Freeze-drying revolution: unleashing the potential of lyophilization in advancing drug delivery systems.

Lyophilization also known as freeze-drying is a technique that has been employed to enhance the long-term durability of nanoparticles (NPs) that are utilized for drug delivery applications. This method is used to prevent their instability in suspension. However, this dehydration process can cause stress to the NPs, which can be alleviated by the incorporation of excipients like cryoprotectants and lyoprotectants. Nevertheless, the freeze-drying of NPs is often based on empirical principles without considering the physical-chemical properties of the formulations and the engineering principles of freeze-drying. For this reason, it is crucial to optimize the formulations and the freeze-drying cycle to obtain a good lyophilizate and ensure the preservation of NPs stability. Moreover, proper characterization of the lyophilizate and NPs is of utmost importance in achieving these goals. This review aims to update the recent advancements, including innovative formulations and novel approaches, contributing to the progress in this field, to obtain the maximum stability of formulations. Additionally, we critically analyze the limitations of lyophilization and discuss potential future directions. It addresses the challenges faced by researchers and suggests avenues for further research to overcome these limitations. In conclusion, this review is a valuable contribution to the understanding of the parameters involved in the freeze-drying of NPs. It will definitely aid future studies in obtaining lyophilized NPs with good quality and enhanced drug delivery and therapeutic benefits.

An In Vitro Small Intestine Model Incorporating a Food Matrix and Bacterial Mock Community for Intestinal Function Testing.

Consumed food travels through the gastrointestinal tract to reach the small intestine, where it interacts with the microbiota, forming a complex relationship with the dietary components. Here we present a complex in vitro cell culture model of the small intestine that includes human cells, digestion, a simulated meal, and a microbiota represented by a bacterial community consisting of E. coli, L. rhamnosus, S. salivarius, B. bifidum, and E. faecalis. This model was used to determine the effects of food-grade titanium dioxide nanoparticles (TiO2 NPs), a common food additive, on epithelial permeability, intestinal alkaline phosphatase activity, and nutrient transport across the epithelium. Physiologically relevant concentrations of TiO2 had no effect on intestinal permeability but caused an increase in triglyceride transport as part of the food model, which was reversed in the presence of bacteria. Individual bacterial species had no effect on glucose transport, but the bacterial community increased glucose transport, suggesting a change in bacterial behavior when in a community. Bacterial entrapment within the mucus layer was reduced with TiO2 exposure, which may be due to decreased mucus layer thickness. The combination of human cells, a synthetic meal, and a bacterial mock community provides an opportunity to understand the implications of nutritional changes on small intestinal function, including the microbiota.

An expanding horizon of complex injectable products: development and regulatory considerations.

There has been a constant evolution in the pharmaceutical market concerning the new technologies imbibed in delivering drug substances for various indications. This is either market-driven or technology-driven to improve the overall therapeutic efficacy and patients' quality of life. The pharmaceutical industry has experienced rapid growth in the area of complex injectable products because of their effectiveness in the unmet market. These novel parenteral products, viz, the nanoparticles, liposomes, microspheres, suspensions, and emulsions, have proven their worth as "Safe and Effective" products. However, the underlying challenges involved in the development, scalability, and characterization of these injectable products are critical. Moreover, the guidelines available do not provide a clear understanding of these complex products, making it difficult to anticipate the regulatory requirements. Thus, it becomes imperative to comprehend the criticalities and develop an understanding of these products. This review discusses various complexities involved in the parenteral products such as complex drug substances, excipients, dosage forms, drug administration devices like pre-filled syringes and injector pens, and its different characterization tools and techniques. The review also provides a brief discussion on the regulatory aspects and associated hurdles with other parenteral products.

Fluid shear stress in a logarithmic microfluidic device enhances cancer cell stemness marker expression.

Fluid shear stress (FSS) is crucial in cancer cell survival and tumor development. Noteworthily, cancer cells are exposed to several degrees of FSS in the tumor microenvironment and during metastasis. Consequently, the stemness marker expression in cancer cells changes with the FSS signal, although it is unclear how it varies with different magnitudes and during metastasis. The current work explores the stemness and drug resistance characteristics of the cervical cancer cell line HeLa in a microfluidic device with a wide range of physiological FSS. Hence, the microfluidic device was designed to achieve a logarithmic flow distribution in four culture chambers, realizing four orders of biological shear stress on a single chip. The cell cycle analysis demonstrated altered cell proliferation and mitotic arrest after FSS treatment. In addition, EdU staining revealed increased cell proliferation with medium to low FSS, whereas high shear had a suppressing effect. FSS increased competence to withstand higher intracellular ROS and mitochondrial membrane potential in HeLa. Furthermore, stemness-related gene (Sox2, N-cadherin) and cell surface marker (CD44, CD33, CD117) expressions were enhanced by FSS mechanotransduction in a magnitude-dependent manner. In summary, these stemness-like properties were concurrent with the drug resistance capability of HeLa towards doxorubicin. Overall, our microfluidic device elucidates cancer cell survival and drug resistance mechanisms during metastasis and in cancer relapse patients.

Injectable hydrogel for co-delivery of 5-azacytidine in zein protein nanoparticles with stem cells for cardiac function restoration.

Heart failure is major cause of mortality associated with mostly Myocardial infarction (MI). Transplanting mesenchymal stem cells (MSC) have exhibited potential role in myocardial regeneration. Secretion of immune-modulatory cytokines and various growth factors after transplantation plays significant role in remodelling process of MI region. However, low retention, higher shear stress during administration and rejection at host infarct environment hinders therapeutic efficacy. Myocardial regeneration demands for accurate spatio-temporal delivery of MSCs with supportive vascular network that leads to improvement of cardiac function. In this study, injectable alginate based microporous hydrogel has been used to deliver 5-Azacytidine (5-Aza) in zein protein nanoparticle with MSCs for attenuating adverse cardiac remodelling after MI. Zein nanoparticles loaded with 5-Aza were prepared by liquid-liquid dispersion, and it was found that 35% of drug was released in 7 days supported with mathematical modelling. The presence of 5-Aza and zein in developed hydrogel supported in vitro MSC proliferation, migration and angiogenesis. Significant increased expression of cardiac specific markers, GATA4, MEF2C, MLC, SERCA and NKX2.5 was observed in vitro. 5-Aza loaded protein nanoparticle with MSCs encapsulated hydrogels in rat MI model also exhibited substantial improvement of functional cardiac parameters such as cardiac output and ejection fraction. Histopathological analysis showed reduced fibrosis, attenuated infarct expansion and cardiac tissue restoration and angiogenesis. In brief, we developed nanocarrier-hydrogel system a promising strategy for co-delivering 5-Aza as cardiac differentiation cue with MSCs to achieve higher cell retention and enhanced improvement in myocardial regeneration after MI.

Recent Advances in Cardiac Tissue Engineering for the Management of Myocardium Infarction.

Myocardium Infarction (MI) is one of the foremost cardiovascular diseases (CVDs) causing death worldwide, and its case numbers are expected to continuously increase in the coming years. Pharmacological interventions have not been at the forefront in ameliorating MI-related morbidity and mortality. Stem cell-based tissue engineering approaches have been extensively explored for their regenerative potential in the infarcted myocardium. Recent studies on microfluidic devices employing stem cells under laboratory set-up have revealed meticulous events pertaining to the pathophysiology of MI occurring at the infarcted site. This discovery also underpins the appropriate conditions in the niche for differentiating stem cells into mature cardiomyocyte-like cells and leads to engineering of the scaffold via mimicking of native cardiac physiological conditions. However, the mode of stem cell-loaded engineered scaffolds delivered to the site of infarction is still a challenging mission, and yet to be translated to the clinical setting. In this review, we have elucidated the various strategies developed using a hydrogel-based system both as encapsulated stem cells and as biocompatible patches loaded with cells and applied at the site of infarction.

Low intermittent flow promotes rat mesenchymal stem cell differentiation in logarithmic fluid shear device.

Bone marrow mesenchymal stem cells are an ideal candidate for bone tissue engineering due to their osteogenic potential. Along with chemical, mechanical signals such as fluid shear stress have been found to influence their differentiation characteristics. But the range of fluid shear experienced in vivo is too wide and difficult to generate in a single device. We have designed a microfluidic device that could generate four orders of shear stresses on adherent cells. This was achieved using a unique hydraulic resistance combination and linear optimization to the lesser total length of the circuit, making the device compact and yet generating four logarithmically increasing shear stresses. Numerical simulation depicts that, at an inlet velocity of 160 µl/min, our device generated shear stresses from 1.03 Pa to 1.09 mPa. In this condition, we successfully cultured primary rat bone marrow mesenchymal stem cells (rBMSCs) in the device for a prolonged period of time in the incubator environment (four days). Higher cell proliferation rate was observed in the intermittent flow at 1.09 mPa. At 10 mPa, both upregulation of osteogenic genes and higher alkaline phosphatase activity were observed. These results suggest that the intermittent shear of the order of 10 mPa can competently enhance osteogenic differentiation of rBMSCs compared to static culture.

Organotypic cancer tissue models for drug screening: 3D constructs, bioprinting and microfluidic chips.

Successful translation of potential cancer chemotherapeutic drugs to the clinic depends on sufficient predictability of response in the human system through in vitro simulations. High expenditure and longer duration in preclinical and clinical research urge the enhancement of effective in vitro drug screening. 3D models emulate cell morphology, cell-cell and cell-matrix interactions and are physiologically more relevant for predicting drug responses for complex heterogenic cancers, widely replacing conventional cultures. Bioprinting and microfluidic technology facilitate tissue mimetic model construction and multifaceted simulation of physiology, respectively, promising more-appropriate predictability of drug interactions. Precisely, organotypic tissue constructs assembled using cell-laden matrices or organ-on-a-chip serve as realistic tissue models. This review projects the progress toward biomimetic tissue model development, highlighting the emergence of bioprinting and microfluidic technology in in vitro cancer drug screening and pertaining challenges.

Long range microfluidic shear device for cellular mechanotransduction studies.

Cells sense external mechanical stimulus and respond to it through mechanotransduction mechanism. Fluid shear stress (FSS) has been found to be an important element among the mechanical stimuli. Recent advancements in microfluidics made mechanotransduction studies possible in near physiological conditions using microfluidic devices. FSS on human cells covers a broad range from very low level experienced due to interstitial flows (0.1 mPa) to very high level in aorta (10 Pa). In the present communication, we have designed a novel microfluidic device which can generate FSS on cells of five different orders with single inflow of fluid which can cover the whole range of physiological fluid shear stresses in one run. The dimensions of the device were calculated taking a resistance model for the micro channels. Flow velocities and wall shear stress were predicted through computer simulation. Shear stress values were analyzed for two different depths of channels and different inlet flow rates ranging from 50 to 0.5 µl/s. FSS was found to increase linearly with inlet flow rate and the stress profile was flatter for lesser depth of channel.