A combinatorial approach for targeted delivery using small molecules and reversible masking to bypass nonspecific uptake in vivo.

We have developed a multi-disciplinary approach combining molecular biology, delivery technology, combinatorial chemistry and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding nonspecific uptake in vivo. We initially used a well-characterized model targeting the asialolglycoprotein receptor in the liver. Using our bilamellar invaginated vesicle (BIV) liposomal delivery system with reversible masking, we increased expression in the liver by 76-fold, nearly equaling expression in first-pass organs using non-targeted complexes, with no expression in other organs. The same technology was then applied to efficiently target delivery to a human tumor microenvironment model. We achieved efficient, targeted delivery by attachment of specific targeting ligands to the surface of our BIV complexes in conjunction with reversible masking to bypass nonspecific tissues and organs. We identified ligands that target a human tumor microenvironment created in vitro by co-culturing primary human endothelial cells with human lung or pancreatic cancer cells. The model was confirmed by increased expression of tumor endothelial phenotypes including CD31 and vascular endothelial growth factor-A, and prolonged survival of endothelial capillary-like structures. The co-cultures were used for high-throughput screening of a specialized small molecule library to identify ligands specific for human tumor-associated endothelial cells in vitro. We identified small molecules that enhanced the transfection efficiency of tumor-associated endothelial cells, but not normal human endothelial cells or cancer cells. Intravenous (i.v.) injection of our targeted, reversibly masked complexes into mice, bearing human pancreatic tumor and endothelial cells, specifically increased transfection to this tumor microenvironment approximately 200-fold. Efficacy studies using our optimized targeted delivery of a plasmid encoding thrombospondin-1 eliminated tumors completely after five i.v. injections administered once every week.

Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides.

This study details a series of conditions that may be applied to ensure 'safe' incorporation of cysteine with minimal racemization during automated or manual solid-phase peptide synthesis. Earlier studies from our laboratories [Han et al. (1997) J. Org. Chem. 62, 4307-4312] showed that several common coupling methods, including those exploiting in situ activating agents such as N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), N-[1H-benzotriazol-1-yl)-(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), and (benzotriazol-1-yl-N-oxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) [all in the presence of N-methylmorpholine (NMM) or N,N-diisopropylethylamine (DIEA) as a tertiary amine base], give rise to unacceptable levels (i.e. 5-33%) of cysteine racemization. As demonstrated on the tripeptide model H-Gly-Cys-Phe-NH(2), and on the nonapeptide dihydrooxytocin, the following methods are recommended: O-pentafluorophenyl (O-Pfp) ester in DMF; O-Pfp ester/1-hydroxybenzotriazole (HOBt) in DMF; N,N'-diisopropylcarbodiimide (DIPCDI)/HOBt in DMF; HBTU/HOBt/2,4,6-trimethylpyridine (TMP) in DMF (preactivation time 3.5-7.0 min in all of these cases); and HBTU/HOBt/TMP in CH(2)Cl(2)/DMF (1:1) with no preactivation. In fact, several of the aforementioned methods are now used routinely in our laboratory during the automated synthesis of analogs of the 58-residue protein bovine pancreatic trypsin inhibitor (BPTI). In addition, several highly hindered bases such as 2,6-dimethylpyridine (lutidine), 2,3,5,6-tetramethylpyridine (TEMP), octahydroacridine (OHA), and 2,6-di-tert-butyl-4-(dimethylamino)pyridine (DB[DMAP]) may be used in place of the usual DIEA or NMM to minimize cysteine racemization even with the in situ coupling protocols.

Increase in fluorescence upon the hydrolysis of tyrosine peptides: application to proteinase assays.

The intrinsic fluorescence of tyrosine increases by a factor of approximately two when the carboxy group is liberated from a peptide bond by hydrolysis. The increase in fluorescence provides a novel way to monitor the hydrolysis of native tyrosine peptides that contain only proteinogenic amino acids. Thus, for example, the hydrolysis by HIV-1 proteinase of a heptapeptide viral protein fragment gag129-135, Ser-Gln-Asn-Tyr-Pro-Ile-Val, was followed continuously at excitation and emission wavelengths 275 and 305 nm. The fluorescence increase is magnified by at least a factor of a thousand when a resonance energy quencher, such as paranitrophenylalanine, is in the vicinity. For example, the peptide Lys-Ala-Arg-Val-Tyr-Phe(p-NO2)-Glu-Ala-Nle-NH2 [Richards et al. (1990) J. Biol. Chem. 265, 7733], widely used for spectrophotometric assays of the HIV-1 proteinase, yields a substrate:product fluorescence ratio greater than 1:1000. Tyrosine-containing substrates of pepsin and trypsin showed similar behavior. The detection limit of the present method is at least one order of magnitude lower than absorbance assays of p-nitrophenylalanine peptides.