Self-assembling responsive protein-polymer conjugates as platform therapeutics

The project will develop the concept of employing Novozymes engineered proteins coupled to polymers, to form conjugates that will self-assemble into supramolecular micellar or vesicular architectures. These materials will contain amphiphilic block co-polymers that self-assemble under certain conditions, but which can disassemble under other conditions, for example by changes in temperature or pH. As a consequence it should be possible to encapsulate drug/biotherapeutic compounds in the interior of the self-assembled conjugates, but also to trigger the release of the therapeutic when conditions disfavour self-assembly. The proposal aims to combine the advantageous properties of both engineered proteins (serum stability, target specificity, biodegradability) with synthetic or modified natural polymers (tailored physical properties, tuneable functionalities) to generate a versatile new class of bioactive materials. The polymer components, will be based in the first instance on the well-known Pluronic series of amphiphilic co-polymers, to enable temperature-triggered assembly/disassembly, or hyaluronic acid, to enable ionic and pH-triggered assembly/disassembly. The other, biomolecular, component of the conjugate will consist of molecules available within Novozyme's product portfolio (including recombinant albumin (recombumin), albufuse, hyaluronic acid and recombinant transferrin (including modified versions for subsequent conjugation)). The latter components will be selected dependent on the functionality desired within the self-assembled complex. For example, transferrin could be utilized for improved cellular targeting and uptake, whereas albufuse would be employed to promote serum stability and half-life. Combinations of these conjugates with other protein-polymer hybrids and/or block co-polymers will thus enable a family of self-assembling materials to be developed, that will display varying degrees of transferrin, or transferrin-type functionality, as well as controllable protein activity dependent on self-assembly state. Most importantly, the controllable assembly/disassembly of the conjugates will allow the encapsulation and release of therapeutic agents/actives in the micellar core or vesicular interiors of the conjugates. As such, the conjugates represent a new materials platform that may be applicable to many areas of therapeutic, flavour and fragrance release. The student will prepare polymer-protein conjugates using techniques well-established in the School of Pharmacy (Chem. Commun. 2008, 4433 - 4435, Int. J. Pharmaceutics 2007, 340, 20-28, J. Am. Chem. Soc. 2004, 126, 13208-13209). A key component of their studies will also be the biophysical characterization of the developed conjugates and in particular their self-assembly/disassembly behaviours. This will be achieved to the nanometre scale using the suite of instruments available within the School (including atomic force microscopy (AFM), Quartz-Crystal Microbalance (QCM-D), CPS-Disc Centrifuge and light light scattering (DLS)). Importantly these tools will also be employed to probe interactions with model cellular membranes (building on recently published studies (Molecular Biosystems 2008, 4, 741-745)), and also cellular interaction and uptake (using scanning ion condictance microscopy (SICM) and confocal microscopy). Encapsulation of actives (for example doxorubicin, nucleic acids) and their release from self-assembled bioconjugates will be evaluated by fluorescence and UV-Vis spectroscopies and/or gel-shift assays for biotherapeutics. To assess the efficacy of materials (e.g. drug encapsulation and release within cell models), the student will work closely with researchers within on-going drug-delivery projects within the School. Importantly the developed conjugates will be evaluated in comparison with conventional drug delivery systems (liposomal doxorubicin, Lipofectamine) as well as protein-nanoaggregate carriers (e.g. albumin-paclitaxel (Abraxane))