Metal-binding cyclodextrins: Synthesis and complexation with Zn2+ and Ga3+ cations towards antimicrobial applications.

Highly resistant bacteria producing metallo-ß-lactamases (MBLs) to evade ß-lactam antibiotics, constitute a major cause of life-threatening infections world-wide. MBLs exert their hydrolytic action via Zn2+ cations in their active center. Presently, there are no approved drugs to target MBLs and combat the associated antimicrobial resistance (AMR). Towards this issue, we have prepared a family of cyclodextrins substituted with iminodiacetic acid (IDA) on their narrow side, while the wider side is either unmodified or per-2,3-O-methylated. The molecules form strong coordination complexes with Zn2+ or Ga3+ cations in aqueous solution. Free and metal-complexed compounds have been thoroughly characterized regarding structures, pH-dependent ionization states, distribution of species in solution, pKa values and metal-binding constants. At neutral pH the multi-anionic hosts bind up to four Zn2+ or Ga3+ cations. In vitro, 50 µΜ of the compounds achieve complete re-sensitization of MBL-producing Gram-negative clinical bacterial strains resistant to the carbapenems imipenem and meropenem. Moreover, the radioactive complex [67Ga]Ga-ß-IDACYD prepared, displays high radiochemical purity, sufficient stability both overtime and in the presence of human plasma apo-transferrin, thus providing an invaluable tool for future biodistribution and pharmacokinetic studies of ß-IDACYDin vivo, prerequisites for the development of therapeutic protocols.

Chemical Tailoring of ß-Cyclodextrin-Graphene Oxide for Enhanced Per- and Polyfluoroalkyl Substances (PFAS) Adsorption from Drinking Water.

We report on the synthesis of ß-cyclodextrin (ßCD) modified graphene oxide (GO) nanosheets, having different sized alkyl linkers (GO-Cn -ßCD) and their exploitation as sorbent of per- and polyfluoroalkyl substances (PFAS) from drinking water. ßCD were functionalized with a pending amino group, and the resulting precursors grafted to GO nanosheets by epoxide ring opening reaction. Loading of ßCD units in the range 12 %-36 % was estimated by combined XPS and elemental analysis. Adsorption tests on perfluorobutanoic acid (PFBA), a particularly persistent PFAS selected as case study, revealed a strong influence of the alkyl linker length on the adsorption efficiency, with the hexyl linker derivative GO-C6 -ßCD outperforming both pristine GO and granular activated carbon (GAC), the standard sorbent benchmark. Molecular dynamic simulations ascribed this evidence to the favorable orientation of the ßCD unit on the surface of GO which enables a strong contaminant molecules retention.

Three-in-one: exploration of co-encapsulation of cabazitaxel, bicalutamide and chlorin e6 in new mixed cyclodextrin-crosslinked polymers.

We explored a series of cyclodextrin (CyD) polymers composed either of a single CyD type or a mixture of two CyD types to encapsulate simultaneously different compounds with potential therapeutic interest for multimodal prostate cancer treatment. New mixed CyD polymers were prepared in alkaline water starting from the naturally occurring monomers and a low-cost crosslinking agent. Batches of 200 g of polymer were easily obtained. By means of optical spectroscopy we proved the co-encapsulation of 3 compounds in the polymers: the drugs cabazitaxel (CBX) and bicalutamide (BIC), and the photosensitizer chlorin e6 (Ce6). pßCyD and mixed pαßCyD polymers performed best for single drug solubilization. In the co-encapsulation of BIC and CBX by pßCyD and pαßCyD, pßCyD stands out in drug solubilization ability. Avoiding the use of organic solvents, it was possible to dissolve up to 0.1 mM CBX with 10 mg ml-1 pßCyD polymer and, with 100 mg ml-1, even 1.7 mM BIC, a 100-fold improvement compared to water. Spectroscopic studies afforded the binding constants of CBX and BIC with pßCyD forming complexes of 1 : 2 stoichiometry (drug : CyD) and CBX displayed significantly higher affinity. Also DFT calculations suggested that the drugs are more stable when complexed by two CyD units. Ce6 could be encapsulated simultaneously with the other two drugs in pßCyD and, most importantly, is able to produce singlet oxygen efficiently. Thanks to a single inexpensive CyD-based polymer we were able to produce a three-in-one platform for future implementation of combined chemotherapy and photodynamic therapy. These achievements are most relevant as nanomedicines are continuously proposed but their potential for translation to the pharma industry is compromised by their limited potential for industrial upscale.

A Cyclodextrin Polymer as Supramolecular Matrix for Scalable Green Photooxygenation of Hydrophobic Substrates in Homogeneous Phase.

In the quest for new therapies targeting hypoxia, aromatic endoperoxides have intriguing potential as oxygen releasing agents (ORAs) able to free O2 in tissues upon suitable trigger. Four aromatic substrates were synthesized and the formation of their corresponding endoperoxides was optimized in organic solvent upon selective irradiation of Methylene Blue, a low-cost photocatalyst, producing the reactive singlet oxygen species. Complexation of the hydrophobic substrates within a hydrophilic cyclodextrin (CyD) polymer allowed their photooxygenation in homogeneous aqueous environment using the same optimized protocol upon dissolution in water of the three readily accessible reagents. Notably, reaction rates were comparable in buffered D2 O and organic solvent and, for the first time, the photooxygenation of highly hydrophobic substrates was achieved for millimolar solutions in non-deuterated water. Quantitative conversion of the substrates, straightforward isolation of the endoperoxides and recovery of the polymeric matrix were achieved. Cycloreversion of one ORA to the original aromatic substrate was observed upon thermolysis. These results hold great potential for the launch of CyD polymers both as reaction vessels for green, homogeneous photocatalysis and as carrier for the delivery of ORAs in tissues.

Implementation of Water-Soluble Cyclodextrin-Based Polymers in Biomedical Applications: How Far Are We?

Cyclodextrin-based polymers can be prepared starting from the naturally occurring monomers following green and low-cost procedures. They can be selectively derivatized pre- or post-polymerization allowing to fine-tune functionalities of ad hoc customized polymers. Preparation nowadays has reached the 100 g scale thanks also to the interest of industries in these extremely versatile compounds. During the last 15 years, these macromolecules have been the object of intense investigations in view of possible biomedical applications as the ultimate goal and large amounts of scientific data are now available. Compared to their monomeric models, already used in the formulation of various therapeutic agents, they display superior behavior in terms of their solubility in water and solubilizing power toward drugs incompatible with biological fluids. Moreover, they allow the combination of more than one type of therapeutic agent in the polymeric system. In this review, a complete state of the art on the knowledge and potentialities of water-soluble cyclodextrin-based polymers as therapeutic agents as well as carrier systems for different types of therapeutics to implement combination therapy is provided. Finally, a perspective on their assets for innovation in disease treatment as well as their limits that still need to be addressed is given.

Helical Nanofibers Formed by Palladium-Mediated Assembly of Organic Homochiral Macrocycles Containing Binaphthyl and Pyridyl Units.

Herein, we report the synthesis and characterization of homochiral macrocycles, in which molecular rigidity, combined with the presence of multiple functional groups, allow for the assembly of helical nanostructures. 1,1'-bi-2-naphthol (Binol) units are used as robust chirality inducers, and pyridyl units embedded within the molecular frameworks allow the assembly, upon coordination with Pd(II) metal ions, of the macrocyclic building blocks. CD and NMR spectroscopies show the formation of ordered 1D assembly in solution. AFM studies indicate that the molecular systems are capable of forming nanoscale structures. The effective transfer of chiral information results in helical nanofibers, with lengths ranging from a few hundreds of nanometers to some micrometers. AFM line profiles reveal a helical longitudinal period of about 50 nm and a transverse width of 25 to 45 nm after deconvolution.

Increased antibiotic efficacy and noninvasive monitoring of Staphylococcus epidermidis biofilms using per-cysteamine-substituted γ-cyclodextrin - A delivery effect validated by fluorescence microscopy.

Limited and poor delivery of antibiotics is cited as one reason for the difficulty in treating antibiotic-resistant biofilms associated with chronic infections. We investigate the effectiveness of a positively charged, single isomer cyclodextrin derivative, octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) to improve the delivery of antibiotics to biofilms. Using multiphoton laser scanning microscopy complemented with super-resolution fluorescence microscopy, we showed that γCys tagged with fluorescein (FITC) is uniformly distributed throughout live S. epidermidis biofilm cultures in vitro and results suggest it is localized extracellularly in the biofilm matrix. NMR spectroscopic data in aqueous solution confirm that γCys forms inclusion complexes with both the antibiotics oxacillin and rifampicin. Efficacy of γCys/antibiotic (oxacillin and rifampicin) was measured in the biofilms. While treatment with γCys/oxacillin had little improvement over oxacillin alone, γCys/rifampicin reduced the biofilm viability to background levels demonstrating a remarkable improvement over rifampicin alone. The strong synergistic effect for γCys/rifampicin is at this stage not clearly understood, but plausible explanations are related to increased solubility of rifampicin upon complexation and/or synergistic interference with components of the biofilm. The results demonstrate that designed cyclodextrin nanocarriers, like γCys, efficiently deliver suitable antibiotics to biofilms and that fluorescence microscopy offers a novel approach for mechanistic investigations.

Formation, characterization and pH dependence of rifampicin: heptakis(2,6-di-O-methyl)-ß-cyclodextrin complexes.

Rifampicin (Rif) is a broad spectrum antibiotic used as a first line agent in the treatment of mycobacterial infections. However, its low solubility and reduced stability in water limit its bioavailability, thus requiring the use of complex formulations. Here, we present a systematic study of Rif in complex with a methylated cyclodextrin, heptakis(2,6-di-O-methyl)-ß-cyclodextrin (DIMEB), in phosphate buffer using a combination of nuclear magnetic resonance (NMR) and steady-state UV-vis spectroscopic methods. An increase in the stability and solubility of Rif in complex with DIMEB was observed in buffered solutions (phosphate, PBS). At neutral pH the presence of three distinguishable binding sites was revealed, demonstrating that DIMEB forms predominantly a stable 1:1 (K∼3000M-1) complex at the piperazine site of Rif, while at acidic pH the binding constant decreases significantly (K∼400M-1) due to protonation of the piperazine, thus inducing a release of Rif. The reported results provide new and relevant information for the stability and solubility of Rif in aqueous solution when forming a complex with DIMEB. Furthermore they contribute to clarify Rif interactions with cyclodextrin carriers, thus providing the basis for the development of new methylated cyclodextrin that can efficiently encapsulate and deliver Rif and derivatives of its family.

Cyclodextrin-mesoporous silica particle composites for controlled antibiotic release. A proof of concept toward colon targeting.

Cyclodextrins (CDs) and mesoporous silica particles (MSPs) have been combined as composite carriers for controlled antibiotic release. CDs were employed as "gatekeeper" agents and grafted onto MSPs to retain drug molecules inside the MSP carrier. A variety of CDs (unfunctionalized, positively charged and carboxymethylated) and three different coupling strategies (covalent binding, electrostatic adsorption and inclusion complexation) were systematically investigated for their ability to control the release of two antibiotic drugs, metronidazole and clofazimine. The drugs had significantly different physicochemical properties (metronidazole - small hydrophilic, clofazimine- large hydrophobic). We report for the first time on the encapsulation and characterization of metronidazole-loaded-MSP. Each CD coating strategy reduced the drug release rate in phosphate buffer compared to unmodified MSP (from 20% to 100% retained drug). Covalent binding and inclusion complex approaches were significantly more effective than electrostatically adsorbed CD. In particular, the novel inclusion complex based on host/guest interaction between benzyl-modified silica surface and α-CD proved to be very effective (60-100% retained drug amount). Using pharmaceutical manufacturing processes, our study shows that CD-MSP composites can retain both hydrophobic and hydrophilic antibiotic compounds with potential translation to triggered release formulation targeting bacterial infections in the colon and lower intestine.

Designed positively charged cyclodextrin hosts with enhanced binding of penicillins as carriers for the delivery of antibiotics: The case of oxacillin.

In an effort to identify the optimal cyclodextrin (CD) host for delivery of penicillins to mammalian cells that will also offer protection against ß-lactamase-induced hydrolysis, nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) have been employed to study the inclusion complexes formed in aqueous solution between designed CD derivatives and two aminopenicillins, ampicillin and amoxicillin, and two antistaphylococcal penicillins, methicillin and oxacillin. Anionic and cationic thioether-substituted-ß- and -γCD derivatives were thus synthesized and compared with the neutral, parent CDs for complexation with the penicillins. The synthesized derivatives were shown to present ∼20% elongated cavity space in solution. Moreover, the cationic ones are >98% protonated at physiological pH. The most efficient host was the positively charged octakis[6-(2-aminoethylthio)-6-deoxy]-γ-CD (γCys) that formed the strongest complex with oxacillin (Kb ∼1700M-1) in an enthalpically and entropically favorable process (ΔHb=-10.5kJ/mol,TΔSb=8.0kJ/mol). In vitro biological tests demonstrated that γCys reduces 2.3-fold the rate of hydrolysis of oxacillin in the presence of oxa-1 ß-lactamase while displaying cell crossing capability and efficient internalization into macrophages as well as a sufficiently safe cytotoxicity profile. Overall, γCys could be considered as a promising vehicle for protection and delivery of oxacillin.

A chiroptical molecular sensor for ferrocene.

A homochiral, square-shaped, D2 symmetrical nanosized metal-linked macrocycle is able to form stable complexes with ferrocene in polar solvents, with detection achieved by means of multiple outputs (optical/chiroptical spectroscopies and cyclic voltammetry). Selective sensing using chiroptical spectroscopy in the presence of interfering analytes is demonstrated.