Daniela Arosio

Cyclic RGD functionalized gold nanoparticles for tumor targeting.

Integrin α(v)β(3) is an adhesion molecule involved in physiological and pathological angiogenesis as well as tumor invasion and metastasis. Therefore, it is considered an important target for molecular imaging and delivery of therapeutics for cancer, and there is a strong interest in developing novel agents interacting with this protein. Nevertheless, the interaction of individual ligands is often still weak for efficient tumor targeting, and many research groups have synthesized multivalent displays in order to overcome this problem. Gold nanoparticles can be considered a smart platform for polyvalent presentation on account of their globular shape, tunable size, facile surface chemistry, and biocompatibility. Moreover, their unique physical properties render gold nanoparticles ideal candidates for tumor diagnosis and therapy. Here, we report the synthesis and characterization of gold nanoparticles functionalized with cRGD integrin ligand and their employment for targeting human cancer cells expressing α(v)β(3) integrin.

Cyclic RGD-Peptidomimetics Containing Bifunctional Diketopiperazine Scaffolds as New Potent Integrin Ligands

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins α(v)β(3) and α(v)β(5), which are involved in tumor angiogenesis. Nanomolar IC(50) values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by (1)H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cβ(Arg)-Cβ(Asp) average distance ≥8.8 Å]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin α(v)β(3) complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.

Advancement in integrin facilitated drug delivery

The research of integrin-targeted anticancer agents has recorded important advancements in ingenious design of delivery systems, based either on the prodrug approach, or on nanoparticle carriers, but for now, none of these has reached a clinical stage of development. Past work in this area has been extensively reviewed by us and others. Thus, the purpose and scope of the present review is to survey the advancement reported in the last 3years, with focus on innovative delivery systems that appear to afford openings for future developments. These systems exploit the labelling with conventional and novel integrin ligands for targeting the interface of cancer cells and of endothelial cells involved in cancer angiogenesis, with the proteins of the extracellular matrix, in the circulation, in tissues, and in tumour stroma, as the site of progression and metastatic evolution of the disease. Furthermore, these systems implement the expertise in the development of nanomedicines to the purpose of achieving preferential biodistribution and uptake in cancer tissues, internalisation in cancer cells, and release of the transported drugs at intracellular sites. The assessment of the value of controlling these factors, and their combination, for future developments requires support of biological testing in appropriate mechanistic models, but also imperatively demand confirmation in therapeutically relevant in vivo models for biodistribution, efficacy, and lack of off-target effects. Thus, among many studies, we have tried to point out the results supported by relevant in vivo studies, and we have emphasised in specific sections those addressing the medical needs of drug delivery to brain tumours, as well as the delivery of oligonucleotides modulating gene-dependent pathological mechanism. The latter could constitute the basis of a promising third branch in the therapeutic armamentarium against cancer, in addition to antibody-based agents and to cytotoxic agents.

Synthesis and Biological Evaluation (in Vitro and in Vivo) of Cyclic Arginine–Glycine–Aspartate (RGD) Peptidomimetic–Paclitaxel Conjugates Targeting Integrin αVβ3

A small library of integrin ligand-paclitaxel conjugates 10-13 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the paclitaxel-RGD constructs 10-13 inhibited biotinylated vitronectin binding to the purified αVβ3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin αVβ3, making them attractive to be tested in in vivo models. cyclo[DKP-f3-RGD]-PTX 11 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 11 exhibited a superior activity compared with paclitaxel, despite the lower (about half) molar dosage used.

Cyclic RGD‐Containing Functionalized Azabicycloalkane Peptides as Potent Integrin Antagonists for Tumor Targeting

Vitronectin receptors αvβ3 and αvβ5 have emerged as potential therapeutic targets for the treatment of osteoporosis, restenosis, ocular disease, tumor‐induced angiogenesis, metastasis, and sickle‐cell anemia. Among a collection of compounds, a new potent integrin antagonist was synthesized, and its binding toward the αvβ3 and αvβ5 receptors was evaluated. This molecule is a suitable candidate as a vector for therapeutics and diagnostics.

Design, Synthesis, and Biological Evaluation of Novel cRGD–Paclitaxel Conjugates for Integrin-Assisted Drug Delivery

The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrin-recognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent α(V)β(3) binding capabilities for most conjugates, while in vitro growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative 21, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for in vivo studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy.

Synthesis of Gd and 68Ga Complexes in Conjugation with a Conformationally Optimized RGD Sequence as Potential MRI and PET Tumor‐Imaging Probes

We report the synthesis of novel chelates of Gd and 68Ga with DPTA, DOTA, HP‐DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin ανβ3. Because ανβ3 is involved in neo‐angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of 68Ga complexation for the promising 68Ga‐PET technique; the AAZTA complex 15(68Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.

Functionalized cyclic RGD peptidomimetics: conjugable ligands for αvβ3 receptor imaging.

The α(v)β(3) integrin is an adhesion molecule involved in physiological and pathological angiogenesis as well as in tumor invasion and metastasis, and therefore, there is a strong interest in developing novel agents interacting with this molecule. We report the synthesis and characterization of fluorescent α(v)β(3) integrin probes and their use to visualize integrin α(v)β(3) expression on human normal and cancer cells. The fluorescent probes we describe here may be of use for noninvasive imaging of α(V)β(3) integrin expression also in vivo.

Mimicking gangliosides by design: mimics of GM1 headgroup.

Detailed knowledge of the three-dimensional structure of ganglioside headgroups has allowed the successful design of structural and functional mimics of ganglioside GM1 oligosaccharide. Our recent work in this area is reviewed in this paper.

Improved synthesis of both enantiomers of trans-cyclohex-4-ene-1,2-dicarboxylic acid

Abstract A facile synthesis of both enantiomers of trans -cyclohex-4-ene-1,2-dicarboxylic acid on a multigram scale, that starts from inexpensive, commercially available compounds, is described.