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D J Hirsh

Publications and source records attributed to D J Hirsh.

7 recordsLinked to original sources
PubMed2000

Novel therapeutic nano-particles (lipocores): trapping poorly water soluble compounds.

The development of stable spherical lipid-coated drug particles that are termed 'lipocores' is reported here. Unlike conventional lipid-based particles (i.e. liposomes, emulsions, micelles), these particles are comprised solely of a core of a poorly water soluble drug surrounded by polyethyleneglycol conjugated lipid (PEG-lipid) and are formed via a 'kinetic' trapping process. These lipocore particles were made with the acyl chain of 16 carbon length (C16) acyl-chain derivatives of paclitaxel or vinblastine and with the polyene antifungal hamycin. Formation of the particles occurred regardless of the type of PEG-phospholipid used (i.e. acyl chain length, chain saturation, and polymer length) and could also be formed with the negatively charged lipid N-glutaryl-dioleoyl-phosphatidylethanolamine (DOPE-GA). Images from both freeze-fracture electron microscopy and electron cryo-microscopy revealed solid spherical structures with no internal lamellae for the PEG-lipid particles made with the C16 derivatives of paclitaxel (BrC16-T) or vinblastine (C16-Vin). From a solute distribution study of lipocores made with BrC16-T and distearoyl-phosphatidylethanolamine-PEG2000 (DSPE-PEG2000), the particles were found to have no measurable aqueous captured volume. Fluorescence anisotropy and order parameter measurements revealed the core material of these particles to be highly immobilized. The mole ratio of BrC16-T:lipid in the lipocores was typically > 90: < 10 and as high as 98:2, and the refrigerated lipocores were stable for several months. BrC16-T/DSPE-PEG2000 lipocores of 50-100 nm particle size were far less toxic than paclitaxel (Taxol) after intraperitoneally (i.p.) or intravenously (i.v.) administration in mice and were active against i.p. and subcutaneously (s.c.) planted human (OvCar3) ovarian carcinoma grown in SCID mice. It is believed the high drug:lipid ratio, the stability, and therapeutic efficacy of these novel particles make them a paradigm for delivery of poorly water soluble drugs and/or their hydrophobic derivatives.

Animals↗
PubMed1998

Doxorubicin physical state in solution and inside liposomes loaded via a pH gradient.

We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed linear, curved, and circular bundles of fibers with no significant interaction/perturbation of the vesicle membrane. The individual DOX fibers are putatively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal array with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observed for DOX-citrate fiber aggregates that had been concentrated from solution at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above (approx. 20 mM internal DOX concentration) indicating that the threshold concentration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fiber bundles can account for the vast majority (>99%) of DOX loaded via a pH gradient into citrate buffered liposomes. DOX loaded into liposomes containing lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentration was approx. 200 mM. This finding suggests that in the citrate containing liposomes the citrate multianion electrostatically bridged adjacent fibers to form the observed bundles. 13C-NMR measurements of [1,5-13C]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'free' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (<4% at 1 h) from liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentration). LBA containing liposomes loaded with disaggregated DOX, where the internal DOX concentration was only approx. 20 mM, experienced an osmotic stress induced vesicle rupture with as much as 18% DOX leakage in less than 10 min. The possible implications for this in vivo are discussed.

Calorimetry, Differential Scanning↗
PubMed1998

A new monofluorinated phosphatidylcholine forms interdigitated bilayers.

16-Fluoropalmitic acid was synthesized from 16-hydroxypalmitic acid using diethylaminosulfur trifluoride. This monofluorinated fatty acid then was used to make 1-palmitoyl-2-[16-fluoropalmitoyl]-phosphatidylcholine (F-DPPC) as a fluorinated analog of dipalmitoylphosphatidylcholine (DPPC). Surprisingly, we found that the phase transition temperature (Tm) of F-DPPC occurs near 50 degrees C, approximately 10 degrees C higher than its nonfluorinated counterpart, DPPC, as judged by both differential scanning calorimetry and infrared spectroscopy. The pretransition observed for DPPC is absent in F-DPPC. A combination of REDOR, rotational-echo double-resonance, and conventional solid-state NMR experiments demonstrates that F-DPPC forms a fully interdigitated bilayer in the gel phase. Electron paramagnetic resonance experiments show that below Tm, the hydrocarbon chains of F-DPPC are more motionally restricted than those of DPPC. X-ray scattering experiments confirm that the thickness and packing of gel phase F-DPPC is similar to that of heptanetriol-induced interdigitated DPPC. F-DPPC is the first phosphoglyceride containing sn-1 and sn-2 ester-linked fatty acyl chains of equal length that spontaneously forms interdigitated bilayers in the gel state in the absence of inducing agents such as alcohols.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1996

Secondary structure and location of a magainin analogue in synthetic phospholipid bilayers.

Magainins are cationic, membrane-active peptides which show broad-spectrum antimicrobial activity. We have investigated the secondary structure and location of an analogue of magainin 2 in synthetic phospholipid bilayers using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Ala19-magainin 2 amide exhibits both alpha-helix and beta-sheet secondary structures in lipid bilayers containing either dipalmitoylphosphatidylglycerol (DPPG) or a 1:1 molar mixture of DPPG and dipalmitoylphosphatidylcholine (DPPC). The combination of FTIR and solid-state NMR results suggests that there are two populations of peptide. The secondary structure of one population is alpha-helix while that of the other population is beta-sheet. We demonstrate that the solid-state NMR technique, rotational-echo double resonance (REDOR), can be used to measure both intra- and intermolecular dipole-dipole interactions in membrane-bound peptides. Our REDOR experiments indicate that alpha-helical Ala19-magainin 2 amide is bound near the phospholipid head groups.

Amino Acid Sequence↗
PubMed1992

Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II.

The stable tyrosine radical YD. (tyrosine 160 in the D2 polypeptide) in photosystem II (PSII) exhibits nonexponential electron spin-lattice relaxation transients at low temperature. As previously reported, the tetranuclear Mn complex in PSII significantly enhances the spin-lattice relaxation of YD.. However, in Mn-depleted PSII membranes, the spin-lattice relaxation transients of YD. are also nonexponential, and progressive power saturation (P 1/2) experiments show that it does not behave like an isolated tyrosine radical. A model is developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a random orientation in a magnetic field. This model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of three relaxation rate constants: the "intrinsic" relaxation rate, the relaxation rate due to scalar exchange, and the relaxation rate due to dipole-dipole interactions. The intrinsic and the scalar exchange relaxation rates are isotropic and together contribute a single rate constant to the spin-lattice relaxation transients. However, the dipolar relaxation rate is orientation dependent. Each orientation contributes a different dipolar relaxation rate constant to the net spin-lattice relaxation rate constant. The result is a superposition of single-exponential recoveries, each with a different net rate constant, causing the observed saturation-recovery transients to be non-(single)-exponential. Saturation-recovery relaxation transients of YD. are compared with those of a model tyrosine radical, generated by UV photolysis of L-tyrosine in a borate glass. From this comparison, we conclude that scalar exchange does not make a significant contribution to the spin-lattice relaxation of YD. in Mn-depleted PSII. We account for the nonexponential relaxation transients obtained from YD. in Mn-depleted PSII membranes in terms of dipolar-induced relaxation enhancement from the non-heme Fe(II). From simulations of the spin-lattice relaxation transients, we obtain the magnitude of the magnetic dipolar interaction between YD. and the non-heme Fe(II), which can be used to calculate the distance between them. Using data on the non-heme Fe(II) in the reaction center of Rhodobacter sphaeroides to model the non-heme Fe(II) in PSII, we calculate a YD.-Fe(II) distance of greater than or equal to 38 A in PSII. This agrees well with the distance predicted from the structure of the bacterial reaction center.

Electron Spin Resonance Spectroscopy↗