title: Circulation persistence and biodistribution of lyophilized liposome-encapsulated hemoglobin: an oxygen-carrying resuscitative fluid.
abstract: OBJECTIVE
  To evaluate the circulation persistence and organ biodistribution of a freeze-dried, oxygen carrying resuscitative fluid: liposome-encapsulated hemoglobin.
  
  
  METHODS
  Randomized, animal studies.
  
  
  METHODS
  Accredited animal research facilities.
  
  
  METHODS
  Normal female Balb/c mice and male New Zealand rabbits.
  
  
  METHODS
  Two groups of normal female Balb/c mice were injected in the tail vein with either lyophilized liposome-encapsulated hemoglobin (n = 9) that was reconstituted just before administration, or with unlyophilized liposome-encapsulated hemoglobin (n = 9) as a comparison. Two groups of male New Zealand rabbits were injected in the ear vein with either lyophilized 99mTc-liposome-encapsulated hemoglobin (n = 6) or unlyophilized 99mTc-liposome-encapsulated hemoglobin as a comparison (n = 6). After injection, mice were anesthetized by brief inhalation of halothane followed by blood sampling through the retro-orbital sinus. Rabbits were anesthetized 30 mins before liposome-encapsulated hemoglobin administration with an intramuscular injection of a 5:1 mixture of ketamine (50 mg/kg) and xylazine (10 mg/kg). Rabbits were then dynamically imaged for 90 mins, housed, and at 20 hrs, imaging again followed by autopsy and tissue sampling to validate imaged organ biodistributions.
  
  
  METHODS
  Circulation persistence in the mouse was measured by removing a blood sample at various time points up to 24 hrs after injection. The blood sample was centrifuged in a hematocrit capillary tube and the disappearance of the sedimented liposome-encapsulated hemoglobin fraction was measured. The change in the sedimented fraction of the liposomes with time was used to generate circulation persistence profiles in mice. The circulation persistence and organ biodistribution of 99mTc-liposome-encapsulated hemoglobin was measured by circling regions of interest on computer-generated gamma camera images. These image intensities were then calculated as a function of total injected dose which was measured from a known volume and activity of 99mTc-liposome-encapsulated hemoglobin. Actual tissue uptake was estimated from images by subtracting blood pool contribution which was measured by injecting 99mTc-labeled rabbit red cells. Imaged organ biodistribution was validated at 20 hrs by measuring activity in weighed portions of tissue after autopsy.
  
  
  RESULTS
  The mean circulation half-life of liposome-encapsulated hemoglobin in mice injected at a dose of 1.0 g phospholipid/kg mouse and 1.95 g hemoglobin/kg was approximately 10.4 +/- 0.5 (SD) hrs. The circulation half-life of lyophilized liposome-encapsulated hemoglobin was 10.7 +/- 0.7 hrs. The circulation profiles demonstrate a rapid removal phase over the first 4 hrs after injection, followed by a secondary slow removal measured up to 24 hrs. The rapid removal phase of liposome-encapsulated hemoglobin and lyophilized liposome-encapsulated hemoglobin in the rabbit (injected at the same dose) indicated that lyophilized liposome-encapsulated hemoglobin persists longer than the unlyophilized form in the first 4 hrs after injection. The organ biodistributions of unlyophilized 99mTc-liposome-encapsulated hemoglobin and lyophilized 99mTc-liposome-encapsulated hemoglobin in the rabbit demonstrate that the reticuloendothelial system is the primary site of removal, with significant uptake of lyophilized 99mTc-liposome-encapsulated hemoglobin by the liver (15.6 +/- 1.0%), bone marrow (12.6 +/- 1.6%), and spleen (9.7 +/- 1.1%). The kidneys showed little accumulation of unlyophilized 99mTc-liposome-encapsulated hemoglobin or lyophilized 99mTc-liposome-encapsulated hemoglobin (1.6 +/- 0.2% and 1.8 +/- 0.1%, respectively), an important result for the efficacy and safety of this hemoglobin-based blood substitute. (ABSTRACT TRUNCATED)
authors.affiliation: Center for Biomolecular Science and Engineering, Naval Research Laboratory, Washington, DC 20375-5000.
authors.forename: A S
authors.initials: AS
authors.lastname: Rudolph
authors.forename: R O
authors.initials: RO
authors.lastname: Cliff
authors.forename: R
authors.initials: R
authors.lastname: Klipper
authors.forename: B
authors.initials: B
authors.lastname: Goins
authors.forename: W T
authors.initials: WT
authors.lastname: Phillips
mesh_terms: D000818:Animals; D001802:Blood Substitutes / Q000378:metabolism / Q000493:pharmacokinetics*; D005260:Female; D006207:Half-Life; D006454:Hemoglobins / Q000378:metabolism / Q000493:pharmacokinetics*; D008081:Liposomes; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010100:Oxygen / Q000378:metabolism*; D011817:Rabbits; D012151:Resuscitation; D014018:Tissue Distribution
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; D008081:Liposomes; D010100:Oxygen