Advances toward More Efficient Targeted Delivery of Nanoparticles in

Advances toward More Efficient Targeted Delivery of Nanoparticles in Vivo: Understanding Interactions between Nanoparticles and Cells Ester Polo,† Manuel Collado,‡ Beatriz Pelaz,*,§ and Pablo del Pino*,§ †

Centre for BioNano Interactions, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), Sergas, E15706 Santiago de Compostela, Spain § Centro Singular de Investigación en Química Biolóxica e Materiais Moleculares (CIQUS), and Departamento de Física de Partículas, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain ‡

ABSTRACT: In this Perspective, we describe current challenges and recent advances in efficient delivery and targeting of nanoparticles in vivo. We discuss cancer therapy, nanoparticle−biomolecule interactions, nanoparticle trafficking in cells, and triggers and responses to nanoparticle−cell interactions. No matter which functionalization strategy to target cancer is chosen, passive or active targeting, more than 99% of the nanoparticles administered in vivo end up in the mononuclear phagocytic system, mainly sequestered by macrophages. Comprehensive studies, such as the one reported by MacParland et al. in this issue of ACS Nano, will help to close the gap between nanotechnology-based drug-delivery solutions and advanced medicinal products.

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with conventional drug formulations with notable pharmacokinetic limitations. Several engineered “smart” nanomaterials have been proposed as carriers for therapeutics, diagnostic agents, and combinations of both (i.e., theranostic agents). Nanotechnology enables researchers to combine the properties of multiple materials (e.g., inorganic and/or organic nanoparticles (NPs), biomolecules, therapeutics, etc.) by producing composite self-assembled architectures, thereby providing novel, multifunctional tools for drug-delivery applications. In general, the ideal nanomedicine should be designed to accomplish the following in vivo: (i) avoid sequestration by the mononuclear phagocytic system (MPS); (ii) avoid major accumulation in liver and spleen; (iii) have prolonged circulation time (avoiding fast renal clearance); (iv) target specific cells and tissues only; and (v) have stimuli-responsive controlled delivery of the treatment, for instance, by a trigger, such as light, ultrasound, magnetic fields, pH, enzymatic catalysis, competitive guests, etc. Although the expectations for achieving translational medical solutions based on nanotechnology are high and the proof-of-concept reports are increasingly in the literature, only a few nanomedicines are currently commercialized, including Doxil (liposomal

n the past decade, we have witnessed dramatic progress in our understanding of cancer, immunotherapy, cell therapy, cell reprogramming, and genome editing, which will help us find better solutions to fight cancer and other genetic disorders. Many of the proposed solutions would likely entail safe and efficient delivery of pharmaceutical formulations to targeted cells and/or tissues. Although most current therapies are typically based on systemic delivery of drug formulations in the bloodstream, the therapies of the future should be “smart”, that is, capable of better release efficiency (including when and where) and dose control. In this way, many secondary and offtarget effects could be minimized or even fully avoided.

In this issue of ACS Nano, MacParland et al. shed some light on hepatic sequestration of nanoparticles, the major nonspecific target organ (together with the spleen) of administered nanoparticles. In drug delivery, nanotechnology is a promising approach for providing safer and more efficient solutions than are possible © 2017 American Chemical Society

Published: March 7, 2017 2397

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“Inflammatory” M1-like macrophages showed an average 40% NP uptake reduction compared to more “regulatory” M2c macrophages. Overall, the work by MacParland et al.1 highlights the importance of understanding NP−cell phenotype interactions, as it might open new solutions for targeted delivery of nanomedicines and bionanotechnology (e.g., cell reprogramming, cell therapy, tissue engineering, etc.). Cancer Therapy. The results of MacParland et al.1 are of particular interest in cancer therapy. As mentioned above, the examples of Doxil, Abraxane, and Ambisome are just the first of new classes of nanomedicines with promising application for the management of cancer. The rich complexity of the tumor microenvironment is now recognized not only as an integral factor contributing to carcinogenesis but also as playing critical roles modulating cancer therapy. Tumor-promoting inflammation and escape from antitumor immunity are considered hallmarks of cancer that emerge in the context of an inflammatory microenvironment.2 Importantly, macrophages are a major component of the immune infiltrate in tumors and play essential, but complex, roles in neoplastic diseases. Indeed, tumor-associated macrophages (TAMs) can have dual positive and negative effects on tumor growth and therapy. The M2polarized macrophages are responsible for the tumorpromoting activity of the cancer-associated inflammation, and according to MacParland et al.,1 they also show preferential uptake of NPs. This observation is important to bear in mind when designing antitumor strategies based on NP delivery of the chemotherapeutic drugs to the tumor because an M2-rich tumor microenvironment could potentially limit the reach of the therapy. In this scenario, functional re-education of TAMs to a pro-inflammatory M1 phenotype could kill two birds with one stone. On the one hand, it would decrease the contribution of pro-tumorigenic M2 macrophages and increase the proinflammatory beneficial M1 macrophages, whereas on the other hand, it would increase the availability of chemotherapeutic agents encapsulated in NPs that could potentially reach their target, the tumor cell. On a similar note, therapies targeting the M2 phenotype might increase the effectiveness of NP delivery of antitumor agents by reducing the possibility of NP loss due to ingestion by M2 macrophages. The results of MacParland et al.1 are of special relevance when considering macrophages themselves as potential targets of a NP-based treatment, a new avenue of antitumor strategies gaining momentum.3 It might be worth considering, especially when TAMs are limiting the antitumor activity of chemotherapy or radiotherapy by nurturing a tumor-promoting environment. In this situation, this preferential uptake by M2 macrophages may be a potential positive outcome of their avidity for NPs that can have beneficial consequences for the anticancer therapy. Antiinflammation therapies could be envisaged based on this higher uptake by M2 macrophages to limit their contribution to cancer development if NPs are loaded with cytotoxic compounds that would be preferentially ingested by M2 macrophages.

doxorubicin), Abraxane (NP albumin-bound paclitaxel), and Ambisome (liposomal amphotericin B). Three primary entangled challenges stand between nanotechnology and achieving advanced medicinal products: (i) at the extracellular level, understanding and controlling the biomolecule−NP (biomolecular corona) in vivo; (ii) at the cellular level, avoiding intracellular degradation of the nanoformulations due to lysosomal/phagolysosome storage, as is the case with many pathogens (viruses, bacteria, and parasites); (iii) in vivo, highly specific targeting of cells and/or tissues, where the treatment should be delivered. In this issue of ACS Nano, MacParland et al.1 shed some light on (iii), more specifically, on hepatic sequestration of NPs, the major nonspecific target organ (together with the spleen) of administered NPs (see Figure 1). MacParland et al.1

Figure 1. Scheme showing the findings reported by MacParland et al.1 regarding the uptake of nanoparticles (NPs) by macrophages depending on (A) NP size, (B) macrophage polarization, and (C) macrophage phenotype.

demonstrate that human macrophage phenotype modulates hard NP uptake by using a library of gold NPs with different sizes and surface charges and a library of human-derived macrophages with different phenotypes and primary Kupffer cells (obtained from deceased donor livers for transplantation). This work points at using well-characterized, reliable in vitro models (i.e., to the phenotype level), in addition to libraries of well-characterized nanomaterials, as key points to extract useful information from studies regarding NP−cell interactions. Based on these results, MacParland et al.1 also introduce the concept of modifying the hepatic microenvironment as a tool to modulate (to favor or to diminish) hepatic sequestration of NPs. The authors used up to six cytokine cocktails to drive isolated circulating monocytes from healthy human donors into different macrophage polarization states: “unpolarized” (considered neither M1 nor M2), M1, M2, M2a, M2b, and M2c.

The rich complexity of the tumor microenvironment is now recognized not only as an integral factor contributing to carcinogenesis but also as playing critical roles modulating cancer therapy. 2398

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Figure 2. Different scenarios and biological barriers in which NPs are present once they are injected in vivo.

Nanoparticle−Biomolecule Interactions. Nanoparticle− cell interactions involve active energy-dependent biological processes driven by biological recognition of specific features of the NP. Thus, NP entry into cells, accumulation in organs, or biological barrier crossing can, in principle, be controlled by specific design features such as specific arrangement and organization, including surface-presented receptor recognition motifs located on the surface of the NP. Understanding the interaction of NPs with cells is essential for the development of nanotheranostic tools and to overcome the limited progress made in active targeting of NP-based therapeutics (see Figure 2). It is well-known that when in contact with the biological milieu, NPs acquire different molecules from their surroundingsmainly proteinsforming a biomolecular corona and giving the NPs a new biological identity.4,5 Key biological interactions are driven by those additional biomolecules residing at the interface between the NP surface and the biological target. The composition of this protein corona is strongly correlated with features such as composition, size, and shape and is primarily modulated by engineering the surface chemistry of the NPs. Given this new biological identity of the NPs, it is critical to understand how this biomolecular corona could affect the interaction within cells. Rather than separately studying each entity involved (NPs, cells), in order to understand the NP−cell interactions, it is important to look at the interface between themthe protein coronaand how the protein composition affects this interaction. By understanding the parameters that affect this interaction, one can modulate the properties of NPs in order to achieve accumulation in certain territories or to attain specific and tunable cellular interactions. Recent evidence in the literature suggests a strong correlation between the nature of the biomolecular corona and the cellular uptake of NPs, in vitro and in vivo. The biomolecular corona and, therein, the protein surface that interacts with the

biological machinery may play an important role in the NP biodistribution and in the accumulation and processing of NPs by the liver. It has been reported for many NP models of different size, shape, and surface chemistry that the liver is the major organ of deposition of circulating NPs. After administration in blood, NPs are rapidly sequestered from the bloodstream via the MPS, which consists of a system of phagocytic cells, macrophages resident in the spleen, lymph nodes, and liver. This rapid sequestering represents the major challenge for effective in vivo targeting of nanoformulations. Investigating the interactions of NPs and macrophages is critical to understanding the accumulation and processing of nanomaterials by the liver, as shown by MacParland et al.1 The adsorption of plasma proteins, such as apolipoproteins, complement components, and immunoglobulins6 on the NP surface facilitates the recognition and clearance from the blood (opsonization) by circulating phagocytes as well as tissue macrophages (mainly the hepatic Kupffer cells and macrophages in the spleen). Some of these proteins mediate NP interactions to specific receptors on the surface of macrophages. In addition, the exposure of certain protein domains, for instance, can potentially trigger specific cellular recognition pathways, resulting in the activation of determined biological processes such as activation of the immune system. Thus, protein conformational changes occurring upon binding to the NP surface can result in the exposure of “cryptic” epitopes that may play a major role in NPs’ recognition by specific cellular receptors.7 Controlling in situ formation on the biomolecular corona by nanomaterial surface engineering (using a variety of different coatings) is, to date, the main strategy used to control or to inhibit the level of adsorbed proteins8 or to modulate the presence of specific residual proteins9 in order to increase nanoformulation circulating time and decrease nonspecific cellular uptake. The biomolecular assemblies on the NP surface contain all of the relevant information to understand the NPs’ 2399

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design of a library of homologous NPs,16 which should be studied in different cell lines. During cellular uptake via receptor-mediated routes, the activation of transmembrane receptors is required. These receptors activate intracellular signaling cascades that control cellular processes such as cellular proliferation, differentiation, migration, or survival. The pathway of cargo internalization will differ depending on the nature of the activated receptor. The epidermal growth factor receptor (EGFR) is among the most interesting of the studied cases. This receptor is used by cells to uptake extracellular entities (e.g., proteins, NPs) via clathrinmediated endocytosis, which can then follow different postendocytic trafficking routes. Interestingly, the trafficking of this receptor is sorted through different pathways, depending on the extracellular ligand concentration. At low ligand density, EGFR will be internalized through clathrin-dependent endocytosis, whereas at high concentrations, the pathway will be raft/ caveolar-dependent (a clathrin-independent process).17 The clathrin-dependent route typically leads to the recycling of the receptor (and the cargo if this is not detached), while the second route leads to the receptor’s degradation. Extracellular vesicles (EVs) are involved in many physiological and pathological processes and play important roles in intercellular communications. There are three main classes of EVs: exosomes, microvesicles, and apoptotic bodies. Exosomes exhibit the narrowest size distribution among the classes of EVs (30−120 nm) and are sorted by involving specific proteins related to the endosomal sorting complex required for transport (ESCRT). The ESCRT mediates the recycling of the EGFR. Interestingly, this strategy is also used by retroviruses (e.g., HIV) to escape from cells during the process known as viral budding. The applicability of lysosome-escaping tactics must be investigated carefully in order to determine the limits of tolerance to ensure cellular health. This is a huge task, considering that the individual cellular identity can vary the sensitivity of cells depending not just on the cellular type. Thus, the development of biomimetic materials envisaged to mimic the viruses’ behavior to escape is gaining more attention. Other strategies to produce biomimetic materials, such as coating NPs with engineered cellular membranes (e.g., leukosomes)18 or producing vesicles with a surface that mimics different cellular compositions, will be the future of NP-based materials for vaccines and drug-delivery nano-based solutions. Triggers and Responses to NP−Cell Interactions. Nature has developed a variety of dynamic stimuli-responsive tactics, including chemical gradients, pH gradients, dynamic host−guest recognitions, etc., to perform biological processes such as development, inflammation, wound healing, cancer metastasis, ATP synthesis, transport across membranes, immunity, neural polarization, etc. Engineered NPs, which are diverse in characteristics such as size, charge, shape, elasticity, hydrophobicity, degradability, etc., are the size of many proteins and foreign entities inside the body. Nanoparticles are expected to trigger manifold biological responses (e.g., protein adsorption, uptake by specific cells, cell death, autophagy, protein denaturation, etc.) depending on their characteristics (see the section on Nanoparticle−Biomolecule Interactions), dose, and the biological entities they encounter, for instance, as shown for the macrophage phenotype versus library of NPs by MacParland et al.1 By engineering NP surfaces, it should, in principle, be possible to control cell responses in vivo. As previously

biological behavior. Current techniques to determine the composition of the corona are mainly based on the use of proteomic approaches and only provide average compositional information, which unfortunately, does not fully account for the complexity of the in vivo NP−corona−cellular receptor interactions. Therefore, characterizing the information encoded at the surface of nanoformulations in a realistic environment and in greater detail is critical to elucidating the links between the organization and presentation of certain molecules and their interactions with cells via specific receptors.10,11 Moreover, different presentations and arrangements of these biomolecules could be interpreted in different ways, leading to varying responses and biological processes. Increasingly detailed understanding of the biomolecular organization at the NP interface will elucidate the mechanistic biological processes induced by it. This understanding will, in turn, enable the design of specific nanoscale architectures to exploit specific cellular pathways of interest. Nanoparticle Trafficking in Cells. The next-to-last barrier that NPs administered in vivo cross is the cellular membrane. Ideally, the function of nanomaterials (e.g., drug release, conformational changes, heat production, etc.) will be carried out in a specific cellular location. Thus, targeting organelles is highly desired. However, targeting in vivo is complex because the target site might be located in a particular organelle from a specific cell type with a specific phenotype. After cellular uptake, receptor-mediated or not, most nanomaterials are internalized via endocytic pathways. There are many mechanisms to induce endosomal escape, but most of them occur through partial or total endosome membrane disruption. The aperture or breakdown of the lysosomes can induce the release of cathepsins and other components, initiating a cascade of reactions that can ultimately trigger apoptosis.12 The direct cytosolic release of cargoes has been accomplished using polymeric-based NPs or nanocapsules. Recently, the cytosolic release of CRISPR/Casp9 ribonucleoprotein has been reported.13 The use of cell-penetrating peptides (CPPs) to introduce NPs directly inside cells has also been explored. However, transport through CPPs normally occurs by more than one internalization pathway, and thus, endosomal escape may still be needed. The efficiency of CPP-mediated transport might also be limited by the cargo (e.g., NP) size. In the last 25 years, our understanding of the different receptor-mediated endocytic pathways has greatly evolved. However, many parameters related to the inherent complexities in the trafficking pathways, such as the possibility that the trafficking process will be altered by the ligand concentration or multivalency, are not fully understood.14 These factors become more dramatic when using ligands attached to NPs, as compared with ligands attached to drugs, perhaps because of the different ways in which NPs interact with cells and tissues.15 Multivalency describes the simultaneous interaction of multiple recognition elements on one molecular entity with multiple receptors on another entity. When designing nanocarriers based on NPs, multivalency is the most commonly used approach. Many critical features have been identified, such as size, shape, valency, flexibility, and orientation of the individual recognition elements. Despite all the research in this area, no guidelines exist as to how to place ligands in the most efficient manner to obtain the desired internalization rate and location or how to minimize the off-target effects that arise from nonspecific binding. Considering the dramatic role of the NP itself, the success of multivalent targeting may come with the 2400

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AUTHOR INFORMATION

discussed, although many important challenges regarding in vivo targeted delivery remain, recent progress in understanding what happens to NPs in vitro and in vivo is swiftly closing our knowledge gap. Nanotechnology has equipped scientists with tools and approaches that enable the investigation of the kinetics and mechanisms of degradation of organic coatings firmly grafted onto NPs by proteolytic enzymes in the liver.19 The use of multifunctional NPs as vaccines is one of the most exciting NP applications in drug delivery. The versatility of nano-based vaccines, for instance, enables the targeted delivery of vaccine cargo to antigen-presenting cells in the lymph nodes,20 while radiolabeling enables single-photon emission computed tomography imaging, which can be used to trace migration from the injection site to regional and nonregional lymph nodes. In the previously discussed example, NPs were injected locally in one lymph node (at a low dose, but enough for parallel eliciting antigen-specific immunity and in vivo imaging) so NP sequestration by the MPS was not an issue, thus highlighting that systemic delivery is not always mandatory.

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ester Polo: 0000-0001-8870-5280 Beatriz Pelaz: 0000-0002-4626-4576 Pablo del Pino: 0000-0003-1318-6839 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS Financial support from MINECO (MAT2015-74381-JIN to B.P., RYC-2014-16962 to P.dP., CP/11/00273 and PI14/ 00554 to M.C.) and Science Foundation Ireland (SFI, 12/IA/ 1422 to E.P.), the Conselleriá de Cultura, Educación e Ordenación Universitaria (Centro singular de investigación de Galicia accreditation 2016-2019, ED431G/09), and the European Regional Development Fund (ERDF) is gratefully acknowledged.

The use of multifunctional nanoparticles as vaccines is one of the most exciting nanoparticle applications in the drug-delivery field.

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So far, we have briefly described some endogenous triggers, which define NP−cell interactions and biological responses. However, materials scientists have put enormous effort into designing nanoplatforms that are responsive to exogenous triggers (e.g., electromagnetic fields and ultrasound) and capable of initiating reactions both outside and inside cells. For instance, by using near-infrared photostimulation of plasmonic NPs trapped inside lysosomes, both necrotic and specific apoptotic signals can be modulated by light.12 Magnetogenetics is another important example of cell control by NPs. In analogy to optogenetics, ion channels can also be genetically modified to become heat-sensitive. Thus, local heating provided by alternating magnetic field excitation of magnetic NPs in the vicinity of ion channels enables control of the function of cells and even brain activity in living animals.21 The previous examples illustrate the potential of engineered NPs as actuators of cell behavior, be it by design (e.g., functionalization) or by use of external triggers.

OUTLOOK We foresee that the gap between proof-of-concept and translational medicine nano-based solutions will close as our understanding of NP−biomolecule interactions (in the extracellular matrix, on the cellular membrane, or inside the cell) evolves. To achieve this greater understanding, comprehensive studies entailing libraries of NPs, biomolecules, and cells (exhaustively characterized), such as the one reported by MacParland et al.1 in this issue of ACS Nano, are greatly needed. We believe that such “basic” studies will pave the way to reducing animal testing and to hastening clinical trials. Furthermore, we believe that advancing toward more efficient targeted delivery will enable clinical testing in the near future of the efficiency of many of the nano-based medicines already developed, without the issue of NP sequestration by the MPS. 2401

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