1. PhotoImmuno-NanoTherapy (PINT) Mark Kester G. Thomas Passananti Professor of Pharmacology Director, Penn State Center for NanoMedicine and Materials

2. PhotoImmuno-NanoTherapy (PINT) Mark Kester Keystone Nano, Inc State College, PA Chief Medical Officer

3. PhotoImmuno-NanoTherapy (PINT) Mark Kester Keystone Nano, Inc State College, PA Chief Medical Officer Harnessing the Power of Light for Theranostics

4. NanoJackets Calcium Phosphate NanoParticles (CPNPs)

5. 5 Nanojackets are Molecular Smart Bombs; Encapsulated components are released as a function of pH NanoJackets

6. TEM of ICG-Doped CPNPs of 16nm mean diameter

7. Limitations of free ICG Limitations of free ICG Fluorescence instability in physiological environmentsFluorescence instability in physiological environments Dimerization leads to fluorescent quenching Dimerization leads to fluorescent quenching Protein binding causes absorption shifts Protein binding causes absorption shifts Rapid elimination from the bodyRapid elimination from the body Plasma t 1/2 = 3-4 minutes Plasma t 1/2 = 3-4 minutes Taken up exclusively by hepatic parenchymal cells Taken up exclusively by hepatic parenchymal cells Subsequently secreted entirely into the bile Subsequently secreted entirely into the bile Benefits of NanoJacket Encapsulation Benefits of NanoJacket Encapsulation Monomer caging prevents aggregate formationMonomer caging prevents aggregate formation Solvent protection improves photostabilitySolvent protection improves photostability Surface passivation affords long-term in vivo circulationSurface passivation affords long-term in vivo circulation Tumor localization via the EPR-EffectTumor localization via the EPR-Effect

8. Colloidal Stability of CPNPs in PBS

9. Absorption and Fluorescence Spectra of Free ICG and ICG-Doped CPNPs in Aqueous Solution

10. Fluorescence Lifetime of Free ICG and ICG-CPNPs

11. Comparative Spectral Effects of Various Solvents on the Emission Response of Free ICG and ICG-CPNPs

12. EPR effect 12

13. In Vivo Administration of PEGylated ICG-CPNPs l Accumulation within tumors via enhanced permeation retention effect l Internalization into tumor cells via endosome pathway

14. Comparative Fluorescence Signal Intensity as Function of Depth in Porcine Muscle Tissue

15. ACB DGFE Figure 13. Near-infrared imaging of systemically delivered ICG-loaded CPNPs targeted to gastrin receptors. Athymic nude mice, with orthotopic BXPC-3 (pancreatic cancer) xenografts, were administered ICG-loaded CPNPs via tail vein injection. All injections contained equivalent ICG concentrations. Mice were imaged for particle localization using a Kodak In Vivo FX imaging station. (A) Both mice received untargeted, PEGylated CPNPs loaded with ICG, and images were taken at 24 hours post-injection. (B) Mice received either pentagastrin-avidin-coupled CPNPs loaded with ICG (left mouse), or gastrin 10 - PEG-coupled CPNPs loaded with ICG (right mouse), and images were taken at 7 hours post-injection. (C) Mice in panel B, however images were taken at 24 hours post-injection. (D) Excised stomach from mouse receiving pentagastrin- avidin-coupled CPNPs loaded with ICG. (E) Excised pancreas from mouse receiving pentagastrin-avidin-coupled CPNPs loaded with ICG. (F) Excised stomach from mouse receiving gastrin 10 -PEG-coupled CPNPs loaded with ICG. (G) Excised pancreas from mouse receiving gastrin 10 -PEG-coupled CPNPs loaded with ICG. Active Targeting

16. Theory of Photodynamic Therapy Requirements: photosensitizer, light, and oxygen Requirements: photosensitizer, light, and oxygen Photosensitizer is excited at appropriate wavelength from ground singlet state to excited singlet state Photosensitizer is excited at appropriate wavelength from ground singlet state to excited singlet state Excited photosensitizer undergoes intersystem crossing to excited triplet state Excited photosensitizer undergoes intersystem crossing to excited triplet state Energy transfer to molecular oxygen (ground triplet state) allows photosensitizer to relax to ground singlet state Energy transfer to molecular oxygen (ground triplet state) allows photosensitizer to relax to ground singlet state Oxygen is now in excited singlet state Oxygen is now in excited singlet state Singlet oxygen is highly reactive Singlet oxygen is highly reactive

17. Current Limitations Low quantum yield of photosensitizers Low quantum yield of photosensitizers Short lifetimes of photosensitizers Short lifetimes of photosensitizers Lack of specific targeting Lack of specific targeting Inability to penetrate sufficient light to targets Inability to penetrate sufficient light to targets Poor understanding of mechanisms linking singlet oxygen generation to cancer cell death Poor understanding of mechanisms linking singlet oxygen generation to cancer cell death

18. Photodynamic Therapy Utilizing ICG-Loaded Calcium Phosphate Nanoparticles Prevents Breast Cancer Growth In Vivo (Single IV Injection and Single NIR Treatment) MDA-MB-231 Tumors (Nude Mice) 410.4 Tumors (BalbCJ Mice)

19. PINT Extends Survival of Leukemic Mice (3 IV Injections and Splenic NIR Treatment) Wehi-GFP Myeloid Leukemia (BalbCJ Mice)

20. In Vitro Photodynamic Therapy With ICG- CPNPs

21. Myeloid Derived Suppressor Cells Decrease 5-Days Post-NIR Treatment Ghost-CPNP-PEG ICG-CPNP-PEG PBS

22. PINT Reduces MDSCs in 410.4 Tumor-Bearing BALB/cJ Mice 22

23. Ghost-CPNP-PEG ICG-CPNP-PEG NK DC ** DC cells moderately increase with treatment PINT Increases Natural Killer Cells in MDA-MB-231 Tumor-Bearing Nude Mice 23

24. PINT Increases Dendritic and Natural Killer Cells in 410.4 Tumor-Bearing BALB/cJ Mice 24

25. Adoptive Transfer

26. Injection of Culture-(PINT) Treated Cells in Tumor-Established Nude Mice 26

27. Conclusion Nanotechnology has the potential to “deliver” the promise of light-based pharmaceutics Nanotechnology has the potential to “deliver” the promise of light-based pharmaceutics

28. Acknowledgements Brian Barth Tom Stover James Kaiser Todd Fox Onar Unal Yasser Haekal Tony Brown Sean O’Neil Kristie Houck Murali Nagarajan Sriram Shamnugavelandyu Lindsey Rylund Tom Loughran Xin Liu Jill Smith James Adair Sarah Rouse Erhan Antinouli Tom Morgan Peter Eklund Peter Butler MK is Founder and CMO for Keystone Nano, Inc NanoJackets have been licensed by Penn State Research Foundation to Keystone Nano, Inc., State College, PA