Manganese@Albumin Nanocomplex and Its Assembled Nanowire Activate TLR4‐Dependent Signaling Cascades of Macrophages

The immunomodulatory effect of divalent manganese cations (Mn2+), such as activation of the cGAS−STING pathway or NLRP3 inflammasomes, positions them as adjuvants for cancer immunotherapy. In this study, it is found that trace Mn2+ ions, bound to bovine serum albumin (BSA) to form Mn@BSA nanocomplexes, stimulate pro‐inflammatory responses in human‐ or murine‐derived macrophages through TLR4‐mediated signaling cascades. Building on this, the assembly of Mn@BSA nanocomplexes to obtain nanowire structures enables stronger and longer‐lasting immunostimulation of macrophages by regulating phagocytosis. Furthermore, Mn@BSA nanocomplexes and their nanowires efficiently activate peritoneal macrophages, reprogramme tumor‐associated macrophages, and inhibit the growth of melanoma tumors in vivo. They also show better biosafety for potential clinical applications compared to typical TLR4 agonists such as lipopolysaccharides. Accordingly, the findings provide insights into the mechanism of metalloalbumin complexes as potential TLR agonists that activate macrophage polarization and highlight the importance of their nanostructures in regulating macrophage‐mediated innate immunity.


DOI: 10.1002/adma.202310979
3] Among them, divalent manganese cations accelerate the enzymatic activity of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) to produce the second messenger cGAMP in the presence of low concentrations or even without cytosolic double-stranded DNA (dsDNA). [4,5]Moreover, Mn 2+ can further strengthen the binding affinity of the stimulator of interferon gene (STING) proteins with cGAMP ligands in innate immune cells and produce type I interferon (IFN) through the cGAS-STING pathway, which is beneficial for priming adaptive immunity. [6]In addition, Mn 2+ can trigger the nod-like receptor 3 (NLRP3) inflammasome and induce cell pyroptosis via the caspase-1 (Casp1) pathway. [7,8]Therefore, Mn 2+ ions are recognized as damage-associated molecular patterns (DAMPs) that trigger type I IFN production and inflammasome activation in innate immune cells or directly induce the prophylactic death of infected cells against pathogenic infection or cancer.[19][20][21] Therefore, developing trace Mn 2+ -containing adjuvants or agonists with excellent biosafety and robust immunostimulatory effects is important for future clinical applications.
[24][25][26][27] The abundant carboxyl, amine, and thiol groups of albumins have a strong affinity for divalent metal cations in the body via electrostatic or coordinative interactions. [28][31][32] Recently, weak immunostimulation of macrophage polarization by naive bovine serum albumin (BSA) was augmented by modification with mitochondria-targeted ligands. [33]A Mn@albuminbased nanoagonist stimulates cGAS-STING signaling in dendritic cells for cancer immunotherapy. [34]This study found that trace Mn 2+ ions bound to BSA to form Mn@BSA NCs efficiently stimulated proinflammatory responses in human-or murinederived macrophages.The immunostimulation of Mn@BSA NCs was mainly dependent on Toll-like receptor 4 (TLR4) signaling cascades.This differed from cGAS-STNG or NLRP3 inflammasome pathways activated by cytosolic Mn 2+ .The underlying mechanism elucidated that TLR4-mediated signaling transducers of nuclear factor kappa-B (NF-B) and activator protein 1 (AP-1) were successfully activated for inflammatory responses, accompanied by the activation of interferon regulatory factor 7 (IRF7) for type I IFN production.Furthermore, larger nanowire (NW) structures of Mn@BSA NCs were assembled through glutathione (GSH) reduction, which boosted proinflammatory responses of macrophages with the upregulation of tumor necrosis factor  (TNF-), interleukin 6 (IL-6), and CXC chemokine ligand 10 (CXCL10) gene and protein expression.Finally, in vivo investigations demonstrated that Mn@BSA NCs and Mn@BSA NWs had better biosafety than the classic TLR4 agonist lipopolysaccharide (LPS), efficiently activated murine peritoneal macrophages and reprogrammed tumor-associated macrophages (TAMs), and inhibited the growth of melanoma tumors.Therefore, we suggest that the Mn@BSA NCs and NWs developed in this study have potential clinical applications as TLR4 agonists for enhanced cancer immunotherapy.

Results and Discussion
2.1.The Formation of Mn@BSA NCs and Mn@BSA NWs Mn 2+ ions at 8.0 μg mL −1 were initially stirred with 3 mg mL −1 BSA solution for 12 h and subsequently dialyzed (molecular weight cut-off [MWCO]: 8.0-14.0kDa) to prepare Mn@BSA NCs (Figure 1A) according to a previous study. [34]Subsequently, the S─S bonds of the prepared Mn@BSA NCs were broken into thiol moieties by reduced GSH. [35]Finally, reduced Mn@BSA NCs were crosslinked via the reformation of S─S bonds to obtain Mn@BSA NWs.Inductively coupled plasma mass spectrometry (ICP-MS) analysis showed that the Mn@BSA NCs had a trace Mn content of ≈0.01%(w/w) (Table S1, Supporting Information).The morphology was observed using transmission electron microscopy (TEM).Mn@BSA NCs had a larger irregular shape with a mean size of 25.9 ± 5.4 nm attributed to Mn 2+ incorporation (Figure 1B) compared with the small rod shape of naive BSA. [36]The TEM images presented the assembly of Mn@BSA NCs into NW structures, with an average diameter of 41.3 ± 3.0 nm, and a length between some hundred nanometers and a few micrometers (Figure 1B and Figure S1, Supporting Information).In contrast, BSA nanospheres (BSA NSs) derived from BSA molecules had an average diameter of 58.1 ± 8.0 nm, similar to other results. [37]This comparative analysis of the morphologies of the Mn@BSA NWs and BSA NSs showed that Mn 2+ incorporation determines the oriented growth of the BSA assembly.Here, we hypothesized that the electrostatic repulsive forces of localized Mn 2+ ions prevented crosslinking of the reduced Mn@BSA NCs.
Serial dilution of BSA, Mn@BSA NCs, BSA NSs, and Mn@BSA NWs in the concentration range of 0-2 mg mL −1 showed that the measured hydrodynamic sizes increased and converged on the limits according to dynamic laser scattering (DLS) analysis (Figure S2A, Supporting Information) owing to the attenuation of multiple scattering. [38]For example, BSA or Mn@BSA NCs at 100 μg mL −1 , or Mn@BSA NCs or Mn@BSA NWs at 20 μg mL −1 approached the limited values; thus, 20 μg mL −1 was reasonable to measure the real hydrodynamic size of these samples in our studies.The hydrodynamic size of Mn@BSA NCs was 316.1 ± 77.7 nm with a polydispersity index (PDI) of 0.3, which was larger than that of BSA (7.7 ± 2.8 nm, PDI = 0.3) (Figure 1C).The hydrodynamic size of BSA NSs or Mn@BSA NWs increased to 357.7 ± 92.9 nm (PDI = 0.3) or 484.2 ± 94.2 nm (PDI = 0.5), respectively, compared with BSA or Mn@BSA NCs.These results further confirmed Mn 2+ -induced BSA aggregation via electrostatic interactions and the assembly of reduced Mn@BSA NCs via S─S binding, in agreement with TEM observations.In addition, the incorporation of trace Mn 2+ ions induced a slight zeta potential change from −11.0 ± 3.17 (BSA) to −7.1 ± 3.15 eV (Mn@BSA NCs) in dispersed samples in deionized (DI) water (pH 5.9) (Figure 1D).In comparison, GSH reduction reversed the zeta potentials of the BSA NSs and Mn@BSA NWs to 8.6 ± 3.3 eV.The isoelectric points (IEP) of these BSA-based samples were further determined given that the surface charge of protein particles is affected by the solution pH [39] (Figure S2B, Supporting Information).The IEP of BSA was ≈4.1 in our studies, which was close to the value of 4.7-5.1 reported in other studies. [40]The IEP (≈5.4) of the Mn@BSA NCs was lower than the pH (≈5.9) of DI water; thus, their zeta potential tended to acquire more negative charges.The IEPs (≈9.0) were greater than the pH of DI water for the BSA NSs and Mn@BSA NWs, resulting in a switch from negative to positive charges.This revealed that the breakage triggered by reduced GSH and the reformation of S─S bonds affected the surface charge density of BSA.The UV-vis spectra displayed a characteristic Figure 1.A) Scheme illustrating the fabrication route, Mn 2+ -induced aggregation, and the orientational assembly of Mn@BSA NCs via GSH reduction.B) TEM images of Mn@BSA NCs, BSA NSs, and Mn@BSA NWs (scale bar = 100 nm).C) Hydrodynamic size, D) zeta potential, and E) UV-vis spectra of BSA, Mn@BSA NCs, BSA NSs, and Mn@BSA NWs.F) Long-term stability of Mn@BSA NWs dispersed in PBS, PBS containing 6 μmol L −1 GSH, and FBS.
absorption peak at 278 nm for the BSA-based samples (Figure 1E), which was assigned to the tryptophan residues of the BSA molecules. [30]Negligible changes in this peak indicated good integrity of the polypeptide chains with Mn 2+ binding and GSH reduction.
Finally, phosphate buffered saline (PBS) solution (pH = 7.4), PBS solution containing 6 μmol L −1 GSH, or fetal bovine serum (FBS) were chosen as an alternative to the blood in the body to evaluate the long-term stability of Mn@BSA NWs in vitro.Mn@BSA NWs were stable in the size range of 560-590 nm (Figure 1F and Figure S2C, Supporting Information).The size decreased from 575 to 544 nm after 24 h and returned to the stable range of 570-590 nm over the next 7 d in the PBS solution plus GSH.In contrast, the size significantly decreased to the stable range of 120-140 nm in FBS owing to stronger multiple scattering of abundant proteins in FBS.Collectively, these results demonstrate that the various components of biological fluids have negligible effects on the long-term stability of Mn@BSA NWs.
We chose 50 μg mL −1 Mn@BSA NCs (Mn@BSA-50) or 100 μg mL −1 (Mn@BSA-100) for incubation with 5 × 10 5 per mL RAW 264.7 cells for 48 h to maintain >70% of living cells.The treatment of BSA at 50 or 100 μg mL −1 , and Mn 2+ at the equivalent concentration of 4 × 10 −2 or 8 × 10 −2 μg mL −1 was also performed considering the concentration ratio (8 × 10 −4 , w/w) of Mn 2+ /BSA to fabricate Mn@BSA NCs.Meanwhile, untreated cells were used as the control group, and those stimulated with 100 ng mL −1 of LPS plus 25 ng mL −1 of IFN- were used as a positive control.Proinflammatory responses of macrophages were assessed by analyzing the gene expression of inflammatory biomarkers of TNF-, IL-6, IL-1, IL-18, inducible nitric oxide synthase (iNOS), cluster of differentiation 86 (CD86), and CC chemokine ligand 2 (CCL2), or type I IFN biomarkers of IFN- and CXCL10 using quantitative real-time polymerase chain reaction (q-PCR) (Figure S3, Supporting Information).For instance, heat-map analysis (Figure 2B) showed that upregulation in the Mn@BSA-50 and Mn@BSA-100 groups occurred at 6 h, like the positive control.Among these biomarkers, mRNA levels of IL-6 and IL-1 appeared to be significantly enhanced, while IL-18 gene expression seemed to be negligibly changed.Statistical analysis further showed that the mRNA level of iNOS, TNF-, IL-6, IL-1, CD86, CCL2, IFN-, and CXCL10 in Mn@BSA groups was much higher than that of BSA or Mn 2+ groups at 6 h (P*** < 0.001) (Figure 2C).The immunostimulation of Mn@BSA NCs also depended on their concentration.The gene expression of IL-18 increased in the Mn@BSA-100 group (P*** < 0.001, red asterisks), but not in the Mn@BSA-50 group (P > 0.05) (Figure S3, Supporting Information).In addition, the supernatants were collected from the culture media to detect the secretion of cytokines and chemokines using a pre-coated enzymelinked immunosorbent (ELISA) assay.The secretion concentration of TNF-, IFN-, IL-6, CCL2, and CXCL10 in the Mn@BSA-100 group was higher (P*** < 0.001) compared with the control, BSA-100, or Mn-100 group (Figure 2D).These results indicated that the synergy between BSA and Mn 2+ primed proinflammatory responses in macrophages.Meanwhile, protein production of IL-1 in the Mn@BSA-100 group was less than that of the Mn-100 group; however, it was still higher compared with the control (P*** < 0.001).Intracellular reactive oxygen species (ROS) direct macrophage proinflammatory polarization; [33] thus, cytosolic ROS production was evaluated by the dihydroethidium (DHE) assay in this study.No significant differences were observed when comparing Mn@BSA-100 with the control, BSA-100, or Mn-100, indicating that intracellular ROS had no significant effect on immunostimulation (Figure S4, Supporting Information).
Finally, the mRNA levels of Mn@BSA-100 and the positive control group were further normalized to analyze the dynamic profiles of the proinflammatory responses (Figure S3, Supporting Information).The gene expression of IFN-, iNOS, CD86, TNF-, and IL-1 rapidly peaked at 6 h in the Mn@BSA-100 group, and gradually decreased to the control level during the next 42 h.The peak expression time of CCL2 was at 24 h (Figure 3).These dynamic profiles were highly similar to those of the positive control, LPS.Mn@BSA NCs induced IL-6 and CXCL10 gene expression, which was faster than those of LPS.Accordingly, the Mn@BSA NCs efficiently elicited acute inflammatory responses in macrophages for over 24 h.

Mn@BSA NCs Elicit TLR4-Dependent Signaling Cascades
LPS is a glycolipid originating from the outer membrane of Gram-negative bacteria that is sensitively detected by membranebound TLR4 in innate immune cells. [41]We hypothesize that Mn@BSA NCs activate TLR4 signaling in macrophages, like the TLR4 agonist LPS.RAW-Dual and RAW-Dual KO-TLR4 cells with stable TLR4 knockouts were comparatively investigated to further verify this hypothesis.RAW-Dual cells generated from RAW 264.7 cells express pattern recognition receptors of TLR2 and TLR4, the cytosolic DNA sensor of cGAS, and the cyclic dinucleotide sensor of STING. [42,43]In addition, RAW-Dual cells stably express two reporter genes encoding secreted embryonic alkaline phosphatase (SEAP) for NF-B activation, and Lucia luciferase (LUCIA) for IRFs activation.RAW-Dual KO-TLR4 cells do not respond to TLR4 agonists, such as LPS or monophosphoryl lipid A, resulting from TLR4 knockout in their parental RAW-Dual cell line.Here, 50 or 100 μg mL −1 BSA, 4 × 10 −2 or 8 × 10 −2 μg mL −1 Mn 2+ , and 50 or 100 μg mL −1 Mn@BSA NCs were added to 2 × 10 5 mL −1 RAW-Dual and RAW-Dual KO-TLR4 cells, respectively.Meanwhile, untreated cells or those stimulated with 100 ng mL −1 of LPS and 25 ng mL −1 of IFN- were used as the control or positive control, respectively.The supernatants were analyzed using a microplate photometer to detect the enzymatic activities of SEAP and LUCIA after 24 h.The activities of SEAP (Figure 4A) and LUCIA (Figure 4B) in the Mn@BSA groups were much higher compared to the control, BSA, and Mn 2+ groups (P*** < 0.001).However, SEAP and LUCIA activities were significantly inhibited (P*** < 0.001) and close to control levels in RAW-dual KO-TLR4 cells.Similar inhibition was observed in the positive control groups treated with LPS  (P*** < 0.001, red asterisks).These results validated that Mn@BSA NCs activated NF-B and IRFs in macrophages via TLR4 signaling, like LPS.TAK-242 (resatorvid) was used as a selective TLR4 inhibitor to inhibit the gene expression of iNOS, TNF-, IL-6, IL-1, CCL2, IFN-, and CXCL10 to further investigate TLR4-mediated proinflammatory responses. [44]Here, 100 μg mL −1 Mn@BSA NCs were incubated with 5 × 10 5 mL −1 RAW 264.7 cells for 6 h in the presence of TAK-242 inhibitor at 0-100 nmol L −1 .Untreated cells were used as a control, and those stimulated with 100 ng mL −1 LPS plus 25 ng mL −1 IFN- were the positive control.The TAK-242 inhibitor markedly reduced the mRNA levels of pro-inflammatory cytokines and chemokines in macrophages stimulated with Mn@BSA NCs (P*** < 0.001) (Figure 4C).Among these biomarkers, the inhibitory effect of TAK-242 on the gene expression of iNOS, TNF-, IL-1, and CXCL10 was dependent on the concentration range of 0-100 nmol L −1 .Similar inhibition was observed in the positive control LPS (Figure S5, Supporting Information).Accordingly, Mn@BSA NCs elicited pro-inflammatory responses in macrophages through TLR4 signaling, like LPS.
TLR4 signaling engages the myeloid differentiation marker 88 (MyD88) adaptor to activate NF-B for downstream inflammatory responses, and TIR domain-containing adaptor inducing IFN- (TRIF) adaptor to activate IRFs for downstream type I IFN production. [45]The underlying mechanism of TLR4 signaling cascades was analyzed by measuring the gene expression of TLR4-related signaling transducers (such as MyD88, NF-B, TRIF, IRF3/7, and AP-1), and other transducers of Casp1, cGAS, STING, NLRP3, nucleotide-binding oligomerization domaincontaining protein 2 (NOD2), and signal transducer and activator of transcription 6 (STAT6) using q-PCR.The heat-map visualization shows that the expression behavior in the Mn@BSA-100 group was similar to that of the positive control of LPS (Figure 5A).Among these intermediators, the gene expression of IRF7, NOD2, NLRP3, NF-B, and TRIF was significantly upregulated, while the mRNA levels of cGAS, STING, and IRF3 remained unchanged (Figure 5B).The mRNA levels of TLR4related intermediators such as MyD88, TRIF, NF-B, AP-1, and IRF7 significantly increased in the Mn@BSA-100 group compared with the BSA-100 or Mn-100 groups (P*** < 0.001).Mn@BSA NCs upregulated the production of MyD88, TRIF, phospho-p65 (p-NF-B), and phospho-IRF7 (p-IRF7) (P*** < 0.001) (but not NF-B and IRF7) compared with the control, BSA-100, or Mn-100 group (Figure 5C and Figure S6, Supporting Information).In comparison, the production of these proteins significantly decreased upon inhibition with 50 nmol L −1 TAK-242 (P*** < 0.001, red asterisks).Mn@BSA NCs upregulated the production of inactive IRF7 and NF-B, which was inhibited by TAK-242 (Figure S6, Supporting Information).Unlike active p-NF-B, the highest production of NF-B expression occurred in the BSA-100 group (P*** < 0.001, green asterisks).However, Mn@BSA NCs strongly stimulated macrophage activation, which highlighted the importance of p-NF-B expression (not inactive NF-B) on proinflammatory responses compared with the weak immunostimulation of BSA (Figure 2).Together, we confirmed that Mn@BSA NCs activated MyD88-NF-B/AP-1 and TRIF-IRF7 (not IRF3) axes through their interactions with membrane-bound TLR4s. [46]OD2, NLRP3, and NOD-like receptors mainly detect cytosolic pathogen-associated molecular patterns (PAMPs) or DAMPs that trigger host defense against viral or bacterial infections.Mn@BSA NCs upregulated the expression of NOD2 and NLRP3 (P*** < 0.001) compared to the BSA or Mn 2+ groups (Figure 5B).NOD expression is beneficial for generating inflammatory cytokines and chemokines through the NF-B pathway. [47]We also found that Mn@BSA NCs upregulated the gene expression of Casp1 (Figure 2D and Figure S7, Supporting Information), which could cleave pro-IL-1 for active IL-1 production. [46]However, in Figure 5B and Figure S7B (Supporting Information), mRNA levels of cGAS, STING, and downstream IRF3 in the Mn-100 group were higher than those in the Mn@BSA group (P** < 0.01, P*** < 0.001, red asterisk).These results showed that free Mn 2+ activated the cGAS-STING pathway to a greater degree than the Mn@BSA NCs.Here, the steric hindrance of Mn@BSA NCs prevented their insertion into cGAS protein, resulting in the deficiency of cGAS−STING pathway activation. [4]The downregulation of STAT6 gene expression in the Mn@BSA-100 group showed no advantage in activating another classic inflammatory pathway of Janus kinase (JAK)-STAT, compared with the BSA-100 group (P*** < 0.001) (Figure S7C, Supporting Information).Therefore, Mn@BSA NCs activated macrophages mainly through TLR4−MyD88−NF-B/AP-1 and TLR4-TRIF-IRF7 signaling axes instead of the cGAS-STING or JAK-STAT pathways.
Finally, the cellular internalization and subcellular distribution of the Mn@BSA NCs were monitored using confocal imaging.Fluorescein isothiocyanate (FITC) conjugation endowed BSA, Mn@BSA NCs, or Mn@BSA NWs with strong green fluorescence at 520 nm (Figure S8, Supporting Information).Subsequently, 5 × 10 5 per mL RAW 264.7 cells were incubated with 100 μg mL −1 FITC-BSA or FITC-Mn@BSA NCs 12 h, respectively.One fluorescent channel (488/564 nm) was chosen to detect green fluorescent signals of FITC, and the other fluorescent channel (405/456 nm) was used for the blue fluorescence of intracellular nuclei labeled with 4′,6-diamidino-2-phenylindole (DAPI).The weak green fluorescence in the BSA-100 group indicated that it was difficult for macrophages to internalize BSA (Figure 6A).In contrast, the cellular uptake of Mn@BSA NCs by macrophages improved to some extent.The pseudopodia morphology (marked by red arrows) of RAW 264.7 cells stimulated by Mn@BSA NCs were clearly observed, indicating the occurrence of macrophage polarization compared with the BSA-100 group.In addition, subcellular distribution was observed with a red fluorescent channel (543/621 nm) for organelle-tracker probes.The green fluorescence of the FITC probes was clearly separated from the red fluorescence of the MitoTracker probes, whereas it mostly overlapped with the red fluorescence of the LysoTracker probes (Figure 6B).According to our previous studies, [33] Pearson's correlation coefficient (PCC) between green and red fluorescence was calculated as 0.30 in lysosomes, which was much higher than −0.22 in mitochondria.This indicated that intracellular Mn@BSA NCs preferentially accumulated in endo-/lysosomes but not in mitochondria.Mn@BSA NCs located in endosomes can trigger activation of the TRIF-dependent pathway via endosomal membranebound TLR4, whereas extracellular Mn@BSA NCs can activate the MyD88-dependent pathway via cell membrane-bound TLR4, according to the classic mechanism of macrophage activation. [46]These two pathways mutually crosstalk to cooperatively prime the pro-inflammatory immune responses of macrophages. [48]
Moreover, the enzymatic activity of SEAP and LUCIA was remarkably increased in the Mn@BSA NWs groups (P*** < 0.001) compared to the control (untreated cells) or Mn@BSA NCs groups after 50 or 100 μg mL −1 Mn@BSA NCs or Mn@BSA NWs were incubated with 2 × 10 5 mL −1 RAW-Dual and RAW-Dual KO-TLR4 cells for 24 h, respectively (Figure 7B).The enzymatic activities were also dependent on the concentration of Mn@BSA NCs or Mn@BSA NWs (P*** < 0.001 or P* < 0.05, green asterisk).As expected, the enzymatic activity of RAW-Dual KO-TLR4 cells was inhibited (P*** < 0.001, red asterisk), and was close to the control levels.The ELISA results showed that RAW 264.7 cells stimulated by Mn@BSA NWs secreted more TNF-, CXCL10, and IL-6, with higher concentrations than those of Mn@BSA NC group (P*** < 0.001) (Figure 7C).Macrophage polarization was further analyzed by labeling multiple surface receptors of CD86, CD80, and CD64, which are biomarkers of the M1 phenotype, with PE-anti-CD80 (488/575 nm), AF488-anti-CD86 (488/519 nm), and APC-anti-CD64 antibodies (633/660 nm) for flow cytometry.Mn@BSA NCs and NWs induced upregulation of M1 biomarkers compared with the control (Figure 7D).The NW structures further promoted the expression of these biomarkers compared with the Mn@BSA NCs.Accordingly, Mn@BSA NWs remarkably potentiated the activation of TLR4-dependent NF-B or IRFs to generate more downstream proinflammatory cytokines and chemokines, resulting in a higher degree of macrophage polarization.TLR4-dependent proinflammatory responses stimulated by Mn@BSA NWs were evaluated by incubating 50 μg mL −1 Mn@BSA NCs or Mn@BSA NWs with 5 × 10 5 mL −1 RAW 264.7 cells plus 50 nmol L −1 of TAK-242 inhibitor for 6 h, respectively.Subsequently, the gene expression of IL-6, CXCL10, TNF-, and IFN- was strongly inhibited by TAK-242 in the Mn@BSA NWs group, with a decrease of ≈60%-90% (Figure 7E).The inhibition of Mn@BSA NWs and Mn@BSA NC groups was similar, except for IFN- (P* < 0.05).This revealed that the pro-inflammatory responses stimulated by Mn@BSA NWs still relied on TLR4 signaling, like that of Mn@BSA NCs.
TLR4 initiates significant changes in gene expression and protein production during pro-inflammatory responses that are involved in actin-dependent phagocytosis. [49]Cytochalasin D was selected to study the role of phagocytosis in macrophage polarization given its efficient inhibition of filamentous actin (Factin) polymerization. [50]Here, 5 × 10 5 RAW 264.7 cells mL −1 were incubated with 50 μg mL −1 Mn@BSA NCs or 50 μg mL −1 Mn@BSA NWs and added with 1 μmol L −1 cytochalasin D for 6 h, respectively.The inhibitory effect of IL-6, CXCL10, and IFN- was ≈70%-90% in the Mn@BSA NW group, which was much higher than the ≈30% inhibitory effect of Mn@BSA NCs (P*** <0.001) (Figure 7F).The inhibition of TNF- was stronger in the Mn@BSA NW group, despite no significant differences.This indicated that the immunostimulation of Mn@BSA NWs was mainly dependent on phagocytosis, in contrast to that of Mn@BSA NCs.Notably, the shape, size, and rigidity of the micro/nanoparticles determine their contact with macrophage surfaces, which play a vital role in surface receptor-mediated endocytosis.For instance, particles with high aspect ratios (such as nanowires or nanoworms) improve their attachment to cell surfaces while evading cellular uptake. [51]A similar phenomenon was observed in this study.Confocal imaging showed that larger Mn@BSA NWs with a high aspect ratio range of ≈10-60 exhibited negligible internalization by nonphagocytic cell lines like murine melanoma cells (B16-F10), human cervical cancer cells (Hela), or human umbilical vein endothelial cells (HUVECs) compared with the smaller sphere shape of Mn@BSA NCs (Figure 8A and Figure S10, Supporting Information).In comparison, incubation of 50 μg mL −1 FITC-Mn@BSA NCs or FITC-Mn@BSA NWs with 5 × 10 5 mL −1 RAW 264.7 cells for 12 h showed that macrophages internalized both Mn@BSA NWs and NCs, which was different from the uptake of Mn@BSA NCs by nonphagocytic cells (Figure 8B).This reveals that the cellular uptake of Mn@BSA NWs by macrophages is mainly mediated by phagocytosis.We suggested that long and flexible Mn@BSA NWs (which are typically >0.5 μm in size) adhered to cell surfaces ("tasting"), interacted with plasma membrane-anchored TLR4 ("feeling"), and subsequently entered macrophages ("swallowing") to interact with TLR4 located on endosome membranes, resulting in stronger proinflammatory responses according to the mechanism of multistage phagocytosis. [49,51]These results highlight the importance of nanostructures of agonists or adjuvants in the activation of innate immunity.

In Vivo Behavior of Mn@BSA NCs and Their NWs
In our previous studies, the peritoneal cavity of mice was selected as the ideal site for studying macrophage activation in the body. [33]In this study, the regulatory effects of Mn@BSA NCs and NWs were also evaluated on murine peritoneal macrophages in vivo.First, 200 μL of PBS, 20.0 mg kg −1 Mn@BSA NCs, 20.0 mg kg −1 Mn@BSA NWs, or 0.5 mg kg −1 LPS were administered into the abdomens of 10-week-old female C57Bl/6 mice via intraperitoneal injection.All mice were sacrificed to harvest macrophages from the peritoneal cavities after 24 h.The secretion concentration of TNF-, CCL2, and CXCL10 was analyzed by ELISA assay after another 24-h culture ex vivo.The production of TNF-, CCL2, and CXCL10 in Mn@BSA NCs or Mn@BSA NWs group increased (P*** < 0.001) compared with the PBS control (Figure 9A).The concentrations of CCL2 and CXCL10 in the Mn@BSA NWs group were higher (P*** < 0.001) compared with the Mn@BSA NCs.These results confirm the enhanced regulatory effect of Mn@BSA NWs on macrophage activation in vivo.
The in vivo biosafety of Mn@BSA NCs and NWs was further evaluated for future clinical applications.Two hundred microliters of PBS, Mn@BSA NCs, Mn@BSA NWs, or LPS were Figure 9. A) Secretion concentration of TNF-, CCL2, and CXCL10 in murine peritoneal macrophages harvested on day 1 post the intravenous injection of 200 μL of 20.0 mg kg −1 Mn@BSA NCs, or 20.0 mg kg −1 Mn@BSA NWs, respectively (n = 3 biologically independent samples).B) Growth curve of mice with the intravenous injection of 200 μL of 20.0 mg kg −1 Mn@BSA NCs, or 20.0 mg kg −1 Mn@BSA NWs during 28 d, respectively (n = 3 biologically independent samples).Data were normalized to the body weight of mice on day 1 in the same group.C) Complete blood analysis of the serum collected through the ocular fundus of all mice on day 1, including the counts of WBC, Lymph, Mon, and Gran (n = 3 biologically independent samples).D) Biochemical analysis of the serum collected from all sacrificed mice on day 28, including the counts of ALT, AST, and ALP (n = 3 biologically independent samples).Error bars were based on the standard errors of the mean.(***P < 0.001, **P < 0.01, or *P < 0.05 by ANOVA with Tukey's post-test).administrated into 10-week-old female C57Bl/6 mice at the same concentration of 20.0 mg kg −1 via intravenous injection.The body weights of all mice were recorded every 2 d during the period of 28 d (Figure 9B).The weight of the Mn@BSA NWs group continued to increase and was similar to that of the PBS group.In comparison, weight loss occurred in the Mn@BSA NC group (days 3−5) and LPS group (days 3−13).These results demonstrate that Mn@BSA NWs have better biosafety for the healthy growth of mice compared with Mn@BSA NCs.Serum samples from all mice were harvested through the ocular fundus for complete blood analysis on day 1 post injection, as described in our previous studies. [53]The white blood cell (WBC), red blood cell (RBC), and platelet (PLT) counts and their derivative indices in the Mn@BSA NCs and Mn@BSA NWs groups were mostly normal, according to the reference range (Table S4A, Supporting Information).The infection-induced acute inflam-mation, WBC, lymphocyte (Lymph), monocyte (Mon), granulocyte (Gran), and PLT counts of the Mn@BSA NCs and Mn@BSA NWs groups were lower than those of the LPS group (P** < 0.01, or P* < 0.05) (Figure 9C).This indicates that the intravenous injection of Mn@BSA NCs and Mn@BSA NWs showed better biosafety in avoiding the occurrence of acute inflammation.Serum samples were collected on day 28 from all sacrificed mice for biochemical analysis.The levels of blood urea nitrogen (BUN), total bilirubin (TBIL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were normal in the Mn@BSA NCs and Mn@BSA NWs groups, indicating no pathological changes in liver and renal functions according to the reference range (Table S4B, Supporting Information).Mn@BSA NCs and Mn@BSA NWs also showed higher hepatic and renal biosafety than LPS (P*** < 0.001, P** < 0.01, and P* < 0.05) (Figure 9D).10.A) Tumor volume curves of all mice from PBS, Mn@BSANCs, and Mn@BSA NWs groups during 20 d (volume = 0.5 × length × width × width, n = 5 biologically independent samples).B) Average tumor volume curves of PBS, Mn@BSANCs, and Mn@BSA NWs groups during 18 d (n = 5 biologically independent samples).C) The weight of tumors harvested from all mice in PBS, Mn@BSANCs, and Mn@BSA NWs groups on day 18 (n = 5 biologically independent samples).D) Population of M1 macrophages labeled by F4/80 + and CD80 + in melanoma tumors treated with Mn@BSANCs and Mn@BSA NWs at day 16 after tumor implantation (n = 3 biologically independent samples).Error bars were based on standard errors of the mean (***P < 0.001 or **P < 0.01 by ANOVA with Tukey's post-test).
The heart, kidney, liver, lung, spleen, and brain tissues of a randomly chosen mouse from all sacrificed mice were harvested on day 28.Tissue sections were prepared for pathological analysis using hematoxylin and eosin (H&E) staining.Negligible pathological changes were observed in the spleen sections in the Mn@BSA NCs, Mn@BSA NWs, and LPS groups compared with the PBS group (Figure S12, Supporting Information).Myocardial degeneration and alveolar destruction (marked with green circles) were observed in the LPS group in contrast to the Mn@BSA NC and NW groups.In addition, Mn@BSA NWs or LPS caused the infiltration of inflammatory cells into the hepatic (marked by yellow circles) and kidney parenchyma (marked by cyan circles), respectively.]54] However, the H&E-stained sections of the brain displayed normal cortical and hippocampal neurons with visible Nissl bodies in the Mn@BSA NCs and Mn@BSA NWs groups.The in vivo biodegradability and biodistribution were further assessed by intravenous injection of 200 μL of PBS, 20.0 mg kg −1 FITC-Mn@BSA NCs, or 20.0 mg kg −1 FITC-Mn@BSA NWs into 10-week-old female C57Bl/6 mice.After 24 h, the hearts, kidneys, livers, lungs, spleens, and brains of all euthanized mice were harvested for fluorescence imaging (465/520 nm).The FITC-Mn@BSA NCs and FITC-Mn@BSA NWs showed strong epifluorescence with a range of intensity (ROI) of 1.9 × 10 10 compared with the PBS solution (ROI of 1.5 × 10 9 , marked by green circles) (Figure S13A, Supporting Information).There were no significant differences in radiant efficiency in the Mn@BSA NCs and Mn@BSA NWs groups compared with the PBS group (Figure S13B,C, Supporting Information).This revealed that most of the intravenously injected Mn@BSA NCs and NWs were efficiently degraded in the body after 24 h.
The prophylactic anticancer efficacy was evaluated by mixing 1 × 10 5 B16-F10 cells with Mn@BSA NCs or Mn@BSA NWs at 20 mg kg −1 , followed by subcutaneous implantation into the right side of the abdomen of every female C57Bl/6 mouse (6-8 weeks old).A total of 24 mice bearing melanoma tumors were randomly divided into three groups: PBS as a control, Mn@BSA NCs, and Mn@BSA NWs.Tumor volume was measured every 2 d during the 18 d (Figure 10A).The average tumor volume further demonstrated that Mn@BSA NCs or Mn@BSA NWs significantly inhibited the growth of B16-F10 tumors compared with the control group of PBS (P*** < 0.001) (Figure 10B).Intriguingly, the Mn@BSA NCs showed higher anticancer efficacy compared with the Mn@BSA NWs (P*** < 0.001).On day 18, all mice were sacrificed, and their tumor weights were measured.Images of these mice and their tumors are shown in Figure S14A,B (Supporting Information).The average calculated tumor weight confirmed the therapeutic effect of Mn@BSA NCs and Mn@BSA NWs on B16-F10 tumors (P*** < 0.001 or P** < 0.01) (Figure 10C).Intracellular Mn-based complexes or nanocomposites induce necrosis of tumor cells via direct DNA damage or highly toxic hydroxyl radical production. [10,55]More Mn@BSA NCs were internalized by B16-F10 cells, leading to better anticancer efficacy compared to Mn@BSA NWs (Figure 8A).TAMs promote the proliferation, angiogenesis, and migration of cancer cells to induce an immunosuppressive microenvironment, tumor growth, and metastasis. [56]M1 polarization of TAMs was assessed by isolating single cell suspensions from tumor tissues, followed by labeling with the fixable viability dye eFluor 450 (405/450 nm), APC-anti-F4/80 (638/660 nm) as a unique marker of murine macrophages, and PECy7-anti-CD80 (561/785 nm) as a marker of the M1 phenotype for flow cytometry.The dual-positive population (CD80 + /F4/80 + ) induced by Mn@BSA NCs was the highest compared with that induced by Mn@BSA NWs or the control (P*** < 0.001) (Figure S15, Supporting Information, Figure 10D, and Table S5, Supporting Information).The number of positive events in the Mn@BSA NWs group was also higher than that in the control (P*** < 0.001).These results confirmed that Mn@BSA NCs and NWs significantly reprogrammed TAMs towards the M1 phenotype.Accordingly, we suggest that the synergistic reinforcement between the proinflammatory polarization of TAMs and the necrosis of tumor cells induced by Mn@BSA NCs or NWs efficiently repressed tumor growth. [57,58]The underlying mechanism of anticancer activity (including tumor microenvironment reshaping) needs to be further explored.Finally, the mice treated with Mn@BSA NCs or Mn@BSA NWs showed normal growth, which indicated the good biosafety of the therapeutic strategy developed in this study (Figure S14C, Supporting Information).

Conclusion
The binding of trace Mn 2+ to BSA efficiently stimulated humanor murine-derived macrophage polarization through TLR4mediated signaling pathways, and the NW structures assembled with Mn@BSA NCs further boosted immunostimulation.In addition, in vivo investigations confirmed that Mn@BSA NCs or NWs had good biosafety and robust immunoregulatory effects on the activation of peritoneal macrophages and TAMs, and exhibited remarkable anticancer activities.Therefore, we suggest that the Mn@BSA NWs or Mn@BSA NCs developed in this study have significant potential as TLR4 agonists to enhance cancer immunotherapy in future clinics.

Experimental Section
Fabrication of Mn@BSA NCs and Mn@BSA NWs: First, BSA solution was prepared by dissolving 30.0 mg of BSA (Sinopharm Chemical Reagent, China) in PBS (10 mL, pH 7.2-7.4)(Beijing Solarbio Science & Technology, China).Three milliliters of MnCl 2 solution (Sinopharm Chemical Reagent, China) at a concentration of 18.9 μg mL −1 was added into BSA solution under stirring for 12 h.Subsequently, the mixture solution was dialyzed (MWCO: 8.0-14.0kDa) with DI water (≥18 MΩ cm resistivity, Millipore) for 24 h, and lyophilized to obtain Mn@BSA NCs.On this basis, 50.0 mg Mn@BSA NCs was dissolved in 1.25 mL DI water and stirred with 50 mmol L −1 l-GSH (Sinopharm Chemical Reagent, China) for 1 h at 37 °C.Subsequently, 1.25 mL of ethanol was added to the mixture at room temperature for 30 min.The mixture was dialyzed (MWCO: 8.0-14.0kDa) with DI water for 24 h and lyophilized to prepare Mn@BSA NWs.The FITC conjugates were prepared by dissolving 50 mg Mn@BSA NCs or Mn@BSA NWs in 10 mL carbonate buffer (pH 9.0-9.5),followed by the dropwise addition of 0.25 mL of 1 mg mL −1 FITC in dimethyl sulfoxide (DMSO), and stirred at 4 °C in the dark.After 24 h, the reaction solution was repeatedly dialyzed, lyophilized, and stored at 4 °C.The morphologies of the samples were observed using a high-resolution TEM (JEM-2100 UHR, JEOL, Japan) at an accelerating voltage of 200 kV.The size and zeta potential of the nanoparticles were analyzed using DLS (Zetasizer Nano ZS, Malvern Instruments, UK) according to the NIST-NCL joint assay protocol (PCC-1, version 1.2). [53]UV-vis absorption spectra were measured using a spectrophotometer (UV-2700, Shimadzu, Japan).Photoluminescence (PL) spectra were recorded using a spectrofluorometer (FS5, Edinburgh Instruments, UK).Trace amounts of Mn were analyzed by ICP-MS (NexION 300X, Perkin Elmer, USA).
Mn@BSA NCs at 100 μg mL −1 were incubated with 5 × 10 5 mL −1 RAW 264.7 cells in six-well plates for 6 h, respectively.Naive BSA at 100 μg mL −1 , and free Mn 2+ at the equivalent concentration of 8 × 10 −2 μg mL −1 were also added.In addition, untreated RAW 264.7 cells were employed as a control, and cells stimulated with 100 ng mL −1 LPS and 25 ng mL −1 IFN- were a positive control.After 6-h incubation, culture supernatants of the treated RAW 264.7 cells were collected by centrifugation at 3 × 10 3 rpm for 15 min for ELISA assays according to the manufacturer's instructions.
Mn@BSA NCs and Mn@BSA NWs at 50 μg mL −1 were incubated with 5 × 10 5 mL −1 RAW 264.7 cells in six-well plates for 48 h, respectively.Untreated RAW 264.7 cells were used as a control.After 48-h incubation, culture supernatants of the above-treated RAW 264.7 cells were collected by centrifugation at 3 × 10 3 rpm for 15 min for ELISA assays.
Intracellular ROS Measurements: Mn@BSA NCs at 100 μg mL −1 (Mn@BSA-100) was incubated with 5 × 10 5 RAW 264.7 cells mL −1 in sixwell plates for 6 h, respectively.Naive BSA at 100 μg mL −1 and free Mn 2+ at the equivalent concentration of 8 × 10 −2 μg mL −1 were also performed.In addition, untreated RAW 264.7 cells were employed as a control, and cells stimulated with 100 ng mL −1 LPS and 25 ng mL −1 IFN- were used as a positive control.After 6 h of incubation, the culture media were removed, and fresh media containing 20 μmol L −1 of DHE (KeyGen Biotech, China) were subsequently added at 37 °C for 20 min.Afterward, the culture media were removed again and washed twice with fresh media for ROS measurements.The fluorescence intensity at 605 nm with an excitation peak at 518 nm was recorded using a multifunctional enzyme marker (Cytation 3, Biotek, USA).Relative ROS production was determined using Equation (3)   Relative ROS production = Fluorescence intensity of the experimental group Fluorescence intensity of the blank control group TLR4-KO Studies: In total, 50 or 100 μg mL −1 BSA, 4 × 10 −2 or 8 × 10 −2 μg mL −1 Mn 2+ , 50 or 100 μg mL −1 Mn@BSA NCs, and 50 or 100 μg mL −1 Mn@BSA NWs were added into 2 × 10 5 mL −1 RAW-Dual and RAW-Dual KO-TLR4 cells (InvivoGen, USA), respectively.Meanwhile, untreated cells or those stimulated with 100 ng mL −1 of LPS + 25 ng mL −1 of IFN- were used as the control or positive control, respectively.After 24 h, cell culture supernatants were analyzed by a microplate photometer (Filter Max F5, USA) using QUANTI-Blue solution (InvivoGen, USA) to detect SEAP activity as NF-B reporters or QUANTI-Luc 4 Lucia/Gaussia solution (InvivoGen, USA) to detect LUCIA activity as IRFs reporters, respectively.
Confocal Imaging and Cytometry Analysis: RAW 264.7 cells were seeded in six-well plates and incubated overnight at a density of 2.5 × 10 5 cells per well.Afterward, 100 μg mL −1 FITC-Mn@BSA NCs were added into RAW 264.7 cells.After 12-h incubation, the cell samples were rinsed twice with PBS and stained with DAPI, MitoTracker Orange, or LysoTracker Red Probes (KeyGEN Biotechnology, China) for confocal microscopy imaging (LSM710 NLO, Zeiss, Germany).In addition, 1.5 × 10 5 Hela, HUVEC, and B16-F10 cells per well, or 1.5 × 10 5 RAW 264.7 cells per well were incubated for 12 h with 50 μg mL −1 FITC-Mn@BSA NCs or 50 μg mL −1 FITC-Mn@BSA NWs, respectively.Subsequently, the cell samples were rinsed twice with PBS and stained with DAPI for confocal imaging to evaluate cellular uptake.
Western Blotting: RAW 264.7 cells were seeded in six-well plates at 5 × 10 5 cells per well and treated with 100 μg mL −1 BSA, 8 × 10 −2 μg mL −1 Mn 2+ , 100 μg mL −1 Mn@BSA NCs with or without 50 nmol L −1 TAK-242 inhibitor and evaluated by western blotting for MyD88, TRIF, NF-B, p−NF-B, IRF7, and p-IRF7 levels.After 6-h incubation at 37 °C, cell samples were washed twice with PBS solution, and the total protein concentration was analyzed using the bicinchoninic acid assay (Biosharp, China), according to the manufacturer's instructions.Proteins were separated by gel electrophoresis (Smart-Lifesciences Biotechnology, China), and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA).The PVDF membranes were incubated with the following primary antibodies: MyD88 (Proteintech Group, USA), TRIF (Proteintech Group, USA), IRF7, p-IRF7 (Affinity Biosciences, USA), p65, p-p65 (Abcam, USA), and actin (Proteintech Group, USA).The PVDF membrane was subsequently incubated with the corresponding secondary antibodies and supersensitive chemiluminescent agents (Biosharp, China) for blot imaging (SH-Focus 523, Shenhua Science Technology, China) and quantitative analysis (Gel-Pro Analyzer 3.0).The relative protein production was determined after the ratio of interest protein to loading control (-actin) was calculated using Equation (4)   Relative production = Ratio of the experimental group Ratio of the blank control group (4) Biosafety, Immunostimulation, and Anticancer Assay In Vivo: Ten-weekold 12 C57BL/6J mice were divided into four groups and intravenously injected with PBS, Mn@BSA NCs (20.0 mg kg −1 ), Mn@BSA NWs (20.0 mg kg −1 ), or LPS solution (20.0 mg kg −1 ).After 1 d, fundus blood was collected from all mice to obtain serum samples for whole-blood analysis ex vivo (WBC, RBC, hemoglobin [HGB], and PLT levels).After 28 d, blood was collected from all sacrificed mice to obtain serum samples for biochemical analysis ex vivo (BUN, TBIL, ALT, ALP, and AST levels).Sections of the heart, kidneys, liver, lungs, spleen, and brain were prepared for H&E staining.After 28 d, the body weights of the mice were recorded every 2 d.
Twelve 10-week-old C57BL/6J mice were divided into four groups and intraperitoneally injected with PBS, Mn@BSA NCs (20.0 mg kg −1 ), Mn@BSA NWs (20.0 mg kg −1 ), or LPS solution (1.0 mg kg −1 ) to study the polarization of the peritoneal macrophages.Macrophages were harvested from the peritoneal lavage one day post-injection.The cells were washed twice with DMEM, resuspended in DMEM supplemented with 10% FBS, and incubated at 37 °C for 6 h.Adherent cells were cultured in a fresh culture medium the following day.Culture supernatants were collected for ELISA assays ex vivo.Biodistribution in the body was assessed by injecting FITC-Mn@BSA NCs or FITC-Mn@BSA NWs at 20 mg kg −1 into mice through the tail vein; PBS was used as the control.After 24 h, heart, liver, spleen, lung, kidney, and brain samples were collected from all mice for fluorescent imaging using an excitation filter of 450-480 nm and emission filter of 515-575 nm (IVIS Lumina LT Series III in vivo imaging system, PerkinElmer, USA).
Prophylactic tumor models were prepared in vivo by injecting 1 × 10 5 B16-F10 cells with Mn@BSA NCs (20 mg kg −1 ) and Mn@BSA NWs (20 mg kg −1 ) into the right flanks of female C57BL/6J mice (6-8 weeks old).The tumor size and body weight of all the mice were measured every 2 d from day 6.On day 16, three mice from each group were randomly selected and sacrificed to prepare single-cell suspensions isolated from the tumors for flow cytometry.After staining with the fixable viability dye eFluor 450 (eBioscience, USA), single cell suspensions were incubated with anti-mouse CD80-PECy7 [BioLegend, USA] and F4/80 monoclonal antibody-APC [eBioscience, USA]) to detect F4/80/CD80 co-expression in macrophages.On day 18, the remaining mice were sacrificed, and their tumors were collected, photographed, and weighed.
Animal Statistical Analysis: All results are presented as the mean ± standard deviation, and at least three biologically independent samples are used in every experiment.All statistical data were processed using SPSS statistics software (version 26.0).A Student's t-test was used to analyze the differences between two groups, and an ANOVA with Tukey's post-test was used to compare the differences between more than two groups (***p < 0.001, **p < 0.01, *p < 0.05, or no significant differences p > 0.05).

Figure 3 .
Figure 3. Dynamic profile of normalized relative gene expression of RAW 264.7 cells treated with 100 μg mL −1 Mn@BSA NCs or 100 ng mL −1 LPS + 25 ng mL −1 IFN- for 48 h, respectively (n = 3 biologically independent samples).Data were normalized to the maximum value of the relative gene expression folder in the same group.Error bars were based on the standard errors of the mean.

Figure
Figure10.A) Tumor volume curves of all mice from PBS, Mn@BSANCs, and Mn@BSA NWs groups during 20 d (volume = 0.5 × length × width × width, n = 5 biologically independent samples).B) Average tumor volume curves of PBS, Mn@BSANCs, and Mn@BSA NWs groups during 18 d (n = 5 biologically independent samples).C) The weight of tumors harvested from all mice in PBS, Mn@BSANCs, and Mn@BSA NWs groups on day 18 (n = 5 biologically independent samples).D) Population of M1 macrophages labeled by F4/80 + and CD80 + in melanoma tumors treated with Mn@BSANCs and Mn@BSA NWs at day 16 after tumor implantation (n = 3 biologically independent samples).Error bars were based on standard errors of the mean (***P < 0.001 or **P < 0.01 by ANOVA with Tukey's post-test).
Licenses and Permissions: All animal experiments were reviewed and approved by the Experimental Animal Committee of KeyGEN Biotechnology Co. Ltd., China.The animal ethics review approval number is IACUC−20230510.All female 10-week-old C57BL/6J mice were obtained from Shanghai Slac Laboratory Animal Co. Ltd., China, with a laboratory animal production license: SCXK (Shanghai) 2018-0003.The laboratory animals in the experiments were used with a permit license: SYXK (Jiangsu) 2017-0040.All mice in each group were fed in a ventilated CP-4 cage (L × W × H: 290 mm × 178 mm × 160 mm) with a 12-h light/dark cycle (07:00-19:00, light; and 19:00-07:00, dark); the temperature was set between 20 and 26 °C, and the relative humidity was 40%-70% in the animal house.The breeding conditions for all the mice used in the experiments were in accordance with the national standard of the People's Republic of China (No. GB14925-2010), including bedding (GB14924.2-2001),feed (GB14924.3-2010,GB14924.2-2001) with the production license of SCXK (Beijing) 2019-0003, and drinking water (GB5749-2006).All mice were allowed ad libitum access to food and drinking water.Finally, surviving mice were euthanized using carbon dioxide. )