Neuroscience Letters, 51 (1984) 231-234 231

Elsevier Scientific Publishers Ireland Ltd.

NSL 02988

LIPOSOME-ENTRAPPED [125I]ANTI-TETANUS IMMUNOGLOBULIN G: EVIDENCE FOR ENTRY INTO SPINAL CORD NEURONS OF THE RAT

BODO NIGGEMANN*, GE OR G ERDMANN** and HANS H. W E L L H O N E R

Zentrum Pharrnakologie und Toxikologie, Abteilung Toxikologie, Medizinische Hochschule Hannover, D-3000 Hannover 61 (F.R.G.)

(Received December 13th, 1983; Revised version received August 2nd, 1984; Accepted August 7th, 1984)

Key words: anti- tetanus IgG - l iposome - spinal cord - autoradiography - rat

lntrathecally administered free anti- tetanus immunoglobul in G (IgG) diffuses through the spinal cord but does not enter nerve cells. In order to facilitate entry into neurons, ~25I-labeled anti-tetanus lgG was

entrapped in liposomes. After injection into the cerebrospinal fluid of rats, however, only a very low specific radioactivity of the spinal cord could be calculated from gross counts and no neuronal labeling was seen in autoradiographs. Therefore, it was assumed that the liposomes were unable to cross the base- ment membrane of the spinal cord surface. To circumvent this harrier the l iposome-entrapped [~25I]IgGs

were injected directly into the grey matter. His toautoradiographs then showed marked accumulat ions of radioactivity in neurons. Direct intraspinal injection of free [lzSI]lgG, on the other hand, failed to pro- duce heavy neuronal labeling.

Tetanus is a disease for which a causal treatment is not available because there has been no way of introducing the specific anti-tetanus immunoglobulin G (IgG) to the toxin's site of action in the spinal cord inhibitory interneurons [1]. Firstly, intravasal IgGs do not penetrate the blood-brain barrier. Secondly, although in- trathecal IgGs enter the intercellular space of the spinal cord, they do not cross neuronal membranes. Since liposomes are capable of introducing encapsulated substances into cells [4], we attempted to use them as vehicles for transporting the specific antidote into the neuronal cell bodies.

Anti-tetanus IgGs were raised in rabbits, purified on diethylaminoethyl (DEAE)- Sephacel [2], labeled with 1251 [3] (spec. act. 2.36 mCi/mg protein) and entrapped in unilamellar reverse-phase evaporation vesicles. The average diameter was 200 nm (as revealed by electron microscopy) and was thus sufficient in comparison to the dimensions of IgG molecules (maximal extension 14.2 nm [8]). The liposomes were

*Present address: Universit~its-Krankenhaus Eppendorf , Kinderklinik, Martinistrasse 52, D-2000 Ham- burg 20, F .R.G. **Author for correspondence.

0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

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prepared from phosphatidylglycerol, phosphatidylcholine and cholesterol in a molar ratio of 1:4:5 (total lipid 50 #mol) by a procedure modified after Szoka and Papahadjopoulos [9]. The lipids were dissolved in 7.5 ml isopropylether-chloro- form (1:1) in a 50 ml round-bot tom flask with a long-extended neck to which 1.25 ml aqueous phase containing 2.5 mg rabbit IgG and 10 mg bovine serum albumin was added. The system was then purged with nitrogen and sonicated with a Braun probe sonicator at 80-100 W for three 1-min periods at room temperature. When the system became a homogeneous opalescent dispersion, the organic phase was removed using a rotary evaporator. The system frothed during evaporation and subsequently formed an aqueous suspension. At this point 3 ml of phosphate- buffered saline (PBS) were added and the suspension was further evaporated to remove solvent traces. The preparation was dialysed against PBS overnight. The precipitate was discarded. The opalescent supernatant was passed through a Sepharose 6B column. The liposome-containing fractions were vacuum-dialysed to reduce the volume.

Sprague-Dawley rats (250 g) were injected with 25 ng tetanus toxin dissolved in 50 #1 PBS into the left gastrocnemius muscle. After 24 and 48 h, respectively, the animals which had developed symptoms of local tetanus were anesthetized by in- traperitoneal administration of pentobarbital (50 mg/kg) and injected with 50-80 /~1 ( = 8-11 x 106 dpm and 1.5-2.1/~g IgG) of the [125I] antitoxin-entrapping liposome

preparation into the cisterna magna. Another 24 h later the animals were again anesthetized as described above and perfused with a glutaraldehyde- and formaldehyde-containing fixative. The spinal cord was dissected and subdivided in- to individual half segments which were assayed for radioactivity (RA). Paraff in sec- tions were prepared f rom segments L6 and L5 and subjected to autoradiography us- ing Ilford K2 emulsion. Gross counts of spinal cord half segments L6 and L5 reveal- ed no difference in radioactivity between the tetanus-affected and the control sides (20,000 cpm/g) . Autoradiographs showed that on both sides more silver grains were found in the spinal grey than in the white matter (ratio 1.5:1). Virtually no grains were found over motoneurons or interneurons of the tetanus-affected side (or con- trol side). It was considered that the liposomes did not reach the neurons because the neural basement membrane on the surface of the spinal cord [6] acted as a diffu- sion barrier. Therefore, in order to circumvent this barrier, 1-3 #1 of the liposome suspension were administered with a capillary injector directly into the spinal cord grey matter of 3 rats. One control animal received 3/~1 of free [~25I]IgG by the same route. One or three hours later the animals were perfused with the fixative and autoradiographs were prepared from spinal cord sections.

At the injection site, labeling of the parenchyma was too heavy as to allow iden- tification of neurons. At a distance of approximately 300 ~m and more, however, accumulations of label could be attributed to individual neurons. After intraspinal injections of l iposome-entrapped or free [~25I]IgG, far more RA had accumulated in neurons of animals injected with the liposome preparat ion (Fig. lb) than in the

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Fig. I. Distribution of radioactivity in the grey matter of the rat spinal cord following the injection into

the spinal cord parenchyma of ~2SI-labeled free and l iposome-entrapped anti- tetanus lgG. a: after applica-

tion of free IgG, labeling over neurons and surrounding tissue is o f the same intensity, b: after the injec- tion of l iposome-entrapped IgG, massive accumulat ions of radioactivity are observed in neurons.

control rat injected with free [125I]IGG where neuronal labeling and labeling of the surrounding tissue were of the same intensity (Fig. la).

Previous investigations [l] showed that radioactive antitoxin injected into the cerebrospinal fluid accumulated around motoneurons from which tetanus toxin was discharged following its intra-axonal ascent f rom the periphery. As our present in- vestigations show, entry of antitoxin into neurons can be achieved when liposomes are used as vehicles of antitoxin transport. This agrees with the findings of Miiller et al. [7] in isolated nerve endings from the electric organ of Torpedo and Magee et al. [5] who worked with HeLa cells: horseradish peroxidase (HRP) entrapped in liposomes, though not free HRP, was readily incorporated into nerve endings and cells.

Intraspinal injections of liposomal [125I]antitoxin showed far better results than liposomes injected by the intrathecal route. A possible explanation for the latter finding is that the basement membrane acted as an efficient barrier for the liposomes but not for the IgG molecules. This may be due to differences either in charge or in size between the liposomes and the IgGs.

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T h e i n t r o d u c t o n o f l i p o s o m e - e n t r a p p e d a n t i b o d i e s i n t o n e r v e cells in c u l t u r e

c o u l d b e a p r o m i s i n g a p p r o a c h to s t u d y t h e a c t i o n s o f b o t h t h e n e u r o t o x i n s a n d

t h e i r a n t i b o d i e s .

T h e a u t h o r s w i sh t o t h a n k D r . B izz in i , I n s t i t u t P a s t e u r , P a r i s , f o r his g e n e r o u s

g i f t o f t e t a n u s t o x i n .

1 Erdmann, G., Hanauske, A. and Wellh6ner, H.H., lntraspinal distribution and reaction in the grey matter with tetanus toxin of intracisternally injected anti-tetanus toxoid F(ab')z fragments, Brain Res., 211 (1981) 367-377.

2 Garvey, J.S., Cremer, N.E. and Sussdorf, D.H., Methods in Immunology, 3rd edn., W.A. Ben- jamin, Reading, MA, 1977, pp. 223-226.

3 Greenwood, F.C., Hunter, W.M. and Glover, J.S., The preparation of ~ll-labeled human growth hormone of high specific radioactivity, Biochem. J., 89 (1963) 114-123.

4 Gregoriadis, G., Liposomes. In G. Gregoriadis and A.C. Allison (Eds.), Drug Carriers in Biology and Medicine, John Wiley and Son, New York, 1979, pp. 287-341.

5 Magee, W.E., Goff, C.W., Schoknecht, J., Smith, M.D. and Cherian, K., The interaction of cationic liposomes containing entrapped horseradish peroxidase with cells in culture, J. Cell Biol., 63 (1974) 492-504.

6 Morse, D.E. and Low, F.N., The fine structure of the pia mater of the rat, Amer. J. Anat., 133 (1972) 349-368.

7 Mfiller, U., Munz, K. and Waser, P.G., Incorporation of small unilamellar liposomes loaded with horseradish peroxidase into isolated nerve endings from electric organ of Torpedo rnarrnorata, J. Neurocytol., 12 (1983) 507-516.

8 Sarma, V.R., Silverton, E.W., Davies, D.R. and Terry, W.D., The three-dimensional structure at 6 A. resolution of a human gamma GI immunoglobulin molecule, .1. biol. Chem., 246 (1971) 3753-3759.

9 Szoka, Jr., F. and Papahadjopoulos, D., Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation, Proc. nat. Acad. Sci. U.S.A., 75 (1978) 4194-4198.