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.

Mechanistic investigation of smart polymer-protein conjugates.

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.

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.

Site-specific polymer-streptavidin bioconjugate for pH-controlled binding and triggered release of biotin.

Low molecular weight copolymers of acrylic acid (AAc) and N-isopropylacrylamide (NIPAAm) have been synthesized with reactive OH groups at one end, using a chain transfer polymerization technique. The copolymer displays both pH and temperature sensitivity over a wide and useful range of pHs and temperatures, which permits both pH and temperature control of polymer conformation. This copolymer has been conjugated to a specific cysteine thiol site inserted by genetic engineering near the recognition site of streptavidin (SAv). In this paper, we demonstrate that this bioconjugate can provide pH control of biotin binding to and triggered release from the mutant SAv. These actions are relevant to affinity separations, biosensors, diagnostics, enzyme processes, and targeted delivery of drugs or chemical agents, labels, and other signals.

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.

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.

Temperature control of biotin binding and release with A streptavidin-poly(N-isopropylacrylamide) site-specific conjugate.

The many laboratory and diagnostic applications utilizing streptavidin as a molecular adaptor rely on its high affinity and essentially irreversible interaction with biotin. However, there are many situations where recovery of the biotinylated molecules is desirable. We have previously shown that poly(N-isopropylacrylamide) (PNIPAAm), a temperature-sensitive polymer, can reversibly block biotin association as the polymer's conformation changes at its lower critical solution temperature (LCST). Here, we have constructed a streptavidin-PNIPAAm conjugate which is able to bind biotin at room temperature or lower and release bound biotin at 37 degrees C. The conjugate can repeatedly bind and release biotin as temperature is cycled through the LCST. A genetically engineered streptavidin mutant, E116C, which has only one cysteine residue, was conjugated site specifically via the sulfhydryl groups with a PNIPAAm that has pendent sulfhydryl-reactive vinyl sulfone groups. The conjugation site is near the tryptophan 120 residue, which forms a van der Waals contact with biotin that is important in generating the large binding free energy. The temperature-induced conformational change of the polymer at position 116 may lead to structural changes in the region of tryptophan 120 that are responsible for the reversible binding between biotin and the conjugated streptavidin.

An AB block copolymer of oligo(methyl methacrylate) and poly(acrylic acid) for micellar delivery of hydrophobic drugs.

An AB block copolymer of oligo(methyl methacrylate) (oMMA) and poly(acrylic acid) (PAAc) has been synthesized. The block copolymer forms micelles in an aqueous medium, as confirmed by a fluorescence probe technique using pyrene. Doxorubicin hydrochloride was incorporated into the micelle and the release profile of doxorubicin hydrochloride was investigated. Slow and prolonged release of doxorubicin hydrochloride from the micelle was observed. The AB block copolymer micelle can be useful for prolonged mucosal drug delivery of hydrophobic drugs.

Control of protein-ligand recognition using a stimuli-responsive polymer.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein.

A genetically-engineered mutant of cytochrome b5, incorporating a unique cysteine residue, was conjugated to maleimide-terminated oligo(N-isopropylacrylamide). The conjugation of the protein by reaction of the cysteine residue, precisely positioned by site-directed mutagenesis techniques, with an activated oligomer containing only one reactive end group in the oligomer chain permits the site-specific and stoichiometric conjugation of the oligomer with the protein. The protein-oligomer conjugate was shown to exhibit lower critical solution temperature (LCST) behavior, similar to the free oligomer. Furthermore, the LCST behavior of the protein-oligomer conjugate is reversible and allows selective precipitation of the conjugate above its LCST.

Novel delivery system for inducing quiescence in intestinal stem cells in rats by transforming growth factor beta 1.

BACKGROUND/AIMS: Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa. METHODS: Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally. Control animals received phosphate-buffered saline only. The size of the villi was measured. Proliferating and mitotic indices were determined by quantifying immunohistochemical staining for proliferating cell nuclear antigen. RESULTS: Alginate beads released no TGF-beta 1 in acid. However, in pH 7.4, TGF-beta 1 was released in an active form. Histomorphometrical analysis showed a marked reduction in villus height (50%-70%) in the intestinal mucosa of animals treated perorally with recombinant TGF-beta 1 in alginate beads. Also, the proliferating and mitotic indices were significantly reduced (P < 0.01) in these animals as compared with controls and other routes of administration. CONCLUSIONS: This study shows that recombinant TGF-beta 1 administered using a novel oral delivery system induces stem cell quiescence in the intestinal mucosa of the rat.

Correlation between corneal epithelial cell outgrowth and monoclonal antibody binding to the cell binding domain of adsorbed fibronectin.

The ability of corneal epithelial cells to attach, spread, and migrate on synthetic surfaces is largely determined by the characteristics of the adsorbed protein layer. In previous studies we have described an in vitro model for quantitating epithelial cell outgrowth from explanted corneal buttons onto synthetic materials (Pettit et al., Invest. Ophthalmol. Vis. Sci., 31, 2269 [1990]). We have also described the role of fibronectin (fn) adsorption and binding strength on epithelial cell outgrowth (Pettit et al., J. Biomed. Mater. Res., 26, 1259 [1992]). In the current study we have used a monoclonal antibody against the RGD cell binding domain of fn (mAb 3E3) to further characterize the role of adsorbed fn in promoting epithelial cell outgrowth. Ten materials of diverse chemical and physical properties were adsorbed with fn (0.1 mg/ml) or mixtures of fn and albumin (concentrations totaling 0.1 mg/ml) and tested for antibody recognition of the cell binding domain. The surface density of bound anti-cell binding domain antibody varied from a low of 0.66 +/- 0.11 for fluorinated ethylene propylene copolymer (FEP) to a high of 1.90 +/- 0.26 for tissue culture polystyrene dish substrates (units are OD at 450 nm measured in the ELISA technique normalized to polyethylene). A general increase in cell outgrowth areas was noted, with increases in recognizable cell binding domain. However, several exceptions to this trend were noted as well (e.g., low cell outgrowth but high antibody recognizability for glass). These results suggest that, although the number of cell binding domains exposed on adsorbed fn molecules may influence cell outgrowth, other characteristics of the adsorbed protein, such as the binding strength to the underlying substrate, may be equally important in characterizing epithelial cell-substrate interactions.

The stabilization of a human IgM monoclonal antibody with poly(vinylpyrrolidone).

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50 degrees C than at 25 degrees C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4 degrees C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50 degrees C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein-protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Attempts to stabilize a monoclonal antibody with water soluble synthetic polymers of varying hydrophobicity.

Proteins are subject to a variety of physical and chemical reactions that lead to a loss of activity. These reactions are a particular problem in controlled-release devices, where temperatures and protein concentrations are high. Current approaches to increasing protein stability include the addition of saccharides, amino acids, or polymers. New synthetic polymers may be promising protein stabilizers because properties such as molecular weight and side-chain composition can be controlled. In this study, the stability of a murine monoclonal antibody, BR96, was evaluated in solution at 37 degrees C. The antibody was incubated in the presence of a series of synthetic polymers that included poly(glucosylethyl methacrylate) (GEMA) and copolymers of N-vinylpyrrolidone (NVP) and methyl methacrylate (MMA). Samples were taken periodically up to 30 days. The formation of precipitated antibody in particulate aggregates was measured with a Coulter counter, and the molecular-weight distribution of soluble antibody was measured by size-exclusion chromatography. Two trends were evident. First, with poly(GEMA) and copolymers of NVP and MMA, protein aggregation increased at higher polymer concentrations. Second, higher molecular weights of the poly(NVP) homopolymer also led to increases in protein aggregation. Effects of polymer hydrophobicity were more complex. A copolymer containing 9 mol% MMA caused immediate protein precipitation, while a copolymer containing 21 mol% MMA did not. The effects of the copolymer containing 21% MMA were strongly concentration dependent. At 1 wt%, the polymer reduced aggregation, but aggregation increased strongly between concentrations of 2 and 3 wt%.

Effects of branching and molecular weight of surface-bound poly(ethylene oxide) on protein rejection.

To understand better the origin of protein rejection observed with surface-bound poly(ethylene oxide) (or PEO), we have measured fibrinogen adsorption for a series of linear and branched, low-molecular-weight PEOs bound to solid polystyrene surfaces. The results show that a dependence on molecular weight is found below 1500 g mol-1 for linear PEO. Branched PEOs are less effective at protein rejection than linear PEOs. The branched PEOs have smaller exclusion volumes (from GPC) than the corresponding linear PEOs, consistent with restriction in conformational freedom for the branched compounds. The protein rejection results are interpreted in terms of entropy changes that result upon protein adsorption. In addition, some practical problems in preparation of PEO glycidyl ethers have been clarified, thus making these PEO derivatives more useful for surface modification.

Activity of horseradish peroxide adsorbed on radio frequency glow discharge-treated polymers.

Horseradish peroxidase (HRP) has been used as a model enzyme in this study of its physical adsorption and residual enzyme activity on radio frequency glow discharge (RFGD)-treated polymers. The specific enzymatic activity of HRP adsorbed on different surfaces was assumed to be an indication of the extent of its conformational alterations on the surfaces. The surfaces studied were poly (ethylene terephthalate) (PET), polytetrafluoroethylene (PTFE), and tetrachloroethylene and tetrafluoroethylene glow discharge-treated PET, abbreviated as TCE/PET and TFE/PET. All surfaces were characterized by electron spectroscopy for chemical analysis (ESCA) and liquid contact angles in air. HRP adsorbs more strongly onto the two discharge-treated surfaces than onto the untreated polymers, as evidenced by the lower amount of HRP eluted by sodium dodecyl sulfate (SDS) from the treated polymers. For example, seventy percent of the HRP adsorbed on TCE/PET or TFE/PET remains on the surface after overnight elution with a 1% solution of SDS. In contrast, untreated PET and PTFE each retains only c. 20% of the absorbed enzyme. The enzymatic activity of HRP adsorbed on the different surfaces was studied using hydrogen peroxide (H2O2) as the substrate. HRP adsorbed on the higher energy surfaces, PET and TCE/PET, retains significantly more activity than the HRP adsorbed on the lower energy surfaces, PTFE and TFE/PET which appear to destroy rapidly almost all of the activity of HRP after it adsorbs. HRP adsorbed on TCE/PET is relatively more stable over time than HRP adsorbed on PET or free HRP in solution. (For example, only 45% of the specific enzymatic activity of HRP adsorbed on TCE/PET was lost after 3 h of storage in phosphate buffer at 37 degrees C, while 70% of that adsorbed on PET was lost.) In summary, when HRP is adsorbed on TCE/PET, it is very tightly bound, and yet it maintains a significant fraction of its initial specific activity and also retains this activity for 3 h in phosphate buffer at 37 degrees C. Thus, tenacious physical adsorption of proteins such as enzymes on TCE glow discharge-treated surfaces may have potential as a new method of immobilization of such molecules, for uses in biosensors, diagnostics, bioseparations, cell culture and bioreactors.

The feasibility of smoking bans on psychiatric units.

We conducted a prospective study of a smoking ban on a general inpatient psychiatry service in response to staff concerns about the feasibility of a proposed hospital-wide ban. Demographic information, smoking history, and DSM III-R diagnoses were obtained for consecutively admitted patients during two study conditions: smoking and nonsmoking. A log of p.r.n. medication, seclusion, restraint, elopement, incident reports, and smoking-related discharges was kept for each patient. Chi-square analysis of 232 patients for whom demographic, smoking, diagnostic, and log data were complete showed no significant differences between study conditions for demographic or diagnostic variables. Two-tailed t-test analysis of the log data for these 232 patients showed no significant difference in disruptive incidents during smoking and nonsmoking conditions (p = 0.183). Fifty staff members answered pre- and post-ban questionnaires. Paired t-test analysis demonstrated a significant change in staff attitude toward supporting the ban. These data indicate that smoking can be stopped on inpatient psychiatry units without increases in unit disruption or adverse effects on staff morale.

Introduction of amine groups on poly(ethylene) by plasma immobilization of a preadsorbed layer of decylamine hydrochloride.

In order to introduce amine groups on poly(ethylene) (PE) surface, PE surfaces were preadsorbed with decylamine hydrochloride (DA.HCl) and subsequently treated with an argon plasma. It was shown by XPS (X-ray Photoelectron Spectroscopy), that approximately half of the preadsorbed (mono)layer was immobilized and that a substantial part (60-70%) of the incorporated nitrogen containing groups were amine groups. The availability of the surface amine groups for reactions was investigated by applying a gas phase reaction with 4-trifluoromethylbenzaldehyde and by a reductive methylation reaction in aqueous solution with 14C formaldehyde. A maximal number of reactive amine groups was found after a plasma treatment time of 2 s. The reductive methylation reaction was used to estimate the surface concentration of amine groups resulting in a typical surface concentration of 1 x 10(-6) mol/m2 after a plasma treatment time of 2 s.

Influence of the substrate binding characteristics of fibronectin on corneal epithelial cell outgrowth. Student Research Award in the Doctoral Degree Candidate Category, Fourth World Biomaterials Congress (18th annual meeting of the Society for Biomaterials), Berlin, Germany, April 24-28, 1992.

The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. Circular corneal buttons (5 mm) were punched from excised rabbit corneas and placed onto bare substrates or substrates preadsorbed with fibronectin (fn), albumin, or binary mixtures of both fn and albumin. Cell outgrowth areas were measured after culturing the buttons for 4 days in serum-free medium. Fibronectin adsorption to the materials was measured from pure and binary solutions with 125I-radiolabeled fibronectin. A parameter thought to be related to the binding strength of fn to polymeric substrates was measured in parallel experiments by partial elution of the adsorbed fn by 3% sodium dodecyl sulfate (SDS). Following pure solution fibronectin adsorption a range of outgrowth areas was measured (from 0.86 +/- 0.03 cm2 for glass to 1.49 +/- 0.03 cm2 for TCPS). On all of the materials tested cell outgrowth areas increased following fn preadsorption and decreased following albumin preadsorption relative to bare surfaces (p less than 0.05). Following preadsorption with binary protein mixtures cell outgrowth areas increased with fibronectin adsorption, however, the outgrowth areas were not determined solely by the concentration of fn adsorbed onto the surfaces. This result suggested that the biological efficiency of the adsorbed fibronectin was substrate-dependent. When the cell outgrowth data were cross-plotted against fn retention following SDS elution, the outgrowth areas were found to increase along with increases in fn retention. Based on these data we suggest that epithelial cell outgrowth may be partially governed by the tightness of binding between the fn molecules and the underlying substrate.

Immobilization of surface active compounds on polymer supports using a gas discharge process.

By applying an argon plasma treatment to a layer of a surface active agent pre-adsorbed on a polymer substrate, it is possible to covalently couple this layer to the substrate. This method offers a direct route to tailor the surface properties of polymers.