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Nanoparticle-sensitized photoporation for intracellular delivery of external compounds usually relies on the use of spherical gold nanoparticles as sensitizing nanoparticles. As they need stimulation with visible laser light, they are less suited for transfection of cells in thick biological tissues. In this work, we have explored black phosphorus quantum dots (BPQDs) as alternative sensitizing nanoparticles for photoporation with a broad and uniform absorption spectrum from the visible to the near infra-red (NIR) range. We demonstrate that BPQD sensitized photoporation allows efficient intracellular delivery of both siRNA (>80%) and mRNA (>40%) in adherent cells as well as in suspension cells. Cell viability remained high (>80%) irrespective of whether irradiation was performed with visible (532 nm) or near infrared (800 nm) pulsed laser light. Finally, as a proof of concept, we used BPQD sensitized photoporation to deliver macromolecules in cells with thick phantom tissue in the optical path. NIR laser irradiation resulted in only 1.3× reduction in delivery efficiency as compared to photoporation without the phantom gel, while with visible laser light the delivery efficiency was reduced 2×.

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.

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 HAuCl 4 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., Competing Interests: The authors declare no conflict of interest.

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20-50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50-60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Exosome-like vesicles (ELVs) are nanovectors released by cells that are endowed with a variety of molecules, including proteins, nucleic acids, and chemicals that reflect the molecular signature of the producing cell. Given their presence in many biofluids, they form an easily accessible biomarker for early disease detection. Previously we demonstrated the possibility of identifying individual ELVs by analyzing their molecular signatures with surface-enhanced Raman scattering (SERS) after functionalization of ELVs with 4-(dimethylamino)pyridine (DMAP)-stabilized gold nanoparticles (AuNP). Although this strategy was capable of distinguishing ELVs from different cellular origins, the quality of the SERS spectra was suboptimal due to high background coming from the DMAP stabilizing molecules at the AuNP surface. In this study we demonstrate that it is possible to eliminate interfering SERS signals from stabilizing molecules at the AuNP surface by overgrowing in situ the ELV-attached AuNPs with a silver layer so as to form a core-shell nanoparticle (Au@AgNPs) directly at the ELV surface. As such it represents the first known strategy to generate clear SERS spectral fingerprints of delicate biological structures without interference of linker molecules that are needed to ensure colloidal stability of the plasmonic NP and to allow them to associate to the ELV surface. This new strategy using core-shell plasmonic NPs as SERS substrate showed higher near-field enhancements than previous approaches, which resulted in SERS spectra with improved signal-to-noise ratio. This allowed us to discriminate individual vesicles derived from B16F10 melanoma cells and red blood cells (RBC) with an unprecedented sensitivity and specificity >90%. Importantly, thanks to the higher near field enhancement the acquisition time could be reduced by 20-fold in comparison to previously reported strategies, paving the way toward high-throughput label-free single ELV identification.

Myopia, diabetes, and aging are the main causes of progressive vitreous collagen aggregation, resulting in vitreous opacities, which can significantly disturb vision. As vitreous opacities, which induce the visual phenomenon of "floaters", are accessible with nanomaterials and light, we propose a nanotechnology-based approach to locally ablate them with highly reduced light energy compared to the more traditional YAG laser therapy. Our strategy relies on the plasmon properties of gold nanoparticles that generate vapor nanobubbles upon pulsed-laser illumination whose mechanical force can ablate vitreous opacities. We designed gold nanoparticles coated with hyaluronic acid (HA), which have excellent diffusional mobility in human vitreous, an essential requirement to reach the vitreous opacities. In addition, we found that HA-coated gold nanoparticles can accumulate extensively on human vitreous opacities that were obtained by vitrectomy from patients with vision-degrading myodesopsia. When subsequently applying nanosecond laser pulses, the collagen aggregates were efficiently destroyed with ∼1000 times less light energy than typically used in YAG laser therapy. This low-energy "floater-specific destruction", which is due to the accumulation of the small gold nanoparticles on the opacities, is attractive, as it may be safer to the surrounding ocular tissues while at the same time being easier and faster to apply compared to YAG laser therapy, where the opacities need to be ablated piece by piece by a tightly focused laser beam. Gold nanoparticle-assisted photoablation may therefore provide a safer, faster, and more reliable destruction of vitreous opacities in the treatment of ophthalmologic diseases.

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.

In this work, we report a simple strategy to obtain ultrasensitive SERS nanostructures by self-assembly and bioconjugation of Au nanospheres (NSs). Homodimer aggregates with an interparticle gap of around 8 nm are generated in aqueous dispersions by the highly specific molecular recognition of biotinylated Au NSs to streptavidin (STV), while random Au NS aggregates with a gap of 5 nm are formed in the absence of STV due to hydrogen bonding among biotinylated NSs. Both types of aggregates depict SERS analytical enhancement factors (AEF) of around 10(7) and the capability to detect biotin concentrations lower than 1 × 10(-12) M. Quite interesting, the AEF for an external analyte, Rhodamine 6G (RH6G), using the dimer aggregates is 1 order of magnitude greater (10(5)) than using random aggregates (around 10(4)). The dependence on the wavelength and the differences of the AEF for Au random aggregates and dimers are rationalized with rigorous electrodynamic simulations. The dimers obtained afford a new type of an in situ self-calibrated and reliable SERS substrate where biotinylated molecules can selectively be "trapped" by STV and located in the nanogap enhanced plasmonic field. Using this concept, powerful molecular-recognition-based SERS assays can be carried out. The capability of the dimeric structures for analytical applications is demonstrated using SPR spectroscopy to detect biotinylated immunoglobulin G at very low concentrations.