Woei Ping Cheng

Dilemmas in the reliable estimation of the in-vitro cell viability in magnetic nanoparticle engineering: which tests and what protocols?

Magnetic nanoparticles [MNPs] made from iron oxides have many applications in biomedicine. Full understanding of the interactions between MNPs and mammalian cells is a critical issue for their applications. In this study, MNPs were coated with poly(ethylenimine) [MNP-PEI] and poly(ethylene glycol) [MNP-PEI-PEG] to provide a subtle difference in their surface charge and their cytotoxicity which were analysed by three standard cell viability assays: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium [MTS], CellTiter-Blue and CellTiter-Glo (Promega, Southampton, UK) in SH-SY5Y and RAW 264.7 cells The data were validated by traditional trypan blue exclusion. In comparison to trypan blue manual counting, the MTS and Titer-Blue assays appeared to have consistently overestimated the viability. The Titer-Glo also experienced a small overestimation. We hypothesise that interactions were occurring between the assay systems and the nanoparticles, resulting in incorrect cell viability evaluation. To further understand the cytotoxic effect of the nanoparticles on these cells, reactive oxygen species production, lipid peroxidation and cell membrane integrity were investigated. After pegylation, the MNP-PEI-PEG possessed a lower positive surface charge and exhibited much improved biocompatibility compared to MNP-PEI, as demonstrated not only by a higher cell viability, but also by a markedly reduced oxidative stress and cell membrane damage. These findings highlight the importance of assay selection and of dissection of different cellular responses in in-vitro characterisation of nanostructures.

The complexation between novel comb shaped amphiphilic polyallylamine and insulin : towards oral insulin delivery

Novel amphiphilic polyallylamine (PAA) were previously synthesised by randomly grafting palmitoyl pendant groups and subsequent quaternising with methyl iodide. The ability of these self-assembled polymers to spontaneously form nano-complexes with insulin in pH 7.4 Tris buffer was evaluated by transmittance study, hydrodynamic size and zeta potential measurements. The transmission electron microscopy images showed that non-quaternised polymer complexes appeared to form vesicular structures at low polymer:insulin concentrations. However, at higher concentrations they formed solid dense nanoparticles. The presence of quaternary ammonium moieties resulted in insulin complexing on the surface of aggregates. All polymers exhibited high insulin complexation efficiency between 78 and 93%. Incubation with trypsin, alpha-chymotrypsin and pepsin demonstrated that most polymers were able to protect insulin against enzymatic degradation by trypsin and pepsin. Quaternised polymers appeared to have better protective effect against trypsinisation, possibly due to stronger electrostatic interaction with insulin. Interestingly, non-quaternised polymers significantly enhanced insulin degradation by alpha-chymotrypsin. All polymers were less cytotoxic than PAA, with the quaternised polymers exhibiting up to 15-fold improvement in the IC(50) value. Based on these results, quaternised palmitoyl graft polyallylamine polymers showed promising potential as oral delivery systems for insulin.

The influence of polymer architecture on the protective effect of novel comb shaped amphiphilic poly(allylamine) against in vitro enzymatic degradation of insulin-Towards oral insulin delivery

Nanocomplexes formed between amphiphilic poly(allylamine) (PAA) and insulin were prepared, characterised and the impact of polymer architecture on the protection of insulin against three enzymes was investigated. PAA previously modified with either cetyl or cholesteryl pendant groups at two levels of hydrophobic grafting and its quaternised derivatives were used to produce polymer-insulin nanocomplexes. Transmittance study, differential scanning calorimetry, hydrodynamic size and zeta potential measurement were conducted and the morphology of the complexes were visualised using transmission electron microscopy. All polymers were found to have an optimal polymer to insulin ratio of 0.4:1 mg mL(-1) with particle size ranging from 88 to 154 nm. Polymer architecture has an impact on the morphology of the complexes produced but has little influence on the complexation efficiency (CE). Almost all polymers were unable to produce complexes with a CE of above 50%. Most polymers demonstrated an ability to reduce insulin degradation by trypsin while the polymer architecture plays a pivotal role against alpha-chymotrypsin and pepsin degradation. Quaternised cholesteryl polymers were able to significantly limit insulin degradation by alpha-chymotrypsin while cetyl polymers were particularly effective against pepsin degradation. These results indicated that a combination of polymers might be required to enhance protection against all three proteolytic enzymes for efficacious oral delivery of insulin.

In vitro and in vivo characterisation of a novel peptide delivery system: Amphiphilic polyelectrolyte―salmon calcitonin nanocomplexes

The cationic peptide, salmon calcitonin (sCT) was complexed with the cationic amphiphilic polyelectrolyte, poly(allyl)amine, grafted with palmitoyl and quaternary ammonium moieties at pH 5.0 and 7.4 to yield particulates (sCT-QPa). The complexes were approximately 200 nm in diameter, had zeta potentials ranging from +20 to +50 mV, and had narrow polydispersity indices (PDIs). Differential scanning calorimetry revealed the presence of an interaction between sCT and QPa in the complexes. Electron microscopy confirmed the zeta-size data and revealed a vesicular bilayer structure with an aqueous core. Tyrosine- and Nile red fluorescence indicated that the complexes retained gross physical stability for up to 7 days, but that the pH 5.0 complexes were more stable. The complexes were more resistant to peptidases, serum and liver homogenates compared to free sCT. In vitro bioactivity was measured by cAMP production in T47D cells and the complexes had EC50 values in the nM range. While free sCT was unable to generate cAMP following storage for 7 days, the complexes retained approximately 33% activity. When the complexes were injected intravenously to rats, free and complexed sCT (pH 5.0 and 7.4) but not QPa reduced serum calcium over 120 min. Free and complexed sCT but not QPa also reduced serum calcium over 240 min following intra-jejunal administration. In conclusion, sCT-QPa nanocomplexes that have been synthesised are stable, bioactive and resistant to a range of peptidases. These enhanced features suggest that they may have the potential for improved efficacy when formulated for injected and oral delivery.

Nano self-assemblies based on cholate grafted poly-L-lysine enhanced the solubility of sterol-like drugs

The physicochemical compatibility between amphiphilic polymers and hydrophobic drugs has been recognized as an important issue for improving the drug solubilisation in polymeric micelle formulations. In this work, poly-L-lysine (PLL) grafted by cholate pendants as the only hydrophobic moiety were synthesized in order to facilitate the solubilisation of sterol drugs. Results showed that micelles formed by cholate grafted PLL encapsulated significantly higher level of prednisolone and estradiol than palmitoylated PLL micelles, whereas the solubilisation capacity of non-sterol drug (griseofulvin) is inefficient for both polymers. This suggests that higher drug-polymer incorporation can be achieved by the inclusion of hydrophobic moieties with similar architecture as the drugs, i.e. ‘drug-like’ functional groups, which will be useful for the future design of colloidal systems for the encapsulation of specific drug.

Poly(allylamine) magnetomicelles for image guided drug delivery.

This work was funded by the Institute of Science and Technology in Medicine and the School of Pharmacy, Keele University. The NMR and ICP-OES analysis was carried out in the Lennard-Jones Laboratories in the School of Physical and Geographical Sciences. The magnetometer used in this research was obtained through the Science City Advanced Materials project: Creating and Characterizing Next Generation Advanced Materials project, with support from Advantage West Midlands (AWM) and part funded by the European Regional Development Fund (ERDF).

Chemically Modified Polyelectrolytes for Intestinal Peptide and Protein Delivery

Publisher Summary The aim of this chapter is to look at the use of amphiphilic polyelectrolytes and chemically modified chitosan for the gastrointestinal delivery of proteins and peptides, and to discuss how the chemical modifications have impacted on the two important challenges in oral protein/peptide delivery, i.e., protection against intestinal enzymatic degradation and promotion of protein/peptide transport across the gastrointestinal mucosa. Chemically modified polyelectrolytes have shown to form nanocomplexes spontaneously with a range of peptides under mild conditions. Most studies showed that they have the ability to facilitate both paracellular and transcellular transport of peptides in vitro by either opening tight junctions via electrostatic or hydrophobic interactions with cells, or internalization via endocytosis. The additional mucoadhesive properties of chitosan that has been modified with quaternary ammonium moieties and/or thiolated groups increase the permeation of the proteins/peptides across Caco-2 cell monolayers in vitro, which have translated to promising oral peptide delivery systems. However, enzyme inhibitors are still needed to obtain a better pharmacokinetic profile. With regard to the protection against enzymatic degradation, most of the polyelectrolytes have only a limited ability to protect against enzymatic breakdown. They can, however, offer some form of electrostatic or hydrophilic shielding of some target sites. The exceptions to this are polystyrene based amphiphilic polymers which were able to entrap and reduce the activity of a range of enzymes, and thiolated chitosan, where the presence of a negative charge allows the chelation of some metal ions, which are essential for enzymatic activity.

Hydrophobic Drug Solubilisation

Poorly soluble drugs have always been a challenge to the pharmaceutical industry. Today, it has been estimated that 40 % of new chemical entities fail in development because of adverse physical properties such as poor aqueous solubility. In this chapter, the authors look at the principals of drug solubility and dissolution and evaluate the use of drug nano-crystals and nano-size delivery systems such as liposomes, polymeric micelles and solid lipid nanoparticles in enhancing drug solubility. Additional benefits such as achieving site-specific delivery and facilitating cellular uptake are also discussed. The key advantages that nanotechnologies are able to offer and the challenges in the exploitation of these technologies are outlined with appropriate examples. Clinical experience is included to demonstrate the successful approach in using nanotechnologies for hydrophobic drug solubilisation.