A SYSTEM ESTABLISHING COMPATIBILITY PROFILES FOR ARTIFICIAL OXYGEN CARRIERS AND OTHER SUBSTANCES

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  • ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH., 29(1), 5770 (2001)

    A SYSTEM ESTABLISHINGCOMPATIBILITY PROFILES FORARTIFICIAL OXYGEN CARRIERS

    AND OTHER SUBSTANCES

    Stephanie Dinkelmann,1 Wolfgang Rohlke,2

    Hasso Meinert,2 and Hinnak Northoff1

    1Institut fur Transfusionsmedizin, Otfried-Mueller-Str. 4/1,D-72076 Tubingen, Germany

    2AG Chemie Biokompatibler Verbindungen,Universitat Ulm, D-89069 Ulm, Germany

    ABSTRACT

    Worldwide, great efforts are being made to develop a clini-cally useful artificial oxygen carrier. Toxicological and immunolog-ical compatibility is generally tested using animal experiments butinflammatory parameters in particular show large species-specificdifferences. Therefore, we developed an in vitro system using hu-man components to establish a compatibility profile of unknowncompounds. The test system comprises induction of hemolysis, ac-tivation of complement (C3a), induction/suppression of cytokineproduction, influence on cell proliferation, direct toxicity on pe-ripheral leukocytes, and phagocytosis of the material under testand of microbes. The test system will be described, along with re-sults of various perfluorocarbon emulsions. When testing lecithin-based perfluorodecalin (PFD) emulsions, and comparing them toPluronic-based PFD emulsions, we could show that Pluronic-basedemulsions were virtually untoxic to peripheral human leukocytes.

    57

    Copyright C 2001 by Marcel Dekker, Inc. www.dekker.com

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    58 DINKELMANN ET AL.

    They neither inhibited cell proliferation nor caused any hemolysis,but caused mild to moderate inhibition of endotoxin-induced cy-tokine production. At the same time, lecithin-based PFD emulsioncaused substantial cytotoxicity in phagocytic cells like monocytes(60100% after 24 h incubation) and granulocytes (1020% after24 h incubation). They also suppressed endotoxin-induced cytokineproduction in monocytes to more than 98% and inhibited cell pro-liferation of an endothelial (ECV 304) and a monocytic cell line(MonoMac6) to more than 95%.

    INTRODUCTION

    During and after the development of artificial oxygen carriers designed forpotential use in humans, and following analysis of their physicochemical proper-ties, we see the importance of determining their biocompatibility profile in vitrobefore they may be tested in animals or humans. On the one hand, many extensiveanimal experiments could be saved; on the other hand, using tests based on humancells may provide a better predictive value for the judgement of in vivo compati-bility in humans. It is possible that a substance can be shown to be intolerable inthe animal model but be without major problems for use in humans. Much morelikely, and abundantly evident from the literature, are situations where extensiveexperiments with rodents seemed to indicate compatibility and beneficial effects,whereas ensuing human trials ended in severe disappointment. Therefore we de-veloped a multitask test system exploring the interference of test substances withvital functions on the cellular level (Fig. 1). We gathered information on directtoxic effects as well as influences on several cellular functions.

    To estimate direct cytotoxicity, data will show the degree of hemolysis in-duced by different perfluorocarbon emulsions (PFC emulsions) and their effects onthe survival of peripheral human leukocytes. To determine the rate of dead leuko-cytes, we used flow cytometry. We prefer this method because many more cells canbe counted in relation to light microscopy. Furthermore, a discrimination betweenmonocytes, lymphocytes, and granulocytes is possible, and for each populationthe rate of dead cells can be measured separately. In addition to cytotoxicity, theinfluence of perfluorocarbon emulsions on cell proliferation is determined. We useonly human cell lines, and especially cells that will be first in contact with drugsfor intravenous application. Therefore, data will be presented with an endothelialcell line (ECV 304) and a monocytic cell line (MonoMac6). The cytokine re-lease is an important cellular function. We focused our tests on interleukin-1 andinterleukin-6, because these are pleiotropic factors that are released by activatedmonocytes, neutrophils, and several lymphocytes. They play a key role in defenseagainst infectious diseases, tissue damage, and inflammation. Massive induction

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    ARTIFICIAL OXYGEN CARRIERS 59

    Figure 1. A multitask system for biocompatibility testing of artificial oxygen carriers.

    of these cytokines, or massive suppression of stimulan-induced cytokines, wouldbe reason for caution. The biocompatibility profile is completed by testing com-plement activation and phagocytosis. These data will be shown elsewhere.

    We investigated various perfluorodecalin emulsions. Perfluorodecalin (PFD)possesses a high solubility for oxygen and carbon dioxide. PFD was developed tobe used as red cell substitute (1,3). PFD alone is not soluble in water and must beemulsified for intravenous application. The block polymer Pluronic and lecithin arewidely used as emulsifiers. The PFD/lecithin emulsion, the PFD/Pluronic emulsionand, in addition, perfluorodecalin and the emulsifiers alone, were tested. Lecithin isa common component of several pharmaceutical preparations (e.g., lipid solutionsfor intravenous application).

    MATERIALS AND METHODS

    Peripheral leukocytes, serum, and erythrocytes were obtained from healthyvolunteer donors.

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    60 DINKELMANN ET AL.

    Cell Lines

    ECV 304 cells are human umbilical cord venous endothelial cells (4). ECV304 cells were purchased from the German Collection of Microorganisms and CellCultures (Braunschweig, Germany). The cells were cultured with Medium 199(Gibco, Eggenstein, Germany) supplemented with 20% heat-inactivated fetal calfserum (Roth, Karlsruhe). MonoMac6 are human acute monocytic leukemia cells(5). They were also purchased from the German collection. The culture mediumfor MonoMac6 contained: RMPI 1640 (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Myoclone Super Plus, Gibco) and 10 mL HEPESbuffer (Gibco) 2.5 mL nonessential amino acids (Gibco), 3.5 mL L-glutamin(Gibco), and 4.5 mL OPI (Sigma, Steinheim, Germany).

    Test Substances

    Ringers lactate (Baxter, Unterschleissheim, Germany) perfluorodecalin(PFD; Fluka # 77264); PFD emulsion with lecithin, containing 18.5% (w/v) per-fluorodecalin and 1.5% (w/v) perfluorodimorpholinopropane (FDMP, Eup0002),emulsified with 2.5% (w/v) lecithin (Lipoid E100, Lipoid KG) in Ringers lactate,homogenized and steam sterilized; initial average droplet size: 130239 nm; PFDemulsion with Pluronic, containing 18.5% (w/v) perfluorodecalin and 1.5% (w/v)perfluorodimorpholinopropane (FDMP, Eup 0002), emulsified with 4% (w/v)Pluronic PE 6800 (BASF, Ludwigshafen, Germany) in Ringers lactate, homog-enized and steam sterilized; initial average droplet size: 290346 nm; lecithin-emulsion, containing 2.5% (w/v) lecithin (Lipoid E100, Lipoid KG) in Ringerslactate; initial average droplet size: 222 nm; 4% (w/v) Pluronic PE 6800 (BASF,Ludwigshafen, Germany) in Ringers lactate.

    Sterility Control

    For all test substances, controls of sterility were employed each time beforeuse. The substances were plated onto blood/agar plates and incubated at 37C,5 vol.-% CO2 in humid atmosphere. After 24 and 48 h, the plates were controlled forbacterial growth. None of the substances used in the tests showed bacterial growth.

    Hemolysis Assay

    A 20% (w/v) erythrocyte suspension was prepared with Ringers lactate. Todetermine the hemolytic activity of the test substances, 50 L of the erythrocyte

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    ARTIFICIAL OXYGEN CARRIERS 61

    suspension was incubated together with 100 L of the test substance plus 100 Lof serum or isotonic saline. Erythrocyte suspension, together with isotonic saline,was used as negative control; erythrocyte suspension with distilled water was usedas positive control. The samples were incubated for 1 h at 37C in a water bath, thenthe samples were centrifuged for 2 min at 2.000g (Heraeus Instruments, Stuttgart,Germany) and the optical density of the supernatants were measured spectrophoto-metrically (SLT Labinstruments, Crailsheim, Germany). A test substance inducedhemolysis when the following quotient was greater than 2:

    ODt+R.L./s+eODt+R.L./s + ODR.L./s+e (1)

    where ODt+R.L./s+e is the optical density of the supernatant consisting of testsubstance (t) plus Ringers lactate or serum plus erythrocytes (e); ODt+R.L./s is theoptical density of the supernatant consisting of test substance plus Ringers lactateor serum; and ODR.L./s+e is the optical density of the supernatant consisting ofRingers lactate or serum plus erythrocytes.

    Cytokine Assay

    Mononuclear leukocytes were prepared with gradient separation (Lympho-flot separation medium, Biotest, Dreieich, Germany) using human blood (buffycoat from routine blood donations). The mononuclear leukocytes were used witha final concentration of 1 106 cells/mL and maintained with 5 vol.-% fetalcalf serum (Roth, Germany) and RPMI (1640, Gibco, Eggenstein, Germany).One hundred micro liters of the cell suspension were incubated for 4 h at 37C,5 vol.-% CO2, humid atmosphere, together with 50 L of the test substance or thecontrols. Then lipopolysaccharide (Salmonella typhimurium, Difco, Augsburg,Germany) was added to a final concentration of 10 ng/mL and the cell suspensionwas incubated for another 20 h. Thereafter, the interleukin-1 and interleukin-6concentrations of the supernatant were measured using the ELISA technique(IL1 Quantikine, DPC, Bad Nauheim, Germany; IL6, Pharmingen, Hamburg,Germany). The degree of suppression or induction of interleukin-1 (IL1) orinterleukin-6 (IL6) production was determined by the following quotients:

    Degree of suppression = 1 [IL1(or IL6)]MNL+test compounds+LPS[IL1(or IL6)]MNL+LPS

    (2)

    where [IL1 (or IL6)]MNL+test compounds+LPS equals concentration of IL1 (IL6)after incubation of mononuclear leukocytes (MNL) with various test compoundsplus lipopolysaccharide (LPS); and [IL1(or IL6)]MNL+LPS equals concentration

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    62 DINKELMANN ET AL.

    of IL1 (IL6) after incubation of mononuclear leukocytes (MNL) with LPS.

    Degree of induction = [IL1(or IL6)]MNL+test compounds[IL1(or IL6)]MNL+LPS

    (3)

    Cell Proliferation Assay

    For this assay, 96-well culture plates (Falcon, Becton, Dickinson, Heidel-berg, Germany) were used. Each well contained 5 104 cells (ECV 304 or Mono-Mac6) in a volume of 100 L. To each well were added 50 L of a test substanceor the control (culture medium). The test substances and the controls were placedsymmetrically and alternately to avoid position effects on the microtiter plate.The culture plates were incubated for 24 h at 37C, 5 vol.-% CO2, humid atmo-sphere. Then 20 L of 3H-thymidine (37 MBq/mL, Amersham) was added toeach well. After another 24 h of incubation at 37C, the culture plates were har-vested using an automated harvester. As the ECV 304 are adherent cells, cultureswere washed and trypsinized (trypsin from Biochrom, Berlin, Germany) beforeharvesting. Cell proliferation was determined by scintillation counting (Betaplate,LKB Wallac/Pharmacia, Finland). The relative 3H-thymidine incorporation wascalculated as follows:

    rel.3H-thymidine incorporation tc [%]

    =3H-thymidine incorporation tc [cpm]

    3H-thymidine incorporation cm [cpm](4)

    where tc is the test compound and cm is the culture medium.

    Toxicity Assay

    For peripheral leukocyte separation, anticoagulated (sodium-Heparin,10 I.E./mL) blood was incubated at ambient temperature for 1 h on a densitygradient (Ficoll 1077, Biochrom, Berlin, Germany) without centrifugation. There-after, the supernatant was obtained. For each sample, 75 L leukocyte suspensionwas incubated together with 50 L test substance at 37C, 5 vol.-% CO2, humidatmosphere. Samples were obtained after 0, 2, 4, 6, 8, 13, 17, and 24 h. The cellswere stained with CD45 FITC (panleukocyte marker, green fluorescence, BectonDickinson, Heidelberg, Germany), CD14 PE (monocyte marker, orange fluores-cence, Becton Dickinson), and propidium iodide (Sigma, Steinheim, Germany,DNA staining, determination of dead cells). The flow cytometric analyses wereperformed on a flow cytometer from Coulter (Coulter Epics XL, Coulter, Krefeld,Germany). Gates were set for the various leukocyte populations; 10,000 cells wereacquired; data were analysed using XL1 and XL2 software (Coulter).

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    ARTIFICIAL OXYGEN CARRIERS 63

    Table 1. Hemolysis Assay

    Hemolysis in the Presence Hemolysis in theTest Compound of Isotonic Saline Solution Presence of Serum

    Ringers lactate (n = 7) Negative NegativePerfluorodecalin (n = 7) Negative NegativeEmulsion lecithin (n = 4) Negative/weak positive NegativeEmulsion Pluronic (n = 5) Negative NegativeEmulsion PFD/lecithin (n = 4) Negative/weak positive NegativeEmulsion PFD/pluronic (n = 4) Negative NegativePFD = Perfluorodecalin, n = number of measurements.

    RESULTS

    Hemolysis Assay

    The results of the hemolysis tests are shown in Table 1. Only lecithin andthe lecithin/perfluorodecaline emulsion cause a weak positive hemolysis in thepresence of isotonic saline. In the presence of serum, none of the test compoundscaused a hemolysis.

    Induction and Suppression of the Interleukin-1Production of Mononuclear Leukocytes

    Lipopolysaccharide (LPS) is a strong stimulus for mononuclear leukocytes(MNL). After incubation of MNL with LPS, they produce (among others)interleukin-1 and interleukin-6. The results of our tests are depicted in Figure 2.We used Ringers lactate as control, because it is also a component of the PFCemulsions. The incubation of MNL with Ringers lactate resulted in a mild decreaseof interleukin-1 concentration. The incubation with perfluorodecalin (PFD) in-duced an average suppression of 26%, a decrease that is not significant. In contrast,lecithin and the PFD/lecithin emulsion suppressed the interleukin-1 concentra-tion to 98%. Finally...

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