Date Published: , 2012
Publisher: A.I. Gordeyev
Author(s): E.S. Kolotova, S.G. Egorova, A.A. Ramonova, S.E. Bogorodski, V.K. Popov, I.I. Agapov, M.P. Kirpichnikov.
Biodegradable polylactide microparticles with encapsulated cytotoxic protein
viscumin were obtained via the ultrasound-assisted supercritical fluid
technique. The size of the microparticles was 10–50 µM, as shown by
electron microscopy. The time course of viscumin release from microparticles was
studied using an immunoenzyme test system with anti-viscumin monoclonal
antibodies. It was found that 99.91% of the cytotoxic protein was incorporated
into polymer microparticles. Only 0.08% of the initially encapsulated viscumin
was released from the microparticles following incubation for 120 h in a
phosphate-buffered saline at neutral pH. Importantly, the method of ultrasonic
dry supercritical fluid encapsulation failed to alter both the cytotoxic potency
and the immunochemical properties of the encapsulated viscumin. Thus, this
procedure can be used to generate biodegradable polylactide microparticles with
encapsulated bioactive substances.
Viscumin, a ribosome-inactivating lectin, occurs in leaf extracts from the parasitic
plant Common Mistletoe ( Viscum album ). Viscumin has a molecular
weight of 60 kDa and consists of two subunits, A and B, which are linked via a
disulfide bond [1, 2]. This protein has found widespread application in anti-tumor
therapy [3–6]. Its efficacy can be
enhanced by encapsulating viscumin into biodegradable polymer microparticles, thus
ensuring its chemical and spatial stabilization, as well as a prolonged release of
the protein into the surrounding tissues. Hence, the toxin will have a prolonged
effect on tumor cells.
Viscumin was kindly provided by Professor U. Pfüller (Institute of Phytochemistry,
University of Witten/Herdecke, Germany). D,L -polylactide PURASORB
PDL 02 (PURAC Biochem bv, Netherlands) with a molecular mass M w ~ 20000
was used as an initial biodegradable polymer. Carbon dioxide of special purity grade
(99.99%, Balashikha Oxygen Plant, Moscow oblast, Russia) was used without any
additional purification. Dry phosphate buffered saline (PBS, Flow Laboratories,
Great Britain), two-component reagent kit for the substrate mixture based on
tetramethylbenzidine (TMB) for ELISA and the streptavidin–peroxidase conjugate
(IMTEK, Russia); and polystyrene plates (Costar, USA) were also used. Monoclonal
antibodies MNA4 and biotinylated MNA9 against various epitopes of the viscumin
A-subunit were obtained earlier [14, 15]. The remaining reagents were purchased from
Sigma-Aldrich Corporation (USA).
The typical SEM microimages of the experimental samples after their removal from the
inlet chamber of the SCF setup are shown in Fig. 2 . It is clear that the samples consist both of
individual microparticles (10–50 µm) and particle agglomerates (up to 200 µm).
Polylactide microparticles are dense bulk particles of irregular shape with an
appreciably smooth surface.