Nucleic acid degradation as barrier to gene delivery: a guide to understand and overcome nuclease activity.

Gene therapy is on its way to revolutionize the treatment of both inherited and acquired diseases, by transferring nucleic acids to correct a disease-causing gene in the target cells of patients. In the fight against infectious diseases, mRNA-based therapeutics have proven to be a viable strategy in the recent Covid-19 pandemic. Although a growing number of gene therapies have been approved, the success rate is limited when compared to the large number of preclinical and clinical trials that have been/are being performed. In this review, we highlight some of the hurdles which gene therapies encounter after administration into the human body, with a focus on nucleic acid degradation by nucleases that are extremely abundant in mammalian organs, biological fluids as well as in subcellular compartments. We overview the available strategies to reduce the biodegradation of gene therapeutics after administration, including chemical modifications of the nucleic acids, encapsulation into vectors and co-administration with nuclease inhibitors and discuss which strategies are applied for clinically approved nucleic acid therapeutics. In the final part, we discuss the currently available methods and techniques to qualify and quantify the integrity of nucleic acids, with their own strengths and limitations.

Selective Replacement of Cholesterol with Cationic Amphiphilic Drugs Enables the Design of Lipid Nanoparticles with Improved RNA Delivery.

The delivery of RNA across biological barriers can be achieved by encapsulation in lipid nanoparticles (LNPs). Cationic amphiphilic drugs (CADs) are pharmacologically diverse compounds with ionizable lipid-like features. In this work, we applied CADs as a fifth component of state-of-the-art LNPs via microfluidic mixing. Improved cytosolic delivery of both siRNA and mRNA was achieved by partly replacing the cholesterol fraction of LNPs with CADs. The LNPs could cross the mucus layer in a mucus-producing air-liquid interface model of human primary bronchial epithelial cells following nebulization. Moreover, CAD-LNPs demonstrated improved epithelial and endothelial targeting following intranasal administration in mice, without a marked pro-inflammatory signature. Importantly, quantification of the CAD-LNP molar composition, as demonstrated for nortriptyline, revealed a gradual leakage of the CAD from the formulation during LNP dialysis. Altogether, these data suggest that the addition of a CAD prior to the rapid mixing process might have an impact on the composition, structure, and performance of LNPs.

Photothermal Nanomaterial-Mediated Photoporation.

Delivering biological effector molecules in cultured cells is of fundamental importance to any study or application in which the modulation of gene expression is required. Examples range from generating engineered cell lines for studying gene function to the engineering of cells for cell-based therapies such as CAR-T cells and gene-corrected stem cells for regenerative medicine. It remains a great challenge, however, to deliver biological effector molecules across the cell membrane with minimal adverse effects on cell viability and functionality. While viral vectors have been frequently used to introduce foreign nucleic acids into cells, their use is associated with safety concerns such as immunogenicity, high manufacturing cost, and limited cargo capacity.For photoporation, depending on the laser energy, membrane permeabilization happens either by local heating or by laser-induced water vapor nanobubbles (VNB). In our first study on this topic, we demonstrated that the physical force exerted by suddenly formed VNB leads to more efficient intracellular delivery as compared to mere heating. Next, we explored the use of different photothermal nanomaterials, finding that graphene quantum dots display enhanced thermal stability compared to the more traditionally used gold nanoparticles, hence providing the possibility to increase the delivery efficiency by repeated laser activation. To enable its use for the production of engineered therapeutic cells, it would be better if contact with cells with nondegradable nanoparticles is avoided as it poses toxicity and regulatory concerns. Therefore, we recently demonstrated that photoporation can be performed with biodegradable polydopamine nanoparticles as well. Alternatively, we demonstrated that nanoparticle contact can be avoided by embedding the photothermal nanoparticles in a substrate made from biocompatible electrospun nanofibers. With this variety of photoporation approaches, over the years we demonstrated the successful delivery of a broad variety of biologics (mRNA, siRNA, Cas9 ribonucleoproteins, nanobodies, etc.) in many different cell types, including hard-to-transfect cells such as T cells, embryonic stem cells, neurons, and macrophages.In this Account, we will first start with a brief introduction of the general concept and a historical development of photoporation. In the next two sections, we will extensively discuss the various types of photothermal nanomaterials which have been used for photoporation. We discriminate two types of photothermal nanomaterials: single nanostructures and composite nanostructures. The first one includes examples such as gold nanoparticles, graphene quantum dots, and polydopamine nanoparticles. The second type includes polymeric films and nanofibers containing photothermal nanoparticles as well as composite nanoscale biolistic nanostructures. A thorough discussion will be given for each type of photothermal nanomaterial, from its synthesis and characterization to its application in photoporation, with its advantages and disadvantages. In the final section, we will provide an overall discussion and elaborate on future perspectives.

In Vitro and Ex Vivo Models for Assessing Drug Permeation across the Cornea.

Drug permeation across the cornea remains a major challenge due to its unique and complex anatomy and physiology. Static barriers such as the different layers of the cornea, as well as dynamic aspects such as the constant renewal of the tear film and the presence of the mucin layer together with efflux pumps, all present unique challenges for effective ophthalmic drug delivery. To overcome some of the current ophthalmic drug limitations, the identification and testing of novel drug formulations such as liposomes, nanoemulsions, and nanoparticles began to be considered and widely explored. In the early stages of corneal drug development reliable in vitro and ex vivo alternatives, are required, to be in line with the principles of the 3Rs (Replacement, Reduction, and Refinement), with such methods being in addition faster and more ethical alternatives to in vivo studies. The ocular field remains limited to a handful of predictive models for ophthalmic drug permeation. In vitro cell culture models are increasingly used when it comes to transcorneal permeation studies. Ex vivo models using excised animal tissue such as porcine eyes are the model of choice to study corneal permeation and promising advancements have been reported over the years. Interspecies characteristics must be considered in detail when using such models. This review updates the current knowledge about in vitro and ex vivo corneal permeability models and evaluates their advantages and limitations.

Macrophages as a therapeutic target to promote diabetic wound healing.

It is well established that macrophages are key regulators of wound healing, displaying impressive plasticity and an evolving phenotype, from an aggressive pro-inflammatory or "M1" phenotype to a pro-healing or "M2" phenotype, depending on the wound healing stage, to ensure proper healing. Because dysregulated macrophage responses have been linked to impaired healing of diabetic wounds, macrophages are being considered as a therapeutic target for improved wound healing. In this review, we first discuss the role of macrophages in a normal skin wound healing process and discuss the aberrations that occur in macrophages under diabetic conditions. Next we provide an overview of recent macrophage-based therapeutic approaches, including delivery of ex-vivo-activated macrophages and delivery of pharmacological strategies aimed at eliminating or re-educating local skin macrophages. In particular, we focus on strategies to silence key regulator genes to repolarize wound macrophages to the M2 phenotype, and we provide a discussion of their potential future clinical translation.

Transient nuclear lamin A/C accretion aids in recovery from vapor nanobubble-induced permeabilisation of the plasma membrane.

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.

Response Surface Methodology to Efficiently Optimize Intracellular Delivery by Photoporation.

Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.

Multicompartmental Microcapsules for Enzymatic Cascade Reactions Prepared through Gas Shearing and Surface Gelation.

Inspired by the structure of eukaryotic cells, multicompartmental microcapsules have gained increasing attention. However, challenges remain in the fabrication of "all-aqueous" (i.e., oil-free) microcapsules composed of accurately adjustable hierarchical compartments. This study reports on multicompartmental microcapsules with an innovative architecture. While multicompartmental cores of the microcapsules were fabricated through gas shearing, a shell was applied on the cores through surface gelation of alginate. Different from traditional multicompartmental microcapsules, thus obtained microcapsules have well-segregated compartments while the universal nature of the surface-gelation method allows us to finely tune the shell thicknesses of the microcapsules. The microcapsules are highly stable and cytocompatible and allow repeated enzymatic cascade reactions, which might make them of interest for complex biocatalysis or for mimicking physiological processes.

Stimuli-responsive nanobubbles for biomedical applications.

Stimuli-responsive nanobubbles have received increased attention for their application in spatial and temporal resolution of diagnostic techniques and therapies, particularly in multiple imaging methods, and they thus have significant potential for applications in the field of biomedicine. This review presents an overview of the recent advances in the development of stimuli-responsive nanobubbles and their novel applications. Properties of both internal- and external-stimuli responsive nanobubbles are highlighted and discussed considering the potential features required for biomedical applications. Furthermore, the methods used for synthesis and characterization of nanobubbles are outlined. Finally, novel biomedical applications are proposed alongside the advantages and shortcomings inherent to stimuli-responsive nanobubbles.

Bio-Nanohybrid Gelatin/Quantum Dots for Cellular Imaging and Biosensing Applications.

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.

Layer by Layer Assembled Chitosan-Coated Gold Nanoparticles for Enhanced siRNA Delivery and Silencing.

Delivery of small interfering RNA (siRNA) provides one of the most powerful strategies for downregulation of therapeutic targets. Despite the widely explored capabilities of this strategy, intracellular delivery is hindered by a lack of carriers that have high stability, low toxicity and high transfection efficiency. Here we propose a layer by layer (LBL) self-assembly method to fabricate chitosan-coated gold nanoparticles (CS-AuNPs) as a more stable and efficient siRNA delivery system. Direct reduction of HAuCl4 in the presence of chitosan led to the formation of positively charged CS-AuNPs, which were subsequently modified with a layer of siRNA cargo molecules and a final chitosan layer to protect the siRNA and to have a net positive charge for good interaction with cells. Cytotoxicity, uptake, and downregulation of enhanced Green Fluorescent Protein (eGFP) in H1299-eGFP lung epithelial cells indicated that LBL-CS-AuNPs provided excellent protection of siRNA against enzymatic degradation, ensured good uptake in cells by endocytosis, facilitated endosomal escape of siRNA, and improved the overall silencing effect in comparison with commercial transfection reagents Lipofectamine and jetPEI®. Therefore, this work shows that LBL assembled CS-AuNPs are promising nanocarriers for enhanced intracellular siRNA delivery and silencing.

Increasing Angiogenesis Factors in Hypoxic Diabetic Wound Conditions by siRNA Delivery: Additive Effect of LbL-Gold Nanocarriers and Desloratadine-Induced Lysosomal Escape.

Impaired wound healing in people with diabetes has multifactorial causes, with insufficient neovascularization being one of the most important. Hypoxia-inducible factor-1 (HIF-1) plays a central role in the hypoxia-induced response by activating angiogenesis factors. As its activity is under precise regulatory control of prolyl-hydroxylase domain 2 (PHD-2), downregulation of PHD-2 by small interfering RNA (siRNA) could stabilize HIF-1α and, therefore, upregulate the expression of pro-angiogenic factors as well. Intracellular delivery of siRNA can be achieved with nanocarriers that must fulfill several requirements, including high stability, low toxicity, and high transfection efficiency. Here, we designed and compared the performance of layer-by-layer self-assembled siRNA-loaded gold nanoparticles with two different outer layers-Chitosan (AuNP@CS) and Poly L-arginine (AuNP@PLA). Although both formulations have exactly the same core, we find that a PLA outer layer improves the endosomal escape of siRNA, and therefore, transfection efficiency, after endocytic uptake in NIH-3T3 cells. Furthermore, we found that endosomal escape of AuNP@PLA could be improved further when cells were additionally treated with desloratadine, thus outperforming commercial reagents such as Lipofectamine® and jetPRIME®. AuNP@PLA in combination with desloratadine was proven to induce PHD-2 silencing in fibroblasts, allowing upregulation of pro-angiogenic pathways. This finding in an in vitro context constitutes a first step towards improving diabetic wound healing with siRNA therapy.

Intracellular Labeling with Extrinsic Probes: Delivery Strategies and Applications.

Extrinsic probes have outstanding properties for intracellular labeling to visualize dynamic processes in and of living cells, both in vitro and in vivo. Since extrinsic probes are in many cases cell-impermeable, different biochemical, and physical approaches have been used to break the cell membrane barrier for direct delivery into the cytoplasm. In this Review, these intracellular delivery strategies are discussed, briefly explaining the mechanisms and how they are used for live-cell labeling applications. Methods that are discussed include three biochemical agents that are used for this purpose-purpose-different nanocarriers, cell penetrating peptides and the pore-foraming bacterial toxin streptolysin O. Most successful intracellular label delivery methods are, however, based on physical principles to permeabilize the membrane and include electroporation, laser-induced photoporation, micro- and nanoinjection, nanoneedles or nanostraws, microfluidics, and nanomachines. The strengths and weaknesses of each strategy are discussed with a systematic comparison provided. Finally, the extrinsic probes that are reported for intracellular labeling so-far are summarized, together with the delivery strategies that are used and their performance. This combined information should provide for a useful guide for choosing the most suitable delivery method for the desired probes.

Faithful Fabrication of Biocompatible Multicompartmental Memomicrospheres for Digitally Color-Tunable Barcoding.

Barcodes have attracted widespread attention, especially for the multiplexed bioassays and anti-counterfeiting used toward medical and biomedical applications. An enabling gas-shearing approach is presented for generating 10-faced microspherical barcodes with precise control over the properties of each compartment. As such, the color of each compartment could be programmatically adjusted in the 10-faced memomicrospheres by using pregel solutions containing different combinations of fluorescent nanoparticles. During the process, three primary colors (red, green, and blue) are adopted to obtain up to seven merged fluorescent colors for constituting a large amount of coding as well as a magnetic compartment, capable of effective and robust high-throughput information-storage. More importantly, by using the biocompatible sodium alginate to construct the multicolor microspherical barcodes, the proposed technology is likely to advance the fields of food and pharmaceutics anti-counterfeiting. These remarkable properties point to the potential value of gas-shearing in engineering microspherical barcodes for biomedical applications in the future.

Surface Functionalization with Polyethylene Glycol and Polyethyleneimine Improves the Performance of Graphene-Based Materials for Safe and Efficient Intracellular Delivery by Laser-Induced Photoporation.

Nanoparticle mediated laser-induced photoporation is a physical cell membrane disruption approach to directly deliver extrinsic molecules into living cells, which is particularly promising in applications for both adherent and suspension cells. In this work, we explored surface modifications of graphene quantum dots (GQD) and reduced graphene oxide (rGO) with polyethylene glycol (PEG) and polyethyleneimine (PEI) to enhance colloidal stability while retaining photoporation functionality. After photoporation with FITC-dextran 10 kDa (FD10), the percentage of positive HeLa cells (81% for GQD-PEG, 74% for rGO-PEG and 90% for rGO-PEI) increased approximately two-fold compared to the bare nanomaterials. While for Jurkat suspension cells, the photoporation efficiency with polymer-modified graphene-based nanomaterial reached as high as 80%. Cell viability was >80% in all these cases. In addition, polymer functionalization proved to be beneficial for the delivery of larger macromolecules (FD70 and FD500) as well. Finally, we show that rGO is suitable for photoporation using a near-infrared laser to reach 80% FD10 positive HeLa cells at 80% cell viability. We conclude that modification of graphene-based nanoparticles with PEG and especially PEI provide better colloidal stability in cell medium, resulting in more uniform transfection and overall increased efficiency.

The Role of Ultrasound-Driven Microbubble Dynamics in Drug Delivery: From Microbubble Fundamentals to Clinical Translation.

In the last couple of decades, ultrasound-driven microbubbles have proven excellent candidates for local drug delivery applications. Besides being useful drug carriers, microbubbles have demonstrated the ability to enhance cell and tissue permeability and, as a consequence, drug uptake herein. Notwithstanding the large amount of evidence for their therapeutic efficacy, open issues remain. Because of the vast number of ultrasound- and microbubble-related parameters that can be altered and the variability in different models, the translation from basic research to (pre)clinical studies has been hindered. This review aims at connecting the knowledge gained from fundamental microbubble studies to the therapeutic efficacy seen in in vitro and in vivo studies, with an emphasis on a better understanding of the response of a microbubble upon exposure to ultrasound and its interaction with cells and tissues. More specifically, we address the acoustic settings and microbubble-related parameters (i.e., bubble size and physicochemistry of the bubble shell) that play a key role in microbubble-cell interactions and in the associated therapeutic outcome. Additionally, new techniques that may provide additional control over the treatment, such as monodisperse microbubble formulations, tunable ultrasound scanners, and cavitation detection techniques, are discussed. An in-depth understanding of the aspects presented in this work could eventually lead the way to more efficient and tailored microbubble-assisted ultrasound therapy in the future.

High Pressure Nebulization (PIPAC) Versus Injection for the Intraperitoneal Administration of mRNA Complexes.

PURPOSE: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a novel technique delivering drugs into the abdominal cavity as an aerosol under high pressure. It is hypothesized to have advantages such as enhancing tissue uptake, distributing drugs homogeneously within the closed and expanded abdominal cavity and higher local concentration of drugs in the peritoneal cavity. However, the clinical trials of PIPAC so far are limited to liquid chemotherapeutic solution, and the applicability of biomolecules (such as mRNA, siRNA and oligonucleotide) is not known. We aimed to investigate the feasibility of administrating mRNA lipoplexes to the peritoneal cavity via high pressure nebulization. METHODS: We firstly investigated the influences of nebulization on physicochemical properties and in vitro transfection efficiency of mRNA lipoplexes. Then, mRNA lipoplexes were delivered to healthy rats through intravenous injection, intraperitoneal injection and PIPAC, respectively. RESULTS: mRNA lipoplexes can withstand the high pressure applied during the PIPAC procedure in vitro. Bioluminescence localized to the peritoneal cavity of rats after administration by IP injection and nebulization, while intravenous injection mainly induced protein expression in the spleen. CONCLUSION: This study demonstrated that local nebulization is feasible to apply mRNA complexes in the peritoneal cavity during a PIPAC procedure.

Challenges for labeling and longitudinal tracking of adoptively transferred autoreactive T lymphocytes in an experimental type-1 diabetes model.

OBJECTIVE: Tracking the autoreactive T-cell migration in the pancreatic region after labeling with fluorinated nanoparticles (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]-perfluoro-15-crown-5-ether nanoparticles, PDP-PFCE NPs) in a diabetic murine model using 19F MRI. MATERIALS AND METHODS: Synthesis of novel PDP-PFCE fluorine tracer was performed for in vitro labeling of T cells. Labeling conditions were optimized using different PDP-PFCE NPs concentrations. For in vivo 19F MRI, mice were longitudinally followed after adoptive transfer of activated, autoreactive, labeled T cells in NOD.SCID mice. RESULTS: Established MR protocols were used for challenging T cell labeling to track inflammation in a model of diabetes after successful labeling of CD4+ and CD8+ T cells with PDP-PFCE NPs. However, T cells were difficult to be detected in vivo after their engraftment in animals. DISCUSSION: We showed successful in vitro labeling of T cells using novel fluorinated liposomal nanoparticles. However, insufficient and slow accumulation of labeled T cells and subsequent T cell proliferation in the pancreatic region remains as limitations of in vivo cell imaging by 19F MRI.

Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy.

Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.

Photothermally Triggered Endosomal Escape and Its Influence on Transfection Efficiency of Gold-Functionalized JetPEI/pDNA Nanoparticles.

Plasmonic nanoparticles for drug delivery have attracted increasing interest over the last few years. Their localized surface plasmon resonance causes photothermal effects on laser irradiation, which allows for delivering drugs in a spatio-temporally controlled manner. Here, we explore the use of gold nanoparticles (AuNP) as carriers for pDNA in combination with pulsed laser irradiation to induce endosomal escape, which is currently considered to be one of the major bottlenecks in macromolecular drug delivery on the intracellular level. In particular, we evaluate nanocomplexes composed of JetPEI (polyethylenimine)pDNA and 10 nm AuNP, which do not exhibit endosomal escape by themselves. After incubating HeLa cells with these complexes, we evaluated endosomal escape and transfection efficiency using low- and high-energy laser pulses. At low laser energy heat is produced by the nanocomplexes, while, at higher laser energy, explosive vapour nanobubbles (VNB) are formed. We investigated the ability of heat transfer and VNB formation to induce endosomal escape and we examine the integrity of pDNA cargo after inducing both photothermal effects. We conclude that JetPEI/pDNA/AuNP complexes are unable to induce meaningful transfection efficiencies because laser treatment causes either dysfunctionality of the cargo when VNB are formed or forms too small pores in the endosomal membrane to allow pDNA to escape in case of heating. We conclude that laser-induced VNB is the most suitable to induce effective pDNA endosomal escape, but a different nanocomplex structure will be required to keep the pDNA intact.