Ângelo M.L. Denadai

Supramolecular self-assembly of cyclodextrin and higher water soluble guest: thermodynamics and topological studies.

The supramolecular interactions between Imipramine hydrochloride (IMI), a tricyclic antidepressant, and beta-cyclodextrin (betaCD) have been investigated by experimental techniques and theoretical calculations. The association between these molecules might be lead to a host/guest compound, in which the physical chemistry properties of the guest molecule, such as high solubility, can be decreased. These new properties acquired by the inclusion phenomena are important to develop a strategy for pharmaceutical formulation. Nuclear magnetic resonance and horizontal attenuated total reflectance provided relevant information on the complex stoichiometries and the sites of interactions between the host and guest molecules. Stoichiometries of 1:2, 1:1, and 2:1 betaCD/IMI have been detected in solution. Self-diffusion coefficient and dynamic light scattering analysis provided information on the self-aggregation of the complex. Also, isothermal titration calorimetry studies indicated the existence of equilibrium between different complexes in solution. In order to determine the preferred arrangement for the inclusion complex formed by the IMI molecule and betaCD, theoretical calculations were performed. Of all proposed supramolecular structures, the 2:1 betaCD/IMI complex was calculated to be the most energetically favorable, in both gas and aqueous phases. The calculations indicated that the intermolecular hydrogen bonds involving the hydroxyl groups of betaCD play a major role in stabilizing the supramolecular 2:1 structure, corroborating experimental findings.

Superstructure based on β-CD self-assembly induced by a small guest molecule

The size, shape and surface chemistry of nanoparticles play an important role in cellular interaction. Thus, the main objective of the present study was the determination of the β-cyclodextrin (β-CD) self-assembly thermodynamic parameters and its structure, aiming to use these assemblies as a possible controlled drug release system. Light scattering measurements led us to obtain the β-CDs critical aggregation concentration (cac) values, and consequently the thermodynamic parameters of the β-CD spontaneous self-assembly in aqueous solution: Δ(agg)G(o) = -16.31 kJ mol(-1), Δ(agg)H(o) = -26.48 kJ mol(-1) and TΔ(agg)S(o) = -10.53 kJ mol(-1) at 298.15 K. Size distribution of the self-assembled nanoparticles below and above cac was 1.5 nm and 60-120 nm, respectively. The number of β-CD molecules per cluster and the second virial coefficient were identified through Debyes plot and molecular dynamic simulations proposed the three-fold assembly for this system below cac. Ampicillin (AMP) was used as a drug model in order to investigate the key role of the guest molecule in the self-assembly process and the β-CD:AMP supramolecular system was studied in solution, aiming to determine the structure of the supramolecular aggregate. Results obtained in solution indicated that the β-CDs cac was not affected by adding AMP. Moreover, different complex stoichiometries were identified by nuclear magnetic resonance and isothermal titration calorimetry experiments.

Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii

ABSTRACT Cryptococcus gattii is the main etiological agent of cryptococcosis in immunocompetent individuals. The triazole drug itraconazole is one of the antifungals used to treat patients with cryptococcosis. Heteroresistance is an adaptive mechanism to counteract the stress of increasing drug concentrations, and it can enhance the ability of a microorganism to survive under antifungal pressure. In this study, we evaluated the ability of 11 C. gattii strains to develop itraconazole heteroresistance. Heteroresistant clones were analyzed for drug susceptibility, alterations in cell diameter, capsule properties, and virulence in a murine model. Heteroresistance to itraconazole was intrinsic in all of the strains analyzed, reduced both the capsule size and the cell diameter, induced molecular heterogeneity at the chromosomal level, changed the negatively charged cells, reduced ergosterol content, and improved the antioxidant system. A positive correlation between surface/volume ratio of original cells and the level of heteroresistance to itraconazole (LHI) was observed in addition to a negative correlation between capsule size of heteroresistant clones and LHI. Moreover, heteroresistance to itraconazole increased the engulfment of C. gattii by macrophages and augmented fungal proliferation inside these cells, which probably accounted for the reduced survival of the mice infected with the heteroresistant clones and the higher fungal burden in lungs and brain. Our results indicate that heteroresistance to itraconazole is intrinsic and increases the virulence of C. gattii. This phenomenon may represent an additional mechanism that contributes to relapses of cryptococcosis in patients during itraconazole therapy.

Development of Sulfadiazine-Decorated PLGA Nanoparticles Loaded with 5-Fluorouracil and Cell Viability

The aim of this work was to synthesize sulfadiazine-poly(lactide-co-glycolide) (SUL-PLGA) nanoparticles (NPs) for the efficient delivery of 5-fluorouracil to cancer cells. The SUL-PLGA conjugation was assessed using FTIR, 1H-NMR, 13C-NMR, elemental analysis and TG and DTA analysis. The SUL-PLGA NPs were characterized using transmission and scanning electron microscopy and dynamic light scattering. Additionally, the zeta potential, drug content, and in vitro 5-FU release were evaluated. We found that for the SUL-PLGA NPs, Dh = 114.0 nm, ZP = −32.1 mV and the encapsulation efficiency was 49%. The 5-FU was released for up to 7 days from the NPs. Cytotoxicity evaluations of 5-FU-loaded NPs (5-FU-SUL-PLGA and 5-FU-PLGA) on two cancer cell lines (Caco-2, A431) and two normal cell lines (fibroblast, osteoblast) were compared. Higher cytotoxicity of 5-FU-SUL-PLGA NPs were found to both cancer cell lines when compared to normal cell lines, demonstrating that the presence of SUL could significantly enhance the cytotoxicity of the 5-FU-SUL-PLGA NPs when compared with 5-FU-PLGA NPs. Thus, the development of 5-FU-SUL-PLGA NPs to cancer cells is a promising strategy for the 5-FU antitumor formulation in the future.

Structural and thermodynamic characterization of doxycycline/β-cyclodextrin supramolecular complex and its bacterial membrane interactions.

Doxycycline is a semi-synthetic antibiotic commonly used for the treatment of many aerobic and anaerobic bacteria. It inhibits the activity of matrix metalloproteinases (MMPs) and affects cell proliferation. In this study, the structural and thermodynamic parameters of free DOX and a DOX/βCD complex were investigated, as well as their interactions and effects on Staphylococcus aureus cells and cellular cytotoxicity. Complexation of DOX and βCD was confirmed to be an enthalpy- and entropy-driven process, and a low equilibrium constant was obtained. Treatment of S. aureus with higher concentrations of DOX or DOX/βCD resulted in an exponential decrease in S. aureus cell size, as well as a gradual neutralization of zeta potential. These thermodynamic profiles suggest that ion-pairing and hydrogen bonding interactions occur between DOX and the membrane of S. aureus. In addition, the adhesion of βCD to the cell membrane via hydrogen bonding is hypothesized to mediate a synergistic effect which accounts for the higher activity of DOX/βCD against S. aureus compared to pure DOX. Lower cytotoxicity and induction of osteoblast proliferation was also associated with DOX/βCD compared with free DOX. These promising findings demonstrate the potential for DOX/βCD to mediate antimicrobial activity at lower concentrations, and provides a strategy for the development of other antimicrobial formulations.

Chlorhexidine/losartan ionic pair binding and its nanoprecipitation: physico-chemical characterisation and antimicrobial activity

Chlorhexidine is a widely used, di-cationic, broad-spectrum antimicrobial agent and losartan is a well-known, anionic-specific antagonist of AT1 renin–angiotensin receptor that acts as an anti-hypertensive agent. The combination of these molecules gives a chlorhexidine di-losartanate (ClxLos2) hydrophobic ion pair that spontaneously aggregates into nanoparticles (NPs). This work investigated the formation of ClxLos2 NPs using the analysis of the solid state by fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry and scanning electron microscopy and in aqueous environment by calorimetric, zeta potential and dynamic light scattering titrations. Furthermore, to demonstrate the potential antimicrobial activity of ClxLos2, in vitro antibacterial tests were conducted against Staphylococcus aureus (ATCC 27664), Streptococcus viridans (ATCC 11563) and Enterococcus faecalis (ATCC 14508). Based on these studies, it is proposed that ClxLos2 could be used for controlled drug release based on ionic dissociation during dilution, thereby avoiding the use of any solid matrix.

Control of size in losartan/copper(II) coordination complex hydrophobic precipitate

Reaction of highly soluble orally active, non-peptide antihypertensive drug losartan with copper(II) leads to the spontaneous formation of a very insoluble 2:1 covalent complex, which self assembles in a hydrophobic supramolecular structure of nanometric dimensions. Thermal analysis showed that Los/Cu(II) complex presents intermediate stability in comparison with its precursors KLos and Cu(OAc)2·H2O. Isothermal titration calorimetry indicated complexation to be a stepwise process, driven by enthalpy and entropy. Zeta potential and DLS measurements showed that it is possible to control the size and charge of nanoprecipitates by adjusting the relative concentration of Los(-) and Cu(II).

Investigação eletroquímica e calorimétrica da interação de novos agentes antitumorais biscatiônicos com DNA

Biscationic amidines bind in the DNA minor groove and present biological activity against a range of infectious diseases. Two new biscationic compounds (bis-α,ω-S-thioureido, amino and sulfide analogues) were synthesized in good yields and fully characterized, and their interaction with DNA was also investigated. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic properties of binding interactions between DNA and these ligands. A double stranded calf thymus DNA immobilized on an electrode surface was used to study the possible DNA-interacting abilities of these compounds towards dsDNA in situ. A remarkable interaction of these compounds with DNA was demonstrated and their potential application as anticancer agents was furthered.

Physicochemical characterization and biological activities of the ethanol extract of Bryophyllum pinnatum (Lam.) Oken incorporated in β-cyclodextrin

In the present work, a phytocomplex formed by ethanol extract of Bryophyllum pinnatum (Lam.) Oken (EEBP) and β-cyclodextrin (βCD) has been prepared at weight proportion of 1:1, characterized through several physical chemistry methods, and incorporated in an oil-in-water emulsion in order to evaluate its topical anti-inflammatory effect. Changes in the spectra of infrared and UV/VIS as well as TGA and DTA curves suggested the existence of interactions between components of extract and βCD. The incorporation of βCD to the extract also promoted an increase in the zeta potential of the aggregates spontaneously formed in water, with consequent reduction of their size. The in vitro antioxidant activity was also evaluated, showing an improvement of the radical-scavenging ability in presence of βCD. Finally, the topical application of EEBP/βCD semi-solid formulation significantly inhibited the ear edema induced by Croton oil if compared with free EEBP, justifying the use of phytotherapic formulation based on B. pinnatum as a remedy for skin disorders.

A long-lasting oral preformulation of the angiotensin II AT1 receptor antagonist losartan

Abstract Losartan (Los), a non-peptidic orally active agent, reduces arterial pressure through specific and selective blockade of angiotensin II receptor AT1. However, this widely used AT1 antagonist presents low bioavailability and needs once or twice a day dosage. In order to improve its bioavailability, we used the host: guest strategy based on β-cyclodextrin (βCD). The results suggest that Los included in βCD showed a typical pulsatile release pattern after oral administration to rats, with increasing the levels of plasma of Los. In addition, the inclusion compound presented oral efficacy for 72 h, in contrast to Los alone, which shows antagonist effect for only 6 h. In transgenic (mREN2)L27 rats, the Los/βCD complex reduced blood pressure for about 6 d, whereas Los alone reduced blood pressure for only 2 d. More importantly, using this host: guest strategy, sustained release of Los for over a week via the oral route can be achieved without the need for encapsulation in a polymeric carrier. The proposed preformulation increased the efficacy reducing the dose or spacing between each dose intake.