MODULATION OF MULTIDRUG RESISTANCE IN CANCER WITH POLYMERBLEND NANOPARTICLES

1
MODULATION OF MULTI-DRUG RESISTANCE IN CANCER
WITH POLYMER-BLEND NANOPARTICLES Lilian E. van
Vlerken1, Stephen Little2, Zhenfeng Duan3,
Michael Seiden3, Robert Langer2, and Mansoor M.
Amiji1 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
Materials and Methods
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 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-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
7030 and 8020 PCL/PLGAPBAE, 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 71.671.33 following treatment with
1 mM PTX and 8.6 mM CER, compared to treatment
with PTX alone at the same dose (40.093.14 cell
death, plt0.001). 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.

• 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 X-ray 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.

Results

• 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).

Introduction
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). (n 8 samples/treatment/cell
type. and indicates a statistically
significant difference at plt0.05 and plt0.001
respectively between nanoparticle and PTX
solution treatment.







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
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