Glycoligand-targeted core-shell nanospheres with tunable drug release profiles from calixarene-cyclodextrin heterodimers

► This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00 | 1 profiles from calixarene-cyclodextrin heterodimers Laura Gallego-Yerga,a Michela Lomazzi,b Francesco Sansone,*, b Carmen Ortiz Mellet,*, a Alessandro Casnati b and José M. García Fernández*,c Stable core-shell nanospheres have been self-assembled in water from heterodimers encompassing a hydrophobic calix[4]arene moiety and a hydrophilic -cyclodextrin head; their potential to encapsulate and provide sustained release of 10 the anticancer drug docetaxel and undergo surface postmodification with glycoligands targeting the macrophage mannose receptor is discussed. The development of well-ordered functional nanostructures filled with the capacity to assemble under dilute conditions in 15 water or buffer media continues to be one of the more fascinating challenges facing modern chemistry, with relevance in areas like imaging, diagnostics, tissue engineering or drug delivery, among others.1 Many of those channels require the supramolecular system to be capable of 20 encapsulating a cargo, targeting specific cell surface receptors and allowing the gradual release of the payload. Although polymeric materials have concentrated most of the efforts in the field, de novo design of monodisperse building blocks appears as a better suited approach for a detailed investigation 25 of the structural features governing self-assembly.2 Due to their capacity to form host-guest superstructures, macrocyclic cavity-featuring members of the calixarene3 (CA) and cyclodextrin4 (CD) families are privileged platforms towards this end. In this work we report the successful construction of 30 nanospheres entangling an inner core formed by hydrophobic calix[4]arene (CA4) units and an external hydrophilic shell exposing -cyclodextrin (CD) motifs by the self-assembly in water of amphiphilic CA4―CD heterodimers (1 and 2). The CA4 scaffold in its cone structure is very well suited to 35 promote tight packing of lipophilic chains installed at the lower rim, providing a lipid matrix where entrapped hydrophobic drugs can undergo sustained release, whereas the presence of CD at the nanosphere surface allows host-guest directed post-modifications, e.g. for targeting purposes. As a 40 proof of concept, the antimitotic agent docetaxel5 (DXT) and the macrophage mannose receptor (MMR), for which trivalent mannosyl dendrons such as 3 have shown a remarkable affinity,6,7 were selected as the drug and target, respectively (Fig. 1). DXT, primarily used to treat prostate, breast, neck 45 and lung cancer, can induce selective apoptosis in myeloidderived suppressor cells overexpressing the MMR, so that targeting is expected to improve cancer immunotherapies.8 Figure 1 Structure of the calix[4]arene―-cyclodextrin heterodimers 1 50 and 2 prepared in this work and schematic representation of their selfassembly in water to afford core-shell nanospheres that can be loaded with a drug (docetaxel; DXT) and/or coated with targeting ligands (3). There have been several reports on the use of CAs as scaffolds for positioning CD hosts, e.g. for sensing purposes.9 55 In some cases the formation of vesicular or fiber-like aggregates from CA-CD couples was observed, but in those examples the CD cavity was uniformly occupied due to intra- or intermolecular inclusion phenomena.10 In our heterodimer prototype, the molecular geometry was conceived to avoid 60 self-inclusion, whereas the compact arrangement of the four hydrophobic tails onto the CA scaffold also prevents the formation of monodimentional oligomers. The driving force that makes the single amphiphiles to self-assemble into coreshell nanospheres arises then from strong hydrophobic 65 interactions involving exclusively the multi-tail CA4 platform, leaving the external CD moieties fully accessible to participate in further supramolecular events. The heterodimers 1 and 2 were synthesized in a convergent way. The isothiocyanate-armed CA4 building blocks 10 and 17 and the 70 amine-functionalized CD derivative 20 were prepared and coupled through thiourea-forming ligation chemistry in the 2 | Journal Name, [year], [vol] , 00–00 This journal is © The Royal Society of Chemistry [year] OO O O X nnnn 5,12;X=NO 2 6, 13;X=NH 2 [H], 93-97% 7,14;X=NCS O O MeO MeO OMe 6 O O MeO MeO OR TFA,quant. 1. CS 2 2. Boc 2 O 93-99% 4, 11; X = H 18, R = Ts BocHN NCS 1. TFA 2. HCl, 95% HNO 3 ,75% CSCl 2 ,92% 99% OO O O nnnn HN N H H N S O O MeO MeO OMe 6 O O MeO MeO S H N S 1, 2 1,4-10;n=1(C 6 ) 2, 11-17; n = 7 (C 12 ) DCM, Et 3 N 87-90% 4 10,17 N HN HNHBoc S 8,15; X = 4 4 N HN HNH 2 S 9,16; X = 4 N HN HNCS S 10,17;X= 19,R = BocHN SH 97% 2 4 SNHBoc 20,R= 4 SNH 2 · HCl Scheme 1. Synthesis of the CA 4 -CD heterodimers 1 and 2. last reaction. The key step in the synthesis of the singly functionalised CA4 derivatives is the mononitration of the corresponding tetra-hexyl and -dodecyl ethers 4 and 11, which 5 proceeded in 75% after treatment with concentrated nitric acid in a mixture of dichloromethane and glacial acetic acid.11 Sequential reduction to the corresponding amines, isothiocyanation, thiourea coupling with mono-Boc-protected butylenediamine, carbamate hydrolysis and final 10 isothiocyanation (→5-10 and 12-17) proceeded with over 90% yield in every step. The CD amine counterpart 20 was prepared in 92% yield from the known mono-O6-tosyl- derivative 1812 after reaction with Boc-protected cysteamine (→19) and final hydrolysis. Thiourea conjugation of 20 with 15 the CA4 partners 10 and 17 provided the target heterodimers 1 and 2 in 87 and 90% yield, respectively (Scheme 1). Blank (unloaded) nanospheres of the two amphiphilic CA4- CD heterodimers 1 and 2 were prepared by the interfacial solvent displacement method.13 Dynamic light scattering 20 (DLS) monitoring of nanoparticle size revealed unimodal distributions of submicronic aggregates of 129 and 189 nm hydrodynamic diameter, respectively (Supplementary information, Table S1). Such colloidal dispersions remained stable for more than 30 days at rt and at 37 ºC. Atom force 25 microscopy (AFM) observation showed that the average size of the aggregates is 100 nm diameter (Figure 2). An ultra-thin structure was inferred in some cases, suggesting that those objects are composed at their turn of smaller entities of about 22-25 nm. Assuming a conical shape for the molecules and a 30 compact hexagonal arrangement filling 90% of the nanosphere volume, an aggregation number of 17-25 molecules per nanosphere can be estimated from geometrical considerations,14 the higher aggregages being composed of about 60 nanospheres. No collapsing or significant flattening 35 of the nanoparticles was observed even after prolonged drying Figure 2. Tapping mode AFM image (5 x 5 m) of the nanospheres obtained from 1 (left). The insert (0.7 x 0.7 m) shows a zoom were the ultra-thin structure can be appreciated. Schematic representations of the 40 aggregates and the individual nanospheres and a 3D molecular model of 1 with the estimated geometry are also depicted (right; CD moiety in blue, CA moiety in red, thiourea connector in orange, aliphatic chains in green). Figure 3. Release profiles of docetaxel (DXT) from loaded nanospheres 45 prepared from 1 and 2 in water. A schematic representation of the processes involved in the initial burst (dissociation from the - cyclodextrin cavities in the external shell) and the further sustained release (diffusion from the calix[4]arene core) is shown. times, discarding a vesicle-type structure and supporting 50 instead their solid lipid nanosphere nature. Nanoprecipitation of the heterodimers 1 and 2 in the presence of DXT afforded assemblies of 35 and 20 nm hydrodynamic diameter, respectively, that remained stable in water (Supplementary Information, Table S2 and Figure S1). 55 The release profiles of the drug from the loaded nanospheres showed an initial burst followed by sustained delivery over 50 or 30 h, respectively (Figure 3). It is presumed that DXT is encapsulated in the nanospheres in two different compartments, the hydrophobic core formed by the substituted 60 CA moieties and the CD cavities. Actually, the aromatic rings in DXT have been shown to promote divalent interactions with CD derivatives, leading to stable inclusion complexes.6 The observation of significant differences in the -potential between blank and loaded nanospheres, from -31 to -13 mV 65 (Supplementary information, Tables S1 and S2), is consistent with nanoparticle surface modification upon DXT loading. Probably, release of DXT into bulk water out from the CD cavity is faster as compared with diffusion from the CA core.15 The prolonged release of DXT from the nanospheres 70 prepared with the hexyloxy (C6) derivative as compared with the dodecyloxy analogue (C12) might then relate to the differences in particle size and compaction of the CA4- templated chains in the core. The data illustrate how the drug Código de campo cambiado This journal is © The Royal Society of Chemistry [year] Journal Name, [year], [vol] , 00–00 | 3 Figure 4. Inhibition of the adhesion of human MMR to immobilized yeast mannan in the presence of increasing concentrations of 3 and of nanosphere formulations prepared from 1 and 2 after postfunctionalization with 10, 30 and 60% of 3. The corresponding IC 50 5 values (in M referred to 3) are indicated. Control experiments with unmodified nanospheres showed no inhibition at the highest assayed concentration. release rate can be adjusted by judicious design of the heterodimer component. 10 The accessibility of the CD cavity in the nanospheres was further exploited for supramolecularly anchoring the trimannosyl dendron 3 at their surface, taking advantage of the strong affinity of adamantane moieties towards the CD cavity.16 Incorporation of the glycoligand antenna (10, 30 and 15 60% in molar bases with respect to 1 or 2) in water was monitored by measuring the increase in the hydrodynamic diameters (about +10 nm) and -potential (about +4 mV) of the aggregates (Supplementary information, Table S3). Through this sequential self-assembling process, a 20 glycocalyx-like corona was created around the core-shell nanospheres. The availability of the mannopyranosyl glycotopes to participate in protein recognition events was confirmed by monitoring the binding affinity towards the human MMR, following a modified enzyme-linked lectin 25 assay (ELLA).17 The results evidenced a strong affinity increase of the 3-decorated nanospheres for the MMR as compared to the individual trimannosyl ligand 3, up to 24- or 27-fold for nanospheres formulated from 1 and 42- or 46-fold in the case of nanospheres formulated from 2 on a mannose 30 molar basis (Figure 4). The data are consistent with the expected reinforcement of the interaction between the sugar and the MMR lectin after multivalent presentation of the former onto the nanoparticle. Interestingly, the MMR binding affinity of the nanosphere-glycodendron formulations was 35 drastically decreased in the presence of a 10-fold excess of adamantane carboxylate sodium salt, supporting that reversible inclusion in the CD cavity is the main mechanism at play.18 The rather small difference in MMR affinity for 30 and 60% dendron-loaded materials reflects the existence of a 40 plateau on the sugar ligand content-dependent multivalent effect. This is interesting since it might allow optimizing simultaneously the content of different functional elements, e.g. for visualization, biocompatibility, furtivity in the blood circulation or cell internalization. 45 In conclusion, we have prepared a new type of core-shell self-assembled nanospheres stable in water solution consisting of only one molecular species that combines a hydrophobic multi-tail calix[4]arene moiety and a hydrophilic - cyclodextrin head. The results illustrate the potential of CA- 50 CD heterodimer-based delivery systems in nanomedicine applications where a guest molecule has to be stabilized, targeted and/or protected in aqueous media. L.G.-Y is an FPU fellow. This study was supported by the Spanish Ministerio de Economía y Competitividad (contract 55 numbers SAF2010-15670 and CTQ2010-15848), the Junta de Andalucía, the Italian Ministry of Instruction, University and Research (MIUR, PRIN2010JMAZML MultiNanoIta), the COST action MULTIGLYCONANO (CM1102) and the European Regional Development Funds (FEDER and FSE). 60 Technical assistance from the research support services of the University of Seville (CITIUS) and Centro Interdipartimentale di Misure (CIM) of Parma University is also acknowledged. Notes and references a Departamento de Química Orgánica, Facultad de Química, Universidad 65 de Sevilla, E-41012 Sevilla, Spain. Fax: +34 9546 24960; Tel: +34 9545 59860; E-mail: mell[email protected] b Dipartimento di Chimica, Università degli Studi di Parma, I-43124 Parma, Italy. Fax: +39 0521 905472; Tel: +39 0521 905458; E-mail: [email protected] 70 c Instituto de Investigaciones Químicas (IIQ), CSIC – Universidad de Sevilla, E-41092 Sevilla, Spain. Fax: +34 9544 60165; Tel: +34 9544 89553; E-mail: [email protected] † Electronic Supplementary Information (ESI) available: Protocols for blank and DXT-loaded nanosphere preparation, characterization by AFM 75 and DLS, determination of DXT release profiles and affinity measurements of glycoligand-decorated nanospheres towards the human MMR, as well as synthetic procedures and copies of the 1 H and 13 C NMR spectra for compounds 1-3 and their precursors. See DOI: 10.1039/b000000x/ 80 1 S. Mura, J. Nicolas and P. Couvreur, Nat. Mater. 2013, 12, 991; K. Petkau-Milroy and L. Brunsveld, Org. Biomol. Chem. 2013, 11, 219. 2 K. Liu, Y. Kang, Z. Wang and X. Zhang, Adv. 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