Magnesium sensing via LFA-1 regulates CD8+ T cell effector function.

The relevance of extracellular magnesium in cellular immunity remains largely unknown. Here, we show that the co-stimulatory cell-surface molecule LFA-1 requires magnesium to adopt its active conformation on CD8+ T cells, thereby augmenting calcium flux, signal transduction, metabolic reprogramming, immune synapse formation, and, as a consequence, specific cytotoxicity. Accordingly, magnesium-sufficiency sensed via LFA-1 translated to the superior performance of pathogen- and tumor-specific T cells, enhanced effectiveness of bi-specific T cell engaging antibodies, and improved CAR T cell function. Clinically, low serum magnesium levels were associated with more rapid disease progression and shorter overall survival in CAR T cell and immune checkpoint antibody-treated patients. LFA-1 thus directly incorporates information on the composition of the microenvironment as a determinant of outside-in signaling activity. These findings conceptually link co-stimulation and nutrient sensing and point to the magnesium-LFA-1 axis as a therapeutically amenable biologic system.

Variable asialoglycoprotein receptor 1 expression in liver disease: Implications for therapeutic intervention.

AIM: One of the most promising strategies for the treatment of liver diseases is targeted drug delivery via the asialoglycoprotein receptor (ASGPR). The success of this approach heavily depends on the ASGPR expression level on parenchymal liver cells. In this study, we assessed the mRNA and protein expression levels of the major receptor subunit, ASGR1, in hepatocytes both in vitro and in vivo. METHODS: In vitro, various liver cancer-derived cell lines were evaluated. In vivo, we screened the ASGR1 mRNA on 59 hepatocellular carcinoma and matched non-neoplastic tissue using RNA microarray. In addition, 350 human liver specimens of patients with hepatocellular carcinoma or non-neoplastic liver diseases were screened for ASGR1 protein level using tissue microarray analysis. RESULTS: Our data reveal that the ASGR1 mRNA expression directly correlates with the protein level. We demonstrate that the ASGR1 expression is upregulated in cirrhotic specimens and is significantly decreased with increasing hepatocellular carcinoma grade. CONCLUSION: Because the ASGR1 expression levels are variable between patients, our findings suggest that ASGPR-based targeting strategies should be combined with ASGPR-companion diagnostics to maximize clinical benefit.

Polyethylenimine (PEI) in gene therapy: Current status and clinical applications.

Polyethlyenimine (PEI) was introduced 1995 as a cationic polymer for nucleic acid delivery. PEI and its derivatives are extensively used in basic research and as reference formulations in the field of polymer-based gene delivery. Despite its widespread use, the number of clinical applications to date is limited. Thus, this review aims to consolidate the past applications of PEI in DNA delivery, elucidate the obstacles that hinder its transition to clinical use, and highlight potential prospects for novel iterations of PEI derivatives. The present review article is divided into three sections. The first section examines the mechanism of action employed by PEI, examining fundamental aspects of cellular delivery including uptake mechanisms, release from endosomes, and transport into the cell nucleus, along with potential strategies for enhancing these delivery phases. Moreover, an in-depth analysis is conducted concerning the mechanism underlying cellular toxicity, accompanied with approaches to overcome this major challenge. The second part is devoted to the in vivo performance of PEI and its application in various therapeutic indications. While systemic administration has proven to be challenging, alternative localized delivery routes hold promise, such as treatment of solid tumors, application as a vaccine, or serving as a therapeutic agent for pulmonary delivery. In the last section, the outcome of completed and ongoing clinical trials is summarized. Finally, an expert opinion is provided on the potential of PEI and its future applications. PEI-based formulations for nucleic acid delivery have a promising potential, it will be an important task for the years to come to introduce innovations that address PEI-associated shortcomings by introducing well-designed PEI formulations in combination with an appropriate route of administration.

High efficiency preparation of monodisperse plasma membrane derived extracellular vesicles for therapeutic applications.

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics.

Non-viral gene delivery of the oncotoxic protein NS1 for treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.

Optimization-by-design of hepatotropic lipid nanoparticles targeting the sodium-taurocholate cotransporting polypeptide.

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by 111In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles.

PDMS-b-PMOXA polymersomes for hepatocyte targeting and assessment of toxicity.

Nanoparticles, such as polymersomes, can be directed to the hepatic asialoglycoprotein receptor to achieve targeted drug delivery. In this study, we prepared asialofetuin conjugated polymersomes based on the amphiphilic di-block copolymer poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PDMS-b-PMOXA). They had an average diameter of 150nm and formed monodisperse vesicles. Drug encapsulation and sustained release was monitored using the hydrophilic model compound carboxyfluorescein. Asialoglycoprotein receptor specific uptake by HepG2 cells in vitro was energy dependent and could be competitively inhibited by the free targeting ligand. Mechanistic uptake studies revealed intracellular trafficking of asialofetuin conjugated polymersomes from early endosomes and to the lysosomal compartment. Polymersomes showed no toxicity in the MTT assay up to concentrations of 500µg/mL. In addition, acute toxicity and tolerability of our PDMS-b-PMOXA polymersome formulations was assessed in vivo using zebrafish embryos as a vertebrate screening model. In conclusion, a hepatocyte specific drug delivery system was designed, which is safe and biocompatible and which can be used to implement liver-specific targeting strategies.

Immobilization of Enzymes on PLGA Sub-Micrometer Particles by Crosslinked Layer-by-Layer Deposition.

Enzyme immobilization is of high interest for industrial applications. However, immobilization may compromise enzyme activity or stability due to the harsh conditions which have to be applied. The authors therefore present a new and improved crosslinked layer-by-layer (cLbL) approach. Two different model enzymes (acid phosphatase and ß-galactosidase) are immobilized under mild conditions on biocompatible, monodisperse, sub-micrometer poly(lactide-co-glycolide) (PLGA) particles. The resulting PLGA enzyme systems are characterized regarding their size, surface charge, enzyme activity, storage stability, reusability, and stability under various conditions such as changing pH and temperature. The developed and characterized cLbL protocol can be easily adapted to different enzymes. Potential future uses of the technology for biomedical applications are discussed. PLGA-enzyme particles are therefore injected into the blood circulation of zebrafish embryos in order to demonstrate the in vivo stability and activity of the designed system.

Biocompatible polymer-Peptide hybrid-based DNA nanoparticles for gene delivery.

Currently, research on polymers to be used as gene delivery systems is one of the most important directions in both polymer science and biomedicine. In this report, we describe a five-step procedure to synthesize a novel polymer-peptide hybrid system for gene transfection. The block copolymer based on the biocompatible polymer poly(2-methyl-2-oxazoline) (PMOXA) was combined with the biocleavable peptide block poly(aspartic acid) (PASP) and finally modified with diethylenetriamine (DET). PMOXA-b-PASP(DET) was produced in high yield and characterized by (1)H NMR and FT-IR. Our biopolymer complexed plasmid DNA (pDNA) efficiently, and highly uniform nanoparticles with a slightly negative zeta potential were produced. The polymer-peptide hybrid system was able to efficiently transfect HEK293 and HeLa cells with GFP pDNA in vitro. Unlike the commonly used polymer, 25 kDa branched poly(ethylenimine), our biopolymer had no adverse effects on cell growth and viability. In summary, the present work provides valuable information for the design of new polymer-peptide hybrid-based gene delivery systems with biocompatible and biodegradable properties.