Gillian Barratt

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

Biodistribution of Long-Circulating PEG-Grafted Nanocapsules in Mice: Effects of PEG Chain Length and Density

AbstractPurpose: To study the pharmacokinetics and biodistribution of novel polyethyleneglycol (PEG) surface-modified poly(rac-lactide) (PLA) nanocapsules (NCs) and to investigate the influence of PEG chain length and content. Methods: The biodistribution and plasma clearance in mice of different NC formulations were studied with [3H]-PLA. PLA-PEG copolymers were used in NC preparations at different chain lengths (5 kDa and 20 kDa) and PEG contents (10% and 30% w/w of total polymer). In vitro and in vivo stability were also checked. Results: Limited [3H]-PLA degradation was observed after incubation in mouse plasma for 1 h, probably because of to the large surface area and thin polymer wall. After injection into mice, NCs prepared with PLA-PEG copolymers showed an altered distribution compared to poloxamer-coated PLA NCs. An increased concentration in plasma was also observed for PLA-PEG NCs, even after 24 h. A dramatic difference in the pharmacokinetic parameters of PLA-PEG 45-20 30% NCs compared to poloxamer-coated NCs indicates that covalent attachment, longer PEG chain lengths, and higher densities are necessary to produce an increased half-life of NCs in vivo. Conclusions: Covalently attached PEG on the surface of NCs substantially can reduce their clearance from the blood compartment and alter their biodistribution.

Therapeutic applications of colloidal drug carriers

Colloidal drug carriers such as liposomes and nanoparticles can be used to improve the therapeutic index of both established and new drugs by modifying their distribution, and thus increasing their efficacy and/or reducing their toxicity. This is because the drug distribution then follows that of the carrier, rather than depending on the physicochemical properties of the drug itself. If these delivery systems are carefully designed with respect to the target and the route of administration, they may provide one solution to some of the delivery problems posed by new classes of active molecules, such as peptides and proteins, genes and oligonucleotides. They may also offer alternative modes for more conventional drugs, such as highly hydrophobic small molecules. This review discusses the use of colloidal, particulate carrier systems (25 nm to 1 µm in diameter) in such applications.

Surface-engineered nanoparticles for multiple ligand coupling.

The design of surface-engineered nanoparticles for targeting to specific sites is a major challenge. To our knowledge, no study in the literature deals with ligand functionalization of biodegradable nanoparticles through biotin-avidin interactions. With the aim of conceiving small-sized nanoparticles which can be easily functionalized with a variety of ligands or mixtures thereof, biotinylated and PEGylated biotin-poly(ethylene glycol)-poly(epsilon-caprolactone) (B-PEG-PCL) copolymers were synthesized and used to prepare nanoparticles of around 100 nm. Avidin, followed by biotinylated wheat germ agglutinin as a model lectin, were coupled to their surface by taking advantage of the strong biotin-avidin complex formation. The cytotoxicity of the nanospheres towards Caco-2 cells in culture was negligible (more than 82% cell survival for nanoparticle concentrations up to 300 microg/well). The amount of radiolabeled poly(lactic acid) (PLA) or PEG-PLA nanoparticles associated with Caco-2 cells was only 0.7% and 1.5% of the amount added, respectively. This value was increased to 8.5% when a sufficient amount of lectin was bound to the PEG-PLA copolymer. After further studies, the biotin-PEG-coated nanoparticles could be helpful tools for studying the interaction between cells and functionalized nanoparticles with various surface characteristics (PEG layer density and thickness, ligand type and density).

Interactions of nanoparticles bearing heparin or dextran covalently bound to poly(methyl methacrylate) with the complement system.

The efficient uptake of injected nanoparticles by cells of the mononuclear phagocyte system (MPS) limits the development of long-circulating colloidal drug carriers. The complement system plays a major role in the opsonization and recognition processes of foreign materials. Since heparin is an inhibitor of complement activation, nanoparticles bearing heparin covalently bound to poly(methyl methacrylate) (PMMA) have been prepared and their interactions with complement evaluated. The particles retained the complement-inhibiting properties of soluble heparin. Nanoparticles bearing covalently bound dextran instead of heparin were weak activators of complement as compared with crosslinked dextran (Sephadex) or bare PMMA nanoparticles. In addition to the specific activity of bound heparin, the protective effect of both polysaccharides is hypothesized to be due to the presence of a dense brush-like layer on the surface of the particles. Such properties are expected to reduce the uptake by MPS in vivo.

Poly(D,L‐Lactide) Nanocapsules Prepared by a Solvent Displacement Process: Influence of the Composition on Physicochemical and Structural Properties

Nanocapsules (NC) were prepared by interfacial deposition of preformed biodegradable polymer (PLA(50)) after a solvent displacement process. The influence of the composition used for the preparation of NC was evaluated in terms of particle size, polydispersity, zeta potential, homogeneity, and structural characteristics of the systems. The nature of the oil phase, polymer molecular weight, type and concentration of different surfactants were investigated to optimize the formulation to obtain NC suitable for intravenous administration. The influence of the physicochemical properties of the different oils used in NC preparation on the NC size was evaluated. The interfacial tension between the oil and water phases seems to have a greater effect on NC size than the oil viscosity. Miglyol 810 and ethyl oleate lead to the formation of smaller NC, probably because of the reduced interfacial tension. The polymer molecular weight plays only a small role in NC surface charge in the presence of lecithin, whereas NC surface charge, size, polydispersity, and short-term stability were highly influenced by lecithin purity. It appears that the absence of poloxamer 188 leads to smaller polydispersity, less contamination with nanospheres, and reduced formation of structures other than NC. Furthermore, electron microscopy and density gradient density techniques were used to examine the structure of the particles formed and their homogeneity. NC formation was evidenced by the bands with intermediate density between nanoemulsion and nanospheres; however, other bands of low intensity were observed. The presence of liposomes and multilayers in NC preparation was confirmed by electron microscopy. The percentage of carboxyfluorescein entrapped in different NC formulations allowed us to estimate the contamination by liposomes. It has been show that, under our experimental conditions, an excess of lecithin is an essential prerequisite for a stable preparation of PLA NC.

Poly (DL-lactide) nanocapsules containing diclofenac: I. Formulation and stability study

Abstract The aim of this work was to formulate nanocapsules prepared from poly(DL-lactide) containing a non-steroidal anti-infammatory drug, diclofenac, and to study their stability during storage at room temperature. The influence of some factors wich could affect stability, namely, the type of oily phase used or/and its concentration, the concentrations of drug and of surfactants, was investigated. The pH of the preparation, the particle size, the quantity of drug remaining (encapsulated and total) and polymer molecular weight were determined at intervals for up to 8 months after nanocapsule preparation. Although colloidal systems which were physically stable over this period could be obtained with either of the two oils tested, polymer degradation was more rapid in the presence of benzyl benzoate than with Miglyol 810®. The optimal concentration of the latter was found to be 3.33%. The highest loading of diclofenac consistent with a stable preparation was 1.00 mg/ml. Stable nanocapsules could be obtained with as little as 0.75% lipophilic surfactant together with a similar concentration of hydrophilic surfactant. These concentrations are considerably lower than those described in the literature for the formulation of this type of colloid.

Nanotechnologies and controlled release systems for the delivery of antisense oligonucleotides and small interfering RNA.

Antisense oligonucleotides and small interfering RNA have enormous potential for the treatment of a number of diseases, including cancer. However, several impediments to their widespread use as drugs still have to be overcome: in particular their lack of stability in physiological fluids and their poor penetration into cells. Association with or encapsulation within nano‐ and microsized drug delivery systems could help to solve these problems. In this review, we describe the progress that has been made using delivery systems composed of natural or synthetic polymers in the form of complexes, nanoparticles or microparticles.

In Vitro Antileishmanial Activity of Amphotericin B Loaded in Poly(ε-Caprolactone) Nanospheres

The activity of formulations for amphotericin B (AmB) associated with poly (ε -caprolactone) nanospheres and coated with variable amounts of a non ionic surfactant poloxamer 188, was evaluated against AmB-susceptible (WT) and AmB-resistant (AmB r) strains of Leishmania donovani amastigotes in thioglycolate-elicited peritoneal macrophages. AmB-nanospheres were more actives than free AmB only against amastigotes of wild strain. The activity was not influenced by the concentration of poloxamer 188 used to stabilize the nanospheres in spite of this surfactant was previously reported to synergy with AmB on the membrane of the resistant parasite. Similarly, this improvement was not mediated through macrophage activation. In fact, these nanoparticle formulations appeared to inhibit both NO and TNF- α production induced by the free drug. Therefore, we suggest that the association of AmB with nanospheres may improve the capability of the drug to interact with ergosterol. This hypothesis appears to be supported by the fact that nanospheres did not show any improvement of the AmB activity against the resistant strain (characterized by the absence of ergosterol).

Interactions between a macrophage cell line (J774A1) and surface-modified poly (D,L-lactide) nanocapsules bearing poly(ethylene glycol).

The interactions of naked and surface-modified poly(D,L-lactic acid) (PLA) nanocapsules (NC), where polyethyleneglycol (PEG) was adsorbed or covalently attached, have been studied with a macrophage-like cell line. The fluorescent oil marker, DiD, was successfully encapsulated in NCs in order to follow their interactions with cells. The cell-associated fluorescence obtained with PEG-PLA NC was about 3- to 13-fold lower than that obtained with naked-PLA NC. The effects of PEG chain length, its content as a percentage of total polymer and NC concentration in the culture medium were evaluated. PEG-PLA NC showed dramatically reduced fluorescence association with cells during an 18 h incubation compared with naked-PLA NC, showing that covalent attachment of PEG is important for the persistence of low uptake. The best results in reducing cell-associated fluorescence were obtained with a surface-modified PEG-PLA NC bearing a chain with 20000 MW. Increasing the percentage of PEG produced a reduction in marker association for a given PEG chain length. Moreover, when the PEG-containing poloxamer was simply adsorbed, marker association was dependent on the extent of dilution and the type of serum in the culture medium. Serum proteins, especially immunoglobulins, increased cell-associated fluorescence for PEG-adsorbed NC, but had very little effect on PEG-PLA NC. Marker association was only partially inhibited in the presence of cytochalasin B. The mechanisms of cell-NC interaction depended on the characteristics of the NC surface in each formulation. When the NC was physically separated from cells no diffusion of fluorescent marker in aqueous medium occurred. Nevertheless, collision-mediated transfer of DiD from NC to J774 cells was a non-negligible route of marker transfer, mainly for naked NC. However, this collision-mediated transfer was reduced for the PEG-PLA NC probably due to the restricted contact between NC and cells afforded by PEG steric hindrance at the surface.