Fluorescent and Magnetic Radical Dendrimers as Potential Bimodal Imaging Probes

Citation: Zhang, S.; Lloveras, V.; Wu, Y.; Tolosa, J.; García-Martínez, J.C.; Vidal-Gancedo, J. Fluorescent and Magnetic Radical Dendrimers as Potential Bimodal Imaging Probes. Pharmaceutics 2023,15, 1776. https://doi.org/10.3390/ pharmaceutics15061776 Academic Editor: Melgardt de Villiers Received: 19 April 2023 Revised: 5 June 2023 Accepted: 9 June 2023 Published: 20 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article Fluorescent and Magnetic Radical Dendrimers as Potential Bimodal Imaging Probes Songbai Zhang 1,2, Vega Lloveras 1,3,* , Yufei Wu 1, Juan Tolosa 4,5 , Joaquín C. García-Martínez 4,5 and JoséVidal-Gancedo 1,3,* 1Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 Bellaterra, Spain; [email protected] or [email protected] (S.Z.); [email protected] (Y.W.) 2State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China 3CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, Campus UAB, 08193 Bellaterra, Spain 4Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Farmacia, Universidad de Castilla-La Mancha, C/JoséMaría Sánchez Ibáñez s/n, 02008 Albacete, Spain; [email protected] (J.T.); [email protected] (J.C.G.-M.) 5Regional Center for Biomedical Research (CRIB), Universidad de Castilla-La Mancha, C/Almansa 13, 02008 Albacete, Spain *Correspondence: [email protected] (V.L.); [email protected] (J.V.-G.) Abstract: Dual or multimodal imaging probes have emerged as powerful tools that improve detection sensitivity and accuracy in disease diagnosis by imaging techniques. Two imaging techniques that are complementary and do not use ionizing radiation are magnetic resonance imaging (MRI) and optical fluorescence imaging (OFI). Herein, we prepared metal-free organic species based on dendrimers with magnetic and fluorescent properties as proof-of-concept of bimodal probes for potential MRI and OFI applications. We used oligo(styryl)benzene (OSB) dendrimers core that are fluorescent on their own, and TEMPO organic radicals anchored on their surfaces, as the magnetic component. In this way, we synthesized six radical dendrimers and characterized them by FT-IR, 1 H NMR, UV-Vis, MALDI-TOF, SEC, EPR, fluorimetry, and in vitro MRI. Importantly, it was demonstrated that the new dendrimers present two properties: on one hand, they are paramagnetic and show the ability to generate contrast by MRI in vitro , and, on the other hand, they also show fluoresce emission. This is a remarkable result since it is one of the very few cases of macromolecules with bimodal magnetic and fluorescent properties using organic radicals as the magnetic probe. Keywords: organic radicals; dendrimers; radical dendrimers; fluorescence; quenching; imaging techniques; magnetic resonance imaging (MRI); optical fluorescence imaging (OFI) 1. Introduction Imaging techniques are decisive in the diagnosis and follow-up of many diseases and have become essential in clinical practice. Dual or multimodal imaging probes have emerged as potent tools that improve accuracy and detection sensitivity in illness diagnosis and treatment. Multimodal imaging probes are designed to overcome the inherent disadvantages of each imaging modality and take advantage of the complementary information they provide. A probe which incorporates dual or multiple imaging properties is preferred to using a mixture of various contrast agents. Among the different imaging techniques, one of the most versatile and used is magnetic resonance imaging (MRI) due to its high spatial resolution, non-ionizing character, and unlimited depth penetration. Nowadays, the most widely used contrast agents (CAs) to induce significantly improved and enhanced tissue contrast in such a technique are those based on Gd(III) chelates. However, they present toxicity concerns, and alternative imaging probes are highly required [1,2]. Pharmaceutics 2023,15, 1776. https://doi.org/10.3390/pharmaceutics15061776 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2023,15, 1776 2 of 15 Our group has been developing organic radicals for different applications for a long time [ 3 – 8 ] and, lately, we have been working with radical dendrimers, i.e., dendrimers fully functionalized by organic radicals, as an alternative to Gd(III) chelates [ 9 – 19 ]. Since they are formed by organic radicals, radical dendrimers present paramagnetic properties such as Gd-based CAs and can act like them mainly decreasing the longitudinal relaxation time (T 1 ) of the water protons and improving the contrast of the image but minimizing concerns about toxic metal accumulation. In fact, organic radicals such as nitroxides have been shown to be nontoxic in vivo [ 20 , 21 ]. Anchoring many organic radicals to a dendrimer scaffold has resulted in a successful strategy since we obtained systems with high relaxivity and with radicals protected from bioreduction. In this way, the two main limitations of isolated organic radicals were overcome, i.e., their low relaxivity and rapid bioreduction. In addition, we demonstrated that radical dendrimers are not toxic either in vitro or in vivo [13,15,18]. On the other hand, another imaging technique with a promising future is optical fluorescence imaging (OFI). Optical fluorescence imaging also has a non-ionizing character and it is commonly used in histologic analysis of cells, to monitor biodistribution, and has drawn interest from the medical community since it has potential for intraoperative use such as in imaging-guided surgery [ 22 ]. OFI has the main advantage of high sensitivity compared with MRI but it is limited by depth penetration because biological tissue attenuates light and scatters photons, and presents lower spatial resolution in vivo [ 23 ]. High spatial resolution and unrestricted depth penetration are advantages of MRI and low sensitivity is its drawback. By combining these two imaging techniques, the imaging result can be substantially improved. In this work, we aimed to prepare metal-free organic species based on dendrimers not only with magnetic properties but also with fluorescent ones, for potential MRI and optical fluorescence imaging applications. MRI/OFI imaging probes with dual modalities have been reported in some literature sources, using, the majority of them, Gd(III) chelates as the magnetic component (although the use of Fe 3 O 4 or 19 F, among others, has also been reported) and different fluorop hores [24–26]. For example, a bimodal fluorescence-magnetic resonance probe was synthesized based on DOTA-Gd(III) chelate and tetraphenylethylene as aggregationinduced emission luminogen (AIEgen), for apoptosis imaging [27]. However, very few examples have been reported on bimodal MRI/OFI imaging probes using organic radicals as the MRI probe. In fact, this is a challenging goal since, in general, when a nitroxyl radical is close to a fluorophore, the fluorescence is quenched. Different mechanisms have been proposed to explain this phenomenon such as exchange-induced relaxation processes [ 28 ], intersystem crossing [ 29 ], energy transfer interactions [ 30 ], or electron transfer [ 31 ]. These very few reported cases use polymers as scaffolds [ 32 – 34 ]. Rajca and Johnson’s group prepared bimodal MRI and fluorescent contrast agents based on organic radicals (nitroxides) and Cy5.5 fluorophores, although both agents could not work at the same time. On branched-bottlebrush polymers, the fluorescence was quenched by the radicals but once the nitroxides were reduced by ascorbate, the fluorescence intensity increased by 2 to 3.5 times [ 33 ]. The same group prepared polymeric nanoparticles with the same active agents but in that case, it could be achieved simultaneously MRI and NIRF imaging in vivo due to the larger distance between the fluorophore and the nitroxides [ 34 ]. Some of the ways used to impart fluorescence to dendrimers are by anchoring or encapsulating fluorescent dyes in the dendrimer’s structure. However, in this work, we used a different strategy, that is, the use of dendrimers that are fluorescent by themselves. With this strategy, additional synthetic steps due to the anchoring of fluorophores can be avoided, and it is, therefore, a less time-consuming method. In addition, at the same time, in this way all the end groups of the branches are free for full functionalization with organic radicals, resulting in a more optimal strategy. We used oligo(styryl)benzene (OSB) dendrimers cores (Figure 1) that present fluorescent properties on their own and are biocompatible [ 35 ]. We synthesized new radical dendrimers based on them, explor- Pharmaceutics 2023,15, 1776 3 of 15 ing their magnetic and fluorescent properties as proof of concept for potential MRI and OFI applications. Pharmaceutics 2023, 15, x FOR PEER REVIEW 3 of 18 organic radicals, resulting in a more optimal strategy. We used oligo(styryl)benzene (OSB) dendrimers cores (Figure 1) that present fluorescent properties on their own and are biocompatible [35]. We synthesized new radical dendrimers based on them, exploring their magnetic and fluorescent properties as proof of concept for potential MRI and OFI applications. Figure 1. Chemical structures of oligo(styryl)benzene dendrimers cores (tri-acid 7, tetra-acid 8, trialdehyde 9, and tetra-aldehyde 10) derivatives used in this work to synthesize radical dendrimers. 2. Materials and Methods 2.1. Materials All reactants were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA) at the highest purity available and used without further purification. THF, CH2Cl2, DMSO, and ethanol (HPLC grade) were obtained from SDS-Carlo Erba (Sabadell, Spain). CH2Cl2 was distilled over CaH2 and THF over metallic sodium. 2.2. Methods Chromatography. Thin Layer Chromatography (TLC) was performed on Merck 60F254 silica plates and were visualized by UV (254 nm), or by ninhydrin. Silica column chromatography was carried out using silica gel 60 (35–70 mesh). Size exclusion chromatography (SEC) analysis was carried out using an Agilent 1260 infinity II liquid chromatography system apparatus equipped with a diode array detector under the following conditions: a PSS SDV pre-column (3 µm, 8 × 50 mm) and a PSS SDV analytical column (3 µm, 1000 Å, 8 × 300 mm) with a diode array detector were used. CHCl3 was used as an eluent at a flow rate of 0.5 mL/min at 35 °C. Radical dendrimers were dissolved in the eluent to reach a final concentration of 1 mg/mL and filtered through a 0.2 µm PTFE filter before injection. Nuclear Magnetic Resonance spectroscopy (NMR) 1H NMR (250 MHz) spectroscopy was performed at Bruker spectrometer Avance DRX-250. Chemical shifts (δ) are expressed in parts per million downfield from tetramethylsilyl chloride. The following abbreviations are used to indicate multiplicity: s: singlet; d: doublet, m: multiplet. Mass spectrometry. Matrix-assisted laser desorption/ionization-time-of-flight mass (MALDI-TOF) BIFLEX spectrometer (Bruker-Franzen Analytik) equipped with a pulsed nitrogen laser (337 nm), using 19 kV acceleration voltage, at UAB. Dithranol was used as a matrix. Electron Paramagnetic Resonance spectroscopy (EPR) spectra were obtained with an X-Band (9.7 GHz) Bruker ELEXSYS E500 spectrometer equipped with an ST8911 Figure 1. Chemical structures of oligo(styryl)benzene dendrimers cores (tri-acid 7 , tetra-acid 8 , tri-aldehyde 9, and tetra-aldehyde 10 ) derivatives used in this work to synthesize radical dendrimers. 2. Materials and Methods 2.1. Materials All reactants were purchased from Sigma Aldrich Inc. (St. Louis, MO, USA) at the highest purity available and used without further purification. THF, CH2Cl2, DMSO, and ethanol (HPLC grade) were obtained from SDS-Carlo Erba (Sabadell, Spain). CH 2 Cl 2 was distilled over CaH2and THF over metallic sodium. 2.2. Methods Chromatography . Thin Layer Chromatography (TLC) was performed on Merck 60F254 silica plates and were visualized by UV (254 nm), or by ninhydrin. Silica column chromatography was carried out using silica gel 60 (35–70 mesh). Size exclusion chromatography (SEC) analysis was carried out using an Agilent 1 260 in finity II liquid chromatography system apparatus equipped with a diode array detector under the following conditions: a PSS SDV pre-column (3 µ m, 8 × 50 mm) and a PSS SDV analytical column (3 µ m, 1000 Å, 8 × 300 mm) with a diode array detector were used. CHCl 3 was used as an eluent at a flow rate of 0.5 mL/min at 35 ◦ C. Radical dendrimers were dissolved in the eluent to reach a final concentration of 1 mg/mL and filtered through a 0.2 µm PTFE filter before injection. Nuclear Magnetic Resonance spectroscopy (NMR) 1 H NMR (250 MHz) spectroscopy was performed at Bruker spectrometer Avance DRX-250. Chemical shifts ( δ ) are expressed in parts per million downfield from tetramethylsilyl chloride. The following abbreviations are used to indicate multiplicity: s: singlet; d: doublet, m: multiplet. Mass spectrometry . Matrix-assisted laser desorption/ionization-time-of-flight mass (MALDI-TOF) BIFLEX spectrometer (Bruker-Franzen Analytik) equipped with a pulsed nitrogen laser (337 nm), using 19 kV acceleration voltage, at UAB. Dithranol was used as a matrix. Electron Paramagnetic Resonance spectroscopy (EPR) spectra were obtained with an X-Band (9.7 GHz) Bruker ELEXSYS E500 spectrometer equipped with an ST8911 microwave cavity, a Bruker variable temperature unit, a field frequency lock system Bruker ER 0 33 M and an NMR Gaussmeter Bruker ER 035 M. The modulation amplitude was kept well below the line width, and the microwave power was well below saturation. Samples were previously degassed with Ar. Pharmaceutics 2023,15, 1776 4 of 15 Fourier-transform infrared spectroscopy (FT-IR) spectra were recorded in a FT/IR4700 spectrophotometer from JASCO (Tokyo, Japan) with an ATR (attenuated total reflectance) accessory, in the 400–4000 cm−1range with 4 cm−1resolution. Ultraviolet-visible spectroscopy (UV-Vis) spectra were recorded in a UV-Vis-Nir JASCO V-780 spectrophotometer (Tokyo, Japan), in the 200–800 nm range. Fluorescence spectroscopy emission spectra were recorded in a Varian Cary Eclipse fluorimeter from the Laboratori de Luminiscència i Espectroscòpia de Biomolècules (LLEB), at the UAB, in the range 320–800 nm, excitation slit 5 nm, emission 5 nm. Magnetic Resonance Imaging (MRI) experiments were carried out in a BioSpec 70/30 Bru ker system using a 7.0 T horizontal-bore superconducting magnet equipped with actively shielded gradients (B-GA12 gradient coil inserted into a B-GA20S gradient system). A quadrature 72 mm inner diameter volume coil was used for in vitro studies. Relaxometric measurements . Longitudinal (r 1 ) relaxivities were determined per concentration of (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO) units. Different concentrations of TEMPO radicals were prepared, from 10 to 0.63 mM. Relaxivity measurements were obtained at room temperature. The software used for the calculations of T 1 relaxations was Paravision 6.0 (Bruker Software). r1relaxivity . T 1 maps were performed with Rapid Acquisition with Relaxation Enhancement (RARE) sequence with a variable repetition time (VTR). Series of axial T 1 -weighted (T 1 W) images were acquired for each concentration of TEMPO to obtain T 1 maps based on a magnetization saturation experiment and the following parameters: Images at 18 different TR values were acquired: TR = 50, 80, 120, 160, 200, 250, 300, 360, 420, 500, 650, 850, 1100, 1600, 2200, 3000, 5000, and 10,000 ms. T 1 measurements were acquired with a reduced echo time of 6 ms to minimize signal loss due to T 2 . Field of view (FOV) = 2.5 × 2.5 cm, averages (Av) = 1, acquisition matrix ( Mtx) = 128 ×128 . The T 1 values were calculated from the mean signal in the region of interest (ROI) for each repetition time, adjusted to the following equation: S=So[1 −(−TR/T1)]. 3. Results and Discussion 3.1. Synthesis and Characterization of Radical Dendrimers Based on Oligo(styryl)Benzenes We first synthesized oligo(styryl)benzene dendrimers with three and four branches ended in carboxylic acid and aldehyde groups, as previously reported [ 35 , 36 ], named in this work tri-acid ( 7 ), tetra-acid ( 8 ), tri-aldehyde ( 9 ) and tetra-aldehyde ( 10 ) (Figure 1). Briefly, the synthesis is based on the Horner-Wadsworth-Emmons reaction for the formation of the double bonds in situ from phosphonate triand tetra-substituted benzenes and the corresponding precursor aldehyde. These compounds are considered the core of a family of dendrimers. Then, we synthesized a series of radical dendrimers based on them by coupling the amino group of 4-amino-TEMPO radical to the carboxyl or aldehyde end groups, leading to amidoand imino-radical dendrimers derivatives, respectively. In addition, the iminoderivatives were reduced to the corresponding amino-derivatives since a secondary amine is a more stable linker (see the synthesis details in the Supplementary Materials). We are using these compounds as a proof of concept for the future higher generations of radical dendrimers that we plan to develop using the same fluorescent core. In this way, we obtained six radical dendrimers ( 1 – 6 , Schemes 1and 2) which were characterized by FT-IR, 1 H NMR, UV-Vis, MALDI-TOF, SEC, and EPR. Moreover, fluorescence and MRI studies were carried out to check whether bimodality has been achieved in these species or not. Finally, we also optimized the geometry structure of the obtained radical species 1 – 6 at UB3LYP-D3(BJ)/6–31G(d) level in the gas phase using Gaussian 16 (see Figure 2for 1–2structures and Scheme S1 for 3 – 6 ones). The optimized structures are similar to those of their dendrimers precursors [ 37 ]. They are constituted by a highly conjugated backbone of π -bonds that confers a high rigidity. In the case of the tri-substituted compounds, the styrylbenzene branches are arranged towards the apexes of a triangle while the tetrasubstituted compounds present a cross conformation of the styrylbenzene branches with a Pharmaceutics 2023,15, 1776 5 of 15 higher conjugation than the three-styrylbenzene system [ 37 ]. The incorporation of TEMPO radicals at the dendrimers’ surface through a flexible functionalization does not affect the conjugated backbone so their arrangement is fixed and determined by the rigid separation of the structure. The distances between the N-O • groups of the radicals in the tri-substituted compounds are larger (ca. 25 Å) than in the four-substituted analogs in which the shortest distance is between 11 and 14 Å. Pharmaceutics 2023, 15, x FOR PEER REVIEW 6 of 18 Scheme 1. Synthesis of tri-amido-TEMPO 1 and tetra-amido-TEMPO 2 radical dendrimers. The IR spectra of tri-acid dendrimer (7) and the corresponding triand tetra-amidoTEMPO (1 and 2) radical dendrimers are shown in Figure S1. We followed up the reaction by IR, by the shift of the C=O stretching band from 1674 cm−1 (-COOH group) to 1632 cm−1 (-CONH amido group) for both compounds, as well as by the disappearance of the very broad OH stretching band at 3250–2500 cm−1 from the -COOH groups, and the appearance of the new NH stretching band from the amido group at 3299 and 3297 cm−1 for triand tetra-amido-TEMPO, respectively. In addition, we can observe the -CH stretching bands of -CH3 and -CH2groups of TEMPO radicals at the range ca. 2854–2971 cm−1, and the bands assigned to the N-O• stretching of the TEMPO radicals at 1362–64 cm−1 [38,39]. In order to characterize by 1H NMR such species, we used phenylhydrazine to reduce the nitroxyl radicals to the corresponding diamagnetic hydroxylamine (Scheme S2). In Figure 3, we can observe both 1H NMR spectra with the corresponding peak labeling. The peaks of the TEMPO protons can be found in the range of 1–1.73 ppm (a and b) and at 4.22 ppm (c) while the protons of the dendrimer structure (d, e, f, g, h) can be found between 7.47 and 8.17 ppm. Moreover, the relative integral values of the 1H resonances were in agreement with the theoretical ones, confirming the number of TEMPO radicals anchored on their structures (3 and 4, respectively). Scheme 1. Synthesis of tri-amido-TEMPO 1and tetra-amido-TEMPO 2radical dendrimers. Pharmaceutics 2023, 15, x FOR PEER REVIEW 8 of 18 SEC was used to check the purity of both triand tetra-amido-TEMPO 1 and 2. Both compounds showed only one narrow size distribution band. The slightly lower retention time obtained for tetra-amido-TEMPO 2 was in agreement with the slightly larger size than tri-amido-TEMPO 1 (Figure 4). Figure 4. SEC chromatograms of (a) tri-amido-TEMPO (1) and (b) tetra-amido-TEMPO (2) radical dendrimers. 3.1.2. Synthesis and Characterization of Imino and Amino Radical Dendrimers Derivatives (3–6) To obtain the imino derivative compounds, triand tetra-aldehyde dendrimers (9 and 10) were subjected to sonification in the presence of alumina gel with an excess of 4-aminoTEMPO in THF [40,41] (Scheme 2). The reaction completion was also followed up by IR, by the disappearance of the aldehyde band. The alumina gel was removed by filtration, and the resulting product precipitated from the reaction mixture by the addition of npentane. In this way, we obtained the radical dendrimers tri-imino-TEMPO (3) and tetraimino-TEMPO (4) in 63% and 85% yields, respectively. N N O N NO N N O N NO N N O N N O N N O tetra-imino-TEMPO 4 tri-imino-TEMPO 3 O O O O O O O tetra-aldehyde 10 tri-aldehyde 9Al 2 O 3 NH 2 NO THF, rt tri-amino-TEMPO 5 HN N O H N NO H N N O tetra-amino-TEMPO 6 NaBH 4 MeOH, CHCl 3 N H NO N H N O H N N O H N N O Scheme 2. Synthesis of tri-imino-TEMPO 3, tetra-imino-TEMPO 4, tri-amino-TEMPO 5, and tetraamino-TEMPO 6 radical dendrimers. On the other hand, the synthesis of triand tetra-amino-TEMPO (5 and 6) derivatives was performed by reduction of triand tetra-imino-TEMPO (3 and 4) with NaBH 4 , as shown in Scheme 2. After their reduction with NaBH 4 overnight in methanol and chloroform, the reaction mixture was extracted with dichloromethane/water three times, and the products were obtained in 97% and 95% yields, respectively. See the synthesis details of 3–6 compounds in the Supplementary Materials. Scheme 2. Synthesis of tri-imino-TEMPO 3 , tetra-imino-TEMPO 4 , tri-amino-TEMPO 5, and tetraamino-TEMPO 6radical dendrimers. 3.1.1. Synthesis and Characterization of Amido Radical Dendrimers Derivatives 1and 2 The coupling of 4-amino-TEMPO to the carboxyl end groups of OSB tri-acid 7 and tetra-acid 8 has been carried out using HATU/DIEA as a coupling agent, as shown in Scheme 1. The synthesis of tri-amido-TEMPO 1 was performed in THF, while for tetra-amido-TEMPO 2 , a small amount of DMSO was added to make tetra-acid 8 soluble in the reaction system. After an overnight reaction at room temperature, the products were first extracted in dichloromethane/water and then purified by column chromatography on silica gel in dichloromethane/EtOH. The obtained yields were 88 and 53%, for tri-amido-TEMPO 1 and tetra-amido-TEMPO 2 , respectively (see the synthesis details of 1–2compounds in the Supplementary Materials). Pharmaceutics 2023,15, 1776 6 of 15 Pharmaceutics 2023, 15, x FOR PEER REVIEW 5 of 18 Finally, we also optimized the geometry structure of the obtained radical species 1– 6 at UB3LYP-D3(BJ)/6–31G(d) level in the gas phase using Gaussian 16 (see Figure 2 for 1– 2 structures and Scheme S1 for 3–6 ones). The optimized structures are similar to those of their dendrimers precursors [37]. They are constituted by a highly conjugated backbone of π-bonds that confers a high rigidity. In the case of the tri-substituted compounds, the styrylbenzene branches are arranged towards the apexes of a triangle while the tetrasubstituted compounds present a cross conformation of the styrylbenzene branches with a higher conjugation than the three-styrylbenzene system [37]. The incorporation of TEMPO radicals at the dendrimers’ surface through a flexible functionalization does not affect the conjugated backbone so their arrangement is fixed and determined by the rigid separation of the structure. The distances between the N-O • groups of the radicals in the tri-substituted compounds are larger (ca. 25 Å) than in the four-substituted analogs in which the shortest distance is between 11 and 14 Å. Figure 2. Optimized geometric structures of radical dendrimers 1–2 at UB3LYP-D3(BJ)/6–31G(d) level in the gas phase. See Scheme S1 for the corresponding optimized geometric structures of compounds 3–6. 3.1.1. Synthesis and Characterization of Amido Radical Dendrimers Derivatives 1 and 2 The coupling of 4-amino-TEMPO to the carboxyl end groups of OSB tri-acid 7 and tetra-acid 8 has been carried out using HATU/DIEA as a coupling agent, as shown in Scheme 1. The synthesis of tri-amido-TEMPO 1 was performed in THF, while for tetraamido-TEMPO 2, a small amount of DMSO was added to make tetra-acid 8 soluble in the reaction system. After an overnight reaction at room temperature, the products were first extracted in dichloromethane/water and then purified by column chromatography on silica gel in dichloromethane/EtOH. The obtained yields were 88 and 53%, for tri-amidoTEMPO 1 and tetra-amido-TEMPO 2, respectively (see the synthesis details of 1–2 compounds in the Supplementary Materials). tri-amido-TEMPO 1 tetra-amido-TEMPO 2 25.35 Å 14.18 Å 24.35 Å Figure 2. Optimized geometric structures of radical dendrimers 1 – 2 at UB3LYP-D3(BJ)/6–31G(d) level in the gas phase. See Scheme S1 for the corresponding optimized geometric structures of compounds 3–6. The IR spectra of tri-acid dendrimer ( 7 ) and the corresponding triand tetra-amidoTEMPO ( 1 and 2 ) radical dendrimers are shown in Figure S1. We followed up the reaction by IR, by the shift of the C=O stretching band from 1674 cm −1 (-COOH group) to 1632 cm −1 (-CONH amido group) for both compounds, as well as by the disappearance of the very broad OH stretching band at 3250–2500 cm −1 from the -COOH groups, and the appearance of the new NH stretching band from the amido group at 3299 and 3297 cm −1 for triand tetra-amido-TEMPO, respectively. In addition, we can observe the -CH stretching bands of -CH 3 and -CH 2 - groups of TEMPO radicals at the range ca. 2854–2971 cm −1 , and the bands assigned to the N-O•stretching of the TEMPO radicals at 1362–64 cm−1[38,39]. In order to characterize by 1 H NMR such species, we used phenylhydrazine to reduce the nitroxyl radicals to the corresponding diamagnetic hydroxylamine (Scheme S2). In Figure 3, we can observe both 1 H NMR spectra with the corresponding peak labeling. The peaks of the TEMPO protons can be found in the range of 1–1.73 ppm (aand b) and at 4.22 ppm (c) while the protons of the dendrimer structure (d,e,f,g,h) can be found between 7.47 and 8.17 ppm. Moreover, the relative integral values of the 1 H resonances were in agreement with the theoretical ones, confirming the number of TEMPO radicals anchored on their structures (3 and 4, respectively). Triand tetra-amido-TEMPO ( 1 and 2 ) were also characterized by UV-Vis spectroscopy (see Figures S2–S4 of the Supplementary Materials). By UV-Vis spectroscopy, we can quantify the number of TEMPO radical units anchored to the dendrimers since the lowintensity nπ * transition band at ca. 450 nm from the TEMPO radical is known to be additive. The molar extinction coefficient ( ε ) of the ca. 450 nm band of TEMPO radical is around 10 M −1 cm −1 , with small variations depending on the solvent used [ 17 ]. The corresponding ε value for tri-amido-TEMPO ( 1 ) in dichloromethane was 30.6 M −1 cm −1 , indicating 3 times higher molar extinction coefficient than the free TEMPO in the same solvent (11.1 M −1 cm −1 , Figure S2) confirming that three radicals were coupled to the dendrimer. Unfortunately, it was not possible to confirm by UV-Vis spectroscopy the degree of substitution in the tetra-amido-TEMPO ( 2 ) since the higher conjugation of the tetra-styrylbenzene dendrimer, compared with the tri-substituted one, resulted in a red shift of the absorption bands which overlapped with the TEMPO absorption band (Figure S4). By MALDI-TOF mass spectrometry, we observed the molecular ion peaks [M + H] + of triand tetra-amido-TEMPO 1 and 2 at m/z= 977.2 and 1277.2, respectively, which are in agreement with the theoretical molecular mass (976.30 and 1275.69 Da, respectively). Also, it was observed a [M + Na]+cluster in both cases (Figure S5). Pharmaceutics 2023,15, 1776 7 of 15 Pharmaceutics 2023, 15, x FOR PEER REVIEW 7 of 18 Figure 3. 1 H NMR spectra of (a) tri-amido-TEMPO (1) and (b) tetra-amido-TEMPO (2) after being reduced with phenylhydrazine (DMSO-d 6 , 250 MHz). Triand tetra-amido-TEMPO (1 and 2) were also characterized by UV-Vis spectroscopy (see Figures S2–S4 of the Supplementary Materials). By UV-Vis spectroscopy, we can quantify the number of TEMPO radical units anchored to the dendrimers since the low-intensity n-π* transition band at ca. 450 nm from the TEMPO radical is known to be additive. The molar extinction coefficient (ε) of the ca. 450 nm band of TEMPO radical is around 10 M −1 cm −1 , with small variations depending on the solvent used [17]. The corresponding ε value for tri-amido-TEMPO (1) in dichloromethane was 30.6 M −1 cm −1 , indicating 3 times higher molar extinction coefficient than the free TEMPO in the same solvent (11.1 M −1 cm −1 , Figure S2) confirming that three radicals were coupled to the dendrimer. Unfortunately, it was not possible to confirm by UV-Vis spectroscopy the degree of substitution in the tetra-amido-TEMPO (2) since the higher conjugation of the tetra-styrylbenzene dendrimer, compared with the tri-substituted one, resulted in a red shift of the absorption bands which overlapped with the TEMPO absorption band (Figure S4). By MALDI-TOF mass spectrometry, we observed the molecular ion peaks [M + H] + of triand tetra-amido-TEMPO 1 and 2 at m/z = 977.2 and 1277.2, respectively, which are in agreement with the theoretical molecular mass (976.30 and 1275.69 Da, respectively). Also, it was observed a [M + Na] + cluster in both cases (Figure S5). Figure 3. 1 H NMR spectra of ( a ) tri-amido-TEMPO ( 1 ) and ( b ) tetra-amido-TEMPO ( 2 ) after being reduced with phenylhydrazine (DMSO-d6, 250 MHz). SEC was used to check the purity of both triand tetra-amido-TEMPO 1 and 2 . Both compounds showed only one narrow size distribution band. The slightly lower retention time obtained for tetra-amido-TEMPO 2 was in agreement with the slightly larger size than tri-amido-TEMPO 1(Figure 4). Pharmaceutics 2023, 15, x FOR PEER REVIEW 8 of 18 SEC was used to check the purity of both triand tetra-amido-TEMPO 1 and 2. Both compounds showed only one narrow size distribution band. The slightly lower retention time obtained for tetra-amido-TEMPO 2 was in agreement with the slightly larger size than tri-amido-TEMPO 1 (Figure 4). Figure 4. SEC chromatograms of (a) tri-amido-TEMPO (1) and (b) tetra-amido-TEMPO (2) radical dendrimers. 3.1.2. Synthesis and Characterization of Imino and Amino Radical Dendrimers Derivatives (3–6) To obtain the imino derivative compounds, triand tetra-aldehyde dendrimers (9 and 10) were subjected to sonification in the presence of alumina gel with an excess of 4-aminoTEMPO in THF [40,41] (Scheme 2). The reaction completion was also followed up by IR, by the disappearance of the aldehyde band. The alumina gel was removed by filtration, and the resulting product precipitated from the reaction mixture by the addition of npentane. In this way, we obtained the radical dendrimers tri-imino-TEMPO (3) and tetraimino-TEMPO (4) in 63% and 85% yields, respectively. N N O N NO N N O N NO N N O N N O N N O tetra-imino-TEMPO 4 tri-imino-TEMPO 3 O O O O O O O tetra-aldehyde 10 tri-aldehyde 9Al 2 O 3 NH 2 NO THF, rt tri-amino-TEMPO 5 HN N O H N NO H N N O tetra-amino-TEMPO 6 NaBH 4 MeOH, CHCl 3 N H NO N H N O H N N O H N N O Scheme 2. Synthesis of tri-imino-TEMPO 3, tetra-imino-TEMPO 4, tri-amino-TEMPO 5, and tetraamino-TEMPO 6 radical dendrimers. On the other hand, the synthesis of triand tetra-amino-TEMPO (5 and 6) derivatives was performed by reduction of triand tetra-imino-TEMPO (3 and 4) with NaBH 4 , as shown in Scheme 2. After their reduction with NaBH 4 overnight in methanol and chloroform, the reaction mixture was extracted with dichloromethane/water three times, and the products were obtained in 97% and 95% yields, respectively. See the synthesis details of 3–6 compounds in the Supplementary Materials. Figure 4. SEC chromatograms of ( a ) tri-amido-TEMPO ( 1 ) and ( b ) tetra-amido-TEMPO ( 2 ) radical dendrimers. Pharmaceutics 2023,15, 1776 8 of 15 3.1.2. Synthesis and Characterization of Imino and Amino Radical Dendrimers Derivatives (3–6) To obtain the imino derivative compounds, triand tetra-aldehyde dendrimers ( 9 and 10 ) were subjected to sonification in the presence of alumina gel with an excess of 4-amino-TEMPO in THF [ 40 , 41 ] (Scheme 2). The reaction completion was also followed up by IR, by the disappearance of the aldehyde band. The alumina gel was removed by filtration, and the resulting product precipitated from the reaction mixture by the addition of n-pentane. In this way, we obtained the radical dendrimers tri-imino-TEMPO ( 3 ) and tetra-imino-TEMPO (4) in 63% and 85% yields, respectively. On the other hand, the synthesis of triand tetra-amino-TEMPO ( 5 and 6 ) derivatives was performed by reduction of triand tetra-imino-TEMPO ( 3 and 4 ) with NaBH 4 , as shown in Scheme 2. After their reduction with NaBH 4 overnight in methanol and chloroform, the reaction mixture was extracted with dichloromethane/water three times, and the products were obtained in 97% and 95% yields, respectively. See the synthesis details of 3–6compounds in the Supplementary Materials. As well as in compounds 1 and 2 , radical dendrimers 3 – 6 were also successfully characterized by IR, 1 H NMR, UV-Vis, MALDI-TOF, and SEC, confirming their structure and purity. See the Supplementary Materials for the full characterization of 3 – 6 with these techniques (Figures S6–S16). 3.1.3. EPR Study of the Radical Dendrimers 1–6 The EPR spectrum of TEMPO free radical at 300 K showed the typical 3-line spectrum of nitroxides, with the same relative intensities 1:1:1, a coupling constant with the 14 N atom a N = 15.7 G, a line width of ∆ Hpp = 1.20 G and a g-factor = 2.0061 (Figure 5). The EPR spectra of the radical dendrimers 1 – 6 at 300 K also showed 3 lines, indicating that they showed negligible spin exchange interaction between their radical units, probably because of the rigidity of the structures that do not allow radicals to approach each other. They presented similar a N and gfactor than the TEMPO free radical (a N ~ 15.4/15.5 G and g between 2.0053 and 2.0064, Table 1). However, the line width of their spectrum lines was slightly broader (~1.60 G) and they presented a selective decrease in the high-field line. These two features are due to the impeded motion of the radicals when they are attached to a large molecule, confirming, thus, the anchoring of the radicals to the dendrimers. Pharmaceutics 2023, 15, x FOR PEER REVIEW 9 of 18 As well as in compounds 1 and 2, radical dendrimers 3–6 were also successfully characterized by IR, 1H NMR, UV-Vis, MALDI-TOF, and SEC, confirming their structure and purity. See the Supplementary Materials for the full characterization of 3–6 with these techniques (Figures S6–S16). 3.1.3. EPR Study of the Radical Dendrimers 1–6 The EPR spectrum of TEMPO free radical at 300 K showed the typical 3-line spectrum of nitroxides, with the same relative intensities 1:1:1, a coupling constant with the 14N atom aN = 15.7 G, a line width of ΔHpp = 1.20 G and a g-factor = 2.0061 (Figure 5). The EPR spectra of the radical dendrimers 1–6 at 300 K also showed 3 lines, indicating that they showed negligible spin exchange interaction between their radical units, probably because of the rigidity of the structures that do not allow radicals to approach each other. They presented similar aN and g factor than the TEMPO free radical (aN ~ 15.4/15.5 G and g between 2.0053 and 2.0064, Table 1). However, the line width of their spectrum lines was slightly broader (~1.60 G) and they presented a selective decrease in the high-field line. These two features are due to the impeded motion of the radicals when they are attached to a large molecule, confirming, thus, the anchoring of the radicals to the dendrimers. Figure 5. EPR normalized spectra of TEMPO, tri-amido-TEMPO 1, tetra-amido-TEMPO 2, tri-iminoTEMPO 3, tetra-imino-TEMPO 4, tri-amino-TEMPO 5 and tetra-amino-TEMPO 6 radical dendrimers in THF at 300 K. Table 1. EPR data of TEMPO and radical species 1–6 at 300 K and at 120 K, in THF. Compound 300 K 120 K g aN (G) ΔHpp (G) d1/d Area (a.u.) TEMPO 2.0061 15.7 1.20 0.53 2.47 × 105 tri-amido-TEMPO (1) 2.0059 15.5 1.61 0.66 7.22 × 105 tri-imino-TEMPO (3) 2.0060 15.4 1.59 0.63 7.12 × 105 tri-amino-TEMPO (5) 2.0057 15.4 1.62 0.60 7.31 × 105 tetra-amido-TEMPO (2) 2.0064 15.5 1.64 0.80 9.49 × 105 tetra-imino-TEMPO (4) 2.0053 15.4 1.56 0.79 9.61 × 105 tetra-amino-TEMPO (6) 2.0057 15.4 1.40 0.75 9.52 × 105 On the other hand, the EPR signal intensity (the area from the double integral) of the triand tetra-compound derivatives was found to be around three and four times higher than the TEMPO free radical (see Table 1). In frozen solution, 120 K (Figure S17), the EPR spectra shape of the radical dendrimer species with three radical units was similar to the obtained with the TEMPO free radical, 3250 3300 3350 3400 H / Gauss TEMPO tri-amido-TEMPO 33 tri-imino-TEMPO 35 tri-amino-TEMPO 37 3250 3300 3350 3400 H / Gauss tetra-amido-TEMPO 34 tetra-imino-TEMPO 36 tetra-amino-TEMPO 38 1 1 3 2 4 6 5 Figure 5. EPR normalized spectra of TEMPO, tri-amido-TEMPO 1 , tetra-amido-TEMPO 2 , tri-iminoTEMPO 3 , tetra-imino-TEMPO 4 , tri-amino-TEMPO 5 and tetra-amino-TEMPO 6 radical dendrimers in THF at 300 K. Pharmaceutics 2023,15, 1776 9 of 15 Table 1. EPR data of TEMPO and radical species 1–6at 300 K and at 120 K, in THF. Compound 300 K 120 K g aN(G) ∆Hpp (G) d1/dArea (a.u.) TEMPO 2.0061 15.7 1.20 0.53 2.47 ×105 tri-amido-TEMPO (1) 2.0059 15.5 1.61 0.66 7.22 ×105 tri-imino-TEMPO (3) 2.0060 15.4 1.59 0.63 7.12 ×105 tri-amino-TEMPO (5) 2.0057 15.4 1.62 0.60 7.31 ×105 tetra-amido-TEMPO (2) 2.0064 15.5 1.64 0.80 9.49 ×105 tetra-imino-TEMPO (4) 2.0053 15.4 1.56 0.79 9.61 ×105 tetra-amino-TEMPO (6) 2.0057 15.4 1.40 0.75 9.52 ×105 On the other hand, the EPR signal intensity (the area from the double integral) of the triand tetra-compound derivatives was found to be around three and four times higher than the TEMPO free radical (see Table 1). In frozen solution, 120 K (Figure S17), the EPR spectra shape of the radical dendrimer species with three radical units was similar to the obtained with the TEMPO free radical, thus, showing weak dipole-dipole interactions among the TEMPO radicals. In fact, the empirical ratio d 1 /dvalues, which indicate the strength of the dipolar interactions, were only slightly higher than for TEMPO free radical (see Table 1). However, in tetrasubstituted derivatives, these interactions were a bit larger, which means radicals are closer in those structures, in agreement with the distances obtained from the optimized structures (Figure 2 ), i.e., shorter radical–radical distance in the four-substituted derivatives than in the three-substituted ones. Under these conditions, all compounds showed a | ∆ m s | = 2 transition at halffield (Figure S18), typical of dipolar coupled spins that demonstrates a high-spin state is present. This signal was more intense in compounds with four radical units than in the tri-substituted analogs. 3.2. Fluorimetry Study of the OSB Precursors 7–10 and Radical Dendrimers 1–6 In order to check the effect of nitroxyl radicals on the fluorescent properties of the OSB derivatives, we measured the fluorescence intensity of OSB precursors 7 – 10 and the corresponding radical dendrimers 1–6in THF, using quinine sulfate as standard. The corresponding UV-Vis spectra and fluorescence emission spectra are shown in Figure 6and Figure S19, respectively, while the calculated quantum yields (QY), λexc. and λem. values are shown in Table 2. See more details about the fluorescence process and the quantum yield calculation method in the Supplementary Materials. According to the spectroscopic results obtained it is observed that the absorption occurs mainly on the part of the oligo(styryl)benzene units since both the absorbance maximum and the shape of the bands between the precursor dendrimers and the radical dendrimers are coincident. The optical properties of OSBs are highly dependent on their peripheral substituents [ 42 , 43 ]. Specifically, for the tri-acid 7 and tri-aldehyde 9 compounds, a previous study indicates that these absorptions correspond mainly to various contributions from S 0→ S 1 and S 0→ S 2 transitions and the attached functional groups participate in these transitions by stabilizing or destabilizing the HOMO or LUMO [ 44 ]. The tetra-substituted derivatives show more red-shifted absorbances. When comparing the tri-amido-TEMPO 1 and tetra-amido-TEMPO 2 derivatives with the starting precursors (tri-acid 7 and tetraacid 8 , respectively), it is observed that there is no substantial variation in the absorbance maximum for each of them. This is because the electronic characteristics of the OSB do not change substantially since the differences between the electronic properties of the acid group and the amido group are not large enough to modify the OSB since both are electron-withdrawing groups. However, when comparing the iminoand amino-radical