International Journal of Pharmaceutics (v.416, #2)

Editorial by Elias Fattal; Julien Nicolas; Nicolas Tsapis (401).

Cationic nanoemulsion as a delivery system for oligonucleotides targeting malarial topoisomerase II by F. Bruxel; S. Cojean; A. Bochot; H. Teixeira; C. Bories; P.-M. Loiseau; E. Fattal (402-409).
A promising strategy based on the antisense oligonucleotides against the Plasmodium falciparum topoisomerase II has been considered using cationic nanoemulsion as oligonucleotide delivery system. Phosphodiester and chemically modified phosphorothioate oligonucleotides bearing negative charges were adsorbed on positively charged emulsion composed of medium chain triglycerides, egg lecithin, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and water, at different +/− charge ratios (positive charges from cationic lipid/negative charges from oligonucleotide): +0.5/−, +2/−, +4/− and +6/−. The physicochemical properties of the complexes were determined, as well as their stability in culture medium. Their interaction with erythrocytes through hemolysis, binding experiments and confocal microscopy were also evaluated. Finally, the in vitro evaluation of parasite growth and reinfection capacity was performed. The overall results showed that antisense oligonucleotides against P. falciparum topoisomerase II gene can be efficiently adsorbed onto a cationic nanoemulsion forming complexes. Whereas unloaded nanoemulsion displayed an hemolytic effect due to the presence of the cationic lipid, this was not the case of loaded nanoemulsion at low +/− ratios. Oligonucleotide-loaded nanoemulsions were found to be located inside the infected erythrocytes, inhibiting efficiently parasite growth (until 80%) and causing a delay in P. falciparum life cycle.
Keywords: Oligonucleotides; Cationic nanoemulsion; DOTAP; Topoisomerase II; Plasmodium falciparum;

Elucidating the molecular mechanism of PAMAM–siRNA dendriplex self-assembly: Effect of dendrimer charge density by Linda B. Jensen; Giovanni M. Pavan; Marina R. Kasimova; Sandra Rutherford; Andrea Danani; Hanne M. Nielsen; Camilla Foged (410-418).
Dendrimers are attractive vehicles for nucleic acid delivery due to monodispersity and ease of chemical design. The purpose of this study was to elucidate the self-assembly process between small interfering RNA (siRNA) and different generation poly(amidoamine) dendrimers and to characterize the resulting structures. The generation 4 (G4) and G7 displayed equal efficiencies for dendriplex aggregate formation, whereas G1 lacked this ability. Nanoparticle tracking analysis and dynamic light scattering showed reduced average size and increased polydispersity at higher dendrimer concentration. The nanoparticle tracking analysis indicated that electrostatic complexation results in an equilibrium between differently sized complex aggregates, where the centre of mass depends on the siRNA:dendrimer ratio. Isothermal titration calorimetric data suggested a simple binding for G1, whereas a biphasic binding was evident for G4 and G7 with an initial exothermic binding and a secondary endothermic formation of larger dendriplex aggregates, followed by agglomeration. The initial binding became increasingly exothermic as the generation increased, and the values were closely predicted by molecular dynamics simulations, which also demonstrated a generation dependent differences in the entropy of binding. The flexible G1 displayed the highest entropic penalty followed by the rigid G7, making the intermediate G4 the most suitable for dendriplex formation, showing favorable charge density for siRNA binding.
Keywords: PAMAM dendrimers; siRNA; Dendriplex; Nanoparticle tracking analysis; Isothermal titration calorimetry; Molecular dynamics simulations;

SiRNA delivery with functionalized carbon nanotubes by Amir Khashayar Varkouhi; Stéphanie Foillard; Twan Lammers; Raymond M. Schiffelers; Eric Doris; Wim E. Hennink; Gert Storm (419-425).
Carbon nanotubes (CNTs) have been studied for drug, antigen and nucleic acid delivery both in vitro and in vivo. Due to their nano-needle structure, they are supposed to cross the plasma membrane and enter directly into the cytoplasm likely upon an endocytosis-independent mechanism without inducing cell death. In this study, two cationically functionalized CNTs (CNT-PEI and CNT-pyridinium) were investigated for siRNA delivery. Both functionalized CNTs complexed siRNA and showed 10–30% silencing activity and a cytotoxicity of 10–60%. However, in terms of reduced toxicity or increased silencing activity, CNT-PEI and CNT-pyridinium did not show an added value over PEI and other standard transfection systems. Probably, the type of functionalization of carbon nanotubes might be a key parameter to obtain an efficient and non-cytotoxic CNT-based delivery system. Nevertheless, in view of the present results and importantly also of the non-degradability of CNTs, preference should currently be given to designing biodegradable carriers which mimic the needle structure of CNTs.
Keywords: Carbon nanotube; SIRNA; Gene silencing; Cytotoxicity;

DC-SIGN mediated antigen-targeting using glycan-modified liposomes: Formulation considerations by Medha D. Joshi; Wendy W.J. Unger; Astrid J. van Beelen; Sven C. Bruijns; Manja Litjens; Louis van Bloois; Hakan Kalay; Yvette van Kooyk; Gert Storm (426-432).
Dendritic cells (DCs) are key antigen presenting cells that have the unique ability to present antigens on MHC molecules, which can lead to either priming or suppression of T cell mediated immune responses. C-type lectin receptors expressed by DCs are involved in antigen uptake and presentation through recognition of carbohydrate structures on antigens. Here we have explored the feasibility of modification of liposomes with glycans for targeting purposes to boost immune responses. The potential of targeting glycoliposomal constructs to the C-type lectin DC-SIGN on DCs was studied using either PEGylated or non-PEGylated liposomes. Our data demonstrate that formulation of the glycoliposomes as PEGylated negatively affected their potential to target to DCs.
Keywords: Liposomes; DC-SIGN; Targeting; Vaccine; PEGylated; Antigen delivery; Binding to dendritic cells;

Improving solubility and chemical stability of natural compounds for medicinal use by incorporation into liposomes by Maria Coimbra; Benedetta Isacchi; Louis van Bloois; Javier Sastre Torano; Aldo Ket; Xiaojie Wu; Femke Broere; Josbert M. Metselaar; Cristianne J.F. Rijcken; Gert Storm; Rita Bilia; Raymond M. Schiffelers (433-442).
Natural bioactive compounds have been studied for a long time for their chemopreventive and therapeutic potential in several chronic inflammatory diseases, including cancer. However, their physicochemical properties generally result in poor chemical stability and lack of in vivo bioavailability. Very few human clinical trials have addressed absorption, distribution, metabolism, and excretion of these compounds in relation to efficacy. This limits the use of these valuable natural compounds in the clinic.In this study, we examined caffeic acid (derivatives), carvacrol (derivatives), thymol, pterostilbene (derivatives), and N-(3-oxo-dodecanoyl)-l-homoserine lactone. These are natural compounds with strong anti-inflammatory properties derived from plants and bacteria. However, these compounds have poor water solubility or are chemically unstable. To overcome these limitations we have prepared liposomal formulations. Our results show that lipophilic 3-oxo-C12-homoserine lactone and stilbene derivatives can be loaded into liposomal lipid bilayer with efficiencies of 50–70%. Thereby, the liposomes solubilize these compounds, allowing intravenous administration without use of solvents. When compounds could not be loaded into the lipid bilayer (carvacrol and thymol) or are rapidly extracted from the liposomes in the presence of serum albumin (3-oxo-C12-homoserine lactone and pterostilbene derivatives), derivatization of the compound into a water-soluble prodrug was shown to improve loading efficiency and encapsulation stability. The phosphate forms of carvacrol and pterostilbene were loaded into the aqueous interior of the liposomes and encapsulation was unaffected by the presence of serum albumin. Chemical instability of resveratrol was improved by liposome-encapsulation, preventing inactivating cistrans isomerization. For caffeic acid, liposomal encapsulation did not prevent oxidation into a variety of products. Still, by derivatization into a phenyl ester, the compound could be stably encapsulated without chemical degradation.Despite the instability of liposome-association of 3-oxo-C12-homoserine lactone and resveratrol, intravenous administration of these compounds inhibited tumor growth for approximately 70% in a murine tumor model, showing that simple solubilization can have important therapeutic benefits.
Keywords: Caffeic acid; Carvacrol; Resveratrol; Thymol; Pterostilbene; N-(3-oxo-dodecanoyl)-l-homoserine lactone; Resveratrol; Liposome; Inflammation;

Doxorubicin-loaded lipid-quantum dot hybrids: Surface topography and release properties by Bowen Tian; Wafa’ T. Al-Jamal; Khuloud T. Al-Jamal; Kostas Kostarelos (443-447).
A few studies have attempted to combine the physicochemical versatility offered by the liposome structure with the superior optical characteristics of quantum dots (QD) for the construction of multifunctional nanoparticles. We are reporting the construction of drug-loaded liposome-QD hybrid vesicles (L-QD) by incorporating TOPO-capped, CdSe/ZnS QD into the two types of lipid bilayers: the ‘rigid’ disteroylphosphatidylcholine (DSPC:Chol:DSPE-PEG2000) and a fluid-phase bilayer of egg PC (EPC:Chol:DSPE-PEG2000). Structural characterization of L-QD hybrid vesicles using atomic force microscopy (AFM) revealed that the incorporation of QD took place by hydrophobic self-association within the membranes. The encapsulation of hydrophilic small molecules in the internal aqueous phase of the L-QD hybrids showed different degrees of carboxyfluorescein (CF) release in buffer and serum, depending on the type of lipid used. The presence of QD in the lipid bilayer increased the CF release from EPC fluid bilayer. On the other hand, (DSPC) L-QD hybrids showed a higher stability under the same conditions with minimal CF leakage. Furthermore, (DSPC) L-QD hybrids showed a stable mean diameter up to three weeks stored at 4 °C, 25 °C, and 40 °C, determined by photo correlation spectroscopy (PCS) analysis. Finally, doxorubicin (Dox) was loaded into L-QD hybrids using the osmotic gradient technique and with at least 97% loading efficiency. The fluorescence spectrum of Dox was simultaneously detected with that of green-emitting QD that indicated the coexistence of QD and Dox in a single vesicle system. In conclusion, the drug-loaded L-QD-Dox hybrid vesicles presented here constitute a promising multifunctional delivery vector capable of transporting combinations of therapeutic and diagnostic modalities.
Keywords: Nanomedicine; Biological imaging; Theranostic; Multimodality; Nanotechnology;

Preparation and characterization of liposomal formulations of neurotensin-degrading enzyme inhibitors by Inge van Rooy; Shin-Ying Wu; Gert Storm; Wim E. Hennink; Heike Dinter-Heidorn; Raymond M. Schiffelers; Enrico Mastrobattista (448-452).
Neurotensin-degrading enzyme (NTDE) inhibitors hold great potential for treating psychotic disorders. However, brain uptake of such compounds in vivo is generally low due to the presence of the blood–brain barrier. In this study, liposomal formulations of two NTDE inhibitors, named compound 1 (C1) and compound 2 (C2) were prepared. Association of these compounds with the liposomal bilayer, subsequent liposomal stability, and compound release in the presence of albumin was studied. Entrapment of the compounds in the liposomal bilayer showed the solubilizing properties of the liposomes. Size and polydispersity index of the compound-entrapped liposomes did not change over 1 month, showing colloidal stability of the liposomal drug formulations. The amount of compounds associated with the liposomes decreased within one day. After this, the association remained stable at 4 °C. For C1, association remained stable at 37 °C in HEPES buffered saline, and the compound was gradually released in the presence of bovine serum albumin. For C2, the release was rapid in both HBS and BSA at 37 °C. In conclusion, the formulation of NTDE inhibitors C1 and C2 in liposomes has been demonstrated and holds promise to deliver NTDE inhibitors in vivo.
Keywords: Liposomes; Neurotensin; NTDE inhibitors; CNS drugs;

Selegiline-functionalized, PEGylated poly(alkyl cyanoacrylate) nanoparticles: Investigation of interaction with amyloid-β peptide and surface reorganization by Benjamin Le Droumaguet; Hayfa Souguir; Davide Brambilla; Romain Verpillot; Julien Nicolas; Myriam Taverna; Patrick Couvreur; Karine Andrieux (453-460).
Alzheimer's disease (AD) is a neurodegenerative disorder for which the research of new treatments is highly challenging. Since the fibrillogenesis of amyloid-β peptide 1–42 (Aβ1–42) peptide is considered as a major cause of neuronal degeneration, specific interest has been focused on aromatic molecules for targeting this peptide. In this paper, the synthesis of selegiline-functionalized and fluorescent poly(alkyl cyanoacrylate) nanoparticles (NPs) and their evaluation for the targeting of the Aβ1–42 peptide are reported. The synthetic strategy relied on the design of amphiphilic copolymers by tandem Knoevenagel–Michael addition of cyanoacetate derivatives, followed by their self-assembly in aqueous solutions to give the corresponding NPs. Different cyanoacetates were used: (i) hexadecyl cyanoacetate (HDCA) to form the hydrophobic core of the NPs; (ii) rhodamine B cyanoacetate (RCA) for fluorescent purposes; (iii) methoxypoly(ethylene glycol) cyanoacetate (MePEGCA) for stealth properties and (iv) selegiline-poly(ethylene glycol) cyanoacetate (SelPEGCA) to obtain the desired functionality. Two different amphiphilic copolymers were synthesized, a selegiline-containing copolymer, P(MePEGCA-co-SelPEGCA-co-HDCA), and a rhodamine-labelled counterpart, P(MePEGCA-co-RCA-co-HDCA), further blended at variable ratios to tune the amount of selegiline moieties displayed at the surface of the NPs.Optimal formulations involving the different amphiphilic copolymers were determined by the study of the NP colloidal characteristics. Interestingly, it was shown that the zeta potential value of the selegiline-functionalized nanoparticles dramatically decreased, thus emphasizing a significant modification in the surface charge of the nanoparticles. Capillary electrophoresis has then been used to test the ability of the selegiline-functionalized NPs to interact with the Aβ1–42 peptide. In comparison with non functionalized NPs, no increase of the interaction between these functionalized NPs and the monomeric form of the Aβ1–42 peptide was observed, thus highlighting the lack of availability of the ligand at the surface of the nanoparticles. A mechanism explaining this result has been proposed and was mainly based on the burial of the hydrophobic selegiline ligand within the nanoparticles core.
Keywords: Alzheimer's disease; Selegiline; Amyloid-β peptide; Poly(alkyl cyanoacrylate) nanoparticles; Functionalization;

The production of stavudine-loaded solid lipid nanoparticles (SLN) for intravenous injection was scaled up from lab scale (40 g) to medium scale (10 kg) and large scale (20/60 kg). The SLN were produced by high pressure homogenization of stavudine lipid melt dispersed in hot surfactant solution (pre-emulsion) applying 800 bar pressure. Employed were piston-gap homogenizers with increasing capacity (APV Gaulin products LAB 40, LAB 60 and Gaulin 5.5, and Avestin C50), using them in the continuous (circulation) and discontinuous mode. Size analysis was performed by photon correlation spectroscopy (PCS), laser diffractometry and light microscopy. At lab scale a PCS size of 53 nm was obtained. At the same pressure, all homogenizers on larger scale yielded a size in the range of the lab scale product (35–70 nm). Differences were found in the size as a function of circulation time (size increase or size reduction with time) and the number of cycles required (1 or 5) for the optimal product. The stavudine SLN formulation (2% lipid content, high surfactant to lipid ratio) showed a different behavior to conventional higher concentrated SLN suspensions or nanoemulsions (10% or 20% lipid/oil, low surfactant to lipid ratio). In general, smallest sizes were obtained in the discontinuous mode after just one homogenization cycle. The continuous production mode was only efficient with a 10 kg batch size using the LAB 60. In addition, the long-term stability over 1 year was monitored at refrigeration, room temperature and at 40 °C to assess a potential effect of the homogenizer type on stability. All batches at room temperature and below were stable, only a negligible increase in size was observed.
Keywords: HIV/AIDS; Solid lipid nanoparticles; Large-scale production; High pressure homogenization; Stability;

Optimizing the therapeutic index of liposomal glucocorticoids in experimental arthritis by Jolanda M. van den Hoven; Wouter Hofkens; Marca H.M. Wauben; Josee P.A. Wagenaar-Hilbers; Jos H. Beijnen; Bastiaan Nuijen; Josbert M. Metselaar; Gert Storm (471-477).
Small-sized (less than 150 nm) long-circulating liposomes (LCL) may be useful as drug-targeting vehicles for anti-inflammatory agents in arthritis, since they selectively home at inflamed joints after i.v. administration. Previously it was shown in experimental arthritis that encapsulation of glucocorticoids (GC) as water-soluble phosphate esters in PEG–liposomes resulted in a strong improvement of the anti-inflammatory effect as compared to the free drug. In the present study, we compared the therapeutic activity and adverse effects induced by 3 different GC encapsulated in LCL in an attempt to further optimize the therapeutic index of liposomal GC in arthritis. Our data showed that with GC (dexamethasone, budesonide) of higher potency than prednisolone, the therapeutic activity of liposomal GC can be increased. However, side effects at the level of body weight and hyperglycemia were noted, related to the sustained free GC level observed after injection of the liposomal GC. An inverse relationship with the clearance rate of the free GC in question was shown. This study stresses the importance of a high clearance rate of the GC to be encapsulated for achieving a maximal therapeutic index with liposomal GC. Therefore high-clearance GC, which until now are only applied in local treatment approaches, may be very useful for the development of novel, highly effective anti-inflammatory preparations for systemic treatment of inflammatory disorders.
Keywords: Liposomes; Glucocorticoids; Rheumatoid arthritis; Therapeutic index; Targetting;

Targeting of liposome-associated calcipotriol to the skin: Effect of liposomal membrane fluidity and skin barrier integrity by Nina Østergaard Knudsen; Lene Jorgensen; Jens Hansen; Charlotte Vermehren; Sven Frokjaer; Camilla Foged (478-485).
Liposomal fluidity affected how the vesicles interacted with barrier-impaired skin. Calcipotriol intercalated into the liposomal bilayer, but was released from the vesicles upon application onto barrier-impaired skin.Many dermal diseases like psoriasis are characterized by major changes in skin barrier function, which challenge the reproducible delivery of drugs into specific layers of diseased skin. The purpose of this study was to elucidate how liposomal bilayer fluidity and barrier integrity affected the delivery of liposome-associated calcipotriol to the skin. Calcipotriol-containing gel state and liquid state dipalmitoylphosphatidyl-choline:dilauroylphosphatidylcholine liposomes were prepared by extrusion. Using Langmuir monolayers, calcipotriol was shown to affect the packing of the lipid membrane. The penetration of radioactively labeled lipid and calcipotriol into pig skin was examined using the Franz diffusion cell model, and tape stripping was applied to impose an impaired barrier. Distorting the skin barrier resulted in an enhanced penetration of lipid from both gel and liquid state liposomes. In addition, increased penetration of lipid from liquid state liposomes was observed compared to gel state liposomes into barrier-impaired skin. For barrier-impaired skin, an elevated calcipotriol-to-lipid ratio was found in the receptor fluid for both liposome compositions indicating that calcipotriol is released from the vesicles. This suggests that the liposome-mediated delivery of calcipotriol to the epidermis of diseased skin is affected by the fluidity of the liposomal membrane.
Keywords: Skin penetration; Barrier-impaired skin; Liposome; Drug delivery; Calcipotriol; Nanomedicine;

Encapsulation of glucocorticoids into long-circulating liposomes provides targeting of these drugs to the inflamed synovium in experimental arthritis and thereby strongly improves their therapeutic index. The goal of this study was to evaluate the effect and mechanisms of intravenous liposomal delivery of prednisolone phosphate (Lip-PLP) on protease mediated cartilage destruction during murine antigen-induced arthritis (AIA). Mice treated with a single injection of Lip-PLP showed a pronounced suppression of synovial immune cell infiltration compared to control, PBS-treated mice. Liposomal PLP also significantly suppressed interleukin 1β (3.6 fold) in the synovium, but not in the blood serum. Furthermore, expression of the proteases MMP-3, -9, -13 and -14 and ADAMTS-4 and -5 was suppressed by Lip-PLP in the synovium, but not within the articular cartilage of the femur and tibia, demonstrating the specific action of Lip-PLP on the synovium. Lip-PLP is phagocytosed by macrophages in vitro and suppresses their expression of IL-1β and MMPs after LPS activation. Moreover, Lip-PLP suppresses destruction of the cartilage matrix during AIA mediated by active MMPs and ADAMTS, as assessed by immunostaining of their respective neoepitopes VDIPEN and NITEGE in various cartilage layers of the knee joint. Conclusion: liposomal delivery of glucocorticoids protects against cartilage matrix destruction during experimental arthritis by inhibiting protease expression and activity in the inflamed synovium.
Keywords: Liposomes; Animal model; Rheumatoid arthritis; Glucocorticoid; Cartilage destruction;

Safety of glucocorticoids can be improved by lower yet still effective dosages of liposomal steroid formulations in murine antigen-induced arthritis: Comparison of prednisolone with budesonide by Wouter Hofkens; Jolanda M. van den Hoven; Gerard J. Pesman; Karin C. Nabbe; Fred C. Sweep; Gert Storm; Wim B. van den Berg; Peter L. van Lent (493-498).
The goal of this study was to compare the effects of liposomal and free glucocorticoid formulations on joint inflammation and activity of the hypothalamic-pituitary-adrenal (HPA) axis during experimental antigen-induced arthritis (AIA). A dose of 10 mg/kg liposomal prednisolone phosphate (PLP) gave a suppression of the HPA-axis, as measured by plasma corticosterone levels in mice with AIA and in naïve mice. In a subsequent dose-response study, we found that a single dose of 1 mg/kg liposomal prednisolone phosphate (PLP) was still equally effective in suppressing joint inflammation as 4 repeated once-daily injections of 10 mg/kg free PLP. Moreover, the 1 mg/kg liposomal PLP dose gave 22% less suppression of corticosterone levels than 10 mg/kg of liposomal PLP at day 14 of the AIA. In order to further optimize liposomal glucocorticoids, we compared liposomal PLP with liposomal budesonide phosphate (BUP) (1 mg/kg). At 1 day after treatment, liposomal BUP gave a significantly stronger suppression of joint swelling than liposomal PLP (lip. BUP 98% vs. lip. PLP 79%). Both formulations also gave a strong and lasting suppression of synovial infiltration in equal amounts. However, at day 21 after AIA, liposomal PLP still significantly suppressed corticosterone levels, whereas this suppression was not longer statistically significant for liposomal BUP. Conclusion: Liposomal delivery improves the safety of glucocorticoids by allowing for lower effective dosing. The safety of liposomal glucocorticoid may be further improved by encapsulating BUP rather than PLP.
Keywords: Rheumatoid arthritis; Glucocorticoid; HPA axis; Liposomes; Side effects;

Macrophages and liposomes in inflammatory disease: Friends or foes? by B.J. Crielaard; T. Lammers; M.E. Morgan; L. Chaabane; S. Carboni; B. Greco; P. Zaratin; A.D. Kraneveld; G. Storm (499-506).
Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohn's disease (CD).In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.
Keywords: Liposomes; Macrophages; Corticosteroids; Crohn's disease; Multiple sclerosis; Inflammatory disease;

The aim of this work was to investigate the stability in vitro, in simulated gastro-intestinal fluids, of beads, made of α-cyclodextrin and soybean oil, and to study the release of progesterone, a model of lipophilic drug. This was evaluated over time by the monitoring of the proportion of intact beads, their volume and the percentage of progesterone dissolved. Their incubation in the simulated gastric fluid provoked a moderate reduction of their number (20%) and a decrease of their volume (50%) after 55 min. Whatever the intestinal medium subsequently introduced, bead number and volume decreased more until bead disintegration that appeared faster in sodium taurocholate rich-medium. In such fluid, the amount of progesterone dissolved increased rapidly between 65 and 180 min, with both beads and emulsion to be equal after 85 min. With soft capsules, the increase was more gradual. In sodium taurocholate free-medium, more progesterone was dissolved from the emulsion than from beads or soft capsules. The release of progesterone from beads resulted from the erosion of their matrix and its partition equilibrium between oily micro-droplets and aqueous phase. The original structure of beads confers to this multiparticulate system interesting properties for the oral delivery of lipophilic drugs.
Keywords: Beads; Cyclodextrins; Oil; Oral route; Progesterone; Stability;

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers by Claudia Di Tommaso; Alicia Torriglia; Pascal Furrer; Francine Behar-Cohen; Robert Gurny; Michael Möller (515-524).
Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.
Keywords: Polymeric micelles; Ocular tolerance; Biocompatibility; Cyclosporin A; Drug delivery;