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Experience with doxorubicin-bound polyisohexylcyanoacrylate nanoparticles on murine alveolar echinococcosis of the liver
Internorional Journal/or Parasirology Vol. 23, No. 3, pp. 427429, Printed in Grear Britain
1993 0
002&7519/93 16.M) + 0.M) Pergamon Press Lrd Sociery for Parasifology
1993 Ausmlinn
RESEARCH NOTE EXPERIENCE WITH DOXORUBICIN-BOUND POLYISOHEXYLCYANOACRYLATE NANOPARTICLES ON MURINE ALVEOLAR ECHINOCOCCOSIS OF THE LIVER M.
TLaboratoire
LIANCE,*
F. NEMATI,t
C. BoRIEst and P.
CoUVREURt
*Laboratoire de Parasitologie, Faculte de Midecine, 8 rue du GBn&ral Sarrail, 94000 Cr&teil, France de Pharmacie galenique et Biopharmacie, URA CNRS 1218, Facultt de Pharmacie, 92296 Malabry, France
Chatenay-
(Received 16 December 1992; accepted 24 January 1993)
Abstract-FIANCE M., NEMATIF., BORIESC. and COUVREUR P. 1993. Experience
with doxorubicin-bound polyisohexylcyanoacrylate nanoparticles on murine alveolar echinococcosis of the liver. Znrernational Journalfor Parasitology 23: 427-429. The parasiticidal properties of doxorubicin against the metacestode of Echinococcus multiloculuris were investigated after binding of that drug to polyisohexylcyanoacrylate nanoparticles, a colloidal biodegradable drug carrier. A reduction of the hepatic parasite development and a reduced viability of the metacestode were observed in mice injected with 5 mg kg-’ body weight-‘, but 7.5 mg kg-’ body weight-’ did not appear more efficient. Free doxorubicin or unbound nanoparticles had no antiparasitic activity. INDEX KEY WORDS: nanoparticles; doxorubicin.
Echinococcus
multiloculuris; murine
hepatic
alveolar
echinococcosis;
Free doxorubicin was obtained from Farmitalia Carlo Erba (Rueil-Malmaison, France), and isohexylcyanoacrylate (IHCA) monomer from Sopar (Start-Dames-Avelynes, Belgium). Doxorubicin-bound nanoparticles were prepared by polymerization of IHCA, according to Couvreur, Roland & Speiser (1982). Unbound PIHCA nanoparticles were prepared in the same way. For doxorubicin-bound nanoparticles 0.1 ml of the suspensions used for animal treatment contained 1.6 mg of nanoparticles, expressed in polymeric compound, and 0.075 mg of entrapped doxorubicin. The size of the nanoparticles ranged from 250 to300 nm, and their number was 1.9 X lOI* ml-‘. Groups of 10 AKR female mice (C.N.R.SC.S.A.L., OrlCans, France), 6 weeks old, received an intrahepatic inoculum of an E. multilocularis metacestode suspension, as previously described (Liance, Vuitton, Guerret-Stocker, Carbillet, Grimaud & Houin, 1984). Treatments were performed by injecting via the tail vein 0.1 ml of the nanoparticle suspensions, or free doxorubicin solution (same dose of doxorubicin). Three administration schedules of bound doxorubicin were established for 30 g infected-mice. Mice of group 1 received a single dose of 2.5 mg kg-’ body weight-’ at day 70 p.i. Those of groups 2 and 3 received additional injections of the
THE potentially unlimited proliferation of Echinococcus multilocularis metacestodes is responsible for alveolar echinococcosis, a neoplastic-like hepatic disease. Chemotherapy of human patients with benzimidazoles is still not yet satisfactory (Eckert, 1991). To date only two experimental therapeutic approaches have been shown partially effective in a rodent model with an induced peritoneal infection. The first, Mitomycin C, is used in the treatment of human cancers (Novak, 1990). It has been suggested that the second one, Isoprinosine, inhibited the larval growth by inducing damages in the nucleic acids synthesis (Sarciron, Al-Nahhas, Walbaum, Raynaud & Petavy, 1991). Following these studies and taking into account the primary localization of the parasite in the liver, we have tested the efficacy of doxorubicin loaded onto polyisohexylcyanoacrylate nanoparticles. In fact, it has been shown previously that this binding resulted in a preferential tissue distribution of the drug to the liver (Verdun, Brasseur, Vranckx, Couvreur & Roland, 1990), with a subsequent reduced toxicity and an increased antitumoral activity (Chiannilkulchai, Driouich, Benoit, Parodi & Couvreur, 1989).
* To whom all correspondence
model;
should be addressed. 427
428
M. LIANCE~~ al.
same dose at day 80 and at days 80 and 90 p.i., respectively. Control groups included animals injected, at days 70, 80 and 90 pi., with unbound nanoparticle suspension (group 4); animals injected, at the same three times p.i., with 2.5 mg kg-’ body weight-’ of free doxorubicin (group 5) and infected untreated animals (group 6). Treated mice were sacrificed under ether anesthesia 21 days after the end of the injections. This time was chosen in order to avoid secondary formation of hepatic lesions from metastases. Untreated mice were sacrificed at the same time as the groups receiving three treatments. TABLE
I-EFFECTS
OF DOXORUBICLN-LOADED NANOPARTKLE NANOPARTICLE SUSPENSION,AND FREE
SUSPENSION,UNLOADED
DOXORUBICINSOLUTION,ONTHE HEPATICGROWTHOF
E. multilocularis
Group
Positive
of
hepatic
Jirds
i.p. infected
hepatic
from
parasite
infection
mice
in mice
No. infected/
Mean cyst
dissected
weight (g) 17.8
1
lo/lo
414
2
4/10
414
6.7
3
S/IO
9.2
4
IO/l0
414 4/3*
20.8
5
IO/l0
414
21.4
6
IO/l0
414
19.4
(I)
2.5
mg
kg-’
body
5 mg kg-’
weight-’
doxorubicin;
(2)
body weight-’
doxorubicin;
(3) 7.5 mg kg-
’ body
doxorubicin;
(4) unloaded
nanoparticles;
weight-
of
nanoparticle
of nanoparticle
’ of
nanoparticle
(5)
7.5 mg kg-’
body weight -’ of
free doxorubicin; (6) untreated mice. * One jird died before autopsy.
Parasitological monitoring showed no significant difference between the six groups regarding the mean weight of both peritoneal parasites and livers. All mice harboured peritoneal metastases. In mice harbouring a hepatic parasite (Table I), the approximate superficial size of this was always lower than 5 mm in diameter in groups 2 and 3, and always higher than 7 mm in the other groups. A viability assay was performed from both hepatic and peritoneal parasites in 2 Meriones unguiculutus jirds, as previously described (Liance, Bresson-Hadni, Vuitton, Bretagne & Houin, 1990). It was performed from two mice randomly selected from each group. Two months p.i., there was no significant difference between the six groups regarding the parasite burden obtained after infection with peritoneal metacestodes. Results obtained after infection with hepatic metacestodes (Table l), showed a reduced viability of larvae removed from mice of groups 2 and 3.
A histopathological examination of the hepatic lesions from two mice randomly selected from each group and different from those used for testing the parasite viability, revealed different patterns. In mice of groups 1, 4 and 6, numerous parasitic vesicles were present, surrounded by a typical polymorphous granuloma, as previously described in this murine strain (Liance et al., 1984). In mice of groups 2, 3 and 5, vesicles were surrounded by necrosis and numerous leucocytes. Hepatocytes located near these lesions harboured a nucleus dystrophy. Lastly, in mice of group 2, numerous portal vessels were infiltrated with leucocytes. In this study, doses of doxorubicin far less than those used to inhibit hepatic sarcoma metastases (Chiannilkulchai et al., 1989) were evaluated. Free doxorubicin appeared without parasiticidal properties, although it modified the peri-parasitic cellular pattern. The efficiency of nanoparticle doxorubicin was demonstrated by inhibiting the hepatic larval growth, and by reducing the parasite viability, exclusively originating from the liver. However investigations of other strategies for the administration of doxorubicin-loaded PIHCA nanoparticles are required. As the more long-term treatment and the lowest dose appeared obviously of low efficiency, it would be of interest to evaluate the effects of higher doses administered during a shorter period of time. On the other hand, as all animals exhibited high peritoneal parasitic burden at the time of autopsies, it would also be of interest to start the treatment at an earlier stage of the larval development. Because atypical periparasitic tissue reactions occurred with either free or bound doxorubicin, such experiments would include an histopathological study in order to determine the effects of the drug on liver samples taken far from the parasite. Those data should encourage further development of anthelminthic compounds bound to biodegradable and colloidal drug carriers, such as PIHCA. Acknowledgements-The for her technical grant
from
Doctorales
authors
assistance.
the Direction du Ministire
wish to thank
D. Rivollet
This work was supported de la Recherche
de 1’Education
by a
et des Etudes
Nationale.
REFERENCES CHIANNILKULCHAIN., DR~OUICH Z., BENOIT J. P., PARODI A. L.
&
COUVREUR P.
1989.
Doxorubicin-loaded
efficiency
in
particles:
increased
murine
metastases.
Selective Cancer Therapeutics 5: 96106.
COUVREUR P., ROLAND M. & SPEISER P. 1982.
U.S.
nanohepatic Patent
No. 4,329%332. ECKERT J. 1991. update.
Experimental
research
on echinococcosis:
In: Archives de la Hidatidosis XXX (Edited
ROSA F.),pp. 31 I-319.
Libbey
& Co. Ltd, London.
by DE
Research LIANCE M., VUII-~ON D. A., GUERRET-STOCKER S., CARBILLET J. P., GRIMAUD J. A. & HOUIN R. 1984. Experimental alveolar echinococcosis. Suitability of a murine model of intrahepatic infection by Echinococcus mulrilocularis for immunological studies. Experientia 40: 1436-1439. LIANCE M., BRESSON-HADNI S., VUIT~ON D., BRETAGNE S. & HOUIN R. 1990. Comparison of the viability and developmental characteristics of Echinococcus multilocularis isolates from human patients in France. International Journal Parasitology 20: for 83-86.
Note
429
NOVAK M. 1990. Efficacy of mitomycin C against alveolar Echinococcus. International Journhl for Parasitology 20: 119-120. SARCIRONM. E., AL-NAHHAS S., WALBAUMS., RAYNAUDG. & PETAVYA. F. 1991. Treatment of experimental alveolar echinococcosis: comparative study of mebendazole, Isoprinosine and a mebendazole Isoprine association. Tropical Medicine and Parasitology 42: 417419. VERDUN C., BRASSEUR F., VRANCKX H., COUVREUR P. & ROLAND M. 1990. Tissue distribution of doxorubicin associated with polyisohexylcyanoacrylate nanoparticles. Cancer Chemotherapy Pharmacology 26: 13-18.
1993 0
002&7519/93 16.M) + 0.M) Pergamon Press Lrd Sociery for Parasifology
1993 Ausmlinn
RESEARCH NOTE EXPERIENCE WITH DOXORUBICIN-BOUND POLYISOHEXYLCYANOACRYLATE NANOPARTICLES ON MURINE ALVEOLAR ECHINOCOCCOSIS OF THE LIVER M.
TLaboratoire
LIANCE,*
F. NEMATI,t
C. BoRIEst and P.
CoUVREURt
*Laboratoire de Parasitologie, Faculte de Midecine, 8 rue du GBn&ral Sarrail, 94000 Cr&teil, France de Pharmacie galenique et Biopharmacie, URA CNRS 1218, Facultt de Pharmacie, 92296 Malabry, France
Chatenay-
(Received 16 December 1992; accepted 24 January 1993)
Abstract-FIANCE M., NEMATIF., BORIESC. and COUVREUR P. 1993. Experience
with doxorubicin-bound polyisohexylcyanoacrylate nanoparticles on murine alveolar echinococcosis of the liver. Znrernational Journalfor Parasitology 23: 427-429. The parasiticidal properties of doxorubicin against the metacestode of Echinococcus multiloculuris were investigated after binding of that drug to polyisohexylcyanoacrylate nanoparticles, a colloidal biodegradable drug carrier. A reduction of the hepatic parasite development and a reduced viability of the metacestode were observed in mice injected with 5 mg kg-’ body weight-‘, but 7.5 mg kg-’ body weight-’ did not appear more efficient. Free doxorubicin or unbound nanoparticles had no antiparasitic activity. INDEX KEY WORDS: nanoparticles; doxorubicin.
Echinococcus
multiloculuris; murine
hepatic
alveolar
echinococcosis;
Free doxorubicin was obtained from Farmitalia Carlo Erba (Rueil-Malmaison, France), and isohexylcyanoacrylate (IHCA) monomer from Sopar (Start-Dames-Avelynes, Belgium). Doxorubicin-bound nanoparticles were prepared by polymerization of IHCA, according to Couvreur, Roland & Speiser (1982). Unbound PIHCA nanoparticles were prepared in the same way. For doxorubicin-bound nanoparticles 0.1 ml of the suspensions used for animal treatment contained 1.6 mg of nanoparticles, expressed in polymeric compound, and 0.075 mg of entrapped doxorubicin. The size of the nanoparticles ranged from 250 to300 nm, and their number was 1.9 X lOI* ml-‘. Groups of 10 AKR female mice (C.N.R.SC.S.A.L., OrlCans, France), 6 weeks old, received an intrahepatic inoculum of an E. multilocularis metacestode suspension, as previously described (Liance, Vuitton, Guerret-Stocker, Carbillet, Grimaud & Houin, 1984). Treatments were performed by injecting via the tail vein 0.1 ml of the nanoparticle suspensions, or free doxorubicin solution (same dose of doxorubicin). Three administration schedules of bound doxorubicin were established for 30 g infected-mice. Mice of group 1 received a single dose of 2.5 mg kg-’ body weight-’ at day 70 p.i. Those of groups 2 and 3 received additional injections of the
THE potentially unlimited proliferation of Echinococcus multilocularis metacestodes is responsible for alveolar echinococcosis, a neoplastic-like hepatic disease. Chemotherapy of human patients with benzimidazoles is still not yet satisfactory (Eckert, 1991). To date only two experimental therapeutic approaches have been shown partially effective in a rodent model with an induced peritoneal infection. The first, Mitomycin C, is used in the treatment of human cancers (Novak, 1990). It has been suggested that the second one, Isoprinosine, inhibited the larval growth by inducing damages in the nucleic acids synthesis (Sarciron, Al-Nahhas, Walbaum, Raynaud & Petavy, 1991). Following these studies and taking into account the primary localization of the parasite in the liver, we have tested the efficacy of doxorubicin loaded onto polyisohexylcyanoacrylate nanoparticles. In fact, it has been shown previously that this binding resulted in a preferential tissue distribution of the drug to the liver (Verdun, Brasseur, Vranckx, Couvreur & Roland, 1990), with a subsequent reduced toxicity and an increased antitumoral activity (Chiannilkulchai, Driouich, Benoit, Parodi & Couvreur, 1989).
* To whom all correspondence
model;
should be addressed. 427
428
M. LIANCE~~ al.
same dose at day 80 and at days 80 and 90 p.i., respectively. Control groups included animals injected, at days 70, 80 and 90 pi., with unbound nanoparticle suspension (group 4); animals injected, at the same three times p.i., with 2.5 mg kg-’ body weight-’ of free doxorubicin (group 5) and infected untreated animals (group 6). Treated mice were sacrificed under ether anesthesia 21 days after the end of the injections. This time was chosen in order to avoid secondary formation of hepatic lesions from metastases. Untreated mice were sacrificed at the same time as the groups receiving three treatments. TABLE
I-EFFECTS
OF DOXORUBICLN-LOADED NANOPARTKLE NANOPARTICLE SUSPENSION,AND FREE
SUSPENSION,UNLOADED
DOXORUBICINSOLUTION,ONTHE HEPATICGROWTHOF
E. multilocularis
Group
Positive
of
hepatic
Jirds
i.p. infected
hepatic
from
parasite
infection
mice
in mice
No. infected/
Mean cyst
dissected
weight (g) 17.8
1
lo/lo
414
2
4/10
414
6.7
3
S/IO
9.2
4
IO/l0
414 4/3*
20.8
5
IO/l0
414
21.4
6
IO/l0
414
19.4
(I)
2.5
mg
kg-’
body
5 mg kg-’
weight-’
doxorubicin;
(2)
body weight-’
doxorubicin;
(3) 7.5 mg kg-
’ body
doxorubicin;
(4) unloaded
nanoparticles;
weight-
of
nanoparticle
of nanoparticle
’ of
nanoparticle
(5)
7.5 mg kg-’
body weight -’ of
free doxorubicin; (6) untreated mice. * One jird died before autopsy.
Parasitological monitoring showed no significant difference between the six groups regarding the mean weight of both peritoneal parasites and livers. All mice harboured peritoneal metastases. In mice harbouring a hepatic parasite (Table I), the approximate superficial size of this was always lower than 5 mm in diameter in groups 2 and 3, and always higher than 7 mm in the other groups. A viability assay was performed from both hepatic and peritoneal parasites in 2 Meriones unguiculutus jirds, as previously described (Liance, Bresson-Hadni, Vuitton, Bretagne & Houin, 1990). It was performed from two mice randomly selected from each group. Two months p.i., there was no significant difference between the six groups regarding the parasite burden obtained after infection with peritoneal metacestodes. Results obtained after infection with hepatic metacestodes (Table l), showed a reduced viability of larvae removed from mice of groups 2 and 3.
A histopathological examination of the hepatic lesions from two mice randomly selected from each group and different from those used for testing the parasite viability, revealed different patterns. In mice of groups 1, 4 and 6, numerous parasitic vesicles were present, surrounded by a typical polymorphous granuloma, as previously described in this murine strain (Liance et al., 1984). In mice of groups 2, 3 and 5, vesicles were surrounded by necrosis and numerous leucocytes. Hepatocytes located near these lesions harboured a nucleus dystrophy. Lastly, in mice of group 2, numerous portal vessels were infiltrated with leucocytes. In this study, doses of doxorubicin far less than those used to inhibit hepatic sarcoma metastases (Chiannilkulchai et al., 1989) were evaluated. Free doxorubicin appeared without parasiticidal properties, although it modified the peri-parasitic cellular pattern. The efficiency of nanoparticle doxorubicin was demonstrated by inhibiting the hepatic larval growth, and by reducing the parasite viability, exclusively originating from the liver. However investigations of other strategies for the administration of doxorubicin-loaded PIHCA nanoparticles are required. As the more long-term treatment and the lowest dose appeared obviously of low efficiency, it would be of interest to evaluate the effects of higher doses administered during a shorter period of time. On the other hand, as all animals exhibited high peritoneal parasitic burden at the time of autopsies, it would also be of interest to start the treatment at an earlier stage of the larval development. Because atypical periparasitic tissue reactions occurred with either free or bound doxorubicin, such experiments would include an histopathological study in order to determine the effects of the drug on liver samples taken far from the parasite. Those data should encourage further development of anthelminthic compounds bound to biodegradable and colloidal drug carriers, such as PIHCA. Acknowledgements-The for her technical grant
from
Doctorales
authors
assistance.
the Direction du Ministire
wish to thank
D. Rivollet
This work was supported de la Recherche
de 1’Education
by a
et des Etudes
Nationale.
REFERENCES CHIANNILKULCHAIN., DR~OUICH Z., BENOIT J. P., PARODI A. L.
&
COUVREUR P.
1989.
Doxorubicin-loaded
efficiency
in
particles:
increased
murine
metastases.
Selective Cancer Therapeutics 5: 96106.
COUVREUR P., ROLAND M. & SPEISER P. 1982.
U.S.
nanohepatic Patent
No. 4,329%332. ECKERT J. 1991. update.
Experimental
research
on echinococcosis:
In: Archives de la Hidatidosis XXX (Edited
ROSA F.),pp. 31 I-319.
Libbey
& Co. Ltd, London.
by DE
Research LIANCE M., VUII-~ON D. A., GUERRET-STOCKER S., CARBILLET J. P., GRIMAUD J. A. & HOUIN R. 1984. Experimental alveolar echinococcosis. Suitability of a murine model of intrahepatic infection by Echinococcus mulrilocularis for immunological studies. Experientia 40: 1436-1439. LIANCE M., BRESSON-HADNI S., VUIT~ON D., BRETAGNE S. & HOUIN R. 1990. Comparison of the viability and developmental characteristics of Echinococcus multilocularis isolates from human patients in France. International Journal Parasitology 20: for 83-86.
Note
429
NOVAK M. 1990. Efficacy of mitomycin C against alveolar Echinococcus. International Journhl for Parasitology 20: 119-120. SARCIRONM. E., AL-NAHHAS S., WALBAUMS., RAYNAUDG. & PETAVYA. F. 1991. Treatment of experimental alveolar echinococcosis: comparative study of mebendazole, Isoprinosine and a mebendazole Isoprine association. Tropical Medicine and Parasitology 42: 417419. VERDUN C., BRASSEUR F., VRANCKX H., COUVREUR P. & ROLAND M. 1990. Tissue distribution of doxorubicin associated with polyisohexylcyanoacrylate nanoparticles. Cancer Chemotherapy Pharmacology 26: 13-18.