ENGEMS was a Network set up in late 1997 by the European Commission under its Training and Mobility of Researchers (TMR) Programme (1994-1998). * It ran until 30 October 2001. The principal scientists involved remain active in the area. It brought together five leading research groups from three European countries in a coordinated interdisciplinary programme, firmly based in Chemistry, designed to produce new vehicles for drug delivery. A key focus was gene therapy - a high-profile special case where DNA is the drug concerned.

Objectives
The primary objective was the creation of novel Gemini surfactants as synthetic vectors for the safe delivery of functional genes into cells and then into the nucleus. This involved the design, synthesis, characterisation and biological testing of many new compounds, and close collaboration between organic, physical and biological chemists. Over the lifetime of the Network we trained a group of a dozen committed young scientists to think and work across the boundaries of conventional disciplines, while making their own individual contribution to this exciting venture.

Gene Therapy
Successful gene therapy - the replacement or addition of a gene - depends crucially on the development of effective techniques, especially for the safe introduction of the selected gene into living cells. Gene transfer "vectors" in common use are mostly based on viruses. The procedures are complicated and expensive, the capacity of the carriers is limited, and there is a danger of residual infectivity. We believe that synthetic vectors, though currently less efficient by orders of magnitude, can provide a viable alternative: and Gemini surfactants show particular promise as potential vehicles for the transport of bioactive molecules. The Network has already produced a large number of new compounds that are efficient transfection agents, and more are currently under test.

Gemini Surfactants
First reported some 25 years ago,1 this new type of dimeric surfactant molecules is made up of two long hydrocarbon chains and two ionic groups linked by a "spacer". The name 'Gemini' was coined by Menger.2 The Figure shows two types of Gemini surfactant: the spacer can be attached directly 1 to the (identical) ionic groups, each of which is in turn bonded to an identical hydrocarbon chain; alternatively (2) two identical amphiphiles are linked midway via the ionic headgroups and the hydrocarbon tails. The spacer can vary in length, hydrophobicity and flexibility.

The Gemini structure can give surface and bulk properties different from those of conventional single-chain monomeric surfactants with the same number of carbon atoms per polar 'head' group. Monomeric surfactants generally form spherical micelles, but Geminis typically form thread-like aggregates. Gemini surfactants are generally active at lower concentrations,and have excellent foaming and wetting properties. Often neutral hydrophobic molecules are more soluble in aqueous solutions of Gemini surfactants, compared to solutions containing their monomeric surfactant counterparts. Varying the hydrophobic/hydrophilic nature of the linker can have dramatic effects on their physicochemical properties, presumably due to changes in the organisation of micellar structures.

Surfactants as Gene Transfection Agents
Over the last ten years liposomes formed from cationic lipids, in combination with a 'helper' (neutral) lipid such as 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been used extensively for the delivery of DNA into mammalian cells in culture. Several cationic lipids are now available commercially, and are used routinely in genetic technologies.

This is very much interdisciplinary science, needing serious input from chemists of several sorts - structural, synthetic, bioorganic - biochemists and chemical geneticists. All our members worked to some extent across these boundaries, with each other and with groups in adjacent areas.

Design. All groups, and all participants were involved in the design of new systems, each contributing their special expertise. Synthesis was the primary responsibility of the Nijmegen and Groningen groups, who have long experience and special expertise in the design and synthesis of molecules with interesting aggregation properties,3,4 and of the Cambridge group, with experience in the synthesis of ionic carbohydrate-based surfactants. The design and efficient synthesis of new compounds was crucial to the success of the project: eventually almost 250 new Gemini surfactants were produced: remarkably the majority of these showed good to very good transfection activity.

Physicochemical investigations of surfactant behaviour were the primary responsibility of the Lund and Groningen groups, with some special techniques applied by Nijmegen and SB. The Lund group worked with Geminis supplied by the synthetic groups, using both classical techniques and modern methods, including cryo-transmission electron-microscopy, small angle X-ray scattering techniques, NMR relaxation measurements and NMR self diffusion techniques for the study of vesicle size. The Groningen group applied their special expertise and experience to the study of higher order aggregation properties of novel Geminis, particularly vesicle formation. Structures of complexes formed between the Geminis and DNA (plasmids), (m)RNA) or drugs were studied under the electron microscope and by SAXS (small angle X-ray scattering) techniques.

Biological testing. Drug permeability and intensive gene transfection studies were carried out by the SB Group, using the Geminis synthesised in Nijmegen, Cambridge and Groningen. The transfection efficiency of novel Gemini surfactants was compared routinely to commercially available agents such as Life Technologies'.Lipofectamine^TM and Lipofectamine Plus^TM.

Principal Scientists
1. University of Cambridge (Prof. A. J. Kirby) GB ajk1@cam.ac.uk
2. SmithKline Beecham plc (Dr. P. Camilleri) GB Patrick_Camilleri@sbphrd.com
3. University of Groningen (Prof. J. B. F. N. Engberts) NL J.B.F.N.Engberts@chem.rug.nl
4. University of Nijmegen (Prof. R. J., M. Nolte) NL nolte@sci.kun.nl
5. University of Lund (Dr. O. Söderman) SE Olle.Soderman@fkem1.lu.se

Publications
Include 8 Patents, plus the following papers in the open literature.

Cambridge-SB-Lund
The Synthesis and Aggregation Properties of a Novel Anionic Gemini Surfactant. K. Jennings, I. Marshall, H. Birrell, A. Edwards, N. Haskins, O. Södermann, A. J. Kirby and P. Camilleri, J. Chem. Soc., Chem. Commun., 1998, 1951-2.

Cambridge-SB
A novel class of cationic Gemini surfactants showing efficient in vitro gene transfection properties. P. Camilleri, A., Kremer, K. H. Jennings, O. Jenkins, I. Marshall, C. McGregor, W. Neville, S. Q. Rice, R. J. Smith, M. J. Wilkinson and A. J. Kirby, J. Chem. Soc., Chem. Comm., 2000, 1253-1254.
Rational Approaches to the Design of Cationic Gemini Surfactants for Gene Delivery. C. McGregor, C. Perrin, M. Monck, P. Camilleri and A. J. Kirby, J. Am. Chem. Soc. 2001, 123, 6215-6220.
Novel Spermine-Based Cationic Gemini Surfactants for Gene Delivery. G. Ronsin, C. Perrin, P. Guedat, A. Kremer, P. Camilleri and A. J. Kirby, J. C. S. Chem. Comm. 2001, 2234-5.
A Preliminary Study on the Synthesis and in vitro Activity of New Bile Acid-Based Gemini Surfactants. G. Ronsin,, P. Camilleri and A. J. Kirby, J. Org. Chem., in the press.

Groningen­SB
Sugar-based tertiary amino gemini surfactants with a vesicle-to-micelle transition in the endosomal pH range mediate efficient transfection in vitro. M. L. Fielden, C. Perrin, A. Kremer, M. Bergsma, M. C. Stuart, P. Camilleri and J. B. F. N. Engberts, Eur. J. Biochem., 2001, 268, 1269-1279

Nijmegen- SB
Cationic Gemini Surfactants based on Tartaric Acid: Synthesis, Aggregation, Monolayer Behaviour, and Interaction with DNA. P. J. J. A. Buynsters, C. L. García Rodríguez, E. L. Willighagen, N. A. J. M. Sommerdijk, J. M.;A. Kremer, P. Camilleri, M. C. Feiters, R. J. M.Nolte and B. Zwanenburg,, Eur. J. Org. Chem., in the press.

For more information contact the Coordinator, or the Principal Scientist of the group you are interested in.

References
1. Menger, F. M. and Littau, C. A. J. Am. Chem. Soc., 1991, 113, 1451-2; ibid., 1993, 115, 10083-10090.
2. Deinega, Yu. F., Ullberg, Z. R., Marochko, L. G., Rudi, V. P. and Denisenko, V. P. Kolloidn. Zh. 1974, 36, 649-53. Chem. Abs., 1975, 82, 45594f.
3. Pestman, J. M., Terpstra, K. R., Stuart, M. C. A. v. Doren, H. A., Brisson, A., Kellogg, R. M. and Engberts, J. B. F. N. 1997. Langmuir. 13:6857-6860.
4. Sommerdijk, N. A. J. M., Hoeks, T. H. L., Synak, M., Feiters, M. C., Nolte, R. J. M. and Zwanenburg, B. 1997. J. Am. Chem. Soc., 1997, 119, 4338-4344