Recent advances in topical therapeutics for vitreoretinal diseases

Eye drops are convenient for patients, but achieving therapeutic doses and maintaining sustained drug release without frequent re-application to treat diseases of the retina has been largely unsuccessful. Topical administration of drugs is hindered by the anatomy, physiology, and biochemistry of the eye and its highly effective defence mechanisms. Advances in nanotechnology have led to the experimental use of topical permeation-enhancing liposomes, emulsions, and microspheres to enhance absorption and penetration of drugs across membranes; allow controlled release of the drug; and to target drugs at distinct tissues to allow sufficient local bioavailability. In the near future it is hoped that improved technologies may provide means of sustained topical drug delivery for retinal therapy, with improved side-effect profiles and reduced cost compared with currently available clinical treatments.

Designing liposomal adjuvants for the next generation of vaccines

Liposomes not only offer the ability to enhance drug delivery, but can effectively act as vaccine delivery systems and adjuvants. Their flexibility in size, charge, bilayer rigidity and composition allow for targeted antigen delivery via a range of administration routes. In the development of liposomal adjuvants, the type of immune response promoted has been linked to their physico-chemical characteristics, with the size and charge of the liposomal particles impacting on liposome biodistribution, exposure in the lymph nodes and recruitment of the innate immune system. The addition of immunostimulatory agents can further potentiate their immunogenic properties. Here, we outline the attributes that should be considered in the design and manufacture of liposomal adjuvants for the delivery of sub-unit and nucleic acid based vaccines.

Grewia gum 2:mucoadhesive properties of compacts and gels

Purpose: To compare the mucoadhesive performance of grewia polysaccharide gum with those of guar gum, carboxymethylcellulose, hydroxypropyl methylcellulose and carbopol 971P. Methods: Grewia polysaccharide gum compacts or gels as well as those of guar gum, carboxymethylcellulose, hydroxypropyl methylcellulose or carbopol 971P were prepared. Texturometric and tensile analysis of the polymer gels and compacts were carried out using a software-controlled penetrometre, TA.XTPlus texture analyzer. The polymer gels were evaluated for hardness, stickiness, work of cohesion and work of adhesion. Furthermore, the detachment force of the polymer compacts from a mucin substrate was evaluated. Results: The work of adhesion of guar gels was significantly greater than that of grewia gels (p < 0.001) but the latter showed a significantly greater work of adhesion than carboxymethylcellulose gels (p < 0.05) and hydroxypropyl methylcellulose gels (p < 0.001). However, the work of cohesion for grewia/mucin gel mixture was significantly greater (p < 0.001) than those of carboxymethylcellulose/mucin, hydroxypropyl methylcellulose/mucin and carbopol 971P/mucin gel blends. The difference between the mucoadhesive performance of grewia compacts and those of hydroxypropyl methylcellulose and carbopol 971P compacts was insignificant (p > 0.05). Conclusion: Grewia polysaccharide gum demonstrated good mucoadhesive properties, comparable to those of carbopol 971P, carboxymethylcellulose, guar gum and hydroxypropyl methylcellulose, and therefore, should be suitable for the formulation of retentive drug delivery devices. © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City.

Localized surface plasmon resonance biosensors for real-time biomolecular binding study

Surface Plasmon Resonance (SPR) and localized surface plasmon resonance (LSPR) biosensors have brought a revolutionary change to in vitro study of biological and biochemical processes due to its ability to measure extremely small changes in surface refractive index (RI), binding equilibrium and kinetics. Strategies based on LSPR have been employed to enhance the sensitivity for a variety of applications, such as diagnosis of diseases, environmental analysis, food safety, and chemical threat detection. In LSPR spectroscopy, absorption and scattering of light are greatly enhanced at frequencies that excite the LSPR, resulting in a characteristic extinction spectrum that depends on the RI of the surrounding medium. Compositional and conformational change within the surrounding medium near the sensing surface could therefore be detected as shifts in the extinction spectrum. This dissertation specifically focuses on the development and evaluation of highly sensitive LSPR biosensors for in situ study of biomolecular binding process by incorporating nanotechnology. Compared to traditional methods for biomolecular binding studies, LSPR-based biosensors offer real-time, label free detection. First, we modified the gold sensing surface of LSPR-based biosensors using nanomaterials such as gold nanoparticles (AuNPs) and polymer to enhance surface absorption and sensitivity. The performance of this type of biosensors was evaluated on the application of small heavy metal molecule binding affinity study. This biosensor exhibited ∼7 fold sensitivity enhancement and binding kinetics measurement capability comparing to traditional biosensors. Second, a miniaturized cell culture system was integrated into the LSPR-based biosensor system for the purpose of real-time biomarker signaling pathway studies and drug efficacy studies with living cells. To the best of our knowledge, this is the first LSPR-based sensing platform with the capability of living cell studies. We demonstrated the living cell measurement ability by studying the VEGF signaling pathway in living SKOV-3 cells. Results have shown that the VEGF secretion level from SKOV-3 cells is 0.0137 ± 0.0012 pg per cell. Moreover, we have demonstrated bevacizumab drug regulation to the VEGF signaling pathway using this biosensor. This sensing platform could potentially help studying biomolecular binding kinetics which elucidates the underlying mechanisms of biotransportation and drug delivery.

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant Ka was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Specific Increase in MDR1 Mediated Drug-Efflux in Human Brain Endothelial Cells following Co-Exposure to HIV-1 and Saquinavir

Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.

Avaliação da atividade anti-Helicobacter pylori e citotóxica in vitro de extratos orgânicos obtidos das folhas de Encholirium spectabile e Syzygium cumini

Helicobacter pylori is a spiral, Gram negative, mobile, and microaerophilic bacteria recognized as a major cause of gastritis, ulcer, gastric cancer, and gastric low grade, B cell, mucosa – associated lymphoid tissue (MALT) lymphoma, constituting an important microorganism in medical microbiology. Its importance comes from the difficulty of treatment because the requirement of multiple drugs use, besides the increasing emergence of resistant and multiresistant strains to antibiotics used in th e clinic. In order to expand safe and effective therapeutic options , chemical studies on medicinal plants by obtaining extracts, fractions, isolated compounds or essential oils with some biological activity has been intensified . Given the above, the objective was to evaluate the inhi bitory activity of organic extracts derived from Syzygium cumini and Encholirium spectabile, with antiulcer history, and the essential oil, obtained from S. cumini, against H. pylori (ATCC 43504) by the disk diffusion method, for qualitative evaluation, an d determination of minimum inhibitory concentration (MIC) using the broth microdilution method, for quantitative analysis. Also was evaluated the extracts in vitro toxicity by a hemolytic assay using sheep red blood cells, and VERO and HeLa cells using the MTT assay to analyze cell viability. The extracts of both plant used in antimicrobial assays did not inhibit bacterial growth, however the essential oil of S. cumini (SCFO) proved effective, showing MIC value of 205 μg/mL (0.024 % dilution of the original oil). In the hemolytic assay, the same oil shows moderate toxicity, by promote 25% hemolysis at 1000 μg/mL. Regarding the cytotoxicity in cell culture, the SCFO, at 260 μg/mL, affected the cell viability around 80% of HeLa and 50% of VERO cells. So the oi l obtained from S. cumini leaves has antimicrobial activity against H. pylori and cytotoxicity potential, suggesting a source of new molecule drug candidates, since new stages of toxicity in vitro and in vivo, as well, chemical characterization be evaluate d. Moreover, the development of a prospective drug delivery system can result in a prototype to be used in preclinical tests.

Produção de estrutura com gradiente de porosidade a partir da sinterização por plasma da mistura de pós Ti-Nb-Sn

Porous structures are being widely investigated for use in biomedical implants, aiming to mechanically integrate and functionally the implant inside the bone tissue. Moreover, this structure is also important for drugs that can be stored and can induce and accelerate the process of osseointegration. With the purpose to investigate this effect, Ti, Nb and Sn metal powders, were sintered by plasma using a hollow cathode discharge. Sintering was performed in argon plasma set at 4 mbar pressure and temperatures of 500 ° C, 600 ° C and 700 ° C. Samples were also sintered in the electrical resistance furnace at 1200 ° C in order to compare plasma sintering with the conventional method. It was observed that plasma samples sintered with the hollow cathode configuration showed a gradient in porosity, while the samples sintered in the resistive furnace did not. Furthermore, differences in the microstructure of the samples were found, were a surface with higher porosity and ales porous core were obtained at different temperatures. The percolation profile of distilled water and the chemical compositions of the porous layers of the plasma treated samples were the main results obtained. Based on these results, we can conclude that this structure is particularly important for application in the biomedical field such as scaffolds for drug delivery and implants

Micropartículas contendo nanopartículas de quitosana usando sulfato e genipina para liberação modificada da triancinolona: obtenção, caracterização e estudo em células tumorais

Chitosan is a polymer biocompatibility and biodegradability widely used in drug delivery systems. The co-crosslinking of chitosan with sodium sulfate and genipin, to form particulate systems is related of making them more resistant to acidic pH and to modulate the release kinetics for the oral route. Triamcinolone is a glucocorticoid with anti-inflammatory and immunosuppressive actions. The nanoparticles were prepared by co-crosslinking and characterized for particle size, PDI, zeta potential, crosslinking degree, encapsulation rate, morphology, infrared spectroscopy, thermal analysis, release kinetics and cells studies. The nanoparticles were prepared initially without genipin with sodium sulphate and the particles parameters were monitored in function of different ratio of drug / polymer, different concentrations of sodium sulfate and polysorbate 80 and the drip mode of crosslinkers on polymers. After optimizing conditions, the chosen system parameters without genipin included mean diameter of 312.20 ± 5.70 nm, PDI 0.342 ± 0.013 and zeta potential of 20.18 ± 2.28 mV. The genipin was introduced into the system analyzing different concentrations (0.5, 1.0 and 2.0 mM) and crosslinking times (3, 6, 12 and 24 h). Evaluating crosslinking time with genipin (0.5 mM) it was showed that varying the genipin reaction time the systems size ranged from 235.1 to 334.4 nm, the PDI from 0.321 to 0.392 and zeta potential 20.92 to 30.39 mV. The crosslinking degree that coud vary from 14 to 30 %. Nanoparticles without genipina, 6 h and 24 h crosslinking time were dried by spray-drying method. Analysis by scanning electron micrograph (SEM) revealed that the microparticles showed spherical morphology. The encapsulation rate was 75 ± 2.3 % using validated HPLC methodology. The infrared analysis showed chemical interactions between the components of the formulation. Thermal analysis showed that systems with a higher degree of crosslinking had a higher thermal stability. On release kinetics, increasing the degree of crosslinking was able to decrease the concentration and rate of release of triamcinolone. In studies with liver cancer cells (HepG2) and colon (HT-29), the microparticulate prepared with triamcinolone and 24 h of crosslinking with genipin showed a potential for antitumor activity in hepatic cell line HepG2. Therefore, a new delivery system for triamcinolone on polymeric nanoparticles of chitosan cocrosslinked with genipin and sodium sulfate was obtained with hepatic antitumor potential.

Obtenção de triacetato de celulose a partir do bagaço de cana-deaçúcar para revestimento de micropartículas de goma gelana e avaliação do seu perfil de liberação in vitro e da mucoadesão ex vivo

Oral route of administration is considered to be the most comfortable, safe and greater adaptation for patients. But, oral route presents some disadvantages such as drugs bioavailability and side effects on the stomach. Some technologies are studied to soften and/or resolve these problems, such as coating with polymeric films, which are able to protect the pharmaceutical form of the acid stomachic environment and to act in the drug release, and mucoadhesive systems, which allow the pharmaceutical form remains a greater time interval in the intestine, increasing the effectiveness of the drug. Cellulose triacetate (CTA) films were produced from cellulose extracted from sugar cane bagasse. The films were prepared with different morphologies (with and without water, acting as non-solvent) and concentrations (3, 6.5 and 10%) of CTA and characterized using scanning electron microscopy (SEM), water vapor permeability (WVP), puncture resistance (PR), enzymatic digestion (DE), and mucoadhesive force evaluation (MF). Microscopy showed the formation of symmetric and asymmetric morphologies. WVP data showed that more concentrated films have higher values for WVP; moreover, asymmetric films had higher values than symmetric films. PR measurements showed that symmetric membranes are more resistant than asymmetric ones. More concentrated films were also more puncture resistant, except for symmetric membranes with CTA concentrations of 6.5 and 10% that did not show significant differences. All of the films presented large mucoadhesive capacities independent of their morphology and CTA concentration. From the results of WVP and RP, a symmetric filme with 6.5% CTA showed better ability and mechanical resistance, therefore, was selected to serve as coating of gellan gum (GG) particles incorporating ketoprofen (KET), which was confirmed by SEM. The selected film presented low values in measurements of the swelling index (SI) and in a dissolution test (DT). TGA analysis showed that the CTA coating does not influence the thermal stability of the particles and there is no incompatibility evidence between CTA, GG and KET. Coated particles released 100% of the ketoprofen in 24 h, while uncoated particles released the same amount in 4 h. The results of this study highlight the potential of CTA in the development of new controlled oral delivery systems.

Phytochemical-loaded mesoporous silica nanoparticles for nose-to-brain olfactory drug delivery

Central nervous system (CNS) drug delivery is often hampered due to the insidious nature of the blood-brain barrier (BBB). Nose-to-brain delivery via olfactory pathways have become a target of attention for drug delivery due to bypassing of the BBB. The antioxidant properties of phytochemicals make them promising as CNS active agents but possess poor water solubility and limited BBB penetration. The primary aim of this study was the development of mesoporous silica nanoparticles (MSNs) loaded with the poorly water-soluble phytochemicals curcumin and chrysin which could be utilised for nose-to-brain delivery. We formulated spherical MSNP using a templating approach resulting in ∼220nm particles with a high surface porosity. Curcumin and chrysin were successfully loaded into MSNP and confirmed through Fourier transformation infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and HPLC approaches with a loading of 11-14% for curcumin and chrysin. Release was pH dependant with curcumin demonstrating increased chemical stability at a lower pH (5.5) with a release of 53.2%±2.2% over 24h and 9.4±0.6% for chrysin. MSNP were demonstrated to be non-toxic to olfactory neuroblastoma cells OBGF400, with chrysin (100μM) demonstrating a decrease in cell viability to 58.2±8.5% and curcumin an IC50 of 33±0.18μM. Furthermore confocal microscopy demonstrated nanoparticles of <500nm were able to accumulate within cells with FITC-loaded MSNP showing membrane localised and cytoplasmic accumulation following a 2h incubation. MSNP are useful carriers for poorly soluble phytochemicals and provide a novel vehicle to target and deliver drugs into the CNS and bypass the BBB through olfactory drug delivery.

Mechanistic Models of Anti-HIV Microbicide Drug Delivery

A new modality for preventing HIV transmission is emerging in the form of topical microbicides. Some clinical trials have shown some promising results of these methods of protection while other trials have failed to show efficacy. Due to the relatively novel nature of microbicide drug transport, a rigorous, deterministic analysis of that transport can help improve the design of microbicide vehicles and understand results from clinical trials. This type of analysis can aid microbicide product design by helping understand and organize the determinants of drug transport and the potential efficacies of candidate microbicide products.

Microbicide drug transport is modeled as a diffusion process with convection and reaction effects in appropriate compartments. This is applied here to vaginal gels and rings and a rectal enema, all delivering the microbicide drug Tenofovir. Although the focus here is on Tenofovir, the methods established in this dissertation can readily be adapted to other drugs, given knowledge of their physical and chemical properties, such as the diffusion coefficient, partition coefficient, and reaction kinetics. Other dosage forms such as tablets and fiber meshes can also be modeled using the perspective and methods developed here.

The analyses here include convective details of intravaginal flows by both ambient fluid and spreading gels with different rheological properties and applied volumes. These are input to the overall conservation equations for drug mass transport in different compartments. The results are Tenofovir concentration distributions in time and space for a variety of microbicide products and conditions. The Tenofovir concentrations in the vaginal and rectal mucosal stroma are converted, via a coupled reaction equation, to concentrations of Tenofovir diphosphate, which is the active form of the drug that functions as a reverse transcriptase inhibitor against HIV. Key model outputs are related to concentrations measured in experimental pharmacokinetic (PK) studies, e.g. concentrations in biopsies and blood. A new measure of microbicide prophylactic functionality, the Percent Protected, is calculated. This is the time dependent volume of the entire stroma (and thus fraction of host cells therein) in which Tenofovir diphosphate concentrations equal or exceed a target prophylactic value, e.g. an EC50.

Results show the prophylactic potentials of the studied microbicide vehicles against HIV infections. Key design parameters for each are addressed in application of the models. For a vaginal gel, fast spreading at small volume is more effective than slower spreading at high volume. Vaginal rings are shown to be most effective if inserted and retained as close to the fornix as possible. Because of the long half-life of Tenofovir diphosphate, temporary removal of the vaginal ring (after achieving steady state) for up to 24h does not appreciably diminish Percent Protected. However, full steady state (for the entire stromal volume) is not achieved until several days after ring insertion. Delivery of Tenofovir to the rectal mucosa by an enema is dominated by surface area of coated mucosa and whether the interiors of rectal crypts are filled with the enema fluid. For the enema 100% Percent Protected is achieved much more rapidly than for vaginal products, primarily because of the much thinner epithelial layer of the mucosa. For example, 100% Percent Protected can be achieved with a one minute enema application, and 15 minute wait time.

Results of these models have good agreement with experimental pharmacokinetic data, in animals and clinical trials. They also improve upon traditional, empirical PK modeling, and this is illustrated here. Our deterministic approach can inform design of sampling in clinical trials by indicating time periods during which significant changes in drug concentrations occur in different compartments. More fundamentally, the work here helps delineate the determinants of microbicide drug delivery. This information can be the key to improved, rational design of microbicide products and their dosage regimens.