ABSTRACT
Porous silicon (pSi) is being extensively studied as an emerging material for use in biomedical applications, including drug delivery, based on the biodegradability and versatile chemical and biophysical properties. We have recently introduced multistage nanoporous silicon microparticles (S1MP) designed as a cargo for nanocarrier drug delivery to enable the loaded therapeutics and diagnostics to sequentially overcome the biological barriers in order to reach their target. In this first report on biocompatibility of intravenously administered pSi structures, we examined the tolerability of negatively (-32.5±3.1mV) and positively (8.7±2.5mV) charged S1MP in acute single dose (10(7), 10(8), 5×10(8) S1MP/animal) and subchronic multiple dose (10(8) S1MP/animal/week for 4 weeks) administration schedules. Our data demonstrate that S1MP did not change plasma levels of renal (BUN and creatinine) and hepatic (LDH) biomarkers as well as 23 plasma cytokines. LDH plasma levels of 145.2±23.6, 115.4±29.1 vs. 127.0±10.4; and 155.8±38.4, 135.5±52.3 vs. 178.4±74.6 were detected in mice treated with 10(8) negatively charged S1MP, 10(8) positively charged S1MP vs. saline control in single and multiple dose schedules, respectively. The S1MPs did not alter LDH levels in liver and spleen, nor lead to infiltration of leukocytes into the liver, spleen, kidney, lung, brain, heart, and thyroid. Collectively, these data provide evidence of a safe intravenous administration of S1MPs as a drug delivery carrier.
Subject(s)
Drug Carriers/toxicity , Drug Delivery Systems , Microspheres , Silicon/toxicity , Animals , Cytokines/blood , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Female , Injections, Intravenous , L-Lactate Dehydrogenase/metabolism , Male , Mice , Nanopores , Silicon/administration & dosage , Silicon/chemistry , Toxicity TestsABSTRACT
Two crystal forms of the hypoxanthine phosphoribosyltransferase from Trypanosoma cruzi were grown and characterized. Proteolytic modification at the C-terminus of the recombinant enzyme yielded monoclinic crystals that diffract X-rays to higher resolution than the original, trigonal crystal form. Data from the monoclinic crystal form enabled determination of the crystal structure for the trypanosomal HPRT to 1.4 A resolution.
Subject(s)
Crystallization , Hypoxanthine Phosphoribosyltransferase/metabolism , Trypanosoma cruzi/enzymology , Amino Acid Sequence , Animals , Crystallography , Crystallography, X-Ray , Endopeptidases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Mass Spectrometry , Molecular Sequence Data , Mutation , Peptide Fragments/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
The hypoxanthine phosphoribosyltransferase (HPRT) from Trypanosoma cruzi, etiologic agent of Chagas' disease, was cocrystallized with the inosine analogue Formycin B (FmB) and the structure determined to 1.4 A resolution. This is the highest resolution structure yet reported for a phosphoribosyltransferase (PRT), and the asymmetric unit of the crystal contains a dimer of closely associated, nearly identical subunits. A conserved nonproline cis peptide in one active-site loop exposes the main-chain nitrogen to the enzyme active site, while the adjacent lysine side chain interacts with the other subunit of the dimer, thereby providing a possible mechanism for communication between the subunits and their active sites. The three-dimensional coordinates for the invariant Ser103-Tyr104 dipeptide are reported here for the first time. These are the only highly conserved residues in a second active-site loop, termed the long flexible loop, which is predicted to close over the active site of HPRTs to protect a labile transition state [Eads et al. (1994) Cell 78, 325-334]. This structure represents a major step forward in efforts to design/discover potent selective inhibitors of the HPRT of T. cruzi.
Subject(s)
Hypoxanthine Phosphoribosyltransferase/chemistry , Trypanosoma cruzi/enzymology , Animals , Binding Sites , Crystallization , Crystallography, X-Ray , Dimerization , Escherichia coli/enzymology , Escherichia coli/genetics , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Hypoxanthine Phosphoribosyltransferase/isolation & purification , Models, Molecular , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
Expression plasmids encoding the hypoxanthine phosphoribosyltransferases (HPRTs) of Plasmodium falciparum, Schistosoma mansoni, Tritrichomonas foetus, and Homo sapiens were subcloned into genetically deficient Escherichia coli that requires complementation by the activity of a recombinant HPRT for growth on semidefined medium. Fifty-nine purine analogs were screened for their abilities to inhibit the growth of these bacteria. Several compounds that selectively altered the growth of the bacteria complemented by the malarial, schistosomal, or tritrichomonal HPRT compared with the growth of bacteria expressing the human enzyme were identified. These results demonstrate that the recombinant approach to screening compounds by complement selection in a comparative manner provides a rapid and efficient method for the identification of new lead compounds selectively targeted to the purine salvage enzymes of parasites.
