Nephelometric Monitoring Growth of Candida albicans Using BMG LABTECH’s NEPHELOstar
U.-C. Hipler, Universitaetsklinikum Jena, Teilkoerperschaft der Friedrich-Schiller-Universitaet Jena
• Laser nephelometry used for fungal growth determination
and for measurement of drug solubility
• Monitoring the growth of Candida albicans in presence of
• Phase solubility diagrams of potential drug presented
Cyclodextrins (CD) have become useful pharmaceutical excipients,
due to their potential to form inclusion complexes with appropriately
sized drug molecules1. The resulting complexes generally offer a
variety of physicochemical advantages over the free drug, including
increased water solubility, enhanced bioavailability, improved
stability, reduced side effects, etc2.
Econazole-nitrate (EC) and ciclopirox-olamine (CI) are well known
antifungal agents suitable for the treatment of many mycotic
infections. Previous studies showed that both dissolution properties
and consequently microbiological activities of econazole, with very
low water solubility (about 3 µg/mL at 25°C), can be improved by
complexation with natural cyclodextrins, particularly with ß-CD3-5.
By increasing the water solubility of the drug it should be possible
to improve its bioavailability, thus enabling improved oral or topical
Laser nephelometry has been shown to be a reliable technique for
the measurement of drug solubility in 96-well plate format6,7. Laser
nephelometry is the measurement of forward scattered light. When a
laser beam is directed through a clear solution, the more particles or
turbid suspensions (fungi in this study) in the solution, the greater the
amount of forward scattered light (measured as units). The energy of
the scattered light is directly proportional to the particle concentration
in the suspension for up to three orders of magnitude6.
Herein we describe the use of laser nephelometry to investigate the
effects of complexation on the drug antimycotic activity using BMG
LABTECH´s NEPHELOstar (Figure 1). Furthermore nephelometry was
used to prepare phase solubility diagrams and to monitor fungal
Materials and Methods
• Candida albicans DSM 11225
• Sterile and clear 96-well plates, Greiner bio-one, Frickenhausen, Germany
• β-Cyclodextrin, Wacker-Chemie, Burghausen, Germany
• NEPHELOstar, BMG LABTECH, Offenburg, München, Germany
Preparation of cultures
C. albicans were grown on SGA (Sabouraud-Glucose-Agar with
gentamycin-chloramphenicol from bioMerieux, Germany) at 30°C
for 24–48 h. Three to five well-isolated colonies of the same
morphological type were selected from an overnight culture using
a sterile wire loop and inoculated in 20 mL (SGB). The suspensions
were incubated with shaking at 250 rpm/30°C for 24 h. Then the
overnight cell cultures were counted using CASY® 1 and adjusted to
a final working concentration of 6×105 cells/mL in SGB (Sabouraud-
Glucose-Bouillon from Oxoid Ltd, UK).
Preparation of antifungal agents
Both EC and CI were independently dissolved in a mixture of
chloroform/methanol 1:1 to achieve a final stock solution containing
20 mg/mL of antifungal agent. The stock solution of EC was diluted
with SGB and adjusted to be 1.25–100 µg/mL while as for CI; it was
in the range of 1.25–10 µg/mL. All solutions were stored at -80°C
Preparation of the inclusion and antifungal complexes
A solution of EC was prepared by dissolving it in a chloroform/
methanol mixture 1:1. CD was dissolved in hot water at 85–90°C.
Equimolar amounts (1:1 molar ratio) of EC and CD solutions were
mixed together with stirring for 30 min at 85–90°C.8 By cooling,
crystallization of the complex was obtained. The complex was filtered
using G3 filter and kept in a desiccator overnight. On the other hand,
the second complex between CI and CD was also prepared according
to the previously mentioned method, in which methanol was used
as a proper solvent for CI. Moreover, a molar ratio of 1:2 of CI:CD
was also used. The antifungal complex of CD–EC was prepared in a
concentration range of 12.5–100 µg/mL using DMSO as a solvent,
while the CD–CI was prepared in a concentration range of
150–400 µg/mL using distilled water.
Phase solubility studies
In this experiment, both of drugs and complexes were diluted in
DMSO. Then the drug and complex solutions were independently
pipetted into PBS-buffer with a concentration of DMSO of 1–5 vol.%.
All samples were measured on the NEPHELOstar at 30°C, with an
integration time of 0.1 s, so that a plate (96 samples) could be scanned
in ~68 s. A gain of 122 and a laser intensity of 1% were set to allow
direct comparison of all results. All raw data were processed using
the BMG LABTECH NEPHELOstar Evaluation software. The scattered
light will remain at a constant intensity until precipitation occurs. At
that point it will increase sharply.
Results and Discussion
Phase solubility diagrams
Solubility diagrams were monitored using laser nephelometry which
determine the solubility of potential drug candidates supplied as
dimethyl sulfoxide (DMSO) solutions in 96-well plates (Figure 2).
In the absence of CD, the solubility of EC is determined to
be ~1.3 mg/mL (graph not shown), while in the presence of CD the
solubility increased up to ~3.1 mg/mL for CD–EC complex.
The solubility results for ciclopirox-olamine (CI) differ from the other antifungal agent.
In the absence of cyclodextrin, the solubility of CI is determined to be
~1.2 mg/mL (graph not shown). Linear line was obtained for CD–CI
complex and no point of precipitation is found which indicated complete
solubility over the concentration ranges (Figure 3).
Influence of complexed antifungal agents on the growth of Candida
Figure 4 shows, that a concentration of 25 µg/mL of CD-EC inhibits
the cell growth while at 50 µg/mL cells are killed in comparison
with the control. These values obtained with the complex is
comparable to the findings for the free EC.
In contrary to CD-EC the complexed ciclopirox was less effective
(Figure 5). At a concentration of 200 µg/mL of the complex, cells were
inhibited and at a concentration of 300 µg/mL of the complex, cells
were killed. The CD-CI complex was also less effective compared to
the free CI that caused cell death already at 10 µg/mL.
This study has proven that laser nephelometry in a 96-well microtiter
plate can be used as a method for the rapid determination of the
solubility of potential drug compounds.
Laser nephelometry can distinguish between the concentration at
which the drug just goes into or just comes out of solution. On the
other hand, this technique can be used efficiently for monitoring and
evaluating the growth of microorganisms like fungi or bacteria.
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