Nanomedicine at the crossroads - A quick guide for IVIVC.

For many years, nanomedicine is pushing the boundaries of drug delivery. When applying these novel therapeutics, safety considerations are not only a key concern when entering clinical trials but also an important decision point in product development. Standing at the crossroads, nanomedicine may be able to escape the niche markets and achieve wider acceptance by the pharmaceutical industry. While there is a new generation of drug delivery systems, the extracellular vesicles, standing on the starting line, unresolved issues and new challenges emerge from their translation from bench to bedside. Some key features of injectable nanomedicines contribute to the predictability of the pharmacological and toxicological effects. So far, only a few of the physicochemical attributes of nanomedicines can be justified by a direct mathematical relationship between the in vitro and the in vivo responses. To further develop extracellular vesicles as drug carriers, we have to learn from more than 40 years of clinical experience in liposomal delivery and pass on this knowledge to the next generation. Our quick guide discusses relationships between physicochemical characteristics and the in vivo response, commonly referred to as in vitro-in vivo correlation. Further, we highlight the key role of computational methods, lay open current knowledge gaps, and question the established design strategies. Has the recent progress improved the predictability of targeted delivery or do we need another change in perspective?

Heparin modulates the cellular uptake of nanomedicines.

Liposomal formulations are used to improve the safety and cellular absorption of conventional drugs by limiting their interaction with phagocytes. The uptake behaviour of these nanocarriers is affected by the blood composition, and accordingly the presence of an anticoagulant in the blood could have a critical impact on the efficiency of nanomedicines. For the negatively charged liposomes, such as AmBisome®, no significant change in the uptake could be observed when co-incubated with heparin and primary phagocytes. Yet, we observed that a peak of the uptake extent of cationic liposomes was reached at a clinically relevant concentration of heparin for phagocytes and cancer cells. Hence, we recommend avoiding treatment of a heparinized patient with cationic nanomedicines because unexpectedly high uptake can occur in phagocytes.

Vitamin C Loaded Polyethylene: Synthesis and Properties of Precise Polyethylene with Vitamin C Defects via Acyclic Diene Metathesis Polycondensation.

A polyethylene-like polymer with an in-chain vitamin C group was synthesized by olefin metathesis polymerization. Here, we describe both the synthesis and a comprehensive physical characterization. Because of the olefin metathesis synthesis, the vitamin C groups are equidistantly arranged in the polyethylene (PE) main chain. Their separation was adjusted to 20 CH2 units. After hydrogenation, a semicrystalline polymer is obtained that is soluble in polar solvents. Because of its size and steric effect, the vitamin C acts as a chain defect, which is expelled from the crystal lattice, yielding a lamellar crystal with a homogeneous thickness corresponding to the interdefect distance. The physical properties were examined by various methods including differential scanning calorimetry, X-ray scattering, and transmission electron microscopy. We show that vitamin C retains its radical scavenger properties despite being incorporated into a polyethylene chain. Furthermore, we demonstrate that it is degrading in alkaline conditions. To complete its suitability as a biocompatible material, cytotoxicity and cell uptake experiments were performed. We show that the polymer is nontoxic and that it is taken up in nanoparticular form via endocytosis processes into the cytoplasm of cells.

From In Silico to Experimental Validation: Tailoring Peptide Substrates for a Serine Protease.

Smart nanocarriers for the transport of drugs to tumor cells are nowadays of great interest for treating cancer. The use of enzymatic stimuli to cleave peptide-based drug nanocapsules for the selective release of nanocapsule cargo in close proximity to tumor cells opens new possibilities in cancer research. In the present work, we demonstrate a methodology for finding and optimizing cleavable substrate sequences by the type II transmembrane serine protease hepsin, which is highly overexpressed in prostate cancer. The design and screening of combinatorial libraries in silico against the binding cavity of hepsin allow the identification of a panel of promising substrates with high-calculated docking scores. In vitro screening verifies the predictions and showed that all substrates are cleaved by hepsin with higher efficiency than the literature known hepsin substrate RQLR↓VVGG. The introduction of d-amino acids on a selected peptide with the highest catalytic efficiency (kcat/Km) renders it resistant to cleavage by plasma or serum while maintaining their susceptibility to hepsin.

Timing of Heparin Addition to the Biomolecular Corona Influences the Cellular Uptake of Nanocarriers.

Few studies have considered the interaction of nanocarriers with drugs and the implications for their individual efficiency. Here, we demonstrate that heparin, a common anticoagulant, interacts with nanocarriers. Hence, nanocarriers, precoated with heparin and plasma in different conditions, were incubated with cancer cells, as well as primary cells from human blood. The relation between the timing of the heparin's addition to the nanocarrier and the cellular uptake extent was assessed by flow cytometry. Through proteomics the effect of heparin on the biomolecular corona composition was determined. We found that HeLa cells, monocytes and macrophages reacted differently to the presence of heparin: the uptake of the precoated nanocarriers decreased for HeLa and primary monocytes, while it increased for macrophages. Heparin induced no obvious change in the protein corona composition; thus, we suggest that heparin itself, through its adsorption on the nanocarrier, was responsible for the change of uptake.

Development of a panel of DNA Aptamers with High Affinity for Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer costs nearly 40,000 lives in the U.S. each year and has one of the lowest survival rates among cancers. Effective treatment of pancreatic ductal adenocarcinoma is hindered by lack of a reliable biomarker. To address this challenge, aptamers were selected by cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) targeting human pancreatic ductal adenocarcinoma (PL45). Five promising aptamers presenting low Kd values and good specificity were generated. Among these five aptamers, one was tailored into a nanostructure carrying a high drug payload for specific drug delivery. The results show a viability of almost 80% for negative cells while only 50% of the target cells remained alive after 48 h incubation. These results lead to the conclusion that further research could reveal protein biomarkers specific to pancreatic adenocarcinoma, with probes available for early detection.

DNA Aptamer Selected against Pancreatic Ductal Adenocarcinoma for in vivo Imaging and Clinical Tissue Recognition.

In this work, we have developed a truncated DNA aptamer, termed XQ-2d, with high affinity and specificity for pancreatic ductal adenocarcinoma (PDAC). Aptamer XQ-2d selectively binds to PL45 cells with a dissociation constant in the nanomolar range, as determined by its recognition of PL45 tumor cells in mice. Moreover, XQ-2d shows better recognition ratio for 40 tissue sections of clinical PDAC samples (82.5%) compared to the initial cell-SELEX selection library (5%). Therefore, XQ-2d can be considered a promising candidate as a tool for PDAC diagnosis and treatment.

Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX.

Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment), have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers.

Evolution of functional six-nucleotide DNA.

Axiomatically, the density of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addition could also add functional groups not found in natural DNA, but useful for molecular performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-ß-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a laboratory in vitro evolution (LIVE) experiment; the GACTZP library was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.

Identification of cell membrane protein stress-induced phosphoprotein 1 as a potential ovarian cancer biomarker using aptamers selected by cell systematic evolution of ligands by exponential enrichment.

In this paper, we describe the elucidation of the target of an aptamer against ovarian cancer previously obtained by cell-SELEX (SELEX = systematic evolution of ligands by exponential enrichment). The target's identity, stress-induced phosphoprotein 1 (STIP1), was determined by mass spectrometry and validated by flow cytometry, using siRNA silencing and protein blotting. Initial oncologic studies show that the aptamer inhibits cell invasion, indicating that STIP1, which is currently under investigation as a potential biomarker for ovarian cancer, plays a critical role in this process. These results serve as an excellent example of how protein target identification of aptamers obtained by cell-SELEX can serve as a means to identify promising biomarker candidates and can promote the development of aptamers as a new drug class to block important oncological processes.