Exploiting Metabolic Defects in Glioma with Nanoparticle-Encapsulated NAMPT Inhibitors.

The treatment of primary central nervous system tumors is challenging due to the blood-brain barrier and complex mutational profiles, which is associated with low survival rates. However, recent studies have identified common mutations in gliomas [isocitrate dehydrogenase (IDH)-wild-type and mutant, WHO grades II-IV; with grade IV tumors referred to as glioblastomas (GBM)]. These mutations drive epigenetic changes, leading to promoter methylation at the nicotinic acid phosphoribosyl transferase (NAPRT) gene locus, which encodes an enzyme involved in generating NAD+. Importantly, NAPRT silencing introduces a therapeutic vulnerability to inhibitors targeting another NAD+ biogenesis enzyme, nicotinamide phosphoribosyl transferase (NAMPT), rationalizing a treatment for these malignancies. Multiple systemically administered NAMPT inhibitors (NAMPTi) have been developed and tested in clinical trials, but dose-limiting toxicities-including bone marrow suppression and retinal toxicity-have limited their efficacy. Here, we report a novel approach for the treatment of NAPRT-silenced GBMs using nanoparticle (NP)-encapsulated NAMPTis administered by convection-enhanced delivery (CED). We demonstrate that GMX1778 (a NAMPTi) can be formulated in degradable polymer NPs with retention of potency for NAMPT inhibition and anticancer activity in vitro, plus sustained drug release in vitro and in vivo. Direct injection of these drugs via CED into the brain is associated with reduced retinal toxicity compared with systemic administration. Finally, we show that CED of NP-encapsulated GMX1778 to NAPRT-silenced intracranial GBM xenografts in mice exhibit significant tumor growth delay and extends survival. These data support an approach to treat gliomas harboring defects in NAD+ metabolism using CED of NP-encapsulated NAMPTis to greatly improve the therapeutic index and treatment efficacy for this class of drugs.

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.

Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection.

Chlamydia trachomatis is the leading cause worldwide of preventable infectious blindness (trachoma) and sexually transmitted disease, including nongonoccocal urethritis and pelvic inflammatory disease. To date, no effective vaccine against C. trachomatis infection has been identified. A monoclonal anti-idiotypic antibody (anti-Id) to the chlamydial exoglycolipid antigen (GLXA) was tested in a murine model of ocular chlamydial infection for its ability to induce systemic immunity, which reduces microbiologic and clinical disease. The anti-Id to GLXA, delivered either systemically in soluble form or orally after encapsulation in poly(lactide) microspheres, induced significant protective immunity against ocular challenge of mice with a human biovar of C. trachomatis. Protection was associated with induction of anti-GLXA antibody and anti-chlamydial neutralizing antibody.

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.

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.

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.

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.

Topical antibody delivery systems produce sustained levels in mucosal tissue and blood.

Immunity at mucosal surfaces, which are ports of entry for many pathogens, is essential in preventing infections. But most current strategies for passive immunization involve injection of antibodies for systemic, not mucosal, protection. We measured mucosal and systemic antibody levels after controlled topical delivery to the vagina. Poly(ethylene-co-vinyl acetate) disks containing 125I-labeled monoclonal IgG or anti-lactate dehydrogenase-C4 antibodies were placed in the vaginas of mice. High antibody levels (0.26-12 micrograms/ml) were maintained at the mucosal surface for 7 days after disk insertion. Antibody molecules also penetrated into the vaginal epithelium, presumably by diffusing through the extracellular space, and entered the circulation. Biologically active antibodies were detected in the blood. The antibody concentration in the blood was approximately 1% of the concentration in the vagina. Although the permeability of the epithelium to macro-molecules is low, high concentrations were maintained at the luminal surface for an extended period, permitting substantial systemic uptake of antibody.

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.

Covalent coupling of methotrexate to dextran enhances the penetration of cytotoxicity into a tissue-like matrix.

For antitumor agents introduced directly into the intracranial space, the extent of penetration into tissue, and hence the effectiveness of therapy, depends on the rate of drug elimination from the tissue. To test the hypothesis that slowly eliminated agents would penetrate further through tissues, methotrexate (MTX)-dextran conjugates were produced by covalently linking MTX to dextran through a short-lived ester bond (MTX-ester-dextran; t1/2 approximately 3 days in buffered saline) and a longer-lived amide bond (MTX-amide-dextran; t1/2 > 20 days in buffered saline). The ability of these agents to kill cells and to penetrate through tissue was evaluated using: (a) human brain tumor (H80) cells in a standard format; (b) H80 cells in a novel three-dimensional format that mimics many characteristics of intracranial tumors; and (c) 9L gliosarcoma in the rat brain. Penetration into three-dimensional tissue-like matrices was performed by suspending H80 cells in agarose gels within a hollow fiber that was permeable to MTX but not dextran and injecting MTX or MTX-dextran conjugates into one end of the fiber. The cytotoxicity of MTX-ester-dextran and MTX-amide-dextran against H80 was equivalent to unmodified MTX (50% inhibitory concentration, approximately 0.01 microgram/ml). When released from a biodegradable polyanhydride polymer matrix, MTX and MTX-dextran conjugates retained their ability to inhibit dihydrofolate reductase activity. When MTX or MTX-dextran was diffused into the three-dimensional tumor cell matrix for 10 days, cytotoxic activity penetrated > 2 cm for MTX-amide-dextran and approximately 1 cm for MTX or MTX-ester-dextran; this enhanced penetration correlated with the stability of the MTX-dextran linkage. Intracranial polymeric delivery of MTX or MTX-amide-dextran to rats with intracranial 9L gliosarcoma produced modest but significant increases in survival; conjugation of MTX to dextran appeared to shift the dose-response curve to a lower dosage.

Pharmacokinetics of interstitial delivery of carmustine, 4-hydroperoxycyclophosphamide, and paclitaxel from a biodegradable polymer implant in the monkey brain.

Polymeric interstitial chemotherapy increases survival of humans with recurrent gliomas and animals with transplanted tumors in the brain, but the relationship between rates of drug release from polymer implants and drug concentration in brain tissue is unknown. This work presents a pharmacokinetic framework for application of this new modality of chemotherapy delivery in primates. Either [3H]carmustine, 4-hydroperoxycyclophosphamide (4-HC), or paclitaxel was encapsulated in a polyanhydride pellet (28-41 microCi/animal, 40 mg/animal), which was implanted intracranially in cynomolgus monkeys (Macaca fascicularis); (n = 17) for up to 30 days. Drug concentrations in the brain, blood, and cerebrospinal fluid were measured by quantitative autoradiography, TLC, and scintillation counting. High drug concentrations (0.5-3.5 mM for carmustine, 0.3-0.4 mM for 4-HC, and 0.2-1.0 mM for paclitaxel) were measured within the first 3 mm from the polymer implant; significant (0.4 microM for carmustine, 3 microM for 4-HC, and 0.6 microM for paclitaxel) concentrations were measured up to approximately 5 cm from the implant as long as 30 days after implantation. Pharmacokinetic analysis indicated that tissue exposure to carmustine area under concentration-time curve achieved by polymeric delivery was 4-1200 times higher than that produced by i.v. administration of a higher dose.

Antibodies to CD18 influence neutrophil migration through extracellular matrix.

Mac-1 (CD11b/CD18) is known to be involved in neutrophil (PMN) adhesion to endothelial cells and extracellular matrix. Although antibodies to CD 18 are being tested for therapy in humans, their role in PMN migration through the extracellular matrix is unknown. We used direct visualization to quantify PMN motility through reconstituted, three-dimensional gels of collagen type I. Gels were prepared with different concentrations of collagen (ranging from 0.1 to 1.0 mg/mL) and PMN migration was examined in the presence and absence of antibodies to CD18 (anti-CD18), with and without stimulation by N-formyl peptides. In low-concentration gels (<0.6 mg/mL), anti-CD18 had a significant influence on PMN migration, increasing motility in unstimulated PMN by 90% at 0.3 mg/mL collagen, and decreasing motility in N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN by 70% at 0.4 mg/mL collagen. But antiCD18 had no effect on the rate of cell migration through high-concentration collagen gels (>0.6 mg/mL). PMN migration through collagen gels is CD18-dependent but only under conditions of high hydration, suggesting that CD18-mediated effects (e.g., adhesion to gel fibers) are only important when the fiber density is relatively low. Anti-CD18 inhibited, but did not eliminate, the adhesion of fMLP-stimulated PMN to the surface of collagen gels, suggesting that cells use multiple mechanisms for gaining traction within the gel. Because of the multiple modes of interaction between motile cells and the deformable fiber matrix, blockade of one component, such as CD18, can enhance the rate of cell migration under one set of conditions, and inhibit under another.

Polymeric controlled delivery for immunization.

Current vaccine technology has limitations; for example, most vaccines require repeat administration for long-term protection, and immunity at mucosal surfaces is difficult to achieve. In animal models, polymeric controlled-release systems provide long-lasting systemic or mucosal immunoprotection, often after a single administration. Polymeric devices that deliver a controlled amount of antibody can provide passive immunity against genital herpes infections in mice; orally administered polymeric-microsphere-based vaccines produce enhanced immune responses in rodents and primates. These new delivery technologies have many desirable features, and so their use in humans could have a substantial impact on worldwide public health.

Novel systems for controlled delivery of macromolecules.

Gene therapy promises new treatments for human disease by alterations in the DNA content of tissues. Methods for efficient and stable introduction of genes into target cells in the body are critically important in this effort. Researchers and physicians now have many years of experience with synthetic polymers for controlled drug delivery; many of these polymers can also be used to deliver macromolecules at controlled rates to tissues. This article reviews the use of polymers in controlled protein delivery and suggests ways that polymer delivery systems may be useful in the delivery of gene transfer agents.

Antibodies for treating and preventing disease: the potential role of polymeric controlled release.

Following the identification of antibodies (Abs) as agents of immunity, it was hypothesized that individuals could be both (1) protected against disease by the transfer of unmodified Ab (passive immunization), and (2) cured of established disease by Ab armed with cytotoxic agents (immunotherapy). The development of monoclonal antibody (mAb) technology in 1975 reinvigorated these ideas. Although passive immunization has been practiced with great success for many years, successful tissue targeting by systemically delivered immunotoxins in humans has been documented in only a few cases. New modes of drug delivery, engineered for mAb-based products, may enable new applications of passive immunization and may provide improved tissue targeting for immunotherapy. By allowing sustained and tissue-localized delivery of mAb-conjugates, controlled-release polymers may play an important role in this effort. This article reviews the use of mAb in treating and preventing human disease, as well as the pharmacokinetics of Ab delivery. Two areas where controlled Ab release may yield new therapies are highlighted: sustained passive immunization of the mucus secretions and immunotherapy of brain tumors.

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.

Macromolecules released from polymers: diffusion into unstirred fluids.

Polymers that release macromolecules may be useful for preventing and treating human disease. In certain applications of polymeric controlled release, like drug therapy of brain disease and immunoprotection of mucus epithelia, effectiveness may be limited by diffusion through an unstirred fluid near the polymer. Using computer-assisted epifluorescence microscopy, we have examined the local distribution of fluorescently labelled macromolecules released from an ethylene-vinyl acetate copolymer matrix into unstirred layers of phosphate-buffered water and mid-cycle human cervical mucus. Diffusion coefficients in the fluid were determined by observing the concentration profiles as a function of time. Diffusion coefficients determined for fluorescein, bovine serum albumin, and three classes of human immunoglobulins (IgG, sIgA and IgM) in phosphate-buffered water were in good agreement with literature values. For fluorescein, albumin and IgG, diffusion in mucus was comparable with diffusion in water: the largest molecule tested was slowed by only a factor of 3.

Nerve growth factor delivery and cell aggregation enhance choline acetyltransferase activity after neural transplantation.

Fetal cell transplantation may improve the treatment of neurodegenerative diseases, but the optimal conditions for cell transplantation have not yet been defined. In this study, we examined the influence of preaggregation of dissociated donor cells and delivery of an appropriate growth factor on cell survival following transplantation. Cell aggregates were produced by culturing dissociated cells in conventional rotation culture or in a rotating-wall bioreactor. Suspensions of dissociated septal cells from E16 embryos, or suspensions containing small aggregates (<40 microm) of these cells, were transplanted into the brains of adult rats. In some cases, transplanted cells were supplied with nerve growth factor (NGF), a neurotrophin known to promote the survival and function of septal cholinergic cells; NGF was delivered to the transplant site by controlled-release polymers or genetically engineered fibroblasts. Cell function after transplantation was determined by measuring the increase in choline acetyltransferase (ChAT) activity in brain tissue sections encompassing the transplant site. Aggregation of cells prior to transplantation enhanced ChAT activity; at 2 months posttransplantation, ChAT levels were always slightly higher in animals receiving aggregated donor cells than in those receiving single cell suspensions. NGF delivery also enhanced ChAT activity; controlled release polymers produced the largest effect 2 weeks posttransplantation, while NGF-secreting fibroblasts produced significant enhancement at 2 months posttransplantation. These different patterns of enhanced ChAT activity appear to be due to differences in the rate of NGF delivery provided by these two techniques. These studies indicate that cell aggregation and NGF delivery systems may provide important new tools for the enhancement of cell function after transplantation.