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.

The regenerative peptide thymosin ß4 accelerates the rate of dermal healing in preclinical animal models and in patients.

Chronic nonhealing cutaneous wounds are a worldwide problem with no agent able to promote healing. A naturally occurring, endogenous repair molecule, thymosin beta 4 (Tß4), has many biological activities that promote dermal repair. It is released by platelets at the site of injury and initiates the repair cascade. Tß4 accelerated dermal healing of full-thickness punch wounds in various animal models, including normal rats and mice, steroid-treated rats, diabetic mice, and aged mice. Furthermore, in two phase 2 clinical trials of stasis and pressure ulcers, it was found to accelerate healing by almost a month in those patients that did heal. Tß4 likely acts to repair and regenerate wounds by promoting cell migration and stem cell mobilization and differentiation, and by inhibiting inflammation, apoptosis, and infection. We conclude that Tß4 is a multifunctional regenerative peptide important in dermal repair.

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.

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.

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 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.

Radiation-mediated control of drug delivery.

Clinical trials of radiotherapy to control drug delivery were initiated in 1999 at Vanderbilt University. The initial studies exploited the findings that platelets are activated in tumor blood vessels after high-dose irradiation as used in radiosurgery and high-dose-rate brachytherapy. Platelets labeled with 111In showed binding in tumor blood vessels. However, the platelet labeling process caused platelets to also accumulate in the spleen. That clinical trial was closed, and subsequent clinical trials targeted protein activation in irradiated tumor blood vessels. Preclinical studies showed that peptide libraries that bind within irradiated tumor blood vessels contained the peptide sequence Arg-Gln-Asp (RGD). RGD binds to integrin receptors (e.g., receptors for fibrinogen, fibronectin, and vitronectin). We found that the fibrinogen receptor (GPIIb/IIIa, alpha2bbeta3) is activated within irradiated tumor blood vessels. RGD peptidemimetics currently in clinical trials include GPIIb/IIIa antagonists and the platelet-imaging agent biapcitide. Biapcitide is an RGD mimetic that is labeled with 99Tc to allow gamma camera imaging of the biodistribution of the GPIIb/IIIa receptor in neoplasms of patients treated with radiosurgery. This study has shown that the schedule of administration of the RGD mimetic is crucial. The peptide mimetic must be administered immediately before irradiation, whereas the natural ligands to the receptor compete for biapcitide binding if biapcitide is administered after irradiation. The authors currently are conducting a dose deescalation study to determine the threshold dosage required for RGD mimetic binding to radiation activated receptor. Radiation-guided clinical trials have been initiated by use of high-dose-rate brachytherapy. In a separate trial, the pharmacokinetics of radiation-inducible gene therapy are being investigated. In this trial, the radiation-activated promoter Egr-1 regulates expression of the tumor necrosis factor alpha gene, which is administered by use of the attenuated adenovirus vector. The Ad.Egr-TNF (ADGV) gene is administered by intratumoral injection of vector followed by irradiation in patients with soft-tissue sarcomas. This review highlights recent findings in these phase I pharmacokinetic studies of radiation-controlled drug delivery systems.

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.

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.

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.

Cationic liposomal delivery of plasmid to endothelial cells measured by quantitative flow cytometry.

Cationic liposomes are potentially important gene transfer vehicles, capable of conjugating with anionic DNA by condensation. Flow cytometry was used to examine quantitatively the incorporation of DNA-liposome complex into murine capillary lung endothelial cells. The plasmid DNA, a pSV-beta-galactosidase vector, was covalently labeled with ethidium monoazide by photoactivation. The cationic liposome consisted of egg phosphatidylcholine (90%), cholesterol (5%), and stearylamine (5%). The number of plasmid molecules contained within each cell as a function of exposure time was estimated from fluorescence intensity. Fluorescently labeled plasmid is detectable after 10 min and increases with continued exposure, but at a decreasing rate, up to 2160 min. After 2160 min each cell, on average, contains approximately 10,000 plasmid molecules. Following transfection, a single cell unimodal population was detected by flow cytometry, suggesting that all cells participate in transfection equally. Furthermore, cell cycle analysis indicates that the entry of DNA-liposome complex is independent of cell cycle.

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.

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.

Isolated hepatocytes in a bioartificial liver: A single group view and experience.

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte-based artificial liver (BAL). It is based on plasma perfusion through a hollow-fiber module seeded with matrix-anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 +/- 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. (c) 1994 John Wiley & Sons, Inc.

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.

Development of a hybrid bioartificial liver.

OBJECTIVE: The authors developed an extracorporeal liver support system and tested its efficacy in experimental animals with liver failure. The first clinical use of this system to treat a patient with liver failure is reported. SUMMARY BACKGROUND DATA: Multiple attempts have been made, ranging from plasma exchange to use of charcoal columns, to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use. There is a need for providing liver support as a "bridge" to transplantation and for treating patients with potentially reversible liver dysfunction. METHODS: A hybrid liver support system has been developed consisting of plasma perfusion through a charcoal column and a porous hollow fiber module inoculated with 5 x 10(9) matrix-attached hepatocytes. The system was tested in dogs with ischemic liver failure (n = 7) who underwent plasmapheresis; a control group (n = 6) underwent charcoal perfusion alone. A patient with liver failure was treated with this hybrid system. RESULTS: After 6 hours of hybrid liver support treatment, animals had significantly decreased serum ammonia and lactate levels, increased glucose level, normal prothrombin time, and increased systolic blood pressure compared with controls treated with charcoal perfusion alone. Use of the system to treat a patient was well tolerated with evidence of clinical improvement. CONCLUSIONS: Plasma perfusion through a system consisting of a charcoal column and matrix-attached porcine hepatocytes had significant beneficial effects in animals with liver failure and was well tolerated by a patient with liver failure.

Development of a bioartificial liver: properties and function of a hollow-fiber module inoculated with liver cells.

We have developed a bioartificial liver support system utilizing hollow-fiber bioreactor, plasmapheresis and microcarrier cell culture technologies. Liver cells were obtained through portal vein perfusion with ethylenediaminetetraacetate or ethylenediaminetetraacetate/collagenase. A mathematical model of mass transport in a hollow-fiber module, at various plasma flow velocities and system configurations, was developed. The bioartificial liver's ability to carry out specific differentiated metabolic liver functions was tested in vitro and in vivo. A reproducible large-animal model of acute ischemic liver failure was developed. Most major first-generation cyclosporine and 19-norterstosterone metabolites were isolated after substrate addition to the bioartificial liver in vitro. After bioartificial liver treatment for 6 hr (with dog or pig liver cells), dogs with acute liver failure had significantly lower serum ammonia and lactate levels and significantly higher serum glucose levels than did control animals treated with a bioartificial liver system inoculated with microcarriers alone. In addition, bioartificial liver-treated animals had significantly higher mean systolic blood pressures than did controls. Liver cell viability at the end of the 6-hr in vivo experiment was greater than 90%.