cancers Article Synthesis and Validation of a Bioinspired Catechol-Functionalized Pt(IV) Prodrug for Preclinical Intranasal Glioblastoma Treatment Xiaoman Mao 1 , Shuang Wu 2 , Pilar Calero-Pérez 2,3 , Ana P. Candiota 2,3,4 , Paula Alfonso 1,4 , Jordi Bruna 5 , Victor J. Yuste 6 , Julia Lorenzo 2,4 , Fernando Novio 1,7, * and Daniel Ruiz-Molina 1, * 1 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain; mmchong7@zju.edu.cn (X.M.); paula.alfonso@icn2.cat (P.A.) 2 Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain; psychews@gmail.com (S.W.); pilar.calero@uab.cat (P.C.-P.); AnaPaula.Candiota@uab.cat (A.P.C.); julia.lorenzo@uab.cat (J.L.) 3 Centro de Investigación Biomédica en Red: Bioingeniería, Biomateriales y Nanomedicina, Cerdanyola del Vallès, 08193 Barcelona, Spain 4 Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain 5 Neuro-Oncology Unit, Bellvitge University Hospital-ICO (IDIBELL), Avinguda de la Gran Via de l’Hospitalet, 199-203, L’Hospitalet de Llobregat, 089098 Barcelona, Spain; jbruna@bellvitgehospital.cat 6 Institut de Neurociències, Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona (UAB), Campus UAB, Cerdanyola del Vallès, 08193 Barcelona, Spain; Victor.Yuste@uab.cat 7 Departament de Química, Universitat Autònoma de Barcelona (UAB), Campus UAB, Cerdanyola del Vallès, 08193 Barcelona, Spain  * Correspondence: fernando.novio@icn2.cat (F.N.); dani.ruiz@icn2.cat (D.R.-M.)  Citation: Mao, X.; Wu, S.; Simple Summary: Glioblastoma (GB) is a type of brain cancer with a poor prognosis and few Calero-Pérez, P.; Candiota, A.P.; improvements in its treatment. One of the greatest difficulties in GB therapy lies in the fact that Alfonso, P.; Bruna, J.; Yuste, V.J.; most of the drugs with high anticancer potential do not reach the brain and exert high therapeutic Lorenzo, J.; Novio, F.; Ruiz-Molina, D. activity while minimizing side effects. To overcome these limitations, we focused on a catechol-based Synthesis and Validation of a Pt(IV) prodrug (able to reverse cisplatin in a cellular environment) with the intention of repurposing Bioinspired Catechol-Functionalized Pt-based drugs as GB chemotherapeutic agents. Our in vitro results have corroborated the therapeutic Pt(IV) Prodrug for Preclinical effect of the synthesized complexes as comparable to cisplatin, and in vivo studies have demonstrated Intranasal Glioblastoma Treatment. Cancers 2022, 14, 410. https:// the potential of nose-to-brain delivery of this Pt(IV) prodrug for GB treatment. doi.org/10.3390/cancers14020410 Abstract: Glioblastoma is the most malignant and frequently occurring type of brain tumors in adults. Academic Editor: Chiara Laezza Its treatment has been greatly hampered by the difficulty to achieve effective therapeutic concentration Received: 25 November 2021 in the tumor sites due to its location and the blood–brain barrier. Intranasal administration has Accepted: 10 January 2022 emerged as an alternative for drug delivery into the brain though mucopenetration, and rapid Published: 14 January 2022 mucociliary clearance still remains an issue to be solved before its implementation. To address these issues, based on the intriguing properties of proteins secreted by mussels, polyphenol and Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in catechol functionalization has already been used to promote mucopenetration, intranasal delivery and published maps and institutional affil- transport across the blood–brain barrier. Thus, herein we report the synthesis and study of complex 1, iations. a Pt(IV) prodrug functionalized with catecholic moieties. This complex considerably augmented solubility in contrast to cisplatin and showed a comparable cytotoxic effect on cisplatin in HeLa, 1Br3G and GL261 cells. Furthermore, preclinical in vivo therapy using the intranasal administration route suggested that it can reach the brain and inhibit the growth of orthotopic GL261 glioblastoma. Copyright: © 2022 by the authors. These results open new opportunities for catechol-bearing anticancer prodrugs in the treatment for Licensee MDPI, Basel, Switzerland. brain tumors via intranasal administration. This article is an open access article distributed under the terms and Keywords: glioblastoma; bioinspired; Pt(IV); prodrug; catechol; platinum drugs; intranasal conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Cancers 2022, 14, 410. https://doi.org/10.3390/cancers14020410 https://www.mdpi.com/journal/cancers Cancers 2022, 14, 410 2 of 15 1. Introduction Glioblastoma (GB) is the most frequent malignant primary brain tumor in adults with the poorest prognosis and a median overall survival below 2 years, even after treatments combining gross macroscopic resections, radiotherapy and chemotherapy [1,2]. This, to- gether with its aggressiveness and almost ensured recurrence, has driven many efforts over recent years to advance its treatment, unfortunately without germane results. The main challenge for GB drugs is to cross the blood–brain barrier (BBB), together with a restricted bioavailability that prevents achieving the right dose, a common limitation for treating neurological CNS disorders [3]. Temozolomide (TMZ) merely showed the ability to cross the BBB and reach the tumor with the required therapeutic levels as demonstrated in a clinical trial back in 2005, especially in the subgroup of MGMT gene promoter methylated GBs [4–6]. Since then, trials with different systemic treatments at maximum tolerated doses have been unsuccessfully tested, leading to a sequence of side effects [7,8], as well as to the rapidly gained resistance of GB, in part related to the requested rest treatment intervals [9]. So, achieving suitable therapeutic doses within brain tumors while avoiding severe sec- ondary effects turned into a challenge, even for aggressive gliomas that compromise BBB integrity [10]. One of the emerging strategies is to overcome this limitation via the non-invasive and easily accessible intranasal (IN) administration [11]. Over the last decade, clinical trials including patients of all group ages, genders and clinical phases experienced a threefold increase compared with the previous 2000–2010 decade (see ClinicalTrial.gov, accessed on 24 November 2021) [12]. In addition to bypassing the BBB, IN-administered drugs with systemic action can be rapidly absorbed through rich vasculature in the submucosa and exhibit a fast onset of action [13], positioning this delivery route into a theoretical privileged position to fight GB [14]. However, its implementation in commercial uses still remains elusive, among others, due to a reduced mucopenetration and rapid mucociliary clearance of most effective drugs tested. Therefore, syntheses of novel therapeutic agents aimed to overcome these limitations are intensely sought. Herein, we hypothesize that this objective can be achieved with the synthesis of GB-active drugs bearing catechol groups, which are expected to enhance the drug mu- copenetration bioinspired by the mussel adhesion properties in seawater environments [15]. Following this hint, films for effective buccal drug delivery [16], chitosan-catechol polymers with long-lasting mucoadhesive properties [17] or polydopamine (PDA) coatings with enhanced mucopenetration and cellular uptake [17,18] have already been reported. This is attributed to their hydrophilic characteristics and negative charge at physiological pH that minimizes the interaction with the negatively charged and hydrophobic pockets in mucus, a prerequisite for an efficient mucopenetration [19–22]. As a proof of concept to validate our approach, we have designed complex 1. While effective for most malignant tumors [23], platinum-based drugs are only used as a residual fourth-line chemotherapeutics for GB, i.e., only when all standard therapeutic protocols fail. Thus far, reported platinum clinical trials have not improved the overall survival of patients with brain tumors [24], neither in combination with radiotherapy [25] nor other chemotherapeutics such as carmustine (BCNU) [26] and TMZ [27]. Therefore, if successful, our work would not only demonstrate the legitimacy of IN administration but also repurpose this family of drugs back to the first line of action. Pt(IV) agents also minimize side effects as they likely become active upon intracellular reduction to the active Pt(II) form, which interacts with DNA [28] and perturbs other pathways related to cisplatin resistance [29] by biological reductants [30], such as reduced nicotine adenine dinucleotide (NADH) [31], ascorbic acid [28] and even reduced glutathione (GSH) [32]. On top of that complex 1 mimics natural products such as bis-catechol gossypol or nordihydroguaiaretic acid (NDGA), which are already used as antioxidants and anti-inflammatories in cell culture and preclinical GB studies [33,34]. In fact, the development of novel bis-catechol with improved and reproducible therapeutic GB efficiencies has become a research line of interest per se; such is the case of terameprocol, which is considered a global transcription Cancers 2022, 14, x 3 of 15 Cancers 2022, 14, 410 3 of 15 inhibitor inhibitor that that affects affects cell cell division, division, apoptosis, apoptosis, drug drug resistance resistance and and radiation radiation resistance resistance in in hypoxia hypoxia [35]. [35]. A A schematic schematic representation representation of of the the approach approach used used for for the the validation validation of of complex complex 11 asas an an efficient IN drug for the treatment of GB is shown in Figure 1. It mainly involves efficient IN drug for the treatment of GB is shown in Figure 1. It mainly involves the the synthesis synthesis ofofcomplex complex1 and 1 andthethe in vitro in vitro testing testing with murine with murine GBusing GB cells, cells,cisplatin using cisplatin (CDDP), (CDDP), NDGA and the precursor complex disuccinatocisplatin NDGA and the precursor complex disuccinatocisplatin (DSCP) for comparison (DSCP) for comparison purposes. purposes. Finally, theFinally, in vivothe in vivo tolerability tolerability test and therapeutic test and therapeutic activity of activity complexof1 complex 1 after after intranasal intranasal administration was positively evaluated to confirm administration was positively evaluated to confirm its efficacy. its efficacy. Figure 1. (a) Chemical formula of complex 1. (b) IN administration for in in vivo vivo tolerability/efficacy tolerability/efficacy evaluation. The substance evaluation. The substance administered administeredvia viaIN INroute routeenters entersthe the brain brain mainly mainly through through (1)(1) thethe olfac- olfactory tory nerve and (2) trigeminal nerve pathways, while a fraction unavoidably would enter the nerve and (2) trigeminal nerve pathways, while a fraction unavoidably would enter the circulation circu- lation through (3) the airway. (c) Possible uptake (mediated by membrane transporters or passive through (3) the airway. (c) Possible uptake (mediated by membrane transporters or passive diffusion) diffusion) and most feasible action mechanism of complex 1. The Pt(IV) prodrug enters the cell, then and most feasible action mechanism of complex 1. The Pt(IV) prodrug enters the cell, then becomes becomes reduced intracellularly, and the activated form binds to DNA to induce cell death. reduced intracellularly, and the activated form binds to DNA to induce cell death. 2. 2. Materials Materials and and Methods Methods 2.1. Chemicals Chemicals and and Instrumentation Unless otherwise noted, materials obtained from suppliers were used as received. 11H, H, 13C 13 and 11H- C and 1 HCOSY H-1H COSYNMR NMRspectra spectrawere wererecorded recordedat at 400, 400, 100,and 100, and400 400MHz, MHz,respectively, respectively, spectrometer. 195 using Bruker DPX 400 MHz spectrometer. 195Pt PtNMR NMR spectrum spectrum waswas performed performed in in aa Bruker Bruker spectrometer operating operatingat at77.42 77.42MHz. MHz. PtPt 195 chemical shifts Avance DRX-360 spectrometer 195 chemical shifts areare relative relative to to external external 1M1 Na M 2Na 2 [PtCl [PtCl 6] in6 ]Din 2OD(02 Oppm). (0 ppm). High-resolution High-resolution mass mass spectra spectra werewere obtained obtained by by direct direct injection injection of the of the sample sample with with electrospray electrospray techniques techniques in ainBruker a Bruker microTOF-Q microTOF-Q in- instrument. strument. FT-IR FT-IR spectra spectra werewererecorded recordedusing usingaaTensor Tensor 27 27 spectrophotometer spectrophotometer (Bruker (Bruker Optik GmbH, Leipzig, Germany) with KBr pellets. Sixty-four Sixty-four scans were collected for each each sample at a resolution of 4 cm −−11 over 400–4000 cm− 1 . PrestoBlue assays were carried out resolution of 4 cm −1 . PrestoBlue assays were carried using a Victor Victor33microplate microplatereaderreader(PerkinElmer, (PerkinElmer, Boston, Boston, MA, USA), MA, Statistical USA), analysis Statistical and analysis IC50 IC and calculations werewere 50 calculations performed performed withwith GraphPad GraphPadPrism (version Prism 7.0, GraphPad (version 7.0, GraphPad Software Soft- Inc., San ware Inc.,Diego, CA, USA). San Diego, CA, USA). DNA concentration DNA waswas concentration determined determined by abyNanoDrop a NanoDrop UV-vis UV- spectrometer (Thermo Scientific, California, CA, USA). ICP-MS vis spectrometer (Thermo Scientific, California, CA, USA). ICP-MS (PerkinElmer, Wal-(PerkinElmer, Waltham, MA, USA) tham, MA, was USA)used wastouseddetermine the metallic to determine concentration. the metallic concentration. 2.2. Synthesis of Complex 1 2.2. Synthesis of Complex 1 The synthesis of the intermediate DSCP was duplicated according to previously The synthesis of the intermediate DSCP was duplicated according to previously re- reported procedures [36]. DSCP (0.5 g, 0.93 mmol), N,N 0 -Dicyclohexylcarbodiimide (DCC, ported procedures [36]. DSCP (0.5 g, 0.93 mmol), N,N′-Dicyclohexylcarbodiimide (DCC, 0.58 g, 2.8 mmol) and N-Hydroxysuccinimide (NHS, 0.43 g, 3.75 mmol) were dissolved in 0.58 g, 2.8 mmol) and N-Hydroxysuccinimide (NHS, 0.43 g, 3.75 mmol) were dissolved in anhydrous dimethylformamide (DMF, 4 mL) under Ar in darkness. After the solution was anhydrous dimethylformamide (DMF, 4 mL) under Ar in darkness. After the solution was stirred overnight, the slurry mixture formed was cooled to 0 ◦ C, and the white precipitate stirred overnight, the slurry mixture formed was cooled to 0 °C, and the white precipitate dicyclohexylurea (DCU) was removed by filtration. A large amount of ethyl acetate dicyclohexylurea (DCU) was removed by filtration. A large amount of ethyl acetate Cancers 2022, 14, 410 4 of 15 (EtOAc) was poured into the filtrate and sonicated to achieve an off-white precipitate, which was washed with cold ether and dried to give the activated ester (0.424 g, 62.2%). This intermediate (0.42 g, 0.58 mmol) and dopamine hydrochloride (0.263 g, 1.39 mmol) were dissolved in anhydrous DMF (3 mL), and N-methylmorpholine (NMM, 2.3 mL, 20.9 mmol) was added slowly. The reaction was tracked by thin-layer chromatography (TLC). After 24 h, the reaction was stopped and lyophilized to give a brownish residue, which was purified by column chromatography (EtOAC/MeOH, 9/1, v/v) to provide complex 1 as an off-white solid (0.195 g, 41.8%). 1 H NMR (400 MHz, MeOD-d4 ): δ = 6.69 (m, 4H), 6.54 (d, 2H), 3.36 (t, 2H) 2.61–2.70 (m, 8H), 2.46 (t, 4H) ppm; 13 C NMR (100 MHz, MeOD-d4 ): δ 180.9, 173.8, 144.7, 143.2, 130.79, 119.7, 115.5, 114.9, 41.1, 34.4, 31.3, 31.0; 195 Pt NMR, δ (ppm) = 1012. HR-ESI-MS (m/z): [M + Na]+ calculated 827.1276, found 827.1182; Pt content by ICP-MS: calculated (%) 24.25, found (%) 24.12. Elem. Anal. Calcd. For C24 H34 O10 N4 Cl2 Pt: C, 35.83; H, 4.26; N, 6.96, found: C, 35.51; H, 4.50; N, 6.51. 2.3. In Vitro Studies 2.3.1. Cell Viability Human cell lines (HeLa, 1Br3G and GL261) were purchased from the American Type Culture Collection (ATCC) and cultured at 37 ◦ C in a humidified 5% CO2 environment in RPMI 1640 (GL261) supplemented with 2 mM of L-glutamine, 2 g/L of D-glucose and 10% fetal bovine serum (FBS); or Dulbecco Modified Eagle’s Medium (DMEM) supplemented with 2 mM of L-glutamine, 4 g/L of D-glucose and 10% FBS. Cells in exponential growth were seeded on 96-well plates (Corning, Tewksbury, MA, USA) at a density of 4000 cell/well. After 24 h incubation, fresh media containing compounds (CDDP, complex 1, DSCP and NDGA) at different concentrations were added, and the plates were incubated for 24 or 72 h. Afterwards, 10 µl of PrestoBlue® (0.15 mg/mL, Thermo Scientific, California, CA, USA) were added into each well, and the plates were incubated for another 4 h before measuring the fluorescence at 572 nm with excitation at 531 nm in a Victor 3 microplate reader (PerkinElmer, Germany). All experiments were carried out in triplicate, and the IC50 value for each compound was determined using GraphPad Prism software (version 7.0, GraphPad Software Inc., San Diego, CA, USA). 2.3.2. Estimation of ROS Formation HeLa, 1Br3G and GL261 cells were seeded on black 96-well microplates with optically clear bottoms (Corning, USA) at a density of 20,000 cells/well overnight. Cells were then washed, and PBS was added with the fluorescent probe DCFCDA at a final concentration of 10 µM for 30 min in the dark. Afterwards, the buffer with a probe was replaced by media or media with compounds (H2 O2 , complex 1 and CDDP) for 24 h. A total of 0.1 mM of H2 O2 was used as a positive control, while other agents were added at their IC50 concentration. The fluorescence increase in each well was estimated by a microplate reader Victor 3 at 530 nm (emission) and 485 nm (excitation) (Perkin Elmer, USA). This experiment was repeated in triplicate independently. 2.3.3. Cellular Internalization GL261 cells were seeded in 6-well plates (Corning, USA) at a density of 300,000 cells per well. After 24 h incubation, the media were replaced by fresh media in absence or presence of CDDP and complex 1 at a final concentration of 10 µM Pt. The cells were allowed to internalize the compounds for different time points, then rinsed with PBS. Then, cells were harvested and digested, and the platinum content determined by ICP-MS. 2.3.4. DNA-Bound Pt To investigate the Pt content associated with the DNA of GL261 cells, similarly to the procedure for cell uptake, cells were harvested after incubation with CDDP and complex 1 at a concentration of 10 µM Pt for 24 h. The cellular pellets were resuspended and incubated on ice for 15 min in lysis buffer (150 mM of Tris-HCl (pH 8.0), 100 mM of NaCl and Cancers 2022, 14, 410 5 of 15 0.5% (w/v) SDS). After centrifuge, RNase A was added at 200 µg/mL and incubated for 1 h at 37 ◦ C. Later, proteinase K was added at 100 µg/mL to incubate for 3 h at 56 ◦ C. Afterwards, a volume of phenol–chloroform (1/1, v/v) was added to the extract. Obtained aqueous phase containing DNA was collected and precipitated with 0.1 volume of 3 M of sodium acetate and 1 volume of absolute ethanol at −20 ◦ C overnight. DNA samples were washed with ethanol, dried and resuspended in TE buffer (10 mM of Tris-HCl, 1 mM of EDTA, pH 8.0). DNA concentration of the sample was determined by spectral photometry (NanoDrop 1000, Thermo Scientific, USA). Quantified DNA was then frozen for analysis by ICP-MS. 2.4. In Vivo Studies 2.4.1. Animal Studies All animal experiments were approved by the local ethics committee (Comissió d’Ètica en l’Experimentació Animal i Humana, https://www.uab.cat/etica-recerca/, accessed on 24 November 2021), and conducted under the principles of regional and state legislations (protocol CEEAH 4859). Healthy female C57BL/6J mice (8–12 weeks, body weight 20–24 g) were used for all in vivo studies in this work and were obtained from Charles River Labo- ratories (Charles River Laboratories International, Saint-Germain-sur-l’Arbresle, France). Mice were housed at Servei d’Estabulari (animal facility) from Universitat Autònoma de Barcelona (UAB), and their health status was supervised by qualified staff. Each mouse was identified by a unique ear notch combination in order to distinguish animals in the same cage. Animals are then attributed an individual numeric code (e.g., tumor-bearing animals have the code CXXXX where XXXX is a sequential number) allowing not only for animal identification but also to know the implantation round used for its generation. 2.4.2. Tolerability Assessment The mice were randomly divided into two groups, control and complex 1, n = 3 each. Anesthetized mice were intranasally administered with PBS or complex 1 using a micropipette (Gilson, Limburg, Germany) in a horizontally supine position. In total 3 doses were given, which increased from 0.9, 1.2 and 1.5 mg Pt/kg body weight every week [37]. The body weights of mice were recorded prior to the first dose of treatment and were monitored 3 times a week thereafter. The animals were closely observed by veterinary staff from the animal facility, in particular food/water consumption, behavior changes and possible signs of suffering [38]. The whole study lasted for 4 weeks. Mice were euthanized for humanitarian reasons when endpoint was reached. 2.4.3. Orthotopic Intracranial GB Model by Stereotactic Injection of GL261 Cells The GL261 GB model was generated by stereotactic injection of GL261 cells into C57BL/6J mice, performed by accredited personnel. Analgesia (Metacam, Ingelheim am Rhein, Germany) at 1 mg/kg was injected subcutaneously to each animal 15 min before anesthesia and also 24 and 48 h after implantation. Mice were anesthetized with a mixture of ketamine (Parke-Davis SL, Madrid, Spain) at 80 mg/kg and xylazine (Carlier, Barcelona, Spain) at 10 mg/kg via intraperitoneal administration. Once anesthetized, the mouse was immobilized on the stereotaxic holder (Kopf Instruments, Tujunga, CA, USA) in a prone position. Next, the head area was shaved, and the incision site was sterilized with iodophor disinfectant solution, a 1 cm incision was made exposing the skull, and a 1 mm hole was drilled 0.1 mm posterior to the bregma and 2.32 mm to the right of the midline using a microdrill (Fine Science Tools, Heidelberg, Germany). A 26G Hamilton syringe (Reno, NV, USA), positioned on a digital push–pull microinjector (Harvard Apparatus, Holliston, MA, USA) was then used for injection of 4 µL of RPMI cell culture medium containing 100,000 GL261 cells at a depth of 3.35 mm from the skull surface at a 2 µL/min. Once the injection was completed, the Hamilton syringe was left untouched for 2 min more before its removal to prevent the cellular liquid leakage out of the skull. Finally, the Hamilton syringe was gently and slowly withdrawn, and the scission site was closed with Cancers 2022, 14, 410 6 of 15 suture silk 6.0 (Braun, Barcelona, Spain). When the implantation was over, the animal was left in a warm environment to recover from anesthesia. 2.4.4. In Vivo MRI Assessment of Tumor Evolution The animals were subjected to MRI acquisitions to measure tumor volumes and evolu- tion. Acquisitions were performed with 7T BioSpec 70/30 USR spectrometer (Bruker BioSpin GmbH, Ettlingen, Germany) at Servei de Ressonància Magnètica from UAB (also part of Unit 25 of ICTS NANBIOSIS, NMR: Biomedical Applications I https://www. nanbiosis.es/portfolio/u25-nmr-biomedical-application-i/, accessed on 24 November 2021). Mice were positioned in a dedicated bed, which allowed the delivery of anesthesia (isoflu- rane, 1.5–2% in O2 at 1 L/min), with an integrated heating circuit for body temperature regulation. Respiratory frequency was monitored with a pressure probe and kept between 60–80 breaths/min. GL261 tumor-bearing mice were scanned to acquire high-resolution T2w images using a rapid acquisition with relaxation enhancement (RARE) sequence to detect the brain tumors. To calculate the tumor volume from MRI acquisitions, ParaVi- sion software was used to generate regions of interest (ROIs) to measure the tumor area in each slice. 2.4.5. Antitumor Efficacy In Vivo The antitumor efficacy was evaluated for orthotopic GL261 GB-bearing mice. All animals included in the study were confirmed to have suitable GB with homogeneous volume using T2w MRI as described previously. Mice were randomly divided into two groups: control and complex 1. All agents were given at a dose of 1.5 mg Pt/kg via intranasal administration. Tumor growth inhibition rate (IRV ) was calculated using the Equation (1), as follows [39]: IRV = (1 − TVt/TVc) × 100% (1) where TVt and TVc are mean tumor volumes of treated and control group, respectively. 2.5. Statistical Analysis Quantitative data were expressed as mean ± SE. The multiple-group comparison was performed using a one-way ANOVA. Specific comparison between groups was carried out with an unpaired two-tailed Student’s t-test. Differences were considered statistically significant at p < 0.05. 3. Results and Discussion 3.1. Synthesis and Characterization The synthesis of complex 1 involves three main steps (see Scheme 1): (1) oxidation of the commercially available Pt(II) cisplatin (CDDP) with hydrogen peroxide to yield c,c,t-Pt(NH3 )2 Cl2 (OH)2 (oxoplatin), (2) addition of succinic anhydride to oxoplatin, using a protocol already described in the literature [36], to yield c,c,t-Pt(NH3)2Cl2(O2CCH2CH2COOH)2 (DSCP) and (3) coupling of dopamine (DA) to DSCP via a typical DCC/NHS coupling using N-methylmorpholine (NMM). Following this synthetic route, complex 1 was obtained and purified with a final overall yield of 24.7%. In spite of several attempts, no single crystals suitable for X-ray diffraction were obtained. However, the high-resolution mass spectrum showed the expected mass of complex 1 (see Figure S1). The FT-IR spectra of initial oxoplatin showed peaks at 559 cm−1 and 3516 cm−1 , characteristic of the Pt-O and O-H vibrations, respectively. These peaks disappeared when carboxylic groups of succinic acid react to generate the DSCP complex [40], while new peaks in the 1630–1734 cm−1 and 1180–1347 cm−1 regions appeared and were assigned to the C-O stretching from the carboxyl groups and succinic acid, respectively. Finally, the carboxyl peak shifted to lower wavenumber (1527–1645 cm−1 ) for complex 1, indicating the formation of an amide bond with dopamine while a new peak at 1198 cm−1 from the C-O stretching of the catechol also appeared (see Figure S2). 1 H-NMR chemical shifts unequivocally identified the proton characteristic of the Pt-O and O-H vibrations, respectively. These peaks disappeared when characteristic carboxylicof the Pt-O groups of and O-H vibrations, succinic acid react to respectively. generate the These DSCP peaks disappeared complex whennew [40], while carboxylic peaks ingroups of succinic the 1630–1734 cm−1acid andreact to generate 1180–1347 the DSCP cm−1 regions complex appeared and[40], werewhile new to assigned peaks in the 1630–1734 cm −1 and 1180–1347 cm−1 regions appeared and were assigned to the C-O stretching from the carboxyl groups and succinic acid, respectively. Finally, the the carboxyl C-O stretching from the peak shifted to carboxyl groups and(1527–1645 lower wavenumber succinic acid, cm−1respectively. ) for complex Finally, the 1, indicating Cancers 2022, 14, 410 carboxyl peak shifted to lower wavenumber (1527–1645 the formation of an amide bond with dopamine while a new peak at 1198 cm from cm −1) for complex 1, indicating −1 7 of the15 the C-O formation of anofamide stretching bond with the catechol dopamine(see also appeared while a new Figure peak S2). at 1198 1H-NMR cm from −1 chemical theun- shifts C-Oequivocally stretching of the catechol also appeared (see Figure S2). 1H-NMR chemical shifts un- identified the proton signals for complex 1; meanwhile, all 12 different carbon equivocally signals signals for identified for the complex the1;proton symmetrical signals all molecule meanwhile, for werecomplex 12 found1;inmeanwhile, different all 12 the 13C signals carbon NMR different spectrum for carbon (see Figure the symmetrical signals S3). for molecule 195 the Pt NMR symmetrical werespectrum molecule found in showed 13 the C aNMR were found singlespectrum in peak at 1012the 13C NMR spectrum (see ppm Figure(seeS3). 195 FigurePtS4).(see NMRTheFigure spectrum chemical S3).stability 195Pt NMR spectrum showed a single peak at 1012 ppm (see Figure S4). The chemical showed a single of peak at complex the resulting 1012 ppm was(see Figurein studied S4). The chemical stability phosphate-buffered salineof(PBS, the resulting pH 7.4) stability complex of thewasresulting studiedcomplex was studied in phosphate-buffered in(see phosphate-buffered saline (PBS, pH 7.4) saline ◦(PBS, at 37massC and pH 7.4) PBS/BSA at 37 °C and PBS/BSA solution using UV-Vis Figure S5), NMR and spectroscopy at 37 °C solutionand PBS/BSA using solution UV-Vis (see using FigureUV-Vis S5), NMR(see Figure and S5), mass NMR and spectroscopy as characterization techniques. The non-variation of the initial spectra denotes a stability massas spectroscopy characterization as characterization techniques. in the solution Thetechniques. non-variation superior Theatnon-variation to that of 24the initial h with of the no spectra sign ofinitial denotes product spectra denotesinathe a stability reduction. stability solution in the solution superior to that at 24 h with no superior to that at 24 h with no sign of product reduction. sign of product reduction. Scheme 1. Synthetic route of complex 1. Scheme Scheme 1. Synthetic 1. Synthetic route route of complex of complex 1. 1. 3.2. In Vitro Studies 3.2. In Vitro Studies 3.2. In Vitro Studies 3.2.1. 3.2.1. Cytotoxicity Cytotoxicity andand Cell Cell Internalization Internalization 3.2.1. Cytotoxicity and Cell Internalization The The cytotoxicity cytotoxicityofofcomplex complex 1 at 1 24 at and 24 and72 h72 was evaluated h was usingusing evaluated the PrestoBlue assay the PrestoBlue inThe thecytotoxicity assay murine in the murine of complex GL261 GB cellGB GL261 1line at 24 and asline cell 72 wellas hin was aswell asevaluated human using cervical in human thecancer cancer cervical PrestoBlue cell HeLa assay and cell HeLa non- and in the murine malignant GL261 human GB cell fibroblastline as cell well 1Br3G,as in for human cervical comparison cancer purposes. cell These non-malignant human fibroblast cell 1Br3G, for comparison purposes. These studies were HeLa and studies non- were re- malignant peated human with fibroblast CDDP as a cell gold 1Br3G, drug for model. comparison Both drugs purposes. exhibited These studies were concentration-dependent repeated with CDDP as a gold drug model. Both drugs exhibited concentration-dependent re- peated with CDDP cytotoxic cytotoxic effects as effects a gold (see Figure drug model. 2a and and Both Figure Figure drugs S6). S6). The exhibited The half concentration-dependent half maximal maximal inhibitory inhibitory concentrations concentrations cytotoxic (IC effects (IC5050),),referred(see referred to Figure to Pt 2a and Pt concentration, Figure concentration, were S6). The half were calculated maximal calculated using inhibitory using GraphPad GraphPad Prismconcentrations Prism (version (version 7.0) 7.0) (IC50and ), referred and are to Pt concentration, are summarized in Table 1. were calculated using GraphPad Prism (version 7.0) and are summarized in Table 1. Figure 2. Figure (a) Representative 2. (a) Representative cytotoxicity cytotoxicity of of cisplatin cisplatin (CDDP) (CDDP) and and complex complex 11 against against GL261 GL261 cells cells for for 24 h2.and Figure 24 72 72 h. h.(b) (b)Cellular (a) Representative uptake uptakeofofCDDP cytotoxicity Cellular ofCDDP and cisplatincomplex and (CDDP) complex at1 different 1and complex at time points 1 against different time in GL261 GL261 points cell, in cells GL261 forwith cell, 24 haand 72 h. (b) Cellular Pt concentration of 10uptake µM. (c)of CDDP and Pt DNA-bound complex 1 at different in different cell lines time after points exposurein GL261 to 10 µMcell, Pt for 24 h. All data represent mean ± SE of three independent experiments. *** for p < 0.0001. Cancers 2022, 14, 410 8 of 15 Table 1. IC50 (µM) a of compounds against different murine and human cell lines. IC50 (µM) Cell Lines Time Compounds 1Br3G HeLa GL261 complex 1 56.09 ± 1.18 29.94 ± 1.04 17.40 ± 1.08 CDDP 45.07 ± 4.60 15.98 ± 1.04 5.61 ± 0.28 24 h DSCP NT b NT b NT b NDGA NT b 164.15 ± 0.02 109.45 ± 1.95 complex 1 10.80 ± 0.60 1.85 ± 0.36 4.17 ± 0.12 CDDP 4.63 ± 0.42 2.34 ± 0.30 2.16 ± 0.26 72 h DSCP NT b NT b NT b NDGA 119.00 ± 14.20 55.65 ± 6.21 81.73 ± 2.40 a The IC50 of the compounds against different cell lines were determined by PrestoBlue assay. Each value represented mean ± SE of three independent experiments. b NT represents non-toxic, which means the viability of cells is close to 100% even at the maximum concentration. At 24 h, complex 1 showed lower IC50 values than CDDP in both Hela and GL261 tumor cell lines (a reduced cytotoxicity was found for both complexes in the non-tumoral cell line 1Br3G), as confirmed with internalization studies in the GL261 cell line (see Figure 2b), showing that internalization of CDDP at 24 h was notably more effective than that of complex 1. This fact was attributed to the more effective cellular uptake of CDDP thanks to the favorable interaction between the negatively charged cellular membrane with the positive charge resulting upon hydrolysis of CDDP (under the same experimental conditions, complex 1 is mostly deprotonated with a negative charge that originates electrostatic repulsions) [41]. Remarkably, toxicities at 72 h were comparable. It is well known that Pt(IV) prodrugs are kinetically inert, thus minimizing unwanted side interactions with biomolecules prior to DNA binding, making them more effective even at lower concentrations [42,43], but they undergo a slower intracellular reduction to produce the active Pt(II) species, as they exhibit more resistance to ligand substitution reaction than Pt(II) centers. Then, the present results suggest that for the first 24 h the activation of Pt(IV) was not very fast, while at 72 h the activation rate appeared sufficient to obtain a similar cytotoxic effect even though the concentration of 1 was comparatively lower. We cannot discard other internalization effects, though the comparative uptake between CDDP and complex 1 at 72 h could not be obtained due to the low cell viability at this time point, which hampers the obtainment of reliable results. It also turns out to be extremely difficult to properly correlate the in vitro and bloodstream biotransformation of Pt(IV) prodrugs as many biomolecules can influence the reaction [44]. Studies using satraplatin, a Pt(IV) complex similar to complex 1, showed a short half-life of only 6.3 min in whole blood as a result of rapid reduction of the Pt(II) species, being much more stable in fresh plasma (t1/2 = 5.3 h) [45]. These results suggest that the cytotoxicity assays could be a reference on how to correlate the cellular uptake with the cytotoxic effect, but the therapeutic potential should be determined by in vivo tests (vide infra). Finally, and worthwhile to mention, the negative effect on internalization only accounts upon comparison with positively charged drugs. However, if the toxicity of complex 1 is compared with that of the DSCP precursor differing in the presence of carboxylic groups instead of the catechol ones, the scenario differs. In this case, the catechol moiety may increase the lipophilicity of the Pt(IV) prodrug and introduce partially ionizable groups at physiological conditions. In fact, studies of medicinally relevant catechol-derivative compounds revealed that partitioning between the octanol–water phases does not correlate well with the partitioning between the lipid membrane and water, especially for weakly or moderately lipophilic compounds with a combination of ionized and polar chemical groups [46]. The fact of carrying a net positive or negative charge in physiological pH increases the interaction of catecholic compounds with the charged moieties of the lipid headgroups. Moreover, dynamics of the interaction with cell membranes include complex Cancers 2022, 14, 410 9 of 15 hydrogen bonding with both lipids and water molecules. In our case, the increase in lipophilic character in addition to the presence of ionizable chemical groups seem to have a notable influence in cellular uptake. Other relevant considerations about the results of Table 1 are that the IC50 values of NDGA were smaller than those of DSCP but still far (with almost one order of magnitude higher) from those of complex 1, pointing out an effective synergism between Pt ions and catechol groups. 3.2.2. Pt Bound to DNA The quantification of Pt bound to DNA can give a direct hint to the anticancer activity. Thus, the three cell lines were treated with complex 1 and CDDP at a concentration of 10 µM (based on Pt content) for 24 h. The DNA concentration was determined by UV-vis spectrometer (Figure S7), while Pt was quantified by ICP-MS. The analysis suggested that in the case of CDDP, the Pt bound to DNA was significantly higher than that found for complex 1 within all cell lines (Figure 2c), reinforcing the trend observed from cellular internalization studies, and its higher anticancer activity for 24 h. As in the case of uptake studies, Pt-DNA binding studies at 72 h were not conclusive due to the low cell viability, which prevented the obtainment of a precise quantification of Pt bound to DNA. An attempt with a lower Pt concentration (1 µM) also failed as measurements were close to the detection limit, resulting in inconclusive values. Additionally, it is worthwhile to mention the accumulation of Pt in nuclei was remarkably higher in GL261 cells than in the fibroblast 1Br3G cells, in line with the low activity of these compounds against 1Br3G cells. As a normal fibroblast cell line, related proteins, i.e., ATP7A and ATP7B, are expressed more in 1Br3G cells than in glial cells, while the expression level of MRP2 is negligible in both [43]. These transporters have been proven to contribute to the efflux of Pt agents and the emergence of drug resistance [42,47]. Different expression levels of these transporters in specific cell lines might account for the distinct Pt accumulation in different cell lines. 3.2.3. Oxidative Stress Assay Typically, NDGA and overall catechol ligands generate reactive oxygen species (ROS) as a byproduct during the process of oxidation. In fact, dopamine and NDGA itself have already been reported to exhibit cytotoxic effects at a high concentration in various cell lines mainly related to the generation of ROS [43,47,48]. So, to discard oxidative stress as the origin of the notable toxicity of complex 1, further ROS assays were performed using a fluorescent probe 20 ,70 -dichlorofluorescin diacetate (DCFCDA) in the same three cell lines. For comparison purposes, we also tested CDDP, NDGA and H2 O2 under the same experimental conditions (Figure 3). Interestingly, the fluorescence emission triggered by complex 1 and CDDP in all three cell lines was similar and much lower in comparison with the positive control H2 O2 , discarding intracellular generation of ROS. Thus, toxicities of both complexes were attributed to DNA binding to exert cell-killing ability which may be the base of the chemotherapeutic effect. It is also remarkable the high ROS response was generated by NDGA in comparison with complex 1 under the same experimental conditions, proving once again the competence of our system. 3.3. In Vivo Studies 3.3.1. Tolerability Assessment in Mice First, in vivo experiments aimed to study the short-term safety and tolerability of complex 1 after IN administration in healthy C57BL/6J female mice (n = 3). Three single- scaled doses, namely 0.9, 1.2 and 1.5 mg Pt/kg body weight, were administered weekly, increasing dosage every week. Even with the highest dosage of 1.5 mg Pt/kg, all mice maintained good health status during the study. The body weights of the mice were tracked three times a week with no significant weight decrease indicative of toxicity (see Figure S8). Moreover, no treatment-related adverse effects on food/water consumption, or other clinical signs of suffering were observed during this period. In addition, no obvious morphological/pathological changes were observed in histopathological analyses. Cancers 2022, Cancers 2022, 14, 14, 410 x 10 10 of of 15 Figure 3. Figure 3. Reactive Reactive oxygen oxygen species species (ROS) (ROS) generation generation triggered triggered in in (a) HeLa, (b) (a) HeLa, (b) 1Br3G 1Br3G and and (c) (c) GL261 GL261 cells. The concentration of H 2O2 was 0.1 mM, and other agents were used at their corresponding cells. The concentration of H2 O2 was 0.1 mM, and other agents were used at their corresponding IC50.. All data represent mean ± SE (standard error) of three independent experiments. * stands for p IC 50 All data represent mean ± SE (standard error) of three independent experiments. * stands for < 0.05, ** for p < 0.001. p < 0.05, ** for p < 0.001. 3.3. InEfficacy 3.3.2. Vivo Studies Evaluation In Vivo 3.3.1.TheTolerability Assessment in vivo efficacy in Mice 1 was evaluated in immunocompetent orthotopic of complex GL261 glioma-bearing First, mice. Theaimed in vivo experiments therapy tostarted study at theday 10 post-implantation short-term (p.i.) with an safety and tolerability of average of 6.15 ± 0.71 3 and no significant differences in tumor volume complextumor 1 aftervolume IN administration in mm healthy C57BL/6J female mice (n = 3). Three single- or bodydoses, scaled weightnamely between the1.2 0.9, control and 1.5 andmgtreated Pt/kggroups (n = 7 and body weight, n = administered were 3, respectively). It was weekly, followed by an every-six-days immune-enhanced metronomic increasing dosage every week. Even with the highest dosage of 1.5 mg Pt/kg, all mice schedule (IMS) (Figure S9), which was based on the mice immune cycle and was demonstrated maintained good health status during the study. The body weights of the mice were to improve therapeutic outcomes tracked three [49,50], timesmost likely a week withdueno to significant spurring the host immune weight decreasesystem for tumor indicative fighting. of toxicity (see Mice were treated with a dose of 1.5 mg Pt/kg body weight, the Figure S8). Moreover, no treatment-related adverse effects on food/water consumption, highest safe dose assessed by tolerability or other clinicalstudies. signs of Evolution suffering were of tumor volumes observed duringwas monitored this period. Inby high-resolution addition, no obvi- T2w MRI acquisition (see Figure S10), and the corresponding ous morphological/pathological changes were observed in histopathological analyses. results are shown in Figure 4. Overall, the results (Figure 4a) suggest that complex 1 exhibited a tumor growth inhibition rate 3.3.2.(IR V ) of 20% Efficacy in comparison Evaluation In Vivowith the control group. The survival averages (Figure 4c) of the control group and complex 1 group were 19.9 ± 0.7 days and 22.7 ± 1.7 days, The in vivo efficacy of complex 1 was evaluated in immunocompetent orthotopic respectively. Whenever mice body weights dropped under 80% of the initial values, GL261 experimentsglioma-bearing were halted, mice. andThe therapy the mice were started at day 10 post-implantation (p.i.) with euthanized. an average tumor volume of 6.15 ± 0.71 mm 3 and no significant differences in tumor vol- In an attempt to improve the therapeutic efficacy, a second round of therapy was ume or body launched. Forweight this, abetween new cohort the control of miceand treated bearing groups GL261 (n = 7was tumors andgenerated n = 3, respectively). (average It was followed by an every-six-days 3 immune-enhanced tumor volume of 5.80 ± 0.94 mm at day 10 p.i.), without significant differences metronomic schedule (IMS) (Fig- in tumor ure S9), which was based on the mice immune cycle and was volume or body weight between treated and control groups (n = 6 each) at the initial time demonstrated to improve therapeutic point. Then,outcomes mice were [49,50], treated most every likely 3 daysduewith to spurring the samethe host safe immune dose, 1.5 mgsystem of Pt/kg for tumor fighting. Mice were treated with a dose of 1.5 mg Pt/kg (Figure S8) [51–53]. In this case, as shown in Figure 4d, tumor growth was significantly body weight, the highest safe dose assessed inhibited even with bya tolerability higher IRV studies.of 37% Evolution in comparison of tumor withvolumes was monitored control group in a shorter by high-resolution T2w MRI acquisition (see Figure S10), and the period of time. However, the average survival of 19.8 ± 0.2 days was shorter than that corresponding results are shown in in Figureset the previous 4. Overall, the results of experiments and(Figure similar4a) suggest to the thatgroup control complex (of 119.9 exhibited ± 0.7 days)a tu- mor growth inhibition rate (IR ) of 20% in comparison with the (Figure 4f), as a total body weight loss equal or superior to 20% total led to euthanasia. V control group. The sur- vival averages (Figure 4c) of the control group and complex Moreover, as can be also seen in Figure 4, the time elapsed for doubling tumor volume 1 group were 19.9 ± 0.7 days and 22.7 (i.e., ± 1.7 days, “doubling respectively. time”) decreased for Whenever mice body both treatment weights schedules indropped comparison under with80% of the controls initial values, (9–11% decreaseexperiments for the time were halted, frame and the mice measured, 10 towere 16/17 euthanized. days p.i.). All these results evidenced the effectiveness of IN treatment of GB with complex 1. Cancers 2022, 14, x 11 of 15 Cancers 2022, 14, 410 11 of 15 Figure Figure 4. In vivo efficacy evaluation in two treatment schedules at different time post injection (p.i.). (a–c) Every-6-days (a–c) Every-6-days IMS for GL261GL261 GB therapy therapy in mice. Complex 1 was administered every 6 days, and tumor volumes were measured by high-resolution T2w and tumor volumes were measured by high-resolution T2w MRI MRI twice twice per per week. week. (a) (a) Tumor volume Tumor volume evolution of mice from control (n = 7) and complex 1 group (1.5 mg Pt/kg) (n = 3). (b) evolution of mice from control (n = 7) and complex 1 group (1.5 mg Pt/kg) (n = 3). (b) Evolution Evolution of body weights of mice on IMS schedule from all groups. (c) Kaplan-Meier survival of body weights of mice on IMS schedule from all groups. (c) Kaplan-Meier survival curves of all curves of all groups. (d–f) Intensive treatment for GL261 GB therapy in mice. complex 1 was IN-administered groups. (d–f) Intensive treatment for GL261 GB therapy in mice. complex 1 was IN-administered every 3 days, and tumor volumes were acquired by high-resolution T2w MRI twice per week. (d) every 3 days, and tumor volumes were acquired by high-resolution T2w MRI twice per week. (d) Tumor volume evolution of mice from control (n = 7) and complex 1 group (1.5 mg Pt/kg) (n = 6). (e) Tumor volumeevolution Body weight evolutionofofmice miceduring from control (n =(f) treatment. 7) and complex 1survival Kaplan-Meier group (1.5 mg Pt/kg) curves (n = 6). of all groups. (e) Body dash Vertical weight evolution lines indicateofthe mice during days treatment. of therapy (f) Kaplan-Meier administration. survival Horizontal curves dash line of all groups. indicates the Vertical dashreduction 20% weight lines indicate point,the dayswhich below of therapy administration. the mice Horizontal had to be euthanized. Datadash line indicates represent mean ± theSE. * stands 20% for reduction weight p < 0.05. point, below which the mice had to be euthanized. Data represent mean ± SE. * stands for p < 0.05. In an attempt to improve the therapeutic efficacy, a second round of therapy was Noteworthily, launched. For this, the a new application cohort ofof cisplatin mice bearingforGL261 GB treatment tumors was is considerably limited generated (average by its poor solubility in aqueous solution, which is ideally 2.53 mg/mL tumor volume of 5.80 ± 0.94 mm at day 10 p.i.), without significant differences in tumor 3 in water at room temperature, but much lower in saline (the concentration of clinical formulation volume or body weight between treated and control groups (n = 6 each) at the initial time of cisplatin is onlyThen, point. 1 mg/mL micein 0.9%treated were saline every comprising 3 daysmannitol) [54]. Given with the same the 1.5 safe dose, lowmgvolume applied of Pt/kg (Fig- for ureintranasal S8) [51–53].administration In this case, as(upshown to 12 µL), the required in Figure concentration 4d, tumor growth was of significantly cisplatin to achieve inhib- therapeutic ited even with effects could IR a higher not be reached. In contrast, higher concentrations can be achieved V of 37% in comparison with control group in a shorter period using the Pt(IV) prodrug, complex of time. However, the average survival 1, with improved of 19.8 ± 0.2solubility days was in saline shorter(around than 15 mg/mL, that in the 5.6-fold of CDDP). Besides, increased mucopenetration, due to the previous set of experiments and similar to the control group (of 19.9 ± 0.7 days) (Figure presence of catechol, can enhance 4f), as the accumulation a total body weight loss equalof complex 1 in to or superior brain 20%andtotalthus in euthanasia. led to GB tumors,Moreover, therefore improving the anticancer efficacy and minimizing the side effects. as can be also seen in Figure 4, the time elapsed for doubling tumor volume (i.e., In addition to the“dou- well- known nephrotoxicity, neurotoxicity and hepatotoxicity induced bling time”) decreased for both treatment schedules in comparison with controls (9–11% by Pt-based anticancer drugs, decrease damage for theintime fast-growing tissues such frame measured, 10 as to mucous membranes 16/17 days p.i.). All has thesebeen widely results studied evidenced and reported [55].ofTherefore, the effectiveness IN treatment theof restricted GB withintranasal complex 1.delivery of Pt-based drugs can be anticipated in the treatment of GB. To date, few studies have reported to evaluate the in vivo Noteworthily, the application of cisplatin for GB treatment is considerably limited by performance of Pt-based agents against GB, although their significance has been recognized its poor solubility in aqueous solution, which is ideally 2.53 mg/mL in water at room tem- to repurpose or reposition the existing chemotherapeutic drugs for GB treatment [56,57]. perature, but much lower in saline (the concentration of clinical formulation of cisplatin In the sparse reports, only two cases administered the Pt agents via IN route, all about is only 1 mg/mL in 0.9% saline comprising mannitol) [54]. Given the low volume applied nanoformulations based on either carboplatin or FePt alloy nanoparticles [58,59]. However, for intranasal administration (up to 12 μL), the required concentration of cisplatin to while no in vivo efficacy was involved in either case, muco- or nasal-penetration were. achieve therapeutic effects could not be reached. In contrast, higher concentrations can be achieved using the Pt(IV) prodrug, complex 1, with improved solubility in saline (around Cancers 2022, 14, 410 12 of 15 To our knowledge, this work is the first report evaluating Pt agents’ in vivo therapeutic efficacy via IN administration, which shall shed some light on further studies. 4. Conclusions We have reported the synthesis and characterization of complex 1, which exhibits comparable cytotoxicities to CDDP in tumor cells, specifically in the GL261 GB cell line, while being less toxic against normal fibroblast cells. Even more relevant was the response to its treatment via the IN route in an immune-competent aggressive preclinical (murine) GB model using two different administration protocols, namely an IMS (every-six-days) or in an intensive every-three-days treatment schedule. Significant inhibition on GB tumor growth was observed in both cases, although it was more evident for the intensive schedule. However, the intensive schedule did not improve overall survival time, probably due to an enhanced toxicity in comparison with IMS schedule. On top of that, the axial catecholic ligands endowed the Pt(IV) prodrug with augmented solubility anfd stability in an aqueous solution, which can help overcome one of the formidable limitations for the application of Pt agents for GB treatment. This may be especially relevant in cases such as IN administration, in which therapy is administered in low volumes. Altogether, our findings confirm the potential of nose-to-brain delivery for GB treatment and repurposing Pt-based drugs as GB chemotherapeutic agents. To our knowledge, this is the first report evaluating the in vivo therapeutic efficacy of Pt-based drugs via IN administration, which shall shed some light on further studies Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cancers14020410/s1, Figure S1: HR-ESI-MS spectrum of complex 1, Figure S2: FT-IR spec- tra of cisplatin, oxoplatin, DSCP and complex 1, Figure S3: 1 H, 13 C, 2D COSY NMR spectra of complex 1, Figure S4: 195 Pt NMR spectrum of complex 1, Figure S5: UV-Vis spectra of complex 1 in phosphate-buffered saline solution (PBS, pH 7.4) at 37 ◦ C, recorded at different time points, Figure S6: Cytotoxicity of complex 1, CDDP, NDGA and DSCP against a panel of cell lines for 24 h and 72 h treatments, Figure S7: Representative absorbance spectra of DNA concentrations, Figure S8: Tolera- bility assessment of mice for complex 1 over four weeks, Figure S9: Immune-enhancing metronomic schedule (IMS) used for GL261 GB therapy in mice, Figure S10: Representative evolution of tumor volume recorded by high-resolution T2w MRI. Author Contributions: Conceptualization, A.P.C., J.B., V.J.Y., D.R.-M., F.N. and J.L.; methodology, A.P.C., D.R.-M., F.N. and J.L.; validation, X.M., P.A., S.W., P.C.-P., A.P.C., D.R.-M., F.N. and J.L.; investigation, X.M., P.A., S.W., P.C.-P., A.P.C., J.B., V.J.Y., D.R.-M., F.N. and J.L.; resources, A.P.C.; D.R.-M., F.N. and J.L.; data curation, X.M., P.A., S.W., P.C.-P., A.P.C., F.N. and J.L.; writing—original draft preparation, A.P.C., D.R.-M., F.N. and J.L.; writing—review and editing, A.P.C., D.R.-M., F.N. and J.L.; supervision, A.P.C., F.N. and J.L.; funding acquisition, A.P.C., D.R.-M. and J.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by grant RTI2018-098027-B-C21, RTI2018-098027-B-C22 and PID2020-113058GB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF’s A way of making Europe as well as with the support from “Metalfármacos multifuncionales para el diagnóstico y la terapia” with grant RED2018-102471-T funded by MCIN/AEI/10.13039/501100011033. The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, and grant SEV-2017-0706 is funded by MCIN/AEI/10.13039/501100011033. COST is kindly acknowledged for stimulating scientific dis- cussion and financial support via the Cost Action CA 17140 “Cancer nanomedicine from the bench to the bedside”. A.P.C. received funding from the ATTRACT project funded by the EC under grant agreement 777222. The work was also supported by Centro de Investigación Biomédica en Red- Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN [http://www.ciber-bbn.es/en, accessed on 24 November 2021], CB06/01/0010), an initiative of the Instituto de Salud Carlos III (Spain). The PhD program of X.M. was supported by the La Caixa-Severo Ochoa International PhD Fellowship. We acknowledge the UAB predoctoral training program (14th Convocatoria PIF-19612, predoctoral fellowships for P.C.-P) and China Scholarship Council (predoctoral fellowship 201606990027 for S.W.). Cancers 2022, 14, 410 13 of 15 Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee Comissió d’Ètica en l’Experimentació Animal i Humana (protocol CEEAH 4859). Informed Consent Statement: Not applicable. Data Availability Statement: Data are available on the request from the corresponding author. 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