Calcium Phosphate Nanocolloids Calcium Phosphate Nanocolloids f Bi i i d D D lif Bi i i d D D lifor Bioimaging and Drug Delivery for Bioimaging and Drug Delivery

James H. Adairwith

E.I. Altinoglu, B. M. Barth, H.S. Muddana, T.T. Morgan, T.J. Russin, M.R. Parette*, J. Kaiser, T. Tabouillot, A. Tabakovic, C. McGovern, S. , , , , ,Shanmugavelandy, P.C. Eklund, J.K. Yun, Y. Heakal, A. Sharma, P.J. Butler, G.P. Robertson, V. Ruiz-Velasco, J. Smith and M. Kester

Materials Science & Engineering, Bioengineering, Physics, Anesthesiology and Surgery, and Pharmacology,

Penn State University, University Park and Hershey, PA

* K t N B l b PA* Keystone Nano, Boalsburg, PA

Cancer Nanotechnology, Going Small for Big Advances- Using Nanotechnology to Advance Cancer Diagnosis, Prevention& Treatment, NIH Publication No. 04-5489 (2004).

Ceramic and Composite Materials CenterAn NSF Industry/University Cooperative Research Center

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OverviewOverviewBackground: Calcium PhosphateCalcium Phosphate NanocompositeCalcium Phosphate Nanocomposite

Particle (CPNP)•in vitro and in vivo Bioimaging and Drugin vitro and in vivo Bioimaging and Drug Delivery – Breast and Pancreatic CancersWhat is Photodynamic Therapy?What is Photodynamic Therapy?What is Deep Tissue PDT?Photodynamic Therapy of Human BreastPhotodynamic Therapy of Human Breast

Cancer and LeukemiaSummary and ConclusionsSummary and Conclusions

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Features of a Useful Nanoparticle Features of a Useful Nanoparticle Bi i i d D D liBi i i d D D liBioimaging and Drug DeliveryBioimaging and Drug Delivery

I h tl t i t i l dInherently non-toxic materials and degradation products

Bi l i ll t i i ll t ll dBiologically or extrinsically controlled release of therapeutic agents

S ll ti l di t 20 200Small particle diameter: 20-200nmColloidally stable in physiological

diti ( H i i t thconditions (pH, ionic strength, macromolecular interactions, and t ttemperature

Can be targeted to cell/tissue of choice 3

Calcium Phosphate Nanocomposite Particle Calcium Phosphate Nanocomposite Particle O iO iOverviewOverview

Calcium phosphosilicate matrix material 20 nm

Imaging agents and or drugs contained in matrix

ICG CPNPsICG‐CPNPs

PEG, Citrate, Avidin, Anti‐Bodies (e.g., anti‐ on exterior permit targeting

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Calcium Phosphosilicate Nanocomposite Particle (CPNPs): A Broad Platform for Seeking, Treating and Tracking Human DiseaseJ.H. Adair, P.J. Butler, P.C. Eklund, M. Kester, J. Smith and collaborators, , , ,Materials Science & Engr., Bioengineering, Physics, Pharamacology, Surgery

20 nmCalcium phosphosilicate matrix Bi i i /bi b th

Features Research Opportunitiesmaterial: bioresorbable, dissolution on demand within cells, hiigh particle number and encapsulated active agent concentrations

Bioimaging/bioassays both in vitro and in vivo of healthy and transformed cells and tissues

ICG‐CPNPs

concentrations

Imaging agents and or drugs contained in non‐porous matrix

Targeted or untargeted (i.e., EPR) delivery of chemo‐therapeutics to cancer (breastICG CPNPs

Colloidally stable for extended times in physiological fluids: phosphate buffered saline, cell culture media blood

therapeutics to cancer (breast, pancreatic cancer, leukemia)

Potentially, long term

Citrate, PEG, Avidin, Anti‐Bodies (e.g., anti‐CD 71), other molecules (e g Gastrin epitopes) bioconjugated

culture media, blood

Deep tissue (>6cm) photo‐

monitoring for cancer in general patient populuion

(e.g., Gastrin epitopes) bioconjugated on surface permitting targeted delivery

Deep tissue (>6cm) photodynamic therapy of cancer with novel ICG‐CPNPs

Coming SoonFluorescent NanoJacketsFluorescent NanoJackets

Dye λex/λem Surface Functionalization

Cascade Blue 400/425COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, Avidin

COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, Fluorescein 475/515

( ), , , ,Avidin

Red (Rh WT) 530/555COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, AvidinRed (Rh WT) 530/555 Avidin

Indocyanine Green 760/875COOH (Citrate), PEG‐OH, PEG‐Maleimide, 4 arm PEG, Avidin

J.H. Adair and M. Kester are CSO and CMO for Keystone Nano, respectively6

ResorbableResorbable Calcium Phosphate Calcium Phosphate -- NanoCompositeNanoComposite ParticlesParticles

ACP

Tailorable Solubility→ Time Release→ Time Release

Tumor

de Groot et al., Chemistry of Calcium Phosphate Bioceramics, 19807

InIn‐‐Vitro Vitro and and inin‐‐VivoVivo Drug Delivery and Fluorescent ImagingDrug Delivery and Fluorescent Imaging

Calcium Phosphate Calcium Phosphate Nanocomposite ColloidsNanocomposite Colloids

H. Muddana, T. Tabouillot, T.M. Morgan, J.H. Adair, P.J. Butler, NanoLetters, 20088

Melanoma Cell Imaging and ApoptosisMelanoma Cell Imaging and Apoptosis(a)

Rh WT CPNPsMerged Rh WT CPNPs with DAPI StainingDAPI Fluorescent 50 μm

(b)(b)

DAPI Fluorescent Rh WT CPNPsMerged Rh WT CPNPs with Cer10and DAPI Staining50 μm

In vitro effect of Cer10‐CPNPs on melanoma cell survival and viability. Representative photomicrographs of culturedmelanoma UACC 903 cells exposed to Rh WT‐CPNPs without (a) and with (b) Cer10. Fluorescent Cer10‐CPNPs, unlikecontrol CPNPs, induce melanoma cell death. (c) MTS cytotoxicity assay demonstrating dosage responsive cytotoxicactions of Cer10 CPNPs Control CPNPs exhibit modest cytotoxicity at the highest particle number concentration Valuesactions of Cer10‐CPNPs. Control CPNPs exhibit modest cytotoxicity at the highest particle number concentration. Valuesare mean ± SEM for three independent experiments, each experiment replicated in triplicate.

M. Kester and G.P. Robertson et al., NanoLetters, 2008.9

Light propagationin biological tissue

• “Therapeutic Window” – Minimum in absorption

• Highly diffuse scatterer!

• How does light propagateHow does light propagatein a dense scattering medium?medium?

• Radiative Transport EquationDiffusion Approximation– Diffusion Approximation

T. J. Russin et al., in preparation.10

CPNPs: BioPhotonicsEklund Russin Altinoglu Adair

• Photodynamic Therapy

Eklund, Russin, Altinoglu, Adair

hν

N d d l f d th li it

ICG (S0 state) Oxygen (T1 state)ICG (S1 state) Oxygen (S0 state)ICG (T1 state)

– Need model for depth limits• Fluorescent imaging

– Targeting to breast tissueTargeting to breast tissue achieved in nude miceAltinoglu et al., ACS Nano, 2008

N d d l t di t– Need a model to predict imaging capabilities

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Dissection 10 minutes post‐injection

ICG‐CPNP‐PEG Sample

Images show exclusive hepatic clearance of PEGylated CPNPs No significant sequestering or uptake by other tissues or membranes

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B.M. Barth, C. McGovern, R. Sharma, E.I. Altinoglu, J.H. Adair, M. Kester, J. Smith

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What is Photodynamic Therapy?• Photodynamic therapy (PDT) is a non‐invasive cancer treatment option with minimal side effects

• Involves activation of a non‐toxic photosensitizer by an appropriate light source in the

f l lpresence of molecular oxygenhttp://www.easternsuburbsderm.com.au/pdt.htm

Current Limitations•Penetration Depth: ~50μm with red light

http://sterileeye.com/

•Systemic Patient Photo-Sensitivity 15

What is PDT?What is PDT?

• Upon irradiation (absorption 5

p ( pof photons), the photosensitizer is excited to a higher energy state and

01235

S1

1235

T1

e ecay

Intersystem crossing

Vibrational relaxations

a higher energy state and upon return to its ground state, transfers this energy

0

Ene

rgy (Singlet Oxygen)

Abs

orpt

ion

Fluo

resc

ence

on-ra

diat

ive

de

Energy transfer

to oxygen

01235

S0

Photosensitizer (ICG) Oxygen

(Molecular Oxygen)

No

Photosensitizer (ICG) Oxygen

• Both unstable radicals and highly reactive singlet oxygen are produced, which cause localized, lethal cellular p , ,damage in milliseconds

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Photosensitizer: Indocyanine Green (ICG)• Near IR emitting fluorophore

Excitation: 785 nm Emission: 820 nm

Photosensitizer: Indocyanine Green (ICG)

– Excitation: 785 nm Emission: 820 nm

• FDA approved, non‐toxic

• First proposed for PDT by Fickweiler• First proposed for PDT by Fickweiler et al. (1997)– Showed ICG more effective thanShowed ICG more effective than Photofrin

– Fickweiler et al., Indocyanine green: Intracellular uptake and phototherapeutic effects in vitro, J Photochem Photobiol B 38 (1997) 178‐183

• NIR allows deeper penetration depths– Biological transmission window in NIR Fig.: Hamblin MR, Demidova TN. In:

Mechanisms for low light therapy HamblinBiological transmission window in NIR Mechanisms for low-light therapy. Hamblin, MR, Waynant, RW, Anders, J (Eds.) (SPIE, Bellingham, WA, 2006) 614001-614013

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Experimental Layout forhPenetration Depth & Bioimaging

Porcine tissue inhanging clamp

25°Photodiode power meter

785 nm laser diode Infrared Camera

25°

785 nm laseron variableangle mount

20 cm

53.6 mmEmitted light

Optical table

solid angle Ω

Porcine tissue

CPNP sample in 50 μL well

Optical table

T. J. Russin et al., in preparation.18

Penetration Depth for h d h

• Indocyanine Green PDTPhotodynamic Therapy

– Radiation dose of 48 J/cm2

– μeff (pork)= 1.44

– μeff (breast)= 0.739 S. Fickweiler et al., J. Photochem. Photobiol. B-Biol., 38 (1997).

19T. J. Russin et al., in preparation.

Deep Tissue Photodynamic TherapyDeep Tissue Photodynamic Therapy (DTPDT)

with ICG‐CPNPs

B. Barth, S. Shanmugavelandy, E.I. l l dAltinoglu, S. Knupp, J.H. Adair,

M. Kester

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Initial Animal TrialsInitial Animal Trials

• MDA‐MB‐231 human breast adenocarcinoma3 u a b east ade oca c o a• Nude mouse model; 5 groups (n=4)

1 ICG-CPNP-PEG 4 Free ICG Control

• 100 uL tail vein injection

1. ICG-CPNP-PEG 4. Free ICG Control2. ICG-CPNP-COOH 5. PBS Blank Control3. Ghost-CPNP-PEG

100 uL tail vein injection– 2.5e‐7 M ICG absorption value

• Single ~0.002 and 50 J/cm2 (3 minutes)Single 0.002 and 50 J/cm (3 minutes) irradiation 24 hrs post injection– 70 mW, 785 nm laser diode

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Initial In Vivo Photodynamic Therapy of Breast Cancer Tumors ~0.002 J/cm2

ICG-CPNP Photodynamic TherapyMDA-MB-231 Breast Cancer Xenografts in Nude Mouse Model

2500

3000

g

1500

2000

ativ

e Tu

mor

Siz

e GhostCOOHPEG

0

500

1000

Rel

a

• Kodak In Vivo FX Imager used to deliver light to tumors

• About 1/25000th of reported photodynamic therapy light doses

0 5 10 15 20 25 30 35 40 45

Days

• About 1/25000th of reported photodynamic therapy light doses

• Light given 24 hours post‐injection22

Second Trial In Vivo Photodynamic Therapy of Breast Cancer Tumors 50 J/cm250 /

40

45

30

35

or S

ize

PBSGhost-CPNP-PEGFree-ICGICG CPNP COOH

20

25

Rel

ativ

e Tu

mo ICG-CPNP-COOH

ICG-CPNP-PEG

10

15

R

0

5

0 5 10 15 20 25 30 35 40 45 50 550 5 10 15 20 25 30 35 40 45 50 55

Days23

Non Solid Tumors ‐ LeukemiaPDT Targeted to Spleen

PDT of KG-1 Xenografted Nude Mice

70Ghost CPNP PEG

50

60

per u

l

Ghost-CPNP-PEG

ICG-CPNP-PEG

C6-CPNP-PEG

20

30

40

1,00

0 W

BC

ICG- + C6-CPNP-PEG

0

10

20X 1

00 5 10 15 20 25

Days 24

SummarySummary

CPNPs encapsulate imaging agents, therapeutics or both, permitting both in vitro and in vivo simultaneous detecting, delivery, and monitoring for cellular uptakedelivery, and monitoring for cellular uptake

Drug delivery of both hydrophobic and hydrophilic drugs is improved by the intercellular protection afforded by the calcium phosphate during transport to the cell combined with the on‐demand dissolution of the CP and intracellular drug delivery

Enhanced permeability and retention (EPR) in solid humanEnhanced permeability and retention (EPR) in solid human breast cancer tumors and pancreatic tumors is observed in vivo for a pegylated and targeted near infra‐red – CPNP with the

i f d ti i i f l ipromise of deep tissue imaging of lesions

Photodynamic therapy with deep soft tissue penetration holds much promise in the treatment of cancer p

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The EndThe EndThank You for Your Patience

Questions?

C t C Si dl kiCourtesy C. Siedlecki

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