Transcript Spectrofluorimetry and Chemometrics for Investigation of

• ENCAPSULAÇÃO E CARACTERIZAÇÃO DE
FÁRMACOS EM SISTEMAS MICRO E NANO
PARTICULADOS
• Lipossomas
• Ciclodextrinas
• Micro/Nanoesferas
Francisco B.T. Pessine
Instituto de Química
Departamento de Físico Química
[email protected]
Spectrofluorimetry and Chemometrics for
Investigation of Norfloxacin Distribution in
Multilamellar Liposomes
Applied Spectroscopy xx, xxx (2005)
Introdução
• Norfloxacin: partially hydrophobic fluoroquinolone antibiotic active
against Gram+/- bacteria, inhibiting the
gyrase enzyme
Topoisomerase II. Good sensitiser of singlet oxygen.
• Highly effective in the treatment of infectious diseases, mainly in the
urinary/respiratory tracts.
• Has a zwitterionic amphoteric nature, being ionized over the whole pH
range, due to -COOH and –NH2 groups. This influence its
distribution/affinity for lipid bilayers and it is also related to the
phototoxic properties of NFX, under solar UV radiation (induction of
dermic tumors in rats, phototoxicity in mamalian cells in vitro,
accumulation in lysosomes of HS68 human skin fibroblasts and other
citoplasmatic organelles)
• Entrapment of NFX in liposomes: can be of therapeutic interest as
they provide different pathways of interaction with bacterial cells,
compared to the normal routes, being useful in the treatment of
infections caused by quinolone resistant/poorly sensitive bacteria to
this drug.
• Investigation on the distribution of NFX in liposomes: provides a
better understanding of its interaction with biological membranes.
•
.
Equilíbrios entre diferentes espécies de NFX
•
Objetivos
• Investigar a distribuição de NFX em bicamadas
lipídicas de lipossomas MLV em pH 7.0 usando:
• supressão de fluorescência
• grau de anisotropia
• quimiometria
Experimental
• MLV containing NFX: prepared with soy-bean phosphatidylcholine
(Epikuron 200SH; 25/45mg), cholesterol (15mg) and NFX
(10.0mol) all dissolved in CHCl3:MeOH (2:1; v:v)
• The solvents were evaporated and the lipid film was hydrated with
10.0mL of 0.010 mol/L HEPES buffer (pH 7.0) at 65oC.
•
Steady state measurements of fluorescence anisotropy of
free and liposomal NFX (pH 7.0) were obtained on a Jobin
Yvon-Spex Spectrofluorimeter, with L geometry.
• This technique is based on the excitation and emission of
polarized light which excites the fluorophores, according to
their orientation relative to the direction of the polarized
excitation. The emission can be depolarized by rotational
diffusion, with an angular displacement during the lifetime
of the excited state. The emitted radiation does not show
the same orientation as the excitation one.
• The degree of anisotropy is:
I II  I 
r 
I II  2 I 
• Excitation source: Xe lamp (=284nm). Fluorescence monitored at 426nm
at constant T. Free and encapsulated NFX incubated in quartz cells at
25oC (<Tm53oC) in the presence of I- and acrylamide as quenchers in
0.010mol/L HEPES buffer (pH 7.0). All solutions contained 0.010mol/L
sodium thiophosphate to prevent oxidation of I- toward I-3.
• Data were analyzed according to Stern-Volmer equation:
•
F0
 1  K D [Q]
F
• F0/F: fluorescence intensities in the absence/presence of quencher. KD:
Stern-Volmer collision constant.
• To distinguish between two populations of fluorophores a modified SternVolmer equation was used:
F0
 [1 /( f a K[Q])]  (1 / f a )
F
• Fa: fraction of the initial fluorescence supressed by the quencher Q; F:
difference between the fluorescence intensity in the absence (F0) and
presence (F) of quencher; KD: Stern-Volmer constant.
•
Quimiometria
• To resolve the overlapped spectra and quenching profiles of the
zwitterionic and neutral forms, the chemometric method “SelfModeling Curve Resolution” was applied. This method uses
Principal Components Analysis, based on kernel Singular Value
Decomposition (SVD). It allows spectral deconvolution
assuming the presence of only two substances. The SMCR
method uses the following assumptions:
• a) the curves must be non-negative
• b) the curves in the data set must be a linear combination of
two linearly independent curves
• c) at least one wavelength must exist for each substance where
just that substance fluoresces.
• The SMCR method was carried out on a matrix constructed of
relative fluorescence spectra, F0/F.
•
Resultados/Discussão
• Anisotropy measurements give qualitative information on the
incorporation of NFX molecules into the lipidic bilayers of
liposomal vesicles. Fluorescence quenching spectra treated with
chemometric methods confirm the existence of two populations
of NFX (neutral and zwitterionic) in MLV liposomes at pH 7.
• Results: the zwitterionic form of NFX was incorporated into
lipid/aqueous interface of the liposomes, and the neutral form
was located toward the center of the bilayers. I- can quench the
fluorescence of both forms of NFX in non-liposomal solution,
but it cannot quench the encapsulated neutral form in the
bilayer because these ions doesn't have access to the interior of
the bilayers. On the other hand, acrylamide can penetrate into
the bilayer, reaching the neutral species and quenching its
fluorescence.
• Anisotropy measurements gives a strong indication of encapsulation. The
results (Table I), allowed the observation of values directly related to the
kind of environment where the fluorophores were distributed. In
hydrophobic environments, such as liposomal lipidic bilayers, higher
values were noted, followed by ones from micelles. This means that the
molecules of NFX penetrate into these two systems, causing a difference
in their rotational diffusion, having a more restricted degree of freedom in
organized systems like liposomes and miceles.
• .
• Free rotations in solution were related to the smallest values of r.
However, NFX showed a stronger interaction in EtOH and CH3Cl than in
aqueous solutions, decreasing its rotational movements, and increasing
the anisotropy values.
• NFX molecules are inserted deeply into the lipidic bilayer of MLV
liposomes. In neutral medium, there is an equilibrium between the neutral
and zwitterionic forms of NFX, where the latter one predominates. The
neutral form is incorporated into the bilayer, while the zwitterionic form is
in contact with the water/lipid interface
•
Tabela 1: Graus de anisotropia
• Solutions of NFX (1.0 x 10-5 mol/L)
•
r
• Deionized water
0.019
• HEPES buffer (pH 7)
0.024
• Citrate buffer (pH 3)
0.025
• Ethanol
0.077
• Chloroform
0.052
• SDS (pH 7)
0.086
• SDS (pH 3)
0.106
• MLV (45mg PC; NFX 1.0x10-4mol/L)
0.378
• MLV (25mg PC; NFX 1.0x10-4mol/L)
0.269
• Fluorescence quenching using I- and acrylamide as quenchers, in free
and in liposomal NFX solutions (pH=7): the concentrations of
quenchers varied from 0 to 0.25mol/L (these concentrations does not
changes the bilayer structure).
• Using Chemometrics: it was possible to resolve the emission spectra
and the fluorescence quenching profiles of the neutral/zwitterionic
forms of NFX separately (Figs. 2 and 3). The quenching constants
were obtained from the experimental spectra and not from the
calculated ones.
•
Stern-Volmer equation: applied for each fluorescence quenched
spectra. It describes the occurrence of dynamic and static quenching.
The Stern-Volmer plots deviated from linearity with upward
curvatures, suggesting the presence of static and dynamic quenching.
• Dynamic quenching: quenchers interacts with the excited state,
decreasing the fluorescence intensity and lifetime.
• Static quenching: the quencher remove the molecules from the
excited state, decreasing only the emission intensity.
•
Fig. 2: Espectros resolvidos e supressão
da fluorescência
Intemis of each specie of NFX (a. u.)
Intemis of each specie of NFX (a. u.)
1.0
0.8
0.6
0.4
0.2
0.0
350
400
450
500
1.0
0.8
0.6
0.4
0.2
0.0
0.00
550
0.05
0.10
0.15
0.20
0.25
-3
wavelength (nm)
[quencher] (mol.dm )
a
b
0.25
0.8
Intemis of each specie of NFX (a. u.)
Intemis of each specie of NFX (a. u.)
•
1.2
0.6
0.4
0.2
0.0
0.20
0.15
0.10
0.05
0.00
0.1
350
400
450
wavelength (nm)
c
500
550
0.2
0.3
0.4
0.5
0.6
0.7
-3
[quencher] (mol.dm )
d
0.8
0.9
Fig. 3: Espectros resolvidos e supressão da
fluorescência
Intemis of each specie of NFX (a. u.)
Intemis of each specie of NFX (a. u.)
1.0
0.8
0.6
0.4
0.2
0.0
350
400
450
500
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.00
550
0.05
0.10
0.15
0.20
0.25
0.20
0.25
-3
wavelength (nm)
[quencher] (mol.dm )
b
a
0.6
Intemis of each specie of NFX (a. u.)
1.0
Int emis of each especie of NFX (a. u.)
•
0.8
1.2
0.8
0.6
0.4
0.2
0.0
350
400
450
wavelength (nm)
500
550
0.5
0.4
0.3
0.2
0.1
0.00
0.05
0.10
0.15
-3
[quencher] (mol.dm )
• Linear Stern-Volmer plot: indicative of a single
fluorophores, all equally accessible to the quencher.
class
of
• The existence of two populations of fluorophores (neutral and
zwitterionic forms) in MLV liposomes at pH 7, which is in
accordance with the partial hydrophobicity of NFX, allowed the use
of the modified Stern-Volmer equation to obtain the rate of
collisional encounters between the fluorophore and the quencher,
called Stern-Volmer constant values (Table 2).
Tabela 2: Constantes de Stern-Volmer
• Sample
Quencher
NFX specie
KD (L/mol)
• NFX
•
I
Zwitterion
Neutral
71.8
21.8
• NFX-MLV
•
I
Zwitterion
Neutral
14.4
• NFX
•
Acrylamide
Zwitterion
Neutral
2.01
3.32
• NFXMLV
•
Acrylamide
Zwitterion
Neutral
3.07
4.82
• Acrylamide quencher: it was observed that, for the free drug in
solution, the neutral form showed better interaction with this
quencher
than
the
zwitterionic
(Kzwit=2.01
and
Kneut=3.32L/mol). Also, a blue shift in the spectrum was observed
for the neutral specie. The ground state of this specie has a
smaller dipole moment than the excited one. Such a state is better
stabilized when interacting with acrylamide, which is a nonpolar
molecule.
• MLV liposomes: both zwitterionic and neutral forms were quenched
by acrylamide, confirming the existence of two populations of NFX
(neutral and zwitterionic) in lipid bilayers.The local insertion of
acrylamide molecules in the lipid bilayer changes the environment
around the NFX, increasing its interactions with the quencher
molecules. There is an widening of the spectral band for the
neutral specie, which is the one that has a better affinity for
acrylamide. The descending behavior of quenching profiles proves
the existence of such specie, free in solution or inserted in the
lipidic bilayers.
•
Conclusions
• According to the modified Stern-Volmer constant values (Table 2),
it is seen that I- quenches both forms of NFX when it is free in
solution.
• I-: can quench the fluorescence of the encapsulated drug only
when the it is in hydrophilic region.
• Acrylamide: hydrophobic molecule, located inside the bilayer and
quench the fluorescence of encapsulated NFX molecules.
• MLV liposomes: only the zwitterionic form was accessible to the
quencher, although at a lower rate than that observed for the free
form. The neutral species was not quenched by I- because it was
located deeper, near the center of the bilayer. This lack of
quenching is due to the inability of the charged and hydrated I- to
enter the non polar interior of the liposome.
• The drug NFX is incorporated in MLV bilayers.