Immunoparalysis is a compensatory and persistent anti-inflammatory response to trauma, sepsis or another serious insult, which increases the risk of opportunistic infections, morbidity and mortality. Here, we show that in cultured primary human monocytes, interleukin-4 (IL4) inhibits acute inflammation, while simultaneously inducing a long-lasting innate immune memory named trained immunity. To take advantage of this paradoxical IL4 feature in vivo, we developed a fusion protein of apolipoprotein A1 (apoA1) and IL4, which integrates into a lipid nanoparticle. In mice and non-human primates, an intravenously injected apoA1-IL4-embedding nanoparticle targets myeloid-cell-rich haematopoietic organs, in particular, the spleen and bone marrow. We subsequently demonstrate that IL4 nanotherapy resolved immunoparalysis in mice with lipopolysaccharide-induced hyperinflammation, as well as in ex vivo human sepsis models and in experimental endotoxemia. Our findings support the translational development of nanoparticle formulations of apoA1-IL4 for the treatment of patients with sepsis at risk of immunoparalysis-induced complications. A nanoparticle integrating a fusion protein of apolipoprotein A1 and interleukin-4 and that targets myeloid-cell-rich haematopoietic organs resolves immunoparalysis in ex vivo and in vivo models of sepsis. As a result of the immune system’s inability to clear a pathogen, patients with sepsis may experience simultaneous hyper-inflammatory and immunosuppressive characteristics, making this condition extremely challenging to manage4. Up to 30–40% of patients with sepsis show an overriding immunoparalysis phenotype. Immunoparalysis is characterized by a persistent anti-inflammatory innate immune response following an insult such as sepsis5, putting patients at high risk of recurrent and secondary infections, frequently leading to organ dysfunction and death6.Sepsis is a serious medical condition caused by a dysregulated host response to infection that frequently results in organ failure and death8, a process termed ‘trained immunity’10, may be able to reverse tolerance and immunoparalysis induced by exaggerated bacterial stimulation11. Although therapeutically induced trained immunity12 is theoretically a compelling strategy to overcome immunoparalysis, a critical need exists for approaches that can be safely and efficiently translated to clinical settings.Therapeutic strategies to rebalance immune responses in sepsis and to improve patient outcomes through immunotherapy are in their infancy. Recent experimental work suggests that the long-term reprogramming of the innate immune cells15, we discovered that IL4 paradoxically induces trained immunity in addition to its known anti-inflammatory effects. We hypothesized that the unique properties of IL4 could be deployed to overcome immunoparalysis in monocytes induced by stimulation with bacterial endotoxin (lipopolysaccharide (LPS)). However, owing to its unfavourable pharmacokinetic properties and the broad IL4 receptor (IL4R) expression, natural IL4 is poorly suitable for therapeutically regulating myeloid cells in vivo. Routing IL4 directly to the myeloid compartment is therefore an attractive therapeutic avenue. In support of this concept, we developed a fusion protein of apolipoprotein A1 (apoA1)—the main protein constituent of high-density lipoprotein—and IL4 and termed this construct ‘apoA1–IL4’17. The apoA1–IL4 fusion protein readily integrated into lipid nanoparticles to generate myeloid cell-avid IL4 nanoparticles. We then evaluated the behaviour of the IL4 nanoparticles in mice and non-human primates using in vivo positron emission tomography (PET) imaging and ex vivo quantitative gamma counting. Finally, we studied the therapeutic potential of the IL4 nanoparticles in multiple translational inflammation and in vivo and ex vivo models of sepsis.In our quest to simultaneously resolve hyper-inflammation and immunoparalysis, we considered interleukin-4 (IL4) for the modulation of trained immunity and tolerance. When re-evaluating the reported direct effects of this cytokine on monocytes in vitro15. Therefore, we first validated several known inhibitory effects of IL4 on inflammation in primary human monocytes . As expected, IL4 potently inhibited the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF) and IL6  axis and the signal transducer and activator of transcription 6 (STAT6) signalling pathway19 Fig. . We perf19 Fig. . IL4 tra19 Fig. . In cont19 Fig. . Thus, eIL6 and IL12B that are known to be involved in trained immunity10. Among the prominent attenuated genes were CCL19 and SOCS2, which are important for lymphocyte trafficking and suppression of cytokine signalling, respectively21.To gain insight into the molecular program induced by IL4 training, we performed transcriptomics analysis on naive and IL4-trained macrophages, both before and after LPS re-stimulation on day 6. Overall, 140 genes were more strongly induced (‘upregulated’) in IL4-trained macrophages, whereas 249 genes were attenuated Fig. . Among tWe next performed transcription factor (TF) motif enrichment analysis Fig.  and gene22. Furthermore, we evaluated histone-3-lysine-9-tri-methylation (H3K9me3)-mediated repression of TNF using chromatin immunoprecipitation (ChIP)–quantitative PCR (qPCR) analyses in IL4-induced trained immunity. Using an area under the curve (AUC) analysis of six primer pairs showed a decrease of H3K9me3 in IL4-induced trained immunity, although this did not reach statistical significance 3-9, enhancer-of-zeste and trithorax-domain containing lysine methyltransferase 7 (SET7)  histone methyltransferase inhibitor, abrogated the induction of trained immunity by IL4 Fig. . SET7 hance Fig. . Togethe23 . ApoA1-based nanoparticles (aNPs) inherently accumulate in haematopoietic organs and efficiently target myeloid cells and their progenitors23 Fig. . Specifi23 Fig. . We used23 Fig. , while w23 Fig. . Due to 23 Fig. .Fig. 3EnKD of apoA1–IL4 against the human IL4 receptor alpha (IL4Rα) using SPR to be 4.5 ± 1.1 nM  and HEK-Blue IL4/IL13 (HEK-IL4) reporter cells—were performed to determine the preservation of biological activity after the extraction, purification and refolding process. We determined the equilibrium dissociation constants  nM Fig. . HEK-IL4 nM Fig. . Althoug16. Nanoparticles of different sizes and morphologies were obtained by varying the formulations’ compositions  analysis revealed the expression of IL4Rα (green) on the membrane. Furthermore, binding of bare apoA1, bare apoA1–IL4, aNPs and IL4-aNPs (red) were confirmed by the rim covering the cell surface. When focusing on a zoomed-in section of the membrane, we found that bare apoA1–IL4 and IL4-aNPs were associated with the IL4Rα, forming enriched co-clusters on the cell surface, which we did not observe for bare apoA1 and conventional aNPs Fig. . Togethe89Zr). Note that these experiments were performed with the human variant of IL4 that does not show biologic activity in mice. PET with computed tomography (PET-CT) imaging at 24 h post intravenous administration showed that 89Zr-IL4 and 89Zr-apoA1–IL4 accumulated mostly in the kidney and liver. In contrast, besides accumulating in the liver and kidney, 89Zr-IL4-aNPs accumulated in relatively higher amounts in immune-cell-rich organs, including the spleen and bone marrow . After injection, dynamic PET-MRI  significantly reduced TNF and IL6 production of LPS-stimulated monocytes , Th2 (CD4+ IL4+), Treg cells (CD4+ IL10+), Th17 (CD4+ IL17+) and cytotoxic T cells (CD8+ Granzyme B+ Perforin+) were observed between trained macrophages and controls  to induce immunoparalysis or PBS (as a control). We intravenously administered IL4m-aNPs (200 µg per dose) at 24 h and 48 h after LPS treatment. We re-challenged the mice with another intraperitoneal injection of LPS (0.1 mg kg−1) 72 h after the first challenge  increased serum IL6 concentrations following LPS re-challenge of tolerized mice  due to heterogeneity in the therapeutic response  are characterized by a dysregulated immune response, creating a therapeutic paradox that requires both managing hyper-inflammatory responses and improving host-defence responses against (opportunistic) secondary infections. To take advantage of these features of IL4, we developed a nanoparticle protein-engineering strategy, thereby overcoming this cytokine’s unfavourable in vivo pharmacokinetic properties. Therefore, we trade the reduced IL4 potency of the fusion protein, a characteristic that can be improved in future studies by optimizing the protein and expression system as we did for the chimeric fusion protein, for the benefit of the substantially improved bioavailability and pharmacokinetics. We show that the aNP strategy favourably alters the blood half-life and biodistribution profile of IL4, resulting in an elevated neutrophil-specific and monocyte-specific accumulation in myeloid cell-rich organs such as the bone marrow and spleen, as earlier observed in models of trained immunity32. Furthermore, these studies have shown that aNPs consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol and apoA1 do not induce trained immunity, emphasizing that our findings are strictly related to the biological effect of IL4 (ref. 33). While we studied the biodistribution of aNPs containing human apoA1-IL4, we additionally developed the murine variant IL4m-aNP to evaluate in vivo efficacy. Indeed, we show that IL4m-aNPs can partially revert immunoparalysis in an LPS-induced sepsis mouse model. Although the data show restored innate immune responses, with a substantial increase of IL6 production and a clear trend towards increased TNF concentrations in the serum of mice treated with IL4m-aNP, full-blown dose-range-finding studies are required to expand the potential of IL4-aNP therapy in a range of immune-mediated diseases that are characterized by concurrent hyper-inflammation and immunoparalysis. It is encouraging that, using the human fusion protein, we showed that IL4-aNPs can reverse the immune tolerance of cells acquired from a human model mimicking clinical immunoparalysis.The ability of IL4 to simultaneously suppress acute inflammation while inducing a trained immunity program, which has been reported to improve host defence34. Myocardial infarction and stroke are also characterized by sterile inflammation followed by immunoparalysis36, and patients with severe trauma suffer from a similar immune-paralytic condition37. In all these situations, reducing inflammation and overcoming immunoparalysis may be beneficial for patient recovery and preventing secondary infections. IL4-aNP technology might be developed into a therapeutic modality for the treatment of all these conditions.We anticipate that the cytokine-nanoparticle technology may find uses in other myeloid-directed applications. The state of immunoparalysis is not unique to sepsis. Immuno-oncological applications might be considered, because cancer is also characterized by local pro-tumour inflammation and the simultaneous suppression of anti-tumour responses (often mediated by myeloid cells)g was used to separate the peripheral blood mononuclear cell (PBMC) interphase. Following three to five washes with cold PBS, PBMC yield and composition were assessed using a haemoanalyser .Buffy coats (Sanquin) or EDTA whole blood from healthy volunteers was acquired after obtaining written informed consent. The material was diluted at least 1:1 with calcium- and magnesium-free PBS (Lonza) and layered on top of Ficoll-Paque . Density-gradient centrifugation for 30 min at 615 Negatively selected monocytes were obtained using MACS, according to the manufacturer’s instructions . Monocyte yield and purity was assessed on a Sysmex haemoanalyser.6 PBMCs were layered on top of a hyper-osmotic Percoll solution  and centrifuged for 15 min at 580 g, room temperature (RT). The interphase was collected, washed once with cold PBS and re-suspended in RPMI.For some experiments (indicated in the text), monocytes were alternatively enriched from PBMCs by hyper-osmotic density-gradient centrifugation over Percoll (Sigma-Aldrich). About 150–200 × 10−1; Centrafarm). This medium is further referred to as RPMI+++. In addition, 10% (v/v) human pooled serum was added to the medium during cell culture .All primary human monocytes and macrophages were cultured in RPMI-1640 with Dutch modifications (Invitrogen) which was further supplemented with GlutaMAX , sodium pyruvate  and gentamicin  for 24 h with one of the stimuli detailed in Supplementary Table To induce trained immunity in primary human monocytes, a previously optimized and published method was usedFor pharmacological inhibition experiments, the monocytes were pre-incubated for 1 h with one of the inhibitors described in Supplementary Table Following the initial 24 h stimulation, the cells were washed with warm PBS, and warm cell culture medium was added. The monocytes were then allowed to rest and differentiate into macrophages for 5 days. On day 6, the induction of trained immunity was assessed. To this end, the cells were typically re-stimulated with LPS for an additional 24 h to elicit cytokine production. The supernatants were collected and stored at −20 °C until further analysis.g, 4 °C and counted before continuing to downstream applications.For most other trained immunity readout methods, the cells were collected as follows: first, the cells were incubated in Versene cell dissociation reagent (Life Technologies) for 30 min in a cell culture incubator. A cell scraper was then used to remove the cells from the culture plates. To maximize the yield, the culture plates were scraped a second time after adding ice-cold PBS. The macrophages were centrifuged for 10 min at 300 −1) and GM-CSF . The cells were differentiated until day 6, with one medium refresh on day 3. On day 6, the non-adherent cells were collected in addition to the adherent cells (as described above). The moDCs and macrophages were then subjected to analysis by flow cytometry as described below.For experiments in which moDCs were compared to macrophages (untrained control or IL4-trained), moDCs were differentiated as follows. First, negatively selected monocytes were obtained as described above. Following 1 h adherence and a PBS wash, they were cultured in RPMI+++ with 10% HPS, further supplemented with IL4  male volunteers with ages ranging from 18 years to 35 years provided written informed consent to participate in experimental endotoxaemia experiments conducted at the research unit of the intensive care department of the Radboud University Medical Center. All study procedures were approved by the local ethics committee (CMO Arnhem-Nijmegen (Radboudumc), registration numbers NL71293.091.19 and 2019-5730) and were conducted in accordance with the latest version of the declaration of Helsinki.40. In short, participants were admitted to the research unit, and an antecubital vein and radial artery were cannulated to allow administration of fluids and endotoxin, and blood sampling and haemodynamic monitoring, respectively. A 3-lead ECG was recorded continuously throughout the experiment. After iso-osmolar pre-hydration (1.5 l NaCl 0.45% and glucose 2.5% administered intravenously in the hour before the start of endotoxin infusion), volunteers were intravenously challenged with a loading dose of 1 ng kg−1 bodyweight endotoxin (Escherichia coli lipopolysaccharide (LPS) type O113, lot no. 94332B1; List Biological Laboratories), directly followed by continuous infusion of 0.5 ng kg−1 h−1 for 3 h. Participants were monitored for 8 h after the endotoxin loading dose after which they were discharged from the research unit.A continuous endotoxin infusion regimen was used, as is described in detail elsewhereFor this project, blood samples were obtained at two timepoints: 1 h before and 4 h after administration of the loading dose. Negatively selected monocytes were acquired as described above. The cells were adhered and stimulated for 24 h with recombinant human IL4, discoidal IL4-aNPs, LPS  or medium only (as a control). Following a PBS wash, the cells were rested in culture medium for 48 h and re-stimulated with LPS for an additional 24 h. Supernatants were collected and stored at −20 °C.−1), lactate oxidase (2 U ml−1), Amplex red ) was added, and the reaction mixture was incubated for 20 min in the dark at RT. Immediately thereafter, fluorescence was measured at 530/25 nm and 590/35 nm. Gen5 software  was used in conjunction with Microsoft Excel to calculate cytokine and lactate concentrations in the original samples.TNF, IL6 and IL1Ra were measured in cell culture supernatants using duoset ELISA kits (R&D Systems), according to the manufacturer’s instructions. For lactate measurements, a fluorometric assay was used. About 30 µl sample, medium control or known standard was added to a black 96-well plate. Then, 30 µl reaction mix ).Macrophages were collected as described above and transferred to a V-bottom 96-well plate for staining. The cells were centrifuged at 400 Fc-receptors were blocked by incubation in PBS supplemented with 10% human pooled serum for 15 min at 4 °C. After washing once more, surface markers and viability were stained in a volume of 50 µl for 30 min at 4 °C, using the antibodies and viability dye described in Supplementary Table −1) + ionomycin (0.5 µg ml−1) for 4 h in the presence of 100 ng ml−1 Brefeldin A, a ‘golgi plug’. The cells were collected and split over two flow-cytometry antibody panels  at an MOI of 1:5. The cells were washed two times with ice-cold PBA and kept on ice to halt the phagocytosis. The cells were stained for CD45 , and the cells were incubated for 1 h at 37 °C in ambient CO2 levels. Oxygen consumption rates and extracellular acidification rates were measured as proxies for glycolytic and mitochondrial metabolism, using a Seahorse XF Glycolysis Stress Test kit or a Seahorse XF Cell Mito Stress Test kit .Macrophages were collected as described above. The cells were re-suspended in RPMI+++ and seeded into overnight-calibrated cartridges at 10Monocytes or macrophages were lysed in RLT buffer (Qiagen) and stored at −80 °C. RNA extractions were performed using RNeasy mini columns (Qiagen) with on-column DNAse I treatment . Preliminary quality control and measurements of concentration were performed using a Nanodrop apparatus. Samples were sent to the Beijing Genome Institute (BGI Denmark) for RNA sequencing using the DNBseq platform.41. Quantification of gene expression levels as RPKM was performed using MMSEQ (v1.0.10)42. Reads and transcripts were normalized using DEseq2, and pair-wise comparisons were performed. Differentially expressed genes were identified using DEseq2 (v1.34.0) with fold change >2 and P < 0.05, with a mean RPKM >1 (ref. 43). To identify genes that were upregulated or attenuated by IL4 training, RPMI-d6 and IL4-d6 macrophages were compared with RPMI-d6+LPS and IL4-d6+LPS samples, respectively. Gene lists were merged and ranked on the basis of IL4-d6+LPS/RPMI-d6+LPS. Gene ontology and TF motif analysis was performed on gene promoters using the HOMER findMotifs tool (v4.11)44.To infer gene expression levels, RNA sequencing reads were aligned to hg19 human transcriptome using Bowtie (v1.2)6 cells per ml in lysis buffer ), sonicated  and centrifuged .Macrophages were collected as described above and re-suspended in RPMI+++. The cells were fixed for 10 min in 1% methanol-free formaldehyde. The reaction was then quenched for 3 min by adding 125 mM glycine. The fixed cells were washed three times with ice-cold PBS, lysed at approximately 15 × 10−1 proteinase K (Qiagen)) for 1 h at 65 °C and run on a 1% agarose gel to confirm target fragment size of 200–800 bp.Aliquots of chromatin were de-crosslinked in 0.5× TBE buffer , and 1 µg of ChIP-grade antibody (Diagenode) was added. The samples were rotated overnight at 4 °C.3, 1% SDS, in Milli-Q) for 20 min, at RT. Input samples were diluted 12 times in elution buffer. After addition of NaCl (0.2 M) and proteinase K (0.1 mg ml−1), all samples were decrosslinked for at least 4 h on a shaking heatblock . MinElute PCR purification columns (Qiagen) were used to purify DNA fragments. DNA fragments were stored at 4 °C until downstream analysis by qPCR.Magnetic protein A/G beads (Dynabeads) were washed two times in dilution buffer supplemented with 0.15% SDS and 0.1% BSA. The washed beads were added to the ChIP samples and rotated at 4 °C for 1 h. The bead-bound chromatin was subsequently washed  as follows: once with low-salt washing buffer , two times with high-salt washing buffer  and two times with no-salt washing buffer . Chromatin was eluted from the beads in elution buffer  supplemented with 100 µg l−1 ampicillin and grown at 37 °C. At the point that absorbance at 600 nm reached >1.5, 1.0 mM isopropyl β-d-thiogalacopyranoside was added to induce pET20b(+)apoA1–IL4 expression, and cells were incubated overnight at 20 °C. Cells were collected by centrifugation before preparation of lysates and purification.ClearColi BL21 (DE3) (Lucigen) were transformed with a pET20b(+)apoA1–IL4 expression vector. Transformed bacteria were inoculated in 40 ml lysogeny broth (Sigma-Aldrich) supplemented with 100 μg lg and 4 °C for 10 min. Collected cells were re-suspended in PBS and centrifuged at 3,500 g and 4 °C for 15 min. Cells were lysed using 20 ml BugBuster Protein Extraction Reagent (Merck) and 20 µl Benzonase nuclease (Merck) per litre culture on a shaker for 30 min at RT. Cell lysates were centrifuged at 39,000 g and 4 °C for 30 min. Insoluble pellets were washed with 10 ml BugBuster per litre and centrifuged at 39,000 g and 4 °C for 20 min. Pellet containing inclusion bodies was re-suspended in extraction buffer  and incubated on a shaker for 15 min at RT. The suspension was centrifuged at 39,000 g and 4 °C for 30 min to remove insoluble fraction. Filtered soluble fraction was loaded on a nickel column and washed with 15 column volumes IMAC wash buffer. ApoA1–IL4 was unfolded and refolded on the nickel column using a linear gradient unfolding buffer 60 ml  to refolding buffer 60 ml  at 2.5 ml min−1. Refolded apoA1–IL4 was eluted from the column with 0.5 M imidazole, 20 mM Tris and 0.5 M NaCl at pH 7.9. Eluate was collected, concentrated and further purified and buffer-exchanged via size exclusion chromatography  equilibrated with PBS storage buffer. Fractions were analysed by SDS–PAGE, pooled, concentrated and snap-frozen in liquid nitrogen before storing at −80 °C. ApoA1–IL4 mass was confirmed by Q-ToF LC-MS (WatersMassLynx v4.1), using MagTran V1.03 for MS.ApoA1–IL4 fusion protein expressing ClearColi cells were collected by centrifugation at 10,880 m, packaging pCMVR8.74 and envelop pMD2.G in Opti-MEM (GIBCO) at 37 °C for 24 h. Cells were washed with DMEM supplemented with 2% heat inactivated FBS and incubated for 48 h. To obtain the lentivirus containing pHR-apoA1–IL4m, supernatant was centrifuged at 875 g to remove cell debris filtered through 0.45 µm PES syringe filter and centrifuged at 50,000 g for 2 h at 4 °C. Pellet containing pHR-apoA1–IL4m lentivirus was re-suspended in culture medium, snap frozen in liquid nitrogen and sorted at −80 °C. HEK293F cells were transduced with pHR-apoA1–IL4m containing lentivirus in transfection medium  for 24 h. Subsequently, cells were cultured in expression medium (50% EX-CELL 293 Serum-Free Medium for HEK293 Cells (Merck) and 50% FreeStyle 293 Expression Medium (Thermo Fisher Scientific), supplemented with Glutamax, 1% Pen-Strep and 1 µg ml−1 doxycycline (Merck) on a shaker at 150 r.p.m. for 3 days at 37 °C. Culture supernatant containing apoA1–IL4m was centrifuged at 3,500 g, 4 °C for 15 min and filtered through 0.22 µm PES syringe filter to remove cell debris. Filtered soluble fraction was loaded on a StrepTactin XT 4flow 5 ml column (Cytiva) and washed with 5 column volumes W-buffer  with flow rate 1–2 ml min−1. ApoA1–IL4m was eluted from the column with W-buffer supplemented with 50 mM biotin. Eluate was collected, concentrated and snap-frozen in liquid nitrogen before storing at −80 °C. ApoA1–IL4m mass was confirmed by Q-ToF LC-MS (WatersMassLynx v4.1), using MagTran V1.03 for MS.HEK293T cells were co transfected with fuGENE (Promega) including transfer vector pHR-apoA1–IL4To confirm fusion of apoA1 and IL4, 100 ng IL4 (BioLegend), apoA1 and apoA1–IL4 were loaded on a 4–20% polyacrylamide gel (Bio-Rad). After gel electrophoresis, samples were transferred to nitrocellulose membranes with blot buffer . Subsequently, membranes were incubated with blocking buffer ) overnight at 4 °C. The blots were incubated with primary monoclonal antibodies monoclonal anti-IL4  and anti-apoA1  for 1 h at 4 °C. After incubation with primary antibodies, membranes were washed and incubated with rabbit anti-mouse IgG (H+L)–HRP conjugate . HRP-conjugated secondary antibodies were detected with TMB (Thermo Fisher Scientific) and visualized using the Image Quant gel imager .2 dilution concentration series consisted of apoA1–IL4 ranging from 200 nM to 6.25 nM and of human IL4 ranging from 20 nM to 0.65 nM. All samples were prepared in HPS-EP buffer  P20 pH 7.4). Association was monitored for 180 s and dissociation for 180 s with a flow rate of 30 μl min−1. Sensor chip was regenerated with glycine 1.5 . Kinetics was determined by fitting the interaction SPR data for 1:1 binding.SPR measurements were performed using a Biacore ×100 SPR system . Human IL4 receptor alpha-FC chimera (Biolegend) was immobilized on a protein G sensor chip . Log4 cells was added per well in a 96-well-plate. Subsequently, 20 μl stimulus or vehicle was added, and cells were incubated for 20–24 h at 37 °C. Subsequently, 180 μl QUANTI-Blue was added per well to a separate 96-well-plate (flat-bottom), and 20 μl of the cell supernatant was added. The plate was incubated for 1–3 h at 37 °C, and absorbance at 640 nm was measured on a Tecan Spark plate reader to determine SEAP levels.HEK-Blue IL4/IL13 cells were purchased from InvivoGen. This cell line has a fully active STAT6 pathway and carries a STAT6-inducible SEAP reporter gene. The HEK-Blue IL4/IL13 cells produce SEAP in response to IL4 and IL13. The levels of secreted SEAP can be determined with QUANTI-Blue (InvivoGen). About 180 μl DMEM with 10% FBS and 1% Pen-Strep containing 5 × 10−1) in chloroform, DMPC (133.5 μl) and cholesterol (Sigma-Aldrich) (7.5 μl), and for spherical aNPs, POPC (66.5 μl), PHPC (17.5 μl), cholesterol (4.5 μl) and tricaprylin (Sigma-Aldrich) (2.79 μl from 0.956 g ml−1 stock) were combined in a glass vial and dried under vacuum. The resulting film was re-dissolved in an acetonitrile and methanol mixture . For formulation based on apoA1, cholesterol (15 μl) was used. Separately, a solution of apoA1 protein in PBS , apoA1–IL4 protein in PBS  or apoA1–IL4m in PBS  was prepared.All phospholipids were purchased from Avanti Polar Lipids. Four different aNPs were formulated. For discoidal aNPs from stock solutions  was added, and the solution was concentrated to 5 ml; this was repeated twice. The washed solution was concentrated to approximately 1.5 ml and filtered through a 0.22 μm PES syringe filter to obtain the finished aNPs. Protein concentration in aNP samples was quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). To formulated fluorescent aNPs, 0.5 mg of DiOC18(3) dye (DiO) (Thermo Fisher Scientific) was dissolved in the chloroform solution used to prepare the lipid film.Both solutions were simultaneously injected using a microfluidic pump fusion 100 (Chemyx) into a Zeonor herringbone mixer  with a flow rate of 0.75 ml minObtained aNP formulations in PBS were filtered through a 0.22 µm PES syringe filter and analysed by DLS on a Malvern Zetasizer Nano ZS analyser. Values are reported as the mean number average size distribution.−1 in DMSO) for 2 h washed three times using a 10 kDa MWCO Vivaspin tubes to remove any unreacted DFO-p-NCS. For radiolabelling, DFO coupled proteins and aNPs were incubated with 89Zr at 37 °C using a thermomixer at 600 r.p.m. for 1 h and washed three times using 10 kDa MWCO Vivaspin tubes to remove any unreacted 89Zr.IL4, apoA1–IL4 and IL4-aNPs were incubated with two molar excesses of DFO-p-NCS (5 mg ml−2 s−1) or 24,000× magnification (dose rate of 11.8 electrons A−2 s−1) and 1 s acquisition time.First, the surface of 200-mesh lacey carbon supported copper grids (Electron Microscopy Sciences) was plasma treated for 40 s using a Cressington 208 carbon coater. Subsequently, 3 ml of IL4-aNPs sample (~1 mg protein per ml) was applied on a grid and vitrified into a thin film by plunge vitrification in liquid ethane by using an automated robot (FEI Vitrobot Mark IV). Cryo-TEM imaging was performed on the cryoTITAN (Thermo Fisher Scientific), equipped with a field emission gun, a post-column Gatan imaging filter (model 2002) and a post-GIF 2k × 2k Gatan CCD camera (model 794).The images were acquired at 300 kV acceleration voltage in bright-field TEM mode with zero-loss energy filtering at either 6,500×  for a few minutes before and during the imaging. The acquisition was performed in total internal reflection fluorescence mode, using ONI Nanoimager (ONI). It is equipped with a 100×/1.4NA oil immersion objective and a sCMOS camera, and in this study, 488 nm (200 mW) and 640 nm (1000 mW) lasers were used. About 10,000 frames were acquired with a 10 ms exposure time with the field of view of 50 × 80 µm. Raw data were processed using the ThunderSTORM software (v1.3)47, yielding images with a spatial resolution of 10 nm.Human monocytes were isolated from a healthy donor’s peripheral blood as described above. About 100,000 monocytes were seeded per well on a cell-culture-treated chambered coverslip . After 2 h of incubation at 37 °C (cell attachment), the cells were incubated for 2 h at 37 °C with Cy5-labelled variant of either bare apoA1 or apoA1–IL4, discoidal aNPs or IL4-aNPs, and spherical aNPs or IL4-aNPs. Subsequently, the cells were washed with PBS and fixed with 4% PFA for 20 min. IL4 receptor was stained with a polyclonal rabbit IgG1 anti-human IL4R  primary antibody for 24 h at 4 °C, followed by a goat anti-rabbit Alexa Fluor 488-conjugated secondary antibody  for 1 h at room temperature. The stained cells were stored in PBS at 4 °C. For the dSTORM, the cells were immersed in GLOXY imaging buffer (40 μg mlMacaca fascicularis) were used, aged 15 years and 16 years. All animals were co-housed in climate-controlled conditions, respectively, at 20–24 °C, 45–65% humidity with 12 h light–dark cycles and provided water ad libitum. Mice were fed a standard chow diet, and non-human primates were fed Teklad Global 20% Protein Primate Diet. Animal care and experimental procedures were based on approved institutional protocols from the Icahn School of Medicine at Mount Sinai. All mice were randomly assigned to experimental groups.Female C57BL/6 J mice (approximately 8–11 weeks old and approximately 20 g) were purchased from Charles River Germany. For non-human primate studies, two male cynomolgus monkeys , apoA1–IL4 (30.1 ± 0.9 μCi), discoidal IL4-aNPs (146.1 ± 46.5 µCi) and spherical IL4-aNPs (108.6 ± 16.9 μCi). Two non-human primates were injected with discoidal 89Zr-labelled IL4-aNPs (1079 μCi and 682 μCi). At predetermined time points, 1, 2, 5, 10 and 30 min and 1, 2, 4, 8 and 24 h for mice and 5, 30 and 90 min and 48 h for non-human primates, after injection blood was drawn and weighed, and radioactivity was measured using a Wizard2 2480 automatic gamma counter (Perkin Elmer). Data were corrected for radioactive decay, and percentage of injected dose per gram of blood (%ID per g) was calculated. Data were fitted using a nonlinear two-phase decay regression in GraphPad Prism, and weighted blood half-life was calculated via the equation (% fast × t1/2 fast + % slow × t1/2)/100. Biodistribution in mice was determined 24 h post injection. After PBS perfusion, tissues of interest were collected and weighed, and radioactivity was measured using a Wizard2 2480 automatic gamma counter (Perkin Elmer). Data were corrected for radioactive decay, and percentage of injected dose per gram of tissue (%ID per g) was calculated.C57BL/6 mice were intravenously injected with 89Zr-labelled IL4 variants, respectively, IL4 (53.6 ± 6.6 μCi), apoA1–IL4 (30.1 ± 0.9 μCi), discoidal IL4-aNPs (146.1 ± 46.5 μCi) and spherical IL4-aNPs (108.6 ± 16.9 μCi). After 24 h, mice were anaesthetized using 1.0% isoflurane in O2 at a flow rate of ∼1.0 l min−1. PET-CT scans were acquired using a Mediso nanoScan PET-CT (Mediso). A whole-body CT scan was executed  followed by a 20 min PET scan. Reconstruction was performed with attenuation correction using the TeraTomo 3D reconstruction algorithm from the Mediso Nucline software v3.04.020.0000. The coincidences were excluded by an energy window between 400 keV and 600 keV. The voxel size was isotropic with 0.4 mm width, and the reconstruction was applied for four full iterations, six subsets per iteration.C57BL/6 mice were injected intravenously with Tissues were placed in a film cassette against a phosphorimaging plate  at −20 °C to determine the radioactivity distribution. The plates were read at a pixel resolution of 25 mm with a Typhoon 7000IP plate reader .For cellular specificity, mice were intravenously injected with DiO-labelled IL4-aNPs that was allowed to circulate for 24 h. Subsequently, mice were killed, and single cell suspensions were created from blood, spleen and bone marrow as previously described. Cell suspensions were incubated with anti-CD115, anti-CD11b, anti-Ly6C, anti-Ly6G, anti-CD19, anti-CD45, anti-CD11c, anti-CD3 and anti-F4/80. Live or dead Aqua was used as viability stain. Cells were subsequently washed and re-suspended in FACS-buffer. All data were acquired on an Aurora 5 l flow cytometer (Cytek Biosciences). DiO-IL4-aNPs were detected in the FITC channel.−1) and dexmedetomidine (0.0075–0.015 mg kg−1). Non-human primates were injected with 1.114 mCi and 0.682 mCi discoidal 89Zr-labelled IL4-aNPs, at a dose of approximately 0.1 mg kg−1. Dynamic PET imaging as performed for 60 min following infusion, and additional static PET-MRI scans were performed at 1 h and 48 h after injection. Furthermore, blood was drawn during imaging at 5 min, 30 min and 120 min after injection. PET and MRI images were acquired using a 3T PET-MRI system . Beginning concurrently with the injection of aNPs, dynamic PET imaging was performed using one bed position covering the chest and abdomen. MR imaging parameters were as follows: acquisition plane, coronal; repetition time, 1,000 ms; echo time, 79 ms; number of slices, 144; number of averages, 4; spatial resolution, 0.5 × 0.5 × 1.0 mm3; and acquisition duration, 42 min and 42 s. After dynamic PET image acquisition, static whole-body PET images were acquired from the cranium to the pelvis, using 4 consecutive bed positions of 15 min each. Simultaneously with each bed, MR images were acquired as described above, except using only 1.4 signal averages, number of slices 160 and spatial resolution 0.6 × 0.6 × 1.0 mm3 . Whole-body PET and MR imaging was also performed at 48 h after injection, using 4 PET bed positions of 30 min each, with MR parameters as follows: acquisition plane, coronal; repetition time, 1,000 ms; echo time, 79 ms; number of slices, 224; number of averages, 2; spatial resolution, 0.6 × 0.6 × 1.0 mm3; and acquisition duration, 29 min and 56 s. Whole-body MR images from each bed were automatically collated together by the scanner. After acquisition, PET raw data from each bed were reconstructed and collated together offline using the Siemens proprietary e7tools with an ordered subset expectation maximization algorithm with point spread function correction for 3 iterations and 24 subsets. Also, Gaussian filter of 4 mm was applied to the images. A three-compartment  attenuation map was used for attenuation.After overnight fasting, non-human primates were anaesthetized using ketamine  were drawn on tissues of interest including spleen, liver, kidneys, lungs, heart, cerebellum and cerebrum, which were traced in their entirety, and bone marrow uptake was determined using three vertebrae in the lumbar spine. For each ROI, mean standardized uptake values (SUVs) were calculated. Discoidal −1 body weight LPS. At 24 h and 48 h, mice were treated intravenously with either 200 µg IL4m-aNPs or PBS. Subsequently, mice were re-challenged with intraperitoneal 0.1 mg kg−1 LPS injection at 72 h. After 90 min, mice were killed, blood was collected for ELISA and single cell suspensions were created from blood, spleen and bone marrow as previously described. For staining protocol, blood samples for ELISA were allowed to clot at RT for 30 min. Serum was taken after centrifugation at 1,000 g for 10 min at 4 °C. Mouse TNF and IL6 ELISAs (Biolegend) were performed according to manufacturer’s protocols. Animal care and experimental procedures were based on approved institutional protocols from the Nijmegen Animal Experiments Committee.For in vivo tolerance model, 11-week-old female C57BL/6 mice were intraperitoneal tolerized with 0.1 mg kgn is indicated in the figure legend. Unless otherwise indicated, statistical analyses were performed in Graphpad Prism . For trained immunity and acute stimulation experiments with primary human monocytes , Wilcoxon signed-rank tests were used. Statistical methods for RNA-sequencing analysis are described above. Two-sided P values under 0.05 were considered statistically significant.Data are shown as mean ± s.d., unless otherwise indicated. Individual data points in graphs are biological replicates, not technical repeats. The number of data points can be clearly discerned in each figure or Further information on research design is available in the Supplementary InformationSupplementary Tables 1–5, and unprocessed SDS–PAGE gels and western blots for Fig. 3.Reporting Summary


Intertrochanteric femoral fractures are commonly treated with intramedullary nails (IMNs). A tip–apex distance (TAD) of more than 20–25 mm is associated with an increased risk of cut-out. The Stryker Adaptive Positioning System (ADAPT) is a computer-assisted navigation system designed to reduce TADs. We aim to assess if the ADAPT reduces the number of outliers with a TAD > 20 mm. All patients with intertrochanteric fractures treated with an IMN between 1 September 2020 and 12 March 2022 were included. Patients were included in three periods: a pre-ADAPT period (55 patients); an ADAPT period (50 patients), where it was compulsory to use the system; and a post-ADAPT period after the discontinuation of the system (59 patients). The TADs and lag screw protrusions beyond the lateral cortex were measured. The median TADs in the three periods were 17.0 mm (8–31 mm), 15.5 mm (9–30 mm), and 18.0 mm (11–32 mm), respectively. The absolute number of outliers with a TAD > 20 mm decreased from 15/55 patients in the pre-ADAPT period to 11/50 patients during the ADAPT period. This observation was not statistically significant, but this is likely due to the lack of power of the present study to show changes of this magnitude. However, our expectation that the ADAPT would diminish outliers markedly or close to zero outliers was not met, as we observed 11/50 = 22% outliers with a TAD > 20 mm when using computer-assisted surgery, i.e., ADAPT and Gamma3 for intertrochanteric fractures. Based on these findings, the use of the ADAPT was discontinued at our level 1 trauma center. Intertrochanteric fractures are among the most common fractures requiring surgery worldwide. The incidence in the US elderly population is approximately 350/100,000 inhabitants yearly . One of Previous studies on the ADAPT have shown promising results in terms of reducing the TAD, operating time, fluoroscopy time ,7,8,9,10The primary aim of this study was to assess whether the use of ADAPT entirely prevents or markedly reduces the number of outliers (TAD > 20 mm), when used in a clinical setting. A secondary aim was to assess whether the use of ADAPT reduces the lateral protrusion of the lag screw, the median TAD, the use of fluoroscopy, and duration of surgery.In this prospective trial of the ADAPT system, all patients admitted to Aarhus University Hospital with AO type 31A2-31A3 intertrochanteric fractures were included in the period 1 September 2020–12 March 2022. To account for a potential learning period, we chose to have three periods: a pre-ADAPT period, an ADAPT period, and a post-ADAPT period. We planned to include approximately 50 patients in each period. Prior to the study, we agreed that if the ADAPT system could not reduce the number of outliers with a TAD > 20 mm after being used 50 times, we would discontinue its use in our department.In the first period, the pre-ADAPT period (1 September 2020 to 27 February 2021), patients were operated on with the Stryker Gamma3 Hip Fracture Nailing System using the technique as described by the manufacturer without the use of computer assistance. In the following ADAPT period, 1 March 2021 to 30 September 2021, the ADAPT 2.1 system was introduced in our department, and it was mandatory for all surgeons to use the ADAPT system when nailing intertrochanteric hip fractures. The post-ADAPT period, 1 October 2021 to 12 March 2022, resembled the pre-ADAPT period, i.e., no ADAPT was available. In the pre- and post-ADAPT periods, we used regular equipment for intramedullary nailing with the Stryker Gamma3 intramedullary nail.The following data were recorded for all patients in a database using Epidata v4.6.0.6 : age, seThere were no exclusion criteria as all patients treated with a Gamma3 nail were considered eligible for operation using the ADAPT system.All patients were scheduled for a three-month follow-up visit in the outpatient clinic, including a clinical examination and radiographic control.The primary outcome was the number of outliers, defined as a TAD > 20 mm and a TAD > 25 mm. The secondary outcomes were the lateral screw protrusion , median The TAD was measured based on perioperative fluoroscopy images using the method described by Baumgaertner et al. , where tMore than 6 months after the last patient was included, we reviewed the patients’ medical files to determine the re-operation rate. We identified the cause of failure, indication for re-operation, and type of re-operation. The number of outliers with a TAD > 20 mm and >25 mm was compared between groups with a Chi square test. The median TAD and lag screw protrusion were analyzed using the Kruskal–Wallis test for non-parametric data with Dunn’s multiple comparison test. Data are presented as the median  or absolute numbers (%). Prism 9.4.1 for macOS was used for the statistical analyses and graphs.The Regional Ethical Committee was consulted prior to commencing this study but stated that as the ADAPT system was introduced in our department as part of our standard care, no ethical approval was required and that the study was regarded as a quality improvement project.During the three periods, we included a total of 163 patients: for the pre-ADAPT, n = 55; for the ADAPT period, n = 50; and for the post-ADAPT, n = 59. Most of the patients were female (70%), and the mean age was 82 (range: 39 to 101) . During p = 0.10) and a TAD > 25 mm (p = 0.78). The main results of the study are depicted as scatter plots in p = 0.62). However, the median TAD was higher in the post-ADAPT period compared to the ADAPT period (p = 0.001)  .p = 0.15) and post-ADAPT period (5.1 (2–18) mm, p = 0.82).The ADAPT system did not significantly reduce the median protrusion of the lag screw of 5.7 (1–12) mm beyond the lateral cortex compared to the pre-ADAPT period (6.8 (2–15) mm, p = 0.21).There was no difference in the median fluoroscopy time when comparing the three periods (pre-ADAPT: 184 (62–786) seconds, ADAPT: 243 (112–532) seconds, post-ADAPT: 248 (86–506) seconds, p = 0.99) or post-ADAPT periods (p = 0.98).We did not find a reduction in the surgery time in the ADAPT period compared to either the pre-ADAPT . Similarly, there were no significant differences in the duration of surgery or fluoroscopy time (p = 0.21).In this prospective trial of the ADAPT system, we included patients with intertrochanteric fractures in three periods: a pre-ADAPT period, where patients were operated on with the standard Gamma3 intramedullary nail technique (n = 55); an ADAPT period, where patients were operated on with the use of the ADAPT system (n = 50); and a post-ADAPT period, where patients were operated on without the use of the ADAPT system (n = 59). The ADAPT system did not prevent outliers entirely. In fact, the number of outliers was still 11/50 = 22% when using the system. This reduction from 15/55 = 27% in the pre-ADAPT period was not statistically significant. The lateral protrusion of the lag screw was not significantly different between the three periods (p = 0.10) or for outliers with a TAD > 25 mm (p = 0.78). This is in line with the randomized controlled trial performed by Lilly et al. [We did not find a significant reduction in outliers when comparing the ADAPT period to the pre- and post-ADAPT periods for outliers with a TAD > 20 mm  but not when comparing the ADAPT period with the pre-ADAPT period (median TAD = 17) (p = 0.62). As the median TAD in all three periods was below 20 mm, this reduction in the TAD might be statistically significant, but it is not clinically relevant.We found a statistically significantly lower median TAD when comparing the ADAPT period (median TAD = 15.5 mm) with the post-ADAPT period (median TAD = 18.0)  pre-ADAPT, 6/50 (12%) ADAPT, and 8/59 (14%) post-ADAPT. Moreover, the ADAPT is designed to be an adjunct for all surgeons.A second limitation to our study is the lack of a power calculation. We did not perform a power calculation prior to this study, as we beforehand agreed that if the ADAPT system did not reduce the number of outliers significantly when used on 50 patients, we would discontinue its use in our department.Based on the data regarding outliers  provided in With a TAD > 20 mm, the combined pre-ADAPT and post-ADAPT rate of outliers was (11 + 4 + 15 + 8)/(55 + 59) = 38 outliers/114 patients = 33%, while the corresponding rate during the ADAPT-period was (8 + 3)/50 = 22%. Based on these findings, the post hoc power of the study  was only 28%. Consequently, a sample size calculation with a power of 0.8, an alpha of 0.05, and group incidences of 33% and 22% would require 257 patients in each group. In this light, the study can be argued to be underpowered.However, the advantage of the ADAPT was thought to be to reduce the outliers completely, i.e., a reduction from approximately a 25–27% rate of outliers (TAD > 20 mm) to 0%. In this case, only 42 patients would be needed in each group to have a power of 0.95 with an alpha of 0.05. The current study revealed that the ADAPT did not eliminate outliers completely. On the contrary, our study documents that outliers were still common despite the use of the ADAPT, i.e., 11/50 = 22%. Therefore, our initial hope and belief that computer-assisted surgery could entirely prevent or markedly reduce the number of outliers was not confirmed. Moreover, neither residents nor consultants liked to use the system . We therComputer-assisted fracture surgery is on the rise and has been extensively investigated in recent years ,20. AdeqIn the present study, TAD outliers (>20 mm) were still present in 11/50 = 22% of hip fracture patients being treated with the use of computer-assisted surgery (ADAPT for Gamma3 hip fracture nailing system). Based on these findings, the use of ADAPT was discontinued at our level 1 trauma center.


The key to the photothermal-reaction mechanism is the difference in the photothermal absorption coefficients of the materials. The device in which graphene and MoS2 were vertically stacked using a laser-based photothermal reaction demonstrated its potential application as a photodetector that responds to light and its stability against cycling. The laser-based photothermal-reaction method for 2D materials will be further applied to various fields, such as transparent display electrodes, photodetectors, and solar cells, in the future.Implementing a heterostructure by vertically stacking two-dimensional semiconductors is necessary for responding to various requirements in the future of semiconductor technology. However, the chemical-vapor deposition method, which is an existing two-dimensional (2D) material-processing method, inevitably causes heat damage to surrounding materials essential for functionality because of its high synthesis temperature. Therefore, the heterojunction of a 2D material that directly synthesized MoS With the development of next-generation devices and the introduction of new functionalities, the primary objective for semiconductor technology is to increase integration density while achieving various form factors through downscaling and ultra-thinning techniques ,2,3. Pio2 with p-type WS2, eliminating the need for a transfer process. Therefore, various heterojunction structures based on 2D materials need to be implemented and validated by exploiting the universal applicability of selective-laser photothermal reactions.Methods such as metal-organic chemical-vapor deposition (MOCVD), molecular-beam epitaxy, and atomic layer deposition have led to significant progress in the synthesis of transition-metal dichalcogenides, which are representative 2D semiconductors, on various substrates ,11,12,132 heterojunction structure by integrating n-type MoS2 on semi-metallic graphene via a laser-assisted selective photothermal-reaction method by optimizing the photothermal and laser parameters. For the synthesis of MoS2, a thermally decomposable (NH4)2MoS4 precursor was mixed with an organic solvent, and a fiber laser n; PMMA), ammonium tetrathiomolybdate((NH4)2MoS4), CAS No. 15060-55-6), and hexamethyldisilazane((CH3)3SiNHSi(CH3)3, CAS No. 999-97-3) were purchased from Sigma-Aldrich . A photoresist  and Developer  were purchased from Merck . Dimethyl sulfoxide ((CH3)2SO, CAS No. 67-68-5) and ammonium persulfate((NH4)2S2O8, CAS No. 7727-54-0) were purchased from Daejung Chemicals .Poly(methylmethacrylate) ((C4 (125 sccm) as a carbon source and H2 (100 sccm) as a reactant gas [2 plasma process  to improve the copper foils’ wettability. Subsequently, graphene was separated by etching the Cu foil in an ammonium persulfate solution (2 g ammonium persulfate in 100 mL deionized (DI) water) for 4 h. Once all of the copper was etched away, only the PMMA/graphene film remained, which was lifted using a glass slide and rinsed twice in DI water. The PMMA/graphene layer was then transferred to a 300 nm SiO2/P+ Si substrate. Finally, to remove PMMA from the graphene, it was immersed in acetone in two steps: first for 30 min and then for an additional 90 min.Monolayer graphene was grown via CVD on 25 µm-thick copper foils  at tempetant gas . The PMM2) at 300 °C for 20 min, ensuring a clean surface [The formation of the graphene-patterned array was achieved using typical photolithography and oxygen plasma etching methods. The graphene was uniformly covered by the PR solution, spinning at 3000 rpm for 30 s, and then baked at 95 °C for 2 min. Subsequently, the sample was exposed to aligner UV light for 15 s, creating a graphene-pattern array with a size of 100 μm × 300 μm. The samples were rinsed with a developer, followed by a rinse with deionized water, and then dried using nitrogen. The exposed graphene regions, not protected by PR, were etched through a reactive ion etching process. The sample was then soaked in acetone for about 30 min to remove the PR from the graphene. As a result, a graphene-pattern array was obtained. To thoroughly remove the PMMA and PR residues that remained after the acetone treatment, the patterned graphene underwent hydrogen annealing 2MoS4 precursor with 6 mL of dimethyl sulfoxide (DMSO). A multistep coating technique ensured uniform coverage of the precursor solution on the graphene. SiO2/Si wafers (with a 300-nm-thick SiO2 layer) underwent two spin-coating stages: an initial 10 s spin at 500 rpm, followed by a 30 s spin at 2500 rpm. Subsequently, the specimen was annealed on a hot plate at 150 °C for 3 min to evaporate any remaining solvent. A fiber laser with a 1.06 μm wavelength and 20 W output was employed to selectively anneal the precursor layer, thereby synthesizing MoS2. The parameters for optimizing MoS2 synthesis via the laser were as follows: (i) laser power (from 1 W to 20 W), (ii) scan speed (from 10 mm/s to 500 mm/s), and (iii) frequency (from 20 kHz to 200 kHz). After the selective synthesis of MoS2, the untreated parts were washed away by immersion in DMSO solvent.To create a vertically stacked MoS−3 Torr) using a probe station connected to a parameter analyzer . To confirm the optoelectronic characteristics, the devices were illuminated using a white-light halogen lamp, and the light intensities were measured using a power meter .The I–V characteristics of the device were measured under low-vacuum conditions  was defined using photolithography and reactive ion etching with O2 plasma [4)2MoS4; ATM) precursor solution was spin-coated onto the patterned graphene. Considering the contrasting nature of the graphene pattern (hydrophobic) and SiO2 surface (hydrophilic), achieving a uniform precursor coating on this nonuniform surface is challenging. The optimization strategies for these coatings are discussed in 2 pattern on the preformed graphene pattern. We chose a fiber laser among the laser sources with various wavelengths because of its low optical absorption in the surrounding materials, such as graphene, SiO2, and Si; however, it has high optical absorption only in the (NH4)2MoS2 precursor. Therefore, only the precursor layer could be selectively heated and thermally decomposed into MoS2 [2, even if MoS2 was synthesized after the precursor was thermally decomposed; therefore, the laser penetrated the MoS2 [2 heterostructure by synthesizing MoS2 without damaging graphene in the lower layer. More importantly, this direct-synthesis method can realize a vertically stacked layer structure without using a conventional transfer method, thereby fundamentally avoiding problems such as wrinkles or contamination traps at the interface. The optimized laser conditions on the SiO2/Si substrate were as follows: a laser power of 10.8 W, scan speed of 100 mm/s, and frequency of 20 kHz. Finally, the source and drain were affixed using shadow masks to analyze the electrical properties of the graphene/MoS2 heterojunction. −1 and ~2700 cm−1, respectively (marked in red). The 2D peak exhibited a sharp, single Lorentzian line shape and was considerably more intense than the G peak, which is a characteristic feature of monolayer graphene. Additionally, the D peak (~1360 cm−1), which indicates defects and imperfections within the graphene crystal, was barely discernible. The Raman spectra of MoS2 synthesized via the laser-based photothermal reaction are shown in blue. Two prominent MoS2 peaks, E2g and A1g, could be distinctly observed at 383 cm−1 and 409 cm−1, respectively. A Raman characteristic of MoS2 is the narrowing of the gap between these two peaks as it transitions from the bulk state to that of a single layer [2 layers present. The gap between these peaks was approximately 26 cm−1, confirming the few-layered nature of MoS2. Atomic force microscopy (AFM) analysis has substantiated that the synthesized molybdenum disulfide (MoS2) exhibits a consistent and uniform film-like structure with a measured thickness of 10 nm. The thickness control of the MoS2 film can be achieved through the regulation of solvent concentration and the adjustment of spin-coating velocity. The critical aspect to consider is that to achieve uniform synthesis of MoS2 in film form, it is necessary to adjust the laser scanning speed in accordance with changes in the thickness of the precursor film  surface roughness of 1.15 nm [2 overlapped 2MoS4 precursor using thermal CVD, and we analyzed its Raman characteristics. A comparison between the Raman peak of the graphene/MoS2 structure synthesized by laser irradiation (marked in green) and that of the graphene/MoS2 structure created by thermal chemical vapor deposition (T-CVD) (marked in black) showed that the Raman characteristics of MoS2 were similar, whereas those for graphene exhibited a pronounced difference. When MoS2 was directly synthesized using general T-CVD, the underlying graphene sustained thermal damage, leading to an amplified D peak and diminished 2D peak. Therefore, our research demonstrates that the laser-based selective thermal-treatment method can be used to directly synthesize MoS2 layers on a graphene monolayer without causing damage. This mechanism, when paired with other 2D materials that are amenable to thermal decomposition, holds promise for the assembly of diverse heterojunction structures.D method ,37, whic2 plasma . During nto MoS2 . In addithe MoS2  without le layer . This alsor film . Specifi 1.15 nm . Interes4)2MoS4 precursor layer on graphene is essential to demonstrate a MoS2/graphene-heterojunction structure. In addition, a uniform and continuous pinhole-free thin film with a large area enabled the fabrication of a device array with a high yield. However, because graphene has a hydrophobic-surface characteristic owing to its unique atomic-crystal [2 substrate and the graphene-transferred SiO2 substrate, as shown in 2/Si substrate, the surface of the wafer was treated with an O2 plasma process  prior to spin-coating the ATM solution to improve the surface wettability. As shown in the optical-microscopy images in 42−) clusters. However, as a result of numerous mixing control experiments between solutions, when DMF + 2-aminoethanol-based solvent was used, obtaining stable coating conditions was difficult as most of the graphene layers rolled after peeling from the substrate as indicated by the arrow in n-butylamine), a similar phenomenon occurred when the solvent penetrated the substrate and graphene, resulting in delamination. As an alternative, the surface tension of the solution was controlled by adding Triton X-100 surfactant to the ATM solution. As a non-ionic surfactant, the hydrophobic group of Triton-X-100 engaged in interactions with the carbonaceous surface, leading to its adsorption onto the graphene surface. This adsorptive phenomenon enhanced the wettability of the graphene substrate, thereby facilitating a more uniform dispersion of the solvents on its surface [2 interface may allow for an even coating of the ATM precursor without peeling off the graphene ; however, after HMDS formation, both peaks shifted down to 1595 cm−1 and 2686 cm−1, respectively (marked in red). The Raman shift indicated that although the surface of graphene had hydrophobic characteristics, the entire surface was covered well with HMDS because some existing defects or oxidized species in graphene played a role in nucleating the SAM [G: 22.2 V) [Fν is the Fermi velocity previously reported as 1.1 × 106 m/s, n is the intrinsic carrier density per volt with a typical value of approximately 7.2 × 1010 cm−2 V−1, and V1 (V2) is the Dirac point of devices without (with) HMDS treatment. The calculation confirmed that the HMDS treatment shifted the Fermi-energy level of graphene by 0.11 eV closer to the vacuum level. The field-effect carrier mobility of graphene was additionally calculated from the maximum slope near the charge-neutrality point, as follows:chL and chW are the channel length and width, respectively, mg is DdI/GdV, oxC is the gate oxide capacitance per area, and DV is the applied drain-source voltage. As plotted in 2/Vs and ~297.9 cm2/Vs, respectively. This represents a significant enhancement in mobility compared to bare graphene (μhole: ~86.5 cm2/Vs and μelec: ~95.4 cm2/Vs). The electrical properties of MoS2, interfaced with graphene, were also analyzed via transfer characteristics  methods, with an observed on/off ratio of 103. These moderate properties can be attributed to the fine-grained structure of the laser-synthesized MoS2. Determining the electronic band structure of the synthesized graphene/MoS2 heterostructure is necessary for analyzing its electrical characteristics and understanding its charge-transfer dynamics. The work function of each material was experimentally measured using ultraviolet photoelectron spectroscopy. The work function was calculated as follows [hν is the energy of the incident UV photons (He I line = 21.21 eV), SECOE is the secondary electron-cutoff energy, and VBE is the onset of the valence band. As expected, bare graphene exhibited significant fluctuations in the analysis value, ranging from 4.3 eV to 5.2 eV depending on the measurement position, whereas HMDS-doped graphene exhibited a stable value of approximately 4.5 eV. Furthermore, MoS2 synthesized by laser treatment exhibited a uniform work function of approximately 4.2 eV, which was in good agreement with that of the few-layered MoS2 [2 heterojunction, delineated according to the positions of the respective band structures. In a thermal-equilibrium state, the band structure bends at the interface due to the difference in work functions between graphene and MoS2, creating a Schottky barrier (ΦB) and built-in potential (biV) on the semiconductor side. For an applied external bias, the Fermi level of graphene shifted downward during the application of a reverse bias owing to the decrease in negative charges moving toward the graphene, whereas the Fermi level of the n-type MoS2 shifted upward, as illustrated in 2 Fermi level relieved the band bending, leading to a decrease in the built-in potential. Conversely, when a forward bias was applied to the graphene/MoS2 Schottky junction, the Fermi level of graphene shifted upward, whereas that of the MoS2 shifted downward. This occurred because more positive charges were localized in the MoS2 and more negative charges were transported to the graphene, as depicted in 2 and graphene easily overcame the lowered built-in potential and transferred to the counter materials beyond the junction. However, in the case of a forward bias, the barrier at the junction was high; therefore, the charges were greatly restricted when moving toward each other. Therefore, owing to the aforementioned charge-transfer dynamics, the graphene/MoS2 junction, which was MoS2 directly synthesized on graphene using a laser-assisted methodology, exhibited rectification characteristics that depended on the applied forward-to-reverse bias. However, when MoS2 was grown via the thermal CVD process on the graphene, the underlying graphene experienced pronounced thermal degradation. These harsh synthesis conditions led to a substantial decline in conductivity, or in certain instances, a failure to establish a Schottky barrier, thereby inhibiting any rectifying behavior, as shown in The electrical-junction properties of the fabricated graphene/MoSiformity ,48. In aiformity  and lase the SAM . Moreoves than 2 , which i 22.2 V) ,52,53. B 22.2 V) :ΔE=ℏvFπneristics . The MoS follows : Φ = hν 2-based Schottky junction was characterized in terms of its optical responsiveness to external light by periodically turning the light on and off. To this end, the optoelectronic device employs the configuration mentioned in 2 patterns spans an area of 100 µm × 100 µm. Detailed specifications of the device dimensions are described in the 2. To clarify, the optoelectronic device we have constructed incorporates two Schottky junctions encompassing both the graphene/MoS2 junction and MoS2/metal electrode junction. We assessed the photocurrent induced in each region utilizing a focused monochromatic laser beam , testing two distinct bias voltages under forward and reverse biases. A larger photocurrent is observed in the case of forward bias. This can be considered to be due to the effect of the internal barrier depending on the direction of the applied bias as mentioned in The developed graphene/MoSrefer to . The restogating . The difτrise) and decay time (τdecay) of the charge carrier, which represent the duration of charge transfer and production of the maximum output current (ranging between 10 and 90% intensity), respectively, we extracted the following values: τrise = 10.3 s (τdecay = 8.49 s) at −0.7 V, 10.96 s (8.9 s) at −0.2 V, 4.6 s (7.26 s) at 0.2 V, and 6.41 s (7.74 s) at 0.7V [2-based junction devices using the CVD method and evaluated their characteristics. Upon analyzing the photoresponse under the same illumination conditions, it was found that the device fabricated using CVD synthesis exhibited negligible photocurrent generation. A detailed examination of the individual components revealed that the graphene had almost entirely lost its conductivity. These findings suggest that the graphene underwent significant damage during the MoS2 synthesis process (process temperature > 700 °C), which accounts for the absence of the rectifying effect as presented in 2-based Schottky diode was highly responsive to the incident pulsed light, with excellent stability and reproducibility over multiple cycles.When considering the rise time ( at 0.7V ,57. From2 on graphene by employing laser-based photothermal treatment, which enabled the direct formation of graphene/MoS2 heterostructures. Notably, this innovative technique offered a means of designing heterostructures from thermally sensitive materials by confining the heat treatment to the intended layer, effectively addressing the challenges that existing T-CVD methodologies cannot overcome. Central to our study is the understanding that the choice of the laser source is crucial. It must efficiently induce the thermal decomposition of the precursor without compromising the structural integrity of the synthesized MoS2 and graphene. This balance was achieved by harnessing the differential light-absorption rates of the ATM precursor and MoS2. Furthermore, we provided solutions for mitigating the surface irregularities introduced by graphene, thereby paving the way for achieving a uniform coating. The introduction of an HMDS buffer layer was a pivotal step that safeguarded the electrical characteristics of graphene throughout the fabrication sequence. Comprehensive evaluation of the electrical properties of the synthesized graphene/MoS2 heterostructures yielded encouraging results. Although this study did not specifically address contact resistance issues, it expects that the optimization of contact resistance between the two-dimensional semiconductor and the metal electrode could significantly enhance the operational efficiency of the photoelectric device [In this study, we introduced a facile approach for the selective synthesis of MoSc device ,61,62,63


The very serious problem of temperature and humidity regulation, especially for small and medium-sized museums, galleries, and private collections, can be mitigated by the introduction of novel materials that are easily applicable and of low cost. Within this study, archive boxes with innovative technology are proposed as “smart” boxes that can be used for storage and transportation, in combination with a nanocomposite material consisting of polyvinyl alcohol (PVA) and graphene oxide (GO). The synthesis and characterization of the PVA/GO structure with SEM, Raman, AFM, XRD, Optical Microscopy, and profilometry are fully discussed. It is shown that the composite material can be integrated into the archive box either as a stand-alone film or attached onto fitting carriers, for example, those made of corrugated board. By applying the PVA/GO membrane this way, even with strong daily temperature fluctuations of ΔT = ±24.1 °C, strong external humidity fluctuations can be reduced by −87% inside the box. Furthermore, these humidity regulators were examined as Volatile Organic Compounds (VOCs) adsorbers since gas pollutants like formic acid, formaldehyde, acetic acid, and acetaldehyde are known to exist in museums and induce damages in the displayed or stored items. High rates of VOC adsorption have been measured, with the highest ones corresponding to formic acid (521% weight increase) and formaldehyde (223% weight increase). Tackling the degradation of cultural heritage requires a global effort. This is why the development of new nanomaterials and methods for the preservation of artwork is of high significance. Nano-science can serve conservation excellently because, in contrast to conventional materials like polymers applied in conservation today, engineered nanomaterials do not affect the chemical and physical properties of the artworks and have a low environmental footprint. Here, the aim of the work is the combination of paper conservation with advanced materials science and nanotechnology in order to provide solutions to temperature and humidity exposure, which are known problems encountered in small and medium-sized museums and galleries that contribute to the degradation of works of art either on display or in storage. The proposed solution entails the usage of “smart” packaging boxes filled with a composite material consisting of graphene oxide and polyvinyl alcohol. The composite material can be exploited either as a stand-alone film or as a coating applied to the corrugated board in the “smart” box. A systematic characterization of the as-obtained composite film by means of Scanning Electron Microscopy, Raman spectroscopy, Atomic Force Microscopy, Optical Microscopy and profilometry is presented here. The proposed material was tested inside a climatic chamber and found to be an efficient temperature and humidity regulator, and furthermore was found to have the ability to capture some of the most-known volatile organic compounds, like formic acid and formaldehyde, detected inside such (micro)-environments .Important paintings, historic library collections, zoological preparations, certificates written on parchment, paper documents, or contemporary art—cultural assets are as different in their natures and meanings as they are in their chemical structures. Since the vast majority of such objects are not on display in controlled environments but in storage, a strong focus is placed on the influence of the storage climate on the materials’ chemistry in order to slow down the aging processes. As a result, maintaining adequate climatic conditions in warehouses or storerooms in order to conserve such movable cultural heritage (CH) objects is an important issue. The degradation of CH artifacts can seriously increase due to exposure to unstable temperatures, relative humidity (RH), light, and environmental pollutants . As an eOne remedial action would be to install air conditioning, which is most reasonable but usually not affordable for small and medium-sized institutions. A plausible alternative to that is to create favorable conditions in each storage package through integration of active materials or modules. However, in addition to their obvious functions, these climate regulators must also meet some important requirements, some of which are that they should be non-toxic, thin/demand little space, easy to implement, easy to use, and cheap.2, silica gel, or diatomaceous earth, which lead to the drying of the surrounding atmosphere until the desiccant’s saturation [2 and SiO2 are primarily drying and will not return moisture to the environment when needed. Further, alkaline earth metal chlorides are known to release highly problematic HCl [In general, temperature control might be challenging because it requires thick insulation panels like those already applied in the building sector. The implementation of such panels into enclosures would lead to an occupation of too much space inside the packages, thus expanding their dimensions and multiplying the required storage space. However, fluctuations in RH, which are also caused by changes in temperature, are easier to control, which demands fewer active materials. Known compositions for that purpose include CaClturation . Howeveratic HCl .3 [Graphene oxide (GO), a graphene derivative, is an abundantly available, low-cost carbon compound which is composed of a cluster of reactive oxygen functional groups . GO can 3 . On the 3 . PVA als3 .There are several reports in the literature on composite materials combining the properties of GO and PVA, especially for monitoring relative humidity and environmental pollutants. It has been reported that graphene flakes can change their crystal structure and be rotated when are efficiently impregnated in polymers , and, alThis paper presents a solution to the aforementioned problem via implementation of PVA and GO modules into archive enclosures as a promising application.2SO4, 96%) and 10 g of natural graphite flakes were added. The reaction mixture was heated to 80 °C when 5 g of potassium persulfate (K2S2O8) and 5 g of phosphorus pentoxide (P2O5) were added. After stirring for 1 h at this temperature, the reaction mixture was left for 5 h at room temperature. At the end of this process, a dark blue solution was obtained. The reaction was terminated by carefully adding deionized water (DW), followed by vacuum filtration and washing with DW until the pH reached the pH of the DW. The pre-oxidized graphite was dried overnight under ambient conditions. This material was then subjected to oxidation via the modified Hummers method where the powder was added to 220 mL of H2SO4 (96%) under continuous stirring. Slowly, 26.7 g of potassium permanganate (KMnO4) was added to the mixture, keeping the reaction temperature below 20 °C. Thereafter, the mixture was heated at 40°C for 2 h, followed by the careful addition of 450 mL of DW. After 15 min, the reaction was terminated by adding 1.35 L of DW and 22 mL of hydrogen peroxide . The bright yellow mixture was filtered and washed with a 10% w/w HCl solution in water to remove most of the metal ions. The solid product of this process was redispersed in DW and subjected to dialysis until the pH became the same as the pH of the DW. Finally, a mixture of single and few layers of GO was collected [−1 corresponds to the vibrations along the C=O bonds of the epoxy groups, while the peak at ~1625 cm−1 corresponds to the asymmetric vibrations of the C=C bonds of the graphene lattice. The bending of the O–H bonds of the hydroxyl groups of the oxidized lattice appears at ~1380 cm−1 and the vibrations along the C–O bonds appear at ~1080 cm−1 [Graphene Oxide (GO) was synthesized from natural graphite flakes  via a two-step oxidation process, based on a modified Hummer’s method ,25. Thisollected ,25 afterollected ,24,25. O080 cm−1 . The XPS080 cm−1 . The relw/w in deionized water) and GO . Initially, to dissolve the PVA in water, the solution was stirred in an oil bath at 90 °C for 4–5 h until a homogeneous polymeric matrix was formed  films, which were examined as relative humidity regulators, were synthesized from a mixture of aqueous solutions of PVA  evaluated from the spectra of the bare substrates.SEM photos were taken using a LEO SUPRA 35 VP, while Raman spectra of the specimens were recorded using an InVia Reflex  MicroRaman equipment using a 514 nm laser excitation. In all experiments, spectra were recorded at several points on each specimen using a Renishaw InVia Raman Spectrometer with a 1200 groove/mm−1 nominal spring constant of the cantilever. X-ray diffraction measurements were performed with the assistance of a Bruker D8 Advance model diffractometer, while for Optical Microscopy, the inspection of the specimen was performed by using a ΝΙΚOΝ Eclipse L150 optical microscope. The roughness of the different substrates used in this study was measured with a profilometer (Bruker DektakXT 2D). The duration of the scan varies from 10 to 150 s and its length from 100 to 1000 μm while the force exerted by the stylus on the substrate is adjusted depending on how soft the sample is. Therefore, the lowest tip forces were applied on the examined substrates, which were fixed to the instrument base by using a minimum quantity of adhesive tape applied at the edge of the specimen. Each material was examined three times to derive an average roughness value. Surface roughness is expressed in terms of Rq (nm), that is the root mean square deviation of the roughness profile [Atomic Force Microscopy (AFM) images were collected in peak-force tapping mode , by using ScanAsyst-Air probes  with a 0.4 N m profile ,31:(1)Rq−9 mbar. An unmonochromatized MgK line at 1253.6 eV and an analyzer pass energy of 36 eV, giving a full width at half maximum (FWHM) of 0.9 eV for the Au 4f7/2 peak, were used in all XPS measurements. The XPS core level spectra were analyzed using a fitting routine, which can decompose each spectrum into individual mixed Gaussian–Lorentzian peaks after a Shirley background subtraction. The sample was mounted onto a Si substrate with dimensions of 1.5 × 1.5 cm2. Finally, the capacitance and impedance of the sensors were recorded by a computer-controlled LCR . Finally, an instrument for measuring the attenuated total reflectance  was used for collecting the analogous spectrum for GO.The XPS measurements were carried out in an ultra-high vacuum system (UHV), which consists of a fast entry specimen assembly, a sample preparation, and an analysis chamber equipped with a dual anode (Al/Mg) X-ray gun and an LH10 electron analyzer. The base pressure in both chambers was 1 × 102O), acetic acid (99 wt.% in H2O), formic acid (85 wt.% in H2O), or acetaldehyde (99.5 wt.% in H2O), was used as the pollutant source each time. For the humidity adsorption tests and creating particular relative humidity conditions, a saturated aqueous solution of specific salt (sodium chloride: 75%RH) and distilled water (100%RH) were used and placed in sealed containers at a stable temperature [The gas adsorption tests were conducted in static conditions in a closed glass desiccator with an excess of pollutant, using a saturated vapor stream at room temperature, as described elsewhere ,14. A toperature . All samOnce the composites reached the saturation point in the volatile gas adsorption, which corresponds to the maximum weight change, the weight of the specimens stabilized. In order to establish a baseline for the specimens’ saturation, after two consequent gravimetric measurements similar to the maximum observed value, the films were considered saturated.3 was purchased from Donau Carbon, while spherical silica gel with a particle size of 40–75 μm was purchased from Merck. These materials were dried and placed inside the desiccators with the same VOC sources. The masses of the absorbents were measured periodically until they reached saturation, and their maximum calculated adsorption capacities are presented in Some of the most common commercial absorbent materials, activated carbon and silica gel, were used for a benchmark study. Activated carbon with an apparent density of 498 kg/mThe specimens for the needs of the current study were prepared following the experimental route that has been already described. In −1 assigned to the D band and G band were observed. The D band indicates the disorder in the structure of GO while the G band is related to the ordered sp2-bonded carbon [−1, respectively. The neat PVA has no characteristic Raman peak for the range of 1000–2000 cm−1, as was already reported [From d carbon . It can reported ,38. Finareported , and a rreported .PVA/GO plate”) or deposited onto a carrier (“PVA/GO board”)—were examined with regard to their suitability for integration in archive boxes. Afterwards, their mode of humidity regulation was studied in an artificial environment that represents very drastic but realistic conditions at the same time.In this section, the above-described PVA/GO films—either as stand-alone films  can be individually plate-shaped and attached to the inside walls of the box. When applied in this way, a protection between the absorbent and object must be installed to prevent direct contact. For that reason, a protective cover is introduced with enough vent openings for air circulation. The so-created compartments between the protective cover and box wall give tight and stable supports for a plate-shaped regulator module. ions see .The lid has only a slightly larger basal plane than the base, resulting in a very tight closure. In the bottom and lid, planar inlays made of thicker corrugated board prevent the inwardly folded flaps from opening, which gives stability without using notches for fastening. This shall prevent an additional convective flow of volatiles into the box to avoid the intrusion of pollutants and a too-early absorbent deactivation. As Obj = 2450 g and an overall volume of VObj = 21 cm × 14.9 cm × 10.4 cm = 3254.2 cm3. Afterwards, the boxes were closed and subjected to artificial cyclical (daily) T and RH changes in a climate chamber. The exterior (climate chamber) and interior (inside the boxes) values for T and RH were monitored. It should be noted that the applied values for temperature and relative humidity are in a range that represents very harsh conditions which are not usually found in museums and archives. However, these external conditions led to the most evaluable results, as the differences in interior relative humidity between the boxes became clearer this way.At first, an empty and paper-filled P2 box and a paper-filled P2C box were stored inside the climate chamber, leading to a T/RH profile as presented in RH inside the boxes from T, which is evidence for an absorption of thermal energy by the paper. This reduced ΔT of 23.3 °C is causal for the remaining ±9.6 %RH inside P2C, since the coating on the corrugated board shields the box’s interior volume from inflowing RH by blocking the diffusion-open surfaces. This is the minimum condition that must be met for regulators to function inside archive boxes, as the following experiment describes.As it becomes clear at the differences in RH span values ΔP2 boxes, little effect could be seen on the RH scale when compared to the case “without regulator” (P2 + CB), see When humidity regulators were added to the paper-filled, uncoated P2C boxes . By applying one PVA/GO board, this value decreased to ±8.3 %RH. However, the current strongest additional fluctuation reduction was measured by applying the PVA/GO membrane as a coating on corrugated board and as a pure plate at the same time (ΔRH = ±6.4 %RH). This means that in this paper-filled active archive box, even with strong daily temperature fluctuations of ΔT = ±24.1 °C, strong external humidity fluctuations can be reduced by −87%. By applying the regulators PVA/GO board + plate, a further reduction of −33% in RH span was recorded when compared to the (inactive) reference CB without the used film swelling as a consequence of the humidity exposure. Additionally, neither film nor film-coated carriers showed any irregularities in structure uniformity or adhesion. This was determined after carrying out several experiments presented in the current study and after storage in a warehouse for one year. Additionally, the films seem quite resistant towards scratching, even though there are some signs of use. With a total 18.47 g of active material, a very small amount of material is necessary to achieve a noticeable effect under very harsh environmental conditions.In general, the insertion of regulator modules led to a further decrease of RH fluctuation Δ2O), acetic acid (CH3COOH), formic acid (CH2O2), and acetaldehyde (C2H4O), which are the most common pollutants in facilities with artworks, as well as water vapor (relative humidity). These gaseous pollutants are emitted by paints, adhesives, furniture, building materials, etc. and have been proved to be significantly harmful after extended and continuous exposure to both humans and artworks [The fabricated composite films were tested for sorption of formaldehyde . Furthermore, both these molecules can form hydrogen bonds and π-π stacking with the conjugated π-network of GO. Therefore, the selectivity of formic acid over acetic acid by the examined films can be attributed to the electron geometry, since the formic acid has a planar configuration  resulting in lower adsorption resistance compared to acetic acid . It is known that the binding energy during the adsorption is primarily determined by the atom which is closer to the adsorbing site, so the smaller the distance between them, the higher the attraction. As for formaldehyde, it has the ability to form hydrogen bonds and contains a π bond, which contribute to its interaction with GO, yet because of its planar configuration, it exhibits lower polarity than the previously mentioned molecules. It is worth mentioning that the as-prepared GO/PVA film demonstrates higher adsorption for 75% and 100% RH formic acid and formaldehyde when compared to the commercial absorbers, while activated carbon and silica gel are superior for the cases of acetic acid and acetaldehyde. Regarding the comparison of the GO/PVA film to other pure GO or pure PLA films for VOC adsorption, it is denoted that for the first category, the synthesized material has comparative [It is important to note here that when developing absorbents for polar pollutants, in this work, all the examined gases and water are polar molecules, especially if selectivity for certain absorbates is needed. In effect, surface functionality becomes more important than pore volume or pore structure. GO interacts with polar VOCs while graphene prefers hydrophobic VOCs. Hence, the proper balance between hydrophobicity, surface area, and functionality should be considered in order to design an efficient adsorbent for VOCs. Except for the polarity of the examined VOCs, the presence of π bonds and hydrogen bonds in their structure, as well as their electron geometry, play a significant role in selective adsorption . It is eparative  or even parative , even thparative , pure PLIt is also worth mentioning that the GO/PVA films can return to their initial dried state after reaching the maximum adsorbed material capacity. It has been observed that after placing the films inside a fume hood for 24 h with moderate ventilation, the weight of the samples reverts to the original value prior to exposure to VOCs and the relative humidity. This observation paves the way for the examination of the reusability of these materials in future.These results show the multi-functional characteristics of these composite materials, paving the way for their immediate exploitation in cultural heritage protection.In conclusion, a “smart” storage enclosure was presented and proved to function as a self-humidity-regulating archive box when equipped with graphene oxide (GO)/polyvinyl alcohol (PVA) composite films. The easy-to-apply and low-cost nanocomposite membrane makes the shown application very attractive for small and medium-sized museums, libraries, or archives. GO is well-dispersed in the PVA matrix and the resultant composite material can reduce the external humidity fluctuations by −87%, even with strong daily temperature fluctuations of ΔT = ±24.1 °C. Additionally, the composite was also found to be a good adsorber of VOCs with especially high rates for formic acid and formaldehyde, the latter being a major concern regarding CH degradation ,44. Apar