Nanomaterials for Natural Killer Cell-Based Immunoimaging and Immunotherapies in Cancer

Abstract

Natural killer (NK) cells are an important component of the tumor immunosurveillance; activated NK cells can recognize and directly lyse tumor cells eliciting a potent antitumor immune response. Due to their intrinsic ability to unleash cytotoxicity against tumor cells, NK cell-based adoptive cell therapies have gained rapid clinical significance, and many clinical trials are ongoing. However, priming and activating NK cells, infiltration of activated NK cells in the immunosuppressive tumor microenvironment, and tracking the infiltrated NK cells in the tumors remain a critical challenge. To address these challenges, NK cells have been successfully interfaced with nanomaterials where the morphology, composition, and surface characteristics of nanoparticles (NPs) were leveraged to enable longitudinal tracking of NK cells in tumors or deliver therapeutics to prime NK cells. Distinct from other published reviews, in this tutorial review, we summarize the recent findings in the past decade where NPs were used to label NK cells for immunoimaging or deliver treatment to activate NK cells and induce long-term immunity against tumors. We discuss the NP properties that are key to surmounting the current challenges in NK cells and the different strategies employed to advance NK cells-based diagnostics and therapeutics. We conclude the review with an outlook on future directions in NP-NK cell hybrid interfaces, and overall clinical impact and patient response to such interfaces that need to be addressed to enable their clinical translation.

1. INTRODUCTION

Natural killer cells (NK) cells are members of the innate lymphoid cell family; they play a critical role in immune surveillance and host defense against tumor cells and viral infections by exerting a potent immune response.^1–3 As effector cells, NK cells also induce antibody-dependent cell-mediated cytotoxicity with the inherent ability to destroy abnormal cells without major histocompatibility complex (MHC)-dependent antigen presentation.² In the presence of stimulating signals, NK cells are activated via membrane receptors and, in the process, secrete lytic granules such as perforin, which are pore-forming proteins, and granzyme B, which are cytotoxic enzymes.⁴ Exocytosis of these lytic granules and death receptor pathways, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, enable NK cells innate ability to destroy tumors.¹ In addition to innate immune response, NK cells are also key regulators of adaptive immunity, which is mediated via crosstalk between NK cells and dendritic cells (DCs) and T cells.⁵ Indeed, DCs have been shown to activate NK cells via interferon-γ (IFN- γ) production⁶ and activating receptors such as NKp46. Interaction of NK cells and DCs also impacts the maturation of DCs to enable a multicell circuitry with T cells (CD4⁺ and CD8⁺) and promote T cell responses in the immune tumor microenvironment (TME).⁷ These innate cytotoxic and cytolytic abilities of NK cells have propelled adoptive cell therapies (ACT), leveraged by several advantages, including (i) NK cells do not need donor matching, (ii) NK cells do not contribute to graft-versus-host disease, and (iii) NK cells are easy to isolate from blood.^8,9 However, despite these merits, NK cell-based ACT and NK cell recruitment in solid tumors have been severely limited by the immunosuppressive TME, which necessitates innovative immunotherapies and immunoimaging approaches to accurately quantify NK cell migration and proliferation during ACT.¹⁰

Nanomaterials have driven a paradigm shift in cancer therapies. Recent findings support that biocompatible nano-particles (NPs) have significantly advanced both in vivo monitoring and ACT of immune cells that include DCs,¹¹ macrophages,¹² and T cells.¹³ Relative to conventional treatment strategies, NPs-mediated therapies have several merits, including enhanced drug stability and dispersion, improved pharmacokinetics, and controlled and targeted release of drugs within the tumor. NPs also benefit from the enhanced permeability and retention (EPR) effect, which allows extravasation from leaky tumor vasculatures and tumor homing.¹⁴ The design considerations of NPs to enable active targeting or passive uptake in NK cells are driven by their size, shape, surface charge, material composition, and surface ligands; these properties ultimately control NPs biodistribution and clearance in vivo. For example, direct in vivo targeting of NK cells recruited in tumors requires size-controlled penetration of NPs in tumors. NPs often infiltrate into the tumor via interendothelial cell gaps across blood vessels (NPs > 10 nm) or via intercellular gaps of normal capillaries (NPs < 10 nm).¹⁵ However, direct targeting of NK cells in vivo has been challenging. Therefore, the indirect labeling of immune cells, that include DCs,¹⁶ macrophages,¹² and NK cells with NPs ex vivo before systemic administration has shown significant promise in the TME. For labeling ex vivo, NP morphology and surface properties are major factors for high labeling efficiency. For example, both the quantity of NPs endocytosed in cells and the efficiency or rate of NP uptake is driven by the endocytosis pathway and is controlled by the NP shape. Whereas spherical NPs remain largely membrane-bound and then undergo cellular excretion via exocytosis, NPs with sharp corners and edges are translocated to the cytoplasm with suppressed exocytosis rate, thereby assisting in long-term tracking.¹⁷ Rod-shaped NPs are also superior in their ability to penetrate through the cell membrane and have rapid endocytosis that suggests such a design aspect is critical in NK cell labeling with NPs.^18,19 The surface characteristics of NPs, including charge and ligands, are also important in improving the overall stability and bioavailability of NPs during endocytosis. Indeed, cationic NPs have already shown high avidity in NK cell uptake.⁸ NPs assembled with targeting ligands or nontoxic hydrophilic polymers, such as polyethylene glycol (PEG), are beneficial for in vivo tracking of NK cells to minimize protein corona formation and reduce uptake by the liver- and spleen-resident macrophages. Among these NP characteristics, the material composition is most relevant in this review as it governs the functionality of the NP for their utility in preclinical and clinical imaging and therapies. The collective tuning of the physicochemical properties of NPs enables multifunctional characteristics, and a wide variety of such NPs has recently been demonstrated in NK cell-based applications (Figure 1). This review highlights recent findings where NPs interfaced with NK cells have transformed the immunoimaging and immunotherapy landscape, with a primary focus on how the design of nanomaterials and resulting properties and functions impact cancer immunotherapies. An in-depth discussion about the various classes of biomaterials for NK cell-based immunotherapies and the immunology of NK cells and corresponding memory responses is beyond the scope of this review. Readers are encouraged to refer to the detailed reviews by Kim et al.,²⁰ Mikelez-Alonso et al.,¹ Han et al.,²¹ and Raza et al.²² Our review sumamrizes the different strategies used in labeling NK cells with NPs for imaging and tracking in vivo and the underlying mechanisms of NPs that control direct or indirect activation of NK cells in cancer therapies. We also provide a list of key papers in the field (Table 1) where NPs have advanced NK cell-based diagnostics and therapeutics.

Figure 1.

Overview showing the utility of a range of nanoparticles (NPs) used in tracking NK cells with an expansive immunoimaging toolbox and NP-mediated NK cell-based cancer therapies.

Table 1.

Nanoparticles Used in Advanced NK Cell-Based Diagnostics and Therapeutics

nanoparticle used	cell source	summary	route of delivery	ref
DOTAP: cholesterol nanovesicles	primary NK cells	NPs were encapsulated with tumor-suppressor candidate 2 (TUSC2) DNA plasmids to inhibit the TUSC2 gene, decrease tumor volumes, and improve survival rates in murine lung carcinoma models.	intravenous	65
cationic polyethylenimine NPs	human NK-92MI and primary NK cells	Cationic NPs enhanced NK cell cytotoxicity attributed to an increase in the NK cell’s ability to recognize and bind to target cancer cells. This was contributed by increased chemokine receptor expression (CCR4 and CXCR4).	in tra turn oral	8
chiral gold NPs	endogenous	Chiral NPs improved the activation of both T cells and NK cells by stimulating DCs. The L-type chiral NPs had a 1.65× higher activation of immune cells than the D-type.	subcutaneous	66
chitosan NPs loaded with plasmids that encoded the NKG2D and IL-21 genes	endogenous	NPs were used for fusion gene delivery. These NPs were easy to synthesize, had high biocompatibility, and had high tumor biodistribution.	intramuscular	3
coated iron oxide nanoparticles	NK92MI and isolated murine NK cells	Positively charged polymer coating allowed the incorporation of nanoparticles into the NK cell membrane. Labeling did not impact NK cell transmigration ability or cytotoxicity.	in vitro study	67
polycaprolactone-poly(ethylene glycol) (PCL–PEG) micelles	endogenous	These pH-responsive self-aggregating NPs were coloaded with doxorubicin and transforming growth factor-β (TGF-β)/Smad3 signaling pathway inhibitor, SIS3. These NPs initiated NK cell proliferation, increased NK cell cytokines, and improved T cell response in the tumor.	intravenous	68
gold nano spheres	NK92MI	Gold NPs were endocytosed in NK cells via passive uptake. The NK cells were then tracked in vivo in xenografts via CT imaging since gold NPs enhance X-ray signal.	intravenous	30
graphene oxide-based biomimetic nanocluster	primary donor NK cells.	The antibody-functionalized nanoclusters targeted CD16 receptors of NK cells and, in the process, activated the cells and increased IFN-γ production.	in vitro study	69
liposomes conjugated with IL-2 and antiCD137 antibodies	endogenous	Liposomes were anchored with IL-2 and anti-CD137 on the surface to potentiate immune response and tumor honing ability while lowering systemic exposure.	intravenous	70
imidazoquinoline-based Toll-like receptor 7/8 agonist loaded in pH-responsive PLGA NPs	CD3-CD56+ NK	PLGA NPs were acidic pH-responsive and localized primarily in the endosome and lysosome of DCs. Accumulation of these organelles facilitated the targeted and selective release of TLR7/8 agonists.	peritumoral	36
iron oxide nanocubes (IONC) coated in PLGA microsphere	endogenous	IONCs served as high-resolution contrast agents for MRI-guided injection. Localized IFNγ delivery improved NK cell recruitment in VX2 orthoptic rabbit liver tumors.	intra-arterial liver trans-catheter	71
lipid nanoparticle containing a STIN-Gagonist.	NKG2D+	STING lipid NPs activated NK cells and reprogrammed immunologically cold tumors to a hot phenotype, which reduced the PD-1/PD-L1 axis and resulted in a strong antitumor effect in a B16–F10 lung metastasis model.	intravenous	72
magnetic NPs coated with a cationic polydopamine layer	NK92MI	Magnetic NPs genetically modified NK92MI cells where the NP-labeled NK cells were imaged in vivo by MRI and fluorescence dual-imaging modalities.	intravenous	29
core–shell nanoparticles coated with NK cell-derived exosomes	endogenous	The NK cell exosome-coated NP targeted tumors by interfacing with the cell membrane of the target cells. The NPs also released miRNA and regulated the genetic circuitry of the cell. Gene delivery and therapeutic effect of NK cell membrane gave rise to combinatorial antitumor effect.	intravenous	73
immune modulating polymer NPs coated with IgG	donor PMBC NK cells and endogenous	These NPs facilitated IgG-mediated NK cell stimulation, which led to greater NK cell recruitment, IFNγ production, and T-cell recruitment in murine tumor models.	intravenous	74
NPs loaded with cancer membrane proteins, CpG, and aHSP70p	endogenous	In this cancer vaccine-like strategy, NPs displayed tumor membrane-derived antigens combined with two different adjuvants: synthetic HSP70p and CpG oligonucleotides. These NP vaccines achieved complete tumor regression of B160VA melanoma murine models, and a long-term immune memory effect was observed when combined with anti-PD-1 antibodies.	intradermal	75
PDA-coated iron oxide nanoparticle	patient PMBC NK cells	These magnetic nanoparticles allowed the use of a magnetic field, which enabled homing of adoptively transferred NK cells and doubled the quantity of NK cells present at the tumor site.	intravenous	76
PEGylated liposome encapsulating drugs.	primary NK cells	PEGylated liposomes were loaded with agonists to stimulate IFNβ. In vivo, these NPs elicited an immune response by enhancing the recruitment of antigen-presenting cells and NK cells at the tumor site.	intratumoral	77
perfluoropolyether nanoemulsion	NK cells extracted from patient PBMCs	The NK cells were labeled with nanoemulsion via passive uptake, and NK cell migration was tracked in vivo in NSG mice from the site of injection to tumors via MR imaging.	subcutaneous and intratumoral	31
photosensitizer TCPP-loaded polymer NPs coated with NK cell membranes	human NK92 and murine NK cells	Cell membrane coating on NPs achieved tumor targeting, and TCPP initiated photodynamic therapy that polarized macrophages to M1 phenotype and enabled combination immunotherapy.	intravenous	55
ruthenium polypyridyl complex	patient PMBC NK cells	Ruthenium complexes downregulate proteins and immunosuppressive molecules in the TME and sensitize tumors to adoptively transferred NK cells. The complexes with NK cells enhance immune response and result in caspase-3-dependent apoptotic cell death of tumor cells.	intravenous	78
selenium nanoparticles	NK-92MI	The selenium NPs suppressed the inhibitory cell receptor human leukocyte antigen E, which augmented the cytotoxic ability of NK cells and improved the response of the chemotherapeutic agent pemetrexed in the nonsmall cell lung cancer model.	intravenous	38
self-assembling Sec (Dod) 2KGPLGVRGRGD selenopeptide	NK-92MI and primary NK cells.	A selenopeptide nanoparticle comprised a ROS-responsive, enzyme-cleavable, and tumor-targetable motif. The nanoparticle increased tumor sensitivity to doxorubicin, which increased NK cell activity via downregulated HLA-E in tumor cells.	intravenous	39
PEG-coated diselenide-bridged mesoporous silica NPs	primary NK cells	Mesoporous silica NPs were coloaded with doxorubicin and methylene blue. Photodynamic therapy triggered controlled drug release in the tumor site and led to increased immune cell recruitment and memory T cell response in a 4T1 murine carcinoma model.	intravenous	54
streptavidin-coated iron oxide NPs bound to NK cells via biotinylation of NK cell surface.	donor cord blood NK cells	NK cell surface was modified with sulfo-NHS-biotin to enable iron oxide NPs to bind to NK cells. Magnetic field application facilitated the infiltration of NP “biohybrid” NK cells into 3D neuroblastoma culture.	in vitro study	28
trispecific PEG–PLGA NP functionalized with antibodies.	primary NK cells	Trispecific NPs targeted EGFR-overexpressing tumors and released chemotherapeutic drugs at the tumor site. The NPs also recruited NK cells through the activation of antibodies in murine tumor models.	intravenous	79

2. NANOPARTICLE-ENABLED TRACKING AND IMAGING OF NK CELLS

The utility of NPs as imaging contrast agents is motivated by their ability to improve the diagnostic specificity and sensitivity of clinical imaging modalities and surmount challenges in conventional histopathology that fail to accurately predict disease stages. In the clinic, the curative effect of NK cell infusions in achieving complete or partial response is based on the response evaluation criteria in solid tumors (RECIST) criteria.²³ However, RECIST criteria is limited when assessing heterogeneous tumors within the same patient or among multiple patients and for nonmeasurable disease such as small lesions in the premetastatic niche.²⁴ Therefore, diagnostic NPs that detect and track immune cells in vivo are expected to have significant clinical potential in (i) monitoring disease progression in real time, (ii) providing treatment response and enabling early intervention, and (iii) measuring dynamic changes in immune cell population in the TME during treatment to ultimately advance our understanding of the innate and adaptive immune response of NK cells. In this section, we will discuss the merits of NP contrast agents for single-modality and multimodal imaging of NK cells and the use of multifunctional NPs that combine targeting, imaging, and therapy in a single platform. Labeling NK cells with NPs can be broadly categorized into (a) ex vivo labeling, where NK cells are labeled ex vivo with NPs and then systemically administered in vivo (Figure 2a) and (b) in situ labeling (Figure 2b), where NPs are functionalized with ligands that can selectively hone into NK cells via passive or active targeting.

Figure 2.

(a) Schematic representation of immunoimaging and tracking NK cells using NPs. (a) Ex vivo labeling of NK cells using different types of NPs, which are then delivered in mice. (b) In situ labeling of NK cells where antibody-labeled NPs are systemically delivered, which then recognizes and targets NK cells in vivo.

2.1. Ex Vivo Labeling of NK Cells with NPs.

NK cells lack phagocytotic functionality and, thus, require external stimuli such as electroporation, transfection agents (e.g., protamine), lipofection, or magnetic homing for ex vivo labeling of NK cells. For example, Su et al. reported a unique approach to label NK cells using NPs designed with clinically approved drugs, including ferumoxytol (an iron-based prescription medicine), heparin, and protamine. NP-labeled NK cells were then delivered via transcatheter intrahepatic arterial infusion and noninvasively tracked via magnetic resonance imaging (MRI), which showed enhanced migration and infiltration of NK cells in tumors.²⁵ In another approach, Park et al. demonstrated that electroporation of human NK-92 cells with functionalized upconverting NPs facilitated long-term tracking of NK cells in vivo via up-conversion luminescence imaging.²⁶ Therefore, the design and resulting functionality of the NPs are key in their ability to effectively track NK cells in vivo. NPs are also passively internalized by NK cells enabling hitchhiking of the NPs to the target site in vivo with migrating NK cells. For example, NK cells have been labeled with lanthanide-based down-conversion NPs and reactive oxygen species (ROS)-sensitive near-infrared (NIR) dye to enable ratiometric NIR-II fluorescence imaging (Figure 3a,b) of NK cell viability after ACT immunotherapies.²⁷ In another innovative approach, an immune cell−NP biohybrid was designed by decorating the NK cell surface with iron oxide NPs to enable tumor homing of the NK cells via an external magnetic field.²⁸ The biohybrid NPs did not require any genetic modification, a property that was leveraged to ensure the NK cells retained their natural properties and improved the NK cells’ therapeutic functions. Diagnostic NPs can also be modified to enable other functionalities, such as genetically manipulating the NK cells to induce protein expression and then tracking in vivo. Kim et al. showed that fluorescently tagged cationic polydopamine-coated magnetic NPs could efficiently deliver plasmid DNA into NK cells and induce the expression of epidermal growth factor receptors for targeting chimeric antigen receptors (Figure 3c).²⁹ These multifunctional NPs could genetically modify the NK cells and were tracked via fluorescence and MRI after systemic administration. Computed tomography (CT), a noninvasive technique clinically used for assessing RECIST criteria, has also been leveraged for the longitudinal tracking of NK cells. Shamalov et al. have labeled NK cells with gold NPs coated with PEG and d-(β)-glucosamine to enable quantitative tracking of NK cells with CT imaging, since gold NPs enhance X-rays and delineate soft tissues.³⁰ The d-(β)-glucosamine coating enhances the intracellular uptake of gold NPs without altering the NK cells’ antitumor therapeutic effect. Upon cell death, the excessive ROS generation degrades the ROS-sensitive NIR dye, thereby retaining the fluorescence signal of the lanthanide NPs that allowed tracking of viable NK cells during ACT.

Figure 3.

(a) Schematic illustration of the synthesis of down conversion nanoparticles coated with IR786s (DCNP@786s) and the ex vivo labeling of NK cells and (b) ratiometric NIR-II fluorescence imaging after systemic administration of NK cells labeled with DCNP@786s. Reproduced with permission from ref 27. Copyright 2021 John Wiley and Sons. (c) Schematic illustration of genetic engineering of NK cells and in vivo tracking of NK cells using cationic magnetic nanoparticles. Reproduced with permission from ref 29. Copyright 2019 Elsevier. (d) T2-weighted ¹H and ¹⁹F MR images of the migration of ¹⁹F-labeled human NK cells in vivo. Reproduced with permission from ref 31. Copyright 2016 Taylor & Francis.

In another approach, NK cells labeled with nonradioisotope fluorine-19 were monitored for 15 days in vivo with MRI (Figure 3d) in a preclinical tumor model that facilitated a straightforward approach for longitudinal tracking of therapeutic response.³¹ The progress in ex vivo labeling has propelled advances in targeted NK cell tracking in vivo enabled by thoughtfully designed NPs and an expansive immunoimaging toolbox (Figure 1). Since each imaging modality has merits and challenges, multimodal NPs that synergistically integrate the strengths of multiple imaging techniques are of specific interest. Such NPs enable a single platform that can achieve whole-body deep-tissue imaging with high sensitivity, specificity, and multiplexing to detect multiple biomarkers. Whereas preclinical mouse models have been extensively used in NK cell monitoring, clinical translation of NP−NK cell hybrids will require clinically relevant large animal models. In a recent study, Sato et al. reported tracking and quantifying adoptively transferred NK cells in vivo using ⁸⁹zirconium-oxine (⁸⁹Zr)-radiolabeled NK cells that were labeled and expanded ex vivo; their migration was monitored in monkeys (rhesus macaques) using positron emission tomography (PET)/CT imaging.³² While NPs were not used, this work lays the foundation for the utility of multifunctional NPs that combine PET/CT with other modalities to achieve diverse functionalities in vivo and ultimately promote both the tracking and recruitment of NK cells in large animal clinical models.

2.2. In Situ Labeling of NK Cells with NPs.

Much of the literature has focused on the ex vivo labeling of NK cells because of the ease of modification by NPs necessary for ACT therapies. Direct in vivo targeting of NK cells is of immense interest in image-guided interventions and the utility of imaging for treatment response. In such “in situ” labeling techniques, NK cells that are either endogenous or are recruited in the TME post-treatment are targeted in vivo via antibodies or other targeting ligands. For example, in a creative approach, bispecific antibodies were assembled on mesoporous ruthenium NPs where one antibody domain is bound to a carcinoembryonic antigen expressed on tumors, and another domain is bound to CD16 expressed on NK cells.³³ The authors hypothesized that the dual-targeting NPs would engage NK cells and tumor cells, and the anticancer agent loaded in the NPs would generate ROS that would trigger an immune response from NK cells for enhanced therapeutic action. Our group has also targeted and tracked immune cells directly in vivo via antibody-conjugated gold nanostars and monitored immune cell recruitment in tumors in response to immunotherapy.³⁴ In our approach, we labeled the multimodal nanostars with radiolabels (⁶⁴Cu) and Raman tags to enable depth-resolved, whole-body imaging via PET/CT and multiplexed detection of immunomarkers via Raman spectroscopy. Such an approach to image infiltration of endogenous NK cells in conjunction with other immune cells in tumors is advantageous in distinguishing responders from nonresponders of various cancer therapies and determining the immune mechanisms that give rise to treatment resistance.

3. NANOPARTICLE-ENABLED NK CELL IMMUNOTHERAPIES

Cancer immunotherapies are designed to harness and augment the patient’s immune system to respond to cancer cells while minimizing toxic side effects on healthy cells. Beyond the conventional CD8⁺ T cell-based ACT, innate immune effector cell-based immunotherapies with NK cells also play a pivotal role in treating cancer. There are multiple ways that NPs can either activate NK cells or adopt the therapeutic characteristics of NK cells to eliminate tumors (Figure 4). For example, tumor-targeting NPs can induce immunogenic cell death (ICD) to release antigens that lead to the activation of DCs and T cells and further crosstalk between these immune cells and NK cells. NPs can also activate the NK cells to express chemokine receptors, which improves the capability of the NK cells to recognize and eliminate the cancer cells. Finally, NPs coated with NK cell membrane retain their therapeutic potential and can target tumors without needing additional surface ligands on the NPs.

Figure 4.

Schematic illustration of nanoparticle-mediated therapies that enable activation, recruitment, and migration of NK cells to ultimately eliminate tumors.

Jiang et al. reported Trp2/CpG in pH/redox dual-sensitive micelles (Figure 5a) coloaded with immune-activating cytokine interleukin-15 (IL-15) that activated and potentiated the tumor-killing ability of NK cells.³⁵ Upon interaction with IL-15 receptors on the surface of NK cells, an enhancement in the secretion of IFN-γ was observed, followed by tumor reduction. This study suggests that the simultaneous activation of NK cells and cytotoxic T cells could enhance antitumor immunity. In another approach, NK cell activation was mediated by stimulating toll-like receptors (TLR)7/8 activity in DCs (Figure 5b).³⁶ TLR7/8 agonist-loaded poly(lactide-co-glycolide) (PLGA) NPs were leveraged to induce DC maturation, followed by the secretion of pro-inflammatory cytokines and upregulation of costimulatory molecules to result in the activation of NK cells. Further, NK cells can be activated with small interfering RNAs (siRNAs) to silence the intrinsic inhibitory NK cell molecules. Giber et al. reported a nonviral liposome encapsulating siRNAs against genes critical in suppressing NK cell activation, namely SH2-domain-containing protein tyrosine phosphatase-1, casitas B-lineage lymphoma B, and casitas B-lineage lymphoma, thereby augmenting the NK cell activity against human leukocyte antigen-matched cancer cells.⁵ The liposomes achieved high selectivity by assembling NKp46 antibodies to target the NK cell activation receptors, which enabled a 7-fold reduction in tumor growth and improved overall survival in a humanized murine model. Such lipid-based NPs are not only excellent in mediating gene knockdown for NK cell activation but also can regulate key signaling pathways that have been associated with tumor proliferation.

Figure 5.

Schematic representation of (a) Trp2/CpG in pH/redox dual-sensitive micelles coloaded with interleukin-15 (IL-15) and lymph node delivery to activate NK cells in tumors and enhance antitumor immunity. Reproduced with permission from ref 35. Copyright 2021 American Chemical Society. (b) Activation of NK cells via maturation of dendritic cells using TLR7/8 agonist-loaded PLGA NPs. Adapted with permission. Reproduced with permission from ref 36. Copyright 2020 American Chemical Society.

In recent reports, stimulators of interferon genes (STING) agonist-loaded lipid NPs were shown to activate NK cells after intravenous administration; the NPs accumulated in the liver and stimulated the STING pathway within the liver macrophages.³⁷ STING activation induces the secretion of type I interferons (IFN-1) by liver macrophages giving rise to systemic NK cell activation; the NK cells then produce IFN-γ. The combined secretion of IFN-1 and IFN-γ induces the expression of programmed cell death-ligand 1 (PD-L1) on cancer cells, which establishes immunosuppression via the programmed death-1 (PD-1)/PD-L1 axis between PD-1⁺ NK cells. A subsequent immune checkpoint blockade with anti-PD-1 antibodies reprograms the TME from an immunosuppressive to an immunoprotective phenotype and reactivates NK cells and primes them to induce cytotoxicity against cancer cells. This work demonstrates that combinatorial immunotherapy with NPs is a key strategy to activate, prime, and recruit NK cells and enhance their cytotoxic effects on tumors. In another approach, Pan et al. demonstrated that a synergistic combination of NK cell immunotherapy with chemotherapy using selenium-containing NPs enhances the antitumor immune response.³⁸ The authors reported that these NPs intracellularly produced selenic acid from the oxidation response of the β-seleno ester, which then translocated to the cell surface and suppressed the inhibitory cell receptor human leukocyte antigen E (HLA-E). This process enhanced the immunocompetence of the NK cells and improved chemotherapy response in mouse models of nonsmall cell lung cancer with the cytotoxic agent pemetrexed. In a similar approach, Wei et al. reported self-assembled selenopeptide NPs (Figure 6a) that augmented the chemoimmunotherapeutic effect in the orthotopic breast tumor model.³⁹ Upon systemic administration, selenopeptide NPs selectively accumulate and penetrate in tumors aided by tumor-targeting motifs, followed by enzymatic cleavage by the overexpressed MMP-2 enzymes. After tumor homing, DOX encapsulated within these NPs is subsequently released because of the oxidation, which increases intracellular ROS and enhances the deselenization of the selenopeptide NPs. These events resulted in the tumor size reduction (Figure 6b) via downregulation of HLA-E expression (Figure 6c) in tumor cells and the activation of NK cells.

Figure 6.

Schematic representation of (a) self-assembled selenopeptide NPs and deselenization of selenopeptide to activate NK cells, (b) tumor volumes, and (c) quantification of HLA-E levels in the tumor. Reproduced with permission from ref 39. Copyright 2022 John Wiley and Sons. (d) Isolation and expansion of NKT cells from murine splenocytes followed by photothermal therapy to enable recruitment and activation of adoptively transferred NKT cells and (e,f) tumor growth profile of primary and distant tumors. Reproduced with permission from ref 40. Copyright 2021 American Chemical Society.

The generation of intracellular ROS, whether through chemotherapeutic agents or as discussed above through selenium-containing NPs, is critical in stimulating NK cells. Recent investigations show that light-assisted treatments such as photothermal therapy (PTT) also produce ROS and activate natural killer T (NKT) cells, a subset of NK cells that share functional characteristics of both NK cells and T cells (Figure 6d). PTT can be induced by a range of NPs that absorb resonant light and subsequently convert to heat. These include gold NPs,^40–42 NIR dye-based NPs,^43–47 and conjugated polymers. Li et al. recently used conjugated polymer PBIBDF-BT (PBT), which absorbs in the NIR and self-assembles with PEG-b-PLGA polymer to generate micelles with strong photothermal properties. Upon laser irradiation, these NPs elicit a strong antitumor immune response to promote the recruitment of NK cells and T cells, which leads to the regression of both primary tumors (Figure 6e) and distant tumors via the abscopal effect (Figure 6f).⁴⁸ The authors also demonstrated that PTT initiates a chemokine gradient which enhances the recruitment of adoptively transferred NKT cells in tumors, followed by activation through antigen presentation by tumor cells and DCs. The NKT cells can directly lyse tumor cells and prime NK cells and CD8⁺ T cells, which elicits a potent antitumor efficacy and tumor reduction. Such combination therapies promote long-term immunological memory, thereby substantially minimizing the risk of tumor recurrence and metastasis. PTT could reprogram immunologically cold tumors to immunoresponsive hot tumors, which enables a promising strategy to treat undruggable and nontargetable tumors and, therefore, has been combined with immunotherapies and chemotherapies for synergistic effects.^49–51 Sun et al. reported multifunctional NPs that simultaneously codelivers a photosensitizer, an anticancer drug, and an immunomodulatory agent to synergize triple-modal therapies.⁵² Photodynamic therapy (PDT) is another light-assisted therapy that elicits an immune response by triggering ICD and subsequent release of tumor antigens and danger-associated molecular patterns (DAMPs).⁵³ Yang et al. demonstrated synergetic antimetastatic therapy in breast cancer tumor models using red-light-responsive and self-destructive diselenide-bridged mesoporous silica NPs.⁵⁴ The ROS generated during PDT initiates diselenide bond cleavage that results in drug release and degradation of the organosilica matrix. NK cells are activated not only through the ICD effects of this combination treatment but also through the degradation of seleninic acid.

In most of these examples, the material composition of the NPs is the driving factor in enabling new properties that have advanced NK cell-based therapies. However, other NP characteristics, such as the surface charge, can also be leveraged to activate NK cells. Kim et al. demonstrated that NK cells could be programmed to be cytotoxic with the utility of cationic NPs by following a straightforward strategy that does not require complex genetic engineering.⁸ When NK cells were exposed to the cationic NPs, the expression of the NK cell membrane-based chemokine receptors CCR4 and CXCR4 were altered, which boosted the ability of NK cells to identify and dock to target cancer cells and induce an antitumor immune response. In addition to modifying the intrinsic NP properties, NPs cloaked with NK cell membrane offer an exciting approach to target tumors by allowing serum stability while leveraging NK cells’ therapeutic benefit. For example, NK cell membrane-cloaked PEG−PLGA NPs loaded with a porphine-based photosensitizer demonstrated selective accumulation in tumors and eradication through PDT via a high concentration of ROS with cytotoxic abilities. In addition to reducing the size of primary tumors, these PEG−PLGA NPs induced ICD that inhibited distant tumors.⁵⁵ Pitchaimani et al. adopted a different design approach and infused the NK-92 cell membrane with the lipid bilayer of liposomes, which resulted in hybrid NPs with both targeting and therapeutic ability.⁵⁶ These hybrid membrane-infused liposomes loaded with chemotherapy drug DOX had multiple merits. These include (i) an enhanced tumor-targeting ability attributable to the functional receptors of the NK-92 cell membrane coating, (ii) nonimmunogenicity and stability under physiological conditions, and (iii) a prolonged circulation half-life with excellent tumor-homing property. These examples demonstrate that isolating NK cell membrane and either coating it on NPs or designing membrane-infused hybrid NPs are promising approaches to leverage the therapeutic benefit of NK cells. Such NPs are not only applicable in cancer treatment but also in understanding fundamental underpinnings unique to the interface of nanomaterials with immune cells.

4. CONCLUSIONS AND FUTURE DIRECTIONS

In summary, this review summarizes the advances in immunoimaging and immunotherapies interfacing NPs with NK cells where the unique physicochemical properties of NPs are leveraged to either modulate resident NK cells in the TME or recruit NK cells into tumors. We discussed how the morphology, surface characteristics, and composition of NPs simultaneously control their uptake and ability to target NK cells. We have summarized the different strategies utilized to label NK cells ex vivo and in vivo to enable longitudinal imaging and NP-based therapies that can successfully activate, prime, and recruit NK cells in the tumors and improve their cytotoxic abilities to eradicate tumors.

Whereas NP−NK cell interfaces have shown tremendous promise in preclinical murine models of cancer therapies, success in canine, porcine, and nonhuman primate models is necessary to understand the clinical potential of NP-based approaches in NK cell manipulation. Further, the impact of other NP properties beyond the traditional size, shape, and surface charge is also relevant. The mechanical properties or stiffness (elastic modulus) of NPs is pivotal in cellular endocytosis via membrane wrapping and tumor penetration.^57,58 Yet, how the modulus of NPs shapes their interactions with NK and other immune cells and their subsequent impact on the immune TME remain underexplored and could be a focus of future studies. Further, other imaging modalities where NPs play a key role, such as surface-enhanced Raman spectroscopy to track NK cells, could further propel the multiplexed detection of multiple immune cells in vivo.^15,59 NP-based ex vivo sensors and devices⁶⁰ that may track circulating NK cell subsets and other immune cells in peripheral blood samples of patients are also of interest in advancing this field.⁶¹ Fundamental studies focused on cocultures of NK cells with cancer cells and other immune cells such as DCs and T cells will reveal NP-mediated crosstalk between these cells and how downstream signaling pathways are activated or suppressed resulting from such crosstalk.

Further, metabolically active NPs that reprogram metabolic pathways and activate NK cells are also of interest.⁶² For example, activated NK cells utilize glucose primarily through aerobic glycolysis to fuel oxidative phosphorylation and adenosine triphosphate production.⁶³ Therefore, glucose-functionalized or glucose-delivering NPs combined with immunotherapies could potentially harness metabolic pathways and other signaling pathways simultaneously, thereby enabling a holistic approach to cancer therapy. Further, several challenges that currently limit NP−NK cell conjugates must be addressed before clinical translation can be achieved. For example, the TME is highly complex where multiple immune cells are interconnected; this implies the modulation of one cell type may irrevocably alter other immune cells. Therefore, comprehensive studies focused on the impact of NP-based therapeutic strategies on multiple cell types in the TME and components of the extracellular matrix is necessary. Recent clinical investigations also show that NK cell ACT has limited treatment efficacy due to systemic level immunosuppression in advanced cancer patients,⁶⁴ and how ACT impacts resident NK cells in patients remains unclear. Therefore, the clinical impact of NK cell therapies on patients must be strongly considered in advancing NP-based approaches for the ACT. In conclusion, both NP characteristics and NK cell-based therapeutic approaches need further development to design highly effective material interfaces that can improve our understanding of the NK cell’s role in modulating the immune tumor microenvironment.