Synergistic antifungal activity against Candida albicans between voriconazole and cyclosporine a loaded in polymeric nanoparticles.

The goal of this work is to investigate if the synergistic antifungal activity between cyclosporine A, CsA, and voriconazole, VRZ, increases when both drugs are encapsulated in a nanocarrier as compared when they are free. The preparation and characterization of blank and VRZ and CsA loaded polymeric based PLGA nanoparticles (PLGA, PLGA-PEG, and PLGA+PEG) was a necessary previous step. Using the more suitable NPs, those of PLGA, the antifungal susceptibility tests performed with VRZ-loaded PLGA NPs, show no significant increase of the antifungal activity in comparison to that of free VRZ. However, the synergistic behavior found for the (VRZ+CsA)-loaded PLGA NPs was fourfold stronger than that observed for the two free drugs together. On the other hand, the investigation into the suppression of C. albicans biofilm formation showed that blank PLGA NPs inhibit the biofilm formation at high NPs concentrations. However, a minor effect or even a slight biofilm increase formation was observed at low and moderate NPs concentrations. Therefore, the enhancement of the biofilm inhibition found for the three tested treatments (CsA alone, VRZ alone, and VRZ+CsA) when comparing free and encapsulated drugs, within the therapeutic window, can be attributed to the drug encapsulation approach. Indeed, polymeric PLGA NPs loaded with CsA, VRZ, or VRZ+CsA are more effective at inhibiting the C. albicans biofilm growth than their free counterparts.

Biocompatible metal-organic frameworks as promising platforms to eradicate HIV reservoirs ex vivo in people living with HIV.

The HIV attacks the immune system provoking an infection that is considered a global health challenge. Despite antiretroviral treatments being effective in reducing the plasma viral load in the blood to undetectable levels in people living with HIV (PLWH), the disease is not cured and has become chronic. This happens because of the existence of anatomical and cellular viral reservoirs, mainly located in the lymph nodes and gastrointestinal tract, which are composed of infected CD4+ T cells with a resting memory phenotype and inaccessible to antiretroviral therapy. Herein, a new therapeutic strategy based on nanotechnology is presented. Different combinations of antiretroviral drugs (bictegravir/tenofovir/emtricitabine and nevirapine/tenofovir/emtricitabine) and toll-like receptor agonists were encapsulated into metal-organic frameworks (MOFs) PCN-224 and ZIF-8. The encapsulation efficiencies of all the drugs, as well as their release rate from the carriers, were measured. In vitro studies about the cell viability, the hemocompatibility, and the platelet aggregation of the MOFs were carried out. Epifluorescence microscopy assays confirmed the ability of ZIF-8 to target a carboxyfluorescein probe inside HeLa cell lines and PBMCs. These results pave the way for the use of these structures to eliminate latent HIV reservoirs from anatomical compartments through the activation of innate immune cells, and a higher efficacy of the triplet combinations of antiretroviral drugs.

A novel biocompatible polymer derived from D-mannitol used as a vector in the field of genetic engineering of eukaryotic cells.

The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from D-mannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid's conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.

Single -and Multi-Walled Carbon Nanotubes as Nanocarriers for the Delivery of 7-Hydroxyflavone.

The research on flavonoids has exponentially grown since their first therapeutic evidence, in 1937. They are effective in vitro in a wide range of human diseases, particularly those mediated by free radicals, such as cancer, atherosclerosis, AIDS, or neuronal diseases. However, their applications have been reduced due to their low solubility, poor absorption, and rapid metabolism. Flavonoid encapsulation in nanocarriers significantly improves their oral absorption, protects the drug against degradation, decreases the first-pass hepatic effect, and makes absorption through the lymphatic system easier. In this work, carbon nanotubes were used as nanocarriers of 7-hydroxyflavone, 7-HF. The encapsulation of 7-HF into pristine single- and multi-walled carbon nanotubes, and into -COOH functionalized single-walled carbon nanotubes has been investigated. The equilibrium association constants were estimated. The structural backbone of 7-HF, two benzene rings linked through three carbon atoms that form a pyran heterocyclic ring containing a keto group, seems to play a key role in the 7-HF/CNT interactions, although other types of interactions are also at work. The in vitro release of 7-HF was studied at three pHs, 2.0, 7.4, and 9.2, mimicking the different biological barriers of the human organism.

Supramolecular Systems for Gene and Drug Delivery.

Several biomaterial-based supramolecular systems (cyclodextrins [...].

Multivalent Calixarene-Based Liposomes as Platforms for Gene and Drug Delivery.

The formation of calixarene-based liposomes was investigated, and the characterization of these nanostructures was carried out using several techniques. Four amphiphilic calixarenes were used. The length of the hydrophobic chains attached to the lower rim as well as the nature of the polar group present in the upper rim of the calixarenes were varied. The lipid bilayer was formed with one calixarene and with the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE. The cytotoxicity of the liposomes for various cell lines was also studied. From the results obtained, the liposomes formed with the least cytotoxic calixarene, (TEAC12)4, were used as nanocarriers of both nucleic acids and the antineoplastic drug doxorubicin, DOX. Results showed that (TEAC12)4/DOPE/p-EGFP-C1 lipoplexes, of a given composition, can transfect the genetic material, although the transfection efficiency substantially increases in the presence of an additional amount of DOPE as coadjuvant. On the other hand, the (TEAC12)4/DOPE liposomes present a high doxorubicin encapsulation efficiency, and a slow controlled release, which could diminish the side effects of the drug.

Cationic Single-Chained Surfactants with a Functional Group at the End of the Hydrophobic Tail DNA Compacting Efficiency.

The interaction between calf-thymus DNA, ctDNA, and various single-chained surfactants with different functional groups at the end of hydrophobic tail was studied with the goal of investigating the influence of the functional group nature on surfactant DNA compacting efficiency. The surfactants investigated were dodecyltriethylammonium bromide (DTEABr), triethyl(1-phenoxydodecyl)ammonium bromide (12PhBr), triethyl(2-naphthoxydodecyl)ammonium bromide (12NBr) and 11-(isonicotinoyloxy)-N,N,N-triethyl-1-undecanaminium bromide (11PyBr). Results made evident that the surfactants' tendencies to self-aggregation is the key factor determining their efficiency to compact the nucleic acid. Subsequently, DOPE/12NBr/pEGFP-C1 lipoplexes, with different cationic surfactant molar fractions (α) and mass ratios (L/D), were prepared and characterized. DOPE is a zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the plasmid pEGFP-C1 carries a GFP coding sequence with the necessary regulatory elements for constitutive expression of the gene in human cells. 12NBr was chosen because it was the most efficient DNA compacting agent among the surfactants investigated. Finally, the cytotoxicity and transfection efficiency (TE) of DOPE/12NBr/pDNA lipoplexes, with different compositions, were investigated.

Potentiometric Study of Carbon Nanotube/Surfactant Interactions by Ion-Selective Electrodes. Driving Forces in the Adsorption and Dispersion Processes.

The interaction (adsorption process) of commercial ionic surfactants with non-functionalized and functionalized carbon nanotubes (CNTs) has been studied by potentiometric measurements based on the use of ion-selective electrodes. The goal of this work was to investigate the role of the CNTs' charge and structure in the CNT/surfactant interactions. Non-functionalized single- (SWCNT) and multi-walled carbon nanotubes (MWCNT), and amine functionalized SWCNT were used. The influence of the surfactant architecture on the CNT/surfactant interactions was also studied. Surfactants with different charge and hydrophobic tail length (sodium dodecyl sulfate (SDS), octyltrimethyl ammonium bromide (OTAB), dodecyltrimethyl ammonium bromide (DoTAB) and hexadecyltrimethyl ammonium bromide (CTAB)) were studied. According to the results, the adsorption process shows a cooperative character, with the hydrophobic interaction contribution playing a key role. This is made evident by the correlation between the free surfactant concentration (at a fixed [CNT]) and the critical micellar concentration, cmc, found for all the CNTs and surfactants investigated. The electrostatic interactions mainly determine the CNT dispersion, although hydrophobic interactions also contribute to this process.

Metallo-Liposomes of Ruthenium Used as Promising Vectors of Genetic Material.

Gene therapy is a therapeutic process consisting of the transport of genetic material into cells. The design and preparation of novel carriers to transport DNA is an important research line in the medical field. Hybrid compounds such as metallo-liposomes, containing a mixture of lipids, were prepared and characterized. Cationic metal lipids derived from the [Ru(bpy)3]2+ complex, RuC11C11 or RuC19C19, both with different hydrophobic/lipophilic ratios, were mixed with the phospholipid DOPE. A relation between the size and the molar fraction α was found and a multidisciplinary study about the interaction between the metallo-liposomes and DNA was performed. The metallo-liposomes/DNA association was quantified and a relationship between Kapp and α was obtained. Techniques such as AFM, SEM, zeta potential, dynamic light scattering and agarose gel electrophoresis demonstrated the formation of lipoplexes and showed the structure of the liposomes. L/D values corresponding to the polynucleotide's condensation were estimated. In vitro assays proved the low cell toxicity of the metallo-liposomes, lower for normal cells than for cancer cell lines, and a good internalization into cells. The latter as well as the transfection measurements carried out with plasmid DNA pEGFP-C1 have demonstrated a good availability of the Ru(II)-based liposomes for being used as non-toxic nanovectors in gene therapy.

Influence of the degree of oligomerization of surfactants on the DNA/surfactant interaction.

The interaction between calf thymus DNA, ctDNA, and a series of oligomeric surfactants derived from N-benzyl-N,N-dimethyl-N-(1-dodecyl)ammonium chloride is investigated. The influence of the surfactants' degree of oligomerization (2, 3 and 4) on the ctDNA/surfactant interaction is studied, as well as the effect of the structure of the spacer group linking the individual surfactant fragments. In particular, the effect of the distance between the positive charges and the hydrophobic chains within the oligomers on these interactions was examined, by using the three positional isomers (i.e., ortho-, meta-, and para-) with the rigid xylidene moiety as spacer. Results show that the dimeric ("gemini") surfactants are much more efficient in the inversion of the nucleic acid charge than the single-chained (monomeric) surfactant. Whereas the ortho - isomer causes a partial condensation, the meta - and para - isomers can completely condense ctDNA. The meta - and para - isomers of the trimeric surfactants can also completely condense the polynucleotide. In contrast, the tetrameric surfactant investigated does not change the morphology of the nucleic acid from an elongated coil into a compacted form, in spite of effectively inverting the nucleic acid's charge in their complex. Accordingly, the capacity for ctDNA compaction of oligomeric surfactants is not simply correlated to their degree of oligomerization, but depends on a complex balance of the number and relative distance of cationic charges and/or hydrophobic tails in the surfactants for effectively interacting with the nucleic acid to form the appropriate complex. This information will help to design more effective cationic surfactants as non-viral vectors for gene therapy.

Assessment of the denaturation of collagen protein concentrates using different techniques.

The use of collagen and gelatin in the field of regenerative medicine is widely extended. However, most of the studies in this topic are focused on the scaffolds' properties, but only a few are related to the properties of the raw material used. The raw material analysis not only consists of a study of the composition, but also of the denaturation degree that can influence the processing and properties of the structure of the scaffold. Thus, the denaturation degree analysis of different collagen proteins was performed and assessed by the comparison of four different methods: differential scanning calorimetry (DSC), Fourier transform Infrared Spectroscopy (FTIR) and circular dichroism (CD) spectra and sulfhydryls content analysis. DSC measurements put forward a glass transition between 88°C and 95°C as well as from the FTIR measurements; the characteristic peaks for proteins are evidenced. However, from the sulfur content, only a small proportion of free sulfhydryls are present with respect to their total amount. In addition, CD spectra allow to estimate the secondary structure of the protein by the analysis of the α-helix and ß-strand and also quantify the denaturation degree with the 'positive/negative ratio' (RPN) from the CD profiles, obtaining values in the range between 25% and 100%.

Optimized Preparation of Levofloxacin Loaded Polymeric Nanoparticles.

In this work, poly(lactic-co-glycolic acid) (PLGA) and chitosan (CS) nanoparticles were synthesized with the purpose of encapsulating levofloxacin (LEV). A thorough study has been carried out in order to optimize the preparation of LEV-loaded polymeric nanoparticles (NPs) suitable for parenteral administration. Changes in the preparation method, in the organic solvent nature, in the pH of the aqueous phase, or in the temperature were investigated. To the authors´ knowledge, a systematic study in order to improve the LEV nanocarrier characteristics and the yield of drug encapsulation has not been carried out to date. The physicochemical characterization of the NPs, their encapsulation efficiency (EE), and the in vitro release of LEV revealed that the best formulation was the emulsion-solvent evaporation method using dichloromethane as organic solvent, which renders suitable LEV loaded PLGA NPs. The morphology of these NPs was investigated using TEM. Their antimicrobial activities against several microorganisms were determined in vitro measuring the minimum inhibitory concentration (MIC). The results show that the use of these loaded LEV PLGA nanoparticles has the advantage of the slow release of the antibiotic, which would permit an increase in the time period between administrations as well as to decrease the side effects of the drug.

Preparation and Characterization of New Liposomes. Bactericidal Activity of Cefepime Encapsulated into Cationic Liposomes.

Cefepime is an antibiotic with a broad spectrum of antimicrobial activity. However, this antibiotic has several side effects and a high degradation rate. For this reason, the preparation and characterization of new liposomes that are able to encapsulate this antibiotic seem to be an important research line in the pharmaceutical industry. Anionic and cationic liposomes were prepared and characterized. All cationic structures contained the same cationic surfactant, N,N,N-triethyl-N-(12-naphthoxydodecyl)ammonium. Results showed a better encapsulation-efficiency percentage (EE%) of cefepime in liposomes with phosphatidylcholine and cholesterol than with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). The presence of cholesterol and the quantity of egg-yolk phospholipid in the liposome increased the encapsulation percentage. The bactericidal activity against Escherichia coli of cefepime loaded into liposomes with phosphatidylcholine was measured. The inhibitory zone in an agar plate for free cefepime was similar to that obtained for loaded cefepime. The growth-rate constant of E. coli culture was also measured in working conditions. The liposome without any antibiotic exerted no influence in such a rate constant. All obtained results suggest that PC:CH:12NBr liposomes are biocompatible nanocarriers of cefepime that can be used in bacterial infections against Escherichia coli with high inhibitory activity.

Family Support and Diabetes: Patient's Experiences From a Public Hospital in Peru.

Family support is crucial for managing chronic conditions but it is often overlooked when designing behavioral interventions in type 2 diabetes mellitus (T2DM). As part of the formative phase of a feasibility randomized control trial (RCT), we conducted 20 semistructured interviews with people with T2DM from Lima, Peru. Based on such results, we describe the support people with T2DM receive from their families and the role that such support has in their efforts to implement diabetes management practices. We learned that participants receive support from family members, but mostly from their spouses and children. Their relatives encourage them and motivate them to fight for their health, they also provide instrumental support by preparing healthy meals, reminding them to take medications, and sharing physical activity. Participants also reported controlling actions which were not always "well received." Thus, any intervention supporting self-management practices need to work with key family members. We support the literature that suggests that interventions should target family members to ensure improved T2DM self-management practices.

Importance of hydrophobic interactions in the single-chained cationic surfactant-DNA complexation.

The goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiency.

Stoppering/unstoppering of a rotaxane formed between an N-hetorycle ligand containing surfactant: ß-cyclodextrin pseudorotaxane and pentacyanoferrate(II) ions.

The assembly of a surfactant-based rotaxane by adding the labile aquopentacyanoferrate(II) ion to the previously formed pseudorotaxane between the surfactant 11-(isonicotinoyloxy)-N,N,N-triethyl-1-undecanaminium bromide and ß-cyclodextrin was investigated by 1H NMR and kinetic measurements. NMR spectroscopy has showed that the rotaxane can be formed through two different mechanisms. The rotaxane can be unstoppered by using the pyridine ligand substitution reaction by the high-field cyanide ligand. In this work a new method is developed for the preparation of several new surfactant-based rotaxanes by changing the hydrophilic and hydrophobic regions of the surfactants and the nature of the macrocycle.

P-Sulfocalix[6]arene as Nanocarrier for Controlled Delivery of Doxorubicin.

Given the high toxicity of the anthracycline antibiotic doxorubicin (DOX), it is relevant to search for nanocarriers that decrease the side effects of the drug and are able to transport it towards a therapeutic target Here, the encapsulation of DOX by p-sulfocalix[6]arene (calix) has been studied. The interaction of DOX with the macrocycle, as well as with DNA, has been investigated and the equilibrium constant for each binding process estimated. The results showed that the binding constant of DOX to DNA, KDNA , is three orders of magnitude higher than that to calix, Kcalix . The ability of calixarenes to encapsulate DOX molecules, as well as the capability of the DOX molecules included into the inner cavity of the macrocycle to bind with DNA have been examined. Cytotoxicity measurements were done in different cancer and normal cell lines to probe the decrease in the toxicity of the encapsulated DOX. The low toxicity of calixarenes has also been demonstrated for different cell lines.

Host-guest interactions between cyclodextrins and surfactants with functional groups at the end of the hydrophobic tail.

The aim of this work was to investigate the influence of the incorporation of substituents at the end of the hydrophobic tail on the binding of cationic surfactants to α-, ß-, and γ-cyclodextrins. The equilibrium binding constants of the 1:1 inclusion complexes formed follow the trend K1(α-CD)>K1(ß-CD)≫K1(γ-CD), which can be explained by considering the influence of the CD cavity volume on the host-guest interactions. From the comparison of the K1 values obtained for dodecyltriethylammonium bromide, DTEAB, to those estimated for the surfactants with the substituents, it was found that the incorporation of a phenoxy group at the end of the hydrocarbon tail does not affect K1, and the inclusion of a naphthoxy group has some influence on the association process, slightly diminishing K1. This makes evident the importance of the contribution of hydrophobic interactions to the binding, the length of the hydrophobic chain being the key factor determining K1. However, the presence of the aromatic rings does influence the location of the host and the guest in the inclusion complexes. The observed NOE interactions between the aromatic protons and the CD protons indicate that the aromatic rings are partially inserted within the host cavity, with the cyclodextrin remaining close to the aromatic rings, which could be partially intercalated in the host cavity. To the authors' knowledge this is the first study on the association of cyclodextrins with monomeric surfactants incorporating substituents at the end of the hydrophobic tail.

Binding of 12-s-12 dimeric surfactants to calf thymus DNA: Evaluation of the spacer length influence.

Several cationic dimeric surfactants have shown high affinity towards DNA. Bis-quaternary ammonium salts (m-s-m) have been the most common type of dimeric surfactants investigated and it is generally admitted that those that posses a short spacer (s≤3) show better efficiency to bind or compact DNA. However, experimental results in this work show that 12-s-12 surfactants with long spacers make the surfactant/ctDNA complexation more favorable than those with short spacers. A larger contribution of the hydrophobic interactions, which control the binding Gibbs energy, as well as a higher average charge of the surfactant molecules bound to the nucleic acid, which favors the electrostatic attractions, could explain the experimental observations. Dimeric surfactants with intermediate spacer length seem to be the less efficient for DNA binding.

Reversibility of the interactions between a novel surfactant derived from lysine and biomolecules.

In this work the novel cationic surfactant derived from lysine (S)-5-acetamido-6-(dodecylamino)-N,N,N-trimethyl-6-oxohexan-1-ammonium chloride, LYCl, was prepared and the physicochemical characterization of its aqueous solutions was carried out. The binding of LYCl to bovine serum albumin, BSA, and to double stranded calf thymus DNA, ctDNA, was investigated using several techniques. Results show that LYCl binding to BSA is followed by a decrease in the α-helix content caused by the unfolding of the protein. LYCl association to ctDNA mainly occurs through groove binding and electrostatic interactions. These interactions cause morphological changes in the polynucleotide from an elongated coil structure to a more compact globular structure, resulting in the compaction of ctDNA. Addition of ß-cyclodextrin, ß-CD, to the BSA-LYCl and ctDNA-LYCl complexes is followed by the refolding of BSA and the decompaction of ctDNA. This can be explained by the ability of ß-CD to hinder BSA-LYCl and ctDNA-LYCl interactions due to the stronger and more specific ß-CD-LYCl hydrophobic interactions. The stoichiometry of the ß-CD:LYCl inclusion complex and its formation equilibrium constant were determined in this work. The reported procedure using ß-CD is an efficient way to refold proteins and to decompact DNA, after the morphological changes caused in the biomolecules by their interaction with cationic surfactants.