The effect of macromolecular conjugates of daunorubicin on nuclear ultrastructure in Trypanosoma brucei rhodesiense

Cell Biology

International

The

effect

nuclear

of

Reports,

Vol. 10, No. 9, September

macromclecular

ultrastructure

conjuqates in

of

Trypanosoma

1

Present

Address:

School of Polytechnic,

daunorubicin

brucei

J.B. Mitchell* L, Golightly, J.E. Brown', Tropical Diseases Chemotherapy Research Sunderland Polytechnic, Sunderland SRl

717

1986

on

rhodesiense

and Unit, 3SD

Pharmacy, Leicester Leicester LEl

J.R.

Brown

9BH

2 To whom

correspondence

should

be addressed

ABSTRACT The effects of free and conjugated daunorubicin on --T-b. rhodesiense in vitro are described. Free drug caused nucleolar lesions ranging fro%-%&-egation to complete fragmentation. At equimolar concentrations a soluble bovine serum albumin conjugate with a stable succinyl linkage (D-BSAS) produced no ultrastructural lesions whereas a conjugate with a labile glutaraldehyde linkage (D-BSAG) and a conjugate linked to large agarose beads (D-ag) produced similar though less severe lesions than free drug. Polyisobutylcyanoacrylate nanoparticles caused trypanosomal lysis both with (D-PICA) and without adsorbed daunorubicin. INTRODUCTION The anthracycline antibiotic daunorubicin has antitumour activity and is used clinically in the treatment of acute nonlymphocytic leukaemia (Wiernik, 1984). It also has a powerful trypanocidal effect against Tryp?nosoma brucei rhodesiense -in vitro but only becomes active in \/iv0 when coupled to -__ macromolecules (Williamson 1981), --et al., Daunorubicin is known to intercalate into DNA (DiMarco, 1967) and to cause free radical damage such as lipid peroxidation (Goodman & Hochstein, 1977). Recent studies with tumour cells suggest that the drug may also ha?e an action at the cell surface (Tritton & Yee, 1982) but preliminary studies on trypanosomes by Williamson et al. (1983) reveal that the principal lesions -produced by macromolecular conjugates of daunorubicin are nuclear. The work described here extends that of Williamson -et -"al (1983) to cover the effects of a range of both soluble and particulate daunorubicin macromolecule conjugates on the ultrastructure of --T.b. rhodesiense. Bovine serum albumin was 0309-I

651 I86109071

7-OSl$O3.OOtO

@ 1986

Academic

Press

Inc.

(London)

used Ltd.

718

Cell Biology

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Reports,

Vol.

10, No. 9, September

1986

for the soluble prepara-Lion, wi?h drug attached either by a glutaraldehyde (D-BSAG) or a succinyl (D-BSAS) linkage. Two particulate preparations were used viz. co-valently linked daunorubicin-agarose (D-as) and daunorubicin adsorbed onto polyisobutylcyanoacrylate nancpar'icles (D-PICA)" MATERIALS

AND METHODS

Blood was taken by cardiac puncture from Swiss albino Porton mice heavily infected with a monomorphic strain (Yorke et al., -1929) of --T-b. rhodesiense. Centrifugation of the titrated blood provided a trypanosome buffy coat layer which was aspirated and washed twice in Krebs phosphate saline buffer, pH 7.4,7containing 0.2% (w/v) glucose. Trypanosomes were resuspended (10 /ml! in 50'50 inactivated bovine serum/Krebs saline-glucose. Free drug (Sigma) or conjugate was added to give daunorubicin concentrations of 0.1, 1.0, and 5.0 M. Apart from D-BSAG, the macromolecular coniuqates were prepared according to methods pret/iously described: Arnold et al. (1983) for D-BSAS, Tritton & Yee (1982) for D-ag, and Couvreur et al. (1982) for D-PICA. D-BSAG was prepared by a modification of the method described by Hurwitz e,t al. (1975). Bovine serum --. albumin (Sigma, in distilled water fractign V) (29) was dissolved (4.0ml) overnight at 4 C. Daunorubicin HCl (Cerubidin, May R Baker) (20mg) was dissolved in distilled water (4.0ml) and this was added to the BSA solution. Glutaraldehyde (1% v/v) (2.0ml) was added dropwise with continuous mixing and reactants incubated at 4'C for 72h, The mixture (lO.Oml) was dialysed three times against 11 of distilled water the7 lyophilised. Drug included

free where

controls, appropriate.

containing

macromolecules

alone,

were

The parasite suspensions were incubated at 37'C for 4h andfien prepared for transmission electron microscopy. Treated trypanosomes were centrifuged and the pellet washed twice before resuspension in a small volume of Krebs saline-glucose. Trypanosomes were then fixed in ice cold 2.5% glutaraldehyde in 50mM sodium gacodylate buffer, pH 7.2, containing 0.22M sucrose, for 18h at 4 C, followed by 3 washes in the same cacodylate buffer and post-fixation in 1% osmium tetroxide in 0.2M After washing in distilled water they s-collidine buffer for lh. were prestained by a ?O min treatment with 0.5% aqueous uranyl The specimens were acetate, pH 5.0, containing 45mg!ml sucrose. dehydrated in a graded series of ethanols, treated with propylene oxide and embedded in E-mix (Medium) resin (EMscope).

knife stained

Sections prepared with were collected on 300 with uranyl acetate

an LKB ultramicrotome and diamond mesh copper grids. These were postand lead citrate and examined on an AEI

Cell Biology International

CD1 transmission

Reports, Vol. 10, No. 9, September

electron

1986

719

nicrosccpe. RESULTS

Daunorubicin 0.1 IJM. Tfr-1) components

Nucleolar ultrastructure ranged to extreme polar segregation of of the nucleolus (fig. 2).

1.0i-1M There were no normal nuclei. occurred but nucleolar fragmentation Cases of extreme fragmentation were peripheral chromatin and lucency of (fig. 3). 5.0~M. Extreme was apparent in unaffected (fig.

nucleolar all nuclei, 4).

Daunorubicin-bovine (For this daunorubicin O,lPMp nucleolar

and

subsequent content of

albumin

Though a few nuclei were components most remained

and

Daunorubicin-bovine 0.1, 1.0 described

beginning normal.

serum

1.0 PM There nuclei remained

to

The to

lucency

(D-BSAG).

refer

show

to

segregation

of

principal lesion was that observed with was present in a

nucleolar fragmentation, together with and nucleoplasmic lucency in most

albumin

and 5.0 IM, None of were observed: the

Daunorubicin-agarose

5.0 iiM. Peripheral membrane

extreme chromatin

linked)

concentrations

1.01-1 M. None of the nuclei were normal. polar segregation of the nucleolus similar 0.1~ M daunorubicin. Nucleolar fragmentation small number of trypanosomes. 5.01~ M. There was loss of peripheral nuclei

nucleop lasmic DNA rema ined

(glutaraldehyde

preparations conjugates).

normality fibrillar

Some nucleolar segregation was the principal lesion. accompanied by loss of the nucleoplasmic matrix

fragmentation but kinetoplast

serum

from apparent granular and

(succinyl

linked)

the ultrastructural nuclei were normal.

(D-BSAS) lesions

so far

(D-ag) was some normal.

evidence

of

nucleolar

segregation

There was slight fragmentat,un in many nuclei. chromatin and nucleoplasmic matrix remained damage was evident (fig. 5 & 6).

but

intact.

most

No

720

Cell Biology

International

FIG. 1. Nuclear region of large nucleolus, neripheral nucleoplasmic matrix.

Reports,

trypanosome chromatin

FIG. 2. Nuclear region of trypanosome for 4h. Note nucleolus showing polar Peripheral and fibrillar components. matrix remain intact.

Vol. 10, No. 9, September

from unincubated and uniform1.y

control. dense

exposed to O.lvM daunorubicin segregation of the granular chromatin and nucleoplasmic

FIG. 3. Nuclear region of trypanosome exposed to l.OvM daunorubicin for 4h. Note fragmented nucleolar material dispersed throughout the nucleus, lack of periaheral chromatin and electron lucent nucleoplasmic matrix.

1986

Note

Cell Biology

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FIG. 4. Kinetoplast daunorubicin for FIG. 5. agarose Peripheral

4h.

Reports,

region Note

Vol. 10, No. 9, September

of trypanosome ultrastructure

1986

721

exposed to 5.OUM is unaffected.

Nuclear region of trypanosome exposed to 5.0~M daunorubicinfor 4h. Note remains of partially fragmented nucleolus. chromatin and nucleoplasmic matrix remain intact.

FIG. 6. Trypanosome and part of agarose bead (ab) 5.Oi:M daunorubicin-agarose for 4h.. Note membrane integrity appear unaffected. FIG. 7. Trypanosome polyisobutylcyanoacrylate disruption of outer

from incubation structure and

after

incubation in O.lpM daunorubicinfor 4h. Note extensive lysis trypanosomal membrane and damage to

involving organelles.

in

722

Cell Biology

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Reports,

Daunorubicin-polyisobutylcyanoacrylate lo-100nm).

Vol. IO, No. 9, September

(D-PICA)

(particle

diameter

0.1 PM. disrupted exterior. cytoplasm

The pellicle and plasma membrane of most trypanosomes and cytoplasmic organelle. s were damaged or lost to In most of those trypanosomes that remained intact was less electron-dense than normal (fig. 7).

1.0 PM. andisolated

There

Unloaded identical

polyisobutylcyanoacrylate to those seen with

In

all

were no intact trypanosomes; organelles remained.

other

controls

only

membrane

(Control). Effects daunorubicin loaded PICA. trypanosome

ultrastructure

1986

was the the

debris

were

remained

normal. DISCUSSION In keeping with the DNA intercalative properties of (Plumbridge & Brown, 1979) the results indicate that the focus of daunorubicin action in -T.b.-. rhodesiense lesions ranging from nucleolar segregation (fig. 2) fragmentation (fig. 3).

daunorubicin the nucleus is in vitro with toextreme

Trypanosomal lesions of similar type have been demonstrated with other cationic drugs (Williamson, 1979). Nucleolar segregation is thought to be a morphological expression of interference with RNA synthesis indicating the blockade of DNA dependent RNA polymerase activity (Bernhard, 1971), whilst fragmentation implies further inhibition of RNA synthesis by mechanisms other than template DNA binding (Faber & Roberts, 1971). Ultrastructural studies of trypanosomes exposed in vivo for 22h to a covalently linked drug-albumin conjugate equiam to 18 mg/kg daunorubicin have shown segregation of the nucleolus similar to that seen with free d+ug in vitro (Williamson --et al., Drug linked in this manner is trypanocidal in vitro and -in 1983). In vitro incubationoftrypanosomes vivo (Williamson 1981). --et al. withincreasing concentrations ofZt?!me, as described here, facilitates a more detailed study of the nuclear lesions produced by covalently linked D-BSA, and allows direct comparison with effects due to unconjugated daunorubicin. Trypanosnmes incubated in-- vitro with daunorubicin covalently bound to serum albunin by means of Schiffs base linkages using glutaraldehyde as a linking agent, showed ultrastructural lesions similar to those caused by free drug. However, the concentration of daunorubicin required to produce both nucleolar segregation and complete fragmentation was greater for the conjugated daunorubicin than free drug.

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Dsllnorubicin (Peterson, --et al., to macromolecules. to a macromolecule of the parasites, studies (in orogress) is accumulated by

Reports,

Vol. 70, No. 9, September

1986

723

is

believed to enter cells by simple diffusion 1980), a process prevented when drug is bound Trypanosomal accumulation of drug when attached therefore relies on the endocytotic capability a slower process than diffusion. Fluorescence that covalently linked D-B% hav e confirmed trypanosomes at a slower rate than free drug.

When daunorubicin is attached by a stable, succinyl linkage to BSA, -in vivo trypanocidal activity is not maintained (Williamson 1981; Hardman et al., 1983). The lack of ultrastructural --et al., lesions in our experiments is in agreement with these results. Nuclear damage and retentbn of trypanocidal activity only when the drug-carrier linkage is labile indicates that drug must be released from the conjugate before it can exert its effect. Tritton & Yee (1982) have shown that daunorubicin irreversibly linked to agarose beads larger than the size of the cell, is cytotoxic to certain tumour cells. They concluded that daunorubicin can be cytotoxic without entering cells, presumably by interaction with the cell membrane. The same complex has been used in this work to ascertain if surface effects are important in the action of daunorubicin against trypanosomes. The agarose beads remained intact and external to the trypanosomes (fig. 6). There was no evidence of gross damage to the external membrane of the trypanosomes but nuclear lesions were detected which were similar in character to, although much less severe than, those of free drug, indicating that some daunorubicin was released from the D-ag conjugate. Lack of lesions demonstrable by electron microscopy is not necessarily an indication of lack of surface activity. Anthracyclines have been shown to interact with negatively charged membrane phospholipids (Goormaghtigh, et al., 1980) and also to -induce changes in the ion transport across some cell membranes (Solie & Yuncker, 1978). Interactions of this type might alter permeability and would be unnoticeable by conventional transmission electron microscopy until substantial cell leakage had occurred. Daunorubicin adsorbed to polyisobutylcyanoacylate (PICA) in colloidal suspension was considered capable of uptake by trypanosomes as the particle size ( 100nm) should be within their endocytotic capability. Host toxicity to daunorubicin bound in this way is considerably reduced (Couvreur 1982). It was --et al., hoped that PICA binding would prolong drug exposure to trypanosomes sufficiently to stimulate a trypanocidal effect to act --in vivo i.e. as an endocytotic delivery device. Drug-loaded nanoparticles caused extensive lysis to trypanosomes in vitro (fig. 7); but as lysis also occurred when trypanosomes wzewsed to PICA without adsorbed daunorubicin PICA is not satisfactory as a carrier in these particular drug targetting studies.

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1986

It is interesting to note that, whilst the principal daunorubicin-induced lesion is brought about by Intercalation of drug with DNA, the effect is restricted to the nucleus; the kinetoplast-DNA remains unaltered (fig. 4) perhaps because the mitochondrion of these bloodstream forms is inactive and its k-DNA In contrast, T. cruzi is not active in transcription. epimastigotes have an active mitochondrion and-treatment with rearrangement of the typical daunorubicin has been shown to cause bilayer structure of the kinetoplast into arched loops (Williamson, 1979). ACKNOWLEDGEMENTS We are indebted on the manuscript. and a project grant

to Dr. J. Williamson The work was supported from the MRC.

for his helpful comments by an SERC Studentship

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RcccGvcd:

70.6.86

Accepted:

17.6.86

J.R.

is