Michael R. Jorgensen

The stereoselectivity of both the Wittig and the Horner-Wadsworth-Emmons reactions allows for the synthesis of ortho­gonally protected fumarates and maleates, respectively, from α-keto esters. This methodology has been shown to be useful in the synthesis of a derivative of the pH sensitive cis-aconitic linker, important for prodrug approaches in drug delivery. We have incorporated this linker into a cholesterol-based PEGylated lipid, for its potential use in liposomal drug and gene delivery. The synthesized lipids have also been shown to be pH degradable using HPLC analyses.

Positively-charged gene delivery agents, such as cationic liposomes, typically prepared by mixing a cationic lipid and a neutral lipid in a 1 : 1 molar ratio, exhibit a fundamental flaw: on the one hand, the charge encourages cell uptake; on the other hand, the charge leads to aggregation in vivo with anionic serum components. We herein report a more phase-stable analogue of the zwitterionic and fusogenic lipid DOPE that allows for the reduction of the cationic lipid component of the liposome from 50 to 9 mol% with almost no apparent loss in transfection activity. This reduction in charge may induce important in vivo stability whilst still imparting high cell uptake and transgene expression.

One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool‐kits of chemical components. First‐generation LMD systems were prepared from DC‐Chol/DOPE cationic liposomes (DC‐Chol=3β‐[N‐(N′,N′‐dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl‐L‐α‐phosphatidylethanolamine), μ peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide‐targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell‐surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target α₉β₁‐integrin proteins predominant on upper‐airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. “Targeted” LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide‐targeted, synthetic, nonviral gene delivery to be realised.

A simple solid-phase assisted strategy for the N-1 functionalization of azamacrocycles is described. Compounds such as cyclen, cyclam and piperazine can be selectively modified by ­temporary attachment to solid-phase resins providing an efficient and clean method to prepare biomedically interesting moieteies.