ transplantation-ineligible R/R to this ADC, there are five main reasons to further develop DLBCL patients. our nanoconjugate: Data Sharing Statement 1. T22-AUR could be more efficient than polatuzumab Data available on request from the authors: The data that vedotin in selectively delivering the MMAE payload support the findings of this study are available from the to target cells, and in subsequently inducing their corresponding author upon reasonable request. Some data elimination, because each nanoconjugate shows may not be made available because of privacy or ethical a multivalent ligand display of around 12 molecules restrictions. to internalize through CXCR4-mediated internaliza­ tion, as compared to the only two epitope-binding Acknowledgments domains from two antigen-binding fragment (Fab) This work was supported by Instituto de Salud Carlos III present in the antibody of ADCs.16,40 (ISCIII, Co-funding from FEDER) [PI18/00650, PIE15/ 2. Each T22-AUR nanoconjugate displays about 228 00028, PI15/00378 and EU COST Action CA 17140 to R. molecules of MMAE linked to the nanocarrier, as M., FIS PI17/01246 and RD16/0011/0028 and FIS PI14/ compared to a mean of 3.65 MMAE molecules 00450 to J.S., and FIS PI15/00272 to E.V.]; Agencia Estatal added to polatuzumab vedotin.41 Thus, lower de Investigación (AEI) and Fondo Europeo de Desarrollo doses of T22-AUR should be more effective than Regional (FEDER) [grant BIO2016-76063-R, AEI/FEDER, polatuzumab vedotin. UE to A.V. and grant PID2019-105416RB-I00/AEI/10.13039/ 3. Our nanoconjugate is mostly uptaken by cancer tar­ 501100011033 to E.V.]; CIBER-BBN [CB06/01/1031 and get cells in the tumor tissue, while only low or neg­ 4NanoMets to R.M., VENOM4CANCER to A.V. and ligible uptake is seen in non-lymphoma infiltrated NANOREMOTE to E.V.]; AGAUR [2017 FI_B 00680 to A. 1886 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress Dovepress Falgàs et al F., 2017- SGR-865 to R.M., 2017-SGR-1395 to J.S. and 2017SGR-229 to A.V.]; Josep Carreras Leukemia Research References Institute [P/AG to R.M.] La Marató TV3 [201941-30-31-32 to 1. Smith A, Crouch S, Lax S, et al. Lymphoma incidence, survival and prevalence 2004–2014: sub-type analyses from the UK’s haematolo­ J.S. and A.V.]; a grant from the Cellex Foundation, Barcelona gical malignancy research network. Br J Cancer. 2015;112(9): to J.S.; a grant from the Generalitat de Catalunya (PERIS) 1575–1584. doi:10.1038/bjc.2015.94 [SLT002/16/00433 to J.S.] and a grant from the Generalitat de 2. Coiffier B, Sarkozy C. Diffuse large B-cell lymphoma: r-CHOP fail­ ure—what to do? Hematol Am Soc Hematol Educ Program. Catalunya CERCA Programme. A.V. received an Icrea 2016;2016(1):366–378. doi:10.1182/asheducation-2016.1.366 Academia Award. U.U was also supported by Grant PERIS 3. Raut LS, Chakrabarti PP. Management of relapsed-refractory diffuse large B cell lymphoma. South Asian J Cancer. 2014;3(1):66–70. SLT006/17/00093 from la Generalitat de Catalunya and doi:10.4103/2278-330X.126531 Miguel Servet fellowship (CP19/00028) from Instituto de 4. González-Barca E, Coronado M, Martín A, et al. Spanish lymphoma Salud Carlos III co-funded by European Social Fund (ESF group (GELTAMO) guidelines for the diagnosis, staging, treatment, and follow-up of diffuse large B-cell lymphoma. Oncotarget. 2018;9 investing in your future). The bioluminescent follow-up of (64):32383–32399. doi:10.18632/oncotarget.25892 cancer cells and toxicity studies has been performed in the 5. Fridrik MA, Jaeger U, Petzer A, et al. Cardiotoxicity with rituximab, cyclophosphamide, non-pegylated liposomal doxorubicin, vincristine ICTS-141007 Nanbiosis Platform, using its CIBER-BBN and prednisolone compared to rituximab, cyclophosphamide, doxorubi­ Nanotoxicology Unit (http://www.nanbiosis.es/portfolio/u18- cin, vincristine, and prednisolone in frontline treatment of patients with nanotoxicology-unit/). Protein production has been partially diffuse large B-cell lymphoma: a randomised phase-III study from the Austrian cancer drug therapy working group [Arbeitsgemeinschaft performed by the ICTS “NANBIOSIS”, more specifically by Medikamentöse Tumortherapie AGMT] (NHL-14). Eur J Cancer. the Protein Production Platform of CIBER-BBN/IBB (http:// 2016;58:112–121. doi:10.1016/j.ejca.2016.02.004 6. Unzueta U, Céspedes MV, Vázquez E, Ferrer-Miralles N, www.nanbiosis.es/unit/u1-protein-production-platform-ppp/). Mangues R, Villaverde A. Towards protein-based viral mimetics for The authors would like to thank Annabel García-León cancer therapies. Trends Biotechnol. 2015;33(5):253–258. doi:10. (IIB-Sant Pau, Barcelona) and Lola Mulero Pérez (histol­ 1016/j.tibtech.2015.02.007 7. Bamrungsap S, Zhao Z, Chen T, et al. Nanotechnology in therapeu­ ogy unit from CMRB, Barcelona) for their technical tics: a focus on nanoparticles as a drug delivery system. support. Nanomedicine (Lond). 2012;7(8):1253–1271. doi:10.2217/nnm.12.87 8. Chen J, Xu-Monette ZY, Deng L, et al. Dysregulated CXCR4 expres­ sion promotes lymphoma cell survival and independently predicts Author Contributions disease progression in germinal center B-cell-like diffuse large All authors contributed to data analysis, drafting or revising B-cell lymphoma. Oncotarget. 2015;6(8):5597–5614. doi:10.18632/ oncotarget.3343 the article, have agreed on the journal to which the article will 9. Moreno MJ, Bosch R, Dieguez-Gonzalez R, et al. CXCR4 expression be submitted, gave final approval of the version to be pub­ enhances diffuse large B cell lymphoma dissemination and decreases patient survival. J Pathol. 2015;235(3):445–455. doi:10.1002/path. lished, and agree to be accountable for all aspects of the 4446 work. Conception and design: AF, EV, RM and IC; 10. Pansy K, Feichtinger J, Ehall B, et al. The CXCR4-CXCL12-axis is Development of methodology: AF, VP, UU, LAC, MAM, of prognostic relevance in DLBCL and its antagonists exert pro-apoptotic effects in vitro. Int J Mol Sci. 2019;20(19):4740. PA and IC; Acquisition of data: AF, VP and YN. doi:10.3390/ijms20194740 Histopathological analysis: AF and AG; Analysis and inter­ 11. Laursen MB, Reinholdt L, Schönherz AA, et al. High CXCR4 expression impairs rituximab response and the prognosis of pretation of data: AF, JS, AV, EV, RM and IC. Drafted the R-CHOP-treated diffuse large B-cell lymphoma patients. manuscript: AF; Review and revision of manuscript: AV, Oncotarget. 2019;10(7):717–731. doi:10.18632/oncotarget.26588 UU, EV, RM and IC. Study supervision: RM, IC and EV. 12. Loong HH, Yeo W. Microtubule-targeting agents in oncology and therapeutic potential in hepatocellular carcinoma. OncoTargets Ther. 2014;7:575–585. Disclosure 13. Bates D, Eastman A. Microtubule destabilising agents: far more than just antimitotic anticancer drugs. Br J Clin Pharmacol. 2017;83 AV, EV, UU, RM, IC are cited as inventors in three (2):255–268. doi:10.1111/bcp.13126 patents: PCT/EP2018/061732, covering Nanostructured 14. Field JJ, Kanakkanthara A, Miller JH. Microtubule-targeting agents Proteins and uses thereof; PCT/EP2018/069303, covering are clinically successful due to both mitotic and interphase impair­ ment of microtubule function. Bioorg Med Chem. 2014;22(18): Therapeutic Nanoconjugates and uses thereof; and PCT/ 5050–5059. doi:10.1016/j.bmc.2014.02.035 EP2012/050513 (WO2012/095527), covering Methods 15. Falgàs A, Pallarès V, Unzueta U, et al. A CXCR4-targeted nanocar­ rier achieves highly selective tumor uptake in diffuse large B-cell and reagents for efficient and targeted delivery of thera­ lymphoma mouse models. Haematologica. 2020;105(3):741–753. peutic molecules to CXCR4 cells, all licensed to doi:10.3324/haematol.2018.211490 Nanoligent. AV, EV, RM are scientific advisors and 16. Pallarès V, Unzueta U, Falgàs A, et al. An Auristatin nanoconjugate targeting CXCR4+ leukemic cells blocks acute myeloid leukemia cofounders of Nanoligent. The authors report no other dissemination. J Hematol Oncol. 2020;13(1):36. doi:10.1186/s130 conflicts of interest in this work. 45-020-00863-9 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 1887 DovePress Falgàs et al Dovepress 17. Li C, Zhang C, Li Z, et al. Clinical pharmacology of vc-MMAE 30. Yao S, Li J, Yao Z, et al. Extranodal involvement in young patients antibody–drug conjugates in cancer patients: learning from eight first- with diffuse large B-cell lymphoma: distribution, prognostic value in-human Phase 1 studies. mAbs. 2020;12(1):1699768. doi:10.1080/ and treatment options. Chin J Cancer Res. 2017;29(1):57–65. 19420862.2019.1699768 doi:10.21147/j.issn.1000-9604.2017.01.07 18. Zhao P, Zhang Y, Li W, Jeanty C, Xiang G, Dong Y. Recent advances 31. Hashwah H, Bertram K, Stirm K, et al. The IL-6 signaling complex is of antibody drug conjugates for clinical applications. Acta Pharm Sin a critical driver, negative prognostic factor, and therapeutic target in B. 2020;10(9):1589–1600. doi:10.1016/j.apsb.2020.04.012 diffuse large B-cell lymphoma. EMBO Mol Med. 2019;11(10): 19. Ma H, Sawas A. Combining biology and chemistry for a new take on e10576. doi:10.15252/emmm.201910576 chemotherapy: antibody-drug conjugates in hematologic 32. Klener P, Klanova M. Drug resistance in non-hodgkin lymphomas. malignancies. Curr Hematol Malig Rep. 2018;13(6):555–569. doi:10. Int J Mol Sci. 2020;21(6):2081. doi:10.3390/ijms21062081 1007/s11899-018-0485-3 33. Cavnar SP, Rickelmann AD, Meguiar KF, et al. Modeling selective 20. Bourbon E, Salles G. Polatuzumab vedotin: an investigational elimination of quiescent cancer cells from bone marrow. Neoplasia. anti-CD79b antibody drug conjugate for the treatment of diffuse 2015;17(8):625–633. doi:10.1016/j.neo.2015.08.001 large B-cell lymphoma. Expert Opin Investig Drugs. 2020;29 34. Cavnar SP, Xiao A, Gibbons AE, et al. Imaging sensitivity of quies­ (10):1079–1088. doi:10.1080/13543784.2020.1800638 cent cancer cells to metabolic perturbations in bone marrow 21. Gerber H-P, Senter PD, Grewal IS. Antibody drug-conjugates target­ spheroids. Tomography. 2016;2(2):146–157. ing the tumor vasculature. mAbs. 2009;1(3):247–253. doi:10.4161/ 35. Sircar A, Chowdhury SM, Hart A, et al. Impact and intricacies of mabs.1.3.8515 bone marrow microenvironment in B-cell lymphomas: from biology 22. Donaghy H. Effects of antibody, drug and linker on the preclinical to therapy. Int J Mol Sci. 2020;21(3):904. doi:10.3390/ijms21030904 and clinical toxicities of antibody-drug conjugates. mAbs. 2016;8 36. Martinez-Veracoechea FJ, Frenkel D. Designing super selectivity in (4):659–671. doi:10.1080/19420862.2016.1156829 multivalent nano-particle binding. Proc Natl Acad Sci. 2011;108 23. Erickson HK, Lambert JM. ADME of antibody–maytansinoid con­ (27):10963–10968. doi:10.1073/pnas.1105351108 jugates. AAPS J. 2012;14(4):799–805. doi:10.1208/s12248-012- 37. Liu M, Apriceno A, Sipin M, et al. Combinatorial entropy behaviour 9386-x leads to range selective binding in ligand-receptor interactions. Nat 24. Han TH, Zhao B. Absorption, distribution, metabolism, and excretion Commun. 2020;11(1):4836. doi:10.1038/s41467-020-18603-5 considerations for the development of antibody-drug conjugates. 38. Sehn LH, Herrera AF, Flowers CR, et al. Polatuzumab vedotin in Drug Metab Dispos. 2014;42(11):1914–1920. doi:10.1124/dmd.11 relapsed or refractory diffuse large B-cell lymphoma. J Clin Oncol. 4.058586 2020;38(2):155–165. doi:10.1200/JCO.19.00172 25. Longmire M, Choyke PL, Kobayashi H. Clearance properties of 39. Malecek M-K, Watkins MP, Bartlett NL. Polatuzumab vedotin for the nano-sized particles and molecules as imaging agents: considerations treatment of adults with relapsed or refractory diffuse large B-cell and caveats. Nanomed. 2008;3(5):703–717. doi:10.2217/17435889. lymphoma. Expert Opin Biol Ther. 2020;Jun:1–9. doi:10.1080/1471 3.5.703 2598.2020.1777979 26. Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through 40. Sanchez-Trincado JL, Gomez-Perosanz M, Reche PA. Fundamentals a tragedy: mitotic catastrophe. Cell Death Differ. 2008;15(7): and methods for T- and B-cell epitope prediction. J Immunol Res. 1153–1162. doi:10.1038/cdd.2008.47 2017;2017:2680160. doi:10.1155/2017/2680160 27. Souza RD, Ayub LC, Yip K. Mitotic catastrophe. In: Apoptosis and 41. Lu D, Lu T, Gibiansky L, et al. Integrated two-analyte population Beyond. John Wiley & Sons, Ltd; 2018:475–510. pharmacokinetic model of polatuzumab vedotin in patients with non- 28. Mitchison TJ. The proliferation rate paradox in antimitotic hodgkin lymphoma. CPT Pharmacomet Syst Pharmacol. 2020;9 chemotherapy. Mol Biol Cell. 2012;23(1):1–6. doi:10.1091/mbc.e10- (1):48–59. doi:10.1002/psp4.12482 04-0335 29. Shi Y, Han Y, Yang J, et al. Clinical features and outcomes of diffuse large B-cell lymphoma based on nodal or extranodal primary sites of origin: analysis of 1,085 WHO classified cases in a single institution in China. Chin J Cancer Res. 2019;31(1):152–161. doi:10.21147/j. issn.1000-9604.2019.01.10 International Journal of Nanomedicine Dovepress Publish your work in this journal The International Journal of Nanomedicine is an international, peer- Journal Citation Reports/Science Edition, EMBase, Scopus and the reviewed journal focusing on the application of nanotechnology in Elsevier Bibliographic databases. The manuscript management system diagnostics, therapeutics, and drug delivery systems throughout the is completely online and includes a very quick and fair peer-review biomedical field. This journal is indexed on PubMed Central, system, which is all easy to use. Visit http://www.dovepress.com/ MedLine, CAS, SciSearch®, Current Contents®/Clinical Medicine, testimonials.php to read real quotes from published authors. Submit your manuscript here: https://www.dovepress.com/international-journal-of-nanomedicine-journal 1888 submit your manuscript | www.dovepress.com International Journal of Nanomedicine 2021:16 DovePress