Transcript Document

MODULATION OF MULTI-DRUG RESISTANCE IN CANCER WITH POLYMER-BLEND
NANOPARTICLES
Lilian E. van
1
Vlerken ,
Stephen
2
Little ,
Zhenfeng
3
Duan ,
Michael
3
Seiden ,
Robert
2
Langer ,
and Mansoor
1
M. Amiji
1Department
of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston MA
2Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA
3Department of Hematology and Oncology, Massachusetts General Hospital, Harvard Medical School, Boston MA
Abstract
Objective
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 nonfunctional 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-co-glycolic 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 71.67±1.33% following treatment with 1 mM PTX
and 8.6 mM CER, compared to treatment with PTX alone at the same dose
(40.09±3.14% 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
polymer-blend nanoparticles to fine-tune release drug profiles, where the
application can chemosensitize not only MDR but also drug-sensitive cancer
phenotypes.
The purpose of this study was develop a novel therapeutic approach
using polymer-blend nanoparticles for controlled co-administration of
ceramide with the chemotherapeutic paclitaxel, to overcome MDR in
ovarian cancer.
Introduction
›
a.
100
p<0.001
80
Tumor intracellular
environment
b.
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›
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Polymer-blend nanoparticles were successfully fabricated with a
mean particle size around 200 nm. While the 70/30 blends do not
show much deviation in zeta-potential, the 80/20 blends do,
suggesting that perhaps the drug-load and/or PBAE is not
entrapped into the particle (Table 1).
Chemical surface composition indicated that although PCL/PBAE
blended evenly, suggested by the surface composition ranging
between that of pure PCL and pure PBAE, PLGA/PBAE blended
unevenly to form a PBAE core or regions, suggested by the surface
composition that mimicked that of pure PLGA (Table 2).
Release studies revealed that the 70%/ 30% PLGA/PBAE blend
showed the most promising release profile, where the drop in pH to
6.5 caused a surge in release of PTX, while CER release followed
slowly (Figure 2).
Efficacy studies determined that the PTX/CER co-therapy mediated
by all nanoparticle formulations resulted in significantly more cell kill
of both the MCF7 and MCF7TR cells than the PTX treatment alone,
although the 80/20 PLGA/PBAE were the least successful
potentially due to their larger size (Figure 3).
(pH ~6.5)
100% (PEO) -PCL
Atomic composition (%)
Zeta-potential (mV)
211.6 ± 1.8
- 31.1 ± 1.5
141.6 ± 0.9
-22.66 ± 7.63
(PEO) -70% PCL/30% PBAE
2.5% PTX, 10% CER
177.7 ± 1.9
-29.32 ± 0.67
(PEO) - 80% PCL/20% PBAE
2.5% PTX, 10% CER
162.1 ± 1.4
(PEO) - 70% PLGA/30% PBAE
2.5% PTX, 10% CER
208.2 ± 5.7
-26.89 ± 5.36
(PEO) - 80% PLGA/20% PBAE
2.5% PTX, 10% CER
439.4 ± 25.3
-6.02 ± 4.01
›
Polymer-blend nanoparticles containing various ratios of
PCL/PLGA to PBAE blending were fabricated by controlled
solvent displacement of 1 part polymer/drug solution in organic
solvent into 10 parts aqueous phase
Nanoparticles were characterized for size by Dynamic Light
Scattering and for surface charge by zeta-potential
measurement on a Brookhaven zeta-PLUS particle analyzer
Chemical analysis of surface composition was performed by Xray positron spectroscopy (XPS)
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. PTX release was
monitored by RP-HPLC, while CER release was monitored by
fluorescence intensity of NBD-CER.
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.
Table 2– Electron spectroscopy for chemical analysis (ESCA)
Size (nm)
100% (PEO) -PLGA
›
-16.93 ± 3.92
High resolution C1 peak area (%)
C
N
O
C-H / C-C
C-O/ C-N
C=O
100% PCL
74.7 ± 0.7
0.0 ± 0.0
24.1 ± 0.6
66.1 ± 0.8
18.5 ± 0.5
15.4 ± 0.3
100% PBAE
73.6 ± 0.7
5.7 ± 0.5
16.6 ± 0.1
81.4 ± 0.2
11.1 ± 0.2
7.5 ± 0.0
100% PLGA
58.5 ±0.1
0.0 ± 0.0
43.6 ± 6.2
29.3 ± 0.2
36.6 ± 0.0
34.2 ± 0.2
70% PCL/ 30% PBAE
78.4 ± 0.8
3.2 ± 0.1
18.2 ± 0.8
72.8 ± 0.1
15.9 ± 0.1
11.3 ±0.1
80% PCL/ 20% PBAE
78.0 ± 0.2
3.7 ± 0.2
18.3 ± 0.1
72.4 ± 0.6
16.2 ± 0.5
11.4 ± 0.1
70% PLGA/ 30% PBAE
61.5 ± 0.3
0.8 ± 0.3
37.8 ± 0.6
32.6 ± 0.7
34.8 ± 0.1
12.8 ± 9.2
80% PLGA/ 20% PBAE
62.8 ± 1.6
1.3 ± 0.3
35.9 ± 1.3
38.5 ± 1.4
32.9 ± 0.7
28.6 ± 0.7
100
a.
140
120
100
80
60
40
20
% cell survival
›
›
›
›
b.
120
100
80
60
40
20
0
5
10
15
20
25
30
0
5
10
time (hrs)
0
10
15
20
time (hrs)
25
25
30
0
5
10
15 20
time (hrs)
Figure 3 – Cell kill efficacy of
the PTX/CER combination
therapy (1 mM PTX, 8.6 mM
CER) administered within
the 70%/30% and 80%/20%
blend
PCL/PBAE
and
PLGA/PBAE formulations on
the drug-sensitive MCF7 (a)
and MDR MCF7TR (b) cells,
compared to cell killl efficacy
of 1 mM PTX alone (without
carrier).
80
60
40
20
**
**
**
**
0
30
100
d.
120
100
80
60
40
20
5
20
time (hrs)
c.
120
100
80
60
40
20
15
% cell survival
›
Results
% drug load release (cumulative)
›
›
›
Table 1 – Polymer blend Nanoparticle size and surface
charge characterization
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,
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.
% cell death
›
Materials and Methods
25
30
Figure 2 – Release profile of PTX (solid line) and CER (dotted line) from polymer blend
nanoparticles, at pH 7.4 for the first 6 hours, then at pH 6.5 for the duration of the study.
a) 70% PCL/ 30% PBAE, b) 80% PCL/ 20% PBAE, c) 70% PLGA/ 30% PBAE, d) 80%
PLGA/ 20% PBAE. The arrow indicates a drop in pH from 7.4 to 6.5
80
*
60
40
(n= 8 samples/treatment/cell
type. * and ** indicates a
statistically
significant
difference at p<0.05 and
p<0.001
respectively
between nanoparticle and
PTX solution treatment.
**
**
20
0
70/30
80/20
PCL/PBAE
70/30
80/20
1 uM PTX
PLGA/PBAE
Conclusion
Acknowledgements
Polymer-blend nanoparticles can be developed to tune release of
combination therapies from a single nanoparticle drug delivery system.
These blend nanoparticles can be used to successfully tune release of
a PTX and CER combination therapy, resulting in significant
chemosensitation 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.
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 NIH grants CA-095522
and CA-119617. 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
60
40
20
0
-20
PTX
(100nM)
PTX
+CER
@ t=0
hrs
PTX
+CER
@ t=24
hrs
Figure 1 – a) Cell kill efficacy of PTX/CER
dosing kinetics on MDR (MCF7TR) cells. b)
Illustration of the polymer-blend nanoparticle
design.
(PCL) or
(PLGA)
Hydrophobic
polymer
Location of
ceramide
(PBAE) pH
responsive
polymer
Location of
paclitaxel
Stealth-shielding
PEO surface
modification