Amphiphilic Chlorin-ß-cyclodextrin Conjugates in Photo-Triggered Drug Delivery: The Role of Aggregation.

Conjugates of chlorins with ß-cyclodextrin connected either directly or via a flexible linker were prepared. In aqueous medium these amphiphilic conjugates were photostable, produced singlet oxygen at a rate similar to clinically used temoporfin and formed irregular nanoparticles via aggregation. Successful loading with the chemotherapeutic drug tamoxifen was evidenced in solution by the UV-Vis spectral changes and dynamic light scattering profiles. Incubation of MCF-7 cells with the conjugates revealed intense spotted intracellular fluorescence suggestive of accumulation in endosome/lysosome compartments, and no dark toxicity. Incubation with the tamoxifen-loaded conjugates revealed also practically no dark toxicity. Irradiation of cells incubated with empty conjugates at 640 nm and 4.18 J/cm2 light fluence caused >50 % cell viability reduction. Irradiation following incubation with tamoxifen-loaded conjugates resulted in even higher toxicity (74 %) indicating that the produced reactive oxygen species had triggered tamoxifen release in a photochemical internalization (PCI) mechanism. The chlorin-ß-cyclodextrin conjugates displayed less-lasting effects with time, compared to the corresponding porphyrin-ß-cyclodextrin conjugates, possibly due to lower tamoxifen loading of their aggregates and/or their less effective lodging in the cell compartments' membranes. The results suggest that further to favorable photophysical properties, other parameters are important for the in vitro effectiveness of the photodynamic systems.

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.

Small anticancer drug release by light: Photochemical internalization of porphyrin-ß-cyclodextrin nanoparticles.

Aiming to engineer simple, neutral, strongly amphiphilic photoactive nanoparticles (NPs) to specifically target cancer cell lysosomes for drug transport and light-controlled release, new conjugates of ß-cyclodextrin with highly hydrophobic triphenylporphyrin bearing different alkyl chains, were synthesized. Although differently sized, all conjugates self-assemble into ~60 nm NPs in water and display similar photoactivity. The NPs target selectively the lysosomes of breast adenocarcinoma MCF-7 cells, embedding in vesicular membranes, as experiments with model liposomes indicate. Either empty or drug-loaded, the NPs lack dark toxicity for 48 h. They bind with differently structured anticancer drugs tamoxifen and gemcitabine as its N-adamantyl derivative. Red light irradiation of cells incubated with drug-loaded NPs results in major reduction of viability (>85 %) for 48 h displaying significant synergy of photo-chemotoxicity, as opposed to empty NPs, and to loaded non-irradiated NPs, in manifestation of photochemical internalization (PCI). Our approach expands the field of PCI into different small molecule chemotherapeutics.

5-ALA Is a Potent Lactate Dehydrogenase Inhibitor but Not a Substrate: Implications for Cell Glycolysis and New Avenues in 5-ALA-Mediated Anticancer Action.

In a course of metabolic experiments, we determined that the addition of δ-aminolevulinic acid (5-ALA) to a panel of glioblastoma multiforme (GBM) cells caused a steep reduction in their glycolytic activity. This reduction was accompanied by a decrease in adenosine triphosphate (ATP) production from glycolysis. These results suggested that 5-ALA is an inhibitor of glycolysis; due to the structural similarity of 5-ALA to the established lactate dehydrogenase (LDH) inhibitors oxamate (OXM) and tartronate (TART), we initially investigated LDH inhibition by 5-ALA in silico. The modelling revealed that 5-ALA could indeed be a competitive inhibitor of LDH but not a substrate. These theoretical findings were corroborated by enzymatic and cell lysate assays in which 5-ALA was found to confer a potent LDH inhibition comparable to that of OXM and TART. We subsequently evaluated the effect of 5-ALA-induced glycolysis inhibition on the viability of GBM cells with diverse metabolic phenotypes. In the Warburg-type cell lines Ln18 and U87, incubation with 5-ALA elicited profound and irreversible cell death (90-98%) at 10 mM after merely 24 h. In T98G, however, which exhibited both high respiratory and glycolytic rates, LD95 was achieved after 72 h of incubation with 20 mM 5-ALA. We additionally examined the production of the 5-ALA photosensitive metadrug protoporphyrin IX (PpIX), with and without prior LDH inhibition by TART. These studies revealed that ~20% of the 5-ALA taken up by the cells was engaged in LDH inhibition. We subsequently performed 5-ALA photodynamic therapy (PDT) on Ln18 GBM cells, again with and without prior LDH inhibition with TART, and found a PDT outcome enhancement of ~15% upon LDH pre-inhibition. We expect our findings to have a profound impact on contemporary oncology, particularly for the treatment of otherwise incurable brain cancers such as GBM, where the specific accumulation of 5-ALA is very high compared to the surrounding normal tissue.

Host-guest inclusion complexes of hydroxytyrosol with cyclodextrins: Development of a potential functional ingredient for food application.

Hydroxytyrosol (HT), a potent phenolic phytochemical, exerts positive health effects due to its antioxidant properties. However, it is highly reactive to oxygen, light, and heat and presents high instability. Alpha- and beta-cyclodextrin (α-CD, ß-CD) have structures that allow them to encapsulate a variety of hydrophobic molecules. The aim of this study was to examine the outcomes of the inclusion of HT into α-CD and ß-CD. Aqueous solutions of HT and either α-CD or ß-CD were prepared and freeze-drying was applied for the encapsulation, in 1:1 and 2:1 molar ratios. The produced solid complexes were studied and characterized using NMR spectroscopy, differential scanning calorimetry (DSC) and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR). Encapsulation efficiency (EE%), stability, and in vitro release of the encapsulated complexes under simulated digestion conditions were also evaluated. In both DSC thermograms and FTIR spectra of the inclusion complexes, absence of the characteristic peaks of HT and shifts of the CDs peaks were observed, showing an interaction between the molecules. NMR suggested a stronger complex formed between ß-CD and HT. The EE% of ß-CD/HT (1:1 and 2:1) complexes and α-CD/HT (1:1) complex was found to be higher (83%, 76%, 78%, respectively), compared to α-CD/HT (2:1) (51%). Data obtained support the encapsulation of HT in both CDs, revealing a potential interaction between them and an improvement in HT's thermal stability. Regarding the in vitro release study, both CD complexes had similar behavior and a controlled release of HT in the intestinal site was observed. PRACTICAL APPLICATION: The encapsulation of hydroxytyrosol in cyclodextrins resulted in white amorphous food-grade powders with no aroma and taste. Incorporation of these powders in foods could lead to an increase in their antioxidant content and offer an additional nutritional value.

A guanidino-γ-cyclodextrin superdimer generates a twin receptor for phosphate dimers assembled by anti-electrostatic hydrogen bonds.

Octakis-6-guadinidino-γ-cyclodextrin (gguan) hydrochloride in the presence of phosphates crystallises from aqueous solution in the unprecedented form of a superdimer (dimer-within-a-dimer). The self-assembly exposes four circular octa-guanidinium regions that bind and stabilise discrete H-bonded phosphate anion dimers. The small (∼2 nm) gguan-phosphate assembly is preorganised and stable in aqueous solution, as demonstrated by DLS and NMR experiments.

Design and Synthesis of Porphyrin-Nitrilotriacetic Acid Dyads with Potential Applications in Peptide Labeling through Metallochelate Coupling.

The need to detect and monitor biomolecules, especially within cells, has led to the emerging growth of fluorescent probes. One of the most commonly used labeling techniques for this purpose is reversible metallochelate coupling via a nitrilotriacetic acid (NTA) moiety. In this study, we focus on the synthesis and characterization of three new porphyrin-NTA dyads, TPP-Lys-NTA, TPP-CC-Lys-NTA, and Py 3 P-Lys-NTA composed of a porphyrin derivative covalently connected with a modified nitrilotriacetic acid chelate ligand (NTA), for possible metallochelate coupling with Ni2+ ions and histidine sequences. Emission spectroscopy studies revealed that all of the probes are able to coordinate with Ni2+ ions and consequently can be applied as fluorophores in protein/peptide labeling applications. Using two different histidine-containing peptides as His6-tag mimic, we demonstrated that the porphyrin-NTA hybrids are able to coordinate efficiently with the peptides through the metallochelate coupling process. Moving one step forward, we examined the ability of these porphyrin-peptide complexes to penetrate and accumulate in cancer cells, exploring the potential utilization of our system as anticancer agents.

Unsymmetrical, monocarboxyalkyl meso-arylporphyrins in the photokilling of breast cancer cells using permethyl-ß-cyclodextrin as sequestrant and cell uptake modulator.

In the search for photosensitizers with chemical handles to facilitate their integration into complex drug delivery nanosystems, new, unsymmetrically substituted, water insoluble meso-tetraphenylporphyrin and meso-tetra(m-hydroxyphenyl)porphyrin derivatives bearing one carboxyalkyl side chain were synthesized. Permethyl-ß-cyclodextrin (pMßCD) was their ideal monomerizing host and highly efficient shuttle to transfer them into water. New assembly modes of the extremely stable (Kbinding > 1012 M-2) 2:1 complexes were identified. The complexes are photostable and do not disassemble in FBS-containing cell culture media for 24 h. Incubation of breast cancer MCF-7 cells with the complexes results in intense intracellular fluorescence, strongly enhanced in the endoplasmic reticulum (ER), high photokilling efficiency (~90%) and low dark toxicity. pMßCD stands out as a very capable molecular isolator of mono-carboxyalkyl-arylporphyrins that increases uptake and modulates their localization in the cells. The most efficient porphyrins are envisaged as suitable photosensitizers that can be linked to biocompatible drug carriers for photo- and chemo-therapy applications.

A Self-locked ß-Cyclodextrin-rhodamine B Spirolactam with Photoswitching Properties.

Supramolecular organization and self-assembly are the pillars of functionality of many nanosystems. The covalent conjugate (6-spirolactam rhodamine B-6-monodeoxy)-ß-cyclodextrin (Rho-ßCD) is assembled as a self-included, rigid nanostructure, identical in the crystal and in aqueous solution, as revealed by detailed X-ray and NMR analyses. Rho-ßCD self-assembly is the result of an interesting reaction pathway, which partially de-aggregates Rho and disturbs the zwitterion↔spirolactone equilibrium. Rho-ßCD is stable at pH 4.6, but displays controllable photoswitching between the colored, fluorescent, zwitterionic and the colorless, non-fluorescent closed structures, during several iterative cycles. After an initial drop in absorbance, the on-off process continues without further changes under our irradiation conditions, a consequence of the specific self-locked arrangement of Rho in the cavity. Rho-ßCD exemplifies a water soluble photoresponsive nanosystem with improved photostability suggesting promising applications in super resolution bioimaging.

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.

Suppressing the Photocatalytic Activity of Zinc Oxide Electron-Transport Layer in Nonfullerene Organic Solar Cells with a Pyrene-Bodipy Interlayer.

Organic solar cells based on nonfullerene acceptors have recently witnessed a significant rise in their power conversion efficiency values. However, they still suffer from severe instability issues, especially in an inverted device architecture based on the zinc oxide bottom electron transport layers. In this work, we insert a pyrene-bodipy donor-acceptor dye as a thin interlayer at the photoactive layer/zinc oxide interface to suppress the degradation reaction of the nonfullerene acceptor caused by the photocatalytic activity of zinc oxide. In particular, the pyrene-bodipy-based interlayer inhibits the direct contact between the nonfullerene acceptor and zinc oxide hence preventing the decomposition of the former by zinc oxide under illumination with UV light. As a result, the device photostability was significantly improved. The π-π interaction between the nonfullerene acceptor and the bodipy part of the interlayer facilitates charge transfer from the nonfullerene acceptor toward pyrene, which is followed by intramolecular charge transfer to bodipy part and then to zinc oxide. The bodipy-pyrene modified zinc oxide also increased the degree of crystallization of the photoactive blend and the face-on stacking of the polymer donor molecules within the blend hence contributing to both enhanced charge transport and increased absorption of the incident light. Furthermore, it decreased the surface work function as well as surface energy of the zinc oxide film all impacting in improved power conversion efficiency values of the fabricated cells with champion devices reaching values up to 9.86 and 11.80% for the fullerene and nonfullerene-based devices, respectively.

Porphyrinoid-Cyclodextrin Assemblies in Biomedical Research: An Update.

Porphyrinoids, well-known cofactors in fundamental processes of life, have stimulated interest as synthetic models of natural systems and integral components of photodynamic therapy, but their utilization is compromised by self-aggregation in aqueous media. The capacity of cyclodextrins to include hydrophobic molecules in their cavity provides porphyrinoids with a protective environment against oxidation and the ability to disperse efficiently in biological fluids. Moreover, engineered cyclodextrin-porphyrinoid assemblies enhance the photodynamic abilities of porphyrinoids, can carry chemotherapeutics for synergistic modalities, and can be enriched with functions including cell recognition, tissue penetration, and imaging. This Perspective includes synthetic porphyrinoid-cyclodextrin models of proteins participating in fundamental processes, such as enzymatic catalysis, respiration, and electron transfer. In addition, since porphyrinoid-cyclodextrin systems comprise third generation photosensitizers, recent developments for their utilization in photomedicine, that is, multimodal therapy for cancer (e.g., PDT, PTT) and antimicrobial treatment, and eventually in biocompatible therapeutic or diagnostic platforms for next-generation nanomedicine and theranostics are discussed.

Homo- and hetero-difunctionalized ß-cyclodextrins: Short direct synthesis in gram scale and analysis of regiochemistry.

The regioselective difunctionalization of cyclodextrins (CDs) leading to derivatives amenable to further transformations is a daunting task due to challenging purification and unambiguous characterization of the obtained regioisomers with similar physicochemical properties. The primary-side homo-difunctionalization of ß-CD can lead to three regioisomers, while the hetero-difunctionalization can generate three pairs of pseudoenantiomers. Previously, approaches with several synthetic steps, expensive reagents, high purification demands and low yields of the products have been employed. Herein we present direct, short and efficient primary-side difunctionalization strategies featuring reproducibility, ease of product purification, scalability of the reactions and versatility of the substituents introduced. Specifically, the prepared ditosylated ß-CDs were separated using preparative reversed-phase column chromatography and their structures were elucidated by NMR experiments. Azidation led to the corresponding pure diazido regioisomers. Direct monotosylation of 6-monoazido-ß-CD or monoazidation of the single regioisomers 6A,6X-ditosyl-ß-CDs afforded hetero-difunctionalized 6A-monoazido-6X-tosyl-ß-CDs in significant yields. Overall, the single regioisomers, 6A,6X-ditosyl-, 6A,6X-diazido- and 6A-monoazido-6X-monotosyl-ß-CD were prepared in one or two steps and purified in multigram scale thus opening the way towards further selective and orthogonal functionalizations of ß-CD hosts.

Stability and reactivity of γ-ΜPTMS silane in some commercial primer and adhesive formulations.

OBJECTIVES: To evaluate the stability and reactivity of γ-methacryloxypropyl trimethoxysilane (MPTMS) in commercially available primers and adhesives. METHODS: Four representative primer formulations [Calibra Silane Coupling Agent/Dentsply (CLB), G-Multi Primer/GC (GMP), Kerr Silane Primer/Kerr (KSP), Monobond Plus/Ivoclar Vivadent (MBP)] and a universal adhesive [Scotchbond Universal/3M ESPE (SBU)] containing MPTMS were analyzed spectroscopically. For the stability study, the silanol content was evaluated in bulk solutions as received (reference-RE) and after aging (AG, 48°C/1month) by 1H, 13C, 31P NMR and in fresh films by transmission FTIR analysis (TIR, films applied on Ge windows after solvent evaporation). The reactivity, as expressed by the siloxane formation capacity of the RE products, was evaluated by micro-multiple internal reflectance FTIR analysis (MIR, films applied on Ge crystals) after drying and ethanol rinsing (t0) and following 1 (t1) and 24h (t24) storage (air/37°C). RESULTS: NMR and TIR showed ∼60% MPTMS silanol groups in RE-CLB, with the other (∼40%) groups being methylated or ethoxylated. In AG-CLB, the silanol peaks further decreased, while ethoxylation and siloxane derivatization increased. In all other products and aging conditions no silanols were traced and formation of small- and large-size MPTMS derivatives was evident. Apart from the 10-MDP molecule, phosphorous impurities were identified in all RE specimens (2-5%), which after AG reached a maximum value of 15% (MBP). MIR analysis showed siloxane formation in all products, regardless the presence of free silanols (t1), which further increased at t24 especially in CLB, GMP and MBP. SIGNIFICANCE: MPTMS silanols are very sensitive to mild thermal aging. Incorporation of MPTMS in the same vials with adhesive and conventional methacrylate monomers, results in derivatization with no detectable silanols, even in fresh materials. The condensates formed may induce additional siloxane formation due to residual activity, which greatly varies among the materials tested. These may have a detrimental effect on MPTMS silanol chemisorption and bonding capacity.

NMR and EPR Structural Analysis and Stability Study of Inverse Vulcanized Sulfur Copolymers.

Sulfur copolymers with high sulfur content find a broad range of applications from Li-S batteries to catalytic processes, self-healing materials, and the synthesis of nanoparticles. Synthesis of sulfur-containing polymers via the inverse vulcanization technique gained a lot of attention due to the feasibility of the reaction to produce copolymers with high sulfur content (up to 90 wt %). However, the interplay between the cross-linker and the structure of the copolymers has not yet been fully explored. In the present work, the effect of the amount of 1,3-diisopropenyl benzene (DIB) cross-linker on the structural stability of the copolymer was thoroughly investigated. Combining X-ray diffraction and differential scanning calorimetry, we demonstrated the partial depolymerization of sulfur in the copolymer containing low amount of cross-linker (<30 wt % DIB). On the other hand, by applying NMR and electron paramagnetic resonance techniques, we have shown that increasing the cross-linker content above 50 wt % leads to the formation of radicals, which may severely degrade the structural stability of the copolymer. Thus, an optimum amount of cross-linker is essential to obtain a stable copolymer. Moreover, we were able to detect the release of H2S gas during the cross-linking reaction as predicted based on the abstraction of hydrogen by the sulfur radicals and therefore we emphasize the need to take appropriate precautions while implementing the inverse vulcanization reaction.

Molecular recognition of N-acetyltryptophan enantiomers by ß-cyclodextrin.

The enantioselectivity of ß-cyclodextrin (ß-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that ß-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only ß-CD-L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex ß-CD-D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the ß-CD-L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the ß-CD-L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host-guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with ß-CD.

Positively charged cyclodextrins as effective molecular transporters of active phosphorylated forms of gemcitabine into cancer cells.

Positively charged cyclodextrins (PCCDs) are molecular carriers of particular interest for their ability to readily enter into cancer cells. Of main interest, guanidino- and aminoalkyl- PCCDs can be conveniently synthesized and form stable and strong inclusion complexes with various active molecules bearing phosphate groups. We have addressed here the challenge to deliver into cancer cells phosphorylated gemcitabine drugs well known for their instability and inability to permeate cell membranes. NMR data corroborated by semiempirical theoretical calculations have shown that aminoalkyl-CDs form sufficiently stable complexes with both mono- and tri-phosphate forms of gemcitabine by simple mixing of the compounds in aqueous solution at physiological pH. Confocal microscopy and radioactivity counting experiments revealed that the developed systems enabled phosphorylated gemcitabine to penetrate efficiently into aggressive human breast cancer cells (MCF7), eventually leading to a substantial reduction of IC50 values. Moreover, compared to free drugs, phosphorylated metabolites of gemcitabine encapsulated in PCCDs displayed improved in vitro activities also on the aggressive human cancer cells CCRF-CEM Ara-C/8 C, a nucleoside transport-deficient T leukemia cell line. The current study offers the proof-of-principle that phosphorylated nucleoside drugs could be efficiently transported by PCCDs into cancer cells.

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.

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.