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Controlling the actuation of therapeutic nanomaterials: enabling nanoparticle-mediated drug delivery The implementation of biofunctionalized nanoparticles (NPs) as potential therapeutic materials has seen exponential growth in recent years due to their unique ability to overcome the constraints of current medicine. This has been largely driven by significant advances on a number of basic research fronts including high-quality NP synthesis, bioconjugation, cellular delivery and the controlled release or ‘actuation’ of NP-associated cargos. Cumulatively, these are the key enabling tools for the full realization of NP-mediated drug delivery. In this review, the authors’ focus is on recent developments in methodologies for the controlled actuation of therapeutic NPs. The authors discuss the critical requirements for their integration into biological systems and highlight examples from the recent literature where controlled NP actuation has been successfully demonstrated. The current state of therapeutic NPs in the clinical setting is summarized and the article concludes with a brief perspective of how we can expect to see this emerging field develop in the coming years. Over the past 10 years there have been significant developments in the interfacing of nanoparticle (NP) materials with biological systems (i.e., cells/tissues/whole organisms) for a variety of applications, spanning from the labeling/imaging of cellular structures [1,2] to the in situ real-time measurement of cellular processes [3,4]. These endeavors have been largely facilitated by progress along a number of basic science research fronts including improved methodologies for: n The synthesis of high quality NP materials; NP biocompatibility and bioconjugation of biologicals (e.g., proteins, peptides, drugs);

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NP delivery to cells and tissues;

is integral and it can take a number of various forms that span a continuum of complexity and sophistication in terms of their construction and use. In the context of this review, we define nanoparticle actuation as any modality that is employed to affect the controlled release of a NP-associated therapeutic cargo(s). To date, several methods for achieving the actuation of therapeutic NP cargos have been demonstrated. In this review, we refer to these various actuation modalities as: n Passive NP actuation; Active/extracellularly triggered NP actuation;

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James B Delehanty*1, Joyce C Breger1, Kelly Boeneman Gemmill1, Michael H Stewart2 & Igor L Medintz1 Center for Bio/Molecular Science & Engineering, Code 6900, US Naval Research Laboratory, Washington, DC 20375, USA 2 Division of Optical Sciences, Code 5600, US Naval Research Laboratory, Washington, DC 20375 USA *Author for correspondence: Tel.: +1 202 767 0291 Fax: +1 202 767 9594 E-mail: [email protected] 1

Active/intracellularly triggered NP actuation.

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The controlled release of NP-appended therapeutic cargos (Figure 1) [5,6]. This latter aspect, referred to herein as ‘controlled actuation’ is central to the rapidly expanding fields of NP-mediated drug delivery (NMDD) and theranostics [7,8]. While the former uses the NP scaffold to mediate the enhanced delivery of therapeutic drugs, the latter seeks to combine both diagnostic and therapeutic drug delivery functions on a single NP vector. Here, the goal is to simultaneously take advantage of NPs’ small size and their large surface area to deliver large drug doses to targeted areas that are not accessible by other materials [9]. In both regimes, the controlled actuation of NP-associated cargos

Passive NP actuation involves the simple efflux of the cargo from within the NP core or release of the drug cargo from the NP surface. The simplest of all of the NP actuation, modalities described to date, passive NP cargo actuation has its roots in the field of cancer chemotherapy where the goal continues to be the NP-facilitated optimization of the therapeutic indexes (i.e., maximal targeted cell killing, minimal non-targeted cell necrosis) of often highly toxic drugs through the modulation of the drug’s release and systemic distribution kinetics. Examples of NP materials used for passive actuation include liposomes [10], dendrimers [11], proteins [12] and solid NP scaffolds [13]. While this NP actuation strategy offers the

10.4155/TDE.13.110

Ther. Deliv. (2013) 4(11), 1411–1429

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ISSN 2041-5990

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Review | Delehanty, Breger, Gemmill, Stewart & Medintz Key Terms

nanoparticle vectors for the enhanced delivery of and improved therapeutic index of therapeutic drugs. Nanoparticles’ small size, large surface area and amenability to controlled drug release schemes have driven the rapid expansion of nanoparticle-mediated drug delivery.

Theranostic: Material that

integrates both a diagnostic and therapeutic functionality into a single scaffold for the simultaneous identification and treatment of diseased cells/tissue. The use of nanoparticles as theranostics is a direct extension of nanoparticle-mediated drug delivery.

Nanoparticle actuation:

The controlled or triggered release of nanoparticleappended or nanoparticleimpregnated drugs (or other therapeutics) by either passive or active means.

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Biocompatibility/ biofunctionalization

Peptides Bioconjugation

Proteins Nucleic acid Drugs

Therapeutic nanoparticle

Cellular delivery/uptake – Cytosolic delivery – Organelle targeting

Controlled actuation of nanoparticle-drug cargos Increasing complexity

Nanoparticle-mediated drug delivery: The use of

– Particle – Active/extracellularly-triggered – Active/intracellularly-triggered

Labeling Imaging Tracking Sensing Drug delivery

Figure 1. Requisite properties for the controlled actuation of therapeutic nanoparticles. Pertinent attributes required for the controlled actuation of therapeutic cargos within biological systems. (A) The controlled bioconjugation of disparate functional biologicals (peptides, proteins, nucleic acids, drug molecules) mediates both nanoparticle (NP) targeting and therapeutic effects. (B) Actuatable NP systems require high quality, biocompatible NP materials that can be functionalized for enhanced stability and targeting to cells/tissues. (C) The controlled actuation of NP-associated cargos represents the current forefront of interfacing NPs with cells/tissues. Actuation modalities span a continuum of complexity ranging from the simplest passive efflux systems to more elegant systems that are triggered by extra- and intracellular-specific stimuli. (D) Targeted cellular delivery and uptake of NPs affords labeling, imaging and sensing combined with therapeutic drug delivery.

least amount of spatiotemporal control over the cargo release, it remains the only modality that has gained US FDA approval for clinical use. Active/extracellularly triggered NP actuation utilizes externally applied stimuli to affect the release of NP–cargo conjugates that have (ideally) been pre-targeted to their intended site of function. Approaches here have taken the form of, for example, the photoactivation of labile linkers [14] or the radiofrequency ablation of NP-embedded cargos [15]. In contrast to passive actuation, these externally triggered systems offer the greatest degree of temporal control and when coupled with pre-targeted NP therapeutics, they can realize fine spatiotemporal control over cargo release. Active/intracellularly-triggered NP actuation is the final modality currently under development. Here, the goal is to rely on inherent intracellular processes (pH gradients, redox potential, enzymatic activity) to mediate the on-command release of therapeutic cargos. In this regime, the use of the low pH inherent to the tumor environment [16,17] and the reduction of dithiol linkages [18,19] have emerged as two of the more popular approaches for intracellular cargo release. These actuation modalities are represented schematically in Figure 2 and it is important to appreciate that, cumulatively, they span a continuum Ther. Deliv. (2013) 4(11)

of complexity and sophistication in terms of both the loading and the actuated release of the NP-associated therapeutic cargo. In this review, the authors examine the current trends in NP actuation of therapeutic cargos. In contrast to other excellent recent reviews that have focused primarily on the active actuation of therapeutic NP materials from outside the cell [20,21], on a single NP material [22,23], or on newly emerging engineering and fabrication techniques for controlled drug delivery [24], our purpose here is to review a full breadth of currently available actuation mechanisms across a variety of NP materials, which are triggered either passively or actively from outside or inside the cell. We begin with a discussion of the requisite parameters necessary for their successful implementation within cells, tissues and whole organism settings. We then highlight recent literature examples of each of the three current modalities, including both in vitro and in vivo applications, and then discuss the current state of therapeutic NPs in the clinical setting and the important role played by rigorous toxicity assessment as a critical stepping stone on the pathway to clinical success. Finally, we offer our own perspective/ vision of the developments we expect to see in the coming years. future science group

Controlling the actuation of therapeutic nanomaterials: enabling nanoparticle-mediated drug delivery Methods for NP–cargo conjugation: introducing the therapeutic cargo to the NP carrier In this review, the term conjugation refers to how the therapeutic cargo is introduced into or onto the NP carrier. Cargos can be conjugated to NP carriers in a variety of ways including covalent and noncovalent methods. Here, the physicochemical properties of both the NP carrier and the drug cargo must be considered [5,6]. The NP scaffold can often impose physical restrictions on how it can host the therapeutic cargo. For example, solid NPs such as semiconductor quantum dots (QDs) and metal oxides are only capable of cargo conjugation through their surfaces or through their solubilizing surface ligands. Conversely, the interior spaces of ‘porous’ NPs including silica, liposomes, micelles and polymer NPs can be used to carry cargo inside their structures in addition to conjugation at their periphery.

| Review

The nature of the therapeutic cargo also influences what type of NP should be used for loading and delivery purposes. Sailor and Park recently developed an excellent macroscopic analogy for this purpose wherein they described the NP as a ship and the drug as a cargo that can be loaded either onto the NP surface (NP as a barge) or within the NP interior (NP as a tanker) [25]. For example, drugs that can react with or are easily degraded by their intended environment should be protected by loading them within the NP interior. Furthermore, any drugs that are not biocompatible, acutely toxic or will decrease the circulation time of the NMDD system should also be masked in the interior spaces of NPs. Conversely, drug cargo can be placed on the surface of NPs if the drug provides a targeting attribute, is biocompatible, does not adversely affect circulation time or requires release through biologically targeted stimuli. Below, we briefly outline examples of drug–NP

Passive actuation Drug loading/release mechanism 1. Surface adsorption/desorption 2. Surface conjugation/desorption 3. Nanoparticle core partitioning/efflux Active/extracellularly triggered actuation

1 3

x

2 Continuum of complexity

Active/intracellularly triggered actuation Drug loading/release mechanism 1. Surface adsorption/cell (tumor low pH, extracellular enzymes) or externally applied stimuli (photo, radiowave) 2. Surface conjugation/cell (tumor low pH, extracellular enzymes) or externally applied stimuli (photo, radiowave) 3. Nanoparticle core partitioning/active intracellularly induced release

Drug loading/release mechanism 1. Surface adsorption/cell (tumor low pH, extracellular enzymes) or externally applied stimuli (photo, radiowave) 2. Surface conjugation/cell (tumor low pH, extracellular enzymes) or externally applied stimuli (photo, radiowave) 3. Nanoparticle core partitioning/active extracellularly induced release 1 Extracellular stimulus

3

x

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1 Intracellular stimulus

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Figure 2. Representation of nanoparticle actuation modalities. The various schemes for the loading and actuated release of nanoparticle (NP)-associated therapeutic cargos. These schemes include passive NP actuation, active/extracellularly triggered actuation and active/intracellularly triggered actuation. The fundamental difference between the active mechanisms is the location of the trigger that drives actuated drug release. Cumulatively, these actuation schemes represent a continuum of complexity and sophistication in terms of both the loading/attachment of the NP cargo and the controlled, actuated release of the therapeutic cargo.

future science group

www.future-science.com

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Review | Delehanty, Breger, Gemmill, Stewart & Medintz conjugation for passive, active/extracellularly triggered and active/intracellularly triggered actuation mechanisms. „„NP

loading for passive actuation Multiple examples of NMDD systems that employ passive actuation appear both in the literature and in the clinical setting. The types of conjugation used for passive actuation are typically noncovalent in nature and often rely on the passive efflux of cargo from within the NP interior or from the NP surface. A prominent example is DOXIL®, a liposome-encapsulated formulation of doxorubicin (DOX) that promotes longer circulation time by evading the body’s immune system. Similarly, hydrophobic drugs can be conjugated to the hydrophobic regions of hydrophilic micelles, dendrimers, polymers and solid NPs (i.e., in the ligand corona) to overcome their insolubility in water. Such hydrophobic interactions allow for passive release in situ. The surfaces of QDs and gold NPs have also been used extensively for drug conjugation and passive actuation. This can be achieved through direct coordination of the drug to the inorganic NP surface [13] or by electrostatic interaction with a charged NP surface [26]. „„NP

loading for active/extracellularly triggered actuation Beyond the mere passive efflux of drug cargos from the NP interior or surface is the more elegant release of therapeutic cargo in response to specific extracellular cues/stimuli. External cellular stimuli (e.g., pH, protease activity) have been employed to mediate the triggered release of liposome-caged cargos. Here, while the cargo is still noncovalently encapsulated in the liposome interior, the integrity of the liposome structure is maintained via functional groups that are compromised when exposed to extracellular cues. These chemical changes result in destabilization of the liposomal structure and release of therapeutic cargo, which has been demonstrated using dye models [27,28]. Another valuable extracellularly controlled actuation method in NMDD systems employs photo-triggered cargo release, which does not necessarily require drug conjugation through covalent photocleavable chemical bonds. You et al. demonstrated this by conjugating DOX to hollow gold nanospheres via electrostatic interactions to form stable conjugates [29]. DOX release from the conjugates was controlled by near-IR laser irradiation at 808 nm, where the hollow 1414

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gold nanospheres have strong surface plasmon absorption. The conversion of the adsorbed light to thermal energy, which was postulated as the release mechanism, was demonstrated in MDAMB-231 cells. Due to the electrostatic nature of the conjugation, DOX release from the hollow gold nanospheres was also demonstrated to be pH-dependent. „„NP

loading for active/intracellularly triggered actuation Therapeutic cargo can also be conjugated to NPs for triggered release by biological stimuli present intracellularly. For example, liposomes have been designed to release encapsulated cargo when triggered by cellular activators present in diseased or cancerous cells, such as matrix metallo­ proteinases and apoptosis-associated caspase proteolytic enzymes [30,31]. The proteases cleave bonds to structural components that hold the liposomes together, resulting in destabilization of the liposomal structure. Naturally occurring reductants found in the cytosol, such as glutathione, can also be used to trigger drug release inside of cells. Kim et al. prepared polymer nanocapsules held together by disulfide bridges, which were cleaved by reductants causing the nanocapsule to rupture [32]. This concept was demonstrated in HepG2 cells (hepatocellular carcinoma), where dye cargo was released from nanocapsules upon cellular uptake with subsequent nanocapsule degradation via reduction. Dye was not released under the same conditions from nanocapsules lacking disulfide bridges. Conjugation methods that are susceptible to changes in pH are also important for intra­ cellular actuation since lower pH conditions exist in tumors and within intracellular endo­ lysosomal compartments. For this purpose, Kievit et al. utilized a pH-sensitive covalent hydrazone linkage to attach DOX to iron oxide NPs to overcome multidrug resistance in rat glioma cells [33]. High drug loading per NP was achieved through the use of polyethylenimine as the docking molecule, wherein DOX was attached to the organic coating on the NPs. Drug release under acidic conditions via hydrazone bond cleavage was reported and the NP– DOX conjugates demonstrated an improvement over multidrug resistance relative to DOX alone. Methods for the delivery of NP–cargo complexes to cells & tissues Paramount for the successful implementation of NPs in drug-delivery regimes is the efficient future science group

Controlling the actuation of therapeutic nanomaterials: enabling nanoparticle-mediated drug delivery delivery of the ensemble NP–drug cargo complex to targeted cells and tissues as these are their ultimate sites of function. As the authors highlight in this section, there are a number of critical requirements for the facile cellular delivery of NPs. Throughout this discussion, it is important to appreciate that there are subtle, yet distinct differences in how these requirements are met depending on whether one is considering a NP–drug cargo complex in an in vitro cell culture system or in the more complex environment of an in vivo whole animal or ultimately a patient setting. First, the NP–cargo assembly should exhibit minimal nonspecific binding to non-targeted cells/tissues while displaying a high degree of specificity and (ideally) high-affinity interactions with the sites of intended function. While this aspect is no doubt important in initial in vitro cell culture testing and development, it becomes a critical determinant of the ultimate therapeutic index of the NP–cargo complex in vivo. Here, biological recognition for specific cell/tissue targeting works in tandem with the minimization of nonspecific binding to off-target sites. To date, targeting has been achieved via a variety of strategies. The enhanced permeability and retention (EPR) effect, utilized primarily in tumor oncology therapy, is a nonspecific targeting modality that relies on the preferentially ‘leaky’ nature of the tumor vasculature compared with normal blood vessels [34,35]. In this approach, the slightly leaky nature of newly forming blood vessels in the actively growing tumor tissue allows for the preferential collection of therapeutic NP materials from the bloodstream within the growing tumor. Conversely, the specific cellular targeting of NPs has been achieved by decorating the NP surface with any of a number of biological or chemical moieties including proteins (e.g., antibodies, growth factors), peptides, sugars and small molecules (e.g., folate) [1]. The issue of nonspecific binding has been addressed in large part by the incorporation of poly(ethylene glycol) (PEG) moieties and other polymeric matrices onto the NP surface [36]. More recently, the implementation of zwitterionic surface ligands has been used to overcome some of the potential steric hindrance effects associated with the use of longer PEG chains and polymer structures [37–39]. The second critical element for the delivery of therapeutic NP constructs is an efficient means of mediating cellular NP uptake and eventual control over intracellular NP location. Here, the use of peptides has become one of the most popular future science group

| Review

modalities for NP delivery. This is cumulatively a product of their: n Small size, which allows for maintenance of a small NP hydrodynamic diameter for effective tissue penetration and access to the blood–brain barrier [40]; Ease of synthesis including the incorporation of non-natural amino acid residues which expands the repertoire of available sequence motifs;

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Amenability to bioconjugation;

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Sufficiently high binding affinity for targeted cell surface receptors;

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Minimal in vivo toxicity.

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To date, a number of peptides that fall under the colloquial heading of ‘cell-penetrating peptides’ (CPPs) have been used for successful NP delivery. Examples of these include those based on the HIV-1 TAT and its derivatives, RGDbased integrin-binding peptides, transportan, penetratin, neuropeptides and peptides derived from the Rabies virus (see [41–43] for reviews). One important characteristic shared by nearly all CPPs is that their mechanism of cellular uptake is via endocytosis [43,44]. As a result, the internalized NPs remain largely sequestered within intracellular vesicles where they are not accessible to the cellular cytosol (or other targets). Such sequestration does not always inhibit the therapeutic potential of a given NP–cargo. For example, DOX delivered as a covalent conjugate to the surface of QDs was able to efficiently efflux from the QD surface, translocate across the endosomal membrane and reach the nucleus to induce apoptosis in primary alveolar macrophages [13]. Still, in other instances, release of the NP vector to the cytosol is required for the full activity of the therapeutic cargo. While certain fusogenic peptide motifs derived from influenza virus haemagglutinin have been demonstrated to be very effective disruptors of endosomal membranes, these peptides often need to be used in conjunction with other targeting peptides for full ensemble NP function [45]. Such a strategy was used by the Brinker group, where ratiometric combinations of targeting and fusogenic peptides were employed to achieve cytosolic delivery of supported membrane bilayers containing cocktails of drug toxins, siRNA and diagnostic QDs [46]. Given the now well-documented importance of peptide valence (minimum number of peptides required to mediate targeting/ www.future-science.com

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Review | Delehanty, Breger, Gemmill, Stewart & Medintz uptake) in cellular NP uptake, this is a potential liability for the cytosolic delivery of NPs at the smaller end of size spectrum (

Controlling the actuation of therapeutic nanomaterials: enabling nanoparticle-mediated drug delivery.

The implementation of biofunctionalized nanoparticles (NPs) as potential therapeutic materials has seen exponential growth in recent years due to thei...
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