PLA-PEG forming worm-like nanoparticles despite unfavorable packing parameter: Formation mechanism, thermal stability and potential for cell internalization.

Most nanoparticles produced for drug delivery purposes are spherical. However, the literature suggests that elongated particles are advantageous, notably in terms of cellular uptake. Thus, we synthesized biocompatible polylactide-b-poly(ethylene glycol) (PLA-PEG) polymers bearing carboxylate moieties, and used them to formulate worm-like nanoparticles by a simple emulsion-evaporation process. Worm-like nanoparticles with variable aspect ratio were obtained by simply adjusting the molar mass of the PLA block: the shorter the molar mass of the PLA block, the more elongated the particles. As PLA molar mass decreased from 80,000 g/mol to 13,000 g/mol, the proportion of worm-like nanoparticles increased from 0 to 46%, in contradiction with the usual behavior of block polymers based on their packing parameter. To explain this unusual phenomenon, we hypothesized the shape arises from a combination of steric and electrostatic repulsions between PEG chains bearing a carboxylate moiety present at the dichloromethane-water interface during the evaporation process. Worm-like particles turned out to be unstable when incubated at 37 °C, above polymer glass transition temperature. Indeed, above Tg, a Plateau-Rayleigh instability occurs, leading to the division of the worm-like particles into spheres. However, this instability was slow enough to assess worm-like particles uptake by murine macrophages. A slight but significant increase of internalization was observed for worm-like particles, compared to their spherical counterparts, confirming the interest of developing biocompatible anisotropic nanoparticles for pharmaceutical applications such as drug delivery.

Pickering emulsions stabilized with biodegradable nanoparticles for the co-encapsulation of two active pharmaceutical ingredients.

Innovative Pickering emulsions co-encapsulating two active pharmaceutical ingredients (API) were formulated for a topical use. An immunosuppressive agent, either cyclosporine A (CysA) or tacrolimus (TAC), was encapsulated at high drug loading in biodegradable and biocompatible poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP). These NP stabilized the oil droplets (Miglyol) containing an anti-inflammatory drug, calcitriol (CAL). The influence of the API on the physico-chemical properties of these emulsions were studied. Emulsions formulated with or without API had a similar macroscopic and microscopic structure, as well as interfacial properties, and they exhibited a good stability for at least 55 days. The emulsions did not alter the viability of human keratinocytes (HaCaT cell line) after 2 and 5 days of exposure to NP concentrations equivalent to efficient API dosages. Thus, these new Pickering emulsions appear as a promising multidrug delivery system for the treatment of chronical inflammatory skin diseases.

Biomaterial-embedded extracellular vesicles improve recovery of the dysfunctional myocardium.

Extracellular vesicles (EV) are increasingly recognized as a therapeutic option in heart failure. They are usually administered by direct intramyocardial injections with the caveat of a rapid wash-out from the myocardium which might weaken their therapeutic efficacy. To improve their delivery in the failing myocardium, we designed a system consisting of loading EV into a clinical-grade hyaluronic acid (HA) biomaterial. EV were isolated from umbilical cord-derived mesenchymal stromal cells. The suitability of HA as a delivery platform was then assessed in vitro. Rheology studies demonstrated the viscoelastic and shear thinning behaviors of the selected HA allowing its easy injection. Moreover, the release of HA-embedded EV was sustained over more than 10 days, and EV bioactivity was not altered by the biomaterial. In a rat model of myocardial ischemia reperfusion, we showed that HA-embedded EV preserved cardiac function (echocardiography), improved angiogenesis and decreased both apoptosis and fibrosis (histology and transcriptomics) when compared to intramyocardial administration of EV alone. These data thus strengthen the concept that inclusion of EV into a clinically useable biomaterial might optimize their beneficial effects on post-ischemic cardiac repair.

Hybrid systems combining liposomes and entangled hyaluronic acid chains: Influence of liposome surface and drug encapsulation on the microstructure.

Mixtures of hyaluronic acid (HA) with liposomes lead to hybrid colloid-polymer systems with a great interest in drug delivery. However, little is known about their microstructure. Small angle neutron scattering (SANS) is a valuable tool to characterize these systems in the semi-dilute entangled regime (1.5% HA) at high liposome concentration (80 mM lipids). The objective was to elucidate the influence of liposome surface (neutral, cationic, anionic or anionic PEGylated), drug encapsulation and HA concentration in a buffer mimicking biological fluids (37 °C). First, liposomes were characterized by SANS, cryo-electron microscopy, and dynamic light scattering and HA by SANS, size exclusion chromatography, and rheology. Secondly, HA-liposome mixtures were studied by SANS. In HA, liposomes kept their integrity. Anionic and PEGylated liposomes were in close contact within dense clusters with an amorphous organization. The center-to-center distance between liposomes corresponded to twice their diameter. A depletion mechanism could explain these findings. Encapsulation of a corticoid did not modify this organization. Cationic liposomes formed less dense aggregates and were better dispersed due to their complexation with HA. Liposome surface governed the interactions and microstructure of these hybrid systems.

Transtympanic injection of a liposomal gel loaded with N-acetyl-L-cysteine: A relevant strategy to prevent damage induced by cochlear implantation in guinea pigs?

Patients with residual hearing can benefit from cochlear implantation. However, insertion can damage cochlear structures and generate oxidative stress harmful to auditory cells. The antioxidant N-acetyl-L-cysteine (NAC) is a precursor of glutathione (GSH), a powerful endogenous antioxidant. NAC local delivery to the inner ear appeared promising to prevent damage after cochlear implantation in animals. NAC-loaded liposomal gel was specifically designed for transtympanic injection, performed both 3 days before and on the day of surgery. Hearing thresholds were recorded over 30 days in implanted guinea pigs with and without NAC. NAC, GSH, and their degradation products, N,N'-diacetyl-L-cystine (DiNAC) and oxidized glutathione (GSSG) were simultaneously quantified in the perilymph over 15 days in non-implanted guinea pigs. For the first time, endogenous concentrations of GSH and GSSG were determined in the perilymph. Although NAC-loaded liposomal gel sustained NAC release in the perilymph over 15 days, it induced hearing loss in both implanted and non-implanted groups with no perilymphatic GSH increase. Under physiological conditions, NAC appeared poorly stable within liposomes. As DiNAC was quantified at concentrations which were twice as high as NAC in the perilymph, it was hypothesized that DiNAC could be responsible for the adverse effects on hearing.

Tuning morphology of Pickering emulsions stabilised by biodegradable PLGA nanoparticles: How PLGA characteristics influence emulsion properties.

In this study, we proved that the stabilisation of Pickering emulsions by polymer nanoparticles (NPs) heavily depends on polymer characteristics. We prepared NPs with four poly(lactide-co-glycolide) polymers (PLGA), of different molar masses (14,000 and 32,000 g/mol) and end groups (acid or alkylester). NPs were either bare (without stabilising polymer) or covered by polyvinyl alcohol (PVA). Pickering emulsions were prepared by mixing NP aqueous suspensions with various amounts of oil (Miglyol 812 N). First, NP wettability was directly affected by PLGA end group: ester-ending PLGA led to more hydrophobic NPs, compared to acid-ending PLGA. This effect of the end group could be slightly enhanced with smaller molar mass. Thus, bare PLGA NPs stabilised different types of emulsions (W/O/W and W/O), following Finkle's rule. However, the effect of PLGA characteristics was masked when NPs were covered by PVA, as PVA drove the stabilisation of O/W emulsions. Secondly, PLGA molar mass and end group also influenced its glass transition temperature (Tg), with spectacular consequences on emulsion formation. Indeed, the shortest ester-ending PLGA exhibited a Tg close to room temperature, when measured in the emulsion. This Tg, easily exceeded during emulsification process, led to a soft solid emulsion, stabilised by a network of NP debris.

In vitro antileishmanial potentialities of essential oils from Citrus limon and Pistacia lentiscus harvested in Tunisia.

Leishmaniasis is a tropical parasitic disease that affects up to 12 million people worldwide. Current chemotherapies have limitations such as toxicity, high cost, and parasite resistance. This work aims to select an essential oil (EssOil) isolated from the Tunisian flora as a new antileishmanial candidate. Two plants were chosen for their antileishmanial potential: Citrus limon (Citrus) and Pistacia lentiscus (Pistacia). Each of these plants was harvested from two different sites (area 1 and area 2). Extracted EssOils were characterized using GC-MS. Their antiparasitic activity against axenic and intracellular Leishmania major amastigotes and their cytotoxicity were assessed. Citrus EssOil from area 1 displayed an interesting activity against L. major intramacrophage amastigotes with IC50 value at 4.2 ± 1.3 µg/mL. Interestingly, this activity was close to that of miltefosine. Moderate activities against intracellular amastigote were observed for Pistacia EssOil from area 1 and Citrus EssOil from area 2. However, low cytotoxicity with high selectivity index was proved only for Citrus EssOil from area 1, revealing its safety for macrophages. This study also demonstrated for the first time the antileishmanial activity of EssOil extracted from Citrus limon leaves. The EssOil interesting activity could be related to the lipophilic properties of terpenes that were shown in literature to contribute to the disruption of parasite intracellular metabolic pathways.

Extracellular Vesicles and Biomaterial Design: New Therapies for Cardiac Repair.

There is increasing evidence that extracellular vesicles (EVs) mediate the paracrine effects of stem cells. Although EVs have several attractive characteristics, they also raise issues related to delivery. For patients with cardiac disease that require a surgical procedure, direct intramyocardial (IM) administration of EVs is straightforward but its efficacy may be limited by fast wash-out, hence the interest of incorporating EVs into a controlled release polymer to optimize their residence time. For patients without surgical indication, the intravenous (IV) route is attractive because of its lack of invasiveness; however, whole-body distribution limits the fraction of EVs that reach the heart, hence the likely benefits of EV engineering to increase EV homing to the target tissue.

Nanocarriers for drug delivery to the inner ear: Physicochemical key parameters, biodistribution, safety and efficacy.

Despite the high incidence of inner ear disorders, there are still no dedicated medications on the market. Drugs are currently administered by the intratympanic route, the safest way to maximize drug concentration in the inner ear. Nevertheless, therapeutic doses are ensured for only a few minutes/hours using drug solutions or suspensions. The passage through the middle ear barrier strongly depends on drug physicochemical characteristics. For the past 15 years, drug encapsulation into nanocarriers has been developed to overcome this drawback. Nanocarriers are well known to sustain drug release and protect it from degradation. In this review, in vivo studies are detailed concerning nanocarrier biodistribution, their pathway mechanisms in the inner ear and the resulting drug pharmacokinetics. Key parameters influencing nanocarrier biodistribution are identified and discussed: nanocarrier size, concentration, surface composition and shape. Recent advanced strategies that combine nanocarriers with hydrogels, specific tissue targeting or modification of the round window permeability (cell-penetrating peptide, magnetic delivery) are explored. Most of the nanocarriers appear to be safe for the inner ear and provide a significant efficacy over classic formulations in animal models. However, many challenges remain to be overcome for future clinical applications.

Pickering emulsions: Preparation processes, key parameters governing their properties and potential for pharmaceutical applications.

An increased interest in Pickering emulsions has emerged over the last 15 years, mainly related to their very attractive properties compared to regular emulsions, namely their excellent stability and their numerous possible applications. In this review, after detailing the interest of Pickering emulsions, their main preparation processes are presented and their advantages and disadvantages discussed. In the third part, the key parameters that govern Pickering emulsions type, droplet size and stability are analyzed. Finally, the interest and the potential of Pickering emulsions for pharmaceutical applications are exposed and discussed, taking all the administration routes into consideration and focusing on organic particles.

Bare and Sterically Stabilized PLGA Nanoparticles for the Stabilization of Pickering Emulsions.

Pickering emulsions were formulated using biodegradable and biocompatible poly(lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) prepared without surfactants or any other polymer than PLGA. A pharmaceutical and cosmetic oil (Miglyol) was chosen as the oil phase at a ratio of 10% w/w. These emulsions were then compared with emulsions using the same oil but formulated with well-described PLGA-poly(vinyl alcohol) (PVA) NPs, i.e., with PVA as NP stabilizers. Strikingly, the emulsions demonstrated very different structures at macroscopic, microscopic, and interfacial scales, depending on the type of NPs used. Indeed, the emulsion layer was significantly thicker when using PLGA NPs rather than PLGA-PVA NPs. This was attributed to the formation and coexistence of multiple water-in-oil-in-water (W/O/W) and simple oil-in-water (O/W) droplets, using a single step of emulsification, whereas simple O/W emulsions were obtained with PLGA-PVA NPs. The latter NPs were more hydrophilic than bare PLGA NPs because of the presence of PVA at their surface. Moreover, PLGA NPs only slightly lowered the oil/water interfacial tension whereas the decrease was more pronounced with PLGA-PVA NPs. The PVA chains at the PLGA-PVA NP surface could probably partially desorb from the NPs and adsorb at the interface, inducing the interfacial tension decrease. Finally, independent of their composition, NPs were adsorbed at the oil/water interface without influencing its rheological behavior, possibly due to their mobility at their interface. This work has direct implications in the formulation of Pickering emulsions and stresses the paramount influence of the physicochemical nature of the NP surface into the stabilization of these systems.

Assessment of the efficacy of a local steroid rescue treatment administered 2 days after a moderate noise-induced trauma in guinea pig.

OBJECTIVES: Intratympanic injection of corticosteroids membrane after noise-induced hearing loss is an accepted alternative to general administration. We investigated the effect on hearing of a hyaluronic acid gel with liposomes loaded with dexamethasone (DexP) administered into the middle ear. METHODS: An acute acoustic trauma was performed to 13 guinea pigs for a period of 1 h on Day -2. Two 2 days after the noise trauma, the animals were then assigned randomly to four experimental groups: control without gel, gel injection, gel-containing free DexP, gel-containing DexP loaded into liposomes. Auditory thresholds were measured with Auditory Brainstem Response before Day -2 and at Day 0, Day 7 and Day 30 after noise trauma. RESULTS: Seven days after, a complete hearing recovery was observed in the control group at all frequencies apart from 8 kHz, and no recovery was observed in the three groups receiving a gel injection. Thirty days after trauma, all of the animals had recovered normal hearing, apart from at the 8-kHz frequency, with similar auditory thresholds. CONCLUSIONS: Local DexP administration 48 h after a mild acoustic trauma did not improve hearing recovery, even with a sustained release in a specific gel formulation designed for inner ear therapy.

Effect of high pressure homogenization on the structure and the interfacial and emulsifying properties of ß-lactoglobulin.

The effect of high pressure homogenization (HPH) on the structure of ß-lactoglobulin (ß-lg) was studied by combining spectroscopic, chromatographic, and electrophoretic methods. The consequences of the resulting structure modifications on oil/water (O/W) interfacial properties were also assessed. Moderated HPH treatment (100 MPa/4 cycles) showed no significant modification of protein structure and interfacial properties. However, a harsher HPH treatment (300 MPa/5 cycles) induced structural transformation, mainly from ß-sheets to random coils, wide loss in lipocalin core, and protein aggregation via intermolecular disulfide bridges. HPH-modified ß-lg displayed higher surface hydrophobicity leading to a faster adsorption rate at the interface and an earlier formation of an elastic interfacial film at Cß-lg = 0.1 wt%. However, no modification of the interfacial properties was observed at Cß-lg = 1 wt%. At this protein concentration, the prior denaturation of ß-lg by HPH did not modify the droplet size of nanoemulsions prepared with these ß-lg solutions as the aqueous phases. A slightly increased creaming rate was however observed. The effects of HPH and heat denaturations appeared qualitatively similar, but with differences in their extent.

Mixtures of hyaluronic acid and liposomes for drug delivery: Phase behavior, microstructure and mobility of liposomes.

Hyaluronic acid liposomal gels have previously demonstrated in vivo their great potential for drug delivery. Elucidating their phase behavior and structure would provide a better understanding of their use properties. This work evaluates the microstructure and the phase behavior of mixtures of hyaluronic acid (HA) and liposomes and their impact on the vesicle mobility. HA concentration and surface properties of liposomes (positively or negatively charged, neutral, with a polyethylene glycol corona) are varied while the liposome concentration remains constant. Below the entanglement concentration of HA (0.4%), the mixtures exhibit a depletion phase separation except for positively charged liposomes that interact with anionic HA through attractive electrostatic interactions. At high HA concentration, no macroscopic phase separation is observed, except a slight syneresis with cationic liposomes. The microstructure shows aggregates of liposomes homogeneously distributed into a HA network except for PEGylated liposomes, which seem to form bicontinuous interpenetrating networks. The diffusion of liposomes is controlled by HA concentration and their surface properties. Finally, PEGylated liposomes display the highest mobility at high HA concentration (2.28%) both macro- and microscopically. The microstructure of HA-liposomes mixtures and the diffusion of liposomes are key parameters that must be taken into account for drug delivery.

Effect of a liposomal hyaluronic acid gel loaded with dexamethasone in a guinea pig model after manual or motorized cochlear implantation.

Goals of cochlear implantation have shifted from complete insertion of the cochlear electrode array towards low traumatic insertion with minimally invasive techniques. The aim of this study was first to evaluate, in a guinea pig model of cochlear implantation, the effect of a motorized insertion technique on hearing preservation. The second goal was to study a new gel formulation containing dexamethasone phosphate loaded in liposomes (DEX-P). Guinea pigs had a unilateral cochlear implantation with either a manual technique (n = 12), or a motorized technique (n = 15), with a 0.4 mm diameter and 4 mm long array trough a cochleostomy. At the end of the procedure, hyaluronic acid gel containing drug-free liposomes, or liposomes loaded with DEX-P, was injected into the bulla. Auditory brainstem responses thresholds were recorded before surgery and day 2 and 7 after surgery. All the animals had increased auditory brainstem responses thresholds after the cochlear implantation. Implanted animals with the motorized insertion tool experienced a partial hearing recovery at day 7 but not in those implanted with the manual insertion procedure (p < 0.001). In the manually implanted animals, a partial recovery was observed when DEX-P contained in liposomal gel was locally administrated (p < 0.0001). Finally, no additive effect with the motorized insertion was noticed. The deleterious effect of manual insertion, during cochlear implantation, can be prevented with local DEX-P administration in the bulla at day 7. The use of a motorized tool performed more atraumatic electrode array insertion for postoperative hearing.

Hyaluronic acid liposomal gel sustains delivery of a corticoid to the inner ear.

The inner ear is one of the most challenging organs for drug delivery, mainly because of the blood-perilymph barrier. Therefore, local rather than systemic drug delivery methods are being developed for inner ear therapy. In this work, we have evaluated the benefit of a hyaluronic acid liposomal gel for sustained delivery of a corticoid to the inner ear after local injection into the middle ear in a guinea pig model. The liposomal gel was easily injectable as a result of the shear-thinning behavior of hyaluronic acid. A prolonged residence time at the site of injection as well as in the round window were achieved without any negative effect on the hearing thresholds of the animals. The presence of liposomes in the formulation resulted in sustained release of the drug in the perilymph for 30days and promoted the conversion of the prodrug loaded within the liposomes (dexamethasone phosphate) into its active form (dexamethasone). In this way, therapeutic doses were attained in the perilymph. A small amount of intact liposomes was visualized in the perilymph, whereas the main proportion of liposomes seemed to be trapped in the round window resulting in a reservoir effect. Thus, the administration of hyaluronic acid liposomal gel to the middle ear is an efficient strategy for delivering corticoids to the inner ear in a sustained manner.

ß-lactoglobulin stabilized nanemulsions--Formulation and process factors affecting droplet size and nanoemulsion stability.

To avoid the toxicological concerns associated to synthetic surfactants, proteins might be an alternative for the stabilization of pharmaceutical nanoemulsions. The present study investigates the use of ß-lactoglobulin (ß-lg) to stabilize oil in water biocompatible nanoemulsions intended for a pharmaceutical use and prepared by high pressure homogenization (HPH). The effects of composition (nature and weight fraction of oil, ß-lg concentration) and of process parameters (pressure and number of cycles) on the droplet size and on the stability of nanoemulsions were thoroughly assessed. The nanoemulsions prepared with ß-lg at 1 wt% and with 5 wt% Miglyol 812 (the oil with the lowest viscosity) displayed a relatively small particle size (about 200 nm) and a low polydispersity when a homogenization pressure of 100 MPa was applied for 4 cycles. These nanoemulsions were the most stable formulations over 30 days at least. Emulsification efficiency of ß-lg was reduced at higher homogenization pressures (200 MPa and 300 MPa). The effect of HPH process on the interfacial properties of ß-lg was evaluated by drop shape analysis. This treatment had an effect neither on the interfacial tension nor on the interfacial dilatational rheology of ß-lg at the Miglyol 812/water interface.

Recent advances in local drug delivery to the inner ear.

Inner ear diseases are not adequately treated by systemic drug administration mainly because of the blood-perilymph barrier that reduces exchanges between plasma and inner ear fluids. Local drug delivery methods including intratympanic and intracochlear administrations are currently developed to treat inner ear disorders more efficiently. Intratympanic administration is minimally invasive but relies on diffusion through middle ear barriers for drug entry into the cochlea, whereas intracochlear administration offers direct access to the colchlea but is rather invasive. A wide range of drug delivery systems or devices were evaluated in research and clinic over the last decade for inner ear applications. In this review, different strategies including medical devices, hydrogels and nanoparticulate systems for intratympanic administration, and cochlear implant coating or advanced medical devices for intracoclear administration were explored with special attention to in vivo studies. This review highlights the promising systems for future clinical applications as well as the current hurdles that remain to be overcome for efficient inner ear therapy.

Effect of liposomes on rheological and syringeability properties of hyaluronic acid hydrogels intended for local injection of drugs.

The aim of this work was to thoroughly study the effect of liposomes on the rheological and the syringeability properties of hyaluronic acid (HA) hydrogels intended for the local administration of drugs by injection. Whatever the characteristics of the liposomes added (neutral, positively or negatively charged, with a corona of polyethylene glycol chains, size), the viscosity and the elasticity of HA gels increased in a lipid concentration-dependent manner. Indeed, liposomes strengthened the network formed by HA chains due to their interactions with this polymer. The nature and the resulting effects of these interactions depended on liposome composition and concentration. The highest viscosity and elasticity were observed with liposomes covered by polyethylene glycol chains while neutral liposomes displayed the lowest effect. Despite their high viscosity at rest, all the formulations remained easily injectable through needles commonly used for local injections thanks to the shear-thinning behavior of HA gels. The present study demonstrates that rheological and syringeability tests are both necessary to elucidate the behavior of such systems during and post injection. In conclusion, HA liposomal gels appear to be a promising and versatile formulation platform for a wide range of applications in local drug delivery when an injection is required.

Proteins, polysaccharides, and their complexes used as stabilizers for emulsions: alternatives to synthetic surfactants in the pharmaceutical field?

Emulsions are widely used in pharmaceutics for the encapsulation, solubilization, entrapment, and controlled delivery of active ingredients. In order to answer the increasing demand for clean label excipients, natural polymers can replace the potentially irritative synthetic surfactants used in emulsion formulation. Indeed, biopolymers are currently used in the food industry to stabilize emulsions, and they appear as promising candidates in the pharmaceutical field too. All proteins and some polysaccharides are able to adsorb at a globule surface, thus decreasing the interfacial tension and enhancing the interfacial elasticity. However, most polysaccharides stabilize emulsions simply by increasing the viscosity of the continuous phase. Proteins and polysaccharides may also be associated either through covalent bonding or electrostatic interactions. The combination of the properties of these biopolymers under appropriate conditions leads to increased emulsion stability. Alternative layers of oppositely charged biopolymers can also be formed around the globules to obtain multi-layered "membranes". These layers can provide electrostatic and steric stabilization thus improving thermal stability and resistance to external treatment. The novel biopolymer-stabilized emulsions have a great potential in the pharmaceutical field for encapsulation, controlled digestion, and targeted release although several challenging issues such as storage and bacteriological concerns still need to be addressed.