Assessment of liposome disruption to quantify drug delivery in vitro

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.

Poly(vinylidene fluoride-trifluoroethylene)/NAY zeolite hybrid membranes as a drug release platform applied to ibuprofen release

Poly(vinylidene fluoride-trifluoroethylene)/NaY zeolite composite membranes were prepared by solvent casting and evaluated as a suitable drug release platform through the evaluation of loading and release of ibuprofen. The membranes were characterized at the morphological, structural and mechanical levels. The 1H-NMR spectra indicate that only the membranes with 16 and 32 % of NaY were useful for IBU encapsulation and the drug release was followed by UV-Vis spectroscopy. The release profile is independent of the zeolite content and can be described by the Korsmeyer-Peppas model. The membrane with 32 % zeolite content releases more than double IBU amount when compared with the membrane with 16 % showing that zeolite content allows tailoring membrane drug release content for specific applications. The drug release platform developed in this work is suitable for other drugs and applications.

Poly(2-hydroxyethyl methacrylate): A new star polymer

Multiarm star polymers are attractive materials due to their unusual bulk and solution properties. They are considered analogues of dendrimers with a wide range of applications, such as drug delivery, membranes, coatings and lithography.1 The advent of controlled polymerization made possible the existence of this unique class of organic nanoparticles (ONPs).2 Two major synthetic strategies are usually employed in the preparation of star polymers, the core-first and arm-first approaches. The core-first approach involves a controlled living polymerization using a multiarm initiator core while the arm-first methodology is based in the quenching of living polymers with multifunctional coupling agent or bifunctional vinyl compounds. Herein, we present the synthesis and characterization of a new star polymer, the multiarm star poly(2-hydroxyethyl methacrylate). The tetra-armed star polymer was prepared by reversible addition fragmentation chain-transfer (RAFT) polymerization using the core-first approach. The RAFT chain-transfer agent (RAFT CTA) pentaerythritol tetrakis[2-(dodecylthiocarbonothioylthio)-2-methylpropionate] was used as multiarm initiator core were 2-hydroxyethyl methacrylate (HEMA) was polymerized using AIBN as radical initiator. Structural characterization was performed by 1H NMR and FTIR. The new polymer is able to uptake large quantities of organic solvents, forming gels. The rheological behavior of these gels was also investigated.

Prediction of drug targets in human pathogens

The identification of new and druggable targets in bacteria is a critical endeavour in pharmaceutical research of novel antibiotics to fight infectious agents. The rapid emergence of resistant bacteria makes today's antibiotics more and more ineffective, consequently increasing the need for new pharmacological targets and novel classes of antibacterial drugs. A new model that combines the singular value decomposition technique with biological filters comprised of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of E. coli has been developed to predict potential drug targets in the Enterobacteriaceae family [1]. This model identified 99 potential target proteins amongst the studied bacterial family, exhibiting eight different functions that suggest that the disruption of the activities of these proteins is critical for cells. Out of these candidates, one was selected for target confirmation. To find target modulators, receptor-based pharmacophore hypotheses were built and used in the screening of a virtual library of compounds. Postscreening filters were based on physicochemical and topological similarity to known Gram-negative antibiotics and applied to the retrieved compounds. Screening hits passing all filters were docked into the proteins catalytic groove and 15 of the most promising compounds were purchased from their chemical vendors to be experimentally tested in vitro. To the best of our knowledge, this is the first attempt to rationalize the search of compounds to probe the relevance of this candidate as a new pharmacological target.

PEGylated ofloxacin nanoparticles render strong antibacterial activity against many clinically important human pathogens.

The rise of bacterial resistance against important drugs threatens their clinical utility. Fluoroquinones, one of the most important classes of contemporary antibiotics has also reported to suffer bacterial resistance. Since the general mechanism of bacterial resistance against fluoroquinone antibiotics (e.g. ofloxacin) consists of target mutations resulting in reduced membrane permeability and increased efflux by the bacteria, strategies that could increase bacterial uptake and reduce efflux of the drug would provide effective treatment. In the present study, we have compared the efficiencies of ofloxacin delivered in the form of free drug (OFX) and as nanoparticles on bacterial uptake and antibacterial activity. Although both poly(lactic-co-glycolic acid) (OFX-PLGA) and methoxy poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (OFX-mPEG-PLGA) nanoformulations presented improved bacterial uptake and antibacterial activity against all the tested human bacterial pathogens, namely, Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae and Staphylococcus aureus, OFX-mPEG-PLGA showed significantly higher bacterial uptake and antibacterial activity compared to OFX-PLGA. We have also found that mPEG-PLGA nanoencapsulation could significantly inhibit Bacillus subtilis resistance development against OFX.

In vitro and in vivo studies of temozolomide loading in zeolite structures as drug delivery systems for glioblastoma

Zeolites Y (faujasite) and MOR (mordonite) were used as hosts for temozolomide (TMZ), a current good-standard chemotherapeutic agent used in the treatment of glioblastoma brain tumors. TMZ was loaded into zeolites by liquid-phase adsorption at controlled pH. FTIR, 1H NMR, MS, SEM, UV/vis and chemical analysis demonstrated the successful loading of TMZ into zeolite hosts. The hydrolysis of TMZ in MTIC (TMZ metabolite) after the preparation of drug delivery systems (DDS) was observed in simulated body fluid. The effect of zeolites and DDS were evaluated on the viability of glioblastoma cell lines. Unloaded Y zeolite presented toxicity to cancer cells in contrast to MOR. In accordance, the best results in potentiation of the TMZ effect was obtained with MOR. We found that mordonite loaded with 0.026 mmol of TMZ was able to decrease the half maximal inhibitory concentrations (IC50) at least 3-fold in comparison to free temozolomide both in vitro and in vivo.

Nanotechnological approaches using curcumin and Withania somnifera: neuroprotection and antimicrobial activities

Tese de Doutoramento em Biologia de Plantas

Development of folate-targeted liposomes for rheumatoid arthritis therapy

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).

Protein-based nanodevices for therapeutic applications

Tese de Doutoramento em Biologia Molecular e Ambiental (área de especialização em Biologia Celular e Saúde).

Micro- and mesoporous structures as drug delivery carriers for salicylic acid

The potential of salicylic acid (SA) encapsulated in porous materials as drug delivery carriers for cancer treatment was studied. Different porous structures, the microporous zeolite NaY, and the mesoporous SBA-15 and MCM-41 were used as hosts for the anti-inflammatory drug. Characterization with different techniques (FTIR, UV/vis, TGA, 1H NMR, and 13C CPMAS NMR) demonstrated the successful loading of SA into the porous hosts. The mesoporous structures showed to be very efficient to encapsulate the SA molecule. The obtained drug delivery systems (DDS) accommodated 0.74 mmol (341 mg/gZEO) in NaY and 1.07 mmol (493 mg/gZEO) to 1.23 mmol (566 mg/gZEO) for SBA-15 and MCM-41, respectively. Interactions between SA molecules and pore structures were identified. A fast and unrestricted liberation of SA at 10 min of the dissolution assay was achieved with 29.3, 46.6, and 50.1 µg/mL of SA from NaY, SBA-15, and MCM-41, respectively, in the in vitro drug release studies (PBS buffer pH 7.4, 37 °C). Kinetic modeling was used to determine the release patterns of the DDS. The porous structures and DDS were evaluated on Hs578T and MDA-MB-468 breast cancer cell lines viability. The porous structures are nontoxic to cancer cells. Cell viability reduction was only observed after the release of SA from MCM- 41 followed by SBA-15 in both breast cancer cell lines.

Propolis: A complex natural product with a plethora of biological activities that can be explored for drug development

The health industry has always used natural products as a rich, promising, and alternative source of drugs that are used in the health system. Propolis, a natural resinous product known for centuries, is a complex product obtained by honey bees from substances collected from parts of different plants, buds, and exudates in different geographic areas. Propolis has been attracting scientific attention since it has many biological and pharmacological properties, which are related to its chemical composition. Several in vitro and in vivo studies have been performed to characterize and understand the diverse bioactivities of propolis and its isolated compounds, as well as to evaluate and validate its potential. Yet, there is a lack of information concerning clinical effectiveness. The goal of this review is to discuss the potential of propolis for the development of new drugs by presenting published data concerning the chemical composition and the biological properties of this natural compound from different geographic origins.

A novel hanging spherical drop system for the generation of cellular spheroids and high throughput combinatorial drug screening

We propose a novel hanging spherical drop system for anchoring arrays of droplets of cell suspension based on the use of biomimetic superhydrophobic flat substrates, with controlled positional adhesion and minimum contact with a solid substrate. By facing down the platform, it was possible to generate independent spheroid bodies in a high throughput manner, in order to mimic in vivo tumour models on the lab-on-chip scale. To validate this system for drug screening purposes, the toxicity of the anti-cancer drug doxorubicin in cell spheroids was tested and compared to cells in 2D culture. The advantages presented by this platform, such as feasibility of the system and the ability to control the size uniformity of the spheroid, emphasize its potential to be used as a new low cost toolbox for high-throughput drug screening and in cell or tissue engineering.

Marine origin polysaccharides in drug delivery systems

Oceans are a vast source of natural substances. In them, we find various compounds with wide biotechnological and biomedical applicabilities. The exploitation of the sea as a renewable source of biocompounds can have a positive impact on the development of new systems and devices for biomedical applications. Marine polysaccharides are among the most abundant materials in the seas, which contributes to a decrease of the extraction costs, besides their solubility behavior in aqueous solvents and extraction media, and their interaction with other biocompounds. Polysaccharides such as alginate, carrageenan and fucoidan can be extracted from algae, whereas chitosan and hyaluronan can be obtained from animal sources. Most marine polysaccharides have important biological properties such as biocompatibility, biodegradability, and anti-inflammatory activity, as well as adhesive and antimicrobial actions. Moreover, they can be modified in order to allow processing them into various shapes and sizes and may exhibit response dependence to external stimuli, such as pH and temperature. Due to these properties, these biomaterials have been studied as raw material for the construction of carrier devices for drugs, including particles, capsules and hydrogels. The devices are designed to achieve a controlled release of therapeutic agents in an attempt to fight against serious diseases, and to be used in advanced therapies, such as gene delivery or regenerative medicine.

Can the dopaminergic-related effects of general anesthetics be linked to mechanisms involved in drug abuse and addiction?

BACKGROUND: General anesthetics (GA) are well known for the ability to induce a state of reversible loss of consciousness and unresponsiveness to painful stimuli. However, evidence from animal models and clinical studies show that GA exposure may induce behavioral changes beyond acute effects. Most research and concerns are focused on changes in cognition and memory. METHODS: We will look at effects of GA on behavior that is mediated by the dopaminergic system. RESULTS: Pharmacological resemblance of GA with drugs of abuse, and the complexity and importance of dopaminergic systems in both reward seeking and addictive illnesses make us believe that it deserves an overview about what is already known and what matters to us as healthcare workers and specifically as anesthesiologists. CONCLUSION: A review of available evidence strongly suggests that there may be a link between the effects of GA on the brain and substance abuse, partly explained by their influence on the dopaminergic system.

Biodegradable drug-eluting stents: Targeting urothelial tumors of upper urinary tract

INTRODUCTION & OBJECTIVES: Urothelial tumors of upper urinary tract are ranked among the most common types of cancers worldwide. The current standard therapy to prevent recurrence is intravesical Bacillus Calmetteâ Guerin (BCG) immunotherapy, but it presents several disadvantages such as BCG failure and intolerance. Another way is to use chemotherapy, which is generally better tolerated that BCG. In this case, drugs such as epirubicin, doxorubicin, paclitaxel and gemcitabine are used. Nevertheless, intravesical chemotherapy only prevents recurrence in the short-term. These failings can be partially attributed to the short residence time and low bioavailability of the drug within the upper urinary tract and the cancer cells, resulting in a need for frequent drug instillation. To avoid these problems, biodegradable ureteral stents impregnated by supercritical fluid CO2 (SCF) with each of the four anti-cancer drugs were produced. MATERIAL & METHODS: Four formulations with different concentrations of gelatin and alginate and crosslink agent were tested and bismuth was added to confer radiopaque properties to the stent. The preliminary in vivo validation studies in female domestic pigs was conducted at the University of Minho, Braga, after formal approval by the institutionâ s review board and in accordance with its internal ethical protocol for animal experiments. Paclitaxel, epirubicin, doxorubicin and gemcitabine were impregnated in the stents and the release kinetics was measured in artificial urine solution (AUS) for 9 days by UV spectroscopy in a microplate reader. The anti-tumoral effect of the developed stents in transitional cell carcinoma (TCC) and HUVEC primary cells, used as control, was evaluated. RESULTS: The in vivo validation of this second-generation of ureteral stents performed was herein demonstrated. Biodegradable ureteral stents were placed in the ureters of a female pigs, following the normal surgical procedure. The animals remained asymptomatic, with normal urine flow. The in vitro release study in AUS of the stent impregnated showed a higher release in the first 72h for the four anti-cancer drugs impregnated after this time the plateau was achieved and the stent degraded after 9 days. The direct and indirect contact of the anti-cancer biodegradable stents with the TCC and HUVEC cell lines confirm the anti-tumor effect of the stents impregnated with the four anti-cancer drugs, reducing around 75% of the viability of the TCC cell line after 72h and no killing effect in the HUVEC cells. CONCLUSIONS: The use of biodegradable ureteral stent in urology clinical practice not only reduce the stent-related symptoms but also open new treatment therapyâ s, like in urothelial tumors of upper urinary tract. Furthermore, we have demonstrated the clinical validation in vivo pig model. This study has thus shown the killing efficacy of the anti-cancer drug eluting biodegradable stents in vitro for the TCC cell line, with no toxicity observed in the control, non-cancerous cells.The direct and indirect contact of the anti-cancer biodegradable stents with the TCC and HUVEC cell lines confirm the anti-tumor effect of the stents impregnated with the four anti-cancer drugs, reducing around 75% of the viability of the TCC cell line after 72h and no killing effect in the HUVEC cells. This study has thus shown the killing efficacy of the anti-cancer drug eluting biodegradable stents in vitro for the TCC cell line, with no toxicity observed in the control, non-cancerous cells.