Differences in immune characteristics, including immune gene expression by peripheral blood mononuclear cells (PBMCs), correlating with herpes labialis and good or poor immune control of herpes simplex virus type 1 (HSV‐1), and how these characteristics change after dosing with squaric acid dibutyl ester (SADBE), were investigated.Candida) and immune gene expression in the presence of HSV‐1 and Candida. On day 1 after blood collection the subjects with frequent outbreaks were dosed topically on the arm once with SADBE, and their PBMCs were collected and tested 8 weeks later.PBMCs were collected from persons positive for IgG against HSV‐1 and having frequent, infrequent, or no herpes labialis outbreaks. The PBMCs were tested for proliferation against HSV‐1 and a fungal antigen (Candida considered together (P < 0.01). (2) Higher expression of IFNG and five other immune‐related genes (P < 0.05 for each) and lower expression of IL5 and two other immune‐related genes (P < 0.05 for each) in PBMCs in vitro stimulated with HSV‐1 virus. The subjects with frequent outbreaks were treated once with SADBE, and 56 days later the PBMCs of these subjects differed from PBMCs from the same subjects taken on day 1 before treatment in exactly the same ways listed above as differences between those with good and poor immune control of HSV‐1, and at the same levels of significance.Those with good immune control of their HSV‐1 infection (fewer outbreaks) differ from those with poorer immune control in these ways: (1) Greater PBMC proliferation in vitro to HSV‐1, HSV‐1‐infected cell extracts, and Higher IFNG and lower IL5 expression by PBMCs in the presence of HSV‐1 correlate with fewer herpes labialis outbreaks, and a single topical dose of SADBE to the arm of subjects with frequent herpes labialis episodes improves immune response to HSV‐1. The virus is later activated from latency by events including fever, stress, cold or flu infection, immunosuppression, and sunlight. Upon activation, the virus migrates down sensory neurons to epithelial cells, typically on the vermillion border of the lip, and causes cell lysis and outbreaks as epidermal lesions there.P = .009).In a previous placebo‐controlled clinical trial, topical application of a 2% solution of squaric acid dibutyl ester (SADBE) in dimethylsulfoxide (DMSO), applied once to the arm of persons who self‐reported 6 or more herpes labialis outbreaks over the previous 12 months, was found to significantly delay the next outbreak. The median time to the next outbreak for the placebo group was 40 days vs more than 122 days for the 2.0% SADBE group, which difference was highly significant P = .00.9 SADBE In this study, we first sought to determine differences in immune function in general, and in immune response to HSV‐1 in particular, between persons who are infected with HSV‐1 and have frequent herpes labialis episodes  as compared to persons who are infected with HSV‐1 and have infrequent herpes labialis episodes  or infected with HSV‐1 and have no herpes labialis episodes . Second, we also sought to determine the effects on the immune system in general and on immune response to HSV‐1 virus in particular of a single topical application of 2% SADBE in DMSO to the arm.The outcomes measured were:Blood cell counts, including lymphocytes, T cells, B cells, CD4, CD8, and natural killer cells.Serum anti‐HSV‐1 IgG quantitative levels.Plasma cytokine levels.Blood levels:HSV‐1‐infected cell extracts (heat inactivated)HSV‐1 virus, cell‐free (heat inactivated)Candida albicans extract .Peripheral blood mononuclear cell (PBMC) proliferation in vitro in response to:Medium only negative controlHSV‐1‐infected cell extracts (heat inactivated)HSV‐1 virus, cell‐free (heat inactivated)Candida albicans extractCytokine and immune‐function gene expression of PBMC in vitro in response to:These assays were performed on all subjects in each of the groups on day 1. The subjects in group A, the frequent cold sore sufferers, were then treated with a single dose of SADBE topically on the arm after their blood collection on day 1. They returned for blood collections on days 15 and 57 to measure the same outcomes.2ClinicalTrials.gov under registration no. NCT03661541.This study was a clinical trial titled: “A Phase 1 Study of the Immune Response to Herpes Simplex Virus Type 1 (HSV‐1) and General Immune Health in Subjects Infected with HSV‐1” conducted at Prism Clinical Research, St. Paul, MN, USA, in accordance with the principles of the Declaration of Helsinki. The study protocol, the investigator's brochure, and other trial‐related information were approved by an independent Institutional Review Board. The study protocol was reviewed, approved, and registered at 2.1Subjects ages 18–64 who were positive for anti‐HSV‐1 IgG were recruited in three groups of 12 subjects each. The groups were approximately age matched and gender composition matched. The groups self‐reported different numbers of herpes labialis episodes over the prior 12 months: (A) 6 or more herpes labialis outbreaks over the previous 12 months, (B) 1 or 2 herpes labialis outbreaks over the previous 12 months, and (C) zero herpes labialis outbreaks over the previous 12 months. Group A was recruited first, and groups B and C were then recruited to be approximately sex and age matched to group A.2.2After a screening visit at which blood was drawn to test for IgG antibody against HSV‐1 and subjects were interviewed for inclusion/exclusion criteria, selected subjects returned for blood draws on day 1. After the blood draw on day 1, subjects in group A only were dosed with SADBE. A petrolatum donut was applied with a cotton swab to form about a 1 cm diameter donut on skin on the inner aspect of the upper arm. Then a separate cotton swab was dipped in a 2% SADBE solution (w/v) in DMSO, and the swab was then used to apply about 10–20 mg of solution over about a 1 cm diameter circle within the petrolatum donut. Immediately after application, the application site was covered with TEGADERM. Subjects were advised to remove the TEGADERM and rinse and wipe the spot 3 h later. The group A subjects then returned for blood draws on days 15 and 57 and were queried about adverse events on those dates.At this study visit where blood was drawn it was tested for blood cell counts, various cytokine levels, and anti‐HSV‐1 IgG quantitative levels.Anti‐HSV‐1 IgG was quantitated with Focus Diagnostics HerpeSelect 1 ELISA IgG assay. Cell counts were obtained by flow cytometry with Miltenyi Biotec 7‐Color Immunophenotyping Kit Human. Plasma cytokines were quantitated with the Invitrogen ProcartaPlex Custom Multiplex Immunoassay using magnetic microsphere technology.Blood was also collected for isolation of peripheral blood mononuclear cells (PBMC) and the PBMC were subsequently isolated the same day and plated the same day for proliferation assays and gene expression assays as described below.Negative control in medium .Medium plus 16 μg/mL protein from heat‐inactivated HSV‐1‐infected Vero cell extracts.Medium plus heat‐inactivated HSV‐1 cell‐free virus at a final 7.5 million pfu/mL.Candida cell extract. .Medium plus Positive control, medium plus 10 μg/mL concanavalin A.PBMC were isolated using SepMate PBMC isolation tubes  and Ficoll‐Paque according to the manufacturer's instructions. PBMC were isolated and suspended in negative control medium at 2 million cells/mL. PBMC suspension (100 μL) was added to 100 μL of medium in quadruplicate in 96‐well plates that had been prepared in advance and stored frozen at −70°C and thawed immediately before cell addition. After cell addition to the plates, the plates were immediately incubated at 37°C. The wells were in these final concentrations after addition of the 100 μL of PBMC suspension in negative control medium:2 atmosphere and then RNA was isolated from the pooled contents of the four wells of a given condition for a specific patient, and the RNA was stored at −70°C and subsequently used for quantitative real time PCR analysis of gene expression of 41 immune‐related genes.Two plates were created for each subject at each collection point. One plate was incubated for 2 days at 37°C in a humidified 5% COA second plate was incubated for 4 days. On the 4th day 1 μCi of tritiated thymidine in 50 μL of fresh negative control medium was added to each well. After 22 h incubation the cells were harvested onto a filter mat and each well counted by scintillation counting for tritium incorporation into DNA as a quantitative measure of cell division.2.3HSV‐1‐infected cell extracts were made by plating 10 million Vero cells in each of three T‐75 flasks on day 0, withdrawing all medium and adding cell‐free HSV‐1 virus (KOS strain) (ATCC strain VR‐1493) in 2 mL medium on day 1 (at a multiplicity of infection of about 3), incubating for 2 h to allow infection, and then adding 13 mL of medium. Then on day 2 cells were scraped off with cell scrapers, pelleted by low‐speed centrifugation, washed twice in PBS, resuspended in 2 mL water total for all three flasks pooled in one snap‐cap tube, and sonicated in a bath type sonicator in an ice‐water bath for 4 min. The resultant cell extracts were stored at −70°C.2.4g for 10 min to pellet cells and cell debris. The supernatant was collected and centrifuged at 23 000g for 2 h to pellet virus. The pellet was resuspended in 1 mL of Vero medium for each T‐75 harvested, and then frozen at −70°C. Virus prepared this way was found to have about 2.4 × 109 pfu/mL.Cell‐free HSV‐1 virus was made by plating 10 million Vero cells per T‐75 flask on day 0. On day 1 medium was removed, and about 10 million virus was added in 2 mL of medium per flask for a multiplicity of infection of about 1. This was incubated with rocking every 2 min for 1–2 h to allow viral adsorption to the cells. On day 4, the flasks were shaken to detach cells, the contents were then withdrawn and centrifuged at 1400Both the HSV‐1‐infected cell extracts and the cell‐free HSV‐1 virus were heated at 70°C for 30 min before adding them to the PBMC plates to heat inactivate virus to prevent it from killing the PBMCs in the plates.2.52 humidified atmosphere. Then 50 μL of a 4:1 mixture of medium and Dojindo cell counting kit reagent was added to each well, and the absorbance at 450 nm read until the O.D. in negative control wells with no virus added reached 2.5. The greatest dilution with loss of more than 50% of cell viability in wells was considered to have 1 pfu added to the well.To determine the number of plaque forming units of HSV‐1 per mL in a preparation, the following assay was done. Vero cells were plated at 5000 cells per well in a 96‐well plate on day 0. On day 1 the HSV‐1 preparation was diluted in a series of 2.5‐fold dilutions from a 10 000‐fold dilution. Forty μL of each of the dilutions was added to wells in triplicate, and the plate incubated 6 more days at 37°C in a 5% CO2.62.6.12 incubator in a 96‐well plate, four wells of a single condition  with cells from a single subject (200 μL in each well) were pooled, and the RNA was immediately isolated with Trizol reagent and chloroform by Qiagen RNeasy MiniElute clean up kit Qiagen cat #74204, according to manufacturer's instructions into a final volume of 13 μL, and then flash frozen on powdered dry ice, and stored at −70°C in aliquots of 3 μL for RNA quantification and 10 μL for PCR analysis.As noted above, after 2 days of growth of the PBMC in a 37°C humidified 5% CORNA was preamplified with the Qiagen RT2 PreAMP cDNA synthesis kit (Ref. 330451) and custom RT2 PreAmp primer mix (Ref. 330141) starting with about 50 ng RNA according to the procedure of RT preamp cDNA synthesis handbook date 03/2011, pages 17‐20. The amplification reactions  were frozen at −70°C and used for qPCR analysis the next day.2.6.2qPCR was done with the Qiagen RT PCR profiler gene analysis kit  using RT2 Sybr Green Rox qPCR Mastermix (24) (Part 330523) and Custom RT2 PCR array plates (Ref. 330171). For this 675 μL 2× RT2 SYBR green mastermix, 51 μL pre‐AMP synthesis reaction, and 623 μL RNase free water were mixed, and then 25 μL of the mixture was added to 48 wells on the left or right half of the custom PCR plate. The PCR instrument was Applied Biosystems 7900HT fast real time PCR. The program was 95°C for 10 min; then 40 cycles of 95°C for 15 s, 60°C for 1:00; then stage 3 denaturation stage of 95°C for 15 s, 60°C for 15 seconds, and 95°C for 15 s. The detector and reporter dye was Sybr, passive reference was Rox. For data analysis a manual Cq of 0.5 was used for all runs.Control genes were ACTB, RPLP0, GAPDH. There were 41 test genes involved in immune function, and three wells for reverse transcriptase control, genomic DNA control, and positive PCR control. A fixed threshold level of 0.5 was used for all plates, and the threshold cycle (Ct) was calculated for each well as the cycle when the signal crossed the threshold.‐ average ΔCt.‐ΔΔCt.‐ average ΔΔCt.ΔCt for each test gene was Ct of the test gene minus average Ct of the three housekeeping genes in the same well. Statistical significance of differences in absolute expression levels was calculated based on ΔCt.2.7P values for comparisons between group A and groups B or C were calculated using unpaired 2‐tailed Student's t‐test, and between group A on day 1 versus days 15 or 57 using the paired 2‐tailed Student's t‐test.33.1The summary statistics of the subjects are in Table 3.2The study drug was well tolerated. Of the 12 subjects treated, two experienced stinging at the application site, and one had nausea that was ruled possibly related to the study drug. No rashes or delayed‐type hypersensitivity reactions were reported, which is sometimes seen with SADBE.3.3Candida—or the positive control stimulus concanavalin A; and the negative control is the medium‐only unstimulated condition.The relative proliferation for each test condition was calculated as (avg. CPM test condition/avg. CPM negative control). The average CPM is the average tritium counts per minute of the four wells in a given test condition for a given collection point of a given patient. Table Candida, and Concanavalin A. In group A, after treatment on day 1, proliferation in the three test conditions and in Con A positive control was basically unchanged on day 15 but was substantially increased on day 57 in all three test conditions and in Con A positive control. The difference between A1 and A57 by paired t‐test was significant for Candida and almost significant for HSV‐1. A paired t‐test was done between the day 57 and day 1 combined relative proliferation values in all of the three test antigens , and the P‐value was 0.0109 . However, to perform a similar comparison between groups C and A1, the relative proliferation values in the three test antigens needed to be normalized because different stimuli were used and the average proliferation was different in the different stimuli. So the relative proliferation for each individual in each stimulus was normalized to a percentage of the average relative proliferation in group C. An unpaired t‐test of normalized relative proliferations in the three specific stimuli in group C versus group A1 (n = 36) give P = 0.0039. When the paired t‐test is done with the normalized relative proliferation of group A57 versus A1, P = 0.0047. So proliferation in the three test antigens taken together is higher in group C than A1 and in group A day 57 than A day 1, and in both cases the difference is highly significant (P < 0.01). (Table On day 1 group C (zero outbreaks) had substantially higher proliferation in all three test conditions and in the concanavalin A positive control. The differences vs group A1 (group A day 1) were almost significant (between 0.05 and 0.10) for HSV‐1, We wondered whether the increased proliferation that we saw in HSV‐1‐infected Vero cell extracts was due to the HSV‐1 proteins or the Vero cell proteins, so we tested proliferation of PBMCs from two donors in group C with uninfected Vero cell extract as well. In those two patients the average cpm in medium‐alone negative control, uninfected Vero cell extracts, and HSV‐1‐infected Vero cell extracts were respectively 300, 526, and 6213 for the first patient and 456, 438, and 12652 in the second patient. So for both patients the proliferation with uninfected Vero cell extracts was essentially the same as in medium‐alone negative control and was very much less than proliferation in HSV‐1‐infected cell extracts. From this, we can conclude that the proliferation we saw with HSV‐1‐infected Vero cell extract was due to stimulation of PBMCs by the HSV‐1 proteins, not by the Vero proteins.3.43.4.1Certain blood cell count parameters were measured in the study—specifically, absolute counts of natural killer cells, B cells, T cells, CD4 cells, CD8 cells, lymphocytes, and percentages and ratios of the above. Almost none of the measures were significantly different between the different groups or on day 15 or 57 versus day 1 in group A. The data where statistical significance was found and helper/cytotoxic ratio (CD4/CD8) where the result was not significant but a possible pattern of lower ratios in groups B and C versus A1 and in A15 and A57 versus A1 was found are shown in Table 3.4.2Plasma cytokine levels of the following cytokines were measured: IL‐gamma, IL‐1‐beta, IL‐10, IL12/IL23 p40, IL‐13, IL17A, IL‐4, IL‐5, IL‐6, IL‐8, tumor necrosis factor alpha, IL‐2, and IL‐2 receptor. For all but IL‐2 and IL‐2R the majority of subjects had undetectable levels. There were no significant differences between groups or on days 15 or 57 versus day 1 in group A. (Data not shown).3.4.3Anti‐HSV‐1 IgG was quantified in serum collected at each study visit. The levels are shown in Figure P = 0.16), which is a move by day 57 toward the characteristic of the groups with fewer herpes labialis outbreaks. Since the antibody level initially went up in group A from day 1 to day 15, the change from day 15 to day 57 in group A was a highly significant decrease in anti‐HSV1 IgG (P < 0.01).Group B had significantly lower anti‐HSV1 IgG antibody than group A at day 1 and groups B and C pooled together had a significantly lower level of anti‐HSV1 IgG than group A at day 1. Group C also had a lower level of anti‐HSV1 antibody than group A, although the difference was not statistically significant. And on day 57, group A's anti‐HSV1 IgG level was lower than it had been on day 0  or with HSV‐1‐infected cell extracts, heat‐inactivated HSV‐1 virus, or with Figure P < 0.05) and in particular all of the changes highlighted in either yellow or orange are statistically significant.Almost all the gene expression changes shown in Figure The two Th1 cytokines3.5.1Figures The three columns to the left of the gene symbol in Figures Figures Candida extract, and Figure Figure Candida extract they were CXCL10, FOXP3, SPP1, STAT1, and STAT4. The genes significantly less expressed in groups B+C than A in HSV‐1 infected cell extracts were DDX58, IL5, LYZ, and MX1; in HSV‐1 virions the genes less expressed in groups B+C than A were GATA3, IL16, and IL5; and in Candida extract they were GATA3 and IL16.IFNG was significantly more expressed in groups B+C combined than group A day 1 in all three stimuli. The other genes besides IFNG significantly more expressed in groups B+C than A in HSV1‐infected cell extracts were CCL3, CXCL5, DDX58, IL1B, IL8, SPP1, and TNF. In HSV‐1 virions they were FOXP3, IL12A, IL8, SPP1, and STAT4; and in every gene with a significant change in A57 versus A1 and in group B+C combined versus A1 the change was in the same direction.Many genes had significant changes in gene expression in group A on day 57 compared to day 1 in the three stimuli, and in essentially every case the same gene's expression was also changed in the same direction in groups B+C compared to A1. In fact, for In the unstimulated negative control condition Figure  the only3.5.2The ratios of day 15/day 1 and day 57/day 1 PBMC absolute gene expression in group A are shown in Figure 3.5.3Table 4The data here show a cellular immune response is more important than humoral and a Th1 immune response more important than Th2 in controlling HSV‐1 outbreaks as herpes labialis. And the data show that a single dose of SADBE changes the immune response of subjects with frequent herpes labialis over 8 weeks to make it more like, and at least in IFNG expression better than, the immune response of subjects with few or no herpes labialis outbreaks.4.1Candida fungal extract or HSV‐1 virus and Candida  is down‐regulated by all three stimuli. That makes sense because it is an antibacterial enzyme and the three stimuli are viral and fungal.Candida.MX1 is a gene involved in antiviral response,Candida. SPP1 is a cytokine that upregulates expression of IFNG and IL12. It is also a regulator of apoptosis.Candida extract.SPP1 is strongly upregulated by HSV‐1‐infected cell extracts, and to a lesser extent by virus, but down regulated by Candida and slightly down regulated by HSV‐1‐infected cell extracts. GATA3 induces differentiation of Th0 cells to the Th2 subtype and suppresses differentiation to the Th1 subtype.GATA3 is significantly down regulated by virus and IL16 is down‐regulated by all three stimuli.4.24.2.1P = 0.08 for HSV‐1 virus and P = 0.08 for Candida, n = 12), but the response to all three stimuli taken together and normalized was highly significantly greater in group C than group A .PBMC proliferation in response to all three stimuli showed the same trends of group C1>B1>A1.  it was closer to group A1). In other words, PBMC from those with better immune control of HSV‐1 proliferated more than those with worse immune control. The difference was not statistically significant for any one stimulus . The PBMC proliferative response to all three stimuli normalized and taken together was significantly greater on day 57 than day 1 in the group A subjects ). Thus, in regard to proliferation of PBMCs in vitro against HSV‐1‐infected cell extracts, HSV‐1 virus, and Candida extract, the group A subjects were more like the group C subjects with good immune control of HSV infection 56 days after SADBE treatment than they had been on day 1 before drug treatment.PBMC proliferation against all three test stimuli for the group A subjects was also greater on day 57 than on day 1 before drug treatment, significantly so for 4.2.2Groups C and B pooled together had significantly lower anti‐HSV‐1 IgG levels than group A. Among group A subjects, anti‐HSV‐1 IgG levels were lower on day 57 than day 1, although not significantly so, making the group A subjects again more like those with fewer outbreaks and better immune control of the virus on day 57 than they had been on day 1.4.2.3Expression in PBMCs upregulated (or downregulated) by HSV‐1 (or other stimulus).Same upregulated gene was more expressed in the presence of HSV‐1 (or other stimulus) in group C and B subjects  than group A subjects . .Same upregulated gene was more expressed in the presence of HSV‐1 (or other stimulus) in group A subjects on day 57 after treatment with SADBE than on day 1 before treatment. .For almost every immune‐related gene where any differences were significant the direction of these changes match:Thus in essentially every case, the SADBE treatment changed the group A subjects by day 57 to make them much more like the group B and C subjects who have better immune control of their HSV‐1 infection than the group A subjects were on day 1.every case, the change in expression is in the same direction: that is, group A on day 57 becomes more like groups B and C than it had been on day 1. And in nearly every case genes with higher expression in groups B+C than A1 and in A57 than A1 were upregulated in the corresponding stimulus and those with lower expression were downregulated by the stimulus. The lone clear exception is MX1, which was less expressed in HSV‐1‐infected cell extracts in groups B+C versus A1 and in A57 versus A1 but was markedly upregulated by the stimulus  was the gene (1) most consistently upregulated by a large amount in all three stimuli versus negative control; (2) most consistently increased by the greatest amount in its expression in groups B+C versus group A day 1 in all three stimuli, and (3) most consistently increased in its expression by the greatest amount in group A day 57 versus day 1 in all three stimuli. IL5 (a Th2 cytokine) (1) was not markedly upregulated by any of the three stimuli and (2) was less expressed in groups B+C than group A day 1 in both HSV‐1 virus and HSV‐1‐infected cell extracts and (3) was less expressed in group A on day 57 than day 1 in both HSV‐1 virus and HSV‐1‐infected cell extracts.These results are consistent with, and tend to confirm, the results of the prior clinical trial that showed a single topical application of 2% SADBE to the arm delayed next cold sore outbreak in persons with frequent cold sores.P‐value is less than 0.20 for comparisons of group A to both groups B and C, and if groups B and C are pooled and compared to group A the P‐value is 0.065, almost significant. This is consistent with prior evidence discussed below that CD8+ T cells are important and effective in controlling HSV recurrences.Others have previously shown that CD8+ T cells are important in controlling herpes labialis outbreaks.lower anti‐HSV‐1 IgG levels correlate with better control of HSV‐1 outbreaks, Spruance et al. also found lower serum anti‐HSV‐1 antibodies in HSV seropositive patients with a history of frequent herpes labialis than in seropositive persons with no history of herpes labialis.Consistent with our finding + T‐cells could prevent HSV‐1 reactivation from latency in excised trigeminal ganglia (TG), and that IFN‐gamma protein was produced by the CD8+ T cells, and that neutralization of IFN‐γ significantly enhanced the rate of HSV‐1 reactivation from latency in TG cultures. Spruance et alSeveral prior reports also found IFN‐gamma to be important in controlling HSV infection and reducing HSV outbreaks. Dobbs et alOur data are consistent with this in linking IFNG expression inversely with frequency of herpes labialis outbreaks. IFNG, the key Th1 cytokine, was at least nine‐fold overexpressed in groups B+C in the stimuli versus negative control, at least three‐fold more highly expressed in all three stimuli in groups B+C than group A on day 1, and at least eight‐fold more expressed in all three stimuli in group A on day 57 than day 1.Conversely, IL5, a Th2 cytokine, was not markedly upregulated by any of the three stimuli versus negative control, was significantly less expressed in groups B+C than group A on day 1 in both HSV‐infected cell extracts and heat‐killed HSV‐1 virus, and in the negative control condition, and was significantly less expressed in both HSV‐infected cell extracts and HSV1‐ virus and in the negative control in group A on day 57 than on day 1. IL‐5 stimulates B cells and antibody production,Thus, the two genes that appear to be most linked to immune prevention of HSV‐1 outbreaks but in opposite directions are IFNG (increased expression in both HSV stimuli) and IL5 (decreased expression in both HSV stimuli). This link of lower IL5 expression or protein level to reduced HSV‐1 recurrences has not previously been shown to our knowledge.SADBE appears to somehow reset the immune system 8 weeks after drug application to more of a type 1 cellular immune response and a larger cellular immune response to the virus—with greater PBMC proliferation against heat‐killed HSV‐1, and greater IFNG expression and lower IL5 expression in response to both the heat‐killed HSV‐1 virus and HSV‐1‐infected cell extracts. These same differences characterize HSV‐1‐seropositive subjects with few or no herpes labialis outbreaks as opposed to those with frequent outbreaks, so SADBE resets the immune response to the virus to more like that of persons with more effective immune control of the virus.Moreover, SADBE dosing did not just ameliorate the defect in group A immune response to HSV‐1, it completely reversed it in the key measure of IFNG expression in the presence of HSV‐1 virus: At day 1 group A had significantly lower IFNG expression than groups B+C, whereas at day 57 group A had significantly higher IFNG expression than groups B+C.Candida extract, even though it is unrelated to herpes—with greater PBMC proliferation to Candida extract and greater expression of upregulated immune genes in Candida extract, including IFNG, and lower expression of down‐regulated immune genes in Candida extract. And interestingly, SADBE application also improved the immune response to the Candida extract in the same ways in subjects with frequent herpes labialis outbreaks 8 weeks after SADBE application. This suggests SADBE application on the arm may also be effective to improve immune response to yeast infection anywhere in the body.Interestingly, those with fewer or no herpes labialis outbreaks also mounted a larger immune response to Stricker et al5P = 0.17, not significant), HSV‐1 virus , Candida extract , and the three stimuli normalized and taken together  in group C than group A.Greater PBMC proliferation in vitro to HSV‐1‐infected cell extracts .Lower levels of anti‐HSV‐1 IgG in serum  in one or more of the three stimuli in groups B+C than group A, all of which are upregulated by the same stimulus versus negative control medium; and significantly lower expression of LYZ, MX1, GATA3, and IL16 in one or more of the three stimuli in groups B+C than group A, all of which except MX1 are also downregulated by the same stimulus versus negative control medium.Those with good immune control of their HSV‐1 infection ; or 1 or 2 outbreaks per year (group B)) differ from those with poorer immune control  in these ways:Treatment of subjects with frequent outbreaks (group A) with a single topical dose of SADBE causes their serum and PBMCs to become more like those of the subjects with infrequent or zero outbreaks 56 days after the a single topical treatment with SADBE in every one of these ways than they had been on day 1 before the treatment, significantly so for each of the genes listed and highly significantly so for the normalized PBMC proliferation to the three stimuli taken together.ClinicalTrials.gov under registration no. NCT03661541. The patients gave a written informed consent for the study.The study protocol, the investigator's brochure, and other trial‐related information were approved by an independent Institutional Review Board—Salus IRB. The study protocol was reviewed, approved, and registered at


Mesocrystals are assemblies of smaller crystallites and have attracted attention because of their nonclassical crystallization pathway and emerging collective functionalities. Understanding the mesocrystal crystallization mechanism in chemical routes is essential for precise control of size and microstructure, which influence the function of mesocrystals. However, microstructure evolution from the nucleus stage through various crystallization pathways remains unclear. We propose a unified model on the basis of the observation of two crystallization pathways, with different ferric (oxyhydr)oxide polymorphs appearing as intermediates, producing microstructures of magnetite mesocrystal via different mechanisms. An understanding of the crystallization mechanism enables independent chemical control of the mesocrystal diameter and crystallite size, as manifested by a series of magnetic coercivity measurements. We successfully implement an experimental model system that exhibits a universal crystallite size effect on the magnetic coercivity of mesocrystals. These findings provide a general approach to controlling the microstructure through crystallization pathway selection, thus providing a strategy for controlling magnetic coercivity in magnetite systems. Transient metastable intermediates play an important role in the crystallization process. Here, the authors unveil the microstructural changes in magnetite mesocrystals that depend on the intermediate polymorphism and the universal crystallite size effect on the magnetic coercivity of mesocrystals. Of many magnetic materials, magnetite (Fe3O4), which is biocompatible and exhibits superparamagnetic–ferrimagnetic transition depending on its size, has been extensively studied as a diagnostic and therapeutic reagent, leading to advances in the biomedical field6. To date, shape, size, and composition are used to control magnetic coercivity for applications such as magnetic particle imaging (MPI), which require nonlinear magnetism or magnetic hyperthermia requiring hysteresis loss. However, remarkable advances currently appear unlikely5. The microstructure provides a breakthrough in the control of coercivity because crystallite size and inter-crystallite magnetic interaction are closely related to coercivity3. To verify this at the nanoscale, the magnetic mesocrystal  is a promising model system for exhibiting collective magnetic coercivity behavior influenced by individual crystallites9. Magnetic mesocrystals have been reported to exhibit magnetic properties dissimilar from those of single-crystalline particles, depending on the structural arrangement and the magnetic coupling between crystallites. However, we still do not understand the correlation between the microstructure of the mesocrystal and its magnetic properties due to the complexity of the phenomenon. Controlling the crystallite size of mesocrystals at the nanoscale would experimentally reveal this correlation, but chemically controlling the microstructure of mesocrystals synthesized in a bottom-up process still remains a challenge.Magnetic coercivity behavior is essential for demonstrating magnetism in fine particles and is also a crucial performance indicator for a wide range of electrical and biomedical applications3O4 mesocrystal and its crystallization pathway is an essential underpinning of this study. Understanding the crystallization mechanism in chemical routes is crucial for precise control of the microstructure of Fe3O4 mesocrystals, which affects magnetic functions. In general, the crystallization mechanism of materials is described by the classical nucleation and growth theory, through which the crystals are spontaneously nucleated in solution and are grown after reaching critical size. For several systems, crystalline phases are not formed directly from solution but by stepwise phase transformations preceded by metastable intermediates12. Formation of metastable intermediates advances with solubility, and are governed by the Ostwald step rule that the phases with a lower kinetic energy barrier are formed prior to the one with a higher barrier13. Recently, several mechanisms besides the classical nucleation and growth model have been observed during the transformation steps of intermediate or crystalline phases15. In particular, depending on the particle attachment model, mesocrystals comprising assemblies of smaller crystallites frequently exhibit a nonclassical pathway in which crystallization proceeds via attachment of nanometric building blocks (including prenucleation clusters or nanoparticles) and not atom accretion17.The correlation between the microstructure of the Fe3), calcium phosphate (Ca3(PO4)2), and calcium sulfate (CaSO4) systems20. The Fe3O4 phase is also commonly formed stepwise through a series of intermediates down to the lowest-energy rather than being formed directly from ionic precursors23. For Fe3O4, various types of ferric (oxyhydr)oxide polymorphs  and ferrihydrite have been observed as intermediates. Because ferric (oxyhydr)oxide polymorphs represent similar solubilities, the types of intermediates appearing before the Fe3O4 phase can be significantly varied based on experimental conditions such as pH, additive content, and Fe3+ and Fe2+ cation concentrations25. Furthermore, nonclassical crystallization mechanisms, which occur in the formation of ferric (oxyhydr)oxide intermediate or Fe3O4, can also affect the type of intermediate30. Baumgartner et al. observed the crystallization process via particle attachment in Fe3O4 formation and proposed that the energetic stability of primary crystallites can influence the presence of ferrihydrite intermediates28. These prior observations provide useful experimental and theoretically indications regarding the role of crystallization pathways. However, we still cannot fully explain the pathways via a universal mechanism, and the evolution of the microstructure during the reaction remains unclear.In these complex processes, the transient metastable intermediate state acts as a precursor to the post-nucleation stage in which the crystalline phase is formed. This intermediate state affects the type and structure of the final crystalline phase, as proposed primarily in calcium carbonate oxide polymorphs appear separately as intermediates on each pathway. We uncover that the microstructure of Fe3O4 mesocrystal is affected by which pathway governs the overall reaction and can be chemically controlled by polymorph selection of iron (oxyhydr)oxide. Based on this understanding, diameter and crystallite size of Fe3O4 mesocrystals are controlled independently. Finally, we report the successful experimental implementation of the crystallite size effect of Fe3O4 mesocrystal on the magnetic coercivity curves as a function of the diameter. Because Fe3O4 mesocrystals represent crystallographically ordered aggregate of spheres, we can discuss the collective coercivity behavior, which is difficult to observe in singular particle.In this study, we provide two crystallization pathways of Fe3O4 phase was formed from different ferric (oxyhydr)oxide polymorphs as intermediates, there would be differences in the microstructure of the Fe3O4 mesocrystal because of the different crystallization mechanisms. If possible, we could chemically control the microstructure by selecting an intermediate among the polymorphs, and eventually, change the crystallite size of the Fe3O4 mesocrystal. To verify our hypothesis, we observe the crystallization pathways of Fe3O4 mesocrystals consisting of different crystallite sizes synthesized using the modified polyol method. We identified two crystallization pathways, shown in the schematic illustration  (Pathway 1)lepidocrocite (γ–FeOOH) → magnetite (Fe3O4)(Pathway 2).lepidocrocite (γ–FeOOH) → goethite (α–FeOOH) → magnetite . In pathway 2, Fe3O4 does not form directly in the lepidocrocite but apparently forms via stepwise transformation from lepidocrocite and goethite, conforming with the Ostwald step rule . Broadly, the entire reaction proceeds concurrently through these classical and nonclassical pathways. However, each pathway produces Fe3O4 mesocrystal at different stages. In pathway 1, the Fe3O4 mesocrystal forms at an early stage in the reaction, while in pathway 2, it is nucleated at a later stage in the reaction. Further, the microstructure is affected by the pathway governing the entire reaction and can be chemically controlled. We confirmed that the size of the crystallites formed in pathway 1 is smaller than that of those formed in pathway 2.In pathway 1, Fe3O4 mesocrystals having the same ~60 nm diameter (D) but different crystallite sizes (C) of 23 and 43 nm (Sample 1 (S1): D = 62 ± 3 nm, C = 23 ± 3 nm; Sample 2 (S2): D = 61 ± 4 nm, C = 43 ± 3 nm) synthesized using Fe:NaOAC:H2O ratios of 2:15:150 and 1:3:200, respectively  results reveal that the cause of this difference in the initiation time of mesocrystal formation is due to the crystallization pathway rather than the reaction rate  before refluxing for 3 h, after which it grows in the tubular intermediate (goethite). Conversely, in S2, most of the nanocrystalline intermediates are transformed into tubular intermediates rather than Fe3O4 mesocrystals in the first 3 h of refluxing, and Fe3O4 mesocrystals began to grow primarily from the tubular intermediate. Both S1 and S2 show a similar crystallization process after 3 h refluxing. Fe3O4 mesocrystals in S1 grow primarily from a nanocrystalline intermediate before refluxing for 3 h (pathway 1), while that of S2 grows from a tubular intermediate after refluxing for 3 h (pathway 2). This indicates that S1 and S2 were grown through different crystallization pathways: S1 through pathway 1 and S2 through pathway 2.In S1, the Fe3O4 mesocrystal over time for S1 and S2. The growth kinetics of S1 shows two-step growth, while that of S2 shows one-step growth. The growth kinetics of each crystallization pathway is clearly distinguishable around 3 h. In S1, the Fe3O4 mesocrystals grew to 53 nm via pathway 1 and then grew an additional 9 nm via pathway 2. In S2, Fe3O4 mesocrystals did not form on pathway 1 but grew after 3 h via pathway 2. The formation of Fe3O4 mesocrystal via pathway 2 begins at similar times in both S1 and S2, which is illustrated by the green region of Fig. This phenomenon is well reflected in Fig. 3O4 [International Center for Diffraction Data (ICDD) no. 01-086-1344] and ferric (oxyhydr)oxide phases coexisted after 1.5 h of refluxing. The broad diffraction peaks are assigned primarily to poorly crystalline lepidocrocite (ICDD no. 00-044-1415). With increasing reflux time, the peaks of the Fe3O4 phase become prominent and sharpened. For S2, we identify the tubular intermediate phase observed primarily in pathway 2  patterns Fig. , we couly 2 Fig. . At 3.5 3O4 mesocrystals, comprising spherical primary crystallites in nanocrystalline lepidocrocite intermediates, appear after 1 h of refluxing. In region 1, the fast Fourier transform (FFT) pattern of the Fe3O4 mesocrystal appears as a spot pattern, indicating that the primary crystallites have slightly tilted and almost identical orientations. The spots closest to the center are indexed as the (220) family of planes of Fe3O4. This is the characteristic phenomenon of mesocrystal formation in which the building units are crystallographically aligned31. Figure 3O4 mesocrystal surface has the same crystallographic orientation as the core. The Fe3O4 mesocrystal and primary crystallites are attached along the direction of the (220) planes. In region 2, spherical intermediates appear and exhibit an unclear FFT pattern attributed to short-range order  after 4.5 h oxide intermediates to the Fe3O4 phase, we can gain more insight into the crystallization of Fe3O4 mesocrystals in S1 and S2 using the Johnson–Mehl–Avrami–Kolmogorov (JMAK) model , b is the dimensionality of the grown phase (0 < b < 3), and c is the growth rate (c = 0.5 or 1)35.As discussed in the preceding sections, two coexisting pathways are competitively responsible for the crystallization process of Fe3O4 mesocrystals originate. Neither S1 nor S2 contains any phase other than Fe3O4 after the reaction is complete. Thus, we regard f as the volume fraction grown at a particular time with respect to the final volume formed at the end of each pathway  and S2 (pathway 2) are similar, which implies that the mechanism of each crystallization pathway is accurately reflected in the classical JMAK model. In S1 (pathway 1), there is spherical growth (b = 3) of Fe3O4 mesocrystals via oriented attachment of primary crystallites. It is hypothesized that a diffusion-controlled process (c = 0.5) determines the growth rate when primary crystallites approach each other. Here, a is deduced to be 1.3–1.4, indicating the accelerated and autocatalytic nucleation rate35. This is in good agreement with the TEM results as the Fe3O4 primary crystallites formed from lepidocrocite gradually increase over time  was confirmed by direct observation of Fe3O4 mesocrystal growth at the interface with goethite. Because the Fe3O4 mesocrystal are formed from one-dimensional tubular goethite intermediate and grow along the orientation of preexisting Fe3O4 mesocrystals, we can thus deduce one-dimensional growth (b = 1) and decreasing nucleation rate in pathway 2 (a = 0.4–0.6).Figure ime Fig. . Fe3O4 p3O4, which differentiates the microstructures of Fe3O4 mesocrystals derived via each crystallization mechanism. We propose that the crystallization pathways of Fe3O4 can be selected by varying the chemical content. Recent experimental and theoretical studies indicate that the solution chemistry affects the free-energy landscape that directly governs polymorph selection37. The free-energy landscape is determined by the ratio of surface to bulk energy, which is a function of solution chemistry. Consequently, even minor variations in reactant type and concentration influence the phase is selected as intermediate among the ferric (oxyhydr)oxide polymorphs, which have different solubilities39. The graphs in Fig. 3·6H2O, NaOAC, and H2O on the diameter and crystallite size of the Fe3O4 mesocrystals. The modified polyol method has been widely used for a decade, but we were able to observe changes in the diameter and crystallite size over a wide range of chemical content and developed a comprehensive understanding of the synthesis process41. Here, we focused on how crystallite size varies with NaOAC/FeCl3·6H2O ratio and H2O content in terms of which crystallization pathway covers the entire reaction (see Supplementary Section 3·6H2O (the Fe precursor) to that of NaOAC and H2O (the OH− sources) can lead to a reduction in crystallite size, implying that pathway 1 gradually accounts for the entire reaction. The reason could be the effect of excess Fe3+ cations, which could be reduced to Fe2+ by the subsequent reaction with ethylene glycol. As the Fe2+ concentration increases, it is adsorbed onto the surface of the iron (oxyhydr)oxide phases, and it transfers electrons to the bulk iron (oxyhydr)oxide, thus promoting nucleation of Fe3O442. As shown in Fig. 2O is added in excess to the NaOAC/FeCl3·6H2O ratio, the Fe3O4 phase is no longer formed, and the reaction is blocked at the intermediate phase. This is because excess H2O interferes with dehydration in the stepwise phase transformation of pathway 243.We show that ferric (oxyhydr)oxide polymorphs play an essential role in determining the crystallization pathway of Fe3O4 mesocrystals oxide intermediates can affect the crystallization mechanism. We demonstrate that the growth mechanism of Fe3O4 mesocrystals from different iron (oxyhydr)oxide polymorphs leads to a variation in the microstructure of the mesocrystal characterized by the JMAK model. The former pathway, following oriented attachment, produces a small crystallite, while the latter, following the classical model by stepwise transformation, produces a large crystallite. These two parallel crystallization pathways operate competitively, and we were able to selectively control the ratio of the pathways that govern the entire reaction depending on the NaOAC/FeCl3·6H2O ratio and H2O content. We hereby propose the Hc model for Fe3O4 mesocrystals 100 nm or less in size, which facilitates fundamental magnetism in fine particles. To the best of our knowledge, this is the first report of Hc variation in Fe3O4 mesocrystals synthesized using the same method as a function of both diameter and crystallite size. The Fe3O4 mesocrystals synthesized via chemical routes, with diameters ranging from a few nanometers to a hundred nanometers and exhibiting soft magnetism, are of emerging technological interests in electrical as well as biomedical applications. This study suggests a reliable design rule where coercivity modulation is desirable.We have discovered a new approach to chemically control the microstructure of Fe35. This method is effective for investigating the prenucleation attachment model of Fe3O4 mesocrystal formation and explaining changes in the geometric and microstructural sizes. The experimental procedure is as follows: we use iron chloride hexahydrate (FeCl3·6H2O), sodium acetate (NaOAC), and ethylene glycol (which are commonly used in the modified polyol method) as the Fe precursor, hydroxyl ion supplier, and solvent, respectively. We do not use any other surfactant or modifier. In a typical synthesis of Fe3O4 mesocrystals, FeCl3·6H2O, NaOAC, and distilled water are completely dissolved in 50 mL of ethylene glycol under vigorous mechanical stirring to form a yellow–brown turbid solution. FeCl3·6H2O , NaOAC , and ethylene glycol  are used as received. We add distilled water (Millipore Direct-Q UV 3) to the weighed FeCl3·6H2O powder to obtain the appropriate concentration. Next, the desired volume of FeCl3·6H2O aqueous solution is injected rapidly into the ethylene glycol solution containing NaOAC. Here, the ratios of FeCl3·6H2O, NaOAC, and distilled water should be carefully controlled because they significantly influence the diameter and crystallite size of the final Fe3O4 mesocrystals. The solution is then refluxed for 8 h, during which period it turns reddish-brown and then slowly becomes black. The temperature is set to 200 °C, but the size of the mesocrystals can be controlled by adjusting the duration of the reaction at temperatures between 70 and 90 °C. After cooling to room temperature, the black sediment is washed upwards of five times using ethanol and distilled water to eliminate any organic and inorganic by-products. To monitor the formation process of the mesocrystals, the experiments are stopped after refluxing for the desired time, and the samples stored at room temperature. For S1, we synthesize the Fe3O4 mesocrystals following the preceding procedure and using 2 mmol of FeCl3·6H2O, 15 mmol of NaOAC, 150 mmol of H2O, and 50 mL of ethylene glycol. Refluxing is performed at 200 °C, and we stop the experiment at 30 min or 1 h intervals. The reaction mixture is cooled to room temperature and stored at 4 °C in a refrigerator. S2 is prepared in the same way as S1, but different amounts of the precursors are used, as follows: 1 mmol of FeCl3·6H2O, 3 mmol of NaOAC, 200 mmol of H2O, and 50 mL of ethylene glycol. We adjust the concentration of each precursor individually to analyze the changes in the diameter and crystallite size of the Fe3O4 mesocrystals according to the OH−/Fe3+ ratio. To analyze the effects of NaOAC, 2, 3, 4, 5, 6, 10, and 15 mmol of NaOAC/FeCl3·6H2O is used, and the amount of FeCl3·6H2O is constant at 1, 2, and 3 mmol. To analyze the effects of Fe3+, 1, 1.5, 2, 2.5, and 3 mmol of FeCl3·6H2O is used, with 6, 9, 12, or 15 mmol of NaOAC. Furthermore, the amount of distilled water is varied from 100 to 300 mmol while the NaOAC/FeCl3·6H2O ratio is maintained at 15, 7.5, or 5. We successfully determined the trends in size change of the mesocrystals and crystallites.We use a modified polyol method that has been widely employed to synthesize metal/metal oxide mesocrystals with a controlled diameter in a wide range3O4 mesocrystals are determined using analytical TEM  at 200 kV. To observe the formation process of Fe3O4 mesocrystals in S1 and S2 at different times while preserving the samples, a crude solution is used for sampling on a TEM grid without washing or dilution. We drop 6 μL of the solution onto a carbon-coated copper grid. Next, the intermediate and Fe3O4 mesocrystals are adsorbed on the grid for approximately 3 h in a vacuum desiccator, and the remaining solution is removed via filter paper. The samples for the TEM experiments are prepared by diluting washed samples using absolute ethanol and then placing a drop of the sample solution on a carbon-coated copper grid. The sizes of n > 500 Fe3O4 mesocrystals and n > 50 primary Fe3O4 crystallites are measured from the TEM images. The mean value is obtained by fitting the counts using Gaussian distributions. The crystal structure and crystallite size of the emerging phases are determined using powder XRD  with a Cu Kα radiation source (λ = 1.5406 Å) at the Korea Basic Science Institute, Seoul Western Center. Measurements are performed in θ–2θ geometry from 20 to 80° using a 45 kV and 40 mA tube. The average crystallite size is calculated using the Williamson-Hall plot, considering the deformation factors of the samples. The crystallite sizes are determined using the Williamson-Hall plot after the profiles of the samples are fitted with a pseudo-Voigt function, and instrumental broadening is corrected. The XRD peak broadening induced by microstrain θ positions are analyzed using the Williamson-Hall plot and the uniform deformation model, assuming that the microstrain is uniform in all crystallographic directions and the crystallite size and strain contribute independently to line broadening. The strain and crystallite size are calculated from the slope and y-intercept of a linear fitting. Samples S1 and S2 have crystallite sizes of 23 and 43 nm, respectively oxide intermediates were obtained with a confocal–Raman spectrometer  equipped with a equipped with a Nd-YAG laser operating at 532 nm and a 1200 lines/mm grating. Vacuum dried samples were mounted onto a glass. The samples were measured at 1 s detector exposure time with 100 spectra accumulations using a low laser power of 1% to avoid any damages in the sample. The magnetic properties of the powdered samples are analyzed by measuring the magnetization curves M(H) using a vibrating sample magnetometer . We measure M(H) at 300 K using an applied field of up to 20 kOe.The morphology and microstructural evolution of the crystallization pathways of the Fe3O4 after the reaction is complete, we regard f as the volume fraction of Fe3O4 mesocrystals transformed from the appropriate intermediate. Thus, the JMAK equation yields the following: f is the volume fraction of the transformed material, t is the transformation time, k is a constant, and n is the Avrami exponent. Vt is the volume of Fe3O4 mesocrystals grown at each transformation time, and Vf is the final volume of Fe3O4 mesocrystals from each intermediate. The Avrami exponent can be written as follows: a is the time-dependent nucleation rate (a > 0), b is the dimensionality of the grown phase (0 < b < 3), and c is the growth rate (c = 0.5 or 1). The value of a indicates the nucleation rate as follows: a = 0 (no nucleation or preexisting nuclei), 0 < a < 1 (decreasing nucleation), a = 1 (constant nucleation), a > 1 (increasing nucleation). The value of c indicates the relationship between time and size based on the growth mode, where c = 0.5 for diffusion-controlled growth and c = 1 for interface-controlled growth. We apply the JMAK model to each pathway separately. This is possible because the Fe3O4 growth from lepidocrocite can be distinguished from that from goethite based on time. When Fe3O4 crystals are derived from lepidocrocite or goethite, the growth kinetics operate differently. For S1, the formation process can be divided into two pathways: an early-stage process (up to 3.5 h), and a late-stage process (from 3.5 to 8 h). As shown in the JMAK model timeline of S1, the Fe3O4 core grows via pathway 1, and then the Fe3O4 shell grows via pathway 2 :3O4, treflux = 3.5–8 h, ttrans, pathway2 = 0–4.5 h):3O4 mesocrystals are formed primarily from goethite, we considered the refluxing time of 3 h at which Fe3O4 crystals start to form, as ttrans = 0. For pathway 2 of S2 :For pathway 1 of S1 (from Lp to FeSupplementary Information


A quadratic decrease of the sublattice magnetization was determined from the temperature dependence of the isotropic hyperfine field. We calculated the magnetization using linear spin wave theory for the incommensurate spiral spin order and compared this outcome with experimental results to estimate the coupling constants. The hyperfine fields at Cr and Se ions provide evidences that the spin polarization of Cr ions is transferred to neighboring Se ions due to the covalent bonding between them, resulting in reduced magnetic moment in the Cr ion. This observation indicates that the Jahn-Teller effect, which leads to distortion inducing spin-lattice coupling, is not completely missing in ZnCr2Se4.The zero field  The antiferromagnetic direct exchange interaction is dominant in ZnCr2O4 given the narrow distances between Cr ions. The tetrahedral structure of Cr ions subsequently predicts the three dimensional geometrical frustration2. At greater distances between the Cr ions, as in HgCr2Se4, the ferromagnetic superexchange interaction is dominant over the direct exchange interaction5. In ZnCr2Se4, the Cr–Cr distance is between these two. The resulting interaction is ferromagnetic as manifested by a positive Curie-Weiss temperature of θcw = 90 K7, but the actual spin order occurring at TN = 21 K8 is incommensurate helical. This discrepancy implies that nearest-neighbor interaction is comparable and opposite to farther neighbor interactions, that is, bond frustration plays an important role.In spinels consisting of a chromium and nonmagnetic ions, ACr2Se4 undergoes a structural transition from cubic to tetragonal (c/a = 0.9992)9 concurrent with the magnetic phase transition. It also exhibits negative thermal expansion8 and high magnetostriction10. All of these observations provide evidence of strong spin-lattice coupling11. This is an interesting feature because three d3 electrons of a Cr3+ ion half-fill the tg2 triplet and the orbital angular momentum is quenched to zero. Therefore, the simultaneous transition of the structure and magnetic orders does not originate from the conventional Jahn-Teller (JT) distortion and magnetostriction effect is not due to spin-orbit coupling. The observed g-factor is in fact quite close to the spin-only value12. On the other hand, spin-lattice coupling in frustrated magnets can be generated by the spin driven JT effect13 that the frustration is released by structural distortion. That is, the strong spin-lattice coupling is the result of the competition between the ferromagnetic nearest neighbor interaction and the antiferromagnetic farther neighbor interactions.ZnCr2Se4, reported values of the strength of the interactions have been inconsistent. The results of a neutron powder diffraction experiment provided suitable explanation with the combination of J1 ~ −1, J2 ~ 0.3 meV14, where Ji is the coupling constant between the i-th nearest Cr neighbors as shown in Fig. J1 ~ −3, J2 ~ 0.03, J3 ~ 0.5, J4 ~ −0.04 meV15. A calculation of the local spin density approximation plus U also predicted that the magnitudes of ferromagnetic J1 and antiferromagnetic J3 are larger than those of J2 and J4 by one order, though estimated values5 were quantitatively different from those in the neutron scattering data.Despite the necessity for information about these interactions to understand the spin-lattice coupling in ZnCr3+ ions has also been the subject of some debate. The fit of susceptibility data to the Curie-Weiss law produced 3 μB per Cr ion16. Contrary to macroscopic measurements, neutron diffraction studies have reported that the magnetic moment of a Cr3+ ion is not greater than 2 μB18 and have suggested that spin fluctuation reduces the average magnetic moment. In contrast, a recent neutron diffraction study claimed that 3 μB was obtained in agreement with a spin-only Cr3+ ion and that smaller values may originate from the non-stoichiometry of samples14. In addition to spin fluctuation and non-stoichiometry, metal-ligand covalence can reduce the magnetic moment of a Cr ion in ZnCr2Se4. It is known that covalent hybridization spreads the spin polarization of a metal ion onto the ligand orbital19. Considering that covalence is relatively strong in selenides, this could in fact be the major cause of the reduced moment. The covalence is related to the spin-lattice coupling either because the changed electron density in Cr ions would induce distortion.The magnetic moment of Cr2Se4 as obtained by zero-field nuclear magnetic resonance (NMR). Temperature dependence of the magnetic moment M(T) was obtained from the isotropic part of the hyperfine field. The sublattice magnetization was calculated by applying linear spin wave theory to the incommensurate helical spin distribution, which was in good agreement with experimental outcomes. The exchange constants were estimated by fitting the theory to the experimental results. The evidences of strong Cr-Se covalence were provided by the isotropic and anisotropic hyperfine fields obtained by Cr and Se NMR.In this paper, we report the coupling constants and the magnetic moment of Cr ions in ZnCr53Cr NMR spectrum obtained from polycrystalline ZnCr2Se4 at 6 K is shown in Fig. 3, the spins pointing in all directions produce such a spectrum21. A similar spectrum is observed when an external magnetic field generates diverse spin directions in powder samples22. Because spectrum was obtained in a zero external field, the observed U-shape is not the characteristics of powder samples but instead represents the incommensurate spin helical structure of ZnCr2Se4.The zero-field iso, Hani, and HNQR represent the isotropic and anisotropic hyperfine interactions, and the nuclear quadrupole interaction, respectively. The ordered electron spins in the d orbital polarize the spins in the inner s-shell, generating a strong isotropic hyperfine field at the nuclei through Fermi contact interaction. The spins in the same and neighboring atoms generate dipolar hyperfine fields which are anisotropic. The spectral shape expected from these interactions for an incommensurate helical spin distribution is derived in the Method.The NMR resonance frequency for magnetic materials is determined by the interactionν0, ν1, and νNQR, which are the resonance frequencies of the isotropic and anisotropic hyperfine fields, and the nuclear quadrupole resonance (NQR) frequency, respectively. The result of the fit drawn together in the Fig. appears to be quite satisfactory. The temperature dependence of ν0, ν1, and νNQR obtained from the fit to the NMR spectra measured at various temperatures are shown in Fig. ν0 shows a monotonic decrease of approximately 15% as the temperature increases from 4 K to 15 K. ν1 drawn in the lower left inset, also decreases by a similar amount. Because hyperfine fields are proportional to the magnetic moment of the electron spins, ν0 and ν1 are proportional to the electron magnetic moment either. Therefore, they should change with the temperature in the same way. The NQR frequency depends on the ion position in a crystal structure and is therefore independent of the temperature. νNQR drawn in the upper right inset remains constant with the temperature, as expected. The same temperature dependence of ν0 and ν1 and the temperature-independent νNQR strongly supports the legitimacy of the analysis.We fit the data in Fig. ν0 is proportional to the electron magnetic moment as mentioned above, the main graph in Fig. 15 ofQ, where Jk is the Fourier transform of Jij.Since Sz> is obtained from the Bose-Einstein distribution of magnons asXk ≡ Jk − JQ and k comes mostly near |k| = |Q| and k = 0. However, it has been reported that magnon-magnon scattering opens gaps at  and 15. Therefore, we need to sum only around  and the Goldstone modes centered at . Then it can be shown that the magnetic moment reduction depends on temperature asα(Jij) is a constant depending on the coupling constants Jij. For example,J1 and J3. Here γ is the angle between the spins in the neighboring ab planes. The magnetic moment decreases in proportion to T2, as in antiferromagnets.The temperature dependence of the magnetic moment <n in Eq.  shows thmentclass2pt{minimαT2 gives α = 33(±1) × 10−3 MHz/K2. The result is depicted as the black solid curve in the figure. It appears that the theoretical model describes the data well. Considering the uncertainty in the estimation of α by the fit, we searched for J1 and J3 pairs which gives |α(Jij) − α|/α < 0.03. All the pairs of J1 and J3 satisfying this condition are depicted as red open circles in Fig. J3 value because the difference is negligible5.The fit of the data in Fig. s α = 33± × 10−3 MJ1| increases, the points approach asymptotically a linear curve that passes through the origin, J1 and antiferromagnetic J3 as long as their ratio is fixed. When only J1 and J3 are considered, the ground-state energy of a helical order of classical spins is given by E(γ) = 4J1(cosγ − 1) + 8J3(cos2γ − 1)5. The ground-state configuration is obtained by requiring (dE/dγ)γ0 = 0. The experimentally observed angle between the spins on the neighboring ab planes γ0 = 42° gives J3/J1 ~ −0.17, which roughly matches with the value estimated from the fit. The black squares in Fig. J1 and J3 values reported in the experimental and theoretical works claiming that J1 and J3 are one order of magnitude stronger than J2 or J415. Although these values are different from each other, they follow the relationship trend we introduce here.As |J1 and J2 instead of J1 and J3. The result shows that the pairs of J1 > |−2.4| meV and J2 > 0.8 meV fit the data similarly. However, the result is incompatible with that of a neutron powder diffraction experiment which was explained with J1 ~ −1 and J2 ~ 0.3 meV14. The NMR result supports the bonding scheme with the ferromagnetic J1 competing with antiferromagnetic J3, rather than that with J1 and J2. We also attempted to fit the data to the form T 3/2exp(−EG/kBT) which is the temperature dependence of magnetization expected when energy gap EG exists in the spin wave dispersion relation. The result was EG = 1 ± 13 meV, indicative of practically no energy gap and, consistent with the neutron scattering experiment.We repeated the same fit procedure with 3+ ions extrapolated to 0 K in Fig. 2Se4 compounds was also observed at a similar frequency23. Cr NMR for ACr2O4 compounds is, however, observed in the frequency range of 68 ± 7 MHz28. The resonance frequency of Mn2+ NMR for manganese compounds is also reduced by about a third when O or F ion ligands are replaced by Se ions27. The difference in the resonance frequencies of the selenides and oxides is attributed to the difference in the magnetic moments and the hyperfine coupling constants. Given that the magnetic moment of Cr3+ ions in chromium oxide spinels is 3 μB, the magnetic moment of the chromium selenide should be less than 3 μB.The NMR center frequency of the Cr3+ ions are also provided by the Se NMR and the anisotropic hyperfine field at a Cr ion. When macroscopic measurements of the magnetic moment gives 3 μB per Cr ion16, and one Cr ion has less than 3 μB, the remaining moment should be found in the other ions of ZnCr2Se4. In general, the covalence increases in metal-ligand bonding as the oxygen ligand is replaced by sulfur, and sulfur by selenium. Covalent hybridization due to the strong overlap between the Cr 3d and Se 4p orbitals is expected to transfer the spin polarization of Cr ions to Se ions27. We measured the transferred hyperfine field at the Se nuclei of ZnCr2Se4 by zero-field Se NMR. The resulting hyperfine field strength is 8 ~ 10T, which is about half of that in Cr nuclei, 18.3 T. Cr and Se NMR for CdCr2Se4 and HgCr2Se4 show the similar results23. This large hyperfine field in nonmagnetic Se ions provides one clear evidence of a large spin polarization transfer from Cr ions. It is known that the intensity of spin fluctuations measured in neutron scattering experiment decreases due to reduced magnetic moment associated with such strong covalence29.Supporting evidences for the reduced magnetic moment of CrL or S at the same or neighboring atoms. In the simple ionic picture in which three d electrons occupy tg2 orbitals, the orbital angular momentum L is quenched and the hyperfine field due to S at the same atom is isotropic because the shape of the half-filled tg2 orbitals is spherical. Therefore, only the dipolar field due to S in the neighboring Cr ions can contribute to the anisotropic hyperfine field. We calculated the dipolar field generated by the helically ordered spins in ZnCr2Se4. Considering up to the third nearest neighbors, the sum of the dipolar field is estimated to be approximately 340 mT. Extrapolation of the anisotropic hyperfine field data in the inset of Fig. The anisotropic hyperfine field is generated by L or that the orbital is not exactly spherical. The g-factor measured along the [111] direction of a single crystal8 is identical to that obtained from poly-crystals12, 1.996. Therefore, the unquenched orbital angular momentum is negligible and the most plausible cause of the anisotropic hyperfine field is the spin in the non-spherical d orbital. The orbital in Cr3+ ions might become non-spherical when the effective number of electrons changes from three by covalence. A small fraction of a pair of up and down electrons is shifted from Se to Cr ions in the covalent bonding, and a similar fraction of one electron is shifted in the opposite direction in the antibonding30. In this way, a net electron is transferred from a Se ion to a Cr ion, while a spin polarization is transferred in the opposite direction. The changed electron density in Cr ions should then induce distortion together with the spin-driven JT effect, generating spin-lattice coupling in ZnCr2Se4 where the JT effect is missing from the pure ionic point of view.Because the dipolar field is only a third of the observed value, the major anisotropic hyperfine field should be generated in the same atom. This means that either there should be an unquenched 2Se4, where the conventional JT effect is missing, can be explained by the spin driven JT effect. That is, the spin-lattice coupling in the frustrated magnet ZnCr2Se4 is the result of competing ferromagnetic and antiferromagnetic couplings. To determine the coupling constants, we obtained M(T) from the temperature dependence of the zero-field Cr NMR spectrum and compared with theory. We derived J1 and J3 are dominant couplings rather than J1 and J2. The result yielded the asymptotic relationship J3 ~ −0.2J1 for large couplings.In summary, the strong spin-lattice coupling in ZnCrd electron spins in a Cr ion is less than three. Second, the anisotropic hyperfine field indicates that the total d electron orbital is not spherical. Third, Se NMR supports a large spin polarization transfer from Cr ions to Se ions. The changed number of electrons can then induce distortion of the lattice in addition to the spin-induced JT distortion. Covalence plays a role in the spin-lattice coupling in ZnCr2Se4.We have shown evidences for the spin polarization transfer from Cr ions to Se ions due to strong covalence. First, the strength of the isotropic hyperfine field implies that the effective number of 2Se4 samples were used because single crystals produced much lower signal intensity levels, most likely due to conductivity. The samples were prepared in a closed quartz ampule using a stoichiometric quantities of Zn (99.99%), Cr (99.999%), and Se (99.999%). The elements were thoroughly mixed, pressed into pellets, and heated to 650 °C for 24 hrs. The product was reground and the same heating procedure was repeated once more. The phase purity of the polycrystalline sample was checked at room temperature via powder X-ray diffraction. All experiments were performed without external magnetic fields. A conventional 90-τ-180 pulse sequence was used to obtain the spin echo signal at temperatures lower than TN. The echo signal was measured in a wide frequency range between 35 and 50 MHz at an interval of 0.1 MHz with rf pulses of 0.25 MHz bandwidth. The full NMR spectrum was obtained by summing the Fourier-transformed signals31.For NMR experiments, polycrystalline ZnCrConsidering all three interactions in Eq. , the NMRα and β represent the angles between the spin direction and the anisotropy axis of the hyperfine field and the principal axis of the NQR, respectively. ν0 and ν1 are the resonance frequencies of the isotropic and anisotropic hyperfine fields, respectively, and the first order NQR frequency for m → m − 1 transition, νNQR = −(3e2qQ/2hI(2I − 1))(m − 1/2)/2. Because the spins of ZnCr2Se4 lie on the ab plane, the spin direction is describable with only the azimuthal angle ϕ. The crystal symmetry implies that the NQR principal axis is along the [111] axis, along which Cr ions are located symmetrically. It is known CdCr2Se4 has the principal axis of NQR in this direction32. The angle dependence of the NQR term, 3cos2β − 1, can then be replaced by sin(2ϕ). Expressing the angle dependence of the anisotropic hyperfine field in terms of ϕ, Eq. (21δ is the azimuthal angle of the axis of the anisotropic hyperfine field. ν0 is the sum of all angle-independent terms and ν1 is the value of νani projected on the ab plane.Here, f ϕ, Eq.  can be rA simple trigonometric calculation of Eq.  gives9\dϕ0 is a constant. The equation shows that the frequency change with the azimuthal angle is minimal at every 90 degrees. In an incommensurate spiral spin distribution, all spins are uniformly distributed in the angle domain. Such a spatially uniform distribution appears as a U-shaped distribution in the frequency domain. NQR gives three different spectral shifts m = 3/2, 1/2, −1/2, to split the spectrum into three. As a result, the superposition of three U-shaped spectra with six peaks is generated.Here, ν0 + ν1/2 was determined according to the symmetry point of the spectrum, and the fitting parameters were ν1, νNQR, and δ. The result of the fit drawn together in Fig. δ = π/4, meaning that the axis of the anisotropic hyperfine field is on the (12Se4, having a crystal structure identical to that of ZnCr2Se4, was also suitably described using the same axis of the anisotropic hyperfine field32.We fit the data in Fig.