Development of a Bladder Cancer-on-a-Chip Model to Assess Bladder Cancer Cell Invasiveness.

We have developed a bladder cancer-on-a-chip model which supports the 3D growth of cells and can be used to assess and quantify bladder cancer cell invasiveness in a physiologically appropriate environment. Three bladder cancer cell lines (T24, J82, and RT4) were resuspended in 50% Matrigel® and grown within a multi-channel organ-on-a-chip system. The ability of live cells to invade across into an adjacent 50% Matrigel®-only channel was assessed over a 2-day period. Cell lines isolated from patients with high-grade bladder cancer (T24 and J82) invaded across into the Matrigel®-only channel at a much higher frequency compared to cells isolated from a patient with low-grade cancer (RT4) (p < 0.001). The T24 and J82 cells also invaded further distances into the Matrigel®-only channel compared to the RT4 cells (p < 0.001). The cell phenotype within the model was maintained as assessed by cell morphology and immunohistochemical analysis of E-cadherin. Treatment with ATN-161, an α5ß1 integrin inhibitor and well-known migrastatic drug, caused a dose-dependent decrease in the invasiveness of the J82 cells (p < 0.01). The combined data demonstrate that our bladder cancer-on-a-chip model supports the retention of the bladder cancer cell phenotype and can be used to reproducibly assess and quantify the invasiveness of live bladder cancer cells.

Intratracheally Administered Peptide-Modified Lipid Admixture Containing Fasudil and/or DETA NONOate Ameliorates Various Pathologies of Pulmonary Arterial Hypertension.

This study examined the therapeutic potential of a combination therapy using fasudil, a Rho-kinase inhibitor, and DETA NONOate (DN), a nitric oxide donor, delivered as a lipid admixture modified with a cyclic homing peptide known as CAR (CAR-lipid mixture) for the treatment of pulmonary arterial hypertension (PAH). CAR-lipid mixtures were initially prepared via a thin-film hydration method and then combined with fasudil, DN, or a mixture of both. The therapeutic efficacy of this drug-laden lipid mixture was evaluated in a Sugen/Hypoxia (Su/Hx) rat model of PAH by measuring RV systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), Fulton indices, and assessing right ventricular (RV) functions, as well as evaluating pulmonary vascular morphology. Rats that received no treatment exhibited increases in RVSP, mPAP, Fulton indices, and changes in RV functional parameters. However, the treatment with the CAR-lipid mixture containing either fasudil or DN or a combination of both led to a decline in mPAP, RVSP, and Fulton indices compared to saline-treated rats. Similarly, rats that received these treatments showed concurrent improvement in various echocardiographic parameters such as pulmonary acceleration time (PAT), tricuspid annular plane systolic excursion (TAPSE), and ventricular free wall thickness (RVFWT). A significant decrease in the wall thickness of pulmonary arteries larger than 100 µm was observed with the combination therapy. The findings reveal that fasudil, DN, and their combination in a CAR-modified lipid mixture improved pulmonary hemodynamics, RV functions, and pathological alterations in the pulmonary vasculature. This study underscores the potential of combination therapy and targeted drug delivery in PAH treatment, laying the groundwork for future investigations into the optimization of these treatments, their long-term safety and efficacy, and the underlying mechanism of action of the proposed therapy.

Catheterization of Pulmonary and Carotid Arteries for Concurrent Measurement of Mean Pulmonary and Systemic Arterial Pressure in Rat Models of Pulmonary Arterial Hypertension.

Pulmonary hypertension (PH) is a group of pulmonary vascular disorders in which mean pulmonary arterial pressure (mPAP) becomes abnormally high because of various pathological conditions, including remodeling of the pulmonary arteries, lung and heart disorders, or congenital conditions. Various animal models, including mouse and rat models, have been used to recapitulate elevated mPAP observed in PH patients. However, the measurement and recording of mPAP and mean systemic arterial pressure (mSAP) in small animals require microsurgical procedures and a sophisticated data acquisition system. In this paper, we describe the surgical procedures for right heart catheterizations (RHC) to measure mPAP in rats. We also explain the catheterization of the carotid artery for simultaneous measurement of mPAP and mSAP using the PowerLab Data Acquisition system. We enumerate the surgical steps involved in exposing the jugular vein and the carotid artery for catheterizing these two blood vessels. We list the tools used for microsurgery in rats, describe the methods for preparing catheters, and illustrate the process for inserting the catheters in the pulmonary and carotid arteries. Finally, we delineate the steps involved in the calibration and setup of the PowerLab system for recording both mPAP and mSAP. This is the first protocol wherein we meticulously explain the surgical procedures for RHC in rats and the recording of mPAP and mSAP. We believe this protocol will be essential for PH research. Investigators with little training in animal handling can reproduce this microsurgical procedure for RHC in rats and measure mPAP and mSAP in rat models of PH. Further, this protocol is likely to help master RHC in rats that are performed for other conditions, such as heart failure, congenital heart disease, heart valve disorders, and heart transplantation.

An evolving perspective on novel modified release drug delivery systems for inhalational therapy.

INTRODUCTION: Drugs delivered via the lungs are predominantly used to treat various respiratory disorders, including asthma, chronic obstructive pulmonary diseases, respiratory tract infections and lung cancers, and pulmonary vascular diseases such as pulmonary hypertension. To treat respiratory diseases, targeted, modified or controlled release inhalation formulations are desirable for improved patient compliance and superior therapeutic outcome. AREAS COVERED: This review summarizes the important factors that have an impact on the inhalable modified release formulation approaches with a focus toward various formulation strategies, including dissolution rate-controlled systems, drug complexes, site-specific delivery, drug-polymer conjugates, and drug-polymer matrix systems, lipid matrix particles, nanosystems, and formulations that can bypass clearance via mucociliary system and alveolar macrophages. EXPERT OPINION: Inhaled modified release formulations can potentially reduce dosing frequency by extending drug's residence time in the lungs. However, inhalable modified or controlled release drug delivery systems remain unexplored and underdeveloped from the commercialization perspective. This review paper addresses the current state-of-the-art of inhaled controlled release formulations, elaborates on the avenues for developing newer technologies for formulating various drugs with tailored release profiles after inhalational delivery and explains the challenges associated with translational feasibility of modified release inhalable formulations.

A Protocol for Fabrication and on-Chip Cell Culture to Recreate PAH-Afflicted Pulmonary Artery on a Microfluidic Device.

Pulmonary arterial hypertension (PAH) is a rare pulmonary vascular disease that affects people of all ethnic origins and age groups including newborns. In PAH, pulmonary arteries and arterioles undergo a series of pathological changes including remodeling of the entire pulmonary vasculatures and extracellular matrices, mis-localized growth of pulmonary arterial cells, and development of glomeruloid-like lesions called plexiform lesions. Traditionally, various animal and cellular models have been used to understand PAH pathophysiology, investigate sex-disparity in PAH and monitor therapeutic efficacy of PAH medications. However, traditional models can only partially capture various pathological features of PAH, and they are not adaptable to combinatorial study design for deciphering intricately intertwined complex cellular processes implicated in PAH pathogenesis. While many microfluidic chip-based models are currently available for major diseases, no such disease-on-a-device model is available for PAH, an under investigated disease. In the absence of any chip-based models of PAH, we recently proposed a five-channel polydimethylsiloxane (PDMS)-based microfluidic device that can emulate major pathological features of PAH. However, our proposed model can make a bigger impact on the PAH field only when the larger scientific community engaged in PAH research can fabricate the device and develop the model in their laboratory settings. With this goal in mind, in this study, we have described the detailed methodologies for fabrication and development of the PAH chip model including a thorough explanation of scientific principles for various steps for chip fabrication, a detailed list of reagents, tools and equipment along with their source and catalogue numbers, description of laboratory setup, and cautionary notes. Finally, we explained the methodologies for on-chip cell seeding and application of this model for studying PAH pathophysiology. We believe investigators with little or no training in microfluidic chip fabrication can fabricate this eminently novel PAH-on-a-chip model. As such, this study will have a far-reaching impact on understanding PAH pathophysiology, unravelling the biological mystery associated with sexual dimorphism in PAH, and developing PAH therapy based on patient sex and age.

Multilayer Soft Photolithography Fabrication of Microfluidic Devices Using a Custom-Built Wafer-Scale PDMS Slab Aligner and Cost-Efficient Equipment.

We present a robust, low-cost fabrication method for implementation in multilayer soft photolithography to create a PDMS microfluidic chip with features possessing multiple height levels. This fabrication method requires neither a cleanroom facility nor an expensive UV exposure machine. The central part of the method stays on the alignment of numerous PDMS slabs on a wafer-scale instead of applying an alignment for a photomask positioned right above a prior exposure layer using a sophisticated mask aligner. We used a manual XYZR stage attached to a vacuum tweezer to manipulate the top PDMS slab. The bottom PDMS slab sat on a rotational stage to conveniently align with the top part. The movement of the two slabs was observed by a monocular scope with a coaxial light source. As an illustration of the potential of this system for fast and low-cost multilayer microfluidic device production, we demonstrate the microfabrication of a 3D microfluidic chaotic mixer. A discussion on another alternative method for the fabrication of multiple height levels is also presented, namely the micromilling approach.

Multicellular Cell Seeding on a Chip: New Design and Optimization towards Commercialization.

This paper shows both experimental and in-depth theoretical studies (including simulations and analytical solutions) on a microfluidic platform to optimize its design and use for 3D multicellular co-culture applications, e.g., creating a tissue-on-chip model for investigating diseases such as pulmonary arterial hypertension (PAH). A tissue microfluidic chip usually has more than two channels to seed cells and supply media. These channels are often separated by barriers made of micro-posts. The optimization for the structures of these micro-posts and their spacing distances is not considered previously, especially for the aspects of rapid and cost-efficient fabrication toward scaling up and commercialization. Our experimental and theoretical (COMSOL simulations and analytical solutions) results showed the followings: (i) The cell seeding was performed successfully for this platform when the pressure drops across the two posts were significantly larger than those across the channel width. The circular posts can be used in the position of hexagonal or other shapes. (ii) In this work, circular posts are fabricated and used for the first time. They offer an excellent barrier effect, i.e., prevent the liquid and gel from migrating from one channel to another. (iii) As for rapid and cost-efficient production, our computer-aided manufacturing (CAM) simulation confirms that circular-post fabrication is much easier and more rapid than hexagonal posts when utilizing micro-machining techniques, e.g., micro-milling for creating the master mold, i.e., the shim for polymer injection molding. The findings open up a possibility for rapid, cost-efficient, large-scale fabrication of the tissue chips using micro-milling instead of expensive clean-room (soft) lithography techniques, hence enhancing the production of biochips via thermoplastic polymer injection molding and realizing commercialization.

3D Printing in Solid Dosage Forms and Organ-on-Chip Applications.

3D printing (3DP) can serve not only as an excellent platform for producing solid dosage forms tailored to individualized dosing regimens but can also be used as a tool for creating a suitable 3D model for drug screening, sensing, testing and organ-on-chip applications. Several new technologies have been developed to convert the conventional dosing regimen into personalized medicine for the past decade. With the approval of Spritam, the first pharmaceutical formulation produced by 3DP technology, this technology has caught the attention of pharmaceutical researchers worldwide. Consistent efforts are being made to improvise the process and mitigate other shortcomings such as restricted excipient choice, time constraints, industrial production constraints, and overall cost. The objective of this review is to provide an overview of the 3DP process, its types, types of material used, and the pros and cons of each technique in the application of not only creating solid dosage forms but also producing a 3D model for sensing, testing, and screening of the substances. The application of producing a model for the biosensing and screening of drugs besides the creation of the drug itself, offers a complete loop of application for 3DP in pharmaceutics.