Wallace W. Muhonen

Release of Liposomal Contents by Cell-Secreted Matrix Metalloproteinase-9

Liposomes have been widely used as a drug delivery vehicle, and currently, more than 10 liposomal formulations are approved by the Food and Drug Administration for clinical use. However, upon targeting, the release of the liposome-encapsulated contents is usually slow. We have recently demonstrated that contents from appropriately formulated liposomes can be rapidly released by the cancer-associated enzyme matrix metalloproteinase-9 (MMP-9). Herein, we report our detailed studies to optimize the liposomal formulations. By properly selecting the lipopeptide, the major lipid component, and their relative amounts, we demonstrate that the contents are rapidly released in the presence of cancer-associated levels of recombinant human MMP-9. We observed that the degree of lipid mismatch between the lipopepides and the major lipid component profoundly affects the release profiles from the liposomes. By utilizing the optimized liposomal formulations, we also demonstrate that cancer cells (HT-29) which secrete low levels of MMP-9 failed to release a significant amount of the liposomal contents. Metastatic cancer cells (MCF7) secreting high levels of the enzyme rapidly release the encapsulated contents from the liposomes.

Characterization of the ATP- and GTP-specific succinyl-CoA synthetases in pigeon. The enzymes incorporate the same alpha-subunit.

Two succinyl-CoA synthetases, one highly specific for GTP/GDP and the other for ATP/ADP, have been purified to homogeneity from pigeon liver and breast muscle. The two enzymes are differentially distributed in pigeon, with only the GTP-specific enzyme detected in liver and the ATP-specific enzyme in breast muscle. Based on assays in the direction of CoA formation, the ratios of GTP-specific to ATP-specific activities in kidney, brain, and heart are ∼7, 1, and 0.1, respectively. Both enzymes have the characteristic α- and β-subunits found in other succinyl-CoA synthetases. Studies of the α-subunit by electrophoresis, mass spectrometry, reversed-phase high performance liquid chromatography, and peptide mapping showed that it was the same in the two enzymes. Characterization of the β-subunits by the same methods indicated that they were different, with the tryptic peptide maps providing evidence that the β-subunits likely differ along their entire sequences. Because the two succinyl-CoA synthetases incorporate the same α-subunit, the determinants of nucleotide specificity must reside within the β-subunit. Determination of the apparent Michaelis constants showed that the affinity of the GTP-specific enzyme for GDP is greater than that of the ATP-specific enzyme for ADP (7 versus 250 μm). Rather large differences in apparentK m values were also observed for succinate and phosphate.

MMP‑9 Responsive PEG Cleavable Nanovesicles for Efficient Delivery of Chemotherapeutics to Pancreatic Cancer

Significant differences in biochemical parameters between normal and tumor tissues offer an opportunity to chemically design drug carriers which respond to these changes and deliver the drugs at the desired site. For example, overexpression of the matrix metalloproteinase-9 (MMP-9) enzyme in the extracellular matrix of tumor tissues can act as a trigger to chemically modulate the drug delivery from the carriers. In this study, we have synthesized an MMP-9-cleavable, collagen mimetic lipopeptide which forms nanosized vesicles with the POPC, POPE-SS-PEG, and cholesteryl-hemisuccinate lipids. The lipopeptide retains the triple-helical conformation when incorporated into these nanovesicles. The PEG groups shield the substrate lipopeptides from hydrolysis by MMP-9. However, in the presence of elevated glutathione levels, the PEG groups are reductively removed, exposing the lipopeptides to MMP-9. The resultant peptide-bond cleavage disturbs the vesicles’ lipid bilayer, leading to the release of encapsulated contents. These PEGylated nanovesicles are capable of encapsulating the anticancer drug gemcitabine with 50% efficiency. They were stable in physiological conditions and in human serum. Effective drug release was demonstrated using the pancreatic ductal carcinoma cells (PANC-1 and MIAPaCa-2) in two-dimensional and three-dimensional “tumor-like” spheroid cultures. A reduction in tumor growth was observed after intravenous administration of the gemcitabine-encapsulated nanovesicles in the xenograft model of athymic, female nude mice.

Ultrasound Enhanced Matrix Metalloproteinase-9 Triggered Release of Contents from Echogenic Liposomes

The extracellular enzyme matrix metalloproteinase-9 (MMP-9) is overexpressed in atherosclerotic plaques and in metastatic cancers. The enzyme is responsible for rupture of the plaques and for the invasion and metastasis of a large number of cancers. The ability of ultrasonic excitation to induce thermal and mechanical effects has been used to release drugs from different carriers. However, the majority of these studies were performed with low frequency ultrasound (LFUS) at kilohertz frequencies. Clinical usage of LFUS excitations will be limited due to harmful biological effects. Herein, we report our results on the release of encapsulated contents from substrate lipopeptide incorporated echogenic liposomes triggered by recombinant human MMP-9. The contents release was further enhanced by the application of diagnostic frequency (3 MHz) ultrasound. The echogenic liposomes were successfully imaged employing a medical ultrasound transducer (4-15 MHz). The conditioned cell culture media from cancer cells (secreting MMP-9) released the encapsulated dye from the liposomes (30-50%), and this release is also increased (50-80%) by applying diagnostic frequency ultrasound (3 MHz) for 3 min. With further developments, these liposomes have the potential to serve as multimodal carriers for triggered release and simultaneous ultrasound imaging.

Quantitation of Human Metallothionein Isoforms: A Family of Small, Highly Conserved, Cysteine-rich Proteins

Human metallothioneins (MTs) are important regulators of metal homeostasis and protectors against oxidative damage. Their altered mRNA expression has been correlated with metal toxicity and a variety of cancers. Current immunodetection methods lack the specificity to distinguish all 12 human isoforms. Each, however, can be distinguished by the mass of its acetylated, cysteine-rich, hydrophilic N-terminal tryptic peptides. These properties were exploited to develop a bottom-up MALDI-TOF/TOF-MS-based method for their simultaneous quantitation. Key features included enrichment of N-terminal acetylated peptides by strong cation exchange chromatography, optimization of C18 reversed-phase chromatography, and control of methionine oxidation. Combinations of nine isoforms were identified in seven cell lines and two tissues. Relative quantitation was accomplished by comparing peak intensities of peptides generated from pooled cytosolic proteins alkylated with 14N- or 15N-iodoacetamide. Absolute quantitation was achieved using 15N-iodoacetamide-labeled synthetic peptides as internal standards. The method was applied to the cadmium induction of MTs in human kidney HK-2 epithelial cells expressing recombinant MT-3. Seven isoforms were detected with abundances spanning almost 2 orders of magnitude and inductions up to 12-fold. The protein-to-mRNA ratio for MT-1E was one-tenth that of other MTs, suggesting isoform-specific differences in protein expression efficiency. Differential expression of MT-1G1 and MT-1G2 suggested tissue- and cell-specific alternative splicing for the MT-1G isoform. Protein expression of MT isoforms was also evaluated in human breast epithelial cancer cell lines. Estrogen-receptor-positive cell lines expressed only MT-2 and MT-1X, whereas estrogen-receptor-negative cell lines additionally expressed MT-1E. The combined expression of MT isoforms was 38-fold greater in estrogen-receptor-negative cell lines than in estrogen-receptor-positive cells. These findings demonstrate that individual human MT isoforms can be accurately quantified in cells and tissues at the protein level, complementing and expanding mRNA measurement as a means for evaluating MTs as potential biomarkers for cancers or heavy metal toxicity.

The compartmentation of nucleoside diphosphate kinase in mitochondria

The compartmentation of nucleoside diphosphate kinase (NDPK) was studied in mitochondria isolated from heart and liver of rat, rabbit, and pigeon. Compartmentation was assessed by determining latencies of enzyme activities, fractionating mitochondria with digitonin, and treating mitochondria with trypsin in the presence and absence of digitonin. NDPK activity in pigeon liver mitochondria was five- and seven-fold higher than in rat and rabbit liver mitochondria. The ratios of NDPK activities in liver vs. heart mitochondria were about 15 for rat, 2 for rabbit, and more than 40 for pigeon. Nearly all NDPK in pigeon liver mitochondria is in the matrix space, but outside the matrix in rat and rabbit liver mitochondria. Most NDPK in pigeon heart mitochondria was located outside the matrix while a significant fraction may be in the matrix of rat and rabbit heart mitochondria. These results are discussed relative to the assumed role that mitochondrial NDPK transfers the phosphoryl group of GTP produced in the Krebs cycle to the adenine nucleotide pool.

RhoA Kinase (Rock) and p90 Ribosomal S6 Kinase (p90Rsk) phosphorylation of the sodium hydrogen exchanger (NHE1) is required for lysophosphatidic acid-induced transport, cytoskeletal organization and migration☆

The sodium hydrogen exchanger isoform one (NHE1) plays a critical role coordinating asymmetric events at the leading edge of migrating cells and is regulated by a number of phosphorylation events influencing both the ion transport and cytoskeletal anchoring required for directed migration. Lysophosphatidic acid (LPA) activation of RhoA kinase (Rock) and the Ras-ERK growth factor pathway induces cytoskeletal reorganization, activates NHE1 and induces an increase in cell motility. We report that both Rock I and II stoichiometrically phosphorylate NHE1 at threonine 653 in vitro using mass spectrometry and reconstituted kinase assays. In fibroblasts expressing NHE1 alanine mutants for either Rock (T653A) or ribosomal S6 kinase (Rsk; S703A) we show that each site is partially responsible for the LPA-induced increase in transport activity while NHE1 phosphorylation by either Rock or Rsk at their respective site is sufficient for LPA stimulated stress fiber formation and migration. Furthermore, mutation of either T653 or S703 leads to a higher basal pH level and a significantly higher proliferation rate. Our results identify the direct phosphorylation of NHE1 by Rock and suggest that both RhoA and Ras pathways mediate NHE1-dependent ion transport and migration in fibroblasts.

Microwave-assisted synthesis of triple-helical, collagen-mimetic lipopeptides

Collagen-mimetic peptides and lipopeptides are widely used as substrates for matrix degrading enzymes, as new biomaterials for tissue engineering, as drug delivery systems and so on. However, the preparation and subsequent purification of these peptides and their fatty-acid conjugates are really challenging. Herein, we report a rapid microwave-assisted, solid-phase synthetic protocol to prepare the fatty-acid conjugated, triple-helical peptides containing the cleavage site for the enzyme matrix metalloproteinase-9 (MMP-9). We employed a PEG-based resin as the solid support and the amino acids were protected with Fmoc- and tert-butyl groups. The amino acids were coupled at 50 °C (25 W of microwave power) for 5 min. The deprotection reactions were carried out at 75 °C (35 W of microwave power) for 3 min. Using this protocol, a peptide containing 23 amino acids was synthesized and then conjugated to stearic acid in 14 h.

Factors affecting the manganese and iron activation of the phospho enol pyruvate carboxykinase isozymes from rabbit

Timed assays in which GTP and GDP were separated and quantitated by HPLC were developed and used to study the metal activation of the mitochondrial and cytosolic isozymes of phosphoenolpyruvate carboxykinase purified from rabbit liver. These assays allowed both directions of catalysis to be studied under similar conditions and in the absence of coupling enzymes. The mitochondrial enzyme is rapidly inactivated by preincubation with Fe2+, as had been shown previously for the cytosolic isozyme. The greatest activation by Fe2+ was obtained by adding micromolar Fe2+ immediately after enzyme to form the complete assay mixture that also contained millimolar Mg2+. In the direction of synthesis of OAA from Pep, the K0.5 values for Mn2+ and Fe2+ were in the 3-7 microM range when a nonchelating buffer, Hepes, was used. The buffer used strongly affected activation by Fe2+ at pH 7.4; activation was eliminated in the case of phosphate and K0.5 increased several-fold over that obtained with Hepes when imidazole was used. In non-chelating buffer, the pH optimum was near 7.4 for both isozymes and for both directions of catalysis. However, the near optimal pH range extended below 7.4 for the direction of oxaloacetate synthesis while the range was above 7.4 for Pep synthesis. In the direction of oxaloacetate synthesis: (1) Both isozymes required the presence of micromolar Mn2+ or Fe2+ in addition to millimolar Mg2+ in order to shown significant activity. (2) Fe2+ was as effective an activator as Mn2+ at pH 7 and below. In the direction of Pep synthesis: (1) Micromolar Mn2+ was a much better activator than Fe2+ at the higher pH values needed for optimal activity in this direction. (2) With increasing pH, decreasing activation was obtained with Fe2+ while the activity supported by Mg2+ alone increased. The results demonstrate the potential for regulation of either isozyme of Pep carboxykinase by the availability of iron or manganese.

High-performance liquid chromatography-based assays of enzyme activities

Interest in using HPLC to assay enzymatic reactions continues to grow as evidenced by the more than 100 papers published during the early 1990s. HPLC can be used for any enzymatic assay that requires separation of substrates and products before quantifying the extent of the reaction. The popularity of HPLC-based assays is due to several reasons: (1) HPLC offers unsurpassed precision, specificity, sensitivity, and reproducibility. (2) Powerful microcomputers and user-friendly software automate the running of samples and collection and processing of data. (3) Current columns, especially C18 packings, separate a very wide variety of samples, and (4) A variety of on-line detectors provide a means to detect virtually any compound. This review surveys recent papers on the development of HPLC-based assays for enzymes that degrade or otherwise modify macromolecules. Methods for assaying enzymes involved in metabolic pathways are also reviewed. Work by the authors in developing HPLC-based assays for mitochondrial enzymes that use GTP/GDP and other nucleotides that cannot be or are not easily assayed by enzyme-coupled assays is discussed. These enzymes include nucleoside diphosphate kinase, succinate thiokinase, and GTP-AMP phosphotransferase. The assays are suitable for determining the submitochondrial compartmentation of enzyme activities. Finally, current and anticipated trends in HPLC technology, including new column packings and the trend toward smaller columns that give faster separations, are reviewed in relation to enzyme assays.