Biodegradable Nanoparticles for Cancer Therapy

1. Biodegradable Nanoparticles for Cancer Therapy Jamboor K. Vishwanatha, Ph.D.Dean and Professor Graduate School of Biomedical Sciences

2. Extensively investigated polyester Numerous assets Release profile can be controlled Nanoparticle size can be controlled Capable of the capture of any therapeutic agent Hydrophobic (ATRA, doxorubicin, 5 fluorouracil) Hydrophilic (DNA, protein, small molecules) Potential for development of targeted or combinational therapies Very low immunogenicity and cytotoxicity High transfection potential Poly D,L lactide-co-glycolide (PLGA)

3. Properties of PLGA Hydroxyl terminus Carboxyl terminus Lactide Glycolide PLGA undergoes acid catalyzed hydrolysis to release cellular metabolites of lactic and glycolic acid PLGA nanoparticle size can be controlled through variations in nanoparticle formulation conditions Variations in lactic acid to glycolic acid ratios effect the degradation profile of the polymer (release rate) Degradation rate is also affected through variations in the intrinsic viscosity (i.v.) of the polymer

4. Nanotechnology Applications • Gene Delivery • Chemotherapeutic Delivery

5. Gene Delivery: Formulation of DNA loaded nanoparticles • Traditional formulation is accomplished through the use a W/O/W double emulsion solvent evaporation technique • Our formulation parameters include the use of a non-solvent. • The addition of a non-solvent accomplishes several goals • Minimization of shear forces • Decreases in particle size

6. What do these particles look like? A C B A C Pictures of PLGA nanoparticles following completion of fabrication (Panel A; Size bar ~100 nm). Panel B is a TEM image PLGA nanoparticles (Size bar ~500nm). Panel C is a TEM image of antibody targeted nanoparticles.Here the nanoparticles appear translucent with colloidal gold labeled anti-mouse antibody as the dark specks (Size bar indicates 100nm). Nanoparticles are stable at 4oC indefinitely and easily resuspend in isotonic buffers or cell media.

7. A B C PLGA nanoparticle size Figure 2: Formulation parameters and their effect on size of plasmid DNA loaded nanoparticles. Through the optimal choice of solvent/non-solvent systems we can control the size of nanoparticles produced. Panel A: Solvent: Chloroform, Non-solvent: Water; size range 100->1000 nm. Panel B: Solvent: Chloroform, Non-solvent: Ethanol; size range 100-400 nm. Panel C: Solvent: Chloroform, Non-solvent: Methanol; size range 51-138 nm. It is important to be able to control the ultimate size of the particles in order to achieve optimal transfection of cells and cross physiological barriers (i.e. blood brain barrier and nuclear pore complexes).

8. Intracellular Uptake Nanoparticles labeled with Nile Red appear red and can be seen within the cells after 1 hour of incubation.

9. Nanoparticle efficiency: Uptake and transfection A B B Transfection ability and cytotoxic effects of nanoparticles. PLGA nanoparticles were dual loaded with sulforhodamine 101 (red) and GFP plasmid DNA (green) and exposed to DU-145 cells. Four days post-transfection cells were visualized under laser confocal microscopy. Greater than 90% of the cells display transcription of GFP encoding plasmid DNA and cellular uptake of the nanoparticles (panel A). Unloaded nanoparticles were evaluated for cytotoxic effects upon cells (panel B). Greater than 90 percent cell viability at the maximal dose of 1 mg/ml can be seen. We have also observed no cytotoxic effects on cells at concentrations up to 3 mg/mL

10. B A C 24 hours F E D 48 hours Migration of DU-145 cells upon administration of plasmid DNA loaded nanoparticles and blank unloaded nanoparticles. Transfection of DU-145 cells was performed for 4 days and visualized 24 and 48 hours after plating of the migration assay. Control cells are seen in panel A and D respectively. There is a tremendous reduction in cellular migration of DU-145 cells treated with plasmid DNA loaded nanoparticles (panel B and E). There is no effect upon migration when treated with blank unloaded nanoparticles (panel C and F). At 48 hours cells have been counter stained with crystal violet to enhance visualization. pDrive-sh AnxA2 loaded nanoparticles can serve to mediate prostate cancer cellular migration

11. pDrive-sh AnxA2 nanoparticles also effect prostate cancer cellular proliferation DU-145 cells were exposed to nanoparticles over an 8 day time course. Control cell growth is indicated by the solid black line. Unloaded blank nanoparticles (dashed line) display no effect upon cell growth or growth rate. pDrive-sh AnxA2 loaded nanoparticles (dotted line) significantly diminish cellular growth and rate of growth.

12. A A B C B C Control HBSS treated: 18 days Blank unloaded nanoparticle treated: 9 days pDrive-sh AnxA2 nanoparticle treated: 27 days In vivo analysis of nanoparticle efficacy

13. In vivo continued Control 21 days Sh treated 27 days Blank 9 days

14. Chemotherapeutic Delivery: ‘Nanocurcumin’ • Polymeric nanoparticles encapsulating Curcumin (anti-cancer drug) • Curcumin: • Diferuloylmethane, a yellow polyphenol extracted from Curcurma longa • Therapeutic agent in traditional Indian medicine

15. Free Curcumin Poorly dispersible in water Reduced Bioavailability Nanocurcumin Dispersible in water Sustained drug release kinetics Improved Bioavailability Improved cellular uptake Improved inhibition of clonogenicity of cancer cell lines Curcumin Vs Nanocurcumin

16. Characterization • Percent Yield : 90-94 • Encapsulation Efficiency: > 95%

17. Particle Size Analysis:

18. Formulation Optimization

19. Surface Morphology: 500nm Transmission Electron Microscopy

20. Confocal Microscopy: Curcumin PLGA Nanoparticles Curcumin nanoparticles were observed under Confocal Microscope (Carl Zeiss LSM 410). For curcumin: λex is 450nmand λem is 488nm

21. In-vitro Release Kinetics Curcumin nanoparticles were incubated in PBS (pH 7.4) and at different time points, the supernatant was analyzed at λ:450nm for cumulative curcumin release

22. Cell Viability Assay

23. We are working on the development of targeted nanotherapeutics The goal of our work is to deliver locally higher concentrations of drug to diseased cells or tissues These nanoparticles are capable of selective attachment of nucleophilic substrates Antibodies Proteins (Transferrin) Peptides (NLS sequences) Small molecules (N-acetyl cysteine) Second generation nanoparticles

24. Schematic diagram for the development of targeted nanoparticles • Using a platform technology we first generate an activated nanoparticle • In a second reaction the targeting agent is conjugated to the outer surface of the nanoparticle

25. Mode of action • The targeted nanoparticle finds the specific cellular target • The nanoparticle binds to the surface of the cell • If the target is internalized (i.e. folate receptors) the nanoparticle is carried to the intracellular environment • If the target is not internalized (i.e. annexin A2) the delivery system has been engineered to release the nanoparticle at the surface of the cell allowing for endocytosis to occur

26. PSMA targeting under co-culture conditions Activated nanoparticles loaded with sulforhodamine 101 (red) were quenched and exposed to PSMA antibody. Following 1 hour, untargeted nanoparticles were exposed to a co-culture of PC-3 and LNCaP C4-2 cells under dymanic motion conditions for 30 minutes. No preferential uptake of nanoparticles is observed.

27. Targeted preferential uptake PSMA targeted nanoparticles were loaded with sulforhodamine 101 (red) and exposed to a co-culture of PC-3 and LNCaP C4-2 cells for 30 minutes under dynamic motion. Samples were fixed in paraformaldehyde and visualized through laser confocal microscopy. PC-3 cells are shown with yellow arrows, LNCaP C4-2 cells are shown with green arrows. It is evident that there is a preferential uptake of targeted nanoparticles to the LNCaP C4-2 cell line.

28. Intravitreal injection Potential convective current for vitreous Anatomy of the eye

29. Nanoparticles are capable of reaching the retinal cell layers Pig retinal section 4 days post-intra vitreal injection of nanoparticles. Nanoparticles were loaded with sulforhodamine 101 (red) and GFP plasmid DNA (green). The concentration of nanoparticles was 1 mg/75 mL. Nuclear visualization was performed using hematoxylin. The section shown is located in the posterior portion of the retina. Ganglion cell layer Outer Nuclear Layer Inner Nuclear Layer Retina

30. The ciliary body is located adjacent to the lens in the anterior portion of the eye One of the functions is the production of vitreal fluid It may be possible to use accumulation in the ciliary body as a drug reservoir for sustained release Issues of drug transport to the retina still remain Investigation of the ciliary body after intra-vitreal injection of nanoparticles Confocal image of the ciliary body from pig retinal sections. It appears that a higher accumulation of nanoparticles is occurring.

31. We are developing multi-phase nanoparticles for protection against oxidative damage to cells. We expect these nanoparticles to provide an immediate scavenging response to cellular oxidative stressors (first phase). In the second phase we are going to provide sustained long-term protection against oxidative damage. We anticipate applications of these nanoparticles in the areas of glaucoma, ischemic recovery (stroke victims) and COPD. Preliminary data suggest that we are able to reduce the effective dose of a known protective agent by 25 fold. Reduction of reactive oxygen species in various disease states

32. IAA is an chemical inducer of reactive oxygen. Treatment was with 8 mM N-acetyl cysteine was administered at a concentration of 5 mM N-acetyl cysteine was conjugated to the surface of the nanoparticle at a concentration of 0.5 mM Visualization was performed 20 hours after IAA induction IAA Control Nanoparticle N-acetyl cysteine Protection of retinal ganglion cells from reactive oxygen

33. Acknowledgements: • Dr. Arthur Braden • Dr. Anindita Mukerjee • Mallika Valapala jvishwan@hsc.unt.edu