RGD Peptide‐Drug Conjugates as Effective Dual Targeting ...

RGD Peptide-Drug Conjugates as Effective Dual TargetingPlatforms: Recent AdvancesLucia Battistini,*[a] Kelly Bugatti,[a] Andrea Sartori,[a] Claudio Curti,[a] and Franca Zanardi*[a]

Dedicated to Professor Franco Cozzi on the occasion of his 70th birthday.

In recent years, targeted therapies have raised increasinginterest as effective strategies to improve therapeutic efficiency,reduce the impact of adverse side effects, and overcome drugresistance, particularly in the field of cancer chemotherapeutics.Many examples of RGD (Arg-Gly-Asp) peptide-drug conjugates(PDCs) have been proposed as targeted drug delivery systems.These molecular hybrids embody high affinity ligands targetingdisease signatures (overexpressed integrin receptors or specificintegrin-related pathways within diseased cells/tissues) con-

nected to recognized therapeutic units by means of carefullydesigned linker-spacer combinations, to ultimately controlstability, physico-chemical properties, timing, and localization ofdrug release. This account has collected and critically surveyedrelevant contributions from the period of 2015 to the presentday, wishing to provide a window open on new synthesisstrategies facing the challenging issues of site-selective drugdelivery and dual drug targeting.

1. Introduction

Precision medicine represents an emerging paradigm for thecurrent biomedical research, which is now witnessing anincreasing interest for targeted therapies.[1–3] Selective andtargeted delivery of drugs to diseased tissues is a majorchallenge, in particular in the oncology field, but also in othertherapeutic domains, where the clinical use of traditionalchemotherapeutics is often limited by their severe toxicity tohealthy tissues and the occurrence of resistance mechanisms. Inthis regard, safety and efficacy of common cytotoxic agents canbe significantly improved by designing drug delivery platforms,which merge the properties of two (or more) differentmolecular entities into one individual compound. In this type ofbifunctional hybrids, a targeting unit, capable to selectivelyaddress pathologic cells, is connected by a linker/spacer to thecytotoxic payload and, optionally, to a visualizing probe tomonitor the accumulation of the drug at the diseased site and/or reveal the treatment efficacy, as in the case of the so-calledtheranostic agents.[4]

The use of antibodies as targeting units in the assembly ofAntibody-Drug Conjugates (ADCs) represents a validated strat-egy to reduce systemic exposure of normal cells to cytotoxic

agents, thus improving their therapeutic indexes, as testified bythe number of ADCs approved in the clinics or currently underclinical development.[5,6]

As an alternative approach to ADCs, Small Molecule-DrugConjugates (SMDCs) and, in particular, Peptide-Drug Conjugates(PDCs), where small peptides or peptidomimetics are used ashoming units,[7] have gained momentum for several reasonsincluding, first of all, better pharmacokinetic properties incomparison to large protein therapeutics, granting rapid andhomogeneous accumulation at the tumor mass and improvingcell permeability, while still maintaining high affinity for thetargeted protein. Other strengths of these small ligand-baseddelivery systems include i) easy accessibility by chemicalsynthesis possibly suitable for large-scale production, ii) accu-racy in controlling both payload-to-ligand ratio and batch-independent product characterization, iii) flexibility of theconjugation chemistry allowing for modulation of in vitro andin vivo stability, and iv) absence of immunogenic effects incomparison with the antibody-based counterparts.[5,8]

The accurate selection and flexible optimization of theindependent modules of these conjugates – the low-molecularweight peptide/peptidomimetic, the active cargo, the linkerand, optionally, interposed spacer(s) – crucially impact theoverall success of the conjugate as a therapeutic tool. Ingeneral, the therapeutic efficacy of the conjugate is mainlydictated by the potency of the active cargo (e.g. the cytotoxicdrug), the drug-release efficiency mainly relies on the linker/spacer features, while the safety of the construct mainlydepends on the affinity and selectivity of the ligand toward thetargeted proteins or specific pathways in the diseased cells. Theoverall physicochemical properties obviously depend on thecontribution of the three modules and are mainly tunable,again, by suitable linker/spacer(s).[2,8]

Leading criteria in the design of optimal targeted drugdelivery systems have been recently reviewed and somesuccessful examples of SMDCs and PDCs, that have entered

[a] Prof. Dr. L. Battistini, Dr. K. Bugatti, Prof. Dr. A. Sartori, Prof. C. Curti,Prof. Dr. F. ZanardiDipartimento di Scienze degli Alimenti e del FarmacoUniversità di ParmaParco Area delle Scienze 27 A, 43124 Parma, ItalyE-mail: [email protected]

[email protected]://personale.unipr.it/en/ugovdocenti/person/18151https://personale.unipr.it/en/ugovdocenti/person/19006

Part of the “Franco Cozzi’s 70th Birthday” Special Collection.

© 2021 The Authors. European Journal of Organic Chemistry published byWiley-VCH GmbH. This is an open access article under the terms of theCreative Commons Attribution License, which permits use, distribution andreproduction in any medium, provided the original work is properly cited.

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http://orcid.org/0000-0002-5341-5547

http://orcid.org/0000-0001-7745-0884

http://orcid.org/0000-0002-9688-6760

http://orcid.org/0000-0002-6117-1503

http://orcid.org/0000-0001-7451-781X

https://personale.unipr.it/en/ugovdocenti/person/18151&TR_RET;https://personale.unipr.it/en/ugovdocenti/person/19006

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https://chemistry-europe.onlinelibrary.wiley.com/doi/toc/10.1002/(ISSN)1099-0690.Franco-Cozzi-70

clinical evaluation, have been listed in comprehensive accountsappeared in the literature during the past five years.[2,3,8–12] Mostof these covalent conjugates are directed to tumor and tumor-related pathologies (angiogenesis), but examples also includeinflammatory diseases (e.g. ostheoarthritis) and others (e.g.malaria, bacterial infections).[13–16]

Concerning the selection of an optimal payload, the drugshould hit a validated biomarker with high potency andspecificity; indeed, most of the ligand-targeted conjugatescurrently undergoing clinical trials contain low-nanomolar orsubnanomolar active drugs. As for the directing ligand, thechoice depends on the targeted receptors/enzymes, whichought to be overexpressed in the diseased cells. The selectivedelivery of anticancer drugs to tumors, for example, has beenaddressed by proper design of specific binders for receptorsoverexpressed in cancer cells.

Among such receptors, certain subtypes of integrin recep-tors have shown upregulated expression in many disease states,including cancer and vascular, inflammatory, and autoimmunediseases. Thus, ligands that recognize specific integrin subtypesrepresent ideal candidates to build covalent conjugates aspharmacodelivery systems. Integrins are transmembrane recep-tors that commonly provide the physical interaction of cellswith the extracellular matrix necessary for cell adhesion,migration, and positioning; they induce signaling eventsessential for cell survival, proliferation, and differentiation. Therecognized role of specific integrin subtypes in blood clotting,angiogenesis, bone remodeling, and immune responses hasprompted the research towards their ligands for the preventionor treatment of many pathologies including thrombosis, cancer,osteoporosis, and inflammatory diseases. Most of the integrinligands that have been developed so far mimic the conservedRGD binding sequence of endogenous protein ligands such as

Lucia Battistini is currently Associate Professorof Organic Chemistry at the University ofParma, Italy (Department of Food and Drug).She received her Ph.D. degree in BioorganicChemistry from the University of Torino, andin the same year she joined the group led byProf. Giovanni Casiraghi at the Department ofPharmacy, University of Parma. Her currentspecific interests are focused on the develop-ment of new classes of peptidomimeticligands for molecular recognition and theiruse in therapeutic and diagnostic applications.

Kelly Bugatti is currently a postdoctoral re-searcher in the bioorganic chemistry researchgroup of Prof. Dr. Zanardi, at the University ofParma, Italy (Department of Food and Drug).She graduated in Pharmaceutical Chemistryand Technology at the University of Parma in2017, and she obtained her Ph.D in Drugs,Biomolecules and Health Products at the sameuniversity (2021) under the supervision ofProf. Dr. Lucia Battistini. Her research interestsare focused on the design and synthesis ofintegrin-targeted small molecule peptidomi-metics and their covalent conjugates forbiomedical applications.

Andrea Sartori is currently an Associate Pro-fessor of Organic Chemistry at the Universityof Parma, Italy (Department of Food andDrug). He graduated in Chemistry at theUniversity of Parma in 1997, and after oneyear of research fellowship at the PhysicsDepartment of the same Institution, he joinedthe group of Prof. D. N. Reinhoudt at theUniversity of Twente (NL), where he receivedhis Ph.D. in 2004. He returned to the Univer-sity of Parma and obtained a position asAssistant Professor in 2010. His work focuseson the development of new asymmetricmethodologies for the synthesis of chiralbioactive molecules, and on the preparationof integrin-targeted ligands and nanoparticlesfor therapeutic and imaging purposes.

Claudio Curti is currently an Associate Profes-sor of Organic Chemistry at the University ofParma, Italy (Department of Food and Drug).He earned his Laurea degree in Pharmaceut-ical Chemistry and Technology in 2002 at theUniversity of Parma. In 2005, he graduatedfrom the postgraduate School of ChemicalSynthesis at the University of Milan, Italy. In2001, he joined the bio-organic synthesisgroup of the Department of Pharmacy (Uni-versity of Parma) under the supervision ofProf. Giovanni Casiraghi, where he obtainedhis current position. His main research inter-ests are in the field of asymmetric synthesisand organic chemistry methodology, focusingon the development of organocatalytic proc-esses to access multifunctional natural andnatural-like compounds, including functional-ized heterocycles and polyphenol metabolites.

Franca Zanardi is Full Professor of OrganicChemistry at the University of Parma (Italy).She received her Laurea degree in Chemistry(1993) and her Ph.D. in bioorganic chemistry(1997) from the University of Parma. In 2017she was the recipient of the Research Prize inOrganic Chemistry-Life Sciences from theOrganic Chemistry Division of the ItalianChemical Society. She currently leads the bio-organic synthesis group of the Department ofFood and Drug at the University of Parma. Herresearch interests concern the developmentof methodologies addressed at the synthesisof biologically relevant chiral nonracemicmolecules in the bio-organic and pharmaceut-ical domains. She is also involved in researchprograms aimed at the synthesis and applica-tion of peptidomimetics and conjugates to beexploited as therapeutic/diagnostic tools inmedicine.


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fibronectin, fibrinogen, and vitronectin, among others. Inparticular, the αV (αVβ3, αVβ5, αVβ6, αVβ8) and the α5β1 integrins,all belonging to the RGD-dependent integrin subfamily, arenumbered among the most investigated receptors for tumor-specific drug delivery. During the past three decades, the Arg-Gly-Asp (RGD) tripeptide-recognition sequence provided thebasis for discovering a huge number of small molecule integrinbinders, as both linear and cyclic peptides andpeptidomimetics.[17–19] Although, up to now, no RGD-ligand hasreached the clinics as anti-integrin drugs, their conjugation todifferent diagnostic and/or therapeutic cargoes has been widelyexplored, as demonstrated by the intense efforts made both inacademic and industrial research in this domain.[9,20–23]

The use of integrin ligands as selective drug carriersrepresents a particularly appealing strategy, since integrins aresurface receptors exhibiting extracellular binding domains;therefore, they are readily accessible by bioactive conjugates inthe extracellular compartment. Besides their favorable location,integrins are known to promote internalization upon ligandbinding, so that the ligand-drug conjugate can enter itstargeted cell by receptor-mediated endocytosis and then trafficto intracellular endosomes and/or lysosomes, allowing the drugto be released into the cytoplasm.In addition, when multiple receptors/enzymes/pathways are

involved in complex diseases stemming from multiple alter-ations (like in cancer and tumor angiogenesis), the conjugationof bioactive modules (effectors) offers the opportunity toconcomitantly hit diverse targets possibly leading to enhanced/synergistic therapeutic effects. These dual-drug approaches aredesigned to take full advantage of spatial and functionalcooperation between receptors/proteins involved in the diseaseprogression (for example by interfering in their crosstalk), andhold promise to overcome the shortcomings of combinedtherapies.Given the growing piece of evidence speaking of strictly

intertwined spatial and functional connections between certainintegrin subtypes and neighboring proteins (e.g. crosstalkbetween αVβ3/VEGFR, αVβ6/PAR2, αVβ3/PAR1),

[24–27] integrinligand-based conjugates may rightly fit into a category of dualplatforms where the targeting unit namely, the RGD-peptideligand, may adopt the double role of both directing agent and

therapeutic tool as “best supporting actor” flanking the leadingactor-role of the attached drug (Figure 1).Like all molecular conjugates, RGD peptide-drug conjugates

(RGD-PDCs) must comply with the general requisites they (atleast ideally) should possess. Whichever the role, whether“simply” targeted or dual-effective conjugates, RGD-PDCs mustbe robust enough to reach the integrin target with minimaldegradation and loss of the cargo in the bloodstream andoptimal extravasation rate/residence time at the diseased tissue,while providing the active drug available at the correspondingbiological target. Depending on the physical localization of thedrug target, different scenarios would open: i) it is exposed inthe extracellular compart and possibly it flanks/cooperates withthe integrin receptor; ii) it is located in the cytoplasm; iii) it ispresent in the nucleus. In the first scenario, for example, anideal condition would be for the conjugate to possess a non-internalizing integrin ligand, with the drug portion availableand active either within the intact conjugate or after ad hoc-cleavage in the extracellular tumor microenvironment. If,instead, the drug target is inside the cell, as in the second andthird scenarios, integrin-mediated endocytosis of the RGD-PDCwould be welcome, with subsequent intracellular release of thecargo; alternatively, after integrin-ligand recognition, ad hocrelease of the cargo in the extracellular diseased microenviron-ment could be an option, followed by passive diffusion of thedrug into the cell.The localization and timing of the drug release (if any), and

consequent therapeutic activity, are mainly responsibility of thelinker/spacer moiety, whose crucial role in the overall perform-ance of the conjugate has been widely recognized and high-lighted. In fact, careful design of the linker/spacer characteristicsdirectly influences the physico-chemical properties (i. e. chem-ical stability in the different compartments, water/membranesolubility, brain permeability) of these platforms and stronglyimpacts on their metabolism. Such design is the result of asubtle compromise between sufficient chemical stability tominimize/avoid premature detachment of the conjugate com-ponents in the circulation and sufficient chemical lability torender the active modules available in a predictable andprogrammed manner, if and where it is necessary.

Figure 1. Pros and cons of RGD/Drug independent administration vs covalent conjugation.




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In this fascinating and rapidly evolving scenario, the aim ofthis mini-review is to collect and critically survey relevantcontributions from the period of 2015 to the present day – theprevious period was well covered by other accounts[9,10,21–23,28] –with a special focus on the design and synthesis of innovativedual conjugates bearing an integrin targeting RGD moiety (or amimetic thereof) covalently linked to a recognized chemo-therapeutic agent, especially (but not limited to) anti-cancerdrugs. Theranostic systems, where dual targeting platforms areconjugated with additional reporter units (imaging probes), willbe considered only when developed as ligand-targeted com-panion diagnostics to readily monitor the delivery/localization/fate of therapeutic agent in vitro and in vivo at preclinical level.Far from a comprehensive covering of this topic, the main

objective of our effort is to highlight the merits and limitationsof such compounds addressing couples of biological markersnamely, integrin receptors and drug target, pointing out,whenever possible, the actual role of the RGD ligand, whetheras a mere integrin targeting drug carrier, or a true therapeuticagent/integrin modulator assuming a synergistic/cooperativerole together with the chemotherapeutic agent.The selected contributions will be organized according to

the design of the linker. Dual conjugates exclusively comprisingrobust linkages (e.g. amides, triazole, (thio)ethers) that resistproteolytic degradation, and capable to exert a dual action onsurface receptors as intact entities, will be grouped as “non-cleavable” conjugates; in this group, molecular conjugatesintended to recognize integrins and to be internalized as wholeconstructs will be also placed, where the expected action of thedrug is independent by its possible detachment from theconjugate (Figure 2). Then, constructs programmed to bespecifically cleaved after integrin recognition by proper stimuli(acidic pH, reducing or proteolytic cues, etc.) will be listed anddiscussed as “cleavable” conjugates, which are recognized toliberate the drug in its active form either in the extracellularcompart or inside the cells.

2. Dual conjugates embedding noncleavablelinkers

As highlighted in the introduction, this section collects papersdescribing the synthesis of robust RGD-PDCs embodying stablecovalent connections and designed to exert their action asuncut molecular entities either in the extracellular space, afterintegrin receptor recognition, or inside cells after integrin-mediated endocytosis (Figure 2, eq. 1). In such conjugates, boththe RGD peptide and the drug modules are meant to retainoptimal bioactivity and affinity for the respective biologicaltargets, without the need of specific activation mechanism fordrug release.To this purpose, suitable connections have been exclusively

used, which include amide and ether/thioether linkages, triazolerings, which are generally stable to degradative processes bothduring circulation and at the extracellular diseased site.Importantly, all the reported case studies feature the

exploitation of easy, tunable, chemoselective, and inexpensiveligation chemistry (e.g. azide-alkyne (3+2)-cycloaddition, oximeligation, thiol-maleimide ligation), in turn enabled by ad hoc-installed orthogonal functionalities within the single modules.Angiogenesis is a well-recognized physiological event also

contributing to pathological phenomena including inflamma-tion, tumor growth, invasion and metastasis spread, so that it isgenerally accepted as an indicator of tumor prognosis. Amongthe protein/receptors sustaining angiogenesis, the αVβ3 integrinplays a prominent role, being overexpressed on activatedendothelial cells, new-born vessels and other tumor cells, whileits low expression in most adult epithelial cells makes it asuitable target for antiangiogenic therapy. Moreover, growingevidences have emerged for the existence of a strict crosstalk ofsignaling pathways between integrins and growth factorreceptors, which is crucial in the insurgence of malignanttransformations as well as cancer progression and tumorresistance. In particular, αVβ3 integrin and tyrosine kinase VEGFRreceptors are known to be spatially and functionally connectedin endothelial and cancer cell types, and interfering with theirintertwined pathways has been the aim of diverse antiangio-genic dual targeted therapies.[29] In this research context,Gennari and collaborators[30] proposed a dual-action covalentconjugate based on the αVβ3 integrin-targeting diketopiper-azine (DKP) ligand and a decapentapeptide known as a VEGFRantagonist (1, Scheme 1). This choice was prompted, on onehand, by the proven ability of peptidomimetic c(DKP-RGD) tobind the αVβ3 integrin with high affinity and to inhibit tubuleformation in HUVEC cells,[31] on the other hand, by thepossibility to target the extracellular segment of the VEGFreceptors by means of peptide or peptidomimetic inhibitorsdistinct from current small-molecule antiangiogenictherapeutics.[32,33] The authors adopted a convergent approachto assemble pseudopeptide 1, by exploiting a click reactionbetween the azido-terminating DKP unit and the α-helical-shaped decapentapeptide[33] possessing an alkyne handle. Theresulting robust 39-atom-long PEGylated spacer was ad hoc-designed to commensurate the conjugate with proper length

Figure 2. General scheme of “noncleavable” vs “ad hoc-cleavable” RGD-PDCs.




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for simultaneous dual action toward the neighboring targetedreceptors in the extracellular space.[29] Circular dichroism studiesin water solution confirmed that both the RGD and VEGFRligand portions within 1 retain their biologically active con-formations. Conjugate 1 was tested in vitro for its ability to bindto both isolated αVβ3 integrin and VEGFR extracellular domain;it showed good affinity towards both receptor targets, althoughwith potency 10–20 times lower than the unconjugated peptideligands (IC50 αVβ3 97.5 nM vs 4.5 nM), possibly due to undesiredsteric interference between the units. Also, conjugate 1 retainedthe ability to inhibit the angiogenic activity in VEGF-stimulatedmorphogenesis assays on HUVEC cells, which resulted as strongas that displayed by the individual peptidomimetic compo-nents. This last finding was interpreted by the authors as the

probable result of a cooperative action of the two covalentlylinked binding peptides, although the lack of a real advantagein the antiangiogenic assays with respect to the independentligands posed questions about possible re-design of theconjugate with no interference between the active units.To address the same intricate crosstalk between VEGFR2

and αVβ3 receptors, crucial for angiogenesis regulation, Zanardiand colleagues proposed a conceptually distinct approachwhere a selective αVβ3 integrin binder was covalently linked tothe antiangiogenic agent sunitinib, a clinically approved smallmolecule tyrosine kinase inhibitor interacting with the cytoplas-mic domain of VEGFR2 (Scheme 2).[34] The 4-aminoproline-basedcyclotetrapeptide c(AmpRGD) was exploited as targeting unit(Scheme 2), which proved to possess selective and remarkableαVβ3 integrin binding affinity in both cell-free and in-cellassays.[35–37] The authors planned and realized the covalentassemblage of an anchorable sunitinib analogue (“sun” portionin light blue, Scheme 2) with the Amp-based RGD peptide bymeans of three distinct linkers, that resulted in dual conjugates2 and 3, carrying a single copy of the pseudopeptide targetingunit, and compound 4, featuring a dimeric RGD presentation.The ad hoc-synthesized linkers were different in length andtype (11–22 bonds, secondary amide vs tertiary amines,saturated carbon vs PEGylated chains) and connected the activeunits via robust triazole/ether/amide linkages. The authorsintention was to exploit the c(AmpRGD) ligand ability toselectively target αVβ3 integrin-overexpressing cells, to enhanceintegrin-mediated cell internalization of the construct, andpossibly interfere with the crosstalk between αVβ3 integrin andVEGFR, by addressing the sunitinib moiety at the respectiveintracellular kinase targets. The designed modular synthesis ofthe three conjugates entailed preparation of the c(AmpRGD)-N3cyclopeptide by solid phase peptide synthesis (SPPS) followedby in-solution cyclization, as well as the synthesis of thesunitinib analogue ready for conjugation to the three differentalkyne-terminating linkers. In all cases, the final step of the

Scheme 1. The DKP-based dual conjugate targeting both αVβ3 integrin andVEGF receptors.

Scheme 2. Molecular structures of c(AmpRGD)-sunitinib dual conjugates.




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synthesis consisted of a copper-catalyzed azide-alkyne clickcycloaddition reaction. To fully characterize conjugates 2–4 andverify their efficiency in impairing angiogenesis in comparisonto the individual components, they were subjected to a batteryof analytical and biological assays entailing evaluation ofplasma stability, cellular uptake in endothelial progenitor cells(EPC), binding affinity and selectivity towards αVβ3 integrin andVEFG/PDGF isolated receptors, inhibition of EPC adhesion tovitronectin, cell proliferation and viability, blocking of tyrosinekinase activity and tubule formation in vitro and in vivo. Theoverall data pointed out that, in all cases, conjugation of thetargeting peptide with the appended drug did not affect theligand binding competence toward the respective receptors;moreover, the kinase inhibitory activity of the constructsremained comparable to that of sunitinib alone. In particular,dimeric conjugate 4 best summarized the structural character-istics, enabling i) the RGD and sunitinib modules to play activeand synergistic roles in the selective targeting of αVβ3-expressing cells, ii) the αVβ3-mediated internalization/accumu-lation of the uncut construct in endothelial cells, and iii) theremarkable antiangiogenic effect both in vitro and in murinemodels implanted with Matrigel plugs, which proved higherthan the independent modules.Later on, the same set of covalent conjugates (compounds

2–4, Scheme 2) was assayed in human melanoma cells and inmelanoma tumor-bearing mice to demonstrate the superiorpotential of these robust covalent constructs with respect tothe combined therapy (i. e. the simultaneous administration ofthe free integrin ligand and sunitinib) in promoting selectivedrug accumulation inside melanoma cells by integrin receptor-mediated endocytosis, as well as in modulating key intracellulartargets and pathways with dosages lower than individual drugregimens.[38] To this end, the authors succeeded in characteriz-ing the dual molecular platforms in a preclinical model ofadvanced human melanoma and, in particular, they demon-strated that the dimeric conjugate 4 was selectively internalizedby human melanoma cells through αVβ3 receptor-mediatedendocytosis. The better internalization performance of dimericconjugate 4 in comparison to monomeric compounds 2 and 3was probably due to its enhanced affinity toward the integrinreceptor and favorable protonation state. Also, this dimericplatform resulted more effective than sunitinib in reducingmelanoma cell proliferation, migration and invasion in vitro,and it was able to drastically reduce growth of melanomaxenografts in tumor-bearing mice, more efficiently than anequimolar dose of sunitinib alone. It is worth noticing thatdimeric compound 4 was proven to undergo only partial cellinternalization and this behavior was judged to be beneficial.The authors speculated that a possible synergy action could beoperative, involving both the extracellular RGD-integrin inter-action (provided by the non-internalized fraction), and thesunitinib-VEGFR2 kinase interaction (provided by the amount ofinternalized compound). Compounds 2 and 3, though structur-ally similar, showed a very different cell uptake profile andoverall biological activity, highlighting how the structure of thelinker could deeply influence the biological properties of theresulting conjugates. Therefore, the subtle differences in the

physico-chemical characteristics of conjugates 2–4 were sup-posed to be crucial for the observed biological behavior, whichalso widely differed from the small-molecule sunitinib. Theproven localization of these conjugates in the cytosol, forexample, was opposed to the compartmentalization of sunitinibin lysosomes of melanoma cells, suggesting a possible mecha-nism for overcoming drug resistance that represents a criticalpoint in conventional sunitinib-based treatments.The efficacy of these c(AmpRGD)-based covalent conjugates

as targeted drug-delivery platforms was definitely confirmed ina study exploiting ovarian carcinoma preclinical models.[39] Thisparticularly lethal cancer is characterized by pro-invasivebehavior and aggressive features in terms of drug-resistanceinsurgence, which limit the efficacy of platinum-based thera-pies. Compounds 2–4 (Scheme 2) were investigated in bothcisplatin-sensitive and cisplatin-resistant ovarian carcinomacells, that are known to express both the VEGFR2 and αVβ3receptors, though at different levels. The results demonstratedthe ability of Amp-based conjugates to inhibit the growth andmigration of cisplatin-sensitive ovarian carcinoma cells at lowmicromolar concentrations and that, again, compound 4 wasmarkedly more potent than sunitinib in reducing migratorypotential of Pt-resistant strains. Interestingly, a decreasedaggressiveness in tumor cell population was observed underchronic treatment with conjugates 2 and 4 as compared tosunitinib alone. The in vivo targeting ability and tumor inhib-ition of these constructs was tested by experiments inxenografted murine models, revealing that the growth ofresistant carcinoma cells implanted in nude mice was signifi-cantly reduced by dual conjugate 4 even at low dosages. Thesedata definitely confirmed the hypothesis that the integrin-recognizing cyclopeptide and the sunitinib moiety act synerg-istically as antiangiogenic and antitumor agents in ovariancarcinoma and other cisplatin resistant cancers, thus openingthe way for the use of these selective conjugates as drugs ableto overcome the TKI-related tumor resistance.Recently, Dockendorff and colleagues, based on preliminary

evidences about the possible synergistic role played by αVβ3integrin and PAR2 (Proteinase Activated Receptor 2) receptorsin mediating anti-inflammatory signals in endothelial cells,proposed a series of dual targeting ligands embodyingmodified αVβ3 integrin binders and known PAR2 agonistsconnected via PEG spacers of variable length (Scheme 3).[26] Acyclopeptide analogue of cilengitide – alias c[RGDf(NMe)V], aknown αVβ3 integrin binder – containing a propargyl glycine inplace of the valine unit, and a linear RGD-mimetic terminatingwith an alkyne group were selected as integrin ligands. Amongthe several reported PAR2 peptide agonists, the authorsselected the peptidomimetic AY77 reported by Fairlie[40] andthe 2-furoyl hexapeptide described by McGuire,[41] which weresuitably modified with robust amino-PEG spacers terminatingwith azido functionalities for practical azide-alkyne cycloaddi-tions. The authors described the synthesis of three differentconstructs embodying the AY77 dipeptide and the cycloRGD-mimic with variable PEG spacers (5a–c, n=8, 10, 23), threecongeners containing the McGuire hexapeptide and the cRGD-mimic (6a–c), and one candidate presenting the linear RGD-




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binder and AY77 (5d). Initially, selected conjugates (5c, 6c, 5d)were tested for their ability to impair HUVEC binding tovitronectin/fibronectin and the results confirmed that the αVβ3integrin binding affinity was preserved and the covalentconjugation with bulky substituents was well tolerated, partic-ularly in case of the cilengitide analogues. Then, the ability ofdual targeting ligands to activate PAR2 by measuring calciummobilization in adherent EA.hy926 and HUVEC endothelial cellswas assayed; ligands containing the McGuire peptide resultedslightly more potent than their unconjugated reference com-pound, while no significant differences in concentration-responses were observed for the remaining conjugates com-pared to the individual components. This behavior wouldsupport the hypothesis of the presence of the αVβ3-PAR2complex, or at least that the two receptors lie in close spatialproximity. However, since no significant increase in potencywas detected even in the presence of inflammatory agents, theauthors concluded that further investigations in suitable cellularmodels of inflammation were needed to definitely proveconcurrent binding of these dual conjugates to both receptors.The development of bioactive metallodrugs with dual

cytotoxic and antiangiogenic properties holds promise as analternative to classical cisplatin-based drugs in chemotherapy.To this aim, Marchán, Ruiz and colleagues[42] proposed thesynthesis and biological evaluation of a robust covalentconjugate (compound 7, Scheme 4) embodying the cilengitideanalogue c(RGDfK) and the cyclometalated Pt(II)-DMBA com-plex, which had previously demonstrated to display antiangio-genic and antitumor activities at sub-micromolarconcentrations.[43] Unfortunately, cytotoxicity studies in vitrorevealed that conjugation to the RGD peptide resulted in adramatic decrease of antiproliferative activity with respect tothe unconjugated Pt-DMBA complex reference, while theantiangiogenic activity was retained at sub-cytotoxic concen-trations and was similar to that of reference cyclopeptide

c(RGDfK) alone. Based on this evidence, the authors concludedthat peptide conjugation seemed to deplete the antiprolifer-ative efficacy of the organoplatinum moiety, without affectingits ability to interfere with the formation of capillary-likestructures.Another example of cyclometalated Pt(II) complex cova-

lently linked to an RGD cyclopeptide vector was proposed bySarli and colleagues[44] under the shape of a targeted conjugateplatform for photodynamic therapy and concomitant monitor-ing of drug delivery. Indeed, apart from the considerableinterest raised as anticancer agents, Pt-based complexes displayinteresting luminescence properties per se, which may beexploited in bioimaging and/or theranostic applications. There-fore, the authors selected the [Pt(II)-N,C,N]-1,3-di(2-pyridyl)-

Scheme 3. Dual ligands targeting αVβ3 and PAR2 receptors.

Scheme 4. Tumor-targeting Pt(II)-based RGD-conjugates.




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benzene complex as the photosensitizer unit, which presentsfavorable photophysical and antiproliferative characteristics,[45,46]

and directly linked this complex to the c(RGDyK) peptidethrough a stable amide bond (compound 8, Scheme 4).Conjugate 8 was tested against various cancer cell linesexpressing αVβ3 integrin to variable extent and revealed amodest cytostatic effect (required growth inhibition concen-trations >100 μM) but promising photophysical properties. Theconjugate also demonstrated rapid cell uptake by integrin-mediated endocytosis and significant reduction of survival ofrat bladder cancer cells upon incubation prior to light exposure,thus indicating the crucial contribution of c(RGDyK) to theanticancer action of the construct. Overall, the authors demon-strated the efficacy of this conjugate that, while remainingnoncleaved, is able to generate the expected ROS species.

3. Dual conjugates embedding cleavablelinkers

This chapter collects and documents examples of RGD-PDCswhere the attached cargo (drug) becomes therapeutically usefulonly when the chemical bridge (linker/spacer) with thetargeting unit is cleaved (Figure 2, eq. 2). Recently, innovativestrategies have been proposed for assembling prodrug con-structs robust enough to allow accumulation at the diseasedsite without premature leakage of the payload into the blood-stream, while conceived for programmed delivery uponendogenous or external stimuli.[10,47]

An important feature of ad hoc-cleavable RGD-PDCs is thatthey should be readily disassembled in close proximity to thetarget cells or upon internalization, resulting in the release ofthe cytotoxic drug. Several linkers (both peptidic and non-peptidic) have been developed to release payloads in thepresence of specific hallmarks of the pathological tissues, forinstance acidic pH, enzyme overexpression, hypoxic andreducing conditions.Many RGD-PDCs hereby presented possess linkers that

release the active moieties under acidic conditions. The low pHfeaturing tumor extracellular medium depends on the inad-equate oxygen supply to tumor tissues prompting fast-replicat-ing cells to produce lactate and pyruvate through the glycolyticpathway. On the other hand, endosomal and lysosomalcompartments are responsible for intracellular acid-promotedhydrolysis of drug delivery conjugate systems. On this premise,ideal linkers should be rapidly hydrolyzed after accumulation atthe acidic diseased site, whilst stability of the constructs atneutral pH should devoid hydrolysis during blood circulationand consequent drug release in healthy tissues. Recentadvances in the field of acid-labile linkers point to maximize thedifference of linker cleavage rates at neutral vs acidic pH.Among such selectively hydrolyzable bonds, sterically hinderedesters, carbamates, Mannich bases, hydrazides and (acyl)hydrazones are included, which are sensitive to acidic con-ditions, while “simple” hydrazones, that have been extensively

employed in the development of first-generation ADCs, havefound limited applications in SMDC and RGD-PDC technology.The tumor environment is also characterized by increased

production of reducing species including cysteine side chains,reduced glutathione, thioredoxin and other antioxidant species.These conditions have inspired the design of linkers sensitive toreducing stimuli such as disulphide bonds and Pt(IV) complexes.Specific peptide sequences, featuring robust amide bonds, canbe recognized and selectively cleaved by intracellular proteo-lytic enzymes associated to the pathology phenotype, such asintracellular endosomal/lysosomal enzymes cathepsins, cas-pases, legumain, or membrane-expressed metalloproteinases(MMPs), elastase, plasmin. In addition to this widely investigateddomain, the exploitation of carbohydrate linkers as glycosidase-responsive triggers of drug release has been described.[48] Anumber of targeted covalent conjugates are hereby disclosedembodying specific peptide sequences and carbohydrate-basedlinkers which are privileged substrates of enzymes overex-pressed in inflammatory, angiogenic and tumor tissues.To complete the picture, particular ancillary units called

“self-immolative spacers” are often constitutive elements ofRDG-PDCs (and in general SMDCs) (Figure 3). They are chemicalmoieties, flanking the cleavable linker, which undergo sponta-neous degradation upon linker cleavage and are used toimprove proteolytic efficiency and pharmacokinetic properties,as well as lower steric hindrance between the active units. Afterbreakage, the spacer undergoes spontaneous eliminationthrough cyclizations or electronic cascades over conjugated π-systems, ultimately releasing the free drug.Similarly to noncleavable RGD-PDCs, cleavable constructs

should possess some general features for optimal feasibility,including easy synthetic accessibility of the single modules,practical and versatile ligation chemistry, and minimal changesof the parent structure required, with the aim of preserving theefficacy of the original drug and the receptor binding ability.All in all, the success or failure of RGD-PDCs depends upon

the selection of the right linker/endogenous stimuli combina-tion, which crucially depends on the expression level andlocalization of the enzyme of choice, features that in turn relyon the disease/tumor type and progression grade.On the bases of encouraging preliminary results in previous

works,[50] in 2015 Manzoni and colleagues synthesized acollection of covalent conjugates (12a, b, and 14, Scheme 5),[51]

where a robust network of amide and/or triazolyl bridgesprovided the connection among the multiple aza-bicycloalkane-RGD cyclopeptide units [c(AbaRGD)], while an hydrolyzableester bond ensured conjugation of the RGD core to the highlycytotoxic microtubule-interfering agent paclitaxel (PTX). Thec(AbaRGD) unit was demonstrated to serve as a specific αVβ3(and αVβ5) integrin ligand with nanomolar binding affinity, so itwas selected as the targeting unit.In the field of integrin recognizing ligands, the development

of multimeric platforms able to simultaneously establish manyinteractions with clustered receptors has long been pursued toimprove targeting performance in both diagnosis andtherapy.[52–54] In this work, the exploitation of multimeric RGDpresentations (as represented by dimeric compounds 12a and




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12b, and tetrameric compound 14) was explored by theauthors with the aim of improving the tumor targetingcapability, and hence the antitumor efficacy. The cleversynthetic approach for the construction of these multimericsystems is based on glutamic acid dendrons: one or moreglutamic acid units constitute the inert branching core (com-pounds 9, 11, 13, Scheme 5) onto which multiple copies of thepeptide ligand and the active unit were grafted. The conjuga-tion steps relied on simple and efficient peptide couplings andcopper-catalyzed alkyne-azide 1,3-dipolar cycloadditions. Thebinding affinity for isolated integrin receptors was determinedin vitro and the tetramer 14 resulted 100-fold more potent thanthe monomeric counterpart, thus demonstrating that multipleinteractions enhanced the binding strength. The antiprolifer-ative activity was tested in vitro towards different tumor celllines and all the conjugates showed remarkable cytotoxicactivity in the nanomolar range. Among these, the dimericconjugate 12b displayed an antitumor activity similar to that ofPTX, inhibiting the tumor growth of the cisplatin-resistantIGROV-1/Pt1 ovarian carcinoma in xenograft murine models atdoses lower than the free PTX. The authors concluded that themultipresentation of the RGD peptide positively influenced theαVβ3 targeting capability, and a cytotoxic effect similar to PTXcould be obtained, with a much more favorable toxicity profile.However, the stability profile of the constructs, as well as theissue of possible premature drug release remained to beinvestigated.Along a similar strategy, Zanda and colleagues[55] proposed

the synthesis and in vitro evaluation of hybrid compound 20

(Scheme 6), where PTX was conjugated to a nonpeptidic RGDmimic ligand (depicted in red, Scheme 6) through robust oximeligation. The triazole-based peptidomimetic 15, featuring nano-molar affinity toward the isolated αVβ3 integrin receptor,

[56] wasselected as the αVβ3-targeting ligand, and was anchored to theremaining conjugate at its triazole core, based on molecularmodeling studies that revealed its suitability for functionaliza-tion without presumably impacting on the integrin recognitioncapability. The authors adopted a convergent approach toaccess conjugate 20, based on the assembly of three buildingblocks: 1) aldehyde 17, which was obtained in two stepsstarting from the iodinated analogue of ligand 15 (compound16, Scheme 6) through a Sonogashira cross coupling to installthe pentynol handle followed by oxidation; 2) the bifunctionallinker 18, obtained in four synthetic steps from 1-bromo-4-chlorobutane; and 3) the NHS-activated PTX-succinyl ester 19,featuring, again, a hydrolyzable ester bond linking the PTXmoiety.The in vitro evaluation of conjugate 20 demonstrated that

the introduction of the bulky appendage to the integrin ligandportion produced a slight decrease of binding affinity towardsimmobilized αVβ3 integrin in comparison to unconjugatedligand 15 (200 nM vs 25 nM). The targeting capability of theconjugate towards αVβ3-positive U87MG cancer cell line vis-à-visαVβ3-negative MCF7 cells was promising, even though thecytotoxic activity of 20 was lower than PTX. The authorsconcluded that the remarkable stability of the conjugate, asassessed by microsome assays, could be the reason for theconsiderable decrease of the antiproliferative activity, indicating

Figure 3. Popular self-immolative spacers flanking linkers sensitive to enzymatic/reducing stimuli. The p-aminobenzyloxycarbonyl (PABC) scaffold is one of themost used and versatile aromatic structure: when disconnected from the linker upon cleavage, the free aniline moiety of PABC rapidly undergoes 1,6-elimination, affording an azaquinone methide metabolite, carbonic anhydride, and releasing the free drug (a); alternatively, after 1,6-eliminative sacrifice ofthe p-aminobenzyl scaffold, an intramolecular nucleophilic attack of the secondary amine forms a cyclic urea (b); the reduction of disulfide linker releases thethiol moiety that undergoes intramolecular ring closing onto a carbonate (or carbamate) unit liberating a cyclic thiolactone and the free drug (c). Veryrecently, a novel (S)-2-(aminomethyl)pyrrolidine self-immolative spacer (not shown) has been reported, which is able to release different types of drugspossessing either phenolic or secondary/tertiary hydroxyl groups through a fast cyclization mechanism involving carbamate cleavage.[49]




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that the taxane moiety is not efficiently released after cellinternalization, despite the presence of a labile ester bond.Glioblastoma multiforme (GBM) is one of the most aggres-

sive brain tumors, and its metastasizing nature renders localtreatments (such as radiotherapy or surgery) non-resolutive.Thus, post-surgical treatments are essential and, in this field,targeted drug delivery as well as combined administration ofmultiple chemotherapeutics have given promising results. Inline with this evidence, and prompted by previous encouragingresults,[57] Chen, Wu et al.[58] planned to build a single covalentconjugate comprising the αVβ3 integrin-targeting c(RGDyK) andthe known ERK pathway-inhibitor PD0325901, connected eachother through robust amide/ether bonds and cleavable esterlinkage (compounds 21a–c, Scheme 7). Conjugates 21a–c,having linkers of distinct nature, were synthesized, character-ized and assayed both in vitro against αVβ3 integrin-over-expressing U87MG cell line, and in vivo in U87MG-xenograftedmice model. PEGylated conjugate 21b turned out to performbetter than the succinyl- and polyglycyl- counterparts 21a and21c. The behavior of compound 21b was also compared tothat observed for the combination of the individual compo-nents PD0325901 and RGD cyclopeptide. Both strategies (theconjugate and the combination) resulted efficient in inhibiting

cancer cell migration and proliferation in vitro and showedsignificant inhibition of tumor growth in vivo due to synergisticeffect arising from the simultaneous blocking of ERK pathwayactivity and αVβ3-dependent cell migration. The authors con-cluded that, for equal performance in the antiproliferativeactivity, the conjugated platform could have an added value byvirtue of its cell-selective delivery and accumulation at thetumor site allowing dose reduction of each drug; last but notleast, the conjugate would inherently overcome the problemsassociated to the distinct pharmacokinetic properties of theindependently administered drugs. This is an interestingexample where the double nature of the RGD portion as bothtargeting unit and antiproliferative agent is shown, which actsin synergy with the attached cytotoxic drug; the precise timingand location of the drug release has however to be demon-strated.In 2016, Gellerman and collaborators described the syn-

thesis of three new RGD-based conjugates (24a, b and 25,Scheme 8) differing each other in the nature of chemicalbridges between the cyclopeptide core and the cytotoxic unit –selected as either chlorambucil (CLB) or camptothecin (CPT) –with the aim to compare their drug release profiles.[59] Thesynthesis of linear RGD peptides of type 22 (Scheme 8) was

Scheme 5. Synthesis of multimeric c(AbaRGD)-paclitaxel conjugates 12a, 12b and 14.




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realized using standard SPPS, followed by cyclization tocompounds 23a, b. The functionalization of hydroxy- or amine-terminating side-chains with the drug moiety was accomplishedon-resin, affording conjugates 24a, b and 25 after removal fromthe resin (Scheme 8). In conjugate 24a, the known αVβ3integrin-targeting peptide c(RGDfK) was connected to CLB (aDNA alkylating agent) by an amide bond, whereas the linkage

to the CPT unit (a topoisomerase 1 inhibitor) within conjugate25 entailed the presence of a carbamate bridge. To explore theproperties of an ester functional group, the lysine residue withinthe cyclopeptide was replaced by a serine in the construction ofc(RGDfS)-CLB conjugate 24b. The metabolic stability of allconjugates was tested in mice liver homogenates indicating, asexpected, that the ester and the carbamate ligations werebiodegraded more rapidly than the amide. The chemicalstability test performed at both physiological pH 7.4 and tumorenvironment acidic pH 5.2 showed that both amide and ester-linked CLB-derivatives were rapidly decomposed without anysignificant release of the intact free drug, while carbamate-linked CPT conjugate 25 was relatively stable at physiologicalpH and capable to slowly release the free drug at the same pH.The in vitro cytotoxicity displayed by conjugate 25 in αVβ3integrin-expressing cancer cells (human NSLC, murine melano-ma) was higher than that of free CTP at doses >100 μM andwas consistent with the supposed integrin-mediated drugtargeting and accumulation. Based on the evidence reported, itis arguable that the conjugation of CPT (and other chemo-therapeutics with poor bioavailability) with RGD peptides via acarbamate unit can be useful in overcoming drug resistance orreducing the off-target toxicities related to high-dosage regi-mens, provided that the premature release and consequentaspecific cytotoxic effect can be excluded at preclinical level.In another contribution, Gellerman and colleagues planned

and put in practice an efficient and general synthetic approachto the preparation of RGD-based drug conjugates (and someNGR congeners) (not shown).[60] The synthesis of a number of

Scheme 6. Synthesis of the RGD peptidomimetic-paclitaxel conjugate 20.

Scheme 7. Structure of RGD-PDCs of type 21 embodying the MEK1/2inhibitor PD0325901.




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Nα-protected drug-loaded amino acid building blocks wasaccomplished by functionalization at the remote ω-site ofamino acids – lysine, serine, tyrosine, threonine, aspartate andglutamate – with anticancer active ingredients such as chlor-ambucil, camptothecin, deacetylcolchicine, among others. FourRGD-cyclopeptides bearing different amino acid-drug unitswere obtained by practical and efficient SPPS, evidencing theirutility in fast parallel synthesis of collections of peptide-drugconjugates for rapid assessment of drug release profiles. Amongthese compounds, the c(RGDfK)-CPT conjugate (coinciding withcompound 25 in Scheme 8) was selected for preliminaryevaluation in vitro, showing selective, although moderate,cytotoxicity against αVβ3-expressing cancer cell lines, consistentwith previous results (vide supra).Going on in this research field, the same authors proposed

the synthesis of four additional conjugates based on thecyclopeptide core c(RGDfK) and loaded with two cytotoxic unitseach (compounds 27a–d, Scheme 8).[61] To this end, the on-resin cyclopeptide 23a was coupled to a “splitter” amino acid –either lysine or serine – that doubled the anchoring sites andenabled the loading of two active agents (same or different)onto a single peptide carrier. Compounds 27a and 27c wereobtained as homodimeric conjugates with CLB-amide and CPT-carbamate junctions, respectively. The use of orthogonallyprotected functionalities within the lysine/serine unit allowedfor the preparation of both homodimer 27b, where CLB wasamide- and ester-linked, and heterodimer 27d, presenting the

two different drugs and two different amide/carbamate chem-ical bridges. The cytotoxic activity of these conjugates wastested against melanoma and non-small lung cancer cell linesand all the evidences pointed out the higher potency of dual-loaded conjugates with respect to the previous monomericanalogues. Molecular dynamic simulations on both monomericconjugate 25 and heterodimer 27d confirmed that theconformational arrangement of the cyclopeptide core was notaffected by the bulky conjugated appendages and wassubstantially similar to that of the bioactive reference cilengitide[c(RGDf(NMe)V], suggesting that the cyclopeptide core fulfillsthe major requirement for targeted drug delivery in allconjugates. According to these results, the authors hypothe-sized that the CLB activity could be restored in CLB-resistantleukemic cells upon its conjugation to the peptide carrier andthat the conjugation of two drugs with different modes ofaction and different release kinetics could enhance theprobability of tumor eradication.Among the platinum-based compounds used in tumor

therapy, cisplatin is one of the most widely used agent, thoughits clinical use is limited due to severe side effects. Moreover,the poor selectivity for cancer cells and the inherent or acquiredresistance to Pt(II) metallo-drugs have limited the scope of theapplication of such cytotoxic drugs. One of the possiblestrategies to address these limitations is the administration ofnon-toxic octahedral Pt(IV) prodrugs, whose intracellular reduc-tion by glutathione or ascorbate produces active square-planar

Scheme 8. On-resin synthetic approach for the preparation of RGD-PDCs 25a–c and 27a–d.




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Pt(II) complexes and restores their latent cytotoxic activity. Inaddition, these platinum prodrugs provide the possibility ofconjugating targeting carriers to increase their selectivitytoward cancer cells. To this purpose, Marchán and colleagues[62]

exploited a Pt(IV) derivative of picoplatin (cpx-1, Scheme 9) toinstall one or more c(RGDfK) cyclopeptides as targeting units atthe detachable axial position of the complex. The authorsaimed to explore the merits of a multimeric presentation of theRGD carrier (compound 30, Scheme 9) in comparison with themonomeric counterpart (compound 28). Derivative 28 wasprepared starting from Pt(IV) picoplatin (cpx-1), which wasfunctionalized at the axial position with a succinyl moiety, andamide-bonded to the RGD cyclopeptide by means of a PEGspacer. On the other hand, tetrameric conjugate 30 wasobtained by exploiting a practical click reaction to install fourcopies of the RGD carrier on the known cyclodecapeptidetemplate RAFT.[63] Conjugates 28 and 30 were evaluated in vitrotoward various human cancer cell lines, either positive ornegative in αVβ3/αVβ5 integrin expression. These experimentsevidenced that the intact Pt-peptide conjugates were internal-ized and accumulated selectively in the integrin-expressingcancer cells (where they are expected to be activated by Pt(IV)-to-Pt(II) reduction), with higher internalization properties dis-played by the RAFT-based conjugate 30 as compared to themonovalent counterpart. Concerning the antiproliferative activ-ity, a superior potency displayed by compound 30 in integrin-expressing cell lines was observed with respect to picoplatin,and efficacy was correlated with both integrin expression levelsand number of RGD ligand copies. Quite surprisingly, the

antiproliferative effects displayed by either the Pt(IV)-conju-gated compound 30 or the platinum-free unconjugated RGD-RAFT analogue were similar, confirming that the interactionwith integrin receptors causing morphological changes and celldetachment was crucial for the proapoptotic activity of thismultimeric conjugate. The authors, however, did not clarify therole actually played by the Pt(IV) moiety in these conjugates.A similar Pt(IV) complex was developed by the same

researchers[64] as photoactivatable tumor targeting prodrug(compound 29, Scheme 9). The inert and non-toxic Pt(IV)complex cpx-2 was conjugated to the known αVβ3/αVβ5targeting c(RGDfK) peptide via a succinyl-PEG linker. It is wellknown that irradiation of such photoactivatable complexes withvisible light triggers the release of cytotoxic Pt(II) species, whichefficiently bind to various biological targets (DNA, RNA,proteins), generating adducts distinct from those generated bycisplatin, thus expanding the applicability of these conjugatesto cisplatin-resistant tumors. The authors evaluated the effi-ciency of photoactivation in the presence of 5’-GMP (guano-sine-monophosphate) as a model of DNA-capturing of Pt(II)species, and the phototoxicity was tested in various cancer celllines. These assays confirmed that the RGD peptide providedthe conjugate 29 with remarkable selectivity towards cellsoverexpressing αVβ3/αVβ5 integrins. The phototoxicity of 29 invarious tumor cell lines was in good agreement with theintegrin expression levels, and the peptide conjugation hadpositive effect on intracellular metal accumulation, whichresulted higher than that of unconjugated control complexes,thus excluding the occurrence of premature Pt-activation or

Scheme 9. Pt-based RGD conjugates targeting αVβ3/αVβ5 integrin-expressing cancer cells.




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hydrolysis of the conjugate. Experimental evidence seemed tosuggest indirectly that the drug is released after αVβ3/αVβ5-mediated cell internalization and that it could exert cytotoxicactivity upon visible light irradiation, although definitiveconclusions on this issue have not been provided by theauthors.Reducible metal complexes have also been exploited by Liu

and colleagues in the preparation of multipurposing RGD-Pt(IV)-prodrugs for photodynamic therapy against cisplatin-resistantcancer cells (compound 31, Scheme 9).[65] Indeed, photodynam-ic therapy exploits photosensitizer (PS) moieties to generatereactive oxygen species (ROS) upon light irradiation that aretoxic to fast-replicating tumor cells. Fluorogens with Aggrega-tion-Induced Emission characteristics (AIE) have attracted greatinterest in biosensing and bioimaging applications, since thesemolecules are non-emissive in a dissolved state, but therestriction of free rotation in the aggregated state induces themto emit bright fluorescence. In this contribution, the authorsreported the synthesis of molecular device 31, a theranosticplatform[66] designed for real-time monitoring of drug activationand simultaneous photodynamic-chemotherapy, where the Pt-(IV)-prodrug was axially linked to both a photosensitizer unitpossessing AIE properties and to a RGD-targeting ligand. Theobtained prodrug was non-emissive in aqueous medium due tothe presence of a poly-aspartate/cyclopeptide appendage, butit was lighted up upon reduction by intracellular GSH.Conjugate 31 was internalized by αVβ3 integrin-expressing cellsand the Pt(IV) reduction led to cisplatin release and emission offluorescence useful for image-guided photodynamic therapy.The antiproliferative effect of conjugate 31 was evaluated in

αVβ3 integrin-positive melanoma cells (MDA-MB-231) upon lightirradiation and resulted in enhanced cell growth inhibition. Theauthors concluded that the intracellular reducing conditionseffectively released the Pt(II)-complex and that construct 31held great promise for combined chemo- and photodynamictreatments to ablate cisplatin-resistant tumor cells.In 2017, Becker, Heffeter and colleagues developed a panel

of targeted multifunctional platforms of type 32 (Scheme 10)embodying two copies of integrin-directed RGD peptide and aPt(IV)-based prodrug connected through a flexible Y-shapedlinker, with the aim to demonstrate the potential of thesemodular assemblies for precise drug delivery in cancertherapy.[67] The modular synthetic approach was designed to setthe stage for screening and rapid optimization of theconjugates, and the use of bioorthogonal functionalities wasenvisaged to maximize the structural variability of affordableplatforms. As a proof of concept, the authors selected RGD-peptide 35 as the αVβ6 integrin-targeting unit, being thisintegrin upregulated in most cancers of epithelial origin andexpressed at low levels in healthy tissues. Peptide 35 wasdecorated with an azido group for the copper-catalyzed clickligation to the PEGylated peptide Y-linker 33, which in turnpossesses a terminal cysteine residue designed to take part inthe thiol-maleimide ligation with the carbamate-linked oxalipla-tin derivative 34 (Scheme 10). The inert PEG27 within the spacerwas introduced to confer hydrophilicity and flexibility to theconjugate, while the dimeric presentation was designed toincrease affinity toward αVβ6 integrin-expressing cells bysimultaneous binding to multiple receptors. In addition, afluorescent label such as biotin (or Cy5, not shown) was

Scheme 10. Schematic representation of modular αVβ6 integrin-targeting Pt(IV)-based conjugate 32.




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introduced, to monitor the cell localization of the conjugate.Both the integrin-targeting peptide 35 and the Y-peptidescaffold 33 were prepared by conventional SPPS and thensubjected to high-yielding and sequential chemoselectiveligations namely, the copper-catalyzed click reaction and thethia-Michael addition reaction with maleimide-bearing Pt-complex 34, ultimately providing the multifunctional conjugate32. A dimeric biotinylated analogue of compound 32 (withoutPt-complex) was tested in colon carcinoma cells transfectedwith β6 integrin and showed interesting binding affinity with anapproximate half-maximal concentration in the nanomolarrange. The β6 integrin-promoted internalization of Pt-basedconjugate 32 was selective and rapid in the same transfectedcell line and the consequent metal accumulation (measured byInductively Coupled Plasma Mass Spectrometry, ICP-MS) couldexplain the cytotoxic activity of the multifunctional platform 32.On the other hand, the RGD peptides themselves did notdisplay inhibition of cell growth, thus excluding any possiblesynergistic antitumor effect from the integrin antagonistcomponent. The authors concluded that further studies wouldbe needed to get insights into the rate at which the Pt(IV)moiety is actually reduced to Pt(II). Anyway, the design of thesemodular covalent platforms has the potential to be modifiedand adapted to combine different targeting moieties, activepayloads and labels, to enable distinct disease-specific therapiesand/or imaging tools.In 2017, Gennari and colleagues exploited the DKP-RGD

peptidomimetic in the preparation of a “theranostic” platform(compound 36, Scheme 11) where the anticancer agentcamptothecin (CPT), the RGD ligand and a fluorescent naph-thalimide probe were joined together through a disulfidebridge, besides other amide, carbamate and carbonatelinkages.[68] This type of reducible linker, though common inSMDCs, has been rarely used to build up integrin targetedconjugates, with the only exception given by Kim in 2012.[69]

Conveniently, the S-S linkage can be selectively cleaved byintracellular exchange with free thiols of glutathione (GSH) or

thioredoxin. The RGD-PDC platform 36 displayed in Scheme 11was designed to release free CPT upon reducing cue andsimultaneously produce red-shifted emission of fluorescence,thus allowing real-time monitoring of the conjugate fate.Conjugate 36, along with a noncleavable congener and ac(RGDyK) analogue, were evaluated in U87 human glioblastomacells (αVβ3+) and in a β3-KO clone by both confocal microscopyand immunofluorescence studies, to get insights on theinternalization process. According to the reported results, thefluorescent conjugate 36 is internalized in both cell linesirrespective to the presence of the αVβ3 receptor, probably viaan active uptake mediated by other integrins (e.g. αVβ5). Theoverall cell assays demonstrated that both reduction of the S-Sbond and CPT release could occur in the extracellular mediumand passive diffusion of CPT inside the cell could be responsiblefor the observed antiproliferative activity.In search of new actively targeting bioconjugates embody-

ing cytotoxic drugs connected to αVβ3 integrin ligands, Gennariand colleagues gave a fundamental contribution launching in2015 a line of research based on the exploitation of linkerpeptide sequences specifically recognized by either lysosomalcysteine proteases (cathepsin B) or extracellular proteolyticenzymes (matrix metalloproteases 2 and 9).[70] The authorschose Val-Ala and Phe-Lys dipeptides as cleavable linkers tocovalently connect the diketopiperazine-based (DKP) αVβ3integrin ligand c(DKP-RGD) to the anticancer drug paclitaxel(PTX). The synthesis of conjugates 37 and 38 (Scheme 12) wasaccomplished with the aim to improve the therapeutic profileof PTX by targeted delivery. A third DKP-PTX conjugate with anon-peptide noncleavable linker (featuring a glutaric acid unit,not shown) was also synthesized to be used as a negativecontrol. All the synthesized compounds maintained the abilityto inhibit biotinylated vitronectin binding to the purified αVβ3integrin receptor at nanomolar concentrations and showedgood stability at both pH 7.4 and pH 5.5. Enzymatic digestion ofthe conjugates followed by HPLC-MS analysis revealed efficientPTX release from compounds 37 and 38, whereas the non-cleavable counterpart remained untouched. To demonstratethe integrin targeting effect displayed by the RGD ligand, theantiproliferative activities of conjugates 37 and 38 wereevaluated against two acute lymphoblastic leukemia cell linesexpressing integrin αVβ3 at different levels (the αVβ3-negativeCCRF-CEM and its αVβ3-positive subclone CCRF-CEM), and werecompared with the intrinsic selectivity of unconjugated PTX.The RGD-Val-Ala-PTX conjugate 37 was found quite effective ininhibiting proliferation of antigen-positive cell line versusantigen-negative isogenic cells (Targeting Index, TI=9.0);[71] thetargeting integrin capability of RGD-Phe-Lys-PTX conjugate 38,instead, was more modest (TI=2.1). The complete lack ofactivity displayed by the noncleavable control confirmed thatthe peptide linker plays a crucial role for achieving the selectiverelease of the cytotoxic payload.Later on, the same authors[72] reported the synthesis of

compound 39 (Scheme 12), the first PTX-conjugate based onc(DKP-isoDGR) (not a RGD) ligand designed for selective releasein αVβ3 integrin-expressing cancer cells. The conjugate wassynthesized by connecting the peptidomimetic diketopipera-

Scheme 11. The RGD-camptothecin conjugate 36 presents a fluorescentprobe and a reducible linker for real-time monitoring of the drug delivery.




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zine with paclitaxel via the Val-Ala dipeptide linker and a self-immolative spacer. The novel conjugate displayed a goodaffinity for the purified αVβ3 integrin receptor (IC50=11.0 nM),similar to that displayed by the RGD-congener 37. The tumortargeting ability of compound 39 was assessed in vitro throughantiproliferative assays on two isogenic human glioblastomacell lines with different integrin expression (U87 αVβ3+and U87αVβ3� ). The resulting targeting index (TI=9.9) was notable, ifcompared with the RGD-based congener 37 (TI=2.4).In 2018, Gennari and collaborators[73] expanded the collec-

tion of DKP-based PTX-conjugates by proposing the synthesisof compounds 40 and 41 (Scheme 12), containing the Gly-Phe-Leu-Gly (GFLG) linker (which is sensitive to lysosomal proteases),to be compared with the previously reported c(DKP-RGD)-Val-Ala-PTX conjugates 37, as well as novel counterpart 42,possessing Val-Ala peptide and a PEGylated triazole spacer suchas S2. As for the previous examples, the evaluation of the abilityto bind isolated αVβ3 integrin receptor demonstrated thatconjugation does not erode the high integrin binding affinity ofthe free ligands. However, cell viability assays performed inboth U87 (αVβ3+) and HT29 (αVβ3� ) cell lines showed that allconjugates are less potent than free PTX, with a lower decreaseshown by PEGylated compounds 41 and 42, probably due totheir improved water solubility and conformational flexibility.The best performing conjugate in terms of targeting capabilitywas the c(DKP-RGD)-PEG4-Val-Ala-PTX 42 (TI=533), which wasalso assayed in αVβ3-positive cell line in the presence of excess

of free c(DKP-RGD) peptide ligand. In this competition experi-ment, the marked decrease of cytotoxic activity emphasized theimportance of a possible αVβ3 integrin-mediated internalizationto explain the cytotoxicity of such conjugates.The DKP-RGD cyclopeptide ligands and their isoDGR

analogues were also employed as αVβ3 integrin targeted carriersin the construction of a series of α-amanitin conjugatesdiffering each other in the nature/cleavability of the linker/spacer units, which were chosen among either a six carbon-long ether, a glutarate-Val-Ala, or a triazole-PEG4-Val-Ala moiety(not shown).[74] The α-amanitin is a bicyclic octapeptide actingas a potent inhibitor of cellular DNA transcription, possessingpoor membrane permeability and high liver toxicity that madeit eligible as a valuable cytotoxic payload in the development oftargeted cancer chemotherapeutics. The series of α-amanitin-DKP conjugates synthesized by Gennari, Piarulli and colleagueswas subjected to antiproliferative assays in tumor cell modelswith different levels of integrin expression affording variableresults. Only the c(DKP-isoDGR) conjugate, presenting a gluta-rate-Val-Ala linker, showed a slight increase in potency withrespect to the free toxin, without any apparent correlation withthe αVβ3 expression level in the tested cell lines. In addition,competition experiments carried out in the presence of excesscilengitide could not definitely shed light on the actual roleexerted by the DKP ligand in the cytotoxic activity displayed bythese conjugates. The overall data pointed out that α-amanitin-

Scheme 12. Schematic representation of DKP-based conjugates bearing taxane active units linked by enzymatically cleavable peptide connections. Allconjugates share a p-aminobenzylcarbamate (PABC) self-immolative unit undergoing 1,6-elimination/intramolecular nucleophilic attack to release the taxanedrug.




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DKP conjugates are possibly cell internalized by a processmediated by integrins different from αVβ3.In 2019, the same authors demonstrated, through confocal

microscopy studies on c(DKP-RGD) ligands labeled with the NIR-probe sulfo-cyanine5 (sCy5), that these ligands fairly accumu-lated on the surface of αVβ3 integrin-positive tumor cells andwere poorly internalized, thus explaining the incomplete intra-cellular activation and scarce potency displayed by a number ofDKP-drug conjugates embodying lysosomally cleavable pepti-des (vide supra). Wishing to overcome this problem, Gennariand colleagues decided to take advantage of the leukocyte-secreted enzymes released in the extracellular space inresponse to tumor-associated inflammation to activate theirconjugates by proteolysis.[75] To this aim, they synthesizedcompound 43 (Scheme 12) bearing the DKP ligand connectedto paclitaxel via the Asn-Pro-Val (NPV) tripeptide sequence,which is a substrate of the neutrophil elastase secreted bytumor infiltrating immune cells. Two additional noncleavableconjugates respectively embedding a triazole-PEG4 linker andthe NPv sequence (not shown), were synthesized as negativecontrols. The antiproliferative activity of all the conjugates wasevaluated towards 786-O cell line (human renal carcinoma cells)upon addition of elastase in the medium. Interestingly, theefficient proteolysis of NPV linkage within compound 43resulted in high inhibition of cell proliferation similar to freePTX (IC50 19.6 nM vs IC50 29.5 nM), while the noncleavableconjugates resulted completely inactive.Another important contribution dates back to 2017 and

reports the synthesis and evaluation of a series of multimericRGD-PDCs possessing two, three, or four copies of DKP integrinligands connected to a taxane moiety through an enzyme-sensitive Val-Ala peptide and a PABC self-immolative spacer(Scheme 13).[76]

The multimeric RGD peptidomimetic-paclitaxel platformsand the monomeric counterparts were designed to release PTXintracellularly by lysosome proteolysis of the Val-Ala peptideand 1,6-elimination of PABC spacer. PEG4 units were introducedto improve water solubility and flexibility of the final com-pounds. The effects of ligand multipresentation were assessedby competitive binding assays on isolated αVβ3 integrinreceptor. The best performing multimeric conjugate was thetrimeric compound 44 displayed in Scheme 13 (IC50 1.2 nM),thus confirming that multipresentation of the targeting ligandcan effectively strengthen the ligand-receptor interactions,although this improvement can be partially counterbalanced bythe increasing steric bulk of the platforms.Moving to the use of monomethyl auristatin E and F

(MMAE/F, Scheme 14) as cytotoxic payloads, some diketopiper-azine-based conjugates were prepared by Piarulli and col-leagues by exploiting cathepsin B-sensitive Val-Ala/PABC linkers(compounds 45–48, Scheme 14).[77] MMAE/F are syntheticpseudopeptide analogues of dolastatin 10, a potent antitubulinagent of marine origin. In this study, the authors reported thesynthesis of a series of conjugates possessing either c(DKP-RGD)or c(DKP-isoDGR) ligands as homing moieties, which wereconnected to cytotoxic agents MMAE or MMAF by means of thelysosomally cleavable linker/spacer Val-Ala/PABC (an alternative

noncleavable bridge was used in a control conjugate, notshown). The straightforward synthetic scheme for all com-pounds entailed a versatile copper-catalyzed azide-alkyne cyclo-addition as the key step. As for all the previously mentionedDKP conjugates, the nanomolar binding affinity of compounds45–48 toward the isolated αVβ3 receptor was retained. Unfortu-nately, the cytotoxic activities evaluated in both humanglioblastoma (U87) and melanoma (M21) cells was markedlyreduced as compared to the free drugs, consistent with thealready described scarce integrin-promoted endocytosis andconsequent incomplete drug release. However, compound 47,featuring the isoDGR sequence, resulted to be the bestperforming conjugate in the series, and this was attributed bythe authors to its ability to promote a more evident internal-ization with respect to the RGD counterpart.The same MMAE chemotherapeutic agent was then con-

jugated to the non-internalizing c(DKP-RGD) ligand by using aβ-glucuronidase-sensitive bridge (compound 49, Scheme 14).[78]

The β-glucuronidase enzyme is known to be secreted in tumorextracellular compartment by infiltrating monocytes and neu-trophils or apoptotic/necrotic tumor cells. The novel synthe-

Scheme 13. The multimeric presentation of DKP-RGD-paclitaxel conjugate44 enhances the αVβ3 integrin binding affinity.




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sized platform 49 comprised a β-glucuronic residue for selectiveextracellular release of the payload upon cleavage of theglycosidic bond and 1,6-eliminative sacrifice of PABC scaffold.Conjugate 49, endowed of a PEG4 spacer, resulted efficient asinhibitor of cell proliferation at low-nanomolar concentrationsin integrin-expressing tumor cell models upon addition of theglycosidic enzyme, whilst its congener lacking the PEG unit wasinactive. This indicated that the quite long hydrophilic PEG4moiety is important for the overall conjugate efficiency,conceivably due to minor steric hindrance around the glycosidicbond.Cryptophycins are potent anticancer agents acting as

inhibitors of tubulin polymerization at picomolar concentrationsand are also efficacious against multidrug resistant cancers. Thepharmacological profile of these cytotoxic agents can beimproved (and severe neurotoxic side effects minimized) byadopting selective targeted delivery strategies. To this aim,Sewald and colleagues[79] designed two conjugates (compounds50 and 51, Scheme 15) based on the known cyclopeptidec(RGDfK) linked to cytotoxic cryptophycin-55 glycinate througha protease-cleavable Val-Cit linker and two different self-immolative spacers namely, the p-aminobenzyloxycarbonyl(PABC) spacer (compound 50), and the Gly-Pro dipeptide unit(compound 51), which decomposes by diketopiperazine for-mation. The synthesis of the linkers was accomplished bystandard SPPS and the decoration with polyethylene glycolspacer containing terminal alkyne was realized on solid support.The linkers were then conjugated to cryptophycin-55 glycinate

and the lysine residue of c(RGDfK) peptide was modified with 3-azidopropanoic acid to allow exploitation of copper-catalyzedclick reactions as the final ligation steps. It was found that thebiological and biochemical properties of the conjugatesstrongly depended on the nature of the linker. Thus, conjugate50, bearing Val-Cit/PABC, proved stable in both mouse andhuman plasma, the linker cleavage was efficient in the presenceof cathepsin B enzyme, free Cry-55 glycinate was released, andthe conjugate showed high antiproliferative activity againstαVβ3 integrin-expressing M21 and αV-negative M21-L humanmelanoma cells (IC50 values in the low-nanomolar range). Onthe other hand, conjugate 51, bearing Val-Cit/Gly-Pro, showedexcellent stability both in mouse and human plasma, butenzymatic cleavage of the linker resulted in incomplete releaseof Cry-55 glycinate; however, conjugate 51 resulted morepotent than 50, exhibiting IC50 values in the sub-nanomolarrange. Both compounds 50 and 51 showed scarce selectivity forcancer cell lines with different αVβ3 integrin expression andthese results seemed to indicate that the RGD-cryptophycinconjugates were internalized via αVβ3-independent (nonspecific)process that could be related, in some way, to the presence ofthe RGD ligand but also to the overall hydrophobic/hydrophilicbalance of the conjugates.In 2020, the same authors reported on the synthesis and

biological evaluation of a multimeric version of the previouslydisclosed conjugates, based on c(RGDfK) integrin ligand andcryptophycin-55 glycinate (compound 52, Scheme 15).[80] Tobuild up the multimeric conjugate 52, the authors adopted the

Scheme 14. DKP-based conjugates containing monomethyl auristatin cytotoxic payloads.




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known RAFT template, to which four copies of the RGD ligandwere conjugated via oxime ligation. The lysosomally cleavableVal-Cit linker and the self-immolative PABC unit were thenchosen to ensure selective release of the cytotoxic payload. Thetetrameric conjugate displayed good cytotoxic activity inin vitro assays toward different cell lines (IC50 2.5 nM in M21melanoma cells and >10 nM in αV-negative M21-L cells), thusconfirming that the multipresentation of RGD ligand canenhance the selectivity and tumor-targeted drug delivery ascompared to the monomeric counterpart 50.Li et al. in 2015[81] proposed the synthesis of conjugate 53

(Scheme 16) with the aim of bringing together in a singlestructure i) a terminal linear sequence RGD as integrin targetingunit, ii) doxorubicin (DOX) as antitumor agent, iii) a carboxy-fluorescein probe for real-time monitoring of drug release, andiv) a fluorescence quencher, Dabcyl. This unit, in fact, canquench the fluorescence of proximity fluorophores throughFörster Resonance Energy Transfer (FRET), and in compound 53this unit is able to quench the fluorescence of both carboxy-fluorescein (FAM) and DOX moieties. DOX was linked throughan acid-labile acyl hydrazone bond that can be cleaved by theacidic microenvironment in both cancer tissues and endosomes;the drug release could be real-time monitored by the increaseof red fluorescence from DOX. FRET analysis, after incubation ofthe samples with caspase-3, revealed that, in 11 hours, nearly90% DOX was released from 53 at pH 5.0 and about 19% wasreleased at pH 7.4. When released, DOX induced cell apoptosisand consequent liberation of caspase-3 in the extracellularenvironment. The presence of the tetrapeptide sequence Asp-

Glu-Val-Asp (DEVD) in the backbone of 53 allowed monitoringthe proapoptotic activity of DOX, the cleavage of this sequencebeing caspase-3-mediated and assessable by the fluorescencerecovery of FAM. The FAM-GCSDEVDSK(Dabcyl)RGD moiety wassynthesized by standard solid phase synthesis, and thencompound 53 was obtained by thiol-maleimide reaction with a6-maleimidocaproylhydrazone derivative of DOX. Conjugate 53was tested on αVβ3 integrin positive U87 cell line (humanglioblastoma cells) and αVβ3 integrin negative COS7 cell line (anAfrican green monkey kidney fibroblast-like cell model) asnegative control, revealing the importance of the RGD targetingunit, since the cellular uptake in U87 cells was much higherthan in COS7 cells. However, conjugate 53 showed reducedcytotoxicity with respect to free DOX, likely due to its slowerendocytosis compared with the fast passive diffusion of freeDOX.Conjugate 54 (Scheme 17), designed by Jin, Ji et al.,[82]

contains the chemotherapeutic drug gemcitabine (GEM), atetraphenylene unit (TPE) as a fluorescent probe, and a terminalAsp-Gly-Arg[83] sequence as the targeting carrier. GEM isconjugated to the system by a reduction-responsive disulfidebond that can be cleaved inside cancer cells, where thereducing potential (high glutathione concentration) is 100–1000 times higher than in blood circulation. TPE is a fluorescentprobe with Aggregation-Induced Emission (AIE) behavior, i. e. itshows weak fluorescence in the molecularly dissolved state, buta strong fluorescence when it is in the aggregated state. Thislast unit is connected to the remaining construct through acathepsin B-cleavable GFLG peptide sequence. The lysosomal

Scheme 15. Monomeric and tetrameric RGD-cryptophycin conjugates for targeted anticancer therapy.




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protease cathepsin B is known to be upregulated in many kindsof cancers, and its hydrolytic activity is responsible for theproduction of the hydrophobic TPE-GF unit that aggregates andexhibits blue fluorescence, thus allowing monitoring the intra-cellular process. The TPE-conjugated polypeptide TPE-GFLG-D5-GKGDGR was prepared via standard SPPS, and then the aminogroup of the lysine residue was conjugated to the acryloylderivative of GEM-disulfide linker unit via Michael additionreaction. BxPC-3 pancreatic cancer cells were used as a model

cancer cell to study in vitro the properties of 54 as bothanticancer drug and specific fluorescence light-up probe. Aftera series of biological assays, including fluorescence imagingstudies and evaluation of cell viability by MTT, the authorsconcluded that the integrin-targeted GEM-prodrug 54 wasmore efficient in inhibiting the proliferation of pancreatic cancercells in comparison to the free GEM and the non-targetedprodrug, providing new opportunities for the development offluorescence light-up antitumor platforms.

Scheme 16. Structure of the dual FRET-based integrin targeting prodrug 53 for real-time monitoring of doxorubicin release and in situ efficacy evaluation.

Scheme 17. Structure of the reduction-activatable gemcitabine prodrug (TPE-GEM-RGD) 54 as anticancer drug and specific fluorescence light-up probe.




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In 2019, Neundorf at al. designed conjugates of type 55(Scheme 18) where a c(DKP-RGD) targeting unit was connectedto a cell-penetrating peptide (CPP) through a noncleavablelinker (a robust triazole unit), with the aim to increase the cellinternalization ability of the construct.[84] The idea of the authorswas to selectively deliver to αVβ3 integrin overexpressing cellsthe antitumor drug daunorubicin (DAU), that was conjugated toa lysine residue of the CPP sequence via oxime ligation. Oximebonds are rapidly cleaved at pH lower than 5.5, which isencountered inside tumor cells. A variant of this conjugate wascompound 56, presenting the sequence GFLG, which isrecognized and cleaved by the lysosomal protease cathepsin B.This tetrapeptide scaffold was inserted between the DAU andlysine moieties with the aim to favor the DAU release insidetumor cells. In vitro studies performed on tumor cell lines withdifferent integrin expression (αVβ3+U87 glioblastoma, αVβ5+/αVβ3� MCF-7 breast cancer, and low level-αVβ3+HT-29 coloncarcinoma cells) did not show any difference in the cytotoxicbehavior of these compounds, and consequently no clearselectivity trend could be defined by the authors. As expected,the cytotoxic effect of DKP-RGD conjugates was definitely lowerthan the free daunorubicin; however, the internalization profilematched with the integrin expression only when the drug-loaded conjugates were incubated with the cells for a veryshort time (15 min). For this reason, the authors proposed a“kiss-and-run”-like model, in which short contact times areneeded for selective targeting by the integrin ligands, followedby internalization mainly mediated by the CPP module, andconcluded that setting up in vivo experiments could confirmthe utility of these new multimodal drug delivery systems.

4. Concluding remarks

The idea that fueled drug discovery until recently – i. e. newdrugs have to be maximally selective in addressing oneindividual target – seems to have been overtaken by theincreasing interest for targeted drug delivery systems, which aremolecular hybrids comprising two or more active units capableto interfere simultaneously with multiple biological targets anddesigned for counteracting the poor selectivity of one of thecomponents. Among advantages of multitarget drugs over thecombined administration of different therapeutic agents (com-bination therapies), the lower off-target toxicities and the morepredictable and safe pharmacokinetic profiles are the mostappreciated. In this sense, recent achievements in the field ofRGD-PDC development are expected to lead to innovativeclinical applications in the near future.Most of the papers here reviewed describe the design and

synthesis of covalent platforms addressed to various types ofcancer and cancer-related phenomena such as inflammationand tumor angiogenesis; in fact, most of the anticancer agentsin clinical use require precise drug vectorization to increasetheir therapeutic index, reduce off-target toxicities and modu-late the pharmacokinetic properties.Despite the remarkable progress done in the field and the

many evidences collected in these studies, the real-world resultsare not always aligned to the expectations of the authors,especially concerning the location and timing of drug releasefrom cleavable conjugates. Thus, conjugates designed to beenzymatically cleaved in cells happen to be poorly internalizedby integrin-mediated endocytosis, compromising the efficacy ofdrug release. In conjugates where a synergistic cooperationbetween the integrin ligand and the appended cargo isexpected, unfavorable interference between the units risks tomutually compromise their action. In some instances, the use of

Scheme 18. Structure of the two conjugates 55 and 56, which follow the so-called “kiss-and-run”-like model.




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poorly selective RGD targeting ligands (i. e. targeting more thanone integrin subtype with high affinity) may lead to misinter-pretation in the cell uptake experiments, while in other cases,“positive” in vitro results are completely controverted by morecomplex in vivo preclinical models. In some other instances,biological assays simply prove not suitable for testing thehypotheses of the authors. In general, the heterogeneity ofbiological assays and the reported data do not always allowdirect comparison and firm evaluation of the efficacy of thesehybrid structures.Another point to be underlined is that the ligands most

commonly used for the construction of RGD-PDCs belong to arather limited group of cyclopeptides – the conjugableanalogues of cilengitide are often used – whose function asintegrin antagonists or their synergistic action with chemo-therapeutic units have not always been certified. Moreover, thetoolbox from which cytotoxic active units are drawn is oftencircumscribed to three or four “famous” chemotherapeuticssuch as PTX, CPT, and cisplatin-derivatives. However, consider-ing the ever increasing number of disease-specific targetsidentified as cancer biomarkers, the broad know-how inconjugation chemistry which exploits innovative linkers andself-immolative spacers, along with the modern tools for high-throughput chemistry that opens access to structural diversityof warheads, it is conceivable that the future and the success ofRGD-PDCs will depend upon the judicious combination ofdifferent novel targeting units with a wide number of activeingredients. Lastly, their success will also depend on thecapability to select the most reliable protocols to validate bothsafety and therapeutic efficiency of new molecularly targetedplatforms aiming to meet the most compelling medical needsof our society.

Conflict of Interest

The authors declare no conflict of interest.

Keywords: Dual targeting · Integrin receptors · RGDpeptidomimetics · Targeted therapy

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Manuscript received: February 25, 2021Revised manuscript received: April 9, 2021Accepted manuscript online: April 15, 2021




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