Optica Applicata, Vol. XXXVI, No. 1, 2006
Deactivation rate of camptothecin
determined by factor analysis
of steady-state fluorescence
and absorption spectra
B LANKA ZIOMKOWSKA1, S TEFAN KRUSZEWSKI1, R YSZARD SIUDA2, M ICHAŁ CYRANKIEWICZ1 1Medical Physics Division, Biophysics Department,
Collegium Medicum of Nicolaus Copernicus University,
ul. Jagiellońska 13, 85-067 Bydgoszcz, Poland
2Institute of Mathematics and Physics, University of Technology and Agriculture,
ul.Kaliskiego 7, 85-796 Bydgoszcz, Poland
Camptothecin is a fluorescent compound exhibiting strong anticancer properties. A serious limitation to clinical application of this compound is its hydrolysis, when biologically active lactone form converts into inactive carboxylate. There are some differences in the shapes of both fluorescence and absorption spectra of the lactone and carboxylate forms of camptothecin.
Therefore, during hydrolysis resultant fluorescence and absorption spectra evolve. Factor analysis of fluorescence/absorption spectra recorded during the hydrolysis process of camptothecin enables one to determine the temporary concentration of the lactone and carboxylate forms and obtain the deactivation rate of this compound.
Keywords: camptothecin, fluorescence, absorption, factor analysis.
1. Introduction
Camptothecin (CPT) is a plant alkaloid exhibiting anticancer properties [1]. The cellular target of CPT is nuclear protein topoisomerase I, an enzyme responsible for solving topological problems arising during the replication process of cells [2]. Cancerous cells replicate much more rapidly, so they can be killed with higher efficiency than healthy cells. Because CPT is a fluorescent compound, methods of fluorescence spectroscopy [3] can be used to study how CPT behaves in physiological conditions.
The lactone ring of CPT undergoes an opening due to hydrolysis in an aqueous solution at neutral and basic pH (Fig.1). Only the lactone form (stable in an acid environment at pH<5.5) has anticancer properties. In blood (pH 7.4) the lactone form easily hydrolyzes to the biologically inactive carboxylate form, which then immediately binds to human serum albumin (HSA), thus lowering the concentration of active
138  B. Z IOMKOWSKA et al.
lactone. After about 2hours the concentration of lactone in blood is equal to about 5% [4], while in plasma or HSA it practically decreases to zero [4,5]. For the potential clinical use of this drug it is important to know the rate of hydrolysis reaction of the lactone form in different fluids, especially in physiological solutions, so as to find ways of keeping the lactone form active in human organisms for as long as possible.
High performance liquid chromatography (HPLC) is a method typically used to analyze the hydrolysis process of CPT [6]. The method allows direct measurement of the rate of hydrolysis, however it has some disadvantages [7]: 1) the solvents used for preparation of the liquid phase can interact with the lactone and carboxylate forms and introduce distortions in determining the rate of hydrolysis, 2) the time needed for sample analysis is quite long and can influence the results.
Optical methods of analysis are free of the above disadvantages and therefore they are worth trying. The base for application of optical methods is that there are differences in the shapes of both emission and excitation fluorescence spectra of the lactone and carboxylate forms. Absorption spectra of lactone and carboxylate forms also exhibit differences in shape. Because of the convertion of the lactone form into the carboxylate form during the hydrolysis process both fluorescence and absorption spectra evolve. C HOURPA et al.[7] proposed a spectroscopic non-invasive method of determining the hydrolysis rate of camptothecins. On the basis of variations of fluorescence intensity for two selected wavelengths during the hydrolysis process the change of concentration of the lactone form against time was determined [7]. The present paper reports results obtained with another approach for determining the rate of lactone hydrolysis. This approach is based on the analysis of the set of evolving fluorescence/absorption spectra with two methods well known in multivariate analysis, i.e., pri
ncipal component analysis (PCA) and factor analysis (FA) [8,9]. Because PCA and FA make use of all information contained in spectra they are less sensitive to the random disturbances present in the spectra and it can, therefore, be expected that they should provide more precise results than the local method proposed by C HOURPA et al. [7]. This paper presents the results of application of PCA and FA to sets of fluorescence and absorption spectra that enable a determination of the rate of camptothecin deactivation in phosphate buffered saline (PBS) and HSA solutions. These results are compared with the results from HPLC.
Deactivation rate of camptothecin determined by factor analysis (139)
2. Materials, experiment, method
2.1. Materials
The samples of camptothecin (National Cancer Institute, Betheseda, USA) were obtained from the laboratory of biotechnology, College of Pharmacy, University of Kentucky, Lexington (USA). A 2mM stock solution of camptothecin was prepared in DMSO (dimethylsulfoxide C2H6OS). Such stock solution contains only a pure lactone form. A 1mM stock carboxylate solution was obtained by dilution of stock lactone solution in PBS at pH10 in a volume ratio 1:1. The PBS was adjusted to the desired va
lue of pH using small quantities of 0.1M KOH or HCl. Human serum albumin (95–97%) was purchased from Sigma-Aldrich (USA – Poland). A 40µM solution of HSA in PBS was prepared. The pH of this solution was kept at 7.4 and temperature was kept at 37°C.
reaction rate
For fluorescence measurements, the concentration of camptothecin in final samples was equal to 1µM. The desirable concentration was obtained by adding the stock solutions to PBS at pH7.4 or to an HSA solution also at pH 7.4. For absorbance measurements the concentration of camptothecin in the sample was equal to 20µM.
2.2. Instrumentation and measurements
To excite the fluorescence of camptothecin the following instrumentation was used: 150W Xenon lamp, monochromator SPM2 (Carl Zeiss Jena, Germany) and quartz lens focusing a light of 370nm on the sample. The fluorescence light was collected by a photographic lens (Sigma, Japan) at focal length 28–105mm and F/2.8-4, into the entrance slit of an emission monochromator (SPM2 monochromator was used) equipped with a photomultiplier M12FQ51. A PC with a measuring card (AMBEX, Poland) and software written in the MATLAB environment [10] was used for monochromator control and to manage data acquisition. The absorption spectra of camptothecin were collected with a spectrophotometer UV MINI1240 (Shimadzu, Japan).
The time needed for recording single absorption or fluorescence spectrum was about 50seconds. The fluorescence and absorbance measurements were carried out at room (23°C) and at elevated temperatures (37°C), in PBS solution at pH7.4. The sample temperature during measurements was stabilized with an ultrathermostat U7c (Medengen, Germany).
The spectra of the pure lactone and carboxylate forms were recorded immediately after mixing an adequate volume of the stock solutions in PBS. Single spectrum sweeps were repeated every 2 minutes during the hydrolysis process.
An SLM 8100 spectrofluorometer (SLM-Aminco, USA) was used for steady-state fluorescence anisotropy measurements. The measurements were performed with instrument in the “T-format”. The 370nm exciting light and 400nm long-pass filters in each emission channel were used.
140  B. Z IOMKOWSKA et al.
2.3. Principal component analysis and factor analysis
The principles of the PCA and FA methods were described in previous papers [11,12]. PCA of the experimentally obtained sets of fluorescence emission, fluorescence excitation and absorption spectra,
proved that only two independent components exist. These two factors can be identified as the spectra of the lactone and carboxylate forms of camptothecin. FA makes it possible to determine the contribution of each factor in each resultant spectrum. Time dependence of the contributions obtained from series of evoluting fluorescence/absorption spectra determine the deactivation rate of camptothecin in PBS and in HSA.
3. Results and discussion
Figure2a presents fluorescence spectra of the lactone and carboxylate forms of camptothecin in PBS buffer at pH 7.4. As one can see the emission fluorescence spectrum of the pure carboxylate form exhibits a lower intensity and is red-shifted in comparison to the spectrum of the pure lactone form. Similar differences are observed in excitation fluorescence spectra of the lactone and carboxylate forms.
The shape of emission and excitation fluorescence spectra recorded during the hydrolysis process change over time. Figure2b presents fluorescence spectra of the pure lactone form (characterized by the highest intensities in both series), selected
a b
Fig.2.Excitation and emission fluorescence spectra of pure lactone and pure carboxylate forms of camptothecin, recorded in PBS at pH7.4 (a). Excitation and emission fluorescence spectra recorded during the hydrolysis process of camptothecin in PBS at pH7.4 (b). The excitation spectra (maximum at about 370nm): the highest intensity spectrum is of the lactone form, the subsequent ones are the spectra recorded 40, 90 and 180minutes after sample preparation; the lowest spectrum (dash-dot line) is the excitation spectrum of the carboxylate form. The emission spectra (maximum at about 450nm): the spectrum of highest intensity is for the lactone form, the subsequent ones are the spectra recorded 20, 50, 100 and 170minutes after sample preparation; the lowest spectrum (dashed line) is the emission spectrum of the carboxylate form.
Deactivation rate of camptothecin determined by factor analysis (141)
Fig.3.Rate of deactivation process of camptothecin obtained by factor analysis of a series of excitation and emission spectra recorded during the hydrolysis process in PBS at pH7.4 and temperature of 23 and 37ºC.
spectra recorded during the hydrolysis process and spectra of the pure carboxylate form (characterized by the lowest intensities in both series). As one can see the intensity of fluorescence decreases and the maximum of the spectra shifts during the hydrolysis process. The shape of evoluting spectra approaches the shape of spectrum of the pure carboxylate form but does not reach it. The observed changes are the result of the convertion of the lactone form into the carboxylate one.
Both emission and excitation fluorescence spectra recorded during the hydrolysis process were analyzed using FA, with the spectra of pure lactone and pure carboxylate forms as the factors. As a result, the fraction of the lactone form over time was obtained. The curves of decay of the lactone form obtained with FA are presented in Fig.3. The measurements carried out at temperatures of 23 and 37°C were repeated several times, so each of the presented curves is the average of at least three independent measurements.
Figure4a presents the absorption spectra of the pure lactone and pure carboxylate form of CPT in PBS
at pH 7.4. The presented spectra were recorded immediately after adding a stock solution of lactone or carboxylate, respectively, into PBS at pH 7.4. There are some differences in the shape of absorption spectra of lactone and carboxylate. In the range near to ultraviolet the absorption spectrum of the pure lactone form exhibits two maxima (350 and 370nm). The spectrum of the pure carboxylate form exhibits only one maximum (about 370nm). Because of the convertion of lactone into carboxylate, the shape of absorption spectra recorded during the hydrolysis process changes over time. Figure4b presents absorption spectra of the pure lactone form, some selected spectra recorded during the hydrolysis process and the spectrum of the pure carboxylate form. As one can see in Fig.4b, the shape of absorption spectra evolves towards the shape of the spectrum of the carboxylate form, but does not reach it. The set of recorded absorption spectra was analyzed by FA, with the absorption

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