Transcript www.stem.neu.edu

Hyaluronic Acid Based Self-assembling
Nanosystems for
Nucleic Acid Delivery in Cancer Therapy
Betelhem Gemechu, YSP Student, Braintree High School
Nathan Pan-Doh, YSP Student, Lincoln-Sudbury Regional High School
Research Mentors: Professor Mansoor Amiji & Dr. Meghna Talekar, Department of
Pharmaceutical Sciences, Northeastern University
Cancer
●
Cancer is when cells abnormally divide without control
●
DNA of a cell can become damaged:
o Mutations occur
o Cells do not die
o New cells form when the body does not need
them
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Extra cells may form a mass of tissue which creates
tumors
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Cancer is the second most common cause of death in
the US with an estimated 1.7 million cases diagnosed
and 0.5 million cancer deaths by 2014.
[1]
[1]
Ovarian Cancer
Types:
●
Epithelial tumors – ~90 percent, grow in
epithelium (tissue that covers ovaries)
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Germ cell carcinoma tumors – ~5 percent,
starts in cells that form eggs
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Form in germ cells → usually benign
[15]
Stages:
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I: Confined to ovaries
II: One or two ovaries and spreads to pelvic
area
III: Continued extension to either lining of
abdomen/abdominal surfaces or lymph Nodes
IV: Metastasis to the liver or lungs
[15]
[15]
Available treatments
Surgery
Chemotherapy
Radiation
[2]
[3]
[4]
Problems: The harmful side effects
These cytotoxic drugs either have side effects/toxicity
problems or stability problems. Thus, they have to be
formulated in delivery systems.
[5]
[6]
Nanoparticle based delivery systems
●
Deliver drugs specifically to tumor
tissues
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Healthy tissue cells tightly bound →
nanoparticles cannot enter
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Tumor tissue blood vessels are leaky
→ nanoparticles accumulate
→
Enhanced
permeation
retention
effect
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EPR effect - method of targeting
drugs to tumor tissue
[7]
RNAi Pathway and siRNA
[8] [12]
Cellular Respiration
Glycolysis
●
Part of cellular respiration, series of reactions
in first phase of most carbohydrate
catabolism
●
Breaks down glucose, forms pyruvate with
the production of two molecules of ATP.
●
End product can be used in anaerobic
respiration or aerobic respiration via the
TCA/Krebs cycle, which yields much more
usable energy.
●
PKM-2 isoform of PKM is highly expressed in
cancer tissues compared to normal tissues →
target for cancer therapy.
[11]
[9]
Objectives
● Encapsulate siRNA in nanoparticles
● Assess cellular uptake of these particles
● Determine transfection efficiency
● Assess the toxicity of the nanoparticles
Methods
● HA-PEI and HA-PEG
synthesis:
Fluorescein labeled siRNA (- charge)
HA-PEI/PEG/siRNA nanoparticle
o 6 mg/mL
o 3 mg/mL
o 1.5 mg/mL
+
PEI
modified
HA
● Tested different
ratios of HAPEI/PEG:siRNA to
assess effect on
transfection
efficiency
Rhodamine and PEG
labeled HA (- charge)
PEI (+ charge)
[12]
HA-PEI/PEG siRNA nanoparticles - size and
charge
Size Distribution for 6 mg/mL
Nanoparticles
Size Distribution for 3 mg/mL
Nanoparticles
Size Distribution for 1.5 mg/mL
Nanoparticles
Sample Name
Z-Ave (d.nm)
PDI
ZP (mV)
HA-PEI/PEG 6mg/mL
134.0
0.2
-32.7
HA-PEI/PEG 3mg/mL
152.7
0.4
-31.0
HA-PEI/PEG 1.5mg/mL
186.3
0.3
-25.6
HA-PEI/PEG siRNA nanoparticles Morphology
6 mg/mL in 100 nM
3 mg/mL in 100 nM
100 nm
100 nm
1.5 mg/mL in 100 nM
100 nm
Cytotoxicity of HA-PEI/PEG siRNA nanoparticle
HA-PEI/PEG uptake in SKOV-3 cells
Confocal Magnification: 40X
HA-PEI/PEG siPKM2 transfection studies in
SKOV3 cells
Conclusion
● HA/PEI/PEG nanoparticles were determined non-toxic
to SKOV-3 cells and have ideal size, charge and shape.
Their size is within the range of 134-186 nm, the charge
of the nanoparticles between -25 and -33 mV, and they
prove to have spherical morphology.
● 3 mg/mL HA/PEI/PEG siPKM2 nanoparticle was the
most effective in down-regulating PKM2 expression,
shown through transfection studies
Future studies
● Trafficking studies can be conducted to test the uptake
and release of the siRNA from these nanoparticles.
● Forster Resonance Emission Transfer (FRET) can be
developed to assess the trafficking of these particles
through cancer cells.
Acknowledgements
Center for STEM Education - Young Scholars Program & Team
● Claire Duggan - Director
● Kassi Stein, Jake Holstein, Chi Tse - YSP Coordinators
● Dan Sullivan - Program Implementation Coordinator
● Mary Howley - Administrative Officer
Northeastern University Department of Pharmaceutical Sciences
● Professor Mansoor Amiji & Dr. Meghna Talekar
● Dr. Huyen Tran, Adwait Oka, Dr. Amit Singh, Srujan Gandham, Oscar
Ouyang, Vanessa Nascimento
References
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"What Is Cancer?." National Cancer Institute. N.p., n.d. Web. 26 July 2014. <http://www.cancer.gov/cancertopics/cancerlibrary/what-is-cancer>.(1)
"Mesothelioma Treatment." HealthLobcom. N.p., n.d. Web. 27 July 2014. <http://healthlob.com/2011/06/mesothelioma-treatment/> (2)
"Lung Cancer Surgery in India." Lung Cancer Surgery India, Low Cost Lung Cancer Surgery India, Lung Cancer Surgery Benefits India. N.p., n.d.
Web. 27 July 2014. <http://www.forerunnershealthcare.com/lung-cancer-surgery-low-cost-benefits-India.html> (3)
"The Latest Research, To Boost Your Health Intelligence." Pretty Mom Guide RSS2. N.p., n.d. Web. 27 July 2014. <http://prettymomguide.com/thelatest-research-to-boost-your-health-intelligence.html> (4)
"Mesothelioma Chemotherapy." Mesothelioma Cancer Alliance. N.p., n.d. Web. 27 July 2014.
<http://www.mesothelioma.com/treatment/conventional/chemotherapy> (5)
"Cancer Protocols Best To Avoid, Chemotherapy, Radiation, Mammograms, Breast Cancer." Cancer Protocols Best To Avoid, Chemotherapy,
Radiation, Mammograms, Breast Cancer. N.p., n.d. Web. 27 July 2014. <http://www.chimachine4u.com/cancer-protocols.html> (6)
"Drug delivery: One nanoparticle, one kill." nature.com. N.p., n.d. Web. 14 July 1929. <http://www.nature.com/nmat/journal/v10/n5/full/nmat3014.html
(7)
"RNA." alnylam.com. N.p., n.d. Web. 14 July 2014. <http://www.alnylam.com/rnai_primer/files/RNAi-therapeutic-mechanism-full.gif> (8)
"Glycolysis." hyperphysics. N.p., n.d. Web. 21 July 2014. <http://hyperphysics.phy-astr.gsu.edu/hbase/biology/glycolysis.html> (9)
Ganesh, Shanthi, Arun K. Iyer, David V. Morrissey, and Mansoor M. Amiji.
"Hyaluronic Acid Based Self-assembling Nanosystems for CD44 Target Mediated SiRNA Delivery to Solid Tumors." Thesis. Northeastern
University, 2013. Print. (10)
"Pyruvate kinase M2-specific siRNA induces apoptosis and tumor regression." Jem.rupress.org. N.p., n.d. Web. 15 July 2014.
<http://jem.rupress.org/content/209/2/217.fulll> (11)
"Result Filters." National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 27 July 2014.
<http://www.ncbi.nlm.nih.gov/pubmed/22271574> (12))
"Statistics - We work to save women's lives.." We work to save women's lives.. N.p., n.d. Web. 26 July 2014.
<http://www.ovariancancer.org/about/statistics> (13)
Jong, Wim, and Paul Borm. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 5 Nov. 1927. Web. 27 July
2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2527668/> (Abstract)
Thank You!
Dr. Meghna Talekar
Any Questions?????
Oscar Ouyang
Professor Mansoor Amiji
Nathan Pan-Doh
Betelhem Gemechu