Monobody adapter for functional antibody display on nanoparticles for adaptable targeted delivery applications.

Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.

Initial evaluation of the use of USPIO cell labeling and noninvasive MR monitoring of human tissue-engineered vascular grafts in vivo.

This pilot study examines noninvasive MR monitoring of tissue-engineered vascular grafts (TEVGs) in vivo using cells labeled with iron oxide nanoparticles. Human aortic smooth muscle cells (hASMCs) were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The labeled hASMCs, along with human aortic endothelial cells, were incorporated into eight TEVGs and were then surgically implanted as aortic interposition grafts in a C.B-17 SCID/bg mouse host. USPIO-labeled hASMCs persisted in the grafts throughout a 3 wk observation period and allowed noninvasive MR imaging of the human TEVGs for real-time, serial monitoring of hASMC retention. This study demonstrates the feasibility of applying noninvasive imaging techniques for evaluation of in vivo TEVG performance.

Small-diameter biodegradable scaffolds for functional vascular tissue engineering in the mouse model.

The development of neotissue in tissue engineered vascular grafts remains poorly understood. Advances in mouse genetic models have been highly informative in the study of vascular biology, but have been inaccessible to vascular tissue engineers due to technical limitations on the use of mouse recipients. To this end, we have developed a method for constructing sub-1mm internal diameter (ID) biodegradable scaffolds utilizing a dual cylinder chamber molding system and a hybrid polyester sealant scaled for use in a mouse model. Scaffolds constructed from either polyglycolic acid or poly-l-lactic acid nonwoven felts demonstrated sufficient porosity, biomechanical profile, and biocompatibility to function as vascular grafts. The scaffolds implanted as either inferior vena cava or aortic interposition grafts in SCID/bg mice demonstrated excellent patency without evidence of thromboembolic complications or aneurysm formation. A foreign body immune response was observed with marked macrophage infiltration and giant cell formation by post-operative week 3. Organized vascular neotissue, consisting of endothelialization, medial generation, and collagen deposition, was evident within the internal lumen of the scaffolds by post-operative week 6. These results present the ability to create sub-1mm ID biodegradable tubular scaffolds that are functional as vascular grafts, and provide an experimental approach for the study of vascular tissue engineering using mouse models.

Polymer nanoparticles for immunotherapy from encapsulated tumor-associated antigens and whole tumor cells.

Encapsulation of tumor-associated antigens (TAA) in polymer nanoparticles is a promising approach to increasing the efficiency of antigen (Ag) delivery for antitumor vaccines. We optimized a polymer preparation method to deliver both defined tumor-associated proteins and the complex mixtures of tumor Ags present in tumors. Tumor Ags were encapsulated in a biodegradable, 50:50 poly(D,L-lactide co-glycolide) copolymer (PLGA) by emulsification and solvent extraction. Two particular Ags were studied, gp100 (a melanoma-associated antigen) and ovalbumin (OVA), as well as mixtures of proteins and lysates of tumor cells. The efficiency of encapsulation was measured by protein assays of dissolved nanoparticles. Ag stability after release from nanoparticles was verified by SDS-acrylamide gel electrophoresis and Western blot analysis. Molecular weight and protein loading interact to define the encapsulation efficiency and release rate of nanoparticles formulated from 50:50 PLGA. A midrange molecular weight polymer had more desirable release properties at 100 mg/mL than at 50 mg/mL protein loading, indicating the need for optimization of nanoparticle formulation for preparations with different particle loadings. Mixtures of proteins derived from cell lysates were reliably encapsulated into nanoparticles, which released the spectrum of proteins contained in lysates. Antigenic proteins were co-encapsulated with cell lysate and released from nanoparticles; these Ags retained their antigenicity and functioned better than soluble Ags when tested in in vitro assays of T cell cytokine formation and in vivo tumor vaccination challenge.

DNA diffusion in mucus: effect of size, topology of DNAs, and transfection reagents.

DNA represents a promising therapeutic and prophylactic macromolecule in treating genetic diseases, infectious diseases and cancers. The therapeutic potential of DNA is directly related to how DNA transports within the targeted tissue. In this study, fluorescence photobleaching recovery was used to examine the diffusion of plasmid DNAs with various size (2.7-8.3 kb), topology, and in the presence of transfection reagents in mucus. We observed that DNAs diffused slower when size of DNAs increased; supercoiled DNAs diffused faster than linear ones; mucus did not reduce the diffusion of linear DNAs but retarded the diffusion of supercoiled DNAs. Diffusion data were fitted to models of a polymer chain diffusing in gel systems. Diffusion of linear DNAs in mucus were better described by the Zimm model with a scaling factor of -0.8, and supercoiled DNAs showed a reptational behavior with a scaling factor of -1.3. Based on the Zimm model, the pore size of bovine mucus was estimated and agreed well with previous experimental data. In the presence of transfection reagents, e.g., liposomes, the diffusion of DNAs increased by a factor of 2 in mucus. By using bovine mucus as a model system, this work suggests that DNA size, topology, and the presence of transfection reagents may affect the diffusion of DNA in tissues, and thus the therapeutic effects of DNA.

Fabrication and characterization of microfluidic probes for convection enhanced drug delivery.

Convection enhanced drug delivery (CED) is a promising therapeutic method for treating diseases of the brain by enhancing the penetration of drugs. Most controlled release delivery methods rely on diffusion from a source to transport drugs throughout tissue. CED relies on direct infusion of drugs into tissue at a sufficiently high rate so that convective transport of drug is at least as important as diffusive transport. This work describes the fabrication and characterization of microfluidic probes for CED protocols and the role diffusion plays in determining penetration. Microfluidic channels were formed on silicon substrates by employing a sacrificial photoresist layer encased in a parylene structural layer. Flow in the microchannels was characterized by applying constant upstream pressures from 35 to 310 kPa, which resulted in flow rates of 0.5-4.5 microL/min. The devices were used to infuse Evans Blue and albumin in hydrogel brain phantoms. The results of these infusions were compared to a simple convection-diffusion model for infusions into porous media. In vivo infusions of albumin were performed in the gray matter of rats at upstream pressures of 35, 70, and 140 kPa. The microfabricated probes show reduced evidence of backflow along the device-tissue interface when compared with conventional needles used for CED.

Transplantation of brain cells assembled around a programmable synthetic microenvironment.

Cell therapy is a promising method for treatment of hematopoietic disorders, neurodegenerative diseases, diabetes, and tissue loss due to trauma. Some of the major barriers to cell therapy have been partially addressed, including identification of cell populations, in vitro cell proliferation, and strategies for immunosuppression. An unsolved problem is recapitulation of the unique combinations of matrix, growth factor, and cell adhesion cues that distinguish each stem cell microenvironment, and that are critically important for control of progenitor cell differentiation and histogenesis. Here we describe an approach in which cells, synthetic matrix elements, and controlled-release technology are assembled and programmed, before transplantation, to mimic the chemical and physical microenvironment of developing tissue. We demonstrate this approach in animals using a transplantation system that allows control of fetal brain cell survival and differentiation by pre-assembly of neo-tissues containing cells and nerve growth factor (NGF)-releasing synthetic particles.

Micron-scale positioning of features influences the rate of polymorphonuclear leukocyte migration.

Microfabrication technology was used to create regular arrays of micron-size holes (2 microm x 2 microm x 210 nm) on fused quartz and photosensitive polyimide surfaces. The patterned surfaces, which possessed a basic structural element of a three-dimensional (3-D) network (i.e., spatially separated mechanical edges), were used as a model system for studying the effect of substrate microgeometry on neutrophil migration. The edge-to-edge spacing between features was systematically varied from 6 microm to 14 microm with an increment of 2 microm. In addition, collagen was used to coat the patterned quartz surfaces in an attempt to change the adhesive properties of the surfaces. A radial flow detachment assay revealed that cell adhesion was the strongest on the quartz surface (approximately 50% cell attached), whereas it was relatively weaker on polyimide and collagen-coated quartz (approximately 25% cell attached). Cell adhesion to each substrate was not affected either by the presence of holes or by the spacing between holes. A direct visualization assay showed that neutrophil migration on each patterned surface could be characterized as a persistent random walk; the dependence of the random motility coefficient (mu) as a function of spacing was biphasic with the optimal spacing at approximately 10 microm on each substrate. The presence of evenly distributed holes at the optimal spacing of 10 microm enhanced mu by a factor of 2 on polyimide, a factor of 2.5 on collagen-coated quartz, and a factor of 10 on uncoated quartz. The biphasic dependence on the mechanical edges of neutrophil migration on 2-D patterned substrate was strikingly similar to that previously observed during neutrophil migration within 3-D networks, suggesting that microfabricated materials provide relevant models of 3-D structures with precisely defined physical characteristics. In addition, our results demonstrate that the microgeometry of a substrate, when considered separately from adhesion, can play a significant role in cell migration.

The anti-idiotypic antibody to chlamydial glycolipid exoantigen (GLXA) protects mice against genital infection with a human biovar of Chlamydia trachomatis.

Despite more than three decades of anti-chlamydial vaccine research and improved vaccine strategies with new technologies, no vaccine candidate has protected against heterologous challenge, nor at more than one site of infection. The majority of experimental anti-chlamydial vaccines to date have targeted the chlamydial major outer membrane protein (MOMP). Many MOMP-directed vaccine candidates have been highly immunogenic, but have failed to protect against infectious challenge. We have extended our previous studies of a different anti-chlamydial vaccine, a monoclonal anti-idiotypic antibody (anti-Id; mAb2) which is a molecular mimic of the chlamydial glycolipid exoantigen (GLXA). The present studies demonstrate that the mAb2 vaccine is protective in a murine genital infection model utilizing a human urogenital strain. After either mucosal (oral or intranasal) or systemic (subcutaneous) immunization with the poly (lactide) encapsulated-mAb2 to GLXA, C3H/HeJ mice were significantly protected against topical vaginal challenge with Chlamydia trachomatis (K serovar; UW-31). Reduced vaginal shedding of organism and genital tract inflammation were associated with GLXA-specific and/or anti-EB neutralizing serum antibody. Our results demonstrate that the anti-Id (mAb2) vaccine is protective against an additional human biovar of C. trachomatis in C3H/HeJ mice, which are allogeneic to the source of mAb2 (BALB/c).

Aggregation enhances catecholamine secretion in cultured cells.

Transplanted cells and tissues have potential uses in the treatment of genetic, geriatric, and metabolic disorders, but optimal conditions for transplantation are not yet known. In this report, PC12 cells were aggregated in rotary and microgravity culture, using serum-free or serum-supplemented medium, and using a multifunctional polymer-peptide aggregation factor. Aggregates and single cells were then encapsulated and cultured within agarose gels, and the dopamine secretion in response to a depolarization buffer was measured using high-performance liquid chromatography combined with electrochemical detection (HPLC-ECD). On a per-cell basis, aggregated cells secreted higher levels of dopamine than did single cells. The size of the aggregates was also a factor in catecholamine secretion; dopamine release from the larger aggregates formed in rotary culture was observed to increase at a faster rate, then achieve a plateau level at an earlier time than did the smaller aggregates. Cells aggregated in microgravity culture exhibited a markedly different behavior, lacking the rapid rise in dopamine secretion characteristic of the rotary-aggregates cells: on a per-cell basis, the dopamine secretion remained at a level corresponding to the plateau level expressed by the rotary-aggregates cells. Dopamine secretion in aggregates may be enhanced by the increase in number of cell-cell contacts, as occurs during high-density culture of PC12 cells. These results provide further evidence that cell-cell contact regulates the behavior of differentiated cells, and therefore is important in tissue engineering.

Simultaneous delivery of an active protein and neutralizing antibody: creation of separated regions of biological activity.

Spatial control over the biological activity of nerve growth factor (NGF) via a novel type of controlled-release device was demonstrated in an in vitro system. Two-layer polymer matrices that simultaneously released NGF and a neutralizing antibody (anti-NGF) from opposite faces were placed in PC12 cell-populated collagen gels. Biological activity in the gels was assessed over the course of 10 days by direct observation of the cells, which extend neuronal processes in the presence of NGF in a dose-dependent manner. The concentrations of both proteins in the gels were determined by ELISA as a function of distance from the polymer matrices at various time points. A boundary in biological activity was established within a few days of the initiation of the cultures; this boundary persisted and became more pronounced throughout the duration of the experiment. ELISA analysis revealed regions of high concentration of both NGF and anti-NGF on their respective sides of the polymer matrix early in the experiment. The theoretical amount of active NGF in the gel sections was calculated on the basis of these ELISA results; the concentration of active NGF in the region adjacent to the polymer correlated with the observed degree of biological response. These experiments suggest that spatial control over the biological activity of a potent agent can be obtained by an appropriately designed controlled-release device.

Synthetic polymers alter the structure of cervical mucus.

Mucosal sites have an innate defense system--which includes immune cells, antibodies, and mucus--to protect the body from opportunistic pathogens. Some sexually transmitted diseases (STDs), such as HIV, utilize host defense mechanisms to evade detection by infecting motile immune cells present at the site. The infected cells migrate through the mucus layer and penetrate the epithelium undetected. A new strategy for preventing STDs could involve inhibiting cell migration through the mucus. One method for inhibiting migration is to alter the barrier property of mucus by modifying its gel structure. Mucin, the structural component of mucus, is a high molecular weight anionic molecule, which forms an entangled fiber network through non-covalent interactions. The addition of nonionic or cationic polymers, such as poly(ethylene glycol) (PEG) or poly(vinyl pyridine) (PVP), altered the overall gel structure as revealed by scanning electron microscopy (SEM), while anionic poly(acrylic acid) had little effect on the structure. Acid residues on mucin associate with PEG through hydrogen bonds to form regions of coalesced fibers within the mucus. PVP, however, interacts with mucin via electrostatic bonds, forming a gel that had areas of aggregated fibers adjacent to regions with virtually no fibers. These results suggest that addition of small amounts of certain synthetic polymers will modify mucus structure; these changes should alter the barrier properties of mucus.

Enhancement of transfection by physical concentration of DNA at the cell surface.

Efficient DNA transfection is critical for biological research and new clinical therapies, but the mechanisms responsible for DNA uptake are unknown. Current nonviral transfection methods, empirically designed to maximize DNA complexation and/or membrane fusion, are amenable to enhancement by a variety of chemicals. These chemicals include particulates, lipids, and polymer complexes that optimize DNA complexation/condensation, membrane fusion, endosomal release, or nuclear targeting, which are the presumed barriers to gene delivery. Most chemical enhancements produce a moderate increase in gene delivery and a limited increase in gene expression. As a result, the efficiency of transfection and level of gene expression after nonviral DNA delivery remain low, suggesting the existence of additional unidentified barriers. Here, we tested the hypothesis that DNA transfection efficiency is limited by a simple physical barrier: low DNA concentration at the cell surface. We used dense silica nanoparticles to concentrate DNA-vector (i.e. DNA-transfection reagent) complexes at the surface of cell monolayers; manipulations that increased complex concentration at the cell surface enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. We predict that manipulations aimed at optimizing DNA complexation or membrane fusion have a fundamental physical limit; new methods designed to increase transfection efficiency must increase DNA concentration at the target cell surface without adding to the toxicity.

Controlling human polymorphonuclear leukocytes motility using microfabrication technology.

We describe a new approach for controlling cell motility on a material surface. Transparent, photosensitive polyimide materials were used to fabricate physical structures on glass; cell motility was then followed over time using optical microscopy. Arrays of pillars and holes with 2 micron square, 4-microm height (or depth) separated by 10 microm were successfully patterned using photolithography. Neutrophils attached and spread on the smooth glass surface and surfaces with pillars. In contrast, cells were rounded and did not adhere to either smooth polyimide film or films with holes. The migration of neutrophils was much faster on holes than on polyimide surface, but it was significantly slower on pillars than on glass. These results suggest that physical patterning may be an effective tool to manipulate cell migration in the design of biomaterials for tissue engineering.

Long-term vaginal antibody delivery: delivery systems and biodistribution.

Topical delivery systems can provide prolonged delivery of antibodies to the vaginal mucosal surface for long-term protection against infectious diseases. We examined the biodistribution of antibodies during 30 days of vaginal antibody delivery in mice. Different antibody preparations (including monoclonal IgG and IgM, as well as several different (125)I-labeled IgGs) were administered by polymer vaginal rings, which were designed to provide continuous antibody delivery. Antibody concentrations remained high in the vaginal secretions for up to 30 days after disk insertion; radiolabeled antibody was also found, at approximately 100 times lower concentration, in the blood and other tissues. The measured concentrations agreed reasonably well with a simple pharmacokinetic model, which was used to calculate mucosal and systemic concentrations as a function of antibody delivery and elimination rates. Results from the model were consistent with previously reported antibody pharmacokinetic measurements: the half-life for antibody elimination for the vagina was approximately 3 h; the half-life for IgG(1) clearance from the blood was >1 day; and the overall permeability constant for vaginal uptake of IgG was approximately 0.01 to 0.03 h(-1). These results provide important information for the design of controlled antibody delivery devices for vaginal use, and suggest that high-dose, long-term vaginal administration of antibodies may be a reasonable approach for achieving sustained mucosal and systemic antibody levels.

Synthetic DNA delivery systems.

The ability to safely and efficiently transfer foreign DNA into cells is a fundamental goal in biotechnology. Toward this end, rapid advances have recently been made in our understanding of mechanisms for DNA stability and transport within cells. Current synthetic DNA delivery systems are versatile and safe, but substantially less efficient than viruses. Indeed, most current systems address only one of the obstacles to DNA delivery by enhancing DNA uptake. In fact, the effectiveness of gene expression is also dependent on several additional factors, including the release of intracellular DNA, stability of DNA in the cytoplasm, unpackaging of the DNA-vector complex, and the targeting of DNA to the nucleus. Delivery systems of the future must fully accommodate all these processes to effectively shepherd DNA across the plasma membrane, through the hostile intracellular environment, and into the nucleus.

Synthesis and biological activity of polyethylene glycol-mouse nerve growth factor conjugate.

Conjugation of poly(ethylene glycol) derivatives with therapeutic proteins is a promising approach for enhancing protein stability and, therefore, effectiveness. An N-hydroxysuccinimidyl ester of fluorescein-PEG 2000 was used for chemical modification of mouse nerve growth factor (mNGF), a dimeric protein with therapeutic potential for Alzheimer's disease. The mNGF-PEG2000-fluorescein conjugate was characterized by RP-HPLC, spectrofluorometry, and SDS-PAGE and was biologically active, as determined by two independent NGF-specific assays (enhancement of ChAT activity in fetal neurons and neurite outgrowth in PC12 cells). The conjugate was not detectable by a standard NGF ELISA, suggesting a fortuitous reduction in protein recognition by antibodies.

Controlled DNA delivery systems.

PURPOSE: Genes are of increasing interest as pharmaceuticals, but current methods for long-term gene delivery are inadequate. Controlled release systems using biocompatible and/or biodegradable polymers offer many advantages over conventional gene delivery approaches. We have characterized systems for controlled delivery of DNA from implantable polymer matrices (EVAc: poly (ethylene-co-vinyl acetate)) and injectable microspheres (PLGA and PLA: poly (D, L-lactide-co-glycolide) copolymer and poly (L-lactide), respectively). METHODS: Herring sperm DNA and bacteria phage lambda DNA were encapsulated as a model system. Released DNA concentration was determined by fluoroassays. Agarose electrophoresis was used to determine the dependence of release rate on DNA size. The Green Fluorescent Protein (GFP) gene was used to determine the integrity and functionality of released DNA. RESULTS: Both small and large DNA molecules (herring sperm DNA, 0.1-0.6 kb; GFP, 1.9 kb; lambda DNA, 48.5 kb) were successfully encapsulated and released from EVAc matrices, and PLGA or PLA microspheres. The release from DNA-EVAc systems was diffusion-controlled. When co-encapsulated in the same matrix, the larger lambda DNA was released more slowly than herring sperm; the rate of release scaled with the DNA diffusion coefficient in water. The chemical and biological integrity of released DNA was not changed. CONCLUSIONS: These low cost, and adjustable, controlled DNA delivery systems, using FDA-approved biocompatible/biodegradable and implantable/injectable materials, could be useful for in vivo gene delivery, such as DNA vaccination and gene therapy.