Kreuter Peer Reviewed Article #2AND HERE'S THE SECOND ARTICLE
Toxicology Letters 126 (2002) 131-141
Toxicological studies of doxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles in healthy rats and rats with intracranial glioblastoma
S.E. Gelperina a, A.S. Khalansky b, I.N. Skidan a, Z.S. Smirnova c,
A.I. Bobruskin a, S.E. Severin a, B. Turowski d, F.E. Zanella d, J. Kreuter e,*
a Moscow Institute of Medical Ecology, Sympheropolsky Blvd. 8, Moscow, Russia
b Institute of Human Morphology, Tsurupa ul. 3, Moscow, Russia
c Blokhin Cancer Research Centre, Kashirskoye sh. 24, Moscow, Russia
d Institute of neuroradiology, J.W. Goethe University, Schleusenweg 2-16, Frankfurt Germany
e Institute of Pharmaceutical Technology, Biocentre, J.W. Goethe University, Marie-Cu, rie-Strasse 9,
D-60439 Franfurt | Main, Germany
Received 9 July 2001; received in revised form 11 October 2001; accepted 15 October 2001
Abstract
Polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles (NP) were shown to enable the transport of a
number of drugs including the anti-tumor antibiotic doxorubicin (DOX) across the blood-brain barrier (BBB) to
the brain after intravenous administration and to considerably reduce the growth of brain tumours in rats. The
objective of the present study was to evaluate the acute toxicity of DOX associated with polysorbate 80-coated NP
in healthy rats and to establish a therapeutic dose range for this formulation in rats with intracranially implanted
101/8 glioblastoma. Single intravenous administration of empty poly(butyl cyanoacrylate) NP in the dose range
100–400 mg/kg did not cause mortality within the period of observation. NP also did not affect body weight or
weight of internal organs. Association of DOX with poly(butyl cyanoacrylate) NP did not produce significant changes
of quantitative parameters of acute toxicity of the anti-tumour agent. Likewise, the presence of polysorbate 80 in the
formulations was not associated with changes in toxicity compared with free or nanoparticulate drug. Dose regimen
of 3 x 1.5 mg/kg on days 2, 5, 8 after tumour implantation did not cause drug-induced mortality. The results in
tumour-bearing rats were similar to those in healthy rats. These results demonstrate that the toxicity of the DOX bound
to NP was similar or even lower than that of free DOX. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Doxorubicin; Glioblastoma; Nanoparticles; Poly(butyl cyanoacrylate); Polysorbate; Toxicology; Rats
1. Introduction
*Corresponding author. Tel.: +49-69-798-29682; fax: + 49-69-798-29724.
E-Mail address: kreuter@em.uni-frankfurt.de (J. Kreuter).
Malignancies of the central nervous system are among the most resistant to systemic chemother-
apy largely due to the presence of the blood-brain barrier (BBB) that limits penetration of antineoplastic drugs into brain tumours. The BBB acts as an anatomical and physiological barrier formed by a monolayer of endothelial cells that exhibit specific properties such as intercellular tight junctions, which prevent paracellular transport. Also the ATP-dependant efflux pump P-gylcoprotein (P-gp) has been shown to be present at the luminal side of the endothelial cells of the BBB (Saunders et al., 1999). As a consequence of the P-gp expression at the BBB interface and overexpression at the tumour cell level, the bioavailability of anticancer agents is extremely low, which explains the failure of brain tumour chemotherapy (Tsuji, 1998; Bossanyi et al., 1997). Different methods have been developed to overcome limited access of the anti-tumour drugs to the brain, such a disruption of the BBB by mannose or bradykinin (Bonstelle et al., 1983; Nomura et al., 1994), or intratumoural administration (Shimura et al., 1996). However, these approaches are rather drastic measures: The disruption of the BBB is accompanied by complications such as cerebral oedema while intratumoral administration is an invasive procedure requiring neurosurgical intervention.
An alternative approach to the problem is the employment of a drug delivery system that enables an improved or even targeted delivery of the anti-tumour agents to the brain. The results of a number of authors have proved that brain delivery of the drugs could be achieved using sterically stabilised colloidal carriers, such as liposomes (Siegal et al., 1995; Boiardi et al., 1999), solid lipid nanoparticles (NP; Fundaro et al., 2000) or polymer NP. Polysorbate 80-coated poly(butyl cyanoacrylate) NP enabled the transport of loperamide, tubocurarine and dalargin across the BBB to the brain after intravenous administration (Alyautdin et al., 1995, 1997, 1998; Schroeder et al., 1998).
In a previous study we showed that anti-tumour antibiotic doxorubicin (DOX) also could be delivered in the brain with polysorbate 80-coated NP after intravenous administration in healthy rats, yielding high whole brain concentrations of 6 µg/g tissue whereas all the controls, DOX in solution form without and with polysorbate 80 or DOX bound to uncoated NP remained below the detection limit (0.1 µg/g; Gulyaev et al., 1999). This formulation also enabled a considerable growth reduction of an experimental rat Glioblastoma, 101/8 (Gelperina et al., 2000). Forty percent of the rats treated with DOX bound to NP overcoated with polysorbate 80 survived for half a year and after sacrifice showed total tumour remission whereas all controls died between 10 and 20 days.
The objective of the present study was to evaluate the acute toxicity of the anthracycline antibiotic DOX associated with polysorbate 80-coated NP in rats and to establish a therapeutic dose range for this formulation in rats with intracranially implanted glioblastomas.
2. Materials and methods
2.1. Chemicals
DOX hydrochloride was a generous gift from Sicor (Milan, Italy), polysorbate 80 and dextran 70.000 were supplied by Sigma (St. Louis, USA), butylcyanoacrylate was obtained from Sichel-Werke (Hanover, Germany). Other reagents used in this study were of analytical grade (Fluka Chemie, Buchs, Switzerland).
2.2. Preparation and characterisation of nano particulate preparations
Nanoparticle-bound doxorubicin (DOX-NP) was prepared by anionic polymerisation (Gulyaev et al., 1999). Briefly, 1% of butylcyanoacrylate was added to 1% dextran solution on 0.001 N HCl under constant stirring. After 40 min DOX was added to the mixture to obtain a final concentration of 0.4%. After 2.4 h the mixture was neutralised, filtered and freeze-dried with addition of 3% manitol as cryoprotector. The particle size was measured by light scattering (Nanosizer Malvern, Great Britain), an average diameter of 270 ± 30 nm was found. The amount of free drug in the preparation was assessed spectrophotometrically after particle separation by centrifugation. It was shown that 80% of the drug in suspension was associated with the NP. For surfactant coating 1% polysorbate 80 was added to the suspension, and the suspension was incubated for 30 min under stirring and then used within 2 h. Empty NP (average diameter of 250 ± 30 nm) were produced using this same technique.
2.3. Animals
The animal experiments were performed in accordance to the Russian Guidelines for Animal Experiments and authorised by the Russian Ministry of Health (1045)-73 and 52-F3-24.04.95).
The toxicity of empty NP and free and NP -DOX in healthy animals was assessed using male Wistar rats weighing 160–195 g. The animals were purchased from the animal production unit of the Russian Academy of Medical Sciences (Moscow, Russia).
The toxicity in tumour-bearing animals was carried out using white male non-inbred rats weighing 200–220 g obtained from Blokhin Cancer Research Centre (Moscow, Russia).
All animals were kept in standard animal facilities and given free access to food and water throughout the study.
2.4. Tumour line
Glioblastoma 101/8 was initially produced by local injection of DMBA and maintained by repeated intracerebral implantation (Yablonovskaya et al., 1997). For long-term storage the tumour cells were kept at -196 *C.
2.5. Tumour implantation
The animal was anaesthetised with nembutal. A small hole was drilled with a spherical dental bore 2 mm from bregma and 3.5 - 4 mm from the sagittal suture in the right parietal bone. With a syringe fixed in a sterotactic device tumour cells (4 x 106) from the frozen stock were introduced in the cavity of the right lateral ventricle. After development of pronounced clinical signs of the disease (usually day 14) the animal was anaesthetised with ether and the brain was immediately removed. The tumour was excised and homogenised with a scalpel, 30 mg of homogenised tissue was implanted into the brain of each experimental animal as described above.
2.6. Toxicological study in healthy animals
Wistar rats (n = 207) were randomised into groups, as follows, n = 9 for placebo; n = 6 for DOX preparations. Intact animals were used as a control (n = 9). The preparations were administered as a single intravenous injection in the tail vein. Empty NP were resuspended in water and injected in the concentration 26 mg/ml (as poly(-butyl cyanoacrylate)) with a rate of 0.1 ml/s at doses 100, 160, 220, 280, 340, or 400 mg/kg. Doses of 280–400 mg/kg were divided in two and injected after a 1 h interval. DOX preparations were dissolved or resuspended in water or 1% polysorbate 80, as described above and injected in the concentration 2.5 mg/ml with a rate of 0.1 ml/s at doses 4.5, 7.5, 9, 12, 15, 18, or 21 mg/kg (corresponding to DOX). The animals were observed for 30 days, then surviving animals were sacrificed and necropsied.
Acute toxicity was estimated by mortality and survival time, as well as by clinical picture of intoxication including behavioural reactions. Animals on study were observed for any adverse events, such as changes in body weight, stool, condition of eyes and nose, motor activity, as well as neuromuscular reactions (tremor, cramps, changes of muscular tonicity), ataxia, etc. All animals were examined for internal abnormalities, size; weight; and appearance of brain, heart, lungs, liver, spleen, kidneys, and testes were assessed at necropsy.
2.7. Toxicological study in tumour-bearing animals
Animals were randomly divided into six groups as follows, (1) untreated, which served as control; (2) treated with blank NP coated with polysorbate 80 (NP-Ps 80); (3) treated with standard DOX formulation in saline (DOX); (4) treated with standard DOX formulation in 1% polysorbate 80 solution (DOX + Ps 80); (5) treated with DOX - NP; (6) treated with DOX bound to NP coated with polysorbate 80 (DOX–NP + Ps 80). Drug preparations were administered intravenously in the following regimens, 1 x 9 mg/kg on day 2; 2 x 4.5 mg/kg on day 2 and day 6; 2.5 mg/kg on day 2, day 5, and day 8; and 1.5 mg/kg on day 2, 5, and 8. Hence, total doses amounted to 9, 7.5 and 4.5 mg/kg.
For analysis of the data on body weights of the experimental groups were compared to the control group using Student's t-test (P < 0.05).
3. Results
3.1. Investigation of acute toxicity of empty nanoparticles and doxorubicin formulations in healthy rats.
Single intravenous injection of NP in the dose range of 100–400 mg/kg did not cause mortality and also did not affect the body weight comparing to the control group within the 30-day observation period (data not shown). Necropsy of the animals at the end of the experiment did not show any macroscopic changes of the organs. The average weight of these organs did not differ from that of the control group.
It should be mentioned, however, that the injection of high doses of NP caused transient reversible changes in behavioural and peripheral reactions of animals within 10–15 min after injection. These changes were manifested as a pronounced increase of motor activity and stereotypy, as well as changes in heart and ventilation rates. After 10–15 min activation changed into inhibition followed by sleep that lasted approximately 1.5 h. After this time the state of the animals was normal and adverse vascular or behavioural reactions were not observed.
It is noteworthy, that the events described above were only observed after administration of nanoparticle doses that considerably exceeded the therapeutic level required for the delivery of DOX, i.e. below 20 mg/kg of poly(butyl cyanoacrylate) NP.
The toxicity of DOX injected in form of an aqueous solution (DOX) or in 1% polysorbate 80 solution (DOX + Ps 80) was dose-dependent (Table 1). Administration of DOX or DOX + Ps 80 in doses that caused total or partial mortality was associated with considerable loss of weight, adynamia, tremor, and piloerection. Diarrhoea was observed from day 3. Animals in extremis showed adynamia, hypodynamia, piloerection, narrowing of the eye-slit and fading of the eye colour; body weight was decreased to cachexia (by 25–45%).
Fig. 1 shows the effect of DOX on rat weight. It can be seen that the effect on both preparations (DOX and DOX + Ps 80) in the dose of 4.5 mg/kg on body weight was not significant. Administration of DOX or DOX + Ps 80 in the dose of 7.5 mg/kg caused a significant decrease in body weight by 16-20% compared with the initial weights at day 0. By day 20 surviving rats regained some weight, but not to control levels. Administration of DOX in the dose of 9 mg/kg caused partial mortality and also inhibited the increase in body weight. The same doses of DOX + Ps 80 caused a 30% decrease in body weight compared with the controls. This decrease was not as pronounced as with DOX alone. After day 10–15 animals treated with 4.5 mg/kg DOX started to regain some weight. In general, these data are consistent with the clinical picture.
Necropsy examination of animals that died because of toxicity of DOX and DOX + Ps 80 revealed plethoric blood vessels in the pericardium and hemopericardium, significantly lower weight for the spleen and collapsed lungs. The stomach was filled with liquid contents, the intestine was collapsed and partially filled with mucus, and the intestine mucosa was thinned and ulcerated. Faded lesions were observed in liver edge and blood vessels in the liver were plethoric. 70% of rats in the group treated with 9 mg/kg of DOX + Ps 80 had serious ascites in the abdominal cavity with 2–3.5 ml of exudate.
When expressed relative to body weight, statistically significant lower weights were seen only for kidneys (two times lower) and testes (3.5 times lower). Weights of other organs did not differ from that of control.
Necropsy of the remaining animals showed signs of recovery, with the exception of kidneys and testes. Weights of these organs were not regained by the end of the study, therefore, kidneys and testes appeared to be most sensitive to DOX toxicity, which correlates with the previously published data (Borovskaya and Goldberg, 2000).
Peaks of death after lethal doses of DOX - NP and DOX-NP + Ps 80 were observed on days 1–4 and 1–2, respectively, (Table 1). Animals in the terminal stage showed hypodynamia, adynamia and narrowing of the eye slit.
Necropsy examination of the animals that died of toxicity showed moderately lower spleen weights, some animals had exudate (up to 1.5 ml) in the pleural cavity. In contrast to DOX, nanoparticulate preparations did not cause pronounced damage of GIT mucosa.
Administration of DOX–NP and DOX–NP + Ps 80 in non-toxic doses or doses that caused partial mortality was associated with hypodynamia within two days after treatment. Diarrhoea or other toxic effects were not observed.
The effects of DOX-NP and DOX-NP + Ps 80 on the body weight were less pronounced compared to DOX (Fig. 2). It can be seen from Fig. 2B that weight of rats treated with 4.5 mg/kg of DOX-NP + Ps 80 did not differ statistically from control.
Exudate in pleural cavity was found in animals that were treated with DOX–NP and died of toxicity, but there was no exudate in the group treated with DOX–NP + Ps 80.
Necropsy examination of surviving animals in the groups treated with DOX–NP did not reveal macroscopic abnormalities. Assessment of weights of organs of remaining animals in the groups treated with DOX–NP and DOX–NP + Ps 80 demonstrated that weights of kidneys and testes were lower by 2.2 and three times, respectively, which is similar to the effect of drug substance. However, the average weight of testes in the group treated with DOX–NP + Ps 80 was significantly higher by 21% compared with the groups treated with DOX and DOX + Ps 80 (data not shown).
Therefore, association of DOX with poly(butyl cyanoacrylate) NP did not produce significant changes of quantitative parameters of the acute toxicity of antibiotic. However, the overall toxicity profile appears to be altered favorably as demonstrated by the absence of pronounced GIT toxicity of nanoparticulate formulations. In addition, as shown previously, the cardiac toxicity may be reduced considerably (Couvreur et al., 1982). Presence of polysorbate 80 in the formulation was not associated with considerable changes of toxicity of free or nanoparticulate drug.
3.2. Investigation of toxicity of doxorubicin formulations in rats with intracranially implanted glioblastoma
The toxicity of DOX formulations using various regimens was investigated in rats with intracanially implanted 101/8 glioblastoma. This tumour is characterised by a monomorphological structure, its transplantability at intracranial implantations exceeds 90%. Glioblastoma 101/8 was extensively used for a variety of studies, especially for investigations of biology and pathology of glia and glial tumours, as well as for experimental chemotherapy of brain malignancies (Smirnova et al., 2000).
The basic parameter for evaluation of the cause of animal death was the presence or absence of tumour at the site of implantation; other parameters included the clinical signs of the disease and the results of the necropsy examination. Death was considered to be due to drug toxicity if a tumour could not be detected at the site of implantation. The data on drug-related toxicity are presented in table 2. Necropsy of all these animals revealed absence of tumour; in some animals occasional lesions (diameter up to 1 mm) of transformed tissue could be seen. Microscopic investigation demonstrated that these lesions contained single cells or cell groups that could be characterised by absence of mitosis, cytoplasm vacuolisation and changed form of outer membrane and nuclei. It is noteworthy that by this time (days 11-13) control animals developed tumours up to 9 mm in diameter. Drug-induced death was associated with considerable loss of body weight and lower spleen weight. Investigation of internal organs of the animals that died after day 13 revealed decreased blood supply of brain and spleen, ulceration of colon mucosa and pathological changes of liver (changes of colour and decreased blood supply).
As seen from Tables 1 and 2, there was a good correlation for DOX toxicity between tumour-bearing and healthy animals for the dose of 9 mg/kg, in both cases adminstration of this dose of DOX resulted in 33% (2/6 vs. 5/15 animals) drug-related mortality. Administration of 2 x 4.5 mg/kg led to an increase in mortality up to 53% (8/15) animals, probably due to cumulative drug toxicity. The same tendency was observed for DOX + Ps (12% and 62%, or 1/8 and 5/8 animals, respectively).
Single administration of 9 mg/kg of DOX–NP + Ps 80 in tumour-bearing animals caused 33% (6/18 animals) of drug-related mortality, which also corresponds to the result obtained with the healthy rats (2/6 animals). Only 12 % (1/8 animals) mortality was observed for the same dose of DOX–NP. Fifty percent (4/8) of the animals died due to drug-related toxicity after administration of 2 x 4.5 mg/kg of DOX–NP, whereas in the group treated in the same regimen with DOX–NP + Ps 80 toxic death was observed only for 12% (12/17) of the animals.
As seen from the data presented in Table 2 the administration of nanoparticulate formulations in dose regimen of 3 x 2.5 mg/kg was associated with minor toxicity (1/24 animals died in the group treated with DOX–NP + Ps 80) and no drug-induced mortality occurred with a dose of 3 x 1.5 mg/kg on days 2, 5, 8 after tumour implantation.
4. Discussion
Polyalkylcyanoacrylate NP hold promise as carriers for the delivery of drugs to solid tumours, and DOX–NP has successfully passed the clinical phase I study (Kattan et al., 1992). Due to the enhanced permeability and retention (EPR) effect first described by Maeda et al. (Matsumura and Maeda, 1986; Maeda and Matsumura, 1989; Seymour, 1992) these particles can passively accumulate in solid tumour tissue. This effect has been attributed to two main factors (Duncan et al., 1996), firstly, tumour vascularisation often has a discontinuous endothelium, which allows extravasation of macromolecules and colloidal particles to a greater extent than via most other endothelial barriers, and secondly, tumours also frequently have a reduced lymphatic drainage leading to the accumulation of the macromolecular material.
However, the data on toxicity and safety of poly(butyl cyanoacrylate) NP are somewhat contradictory. Kante et al. found LD50 of 230 mg/kg for poly(butyl cyanoacrylate) NP injected intravenously in mice (Kante et al., 1982). Olivier et al. (1999) observed a 30–40% mortality in mice after injection of 166 mg/kg of poly(butyl cyanoacrylate) NP, while some animals died already at lower doses. At the same time Simeonova et al., who investigated the immunomodulating properties of poly(butyl cyanoacrylate) NP in mice, did not report any signs of toxicity at doses up to 200 mg/kg (Simeonova et al., 1998). A much higher LD50 of 585 mg/kg was found for NP made of a similar polymer, poly(hexyl cyanoacrylate) (Couvreur et al., 1986). Moreover, Couvreur et al. did not observe any significant toxicity after multiple injections of poly(isohexyl cyanoacrylate) NP at doses of 8 x 20 mg polymer/kg body weight or 16 x 20 mg polymer/kg body weight over a time period of 30 or 105 days, respectively (Couvreur et al., 1986). However, poly(isohexyl cyanoacrylate) possesses pronounced bone marrow toxicity (Gibaud et al., 1999).
In view of this result, the observation that DOX also possesses considerable bone marrow toxicity (Sundman-Engberg et al., 1998), and because poly(butyl cyanoacrylate) was employed in the most promising studies on brain delivery with NP, we used poly(butyl cyanoacrylate) NP in the present study.
Administration of empty poly(butyl cyanoacrylate) particles in doses up to 400 mg/kg was not associated with mortality. Assessment of the body weight compared with the control group or macroscopic investigation of the organs also did not reveal any apparent signs of toxicity.
Association of DOX with poly(butyl cyanoacrylate) NP did not produce significant changes in the observed quantitative parameters of acute toxicity of the antibiotic. Also the presence of polysorbate 80 in the formulation was not associated with any considerable changes of toxicity of free or nanoparticle-bound drug. In contrast, polysorbate 80 seemed to even reduce toxic effects, although these differences were mire and not at all significant. However, it may be expected that the overall toxicity profile of DOX could be altered by binding to NP: we observed the absence of pronounced GIT toxicity of nanoparticulate formulations, and Couvreur et al. reported a very considerable reduction in heart accumulation of DOX bound to similar polyalkylcyanoacrylate NP (Couvreur et al., 1982). The latter finding is especially important since the heart toxicity is a paramount problem in DOX chemotherapy. Couvreur et al. also investigated the toxicity of DOX bound to poly(ishexyl cyanoacrylate) NP after multiple intravenous injections of various doses in mice (Couvreur et al., 1982). Administration of nanoparticulate DOX was associated with a considerably lower mortality and loss of body weight was less pronounced, compared with the free drug. These facts allowed these authors to conclude that DOX toxicity could be decreased using NP. It has to be mentioned, however that the toxicity of free DOX as assessed in Couvreur's study was considerably lower compared to our results, i.e. daily injection of antibiotic in dose of 15 mg/kg in the course of 3 days led to only a partial mortality.
In general, the experiments in the tumour-bearing animals showed a good correlation with the data obtained for the healthy animals. The maximum tolerated dose for the DOX formulations was close to 7.5 mg/kg. The dose regimens of 3 x 2.5 and 3 x 1.5 mg/kg on days 2, 5, 8 after tumour implantation are used in an ongoing study of the anti-tumour efficacy of DOX formulations in rats with intracranially implanted glioblastoma.
5. Conclusions
The single intravenous administration of empty poly(butyl cyanoacrylate) NP in the dose range 100–400 mg/kg did not cause mortality within the period of observation. NP also did not affect body weight or weight of internal organs. The only adverse effect manifested as behavioural and peripheral reactions only could be observed at NP doses that considerably exceeded doses that were required to achieve a therapeutic level of DOX. Moreover, this effect was transitory and reversible (within 10–15 min after injection).
Association of DOX with poly(butyl cyanoacrylate) NP did not produce significant changes of quantitative parameters of acute toxicity of the anti-tumour agent. Also the presence of polysorbate 80 in the formulation was not associated with considerable changes of toxicity of free or nanoparticulate drug.
The toxicological results obtained after multiple administration of nanoparticle-bound DOX in glioblastoma-bearing rats in the present study allow an estimation of the maximal therapeutic dose levels for brain delivery.
Acknowledgements
The authors wish to gratefully acknowledge that this study in part was enabled by a generous donation by the Suntrup and Fredebeul Families, Ennigerloh, Germany, in commemoration of their mother, Magret Fredebeul, who died of a brain tumour and by a generous gift of DOX by the Sicor company, Milan, Italy. This study also was supported by travel grants for two of the authors (S.E. Gelperina A.S. Khalansky) by DFG (Deutsche Forschungsgemeinschaft).
References
Alyautdin, R.N., Gothier, D., Petrov, V.E., Kharkevich, D.A., Kreuter, J., 1995. Analgesic activity of the hexapeptide dalargin adsorbed on the surface of polysorbate 80-coated poly(butylcyanoacrylate) nanoparticles. Eur. J. Pharm. Biopharm. 41, 44–48.
Alyautdin, R.N., Petrov, V.E., Langer, K., Berthold, A., Kharkevich, D.A., Kreuter, J., 1997. Delivery of loperamide across the blood-brain barrier with polysorbate 80-coated polybutylcyanoacrylate nanoparticles. Pharm. Res. 14, 325-328.
Alyatdin, R.N., Tezikov, E.B., Ramge, P., Kharkevich, D.A., Begley, D.A., Kreuter, J., 1998. Significant entry of tubocurarine into the brain of rats by adsorption to polysorbate 80-coated polybutylcyanoacrylate nanoparticles. J. Microencapsul. 15, 67–74.
Boiardi, A., Pozzi, A., Salmaggi, A., Eoli, M., Zucchetti, A., Silvani, A., 1999. Safety and potential effectiveness of daunorubicin-containing liposomes in patients with advanced recurrent malignant CNS tumors. Cancer Chemother. Pharmacol. 43, 178–179.
Bonstelle, C.T., Kori, S.H., Rekate, H., 1983. Intracarotid chemotherapy of glioblastoma after induced blood-brain barrier disruption. Am. J. Neuroradiol. 4, 810-812.
Barovskaya, T.G., Goldberg, E.D., 2000. Influence of antracycline antibiotics on functional status of reproductive system in rats. Bjull. Eksp. Biol. Med. 130, 527–530 (Russian).
Bossanyi, P., Diete, S., Dietzmann, K., Warich-Kirches, M., Kirches, E., 1997. Immunohistochemical expression of P-glycoprotein and glutathione S-transferases in cerebral gliomas and response to chemotherapy. Acta Neuropathol. 94, 605–611.
Couvreur, P., Kante, B., Grislain, L., Roland, M., Speiser, P., 1982. Toxicity of polyalkylcyanoacrylate nanoparticles III: doxorubicin-loaded nano particles. J. Pharm. Sci. 71, 790–792.
Couvreur, P., Grislain, L., Lenaerts. V., Brasseur, P., Guiot, P., Biernacki, A., 1986. Biodegradable polymeric nanoparticles as drug carrier for antitumor agents. In: Guiot, P., Couvreur, P. (Eds.), Polymeric Nanoparticles and Microspheres. CRC Press, Boca Raton, pp. 27–93.
Duncan, R., Dimitrijevic, S., Evagorou, E.G., 1996. The role of polymer conjugates in the diagnosis and treatment of cancer. S.T. P. Pharma Sci. 6. 237–263.
Gelperina, S.E., Smirnova, Z.S., Khalansky, A.S., Skidan, I.N., Bobruskin, A.I., Kreuter, J., 2000. Chemotherapy of brain tumors using doxorubicin bound to polysorbate 80-coated nanoparticles, Proceedings 3rd World Meeting APV/SPGI, Berlin, 3/6 April 2000, pp. 441–443.
Gibaud, S., Weingarten, C., Andreux, J.-P., Couvreur, P., 1999. Ciblage de la moelle osseuse á l'aide de nanoparticules de polycyanoacrylate. Ann. Pharm. Fr. 57, 324–331.
Gulyaev, A.E., Gelperina, S.E., Skidan, I.N., Antropov, A.S., Kivman, G.Y., Kreuter, J., 1999. Significant transport of doxorubicin into the brain with polysorbate 80-coated nanoparticles. Pharm. Res. 16, 1564–1569.
Fundaro, A., Cavalli, R., Bargoini, A., Vighetto, D., Zara, G.P., Gasco, M.R., 2000. Non-stealth and stealth solid lipid nanoparticles (SLN) carrying doxorubicin: pharmacokinetics and tissue distribution after i.v. administration to rats. Pharmacol. Res. 42, 337–343.
Kante, B., Couvreur, P. Dubois-Krack, G., De Meester, C., Guiot, P., Roland, M., Mercier, M., Speiser, P., 1982. Toxicity of polyalkylcyanoacrylate nanoparticles I: free nanoparticles. J. Pharm. Sci. 71, 786–790.
Kattan, J., Droz, J.-P., Couvreur, P., Marinao, J.-P., Boutan-Laroze, A., Rougier, P., Brault, P., Vranckx, H., Grognet, J.-M., Morge, X., Sancho-Garnier, H., 1992. Phase I clinical trial and pharmacokinetic evaluation of doxorubicin carried by polyisohexylcyanoacrylate nanoparticles. invest. New Drugs 10, 191–192.
Maeda, H., Matsumura, Y. 1989. Tumoritropic and lymphotropic principles of macromolecular drugs. CRC Crit. Rev. Drug Carrier Syst. 46, 193–210.
Matsumura, Y., Maeda, H., 1986. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS. Cancer Res. 6, 6387–6392.
Nomura, T., Inamura, T., Black, K.L., 1994. Intracarotid infusion of brandykinin selectively increases blood-tumour permeability in 9L and C6 brain tumors. Brain Res. Brain Res. 659, 62–66.
Olivier, J.C., Fenart, L., Chauvet, R., Pariat, C., Cecchelli, R., Couet, W., 1999. Indirect evidence that drug brain targeting using polysorbate 80-coated polybutylcyanoacrylate nanoparticles is related to toxicity. Pharm. Res. 16, 1836–1842.
Saunders, N.R., Habgood, M.D., Dziegielewska, K.M., 19999. Barrier mechanisms in the brain. I. Adult brain. Clin. Exp. Pharmacol. Physiol. 26, 11–19.
Schroeder, U., Sommerfeld, P., Ulrich, S., Sabel, B.A., 1998. Nanoparticle technology for delivery of drugs across the blood-brain barrier. J. Pharm. Sci. 87, 1305–1307.
Seymour. L.W., 1992. Passive tumour targeting of soluble macromolecules and drug conjugates. CRC Crit. Rev. Drug Carrier Syst. 9. 135–187.
Shimura, T., Teramoto, A., Nakazawa, S., Aihara. K., 1996. A clinicopathological study of malignant glioma done after local administration of chemotherapeutic agents. Clin. Neuropathol. 15, 119–124.
Siegal, T., Horowitz, A., Gabizon, A., 1995. Doxorubicin encapsulated in sterically stabilised liposomes for the treatment of a brain tumor model: biodistribution and therapeutic efficacy. J. Neurosurg. 83, 1029–1037.
Simeonova, M., Chorbadjiev, K., Antcheva, M.; 1998 Study of the effect of polybutylcyanoacrylate nanoparticles and their metabolites on the primary immune response in mice to sheep red blood cells. Biomaterials 19, 2187–2193.
Smirnova Z.S., Gerassimova G.K., Rodionova Y.V., Gershtein E.S., Khalansky A.S., 2000. Experimental brain tumours for preclinical trial of experimental anticancer drugs., Proceedings V Russian Symposium Oncology, Kalzan. 4-7 October 2000, pp. 214–217 (Russian).
Sundman-Engberg, B., Tidefelt, U., Paul, C., 1998. Toxicity of cytostatic drugs to normal bone marrow cells in vitro. Cancer Chemother. Pharmacol. 42, 17–23.
Tsuji, A., 1998. P-glycoprotein-mediated efflux transport of anticancer drugs at the blood-brain barrier. Ther. Drug Monit. 20, 588–590.
Yabonovskaya, A.Y., Spryshkova, N.A. , 1997. Morphological and biological evaluation of rat experimental cerebellum tumours. Arch. Pathol. 33, 50–52 (Russian).