MRI Assessment of Prostate-Specific Membrane Antigen (PSMA

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MRI Assessment of Prostate-Specific Membrane Antigen (PSMA) Targeting by a PSMA-Targeted Magnetic Nanoparticle: Potential for Image-Guided Therapy Ethel J Ngen, Babak Behnam Azad, Srikanth Boinapally, Ala Lisok, Mary Brummet, Desmond Jacob, Martin G Pomper, and Sangeeta R. Banerjee Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Molecular Pharmaceutics

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MRI Assessment of Prostate-Specific Membrane Antigen (PSMA)

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Targeting by a PSMA-Targeted Magnetic Nanoparticle: Potential for

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Image-Guided Therapy

4 5

Ethel J. Ngen1, Babak Benham Azad1, Srikanth Boinapally1, Ala Lisok1, Mary Brummet1,

6

Desmond Jacob1, Martin G. Pomper1,2, Sangeeta R. Banerjee1,2,3*

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1

The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins

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University School of Medicine, Baltimore, MD 21287, USA.

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2

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Medicine, Baltimore, MD 21231, USA.

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3

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Institute, Baltimore, Maryland 21205, USA.

The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of

The F. M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Research

15 16 17

*Correspondence

should be addressed to: Sangeeta R. Banerjee, Ph.D. ([email protected])

18 19 20 21 MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Table of Content (TOC) Graphic

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MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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Abstract

2

Magnetic nanoparticle (MNP)-induced hyperthermia is currently being evaluated for

3

localized prostate cancer. We evaluated the feasibility of tumor-selective delivery of prostate-

4

specific membrane antigen (PSMA)-targeted MNPs in a murine model with high-resolution

5

magnetic resonance imaging (MRI) after intravenous administration of MNPs at a concentration

6

necessary for hyperthermia. A PSMA-targeted MNP was synthesized and evaluated using T2-

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weighted MRI after intravenous administration of 50 mg/kg of the MNP. Significant contrast

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enhancement (P < 0.0002, n = 5) was observed in PSMA(+) tumors compared to PSMA(-) tumors

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24 h and 48 h after contrast agent administration. Mice were also imaged with near-infrared

10

fluorescence imaging, to validate the MRI results. Two-photon microscopy revealed higher

11

vascular density at the tumor periphery in keeping with peripheral accumulation of PSMA-targeted

12

MNPs. These results suggest that the delivery of PSMA-targeted MNPs to PSMA(+) tumors is

13

both actively targeted and passively mediated.

14 15 16

Keywords: prostate cancer, MNP, magnetic resonance imaging, optical imaging, magnetic

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hyperthermia therapy

18 19 20




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1. Introduction

2

Prostate cancer (PC) is the most common non-cutaneous cancer among men in the United

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States.1 Although PC screening via the measurement of prostate specific antigen (PSA) levels in

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serum has improved PC detection, it has also resulted in over-treatment, especially in patients with

5

a low risk of developing aggressive and metastatic disease.2, 3 Generally, PC confined within the

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prostate capsule is treated by radical prostatectomy and radiation therapy.4 However, despite the

7

development of nerve-sparing radical prostatectomy and stereotactic radiotherapy, therapeutic

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interventions still result in substantial morbidity.5

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Although active surveillance has been effective in monitoring and deferring treatment for

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patients with low-risk disease, those with intermediate-risk disease enrolled in active surveillance

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programs may have compromised survival outcomes compared to intermediate-risk patients who

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receive treatment.6, 7 Accordingly, minimally invasive and less morbid focal therapies are being

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developed for treatment of patients with intermediate-risk PC. Focal therapies are also being

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developed for patients with recurrent, aggressive, castration-resistant PC localized within the

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prostate capsule.8, 9

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A hallmark of focal therapy is targeted destruction of tumors and preservation of

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surrounding healthy tissues.8,

9

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therapy that is being evaluated to address localized, aggressive PC.10,

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hyperthermia, MNPs provide a means to deliver high concentrations of encapsulated/conjugated

20

chemotherapeutic agents and genetic material specifically to the tumor microenvironment, while

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minimizing systemic toxicity.12, 13 MNPs also provide a means of image guidance by magnetic

22

resonance imaging (MRI) during therapy.14, 15 To ensure localized therapy, targeted MNPs with

23

appropriate pharmacokinetics are necessary.16, 17

Magnetic nanoparticle (MNP)-induced hyperthermia is a focal 11

In addition to



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Prostate-specific membrane antigen (PSMA) is a type II integral membrane glycoprotein

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that is overexpressed in PC, especially aggressive, castration-resistant disease.18, 19 Several groups

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have exploited different strategies to target PSMA expression in aggressive PC for imaging and

4

therapy. Those strategies include: PSMA-targeted monoclonal antibodies

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fragments such as single-chain antibody fragments 22, 23 and nanobodies 24 as well as aptamers.25,

6

26

7

been effective20, 27, antibody-drug conjugates have disadvantages compared to low-molecular-

8

weight PSMA-targeting ligands.27 Those disadvantages include: potential immunogenicity and

9

slow clearance rates, which could lead to systemic toxicity.28, 29 Previously, we and others have

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demonstrated both preclinically and clinically the feasibility of using low-molecular-weight

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targeting ligands such as Glu-Lys-ureas to access PSMA expression in PC.30-33 Here, we

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hypothesize that PSMA targeting using a low-molecular-weight ligand, previously tested Glu-Lys-

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urea-suberate ((((S)-1-carboxy-5-(7-carboxyheptanamido)pentyl)carbamoyl)-L-glutamic acid),

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could also be used to enhance the delivery and retention of MNPs to PC at MNP concentrations

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necessary for MRI detection and hyperthermia.

20, 21

and antibody

Although PSMA targeting using high-molecular-weight targeting entities such as antibodies has

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Recently, direct correlations have been made between the iron content and the thermal and

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magnetic properties of MNPs.34, 35 MNPs ranging between 100 - 200 nm in diameter, with high

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iron content, and capable of specifically targeting tumors are highly desirable for MRI-guided

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hyperthermia.36

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We previously developed a PSMA-targeted MNP and demonstrated the feasibility of

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preferentially targeting PSMA(+) tumors compared to PSMA(-) tumors at low MNP

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concentrations.37 Here, we used the high spatial resolution of MRI to evaluate the feasibility of

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this MNP construct (Figure 1) to serve as a PSMA-targeted MRI contrast agent and preferentially MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



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accumulate in PSMA(+) tumors compared to PSMA(-) tumors, after the systemic administration

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of a high MNP dose (50-fold higher than we previously reported), needed for effective MRI

3

detection and hyperthermia.11 We also used the high spatial resolution of MRI to determine the

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intra-tumoral biodistribution pattern of the delivered PSMA-targeted MNPs. Finally, we used

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high-resolution multi-photon microscopy to elucidate the effects of intra-tumoral vascular patterns

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on the intra-tumoral delivery and biodistribution of PSMA-targeted MNP.

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8 9 10

Figure 1. Structure of a PSMA-targeted MNP. A low-molecular-weight PSMA-targeting ligand (T), Glu-Lys-urea-suberate, was used to target PSMA.

11



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2. Experimental Section

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2.1 Materials and Reagents

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Starch-coated, amine-surface-modified bionized nanoferrite (BNF) particles (80 nm in

5

diameter) were obtained from Micromod Partikeltechnologie GmbH (Rostock, Germany) and

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were used as the magnetic nanoparticle (MNP) core. LI-COR IR Dye® 800CW, obtained from LI-

7

COR Biosciences (Lincoln, NE), was used as an optical imaging moiety. All magnetic separations

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were carried out using a DynaMag™-Spin magnet, obtained from Thermo Fisher Scientific,

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(Waltham, MA).

10 11

2.2 Instrumentation

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Size and ζ-potential measurements were performed using a Malvern ζ-sizer Nano ZS-90,

13

obtained from Malvern Instruments Ltd (Worcestershire, UK). MRI scans were performed on a

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Bruker Biospec 11.7T, horizontal bore scanner, equipped with a quadrature proton resonator

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radiofrequency coil (RF RES 500 1H 075/040 QSN TR), for 500 MHz MR systems (Billerica,

16

MA). Paravision 6.1.0 software was used for image acquisition. Phantom and small animal optical

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images were acquired using a LI-COR Pearl® Trilogy imaging system (Lincoln, NE). Optical

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microscopy images were acquired using a Nikon Eclipse 80i microscope, equipped with either a

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Nikon DS-Fi1 bright field camera or a Nikon DS-Qi1Mc dark field camera. NIS-Elements BR 3.2

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software and National Institute of Health (NIH) ImageJ software were used for image processing.

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Multi-photon microscopy experiments were performed on an Olympus Laser Scanning FV1000

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microscope equipped with a 25xw/1.05XLPLN MP lens and obtained from Olympus Corporation

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(Center Valley, PA). MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



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2.3 PSMA-Targeted Magnetic Nanoparticle (MNP) Functionalization PSMA-targeted MNPs were functionalized by conjugating amine-surface-modified bionized

3

nanoferrite

particles

to

((((S)-1-carboxy-5-(8-((2,5-dioxopyrrolidin-1-yl)oxy)-8-

4

oxooctanamido)pentyl)carbamoyl)-L-glutamic acid via NHS-ester/amine chemistry in an aqueous

5

buffer (Supplementary Scheme S1), as previously described.37 Briefly, starch-coated and amine-

6

surface-modified MNPs, (amine density = 11 - 12 nmol/mg of Fe) were added to 0.01 M phosphate

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buffered saline (PBS) at pH 7.0 to a final concentration of 6.0 mg of Fe/mL. A 50-fold molar

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excess

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oxooctanamido)pentyl)carbamoyl)-L-glutamic acid was then added to the MNP suspension and

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swirled on a rotary shaker for 2 h at room temperature. The urea-conjugated MNPs were then

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purified by magnetic separation using a DynaMag™-spin magnet and rinsed thrice with 0.01 M

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PBS. The MNPs were next reformulated in 0.01 M PBS at a concentration of 6.0 mg of Fe/mL.

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Next the urea-conjugated MNPs were PEGylated via conjugation to an N-hydroxylsuccinimide

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(NHS) bifunctionalized polyethylene glycol linker (NHS-PEG1000-NHS) obtained from Creative

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PEG Works (Durham, NC). Briefly, a 50-fold molar excess of the NHS-PEG1000-NHS

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crosslinker was added to the MNP-urea conjugates in 0.01 M PBS and the suspension was swirled

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on a rotary shaker for 2 h at room temperature. The PEGylated-urea-conjugated MNPs were then

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purified by magnetic separation as described above. A LI-COR IR Dye® 800CW was next

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conjugated to the MNP-urea-PEGylated construct via NHS/amine chemistry, under the same

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conditions stated above. The MNPs were then purified by magnetic separation and reformulated

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in 0.01 M PBS at a final concentration of 2.5 mg of Fe/mL. Non-targeted MNPs were also

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functionalized with PEG as control MNPs (Supplementary Methods S1 and Supplementary

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Scheme S2), as previously described.37

of

(((S)-1-carboxy-5-(8-((2,5-dioxopyrrolidin-1-yl)oxy)-8-



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2.4 Nanoparticle Characterization

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The hydrodynamic diameter (Z-average), polydispersity index (PDI) and the ζ-potential of

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the functionalized nanoparticles were measured using a Malvern ζ-sizer Nano ZS-90. For size

4

measurements, the nanoparticles were suspended in water at 10 µg/mL and both the hydrodynamic

5

diameter and the PDI were measured at 25 °C, using disposable square polystyrene cuvettes. The

6

hydrodynamic diameter and PDI results were represented as the average value of three

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measurements, with each measurement consisting of 30 runs. The ζ-potentials of the respectively

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functionalized nanoparticles suspended in water (10 µg/mL) were next measured using disposable

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capillary cells. The results were represented as the average value of three measurements, with 20

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- 25 runs within each measurement.

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2.5 Phantom Magnetic Resonance Imaging (MRI) and Optical Imaging

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MRI phantoms of the PSMA-targeted MNPs were prepared by mixing nanoparticles in 0.01

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M PBS (100 µL) at different concentrations in 200 µL eppendorf tubes. The PSMA-targeted MNP

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suspensions were then imaged with both T2-weighted (T2-W) MRI and optical imaging. Phantom

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MRI experiments were performed on a Bruker Biospec 11.7T horizontal bore scanner, equipped

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with a quadrature proton resonator radiofrequency coil. T2-W images were acquired using a spin

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echo pulse sequence: rapid acquisition with refocused echoes (RARE); echo time (TE) = 10 ms;

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effective echo time (TEEff) = 30 ms; RARE factor = 8; repetition time (TR) = 2000 ms; number of

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averages (NA) = 2; field of view (FOV) = 25 × 25 mm; matrix size (MS) = 128 × 128 pixels; and

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slice thickness = 0.5 mm. Final image analyses were performed using the NIH ImageJ software.

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The amount of T2-W contrast enhancement from the MNPs in each phantom was calculated

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relative to that of a 0.01 M PBS phantom without MNPs. Phantom optical imaging experiments MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



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were performed using a LI-COR Pearl® Trilogy imaging system. Briefly, LI-COR IR Dye®

2

800CW on the PSMA-targeted MNPs was detected, using a fixed excitation wavelength of 785

3

nm and emission wavelength of 800 nm. The images were acquired at a resolution of 170 μm. The

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fluorescence signal from each region of interest on the original image, was then quantified using

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the LI-COR Pearl® Trilogy Imaging Software Version 2.0. The amount of optical contrast

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enhancement from the MNPs in each optical phantom was calculated relative to that of a 0.01 M

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PBS phantom without MNPs.

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2.6 Cell Lines

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Cell sublines derived from an androgen-independent PC3 human prostate tumor xenograft

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were used. Those sublines were genetically engineered either to express high levels of PSMA (PC3

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PIP cells) or not to express PSMA (PC3 flu cells), and were generously provided by Dr. Warren

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Heston (Cleveland Clinic).38, 39 Both PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer cell

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lines were cultured in RPMI 1640 medium (Corning Cellgro, Manassas, VA) containing 10% fetal

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bovine serum (FBS) (Invitrogen) and 1% penicillin-streptomycin (Corning Cellgro, Manassas,

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VA) as previously described.32, 38, 40 All cell cultures were maintained in 5% carbon dioxide (CO2),

17

at 37 °C in a humidified incubator.

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2.7 Mouse Model

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Animal experiments were conducted in accordance with guidelines of the Johns Hopkins

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University Animal Care and Use Committee. Six-to-eight week-old, male non-obese diabetic

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severe-combined immune-deficient (NOD-SCID) mice (Charles River Laboratories, Wilmington,

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MA), were used. Mice were subcutaneously inoculated with 1 × 106 PSMA(+) PC3 PIP and MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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PSMA(−) PC3 flu cells at the right and left upper flanks, respectively. Tumor growth was closely

2

monitored until the xenografts reached 8 to 10 mm in diameter, in at least one direction at which

3

time the mice were imaged.

4 5

2.8 In vivo MRI and Optical Imaging

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To determine the best time point for MRI acquisition, a small cohort of mice (n = 3), were

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catheterized and imaged before administration of the PSMA-targeted MNP. The PSMA-targeted

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MNP was then administered and the mice were imaged sequentially over 4 h, then at 24 h, 48 h

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and 72 h after contrast administration. Based on results of the preliminary study, the optimum

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imaging time points were obtained (0 h, 24 h and 48 h). A larger cohort of mice (n = 10), each

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bearing both a PSMA(+) PC3 PIP and a PSMA(−) PC3 flu tumor in the upper flanks, was then

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imaged before (0 h), and 24 h after the administration of the PSMA-targeted MNPs with MRI and

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optical imaging. Following in vivo imaging at 24 h, five of the mice were sacrificed and imaged

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ex vivo with optical imaging. The remaining five mice were imaged in vivo with both MRI and

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optical imaging, 48 h after the administration of the PSMA-targeted MNPs. Following in vivo

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imaging at the 48 h time point, the mice were sacrificed and imaged ex vivo with optical imaging

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at the 48 h time point.

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Animal MRI procedures were carried out under anesthesia using 2% isoflurane in an

19

oxygen and air mixture. Contrast enhanced T2-weighted (T2-W) MRIs were acquired following

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the bolus intravenous administration of the nanoparticles formulated in 0.01 M PBS and

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administered at a dose of 50 mg/kg. T2-W MRIs were acquired using a spin echo pulse sequence.

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T2-W MRI sequence: rapid acquisition with refocused echoes (RARE); echo time (TE) = 10 ms;

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effective echo time (TEEff) = 30 ms; RARE factor = 8; repetition time (TR) = 4000 ms; number of MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



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averages (NA) = 2; field of view (FOV) = 25 × 25 mm; matrix size (MS) = 128 × 128 pixels; and

2

slice thickness = 0.5 mm. Final image analyses were performed using NIH ImageJ software.

3

To evaluate the ability of PSMA-targeted MNPs to accumulate in PSMA(+) tumors

4

compared to PSMA(-) tumors (which served as internal negative controls for active PSMA

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targeting), we compared the ratio of contrast enhancement in PSMA(+) tumors to PSMA(-) tumors

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for each mouse, before the administration of the contrast agent (0 h), to that after the administration

7

of the contrast agent (at the 24 h and 48 h time points, respectively).The amount of contrast

8

enhancement in the respective tumors of each mouse, at each time-point, was analyzed using a

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modified black pixel analyses, as previously reported 41 and described in Supporting Methods S2

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and Supporting Figures S1.

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In vivo MRI findings were validated with optical imaging at each time point after MRI.

12

Mice were imaged using a LI-COR Pearl® Trilogy Small Animal Imaging system. Briefly, LI-

13

COR IR Dye® 800CW on the PSMA-targeted MNPs was detected, using a fixed excitation

14

wavelength of 785 nm and emission wavelength of 800 nm. The images were acquired at a

15

resolution of 170 μm. The fluorescence signal from each region of interest on the original image,

16

was then quantified using the LI-COR Pearl® Trilogy Imaging software version 2.0.

17 18

2.9 Immunohistochemistry

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Tumors were harvested from the mice after in vivo imaging. Tumors were fixed in 10%

20

neutral buffered formalin, paraffin-embedded and sectioned into 30 µm slices. Adjacent tumor

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sections were then stained with hematoxylin and eosin (H & E) and for PSMA expression. A

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standard immunohistochemistry procedure was used for PSMA staining. Slides were

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deparaffinized using a xylene rinse and an alcohol gradient rinse. The slides were then treated with MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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antigen retrieval buffer in a steam chamber. Next, the slides were blocked with 10% fetal bovine

2

serum for 30 min, then incubated overnight in a humidified chamber at 4°C, with an anti-PSMA

3

primary antibody (1:50 dilution, Clone 3E6, Dako, Carpinteria, CA). The slides were then rinsed

4

with PBS and incubated with an Alexa Fluor 569 goat anti-mouse secondary antibody (1:1000,

5

Invitrogen) for 60 min at room temperature. Next, the slides were rinsed with PBS and counter

6

stained with 4, 6-diamidino-2-phenylindole (DAPI).

7 8

2.10 Multi-photon Microscopy

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Mice bearing both PSMA(+) PC3 PIP and PSMA(−) PC3 flu tumors subcutaneously were

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intravenously administered 50 µL of a 2,000 kDa Texas Red conjugated dextran polymer (Sigma

11

Aldrich), prepared at a concentration of 1 µg/µL. The mice were then euthanized and the tumors

12

excised immediately, sectioned into 1 mm slices and mounted on an in-house built mounting slide.

13

The sections were then imaged using an Olympus Laser Scanning FV1000 MPE microscope using

14

an excitation wavelength of 820 nm and an emission of 615 nm.42 Each tumor slice was divided

15

into two main regions (Supplementary Figure S2): 1) The tumor periphery (< 2 mm from the tumor

16

margin) and 2) the tumor center (˃ 2 mm from the tumor margin). At least three fields of views

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(FOVs) were acquired from each of the two regions per tumor slice. A 25× objective was used to

18

acquire confocal z-stacks with FOV 508 × 508 μm2, and z-intervals of 5 μm.

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2.11 Statistical Analyses

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Data were presented as the mean ± standard deviation of at least three independent

22

experiments (technical replicates). Statistical comparisons were made using appropriate statistical




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tests. The results were considered statistically significant at P < 0.05, except when otherwise

2

stated.

3 4 5

3. Results

6 7

3.1 Characterization of the Functionalized Nanoparticles

8

PSMA-targeted MNPs were successfully functionalized, as previously reported.37 PSMA-

9

targeted MNPs possessed a hydrodynamic diameter of 147 ± 8 nm and a ζ-potential of -10.9 ± 0.3

10

mV (Table 1). These values were different from those obtained from the unmodified MNPs, with

11

a hydrodynamic diameter of 133 ± 1 nm and a ζ-potential of +24.0 ± 2.0 mV (Table 1). Diameter (nm)

Poly Dispersity Index

ζ-Potential (mV)

PSMA-Targeted MNP

147 ± 8

0.08 ± 0.03

-10.9 ± 0.3

Unmodified MNP

133 ± 1

0.15 ± 0.07

+24.0 ± 2.0

12 13

Table 1. Hydrodynamic diameters, polydispersity indices and ζ-potentials of PSMA-targeted

14

MNPs and unmodified MNPs, measured using a Malvern ζ-sizer.

15 16

3.2 Phantom MRI and Optical Imaging

17

MRI contrast enhancement was detected in the phantoms, at MNP concentrations as low as

18

0.07 µg/µL (0.039 μg of Fe/µL), using T2-W MRI (Figure 2). An exponential correlation was

19

observed between the T2-W MRI contrast enhancement and the MNP concentration, at high MNP

20

concentrations. Optical contrast enhancement was also detected in the phantoms, at MNP MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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concentrations as low as 0.07 μg/µL (Figure 2). Similarly, an exponential correlation was observed

2

between the optical contrast enhancement and the MNP concentration, at high MNP

3

concentrations. This similarity in the T2-W MRI contrast enhancement trend and the optical

4

imaging contrast enhancement trend suggested that the T2-W MRI signal obtained from the

5

PSMA-targeted MNP could be validated with its optical signal.

6

7 8

Figure 2. a) T2-W MRIs (axial view) and optical images of phantoms containing PSMA-targeted

9

MNPs at different nanoparticle concentrations, respectively. b) Graph of normalized contrast

10

enhancement generated by the PSMA-targeted MNPs present in the phantoms. The graph




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represents the normalized amount of contrast enhancement in each phantom relative, to that from

2

a phantom without nanoparticles (blank phantom).

3 4

3.2 In vivo MRI

5

Significant T2-W MRI contrast enhancement was detected in PSMA(+) tumors compared

6

to PSMA(-) tumors, 24 h (P < 0.0001; n = 10) and 48 h (P < 0.0002; n = 5) after the administration

7

of the PSMA-targeted MNPs (Figure 3a). This contrast enhancement was quantified by comparing

8

the ratio of contrast enhancement in PSMA(+) tumors to PSMA(-) tumors for each mouse, before

9

the administration of the contrast agent (at the 0 h time point), to that after the administration of

10

the contrast agent at the 24 h and 48 h time points, respectively (Figure 3, Supplementary Methods

11

S2, and Supplementary Figure S1). A 287% contrast enhancement was calculated for the PSMA(+)

12

tumors compared to the PSMA(-) tumors at the 24 h time point, relative to that at the 0 h time

13

point (before contrast administration). That enhancement in the PSMA(+) tumors compared to

14

PSMA(-) tumors, increased to ~ 330% at the 48 h time point, relative to that at the 0 h time point

15

(before contrast administration).

16

Non-targeted MNPs on the other hand displayed no statistically significant T2-W MRI

17

contrast enhancement in either PSMA(+) or PSMA(-) tumors, at any given time point (n = 3,

18

Supplementary Figures S4 and S5). Nevertheless, moderate contrast enhancement was detected in

19

both PSMA(+) and PSMA(-) tumors, from the non-targeted MNPs.

20

Using the high spatial resolution of MRI, we assessed the intra-tumoral contrast

21

enhancement patterns from the delivered PSMA-targeted MNPs in both PSMA(+) and PSMA(-)

22

tumors. The MRIs revealed preferential contrast enhancement at the tumor peripheries of both

23

PSMA(+) and PSMA(-) tumors, as opposed to homogenous contrast enhancement throughout the MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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[Ngen] Page 17 1

tumors (Figure 4). This suggested preferential delivery of the PSMA-targeted MNPs to the tumor

2

peripheries.

3 4

Figure 3. Active Targeting Component. a) T2-W MRIs (gray scale and color coded) of a

5

representative male NOD/SCID mouse bearing both human PSMA(+) PC3 PIP and human

6

PSMA(-) PC3 flu prostate tumor xenografts. The MRIs were acquired before contrast

7

administration; 24 h and 48 h after administration of the PSMA-targeted MNP. Pixel intensity

8

histograms of PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate tumor xenografts; b) before

9

contrast administration; c) 24 h after administration of the PSMA-targeted MNP; and d) 48 h after

10

administration of the PSMA-targeted MNP. e) The ratio of contrast enhancement in PSMA(+) PC3

11

PIP tumors versus PSMA(-) tumors 0 h, 24 h, and 48 h after administration of the PSMA-targeted MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



Page 18 of 44

[Ngen] Page 18 1

MNP. A significant difference of contrast enhancement was observed in PSMA(+) tumors 24 h (P

2

< 0.0001; n = 10) and 48 h (P < 0.0002; n = 5) after administration of the PSMA-targeted MNP

3

compared to 0 h using a one-sample paired two-tailed t-test analysis.

4 5

Figure 4. Passive Targeting Component. T2-W MRI assessment of the intra-tumoral contrast

6

enhancement pattern generated from the delivered PSMA-targeted MNP in human PSMA(+) PC3

7

PIP and human PSMA(-) PC3 flu prostate tumor xenografts. T2-W MRI (gray scale and color

8

coded) before the administration of the PSMA-targeted MNP and 24 h after the administrations of

9

the PSMA-targeted MNP revealed greater contrast enhancement in the tumor periphery, in both

10

PSMA(+) PC3 PIP and PSMA(-) PC3 flu tumor xenografts, as opposed to homogeneous contrast

11

enhancement throughout the tumors. This suggested peripheral intra-tumoral distribution of the

12

PSMA-targeted MNP.

13 14 MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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[Ngen] Page 19 1

3.3 In vivo Optical Imaging

2

In vivo optical imaging of mice 24 h and 48 h post-contrast administration revealed a

3

significant difference in the uptake of the PSMA-targeted MNPs in PSMA(+) tumors compared to

4

PSMA(-) tumors, at both the 24 h time point and the 48 h time point, compared to the 0 h time

5

point (Figure 5 and Supplementary Figure S6). Ex vivo optical imaging also showed that there is a

6

statistically significant difference in the uptake of the PSMA-targeted MNPs in PSMA(+) tumors

7

versus PSMA(-) tumors, after the administration of the PSMA-targeted MNPs (Supplementary

8

Figure S7).

9

The PSMA-targeted MNPs were also detected in organs of the reticuloendothelial system,

10

such as the liver, spleen and kidney (Figure 5). Weight measurements of the excised tumors

11

confirmed that both PSMA(+) and PSMA(-) tumors were of comparable sizes (Supplementary

12

Figure S8).

13




Page 20 of 44

[Ngen] Page 20

1 2

Figure 5. a) In vivo near-infrared fluorescence images of a representative male NOD/SCID mouse

3

bearing both human PSMA(+) PC3 PIP and human PSMA(-) PC3 flu prostate tumor xenografts,

4

0 h, 24 h and 48 h after administration of PSMA-targeted MNPs. Ex vivo fluorescence images of

5

the organs of a representative male NOD/SCID mouse bearing both human PSMA(+) PC3 PIP

6

and human PSMA(-) PC3 flu prostate tumor xenografts 24 h and 48 h after the administration of

7

PSMA-targeted MNPs. b) Quantification of the in vivo fluorescence signal ratio from the

8

PSMA(+) tumors to the PSMA(-) tumors in each mouse, 0 h, 24 h and 48 h after the administration

9

of PSMA-targeted MNPs. A statistically significant difference in the uptake of the PSMA-targeted

10

MNPs in PSMA(+) tumors compared to PSMA(-) tumors, at both the 24 h time point (P = 0.002; MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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[Ngen] Page 21 1

n = 10) and the 48 h time point (P = 0.024; n = 5), compared to the 0 h time point, were calculated

2

using a one-sample paired two-tailed t-test statistical analysis. c) Immunohistochemistry of PSMA

3

expression in PSMA(+) PC3 PIP and PSMA(−) PC3 flu tumors. The fluorescence images indicate

4

high levels of cell surface PSMA expression in PSMA(+) PC3 PIP tumors and no PSMA-

5

expression in PSMA(−) PC3 flu tumors. The scale bar represents 20 µm.

6 7

3.4 Immunohistochemistry

8

Immunohistochemistry of the excised tumors confirmed high PSMA expression levels in the

9

PSMA(+) PC3 PIP tumor cell surfaces and no PSMA expression on the PSMA(-) PC3 flu tumor

10

cell surfaces (Figure 5c), as was expected. H&E staining revealed that both PSMA(+) and PSMA(-

11

) tumors were more cellular at the tumor periphery and less so at the tumor center. That suggested

12

necrotic tumor cores (Figure 6 and Supplementary Figure S9).

13




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[Ngen] Page 22

1 2

Figure 6. a) Hematoxylin and eosin (H&E) staining of human PSMA(+) PC3 PIP and human

3

PSMA(-) PC3 flu prostate tumors, excised from mice after in vivo imaging. The scale bar

4

represents 20 µm. b) Quantification of the ratio of loss of cellularity at the tumor center compared

5

to the tumor periphery. The images revealed that both tumor types were more cellular at the tumor

6

peripheries compared to the tumor centers.

7 8 9 10 11 MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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[Ngen] Page 23 1

3.5 Two-photon Microscopy

2

High resolution two-photon microscopy revealed a higher density of blood vessels at the

3

tumor peripheries compared to the tumor centers, in both PSMA(+) tumors and PSMA(-) tumors

4

(Figure 7). Additionally, blood vessels of larger diameters were found at the tumor peripheries,

5

while blood vessels of smaller diameters were found at the tumor centers, in both PSMA(+) tumors

6

and PSMA(-) tumors (Figure 7).

7 8

Figure 7. a) Two-photon microscopy images of both human PSMA(+) PC3 PIP and human

9

PSMA(-) PC3 flu prostate tumors, excised from mice after the intravenous administration of a

10

2,000 kDa Texas Red conjugated dextran polymer, revealed higher tumor vascular densities at the MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



Page 24 of 44

[Ngen] Page 24 1

tumor peripheries compared to the tumor centers, of both PSMA(+) PC3 PIP and PSMA(-) PC3

2

flu prostate tumors. The scale bar represents 50 µm. b) Quantification of tumor blood vessel

3

diameters, at the tumor peripheries and the tumor centers of both PSMA(+) PC3 PIP and PSMA(-)

4

PC3 flu prostate tumors. Blood vessels of larger diameters were found at the tumor peripheries,

5

while blood vessels of smaller diameters were found at the tumor centers (P = 0.008; n = 3 for

6

PSMA(-) PC3 flu tumors and P = 0.021; n = 3 for PSMA(+) PC3 PIP tumors). c) Quantification

7

of the ratio of the surface area occupied by blood vessels at the tumor periphery to the surface area

8

occupied at the tumor center, in PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate tumors, revealed

9

no statistically significant difference between both tumor types.

10 11

4. Discussion

12

Current clinical practices for the delivery of MNPs to PC for MNP-induced hyperthermia

13

involve directly injecting the MNPs into the tumors.11 However, that is an invasive procedure,

14

which might also miss remote or even nearby lesions. We have chosen to develop targeted MNPs

15

capable of accumulating in tumors at concentrations necessary for MRI-guided hyperthermia after

16

systemic administration. Our previous report described the synthesis, optimization of surface

17

functionalization and the full characterization of the MNP formulation used here.37 We empirically

18

determined and demonstrated that PSMA targeting by the synthesized MNP could be optimized

19

by controlling the stoichiometry of the PSMA-targeting urea relative to the free amines on the

20

nanoparticle surface. In this report we examined the high spatial resolution of MRI and optical

21

imaging to evaluate the feasibility of PSMA-specific delivery of MNPs to PC in vivo at

22

concentrations necessary for MRI-guided hyperthermia.



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[Ngen] Page 25 1

The PSMA-targeted MNP was administered intravenously to mice at a dose of 50 mg/kg (30

2

mg of Fe/kg) to enable T2-W MRI detection. That dose is approximately three times higher than

3

that reported for the iron oxide nanoparticle (ferumoxytol) used clinically for the treatment of iron

4

deficiency anemia.43,

5

nanoparticles currently in clinical trials.45, 46 Importantly, that dose is 50-fold higher that what we

6

previously reported for PSMA-targeting.37 We previously showed that mice treated with these

7

PSMA-targeted MNPs at doses up to 200 mg/kg, were healthy and showed no signs of distress.37

8

We detected significant T2-W MRI contrast enhancement in PSMA(+) tumors compared to

9

PSMA(-) tumors, 24 h after contrast agent administration indicating target specificity. That

10

enhancement persisted up to 48 h. Using the high spatial resolution of T2-W MRI, we assessed the

11

intra-tumoral distribution of the delivered PSMA-targeted MNP and observed that the

12

nanoparticles preferentially accumulated at the tumor periphery compared to the tumor centers, in

13

both PSMA(+) and PSMA(-) tumors. Two-photon microscopic images revealed greater tumor

14

vascular density at the periphery compared to centrally for both PSMA(+) and PSMA(-) tumors.

15

By comparing the intra-tumoral distribution pattern of the PSMA-targeted MNP to the intra-

16

tumoral vascular distribution patterns, we inferred that the delivery and retention of PSMA-

17

targeted MNP to PSMA(+) tumors was mediated by two main factors: (1): Presence of high tumor

18

vascular density, with resulting high tumor perfusion; and, (2) PSMA expression in the tumor

19

epithelium. In PSMA(+) tumors, where PSMA expression levels were high on the cell surface, the

20

delivery and retention of the PSMA-targeted MNPs was greater at the periphery, which was more

21

vascularized and more cellular than at the tumor center. That suggested that the delivery and

22

retention of the PSMA-targeted MNP to PSMA(+) tumors, was mediated by a combination of both

44

It is also ~ 2.5 times higher than the dose reported for several other




Page 26 of 44

[Ngen] Page 26 1

passive tumor targeting through the enhanced permeability and retention (EPR) effect and through

2

active PSMA targeting.

3

We also noticed that the extravasation and accumulation of the PSMA-targeted MNPs was

4

affected by both the location of tumor implantation and the size of the tumors (Supplementary

5

Figure S10). In small tumors (~ 50 mm3), irrespective of their location, minimal accumulation of

6

the MNPs was detected. However, in larger tumors (8 to 10 mm in diameter in at least one

7

direction, ~250 mm3), accumulation of nanoparticles could be detected. The tumor sizes used in

8

our current study were larger than what we have typically used in our studies with low-molecular-

9

weight PSMA-targeted agents.32,

38

These findings related to tumor size suggest that the

10

accumulation of the PSMA-targeted MNPs is greatly affected by both the blood supply and the

11

EPR effect. Because in small tumors the EPR effect is less pronounced, MNP extravasation is

12

reduced. In larger tumors where the EPR effect is more pronounced, MNP extravasation is also

13

greater and more PSMA-targeting by the PSMA-targeted MNP can be achieved. However, in these

14

larger tumors with improved EPR, there is also a greater possibility of necrosis at the tumor core

15

(Figure 6 and Supplementary Figure S9). These data underscore the important contribution of the

16

EPR effect to active PSMA-targeting.

17

Based on the organ distribution ratio obtained from the ex vivo fluorescence images, we

18

estimated that ~ 6.2% ID/g of the administered PSMA-targeted MNPs is present in the PSMA(+)

19

tumors vs ~ 5.2% ID/g in the PSMA(-) tumors; and 76.4% ID/g in the liver; 8.2% ID/g in the

20

spleen; and 4.0% ID/g in the kidney at the 48 h time point (Supplementary Table S5 and S6).

21

Those biodistribution ratios are similar to our previous reports.37, 40 These findings also suggest

22

that although increasing the administered dose of the PSMA-targeted MNPs increases their

23

presence in the PSMA(+) tumors, it also increases non-specific uptake in the PSMA(-) tumors via MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle


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[Ngen] Page 27 1

the EPR effect. Alternative strategies are needed to increase the accumulation of targeted MNPs

2

specifically for enhanced therapy. We and others are currently developing targeted tumor priming

3

strategies to achieve such enhancement.47

4

Our current findings have several implications, especially since targeted nanoparticle

5

delivery to tumors remains a challenge.48 Since the delivery and intra-tumoral biodistribution of

6

the PSMA-targeted MNPs can be detected with MRI, MRI could be used to monitor tumor

7

response patterns noninvasively during and after therapy, while taking into account the MNP

8

pattern of intra-tumoral distribution.49 That will be particularly significant given the higher spatial

9

resolution of MRI compared to optical imaging and radionuclide imaging. Finally, current clinical

10

practice for the delivery of MNPs involve direct injection of the MNPs into the tumor site.11

11

However, that is invasive and precludes targeting of remote or even nearby lesions. Accordingly,

12

understanding the roles of vascular supply EPR in the delivery and intra-tumoral biodistribution

13

patterns of these PSMA-targeted MNPs could aid in better design strategies to optimize intra-

14

tumoral MNP delivery and biodistribution.47, 50

15 16

5. Conclusion

17

Here, we evaluated the feasibility of selectively delivering PSMA-targeted MNPs to

18

PSMA-expressing human prostate tumor xenografts in a preclinical murine model, with optical

19

imaging and high resolution T2-W MRI, in concentrations necessary for MNP-induced

20

hyperthermia therapy to be accomplished. Through a number of complementary techniques,

21

including the high spatial resolution of T2-W MRI and optical imaging, we demonstrated that the

22

delivery of the targeted MNPs is due not only to active PSMA targeting – as is the dominant mode

23

of delivery of the low-molecular-weight agents we have developed for imaging – but also includes MRI Assessment of PSMA Targeting by a PSMA-Targeted Magnetic Nanoparticle



Page 28 of 44

[Ngen] Page 28 1

a passive, EPR-mediated effect that resulted in inhomogeneous distribution of MNPs within the

2

tumors. This inhomogeneous MNP distribution seemed to be influenced by the intra-tumoral blood

3

vessel density as demonstrated with multi-photon microscopy. Accordingly, we anticipate that this

4

strategy could be used to deliver PSMA-targeted drug-loaded magnetic nanoparticles to localized,

5

aggressive, PSMA-expressing, castration-resistant prostate tumors, for enhanced MRI-guided

6

hyperthermia and sustained drug release.

7 8

Supporting Information.

9

Synthetic schemes; MRI analyses; Two-photon microscopy; MRIs; In vivo fluorescence images;

10

ex vivo fluorescence images; histology.

11 12

Acknowledgements

13

We would like to thank Dr. Jiadi Xu and Ms. Kazi Akhter for assistance with the MRI

14

acquisitions. This research was sponsored by the National Institutes of Health (grant numbers:

15

R01CA134675, U01CA183031, P50CA058236, P41EB024495 and K25CA148901).

16 17 18 19 20 21



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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



TOC


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MRI Assessment of Prostate-Specific Membrane Antigen (PSMA

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