pharmaceutics Article Intracellular Delivery of Anti-SMC2 Antibodies against Cancer Stem Cells Sara Montero 1,2,† , Joaquin Seras-Franzoso 1,† , Fernanda Andrade 1,3,4 , Francesc Martinez-Trucharte 1 , Mireia Vilar-Hernández 1 , Manuel Quesada 1 , Helena Xandri 1 , Diego Arango 5,6 , Ibane Abasolo 1,2,7, *, Diana Rafael 1,2, * and Simo Schwartz, Jr. 1,2, * 1 Drug Delivery and Targeting Group, Molecular Biology and Biochemistry Research Centre for Nanomedicine (CIBBIM-Nanomedicine), Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; sara.montero@vhir.org (S.M.); joaquin.seras@vhir.org (J.S.-F.); fernanda.silva@vhir.org (F.A.); francesc.martinez@vhir.org (F.M.-T.); mireia.vilar@alumni.vhir.org (M.V.-H.); mbenque@correo.ugr.es (M.Q.); helena.xandri@alumni.vhir.org (H.X.) 2 Networking Research Centre for Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III, 28029 Madrid, Spain 3 i3S—Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-180 Porto, Portugal 4 INEB—Instituto Nacional de Engenharia Biomédica, Universidade do Porto, Rua Alfredo Allen 208, 4200-180 Porto, Portugal 5 Biomedical Research in Digestive Tract Tumors, CIBBIM-Nanomedicine, Vall d’Hebron Institut de Recerca, Universitat Autonoma de Barcelona, 08035 Barcelona, Spain; diego.arango@vhir.org 6 Immune Regulation and Immunotherapy, CIBBIM-Nanomedicine, Vall d’Hebron Institut de Recerca, Universitat Autonoma de Barcelona, 08035 Barcelona, Spain 7 Functional Validation and Preclinical Research, Molecular Biology and Biochemistry Research Centre for Nanomedicine (CIBBIM-Nanomedicine), Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain * Correspondence: ibane.abasolo@vhir.org (I.A.); diana.fernandes_de_so@vhir.org (D.R.); simo.schwartz@vhir.org (S.S.J.); Tel.: +34-934893375 (I.A.); +34-934894056 (D.R.); +34-934894055 (S.S.J.) † These authors contributed equally to this work.  Received: 10 February 2020; Accepted: 20 February 2020; Published: 21 February 2020  Abstract: Structural maintenance of chromosomes protein 2 (SMC2) is a central component of the condensin complex involved in DNA supercoiling, an essential process for embryonic stem cell survival. SMC2 over-expression has been related with tumorigenesis and cancer malignancy and its inhibition is regarded as a potential therapeutic strategy even though no drugs are currently available. Here, we propose to inhibit SMC2 by intracellular delivery of specific antibodies against the SMC2 protein. This strategy aims to reduce cancer malignancy by targeting cancer stem cells (CSC), the tumoral subpopulation responsible of tumor recurrence and metastasis. In order to prevent degradation and improve cellular internalization, anti-SMC2 antibodies (Ab-SMC2) were delivered by polymeric micelles (PM) based on Pluronic® F127 amphiphilic polymers. Importantly, scaffolding the Ab-SMC2 onto nanoparticles allowed its cellular internalization and highly increased its efficacy in terms of cytotoxicity and inhibition of tumorsphere formation in MDA-MB-231 and HCT116 breast and colon cancer cell lines, respectively. Moreover, in the case of the HCT116 cell line G1, cell-cycle arrest was also observed. In contrast, no effects from free Ab-SMC2 were detected in any case. Further, combination therapy of anti-SMC2 micelles with paclitaxel (PTX) and 5-Fluorouracil (5-FU) was also explored. For this, PTX and 5-FU were respectively loaded into an anti-SMC2 decorated PM. The efficacy of both encapsulated drugs was higher than their free forms in both the HCT116 and MDA-MB-231 cell lines. Remarkably, micelles loaded with Ab-SMC2 and PTX showed the highest efficacy in terms of inhibition of tumorsphere formation in HCT116 cells. Accordingly, our data clearly suggest an effective intracellular release of antibodies targeting SMC2 in these cell models and, further, strong cytotoxicity against CSC, alone and in combined treatments with Standard-of-Care drugs. Pharmaceutics 2020, 12, 185; doi:10.3390/pharmaceutics12020185 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 185 2 of 20 Keywords: SMC2; condensin complexes; nanomedicine; polymeric micelles; cancer stem cells; 5-FU; PTX; breast cancer; colon cancer; antibody intracellular delivery 1. Introduction Protein therapy has emerged as a promising therapeutic alternative due to its high specificity and reduced off-target effects [1]. Among protein-based therapies, the use of antibodies have been largely explored and numerous treatments are currently available for a broad spectrum of clinical indications, from transplant rejection to cancer [2]. One of the most interesting features of antibody-based therapies is their potential to reach a wide range of targets, including those that are usually unattainable by other strategies (i.e., undruggable targets). However, because most antibodies are unable to easily cross the extracellular membrane, their applications are often limited to extracellular targets [3,4]. In the last years, several research groups have explored the use of antibodies against intracellular targets. Straightforward techniques for antibody delivery, such as microinjection, electroporation and antibody modification with cell penetration peptides (CPPs), have been tested. Unfortunately, even though experimentally effective in vitro, they have not been efficiently translated to in vivo models or into clinical applications [5,6]. In addition, although different nanoparticles, mainly metallic and lipidic, have been developed for intracellular delivery of antibodies, only few of them showed efficient intracellular delivery [7,8]. The current drawbacks are related with limited cargo for hydrophilic substances, un-adequate endosomal escape, in vivo instability and a short half-life circulation time. Thus, intracellular delivery of antibodies is still a considerable challenge [9,10]. On the other hand, although extensively investigated and besides constant development of new therapies, cancer often remains as an incurable disease. Insights into cancer physiology have shown the complexity of cancer regulatory networks in which a large variety of different cell types are involved and largely interconnected to determine disease progression. In this regard, particular attention should be addressed to metastatic spreading, resistance to treatment and tumor recurrence. These features are related with the presence of a cancer cell subpopulation known as cancer stem cells (CSC). CSC display stemness properties, such as self-renewal, quiescence and the capability to remain in an undifferentiated state [11,12]. Besides, CSC have been shown to exhibit high expression levels of drug efflux transporters and unregulated DNA repair machinery, conferring them drug resistance potential. In this context, CSC have become a highly appealing target in order to improve current anti-cancer therapies [13,14]. Although the number of new drugs with therapeutic potential against CSC is rising and several products are already in clinical trials, there is still strong need to broaden the catalogue of therapeutic options. In this sense, the Structural Maintenance of Chromosomes 2 (SMC2) protein could be a good therapeutic target candidate. SMC2 forms part of the condensin complex that has a central role in many aspects of chromosome biology, including the segregation of sister chromatids and compaction of chromosomes during cell division, as well as the regulation of gene expression during the interphase [15–18]. Importantly, SMC2 protein has been mostly located at the cell cytoplasm during cell interphase with only a minor amount remaining associated to chromatin in the nucleus. The condensin complex is formed by five subunits and requires the initial arrangement of a SMC2/SMC4 heterodimer. The condensin complex is completed by the union of a subcomplex, formed by the three non-SMC subunits (Cnd1, Cnd2 and Cnd3), to the heterodimer of SMC2/SMC4 [15–17]. Of note, SMC2 has been found to be over-expressed in a significant number of patients with colorectal cancer, gastric cancer, lymphoma and some types of neuroblastoma [19,20], and has been suggested as a risk biomarker in pancreatic cancers [21]. Moreover, when SMC2 expression was knocked down it drastically reduced tumor growth in colorectal cancer mice models [18]. This data and the central role of the condensin complex in cell division suggested that an effective inhibition of the activity of this complex would Pharmaceutics 2020, 12, 185 3 of 20 prevent cancer cells from dividing. Under this premise, directing SMC2-targeted inhibitory molecules into the cell cytoplasm should ensure SMC2 protein blockade and serve as a novel therapeutic strategy. Hereby we propose a polymeric micelle delivery system (PM) based on amphiphilic polymers, namely the Pluronic® F127 as a nanoplatform to cluster the antibodies directed against the SMC2 protein. Because of its small size, biocompatibility, stealth properties and amphiphilic nature, this is an ideal system to overcome major drawbacks related to intracellular delivery of antibodies. Furthermore, poloxamer polymers like Pluronic® F127 have shown to interfere with the function of P-glycoprotein, being therefore helpful to further overcome drug resistance and reduce cancer recurrence [22–24]. Moreover, taking advantage of the plasticity provided by amphiphilic PM we have also explored potential combinatorial therapeutic strategies with 5-FU and PTX, drugs commonly used in colon and breast cancer, respectively. 2. Materials and Methods 2.1. Cell Lines and Culture Conditions The HCT116 (ATCC® CCL-247™) colon cancer cell line, MDA-MB-231 (ATCC® HTB-26™) breast cancer cell line and PANC-1 (ATCC® CRL-1469™) pancreas cancer cell line were obtained from American Type Culture Collection (ATTC® , LGC Standards, Barcelona, Spain). Cells were cultured in RPMI medium (ThermoFisher Scientific, Madrid, Spain supplemented with 10% Fetal Bovine Serum (FBS) (ThermoFisher Scientific, Madrid, Spain), a 1% penicillin–streptomycin mixture (ThermoFisher Scientific, Madrid, Spain), 1% L-glutamine (Lonza, Basel, Switzerland) and 1% Non-Essential Amino Acids (NEAA) (ThermoFisher Scientific, Madrid, Spain). All cell lines were maintained at 37 ◦ C under a 5% CO2 -saturated atmosphere. The medium was changed every two days, and upon sub-confluence, cells were harvested from plates with 0.25% trypsin-EDTA and subcultured (ThermoFisher Scientific, Madrid, Spain). Cells were also cultured in low attachment conditions as tumorspheres in serum-free media and seeded in ultra-low attachment surface plates (Corning Life Sciences, Chorges, France). The medium for breast and pancreas sphere growth was supplemented with glucose, 60 mg/mL (Merck Life Science S.L.U., Madrid, Spain), as well as 10 µL/mL L-Glutamax (Merck Life Science S.L.U., Madrid, Spain), 10 µL/mL antibiotic–antimitotic mixture (Merck Life Science S.L.U., Madrid, Spain), 4 µg/mL heparin (Merck Life Science S.L.U., Madrid, Spain), 2 mg/mL BSA (Merck Life Science S.L.U., Madrid, Spain), 0.02 µg/mL of human recombinant EGF (Merck Life Science S.L.U., Madrid, Spain), 0.01 µg/mL of human recombinant bFGF (ThermoFisher Scientific, Madrid, Spain) and 10% of Hormone Mix: 10 µg/mL putrescin, 20 µM progesterone (both from Merck Life Science S.L.U., Madrid, Spain), 0.1 mg/mL apo-transferrin, 25 µg/mL insulin and 30 µM selen (ThermoFisher Scientific, Madrid, Spain). Tumorspheres from the HCT116 cell line were also cultured in serum-free media supplemented with a 1% antibiotic–antimitotic mixture, 10 ng/mL of human recombinant EGF, 20 ng/mL of human recombinant bFGF and 10 ng/mL of LIF Recombinant Human Protein (TermoFisher Scientific, Madrid, Spain). 2.2. Phase Contrast Microscopy The morphology and structure of adherent cells and spheres were routinely assessed by phase contrast microscopy imaging after treatment with the distinct compounds. Images were acquired at 10× amplification using a Nikon Eclipse TS100 (Nikon Instruments Europe BV, Amsterdam, Netherlands) inverted microscope and further edited with ImageJ 1.49v software. Pharmaceutics 2020, 12, 185 4 of 20 2.3. Cell Transfection with siRNA 2.3.1. Gene Expression Determination 105 cells were seeded in 6-well plates and transfected with SMC2-siRNA (SI02654260, Qiagen, Hilden, Germany) using Lipofectamine® 2000 (ThermoFisher Scientific, Madrid, Spain) according to the manufacturer’s instructions. A nonspecific sequence was used as the control (siControl). The culture medium was changed 6 h after transfection and cells harvested after 72 h of incubation. Samples were further processed for RNA extraction as detailed below in Section 2.4. 2.3.2. Cell Growth Assay After cell transfection, cells were washed in PBS and harvested by trypsin digestion following standard procedures. Cells were counted in a CountessTM II (ThermoFisher Scientific, Madrid, Spain). 2.3.3. Sphere Formation Assay SMC2 knock-down was performed following a 2-times siRNA shot strategy in order to guarantee an efficient gene expression inhibition along the assay. Briefly, a first treatment with SMC2-siRNA using Lipofectamine® 2000 (ThermoFisher Scientific, Madrid, Spain) was performed as described above. After 72 h an identical SMC-siRNA shot was performed. After 6 h upon the second siRNA transfection, cells were harvested and washed 3 times in serum free media. Then, 1000 cells were seeded in ultra-low attachment 96-well plates, adding sphere growth media for assessing sphere formation (as described in the cell viability assay Section 2.10.1). 2.4. RNA Extraction and Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Total RNA was extracted from cells using RNeasy Micro Kit (Qiagen, Hilden, Germany), and the obtained RNA was reverse transcribed with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Madrid, Spain), according to the manufacturer’s instructions. The cDNA reverse transcription product was amplified with specific primers for SMC2 (hSMC2 F: 50 AAT GAG CTG CGG GCT CTA GA 30 ; hSMC2 R: 50 TTG TTG CTT GTG ATA TGA GCT TTG 30 ); GADPH (hGADPH F: 50 ACC CAC TCC TCC ACC TTT GAC; hGADPH R: 50 CAT ACC AGG AAA TGA GCT TGA CAA 30 ) and Actin (hActin F: 50 CAT CCA CGA AAC TAC CTT CAA CTC C 30 ; hActin R: 50 GAG CCG CCG ATC CAC AC 30 ) by qPCR using SYBR Green (ThermoFisher Scientific, Madrid, Spain) to fluorescently label double stranded DNA. The reaction was performed on a 7500 Real time PCR system (Applied Biosystems, Madrid, Spain). At least three biological replicates, each comprising two technical replicates, were performed. Relative normalized quantities (NRQ) of mRNA expression were calculated using the comparative Ct method (2e-∆∆Ct) with two reference genes (hGAPDH and hActin) used as endogenous controls through Qbase™ software v3.2. 2.5. Protein Extraction and Western Blotting Cell pellets were lysed with the Cell Lytic M reagent (Merck Life Science S.L.U., Madrid, Spain) containing a protease inhibitor cocktail (Roche cOmplete™, Merck Life Science S.L.U., Madrid, Spain). Proteins in the crude lysates were quantified using the PierceTM BCA protein quantification kit (ThermoFisher Scientific, Madrid, Spain) following the manufacturer’s instructions. A total of 20 µg of whole-cell lysates were separated by SDS-PAGE and transferred onto PVDF membranes. Blots were probed using primary antibodies anti-SMC2 (Merck Life Science S.L.U., Madrid, Spain) and β-Tubulin (Invitrogen, ThermoFisher Scientific, Madrid, Spain). Proteins were detected using HRP-conjugated secondary antibodies (Dako, Palex Medical SA, Barcelona, Spain) and developed after appropriate incubation in an Immobilion® Western reagent (Merck Life Science S.L.U., Madrid, Spain) using an Odissey FC imaging system (LI-COR Biotechnology GmbH, Bad Homburg, Germany) for chemiluminescence detection. Pharmaceutics 2020, 12, 185 5 of 20 2.6. Production of Polymeric Micelles Pluronic® F127 was kindly provided by BASF (Ludwigshafen, Germany). PM were prepared using the thin-film hydration technique [22]. Pluronic® F127 carboxylation was performed as previously reported [25]. Briefly, F127 and F127:COOH polymers were individually weighed in an 8:2 (w/w) ratio and dissolved in a mixture of methanol:ethanol (1:1) (Merck Life Science S.L.U., Madrid, Spain). For the formation of loaded micelles, the drug, 5-FU or PTX (both from Merck Life Science S.L.U., Madrid, Spain) were added to the organic solution at the desired concentration. Then, the solvent was removed under vacuum in a rotary evaporator (bath temperature 60 ◦ C, 200 rpm), and the resulting film left to dry overnight at room temperature to eliminate any remaining solvent. The film was then hydrated with PBS and vortexed for 5 min at full speed. For the PM encapsulating SMC2 antibodies (PM:SMC2), the antibody (Anti-SMC2/hCAP-E Antibody, Merck Life Science S.L.U., Madrid, Spain) was added at the aqueous phase during the rehydration step. For PM functionalized with SMC2 antibodies (PM-CON:SMC2), an adequate amount of EDC (polymer:EDC ratio 1:1.5) (Merck Life Science S.L.U., Madrid, Spain) was incubated with the formulation during 30 min at RT. Afterwards, the SMC2 antibody solution was added and incubated under stirring during 2 h at RT. Samples were freeze-dried for long-term storage using a VirTisBenchTop Freeze-Dryer (SP Scientific, Ipswich, UK) when required. 2.7. Physicochemical Characterization of the Polymeric Micelles 2.7.1. Zeta Potential Measurements Zeta potential was assessed by laser Doppler microelectrophoresis using a NanoZS (Malvern Instruments, Malvern, UK) with an angle of 173◦ . Samples were diluted 1:5 in Milli-Q® water (18.2 MΩ·cm at 25 ◦ C) in order to obtain an adequate nanoparticle concentration. Data obtained from each formulation were represented as mean values and measured at least in triplicate. 2.7.2. Transmission Electron Microscopy (TEM) Particle shape and morphology were observed by TEM, using a JEM-1400 Electron Microscope (JEOL Ltd., Croissy-sur-Seine, France) with an applied voltage of 120 kV. For that, a drop of sample (previously diluted 1:20 in Milli-Q® water) was placed on a carbon 400-mesh copper grid, the liquid excess removed, and the sample contrasted with uranyl acetate before visualization. Image J 1.49v software was used to process information and determine diameter measures (n > 200) from TEM images, while histogram plots from nanoparticles size distribution were generated by GraphPad Prism 6. The dispersion index (d) was determined by Equation (1). Standard Deviation (SD)/Particle Size Arithmetic Mean (1) 2.7.3. Loading/Association Efficiency Determination The efficacy of SMC2 loading in the case of PM:SMC2 and association efficiency in the case of PM-CON:SMC2 was assessed by BCA protein assay. Briefly, the amount of free SMC2 antibody in the aqueous phase of the PM was separated by centrifugation with filtration (10,000 g, 10 min at RT) using 300k membrane centrifugal devices (Nanosep® Centrifugal Devices, Pall España, Madrid, Spain). Then, the protein level was measured from the flow using a PierceTM BCA protein assay kit (ThermoFisher Scientific, Madrid, Spain). The Loading/Association efficiency percentage was determined according to Equation (2): Total amount of SMC2 (unfiltered PM) − free SMC2 in filtrate/Total amount of SMC2 (2) (unfiltered PM) × 100 Pharmaceutics 2020, 12, 185 6 of 20 2.7.4. Fourier-Transform Infrared Spectroscopy (FTIR) The conjugation of the SMC2 antibody at the PM surface was also confirmed by FTIR analysis. FTIR was carried out using a spectrometer Perkin-Elmer Spectrum One (energy range: 450–4000 cm−1 ) equipped with a Universal Attenuated Total Reflectance Accessory (U-ATR, Perkin-Elmer, Madrid, Spain). Prior to analysis, samples were freeze-dried using a VirTisBenchTop Freeze-Dryer (SP Scientific, Ipswich, UK). 2.8. Micelles Internalization: Flow Cytometry and Confocal Microscopy Cell internalization of the different formulations was assessed in HCT116 and MDA-MB-231 using 5-DTAF (Merck Life Science S.L.U., Madrid, Spain) fluorescently labeled PM [25,26]. 2.8.1. Flow Cytometry Briefly, 20,000 cells were seeded in complete RPMI medium in 96-well plates and left to attach for 24 h. PM were added to cells (10 mg/mL) and incubated for 15, 30, 60, 180, 360 and 1440 min, respectively. Then, cells were washed with 1× PBS, trypzinized and neutralized with PBS supplemented with 10% FBS and 1 µg/mL DAPI (Merck Life Science S.L.U., Madrid, Spain) used for vital staining. The plate was read in a cytometer Fortessa (BD Biosciences, Madrid, Spain) and data was analyzed with FCS Express 4 Flow Research Edition software (De Novo Software, Los Angeles, USA, Version 4). Three biological replicated were performed for each condition and only DAPI negative cells were admitted for the analysis, while cell debris or possible aggregates were removed by forward and side scatter gating. For each sample, at least 10,000 individual cells were collected, and the percentage of fluorescent cells evaluated. 2.8.2. Confocal Microscopy A total of 50,000 cells were seeded in complete RPMI medium in 8-well chambered coverglass (ThermoFisher Scientific, Madrid, Spain) and incubated overnight at 37 ◦ C and 5% CO2 to allow cell adhesion. Cells were incubated with 5-DTAF-fluorescently labelled PM (10 mg/mL) for 6 h. Lysosomes were stained with 1 µM Lysotracker® Red DND-99 (ThermoFisher Scientific Madrid, Spain) for 30 min at 37 ◦ C while the cell membrane was stained with 5 µg/mL CellMask™ (Invitrogen, ThermoFisher Scientific, Madrid, Spain) for 15 min at 37 ◦ C. Subsequently, cells were fixed in 4% PFA (Merck Life Science S.L.U., Madrid, Spain) at 4 ◦ C for 20 min followed by nuclei staining with DAPI (1 µg/mL) for 5 min at RT in the dark. Cells were viewed under a Spectral Confocal Microscope MFV1000 Olympus (Olympus Iberia, S.A.U., L’Hospitalet de Llobregat, Spain). The 561 nm excitation wavelength of the green laser (10 mW) was used for selective detection of the red fluorochromes (Lysotracker® Red and CellMask™). The 488 nm excitation wavelength of Argon multiline laser (40 mW) was used for selective detection of the green fluorochrome (5-DTAF). The nuclear staining DAPI was excited at 405 nm with a violet laser (6 mW). Minimal single optical sections were collected for each fluorochrome sequentially. Images were merged and analyzed with Image J 1.49v software. 2.9. Cell Cycle Assay by Flow Cytometry A flow cytometric analysis of cell cycle with propidium iodide DNA staining was carried out. Briefly, 40,000 cells of HCT116 cells and 30,000 cells of MDA-MB-231 cells were seeded in 24-well plates and left to adhere during 24 h at 37 ◦ C and a 5% CO2 -saturated atmosphere. Then, cells were treated and incubated with empty PM (control PM) (5 mg/mL) and PM-CON:SMC2 (5 mg/mL polymer and 32.9 µg/mL of SMC2 antibody) for 48 h. Untreated cells were used as the negative control. Afterwards, cells were harvested, centrifuged (8000 rpm, 5 min at 4 ◦ C) and fixed in cold 70% ethanol for 30 min at 4 ◦ C. After centrifugation, cells were washed two times with PBS and the cell pellets resuspended in 250 µL of the staining solution containing 100 µg/mL of ribonuclease PureLink™ RNase A (ThermoFisher Pharmaceutics 2020, 12, 185 7 of 20 Scientific, Madrid, Spain) and 50 µg/mL of propidium iodide (Merck Life Science S.L.U., Madrid, Spain) in order to ensure that only DNA, not RNA, was stained. Cell cycle evaluation was performed using an FACS Calibur (BD Biosciences, Madrid, Spain) flow cytometer and the resulting histograms analysed with FCS Express 4 Flow Research Edition software (De Novo Software, Version 4). Three biological replicates were performed and at least 5000 individual cells were collected for the analysis once debris were removed by forward and side scatter gating. The multicycle option in autofit mode was performed in order to automatically quantify the percentage of cells in each cell cycle phase. By this method a Gaussian curve was fitted to each phase allowing the precise determination of the area under the curve. In addition, the G2/G1 ratio was also represented, scoring around 2 in all the cases, in compliance with the accepted quality standards for cell-cycle assays. 2.10. Tumor Cell Viability Assays 2.10.1. Viability Assay in Adherent Cell Cultures 3000 cells of the HCT116 cell line and 5000 cells of the MDA-MB-231 cell line were seeded in 96-well plates and incubated in overnight to allow adhesion. Then, cells were treated and incubated during 72 h with crescent concentrations of the different formulations (PM, PM:SMC2 and PM-CON:SMC2) at 5 mg/mL of polymer and 32.9 µg/mL of SMC2 antibody. To determine the cytotoxicity of free 5-FU and free PTX (Merck Life Science S.L.U., Madrid, Spain), cells were incubated during 48 h with a range concentration of 5-FU and PTX of 384.40 µM to 0.023 µM and 1 µM to 0.0078 µM, respectively. Complete medium was used as negative control and 10% DMSO as positive control of toxicity. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Merck Life Science S.L.U., Madrid, Spain). The absorbance of each well was read on an absorbance microplate reader ELx800 (BioTek, Colmar, France), at 590 and 630 nm for 5-FU and PTX, respectively. The half-maximal inhibitory concentration (IC50 ) was determined by nonlinear regression of the concentration-effect curve fit using Prism 6.02 software (GraphPad Software, Inc.). 2.10.2. Tumorsphere Viability Assays Studies were performed using ultra-low attachment surface plates (Corning Life Sciences, Chorges, France). Cells were cultured in serum free media supplemented differently for HCT116 colon cell line and MDA-MB-231 breast cancer cell line, as mentioned above. HCT116, MDA-MB-231 and Panc-1 cells were transfected with siRNA anti-SMC2, as previously described. A total of 1000 cells were seeded and cultured for 6 days in ultra-low attachment plates. For PM and drug efficacy assays, 1000 cells from the HCT116 cell line and 2000 cells from the MDA-MB-231 cell line were seeded in 96-well ultra-low attachment plates in serum-free media, supplemented as described above. After overnight incubation, cells were treated with different PM formulations (PM, PM-CON:SMC2, PM/5-FU, PM-PTX, PM-CON:SMC2/5-FU, PM-CON:SMC2/PTX) (1 mg/mL of polymer), the free drugs (5-FU, 3.84 µM and PTX, 0.1 µM) and free SMC2 antibody (6.58 µg/mL) for 7 days. The IC50 of the free tested drugs were also determined in low attachment conditions. Cells were treated with serial 1:2 dilutions of 5-FU (from 768.80 µM to 0.047 µM) and PTX (from 10 µM to 0.078 µM) for 7 days. Free serum medium was used as negative control and 10% DMSO as positive control of toxicity. In all cases, sphere formation was monitored with a Nikon Eclipse TS100 inverted microscope (Nikon Instruments Europe BV, Amsterdam, Netherlands) and quantified by Presto Blue® Reagent assay (ThermoFisher Scientifics, Madrid, Spain). The absorbance of each well was read on a MultiskanTM FC Microplate Photomer (ThermoFisher Scientific, Madrid, Spain), at 570 and 620 nm, and the data were processed by GraphPad Prism 6 software. 2.11. Statistical Analysis At least three batches of each PM were produced and characterized. The results were expressed as the mean ± standard deviation. For biological studies, at least 3 replicates, each involving at least Pharmaceutics 2020, 12, 185 8 of 20 Pharmaceutics 2020, 12, x 8 of 20 two technical replicates, were involved in the final results expressed as the mean ± standard deviation. Statistical analysis was performed in GraphPad Prism 6 software using a non-parametric Dunn test for standard deviation. Statistical analysis was performed in GraphPad Prism 6 software using a non- multiple comparisons and Mann–Whitney U test for simple comparisons. Differences were regarded parametric Dunn test for multiple comparisons and Mann–Whitney U test for simple comparisons. as statistically significant when the p-value was smaller than 0.05. Differences were regarded as statistically significant when the p-value was smaller than 0.05. 3. Results 3. Results 3.1. SMC2 Silencing Causes Strong Reduction of Tumorsphere Formation in Cancer Cells 3.1. SMC2 Silencing Causes Strong Reduction of Tumorsphere Formation in Cancer Cells SMC2 siRNA efficiently reduces the expression of the gene at mRNA and protein level in the three SMC2cell different siRNA lines.efficiently reduces the Namely, colorectal expression cancer cell lineofHCT116, the genetriple at mRNA andbreast negative protein levelcell cancer in the line three different cell MDA-MB-231 andlines. Namely, pancreatic colorectal cancer cell linecancer PANC-1 cell(Figure line HCT116, 1a,b and triple negative Figure S1a,b).breast cancer Interestingly, cell the line effectMDA-MB-231 of SMC2 silencingand pancreatic resulted in cancer a clear cell line PANC-1 decrease (Figurein 1a,b in cell viability and whereas HCT116, Figure S1a,b). only a Interestingly, the effect of SMC2 silencing resulted in a clear decrease trend to reduce the number of cells was observed for MDA-MB-231 (Figure 1c); no difference in cell viability in HCT116, could be whereas detectedonly a trend in other tumorto reduce the number types tested as in theof case cells of was observed PANC-1 for MDA-MB-231 (Figure (Figure S1c). Importantly, when1c);cells no difference could be detected in other tumor types tested as in the case of PANC-1 were serum deprived and forced to grow in absence of cell-surface contacts, a remarkable decrease of (Figure S1c). Importantly, tumorspheres when cells were proliferation wasserum observeddeprived andlines in all cell forced to grow treated with SMC2in absence siRNAof cell-surface (Figure 1d and contacts, a remarkable decrease of tumorspheres proliferation was observed in all Figure S1d). Of note, as previously reported by our group and others [27,28], cells growing as spheres cell lines treated with SMC2 display siRNAfeatures CSC-like (Figure(i.e., 1d and HCT116Figure cellS1d). line; Of note,S2). Figure as previously reported by our group and others [27,28], cells growing as spheres display CSC-like features (i.e., HCT116 cell line; Figure S2). Figure 1. SMC2 siRNA inhibition. (a) Relative mRNA levels after SMC2 silencing for HCT116 and Figure 1. SMC2cell MDA-MB231 siRNA lines inhibition. (a) Relative (siSMC2). Fold changemRNA levels after is represented withSMC2 respectsilencing to mRNA forlevels HCT116 and obtained MDA-MB231 cell lines (siSMC2). Fold change is represented with respect to mRNA from non-relevant siRNA-treated cells (siControl); (b) SMC2 and beta tubulin proteins detected by levels obtained from Westernnon-relevant blot aftersiRNA-treated SMC2 silencing cells (siControl); (siSMC2) (b) SMC2 and and compared beta tubulinsiRNA-treated to non-relevant proteins detected cellsbyC; Western blot after (c) cell viability SMC2 assay silencingand for HCT116 (siSMC2) and compared MDA-MB-231 adherenttocells non-relevant siRNA-treated after treatment with siRNAcells C; against (c) cell (siSMC2) SMC2 viability and assay foraHCT116 with scrambled and MDA-MB-231 control adherentand siRNA (siControl); cells (d) after treatment cell viability with assay siRNA for HCT116 against SMC2 (siSMC2) and MDA-MB-231 and with growing a scrambledconditions in non-adherent control siRNA (siControl); and as tumorspheres, after(d) cell viability treatment assay with siRNA for against SMC2 (siSMC2) and with a scramble control siRNA (siControl). * p < 0.05, *** p < 0.001. after HCT116 and MDA-MB-231 growing in non-adherent conditions as tumorspheres, treatment with siRNA against SMC2 (siSMC2) and with a scramble control siRNA (siControl). * p < 3.2. 0.05, Physicochemical *** p < 0.001. Characterization of Polymeric Micelles with Conjugated or Encapsulated SMC2 Antibodies 3.2. Physicochemical Characterization In order to develop of Polymeric a drug delivery systemMicelles able to with Conjugated target or Encapsulated SMC2 protein SMC2 intracellularly, anti-SMC2 Antibodies antibodies (Ab-SMC2) were successfully conjugated onto PM using two different approaches: (1) encapsulation In order toby affinityainto develop drugthedelivery PM hydrophilic shellto(PM:SMC2) system able target SMC2 and (2) by intracellularly, protein covalent conjugation anti- between the –COOH terminals of the PM and the -NH2 groups present SMC2 antibodies (Ab-SMC2) were successfully conjugated onto PM using two different in Ab-SMC2 (PM-CON:SMC2). Particles TEM approaches: 1) analysis revealedby encapsulation spherical affinitymorphologies into the PMinhydrophilic all cases andshell displayed rather homogenous (PM:SMC2) and 2) by populations covalent in size (Figure conjugation between 2b). theThe PM obtained –COOH terminalsby of thethe first PM approach and the presented -NH2 groupsa mean diameter present in Ab-of SMC2 (PM-CON:SMC2). Particles TEM analysis revealed spherical morphologies in all cases and displayed rather homogenous populations in size (Figure 2b). The PM obtained by the first approach presented a mean diameter of 35.99 ± 0.39 nm with a dispersity index of 0.16, and a Pharmaceutics 2020, 12, 185 9 of 20 Pharmaceutics 2020, 12, x 9 of 20 35.99 ± 0.39 nm with a dispersity index of 0.16, and a slightly negative superficial charge measured byslightly NanoZSnegative superficial (Figure 2c). chargeproduced The micelles measuredaccording by NanoZS (Figure to the 2c). second The micelles approach produced presented a mean according to the second approach presented a mean diameter of (35.04 ± 0.42 nm), a d value of 0.18 diameter of (35.04 ± 0.42 nm), a d value of 0.18 and are faintly negatively charged (Figure 2c). and are faintly negatively charged (Figure 2c). Figure Figure 2. PM-Ab-SMC2 2. PM-Ab-SMC2 characterization. characterization. (a) Schematic (a) Schematic representation representation of the of twothe two strategies strategies employed foremployed for PM PM synthesis synthesis loaded with loaded withantibody, anti SMC2 anti SMC2namely antibody, namely encapsulation encapsulation and surfaceand surface conjugation. (b)conjugation. (b) TEMofmicrographs TEM micrographs of the PM the four distinct fourformulations. distinct PM formulations. The panels The abovepanels above the the micrographs micrographs correspond correspond to the to the size size distribution distribution of the of the particles particles obtained by TEM obtained by TEM image analysis. image Scale baranalysis. represents Scale bar represents 200 nm. (c) Summary table of mean diameter (Md), Dispersity 200 nm. (c) Summary table of mean diameter (Md), Dispersity Index (d), Zeta Potential (ZP) Index (d), Zeta and Potential (ZP) and (d) FTIR spectra, displaying the appearance−1 of a peak at 1646 cm −1 and (d) FTIR spectra, displaying the appearance of a peak at 1646 cm and corresponding to amide I corresponding bond formation. to amide I bond formation. Pharmaceutics 2020, 12, 185 10 of 20 Regarding the antibody loading/association efficiency, it was determined by the measurement of free antibody in the micelle’s supernatant after 300 KDa filtration. PM:SMC2 presented an SMC2 loading efficiency of 68.02% ± 1.01% and PM-CON:SMC2 showed an association efficiency of 61.49% ± 3.11%. Moreover, the loading/association of Ab-SMC2 at the micelles surface were confirmed by FTIR (Figure 2d). Regarding FTIR analysis, control PM-COOH showed a peak at 1727 cm−1 corresponding to the –C=O stretching of the –COOH groups of the carboxylated Pluronic® F127. As expected, this peak is absent in control PM composed only by unmodified Pluronic® F127 [25]. Ab-SMC2 showed a peak at 1654 cm−1 corresponding to the C=O stretching vibrations of the peptide bond of the amide I and also a peak at 1545 cm−1 related to the N–H bending vibration/C–N stretching vibration of the amide II [29]. In the case of PM:SMC2, it was possible to observe a small shift of the peak corresponding to the amide I of Ab-SMC2 from 1654 to 1648 cm−1 , possibly due to non-covalent interactions/hydrogen bonding between the protein and the Pluronic® F127 [30]. In the case of PM-CON:SMC2, a small shift together with an increase in the intensity of the peak of the amide I (1646 cm−1 ) were detected, probably due to the conjugation of the –NH2 of Ab-SMC2 with the –COOH from carboxylated Pluronic® F127 and the formation of new bonds. A small peak shift (1726 cm−1 ) accompanied by a drop of intensity of the –COOH peak also suggested a reduction of the availability of these free groups due to conjugation with –NH2. Altogether, these results suggest a covalent conjugation of Ab-SMC2 to the surface of the PM. In order to better confirm the peaks, PM-CON:SMC2 were produced with 100% of carboxylated Pluronic® F127 (Figure S3). A peak at 1728 cm−1 , corresponding to the –C=O stretching of the –COOH groups, and a peak at 1653 cm−1 , corresponding to the C=O stretching vibrations of the peptide bond of the amide I, were observed. The lower shift of the amide I peak could be a result of the absence of the –OH groups from the unmodified Pluronic® F127 and, consequently, lower hydrogen bonding interactions. 3.3. In Vitro Efficacy of PM-SMC2 and PM-CON:SMC2 in Adherent Cultures Cell viability assays were performed in HCT116 colon and MDA-MB-231 breast cancer cells. PM-SMC2 and PM-CON:SMC2 formulations were compared with the free SMC2 antibody and control PM. As expected, control PM and the free antibody did not show cell cytotoxicity at the maximum tested dose, namely 32.9 µg/mL for Ab-SMC2 and 5 mg/mL for Pluronic® 127 (Figure 3a,b). On the other hand, the two formulations presented a significant cytotoxic effect in both cell lines at 72 h of incubation (Figure 3b). Nonetheless, PM-CON:SMC2 displayed a slight advantage in terms of effectiveness comparing to PM:SMC2. Thus, we decided to continue with the conjugated formulation of Ab-SMC2. Further, we analyzed whether PM-CON:SMC2 might also cause changes in cell morphology and cell distribution in HCT116 and MDA-MB-231 models. Our data show a dramatic change in cell morphology in HCT116 cells. Cells treated with PM-CON:SMC2 showed a highly stretched shape and formed significantly less cell clusters than free Ab-SMC2 and empty PM (control PM). For fibroblast-shaped MDA-MB-231 cultures, cells treated with PM-CON:SMC2 displayed similar morphology and distribution than controls. Interestingly, a significant number of vacuoles were observed in samples incubated with PM-CON:SMC2 whereas no such structures were detected with free Ab-SMC2 and control PM (Figure 3a). These results show a biological activity of Ab-SMC2 when administered in PM that is not observed when PM are not employed. Pharmaceutics 2020, 12, 185 11 of 20 Pharmaceutics 2020, 12, x 11 of 20 Figure 3. In vitro efficacy of Ab-SMC2 in tumor cell lines. (a) Phase contrast images of HCT116 (above Figure 3. In vitro efficacy of Ab-SMC2 in tumor cell lines. (a) Phase contrast images of HCT116 panel) and MDA-MB-231 (below) after 48 h incubation with free SMC2 antibody, control PM and (above panel) and MDA-MB-231 (below) after 48 h incubation with free SMC2 antibody, control PM PM-CON:SMC2. Red arrows point at vacuoles appeared after PM-CON:SMC2 treatment. Scale bar and PM-CON:SMC2. Red arrows point at vacuoles appeared after PM-CON:SMC2 treatment. Scale correspond to 100 µm; (b) cell viability representation of HCT116 (above panel) and MDA-MB-231 bar correspond to 100 µ m; (b) cell viability representation of HCT116 (above panel) and MDA-MB- (below) after 72 231 (below) h of72incubation after with anti h of incubation SMC2 with antiantibody, control PM, SMC2 antibody, PM:SMC2 control PM, and PM-CON:SMC2. PM:SMC2 and PM- The concentrations of the antibody andantibody ® Pluronic and127Pluronic were 32.9 CON:SMC2. The concentrations of the ® 127 wereand µg/mL 32.95µmg/mL, respectively. g/mL and 5 mg/mL, These concentrations respectively. were also maintained These concentrations were alsoinmaintained both formulations: PM:SMC2 and in both formulations: PM-CON:SMC2. PM:SMC2 and PM- ** p < 0.01, *** p < 0.001. CON:SMC2. ** p < 0.01, *** p < 0.001. 3.4. PM-CON:SMC2 Micelles Show Faster Cellular Uptake than Control PM 3.4. PM-CON:SMC2 Micelles Show Faster Cellular Uptake than Control PM Cellular internalization and intracellular localization assessment of PM decorated with Ab-SMC2 Cellular internalization and intracellular localization assessment of PM decorated with Ab- were carried out at several time-points by flow cytometry. Accordingly, 5-DTAF fluorescently labeled SMC2 were carried out at several time-points by flow cytometry. Accordingly, 5-DTAF PM-CON:SMC2 were incubated with HCT116 and MDA-MB-231 cells. Figure 4a shows that the fluorescently labeled PM-CON:SMC2 were incubated with HCT116 and MDA-MB-231 cells. Figure conjugated nanoparticle (PM-CON:SMC2) presented a faster uptake profile than PM in both cell lines. 4a shows that the conjugated nanoparticle (PM-CON:SMC2) presented a faster uptake profile than Further, we could also observe that the MDA-MB-231 cell line exhibited faster uptake profiles than PM in both cell lines. Further, we could also observe that the MDA-MB-231 cell line exhibited faster HCT116 cells, which indicates that internalization efficiency is largely dependent on the cell type. uptake profiles than HCT116 cells, which indicates that internalization efficiency is largely Fluorescently labelled PM were also employed for confocal analysis, after 6 h of incubation with dependent on the cell type. HCT116 and MDA-MB-231 cells. Acidic vesicles were labelled with Lysotracker® Red to discriminate Fluorescently labelled PM were also employed for confocal analysis, after 6 h of incubation whether particles ended up into the lysosomes or were able to escape, at least partially, ®from the with HCT116 and MDA-MB-231 cells. Acidic vesicles were labelled with Lysotracker Red to endosomal compartments. For all the tested formulations, we observed yellow dots corresponding to discriminate whether particles ended up into the lysosomes or were able to escape, at least partially, the merge of the green signal provided by DTAF and the red signal produced by the Lysotracker® from the endosomal compartments. For all the tested formulations, we observed yellow dots labelling late endosomes, lysosomes and other membrane structures with an acidic pH (Figure 4b). corresponding to the merge of the green signal provided by DTAF and the red signal produced by These results indicate that at least part of the formulated PM was endocyted and finally processed in the Lysotracker® labelling late endosomes, lysosomes and other membrane structures with an acidic the lysosomes. Nevertheless, because an effective blockage of SMC2 will require a cytosolic delivery pH (Figure 4b). These results indicate that at least part of the formulated PM was endocyted and of Ab-SMC2, we also measured the green fluorescence intensity at the cytoplasm of the HCT116 and finally processed in the lysosomes. Nevertheless, because an effective blockage of SMC2 will MDA-MB-231 cells after their incubation with PM-CON:SMC2. For this, we used cell mask membrane require a cytosolic delivery of Ab-SMC2, we also measured the green fluorescence intensity at the staining and DAPI nucleus labelling to define a region of interest, namely the cellular cytosol. Excluding cytoplasm of the HCT116 and MDA-MB-231 cells after their incubation with PM-CON:SMC2. For from the analysis bright green dots as PM accumulation within endosomal vesicles, we could detect an this, we used cell mask membrane staining and DAPI nucleus labelling to define a region of increase of green fluorescence in the cell cytoplasm of cells treated with PM-CON:SMC2, suggesting interest, namely the cellular cytosol. Excluding from the analysis bright green dots as PM that this formulation was able to reach the cell cytoplasm (Figure 4c). accumulation within endosomal vesicles, we could detect an increase of green fluorescence in the cell cytoplasm of cells treated with PM-CON:SMC2, suggesting that this formulation was able to reach the cell cytoplasm (Figure 4c). Pharmaceutics 2020, 12, 185 12 of 20 Pharmaceutics 2020, 12, x 12 of 20 3.5. G1/S Cell Cycle Arrest Induced by PM-CON:SMC2 3.5.Finally, G1/S CelltoCycle Arrest further Induced these validate by PM-CON:SMC2 results, we performed cell cycle assays in HCT116 and MDA-MB-231 cells treated with PM and PM-CON:SMC2. Finally, to further validate these results, we performed Of note, an effective cell cycle assays intargeting HCT116ofand intracellular MDA- SMC2 MB-231should cells have treatedanwith impactPM inandthe cell cycle and Of PM-CON:SMC2. reduce note, the number targeting an effective of cells into G2/M phase. of intracellular AsSMC2 it can should be observed have an in impact Figure in4d,the HCT116 cells cell cycle andtreated reducewith PM-CON:SMC2 the number displayed of cells into a markedly G2/M phase. As it can be higher observed of percentage in cells Figure in 4d, G1 HCT116 cells treated in comparison with PM-CON:SMC2 to untreated cells and cellsdisplayed incubateda with markedly PM as higher In controls. percentage addition,of thecells in G1 in of percentage comparison cells in G2/Mto untreated cells andincells was also reduced the incubated with PM as PM-CON:SMC2-treated controls. For samples. In addition, MDA-MB-231 the percentage of cells the response in G2/M pattern waswas also reduced similar in the PM-CON:SMC2- when comparing untreated and treated samples. For MDA-MB-231 PM-CON:SMC2-treated the response cells, exhibiting even higherpattern was similar differences when comparing regarding the decreaseuntreated of G2/M percentage. Unexpectedly, control PM also showed an impact in cell-cycle arrest at G1 in this cellof and PM-CON:SMC2-treated cells, exhibiting even higher differences regarding the decrease line G2/M (see percentage. Figure Unexpectedly, S3 for cell-cycle controlexamples). curve fitting PM also showed an impact in cell-cycle arrest at G1 in this cell line (see Figure S3 for cell-cycle curve fitting examples). Figure Figure4. 4.PM-CON:SMC2 PM-CON:SMC2 uptake uptake and and intracellular fate. (a) intracellular fate. (a)Flow Flowcytometry cytometrygraphsgraphsdisplaying displaying thethe percentage percentage ofoffluorescent fluorescentcells cellsafter afterHCT116 HCT116 and MDA-MB-231 MDA-MB-231cell cellincubation incubationwithwith 5 mg/mL 5 mg/mL PM, PM, PM:SMC2 PM:SMC2 andand PM-CON:SMC2. PM-CON:SMC2. (b)(b) Confocal Confocal images images showing showing either either PMPM or PM-CON:SMC2 or PM-CON:SMC2 in in green, green, acidic acidic in vesicles vesicles red andin nuclei red and in nuclei blue forin HCT116 blue for and HCT116 and MDA-MB-231 MDA-MB-231 cells aftercells 6 h after 6 h incubation incubation with 5 mg/mLwith PM.5Scale mg/mLbarPM. Scale bar represent represent 10 µm. 10 µ m. images (c) Confocal (c) Confocal imagesPM-CON:SMC2 displaying displaying PM- in CON:SMC2 green, plasma in green, plasma membrane in redmembrane and nucleiin red and nuclei in blue, in blue,and for HCT116 for MDA-MB-231 HCT116 and MDA-MB-231 cells after 6 h cells after 6 h incubation with 5 mg/mL PM. Scale bar represent incubation with 5 mg/mL PM. Scale bar represent 20 µm. Side panels, graphical 20 µ m. Side panels, graphical of representations representations of green fluorescence measures in the cytoplasm. (d) Diagrams green fluorescence measures in the cytoplasm. (d) Diagrams of cell cycle assay performed for HCT116 of cell cycle assay andperformed MDA-MB-231 for HCT116 and 48 cells after MDA-MB-231 h of incubationcellswith after548 h of incubation mg/mL with 5 mg/mL PM, PM-CON:SMC2 PM, (32.9 PM- of µg/mL antibody) and their respective untreated control. Percentages of cells at distinct cell cycle phases:atG1, CON:SMC2 (32.9 µ g/mL of antibody) and their respective untreated control. Percentages of cells distinct S and G2/M cell arecycle phases: G1, displayed. The SG2/G1 and G2/M ratio are displayed. is shown The inside theG2/G1 circle.ratio ** pis