Interplay between mucus mobility and alveolar macrophage targeting of surface-modified liposomes.

Alveolar macrophages play a crucial role in the initiation and resolution of the immune response in the lungs. Pro-inflammatory M1 alveolar macrophages are an interesting target for treating inflammatory and infectious pulmonary diseases. One commune targeting strategy is to use nanoparticles conjugated with hyaluronic acid, which interact with CD44 overexpressed on the membrane of those cells. Unfortunately, this coating strategy may be countered by the presence on the surface of the nanoparticles of a poly(ethylene glycol) corona employed to improve nanoparticles' diffusion in the lung mucus. This study aims to measure this phenomenon by comparing the behavior in a murine lung inflammation model of three liposomal platforms designed to represent different poly(ethylene glycol) and hyaluronic acid densities (Liposome-PEG, Liposome-PEG-HA and Liposome-HA). In this work, the liposomes were obtained by a one-step ethanol injection method. Their interaction with mucin and targeting ability toward pro-inflammatory macrophages were then investigated in vitro and in vivo in a LPS model of lung inflammation. In vitro, poly(ethylene glycol) free HA-liposomes display a superior targeting efficiency toward M1 macrophages, while the addition of poly(ethylene glycol) induces better mucus mobility. Interestingly in vivo studies revealed that the three liposomes showed distinct cell specificity with alveolar macrophages demonstrating an avidity for poly(ethylene glycol) free HA-liposomes, while neutrophils favored PEGylated liposomes exempt of HA. Those results could be explained by the presence of two forces exercising a balance between mucus penetration and receptor targeting. This study corroborates the importance of considering the site of action and the targeted cells when designing nanoparticles to treat lung diseases.

Nose-to-brain drug delivery mediated by polymeric nanoparticles: influence of PEG surface coating.

Intranasal administration of mucus-penetrating nanoparticles is an emerging trend to increase drug delivery to the brain. In order to overcome rapid nasal mucociliary clearance, low epithelial permeation, and local enzymatic degradation, we investigated the influence of PEGylation on nose-to-brain delivery of polycaprolactone (PCL) nanoparticles (PCL-NPs) encapsulating bexarotene, a potential neuroprotective compound. PEGylation with 1, 3, 5, and 10% PCL-PEG did not affect particle diameter or morphology. Upon incubation with artificial nasal mucus, only 5 and 10% of PCL-PEG coating were able to ensure NP stability and homogeneity in mucus. Rapid mucus-penetrating ability was observed for 98.8% of PCL-PEG5% NPs and for 99.5% of PCL-PEG10% NPs. Conversely, the motion of non-modified PCL-NPs was markedly slower. Fluorescence microscopy showed that the presence of PEG on NP surface did not reduce their uptake by RMPI 2650 cells. Fluorescence tomography images evidenced higher translocation into the brain for PCL-PEG5% NPs. Bexarotene loaded into PCL-PEG5% NPs resulted in area under the curve in the brain (AUCbrain) 3 and 2-fold higher than that for the drug dispersion and for non-PEGylated NPs (p < 0.05), indicating that approximately 4% of the dose was directly delivered to the brain. Combined, these results indicate that PEGylation of PCL-NPs with PCL-PEG5% is able to reduce NP interactions with the mucus, leading to a more efficient drug delivery to the brain following intranasal administration. Graphical abstract.

Antioxidant-mediated control of degradation and drug release from surface-eroding poly(ethylene carbonate).

Surface-eroding polymers are of significant interest for various applications in the field of controlled drug delivery. Poly(ethylene carbonate), as an example, offers little control over the rate of degradation and, thus, drug release, which usually conflicts with the requirements for long-acting medications. Here, we challenged an option to decelerate the degradation of poly(ethylene carbonate) in vitro and in vivo. When polymer films loaded with distinct antioxidants (vitamins) along with the model drugs leuprorelin and risperidone were incubated in superoxide radical solution and phagocyte culture, the mass loss and drug release from the delivery vehicle was a function of the type and dose of the utilized antioxidant. Once the polymer surface was "attacked" by reactive oxygen species, the antioxidants were released on demand quenching the polymer-degrading radicals. Accordingly, specific combinations of polymer and radical scavengers resulted in controlled release medications with an extended "life-time" of one month or longer, which is difficult to achieve for poly(ethylene carbonate) in the absence of antioxidants. A comparable degradation and drug release behavior was observed when antioxidant-loaded poly(ethylene carbonate) films were implanted in rats. Furthermore, linear correlations were obtained between the mass loss of the polymer films and the released fraction of drug (with slopes close to 1), a clear indication for the surface erosion of poly(ethylene carbonate) in vitro and in vivo. Overall, an addition of antioxidants to poly(ethylene carbonate)-based controlled drug delivery vehicles represents a reasonable approach to modify the performance of long-acting medications, especially when a "life time" of weeks to months needs to be achieved. STATEMENT OF SIGNIFICANCE: Surface-eroding poly(ethylene carbonate) (PEC) is of significant interest for long-acting injectable formulations. However, PEC offers only little control over the rate of degradation and, thus, drug release kinetics. We describe an option to decelerate the degradation rate of PEC in vitro and in vivo. When polymer films loaded with distinct antioxidants along with model drugs were incubated in superoxide radical solution, phagocyte culture and implanted in rats, their mass loss and drug release was a function of the type and dose of the utilized antioxidant. Accordingly, specific combinations of polymer and radical scavengers resulted in controlled release medications with an extended "life-time" of one month or longer, which is difficult to achieve for PEC in the absence of antioxidants.

In Vivo Biodistribution of Respirable Solid Lipid Nanoparticles Surface-Decorated with a Mannose-Based Surfactant: A Promising Tool for Pulmonary Tuberculosis Treatment?

The active targeting to alveolar macrophages (AM) is an attractive strategy to improve the therapeutic efficacy of 'old' drugs currently used in clinical practice for the treatment of pulmonary tuberculosis. Previous studies highlighted the ability of respirable solid lipid nanoparticle assemblies (SLNas), loaded with rifampicin (RIF) and functionalized with a novel synthesized mannose-based surfactant (MS), both alone and in a blend with sodium taurocholate, to efficiently target the AM via mannose receptor-mediated mechanism. Here, we present the in vivo biodistribution of these mannosylated SLNas, in comparison with the behavior of both non-functionalized SLNas and bare RIF. SLNas biodistribution was assessed, after intratracheal instillation in mice, by whole-body real-time fluorescence imaging in living animals and RIF quantification in excised organs and plasma. Additionally, SLNas cell uptake was determined by using fluorescence microscopy on AM from bronchoalveolar lavage fluid and alveolar epithelium from lung dissections. Finally, histopathological evaluation was performed on lungs 24 h after administration. SLNas functionalized with MS alone generated the highest retention in lungs associated with a poor spreading in extra-pulmonary regions. This effect could be probably due to a greater AM phagocytosis with respect to SLNas devoid of mannose on their surface. The results obtained pointed out the unique ability of the nanoparticle surface decoration to provide a potential more efficient treatment restricted to the lungs where the primary tuberculosis infection is located.

Impact of drug loading in mesoporous silica-amorphous formulations on the physical stability of drugs with high recrystallization tendency.

In this study, a method is described to determine the monolayer loading capacity (MLC) of the drugs naproxen and ibuprofen, both having high recrystallization tendencies, in mesoporous silica (MS), a well known carrier that is able to stabilize the amorphous form of a drug. The stabilization has been suggested to be due to direct absorption of the drug molecules onto the MS surface, i.e. the drug monolayer. In addition, drug that is not in direct contact with MS surface can fill the pores up to its pore filling capacity (PFC) and is potentially stabilized by confinement due to the pore size being smaller than a crystal nuclei. For drugs with high recrystallization tendencies, any drug outside the pores crystallizes due to its poor physical stability. The drug monolayer does not contribute to the glass transition temperature (Tg ) in the DSC, however, the confined amorphous drug above MLC has a Tg and the heat capacity (ΔC p) over the Tg increases with an increasing fraction of confined amorphous drug. Hence, several drug loading values above the MLC were investigated towards the presence of a Tg and ΔC p using differential scanning calorimetry (DSC). A linear correlation between the amount of confined amorphous drug and its ΔC p was identified for the mixtures between the MLC and PFC. By subsequent extrapolation to zero ΔC p the experimental MLC could be determined. Using theoretical density functional theory (DFT) and ab initio Molecular Dynamics (AIMD), the binding energies for the monolayer suggested that the monolayer in fact is thermodynamically more favorable than the crystalline form, whereas the confined amorphous form is thermodynamically less favorable. Consequently, a physical stability study showed that the confined amorphous drugs above the MLC were thermodynamically unstable and consequently flowing out of the pores in order to crystallize, whereas the monolayer remained physically stable.

Increased Nose-to-Brain Delivery of Melatonin Mediated by Polycaprolactone Nanoparticles for the Treatment of Glioblastoma.

PURPOSE: Intranasal administration has been extensively applied to deliver drugs to the brain. In spite of its unfavorable biopharmaceutic properties, melatonin (MLT) has demonstrated anticancer effects against glioblastoma. This study describes the nose-to-brain delivery of MLT-loaded polycaprolactone nanoparticles (MLT-NP) for the treatment of glioblastoma. METHODS: MLT-NP were prepared by nanoprecipitation. Following intranasal administration in rats, brain targeting of the formulation was demonstrated by fluorescence tomography. Brain and plasma pharmacokinetic profiles were analyzed. Cytotoxicity against U87MG glioblastoma cells and MRC-5 non-tumor cells was evaluated. RESULTS: MLT-NP increased the drug apparent water solubility ~35 fold. The formulation demonstrated strong activity against U87MG cells, resulting in IC50 ~2500 fold lower than that of the free drug. No cytotoxic effect was observed against non-tumor cells. Fluorescence tomography images evidenced the direct translocation of nanoparticles from nasal cavity to the brain. Intranasal administration of MLT-NP resulted in higher AUCbrain and drug targeting index compared to the free drug by either intranasal or oral route. CONCLUSIONS: Nanoencapsulation of MLT was crucial for the selective antitumoral activity against U87MG. In vivo evaluation confirmed nose-to-brain delivery of MLT mediated by nanoparticles, highlighting the formulation as a suitable approach to improve glioblastoma therapy.

Ascorbic acid encapsulated into negatively charged liposomes exhibits increased skin permeation, retention and enhances collagen synthesis by fibroblasts.

Ascorbic acid (AA) is widely used in cosmetic formulations due to its antioxidant property and ability to increase collagen synthesis. Here, we encapsulated AA in vesicles with different lipid compositions. Negative liposome charge favored AA skin retention, with accumulation of 37 ± 12 and 74 ± 23 µg/cm2 in the epidermis and dermis, respectively, after 6 hours. Drug flux was influenced by the formulation composition, and both the presence of cholesterol and the liposomes surface charge were able to increase the amount of AA crossing the skin. The formulation was stable for at least 30 days and promoted a 7-fold increase in flux compared to free AA. Additionally, liposomes were able to interact better with keratinocytes and fibroblasts membranes. In vitro efficacy studies demonstrated that associating AA to these liposomes resulted in increased effectiveness of type I collagen synthesis by fibroblasts and regeneration of UVA-induced damage in keratinocytes. Our results demonstrate the applicability of AA-negatively charged liposomes in promoting AA cutaneous permeation and increasing the retention and flux of this molecule in the skin. This formulation also increased AA stability and effectiveness, opening new perspectives for its application in view of reducing certain skin ageing outcomes.

Efflux Inhibitor Bicalutamide Increases Oral Bioavailability of the Poorly Soluble Efflux Substrate Docetaxel in Co-Amorphous Anti-Cancer Combination Therapy.

Many anti-cancer drugs are difficult to formulate into an oral dosage form because they are both poorly water-soluble and show poor permeability, the latter often as a result of being an intestinal efflux pump substrate. To obtain a more water-soluble formulation, one can take advantage of the higher solubility of the amorphous form of a given drug, whereas to increase permeability, one can make use of an efflux pump inhibitor. In this study, a combination of these two strategies was investigated using the co-amorphous approach, forming an amorphous mixture of two anti-cancer drugs, docetaxel (DTX) and bicalutamide (BIC). The efflux substrate, DTX, was combined with the efflux inhibitor, BIC, and prepared as a single phase co-amorphous mixture at a 1:1 molar ratio using vibrational ball milling. The co-amorphous formulation was tested in vitro and in vivo for its dissolution kinetics, supersaturation properties and pharmacokinetics in rats. The co-amorphous formulation showed a faster in vitro dissolution of both drugs compared to the control groups, but only DTX showed supersaturation (1.9 fold) compared to its equilibrium solubility. The findings for the co-amorphous formulation were in agreement with the pharmacokinetics data, showing a quicker onset in plasma concentration as well as a higher bioavailability for both DTX (15-fold) and BIC (3-fold) compared to the crystalline drugs alone. Furthermore, the co-amorphous formulation remained physically stable over 1.5 years at 4 °C under dry conditions.

Antiproliferative Activity and VEGF Expression Reduction in MCF7 and PC-3 Cancer Cells by Paclitaxel and Imatinib Co-encapsulation in Folate-Targeted Liposomes.

Co-encapsulation of anticancer drugs paclitaxel and imatinib in nanocarriers is a promising strategy to optimize cancer treatment. Aiming to combine the cytotoxic and antiangiogenic properties of the drugs, a liposome formulation targeted to folate receptor co-encapsulating paclitaxel and imatinib was designed in this work. An efficient method was optimized for the synthesis of the lipid anchor DSPE-PEG(2000)-folic acid (FA). The structure of the obtained product was confirmed by RMN, FT-IR, and ESI-MS techniques. A new analytical method was developed and validated for simultaneous quantification of the drugs by liquid chromatography. Liposomes, composed of phosphatidylcholine, cholesterol, and DSPE-mPEG(2000), were prepared by extrusion. Their surface was modified by post-insertion of DSPE-PEG(2000)-FA. Reaction yield for DSPE-PEG(2000)-FA synthesis was 87%. Liposomes had a mean diameter of 122.85 ± 1.48 nm and polydispersity index of 0.19 ± 0.01. Lyophilized formulations remained stable for 60 days in terms of size and drug loading. FA-targeted liposomes had a higher effect on MCF7 cell viability reduction (p < 0.05) when compared with non-targeted liposomes and free paclitaxel. On PC-3 cells, viability reduction was greater (p < 0.01) when cells were exposed to targeted vesicles co-encapsulating both drugs, compared with the non-targeted formulation. VEGF gene expression was reduced in MCF7 and PC-3 cells (p < 0.0001), with targeted vesicles exhibiting better performance than non-targeted liposomes. Our results demonstrate that multifunctional liposomes associating molecular targeting and multidrug co-encapsulation are an interesting strategy to achieve enhanced internalization and accumulation of drugs in targeted cells, combining multiple antitumor strategies.

Effect of hyaluronic acid-binding to lipoplexes on intravitreal drug delivery for retinal gene therapy.

Intravitreal administration of nanomedicines could be valuable for retinal gene therapy, if their mobility in the vitreous and therapeutic efficacy in the target cells can be guaranteed. Hyaluronic acid (HA) as an electrostatic coating of polymeric gene nanomedicines has proven to be beneficial on both accounts. While electrostatic coating provides an easy way of coating cationic nanoparticles, the stability of electrostatic complexes in vivo is uncertain. In this study, therefore, we compare electrostatic with covalent coating of gene nanocarriers with HA for retinal gene therapy via intravitreal administration. Specifically, DOTAP:DOPE/plasmid DNA lipoplexes coated with HA are evaluated in terms of intravitreal mobility using a previously optimized ex vivo model. We find that both electrostatic and covalent HA coating considerably improve the mobility of the lipoplexes in the vitreous humor of excised bovine eyes. In addition we evaluate in vitro uptake and transfection efficiency in ARPE-19 cells. Contrary to PEGylated lipoplexes it is found that HA coated lipoplexes are efficiently internalized into ARPE-19 cells. Covalent HA-coated lipoplexes had an 8-fold increase of transgene expression compared to the uncoated lipoplexes. We conclude that covalent HA-coating of gene nanomedicines is a promising approach for retinal gene therapy by intravitreal administration.

Lipid-based nanosystems for CD44 targeting in cancer treatment: recent significant advances, ongoing challenges and unmet needs.

Extensive experimental evidence demonstrates the important role of hyaluronic acid (HA)-CD44 interaction in cell proliferation and migration, inflammation and tumor growth. Taking advantage of this interaction, the design of HA-modified nanocarriers has been investigated for targeting CD44-overexpressing cells with the purpose of delivering drugs to cancer or inflammatory cells. The effect of such modification on targeting efficacy is influenced by several factors. In this review, we focus on the impact of HA-modification on the characteristics of lipid-based nanoparticles. We try to understand how these modifications influence particle physicochemical properties, interaction with CD44 receptors, intracellular trafficking pathways, toxicity, complement/macrophage activation and pharmacokinetics. Our aim is to provide insight in tailoring particle modification by HA in order to design more efficient CD44-targeting lipid nanocarriers.

Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor.

The mechanism by which hyaluronic acid (HA)-bearing lipoplexes target the A549 lung cancer cell line was evaluated. For this purpose, cationic liposomes targeting the CD44 receptor were designed thanks to the incorporation in their composition of a conjugate between high molecular weight HA and the lipid DOPE (HA-DOPE). Liposomes containing HA-DOPE were complexed at different lipids:DNA ratios with a reporter plasmid encoding the green fluorescent protein (GFP). Diameter, zeta potential, lipoplex stability and DNA protection from nucleases have been determined. Lipids:DNA ratios of 2, 4 and 6 provided a diameter around 250 nm with a zeta potential of -30 mV. The strength of lipids:DNA interaction and the fraction of DNA protected from enzymatic degradation increased with the lipids:DNA ratio. 2D-immunoelectrophoresis demonstrated the low capacity to activate the C3 fraction of the complement system of any of these three ratios, with and without HA-DOPE. Transfection efficiency in the presence of 0, 10 and 15% of HA-DOPE or unconjugated HA, was determined on the CD44-expressing A549 cells by flow cytometry. Lipoplexes at a lipids:DNA ratio of 2 containing 10% (w/w) of HA-DOPE were the most efficient for transfection. The maximal level of GFP expression was obtained after 6h of incubation demonstrating a slow transfection kinetics of lipoplexes. Finally, lipoplex cellular uptake, measured indirectly by the level of transfection using flow cytometry and validated by fluorescence microscopy, was shown to be mediated by the CD44 receptor and caveolae. These results demonstrate the strong specificity of DNA targeting through the CD44 receptor using HA of high molecular weight as a ligand.