Amphiphilic poly-N-vinylpyrrolidones: synthesis, properties and liposome surface modification.

Certain amphiphilic water-soluble polymers including amphiphilic derivatives of polyvinyl pyrrolidone (PVP) were found to be efficient steric protectors for liposomes in vivo. In this study, we have tried to develop synthetic pathways for preparing amphiphilic PVP and to investigate the influence of the hydrophilic/hydrophobic blocks on some properties of resulting polymers and polymer-coated liposomes. To prepare amphiphilic PVP with the end stearyl (S) or palmityl (P) residues, amino- and carboxy-terminated PVP derivatives were first synthesized by the free-radical polymerization of vinyl pyrrolidone in the presence of amino- or carboxy-mercaptans as chain transfer agents, and then modified by interaction of amino-PVP with stearoyl chloride or palmitoyl chloride, or by dicyclohexyl carbodiimide coupling of stearylamine with carboxy-PVP. ESR-spectra of the hydrophobic spin-probe, nitroxyl radical N-oxyl-2-hexyl-2-(10-methoxycarbonyl)decyl-4,4'-dimethyl oxazoline, in the presence of amphiphilic PVP demonstrated good accessibility of terminal P- and S-groups for the interaction with other hydrophobic ligands. Spontaneous micellization and low CMC values (in a low micromolar range) were found for amphiphilic PVP derivatives using the pyrene method. In general, S-PVP forms more stable micelles than P-PVP (at similar MW, CMC values for S-PVP are lower than for P-PVP). It was found that amphiphilic PVP incorporated into negatively charged liposomes effectively prevents polycation(poly-ethylpyridinium-4-vinylchloride)-induced liposome aggregation, completely abolishing it at ca. 10 mol% polymer content in liposomes. Additionally, the liposome-incorporated PVP prevents the fluorescence quenching of the membrane-incorporated hydrophobic fluorescent label [N-(4-fluoresceinthiocarbamoyl)dipalmitoyl-PE] by the free polycation. PVP-modified liposomes were loaded with a self-quenching concentration of carboxyfluorescein, and their destabilization in the presence of mouse serum was investigated following the release of free dye. Amphiphilic PVP with MW between 1,500 and 8,000 provides good steric protection for liposomes. The degree of this protection depends on both polymer concentration and molecular size of the PVP block.

p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment of specific ligands, including monoclonal antibodies, to distal ends of PEG chains via p-nitrophenylcarbonyl groups.

We have attempted to simplify the procedure for coupling various ligands to distal ends of liposome-grafted polyethylene glycol (PEG) chains and to make it applicable for single-step binding of a large variety of a primary amino group-containing substances, including proteins and small molecules. With this in mind, we have introduced a new amphiphilic PEG derivative, p-nitrophenylcarbonyl-PEG-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (pNP-PEG-DOPE), synthesized by reaction of DOPE with excess of bis(p-nitrophenylcarbonyl)-PEG in a chloroform/triethylamine mixture. pNP-PEG-DOPE readily incorporates into liposomes via its PE residue, and easily binds primary amino group-containing ligands via its water-exposed pNP groups, forming stable and non-toxic urethane (carbamate) bonds. The reaction between the pNP group and the ligand amino group proceeds easily and quantitatively at pH around 8.0, and remaining free pNP groups are promptly eliminated by spontaneous hydrolysis. Therefore, pNP-PEG-DOPE could serve as a very convenient tool for protein attachment to the distal ends of liposome-grafted PEG chains. To investigate the applicability of the suggested protocol for the preparation of long-circulating targeted liposomes, we have coupled several proteins, such as concanavalin A (ConA), wheat germ agglutinin (WGA), avidin, monoclonal antimyosin antibody 2G4 (mon2G4), and monoclonal antinucleosome antibody 2C5 (mon2C5) to PEG-liposomes via terminal pNP groups and studied whether the specific activity of these immobilized proteins is preserved. The method permits the binding of several dozens protein molecules per single 200 nm liposome. All bound proteins completely preserve their specific activity. Lectin-liposomes are agglutinated by the appropriate polyvalent substrates (mannan for ConA-liposomes and glycophorin for WGA-liposomes); avidin-liposomes specifically bind with biotin-agarose; antibody-liposomes demonstrate high specific binding to the substrate monolayer both in the direct binding assay and in ELISA. A comparison of the suggested method with the method of direct membrane incorporation was made. The effect of the concentration of liposome-grafted PEG on the preservation of specific protein activity in different coupling protocols was also investigated. It was also shown that pNP-PEG-DOPE-liposomes with and without attached ligands demonstrate increased stability in mouse serum.

Poly(Hpma)-coated liposomes demonstrate prolonged circulation in mice.

Surface modification of liposomes with amphiphilic flexible polymers significantly prolongs their circulation time in blood and reduces uptake by cells of the reticuloendothelial system (RES). Several polymers have already been shown to provide steric protection to liposomes. Still more polymers are expected to serve this purpose, thus broadening the variability of properties of long-circulating liposomes. Poly[N-(2-hydroxypropyl)methacrylamide] (poly (HPMA)) seems to have some properties similar to polyethylene glycol (PEG), the most widely used polymer in liposome surface modification, including flexibility, hydrophilicity and low immunogenicity, which suggest that it may also function as an efficient steric protector of liposomes. Semitelechelic poly(HPMA) with single- or double-oleic acid hydrophobic terminus were synthesized and incorporated into the surface of liposomes composed of phosphatidylcholine and cholesterol. These poly(HPMA)-modified liposomes provided strong steric protection for liposomes, increasing their circulation time and decreasing liver accumulation in experimental mice. Poly(HPMA)-modified liposomes may become a useful addition to a family of long-circulating liposomes with potential to be used as a drug delivery system.

Indicator measurement in tissues: CT with iohexol versus storage-phosphor autoradiography with carbon-14-labeled inulin.

RATIONALE AND OBJECTIVES: Computed tomography (CT) provides accurate measurement of blood iodine concentration in vivo, as well as in phantoms simulating tissue; however, its ability to measure radiopaque agents in biologic tissues in comparison with a standard technique does not seem to have been demonstrated. To validate the performance of CT imaging for quantification of contrast media in a variety of biologic tissues in vivo, a comparison between CT imaging with an iodinated contrast agent (iohexol) and the reference tracer quantification technique (storage-phosphor autoradiography with carbon-14-labeled inulin) was performed. MATERIALS AND METHODS: Six New Zealand White rabbits were injected intravenously with a cocktail of iohexol and C-14-labeled inulin at different dose ratios and sacrificed shortly after injection to arrest blood flow at different stages of tissue tracer distribution. One rabbit received no iohexol-inulin mixture and provided baseline data. Liver, spleen, kidneys, testis, and heart were excised and rapidly frozen. Each organ was scanned with CT (1-mm contiguous sections) to determine tissue iodine distribution. Twenty-micrometer tissue slices were made in the same planes in which the CT images had been acquired, and storage-phosphor screen autoradiography was performed to quantify C-14-labeled inulin distribution. RESULTS: Digital image analysis of CT images and autoradiograms was performed on spatially matched regions, and resultant tracer concentrations were compared. Tracer concentrations were highly correlated, with resultant R2 values exceeding 0.9 in all tissues. CONCLUSION: The highly correlated results for iodinated tracer quantification in tissues for CT versus those obtained with the reference technique validate the performance of CT as an accurate means of measuring concentration of radiopaque agent in tissue, independent of tracer dose.

Accumulation of protein-loaded long-circulating micelles and liposomes in subcutaneous Lewis lung carcinoma in mice.

PURPOSE: The purpose of our work was to compare the biodistribution and tumor accumulation of a liposome- or micelle-incorporated protein in mice bearing subcutaneously-established Lewis lung carcinoma. METHODS: A model protein, soybean trypsin inhibitor (STI) was modified with a hydrophobic residue of N-glutaryl-phosphatidyl-ethanolamine (NGPE) and incorporated into both polyethyleneglycol(MW 5000)-distearoyl phosphatidyl ethanolamine (PEG-DSPE) micelles (< 20 nm) and PEG-DSPE-modified long-circulating liposomes (ca. 100 nm). The protein was labeled with 111In via protein-attached diethylene triamine pentaacetic acid (DTPA), and samples of STI-containing liposomes or micelles were injected via the tail vein into mice bearing subcutaneously-established Lewis lung carcinoma. At appropriate time points, mice were sacrificed and the radioactivity accumulated in the tumor and main organs was determined. RESULTS: STI incorporated into PEG-lipid micelles accumulates in subcutaneously established Lewis lung carcinoma in mice better than the same protein anchored in long-circulating PEG-liposomes. CONCLUSIONS: Small-sized long-circulating delivery systems, such as PEG-lipid micelles, are more efficient in the delivery of protein to Lewis lung carcinoma than larger long-circulating liposomes.

Massage-induced release of subcutaneously injected liposome-encapsulated drugs to the blood.

Liposome-based, externally regulated drug delivery system is described in which liposome-encapsulated bioactive molecules can be delivered into the blood in response to simple mechanical action. Without any mechanical stimulation, subcutaneously injected 200 mm liposomes are usually trapped in the interstitial for prolonged time. However, upon lymphotropic stimulation (such as manual massage of the injection site), the liposomes can be mobilized into the blood via lymphatic pathway. Up to 40% of the injection dose can be delivered to the blood via lymphatic pathway from the injection site at the rabbit's front paw dorsum during 5 min manual massage cycle. Using vasoconstricting hormone angiotensin II as liposome-encapsulated pharmacological marker, we demonstrated that physiological response to encapsulated drug (average blood pressure increase) can also induced and modulated by massage. Massage itself was found to have no effect on the blood pressure. Modification of liposome surface with polyethylene glycol was found to increase blood localization of the liposome-encapsulated drug presumably due to decreasing the uptake of the drug carrier by lymph node macrophages. Pressure-dependent gaps between lymphatic capillary endothelial cells are thought to play the role of the size discrimination device allowing larger particulates into the lymphatics and, eventually into the blood after increase of interstitial pressure caused by injection site massage.

Retrograde nailing of the femur using an intercondylar approach.

The standard of care for femoral fractures is an intramedullary nail, placed in an antegrade approach. This has produced high rates of healing and low complication rates. The use of retrograde nailing for femoral shaft fractures is a relatively new technique, previously described as an extraarticular approach. Forty-one patients with 45 fractures were available for assessment after retrograde femoral nailing using an intracondylar approach. There were 2 nonunions, which healed with revision surgery, in addition to 5 other complications including: reflex sympathetic dystrophy, an ileus, deep vein thrombosis, skin loss about the knee, and malrotation noted after initial nailing. There were no postoperative infections, no arthrosis was noted, and flexion of the knee averaged 129 degrees. Retrograde femoral nailing may be used to treat fractures in patients who present with multisystem trauma; multiskeletal trauma, especially peritrochanteric and acetabular injuries; floating knee fractures; bilateral femur fractures; and patients with morbid obesity.

Stable polymeric micelles: lymphangiographic contrast media for gamma scintigraphy and magnetic resonance imaging.

RATIONALE AND OBJECTIVES: Amphiphilic biocompatible polyoxyethylene (PEO)-based polymers form particles (micelles) that are 10-50 nm in diameter. In the current research, we successfully incorporated amphiphilic indium-111 (111In) and gadolinium chelates into these particles and used them as particulate contrast media in percutaneous lymphography. METHODS: Micelles of amphiphilic PEO-lipid conjugates were loaded with 111In and gadolinium diethylenetriamine pentaacetic acid-phosphatidylethanolamine (Gd-DTPA-PE) and were injected subcutaneously into the rabbit's paw. Corresponding images of local lymphatics were acquired using a gamma camera and a magnetic resonance (MR) imager. RESULTS: The entire lymphatic chain from the paw to the thoracic duct could be visualized using 111In micelles after injection site massage. T1-weighted MR images of the primary lymph node and collecting vessels were obtained within 4 min after administration of gadolinium micelles and massage. CONCLUSION: Polymeric PEO-containing micelles can be loaded with diagnostic metals and, on subcutaneous injection, can visualize elements of lymphatic system. The major fraction of injected micelles stays within the lymph fluid, thus serving as lymphangiographic agents for indirect MR or gamma lymphography.

New synthetic amphiphilic polymers for steric protection of liposomes in vivo.

Carboxy group-terminated synthetic polymers--branched poly(ethylene glycol), poly(acryloylmorpholine), and poly(vinylpyrrolidone)--were made amphiphilic by derivatization with phosphatidyl ethanolamine via the terminal carboxy group and then incorporated into lecithin-cholesterol liposomes prepared by the detergent dialysis method. Following the biodistribution of liposomes in mice, all three polymers were shown to be effective steric protectors for liposomes and were able to sharply increase liposome circulation times in a concentration-dependent manner. The accumulation of liposomes in the liver decreases. The effects observed are similar to those found for liposomes modified with linear poly(ethylene glycol). At low polymer concentration, amphiphilic branched poly(ethylene glycol) seems to be the most effective liposome protector, most probably, because at the same molar content of anchoring groups, each attachment point carries two polymeric chains and doubles the quantity of liposome-grafted polymer comparing to linear poly(ethylene glycol).

Effects of continuous lateral rotation therapy on pulmonary complications in liver transplant patients.

BACKGROUND: When liver transplant candidates and recipients suffer from pulmonary complications of immobility, the results can be life-threatening. Continuous lateral rotation therapy has been reported to decrease complications of immobility. OBJECTIVES: To determine whether continuous lateral rotation therapy decreases the duration of mechanical ventilation, intensive care unit length of stay, incidence or resolution of atelectasis, incidence or onset time of lower respiratory tract infection and pneumonia. METHODS: Sixty-nine subjects admitted to a liver transplant intensive care unit at a university teaching hospital were randomly assigned to continuous lateral rotation therapy or a stationary bed. All subjects were mechanically ventilated for 24 hours and had a Glasgow Coma Scale score of 11 or less upon admission to the study. Subjects were followed until out of bed, unable to rotate for 3 consecutive days, or transferred from the intensive care unit. Data and chest roentgenogram results were collected on admission and daily during the study. Sputum culture results were obtained if available as part of normal patient care. RESULTS: Incidence of lower respiratory tract infection was significantly lower and length of time to occurrence of lower respiratory tract infection was significantly longer in the continuous lateral rotation therapy group than in the stationary bed group. CONCLUSIONS: Although continuous lateral rotation therapy did not affect duration of mechanical ventilation, length of stay, or incidence of atelectasis, it was effective in decreasing the incidence of, and increasing onset time to, lower respiratory tract infection in the liver transplantation population.

Amphiphilic vinyl polymers effectively prolong liposome circulation time in vivo.

Newly synthesized amphiphilic polyacrylamide and poly(vinyl pyrrolidone), single terminus-modified with long-chain fatty acyl groups, are able to incorporate into the liposomal membrane, and similar to poly(ethylene glycol) prolong liposome circulation in vivo and decrease liposome accumulation in the liver. Protective efficacy of modified polymers increases with the increase in the length of acyl moiety and decreases for higher molecular weight polymers. The data on amphiphilic polymer-modified liposome biodistribution are presented.

Using clinical innovations for research-based practice.

Making choices about patient-care interventions pervades critical care nursing practice. Research utilization activities provide the reasoning by which assessment parameters are established, preventative actions are identified, and interventions are evaluated in the clinical setting for positive effects on patient outcomes. For research results to be directly applicable, they must be transformed into clinical innovations specific to a patient population, clinical situation, or institutional setting. A brief summary of using research findings to design clinical innovations is provided. Examples of selected clinical innovations are included to illustrate the steps of the research utilization process. Clinical innovations are intended to improve or validate patient outcomes and are considered the key to quality patient care.