Early treatment with Fibrinogen γ-chain peptide-coated, ADP-encapsulated Liposomes (H12-(ADP)-liposomes) ameliorates post-partum hemorrhage with coagulopathy caused by amniotic fluid embolism in rabbits.

BACKGROUND: Amniotic fluid embolism is an unpredictable and sometimes lethal complication of childbirth. Fibrinogen γ-chain peptide-coated, ADP-encapsulated Liposomes (H12-(ADP)-liposomes), which were developed as a platelet substitute, may be useful to control postpartum hemorrhage with consumptive coagulopathy. OBJECTIVE: This study aimed to establish a hemodynamically stable amniotic fluid embolism animal model and evaluate the efficacy of H12-ADP-liposome infusion in the initial management of postpartum hemorrhage complicated with amniotic fluid embolism-involved coagulopathy. STUDY DESIGN: Pregnant New Zealand white rabbits (28th day of pregnancy or normal gestation period of 29-35 days) underwent cesarean delivery, followed by intravenous administration of amniotic fluid (a total of 3.0 mL administered in 4 doses over 9 minutes). Thereafter, uncontrolled postpartum hemorrhage was induced by transecting the right midartery and concomitant vein in the myometrium. After initial bleeding for 5 minutes, rabbits received isovolemic fluid resuscitation through the femoral vein with an equivalent volume of blood loss every 5 minutes for 60 minutes. The transfusion regimens included platelet-rich plasma, platelet-poor plasma, and a bolus administration of H12-ADP-liposomes followed by platelet-poor plasma transfusion (8 rabbits per group). Moreover, 60 minutes after initiation of bleeding, rabbits received surgical hemostasis by ligation of bleeding vessels, except in cases with spontaneous hemostasis. RESULTS: The administration of amniotic fluid caused thrombocytopenia (56±3 × 103/µL) and prolonged both clotting time (before administration: 130.0±3.0 to 171.0±5.0 seconds) and prothrombin time (4.5±0.1 to 4.7±0.1 seconds). After the initial 5-minute bleeding in the rabbits, the mean arterial pressure fell to 43±2 mm Hg. Platelet-poor plasma transfusion alone further prolonged clotting time and prothrombin time at 60 minutes (192.0±10.0 and 5.2±0.1 seconds, respectively) with decreasing mean arterial pressure to <40 mm Hg. By contrast, the administration of H12-ADP-liposomes followed by platelet-poor plasma transfusion reduced the prolonged clotting time (153.0±5.0 seconds) and prothrombin time (4.9±0.1 seconds) similar to platelet-rich plasma transfusion (154.0±11.0 and 4.9±0.1 seconds, respectively) at 60 minutes. These rabbits maintained a mean arterial pressure of >45 mm Hg throughout the experiment. H12-ADP-liposome infusion and platelet-poor plasma transfusion and platelet-rich plasma transfusion yielded spontaneous hemostasis in 4 of 8 rabbits, whereas platelet-poor plasma transfusion did not stop bleeding in any of the rabbits. The total blood loss was 59±17 mL in the H12-ADP-liposomes and platelet-poor plasma group, which was half of that in the platelet-poor plasma group (124±10 mL). CONCLUSION: H12-ADP-liposome infusion may be effective in the initial management of postpartum hemorrhage complicated with amniotic fluid embolism, resulting in mitigation of consumptive coagulopathy.

Development of the observation of membrane fusion with label-free liposomes by calcium imaging.

Liposomes are artificial vesicles composed of lipid bilayers that have enabled drugs to be encapsulated and delivered to tumor tissue. Membrane-fusogenic liposomes fuse with the plasma membranes of cells to deliver encapsulated drugs directly to the cytosol, which makes it a promising method for rapid and highly efficient drug delivery. In a previous study, liposomal lipid bilayers were labeled with fluorescent probes, and colocalization of labeled lipids with plasma membrane was observed under a microscope. However, there was concern that fluorescent labeling would affect lipid dynamics and cause liposomes to acquire membrane fusogenic ability. In addition, encapsulation of hydrophilic fluorescent substances in the inner aqueous phase sometimes requires an additional step of removing unencapsulated substances after preparation, and there is a risk of leakage. Herein, we propose a new method to observe cell interaction with liposomes without labeling. Our laboratory has developed two types of liposomes with different cellular internalization pathways, i.e., endocytosis and membrane fusion. We found that cytosolic calcium influx would be triggered following the internalization of cationic liposomes, and different cell entry routes led to different calcium responses. Thus, the correlation between cell entry routes and calcium responses could be utilized to study liposome-cell interactions without fluorescent labeling lipids. Briefly, liposomes were added to phorbol 12-myristate 13-acetate (PMA)-primed THP-1 cells, and calcium influx was measured by time-lapse imaging using a fluorescent indicator (Fura 2-AM). Liposomes with high membrane fusogenic ability elicited a strong transient calcium response immediately after adding liposomes, whereas those taken up mainly by endocytosis elicited multiple weak calcium responses. In order to verify the cell entry routes, we also tracked the intracellular distribution of fluorescent-labeled liposomes in PMA-primed THP-1 cells using a confocal laser scanning microscope. It was shown that for fusogenic liposomes, colocalization with plasma membrane occurred at the same time as calcium elevation, whereas for liposomes with a high endocytosis potential, fluorescent dots were observed in the cytoplasm, suggesting the cell internalization by endocytosis. These results suggested that the calcium response patterns correspond to cell entry routes, and membrane fusion can be observed by calcium imaging.

Temperature-Responsive Liposome-Linked Immunosorbent Assay for the Rapid Detection of SARS-CoV-2 Using Immunoliposomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the etiological agent of coronavirus disease 2019 (COVID-19), has infected more than 340 million people since the outbreak of the pandemic in 2019, resulting in approximately 55 million deaths. The rapid and effective diagnosis of COVID-19 patients is vital to prevent the spread of the disease. In a previous study, we reported a novel temperature-responsive liposome-linked immunosorbent assay (TLip-LISA) using biotinylated-TLip that exhibited high detection sensitivity for the prostate-specific antigen. Herein, we used immunoglobulin-TLip (IgG-TLip), in which the antibodies were directly conjugated to the liposomal surface to simplify pretreatment procedures and reduce the detection time for SARS-CoV-2. The results indicated that TLip-LISA could detect the recombinant nucleocapsid protein and the nucleocapsid protein in inactivated virus with 20 min incubation time in total, and the limit of detection was calculated to be 2.2 and 1.0 pg/mL, respectively. Therefore, TLip-LISA has high potential to be used in clinic for rapid diagnosis and disease control.

Arginine-based cationic liposomes accelerate T cell activation and differentiation in vitro.

Cationic liposomes are versatile lipid nanocarriers to improve the pharmacological properties of drug payloads. Recent advantages include the application of their intrinsic immunostimulatory effects to enhance immune activation. Herein, we report for the first time the structural effect of cationic lipids in promoting T cell activation and differentiation in vitro. Two types of cationic liposomes R3C14 and R5C14 were prepared from single type of lipids Arg-C3-Clu2C14 or Arg-C5-Clu2C14, which bear arginine head group and ditetradecyl tails but vary in the carbon number of the spacer in between. Murine CD8 or CD4 T cells were pretreated with 50 µM of each type of liposomes for 2 h, followed by stimulation with anti-CD3/CD28 antibodies for 24 h. In comparison to liposome-untreated T cells, R5C14-pretreatment induced a robust T cell activation (IL-2, CD25+) and differentiation into effector cells (CD44high, CD62Llow), whereas R3C14 did not show comparable effect. Furthermore, a weak activation of nuclear factor of activated T cells (NFAT) was detected in Jurkat-Lucia NFAT cells (InvivoGen), suggesting a potential signaling pathway for the liposomal effect. Although R5C14 liposomes did not activate T cells without subsequent CD3/CD28 stimulation, this study implied a recessive effect of some cationic adjuvant in priming T cells to enhance their responsiveness to antigens.

H12-(ADP)-liposomes for hemorrhagic shock in thrombocytopenia: Mesenteric artery injury model in rabbits.

BACKGROUND: Damage control resuscitation improves patient outcomes after severe hemorrhage and coagulopathy. However, effective hemostasis methods for these critical situations are lacking. OBJECTIVE: We evaluated the hemostatic efficacy of fibrinogen γ-chain (HHLGGAKQAGDV, H12)-coated, adenosine-diphosphate (ADP)-encapsulated liposomes (H12-[ADP]-liposomes) in thrombocytopenic rabbits with hemorrhagic shock. METHODS: Acute thrombocytopenia (80%) was induced in rabbits that also received mesenteric vessel injury, leading to hemorrhagic shock. Five minutes after injury, subjects received intravenous bolus injection with H12-(ADP)-liposomes (20 mg/kg), followed by isovolemic transfusion with stored red blood cells (RBCs)/platelet poor plasma (PPP) (RBC:PPP = 1:1 [vol/vol]), or lactated Ringer solution every 5 min to compensate blood loss. One group received H12-(phosphate buffered saline [PBS]) liposomes followed by RBC/PPP. Additional groups were received isovolemic transfusion with RBC/platelet rich plasma (PRP) (RBC:PRP = 1:1 [vol/vol]), RBC/PPP, PPP alone, or lactated Ringer solution. RESULTS: Treatment with H12-(ADP)-liposomes followed by RBC/PPP transfusion and RBC/PRP transfusion effectively stopped bleeding in all thrombocytopenic rabbits. In contrast, three of 10 rabbits treated with RBC/PPP failed hemostasis, and no rabbits receiving lactated Ringer solution stopped bleeding or survived. Twenty-four hours after hemorrhage, 80% of rabbits receiving H12-(ADP)-liposome followed by RBC/PPP transfusion survived and 70% of rabbits receiving RBC/PRP transfusion also survived, although RBC/PPP-transfused rabbits showed 40% survival. Rabbits receiving H12-(ADP)-liposomes followed by lactated Ringer solution showed a transient hemostatic potential but failed to survive. H12-(PBS)-liposomes showed no beneficial effect on hemostasis. Neither the PPP group nor the lactated Ringer group survived. CONCLUSION: H12-(ADP)-liposome treatment followed by RBC/PPP may be effective in lethal hemorrhage after mesenteric vessel injury in coagulopathic rabbits.