International Journal of Pharmaceutics (v.298, #2)

Introduction by A.T. Florence (273).

The major problem in drug delivery to the brain is the presence of the blood–brain barrier (BBB) which limits drug penetration even if in certain pathological situations the BBB is partly disrupted. Therefore, various strategies have been proposed to improve the delivery of drugs to this tissue. This review presents the status of the BBB in healthy patients and in pathologies like neurodegenerative, cerebrovascular and inflammatory diseases. The second part of this article aims to review the invasive and non-invasive strategies developed to circumvent the BBB and deliver drugs into the brain. The use of nanotechnologies (liposomes, nanoparticles) is especially discussed in the ultimate part of the review evidencing their potentiality as non-invasive technique in the brain delivery of drugs with the possibility to target specific brain tissue thanks to ligand linked to carrier surface.
Keywords: Blood–brain barrier; Brain pathologies; Drug delivery; Colloidal carriers;

Role of gut macrophages in mice orally contaminated with scrapie or BSE by Thomas Maignien; Monjed Shakweh; Pilar Calvo; Dominique Marcé; Nicole Salès; Elias Fattal; Jean-Philippe Deslys; Patrick Couvreur; Corinne Ida Lasmezas (293-304).
While there is a growing consensus on the understanding of the propagation pathways after oral infection of transmissible spongiform encephalopathy (TSE) agents and even if the central role of follicular dendritic cells is identified, little is known about the key players in the first steps of the infection and about the site of the disease development. We investigated the role of gut macrophages, which are capable of capturing aggregates of the prion protein. PLGA particles containing clodronate were designed in order to be orally administered and to target Peyer's patches for inducing gut-associated macrophages suicide in mice. Mice were subsequently infected with scrapie or BSE by the oral route. It was found that the efficacy of macrophage suppression in the Peyer's patches correlated well with an earlier appearance of PrPres in these formations and with a higher amount of PrPres at a later stage of the infection. Thus, the capture of infectious particles that have crossed the epithelial gut barrier and their elimination by macrophages seems to be a key event to restrict the amount of agent initiating the infection.
Keywords: Transmissible spongiform encephalopaties; Macrophage-suicide technique; Clodronate; Peyer's patches; Intestinal uptake; Nano- and microparticles;

Strategies for cytosolic delivery of liposomal macromolecules by Marjan M. Fretz; Enrico Mastrobattista; Gerben A. Koning; Wim Jiskoot; Gert Storm (305-309).
Potential approaches to achieve cytosolic delivery of liposomal macromolecules are presented. These approaches include: (1) the co-encapsulation of fusogenic peptides into targeted drug-containing liposomes (2) coupling of the HIV-1-derived cell-penetrating peptide TAT to the surface of liposomes and (3) photochemical internalization, based on photochemically inducible permeabilization of endocytic vesicles.
Keywords: Cytosolic delivery; Liposomes; Endosomal escape; Cell-penetrating peptides; Photochemical internalization;

A methodology to study intracellular distribution of nanoparticles in brain endothelial cells by Elizabeth Garcia-Garcia; Karine Andrieux; Sophie Gil; Hyun Ryoung Kim; Trung Le Doan; Didier Desmaële; Jean d’Angelo; Frédéric Taran; Dominique Georgin; Patrick Couvreur (310-314).
Cell internalisation and intracellular distribution of PEG-coated polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles in rat brain endothelial cells (RBEC) have been investigated. A cell fractionation method has been developed based on the selective permeabilisation of RBEC plasma membrane by digitonin. By interacting with membrane cholesterol, digitonin creates pores allowing the release of soluble and diffusible species outside the cell. The selectivity of plasma membrane permeabilisation was controlled by using compartment markers such as lactate dehydrogenase (LDH) for cytoplasm and cathepsin B for lysosomes. An optimal digitonin concentration of 0.003% (w/v) has been identified to induce a pattern of membrane permeabilisation corresponding to the extraction of 72% LDH and less than 15% of Cathepsin B. Membrane permeabilisation at this digitonin concentration allows one to distinguish between the cell cytoplasm and its endo/lysosomal fraction.This methodology was applied to investigate the intracellular distribution of the nanoparticles after their incubation with the RBEC. The results showed that PEG-PHDCA nanoparticles were able to be internalised to a higher extent than PHDCA nanoparticles (after 20 min incubation). Additionally, these nanoparticles displayed different patterns of intracellular capture, depending on their specific surface composition: PEG-PHDCA nanoparticles were 48% in the plasma membrane, 24% in the cytoplasm, 20% in vesicular compartments and 8% associated with the fraction of the nucleus, the cytoskeleton and caveolae suggesting that PEG-PHDCA nanoparticle uptake by RBEC is specific and presumably due to endocytosis. Confocal microscopy studies confirmed the cellular uptake of PEG-PHDCA nanoparticles.
Keywords: Pegylated-nanoparticles; Intracellular distribution; Brain endothelial cells; Digitonin;

Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model by Camilla Foged; Birger Brodin; Sven Frokjaer; Anne Sundblad (315-322).
Current vaccine development includes optimization of antigen delivery to antigen presenting cells, such as dendritic cells (DC). Particulate systems have attracted increasing attention in the development of vaccine delivery systems. In the present study, we investigated DC uptake of model fluorescent polystyrene particles with a broad size range and variable surface properties. Localization of particles was investigated using confocal laser scanning microscopy and uptake was quantified by flow cytometry. Immature DC were generated from mononuclear cells isolated from human blood. The polystyrene particles interacted with the DC throughout the tested diameter range of 0.04–15 μm in a time- and concentration-dependent manner. The optimal particle diameter for fast and efficient acquisition by a substantial percentage of the DC was 0.5 μm and below. The surface of 1 and 0.1 μm polystyrene particles was covalently modified with different polyaminoacids/proteins, yielding particles with varying surface charge. Uptake of 1 μm particles was greatly enhanced when particles displayed a positive surface charge. In general, the present findings establish that particle diameters of 0.5 μm and below were optimal for DC uptake; however uptake of larger particles could be greatly enhanced by rendering the particle surface positive. Whether increased particle uptake is correlated with increased immune responses, remains to be established.
Keywords: Dendritic cells; Antigen delivery; Particle uptake; CLSM; Flow cytometry;

Nanoencapsulation of a crystalline drug by Anne-Magali Layre; Ruxandra Gref; Joël Richard; Denis Requier; Hélène Chacun; Martine Appel; Abraham J. Domb; Patrick Couvreur (323-327).
The aim of this work was to assess the influence of various formulation parameters on the incorporation of a poorly water-soluble crystalline drug into nanoparticles. For this purpose, the influence of the polymer (polylactic acid, polysebacic acid terminated with lithocholic acid, and polysebacic acid-co-lithocholic acid) as well as the effect of the dispersion medium (aqueous phases at different temperatures, saline medium and ethanol) on the encapsulation was investigated. 3H-labelled drug was used in order to determine the loading efficiency by liquid scintillation counting. The solubility of the drug in the various polymer materials was assessed by differential scanning calorimetry (DSC). The solubility of the drug in the different dispersion media was then determined by gas chromatographic–mass spectrometric measurements. The highest loading ratios were obtained using poly (lactic acid) (PLA). However, the drug solubility in the polymers, determined by DSC analysis, cannot be considered as predictive for encapsulation efficiency. The study of the influence of the liquid outer phase showed that the encapsulation efficiency increased when the drug solubility in the dispersion medium (before acetone evaporation) decreased. These experiments made it possible to propose a mechanism to account for the leakage of the crystalline drug during the nanoprecipitation process. So, when acetone is eliminated by evaporation, the drug solubility in the dispersion medium decreases, leading to the formation of crystals. During nanoparticles storage, the crystals continue to grow, the nanoparticles serving as drug reservoirs. These findings highlight the importance of using a polymer with a specific affinity for the drug, and a dispersion medium with the lowest drug solubility to achieve an efficient encapsulation of a crystalline drug.
Keywords: Nanoparticles; Poorly water-soluble crystalline drug; PLA; Polyanhydrides; DSC;

Enzymatic characterization of lipid-based drug delivery systems by Helena Ljusberg-Wahren; Flemming Seier Nielsen; Mattias Brogård; Emma Troedsson; Anette Müllertz (328-332).
The present work introduces a simple and robust in vitro method for enzymatic characterisation of surface properties of lipid dispersions in aqueous media. The initial lipolysis rate in biorelevant media, using pancreatic lipase and a self-microemulsifying formulation (SMEDDS) containing digestible lipids as substrate, was determined. The impact of incorporating two sparingly water soluble model drugs, probucol and halofantrine, into the SMEDDS was studied. It was found that both model drugs reduced the initial rate of lipolysis compared with the vehicle, probucol having a larger effect than halofantrine. The reduction of initial lipolysis rate indicates that probucol and halofantrine are bound in the water/emulsion interface limiting the substrate availability.
Keywords: Lipolysis; Self-microemulsifying drug delivery system; Probucol; Halofantrine;

Calorimetric study of bovine serum albumin dilution and adsorption onto polystyrene particles by M.J. Pollitt; G. Buckton; S. Brocchini; H.O. Alpar (333-338).
Titration calorimetry was used to investigate the interaction between a model antigen, bovine serum albumin (BSA), and a model particulate carrier, polystyrene (PS). The binding enthalpy was much higher than reported in the literature for a similar system and did not display a sigmoidal binding curve. These experiments may have accessed low coverage surface sites due to the irreversible nature of protein binding and stepwise titration. An important correction is the heat of dilution of the protein solution. Two regimes were observed: at low concentrations of BSA (below ca. 0.3% (w/v)) an exothermic dilution enthalpy of ca. −100 mJ mg−1 was determined, whereas at higher concentrations of BSA values of ca. −20 mJ mg−1 were obtained. Solution rheological data also showed a change at 0.3% (w/v) BSA, so we hypothesise that the fraction of the BSA as monomers, dimers and polymers in solution changes at approximately 0.3% (w/v).
Keywords: Adsorption; Dilution; Titration calorimetry; Bovine serum albumin; Protein; Binding;

Cholesterol–bile salt vesicles as potential delivery vehicles for drug and vaccine delivery by C. Martin; J. Thongborisute; H. Takeuchi; H. Yamamoto; Y. Kawashima; H.O. Alpar (339-343).
The aim of this study was to further investigate the interactions between cholesterol (CH) and mixed bile salts (BS) (sodium cholate and sodium deoxycholate) and their suitability for drug and vaccine delivery. Insulin was used as a model protein to assess the ability of CH:BS vesicles to entrap a therapeutically relevant macromolecule. The association of protein (FITC–insulin) with the CH:BS structure was confirmed with fluorescence microscopy, and the overall morphology of the vesicles was examined with atomic force microscopy (AFM). Results demonstrate that the nature of the vesicles formed between CH and BS is dependent not only on the concentration of BS but also on the increasing CH concentration leading to CH crystal formation.
Keywords: Cholesterol; Bile salts; Vaccine delivery;

Effect of Vitamin E TPGS on immune response to nasally delivered diphtheria toxoid loaded poly(caprolactone) microparticles by S. Somavarapu; S. Pandit; G. Gradassi; M. Bandera; E. Ravichandran; Oya H. Alpar (344-347).
The nasal mucosa has many advantages as a potential site for drug and vaccine delivery. The present study has sought to exploit this route of delivery using microparticles composed of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) as a matrix material blended with poly(caprolactone) for nasal immunisation with diphtheria toxoid. Particles were prepared by a double emulsion method, followed by spray drying and the effect of TPGS on size, zeta potential, loading and release of antigen was assessed. Particles composed of TPGS–PCL blends were spherical, smooth and monodisperse, displaying increasing yields after spray drying with increasing concentrations of TPGS. The immune response to diphtheria toxoid loaded PCL-TPGS microspheres after nasal administration was shown to be higher than that achieved using PCL microspheres alone. We conclude that TPGS shows significant potential as a novel adjuvant either alone or in combination with an appropriate delivery system.
Keywords: Microspheres; Vitamin E TPGS; PCL; Emulsification; Spray drying; Diphtheria toxoid; Intra nasal;

Hydrophobic dendrimer-derived nanoparticles by Baljit Singh; Alexander T. Florence (348-353).
Lipidic polylysine dendrimers, synthesized using Fmoc solid phase peptide techniques, have been formulated as nanoparticles by precipitation from solution in dichloromethane. The effect of concentration on the diameter and stability of nanoparticles formed from two short homologous series of dendrimers – one fifth generation and one sixth generation series and with surface C4, C10 or C12 groups – was investigated using photon correlation spectroscopy. The increase in generation from fifth to sixth resulted in increased diameter for each chain length. An increase in the surface lipidic chain length from C4 to C12 had no effect on the particle diameter of aggregates derived from fifth generation dendrimers, and a small and variable effect on the sixth generation derived nanoparticles. Using pyrene (excitation 340 nm) as a hydrophobic fluorescent probe, a decrease in intensity peak I 1 (374 nm)/I 3 (385 nm) in the emission spectra (340–600 nm) was observed in the two dendrimers studied, fifth generation dendrimers with C10 or C12 surface lipidic chains, as the dendrimer concentration increased, reaching a plateau at higher concentrations, indicating that a more compact form of the aggregates with a more hydrophobic interior was obtained. Apart from the hydrophobicity of the dendrimers and dendrimer concentration, the flexibility of the dendrimers might have a significant effect in determining nanoparticle size. The aggregates derived from the fifth generation dendrimers with C10 or C12 surface lipidic chains are stable in purified intestinal fluid but not in purified stomach fluid, in which further aggregation of the nanoparticulate dendrimer aggregates occurs as an effect of pH, salts, proteins and enzymes in these fluids. This study demonstrates, inter alia, the importance of testing nanoparticulate delivery systems in relevant physiologically based fluids prior to their use in vivo.
Keywords: Dendrimers; Nanoparticles; Aggregation;

Release of DNA from dendriplexes encapsulated in PLGA nanoparticles by Suzie Ribeiro; Nasir Hussain; Alexander T. Florence (354-360).
Biodegradable PLGA particles of less than 1 μm can encapsulate DNA and DNA–dendron complexes (dendriplexes) providing sustained DNA release for transfecting cells in gene delivery. Two polylysine-based dendrons prepared by solid state peptide synthesis were used to condense pRedN-1 DNA (7.5 kbp), a fluorescent protein vector. The dendrons had 16 free surface amino groups attached to seven lysine groups, bound to a lipid core, one containing three C18 chains and the other a single C10 chain. Increased lipophilicity and molar charge ratios are key factors in producing compact and reproducible dendriplexes, shown by the hydrodynamic diameter which is of the order of 800 nm (p.d. > 0.5) at a 2:1 molar charge ratio, a value which decreases to around 200 nm at a 5:1 charge ratio. At lower charge ratios the dendriplexes are negative and have a zeta potential in order of −18 mV. As the ratio increases (5:1, 10:1) the complexes bear a positive potential (13 ± 2 mV). This suggests that at the 2:1 ratio the DNA is not fully condensed. The DNA was radiolabelled with 35S dCTP (deoxycytidinetriphosphate) with the removal of the un-incorporated radiolabelled nucleotides. The encapsulation efficiency of dendriplexes in PLGA particles is higher than that for uncomplexed DNA. When the results are normalised for DNA content and particle surface area, complexation of the DNA was found to decrease release rate.
Keywords: Dendron; pDNA; Encapsulation; PLGA; Dendriplexes;

The diffusion of latex nanospheres and the effective (microscopic) viscosity of HPMC gels by Pakatip Ruenraroengsak; Alexander T. Florence (361-366).
Dynamic light scattering (DLS) has been used to measure the diffusion coefficients of 108 ± 7 (±S.D.) and 495 ± 23 nm positively charged amino latex nanospheres (ALNs) and negatively charged carboxyl latex nanospheres (CLNs) (48 ± 7, 91 ± 9.8 and 483 ± 10 nm) in three different media, water, glycerol aqueous solutions and hydroxypropyl methylcellulose (HPMC) gels. The translational diffusion coefficients (D) of these latex spheres in water were found to be 13.00 (±0.12), 5.11 (±0.06), 0.89 (±0.01) μm2/s for 48, 91 and 483 nm CLN, and 3.26 (±0.01) and 0.88 (±0.03) μm2/s for 108 and 495 nm ALN, respectively. In Newtonian glycerol aqueous solutions as anticipated the diffusion could be predicted by the Stokes–Einstein relationship over a range of system viscosities. In HPMC gels the results show the deviation of the diffusion coefficient from the Stokes–Einstein equation when the viscosity of the medium is increased. In addition, there was an increase in the polydispersity index (PI) from 0.217 to 0.928 with 108 nm ALN on increasing HPMC concentrations from 0.2% to 0.8% (w/v), which implied an interaction between the positively charged nanospheres and the gel. From the D values, the “effective” or “microscopic” viscosities of the HPMC medium were calculated, and ranged from 0.899 to 0.925 mPa s.
Keywords: Diffusion coefficient; Effective viscosity; Nanospheres; Diffusion in gels;

Novel anhydrous emulsions: Formulation as controlled release vehicles by Orawan Suitthimeathegorn; Vikas Jaitely; Alexander T. Florence (367-371).
Novel anhydrous emulsions, which may offer some advantages as depot or reservoir vehicles for lipophilic drugs in controlled delivery systems, were formulated using castor oil as the disperse phase and dimethicone or cyclopentasiloxane as the continuous phase. Among the emulsifiers studied only silicone surfactants (cyclomethicone/dimethicone copolyols) which were miscible in silicone oil stabilized the emulsions. Cyclomethicone/PEG/PPG-18/18 Dimethicone and Cyclopentasiloxane/PEG/PPG-18/18 Dimethicone were more effective in lowering the interfacial tension between castor oil and both dimethicone and cyclopentasiloxane. Emulsions formulated using either of these two surfactants were found to be stable against phase separation and exhibited least globule growth over 168 h. The average particle size was found to be 2–6 μm in these systems formed by probe sonication. Slow release patterns of 3H-dehydroepiandrosterone (DHEA) and 3H-dexamethasone solubilized in the disperse castor oil phase into an aqueous dialyzing medium were observed over 48 h.
Keywords: Emulsions; Non-aqueous emulsions; Interfacial tension; Silicone surfactants; Non-ionic surfactants; Depot; Controlled release;

The relative flow of the walls of phospholipid tether bilayers by Behrooz Nasseri; Alexander T. Florence (372-377).
Lipid nanotubes or “tethers” can be formed from liposomes or niosomes, pulled from the parent vesicles by micromanipulation. The tethers are cylindrical multibilayer tubes. Here, we describe the movement of these multilamellar walls, initiated by creating a surface tension gradient along the tether. The movement of lipid can give rise to a visible moving boundary. In the case of bilayer membranes, a tangential gradient in surface tension produces membrane bulk flow toward regions of higher surface tension. The flow of the bilayers comprising the tether nanotubes seems to be restricted to the inner bilayers, creating a velocity gradient in the bilayers. In this study, we discuss the implementation of tension-driven flows as a transport method in a tether-vesicle network. Interactions between fluid within the tether channels and the lipid layers are important, leading to anomalies in the transport of fluids and particles compared to bulk systems.
Keywords: Liposome; Tether; Nanotube; Soft-matter; Nanofluidics;

Preparation and in vitro evaluation of chitosan nanoparticles containing a caspase inhibitor by Yeşim Aktaş; Karine Andrieux; Maria Jose Alonso; Pilar Calvo; R. Neslihan Gürsoy; Patrick Couvreur; Yılmaz Çapan (378-383).
The aim of this work was to develop a formulation for Z-DEVD-FMK, a peptide which is a caspase inhibitor and has been used in experimental animal studies for a decade. Peptide loaded chitosan nanoparticles were obtained by ionotropic gelation process and Z-DEVD-FMK was quantified by an HPLC method. The influence of the initial peptide concentration on the nanoparticle characteristics and release behavior was evaluated. The CS nanoparticles have a particle diameter (Z-average) ranging from approximately 313–412 nm and a positive zeta potential (20–28 mV). The formulation with the initial peptide concentration of 400 ng/ml provided the highest loading capacity (0.46%) and the highest extent of release (65% at 24 h) suggesting the possibility to achieve a therapeutic dose. According to the data obtained, this chitosan-based nanotechnology opens new and interesting perspectives for anticaspase activity.
Keywords: Chitosan; Nanoparticles; Z-DEVD-FMK; HPLC;

Noticeboard (384-386).