Biological evaluation of liposome-encapsulated hemoglobin surface-modified with a novel PEGylated nonphospholipid amphiphile

Abstract

Traumatic injury is often associated with hemorrhagic shock. Liposome-encapsulated hemoglobin (LEH) is being developed as an artificial oxygen carrier to address post-hemorrhage oxygen and volume deficit. Here, we report a new composition of LEH based on the use of polyethylene glycol (PEG2K ) conjugated with nonphospholipid hexadecylcarbamoylmethylhexadecanoate (HDAS) to modify the surface of LEH particles. LEH was manufactured by the high-pressure homogenization method using dipalmitoylphosphatidylcholine (∼38 mol%), cholesterol (∼38 mol%), HDAS (∼20 mol%), and highly purified stroma-free human hemoglobin. HDAS-PEG2K was postinserted into the resultant LEH to generate HDAS-PEG2K -LEH. We investigated the potential immune response to HDAS-PEG2K -LEH in a mice model. At the same time, the preparation was tested in a rat model to study the effect of repeated HDAS-PEG2K -LEH injection over 4 weeks. We found that HDAS-PEG2K modification substantially reduced the circulating levels of anaphylatoxins C3a and C5a, as well as plasma levels of thromboxane B2, in mice. Repeated injections of HDAS-PEG2K -LEH in rats did not appear to alter its clearance profile after 4 weeks of treatment. No antibody response against human hemoglobin or PEG was detected in rat plasma. Histological observations of lung, liver, spleen, and kidney were not significantly different between saline-treated rats and HDAS-PEG2K -LEH-treated rats. Immunohistochemical staining for rat heme oxygenase-1 (HO-1) did not show induced expression of HO-1 in these organs. These results suggest that the new surface modification of LEH is immune-neutral and does not adversely affect histology even after repeated administration.

Keywords: Complement; Liposome-encapsulated hemoglobin; Oxygen delivery.

Figure 1

(A and B) SEM of LEH surface-modified with HDAS-PEG_2K. (C) A confocal micrograph of HDAS-PEG_2K-LEH showing the presence of PEG on the surface of the liposome. HDAS-PEG_2K-LEH was labeled with FITC-labeled anti-PEG antibody.

Figure 2

(A) and (B) are the levels of complement proteins C3a and C5a, respectively. The complement proteins were estimated by ELISA in BALB/c mice serum (n = 3/group). Treatment with human Hb was taken as a positive control. (C) Plasma TxB2 concentrations (mean ± standard error of mean) measured in CF1 mice after 60 min of plain liposome, DSPE-PEG_2K-LEH, and HDAS-PEG_2K-LEH infusion.

Figure 3

Clearance profile of HDAS-PEG_2K-LEH in plasma with respect to (A) PEG and (B) human Hb. The rats were administered saline or HDAS-PEG_2K-LEH every week for 3 weeks. On fourth week (28^th day), all the rats were injected HDAS-PEG_2K-LEH. Blood was collected to estimate the amount of PEG and human Hb by ELISA.

Figure 4

(A) Anti-PEG and (B) anti-human Hb antibody response in rats injected with HDAS-PEG_2K-LEH or saline over 4 weeks (0, 7, 14, 21 days). Rat serum analyzed by ELISA. One group received weekly injections of HDAS-PEG_2K-LEH at a dose rate of 10 ml/kg, whereas the other group was given equal volume of saline (control).

Figure 5

(A) Rat organ weights (lung, liver, kidney, and spleen) after HDAS-PEG_2K-LEH and saline administration for 4 weeks (0, 7, 14, 21 days) at a dose rate of 10 ml/kg. The values are mean ± SEM. (B) Histology of lung, liver, spleen, and kidney. One group received repeated weekly injections of HDAS-PEG_2K-LEH for 4 weeks (10 ml/Kg), whereas the other group was given equal volume of saline (control).

Figure 6

Immunohistochemical staining for HO-1 in rat lung, liver, spleen, and kidney. The treatment groups are the same as defined in figures 3–5. The tissues were stained (brown-color) with antibody against rat HO-1.