We analyse the impact of the substrate on the initial MLD reactions in titanicone film growth using three different surface models: anatase TiO2, rutile TiO2 and Al2O3. Calculated energetics show that while TiCl4 is reactive towards the anatase and rutile TiO2 surfaces, it is not reactive towards the Al2O3 surface. Ti(DMA)4 is reactive towards all surfaces. This is attributed to the stronger Ti–Cl bonds in TiCl4 compared to Ti–N bonds in Ti(DMA)4. Ti(DMA)4 also shows high reactivity to the organics compared to TiCl4. Double reactions of EG and GL with the TiCl3 species from TiCl4 and TiDMA species from Ti(DMA)4 are also explored to better understand the origin of the different thicknesses of EG–titanicone and GL–titanicone films observed in experimental work. We find that EG and GL coupled with TiCl4 can orient in a flat lying configuration on anatase while on rutile, the preferred orientation is upright. When combined with Ti(DMA)4, EG and GL prefer the flat lying configuration on all surfaces. This work shows that the choice of the surface and the metallic precursor has a major impact on the behaviour of organic species. DFT findings provide motivation to develop a low temperature rutile TiO2/titanicone film suggesting that the desired film growth could be achieved.The development of hybrid inorganic–organic films with well-controlled properties is important for many applications. Molecular layer deposition (MLD) allows the deposition of these hybrid films using sequential, self-limiting reactions, similar to atomic layer deposition (ALD). In this paper, we use first principles density functional theory (DFT) to investigate the growth mechanism of titanium-containing hybrid organic–inorganic MLD films, known as “titanicones”. We investigate in detail the chemistry between the most common Ti precursors, namely titanium tetrachloride (TiCl Molecular layer deposition (MLD), a thin film deposition technique, has attracted significant attention in recent years as a suitable approach for the deposition of organic–inorganic hybrid films for applications in several technological application areas, including packaging/encapsulation, electronics, batteries and biomedical applications –4. MLD iThe advantages offered by hybrid organic–inorganic MLD films are that they are ultrathin films with high flexibility, have tunable properties and excellent mechanical and electronic properties resulting from the combination of the individual properties of the organic and inorganic components that are incorporated into the film –213–15.2x)y–O–M–O. Metalcones are known to be flexible in nature due to the flexible organic backbones present in their architectures and with excellent mechanical properties at the atomic and molecular level arising from the organometallic precursors [A special class of hybrid organic–inorganic films are so-called metalcones, which are fabricated via MLD using organometallic precursors and organic alcohols. These films are described as O–M–O–, with organic alcohols such as ethylene glycol (EG) [3)2)4), henceforth denoted Ti(DMA)4) was also successfully employed as Ti source and combined with EG and GL to deposit titanicone films [One of the most extensively researched metalcones are titanicones. Titanicones are deposited by coupling a titanium inorganic precursor, such as titanium tetrachloride (TiClcol (EG) –31, glyccol (EG) –31 or fucol (EG) . Tetrakine films .4 as metal source and EG or GL as organic precursors. TiCl4–EG films and TiCl4–GL films were deposited on Si(100) wafers. XRR analysis found a growth per cycle (GPC) of ≈4.6 Å/cycle for TiCl4–EG films at 90 °C which decreases to 1.5 Å/cycle at 135 °C. It was assumed that this drop could be related to the desorption of unreacted TiCl4 species at 135 °C. In addition, it was proposed that the reduction in growth rate could also be caused by the desorption of Ti(–O(CH2)2O–)2 species or double reactions of the organic precursor in which the molecule lies flat with both termini binding to Ti sites.In ref  titanico4–EG films was similar to the thickness and thickness reduction observed for alucone films grown using trimethylaluminium (TMA) and EG (4.0 Å/cycle at 85 °C to 0.4 Å/cycle at 175 °C) [The thickness and thickness reduction with temperature increment observed for TiCl 175 °C)  and zinc 175 °C) .4–GL films the growth rate was 2.8 Å/cycle at 130 °C and it decreased to 2.1 Å/cycle at 210 °C. The thickness of TiCl4–GL films was similar to the thickness of alucone films grown using TMA and GL (2.5 Å/cycle) [4–EG and TiCl4–GL films were achieved also in ref. [4–EG: 4.5 Å/cycle at 115 °C; TiCl4–GL: 2.2 Å/cycle at 150 °C).For TiClÅ/cycle) . Similar in ref.  4 was combined with EG and GL to deposit titanicone films in a temperature range from 80 °C to 160 °C on an Si substrate with 100 nm thermal oxide. In situ ellipsometry revealed for EG-based titanicones that the growth initiates but terminates after only 5 to 10 cycles, while for GL-based titanicones the growth proceeds and films have a GPC of 0.9 Å/cycle to 0.2 Å/cycle as the temperature increases. This is due to the double reaction phenomenon, which for EG removes both the reactive OH groups required to couple with the Ti precursor, while even with a double reaction, GL has a third OH group available to couple to the Ti(DMA)4 precursor in the next cycle [4 inorganic precursor is well known and widely used in ALD for the deposition of TiO2 and TiN films by thermal and plasma-enhanced processes [Ti(DMA)xt cycle . Ti(DMA)rocesses –36.4 and Ti(DMA)4 were successfully employed in MLD of titanicone films [4 is a small halide molecule that has Ti–Cl bonds, Ti(DMA)4 is a bulkier molecule with Ti–N bonds. Bond dissociation energy values for the breakage of the Ti–Cl and Ti–N bonds serve as a measure of the stability of these precursors and these are 494 kJ/mol and 464 kJ/mol for Ti–Cl and Ti–N, respectively. Bond dissociation energies indicate that a lower energy is needed to break the Ti–N bond compared to the Ti–Cl bond and hence a higher reactivity for the Ti(DMA)4 precursor towards the surface and towards the co-reactant is expected.As described above, TiClne films ,33. Whil4, the “reservior” effect, which is very common for small molecules such as TiCl4 [4 process yield H–N(CH3)2 by-products. In contrast this is non-corrosive compared to HCl by-products released during the TiCl4 process. For TiCl4 this is considered a significant drawback [In addition, due to the bulky DMA ligands of Ti(DMA)as TiCl4 , DEZ [21as TiCl4  and whicdrawback .4 on an Si substrate in a temperature range of 180 °C to 350 °C. A temperature-dependent growth characteristic was observed with the growth rate decreasing from 1.10 Å/cycle at 180 °C to 0.49 Å/cycle at 300 °C. This reduction was attributed to the increased thermal motion and desorption of molecules on the growth surface. FTIR spectra indicated that the hybrid film shows a stable bridging bonding mode between Ti and the acid group at temperatures under 200 °C and a high bridging/bidentate mixed bonding mode at temperatures over 250 °C and 300 °C [Fumaric acid (FC) is another alcohol organic precursor that was used to deposit titanicone films using TiCld 300 °C .Many studies show that the desired properties and the target thickness of a metalcone MLD film are not actually achieved. To help understand this, first principles density functional theory (DFT) calculations have been employed to explore the reaction mechanisms, energetics and the role of the organic precursors on the growth of hybrid films ,39–41. D2; EG still prefers to orient in a flat configuration and react twice with the surface, while GL prefers to lie in an upright configuration. This yields thicker GL-based magnesicone films, consistent with the experimental observations [However, DFT results also show that this scheme depends on the metal precursor and the surface as the same aliphatic precursors behave differently when combined with Mg(Cp)rvations .DFT studies and experimental work show that aromatic organics can be a better option for the growth of thicker, more flexible and more stable hybrid films. This because aromatic molecules, due to their stiff backbone prefer to orient in a vertical configuration and avoid the double reactions ,42. Such4 was coupled with 4-aminophenol (AP) to deposit Ti–(O–C6H4–N=) thin films with an essentially ideal growth rate of 10–11 Å/cycle at a temperature range of 120–160 °C. The deposited films were of high quality and stable in air. The high growth rates are attributed to the rigid structure of AP and the fact that Cl ligands of TiCl4 are small and do not cause steric hindrance.Many Ti–organic MLD processes have been developed by using different aromatic molecules as organic precursors. In ref , TiCl4 w4 and the homo/heterobifunctional aromatic molecules hydroquinone (HQ), 4-aminophenol (AP), p-phenylenediamine (PDA) and 4,4′-oxydianiline (ODA). All films were deposited on a Si surface. All four processes yielded amorphous Ti–organic films. A growth rate of 10–11 Å/cycle was achieved for the TiCl4–AP process, which is higher when compared to the growth rates 4.3 Å/cycle, 1.2 Å/cycle and 1.4 Å/cycle for TiCl4–HQ, TiCl4–PDA and TiCl4–ODA processes, respectively. The results are attributed the higher reactivity of the OH group with TiCl4 in comparison to the NH2 group and the higher tendency of the heterobifunctional organic precursor to orientate in an upright configuration and avoid unwanted double reactions on the surface [In another study, Ti–organic films were deposited using TiCl surface .4 was also coupled with 4,4′-oxydianiline (ODA) to deposit the (–Ti–N–C6H4–O–C6H4–N–)n thin films. Films were deposited on an Si surface in two temperature ranges, 160–230 °C and 250–490 °C. The growth rate increases with increasing temperature, from 0.3 Å/cycle at 160 °C to 1.1 Å/cycle at 490 °C. Films deposited at a higher temperature were also more stable in atmosphere compared to films deposited at low temperatures [TiCleratures .2 ALD films, titanicone films are promising for electronics and solar applications [2 films have catalytic and photocatalytic properties [2 frameworks formed by the annealing of titanicone films may serve as catalytic supports [2/carbon composite films with important electrochemical applications as electrodes for Li ion batteries or pseudocapacitance supercapacitors [Because of the similarity with TiOications  and may operties  porous Tsupports . Titanicpacitors . These fpacitors .As described above, different titanicone and Ti–organic MLD processes have been developed and although first principles density functional theory (DFT) simulations have recently been used to explore the reaction mechanism in Ti–organic MLD film growth , such a 2, rutile TiO2 and Al2O3 surfaces using TiCl4 or Ti(DMA)4 as Ti source and EG or GL as organic components. Calculated energetics suggest a higher reactivity of Ti(DMA)4 towards the selected surfaces and the organic molecules compared to TiCl4. This is due to the stronger Ti–Cl bonds in TiCl4 compared to Ti–N bonds in Ti(DMA)4. We also found that the choice of surface and inorganic precursors can influence the behavior of organic molecules by allowing or preventing undesirable double reactions. Based on the calculated energetics we propose that a low temperature rutile TiO2/TiCl4–EG process and rutile TiO2/TiCl4–GL process can lead to thicker hybrid films.In this study we investigate the molecular mechanism of formation of titanicone films on anatase TiO2 (101) surface with a coverage of 1 ML OH, rutile TiO2 (110) surface with a coverage of 0.75 ML OH, and an Al2O3 (0001) surface at a coverage 0.50 ML OH [4) and tetrakis(dimethylamido)titanium (Ti(DMA)4) inorganic precursors and then ethylene glycol (EG) and glycerol (GL) organic co-reactants.All DFT calculations in this work were performed using the Vienna Ab initio Simulation Package (VASP) version 5.4 . Titanic50 ML OH . These s34s1, Al: 3s23p1, O: 2s22p4, Cl: 3s²3p⁵, C: 2s22p2 and H: 1s1. Calculations were performed using the Perdew–Burke–Ernzerhof (PBE) exchange–correlation functional [−4 eV while that for the forces was −2 × 10−2 eV/Å. The geometry was optimized by relaxing the ionic positions using an energy cut-off of 400 eV as well as a Monkhorst−Pack k-point sampling grid of (3 × 3 × 1). The lattice parameters are a = 20.612, b = 15.164, c = 24.399 and α = β = γ = 90° for the anatase TiO2 surface model, a = 11.846, b = 13.051, c = 33.870 and α = β = γ = 90° for the rutile TiO2 surface model and a = b = 19.228, c = 40.627 and α = β = 90°, γ = 120° for the Al2O3 surface model. The surfaces are 2, 4 and 5 layers thick for anatase (101), rutile (110) and Al2O3 (0001).The valence electron–core electron interactions are described by projector augmented wave potentials (PAW)  and the nctional . The empReaction energetics were calculated using:Ep is the energy of products and Er is the energy of reactants.Here 4 adsorbing on the hydroxylated surfaces the energy was calculated as follows:For the example of TiCl is the total energy of TiCl4 adsorbed on hydroxylated surface,  is the total energy of free TiCl4 molecule and EHO–surface is the total energy of the hydroxylated surface. Energetics of ligand elimination can be calculated from:where 2 on the TiO2 surface.where a HCl molecule is eliminated. A similar equation is used for elimination of another HCl to give TiCl3-terminated surface the energy was calculated as follows:For the example of EG interacting on a TiCl is the total energy of EG bound to TiCl4 on the hydroxylated surface, EHCl is the total energy of a free HCl molecule released as by-product, EEG is the total energy of the EG molecule and  is the total energy of TiCl3-terminated hydroxylated surface. In computing the energies in 4 precursor.where The underlying data files are available at https://github.com/ArbreshaMuriqi/Titanicone.2/titanicone, rutile TiO2/titanicone and Al2O3/titanicone film formation. This is a common approach in modelling MLD chemistry [2, hydroxylated rutile TiO2 and hydroxylated Al2O3 surfaces with TiCl4 and EG or GL precursors. The hydroxylated surfaces that results from the interactions with water and before the introduction of TiCl4 are taken from previous studies [As oxide/metalcone films are of high interest, we explored the feasibility of anatase TiOhemistry ,39–41. W studies –51.4 molecule on the selected surfaces. These energies are −0.29 eV for the anatase TiO2, −0.22 eV for the rutile TiO2 and 0.87 eV for the Al2O3. These energies indicate that TiCl4 will adsorb favourably, although with a small energy gain, on the anatase TiO2 surface and rutile TiO2 surface and will not adsorb on the Al2O3 surface. A previous study reports that TiO2 films grow well using TiCl4 and H2O on amorphous Al2O3 [2O3 surface model we use is crystalline. It is well known that in an amorphous surface the molecular mobility is significantly higher than in any corresponding crystalline form and there is a lower coordination number for atoms in the surface which gives rise to enhanced chemical reactivity of the amorphous surface. These results show that the choice of the surface can have a major impact on the initial deposition steps.In the first calculations we have calculated the interaction energies of the TiClus Al2O3 . However4 was not favourable on the Al2O3 surface, we continued with only the anatase TiO2 surface models and the rutile TiO2 surface models.As we found that the adsorption of TiCl4, which involve the proton transfer from the surface OH groups to the Cl ligands of TiCl4 to form HCl as by-product, and the formation of new Ti–O bonds between the TiCl4 molecule and surface oxygen. During the first ligand loss reaction, TiCl4 forms one new Ti–O bond with the surface and one HCl molecule is released. This reaction leaves the surface covered with three Cl ligands, TiO2–TiCl3. During the second ligand loss reaction TiCl4 forms a second new Ti–O bond with the surface and a second HCl molecule is released. After this reaction the surface is left covered with two Cl ligands, TiO2–TiCl2. The coordination number of Ti in the TiCl4 molecule is four and remains unchanged during the first and second ligand loss reactions. Atomic structures of the anatase TiO2 surface and rutile TiO2 surface after adsorption of one TiCl4 molecule and the elimination of the first and second ligand of TiCl4 are presented in Figure 1. Calculated interaction and ligand loss energies of TiCl4 on the anatase TiO2 surface and rutile TiO2 surface are presented in Table 1. Energetics for the ligand loss reactions are calculated relative to the first model of TiCl4 interacting with the surface, and present an overall reaction energy. The overall energy for TiCl4 to lose Cl ligands and bind on either TiO2 surface is negative, although there is a notable energy cost for losing the second Cl ligand from surface bound TiCl3. We do not explore further loss of HCl to give adsorbed TiCl since XPS analysis from previous experimental work showed that chlorine impurities are still present in the film [Next, we investigated the first and second ligand loss reactions of TiClthe film , indicat4 and both TiO2 surfaces during the first and second ligand loss reactions is presented in Table S1  is ≈4.97 Å. This distance is similar with the achieved growth rate of ≈4.5 Å/cycle to 6 Å/cycle for TiCl4–EG films in a temperature range of 100–120 °C on a SiO2 membrane [The EG and GL molecules interact also favourably in an upright configuration with the 2TiClmembrane  and ≈4.6membrane . We notemembrane ,30 which3–GL model, the distance between Ti and O in the Ti–O–CH2CH2OHCH2–O fragment (GL in the upright configuration) is ≈6.26 Å. However, the achieved growth rate of TiCl4–GL films in experimental work is much smaller, 2.8 Å/cycle at 130 °C to 2.1 Å/cycle at 210 °C when deposited in Si wafers [3–GL films in experimental work. Although the lower growth per cycle of TiCl3–GL films achieved in these experimental studies are most consistent with a flat lying orientation of GL when deposited on Si surfaces, DFT shows that the desired upright orientation of GL could be energetically favourable on a rutile TiO2 surface and this provides motivation to develop a lower temperature rutile TiO2/TiCl4–GL process where higher growth per cycle could be achieved. We note that the experimental data are not immediately comparable since different deposition temperatures are used, with significantly higher temperatures for the GL process compared to the EG process which may favour the flat-lying configuration.For the TiCli wafers . The dis2 surface the organic molecules EG and GL can orient in both configurations, upright and lying flat, in the rutile TiO2 surface these molecules could prefer more the upright configuration, although the flat-lying configuration may also be stabilised. This indicates that the surface can also have an important role on the orientation of the organic species.In summary, DFT calculations show that while in a anatase TiO2 surface (0.01 Å longer). On the other hand, on rutile, the Ti–O bonds to the organic molecules in the flat lying configuration are 0.03 Å to 0.06 Å longer compared to Ti–O bonds of these molecules in the upright configuration. The increment of these bonds when EG and GL change their configuration from upright to lying flat on the rutile TiO2 surface also indicates the lower stability of these molecules in the flat configuration on the TiCl3 terminated rutile TiO2 surface.The Ti–O distances to the EG and GL in the upright and flat lying configurations are presented in Table S3 . The com2 surface, the Ti–O distance between Ti of TiCl4 and the oxygen atom of the anatase TiO2 surface is 1.77 Å. This distance decreases to 1.75 Å when EG and GL change their configuration to lying flat. For EG in the upright and flat laying configuration on the rutile TiO2 surface the Ti–O distance between Ti of TiCl4 and the oxygen atom is 1.77 Å. For GL in the upright and flat lying configuration on the rutile TiO2 surface this distance is 1.76 Å.For the upright EG and GL on the anatase TiO4 is another Ti precursor that has been used for the deposition of TiO2 [4 is a metalorganic precursor containing Ti–N bonds to the organic ligands, dimethylamino 2), and with a much larger molecular size when compared to TiCl4. It offers some advantages as a precursor, including the noncorrosive nature of by-products of ligand elimination, higher reactivity due to the weak Me–N bonds and good thermal stability [4 and EG or GL as MLD precursors and to investigate the molecular mechanism behind the possible growth. The hydroxylated anatase TiO2 surface, hydroxylated rutile TiO2 surface and hydroxylated Al2O3 surface were selected as surface models.Ti(DMA) of TiO2  and TiN  of TiO2  ALD film of TiO2 . Ti(DMA)tability . We perf4 precursor towards the selected surfaces. The calculated interaction energies on the anatase TiO2, rutile TiO2 and the Al2O3 surfaces are −0.88 eV, −0.74 eV and −1.47 eV, respectively. Thus, Ti(DMA)4 adsorbs favourably on the three oxide surfaces.In the first calculations, we calculate the reactivity of the Ti(DMA)4 precursor are examined. These reactions take place with a proton transfer from surface OH groups to the DMA ligands of Ti(DMA)4 to form protonated molecules, H–DMA, released as by-products and the formation of new Ti–O bonds between Ti(DMA)4 and oxygen atoms on the surface. After the third ligand loss reaction, Ti of Ti(DMA)4 is bonded to three surface oxygens and to one DMA ligand that can exchange with one organic molecule during the organic precursor pulse. The coordination number of Ti of the Ti(DMA)4 molecule is four and remains unchanged during the ligand loss reactions.Next, the thermodynamics of the first, second and third ligand loss reactions of the Ti(DMA)4 is presented in 2, rutile TiO2 and the Al2O3 surfaces interacting with Ti(DMA)4 and after the elimination of the first, second and third ligand of Ti(DMA)4 are presented in 4 are presented in 4 interacting with the surface, and present an overall reaction energy. Ligand loss energies show that the overall energy for Ti(DMA)4 to lose DMA ligands and bind on either TiO2 surface or Al2O3 surface is favourable.The ligand loss reaction mechanism of Ti(DMA)4 interacting and binding on the same selected surfaces, we see that Ti(DMA)4 interacts and binds more favourably with the surfaces as the calculated reaction energies are much larger. This is consistent with bond dissociation energy values for the breakage of the Ti–Cl and Ti–N bonds  which show that the Ti–Cl bond is stronger and thus more difficult to break.When we compare these energies to those calculated for TiCl4 with the surface and the three ligand loss reactions is the largest on the rutile TiO2 surface, −2.53 eV, which is −0.53 eV larger when compared to the Al2O3 surface and −1.73 eV larger when compared to the anatase TiO2 surface.The overall reaction energy which includes the interaction of Ti(DMA)4 to the Al2O3 surface are 0.03–0.08 Å shorter, when compared to those formed between Ti(DMA)4 and the anatase TiO2 surface or rutile TiO2 surface, Table S4 (The new Ti–O bonds of Ti(DMA)Table S4 .4 molecules. The reactions when a second Ti(DMA)4 molecule is adsorbed are associated with the release of three H-DMA molecules as by-products and the surfaces are left covered with two TiDMA species, anatase–2TiDMA, rutile–2TiDMA and Al2O3–2TiDMA. Calculated energetics for the adsorption of the second Ti(DMA)4 precursors are −1.05 eV for the anatase TiO2 surface, −1.29 eV for the rutile TiO2 surface, −2.06 eV for the Al2O3 surface. The distance between Ti atoms of the two TiDMA species is 7.2 Å on the anatase TiO2 surface and rutile TiO2 surface and 7.1 Å on the Al2O3 surface.In order to investigate double reactions of EG and GL we built also the surface models after the adsorption of two Ti(DMA)4 and EG or GL, the reaction between Ti(DMA)4 and EG or GL requires the transfer of one proton from the terminal OH group of the organic molecule to one DMA ligand of Ti(DMA)4 to release a H–DMA molecule as a by-product coupled with the formation of a new Ti–O bond between Ti and the organic molecule. Next, we analysed MLD reactions using EG and GL as organic reactants where the organic molecules are modelled in both upright and flat lying configurations. Similar to the reaction between TiCl-terminated anatase TiO2, rutile TiO2 and Al2O3 surfaces with EG and GL is presented in 2 followed by rutile TiO2.The overall energy change for the reactions between the TiDMA2 and rutile TiO2 surfaces are 0.06 Å to 0.08 Å shorter when compared to those formed between the EG and GL and the TiDMA-terminated Al2O3 surface, which is consistent with the more favourable interaction energies.Ti–O distances between Ti and the organic molecules are shown in Table S5 . Ti–O boWe also examined the change in the Ti–O distances to the surface oxygens after the introduction of the EG and GL, and these are presented in Table S6 . The com2O3–2DMA, where EG and GL are modelled in the upright and flat lying configurations. In the upright configuration organic molecules bind to Ti sites through one terminal OH group and one H–DMA molecule is released while in the flat configuration organic molecules will bind through two terminal OH groups with two neighbouring Ti sites and two H–DMA molecules are released. 2, rutile TiO2 and Al2O3 surfaces with the upright and flat lying EG and GL. Calculated energetics for the reactions of EG and GL in the upright and flat laying configuration with the 2TiDMA-terminated surfaces are shown in We investigated also the double reactions of EG and GL on the anatase-2TiDMA surface, rutile–2TiDMA surface and Al2, rutile TiO2 and Al2O3 surfaces are negative, meaning that the reactions are exothermic and therefore favourable. The energies for the reactions between the upright EG and GL and the 2TiDMA-terminated anatase TiO2, rutile TiO2 and Al2O3 surfaces are calculated relative to the energy for the adsorption of two Ti(DMA)4 molecules on the relevant surface. The energies for the flat configuration are calculated relative to the upright structures of EG and GL. A negative energy gain was calculated for all reactions when EG and GL change their configuration from upright to flat lying and one H–DMA molecule is released, regardless of the surface.Calculated energetics for the reactions between upright EG and GL with the 2TiDMA-terminated anatase TiO4 in the next cycle and the growth proceeds.Calculated energetics show that while the EG and GL can bind to the Ti sites via formation of Ti–O bonds and loss of H–DMA, it is most favourable for the organic precursors to lie flat and create the double reactions through the two terminal OH groups. This phenomenon removes all active sites for EG and the growth will be less favourable while GL has an extra OH group compared to EG which reacts with Ti(DMA)4 in a anatase TiO2, rutile TiO2 and Al2O3 surface behave similarly as when combined with Ti(DMA)4 on a Si substrate [4–EG films deposited on a Si substrate in a temperature range of 80–150 °C the growth starts but it stops after 5–10 cycles, and this most probably due to the favourable double reactions of EG molecules with the Ti species. For Ti(DMA)4–GL films the growth proceeds even when the molecule reacts twice with the surface due to the extra OH group which reacts with Ti(DMA)4 in the next cycle. However, although the growth of Ti(DMA)4–GL films proceeds, these double reactions lead to small GPCs, 0.9 Å/cycle to 0.2 Å/cycle when deposited in a Si substrate in a temperature range of 80–160 °C. The stability and GPC of the Ti(DMA)4–GL-based film may also depend on the strength of interaction with GL, which is less favourable than with EG.Calculations energetics suggest that organic molecules EG and GL combined with Ti(DMA)ubstrate . Experimubstrate  show tha2 and rutile TiO2 surfaces are lengthen from 0.05 Å to 0.25 Å when EG and GL change their configuration from upright to lying flat.The Ti–O distances to the EG and GL molecules in the upright and flat lying configurations are presented in Table S7 . The com4 on the anatase TiO2, rutile TiO2 and Al2O3 surfaces and with TiCl4 in a anatase TiO2 surface but they do not prefer to lie flat when combined with TiCl4 on a rutile TiO2 surface. The interactions with TiCl4 precursor are also less favourable than with Ti(DMA)4, partially explained by the strong Ti–Cl bond compared to Ti–N.In summary, DFT calculations show that the chemistry of the organic molecules will depend on the inorganic precursor used and on the surface onto which the films are deposited. We see that EG and GL prefer to lie flat when combined with Ti(DMA)4 or Ti(DMA)4 inorganic precursors and EG or GL organic molecules. We used anatase TiO2, rutile TiO2 and Al2O3 surface models.In this study, we used first principles density functional theory (DFT) to investigate the atomistic mechanism of the growth of titanium containing hybrid films known as “titanicones” deposited by MLD. We investigated in detail the chemistry of the MLD process between the TiCl4 or Ti(DMA)4 inorganic precursors towards the selected surfaces and towards EG and GL organic molecules and predicted the preferred orientation of these organic molecules when combined with TiCl4 and Ti(DMA)4 in a anatase TiO2, rutile TiO2 and Al2O3 surface.Through DFT we calculated the reactivity of TiCl4 interacts and binds favourably with the anatase TiO2 and rutile TiO2 surfaces via Ti–O bonds and release of HCl, the interaction with the Al2O3 surface is not favourable. Ligand loss reactions of TiCl4 on the anatase TiO2 and rutile TiO2 surface are favourable, although there is a notable energy cost for losing the second Cl ligand from surface bound TiCl3. Double reactions of EG and GL molecules with TiCl3 species are also closely explored. DFT findings show that these molecules can bind with TiCl3 species in the anatase TiO2 surface in the upright configuration with one terminal OH group and in the flat configuration with two terminal OH groups. In the rutile TiO2 surface on the other hand, the preferred orientation of EG and GL molecules is upright. Therefore, we consider the rutile TiO2 surface as a suitable surface for the growth of TiCl4–EG and TiCl4–GL titanicone films where the desired GPCs could be achieved.Calculated energetics show that while TiCl4 also interacts and binds favourably with the anatase TiO2, rutile TiO2 and the Al2O3 surface via Ti–O bonds. The ligand loss reactions of Ti(DMA)4 are associated with the release of H–DMA (H–N(CH3)2) by-products. A higher reactivity of Ti(DMA)4 compared to TiCl4 was calculated and this is most probably due to the stronger Ti–Cl bonds present in TiCl4 compared to Ti–N bonds present in Ti(DMA)4. We show that EG and GL bind favourably with Ti species of Ti(DMA)4 via Ti–O bonds and release of H–DMA by-products. However, reaction energetics indicate that these molecules can lie flat and create the unwanted double reactions through the reaction of the two terminal OH groups with the surface fragments. For EG this phenomenon removes active OH groups from the surface and the growth will be less favourable while for GL the third OH group is available and growth proceeds. This analysis supports experimental data on Ti(DMA)4–EG and Ti(DMA)4–GL film growth and clarifies why for the Ti(DMA)4–EG process, the growth stops after 5–10 cycles while for the Ti(DMA)4–GL process, the growth proceeds [DFT calculations show that Ti(DMA)proceeds .Temperature is a very important factor for the successful deposition of MLD films. It significantly affects the chemistry between the MLD precursors and especially the behaviour of the organic molecules in the hybrid films. Therefore, we consider this aspect very interesting and worthy of investigating in future work.File 1Additional data on the geometry of the structures.


The glass sets (A) with a molar composition of 40MO–60P2O5 and (B) with a molar composition of 40MO–20B2O3–40 P2O5 are investigated, both with M =  and all doped with 0.1 mol% Dy2O3. Raman and fluorescence spectroscopy are used to analyse the structure and optical characteristics of these glasses. Four typical Dy3+ emission bands in the yellow (572 nm), blue (483 nm) and red (633 and 752 nm) regions of the spectrum are observed in both sets. The fluorescence lifetimes in each glass set are correlated to the network modifier's ionic field strength. The Mg2+ and Zn2+ containing glasses have the longest fluorescence lifetimes. The yellow to blue emission intensity ratio of the respective bands can be used to indicate a symmetric environment around Dy3+ ions and varies with the ionic field strength of the modifier cations: a higher ionic field strength leads to a higher yellow to blue ratio, which in turn indicates a higher asymmetrical local coordination environment of Dy3+ ions in the glassy host network.The optical characteristics of Dy Particularly LEDs, which can be solid-state LEDs, organic LEDs, or polymer LEDs, have clear advantages over the other two such as a greater energy efficiency and a longer working life which result in a greater environmental friendliness. These advantages result from the spontaneous light emission in semiconductors caused by the radiative recombination of excess electrons and holes created in the presence of an electric current which resolves many constraining factors in other light sources. The nearly monochromatic emission of classic LEDs can be used to excite other phosphors which in return are able to emit white light. The combination of these two elements is the basis for "solid-state illumination" using white LEDs (WLEDs). The first commercial solid-state lighting WLED was produced in 1996 based on InGaN semiconductors and the inorganic phosphor (YAG:Ce). It opened the door for the further development of WLEDsRare earth (RE) ion-doped materials are widely used in photonic devices and other lighting applications because of their superior optical performance and a long and stable lifetime. WLEDs can be produced by using RE phosphors to generate blue or near-UV light and balancing it with a yellow emission or a complementary package of red, green, and blue emissions.4. Among the RE ions in glass hosts, Dy3+ has a broad emission spectrum and a high luminescence efficiency. This is caused by two main emission bands in the blue and yellow regions of the visible spectrum, which correspond to the transitions 4F9/2 → 6H15/2 (magnetic dipole) and 4F9/2 → 6H13/2 (electric dipole). Due to a hypersensitivity of the yellow transition, its emission intensity is greatly impacted by the local field environment5, whereas the blue transition is less sensitive to the host. These emission bands enable to adjust the stimulation of white light by tuning the yellow to blue emission intensity ratio by changing the host composition. Furthermore, the ratio of the yellow to blue emission intensity is considered to be a measure for the Dy3+ environment distortion; a yellow to blue ratio ≥ 1 implies a high local symmetry around the Dy3+ ion6. It is possible to prolong the emission lifetime by increasing the yellow to blue ratio as well as the symmetric distortion of the host matrix6.RE ion emission characteristics significantly depend on the electronic structure of the RE ion, the host matrix, the network modifiers and the doping concentration4 tetrahedral units which are connected by P-O covalent bonds. The degree of bonding varies with the composition of the glass7. The phosphate structures in the glass network are defined by the Qi terminology, in which i represents the number of bridging oxygen atoms. Borate glasses, on the other hand, are composed of both trigonal BO3 and tetragonal BO4 units whose relative amounts strongly depend on the glass composition8. Adding B to the phosphate glass enables to obtain a variety of structural arrangements by linking [PO4] units with [BO3] and/or [BO4] units10. The potential of Dy3+ doping in different glass hosts has been demonstrated for borophosphate (BP), Li borate-, Sr-Li-Bi borate-, and fluorozirconate glasses19.A phosphate glass network is composed of PO3+ emission resulting from adding B2O3 to Dy3+-doped phosphate glasses and incorporate the divalent network modifiers Mg2+, Ca2+, Sr2+, Ba2+, or Zn2+.The aim of this study is to monitor the Dy3BO3, NH4H2PO4 and ZnO , BaCO3 , CaCO3 , MgCO3 , Dy2O3 , and SrCO3 . The molar composition of set (A) is 40 MO-60 P2O5 + 0.1 mol % Dy2O3 while the composition of set (B) is 40 MO-20 B2O3-40 P2O5 + 0.1 mol % Dy2O3, with M = Ba2+, Ca2+, Mg2+, Sr2+, or Zn2+. The nominal glass compositions are summarised in Table Two glass sets were prepared by conventional melt-quenching using the chemicals Hg) + 10 °C. The respective Tg were estimated based on previous research2. After 0.5 h, the furnace was switched off and the samples slowly cooled to room temperature (RT).Batches for 50 g of glass were homogenised for 1 h in a rotating homogenizer and melted in alumina crucibles using an electric furnace . The batches were heated to 450 °C and kept there for 12 h. Subsequently the temperature was slowly increased to 1400 °C over the course of 7 h and held for 2 h to homogenize the melt. The melts were poured into graphite moulds and transferred to a furnace  preheated to a temperature of the corresponding glass transition temperature . Photoluminescence excitation (PLE) and emission spectra (PL) were measured in the front-face configuration at an excitation wavelength of 349 nm using a continuous Xenon lamp (450 W) as the excitation light source. Appropriate cut-off filters were used to eliminate the higher-order reflection artefacts in the photoluminescence spectra. The PL and the PLE spectra were measured under the same conditions, i.e. slit width, integration time and excitation/monitored wavelength. The PL spectra were corrected for the spectrometer optics and the excitation lamp response, whereas the PLE spectra were only corrected for the spectrometer optics.Lifetime decay curves were obtained by exciting the samples using a Xenon flash lamp at 349 nm at RT and the blue (483 nm) and yellow (572 nm) emission wavelengths were monitored. The decay curves were normalized and fitted using a double-exponential function. All produced glasses contained a few bubbles and were colourless and transparent to the naked eye.2O32. The spectra of set (A) are dominated by two distinct bands at ~ 700 and 1200 cm−1 which can be assigned to the symmetric stretching vibrations of the bridging oxygen atoms connecting two PO4 tetrahedra (νs(P-O-P)) and to the symmetric stretching vibrations of the phosphate Q2 groups (νs(PO2))20. The minor feature at ~ 1270 cm−1 can be attributed to the asymmetric stretching vibrations of the non-bridging oxygen atoms of PO2 units7. The νs(P-O-P) shifts to lower wavenumbers as the field strength of the network modifier decreases, i.e. from 705 cm−1 (Zn2+) to 690 cm−1 (Ba2+). The same trend is observed for the νs(PO2), i.e. the vibration shifts from 1209 cm−1 (Zn2+) to 1163 cm−1 (Ba2+) accompanied by an increasing band width and a shoulder at ~ 1210 cm−1. These results are in good agreement with previous results on alkaline earth (AE) phosphate glasses23.The normalised Raman spectra of the sets (A) and (B) with various network modifiers are presented in Fig. 2O5 is partially substituted by B2O3 as is discernible in Fig. 2 groups (νs(PO2)) shifts to lower wavenumbers when B2O3 is introduced, e.g. from 1209 cm−1 to 1161 cm−1 for Zn2+ containing glasses of set (A) and set (B), and increases its bandwidth, e.g. from 31 to 115 cm−1  for the same glasses. This is due to a change in the next nearest neighbours of the Q2 groups and the formation of P21B and P22B structural units accompanied by an increasing variety of P-O bond angles and bond lengths27. The peak of the νs(PO2) also shifts to lower wavenumbers as the ionic field strength of the network modifier decreases, i.e. 1161 (Zn2+), 1176 (Mg2+), 1108 (Ca2+), 1121 (Sr2+), and 1093 cm−1 (Ba2+) for glass set (B). The symmetric stretching vibration of P-O-P at around 700 cm−1 in the pure phosphate glasses is substituted by two overlapping bands at around 680 and 750 cm−1, which can be assigned to the P-O-B stretching vibrations7 and the breathing of borophosphate rings28. These bands are more distinct in the Mg2+ and Zn2+ containing glasses. The variation of the Raman shift within one kind of modifier is strongest for a B2O3 content of 20 mol% and the peak width also increases with a higher B2O3 content. The Raman shift of the maximum of the νs(PO2) as a function of the ionic field strength of the network modifier is presented in Fig. The Raman spectra become more complex when P2, where Z is the charge of the specific cation and a is the ionic distance for oxides29. The νs(PO2) increases with increasing ionic field strengths and decreasing B2O3 contents. It is caused by the more covalent character of the P-O bonds, leading to an overall strengthened glass network.The ionic field strength of a cation is related to the ionicity of the cation-oxygen bond and can be calculated by Z/a6H15/2 to the different excited states 6P3/2 (324 nm), 4I9/2 (337 nm), 6P7/2 (349 nm), 6P5/2 (364 nm), 4K17/2 (386 nm), 4G11/2 (425 nm), 4I15/2 (453 nm), and 4F9/2 (473 nm)30. The most intense band of all glasses at ≈349 nm is assigned to the 6H15/2 → 6P7/2 transition. Thus, this wavelength was used as an excitation wavelength for recording the emission spectra of both glass sets. The peak intensities vary if the network modifiers Mg2+, Ca2+, Sr2+, Ba2+, or Zn2+ are alternated while their positions (emission wavelengths) remain constant. The peak intensities also differ for the two host matrices: the peak intensity for glass set (A) decreases in the order Zn2+  > Mg2+  > Ca2+  > Sr2+  > Ba2+, while a different trend Ca2+  > Ba2+  > Sr2+  > Mg2+  > Zn2+ is observed for the glass set (B).The excitation spectra monitored at two prominent emission bands, 483 nm and 572 nm, are presented in the Fig. 3+-doped glasses in the visible region of 400–800 nm. Four similarly shaped emission bands at 4F9/2 → 6H15/2 (483 nm), 6H13/2 (572 nm), 6H11/2 (663 nm) and 6H9/2 (752 nm) are observed for both glass sets. The 4F9/2 → 6H13/2 (yellow) and 4F9/2 → H15/2 (blue) transitions are the two most intense and respectively correspond to the electric or magnetic dipole transitions. In general, the yellow emission at 572 nm is roughly four times as intense as the blue emission at 483 nm, particularly when excited with a wavelength of 349 nm.Figure 3+ ion, while the yellow emission is hypersensitive and strongly influenced by the local environment of Dy3+. The yellow to blue intensity ratio can be used as a spectroscopic reference to probe the local symmetry around Dy3+ and the covalency of the Dy-O bonds32, as a higher asymmetry should increase the transition probabilities of the hypersensitive bands. A high yellow to blue ratio results from an increased local asymmetry and a high covalency of the Dy-O bond. Figure 2.The blue transition with its magnetic dipole character is not significantly affected by the local environment around the Dy2+ containing glasses. It increases slightly when modifiers with a low ionic field strength occur in the matrix, ranging from ~ 2.75 to ~ 3.25 between Ba and Ca, respectively. Larger changes in the yellow to blue ratio are observed for the high field strength ions Mg2+ and Zn2+. The yellow to blue ratio initially increases with the substitution of P2O5 by B2O3 and shows a maximum at 10 mol % B2O3. This is the opposite yellow to blue ratio trend of that reported for the effect of different AE ions on the luminescence properties of Dy3+ in glasses with the general molar composition 50Li2O-20MO-29.5B2O3-0.5Dy2O3 and M = Mg2+, Ca2+, Sr2+ or Ba2+ where Ba2+ showed the highest and Mg2+ the lowest yellow to blue ratio3. It was concluded that the covalency degree of the Dy-O bond increases with the atomic weight of the AE ion based on a Judd–Ofelt analysis3. This disagreement to the results presented here could be caused by the differing glass-forming system as well as the modifier types and contents. Any change in the composition of the glass system and the structure of the network would thus have a direct effect on the Dy3+ ion surroundings.These results show that the yellow to blue ratio increases with the increasing ionic field strength of the network modifier and has its maximum in the Mgth of the host matrix is presented in Fig. th values were calculated according to Eq. (a/2), Λ(BOb/2), … representing the optical basicity of the oxides AOa/2, BOb/2, … and ΧAOa/2, ΧBOb/2, … the equivalent fractions, i.e. the proportion of oxygen atoms they contribute to the network35. Λth increases when P2O5 is substituted by B2O3, e.g. in Mg2+ containing glasses from 0.38 (pure phosphate glass) to 0.41 (20 mol% B2O3), and increases within one set of glasses when the ionic field strength of the network modifier decreases, e.g. in set (A) from 0.38 (Mg2+) to 0.43 (Ba2+). The trend of the yellow to blue ratio over Λth is almost linear within each glass set for Ba2+, Sr2+ and Ca2+ but changes significantly when Mg2+ or Zn2+ are present, indicating a decreasing electron donor power of the host matrix with a high field strength of the network modifiers.In order to reach a common basis for the elucidation of the yellow to blue ratio behaviour, the dependency of the yellow to blue ratio to the calculated optical basicity of the glasses will now be explored. The evolution of the yellow to blue ratio over the theoretical optical basicity Λg to Eq. :1\documeth and the yellow to blue ratios with the information obtained from the Raman spectra are presented in Fig. th over the Raman shift of all glass sets and the yellow to blue ratio obtained from the emission spectra of the glasses are presented in Fig. th decreases linearly with an increasing Raman shift of the νs(PO2) (and increasing ionic field strength of the network modifier) within the AE ions, but the Zn2+ containing glasses do not follow this trend. The yellow to blue ratio increases linearly with an increasing Raman shift of the νs(PO2). The Zn2+ containing glasses again do not follow the described trend.The correlation of the Λ4F9/2 → 6H13/2 (572 nm) and 4F9/2 → 6H15/2 (483 nm) transitions and were fitted using the bi-exponential function Eq.  is calculated according to Eq. (39:The luminescence decay curves of set (A) are presented in Fig. All lifetime values and weighting factors of the analysed glasses are listed in Table 2+ or Zn2+ containing pure phosphate glasses show the highest emission intensity, followed by the Ca2+, Sr2+ and Ba2+ containing glasses. The intensities are highest for Ca2+ and lowest for Zn2+ or Mg2+ in the case of glass set (B). The overall trends are the same for both emission intensities.The decay curves of the two highest emission intensities at 483 and 572 nm show that the ionic field strength impacts the emission lifetime. The Mg3+ ions in the Mg2+ or Zn2+ containing BP glasses of set (B) have a longer lifetime than those in the pure phosphate glasses of set (A). However, low ionic field strength glasses containing the modifiers Ba2+, Sr2+ or Ca2+ show longer light emissions in set (A) than in set (B). Divalent metal ions have a significant effect on the luminescence lifetimes of glass products because of their different ionic field strengths. Higher cationic field strengths lead to more stable M–O bonding and hence a longer emission lifetime of the active ion (Dy3+). This trend is better observed for high ionic field strength modifiers (such as Mg2+ and Zn2+) than for the lower ionic field strength modifiers Ca2+, Sr2+ or Ba2+. However, the results presented above do not show a clear trend in the emission profiles when changing M2+ ions within a glass set.Figure 11. Shamshad et al. investigated the influence of different AE ions on the Dy3+ emission properties in Li borate glasses3 and found an opposite correlation of ionic field strength and emission lifetime: to the best of our knowledge, they measured the longest lifetime in the Ba2+ containing glass. Their comparably short lifetimes might be caused by concentration quenching, as they used a doping concentration of 0.5 mol% Dy2O3. A previous study on BP glasses with the general composition 40MO-10B2O3-50P2O5 found that a similar trend exists for the correlation of the Dy3+ emission lifetime with the ionic field strength of the network modifier2. These values are included in Fig. 2+ containing glass2. Considering these previous results enables to conclude that the emission lifetime increases when P2O5 is partially substituted by B2O3 and then decreases again, when the boron content exceeds a certain threshold.The obtained lifetime values of this study are in good agreement with literature data summarised in Table 3+ ion. It is well described by the yellow to blue ratio, where the yellow intensity varies with the electronic field strength but the blue intensity, affected by the magnetic field strength, remains nearly constant. Furthermore, the yellow to blue ratio can be a measure of the strength of the covalent or ionic bonding between the Dy3+ ions and the surrounding ligands31. As the yellow to blue ratio increases with increasing ionic field strength, the covalent character of the Dy-O bond also increases. This increase is present in all glass sets of this study and is independent of the B2O3 content. Figure 2] groups increases when network modifiers with a high ionic field strength are present, indicating that the glass network becomes strengthened. The highest shift is observed for Mg2+ containing glasses which also show the highest values for the yellow to blue ratio, proving that the P-O bond strength directly influences the Dy-O bonds and thus the emission properties of the Dy3+ ions.As discussed above, the hypersensitive transition in the host matrix is considered to be an indicator of the environment around the Dy3+ doped P and BP glasses under 349 nm excitation were calculated from the emission spectra and are summarised in Table The emission color coordinates of the Dy3+ containing phosphate and borophosphate glasses was investigated and the emission characteristics of the Dy3+ ion were analysed. The Raman spectra reveal a progressive structural modification when B is introduced into the phosphate glass network. Under the 349 nm excitation, Dy3+ emits four typical bands in both glass environments. With increasing ionic field strength of the network modifier, the yellow to blue emission intensity ratio also increases. Substituting P2O5 by B2O3 leads to a progressive distortion of the local symmetry around the Dy3+ ions and causes not only increasing yellow to blue ratios but also longer emission lifetimes. Beyond a certain content, B2O3 in the glasses enables formation of P-O-B bonds in the network, which create a more symmetric coordination environment around the Dy3+ ions.The structure of Dy


The degree of interpenetrationof the van der Waals crusts of twoatoms, represented by a penetration index, is defined to better quantifythe meaning of the nonbonding contact distances between two atoms,which should allow us to compare different atom pairs on the samefooting. The structural trends of the intermolecular contacts betweenthe tetramethylammonium cation (TMA) and halogen atoms are reviewed,and a computational study of model X···TMA ion pairs is presented. The results disclose two energyminima, in each of which the anion simultaneously interacts with threehydrogen atoms. The bonding mechanisms in the two cases are discussedbased on the results of the tools of the trade that provide a consistentpicture in which a distribution of charges significantly varies notonly around each different atom but is also strongly dependent onthe distance to the central N atom. This behavior, together with somenon-negligible covalent character of the interionic interaction, isnot predicted from a single-molecular electrostatic potential mapof the TMA cation. Twointeraction topologies sustain interactions betweenthe tetramethylammonium cation and halide (or auride) anions. A simpledependence of the interaction energy on the anion’s atomicradius is found, as a result of a refined combination of ionic (majorcomponent), covalent, and dispersion forces, in which the interpenetrationof the van der Waals crusts of the interacting atoms seems to havea role. On the one hand, wide research has been conductedin the last few decades in an attempt to design encapsulating organiccations as hosts of anions, referred to as “ammonium-basedanionic receptors,”3 although it has focusedmainly on the R3N-H+ group to form hydrogenbonds with anions, where R3 represents three branches ofan encapsulating polycyclic compound. An interesting case is the designof a macrocyclic receptor that recognizes ion pairs formed by a methylammoniumcation and a halide.4Understanding and harnessingthe forces that bond 5 and have still today an astounding number of applications in detergents,shampoos, cosmetics, artificial tears, household products, and thepopular hydrogels for hand sanitation. Almost half (46.4%) of the580 disinfectants considered by the US Environmental Protection Agencyas effective against COVID-19 contain a QAS in its formulation. Themost common QAS contains one benzyl group, a long even-numbered straightalkyl chain, and two methyl groups (1), referred to genericallyas benzalkonium chlorides (BCs), and an example of a new generationof QAS is didecyldimethylammonium chloride .On the otherhand, the quaternary ammonium cations (QACs) presentin quaternary ammonium salts (QAS) have been known to act as biocidesand disinfectants for more than one century3).6 In general, the mode of actionof QACs plausibly takes advantage of their amphiphilic character,binding anionic species such as phospholipids through the dimethylammoniumfunctions, and to the hydrophobic core of the plasma membrane viathe alkyl chains.The Gemini QACs are designed byconnecting two conventional QACunits through a bridge, resulting in a dication with a slightly differentdesign, with two hydrophilic heads and two hydrophobic tails  has been shownto self-assemble to produce micelles that give rise to the formationof uniform intracrystalline mesoporosity in zeolites. The mesoporousmaterials thus created present enhanced activity as fuel crackingcatalysts.n = 1–4) and halide anions, carried out byus in the CSD and ICSD structural databases, shows that they presenta variety of topologies schematically represented in 4–9, as well as intermediate situations. To distinguishthose topologies, we name them using the kappa convention establishedby the IUPAC to indicate through which donor atom (or atoms) is aparticular ligand coordinated to a metal.8 Thus, binding of a methyl group to an anion through n atoms is represented by the Greek letter kappa with n as a superindex, followed by the symbol(s) of the interacting atom(s),e.g., κ1H (4). An ambiguity appearsfor the topology with the anion at the same distances to the threehydrogen atoms of the same methyl group (3H (6) or κ1C (7), an issue to be discussed in this paper. For simplicity, however,we will consistently use the κ1C notation that willbe shown to be in better agreement with the nature of the interaction.In many instances, an anion is linked to one H atom of two (8) or three (9) different methyl groups of thesame ammonium cation, a situation that is indicated by a numeric prefixand the atomic symbols of the linked atoms, e.g., 3κ1H,H′,H″, which will be shortened throughout this paperto 3κ1H, given the equivalence of the three H atoms.Examples of the binding topologies that can be found in several simple n-methylammonium salts are presented in A structural analysisof contacts between n-methylammonium cations andtwo long (3.36 Å) Br···H distances, while thefour cations at the lower, narrower part have contact topologies intermediatebetween κ2 and κ3, each cation havingtwo Br···H distances of 3.48 Å and one distanceof 3.58 Å. The same topological pattern is found in the structureof the TMA auride.10Twoattractive examples of the sophisticated supramolecular architecturesthat can be assembled around a single TMA cation, highlighted earlierby one of us,vA and vB, are fuzzy. To make it clear, let us recallthat the van der Waals radii are statistical constructs deduced froman approximately Gaussian distribution of experimental distances distinctlylonger than the sum of covalent radii rA and rB. It has been stressed that noncovalentinteractions may appear roughly within ±0.7 Å of the vander Waals radii sum.12 A similar reflectionapplies to the covalent bond distances since the covalent radii havestandard deviations that range from 0.01 to 0.12 Å.12When dealing with noncovalent interactions,we must take into account that the frontiers between an A···Bpair of nonbonded and weakly bonded atoms, generally marked by thesum of their van der Waals radii dAB is compared with the sum of the van der Waalsradii, but to compare different contacts, often a corrected or “normalized”distance is used, simply defined as the difference between the vander Waals sum and the distance. As an alternative parameter, Lommerseand co-workers16 proposed the quotientof the distance over the van der Waals sum, λ = dAB/(νA + νB) that adopts values smaller than 1 for shorter, andlarger than 1 for longer, distances than the van der Waals sum. Wethink that it is more informative to compare the contact distancewith both the covalent and van der Waals radii sums. Our reasoninggoes as follows  and a sphere of radius rA  that encloses most of its valenceelectron density. The van der Waals crust has therefore a width wA = vA – rA. Then, we consider that two atoms A and Bat a distance dAB, at which the interpenetrationbetween their van der Waals crust can vary between 0 (for dAB = vA + vB) and 100% (for dAB = rA + rB), the two canonical situations which we would conventionally defineas a van der Waals contact and a chemical bond, respectively. Thus,to convert an interatomic distance in a percentage of interpenetrationof the van der Waals cores, we need only divide the difference betweenthe van der Waals sum and the distance by the sum of the widths ofthe two crusts, as done in pAB that indicates the percentage of interpenetration ofthe van der Waals crusts of A and B from 0%  to 100% . A preliminary explorationof the various types of noncovalent interactions indicates that thismight be a useful tool to classify them according to the levels ofpenetration they can achieve.To evaluate the relevance of an intermolecularA···B contact, usually, the interatomic distance  follows 3. We firpAB indicatesthat the fuzzy van der Waals spheres are conventionally not interpenetrated,although weak attractive interactions cannot be ruled out. The absolutevalue of a negative penetration index provides a qualitative descriptionof the interaction as probably weakly attracting or as noninteracting or repulsive . Conversely,values of pAB larger than 100% indicatecovalent bonding and provide a qualitative indication of the strengthof the bond, including a differentiation of single and multiple bonds,as will be shown in a forthcoming publication. In summary, the valuesof the penetration index are intended to provide semiquantitativeinformation about interactions within the van der Waals crust regionsand should be used only as a qualitative indicator beyond the limitsof 0 and 100%. A preliminary exploration of the various types of noncovalentinteractions indicates that this might be a useful tool to classifythem according to the levels of penetration they can achieve.Note that a negative value of n-methylammonium cations and halide anionsconsidering both the X···H and X···Ccontacts of the methyl groups. Since in many crystal structures onefinds more than one of the bonding modes 4–9, as well as intermediate situations, we have carried outsearches in the CSD22 for contact distancesof up to 3.6 Å (X···H) and 3.8 Å (X···C),imposing restrictions on the X···C–N and/orX···H–C angles for κ1H , κ2H(5), and κ3H (6 or 7) contact topologies. From the representation of the distancesas a function of the X···C–N angle, we extractthe points that represent approximately the highest penetration foran interval of that angle and a least-squares fitting provides uswith a simplified view of the whole set of compounds. This procedureis illustrated in 1-H···Brcontacts. Such a simplified view of the X···H and X···Ccontacts between halides and methyl groups of n-methylammoniumcations is presented in 1), two (κ2), or three (κ3) contacts, respectively.Inthis section, we analyze the penetration of the van der Waalscrusts of (1)1H contacts is higher for all of the halidesthan that of the carbon atom, an unsurprising result given the restrictionto nearly linear X···H–C units in the structuralsearches.The penetration of the hydrogen crustin the κ(2)1 contacts appears for all halides at an X···C–Nangle of 110°, i.e., the tetrahedral angle that corresponds toa linear X···H–C arrangement.The greatestpenetration indices amongκ(3)1Htopology, penetrations of up to 60% are seen for heavier halides.While there is practically no interpenetrationof the van der Waals crusts of F and H in the κ(4)1H systems, the κ3H ones present higher penetrationsof X with the C than with the H atoms, reaching penetration indicesof up to 25 and 3%, respectively. This suggests that it would be moreadequate to term those interactions κ1C, as willbe done from here on.Different from κ(5)2H binding presentssimilar penetrations (up to 25%) of the halide with carbon and hydrogenatoms, thus suggesting that the interaction is significantly delocalizedover both C and H.The κThe analysis of the penetrationplots of Vs,max = +12.4kcal/mol) at the center of the outer tetrahedral face of each of themethyl groups, which is consistent with the abundance of κCcontacts of halides with methyl groups of alkylammonium cations sincesuch an arrangement maximizes Coulombic attraction between the twoions. On the other hand, there is a region of negative electrostaticpotential in TMA , associated with the more electronegative nitrogen atom,which may generate some degree of electrostatic repulsion in the 3κHcontacts, although it should be expected to be rather weak due tothe long X···N distances in those ion pairs , which are found to be in allcases significantly longer than the sum of van der Waals radii: 2.86,3.48, 3.49, and 3.70; i.e., penetrations of 14, 0, 2, and 0% for F,Cl, Br, and I, respectively.The molecular electrostatic potential(MEP) of the TMA cation 6 shows c1C and 3κ1H adductsH···Cl,23 in which there is only one hydrogen atom available.Our structural analysis indicatedthat two types of noncovalent interactions are mainly responsiblefor the crystal packing of (TMA)X compounds. Accordingly, we havecreated two simple models to comprehensively analyze the nature andstrength of the κ adducts7. Five m26 and differ by less than 3 kcal/mol from those reported for the twotopologies of the chloride and bromide by Denisov et al.27 It is noteworthy that the binding energy ofevery 3κH ion pair is systematically higher  than for the corresponding κC adduct.The main results forthe optimized models with these interaction topologies are summarizedin Table S2 in the Supporting Information)indicate a slight stabilization of the two independent ions and acorresponding increase of the binding energies of both d 3κ1H adducts by 2–5 kcal/mol. The relative stability ofthe two binding modes, however, is practically unaltered, and theκ1C ion pair is slightly more stable  in the presence of water, the most polar solvent, than inthe gas phase .To test the effect of the solventon the calculated binding energies,we recalculated them for the case of (TMA)Cl within the continuouspolarizable model (PCM) with three solvents of increasing polarities,chloroform, dimethylformamide, and water. As expected, the results(28 of the anion is thus found for both the κ1C (Eint = −123.8 + 55.98rcov – 14.24rcov2) and 3κ1H ion pair families, (Eint = −161.4 + 80.01rcov – 19.24rcov2), as seen in The pairing energiesof TMA with the different anions 2 are clepXH penetration indices.The analysis of the optimizedstructures of the ion pairs showsthat the optimized interatomic X···H contact distancesare significantly shorter than the van der Waals radii sum in allcases, yet there are differences between the two topologies, withthe κC pairs having distances about 0.3 Å shorter to agiven anion than the 3κH ones. An obvious consequence of theshort distances is that in all cases, there are positive 7 and 9). Effectively,the X···C–N angle in the κC adducts andthe X···N–C angle in the 3κH ones allare within the range of 178.6–180.0°. In the case of theκC ion pairs, thus, the anion X sits in the region of the maximumpositive electrostatic potential  clearlypenetrate the van der Waals crust of the methylic carbon, but farfrom the full penetration (100%) required to form a bond. Second,the degree of penetration decreases as we go down the group of halogens.Third, a comparison with the related X–C bonds in methyl halidesclearly illustrates the difference between a bond and a noncovalentinteraction. Surprisingly, the dependence of the penetration indexon the position of the halogen atom in the group is just the oppositefor the bonds than for the ion pairs. The penetration of the H’scrust by the halides is significantly smaller but non-negligible inmost cases  or negative,indicative of no interpenetration, except for X = F, for which thereis a penetration of 12% despite its smaller size.In the alternative 3κH arrangement, the penetrationof thehydrogen by a given anion is roughly between 2 and 3 times higherthan in the κC interactions. On the contrary, the penetrationof the carbon atoms is from 2 to 10 times smaller, as can be seenin pC–Cl, drops from 43% in the gas phaseto 18% in the presence of water, while in the 3kH chloride, it isthe pH–Cl index that falls from40 to 21% .The calculationsincluding the solvent effect via the PCM modelfor the case of (TMA)Cl result in larger cation–anion separations,reflected in smaller Cl···C and Cl···Hpenetration indices, in better agreement with the values found fromX-ray crystallography data 5. The hi3CNMe3, for which the three contacting hydrogensare bent away from the fluoride by 2°, and 3κH F···Me3NCH3, in which the interacting H atoms form smallerN–C–H bond angles, 4° smaller than the tetrahedralangle, to get closer to the fluoride.The σ-hole interactions are characterizedby the existenceof electron density donation from a Lewis base to an empty σ*orbital corresponding to a bond trans to the noncovalent interaction,in the present case, the C–N bond. As a consequence, one shouldexpect some weakening of that bond. The relevant changes in bond distancesupon ion pair formation are summarized in Table S1 and Figure S1). The first thing that must be said is thatthe calculated X···C distances in these ion pairs aresignificantly shorter—and the penetration indices accordinglylarger—than in the experimental structures, a trend that canbe attributed to the fact that such a cation in the solid state doesnot form ion pairs but establishes similar interactions with a largernumber of counterions is distributed among the outer shell of 12 hydrogen atoms, providingus with a raw description of the TMA cation as formed by a sphereof negative charge circumscribed in a spherical shell of positivecharge ,even if the numerical values should be taken with a grain of salt.The net atomic charges for the independent TMA cation 12a show echarge 12b.Figure S2 inthe Supporting Information) aremodified within the ranges shown in Upon ion pair formation, the chargesof the different atoms  ΔEPauli, corresponding to the repulsion between paired electronsof the two interacting fragments, which includes as well the core–corerepulsions; (b) an electrostatic term ΔEelec; (c) a charge transfer term ΔECT corresponding to donor–acceptor interactions; (d)ΔEpol, a polarization term resultingfrom mixing of occupied and empty orbitals of one fragment inducedby the presence of the other fragment; and (e) a dispersion term ΔEdisp, reflecting the London forces acting betweenthe two fragments. Since the charge transfer and polarization termsinvolve both modifications of the molecular orbitals upon interaction,we will refer to the sum of the two contributions as an overall orbitalcontribution ΔEorb, while the termsrelated to the Pauli and electrostatic interactions will be summedup in a single Pauli + electrostatic term, ΔEPe. The results of the energy decomposition analysis (EDA)are presented in An energy decomposition schemeConsistentlywith the electrostatic interaction expected from the charge distributionin the case of the κC ion pairs, we can see that for every anion,the stabilizing contribution of the electrostatic attractive termis more than 10 kcal/mol higher for the alternative 3κH topology.(1)For a given anion, the 3κH interactingmode is significantly more stabilizing than the κC one.(2)For the same interactiontopology,the stability of the ion pair increases as the monoatomic anion becomessmaller .(3)The most important contribution inboth interaction modes comes from the sum of the Pauli and electrostaticterms, as expected for an ionic interaction.(4)The orbital contribution is much smallerbut non-negligible, and significantly higher in the 3κH interactingmode.(5)The dispersioncontribution is rathersmall and practically independent of the nature of X.(6)The orbital and Pauling + electrostaticcontributions show a similar dependence on the nature of X than thetotal interaction energy.By looking at the interaction energies and their EDA results 15, severE(2) from 7.87 to 18.08 kcal/mol (Eint = −55.3–2.274 E(2), r2 = 0.996).Such an electron density donation is reflected in a lengthening ofthe C–N bond as the interaction becomes stronger, showing anice correlation for the TMA halides  analysis of the κC ion pairsdiscloses the existence of one donor–acceptor orbital interactionfrom an X lone pair to a C–N antibonding orbital, LP(X) →σ*(C–N), with an associated stabilization energy kcal/mol 3, with a halides 16a. Sinc halides 16b, whil halides 17.E(2) MOs. The calculated stabilization energies E(2) 3 indicatE(2) 17 and thE(2) 13. The sE(2) 3.3CNMe3  ion pairs, a Bader’squantum theory of atoms in molecules (QTAIM) analysis was performed.The topological parameters such as electron density (ρ), Laplacianof electron density (∇2ρ), total energy density(H), and kinetic energy density over electron density(G/ρ) were computed and are provided in theSupporting Information (Table S3). TheQTAIM results evidence that both the halides and auride anions areindeed connected to the carbon atom of the methyl group by a bondpath, consistent with the presence of an X···C σ-holeinteraction  ion pairs. The H···Xinteractions appear in the topological analysis of the electron densitiesas bcp’s in bond paths connecting the anion and the H atoms(pXN ≤ 12%), compared to those of the three electrostaticallyattractive X···H interactions (32 ≤ pXH ≤ 50%), the net electrostatic contributionto the interaction energy is much more attractive than in the κ1C case, according to the EDA , whichfor the halides follows the same order as the penetration indices.These densities are as well correlated with the orbital contributionfound in the EDA  and practicallyindependent of the nature of X.p orbitals compared to that of the occupied np orbitals (n = 2–5) of the halides. Indeed,the NBO results indicate that the lone pair orbitals of the halidesinvolved in the donor–acceptor interactions with TMA have ap orbital contribution of 87–92%, whereas in auride, the interactinglone pair has 88% d character.The NBO analysis reveals theexistence of relevant donor–acceptorinteractions from the anion to a σ*(C–N) orbital in theκC case, and to a σ*(H–C) orbital in the 3κHgeometry. Changes in the charge distributions and bond distances withinthe cation upon ion pair formation are consistent with the existenceof such orbital interactions. Complementary, a QTAIM analysis disclosesbond paths and bond critical points between X and the closest C atomin the κC ion pairs, and between X and the three closest H atomsin the 3κH adducts, while NCI isosurfaces are located betweenthe two interacting atoms and coincident with the bcp’s. Onthe other hand, the Laplacian of the electron density and the kineticenergy density over electron density are positive which is an indicationof the existence of strong electrostatic interactions. The electrondensity at the BCP increases in the order I < Br < Cl ≪F, and are as well correlated with the covalent contribution foundin the EDA 19 for thpXC and pXH, respectively. However, the charge transfer contributionsthat imply the interaction between a filled MO of the anion and anempty one of the cation show no clear dependence on the position ofthe latter within the group of the halogens, contrary to the orbitalinteraction rules.33 In fact, the strengthof an orbital interaction is favored by a small energy differencebetween the interacting orbitals and by a strong overlap. We may assume that the overlap between thevalence orbitals of the interacting atoms can be roughly representedby the penetration index since it takes into account both the interatomicdistance and the size of the atoms, essentially determined by thesize of its valence orbitals. Then, the charge transfer interactionsare favored for the heavier halogens with electronegativities closerto those of C and H, whereas they are disfavored due to the smallerpenetration and the corresponding small overlap. Both factors seemthus to compensate, resulting in the very small variation of the ΔECT terms observed. Indeed, we can obtain a goodbilinear correlation for the five 3κH ion pairs , or between H and X (for the 3κH ones), and by thepenetration indices pXC, gives poorer results. We recall, however,that in this topology, the X···H penetration, althoughsmaller, is not negligible, and interaction with the H atoms may somewhatdecrease electron donation to the C one. If we include both pXC and pXH in theregression, a reasonable expression results , version5.43 (November 2021).35 for all atoms. This functional has shown very good performance dealingwith noncovalent interactions in previous benchmark reports.37 Interaction energies were calculated via the supermolecule approachand corrected for the BSSE by means of the counterpoise method.38 MEP maps were built on the 0.001 Å electrondensity isosurface with GaussView.39 Thepolarizable continuum model (PCM) implemented in Gaussian16 was usedto perform the calculations in the presence of different solvents.The dielectric constants employed for water, N,N-dimethylformamide,and chloroform were 78.355, 37.219, and 4.771, respectively. QTAIMand NCI topological analyses of the electron density were performedusing the Multiwfn 3.7 program40 on thecorresponding DFT wave functions and represented using VMD.41 NBO analysis was done with the NBO 3.1 software42 as implemented in Gaussian1643 at the DFT level. For the decomposition of the interactionenergy, we employed the second-generation ALMO-EDA method44 implemented in Q-Chem 5.3.45 All other DFT calculations were done with Gaussian16.43DFT calculations were carried out with theM062X functional and the def2-TZVP basis set


The M1 polarization of microglia and neuroinflammation restrict the treatment of acute spinal cord injury (ASCI), and long non‐coding ribonucleic acid (lncRNA) maternally expressed gene 3 (MEG3) expression is lessened in ASCI. However, the function and mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI are unclear. The expressions of lncRNA MEG3 in ASCI mouse spinal cord tissues and lipopolysaccharide (LPS)‐treated primary microglia and BV2 cells were quantified through a quantitative real‐time polymerase chain reaction. In‐vitro assays were conducted to explore the function of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI. RNA degradation, RNA immunoprecipitation, RNA pull‐down, cycloheximide‐chase, and ubiquitination analyses were carried out to probe into the mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation in ASCI. The lncRNA MEG3 expression was lessened in the ASCI mouse spinal cord tissues and LPS‐treated primary microglia and BV2 cells, and the overexpression of lncRNA MEG3 restrained the M1 polarization of microglia and the neuroinflammation by regulating the NF‐κB signaling pathway. For the investigation of the potential mechanism of such, the overexpression of lncRNA MEG3 restrained the M1 polarization of microglia through the HuR/A20/NF‐κB axis and boosted the motor function recovery and neuroinflammation relief in the mice with SCI. The overexpression of lncRNA MEG3 restrained the M1 polarization of microglia through the HuR/A20/NF‐κB axis. A working model was used to reveal the underlying mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation. The Microglia are the key immune cells in the central nervous system , and maiLong non‐coding RNAs (lncRNAs) are generally defined as transcripts greater than 200 nucleotides in length that do not have coding capabilities  and are In the current study, we mainly detected the expression of lncRNA MEG3 in the ASCI mouse spinal cord tissues and LPS‐treated primary microglia and BV2 cells and analyzed the correlation between the expression of lncRNA MEG3 in the serum samples from patients with traumatic SCI and the levels of the TNF‐α, IL‐6, and IL‐1β inflammatory factors. We further probed into the possible mechanism and related signaling pathways of lncRNA MEG3 in regulating the M1 polarization of the microglia in ASCI.22.1n = 36) and healthy controls (n = 36) after obtaining such patients' and healthy controls' written informed consent, and the serum samples were centrifuged at 3000 rpm for 10 min. This study was approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University.Peripheral blood samples were collected from patients with traumatic SCI . All the mice were kept in an environment with a 22°C–24°C temperature and 50% humidity and were provided food and water ad libitum. All animal experiment protocols were approved by the Animal Care and Use Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. The study was carried out in compliance with the ARRIVE guidelines.The acute SCI mouse model was established based on the method reported in the previous literature . The micMoreover, to evaluate the effect of lncRNA MEG3 on mice with SCI, pcDNA‐MEG3 and pcDNA were injected into such mice via intrathecal injection. The mice were divided into the SCI + pcDNA group and the SCI + pcDNA‐MEG3 group, and 10 mice were randomly assigned to each group.2.3At SCI days 1, 3, 7, 14, 21, and 28, the motor function of the mice was evaluated by Basso Mouse Scale (BMS) score. The scores range from 0 to 9, with 0 representing complete paraplegia and 9 representing a normal function. Each mouse's motor function was analyzed by two independent inspectors who were unaware of the treatment regimen.2.4The spinal tissues surrounding the SCI were isolated and fixed with 4% paraformaldehyde. They were then embedded in paraffin wax and sliced into 4‐μm‐thick pieces. The samples were treated with Nissl staining  for nearly 5 min, were fixed with neutral balsam , and were observed through a microscope .2.5−ΔΔCt method was used to measure the relative expressions of lncRNA MEG3, TNF‐α, IL‐6, IL‐1β, A20, and HuR.On the basis of the method reported in the previous literature , quantit2.6The concentrations of TNF‐α, IL‐1β, and IL‐6 in the serum samples were detected by TNF alpha human enzyme‐linked immunosorbent assay (ELISA) Kit (Invitrogen), Human IL‐1β ELISA kit  or Mouse IL‐6 ELISA Kit (Abcam) according to the manufacturer's instructions. Optical density (OD) was measured at 450 nm using a microplate reader (Thermo Scientific).2.75 cells/ml) in an incubator in 5% CO2 at 37°C. The medium was replaced with fresh ingredients once a week, and the mixed glial cell cultures were treated with diluted trypsin to obtain more‐than‐98%‐pure microglial cell cultures.The mouse primary microglia were isolated from the spinal cord tissues using the methods reported in the previous literature . In brie2.8The membrane proteins CD16/32 (M1) and CD206 (M2) in the primary microglia were detected by flow cytometry assay. The cells were first digested using trypsin, and then washed and re‐suspended with PBS. The cells were incubated with PE‐conjugated monoclonal mouse CD206 antibodies or APC‐conjugated monoclonal mouse CD16/32 antibodies at room temperature in the dark for 30 min. After that, the cells were washed with PBS and re‐suspended. APC and PE‐conjugated monoclonal antibodies with irrelevant specificity were used as negative controls. Each sample were detected by flow cytometry .2.92 at 37°C.The BV2 cells that were used in this study were purchased from ATCC (USA). They were cultured in Dulbecco's Modified Eagle Medium (DMEM) with the addition of 10% fetal bovine serum  and 100 μg/ml penicillin–streptomycin  in 5% CO2 at 37°C.SH‐SY5Y human nerve cells were purchased from ATCC (USA) and were cultured in a 1:1 mixture of DMEM and F12 medium  with the addition of 10% FBS  in 5% CO2.10g. After the supernatant was discarded, the precipitate was obtained as the nucleus and was then suspended in 1 ml Trizol for RNA isolation. qRT‐PCR was conducted to test the expressions of lncRNA MEG3 in the cytoplasm and nucleus.Given the previously described method , the cyt2.11To probe into the effects of lncRNA MEG3 on the M1 polarization of microglia and neuroinflammation, the pcDNA‐MEG3 (overexpressing MEG3) or pcDNA (negative control) was transfected into BV2 cells or primary mouse microglia. Specifically, through the standard procedure indicated by the reagent manufacturer, pcDNA‐MEG3 or pcDNA was transfected into BV2 cells or primary mouse microglia using Lipofectamine 2000 . After 48 h, qRT‐PCR was conducted to verify the transfection efficiency. LPS (1 μg/ml) was also applied to stimulate the cells for approximately 24 h to induce neuroinflammation.To determine if lncRNA MEG3 affected the M1 polarization of the microglia through the NF‐κB signaling pathway, the BV2 cells or primary mouse microglia transfected with pcDNA‐MEG3 were treated with the NF‐κB signaling pathway activator phorbol myristate acetate (PMA)  and thenTo probe into the effect of lncRNA MEG3 on HuR expression, si‐MEG3 was transfected into BV2 cells or primary mouse microglia. To determine if lncRNA MEG3 regulated A20 expression through HuR, pcDNA‐MEG3 and/or pcDNA‐HuR was transfected into BV2 cells or primary mouse microglia. After 48 h, the treatment of the cells with LPS was continued for approximately 24 h.To determine if lncRNA MEG3 affected the M1 polarization of the microglia through the Hur/A20/NF‐κB pathway, pcDNA‐MEG3 and/or pcDNA‐HuR was transfected into BV2 cells, and the cells were treated with the BAY 11‐7082 NF‐κB pathway inhibitor and were further treated with LPS for approximately 24 h.2.12Through the previously described method , Western2.13The spinal cord tissues of the mice and BV2 cells with different treatments were collected. The BV2 cells were fixed with 4% paraformaldehyde and then blocked with 5% bovine serum albumin at room temperature for about 1 h. After that, the cells were incubated overnight with anti‐Iba1  and anti‐iNOS  at 4°C. The cells were further incubated with the secondary antibodies and were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) for approximately 15 min. A fluorescent confocal microscope  was used to assess all the images.In addition, the spinal cord tissues were sliced into 5‐μm‐thick pieces and were further incubated overnight with anti‐Iba1  and anti‐iNOS  at 4°C. The rest of the experimental operations were conducted in the same way that they were previously conducted.2.144) were cultured on transwell inserts in a 96‐well plate placed above the SH‐SY5Y neuronal layer.Referring to the previously described methods , a trans2.154) were added to the 96‐well plates and were cultured overnight, and the medium alone was added as the blank control. Through the standard procedure indicated by the reagent manufacturer, CCK‐8  was used to assess cell proliferation. A 10 μl CCK‐8 solution was added to each well, and the cells were incubated at 37°C for approximately 2 h. A microplate reader  was used to measure the absorbance values at 450 nm.The proliferation ability of the SH‐SY5Y human nerve cells was assessed through cell counting kit‐8 (CCK‐8) assay. Specifically, SH‐SY5Y cells  Kit  was applied to perform RIP assay. The BV2 cell lysate was incubated with a RIP buffer containing magnetic beads conjugated with the anti‐HuR antibody , and normal mouse IgG  was used as a negative control. qRT‐PCR was performed to test the relative expression of lncRNA MEG3.2.18RNA pull‐down assay was conducted to verify the interaction between lncRNA MEG3 and HuR. The details of the experiment were as follows: a large amount of biotin‐labeled MEG3 was synthesized, and the biotinylated MEG3 was captured using streptavidin magnetic beads, through the standard procedure indicated by the reagent manufacturers, and was incubated with BV2 cell lysate. The magnetic beads were washed and the eluted proteins were tested via Western blot.2.19Cycloheximide (CHX) is a commonly used protein synthesis inhibitor  and a CH2.20After ubiquitin  and si‐MEG3 were transfected into 293T cells for 36 h, the cells were further treated with 2 mM MG132 for 16 h, followed by protein extraction for Western blot assay. The cell lysates and antibodies were then incubated in immunoprecipitation buffer for 4 h and then incubated with immunomagnetic beads  overnight. The eluted protein was tested via Western blot.2.21The spinal cord tissues of mice were harvested and sliced into 5‐μm‐thick pieces, and the slices were deparaffinized and permeabilized. The slices were then incubated with anti‐Ki‐67 antibody  and anti‐p65  overnight at 4°C. The slices were further incubated with the secondary antibody (Abcam). After that, the slices were stained with DAPI for approximately 15 min, and the stained slices were imaged using a microscope .2.22The spinal cord tissues of mice were sliced into 5‐μm‐thick pieces. In accordance with the standard protocol of the reagent manufacturer, a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit  was applied to assess the cell apoptosis in the spinal cord tissues. The spinal cord slices were incubated with a mixture of 50 μl TUNEL at 37°C for approximately 1 h, and the mice spinal cord tissue slices were then stained with DAPI. Microscopy  was conducted for observation and analysis.2.23RNA fluorescence in situ hybridization (RNA FISH) was conducted using a RiboTM fluorescent in situ hybridization kit . After blocking with pre‐hybridization buffer for 30 min, fresh kidney tissue samples were incubated with a hybridization buffer containing 2.5 μM Cy3‐labeled mouse‐Hottip FISH probe  at 37°C overnight. On the second day, the coverslips were counterstained with DAPI for 10 min and photographed under confocal microscopy.2.24t‐test, and the differences among multiple groups were assessed via one‐way analysis of variance (ANOVA), followed by Tukey's test. Statistical significance was set at P < 0.05.SPSS v19.0 was applied to assess all the results of the statistical analyses. At least three independent assays were conducted for each group, and all the values were expressed as mean ± standard deviation. The differences between the two groups were assessed via Student's 33.1To determine the potential function of lncRNA MEG3 in SCI, we constructed a mouse model of acute SCI and evaluated the motor function of mice after acquiring SCI using the BMS score. The results indicated that the BMS score became lower in the SCI group Figure . The resAs in the in vitro experiments, after the mouse primary microglia and mouse microglia cell line BV2 were treated with LPS for different times, the qRT‐PCR analysis revealed that the lncRNA MEG3 expression was gradually lessened with the extension of the LPS treatment time Figure . Moreove3.2To investigate the effect of lncRNA MEG3 on LPS‐induced microglial polarization and neuroinflammation, pcDNA‐MEG3 was transfected into BV2 cells and spinal primary microglia, and then the cells were treated with LPS. As shown in Figure Iba‐1 is a routine marker for microglia . The imm3.3A previous study has shown that the nuclear transcription factor NF‐κB plays a key role in the process of M1 polarization of the microglia to cytokine release . On the A20 is a common inhibitor of the NF‐κB signaling pathway , which p3.4To further probe into the potential mechanisms, pcDNA‐HuR was transfected into BV2 cells and primary microglia for 48 h, and then the cells were treated with LPS for 24 h. The qRT‐PCR and Western blot results showed that the overexpression of HuR lowered the mRNA and protein levels of A20 Figure . ActinomCombining the finding shown in Figure Furthermore, pcDNA‐MEG3 and/or pcDNA‐HuR was transfected into BV2 cells, and the cells were treated with LPS for 24 h. The qRT‐PCR analysis expounded that the overexpression of lncRNA MEG3 raised the lncRNA MEG3 expression while the overexpression of HuR did not affect the lncRNA MEG3 expression. Furthermore, the Western blot assay indicated that the overexpression of lncRNA MEG3 lowered the HuR protein level while the overexpression of HuR reversed this trend Figure . The ove3.5To verify if lncRNA MEG3 regulated the M1 polarization of microglia via the HuR/A20/NF‐κB axis, pcDNA‐MEG3 and/or pcDNA‐HuR was transfected into BV2 cells, and the cells were treated with the BAY 11–7082 NF‐κB pathway inhibitor and were further treated with LPS for 24 h. As shown in Figure BV‐2 cells were co‐incubated with the SH‐SY5Y human nerve cells for 12 h. As shown in Figure 3.6Next, we verified the role of lncRNA MEG3 in motor function recovery and neuroinflammation alleviation in mice with SCI. As shown in Figure Ki‐67 is a widely applied marker for proliferation . The imm4The current study aimed to reveal the function and underlying mechanism of lncRNA MEG3 in the M1 polarization of microglia and neuroinflammation. In this study, we discovered that the lncRNA MEG3 expression was lessened in the ASCI mouse spinal cord tissues and LPS‐treated microglia. Based on this finding, we further demonstrated that the overexpression of lncRNA MEG3 restrained the M1 polarization of the microglia and the neuroinflammation caused by LPS. The investigation of the underlying mechanisms expounded that lncRNA MEG3 regulated the M1 polarization of the microglia through the HuR/A20/NF‐κB axis and that the overexpression of lncRNA MEG3 boosted the motor function recovery and neuroinflammation relief in the mice with SCI .As is widely known, the imbalance of the M1 polarization of microglia and abnormal neuroinflammation play pivotal functions in ASCI , 39. ThuHuR, also named Elavl1, is an RNA‐binding protein that can bind to the AU‐rich elements (AREs) in the 3′ prime untranslated region (3′‐UTR) of its target mRNAs and can raise mRNA stability and translation by competing for ARE occupancy against mRNA‐destabilizing modulators , 45. PreA20 is a zinc finger protein that is a potent regulator of ubiquitin (Ub)‐dependent signals . StudiesNotably, there are still some limitations in this study. It is difficult to collect clinical samples, so the sample size is not sufficient. We will expand clinical data in future studies, so that the conclusions of this article will be more evidence‐based. In general, the data that we obtained in this study showed that lncRNA MEG3 functioned in the M1 polarization of microglia and neuroinflammation, and provided a novel regulation axis in ASCI: lncRNA MEG3/HuR/A20/NF‐κB. Our study might provide novel molecules and insights for the restraint of the M1 polarization of microglia and of neuroinflammation to relieve ASCI.The authors have no conflicts of interest to disclose.Figure S1 Peripheral blood samples were collected from patients with traumatic spinal cord injury  and healthy control (n = 36) and the serum samples were centrifuged at 3000 rpm for 10 min. (A) The expression of lncRNA MEG3 was tested by qRT‐PCR. (B) The concentrations of TNF‐α, IL‐6, and IL‐1β were detected by ELISA assay. (C) Correlation analysis was conducted between the expression of lncRNA MEG3 and the levels of inflammatory factors TNF‐α, IL‐6, and IL‐1β. *P < 0.05, *** P < 0.001 versus Healthy. Data are represented as the mean ± SD of three independent assays.Click here for additional data file.