The development of recombinant DNA technology and biotechnology has allowed for the industrial-scale production of proteins, which has elevated their use to a new level. Indeed, due to their selectivity and therapeutic efficacy, protein drugs have become the first choice of treatment for numerous diseases. The route of administration of protein drugs into the body is mainly limited to parenteral delivery, as there is little or no protein absorption from the gastrointestinal tract due to their structural properties (i.e., molecular size, hydrophilicity, and physical, chemical and proteolytic instability). On this basis, and from the patient perspective, the development and use of advanced delivery systems for oral administration of protein drugs have become increasingly important. Due to their specific properties, nanoparticles (NPs) offer a promising way forward for the delivery of protein drugs. As an advanced delivery system, NPs offer a promising way forward to improve the stability and bioavailability of protein drugs. The mechanism of protein drug permeability at the membrane level is not yet fully understood, and opinions in the field differ in terms of the importance of the carrier materials on drug loading, and consequently on the properties and permeability of NPs therefore this was also the aim of the research carried out in the framework of this Doctoral thesis. The Introduction of this thesis presents the basic characteristics, design, preparation and in-vitro and in-vivo evaluation of various protein drugs and NPs. This is followed by the study of human recombinant erythropoietin (EPO), its synthesised modified conjugate with caproic acid (mC6EPO), recombinant granulocyte colony stimulating factor (GCSF), and ovalbumin. The selected biocompatible polyelectrolytes used in this study were: chitosan (CS); trimethylchitosan (TMC); a copolymer of polylactic and aspartic acid; a newly synthesised and evaluated chemically modified chitosan -graft-poly(L-glutamate) (CS-g-PGlu); alginate; and chondroitin sulphate. The crosslinker used was tripolyphosphate (TPP). The NPs were prepared by complexation of the protein drugs with the polyelectrolytes in an aqueous medium, which allowed spontaneous formation of NPs under mild preparation conditions. The optimised NP preparation process allowed formation of NPs with hydrodynamic diameters of 200 nm to 300 nm and a polydispersity index <0.3, accompanied by preserved biological activity of the loaded protein drugs. In the preformulation study, the surface properties of free EPO and NP-loaded EPO were investigated, to monitor the relative hydrophobicity for correlation with membrane permeability. The surface properties of EPO were determined by varying the fluorescence intensity of the 4,4’-bis-1-anilinonaphtalene-8-sulfonate (bis-ANS) dye. This demonstrated that the surface hydrophobicity of the protein drug EPO was changed by varying the pH of its environment and after its complexation with the polyelectrolyte(s). EPO-loaded CS NPs showed the highest surface hydrophobicity, followed by free EPO, and EPO-loaded CS-TMC NPs. The highest permeability coefficient of EPO for permeation through a monolayer of Caco-2 epithelial cells was seen for the CS NPs, which also had the highest hydrophobicity. We show that polyelectrolyte complexation of hydrophilic proteins with pH-sensitive polyelectrolytes reduces the protein charge density, which thus increases the protein surface hydrophobicity. Comparable behaviour was seen for CS/ mC6EPO/ TPP NPs, CS/ ovalbumin/ poly(lactic-co-aspartic acid) NPs, and alginate/ ovalbumin/ CS NPs. Newly synthesised CS-g-PGlu copolymers where CS was modified with 5-, 10- or 15-molecule-long chains of glutamic acid were used to prepare NPs with GCSF, and were coated with TMC. We show that the length of the attached glutamic acid chains significantly increased the capacity of the polyelectrolyte for complexation with GCSF. We show that for the interaction of protein drugs with polyelectrolytes, the charge density and the structural accessibility of the functional groups on the polyelectrolytes are important. Loading of GCSF into these NPs of up to 45% was obtained, and the NPs were thermally stable from 25 °C to 39 °C, and showed longer pH-dependent stability below the isoelectric point of GCSF (pI, 6.1).
The modified EPO conjugate that was prepared, mC6EPO, increased its loading into the NPs, partially protected mC6EPO against acid and enzymatic degradation, and increased its permeability through the Caco-2 cell monolayers, all compared to free EPO. The addition of the penentration enhancer CYMAL into NPs improved the permeability of both proteins, (i.e., EPO, mC6EPO) further, where the enhancement ratio of Papp of the NPs relative to that of free EPO in the some medium were 1186 for CS/ CYMAL-EPO/ TPP and 927 for CS/ CYMAL-mC6EPO/ TPP). We can conclude that these NPs protected EPO against enzymatic degradation and preserved its biological activity, which was confirmed by the pharmacological responses of EPO and mC6EPO in tests on rats after oral administration. Storage of NPs with sensitive protein drugs over long periods of time presents a challenge for researchers. It has been demonstrated that the selection of suitable cryo/lyoprotectants (those optimal were trehalose and mannitol) allows the production of NPs with characteristics that are comparable to freshly prepared dispersions of NPs. As a first, we introduced the spraying of freshly prepared NP dispersions into liquid nitrogen before lyophilisation (known as spray-freeze drying) to prepare dry bulk powders of NPs with loaded protein drugs with preserved biological activities. The results in the framework of this Doctoral thesis demonstrate the crucial importance of polyelectrolyte selection for the preparation of NPs for selected protein drugs. Mild preparation conditions, NP size, high protein drug entrapment efficiency into complexes, altered surface polarity of NPs, and pH-dependent release all provide promising opportunities for development of an appropriate delivery system for per-oral administration of protein drugs. Such polyelectrolyte NPs have positive influences in the formation of protein drug delivery systems, compared to conventional dosage forms and the limitations of known technological approaches. The aim of the research carried out in the framework of this Doctoral thesis was to integrate our knowledge about the mechanisms of protein drug loading into polymeric NPs, the influence of such polymeric NPs on protein permeability across biological membranes, and the conversion of liquid NP dispersions into a stable and dry state. Together, these aspects constitute thebasis for more effective design of therapeutic proteins in NPs.
|
