Saccharomyces cerevisiae strains that express each as their sole source of actin. Both β- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-β4 (Tβ4). Notably, Tβ4 and profilin bind to β- or γ-actin with higher affinity than to α-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human β- or γ-actin (i.e. cytoplasmic actins), we developed  Summary: Generation and characterization of human β- and γ-actin from budding yeast as an accessible and low-cost way to study the regulation of specific actin isoforms. ACTA1, ACTA2, ACTC1 and ACTG2), which occur predominantly in skeletal, cardiac and smooth muscle cells, and β (ACTB) and γ1  found predominantly in non-muscle cells and considered cytoplasmic isoforms of actin. Human β- and γ-actin are structurally divergent  and fascin. Thus, these engineered strains provide convenient sources of pure β- or γ-actin for biochemical studies where conventional α-actin sources might be unsuitable.Here, we have taken a different approach to generate pure human β-or γ-actin, engineering the yeast nt actin . A follont actin . We haveSaccharomyces cerevisiae strains lacking the sole yeast actin gene (ACT1). The strain expressing human β-actin grew slowly . Unlabeled 1 µM β-actin, γ-actin or control α-actin (RMA) was visualized with 10% fluorescently labeled RMA (P≥0.45) (−1 µM−1 (mean±s.e.m.), consistent with other studies   B. Contro studies C (Liu et studies . β-actinectively C. The me P=0.02) C.KD=1.05±0.30 nM (mean±s.e.m.)] and γ-actin (KD =0.94±0.09 nM) with similar affinities, which were each significantly stronger than skeletal muscle actin   A (Pimm eP<0.002) . This reP≤0.01) (P>0.99)  C (AntokuP≤0.01) . However(P>0.99) D,E.P>0.91) (−1 µM−1 (mean±s.e.m.), the mean elongation rate for β-actin and γ-actin with profilin and formin was not significantly slower at 59.3±4.2 or 61.2±4.5 subunits s−1 µM−1, respectively (P≤0.051) (Movie 2). In summary, α-actin from muscle and both cytoplasmic actins display similar properties in the presence of profilin and the formin mDia1, albeit to different levels.When present with the actin polymerization-stimulating formin (mDia1), profilin enhances actin filament elongation . Thus, tP>0.91) D,E and sP>0.91) , with γ-P≤0.051) , Movie 2P=0.98) (P<0.01)  B. In con(P<0.01) C. In conWe developed budding yeast strains for the expression and purification of human cytoplasmic β- and γ-actin, and have demonstrated that these actin isoforms polymerize and interact with several canonical actin-binding proteins. There are several benefits to purifying recombinant actin from this system including: yield (0.5–1 mg/l starting culture), no special growth or tissue culture requirements, use of conventional purification reagents and protocols, no additional post-purification processing (e.g. cleavage of fusion tags), and no contaminating ‘host’ actin. Even with these advantages, we note that for both yeast and human actin purifications, a significant amount of protein (>50%) is lost following post-elution dialysis. This likely reflects the denaturing activity of the formamide used to elute actin from the DNaseI column and might be improved by supplementing our approach with an alternative 6×His–gelsolin fragment-based purification .Comparisons of actin isoforms or mixes that reflect other species and non-muscle cells are crucial to our understanding of how actin and its regulators truly function. For example, the formin FHOD1 displays differential interactions with actin from different sources, specifically enhancing the nucleation phase of actin assembly with cytoplasmic actin but preventing the assembly of filaments generated from RMA . ProfiliOver 100 post-translational modifications (PTMs) of cytoplasmic and muscle actins have been described, with common reports of acetylation, arginylation, methylation and phosphorylation . Post-trMost mammalian actin is processed to remove the N-terminal methionine and then acetylated or less commonly (and only for β-actin) arginylated at Asp3 . StudiesUnless otherwise specified, chemicals and supplies were purchased from Thermo Fisher Scientific . DNaseI for affinity columns was purchased from Worthington Biochemical . Cloning reagents were obtained as follows: restriction enzymes and DNA ligase ; Primestar HS DNA polymerase ; and oligonucleotides .ACTA1; NCBI Gene ID: 58) and β-actin  genes flanked by BamHI and HindIII sites were codon-optimized for expression in budding yeast . Codon-optimized γ1-actin  was generated from the ACTB sequence with the following mutagenic primer (5′-CTCGGATCCATGGAAGAAGAAATTGCTGCATTGGTTATTGATAATGGTTCTGGCATGTG-3′). The TDH3 promoter was PCR amplified as an EcoRI-BamHI fragment from yeast genomic DNA with 5′-GTGAGAATTCTCAGTTCGAGTTTATCATTA-3′ and 5′-CTCGGATCCTTTGTTTGTTTATGTGTGTTT-3′. TDH3 and actin genes were inserted into YEp351 (ACT1/act1Δ heterozygous diploid that carried the β-actin expression plasmid. The yeast strain that expresses only γ1-actin (BHY848) was generated by introducing the γ1-actin expression plasmid into a haploid act1Δ strain carrying the yeast ACT1 gene on a URA3-based plasmid (ACT1 (URA3) plasmid but remained viable. We were unable to recover yeast expressing human α1-actin, consistent with previous reports  supplemented with protease inhibitors [0.1 mM PMSF and 1:500 Cocktail IV ] and lysed by two passes through a French press at 1000 psi (6.9 MPa). Lysates were bound to 3 ml DNaseI–Sepharose, then beads were washed in five column volumes of each of the following: (1) 10% formamide in G-buffer, (2) 0.2 M NH4Cl in G-buffer, and (3) G-buffer alone. Actin was eluted with 50% formamide in G-buffer, and then immediately diluted with 1–2 ml G-buffer and dialyzed overnight against 2 l G-buffer. To remove contaminating proteins, actin underwent at least two polymerization–depolymerization cycles alternating between 0.6 M KCl and pipetting with dialysis against G-buffer.Human β- and γ-actin were prepared as described in Rabbit muscle actin , mDia1(FH1-C) (amino acids 571–1255), GFP–Tβ4, profilin-1 and fascin used in TIRF or anisotropy assays were purified as described previously . Actin wSample preparation, operation and mass spectrometry (MS) analyses were performed by the Upstate Medical University Proteomics and Mass Spectrometry Core Facility. Samples (15–30 µg) were treated with 5 mM tris(2-carboxyethyl)phosphine (TCEP) and then 15 mM iodoacetamide for 30 min in the dark. Trypsin digestion was performed at 37°C with 0.66 µg Trypsin Platinum  overnight. Samples were acidified and then desalted using 2-core MCX stage tips . PeptideS. cerevisiae , Rabbit O. cuniculus , a list of common contaminants , and a custom list of human β-actin, γ-actin and rabbit α-actin. Additional entries for each actin were made with truncations of the first one, two and three N-terminal residues. Enzyme specificity was set to semi-tryptic with up to two missed cleavages. Precursor and product ion mass tolerances were set to 10 ppm and 0.02 Da. Cysteine carbamidomethylation was set as a fixed modification and the following modifications were set as variable: methionine oxidation, protein N-terminal acetylation, peptide N-terminal arginylation, lysine acetylation or methylation of lysine, histidine and arginine. The output was filtered using the Percolator algorithm. We visualized datasets with software at http://www.interactivepeptidespectralannotator.com/PeptideAnnotator.html (PXD040174).The core facility used SequestHT in Proteome Discoverer  to search three MS databases: tor.html . Full da2, 1 mM EGTA, 0.5 mM ATP). Samples were incubated for 30 min at room temperature, then subjected to centrifugation at 200,000 g for 30 min at 20°C. Supernatants were removed and pellets and supernatant samples were brought to equal volumes in protein loading buffer. Equal volumes were loaded on SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. Indicated concentrations of profilin-1 were mixed with 2 µM actin monomers in G-buffer and then incubated for 30 min at room temperature. Actin polymerization was then induced with F-buffer for an additional 30 min prior to centrifugation as above.Polymerization of 1 or 2 µM actin was induced with the addition of concentrated (20×) F-buffer , 25 mM KCl, 4 mM MgClActin isoform co-polymerization reactions were assessed by epifluorescence microscopy . 2 µM unlabeled or OG-labeled β- or γ-actin were polymerized in F-buffer, either individually or as a 1:1 mixture, then bound to 1.1 µM rhodamine–phalloidin. Samples were viewed individually or as mixtures bound to phalloidin prior to mixing. Samples were flowed into slide chambers prepared by laying a cover glass onto a slide with two intervening strips of double-sided tape, then visualized with DsRed and FITC filter sets. Views were obtained near fluid–air boundaries.2, 1 mM EGTA, 0.2 mM ATP, 10 mM DTT, 40 mM glucose and 0.25% methylcellulose (4000 cP)], with minimal (5–7%) labeled RMA. Some TIRF experiments with β- or γ-actin were supplemented with 10% RMA to enable visualization of filaments, as noted in figure legends. Experiments performed with fascin used unlabeled actin isoforms and were visualized with 130 nM Alexa Fluor 488-conjugated phalloidin. Filament elongation rates (subunits s−1 µM−1) were determined by measuring filament lengths from at least five frames, with a conversion factor of 370 subunits/µm  as in units/µm . Total funits/µm . The skeunits/µm .Fluorescence polarization determination of GFP–Tβ4 binding to actin  was carrP-values presented are those from ANOVA tests across tested conditions (under the line), except for P≤0.05) was used to determine significance throughout this work. Specific comparisons are described in each figure legend. Figures were made in Adobe Illustrator 2023 .GraphPad Prism  was used to plot all data and perform statistical tests. All experiments were repeated at least three times. Individual data points are presented as dots in each figure and histograms represent mean±s.e.m. (unless noted otherwise). All one-way ANOVA tests performed compared all means with Tukey post-hoc analysis and passed tests for normality. Click here for additional data file.10.1242/joces.260540_sup1Supplementary informationClick here for additional data file.


Here, we report the design, synthesis, physicochemicaland optoelectronic characterizations, and OFET performances of a libraryof solution-processable, low-LUMO (−4.20 eV) 2,2′-indenofluorene-6,12-diylidene)dimalononitrile small molecules, β,β′-Cn-TIFDMTs, having varied alkyl chain lengths . An intriguing correlation is identifiedbetween the solid–isotropic liquid transition enthalpies andthe solubilities, indicating that cohesive energetics, which are tunedby alkyl chains, play a pivotal role in determining solubility. Thesemiconductors were spin-coated under ambient conditions on denselypacked (grafting densities of 0.19–0.45 chains/nm2) ultrathin (∼3.6–6.6 nm) polystyrene-brush surfaces.It is demonstrated that, on this polymer interlayer, thermally induceddispersive interactions occurring over a large number of methyleneunits between flexible alkyl chains  are criticalto achieve a favorable thin-film crystallization with a proper microstructureand morphology for efficient charge transport. While C8 and C16 chains show a minimal zipper effect upon thermalannealing, C12 chains undergo an extended interdigitationinvolving ∼6 methylene units. This results in the formationof large crystallites having lamellar stacking ((100) coherence length∼30 nm) in the out-of-plane direction and highly favorablein-plane π-interactions in a slipped-stacked arrangement. Uninterruptedmicrostructural integrity -oriented crystallites)was found to be critical to achieving high mobilities. The excellentcrystallinity of the C12-substituted semiconductor thinfilm was also evident in the observed crystal lattice vibrations (phonons)at 58 cm–1 in low-frequency Raman scattering. Two-dimensionalmicrometer-sized (∼1–3 μm), sharp-edged plate-likegrains lying parallel with the substrate plane were observed. OFETsfabricated by the current small molecules showed excellent n-channelbehavior in ambient with μe values reaching ∼0.9cm2/(V·s), Ion/Ioff ∼ 107–108, and Von ≈ 0 V. Our study notonly demonstrates one of the highest performing n-channel OFET devicesreported under ambient conditions via solution processing but alsoelucidates significant relationships among chemical structures, molecularproperties, self-assembly from solution into a thin film, and semiconductingthin-film properties. The design rationales presented herein may openup new avenues for the development of high-electron-mobility novelelectron-deficient indenofluorene and short-axis substituted donor–acceptorπ-architectures via alkyl chain engineering and interface engineering.The development of solution-processable n-type molecularsemiconductorsthat exhibit high electron mobility (μ These improvements were revealed to arise from advantageousnanostructure formation with strong π-interactions and bettergrain interconnectivity in the p-type semiconductor layer. Therefore,although it has been less explored, it is reasonable to expect thatdense ultrathin polymer interlayers would enhance the electron mobilitiesof n-type semiconductors, particularly in the context of urgentlyneeded solution-processed and ambient-stable OFETs.N-type (electron-transporting) molecularsemiconductors that aresolution-processable and ambient-stable are relatively rare, particularlythose that meet the criteria of high electron mobility dimalononitrile (IFDM). Thestructure for IFDM is shown as the acceptor π-unitin b]fluorene π-core is functionalizedwith electron-withdrawing dicyanovinylene moieties at the 6,12-positions.Employing this π-core, our group has previously designed andsynthesized a number of low-LUMO n-type semiconductors, 2,2′-ethynyl)indenofluorene-6,12-diylidene)dimalononitrile (TIPS-IFDM),26 2,2′--2,2′-bithiophen-5-yl)indenofluorene-6,12-diylidene)dimalononitrile (2OD-TTIFDMTT),27 and 2,2′-thiophen-2-yl)indenofluorene-6,12-diylidene)dimalononitrile ,28 yet all with electron mobilitiesof ∼0.01–0.1 cm2/(V·s) . Among thesesemiconductors, the thienyl-terminated IFDM molecularframework, 2,2′-indenofluorene-6,12-diylidene)dimalononitrile  have critical functions in determining crystalmotifs with varied degrees of interdigitation, lamellar stacking,and π-interactions.35 Our previous studydemonstrated that β-substitution is more effective than α,ω-substitutionfor solubility in indenofluorenes.36 Unlikearylene-based molecular semiconductors previously reported, whereβ-alkylation resulted in a marked reduction in thin-film crystallinityand charge transport,37 β-alkyl substitutionon the TIFDMT D-A-D π-system promotes high crystallinityand effective electron transport. Given the significance of alkylchain engineering and the semiconductor–dielectric interfacein determining OFET performance, the aforementioned rationales ledus to examine the low-LUMO β,β′-Cn-TIFDM π-corewith three different even-numbered linear alkyl chains of octyl (−C8H17), dodecyl (−C12H25), and hexadecyl (−C16H33) on high-density,ultrathin polymer interlayer brushes. This study has resulted in thedevelopment of two new molecular structures, β,β′-C8-TIFDMT and β,β′-C16-TIFDMT , Ion/Ioff ≈ 107–108, and Von ∼ 0 V.As pioneeredby several research groups in the early 2000s, π-conjugatedladder-type indenofluorene scaffolds have attracted a great deal ofattention to develop electron-transporting semiconductors for OFETsin the last two decades.1H NMR 400 MHz/13C NMR 100MHz). High-resolution mass spectrum measurements were carried outon a Advion Expression CMS-L spectrometer by using the atmosphericpressure chemical ionization (MS-APCI) method or on a Bruker Daltonics(microflex LT) spectrometer by using the matrix-assisted laser desorption/ionization-time-of-flight(MALDI-TOF) method. Conventional melting temperature measurementswere performed using an Electrothermal IA 9000 Series melting pointapparatus. Differential scanning calorimetry (DSC) and thermogravimetricanalysis (TGA) were performed on Mettler Toledo DSC822e and TGA/SDTA851einstruments, respectively. Indium and zinc standards  were used for calibration in DSC. UV–visoptical absorption measurements were performed via a Shimadzu UV-1800spectrophotometer in solution and as spin-coated thin films on quartz.Cyclic voltammetry measurements were performed in dichloromethaneon a C3 cell stand with a BASi LC Epsilon electrochemical analyzer.In the CV instrument, the counter and working electrodes were platinum(Pt) and the reference electrode was Ag/AgCl (3 M NaCl). The calibrationsof all potentials were done with reference to the standard ferrocene/ferroceniumredox couple (Fc/Fc+: E1/2 =+0.40 V measured in the same CV measurement setup). The optimizationsof the molecular geometries were carried out using density functionaltheory (DFT) at the B3LYP/6-31G** level with Gaussian 09.38The reactions were conductedunder nitrogen by using conventional Schlenk techniques. All chemicalswere purchased from commercial sources and used as received exceptwhere noted. Silica gel with a particle size of 230–400 meshand a pore size of 60 Å was used as the stationary phase forcolumn chromatography. NMR measurements were conducted using a Bruker400 spectrometer and malononitrile  was dissolved in dry chlorobenzene(35.0 mL) under nitrogen and stirred at 35 °C for 15 min. Then,anhydrous pyridine  and TiCl4  were added, and the reaction mixture was stirred at110 °C for 5 h under nitrogen. The resulting reaction mixturewas cooled to room temperature, and it was quenched with water andextracted with chloroform. The organic phase was washed with water,dried over Na2SO4, filtered, and evaporatedto dryness to give a crude product, which was purified by column chromatographyon silica gel using CHCI3:hexanes (8:2 (v/v)) as the eluent.Finally, the product was washed with methanol and filtered to affordthe final pure product as a dark green solid . 1H NMR (CDCl3): δ 8.57 , 8.50 ,7.68 , 7.63 , 7.31 , 7.03  2.72 , 1.27, 0.86  ppm. 13C NMR (CDCl3): 14.1, 22.7, 29.0, 29.3, 29.4, 29.5, 30.8,31.8, 78.6, 112.6, 113.1, 118.2, 121.2, 124.9, 127.5, 130.0, 134.4,135.5, 135.8, 137.1, 139.3, 140.3, 143.4, 159.7 ppm. Mp: 237–238°C. MS (MALDI-TOF) m/z (M+): calcd for C50H46S2N4, 767.32; found, 767.156. Anal. Calcd for C50H46S2N4: C, 78.29; H, 6.04; N, 7.30. Found:C, 78.42; H, 6.17; N, 7.52.A mixture of β,β′-C12-TIFDKT and malononitrile  was dissolved in dry chlorobenzene(35.0 mL) under nitrogen and stirred at 35 °C for 15 min. Then,anhydrous pyridine  and TiCl4  were added, and the reaction mixture was stirred at110 °C for 5 h under nitrogen. The resulting mixture was allowedto cool to room temperature, and it was quenched with water and extractedwith chloroform. The organic phase was washed with water, dried overNa2SO4, filtered, and evaporated to drynessto give a crude product, which was purified by column chromatographyon silica gel using CHCI3:hexanes (8:2 (v/v)) as the eluent.Finally, the product was washed with methanol and filtered to affordthe final pure product as a dark green solid . 1H NMR (CDCl3): δ 8.61 , 8.52 ,7.70 , 7.64 , 7.31 , 7.03  2.70 , 1.24, 0.85  ppm. 13C NMR (CDCl3): 14.1, 22.7, 29.0, 29.3, 29.4, 29.5, 30.8,31.8, 78.6, 112.6, 113.1, 118.2, 121.2, 124.9, 127.5, 130.0, 134.4,135.5, 135.8, 137.1, 139.3, 140.2, 143.1, 159.6 ppm. Mp: 231–232°C. MS (MALDI-TOF) m/z (M+): calcd for C58H62S2N4, 878.44; found, 879.029. Anal. Calcd for C58H62S2N4: C, 79.23; H, 7.11; N, 6.37. Found:C, 79.44; H, 7.21; N, 6.56.A mixture of β,β′-C16-TIFDKT and malononitrile  was dissolved in dry chlorobenzene(25.0 mL) under nitrogen and stirred at 35 °C for 15 min. Then,anhydrous pyridine  and TiCl4  were added, and the reaction mixture was stirred at110 °C for 5 h under nitrogen. The resulting mixture was allowedto cool to room temperature, and it was quenched with water and extractedwith chloroform. The organic phase was washed with water, dried overNa2SO4, filtered, and evaporated to drynessto give a crude product, which was purified by column chromatographyon silica gel using CHCl3:hexanes (8:2 (v/v)) as the eluent.Finally, the product was washed with methanol and filtered to affordthe final pure product as a dark green solid . 1H NMR (CDCl3): δ 8.61 , 8.53 ,7.70 , 7.64 , 7.31 , 7.03  2.70 , 1.24, 0.87  ppm. 13C NMR (CDCl3): 14.1, 22.7, 29.0, 29.4, 29.7, 30.8, 31.9,78.8, 112.8, 118.2, 120.5, 121.2, 122.5, 124.8, 127.5, 130.0, 134.8,135.9, 136.9, 137.1,140.2, 143.3, 159.6 ppm. Mp: 221–222 °C.MS (MALDI-TOF) m/z (M+): calcd for C66H78S2N4, 990.57; found, 991.598. Anal. Calcd for C66H78S2N4: C, 79.95; H, 7.93; N, 5.65. Found: C,79.85; H, 8.02; N, 5.79.A mixture of msemiconductor) and the total amount of solvent (Vsolvent) used based on the equation solubility= msemiconductor/Vsolvent. The final completely dissolved semiconductor solutionwas filtered through a PTFE syringe filter  and then evaporated to dryness with a rotaryevaporator. The recovered semiconductor was weighed again in orderto double check the dissolved semiconductor amount in Vsolvent; the difference between the originally weighedand the recovered semiconductor solids was typically less than 3–4%.The solubility of the currentsemiconductors was measured by using a gravimetric method. A smallamount (2.0–3.0 mg) of organic semiconductor solid was preciselyweighed into a vial, and incremental volumes (in 50–100 μLportions) of chloroform were added via micropipet. After each addition,the solution was stirred/sonicated for 10–20 min at room temperatureand heated to ∼50 °C to kinetically aid the dissolutionprocess. The solution was cooled to room temperature for visual observation,and solvent addition was continued until complete dissolution wasvisually confirmed at room temperature. The solubility was calculatedbased on the initial semiconductor solid weight for 3 min was applied to the substrates to activate their surfaces.The air plasma treated SiO2 layer was modified by usinghydroxyl-terminated polystyrene (PS-OH) with molecular weights of Mn = 5 kDa and Mn = 22 kDa via a “grafting-to”method. In this method, PS-OH in a 0.5 wt % toluene solution was spin-coatedon top of the SiO2 layer, followed by a thermal treatmentat 170 °C for 48 h in a vacuum oven. This annealing process allowedPS-OH chains to tether covalently from their hydroxyl end groups tothe hydroxylated SiO2 dielectric surface. After annealing,PS-brush-treated substrates were cleaned in an ultrasonic bath byusing toluene in order to remove any free PS-OH chains. The contactangle measurement was carried out for the PS-brush-treated substratewith a Biolin Scientific Attension Theta Lite instrument. The thicknessmeasurements for the PS-brush layers were performed using an ellipsometer, and the surface morphology was evaluatedby atomic force microscopy . The UV–visdiffuse reflectance spectra of the semiconductor thin films on thePS-brush interlayer were collected on a Shimadzu UV-3600i Plus spectrophotometerusing an ISR-603 integrating sphere.A heavily p-doped Siwafer with a thermally grown 300 nm thick SiOβ,β′-Cn-TIFDMT solution in CHCl3 (4.0 mg/mL)at 1100 rpm under ambient conditions, followed by thermal annealingat 100, 120 and 150 °C (for 30 min) in a vacuum oven. The OFETdevice structure was completed with thermal evaporation of 50 nm thickAu source-drain electrodes (growth rate 0.2 Å/s) to give channellengths (L) and widths (W) of 30,40, 50, 60, 80, and 1000 μm, respectively. The electrical characteristicsof the OFETs were measured under ambient conditions  using a Keithley 2614B source-measureunit in an Everbeing BD-6 probe station. The field effect mobilitywas calculated from the ISD1/2 vs VG plot by using an equation derivedfor the saturation regionISD is thesource-drain current, VG and VT are the gate and threshold voltages, respectively, and Cox is the specific capacitance of the gate dielectricwith PS-brush interlayer per unit area (taken as 10.5 nF/cm2).23 The surface morphologies and microstructuresof the semiconductor thin films were studied by atomic force microscopyon a NanoSurf FlexAFM C3000 instrument, and by grazing incidence X-raydiffraction (GIXRD) on a Malvern Panalytical Empyrean diffractometer.A confocal Raman spectrometer (Jasco NRS-4500) with an excitationwavelength of 785 nm was used for the Raman investigation of semiconductorthin films. All Raman spectra were collected using a 20× microscopeobjective, an optical power of 75 mW, and an acquisition time of 60s.Semiconducting thin films (∼40–45 nm) were depositedonto the PS-brush-treated substrates by spin-coating the corresponding b]fluorene-6,12-diylidene)dimalononitrile small molecules β,β′-Cn-TIFDMTs. The dibrominated indenofluorene-6,12-dione π-core, IFDK-Br2, is a crucial intermediate for producingthese molecules and was obtained via a two-step synthesis  in accordance with our group’spreviously reported procedure.36 The trimethylstannanereagents 1–3 were synthesized foreach molecule with a different alkyl chain , using a synthetic route involving Kumada coupling,bromination, and stannylation steps . IFDK-Br2 was reactedwith the corresponding reagents 1–3 following a Pd(PPh3)2Cl2-catalyzedStille cross-coupling protocol in DMF at 125 °C. A highly polarsolvent at an elevated temperature was used in these cross-couplingreactions in order to partially solubilize IFDK-Br2. Note that IFDK-Br2 exhibits a very low solubility in common organic solvents.2,8-Bisindenofluorene-6,12-dionecompounds β,β′-C8-TIFDKT, β,β′-C12-TIFDKT, and β,β′-C16-TIFDKT were obtained in 9–45%yields. Finally, these dicarbonyl-functionalized indenofluorene compoundsunderwent a Knoevenagel condensation reaction with malononitrile inchlorobenzene in the presence of a pyridine base and TiCl4 Lewis acid to yield the target compounds β,β′-C8-TIFDMT, β,β′-C12-TIFDMT, and β,β′-C16-TIFDMT in 35–51% yields.The molecular solids were soluble in common nonprotic organic solvents,which allowed for convenient chromatographic purifications. The chemicalstructures and the purities were confirmed via 1H and 13C NMR , elemental analysis, and mass spectrometry and matrix-assistedlaser desorption/ionization (MALDI-TOF) techniques) .β,β′-Cn-TIFDMTs were investigated by using thermogravimetricanalysis (TGA), differential scanning calorimetry (DSC), and conventionalmelting point measurements. As shown in 8, ∼57% for C12,and ∼62% for C16, which were attributed to the thermaldecomposition of alkylthienyl end units from the β,β′-Cn-TIFDMT compounds. This yielded the IFDM π-coreas the final solid. On the other hand, sharp thermal transitions ofsolid–isotropic liquid origin were observed by using a conventionalmelting point apparatus. The melting temperature (Tmp) was found to gradually decrease as the alkyl chainlength increased (Tmp = 237–238°C → 231–232 °C → 221–222 °Cfor C8 → C12 → C16). These melting transitions, alongwith some prior endothermic transitions, were evident in the DSC scans(HS→I) for solid–isotropic liquidtransitions. In sharp contrast to the observed melting temperaturetrend, ΔHS→I was found toincrease with the alkyl chain length as 44.07 kJ/mol for C8, 87.73 kJ/mol for C12, and 118.41 kJ/mol for C16 (Table S2). Since D-A-D π-backbonestructural properties 36 are expected to be the same in all three molecules, the observedΔHS→I increase with alkyllength is attributed to enhanced solid-state cohesive energetics viadispersion interactions between extended methylene units (−(CH2)n–CH3, n = 7 → 11 → 15).40 The observed endothermic transitions for C12 and C16 prior to melting suggest the presence of a liquid crystalphase 41 in certaintemperature ranges. The relationship between solubility and cohesiveenergetics has been initially established by Hildebrand and Scatchard,43 and solubility is correlated with cohesive energetics based on bothideal and regular solution theories.42 Thesolubilities of the present molecules were measured by using a gravimetricmethod, which closely follow the corresponding total enthalpy changes.40 While β,β′-C8-TIFDMT exhibits a solubility of 10.3 mg/mL (0.0134M) in chloroform at room temperature, β,β′-C12-TIFDMT and β,β′-C16-TIFDMT exhibit decreased(∼2–6×) solubilities of 6.9 mg/mL (7.85 ×10–3 M) and 2.5 mg/mL (2.52 × 10–3 M), respectively. A similar solubility dependence on alkyl chainlength and cohesive energetics was previously demonstrated for fusedthienoacene compounds.40 It is noteworthy that the completelydissolved solution of β,β′-C16-TIFDMT at concentrations of >3 mgmL–1 starts to aggregate upon prolonged waiting(>2–3h), probably through enhanced dispersion interactions between relativelylong linear −C16H33 chains. Therefore,thin-film processing (vide infra) for β,β′-C16-TIFDMT was performed by usingslightly heated solutions (∼40–50 °C). On the otherhand, for complete dissolution of β,β′-C12-TIFDMT, heating at ∼40–50°C was required; however, the solid remains in solution uponcooling to room temperature. This suggests that the thermal treatmentin the case of β,β′-C12-TIFDMT kinetically drives the initial dissolutionprocess for better solvent–solute interactions, rather thana pure thermodynamic effect.42 BecauseΔHS→I increases and Tmp decreases with the increased alkyl length,the corresponding total entropy changes for solid–isotropicliquid transitions also showed clear increases (ΔSS→I = 85.91 → 198.47 → 273.3 J/(Kmol) for C8 → C12 → C16, Table S2; see the Supporting Information for further details). This entropyincrease is undoubtedly endorsed by enhanced degrees of freedom (gauche and trans conformations)44 for long alkyl substituents via internal bondrotations in the melted state, adding an entropy value of ∼10J/(K mol) for each additional methylene unit, which is consistentwith earlier studies.40 For each alkyl substituent, the β,β′-Cn-TIFDMT molecule exhibits an ∼70–80°C increased melting temperature compared to the corresponding β,β′-Cn-TIFDKT molecule. This reflects enhanced intermolecularinteractions through increased local dipoles, greater π-delocalization,and increased D-A-D electronic structure going from carbonyl (C=O)to the dicyanovinylene unit (C=C(CN)2).The thermal properties of the present ompoundsS25. Thisβ,β′-Cn-TIFDMT molecules.47 The first and second half-wave reduction potentials are locatedat low voltages of −0.24 and −0.54 V, respectively.On the other hand, one main optical absorption peak at 342 nm, alongwith an extremely weak peak at 656 nm, was observed in dichloromethanesolutions , which corresponds to a vibronic coupling withthe C=C stretching modes41 and originatesfrom the rigid TIFDMT π-backbone. The optical band gaps in solutionare estimated to be 1.55 eV from the low-energy band edges. From theCV and UV–vis data, the HOMO/LUMO energy levels are estimatedto be −5.75–-4.20 eV for the present β,β′-Cn-TIFDMT molecules,which appear to be minimally affected by the length of the alkyl chain.The optical and electrochemical characterizationsof the presentmolecules were performed by UV–vis absorption/diffuse reflectancespectroscopy and cyclic voltammetry (CV). As shown in β,β′-Cn-TIFDMT thin films .48 When the thermal annealing temperature was raised to 150 °C on the p++-Si/SiO2/PS-brush (Mn = 5 kDa) substrateare transformed to pseudoabsorption data using the Kubelka–Munkfunction.46 Amazingly, these absorptionspectra (green lines in max = +85–90nm), narrow absorptions at λmax = 427–430nm (fwhm ≈ 20 nm). These spectral changes clearly point atthe formation of J-aggregation on the PS-brush interlayer, in whichthe D-A-D π-frameworks are in a slipped-stacked arrangement.50 This arrangement facilitates favorable intermolecular D···Ainteractions between thienyl (donor) and IFDM (acceptor) units, whichalso manifests itself in the significantly enhanced, broad low-energyHOMO → LUMO transitions at ∼600–800 nm. The structuralordering in the solid state on the PS-brush interlayer is consistentwith the thin-film XRD and AFM findings (vide infra).Going from solution to spin-coated thin films on quartz, for 100°C annealed in films S26 on thMn =5 and 22 kDa) in our study, and the PS-brush interlayers were preparedfollowing a “grafting-to” methodology.21 This is because high-molecular-weight (Mw > 100 kDa) polymers have been reported to yield alowareal grafting density (σ ≪ 0.1 chains/nm2) with pancake-like polymer interlayer structures, which can impedethe semiconductor molecular self-assembly, leading to an unfavorablepacking/microstructure for efficient charge transport.23 Both PS-5 and PS-22 surfaces are found to bemorphologically very smooth and pinhole-free, showing a low root-mean-square(Rq) roughness value of 0.17–0.18nm for a 10 × 10 μm2 area  according to Wu’s method,52 and they are much lower than that of the silanol surface(γ > 87 mJ/m2). The areal grafting density (σ)is the key parameter when considering the surface coverage and thepositional arrangement of polymer brushes with respect to each other. It can be calculated by using theequationPS = 1.05 g/cm3),23do is the brush height (polymerinterlayer film thickness), and NA and Mn represent Avogadro’s constant and thenumber-average molecular weight of the polymer, respectively. Basedon the polymer interlayer film thicknesses (do = 3.6 ± 0.2 nm for PS-5 and do = 6.6 ± 0.2 nm for PS-22) measured via ellipsometry, excellentgrafting densities of 0.45 and 0.19 chains/nm2 were calculatedfor PS-5 and PS-22, respectively. These high densities (σ >0.1 chains/nm2), along with low surface energy and smoothness,could hamper any undesired molecular diffusion into the polymer interlayer,which in turn can minimize any microstructural or morphological disordersat the semiconductor–dielectric interface (vide infra). Amazingly, the surface prepared in this study by the lower-molecular-weight(Mn = 5 kDa) chains show one of the finestgrafted-PS-brush arrangements of those given in the literature,22 and it offers a highly favorable dielectricsurface for β,β′-Cn-TIFDMT depositions.We employed hydroxyl-end-functionalized PS polymers (PS-OH) withrelatively low molecular weights  OFETs by spin-coating  were annealed at different temperaturesof 100, 120, and 150 °C. Higher annealing temperatures were foundto have negligible effects. As a result of their highly stabilizedLUMOs, all β,β′-Cn-TIFDMT molecules functionedas n-type semiconductors under ambient conditions with excellent electronmobilities of as high as 0.21 cm2/(V·s) (μeavg = 0.12 cm2/(V·s)) for β,β′-C8-TIFDMT, 0.87 cm2/(V·s) (μeavg = 0.60 cm2/(V·s)) for β,β′-C12-TIFDMT, and 0.29 cm2/(V·s) (μeavg = 0.14 cm2/(V·s)) for β,β′-C16-TIFDMT. Typical transfer and outputplots are shown in Figure S27, respectively.In addition to high electron mobilities, excellent current modulationcharacteristics with very large Ion/Ioff values of 107–108 and near-zero turn-on voltages were realized in our solution-processedOFETs. This is attributed to the well-tuned LUMO energy level (−4.20eV) of the TIFDMT π-system, which, while providingambient stability for electron transport, impedes undesired semiconductorelectron doping in the off state.9 While β,β′-C8-TIFDMT-based OFETs showed similar n-channel semiconductivities with μeavg values of 0.10–0.12 cm2/(V·s)at 120 and 150 °C annealing temperatures  → 0.60 cm2/(V·s) (2/(V·s) → 0.14 cm2/(V·s) (eavg < 10–3 cm2/(V·s)).The semiconductor properties of the current -coating 1. In ordβ,β′-Cn-TIFDMT thin films were studied by atomic forcemicroscopy (AFM) and out-of-plane grazing-incidence X-ray diffraction(GIXRD) techniques. The GIXRD technique at an incidence angle of 0.5°was preferred over a Bragg–Brentano geometry in order to revealsubtle microstructural differences between varied β,β′-Cn-TIFDMT thinfilms. As shown in β,β′-C12-TIFDMT and β,β′-C16-TIFDMT thin films, a similarlyprepared β,β′-C8-TIFDMT thin film showed relatively smaller grains(∼300–400 nm). A sufficient alkyl chain length (n ≥ 12) seems to be a key factor herein to achievemicrometer-sized crystalline domains.The morphologies and microstructures for spin-coated β,β′-Cn-TIFDMT thin-filmsexhibit one low-angle  (100) diffraction peak for each molecule(12 and (200)for −C16) develop. All these changes point to athermally induced thin-film crystallization behavior on the low-energyPS-brush interlayer, which is consistent with the aforementioned morphologicalimprovements. The d spacings for the 150 °Cannealed thin films were calculated to be 21.90 Å for β,β′-C8-TIFDMT, 25.88 Å for β,β′-C12-TIFDMT, and 29.61 Å for β,β′-C16-TIFDMT. Upon comparing d spacings with the geometry-optimized, computed moleculardimensions of β,β′-Cn-TIFDMT , we see that although they are longer than theTIFDMT π-length of 20.51 Å, they can be related to themolecular lengths perpendicular to the long π-axis. Moreover,the d spacings gradually increase with the alkylchain length (n = 8 → 12 → 16). Theseindicate that the present thin films have a long-range ordered edge-onlamellar packing with alternating σ- and π-layers, andthe alkyl substituents extend along the out-of-plane direction asillustrated in 53 It is worth noting that the highμe values observed in the β,β′-Cn-TIFDMT thinfilms provide additional evidence for edge-on lamellar packing. Thisis because such packing allows for strong interlamellar π-interactionsalong the substrate surface, facilitating an efficient source-to-drainelectron transport.54 As evidenced by optical characterizations (vide supra), the π-interactions appear to be in a J-aggregate-like slipped-stackedarrangement, which could also maximize the utilization of these dispersiveinteractions between alkyl chains of the adjacent lamellae. d spacings of thesemiconductor against the number of carbon atoms in its alkyl substituent,the resulting slope was determined to be 0.96 Å per methylene(−CH2−) unit. This value is lower than thecomputed increase in all-trans alkyl substituent length (1.24 Å/methylene).The difference points to a tilt angle of ∼39° for thealkyl substituents from the substrate surface normal, which matcheswell with the geometry-optimized, computed molecular structures havingall-trans alkyl substituents at an angle of ∼37–42°with respect to the long molecular axis . The observed d spacings for −C8 (24.51 Å) and −C12 (32.32 Å)at 100 °C annealing temperature compare well with the maximumpossible computed molecular lengths  with all-trans alkyl substituents.This also suggests that alkyl chains are not interdigitating at 100°C annealed thin films.The XRD profiles of the present molecule5 insets.n-TIFDMT S28, we sn-TIFDMT S28, we sd spacing successively decreases as 24.51 Å →21.90 Å → 21.90 Å for β,β′-C8-TIFDMT, 32.32 Å →31.52 Å → 25.88 Å for β,β′-C12-TIFDMT, and 31.07 Å→ 30.22 Å → 29.61 Å for β,β′-C16-TIFDMT. In view of the factthat the d spacing shortenings are accompanied bya strong thin-film crystallization and an increase in OFET electronmobilities, alkyl chain interdigitation is undoubtedly the key element.This is because a simple molecular tilt on the surface, which couldbe the only other option for the d spacing shortening,is highly unlikely to promote a large crystallization to yield micrometer-sizedgrains. Also, a molecular tilt may impede electron mobility, as thealkyl chains could adopt in-plane conformations and become more likelyto disrupt source-to-drain charge transport.56 On the other hand, dispersive interactions that could form overa large number of methylene units between flexible alkyl chains couldinduce a strong crystallization behavior via the so-called “fastener”(or “zipper”) effect.57 These interactions not only occur within the lamellaeto increase crystallite size in the out-of-plane direction but alsopromote in-plane crystal growth as evidenced in the AFM morphologies.We note that the cohesive energetics for an interdigitation of sixmethylene units, as we observed in the case of β,β′-C12-TIFDMT, could promote a largecrystallization energy of ∼50–60 kJ mol–1 of a molecule (8.9 ± 0.6 kJ mol–1 per eachmethylene unit),59 which can rearrange the in-planeπ-interactions over large domains. Moreover, this type of interdigitationeffectively maintains the insulating σ-alkyl chains within thelamellar plane, preventing them from hindering the charge transportbetween the source and drain. The degree of interdigitation was foundto increase with the alkyl chain length; while final interdigitationsof ∼6 and ∼10 methylene units were estimated for β,β′-C12-TIFDMT and β,β′-C16-TIFDMT, respectively, it was found to be more limitedfor β,β′-C8-TIFDMT involving ∼2 methylene units. Interestingly,the interdigitation for all semiconductors seems to propagate in theσ-layer of the lamellae until the first six methylene units(−(CH2)6–TIFDMT–(CH2)6−) are attached to the π-system.At this point, our attention is directed toward the alteration inthe degree of interdigitation upon thermal annealing, rather thanthe ultimate magnitude of interdigitation. This alteration should,indeed, be the pivotal mechanism driving the process of thermallyinduced thin-film crystallization. Among the current thin films, thelargest decrease in d spacing occurred for β,β′-C12-TIFDMT. While the d spacing shortening for −C8 and −C16 is only ∼1.5–2.5Å, −C12 shows a larger effect with ∼6.5Å, indicating that a larger number of methylene units are interdigitating(enhanced cohesive energetics)60 to drive the crystallization process. This effect was also apparentin the (100) coherence lengths (L) , which were estimated usingthe Scherrer equation61K is the Scherrerconstant (0.9), λ is the wavelength of the radiation for theCu Kα X-ray source (0.15406 nm), and β and θ arethe fwhm and the peak position values for the diffraction peaks, respectively,in radians. As shown in 12 when all (100)–(400) diffractionspeaks were taken into consideration, whereas −C8 and −C16 showed relatively smaller lengths of17.95 and 14.83 nm, respectively . In addition, higher order diffraction peaksup to the fourth order appear only for β,β′-C12-TIFDMT thin films, confirmingthe presence of a long-range crystal ordering.As shown in β,β′-C8-TIFDMT and C16-TIFDMT thin films show a broad peak at 2θ ≈ 20–21°at all annealing temperatures, which is assigned to short-range interlamellarπ-interactions (∼4.1 Å) between (010) planes. Thesame (010) peak was also evident for β,β′-C12-TIFDMT at 100 and 120 °Cannealed thin films. However, this peak completely disappears forthe β,β′-C12-TIFDMT thin film after 150 °C annealing  interruptions on the PS-brush interlayer. While going from −C8 to −C12 is apparently an effective strategyfor small molecules to realize additional crystallization via alkylchain interdigitation, much longer −C16 chains seemto induce extended alkyl chain interactions even at lower annealingtemperatures without leaving additional room for further interdigitationand, thus, crystallization. On the other hand, it is noteworthy thatthe annealing temperature of 150 °C employed for the present β,β′-Cn-TIFDMT semiconductors, which yielded the bestsemiconductor thin-film morphology/microstructure and the highestelectron mobility, is above the reported glass transitions of low-molecular-weightpolystyrene-based thin films63 and brushes.64 One could expect a deteriorating effect on chargetransport efficiency due to interfacial viscoelasticity of the polymerinterlayer at elevated temperatures.65 In contrast, the increased μe values(>100–1000×) measured in our case clearly suggeststhatthe thermally induced, strong crystallization behavior in the alreadyexisting, top-lying semiconductor layer (∼40–45 nm thicknessvs 3.6–6.6 nm for polymer interlayer) minimizes the interfacialviscoelasticity effect and governs the microstructural and morphologicalarrangement, as evidenced in our characterizations (vide supra). Note that, for the previously reported PS-brush interlayers, thesemiconductor layer was slowly deposited via thermal evaporation,which allows for the polymer interlayer viscoelasticity to interferewith the semiconductor crystallization during the early stages offilm growth.65 The excellent crystallinity for β,β′-C12-TIFDMT on the PS-brush interlayer even allowed us to observe external crystallattice vibrations (phonons) of the present organic thin film by usinglow-frequency Raman (<200 cm–1) scattering. Dueto the large molecular weight and weakness of intermolecular forces,these lattice vibrations typically occur at low frequencies for organiccrystals.67 As shown in β,β′-C12-TIFDMT thin film, after annealingat 150 °C, exhibited a clear, high-intensity peak at 58 cm–1 while the underlying substrate-based signal becamesuppressed . Note that this low-frequency peak did not exist in the 100°C annealed semiconductor thin film, as well as in the p++-Si/SiO2/PS-brush substrate. On the other hand,when the current β,β′-Cn-TIFDMT semiconductorthin films were fabricated on the Mn =22 kDa PS-brush , while loweraverage electron mobilities of 0.06–0.11 cm2/(V·s)were measured at an annealing temperature of 150 °C for −C8 and −C16, β,β′-C12-TIFDMT thin films maintainedtheir high average electron mobility of 0.48 cm2/(V·s)(μemax = 0.81 cm2/(V·s)).This clearly suggests that a relatively lower grafting density ofPS-22 (0.19 chains/nm2 vs 0.45 chains/nm2 ofPS-5) still offers a highly favorable polymer interlayer surface fora high-mobility n-type semiconductor, whereas it somewhat affectsthe charge-transport characteristics (Δμe ≈3–4×) for β,β′-C8-TIFDMT and β,β′-C16-TIFDMT. The AFM and XRD characterizationson PS-22  compare well withthose on PS-5, explaining the observed high electron mobility forthe β,β′-C12-TIFDMT thin film even on the PS-22 interlayer surface.Finally, we note that the output curves  exhibited a certain level of contact resistance, possiblyarising from an energetic mismatch between the work function of theAu electrode and the unoccupied transport states of β,β′-Cn-TIFDMTs ordue to electronic/morphological interfacial effects between the organicsemiconductor and the metallic electrode.69 Future investigations that explore alternative metallic/organicsource-drain contacts, utilize self-assembled monolayers on the electrodesurface, and incorporate charge injection layers or contact dopantshave the potential to reduce contact resistance and even further enhanceelectron mobility.70Finally, a subtledifference was evident in the GIXRDs. As shownin nnealing 6c inset.nnealing 6d. The eb]fluorene-6,12-diylidene)dimalononitrile small molecules, β,β′-Cn-TIFDMTs,having varied alkyl chain lengths has been designed and synthesized. The physicochemical and optoelectronicproperties of the new molecules have been studied in detail. The decreasedsolubility as the alkyl chain length increases was found to correlatewell with the increased solid–isotropic liquid transition enthalpies.This correlation strongly indicates that cohesive energetics, tunedvia alkyl chains, play a pivotal role in determining solubility. Moreover,it has been observed that all molecules undergo partial thermolysisof the terminal alkylthienyl end units at temperatures of 500–600°C. The HOMO and LUMO energies were estimated as −5.75and −4.20 eV, respectively. The semiconductor thin films wereprepared under ambient conditions via spin-coating on densely packed,ultrathin PS-brush surfaces. Going from solution to the solid-state, while optical characterizationssuggested J-aggregate formation in thin films, morphological characterizationsexhibited two-dimensional, micrometer-sized (∼1–3 μm),sharp-edged plate-like grains lying parallel with the substrate plane.OFETs fabricated by the current β,β′-Cn-TIFDMT moleculesshowed excellent n-channel behavior under ambient with μe values reaching ∼0.9 cm2/(V·s), Ion/Ioff ≈107–108, and Von ≈ 0 V. Detailed microstructural and morphological characterizationshave given us key insights into the present thin-film crystallizationmechanisms. Our findings revealed that thermally induced dispersiveinteractions occurring over a large number of methylene units betweenflexible alkyl chains  are critical to achievea favorable thin-film crystallization on the current PS-brush interlayer.While going from −C8 to −C12 isapparently an effective strategy for small molecules to realize additionalcrystallization via alkyl chain interdigitation, much longer −C16 chains seem to induce extended alkyl chain interactionseven at lower annealing temperatures without leaving additional roomfor further interdigitation and, thus, crystallization. Therefore,the optimal zipper effect was evident with C12 chains,which resulted in the formation of large crystallites having lamellarstacking ((100) coherence length ∼30 nm) in the out-of-planedirection with no face-on (010)-oriented crystallites. This particularmicrostructure could induce very efficient in-plane S → D chargetransport and lies at the origin of the impressive electron mobilitywe have observed with the current β,β′-C12-TIFDMT-based OFETs. The excellentcrystallinity of the β,β′-C12-TIFDMT thin film was also evident inthe observed crystal lattice vibrations (phonons) at 58 cm–1 in low-frequency Raman scattering. Our study, by combining all threekey transistor characteristics , not only demonstrates one of the finest solution-processedn-channel OFET devices reported under ambient conditions but alsohighlights the significant potential of electron-deficient indenofluoreneπ-cores for the development of high-performance semiconductors.In a broader context, the utilization of donor–acceptor typeπ-systems with alkyl substituents positioned along the shortmolecular axis exhibits great potential as high-performance semiconductorsin future research endeavors. This architecture possesses a naturalvoid space created by the alkyl substituents, allowing for interdigitationof alkyl chains. By carefully tuning the length of the alkyl substituentsand the underlying surface properties, optimal chain interdigitationand cohesive energetics can be achieved, leading to favorable microstructureand morphology for highly efficient electron transport.In summary, a library of solution-processable,low-LUMO (−4.20eV) 2,2′-indeno[1,2-


Furthermore, the natural bond orbital (NBO) analysis of the Fock matrix showed that in the dimer model, the (N8-H9···O24) bond is responsible for the stabilization of the salt with an energy value of 13.44 kcal mol−1. The frontier molecular orbitals (FMOs) analysis showed that NIC-OXA (form I) salt is more reactive and less stable than NIC, as the energy gap of NIC-OXA (form I) salt is less than that of NIC. The global and local reactivity descriptor parameters were calculated for the monomer and dimer models of the salt. The electrophilic, nucleophilic, and neutral reactive sites of NIC, OXA, monomer, and dimer models of salt were visualized by plotting the molecular electrostatic potential (MESP) surface. The study provides valuable insights into combining both experimental and theoretical results that could define the physicochemical properties of molecules.In the present work, nicotinamide–oxalic acid  salt was crystallized by slow evaporation of an aqueous solution. To understand the molecular structure and spectroscopic properties of NIC after co-crystallization with OXA, experimental infrared (IR), Raman spectroscopic signatures, X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC) techniques were used to characterize and validate the salt. The density functional theory (DFT) methodology was adopted to perform all theoretical calculations by using the B3LYP/6-311++G  functional/basis set. The experimental geometrical parameters were matched in good correlation with the theoretical parameters of the dimer than the monomer, due to the fact of covering the nearest hydrogen bonding interactions present in the crystal structure of the salt. The IR and Raman spectra of the dimer showed the red (downward) shifting and broadening of bands among (N15-H16), (N38-H39), and (C13=O14) bonds of NIC and (C26=O24), (C3=O1), and (C26=O25) groups of OXA, hence involved in the formation of NIC-OXA salt. The atoms in molecules (AIM) analysis revealed that (N8-H9 Pepinsky proposed the term “crystal engineering” to denote the process of constructing crystalline materials from molecules or ions using non-covalent synthesis that intended, nowadays, to evaluate or manipulate the structure–property relationship . This apNicotinamide  has attracted a lot of attention from researchers, as it is a cofactor and precursor of nicotinamide adenine dinucleotide (NAD), which is responsible for cell function, growth, survival, and metabolism . NIC is In continuation to the previously published work of nicotinamide–oxalic acid (form II) salt  and nicoP21/c space group with one NIC cation and one oxalate anion (singly deprotonated).The NIC-OXA (form I) salt was prepared by mixing an equimolar ratio of NIC and OXA in Millipore water. Initially, a supersaturation solution was prepared at room temperature and under-saturated by adding water to it. The solution was filtered using Whatman filter paper, and the crystalline product was isolated at room temperature by slow evaporation. NIC-OXA (1:1 or form I) salt was crystallized from water by slow evaporation . The pre−1 were obtained by averaging 20 scans with an optical resolution of 4 cm−1.Infrared (IR) measurements were performed using a Bruker Alpha-P ATR-spectrometer equipped with a diamond crystal  in an attenuated total reflection configuration. Data evathluation was carried out with the OPUS program . A single-beam background without the sample and single-beam spectra of the powered samples in a range of 400–4,000 cm−1 with a spectral resolution of 2 cm−1 for obtaining the spectra.Raman spectra were collected using a LabRAM HR 800 Raman spectrometer  equipped with an Nd:YAG laser operating at 532 nm. Samples were placed on microscope glass slides and were exposed to a laser power of 0.8 mW through an objective lens with ×50 magnification. Typically, 50 scans were accumulated with 1–2 s exposure times from 100 to 4,000 cmα1 radiation X-Ray tube (λ = 1.54059 Å). The tube voltage and amperage were set at 45 kV and 40 mA, respectively. The samples were measured on silicon wafer-based zero-background holders. The white product solid sample was scanned in the 2θ range of 5–40°, increasing at a step size of 0.02.XRPD patterns for the sample were collected using an Empyrean X-ray diffractometer  equipped with a PIXcel detector and a monochromatic Cu–KDSC analysis of samples was performed on the Q1000 DSC of TA Instruments. Samples (1–2 mg) were crimped in non-hermetic aluminum pans and scanned at a heating rate of 10°C/min under a continuously purged dry nitrogen atmosphere (flow rate 50 mL/min) using a similar empty pan as a reference.k, sk, and ωk) can predict the atoms suitable for nucleophilic, electrophilic, and radical attacks  were computed by the DFT/B3LYP/6-311++G   methodology  using Ga attacks .−1, respectively.The structures of NIC, OXA, and NIC-OXA (form I) salt in their crystalline states  are showA comparison was performed between the optimized parameters of NIC and the monomer model of NIC-OXA (form I) salt, which shows that calculations do not differ more than by 0.0030/0.0054 Å, 3.6/2.4°, 1.6/1.0° in experimental/theoretical bond lengths, bond angles, and dihedral angles, respectively, except for the bonds attached with the hydrogen atom. However, the major variations of 0.0044/0.0101 Å in experimental/theoretical values are noticed among the (C10-N8) bond in NIC-OXA (form I) salt, directing the formation of hydrogen bonds: (N8-H9···O2 =C3) and (N8-H9···O6-C4), respectively, between NIC (API) and OXA (coformer)  salt, major changes of 0.0406/0.0292 Å, 8.2/6.7° and 2.6/2.0° in bond lengths, bond angles, and dihedral angles, respectively, were found, except for the bonds possessing hydrogen atoms and which are involved in intermolecular hydrogen bonds. A noticeable difference of 0.0862/0.1183 Å was observed in the experimental/optimized bond length parameter of the (O1=C3) bond from OXA to monomer NIC-OXA (form I) salt. This is due to the fact that in individual OXA, a hydrogen atom H8 is attached to O5 of one carboxylic group, while in the NIC-monomer of OXA (form I) salt, this hydrogen was transferred to the N atom of the pyridine ring of NIC, resulting in the (N8-H9) bond. Similarly, the corresponding changes are observed in bond angles and dihedral angles, leading to the presence of intermolecular hydrogen bonds (N8-H9···O2=C3) in the monomer model of salt (form I) (as shown in The optimized parameters of monomer/dimer models of NIC-OXA (form I) salt  were alsIt can be seen that the variations in the bond angle of the C–N–C bond from experimental/monomer and dimer models were noticed to be 123.04°/123.29° and 123.60°, respectively, which confirmed the charge transfer phenomenon in NIC-OXA (form I) salt as reported by NIC, OXA, and NIC-OXA (monomer/dimer) salt contain 15, 8, and 23/46 atoms, respectively; hence, they possess 39, 18, and 63/132 normal modes of vibrations, active in both IR and Raman spectra. Combined experimental and theoretical vibrational assignments and wavenumbers of two constituents NIC, OXA, and NIC-OXA (monomer/dimer) salt are given in Comparisons between experimental and calculated IR and Raman spectra for NIC/OXA are shown in −1  salt. In NIC, the (C6-H12) stretching vibrations were calculated at 3,010 cm−1, correspondingly assigned at 3,013/3,010 cm−1 in the IR/Raman spectra. The (C-H) in-plane and out-of-plane bending deformation was calculated at 1,341 and 980 cm−1, correspondingly observed at 1,340/1,346 and 979/972 cm−1, respectively. In the monomer/dimer, the (C10-H11) stretching vibrations were calculated at 3,038/3,056 cm−1, which corresponds to the Raman peak at 3,023 cm−1. The asymmetric torsion of the ring was calculated at 418 cm−1 and observed at 411 cm−1 in IR and 415 cm−1 in the Raman spectrum region  stretching mode in the monomer/dimer was calculated at 1726/1,668 cm−1 and observed experimentally at 1,689/1,688 cm−1 in the IR/Raman spectra. It is clear from the aforementioned wavenumber that this carbonyl group was free of bonding in NIC and the monomer; therefore, there is a large difference in the simulated and observed wavenumber values with no reduction in wavenumbers of NIC and the monomer. In the dimer model, there was a reduction of 44 cm−1 with an increment in the calculated bond length value of 0.0072 Å. Hence, this group in the dimer was involved in intermolecular hydrogen bonding with neighboring NIC molecules  and (N15-H17) modes were calculated at 3,520 and 3,411 cm−1, respectively. The stretching vibrations of (N38-H40) and (N15-H16)/(N38-H39) were calculated at 3,469 and 3,392/3,141 cm−1 and observed at 3,335 cm−1 in the IR spectrum and 3,139 cm−1 in the Raman spectrum, respectively. It is clear that NH2 group in isolated NIC and monomer were free from bonding. While, in the dimer, the reduction of wavenumbers of 54 and 269 cm−1, with increment in bond lengths of 0.0016 and 0.0225 Å, respectively, in (N38-H40) and (N16-H17)/(N38-H39) bonds, showed the presence of intermolecular hydrogen bonding (N38-H40···O1=C3), (N15-H16···O1=C3), and (N38-H39···O14=C13). Therefore, the dimer has values similar to experimental values.NIC consists of a carboxamide group attached to the pyridine ring of the carbon atom. In individual NIC, the stretching vibration of the carbonyl (C4=O1) group was calculated at 1712 cmshown in . The strshown in . In the −1 /(C26=O24) were calculated to be at 1722/1,652 cm−1, corresponding to the peak observed at 1,647/1,646 cm−1 in the IR/Raman spectra, which showed that in both monomer/dimer models of salt, there were reductions in calculated wavenumbers of 66/136 cm−1 from OXA, along with the increment in bond lengths of 0.0693/0.0568 Å, respectively, hence revealing the presence of intermolecular hydrogen bonds (N8-H9···O2=C3) and (N8-H9···O24=C26), respectively, directing salt formation  group lie within the range of 1780–1,680 cm−1 . In the The XRPD analysis  was perfTo evaluate the presence of traces of physiosorbed water molecules that are not found XRPD data, differential scanning calorimetry analysis (DSC) was performed. b) and Laplacian of electron density (∇2ρb) at bcp, are used to provide the strength and nature of the presence of hydrogen bonds. Koch and Popelier suggested that the presence of hydrogen bonds for (donor proton···acceptor) depends on ρb within the range of (0.002–0.040 a. u.) and (∇2ρb) of 0.024–0.139 a. u. (2ρb) < 0 and Hb < 0 for strong and covalent nature; (ii) (∇2ρb) > 0 and Hb < 0 for medium and partially covalent nature; and (iii) (∇2ρb) > 0 and Hb > 0 for weak and electrostatic nature (Bader’s quantum theory of atoms in molecules (QTAIM) suggests that a bond path between an atomic pair shows an intermolecular hydrogen bonding interaction . DFT or 39 a. u. . Additioc nature . Furtherc nature .The QTAIM molecular graphs given for the optimized geometries of monomer and dimer models of salt (form I) are given in −1 possesses medium and partially covalent nature based on the Koch and Popelier criteria. The intermolecular hydrogen bond interaction (C10-H11···O2) with energy value −5.8986 kcal mol−1 was found to be of the same aforesaid and non-conventional nature. Another hydrogen bond (N8-H9···O6) with the calculated interaction energy value of −2.9493 kcal mol−1 also possesses a medium  nature , which are listed in ) nature .In the dimer model of salt (form I), the order of conventional intermolecular hydrogen bonds based on energy values are (N8-H9···O24) > (N38-H40···O1) > (N15-H16···O1) > (N38-H39···O14), which shows the medium and partially covalent nature, while (C22-H23···O25) > (C41-H42···O2) > (C41-H42···O1) are non-conventional intermolecular hydrogen bonds and direct the formation of NIC-OXA (form I) salt given in .NBO analysis is a powerful tool to examine the conjugative or hyper-conjugative charge transfer interaction phenomenon and provides a detailed study of intramolecular-and intermolecular hydrogen bonding in a molecular system . The ele(2) values of 82.84 and 63.45 kcal mol−1, respectively, which stabilize the molecule and ring R1, respectively. The charge transfer from [unit 1 to unit 2] and [unit 2 to unit 1]; [LP (2)O2 → σ *(N8-H9)] and [σ(C10-H11) → σ *(O2=C3)] with E(2) of 4.50 and 0.25 kcal mol−1, respectively, confirms the presence of strong intermolecular hydrogen bonds (N8-H9···O2) and (C10-H11···O2=C3), respectively  unit 2 (NIC) was [LP (3)O2 → π*(O1=C3)] and [LP (1)N15→ π *(C13=O14)] from maximum E(2) values of 88.62, 194.72, 101.58, and 297.50 kcal mol−1, respectively, confirm the intramolecular charge transfer interactions and stability of NIC-OXA (form I) salt. While other intermolecular charge transfer interactions, such as [LP (1) O1 → σ *(N15-H16)], [LP (1) O1 → σ *(N38-H40)], [σ(N15-H16) → σ *(O1=C3)], [σ(C22-H23) → σ *(O25=C26)], [LP (2) O14 → σ *(N38-H39)], [LP (2) O24 → σ *(N8-H9)], and [σ (N38-H40) → σ *(O1 =C3)], with maximum E(2) values of 10.18, 12.17, 0.18, 0.06, 0.95, 13.44, and 0.36 kcal mol−1, respectively, reveal the strong intermolecular hydrogen bonds: (N15-H16···O1), (N38-H40···O14), (N15-H16···O1=C3), (C22-H23···O25=C26), (N38-H39···O14), (N8-H9···O24), and (N38-H40···O1=C3), and hence the formation of salt (form I) , 2 (NIC), 3 (OXA), and 4 (NIC) [LP (3) O1 → π *(O2=C3)], [LP (1) C12 → π *(N8-C10)], [LP (3) O24 → π *(O25=C26)], and [LP (1) C35 → π *(N31-C33)] with EFMOs are widely used to explain the optical and electronic properties of a molecular system . The higThe chemical reactivity descriptors are focused to study the global and local reactivity of any molecule, as discussed in the following section.Global reactivity descriptors are useful to provide insight into the chemical reactivity and stability of chemical entities. Various global reactivity descriptors, such as electronegativity (χ), global hardness (η), global softness (S), chemical potential (μ), and electrophilicity index (ω), can define the global reactivity trends, which are calculated by using energies of frontier molecular orbitals (HOMO and LUMO). The parameter χ defines the electron attracting ability of molecules toward themselves and is an important parameter to provide fruitful information on active sites of an electrophile. According to Pearson, the lowest reactivity of molecules is due to the requirement of a larger resistance value to change its electronic distribution and harder nature . For anyvice versa for a good nucleophile. Additionally, the stable molecule must have the lowest value of ω. The values of ω lie in the range of NIC < OXA < monomer of NIC-OXA  < dimer of NIC-OXA . So, it is clear that NIC is most stable among OXA, monomer, and dimer models of salt (form I).As shown in The Fukui function (FF), local softness, and electrophilicity indices are useful tools to infer nucleophilic/electrophilic reactivity trends . Three kFrom the reported values of 2 functional group. Additionally, MESP of OXA displayed that positive potential was localized over the hydroxyl (−OH) group, one of which participated in the transfer of a proton from OXA to NIC and negative potential over two carbonyl (C=O) groups in the formation of salt. MESP diagrams of the monomer  onto the electron density (ED) surfaces, which is taken as constant. The blue  and red  color represent the electron-accepting and -donating ability of a molecule . MESP to monomer  and dime monomer  of NIC-O···O2 and N8-H9···O6), displaying a neutral (green) region  region . The othIn the dimer model, the MESP surface suggests that (N31-H32) was in the blue region (suitable for nucleophilic attacks) and (C27=O28) was in the red area (susceptible to electrophilic attack), shown in ···O24) is maximum with an interaction energy value −12.1 kcal mol−1 that is of medium and partially covalent nature. Furthermore, NBO results showed that the charge transfer interaction from OXA → NIC; [LP (2) O24 → σ *(N8-H9)] is responsible for the stabilization of NIC-OXA (form I) salt. The smaller HOMO–LUMO energy gap of the dimer (1.5896 eV) < monomer (2.2044 eV) < NIC (5.5356 eV) ensured that the chemical reactivity of NIC has been enhanced after formation of NIC-OXA (form I) salt. Hence, the salt is softer and less stable than NIC. The local reactivity parameters defined the most reactive nucleophilic and electrophilic attacking sites for O25 and C41 atoms, respectively, in the dimer model. In the monomer, the nucleophilic and electrophilic reactivities were mainly found around NH2 of NIC and (O1 and O5) of OXA, respectively, as viewed from the MESP map. These sites were neutralized in the dimer model, due to the involvement in intermolecular hydrogen bonds. Thus, this study provided the usefulness of combined experimental and theoretical results to define structure-reactivity and hydrogen bonding interactions, which could further be useful to understand other pharmaceutical molecules.The molecular structures, geometries, hydrogen bonding interactions, and reactivities of NIC-OXA (form I) salt have been studied by using experimental techniques (IR and Raman) combined with DFT calculations. The structural and vibrational spectroscopic signatures revealed that in the dimer model, (N8-H9) of the pyridine ring of NIC was involved in intermolecular hydrogen bonding with the (C26=O24) group of OXA and possessed a red shifting in wavenumber along with increase in bond length of (C26=O24) of dimer from that of individual OXA. It is found that the vibrational wavenumbers and bond length values of the dimer model of salt match well with the experimental results due to consideration of the nearest possible hydrogen bonding interactions than the monomer. XRPD and DSC analyses suggested that the obtained NIC-OXA (form I) salt has a single-crystalline pure phase. The AIM analysis depicted that the strength of the hydrogen bond (N8-H9


To assess the effectiveness and safety of the intravitreal fluocinolone-acetonide implant (FAc-i) in patients with chronic diabetic macular edema who did not sufficiently respond to other available therapies.This was a multicenter, prospective, non-randomized, and phase-IV observational study conducted on patients with recurrent-DME who were insufficient responders to currently available therapies (REACT-Study). The primary end-point was the mean change in best-corrected-visual-acuity from baseline to month-24 values.p = 0.0510). The eyes with a baseline BCVA < 70 ETDRS letters had a significant improvement in BCVA from 53.2 ± 10.2 letters at baseline to 61.5 ± 17.9 letters at month-24 (p = 0.0165). In the overall study population, central-subfoveal-thickness (CST) was significantly reduced from 474.0 ± 135.1 µm at baseline to 333.4 ± 135.6 at month-24 (p < 0.0001). Similarly, macular-volume (MV) was significantly reduced from 10.7 ± 2.7 mm3 at baseline to 9.6 ± 2.9 mm3 (p = 0.0027) at month-24. Among the 31 study eyes, 19 (61.3%) required an additional treatment for DME. Throughout the study, 9 (29.0%) eyes required ocular hypotensive medication for controlling their intraocular-pressure and 5 (16.1%) eyes underwent cataract surgery.Thirty-one eyes from 31 patients were included in the study. Mean age was 68.0 ± 7.7 years, and 10 (32.3%) were women. Study patients had received 5.3 ± 7.3 previous DME treatments before starting the study. In the overall study sample, BCVA improved from 56.1 ± 12.3 letters at baseline to 62.4 ± 17.0 letters at month-24 (In DME eyes who did not sufficiently respond to previous therapies, the FAc-i was associated with an improvement in visual and anatomic outcomes. There were no unexpected adverse-events.EudraCT identifier: 2016-001680-37.The online version contains supplementary material available at 10.1007/s10792-023-02864-2. Diabetic macular edema (DME) is a prevalent and disabling disease that directly impairs central vision of patients with diabetic retinopathy (DR), making diabetes the leading cause of severe vision impairment in working-age populations of developed countries .Among the different treatment options currently available for patients with DME, vascular endothelial growth factor inhibitors (anti-VEGF) are considered first-line treatments . However®; Alimera Sciences, Hampshire, UK) is a non-erodible implant, which can deliver a low daily dose (0.2 µg per day) sustainedly of fluocinolone acetonide over a 3-year period [Fluocinolone acetonide sustained-release intravitreal implant (FAc-i) .This study was in accordance with the ICH guidelines and guidelines for Good Clinical Practice (GCP), with the Declaration of Helsinki   and the The study protocol was approved by the ethics committee of Bellvitge University Hospital and registered in the European Union Clinical Trials Register (EudraCT identifier: 2016-001680-37).The study began on September 26, 2017, and ended on February 16, 2022.Patients ≥ 18 years of age; diagnosed with DME according to investigator’s clinical evaluation and demonstrated using fundoscopic photography and spectral domain optical coherence tomography (SD-OCT); who were considered as insufficiently responsive to currently available therapies; and willing to comply with the investigators and protocol indications were included in the study.The main inclusion/exclusion criteria are listed in Table S1.®; Alimera Sciences, Hampshire, UK) in applicator with an initial release rate of 0.2 µg per day. The implant was administered by injection according to the method of administration defined in the Summary of Product Characteristic [All eligible patients with a signed informed consent received a 190 µg FAc-i ; one baseline visit (administration of FAc-i) and 10 follow-up visits performed at week 1 ± 2 days; month 1 ± 7 days; months-3, -6, -9, and − 12 ± 15 days; and months-15, - 18; -21; and − 24 ± 30 days.The primary end-point was the mean change in BCVA from baseline values.The secondary end-points included the mean changes in central subfield thickness (CST) and macular volume (MV) assessed using SD-OCT; the incidence of adverse events; and the results of a Quality-of-Life Analysis (VFQ-25 questionnaire).© 2023 SAS Institute Inc.A standard statistical analysis was performed using SAS/STAT software, Version 9.4 of the SAS System for Windows. CopyrightPrior to the study, it was planning to include 40 patients, 5 patients in each clinical center. Because it was a pilot study, no sample size was estimated.Continuous variables were described by mean; median; standard deviation (SD); inter-quartile range (IqR); minimum; and maximum, as appropriate, while categorical variables were summarized by number and percentages.No data were excluded due to protocol violations.Intent-to-treat analysis included all patients who received study medication.t test or the Wilcoxon test, as appropriate, were used to assess changes in BCVA, CST, MV, and intraocular pressure (IOP).The two-tailed paired sample p value of less than 0.05 was considered significant.Categorical variables were compared using a Chi-square test and a Fisher`s exact test, as needed. In order to evaluate the influence of the initial BCVA on the treatment effectiveness, the eyes were stratified according to their baseline BCVA (BCVA < 50 ETDRS letters versus BCVA ≥ ETDRS 50 letters).Thirty-one eyes from 31 patients were included in the study. Eleven patients were lost of follow-up 10 for personal reasons (due to the lockdown measures taken during the SARS-COV-2 pandemic) and one patient due to an adverse event (Vitreous hemorrhage). The study flowchart is shown in Figure S1.Mean age was 68.0 ± 7.7 years, and 10 (32.3%) were women. The median (IqR) follow-up was 35.9 (23.6 to 37.5) months.Study patients had received 5.3 ± 7.3 previous DME treatments before starting the study.The main demographic and clinical characteristics are summarized in Table p = 0.0510)  ; BCVA was significantly greater at week-1 (p = 0.0085), month-1 (p = 0.0297), month-3 (p = 0.0079), and month-6 (p = 0.0432) in the eyes with a baseline BCVA ≥ 50 ETDRS letters  eyes had a BCVA ≥ 50 ETDRS letters and 8 (25.8%) ones had a BCVA < 50 ETDRS letters. Besides baseline BCVA, which was significantly lower in the eyes with BCVA < 50 ETDRS letters .p = 0.0001); 386.8 ± 132.4 µm (p = 0.0006); 361.4 ± 125.0 µm (p < 0.0001); 376.9 ± 154.4 µm (p = 0.0009); 364.0 ± 126.4 µm (p = 0.0003); 363.2 ± 162.6 µm (p = 0.0020); 368.7 ± 154.6 µm (p = 0.0012); 377.5 ± 146.6 µm (p = 0.0240); 345.6 ± 158.8 µm (p = 0.0011); and 333.4 ± 135.6 µm p < 0.0001) at week-1; and months 1, 3, 6, 9, 12, 15, 18, 21, and 24, respectively  .An analysis of the anatomical response based on the baseline BCVA revealed that, in the eye with a baseline BCVA ≥ 50 letters, CST was significantly reduced from 464.30 ± 133.4 µm at baseline to 321.05 ± 121.7 µm at month-24 (p = 0.0625) .Although in eyes with a baseline BCVA < 50 letters the CST was reduced from 501.75 ± 145.3 µm at baseline to 375.40 ± 185.6 µm at month-24, such a difference was not statistically significant (3 at baseline to 10.2 ± 2.6 mm3 (p = 0.0002); 10.0 ± 2.7 mm3 (p = 0.0017); 10.1 ± 2.0 mm3 (p = 0.0001); 10.1 ± 1.9 mm3 (p = 0.0172); 10.1 ± 1.8 mm3 (p = 0.0209); 10.1 ± 2.4 mm3 (p = 0.0310); 10.5 ± 2.7 mm3 (p = 0.0084); 10.4 ± 2.4 mm3 (p = 0.0306); 10.0 ± 2.3 mm3 (p = 0.0080); and 9.6 ± 2.9 mm3 (p = 0.0027) at week 1; and months 1, 3, 6, 9, 12, 15, 18, 21, and 24, respectively  required an additional treatment for DME. Fifteen (78.9%) eyes underwent intravitreal anti-VEGF and 11 (57.9%) an intravitreal corticoid (Table S3).Over the course of the study, 33 adverse events (AEs) were reported in 17 patients. Among them, 8 (24.2%) AEs were possible-definitive related to the study medication.Six (18.2%) AEs were classified as serious, namely one retinal detachment, one colorectal cancer, one patient who underwent intestinal surgery, and 3 eye who developed cataract in the treated eye. Fifteen (45.5%) AEs were considered as moderate-to-severe. Additionally, 6 (19.4%) patients underwent cataract surgery throughout the study, 5 (16.1%) of them in the study eye.An overview of the different AEs is shown in Table p = 0.0086). Five (16.1%) eyes had an increase in IOP ≥ 10 mmHg.There was statistically significant increase in mean IOP from 14.6 ± 3.0 mmHg at baseline to 16.8 ± 2.7 mmHg  eyes required ocular hypotensive medication for controlling their IOP . Regarding the ocular hypotensive medications administered during the study, 5 (16.1%) eyes received timolol maleate 0.5%; 4 (12.9%) eyes received dorzolamide/timolol fixed combination; 3 (9.7%) eyes received brimonidine/timolol fixed combination; 3 (9.7%) eyes received latanoprost 0.005%; 3 (9.7%) eyes received brimonidine; and 2 (6.5%) eyes receive apraclonidine; and 2 (6.5%) eyes received treatment with brinzolamide.Although DME is a disabling disease, which significantly impacts on patients’ quality of life, there are currently diverse therapeutic strategies for its management .The advent of anti-VEGF meant a change in the paradigm of DME treatment . HoweverThe increasingly relevance that inflammation plays in the pathophysiology of DME has led to corticosteroids gaining importance as an alternative therapeutic strategy, particularly in recurrent DME and in patients resistant to anti-VEGF treatment , 22.Sustained-release corticoid formulations have emerged as a therapeutic strategy that allows a gradual corticoid release, with the subsequent reduction in the patient’s treatment burden .®, AbbVie) and the fluocinolone acetonide intravitreal implant 0.19 mg .There are two sustained-release corticosteroid implants currently approved in Spain for treating patients with DME, namely the dexamethasone intravitreal implant 0.7 mg .Nevertheless, in those eyes with a baseline BCVA < 70 ETDRS letters, BCVA improvement at month 24 was statistically significant  eyes required an additional treatment for DME during the study. This result was greater than that reported by the FAME study (15.2%) , 10 and Regarding safety, the REACT study has found no unexpected adverse events. Throughout the study, 33 adverse events (AEs) were reported in 17 patients, with 6 AEs considered as serious.Over the course of the study, 8 (25.8%) eyes started ocular hypotensive medication for controlling their IOP, although none of them required IOP-lowering surgery.Among the phakic eyes, 5 (50.0%) eyes underwent cataract surgery throughout the study.These findings did not significantly differ from those published in the literature , 25–36.This study has some limitations that need to be considered when interpreting its results. The main one was the sample size. Although it was originally planned to include 40 eyes (5 eyes per center), only 31 eyes were included in the study. This could have motivated the lack of statistical significance in the BCVA improvement of the BCVA achieved at 24 months.FAc-i was associated with a trend toward improvement in BCVA at month 24 in the overall study sample. Moreover, in those eyes with a baseline BCVA < 70 ETDRS letters, there was a statistically significant improvement on BCVA at month 24 after FAc-i injection.The anatomic outcomes, both the CST and the MV, improved significantly after the administration of the FAc-i.Regarding safety, no unexpected adverse events were reported. Five (16.7%) eyes required cataract surgery, and 8 (25.8%) eyes started ocular hypotensive medication for controlling their IOP.Study Flowchart. 31 patients were included in the study from September 26, 2017, to October 30, 2018, and all patients were treated. The patients participated in this study between 26 September 2017 (first consent) and 16 February 2022 (last follow-up visit). Eleven did not complete the study: 10 loss of follow-up and 1 due to an adverse event.Mean change in best corrected visual acuity (BCVA) throughout study follow-up in the overall study population (A) and in the eyes with a baseline BCVA < 70 ETDRS letters. Statistically significance was calculated by using paired sample two-tailed t test or Wilcoxon test, as appropriate. BCVA: Best corrected visual acuity; ETDRS: Early Treatment Diabetic Retinopathy Study; ns: Not significant; W: Week; M: Month.p < 0.01 from baseline. *p < 0.005 from baseline. **p < 0.001 from baseline.A comparison of the mean central subfoveal thickness CST throughout study follow-up in the eyes with a baseline BCVA < 50 ETDRS letters  and those with a baseline BCVA ≥50 ETDRS letters . Intragroup statistically significance was calculated by using the Wilcoxon test. Between group differences were calculated with the independent-samples t test. CST: Central subfoveal thickness; MV: Macular volume; W: Week; M: Month; ns: Not significant. a Between group significance, †p<0.05 from baseline. ‡Supplementary file4 (DOCX 15 kb)Supplementary file5 (DOCX 17 kb)Supplementary file6 (DOCX 13 kb)Below is the link to the electronic supplementary material.