Transcript Document

DESIGN AND DEVELOPMENT OF A POLYMER-BLEND NANOPARTICLE DRUG DELIVERY SYSTEM TO OVERCOME MULTIDRUG RESISTANCE IN CANCER
Lilian E. van Vlerken1, Steven R. Little2, Zhenfeng Duan3, Michael V. Seiden3, Robert Langer4, and Mansoor M. Amiji1
1Department
of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115; [email protected]
2Department of Hematology and Oncology, Massachusetts General Hospital, Boston, MA 02114
3Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, PA 15261
4Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139
Objective
Abstract
Results
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p<0.001
% cell death
120
80
0
PTX
PTX +
PTX + CER
CER +
CER
@ t = 6hrs
PTX @ t
sol'n
= 6 hrs
Figure 1 – Temporal relationship between paclitaxel (PTX) and ceramide (CER)
administration to chemosensitize MDR cancer cells
ceramide
60
40
Drop in pH
to 6.5
20
c.
b.
d.
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**
**
80
a.
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Polymer-blend nanoparticles containing various ratios of PCL/PLGA to PBAE blending
were fabricated by controlled solvent displacement
Nanoparticles were characterized for size and shape by SEM and TEM
pH-sensitive pocket formation and drug compartmentalization in the nanoparticle interior
was modeled by casting the polymer-blends as films for imaging under light microscopy.
Fluorescent derivatives of paclitaxel (rhodamine) and ceramide (NBD) were loaded into
the polymer-blends to simulate drug compartmentalization within the polymer-blend
nanoparticles
Drug release was performed into PBS containing 1% Tween-80 at pH 7.4 for the first 6
hours, then replaced by release medium at pH 6.5 for the duration of the study.
Paclitaxel release was monitored by RP-HPLC, while ceramide release was monitored
by fluorescence intensity of NBD-ceramide.
Human breast adenocarcinoma cells (MCF7) were cultured alongside their respective
MDR subculture (MCF7TR) that had been selected for resistance in the presence of
increasing concentrations of PTX.
Efficacy studies were perfomed on MCF7 and MCF7TR cells by treating the cells with
the nanoparticle formulations alongside adequate controls for 6 days, after which
remaining cell viability was quantitated using the MTT assay.
$$
60
40
20
brightfield
0
5
10
15
20
25
paclitaxel
ceramide
merged
30
time (hrs)
(PBAE) pH responsive
polymer
Location of paclitaxel
20
100
paclitaxel
500 nm
Figure 4 – scanning electron
microscopy (SEM) and transition
electron microscopy (TEM – inset)
images of a) 70% PCL:30% PbAE
and b) 70% PLGA: 30% PbAE
nanoparticles
MCF7
100
b.
(PCL) or (PLGA)
Hydrophobic polymer
Location of ceramide
Figure 2 – illustration of the polymer blend
nanoparticle design
MCF7TR
MCF7
Figure 7 – a) cumulative drug release from the 70% PLGA: 30% PbAE nanoparticles exhibiting
temporal control. b) Increase in cell kill efficacy of the 70% PLGA: 30% PbAE nanoparticles (NP)
delivering a dose of 1 mM paclitaxel (PTX) and 8.6 mM ceramide over treatment with 1 mM PTX
alone. ** indicates a statistically significant difference (p<0.001) between NP and PTX treatment,
and $$ indicates a statistically significant difference (p<0.001) between MCF7TR and MCF7 cells in
response to PTX treatment (n=8/group)
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60
40
500 nm
Figure 3 – Cell kill efficacy of the paclitaxel (PTX) and ceramide (CER)
combination therapy on MDR breast cancer (MCF7TR) as well as wildtype breast cancer (MCF7). ** indicates a statistically significant
difference (p<0.001) between PTX + CER and PTX alone (n=8/group)
p<0.05
80
›
**
40
MCF7TR
(PEO) surface
modification
100
60
0
0
Multidrug Resistance (MDR) refers to the development of a cross-resistance to a
multitude of structurally and functionally unrelated drugs.
Among the many mechanisms responsible for development of MDR in the cancer cell,
alterations in apoptotic signaling appears to greatly contribute to the phenomenon,
whereby the overexpressed enzyme glucosylceramide synthase (GCS) converts the
apoptotic signaling mediator ceramide to an inactive form (glucosylceramide),
Previous work demonstrated that administering ceramide as a combination therapy
with a chemotherapeutic (paclitaxel) to MDR breast and ovarian human cancer cells
could overcome this blockade and reinstate apoptotic signaling initiated by
chemotherapeutic stress.
Additionally, the work revealed that the ceramide/paclitaxel combination was optimally
effective when ceramide was administered several hours following paclitaxel
administration (Figure 1),
For optimal therapeutic efficacy, we developed a polymer blend nanoparticle allowing
for simultaneous delivery of the combination therapy, but with controlled release of the
two therapeutics. The chemotherapeutic paclitaxel was placed within pH-responsive
PBAE pockets that released their load immediately upon internalization of the
nanoparticle into the acidic tumor environment, while ceramide was placed within the
hydrophobic matrix, composed of PCL or PLGA, which degraded slowly to release
their load in a much delayed manner (Figure 2).
% cell survival
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**
80
20
a.
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›
100
% cell death
Introduction
The purpose of this study was develop a novel therapeutic approach using polymer-blend
nanoparticles for temporally-controlled co-administration of ceramide with the
chemotherapeutic drug, paclitaxel, to overcome MDR in cancer.
% drug load released
(cumulative)
The development of multi-drug resistance (MDR) is a major barrier to anti-cancer therapy, since this phenotype
renders the tumor unresponsive to a multitude of chemotherapeutic options. Alterations in apoptotic signaling
have emerged as a common strategy for MDR development, whereby glucosylceramide synthase (GCS) causes
bioactivation of the pro-apoptotic mediator ceramide to a non-functional moiety. The objective of this work is to
overcome MDR through a nanoparticle-based therapy that administers ceramide (CER) in combination with the
chemotherapeutic drug paclitaxel (PTX), to restore apoptotic signaling. For optimal therapeutic efficacy, we
have engineered long-circulating polymeric nanoparticles composed of pH responsive poly(beta-amino ester)
(PBAE) blended into a hydrophobic matrix consisting of poly(epsilon-caprolactone) (PCL) or poly(lactic-coglycolic acid) (PLGA). By regulating the blending ratio of the two polymers, we could tune release of the
combination therapy, specifically tailored to the tumor environment. Efficacy of the formulation was tested on a
breast (MCF7) model of MDR cancer. Optimal size and stability of the nanoparticle formulation was found at a
blend of 70%:30% and 80%:20% PCL/PLGA:PBAE, and a drug load of 2.5% PTX and 10% CER. Release
studies revealed that the blend composition is pH responsive, where a surge in release occurred when spiked to
pH 6.5. Moreover, release of PTX vs. CER could be tuned, where, compared to the 70/30 composition, CER
release from the 80/20 PCL/PBAE particle was delayed while PTX release was accelerated. Unlike the other
three formulations, the 70/30 PLGA/PBAE formulation released PTX rapidly, upon a drop in pH to 6.5, with a
slow sustained release of CER. Efficacy studies then revealed the ability of this tuned therapeutic strategy to
greatly chemo-sensitize the MDR cancer type, shown by an increase in cell death up to 79% following treatment
with 1 mM PTX and 8.6 mM CER, compared to treatment with PTX alone at the same dose (27% cell death,
p<0.001). Remarkebly, the novel therapeutic approach showed and equally successful chemosensitation profile
with the drug-sensitive MCF7 cells. The results demonstrate a promising potential for use of these polymerblend nanoparticles to fine-tune release drug profiles, where the application can chemosensitize not only MDR
but also drug-sensitive cancer phenotypes.
Materials and Methods
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PCL: PbAE
Figure 5 – Microscopy images of polymer films of a)
70% PLGA:30% PbAE and b) 70% PCL:30% PbAE, and
degradation c) 70% PLGA:30% PbAE and d) 70%
PCL:30% PbAE after exposure to pH 6.5 conditions
(200x magnification)
Drug sensitive (wild-type) cells show the same amount of cell death at a 10-fold lower
concentration of paclitaxel than the MDR cells, verifying the MDR phenotype of the model. A
combination therapeutic approach using ceramide then as a co-therapy aside the
chemotherapeutic paclitaxel results in significantly increased cell death in not only the MDR,
but also the drug sensitive MCF7 breast cancer population (Figure 3)
Polymer-blend nanoparticles using a blend of either 70% PCL:30% PbAE or 70%
PLGA:30% PbAE display a spherical appearance and nanometer size-range as indicated by
SEM and TEM (Figure 4)
PLGA:PbAE and PCL:PbAE polymer blends cast into films reveal that the two types of
polymer-blends display strikingly different morphologies. Moreover, while the PCL:PbAE
blend appears to etch away evenly when exposed to acidic solution, only parts of the
PLGA:PbAE blend etch away, while leaving the remainder of the film untouched (Figure 5)
Drug compartmentalization into the PCL:PbAE and PLGA:PbAE films is revealed through
the use of fluorescent-derivatives of the drugs. While in the PCL:PbAE blend the green and
red fluorescence merge completely to suggest that the two drugs are evenly distributed
thoughout the blend, in the PLGA:PbAE blend, the green and red fluorescence does not
merge, suggesting that the drugs compartmentalize within PLGA:PbAE polymer blend
nanoparticle. Moreover, when comparing to Figure 4, the red fluorescent paclitaxel appears
to localize itself into the pH sensitive region that dissolves out when the pH changes to 6.5.
This supports the hypothesis that paclitaxel localizes into the pH-sensitive pockets of the
nanoparticle to allow for temporal release prior to release of ceramide. (Figure 6)
Drug release behavior shows that the PLGA:PbAE blend nanoparticle releases paclitaxel
and ceramide with temporal control, where paclitaxel is rapidly released first, followed by a
slow sustained release of ceramide. Efficacy studies show that this nanoparticle is then
able to significantly enhance chemosensitization of not only MDR, but also drug sensitive
cancer cells (Figure 7).
PLGA: PbAE
Figure 6 – Drug compartmentalization in the 70% PCL:30% PbAE polymer blend (top row) and the 70% PLGA:30% PbAE polymer
blend (bottom row). Brightfield microscopy image with corresponding fluorescence images of red-fluorescent rhodaminepaclitaxel and green-fluorescent NBD-ceramide (200x magnification)
Conclusion
Polymer-blend nanoparticles can be developed to tune release of combination therapies from a
single nanoparticle drug delivery system. In this case, these blend nanoparticles can be used to
compartmentalize the drugs inside the nanoparticle to successfully tune release of a paclitaxel
and ceramide combination therapy. The overall result is a significant chemosensitization of
MDR as well as regular drug sensitive MCF7 breast cancer cells. These results show promising
use of this therapeutic strategy to overcome MDR and enhance anti-cancer therapy.
Acknowledgements
L.E. van Vlerken is a recipient of an NSF IGERT fellowship in Nanomedical Science and
Technology co-sponsored by the NSF and NCI. This study was further supported by the
Nanotechnology Platform grant from the NCI (R01-CA119617). Special thanks to Dr. Lara
Gamble for the University of Washington NESAC/BIO center for her help with XPS studies,
supported by NIBIB grant EB-002027