Detection of the Immune Response during in vivo mapping of the Lymphatic system: A profile of 99mTc-Antimony Trisulfide Colloid and other Radioactive-Nanoparticles

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Critical Review

Detection of the Immune Response during in vivo mapping of the Lymphatic system: A profile of 99mTc-Antimony Trisulfide Colloid and other Radioactive-Nanoparticles

Corresponding author: Chris Tsopelas PhD, RAH Radiopharmacy, Royal Adelaide Hospital, Nuclear Medicine Department, Adelaide, Australia, Email: Chris.Tsopelas@health.sa.gov.au

Abstract


Since the 1970’s, nuclear medicine clinics around the world have employed radioactive-nanoparticles for many different diagnostic studies in man. Currently the most important application of γ-emitting nanoparticles is to map the lymphatic system in cancer patients, and the β-emitting particulate agents are for treating arthritic joints. This review summarizes the current developments in diagnostic lymphoscintigraphy that employ radioactive nanoparticles, with a special emphasis on 99mTc-antimony trisulfide colloid, and it also describes the key cellular and molecular events
in vivo during an immune response.

Keywords: radiocolloid; nanoparticles; lymphoscintigraphy; nuclear medicine; immune system

Introduction

Lipid-based nanoparticle formulations have been actively investigated in nanomedicine research, as carriers to deliver drug with enhanced stability or in a controlled release man- ner, and for targeted therapy[1]. For radioactive-nanoparti- cles of synthetic or biological origin (non-lipid), their trans- lation into nanomedicine[2] and specifically within the field of nuclear medicine, has resulted in a variety of different di- agnostic and therapeutic procedures conducted in the clin- ic daily. The diagnostic tests currently include liver/spleen scans, infection/inflammation scans, assessing colonic mo- tility function, bone marrow imaging and lymphoscintigra- phy. Radiosynovectomy is an example procedure for treating rheumatoid arthritis/joint-related disease from the β-emis- sions of the decaying isotope. The particle size of radioac- tive-nanocolloids makes them suited for imaging the retic- uloendothelial system, and especially the lymphatic system after a dose is administered subcutaneously. Lymphoscintig- raphy is a common technique employed to map lymphatic drainage from a primary tumor in cancer patients, and less frequently to evaluate the patency of lymph flow in patients with edematous limbs. It is based on the in vivo detection of gamma rays emitted from the radiopharmaceutical in lymph, and aims to identify the first draining sentinel node(s) that potentially harbors cancer cells during their metastasis. There are a small number of regulatory approved techne- tium[99mTc]-labelled colloids available for this indication (Table 1). The choice of radiotracer does vary according to country, such as 99mTc-antimony trisulfide colloid (ATC) in Australia, 99mTc-tin fluoride or 99mTc-calcium-phytate in Ja- pan, 99mTc-human-albumin and 99mTc-rhenium sulfide in Eu- rope, or 99mTc-(filtered)sulfur colloid in the USA.

At the imaging clinic, the radiopharmaceutical is usually injected intradermally over a number of sites around the tumor, or if the periareolar technique is used, in the quad- rant containing a tumor of the breast. While the patient preferably lays supine, a γ-camera acquires dynamic im- ages within a time frame of 30-60 minutes for the smaller radiocolloids. Lymph flow differs according to region of the body, the rate is influenced by muscular contraction, envi- ronmental temperature or occlusion from advanced met- astatic deposits, and massaging the injection site for a few minutes also helps facilitate radiotracer movement. The ideal radiopharmaceutical colloid should be a stable ag  agent that comprises of high specific activity 99mTc-particles near to but not less than 4-5 nm in diameter, to avoid blood capil- lary penetration, reduce radioactivity retention and radiation burden at the injection site, and to achieve fast flow and high retention in the lymph nodes. In some imaging departments, larger particles are used to simplify the lymph node map. With the aid of the scintigraphic scan, the nuclear physician uses a pen to mark on the patient’s skin where the concentration of γ-counts corresponds to the sentinel lymph node(s). This oc- curs prior to the patient’s transfer into theatre for surgery. At the time of an operation, the surgeon injects a blue or green dye around the tumour, to aid in the visual identification of an afferent lymphatic channel and node. A portable γ-probe detector is also used to guide the dissection by pinpointing counts in the node. A positive identification is made from si- multaneous signals of radioactivity and color, when the hot sentinel node is stained with dye and at least one afferent lymphatic channel, these selective criteria distinguish it from other lymph nodes in the basin. Initially the surgeon removes the primary tumor, and then the draining sentinel node(s) as part of the sentinel node biopsy procedure. The harvested tissue sample is subsequently examined by a pathologist to identify any tumor cells, their absence indicates the lymphat- ic system is likely to be free of disease further downfield. Un- der this condition the operation is then completed, because it supersedes the decision to commence invasive surgery that removes more contiguous lymphatic tissue. A frequent com- plication for the patient after a wide surgical excision is sec- ondary lymphedema, an outcome which is difficult to treat and manage successfully.

The lymphatic system absorbs interstitial fluid containing waste products, cell debris, proteins or antigens, and lymph contains white blood cells. It is an integral part of immunity

especially in the vicinity of an infectious lesion or an inflamma- tion that harbors antigen material. As radioactive particles are delivered to the lymphatic system, they interact with the living tissue, white cells and fluids, becoming involved in residential immune functions rather than just passively percolating along the network until their exit from the thoracic duct into blood. While complex and sophisticated events occur at the cellular and molecular levels, the lymphoscintigraphy technique gross- ly detects an immune response toward a nanoparticle-antigen containing a γ-signal, within the lymphatic system. This review focuses on radioactive nanoparticles with a special emphasis on 99mTc-antimony trisulfide colloid, their radiolabeling chem- istry, and how these tracers are handled in vivo by the immune elements comprising the lymphatic physiology.

Radionuclide bond to nanoparticles

The chemistry of 99mTc-ATC and other particles

The structure of antimony trisulfide in the solid state is a poly- mer-ribbon lattice comprised of interlocking SbS3 and Sb3S units, described by the nominal monomer formula Sb2S3[3]. When these particles are dispersed in an aqueous solvent, their composition is similar to that of arsenic trisulfide[4] with an electrical double layer of excess thiol and sulfide groups at the surface[5]. The pharmaceutical product is available in a composite pack format[6], and one of the vials contains the aqueous non-radioactive formulation of orange antimony tri- sulfide colloid.

These pre-formed ~10 nm diameter particles can be labelled in situ using the readily available 99mTc-pertechnetate pharma- ceutical. This dispersion does not contain a purposely added reductant, such as stannous chloride that commonly features

Table 1. Physical properties of common radiopharmaceutical colloids used in the imaging clinic.

name type size (nm) Particl e

cold kit 99mTc-colloid pre- 1 formed cold particles 99mTc-particles formula

formed

in situ2 (monomer)

Lymph Flo Kit antimony trisulfide Leucocyte Labelling Kit tin fluoride Phytate calcium phytate

+ – 9 ± 4 nm 90% <20 nm 99mTc-Sb2S3

– + 2100 nm 96% 1000-3000 nm 99mTc-Sn(O )F2

– + 100- 1000 nm – 99mTc-Ca-phytate 400 nm (shaken)

Amerscan Hepatate tin fluoride + 1200 nm (static), 99mTc-Sn(O )F2
Sulfur Colloid sulfur + 74% <200 nm 99mTc2S7
Nanocoll human albumin + 95% <80 nm 90% 100-220 nm 99mTc-albumin
NanoCIS rhenium sulfide + ~100 nm 99mTc-Re2S7
Senti-Scint human albumin + 100-600 nm 99mTc-albumin

millimicroaggregate

1 = 99mTc-coordination directly on particle surface; 2 = 99mTc reacts with particle during growth phase, bound within the matrix in many other 99mTc-kit products. Under the radiolabeling con- ditions when particles are heated in mineral acid at 100oC for 30 minutes, they are resistant to hydrolysis because the dis- persant protects against flocculation. Antimony (III) ions in- hibit the redox reduction of 99mTc-pertechnetate, but these were not detected in the vial contents. Volatile hydrogen sul- fide gas formed but did not participate in the radiolabeling reaction, nor did 99mTcOCl4- anion, and yet the binding mecha- nism was proposed to involve oxidation of thiol groups on the particle surface by 99mTc-pertechnetate into sulfonic, sulfenic or sulfinic acids[5]. The resulting 99mTc(V) and/or 99mTc(III) ions coordinate to abundant thiol groups over the surface (Fig- ure 1). 99mTc-ATC product is easily prepared in high efficiency with >95% radiochemical purity, 90% of the 99mTc-particles are <20 nm by the membrane filtration technique[7]. It was reported an antimony trisulfide formulation contained 5 x 1013 cold particles per mL[8], and after radiolabeling, few techne- tium atoms were likely to be bound per particle[7]. The op- tion of preparing a higher specific activity radiotracer assisted in the earlier identification of a lymph node during a scan[9]. This colloid was also labelled with 111In, the different indium chemistry resulted in a shorter reaction time at room tempera- ture to yield a high purity 111In-ATC product of <20 nm that was stable for 5 days[10]. An 90Y version was also prepared recent- ly[11]. Antimony trisulfide, human nanoaggregated albumin and rhenium sulfide are all pre-formed particles that bind the isotope on their surface. These products are easily prepared in a radiopharmacy, and the final product formulation can be made available for patient injection within a convenient time frame of 3-60 minutes.

nanoaggregated albumin and rhenium sulfide are hydrophobic colloids. For 99mTc-sulfur colloid, 99mTc-tin fluoride colloid and 99mTc-calcium phytate, particles are formed simultaneously during the radiolabeling step, the isotope becomes incorporat- ed indiscriminately throughout the particle matrix[3].

There are a variety of commercial kits available to prepare 99mTc-phytate in which calcium chloride excipient yields 99mTc- Ca-phytate colloid in vitro, or without the excipient the same product is yielded in vivo from endogenous circulating calcium ions. Almost all of the cold kit formulations contain the solu- ble stannous-phytate complex, acting as the reducing agent for 99mTc-pertechnetate, as well as a chelator of reduced radiome- tal. The soluble 99mTc-phytate changes into a dispersion when calcium ions are added. It was shown that radiocolloid size increased with an increasing Ca2+ concentration during the re- constitution step in vitro[16,17], or in the presence of serum calcium to give a particle size range of 5-1000 nm[18].

New generation radiotracers for lymphatic mapping

Some of the newer lymphatic imaging agents currently in the research and development domain, include the so-called hy- brid-tracers, or colloidal particles capable of emitting two or more detectable signals (Table 2). The particle matrix usually binds the signal molecule (ie: indocyanine dye that is stimu- lated into emitting fluorescence) and/or isotope, however the particle itself can be the signal source (ie: magnetic SPIONs for magnetic resonance imaging[19]).

Figure 1. 99mTc-reaction at the surface of an antimony trisulfide particle

Tin fluoride colloid is a hydrophilic colloid, prepared by ‘grow- ing’ the particles at room temperature. The particle mecha- nism most likely involves an association of smaller stannous fluoride particles connected by Sn-O bonds formed after hy- drolysis, during a concurrent reduction reaction and coordi- nation of 99mTc[12]. The synthesis of this radiocolloid has been reviewed elsewhere[13]. Other radiometals such as 90Y[14] and 188Re[15] were coordinated to tin fluoride particles for ap- plication as radiosynovectomy agents. Sulfur colloid, human

If a radiotracer can bind to receptors on cancer cells, white cells etc, then the discrimination of normal lymph nodes from diseased nodes could potentially enhance the established im- aging technique[20]. For lymphatic mapping particularly, the preference is to use smaller labelled-particles that predomi- nantly enter into the lymphatic system rather than remaining at the injection site or entering the bloodstream. Particles and

large complex molecules at the nanometer size also have the advantage of a large surface for covalent bonding/attachment of an isotope, conjugating groups, fluorescent dyes etc. Other experimental nanoparticles such as the lipid-based micelles, liposomes, carbon nanotubes and quantum dots were also widely investigated with some success[21], some were bound to isotopes that decayed by electron, positron or gamma emis- sions[22]. The desirable characteristics of a hybrid lymphatic tracer include: (i) chemical functionalization at the particle surface is possible; (ii) the particle and signal molecule are both integral and stable in vitro and in vivo; (iii) signal mole- cules are capable of simultaneously providing enough signal strength for detection, from the time of initial diagnosis until surgery; (iv) evidence of final product safety before human use; and (v) the final product is efficacious, it achieves a high specificity as well as a high target to non-target ratio.

When radioantigen and immune cells meet

Radioactive-nanoparticles delivered to tissue

Soon after the syringe needle has pierced the epidermis and a bolus of radiocolloid is delivered to the extracellular matrix, the immune system is already involved at the injection site. The defense response during the acute phase, involves naïve dermal dendritic cells becoming primed after they encounter and internalize some of the radioantigen, for the purpose of creating major histocompatibility complex molecules. These cells migrate to the nearest lymphatic network in a regular strategy, then they differentiate into a mature state in the lymph nodes when tightly bound complexes are expressed on the outer membrane surface.

Table 2. Physical properties of experimental radioactive-nanoparticles.

These and other message molecules interact with T cells or B cells in lymph nodes to stimulate an attack sequence on the invading antigen. Meanwhile, pro-inflammatory cytokines (ie: tumor necrosis factor-γ, interferon-γ, interleukins, etc) are expressed by local cells to recruit polymorphonuclear cells (PMNs) from the bloodstream toward the inflammatory site harboring bulk radioantigen. Among those, mast cells at the site release histamines that cause vasodilation of blood cap- illaries and encourages gaps between endothelial cells, the endothelial cells also emit molecular cues for neutrophils and other PMNs to participate in tethering, rolling, adhesion and transmigration into the invaded interstitium, in a sequence known as the multistep adhesion and migration cascade (Fig- ure 2)[37-40].

The chronic phase of the immune response follows with a cascade of simultaneous attack events involving both adap- tive and innate systems. An onslaught of such events by the leukocyte milieu occurs on the 99mTc-ATC particles, where there is an orchestra of intense cell-cell signaling to recruit more PMNs, with numerous direct and indirect actions attempting to control, degrade and remove the antigen. Among those, macrophages and T helper cells are stimulated into releasing inflammatory mediators, destructive enzymes (ie: myeloper- oxidase, xanthine oxidase, etc) and oxidizers (ie: superoxide anions, nitric oxide, hydrogen peroxide, N-chloro analogues). The activated complement system participates via an increase in immunoglobulin production and chemotactic mediators to recruit more neutrophils. Eventually the chronic phase abates when resolution of the inflammation is finally achieved after antigen removal from the injection site.

nanoparticle radioisotope radiosynthesis particle signal/detector application[ref] name t½ (hr) c-group (% yield) size (nm) -ray CT MRI fluorescence animals humans

89Zr-ferumoxytol 78 DFO C (>90%) 17-31 + – + +[23]
68Ga-SPION 1.1 PEG C (97%) 30 + – + Cherenkov L +[24]
67Ga-SPION 78 PEG C (>96%) ~5 + – + +[25]
99mTc-AF-SPION 6 C (100%) 11 ± 2 + – + nIR670- 680 nm +[26]
99mTc-SPION 6 PEG S (99%) 13 ± 2 + – + +[27]
99mTc-SPION-bubbles 6 DTPA, C (52-85%) 4-110 + – + +[28]
NOTA
99mTc-ICN-NC 6 C (nr) <80 + – – nIR +[29,30]
IRDye-800CW-NC C (50%) 15 – – nIR790-1000 nm +[31]
89Zr-NC 78 DFO C (70-75%) 13 ± 1 + – – +[32] +[33,34]
89Zr-xl-dextran 78 DFO S (100%) 13 + – – +[35]
99mTc-ICN-silica gel 6 PAMAM C (nr) 20-50 + – – nIR800-2500 nm +[36]

ref = reference; t½ = half-life; c-group = conjugator or functional group that coordinates with radiometal; CT = x-ray computed tomography; MRI = magnetic resonance imaging; DFO = desferrioxamine; S = simple preparation involves one radioisotope reconstitution step/withdrawal of final patient dose(s); C = com- plex preparation involving multi-steps of: reconstitution/heating/other manipulations/purification/withdrawal of final patient dose(s); SPION = superpara- magnetic iron oxide nanoparticles; L = luminescence; PEG = polyethyleneglycol coating; AF = Alex fluorophore 647 dye; DTPA = diehtylenetriaminepentaacetic acid; NOTA = 1,4,7-triazacyclononane-N,N´,N´’-triacetic acid; ICN = indocyanine green; NC = nanocolloidal albumin; nIR = near infrared; nr = not reported; xl = cross-linked; PAMAM = poly(amidoamine) dendrimer

Cite this article: Chris Tsopelas. Detection of the Immune Response During in vivo Mapping of the Lymphatic System: A Profile of 99mTc-Antimony trisulfide Colloid and other Radioac- tive-Nanoparticles. J J Nanomed Nanotech. 2016, 1(1): 005.

Figure 2. Annotated diagram of an immune response to antigen particles (reprinted with permission © Chris Tsopelas). Tethering: P-selectins bind to PSGL-1 and tether PMN; Rolling: P-selectins bring PMN near to E-selectins, slow rolling commences; Chemokine signalling: slow rolling allows IL-8 binding to CXCR1, then activation signal to integrins from internalized chemokine receptor; Adhesion: activated LFA-1 binds ICAM- 1 or – 2 for firm adhesion; Diapedesis: PMN begins diapedesis by exchanging tight junction molecules with endothelial cells; Migration: PMN follows a gradient of inflammatory chemokines to pathogens; Attack: complement, the incoming antigens, mast cells and macrophages interact with antigen particles, toll-like receptor molecules trigger inflammation, DCs migrate to the nearest lymph node and express MHC molecules, T lymphocytes are activated and neutrophils recruited to the attack site.

PMN = polymorphonuclear neutrophil; CXCR/CCR = chemokine receptor; s-Le-x = sialyl-Lewis X; LFA = lymphocyte function-associated antigen; PSGL = P-selec- tin glycoprotein ligand; IL = interleukin; CD = cluster of differentiation; CD11a/CD18 = integrins; RBC = red blood cell; JAM = junctional adhesion molecule; PE- CAM = platelet endothelial cell adhesion molecule; DC = dendritic cell; MHC = major histocompatibility complex; T cell = T lymphocyte; IP-10 = interferon-gamma induced protein -10; MIP = macrophage inflammatory protein; RANTES = regulated on activation, normal T cell expressed & secreted (CCL5 chemokine); TNFα

= tumor necrosis factor α; C3a, C5a = complement components 3a, 5a; LTB4 = leukotriene B4

The ensuing healing phase is initiated with mast cells, T help- er cells and basophils that release interleukin signals, mac- rophages are stimulated into expressing tissue remodeling molecules as well as angiogenic growth factors. There is a pro- motion of fibroblast cells growth, these cells accumulate at the wound site and then proliferate into myofibroblasts followed by collagen deposition. T lymphocytes and monocytes are re- cruited there, macrophages alternate their functional respons- es, and angiogenesis is promoted during repair.

The drive into lymph and other key interactions

At the injection site, nanocolloid in the bleb is presumably dis- persed in its original aqueous solvent temporarily, then water rapidly diffuses away into the surrounding interstitial fluid and ultimately exchanges with the bloodstream[41], leaving a higher concentration of non-dispersed particles at the depot.

As the immune cells influx at the invasion site, there is a de- creasing concentration of 99mTc-ATC at the depot that is also influenced from an interstitial pressure created by the dose. The size, shape and surface charge of the nanoparticles affect the rate of convective transport through the interstitium, the negatively charged particles seem to move more swiftly[42]. A substantial proportion of these small particles have already migrated towards a lymphatic vessel and then they diffuse through the lymphatic endothelial cell layer (LE) between gap-junctions, to enter into the luminal space.

Larger particles such as 99mTc-tin colloid and 99mTc-sulfur col- loid very slowly diffuse along the interstitial cell matrix and they rely on mechanical factors to encourage their movement. The initial lymphatic sacs are end capillaries anchored to the extracellular matrix, and these comprise of a discontinuous basement membrane (BM) with preformed portals, overlay-

ing an oak-leaf-shaped endothelial cell layer interconnected by buttons (Figure 3). It is the peripheral lobes of the oak-shaped cells between the buttons that are flexible enough to flap in- wards when dendritic cells intravasate[43]. Chemokine mol- ecules such as CCR7 and CCL21 guide interstitial migration of peripheral DC[44] and portal entry through LE can occur without integrin mediated contact, in contrast to vascular en- dothelium.

Figure 3. Annotated diagram and expanded view of an initial lym- phatic vessel with a cut-away of the BM surrounding the LE cells (smooth muscle cells layer not shown). The BM is comprised of a blend of collagen IV, laminins, perlecan and nidogen, as a sheet that is intimately connected with the LE layer below. Along the BM are abun- dant changeable perforations that allow first access to crawling PMNs to enter into the luminal space. Numerous anchoring filaments local- ize the vessel to the extracellular matrix, the bundles of microfibrils primarily comprise of fibrillin, and the attachments contain integrin molecules. Beneath the BM, the end sac is predominantly comprised of oak-leaf shaped LE cells with characteristic button-like junctions, in which the end projection of the cell (flap) is malleable enough to move inward (valve). These flap valves are entry points for dendritic cells to push onto and allow them to crawl directly into the lymphatic vessel lumen (inset).

A simplified explanation of the locomotive mechanism used by a PMN, especially through LE portals, involves polymerization of actin at the front end of a cell that coincides with a forward propulsion, and this is supported by actomyosin contractions at the rear end of the malleable cell structure[43]. Luminal DCs within the lymphatic capillary crawl along the inner LE wall surface until their entry into the collecting lymphatics where they drift freely in lymph[45]. Lymph is intrinsically propelled forward by the rhythmical contraction of lymphangion units containing an outer collagen mesh with inner elastic leaflets that act as one-way valves[46]. Lymphangions are abundant in the collecting vessels that contain a low number of BM perfora- tions[43], different to the simple bicuspid one-way valves oc- casionally found at the initial lymphatics comprising a discon-

tinuous basal lamina[47]. Once inside the lymphatic capillary, the ‘naked’ radiocolloid-antigen becomes opsonized in lymph with complement proteins such as C3, C4 or Fc-γ binding mol- ecules[48-51]to yield immune complexes[52], these predomi- nantly continue flowing downfield toward the larger collecting vessels and into a contiguous lymph node.

The opsonins C3b and C4b are known to form covalent bonds with foreign material such as antigen particles, pathogens, error-assembled cellular materials, senescent or apoptotic-en- dogenous cells, by an exposed thioester bond[53] (Figure 4). When X = sulfur such as in the antimony trisulfide structure, a transthioesterification reaction can occur[54] in which an opsonin bonds to the particle surface. The surface bound C3b and C4b are capable of binding to the circulating antibodies IgG and IgM in the immediate vicinity, another signal visible to immune cells containing the Fcγ receptor. Germline antibod- ies can also bind directly to the antigen particles, with their characteristic variable binding sites, in advance of somatic hypermutation and B cell clonal selection of the optimal-af- finity matured antibodies[55]. The presence of the lymph glycoprotein fibrinogen, enhances the binding of antibody to antigen[56]. Anaphylatoxins such as C3a, C4a, C5a are strong pro-inflammatory molecules via G-protein receptors, that sig- nal antigen-presenting cells and lymphocytes. Downstream in a lymph node, macrophages recognize and bind these op- sonins via their C3aR, C5aR/FcγR (I/III or IIB) receptors, and it is this reaction at the interface that defines an attachment of particulate antigen (ie: 99mTc-ATC) to the macrophage cell surface. The complement receptors CR3 and CR4 (also known as CD11c/CD18 or integrin αxβ2 respectively) are abundant- ly expressed by myeloid leukocytes such as neutrophils, DCs, monocytes, macrophages and NK cells. After they are bound by complement ligands including C3b, conformational changes on the exterior cell surface transmit a signal inwards through the membrane to the cytoplasm, culminating in events such as actin remodeling, degranulation or cytokine signaling[57]. Tyrosine kinases from the Src- and Syk-families become acti- vated and associate with specific recognition sequences (tyro- sine-moieties) within the FcγR subunits, a critical signal impli- cated in phagocytosis[58].

Other non-opsonic receptors are also expressed by phagocytic cells, the pattern recognition receptors (PRR), that include the C-type lectin receptors[59], CD14[60], dectin-1[61] and mac- rophage receptor with collagenous structure (MARCO)[62]. These receptors mediate the uptake of particulate antigens, and also influence the recruitment, activation and intracellu- lar signaling of other PRRs during phagocytosis, as well as the degradation of antigen cargo[63]. The C-type lectin receptor recognizes carbohydrate moieties from fungi, yeast, platyhel- minthes, house dust mites or bacteria, and is capable of induc- ing phagocytosis, cytokine production and oxidative burst. It is an activating receptor in phagocytosis through the association with the adaptor protein FcεRIγ. Nanoparticles with project-

ed polymeric surfaces but absent of sugar composition, have also shown to trigger the lectin pathway[64]. Plasma mem- brane localized CD14 binds antigen such as bacterial cell de- bris (ie: lipopolysaccharide, LPS), or endogenous molecules like intercellular adhesion molecule ICAM-3 on the surface of apoptotic cells, amyloid peptide, ceramide and urate crys- tals. It is a critical entity in the signal transduction process by signaling toll-like receptor 4 (TLR4), and then a Syk/PLCγ2-me- diated endocytosis forms a loaded endosome, that can initiate

Transit through the lymph node

Immune cells

The special anatomical architecture of a lymph node allows for the ingress of: antigen radioactive-nanoparticles/immune cells such as DCs or some naïve T cells from the previous node, via afferent lymph; and, immune cells such as T or B cells, some DCs and NK cells via high endothelial venules (HEV). The mi- gratory DCs carrying antigen from the peripheral site, flow into

Figure 4. Covalent bond between an opsonin-thioester group and nucleophilic atom of a particle.

pro-inflammatory gene transcription (ie: interferons) and the NFκB pathway[60]. Macrophages activated by IFN-γ dis- play increased receptor mediated phagocytosis[65]. CD14 serves as a receptor involved in the phagocytosis of apoptotic cells[66]. Dectin-1 recognizes the major component of the out- er fungal cell wall (1,3-β-glucan), and becomes a dimer after ligand binding. Phosphorylation of the dimer tail in the cyto- plasm by a Src-family enzyme enables the activation of Syk, Syk then mediates the mitogen-activated protein kinases (MAPK) response, it activates the NFκB pathway, and this stimulates Ca2+ flux that drives calcineurin dephosphorylation of nuclear factor of activated T cells (NFAT). This NFAT signaling is con- nected with phagocytosis[63].

Bacterial LPS was shown to induce dectin-1 expression in hu- man monocytes, that induced IL-23 production and enhanced phagocytosis of heat-killed Candida albicans[61]. MARCO is un- able to initiate pro-inflammatory signaling itself, but interacts with other PRRs to influence the cell responses to particulate antigens. For example intracellular signaling with TLR3 occurs in response to PolyIC antigen, or TLR2/CD14 in response to mycobacterial cell wall glycolipid trehalose-6,69-dimycolate, and receptor clustering during cytoskeletal rearrangement in activated DCs[63].

the node with the aim of presenting its cargo-message to the lymph node resident immune cells. The egress of antigen and lymphocytes occurs in efferent lymph along the hilus, within a whole organ structure that is subdivided into the cortex, para- cortex and medulla (Figure 5).

The cortex is the region closest to the afferent vessels that drain into the subcapsular sinus (SCS), and it also contains B cell follicles, or discrete zones in which there are closely packed B cells, follicular DCs and marginal reticular cells[40]. Local macrophages in the SCS floor are presented with antigen in afferent lymph. The SCS is a space between the collagenous capsule roof enveloping the lymph node and the SCS floor, that mainly follows the periphery through the paracortex and me- dulla to meet with the widely branched medullary sinuses and cords on the hilar side. The SCS floor is composed of extracellu- lar matrix and a lining of endothelial cells, above the marginal reticular cells that define a B cell follicle boundary. In the gaps between cells that make up the lining, reside SCS-macrophages with a ‘head’ projection into the sinus and a ‘tail’ projection into the underlying follicle. These sessile cells positioned tran- scellularly are the ‘gatekeepers’ of the lymph node, designed to avidly capture newly presented antigens in flowing lymph, es- pecially those that are opsonised, yet with low internalisation of the cargo[67]. These macrophages can nevertheless present antigen particles to B cells, not just by internalisation and then expression, but also by movement along the macrophage sur- face from the SCS lumen to the follicles[68].

Figure 5a. Annotated diagram of a whole lymph node anatomy.

Beyond the medullary sinuses toward the paracortex is the T cell zone that harbors T cells, and this zone extends further into the cortex between the B cell follicles. DCs enter the T cell zone from the afferent side of the SCS, they can pass through the floor without integrins, arriving mainly at interfollicular areas. In the T zone, naïve T cells await a cognitive interaction with primed DCs to become primed themselves, into a mature state. There is positioning and clustering of T cell subsets in- cumbent for attack events on antigen, and memory cell func- tions[69]. T cells can enter the lymph node from the circulation via the unique post-capillary HEV, comprised of characteristic cuboid endothelial cells that bulge into the vascular lumen. HEVs are able to stack and hold lymphocytes, before they gain access into the lymph node parenchyma[70]. The multistep adhesion and migration cascade is used by lymphocytes to penetrate through the HEV endothelium and then exit through fibroblastic reticular cells that form ramps. Some lymphocytes accumulate transiently in ‘HEV pockets’ between endothelial and fibroblast layers. Lymphocytes and DCs migrate within the parenchyma by crawling along a fibroblastic reticular cell con- duit[71] and/or fibroblastic dendritic cell conduit, involving cues defined by chemokine signalling or haptotactic gradients. The reticular network that exists at the interface between B cell follicles and the T cell zones contains multiple HEVs, in a zone known as the cortical ridge, and this is where key interactions occur between primed dendritic cells and lymphocytes[72]. The RN in the T cell zone of a mature lymph node, differs in the FRCs that have been abundantly replaced by FDCs, the FDCs are capable of capturing (soluble) antigens even in the absence of antigen-specific antibodies[73]. This dense FDC network is

also found in the B cell follicles. Each distinct organisation of the RN within the lymph node parenchyma serves to guide the different cohorts of lymphocytes migrating toward their des- tination via haptotactic gradients, the entire conduit actively propagates adaptive immune responses[69]. Those naïve T cells that have entered the node from afferent lymph, eventu- ally access the T cell zone via the medullary sinuses (MS).

Figure 5b. Annotated diagram and expanded view of a collecting lym- phatic vessel with a cut-away of the top surface of LE cells (BM layer and smooth muscle cells not shown). The over-lapping LE cells are connected by zipper-like junctions in which the gaps allow an in-flow of interstitial fluid. Below the cut-away, LE cells are organized in the lumen to form a valve (closed position) that is part of a lymphangion. The lymphangion valves open or close to propel lymph forward in ac- cordance to movements from the surrounding tissue.

On the efferent side of the lymph node, the labyrinth-ar- ranged medullary sinuses contain fluid and numerous macro- phages attached to the sinus walls, as well as reticular fibers within the lumen. The tissue between the medullary sinuses are the medullary cords, packed with lymphocytes includ- ing naïve T cells[40] or plasma cells, macrophages and den- dritic cells, and usually contains a blood vessel. During an infection or inflammation, the medulla becomes enlarged at the peak of the plasma cell response when a vast number of egressing cells accumulate, in advance of their exit into ef- ferent lymph. The primary function of the lymph node me- dulla is for macrophages with enhanced phagocytic capacity to extract pathogens and particulate antigens from lymph, to support the survival of short-lived plasma cells, as well as to provide a traffic path for egressing cells and antibodies[67]. The macrophage passively binds particles that spontaneous- ly diffuse toward the plane of a membrane, and actively, by probing the immediate environment with actin-driven mem- brane protrusions that involve Rac proteins and phospho- noinositides[74]. Both strategies are used by macrophages to optimize the efficient capture of particulate antigens. The egress of both cognate T and B cells into the cortical sinus central branches depends on haptotactic gradients of sphin- gosine-1-phosphate (SIP) and the expression of the SIP recep- tor, after which these cells become rounded and flow unidirec- tionally into the medullary sinuses and beyond the hilum[40].

Figure 5c. Annotated diagram and expanded view of cortical/paracortical areas within a lymph node. Afferent lymph carries antigen particles and cells flowing into the SCS. Opsonized antigen or immune complexes interact with local cells, then antigen presentation occurs between macrophages and B lymphocytes in the interfollicular region (IFR), as well as dendritic cells and T lymphocytes in the cortical ridge. B cells are clustered as follicles in the IFR, and in its germinal centre these cells undergo proliferation, somatic hypermutation and class switching. Primed dendritic cells enter the IFR through the SCS floor, then move along the fibroblastic reticular cells (FRC) conduit to the cortical ridge, a key zone where antigen presentation occurs to T cells. The FRC conduit and the follicular dendritic cells (FDC) conduit comprise the reticular network (RN) that actively expresses chemokines to guide cell movements. The conduit is made up of lengthy collagen bundles encased within FRC or FDC. In the B cell follicles, cortical ridge and T cell zone, FDCs replace FRCs. HEVs are common in the cortical ridge and usually contain a blood vessel. T cells can enter the lymph node via HEVs and then migrate along the RN to enter into the T cell zone located within the paracortical region.

Jacobs Publishers 10

Radioactive-nanoparticles

From the aspect of the particulate antigen, when afferent lymph enters into the SCS of a lymph node, a bolus of (opsonized) ra- dioactive-nanoparticles becomes exposed to the gatekeeper macrophages, in which a proportion of them bind to cell mem- brane surfaces and the remainder dynamically percolate on- ward through to the medulla. At the medullary sinuses, some particles bind to MS-macrophages of high phagocytic capac- ity, and the remaining concentration flows in efferent lymph onto the next node along the draining chain. If there are fewer macrophages than opsonized-99mTc-ATC particles in lymph at the SCS of a node, then two or more particles could potentially accumulate on a region of the cell surface as a cluster of ≥25 nm, a size threshold above which there is optimal endocyto- sis[75-77]. The interaction between opsonized-radioantigen and the cell surface is dependent on the phagocyte cell re- ceptor’s recognition of the opsonin (ie: C3a, C5a, Fcγ) as de- scribed earlier. Ligand-receptor binding triggers intracellular signaling, to eventually result in remodeling of the actin cyto- skeleton, when the cell membrane morphologically envelopes the particle during the formation of a phagocytic cup[78]. Fcγ receptor-mediated phagocytosis requires the presence of Rho family proteins Rac, Cdc42 GTPases, and a Ca2+-dependent GT- Pase-activating protein CAPRI that is a molecular switch on Ras-dependent cellular processes[79]. The dynamic sequence of bonds formed between cell surface receptor and ligands on the particle surface is known as the zipper mechanism[78]. Eventually the leading edge of the cup becomes closed, a mem- brane vesicle enclosing an antigen particle or phagosome is formed, and then it moves into the cytoplasm to fuse with oth- er vesicles containing deactivation chemicals such as enzymes, acids or oxygenated radicals. It was previously shown that the retention of 99mTc-ATC in lymph nodes is unlikely to involve significant internalization by macrophages within a short time of 30 minutes after injection, however during the course of a patient lymphoscintigram, radioantigen attachment to the sur- face of resident cells explains a retention mechanism in vivo[9].

Detection of the γ-ray signal

For newly designed radiotracers, a formulation is usually opti- mized in vitro for radiolabeling efficiency, radiochemical purity and stability, then further examination of efficacy in vivo oc- curs using small or large animal models (pre-clinical). The typ- ical in vivo test requires administration of a radioactive dose to a rodent by intravenous injection, then allowing the radio- tracer distribution to organs after a defined time. The test is a quantitative assay in which the animal is euthanized to harvest internal organs, then each sample is counted to determine its

% uptake as a proportion of the total radioactive counts. Ra- diocolloids primarily localize in the reticuloendothelial organs such as liver, spleen and bone marrow, where the endogenous phagocytic white cells extract γ-emitting particles from the circulation. If the nanoparticles have agglomerated into larger

units, then they tend to be trapped by the pulmonary vascu- lature. Regulatory-approved radiocolloids indicated for a clin- ical use, require a prospective biodistribution test in normal rodents as part of their quality control release specification. The pharmacopoeial test for a typical radiocolloid specifies the percentage of injected dose at 20 minutes after intravenous in- jection, as ≥ 80% of radioactive uptake in the liver plus spleen, and <5% in the lungs.

Figure 6. Lymphatic drainage by three routes from the tail of a Sprague-Dawley rat. The whole body scan was taken 30 min after subdermal injection of high specific activity 99mTc-ATC[9]. Lymph drainage[80] followed three known routes[81] as per annotated map. Route 1: drainage followed the caudal channels into the sciatic fora- men but in the absence of sciatic nodes, entered the nearest popliteal nodes. Lymph flowed further into the sacral node, onto iliac nodes and then from the respective renal nodes into the cisternal node. The left renal node may empty into the cisternal chili directly, but more frequently an efferent lymph trunk runs across the great vessels to join the right renal node; the cisternal node group is linked with other para-aortic nodes in the thorax. Route 2: drainage along an efferent trunk into the sacral node then the para-aortic chain as per Route 1. Route 3: other vessels from the tail coursed laterally across the groin to an inguinal node. Minor tributary vessels from popliteal nodes are known to connect to inguinal nodes. A large efferent inguinal lym- phatic trunk coursed cephalad along the nipple line to the right ax- illiary node.

The cellular and molecular events involving radioac- tive-nanoparticle-antigen and immune cells in the lymphatic system can be visualized as an image or scan, created after de- tecting the γ-ray energy emissions from the body with a spe- cial γ-camera. As well as the pharmacopoeial biodistribution test, it is common for researchers to evaluate radiocolloid mi- gration in lymphatics after subdermal injection, in which the radioactive signal is followed over the anatomical region of in- terest by acquiring many short image frames (ie: 30 seconds/ frame) over a longer time (ie: 30 minutes). Software ‘stitch- ing’ of the static frames results in a video output, or dynamic. A dynamic image sequence can show the direction of lymph flow and the accumulation of radioactive counts in a lymph

Cite this article: Chris Tsopelas. Detection of the Immune Response During in vivo Mapping of the Lymphatic System: A Profile of 99mTc-Antimony trisulfide Colloid and other Radioac- tive-Nanoparticles. J J Nanomed Nanotech. 2016, 1(1): 005.

node, without euthanizing the animal, and a semi-quantitative analysis of the whole body images can be used to determine the % organ uptake as a proportion of total tissue counts. 99mTc-ATC has been examined in normal animals that are small (ie: rodents, lizards) and large (ie: rabbits, sheep). Ex- amples of lymphatic drainage from the animal tail are shown as scans in Figures 6 and 7, or from a hind limb in Figure 8.

Figure 7. Whole body scans of the gecko Cristinus marmoratus as: (a) digital, with a regenerated tail before radiotracer injec- tion; (b) scintigram, 60 min after subdermal injection of 99mTc- ATC in the tail. This squamate can regenerate its tail, and it was investigated as a model of lymphangiogenesis [82]. In this col- laborative study, lymphoscintigraphy identified a functional lym- phatic network was formed at six weeks after tail autotomy, a regen- erated tail differed morphologically from the original tail, yet it was associated with up-regulated reptilian VEGF-C/D. Within 30 min, 99mTc-ATC rapidly migrated along the major ventral lymphatic vessel (trunk) from the from the tail injection to enter to enter the blood circulation at the cloacal plexus. At 60 min there was also low visible uptake by the heart (H) and liver (L).

Figure 8. Scintigraphic scans of the left hind limb of a sheep in the supine position (injection site above hoof is beyond the field of view), with 30 sec frames acquired at 3 min, 5 min, 9 min and 21 min. An example of complex drainage from the injection (i) site in a sheep[83]. A superficial inguinal node 1 (c) was detected as early as 3 min, that linked to superficial inguinal node 2 (d). Radioactive uptake of (c) was lower than popliteal (a) and iliac (b) lymph nodes at 9 min, indicating that the latter drainage pathway was the more important route. This complex drainage pattern resulted in images that were difficult to in- terpret. The main pathway was directed via the popliteal fossa and onto the first deep iliac node (based on intensity of counts at 21 min). However without surgery, it was not readily obvious that a second route to superficial inguinal nodes was present and drained across the first, at a different tissue plane around the groin. This variable anatomy complicated the scan assessment. The first draining (sen- tinel) nodes were hotter than the next tier lymph nodes in the 99mTc- ATC scans of this sheep.

For cancer patients however, nuclear medicine maps a poten- tially compromised lymphatic system due to invading tumor cells (<0.2 mm), micrometastases (<2 mm), even tumor depos- its (<10 mm)[84] located adjacent to/within lymph nodes or channels. Consequently the invaded tissue architecture is dis- organized, the occult cell activities may dominate to substan- tially alter normal lymphatic function, as well as that of local immune cells[85]. Many types of human cancers use the lym- phatic system, moving cells to another location or niche that favors their proliferation. The radioactive nanocolloids are not tumor cell-specific, but their particulate property and mode of administration into tissue makes them essentially specific for the lymphatic system. From a nuclear imaging perspective, a radioactive-nanoparticle loosely represents a tumor cell in terms of its migratory aptitude. Such a particle does not emit any chemotactic cues, but endogenous molecules/cells from the local environment nevertheless interact with it. In con- trast, a tumor cell avidly interacts with the local environment to serve its purpose[85]. The essence of lymphoscintigraphy is to map the potential location of tumor cells in a patient’s lymphatic system, and this ultimately guides the surgeon to provide treatment using the sentinel node biopsy technique. The lymphoscintigraphy technique has employed radioac- tive-nanocolloids in the clinic for decades, primarily in pa- tients with early to advanced disease of cutaneous melanoma, breast carcinoma and lymphedema (see examples in Figure 9 below). These have been extensively reviewed elsewhere[8], as well as cancers of the vulva[86], esophageal region[87], uri-

nary bladder[88], penile[89,90] and of the prostate[91]. It is also employed in patients after excision of the primary tumor to confirm the residual drainage path.

Figure 9a. Scintigraphic scan of a patient with a primary melanoma on the back, after peritumoral injections of 99mTc-ATC. In the prone position, the posterior view of the patient at 5 min after injection showed a cauliflower-shaped lesion drained radiocolloid along two opposite paths. Two sentinel nodes were observed (one per axilla) in the anterior view at 13 min, when the patient was supine with raised hands. The position of these nodes within the body outline was de- termined from a transmission scan (anterior view) using a low level γ-emitting 57Co-sheet source. Note the leftward path from the tumor drained radiocolloid to the left axilliary node, and the rightward path from the same tumor to the right axilliary node.

Figure 9b. Scintigraphic scan of a patient’s legs 30 min after injection of 99mTc-ATC into a web space of each foot. The right leg showed reg- ular lymphatic drainage of radioactivity to the inguinal lymph node. Delayed radiocolloid flow in the left leg was evidenced from inguinal node counts appearing later and lower than the right side. In the 2 hr scan (not shown) there was dermal back flow in the main channels indicating functional obstruction, consistent with lymphedema.

Conclusion

The major discoveries during the early 1900s of radionuclides with potential medical applications, was the original work that ultimately developed into the growing fields of diagnostic im- aging and radiation therapy. Over that time there were advanc- es in radiolabeling chemistry to optimize how an isotope could be efficiently bound to a particle matrix, the implementation of good manufacturing practice principles for higher safety, and the validation of in vitro/in vivo tests for evaluation of drug ef- ficacy. This international effort has translated into a handful of commercially available radioactive-particle formulations that have successfully claimed their status as radiopharmaceuticals for the detection and treatment of indicated patient disorders. Within the genre of particles, the γ-emitting nanocolloids have been valuable for identifying infectious/inflammatory lesions, liver, spleen or intestine function, lymphoscintigraphy, and the γ-emitters for treating focal arthritis of the joints. This re- view profiled the lymphoscintigraphic agent 99mTc-antimony trisulfide colloid, a regulatory-approved product with a long history in Australian nuclear medicine clinics. In terms of how the normal cellular physiology handles a radio-nanocol- loid for a lymphatic scan, the immune system is immediately activated after its delivery to the injection site.

Figure 9c. Scintigraphic planar scan of a patient with breast cancer, after subareolar injections of 99mTc-ATC in the lower-left quadrant. The anterior (ANT) views of the patient at 10 min and 20 min post injection showed one faint sentinel node inferior to the right pectoral muscle and deep, also highlighted in the transmission scan. A lateral (LAT) scan acquired after 70 min identified another lymph node, located inferior to the axilla lateral thoracic, and deep. The exact location of counts within the anatomy was later confirmed by 3D SPECT-CT scans, presented below as selected fusion scans (an accurate overlay of the nuclear scan on the radiology scan). In both coronal (COR) and axial (AXL) slices, the crosshair on thoracic node depicted a highly intense white centre plus a halo of less intense blue, beyond the larger concentration of injection site counts. The AXL slice showed a lymph node beneath the pectoral muscle with low intensity and it was blue colored.

The biological events that ensue, involve the movement of im- mune cells and radiocolloid-antigen into the lymphatic sys- tem, where they interact or bind to elements of lymph and/ or anatomical structures within the lymph node. It is the con- centration of radioactive counts in the lymph node, because of an interaction between radio-antigen and the local leuko- cyte population, that is ultimately detected as a hot spot by the nuclear medicine gamma camera. The hot spot indicates the sentinel lymph node that first drains the primary tumor, and it mimics the site where a migrating cancer cell is likely to reside. Currently 99mTc-nanocolloids preferably dominate the clinical domain, however a new generation of ‘hybrid’ radiotracers for lymphoscintigraphy are rapidly emerging, and these combine two or more different signals, offering the prom- ise of enhancing the accuracy of this vital diagnostic technique.

Acknowledgements

Ms Deborah Kate Calnan for providing the patient scans ac- quired in this Nuclear Medicine Department.

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