Highly Sensitive Ratiometric Fluorescent Fiber Matrixes for Oxygen Sensing with Micrometer-Spatial Resolution

Bio-Design and Manufacturing (2024) 7:292–306 https://doi.org/10.1007/s42242-024-00277-3 RESEARCH ARTICLE Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution Giuliana Grasso1·Valentina Onesto1·Stefania Forciniti1·Eliana D’Amone1·Francesco Colella1,2 · Lara Pierantoni3,4 ·Valeria Famà1·Giuseppe Gigli1,5 ·Rui L. Reis3,4 ·J. Miguel Oliveira3,4 · Loretta L. del Mercato1 Received: 27 October 2023 / Accepted: 2 April 2024 / Published online: 29 April 2024 © The Author(s) 2024 Abstract Oxygen (O2)-sensing matrices are promising tools for the live monitoring of extracellular O2consumption levels in long-term cell cultures. In this study, ratiometric O2-sensing membranes were prepared by electrospinning, an easy, low-cost, scalable, and robust method for fabricating nanofibers. Poly(ε-caprolactone) and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, which was used as the O2-sensing probe, and rhodamine B isothiocyanate, which was used as the reference dye. The functionalized scaffolds were morphologically characterized by scanning electron microscopy, and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy, thermogravimetric analysis, and water contact angle measurement. The sensing capabilities were investigated by confocal laser scanning microscopy, performing photobleaching, reversibility, and calibration curve studies toward different dissolved O2(DO) concentrations. Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5% to 20% and good stability under ratiometric imaging. In addition, the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines, namely metastatic melanoma cell line SK-MEL2, breast cancer cell line MCF-7, and pancreatic ductal adenocarcinoma cell line Panc-1, thus recreating a suitable biological environment in vitro.These O2-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O2content during drug testing/validation and tissue regeneration processes. Graphic abstract Keywords Electrospinning ·Ruthenium(II) dichloride ·Oxygen sensors ·Ratiometric imaging ·Fluorescence Giuliana Grasso and Valentina Onesto have contributed equally to this work. Extended author information available on the last page of the article Introduction Hypoxia, a condition in which a tissue gradually decreases in oxygen (O2) levels, is generally recognized as a prodromic 123 Bio-Design and Manufacturing (2024) 7:292–306 293 event in cancer growth and progression. The master regulator driving hypoxia is hypoxia-inducible factor-1 [1], which is usually silent in normoxic conditions (5%–10% O2)but strongly upregulated during anoxia (<1.5% O2) in several cancer types, such as melanoma [2], breast cancer [3], and pancreatic ductal adenocarcinoma [4]. The pharmacological and clinical relevance of O2concentration makes it a wellrecognized cancer biomarker [5]. Over the last few years, many strategies have been developed for sensing and imaging O2[6]. Optical O2sensors have become attractive because of various features such as reversibility, selectivity, and miniaturization. Moreover, optical sensors allow noninvasive measurements and image O2at high spatial resolutions (micrometer scale) [7,8]. In recent years, many transition metal complexes (Ru2+, Os2+, and Ir3+) and metalloporphyrins (Pt2+ and Pd2+)have been used for producing optical O2sensors because of their capability to be dynamically quenched in the presence of dissolved O2(DO) [9]. Combining these O2-sensing fluorescent probes with nonsensitive O2fluorophores, ratiometric optical systems that measure fluorescent signals at two different wavelengths can be produced. Ratiometric systems overcome some sensitivity limitations usually encountered in traditional optical sensors that measure fluorescence intensity only at a single wavelength (e.g., interference from analyte-independent factors, including instrumental parameters, local microenvironment, concentration of the probe, and photobleaching) [10–12]. Ratiometric O2-sensing systems based on semiconducting quantum dots, nanoparticles and microparticles, and electrospun fibers prepared with natural [13] and synthetic [14] polymers have been successfully produced for monitoring intracellular O2concentration changes [15,16]. For example, Xu et al. reported dualemissive ratiometric nanoscale metal–organic frameworks (NMOFs) for intracellular quantification of O2, showing that NMOFs were taken up by mouse colon carcinoma CT26 cells, allowing reliable measurements of cellular O2levels by confocal laser scanning microscopy (CLSM) [17]. In a different approach, Wen et al. reported a challenging protocol to engineer ratiometric afterglow/fluorescence dual-emissive O2polystyrene nanoparticles to explore the hypoxia environment in solid tumors when subcutaneously injected in mice bearing mouse sarcoma cell-derived S180 [18]. In addition, Gkika et al. designed poly-l-lysine-coated polystyrene particles encapsulating a new lipophilic and O2-responsive ruthenium(II) (Ru(II)) tris-heteroleptic polypyridyl complex together with a reference BODIPY dye to sense normoxic and O2-deprived (hypoxia) conditions after uptake in A549 lung carcinoma and HeLa cells [19]. Zhao et al. incorporated a highly efficient hydrophobic O2probe meso-5,10,15,20tetrakis(pentafluorophenyl)porphyrinatoplatinum (PtTFPP) conjugated to a reference fluorescence resonance energy transfer donor inside the core of micelles to measure O2concentration in HeLa cells [20]. Sensing extracellular changes of O2is fundamental because it is associated with extracellular matrix (ECM) remodeling and enhanced invadopodia and metastatic invasion [21,22]. However, few examples have been reported in the literature regarding the production of diverse O2sensors based on the ratiometric approach to measure extracellular changes of O2.For instance, Xue et al. demonstrated the extracellular detection of O2changes by producing electrospun core–shell poly(ε-caprolactone)/poly(dimethyl)siloxane (PCL/PDMS) nanofibers embedding O2-quenching Ru complexes or platinum metalloporphyrin dyes in the PDMS core. Based on a single-wavelength detection approach, the fluorescent fiber matrices showed O2-sensing properties and biocompatibility with glioma and glioma-derived primary cells [23]. In this study, an original protocol was proposed for the fabrication of PCL/PDMS nanofibers embedding an O2-sensing tris(4,7-diphenyl-1,10-phenanthroline) Ru(II) dichloride (Ru(dpp)32+) probe and the O2nonsensitive dye rhodamine B isothiocyanate (RBITC) for the ratiometric sensing of microenvironmental changes of DO (Fig. 1). The homogenous blending of PDMS, PCL, Ru(dpp)32+, and RBITC was employed in the electrospinning technique, and fibers were obtained by a single ratiometric fluorescent polymeric solution, resulting in a low-cost and time-saving fabrication method compared to the previously reported core–shell setup [23,24]. Moreover, the continuous ratiometricfluorescentpolymerphaseenabledthedirectexposure of the O2-sensing dye (Ru(dpp)32+) together with the O2permeable material (PDMS) not only within the lumen of the fiber but also at the interface with the surrounding environment, thus avoiding their confinement in the inner core of the fibers enclosed by an O2nonpermeable polymer shell. In addition, this study optimized ratiometric systems for sensing O2, whose fluorescent signals are not affected by analyte-independent factors [12] usually encountered in fibrous platforms based on single-wavelength optical methods. Owing to their excellent cytocompatibility, these ratiometric O2-sensing biomaterials are a promising platform for future applications in the live monitoring of O2 consumption in biological environments and tissue engineering applications. Materials and methods Materials RBITC (CAS no. 36877–69-7) mixed isomers and PCL (#440,744; Mav=80.000) were purchased from SigmaAldrich (Merck KGaA, Darmstadt, Germany). Sylgard®184, a two-part silicone elastomer kit (PDMS), was purchased from Dow Corning Corp. (Midland, MI, USA). [Ru(dpp)32+] 123 294 Bio-Design and Manufacturing (2024) 7:292–306 Fig. 1 Morphology of ratiometric O2-sensing fibers. a,bSEM micrographs of blended PCL/PDMS/Ru(dpp)32+/RBITC fibers at a×2.5 and b×5 magnifications. Scale bars: 10 μm (a) and 2 μm(b). cSize distribution studies. d–fRepresentative CLSM micrographs of PCL/PDMS/Ru(dpp)32+/RBITC fibers. The individual dyellow (false color; Ru(dpp)32+:λexc=405 nm, λem=550–650 nm) and ered (RBITC: λexc=561 nm, λem=570–620 nm) channels are shown, followed by fan overlay of the two channels (SP8, Leica, HC PL FLUOTAR 20×/0.50 dry objective, zoom 2.5; scale bar: 20 μm). SEM: scanning electron microscopy; PCL: poly(ε-caprolactone); PDMS: poly(dimethyl)siloxane; Ru(dpp)32+: tris(4,7-diphenyl-1,10phenanthroline) ruthenium(II) dichloride; RBITC: rhodamine B isothiocyanate; CLSM: confocal laser scanning microscopy; λexc: excitation wavelength; λem: emission wavelength 2Cl−(CAS no. 36309-88-3) were purchased from Alfa Aesar by Thermo Fisher Scientific (Haverhill, MA, USA). Methanol (MeOH; Reag. USP, Ph. Eur., for analysis, ACS, ISO; CAS no. 67-56-1), chloroform (CHCl3; Reag. USP, Ph. Eur., for analysis, ACS, ISO; CAS no. 67-66-3), acetone (Reag. USP, Ph. Eur., for analysis, ACS, ISO; CAS no. 67-64-1), 2-propanol (IPA; Reag. USP, Ph. Eur., for analysis, ACS, ISO; CAS no. 67-63-0), and toluene (Reag. USP, Ph. Eur., for analysis, ACS, ISO; CAS no. 108-88-3) were purchased from PanReac AppliChem (Milan, Italy). N,Ndimethylformamide (DMF; anhydrous, 99.8%; CAS no. 68-12-2) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Tetrahydrofuran (THF; EMSURE® ACS, Reag. Ph. Eur., for analysis; CAS no. 109-99-9) was purchased from VWR Chemicals (Milan, Italy). Fabrication of ratiometric O2-sensing fibers For electrospinning of ratiometric O2-sensing fibers, PCL/PDMS blends containing dissolved Ru(dpp)32+ and RBITC fluorophores were mixed in a solvent mixture and gently stirred in the dark at room temperature (RT) to obtain a homogeneous phase. PCL solutions (12% (0.12 g/mL)) were prepared 24 h in advance to allow for the full dissolution and homogenization of the polymer. To prepare PDMS, the elastomer and curing agent were weighed in a 10:1 ratio, mixed, and degassed in a vacuum pump for 20 min. The fluorophores Ru(dpp)32+ and RBITC were prepared at a concentration of 1 mg/mL in each solvent system used in the study. Therefore, the electrospinning solution was composed of the following ratio: 1 part of PDMS (mass fraction) and 9 parts of 12% PCL, and a total amount of dyes representing the 0.10% (1 mg/mL) of the polymeric formulation. The solution was stirred (300 r/min) at RT for 2 h before electrospinning in the dark. For the electrospinning process, the solutionwastransferredintoa1mLsyringe(HenkeSass Wolf, Tuttlingen, Germany) equipped with a 21-gauge stainless steel blunt needle (Sterican®, B-Braun, Milan, Italy). The syringe was placed on a syringe pump (E-fiber, SKE Research Equipment®, Bollate, Italy), and the electrospun fibers were deposited directly onto the surface of square glasses (10 mm×10 mm) positioned on a stationary collector. The optimized parameters were as follows: flow rate, 0.8 mL/h; voltage, +14/ −6 kV; tip–collector plate distance, 20 cm; time of deposition, 1 min. The environmental conditions were as follows: temperature range from 25 to 30 °C and relative humidity from 10% to 20%. The electrospun 123 Bio-Design and Manufacturing (2024) 7:292–306 295 fiber mat was finally exposed to the thermal polymerization process[25]bytransferringthemattoalaboratoryovensetup at 45 °C for 12 h. The samples were stored in the dark at RT in a vacuum desiccator until further use. Characterization of ratiometric O2-sensing fibers The morphology of ratiometric O2-sensing fibers was analyzed by scanning electron microscopy (SEM; Zeiss Sigma 500, Carl Zeiss, Germany). Before imaging, the fibers were sputtered-coated(compact coating unit CCU-010, SafeMatic GmbH, Zizers, Switzerland) with 10 nm gold (Target Au Ø54mm×0.2 mm; purity, 99.99%). Images were acquired using an accelerating voltage of 5 kV and a secondary electron detector (SE2) with magnifications of ×2500, ×5000, and ×10,000. The diameters of the fibers were then extracted by drawing linear regions of interest along the minor axis of the fibers in ImageJ (version 64-bit Java 1.8.0_172) [26]. Images of the most representative electrospun fibers are shown in Fig. S1 (Supplementary Information). Thermogravimetric analysis (TGA) was carried out using a simultaneous thermal analyzer DSC/TGA STD Q600 (TA Instruments, Waters™, New Castel, DE, USA), and the samples were analyzed in an alumina pan after tare heating at 10 °C/min, from 25 to 800 °C, under N2atmosphere. Data were plotted and analyzed using the TA Universal Analysis 2000 version 4.5A (TA Instruments, Waters™, USA). The chemical groups exposed on the fiber mat were observed using Fourier transform infrared (FTIR) spectroscopy via an FT/IR-6300 type A spectrophotometer (JASCO, Easton, MD, USA), set up in attenuated total reflectance ATR mode, with a resolution of 4 cm−1,inthe range from 500 to 4000 cm−1. The samples were prepared by electrospun deposition on a piece of silicon wafer (previously washed in IPA and acetone and dried under a flux of N2) and polymerized in a laboratory oven at 45 °C for 12 h before analysis. The hydrophilicity of the fibers was calculated using a CAM 200 (KSV Instruments Ltd., Finland) instrument to measure the static water contact angles (WCAs; θ). For the experimental setup, the liquid (heavy phase) was represented by water (density: 0.9986 g/cm3), and the liquid (light phase) was air (density: 0.0013 g/cm3). The volume of the drop employed for the measurements and charged on the needle tip was 5 μL. The WCA was calculated using Laplace-Young fitting, and data were the average of three measurements performed on different areas of each sample. To functionalize the fibers, a Tergeo Plus plasma cleaner (PIE Scientific LLC, Union City, CA, USA) was used. The parameters adopted were 50% O2flow rate and 50% power for 0.3 min. To measure the sensing performance of ratiometric O2sensing fibers to DO by CLSM imaging, the fiber samples were placed in a four-well Ibidi®chamber plate, and 400 μL of double distilled water (DDW) was added to each well. Calibrations were performed via CLSM (SP8 Leica Microsystems, Manheim, Germany) in the O2concentration range of 0.5% to 20%, obtained by purging N2directly into DDW while measuring the O2content in water using a commercial O2meter (Vernier Go Direct®optical dissolved oxygen probe,VernierScienceEducation,Beaverton,OR,USA),and about 30 min time was set to ensure that equilibrium was reached before measurement. Images were acquired using an HC PL FLUOTAR 20×/0.50 dry objective and 2.5 zoom (232.5 μm×232.5 μm). The emission wavelength (λem) of Ru(dpp)32+ and RBITC dyes was sequentially collected at λem=550–650 nm (excitation wavelength λexc=405 nm) and λem=570–620 nm (excitation wavelength λexc=561 nm), respectively. The CLSM images were processed with a custom algorithm written in GNU Octave (version 6.2.0) [27–29], opportunely modified to automatically quantify the fiberfluorescenceintensitiesforratiometricanalyses.Briefly, the RBITC channel images were first converted to grayscale and binarized. Then, morphological opening with a diskshaped element was performed to remove any small white noises in the image, and morphological closing was performed to remove any small holes in the object. In this way, the fibers were identified in the binarized reference channel image, which was used as a mask to store pixel locations and the corresponding fluorescence intensities IRu(dpp)2+ 3/IRBITC (obtained as the pixel-by-pixel ratio between fluorescence intensities of the original Ru(dpp)2+ 3and RBITC images, respectively). The mean and standard deviation from four different images were finally extracted. Ratiometric measurements To develop the sensing fibrous scaffold, Ru(dpp)32+ and RBITC were employed as an O2-sensing reporter and nonsensitive O2dye, respectively, for ratiometric analyses. Whereas RBITC does not respond to O2concentration changes [30], the O2quenching mechanism of Ru complexes has been extensively reported in the literature [31,32]. The linear dependence of the emission intensity behavior of Ru(dpp)32+ on the quencher concentration is governed by the Stern–Volmer equation (Eq. (1)) [33], reported as follows: I0 I=1+Ksv[O2],(1) where I0and Iare the fluorescence intensities in the absence and presence of the quencher, respectively; Ksv is the Stern–Volmer quenching constant; [O2] is the gaseous or dissolved oxygen concentration. In ideal conditions, the Stern–Volmer equation, plotted with the fluorescence intensities against O2concentrations, leads to a linear correlation 123 296 Bio-Design and Manufacturing (2024) 7:292–306 with a slope equal to Ksv and an intercept equal to 1, also providing information on the sensitivity of the sensor in relationship to O2quenching effect. In the ratiometric O2sensing fiber system in this study, the fluorescent response of the Ru(dpp)32+ complex and RBITC toward different O2 concentrations is derived from the ratio (R) of the maximum fluorescence intensity of Ru(dpp)32+ metal complex IRu(dpp)2+ 3tothemaximumfluorescenceintensityofO2insensitive RBITC dye (IRBITC), as follows: R= IRu(dpp)2+ 3 IRBITC .(2) The Stern–Volmer equation is transformed by substituting I0/Iwith R0/R, representing the response of the ratiometric sensor, as follows: R0 R=1+Ksv[O2],(3) where R0is the fluorescent signal in the absence of the quencher. Thus, it was possible to evaluate the ratiometric calibration curve of the developed sensing system. Biocompatibility of ratiometric O2-sensing fibers ThebiocompatibilityofratiometricO2-sensingscaffoldswas evaluated by in vitro cytotoxicity assays using the melanoma cell line SK-MEL2 (HTB-68™; ATCC, Rockville, MD, USA), human pancreatic cancer cell line Panc-1 (CRL1469™; ATCC, Rockville, USA), and breast cancer cell line MCF-7 (HTB-22™; ATCC, Rockville, USA) cultured at 37 °C in a humidified 5% CO2incubator. SK-MEL2 cells were routinely cultured in Eagle’s minimum essential medium (Sigma-Merck KGaA, Darmstadt, Germany), whereas Panc-1 and MCF-7 cells were grown in Dulbecco’s modified Eagle’s medium (Sigma-Merck KGaA, Darmstadt, Germany), both supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA), 2 mmol L-glutamine, and 100 U/mL penicillin and streptomycin (Sigma-Merck KGaA, Darmstadt, Germany). Before cell culture experiments, O2-sensing fibers, deposited on 1cm×1 cm glass slides, were transferred to 24-well plates, sterilized by ultraviolet exposure for 30 min, and functionalized with 0.1 mg/mL fibronectin (Sigma-Merck KGaA, Darmstadt, Germany) for 30 min. Cells (4×104per well) were seeded in each well, and their viability was evaluated using PrestoBlue cell viability reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) on Days 1, 3, and 6. Briefly, at each time point, 5 μL PrestoBlue reagent was added to each well and incubated for 1 h at 37 °C and 5% CO2. The fluorescent signal was obtained using a microplate reader (ClarioStarPlus, BMG Labtech, Germany) at an excitation wavelength of 535 nm and an emission wavelength of 590 nm. Cells grown directly in the well plate were used as controls. Statistical analyses All experiments were performed in triplicate, and the results were reported as the mean±standard error unless otherwise stated. Statistical differences were considered significant at p<0.05 using two-way analysis of variance. Data analyses and graphing were performed using Microsoft Excel 365 and GraphPad Prism (version 8.4.2-2018). Results and discussion Ratiometric O2-sensing fiber preparation and morphological observations The fabrication of O2-sensing fibers was successfully assessed by coupling the advantages deriving from two polymers, namely PDMS and PCL. PDMS shows a high O2permeability coefficient (P=6.95×1015 cm3) resulting from the product of the diffusion constant and O2solubility coefficient [34]. However, PDMS lacks electrospinnability properties [35]. For this reason, PCL addition in the polymeric solution preparation was evaluated to allow the electrospinning of monolithic hybrid fibers. Notably, two fundamental aspects influenced the electrospinning of the hybrid polymeric matrix, particularly the swelling of PDMS in the solvent and the phenomenon of the partition of the solutes between the solvent and PDMS matrix [36]. Focusing on the PDMS swelling, using high-solubility solvents alone (e.g., chloroform and toluene), it is generally not compatible with the fabrication of PDMS-based devices because it causes marked swelling of the polymer. For this reason, it is usually recommended to prepare PDMS using a highsolubility solvent mixed with another low-solubility solvent (moderately to highly polar, e.g., water, alcohols, amides, and sulfoxides) to produce a mixture that does not swell PDMS or that at least reduces the swelling related to the use of high-solubility solvents. As anticipated, the other aspect considered during the preparation of the polymer matrix is the partitioning of the fluorophores (Ru(dpp)32+ and RBITC) in the PDMS phase. This represents a key point for the preparation of O2-sensitive devices. Therefore, to prepare a homogeneous mixture of the solutes in the composite matrix composed of PCL and PDMS, it is necessary to use a low-solubility solvent capable of solvating both fluorophores, which neither reacts with the fluorophores nor quenches their fluorescence and, at the same time, does not causecrosslinkingof thePDMS-PCLprepolymerblend [36]. 123 Bio-Design and Manufacturing (2024) 7:292–306 297 Table 1 Details of the polymeric solution preparation to assess the electrospinning process and fiber fabrication Sample codes Solvent mixture Polymer concentrations (%) and polymer ratio (pr) Polymerization temperature (°C) Morphology F1A DMF/THF (1:1) PCL 12% 45 Fibers F1B DMF/THF (1:1) PCL 12% RT Fibers F2A MeOH/CHCl3(1:1) PCL 12% 45 Fibers F2B MeOH/CHCl3(1:1) PCL 12% RT Fibers F3A DMF/THF (1:1) PCL 12% +PDMS (pr 5:5) 45 Film forming F3B DMF/THF (1:1) PCL 12% +PDMS (pr 5:5) RT Film forming F4A MeOH/CHCl3(1:1) PCL 12% +PDMS (pr 5:5) 45 Film forming F4B MeOH/CHCl3(1:1) PCL 12% +PDMS (pr 5:5) RT Film forming F5A DMF/THF (1:1) PCL 12% +PDMS (pr 6:4) 45 Film forming F5B DMF/THF (1:1) PCL 12% +PDMS (pr 6:4) RT Film forming F6A MeOH/CHCl3(1:1) PCL 12% +PDMS (pr 6:4) 45 Film forming F6B MeOH/CHCl3(1:1) PCL 12% +PDMS (pr 6:4) RT Film forming F7A DMF/THF (1:1) PCL 12% +PDMS (pr 7:3) 45 Fibers F7B DMF/THF (1:1) PCL 12% +PDMS (pr 7:3) RT Fibers F8A MeOH/CHCl3(1:1) PCL 12% +PDMS Sylgard®(pr 7:3) 45 Film forming F8B MeOH/CHCl3(1:1) PCL 12% +PDMS Sylgard®(pr 7:3) RT Film forming F9A DMF/THF (1:1) PCL 12% +PDMS (PDMS dil 7:3) 45 Fibers F9B DMF/THF (1:1) PCL 12% +PDMS (PDMS dil 7:3) RT Fibers F10A MeOH/CHCl3(1:1) PCL 12% +PDMS (PDMS dil 7:3) 45 Fibers F10B MeOH/CHCl3(1:1) PCL 12% +PDMS (PDMS dil 7:3) RT Film forming F11A CHCl3/toluene (1:1) PCL 12% +PDMS (PDMS dil 7:3) 45 Electrospray F11B CHCl3/toluene (1:1) PCL 12% +PDMS (PDMS dil 7:3) RT Electrospray F12A DMF/THF (1:1) PCL 12% +PDMS (PDMS dil 8:2) 45 Film forming F12B DMF/THF (1:1) PCL 12% +PDMS (PDMS dil 8:2) RT Film forming F13A MeOH/CHCl3(1:1) PCL 12% +PDMS (PDMS dil 8:2) 45 Film forming F13B MeOH/CHCl3(1:1) PCL 12% +PDMS (PDMS dil 8:2) RT Film forming F14A CHCl3/toluene (1:1) PCL 12% +PDMS (PDMS dil 8:2) 45 Electrospray F14B CHCl3/toluene (1:1) PCL 12% +PDMS (PDMS dil 8:2) RT Electrospray F15 DMF/THF (1:1) PCL 12% +PDMS (pr 9:1) +dyes 45 – F16 MeOH/CHCl3(1:1) PCL 12% +PDMS (pr 9:1) +dyes 45 Fibers F17 CHCl3/toluene (1:1) PCL 12% +PDMS (pr 9:1) +dyes 45 Electrospray Morphology based on SEM analysis. All figures and size distribution studies of the electrospun samples are reported in Fig. S1 (Supplementary Information). SEM: scanning electron microscopy; DMF: N,N-dimethylformamide; THF: tetrahydrofuran; MeOH: methanol; CHCl3: chloroform; PCL: poly(ε-caprolactone); PDMS: poly(dimethyl)siloxane; RT: room temperature; dil: dilution Considering this knowledge, the experimental planregarding the manufacturing of O2-sensing fibers involved the preparation of a polymeric blend based on PCL and PDMS in different couples of solvent systems (namely, DMF/THF, CHCl3/toluene, and MeOH/CHCl3) to identify the best combination in terms of uniformity of the solution and good processability (Table 1). The first step of this study involved the optimization of the electrospinning settings, including the voltage (+14/ −6kV), flow rate (0.8 mL/h), the distance between the needle tip and static collector (20 cm), and atmospheric temperature (T=25–30 °C) and relative humidity (RH=30%–40%). Another important optimized parameter was the polymerization temperature of the composite matrix [37] that had to consider the necessity of (i) not overcoming the low melting point of PCL, (ii) being optimal to crosslink the blend, and (iii) starting and lasting in a range of time that allows preserving the fiber shape. For these reasons, both RT and 45 °C, and 12 h as crosslinking time were chosen as study conditions. PCL, prepared for this work at the concentration of 12% (0.12 g/mL), was easily electrospun, resulting in defect-free and quite homogeneous fibers, in DMF/THF and MeOH/CHCl3, although fibers left drying at RT were plain (Fig. S1, F1B and F2B in Supplementary Information) 123 298 Bio-Design and Manufacturing (2024) 7:292–306 compared to those dried in the oven at 45 °C (Fig. S1, F1A and F2A in Supplementary Information). Then, a study on the polymer ratio was conducted. The polymer blend in the solution system CHCl3/toluene was not electrospun because a phase separation was obtained between the two polymers PCL and PDMS. An equivalent amount of PCL and PDMS, at polymer ratio 5:5, determined the emulsion solution and the formation of foaming fibers (Fig. S1, F3A,F3B,F4A, and F4B in Supplementary Information). Similarly, the polymer ratio 6:4 (Table 1, F5A, F5B, F6A, and F6B) could not be electrospun at all, ascribed to the incompatibility of a high PDMS concentration, namely more than 30% of the totalpolymerblend weight percentage,intheelectrospinning technique. The increase in PCL and decrease in PDMS in the polymer blend (7:3) formed straight fibers in DMF/THF solvent system (Fig. S1, F7A and F7B in Supplementary Information), whereas a wet glaze was observed around the central core of the fibers deriving from MeOH/CHCl3blend (Fig. S1, F8A and F8B in Supplementary Information). To enhancetheelectrospinnability andmorphologyofthe fibers, PDMS was further diluted. In this step, the solution system CHCl3/toluene was included in the study, considering the possibility of reducing the force at the interface of the polymer blend deriving from a diluted system. Therefore, the dilution of PDMS at the 7:3 ratio resulted in a heterogeneous population of fibers with different diameters in DMF/THF (Fig. S1, F9A and F9B in Supplementary Information) and unimodal fibers in MeOH/CHCl3with 5 μm diameter (Fig. S1, F10A and F10B in Supplementary Information), with a more rounded shape when polymerization occurred at 45 °C. In contrast, more diluted PDMS, at the ratio of 8:2, in the final polymer blend resulted in wet fibers with film foaming in both solvent systems of DMF/THF (Fig.S1, F12A and F12B inSupplementaryInformation)and MeOH/CHCl3(Fig. S1, F13A and F13B in Supplementary Information), due to the non-rapid evaporation of the solvents after fiber deposition. In contrast, the polymer blend in CHCl3/toluene determined an electrospray phenomenon in the Taylor cone, again with no possibility of obtaining electrospun fibers (Table 1, F11A, F11B, F14A, and F14B) in 7:3 and 8:2 PDMS dilutions. The reason for this can be ascribed totheswellingeffectofthecoupleofsolvents CHCl3/toluene on PDMS [36]. Considering the film forming fibers derived fromanexcessofsolventanditsnon-fastevaporationprocess at both RT and 45 °C, as last chance three polymer blends were prepared using PCL/PDMS (polymer ratio 9:1) with the addition of the dyes: (i) a polymer blend in MeOH/CHCl3, resulting in a homogeneous phase (Table 1, F16); (ii) a polymer blend in CHCl3/toluene, obtaining a phase separation (after vortexing and removal of the supernatant, electrospray phenomenon did not allow fiber fabrication) (Table 1, F17); and (iii) a polymer blend in DMF/THF, which gave fluorophore quenching (for this reason, it was excluded from this study) (Table 1, F15). Considering the aim of validating a simple composite matrix and a feasible electrospinning method to prepare ratiometric O2-sensing fibers, the polymer blend represented by the MeOH/CHCl3solvent system was selected. Once the polymer ratio and electrospinning parameters were optimized, the influence of dye loading on the electrospinnability of the polymeric solution and the morphology of the resulting fibers were tested, together with their sensing performances. With this aim, comparative studies on the fabricated scaffolds were conducted to find the best concentration of each fluorescent component to obtain a final ratiometric system that guaranteed the successful entrapment of the luminescent probes within the fibers. Ru(dpp)32+ and RBITC stock solutions were prepared at 1 mg/mL concentration in MeOH/CHCl3(1:1) and used for the following study. Three concentrations of the reference dye RBITC and three concentrations of fluorophore Ru(dpp)32+ were charged in the composite polymer blend (Table S1 in Supplementary Information), and the effect of the probe cargo was detected via CLSM imaging. As expected, dye loading influenced the production protocol and the morphology of the scaffolds. Indeed, although the optimized fabrication parameters were applied to all solutions, the electrospinnability resulted in perturbation with the appearance of the electrospray phenomenonandcurrentinstabilitieswhenconditionsRS1,RS2, RS4,andRS6(Table S1inSupplementaryInformation)were adopted. In Fig. S2 (Supplementary Information), keeping the concentration of the sensing Ru-based complex constant (22 μg/mL), the increase in the cargo of the reference dye RBITC (conditions RS1 and RS2, Table S1 in Supplementary Information) determined a change in the pattern of the fibrous scaffold, which appeared nonhomogeneous in size andshape(Figs.S2aandS2binSupplementaryInformation). Moreover, the fluorescent signals of RBITC appeared confined at the border of the electrospun strings, which instead presented a Ru(dpp)32+-rich core (Fig. S2b in Supplementary Information). In addition, the fibers appeared wet and oily, likely due to a slowing down or interruption of the curing process of the two PDMS components (elastomer and curing agent) within the polymeric mixture. These results agreed with those of a previous report [23] and did not allow the performing of any DO sensing test on the fabricated scaffolds. Thus, lowering the amount of the reference probe from 375 to 75 μg/mL (condition RS3, Table S1 in Supplementary Information) resulted in the colocalization of the ratiometric fluorescent signals that uniformly illuminated the entire fiber mat (Fig. S2c in Supplementary Information) upon laser excitation. Having established the RBITC cargo (75 μg/mL), Ru(dpp)32+ concentrations were also varied in the polymer solution to identify the optimal indicator loading amount for 123 Bio-Design and Manufacturing (2024) 7:292–306 299 ratiometric measurements. At this point, morphology and DO sensing ability were thoroughly investigated through confocal imaging analysis. An at least twofold increase in Ru(dpp)32+ to 90 or 360 μg/mL (conditions RS4 and RS6, Table S1 in Supplementary Information) caused difficulties in the application of the optimized electrospinning parameters, resulting in fibers losing their form and size in both cases (Figs. S3a and S3c in Supplementary Information), as observed previously. In addition, in the highest Ru(dpp)32+ concentration case (condition RS6, Table S1 in Supplementary Information), some fluorescent polymeric clusters in the blended electrospun fibers indicated an excessive amount of dye, resulting in molecular aggregation (Fig. S3c in Supplementary Information). Therefore, an intermediate amount of 180 μg/mL of the Ru-based complex (condition RS5, Table S1 in Supplementary Information) within the polymer blend yielded bead-free, fluorescent, and microscaled scaffolds (Fig. S3b in Supplementary Information). The results from sensor sensitivity experiments at different DO concentrations are shown in Fig. S3d (Supplementary Information). Fluorescent emission responses were collected under the same excitation conditions and analyzed via image segmentation. For all sensing systems, intensity ratios in conditions RS4, RS5, and RS6 were linearly correlated with the DO concentrations in the surrounding environment with similar slopes and regression coefficients. These results agreed with the expectations because although the amount of the sensing dye within the polymer solutions varied, the final scaffolds maintained the same sensing ability, being ratiometric platforms [12,38,39]. Clearly, the calibration curves reported higher intensity ratios when the amount of the Ru-based probe was increased while keeping the RBITC concentrationconstant.Althoughthe intensity ratio (IRu(dpp)2+ 3/IRBITC) was maximized in correspondence with an excessive cargo of the fluorophores (≥0.10% (mass fraction) of the polymer blend), the sensing capacity was slightly reduced. Indeed, a diminished slope of the calibration curve of condition RS6, compared to RS4 and RS5 scaffolds, was obtained (Fig. S3d in Supplementary Information). Moreover, the high standard error during pixel-by-pixel analysis was a consequence of the nonhomogeneous distribution of the fluorescent probes within the fibers. Therefore, the fabrication condition RS5, with dyes representing 0.10% (mass fraction) of the polymeric formulation, was selected as the optimal balance to obtain a ratiometric optical sensing fibrous mat. The microfiber morphology, investigated through SEM, revealedrandomlyorientedfibers withacontinuous, smoothsurface, and bead-free structure with a string-like shape. Size distributionanalysesevidenceduniformfiberdiameters,with an average value of (1.18±0.20) μm (Figs. 1a–1c). The uniformdistributionofbothfluorophores withinthelumenofthe fibers, as visualized using CLSM, confirmed the promising ratiometric sensing platform (Figs. 1d–1f). Ratiometric O2-sensing fiber structural characterization The surface chemistry of the composite fibers was studied by FTIR in the ATR mode. The spectrum of blended PCL/PDMS fibers was compared with the spectra of PCL fibersandcrosslinkedPDMSsolutionbecausenofiberscould be obtained from PDMS (Fig. 2a). From the FTIR spectrum of the blended fibers, it was possible to identify the typical bands of PCL, such as the carbonyl (C=O) overtone at 3446 cm−1, the symmetric CH2stretching at 2938 and 2860 cm−1, the carbonyl (C=O) stretching at 1725 cm−1 together with C–O and C–C stretching, the asymmetric C–O–C stretching at 1290 cm−1, and O–C–O stretching at 1185 cm−1[40,41]. The proof of the blending solution came from the several characteristic bands of PDMS, such as the –CH3deformation in Si–CH3at 1260 cm−1,the –C–H bending corresponding to the peaks between 1400 and 1420 cm−1, the Si–O–Si stretching at 1100–1000 cm−1, and the Si–C rocking in the fingerprint region between 825 and 865 cm−1[37,42]. At 700–500 cm−1, it was possible to find C–H stretching bands of adjacent aromatic and heterocyclic systems, typical of Ru(dpp)32+ and RBITC. All bands are reported in Table S2 (Supplementary Information). The thermal properties of the polymer matrices were evaluated by TGA and differential scanning calorimetry (DSC) under a nitrogen atmosphere (Table 2). As established in the literature [43], the semicrystalline polymer PCL has a melting point (Tm) of about 60 °C. In contrast, PDMS is a noncrystalline material and thus, has no melting point. However, the crosslinking process, occurring during the curing step of the microfibers at 45 °C, determined a slight shift of the composite matrix glass transition to a higher temperature (64.52 °C), as shown in the DSC curve (Fig. S4 in Supplementary Information). Results in the TGA curves (Fig. 2b) demonstrated that from 350 to 450 °C, PCL rapidly decomposed with complete weight loss (99.36%). In contrast, only 47.83% weight loss was observed for PDMS up to 800 °C, indicating that PDMS had better thermal stability than PCL. Therefore, the preparation of composites based on these two polymers resulted in fiber matrices with higher thermal stability. Notably, the derivative thermogravimetric (DTG) curves confirmed the TGA results. The blend presented three degradation steps: the first decomposition peak was recorded at about 350–440 °C, and this corresponded to PCL degradation; the second peak, at 440–500 °C, was related to the fluorophores Ru(dpp)32+ and RBITC; and the third peak, at 530–630 °C, was proper of PDMS breakdown of its crosslinked structure [44]. 123 300 Bio-Design and Manufacturing (2024) 7:292–306 Fig. 2 Structural characterization of ratiometric O2-sensing fibers. aFTIR spectra of blended PCL/PDMS/Ru(dpp)32+/RBITC (orange line), PCL (blue line), and PDMS (green line). bTGAandDTGcurves of PCL (straight and dot blue lines), PDMS (straight and dot green lines), and PCL/PDMS/Ru(dpp)32+/RBITC (straight and dot orange lines). cWCA analysis. Data are presented in mean±standard deviation (n=3), ∗∗∗∗p≤0.0001. FTIR: Fourier transform infrared; PCL: poly(εcaprolactone); PDMS: poly(dimethyl)siloxane; Ru(dpp)32+: tris(4,7diphenyl-1,10-phenanthroline) ruthenium(II) dichloride; RBITC: rhodamine B isothiocyanate; TGA: thermogravimetric analysis; DTG: derivative thermogravimetric analysis; WCA: water contact angle Table 2 TGA of PCL, PDMS, and PCL/PDMS/Ru(dpp)32+/RBITC fibers Sample Tm(°C) Tidecomp. (°C) Tmax decomp. (°C) Weight loss (%) Residue (%) PCL 61.59 350 410 99.36 0.64 PDMS – 460 540 47.83 52.17 PCL/PDMS/Ru(dpp)32+/RBITC 64.52 360 570 83.20 16.80 TGA: thermogravimetric analysis; PCL: poly(ε-caprolactone); PDMS: poly(dimethyl)siloxane; Ru(dpp)32+: tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride; RBITC: rhodamine B isothiocyanate; Tm: temperature of melting; Tidecomp.: initial decomposition temperature: Tmax decomp.: maximum decomposition temperature The overall performance of the developed scaffolds is dependent on the hydrophobicity of the fibrous mat, since this property can hinder the application of the sensing platform in vitro or in vivo [45,46]. Therefore, the optimized ratiometric O2-sensing fibers were subjected to measuring the WCA using water as a liquid heavy phase (Fig. 2c). The drop of water deposited on the polymeric electrospun mats recorded a WCA θ≥110°±7°, indicating high hydrophobicity. To increase the wettability of the fibers, a functionalization step was performed with O2plasma (50% O2flow rate, 50% power, for 0.3 min), which increased the hydrophilicity of the surface due to the exposure of carboxylic and hydroxyl groups [45], endowing PCL/PDMS/Ru(dpp)32+/RBITC fibers with a WCA θ≤20°±6°. Ratiometric O2-sensing fibers: dose-response curve studies, photobleaching, and reversibility One of the main aspects to consider during real-time DO concentration measurements in living systems is the sensitivity and stability of the analytical platform. As theoretically stated, the luminescence of Ru(II)-based polypyridyl complexes is governed by the metal–ligand charge transfer phenomenon. This latter process determines the transition of an electron from a metal dorbital to a ligand π*orbital. Consequently, upon excitation, the exited singlet state is converted to the lowest triplet state through intersystem crossing, generating the emissive fluorescence intensities of Ru polypyridyl complexes in a sensing system, which in turn is quenched by the presence of O2in the close environment [32]. In detail, two main reactions occur when O2interacts with Ru(dpp)32+ complexes, thus quenching their fluorescence. Equation (4) describes the transition of an electron from Ru(dpp)32+ complex (RuL) to give a single oxygen (O2:) and the ground state of RuL, as shown below: RuL2+· 3+O2→RuL2+ 3+O· 2.(4) The second reaction involves the oxidation of the RuL operated by the transfer of an electron with subsequent production of the superoxide anion (O2:−), as indicated below: RuL2+· 3+O2→RuL3+ 3+O·− 2.(5) Therefore, when the 405 nm CLSM laser light excited the Ru(dpp)32+ probe incorporated in the sensing scaffold, the absorption of light determined the energy gain of the excited electron, which, to restore its ground state, promoted energy release through fluorescence emission at higher wavelengths. Thus, the fluorescence behavior of Ru(dpp)32+ was studied as a function of the DO concentration and resulted in the quenching phenomenon described 123