Antonino Mazzaglia

The intracellular effects of non-ionic amphiphilic cyclodextrin nanoparticles in the delivery of anticancer drugs

The aim of this study was to develop nanoparticles made of the amphiphilic cyclodextrin heptakis (2-O-oligo(ethyleneoxide)-6-hexadecylthio-)-beta-CD (SC16OH) entrapping docetaxel (Doc) and establish their in vivo potential. Doc-loaded SC16OH nanoparticles were prepared by the emulsion-solvent evaporation technique and fully characterized for size, zeta potential, amount of entrapped drug, release rate and degradation rate. Spherical vesicular nanoparticles displaying a hydrodynamic radius of about 95 nm which did not change upon storage as an aqueous dispersion, a negative zeta potential and entrapment efficiency of Doc very close to 100% were produced. DSC study highlighted the crystalline nature of SC16OH, unloaded and Doc-loaded SC16OH nanoparticles which resulted in their very slow dissolution during release stage and well-modulated release of entrapped Doc for about 8 weeks. Doc-loaded SC16OH nanoparticles were not hemolytic toward red blood cells as compared to a commercial Doc formulation (Taxotere) which shows a dose-dependent toxicity. After exposure of HEp-2 cells to equivalent doses of free Doc and Doc-loaded SC16OH nanoparticles, superior cell killing and cell damage were observed for nanoparticles. Finally, cell damage was attributed to aberrant mitosis which was found to be significantly higher for HEp-2 cells treated with Doc-loaded SC16OH nanoparticles as compared to free Doc likely due to the ability of nanoparticles to slowly release the drug allowing prolonged cell arrest in mitosis. Taken together, these results highlights a great potential of nanoparticles based on SC16OH in solid tumors therapy.

A Cyclodextrin‐Based Nanoassembly with Bimodal Photodynamic Action

We have developed a supramolecular nanoassembly capable of inducing remarkable levels of cancer cell mortality through a bimodal action based on the simultaneous photogeneration of nitric oxide (NO) and singlet oxygen ((1)O(2)). This was achieved through the appropriate incorporation of an anionic porphyrin (as (1)O(2) photosensitizer) and of a tailored NO photodonor in different compartments of biocompatible nanoparticles based on cationic amphiphilic cyclodextrins. The combination of steady-state and time-resolved spectroscopic techniques showed the absence of significant intra- and interchromophoric interaction between the two photoactive centers embedded in the nanoparticles, with consequent preservation of their photodynamic properties. Photodelivery of NO and (1)O(2) from the nanoassembly on visible light excitation was unambiguously demonstrated by direct and real-time monitoring of these transient species through amperometric and time-resolved infrared luminescence measurements, respectively. The typical red fluorescence of the porphyrin units was essentially unaffected in the bichromophoric nanoassembly, allowing its localization in living cells. The convergence of the dual therapeutic action and the imaging capacities in one single structure makes this supramolecular architecture an appealing, multifunctional candidate for applications in biomedical research.

Multivalent binding of galactosylated cyclodextrin vesicles to lectin

Amphiphilic beta-cyclodextrins with alkylthio chains at the primary-hydroxyl side and galactosylthio-oligo-(ethylene glycol) units at the secondary-hydroxyl side, which form nanoparticles and vesicles, show multivalent effects in their binding to lectin.

Nanostructures of cationic amphiphilic cyclodextrin complexes with DNA.

Complexes of cationic amphiphilic cyclodextrins heptakis[2-(ω-amino-oligo(ethylene glycol))-6-deoxy-6-hexadecylthio]-β-cyclodextrin and heptakis[2-(ω-amino-oligo(ethylene glycol))-6-deoxy-6-dodecylthio]-β-cyclodextrin with DNA were examined by small-angle X-ray scattering and dynamic as well as electrophoretic light scattering. The first cyclodextrin forms bilayer vesicles in water, which, in the presence of calf thymus DNA, transform to a multilamellar complex. In this complex, the DNA lies between the two polar layers of the cyclodextrins protonated amino groups in alternation with the lipidic bilayers. The cyclodextrin with shorter lipid chains, in contrast, forms micelles in water, and electrostatic clustering of these about DNA does not affect their intrinsic structure. These results are relevant to the potential of such cyclodextrins in therapeutic gene delivery, showing that their self-assembly modes in isolation influence their complex formation with DNA and possibly their efficiency in promoting cell transfection.

Physico-chemical characterization of an amphiphilic cyclodextrin/genistein complex.

Specific recognition of cell-targeting systems as host-carriers modified with receptor targeting groups, is a major ambition in the application of supramolecular science to medicine and life science. Genistein (Gen), an isoflavone belonging to the class of phytoestrogens, is of great interest because it has been considered as potential remedy for many kinds of disease. In this work, genistein in aqueous medium and in the presence of an host nanocarrier as amphiphilic cyclodextrin (CyD) modified in the upper rim with oligoethylene hydroxyl groups [(2-oligo(ethyleneoxide)-6-hexylthio)-beta-CyD, SC6OH] at 1:1 molar ratio, has been firstly investigated by UV-vis measurements coupled with circular dichroism data, in order to characterize the drug/macrocycle binding affinity through the formation of the complex. Furthermore, FTIR-ATR technique has been used to detect the complex formation in solid phase and to characterize the functional groups responsible of the solid Gen/SC6OH complex stability. The infrared absorbance spectra of the complex, collected in a wide range of wavenumber and around the physiological temperature, have been analysed and compared with the spectra of the pure compounds and their physical mixture. By monitoring the most significant changes in the shape and position of the absorbance bands of the Gen functional groups, we showed that the formation and/or modification of polar bonds play the main role in the interaction of the drug with the amphiphilic CyD. From the results, Gen is shown to be entangled in SC6OH nanoaggregates, establishing hydrogen bonding with the hydrophilic PEG chains.

Inclusion of 5-[4-(1-Dodecanoylpyridinium)]-10,15,20-triphenylporphine in Supramolecular Aggregates of Cationic Amphiphilic Cyclodextrins: Physicochemical Characterization of the Complexes and Strengthening of the Antimicrobial Photosensitizing Activity

Recent findings suggest that visible light-promoted photooxidative processes mediated by sensitizers of appropriate chemical structure could represent a useful tool for properly addressing the problem of the increasing occurrence of infectious diseases caused by multiantibiotic-resistant microbial pathogens. The monocationic meso-substituted porphyrin 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphine (TDPyP) complexed into supramolecular aggregates of cationic amphiphilic beta-cyclodextrin (SC(6)NH(2)) (mean diameter = 20 nm) appeared to be endowed with favorable properties to act as a photosensitizing agent, including a very high quantum yield (Phi(Delta) = 0.90) for the generation of the highly reactive oxygen species, singlet oxygen ((1)O(2)). Although the yield of (1)O(2) generation was comparable to that obtained after TDPyP incorporation into cationic unilamellar liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) SC(6)NH(2)-bound TDPyP was more active than DOTAP-bound TDPyP in photosensitizing the inactivation of the Gram-positive methicillin-resistant bacterium Staphylococcus aureus (MRSA). At variance with DOTAP-bound TDPyP, photoactivated SC(6)NH(2)-bound TDPyP was efficient also in photokilling Gram-negative bacterial pathogens, such as Escherichia coli . These observations are in agreement with the well-known photobactericidal effect of positively charged porphyrin derivatives, which can be markedly enhanced after incorporation into carriers with multiple positive charges. In addition, transmission electron microscopy studies revealed that potentiation of the TDPyP-mediated photobactericidal effect by incorporation into SC(6)NH(2) is a consequence of the carriers ability to promote an efficient crossing of the very tightly organized three-dimensional architecture of the bacterial outer wall by the embedded porphyrin so that a prompt interaction between the short-lived photogenerated (1)O(2) and the nearby targets, whose integrity is critical for cell survival, can take place.

Nitric oxide photocaging platinum nanoparticles with anticancer potential

In this contribution we report the design, fabrication and properties of hydrosoluble platinum nanoparticles decorated with a nitric oxide (NO) caging compound. Direct monitoring of NO through an ultrasensitive NO electrode demonstrate that the nanoparticles are stable in the dark but supply NO at nanomolar levels exclusively upon light excitation. The biocompatibility of these nanohybrid systems and their potential in photoactivated anticancer therapy have been explored by in vitro experiments using tumor cell lines. Overall these nanoparticles meet a combination of ideal prerequisites in the context of biomedical applications. Indeed, they associate small sizes, good water solubility and thermal stability under physiological conditions with excellent biocompatibility and appreciable tumor cell mortality upon irradiation with visible light. All these features make our photoactivable nanoparticles very appealing point sources of NO from the viewpoint of practical application in the emerging field of nanomedicine.

Probing specific protein recognition by size-controlled glycosylated cyclodextrin nanoassemblies

The balance between hydrophobic and hydrophilic components in amphiphilic β-cyclodextrins, targeted by receptor specific groups (SC6CDGlc, SC6CDGal, SC16CDGlc, SC16CDGal), sensitively influences the structural properties of these systems. The different amphiphilic features of single cyclodextrins generate micellar aggregates and vesicles with an internal aqueous compartment able to encapsulate guests, such as rhodamine 6G. Small-angle light scattering (SAXS), cryo-TEM and AFM investigations describe the size and shape of these self-organized glycoligands. Recognition of the nanoassemblies by a specific receptor has effectively been demonstrated by means of time resolved fluorescence and is addressed in water by the morphological properties of cyclodextrin aggregates. Exclusively galactosylated thiohexyl-cyclodextrin binds specifically lectin from Pseudomonas aeruginosa. β-D-Galactose competes with galactosylated cyclodextrin aggregates by inhibiting lectin binding but does not affect the mesoscopic environment of the protein. The better selectivity of the less hydrophobic cyclodextrins towards lectin should probably be ascribed to the morphology (size and shape) of these cyclodextrin aggregates. The recognition properties of this particular cyclodextrin (SC6CDGal) are probably due to the presence of small micelles which interact more efficiently with the lectin binding site. The modulation of the hydrophobic–hydrophilic balance of the macrocycle labelled with targeting groups allows the design of “active” nanosized carriers for drug delivery.

Supramolecular Assemblies Based on Complexes of Nonionic Amphiphilic Cyclodextrins and a meso-Tetra(4-sulfonatophenyl)porphine Tributyltin(IV) Derivative: Potential Nanotherapeutics against Melanoma

Amphiphilic cyclodextrin (ACyD) provides water-soluble and adaptable nanovectors by modulating the balance between the hydrophobic and hydrophilic chains at both CyD sides. This work aimed to design nanoassemblies based on nonionic and hydrophilic ACyD (SC6OH) for the delivery of a poor-water-soluble organotin(IV)-porphyrin derivative [(Bu3Sn)4TPPS] to melanoma cancer cells. To characterize the porphyrin derivatives under simulated physiological conditions, a speciation was performed using complementary techniques. In aqueous solution (≤ 20 μM), (Bu3Sn)4TPPS primarily exists as a monomer (2 in Figure 1), as suggested by the low static anisotropy (ρ ≈ 0.02) with a negligible formation of porphyrin supramolecular aggregates. MALDI-TOF spectra indicate the presence of moieties (i.e., [(Bu3Sn)3TPPS](-)) that are derivatives of the monomeric species. Spectrofluorimetry coupled with potentiometric measurements primarily assesses the presence of the hydrolytic [(Bu3Sn)4TPPS (OH)4](4-) species under physiological conditions. Nanoassemblies of (Bu3Sn)4TPPS/SC6OH were prepared by dispersion of organic films in PBS at pH 7.4 and were investigated using a combination of spectroscopic and morphological techniques. The UV-vis and emission fluorescence spectra of the (Bu3Sn)4TPPS/SC6OH reveal shifts in the peculiar bands of the organotin(IV)-porphyrin derivative due to its interaction with the ACyD supramolecular assemblies in aqueous solution. The mean size was within the range of 100-120 nm. The ξ-potential was negative (-16 mV) for the (Bu3Sn)4TPPS/SC6OH nanoassemblies, with an entrapment efficiency of approximately 67%. The intracellular delivery, cytotoxicity, nuclear morphology and cell growth kinetics were evaluated via fluorescence microscopy on A375 human melanoma cells. The delivery of (Bu3Sn)4TPPS by ACyD with respect to free (Bu3Sn)4TPPS increases the internalization efficiency and cytotoxicity to induce apoptotic cell death and, at lower concentrations, changes the cellular morphology and prevents cell proliferation.

Photoresponsive multilayer films by assembling cationic amphiphilic cyclodextrins and anionic porphyrins at the air/water interface

Densely packed hybrid monolayers of amphiphilic cyclodextrins incorporating hydrophilic porphyrins are formed at the air/water interface through electrostatic interaction and can be transferred onto quartz substrates by Langmuir–Schafer deposition. The resulting multilayers exhibit a good response to light excitation as proven by fluorescence emission, triplet–triplet absorption and singlet oxygen photogeneration.