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Targeted nanoscale magnetic hyperthermia: challenges and potentials of peptide-based targeting “
Recent reports suggest that diagnosis and therapy of other malignancies using superparamagnetic iron oxide nanoparticles is feasible through active targeting and accumulation of the magnetic nanoparticles to tumor sites.
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Keywords: cancer • cell death • hyperthermia • magnetic field • peptide • receptor • targeting
Magnetic nanoparticles (MNPs) are the subject of an increasing attention in oncology. Indeed, MNPs enable MRI of malignancies and, when exposed to a high frequency alternating magnetic field, generate hyperthermia. Superparamagnetic iron oxide nanoparticles (SPION) have been used as contrast agent for liver, spleen and lymph node MRI imaging. In such clinical applications, intravenously administrated SPIONs accumulate preferentially to malignant tissues because of capture by reticuloendothelial system. Recent reports [1–3] suggest that diagnosis and therapy of other malignancies using SPIONs is feasible through active targeting and accumulation of the MNPs to tumor sites. Active targeting exploits the fact that cells composing tumors (tumor and microenvironment cells) express and even overexpress biological markers that are not expressed, or less expressed in normal cells. Ligand molecules recognizing biological markers of the tumor are thus grafted at the MNP surface so that intravenously injected MNPs can reach tumor cells and accumulate in these cells. The strategy reported with EGF- and gastrin-grafted SPIONs [1–3] presents the originality of using the physiological process of ligand-induced internalization of receptors and their sorting to lysosomes. It is assumed that exposure, to a high frequency alternating magnetic field, of cells containing minute amounts of MNPs in their lysosomes, generates nanoscale hyperthermia at the surface of MNPs, leading to activation of death signal-
10.2217/NNM.14.236 © 2015 Future Medicine Ltd
ing pathways originating from the interior of the lysosomes [4] . At the difference of magnetic fluid hyperthermia experienced with nontargeted SPIONs directly injected into glioma [5] and prostate cancers [6] , targeted nanoscale hyperthermia requires minute amounts of MNPs, making this approach an attractive therapeutic option. However, it remains to establish if targeted nanoscale hyperthermia generated inside lysosomes of tumor cells will be sufficient to effectively eradicate tumors in vivo. So far, only SPIONs with a low thermal power were used to generate nanoscale hyperthermia. We assume that substitution of SPIONs with MNPs displaying an enhanced thermal power [7] will improve the outcome of treatment. Beyond the nature of the MNP magnetic core, one crucial aspect in the strategy of targeted nanoscale hyperthermia is the design of the targeting moiety of the MNPs. In this respect, receptors for peptidic hormones and growth factors being frequent cancer markers, the grafting of peptides to MNPs represents a usual way to target MNPs to tumors. Peptide grafting of MNPs offers a large spectrum of opportunities and also presents some potential problems. Efficient covalent attachment of peptidic ligands to MNPs is made possible, thanks to several types of functionalization and coupling chemistry [8] . The choice of coupling strategy must however take into account the requirement of maintaining the integrity of receptor-binding and -activation domain(s)
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Daniel Fourmy Author for correspondence: University of Toulouse 3, EA4552 Receptor & Therapeutic Targeting of Cancers, Toulouse, France Tel.: +33 561 323 057 Daniel.Fourmy@ inserm.fr
Julian Carrey Université de Toulouse 3, INSA, CNRS UMR5215, Laboratoire de Physique et Chimie des Nano-Objets (LPCNO), Toulouse, France
Véronique Gigoux University of Toulouse 3, EA4552 Receptor & Therapeutic Targeting of Cancers, Toulouse, France
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Editorial Fourmy, Carrey & Gigoux of the peptidic ligand. For most targeted surface receptors, this information is available from structure– activity relationship studies and/or from 3D structure determination of liganded receptors. In fact, depending on the targeted receptor, binding and activation domains can be contained in the same peptidic fragment, which is sometimes composed of a relatively small number of amino acids, or they can be partly distinct. Having in mind these data, next challenge will be to orientate the coupling reaction so that no or minimal undesired chemical reaction could occur with amino acid side chains of the binding and activation domain(s). By using adequate coupling chemistry [8] and eventually, by modifying peptide sequence of the ligand in a way that preserves its pharmacophore, efficient coupling of peptidic ligands to MNPs can be expected. An additional major point to consider is the steric hindrance. Indeed, the ligand’s pharmacophore must remain sufficiently away from the MNP and freely exposed to its environment to enable interactions in the binding cavity of the cell surface receptors. This is generally achieved by coupling the ligands to the MNPs via functional groups located at the extremity of poly(ethyleneglycol) coating (PEG 2000) used to enhance circulation time of the MNPs. However, such traditional approach may not be optimal for all targeting purposes, especially in the case of ligands having moderate to low affinities for their targeted receptor. For example, a study aimed at targeting HER-2-overexpressing cancer cells revealed that coating of MNPs with PEG350 and ligand grafting through a linker equivalent to PEG500 significantly enhance specific MNP uptake by cells [9] .
“...such traditional approach may not be optimal for all targeting purposes, especially in the case of ligands having moderate to low affinities for their targeted receptor.
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The ligand density at MNP surface is an additional critical point conditioning both effectiveness and specificity of the targeting. An optimal ligand density is that leading to the highest amount of MNPs accumulation in cells expressing variable levels of receptors while keeping the amount of added MNPs as low as possible for minimal nonspecific uptake and toxicity. Successful targeting of cell expressing EGF receptor was documented with SPIONs bearing an average of 20–50 EGF molecules per MNP. For CCK2R targeting, a maximum uptake and accumulation in lysosomes was achieved with MNPs bearing 100 ligand molecules per MNP. In the later example, it was also noticed that association kinetic of the targeted MNPs to the cells was dramatically slowed relative to the free
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ligand. Such slow binding, which is probably due to the size and solid nature of the MNPs, could negatively influence MNP uptake by tumors in vivo. Interestingly, multivalency resulting from grafting of a large number of ligands can counterbalance ligand affinity loss caused by ligand coupling, especially in the case of very small ligand. This was illustrated for angiotensin II receptor type 1 and αvβ integrins targeting [10,11] . Ligand multivalency at the MNP surface can also favor receptor clustering or oligomerization, which are molecular events required for, or contributing to receptor activation and internalization. Furthermore, magnetic field can cause clustering of MNP-bound receptors, as reported with EGF receptors [12] , T-cell receptors [13] and death receptor 4 [14] . In the two last examples, receptor clustering was used to cause tumor cell death. The pharmacological profile of the peptidic ligand used to target MNPs to tumor cell surface receptors is an additional critical point which must be considered with regard to the signaling pathways that provide advantage in the uptake of the MNPs. Peptidic antagonists of G-protein-coupled receptors recognize equally both coupled and uncoupled states of the receptors. Consequently, they generally show enhanced binding activity relative to agonists. However, since they do not trigger cell signaling involved in receptor internalization, they remain at the cell surface. Peptidic antagonists are therefore adequate to deliver MNPs at the surface of tumor cells but not in their lysosomes. The concept of biased ligands of G-protein-coupled receptors which are ligands having distinct efficacy and potency on the different signaling pathways of the targeted receptors makes theoretically possible the design of peptide-targeted MNPs with more predictable activity. Peptide-targeted MNPs present also the particularity of being potentially sensitive to proteases, even though MNP-grafted peptides are presumably less sensitive than free peptides. The stability of the grafted peptide is an important parameter which can be either detrimental or beneficial. In the case of ligand cleavage in the blood, the targeting advantage is lost, even if the MNPs are designed to have a long circulating time. On the contrary, if peptide cleavage occurs in endocytosic vesicles following receptor-mediated internalization of the ligand-targeted MNPs, ligand (and MNP) dissociation from the receptor can be expected, eventually leading to receptor resensitization and recycling to the cell surface [15] . Therefore, cancer cell targeting with MNPs grafted with peptidic ligands sensitive to endosomal proteases can be a way to enhance MNP accumulation in tumors since this formulation permits at the same time internalization of the MNPs
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Targeted nanoscale magnetic hyperthermia
and reappearance of the targeted receptors at the cell surface. In vivo studies on animal models of cancers have consistently shown that uptake of targeted MNPs by solid tumors is not only dependent on effectiveness of targeting measured in vitro, but is also conditioned by the ability of the MNPs to escape capture by the reticuloendothelial system, remain in the blood for enough time and cross biological barriers existing between the blood stream and tumor cells [16] . It has been appreciated that MNP coating by PEG or dextran significantly increases their stealthiness and circulating time. Whether the presence of peptides on MNP surface affects the nature of the protein corona surrounding MNPs in biological fluids and circulating time is case dependent and therefore requires specific investigations. Concerning the ability of the targeted MNPs to cross tumor microenvironment and to pene trate deeply in the tumor, recent findings suggest that their decoration with peptides carrying motifs targeting αvβ3/5 integrins and neuropilin-1, which have reported to enhance tumor homing and penetration of pharmaceutics, represents an attractive option [17] . Anticancer therapy with peptide-targeted MNPs also offers the possibility of minimizing undesired accumulation of the MNPs in healthy tissues expressing the targeted receptor, albeit at low levels relative to tumor cells. This can be achieved by coupling ligands aimed at increasing homing of the MNPs near the tumor (for instance, RGD motifs targeting αvβ integrins) or by taking advantage of acidosis of tumor microenvironment. The introduction of a chemical
moiety protecting the ligand from receptor recognition in neutral environment but enabling ligand deprotection in acidic pH represents an original approach [18] .
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“Peptide-targeted magnetic nanoparticles have
the potential to enable personalized anticancer therapy since ligands recognizing several targets can be simultaneously coupled to magnetic nanoparticles.
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To conclude, we believe that the different opportunities and constraints briefly summarized here highlight how peptides are ideal ligands for targeting MNPs to tumors. Peptides enable engineering of MNPs to perform cooperative functions such as self-amplified homing and penetration of the MNPs, amplification of the targeting by ligand combination and enhanced tumor uptake while minimizing undesired targeting and uptake. Peptide-targeted MNPs have the potential to enable personalized anticancer therapy since ligands recognizing several targets can be simultaneously coupled to MNPs. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
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Domenech M, Marrero-Berrios I, Torres-Lugo M, Rinaldi C. Lysosomal membrane permeabilization by targeted magnetic nanoparticles in alternating magnetic fields. ACS Nano 7(6), 5091–5101 (2013). Iovino N, Bohorquez Ac, Rinaldi C. Magnetic nanoparticle targeting of lysosomes: a viable method of overcoming tumor resistance? Nanomedicine (Lond.) 9(7), 937–939 (2014). Silva Ac, Oliveira Tr, Mamani Jb et al. Application of hyperthermia induced by superparamagnetic iron oxide nanoparticles in glioma treatment. Int. J. Nanomed. 6, 591–603 (2011).
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a comparative study for the elaboration of multimodal superparamagnetic nanoparticles targeting alphavbeta3 integrins. Langmuir 29(47), 14639–14647 (2013).
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Bharde AA, Palankar R, Fritsch C et al. Magnetic nanoparticles as mediators of ligand-free activation of EGFR signaling. PLoS ONE 8(7), e68879 (2013).
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Lammers T, Kiessling F, Hennink WE, Storm G. Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress. J. Control. Release 161(2), 175–187 (2012).
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Perica K, Tu A, Richter A, Bieler JG, Edidin M, Schneck Jp. Magnetic field-induced T cell receptor clustering by nanoparticles enhances T cell activation and stimulates antitumor activity. ACS Nano 8(3), 2252–2260 (2014).
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Ruoslahti E. Peptides as targeting elements and tissue penetration devices for nanoparticles. Adv. Mater. 24(28), 3747–3756 (2012).
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