Bacterial ghosts as novel efficient targeting vehicles for DNA delivery to the human monocyte-derived dendritic cells

Recombinant bacterial ghosts loaded with plasmids were tested as an antigen delivery system and as a potential mediator of maturation for human monocyte-derived dendritic cells (DCs). Bacterial ghosts are cell envelopes derived from Gram-negative bacteria; the intracellular content is released by the controlled expression of plasmid-encoded lysis gene E of PhiX174. All the cell surface structures of the native bacteria, including the outer membrane proteins, adhesins, LPS, lipid A, and peptidoglycans, are preserved. Co-incubation of immature DCs with ghosts resulted in decreased expression of CD1a, CD80, and CD83 molecules, while addition of maturation mix (TNF-alpha, IL-1 beta, IL-6, and PGE2) to the cultures enhanced expression of these molecules. No marked changes were observed in the expression of the CD11c, CD40, and CD86 surface molecules. The exposure of DCs to ghosts in combination with maturation mix resulted in a nonsignificant increase in their ability to activate T cells. DCs co-incubated with bacterial ghosts carrying plasmids encoding GFP in combination with maturation mix exhibited high expression levels of GFP (up to 85%). These results indicate that in addition to their well-established use as vaccines, bacterial ghosts can also be used as carriers of nucleic acid-encoded antigens.     

Transport of human growth hormone across Caco-2 cells with novel delivery agents: evidence for P-glycoprotein involvement.

Emisphere Technologies, Inc. has synthesized a series of small molecules which have been shown to improve protein absorption through mucosal tissue. This enhancement is specific between protein and a particular delivery agent. Despite the specificity of interaction, the mechanism of enhanced tissue penetration is still unclear. The purpose of this work is to understand the enhancement mechanism(s) of these delivery agents by using Caco-2 cells as a model membrane. It was found that the bidirectional transepithelial fluxes of human growth hormone (hGH) in the presence of these delivery agents across human intestinal epithelial Caco-2 cell line showed marked asymmetry. Average permeability coefficient values obtained in the apical (AP) to basolateral (BL) direction were lower than those of the reverse (BL to AP) direction. On the other hand, the fluxes for human growth hormone alone were symmetric. When P-glycoprotein inhibitors were included in the transport medium, the permeability coefficient values of BL to AP direction were significantly decreased while the transport was increased in the reverse direction in the presence of delivery agents. P-glycoprotein inhibitors had no effect on the transport of human growth hormone alone. This study shows that human growth hormone alone can be transported across Caco-2 cells in very limited quantities by passive diffusion, but in the presence of delivery agents, human growth hormone can be effluxed in a P-glycoprotein-mediated fashion. This also indirectly shows that the human growth hormone has become more lipophilic in the presence of delivery agents.     

Analysis of drug transporter expression in human intestinal Caco-2 cells by real-time PCR

Expression of drug transporters corresponds to a crucial parameter in intestinal Caco-2 cells widely used for investigating drug absorption. In order to characterize it in an accurate, reproducible and comparative manner, we analyzed mRNA levels of 19 influx and efflux drug transporters through real-time quantitative polymerase chain reaction assays combined with the use of a total RNA reference standard. Profiles of transporter expression were found to be significantly correlated in two independent Caco-2 cell clones and in human small intestine, which may support the use of Caco-2 cells for investigating intestinal drug transport. Several transporters were nevertheless quantitatively expressed at higher (MRP2, MRP3, MRP4, MRP5, MRP6, OATP-A, OATP-B, OCT1 and MCT1) or lower (BCRP) levels in Caco-2 cells comparatively to small intestine. Moreover, MDR1, MRP2, OATP-A and PEPT1 mRNA relative expression were found to differ in the two analyzed Caco-2 cell clones by at least a twofold factor, highlighting that some variations in transporter expression may occur in Caco-2 cells depending on cell origin, and therefore underlining the interest of carefully characterizing transporter levels in any Caco-2 cell clone before its use for drug transport assays.     

Calcium phosphate cements as bone drug delivery systems: a review.

Since calcium phosphate cements were proposed, several formulations have been developed, some of them commercialised, and they have proven to be very efficient bone substitutes in different applications. Some of their properties, such as the injectability, or the low-temperature setting, which allows the incorporation of different drugs, make them very attractive candidates as drug carriers. In this article, the performance of calcium phosphate cements as carriers of different types of drugs, such as antibiotics, analgesics, anticancer, anti-inflammatory, as well as growth factors is reviewed.     

Assemblage of novel release modules for the development of adaptable drug delivery systems.

The aim of this paper was to study the applicability of the release module assemblage technology for an adaptable control of drug delivery rate and site. The elementary release module was a swellable matrix having one base convex and the other concave, named Dome Matrix. The swelling and release behavior of the release module was studied. The presence of the convex and concave bases in the swellable matrix slightly changed the overall drug delivery kinetics exhibited by a flat base cylindrical matrix having the same weight and composition. The swelling and drug release of the individual bases of the matrix was also studied to investigate the effect of the surface shape. The concave, convex and flat bases exhibited different swelling and release kinetics. The convex base released drug at faster rate than the concave base, whereas the flat base was intermediate. The release mechanisms of convex and concave bases were significantly different. The Dome Matrix module was selected for assembling several modules in a delivery system obtained by a guided insertion of the convex base into the concave base or by concave/concave base sticking. The module assemblage shows different drug release behavior depending on the geometry of assembled systems.     

Evaluation of the antiproliferative activity of 2-amino thiophene derivatives against human cancer cells lines

In spite of great progress in understanding cancer biology, current therapeutic procedures remain unsatisfactory. Chemotherapy is often followed by secondary effects with cellular toxicity negatively affecting the results. The discovery and development of new safe and efficient antitumor agents is necessary. Derivatives of 2-amino thiophene have been a topic of constant investigation due to their versatile synthetic applicability and broad spectrum of biological applications; among which are antifungal and antiproliferative activity shown in prior studies of our group. In the current study, compounds 6CN09, 6CN10, 6CN12, 6CN14, 7CN09 and 7CN11 were analyzed as to antiproliferative effect in human cells of cervical adenocarcinoma (HeLa), human pancreatic adenocarcinoma (PANC-1) and mice fibroblasts (3T3), which were exposed to the compounds in concentrations of 5, 10, 25 and 50 μM during 24 and 48 h. They were submitted to MTT assay. In order to elucidate the action mechanism of antitumor thiophene derivatives flow cytometry was performed to evaluate cell death and cell cycle analysis. The results showed that thiophene derivatives demonstrated great antiproliferative potential in the HeLa and PANC-1 cell lines when compared with the control, and the percentage of cell proliferation inhibition approximated or was higher than the standard drug used; doxorubicin (Dox). In highlight were the derivatives 6CN14 and 7CN09 that showed greater efficiency in the antiproliferative evaluation. Further, all compounds had a protective effect on the non-tumor 3T3 cell line. The flow cytometry analysis showed few cells in apoptosis in both the HeLa and PANC-1 lines, although the compounds interfered with the progression of the cell cycle, and avoided cell growth and multiplication in the HeLa tumor line. These thiophene derivatives demonstrated cytostatic and antiproliferative effects and may be considered as promising molecular candidates for anticancer drugs.     

Rheological, mechanical and membrane penetration properties of novel dual drug systems for percutaneous delivery.

In this study it has been demonstrated that mixtures of two solid drugs, ibuprofen and methyl nicotinate, with different but complementary pharmacological activities and which exist as a single liquid phase over a wide composition range at skin temperature, can be formulated as o/w emulsions without the use of an additional hydrophobic carrier. These novel dual drug systems provided significantly enhanced in vitro penetration rates through a model lipophilic barrier membrane compared to conventional individual formulations of each active. Thus, for ibuprofen, drug penetration flux enhancements of three- and 10-fold were observed when compared to an aqueous ibuprofen suspension and a commercial alcohol-based ibuprofen formulation, respectively. Methyl nicotinate penetration rates were shown to be similar for aqueous gels and emulsified systems. Mechanisms explaining these observations are proposed. Novel dual drug formulations of ibuprofen and methyl nicotinate, formulated within the liquid range at skin temperature, were investigated by oscillatory rheology and texture profile analysis, demonstrating the effects of drug and viscosity enhancer concentrations, and disperse phase type upon the rheological, mechanical and drug penetration properties of these systems.     

Transport characteristics of diphenhydramine in human intestinal epithelial Caco-2 cells: contribution of pH-dependent transport system.

Transport characteristics of diphenhydramine, an antihistamine, were studied in cultured human intestinal Caco-2 cell monolayers to elucidate the mechanisms of its intestinal absorption. Diphenhydramine accumulation in the monolayers increased rapidly and was influenced by extracellular pH (pH 7.4 > 6.5 > 5.5). Diphenhydramine uptake was temperature dependent, saturable, and not potential sensitive. Kinetic analysis revealed that the apparent Km values were constant (0.8-1.0 mM) in all pH conditions tested, whereas Vmax values decreased at the lower pH. The initial uptake of diphenhydramine was competitively inhibited by another antihistamine, chlorpheniramine, with a Ki value of 1.3 mM. On the other hand, cimetidine and tetraethylammonium, typical substrates for the renal organic cation transport system, had no effect. Moreover, biological amines and neurotransmitters, such as histamine, dopamine, serotonin, and choline, also had no effect on the diphenhydramine accumulation. Finally, diphenhydramine uptake was stimulated by preloading monolayers with chlorpheniramine (trans-stimulation effect). These findings indicate that diphenhydramine transport in Caco-2 cells is mediated by a specific transport system. This pH-dependent transport system may contribute to the intestinal absorption of diphenhydramine.     

Effects of polyether-modified poly(acrylic acid) microgels on doxorubicin transport in human intestinal epithelial Caco-2 cell layers.

Novel microgels composed of cross-linked copolymers of poly(acrylic acid) and Pluronics were evaluated as possible permeation enhancers for doxorubicin transport using Caco-2 cell monolayers as a gastrointestinal model. Pluronic, triblock copolymers of ethylene oxide (EO) and propylene oxide (PO), were chosen to represent the most hydrophobic (Pluronic L61 and L92 with average compositions of EO(3)PO(30)EO(3) and EO(8)PO(52)EO(8), respectively) and the relatively hydrophilic (Pluronic F127 with average formula EO(99)PO(67)EO(99)) extremes of this class of block copolymers. The weight ratio of Pluronic to poly(acrylic acid) in the microgels was set at 45:55. By inhibiting the P-glycoprotein (P-gp)-mediated doxorubicin efflux from the cells and enhancing the passive influx, the microgels were shown to enhance the overall cell absorption of doxorubicin. The enhancement effect was more pronounced than with a known penetration enhancer, Pluronic L61, and was comparable to that of Pluronic L92. Microgels exhibited synergism of the doxorubicin transport enhancement with Verapamil, a known inhibitor of the P-gp. The effects of the microgels were studied using the hydrophilic marker ([14C]mannitol) test and the MTT assay. Transepithelial electrical resistance (TEER) studies demonstrated that the microgels decreased TEER to about 80% of initial values, but these minor effects were fully reversible, indicating viability of the cells after incubation with microgels. No significant enhancement of [14C]mannitol transport by microgels was observed, relative to Carbopol 934NF (control polymer). Cytotoxicity studies confirmed that the transport-enhancing properties of the microgels were not due to damage of the Caco-2 cell monolayers.     

Chemically modified polyacrylamide-g-guar gum-based crosslinked anionic microgels as pH-sensitive drug delivery systems: preparation and characterization.

New spherically shaped cross-linked hydrogels of polyacrylamide-grafted guar gum were prepared by the emulsification method. These were selectively derivatized by saponification of the -CONH2 group to the -COOH group. The derived microgels were characterized by FTIR and elemental analyses. The derivatized microgels were responsive to pH and ionic strength of the external medium. The swelling of microgels increased when the pH of the medium changed from acidic to alkaline. Transport parameters, viz., solvent front velocity and diffusion coefficients were calculated from a measurement of the dimensional response of the microgels under variable pH conditions. The variation in pH changed the transport mechanism from Case II (in 0.1 N HCl) to non-Fickian (in pH 7.4 buffer), and these processes are relaxation-controlled. Ionic strength exerted a profound influence on the swelling of the microgels. Swelling was reversible and pulsatile with the changing environmental conditions. The pH-sensitive microgels were loaded with diltiazem hydrochloride and nifedipine (both antihypertensive drugs) and their release studies were performed in both the simulated gastric and intestinal pH conditions. The release was relatively quicker in pH 7.4 buffer than observed in 0.1 N HCl; the release followed non-Fickian transport in almost all the cases.     

New polymers for drug delivery systems in orthopaedics: in vivo biocompatibility evaluation.

The use of biodegradable polymers for drug delivery systems excluded the need for a second operation to remove the carrier. However, the development of an avascular fibrous capsule, reducing drug release, has raised concern about these polymers in terms of tissue-implant reaction. Five novel polymers were evaluated in vivo after implantation in the rat dorsal subcutis and compared to the reference polycaprolactone (PCL). Poly(cyclohexyl-sebacate) (PCS), poly(L-lactide-b-1,5-dioxepan-2-one-b-L-lactide) (PLLA-PDXO-PLLA), two 3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (D400G and D600G), and a poly(organo)phosphazene (POS-PheOEt:Imidazole) specimens were histologically evaluated in terms of the inflammatory tissue thickness and vascular density at 4 and 12 weeks from surgery. The highest values of inflammatory tissue thickness were observed in D600G (P < 0.01), PCS (P < 0.001) and PLLA-PDXO-PLLA (P < 0.001) at 4 weeks, while POP-PheOEt:Imidazole showed the lowest value of inflammatory tissue thickness (P < 0.05) at 12 weeks. D400G, D600G, PLLA-PDXO-PPLA and POP-PheOEt:Imidazole showed higher (P < 0.001) values of vascular density near the implants in comparison to PCL at 4 weeks. Finally, D400G and D600G increased their vessel densities while POP-PheOEt:Imidazole and the synthetic polyester PLLA-PDXO-PLLA presented similar vessel density values during experimental times. These different behaviours to improve neoangiogenesis without severe inflammatory tissue-responses could be further investigated with drugs in order to obtain time-programmable drug delivery systems for musculoskeletal therapy.     

Cationic cellulose hydrogels: kinetics of the cross-linking process and characterization as pH-/ion-sensitive drug delivery systems.

The cross-linking process of two cationic hydroxyethylcelluloses of different hydroxyethyl and ammonium group contents, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), with ethylenglycol diglycidylether (EGDE) was characterized and optimized through rheometric analysis of the forming network. The influence of NaOH concentration, temperature, and EGDE concentration on the cross-linking rate were studied. The evolution of the elastic (G') and viscous (G") moduli, recorded in time-sweep experiments carried out at a fixed angular frequency, showed that the cross-linker requires a minimum of 0.05 M NaOH and 30 degrees C to be active. The increase in G' and G" followed first order kinetics, the slopes of G' being higher than those corresponding to G". The gel time, i.e. the time at which the crossover of G' and G" occurs, decreases exponentially when temperature increases from 30 to 60 degrees C. Apparent activation energies, estimated from the gel times, ranged between 70 and 90 kJ/mol. The cross-linking rate was greater in PQ-4 than in PQ-10 owing to the initial lower viscosity and higher content in hydroxyethyl groups of the former. However, IR spectra of the final hydrogels suggest the formation of a similar number of cross-linking junctions in both polymer systems. The optimum conditions for hydrogel preparation were 60 degrees C in 0.10 M NaOH medium, and no depolymerization was observed. Such hydrogels were transparent, presented a smooth, continuous surface, and were superabsorbent in water. After drying in an oven, the degree of swelling was lower than that of freshly prepared hydrogels; the behavior of water uptake being Fickian. The hydrogels presented a significant loading capacity of diclofenac sodium, with which they interact through ionic and hydrophobic bonding. The affinity is kept at an acidic pH, preventing drug release. In contrast, at pH 8 the interactions are broken and the release process is sustained for more than 4 h. The results also indicate that the ionic strength as well as the initial pH of the medium, when the release was evaluated switching the pH from acidic to basic, are two critical factors which have to be considered to extract conclusions about the behavior of the hydrogels as site-specific delivery systems under in vivo conditions.     

The use of FT-IR imaging as an analytical tool for the characterization of drug delivery systems.

FT-IR imaging spectroscopy is well suited for studying dynamic processes occurring in multi-component systems. Each component is resolved spatially based on the spectral response at each detector element. Additionally, the sequential collection of images tracks the movement of each component over time. In this study, the delivery characteristics of the drug, testosterone, suspended in a poly(ethylene oxide) (PEO) matrix was observed using this technique. Drug release occurred as the hydrophilic, erodible polymer underwent controlled dissolution, exposing the drug to the aqueous environment. The subsequent conversion of the drug into the therapeutic aqueous form completed the delivery process. Qualitative evaluation of the false color composite infrared images led to the elucidation of two distinct delivery mechanisms, dependent on the degree of drug loading. The spatially embedded spectral features led to the quantification of the drug release rates as well as the rates of polymer dissolution. The rates for both polymer dissolution and drug release were evaluated using well-established models. Additionally, the homogeneity of the drug dispersion for different loadings was characterized. The roles of chemical interactions across the solvent interface of species were also investigated. Changes in each component from the bulk to the solvated region were investigated, revealing changes in concentration and polymer orientation as well as inter-species interactions.     

Controlling chronic pain in the workplace: nerve stimulation and intrathecal drug delivery systems.

Chronic pain can have a major effect on both workers and the workplace. Collaboration among workers, occupational health nurses, and specialist providers is important for ongoing management of mechanism-based therapies for chronic pain. This article examines collaborative care issues relative to nerve stimulation and intrathecal drug delivery systems.     

Formulation aspects in the development of osmotically controlled oral drug delivery systems.

Osmotically controlled oral drug delivery systems utilize osmotic pressure for controlled delivery of active agent(s). Drug delivery from these systems, to a large extent, is independent of the physiological factors of the gastrointestinal tract and these systems can be utilized for systemic as well as targeted delivery of drugs. The release of drug(s) from osmotic systems is governed by various formulation factors such as solubility and osmotic pressure of the core component(s), size of the delivery orifice, and nature of the rate-controlling membrane. By optimizing formulation and processing factors, it is possible to develop osmotic systems to deliver drugs of diverse nature at a pre-programmed rate. In the present review, different types of oral osmotic systems, various aspects governing drug release from these systems, and critical formulation factors are discussed.