Poly(amino acid)s - next-generation coatings for long-circulating liposomes

Incorporation of a lipid conjugate of a water-soluble polymer into liposomes can reduce the adhesion of plasma proteins that would otherwise cause rapid recognition and removal of the liposomes by phagocytes. Such polymer-coated liposomes show prolonged circulation property and passive targeting to pathological tissues such as tumors or inflamed sites. This thesis describes the evaluation of two poly(amino acid) (PAA)-lipid conjugates for the coating of long-circulating liposomes as an alternative to poly(ethylene glycol)-phosphatidylethanolamine (PEG-PE), which is the polymer-conjugate of choice for the preparation of long-circulating liposomes. Two conjugates based on poly(hydroxyethyl L-glutamine) (PHEG) and poly(hydroxyethyl L-asparagine) (PHEA) were investigated. A 1H-NMR method was developed to quantify grafted poly(amino acid)-conjugates on the liposome surface. Pharmacokinetic properties of long-circulating liposomes are dependent on the grafting density of the coating polymer on the liposome surface, and therefore quantification methods are highly needed. The pharmacokinetic behavior of PAA-coated liposomes was studied in rats, when liposome characteristics were varied. The influence of PAA grafting density, bilayer fluidity, inclusion of charged lipids and particle size was evaluated. It was demonstrated that the prolonged circulation kinetics of PAA-liposomes can be maintained upon variation of the liposomes characteristics and the lipid dose given. For PEG-liposomes, rapid clearance from the circulation has been reported when administered at low lipid dose and upon repeated administration. PAA-liposomes, however, showed unaltered pharmacokinetic behavior in rats at low lipid doses, which are applied for e.g. diagnostic imaging of tumors and sites of inflammation. Furthermore, PHEA-liposomes show superior circulation kinetics when compared to PEG-liposomes upon repeated administration. Initial results on the activation of the complement system by PAA-liposomes in vitro and in a porcine model of complement-mediated cardiopulmonary distress show that, like other types of liposomes, also PHEA- and PHEG-coated liposomes are activators of the complement system. PHEG was proven to be degradable by the model proteases papain, pronase E and the lysosomal cathepsin B when it is in solution and also when it is grafted to a liposome surface. Such enzymatic degradability reduces the risk of intracellular polymer accumulation and possible side effects upon repeated exposure. Furthermore, removal of the coating from the liposome surface (shedding) by enzymatic degradation can be used to facilitate target cell interaction of the nanoparticle and/or drug release. It was investigated whether liposomes composed of the fusogenic, non-bilayer-forming lipid DOPE and the sheddable PHEG-lipid conjugate can be used as a passively targeted liposome system, which efficiently releases the entrapped contents upon arrival at the target site. Enzymatic degradation of PHEG led to destabilization of the liposomes and concomitant release of an encapsulated fluorescent dye. One can envisage triggered drug release at the target site, either extracellularly by the action of proteases in the extracellular space of tumors and sites of inflammation or intracellularly after PHEG-degradation in the endo/lysosomal compartment and possibly endosomal escape. In conclusion, due to their favorable properties as described in this thesis, poly(amino acid)-conjugates represent next-generation coatings for the preparation of long-circulating liposomes for the systemic delivery of therapeutic agents.