Biocompatibility of Antifogging SiO-doped Diamond-Like Carbon Laparoscope Coatings.

Laparoscopes can suffer from fogging and contamination difficulties, resulting in a reduced field of view during surgery. A series of diamond-like carbon films, doped with SiO, were produced by pulsed laser deposition for evaluation as biocompatible, antifogging coatings. DLC films doped with SiO demonstrated hydrophilic properties with water contact angles under 40°. Samples subjected to plasma cleaning had improved contact angle results, with values under 5°. Doping the DLC films with SiO led to an average 40% decrease in modulus and 60% decrease in hardness. Hardness of the doped films, 12.0 - 13.2 GPa, was greater than that of the uncoated fused silica substrate, 9.2 GPa. The biocompatibility was assessed through CellTiter-Glo assays, with the films demonstrating statistically similar levels of cell viability when compared to the control media. The absence of ATP released by blood platelets in contact with the DLC coatings suggests in vivo hemocompatibility. The SiO doped films displayed improved transparency levels in comparison to undoped films, achieving up to an average of 80% transmission over the visible spectrum and an attenuation coefficient of 1.1 × 104 cm-1 at the 450 nm wavelength. The SiO doped DLC films show promise as a method of fog prevention for laparoscopes.

Quantitative measurement of multifunctional quantum dot binding to cellular targets using flow cytometry.

Semiconductor nanocrystals such as quantum dots (QDs) are a potentially powerful resource in the fields of flow cytometry and fluorescence microscopy. QD size and fluorescence characteristics offer attractive features for use in targeted delivery systems and detection by flow cytometry. While quantitative measurements of a variety of fluorescent molecules are routinely performed, fluorophores for which no calibration standards exist, such as QDs, pose a problem for quantitation in flow cytometry. Our goal was to develop a targeted nanoparticle delivery platform as well as a corresponding method to accurately and quantitatively assess the performance of this system. We synthesized surface-modified QD probes targeted to cellular surface receptors and measured the MFI of the resulting cell-probe conjugates by flow cytometry. MFI was converted to mean equivalent R-PE intensity (MEPE) using standard calibration microspheres. Known concentrations of both R-PE and QD probes were measured by fluorometry to relate R-PE and QD fluorescence. Fluorometry results were then used to translate MEPE measurements to the number of bound QD probes. The targeted probes exhibited superior binding characteristics over unmodified and untargeted particles. This binding interaction was shown to be specific and mediated by the NGR targeting peptide tethered to the QD surface. The calibration method developed to assess this system proved successful at converting raw fluorescence data to quantitative probe binding values. We demonstrate the synthesis and performance of a highly modular nanoparticle system capable of targeted binding and fluorescent imaging. The calibration method implemented to quantify the performance of this system represents a potentially powerful tool to utilize truly quantitative flow cytometry measurements with an array of fluorescent molecules, including QDs.

Shear stress-facilitated calcium ion transport across lipid bilayers.

Small unilamellar liposomes were used in this study of shear stress effects on the trans-bilayer flux of calcium ions (Ca2+). Liposome suspensions were prepared from 99% egg phosphatidylcholine by a microporous filter extrusion technique. The inner aqueous phase of the unilamellar liposomes contained indo-1(5-), a fluorescent indicator of free Ca2+. The external aqueous phase was composed of Hepes-buffered saline containing normal physiological levels of common ionic species. Calcium ion levels were set at 100 nM and 1 mM in the inner and outer aqueous phases, respectively. Liposome suspensions were exposed to graded levels of uniform shear stress in an optically modified rotational viscometer. Intraliposome Ca2+ concentration was estimated from continuous measurement of indo-1(5-) fluorescence. Electronically measured particle size distribution was used to determine liposome surface area for estimation of trans-bilayer Ca2+ flux. Trans-bilayer Ca2+ flux increased linearly with applied shear rate from 27 s-1 to 2700 s-1. Diffusional resistance of the lipid bilayer, not the convective resistance of the surrounding fluid, was the limiting step in the transport of Ca2+. Liposome permeability to Ca2+ increased by nearly two orders of magnitude over the physiologically relevant shear rate range studied. Solute transport in injectable liposome preparations may be dramatically influenced by cardiovascular fluid stress. Solute delivery rates determined in liposomes exposed to static conditions may not accurately predict in vivo, cardiovascular solute transport.

The effect of bilayer composition on calcium ion transport facilitated by fluid shear stress.

Passive calcium ion permeability across liposome bilayers is increased during exposure to fluid shear forces attainable in the mammalian vasculature. In this study, liposomes prepared from three different lipid mixtures (phosphatidylcholine alone; phosphatidylcholine and cholesterol; a mixture of anionic and cationic phospholipids plus cholesterol) are exposed to uniform shear stress in a rotational viscometer. Liposome permeability to calcium ion is estimated from continuous measurement of free intraliposome calcium ion concentration using a fluorescence technique. Calcium ion permeability in the absence of fluid force and susceptibility to shear-induced permeability modulation are positively correlated with estimated bilayer compressibility. Fluid shear forces are presumed to influence bilayer packing and modulate defect formation in proportion to bilayer compressibility. Bilayer defects produced by fluid forces may increase liposome permeability.

Mitosis enhances transgene expression of plasmid delivered by cationic liposomes.

A critical requirement of gene therapy is expression of the delivered transgene. Transgene expression is facilitated by access to the transcription mechanism found primarily in the nucleus. Factors modulating the interactions between intracellular plasmid and nuclear access are not well understood. In this study, the effect of mitosis on transgene expression was examined by quantitative flow cytometry. Transfection of HeLa cells synchronized at late G1 phase or G2/M phase was performed using a liposomal vector containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dioleoyl-phosphatidylethanolamine (DOPE) (1:1 mol/mol). Cell samples were transfected and subsequently maintained in G1 phase for various durations to modulate the time between plasmid entry and mitosis. The plasmid contains the sequence for a mutated green fluorescent protein (GFP(S65T)) that was used to examine transgene expression. Ethidium monoazide-labeled plasmid was employed to examine the association of plasmid with the cell membrane. The percentage of cells expressing GFP(S65T) increased sharply as the synchronized cell population passed through M phase, suggesting that an event associated with mitosis is essential for transgene expression. Expression levels of the transgene then declined 18 h after mitosis irrespective of transfection strategy. All transfection strategies resulted in the same maximum percentage of GFP(S65T) positive cells (40%) and average GFP(S65T) expression level (3.14x106 molecules per positive cell). Association of plasmid with the cell membrane at late G1 phase was 1.5-fold of that at G2/M phase. These data are evidence for control of transgene expression triggered by events associated with cell cycle.

Cell-based screening: a high throughput flow cytometry platform for identification of cell-specific targeting molecules.

Targeting of drugs and genes to specific cell types is an emerging paradigm in the treatment of many medical conditions. However, targeting structures such as peptides are susceptible to rapid inactivation in vivo. To address this problem, novel targeting molecules can now be rapidly synthesized using a combinatorial approach. Methods to screen the large libraries created in this process are often lacking or compatible only with solution-based screening. This report describes a high-throughput cell-based method utilizing flow cytometry, capable of rapidly screening large libraries of molecules simultaneously for biological functionality and stability. In this method, each library molecule is attached to a microsphere exhibiting a unique set of optical properties, or "fingerprint", conferring modularity and multiplex capability. We investigated the multiplex capability of our flow cytometric method to determine its capacity for high-throughput screening. Current instrumentation in our laboratory allows the screening of at least 75 unique compounds in a single well, a number comparable to available solution-based assays. In state-of-the-art configuration, however, this methodology can support the screening of up to 1875 compounds per well, achieving high-throughput potential in a single multiwell plate. We also investigated the binding capability of targeted microspheres to adherent target cells. These microspheres exhibited a 12-fold increase in binding over control, untargeted microspheres. Competitive inhibition experiments with soluble ligand confirmed the specificity of microsphere binding. Overall, the methodology proposed here is capable of quickly and effectively screening large libraries of targeting molecules using instrumentation readily available to the greater research community.

A note on the use of Quin2 in studying shear-induced platelet aggregation.

Quin2, a calcium ion chelator which can penetrate plasma membranes, was used to study the role of intracellular calcium ion concentration in mediating shear-induced platelet activation. Washed platelet suspensions were subjected to various levels of uniform, known shear stress in a cone and plate viscometer in the absence of added agonists. Additional samples were aggregated in response to chemical platelet agonists in a conventional aggregometer. The aggregometer response of Quin2-containing platelets to collagen, thrombin and ADP exhibited increased lag time and reduced maximum rate of aggregation in comparison to controls. However, the extent of aggregation of the Quin2-containing platelets eventually reached the same level as that of the controls. Very different results were obtained for aggregation by shear stress in the viscometer. Shear-induced aggregation was significantly suppressed by Quin2 treatment at both short (30 seconds) and long (300 seconds) times of exposure to the shear field. Shear-induced dense granular release and cellular lysis were unaltered by Quin2 treatment at 30 second exposure times, but both were significantly increased by Quin2 treatment at 300 second exposure times. These results suggest that intracellular calcium ion mobilization is an important early step in shear-induced platelet activation. Additionally, Quin2 appears to have effects resulting in increased platelet fragility. Thus, the findings raise questions on the suitability of Quin2 as an intracellular calcium ion probe in studies in shear fields.

SC-49992--a potent and specific inhibitor of platelet aggregation.

Fibrinogen binding is required for platelet aggregation and subsequent thrombus formation. SC-49992 (SC), an RGDF mimetic, is a potent and specific inhibitor of the binding of fibrinogen to its receptor on activated platelets, glycoprotein IIb/IIIa (IC50 0.7 microM). SC was more potent (1-5 microM) than either RGDS, RGDF or the gamma chain dodecapeptide in blocking platelet aggregation to a variety of agonists in both dog and human platelet rich plasma. SC was more potent as an inhibitor of GP IIb/IIIa on platelets than it was against other integrin and non-integrin receptors, including the RGD-dependent vitronectin receptor and other non-RGD-dependent integrins such as CDII/CD18. SC had little effect on ristocetin induced agglutination. SC blocked ex vivo collagen induced aggregation in dogs and collagen induced thrombocytopenia in rats. These data suggest that elimination of the Arg-NH2 and the Arg-Gly amide bond of RGDF provided increased inhibitory potency and specificity. This structural modification may be of value in the development of other more potent RGDF mimetics for the inhibition of platelet aggregation.

Mass transfer in a hollow fiber device used as a bioartificial liver.

The transport of albumin, glucose and fluid in a hollow fiber bioartificial liver (BAL) was predicted by theory and measured experimentally. Results from the experiment were used in a three compartment transport model to estimate the transfiber fluid flux. Fluid convection driven by the transfiber pressure gradient transported solutes across the semi-permeable fibers of the BAL. Diffusion contributed significantly to the transfiber transport of both glucose and albumin. Modification of the BAL system hydraulic geometry significantly increased transfiber pressure gradient with corresponding increase in transfiber fluid flux. Transfiber solute transport was increased by osmotically driven transport which resulted from the revised flow geometry. Substantial delivery of hepatocyte synthesis products is possible with careful control of the physical parameters and operating conditions of the BAL.

A cone and plate viscometer for the continuous measurement of blood platelet activation.

A cone and plate viscometer was modified to permit the continuous study of platelet response during shear stress exposure times on the order of one second to 180 seconds. Platelets may be stimulated by uniform, controlled shear stress alone or with the addition of chemical platelet agonists. The time course of platelet aggregation is interpreted from alterations in the apparent optical density of the platelet suspension. The rate and extent of platelet dense granule release is estimated from the intensity of the luminescent reaction of platelet released ATP with firefly luciferase and luciferin. Intracellular calcium ion concentration is determined as a function of shear exposure time through the fluorescence intensity of indo-1(5-), a membrane-permeant pentacarboxylate calcium ion chelator.

The effects of elongational stress exposure on the activation and aggregation of blood platelets.

Hemodynamic shear is known to stimulate blood and endothelial cells and induce platelet activation. Many studies of shear-induced platelet stimulation have employed rotational viscometers in which secondary flow effects are assumed to be negligible. Shear induced platelet activation occurs at elevated shear rates where secondary flows may contribute a significant percentage of the total hydrodynamic force experienced by the sample. Elongational stress, one component of this secondary flow, has been shown to alter transmembrane ion flux in intact cell and the permeability of synthetic membrane preparations. Elongational flow also occurs in the vasculature at sites of elevated shear stress. Secondary flow components may contribute to platelet activation induced during shear stress application in rotational viscometry. A unique 'constrained convergence' elongational flow chamber was designed and fabricated to study platelet response to elongational stress exposure. The elongational flow chamber was capable of producing an elongation rate of 2.1 s-1 with a corresponding volume averaged shear rate of 58.33 s-1. Significant changes were observed in the total platelet volume distribution and measured response to added chemical antagonists after elongational stress exposure. The total platelet volume histogram shifted toward larger particle sizes, suggesting the formation of large aggregates as a result of elongational stress exposure. Platelets exposed to elongational stress demonstrated a dose dependent decrease in added ADP-induced aggregation rate and extent of aggregation.

Convective flow increases lipoplex delivery rate to in vitro cellular monolayers.

The mass transport characteristics of cationic, nonviral liposome-DNA plasmid complexes (lipoplexes) were evaluated over a range of fluid shear stresses. The typical case of stagnant flow transfection was expanded to include controlled fluid convection provided by constant flow through a parallel plate flow chamber. Equations describing the transport of lipoplex by sedimentation and convection were derived from theory and solved numerically. Instantaneous lipoplex delivery rate and total lipoplex surface delivery during a 72-h transfection were estimated for two shear stress levels and for static conditions. Theory predicted that lipoplex is delivered to the cell surface more than 12- to 19-fold faster through the addition of convection, at least for physiologic shear stresses of 2.3-9.7 dyn/cm2, respectively. These calculations were tested experimentally using a cell line (ECV-304) transfected with fluorescently labeled plasmid DNA formulated into a lipoplex. Transfections were conducted during cellular exposure to the same known, uniform levels of fluid shear stress presumed in theoretical calculations. Lipoplex delivery was increased by more than nine-fold at 2.3 dyn/cm2 compared to the static case as assessed by flow cytometric measurement. Lipoplex delivery was modestly reduced at the highest fluid shear stress, to six-fold of the static case, consistent with the disruption of lipoplex-cell binding mediated by hydrodynamic forces. The complicated relationship between fluid convection and lipoplex delivery has important implications for nonviral gene therapy.

Quantitation and kinetics of CD51 surface receptor expression: implications for targeted delivery.

The CD51 integrin subunit is important in many functions ranging from mediation of adenovirus attachment and internalization to facilitation of angiogenesis. CD51 has also gained interest as an attractive ligand for directed therapy studies, including those for targeted gene delivery. While the function and importance of several CD51-specific targeting molecules have been examined, studies are often carried out without quantitative assessment of the receptor. A lack of this data complicates further mathematical analysis of targeting data and elucidation of the mechanism(s) underlying specific targeting. We performed a quantitative evaluation of CD51 receptors on the surface of HeLa cells, a common cell line utilized in many receptor-based studies, and compared them to other similar and dissimilar cell types. Unstimulated HeLa cells strongly express the CD51 receptor at a level of 212,700 +/- 12,000 (mean +/- SD) antibody binding capacity (ABC) cell(-1) (n = 3). Following irradiation with 3 Gy, receptor expression increases dramatically, peaking at a value of 403,700 +/- 26,400 (n = 4). The utility of this quantitative information is highlighted by the application of our data to targeting studies from the literature. Taken together, the results yield more detailed information than is available with experimental targeting data alone.

Nuclear-associated plasmid, but not cell-associated plasmid, is correlated with transgene expression in cultured mammalian cells.

Intracellular plasmid is rapidly incorporated into the nucleus of HeLa cells following cationic lipoplex transfection. CV1 cells are less effective in translocating plasmid to the nucleus and also express less transgene than HeLa cells. Cultured HeLa and CV1 cells and corresponding isolated nuclei were analyzed after transfection of a Cy3-labeled pGreenLantern plasmid (Cy3-pGL). Flow cytometry was used to measure both plasmid delivery and transgene expression from the plasmid encoding a CMV promoter-driven green fluorescent protein. During transfection, HeLa cells rapidly incorporated the plasmid, reaching a maximum of 80% Cy3-pGL positive cells 8 h posttransfection. The average Cy3-pGL-positive HeLa cell contained approximately 2470 plasmid copies. Forty-eight percent of the nuclei isolated from the transfected HeLa cells were positive for the plasmid marker after 8 h. In contrast to HeLa cells, fewer CV1 cells and CV1 nuclei incorporated plasmid DNA with peak transfection occurring after 12 h for 36% of the cells and after 8 h for 12% of the nuclei. However, the average Cy3-pGL-positive CV1 cell did not have a significantly different number of total cellular plasmid copies than the average positive HeLa cell. CV1 nuclei, however, had half as much nuclear associated plasmid as HeLa nuclei. HeLa cells are more efficient than CV1 cells at transporting plasmid from the cytoplasm to the nucleus. This study demonstrates the use of a novel quantitative method to study plasmid transport from the cytoplasm to the nucleus and the effect on transgene expression.

Transfection by cationic liposomes using simultaneous single cell measurements of plasmid delivery and transgene expression.

Cationic liposomes are potentially important gene transfer vehicles, although their application has been limited by relatively low efficiency of transgene expression. Single cell quantitative methods, such as those used in this study, should permit a more detailed understanding of the relationships between delivered plasmid and transgene expression. Intracellular plasmid delivery and transgene expression were measured simultaneously using photoconjugated ethidium monoazide as an intracellular plasmid delivery marker and green fluorescent protein (GFP(S65T)) as a transgene expression marker. Quantitative flow cytometry was used to estimate plasmid copy number and GFP(S65T) molecules in single cells. The plasmid was delivered to HeLa cells with a cationic liposome vehicle containing 1,2-dioleoyloxy-3-trimethylammonium-propane and dioleoylphosphatidylethanolamine (1:1 mol/mol). Treatment was carried out continuously for 24 h. Flow cytometry measurements on 20, 000 cells were performed during treatment and for 48 h post-treatment. On a single cell basis, transgene expression efficiency and average GFP(S65T) expression level increased with intracellular plasmid copy number. After 3-h exposure to the liposomal vector, more than 95% of the cells were positive for plasmid entry, but none had detectable transgene expression. Maximum transgene expression was achieved at 24 h and remained unchanged at the 72-h measurement. At 24 h, the average positive cell contained 1.6 x 10(5) plasmid copies and 2.3 x 10(6) GFP(S65T) molecules. Importantly, the measurement strategies revealed that transgene expression varied widely within the entire cell population. Although only 30% of all cells expressed transgene, the subpopulation of cells that rapidly incorporated the vector demonstrated 100% efficiency in transgene expression. This study identifies parameters that modulate highly efficient transgene expression from plasmid delivery by cationic liposomes.

A model for the analysis of nonviral gene therapy.

Further understanding of the mechanisms involved in cellular and intracellular delivery of transgene is needed to produce clinical applications of gene therapy. The compartmental and computational model designed in this work is integrated with data from previous experiments to quantitatively estimate rate constants of plasmid translocation across cellular barriers in transgene delivery in vitro. The experimental conditions between two cellular studies were held constant, varying only the cell type, to investigate how the rates differed between cell lines. Two rate constants were estimated per barrier for active transport and passive diffusion. Translocation rates of intact plasmid across the cytoplasmic and nuclear barriers varied between cell lines. CV1 cells were defined by slower rates (0.23 h(-1) cytoplasmic, 0.08 h(-1) nuclear) than those of the HeLa cells (1.87 h(-1) cytoplasmic, 0.45 h(-1) nuclear). The nuclear envelope was identified as a rate-limiting barrier by comparing the rate of intact plasmid translocation at each barrier. Slower intact plasmid translocation in CV1 cells was correlated with a reduced absolute capacity for transgene efficiency in comparison with HeLa cells. HeLa cells were three times more efficient than CV1 cells at producing green fluorescent protein per intact plasmid delivered to the nucleus. Mathematical modeling coordinated with experimental studies can provide detailed, quantitative understanding of nonviral gene therapy.