Decellularized kidney extracellular matrix-based hydrogels for renal tissue engineering.

Kidney regeneration is hindered by the limited pool of intrinsic reparative cells. Advanced therapies targeting renal regeneration have the potential to alleviate the clinical and financial burdens associated with kidney disease. Delivery systems for cells, extracellular vesicles, or growth factors aimed at enhancing regeneration can benefit from vehicles enabling targeted delivery and controlled release. Hydrogels, optimized to carry biological cargo while promoting regeneration, have emerged as promising candidates for this purpose. This study aims to develop a hydrogel from decellularized kidney extracellular matrix (DKECM) and explore its biocompatibility as a biomaterial for renal regeneration. The resulting hydrogel crosslinks with temperature and exhibits a high concentration of extracellular matrix. The decellularization process efficiently removes detergent residues, yielding a pathogen-free biomaterial that is non-hemolytic and devoid of α-gal epitope. Upon interaction with macrophages, the hydrogel induces differentiation into both pro-inflammatory and anti-inflammatory phenotypes, suggesting an adequate balance to promote biomaterial functionality in vivo. Renal progenitor cells encapsulated in the DKECM hydrogel demonstrate higher viability and proliferation than in commercial collagen-I hydrogels, while also expressing tubular cells and podocyte markers in long-term culture. Overall, the injectable biomaterial derived from porcine DKECM is anticipated to elicit minimal host reaction while fostering progenitor cell bioactivity, offering a potential avenue for enhancing renal regeneration in clinical settings. STATEMENT OF SIGNIFICANCE: The quest to improve treatments for kidney disease is crucial, given the challenges faced by patients on dialysis or waiting for transplants. Exciting new therapies combining biomaterials with cells can revolutionize kidney repair. In this study, researchers created a hydrogel from pig kidney. This gel could be used to deliver cells and other substances that help in kidney regeneration. Despite coming from pigs, it's safe for use in humans, with no harmful substances and reduced risk of immune reactions. Importantly, it promotes a balanced healing response in the body. This research not only advances our knowledge of kidney repair but also offers hope for more effective treatments for kidney diseases.

Microfluidic Devices: A Tool for Nanoparticle Synthesis and Performance Evaluation.

The use of nanoparticles (NPs) in nanomedicine holds great promise for the treatment of diseases for which conventional therapies present serious limitations. Additionally, NPs can drastically improve early diagnosis and follow-up of many disorders. However, to harness their full capabilities, they must be precisely designed, produced, and tested in relevant models. Microfluidic systems can simulate dynamic fluid flows, gradients, specific microenvironments, and multiorgan complexes, providing an efficient and cost-effective approach for both NPs synthesis and screening. Microfluidic technologies allow for the synthesis of NPs under controlled conditions, enhancing batch-to-batch reproducibility. Moreover, due to the versatility of microfluidic devices, it is possible to generate and customize endless platforms for rapid and efficient in vitro and in vivo screening of NPs' performance. Indeed, microfluidic devices show great potential as advanced systems for small organism manipulation and immobilization. In this review, first we summarize the major microfluidic platforms that allow for controlled NPs synthesis. Next, we will discuss the most innovative microfluidic platforms that enable mimicking in vitro environments as well as give insights into organism-on-a-chip and their promising application for NPs screening. We conclude this review with a critical assessment of the current challenges and possible future directions of microfluidic systems in NPs synthesis and screening to impact the field of nanomedicine.

Size-Dependent Polymeric Nanoparticle Distribution in a Static versus Dynamic Microfluidic Blood Vessel Model: Implications for Nanoparticle-Based Drug Delivery.

Nanoparticles (NPs) have been widely investigated in the nanomedicine field. One of the main challenges is to accurately predict the NP distribution and fate after administration. Microfluidic platforms acquired huge importance as tools to model the in vivo environment. In this study, we leveraged a microfluidic platform to produce FITC-labeled poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-PEG) NPs with defined sizes of 30, 50, and 70 nm. The study aimed to compare the ability of NPs with differences of 20 nm in size to cross an endothelial barrier using static (Transwell inserts) and dynamic (microfluidic perfusion device) in vitro models. Our results evidence a size-dependent NP crossing in both models (30 > 50 > 70 nm) and highlight the bias deriving from the static model, which does not involve shear stresses. The permeation of each NP size was significantly higher in the static system than in the dynamic model at the earliest stages. However, it gradually decreased to levels comparable with those of the dynamic model. Overall, this work highlights clear differences in NP distribution over time in static versus dynamic conditions and distinct size-dependent patterns. These findings reinforce the need for accurate in vitro screening models that allow for more accurate predictions of in vivo performance.

On the size-dependent internalization of sub-hundred polymeric nanoparticles.

The understanding of the interaction between nanoparticles (NPs) and cells is crucial to design nanocarriers with high therapeutic relevance. In this study, we exploited a microfluidics device to synthesize homogeneous suspensions of NPs with ≈ 30, 50, and 70 nm of size. Afterward, we investigated their level and mechanism of internalization when exposed to different types of cells (endothelial cells, macrophages, and fibroblasts). Our results show that all NPs were cytocompatible and internalized by the different cell types. However, NPs uptake was size-dependent, being the maximum uptake efficiency observed for the 30 nm NPs. Moreover, we demonstrate that size can lead to distinct interactions with different cells. For instance, 30 nm NPs were internalized with an increasing trend over time by endothelial cells, while a steady and a decreasing trend were observed when incubated with LPS-stimulated macrophages and fibroblasts, respectively. Finally, the use of different chemical inhibitors (chlorpromazine, cytochalasin-D, and nystatin), and low temperature (4 °C) indicated that phagocytosis/micropinocytosis are the main internalization mechanism for all NPs sizes. However, different endocytic pathways were initiated in the presence of particular NP sizes. In endothelial cells, for example, caveolin-mediated endocytosis occurs primarily in the presence of 50 nm NPs, whereas clathrin-mediated endocytosis substantially promotes the internalization of 70 nm NPs. This evidence demonstrates the importance of size in the NPs design for mediating interaction with specific cell types.

Intracellular Trafficking of Size-Tuned Nanoparticles for Drug Delivery.

Polymeric nanoparticles (NPs) are widely used as drug delivery systems in nanomedicine. Despite their widespread application, a comprehensive understanding of their intracellular trafficking remains elusive. In the present study, we focused on exploring the impact of a 20 nm difference in size on NP performance, including drug delivery capabilities and intracellular trafficking. For that, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PLGA-PEG) NPs with sizes of 50 and 70 nm were precisely tailored. To assess their prowess in encapsulating and releasing therapeutic agents, we have employed doxorubicin (Dox), a well-established anticancer drug widely utilized in clinical settings, as a model drug. Then, the beneficial effect of the developed nanoformulations was evaluated in breast cancer cells. Finally, we performed a semiquantitative analysis of both NPs' uptake and intracellular localization by immunostaining lysosomes, early endosomes, and recycling endosomes. The results show that the smaller NPs (50 nm) were able to reduce the metabolic activity of cancer cells more efficiently than NPs of 70 nm, in a time and concentration-dependent manner. These findings are corroborated by intracellular trafficking studies that reveal an earlier and higher uptake of NPs, with 50 nm compared to the 70 nm ones, by the breast cancer cells. Consequently, this study demonstrates that NP size, even in small increments, has an important impact on their therapeutic effect.

Microfluidic-driven mixing of high molecular weight polymeric complexes for precise nanoparticle downsizing.

Chitosan (CHIT) and hyaluronic acid (HA) are two polysaccharides (PSs) with high value in several biomedical applications. In this study, we present a microfluidic method to synthetize CHIT-HA NPs to overcome the disadvantages of the dropwise approach generally used for nanoprecipitation of polyelectrolyte complexes. The proposed microfluidic approach enables to generate monodisperse suspensions of NPs with ≈100 nm of size compared to the dropwise method that generated ≈2 times bigger NPs. Finally, we evaluated the potential of obtained NPs in an inflammatory scenario. The treatment with NPs led to the reduction of the main inflammatory molecules produced by macrophages (PGE2, IL-6, IL-8, MCAF and TNF-α) and fibroblasts (IL-1 α, PGE2, TNF-α) stimulated with lipopolysaccharide or conditioned medium, respectively. This study demonstrates that our approach can be used to enhance the synthesis of nanocarriers based on bioactive macromolecules.

Microfluidic mixing system for precise PLGA-PEG nanoparticles size control.

In this study, a microfluidic device was employed to produce polymeric nanoparticles (NPs) with well-controlled sizes. The influence of several parameters in the synthesis process, namely, polymer concentration, flow rate and flow rate ratio between the aqueous and organic solutions was investigated. To evaluate the NPs size effect, three diameters were selected (30, 50 and 70 nm). Their cytocompatibility was demonstrated on endothelial cells and macrophages. Additionally, their efficacy to act as drug carriers was assessed in an in vitro inflammatory scenario. NPs loaded and released diclofenac (DCF) in a size-dependent profile (smaller sizes presented lower DCF content and higher release rate). Moreover, 30 nm NPs were the most effective in reducing prostaglandin E2 concentration. Therefore, this study demonstrates that microfluidics can generate stable NPs with controlled sizes, high monodispersity and enhanced batch-to-batch reproducibility. Indeed, NPs size is a crucial parameter for drug encapsulation, release and overall biological efficacy.

Monitoring the Fate of Orally Administered PLGA Nanoformulation for Local Delivery of Therapeutic Drugs.

One of the goals of the pharmaceutical sciences is the amelioration of targeted drug delivery. In this context, nanocarrier-dependent transportation represents an ideal method for confronting a broad range of human disorders. In this study, we investigated the possibility of improving the selective release of the anti-cancer drug paclitaxel (PTX) in the gastro-intestinal tract by encapsulating it into the biodegradable nanoparticles made by FDA-approved poly(lactic-co-glycolic acid) (PLGA) and coated with polyethylene glycol to improve their stability (PLGA-PEG-NPs). Our study was performed by combining the synthesis and characterization of the nanodrug with in vivo studies of pharmacokinetics after oral administration in mice. Moreover, fluorescent PLGA-nanoparticles (NPs), were tested both in vitro and in vivo to observe their fate and biodistribution. Our study demonstrated that PLGA-NPs: (1) are stable in the gastric tract; (2) can easily penetrate inside carcinoma colon 2 (CaCo2) cells; (3) reduce the PTX absorption from the gastrointestinal tract, further limiting systemic exposure; (4) enable PTX local targeting. At present, the oral administration of biodegradable nanocarriers is limited because of stomach degradation and the sink effect played by the duodenum. Our findings, however, exhibit promising evidence towards our overcoming these limitations for a more specific and safer strategy against gastrointestinal disorders.

Repeated administration of the food additive E171 to mice results in accumulation in intestine and liver and promotes an inflammatory status.

Titanium dioxide (TiO2) is widely used in pharmaceuticals preparations, cosmetics, and as a food additive (E171). It contains microparticles and a fraction of nanoparticles (NPs) which can be absorbed systemically by humans after ingestion. Increasing concern has been aroused about the impact of oral exposure to TiO2 NPs from dietary and non-dietary sources on human health. In spite of several toxicological studies conducted in recent years, a solid risk assessment of oral exposure to E171 has not been satisfactorily achieved. We investigated whether repeated oral administration of E171 to mice at a dose level (5 mg/kg body weight for 3 days/week for 3 weeks) comparable to estimated human dietary exposure, results in TiO2 deposition in the digestive system and internal organs, and in molecular and cellular alterations associated with an inflammatory response. To reproduce the first phase of digestion, a new administration approach involving the dripping of the E171 suspension into the mouth of mice was applied. Significant accumulation of titanium was observed in the liver and intestine of E171-fed mice; in the latter a threefold increase in the number of TiO2 particles was also measured. Titanium accumulation in liver was associated with necroinflammatory foci containing tissue monocytes/macrophages. Three days after the last dose, increased superoxide production and inflammation were observed in the stomach and intestine. Overall, the present study indicates that the risk for human health associated with dietary exposure to E171 needs to be carefully considered.

Dexamethasone Conjugation to Biodegradable Avidin-Nucleic-Acid-Nano-Assemblies Promotes Selective Liver Targeting and Improves Therapeutic Efficacy in an Autoimmune Hepatitis Murine Model.

Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acid-labile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS-Hz-Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS-Hz-Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders.