Nanomedicines for Diagnosis and Treatment of Prostate Cancer

Nanomedicines for Diagnosis and Treatment of Prostate Cancer

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Nazila Kamaly, Archana Swami, Ryan Wagner, and Omid Cameron Farokhzad

Nanomedicines for Drug Delivery Nanotechnology for healthcare applications involves the development of nanomedicines, which to date have had a major impact in the fields of drug delivery and diagnostics. Nanomedicines are small structures within the typical range of 1–100 nm that are capable of transporting thousands of therapeutic drug and/or imaging agent molecules either encapsulated or covalently bound within their nanostructures to sites of disease. The impact of nanotechnology on healthcare is now rapidly recognized as funding and investment from both government and private sectors in this area continue to increase at a fast pace [1, 2]. Research at the nanoscale has led to the development of several major nanomedicine platforms for drug delivery which include liposomes, polymeric nanoparticles (NPs), micelles, and dendrimers [3]. Lipid-based liposomes were the first nanomedicines developed to improve the pharmaceutical efficacy and dosage of preexisting N. Kamaly, Ph.D. • A. Swami, Ph.D. • R. Wagner Department of Anesthesiology, Brigham and Women’s Hospital, 75 Francis Street, Medical Research Building, Boston, MA 02115, USA e-mail: [email protected]; [email protected]; [email protected] O.C. Farokhzad, M.D. (*) Department of Anesthesiology, Brigham and Women’s Hospital, 75 Francis Street, Neville House, Boston, MA 02115, USA e-mail: [email protected]

approved cytotoxic drugs. An example of this is the FDAs approval of the first NP product, Doxil (doxorubicin-liposome), in 1995 which was used for the treatment of AIDS-related Kaposi’s syndrome [4]. Through encapsulation of doxorubicin, the pharmacokinetics and biodistribution of this drug were greatly enhanced, leading to higher tumor drug concentrations and therefore lower side effects. This initial achievement in nanodrug delivery paved the path for the development of more effective nanomedicines, which with their unique designs and properties are set to contribute in a major way to healthcare and drug delivery in the future [5]. The packing of drugs inside nanocarriers has further advantages from a pharmaceutical research and development point of view, since nanomedicines are commonly judged and validated against the preexisting free form of their drug load, and as such pharmaceutically suboptimal drugs can be reevaluated and their clinical use extended in this manner [6]. Given the systemic toxicity of common chemotherapies and damage to healthy tissue by radiotherapies, and the unfavorable biodistribution of drugs to achieve maximal doses at tumor sites, there is clearly a need for the investigation of more efficient drug delivery approaches. In particular, targeted nanomedicines that are specific to PCa cells are needed in order to deliver cytotoxic drugs with minimal damage to the healthy structures which surround the prostate such as neurovascular bundles, the sphincter, and the rectum.

T.J. Polascik (ed.), Imaging and Focal Therapy of Early Prostate Cancer, Current Clinical Urology, DOI 10.1007/978-1-62703-182-0_15, © Springer Science+Business Media, LLC 2013

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204 Table 15.1 Nanomedicines in clinical development for the treatment of PCa and solid tumors NP name BIND-014

CALAA-01 CRLX101

C32/DT-A

Doxil and Taxotere

MFL AS

Paclitaxel and Lapatinib

Status (trial status) Phase I

System Polymeric NP loaded with docetaxel targeted to prostate-specific membrane antigen Cyclodextrin-containing polymer with siRNA Camptothecin (CPT) conjugated cyclodextrinbased polymer

Indication Solid tumor cancers

Toxin-based suicide genes Cationic polymer NP -poly(b-amino ester) polymer Doxil – PEGylated liposome encapsulating doxorubicin

Benign prostatic hyperplasia (BPH) and localized PCa

Magnetic NPs in combination with LDR brachytherapy Paclitaxel albumin-stabilized NP formulation

Localized PCa

Solid tumor cancers Advanced solid tumor cancers

Advanced AndrogenIndependent PCa

Advanced solid tumor cancers

Advances in nanotechnologies have led to the development of a range of polymeric and liposomal nanomedicines for the treatment of prostate tumors. Table 15.1 presents a list of nanomedicines that are currently investigated in clinical trials for the treatment of PCa. Perhaps one of the most attractive advantages of nanomedicines is the fact that they can be developed and manufactured in a versatile manner, allowing for their physical properties to be easily altered and tuned to a specific application. NP properties such as composition, size, surface charge, load, specificity, toxicity, and degree of selectivity can be modified according to the nature of the organ, tissue, or cell population identified for therapy. Therefore, NPs can be envisaged as a tool-kit technology with tuneable properties toward various applications involving therapeutic drug delivery and/or diagnostic imaging. Nanomedicines can be utilized in both a targeted and a non-targeted manner, and more recently

Phase I

Company or center Bind biosciences

References [59]

Calando pharmaceuticals Cerulean Pharma Inc.

[60]

Phase I (Phase II for Non-small Cell Lung Cancer) Preclinical None

[61]

Doxil – Phase II Taxotere – Phase I Phase II

James Graham Brown Cancer Center

[63]

MagForce Nanotechnologies

[64]

Phase I

National Cancer [65] Institute, University of California

[62]

interest in the development of targeted NPs has increased and efforts have been made to enhance NP retention at sites of disease through the conjugation of targeting ligands to their surfaces. This has become an effective strategy for increasing the concentration of therapeutic NPs at tumor sites, owing to the high surface-area-to-volume ratios of NP surfaces which accommodate high ligand densities, in addition to research into the identification and isolation of targeting ligands with high affinities to extracellular domains [7]. In summary, specific to drug delivery applications, NPs provide the following advantages: (1) the encapsulation and delivery of poorly soluble drugs, (2) the reduction of systemic toxicity due to free drug, (3) spatial and temporal controlled release of drugs, (4) highly localized release of drug due to the incorporation of targeting elements onto the NP, (5) the co-delivery of two or more types of drugs to sites of action for combination therapy, (6) the visualization and

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quantitation of drug delivery and/or therapeutic response, (7) the delivery of plasma-sensitive nucleic acids such as siRNA or miRNA in an intracellular manner, and (8) the extension of drug life-cycles post-patent expiration [2, 6].

Polymeric Nanomedicines Polymeric NPs represent a highly effective nanoplatform for drug delivery [8]. An attractive feature of polymer-based drug delivery platforms is their controlled-release properties which is attainable via the use of biodegradable monomers [9]. Polymer NPs are capable of drug encapsulation and release in a temporally controlled manner, a property which can be achieved through surface or bulk erosion of the polymeric NPs, diffusion of drugs out of the polymer mesh network, or swelling of the polymer matrix and subsequent diffusion [10]. Drug release in polymeric systems may also be achieved in a triggered or smart manner in addition to temporal release, which can be manifested by response of the polymers to various triggers in their environment such as pH, heat, enzymes, or exposure to other appropriate sources of energy or chemicals that can break bonds or displace the drug molecules [10]. Their property of longitudinal controlled-drug release renders polymeric nanomedicines highly appealing as drug delivery vehicles, since drug release can be optimally achieved at the site of action for over long periods, without the patient requiring repeat dosing. Examples of drug delivery and imaging applications of polymeric NPs will be discussed in the subsequent sections of this chapter.

Nontargeted Nanomedicines for PCa Therapy Oncology is one of the areas where nanomedicine has had the most impact to date, in particular with many NPs developed for the treatment and imaging of PCa [11, 12]. Both actively and passively targeted nanomedicines have been extensively utilized for oncology applications, in particular for drug delivery to solid tumors.

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If drug delivery NPs are to successfully reach the tumor and effectively deliver their drug load to cancer cells, they must be capable of effectively maneuvering the complex in vivo environment, and in particular avoiding clearance by the host phagocytic cells post-systemic administration. Indeed the dose of NPs that reach the tumor is dependent on a number of biophysicochemical properties of the NPs. These include the chemical signatures of the constituent material, be it lipids, polymers, or other inorganic components; the nature of the encapsulated drugs; the size and shape of the NP; and surface charge and hydrophilicity. In addition to these NP parameters, the tumor microenvironment, such as the degree of vascularization, necrosis, and size can also be factors influencing the navigation of NPs within tumors and hence their therapeutic efficacy. In the case of nontargeted NP-mediated drug delivery, the non-heterogeneous nature of the tumor microenvironment and the presence of abnormal leaky vasculature, termed the enhanced permeability and retention (EPR) effect, are beneficial as NPs can effectively extravasate through these distorted blood vessels and accumulate in tumor tissues at high concentrations. The degree of extravasation is inversely proportional to NP size, and smaller particles with sizes < 150 nm are deemed to be most effective at trans-endothelial passage into tumor tissue [13]. Considering that docetaxel (Dtxl) is the common first-line chemotherapeutic treatment in castration-resistant PCa and given the adverse effects of Taxotere, which is the clinically approved formulation of Dtxl, Cervin et al. formulated liquid crystal nanoparticles (LCNPs) and showed these NPs to be effective at inhibiting PC3-induced tumors in SCID mice [14]. These 80–90 nm NPs were formulated with the lipids phosphatidyl choline, glycerol dioleate, and polysorbate 80, and their efficacy on tumor growth post-systemic administration was compared to that of Taxotere and empty LCNPs. In one study, the results from these nontargeted and passively accumulating NPs showed that the LCNP/Dtxl formulation led to the highest level of tumor regression of volumes decreasing to 10% or less in comparison to 18% and 70%

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for Taxotere, 10 days post-administration (total Dtxl dose of 1.62 mg used for entire course of study) [14]. The EPR effect can be successfully utilized for drug delivery to solid tumors, and other disease indications that lead to impaired lymphatic drainage and aberrant vasculature. However, active targeting using affinity ligands may lead to more efficient retention of nanomedicines within tumor sites [15].

Targeted Nanomedicines for PCa Diagnosis and Therapy Targeted NPs have the advantage of faster retention within tumors due to selective binding to overexpressed receptors on the surface of cancer cells. It is envisaged that the development of targeted NPs which represent the next generation of nanomedicines entering the clinic will lead to more effective therapeutic and imaging agents for PCa [16–18]. These vehicles can be engineered to recognize biophysical characteristics that are unique to the cancer cells. Most commonly, this recognition process involves the binding of vehicles to antigens that are expressed on the plasma membrane of the targeted cells. Small molecule ligands, antibodies, antibody fragments, and peptides have been used as targeting moieties in targeted nanoparticle development. A particular class of targeting ligands with high affinity to PCa cells is aptamers (Apts). Aptamers are single-stranded DNA or RNA oligonucleotides that fold into well-defined 3D structures that are capable of high affinity toward proteins, phospholipids, sugars, and nucleic acids [19]. Aptamers are ideal targeting ligands as they are non-immunogenic and exhibit remarkable stability in a wide range of pH (4–9), temperature, and organic solvents, with minimal loss of activity [20]. Furthermore, Apts can be chemically synthesized without the need for biological production, leading to reduction in batch-to-batch variability, which is one major advantage of Apts in comparison to larger and bulkier antibodies that can elicit toxic immune responses and are not easily produced on large scales [13].

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We have shown that the generation of NP–Apt bioconjugates with nuclease-stabilized A10 2-fluoropyrimidine RNA Apts that bind to the prostate-specific membrane antigen (PSMA) efficiently targets and enters prostate epithelial cells which express the PSMA protein [21]. PSMA is a well-known characterized transmembrane protein that is overexpressed on PCa epithelial cells, is involved in membrane recycling, and becomes internalized through ligand-induced endocytosis [22]. Using the FDA approved biodegradable drug delivery polymer poly(d,l-lactic-co-glycolic acid) (PLGA) as a controlled release polymer, we were able to develop NPs with surfaces decorated with Apts that were targeted to the PSMA on the surface of PCa cells [20]. The developed Apt-targeted polymer NPs demonstrated a 77-fold increase in binding to prostate LNCaP cells in comparison to nontargeted polymeric NPs. To protect these particles from macrophages and increase their circulation time in vivo, the particles were also functionalized with the widely used poly(ethylene glycol) (PEG) polymer. These proof-of-principle studies using Apts as targeting ligands led us to develop and pioneer Apt-conjugated polymeric NPs to improve the therapeutic index of drugs by targeted delivery and controlled release to PCa cells [21, 23]. To achieve differential cytotoxicity against PCa cells, Apt-conjugated polymeric NPs were loaded with Dtxl, which has been demonstrated to prolong survival of patients with hormone-resistant PCa [20]. Figure 15.1 presents the general procedure used to prepare Dtxlencapsulated pEGylated PLGA NP–Apt bioconjugates, which were formulated using a nanoprecipitation technique. The targeted polymeric NPs were formulated by first co-precipitating Dtxl with the diblock polymer poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG), followed by surface functionalization with the A10 Apt, with affinity to the extracellular domain of PSMA [23]. Using this method, the hydrophilic PEG polymer protrudes outward on the surface of the polymeric NP core, and the terminal carboxy functionalities can then be conjugated with the A10 PSMA Apt targeting ligand. Additionally, the negative charge of the carboxy-

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Fig. 15.1 Development of Dtxl-encapsulated PEGylated PLGA NP-Apt bioconjugates. (a) Synthetic scheme and (b) representative SEM image of the targeted NPs. From

Farokhzad et al. [20], with permission. Copyright 2006 National Academy of Sciences USA

late groups prevents the nonspecific interaction of the Apt molecules with the surface of the NPs [20]. The efficacy of the Apt NPs was investigated using a mouse xenograft model of PCa and the NPs were injected intratumorally in LNCaP generated solid tumors (Fig. 15.2). PCa was induced in mice by implanting LNCaP prostate epithelial cells s.c. in the flanks of nude mice and allowing the tumors to develop to appropriate sizes (~300 mm3). The tumor size and weight were monitored for up to 109 days and results revealed that a single intratumoral administration of Dtxl– NP–Apt Bioconjugate NPs was significantly effective at tumor size reduction compared to the nontargeted NP controls. The Apt conjugated NPs are believed to bind to PSMA on the surface of

the LNCaP cells and become endocytosed, allowing their cytotoxic Dtxl cargo to be delivered post-endosomal release. This study demonstrated how the therapeutic index of Dtxl can be improved with the Dtxl-encapsulated PLGA-PEG-Apt NPs showing almost complete tumor reduction and 100% survival, compared with the survivability of 57% for nontargeted PLGA-PEG NPs and 14% for Dtxl alone (Fig. 15.2) [23]. The materials used in the development of these bioconjugated NPs are FDA approved and the Apts are small in size, relatively stable, nonimmunogenic, and easy to synthesize, which can facilitate the translation of these nanomedicines into clinical practice [23]. However, it is important to note that, akin to other nucleic acid-based ligands, Apts may also suffer from nonspecific interactions after systemic

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Fig. 15.2 In vivo efficacy study of Apt–NP bioconjugates and controls. (a) Comparative efficacy study of a single intratumoral injection of Apt–NP bioconjugates and controls (40 mg/kg total Dtxl dose). (b) Representative mice at each end point (Left) alongside images of excised tumors (Right). Black arrows; position of the implanted tumor on each mouse. (c) Plot of outcomes for each of the

treatment groups, complete tumor regression (blue), incomplete tumor regression (red), tumor growth (yellow), and mortality (black). (d) Kaplan–Meier survival curve (end points defined as tumor load of 800 mm3 or BWL ~20%). From Farokzad et al. [20], with permission. Copyright 2006 National Academy of Sciences USA

administration and thus it is important to engineer optimal NP surface properties and ligand densities that lead to effective targeting of nanomedicines at sites of disease. In addition to Dtxl delivery, Apt–NP bioconjugates have also been developed for the delivery of cisplatin to PCa cells [24]. Cisplatin is the most potent member of the Pt anticancer drug family, and its use in PCa therapy is therefore highly attractive. We devised a strategy for cisplatin therapy in PCa that employed Pt chemistry and NP delivery vehicles [24]. In this study, a hydrophobic platinum (IV) prodrug was synthesized for encapsulation into polymeric PLGA-bPEG NPs using the nanoprecipitation method, which resulted in highly loaded NPs of appropriate size. The NPs were then targeted to PSMA by decorating the surface of the particles with A10 Apts that specifically bound to the extracellular domain of PSMA. The Apt-facilitated cellular

uptake of the Pt(IV)-encapsulated NPs by PSMA expressing LNCaP cells via endocytosis was demonstrated using an antibody specific for endosome formation. The Apt-derivatized Pt(IV)encapsulated NPs were shown to be significantly superior to cisplatin or nontargeted NPs in inhibiting LNCaP cellular growth. Moreover, by encapsulating a cisplatin prodrug, PLGA-PEGApt NPs displayed significant dose-sparing, with equivalent antitumor efficacy in LNCaP xenografts achieved at only a third of the conventional administered dose of free cisplatin (0.3 mg/kg vs. 1 mg/kg) [25].

Nanoparticles for Sensitive Diagnosis of PCa Novel targeted NPs for the detection of extremely small amounts of PSA were recently developed

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by Mirkin and coworkers that are capable of detecting PSA at femtogram levels [26, 27]. This system utilizes magnetic microparticles conjugated with antibodies that extract trace levels of PSA from patient’s blood, and then PSA levels are detected using gold NPs that posses PSAspecific antibodies and short DNA sequences that act as “barcodes.” This development can lead to more efficient detection and monitoring of PSA levels posttreatment and therefore PCa outcomes.

Modular Self-assembly of Targeted Nanomedicines for PCa Conventional methods of synthesizing targeted NPs involve serial chemical processing of particles, whereby drug-encapsulated NPs are first formed, followed by the conjugation of targeting ligands to their surface. This post-conjugation requires the addition of an excess amount of reactants to ensure high coupling efficiencies, after which the ligand-conjugated NPs need to be further purified by removing the excess reactants. This added complexity makes it difficult to adjust the NP surface properties in a reproducible manner and the multistep processing contributes to premature drug release from particles, resulting in batch-to-batch variability of NP surface properties and drug loading and release characteristics. To precisely engineer targeted NPs in a simple and scalable manner, an innovative strategy was developed by first pre-functionalizing polymer components with targeting ligands, and then self-assembling the polymers into NPs [28]. Figure 15.3 presents the development and characterization of PLGA-PEG-Apt triblock polymers, and the self-assembly of targeted NPs simply by nanoprecipitating the mixture of PLGA-PEG, PLGA-PEG-Apt, and drug. In this manner, by using distinct ratios of PLGA-PEGApt and PLGA-PEG during NP formulation, the Apt surface density can be precisely tuned. Thus, this approach could eliminate the need for postparticle modification and purification, enabling the formulation of distinct targeted NP libraries. Other types of polymer conjugates have also been used effectively for drug delivery to PCa.

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The delivery of Dtxl using actively targeted N-(2hydroxypropyl)methacrylamide (HPMA) copolymer conjugates consisting of covalently bound Dtxl and the RGDfk targeting ligand was recently demonstrated by Ray et al. [15]. Their small sized RGDfk bioconjugate polymeric nanoparticles were shown to inhibit the proliferation of human prostate cancer DU145 and PC3 cells in vitro and effectively lead to the regression of DU145 tumor xenografts in nu/nu mice in vivo, following a single dose of either 20 mg/kg or 40 mg/kg of Dtxl [15]. With advances in nanoengineering technologies for the development of targeted NPs and their high-throughput screening, the potential to rapidly develop targeted NPs and accelerate their clinical translation is now therefore feasible.

Novel Nanomedicine Approaches for PCa Treatment and Monitoring Newly developed nanomedicines have shown promising results in the treatment of PCa, by achieving an improvement in drug biodistribution and therapeutic index. In particular, targeted NP delivery has led to the improvement of sitespecific drug delivery. In addition to exploring further specific prostate cell-surface antigens and the delivery of improved PCa drugs, other avenues such as combination therapies, precisely engineered nanomedicines, and the use of smart theranostic NPs also present exciting and promising avenues for PCa treatment and monitoring in the future.

Combination Therapies Combination therapy by co-delivering multiple drugs via targeted polymeric NPs was proposed to address the challenges of single-agent chemotherapy [29], with several advantages which include the following: (1) co-delivery of precise drug ratios to targets of interest for synergistic therapeutic effects, (2) control over drug resistance, and (3) control of co-drug exposure in a temporal manner. In a proof-of-concept study,

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Fig. 15.3 Development of self-assembled targeted NPs. (a, b) Synthesis and characterization of PLGA-PEG-Apt triblock polymer. (c) In situ self-assembly of PLGA-PEG-

Apt NPs by the nanoprecipitation method. From Gu et al. [28], with permission. Copyright 2008 National Academy of Sciences USA

cisplatin and Dtxl were effectively co-delivered to PCa cells using a targeted PLGA-PEG NP platform with synergistic cytotoxicity [29]. The hydrophilic Pt(IV) cisplatin prodrug was first conjugated to a polylactide polymer derivative with pendant hydroxyl groups (PLA-OH) to yield a PLA-Pt(IV) copolymer, and subsequently blended with PLGA-PEG and Dtxl by a nanoprecipitation process (Fig. 15.4) [29]. The dual-drug encapsulated NPs were then conjugated with the A10 Apt to develop a targeted co-delivery NP platform [29]. In vitro studies demonstrated that the Apt-targeted, dual-drug encapsulated NPs were ~ 5.5-10 times more cytotoxic than respective single drug encapsulating NPs (PLA-Pt-NPApt and Dtxl-NP-Apt) [29]. In addition to delivering multiple types of drugs using Apt–NP bioconjugates, NP systems

have also been developed that are capable of simultaneous delivery of both hydrophobic and hydrophilic drugs. A novel targeted drug delivery system consisting of NP–Apt bioconjugates that can simultaneously deliver both a hydrophobic taxane and a hydrophilic nucleic acid intercalating drug to cancer cells was recently developed by Zhang et al. [30]. PSMA expressing LNCaP prostate adenocarcinomas were chosen as the target cell line for in vitro testing, and PC3 prostate adenocarcinomas, which do not express the PSMA antigen, were employed as a negative control [30]. To visualize cell uptake of drugs using fluorescence microscopy, a hydrophobic fluorescent probe, NBD cholesterol, was encapsulated within the PLGA-b-PEG NPs since this dye has properties similar to a hydrophobic drug, and Dox was chosen due to its fluorescence

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Fig. 15.4 Co-delivery of Dtxl and Pt(IV)-monosuccinate prodrug. From Kolishetti et al. [29], with permission. Copyright 2010 National Academy of Sciences USA

emission spectrum which is in the red region and which can therefore be used for visualization of the NPs in the cells [30]. Both NBD and Dox were effectively delivered to LNCaP cells by NP-Apt bioconjugates with minimal signal observed from the control PC3 cells. In vitro cellular cytotoxicity of targeted NP-Apt bioconjugates carrying both Dtxl and Dox [(Dtxl)Apt(Dox)], Dtxl alone [(Dtxl)–Apt], Dox alone [NP-Apt(Dox)], or no drug [NP-Apt] on LNCaP and PC3 cell lines was then investigated [30]. MTT cell proliferation assay results revealed that for LNCaP cells treated with the same dose of drugs, the [(Dtxl)-Apt(Dox)] NPs were most cytotoxic in comparison to the other controls [30]. These results demonstrated that co-delivery of Dtxl and Dox could potentially be more cytotoxic than the single delivery of either drug alone. Further studies are required to investigate the release kinetics of each individual drug in combination therapies and the results of this action on combinatorially administered drug encapsulating NPs. As such the further optimization of targeted NPs may allow co-delivery of two distinct classes of drugs with varying properties for PCa therapy, and could potentially allow the delivery of different drugs to distinct subcellular compartments.

The concurrent administration of chemotherapy and radiotherapy, which is termed chemoradiation, has led to significant improvements in local tumor regression and, therefore, survival rates. However, chemoradiation has the disadvantage of high toxicity, thereby limiting patients with poor health from undergoing treatment. To improve efficacy and lower toxicity of this combination therapy, lipid–polymer hybrid NPs were developed for the co-delivery of chemotherapeutics and radiotherapeutics (ChemoRad NP) [31]. These lipid–polymer hybrid NPs, which could benefit from the unique properties of both liposomes and polymeric NPs while overcoming some of their limitations, were prepared by nanoprecipitation and self-assembly of PLGA polymers and biocompatible lipids [32]. Compared to PLGA-PEG NPs, the hybrid NPs present several advantages such as higher drug loading and slower drug release, which are mainly attributed to the existence of a lecithin monolayer at the interface of the PLGA core and PEG shell. For targeted co-delivery of chemotherapeutics (Dtxl) and radiotherapeutics (yttrium90), the ChemRad NPs were engineered by self-assembling PLGA, lecithin, DSPE-PEG, DSPE-PEG-Apt, and DMPE-DTPA in a single-step manner [31]. It was proposed that the DMPE-DTPA mono-

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layer can efficiently chelate with radioisotopes, while the PLGA core can carry Dtxl with a high loading efficiency [31]. The targeted ChemoRad NPs showed much higher therapeutic efficacy than respective chemotherapy and radiotherapy treatments [31], suggesting the potential of these NPs for clinical translation and improvement of chemoradiation therapies.

Theranostic Nanomedicines NPs that are capable of both carrying a drug load to treat tumors and imaging agents that allow the visualization of cancer can have a major impact on cancer treatment and monitoring. These types of particles are termed “theranostic” and can facilitate disease diagnosis, treatment, and monitoring via a single NP [33–36]. It is envisaged that nanotheranostics will facilitate a point-ofcare approach toward the treatment of cancers such as PCa. One of the most useful advantages of nanotheranostic agents is their utilization in the optimization of drug delivery systems where information such as biodistribution of the NPs and the effect of their therapeutic payload at the target site can be monitored in a spatiotemporal and longitudinal manner. Additionally, “smart” nanotheranostic agents currently in development provide the added benefit of triggered drug release, allowing for drug release at an optimal time point in the biodistribution of the nanomedicine and suited to a patient’s disease progression stage [33, 37]. For example, recently a smart CdSe/ZnS core-shell quantum dot (QD)–Dox– Apt system was engineered that was capable of sensing drug release in a simple and easily detectable manner [38]. In this theranostic system, the fluorescence of both QD and Dox can be quenched by the intercalation of Dox within the A10 Apt (“OFF” state), through a bi-fluorescence resonance energy transfer (Bi-FRET) mechanism [38]. Upon the specific uptake of QD–Dox–Apt conjugates into target cancer cells via receptormediated endocytosis, the release of Dox from the conjugates induces the recovery of fluorescence from both QD and Dox (“ON”

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state), thereby sensing the intracellular release of Dox and enabling the simultaneous fluorescent localization and destruction of cancer cells. Nanotheranostic agents based on superparamagnetic iron oxide NPs were developed with Apt targeting by Yu et al. [39]. These particles which contained a CG-rich duplex containing PSMA Apt-conjugated to iron oxide nanoparticles (termed Apt-hybr-TCL-SPION) showed affinity toward LNCaP prostate cancer cells that overexpressed PSMA, both in vitro and in vivo [39]. This binding was measured using T2 magnetic resonance imaging (MRI). These NPs also exhibited selectivity toward LNCaP xenograft tumors in vivo, demonstrating their potential use as novel PCa targeted nanotheranostics. Dual-imaging theranostic nanoparticles that were capable of targeted noscapine delivery to uPAR overexpressing PCa cells were investigated by Abdalla et al. [40]. In this study, noscapine which is a tubulin binder and tumor growth inhibitor was adsorbed onto polymeric iron oxide NPs, to which was conjugated a Cy5.5-labeled humantype 135 amino-acid amino-terminal fragment (hATF) of urokinase plasminogen activator (uPA), which is considered a high-affinity natural ligand for the urokinase plasminogen activator receptor (uPAR). These uPAR targeted duallabeled drug-loaded theranostic NPs were capable of selective binding to PC-3 cells and up to 6-fold more cytotoxic to these cells in comparison to free noscapine. These NPs have the potential to more effectively deliver the antitussive agent noscapine, in addition to facilitating tumor imaging and treatment response monitoring for PCa therapy.

Precise Engineering of Nanomedicines Using Microﬂuidics One major consideration for the successful development of targeted NPs is the ability to identify and screen optimal NP biophysicochemical characteristics that could result in an enhanced biodistribution and specific delivery. It is evident that particle size and surface properties play a major

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role in NP uptake by the mononuclear phagocyte system (MPS) cells in various organ systems [41]. Studying the effect of targeting ligand density has also revealed a relatively narrow window of ligand density that could result in the most favorable biodistribution of targeted NPs [28]. We have recently developed a microfluidic technology that enables reproducible preparations of small and homogeneous PLGA-PEG NPs, hybrid lipid–polymer NPs, and lipid-QD NPs through rapid mixing [42, 43]. By simply varying the flow rates, particle compositions, and precursor concentrations into the microfluidic device, the properties of the resulting NPs can be systematically and reproducibly controlled, which presents an opportunity to develop a high-throughput platform to rapidly synthesize libraries of distinct targeted NPs. Beyond NP synthesis, microfluidic systems can also be applied to optimize targeted NPs with high-throughput capability. Microfluidic channels lined with cells were used as a model of microcirculation to screen parameters that affect the interactions between targeted NPs and cells [44]. The development of such microfluidic systems that provide fluid flow conditions has the advantage that conditions for NP screening and studying NP–cell interactions are more representative of the biological microvasculature. In addition, more comprehensive biomimetic microfluidic systems, and “organ-on-a-chip” systems [45], could also be explored for the evaluation and screening of targeted NP systems. As a model system, we studied the interaction of targeted polymeric NPs and microparticles with two prostate cell lines that differ in their expression of the transmembrane prostate PSMA protein [46]. In this study, PEGylated poly(lactic acid) (PLA) NPs or microparticles conjugated to aptamers that recognize the PSMA protein were utilized [46]. The microfluidic channels were seeded with LNCaP or PC3 as model PSMA expressing or nonexpressing cell lines, respectively [46]. Binding to these cells was evaluated with respect to changes in shear stress, the presence or absence of PSMA on target cells, and the particle size, and it was shown that NP–Apt bioconjugates adhered to LNCaP cells but not to

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PC3 cells under various flow rates. In contrast, non-targeted NPs or microparticles failed to demonstrate any significant binding to LNCaP or PC3 cells under any flow rate. We believe that similar systems may be used for a more comprehensive in vitro characterization and optimization of cell particle interactions in order to potentially optimize and engineer these systems prior to animal studies. Depending on the specific application, the ideal vehicle should be able to adhere to its target cell at physiologically relevant shear rates. Therefore, these microfluidic systems can be used to determine the ideal particle size and ligand density on particle surfaces that lead to optimal PCa cellular binding and retention under fluid flow conditions. NPs with smaller sizes are more effective at evading uptake by macrophages and remain longer in the bloodstream. We previously reported a microfluidic technology that enables reproducible preparation of distinct, homogeneous, PLGA-PEG NPs by rapid mixing through a method known as hydrodynamic flow focusing (HFF) [29, 47]. By varying the flow rates of different polymeric precursors into the microfluidic device, NP properties can be systematically controlled in a reproducible manner, allowing for the ability to develop a platform technology to rapidly synthesize libraries of distinct NPs. To reduce NP size, we demonstrated using HFF that polymeric precursors dissolved in an organic solvent miscible with water can be mixed with an antisolvent (i.e., water) in a microfluidic device. This leads to the rapid and homogeneous mixing of acetonitrile with water, which results in a controlled nanoprecipitation process. These NPs have smaller sizes and higher drug loading capabilities than those obtained from bulk nanoprecipitation methods.

Nanomedicines in Focal Therapies and Ablative Technologies for PCa Therapy Focal therapy has been well established for treatment of breast and kidney tumors and is now considered as a significant treatment for early stage

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PCa. Focal treatment is described as “individualized treatment that selectively ablates known disease and preserves existing functions, with the overall objective of minimizing lifetime morbidity without compromising life expectancy” [48]. Focal therapy is capable of completely ablating the clinically significant cancer foci within the prostate using a minimally invasive technique with preservation of the sphincter, normal gland tissue, and the neurovascular bundles [49]. The advantage of this approach is that it maintains the efficacy of current therapies of whole-gland treatment while minimizing treatment and diseaseassociated morbidity and costs [50]. Focal therapy can be achieved using a number of devices or techniques, including thermo-ablative methods, radiation techniques such as brachytherapy, or chemical methods such as regional injections. Currently there are four leading technologies employed for focal therapy: high-intensity focused ultrasound (HIFU), cryotherapy, laser ablation, and photodynamic therapy (PDT). Magnetic resonance hyperthermias using magnetic NPs, as well as electroporation are new approaches that may also hold promise. Internal radiation therapy termed brachytherapy is also utilized for PCa treatment. Selective internal radiation therapy (SIRT) is a widely used brachytherapy in the case of patients with unresectable PCa [51]. In this procedure radioactive microspheres (50–100 mm) are injected into the arteries which supply the prostate tumors. Two different types of microspheres are currently used termed TheraSphere and SIR-Spheres which utilize 125I or 103Pd and 90Y radiation, respectively [51]. However these spheres do not possess affinity or retentive properties to the tumor vasculature and have limited utility in tumor penetration due to their large sizes. Recently, new approaches using NP-based delivery systems for the administration of therapeutic radioactive isotopes have been explored. Kannan et al. investigated the use of Gum Arabicfunctionalized radioactive gold NPs (GA-198AuNP) as agents with high affinity for PCa tumor vasculature and showed that the intratumoral delivery of GA-198AuNP could allow for the delivery of a

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therapeutic radioactive payloads leading to tumor ablation [52]. In a study by Schwartz et al. the utility of NP directed photothermal treatment for PCa was investigated [53]. These NPs were directly injected into exposed canine prostates which were exposed by surgical laparotomy and subsequently irradiated using a NIR laser source delivered using a fiber optic catheter and diffuser. The size of the ablative thermal lesion was found to be within ~4 mm of the optical fiber radius. These authors suggest the potential of this technique for PCa treatment leading to more precise and minimally invasive percutaneous ablation of prostate tumor tissue. In another study, Cadeddu and coworkers investigated the thermal dose range for PCa ablation therapy using quantum dot (QD) fluorescence thermometry [54]. In this study, PC-3 cells with gold nanoshells (GNS) and QDs were exposed to a near-infrared laser and QD excitation light. The cells were heated using the GNS, while local temperature was monitored and measured using the temperature-dependent fluorescence intensity of the QDs. PC-3 cell death which was proportional to thermal energy and was measured with QD-mediated thermometry was investigated and it was shown that cell death could reach ~90% in 120 s. This study shows that QD fluorescence thermometry can accurately monitor PC-3 cell death by laser-heated gold nanoshells (LGNS) ablation. This approach could potentially lead to improvements of thermal ablation procedures in clinical practice. Multiwalled carbon nanotubes (MWNTs) responsive to laser irradiation were used to enhance the treatment of cancer cells using controlled thermal deposition and led to increased tumor injury and diminished heat shock protein (HSP) expression [55]. These MWNTs were capable of greater temperature elevation following laser heating which was used to determine the efficacy of laser treatment alone or in combination with MWNTs. The investigators showed that MWNTs dramatically decreased cell viability and HSP expression in combination with laser irradiation. In another study, the complete eradication

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of PC3 xenograft tumors was achieved in nude mice with MWNTs following near-infrared (NIR) irradiation, after a single intratumoral injection of MWNTs followed by laser irradiation at 1,064 nm, 2.5 W/cm2 [56]. The photodynamic activity of chloro(5,10,15,20tetraphenylporphyrinato)indium(III) loadedpoly(lactide-co-glycolide) NPs in LNCaP prostate tumor cells was investigated by da Silva et al. [57]. In this study, it was shown that the cell viability of LNCaP cells was significantly reduced for the indium-loaded NPs in comparison to the case when the drug is administered in its free form. Prostate focal therapy has been praised as a promising emerging treatment for patients with low risk PCa. However, better imaging techniques and understanding of PCa lesions are required in order to reap the full benefits of this technique compared to whole-gland therapies [58].

Conclusion and Future Perspectives Nanomedicine approaches that allow for a constant dose of chemotherapy to be specifically delivered to cells over extended periods can result in improved therapeutic outcomes for early stage PCa. However, a considerable amount of research and development is necessary from the proof-of-principle stage of developing novel targeted nanomedicines to their bench-to-bedside translation since there is still work to be done in order to create clinical toxicity evaluation protocols that can be used as benchmarks for the investigation of novel nanomedicines and nanomaterials. Nonetheless, nanomedicine is set to make a significant impact on urological clinical practice and thus should be embraced as a viable therapeutic mechanism. Acknowledgments This work was supported by National Institutes of Health (NIH) grants CA151884, EB003647, and N01 HV-08236, and the David Koch— Prostate Cancer Foundation Award in Nanotherapeutics. Dr. Farokhzad declares financial interests in BIND Biosciences and Selecta Biosciences. The rest of the authors declare no conflict of interest.

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