![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
NEUROPHARMACOLOGY
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska (E.V.K., S.L., S.V.V., D.W.M., A.V.K.); and Supratek Pharma Inc., Laval, Quebec, Canada (V.Y.A.)
Received August 27, 2002; accepted October 14, 2002.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
;
Alakhov and Kabanov, 1998;
Kwon and Okano, 1999). An
important and promising example of these systems is Pluronic block copolymers,
which were shown to enhance drug performance by acting as biological
response-modifying agents. Specifically, they sensitize multidrug-resistant
cells by inhibiting drug efflux transporters
(Venne et al., 1996).
Moreover, Pluronic formulations were shown to enhance transgene expression in
the body (Lemieux et al.,
2000). One particular composition, Pluronic block copolymer P85
(P85), was found to improve the transport of select solutes across the BBB in
vitro (Batrakova et al., 1998,
1999) and in vivo
(Kabanov et al., 1989;
Batrakova et al., 2001b). Using
monolayers of polarized bovine brain microvessel endothelial cells (BBMECs) as
an in vitro model of the BBB it was demonstrated that coadministration of P85
significantly increased the transport of various Pgp substrates across brain
microvessels by inhibiting the Pgp efflux transport system (Batrakova et al.,
1998,
1999). Similar responses were
observed in vivo because administration of P85 significantly enhanced the
brain penetration of the Pgp substrate digoxin in wild-type mice expressing
functional Pgp to levels that were similar to those observed in Pgp-deficient
knockout mice (Batrakova et al.,
2001b). The mechanism of the effect of this copolymer on the drug
efflux system in the BBB has been discussed recently
(Batrakova et al., 2001a). It
was found that the P85 treatment caused membrane fluidization leading to
decreases in Pgp ATPase activity (Batrakova
et al., 2001a). Furthermore, P85 caused significant depletion of
cellular ATP in BBMEC monolayers that affected the ATP-dependent Pgp efflux
transport system and increased drug diffusion across the blood-brain barrier
cell monolayers. Consequently, both membrane fluidization (inhibiting Pgp
AT-Pase activity) and energy depletion (decreasing the ATP pool available for
Pgp) were found to be critical factors contributing to the activity of the
block copolymer in the BBB.
Pluronic block copolymers consist of ethylene oxide (EO) and propylene
oxide (PO) blocks arranged in a basic A-B-A structure:
EOx-POy-EOx. Due
to their amphiphilic nature, Pluronic block copolymers are able to
self-assemble into micelles in aqueous solutions above critical micelle
concentration (CMC). Below the CMC, Pluronic copolymers exist in solution in
the form of a molecular dispersion of individual block copolymer molecules
termed "unimers" (Alexandridis
et al., 1994). Variations in the number of hydrophilic EO units
(x) and lipophilic PO units (y) result in copolymers with different molecular
mass and distinct hydrophilic-lipophilic balance (HLB). Copolymers with a
short hydrophilic poly-EO block or/and an extended lipophilic poly-PO block
(such as Pluronic L121 and L101) are highly lipophilic and are characterized
by a relatively low CMC and low HLB. In contrast, copolymers with an extended
hydrophilic poly-EO block or/and short lipophilic poly-PO block (such as
Pluronic F108 and F88) are hydrophilic and are characterized by relatively
high CMC and high HLB. Pluronic compositions such as P85 or P103 are
intermediate in their lipophilicity and have CMC and HLB values that fall
between the two extremes identified above.
Although most experiments examining the effects of Pluronic on BBB permeability were performed with Pluronic P85, there is an entire series of Pluronic block copolymers with differing molecular properties. The availability of a wide variety of Pluronic compositions provides a unique opportunity to identify those structural features that are important for the effects of the block copolymer on drug efflux transporter activity in BBB. In the current study, a series of Pluronic block copolymers with a wide range of HLB were used to identify 1) those composition with the best transporter activity profiles in brain endothelial cells, and 2) the impact of copolymer composition on critical factors (i.e., energy depletion and membrane fluidization) known to influence Pgp transporter activity in BBMEC monolayers.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Briefly, Pluronic block copolymers were activated with
1,1'carbonildiimidazole, and then modified with excess of
ethylenediamine and purified by gel filtration. Amino-modified copolymers were
conjugated with fluorescein isothiocyanate according to manufacturer's
protocol. A gel-permeation chromatography was used to ensure that FITC was
attached to the block copolymer. Two-step separation was performed: 1) rough
separation on Sephadex PD-10 column (Sigma-Aldrich, St. Louis, MO) in
acetonitrile/water (1:1) phase, and 2) precise separation on Sephadex LH-20
(Sigma-Aldrich) column in ethanol/water (1:1) phase. FITC-labeled Pluronic was
eluted in the first distant peak, and detected by spectrofluorimeter (for
FITC) and iodine test (for Pluronic). Second peak of nonconjugated FITC and
low molecular products was eluted with a substantial delay. The yield of
FITC-Pluronic conjugation was ca. 70%.
|
Cell Isolation and Culture. BBMECs were isolated from fresh cow
brains using a combination of enzymatic digestion and density centrifugation
as described previously (Miller et al.,
1992). The cells were maintained in minimal essential medium/F-12
culture medium supplemented with 10% horse serum, 100 µg/ml heparin
sulfate, 2.5 µg/ml amphotericin B, and 50 µg/ml gentamicin. Tissue
culture media were obtained from Invitrogen (Carlsbad, CA), and serum and
medium supplements were purchased from Sigma-Aldrich. Isolated BBMECs were
seeded at a density of 50,000 cells/cm2 in 24-well plates and were
used for the ATP assay and R123 accumulation studies after reaching confluence
(typically within 14 days).
Characterization of Pgp Expression in BBMEC Monolayers.
Identification of Pgp was done using immunoblot technique described previously
(Miller et al., 1996). The
monoclonal antibodies to Pgp, C219 (DAKO, Carpinteria, CA), and 
-actin,
anti--1-chicken Integrin (Sigma-Aldrich), were used at 1:100 and 1:200
dilutions, respectively. The secondary horseradish peroxide anti-mouse Ig
antibodies (1:1500 dilution) were purchased from Amersham Biosciences, Inc.
(Cleveland, OH). The specific protein bands were visualized using a
chemiluminescence kit (Pierce Chemical, Rockford, IL). The level of Pgp
expression was quantified by densitometry (Nucleo Vision; Nucleo Tech,
Curitiba-Pr., Brazil). To correct for loading differences, the level of the
protein was normalized to constitutively expressed -actin. The relative
amount of the protein in the Pgp-overexpressing human oral epidermal carcinoma
(KBv) derived by selection with vinblastine were used as a positive control,
and human umbilical vein endothelial cells (HUVECs) was used as a negative
control.
R123 Accumulation Studies. R123 accumulation in BBMECs was studied
as described previously (Miller et al.,
1997). Briefly, confluent cell monolayers were preincubated with
the assay buffer for 30 min at 37°C, and then the assay buffer was removed
and cell monolayers were exposed to 3.2 µM R123 in either assay buffer or
Pluronic solutions at different concentrations for 90 min. After the
incubation the dye solutions were removed, the cell monolayers were washed
three times with ice-cold PBS, and solubilized in Triton X-100 (1.0%).
Aliquots were removed for determination of the cellular dye using an RF5000
fluorescent spectrophotometer (
ex = 505 nm,
em = 540 nm) (Shimadzu, Kyoto, Japan) and cellular protein
using the Pierce bicinchoninic acid assay. All experiments were carried out in
quadruplicate.
The effects of Pluronic compositions on Pgp activity were expressed as the R123 enhancement factor (maximal R123 accumulation levels in the presence of Pluronic versus those observed in the control groups in the absence of the block copolymer).
ATP Assay. To examine the effects of Pluronics on ATP intracellular
levels the confluent BBMEC monolayers were pretreated with assay buffer for 30
min after which the cells were incubated with various concentrations of
Pluronic solutions for 2 h. After treatment, the cells were washed two times
with ice-cold PBS, solubilized in Triton X-100 (1.0%), and immediately frozen
for subsequent ATP quantification (conducted within 24 h after the sample
collection). Cellular ATP was determined using a luciferin/luciferase assay
(Garewal et al., 1986). For
this purpose, 100-µl aliquots of cell lysate were mixed with 100 µl of
ATP assay mix (FL-AAM; Sigma-Aldrich). Light emission was measured with a
luminometer (model 20/20; Turner Designs, Inc., Sunnyvale, CA). Raw data were
collected as relative light units integrated over 20 s for samples and
converted to ATP concentrations with the aid of a standard calibration curve
obtained using ATP standard (FL-AAS; Sigma-Aldrich). Cellular ATP levels were
normalized for protein content and each data point represented the mean
± S.E.M. of a minimum of four replicates.
Pgp ATPase Assay. Membranes from Pgp-overexpressing cells were used
to evaluate effects of P85 on Pgp ATPase activity (BD Gentest, Woburn, MA). A
0.06-ml reaction mixture containing 40 µg of membranes, 20 µl of the
various Pluronic compositions or assay buffer, and 3 to 5 mM MgATP, in a 50 mM
Tris-MES buffer containing 2 mM EGTA, 50 mM KCl, 2 mM dithiothreitol, and 5 mM
sodium azide, pH 6.8. The membrane samples were incubated at 37°C for 20
min. An identical reaction mixture containing 100 µM sodium orthovanadate
was assayed in parallel. Orthovanadate inhibits Pgp by trapping MgADP in the
nucleotide-binding site. Thus, ATPase activity measured in the presence of
orthovanadate represents non-Pgp ATPase activity and can be subtracted from
the activity generated without orthovanadate to yield vanadate-sensitive
ATPase activity (Pgp ATPase activity). The reaction was stopped by the
addition of 30 µl of 10% SDS with Antifoam A. Aliquots (200 µl) of
ammonium molybdate in 15 mM zinc acetate/10% ascorbic acid (1:4) were added to
each sample and incubated for an additional 20 min at 37°C. The liberation
of inorganic phosphate was detected by its absorbance at 630 nm and
quantitated by comparing the absorbance to a phosphate standard curve
(Druekes et al., 1995;
Shepard et al., 1998).
DPH and 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH)
Labeling of BBMECs. DPH was used as a probe to examine the fluidity
properties of the hydrocarbon region of the cell membranes. DPH is a
hydrophobic fluorescent compound that spontaneously incorporates in the
hydrocarbon regions of lipid membranes
(Shinizky and Inbar, 1967;
Laat et al., 1977). Transfer
of DPH from the aqueous environment into the cell membranes results in a
drastic increase of the fluorescence emission for this probe. Furthermore,
once the probe is incorporated into lipid membranes, its fluorescence
polarization is strongly dependent on the microenvironment, with decreases in
membrane microviscosity resulting in increased fluorescent polarization. It
should be noted that DPH binds not only with the plasma membranes but also
with other membranes within the cells, thus the polarization value obtained
reflects the overall membrane microviscosity of the cells
(Pagano et al., 1977).
Consequently, once the polarization changes are observed, it is difficult to
discriminate which membranes (i.e., plasma or intracellular organelle) are
affected. To examine effects of Pluronics on plasma membranes of BBMECs, the
cationic analog of DPH, TMA-DPH, was used. The cationic charge of this probe
ensures that TMA-DPH is anchored at the lipid-water interface, whereas the DPH
moiety is intercalated between the upper portions of the lipid milieu
(Prendergast et al., 1981).
For these studies, the BBMEC suspension was washed twice with PBS and
incubated with 2 µM DPH (Sigma-Aldrich) dispersion for1hat37°C. For
TMA-DPH studies, cells were incubated with 2 µM TMA-DPH (Molecular Probes,
Eugene, OR) for 10 min at 37°C. Then, the cells were washed twice with PBS
to remove extracellular probe, and resuspended in an appropriate volume of
PBS. To evaluate the kinetic effects of Pluronics in BBMECs, four different
Pluronic compositions representing each group were added at concentrations
producing maximal inhibition of the Pgp efflux system in BBMECs, and changes
in fluorescent polarization were recorded.
Fluorescence Polarization Measurements. Fluorescence intensities
were measured with a Hitachi F5000 spectrophotometer equipped with a polarizer
set. This instrument detects fluorescence intensity (I) with the
relative position of the polarizer and analyzer (parallel,
I||, or perpendicular, I
) and
fluorescence anisotropy r, was calculated according to eq.1:
 | (1) |
of unlabeled samples from those of identical but
labeled samples.
Microviscosities (
) were derived as described previously for DPH
(Shinizky and Inbar, 1967;
Laat et al., 1977) and TMA-DPH
(Chazotte, 1994) by the method
based on the Perrin equation (eq. 2) for rotational depolarization of a
nonspherical fluorophore:
 | (2) |
is the exited state lifetime. The value of r0 used for
both probes was 0.362; values were 10 and 7 ns for DPH and TMA-DPH,
respectively. C(r) is a molecular shape parameter equal to
8.6 x 105 poise · deg-1
s-1 and 15.3 x 105 poise ·
deg-1 s-1 for DPH and TMA-DPH,
respectively. Fluorescent Microscopy. BBMECs grown on chamber slides (Fisher Scientific Co., Fair Lawn, NJ) were incubated with 0.1% F108-FITC, P85-FITC, L35-FITC, and L121-FITC in assay buffer for 2 h at 37°C. After this period, the loading solutions were removed, and the cell monolayers were washed three times with ice-cold PBS containing 1% bovine serum albumin and examined using an ACAS-570 (Meridian Instruments, Okimos, MI) confocal laser microscope.
Cytotoxicity Assay. To examine the possible cytotoxic effect of
studied block copolymers, BBMEC were seeded in 96-well plates at a density of
5000 cells/well and allowed to reattach overnight. Then, the cells were
exposed to various concentrations of Pluronic solutions for 2 h at 37°C.
After this treatment cells were washed three times and cultured for 3 days in
the media. The cytotoxic effects were determined using a standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay
(Ferrari et al., 1990). All
experiments were repeated eight times. No cytotoxic effects of Pluronic block
copolymers were observed over entire range of concentrations used in this
study.
Statistical Analysis. All statistical tests were performed by Microsoft Excel 97 SR-1 program using the two-tailed heteroscedastic t tests. A minimum p value of 0.05 was estimated as the significance level for all tests. S.E.M. values for R123 accumulation levels, microviscosity, and ATP measurements were less than 10% of the mean.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Lee et al., 1994,
Fontaine et al., 1996). In the
preliminary study, we examined the level of Pgp expression in BBMEC monolayers
by Western blot. The determined amount of the protein was normalized to the
amount of constitutively expressed -actin. Pgp-overexpressing human oral
epidermal carcinoma (KBv) cells derived by selection with vinblastine were
used as a positive control for the drug transporter expression, whereas the
HUVECs were used as a negative control. The relative amounts of Pgp in KBv,
BBMEC, and HUVEC cells were 1.33, 0.81, and 0.07, respectively, which confirms
substantial Pgp expression in BBMECs. To estimate the ability of various Pluronics to inhibit the Pgp efflux system in BBMEC monolayers, R123 accumulation studies in the presence and absence of 12 different Pluronic compositions were performed.
The concentration-dependent effects of four selected Pluronic compositions
on R123 accumulation in BBMEC monolayers are presented in
Fig. 1A. As it seen in the
figure, one pattern observed for all the block copolymers is that accumulation
of R123 reaches maximal levels at or near the respective copolymer CMC, and
then decreases at concentrations above the CMC. This result is consistent with
previous reports suggesting that the unimers of Pluronic (i.e., single
molecular chains of block copolymer) are responsible for the inhibition of the
Pgp efflux system in these cells (Batrakova et al.,
1998,
2001a). The effect of high
Pluronic concentrations is believed to be due to incorporation of the probe in
the micelles, resulting in a decrease in the amount of free probe available
for diffusion into the cells.
|
The effects of the various Pluronic compositions on R123 accumulation were plotted as a function of length of PO block (Fig. 1B). This parameter was chosen as an index of polymer lipophilicity. As seen in Fig. 1B, the effects of the various Pluronic block copolymers on R123 accumulation in BBMEC monolayers were dependent on the composition of the polymer. The hydrophilic copolymers with HLB ranging from 20 to 29 (group I) had little affect on Pgp functional activity. In contrast, the lipophilic copolymers (HLB <20) with intermediate-length PO blocks (i.e., 30–60 units) (group II) were very effective at inhibiting of Pgp activity in BBMEC monolayers (Fig. 1B). Those lipophilic copolymers with the PO blocks less than 30 (group III a) and longer than 60 (group III b) had little if any effect on R123 accumulation. These groups are presented in Fig. 2 showing a grid of Pluronic block copolymers with different number of PO blocks (NPO) and HLB. This figure also depicts selective copolymer representative of each group.
|
Effects of Pluronics on the Total Membrane Microviscosity in BBMECs.
It has been shown recently that Pluronic P85 treatment causes significant
changes in membranes microviscosity and that these effects correlate with the
degree of inhibition of the Pgp efflux system in BBMECs
(Batrakova et al., 2001a). To
examine the effects of Pluronic structure on membrane microviscosity, the
interactions of various Pluronic compositions with BBMEC membranes were
studied using the DPH fluorescence polarization method. This compound has been
used extensively as a membrane probe for assaying the microenvironment in the
hydrocarbon regions of the lipid bilayer
(Laat et al., 1977). It has
been shown that DPH binds with plasma membranes as well as with other
membranes within the cells, and thus the data obtained with DPH reflect the
net polarization value of all cell membranes
(Pagano et al., 1977). We
examined the time-dependent changes in fluorescence polarization of DPH in
BBMECs after exposure to the various Pluronic block copolymers. All Pluronics
were added at concentrations producing maximal inhibition of the Pgp efflux
system in BBMECs. The microviscosity values were calculated from the
polarization measurements, as described under Materials and Methods.
The changes in the total microviscosity of BBMECs exposed to representative
Pluronic compositions from each group are shown in
Fig. 3A.
|
There are two distinct effects caused by Pluronics in BBMEC cellular membranes. The hydrophilic block copolymer F88 (group I) and the lipophilic block copolymer with a short lipophilic PO block L35 (group III a) caused solidification, i.e., increasing of the membrane microviscosity (Fig. 3A), suggesting that molecules of these Pluronics adhere on the cellular surface and limit the lateral mobility of the membrane lipids. In contrast, the lipophilic copolymers with either long PO block L121 (group III b) or intermediate PO block P85 (group II) decreased the microviscosity in BBMECs (Fig. 3A), indicating their incorporation into the lipid bilayer and subsequent increase in membrane fluidization. All changes in microviscosity for each Pluronic were observed within the first 20 to 40 min after addition of the block copolymer to the cell suspension. After that time period, the microviscosity leveled off and remained constant throughout the duration of the experiment.
The microviscosity data for each Pluronic were used for calculating the total microviscosity factor (value of total cellular membrane microviscosity in the control groups in the absence of the block copolymer versus those observed in the presence of the Pluronic). After that, the microviscosity factor values were plotted versus length of lipophilic PO block for each Pluronic (Fig. 3B). As seen in the figure, the graph is qualitatively similar to those results obtained with the R123 enhancement factor (Fig. 1B). There are two separate phenomena: a bell-shaped curve corresponding to the lipophilic Pluronics and a linear dependence corresponding to the hydrophilic Pluronics. The similarities between the dependence of the rhodamine enhancement factor versus length of PO block and dependence of the microviscosity factor versus length of PO block suggest that there is a strong relationship between the effects of Pluronics on the membrane microviscosity and their ability to inhibit the Pgp efflux system in BBMECs.
Effects of Pluronics on Pgp ATPase Activity. The effect of various Pluronic compositions on Pgp ATPase activity was also examined (Fig. 4). All Pluronics were added at concentration producing maximal inhibition of the Pgp efflux system in BBMECs. The effects of four different Pluronic compositions representing each group on Pgp ATPase activity are shown in Fig. 4A.
|
Similar to the microviscosity data (Fig. 3A) there were two distinct effects on Pgp ATPase activity caused by Pluronics. The hydrophilic block copolymer F108 (group I) and the lipophilic block copolymer with a short lipophilic PO block L35 (group III a) increased the Pgp ATPase activity. In contrast, the lipophilic copolymers with the long PO block L121 (group III b) or intermediate PO block P85 (group II) decreased the Pgp ATPase activity in Pgp-overexpressing membranes. With regard to the data described in the previous section, it indicates that membrane solidification, i.e., decreasing the mobility of membrane lipids caused by the hydrophilic Pluronics, results in an increase in Pgp ATPase activity. In contrast, membrane fluidization, caused by the lipophilic block copolymers, results in a decrease in Pgp ATPase activity.
Despite these similarities, there is a major difference between the effects of various Pluronics on the membrane microviscosity and Pgp ATPase activity. Basically, the most significant membrane fluidization was caused by the intermediate lipophilic Pluronics (group II) (Fig. 3A), although the most efficient inhibition of Pgp ATPase activity was caused by the extremely lipophilic Pluronics (group III b) (Fig. 4A). This is more clearly observed by plotting the Pgp ATPase activity factor (values of Pgp ATPase activity in the control membranes in the absence of the block copolymer versus those observed in the presence of the Pluronic) versus a function of length of lipophilic PO block for each Pluronic (Fig. 4B).
As is seen in the figure, the S-shape curve for the Pgp ATPase activity factor (Fig. 4B) differs from the bell-shape curves observed for the microviscosity and R123 enhancement factors (Figs. 1B and 3B) corresponding to the lipophilic Pluronics. These differences may be due to differences in experimental protocols. Whole cells were used for the microviscosity experiments, whereas cell membranes were used for the Pgp ATPase activity studies. Therefore, the changes in the total membrane microviscosity caused by Pluronics might depend on transport of the block copolymer inside the cells. In contrast, changes in the Pgp ATPase activity in the Pgp membranes should not be affected by this factor. To prove this suggestion, the effects of various Pluronics on the plasma membrane microviscosity of BBMECs were examined.
Effects of Pluronics on the Microviscosity of Plasma Membranes in
BBMECs. Changes in the dynamics of the plasma membrane in BBMECs caused by
various Pluronics were studied by a fluorescence polarization method using
TMA-DPH. This cationic probe at early time points interacts with head groups
of phospholipids and intercalates into the outer surface leaflet of the plasma
membrane of cells, but not into the intracellular compartments
(Prendergast et al., 1981).
All Pluronics were used at the concentration that caused the most efficient
inhibition of the Pgp efflux system in BBMECs.
Similar to the results obtained from the DPH polarization measurements, two distinct effects of block copolymers on TMA-DPH polarization in BBMECs were observed: the plasma membrane solidification caused by the Pluronics F88 and L35 (groups I and III a) and the plasma membrane fluidization caused by the block copolymers P85 and L121 (groups II and III b) (Fig. 5A). The changes in the plasma membrane microviscosity in Pluronic-treated BBMECs occurred faster (within the first 3–10 min after addition of the block copolymer to the cell suspension) (Fig. 5A) than the changes in the total membrane microviscosity (Fig. 3A). The shorter time frame with TMA-DPH suggests that the insertion of Pluronics into the plasma membrane and the effect on the mobility of plasma membrane lipids occurs more quickly than transport of Pluronics into the cells and the resulting effects on total membrane microviscosity.
|
The plasma membrane microviscosity factor for each Pluronic composition was calculated, and the resulting data were plotted as a function of length of PO block for each Pluronic (Fig. 5B). An S-shaped curve corresponding to the lipophilic Pluronics and a linear dependence corresponding to the hydrophilic Pluronics were observed. It is noteworthy, that the S-shaped dependence of plasma membrane microviscosity factor (Fig. 5B) is analogous to the S-shaped dependence of the Pgp ATPase activity factor (Fig. 4B).
Effects of Pluronics on the Intracellular ATP Content in BBMECs. The
effects of various Pluronic compositions on intracellular ATP content were
measured by luciferin-luciferase assay
(Garewal et al., 1986). The
concentration-dependent effect of four different Pluronic compositions from
each major group on cellular ATP is shown in
Fig. 6A. The BBMEC monolayers
treated with the hydrophilic Pluronic F88 (group I) and lipophilic block
copolymer with a short lipophilic PO block L35 (group III a) increased
intracellular ATP content. In contrast, incubation of the cell monolayers with
the lipophilic Pluronic L121 (group III b) caused substantial energy depletion
in BBMECs. Taking into account that hydrophilic Pluronics corresponding to
group I and III a caused a significant membrane solidification
(Fig. 3A), whereas the
lipophilic Pluronics (groups II and III b) caused membrane fluidization, it
suggests the relationship between the status of the membranes and the
intracellular ATP level in BBMECs. Generally, Pluronics that increase the
membrane microviscosity elevate the ATP content, and vise versa, block
copolymers that decrease membrane microviscosity case energy depletion in the
blood-brain barrier cells. The reason of this phenomenon is unknown.
Intermediate Pluronic P85 (group II) reduced the intracellular ATP level,
identically to lipophilic L121, but at significantly higher extent (less than
10% of Pluronic nontreated control cells). This result is consistent with our
previous observation showing the most efficient membrane fluidization by the
lipophilic block copolymers with the intermediate length of PO block.
|
Finally, the ATP depletion factor (ATP intracellular levels in the absence of Pluronic versus those observed in the presence of Pluronic) was calculated and plotted versus the length of PO block for the each copolymer (Fig. 6B). There are two separate dependences: a bell-shaped curve corresponding to the lipophilic Pluronics and a linear dependence corresponding to the hydrophilic block copolymers. The lipophilic Pluronics with the intermediate length of PO block caused the most significant energy depletion in BBMECs.
Confocal Microscopy Studies of FITC-labeled Pluronics Transport into BBMEC
Pluronic F108, L35, P85, and L121 were labeled with FITC, as described
previously (Beauchamp et al.,
1983) and used to determine the cellular distribution in BBMEC
monolayers. Figure 7 shows the
confocal fluorescence photomicrographs of BBMECs after a 2-h (37°C)
exposure to FITC-labeled Pluronics. As is seen in the figure, the FITC-labeled
hydrophilic Pluronic F108 (group I) shows a poor internalization into the
cells, with most of the internalized Pluronic confined to what is presumed to
be endocytic compartments (Fig.
7A). In contrast, lipophilic Pluronic L35 with short PO block
(group III a) and intermediate Pluronic P85 (group II) accumulated throughout
the cells, including the cytoplasm, the cellular organelles, and to some
extent the nuclei (Fig. 7, B and
C). The cellular accumulation of highly lipophilic Pluronic L121
with the long PO block (group III b) was dramatically different from that
observed with Pluronics L35 and P85 (Fig.
7D). The fluorescent microphotograph shows L121 localized
presumably in the endocytic compartments. This suggests that the highly
lipophilic Pluronic L121 could not cross out the BBMEC membranes, probably,
due to its strong interaction with the lipid bilayer. Thus, L121 effectively
decreases the microviscosity of the plasma membranes
(Fig. 5B), but not the
intracellular membranes. These data provide an explanation for the effects of
various Pluronics on the total membrane microviscosity in BBMEC. Because DPH
polarization reflects changes in the microenvironment of all membranes, the
total effect of L121 on the net microviscosity in BBMECs is less than the
effect of intermediate Pluronics (group II), which explains the bell-shaped
dependence for the total microviscosity on the length of PO block
(Fig. 3A).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Hydrophilic block copolymers F38, F88, F108, and F127 (group I) showed no
or little inhibition of the Pgp efflux system in BBMEC monolayers. Confocal
microphotographs showed a poor cellular internalization of hydrophilic
Pluronics, with intracellular accumulation mainly restricted presumably to
endocytic compartments. Molecules of these block copolymers adhere to the
surface plasma membrane of the cells and limit the lateral mobility of
membrane lipids, causing membrane solidification. The increased membrane
microviscosity could be a reason for the elevated Pgp ATPase activity observed
with these block copolymers. It has been found recently that membrane
fluidization by various agents, including nonionic surfactants, abolishes Pgp
ATPase activity (Regev et al.,
1999). Moreover, a mutational analysis of Pgp showed that
interaction between the two ATP binding sites in the efflux protein is
essential for the ATP hydrolysis (Ambudkar
et al., 1999). Therefore, we suggest that membrane solidification
caused by hydrophilic Pluronics may increase interaction between the main
functional domains of Pgp and enhance the Pgp ATPase activity.
Hydrophilic Pluronic compositions also increase intracellular ATP levels in BBMECs. The mechanism of this effect remains unclear and needs further investigation. Taken together, Pluronics in group I have an extended hydrophilic ethylene oxide block, do not incorporate into lipid bilayers, undergo limited transport into the cells, and as a result, have negligible effect on Pgp efflux activity in BBMECs (Fig. 8).
|
Pluronic compositions in group II consist of lipophilic copolymers with
intermediate length of PO block (Pluronics L64, P85, L81, and P105). Members
of this group rapidly adhered to the cell membranes and incorporated into
them, resulting in an increased fluidization. This is also supported by the
confocal microphotographs demonstrating that group II Pluronics spread
throughout the cells into the cytoplasm, cellular organelles, and even some
extend into the nuclei. Our recent studies indicated colocalization of
Pluronic P85 with a mitochondrial marker in BBMEC monolayers
(Batrakova et al., 2001a),
indicating possible interactions of P85 and mitochondria membranes. The
membrane distribution of P85 and related polymers has a 2-fold effect; causing
1) a decrease of Pgp ATPase activity due to changes in the lipid
microenvironment of Pgp, and 2) an inhibition of ATP synthesis due to changes
in the electron transport in the mitochondria membranes. The evidence
supporting the latter is that intermediate Pluronic P105 decreased the
activity of the electron transport chains in the mitochondria from HL-60 cells
(Rapoport et al., 200). Finally, the reduced ability of Pgp to consume
intracellular ATP, along with energy depletion, leads to a dramatic inhibition
of Pgp efflux transport system, facilitating drug transport to the brain
(Batrakova et al., 2001a)
(Fig. 8). There may be other
mechanisms by which Pluronics increase drug transport into the cells. For
example, it was shown recently that intermediate Pluronics enhanced the
transport of doxorubicin by accelerating the processes of solute diffusion
within lipid bilayers (Erukova et al.,
2000). However, the inhibition of efflux transport systems is
believed to be the most important in the facilitation of drug transport across
the BBB.
Lipophilic copolymers with short PO blocks Pluronics L35 and L43 (group III a) could be placed between the hydrophilic Pluronics (group I) and the intermediate lipophilic Pluronics (group II) with respect to their effect on Pgp activity in BBMEC monolayers. Similar to the hydrophilic block copolymers, molecules of these Pluronics adhere on the surface membranes of BBMECs causing the membrane solidification and increasing Pgp ATPase activity. However, in contrast to the hydrophilic block copolymers and similar to the intermediate lipophilic Pluronics, they easily spread throughout the cells into cytoplasm and reach intracellular compartments, including nuclei. In spite of their effective transport into the BBMECs, they practically do not affect ATP content in the cells. The lack of effect on ATP intracellular levels is likely due to absence of membrane fluidization, particularly, in mitochondria membranes.
Finally, extremely lipophilic copolymers with long PO blocks Pluronics L121
and L101 (group III b) are the most membranotropic block copolymers. They
cause the highest fluidization effect on plasma membranes and the most
efficient inhibition of Pgp ATPase activity in Pgp-containing membranes.
Because of such high membranotropic properties these block copolymers anchor
in the plasma membranes and remain there for an extended period of time. As a
result, they are less efficiently transported into the intracellular
compartments than intermediate Pluronics (group II)
(Fig. 8). Therefore, the
extremely lipophilic Pluronics cause less energy depletion and, consequently,
have less effect on Pgp efflux system in blood-brain barrier cells than the
intermediate block copolymers. An additional consideration with the very
lipophilic Pluronic compositions is the low CMC. It has been shown previously
that the effect of Pluronics is mediated by the copolymer single chain
unimers, rather than by the micelles
(Miller et al., 1997).
Extremely lipophilic Pluronics tend to form micelles at low concentrations of
the copolymer in water solutions. Thus, the micelle formation decreases the
ability of Pluronic molecules to enter the cells and reduces the influence of
the copolymer on all systems in the barrier cells.
All in all, a delicate balance between hydrophilic and lipophilic components in the Pluronic molecule should be accomplished to provide the best interactions and the most significant impact of the block copolymer on the endothelial cell transport.
Present studies strongly indicate the key roles of membrane fluidization and energy depletion caused by Pluronics on the inhibition of the Pgp efflux system in BBMECs. To make this statement clear the dependence of R123 accumulation factor versus total microviscosity factor (Fig. 9A) and R123 accumulation factor versus ATP depletion factor (Fig. 9B) were plotted for all studied block copolymers. As is seen in the figure, both dependences are linear. This supports the importance of membrane fluidization and energy depletion, in the effects of Pluronics on Pgp efflux transport activity in BBMECs. Overall, Pluronics with the intermediate hydrophilic-lipophilic properties are believed to have a remarkable potential use for the delivery of therapeutic agents to the brain.
|
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: BBB, blood-brain barrier; BBMEC, bovine brain microvessel endothelial cell; EO, ethylene oxide; PO, propylene oxide; CMC, critical micelle concentration; HLB, hydrophilic-lipophilic balance; FITC, fluorescein isothiocyanate; HUVEC, human umbilical vein endothelial cell; R123, rhodamine 123; PBS, phosphate-buffered saline; Pgp, P-glycoprotein; MES, 4-morpholineethanesulfonic acid; DPH, 1,6-diphenyl-1, 3,5-hexatriene; TMA, 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene.
Address correspondence to: Dr. Alexander V. Kabanov, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025. E-mail: akabanov{at}unmc.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Alakhov VY and Kabanov AV (1998) Block copolymeric biotransport carriers as versative vehicles for drug delivery. Expert Opin Investig Drugs 7: 1453–1473.
Alexandridis P, Holzwarth JF, and Hatton TA (1994) Micellization of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers in aqueous solutions: thermodynamics of copolymer association. Macromolecules 27: 2414–2425.[CrossRef]
Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, and Gottesman MM (1999) Biochemical, cellular and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 39: 361–398.[CrossRef][Medline]
Batrakova EV, Han HY, Miller DW, and Kabanov AV (1998) Effects of Pluronic P85 unimers and micelles on drug permeability in polarized BBMEC and Caco-2 cells. Pharm Res 15: 1525–1532.[CrossRef][Medline]
Batrakova EV, Li S, Miller DW, and Kabanov AV (1999) Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers. Pharm Res 16: 1368–1374.
Batrakova EV, Li S, Vinogradov SV, Alakhov VY, Miller DW, and
Kabanov AV (2001a) Mechanism of pluronic effect on P-glycoprotein
efflux system in blood brain barrier: contributions of energy depletion and
membrane fluidization. J Pharmacol Exp Ther
299:
483–493.
Batrakova EV, Miller DW, Li S, Alakhov VY, Kabanov AV, and Elmquist WF (2001b) Pluronic P85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies. J Pharmacol Exp Ther 296: 556–562.
Beauchamp CO, Gonias SL, Menapace DP, and Pizzo SV (1983) A new procedure for the synthesis of polyethylene glycol-protein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin and alpha 2-macroglobulin. Anal Biochem 131: 25–33.[CrossRef][Medline]
Chazotte B (1994) Comparisons of the relative effects of polyhydroxyl compounds on local versus long-range motions in the mitochondrial inner membrane. Fluorescence recovery after photobleaching, fluorescence lifetime and fluorescence anisotropy studies. Biochim Biophys Acta 1194: 315–328.[Medline]
Druekes P, Schinzel R, and Palm D (1995) Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates. Anal Biochem 230: 173–177.[CrossRef][Medline]
Erukova V, Yu, Krylova OO, Antonenko, Yu N, and Melik-Nubarov NS (2000) Effect of ethylene oxide and propylene oxide block copolymers on the permeability of bilayer lipid membranes to small solutes including doxorubicin. Biochim Biophys Acta 1468: 73–86.[Medline]
Ferrari M, Fornasiero MC, and Isetta AM (1990) MTT calorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 131: 165–172.[CrossRef][Medline]
Fontaine M, Elmquist WF, and Miller DW (1996) Use of rhodamine 123 to examine the functional activity of P-glycoprotein in primary cultured brain microvessel endothelial cell monolayers. Life Sci 5: 1521–1531.
Garewal HS, Ahmenn FR, Shifman RB, and Celniker A (1986) ATP assay: ability to distinguish cytostatic from cytocidal anticancer drug effects. J Natl Cancer Inst 77: 1039–1045.
Jancis EM, Carbone R, Loechner KJ, and Dannies PS (1993) Estradiol induction of rhodamine 123 efflux and the multidrug resistance pump in rat pituitary tumor cells. Estradiol induction of rhodamine 123 efflux and multidrug resistance pump in rat pituitary tumor cells. Mol Pharmacol 43: 51–56.[Abstract]
Kabanov AV, Chekhonin VP, Alakhov VY, Batrakova EV, Labedev AS, Melik-Nubarov NS, Arzhakov SA, Lavashov AV, Morozov GV, Severin ES, et al. (1989) The neuroleptic activity of haloperidol increases after its solubilization in surfactant micelles. FEBS Lett 258: 343–345.[CrossRef][Medline]
Kwon GS and Okano T (1999) Soluble self-assembled block copolymers for drug delivery. Pharm Res 16: 597–600.[CrossRef][Medline]
Laat SW, Saag PT, and Shinitzky M (1977) Microviscosity modulation during the cell cycle of neuroblastoma cells. Proc Natl Acad Sci USA 10: 4458–4461.
Lee JS, Paull K, Alvarez M, Hose C, Monks A, Grever M, Fojo AT, and Bates SE (1994) Rhodamine efflux patterns predict P-glycoprotein substrates in the national cancer institute drug screen. Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen. Mol Pharmacol 46: 627–638.[Abstract]
Lemieux P, Guerin N, Paradis G, Proulx R, Chistyakova L, Kabanov A, and Alakhov V (2000) A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle Gene Ther 7: 986–991.[CrossRef][Medline]
Miller DW, Audus KL, and Borchardt RT (1992) Application of cultured bovine brain endothelial cells of the brain microvasiculature in the study of the blood-brain barrier. J Tissue Cult Methods 14: 217–224.[CrossRef]
Miller DW, Batrakova EV, Waltner TO, Alakhov VY, and Kabanov AV (1997) Interactions of Pluronic block copolymers with brain microvessel endothelial cells: evidence of two potential pathways for drug absorption. Bioconj Chem 8: 649–657.[CrossRef][Medline]
Miller DW, Fontain M, Kolar C, and Lawson T (1996) The expression of multidrug resistance-associated protein (MRP) in pancreatic adenocarcinoma cell lines. Cancer Lett 107: 301–306.[CrossRef][Medline]
Pagano R, Ozato K, and Ruysschaert J-M (1977) Intracellular distribution of lipophilic fluorescent probes in mammalian cells. Biochem-Biophys Acta 465: 661–666.[Medline]
Prendergast FG, Haugland RP, and Callahan PJ (1981) 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene: synthesis, fluorescence properties and use as a fluorescence probe of lipid bilayers. Biochemistry 20: 7333–7338.[CrossRef][Medline]
Regev R, Assaraf YG, and Eytan GD (1999) Membrane fluidization by ether, other anesthetics and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells. Eur J Biochem 259: 18–24.[Medline]
Shepard RL, Winter MA, Hsaio SC, Pearce HL, Beck WT, and Dantzig AH (1998) Effect of Modulators on the ATPase activity and vanadate nucleotide trapping of human P-glycoprotein. Biochem Pharmacol 56: 719–727.[CrossRef][Medline]
Shinizky M and Inbar M (1967) Microviscosity parameters and protein mobility in biological membranes. Biochim Biophys Acta 433: 133–149.
Venne A, Li S, Mandeville R, Kabanov AV, and Alakhov VY
(1996) Hypersensitizing effect of Pluronic L61 on cytotoxic
activity, transport and subcellular distribution of doxorubicin in multiple
drug-resistant cells. Cancer Res
56:
3626–3629.
Yokoyama M (1992) Block copolymers as drug carriers.
Crit Rev Ther Drug Carrier Syst
9:
213–248.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Bello-Roufai, O. Lambert, and B. Pitard Relationships between the physicochemical properties of an amphiphilic triblock copolymers/DNA complexes and their intramuscular transfection efficiency Nucleic Acids Res., February 16, 2007; 35(3): 728 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Moghimi, A. C. Hunter, and J. C. Murray Nanomedicine: current status and future prospects FASEB J, March 1, 2005; 19(3): 311 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. Jakate, C. M. Einhaus, A. P. DeAnglis, G. S. Retzinger, and P. B. Desai Preparation, Characterization, and Preliminary Application of Fibrinogen-Coated Olive Oil Droplets for the Targeted Delivery of Docetaxel to Solid Malignancies Cancer Res., November 1, 2003; 63(21): 7314 - 7320. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Gebhart and A. V. Kabanov Perspectives on Polymeric Gene Delivery Journal of Bioactive and Compatible Polymers, March 1, 2003; 18(2): 147 - 166. [Abstract] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
