Cell Volume (3D) Correlative Microscopy Facilitated by Intracellular Fluorescent Nanodiamonds as Multi-Modal Probes

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets.Peer reviewe

Microfluidic-Assisted Fabrication of Dual-Coated pH-Sensitive Mesoporous Silica Nanoparticles for Protein Delivery

Microfluidics has become a popular method for constructing nanosystems in recent years, but it can also be used to coat other materials with polymeric layers. The polymeric coating may serve as a diffusion barrier against hydrophilic compounds, a responsive layer for controlled release, or a functional layer introduced to a nanocomposite for achieving the desired surface chemistry. In this study, mesoporous silica nanoparticles (MSNs) with enlarged pores were synthesized to achieve high protein loading combined with high protein retention within the MSN system with the aid of a microfluidic coating. Thus, MSNs were first coated with a cationic polyelectrolyte, poly (diallyldimethylammonium chloride) (PDDMA), and to potentially further control the protein release, a second coating of a pH-sensitive polymer (spermine-modified acetylated dextran, SpAcDEX) was deposited by a designed microfluidic device. The protective PDDMA layer was first formed under aqueous conditions, whereby the bioactivity of the protein could be maintained. The second coating polymer, SpAcDEX, was preferred to provide pH-sensitive protein release in the intracellular environment. The optimized formulation was effectively taken up by the cells along with the loaded protein cargo. This proof-of-concept study thus demonstrated that the use of microfluidic technologies for the design of protein delivery systems has great potential in terms of creating multicomponent systems and preserving protein stability

Cell Volume (3D) Correlative Microscopy Facilitated by Intracellular Fluorescent Nanodiamonds as Multi-Modal Probes

Three-dimensional correlative light and electron microscopy (3D CLEM) is attaining popularity as a potential technique to explore the functional aspects of a cell together with high-resolution ultrastructural details across the cell volume. To perform such a 3D CLEM experiment, there is an imperative requirement for multi-modal probes that are both fluorescent and electron-dense. These multi-modal probes will serve as landmarks in matching up the large full cell volume datasets acquired by different imaging modalities. Fluorescent nanodiamonds (FNDs) are a unique nanosized, fluorescent, and electron-dense material from the nanocarbon family. We hereby propose a novel and straightforward method for executing 3D CLEM using FNDs as multi-modal landmarks. We demonstrate that FND is biocompatible and is easily identified both in living cell fluorescence imaging and in serial block-face scanning electron microscopy (SB-EM). We illustrate the method by registering multi-modal datasets

Targeted modulation of cell differentiation in distinct regions of the gastrointestinal tract via oral administration of differently PEG-PEI functionalized mesoporous silica nanoparticles

Targeted delivery of drugs is required to efficiently treat intestinal diseases such as colon cancer and inflammation. Nanoparticles could overcome challenges in oral administration caused by drug degradation at low pH and poor permeability through mucus layers, and offer targeted delivery to diseased cells in order to avoid adverse effects. Here, we demonstrate that functionalization of mesoporous silica nanoparticles (MSNs) by polymeric surface grafts facilitates transport through the mucosal barrier and enhances cellular internalization. MSNs functionalized with poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and the targeting ligand folic acid in different combinations are internalized by epithelial cells in vitro and in vivo after oral gavage. Functionalized MSNs loaded with γ-secretase inhibitors of the Notch pathway, a key regulator of intestinal progenitor cells, colon cancer, and inflammation, demonstrated enhanced intestinal goblet cell differentiation as compared to free drug. Drug-loaded MSNs thus remained intact in vivo, further confirmed by exposure to simulated gastric and intestinal fluids in vitro. Drug targeting and efficacy in different parts of the intestine could be tuned by MSN surface modifications, with PEI coating exhibiting higher affinity for the small intestine and PEI-PEG coating for the colon. The data highlight the potential of nanomedicines for targeted delivery to distinct regions of the tissue for strict therapeutic control

Circumventing drug treatment? Intrinsic lethal effects of polyethyleneimine (PEI)-functionalized nanoparticles on glioblastoma cells cultured in stem cell conditions

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood–brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.</p

The International Natural Product Sciences Taskforce (INPST) and the power of Twitter networking exemplified through #INPST hashtag analysis

Background: The development of digital technologies and the evolution of open innovation approaches have enabled the creation of diverse virtual organizations and enterprises coordinating their activities primarily online. The open innovation platform titled "International Natural Product Sciences Taskforce" (INPST) was established in 2018, to bring together in collaborative environment individuals and organizations interested in natural product scientific research, and to empower their interactions by using digital communication tools. Methods: In this work, we present a general overview of INPST activities and showcase the specific use of Twitter as a powerful networking tool that was used to host a one-week "2021 INPST Twitter Networking Event" (spanning from 31st May 2021 to 6th June 2021) based on the application of the Twitter hashtag #INPST. Results and Conclusion: The use of this hashtag during the networking event period was analyzed with Symplur Signals (https://www.symplur.com/), revealing a total of 6,036 tweets, shared by 686 users, which generated a total of 65,004,773 impressions (views of the respective tweets). This networking event's achieved high visibility and participation rate showcases a convincing example of how this social media platform can be used as a highly effective tool to host virtual Twitter-based international biomedical research events

Multimodal imaging probes and delivery systems for cancer nanomedicine

Nanoparticles have emerged as one of the most promising tools for addressing central challenges in cancer diagnostics and therapy. This thesis presents the design, surface functionalization, biocompatibility, intracellular interactions and applicability of nanoparticles as potential tools for cancer diagnosis and RNAi therapeutics. The thesis is divided into two parts; 1) cancer cell imaging and 2) siRNA delivery. In part 1, studies were performed with inherently fluorescent carbon-based nanoparticles (nanodiamonds, NDs, and nanographene oxide, nGO) in order to evaluate their suitability for cancer cell imaging (in vivo and in vitro). A novel application of NDs in super resolution correlative light and electron microscopy is presented here, whereby NDs are used as a dual-purpose fluorescent and electron dense probes for correlative multi-modal microscopy. Further, the intracellular interactions of NDs are studied to understand the biocompatibility of nondegradable NDs and the reasons underlying the biocompatibility. Moreover, this thesis elucidates the role of organic surface modifications for enhancing the optical properties of nanographene oxide (nGO) for in vivo imaging. When nGO was surface functionalized with the organic polymer PEG-PEI (polyethyleneimine – a polyethylene glycol co-polymer), and attached to the cancer cell affinity ligand FA (folic acid), its dispersibility, cellular internalization and quantum efficiency were improved compared to the non-modified nGO. The surface functionalized nGOs were further applied as optical markers for the non-invasive in vivo imaging of cancer cells in a model organism. The nGOs were found to be well suited for detecting cancer cells over the studied 1-week period. In part 2, systems for the efficient delivery of therapeutic cargo to cancer cells were studied using a nanodiamond (ND) - silica (MSN) composite (ND@MSN) and mesoporous silica nanoparticles (MSN) with redox responsive linkers. ND@MSN, a novel composite material was synthesized by taking advantage of the properties of both the ND (photoluminescence) and the MSN (drug-delivery). The validation of ND@MSN for drug delivery was performed by surface functionalizing the composite particles with the co-polymer PEG-PEI and by loading the ND@MSN with a hydrophobic luminescent dye, which acted as a model drug. The intracellular delivery of a hydrophobic drugs is generally challenging, but the ND@MSN surface functionalized with co-polymers (Cop) demonstrated excellent efficiency for the intracellular delivery of the hydrophobic model drug. Other noteworthy features related to the use of ND@MSN-Cop were that no premature extracellular release of the dye was observed, that the endosomal escape and intracellular release of the cargo were achieved and that the subsequent tracking of the NDs in the cell was possible. For the delivery of siRNAs into cancer cells, the MSN nanocarriers were synthesized, in attempt to overcome challenges associated with the in vivo delivery of siRNA. Hyperbranched PEI and redox-responsive intracellular triggerable bonds were thus incorporated into the developed MSN nanocarriers. The design of the MSN nanocarriers took into consideration important aspects of in vivo delivery, such as the high loading of siRNA, intracellular cleavable linkers, high cellular uptake, and a large pore size to host the siRNA molecules and to protect them from degradation. In the experimental set-up, the MSN nanocarriers demonstrated the sustained intracellular release of the siRNA (120h), while offering protection from enzymatic degradation. The gene knockdown efficiency was further evaluated using a transfection control siRNA. The MSN nanocarriers performed remarkably well and showed excellent transfection efficiency