production of selected secondary metabolites in transformed ...

copolymers were additionally functionalized by itaconic acid (ITA), which can be gained
from renewable resources by pyrolysis of citric acid or by fermentation of polysaccharides.
ITA brings reactive double bonds and functional carboxylic acid groups to the end of
copolymer resulting in preparation of biodegradable ITA/PLGA-PEG-PLGA/ITA
macromonomer. Successful end-capping of ITA to PLGA-PEG-PLGA copolymer was proved
by 1H NMR and FT-IR analysis followed by characterization with GPC method. The above
mentioned thermosensitive polymers are soluble in water forming free-flowing solution that
spontaneously gels as the temperature increases generating a water-insoluble physical
hydroge. The sol-gel transition behaviors were studied by the test tube inverting method. The
tests of degradation behaviors of PLGA-PEG-PLGA copolymer were carried out in vitro in
the phosphate buffer medium at 37 °C.
EXPERIMENTAL WORK
Materials
The triblock copolymers of PLGA-PEG-PLGA and ITA- PLGA-PEG-PLGA-ITA were
synthesized at our faculty by Dr. Lucy Vojtová.
Tetrahydrofurane for GPC analyses (THF for HPLC, gradient grade, Merck, Germany),
was used as received. Polystyrene standards (Mp= 316 500 – 162) were purchased from
Polymer Laboratories, Germany. Milli-Q water, phosphoric acid (p.a., 85 %, Czech Republic
and potassium phosphate dibasic (p.a., for HPLC, Fluka, USA) were used for the polymer
degradation studies.
Method
The molecular weight and the molecular weight distribution of the copolymers were
determined by GPC method using Agilent Technologies 1100 Series instrument equipped
with a refractive index detector, PLgel Mixed C column of 300 x 7.5 mm with particle size 5
μm, degasser, pump, auto sampler and fraction collector. Tetrahydrofurane was used as the
mobile phase at a flow rate equal to 1 ml.min -1 . The average molecular weight was calculated
using a series of polystyrene standards (Mp = 316 500 – 162). Samples of triblock copolymers
were prepared in the concentration of 0.5 mg/ml in tetrahydrofurane for HPLC.
The sol-gel transition was determined by the test tube inverting method. 4 ml vials
containing 1 ml of the triblock copolymer were heated from 10 to 60 °C in a water bath. The
transition temperatures were determined by a flow (sol) – no flow (gel) criterion when the vial
was inverted with a temperature increment of 1 °C per step.
The degradation behavior study was performed using 0.3 ml of 23 w% polymer aqueous
solutions. The 1.8 ml vials with polymer solutions were put into an incubator at 37 °C (the
temperature of a human body) to formed the hydrogels. Consequently, 0,5 ml of the
phosphate buffer (pH 7.4, 37 °C) was added to the vials and the samples were placed into the
incubator for 10 days with a view to degrade during this time. At regular intervals, the
samples were withdrawn from of the incubator, lyophilized and analyzed.
RESULTS AND DISCUSSION
The sol-gel transition diagrams of the triblock copolymers (PLGA-PEG-PLGA, ITA-
PLGA-PEG-PLGA-ITA) were created on the base of the test tube inverting method (Fig. 2).
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2006, strana 219