In vitro and in vivo evaluation of a non-carbohydrate targeting platform for lysosomal proteins.
ABSTRACT Lysosomal storage diseases arise from a genetic loss-of-function defect in enzymes mediating key catabolic steps resulting in accumulation of substrate within the lysosome. Treatment of several of these disorders has been achieved by enzyme replacement therapy (ERT), in which a recombinant version of the defective enzyme is expressed in vitro and administered by infusion. However, in many cases the biodistribution of the administered protein does not match that of the accumulated substrate due to the glycosylation-mediated clearance of the enzymes from circulation, resulting in poor or absent substrate clearance from some tissues. To overcome this limitation, we have evaluated several peptide-based targeting motifs to redirect recombinant human alpha-galactosidase (rhalphaGal) to specific receptors. A reversible thiol-based PEGylation chemistry was developed to achieve multivalent peptide display with lysosomal release. In vitro, cell uptake was peptide dependent and independent of the normal mannose-6-phosphate receptor mediated pathway. Surprisingly, despite increased plasma half-life and decreased liver uptake, none of the peptide conjugates showed significantly altered biodistribution in alphaGal-knockout mice. This suggests that these peptide-based targeting motifs are unlikely to provide substantial therapeutic benefit likely due to the complexity of factors affecting PK and biodistribution.
Clinical Therapeutics 01/2009; 31. DOI:10.1016/S0149-2918(09)80013-1 · 2.59 Impact Factor
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ABSTRACT: Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes.Journal of Membrane Biology 04/2014; 247(9-10). DOI:10.1007/s00232-014-9655-y · 2.17 Impact Factor
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ABSTRACT: Dendrimers have emerged as promising carriers for the delivery of a wide variety of pay-loads including therapeutic drugs, imaging agents and nucleic acid materials into biological systems. The current work aimed to develop a novel mitochondria-targeted generation 5 poly(amidoamine) (PAMAM) dendrimer (G(5)-D). To achieve this goal, a known mitochondriotropic ligand triphenylphosphonium (TPP) was conjugated on the surface of the dendrimer. A fraction of the cationic surface charge of G(5)-D was neutralized by partial acetylation of the primary amine groups. Next, the mitochondria-targeted dendrimer was synthesized via the acid-amine-coupling conjugation reaction between the acid group of (3-carboxypropyl)triphenyl-phosphonium bromide and the primary amines of the acetylated dendrimer (G(5)-D-Ac). These dendrimers were fluorescently labeled with fluorescein isothiocyanate (FITC) to quantify cell association by flow cytometry and for visualization under confocal laser scanning microscopy to assess the mitochondrial targeting in vitro. The newly developed TPP-anchored dendrimer (G(5)-D-Ac-TPP) was efficiently taken up by the cells and demonstrated good mitochondrial targeting. In vitro cytotoxicity experiments carried out on normal mouse fibroblast cells (NIH-3T3) had greater cell viability in the presence of the G(5)-D-Ac-TPP compared to the parent unmodified G(5)-D. This mitochondria-targeted dendrimer-based nanocarrier could be useful for imaging as well as for selective delivery of bio-actives to the mitochondria for the treatment of diseases associated with mitochondrial dysfunction.Biomaterials 03/2012; 33(18):4773-82. DOI:10.1016/j.biomaterials.2012.03.032 · 8.31 Impact Factor