PhotoImmuno-NanoTherapy (PINT)PhotoImmuno-NanoTherapy (PINT)

Mark KesterMark Kester

G. Thomas Passananti Professor of PharmacologyG. Thomas Passananti Professor of PharmacologyDirector, Penn State Center for NanoMedicine and MaterialsDirector, Penn State Center for NanoMedicine and Materials

PhotoImmuno-NanoTherapy (PINT)

Mark Kester

Keystone Nano, Inc

State College, PA

Chief Medical Officer

PhotoImmuno-NanoTherapy (PINT)

Mark Kester

Keystone Nano, Inc

State College, PA

Chief Medical Officer

Harnessing the Power of Light for Theranostics

NanoJacketsNanoJacketsCalcium Phosphate NanoParticles (CPNPs)Calcium Phosphate NanoParticles (CPNPs)

5

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

NanoJackets

TEM of ICG-Doped CPNPs of 16nm mean diameter

Limitations of free ICGLimitations of free ICG

• Fluorescence instability in physiological environmentsFluorescence instability in physiological environments Dimerization leads to fluorescent quenchingDimerization leads to fluorescent quenching Protein binding causes absorption shiftsProtein binding causes absorption shifts

• Rapid elimination from the bodyRapid elimination from the body Plasma tPlasma t1/21/2

= 3-4 minutes= 3-4 minutes Taken up exclusively by hepatic parenchymal cellsTaken up exclusively by hepatic parenchymal cells Subsequently secreted entirely into the bileSubsequently secreted entirely into the bile

Benefits of NanoJacket EncapsulationBenefits of NanoJacket Encapsulation

• Monomer caging prevents aggregate formationMonomer caging prevents aggregate formation• Solvent protection improves photostabilitySolvent protection improves photostability• Surface passivation affords long-term Surface passivation affords long-term in vivoin vivo circulation circulation• Tumor localization via the EPR-EffectTumor localization via the EPR-Effect

Colloidal Stability of CPNPs in PBS

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

Fluorescence Lifetime of Free ICG and ICG-CPNPs

Comparative Spectral Effects of Various Solvents on the Emission Response of Free

ICG and ICG-CPNPs

EPR effectEPR effect

12

In Vivo Administration of PEGylated In Vivo Administration of PEGylated ICG-CPNPsICG-CPNPs

Accumulation within tumors via enhanced permeation retention effect

Internalization into tumor cells via endosome pathway

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

A CB

D GFE

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 gastrin10-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 gastrin10-PEG-coupled CPNPs loaded with ICG. (G) Excised pancreas from mouse receiving gastrin10-PEG-coupled CPNPs loaded with ICG.

Active Targeting

Theory of Photodynamic Theory of Photodynamic TherapyTherapy

Requirements: photosensitizer, light, and oxygenRequirements: photosensitizer, light, and oxygen

Photosensitizer is excited at appropriate wavelength Photosensitizer is excited at appropriate wavelength from ground singlet state to excited singlet statefrom ground singlet state to excited singlet state

Excited photosensitizer undergoes intersystem Excited photosensitizer undergoes intersystem crossing to excited triplet statecrossing to excited triplet state

Energy transfer to molecular oxygen (ground triplet Energy transfer to molecular oxygen (ground triplet state) allows photosensitizer to relax to ground singlet state) allows photosensitizer to relax to ground singlet statestate

Oxygen is now in excited singlet stateOxygen is now in excited singlet state

Singlet oxygen is highly reactiveSinglet oxygen is highly reactive

Current LimitationsCurrent Limitations

Low quantum yield of photosensitizersLow quantum yield of photosensitizers

Short lifetimes of photosensitizersShort lifetimes of photosensitizers

Lack of specific targetingLack of specific targeting

Inability to penetrate sufficient light to Inability to penetrate sufficient light to targetstargets

Poor understanding of mechanisms linking Poor understanding of mechanisms linking singlet oxygen generation to cancer cell singlet oxygen generation to cancer cell deathdeath

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)

PINT Extends Survival of Leukemic Mice

(3 IV Injections and Splenic NIR Treatment)

Wehi-GFP Myeloid Leukemia(BalbCJ Mice)

In Vitro Photodynamic Therapy With ICG-In Vitro Photodynamic Therapy With ICG-CPNPsCPNPs

Myeloid Derived Suppressor Cells Decrease

5-Days Post-NIR TreatmentG

ho

st-

CP

NP

-PE

GIC

G-C

PN

P-P

EG

PB

S

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

22

Ghost-CPNP-PEG

ICG-CPNP-PEG

NK

NK

DC

DC

** DC cells moderately increase with treatment

PINT Increases Natural Killer Cells in MDA-MB-231 Tumor-Bearing Nude Mice

23

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

24

Adoptive Transfer

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

26

ConclusionConclusion

Nanotechnology has the Nanotechnology has the potential to “deliver” the potential to “deliver” the promise of light-based promise of light-based pharmaceuticspharmaceutics

AcknowledgementsAcknowledgementsBrian BarthBrian Barth

Tom StoverTom StoverJames Kaiser James Kaiser Todd FoxTodd FoxOnar UnalOnar UnalYasser HaekalYasser HaekalTony BrownTony BrownSean O’NeilSean O’NeilKristie HouckKristie HouckMurali NagarajanMurali NagarajanSriram ShamnugavelandyuSriram ShamnugavelandyuLindsey RylundLindsey Rylund

Tom LoughranTom LoughranXin LiuXin LiuJill SmithJill Smith

James AdairJames Adair

Sarah RouseSarah RouseErhan AntinouliErhan AntinouliTom MorganTom MorganPeter EklundPeter EklundPeter ButlerPeter Butler

MK is Founder and CMO for Keystone MK is Founder and CMO for Keystone Nano, Inc Nano, Inc

NanoJackets have been licensed by NanoJackets have been licensed by Penn State Research Foundation Penn State Research Foundation to Keystone Nano, Inc., State to Keystone Nano, Inc., State College, PACollege, PA