Transdermal photopolymerization for minimally invasive implantation.
Photopolymerizations are widely used in medicine to create polymer networks for use in applications such as bone restorations and coatings for artificial implants. These photopolymerizations occur by directly exposing materials to light in "open" environments such as the oral cavity or during invasive procedures such as surgery. We hypothesized that light, which penetrates tissue including skin, could cause a photopolymerization indirectly. Liquid materials then could be injected s.c. and solidified by exposing the exterior surface of the skin to light. To test this hypothesis, the penetration of UVA and visible light through skin was studied. Modeling predicted the feasibility of transdermal polymerization with only 2 min of light exposure required to photopolymerize an implant underneath human skin. To establish the validity of these modeling studies, transdermal photopolymerization first was applied to tissue engineering by using "injectable" cartilage as a model system. Polymer/chondrocyte constructs were injected s.c. and transdermally photopolymerized. Implants harvested at 2, 4, and 7 weeks demonstrated collagen and proteoglycan production and histology with tissue structure comparable to native neocartilage. To further examine this phenomenon and test the applicability of transdermal photopolymerization for drug release devices, albumin, a model protein, was released for 1 week from photopolymerized hydrogels. With further study, transdermal photpolymerization potentially could be used to create a variety of new, minimally invasive surgical procedures in applications ranging from plastic and orthopedic surgery to tissue engineering and drug delivery. (+info
Enhancement of fluid filtration across tumor vessels: implication for delivery of macromolecules.
Cancer therapies using genes and other macromolecules might realize their full clinical potential if they could be delivered to tumor tissue in optimal quantities. Unfortunately, the compromised circulation within tumors poses a formidable resistance to adequate and uniform penetration of these agents. Previously, we have proposed elevated interstitial fluid pressure (IFP) as a major physiological barrier to delivery of macromolecules. Here we postulate that modulation of tumor microvascular pressure (MVP) and associated changes in IFP would enhance macromolecular delivery into a solid tumor. To test our hypothesis, we altered tumor MVP by either periodic injection or continuous infusion of angiotensin II (AII) and measured the resulting changes in IFP and uptake of macromolecules. We used the nicotinyl hydrazine derivative of human polyclonal IgG (HYNIC-IgG) as a nonspecific macromolecule and CC49 antibody as a specific macromolecule. We found that both chronic and periodic modulation of tumor MVP enhances transvascular fluid filtration, leading to a 40% increase in total uptake of the specific antibody within 4 hr of its administration. Conversely, neither continuous nor periodic infusion of AII induced any increase in uptake of nonspecific antibodies. Strategies to improve delivery of macromolecules and limitations of this approach are identified. (+info
Oxidized low-density lipoprotein as a delivery system for photosensitizers: implications for photodynamic therapy of atherosclerosis.
Photodynamic therapy is a promising new strategy in the treatment of cardiovascular diseases. Photodynamic therapy for vascular diseases may be improved by the specific delivery of photosensitizers to the atherosclerotic lesion. In this study, we studied whether oxidatively modified low-density lipoprotein (OxLDL) could be used as a specific carrier for photosensitizers, thereby using the scavenger receptor expressed on macrophages as a target. The photosensitizer aluminum phthalocyanine chloride (AlPc) was incorporated into OxLDL, and its photodynamic effects were studied. Macrophages (RAW 264.7) were incubated with various concentrations of OxLDL-AlPc for different periods. After illumination of the cells with red light, cytotoxicity was observed that was dependent on the time of illumination and incubation. Macrophages incubated with OxLDL-AlPc that were not illuminated revealed no cytotoxicity. The uptake of the OxLDL-AlPc complexes was mediated by scavenger receptors expressed on macrophages. In the presence of the polyanion polyinosinic acid, a specific ligand for scavenger receptors, no cytotoxicity could be observed. Serum incubations of the OxLDL-AlPc complexes revealed that these complexes stay intact after incubation. No redistribution of AlPc to other plasma (lipo-) proteins could be detected, and 80-90% of the AlPc remained associated with the OxLDL particle. These results indicate that OxLDL may function as a specific delivery system for photosensitizers to the scavenger receptors expressed on the macrophages in the atherosclerotic lesion, increasing the beneficial effects of photodynamic therapy for cardiovascular diseases. (+info
Early use of inhaled nedocromil sodium in children following an acute episode of asthma.
BACKGROUND: Current guidelines on the treatment of childhood asthma recommend the introduction of an anti-inflammatory drug in children who have persistent symptoms and require regular treatment with a bronchodilator. The efficacy and safety of inhaled nedocromil sodium (Tilade Mint aerosol) administered using a Fisonair spacer at a dose of 4 mg three times daily was compared with placebo in the treatment of asthmatic children aged 6-12 years who are symptomatic and recovering from an acute exacerbation of asthma. METHODS: A group comparative, double blind, placebo controlled trial was performed in children who were recovering from an acute episode of asthma following treatment in the emergency department of the hospital or in children referred from their general practitioner following a wheezing episode and documented evidence of at least two previous episodes of wheezing. A two week baseline period on existing bronchodilator treatment was followed by a 12 week treatment period on either nedocromil sodium (2 mg/puff) or placebo. Both treatments were administered using a Fisonair spacer at a dose of two puffs three times daily. Changes from baseline values in daytime asthma and night time asthma symptom scores, usage of rescue bronchodilators, mean peak expiratory flow (PEF) recorded twice daily on diary cards, patients' opinion of treatment, and withdrawals due to treatment failure were measured during the primary treatment period (last six weeks of treatment). RESULTS: One hundred and forty two children aged 6-12 years entered the baseline period. Sixty three were withdrawn due to failure to meet the entry criteria (18) or the criteria for asthma symptom severity (15) or reversibility (9), because they developed uncontrolled asthma (2), because they took disallowed treatment (2), or for other non-trial related reasons (17). Seventy nine patients (46 boys) of mean age 8. 8 years entered the treatment period. There were significant differences in the changes from baseline values during the last six weeks of treatment in favour of nedocromil sodium compared with placebo in the primary variables of daytime asthma and night time asthma, morning and evening PEF, and the usage of rescue inhaled bronchodilators; 53% of patients reported nedocromil sodium to be very or moderately effective compared with 44% placebo. Improvement in asthma symptoms, PEF, and reduction in use of rescue bronchodilators did not reach statistical significance until after six weeks of treatment. Twenty two patients were withdrawn or dropped out during the treatment phase, 12 due to uncontrolled asthma or persistence of asthma symptoms, four due to suspected adverse drug reactions (nedocromil sodium 3 (headaches 2, angio-oedema/urticaria 1), placebo 1(persistent cough)), and six due to non-treatment related reasons. Seventy one adverse events were reported by 27 patients in the nedocromil group and 75 by 30 patients in the placebo group. CONCLUSIONS: Asthma symptoms, use of bronchodilators, and lung function can be improved significantly in children recovering from an acute exacerbation of asthma or wheeze and currently receiving treatment with bronchodilators alone by the addition of inhaled nedocromil sodium at a dose of 4 mg three times daily administered using a Fisonair holding chamber. (+info
Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.
The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment. (+info
Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor.
Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides. (+info
Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery.
Poly(ethylenimine) (PEI) is one of a number of polycations that has been used successfully to transfer genes into living cells. Although PEI shows promise in the field of gene therapy, to date no rigorous proof of mechanism has been published regarding the fate of PEI/DNA administered for transfection. Here we show, by using fluorescent labeling and confocal microscopy, the paths of PEI/DNA complexes from endocytosis to gene expression. We found that complexes attach to cell surfaces and migrate into clumps that are endocytosed. The endocytotic vesicles grow in number and size and are occasionally seen to lyse. Most interesting is the fact that endocytosed PEI, whether administered with or without DNA, undergoes nuclear localization in the form of ordered structures. (+info
Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin.
We report an intracellular peptide delivery system capable of targeting specific cellular compartments. In the model system we constructed a chimeric protein consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (EtxB) fused to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. Viral DNA synthesis takes places in the nucleus and requires the interaction with an accessory factor, UL42, encoded by the virus. The peptide, designated Pol, is able to dissociate this interaction. The chimeric protein, EtxB-Pol, retained the functional properties of both EtxB and peptide components and was shown to inhibit viral DNA polymerase activity in vitro via disruption of the polymerase-UL42 complex. When added to virally infected cells, EtxB-Pol had no effect on adenovirus replication but specifically interfered with herpes simplex virus 1 replication. Further studies showed that the antiviral peptide localized in the nucleus, whereas the EtxB component remained associated with vesicular compartments. The results indicate that the chimeric protein entered through endosomal acidic compartments and that the Pol peptide was cleaved from the chimeric protein before being translocated into the nucleus. The system we describe is suitable for delivery of peptides that specifically disrupt protein-protein interactions and may be developed to target specific cellular compartments. (+info