PEGylated Ultrasound Contrast Agents for Drug Delivery to Pancreatic
Tumor Cells
Timothy Hoang, Averie M. Palovcak, Jaap Patel, Rebecca Sheridan, Stephen Zachariah, Lauren J. Jablonowski, and Margaret A. Wheatley, Ph.D.
School of Biomedical Engineering Science and Health Systems, Drexel University, Philadelphia, PA 19104
Immune Response
Stroma Penetration
Overview
•  GEM limited effectiveness due to inability to penetrate stroma (connective tissue and
interstitium) around tumor [1]
•  PDA involves stroma proliferation (desmoplasia) •  Stroma separates blood vessels and tumor [2]
•  Upon ultrasound exposure, MB will shatter and produce GEM-loaded, nano-sized
fragments
•  Ultrasound interrogation can also open up angiogenic blood vessels around
stroma
•  Direct, targeted drug delivery in tumor region
Pancreatic cancer treatment is ineffective due to excessive interstitium and connective tissue (stroma) surrounding
the tumor, impeding drug access. Ultrasound (US) irradiation used with drug-loaded contrast agents provides a
means of combating this by targeted delivery of the drug gemcitabine (GEM), increasing tumor vasculature
permeability and loosening the stroma. The drug-delivery platform consists of micron-sized, biodegradable
poly(lactic acid) (PLA) microbubbles (MB) that shatter into nano-sized drug-loaded fragments (n-Sh). However in
circulation, MB and n-Sh are recognized as foreign and are quickly tagged by the complement system for immune
clearance, reducing the potential for maximal drug delivery. This work seeks to design MB that retain the properties
of traditional US contrast agents (echogenicity and capacity to shatter), incorporate GEM into the shell, and
manifest poly(ethylene glycol) (PEG) chains on the surface to create a steric barrier that obstructs surface binding
of complement proteins. Based on results from previous studies, concentration ranges for maximal GEM (3 and 6
wt%) and PEG (2, 5, and 10 wt%) incorporation were tested. GEM-loaded MB were evaluated for acoustic
enhancement and stability in the US beam, surface morphology, average diameter, and zeta potential. C3
complement activation in serum was assessed by ELISA. Acoustic evaluation and SEM suggest that GEM
encapsulation does not significantly affect MB morphology and n-Sh production upon US exposure. All groups
reflected a clinically-relevant signal, and GEM-loaded MB reduced in stability compared to unPEGylated controls
suggesting they are more likely to shatter into n-Sh. The C3 assay results show that PEGylation successfully
reduced MB immunogenicity compared to unPEGylated control MB. It was found that the 5 wt% PEG and 3 wt%
GEM MB best met the design criteria, providing the most viable agent for targeted pancreatic cancer drug delivery. •  MB and n-Sh tagged by immune complement system for removal or
phagocytosis
•  Main complement proteins (antibodies) involved [3]
•  C3b: adsorbs to MB surface
•  C3a: macrophage recruitment
•  Enhanced permeability and retention (EPR) effect [4]
•  Leaky tumor vasculature due to rapid formation
•  Allows unrecruited, drug-loaded n-Sh to reach tumor and accumulate
•  Secondary delivery (shown below)
•  MB and n-Sh tumor access hindered by immune system
•  Design: attenuate immunogenicity by reducing C3 adsorption and recruitment
•  Polyethylene glycol (PEG) on MB surface: current stealth gold standard
[5]
•  Displacement of adsorbed antibodies in blood, including C3 [6]
•  PEG-PLA MB must also retain echogenicity, shattering capacity, ability
to incorporate GEM
[2]
Microbubbles were mounted onto an aluminum stub and sputter coated with platinum-palladium (Pt-Pd), then imaged with a Philips FEI XL30
Environmental Scanning Electron Microscope.
Imaging parameters: acceleration voltage 5kV, spot size 3, and 2500x magnification, size bar 5µm.
10% PEG-PLA MB
•  Significant deformities and few intact
MB
•  Overloading of PEG compromised
shell
5% PEG-PLA 3% GEM MB
•  Spherical MB morphologies with
minor concavities •  GEM loading did not significantly
interfere with MB properties
PEG-PLA MBs
GEM PEG-PLA MBs
[4]
Zeta Potential
In order to circumvent sizing constraints,
bubbles cannot show
significant clumping. For the testing, 1 mg of each MB sample was
Scanning Electron
Microscopy (SEM)
2% PEG-PLA MB
•  Spherical morphology comparable
to unPEGylated sample
•  Sizing averages 2 μm in diameter insonated with a 5MHz transducer through an acoustically transparent window, results
were recorded using LabView.
unPEGylated PLA MB
•  Showed spherical morphology
•  Sizing around 2 μm in diameter
consistent with zetasizer data Acoustic Enhancement and Stability
Acoustic tests were preformed in a sample vessel
in a 37°C water bath. The sample was
5 wt% PEG-PLA MB
•  MB shown minor concavities and
bursting but significant amount of
spherical, intact MB
5% PEG-PLA 6% GEM MB
•  Large population of oversized and
broken MB
•  Shows overloading of GEM in MB
shell
Sizing of MB and Nanoshards
C3 Immune Complement Assay
suspended and in distilled water, vortexed, and subjected to charge
testing via ZetaSizer. Intact MB should be smaller than 7 μm in diameter to avoid blockage
in capillaries. All intact MB populations showed average diameter
sizes less than 2.5 μm. Nanoshards should show sizes less than 400
A specialized Sandwich C3 ELISA kit was used to quantify
nm to take advantage of the leaky tumor vasculature and escape
immunogenicity. Specifically, C3a levels were determined from
All MB populations showed negative zeta potentials, meaning the net
through the pore to congregate for secondary delivery. All samples
corresponding detection antibody absorbance values read at 450 nm.
repulsion did not cause MB aggregation or clumping.
except the 5% PEG 3% GEM met this sizing constraint.
Future Work
Conclusions and Clinical Relevance
•  In vitro testing on MIA PaCa-2 pancreatic cancer cell line
•
Agents retain potential for in situ generation of nanoshards for targeted therapy
•  High performance liquid chromatography (HPLC) for quantification of GEM encapsulation
•  The 5 wt% PEGylated MBs showed the best acoustic enhancement (18 dB) comparable to
•  In vivo testing on PDA mouse model
unPEGylated MBs
•  The 6 wt% GEM encapsulated MBs showed significant deformations due to overloading of drug
•  The 5 wt% PEG and 3 wt% GEM MBs showed optimal GEM and PEG loading
References
[1] Maeda H. (2001). Adv Enzyme Regul. 41:189-207.
[2] Korc, M. (2007). Am J Surg. 194(4 Suppl), S84-S86. •  C3 assay showed an 70% reduction when compared to unPEGylated MB in the 5 wt% PEG-PLA and
[3] S.I. Jeon, J.D. Andrade, J. Colloid Interf. Sci. 142 (1991) 159–166.
3 wt% GEM
[4] Ishida, T, et al. (2007). J. Control. Release. 122(3): 349-355
[5] Li, J et al. (2013). Nanoscale Res Lett., 8(1), 176 [6] Malekigorji, M., et al. (2014). J. Nanomedicine Res. 1(1): 4-16. •  PEGylated GEM loaded MBs can potentially revolutionize cancer treatment, especially in sensitive
patient populations
•  Potential to greatly reduce, or even eliminate, need for toxic systemic chemotherapy
Acknowledgements
•  Real-time treatment monitoring, gathering continuous diagnostic information with each treatment
•
Drexel University Senior Design Program
•  Increased immune shielding allows for increased circulation time and greater drug payload
•  Dolores Conover for training on laboratory equipment and cell culture •  Drexel University Centralized Research Facility for SEM
•  Lauren Jablonowski and Tarn Teraphongphom