ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH., 23(1), 135-139 (1995)

HAEMOLYTIC PROPERTIES OF PLURONIC SURFACTANTS AND EFFECTS OF PURIFICATION

Kenneth C. Lowe, Barbara A. Furmidge and Stephen Thomas Department of Life Science. University of Nottingham,

University Park, Nottingham NG7 2RD, U.K.

ABSTRACT

The effects of incubating blood from mice, rats, rabbits or hamsters with either a commercial grade or a silica-purified fraction of Pluronic F-68 or Pluronic F-38 have been studied. Incubation of blood with up to 4.0% (w/v) of commercial or purified Pluronic F-68 produced no detectable haernolysis ( < 0.1 %). Haernolysis did occur with concentrations of commercial Pluronic F-68 above 4.0% (w/v). This was maximal with rat blood incubated with 10.0% (w/v) Pluronic F-68, where the mean haemolysis was 4.7 % 1.5%; the mean haemolysis in rat blood was reduced to 0.5 5 0.3% (P < 0.05) following incubation with the purified Pluronic F-68 fraction. Neither commercial grade or purified Pluronic F- 38 produced any significant haemolysis when incubated with rat or hamster blood. Incubation of rabbit blood with 10.0% (w/v) commercial Pluronic F-38 produced only 0.5% haemolysis.

INTRODUCTION

Pluronic F-68 (Poloxamer 188) is a non-ionic, poly(oxyethy1ene)- poly(oxypropy1ene) co-polymer surfactant which has industrial and potentially valuable biomedical uses [ I , 21. I t has, for example, been used for priming cardiac bypass pump oxygenators to reduce blood viscosity and platelet adhesiveness 131 and to lower blood "sludging" during cold injury [4] . Pluronics have also been studied as immunological adjuvants [ S . 61. There is interest in the biological properties of Pluronic F-68 because of its use as a surfactant in the oxygen-carrying perfluorochernical emulsion "blood substitute". FfuosolTv (Alpha Therapeutic), which has been approved for clinical use as an adjunct to coronary balloon angioplasty 17. 81. A related area of interest concerns the potential therapeutic applications of surfactant solutions for improving blood rheology. In this regard, a Pluronic F-68-based formulation. RheothRx.IM (Burroughs-Wellcome Co.). has been tested in

135

Copyright 0 1995 by Marcel Dekkcr. Inc

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136 LOWE, FURMIDGE, AND THOMAS

preliminary clinical trials with encouraging results [9. lo]. A pre- requisite to such uses. however, is an assessment of the effects of Pluronic surfactants on blood cells. The effects have therefore been studied of incubating mammalian blood with Pluronic F-68 or Pluronic F-38 or their fractions in vitro.

MATERIALS AND METHODS

Pluronic Dreoarations and their purification

Commercial grade Pluronic F-68 and Pluronic F-38 were donated by the BASF Corporation (Michigan, USA). 10% (wlv) solutions of each compound were prepared i n isotonic saline (0.9% wlv NaCI) and then purified by passing 3 times through a glass column packed with silica resin [ I I ] . The removal of contaminants was confirmed by measurement of ultraviolet (UV) absorption (200-400 nm) using a Kontron Uvikon 860 spectrophotometer following each passage through the column. Concentrations of Pluronic F-68 or Pluronic F-38 before and after column purification were measured by turbidimetric assay based on absorbance at 492 nm (A492) [ 121.

Collection of blood samdes and haemolvsis test

Blood samples were obtained by venepuncture from Wistar rats, CFLP mice. DSN hamsters or New Zealand White rabbits and placed i n plastic tubes containing heparin (Sarstedt. U.K.). SO pl aliquots of blood were added to 5 ml of 0,025-10.0% ( w h ) solutions of either commercial or purified Pluronic F-68 or Pluronic F-38 in 0.9% (wlv) NaCI. Samples were incubated in a water bath at 37'C for 60 min., centrifuged at 3000 rpin for S min. and the supernatants aspirated. Haemolysis was measured by conventional spectrophotonietric assay [ 131 using an Ultrospec I 1 4050 spectrophotometer.

Statistical analvses

Statistical analyses were performed according to the methods of Snedecor and Cochran 1141. Means and standard deviations (S.D.) have been used throughout. Statistical significance between mean values was assessed using a conventional Student's f-test and a probability of P < 0.05 was considered significant.

RESULTS

A 4.0% (wlv) solution of commercial grade Pliironic F-68 showed an absorption band with peaks at 210-215 nm (absorbance 2.7 A.U.) and 270-280 nm (absorbance 0.4 A.U.). consistent with previous findings [ 1 I]. Additionally. a 4.0% (wlv) solution of commercial grade Pluronic F-38 had a similar absorption band with peaks at 205-210 nm

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Tab

le 1

. w

ith

4.0-

10.0

% (

w/v

) of

ei

ther

co

mm

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al

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e or

pur

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. H

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%)

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37

OC

-__--

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I

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UR

ON

IC F

-68

SPE

CIE

S

(%

wlv

) M

OU

SE

RA

T R

AB

BIT

H

AM

STER

C

ON

CEN

TRA

TIO

N

__

_

__

4.0%

Com

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cial

gr

ade

< 0

.1

< 0

.1

< 0

.1

< 0

.1

Puri

fied

fr

acti

on

< 0

.1

< 0.

1 <

0.1

<

0.1

6.0%

Com

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.1

0.4

2 0.

2 0.

2 2

0.1

0.7

5 0

.4

Puri

fied

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acti

on

< 0

.1

< 0

.1

< 0

.1

< 0.

1

8.0%

Com

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2.8

2 1

.2

0.6

2 0.

3 1.

5 2 0

.3

Puri

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< 0

.1

< 0

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1"

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%

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4.7

2 1

.5

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.9

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.1

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.3'

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5 0

.2*

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n 2 S.D. (

n =

3 t

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*P <

0.0

5.

$1 e

W 4

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138 LOWE, FURMIDGE, AND THOMAS

(absorbance 2.4 A.U.) and 240-290 nm (absorbance 0.2 A.U.). After one passage through the silica column, the higher UV absorption band for both compounds was no longer detectable (< 0.005 A.U.). Plots of A492 for both Commercial or purified Pluronics were similar, with niaximum differences between mean values of < 6%.

Incubation of blood with up to 4.0% (w/v) of either commercial or purified Pluronic F-68 produced no detectable haemolysis (data not shown). However. haeniolysis occurred with commercial Pluronic F-68 concentrations above 4.0% (w/v). with a species-dependent response (Table I ) . This was most pronounced with rat blood where. for example, incubation with 10.0% (w/v) commercial Pluronic F-68 produced 4.7 1.5 % (n = 3) haemolysis. but this was significantly (P < 0.05) reduced when the same concentration of purified Pluronic F-68 was used (0.5 5 0.3 %). In contrast, incubation of rat or hamster blood with either commercial or purified Pluronic F-38 produced no significant haemolysis (< 0.1 %). irrespective of' concentration tested. Incubation of rabbit blood with commercial Pluronic F-38 produced a maximum of 0.5% haemolysis at 10.0% (wlv); this was only marginally lower when the purified Pluronic F-38 fraction was used (data not shown).

DISCUSSION

These results show that incubation of mammalian whole blood with either a commercial grade or a silica-purified fraction of Pluronic F-68 produced no detectable haemolysis at concentrations of up to 4.0% (w/v). The present results also show that haemolysis occurred when blood was incubated with commercial Pluronic at >4.0% ( w h ) but that this was reduced with the purified fraction. While caution is needed i n extrapolating from in vitro to in viva systems. these findings suggest that the purified fraction may be preferable in Pluronic F-68 formulations for intravascular applications. This supports previous findings (I I ] that purification of Pluronic F-68 reduces liver weight increases in rats injected with this compound. The species variability in red cell responses to Pluronic F-68 seen here was consistent with earlier work using poly(oxyethy1ene) ether surfactants [ I 3 1 and most probably reflects differences in erythrocyte membrane composition. The finding that neither commercial nor purified Pluronic F-38 had significant haemolyt ic act ivi ty is diff icul t to explain given that i t s poly(oxypropy1ene) content is identical to that of Pluronic F-68 [ I , 21 and that both compounds have similar permeabilizing effects on yeast [ 151. One possible explanation is differences in haemolytically-active impurities between the commercial grades of Pluronic F-68 and Pluronic F-38. but this needs clarification.

REFERENCES

1 . Schmolka. I.R. J . Am. Oil Chein. Soc., 54, 110-1 16 (1977).

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HAEMOLYTIC PROPERTIES OF PLURONIC SURFACTANTS 139

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