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A replication-deficient lentiviral vector encoding the tumor antigen gene NY-ESO-1 was characterized in terms of vector morphology, particle size range, concentration, and zeta potential using a variety of physical methods. Environmentally stressed vector samples were then evaluated in terms of viral vector particle size and concentration by nanoparticle tracking analysis (NTA). These NTA stability results correlated reasonably well with a quantitative polymerase chain reaction assay for quantitation of viral genome copy number (r2 = 0.80). Approximately 40 pharmaceutical excipients were examined for their ability to stabilize the vector against exposure to an adsorptive container surface (glass) as well as freeze-thaw cycling using NTA as the screening method. Stabilizing additives that inhibited viral vector particle loss under these conditions included proline, lactose, and mannitol. Several candidate frozen liquid formulations that contained a combination of these lead excipients and various buffering agents were further evaluated for their ability to stabilize the viral vector. The additional benefit of lowering the Tris buffer concentration was observed. This study highlights the use of physical particle assays such as NTA for initial screening of stabilizing excipients to minimize vector loss due to container adsorption and freeze-thaw cycling to facilitate early formulation development of viral vector candidates in frozen liquid formulations.

This chapter reviews the preparation and surface characterization of hydrogels. It discusses applications of hydrogels containing immobilized biomolecules and cells. Biomolecules and cells can be immobilized on and within many different supports using a variety of techniques. There are three ways to prepare hydrogels for the immobilization of biomolecules and cells: bulk polymerization, grafting to a support, and conversion of existing polymers. There are several techniques for the immobilization of biomolecules and cells on and within hydrogels. They include physical entrapment, electrostatic attraction, physical adsorption with or without cross-linking, and chemical bonding. The entrapment method of immobilization is based on the occlusion of biomolecules or cells within a constraining hydrogel which has a tight enough structure to prevent the species from diffusing rapidly into the surrounding medium. Immobilized biomolecules and cells on and within hydrogels can be used in synthetic reactions for production of bioenergy, organic acids, amino acids, and drugs.

Polymeric micelles formed by the self-assembly of amphiphilic block copolymers can be used to encapsulate hydrophobic drugs for tumor-delivery applications. Filamentous carriers with high aspect ratios offer potential advantages over spherical carriers, including prolonged circulation times. In this work, mixed micelles comprised of poly (ethylene oxide)-poly-[(R)-3-hydroxybutyrate]-poly (ethylene oxide) (PEO-PHB-PEO) and Pluronic F-127 (PF-127) were used to encapsulate a near-infrared fluorophore. The micelle formulations were assessed for tumor accumulation after tail vein injection to xenograft tumor-bearing mice by non-invasive optical imaging. The mixed micelle formulation that facilitated the highest tumor accumulation was shown by cryo-electron microscopy to be filamentous in structure compared to spherical structures of pure PF-127 micelles. In addition, increased dye loading efficiency and dye stability was attained in this mixed micelle formulation compared to pure PEO-PHB-PEO micelles. Therefore, the optimized PEO-PHB-PEO/PF-127 mixed micelle formulation offers advantages for cancer delivery over micelles formed from the individual copolymer components.

Background: ZVex is a novel, integration-deficient, dendritic cell-targeting lentiviral vector platform currently being evaluated in clinical trials in sarcoma patients. The vector is capable of delivering multiple full-length genes simultaneously. Here, we describe ZVex preparations encoding the human cancer testis antigens MAGE-A1, 3, 4, 10 and Interleukin 12. Because lentiviral vectors package two RNA molecules each, this ZVex approach results in the generation of 15 possible permutations of homozygous or heterozygous vector genotypes. The homologous nature of the MAGEA family members (50-80% sequence identity) poses significant challenges to their specific detection and quantification in a MAGEA gene pool. Here, we describe highly specific qPCR assays for detection of the 4 MAGEA genes and IL12 for characterization and identity testing of multigenome ZVex preparations. Materials and Methods: Genes were codon-modified to enable specific detection of vector-expressed genes over cellular sequences. The entire IL12B and short, non-overlapping, divergent target regions in each of the 4 MAGEA genes were carefully selected as templates for primer/probe design. Primer-blast was used to predict absence of off-target detection of cross-reacting genomic sequences. To further enhance the specificity of the primers/probes to each of their target MAGEA, potential off-target amplicons that can be generated in the other 3 MAGEA genes due to base pair mismatch were further sequence-modified to reduce sequence identities. ZVex vectors were generated by transfecting 4 or 5 different plasmids encoding the codon modified full-length MAGEA and/or IL12 genes in a producer cell line along with essential vector packaging plasmids. Single/multiplex qPCR assays were performed to analyze vector RNA/reverse-transcribed DNA. Results: The primer/probe combinations were highly specific for each of the 4 MAGEA genes and IL12 as indicated by melt curve analysis and producing a single qPCR product at the expected size. The primer/probes specifically detected from 10 to 107 copies of their target MAGEA with >=93% efficiency and specifically detected reverse-transcribed vector DNA in a cell-based vector transduction assay. The assays demonstrated relative gene specific titers that correlate with transfected plasmid ratios. MAGEA/IL12 specific qPCR assays benchmark well against previously established “gold standard” qPCR assays used for quantifying total ZVex vector genome titers. Conclusions: Component-specific qPCR assays were developed for the specific assessment of constituent genes in multigenome ZVex preparations. These assays demonstrate inter/intragene specificity, are amenable to multiplexing, and can be used for quality control, titration, characterization, and identity testing of ZVex lots in a cGMP setting. Citation Format: Anshika Bajaj, Tsai-Yu Lin, Lisa Y. Ngo, Michele Murphy, Brenna Kelley-Clarke, Wayne R. Gombotz, Jan H. ter Meulen, Peter Berglund. Component-specific qPCR assays for characterization and identity testing of multigenome ZVex®, a dendritic cell-targeting lentiviral vector platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5919.

Indocyanine green (ICG), an FDA-approved near infrared (NIR) dye, has potential application as a contrast agent for tumor detection. Because ICG binds strongly to plasma proteins and exhibits aqueous, photo, and thermal instability, its current applications are largely limited to monitoring blood flow. To address these issues, ICG was encapsulated and stabilized within polymeric micelles formed from the thermo-sensitive block copolymer Pluronic F-127, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), to increase the stability and circulation time of ICG.ICG-loaded Pluronic micelles were prepared at various concentrations of Pluronic and ICG and characterized by determining particle sizes, dye loading efficiency, and the kinetics of dye degradation. Förster resonance energy transfer spectroscopy was employed to monitor the stability of Pluronic micelles in physiological solutions. The plasma clearance kinetics and biodistribution of ICG-loaded micelles was also determined after intravenous delivery to CT-26 colon carcinoma tumor-bearing mice, and NIR whole-body imaging was performed for tumor detection.The Pluronic F-127 micelles showed efficient ICG loading, small size, stabilized ICG fluorescence, and prolonged circulation in vivo. Solid tumors in mice were specifically visualized after intravenous administration of ICG-loaded micelles.These materials are therefore promising formulations for noninvasive NIR tumor imaging applications.

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.

We present here the purification and the characterization of the isoforms of PIXY321, a genetically engineered fusion of granulocyte-macrophage-colony stimulating factor and interleukin-3 expressed in yeast. The isoforms of PIXY321 were isolated using preparative isoelectric focusing (IEF) on immobilized pH gradients. Analysis of the collected fractions on analytical IEF gels showed that PIXY321 was resolved into four discrete isoforms of isoelectric point (pI) 5.0, 5.1, 5.2 and 5.3 with excellent yields. Subsequent analysis of purified isoforms of PIXY321 by peptide mapping and mass spectrometry linked the microheterogeneity of the original molecule to three parameters, the presence of deamidated residues, charged glycans and the pattern of O-linked glycosylation along the peptide sequence. This last parameter emphasizes the role of conformational aspects as key factors influencing the apparent isoelectric point of protein isoforms.

This study elucidates the importance of thermal reversibility as it pertains to the minimization of recombinant human Flt3 ligand aggregation and its potential role for determining solution conditions that can achieve the greatest long-term storage stability. Both thermal reversibility and Tm were evaluated as microcalorimetric parameters of stability within the range extending from pH 6 to 9, where the Tm was shown to plateau near 80 degrees C. Within this region, the reversibility was shown to decrease from 96. 6% to 15.2% while the pH was increased from 6 to 9, respectively. Accelerated stability studies conducted at 50 degrees C exhibited rates of aggregation augmented by pH that inversely correlated with the thermal reversibility data. Namely, high thermal reversibility at the Tm plateau correlated with slower rates of aggregation. Enthalpic calorimetric to van't Hoff ratios (DeltaH1/DeltaHv) yielded results close to unity within the plateau region, suggesting that the unfolding of rhFlt3 ligand was approximately two-state. Evidence that unfolding preceded the formation of the aggregate was provided by far-UV CD data of a soluble islolate of the aggregated product exhibiting a 28% loss of alpha-helix offset by a 31% gain in beta-sheet. This information combined with the thermal reversibility data provided compelling evidence that unfolding was a key event in the aggregation pathway at 50 degrees C. Minimization of aggregation was achieved at pH 6 and corroborated by evidence acquired from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion data. Correspondingly, the bioactivity was found to be optimal at pH 6. The findings link thermal reversibility to the propensity of Flt3 ligand to aggregate once unfolded in the Tm plateau region and provide a basis for relating the reversibility of thermal denaturation to the prediction of long-term storage stability in aqueous solution.

The desire to deliver protein and peptide biopharmaceuticals conveniently and effectively has led to intense investigation of site-specific drug-delivery systems. Despite challenges, progress towards the convenient noninvasive delivery of proteins and peptides has been achieved through specific routes of administration. In addition, the delivery of proteins and peptides to specific sites of action has been utilized to lower the total delivered dose, to gain access to specific organs or body compartments and to concentrate a therapeutic dose at a specific site of pharmacological action.

There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins. Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent. Alginate, a naturally occurring biopolymer extracted from brown algae (kelp), has several unique properties that have enabled it to be used as a matrix for the entrapment and/or delivery of a variety of biological agents. Alginate polymers are a family of linear unbranched polysaccharides which contain varying amounts of 1,4'-linked beta-D-mannuronic acid and alpha-L-guluronic acid residues. The residues may vary widely in composition and sequence and are arranged in a pattern of blocks along the chain. Alginate can be ionically crosslinked by the addition of divalent cations in aqueous solution. The relatively mild gelation process has enabled not only proteins, but cells and DNA to be incorporated into alginate matrices with retention of full biological activity. Furthermore, by selection of the type of alginate and coating agent, the pore size, degradation rate, and ultimately release kinetics can be controlled. Gels of different morphologies can be prepared including large block matrices, large beads (>1 mm in diameter) and microbeads (

Purpose. To elucidate the solution conditions that confer stability of aqueous IL-1R using differential scanning calorimetry (DSC).

The stability of thiotepa in a new formulation of the drug was studied. Vials of Thioplex (Immunex), a relatively new lyophilized formulation of thiotepa, were reconstituted with sterile water and diluted with 0.9% sodium chloride injection in polyvinyl chloride infusion bags to thiotepa concentrations of 0.5, 1, and 3 mg/mL. The solutions were stored at 8 and 25 degrees C in ambient light and analyzed at 0, 8, 24, and in most cases 48 hours for thiotepa concentration and chloro-adduct formation by stability-indicating high-performance liquid chromatography. Thiotepa 1 and 3 mg/mL was stable for 48 hours at 8 degrees C and for 24 hours at 25 degrees C. Thiotepa 0.5 mg/mL was not stable at either temperature. Storage at 8 degrees C slowed but did not prevent chloro-adduct formation and loss of potency. The pH tended to increase with time; turbidity remained low. Thiotepa (lyophilized) 1 and 3 mg/mL in 0.9% sodium chloride injection was stable for 48 hours at 8 degrees C and for 24 hours at 25 degrees C; the drug was unstable when diluted to 0.5 mg/mL and stored under the same conditions.

Purpose. This study describes the preparation and characterization of a controlled release formulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) encapsulated in poly(glycolide-co-D,L-lactide) (PLGA) and poly(D,L-lactide) (PLA) microspheres.

Interleukin (IL)-15 is a multifunctional cytokine that shares many biological activities with IL-2. This functional overlap, as well as receptor binding subunits shared by IL-15 and IL-2, suggests tertiary structural similarities between these two cytokines. In this study, recombinant human IL-15 was PEGylated via lysine-specific conjugation chemistry in order to extend the circulation half-life of this cytokine. Although PEGylation did extend the beta-elimination circulation half-life of IL-15 by greater than 50-fold, the biological activity of polyethylene glycol (PEG)-IL-15 was significantly altered. Specifically, PEG-IL-15 lost its ability to stimulate the proliferation of CTLL but took on the properties of a specific IL-15 antagonist in vitro. In comparing sequence alignments and molecular models for IL-2 and IL-15, it was noted that lysine residues resided in regions of IL-15 that may have selectively disrupted receptor subunit binding. We hypothesized that PEGylation of IL-15 interferes with beta but not alpha receptor subunit binding, resulting in the IL-15 antagonist activity observed in vitro. The validity of this hypothesis was tested by engineering site-specific mutants of human IL-15 as suggested by the IL-15 model (IL-15D8S and IL-15Q108S block beta and gamma receptor subunit binding, respectively). As with PEG-IL-15, these mutants were unable to stimulate CTLL proliferation but were able to specifically inhibit the proliferation of CTLL in response to unmodified IL-15. These results supported our model of IL-15 and confirmed that interference of beta receptor subunit binding by adjacent PEGylation could be responsible for the altered biological activity observed for PEG-IL-15.

The impaired wound healing associated with aging may reflect inadequate secretion or delivery of cytokines. Transforming growth factor-beta(1) is a mitogenic polypeptide with beneficial effects on wound healing. In the present study we questioned whether topical administration of transforming growth factor-beta(1) could improve the wound healing process in aged rats in vivo. Wound repair (from 1 to 14 days) was analyzed in full-thickness incisional wounds from 2-year-old rats with or without a single topical application of transforming growth factor-beta(1) (1 microg/wound) at the time of wounding. Identical wounds from 3-month-old, untreated rats served as controls. Histologic analysis showed a marked delay in several aspects of wound repair in the aged rats in comparison with that noted in the younger animals. Immunostaining of the wounds for proliferating cell nuclear antigen showed a reduction in the number of cycling fibroblasts in old rats. In addition, the number of capillaries per unit area of the wound as determined by a stain for Griffonin (Bandeiraea) simplicifolia lectin, and the number of inflammatory cells as identified by an antibody specific for macrophages, were also reduced in the wound area in old rats. Treatment with transforming growth factor-beta(1) resulted in marked enhancement of the following parameters: cell proliferation, inflammatory cell and fibroblast influx, wound closure, and angiogenesis. As seen with in situ hybridization, a similar temporal pattern of expression of messenger RNAs corresponding to type I procollagen and Secreted Protein, Acidic and Rich in Cysteine (osteonectin), known to be prevalent in healing wounds, was observed in both young and aged rats. However, the levels of mRNA corresponding to these secreted proteins appeared to be reduced in wound tissue from aged rats. Treatment with transforming growth factor-beta(1) subsequently resulted in an increase in the expression of both type I procollagen and Secreted Protein, Acidic and Rich in Cysteine mRNA in the wound tissue from aged rats. In summary, a single topical application of transforming growth factor-beta(1) to the wounds of aged rats at the time of wounding was associated with a healing response that, in all the parameters of wound repair examined, was similar to that of young rats. Topical transforming growth factor-beta(1) might therefore be beneficial in the treatment of dermal wounds in the aged.

We have reviewed a large cross-section of degradable polymeric delivery systems for protein and peptide pharmaceuticals. These systems include monolithic type devices in which the drug is dispersed throughout the polymer and protein-polymer conjugates where the drug is covalently bound to the polymer. These delivery systems have unique challenges associated with their development that are related to both protein stability and protein release kinetics. Despite numerous reports in the scientific literature which include many encouraging results in preclinical models, very few of these systems have been developed into viable products. The products that have made it to market, however, have proven to be very successful and demonstrate the significant advantages that these systems can provide. The continuous advances in biotechnology will produce more proteins and peptides that will be difficult to administer by conventional means, and an increased demand for controlled or site-specific delivery systems is anticipated.

Transforming growth factor-beta 1 (TGF-beta 1) has beneficial effects on wound healing. However, the ideal method for its administration to the wound site remains unknown. Our aim was to analyze the release of TGF-beta 1 from different formulations and to study whether the changes in wound healing by TGF-beta 1 depend on its topical delivery system. For the studies the TGF-beta 1 was incorporated into phosphate-buffered saline, into a polyoxamer gel, into DuoDERM hydroactive paste, and into a poly(ethylene oxide) hydrogel. The release of 125I-labeled TGF-beta 1 from carriers was measured in full-thickness wounds in rats and the healing of the wounds was analyzed by histology and wound area measurements. The TGF-beta 1 was released from all formulations at a different rate and in an active form as determined by growth inhibition assay. Wound size measurements and the analysis on the amount of cellular influx, fibroplasia, and granulation tissue showed that a single dose (1 microgram/wound) of locally administered TGF-beta 1 significantly (P0.01) enhanced the wound healing. This effect was most prominent with polyoxamer gel formulation, which provided the most sustained release of TGF-beta 1. Our finding that the enhancement in wound healing by TGF-beta 1 was significantly dependent on the carrier used for its topical delivery to the wound site is novel and shows the importance of using adequate delivery systems when growth factors are used to enhance wound repair.

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%.

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.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.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 (P0.01) in these animals as compared with controls and other routes of administration.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.

The susceptibility of the gastrointestinal tract to the toxic effects of chemotherapeutic drugs remains a complication in chemotherapy. Recent studies have suggested that transforming growth factor-β1 (TGF-β1) can be used as a cytoprotectant against cell cycle specific drugs. This work describes the use of alginate beads as a potential oral delivery system for TGF-β1 designed to release the drug in the lumen of the small intestine. TGF-β1 encapsulation and extent of release from alginate beads approached 100% as determined by 125I-labelled TGF-β1. However, when assayed by ELISA and a growth inhibition assay, nearly all immunoreactivity and bioactivity was lost, apparently due to a very high affinity of the alginate for TGF-β1. This limitation was overcome by two novel methods: (1) incorporation of selected polyanions within the alginate beads to ‘shield’ TGF-β1 from interaction with alginate and (2) exposure of the alginate beads containing TGF-β1 to 0.1 N HCl (acid treatment) to simultaneously reduce the molecular weight of the alginate and its ability to interact with TGF- β1. If the beads were only acid treated, just 8% of the immunoreactivity ofTGF-β1 was retained. If polyacrylic acid (90 kDa) was added to the beads, 50% of the immunoreactivity of TGF-β1 was retained. However, when TGF-β1 was released from acid-treated beads also containing polyacrylic acid, more than 80% of the TGF-β1 remained immunoreactive and bioactive. The retained TGF-β1 activity after release from the beads was found to continue to increase with increasing concentrations of polyacrylic acid, until a concentration was reached where beads would not form. The dramatic increase in retained TGF-β1 activity is attributed to the ability of polyacrylic acid to shield TGF-β1 from interaction with lower molecular fragments of alginate.

Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-beta 1 is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-beta 1 to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-beta 1 to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-beta 1 comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-beta 1 released was determined using several methods including 125I-labeled TGF-beta 1 as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-beta 1 released was directly proportional to both the TGF-beta 1 loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-beta 1 released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50°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°C than at 25°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°C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50°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. Low concentrations of less than 1.0% PVP can be used to stabilize proteins against heat-induced aggregation, but care should be exercised, since even slightly higher concentrations of PVP can also lead to protein destabilization.

We have reviewed a large cross-section of degradable polymeric delivery systems for protein and peptide pharmaceuticals. These systems include monolithic type devices in which the drug is dispersed throughout the polymer and protein-polymer conjugates where the drug is covalently bound to the polymer. These delivery systems have unique challenges associated with their development that are related to both protein stability and protein release kinetics. Despite numerous reports in the scientific literature which include many encouraging results in preclinical models, very few of these systems have been developed into viable products. The products that have made it to market, however, have proven to be very successful and demonstrate the significant advantages that these systems can provide. The continuous advances in biotechnology will produce more proteins and peptides that will be difficult to administer by conventional means, and an increased demand for controlled or site-specific delivery systems is anticipated.

Surfaces containing poly(ethylene oxide) (PEO) are interesting biomaterials because they exhibit low degrees of protein adsorption and cell adhesion. In this study different molecular weight PEO molecules were covalently attached to poly(ethylene terephthalate) (PET) films using cyanuric chloride chemistry. Prior to the PEO immobilization, amino groups were introduced onto the PET films by exposing them to an allylamine plasma glow discharge. The amino groups on the PET film were next activated with cyanuric chloride and then reacted with bis-amino PEO. The samples were characterized by scanning electron microscopy, water contact angle measurements, gravimetric analysis, and electron spectroscopy for chemical analysis (ESCA). The adsorption of 125I-labeled baboon fibrinogen and bovine serum albumin was studied from buffer solutions. Gravimetric analysis indicated that the films grafted with the low-molecular-weight PEO contained many more PEO molecules than the surfaces grafted with higher-molecular-weight PEO. The high-molecular-weight PEO surfaces, however, exhibited greater wettability (lower water contact angles) and less protein adsorption than the low-molecular-weight PEO surfaces. Adsorption of albumin and fibrinogen to the PEO surfaces decreased with increasing PEO molecular weight up to 3500. A further increase in molecular weight resulted in only slight decreases in protein adsorption. Protein adsorption studies as a function of buffer ionic strength suggest that there may be an ionic interaction between the protein and the allylamine surface. The trends in protein adsorption together with the water contact angle results and the gravimetric analysis suggest that a kind of "cooperative" water structuring around the larger PEO molecules may create an "excluded volume" of the hydrated polymer coils. This may be an important factor contributing to the observed low protein adsorption behavior.

This chapter has reviewed the multifunctional role that the TGF-β play with diverse proliferative and suppressive effects on many different cell types. The various in vitro and in vivo effects of TGF-β was discussed with emphasis on the therapeutic potential of this growth factor in areas such as wound healing and bone regeneration. The structure and properties of TGF-β were presented, followed by a more detailed description of the characterization and stabilization of TGF-β1) in both the liquid and lyophilized state. The chapter concluded with a review of some of the different controlled release systems that have been utilized for the delivery of TGF-β1 to various preclinical models. Clearly the therapeutic potential for TGF-β1) is great. Both the formulation and delivery of this growth factor will play an important role in its eventual clinical success

The CD40 ligand molecule is unique, consisting of a receptor-binding domain anchored by an isoleucine zipper moiety. Exact determination of the multimeric state and its tendency to form molten globules has not been elucidated. Corroborating evidence of a trimerized molecule in aqueous solution was obtained from size-exclusion chromatography, laser light scattering, and analytical ultracentrifugation. A reversible acid-denatured molten globule state was observed from circular dichroism and fluorescence spectroscopy data. The molten globule state was characterized by a loss of tertiary structure with associated retention of secondary structure near pH 3. Once returned to pH 7, the acid-denatured state refolded over the course of 7 days resulting in approximately 90% recovery of the native structure. The molten globule state was characterized by a broadening of structural features in the second-derivative spectra of Fourier transform infrared spectroscopy. A component band at 1650 cm(-1) was shown to be alpha-helix and originate from amide carbonyl vibrations of the isoleucine zipper. Differential scanning calorimetry measurements characterized the pH-sensitive molten globule state at pH 3.3 as one lacking a well-defined unfolding transition with an accompanying baseline shift at 58 degrees C (a consequence of increased heat capacity). The tendency to form molten globules during acid denaturation stress permits an opportunity to study the process of partial protein unfolding with implications concerning stability. Although reversible molten globules can be formed, it is important to recognize the unusual nature since the molten globule state is formed exclusively within the beta-sheet receptor-binding region.

BACKGROUND: The toxicity of chemotherapeutic drugs is often the limiting factor in patient dosing and can be the source of severe discomfort and suffering for patients undergoing chemotherapy. For example, the anthracycline antibiotic doxorubicin (DOX) is a potent chemotherapeutic but prolonged use can also lead to permanent heart damage. Tumors can also develop resistance to DOX by increased expression of drug efflux pumps. Nanoparticle formulations of DOX have been reported to improve tumor-specific delivery via the enhanced permeability and retention (EPR) effect. METHODS: In this work, we have encapsulated DOX in polymeric micelles formed from mixtures of amphiphilic triblock copolymers. The copolymers are composed of a hydrophobic polymer segment flanked by two hydrophilic blocks. Two different copolymers were used to form the mixed micelles: a copolymer of poly(ethylene oxide) and poly(hydroxybutyrate) (PEO-PHB-PEO), and a copolymer of poly(ethylene oxide) and poly(propylene oxide) (Pluronic F127). We hypothesize that forming mixed micelles will improve drug loading and enable preparation at room temperature (where Pluronic F127 micelles are highly unstable), while facilitating drug delivery by utilizing the stability of Pluronic F127 micelles at physiological temperatures. RESULTS: We have demonstrated that the micelles formed from the mixtures of PEO-PHB-PEO and Pluronic F127 triblock copolymers provide a combination of high drug loading content and drug delivery capabilities. DOX loading for mixed micelles was greater than that for Pluronic F127-only micelles (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4770. doi:1538-7445.AM2012-4770

The ability to transforming growth factor-beta 1 (TGF-beta 1), to stimulate bone healing was evaluated in a rat critical calvarial defect model. Both a low dose and a high dose of TGF-beta 1 were incorporated into two different types of implants: one made from a composite of poly(lactic-co-glycolic acid) (PLPG) (50:50) and demineralized bone matrix (DBM), and the other from calcium sulfate (CaSO 4). Scanning electron microscopy showed that the CaSO 4 implants were more porous than the PLPG/DBM samples. Both types of implants released biologically active TGF-beta 1 for over 300 h in vitro. The samples were implanted in a 9-mm diameter rat calvarial defect for 6 weeks along with contralateral control implants containing no TGF-beta 1. Microradiography and histological analysis were used to assess the bone healing in the defects. Microradiography revealed that the greatest amount of calcified bone (67.5%) was present in in the CaSO 4 implants containing a high dose of TGF-beta 1 while minimal new bone formation occurred in the PLPG/DBM implants. Histologically, the PLPG/DBM implants exhibited an inflammatory response with little mineralization or bone formation. The defects containing the PLPG/DBM implants consisted of a connective tissue stroma with large void spaces. Giant cells and numerous polymorphonuclear leukocytes were present throughout the implants. In contrast, the CaSO 4 implants had only a few inflammatory cells and the presence of mineralization and true bone was a more consistent feature. These preliminary studies show that TGF-beta 1 is capable of inducing new bone formation. Furthermore, the materials used to deliver the growth factor can play a significant role in the bone healing process.

The goal of this study is to develop formulations for non-invasive, near-infrared detection of tumors. Indocyanine green (ICG) is an FDA-approved near-infrared (NIR) imaging agent that is clinically used for angiography and evaluation of liver function. ICG could potentially be used as a NIR fluorescence imaging agent for shallow tumors such as breast cancers. To fulfill this potential, formulations that improve the stability of ICG and direct its accumulation in tumors are necessary. We have previously described Pluronic F127 copolymer micelles that stabilize ICG, enhance its stability, and facilitate tumor accumulation in vivo after systemic administration. A major advantage of Pluronic F127 is its temperature-sensitive behavior that results in more stable micelle formulations at body temperature. However, Pluronic F127 micelles are not stable at room temperature, making micelle preparation and handling challenging. We hypothesized that mixed micelles formed from both Pluronic F127 and a temperature insensitive triblock copolymer composed of poly(ethylene oxide) and poly(3-hydroxybutyrate) (PEO-PHB-PEO), would result in micelles that are both easy to formulate at room temperature and that demonstrate increased stability at body temperature. Micelle formulations with various ratios of Pluronic F127: PEO-PHB-PEO were prepared and loaded with ICG. The micelle stability was determined by assessment of critical micelle concentration measurements, and time-course fluorescence assays were conducted to assess the ability of each micelle formulation to stabilize ICG fluorescence in buffer conditions. The ICG-loaded micelles were administered to tumor-bearing mice by tail vein injection and tumor localization was monitored by non-invasive fluorescence imaging in live mice. The polymer ratio used in micelle formulation was found to play a significant role in the ability of each preparation to stabilize ICG fluorescence Mixed micelles were found to enhance the stability of ICG fluorescence in buffer conditions, and CMC was shown to be dependent on polymer ratios. In vivo, mixed micelles were found to be more effective at maintaining ICG fluorescence in the bloodstream and achieved higher fluorescence signals in the tumor tissues compared with either PEO-PHB-PEO or Pluronic 127 micelles. These results suggest that the mixed micelle formulations offer a promising route to stabilizing these nanoparticles for improved delivery of NIR fluorescent contrast agents to tumors for imaging applications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5293. doi:10.1158/1538-7445.AM2011-5293

Poly(ethylene oxide) (or PEO) surfaces represent an important class of biomaterials because of their low capacity for protein adsorption. A potentially wide range of applications exists for PEO surfaces including blood contacting devices, drug delivery systems, contact lenses, intraocular lenses, vascular grafts, catheters, immunoassays, biosensors, and media for protein and cell separations, to name a few.

The enzyme l -asparaginase has been used as a drug to treat acute lymphocytic leukemia. However, its effectiveness is limited as a free drug and it is more useful when immobilized. In this study, l -asparaginase has been immobilized onto the porous polypropylene (PP) hollow fibers of a plasma filter. The fibers were first radiation grafted with polymethacrylic acid (poly(MAAc)) which introduced carboxyl groups onto their surface, and within their pores. l -asparaginase was then covalently bound to the fibers using carbodiimide and N-hydroxy succinimide (NHS). In vitro tests were performed on the plasma filters to determine the immobilized enzyme's stability over time, Km, pH optimum and activity dependence on flow rate. In vivo tests have also been carried out using a sheep as an animal model. We concluded that plasmapheresis using an immobilized l -asparaginase plasma filter could become a useful clinical tool for the treatment of acute lymphocytic leukemia.

The enzyme asparaginase was immobilized onto a porous hollow polypropylene (PP) fiber blood plasma exchange device for the treatment of acute lymphocytic leukemia. The devices were first radiation grafted with polymethacrylic acid (poly(MAAc)). This introduces carboxyl groups onto the surface of the fibers. Several variables were studied in the grafting reaction including the effects of solvent type and monomer concentration. The carboxyl groups were activated with N-hydroxy succinimide (NHS) using carbodiimide chemistry. Asparaginase was then covalently immobilized on the activated surfaces. Quantitative relationships were found relating the percent graft to the amount of immobilized enzyme which was active. The enzyme reactor was tested both in vitro and in vivo using a sheep as an animal model.

Poly(ethylene oxide) (PEO) has been covalently immobilized on poly(ethylene terephthalate) (PET) films using a radio frequency glow discharge polymer deposition process, followed by chemical coupling. Amino or hydroxyl groups were introduced onto the surface of the PET by exposing the films to allylamine and allyl alcohol plasmas. These functional groups were activated with cyanuric chloride, and then they were reacted with PEO. ESCA and water contact angle studies were used to characterize the surfaces of these films during the different stages of the reaction. The films containing the higher molecular weight PEO exhibited an increase in the CO peak of the Cls ESCA spectrum and an increase in oxygen content on the film surfaces. Increasing the molecular weight of the PEO attached to the PET also resulted in an increased wettability of the films.

Pre-irradiation and mutual radiation grafting were employed to produce poly(methacrylic acid) (MAAc) hydrogels on polypropylene/polyethylene (PP/PE) copolymer films, PP films and porous PP fibers of a plasma filter. A diphenyl picryl hydrazyl (DPPH) assay was developed to measure the surface peroxide concentration of the pre-irradiated PP/PE films prior to grafting. Mutually grafted porous PP fibers were used for subsequent immobilization of L-asparaginase while the mutually grafted PP/PE films were used to immobilize a schistosoma monoclonal antibody.