Hanne Mørck Nielsen

Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

The hydrophilic nature of peptides and proteins renders them impermeable to cell membranes. Thus, in order to successfully deliver peptide and protein-based therapeutics across the plasma membrane or epithelial and endothelial barriers, a permeation enhancing strategy must be employed. Cell-penetrating peptides (CPPs) constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB). CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate cell membranes are believed to involve both endocytosis and direct translocation, but are still widely investigated and discussed. The fact that multiple factors influence the mechanisms responsible for cellular CPP internalization and the lack of sensitive methods for detection of the CPP, and in some cases the cargo, further complicates the design and conduction of conclusive mechanistic studies.

Spray drying of siRNA-containing PLGA nanoparticles intended for inhalation

Abstract Local delivery of small interfering RNA (siRNA) to the lungs constitutes a promising new area in drug delivery. The present study evaluated parameters of importance for spray drying of siRNA-loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) into nanocomposite microparticles intended for inhalation. The spray drying process was optimised using a statistical design of experiment and by evaluating powder characteristics upon systematic variation of the formulation parameters. Concentration, carbohydrate excipient (trehalose, lactose and mannitol) and the ratio of NP to excipient were varied to monitor the effects on moisture content, particle morphology, particle size and powder yield. The identified optimum conditions were applied for spray drying of siRNA-loaded nanocomposite microparticles, resulting in a product with a low water content (0.78% w/w) and an aerodynamic particle diameter considered suitable for inhalation. The use of mannitol in the formulation allowed a significantly lower moisture content than trehalose and lactose. The inclusion of 50% (w/w) or higher amounts of NPs resulted in a marked change in the surface morphology of the spray-dried particles. Importantly, the integrity and biological activity of the siRNA were preserved during the spray drying process. In conclusion, the present results show that spray drying is a suitable technique for producing nanocomposite microparticles comprising siRNA-containing PLGA NPs for potential use in inhalation therapy.

High loading efficiency and sustained release of siRNA encapsulated in PLGA nanoparticles: quality by design optimization and characterization.

Poly(DL-lactide-co-glycolide acid) (PLGA) is an attractive polymer for delivery of biopharmaceuticals owing to its biocompatibility, biodegradability and outstanding controlled release characteristics. The purpose of this study was to understand and define optimal parameters for preparation of small interfering RNA (siRNA)-loaded PLGA nanoparticles by the double emulsion solvent evaporation method and characterize their properties. The experiments were performed according to a 2(5-1) fractional factorial design based on five independent variables: The volume ratio between the inner water phase and the oil phase, the PLGA concentration, the sonication time, the siRNA load and the amount of acetylated bovine serum albumin (Ac-BSA) in the inner water phase added to stabilize the primary emulsion. The effects on the siRNA encapsulation efficiency and the particle size were investigated. The most important factors for obtaining an encapsulation efficiency as high as 70% were the PLGA concentration and the volume ratio whereas the size was mainly affected by the PLGA concentration. The viscosity of the oil phase was increased at high PLGA concentration, which explains the improved encapsulation by stabilization of the primary emulsion and reduction of siRNA leakage to the outer water phase. Addition of Ac-BSA increased the encapsulation efficiency at low PLGA concentrations. The PLGA matrix protected siRNA against nuclease degradation, provided a burst release of surface-localized siRNA followed by a triphasic sustained release for two months. These results enable careful understanding and definition of optimal process parameters for preparation of PLGA nanoparticles encapsulating high amounts of siRNA with immediate and long-term sustained release properties.

Preparation and characterization of poly(dl-lactide-co-glycolide) nanoparticles for siRNA delivery

Synthetic short interfering RNA (siRNA) is promising for specific and efficient knockdown of disease-related genes. However, in vivo application of siRNA requires an effective delivery system. Commonly used siRNA carriers are based on polycations, which form electrostatic complexes with siRNA. Such poly- or lipoplexes are of limited use in vivo due to severe problems associated with toxicity, serum instability and non-specific immune-responses. The aim of the present study was to prepare uniformly sized nanoparticles (NPs) with a high load of siRNA by use of the safe and biodegradable poly-(DL-lactide-co-glycolide) (PLGA) polymer without including polycations. The siRNA was encapsulated in the core of NPs by the double emulsion solvent evaporation method. To optimize the NP formulation, the effects of important formulation and processing parameters were investigated systematically. Generally, spherical siRNA-loaded NPs (<300 nm, PDI<0.2, zeta potential -40 mV) were obtained. An encapsulation efficiency of up to 57% was achieved by adjusting the inner water phase volume, the PLGA concentration, the first emulsification sonication time, and stabilization of the water-oil interface with serum albumin. The integrity of siRNA was preserved during the preparation. Preparation of core-loaded siRNA-NPs based on PLGA and no cationic excipient seems possible and promising for delivery of siRNA.

Design of an inhalable dry powder formulation of DOTAP-modified PLGA nanoparticles loaded with siRNA

Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue.

Antimicrobial, Hemolytic, and Cytotoxic Activities of β-Peptoid–Peptide Hybrid Oligomers: Improved Properties Compared to Natural AMPs

While natural host-defense antimicrobial peptides (AMPs) and analogues thereof have been investigated intensely in the last two decades with the purpose of combating the still increasing threat from emerging multiresistant pathogenic microbes, 2] compounds with peptidomimetic backbones have received considerable attention due to their superior stability against proteolytic enzymes. 4] Typically, studies of peptidomimetic AMP analogues have involved a brief microbiological evaluation of an array of oligomers, and only occasionally has testing been performed across a broader range of microorganisms or involved systematic structure–activity relationship (SAR) studies. Such investigations have proven fruitful for a-peptidic AMPs, 18] and might reveal unexpected lead structures and selectivity profiles when applied to peptidomimetics as well. Therefore, we have performed a more rigorous microbiological evaluation as well as toxicity profiling of a series of oligomers based on our b-peptoid–peptide hybrid backbone architecture. 20] Antimicrobial activities were determined alongside the archetypal cationic AMP magainin-2 and its clinically tested derivative pexiganan against a series of five important pathogens belonging to different classes. The obtained SAR data were subsequently correlated with various measurements of toxicity towards mammalian cells. Thereby we were able to derive useful trends for the future design of antibacterial and antifungal peptidomimetic constructs with potential for enhanced selectivity. Three subclasses 1 a–3 d (Scheme 1) were originally designed to address the general effects of length, type of cationic side chains, and presence of a-chirality in the b-peptoid residues. These series had previously been confirmed to possess membrane activity, as indicated by testing for hemolytic and prehemolytic effects, as well as by calcein release experiments with model liposomes, albeit the interaction of these compounds with intracellular targets cannot be ruled out based on our data. The all-aliphatic compound 4 and the mixed aromatic–aliphatic chimera 5 were included to address the importance of lipophilicity and type of cationic residue. Finally, we included three 5/6-carboxyfluorescein-labeled oligomers (6–8) to assess the influence of the presence of this widely used fluorophore on the antimicrobial activity, which might have important implications for confocal fluorescence microscopy studies of the interaction of labeled compounds with live bacteria. The chimeras 4–8 were of dodecamer length to minimize undesired mammalian cell toxicity that might be observed with increasing length. This compound collection was tested against a variety of clinically relevant pathogens and human red blood cells (Table 1). Scheme 1. Chemical structures of the examined hybrid oligomers. The abbreviations used for the b-peptoid units were adapted from the abbreviations commonly used for peptoids (i.e. , N-alkylglycines), 7] by adding the b-prefix. bNspe = N-(S)-1-phenylethyl-balanine, bNphe =b-N-phenylalanine, bNsce = N-(S)-1-cyclohexylethyl-b-alanine, hArg = homoarginine, CF = 5/6-carboxyfluoresceinoyl.

Nicotine permeability across the buccal TR146 cell culture model and porcine buccal mucosa in vitro: effect of pH and concentration

The present study was conducted to investigate and compare the effect of pH and drug concentration on nicotine permeability across the TR146 cell culture model and porcine buccal mucosa in vitro. As a further characterization of the TR146 cell culture model, it was explored whether the results were comparable for bi-directional and uni-directional transport in the presence of a transmembrane pH gradient. Nicotine concentrations between 10(-5) and 10(-2) M were applied to the apical side of the TR146 cell culture model or the mucosal side of porcine buccal mucosa. Buffers with pH values of 5.5, 7.4 and 8.1 were used to obtain different fractions of non- and mono-ionized nicotine. The apparent permeability (P(app)) of nicotine across both models increased significantly with increasing pH, and the P(app) values obtained with the two models could be correlated in a linear manner. With increasing concentrations of nicotine, the P(app) values decreased, which can partly be explained by an effect on the paracellular pathway. Similar results were also obtained when using the models for bi-directional as well as for uni-directional studies. The TR146 cell culture model may be used as model for buccal epithelium in studies with ionized drugs and a transmembrane pH gradient.

Elucidating the molecular mechanism of PAMAM-siRNA dendriplex self-assembly: effect of dendrimer charge density.

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.

Corticosteroid solubility and lipid polarity control release from solid lipid nanoparticles

Solid lipid nanoparticles (SLN) show promise as a drug delivery system for skin administration. The solid state of the lipid particle enables efficient drug encapsulation and controlled drug release. The present study addresses the influence of lipid composition and drug substance lipid solubility on the in vitro release profile of corticosteroids from SLN for topical administration. Firstly, the effect of lipid composition on the lipid solubility and in vitro release of betamethasone-17-valerate (BMV) was determined by varying the lipid monoglyceride content and the chain length of the fatty acid moiety. Secondly, the effect of drug substance physicochemical properties was determined by studying five different corticosteroid derivatives with different lipophilicity. A high concentration of monoglyceride in SLN increased the amount of BMV released. The corticosteroid release rate depended on the drug substance lipophilicity and it was clear that the release profiles depended on drug partitioning to the aqueous phase as indicated by zero order kinetics. The results emphasize that the corticosteroid solubility in the lipid phase greatly influence drug distribution in the lipid particles and release properties. Thus knowledge of drug substance solubility and lipid polarity contributes to optimize SLN release properties.

Metabolic cleavage of cell-penetrating peptides in contact with epithelial models: human calcitonin (hCT)-derived peptides, Tat(47–57) and penetratin(43–58)

We assessed the metabolic degradation kinetics and cleavage patterns of some selected CPP (cell-penetrating peptides) after incubation with confluent epithelial models. Synthesis of N-terminal CF [5(6)-carboxyfluorescein]-labelled CPP, namely hCT (human calcitonin)-derived sequences, Tat(47-57) and penetratin(43-58), was through Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry. Metabolic degradation kinetics of the tested CPP in contact with three cell-cultured epithelial models, MDCK (Madin-Darby canine kidney), Calu-3 and TR146, was evaluated by reversed-phase HPLC. Identification of the resulting metabolites of CF-hCT(9-32) was through reversed-phase HPLC fractionation and peak allocation by MALDI-TOF-MS (matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry) or direct MALDI-TOF-MS of incubates. Levels of proteolytic activity varied highly between the investigated epithelial models and the CPP. The Calu-3 model exhibited the highest proteolytic activity. The patterns of metabolic cleavage of hCT(9-32) were similar in all three models. Initial cleavage of this peptide occurred at the N-terminal domain, possibly by endopeptidase activity yielding both the N- and the C-terminal counterparts. Further metabolic degradation was by aminopeptidase, endopeptidase and/or carboxypeptidase activities. In conclusion, when in contact with epithelial models, the studied CPP were subject to efficient metabolism, a prerequisite of cargo release on the one hand, but with potential for premature cleavage and loss of the cargo as well on the other. The results, particularly on hCT(9-32), may be used as a template to suggest structural modifications towards improved CPP performance.