EUROPEAN  TISSUE  REPAIR  SOCIETY

4. Drugs to Augment Cell Wound Healing

Amphiphilic and Hydrophilic
Co-polymers The possibility of augmenting repair of cell membrane wounds using synthetic surfactants is now well established although the exact mechanisms of repair (i.e., sealing) is still under investigations. (Lee et al., 1994) Known effective agents include the surfactant class of poloxamers, representing a group of tri-block copolymers. Poloxamer 188 (P188) made by BASF Corporation (Pluronic F68), was initially shown to seal cells against loss of carboxy-fluorescein dye after electroporation as seen in Figure 1. (Lee et al., 1992) In the following years, it has been demonstrated that P188 can also seal membrane pores in skeletal muscle cells after heat shock (Padanilam et al. 1994) and enhance the functional recovery of lethally heat-shocked fibroblasts (Merchant et al., 1998) and following high-dose ionizing radiation (Greenebaum, in press). Very recently, P188 has been shown to protect embryonic hippocampal neurons against death due to neurotoxic induced loss of membrane integrity (Marks et al., 1998).

Figure 1: Kinetics of flourescent dye loss from
electroporated muscle cells (control) shown.
Dye loss was retarded by neutral 10 kilodalton
dextran and was stopped by P188.
(from Lee et al., 1992)

Other surfactants, such as Poloxamine 1107, of a similar class of quad-block copolymers, have been shown to reduce testicular ischemia-reperfusion injury (Palmer et al. 1998) and hemoglobin leakage from erythrocytes after ionizing radiation (Hannig et al., 1999). In all the aforementioned investigations the observed phenomena were attributed to sealing of permeabilized cell membranes by the surfactants. In addition, the effect of P188 infusions in reducing duration and severity of acute painful episodes of sickle cell disease is presently also explained by beneficial surfactant-erythrocyte membrane interactions (Adams-Graves et al., 1997).

Poloxamers and poloxamines belong to a class of water-soluble multi-block copolymers that have important 'surface-active' properties. Poloxamer 188 is a tri-block copolymer often abbreviated as POE-POP-POE with POE and POP representing poly(oxyethylene) and poly(oxypro-pylene) respectively. The POE chains are hydrophilic due to their short carbon unit between the oxygen bridges whereas the POP center is hydrophobic due to the larger propylene unit (see Figure 2).

Figure 2: Chemical structure of poloxamers.
The series of different poloxamers is
constituted through varying numbers and
ratios for a and b.

The poloxamer series covers a range of liquids, pastes and solids, with molecular weights varying from 1100 to about 14,000. The ethylene oxide propylene oxide weight ratios range from about 1:9 to about 8:2. Poloxamer 188 (P188) has an average molecular weight of about 8400. It is prepared from a 1750 average molecular weight hydro-phobe and its hydrophile comprises about 80% of the total molecular weight. The length of the hydrophilic chain might influence the strength of the interaction between the permeabilized membrane and the surfactant and thereby influence the effectiveness of a surfactant in sealing permeabilized membranes. The poloxamine series is slightly different from the poloxamer series in its chemical structure. The hydrophobic center consists of two tertiary amino groups carrying both two hydrophobic poly(oxypro-pylene) chains of equal length each followed by a hydrophilic poly(oxyethylene) chain. Thus, it still can be described as a tri-block copolymer but is much bulkier than poloxamers (see Figure 3).

Most often, P188 is used at a sub-critical micelle concentration (sub-CMC) of 0.1mM to1.0mM for membrane repair in vitro (Lee et al., 1992). Above their CMC sur-factants self-aggregate to micelles causing the (active) surfactant monomer concentration to remain constant (= CMC) independently of the total surfactant concentration (Kabanov et al., 1995). The capability of these amphi-philic copolymers to repair cell membranes at millimolar concentrations distinguishes the sealing capability of this copolymer from purely hydrophilic polymers such as poly(-ethyleneglycol) (PEG) which require molar concentrations (Shi et al., 1999)

PEG has a long history in cell-cell fusion applications and is also very well investigated in the fusion of model membranes (Lee and Lentz, 1997). It is hypothesized that PEG can force very close contact between vesicle membranes by lowering the activity of water adjacent to the membrane (Arnold et al., 1990). But even at the required high concentrations (e.g., 17.5%, Lee and Lentz 1997), PEG-mediated vesicle fusion only occurs when the organization of lipid in the bilayers are substantially perturbed from their equilibrium values. It is our working hypothesis that the tri-block copolymers with their hydrophobic center chains act like membrane defect targeted PEG molecules, thus requiring much lower concentrations to achieve fusion (sealing) of a permeabilized cell membrane.The membrane repair mechanism of these surfactants is widely discussed. It is not yet understood whether the surfactants interact only with the disrupted parts of the membrane to seal the membrane wounds or whether their integration and interaction with the entire bilayer alters the membrane properties in a way to repair itself (e.g., decreased fluidity) (Sharma et al., 1996, Baekmark et al., 1997).