Ascorbyl-dipalmitate-stabilised nanoemulsions as a potential localised treatment of inflammatory bowel diseases.

Current efforts on inflammatory bowel diseases (IBD) treatment are focused on strategies for localised drug delivery at the intestinal mucosa. Despite the potential of curcumin (CC) for IBD treatment, its low solubility and stability limit its application. Thus, the design of nanocarriers that focus CC delivery at the intestinal epithelium is an area of interest. This work proposes α-tocopherol nanoemulsions (NE) stabilised by ascorbyl-2,6-dipalmitate (ADP) as intestinal CC-carriers. The antioxidant capacity of α-tocopherol and ADP could have a synergistic effect on IBD-affected tissues, characterised by an oxidative environment. We obtained nanoemulsions (NE-ADP) with size below 200 nm, negative surface charge, stable in gastrointestinal media and no toxic in the Caco-2 cell model. Intracellular retention of NE-ADP in Caco-2 cells was observed by confocal microscopy. The extremely low Papp values obtained for CC and α-tocopherol indicated the lack of transport across the Caco-2 monolayer. Control nanoemulsion stabilised by lecithin (NE-L) was greatly transported across the Caco-2 cells monolayer, confirming the relevance of ADP on the cellular retention of NE-ADP. The therapeutic potential of NE-ADP was shown by the significant decrease of intracellular ROS levels. Altogether, these results indicate the potential of NE-ADP as a novel approach for the treatment of IBD.

Spontaneously self-assembled micelles from poly(ethylene glycol)-b-poly(epsilon-caprolactone-co-trimethylene carbonate) for drug solubilization.

Di-block copolymers composed of polyethylene glycol (PEG) and a second block of (co)polyesters of epsilon-caprolactone (CL) and/or trimethylene carbonate (TMC) were synthesized and characterized. Tin octoate was used as catalyst and polymerization were completed over a period of 24 h with high conversion (> 95%). Self-assembling properties in water were evaluated. All di-block copolymers behave similarly except when PCL served as the second block. Stable crew-cut micelles of about 20 nm were obtained by direct dissolution of the liquid di-block copolymers in water at room temperature. When PCL was present as the second block, no solubilization occurred. Drug encapsulation of poorly water-soluble drugs belonging to biopharmaceutics classification system (BCS) class II (ketoprofen and furosemide) was evaluated. Experimental solubility for these two drugs shows a significant enhancement such that a maximum value of 23.4 mg/ml was obtained for ketoprofen in a 10% w/v micellar solution as compared to 0.14 mg in water. In the case of furosemide, the solubility increased from 0.04 mg/ml in water to about 3.2 mg/ml in a 10% w/v micellar solution. Enzymatic degradation of diblock copolymers was also studied in the presence of Pseudomonas lipase in a phosphate buffer solution (pH 7.4). Results indicated rapid degradation of copolymers containing relatively higher amounts of CL compared to TMC suggesting the potential in vivo degradation.

Characterization of self-assembling copolymers in aqueous solutions using Electron Paramagnetic Resonance and Fluorescence spectroscopy.

Electron Paramagnetic Resonance and fluorescence spectroscopy have been used to determine the micropolarity and microviscosity of self-assembling systems based on mmePEG-p(CL-co-TMC) having different PEG chain lengths and different CL/TMC ratios and PEG/MOG/SA (45/5/50) polymers with different PEG chain lengths. Four reporter probes have been used: two spin probes, 16-doxyl stearic acid and 5-doxylstearic acid, and two fluorescent probes, pyrene and 1,3-bis(1-pyrenyl) propane (P3P). We found that the micelles based on mmePEG-p(CL-co-TMC) polymers are of a biphasic nature. The micelles are made of a hydrophilic corona with low viscosity while the core of the micelle is more hydrophobic and more viscous. The outer shell is made up of PEG chains, the hydrophobic part of the chains making the core. The partial hydration of the shell seems to lead to a looser chain network than that associated with deeper domains in the micelles. By contrast, in micelles composed of PEG/MOG/SA, there is no clear domain separation. This is consistent with a spatial configuration of random polymeric chains forming a loose network. In these micelles, the microviscosity is low and the hydrophobicity is high.

Prediction of drug solubility in amphiphilic di-block copolymer micelles: the role of polymer-drug compatibility.

The goal of the current study was to assess the value of predictive computational approaches for estimating drug solubility in hydrated micelles formed from di-block copolymers of polyethylene glycol (PEG) and random copolyesters of epsilon-caprolactone (CL) and trimethylene carbonate (TMC) using drug-polymer compatibility as assessed through the Flory-Huggins interaction parameter (chi). In order to accomplish this, the compatibility of several well-known model drugs (associated with the four biopharmaceutics classification system (BCS) classes) was assessed with both segments of the amphiphilic di-block copolymer PEG-b-P(CL-co-TMC). Compatibilities were estimated based on the Hansen modification of the Hildebrand approach using Molecular Modeling Pro software. Experimental solubilities for model drugs were determined using a shake-flask technique at various polymer concentrations. The solubilities of 8 compounds in 10% w/v micelle solutions were in relatively good agreement with the predicted drug-polymer compatibility. In addition, the approach allows for the selection of a suitable di-block copolymer for optimal solubilization of a specific drug. Furosemide was assessed as a model with results suggesting that it can be best entrapped in a di-block copolyester containing a relatively high CL content. The data suggests that prediction of drug solubilization of block copolymer-based micelles may be facilitated by assessing the compatibility of the drug for the component polymeric domains.

Injectable nanomedicine hydrogel for local chemotherapy of glioblastoma after surgical resection.

Glioblastoma (GBM) treatment includes, when possible, surgical resection of the tumor followed by radiotherapy and oral chemotherapy with temozolomide, however recurrences quickly develop around the resection cavity borders leading to patient death. We hypothesize that the local delivery of Lauroyl-gemcitabine lipid nanocapsule based hydrogel (GemC12-LNC) in the tumor resection cavity of GBM is a promising strategy as it would allow to bypass the blood brain barrier, thus reaching high local concentrations of the drug. The cytotoxicity and internalization pathways of GemC12-LNC were studied on different GBM cell lines (U251, T98-G, 9L-LacZ, U-87 MG). The GemC12-LNC hydrogel was well tolerated when injected in mouse brain. In an orthotopic xenograft model, after intratumoral administration, GemC12-LNC significantly increased mice survival compared to the controls. Moreover, its ability to delay tumor recurrences was demonstrated after perisurgical administration in the GBM resection cavity. In conclusion, we demonstrate that GemC12-LNC hydrogel could be considered as a promising tool for the post-resection management of GBM, prior to the standard of care chemo-radiation.

Intestinal uptake and biodistribution of novel polymeric micelles after oral administration.

To determine the fate of polymeric micelles after oral administration, we investigated the possible transport of polymeric micelles across Caco-2 monolayers and their biodistribution in rats after per os administration of [14C]-labelled mmePEG750P(CL-co-TMC) micelles containing risperidone (BCS Class II drug). mmePEG750P(CL-co-TMC) was able to cross Caco-2 monolayer via a saturable transport mechanism. The oral bioavailability of the polymer was 40%. Polymeric micelles based on mmePEG750P(CL-co-TMC) showed very low clearance by the reticuloendothelial system (RES) and a renal excretion. A sustained release of risperidone was observed.

Encapsulation of amphotericin B in poly(ethylene glycol)-block-poly(epsilon-caprolactone-co-trimethylenecarbonate) polymeric micelles.

The aim of this work was to evaluate the potential of self-assembling poly(ethyleneglycol)(750)-block-poly(epsilon-caprolactone-co-trimethylenecarbonate)(4500) 50/50 copolymers (PEG-p(CL-co-TMC)) to solubilize amphotericin B in polymeric micelles and to disaggregate the drug to the less toxic monomeric form. Amphotericin B was encapsulated in the micelles upon dilution of a mixture of the liquid polymer and the drug in water. Its solubility was increased by two orders of magnitude depending on polymer concentration. The aggregation state of amphotericin B was decreased by PEG-p(CL-co-TMC). The preparation method and the loading of the polymeric micelles influenced it. The antifungal activity of the drug was reduced by encapsulation in the polymeric micelles whereas the onset of amphotericin B-induced hemolysis was delayed. PEG-p(CL-co-TMC) micelles could be an easy method for amphotericin B encapsulation.

Patterns of ligand binding to normal, regenerating, preneoplastic, and neoplastic rat hepatocytes.

The binding of epidermal growth factor, asialoorosomucoid, and apoprotein E-rich lipoproteins to isolated hepatocytes was investigated at various time intervals during the step-by-step development of liver cancer in rats. The degree of binding of the three ligands showed a progressive reduction in early persistent and late persistent putative preneoplastic hepatocyte nodules. This was further decreased in hepatocytes isolated from unequivocal hepatocellular carcinomas. Regenerating liver hepatocytes bound lesser amounts of epidermal growth factor and asialoorosomucoid than did hepatocytes from control resting liver but increased amounts of apoprotein E-rich lipoproteins. The progressive decrease in ligand binding during the precancerous phase of hepatocarcinogenesis, the nodule-to-cancer sequence, may render nodules less responsive to the influences of their external environments.

Anticancer drug-loaded hydrogels as drug delivery systems for the local treatment of glioblastoma.

Among central nervous system tumors, Glioblastoma (GBM) is the most common, aggressive and neurological destructive primary brain tumor in adults. Standard care therapy for GBM consists in surgical resection of the accessible tumor (without causing neurological damage) followed by chemoradiation. However, several obstacles limit the assessment of tumor response and the delivery of cytotoxic agents at the tumor site, leading to a lack of effectiveness of conventional treatments against GBM and fatal outcome. Despite the efforts of the scientific community to increase the long-term benefits of GBM therapy, at the moment GBM remains incurable. Among the strategies that have been adopted in the last two decades to find new and efficacious therapies for the treatment of GBM, the local delivery of chemotherapeutic drugs in the tumor resection cavity emerged. In this review, our aim is to provide an overview on hydrogels loaded with anticancer drugs for the treatment of GBM recently used in preclinical and clinical studies, their advantages and major limitations for clinical translation. This review is divided in three parts: the first one describes the context of GBM and its current treatments, with a highlight on the role of local delivery in GBM treatment and the development of GBM resection murine models. Then, recent developments in the use of anticancer drug-loaded hydrogels for the treatment of GBM will be detailed. The final section will be focused on the limitations for in vivo studies, clinical translation and the clinical perspectives to the development of hydrogels.

Lauroyl-gemcitabine-loaded lipid nanocapsule hydrogel for the treatment of glioblastoma.

The local delivery of chemotherapeutic agents is a very promising strategy for the treatment of glioblastoma (GBM). Gemcitabine is a chemotherapeutic agent that has a different mechanism of action compared to alkylating agents and shows excellent radio-sensitizing properties. So, we developed an injectable gel-like nanodelivery system consisting in lipid nanocapsules loaded with anticancer prodrug lauroyl-gemcitabine (GemC12-LNC) in order to obtain a sustained and local delivery of this drug in the brain. In this study, the GemC12-LNC has been formulated and characterized and the viscoelastic properties of the hydrogel were evaluated after extrusion from 30G needles. This system showed a sustained and prolonged in vitro release of the drug over one month. GemC12 and the GemC12-LNC have shown increased in vitro cytotoxic activity on U-87 MG glioma cells compared to the parent hydrophilic drug. The GemC12-LNC hydrogel reduced significantly the size of a subcutaneous human GBM tumor model compared to the drug and short-term tolerability studies showed that this system is suitable for local treatment in the brain. In conclusion, this proof-of-concept study demonstrated the feasibility, safety and efficiency of the injectable GemC12-LNC hydrogel for the local treatment of GBM.

DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse.

Electroporation is an effective alternative to viral methods to significantly improve DNA transfection after intradermal and topical delivery. The aim of the study was to check whether a combination of a short high-voltage pulse (HV) to permeabilize the skin cells and a long low-voltage pulse (LV) to transfer DNA by electrophoresis was more efficient to enhance DNA expression than conventional repeated HV or LV pulses alone after intradermal injection of DNA plasmid. GFP and luciferase expressions in the skin were enhanced by HV+LV protocol as compared to HV or LV pulses alone. The expression lasted for up to 10 days. Consistently, HV+LV protocol induced a higher Th2 immune response against ovalbumin than HV or LV pulses. Standard methods were used to assess the effect of electric pulses on skin: the application of a combination of HV and LV pulses on rat skin fold delivered by plate electrodes was well tolerated. These data demonstrate that a combination of one HV (700 to 1000 V/cm; 100 micros) followed by one LV (140 to 200 V/cm; 400 ms) is an efficient electroporation protocol to enhance DNA expression in the skin.

Stability study of nanoparticles of poly(epsilon-caprolactone), poly(D,L-lactide) and poly(D,L-lactide-co-glycolide).

The objective was to evaluate the stability of nanoparticles prepared with poly(epsilon-caprolactone), poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers and stored at different temperatures and in different media. The stability parameters studied were molecular weight and crystallinity of the polymer, nanoparticle size and pH. The results show that the stability of polymeric nanoparticles depends on (i) the type of polymers with the following increasing order of polymer stability: PLA25GA50 < PLA37.5GA25 < PLA50 = PCL, (ii) the storage temperature: PCL and PLA50 nanoparticles can be kept at 4 degrees C and RT during one year, while PLA37.5GA25 and PLA25GA50 nanoparticles have to be stored at 4 degrees C, and (iii) the storage conditions: buffering or freeze-drying nanoparticles improves stability.

Polymeric nanoparticles as delivery system for influenza virus glycoproteins.

The objective of this work was to develop a new delivery system which could enhance the mucosal immune response to influenza virus antigens. Poly(D,L-lactide-co-glycolide) nanoparticles of about 200 nm containing hemagglutinin were chosen as the delivery system. Due to the amphiphilic nature of hemagglutinin (hydrophilic HA1 and hydrophobic HA2), nanoparticles were prepared by both classical oil in water solvent evaporation technique as well as by a [(water-in-oil) in water] solvent evaporation technique. Hemagglutinin was well encapsulated in nanoparticles prepared by both techniques. Molecular weight and antigenicity of entrapped hemagglutinin were not affected by the entrapment procedure.

Transdermal permeation of neutral molecules by skin electroporation.

Electroporation of skin has recently been shown to enhance transport of charged molecules across skin by up to four orders of magnitude. This study demonstrates that high-voltage pulses can also increase transdermal permeation of two neutral model solutes, i.e. mannitol and water, up to 100-fold. The elevated flux results from the persistent increase in skin permeability following electroporation, rather than from electro-osmosis during pulsing. Control on transport was achieved by controlling the electrical parameters of the pulse, i.e. the pulse voltage, time constant and number.

Influence of formulation excipients and physical characteristics of inhalation dry powders on their aerosolization performance.

The objective of this study was to determine the effects of formulation excipients and physical characteristics of inhalation particles on their in vitro aerosolization performance, and thereby to maximize their respirable fraction. Dry powders were produced by spray-drying using excipients that are FDA-approved for inhalation as lactose, materials that are endogenous to the lungs as albumin and dipalmitoylphosphatidylcholine (DPPC); and/or protein stabilizers as trehalose or mannitol. Dry powders suitable for deep lung deposition, i.e. with an aerodynamic diameter of individual particles <3 microm, were prepared. They presented 0.04--0.25 g/cm(3) bulk tap densities, 3--5 microm geometric particle sizes, up to 90% emitted doses and 50% respirable fractions in the Andersen cascade impactor using a Spinhaler inhaler device. The incorporation of lactose, albumin and DPPC in the formulation all improved the aerosolization properties, in contrast to trehalose and the mannitol which decreased powder flowability. The relative proportion of the excipients affected aerosol performance as well. The lower the bulk powder tap density, the higher the respirable fraction. Optimization of in vitro aerosolization properties of inhalation dry powders can be achieved by appropriately selecting composition and physical characteristics of the particles.

Transdermal delivery of fentanyl: rapid onset of analgesia using skin electroporation.

Skin electroporation has recently been shown to increase transdermal transport of small-size drugs as well as considerably larger molecules by up to 4 orders of magnitude in vitro. Nevertheless, no in vivo studies have proven that high-voltage pulses can induce therapeutic plasma levels of drug. The aim of the present report was precisely to study the potential of skin electroporation in transdermal delivery of fentanyl in vivo. Fentanyl was transdermally delivered to hairless rats using high-voltage pulsing. Following the administration, the pharmacokinetics and pharmacodynamics were assessed. Significant fentanyl plasma concentrations were rapidly achieved using skin electroporation. Immediately after the 5 min pulsing, fentanyl plasma levels reached one third of the maximal plasma concentration of approximately 30 ng/ml, the peak occurring 30 min after the electroporation. Deep analgesia and supraspinal effects were achieved, antinociception lasting for an hour. The magnitude of the effects was, however, dependent on the electrical parameters of the pulses.

Transdermal alniditan delivery by skin electroporation.

The aim of this study was to evaluate the transdermal permeation of alniditan by electroporation and to compare with iontophoretic delivery. The influence of the electrical parameters of electroporation was investigated in vitro using a factorial design study. The transdermal flux of alniditan was enhanced by two orders of magnitude by application of high voltage electrical pulses. The electrical parameters of electroporation-i.e. the voltage, the duration and the number of pulses-allowed a control of drug permeation. Both transport during and after pulsing were shown to be important for alniditan transdermal delivery by electroporation. Electroporation was found more efficient in promoting alniditan permeation than an iontophoresis transferring the same amount of charges.

Comparison of the effects of short, high-voltage and long, medium-voltage pulses on skin electrical and transport properties.

High-voltage pulses have been shown to increase rates of transport across skin by several orders of magnitude on a time scale of minutes to seconds. Two main pulse protocols have been employed to promote transport: the intermittent application of short ( approximately 1 ms) high-voltage (approximately 100 V across skin) pulses and a few applications of long (=100 ms) medium-voltage (>30 V across skin) pulses. In order to better evaluate the benefits of each protocol for transdermal drug delivery, we compared these two protocols in vitro in terms of changes in skin electrical properties and transport of sulforhodamine, a fluorescent polar molecule of 607 g/mol and a charge of -1. Whereas both protocols induced similar alterations and recovery processes of skin electrical resistance, long pulses of medium-voltage appeared to be more efficient in transporting molecules across skin. Skin resistance decreased by three (short pulses) and two (long pulses) orders of magnitude, followed by incomplete recovery in both cases. For the same total transported charge, long pulses induced faster and greater molecular transport across skin than short pulses. In addition, a greater fraction of the aqueous pathways created by the electric field was involved in molecular transport when using long pulse protocols. Transport was concentrated in localized transport regions (LTRs) for both protocols but LTRs created by long pulses were an order of magnitude larger than those formed by short pulses and the short pulses created an order of magnitude more LTRs. Overall, this study is consistent with the creation of fewer, but larger aqueous pathways by long, medium-voltage pulses in comparison to short, high-voltage pulses.

Electroporation-mediated delivery of 3'-protected phosphodiester oligodeoxynucleotides to the skin.

The feasibility of topical delivery in the skin of 3' end modified phosphodiester oligonucleotides using electroporation was investigated. Experiments were performed in vitro, using hairless rat skin. Five pulses of (200 V, 450 ms) were applied. The 3' end modifications of the 15 mer oligonucleotide were: (1) 3'-aminohexyl, (2) biotin, with a triethyleneglycol arm, (3) methylphosphonate links between nucleotides 13, 14 and 15, and (4) 2-O-methyl nucleotides at 13, 14 and 15 positions. All the modifications were efficient to protect the oligonucleotides against degradation in the skin. Electroporation increased the topical delivery of the 3' end-modified phosphodiesters by two orders of magnitude compared to passive diffusion, without significant differences between the derivatives. Oligonucleotide concentrations in the range of 1 microm could be achieved in the viable skin. The delivery of a phosphorothioate congener was lower than phosphodiester delivery due to the interaction of phosphorothioate with the stratum corneum. Consequently, 3' end-protected phosphodiesters could be an interesting alternative to phosphorothioate oligonucleotides for topical treatment of cutaneous diseases.

On the mechanism of the hepatocarcinogenicity of peroxisome proliferators.

The absence of a genotoxic action in the rat of several peroxisome proliferators (PP) has been confirmed by measuring gross degradation, unscheduled DNA-synthesis (UDS), as well as by measurement of single strand breaks using alkali unwinding in absence and presence of inhibitors of DNA-repair. Similar results were obtained even after drastically lowering the glutathione content of liver. Further, after oral administration of ciprofibrate, no potentiating effect was found in vivo on the generation of micronuclei in hepatocytes by ionizing radiation. The metabolically inert PP, perfluorooctanoic acid, was found to act as a promoter of liver tumors in the rat induced by diethylnitrosamine in an initiation-selection-promotion protocol. The results are discussed in light of available information concerning the mechanism of action of PPs.