European Journal of Pharmaceutics and Biopharmaceutics (v.79, #1)

APV Diary (I).

Editorial by Robert Gurny; Jean Christophe Leroux (1-2).

Gellan gum blended PEI nanocomposites as gene delivery agents: Evidences from in vitro and in vivo studies by Ritu Goyal; S.K. Tripathi; Shilpa Tyagi; K. Ravi Ram; K.M. Ansari; Y. Shukla; D. Kar Chowdhuri; Pradeep Kumar; K.C. Gupta (3-14).
In this study, blending of gellan gum (GG) with polyethylenimine (PEI) was performed electrostatically to form GP nanocomposites and investigated then as gene delivery agents in vitro and in vivo.Branched Polyethylenimine, 25 kDa (PEI), was blended with gellan gum, an anionic heteropolysaccharide, for partial neutralization of its excess positive charge to form gellan gum-polyethylenimine (GP) nanocomposites (NCs). Subsequently, we manipulated the amount of gellan gum for obtaining a series of NCs and characterized them for their size, charge and morphology. Among all the NCs, one member, named GP3, showed the best transfection efficiency in tested cell lines in comparison with the rest of the series, PEI, Lipofectamine and other commercial transfection agents and also exhibited minimum cytotoxicity. It was found to transfect primary cells of mouse skin with better efficiency than PEI and Lipofectamine and was able to protect the plasmid DNA from nucleases and serum proteins present in the blood. GP3 exhibited efficient intracellular delivery of plasmid as revealed by confocal studies while its intracellular presence was also confirmed by the knockdown of GFP expression (using GFP specific siRNA) and JNKII by quantifying proteins in cell lysates and by western blotting and hybridization, respectively. In vivo cytotoxicity studies in Drosophila showed lack of induction of stress response in the exposed organisms. Further, exposed organisms did not show any developmental delay or mortality and no morphological defects were observed in the emerged flies. In vivo gene expression studies in Balb/c mice revealed maximum expression of luciferase enzyme in spleen. The study suggests that GP3 may act as an efficient non-viral gene carrier with diverse biomedical applications.
Keywords: Gellan gum; PEI; Primary mouse keratinocytes; Drosophila melanogaster; Gene delivery; In vivo;

Preparation of phytantriol cubosomes by solvent precursor dilution for the delivery of protein vaccines by S.B. Rizwan; D. Assmus; A. Boehnke; T. Hanley; B.J. Boyd; T. Rades; S. Hook (15-22).
Cubosomes are traditionally prepared using fragmentation. However, this approach may not be practical for scale up and can lead to degradation of labile actives. Here we describe a simple process whereby a liquid precursor mixture consisting of the liquid crystal forming lipid (phytantriol), the polymer (F127) and a hydrotrope (propyleneglycol) when diluted in water and with vortex mixing result in the formation of cubic nanoparticles within the submicron size range. Furthermore, fluorescently labelled ovalbumin can be encapsulated efficiently within the cubosome particles.Different delivery strategies to improve the immunogenicity of peptide/protein-based vaccines are currently under investigation. In this study, the preparation and physicochemical characterisation of cubosomes, a novel lipid-based particulate system currently being explored for vaccine delivery, was investigated. Cubosomes were prepared from a liquid precursor mixture containing phytantriol or glycerylmonooleate (GMO), F127 for particle stabilisation, and a hydrotrope (ethanol or polyethylene glycol (PEG200) or propylene glycol (PG)). Several liquid precursors were prepared, and the effect of varying the concentrations of F127 and the hydrotrope on cubosome formation was investigated. Formulations were prepared by fragmentation for comparison. The model protein ovalbumin (Ova) was also entrapped within selected formulations. Submicron-sized particles (180–300 nm) were formed spontaneously upon dilution of the liquid precursors, circumventing the need for the preformed cubic phase used in traditional fragmentation-based methods. The nanostructure of the phytantriol dispersions was determined to be cubic phase using SAXS whilst GMO dispersions had a reverse hexagonal nanostructure coexisting with cubic phase. The greatest entrapment of Ova was within phytantriol cubosomes prepared from liquid precursors. Release of Ova from the various formulations was sustained; however, release was significantly faster and the extent of release was greater from fragmented dispersions compared to liquid precursor formulations. Taken together, these results suggest that phytantriol cubosomes can be prepared using liquid precursors and that it is a suitable alternative to GMO. Furthermore, the high entrapment and the slow release of Ova in vitro highlight the potential of phytantriol cubosomes prepared using liquid precursors as a novel vaccine delivery system.
Keywords: Cubosomes; Phytantriol; Vaccines; Liquid precursor; Proteins; Glyceryl monooleate;

Drug delivery into the skin by degradable particles by Wing Cheung Mak; Heike Richter; Alexa Patzelt; Wolfram Sterry; Kwok Kei Lai; Reinhard Renneberg; Jürgen Lademann (23-27).
Introduction of a two-component drug delivery system, consisting of degradable particles loaded with a model drug and a separate protease formulation for degradation of the particles after follicular penetration.Recently, it was demonstrated that particles could be utilized as carrier systems for drugs into the hair follicles. In the present study, a two-component drug delivery system is presented consisting of degradable particles loaded with fluorescein isothiocyanate and a separate protease formulation for degradation. The particles were applied alone, 30 min previous to the protease application and simultaneously with the protease onto porcine skin. Subsequently, biopsies were removed, and the penetration depths of the particles were analyzed using laser scanning microscopy.The obtained results demonstrate that the particles alone achieved a penetration depth of around 900 μm. Similar results were obtained for the successive application of particles and protease, whereas a release of the fluorescent dye was only observed in the upper 250 μm corresponding to the penetration depth of the protease. In the case of the simultaneous application, the particles were partly dissolved before application, leading to a reduced particle size and diminished penetration depth.The results revealed that degradable particles are a promising tool for drug delivery into the skin.
Keywords: Hair follicles; Penetration; Protease; Acne therapy; Topical vaccination;

A new approach for the evaluation of niosomes as effective transdermal drug delivery systems by Rita Muzzalupo; Lorena Tavano; Roberta Cassano; Sonia Trombino; Teresa Ferrarelli; Nevio Picci (28-35).
Schematic representation of the synthesis of modified L64ox and P105ox Pluronic surfactants and typical photomicrographs of obtained niosomal formulations as seen by TEM..The central motivation for this study was to evaluate if the increased hydrophilic drug permeation across the skin, which is always observed in presence of vesicular systems, is dependent on the structural organization of niosomes, that are used to transport the active molecules, or if it is only dependent on the surfactant dual nature. To answer this question, non-ionic surfactants belonging to the class of Pluronic and sucrose esters were used both as components of niosomal systems or in the form of sub-micellar solutions. The obtained niosomes were characterized by their entrapment efficiency, size and morphology.The enhancing effect of niosomes on the ex vivo percutaneous penetration of a model drug was investigated using a Franz-type diffusion chamber and compared to that obtained by using sub-micellar solution of surfactant or achieving pretreatment of the skin with surfactants’ sub-micellar solution or empty niosomes.The results suggest that the surfactants used in this study could be considered as percutaneous permeation enhancers only when they are in the form of drug-loaded vesicular systems: no percutaneous promotion was achieved by using sub-micellar solution containing free Sulfadiazine sodium salt or performing pretreatment with empty niosomes or sub-micellar solutions of the surfactant. In our experiments, only niosomes act as effective transdermal drug delivery systems.
Keywords: Pluronic; Sucrose cocoate; Niosomes; Sulfadiazine sodium salt; Percutaneous permeation;

Lipid nanocarriers are efficient transdermal drug delivery systems while polymeric nanoparticles are better suited for local effects on the skin.Lipid nanocapsules (LNC) are colloidal carriers providing controlled release profiles and improved bioavailability for many drug substances and diverse administration routes. However, they have not been explored before for transdermal application. Here, we study the behavior of LNC as a transdermal drug delivery system using ibuprofen as a model drug. A comparison to other lipid nanocarriers such as solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) and polymeric nanocarriers has been made. It was found that LNC could increase the flux rate of ibuprofen 21.9 ± 0.5 compared to 5.8 ± 0.4 μg/cm2  h in case of drug solution. Similar flux rates were obtained for SLN and NLC with average values of 22.9 ± 0.5 and 22.5 ± 2.0 μg/cm2  h, respectively. On the other side, comparison to polymeric nanoparticles showed that the polymer-based carriers of the same particle size had lower permeation-enhancing effect with a flux rate of 10.62 ± 1.84 μg/cm2  h. Polymeric carriers had fourfold higher accumulation in the skin compared to that of the LNC and twice the accumulation of SLN and NLC. These results would suggest that the LNC can be considered as efficient as SLN and NLC for the transdermal drug delivery while polymeric nanoparticles are more suitable for localized drug delivery to the skin.
Keywords: Lipid nanocapsules; Skin; Transdermal; Nanocarriers; Polymer nanoparticles;

Skin penetration behaviour of liposomes as a function of their composition by A. Gillet; F. Lecomte; P. Hubert; E. Ducat; B. Evrard; G. Piel (43-53).
Schematic representation of fluorescently labelled liposomes encapsulating calcein in their inner cavity (A), liposomes encapsulating NBD-PC in their lipid bilayer (B) and liposomes encapsulating rhodamine B and NBD-PC in their lipid bilayer (C). Skin penetration of these liposomes is shown in the corresponding confocal images.Deformable liposomes have been developed and evaluated as a novel topical and transdermal delivery system. Their mechanism of drug transport into and through the skin has been investigated but remains a much debated question. The present study concerns ex vivo diffusion experiments using pig ear skin in order to explain the penetration mechanism of classical and deformable liposomes. Classical and deformable vesicles containing betamethasone in the aqueous compartment through the use of cyclodextrin inclusion complexes were compared to vesicles encapsulating betamethasone in their lipid bilayer. Deformable liposomes contained sodium deoxycholate as the edge activator. Liposomes were characterised by their diameter, encapsulation efficiency, deformability, stability (in terms of change in diameter) and release of encapsulated drug. Ex vivo diffusion studies using Franz diffusion cells were performed. Confocal microscopy was performed to visualise the penetration of fluorescently labelled liposomes into the skin. This study showed that liposomes do not stay intact when they penetrate the deepest layers of the skin. Betamethasone is released from the vesicles after which free drug molecules can diffuse through the stratum corneum and partition into the viable skin tissue.
Keywords: Liposome; Cyclodextrin; Betamethasone; Franz cells; Pig skin;

A new drug nanocarrier consisting of polyarginine and hyaluronic acid by Felipe A. Oyarzun-Ampuero; Francisco M. Goycoolea; Dolores Torres; Maria J. Alonso (54-57).
A new nanoparticulate system was obtained by simply mixing polyarginine and hyaluronic acid aqueous solutions. The nanoparticles surface charge could be conveniently modulated as a function of the charge ratio of the polymers.The purpose of this study was to produce and characterize a variety of nanostructures comprised of the polyaminoacid polyarginine (PArg) and the polysaccharide hyaluronic acid (HA) as a preliminary stage before evaluating their potential application in drug delivery. PArg was combined with high- or low-molecular-weight HA (HMWHA or LMWHA, respectively) to form nanoparticles by simply mixing polymeric aqueous solutions at room temperature. The average size of the resulting nanocarriers was between 116 and 155 nm, and their zeta potential value ranged from +31.3 to −35.9 mV, indicating that the surface composition of the particle could be conveniently modified according to the mass ratio of the polymers. Importantly, the systems prepared with HMWHA remained stable after isolation by centrifugation and in conditions that mimic the physiological medium, whereas particles that incorporated LMWHA were unstable. Transmission electron microscopy showed that the nanostructures made with HMWHA were spherical. Finally, the systems were stable for at least three months at storage conditions (4 °C).
Keywords: Nanoparticles; Hyaluronic acid; Polyarginine; Drug delivery;

Development of sucrose stearate-based nanoemulsions and optimisation through γ-cyclodextrin by Victoria Klang; Nadejda Matsko; Karoline Raupach; Nivine El-Hagin; Claudia Valenta (58-67).
The graphical abstract depicts the change in nanoemulsion microstructure after addition of γ-CD as observed with TEM. The left-hand pictures show nanoemulsions with sucrose stearate or lecithin. The right-hand side pictures show the corresponding formulations with additional CD.Nanoemulsions aimed at dermal drug delivery are usually stabilised by natural lecithins. However, lecithin has a high tendency towards self-aggregation and is prone to chemical degradation. Therefore, the aim of this study was to develop nanoemulsions with improved structure and long-term stability by employing a natural sucrose ester mixture as sole surfactant. A thorough comparison between the novel sucrose stearate-based nanoemulsions and corresponding lecithin-based nanoemulsions revealed that the sucrose ester is superior in terms of emulsifying efficiency, droplet formation as well as physical and chemical stability. The novel formulations exhibited a remarkably homogeneous structure in cryo TEM investigations, as opposed to the variable structure observed for lecithin-based systems. The in vitro skin permeation rates of lipophilic drugs from sucrose stearate nanoemulsions were comparable to those obtained with their lecithin-based counterparts. Furthermore, it was observed that addition of γ-cyclodextrin led to enhanced skin permeation of the steroidal drug fludrocortisone acetate from 9.99 ± 0.46 to 55.10 ± 3.67 μg cm−2 after 24 h in the case of sucrose stearate-based systems and from 9.98 ± 0.64 to 98.62 ± 24.89 μg cm−2 after 24 h in the case of lecithin-based systems. This enhancement effect was significantly stronger in formulations based on lecithin (P  < 0.05), which indicates that synergistic mechanisms between the surfactant and the cyclodextrin are involved. Cryo TEM images suggest that the cyclodextrin is incorporated into the interfacial film, which might alter drug release rates and improve the droplet microstructure.
Keywords: Lecithin; Sucrose stearate; Nanoemulsion; Cryo TEM; γ-Cyclodextrin; Fludrocortisone acetate;

The relative amount of the corticosteroid betamethasone 17-valerate (BMV) penetrated into the different skin layers after applying BMV in an ointment and in three different SLN, respectively, onto intact and barrier-impaired skin for 24 h. The skin permeation of BMV increased when the barrier was impaired but it was shown that a significantly higher amount of BMV remained in the skin, intact as well as barrier impaired, when SLN was used as a vehicle. The results are relative to the total amount of BMV recovered. Mean ± SD (n  = 8).Treatment of skin diseases implies application of a drug to skin with an impaired epidermal barrier, which is likely to affect the penetration profile of the drug substance as well as the carrier into the skin. To elucidate this, the effect of skin barrier damage on the penetration profile of a corticosteroid applied in solid lipid nanoparticles (SLN) composed of different lipids, varying in polarity, was studied. The studies were carried out in vitro using impaired and intact porcine ear skin, and the SLN were compared with a conventional ointment. It was shown that a significantly higher amount of corticosteroid remained in the skin, intact as well as barrier impaired, when SLN was used as a vehicle. In general, the penetration profile of the drug substance into the skin was affected by the type of lipid used in the formulation and related to lipid polarity and drug substance solubility. When formulated in SLN and applied to intact skin, the permeation of the drug substance across the skin was significantly reduced, as compared to the ointment. Altogether, in both barrier-impaired and intact skin, a higher amount of drug substance remained in the skin during application of SLN for 6, 16, and 24 h, as compared to the ointment. These results emphasize the applicability of SLN to create a drug reservoir in skin, with the drug localized distinctively in the stratum corneum.
Keywords: Solid lipid nanoparticles; SLN; Barrier-impaired skin; Tape stripping; Skin penetration; Corticosteroid;

Cryo TEM micrographs of DPPC and Chitosan-DPPC liposomes.The stratum corneum (SC), top layer of the epidermis, is comprised mostly of lipids that are responsible for the permeability properties of the SC and which protect the body from external agents. Changes in these skin microconstituents can be understood by instrumental methods such as attenuated total reflectance Fourier-transform infrared (ATR–FTIR) spectroscopy. The present work shows that different types of analyzed skin, dermatomed abdominal porcine skin, pig ear skin, and human heat separated skin, influenced both the shape and the intensity of recorded spectra. The typical FTIR spectral bands of the conformation of the lipid aliphatic chains in the skin samples were altered after treatment with pure DPPC liposomes and chitosan (CS) coated DPPC liposomes, but not with aqueous CS-solution. The conformational change could be the reason for the variable permeability of the skin. This was confirmed by tape stripping on pig ear skin (imitating in vivo studies): the amount of aciclovir penetrating from polymer coated and polymer free liposomes was significantly higher under the skin surface in comparison with the aqueous CS-solution. Moreover, the addition of the polymer to liposomes induced a higher skin penetration than pure liposomes. One explanation might be the CS’s stronger adhesion to the skin.
Keywords: FTIR; Tape stripping; Aciclovir; DPPC liposomes; Cryo-TEM;

Modified nanolipid carrier (MNLC) involving the use of lipophilic solubilizers offered much more opportunity to load drugs with poor solubility, such as tacrolimus. It allowed remarkable flexibility in the modulation of drug release with advantages of enhanced effectiveness and improved performance in terms of stability and skin localization of tacrolimus for topical application resulting in a new, effective, and interesting alternative to the currently available marketed products.Low solubility of tacrolimus in carrier matrix and subsequent poor in vivo bioavailability was overcome by constructing modified nanolipid carrier (MNLC) as a novel approach. The aim of this study was to develop MNLC with enhanced drug solubility in carrier lipid matrix using lipophilic solubilizers for topical delivery. Comprehensive characterization of tacrolimus-loaded MNLC (T-MNLC) was carried out for particle size, morphology, and rheology. Lipid modification resulted in the formation of less perfect crystals offering space to accommodate the dissolved drug leading to high entrapment efficiency of 96.66%. Compatibility and mixing behavior of carrier constituents was evaluated using DSC, FT-IR, and 1H NMR. T-MNLC displayed sufficient stability that could be attributed to possibility to reduce total lipid concentration in carrier. T-MNLC-enriched gels showed significantly higher in vitro drug release, skin permeation, and in vivo bioavailability with dermatopharmacokinetic approach in guinea pigs compared to commercial ointment, Protopic® as reference. Penetration-enhancing effect was confirmed using gamma scintigraphy in vivo in rats. Radioactivity remained localized in skin at the application site avoiding unnecessary biodisposition to other organs with prospective minimization of toxic effects. Skin irritation studies showed T-MNLC to be significantly less irritating than reference. Research work could be concluded as successful development of novel T-MNLC using lipophilic solubilizers to increase the encapsulation efficiency of colloidal lipid carriers with advantage of improved performance in terms of stability and skin localization.
Keywords: Modified carrier; Tacrolimus; Entrapment efficiency; Improved skin permeation; Skin localization;

Improved photostability and reduced skin permeation of tretinoin: Development of a semisolid nanomedicine by Aline Ferreira Ourique; Ana Melero; Cristiane de Bona da Silva; Ulrich F. Schaefer; Adriana Raffin Pohlmann; Silvia Stanisçuaski Guterres; Claus-Michael Lehr; Karl-Heinz Kostka; Ruy Carlos Ruver Beck (95-101).
Graphical representation of the reduced skin permeation profile of nanoencapsulated tretinoin from hydrogels.The aims of this work were to increase the photostability and to reduce the skin permeation of tretinoin through nanoencapsulation. Tretinoin is widely used in the topical treatment of various dermatological diseases such as acne, psoriasis, skin cancer, and photoaging. Tretinoin-loaded lipid-core polymeric nanocapsules were prepared by interfacial deposition of a preformed polymer. Carbopol hydrogels containing nanoencapsulated tretinoin presented a pH value of 6.08 ± 0.14, a drug content of 0.52 ± 0.01 mg g−1, pseudoplastic rheological behavior, and higher spreadability than a marketed formulation. Hydrogels containing nanoencapsulated tretinoin demonstrated a lower photodegradation (24.17 ± 3.49%) than the formulation containing the non-encapsulated drug (68.64 ± 2.92%) after 8 h of ultraviolet A irradiation. The half-life of the former was seven times higher than the latter. There was a decrease in the skin permeability coefficient of the drug by nanoencapsulation, independently of the dosage form. The liquid suspension and the semisolid form provided K p  = 0.31 ± 0.15 and K p  = 0.33 ± 0.01 cm s−1, respectively (p  ⩽ 0.05), while the samples containing non-encapsulated tretinoin showed K p  = 1.80 ± 0.27 and K p  = 0.73 ± 0.12 cm s−1 for tretinoin solution and hydrogel, respectively. Lag time was increased two times by nanoencapsulation, meaning that the drug is retained for a longer time on the skin surface.
Keywords: Hydrogels; Lipid-core nanocapsules; Nanomedicines; Photostability; Skin permeation; Tretinoin;

Preparation and characterization of triclosan nanoparticles intended to be used for the treatment of acne by Clara Luisa Domínguez-Delgado; Isabel Marlen Rodríguez-Cruz; José Juan Escobar-Chávez; Iván Omar Calderón-Lojero; David Quintanar-Guerrero; Adriana Ganem (102-107).
Triclosan retained in pig ear skin/vehicle concentration (RA) at the end of the permeation experiments. (▪) solution (n  = 6); (■) emulsion (n  = 4); (□) nanoparticles dispersion (n  = 6).This work focuses on the preparation and characterization of nanoparticles containing triclosan. Additionally, in vitro percutaneous permeation of triclosan through pig ear skin was performed, and comparisons were made with two commercial formulations: An o/w emulsion and a solution, intended for the treatment of acne. The nanoparticle suspensions were prepared by the emulsification–diffusion by solvent displacement method, using Eudragit® E 100 as polymer. All batches showed a size smaller than 300 nm and a positive Zeta potential, high enough (20–40 mV) to ensure a good physical stability. Differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) studies suggested that triclosan was molecularly dispersed in the nanoparticle batches containing up to 31% of triclosan, with good encapsulation efficiency (95.9%). The results of the in vitro permeation studies showed the following order for the permeability coefficients: Solution > cream ≈ nanoparticles; while for the amount retained in the skin, the order was as follows: cream > nanoparticles ≈ solution. Nanoparticles, being free of surfactants or other potentially irritant agents, can be a good option for the delivery of triclosan to the skin, representing a good alternative for the treatment of acne.
Keywords: Skin; Acne; Triclosan; Nanoparticles; Emulsion; Percutaneous permeation;

Antimicrobial PLGA ultrafine fibers: Interaction with wound bacteria by Somiraa S. Said; Affaf K. Aloufy; Omar M. El-Halfawy; Nabila A. Boraei; Labiba K. El-Khordagui (108-118).
The dynamic interaction of antimicrobial-loaded ultrafine fibers with the wound milieu results in bacterial colonization and biofilm formation. This, in turn, affects the structural integrity of the fibers resulting in enhancement of the antimicrobial release, leading to drastic bacterial killing.The structure and functions of polymer nanofibers as wound dressing materials have been well investigated over the last few years. However, during the healing process, nanofibrous mats are inevitably involved in dynamic interactions with the wound environment, an aspect not explored yet. Potential active participation of ultrafine fibers as wound dressing material in a dynamic interaction with wound bacteria has been examined using three wound bacterial strains and antimicrobial fusidic acid (FA)-loaded electrospun PLGA ultrafine fibers (UFs). These were developed and characterized for morphology and in-use pharmaceutical attributes. In vitro microbiological studies showed fast bacterial colonization of UFs and formation of a dense biofilm. Interestingly, bacterial stacks on UFs resulted in a remarkable enhancement of drug release, which was associated with detrimental changes in morphology of UFs in addition to a decrease in pH of their aqueous incubation medium. In turn, UFs by allowing progressively faster release of bioactive FA eradicated planktonic bacteria and considerably suppressed biofilm. Findings point out the risk of wound reinfection and microbial resistance upon using non-medicated or inadequately medicated bioresorbable fibrous wound dressings. Equally important, data strongly draw attention to the importance of characterizing drug delivery systems and establishing material–function relationships for biomedical applications under biorelevant conditions.
Keywords: Ultrafine fibers; Fusidic acid; Electrospinning; Drug release; Wound bacteria; Interaction;

Edaravone-loaded liposomes for retinal protection against oxidative stress-induced retinal damage by Kohei Hironaka; Yuta Inokuchi; Takuya Fujisawa; Hiroki Shimazaki; Mai Akane; Yuichi Tozuka; Kazuhiro Tsuruma; Masamitsu Shimazawa; Hideaki Hara; Hirofumi Takeuchi (119-125).
Edaravone-loaded liposomes significantly reduced N-methyl-d-aspartate(NMDA)-induced retinal damage compared with free edaravone after intravitreal injection.To optimize the retinal protective effects of submicron-sized liposomes (ssLips) containing edaravone for intravitreal administration, we investigated the effects of liposomal formulation on the pharmacological effects. Loading of edaravone into ssLips of around 50% entrapment efficiency was achieved by a calcium acetate gradient method. The in vitro radical-scavenging capacity of edaravone-loaded ssLip based on egg phosphatidylcholine (EPC-ssLip) and l-α-distearoyl phosphatidylcholine (DSPC-ssLip) was determined in RGC-5, a neuronal precursor cell line that can be differentiated to resemble retinal ganglion cells. Edaravone-loaded EPC-ssLip scavenged intracellular H2O2 radical more strongly than DSPC-ssLip, although there was only a small difference in cellular uptake of edaravone into RGC-5. An in vivo N-methyl-d-aspartate (NMDA)-induced disease model was used to investigate the retinal protective effects in mice. The edaravone-loaded EPC-ssLip significantly reduced NMDA-induced ganglion cell layer (GCL) cell death compared with free edaravone. Such protective effect was small in the case of DSPC-ssLip. These results may be related to the release profile of the edaravone from ssLips across the inner layers of the retina including GCL, indicating effective retinal protection of EPC-ssLip compared to that of DSPC-ssLip. EPC-ssLip is a promising carrier for edaravone in treating oxidative stress-induced retinal diseases.
Keywords: Edaravone; Liposome; Calcium acetate gradient method; Intravitreal injection; Retina;

A novel nanomatrix system consisted of colloidal silica and pH-sensitive polymethylacrylate improves the oral bioavailability of fenofibrate by Zengrong Jia; Ping Lin; Yu Xiang; Xueqing Wang; Jiancheng Wang; Xuan Zhang; Qiang Zhang (126-134).
A novel nanomatrix drug delivery system in combination of mesoporous colloidal silica and pH-sensitive polyacrylate improves the oral bioavailability of insoluble drug fenofibrate in rats.A novel solid particle system with a nanomatrix structure and without surfactant for the oral delivery of insoluble drugs was prepared. This used a combination of pH-sensitive polymethylacrylate and nano-porous silica, in order to improve the drug absorption using only pharmaceutical excipients and a relative simple process. The in vitro drug dissolution and in vivo oral bioavailability of this formulation, using fenofibrate as the model drug, were compared with other reference formulations such as a suspension, micronized formulation or self microemulsion drug delivery system (SMEDDS). The supersaturation stabilizing effect of different polymers was evaluated and the physicochemical characterization of the optimal formulation was conducted by SEM, TEM, surface area analysis, DSC, and XRD. The optimized formulation prepared with polymethylacrylate (Eudragit®L100-55) and silica (Sylysia®350) markedly improved the drug dissolution compared with other reference preparations and displayed a comparative oral bioavailability to the SMEDDS. Fenofibrate existed in a molecular or amorphous state in the nanomatrix, and this state was maintained for up to 1 year, without obvious changes in drug release and absorption. In conclusion, the nanomatrix formulation described here is a promising system to enhance the oral bioavailability of water-insoluble drugs.
Keywords: Fenofibrate; Oral bioavailability; Nanomatrix; Silica; Polymethylacrylate;

Baclofen-loaded solid lipid nanoparticles: Preparation, electrophysiological assessment of efficacy, pharmacokinetic and tissue distribution in rats after intraperitoneal administration by Lorenzo Priano; Gian Paolo Zara; Nadia El-Assawy; Stefania Cattaldo; Elisabetta Muntoni; Eva Milano; Loredana Serpe; Claudia Musicanti; Chantal Pérot; Maria Rosa Gasco; Giacinta Miscio; Alessandro Mauro (135-141).
We studied efficacy, pharmacokinetic and tissue distribution of baclofen incorporated in solid lipid nanoparticles (SLN), after intraperitoneal administration in rats. SLN are able to give a sustained release and targeting the CNS. Our study demonstrated prolonged efficacy of this novel formulation of baclofen, even if high baclofen concentrations in brain tissue and sedation require optimization of dosages for clinical purposes.Intrathecal baclofen administration is the reference treatment for spasticity of spinal or cerebral origin, but the risk of infection or catheter dysfunctions are important limits. To explore the possibility of alternative administration routes, we studied a new preparation comprising solid lipid nanoparticles (SLN) incorporating baclofen (baclofen-SLN). We used SLN because they are able to give a sustained release and to target the CNS. Wistar rats were injected intraperitoneally with baclofen-SLN or baclofen solution (baclofen-sol group) at increasing dosages. At different times up to 4 h, efficacy was tested by the H-reflex and two scales evaluating sedation and motor symptoms due to spinal lesions. Rats were killed and baclofen concentration determined in blood and tissues. Physiological solution or unloaded SLN was used as controls. After baclofen-SLN injection, the effect, consisting in a greater and earlier reduction of the H/M ratio than baclofen-sol group and controls, was statistically significant from a dose of 5 mg/kg and was inversely correlated with dose. Clinical effect of baclofen-SLN on both the behavioral scales was greater than that of baclofen-sol and lasted until 4th hour. Compared with baclofen-sol, baclofen-SLN produced significantly higher drug concentrations in plasma from 2nd hour until 4th hour with a linear decrement and in the brain at all times. In conclusion, our study demonstrated the efficacy of a novel formulation of baclofen, which exploits the advantages of SLN preparations. However, for clinical purposes, high baclofen concentrations in brain tissue and sedation may be unwanted effects, requiring further studies and optimization of dosages.
Keywords: Baclofen; Solid lipid nanoparticles; Intraperitoneal; Rats; Spasticity; H-reflex;

Cisplatin-loaded gelatin-poly(acrylic acid) nanoparticles: Synthesis, antitumor efficiency in vivo and penetration in tumors by Dan Ding; Zhenshu Zhu; Qin Liu; Jing Wang; Yong Hu; Xiqun Jiang; Baorui Liu (142-149).
CDDP-loaded gelatin-poly(acrylic acid) nanoparticles exhibit significantly superior in vivo antitumor effect than the commercially available CDDP.Cisplatin (CDDP)-loaded gelatin-poly(acrylic acid) (GEL-PAA) nanoparticles were successfully prepared by polymerizing acrylic acid in the presence of gelatin in aqueous solution followed by incorporating CDDP into the formed GEL-PAA nanoparticles through polymer–metal complex formation of CDDP with carboxylic groups in the nanoparticles. The obtained nanoparticles had a spherical shape, with a mean size of about 100 nm, and high drug payload as well as stability. It is found that CDDP can be released from the nanoparticles in a sustained manner with a small initial burst release. In vitro cytotoxicity revealed that CDDP-loaded nanoparticles had similar cytotoxicity to free CDDP after 48 h co-incubation with human colorectal cancer cell line LoVo. In vivo antitumor activity indicated that the nanoparticle formulation was superior in anticancer effect to free CDDP on murine hepatic H22 tumor-bearing mice model through intraperitoneal (i.p.) administration and displayed a dose-dependent antitumor efficacy. Further, the penetration examination of the nanoparticles through tumor tissue revealed that the CDDP-loaded GEL-PAA nanoparticles could only affect the cells near the tumor vasculature after they entered into the tumor tissue.
Keywords: Polymer nanoparticles; Drug delivery; Antitumor; Cisplatin; Gelatin;

The physical state of lipid nanoparticles influences their effect on in vitro cell viability by Silvia Petersen; Frank Steiniger; Dagmar Fischer; Alfred Fahr; Heike Bunjes (150-161).
Crystalline (platelet-like) lipid nanoparticles reduce the viability of mouse fibroblasts to a higher extent than corresponding liquid crystalline (barrel-shaped) or liquid (spherical) lipid nanoparticles.Although lipid nanoparticles represent potent drug carriers, for many formulations toxicity data are rare. Thus, in this study, the effect of different lipid nanoparticles on the cell viability of L929 mouse fibroblasts was systematically investigated using the MTT assay. The formulations were composed of trimyristin, tristearin or cholesteryl myristate stabilized with poloxamer 188, polysorbate 80, polyvinyl alcohol or a blend of soybean phospholipid and sodium glycocholate. Depending on lipid and storage conditions, the nanoparticles were prepared in different physical states or crystal modifications leading to different particle shapes. The cell viability was influenced considerably by the physical state of the particle matrix with crystalline nanoparticles causing a stronger decrease in viability than the corresponding liquid or liquid crystalline particles. Effects on the cell viability were also related to the type of matrix lipid, stabilizer and the particle shape. However, the effects of differently shaped particles of different polymorphic modifications of crystalline tristearin were comparable. The low viability caused by poloxamer 188-stabilized particles could be correlated with a strong cell uptake which was investigated by confocal laser scanning microscopy.
Keywords: Cell viability; Cytotoxicity; L929 mouse fibroblasts; Lipid nanoparticles; Physical state; Particle shape;

Cytotoxicity and cellular uptake of newly synthesized fucoidan-coated nanoparticles by M.C.B. Lira; N.S. Santos-Magalhães; V. Nicolas; V. Marsaud; M.P.C. Silva; G. Ponchel; C. Vauthier (162-170).
Cytotoxicity and internalization of nanoparticles interacting with J774 cells. Top: nanoparticles obtained by anionic emulsion polymerization with 0 (A), 25 (B), 100 (C) fucoidan. Bottom: nanoparticles obtained by redox radical emulsion polymerization with 0 (D) and 25% (E) fucoidan.The aim was to synthesize and characterize fucoidan-coated poly(isobutylcyanoacrylate) nanoparticles. The nanoparticles were prepared by anionic emulsion polymerization (AEP) and by redox radical emulsion polymerization (RREP) of isobutylcyanoacrylate using fucoidan as a new coating material. The nanoparticles were characterized, and their cytotoxicity was evaluated in vitro on J774 macrophage and NIH-3T3 fibroblast cell lines. Cellular uptake of labeled nanoparticles was investigated by confocal fluorescence microscopy. Results showed that both methods were suitable to prepare stable formulations of fucoidan-coated PIBCA nanoparticles. Stable dispersions of nanoparticles were obtained by AEP with up to 100% fucoidan as coating material. By the RREP method, stable suspensions of nanoparticles were obtained with only up to 25% fucoidan in a blend of polysaccharide composed of dextran and fucoidan. The zeta potential of fucoidan-coated nanoparticles was decreased depending on the percentage of fucoidan. It reached the value of −44 mV for nanoparticles prepared by AEP with 100% of fucoidan. Nanoparticles made by AEP appeared more than four times more cytotoxic (IC50 below 2 μg/mL) on macrophages J774 than nanoparticles made by RREP (IC50 above 9 μg/mL). In contrast, no significant difference in cytotoxicity was highlighted by incubation of the nanoparticles with a fibroblast cell line. On fibroblasts, both types of nanoparticles showed similar cytotoxicity. Confocal fluorescence microscopy observations revealed that all types of nanoparticles were taken up by both cell lines. The distribution of the fluorescence in the cells varied greatly with the type of nanoparticles.
Keywords: Poly(isobutylcyanoacrylate); Fucoidan; Core–corona nanoparticles; Emulsion polymerization; Cellular uptake;

Uptake of phenothiazines by the harvested chylomicrons ex vivo model: Influence of self-nanoemulsifying formulation design by Gul Shahnaz; Markus Hartl; Jan Barthelmes; Katharina Leithner; Federica Sarti; Fabian Hintzen; Deni Rahmat; Willi Salvenmoser; Andreas Bernkop-Schnürch (171-180).
Schematic diagram illustrating the mechanism of intestinal drug transport from self-nanoemulsifying drug delivery systems (SNEDDS).The aim of this study was to examine the potential of self-nanoemulsifying drug delivery systems (SNEDDS) on the uptake of the lipophilic and poorly water soluble phenothiazines thioridazine and chlorpromazine with the isolated plasma derived chylomicron (CM) ex vivo model. The multi-component delivery systems were optimized by evaluating their ability to self-emulsify when introduced to an aqueous medium under gentle agitation. The uptake of phenothiazines by isolated plasma derived chylomicrons was investigated with short chain triglyceride (SCT) SNEDDS, medium chain triglyceride (MCT) SNEDDS, and long chain triglyceride (LCT) SNEDDS. SNEDDS were also evaluated for their stabilities, dispersibilities, percentage transmittances and by particle size analyses. For thioridazine a 5.6-fold and for chlorpromazine a 3.7-fold higher CM uptake could be observed using a LCT-SNEDDS formulation compared to the drugs without formulation. In contrast, ex vivo uptake by isolated CM was not significantly increased by SNEDDS formulations based on MCT and SCT. Compared with isolated CM, the CM sizes were increased 2.5-fold in LCT-SNEDDS, whereas in MCT-SNEDDS or SCT-SNEDDS only a small, non-significant (P  < 0.05) increase in CM size was observed. These results show that distinct SNEDDS formulations containing phenothiazines are efficiently uptaken by plasma derived chylomicrons ex vivo.
Keywords: Chylomicrons (CM); Self-nanoemulsifying drug delivery system (SNEDDS); Phenothiazines; Uptake;

A new formulation of Sn38 nanocapsules was developed with high drug payload; it was also stable in gastrointestinal media and enhanced permeability of Sn38 across Caco-2 cells.The purpose of this work was to encapsulate 7-Ethyl-10-hydroxy-camptothecin (Sn38) in lipid nanocapsules (LNCs) using phase inversion-based method in order to deliver Sn38 by oral route. LNCs were prepared by a low-energy emulsification method and were characterized for size, polydispersity index (PDI), surface charge, drug payload, in vitro drug release, and storage stability. Moreover, in view of an oral administration, in vitro stability in gastrointestinal fluid and permeability across Caco-2 cells were tested. Sn38-loaded LNCs with a mean particle size of 38 ± 2 nm were obtained. The particles displayed a narrow size distribution and a drug payload of 0.40 ± 0.07 mg/g of LNC dispersion. In vitro stability in simulated gastric and intestinal media was also observed. Finally, Sn38-loaded LNCs improved permeability of Sn38 across Caco-2 cells (5.69 ± 0.87 × 106  cm s−1 at 6 h vs 0.31 ± 0.02 × 106  cm s−1) and intracellular concentration compared with free Sn38. In conclusion, Sn38 nanocarriers have been developed and display a strong potential for oral administration.
Keywords: Lipid nanocapsule; Sn38; Caco-2; Stability; Oral administration; Nanotechnology;

Development of topotecan loaded lipid nanoparticles for chemical stabilization and prolonged release by L.G. Souza; E.J. Silva; A.L.L. Martins; M.F. Mota; R.C. Braga; E.M. Lima; M.C. Valadares; S.F. Taveira; R.N. Marreto (189-196).
Topotecan is a cytotoxic drug that can be hydrolyzed in vivo, decreasing the drug’s therapeutic efficacy. Solid lipid nanoencapsulation sustained topotecan release and improved its chemical stability and cytotoxicity.Topotecan is an important cytotoxic drug that has gained broad acceptance in clinical use for the treatment of refractory ovarian and small-cell lung cancer. The lactone active form of topotecan can be hydrolyzed in vivo, decreasing the drug’s therapeutic efficacy. Lipid encapsulation may promote in vivo stabilization by removing topotecan from aqueous media. Earlier reports of topotecan lipid nanoencapsulation have focused on liposomal encapsulation; however, the higher stability and cost-effectiveness of solid lipid nanoparticles (SLN) highlight the potential of these nanoparticles as an advantageous carrier for topotecan. The initial motivation for this work was to develop, for the first time, solid lipid nanoparticles and nanostructured lipid carriers (NLC) with a high drug loading for topotecan. A microemulsion technique was employed to prepare SLNs and NLCs and produced homogeneous, small size, negatively charged lipid nanoparticles with high entrapment efficiency and satisfactory drug loading. However, low recovery of topotecan was observed when the microemulsion temperature was high and in order to obtain high quality nanoparticles, and precise control of the microemulsion temperature is critical. Nanoencapsulation sustained topotecan release and improved its chemical stability and cytotoxicity. Surprisingly, there were no significant differences between the NLCs and SLNs, and both are potential carriers for topotecan delivery.
Keywords: Microemulsion; Nanoparticles; Topotecan; Lipid matrix; Chemical degradation; Mass spectrometry;

Reverse micelle-loaded lipid nanocarriers: A novel drug delivery system for the sustained release of doxorubicin hydrochloride by Sandy Vrignaud; Nicolas Anton; Pascal Gayet; Jean-Pierre Benoit; Patrick Saulnier (197-204).
Hydrophilic anticancer drug, doxorubicin hydrochloride, is encapsulated into lipid nano-emulsions droplets through its solubilization in reverse micellar system in oil. This multiscaled nano-system also shown the sustained diffusion-based release of the drug.▪In this study, we are pioneering new nanotechnology for the encapsulation of anticancer drugs (doxorubicin (DOX) and/or docetaxel (DOCE)), whatever their solubility and water affinity. The purpose of this study is to highlight the potential of this recently patented technology, by carrying out a thorough physicochemical characterisation of these multiscaled nanocarriers, followed by the study of an encapsulation and release model of hydrophilic anticancer drug. The formulation process is based on a low-energy nano-emulsification method and allows the generation of a structure composed of oil-based nanocarriers loaded with reverse micelles. Thanks to this, hydrophilic contents can be solubilised in the oily core of this kind of nano-emulsion along with lipophilic content. The results emphasise some original structure particularities due to the multistep formulation process, and the diffusion-based behaviour revealed for the DOX release profile that is shown to be intimately linked to the morphology of the particles.
Keywords: Nanoparticle; Reverse micelle; Low-energy emulsification; Doxorubicin; Docetaxel;