Award details

Bioresponsive polymer-peptide conjugates for enhanced intracellular delivery of macromolecules.

Reference	BB/D013038/1
Principal Investigator / Supervisor	Professor Arwyn Jones
Co-Investigators / Co-Supervisors	Dr Dirk Schmaljohann
Institution	Cardiff University
Department	Welsh School of Pharmacy
Funding type	Research
Value (£)	245,304
Status	Completed
Type	Research Grant
Start date	20/09/2006
End date	19/06/2010
Duration	45 months

Abstract

This multidisciplinary application combines cell biology, peptide and polymer chemistry and aims to design new chemical entities to deliver therapeutic molecules across biological barriers. The basis for the application is new data regarding the cellular dynamics of protein transduction domains and the identification that novel peptides containing protein transduction and proapoptotic domains influence the integrity of cellular membranes. The main objective of this application is to design bioinert polymer-peptide molecules that are internalised by endocytosis, activated on endocytic pathways to mediate an intrinsic biological effect or to deliver a therapeutic entity. Initially, combinations of peptides, bioresponsive polymers and polymer-peptide conjugates will be designed, synthesised and assessed for their interaction with cells and abilities to promote apoptosis under defined conditions. Further experiments will determine whether co-internalisation of alternative polymer-peptide conjugates with fluorescently labelled macromolecules enhances their cytosolic delivery. These will then be supported by parallel functional assays employing reporter genes as translocation markers. Cellular dynamics of fluorescent conjugates of polymer-peptide molecules will be analysed by double labelling live-cell imaging confocal microscopy. This will be performed with molecules under study in cells co-incubated with endocytic probes or those expressing fluorescent Rab proteins that define specific endosomal locations. These experiments will pinpoint more exactly the locations on the endocytic pathway that is sensitive to membrane active peptides. Live cell confocal microscopy will be performed in house and via an existing collaboration with the Advanced Light Microscopy Facility at EMBL, Heidelberg. All these studies will be supported by FACS analysis, apoptosis and endosome translocation assays. An in vitro endosome-endosome fusion assay will be utilised to characterise the relationship between endosome integrity and specific peptides, polymers and polymer peptide conjugates. Peptides will be generated via an existing collaboration with Professor Shiroh Futaki, Kyoto University, and in-house using a Protein Technologies, Symphony Quartet peptide synthesiser. An orthogonal synthesis strategy will be adopted for side-specific conjugation of the peptide to a polymer. Polymers such as poly(ethylene glycol), poly(2-hydroxypropylmethacrylamide), poly(N-isopropylacrylamide) and their functional copolymers will be synthesised in the Centre for Polymer Therapeutics and characterised by NMR, GPC, mass spectrometry, HPLC, FPCL, FTIR, UV Spectroscopy. The base polymers will be functionalised for selected conjugation to the peptide via an acid labile linker. Molecular masses will be further determined in collaboration with the School of Chemistry at Cardiff University and if necessary at the EPSRC Mass Spectrometry Service Centre.

Summary

When the body is unable to deal with a disease such as cancer or serious infection there is a requirement for effective drugs to alleviate the symptoms and cure the condition. In this millennium we are faced with an understanding of the causes of diseases, the effects they have on the human body and, on paper at least, how we could intervene via administration of drugs. For effective therapy there is an absolute requirement to target only the 'diseased' cells of the body and then deliver the drug to the right location inside those cells. This may be to kill a cancer cell or to deliver an agent such as a gene to correct a defect thus benefiting the body as a whole. Cells are protected by a number of barriers called membranes, and these include the external surface plasma membrane and a number of internal membranes lining important structures such as the nucleus. For the good of the cell, this all ensures that the right material gains entry to the right compartment; nature has of course devised clever strategies for navigating through these barriers. For example the cells nutrients is partly supplied via a highly efficient processes where part of the plasma membrane is internalised into the cell and external material is then trapped inside the cell in small membrane bags called endosomes. The material is then directed (trafficked) to a specific part of the cell where it is required. A goal of this application is to use this system called endocytosis to deliver drugs to diseased cells. Bacteria and viruses are experts at cell entry. In many cases this involves attachment to the cell surface and then high-jacking the cells uptake mechanisms /endocytosis- to gain entry. Viruses have additional tricks that enable them to escape from endosomes before they are delivered to the hostile environment of the cell stomach called the lysosome. We, as researchers interested in drug delivery, wish to learn from these organisms and use virus tools and tricks to safely deliver therapeutic molecules into cells. The applicants in this study are experts in cell biology of endocytosis and chemistry. The chemist has expertise in designing and synthesising polymer compounds, very large molecules that have for several years been used as drug carriers. Together they will design specific molecules, that like viruses, enter cells by endocytosis and then escape from the endocytic pathway to mediate a therapeutic response. We will synthesise small proteins called peptides that are based on a HIV virus protein called TAT. This molecule is famous for entering cells by endocytosis and then escaping before reaching the lysosome. The principal applicant has extensive knowledge of the interactions and movements of these peptides in cells. These molecules will then be attached to another peptide to make a molecule that we have recently discovered is able to penetrate cell membranes and kill cells. The focus of the application however is to mask their toxicity by attaching to them to specific endosome-reactive polymers. Endosomes have unique environments and the polymers are designed to change their conformation in endosomal conditions thus exposing the 'dangerous' peptide to the membrane of the endosomes. Thus the polymer-peptide molecules will be inactive unless they are internalised by a cell via endocytosis. This will result in the release of the peptide-polymer before it reaches the lysosome. If lysosome-fragile therapeutic molecules such as genes for gene therapy are directly or indirectly attached to these polymer-peptides then they will also be released from the endosomes and will be able to reach their targets. A number of different polymer-peptide structures will be manufactured and tested for their effects on cells. Finally their ability to deliver genes and proteins into cells and out of endosomes will be analysed. The best will then go on to further research and development with the hope that they will some day be used in patients.

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	Pharmaceuticals, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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