Medical Device Industry Approaches for Addressing Sources of Failing Cytotoxicity Scores.

To ensure patient safety, medical device manufacturers are required by the Food and Drug Administration and other regulatory bodies to perform biocompatibility evaluations on their devices per standards, such as the AAMI-approved ISO 10993-1:2018 (ANSI/AAMI/ISO 10993-1:2018).However, some of these biological tests (e.g., systemic toxicity studies) have long lead times and are costly, which may hinder the release of new medical devices. In recent years, an alternative method using a risk-based approach for evaluating the toxicity (or biocompatibility) profile of chemicals and materials used in medical devices has become more mainstream. This approach is used as a complement to or substitute for traditional testing methods (e.g., systemic toxicity endpoints). Regardless of the approach, the one test still used routinely in initial screening is the cytotoxicity test, which is based on an in vitro cell culture system to evaluate potential biocompatibility effects of the final finished form of a medical device. However, it is known that this sensitive test is not always compatible with specific materials and can lead to failing cytotoxicity scores and an incorrect assumption of potential biological or toxicological adverse effects. This article discusses the common culprits of in vitro cytotoxicity failures, as well as describes the regulatory-approved methodology for cytotoxicity testing and the approach of using toxicological risk assessment to address clinical relevance of cytotoxicity failures for medical devices. Further, discrepancies among test results from in vitro tests, use of published half-maximal inhibitory concentration data, and the derivation of their relationship to tolerable exposure limits, reference doses, or no observed adverse effect levels are highlighted to demonstrate that although cytotoxicity tests in general are regarded as a useful sensitive screening assays, specific medical device materials are not compatible with these cellular/in vitro systems. For these cases, the results should be analyzed using more clinically relevant approaches (e.g., through chemical analysis or written risk assessment).

Cell-penetrating peptide secures an efficient endosomal escape of an intact cargo upon a brief photo-induction.

Since their discovery, cell-penetrating peptides (CPPs) have provided a novel, efficient, and non-invasive mode of transport for various (bioactive) cargos into cells. Despite the ever-growing number of successful implications of the CPP-mediated delivery, issues concerning their intracellular trafficking, significant targeting to degradative organelles, and limited endosomal escape are still hindering their widespread use. To overcome these obstacles, we have utilized a potent photo-induction technique with a fluorescently labeled protein cargo attached to an efficient CPP, TP10. In this study we have determined some key requirements behind this induced escape (e.g., dependence on peptide-to-cargo ratio, time and cargo), and have semi-quantitatively assessed the characteristics of the endosomes that become leaky upon this treatment. Furthermore, we provide evidence that the photo-released cargo remains intact and functional. Altogether, we can conclude that the photo-induced endosomes are specific large complexes-condensed non-acidic vesicles, where the released cargo remains in its native intact form. The latter was confirmed with tubulin as the cargo, which upon photo-induction was incorporated into microtubules. Because of this, we propose that combining the CPP-mediated delivery with photo-activation technique could provide a simple method for overcoming major limitations faced today and serve as a basis for enhanced delivery efficiency and a subsequent elevated cellular response of different bioactive cargo molecules.

Retro-inversion of certain cell-penetrating peptides causes severe cellular toxicity.

Cell-penetrating peptides (CPPs) are a promising group of delivery vectors for various therapeutic agents but their application is often hampered by poor stability in the presence of serum. Different strategies to improve peptide stability have been exploited, one of them being "retro-inversion" (RI) of natural peptides. With this approach the stability of CPPs has been increased, thereby making them more efficient transporters. Several RI-CPPs were here assessed and compared to the corresponding parent peptides in different cell-lines. Surprisingly, treatment of cells with these peptides induced trypsin insensitivity and rapid severe toxicity in contrast to L-peptides. This was measured as reduced metabolic activity and condensed cell nuclei, in parity with the apoptosis inducing agent staurosporine. Furthermore, effects on mitochondrial network, focal adhesions, actin cytoskeleton and caspase-3 activation were analyzed and adverse effects were evident at 20 µM peptide concentration within 4 h while parent L-peptides had negligible effects. To our knowledge this is the first time RI peptides are reported to cause cellular toxicity, displayed by decreased metabolic activity, morphological changes and induction of apoptosis. Considering the wide range of research areas that involves the use of RI-peptides, this finding is of major importance and needs to be taken under consideration in applications of RI-peptides.

Peptide-mediated protein delivery-which pathways are penetrable?

The growing number of reports on the effective cargo delivery by cell-penetrating peptides (CPPs) has extensively widened our knowledge about the mechanisms involved in CPP-mediated delivery. However, the data available on the internalization mode of CPP-cargo complexes are often conflicting and/or equivocal. Moreover, the intracellular trafficking of CPP-cargo complexes is, to date, relatively unexplored resulting in only minimal information about what is really happening to the complexes inside the cell. This review focuses on defining the endocytic pathways engaged in the transduction of CPP-cargo complexes and seeks to determine the extent of different endocytic routes required for effective uptake. In addition, the intracellular pathways utilized during the trafficking and sorting of CPP-cargo complexes as well as the ultimate fate of the complexes inside cells will be discussed.

Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads.

Lumen morphogenesis results from the interplay between molecular pathways and mechanical forces. In several organs, epithelial cells share their apical surfaces to form a tubular lumen. In the liver, however, hepatocytes share the apical surface only between adjacent cells and form narrow lumina that grow anisotropically, generating a 3D network of bile canaliculi (BC). Here, by studying lumenogenesis in differentiating mouse hepatoblasts in vitro, we discovered that adjacent hepatocytes assemble a pattern of specific extensions of the apical membrane traversing the lumen and ensuring its anisotropic expansion. These previously unrecognized structures form a pattern, reminiscent of the bulkheads of boats, also present in the developing and adult liver. Silencing of Rab35 resulted in loss of apical bulkheads and lumen anisotropy, leading to cyst formation. Strikingly, we could reengineer hepatocyte polarity in embryonic liver tissue, converting BC into epithelial tubes. Our results suggest that apical bulkheads are cell-intrinsic anisotropic mechanical elements that determine the elongation of BC during liver tissue morphogenesis.

Adenosine-oligoarginine conjugate, a novel bisubstrate inhibitor, effectively dissociates the actin cytoskeleton.

Aberrant regulation of protein kinases impairs normal cellular functioning and may lead to disease. The protein kinase involved in the regulation of the dynamics of the actin cytoskeleton, Rho-kinase (ROCK), phosphorylates various substrates (e.g. myosin light chain, myosin phosphatase), causing the formation of actin fibers and tension inside cells. Hyperactivation of ROCK, for example, causes hypertension and cardiovascular disorders. Thus, the design of highly specific protein kinase inhibitors is of the utmost importance. To date, the majority of inhibitors investigated have been found to mimic and compete with ATP. However, in the present study we characterized the cellular effects of a novel bisubstrate inhibitor -- adenosine-oligoarginine conjugate (ARC) -- designed to interfere simultaneously with the ATP site and the substrate-binding pocket of basophilic kinases. ARC effectively pulled down ROCK from cell lysates, showed no cytotoxicity and suppressed the assembly of the actin cytoskeleton (especially central actin bundles) as the result of interference with the activity of the kinase. Combination of ARC with chloroquine yielded a stronger inhibitory effect and gave results similar to treatment with Y-27632. However, treatment with ARC produced more actin fragments and yielded a longer-lasting effect than treatment with Y-27632. Additionally, quantification of phosphorylated myosin light chain levels in ARC-treated or Y-27632-treated cells implies that ARC is more effective than Y-27632 in suppressing the phosphorylation of at least one of the substrates of ROCK. We believe that the described bisubstrate strategy could be a useful lead for designing novel, highly specific inhibitors for different protein kinases.

Mapping of protein transduction pathways with fluorescent microscopy.

The number of various cargo delivered into cells by CPPs demonstrates the effective transport abilities of these short-peptidic sequences. Over the years of research, the translocation process of CPP-cargo complexes has been mapped to being of mostly endocytic nature, however, there is still no consensus on which of the endocytic routes is prevalent and to which extent the interplay between different modes of endocytosis is taking place. The intracellular trafficking of CPPs attached to a cargo molecule is even less understood. Therefore, the internalization and the subsequent intracellular targeting of complexes need clarification in order to define cellular destinations and improve the targeting of the cargo molecule to specific cellular compartments depending on the cargo attached to the transporting vector. This chapter focuses on describing the methods for visualizing the CPP-protein complexes in relation to different endocytic markers, for example transferrin (marker for clathrin-mediated endocytosis) and cholera toxin (ambiguous marker for clathrin-, caveolin-, and flotillin-mediated, but also clathrin- and caveolin-independent endocytosis) to determine the role of the respective pathways during entry to cells, and to different intracellular targets, for instance the lysosomal organelles or the Golgi apparatus. Additionally, antibody staining of respective endocytic vesicles following the internalization of CPP-protein complexes will be discussed.

CPP-protein constructs induce a population of non-acidic vesicles during trafficking through endo-lysosomal pathway.

The major limitation in the application of bioactive molecules is their low permeation across plasma membrane. Effective transporters - cell-penetrating peptides (CPPs) - are utilized to enhance uptake of various cargo upon attachment to its sequences. Still, information about relevance of different endocytic routes during CPP-cargo internalization is ambiguous and underlying mechanism(s) of intracellular trafficking is even less understood. We first defined involvement of recycling pathway in trafficking of 3 different CPPs - transportan, oligoarginine and Tat - complexed to avidin-TexasRed in Cos-7 cells in relation to trans-Golgi network spatially constraining recycling endosomes. By confocal microscopy, only a negligible fraction of complexes-containing vesicles were found inside trans-Golgi ring suggesting its marginal role in CPP-mediated delivery. Secondly, we characterized engagement of endo-lysosomal pathway to assess acidity of complexes-containing vesicles. CPPs induced 3 different populations of complexes-containing vesicles which size and proportion depended on CPP, time and concentration. In time, more complexes were targeted to low-pH structures. However, a population of complexes-containing vesicles was observed to retain rather neutral pH. Induction of vesicles with non-acidic pH generated i.e. by caveolin-dependent endocytosis or by CPPs themselves during intracellular trafficking could be the key step in inducement of escape of complexes from endosomal structures, a limiting step in effective cargo delivery by CPPs.