Symposium SB03—Transformative Nanostructures with Therapeutic and Diagnostic Modalities

Design of theranostic nanocomplex has gained a lot of attention because its capability for simultaneous imaging, detection and therapy. In particular, high selectivity nanocomplex for photodynamic therapy and drug delivery are important tools to fight the cancer problem. Here, we report recent results on the design of theranostic complex based on the use of upconversion nanoparticles (UCNP) which convert near infrared (NIR) excitation at 970 nm into blue, green, red and NIR emission tuned with the lanthanide doping ion. Such nanoparticles are ideal for overpassing the limited penetration depth of conventional photodynamic therapy (PDT), also useful for imaging and detection. Green and red emissions were obtained with the introduction of Er³⁺ ion. The green and red emission is used as an excitation source for zinc phtalocyanine (ZnPc), a photosensitizer (PS) bounded to the surface of UCNP. PS transfers energy to molecular oxygen in the surroundings to produce reactive oxygen species (ROS) able to induce death of breast cancer cells (BCC). Selectivity was obtained by the conjugation with Trastuzumab (Tras), a specific monoclonal antibody for selective detection and treatment of HER2-overexpresing malignant BCC. With the use of nanocomplex UCNP-ZnPC-Tras, was observed selective tracking of SKBR-3 HER2+ BCC by using a confocal microscope and reducing cells viability to 80% upon 200mg/ml load and 5 minutes of irradiation. Furthermore, PS was replaced for a photocatalytic TiO₂/ZrO₂ shell which is excited with blue light coming from Tm³⁺ ions enhancing the ROS production and then killing cancer cells up to 88%. In this case, emission band at 801 nm was used for tracking cells at deep tissue. Indeed, we report the fabrication of inmunoliposomes nanocomplex loaded with UCNP, PS for PDT and drugs for chemotherapy. Results shows a combined chemo-photodynamic synergistic effect killing cells up to 80% after 10mM load and 5 minutes of irradiation. The specificity of our nanocomplex was achieved by conjugating a newly discovered anti-HER2 peptide screened from a phage display peptide library. The high selectivity of the peptide-conjugated was confirmed by confocal imaging of SKBR-3 (HER2-positive) and MCF-7 (HER2-negative) breast cancer cell lines, illustrating its target-specific nature. Results suggest great potential for multifunctional theranostic nanocomplex for detection, imaging and therapy of cancer.

We acknowledge financial support from UDLSB and CONACYT through grant 259192.

Mesoporous silica nanoparticles (MSN) exhibit highly beneficial features for devising nanosystems applicable in cancer therapy, imaging, or diagnostics. The high surface area of MSN, which can be easily postsynthetically functionalized with different organosilanes, in addition to their structured porosity available for loading cargo molecules, allows plethora of opportunities for devising multifunctional and multipurpose nanostructures.
In this context, our research focuses on devising MSN-based cancer theranostics, containing different modalities for targeting specific cancer microenvironment, and the capabilities for delivering cargo therapeutic molecules upon exposure to the specific conditions of this environment, such as tissue acidosis or elevated concentration of glutathione and other cancer-related biomolecules. Thus, specific surface modification and controlling the release process of therapeutic molecules enables utilization of MSNs as unique facilitators toward enhancing the efficacy and precision of cancer treatment, imaging and sensing.

Vaccines can take one of several forms, but those based on subunit antigens (representative subunits of the pathogen for which immunity is desired) offer the greatest safety profile and scalability, but generally elicit weaker and less durable immune responses. Failure to elicit a sufficiently strong response likely arises from inappropriate temporal control over antigen/adjuvant presentation and immune cell activation. Either short-term presentation of these signals, or misalignment of their presentation along different timelines, results in poor affinity maturation of antibodies and poor immune memory responses. When considering the iterative selection process occurring during somatic hypermutation and antibody affinity maturation in B lymphocytes, it is intuitive that prolonged lymphocyte activation and antigen exposure would lead to the generation of higher-affinity antibodies. Moreover, the immune system is highly spatially organized and so spatiotemporal control over vaccine exposure is crucial to potent and safe responses. In this work we will discuss novel technologies for improving the spatiotemporal presentation of vaccine components to the immune system. In particular we will describe an injectable hydrogel platform providing unique drug delivery capabilities for the long-term co-delivery of antigen and adjuvant in subunit vaccines and discuss the impact of prolonged vaccine delivery on humoral immune responses. We demonstrate that prolonged hydrogel-based immunizations greatly enhance the magnitude and duration of antibody responses, enhance and prolong germinal responses, and lead to 1000-fold enhancement in antibody affinity maturation when compared to the same vaccine delivered in bolus. These advanced materials technologies, therefore, have the potential to provide vastly superior vaccine performance through the precise and sustained delivery of subunit vaccines that take advantage of natural immune mechanisms for building long-lasting and potent immunity.

Supramolecular hydrogels are promising materials to address challenges associated with the formulation of biotherapeutic drugs, such as antibodies, which often suffer from a lack of stability in formulation and poor pharmacokinetics. To address these challenges, we have developed an injectable polymeric materials platform to encapsulate and deliver macromolecular drug cargo. Our modular, tunable supramolecular hydrogel system is comprised of a polymer matrix that is physically cross-linked by polymeric nanoparticles, resulting in a material which exhibits shear-thinning and self-healing properties, both desirable characteristics for an injectable drug delivery vehicle. In vitro drug release assays and diffusion measurements indicate that our materials dramatically prolong the release of antibodies and improve formulation stability under severe stressed aging conditions. To investigate the performance of these materials in vivo, antibodies against SARS-CoV-2 were delivered in mice to assess the pharmacokinetics of an antibody delivered in our hydrogel compared to standard routes of administration. In addition, a model was implemented to further understand how the materials design impacts the ability of the hydrogel to modulate pharmacokinetics in vivo, enabling customization of the hydrogel properties to achieve the target pharmacokinetic profile based on properties of both the hydrogel formulation and the specific antibody drug. The model will help inform future materials design efforts to generate long-acting antibody-based drug products for a variety of important therapeutic applications.

Long non-coding RNAs (lncRNAs) are emerging as a new player in the field of cancer research. They show tissue -and sometimes even cell-specific expression and have potential in therapeutic and diagnostic settings. Whilst research in this area is still in its early stages, promising lncRNA candidates have been identified. Therapeutically, the most widely used approach is to silence cancer-associated lncRNAs with RNAse H competent antisense oligonuclotides (ASOs). While this approach is promising, in vivo ASO delivery to tumors remains a hurdle. Different advances in nano compositions of lipid nanoparticles (LNPs), lipoplexes and exosomes offer some possibilities to maximize tumoral and minimize hepatic delivery. Here, I will present on the role that these nanotechnologies, specifically LNPs, could play in the development of novel ASO-based cancer therapeutics and will also discuss remaining problems with this approach and next steps that have to be taken in order to move such novel drugs towards the clinic. I will also explore possibly targetable lncRNAs associated with different cancers. It is expected that collaborative efforts in nanotechnology and cancer biology may lead to therapeutic platforms that could usher in a new era of therapeutics.

Introduction: Efficient encapsulation of therapeutic agents is a key factor when designing a nanoparticle (NP) drug delivery platform. For some hydrophobic small molecules, encapsulation within polymer-lipid core-shell NPs is more efficient than polymer- or lipid-based NPs. Most well-studied core-shell formulations incorporate poly(ethylene glycol) (PEG) as a stealth molecule to extend circulation time, but there are disadvantages to PEG as well, including limited uptake into cancer cells and the potential for allergic reactions. In addition, surface PEG limits the ability to perform layer-by-layer (LbL) functionalization.
Objectives: We sought to develop a stable core-shell NP without PEG that can efficiently load hydrophobic drugs and serve as a biocompatible anionic substrate for layer-by-layer (LbL) functionalization. LbL assembly involves the iterative adsorption of oppositely charged polyelectrolytes onto charged NP cores in a simple and scalable water-based system. LbL NPs have key advantages including preserving the biological activity of therapeutic agents, controlling drug release behavior, and adding functional targeting groups to the surface. Here we report a new polymer-lipid core-shell NP formulation with a poly(lactic-co-glycolic acid) (PLGA) core and a lipid shell, with the eventual goal of loading a class of poorly soluble small molecule inhibitors for a targeted cancer application.
Methods: We first tested a suite of lipids with anionic head groups for solubility in water-miscible organic solvents to maintain compatibility with the nanoprecipitation method. Next, lead lipid candidates were combined with soy lecithin in the aqueous phase, and a panel of core-shell NPs were formed via nanoprecipitation with 50:50 esterified PLGA in the organic phase (protocol adapted from Chan et al. Biomaterials, 30, (2009), 1627). NPs were characterized for dynamic light scattering size, polydispersity, and zeta potential. Transmission electron microscope (TEM) imaging was used to investigate NP morphology. Finally, lead formulations were used as core templates for layering, with cationic poly-L-arginine (PLR) as the first layer and anionic poly-L-aspartic acid (PLD) as the terminal layer.
Results: We identified two lipids with anionic head groups that were soluble in ethanol, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (16:0 PG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (16:0-18:1 PG), making them ideal for the nanoprecipitation method. They had higher solubility than analogously structured phosphoethanolamine (PE)-based lipids, likely due to the PG head group bearing more negative charge. Incorporating 16:0 PG and 16:0-18:1 PG with soy lecithin at a molar ratio of 3:7, we were able to form NPs with favorable properties for drug delivery. The z-averaged diameter, number mean diameter, and zeta potential of the formulations are as follows: 186 nm, 130 nm, -53 mV (16:0 PG formulation); 175 nm, 117 nm, -61 mV (16:0-18:1 PG formulation); 157 nm, 89 nm, -52 mV (1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG) control). 16:0-18:1 PG was chosen as the lead candidate after dry-state TEM imaging confirmed the core-shell structure. This formulation also formed stable LbL NPs, with surface charge reversal after layering with PLR (63 mV) and PLD (-56 mV).
Discussion: Our data suggests that polymer-lipid core-shell nanoparticles without PEG are a promising formulation to improve hydrophobic drug loading, avoid clinical disadvantages of PEG, and take advantage of the layering capabilities of LbL technology. Ongoing work with this formulation includes drug loading, release characterization, and the generation of an expanded LbL NP library with functionalization for specific cancer applications.

Duchenne Muscular Dystrophy (DMD) is one of the most common forms of muscular dystrophy caused by mutations in the dystrophin gene that result in absent or insufficient functional dystrophin, a cytoskeletal protein that enables the strength, stability, and functionality of myofibres. Progressive muscular damage and degeneration occurs in people with DMD, resulting in muscular weakness, associated motor delays, loss of ambulation, respiratory impairment, and cardiomyopathy. Up to now there is no cure for DMD.
Recently, promising outcomes are revealed from the therapeutic use of exosomes: small extracellular vesicles secreted by cells and found in body fluids. Exosomes can be seen as exciting biological carriers, thanks to their ability to traverse biological barriers, to transport functional RNAs between cells and to be better tolerated by the immune system. They can be considered as surrogate for cell-based therapy with a stronger safety, for their well-documented active therapeutic role.
In this context, our aim is the development of a stable, therapeutic, tissue-specific exosome-based approach in the field of DMD, supported by the use of advanced multimodal probes, in order to track and specifically direct the exosomes to dystrophic muscles. For this reason, we combined fluorescent, labeling and ferromagnetic functionalities on single nano-object through self-assembly interactions to obtain a multimodal probe for high contrast non-invasive in vitro imaging and efficient in vivo tracking of exosomes in dystrophic mice. The multimodal probe is made of inert and biocompatible chrysotile nanotubes (NTs). The positive surface potential of NTs could be exploited for anchoring anionic species, exosomes included, in aqueous environment, following an ionic self-assembly scheme. A magnetic functionality is introduced through the interaction of NTs with anionic functional groups of the TAU capping ligand of ferrite magnetic nanoparticles (MAG NPs). Moreover, the fluorescent functionality is introduced by a red emitting chromophore. Side-by-side fluorescence imaging and magnetic resonance (MRI) experiments have confirmed the effectiveness of fluormagnetic Nanotubes as multimodal contrast agents.
In particular, the strong ferromagnetic properties of these probes will enable the driving of regenerative exosomes under high magnetic field by a clinically applicable permanent magnet, for a specific targeted in vivo delivery towards interested tissue. The possibility to specifically direct exosomes to dystrophic muscle will allow the development of a controlled release of sufficient exosome amount at disease target site for a clinical outlook of personalized highly efficient and safe nanomedicine.

¹ C. Villa, M. Campione, B. Santiago-González, F. Alessandrini, S. Erratico, I. Zucca, M. G. Bruzzone, L. Forzenigo, P. Malatesta, M. Mauri, E. Trombetta, S. Brovelli, Y. Torrente, F. Meinardi, and A. Monguzzi Self-Assembled pH-Sensitive Fluoromagnetic Nanotubes as Archetype System for Multimodal Imaging of Brain Cancer Adv. Funct. Mater. 2018, 1707582

TRAIL (Tumor Necrosis Factor-related apoptosis-inducing ligand), a cytokine belonging to the TNF superfamily, attracted major interest in oncology owing to its selective antitumor properties [1]. Therapeutic clinical trials using soluble TRAIL or antibodies targeting the two main agonist receptors (TRAIL-R1 and TRAIL-R2) have, however, failed to demonstrate their efficacy [2]. Attempts to increase its antitumor activity include, amongst others, its functionalization by nanoparticles [3], particularly magnetic nanoparticles (MNPs). Such carriers allow concentrating TRAIL [4] which can be driven to a defined target site by local application of an external DC-magnetic field. They also offer the possibility of monitoring TRAIL delivery by magnetic resonance imaging (MRI) and the possibility to increase antitumor TRAIL functionality by magnetic hyperthermia [5]. Magnetic iron oxide MNPs, magnetite and maghemite, have the right magnetic properties [6] and exhibit relatively low toxicity [7]. For this reason, they were specifically produced with different average sizes, i.e. 10 and 100 nm (magnetic single core versus magnetic multicore) [4], and were attached to TRAIL through peptide coupling, engaging respectively carboxylic or amino protein group to evaluate both the effect of their size and their chemical bounding on TRAIL antitumoral activity on malignant mammal cells. They were also subsequently submitted to an external AC-magnetic field to confirm their capability to eradicate residual TRAIL resistant tumoral cells through local heating [5]. Clearly, the properties of TRAIL and the multimodal characteristics of MNPs hold great promise for the treatment of cancer and pave the way for the generation of new and efficient drugs.

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[5] H. Belkahla, E. Mazarío, J. S. Lomas et al., Theranostics, 2019, 9, 5924–5936.
[6] S. Laurent, S. Dutz, U. O. Hafeli et al., Adv. Colloid Interface, 2011, 166, 8–23.
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Luminescent materials are used in a variety of applications, from lighting and display technologies to scintillators for high-energy radiation detection. ^[1] A particular interest has grown towards nanoscale phosphors, proposed also for innovative bio-imaging excited by infrared radiation or photosensitizers for cancer self-lighting photodynamic therapy. ^[2]
The employment of nanoparticles in these applications relies on the ability to tailor their photo-physics, which is based on the control over structural features like their size, composition and crystal symmetry able to impact their optical properties. Moreover, to be suitable for specific applications, the surface chemistry of these materials must be adapted to provide homogeneous stable dispersions/inks and thus facilitate handling and deposition, as well as efficient cellular uptake.
Colloidal nanocrystals of HfO₂ can be synthesized from molecular precursors by a solvothermal route, where both fluorescence and structural control can be achieved thanks to the multifunctional role of rare earth (RE) doping. ^[3] While RE dopant ions activate the visible luminescence of nanocrystals, at the same time, their incorporation can also stabilise the cubic polymorph of HfO₂ at room temperature. Such a structural change can significantly affect the optical quality of sintered polycrystalline scintillator ceramics as well as the performance of RE-based upconversion. In addition, the native surface chemistry of such nanoparticles allows their further adaptation to either organic solvents or, alternatively, to aqueous biological environments, enabling cellular uptake and making them suitable as non-toxic probes for optical imaging, and possibly therapy. ^[4]
In this talk the control over the structure/function relationship in HfO₂ doped nanoparticles is discussed, together with its implications towards and functional inks for innovative deposition/assembly of nanoparticle based optical materials as well as for advanced bio-imaging.

[1] S. V. Eliseeva, et. al., New J. Chem. 35, 1165 (2011).
[2] B. del Rosal, et. al., Adv. Funct. Mater. 28, 1803733 (2018).
[3] A. Lauria, et. al., ACS Nano 7, 7041 (2013).
[4] I. Villa, et. al., Nanoscale 10, 7933 (2018).

The mammalian nervous system spans spatial scales from centimeters to nanometers, and functions at time scales from microseconds to decades. To access this spatiotemporal complexity, we envision tools capable of modulating individual receptors at the nanoscale in behaving organisms at the macroscale. Weak magnetic fields offer unprecedented access to the arbitrary locations within the body due to the low conductivity and negligible magnetic permeability of biological matter. To convert weak magnetic fields into biological signals, we design and synthesize a range of nanotransducers. Based on their composition and geometry, these nanotransducers can be tailored to dissipate heat or apply force or torque. We leverage magnetothermal and magnetomechanical transduction mechanisms to enable wireless remote control of a variety of receptors in neurons and thus modulate animal behavior and physiology.

Colloidal magnetic nanoparticles (MNPs) are an important class of nanomaterials being investigated for use in a host of therapeutic and diagnostic modalities such as medical imaging, remote sensing, drug delivery, and hyperthermia. These applications exploit the very soft magnetic behavior found in certain materials, often ferrites, when they are confined in size to tens of nanometers in diameter or less. Such nanoscale magnets produce a strong collective response to applied alternating (AC) magnetic fields, while simultaneously remaining dispersed in heterophasic liquid media. Further, the synthesis of these magnetic colloids employs inexpensive, scalable, highly tailorable solution chemistry routes that both allow fine control of their properties and afford a practical route to commercialization.

Recently, we have been developing remote magnetic thermal imaging as a potential diagnostic application of colloidal MNPs. This measurement will employ dispersed particles to construct a 3D visual representation of temperature throughout a volume. The technique is based on the temperature-dependent collective response of MNPs to applied AC magnetic fields coupled with simultaneously applied DC fields to afford spatial control over the region in which the measurement is conducted. However, significant challenges remain in the development of this metrology, including a need to increase both the MNPs’ magnetic thermosensitivity and magnitude of AC field response.

Here, we report on our development of MNPs with a robust liquid-phase response to applied AC magnetic fields, specifically for solution-phase remote thermometry and other sensing applications. First, a series of colloidal nanocrystals based on ferrites were synthesized via solution routes in a variety of sizes and compositions. These MNPs were then systematically investigated using liquid-phase AC and DC field magnetometry measurements. An arbitrary-wave magnetic particle spectrometer was employed to measure magnetic AC susceptibility, allowing for a rigorous analysis of the temperature- and frequency-dependent AC response from 1 Hz to 30 MHz. These results were cross-correlated with detailed structural characterization, including electron microscopy, optical spectroscopy, and X-ray/electron diffraction, which underpins the inherent magnetic properties of the particles.

We find the relaxation dynamics in solution are strongly dictated by the structural and compositional properties of the MNPs. Specifically, particle size and compositional doping act as levers to optimize the liquid-phase AC response through the manipulation of interparticle and intraparticle magnetic spin interactions. Using these guidelines, we find the set of structural parameters that produce the most robust thermosensitive magnetic response. Collectively, these studies illuminate the complex behavior of MNPs under AC driving fields, reveal extensive correlations between nanoscale atomic structure and their liquid-phase magnetic response, and provide guidelines for the design of MNPs used in magnetic thermal imaging and other remote sensing applications.

Magnetic nanoparticles are extremely useful tools for purifying biological targets in complex mixtures. Magnetofluorescent nanoparticles have additional benefits allowing for sensing and multiplexing. Although the chemical coupling of conventional organic dyes to silicon dioxide shells is straightforward, densely packing can lead to quenching and non-radiative decay pathways. These unwanted side effects, significantly reduce the overall brightness of a given particle. To circumvent this issue, we implement a new method to sparsely distribute dye labeling on multiple coating layers, yielding distinctively bright magnetic silica-coated fluorescent particles, which we termed MagSiGlow. In this study, we describe the design and synthesis of MagSiGlow particles using novel and commercially available fluorophores detectable in different color channels. Additionally, we discuss experiments where implementing MagSiGlow particles facilitates detection of extracellular vesicles and visualization of living cells using flow cytometer and confocal microscopy.

The year 2018 Nobel Prize in Physics was awarded for Optical Tweezers and their application in biological systems. Optical tweezers have emerged as a powerful tool for the non-invasive trapping and manipulation of colloidal particles and biological cells. However, the stable trapping of nanometer-scale biological objects such as proteins, small extracellular vesicles, and virions has been met with challenges due to the diffraction limit of light. Significantly increasing the laser power can provide sufficient trapping potential depth to trap nanoscale objects. Unfortunately, the significant optical intensity required predisposes the trapped biological specimens to photo-toxicity and thermal stress. I will discuss a new paradigm for optically controlled nanotweezers termed Opto-Thermo-Electrohydrodynamic Tweezers (OTET) that enables the stable trapping and dynamic manipulation of nanoscale biological objects such as EVs with negligible photothermal heating and light intensity. The OTET platform employs a finite array of plasmonic nanoholes illuminated with light in conjunction with an applied a.c. electric field to create the spatially varying electrohydrodynamic potential that can rapidly trap single nanoscale EVs on-demand. I will also discuss potential translational biomedical applications of this new technology for early cancer detection via EV-based liquid biopsy. This novel non-invasive optical nanotweezer is expected to open new horizons in life science and medicine by offering an unprecedented level of control of tiny nano-sized biological objects in solution without photo-induced damage.

Large molecules such as insulin are too big to be able to cross the skin and reach the blood flow through passive diffusion, unlike smaller molecules such as nicotine for example. Needles are thus usually required to cross this natural barrier. In order to improve the life quality of patients using regular hypodermal injections, alternative delivery methods are needed. Carbon-based nanoparticles, free or associated with a polymer matrix, are increasingly used in innovative biomedical applications [1]. We are developping a non-invasive transdermal delivery route, using a two-in-one nanocomposite device and the reversible permeabilisation of the skin, thanks to electroporation. The device is a nanocomposite with a biocompatible hydrogel matrix containing carbon nanotubes (CNTs), which combines both purposes of assisting the permeabilisation and storing the molecule to be delivered. The inclusion of CNTs in the hydrogel matrix is also intended to prevent their release during operation of the device, with a safe(r) by design strategy in mind. Previous work from our group described an enhanced drug delivery of dextran FITC through permeabilised skin for devices containing CNTs [2]. Later, the electrical characterisation of those devices was performed [3]. However, although the proof of concept was validated, such devices are still far from practical applications and the optimisation of electroporation parameters, depending on the intended use, is still in progress. We will describe and discuss our most recent advances.

References:

[1] Simon, J.; Flahaut, E.; Golzio, M. Overview of Carbon Nanotubes for Biomedical Applications. Materials 2019, 12(4), 624. https://doi.org/10.3390/ma12040624

[2] Guillet, J.-F.; Flahaut, E.; Golzio, M. A Hydrogel/Carbon-Nanotube Needle-Free Device for Electrostimulated Skin Drug Delivery. ChemPhysChem 2017, 18 (19), 2715–2723. https://doi.org/10.1002/cphc.201700517

When properly deployed, the immune system can render deadly pathogens harmless, eradicate even metastatic cancers, and provide long-lasting protection from diverse diseases. However, realizing these remarkable capabilities is inherently risky, as disruption to immune homeostasis can lead to dangerous complications and autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. We previously described a polymer-nanoparticle (PNP) based hydrogel that is biocompatible, long lasting, and injectable^{1, 2}, traits that we hypothesized would improve controlled local delivery of immunotherapy.

To test this hypothesis, we explored PNP hydrogel-mediated delivery of CD40 agonist antibody (aCD40), a potent immunostimulant that has been shown to improve outcomes in combination immunotherapy, but poses considerable dose-limiting toxicity.³ PNP hydrogels were formed by mixing CD40 agonist antibody (CD40a), dodecyl-modified hydroxymethylcellulose (HPMC-C₁₂), and poly(ethylene glycol)-b-poly(lactic acid) nanoparticles (PEG-PLA NPs) to generate gels with 2 wt% HPMC-C₁₂, 10 wt% PEG-PLA NPs, and the desired dose of CD40a (ranging from 10-100 mg). HPMC-C₁₂ and PEG-PLA NPs were prepared as described previously¹.

Using positron emission tomography (PET) imaging, we quantitatively tracked how delivery strategy (e.g., hydrogel vs. local bolus) shaped aCD40 pharmacokinetics over the course of 12 days. PET studies demonstrated that hydrogels substantively altered antibody pharmacokinetics to favor drug exposure in target tissues. Our data indicate that hydrogels increased exposure at the injection site by 96 ± 13% (q = 4 x 10-6) and at the tumor draining lymph node by 35 ± 19% (q = 5 x 10^-3) over the course of the 12-day study. Locoregional hydrogel delivery also circumvented dose-limiting toxicity commonly associated with aCD40 therapy (e.g., acute weight loss and hepatotoxicity). Using Luminex multiplexed cytokine assays, we also evaluated how delivery strategy shaped the induction of effector cytokines in systemic circulation and in tumor draining lymph nodes. We found that hydrogels substantively attenuated the induction of systemic cytokines, particularly across a number of pro-inflammatory cytokines (IFNy, TNFa, IL6, and IL1b) commonly associated with cytokine storm syndrome, another common side effect of aCD40 therapy. Despite reducing cytokine levels systemically, hydrogel delivery generated a more potent immune response, as measured by effector cytokine (IFNy, CXCL10) levels within tumor-draining lymph nodes (TdLN). Correspondingly, hydrogel-mediated locoregional aCD40 therapy was more tolerable and effective than even locoregional bolus injections, and synergized with PD-L1 blockade (40% survivors in combo-therapy arms vs. 0% survivors in monotherapy arms, n=10, p_adj<0.03).

Overall, this study provides compelling evidence that PNP hydrogels can improve aCD40 pharmacokinetics and pharmacodynamics, leading to greater tolerability and therapeutic efficacy in the B16F10 model of melanoma. Notably, PNP hydrogels mediated these effects without the need to modify their cargo in any way. Therefore, we anticipate that this approach may also improve the safety of other potent immunostimulants, and is thus a highly translatable solution to the problem of immune-related adverse effects.

References:
1. Appel, E.A. et al. Nat Commun 6, 6295 (2015).
2. Stapleton, L.M. et al. Nat Biomed Eng 3, 611-620 (2019).
3. O'Hara, M.H. et al. The Lancet Oncology 22, 118-131 (2021).
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The high activity of specific enzymes in cancer has been utilized as tumor-specific trigger for anticancer drug delivery. NAD(P)H:quinone oxidoreductase-1 (NQO1), an overexpressed enzyme in certain tumor types, maintains homeostasis and inhibits oxidative stress caused by elevated reactive oxygen species (ROS) in tumor cells. The activity of NQO1 in certain types of cancer cells is increased compared to that in normal cells. Interestingly, NQO1 catalyze the reaction of trimethyl-locked quinone propionic acid (QPA) to a lactone-based group via intramolecular cyclization. Toward this objective, we synthesized amphiphilic block copolymer (QPA-P) composed of QPA-locked polycaprolactone (PCL) as a hydrophobic block and poly(ethylene glycol) (PEG) as a hydrophilic block. This polymer formed a self-assembled micelle via hydrophobic interaction. QPA-locked PCL block was depolymerized by NQO1 via a cascade two-step cyclization process, which eventually mediated the disassembly of micelle and released anticancer drugs at the target cancer cells. The micelles showed NQO1-responsive intracellular drug release and enhanced anticancer effects in vivo. These results indicate that enzyme-responsive depolymerization process could be a promising strategy for improving therapeutic efficacy of polymer-based anticancer drug delivery carrier.

Detection of hydrogen peroxide (H₂O₂) possess an essential role in both analysis and applications (chemical synthesis, cosmetics, biology, medicine, food, paper and textile industries) in bioelectronics. Electrochemical detection of H₂O₂ seems to be the highly promising due to its simplicity, fast response and low cost. Enzyme-free electrochemical sensors were demonstrated utilizing metal nanostructures, particularly silver (Ag) based nanostructures, for the detection of H₂O₂. Oxidation tendency of Ag together with its degradation in the presence of H₂O₂ necessitates use of a protective coating for Ag based amperometric sensors. In this work, a platinum (Pt) shell layer was deposited onto Ag NWs to improve the oxidation stability so then they can be used for the detection of H₂O₂. Ag-Pt core-shell NW electrodes were used for the detection of H₂O₂ with different concentrations (10, 50, 100 mM) at an applied potential of +0.8 V (vs. Ag/AgCl). The fabricated Ag-Pt core-shell NWs electrode showed a high sensitivity of 0.04 μA/μM over a wide linear range of concentrations (16.6 - 990.1 μM) with a low detection limit (10.95 μM). the regression equation was determined as I (μA) = 1.61 + 0.04 × [H₂O₂]/μM with a correlation coefficient R² of 0.9947. Using the 90% initial step change in the current, the response time of the sensor was 4.74 seconds. Fabricated sensors gave response only to H₂O₂ which proved interference selectivity. A simple and highly reproducible method for the fabrication of H₂O₂ sensors with highly repeatable response was demonstrated, which can be further expanded to other bioelectronics applications requiring electrochemical stability.

This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) under grant no 117E539.

Ubiquitous, autonomous, and real-time chemical sensing can provide abundant information related to the warfighter health and performance status. For example, volatile organic compounds (VOCs) found in exhaled breath are critical biomarkers for assessing human health and physiological status. Biorecognition elements (BREs) are biological materials exhibiting specific affinity to the target molecules. Short peptides (generally composed of 5-15 amino acid residuals) are promising BREs for selective VOC detection. They provide chemical stability and design flexibility to sensing the targeted biomarker. Peptide BRE-coated CNT chemiresistor has emerged as a miniaturized sensor platform for wearable applications. Previous peptide-functionalized CNT gas sensors have shown detecting explosives and bacterial food contamination byproducts. However, the peptide sensing mechanism of a peptide-CNT pair is yet unclear. We presumed that the conformation of the peptide on the CNT surface affects the molecular accessibility to the BRE-CNT interface. Here, we present a peptide conformation model to investigate peptides' affinity towards VOC on a CNT chemiresistor platform. This model predicts the conformation of the peptide on the CNT surface using peptide-CNT interaction energy obtained from a molecular dynamics simulation. A near-edge X-ray absorption fine structure (NEXAFS) spectroscopy experimentally validates the peptide conformation model by observing the peptide alignment to the CNT. Then, a peptide-functionalized CNT chemiresistor was tested to various gases to investigate how the conformation affects VOC sensing. The result shows that the vertically oriented peptide on the CNT surface hinders VOC access to the peptide-CNT interface, resulting in a significantly low sensor signal than the CNT chemiresistor with the horizontally oriented peptide. Our results strongly indicate that molecular accessibility is a key factor detrimental to the VOC sensing from peptide-CNT devices. This finding is crucial for designing the peptide sequence specific to target VOC, and ultimately, developing sensitive CNT-based VOC sensors.

Aptamers, nucleic acid analogs of antibodies, have shown promise in molecular diagnostics capitalizing on their programmable nature and high affinity/selectivity towards cognate ligands. Despite the breadth of biomolecular targets ranging from biofluid biomarkers to cellular analytes, a biologically deliverable aptasensor that enables to accurately monitor endogenous biomolecular abundance still remains elusive. DNA nanoassembly aptasensors built from short staple DNA stands have shown particular success based on their precise Förster resonance energy transfer (FRET)-based operations; nonetheless, challenges are often associated with relatively low structural stability, its labor-/time-intensive fabrication, and the complex sensor operations that limit the design generalizability and necessitate computational modeling. Here, we present high-throughput biomimetic crystallization-driven DNA self-assembly as a route to tackle the challenges and produce a highly programmable and agile intracellular ratiometric aptasensor.[1] The probes are formulated by rolling circle amplification (RCA), whereby multifunctional elements-encoded polymeric DNA strands are enzymatically produced and self-compacted into DNA–inorganic hybrid nanoarchitectures. Harnessing the programmability of RCA template, the obtained DNA nanoparticles are intrinsically encoded with three functional features: 1) Tumor targetability using cell-targeting aptamer, 2) Ratiometric aptasensing of cellular analytes empowered by a tripartite duplexed aptasensor and 3) Synergistic assistance of cellular internalization by cholesterol decoration. The resultant probes possess biocompatibility, structural integrity and sequence-driven multifunctionalities which can be readily customized using the template without the need of computational modeling or any modification of fabrication procedures. Taking LNCaP human prostate adenocarcinoma cells and intracellular adenosine triphosphate as an exemplar target system, we illustrate the targeted delivery of our probe and subsequent ratiometric signaling triggered by intracellular target analyte recognition events. The limitations often associated with intensiometric-signaling intracellular probes are elegantly leveraged by our approach, where the quantitative detection is demonstrated with drug-treated cells and corresponding ratiometric FRET signal changes from internalized aptasensors. To the best of our knowledge, this represents the first demonstration of harnessing biomimetic crystallization to construct a DNA nanosensor that is capable of target-responsive biomolecular detection within intracellular environments. We envision that the proposed probe has substantial promise as a biologically deliverable probe that can be readily tailored across wide-ranging target systems, being capable of the quantitative monitoring of biomolecular profiles.

[1] N. Kim, E. Kim, H. Kim, M. R. Thomas, A. Najer, M. M. Stevens, Tumor-Targeting Cholesterol-Decorated DNA Nanoflowers for Intracellular Ratiometric Aptasensing, Advanced Materials, 2021, 33, 2007738.

All-optical ultrasound imaging has emerged as an imaging paradigm well-suited for minimally invasive surgical procedures. With this modality, ultrasound is generated when pulsed or modulated light is absorbed within a coating material. The ensuing heat rise causes a corresponding pressure rise which propagates as ultrasound. Using optic fibres to deliver light for ultrasound generation and reception allows complementary therapeutic and imaging modalities to be integrated to create highly miniaturised devices. One area of interest for these devices is combined imaging and optical ablation, to provide real-time monitoring of ablation lesion depth. However, combining modalities requires carefully engineered composite materials with wavelength-selective absorption. Previous research has demonstrated the use of gold nanoparticles and crystal violet dye for co-registered ultrasound and photoacoustic imaging [1]. However, these materials were limited by material degradation with prolonged exposure and limited ultrasound pressures and bandwidths.

We present the optimisation of a nanocomposite material that comprises a near infrared absorbing dye in a polydimethylsiloxane (PDMS) host on the distal tip of optical fibres. The optical absorption of this composite has a pronounced peak at 1064 nm, allowing the absorption of laser light for ultrasound generation. Optical transmission through the composite was low for wavelengths below 900 nm allowing for the transmission of laser light at 808 nm for tissue ablation. Crucially, the composite demonstrates a high photostability to laser exposure, allowing continuous ultrasound imaging for larger periods of time (several hours). Varying the dye concentration allows for strong optical absorption for ultrasound generation, whilst maintaining higher transmission at shorter wavelengths for complementary modalities.

Our studies demonstrate that ultrasound pressures in excess of 1 MPa can be generated, sufficient for M-mode imaging at centimetre scale depths and consistent with other ultrasound generating materials [1,2]. Further, the corresponding ultrasound bandwidths (25 MHz), will enable high resolution imaging. We have used this composite to provide ultrasound imaging for the monitoring laser ablation of tissue in real-time, allowing the tracking of lesion depth. By optimising the optical absorption of the composite, light delivery for laser ablation is possible through the same optical fibre used for ultrasound generation. This would allow for device miniaturisation and coaxially aligned imaging and tissue ablation.

[1] S. Noimark et al., “Polydimethylsiloxane Composites for Optical Ultrasound Generation and Multimodality Imaging,” Adv. Funct. Mater., vol. 28, no. 9, p. 1704919, Feb. 2018, doi: 10.1002/adfm.201704919.
[2] S. Noimark et al., “Carbon-Nanotube-PDMS Composite Coatings on Optical Fibers for All-Optical Ultrasound Imaging,” Adv. Funct. Mater., vol. 26, no. 46, pp. 8390–8396, Dec. 2016, doi: 10.1002/adfm.201601337.

The upregulation of glutathione (GSH) in cancer contributes to the resistance of cancer cells against oxidative stress and apoptosis. Thus, it is considered a crucial barrier that inhibits the therapeutic efficacy of reactive oxygen species-mediated cancer therapy. Herein, we develop glutathione-depleting nanocomposites (dGSH-NC) for amplifying the oxidative stress in sonodynamic therapy of cancer. dGSH-NC is composed of titanium dioxide nanoparticles and manganese dioxide surface for providing ultrasound-responsive ROS generation and GSH depletion. Further, GSH reduces manganese dioxide to manganese(II) ion that induces additional intracellular ROS generation. In SCC7 cells, dGSH-NC remarkably inhibits GSH of SCC7 cells and produces reactive oxygen species to amplify the cellular oxidative stress. In the presence of ultrasound, dGSH-NC exhibited an improved cytotoxic effect than that of bare titanium dioxide nanoparticles, and exposed cells presented necrotic cell death features.

Surface enhanced Raman spectroscopy (SERS) is gaining popularity as a diagnostic technique, due to its ability for multiplexed detection and ultra-high sensitivity. So far, in the majority of its clinical applications, it has relied on the use of nanoprobes for indirect detection. In general, the nanoprobe brightness decreases exponentially as the Raman reporter molecule moves away from the nanoparticle surface, and local enhancement factors can vary dramatically on specific locations of the nanoparticle due to the presence of “hot spots”. In order to develop a SERS based diagnostic platform with maximum sensitivity and reliability, it is imperative to understand the orientation and binding behavior of the Raman reporter on the nanoparticle surface, as well as under what conditions its orientation may change. Herein, we investigate analyte orientation and interaction on the nanoparticle surface using two different nanostar morphologies, surfactant-free nanostars and 6-branched nanostars, and three different analytes, thiophenol, 4-aminothiophenol, and crystal violet. Nanoprobe-analyte interactions are characterized by Raman spectroscopy and nanostar morphologies by transmission electron microscopy (TEM). Molecular dynamics simulations were conducted to verify the changes in analyte-nanoprobe interaction. It was found that signal amplification provided by surfactant-free nanostars and 6-branched nanostars can follow dramatically different trends depending on the analyte molecular structure. Molecular orientation on the nanoprobe surface was demonstrated to vary with changing analyte concentration.

Antibody and nanoparticle mediated drug delivery to solid tumors can improve pharmacokinetics and dynamics of local payloads and decrease systemic toxicity. Despite these obvious advantages, there remains considerable room for improvement. We have previously shown that localized bursts of radiation can enhance local myeloid cell delivery and result in improved anti-tumor effects. To further enhance target specificity, we hypothesized that radiation induced drug release could represent a powerful complementary approach. Here, we developed scintillating yttrium oxide nanoparticles that emit UV light upon excitation with external X-ray irradiation, resulting in the release of attached UV-activatable prodrugs. Using Fourier-transform infrared spectroscopy, zeta potential and dynamic light scattering measurements, we have demonstrated and optimized the covalent modification of the nanoparticle surface with a prodrug. The release kinetics and total drug release were then observed by X-ray irradiation, followed by drug quantification using liquid chromatography. Likewise, the in vitro activation of the prodrugs has been demonstrated in culture, following X-ray irradiation. We show that these X-ray activatable nanoparticles efficiently release active cytotoxic agent with revise control due to the external radiation beam.

Photodynamic therapy is increasingly being recognized as a non-invasive feature and local treatment. Especially, inorganic photocatalysts, such as titanium dioxide (TiO₂), are regarded alternative materials for organic photosensitizer due to their high photo-stability, biocompatibility and significant photocatalytic ability. However, inorganic photocatalysts have critical threshold in the absorption of low energy photons to introduce in cancer therapy owing to their large band gap.
Meanwhile, plasmonic noble metal-based nanomaterials has drawn important attraction in recent years due to their broadly tunable localized surface plasmon resonance properties derived from the collective oscillation of surface free electrons under incident light. According to recent studies, anisotropic gold nanomaterials with tunable aspect ratio can generate strong electric fields at their tips. Particularly, gold nanobipyramids possessing sharp-tip morphology induce much stronger electric fields against gold nanorods commonly used in photothermal therapy. With suitable biocompatibility, plasmonic gold nanomaterials were investigated for cancer treatment and became a promising candidate to redeem the limitations of TiO₂ in biomedical applications. Upon strategic coupling with TiO₂, LSPR-induced hot carriers of plasmonic Au nanomaterials are injected into the conduction band of TiO₂ over the Schottky barrier, which can be consumed for the reduction of oxygen.
Despite of these advantages, Au NBP is hard to utilized due to their low thermal stability. To date, the synthesis of Au/TiO₂ heterostructures with crystalline TiO₂ requiring annealing steps at high temperature after amorphous TiO₂ coating. In order to convert the amorphous phase of TiO₂ to anatase phase, high temperature treatment is necessary. However, sharp tip gold nanomaterials such as Au NBP lose their tip during annealing process. Therefore, undesirable morphological disruption is occurred during annealing step, leading to LSPR peak changes. Moreover, TiO₂ shell agglomeration could not be avoided. These leads to low photocatalytic effects during NIR laser irradiation.
Herein, we newly developed anatase TiO₂-decorated Au NBP (Au NBP/a-TiO₂) as a novel metal/semiconductor core/shell colloidal heterostructure for NIR-responsive phototherapy. This is the first time that introducing low temperature hydrothermal method to crystalline anatase TiO₂ onto Au NBP surface, while preserving a well-defined Au NBP/a-TiO₂ morphology with sharp-metal tips without any aggregation. Strategically, the highly energetic LSPR-derived hot electrons generated by incident NIR light were injected directly into the conduction band of the anatase TiO₂ nanoclusters, inducing the reduction of oxygen to superoxide and finally producing hydroxyl radical species. Simultaneously, the LSPR-induced photothermal effect was investigated for combinational photothermal and photodynamic cancer therapy. Our work emphasizes the facile and mild fabrication of anatase TiO₂ decorated anisotropic plasmonic nanostructures, while expanding the promise of novel NIR-responsive nanostructures with remarkably superior efficiency for synergistic photo-induced cancer treatments.

Lanthanide-doped upconversion nanoparticles (UCNP) have been attracted great attention with the unique ability to convert low-energy NIR radiation to high-energy UV-vis light in biomedical applications. UCNP additionally possesses unique properties, including good photostability with biological transparent excitation wavelength, tunable emission band, and low cytotoxicity. However, the UCNP showcases the intrinsic low quantum efficiency for practical applications as a result of insufficient absorption cross-section area and surface defects. To overcome this limitation, the incorporation of localized surface plasmon resonance (LSPR) has been suggested as a promising way. In this work, anatase titania-coated gold nanorods (Au NR@aTiO₂) were decorated with UCNP. aTiO₂ was integrated as a spacer between plasmonic Au NR and UCNP to avoid the quenching of UCL. Au NR and UCNP components showed characteristic absorbance in the NIR region, detailed characterization of the resulting core@shell nanostructures revealed enhanced UCNP-derived UV-vis emission due to the plasmonic properties of Au NR.¹ In presence of UCNP, the emitted UV light was proposed to excite the aTiO₂ semiconductor and play a key role in the generation of reactive oxygen species (ROS). In addition, emitted visible light from UCNP was re-absorbed by Au NR which was well matched with the transverse absorption peak of Au NR to further generate the hot carriers. The resultant hot carriers could inject into the conduction band of aTiO₂ to further generate the ROS. LSPR-induced photo-thermal effect was also investigated by temperature-rise profile. In vitro and in vivo phototherapy effect was induced on U87MG (Glioblastoma astrocytoma). Interestingly, the overall therapeutic effects of our hybrid nanostructures were enhanced under laser irradiation, confirming the combinational photothermal and photodynamic effects. Thus, serving as a multifunctional phototherapy agent, the hybrid nanostructures herein introduced are suggested to be a valuable addition in deep-range noninvasive cancer phototherapy studies.

The immune checkpoint blockade (ICB) for tumor therapy is promising in the clinic. Among some ICB drug, Anti-PD-L1 antibody (aPD-L1 antibody) blocks the overexpressed PD-L1 of the tumor cell membrane and enhance the antitumor effect of cytotoxic T cell. However, these antibody therapies still have several challenges of poor delivery efficiency and immune-related adverse events. Herein, we designed the delivery platform for systemic controlled release of aPD-L1 antibody. Through the formation of pH-responsive phenylboronic ester between PBA group on polymeric phenylboronic acid (pPBA) and diol on inherent glycosylation site of the antibody, a polymeric phenylboronic acid-antibody (pPBA-Ab) nanocomplex was prepared by simply mixing pPBA and antibody. As the basic key point of effective delivery, we focused on the property of antibody nanocomplex, which are protection from external attack and release at tumor lesion. In various in vitro assays, Our antibody nanocomplex protected antibody by shielding effect of polymer shell and released antibody from acidic pH which are known as hallmarks of tumor. Furthermore, antibody complex exhibited longer circulation and enhanced tumor accumulation in vivo, which led to a significant antitumor effect when compared to free antibody. Overall, we propose the potential of our antibody nanocomplex platform as a successful delivery system for a therapeutic application that improved antitumor immunity.

Highly optically absorbing nanocomposites have broad versatility with applications ranging from solar cell technology to biomedical imaging. One area in which these nanocomposites have shown particular promise, is optical ultrasound generation. We report the development of CuInS₂ quantum dot (CIS QD)-polydimethylsiloxane (PDMS) nanocomposites with wavelength-selective optical absorption properties for all-optical multimodality imaging. Using bottom-up fabrication methods, micron-scale thickness CIS QD-PDMS nanocomposites were created on highly miniature optical fibre substrates. These nanocomposites were engineered to exhibit high optical absorption (> 90%) at 532 nm for optical ultrasound generation and high optical transmission (> 95 %) at wavelengths greater than 700 nm, enabling other modalities such as photoacoustic imaging and spectroscopy.

Pulsed laser excitation light at 532 nm transmitted by the optical fibre was absorbed by the coating and ultrasound generation was achieved via the photoacoustic effect. These CIS QD-PDMS nanocomposites generated high ultrasound pressures of up to 3.7 MPa (laser fluence: 153.4 mJ/cm², distance: 1.5 mm) with corresponding -6 dB ultrasound bandwidths of 18 MHz. Using 532 nm and 1064 nm laser excitation sources for ultrasound and photoacoustic imaging respectively, a co-registered image of a carbon black ink-filled tube gel wax phantom was obtained with good correspondence between the ultrasound image and photoacoustic signals. Due to their wavelength-selective optical absorption properties, these high-performance fibre-based ultrasound transmitters show promise for diagnostic and therapeutic clinical applications such as the imaging of atherosclerotic plaques in coronary arteries, or use in photodynamic therapy for cancer treatment.

Photo dynamic therapy (PDT) is one of the important therapeutic methods for cancer treat especially for skin cancer. Currently, a sort of dye, such as chlorin e6, are used for generating singlet oxygen for killing cancer by irradiating visible light. However, the penetration depth of the visible excitation light is only limited to be less than a millimeter. We proposed to use near infrared (NIR) light for the excitation to penetrate into tissues with centimeter order depth. Upconversion (UC) luminescence is a specific phenomenon observable for the rare-earth ions in ceramic hosts to emit green and red visible light under NIR excitation. We had reported a nano-structure by combining the UC ceramic nano phosphors with red emission (660 nm) to generate singlet oxygen by exciting the chlorin e6 on the surface. Simultaneous emission of NIR luminescence (1550 nm) can be used for NIR fluorescence imaging for diagnosis. The structure can kill two birds with one stone [M. Kamimura et al., Chem. Lett. 2017, 46, 1076−1078(doi: 10.1246/cl.170322)]. Rose Bengal (RB) is another known dye for the PDT to be excited with green light. The UC nano phosphor can also be designed to emit 550-nm green light efficiently. Here, we designed a new nanostructure for using the RB for the NIR-excited PDT agent. Basically the position of the RB is important. The RB is known to have toxicity. If the RB is exposed to the surface of the structure, the toxicity cannot be avoided. We designed to form a nano micelle structure with hydrophobic core with PLGA and PEGylated hydrophilic shell. The RB was installed to the border position of the border of the PLGA core and PEG shell. For installing the RB at a proper position, C₁₈RB was used for sticking the C18 part to the hydrophobic core to locate the RB on the border. Singlet oxygen generation effectively killed cancer cells with 980-nm NIR excitation. As well, NIR imaging was successfully performed by observing 1550-nm luminescence. Also, the toxicity of the RB was effectively suppressed. Therapy, diagnosis and toxicity suppression are the three birds. One stone killed three birds [K. Tezuka et al., ACS Appl. Bio Matter, 2021, 4 4462-4469 (doi: 10.1021/acsabm.1c00213)]! The nanostructure for utilizing the merits of transparency of the NIR photonics [K. Soga et al. ed., “Transparency in Biology,” Springer, 2021(doi: 10.1007/978-981-15-9627-8)] will be furthermore discussed.

Nanoparticles (NP) exhibit some interesting bio-medical applications and the development are in high degree determined by new facilities opened by the NP’s multifunctionalities. Among all developed nanoparticles, nanodiamond (ND) and ND-based hybrid complexes are promising due to their biocompatibility, variable sizes, structure, surface chemistry, and physical properties. Recent works have demonstrated exciting advantages of ND for theranostic applications, including bio imaging and drug delivery. ND’s surface chemistry allows advanced methods of functionalization with the molecules of interest extending the applications, for example, ND enhanced drug delivery and efficiency has been observed, including effects on chemoresistant tumors and direct delivery across blood-brain barrier.
In this presentation, nanodiamond-gold core-shell (ND@Au) hybrid nanoparticles are synthesized combining properties and advantages of Si-doped nanodiamond and nanogold. It is shown that the ND@Au nanoparticles are biocompatible. NDs are demonstrated as imaging agent for Raman mapping, one-photon Fluorescence imaging, two-photon Fluorescence Lifetime Imaging, and high-resolution X-ray microscopy utilizing absorption properties of Au in the nanoshell. This demonstrated the synergistical effects of combining gold and nanodiamond render then useful markers in bio tracing and imaging.
When drug molecules are conjugated onto ND, the composed ND-X complexes are demonstrated in a 3D cellular model (multicellular tumor spheroid, MCTS), consisted of large collection of cells in a three dimensional format extend about 0.7 mm in diameter; resemble the configuration of a tumor, ideal for evaluating the cytotoxicity and cytotoxicity mechanism of ND-drug complex and their efficacy. The drug molecules can be released in a pH dependent manner and enhanced drug efficacy can be achieved. Although this work presents only few examples, they demonstrate wide perspectives of development of NP with new synergy properties and of their use.

The well-controlled synthesis of nanoscale materials is arguably one of the most important achievements of material science in the past decades. With the push to simplify biomedical material designs, inorganic nanomaterials have regained interest. Especially metal and metal oxide nanomaterials have attracted significant attention due to the scalability and robustness of their synthesis and their tailorable composition and architecture. While fascinating results have been achieved by loading inorganic nanoparticles with organic molecules, my presentation will focus on purely inorganic systems.
In the first part, I will present an approach to unite tissue adhesion, based on nano-bridging, with bioactivity for wound healing applications. Uniting these properties requires control over nanoparticle architecture and freedom of choice in materials. Liquid-feed flame spray pyrolysis (LF-FSP) fulfills these requirements, while offering scalable and sterile synthesis. By utilizing the versatility of LF-FSP, we have united the wound closure properties of bioglass with the anti-inflammatory properties of ceria in one nanoparticle hybrid system. Careful selection of the bioglass composition and the hybrid architecture gives access to hard and soft tissue healing properties. In addition, by adjusting LF-FSP process parameters, we are able to control the oxidation state of the ceria, which governs anti-oxidative and anti-microbial properties of the hybrid. By tailoring the architecture of the hybrid nanoparticles, temporal control of the material bioactivities can be achieved in order to optimally address the different phases of the wound healing cascade.
In the second part, I will show how LF-FSP gives assess to ultra-small, near-monodisperse hafnium dioxide nanoparticles with excellent radio-enhancement properties. Such radio-enhancers can be employed to increase the x-ray absorption cross-section of tumor compared to healthy tissues. Direct benchmarking of the LF-FSP-made hafnia against gold nanoparticles, the current gold standard in the field, rationalizes their use based on their superior radio-enhancement performance. The competitive radio-enhancement properties for near-monodisperse nanoparticles produced by scalable and sterile flame spray synthesis offer a route to overcoming key roadblocks in the translation of nanoparticle-based radio-enhancers.

Matrices for matrix assisted laser desorption/ionization mass spectrometry (MALDI MS) is a standard tool to investigate high molecular weight (HMW) compounds, e.g. proteins and polymers. Small organic matrices (SOMs) generally consist of an aromatic ring to absorb the laser energy and transfer it to the HMWC analyte, and an ionizing functional group to transfer charge. Now the MALDI analysis of low molecular weight (LMW) compounds is coming into focus [1], to use this fast and reliable method to e.g. trace metabolomic changes in tumor development. Here, SOMs are problematic as they give rise to noise in the LMW part of the spectrum (are not “MALDI-silent”), are generally not “dual mode” (i.e. supporting measurements in both ion modes) and in MALDI MS imaging (MSI), their uneven surface coverage limits spatial resolution.

We develop polymeric matrices to enable MALDI analytics of LMW compounds, thereby turning the initial MALDI MS concept of “small matrices – big analytes” on its head.

For conjugated polymers (CPs), the conjugated backbone translates into a strong optical absorption, alkyl side chains make the CPs solution-processable and allows to coat uniform thin films. We tested several CPs as matrices and found that they are MALDI silent in the full measurement range (>m/z= 150k) except at high laser intensities, when fragmentation of the solubilizing sidechains starts [2]. Most importantly, the CPs allow the analysis of noise-free analysis of LMW analytes. Also, the CPs are dual-mode matrices, and the analyte ionization ability is competitive to standard SOMs in both positive and negative mode. MALDI MSI experiments were carried out visualize morphologies of rat brain sections, and the uniform thin films allowed a spatial resolution of 10 mm, which is high for MSI and was limited by the resolution of the instrument. SEM analyses of the films indicates that using more advanced instruments, even higher resolutions are possible using CPs.

It is a general assumption that high crystallinity is a crucial prerequisite for MALDI matrices. Yet polymers are only capable of adopting a semi-crystalline morphology, i.e., forming crystalline and amorphous domains. The analytes are therefore expected to be incorporated in the crystalline domains, and efficiency is expected to depend on the polymer’s degree of crystallinity. To explore this, we synthesized two fully amorphous NDI(T2) and fluorene co-polymers carrying long and branches sidechains on both monomers [3]. Both turned out to be excellent MALDI silent dual-mode matrices, suggesting that in CP matrices analyte incorporation takes place in the amorphous phase.

Finally, the polymerization of high-performing SOMs can retain the advantageous properties of the SOM while increasing their vacuum stability and making them MALDI silent in the LMW area. The polymerized SOMs were used in MALDI MSI measurements of breast cancer xenografts, and showed excellent ion extraction abilities [4].

References:
[1a] Calvano, Monopoli, Cataldi, Palmisano: Anal. Bioanal. Chem. 2018, 410, 4015; [1b] Qiao, Lissel: Chem. Asian J. 2021, doi:10.1002/asia.202100044.
[2] Horatz, Giampa, Karpov, Sahre, Bednarz, Kiriy, Voit, Niehaus, Hadjichristidis, Michels, Lissel. J. Am. Chem. Soc. 2018, 140 (36), 11416.
[3] Horatz, Ditte, Prenveille, Zhang, Jehnichen, Kiriy, Voit, Lissel: ChemPlusChem 2019, 84 (9), 1338.
[4] Horatz, Giampa, Qiao, Mostue, Lissel: ACS Appl. Polym. Mater. 2021, accepted.

Communication between biological components is critical for homeostasis maintenance among the convergence of complicated bio-signals. For therapeutic nanoparticles (NPs), the general lack of effective communication mechanisms with the external cellular environment causes loss of homeostasis, resulting in deprived autonomy, severe macrophage-mediated clearance, and limited tumor accumulation. Here, we develop a multistage signal-interactive system on porous silicon particles through integrating the Self-peptide and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide into a hierarchical chimeric signaling interface with ‘‘don’t eat me’’ and ‘‘eat me’’ signals. This biochemical transceiver can act as both the signal receiver for amantadine to achieve NP transformation and signal conversion as well as the signal source to present different signals sequentially by reversible self-mimicking. Compared with the non-interactive controls, these signal-interactive NPs loaded with AS1411 and tanespimycin (17-AAG) as anticancer drugs improve tumor targeting 2.8-fold and tumor suppression 6.5-fold and showed only 51% accumulation in the liver with restricted hepatic injury.
Here, we demonstrate that constructing a signal-interactive NP system improves NP-cell communication efficiency. Moreover, we show that the functional chimeric peptide design enables orderly integrating of multiple signal modules and the signal-interactive NPs reduce liver accumulation and promote tumor targeting in an animal cancer model.

The freshness of fish meat is the most important criterion in the pre-consumption quality control process. Complex microbiological processes lead to the loss of freshness and subsequent spoilage. Six hours after death, autolytic decomposition of adenosine triphosphate (ATP, the energy-storing molecule found in cells) leads to xanthine (XA) production. XA concentration increases with storage time, and thus it can be used as an indicator of fish freshness.
Current analytical methods of XA determination that are based on chromatography, colorimetry, spectrophotometry or mass spectrometry are time consuming, expensive and require experienced operators. Electrochemical sensor platforms on the other hand are attractive due to their simple operation, low cost, low sample volume requirements, and speed of analysis. In recent years, enzyme-based electrochemical biosensors, employing xanthine oxidase (XO) for selective recognition and catalysis of XA, have become promising for XA determination in fish and meat samples because of their low detection limits, high selectivity and sensitivity.
The working electrode material is a key factor affecting the analytical performance of electrochemical biosensors, and integration of nanomaterials in the working electrode has previously improved sensor performance. Though carbon based nanomaterials, metal and metal oxide nanostructures that have favorable electrical properties and biocompatibility have been reported for enzymatic biosensor fabrication, mesoporous NiO electrodes are rarely used for electrochemical enzymatic detection of XA. Mesoporous NiO electrodes are ideal for electrochemical biosensor fabrication owing to their large surface area for enzyme immobilization, high isoelectric point, biocompatibility, chemical stability and fast electron transfer features. Moreover, NiO has a redox couple of its own (Ni³⁺/Ni²⁺), paving the way for reagentless biosensing. In this work, we fabricate mesoporous NiO electrodes to immobilize XO, and we demonstrate sensitive and selective amperometric determination of XA. Electrodes were formed using the glancing angle deposition (GLAD) technique, and the large surface area and internal porosity of the GLAD films should allow immobilization of large quantities of the XO enzyme, which combined with the excellent electrical properties of the GLAD electrode allowed amperometric detection of XA at +0.5 V vs Ag/AgCl with excellent sensitivity, detection limit and response time. The sensor was also used to measure XA in a fish sample.
Sensor fabrication and performance
Nanocolumnar and mesoporous NiO films (thickness ~500 nm) were deposited on ITO using GLAD. XO was physisorbed into the pores of activated GLAD NiO electrode by incubating in XO-loaded PBS buffer, pH 7.4. In brief, the NiO electrode was first electrochemically activated using 0.1 N NaOH for 25 cycles at 10 mV/s. 20 µl 0.18 U/mg XO was then immobilized in the activated NiO electrode for 4 hours at room temperature. The modification of the NiO GLAD film with XO was characterized by electrochemistry and FTIR techniques. XO catalyzes the oxidation of XA to uric acid and hydrogen peroxide (H₂O₂), and therefore, using an optimized measurement condition, current was monitored by applying +0.5 V vs Ag/AgCl to the electrode. This current was proportional to the concentration of XA. The amperometric sensor rendered a dynamic range of 60 nM to 3.5 µM, with a limit of detection of 2 nM, limit of quantitation of 6 nM and a sensitivity of 0.01 μA/nM. The sensor showed little interference from common fish sample matrices (such as glucose, uric acid, hypoxanthine) and the common fish preservative (sodium benzoate). The sensor could also be stored in buffer at 4^oC for over a week without losing its performance.

In recent years, nanomaterials (defined as materials < 100 nm in a single dimension) have garnered significant interest for the development of novel applications in the physical and life sciences. This is especially true for luminescent nanoparticles, which have been investigated for the development of sensors, imaging and diagnostic probes. Recently, a relatively new class of luminescent nanomaterials, namely carbon dots, has come to light. Carbon dots, sometimes known as carbongenic dots, are carbon, oxygen, nitrogen and hydrogen containing materials that are typically water dispersible and can be prepared from an abundant number of inexpensive sources including small molecules such as citric acid, amino acids and sugars. Of particular interest are their optical properties, which can be tailored via careful selection of the starting precursors and the desired synthesis route resulting in the ability to generate fluorescence from the blue to the near infrared regions of the spectrum. In addition to their versatile optical properties, these carbon dots are generally known to have low cytotoxicity and good biocompatibility. Combined with their small size and versatile optical properties, developing carbon dots as a nanoplatform can be achieved as these nanodots lend themselves for integration in a myriad of applications most notably in bio-imaging, sensing and drug delivery, among others. Our work focuses on achieving a fundamental understanding of the synthesis of these carbon dots in order to control their size and achieve homogenous surface chemistry and optical properties. We can also endow them with chirality during synthesis opening up novel avenues for chiral sensing applications. As they are fluorescent, we have investigated their development as bimodal imaging probes for optical and magnetic resonance imaging applications. We have also exploited their temperature and pH dependent optical properties in order develop novel tools that can be used for intracellular sensing applications.

The therapeutic index of established nuclear medicine procedures for an aggressive type of cancer such as triple negative breast cancer, (TNBC, lacking estrogen, progesterone receptors, and excess HER2 protein) is typically low due to the lack of targeted therapies and limited stability of radiolabeled carrier molecules in physiological environment. In this study, we developed a potential dual-purpose therapeutic nanocarrier (mesoporous silica particles) by integrating complementary functionalities (folate ligand, DOTA chelator) and payloads (doxorubicine, radionuclides ⁶⁸Ga/¹⁷⁷Lu) into one nanocarrier through covalent conjugation strategies.The nanocarriers, [⁶⁸Ga]Ga-DOTA-FA and [¹⁷⁷Lu]Lu-DOTA-FA demonstrated an outstanding radiochemical yield of > 98 % showing successful incorporation of radionuclides to DOTA cavity. Moreover, radiolabeled nanocarrier exhibited excellent stability in PBS and human serum during 3 h (⁶⁸Ga) and 120 h (¹⁷⁷Lu). In addition, the labeled FA-DOTA@mSiO₂ exhibited a favorable partition coefficient (log P) of -3.29 ± 0.08, suggesting highly hydrophilic carriers. In vitro studies using 3 different TNBC cell lines with FA-DOTA@mSiO₂ carriers demonstrated a significant cell internalization suggesting specific targeting of folate receptor in TNBC cells. Interestingly, FA-capped nanocarrier revealed a significant time dependent cell uptake after labeling with ¹⁷⁷Lu in SUM149PT (33 %) MDA-MB-231 (9%), BT20 (28%) compared to nanocarrier without FA. In Annexin-V based apoptosis studies, combined delivery of ¹⁷⁷Lu radiations and DOX induced significant cell death to TNBC cells earlier at different time points. In ovo studies using fertilized chicken eggs, exhibited higher uptake of dual-purposed nanocarriers (PET imaging). Our data suggests that dual functions nanocarriers has great potential in delivering radiation and drug doses for TNBC tumor treatment and highlight further studies to evaluate its preclinical therapeutic efficacy.

Nanoparticles that block fundamental bacterial functions like quorum sensing could potentially replace conventional antibiotic therapies. Vesicles that bind interfacially to charged biomolecules could be used to block these quorum sensing pathways. Towards this goal, dendronized vesicles (DVs) consisting of polyamidoamine dendron-grafted amphiphiles (PDAs) and dipalmitoyl-sn-glycero-3-phosphocholine lipids are investigated. The molecular dynamics simulation technique in conjunction with an explicit solvent coarse-grained force field is employed. The key physical factors responsible for the stability of DVs as a function of the dendron generation and relative concentration are identified. The threshold concentration of each dendron generation that yields stable DVs is determined. Lower dendron generations rupture the DVs at high relative concentrations due to the electrostatic repulsions between the terminally protonated amines. Whereas, intermediate dendron generations demonstrate a mushroom-to-brush transition. Such conformational changes in the dendrons expand the outer DV surface, resulting in instability in the DV bilayer. DVs encompassing dendrons with higher generations incur stresses on the bilayer due to their high charge density and spontaneous curvature. Minimization of asymmetric stresses across the bilayer are understood by studying the self-organization of PDAs on the DV surface. A conducive set of conditions is determined for the formation of a single cluster of PDAs that decorates the DV surface like a mesh. Results from this study can potentially guide the design and synthesis of nanoparticles which target quorum sensing pathways in bacteria. Beyond applications in prevention of bacterial infections, these nanoparticles can be used in diverse applications, e.g. biomedicine, energy or bioelectronics, that require synthetic dendronized cells or the adsorption and transport of charged species.

Immunotherapies have driven a paradigm shift in the landscape of cancer treatment and currently many immune active drugs are in FDA trial for cancer therapies. Yet, the distribution of many of these drugs remains low in the tumor due to rapid in vivo degradation or low accumulation. Further, which patients are good candidates for immunotherapies remains a challenge incurring high costs of unsuccessful therapies and unnecessary side effects. In this talk I will show the utility of inorganic nanoprobes (gold nanostars) designed in my lab for rapid and noninvasive detection of multiple immunomarkers of cancer to enable patient selection for immunotherapies, as well as response to treatment after immunotherapy. We combined a clinical and pre-clinical imaging technique, positron emission tomography with surface-enhanced Raman spectroscopy (ImmunoPET-SERS) in vivo by labeling gold nanostars with radiolabels, Raman reporter molecules, and targeting antibodies. Multimodal ImmunoPET-SERS seamlessly integrates depth-resolved whole-body imaging and high sensitivity of PET with high spatiotemporal resolution and multiplexing of SERS providing dynamic immunomarkers profiling in vivo. ImmunoPET-SERS was used to determine the immunomarker status of mice treated with combinatorial immunotherapy; we demonstrated real-time feedback of CD8+ infiltration and PD-L1 status in tumors which was confirmed with IHC ex vivo. We are also tracking both CD8+ T cells and Natural Killer (NK) cells in vivo to examine repose to immunotherapies. I will also show the utility of organic nanoprobes (liposomes) for highly effective combinatorial immunotherapies integrating mild hyperthermia and hypoxic drugs with immune checkpoint blockade for enhanced treatment outcome in aggressive breast tumors. In summary, my talk will show nanoscale probes can be engineered to have multifunctional properties from therapeutic response to monitoring treatment efficacy in vivo.

Recent experiments have shown that certain molecular nano-agents can selectively penetrate and aggregate in bacterial lipid membranes, leading to their permeability and rupture, and as such can effectively address the looming crisis on antimicrobial resistance by killing bacteria even in their dormant state - the so-called “persisters”. So far, the discovery of these agents is still mainly restricted to empirical screening with limited physical understanding. To help reveal and understand the underlying mechanisms, here we establish a theory to show that the deformation energy of the membrane tends to limit the growth of molecular domains on a lipid membrane, resulting in a characteristic domain size, and that the domain aggregation significantly reduces the energy barrier to pore growth. Coarse-grained molecular dynamics simulations are performed to validate such domain aggregation and associated pore formation. This study sheds light on how lipid membranes can be damaged through molecular domain aggregation and contributes to establish a theoretical foundation for the next-generation membrane-targeting nanomedicine to effectively target bacteria with high antimicrobial resistance against conventional antibiotics.

Since the DoE funded TEAM project ended in the United States with the development of spherical and chromatic aberration correction in 2009 single atom sensitivity is established for transmission electron microscopy (TEM) imaging. Theoretically, every material could be imaged in full atomic resolution, and every atom could be resolved within all material structures. In an experimental setup, however, electron beam sensitivity of a given material determines which original structures can be resolved in reality. Therefore, our focus in the last decade has been on minimizing electron beam induced material damage, and to find alternative ways to image beam sensitive materials without altering its original structure. At least, if a material is altered under the electron beam the dynamics of this behavior should be recorded, too. Such dynamic considerations led to the development of current-controlled TEM.

Low dose rate and pulsed electron beams were applied to image pristine properties of MgCl₂, a Ziegler-Natta catalyst which is both air and electron beam sensitive. The particular choice of pulse and delay times allowed to mitigate damage caused by phonon accumulation. The total electron dose can be extended more than ten times before material alterations set in. Changing electron currents while imaging polymers or various catalytic nano-crystals by varying dose rates and / or the irradiated areas had similar effects. Using direct electron detectors facilitated those experiments, and allowed for the use of ultra-low currents where single electrons are delivered to interact with the material under investigation. We find that the beam current limits material alterations in the ultra-low current regime rather than the deposition of electron charge.

With the application of current-controlled TEM the onset of material changes is found to be generally delayed. The experimental setup may be used in combination with cryo-microscopy, and could further postpone material alterations, even in soft materials.

Work at the Molecular Foundry was supported by the Office of Science, Office of Basic Energy Sciences, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.

Surface plasmon resonance (SPR) is characteristic of strong light harvesting, enhanced localized electromagnetic filed and tunable energy donation. As a result, SPR has a great potential in biosensing, drug delivery and theranostics. In this work, SPR has been used to develop new fluorescence and surface-enhanced Raman scattering (SERS) sensors. In particular, SPR enables sensitive detection of analytes in human fluidic samples in the biological transparency window in the near-infrared spectral range. In addition, SPR can be incorporated with inorganic semiconductors to create new stable agents for photodynamic therapy.

The rapid and efficient purification of lysosomes is the key to uncover the mechanistic insights of lysosomal activity using proteomics analysis. The traditional method for isolating lysosomes such as the density-gradient ultracentrifugation technique often takes a long time and the intact structure of lysosomes is not preserved.^[1] Therefore, it may lead to a change of protein composition in labile lysosomes as well as detach and/or denature of membrane proteins on it. Recently, the nanoparticle-based fractionation method which uses superparamagnetic iron oxide nanoparticles (SPIONs) for targeting lysosomes through the endocytosis pathway has been used for the isolation of lysosomes to identify disease-related alternation in protein composition.^[2] The versatility of SPIONs together with the quick and gentle nature of magnetic isolation are advantages of this technique. However, to obtain a highly-purified lysosomal fraction with high yield, it is important to determine the appropriate timing for performing magnetic separation, since the time-dependent localization of SPIONs in different organelles (i.e. early endosome, late endosome, and lysosome) depends on cell type, loading condition, incubation time, etc. To screen localization of SPIONs in a large number of cells, they are often labeled with the fluorescent dye to be monitored by fluorescent microscopy. However, besides the photobleaching problem, dye labeling of nanoparticles could have dye-leaking or influence on nanoparticle uptakes.^[3] Therefore, to further sophisticate the SPIONs-based isolation technique, it has been demanded to develop a new type of magnetic probes with intrinsic imaging capability to both precisely visualize the intracellular transport and quickly obtain high purified isolation fraction.
Herein, to satisfy those requirements, we have developed magnetic-plasmonic Ag/FeCo/Ag core/shell/shell nanoparticles (MPNPs) for lysosome isolation. Since dextran has been well-known as a fluid phase cargo, which is internalized via pinocytosis rather than receptor-mediated endocytosis and eventually delivered to lysosomes. In this study, the MPNPs were encapsulated in phospholipid micelles followed by conjugation with amino dextran (aDxt). The hydrodynamic size of aDxt-conjugated MPNPs (aDxt-MPNPs) was ~50 nm at pH in the range of 4~10, which is suitable for efficient cellular uptake through various pathways. Owing to plasmonic property, the intracellular trafficking of aDxt-MPNPs could be simply visualized by confocal laser scanning microscopy (CLSM). After determining the intracellular fate of aDxt-MPNPs, lysosomes were quickly isolated using a MACS separator (Miltenyi Biotec). The purity and the intactness of the lysosome fraction were confirmed by western blot and CLSM images. Furthermore, we will discuss the influence of the elapsed time from homogenization to the magnetic separation on the protein composition in lysosomes.

References:
B. D. Beaumelle et al., J. Cell. Biol. 111, 1811-1823 (1990).
A. K. Tharkeshwar et al., Sci. Rep. 7, 41408 (2017).
S. Snipstad et al., Cytometry A 91, 760-766 (2017).

In biomedical research, metal-organic-frameworks (MOF) have attracted increasing interest over the past decade. MOFs hold promises as drug delivery agents, radioenhancers and application in theranostic and personalized medicine. Zeolite-imidazole-framework-8 nanoparticles (ZIF-8) are amongst the most extensively studied MOFs featuring anti-inflammatory and anti-microbial properties when doped with ceria.^1,2 Alternatively, hafnium-based MOFs have been shown to enhance radiation therapy damage in tumor cells via reactive oxygen species creation.³ However, clinical translation for such novel materials remains challenging and tight control over nanomaterial chemistry during nanomedical life-cycle is needed. While stability and dissolution have been studied in the past for several MOFs, a detailed understanding of the intracellular behaviour and potential phase-transformation and/or generation of secondary nanomaterials is missing. Compared to their metal oxide counterparts, MOFs typically exhibit higher instability, making them prone to degradation/biotransformation. For biomedical applications, the fate of these new materials is, next to efficacy and toxicity, a major determinant of clinical success.

To overcome the gap between material engineering and medical application, and enable more rationalized safe-by-design material development, we investigated the environment-dependent biochemical-transformation of different nano-particulate MOFs containing essential (Zn, Fe) and non-essential (Hf, Ce) metal ions in physiologically relevant liquids and in-vitro systems. We present biotransformation kinetics of a biomedically relevant set of MOFs, including findings of particle transformation with changes in elemental distribution and morphological appearance with potential formation of nanomaterial from degradation products. Taken together, our nano-analytical characterization approach offers a route to follow the biological fate of MOF-based nanomedicines.

References
[1] Feng S, Zhang X, Shi D, Wang Z. Front Chem Sci Eng. 15 (2020) 221; [2] Li X, Qi M, Li C, et al. J Mater Chem B. 44 (2019) 6955; [3] Ni K, Lan G, Chan C, et al. Nat Commun. 1 (2018) 2351

To improve the efficiency of current medical practices, more significant data from technology is needed. One lever to optimize medical decision time is to shorten the delay from biological test to results. In the field of fast biological tests, biosensors have received increasing attention over the last decades and several technologies have been developed to deliver portable, cheap and sensitive analytical systems. Nevertheless, two main obstacles prevent the marketing of these biosensors: 1) an accurate analysis of the market in terms of real needs for medical practices and 2) perfect matching with current medical recommendations We identify several obstacles that prevent the marketing of biosensors today: 1/ prior precise study of the market (needs for medical practices that fit perfectly into the current recommendations); 2/ technological realization that can meet this need; 3/ Knowledge of standards and validation and calibration procedures. Our study is divided into 5 phases: 1/ Determination of the use case(s) and the biomarkers of interest with an international opinion survey of physicians from all specialties; 2/ Design of a laboratory prototype; 3/ Manufacturing of a more advanced prototype suitable for the first steps of clinical validation phase; 4/ Interconnection with artificial intelligence and biological gold standard; 5/ Multicentric study for validation as a medical device. For this purpose, we have assembled an interdisciplinary team with medical doctors, engineers, and experts on market validation with the goal of identifying the most promising use cases in human medicine and creation of the most suitable biosensors anchored in medical practices.
In this work, we first determine the use case(s) and the biomarkers of interest through an international opinion survey conducted among physicians from a broad range of specialties. We distributed a questionnaire via our networks and professional social networks for a voluntary survey of physicians about existing needs for rapid biological diagnosis and what this might change in their daily practice. More than 200 physicians from 10 different countries and 19 different medical specialty responded to our survey. 48,5% of participants think that it would save medical time (fluidity in the hospital environment, saving time in ambulatory care), 55,5% think it could improve their medical practice (guide a course of action, help a therapeutic decision, avoid unnecessary treatment) and 31,5% think that this could lead to faster and easier patient referrals.
As a result, the main biomarkers of interest identified by our panel are troponin (50%), PCR (30,5%), d-dimères (26,5%), BNP or NT-proBNP (12,5%), and respiratory viruses (9,5%).
Our study is based on the creation of biosensors, in a trans and interdisciplinary manner, so that they can be perfectly integrated into daily medical practice and respect all the issues at stake, in terms of diagnostic sensitivity, cost/efficiency and service to the patient. The biosensors are based on AlGaN/GaN High Electron Mobility Transistors (HEMTs). GaN-based HEMTs have demonstrated high sensitivity as detectors, sensors and biosensors and have the advantage of extreme chemical stability and are biocompatible. Furthermore, the basic technology is widely commercialized and mature, making for rapid industrial upscaling
In this presentation, we will describe the interest of the various biomarkers that were identified in our study and we will compare medical practices, literature, recommendations of good practices and technological possibilities, in order to define the best use case for biosensors.
This study is of importance in terms of future public health, making it possible to provide the medical community and the patient with one or more relevant tools for the fluidity of care.

Dental implant failures arise from poor integration of the implant with surrounding soft and hard tissues. Osseointegration and periointegration are crucial to their prevention. A persistent challenge is the inconsistency between tissue surrounding an implant and natural periodontal tissue around natural teeth. This increases vulnerability to peri-implant diseases, affecting soft and hard tissue integration and highlighting the need for implants with improved osseointegration and periointegration potential. Implant surface nanostructures promote osseointegration; however, designs which improve the soft tissue response have yet to be identified. We present novel nanoparticle coatings applied to surfaces in a single step technique driven by liquid-feed flame spray pyrolysis, which uniquely permits the construction of bespoke nanoparticle architectures from a breadth of inorganic precursors. This highly scalable technique has the potential to create multi-layer nanostructures of differing thicknesses, diverse patterns, and pore sizes – qualities as diverse as the inorganic building blocks of which these nanoparticle layers are composed. Novel nanoparticle-based coatings with bioactive inorganic architectures can enhance osseointegration, reduce the inflammatory response, and possess exemplary process versatility and stability, permitting precise control over the synthesis process, and so too, evaluation of the concomitant soft tissue bioresponse.

Drug delivery technologies have been quickly innovated in the last few decades, especially in aspect of combination with nanotechnology to advance into multifunctional drug carriers, which can accomplish targeting a tumor, delivering therapeutic molecules, imaging and monitoring drug response. Many models were built and tested; still the challenge is at the choice of the appropriate materials and the facilitation of carrier preparation to achieve both safety and efficacy. Reflecting such needs, silk protein was chosen to provide a biodegradable and biocompatible matrix for carrying drug molecules. Such system with controllable structures and compositions can be an advanced type of drug carriers and further contribute to the improvement of cancer therapy.
Silk protein originated from Bombyx mori silkworm was dissolved in various types of aqueous solution, including LiBr [1], CaCl₂/Ethanol [2], and formic acid solutions. In this research, the fabrication of silk NPs from the obtained silk protein solution was carefully monitored to achieve various morphologies and structures, through the reprecipitation method. Silk NPs were obtained in narrow size distribution, with the average diameter can be tuned from 10 to 100 nm. The surface charges of as-synthesized silk nanoparticles can be either negative or positive, depending on preparation conditions, brings out the possibility of loading different types of drugs. The obtained silk NPs showed good drug uptakes, adequate stability in aqueous and almost no toxicity at cellular level. The research also provides a NP library with diverse structures and properties, which can be used to manipulate the drug delivery performance. The results are discussed in terms of UV-Vis, DLS, XRD, TEM, XPS, and CLSM.
[Acknowledgement] This work was supported by JSPS KAKENHI for Early-Career Scientists Grant Number JP19K15388.
[References] [1] A. Matsumoto J. Phys. Chem. B 110 (2006) 21630; [2] Anh T.N. Dao et al. Polym. Degrad. Stab. 153 (2018) 37.

Gallium oxide nanoparticles (GaO-(OH)) have been proposed for biomedical applications such as diagnostics and therapeutic approaches, like cancer treatments. Understanding its interaction with biological components, like proteins, is an important factor for the development of new nanomaterials. Gallium oxide nanoparticles conjugated with β-cyclodextrin (GaO-CD) were synthesized using a wet-chemical route, characterized by molecular spectroscopic and imaging techniques, and further used to probe equilibrium interactions with human serum albumin (HSA). In this work, we have used the sensitivity of the single tryptophan (Trp) fluorescence of HSA to monitor the interaction between GaO-CD and the protein at different temperatures. The binding and quenching constants were determined using Stern-Volmer analysis. Also, the thermal stability of the complex was followed by fluorescence spectroscopy using a simple two-state model. A weak interaction of HSA with GaO-CD, relative to other nanomaterials, like gold nanoparticles, was observed. In addition, results show no change in temperature of unfolding (T_m) of the protein, implying that GaO-CD does not affect HSA tertiary structure.

The remarkable optomagnetic properties of the rare-earths (RE) make RE-based materials ideal for biomedical applications, including diagnostic (e.g., imaging, nanothermometry) and therapeutic (e.g., drug delivery, photodynamic therapy) approaches. This is due the unique electronic properties of the f-elements allowing for upconversion and near-infrared emission under near-infrared excitation as well as high magnetic moments.
Yet, challenges remain; low emission intensity and efficiency of small nanoparticles (NPs), and reliable, fast synthesis routes. As material chemists, we tackle these challenges with new designs of RE-NPs by chemically controlled synthesis, application-oriented surface chemistry, and understanding of structure-property-relationships. Sodium rare-earth fluorides (NaREF₄) are our favorite materials, and we developed a fast and reliable microwave-assisted synthesis approach allowing crystalline phase and size control in the sub 15nm realm. Such control is crucial for the understanding of fundamental structure-property relationships and to optimize their optical and magnetic properties, when aiming for the design of next-generation optical probes or contrast agents for magnetic resonance imaging. For instance, NaGdF₄ NPs are gaining interest as alternative MRI contrast agent, while co-doping with RE³⁺ ions renders them excellent candidates for photoluminescent optical probes. The hexagonal crystalline phase of NaGdF₄ is known as the more efficient host material for upconversion emission, yet interestingly, it was found that its cubic counterpart shows superior performance as MRI contrast agent. Having a fast and reliable synthesis route towards NaREF₄ NPs on hand, we now explore various nanoparticle architectures and compositions with the goal to optimize their optomagnetic properties, ultimately resulting in the design of biocompatible multimodal bioprobes.
This presentation will shed light on recent results and remaining challenges in the field of RE-based nanostructures with respect to their microwave-assisted synthesis as well as structural and optomagnetic properties, seeking biomedical application, while also touching on hyperspectral imaging as an emerging analytical tool offering spatio-spectral information about RE-based materials.

Magnetic separation offers an elegant and efficient route to selectively capture and remove specific compounds from blood (or other body fluids), which are inaccessible to current blood cleansing technologies, such as dialysis and hemosorption [1]. However, currently available superparamagnetic iron oxide nanoparticles (SPIONs) impose significant performance limits arising from their physical properties. Their low saturation magnetization coming from their oxidic nature results in a poor magnetic response, which severely limits performance.
The development of disk-shaped nanoparticles fabricated by lithographic methods and thin film deposition enables a precise control of size, geometry, material composition and magnetic properties such as interlayer exchange coupling and magnetic anisotropies.
In this work, we develop magnetic thin film systems for synthetic antiferromagnetic (SAF) coupled disk shaped particles with tunable in-plane anisotropies. The antiferromagnetic coupling is obtained by the stray field interaction of the two ferromagnetic layers. These SAF particles are designed to show zero net-magnetization in absence of a magnetic field – hence allowing perfect colloidal stability. Upon application of a magnetic field overcoming the antiferromagnetic coupling, a high net-magnetization can then be obtained, which translates into a large magnetic moment and magnetic force, acting on the particles in a gradient field allowing efficient capturing.
In order to synthesize such synthetic antiferromagnets, we evaluated strategies to obtain the required magnetic anisotropies. We report results obtained by deposition of soft magnetic CoFeB layers on Ta seed layers grown by oblique sputtering and compare the resulting anisotropies to those obtained by deposition of the magnetic layer in an applied field. Moreover, for the CoFeB layers on the oblique-sputtered seeds we compare the anisotropies obtained for different CoFeB-thicknesses to distinguish between a surface or interface induced anisotropy or a bulk anisotropy term. While with oblique sputtering, a wide range of in-plane anisotropies can be achieved (2kJ/m3 – 25kJ/m3), the anisotropy remains much smaller when CoFeB films are deposited in field(1.35kJ/m3).
Our work shows that in-plane anisotropies in magnetically soft systems can be tuned over a wide range by oblique seed-layer depositions or in-field depositions. These thin film systems open up the path for a new kind of magnetic disk shaped nanoparticles for prospective use in magnetic blood purification.

[1] Anthis AHC, Matter MT, Keevend K, Gerken LRH, Scheiber S, Doswald S, Gogos A, Herrmann IK, Tailoring the Colloidal Stability, Magnetic Separability, and Cytocompatibility of High-Capacity Magnetic Anion Exchangers, ACS Applied Materials and Interfaces, 2019.

Gene and gene editing therapies have been widely investigated for the treatment of inherited or acquired genetic diseases. Efficient delivery of therapeutic agents has become a significant barrier in clinical applications due to the toxicity and instability of the vectors in the complex intracellular environment. Among non-viral vectors, individual inorganic nanoparticles (NPs) have become a popular strategy for nucleic acid delivery. However, the nanoshell geometry of viruses that creates a compartment to protect the cargo from its environment is still advantageous. Recreation of these compartments’ formation has both technological and fundamental importance. In this study, we show that L-cysteine stabilized iron sulfide NPs can self-assemble into multi-compartment supraparticles (SPs) with nanoshell geometry. The transmission electron microscopy results showed that constituent NPs initially form ~55nm cup-like structures and then mature into positively charged ~75nm SPs with ca.190 interconnected compartments. The results of selected area electron diffraction and energy dispersive X-ray spectroscopy confirm SPs are formed from FeS₂ and Fe₂O₃ NPs. Elemental mapping showed that Fe₂O₃ NPs are located on the surface of the SP and protecting FeS₂ and compartments from the surroundings. DNA was loaded in the compartments during the formation of supraparticles. We tested these complexes in circular dichroism, UV-Vis spectroscopy, electrophoretic mobility shift, and protection assays. Our results show successful DNA encapsulation in compartments. These pDNA-SP complex protect DNA against degradation, penetrate through cellular membranes and facilitate endolysosomal escape. Therefore, the development of these particles can be used as an effective cargo delivery tool for gene and gene-editing therapies.

Bulk mixed halides constitute an important class of optical materials used as detection and storage phosphors in medicine. However, the potential of their nanocrystalline counterparts in biophotonic applications such as imaging and sensing is largely unexplored. In this talk I will highlight the potential of mixed-halide nanocrystals as luminescent thermometers capable of operating in a broad temperature range, from cryogenic to physiological temperatures. First, I will describe the chemistry that enables the synthesis of size- and shape-controlled mixed-halide nanocrystals with sizes ranging from 20 to 100 nm. Then, I will show how controlled amounts of rare-earth ions may be incorporated into these nanocrystals to generate a family of color-tunable NIR-to-visible upconverters. Finally, I will demonstrate that rare-earth-doped mixed halide nanocrystals serve as optical temperature sensors in the 100–400 K range. These luminescent thermometers can be excited within the first biological window.