Theranostic nanoparticles for detection and targeted therapy of prostate cancer and lymph node metastases

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Thesis

Reference

Theranostic nanoparticles for detection and targeted therapy of prostate cancer and lymph node metastases

MAUDENS, Stella-Saphira

Abstract

The aim of this thesis was to explore iron oxide nanoparticles (IONPs) for ultimate detection and co-treatment of early prostate cancer (PC) lymph node metastases. IONPs previously optimized as a theranostic tool for the combined use in MRI and magnetic hyperthermia were functionalized with an RNA aptamer or a small molecule, both actively targeting the prostate-specific membrane antigen (PSMA). Functionalized IONPs were developed to meet the requirements for lymphatic targeting and specific binding to PSMA-expressing PC cells while preserving high MRI relaxivity and heating properties. In a second project, we designed and tested an in situ forming implant entrapping IONPs as a heating source for magnetic hyperthermia with intended application in localized PC. Besides appropriate syringeability and implant formation upon injection, we confirmed homogeneous intrinsic radiopacity in micro-CT in vivo. When applying magnetic field strengths and frequencies eligible for use in humans, the implant reached the hyperthermia target temperature of 48 °C.

MAUDENS, Stella-Saphira. Theranostic nanoparticles for detection and targeted therapy of prostate cancer and lymph node metastases. Thèse de doctorat : Univ.

Genève, 2018, no. Sc. 5273

DOI : 10.13097/archive-ouverte/unige:125025 URN : urn:nbn:ch:unige-1250258

Available at:

http://archive-ouverte.unige.ch/unige:125025

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section des sciences pharmaceutiques Docteur Olivier Jordan Laboratoire de Biopharmacie Professeur Gerrit Borchard

Theranostic Nanoparticles

for Detection and Targeted Therapy

of Prostate Cancer and Lymph Node Metastases

THÈSE

présentée à la Faculté des sciences de l’Université de Genève

pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques

par

Stella-Saphira Maudens (Ehrenberger) de

Sinsheim (Allemagne)

Thèse N°: 5273

Genève 2018

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UNIVERSITE DE GENEVE

FACUL TE DES SCIENCES

DOCTORATES SCIENCES,

MENTION SCIENCES PHARMACEUTIQUES These de Madame Stella-Saphira MAUDENS

intitulee

«Theranostic Nanoparticles for Detection and Targeted Therapy of Prostate Cancer and Lymph Node Metastases»

La Faculte des sciences, sur le preavis de Monsieur 0. JORDAN, docteur et directeur de these (Section des sciences pharmaceutiques, Ecole de pharmacie Geneve-Lausanne), Monsieur G. BORCHARD, professeur ordinaire et codirecteur de these (Section des sciences pharmaceutiques), Monsieur E. ALLEMANN, professeur ordinaire (Section des sciences pharmaceutiques, Ecole de pharmacie Geneve, Lausanne), Madame D. FISCHER, professeure (Department of pharmaceutical technology and biopharmaceutics, University of Jena, Germany), Monsieur T. LAMMERS, professeur (Department of Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, Rheinisch-Westfalische Technische Hochschule Aachen, Germany), autorise

!'impression de la presente these, sans exprimer d'opinion sur les propositions qui y sont enoncees.

Geneve, le 24 octobre 2018 These - 5273 -

Le Doyen

N.B. - La these doit porter la declaration precedente et remplir les conditions enumerees dans les "Informations relatives aux theses de doctorat

a

l'Universite de Geneve".

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To Edouard and Pierre

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Table of contents

Abbreviations

ACTB β-actin

ACUPA S, S-2-[3-[5-Amino-1-carboxypentyl]-ureido]-pentanedioic acid

ACUPA-IONPs ACUPA-functionalized cysteine-coated iron oxide nanoparticles

AMF Alternating magnetic field

Apt-IONPs Aptamer-functionalized cysteine-coated iron oxide nanoparticles

ATCC American Type Culture Collection Au-NPs Gold nanoparticles

bp Base pairs

Casp3 Caspase-3

CIE Clathrin- and caveolin-independent endocytosis CK18 Cytokeratin-18

CME Clathrin-mediated endocytosis CRPC Castration-resistant prostate cancer

CT Computed tomography

Cy5 Cyanine 5

Cy5-aptamer Cyanine 5-labeled aptamer Cy5-apt-IONPs Cyanine 5-labeled apt-IONPs Cys-IONPs = Cys-IONPsA

Cys-IONPsA Cysteine-coated iron oxide nanoparticles, prepared in acidic medium

Cys-IONPsN Cysteine-coated iron oxide nanoparticles, prepared in pH neutral medium

DBCO Dibenzocyclooctyne

dh Hydrodynamic diameter

DIPEA N,N-Diisopropylethylamine

DMEM Dulbecco’s Modified Eagle’s Medium DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

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Abbreviations

2

ESI-MS Electrospray ionization mass spectrometry

ƒ Frequency

FTIR Fourier transform infrared

H Magnetic field strength

HA Calcium hydroxyapatite

HBSS Hank’s Balanced Salt Solution

HE Hematoxylin and eosin

ICP-OES Inductively coupled plasma optical emission spectroscopy IONPs Iron oxide nanoparticles

IVIVC In vitro – in vivo correlation KD Equilibrium dissociation constant LAL Limulus amoebocyte lysate

LNCaP Cell line derived from human prostate cancer lymph node metastases

MAT-LyLu Cell line derived from serial passages of the Dunning R 3327 AT tumor resulting in a metastatic AT tumor disseminating to lymph nodes and lung

mCRPC Metastatic castration-resistant prostate cancer MRI Magnetic resonance imaging

mRNA Messenger ribonucleic acid

MTIB-PVA Mono- and triiodobenzylether-polyvinylalcohol NCBI National Center for Biotechnology Information

NHS N-hydroxysuccinimide

NMR Nuclear magnetic resonance

NP Nanoparticle

PB Prussian blue

PC Prostate cancer

PC3 Cell line derived from grade IV human prostate cancer bone metastases

PET Positron emission tomography PSA Prostate-specific antigen

PSMA Prostate-specific membrane antigen

RNA Ribonucleic acid

RPMI Roswell Park Memorial Institute medium

RT-PCR Reverse transcriptase-polymerase chain reaction

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Abbreviations

3

SC Subcutaneously

ScrApt-IONPs Scrambled aptamer-functionalized cysteine-coated iron oxide nanoparticles

SEM Scanning electron microscopy

SLP Specific loss power

SPECT Single-photon emission computed tomography SPIONs Superparamagnetic iron oxide nanoparticles TEM Transmission electron microscopy

TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling

UHPLC Ultra-high performance liquid chromatography XPS X-ray Photoelectron spectroscopy

ζ-potential Zeta potential

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Preface

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Preface

7

Preface

Iron oxide nanoparticles (IONPs) are widely studied for biomedical applications in magnetic resonance imaging (MRI), local hyperthermia, drug delivery, magnetic targeting and combinations thereof. Developing a successful nanoparticulate system based on iron oxide requires optimization of a multitude of parameters, which necessitates a multi-disciplinary approach at the interface of material science, pharmaceutical and medical research.

Besides core and surface properties, the performance of IONPs mainly depends on their formulation adapted for the desired application and route of administration as well as the setting in which they are tested. IONPs as MRI contrast agents require a high relaxivity and surface modifications allowing to reach and accumulate at the target site to obtain a sufficient contrast. Accumulation at the site of interest and appropriate cargo release are also necessary for their use as drug delivery systems. In magnetic hyperthermia, when IONPs serve as heating source to provoke cell death of the surrounding tissue, IONPs with elevated intrinsic heating properties are needed. An additional challenge represents the delivery of a considerable amount of IONPs to the site of interest in order to reach the target temperature while applying clinically accepted alternative magnetic field strengths.

In this context, the presented work in this thesis was based on IONPs optimized for the combined use in MRI and magnetic hyperthermia and aimed to address some of the aforementioned challenges towards clinical translation.

The first goal was to exploit these IONPs as a theranostic tool by designing functionalized nanoparticles for the detection of early prostate cancer (PC) lymph node metastases and targeted hyperthermia co-treatment. For diagnosis of early metastatic spread, extended lymph node removal still represents the gold standard due to the lack of sensitivity of conventional computed tomography (CT) and MR imaging. While promising radiotracers are in the pipeline for the detection of PC metastases, IONPs as MRI contrast agent may represent an alternative in clinical routine, especially when positron emission tomography (PET)/CT or single-photon emission computed tomography (SPECT)/CT scanners are not available. Local hyperthermia would offer a co-treatment option for inaccessible metastatic lymph

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Preface

8

nodes, for example in combination with chemotherapy. For the design of the nanoparticles, attention was therefore given to meet the requirements for lymphatic targeting while preserving the high MRI relaxivity and heating properties of the IONP core. Considering different surface markers on PC cells, the prostate-specific membrane antigen (PSMA) was selected as a molecular target due to its reliable overexpression in advanced and metastatic PC. Two promising PSMA-ligands were selected in order to compare their performance as targeting moieties of IONPs.

A secondary aim of this thesis was to investigate a formulation for local administration of these IONPs as heating source for magnetic hyperthermia with intended application in primary, localized PC. Especially in elderly PC patients, local hyperthermia may offer a less aggressive treatment option with very few side effects to fill the gap between radical prostatectomy and watchful waiting.

Prostate cancer pathology, challenges in the disease management and the advantages of PSMA as a molecular target for imaging and therapy at different stages of PC and other malignancies are introduced in CHAPTER I. Scientific publications were reviewed to give an overview of recent developments in the field of nanoparticulate systems targeting PSMA.

CHAPTER II presents the development of aptamer-functionalized IONPs (apt- IONPs). First, L-cysteine was evaluated as surface coating for IONPs in terms of colloidal stability, biocompatibility and interaction with PC cells. In a second step, cysteine-coated IONPs were functionalized with the PSMA-targeting RNA aptamer A10 in a copper-free click chemistry approach at different surface densities. Coating and functionalization processes were performed in aqueous media respecting aseptic conditions with the aim of reducing the use of organic solvent and to obtain endotoxin-free particles. Besides a detailed physicochemical characterization, we investigated MRI relaxivities and heating properties. PSMA-positive and PSMA- negative human PC cell lines were used in vitro to test biocompatibility, specific PSMA-targeting in comparison to scrambled apt-IONPs and the elucidation of uptake mechanisms. A first imaging study was performed in the metastasizing rat PC model MAT-LyLu.

In CHAPTER III, the PSMA-targeting urea-based small molecule ACUPA was evaluated as targeting ligand. An ACUPA-PEG-spacer was synthesized and

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Preface

9

“clicked” on the surface of cysteine-coated IONPs. The physicochemical properties of ACUPA-functionalized IONPs and their performance in binding to PSMA-positive PC cells in vitro were compared to apt-IONPs in order to select the most promising candidate.

The study presented in CHAPTER IV is based on the need of an appropriate animal model to test the developed IONPs in vivo. Regarding the ultimate goal of targeting lymph node metastases of PC, the cancer cells used for the animal model should express PSMA on their membrane surface and reliably metastasize to lymph nodes in an appropriate time frame. For MR imaging, we required animals with a lymph node size detectable in a clinical 3.0 T scanner, which excluded the use of mice. The metastasizing rat PC model MAT-LyLu met all mentioned requirements, apart from its unknown PSMA expression status. We therefore tested MAT-LyLu cells for PSMA mRNA expression and evaluated specific binding and uptake of the PSMA-targeting aptamer in vitro.

In preparation of the main study, a preliminary in vivo study was performed by Richter et al. to compare injection of MAT-LyLu cells in the hock versus the footpad regarding tumor progression and appearance of metastases in the popliteal lymph nodes. Being involved in this study, it is placed in APPENDIX I.

CHAPTER V is dedicated to the therapeutic use of IONPs for local, magnetic hyperthermia. In this study, we sought to incorporate a high amount of IONPs in an intrinsically radiopaque polymer. Upon contact with body or tumor tissue, the polymer in the liquid injectable formulation is precipitating as a compact implant entrapping the IONPs at the injection site. Besides appropriate implant formation, one of the most important criteria during in vitro characterization was to reach the target temperatures needed for successful hyperthermia under low magnetic field strengths, which can be applied to humans. After injection in a rat liver lobe, the nanocomposite was evaluated for its radiopacity by micro-CT, its heating performance and the impact of the released heat on surrounding hepatocytes to evaluate its potential for hyperthermia treatment.

To summarize the main findings of the presented studies, this thesis ends with general conclusions and perspectives aiming to contribute to future progress in the use of IONPs in MR imaging and hyperthermia towards clinical translation.

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Chapter I

Current Status of Prostate-specific Membrane

Antigen-Targeted Nanoparticulate Systems in

Preclinical Research

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Chapter I

13

Current Status of Prostate-specific Membrane Antigen-Targeted Nanoparticulate Systems

in Preclinical Research

Stella-Saphira Maudens, Gerrit Borchard, Olivier Jordan

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneva 4, Switzerland

Abstract

Prostate cancer (PC) is the most commonly diagnosed cancer in men in developed countries. While PC can progress slowly without the need of imminent treatment, the advanced metastatic state, recurrence or castration resistance represent important clinical challenges. PC patients would greatly benefit from more precise detection methods and more effective therapy options with less systemic toxicity. Nanoparticulate systems in oncology have already proven to enhance drug targeting while reducing side effects. To further improve specific tumor cell uptake, nanoparticles (NPs) have been conjugated to ligands targeting a specific biomarker overexpressed in cancer cells. A promising biomarker for PC represents the prostate-specific membrane antigen (PSMA), which is reliably overexpressed in advanced PC. With one formulation already tested in clinical trials, a multitude of PSMA-targeted NPs is currently in preclinical evaluation to explore the significant potential of the combination of nanosystems and targeting of PSMA for imaging and therapy. With this review, we aimed at providing a comprehensive overview over PSMA-targeted NPs in preclinical research as well as important parameters and challenges during development.

Key words

Nanoparticles, targeting of PSMA, prostate cancer, imaging, therapy

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PSMA-Targeted Nanoparticulate Systems in Preclinical Research

14

1. Introduction

With an estimated 14.1 million new cases diagnosed and 8.2 million deaths worldwide each year (data from 2012), the global cancer burden continues to rise [1]. Despite tremendous efforts and progress to improve cancer management, numerous challenges still have to be resolved to decrease cancer-related mortality.

Early diagnosis of cancer and metastatic spread are of outmost importance to initiate the adapted therapy. Among well-established treatment options like surgery, hormone and radiation therapy, chemotherapy is considered as one of the most powerful. Still, the associated systemic toxicity leading to potentially serious side effects can render chemotherapy inaccessible for cancer patients. This also applies to prostate cancer (PC), the most commonly diagnosed cancer in men in developed countries [2]. While chemotherapy is not considered as first-line treatment at an early stage, docetaxel treatment delays progression and increases overall survival at advanced stages of the disease including metastatic hormone-naïve and castration-resistant PC, the lethal form of the disease [3, 4]. However, age being a prominent risk factor for PC, men have to be fit enough and comorbidities have to allow docetaxel therapy [5]. This challenge, together with early detection of metastases and localization of PC recurrence might be met by the application of nanomedicine, aiming at increasing drug accumulation in the tumor microenvironment to enhance imaging specificity, sensitivity and therapeutic efficacy while reducing systemic toxicity [6].

Since its discovery over 30 years ago, the prostate-specific membrane antigen (PSMA) has been widely explored as molecular target due to its significant overexpression pattern in PC and the neovasculature of a variety of solid tumors [7].

Numerous comprehensive reviews exist on the use of PSMA-ligands in oncology for imaging and therapy [7-11]. The combination of PSMA-targeting for tumor- specific accumulation with nanoscale drug carriers or imaging agents has the potential to overcome some of the limitations in the management of PC or to offer alternative treatment and imaging options.

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The scope of this review therefore focused on nanoparticle (NP) formulations functionalized with a PSMA-targeting ligand in preclinical research, notably polymeric NPs, liposomes, micelles, inorganic NPs and polymeric dendrimers. The goal was to give an overview of PSMA-targeted NPs recently developed to address different stages of PC for therapy, imaging or combinations thereof (theranostics).

We aimed to present the most commonly used targeting moieties for NPs bearing PSMA-ligands as well as PC cell lines used to assess specific binding and uptake in vitro and in vivo by a multitude of different assays. Finally, we intended to identify challenges and pitfalls in the development of NPs for the purpose of targeting PSMA.

2. Prostate cancer (PC)

PC represents the most common cancer in men in developed countries and the second most diagnosed cancer in men worldwide with an estimated 1.1 million cases per year [2]. This discrepancy in incidence is attributable to the risk factor age and the routinely performed prostate-specific antigen (PSA)-screening and subsequent biopsy sampling in more developed countries, which results in a higher detection rate [2, 12]. Consequently, the mortality rate varies less than the incidence rate and identifies PC as the fifth leading cause of death from cancer in men [2].

Age, family history of PC and ethnicity (African American origin) are well-established risk factors [13]. The mentioned risk factors, blood PSA levels, results of digital rectal examination and a previous prostate biopsy are the predictive factors with the strongest association to develop clinically significant PC [3, 14]. Disease progression spans from locally defined, asymptomatic disease to highly aggressive, metastasizing PC. Treatment options are established based on tumor staging, which classifies PC as low, intermediate or high risk localized PC (based on PSA level, Gleason score, clinical stage) or locally advanced disease with the tumor extending through the prostate capsule or the presence of lymph node metastases [3]. The transition of hormone-naïve to castration-resistant PC (CRPC) or metastatic CRPC (mCRPC), the lethal form of PC, is defined by disease progression despite androgen-deprivation therapy. PC relapse after prostatectomy is commonly detected by a rise in PSA levels and is therefore also termed biochemical failure

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Chapter I

17 [15]. However, identifying the site of recurrence remains challenging and current routine imaging modalities lack sensitivity and specificity. Further urgent unmet medical needs include early detection of lymph node and distant metastases and therapies to delay progression to castration-resistant PC and to prolong survival in mCRPC [15].

2.1 Role of PSMA in targeting PC

PSMA, first identified by Horoszewicz et al. [16], is a type II transmembrane glycoprotein composed of 750 amino acids and a molecular weight of 84 kDa [17, 18]. The N-terminal cytosolic amino acids 1-18 follows a single membrane-spanning segment of 25 amino acids and the C-terminal extracellular part of amino acids 44 to 750 [9, 19]. PSMA is physiologically expressed in lacrimal and salivary glands, proximal renal tubules, within the central and peripheral nervous system and the small intestine [20]. In the healthy prostate, PSMA is expressed at a low level. Until today, N-acetyl-aspartyl-glutamate (NAAG) and dietary poly-γ-glutamyl folates have been identified as endogenous ligands in the central nervous system and the small intestine, respectively [19]. For this reason, PSMA is also known as glutamate carboxypeptidase II, NAAG-peptidase, N-acetyl-α-linked acidic dipeptidase I (Naaladase I) or folate hydrolase. However, the specific role of PSMA in other tissues has not been completely elucidated to date.

In PC, PSMA expression in tumor cells rises by 100 to 1’000-fold and increases along with disease progression regardless of the androgen status [18, 21, 22]. PSMA therefore represents an excellent target and holds promise to provide solutions for some of the challenges and unmet needs in the management of PC.

For example, the use of ⁶⁸Ga-PSMA PET/CT improved sensitivity and specificity in comparison to bone scan and abdominal-pelvic CT for localization of the relapse site after biochemical failure and has shown superiority in identifying metastases for initial staging at primary diagnosis compared to CT, MRI or bone scan [23]. The European Association of Nuclear Medicine (EANM) and the Society of Nuclear Medicine and Molecular Imaging (SNMMI) recently released a joint procedure guideline recommending the use of ⁶⁸Ga-PSMA PET/CT for these indications [23].

Apart from PC, PSMA is also overexpressed in the neovasculature of several types

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of solid tumors including breast, kidney, bladder, colon cancer and glioblastoma [20, 24, 25].

Another important aspect qualifying PSMA as valuable molecular target is the internalization of ligands and their cargo after binding to the active center of the extracellular domain [10]. The enhanced uptake into cancerous cells increases imaging quality for diagnostic procedures and the local dose for therapeutic applications [10]. The first and only FDA approved PSMA-targeted imaging agent to date is Indium 111 capromab pendetide (ProstaScint^®, Aytu Bioscience, Englewood, CO, USA) to detect soft tissue metastases and recurrence of PC after biochemical failure. While ProstaScint^®, a mouse monoclonal antibody (mAb) 7E11- C5 conjugate, is binding to an intracellular PSMA epitope [26], ligands currently exploited are targeting its extracellular domain [27-29].

3. PSMA-targeting ligands

After the approval of mAb 7E11 in 1996, antibodies targeting the extracellular domain of PSMA have been developed [27-29]. The most extensively studied mAb is the humanized mAb J591, which has been tested in multiple clinical trials as radionuclide conjugate for both imaging and therapy in PC, most notably in mCRPC, and non-prostatic tumor neovasculature [30, 31]. In the attempt to reduce circulation half-life, the time to reach the tumor site and to improve tumor penetration, smaller sized PSMA-targeting antibody fragments, minibodies, peptides, aptamers and small molecules emerged [32]. Most notably, small molecules are broadly studied in preclinical and clinical research and constitute the targeting moiety of the aforementioned ⁶⁸Ga-PSMA PET-tracer. Mimicking the natural substrate N-acetyl- L-aspartyl-L-glutamate, several families of small molecules were developed in a structure-activity based design approach [11]. Among phosphorus-, thiol-, and urea- based small molecules, the latter are the most widely studied for PSMA targeting [33]. As a famous example, the urea-based small molecule ACUPA represents the ligand of the only PSMA-targeted nanoparticulate system that entered clinical trials to date [34]. These docetaxel-loaded polymeric NPs (BIND-014) were tested for the treatment of advanced solid tumors [35] and mCRPC [36, 37]. Initial preclinical results were promising showing increased plasma circulation, intratumoral drug

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Chapter I

19 concentration and safety compared to docetaxel alone owing to PSMA-targeting and controlled release of docetaxel in the tumor microenvironment [34]. Clinical trials revealed some effect in mCRPC and non-small lung cancer [38], but were not convincing in showing improved efficacy or reduced toxicity compared to docetaxel and were not further pursued.

Interestingly, for the majority of nanoparticulate systems targeting PSMA in current preclinical research, two types of targeting ligands are used: aptamers and urea-based small molecules. Their advantages in comparison to antibodies are the chemical production, which is generally less costly, and the lack of immunogenicity, while maintaining a comparative affinity. Their structures, allowing orthogonal attachment to the NPs’ surface to preserve PSMA binding, and ease of handling are especially attractive for use as NP-ligands. While their smaller size greatly influences the blood clearance for radiotracers, this applies less for NPs, since the diameter of the NPs largely outweighs the size of the ligand. Exceptions are NPs in the lower nanometer range like quantum dots or dendrimers. For applications such as lymph node targeting, in which a diameter below 100 nm needs to be respected to allow interstitial transfer, the use of low molecular weight ligands is also preferred.

For PSMA-targeted NPs, the two most commonly used aptamers A9 and A10 are RNA nucleotides and therefore chemically modified to render them resistant to degradation by nucleases [28]. Mostly, the 2’-position is replaced with a fluoro-, amino-, or O-methyl group and the 3’-end contains an inverted nucleotide (cap) [39].

Aptamers are typically attached to the NP surface by their 5’-end, which contains either a spacer or the desired chemical group needed for attachment to the NP surface. The most commonly used small molecule for PSMA-targeting of NPs, ACUPA, is attached via its amino group to preserve the three carboxylic groups required for binding to the active site of PSMA [29].

Apart from the enzymatic function of PSMA as folate hydrolase cleaving poly- γ-glutamyl folates, it was suggested that PSMA also acts as a folate transporter [40].

The cellular uptake of folate-linked NPs via PSMA was first proposed by Hattori et al. [41]. As a consequence, folates have been studied as targeting moieties of NPs for PSMA-targeting on LNCaP and PC3 cells, the most commonly used cell lines to test specific binding of PSMA, which do not overexpress folate receptors [41, 42].

Flores et al. recently showed the preferential uptake of activatable folate-S-S-

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doxorubicin fluorescent probes in LNCaP and PSMA-positive PC3 cells in comparison to PSMA-negative PC3 cells and confirmed this result with folate- conjugated hyperbranched polymeric NPs [43]. Higher uptake of folate-conjugated NPs in LNCaP cells was also suggested for cyclodextrin NPs encapsulating RelA siRNA [44], liposomes co-entrapping a mitomycin C prodrug and doxorubicin [45], gold NPs for siRNA delivery [46] and iron oxide NPs [47]. However, preclinical biodistribution and targeting studies are needed to show the potential of folates for targeting PSMA-expressing tumors in vivo. Noteworthy, folates have been extensively used to target folate receptor-α, which is overexpressed in a variety of solid tumors including ovarian, breast, pancreatic and lung cancer. The folate receptor-β on activated macrophages in inflammatory pathologies and autoimmune disease including psoriasis, systemic lupus erythematosus, arteriosclerosis, rheumatoid arthritis or Crohn’s disease is also studied as molecular target to target these pathologies [48, 49]. Folate receptors are expressed at low levels in most normal tissues [49, 50].

4. In vitro and preclinical evaluation of PSMA-targeted NPs

One of the first steps in evaluating the interaction of targeted NPs with PSMA on the cell surface are in vitro assays. Besides the three classic human PC cell lines PC3, LNCaP and DU145 [51], numerous cell lines have been established with a variety of gene and marker expression patterns [52]. For an exhaustive overview of cell lines for PC in vitro and in vivo research, we refer to the 2-part review of Sobel et al. [53, 54]. Commonly used human PC cell lines in preclinical research to test cellular uptake of PSMA-targeted NPs were identified based on the reviewed literature and are presented in Table 1. Among these, the most frequently used are the PSMA-positive LNCaP and PSMA-negative PC3 cell lines. Other PSMA-positive cells used for targeting evaluation are the CWR22RV1 and C4–2 cells. The latter being an androgen-independent subline of LNCaP cells was established by co- injection with stromal cells and serial passages in castrated hosts to better study the castration-resistant state of the disease [55, 56]. PC3-PIP (PSMA-IRES-Puromycin) and PC3-flu cells are genetically modified PC3 cells with PSMA-positive (PC3-PIP) or -negative control vectors (PC3-flu) [57, 58]. When comparing PSMA-positive cell

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Chapter I

21 lines, LNCaP showed the highest antigen expression, followed by PC3-PIP and 22RV1 [59]. In vitro, PSMA-targeting NPs are typically tested in PSMA-positive and –negative cells and in comparison to non-targeted NPs (see Table 3 and Table 4).

To demonstrate binding specificity, non-targeted NPs are ideally functionalized with a non-binding scrambled version of the binding ligand. Targeting and non-targeting NPs should at least display a similar hydrodynamic diameter and surface charge, since both are influencing the interaction with cells. If different cell media are used, attention has to be given to potential variations in the agglomeration state of NPs, which also influences cell interaction. Non-binding scrambled controls are equally important for in vivo studies to rule out tumor accumulation solely due to passive accumulation related to the leakiness of tumor vasculature or increased circulation half-life (as for NPs) known as the enhanced permeability and retention (EPR) effect [60, 61]. In order to evaluate NP targeting of PSMA in vivo, xenograft mouse models of the cell lines presented in Table 1, except DU145, are commonly used.

Table 1. Most commonly used human PC cell lines in preclinical research of PSMA- targeted NPs. Invasiveness was assessed by quantification of cells, which invaded through a Matrigel™ matrix [59].

Cell line Origin Androgen

sensitivity

PSMA expression

[59]

Tumorigenicity (Bone metastasis phenotype [62])

Invasive- ness in vitro [59]

LNCaP

Derived from left supraclavicular lymph node metastasis of a 50- year-old man [63]

+ ++ Very low [64]

(osteoblastic) Low C4-2

Subline derived from LNCaP after in vivo passages in castrated hosts [56]

- + Low [64]

(osteoblastic)

Not tested

22RV1

Cell line derived from the xenograft line CWR22R (Relapse from CWR22 tumor, patient with osseous metastases, Gleason grade 9) [65]

+ +

Low (CWR22 tumor)

[64]

(osteolytic)

Low

PC3-PIP

PC3 cells transduced with a retrovirus encoding PSMA [57, 59]

- + Low Low

PC3

Derived from vertebral metastasis of a 62-year-old man [66]

- - High [64]

(osteolytic) High DU145 Derived from brain metastasis of

a 60-year-old man [67] - - High [68]

(osteolytic) High

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5. PSMA-targeted NPs for imaging and therapy of PC

Particles in the nanosize range offer a variety of advantages for imaging and therapy, notably increased bioavailability, higher efficacy, improved safety and toxicological profiles [6]. Since the FDA approval of lipid NPs encapsulating doxorubicin (Doxil^®) in 1995, numerous NPs were tested in clinical trials and entered the market. Excellent reviews exist which further detail the benefits of nanoparticulate systems [6, 69, 70]. Accumulation of NPs at the tumor site is, depending on the type of tumor, suggested to be mainly driven by the EPR effect [71]. Combining the advantages of PSMA-targeting with NPs has the potential to further increase therapeutic efficacy, specificity and sensitivity for imaging by PSMA- driven internalization enhancing preferential accumulation in PC cells [72, 73].

However, this concept cannot always be successfully translated, e.g., because the targeting ligand or ligand-NP conjugation can be unstable after injection or easily shielded by the formation of a protein corona [74].

Hereinafter, recent preclinical developments of NPs functionalized with a PSMA-ligand are presented and evaluated for the additional benefit of PSMA-driven cell uptake for specific targeting. Even though development is still at an early stage, addressed approaches for current challenges and unmet medical needs in the management of PC or alternative treatment and imaging options are summarized in Table 2. This review focuses on the evaluation of polymeric NPs, liposomes, micelles, inorganic NPs and polymeric dendrimers.

Table 2. Indications, challenges and medical needs in the management of PC, which are addressed in the development of PSMA-targeted NPs.

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Chapter I

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5.1. PSMA-targeted NPs developed for imaging

To enhance specificity and sensitivity in MRI for early detection of PC, metastases, relapse sites and monitoring of drug delivery and treatment response, a multitude of PSMA-targeted contrast agents based on superparamagnetic iron oxide NPs (SPIONs) were studied. Details of the reviewed studies are summarized in Table 3.

For antibody-mediated targeting, higher uptake of J591-labeled SPIONs by LNCaP cells in comparison to DU145 cells and non-targeted SPIONs was shown by measuring intracellular iron concentrations and MRI relaxation rates by Abdolahi etal. [75]. Mukherjee et al. studied the influence of mAb J591 surface densities on silica-coated SPION surface on PSMA targeting. Interestingly, intermediate ligand density provided the highest specific cell binding, probably due to decreased steric hindrance, while high densities almost completely suppressed PSMA-binding. A concept also reported for other ligands, including HER2-affibody-conjugated SPIONs [76]. For the study of Mukherjee et al., the lead candidate showed a more than 5-fold higher uptake compared to non-targeted NPs and PSMA-negative cells in vitro [77]. Tse et al. confirmed higher uptake of J591-SPIONs by PSMA-positive cells compared to non-targeted SPIONs and showed enhanced contrast in a pilot study in an orthotopic LNCaP mouse model compared to non-targeted SPIONs [78].

Taylor et al. encapsulated Pt-doped SPIONs together with paclitaxel in micelles functionalized with J591. Significantly more paclitaxel and platinum were retained in C4-2PC mouse xenografts compared to non-targeted NPs [79]. With the intent to deliver the treatment adapted for the disease stage with the same NPs used for initial MRI diagnosis, Kaittanis et al. proposed a nanoplatform based on functionalized SPIONs with a small cyclic PSMA-targeting peptide. A prominent difference in uptake in vitro between PC3 and PSMA-expressing PC3 cells and of corresponding xenograft mouse models in vivo was proved [80]. Co-delivery combinations of doxorubicin, paclitaxel, enzalutamide, BEZ235, cabazitaxel and riluzole as treatments for the different stages of PC were then tested in different in vitro cell lines and in vivo xenografts adapted to the targeted disease progression.

Maudens et al. functionalized SPIONs, which were optimized for MRI and hyperthermia treatment, with the PSMA-targeting aptamer A10 using a click- chemistry approach for detection of early lymph node metastases and hyperthermia

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treatment with the same particles [81]. The aptamer surface density greatly influenced the colloidal stability of the SPIONs in this study. Aptamer-SPIONs resulted in a significantly higher uptake compared to scrambled aptamer-SPIONs and PSMA-negative cells in vitro. A pilot study using the metastasizing MAT-LyLu rat PC model confirmed the detectability of apt-SPIONs by MRI [81]. SPIONs with thermally cross-linked surface coating were studied for PC imaging owing to their interesting properties, such as firm and stable polymer coating, good dispersibility in physiological medium and reported anti-biofouling properties, which prevent the adsorption of plasma protein on their surface [82]. Yu etal. used the intercalation of doxorubicin with aptamers on the surface of thermally cross-linked SPIONs to deliver the drug, which decreased the T2 signal and cell viability in LNCaP cells compared to PC3 cells and non-targeted SPIONs [83]. In LNCaP mouse xenografts, the increased tumor accumulation of aptamer-targeted versus scrambled aptamer- targeted SPIONs could be demonstrated in terms of MRI contrast and tumor growth.

The same approach of intercalating doxorubicin with aptamers was used by Kim et al. to target gold-NPs (Au-NPs) for combined CT imaging and therapy [84]. A deliberate strategy to target PC cells independent of PSMA-expression was explored by Min etal. who used aptamer-doxorubicin conjugates for thermally cross- linked SPIONs. Moreover, the DUP-1 peptide aptamer was added for a double targeting approach of PSMA-positive and PSMA-negative PC cells [85]. The DUP- 1 peptide was identified by phage display on PSMA-negative DU145 cells and cross-tested in PC3 cells and corresponding mouse xenografts [86]. Cell viability indeed decreased in vitro for both LNCaP and PC3, but not for HeLa cells.

Considering the reliable overexpression of PSMA at virtually all stages of PC, the need for a double targeting approach remains debatable and in vivo results using this approach would be of high interest. Leach etal. used SPIONs as a drug delivery system with intercalated doxorubicin in developed aptamer RNA-DNA hybrids on the surface, showing decreased viability for targeted SPIONs in LNCaP compared to PC3 cells and non-targeted SPIONs in vitro [87]. However, untargeted SPIONs were devoid of a non-binding ligand and no information was provided about differences in size or zeta potential between these two probes. Both parameters are known to influence intracellular uptake. Multifunctional nanospheres with SPIONs, quantum dots and paclitaxel, embedded in a PLGA layer at the surface of the

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Chapter I

25 spheres, and functionalized with a PSMA-antibody for PC diagnosis and therapy were described by Cho etal. [88]. Higher uptake in LNCaP cells was shown in vitro and a preliminary in vivo study revealed a potential preferential tumor uptake. Still, results of only a single treated mouse and untreated control mouse are presented, impeding conclusions on the contribution of active targeting in comparison to passive tumor accumulation.

Towards the combination of PET and MRI, Moon etal. developed a promising dual-mode imaging probe of ⁶⁸Ga-labeled SPIONs functionalized with the small molecule targeting ligand ACUPA showing specific binding in vitro and in vivo [89].

Behnam-Azad et al. also demonstrated the benefit of ACUPA-functionalization for targeting of PSMA-PIP tumors by starch-coated SPIONs [90]. To compare preferential tumor uptake due to active targeting in PSMA-positive and PSMA- negative tumors, this tumor model offers the advantage of the presence of both tumors, PC3-PIP and PC3-flu, in the opposite flanks of the same mouse, as shown in this study. During the first 24 h, similar accumulation appeared for targeted SPIONs in both tumors due to a combination of passive and active targeting.

However, while PC3-flu accumulation reached a plateau, it further increased in PC3- PIP tumors resulting in a 5-fold higher signal after 48 h revealed by SPECT/CT imaging. Non-targeted SPIONs did not reveal a difference in specific uptake. These results underline the important contribution of PSMA-targeting and internalization of NPs additionally to passive accumulation. Biodistribution by optical imaging additionally confirmed this preferential uptake. As described before, Benham-Azad et al. also showed that an intermediate ACUPA density on the surface of NPs was preferred over high densities, avoiding steric hindrance for PSMA-binding.

Besides the study published by Kim et al. [84], preferential uptake in PSMA- positive cells was shown for PSMA-targeting Au-NPs functionalized with aptamers by Javier etal. [91] and the targeting, phosphorus-based small molecule CTT54 by Kasten et al. [92]. Mangadlao et al. recently described a 4-fold higher Au accumulation, revealed by optical imaging, in PCR-PIP tumors compared to PC3 tumors for small molecule PSMA-1 targeted Au-NPs. The NPs were loaded with the fluorescent photodynamic therapy drug Pc4 for surgical guidance during prostatectomy or therapeutic intervention when surgery is insufficient. [93].

Fluorescent ACUPA-functionalized Au-NPs for supporting surgery by intraoperative

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optical imaging and as diagnostic tool for fluorescence imaging and CT were also explored in LNCaP and PC3 xenografts by Pretze etal. [94]. While the authors claim at least partly receptor-specific tumor uptake, they also refer to the higher tumor vascularization in LNCaP tumor, which NPs can infiltrate better and more deeply, in comparison to PC3 xenografts, independently of active targeting.

To summarize this paragraph, different strategies implying either mAb, aptamers, peptides or small molecules for PSMA-targeting have shown promise as imaging probes. Among these targeting ligands, mAb and aptamers are preferentially used for SPIONs with mAb-NPs generally resulting in a larger size. No striking difference between these two types of ligands in terms of specific targeting could be identified based on the reviewed studies. Ligand density at the surface influences colloidal stability and targeting ability in vitro and in vivo and should therefore be taken into account during preparation of targeted NPs. Concerning in vivo imaging studies in mice, the advantages of the PSMA-PIP and PSMA-flu tumor model to evaluate the contribution of specific targeting in comparison to passive accumulation, together with biodistribution, were highlighted in different studies.

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Table 3. Overview of PSMA-targeted NPs investigated in preclinical studies for imaging. t = targeted NPs, nt = non-targeted NPs, PB = Prussian blue. All in vivo studies were performed in xenograft mouse models if not otherwise stated. NPSynthesis method

Drug/ imaging agentLiganddh [nm] ζ-potential [mV] PathologyTherapy/ imaging modalityCell lineMain findingsRef. SPIONsCommercial (nanomag®-D- spio)ØmAb J59124.68 ± 0.22ØEarly PCMRI In vitro: LNCaP, DU145

1.45 (t) vs 0.33 mM (nt) in LNCaP, 0.23 (t) vs 0.30 mM (nt) in DU145 (intracellular Fe concentration)

Abdolahi et al. 2013 [75] SPIONsØØmAb J591111-9PCImaging and therapyIn vitro: LMD- MB-231, LMD- MB-231-PSMA

5-fold higher uptake (t vs nt in LMD-MB-231- PSMA, ICP-MS)

Mukhherje e et al. 2017 [77] SPIONsSolvent evaporation methodØmAb J591110ØPCMRI

In vitro: C4-2B, LNCaP-LN3 In vivo: LNCaP-luc ortothopic

Higher uptake in t compared to nt (confocal, PB) Specific targeting vs untargeted (T2 signal)

Tseet al. 2015 [78] Pt-doped SPION micellesThin-film methodPaclitaxel mAb J59145 ± 25ØPCMRI, chemotherapyIn vitro and in vivo: C4-2

Increase in cyto- toxicity (t vs nt) In vivo: 4 / 3 times more pactitaxel / Pt in tumor, contrast enhancement (t vs nt)

Taylor et al. 2012 [79, 95] SPIONs

Commercial (Ferumoxytol, AMAG Pharmaceuticals, Lexington, MA)

Doxorubicin, paclitaxel, enzalutamide, BEZ235, cabazitaxel, riluzole

Small cyclic peptide36-40Different stages of PC MRI staging, therapy adapted to disease stage

In vitro and in vivo: LNCaP, PC3-PSMA, PC3

Significant difference in vitro (fluorescence and T2 signal, PB) and in vivo PC3 vs PC3-PSMA

Kaittanis et al. 2017 [80] SPIONsCo-precipitation and hydrothermal treatment ØAptamer A1072 ± 10-35.5 ± 1.1

Early PC lymph node metastases

MRI, hyperthermiaIn vitro: LNCaP, PC3 In vivo: MAT-LyLu (rat) 4.5-fold higher uptake (LNCaP vs PC3in- vitro, PB, fluorescence), MRI detectability in vivo

Maudens et al. 2018 [81] SPIONsThermally cross- linkedDoxorubicinAptamer 65 ± 12 (w/o doxorubicin)-23 ± 1PCMRI, chemotherapyIn vitro: LNCaP, PC3 In vivo: LNCaP

2-fold decreased cell viability in LNCaP vs PC3, decrease in T2 In vivo: tumor growth after 25 d: 3.4-fold (t) vs 5.2-fold (nt, scr.apt.) Yuet al. 2011 [83]

Chapter I

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