Biomedical subjects

A S Hoffman

Publications and source records attributed to A S Hoffman.

At least 19 recordsLinked to original sources

PubMed2005

'Smart' delivery systems for biomolecular therapeutics.

OBJECTIVE: There is a strong need for drug delivery systems that can deliver biological signals from biomaterials and tissue engineering scaffolds, and a particular need for new delivery systems that can efficiently deliver biomolecules to intracellular targets. Viruses and pathogens have evolved potent molecular machinery that sense the lowered pH gradient of the endosomal compartment and become activated to destabilize the endosomal membrane, thereby enhancing protein or DNA transport to the cytoplasmic compartment. A key feature of many of these biological delivery systems is that they are reversible, so that the delivery systems are not directly toxic. These delivery systems have the ability to change their structural and functional properties and thus display remarkable 'smart' material properties. The objective of this presentation is to review the initial development of smart polymeric carriers that mimic these biological delivery systems and combine similar pH-sensitive, membrane-destabilizing activity for the delivery of therapeutic biomolecules. DESIGN: We have developed new 'smart' polymeric carriers to more effectively deliver and broaden the available types of biomolecular therapeutics. The polymers are hydrophilic and stealth-like at physiological pH, but become membrane-destabilizing after uptake into the endosomal compartment where they enhance the release of therapeutic cargo into the cytoplasm. They can be designed to provide a range of pH profiles and membrane-destabilizing activities, allowing their molecular properties to be matched to specific drugs and loading ranges. A versatile set of linker chemistries is available to provide degradable conjugation sites for proteins, nucleic acids, and/or targeting moieties. RESULTS: The physical properties of several pH-responsive polymers were examined. The activity and pH profile can be manipulated by controlling the length of hydrophobic alkyl segments. The delivery of poly(propyl acrylic acid) (PPAA)-containing lipoplexes significantly enhanced wound healing through the interconnected effects of altered extracellular matrix organization and greater vascularization. PPAA has also been shown to enhance cytoplasmic delivery of a model protein therapeutic. Polymeric carriers displaying pH-sensitive, membrane-destabilizing activity were also examined. The pH profile is controlled by the choice of the alkylacrylic acid monomer and by the ratio of the carboxylate-containing alkylacrylic acid monomer to alkylacrylate monomer. The membrane destabilizing activity is controlled by the lengths of the alkyl segment on the alkylacrylic acid monomer and the alkylacrylate monomer, as well as by their ratio in the final polymer chains. CONCLUSION: The molecular mechanisms that proteins use to sense and destabilize provide interesting paradigms for the development of new polymeric delivery systems that mimic biological strategies for promoting the intracellular delivery of biomolecular drugs. The key feature of these polymers is their ability to directly enhance the intracellular delivery of proteins and DNA, by destabilizing biological membranes in response to vesicular compartment pH changes. The ability to deliver a wide variety of protein and nucleic acid drugs to intracellular compartments from tissue engineering and regenerative scaffolds could greatly enhance control of important processes such as inflammation, angiogenesis, and biomineralization.

Biocompatible Materials↗

PubMed2001

New antibody purification procedure using a thermally responsive poly(N-isopropylacrylamide)-dextran derivative conjugate.

Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble-insoluble poly(N-isopropylacrylamide)-dextran derivative conjugate, named poly(NIPAAm)-DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)-DD conjugation and characterization. On the other hand, the poly(NIPAAm)-DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)-DD. In contrast, they did not bind to native poly(NIPAAm) or poly(NIPAAm) conjugated with another anionic dextran. We conclude that the conjugation of a polysaccharide to poly(NIPAAm) leads to an original and efficient chromatographic method to purify antibodies. Moreover, this novel method of purification is rapid, sensitive, inexpensive and could be used to purify various types of antibodies.

Acrylamides↗

PubMed2001

Release of PEGylated granulocyte-macrophage colony-stimulating factor from chitosan/glycerol films.

We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC). The amount of PEG-(GM-CSF) released from the films decreased with an increase in the amount of glycerol present in the film. In parallel with this, films with higher glycerol content exhibited a lower degree of equilibrium swelling when immersed in release media. pH measurements of the release media and analysis of the dried films by Fourier-transform infrared spectroscopy (FTIR) suggested that the amount of residual acetic acid in the dry films decreased as the glycerol content increased. This indicates that glycerol may act by displacing and releasing bound acetic acid from the chitosan molecules, resulting in chitosan--glycerol hydrogen bond formation as the film dries. Further, it was found that the release rate and the amount of PEG-(GM-CSF) released decreased with increasing molecular weight of the conjugated PEG. This effect was not observed with films containing physical mixtures of PEG and GM-CSF. The decrease in the fraction of PEG-(GM-CSF) released with increasing PEG molecular weight is believed to be due to the increased steric hindrance of the PEGylated protein molecule during its diffusion out of the swollen chitosan/glycerol film.

Biocompatible Materials↗

PubMed2001

Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield.

Many medical and biotechnological processes rely on controlling and manipulating the molecular-recognition capabilities of proteins. This can be achieved using small molecules capable of competing for protein binding or by changing environmental parameters that affect protein structure and hence binding. An alternative is provided by stimuli-responsive polymers that change reversibly from a water-soluble expanded coil to a water-insoluble collapsed globule upon small changes in temperature, pH or light intensity: when attached to proteins in the vicinity of their binding sites, they reversibly block and release small ligands. Here we show how this approach can be extended to achieve size-selective binding of large, macromolecular ligands. We use the thermally responsive polymer poly(N,N-diethylacrylamide) (PDEAAm), and attach it to the protein streptavidin approximately 20 A from the binding site for biotinylated proteins. Below the lower critical solution temperature of PDEAAm, the polymer is in its extended state and acts as a 'shield' to block the binding of large biotinylated proteins; above this temperature, it collapses and exposes the binding site, thereby allowing binding. We find that the degree of shielding depends on both the size of the biotinylated protein and the size of PDEAAm, suggesting that 'smart' polymer shields could be tailored to achieve a wide range of size-dependent ligand discrimination for use in affinity separations, biosensors and diagnostics technologies.

Acrylamides↗

PubMed2001

Control of shape and size of vascular smooth muscle cells in vitro by plasma lithography.

The ability to control the shape and size of cells is an important enabling technique for investigating influences of geometrical variables on cell physiology. Herein we present a micropatterning technique ("plasma lithography") that uses photolithography and plasma thin-film polymerization for the fabrication of cell culture substrates with a cell-adhesive pattern on a cell-repellent (non-fouling) background. The micron-level pattern was designed to isolate individual vascular smooth muscle cells (SMC) on areas with a projected area of between 25 and 3600 microm(2) in order to later study their response to cytokine stimulation in dependence of the cell size and shape as an indication for the phenotypic state of the cells. Polyethylene terephthalate substrates were first coated with a non-fouling plasma polymer of tetraglyme (tetraethylene glycol dimethyl ether). In an organic lift-off process, we then fashioned square- and rectangular-shaped islands of a thin fluorocarbon plasma polymer film of approximately 12-nm thickness. Electron spectroscopy for chemical analysis and secondary ion mass spectroscopy were used to optimize the deposition conditions and characterize the resulting polymers. Secondary ion mass spectroscopy imaging was used to visualize the spatial distribution of the polymer components of the micropatterned surfaces. Rat vascular SMC were seeded onto the patterned substrates in serum-free medium to show that the substrates display the desired properties, and that cell shape can indeed be controlled. For long-term maintenance of these cells, the medium was augmented with 10% calf serum after 24 h in culture, and the medium was exchanged every 3 days. After 2 weeks, the cells were still confined to the areas of the adhesive pattern, and when one or more cells spanned more than one island, they did not attach to the intervening tetraethylene glycol dimethyl ether (tetraglyme) background. Spreading-restricted cells formed a well-ordered actin skeleton, which was most dense along the perimeter of the cells. The shape of the nucleus was also influenced by the pattern geometry. These properties make the patterned substrates suitable for investigating if the phenotypic reversion of SMC can be influenced by controlling the shape and size of SMC in vitro.

Animals↗

PubMed2001

A reverse microemulsion polymerization method for preparation of bioadhesive polyacrylic acid nanoparticles for mucosal drug delivery: loading and release of timolol maleate.

Polyacrylic acid nanoparticles were successfully synthesized using a reverse microemulsion polymerization process. They had a narrow size range, averaging approximately 50 nm, and were stable in buffer. The particles were isolated and lyophilized in dry powder form, and were redispersible as individual particles in buffer. The drug timolol maleate was loaded into the nanoparticles from aqueous drug solutions and, when the drug-loaded particles were dispersed in a phosphate buffer solution, the drug slowly released over several hours from the nanoparticles.

Acrylic Resins↗

PubMed2001

Hydrogels for biomedical applications.

This paper reviews the composition and synthesis of hydrogels, the character of their absorbed water, and permeation of solutes within their swollen matrices. The most important properties of hydrogels relevant to their biomedical applications are also identified, in particular for use of hydrogels as drug and cell carriers, and as tissue engineering matrices.

Biocompatible Materials↗

PubMed2001

Plasma lithography--thin-film patterning of polymeric biomaterials by RF plasma polymerization I: Surface preparation and analysis.

Plasma lithography, combining plasma deposition with photolithography, is described as a versatile method to manufacture all-polymeric substrates with thin-film patterns for applications in biomedical engineering. Patterns of a hydrophobic fluorocarbon plasma polymer with feature sizes between 5 and 100 microm were deposited on a base substrate in a lift-off process: an intermediate tetraglyme plasma polymer layer provides non-fouling properties to the base substrate. Careful analysis of critical process parameters identified the narrow window of process conditions that led to the formation of functional surface patterns. High pattern fidelity, aspect ratios, and resolution of the patterns are demonstrated by atomic force microscopy. Electron spectroscopy for chemical analysis (ESCA) and secondary ion mass spectroscopy (SIMS) were used to characterize the surfaces, showing good retention of the original chemical structure of the pattern components throughout the process. SIMS imaging was used for specific chemical imaging of the components. Potential applications for the patterned polymer films, e.g., for studying cell behavior in vitro in dependence of shape and size of adhering cells, are discussed.

Biocompatible Materials↗

PubMed2001

Plasma lithography--thin-film patterning of polymers by RF plasma polymerization II: Study of differential binding using adsorption probes.

In this study we present methods to physico-chemically modify micropatterned cell culture substrates that were manufactured using plasma lithography to incorporate affinity structures for specific cell binding. The surfaces consist of a pattern of a fluorocarbon plasma polymer with feature sizes between 5 and 100 microm on a background of a non-fouling tetraglyme (tetraethylene glycol dimethyl ether) plasma polymer. The tetraglyme polymer blocks virtually all non-specific binding of proteins, and it is non-adhesive for a fluorocarbon-polyethylene glycol (FC-PEG) surfactant designed to act as a 'hydrophobic anchor' for peptides. The surfactant shows a strong affinity for the fluorocarbon polymer pattern, thus enabling us to form a pattern of the surfactant-conjugated peptide. To verify this, we have synthesized a conjugate between histamine (as a model for a more complex peptide) and a commercially available FC-PEG surfactant. Disuccinimidyl carbonate was used to activate the terminal -OH group of the polyethylene glycol headgroup for the reaction with the amine-containing molecule. Affinity pattern formation can easily be achieved by immersion of the patterned substrates in a solution of the peptide-surfactant conjugate. Time of flight secondary ion mass spectroscopy in the imaging mode was used to verify that the surfactant localizes on the pattern, while the background remains bare. A model protein, bovine serum albumin, showed the same behavior. This suggests that these surfaces can be used for the formation of patterns of cell-adhesive proteins. These substrates will be used to investigate the influence of the cell size and shape of vascular smooth muscle cells on their physiology.

Adsorption↗

PubMed2001

Isolation, quantitation, and characterization of a stable complex formed by Lp[a] binding to triglyceride-rich lipoproteins.

Lipoprotein [a] (Lp[a]) is a cholesterol-rich lipoprotein resembling LDL to which a large polymorphic glycoprotein, apolipoprotein [a] (apo[a]), is covalently coupled. Lp[a] usually exists as a free-standing particle in normolipidemic subjects; however, it can associate noncovalently with triglyceride-rich lipoproteins in hypertriglyceridemic (HTG) subjects. In this study, 10-78% of the Lp[a] present in five HTG subjects was found in the triglyceride-rich lipoprotein (TRL) fraction. The Lp[a]-TRL complex was resistant to dissociation by ultracentrifugation (UCF) alone, but was quantitatively dissociated by UCF in the presence of 100 mM proline. Of this dissociated Lp[a], 70-88% was in the form of a lipoprotein resembling conventional Lp[a]. Incubation of Lp[a]-depleted TRL with native Lp[a] resulted in a reconstituted Lp[a]-TRL complex that closely resembled the native isolates in all examined properties. Complex formation was inhibited by several compounds in the order proline > tranexamate > epsilon-aminocaproate >> arginine > lysine. Neither plasminogen nor LDL inhibited binding of Lp[a] to TRL. We observed the preferential binding of Lp[a] containing higher apparent molecular weight apo[a] polymorphs to TRL both in native and reconstituted Lp[a]-TRL complexes. A disproportionate amount of Lp[a] was bound to the larger TRL particles. Although most apo[a] bound to TRL was in the form of conventional Lp[a] particles, lipid-free recombinant apo[a] was observed to bind TRL. These results provide unequivocal evidence of the existence of an Lp[a]-TRL complex under pathophysiologic conditions. The metabolic fate of the Lp[a]-TRL complex, which is more abundant in hypertriglyceridemia, may be different from that of conventional Lp[a], and may contribute uniquely to the progression or severity of cardiovascular disease.

Aminocaproic Acid↗

PubMed2000

Founder's Award, Society for Biomaterials. Sixth World Biomaterials Congress 2000, Kamuela, HI,May 15-20, 2000. Really smart bioconjugates of smart polymers and receptor proteins.

Over the past 18 years we have been deeply involved with the synthesis and applications of stimuli-responsive polymer systems, especially polymer-biomolecule conjugates. This article summarizes our work with one of these conjugate systems, specifically polymer-protein conjugates. We include conjugates prepared by random polymer conjugation to lysine amino groups, and also those prepared by site-specific conjugation of the polymer to specific amino acid sites that are genetically engineered into the known amino acid sequence of the protein. We describe the preparation and properties of thermally sensitive random conjugates to enzymes and several affinity recognition proteins. We have also prepared site-specific conjugates to streptavidin with temperature-sensitive polymers, pH-sensitive polymers, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed in this article.

Acrylamides↗

PubMed2000

Mucoadhesive drug carriers based on complexes of poly(acrylic acid) and PEGylated drugs having hydrolysable PEG-anhydride-drug linkages.

We have designed a new mucoadhesive drug delivery formulation based on H-bonded complexes of poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA) with the poly(ethylene glycol) (PEG), of a (PEG)-drug conjugate. The PEGylated prodrugs are synthesized with degradable PEG-anhydride-drug bonds for eventual delivery of free drug from the formulation. In this work we have used indomethacin as the model drug which is PEGylated via anhydride bonds to the PEG. The complexes are designed first to dissociate as the formulation swells in contact with mucosal surfaces at pH 7.4, releasing PEG-indomethacin, which then hydrolyses to release free drug and free PEG. We found that as MW of PAA increases, the dissociation rate of the complex decreases, which results in decreased rate of release of the drug. On the other hand, the drug release from PEG-indomethacin alone and from solid mixture of PEG-indomethacin+PAA was much faster than that from the H-bonded complexes. Due to the differences in the thermal stability, PMAA complex exhibited slightly faster drug release than that of the PAA complex of comparable MW. These H-bonded complexes of degradable PEGylated drugs with bioadhesive polymers should be useful for mucosal drug delivery.

Acrylic Resins↗

PubMed2000

The stabilization and release of hirudin from liposomes or lipid-assemblies coated with hydrophobically modified dextran.

Hirudin is a 65-amino acid peptide and the most potent and specific known inhibitor of thrombin (K(i) = 0.2 pM). The short elimination half-life of hirudin from the body (1 hour) necessitates the use of a sustained and controlled delivery system. A proliposome method was used to entrap hirudin in liposomes coated with palmitoyl dextran-coated liposomes and lipid-assemblies. In vitro release studies of hirudin were performed using the lipid systems enclosed in dialysis membranes or deposited in the pores of a vascular graft. The activity of hirudin and released hirudin was measured using a thrombin chromogenic substrate assay. Entrapment efficiencies of hirudin in lipid-assemblies approached 100%, however, the release of hirudin from these systems was rapid with 90% released in 17 hours. Entrapment efficiencies of hirudin in coated-liposomes ranged from 5% to 55% and were dependent on several variables. Palmitoyl dextran- coated-liposomes showed a burst of 30% hirudin released in 5 hours with an additional 10% to 35% released over the next 600 hours. In all samples, 30-40% of the hirudin remained associated with the lipid-systems even after 600 hours. The released hirudin retained only 33% of its ability to inhibit thrombin when released from uncoated liposomes. However, hirudin retained 95% of its thrombin inhibitory activity when released from palmitoyl dextran-coated liposomes. Coated liposomes were found to stabilize hirudin and result in greater retention of hirudin's ability to inhibit thrombin's enzymatic activity, although the mechanism is not yet understood.

Dextrans↗

PubMed2000

Molecular engineering of proteins and polymers for targeting and intracellular delivery of therapeutics.

There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.

Antibodies↗

PubMed2000

Blocking adhesion to cell and tissue surfaces by the chemisorption of a poly-L-lysine-graft-(poly(ethylene glycol); phenylboronic acid) copolymer.

A family of graft copolymers that can sterically inhibit interactions between biological surfaces was developed. These copolymers contained phenylboronic acid (PBA) groups as saccharide-binding moieties on a poly-(L-lysine) backbone and poly(ethylene glycol) (PEG) grafted as adhesion-resisting side chains. These copolymers spontaneously chemisorbed to a saccharide-containing resin, and this binding was sterically controlled by the PEG grafting ratio. Copolymers with optimal grafting ratios spontaneously assembled on red blood cell surfaces and sterically prevented their agglutination by lectins and by antibodies to blood groups. The simple conjugation scheme created a PBA moiety with a pKa ca. 6, which can bind cis-diols much more strongly at physiological pH than typical PBA moieties, whose pKas are typically greater than 8. These surfactant copolymers can be employed to PEGylate cell or tissue surfaces by simply incubating the surfaces with an aqueous polymer solution, and have many potential applications such as preventing antibody binding to transplanted cells.

Adsorption↗

PubMed2000

Insoluble ionic complexes of polyacrylic acid with a cationic drug for use as a mucoadhesive, ophthalmic drug delivery system.

We have developed a new mucoadhesive drug delivery formulation based on an ionic complex of partially neutralized poly(acrylic acid) (PAA) and a highly potent beta blocker drug, levobetaxolol x hydrochloride (LB x HCl), for use in the treatment of glaucoma. PAA was neutralized with sodium hydroxide to varying degrees of neutralization. Aqueous solutions containing concentrations of LB x HCl equivalent to the degree of PAA neutralization were added to the PAA solutions and formed insoluble complexes, which were isolated. The complex formation was followed by turbidimetric titration, and the complexes were characterized by IR and 1H NMR spectroscopy. Complexes were prepared with varying degrees of drug loading, such that the same PAA chain would have free -COOH groups for mucoadhesion along with ionic complexes of LB x H+ with COO- groups. Thin films of the complexes dissociated to release the drug by ion exchange with synthetic tear fluid. The films shrunk continuously during release of the drug and dissolved completely in 1 h. Solid inserts of these films could be useful as a mucoadhesive ophthalmic drug delivery system.

Acrylic Resins↗

PubMed2000

Bioconjugates of intelligent polymers and recognition proteins for use in diagnostics and affinity separations.

Polymers that respond to small changes in environmental stimuli with large, sometimes discontinuous changes in their physical state or properties are often called "intelligent" or "smart" polymers. We have conjugated these polymers to different recognition proteins, including antibodies, protein A, streptavidin, and enzymes. These bioconjugates have been prepared by random polymer conjugation to lysine amino groups on the protein surface, and also by site-specific conjugation of the polymer to specific amino acid sites, such as cysteine sulfhydryl groups, that are genetically engineered into the known amino acid sequence of the protein. We have conjugated several different smart polymers to streptavidin, including temperature-, pH-, and light-sensitive polymers. The preparation of these conjugates and their many fascinating applications are reviewed here.

Acrylamides↗

PubMed1999

Smart and biofunctional streptavidin.

The high affinity recognition of biotin and biotinylated molecules has made streptavidin one of the most important components in diagnostics and laboratory kits. While it is extremely useful as the native protein, there are many applications where its function can be improved re-engineering the subunits. We review here our efforts to construct streptavidin tetramers that have 'smart' recognition capabilities, and which display functional peptide sequences. These smart and biofunctional streptavidin derivatives can 'talk' to cells, and 'listen' to external signals which control capture and release of biotinylated molecules.

Affinity Labels↗