Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: Schematic illustration of in vivo tumor immunotherapy enhanced by mRNA/HNPs through intravenous injection. H18 lipid, DOPE, cholesterol, DMG-PEG 2000 and mRNA were mixed to form mRNA/H 18 NPs with the special multilamellar concentric nanostructures. Following intravenous administration, mRNA/H 18 NPs demonstrated preferential adsorption of complement C3 proteins to form a characteristic protein corona, resulting in specific mRNA transfection in the spleen, especially in splenic dendritic cells. When encapsulating tumor antigen-encoding mRNA, the mRNA/H 18 NPs achieved precise transfection of the antigen mRNA in splenic dendritic cells. This targeted delivery stimulated dendritic cell maturation and subsequent antigen presentation, initiating robust T cell priming. The activated antigen-specific cytotoxic T lymphocytes then infiltrated into tumor tissues, ultimately inducing tumor cell elimination.

Article Snippet: Mouse Complement C3 was purchased from MedChemExpress.

Techniques: In Vivo, Injection, Adsorption, Transfection, Immunopeptidomics

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: In vivo splenic DC-specific transfection of mRNA/H 18 NPs and in vitro protein corona analysis of mRNA/H 18 NPs. (A) EGFP protein expression in splenic cell subsets of C57BL/6J mice 24 h post intravenous injection of different formulations. (B) The top 5 most abundant plasma proteins adsorbed on mRNA/H 18 NPs (C3: Complement C3; Ighm: Immunoglobulin heavy constant mu; Hbat1: Alpha-globin; Itih4: Inter alpha-trypsin inhibitor, heavy chain 4; Cnn2: Calponin). (C) Heatmap plot of major proteins in the protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. PBS group was used as a negative control. (D) Quantification of major adsorbed protein categories of different formulations. (E) Complement C3 abundance in protein corona adsorbed on mRNA/MC3-LNPs and mRNA/H 18 NPs. (F) Bioluminescence images of major organs and (G) Quantification of total bioluminescence flux in the spleen from C57BL/6J mice 6 h after intravenous injection of mLuc/H 18 NPs (mLuc dose of 0.25 mg kg −1 ). Mice were pre-treated with cobra venom factor (CVF) or PBS. (H) Fluorescence quantification of Cy5 mRNA delivered by uncoated or complement C3-coated Cy5-mRNA/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. (I) Bioluminescence intensity of luciferase protein translated from mRNA delivered by uncoated or complement C3-coated mLuc/H 18 NPs in BMDCs. BMDCs were pre-incubated with anti-CD11b (CR3) or anti-IgG blocking antibody. Data were shown as mean ± SD (n = 3).

Article Snippet: Mouse Complement C3 was purchased from MedChemExpress.

Techniques: In Vivo, Transfection, In Vitro, Expressing, Injection, Clinical Proteomics, Negative Control, Combined Bisulfite Restriction Analysis Assay, Fluorescence, Incubation, Blocking Assay, Luciferase

Journal: Blood Vessels, Thrombosis & Hemostasis

Article Title: A second generation of C3 humanized rats for preclinical evaluation of human C3 inhibitors

doi: 10.1016/j.bvth.2026.100138

Figure Lengend Snippet: C3 inhibitor AMY-101 inhibits the alternative but not classical pathway complement-mediated hemolysis in the second-generation hC3 rats in vitro. (A) The C3 inhibitor AMY-101 (0.3-20μM) was incubated with rabbit RBCs in the presence of 80% WT rat serum, or second-generation hC3 rat serum in Mg 2+ -EGTA buffer at 37°C for 30 minutes, then hemolysis was quantitated, showing that AMY-101 almost completely inhibited the second-generation hC3 rat alternative pathway-mediated hemolysis at a concentration as low as 2.5μM while did not affect the WT rat alternative pathway-mediated hemolysis at a concentration as high as 20μM. (B) The C3 inhibitor AMY-101 (0.3-10μM) were incubated with antibody-sensitized sheep RBCs in the presence of 10% second-generation hC3 rat serum in GVB ++ buffer at 37°C for 30 minutes, then hemolysis was quantitated, showing that AMY-101 did not have any effect on the second-generation hC3 rat classical pathway-mediated hemolysis at a concentration as high as 10μM. 2nd gen, second generation.

Article Snippet: In these assays, human and rat C3 proteins were detected using a complement C3 polyclonal antibody (MP Biomedicals, catalog no. 0855117) and a secondary anti-goat immunoglobulin G (IgG) (heavy chain+light chain [H+L]) polyclonal antibody (SouthernBiotech, catalog no. 6160-05).

Techniques: In Vitro, Incubation, Concentration Assay

Journal: Journal of Extracellular Vesicles

Article Title: Yersinia enterocolitica O:3 Outer Membrane Vesicles as a Platform for Complement Activation

doi: 10.1002/jev2.70270

Figure Lengend Snippet: (A) The impact of OMVs on germicidal activity of serum against YeO3‐c bacteria. The influence of OMVs‐associated with: LPS polysaccharide chain length (a) and pYV‐coded factors (b). Data from one of at least two experiments with the use of separately prepared culture supernatants with similar results are presented. 1 sterile cell culture supernatants (secreted by bacteria grown to OD 600 = 0.6) were used as a source of OMVs; 2 expression at 37°C only; 3 NHS inactivated 30 min at 56°C. (B) Y. enterocolitica O:3 OMV‐induced complement activation in the presence of calcium and magnesium chelators. ELISA plates were coated with 10 8 of OMVs secreted by YeS‐c bacteria grown at 4°C, 22°C and 37°C and with OMVs of YeRa‐c, YeRd1‐c and YeRe‐c variants propagated at 37°C. After incubation with/without EDTA, EGTA or EGTA/Mg 2+ (EGTA supplemented with Mg 2+ ions), products of C3 activation were detected with specific antibodies. Data from one of two experiments with similar results are presented. (C) Comparison of complement activation and MBL binding by Yersinia enterocolitica O:3 bacterial cells, LPS and OMVs. ELISA plates were coated with 50 ng/well of bacteria, LPS or OMVs. The deposition of C3 (a, possible AP, CP and LP involvement), C4 (b, possible CP and LP involvement) or C4 LP‐dependent (d) activation products, TCC formation (c, possible AP, CP and LP involvement) and MBL‐binding (e) was analyzed after preincubation with normal human serum (filled columns) or with EDTA‐treated NHS (stripped columns) or without serum (open columns, negative control). Data from one of two experiments with similar results are presented. Dots represent individual OD values for each well. (D) Recognition of Y. enterocolitica O:3 OMVs by human mannose‐binding lectin. YeS‐c_37°C bacteria (1), OMVs (2) and LPS (3) were separated in SDS‐PAGE. After transfer to nitrocellulose and incubation with NHS, the bound MBL was detected with specific mAb (a). The experiment was performed at least 5 times. To control the loading of bacteria, LPS or OMVs the presence of OPS in separated samples was confirmed with anti‐6‐deoxy‐L‐altropyranose mAbs (b).

Article Snippet: Depletion of functional C3 from serum was verified in Western blot [acc. to Younger et al. ( )], using goat antibodies against mouse C3 (MP Biomedicals, USA), HRP‐conjugated anti‐goat Ig (Dako) for detection of intact C3 α‐chain and ECL detection system.

Techniques: Activity Assay, Bacteria, Sterility, Cell Culture, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Binding Assay, Negative Control, SDS Page, Control