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: The second-generation hC3 rats express human, but not rat C3 in their plasma and tissues, at levels higher than those in the first-generation hC3 rats. (A) Plasma from both human and rat samples were collected, and the presence of human (hC3) and rat C3 (rC3) were evaluated by western blots. Purified human C3 (hC3), rat C3 (rC3), human C3 depleted serum (hC3-dpl), and C3 knockout rat serum rat plasma (rC3-KO) were used as controls. Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls. 1st gen, first generation; 2nd gen, second generation.

Article Snippet: Blood levels of human C3 protein were measured in the identified human C3 Tg rats using an enzyme-linked immunosorbent assay (ELISA) kit (Hycult, catalog no. HK366) to identify the founder rats with close-to-physiological expression levels of human C3.

Techniques: Clinical Proteomics, Western Blot, Purification, Knock-Out, Enzyme-linked Immunosorbent Assay, Reverse Transcription

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: To evaluate the inhibitory effect of the C3 inhibitor AMY-101 (MedChemExpress, catalog no. HY-P1717) on alternative pathway activation in the new hC3 rat serum, 80% hC3 rat serum or WT rat serum was incubated with rabbit RBCs in the presence of 0.3 to 20μM AMY-101 in Mg 2+ -EGTA GVB buffer at 37°C for 30 minutes.

Techniques: In Vitro, Incubation, Concentration Assay

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: AMY-101 significantly prolongs the survival of rabbit RBCs in the second-generation hC3 rats in vivo. Rabbit RBCs (2 × 10 9 ), labeled with Vybrant Dil dye, were IV injected into second-generation hC3 rats in the presence or absence of 1 mg/kg AMY-101. Blood samples were collected at 0.5, 1, 2, 5, 10, 20, and 30 minutes after injection and the survived circulating fluorescence-labeled rabbit RBCs were quantitated by flow cytometry. (A) Representative flow cytometry data showing the circulating rabbit RBCs without and with AMY-101 treatment at different time points. (B) Summary of the rabbit RBC (ER) survival data over the 0.5-30 minute course in the second-generation hC3 rats with or without AMY-101 treatment. Two-way analysis of variance, ∗∗∗∗ P < .001; ∗∗∗ P < .001; ∗ P < .05. FSC, forward scatter; ER, rabbit erythrocytes; PE, phycoerythrin.

Article Snippet: To evaluate the inhibitory effect of the C3 inhibitor AMY-101 (MedChemExpress, catalog no. HY-P1717) on alternative pathway activation in the new hC3 rat serum, 80% hC3 rat serum or WT rat serum was incubated with rabbit RBCs in the presence of 0.3 to 20μM AMY-101 in Mg 2+ -EGTA GVB buffer at 37°C for 30 minutes.

Techniques: In Vivo, Labeling, Injection, Fluorescence, Flow Cytometry

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: Anti-rabbit RBC IgM/IgG antibodies exist in the rats, and the AMY-101–treated rats exhibit classical pathway hemolytic activities comparable to the mock-treated rats. Rabbit RBCs were incubated with second-generation hC3 rat serum (+EDTA) for 30 minutes on ice, then washed. The surface binding rat IgM (A) and IgG (B) on the rabbit RBCs were detected by flow cytometry. To evaluate classical pathway hemolytic activity, antibody-sensitized sheep RBCs were incubated with 10% serum from the AMY-101 or mock-treated second-generation hC3 rats in the presence of Ca 2+ and Mg 2+ for 20 minutes, and then hemolysis was quantitated (C). (D) In a different experiment, additional AMY-101 (0-10μM) was added into the AMY-101–treated rat serum, and the classical pathway hemolytic activities were measured again.

Article Snippet: To evaluate the inhibitory effect of the C3 inhibitor AMY-101 (MedChemExpress, catalog no. HY-P1717) on alternative pathway activation in the new hC3 rat serum, 80% hC3 rat serum or WT rat serum was incubated with rabbit RBCs in the presence of 0.3 to 20μM AMY-101 in Mg 2+ -EGTA GVB buffer at 37°C for 30 minutes.

Techniques: Incubation, Binding Assay, Flow Cytometry, Activity Assay

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: The second-generation hC3 rats express human, but not rat C3 in their plasma and tissues, at levels higher than those in the first-generation hC3 rats. (A) Plasma from both human and rat samples were collected, and the presence of human (hC3) and rat C3 (rC3) were evaluated by western blots. Purified human C3 (hC3), rat C3 (rC3), human C3 depleted serum (hC3-dpl), and C3 knockout rat serum rat plasma (rC3-KO) were used as controls. Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls. 1st gen, first generation; 2nd gen, second generation.

Article Snippet: Arrows pointed to the human or rat C3 band. (B) Plasma hC3 levels in the second-generation hC3 rats (both male and female) were evaluated using a human C3 ELISA kit (Hycult) and compared to the levels in the first-generation hC3 rat plasma. (C) Total RNA was extracted from the liver, retina, spleen, kidney, and brain of WT, second-generation, and first-generation hC3 rats, and then semiquantitative reverse transcription PCR was used to detect and quantitate levels of human C3 (hC3) transcripts using the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal controls.

Techniques: Clinical Proteomics, Western Blot, Purification, Knock-Out, Enzyme-linked Immunosorbent Assay, Reverse Transcription