A pharmaceutical answer to nonadherence: Once weekly oral memantine for Alzheimer's disease.

Adherence to medication regimens is a major barrier to effective treatment in many disease areas, notably in dementia which causes cognitive impairment that reduces patients' awareness of non-adherence and their ability to manage medication. The development of oral dosage forms that can be infrequently dosed, and therefore improve adherence rate and facilitate direct observed therapy, has been a goal for decades. We describe the first demonstration of an oral formulation that achieves >7-day gastric retention and sustained pharmacokinetics in the challenging dog model. Gastric retention requires physical resistance of the dosage form to gastric emptying forces, which are known to be stronger in dogs than in humans, making successful gastric retention in dogs a stringent test for predicting human translatability. This formulation of memantine hydrochloride is the first oral dosage form that achieves multi-day drug release with near zero-order kinetics and efficient delivery. In the dog model, relative memantine bioavailability approaches 100% with sustained plasma levels of memantine over seven days and profiles that can be tuned by varying components of the formulation. A single gastric resident dosage form achieves an AUC equivalent to 7 daily treatments with the marketed daily capsule, with a Cmax that is no higher than the daily product. PK modeling predicts that the gastroretentive formulation will maintain therapeutic blood levels in humans when administered once weekly. The formulation methodology presented here is applicable to many water soluble drugs and may enable the development of long-acting oral therapies for a wide variety of conditions.

Label-Free and Regenerative Electrochemical Microfluidic Biosensors for Continual Monitoring of Cell Secretomes.

Development of an efficient sensing platform capable of continual monitoring of biomarkers is needed to assess the functionality of the in vitro organoids and to evaluate their biological responses toward pharmaceutical compounds or chemical species over extended periods of time. Here, a novel label-free microfluidic electrochemical (EC) biosensor with a unique built-in on-chip regeneration capability for continual measurement of cell-secreted soluble biomarkers from an organoid culture in a fully automated manner without attenuating the sensor sensitivity is reported. The microfluidic EC biosensors are integrated with a human liver-on-a-chip platform for continual monitoring of the metabolic activity of the organoids by measuring the levels of secreted biomarkers for up to 7 d, where the metabolic activity of the organoids is altered by a systemically applied drug. The variations in the biomarker levels are successfully measured by the microfluidic regenerative EC biosensors and agree well with cellular viability and enzyme-linked immunosorbent assay analyses, validating the accuracy of the unique sensing platform. It is believed that this versatile and robust microfluidic EC biosensor that is capable of automated and continual detection of soluble biomarkers will find widespread use for long-term monitoring of human organoids during drug toxicity studies or efficacy assessments of in vitro platforms.

Multisensor-integrated organs-on-chips platform for automated and continual in situ monitoring of organoid behaviors.

Organ-on-a-chip systems are miniaturized microfluidic 3D human tissue and organ models designed to recapitulate the important biological and physiological parameters of their in vivo counterparts. They have recently emerged as a viable platform for personalized medicine and drug screening. These in vitro models, featuring biomimetic compositions, architectures, and functions, are expected to replace the conventional planar, static cell cultures and bridge the gap between the currently used preclinical animal models and the human body. Multiple organoid models may be further connected together through the microfluidics in a similar manner in which they are arranged in vivo, providing the capability to analyze multiorgan interactions. Although a wide variety of human organ-on-a-chip models have been created, there are limited efforts on the integration of multisensor systems. However, in situ continual measuring is critical in precise assessment of the microenvironment parameters and the dynamic responses of the organs to pharmaceutical compounds over extended periods of time. In addition, automated and noninvasive capability is strongly desired for long-term monitoring. Here, we report a fully integrated modular physical, biochemical, and optical sensing platform through a fluidics-routing breadboard, which operates organ-on-a-chip units in a continual, dynamic, and automated manner. We believe that this platform technology has paved a potential avenue to promote the performance of current organ-on-a-chip models in drug screening by integrating a multitude of real-time sensors to achieve automated in situ monitoring of biophysical and biochemical parameters.

Engineering Immunomodulatory Biomaterials To Tune the Inflammatory Response.

Current state-of-the-art biomedical implants and tissue engineering methods promise technologies to improve or even restore the function of diseased organs. However, one of the biggest challenges to clinical success is the lack of functional integration. A series of cellular and molecular events following biomaterial implantation poses an important bottleneck for developing breakthrough solutions. With inflammation increasingly recognized as a crucial component influencing regeneration, immunomodulation or immuno-engineering has emerged as a potential solution to overcome this key challenge in regenerative medicine. We postulate possibilities to utilize biomaterial physicochemical modifications to modulate the host inflammatory response and develop strategies for effective biomaterial integration. Biomaterial-based immunomodulation strategies can significantly ameliorate the outcomes of medical implants and tissue engineering therapies.

Quantification of cellular and nuclear uptake rates of polymeric gene delivery nanoparticles and DNA plasmids via flow cytometry.

UNLABELLED: Non-viral, biomaterial-mediated gene delivery has the potential to treat many diseases, but is limited by low efficacy. Elucidating the bottlenecks of plasmid mass transfer can enable an improved understanding of biomaterial structure-function relationships, leading to next-generation rationally designed non-viral gene delivery vectors. As proof of principle, we transfected human primary glioblastoma cells using a poly(beta-amino ester) complexed with eGFP plasmid DNA. The polyplexes transfected 70.6±0.6% of the cells with 101±3% viability. The amount of DNA within the cytoplasm, nuclear envelope, and nuclei was assessed at multiple time points using fluorescent dye conjugated plasmid up to 24h post-transfection using a quantitative multi-well plate-based flow cytometry assay. Conversion to plasmid counts and degradation kinetics were accounted for via quantitative PCR (plasmid degradation rate constants were determined to be 0.62h(-1) and 0.084h(-1) for fast and slow phases respectively). Quantitative cellular uptake, nuclear association, and nuclear uptake rate constants were determined by using a four-compartment first order mass-action model. The rate limiting step for these poly(beta-amino ester)/DNA polyplex nanoparticles was determined to be cellular uptake (7.5×10(-4)h(-1)) and only 0.1% of the added dose was taken up by the human brain cancer cells, whereas 12% of internalized DNA successfully entered the nucleus (the rate of nuclear internalization of nuclear associated plasmid was 1.1h(-1)). We describe an efficient new method for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles using flow cytometry to improve understanding and design of polymeric gene delivery nanoparticles. STATEMENT OF SIGNIFICANCE: In this work, a quantitative high throughput flow cytometry-based assay and computational modeling approach was developed for assessing cellular and nuclear uptake rates of non-viral gene delivery nanoparticles. This method is significant as it can be used to elucidate structure-function relationships of gene delivery nanoparticles and improve their efficiency. This method was applied to a particular type of biodegradable polymer, a poly(beta-amino ester), that transfected human brain cancer cells with high efficacy and without cytotoxicity. A four-compartment first order mass-action kinetics model was found to model the experimental transport data well without requiring external fitting parameters. Quantitative rate constants were identified for the intracellular transport, including DNA degradation rate from polyplexes, cellular uptake rate, and nuclear uptake rate, with cellular uptake identified as the rate-limiting step.

A liver-on-a-chip platform with bioprinted hepatic spheroids.

The inadequacy of animal models in correctly predicting drug and biothreat agent toxicity in humans has resulted in a pressing need for in vitro models that can recreate the in vivo scenario. One of the most important organs in the assessment of drug toxicity is liver. Here, we report the development of a liver-on-a-chip platform for long-term culture of three-dimensional (3D) human HepG2/C3A spheroids for drug toxicity assessment. The bioreactor design allowed for in situ monitoring of the culture environment by enabling direct access to the hepatic construct during the experiment without compromising the platform operation. The engineered bioreactor could be interfaced with a bioprinter to fabricate 3D hepatic constructs of spheroids encapsulated within photocrosslinkable gelatin methacryloyl (GelMA) hydrogel. The engineered hepatic construct remained functional during the 30 days culture period as assessed by monitoring the secretion rates of albumin, alpha-1 antitrypsin, transferrin, and ceruloplasmin, as well as immunostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. Treatment with 15 mM acetaminophen induced a toxic response in the hepatic construct that was similar to published studies on animal and other in vitro models, thus providing a proof-of-concept demonstration of the utility of this liver-on-a-chip platform for toxicity assessment.

Organ-on-a-chip platforms for studying drug delivery systems.

Novel microfluidic tools allow new ways to manufacture and test drug delivery systems. Organ-on-a-chip systems - microscale recapitulations of complex organ functions - promise to improve the drug development pipeline. This review highlights the importance of integrating microfluidic networks with 3D tissue engineered models to create organ-on-a-chip platforms, able to meet the demand of creating robust preclinical screening models. Specific examples are cited to demonstrate the use of these systems for studying the performance of drug delivery vectors and thereby reduce the discrepancies between their performance at preclinical and clinical trials. We also highlight the future directions that need to be pursued by the research community for these proof-of-concept studies to achieve the goal of accelerating clinical translation of drug delivery nanoparticles.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Organs-on-a-chip: a new tool for drug discovery.

INTRODUCTION: The development of emerging in vitro tissue culture platforms can be useful for predicting human response to new compounds, which has been traditionally challenging in the field of drug discovery. Recently, several in vitro tissue-like microsystems, also known as 'organs-on-a-chip', have emerged to provide new tools for better evaluating the effects of various chemicals on human tissue. AREAS COVERED: The aim of this article is to provide an overview of the organs-on-a-chip systems that have been recently developed. First, the authors introduce single-organ platforms, focusing on the most studied organs such as liver, heart, blood vessels and lung. Later, the authors briefly describe tumor-on-a-chip platforms and highlight their application for testing anti-cancer drugs. Finally, the article reports a few examples of other organs integrated in microfluidic chips along with preliminary multiple-organs-on-a-chip examples. The article also highlights key fabrication points as well as the main application areas of these devices. EXPERT OPINION: This field is still at an early stage and major challenges need to be addressed prior to the embracement of these technologies by the pharmaceutical industry. To produce predictive drug screening platforms, several organs have to be integrated into a single microfluidic system representative of a humanoid. The routine production of metabolic biomarkers of the organ constructs, as well as their physical environment, have to be monitored prior to and during the delivery of compounds of interest to be able to translate the findings into useful discoveries.

Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

BACKGROUND: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. METHODS: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. RESULTS: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. CONCLUSION: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming.

Evaluation of polymeric gene delivery nanoparticles by nanoparticle tracking analysis and high-throughput flow cytometry.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases(1) and as a technology for regenerative medicine(2). Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery(1,3) . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers(4) that are hydrolytically degradable(5,6) and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)(7), mouse mammary epithelial cells(8), human brain cancer cells(9) and macrovascular (human umbilical vein, HUVECs) endothelial cells(10). A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained(11). In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

Drug delivery strategies for therapeutic angiogenesis and antiangiogenesis.

INTRODUCTION: Angiogenesis is essential to human biology and of great clinical significance. Excessive or reduced angiogenesis can result in, or exacerbate, several disease states, including tumor formation, exudative age-related macular degeneration (AMD) and ischemia. Innovative drug delivery systems can increase the effectiveness of therapies used to treat angiogenesis-related diseases. AREAS COVERED: This paper reviews the basic biology of angiogenesis, including current knowledge about its disruption in diseases, with the focus on cancer and AMD. Anti- and proangiogenic drugs available for clinical use or in development are also discussed, as well as experimental drug delivery systems that can potentially improve these therapies to enhance or reduce angiogenesis in a more controlled manner. EXPERT OPINION: Laboratory and clinical results have shown pro- or antiangiogenic drug delivery strategies to be effective in drastically slowing disease progression. Further research in this area will increase the efficacy, specificity and duration of these therapies. Future directions with composite drug delivery systems may make possible targeting of multiple factors for synergistic effects.

The relationship between terminal functionalization and molecular weight of a gene delivery polymer and transfection efficacy in mammary epithelial 2-D cultures and 3-D organotypic cultures.

Non-viral gene delivery vectors were developed for efficient gene transfer to hard-to-transfect mouse mammary epithelial cells. Ten modified versions of the same base poly(beta-amino ester), poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol), were tested in both traditional 2-D monolayer and in 3-D organotypic cultures. The polymers self-assembled with plasmid DNA encoding enhanced green fluorescent protein to form nanoparticles (approximately 100 nm) used to transfect the cells. Nanoparticle transfection efficacy was tuned by changes in synthesis and fabrication conditions and the transfection efficacy was analyzed using confocal microscopy and flow cytometry. The best performing polymeric nanoparticles transfected 57 +/- 6% of the cells in 2-D culture and 6 +/- 1% of the cells in 3-D culture. Small modifications to the polymer end-capping molecules and tuning of polymer molecular weight could either significantly enhance the transfection efficacy up to 6-fold or instead abolish efficacy completely. The efficacy of leading polymers was higher than that of the commercial transfection agent FuGENE HD by a factor of 13 in 2-D and 2 in 3-D. These non-viral nanoparticles may be useful as delivery reagents or targeted therapeutics for breast cancer. This gene delivery strategy is also a promising approach for studying the normal development of the mammary gland.

Degradable polymers for gene delivery.

Degradable polymers were synthesized that self-assemble with DNA to form particles that are effective for gene delivery. Small changes to polymer synthesis conditions, particle formulation conditions, and polymer structure led to significant changes to efficacy in a cell-type dependent manner. Polymers presented here are more effective than Lipofectamine 2000 or polyethylenimine for gene delivery to cancerous fibroblasts or human primary fibroblasts. These materials may be useful for cancer therapeutics and regenerative medicine.

Synergistic effect of HIF-1alpha gene therapy and HIF-1-activated bone marrow-derived angiogenic cells in a mouse model of limb ischemia.

Ischemia induces the production of angiogenic cytokines and the homing of bone-marrow-derived angiogenic cells (BMDACs), but these adaptive responses become impaired with aging because of reduced expression of hypoxia-inducible factor (HIF)-1alpha. In this study, we analyzed the effect of augmenting HIF-1alpha levels in ischemic limb by intramuscular injection of AdCA5, an adenovirus encoding a constitutively active form of HIF-1alpha, and intravenous administration of BMDACs that were cultured in the presence of the prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG) to induce HIF-1 expression. The combined therapy increased perfusion, motor function, and limb salvage in old mice subjected to femoral artery ligation. Homing of BMDACs to the ischemic limb was dramatically enhanced by intramuscular AdCA5 administration. DMOG treatment of BMDACs increased cell surface expression of beta(2) integrins, which mediated increased adherence of BMDACs to endothelial cells. The effect of DMOG was abolished by coadministration of the HIF-1 inhibitor digoxin or by preincubation with a beta(2) integrin-blocking antibody. Transduction of BMDACs with lentivirus LvCA5 induced effects similar to DMOG treatment. Thus, HIF-1alpha gene therapy increases homing of BMDACs to ischemic muscle, whereas HIF-1 induction in BMDACs enhances their adhesion to vascular endothelium, leading to synergistic effects of combined therapy on tissue perfusion.