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1.
Assay Drug Dev Technol ; 5(3): 343-54, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17638534

ABSTRACT

Development of high throughput screening (HTS) assays for evaluation of a compound's toxicity and potential for drug-drug interactions is a critical step towards production of better drug candidates and cost reduction in the drug development process. HTS assays for drug metabolism mediated by cytochrome P450s are now routinely used in compound library characterization and for computer modeling studies. However, development and application of HTS assays involving UDP-glucuronosyltransferases (UGTs) are lagging behind. Here we describe the development of a fluorescence-based HTS assay for UGT1A1 using recombinant enzyme and fluorescent substrate in the presence of an aqueous solution of PreserveX-QML (QBI Life Sciences, Madison, WI) polymeric micelles, acting as a stabilizer and a blocker of nonspecific interactions. The data include assay characteristics in 384-well plate format obtained with robotic liquid handling equipment and structures of hits (assay modifiers) obtained from the screening of a small molecule library at the University of Wisconsin HTS screening facility. The application of the assay for predicting UGT-related drug-drug interactions and building pharmacophore models, as well as the effects of polymeric micelles on the assay performance and compound promiscuity, is discussed.


Subject(s)
Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Glucuronosyltransferase/antagonists & inhibitors , Chromatography, High Pressure Liquid , Drug Interactions , Fluorescence , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/metabolism , Kinetics , Micelles
2.
Gene Ther ; 10(3): 261-71, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12571634

ABSTRACT

The intravenous delivery of plasmid DNA complexed with either cationic lipids (CL) or polyethyleneimine (PEI) enables high levels of foreign gene expression in lung. However, these cationic DNA complexes cause substantial toxicity. The present study found that the inclusion of polyacrylic acid (pAA) with DNA/polycation and DNA/CL complexes prevented the serum inhibition of the transfection complexes in cultured cells. The mechanism mediating this increase seems to involve both particle size enlargement due to flocculation and electrostatic shielding from opsonizing serum proteins. The use of pAA also increased the levels of lung expression in mice in vivo substantially above the levels achieved with just binary complexes of DNA and linear PEI (lPEI) or CL and reduced their toxicity. Also, the use of a "chaser" injection of pAA 30 min after injection of the ternary DNA/lPEI/pAA complexes further aided this effort to reduce toxicity while not affecting foreign gene expression. By optimizing the amount of pAA, lPEI, and DNA within the ternary complexes and using the "chaser" injection, substantial levels of lung expression were obtained while avoiding adverse effects in lung or liver. These developments will aid the use of cationic DNA complexes in animals and for eventual human gene therapy.


Subject(s)
Genetic Therapy/methods , Lung Diseases/therapy , Lung/metabolism , Transfection/methods , Acrylic Resins , Animals , Anions , Gene Expression , Genetic Engineering , Liposomes , Luciferases/genetics , Male , Mice , Mice, Inbred ICR , Microinjections , Polyethyleneimine , Tumor Cells, Cultured , beta-Galactosidase/genetics
3.
Br J Cancer ; 82(9): 1513-8, 2000 May.
Article in English | MEDLINE | ID: mdl-10789717

ABSTRACT

Molecular charge is one of the main determinants of transvascular transport. There are, however, no data available on the effect of molecular charge on microvascular permeability of macromolecules in solid tumours. To this end, we measured tumour microvascular permeability to different proteins having similar size but different charge. Measurements were performed in the human colon adenocarcinoma LS174T transplanted in transparent dorsal skinfold chambers in severe combined immunodeficient (SCID) mice. Bovine serum albumin (BSA) and IgG were fluorescently labelled and were either cationized by conjugation with hexamethylenediamine or anionized by succinylation. The molecules were injected i.v. and the fluorescence in tumour tissue was quantified by intravital fluorescence microscopy. The fluorescence intensity and pharmacokinetic data were used to calculate the microvascular permeability. We found that tumour vascular permeability of cationized BSA (pI-range: 8.6-9.1) and IgG (pI: 8.6-9.3) was more than two-fold higher (4.25 and 4.65x10(-7) cm s(-1)) than that of the anionized BSA (pI approximately 2.0) and IgG (pI: 3.0-3.9; 1.11 and 1.93x10(-7) cm s(-1), respectively). Our results indicate that positively charged molecules extravasate faster in solid tumours compared to the similar-sized compounds with neutral or negative charges. However, the plasma clearance of cationic molecules was approximately 2x faster than that of anionic ones, indicating that the modification of proteins enhances drug delivery to normal organs as well. Therefore, caution should be exercised when such a strategy is used to improve drug and gene delivery to solid tumours.


Subject(s)
Capillary Permeability , Neoplasms, Experimental/blood supply , Neoplasms, Experimental/chemistry , Animals , Cattle , Humans , Male , Mice , Mice, SCID , Microscopy, Fluorescence , Models, Theoretical , Transplantation, Heterologous
4.
Nucleic Acids Res ; 27(15): 3090-5, 1999 Aug 01.
Article in English | MEDLINE | ID: mdl-10454604

ABSTRACT

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.


Subject(s)
Anions/metabolism , Cations/metabolism , DNA/chemistry , DNA/metabolism , Electrolytes/metabolism , DNA/genetics , DNA/isolation & purification , Drug Carriers , Flocculation , Microscopy, Atomic Force , Molecular Weight , Nucleic Acid Conformation , Plasmids/chemistry , Plasmids/genetics , Plasmids/isolation & purification , Plasmids/metabolism , Polylysine/analogs & derivatives , Polylysine/metabolism , Solubility , Ultracentrifugation , Water
5.
Bioconjug Chem ; 10(4): 624-8, 1999.
Article in English | MEDLINE | ID: mdl-10411460

ABSTRACT

The assembly of DNA into compact particles that do not aggregate in physiologic salt solution occurs naturally in chromatin and viral particles but has been challenging to duplicate using artificial constructs. Cross-linking amino-containing polycations in the presence of DNA with bisimidoester cross-linker leads to the formation of caged DNA particles that are stable in salt solutions. This first demonstration of caged DNA provides insight into how natural condensation processes avoid aggregation and a promising avenue for developing nonviral gene therapy vectors.


Subject(s)
DNA/chemistry , Cross-Linking Reagents , Cyclohexanes/chemistry , DNA/genetics , Indicators and Reagents , Microscopy, Electron , Osmolar Concentration , Particle Size , Plasmids/chemistry , Plasmids/genetics , Polyamines/chemistry , Solutions , Transfection , Ultracentrifugation
6.
Anal Biochem ; 267(2): 309-13, 1999 Feb 15.
Article in English | MEDLINE | ID: mdl-10036135

ABSTRACT

A fluorescent method is proposed for assessing DNA condensation in aqueous solutions with variety of condensing agents. The technique is based on the effect of concentration-dependent self-quenching of covalently bound fluorophores upon DNA collapse. The method allows a more precise determination of charge equivalency in titration experiments with various polycations. The technique's ability to determine the number of DNA molecules that are condensed together in close proximity is under further investigation.


Subject(s)
DNA, Circular/chemistry , Fluorescence , Polylysine/pharmacology
7.
J Drug Target ; 6(1): 45-52, 1998.
Article in English | MEDLINE | ID: mdl-9769020

ABSTRACT

The high-molecular-weight soluble aggregates of Fab fragments of murine antibodies against cardiac myosin were prepared as a potential long-circulating and low immunogenic pharmaceutical carriers by conjugation of thiolated Fab and Fab modified with succinimidyl 3-(2-pyridyldithio)propionate. The clearance time and biodistribution of 111In-radiolabeled aggregates were studied in normal and nude-mice bearing human breast tumor implant and in rabbits with experimental myocardial infarction. The aggregates had a prolonged circulation time (half clearance time ca. 3-5 h) and ability to concentrate in the tumor and in the necrotic area of infarcted myocardium. Similar tumor-to-normal and infarct-to-normal accumulation ratios (ca. 3 h in both cases) suggest that combination of long circulation with impaired filtration in necrotic tissues is responsible for this accumulation rather than a specific interaction. The aggregates prepared may serve as long-circulating drug carriers able to deliver pharmaceuticals into areas with affected and leaky vasculature.


Subject(s)
Breast Neoplasms/metabolism , Cross-Linking Reagents/chemistry , Disulfides/chemistry , Drug Delivery Systems , Immunoglobulin Fab Fragments/pharmacology , Myocardial Infarction/metabolism , Animals , Chromatography, High Pressure Liquid , Disease Models, Animal , Drug Carriers , Enzyme-Linked Immunosorbent Assay , Female , Humans , Indium Radioisotopes , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Neoplasm Transplantation , Rabbits
8.
Nucleic Acids Res ; 26(18): 4178-85, 1998 Sep 15.
Article in English | MEDLINE | ID: mdl-9722638

ABSTRACT

The self-assembly of supramolecular complexes of nucleic acids and polymers is of relevance to several biological processes including viral and chromatin formation as well as gene therapy vector design. We now show that template polymerization facilitates condensation of DNA into particles that are <150 nm in diameter. Inclusion of a poly(ethylene glycol)-containing monomer prevents aggregation of these particles. The DNA within the particles remains biologically active and can express foreign genes in cells. The formation or breakage of covalent bonds has until now not been employed to compact DNA into artificial particles.


Subject(s)
DNA/chemistry , Polyethylene Glycols/chemistry , 3T3 Cells , Acrylates , Animals , Antigens, Polyomavirus Transforming/genetics , Binding Sites , Cross-Linking Reagents , DNA/genetics , DNA/ultrastructure , Dimerization , Genes, Reporter , Luciferases/biosynthesis , Luciferases/genetics , Mice , Microscopy, Electron , Plasmids/chemistry , Plasmids/genetics , Plasmids/ultrastructure , Recombinant Proteins/biosynthesis , Simian virus 40/genetics , Templates, Genetic , Transfection
9.
J Control Release ; 50(1-3): 13-9, 1998 Jan 02.
Article in English | MEDLINE | ID: mdl-9685868

ABSTRACT

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.


Subject(s)
Drug Delivery Systems , Massage , Angiotensin II/administration & dosage , Angiotensin II/blood , Animals , Drug Carriers , Injections, Subcutaneous , Liposomes , Rabbits
11.
J Drug Target ; 4(6): 381-8, 1997.
Article in English | MEDLINE | ID: mdl-9239578

ABSTRACT

In order to obtain small, polymer-stabilized particulate carriers for organic iodine to serve as a contrast agent for X-ray computed tomography (CT) an attempt was made to design a carrier based on polymeric micelles. Here we describe the synthesis of an iodine-containing amphiphilic block-copolymer which can micellize in aqueous solutions. The two blocks of the copolymer consisted of methoxypoly(ethyleneglycol) and poly[epsilon,N-(triiodobenzoyl)-L-lysine]. Upon dispersion in water, the block copolymer formed particles with average diameter 80 nm and iodine content up to 44.7%. The particles start to dissociate to the individual polymeric chains in the concentration range of 0.05-0.5 microM in water at 23 degrees C. Upon intravenous injection at 250 mg of iodine/kg (570 mg of the agent/kg) in rabbits the medium demonstrated exceptional 24 hr half-life in the blood substantiating corona/core structure of the particles with PEG chains protecting the iodine-containing core. The possible use of these particulates as contrast medium for X-ray computed tomography is discussed.


Subject(s)
Contrast Media/chemical synthesis , Drug Design , Iodine/administration & dosage , Polyethylene Glycols/chemistry , Polylysine/chemistry , Polymers/chemical synthesis , Tomography, X-Ray Computed , Chromatography, High Pressure Liquid , Drug Carriers , Polyethylene Glycols/chemical synthesis , Polylysine/analogs & derivatives , Polylysine/chemical synthesis , Triiodobenzoic Acids/chemical synthesis , Triiodobenzoic Acids/chemistry
12.
Acad Radiol ; 3(3): 232-8, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8796670

ABSTRACT

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.


Subject(s)
Contrast Media , Gadolinium DTPA , Lymph Nodes/anatomy & histology , Lymph Nodes/diagnostic imaging , Magnetic Resonance Imaging , Micelles , Polyethylene Glycols , Animals , Indium Radioisotopes , Pentetic Acid/analogs & derivatives , Phosphatidylethanolamines , Rabbits , Radionuclide Imaging
13.
J Pharm Sci ; 84(9): 1049-53, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8537880

ABSTRACT

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).


Subject(s)
Liposomes/chemistry , Polymers/chemistry , Animals , Detergents , Liposomes/pharmacokinetics , Liver/metabolism , Mice , Molecular Weight , Morpholines/chemical synthesis , Morpholines/chemistry , Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/chemistry , Polymers/chemical synthesis , Povidone/chemical synthesis , Povidone/chemistry , Tissue Distribution
15.
Magn Reson Imaging ; 13(1): 31-7, 1995.
Article in English | MEDLINE | ID: mdl-7534860

ABSTRACT

Surface modification of liposomes containing Gd-diethylenetriaminepentaacetyl-phosphatidylethanolamine (Gd-DTPA-PE) as an amphiphilic paramagnetic label influences their contrast properties as MRI agents. Liposomes with the mean diameter ca. 220 nm and 10% mol content of Gd-DTPA-PE were modified with dextran (molecular weight 6 kDa) or polyethyleneglycol (PEG, molecular weight 5 kDa). The preparations obtained were used for MR visualization of axillary and subscapular lymph nodes after SC injection into rabbit's paw. Dextran-modified liposomes were found to have limited or no effect on lymph node/muscle MR signal intensity ratio during the first 2 h after the administration when compared to nonmodified plain liposomes, whereas the liposomes modified with PEG demonstrated a 3-3.5-fold enhancement of lymph node signal. However, the partial biodistribution studies with the 111In-labeled liposomes revealed that PEG-modified vesicles accumulated in the axillary lymph node at half the amount comparing with nonmodified and dextran-modified liposomes. The phenomenon found might be explained by noticeably increased relaxivity of PEG-modified Gd-liposomes.


Subject(s)
Contrast Media/administration & dosage , Gadolinium DTPA , Lymph Nodes/anatomy & histology , Magnetic Resonance Imaging/methods , Pentetic Acid/analogs & derivatives , Phosphatidylethanolamines , Animals , Dextrans , Liposomes , Polyethylene Glycols , Rabbits , Surface Properties
16.
J Nucl Cardiol ; 2(1): 26-34, 1995.
Article in English | MEDLINE | ID: mdl-9420759

ABSTRACT

BACKGROUND: Cell surfaces and intercellular matrixes contain acidic residues, making them negatively charged. Antibodies are basic, positively charged glycoproteins. Therefore the potential for nonspecific ionic interaction exists, which could increase the background activity. Modification of antibodies with negatively charge-modified polymers have been shown to reduce this nonspecific background activity. This study was performed to investigate the appropriateness of different cross-linkers used covalently to link the chelating negatively charge-modified polylysine to antimyosin Fab (AM-Fab). The cross-linking was performed through peptide (AM-I) or thioether (AM-II) bonds. The in vitro evaluation of the immunointegrity and the in vivo assessment were performed to investigate the potential for reduction of nontarget background activity. Furthermore, the role of the charge of the polymers (whether completely negatively charge modified by succinylation [AM-IIs] or only partially negatively charge modified [AM-IIns]) was also assessed. METHODS AND RESULTS: All polymer-modified preparations (AM-I, AM-IIs, and AM-IIns) retained the immunoreactivities relative to the unmodified or conventional diethylenetriaminepentaacetic acid-coupled AM-Fab as assessed by radioimmunoassay or enzyme-linked immunosorbent assay. These polymer-modified preparations labeled with 111In were assessed in 13 rabbits with acute experimental myocardial infarction. Acute infarcts were produced by 40 minutes of left anterior descending coronary artery occlusion followed by reperfusion. At between 10 and 30 minutes of reperfusion, 10.4 +/- 1.8 mBq 111In-AM-I (10 to 20 micrograms; n = 7) or 11.4 +/- 2.3 mBq 111In-AM-II (n or ns) (20 to 25 micrograms; n = 6) was administered intravenously. Gamma imaging was performed in the left lateral position and arterial blood samples were withdrawn serially for the next 3 hours. At the end of the final imaging session, AM-I uptake was determined to be 1.09% +/- 0.11% (mean percent injected dose per gram myocardium +/- SEM) in 20 infarcted myocardial segments from seven rabbits, compared with 0.031% +/- 0.003% in 20 normal myocardial segments (infarct/normal myocardial ratio 53.9 +/- 18.41). The mean percent injected dose of 111In-labeled thioether-linked AM-Fab preparations in nine infarcted myocardial segments from each group was 0.067% +/- 0.008% (infarct/normal myocardial ratio 9.0 +/- 1.5) and 0.144% +/- 0.011% (infarct/normal myocardial ratio 10.2 +/- 1.9) with AM-IIs (n = 3) and AM-IIns (n = 3), respectively (p < 0.0001). The non-target organ distribution of the AM-I and AM-IIs was similar. AM-IIns preparation resulted in high non-target organ activities. CONCLUSIONS: This study shows that the charge of the antibody can be manipulated favorably by cross-linking with negatively charged polymers, which results in the reduced in vivo non-target organ activities. Charge modification does not adversely affect the apparent affinity of the antibody. However, the type of cross-linkers used may significantly influence the in vivo stability of the modified antibody preparations for target organ visualization. These data may find potential application in future clinical imaging protocols.


Subject(s)
Immunoglobulin Fab Fragments , Indium Radioisotopes , Myocardial Infarction/diagnostic imaging , Myosins/immunology , Radioimmunodetection , Animals , Male , Rabbits , Tissue Distribution
17.
Biochim Biophys Acta ; 1195(1): 11-20, 1994 Oct 12.
Article in English | MEDLINE | ID: mdl-7918551

ABSTRACT

The hypothetical model is built explaining the molecular mechanism of protective action of poly(ethylene glycol) on liposomes in vivo. The protective layer of the polymer on the liposome surface is considered as a statistical 'cloud' of polymer possible conformations in solution. Computer simulation was used to demonstrate that relatively a small number of liposome-grafted molecules of hydrophilic and flexible polymer can create a dense protective conformational cloud over the liposome surface preventing opsonizing protein molecules from contacting liposome. A more rigid polymer fails to form this dense protective cloud, even when hydrophilic. Computer simulation was also used to reveal possible heterogeneity of reactive sites on a polymer-coated liposome surface, and to estimate the optimal polymer-to-lipid ratio for efficient liposome protection. Experiments have been performed with the quenching of liposome-associated fluorescent label (nitrobenzoxadiazole or fluorescein) with protein (rhodamine-ovalbumin or anti-fluorescein antibody) from solution. It was shown that poly(ethylene glycol) grafting to liposomes hinders protein interaction with the liposome surface, whereas liposome-grafted dextran (more rigid polymer) in similar quantities does not affect protein-liposome interaction. Highly-reactive and low-reactive populations of chemically identical reactive sites have been found on polymer-coated liposomes. Experimental data satisfactory confirm the suggested mechanism for the longevity of polymer-modified liposome.


Subject(s)
Liposomes/chemistry , Polyethylene Glycols/analysis , Polymers/chemistry , Binding Sites , Computer Simulation , Drug Stability , Fluorescent Dyes , Molecular Conformation , Surface Properties
18.
Biochim Biophys Acta ; 1195(1): 181-4, 1994 Oct 12.
Article in English | MEDLINE | ID: mdl-7918561

ABSTRACT

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.


Subject(s)
Liposomes/pharmacokinetics , Polyvinyls/chemistry , Animals , Drug Carriers , Liposomes/chemistry , Liver/metabolism , Mice , Mice, Inbred BALB C , Polyethylene Glycols/chemistry , Tissue Distribution
19.
Bioconjug Chem ; 4(4): 251-5, 1993.
Article in English | MEDLINE | ID: mdl-8218480

ABSTRACT

Murine antimyosin Fab fragment was conjugated with 111In-labeled N-terminal-modified DTPA-polylysine using three bifunctional reagents: N-hydroxysuccinimide esters of 3-(2-pyridyldithio)propionic acid (SPDP conjugate), 4-(maleimidomethyl)cyclohexanecarboxylic acid (SMCC conjugate) and bromoacetic acid (BrAc conjugate) for potential localization of experimental myocardial infarction. Using various antibody preparations and a rabbit acute myocardial infarction model the following parameters were observed: (1) an in vitro antigen binding activity of SPDP conjugate = SMCC conjugate > BrAc conjugate, (2) a blood clearance rate of SPDP conjugate > BrAc conjugate > SMCC conjugate, (3) a liver and splenic accumulation of SPDP conjugate > BrAc conjugate > SMCC conjugate, and (4) the infarcted tissue activity showed an accumulation of SMCC conjugate > SPDP conjugate > BrAc conjugate. This study exemplifies the importance of rational chemical design of antimyosin Fab-chelating polymer conjugate for improved target tissue localization in vivo.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin Fab Fragments/chemistry , Myocardial Infarction/metabolism , Myosins/immunology , Animals , Antibodies, Monoclonal/blood , Antibody Specificity , Antigen-Antibody Reactions , Chelating Agents/chemistry , Cross-Linking Reagents/chemistry , Immunoglobulin Fab Fragments/blood , Indium Radioisotopes/blood , Metabolic Clearance Rate , Mice , Myocardium/metabolism , Pentetic Acid/chemistry , Polylysine/blood , Polylysine/chemistry , Rabbits , Tissue Distribution
20.
Infect Immun ; 60(9): 3897-903, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1500196

ABSTRACT

To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.


Subject(s)
Lipopolysaccharides/administration & dosage , Neisseria meningitidis/pathogenicity , Animals , Antibodies, Bacterial/analysis , Fever/chemically induced , Limulus Test , Lipopolysaccharides/immunology , Lipopolysaccharides/toxicity , Liposomes/administration & dosage , Mice , Mice, Inbred CBA , Rabbits , Viral Plaque Assay
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