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Introduction Retinal disease affects millions of people worldwide, generating a massive social and economic burden. Current clinical trials for retinal diseases are dominated by gene augmentation therapies delivered with recombinant viruses as key players. As an alternative, nanoparticles hold great promise for the delivery of nucleic acid therapeutics as well. Nevertheless, despite numerous attempts, ‘nano’ is in practice not as successful as aspired and major breakthroughs in retinal gene therapy applying nanomaterials are yet to be seen. Areas covered In this review, we summarize the advantages of nanomaterials and give an overview of nanoparticles designed for retinal nucleic acid delivery up to now. We furthermore critically reflect on the predominant issues that currently limit nano to progress to the clinic, where faulty study design and the absence of representative models play key roles. Expert opinion Since the current approach of in vitro – in vivo experimentation is highly inefficient and creates misinformation, we advocate for a more prominent role for ex vivo testing early on in nanoparticle research. In addition, we elaborate on several concepts, including systematic studies and open science, which could aid in pushing the field of nanomedicine beyond the preclinical stage.

International audience; Reaching the corneal endothelium through the topical administration of therapeutic drugs remains a challenge in ophthalmology. Besides, endothelial cells are not able to regenerate, and diseases at this site can lead to corneal blindness. Targeting the corneal endothelium implies efficient penetration through the three corneal layers, which still remains difficult for small molecules. Carbon quantum dots (CQDs) have demonstrated great potential for ocular nanomedicine. This study focuses on the corneal penetration abilities of differently charged CQDs and their use as permeation enhancers for drugs. Excised whole bovine eyes were used as an ex vivo model to investigate corneal penetration of CQDs derived from glucosamine using β-alanine, ethylenediamine, or spermidine as a passivation agent. It was found that negatively charged CQDs have limited corneal penetration ability, while positively charged CQDs derived from glucosamine hydrochloride and spermidine (CQD-S) penetrate the entire corneal epithelium all the way down to the endothelium. CQD-S were shown to enhance the penetration of FITC-dextran 150 kDa, suggesting that they could be used as efficient penetration enhancers for therapeutic delivery to the corneal endothelium.

The bio-nanohybrid gelatin protein/cadmium sulfide (Gel/CdS) quantum dots (QDs) have been designed via a facile one-pot strategy. The amino acids group of gelatin chelate Cd2+ and grow CdS QDs without any agglomeration. The 1H NMR spectra indicate that during the above process there are no alterations of the gelatin protein structure conformation and chemical functionalities. The prepared Gel/CdS QDs were characterized and their potential as a system for cellular imaging and the electrochemical sensor for hydrogen peroxide (H2O2) detection applications were investigated. The obtained results demonstrate that the developed Gel/CdS QDs system could offer a simple and convenient operating strategy both for the class of contrast agents for cell labeling and electrochemical sensors purposes. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:19 ispartof: location:Switzerland status: published

Listeria monocytogenesis a foodborne intracellular bacterial pathogen leading to human listeriosis. Despite a high mortality rate and increasing antibiotic resistance no clinically approved vaccine againstListeriais available. AttenuatedListeriastrains offer protection and are tested as antitumor vaccine vectors, but would benefit from a better knowledge on immunodominant vector antigens. To identify novel antigens, we screened forListeriaepitopes presented on the surface of infected human cell lines by mass spectrometry-based immunopeptidomics. In between more than 15,000 human self-peptides, we detected 68Listeriaepitopes from 42 different bacterial proteins, including several known antigens. Peptide epitopes presented on different cell lines were often derived from the same bacterial surface proteins, classifying these antigens as potential vaccine candidates. Encoding these highly presented antigens in lipid nanoparticle mRNA vaccine formulations resulted in specific CD8+ T-cell responses and high levels of protection in vaccination challenge experiments in mice. Our results pave the way for the development of a clinical mRNA vaccine againstListeriaand aid to improve attenuatedListeriavaccines and vectors, demonstrating the power of immunopeptidomics for next-generation bacterial vaccine development.

Vapor nanobubble (VNB) photoporation is a physical method for intracellular delivery that has gained significant interest in the past decade. It has successfully been used to introduce molecular cargo of diverse nature into different cell types with high throughput and minimal cytotoxicity. For translational purposes, it is important to understand whether and how photoporation affects cell homeostasis. To obtain a comprehensive view on the transcriptional rewiring that takes place after VNB photoporation, we performed a longitudinal shotgun RNA-sequencing experiment. Six hours after photoporation, we found a marked upregulation of LMNA transcripts as well as their protein products, the A-type lamins. At the same time point, we observed a significant increase in several heterochromatin marks, suggesting a global stiffening of the nucleus. These molecular features vanished 24 h after photoporation. Since VNB-induced chromatin condensation was prolonged in LMNA knockout cells, A-type lamins may be required for restoring the nucleus to its original state. Selective depletion of A-type lamins reduced cell viability after VNB photoporation, while pharmacological stimulation of LMNA transcription increased the percentage of successfully transfected cells that survived after photoporation. Therefore, our results suggest that cells respond to VNB photoporation by temporary upregulation of A-type lamins to facilitate their recovery.

Many groundbreaking therapies for the treatment of blindness require delivery of biologics or cells to the inner retina by intravitreal injection. Unfortunately, the advancement of these therapies is greatly hampered by de-livery difficulties where obstruction of the therapeutics at the inner limiting membrane (ILM) represents the dominant bottleneck. In this proof-of-principle study, we explore an innovative light-based approach to locally ablate the ILM in a minimally invasive and highly controlled manner, thus making the ILM more permeable for therapeutics. More specifically, we demonstrate that pulsed laser irradiation of ILM-bound indocyanine green (ICG), a clinically applied ILM dye, results in the formation of vapor nanobubbles which can disrupt the bovine ILM as well as the extraordinary thick human ILM. We have observed that this photodisruption allows for highly successful retinal delivery of model nanoparticles which are otherwise blocked by the intact ILM. Strikingly, this treatment is furthermore able of enhancing the efficacy of mRNA-loaded lipid nanoparticles within the bovine retina by a factor of 5. In conclusion, this study provides evidence for a light-based approach to overcome the ILM which has the potential to improve the efficacy of all retinal therapies hampered by this delivery barrier.

Nanotechnology has made an important contribution to oncology in recent years, especially for drug delivery. While many different nano-delivery systems have been suggested for cancer therapy, selenium nanoparticles (SeNPs) are particularly promising anticancer drug carriers as their core material offers interesting synergistic effects to cancer cells. Se compounds can exert cytotoxic effects by acting as pro-oxidants that alter cellular redox homeostasis, eventually leading to apoptosis induction in many kinds of cancer cells. Herein, we report on the design and synthesis of novel layer-by-layer Se-based nanocomplexes (LBL-Se-NCs) as carriers of small interfering RNA (siRNA) for combined gene silencing and apoptosis induction in cancer cells. The LBL-Se-NCs were prepared using a straightforward electrostatic assembly of siRNA and chitosan (CS) on the solid core of the SeNP. In this study, we started by investigating the colloidal stability and protection of the complexed siRNA. The results show that CS not only functioned as an anchoring layer for siRNA, but also provided colloidal stability for at least 20 days in different media when CS was applied as a third layer. The release study revealed that siRNA remained better associated with LBL-Se-NCs, with only a release of 35% after 7 days, as compared to CS-NCs with a siRNA release of 100% after 48 h, making the LBL nanocarrier an excellent candidate as an off-the-shelf formulation. When applied to H1299 cells, it was found that they can selectively induce around 32% apoptosis, while significantly less apoptosis (5.6%) was induced in NIH/3T3 normal cells. At the same time, they were capable of efficiently inducing siRNA downregulation (35%) without loss of activity 7 days post-synthesis. We conclude that LBL-Se-NCs are promising siRNA carriers with enhanced stability and with a dual mode of action against cancer cells.

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.

Microgels are promising materials in drug delivery and biomedicine. Although monodisperse microgels would offer considerable advantages, most microgels investigated and used today are polydisperse in size. We report on the fabrication of 10 mum sized monodisperse microgels by emulsifying an aqueous dextran-hydroxyethyl methacrylate (dex-HEMA) phase within an oil phase at the junction of microfluidic channels. Dex-HEMA microgels are biodegradable and are ideally suited for the controlled delivery of proteins.

Phenylboronic acids are known to form covalent complexes with polyol compounds such as glucose. A novel polyelectrolyte, containing phenylboronic acid as a glucose-sensitive moiety, has been synthesized and used for the fabrication of glucose-sensitive hollow polyelectrolyte capsules using the layer-by-layer technique. The response to glucose was observed as a rather fast dissolution of the capsules when brought into contact with a glucose-containing medium. These polyelectrolyte capsules are the first polyelectrolyte capsules able to respond to a stimulus that can be provided by the human body (i.e., an increase in glucose concentration). Therefore, the concept we present has promising applications in the biomedical field for the controlled delivery of insulin.

Developing effective cancer vaccines is thought to require the strong activation of cytotoxic T cells (CTLs) to kill transformed cells. Initiation of such CTL responses requires DCs presenting the antigen via the MHCI route, a feature hardly achievable using soluble antigens. Internalization of particulate antigens in contrast has been shown to strongly promote MHCI mediated antigen presentation. In this study, ovalbumin (OVA) was encapsulated in polyelectrolyte microcapsules (PEM), a new class of particles allowing efficient antigen encapsulation under non-denaturing conditions. OVA-loaded PEM strongly promoted MHCI mediated OVA presentation by DCs in vitro and in vivo. Being attractive tools to enhance antigen presentation, PEM however fail to activate DCs, a prerequisite for the initiation of potent CTL responses. To cope with this issue, we coated the surface of cationic PEM with oligonucleotides containing unmethylated CpG motifs, which are strong activators of DCs by triggering TLR9. Co-delivery of CpG and OVA-loaded microcapsules synergistically increased the strength of the CTL response, indicated by a dramatic increase in the number of IFN-γ secreting CTLs and a strongly enhanced killing of OVA peptide-pulsed splenocytes after adoptive transfer. Finally, immunisation with CpG-modified capsules was able to confer protective immunity against a lethal challenge with the highly aggressive OVA-transduced B16 melanoma, clearly demonstrating the potential of this approach.

Designing therapeutic vaccines has been a major focus in cancer immunotherapy. We have developed Galsomes, a cancer vaccine consisting of a lipid nanoparticle that incorporates tumor antigen mRNA and the glycolipid -galactosylceramide. Consequently, vaccination with Galsomes activates two types of adaptive and innate immune killer cells: cytotoxic T lymphocytes (CTLs), natural killer T (NKT) cells, respectively. To fully capitalize on these cells’ ability to eradicate cancer cells, it is critical to understand which barriers within the tumor-bearing host might form obstacles for their activity. Programmed death-ligand 1 (PD-L1) is such a barrier that can act on CTLs and NKT cells both at the time of activation as at the time of their effector function. There are indications that Galsome vaccination triggers expression of PD-L1, but up to now, no detailed information on the timing and location of this checkpoint expression is available. We previously developed a nanobody-mediated strategy to noninvasively image PD-L1. In this study, we used this nanobody-mediated SPECT/CT imaging strategy to determine the spatio-temporal expression of PD-L1 upon Galsome vaccination in the B16-OVA mouse melanoma tumor model. Moreover, ex vivo analysis was performed to support the SPECT/CT images. We also studied how Galsome vaccination combined with PD-L1 blockade impacts on the therapy outcome. Noninvasive, nanobody-mediated imaging of PD-L1 and ex vivo analysis was performed at several time points after Galsome vaccination in B16-OVA bearing mice, showing upregulation of PD-L1 expression as soon as one day after vaccination in organs targeted by the vaccine (lung, spleen, lymph node), while PD-L1 upregulation in the tumor environment occurred at later time points. Corroborating these findings, we showed that Galsome vaccination combined with anti-PD-L1 monoclonal antibody therapy significantly improved therapy outcome. In conclusion, Galsome vaccination in combination with PD-L1 blockade represents a promising, more effective treatment regimen for melanoma, a treatment regimen that is supported by noninvasive imaging of PD-L1 upon Galsome vaccination.