undergraduate research presentation
TRANSCRIPT
Study of Cationic Polylactides as a Gene Delivery System to Combat Against Prostate
Cancer
CE 498 Undergraduate Research and Creative Activity David Huang April 26th, 2013
Introduction What is prostate cancer?
Cancer of the prostate gland Effects:
Impotence and infertility Incontrollable urine flow Weakening of bone structure Death
Angiogenesis Cause of growth of aggressive tumors
Provides oxygen and nutrition Stimulators induce angiogenesis
Interleukin-‐8 (IL-‐8) Vascular endothelial growth (VEGF) Transforming growth factor (TGB)-‐β
Introduction What is a polylactide (PLA)?
Polymer of lactic acid and other derivatives Main monomers: lactic acid and cyclic di-‐ester (lactide)
Eco-‐friendly material Biodegradable Derived from renewable resources
Form typically by ring-‐opening polymerization with metal catalysts
Wide Range of Uses Medical supplies: degradable stitches, screws,
pins, rods Compostable packaging Clothes
Gene Therapy Use of gene therapy to combat cancer
Recent popularity High specificity
siRNA used to silence molecular pathways
Cons of gene therapy Inadequate cellular absorption Small retention time in body Susceptible to breakdown
Needs delivery system
Use of Well-‐Defined Cationic Polylactides for siRNA Delivery Protects siRNA from biological harm
Improve cell uptake Positive charges of cationic polylactides enhances endocytosis
Adjustable degradation rate
Low toxicity
Synthesis of Well-‐Defined Cationic Polylactides
Achieved using organocatalyzed ring opening polymerization and thiol-‐ene click reaction
Step 1: ROP of allyl-‐functionalization lactide (LA) with L-‐Lactic Acid Benzyl alcohol (BnOH): initiator 4-‐dimethylaminopyridine (DMAP) as
organocatalyst Reaction done in dichloromethane
(DCM) Reaction Conditions:
35 degrees Celsius Time: One week 90% conversion
Synthesis of Well-‐Defined Cationic Polylactides
Step 2: UV-‐induced thiol-‐ene click reactions to attach tertiary amine group Tertiary amine group: 2-‐(diethylamino)ethanethiol hydrochloride,
(DEAET) Photo-‐initiator: 2,2’-‐dimethoxy-‐2-‐phenylacetophenone (DMPA) Reaction Conditions
UV irradiation Room temperature Time: 30 minutes
Synthesis of Well-‐Defined Cationic Polylactides
Adjust [ene]0:[SH]0:[DMPA]0 ratio to produce cationic polylactides with different amount of tertiary amine groups
Four CPLAs with different mole % of amine-‐polymer backbone units Composition determined by 1H NMR
CPLA-‐9, CPLA-‐18, CPLA-‐30, CPLA-‐50 Suffix number=mole fraction
Confirmation of Well-‐Defined Cationic Polylactides Structure
1H NMR analysis of polylactide made in first step
1H NMR analysis of well-‐defined CPLA made in 2nd step
Confirmation of Well-‐Defined Cationic Polylactides Structure
Gel permeation chromatography Used to check change in hydrodynamic
volume between PLA and CPLA Results of GPC:
Showed no significant change in volume
Concluded no crosslinking
Concluded no side reactions
Study of CPLA’s Degradation
Tests done at samples of 1.0 mg/ml
Tests done with continual GPC analysis Ran at two temperatures
37 degrees Celsius 25 degrees Celsius
Data collected at 4 hour intervals from 1st -‐13th hour and at the 168th hour
Study of CPLA’s Degradation
Results of Tests Faster Degradation Rate at 37oC Faster Degradation Rate at higher amine mole %
Study of CPLA’s Toxicity Tests conditions
PC3 cells treated with all 4 different CPLAs Time incubated: 48 hours
• Tests Results – Low toxicity – Most toxic was CPLA-‐50 at highest dosage
Use of CPLA for Gene Delivery
Successful Nanoplexes Formed
Nanoplexes formed by electrostatic interaction CPLA/siRNA mass ratio 20:1 Time: 30 minutes
TEM image of CPLA-‐50-‐IL5 siRNA Nanoplex
Conclusion
Synthesizing CPLA with low toxicity is possible
Degradation rate can be altered to control gene release
Use of Well-‐Defined CPLA with encapsulated IL-‐8-‐siRNA can provided an alternative treatment for prostate cancer