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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The inner limiting membrane (ILM) represents a major bottleneck hampering efficient drug delivery to the retina after intravitreal injection. To overcome this barrier, we intend to perforate the ILM by use of a light-based approach which relies on the creation of vapor nanobubbles (VNBs) when irradiating photosensitizers with high intensity laser pulses. Upon collapse of these VNBs, mechanical effects can disrupt biological structures. As a photosensitizer, we explore indocyanine green (ICG) loaded nanoparticles (NPs) specifically designed for our application. In light of this, ICG liposomes and PLGA ICG NPs were characterized in terms of physicochemical properties, ICG incorporation and VNB formation. ICG liposomes were found to encapsulate significantly higher amounts of ICG compared to PLGA ICG NPs which is reflected in their VNB creating capacity. Since only ICG liposomes were able to induce VNB generation, this class of NPs was further investigated on retinal explants. Here, application of ICG liposomes followed by laser treatment resulted in subtle disruption effects at the ILM where zones of fully ablated ILM were alternated by intact regions. As the interaction between the ICG liposomes and ILM might be insufficient, active targeting strategies or other NP designs might improve the concept to a further extent. [ABSTRACT FROM AUTHOR]

Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacterial resistance to antibiotics threatens the ability to treat life-threatening bloodstream infections. Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) able to inhibit essential genes can become an alternative to traditional antibiotics, as long as they are safely transported in human serum upon intravenous administration and they are carried across the multilayered bacterial envelopes, impermeable to ONs. In this study, fusogenic liposomes were considered to transport the ONs and promote their internalization in clinically relevant bacteria. Locked nucleic acids and 2′-OMethyl RNA were evaluated as model NAMs and formulated into DOTAP–DOPE liposomes followed by post-PEGylation. Our data showed a complexation stability between the post-PEGylated liposomes and the ONs of over 82%, during 24 h in native human serum, as determined by fluorescence correlation spectroscopy. Quantification by a lipid-mixing assay showed that liposomes, with and without post-PEGylation, fused with all bacteria tested. Such fusion promoted the delivery of a fraction of the ONs into the bacterial cytosol, as observed by fluorescence in situ hybridization and bacterial fractionation. In short, we demonstrated for the first time that liposomes can safely transport ONs in human serum and intracellularly deliver them in both Gram-negative and -positive bacteria, which holds promise towards the treatment of bloodstream infections. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis. [ABSTRACT FROM AUTHOR]

The modification of CD4+ T cells with exogenous nucleic acids or proteins is a critical step in several research and therapeutic applications, such as HIV studies and cancer immunotherapies. However, efficient cell transfections are not always easily achieved when working with these primary hard-to-transfect cells. While the modification of T cells is typically performed by viral transduction or electroporation, their use is associated with safety issues or cytotoxicity. Vapor nanobubble (VNB) photoporation with sensitizing gold nanoparticles (AuNPs) has recently emerged as a new technology for safe and flexible cell transfections. In this work, we evaluated the potential of VNB photoporation as a novel technique for the intracellular delivery of macromolecules in primary human CD4+ T cells using fluorescent dextrans as model molecules. Our results show that VNB photoporation enables efficient delivery of fluorescent dextrans of 10 kDa in Jurkat (>60% FD10+ cells) as well as in primary human CD4+ T cells (±40% FD10+ cells), with limited cell toxicity (>70% cell viability). We also demonstrated that the technique allows the delivery of dextrans that are up to 500 kDa in Jurkat cells, suggesting its applicability for the delivery of biological macromolecules with a wide range of molecular weights. Altogether, VNB photoporation represents a promising technique for the universal delivery of macromolecules in view of engineering CD4+ T cells for use in a wide variety of research and therapeutic applications. [ABSTRACT FROM AUTHOR]

Impaired penetration of antibiotics through bacterial biofilms is one of the reasons for failure of antimicrobial therapy. Hindered drug diffusion is caused on the one hand by interactions with the sticky biofilm matrix and on the other hand by the fact that bacterial cells are organized in densely packed clusters of cells. Binding interactions with the biofilm matrix can be avoided by encapsulating the antibiotics into nanocarriers, while interfering with the integrity of the dense cell clusters can enhance drug transport deep into the biofilm. Vapor nanobubbles (VNB), generated from laser irradiated nanoparticles, are a recently reported effective way to loosen up the biofilm structure in order to enhance drug transport and efficacy. In the present study, we explored if the disruptive force of VNB can be used simultaneously to interfere with the biofilm structure and trigger antibiotic release from light-responsive nanocarriers. The antibiotic tobramycin was incorporated in two types of light-responsive nanocarriers—liposomes functionalized with gold nanoparticles (Lip-AuNP) and graphene quantum dots (GQD)—and their efficacy was evaluated on Pseudomonas aeruginosa biofilms. Even though the anti-biofilm efficacy of tobramycin was improved by liposomal encapsulation, electrostatic functionalization with 70 nm AuNP unfortunately resulted in premature leakage of tobramycin in a matter of hours. Laser-irradiation consequently did not further improve P. aeruginosa biofilm eradication. Adsorption of tobramycin to GQD, on the other hand, did result in a stable formulation with high encapsulation efficiency, without burst release of tobramycin from the nanocarriers. However, even though laser-induced VNB formation from GQD resulted in biofilm disruption, an enhanced anti-biofilm effect was not achieved due to tobramycin not being efficiently released from GQD. Even though this study was unsuccessful in designing suitable nanocarriers for simultaneous biofilm disruption and light-triggered release of tobramycin, it provides insights into the difficulties and challenges that need to be considered for future developments in this regard. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Selectively targeted nanoscale drug delivery systems have recently emerged as promising intravenously therapeutic option for most chronic joint diseases. Here, a newly synthetized dodecapeptide (GE11)-polylactide-co-glycolide (PLGA)-based conjugate was used to prepare smart nanoparticles (NPs) intended for intra-articular administration and for selectively targeting Epidermal Growth Factor Receptor (EGFR). GE11-PLGA conjugate-based NPs are specifically uptaken by EGFR-overexpressed fibroblast; such as synoviocytes; which are the primarily cellular component involved in the development of destructive joint inflammation. The selective uptake could help to tune drug effectiveness in joints and to decrease local and systemic side effects. Dexamethasone (DXM) is a glucorticoid drug commonly used in joint disease treatment for both systemic and local administration route. In the present research; DXM was efficiently loaded into GE11-PLGA conjugate-based NPs through an eco-friendly nanoprecipitation method set up for this purpose. DXM loaded GE11-PLGA conjugate-based NPs revealed satisfactory ex vivo cytocompatibility; with proper size (≤150 nm) and good dimensional stability in synovial fluid. Intra-articular formulation was developed embedding DXM loaded GE11-PLGA conjugate-based NPs into thermosetting chitosan-based hydrogel; forming a biocompatible composite hydrogel able to quickly turn from liquid state into gel state at physiological temperature; within 15 min. Moreover; the use of thermosetting chitosan-based hydrogel extends the local release of active agent; DXM. [ABSTRACT FROM AUTHOR]