Nano-chemotherapeutics: maximising lymphatic drug exposure to improve the treatment of lymph-metastatic cancers.

Journal of Controlled Release 193 (2014) 241–256

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Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel

Nano-chemotherapeutics: Maximising lymphatic drug exposure to improve the treatment of lymph-metastatic cancers Gemma M. Ryan, Lisa M. Kaminskas, Christopher J.H. Porter ⁎ Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Pde, Parkville, VIC 3052, Australia

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Article history: Received 11 March 2014 Accepted 23 April 2014 Available online 5 May 2014 Keywords: Drug delivery systems Lymphatic Metastasis Chemotherapeutic Lymph node

a b s t r a c t Nano-sized drug delivery systems incorporating chemotherapeutic drugs (“nano-chemotherapeutics”) have been widely employed for the treatment of solid tumours. The dimensions of nanoparticulate drug delivery systems also make them ideal vectors for improving drug exposure to the lymphatic system, potentially enhancing the treatment of lymph-resident metastases. This review examines the physical properties of nanoparticulate drug delivery systems that promote lymphatic exposure and lymph node retention, and discusses methods for improving lymphatic access. Drug delivery systems that have been investigated for the treatment of lymph node metastasis are also reviewed, and recent advances towards active targeting approaches for lymphatic metastases highlighted. © 2014 Elsevier B.V. All rights reserved.

1. Current treatment strategies for lymphatic metastases and the limitations of these approaches Tumour metastasis is the primary cause of cancer-related mortality. Wide intercellular pores between adjacent lymphatic endothelial cells and an incomplete underlying basement membrane impart the lymphatic system with properties that facilitate the invasion and metastatic spread of cancer cells. As such, the network of lymphatic vessels is a primary route for the metastatic dissemination of a wide variety of cancers and the arrest of disseminated cancer cells within sentinel lymph nodes draining a solid tumour is of prognostic significance in a large number of cancers [1,2]. Lymph node-resident metastases also have the potential to act as a reservoir for cancer cells, resulting in spread beyond the initial metastatic site and advancement of the malignancy [3–5]. The eradication of cancers within the lymphatic system is therefore a key treatment goal in a range of cancers and an important determinant of patient prognosis. Treatments for lymph node metastases vary, but commonly involve surgical removal of the sentinel lymph nodes (lymphadenectomy) and radiotherapy. These are invasive procedures and are associated with side effects including pain, some loss of movement and lymphedema [6]. Nonetheless, the relatively limited range of effective treatment options for metastatic cancer dictates that collateral effects such as these are usually deemed acceptable. The major limitation of lymphadenectomy, however, is that data supporting the effectiveness of this approach is limited and in most cases there is a poor correlation between the removal of the sentinel lymph nodes draining a tumour and improved ⁎ Corresponding author. Tel.: +61 3 99039649; fax: +61 3 99039583. E-mail address: [email protected] (C.J.H. Porter).

http://dx.doi.org/10.1016/j.jconrel.2014.04.051 0168-3659/© 2014 Elsevier B.V. All rights reserved.

survival [7]. This is particularly true in advanced cancers where metastases have spread beyond the sentinel nodes. A further obstacle to the treatment of lymph node-resident cancers is the delivery of sufficient quantities of a chemotherapeutic to the lymphatics and lymph nodes in order to facilitate cancer killing, whilst limiting toxicity elsewhere. In this regard, the biopharmaceutical properties of small-molecule chemotherapeutics commonly dictate poor lymphatic uptake [8,9], precluding effective treatment of lymph node resident tumours. For example, a recent study in rats has shown that the maximum concentration of doxorubicin in the lymph of animals given a 1 min intravenous (IV) infusion of the drug is approximately 8-fold lower than in plasma [8]. Drug formulations or delivery systems that promote lymphatic targeting therefore provide significant opportunity for improved treatment options in metastatic cancer. This concept is supported by recent work showing that improving the lymphatic exposure of small molecule chemotherapeutics or proteins may lead to a significant reduction in the growth rates of lymphatic metastases [9,10]. Nano-chemotherapeutics have been widely investigated for their use in cancer therapy to improve drug delivery to solid tumours [11], and several formulations are approved for clinical use [12]. In most cases, the advantages of nanoparticulate approaches to tumour delivery stem from their ability to enhance plasma circulation time (often as a result of avoidance of clearance by the mononuclear phagocyte system (MPS) due to the presence of polyethylene glycol (PEG) polymer chains on the particle surface) and to simultaneously reduce free drug concentrations in the systemic circulation. In the vicinity of most solid tumours, angiogenesis is increased in order to maintain nutritional supply to rapidly dividing cells. As such tumour associated blood vessels are often poorly formed and have relatively wide fenestrations. This allows long

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circulating drug delivery systems to more effectively extravasate at the tumour site and forms the basis for the enhanced permeability and retention (EPR) effect, first described by Matsumura and Maeda in 1986 [13] and since exploited by many research groups to enhance delivery to solid tumours [14]. However, dispersed cancer cells and lymphatic micrometastases with volumes less than 10 mm3 are poorly vascularized and therefore passive targeting by EPR is difficult [15]. In some cases, nano-chemotherapeutics may also assist in overcoming multi-drug resistance (MDR), a property that many cancers develop following prolonged exposure to high drug doses. The mechanisms by which MDR develops include increased expression of drug efflux proteins (such as P-glycoprotein) that ‘pump’ drug out of cancer cells, reducing intracellular concentrations below lethal levels [16,17]; decreased expression of the drug uptake transporters required to deliver drugs to the intracellular environment, thereby reducing drug internalisation [18] and inhibiting intracellular pathways; and activation/deactivation of gene mutations, resulting in cellular resistance to apoptosis [17]. Nano-chemotherapeutics enhance the responsiveness of MDR cancer cells to drug therapy by multiple mechanisms [19], including the inhibition of, and competitive binding to, drug efflux pumps such as P-gp [20]; direct interaction with cell membranes inducing structural changes such as greater fluidity and differences in the composition of lipid rafts [21] leading to enhanced cellular drug entry; and promotion of drug uptake via endocytotic mechanisms [22] that avoid efflux process. The utility of nanoparticle-based delivery systems can also be enhanced by the attachment of targeting ligands to the surface of the particle, enhancing cellular specificity and in most cases stimulating receptor mediated internalisation into cancer cells. Nanoparticulate drug delivery systems therefore provide a range of potential advantages for systemic chemotherapy [23]. Many of these advantages are equally beneficial for the treatment of lymph-resident metastases, however, the physical dimensions of nanomaterials also provide an additional benefit since they inherently promote drainage into the lymphatic capillaries after interstitial (subcutaneous (SC), intradermal (ID), intramuscular (IM) etc.) administration, and in some cases appear to promote lymph access across systemic capillary beds after IV administration. Several approaches to enhanced drug delivery to the lymph have been described including access to the intestinal lymphatics after oral administration [24,25] and access to the peripheral lymphatics after parenteral administration [8,25]. With the potential exception of some gastrointestinal or mesenteric cancers, parenteral approaches to lymphatic targeting are of most utility for the treatment of localised or disseminated metastatic tumours and are the focus of this review. Some of the published work to this point has explored non-drug-loaded delivery systems [26,27], however the goal of these studies, i.e. to achieve greater concentrations of therapeutic within the lymphatic system, is consistent with improved therapeutic endpoints. As such, this review describes recent advances in lymphatic targeting using nano-sized drug delivery systems and highlights approaches that have led to enhanced treatment wherever possible. 2. The lymphatic system The lymphatic system has some similarities to the vascular system, but unlike the blood circulation, lymph flow is unidirectional and retrieves excess extracellular fluid, macromolecules, foreign cells and antigens from the periphery and returns them to the systemic circulation. The lymphatics also provide a conduit for the trafficking of lymphocytes. Thus, as blood circulates throughout the capillary beds, fluid and plasma proteins extravasate from the vasculature into the surrounding interstitium. Although some fluid re-enters the systemic circulation via the post capillary venules [28], a proportion is collected by blind-ended initial lymphatic capillaries. These vessels comprise a single layer of endothelial cells that lack a prominent basement membrane. The initial lymph capillaries contain no muscle fibres, but are bound to the surrounding

extracellular matrix by anchoring filaments. This forms a dynamic relationship between the lymphatics and the surrounding environment, facilitating the opening of cellular junctions and fluid entry into the lymphatics with movement and increases in interstitial pressure (Fig. 1). From the lymph capillaries, lymph fluid drains into lymphatic collecting vessels where it passes through at least one, and usually many, lymph nodes. Lymph enters the nodes through one of many afferent channels, is filtered via node-resident macrophages, lymphocytes and reticular fibres, and exits via a single efferent vessel. Collecting vessels converge into lymphatic trunks, which in turn empty into one of two lymphatic ducts that deposit lymph directly into the systemic circulation at the junctions of the left and right subclavian and internal jugular veins in the neck. Lymph is transported towards the neck via the contraction of smooth muscle that is present in the walls of all lymphatic vessels (excluding the initial lymphatics) and indirectly via contraction of skeletal muscles and by respiration [29]. Drug molecules that are introduced into the interstitial environment by e.g. SC injection therefore have the potential to be taken up by not only the blood capillaries, but also by the lymphatic capillaries from where lymph flow transports drug (or a drug delivery system) back to the systemic circulation via a series of lymph nodes. 3. Mechanisms of lymph node metastasis The mechanisms of lymph node metastasis are still incompletely understood. Several hypotheses have been proposed, however most remain controversial with differences between tumour types complicating conclusions. In spite of these uncertainties, there are a number of factors that are acknowledged to play key roles in the spread of malignant disease. In the metastatic cascade, cells must first disseminate from the primary tumour, and migrate towards blood or lymphatic vessels. Cells then travel towards distal organs, and must finally arrest and adhere at distal sites. Initial metastatic arrest within lymph nodes is typically within the nodes sentinel to the primary tumour, followed by secondary nodes and then increasingly towards distal nodes. As such, sentinel node biopsy is often used as a predictive factor for lymph node involvement in cancer. As the lymph nodes are often the initial point of metastatic cell growth, sentinel node biopsy is also useful in many cases for determining the malignancy of the disease. The preference for cells to disseminate via the blood or the lymph may reflect one of many factors, including mechanisms attracting cells to a particular vascular or lymphatic environment (chemokine signalling), the viability of cells under high shear forces within the vasculature vs lower shear forces in the lymph, and the physical availability of blood versus lymph capillaries [30]. The lymphatic system may provide a more favourable route for metastatic spread than the vascular system for reasons that include preferential cell access across the more permeable lymphatic endothelium, wider lymph vs blood vessels, and slower flow rates and reduced pressure in the lymph when compared to the blood circulation, reducing cell damage by shear stress and mechanical deformation. In addition, cell viability may be higher in the lymph, which is comprised of similar components to the interstitial fluid, whereas serum toxicity can reduce cancer cell viability in the blood [28] (Fig. 2). 3.1. Lymphangiogenesis In the case of lymph node metastasis, the formation of new lymphatic vessels (lymphangiogenesis) is a common (but not essential) precursor to malignant spread. Lymphangiogenesis occurs not only at the primary tumour but also at metastasis positive lymph nodes and sentinel lymph nodes prior to the arrival of metastasising cancer cells [31–33]. Lymphangiogenesis at the sentinel lymph node has been correlated with an increased incidence of lymph node metastasis [31].


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Fig. 1. The blood and lymphatic vasculature. A. Lymph from the upper right quadrant of the body drains into the right thoracic duct, and lymph from all other parts of the body drains into the left (major) thoracic duct and from there enters the systemic circulation at the junction of subclavian and internal jugular veins. B. Molecules that access the interstitium by extravasation from the blood vessels or via direct interstitial access via injections may drain into either blood or lymph capillaries. Access of high molecular weight materials across the vascular endothelium is restricted by tight junctions between adjacent endothelial cells and an underlying basement membrane. In contrast access to the lymph vessels is less hindered by virtue of larger interendothelial junctions. Lymphatic endothelial cells are connected to the surrounding interstitium by anchoring filaments that promote the opening of interendothelial gaps as the interstitium moves or in response to increases in interstitial pressure. C. Multiple afferent lymphatic vessels deliver lymph fluid to lymph nodes. Lymph flows through the node and exits via a single efferent vessel.

Lymphatic vessels may also increase in diameter, making the lymphatics more favourable for metastatic spread. Many factors have been shown to play a role in the control of tumour lymphangiogenesis, in particular vascular endothelial growth factors-C and-D (VEGF-C and

VEGF-D) that are released by the tumour and tumour-stromal cells and bind to the tyrosine-kinase receptor VEGFR-3 [31,34]. Accordingly, VEGFR-3 inhibitors have been investigated for the prevention of lymph node metastasis [35–37]. Whilst studies have shown the presence of intratumoural lymphatic vessels in some but not all tumours, uncertainty exists around their functionality, or lack thereof [38]. These lymphatics are generally small and irregular in shape, and many researchers believe that high intratumoural pressures compress and constrict lymphatic capillaries, rendering them non-functional [39]. It is therefore unknown whether the movement of tumour cells into the lymphatics occurs primarily through the formation of intratumoural lymphatics or through the movement of cells towards peritumoural lymphatics. In the later mechanism, it is proposed that as the primary tumour grows, increasing tumoural pressure drives excess fluid and disseminated tumour cells into the surrounding interstitium. The subsequent increase in interstitial pressure drives the opening of peritumoural lymphatic pores that drain away excess interstitial fluid and disseminated cells [40,41]. 3.2. Cytokines

Fig. 2. Secretion of chemokines (e.g. CCL21) from a sentinel lymph node. Metastasising cancer cells expressing the corresponding chemokine receptor (e.g. CCR7) are subsequently attracted to the lymph node down the chemokine concentration gradient in a process termed chemotaxis.

A substantial proportion of cancer cells that traffic towards regional lymph nodes do so in response to chemokines interacting with Gprotein coupled receptors (GPCRs) that are frequently overexpressed on malignant cell surfaces. Organs to which cancers metastasise often secrete chemokines that interact with GPCRs such as the chemokine

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receptors to promote chemotaxis [42,43]. In normal functioning cells, the primary role of chemokines is to direct leukocyte migration between sites of inflammation and lymphoid organs [44]. For cancer cells, the expression of chemokine receptors can be up- or down-regulated, or remain at comparable levels to normal tissue [42]. Cells expressing chemokine receptors migrate towards higher concentrations of chemokines at the site of release via a ‘chemokine concentration gradient’ [44,45] (Fig. 2). Sites of metastasis are therefore often dictated by the nature of the chemokine receptors expressed on the migrating cell. Although many chemokines and chemokine receptors may be involved in chemotaxis, the chemokine receptors CXCR4 and CCR7 and their ligands, CXCL12 and CCL21 respectively, are most commonly implicated in metastatic spread to regional lymph nodes [46–48]. In particular, amplified expression of the chemokine receptor CCR7 has been linked to the increased incidence of lymph node metastasis in multiple cancers [46–49]. Inhibitors of chemokine receptors are being investigated as a means of reducing metastatic spread [50–53]. 3.3. Integrins The integrin family plays a pivotal role in the adhesion of disseminated cancer cells to distal endothelia. Through binding to a range of extracellular matrix proteins such as laminin, trombospondin, vitronectin, fibronectin and various collagens [54], integrins are responsible for the majority of adhesion complexes between cancer cells and the extracellular matrix, and also for a large proportion of cell–cell adhesion processes in normal physiology [55]. Integrins are expressed not only on cancer cells, but also on a variety of tumour stromal cells, and are heavily implicated in multiple stages of cancer metastasis by regulating adhesion between these cell types [56]. Integrin subtype αvβ3 is of particular interest in cancer and metastasis. Integrin αvβ3 is up-regulated in many tumour types, and is prevalent on activated endothelial cells, newly developed vessels, osteoclasts and some tumour cells, but is not expressed in healthy organs or resting endothelial cells [57,58]. αvβ3 is implicated in many components of the metastatic cascade including angiogenesis [59,60], and is involved in the regulation of tumour proteolytic enzymes such as matrix metalloproteinase 2 (MMP-2) [58,61]. Several novel therapeutics have been developed in an attempt to target αvβ3 and inhibit angiogenesis [56,57], and these also have the potential to hinder lymph node metastasis. Using cryostat sections of human lymph nodes, Nip et al. [62] showed that the binding of various metastatic cell lines to lymph nodes was largely mediated by αvβ3. The authors concluded that cancer cell lines expressing high levels of αvβ3 may have a greater potential for lymphatic metastasis. This was confirmed by Gehlsen et al. who found a positive correlation between the expression of αvβ3 integrins on the surface of various human melanoma cell lines and the metastatic potential of each cell line [63]. 4. Routes of administration Conventional chemotherapy typically involves administration via IV infusion. However, in order for drugs to gain access to the lymph after IV administration they must first extravasate from the systemic circulation, convect through the interstitium and finally traverse the lymphatic endothelium. In this way, redistribution of therapeutics from the systemic circulation into the lymphatic system can occur throughout the body. This has potential advantage for delivery to dispersed lymphatic sites of metastasis, but limits opportunities to specifically direct delivery to a particular metastasis-containing lymph node. Conversely, interstitial administration (SC, IM, ID) provides a means by which relatively high quantities of therapeutics can be delivered to the local lymphatics that drain the injection site [64]. Whilst drug molecules can be absorbed from a SC injection site by both the lymphatic and vascular systems, the physical properties of the delivery system can be manipulated to promote preferential absorption via the

lymphatic system. In general this involves the assembly of macromolecular or particulate systems, the dimensions of which preclude ready diffusion across the vascular endothelium, but permit access across the more permeable lymphatic endothelium. SC administration of a delivery system directly upstream (lymphatically) of a target/metastasiscontaining lymph node is expected to deliver high drug concentrations to that node by virtue of unidirectional and converging lymphatic flow. Therapeutics that are administered via direct intratumoural injection also have potential for downstream lymphatic transport. This route of administration delivers high concentrations of therapeutic directly into the primary tumour, and it has been suggested that lymphatic drainage of an injected delivery system will follow the same drainage patterns as that of disseminated tumour cells that exit the primary tumour, thereby enhancing delivery to sentinel, metastasis-containing lymph nodes [65]. However, although lymph vessels are present within tumours, there is debate over whether these vessels are functional or whether high intratumoural pressure compresses lymph vessels leaving them non-functional [39]. For example, Harrington et al. reported that the relative recovery of DTPA encapsulated within liposomes was up to 19 times greater in ipsilateral lymph nodes when compared with contralateral lymph nodes for rats dosed via intratumoural injection, whereas recovery was up to 121 times greater for SC-dosed rats (nontumour bearing). This suggests that more efficient delivery of therapeutics to regional lymph nodes may be achieved via SC administration adjacent to a primary tumour, rather than by intratumoural administration [65]. In the same study, lymph node recovery after IV administration was lower than that observed after SC or intratumoural administration and there was no difference between the ipsilateral and contralateral lymph nodes, consistent with the lack of specificity of this administration route [65]. Lymphatic access of nano-chemotherapeutics has recently also been described following oral administration. In these studies, docetaxel was formulated inside nanocapsules encapsulated within microparticles and delivered to rats and pigs via oral administration [66,67]. The oral bioavailability of docetaxel in plasma was enhanced following formulation within nanoparticles when compared to a docetaxel solution formulation, and the orally administered nanoparticle formulation achieved greater plasma concentrations than those following IV administration of a solution formulation. The authors hypothesised that following internalisation within enterocytes, the nanoparticles were coated with apoproteins and phospholipids prior to exocytosis from enterocytes in an analogous fashion to chylomicrons and therefore facilitated delivery to the systemic circulation via the lymphatic system. This hypothesis was supported by reduced systemic exposure of docetaxel nanoparticles when animals were pretreated with an inhibitor of chylomicron exocytosis, but direct evidence of lymphatic transport was not reported. 5. Targeting the lymphatic system following interstitial administration The ability of nanoparticulate delivery systems to overcome biopharmaceutical limitations of drug molecules such as rapid clearance, toxicity and lack of target specificity has long been exploited in the design of drug delivery systems. Similarly, nanoparticles can be used to promote lymphatic access after interstitial administration, where their physical size provides inherent advantage in avoiding drainage into the blood and promoting uptake into the lymph (Fig. 3). 5.1. Size Drug absorption from the interstitium is effectively a competitive process between the blood and lymph vessels. The significantly faster rate of blood flow through vascular capillaries (100–500 times greater than lymph flow [28]) and improved sink conditions within these vessels when compared to lymphatic capillaries typically favours drug absorption into blood vessels when compared to lymphatic vessels. However, for molecules of increasing molecular weight, tight junctions


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of high molecular weight aggregates that were poorly absorbed from the injection site [76].

5.3. Site of interstitial administration

limit paracellular access across the vascular endothelium and increasing molecular weight also precludes facile transcellular access. Under these circumstances the larger interendothelial gaps between lymphatic endothelial cells promote preferential lymphatic access. It has been suggested that particles smaller than 10 nm in diameter are preferentially absorbed from the interstitial space by the blood [28,68], thereby limiting lymphatic access, although it is also apparent that some proteins with apparent hydrodynamic diameters smaller than 10 nm are effectively taken up by the lymph [9,69]. It is also evident that decreasing the size of an administered macromolecule will increase the rate of drainage towards lymph vessels by improving convection through the interstitial space, potentially increasing access to the lymph vessels [28,70]. Once within the lymphatics, constructs smaller than approximately 10 nm also have a greater potential to diffuse back out into the interstitium when compared to larger particles that are retained to a greater degree [71]. Interstitial aqueous channels of approximately 100 nm in diameter provide the primary route of transport away from an injection site for SC administered compounds [72]. As such, injected particles with a diameter greater than 100 nm are increasingly retained at the injection site in a size-dependent manner [73,74]. 5.2. Charge and hydrophobicity The interaction of macromolecular nanomedicines with the interstitium and subsequent access to the lymphatics is highly influenced by charge. Due to the presence of glycosaminoglycans, the interstitium carries an overall negative charge [75]. Thus, nanomedicines that carry no overall charge (or in some cases an anionic charge [27]) are generally better able to gain access to the regional lymphatics by avoiding the electrostatic interactions that may hinder drainage from the injection site [76]. In contrast, cationic materials are typically more avidly retained at the injection site [77]. In some cases, however, this trend is not observed. For example, SC administration of a polyanionic dendrimer has been shown to result in increased retention at the injection site when compared to a similar but uncharged PEGylated dendrimer. This was speculated to be due to interaction of the poly-arylsulphonated dendrimer with proteins at the injection site, leading to the formation

5.4. Enhancing lymphatic uptake of macromolecules and drug carriers Improving injection site drainage via increased interstitial pressure has been suggested as a means to enhance lymphatic absorption. For example, Feng et al. [79] explored the use of dextrans to enhance the lymphatic absorption of SC administered liposomes. By co-administering a polycationic dextran with a liposomal formulation, the authors were able to decrease the proportion of the liposome dose remaining at the injection site (footpad) 24 h following SC administration in Sprague Dawley rats. As a result, absorption of the liposome into the lymphatics was increased. The authors postulated that the observed enhancement in lymphatic absorption occurred in response to the positively charged dextran binding to the interstitium, increasing interstitial pressure and providing an additional driving force for the liposomes to enter the lymphatics. Local massage [80] and heating [69] also enhance lymphatic uptake and transport by increasing interstitial pressure and dilating vessels respectively.

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Fig. 3. The pathway of trafficking of parenterally administered nanoparticles to a metastasis-containing lymph node. (1) Interstitial (e.g. SC) injection of a drug delivery system produces an increase in interstitial pressure, the opening of interendothelial junctions or lymphatic pores (nanometers to several microns in size) and facilitates the drainage of drug delivery systems into the lymph. The wide interendothelial junctions and incomplete basal lamina (2) in lymphatic vessels allows drug delivery systems to traverse the lymphatic vasculature. In contrast, (3) the tight junctions between endothelial cells lining the vascular endothelium limit uptake into the blood. (4) Interstitial injection of nanoparticulate drug delivery systems upstream of target lymph nodes allows for targeted exposure of chemotherapeutics to metastases present within lymph nodes. To gain access to the lymph from the systemic circulation following IV administration (5), drugs or drug delivery systems must extravasate from the systemic circulation (6), traverse the interstitium (7) and cross the lymphatic endothelium (8). This provides for broader lymphatic access but at lower concentrations than are possible from directed delivery through local interstitial administration. In both cases, lymph resident delivery systems either lodge within draining lymph nodes, or the unidirectional flow of the lymph (9) results in drainage back into the system circulation (10).

The lymphatic uptake of macromolecules injected into an interstitial site is typically greater following administration into an area of high interstitial pressure (such as the leg or foot) when compared to an area of lower pressure (such as the abdomen). Thus, Oussoren et al. found that a significantly larger fraction of an injected liposomal formulation was absorbed by the lymphatics following SC injection into the footpad or the dorsal side of the foot when compared to an injection into the flank in rats (Fig. 4). The authors suggested that the greater proportion of adipose tissue present in the flank facilitated the spread of the liposome dose from the injection site, decreasing interstitial pressure and removing part of the driving force for the formation of lymph. The rate of removal from the interstitium was also greater following SC injection into the footpad in comparison to the dorsal side of the foot, likely reflecting increased interstitial pressure within the footpad, which has less ‘give’ when compared to areas with looser skin [64]. Similar results have been reported by Kota et al. for large proteins in sheep [78].

1.2 1.0 0.8 0.6 0.4 0.2 0.0 flank

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Fig. 4. Recovery of % administered liposome label from regional lymph nodes after SC administration of liposomes at three different sites of injection, 52 h post injection. A single dose of radiolabelled liposome (mean diameter 100 nm) was injected SC in the flank, the dorsal side of the foot and the footpad of rats. Liposome label was recovered in the brachial (red checked bars) and axillary (blue vertically striped bars) lymph nodes in the case of SC injection into the flank, and popliteal (red checked bars) and iliac (blue vertically striped bars) lymph nodes in the case of SC injection into the foot. Total recovery was calculated by adding up the values of both regional lymph nodes (green horizontally striped bars). Data represent mean ± s.e.m. (n = 4 rats). Figure adapted from reference [64].


In addition to promoting drug delivery into the lymph, benefit in treating lymph node metastases is expected to be gained by the retention of drugs (or drug delivery systems) within lymph nodes. The properties that promote lymph node retention of drug delivery systems, however, either by physical filtration (increased size) or phagocytic engulfment (increasing hydrophobicity, receptor interactions) are largely in contrast to those required for efficient drainage from the SC injection site. For example, Moghimi et al. [82] observed that coating polystyrene nanoparticles with hydrophilic PEG-containing polymers resulted in faster drainage kinetics from a SC injection site, but that the coated nanosphere with the fastest drainage from the injection site also had the poorest retention within regional lymph nodes. Conversely, the uncoated nanospheres that were poorly absorbed from the injection site resulted in the greatest lymph node recovery. The authors suggested that the nanospheres coated with polymers containing the longest PEG chain resulted in rapid and complete drainage from the injection site as a result of the steric barrier the polymers provided between the nanospheres and injection site interstitium. The steric barrier was also hypothesised to preclude opsonisation and phagocytosis of the nanospheres by lymph node macrophages, resulting in limited lymph node retention. Somewhat different results have been described for mitomycin Cdextran conjugates of various molecular weights [83]. In this case, drainage from an intramuscular injection site was greatest for unconjugated mitomycin C or mitomycin C conjugated to low molecular weight dextrans, whereas recovery within lymph nodes was greatest for mitomycin C conjugated to dextrans with higher molecular weights. Similarly, Oussoren et al. reported that the rate of clearance of liposomes from a SC injection site was inversely related to liposome diameter, with smaller liposomes having the fastest clearance kinetics [74], whereas the proportion of the absorbed dose that was retained within the local lymph nodes was greatest for larger liposomes. The combination of these two conflicting phenomena led to a similar proportion of the total dose being recovered within lymph nodes for all sizes studied (Fig. 5). When designing drug delivery systems for the purpose of treating lymph node metastases, it is therefore likely that a balance will be required between maximal lymphatic exposure and optimal lymph node retention. Much of the available literature evidence also suggests that retention of macromolecules within lymph nodes is driven by interactions with lymph node resident macrophages [84–86]. Targeting lymph node macrophages may therefore prove beneficial in delivering therapeutics to lymph node metastases, although this strategy is complicated by the realisation that for most particulate delivery systems the first step in achieving site specific targeting after IV administration is avoidance of macrophage interactions in the liver. After interstitial administration, however, the issues of rapid clearance by the liver before reaching the target site are, at least on first pass, removed since the lymph draining a SC injection site passes directly to the sentinel lymph nodes. The broader complication of this approach is the subsequent fate of the drug delivery system within the lymph nodes and whether phagocytosis by node-resident macrophages is able to both retain a delivery system in the node and also stimulate drug release, or whether macrophage phagocytosis sequesters drug, hindering drug release. There is evidence, however, that the release of chemotherapeutics from macrophages following the engulfment of drug delivery devicechemotherapeutic complexes is possible [87].

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For some macromolecules, significant redistribution into the lymphatics can occur from the systemic circulation. This process has been described for both macromolecular carriers such as dendrimers [76] and also for proteins [81]. In this case, however, the concentration of drug carrier or protein in the lymph is typically lower than that which can be achieved after SC administration.

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mean size (nm) Fig. 5. Biodistribution of variously sized, 3H-labelled liposomes associated with regional lymph nodes or injection site tissue at 52 h following SC administration to the dorsal side of the right foot. Liposomes within samples were quantified by radioactivity counting. Panel A represents the % of dose recovered at the injection site, Panel B represents the % of dose recovered per g from the regional lymph nodes, and Panel C represents the % of the absorbed dose recovered per g from the regional lymph nodes. Data represent mean ± s.d. (n = 4 rats). Figure adapted from reference [74].

5.6. PEGylation for improved lymphatic exposure PEG is commonly used to modify the pharmacokinetics of therapeutic macromolecules by improving biocompatibility and/or reducing toxicity and immunogenicity [88–90]. By increasing size, systemic circulation times and limiting tissue interaction, PEGylation has also been reported to enhance the lymphatic exposure of several therapeutics [82]. For example, a significant increase in the recovery of a polylysine dendrimer within thoracic lymph was achieved through surface


capping the cationic dendrimer with either 570 Da or 2000 Da PEG chains in a rat model [76]. In this study, PEGylation increased the systemic bioavailability of the dendrimer following SC administration from 26% for the uncapped dendrimer to approximately 100% for the fully PEGylated dendrimer. The increase in lymphatic transport therefore likely reflected both an increase in bioavailability and an increase in the proportion of the absorbed dose that was taken up into lymphatic rather than blood capillaries. An increase in bioavailability was also seen when the dendrimer was capped with smaller 200 Da PEG groups, but in this case lymphatic recovery was unchanged. It is likely that this was due to the only modest increase in dendrimer hydrodynamic radius resulting from conjugation with the lower molecular weight PEG (5.6 nm diameter for the uncapped dendrimer, 6 nm for the PEG capped dendrimer) such that absorption into the blood remained relatively unhindered. Conversely, dendrimers capped with the 570 Da and 2000 Da PEG chains were larger (11.4 nm and 13.4 nm respectively) and presumably less readily permeable across the vascular endothelium. In a separate study, PEGylation of the protein interferon α2 with 12 and 40 Da PEG polymers also resulted in increased recovery within the thoracic lymph of rats following both IV and SC administration [9]. Consistent with the above study, increasing amounts of PEGylatedinterferon were recovered in the lymph with increasing PEG MW following IV administration, however no change in recovery was observed with increasing PEG MW following SC administration (Fig. 6).

Cumulative recovery in lymph (%)

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A

Masking the charge associated with a drug delivery system by PEG conjugation is therefore favourable for improving injection site drainage, and where particle size is sufficiently large may also enhance lymphatic uptake. However PEGylation is also likely to inhibit lymph node retention by limiting macrophage uptake, and masking electrostatic interactions with node resident cells or stroma such as reticular fibres. The difficulties faced when using PEG to improve lymphatic access and at the same time attempting to retain properties favourable for lymph node retention are not dissimilar to “the PEG dilemma” faced when targeting nanoparticulate systems to systemic tumours. Thus, it has long been observed that PEGylation of drug delivery systems typically enhances accumulation at tumour sites via the EPR effect. However, several investigators have subsequently reported incidences of decreased in vivo efficacy for drug loaded PEGylated drug delivery systems when compared to the equivalent non-PEGylated systems in spite of their greater accumulation at the tumour site [91]. Again, the likely explanation for this phenomenon lies in the realisation that the steric barrier provided by PEG coverage limits the interaction of the delivery system with tumour cells and restricts uptake into the cell. Furthermore, even for particles that are endocytosed, the PEG barrier is also likely to limit interactions between the drug carrier and endosomal lipids and reduce endosomal escape [91]. In systemic therapy, attempts to overcome this dilemma have focussed on the use of cleavable linkers that release PEG from the drug delivery system in response to a (generally cancer specific) external stimuli, such as low pH [92,93], reducing agents [94], cathepsins [95], esterases [96] or matrix metalloproteinases [97–99]. Post PEG release, the particle is more amenable to cellular uptake. A similar approach could be envisaged to harness the beneficial aspects of PEGylation in promoting drainage from SC injection sites and entry into the lymphatics, but where subsequent PEG cleavage enhances lymph node retention and promotes cellular uptake and drug release.

30

6. Drug delivery devices for targeting lymph node metastasis As described above, formulation of therapeutics within colloidal or nanoparticulate drug delivery systems has the potential to limit toxicity by reducing free concentrations in blood and simultaneously promoting access to the lymph and lymph nodes. Whilst this general property holds true across a range of particulate systems, differences in behavior are evident across different systems. These are described below.

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25

30

B

30

20

10

0 0

5

10

15

20

25

30

Time (h) Fig. 6. Cumulative recovery of interferon α2 (green squares), interferon α2 conjugated to a single 12 kDa PEG chain (blue circles) and interferon α2 conjugated to a single 40 kDa PEG chain (red triangles) in thoracic lymph following IV infusion (panel A) or SC injection into the left hind leg (panel B) of thoracic duct-cannulated rats. Data represent mean ± s.d. (n = 4–5 rats). Figure adapted from reference [9].

Liposomes are self-assembled colloidal delivery systems based on a phospholipid bilayer. Therapeutics are generally encapsulated within the liposome core. Liposomes are becoming more commonly employed as vehicles in anti-cancer therapy, to allow solubilisation of poorly water soluble actives [100], to improve tumour delivery and to reduce the concentration of free drug in blood [101–104]. Commonly prescribed chemotherapeutic liposomal formulations include Myocet® (non-PEGylated) and Caelyx® (PEGylated), both liposomal formulations of the anthracycline, doxorubicin. These formulations impart favourable properties onto doxorubicin, including a substantially longer apparent plasma half-life, greater accumulation within tumours and lower systemic toxicity [23,105]. The plasma half-life of the PEGylated liposome, however, is substantially longer than that of the nonPEGylated variant. The versatility for functional group conjugation coupled with the diversity of achievable sizes (nanometers to micrometers in diameter), has also seen liposomes widely utilised to characterise lymphatic absorption, particularly from SC injection sites, and to promote the targeting and treatment of lymph node metastasis [26,64,74,85,106, 107]. For example, Caelyx has been shown to enhance the recovery of doxorubicin in lymph by up to 165-fold following SC and IV administration when compared to an equivalent dose of doxorubicin in solution

248


[8]. Interestingly, however, for Caelyx there was a disconnect between very high recovery of doxorubicin within the lymph fluid and relatively modest recovery within lymph nodes. As such, recovery of doxorubicin within lymph nodes sentinel to the SC injection site was only 1.8 times greater for liposomal doxorubicin when compared to a solution formulation of doxorubicin. Thus, approximately 11.7% of the doxorubicin dose was recovered in lymph after SC injection of Caelyx, but only 0.34% of the quantity of doxorubicin recovered in lymph was retained within the lymph nodes. In contrast, only 0.08% of the doxorubicin solution dose was recovered in lymph, but of this small amount a relatively large proportion (approximately 25%) was retained within lymph nodes. This is likely the result of the highly PEGylated liposome formulation enhancing transport through the interstitial space to the lymphatics (and therefore enhancing lymphatic recovery), but the PEG surface subsequently preventing the interactions required to promote retention within lymph nodes. Encapsulation of doxorubicin within liposomes has also been shown to reduce injection site irritation and therefore to facilitate injection SC or into the gastric mucosa adjacent to primary tumours. This resulted in the delivery of higher concentrations of doxorubicin to the sentinel, draining lymph nodes when compared to conventional IV administration of un-encapsulated doxorubicin [104,108]. In both of the studies described above, the liposomal preparation also resulted in improved cytotoxic effects in metastasis-containing lymph nodes when compared to animals administered free drug. Encapsulation of methotrexate within liposomes has also been shown to increase drug retention within lymph nodes adjacent to an intramuscular injection site for up to 24 h, with an increase in drug exposure to the node of up to 18 times that obtained by administration of a methotrexate solution formulation [102]. Similarly, encapsulation of radiolabelled diethylenetriaminepentaacetic acid (DTPA) within PEGylated liposomes significantly increased the recovery of the injected dose (following IV, SC and intratumoural administration) within regional lymph nodes compared to free DTPA for up to 192 h [65]. 6.2. Micelles Micelles are self-assembled aggregates of surfactants or surfactantlike polymers in solution and are typically 2–20 nm in diameter. The polar ‘head groups’ of micelle-forming monomers arrange at the aggregate surface in contact with the aqueous environment, and the hydrophobic ‘tails’ form the inner core of the micelle where drugs are often encapsulated. Due to the in vivo instability of many ‘dynamic’ micellar systems, chemical cross-linking of micelle cores is becoming increasingly popular as a means of increasing longevity and results in micellar aggregates of up to 100 nm in diameter. The use of covalent cross linkers and conjugates adds complexity to the synthesis of these otherwise self-assembled systems but provides for greater control over drug release kinetics. Similarly to liposomes, micelles have been explored as potential drug delivery vectors for chemotherapeutics [109,110], and to enhance delivery to lymph node metastases. For example, Qin et al. showed that indocyanine green (ICG) delivered alone or encapsulated within PEG-PE copolymer micelles accumulated within mouse lymph nodes to a similar extent at 1 h following IV injection, but was higher for the micellar preparation after 3 h, suggesting that the micelle formulation enhanced lymph node retention in vivo (Fig. 7) [111]. Subsequent studies showed that encapsulation of vinorelbine within the same PE-PEG micelle system reduced the incidence of lymph node metastasis in a syngeneic model of breast cancer [111]. In a similar study, Rafi et al. encapsulated the chemotherapeutic dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt) within a PEG-L-glutamic acid block copolymer micelle (30 nm) and administered the formulation IV to mice with metastatic gastric carcinomas. The fluorescently labelled micelles accumulated within the primary tumour and metastasis-containing lymph nodes (but not in metastasis-free nodes) and subsequently reduced the growth of both

Fig. 7. Ex vivo spectral fluorescence images of lymph nodes 1 h and 3 h after IV injections of micelle-encapsulated ICG (Micelle ICG) or ICG in solution (Free ICG) in mice. Image adapted from reference [111].

the primary tumour (as determined by bioluminescent imaging) and the lymph node metastasis (determined by lymph node weight) when compared to mice treated with oxaliplatin (the active, daughter form of DACHPt) alone [112].

6.3. Dendrimers Dendrimers (4–20 nm diameters) are comprised of multiple generations of monomeric units around a central core [113,114]. The resultant globular polymers resemble the 3-dimensional structure of proteins and can be designed with either stable or biodegradable bonds within the scaffold. The reactive, polyfunctional surface and interior scaffold enables drugs to be either covalently conjugated to the surface or entrapped via non covalent H-bonding, ionic or hydrophobic interactions within the scaffold [114]. As a result of the stepwise synthetic methods used to generate the structures, dendrimers are well defined in terms of molecular weight and branching, and typically have low polydispersity [113,115]. Dendrimer multi-valency also allows tailoring of pharmacokinetic properties by readily modifying the physicochemical and surface properties [76,114,116–123]. Dendrimers have been shown to have significant potential as chemotherapeutic delivery vectors for passive and targeted drug delivery to tumours [124–127]. For example, folic acid conjugation to chemotherapeutic-associated dendrimers has been observed to enhance the delivery of the dendrimer system to cancer cells that over-express folic acid receptors [128]. Conjugation of an αVβ1 integrin inhibitor peptide (PHSCN) to the surface of a poly-lysine dendrimer has also been shown to promote inhibition of αVβ1-mediated invasion of breast cancer cells to basement membranes in vitro when compared to administration of the peptide alone [59]. The patterns of lymphatic access and transport of dendrimers have been extensively investigated [86], in particular as potential lymph imaging agents, and dendrimers have been shown to significantly enhance the delivery of MRI contrast agents such as gadolinium to the lymphatic system for imaging of the lymphatic vasculature and nodes [86, 129–131]. As described in Section 6.6, studies from our group have shown that increasing degrees of PEGylation and molecular weight appear to enhance drainage from a SC injection site and redirect absorption towards the lymph rather than the blood, and that large PEGylated dendrimers appear to redistribute into the lymph after IV administration, presumably via extravasation and lymphatic absorption in peripheral capillary beds [76]. Our more recent studies have further shown that PEGylated poly-lysine dendrimers enhance the lymphatic exposure of doxorubicin when compared with a solution formulation of doxorubicin, doxorubicin encapsulated within simple PEGylated micelles or doxorubicin within PEGylated liposomes (Doxil) following IV and SC administration in a rat model [8] (Fig. 8). Interestingly, in this case, lymph node retention of the dendrimer was also significantly


Cumulative % lymph recovery

50

A

40

30

20

10

*#

0 0

Cumulative % lymph recovery

50

5

10

15

20

25

30

#

B

40

30

20

* 10

*#

0 0

5

10

15

20

25

30

Time (h) Fig. 8. Cumulative % recovery of total dosed doxorubicin in the thoracic lymph following IV infusion (panel A) or SC injection in the hind left leg (panel B) of rats. Doxorubicin was administered dissolved in saline (squares), as the liposomal formulation Doxil®/Caelyx® (triangles) or conjugated to a poly-lysine dendrimer (circles). Data represents mean ± s.e.m. (n = 4–5 rats). *Represents p b 0.05 cf. the recovery in lymph of rats dosed with D-DOX. #Represents p b 0.05 cf. the recovery in lymph of rats dosed with L-DOX. Figure adapted from reference [8].

higher than that of the solution or liposomal formulation, suggesting that PEGylation did not preclude lymph node uptake.

249

polypropylene sulfide nanoparticles were taken up by the lymphatics following IM and ID administration [133]. In tumour-bearing mice, nanoparticles were able to successfully target immune cells capable of suppressing anti-tumour responses in tumour draining lymph nodes, indicating potential for delivery to cancer-relevant cells types. In further studies, loading of these nanoparticles with ligands for toll like receptors produced an inflammatory response in tumour draining lymph nodes, and was subsequently shown to slow tumour growth [134]. This growth inhibition was only evident when the formulation was administered SC on the tumour-bearing side of the mice. Conversely, no inhibition of tumour growth was observed in mice administered the formulation on the side contralateral to the tumour, highlighting the potential role of the lymphatics in drug activity. Newer technology has also seen the incorporation of stimuliresponsive components into nanoparticles for greater control over drug release. For example, Khullar et al. demonstrated delivery of paclitaxel encapsulated in polymeric ‘expansile’ nanoparticles (nanoparticles that increase in volume when exposed to an acidic environment) to regional lymph nodes following ID injection in a pig [73]. The authors initially characterized the lymphatic transport of 100 nm and 50 nm fluorescently labelled nanoparticles, and found that 50 nm nanoparticles were able to deliver a payload to regional lymph nodes, whereas the 100 nm particles remained at the injection site. Fluorescent imaging was then used to map the ability of the 50 nm nanoparticle to deliver a payload of fluorescently labelled paclitaxel to the local lymph node basin, where the paclitaxel was found to localise primarily within the subcapsular sinus, a common site of lymph node metastasis. The efficacy of paclitaxel formulated within a polymeric expansile nanoparticle in treating lymph node metastasis was subsequently investigated [10]. Paclitaxel was either loaded into nanoparticles or formulated in a solution of cremophor and ethanol. Both formulations were administered SC into the mammary fat pad of female nude mice. Formulation of paclitaxel within nanoparticles resulted in a 9-fold higher recovery of paclitaxel within the axillary lymph nodes 4 days following treatment. Additionally, nude mice bearing bioluminescent human breast tumours in the mammary fat pad were imaged 5 weeks following peritumoural injection of nanoparticle-paclitaxel or cremophor–paclitaxel formulations to determine the relative ability of each formulation to treat lymph node resident metastases. Formulation of paclitaxel within the nanoparticle resulted in significantly less regional nodal metastasis when compared with the paclitaxel–cremophor treatment group (Fig. 9) [10].

6.4. Polymeric nanoparticles

6.5. Other drug delivery systems

Polymeric nanoparticles typically comprise polymeric matrices containing one or multiple polymers and have particle sizes of b 1 μm (more commonly 10–200 nm). Depending on the method of synthesis, therapeutics may be entrapped within or conjugated to nanoparticles, offering varying levels of control over drug release kinetics. It is becoming increasingly common for nanoparticles to be comprised of biodegradable polymers, and as a result, nanoparticles often have low toxicities and have been used to deliver therapeutics to the lymphatics via multiple administration routes. For example, Shoa et al. described the delivery of a Coumarin 6 loaded PEG-poly(lactic acid) nanoparticle to deep cervical lymph nodes following intranasal administration [132]. The concentration of Coumarin 6 within lymph nodes was greater than that within blood from 5 min to 12 h following intranasal administration, whereas concentrations were greater in the blood following systemic administration, highlighting the potential for lymphatic targeting via intranasal delivery. Furthermore, surface conjugation of Concanavalin A to nanoparticles enhanced both the systemic bioavailability and the amount of drug within the lymph nodes. The reason for the enhanced lymph node accumulation, however, was not clear. Polymeric nanocarriers have also been investigated for cell specific targeting within lymph nodes. For example, pluronic stabilized,

A range of other drug delivery systems have been investigated for the treatment of lymph node metastasis. Activated carbon nanoparticles, for example, have been shown to enhance the amount of drug recovered within tumour draining lymph nodes [135–138]. In one study in humans, carboplatin was adsorbed to the surface of activated carbon nanoparticles and administered via SC injection adjacent to primary tumours in breast cancer patients [139]. The recovery of carboplatin in axillary lymph nodes was significantly greater in these patients than in the nodes of patients administered an equivalent IV dose of a solution formulation of carboplatin. Carbon nanotubes have also been explored for use in targeting and treating lymph node metastases. In one example, the chemotherapeutic gemcitabine was loaded into magnetic carbon nanotubes functionalized with poly(acrylic acid), and administered to mice with established highly metastatic footpad tumours. In these studies, the nanotubes were made magnetic by loading with magnetite nanoparticles. Prior to drug administration, multiple dosing groups had magnets surgically implanted near the popliteal lymph node (sentinel lymph node). Treatment with the functionalized nanotubes resulted in smaller tumours in draining lymph nodes at 15 days following initial treatment when compared to nanotube alone or gemcitabine alone. Interestingly,

250


Fig. 9. Peritumoural administration of paclitaxel encapsulated within expansile nanoparticles (Pax-eNP) decreases the incidence of lymph node metastases. A represents the in vivo experimental design. B represents bioluminescent images of bilateral axillary lymph nodes ex vivo 5½ weeks following the peri-tumoural formulation administration on the ipsilateral side (local) with Pax-eNP, unloaded nanoparticle (Unloaded-eNP), or paclitaxel in a cremophor/ethanol solution (Pax-C/E), or systemic paclitaxel intraperitoneal therapy (IP). Metastatic disease was evident in all treatment groups except Pax-eNP treated animals. C represents incidence of LN metastases as determined via detection of bioluminescent signal within ipsilateral LN. Pax-eNP was the only treatment to significantly decrease the incidence of lymph node metastases (33%) compared to 100% incidence in the unloaded-eNP group (p b 0.005). D represents a comparison of bioluminescent signal within ipsilateral LN as a function of paclitaxel therapy, demonstrating significantly less metastatic nodal disease in Pax-eNP treated animals, (p b 0.05). Figure reproduced from reference [10], Copyright 2013, with permission from Elsevier.

magnet implantation did not result in improved efficacy as was originally hypothesised [140]. Hyaluronan (HA) is a polysaccharide component of the extracellular matrix that is cleared primarily via the lymphatic system [141]. As such, HA has been exploited to enhance the access of chemotherapeutics to the lymphatics from a SC injection site. Cisplatin conjugated to HA has been shown to have greater lymph node biodistribution following SC injection in rats than unconjugated cisplatin, whilst at the same time retaining similar in vitro cytotoxicity against cancer cells as unconjugated cisplatin [142,143]. However, it is not clear whether the preferential lymphatic drainage of HA from the interstitial space was a result of the high molecular weight of HA, precluding uptake into the blood, or whether this was a HA specific targeting event [9,83,144]. Nonetheless, HA may be an attractive polymer for lymph targeting as it is expected to be highly biocompatible with the interstitium. A broader list of published examples of nanoparticle-based lymphatic targeting studies is provided in Table 1.

can be tailored to release a payload specifically within the tumour, avoiding widespread exposure. Drug release chemistries that are specific to lymph nodes have, to this point, not been described, however more generic tumour-specific drug release systems such as those that rely on low pH [126,170,171] and enzymes that are overexpressed in the tumour environment [172,173] have been widely described and would likely be beneficial in the setting of lymph node metastasis. For delivery systems that rapidly target to, and are retained in lymph nodes, relatively ‘leaky’ delivery systems may be sufficient to allow non-specific liberation of chemotherapeutics in the vicinity of a lymphatic micrometastasis. For drug delivery systems that are retained in lymph nodes via uptake into node-resident macrophages, liberation of drug must also be accompanied by egress from the macrophage. In this case effects on node macrophages are also likely including reduced macrophage function or apoptosis. The net effect of this on metastatic growth is hard to predict since macrophages can either promote tumour growth or regression [174–177].

6.6. Drug liberation from drug delivery systems

7. Targeted approaches for lymph node metastasis

Colloidal and nanoparticulate delivery systems have the potential to enhance delivery to the lymphatic system. Some of the dose, however, is likely to be absorbed directly from the injection site into the systemic circulation and even that which is absorbed into the lymph is ultimately returned to the systemic circulation via the thoracic duct. This may be beneficial as these materials have the potential to enter the primary tumour and sites of metastasis by EPR from the blood (supplementing direct access to metastatic sites from the lymph), but also raises the prospect of systemic toxicity. Many drug-carrier systems are therefore designed with stimuliresponsive drug release mechanisms as a means of reducing unintended release at non-target sites. By exploiting properties of the tumour environment that differ from normal tissues, drug delivery systems

The multivalent nature of many nano-carriers provides an opportunity for the conjugation of active targeting ligands as a means to enhance site specific delivery to tumours. The targeting molecules are generally ligands for receptors that are over-expressed by tumour cells. Targeting ligands that are specific for the lymphatics or for lymph node metastases are uncommon, but have been described, and may offer the advantage of more specific tumour targeting, limiting healthy tissue exposure and systemic toxicity. 7.1. Lyp-1 Using phage display screening, Laakkonen et al. identified a 9 amino acid peptide that binds to a receptor that is present on tumour cells and

Table 1 Nano-chemotherapeutic systems for the targeting and treatment of lymph node metastasis. Chemotherapeutic

Particle size (nm)

Administration route

Species for lymphatic distribution

Species for lymph node metastasis efficacy

Ref.

Liposome Liposome Liposome Liposome Liposome Liposome Liposome Liposome Liposome Magnetite liposome Micelle Micelle

Doxorubicin Methotrexate Doxorubicin 9-Nitrocamptothecin, α-TEA Honokiol Doxorubicin Doxorubicin Doxorubicin Melphalan Hyperthermia Doxorubicin, vinorelbine (1,2-diaminocyclohexane)platinum(II) (DACHPt) Doxorubicin None None Paclitaxel Lyp-1 Doxorubicin Paclitaxel, CpG oligonucleotide Melanocyte-associated antigen gp100 protein Paclitaxel Paclitaxel Mitoxantrone Tyrosinase-related protein 2

120 Not provided Not provided Not provided 80–130 82–94.2 100 Not provided 34.3–250 94.1 14.5 30

SC, IV IM Foot pad injection Aerosol IP SC IP, IV IP, IV SC Peritumoural injection IV IV

– Rat – – Mouse Mouse Rat Rat Rabbit Mouse Mouse

Rabbit (rabbit breast cancer) – Mouse (mouse leukaemia) Mouse (mouse mammary cancer) Mouse (mouse lung carcinoma) Mouse (Human lung cancer) Mouse (Human lymphoma) – Rat (rat mammary adrenocarcinoma) Rabbit (rabbit squamous cell carcinoma) Mouse (Mouse mammary cancer) Mouse (human gastric carcinoma)

[108,145] [102] [146] [100] [147] [148] [149] [150,151] [152,153] [154] [111] [112]

12–100 30 149.4–158.6 50–100 83.1–91.3 100–120 30 258

IV, SC ID, IM Intranasal ID Nail pad injection IV ID Topical

Rat Mouse Rat Pig Mouse – Mouse Mouse

– – – – – Mouse (human pancreatic cancer) Mouse (mouse melanoma) –

[8] [133] [132] [73] [155] [156] [134] [157]

100–1000 238.3 61 30–45

Mammary fat pad injection IP SC SC

Mouse Rat Rat Mouse

Mouse (human breast cancer) Rat (rat ovarian carcinoma) Mouse (human breast cancer) –

[10] [158] [159] [160]

siRNA

50–60

Intratumoural injection

–

[161,162]

Magnetic nanoparticle Activated carbon nanoparticle

Anti miR-10b oligonucliotide Aclarubicin

23.5 20

Mouse Human

Magnetic carbon nanotube Polymeric microbubble Carbon nanohorn Dextran PEGylation Polymer-conjugation Hyaluronan-conjugation Hyaluronan-conjugation

Gemcitabine Doxorubicin Doxorubicin Mitomycin C Interferon α2 Cisplatin Doxorubicin Cisplatin

30–60 800–900 80–100 Not provided Not provided Not provided Not Provided 7.55–25.20

IV SC, Intratumoural, Peritumoural SC IV Intratumoural injection IM IV, SC Interstitial injection SC SC, IV

Mouse (human gastric cancer and human colon adenocarcinoma) Mouse (Human breast cancer) Mouse (mouse leukaemia)

Dendrimer, Liposome Polymeric nanoparticle Polymeric nanoparticle Polymer nanoparticle Polymeric nanoparticle Polymeric nanoparticle Polymeric nanoparticle Polymeric nanoparticle Polymeric nanoparticle Polymeric nanoparticle Lipid nanoparticle Lipid-calcium-phosphate nanoparticle Calcium carbonate nanoparticle

Mouse Rabbit Mouse Rat Rat – Mouse Rat

Mouse (human pancreatic cancer) Rabbit (Rabbit squamous carcinoma cell line) – Mouse (mouse leukaemia) Mouse Rabbit (rabbit breast cancer) – Mouse (human oral squamous carcinoma, human melanoma)


Drug delivery system

[163] [138,164] [140] [165] [166] [83] [9] [144] [167] [142,143,168,169]

251

252


tumour lymphatics associated with a range of cancers that metastasise to lymph nodes in vivo, but not on non-tumour associated lymphatics [178]. The peptide (CGNKRTRGC), ‘lyp-1’, inhibits the growth of tumour associated lymphatic vessels by internalisation into cells upon receptor binding, followed by localisation to the cell nucleus where apoptosis ensues [179]. Soon after the identification of lyp-1, Fogal et al. identified the receptor for lyp-1 as the p32 protein, a trimer located primarily within mitochondria, but also at cell surfaces and in nuclei [180]. Consistent with the work of Laakkonen [179], Fogal noted that p32 was located in nutrient deprived regions of tumours [180]. Tumour associated macrophages were also found to express p32 at high levels [180]. The potential utility of lyp-1 as a targeting moiety is supported by in vivo data indicating that following IV injection into mice, lyp-1 accumulates within p32 positive tumours up to 40 times more avidly than a control peptide, but does not significantly accumulate in other tissues [179]. The ability of lyp-1 to enhance the tumour and metastatic lesion uptake of a lyp-1 conjugated nanoparticle in vivo has been demonstrated by Luo et al. [155]. The in vivo activity of lyp-1 conjugated nanomedicines has also been demonstrated in pre-clinical cancer models. Karmali et al. conjugated lyp-1 to the chemotherapeutic Abraxane [181] and reported that the Lyp-1 conjugated system significantly reduced the growth of MDAMB-435 tumours in nude mice when compared with unconjugated Abraxane or lyp-1 alone. Similarly, Yan et al. compared the efficiency of lyp-1 conjugated, doxorubicin-containing liposomes to a non-lyp-1 conjugated liposome in targeting and killing lymph node metastasis in a mouse model. They found that the lyp-1 liposomes significantly increased the cytotoxicity of the doxorubicin-containing liposomes against SPC-A1 lung adrenocarcinoma cells in vitro, and significantly inhibited tumour growth in mice bearing SPC-A1 lymph node metastases in vivo [148] (Fig. 10). In an attempt to improve upon the stability of 1yp-1 peptides, Li et al. synthesised a ‘syp-1’ peptide, a cyclic analogue of lyp-1 containing selenocysteine in place of both cysteine residues in order to replace the inherently unstable disulphide bond with a more stable diseleno bond [182]. The syp-1 peptide gave enhanced in vivo stability and syp-1 conjugated, doxorubicin-containing liposomes displayed greater efficacy against a p32 positive mouse melanoma model when compared to an equivalent lyp-1 conjugated system. Efficacy against a lymph node metastasis was not investigated.

7.2. TMTP1 The peptide NVVRQ, or “TMTP1” was discovered using a FliTrx bacterial peptide display assay and found to bind to various highly metastatic cancer cell lines but not to non-metastatic cell lines in vitro and in in vivo mouse models of cancer [183]. Upon binding, TMTP1 is

internalised within the cell, but to this point its receptor has not been identified [183]. To further improve targeting and anti-tumour efficacy, TMTP1 was investigated following conjugation to the cell penetrating peptide TAT, and the NF-κB suppressor peptide, NBD [184]. Conjugation resulted in greater cellular uptake by a metastatic breast cancer cell line in vitro when compared to control peptides. Furthermore, the TMTP1-TATNBD peptide showed greater in vivo accumulation in tumour and metastatic legions, and greater efficacy against breast cancer and its metastases in a mouse model [184]. A 10 amino acid derivative of TMTP1, GCGNVVRQGC (or “TMT”, tumour metastasis targeting peptide) has also been described and conjugated to a PEGylated liposome encapsulating doxorubicin. In vitro studies demonstrated that highly metastatic cancer cells had a greater viability when incubated with non-TMT targeted liposomes, compared with cells incubated with TMT-targeted liposomes. This was shown to be a result of enhanced endocytosis of the targeted liposome into the cell. Targeted doxorubicin-containing liposomes also showed greater in vivo efficacy in treating various metastatic tumours in mouse models when compared with untargeted liposomes and free doxorubicin [185, 186]. IV-administered TMT-targeted doxorubicin liposomes also had greater efficacy in reducing the incidence of metastasis and prolonged survival in a mouse model of metastatic breast cancer (within the lung) when compared with untargeted liposomes and free doxorubicin [185]. 7.3. RGD As outlined in Section 3.3, integrins play a central role in cancer biology, and the integrin αvβ3 is of particular importance in the progression of lymph node metastasis. All αV integrin subtypes and about one third of all human integrins bind to ligands via the peptide sequence Arg-GlyAsp (RGD) [55,187]. The RDG peptide has a very short half-life in vivo, and binding efficiencies are 10–100 times greater for cyclic RGD peptides [188]. Although αVβ3 binds to other peptide sequences, the majority of its interactions are with RGD motifs. Upon RGD binding, the ligand is internalised via receptor-mediated endocytosis [189]. As such, the RGD peptide has been examined at length for its ability to target drug delivery systems to tumours and metastases for the purposes of tumour imaging [190–192], anti-tumour therapy [163,193,194], and enhancement of endocytosis of drug delivery systems into cells [195]. For example, doxorubicin-loaded nanoparticles, surface modified with cyclic RGD, have been shown to stimulate an enhanced anti-angiogenic response in vivo when compared to non-RGD conjugated controls [156]. This study also described preferential accumulation of the nanoparticle in tumour vasculature and a decrease in the size of a primary pancreatic tumour in a mouse model. Importantly, metastasis to the sentinel lymph node was significantly reduced following treatment with the RGD-doxorubicin nanoparticle at a doxorubicin concentration 15-fold less than that required for free doxorubicin and without producing the associated weight loss (Fig. 11). 8. Conclusion

Fig. 10. Biodistribution of fluorescent dye (DiR) in metastases-containing popliteal lymph nodes of nude mice as determined by in vivo fluorescence imaging. DiR was encapsulated in PEGylated liposomes with lyp-1 modification (right in inset) or without Lyp-1 modification (left in inset) and administered to mice via SC injection in the hind footpad. Data represents mean ± s.d. (n = 3). Figure reproduced from reference [148] Copyright 2012, with permission from Elsevier.

Chemotherapy for lymph node metastasis generally involves IV infusion of conventional small molecule therapeutics that typically display poor lymphatic specificity. To provide sufficient exposure to lymph node-resident metastases, this requires the maintenance of high systemic drug levels, and often results in toxicity. Association of chemotherapeutics with nano-sized drug delivery systems via encapsulation or conjugation provides one means by which specificity to the lymph and lymph nodes may be enhanced. Nano-chemotherapeutics promote lymphatic absorption from interstitial injection sites by limiting entry into the blood and may enhance retention within lymph nodes by physical entrapment or uptake into lymph resident macrophages. The multivalent nature of many drug delivery devices also offers sites for


Hilar lymph node tumour (mg)

40 [9]

30 [10]

20 [11] [12]

10

*

[13]

-N P R G D -D ox

-N P R AD -D ox

D ox

PB

S

0

Fig. 11. RGD-conjugated nanoparticles encapsulating doxorubicin (RGD-Dox-NP) prevent metastasis to the hepatic hilar lymph node. Following surgical implantation of syngeneic pancreatic tumour cells in the pancreas, mice were injected on days 5, 7 and 9 with RGD-DOX-NP, RAD (control, non-targeted peptide)–conjugated doxorubicin nanoparticle (RAD-Dox-NP), phosphate buffered saline (PBS) or doxorubicin in solution (Dox) (each with 1 mg/kg doxorubicin per dose). On day 11, the hepatic lymph nodes were dissected and weighed. Data represent mean ± sem. *represents p b 0.02 cf. PBS. Figure adapted from reference [156].

[14]

[15]

[16]

[17] [18]

[19]

conjugation of polymers such as PEG and lymph specific targeting moieties. PEG conjugation typically improves drainage from the injection site and by virtue of an increase in molecular size also promotes uptake into the lymph, but may reduce the cellular interactions that drive retention in the node. Whilst largely in its infancy, the conjugation of lymph node metastasis-targeting molecules such as lyp-1 or TMTP1 to nano-chemotherapeutics provides a potential avenue for actively enhancing site-specific chemotherapy and promotion of internalisation, although the therapeutic benefits of these approaches remain largely unproven. In summary, active and passively targeted nanochemotherapeutics provide the potential to significantly enhance lymphatic access and lymph node retention and to reduce systemic toxicity, and provide hope for future improvements in the treatment of lymph node metastasis.

[20]

[21]

[22]

[23] [24]

[25] [26]

Acknowledgements

[27]

LMK was supported by an NHMRC Career Development fellowship. GMR was supported by a Victorian Cancer Council PhD scholarship.

[28] [29]

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