Innovations in Lymph Node Targeting Nanocarriers

Abstract

Lymph nodes are secondary lymphoid tissues in the body that facilitate the co-mingling of immune cells to enable and regulate the adaptive immune response. They are also tissues implicated in a variety of diseases, including but not limited to malignancy. The ability to access lymph nodes is thus attractive for a variety of therapeutic and diagnostic applications. As nanotechnologies are now well established for their potential in translational biomedical applications, their high relevance to applications that involve lymph nodes is highlighted. Herein, established paradigms of nanocarrier design to enable delivery to lymph nodes are discussed, considering the unique lymph node tissue structure as well as lymphatic system physiology. The influence of delivery mechanism on how nanocarrier systems distribute to different compartments and cells that reside within lymph nodes is also elaborated. Finally, current advanced nanoparticle technologies that have been developed to enable lymph node delivery are discussed.

1. Introduction

Nanoparticles (NPs) are colloids with a size from 10 to 1000 nm [1]. The development and application of NPs to biomedical science was sparked by the discovery of the enhanced permeability and retention effect in solid tumors [2]. Since then, NP use has been extended to applications in the diagnosis and treatment of various diseases including cancer, neurodegenerative diseases, human immune deficiency virus, ocular diseases, and respiratory diseases, as well as diseases underpinned by immune dysregulation such as atherosclerosis, type 1 diabetes, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and so on [3,4], owing to the potential for NPs to enable more efficient and controlled spatiotemporal delivery of bioactive and imaging agents [5]. Chemical functionalization, hydrophobic interactions, hydrogen bonding, and electrostatic interactions between NPs and co-formulated drugs have been shown to improve drug bioavailability, solubility, and stability [6]. Due to their nanoscale size, NPs improve drug pharmacokinetics and biodistribution by preventing rapid elimination by the reticuloendothelial system and enhancing blood circulation time [7]. The high surface-to-volume ratio of NPs allows efficient surface functionalization, which not only enables tuning their physicochemical properties on demand, but also facilitates their active targeting to directed tissues or cells via receptor-ligand interactions when the surface is modified with targeting ligands [8]. Taking advantage of NPs, about 30 nanomedicines and nano-imaging agents have been clinically approved by United States Food and Drug Administration and European Medicines Agency as vaccines, imaging agents, iron replacement agents, anticancer drugs, antifungal treatments, and macular degeneration treatments [9]. NPs thus have established as well as yet-to-be-fully-realized potential broadly in biomedical applications.

Lymph nodes serve as concentrated tissue environments distributed throughout the body where adaptive immune responses are mounted and regulated, in which leukocytes recruited from the circulation screen antigen-laden lymph draining from the body’s tissues [10,11]. As a result, vaccines require lymph nodes for their effects [4,10–12]. Lymph nodes that drain primary tumors, termed sentinel lymph nodes, are used diagnostically and prognostically in cancer, and have been considered as major targets of antitumor vaccines and immunotherapies [4,10–12]. Lymph nodes furthermore play important roles in mediating transplant acceptance versus rejection, and their modulation in autoimmune disease has demonstrated therapeutic benefit in clinical contexts [13,14]. As such, the application of nanomedicine principles in enabling the potential of lymph nodes as biomedical targets has begun to emerge. For example, radio-labelled technetium sulfur colloid NPs are used for sentinel lymph node mapping in the United States [15]. Gardasil^® and Cervarix^®, United States Food and Drug Administration-approved vaccines against human papillomavirus infections that are linked to cervical cancer risk, consist of virus-like protein nanoparticles, which allow enhanced access to and immune responses within lymph nodes compared to free viral antigens [16]. Of particular recent relevance, Pfizer’s COVID-19 vaccine comprised of mRNA and lipid NPs has also been shown to allow robust immune response by inducing persistent germinal center B cell response in the axillary lymph node draining the intramuscular arm administration site [17–19]. The development of NPs for lymph node-directed therapies and imaging therefore is already widely used and has high potential for increased implementation in various immunotherapy, vaccination, and diagnostic applications.

There have been numerous reports and reviews that emphasize the potential of lymphatic-targeting NPs for the purpose of treating metastasis, diagnosing disease, and immunotherapy applications [20–33]. Nevertheless, the development of NPs for efficient delivery into lymph nodes remains challenging, due to their complex, often overlooked anatomy and physiology [4,34]. Building upon established approaches and design criteria for lymphatic drug delivery systems [21], we describe in this review the lymphatic physiology relevant to lymph node-targeting nanocarrier design, the engineering principles that underlie those design principles, and recent advanced NP systems engineered to achieve lymph node delivery and effects.

2. Physiology Underlying Lymphatic Transport of Nanoparticles to Lymph Nodes

Lymph nodes have regulated structures that organize the spatial and temporal interactions of antigens, antigen presenting cells (APCs), and lymphocytes within a supporting network comprised of non-leukocytic stromal cells [35,36]. Stromal cell subsets, including lymphatic endothelial cells (LECs), blood endothelial cells, and fibroblastic reticular cells, can play roles in leukocyte homing and function [37–40]. This section focuses on their influences as structural features that may either enable or act as a barrier to the transport of fluids, solutes, and biomolecules, as well as, pertinent to this review, nanomaterials to the lymph node and within its borders.

2.1. Physiology of the lymphatic system

The lymphatic vasculature provides a unidirectional pathway for fluids, solutes, and cells to transit from peripheral tissues to lymph nodes [21]. It is comprised of blind-ended initial lymphatic capillaries composed of overlapping LECs that have incomplete intercellular junctions, which allow the transcapillary transport of interstitial fluid through their expansion resulting from skeletal muscle motion and lymph drainage [41,42] (Figure 1). Once inside the lymphatic vasculature, fluid is termed lymph, which is drained into larger collecting lymphatic vessels. The driving force for this flow is the propulsive motion of smooth muscle cells that line the collector and synchronize pumping along vessel segments termed lymphangions [43] that are bounded by one-way valves that limit backflow and direct net flow unidirectionally away from the tissue drainage site [44]. While this active pumping is the dominant force driving fluid flow, skeletal muscle movement also results in lymph drainage [45]. Along this network of lymphatic vessels are a series of lymph nodes, in which immune cells sample and filter lymph-borne antigens before lymph is eventually returned to the blood at the subclavian vein [46]. It is estimated that 40–90% of lymph-borne proteins are filtered from lymph at the first draining lymph node [47].

Figure 1. Schematic diagram depicting transport mechanisms and barriers in the tissue interstitium, lymphatic vasculature, and lymph nodes.

Small molecules and NPs in tissue interstitium can be transported to the SCS of lymph nodes via size-exclusive passive transport and lymph homing cells in interstitial flow. Small molecules lower than 70 kDa can access to lymph node parenchyma via lymph node conduits.

The structure of lymph nodes that directs the focused and timely interactions of antigens, APCs, and lymphocytes likewise has the capacity to influence how malignant cells, pathogens, or NPs distribute within lymph nodes after their arrival in lymph. Specifically, lymph arrives within the lymph node through afferent lymphatic vessels at the subcapsular sinus (SCS), a luminal structure bounded by monolayer-forming LECs interspersed with lymph-sampling macrophages and dendritic cells [48,49]. This cell monolayer acts as a barrier to free lymph access to the lymph node parenchyma, resulting in lymph being dispersed within the lymph node primarily through the sinuses; from the SCS, which connects to medullary, transverse, and cortical sinus structures also lined with LECs [40], it then flows out of the lymph node via the efferent lymphatic vessels [50]. However, a conduit system composed of FRCs supported by a network of collagen fibers exists that allows a portion of lymph and solutes within the SCS deeper access to the lymph node parenchyma, where the majority of resident leukocytes reside [21,35,40]. Tracer studies suggest that for solutes that gain conduit entrance, their transit through the conduits is rapid (~1 min) [51]. This conduit system delivers lymph and low molecular weight solutes to the lumen of the specialized vascular structures of the lymph node known as high endothelial venules (HEVs) [52–54]. HEVs are the specialized areas of lymph nodes that facilitate lymphocyte homing to the lymph node from the blood circulation at steady state, mediated by a cascade of adhesion and signaling molecules involving the interaction of HEV-expressed adhesion molecules with lymphocyte-expressed homing receptors [55]. That is, the conduits serve as a pathway for chemokines and other molecularly sieved molecules in lymph to directly access and influence the extravasation and immune response of circulating leukocytes entering the lymph node [52,53,56]. Lymph nodes are thus structured to enable not only rapid antigen scavenging and presentation by sinus-proximal and conduit-lining APCs or infiltration by APCs migrating from peripheral tissues, but also to direct infiltration by circulating immune cells in response to pathogenic insult or disease.

2.2. NP transport to lymph nodes after locoregional injection

As elaborated above, nanocarriers offer numerous advantages with respect to opportunities to co-formulate drugs at high levels and direct their distribution profiles after administration in the body. Principles of nanocarrier design that enable lymphatic-mediated delivery to lymph nodes have been well-studied, such that consensus principles on their design and the tissue barriers that regulate these delivery profiles are now established. The extent of uptake into the lymphatic system is generally enhanced by increased half-life of retention in the injected tissue (Figure 1) [21,22]. Put another way, small molecules that have low retention in the tissue site of injection typically accumulate within lymphatic vessels and draining lymph nodes only to low extents. Increasing molecular weight, or co-formulation in a nanocarrier that effectively achieves the same effect, generally increases cargo retention within the injection site and therefore increases overall accumulation in lymph. This section will describe these principles, how nanocarriers influence the way drugs are delivered to lymph nodes when accessed through the lymphatic system after locoregional injection (intramuscular, intradermal, etc.) with respect to the time scales of delivery, and how carrier properties can be tuned to alter these profiles.

The transport of molecules and particles is driven by diffusion in combination with bulk fluid flow [57], the balance of each being heavily dependent upon physicochemical properties of the solute, including size, shape, and surface charge as well as the fluid flow rate. Net transport by diffusion is driven down a concentration gradient, but diffusion times scale with distance squared [58], so diffusion is rapid over short but not long distances [59]. The typical distance of transport from blood vessels to initial lymphatics is on the order of a few hundred micrometers [60]. The transport time of solutes or small molecules with diffusivities ranging from 10⁻⁵ – 10⁻⁶ cm²/s [61–63] across this distance is estimated on the order of one minute. As diffusivity is inversely proportional to hydrodynamic size, species with lower diffusion coefficients (~10⁻⁷–10⁻⁹ cm²/s [64–66]), such as macromolecules and particles, have dramatically higher diffusion times across this same distance (estimated time scales of a few to tens of hours). As a result of these long timescales of diffusion, fluid flow within tissues, termed interstitial flow, that is generally directed towards initial lymphatics due to their lower fluid pressures dominates the manner in which nanocarriers transit the tissue injection site. These flows have been measured at approximately 0.6 μm/s [67] and 0.1–10 μm/s in different tissues [68]. This corresponds to transit times of ~10 minutes through the tissue intracapillary distance, a time length that is far shorter than what would be achieved by diffusion alone. As in the tissue interstitium, molecular and particle transport within the lymphatic vasculature is dominated by the effects of lymphatic flows. Average flow rates in initial lymphatics of mice have been measured at approximately 3–4 μm/s [69], while other studies have reported average lymphatic flow velocities in initial lymphatics reaching substantially higher values of 53 μm/s [70]. In studies of larger collecting lymphatics, average velocities have been measured at 280–1350 μm/s in mice [71], 870 μm/s in rats [72], and 250–1700 μm/s [73] or 2200–16600 μm/s in humans [74]. Considering the collectors only, this would correspond to a transit time of ~10 minutes approximating their length as ~1 m in humans. The major rate-limiting step of lymphatic mediated transport out of the tissue injection site to the lymph node can be considered the ability to transit the interstitium and be taken up into the lymphatic vasculature. The relevance of the competing effects of diffusivity and fluid flow on interstitial transport of solutes versus biomolecules was elegantly elaborated [57] and their relevance holds in the context of nanocarrier design for lymph node delivery after locoregional injection. Namely, the speed at which nanocarriers transit to lymph nodes can be expected to be influenced by rates of interstitial flow [75,76].

With respect to carrier design to increase the extent of delivery to draining lymph nodes, NP size is now well established as being the most influential parameter. Nanocarriers 10–50 nm in hydrodynamic size, and in some contexts as high as 100 nm, generally exhibit the most extensive accumulation within lymph nodes after locoregional injection. This has been shown in the context of macromolecular dextrans, protein-based micelles, gold NPs, lipid NPs, PEG-PLGA NPs, and even polystyrene particles using intradermal, subcutaneous, and intramuscular injection [34,77–120]. Generally, decreasing accumulation is seen for particles with higher hydrodynamic sizes, an effect presumably due to restriction of free particle movement within the tissue extracellular matrix [77,78,90,117–120]. At sizes larger than 50–100 nm, particles remain largely entrapped directly at the injection site [77,78,117].

In addition to size, NP formulations can be engineered to alter their interstitial transport by adjusting surface charge and material properties. It has been shown that carboxylate modified polystyrene particles are lowly diffusive in a model matrix formed from Matrigel^® but are able to penetrate into the scaffold to an extent inversely related to nanoparticle size when placed in convective flow [121]. Velocity of solute transport through the interstitium also depends on solute properties and interactions with the extracellular matrix (ECM) [122]. For uncharged macromolecule dextran ranging in molecular weight from 3–71 kDa, transport through the ECM was shown to exhibit a size exclusion effect that enhanced the velocity of larger macromolecules as a result of these molecules being restricted to movement only through the larger pores of mouse tail ECM in vivo [122]; however, larger dextrans displayed reduced transport in this in vivo system, presumably due to restriction from movement through ECM pores. Negative surface charge was also shown to enhance transport in vivo, presumably due to electrostatic repulsion with the negatively charged ECM [122]. Indeed, lipid NPs with negative-charged surfaces (−11.9 ± 1.0 mV, 33.5 ± 1.7 nm) were delivered in greater quantity to lymph nodes draining the subcutaneous site of injection than neutral (2.4 ± 0.6 mV, 29.6 ± 0.9 nm) and positively charged (14.5 ± 1.5 mV, 24.8 ± 0.5 nm) lipid NPs [119]. Chitosan coated lipid NPs with negatively charged surfaces (−10.4 ± 3.9 mV, 245.1 ± 5.4 nm) also exhibited significantly higher levels of accumulation within intestinal lymph nodes compared to positively charged (27.5 ± 2.1 mV, 182.0 ± 6.7 nm) N-carboxymethyl chitosan-coated lipid NPs delivered orally [123]. The type of functional groups that govern surface charge also affect the efficacy of lymph node accumulation of NPs. For example, generation-5 (G5) poly(amidoamine) (PAMAM) dendrimer (7–8 nm) with terminal phosphate groups (−12 mV) accumulated in the draining lymph node in more significant quantity than ones with terminal sulfonyl (−1.4 mV) and carboxyl (−10 mV) groups after intradermal injection [124]. Surface charges and polymer-terminating functional groups thus regulate how NPs pass through ECM.

In this regard, polyethylene glycol (PEG) benefits NP transport within ECM. PEG is the most widely used polymer in the field of drug delivery, which is intended to be used not only to increase the bioavailability of drugs, but also to prevent non-specific interactions with in vivo components and rapid clearance from the body [125]. PEG conjugation allows the charge neutralization of cationic and anionic carriers, which enhances their transport in tissues [126,127]. Indeed, in model collagen and Matrigel^® ECM, coatings of increasing PEG density on cationic silica NPs were shown to increase their mobility and diffusivity, which was correlated with the reduction in zeta potential that PEG coating affords [128]. In addition, hydrogel NPs conjugated to ovalbumin (OVA) directly or via PEG 500 Da or PEG 5,000 Da showed higher lymph node accumulation than NPs without PEGs [129]. Overall, size and NP surface properties appear to be the major factors to determining the extent of NP transport from the tissue injection site into the lymphatic vasculature.

Transit from the tissue site of injection is also mediated by APCs that take up NPs and traffic to lymph nodes (Figure 1). This represents a major mechanism by which vaccine particles have been explored in various bioengineering approaches to improve vaccine design [12]. Indeed, even particles that become entrapped in the tissue interstitium site of injection because of their ECM pore size-exceeding hydrodynamic size can be transported to lymph nodes via APCs [78,117,128,130,131]. Although the rate of cell migration through the interstitium is cell type dependent [132], cell-mediated delivery to the lymph node generally occurs over the course of hours to days, in contrast to NP transport directed by interstitial flow that occurs over minutes [133]. Specifically, upon encountering antigens, APCs will migrate to and enter the initial lymphatics [134]. After a brief period of crawling within the lymphatics, APCs detach and enter downstream lymph nodes carried by lymph flow [135–137].

Migratory dendritic cells have been measured moving at 1200 μm/min in the collecting lymphatics carried by lymphatic flow, which is approximately 200 times faster than their rate of migration in the initial lymphatics [135]. Thus, despite the collecting lymphatic vessel being the longest distance to be traversed along lymphatic delivery to the lymph node, the processes of particle uptake, APC-intrinsic cues triggering cell emigration from the peripheral interstitium, and transit through and detachment from initial lymphatics predominantly regulate the extent and time scale of APC-mediated transport to lymph nodes.

NPs can be designed to enhance or control delivery to the lymph node by leveraging the migratory functions of APCs in various ways. NPs engineered to interact with specific leukocyte subsets with intrinsic lymph node homing ability [37–40,55] can deliver cargos into the lymph nodes more efficiently and selectively than non-engineered NPs. As one example, liposomes (157.7 ± 14.4 nm, 18.1 ± 4.2 mV) [138], AuNPs (21.61 ± 12.32 nm, −37.1 ± 9.92 mV) [139], and superparamagnetic iron oxide (SPION) NPs (46.2 ± 1.9 nm, −8.88 ± 6.96 mV [140], 37.3 ± 5.6 nm, −8.88 ± 6.96 mV [141], 22.2 ± 5.4 nm, ~20 mV [142]) modified with mannose and mannan, and mannan nanocapsules (226 ± 13 nm, −10 ± 1 mV) [143] that have the ability to interact with the receptors on the surface of APCs showed enhanced accumulation of cargos in lymph nodes, leading to the improved activation of APCs or MR imaging. Hyaluronic acid, hyaluronan, and aCD40 have also been similarly explored for their potential to mediate targeting of peripheral tissue APCs [144–146]. NP physiochemical properties can thus be engineered not only to optimize NP transit to lymph nodes via lymph drainage but also for their capacity to leverage the migration behaviors of lymph node homing leukocytes.

2.3. Transport of lymph-draining NP within lymph nodes

NP distributions within lymph nodes are highly regulated by the way particles are transported to the tissue. When arriving via afferent lymphatic vessels in the SCS, NPs greater than 70 kDa in size are excluded from conduits. During their transit through lymph node sinuses, NPs can be sampled by sinus-lining LECs and scavenging APCs [54,147]. In addition to direct uptake by sinus-lining cells, NPs can penetrate the parenchyma by diffusion [147]. This includes from the SCS into the cortex and intrafollicular zone [148], as well as from conduits into the paracortex [149]. As diffusivities are higher for hydrodynamically smaller molecules and particles, penetration into these regions is more rapid and transpires to greater extents for smaller particles and molecules [121]. Furthermore, only the smallest particle systems have access to the latter structures, given the exclusion of molecules above 70 kDa in molecular weight [147]. However, these size-specific exclusion profiles have been shown to be altered in states of disease [150,151].

Lymph-borne NPs can be transported into lymph nodes in ways that leverage the scavenging functions of sinus-lining cells, despite their general restriction from free access to the lymph node parenchyma. This is because SCS macrophages not only limit the deeper access and systemic spread of lymph-borne pathogens, but also mediate immune responses by producing pro-inflammatory cytokines to recruit other immune cells [137,152,153]. Recently, the exclusion effects of sinus-lining macrophages have been subverted as a means to increase NP delivery into lymph nodes. This approach is based on observations of infection or adjuvant stimulation resulting in their dissociation from sinus lining [137,154,155]. To this end, disruption of SCS macrophages within lymph nodes by administration of lymph-draining clodronate liposomes facilitated the higher access and retention of tumor-derived extracellular vesicles (60–100 nm) [155] and OVA-AuNP (15 and 100 nm) [156] into lymph node follicles, which improved the humoral immune response [156].

LECs represent a major barrier at the lymph node SCS, but also sample lymph-borne antigens [157,158], as well as facilitate molecular transport into the parenchyma of lymph nodes by transcytosis [159]. In this manner, antibodies delivered into lymph after locoregional injection accumulate rapidly with the SCS-proximal regions of lymph nodes draining the site of injection, a process that appears dependent on the Ig molecular weight [160]. Accordingly, a strategy to enhance the transcytosis by or permeability of a lymph node sinus-lining LEC monolayer to improve NP transit into lymph nodes has been explored [97]. Lymph-draining, nitric oxide releasing NPs were found to not only efficiently drain into the lymph nodes [98], but also enable the enhanced penetration of macromolecular dextrans into the (para)cortex of lymph nodes that would otherwise be excluded, increasing their delivery to dendritic cells and B cells [97]. Although it is not yet clear how nitric oxide increases NP access to lymph node cells, nitric oxide-mediated enhanced uptake of the macromolecules by LECs implies potential nitric oxide influences on LEC transcytosis or intracellular gap junctions [161–163].

Targeting specific migratory cell types in the peripheral tissue injection site influences how the injected particles are distributed within the lymph node and the resultant lymph node-resident cell types the NP interacts with, as it has been shown that different types of APCs traffic to specific areas of lymph nodes [164]. For example, while both are present in the skin, dermal dendritic cells migrating to the lymph nodes traffic to the cortex and follicle areas where they predominantly prime B cells and CD4⁺ T cells, whereas Langerhans cells migrate to the inner paracortex where they interact more with CD8⁺ T cells [107, 164]. In addition to APCs, NPs targeted at other migratory immune cell subsets have successfully been used for lymph node targeting and immunomodulation [165–167]. These approaches are presumably driven by the chemotactic behaviors intrinsic to the emigrating cell type rather than the carrier itself, which present interesting opportunities for cargo targeting. However, the extent to which these cargos may be restricted to the intracellular environments of trafficking cells or be made available on the cell surface to interact with other cells must be considered in the breadth of potential applications.

2.4. How NPs are transported to lymph nodes influences their access and effects on lymph node cells

The mechanisms underlying NP transport to lymph nodes as mediated by the lymphatic system elaborated above influence how NPs distribute to lymph node-resident cells (Figure 2). Most apparent perhaps is the contrast between NPs that are delivered into lymph directly versus those trafficked by tissue emigrating APCs that arrive in lymph nodes (Figure 2). Especially in the case of the former, what cells the NPs associate with is tightly regulated by the lymph node structure itself (Figure 2).

Figure 2. Extent, timing, and spatial distribution of delivery to the lymph node are influenced by mechanism of transport and lymph node structural barriers.

(A) Size-varied fluorescent tracer system facilitates (B) study of transport mechanisms to the lymph node. (C–E) Spatial distribution of each tracer within the lymph node 4 and 72 h post-intradermal administration. (C) Representative draining lymph node images, (D) Quantification of penetration depth into the lymph node for each tracer 4 and 72 h post-administration, (E) Average distance of tracer from lymph node capsule. (F) Extent of delivery of each tracer to cells within lymph node measured by frequency of association through flow cytometry. (G) Spatial distribution of leukocyte subsets within lymph node structure. (H) Quantity of each marker-positive cell type within CD45⁺ cells that is tracer positive 4, 24, and 72 h post-administration, as measured by flow cytometry. Scale bars, 200 um; * indicates significance by two-way analysis of variance (ANOVA) (D–F) or one-way ANOVA (H) with Tukey’s comparison (* indicates P < 0.05, *** indicates P < 0.005, **** indicates P < 0.001). n = 5 to 8 mice. Figures adapted and modified with permission from [117,131].

Our group has evaluated in a series of studies how transport mechanism and lymph node structural barriers influence the extent and timing of accumulation in lymph nodes by nanoscale material systems [77,78,97,117,131,151]. By implementing a size-varied system of fluorescent nanoscale tracers (Figure 2), the influence of lymph drainage and cell-mediated transport mechanisms on lymphatic delivery to lymph nodes was studied to understand how nanocarrier design regulates distributions to lymph node leukocytes. Results corroborate established paradigms of how lymphatic transport and intra-lymph node transport are regulated by hydrodynamic size, with 30 nm tracer exhibiting the most rapid and greatest extent of lymph node accumulation [77,78,97,117,131,151] but simultaneous restriction within the lymph node sinus [117]. As a result, despite prodigious accumulation within lymph nodes, 30 nm tracers associate lowly with local leukocytes [131]. Tracers smaller in hydrodynamic size but larger than the blood capillary permeability limit (10 nm) also rapidly drain to lymph nodes after injection but display a lower overall extent of accumulation but greater access to paracortex-resident leukocytes, an effect presumably resulting from conduit access and higher diffusivity [77,117]. These results indicate lymph-draining tracers below the hydrodynamic size limit of the lymph node conduit system associate with paracortex- and cortex-resident lymphocytes to greater extents than tracers that are sinus-restricted [117,131]. In sharp contrast to these nanoscale tracers 10–30 nm in hydrodynamic size that rapidly and prodigiously drain into lymph directly, 500 nm polystyrene particles that reach the lymph node do so to much lower extents (1000x) and at slower rates (over days rather than ~ 1 hour) after injection, presumably due to their transit via migratory APCs [77,78,97,117,151]. As a result, they also associate lowly with local leukocytes [117,131]. However, rather than being found primarily within the lymph node sinuses as seen with tracers that directly drain into lymph, they instead accumulate within the parenchyma [117].

Functional implications of these differences in transport mechanisms that varied the extent, timing, and targeted cells resulting from delivery were tested by surface-modifying nano- and microparticles (30 versus 500 nm in hydrodynamic size) with antigen and assessing the resultant antigen-specific CD8 T cell response [117]. These studies revealed that despite reaching the lymph node in lower extents and rates, antigen delivered on 500 nm microparticles that reached the lymph node via migratory APCs locally induced tumor control-eliciting cytotoxic CD8⁺ T lymphocytes to greater extents than when delivered on lymph-draining, sinus-restricted 30 nm NPs [117]. In contrast, antigens delivered on lymph-draining NPs locally expanded the pool of stem-like CD8⁺ T cells implicated in the therapeutic response to immune checkpoint blockade cancer immunotherapy to greater extents than that seen by microparticles [117]. How nanocarriers distribute to lymph nodes thus exerts strong influences on the resulting immune modulatory effects they can elicit. This concept was proposed in the context of response to infection or endogenous particles as elaborated in an elegant series of papers [168,169]. These principles no doubt apply to nanoengineering approaches for delivery into lymph nodes. How the extent, rate, and quality of leukocyte association may be differently balanced in a given immunotherapy, imaging, or other application may be tuned by advanced NP delivery system design.

3. Advanced Drug Delivery Systems for Lymphatic Delivery

Methods of cargo delivery to lymph nodes have been enhanced by advanced NP systems [77–120]. As already discussed, NP physicochemical properties can be tuned to optimize transit through the peripheral tissue site of injection [77,78,117–120,122–124,128,129]. Optimizing around the physiology of the lymphatic system and lymph nodes also represents an intriguing alternative approach. Below, recent advancements in nanocarrier design that either overcome or leverage these unique attributes are elaborated.

3–1. Proteins and their assemblies for enhanced lymph node delivery

Endogenous physiological proteins that pass through and accumulate in lymph nodes have been exploited as a method to achieve efficient cargo delivery to lymph nodes (Figure 3A). The best described example is albumin, a 66 kDa endogenous protein 5–10 nm in hydrodynamic diameter, which not only efficiently transits to lymph nodes [170–177], but is also allowed to enter the lymph node parenchyma [147,178]. Inspired by the roles of albumin in the transport of fatty acids, various hydrophobic agents, lipids, and peptides have been identified as albumin-binding domains, which have long been widely used in imaging agent technologies [170–173]. Albumin hitchhiking to lymph nodes was achieved by increasing the lipophilicity of peptide and adjuvant for vaccine applications. In so doing, higher systemic CD8⁺ T cell responses could be elicited that improved antitumor effects compared to the free formulations of CpG and antigen peptides [174]. In addition to the hitchhiking strategy [170,174–177], albumin can be fused with small therapeutic proteins to enhance their lymphatic delivery by increasing their molecular weight, such that they are not so easily absorbed from the injection site into the systemic circulation and cleared [179–181]. Albumin fusion with interleukin (IL)-4 [180] and IL-10 [181] resulted in higher lymph node uptake than free IL-4 and IL-10, which ameliorated experimental autoimmune encephalomyelitis and rheumatoid arthritis, respectively.

Figure 3. Schematic diagram depicting strategies of advanced NPs rationally engineered for delivery to lymph nodes.

(A) Proteins are engineered to efficiently transport to lymph nodes via size-dependent lymph drainage. (B) Trojan horse NPs efficiently transport into lymph due to their optimal size for lymphatic uptake and then achieve efficient release of small molecular cargos into the lymph node parenchyma using release from the carrier into lymph. (C) Cargos loaded in PNAd-targeting ADCs and NPs are transported to the lymph node after uptake by the HEVs after administration into the systemic circulation. (D) Magnetic NPs efficiently transport to lymph nodes when external magnetic is applied near the targeted lymph nodes. (E) NPs camouflaged with lymph-homing leukocyte cell membranes accumulate within lymph nodes due to both their optimal size for uptake into lymph and NP surface-coating chemokines, L-selectin, and integrins.

In addition to albumin, several other proteins have been self-assembled into NPs with optimal size for efficient lymphatic delivery [182]. Self-assembled human ferritin heavy chain particle (hFTN, 11.74 ± 0.8 nm, −5.69 ± 0.44 mV), Escherichia coli DNA binding protein (9.5 ± 1.2 nm, −5.63 ± 0.33 mV), Thermoplasma acidophilum proteasome (13.4 ± 2.1 nm, −2.13 ± 0.27 mV), and hepatitis B virus capsid (32.3 ± 1.9 nm, −7.50 ± 0.43 mV) have been found to efficiently drain into the lymph nodes after injection [182]. Among these protein NPs, hFTN exhibited the highest extent of lymph node delivery, for reasons not elaborated. When hFTN was genetically engineered to include model tumor antigen (red fluorescence protein) that increased its hydrodynamic size increased to 26 nm, it elicited antitumor effects on red fluorescence protein-expressing tumors associated with CD8⁺ T cell expansion in the draining lymph nodes after administration [182].

3–2. Trojan Horse NPs for efficient cargo delivery into the lymph node parenchyma

Despite improving overall extents of lymph node accumulation, increasing cargo size by formulation into a nanocarrier can diminish its access to cells within the paracortex of targeted lymph nodes, as elaborated above [21,113,120]. With a concept long explored in the context of tumor-directed delivery wherein the tumor microenvironment can restrict NP penetration and effects [183], the transformation of NPs with optimal lymphatic delivery (10–100 nm) to a smaller size or to release their cargo upon uptake into lymph instead has the potential to leverage the benefits of NPs in lymphatic uptake and delivery to lymph nodes but nevertheless achieve efficient delivery of therapeutics into cortex and paracortex of lymph nodes. This general concept has been extensively explored in the context of tumor-directed therapies, wherein numerous pH responsive drug delivery and imaging systems have been developed to exploit the unique chemical microenvironment of tumors that exhibit alterations in pH, redox potential, reactive oxygen and nitrogen species, proteinase, and so on [184,185]. As the lymph node is a primary bridgehead for metastasis of cancers [186], ultra-pH-sensitive micellar NPs (23.4 ± 2.5 nm) have been designed that emit amplified near-infrared fluorescence when they are disrupted in the presence of tumor microenvironmental pH, creating a signal that can distinguish between lymphatic tumor metastases and normal lymph nodes [187]. However, in contrast to tumors, chemical stimuli within lymph nodes, or more specifically the non-malignant lymph node microenvironment, has never been clearly identified.

To address this limitation to existing lymph node-directed therapies, we recently demonstrated how intra-lymph node delivery can be dramatically enhanced by engineering the release of cargo formulated in lymph-draining nanocarriers into lymph (Figure 3B). This study that demonstrated the generalizable principle of multistage delivery as facilitating robust delivery to lymph node leukocytes employed various lymph-draining nanocarriers, including 30 nm Pluronic-stabilized poly(propylene sulfide) NPs and 60 nm protein-based virus-like particles, and multiple release mechanisms, including release by diffusion after passive encapsulation into the nanocarrier or reversible chemical linkages. The latter included interesting oxanorbornadienes that degrade via first-order retro-Diels–Alder fragmentations in a manner independent of pH and solvent conditions with adjustable half-lives ranging from hours to days [188]. When married with lymph-draining carriers, delivery into lymph node regions normally restricted from cargo access when tethered to NPs using a permanent linkage was dramatically altered, increasing >1000 fold cell access by resident leukocytes, and of special interest, achieving high levels of cargo delivery to many lymphocyte subtypes (Figure 3B). By varying the linker half-life, the timing of delivery could be tuned, an attribute attractive for applications when serial delivery of multiple agents from a single dose might be desired. When this multistage delivery approach was implemented in the context of therapeutic agents, elimination of lymph node B cells by small molecule chemotherapeutic irinotecan was enhanced. The effects of Toll-like receptor ligand CpG used as an in situ vaccine at low dose in lymphomas formed within targeted lymph nodes as well as abscopal sites were greatly enhanced. Multistage delivery into lymph represents a powerful new way to deliver therapeutics with the potential to orchestrate the complex immunological functions of lymph node leukocytes.

3–3. NPs targeting lymph node HEVs

While the systems discussed so far [170,174–177,180–182,187,188] achieved lymph node delivery by leveraging lymphatic transport mechanisms, advanced NP systems have also been developed to improve therapeutic delivery through the blood circulation (Figure 3C) [189–192]. HEVs located in the lymph nodes constitutively express peripheral node addressin (PNAd) that facilitates the access of lymphocytes into the lymph nodes [55,56]. Accordingly, a form of conjugates consisting of therapeutics and a PNAd-targeting antibody (MHA112) enable the efficient delivery of cargos to lymph nodes after systemic administration [190]. When conjugated with IR800 fluorescent dye via a peptide bond, MHA112 administered intravenously exhibited preferential accumulation in the lymph nodes compared to free IR800. Interestingly, the MHA112 was localized within the HEV cells, while IR800 was found mainly to associate with lymph node dendritic cells and to a lesser extent with macrophages and fibroblastic reticular cells. These results imply that MHA112-IR800 conjugates were first taken up by HEV cells, and then underwent intracellular cleavage of the peptide linker, which released cargos extracellularly to be accessed by lymph node resident cells (Figure 3C). As HEVs are distributed throughout lymph nodes, some types of tumors, and metastasized tumors in tumor-draining lymph nodes, and are expanded in metastatic tumor-draining lymph nodes, MHA112-IR800 allowed the diagnosis of early metastasis of tumors in the tumor-draining lymph nodes, and MHA112-paclitaxel conjugates led to reduced tumor growth as well as reduced metastasis of tumors to tumor-draining lymph nodes [190]. In particular, MHA112-paclitaxel exhibited the expansion of activated T cells (CD8⁺CD44^hi) and cytotoxic T cells (CD8⁺TNF-α⁺), and the reduction of regulatory T cells (CD4⁺CD25⁺FoxP3⁺) in tumors and tumor-draining lymph nodes, supporting that those therapeutic effects were attributed to the improved delivery of paclitaxel into the lymph nodes via the interaction between MHA112 and PNAd. The strategy of targeting PNAd in HEVs [190,191] could potentially also be extended to the development of various NPs systems [192] for diagnosis as well as immunotherapy associated with lymph nodes.

3–4. External forces-guided NPs for lymph node homing

The lack of stimuli specifically identified for lymph node microenvironment [188] has limited the utilization of the library of numerous stimuli-responsive NPs developed during several decades of study in efficient lymph node delivery. Instead, NPs capable of being guided on demand by external forces have been explored to improve the delivery efficacy and specificity of cargos into lymph nodes. Superparamagnetic properties of SPION have been utilized for MR imaging as well as magnetic guided targeting [193–195]. Indeed, cargos have been shown to efficiently accumulate in the lymph nodes when magnetic fields are applied near the targeted lymph nodes after injection of cargo-loaded SPION NPs (Size and zeta not reported) (Figure 3D) [194,195]. Furthermore, lipid-SPION hybrid NPs (24.6 ± 1.6 nm) pre-incubated with bone-marrow DCs (BMDCs) to create SPION-decorated BMDCs showed significantly higher accumulation of NPs in the deeper regions of lymph nodes when magnetic fields were directed toward the lymph nodes of interest [196]. These suggest the potential of NPs to be guided by external magnetics to improve lymph node accumulation, which can be further enhanced by having NPs interact with lymph node homing leukocytes. Despite a lack of extracellular stimuli for lymph node delivery, the potential of stimuli-responsive NPs has been explored for the efficient intracellular delivery of cargos to specific intracellular compartments; e.g. reactive oxygen species sensitive NPs for endo-lysosomal escape of antigens and adjuvants in DCs [144].

3–5. Immune cell membranes camouflaging NPs for lymph node homing

There have been numerous NPs camouflaged with membranes of various cells including red blood cells, leukocytes, cancer cells, NK cells, MDSCs, and platelets, which facilitate prolonged circulation, tumor targeting, macrophage polarization, and so on [197]. This approach can also be applied to lymphatic delivery of NPs; efficient lymph node homing ability of DCs via chemokines (i.e. CCR7), L-selectin, and integrins on the membrane [198] has inspired the development of NPs camouflaged with DC membranes for antitumor immunotherapy by modulating lymph nodes (Figure 3E) [199–201]. Histidine-modified stearic acid-grafted chitosan (HCtSA) NPs loaded with OVA proteins and camouflaged by dendritic cell membranes (149.93 ± 7.85 nm, −5.63 ± 0.31 mV) [199], as well as imiquimod (Imq) loaded PLGA NPs camouflaged by anti-CD3ε conjugated dendritic cell membranes (~ 180 nm, −12.87 ± 0.60 mV) [200] exhibited significantly higher accumulation in lymph nodes than their DC membrane-uncoated controls when subcutaneously administered, facilitating an antitumor immune response by activating DCs. In addition, NPs camouflaged by fused membranes composed of dendritic and cancer cell membranes showed significantly higher lymph node homing ability than NPs camouflaged by either cell membrane type individually, perhaps because fused cell membranes contained tumor antigens as well as upregulated chemokines, L-selectin and integrins, allowing them to be efficiently delivered into the lymph nodes [201]. Despite the high efficacy, a limitation of these strategies exploiting the intrinsic lymph node homing ability of immune cell membranes is the potential burden of production scale-up and cost-ineffectiveness.

4. Concluding remarks and perspectives

Interest in lymph nodes as therapeutic and diagnostic targets has dramatically expanded in recent years with the emerging prevalence of immunotherapies for the treatment of a variety of diseases and improvements in imaging methods. Numerous nanotechnologies have now been developed for lymph node delivery, with designs varying based on intended target or application. Numerous paradigms of the nanocarrier physiochemical properties optimal for achieving delivery to lymph nodes after administration in a locoregional tissue bed are now established. The utilization of NP systems for diagnostic or therapeutic applications will undoubtedly continue to unfold. Continued developments that further improve or augment carrier delivery include the application of scaffolds, hydrogels, and microneedles to sustain nanocarrier delivery to provide an additional way to modulate the pharmacokinetics of cargo delivery in the lymph nodes [202–204]. Furthermore, NP targeting through functionalization with ligands specific to target cell types, such as hyaluronic acid, hyaluronan, aCD3, aPD-1, aCTLA-4, and so on [144,145,166,167] has the potential to further improve delivery to target cell populations. Approaches that modulate the tissue, either at the level of the ECM, lymphatic vasculature, or the lymph node structure also have been proposed as potential methods to further adjust profiles of NP delivery [23,130]. Recent and continued innovations continue to develop the potential for nanotechnologies to improve diagnostic and therapeutic potential of NPs in lymph nodes.

Highlights.

• Describes lymphatic physiology and lymph node structures that influence nanocarrier delivery to lymph nodes

• Delineates fundamentals of nanocarrier transport to lymph nodes and the influence of physicochemical properties of nanoparticles on these pathways

• Elaborates advanced nanoparticle designs for lymph node delivery