In Vivo Endothelial Cell Gene Silencing by siRNA-LNPs Tuned with Lipoamino Bundle Chemical and Ligand Targeting.

Although small-interfering RNAs (siRNAs) are specific silencers for numerous disease-related genes, their clinical applications still require safe and effective means of delivery into target cells. Highly efficient lipid nanoparticles (LNPs) are developed for siRNA delivery, showcasing the advantages of novel pH-responsive lipoamino xenopeptide (XP) carriers. These sequence-defined XPs are assembled by branched lysine linkages between cationizable polar succinoyl tetraethylene pentamine (Stp) units and apolar lipoamino fatty acids (LAFs) at various ratios into bundle or U-shape topologies. Formulation of siRNA-LNPs using LAF4-Stp1 XPs as ionizable compounds led to robust cellular uptake, high endosomal escape, and successful in vitro gene silencing activity at an extremely low (150 picogram) siRNA dose. Of significance is the functional in vivo endothelium tropism of siRNA-LNPs with bundle LAF4-Stp1 XP after intravenous injection into mice, demonstrated by superior knockdown of liver sinusoidal endothelial cell (LSEC)-derived factor VIII (FVIII) and moderate silencing of hepatocyte-derived FVII compared to DLin-MC3-DMA-based LNPs. Optimizing lipid composition following click-modification of siRNA-LNPs with ligand c(RGDfK) efficiently silenced vascular endothelial growth factor receptor-2 (VEGFR-2) in tumor endothelial cells (TECs). The findings shed light on the role of ionizable XPs in the LNP in vivo cell-type functional targeting, laying the groundwork for future therapeutic applications.

Continuous Perfusion Experiments on 3D Cell Proliferation in Acoustic Levitation.

An acoustofluidic trap is used for accurate 3D cell proliferation and cell function analysis in levitation. The prototype trap can be integrated with any microscope setup, allowing continuous perfusion experiments with temperature and flow control under optical inspection. To describe the trap function, we present a mathematical and FEM-based COMSOL model for the acoustic mode that defines the nodal position of trapped objects in the spherical cavity aligned with the microscope field of view and depth of field. Continuous perfusion experiments were conducted in sterile conditions over 55 h with a K562 cell line, allowing for deterministic monitoring. The acoustofluidic platform allows for rational in vitro cell testing imitating in vivo conditions such as cell function tests or cell-cell interactions.

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

Single Pulse Nanosecond Laser-Stimulated Targeted Delivery of Anti-Cancer Drugs from Hybrid Lipid Nanoparticles Containing 5 nm Gold Nanoparticles.

Encapsulating chemotherapeutic drugs like doxorubicin (DOX) inside lipid nanoparticles (LNPs) can overcome their acute, systematic toxicity. However, a precise drug release at the tumor microenvironment for improving the maximum tolerated dose and reducing side effects has yet to be well-established by implementing a safe stimuli-responsive strategy. This study proposes an integrated nanoscale perforation to trigger DOX release from hybrid plasmonic multilamellar LNPs composed of 5 nm gold (Au) NPs clustered at the internal lamellae interfaces. To promote site-specific DOX release, a single pulse irradiation strategy is developed by taking advantage of the resonant interaction between nanosecond pulsed laser radiation (527 nm) and the plasmon mode of the hybrid nanocarriers. This approach enlarges the amount of DOX in the target cells up to 11-fold compared to conventional DOX-loaded LNPs, leading to significant cancer cell death. The simulation of the pulsed laser interactions of the hybrid nanocarriers suggests a release mechanism mediated by either explosive vaporization of thin water layers adjacent to AuNP clusters or thermo-mechanical decomposition of overheated lipid layers. This simulation indicates an intact DOX integrity following irradiation since the temperature distribution is highly localized around AuNP clusters and highlights a controlled light-triggered drug delivery system.

Functionalized Hybrid Iron Oxide-Gold Nanoparticles Targeting Membrane Hsp70 Radiosensitize Triple-Negative Breast Cancer Cells by ROS-Mediated Apoptosis.

Triple-negative breast cancer (TNBC) a highly aggressive tumor entity with an unfavorable prognosis, is treated by multimodal therapies, including ionizing radiation (IR). Radiation-resistant tumor cells, as well as induced normal tissue toxicity, contribute to the poor clinical outcome of the disease. In this study, we investigated the potential of novel hybrid iron oxide (Fe3O4)-gold (Au) nanoparticles (FeAuNPs) functionalized with the heat shock protein 70 (Hsp70) tumor-penetrating peptide (TPP) and coupled via a PEG4 linker (TPP-PEG4-FeAuNPs) to improve tumor targeting and uptake of NPs and to break radioresistance in TNBC cell lines 4T1 and MDA-MB-231. Hsp70 is overexpressed in the cytosol and abundantly presented on the cell membrane (mHsp70) of highly aggressive tumor cells, including TNBCs, but not on corresponding normal cells, thus providing a tumor-specific target. The Fe3O4 core of the NPs can serve as a contrast agent enabling magnetic resonance imaging (MRI) of the tumor, and the nanogold shell radiosensitizes tumor cells by the release of secondary electrons (Auger electrons) upon X-ray irradiation. We demonstrated that the accumulation of TPP-PEG4-FeAuNPs into mHsp70-positive TNBC cells was superior to that of non-conjugated FeAuNPs and FeAuNPs functionalized with a non-specific, scrambled peptide (NGL). After a 24 h co-incubation period of 4T1 and MDA-MB-231 cells with TPP-PEG4-FeAuNPs, but not with control hybrid NPs, ionizing irradiation (IR) causes a cell cycle arrest at G2/M and induces DNA double-strand breaks, thus triggering apoptotic cell death. Since the radiosensitizing effect was completely abolished in the presence of the ROS inhibitor N-acetyl-L-cysteine (NAC), we assume that the TPP-PEG4-FeAuNP-induced apoptosis is mediated via an increased production of ROS.

Multiplexed Plasmonic Nano-Labeling for Bioimaging of Cytological Stained Samples.

Reliable cytopathological diagnosis requires new methods and approaches for the rapid and accurate determination of all cell types. This is especially important when the number of cells is limited, such as in the cytological samples of fine-needle biopsy. Immunoplasmonic-multiplexed- labeling may be one of the emerging solutions to such problems. However, to be accepted and used by the practicing pathologists, new methods must be compatible and complementary with existing cytopathology approaches where counterstaining is central to the correct interpretation of immunolabeling. In addition, the optical detection and imaging setup for immunoplasmonic-multiplexed-labeling must be implemented on the same cytopathological microscope, not interfere with standard H&E imaging, and operate as a second easy-to-use imaging method. In this article, we present multiplex imaging of four types of nanoplasmonic markers on two types of H&E-stained cytological specimens (formalin-fixed paraffin embedded and non-embedded adherent cancer cells) using a specially designed adapter for SI dark-field microscopy. The obtained results confirm the effectiveness of the proposed optical method for quantitative and multiplex identification of various plasmonic NPs, and the possibility of using immunoplasmonic-multiplexed-labeling for cytopathological diagnostics.

Antibody-Functionalized Gold Nanostar-Mediated On-Resonance Picosecond Laser Optoporation for Targeted Delivery of RNA Therapeutics.

The rapid advances of genetic and genomic technology indicate promising therapeutic potential of genetic materials for regulating abnormal gene expressions causing diseases and disorders. However, targeted intracellular delivery of RNA therapeutics still remains a major challenge hindering the clinical translation. In this study, an elaborated plasmonic optoporation approach is proposed to efficiently and selectively transfect specific cells. The site-specific optoporation is obtained by tuning the spectral range of a supercontinuum pulsed picosecond laser in order for each individual cell binding gold nanostar with their unique resonance peak to magnify the local field strength in the near-infrared region and facilitate a selective delivery of small interfering RNA, messenger RNA, and Cas9-ribonucleoprotein into human retinal pigment epithelial cells. Numerical simulations indicate that optoporation is not due to a plasma-mediated process but rather due to a highly localized temperature rise both in time (few nanoseconds) and space (few nanometers). Taking advantage of the numerical simulation and fine-tuning of the optical strategy, the perforated lipid bilayer of targeted cells undergoes a membrane recovery process, important to retain their viability. The results signify the prospects of antibody functionalized nanostar-mediated optoporation as a simple and realistic gene delivery approach for future clinical practices.

Insights into Theranostic Properties of Titanium Dioxide for Nanomedicine.

Titanium dioxide (TiO2) nanostructures exhibit a broad range of theranostic properties that make them attractive for biomedical applications. TiO2 nanostructures promise to improve current theranostic strategies by leveraging the enhanced quantum confinement, thermal conversion, specific surface area, and surface activity. This review highlights certain important aspects of fabrication strategies, which are employed to generate multifunctional TiO2 nanostructures, while outlining post-fabrication techniques with an emphasis on their suitability for nanomedicine. The biodistribution, toxicity, biocompatibility, cellular adhesion, and endocytosis of these nanostructures, when exposed to biological microenvironments, are examined in regard to their geometry, size, and surface chemistry. The final section focuses on recent biomedical applications of TiO2 nanostructures, specifically evaluating therapeutic delivery, photodynamic and sonodynamic therapy, bioimaging, biosensing, tissue regeneration, as well as chronic wound healing.

Anodic Titanium Dioxide Nanotubes for Magnetically Guided Therapeutic Delivery.

Hollow titanium dioxide (TiO2) nanotubes offer substantially higher drug loading capacity and slower drug release kinetics compared to solid drug nanocarriers of comparable size. In this report, we load TiO2 nanotubes with iron oxide nanoparticles to facilitate site-specific magnetic guidance and drug delivery. We generate magnetic TiO2 nanotubes (TiO2NTs) by incorporating a ferrofluid containing Ø ≈ 10 nm iron oxide nanoparticles in planar sheets of weakly connected TiO2 nanotubes. After thermal annealing, the magnetic tubular arrays are loaded with therapeutic drugs and then sonicated to separate the nanotubes. We demonstrate that magnetic TiO2NTs are non-toxic for HeLa cells at therapeutic concentrations (≤200 µg/mL). Adhesion and endocytosis of magnetic nanotubes to a layer of HeLa cells are increased in the presence of a magnetic gradient field. As a proof-of-concept, we load the nanotubes with the topoisomerase inhibitor camptothecin and achieve a 90% killing efficiency. We also load the nanotubes with oligonucleotides for cell transfection and achieve 100% cellular uptake efficiency. Our results demonstrate the potential of magnetic TiO2NTs for a wide range of biomedical applications, including site-specific delivery of therapeutic drugs.

Delivery of Flightless I siRNA from Porous Silicon Nanoparticles Improves Wound Healing in Mice.

Flightless I (Flii), a cytoskeletal actin remodelling protein, is elevated in wounds and is a negative regulator of wound healing. Gene silencing using small interfering RNA (siRNA) is an attractive approach to antagonize Flii, and therefore holds significant promise as a therapeutic intervention. The development of siRNA therapeutics has been limited by an inability of the siRNA to cross the cell surface plasma membrane of target cells and also by their degradation due to endogenous nuclease action. To overcome these limitations, suitable delivery vehicles are required. Porous silicon (pSi) is a biodegradable and high surface area material commonly used for drug delivery applications. Here we investigated the use of pSi nanoparticles (pSiNPs) for the controlled release of Flii siRNA to wounds. Thermally hydrocarbonized pSiNPs (THCpSiNPs) were loaded with Flii siRNA and then coated with a biocompatible chitosan layer. Loading regimens in the order of 50 µg of Flii siRNA per mg of pSi were achieved. The release rate of Flii siRNA was sustained over 35 h. With addition to keratinocytes in vitro, reduced Flii gene expression in conjunction with lowered Flii protein was observed, in concert with increased cell migration and proliferation. A significant improvement in the healing of acute excisional wounds compared to controls was observed from day 5 onward when Flii siRNA-THCpSiNPs were intradermally injected. THCpSiNPs therefore are an effective vehicle for delivering siRNA, and nanoparticle-based siRNA delivery represents a promising therapeutic approach to improve wound healing.