Plasmonic gold bipyramids (AuBipy) encapsulated within a NU-1000 metal-organic framework (MOF) create robust core-shell nanocomposites. These porous nanoassemblies combine the near-infrared (NIR) plasmonic resonance of the metallic core with the intrinsic photoluminescence of the pyrene-based organic linkers. Comprehensive optical characterization reveals efficient photothermal heating under NIR irradiation and robust surface-enhanced Raman scattering (SERS) capabilities in colloidal dispersions. For biological applications, AuBipy@NU-1000 nanocomposites exhibit good colloidal stability and maintain high cell viability in A549 cells up to a 200 pM particle concentration (equivalent to 152 µM of Au and 302 µM of Zr). Wide-field hyperspectral microscopy enables label-free intracellular mapping of the distinct optical signatures of both components after cellular internalization. Spatial analysis of these signals supports preservation of the core-shell architecture inside cells. Consequently, these nanomaterials provide a versatile platform for intracellular optical readout, with additional potential for chemical sensing and photothermal applications.

Background: Recombinant tissue plasminogen activator (rtPA) remains the standard thrombolytic treatment for ischemic stroke. Different types of nanoparticles have emerged as promising tools to improve the benefits and decrease the drawbacks of this therapy. Among them, cell membrane-derived (CMD) nanomedicines have gained special interest due to their capability to increase the half-life of particles in blood, biocompatibility, and thrombus targeting. In order to update and evaluate the efficacy of these nanosystems, we performed a meta-analysis of the selected in vivo preclinical studies. Methods: Preclinical in vivo studies in ischemic stroke models have been identified through a search in the Pubmed database. We included studies of rtPA-nanoparticles, which assessed infarct volume and/or neurological improvement. Nanosystems were compared with free (non-encapsulated) rtPA treatment. Standardized mean differences were computed and pooled to estimate effect sizes for lesion volumes and neurological scores. Subgroup analyses by the risk of bias, type of nanoparticle, and time of administration were also performed. Results: A total of 18 publications were included in the meta-analysis. This was based on defined search inclusion criteria. Our analysis revealed that rtPA-nanoparticles improved both lesion volume and neurological scores compared with the free rtPA treatment. Moreover, CMD nanomedicines showed better evolution of infarct volume compared to the other nanoparticles. Funnel plots of lesion volume exhibited asymmetry and publication bias. Heterogeneity was generally high, and the funnel plot and Egger test showed some evidence of publication bias that did not achieve statistical significance in the trim-and-fill analysis. Conclusions: rtPA-encapsulating nanosystems were shown to decrease infarct volume and improve neurological scales compared to the standard treatment, and CMD nanomedicines had the greatest beneficial effect.

Metal nanoparticles are established tools for biomedical applications due to their unique optical properties, primarily attributed to localized surface plasmon resonances. They show distinct optical characteristics, such as high extinction cross-sections and resonances at specific wavelengths, which are tunable across the wavelength spectrum by modifying the nanoparticle geometry. These attributes make metal nanoparticles highly valuable for sensing and imaging in biology and medicine. However, their widespread adoption is hindered due to challenges in consistent and accurate nanoparticle fabrication and functionality as well as due to nanotoxicological concerns, including cell damage, DNA damage, and unregulated cell signaling. In this study, we present a fabrication approach using nanoimprint lithography in combination with thin film deposition which yields highly homogenous nanoparticles in size, shape and optical properties with standard deviations of the main geometry parameters of less than 5% batch-to-batch variation. The measured optical properties closely match performed simulations, indicating that pre-experimental modelling can effectively guide the design of nanoparticles with tailored optical properties. Our approach also enables nanoparticle transfer to solution. Particularly, we show that the surface coating with a PEG polymer shell ensures stable dispersions in buffer solutions and complex cell media for at least 7 days. Furthermore, our in vitro experiments demonstrate that these nanoparticles are internalized by cells via endocytosis, exhibit good biocompatibility, and show minor cytotoxicity, as evidenced by high cell viability. In the future, our high-precision nanoparticle fabrication method together with tunable surface plasmon resonance and reduced nanotoxicity will offer the possibility to replace conventional nanomaterials for biomedical applications that make use of an optical response at precise wavelengths. This includes the use of the nanoparticles as contrast agents for imaging, as probes for targeted photothermal cancer therapy, as carriers for controlled drug delivery, or as probes for sensing applications based on optical detection principles.

Nanosized microporous metal-organic-frameworks (NMOFs) serve as versatile drug delivery systems capable of navigating complex microenvironments and interacting with cells in specific tissues. The physicochemical properties of NMOFs, such as size, composition, porosity, colloidal stability, and external surface functionalization are essential for their success as efficient carriers. This study introduces a flexible, clickable coating using an amphiphilic polymer derivatized with dibenzo cyclooctyne groups as a universal, postsynthetic functionalization tool. To prove its universality, nanosized MOFs with different structure and composition (UiO-67, NU-1000, PCN-222, and ZIF-8) were produced with high monodispersity and were coated with a clickable, amphiphilic polymer. The resulting polymer-coated NMOFs display exceptional colloidal and structural stability in different biologically relevant media. For comparative purposes, we selected two size-equivalent NMOFs, ZIF-8 and UiO-67, which were functionalized with a library of biologically relevant azide-derivatized (macro)molecules, including poly(ethylene glycol), mannose, and a dynein-binding cell-penetrating peptide, using a bioorthogonal reaction. The choice of ZIF-8 and UiO-67, both 150 nm in size but with distinct coordination and surface chemistries, is pivotal due to their differing acid and base stability characteristics, which may potentially influence their performance in cellular environments. To track their performance in vitro, the NMOFs were loaded with cresyl violet, a common histological stain and lysosomal marker. Cellular internalization of the surface-functionalized NMOFs was markedly governed by their distinct (macro)molecule characteristics. This demonstrates that surface properties critically influence uptake efficiency, while also highlighting the versatility and effectiveness of the proposed coating strategy. In particular, the one functionalized with the dynein-binding peptide demonstrated a markedly higher rate of cellular internalization compared to other NMOFs. In contrast, derivatizations with mannose and poly(ethylene glycol) are associated with a substantial reduction in cellular uptake, suggesting stealth behavior. These results provide a bioorthogonal and versatile alternative for the external surface engineering of NMOFs, aiming to improve targeted drug delivery effectiveness.

This investigation demonstrates the development and functionality of cell membrane-cloaked UiO-67 nanosized metal-organic frameworks (NMOFs), which are engineered for precise intracellular delivery of encapsulated cargoes. Utilizing the robust and porous nature of UiO-67, we enveloped these NMOFs with fusogenic cell membrane-derived nanovesicles (FCSMs) sourced from adenocarcinomic human alveolar basal epithelial (A549) cells. This biomimetic coating enhances biocompatibility and leverages the homotypic targeting capabilities of the cell-derived coatings, facilitating direct cytoplasmic delivery and avoiding endolysosomal entrapment. Our nanoplatform exhibited high efficiency in loading and releasing two model cargoes: cresyl violet (CV), a staining agent, and carboplatin (CbPt), a chemotherapeutic agent. This approach demonstrated substantial potential in drug delivery in both 2D cell cultures and 3D spheroids. This study underscores the enhanced cellular uptake facilitated by the engineered biomimetic shell and highlights the system's capacity for sustained release, offering a promising strategy for targeted drug delivery.

Background Intravenous administration of fibrinolytic drugs, such as recombinant tissue plasminogen activator (rtPA) is the standard treatment of acute thrombotic diseases. However, current fibrinolytics exhibit limited clinical efficacy because of their short plasma half-lives and risk of hemorrhagic transformations. Platelet membrane-based nanocarriers have received increasing attention for ischemic stroke therapies, as they have natural thrombus-targeting activity, can prolong half-life of the fibrinolytic therapy, and reduce side effects. In this study we have gone further in developing platelet-derived nanocarriers (defined as cellsomes) to encapsulate and protect rtPA from degradation. Following lyophilization and characterization, their formulation properties, biocompatibility, therapeutic effect, and risk of hemorrhages were later investigated in a thromboembolic model of stroke in mice. Results Cellsomes of 200 nm size and loaded with rtPA were generated from membrane fragments of human platelets. The lyophilization process did not influence the nanocarrier size distribution, morphology, and colloidal stability conferring particle preservation and long-term storage. Encapsulated rtPA in cellsomes and administered as a single bolus showed to be as effective as a continuous clinical perfusion of free rtPA at equal concentration, without increasing the risk of hemorrhagic transformations or provoking an inflammatory response. Conclusions This study provides evidence for the safe and effective use of lyophilized biomimetic platelet-derived nanomedicine for precise thrombolytic treatment of acute ischemic stroke. In addition, this new nanoformulation could simplify the clinical use of rtPA as a single bolus, being easier and less time-consuming in an emergency setting than a treatment perfusion, particularly in stroke patients. We have successfully addressed one of the main barriers to drug application and commercialization, the long-term storage of nanomedicines, overcoming the potential chemical and physical instabilities of nanomedicines when stored in an aqueous buffer.

The blood enzyme glutamate-oxaloacetate transaminase (GOT) has been postulated as an effective therapeutic to protect the brain during stroke. To demonstrate its potential clinical utility, a new human recombinant form of GOT (rGOT) was produced for medical use. We tested the pharmacokinetics and evaluated the protective efficacy of rGOT in rodent and non-human primate models that reflected clinical stroke conditions. We found that continuous intravenous administration of rGOT within the first 8 h after ischemic onset significantly reduced the infarct size in both severe (30%) and mild lesions (48%). Cerebrospinal fluid and proteomics analysis, in combination with positron emission tomography imaging, indicated that rGOT can reach the brain and induce cytoprotective autophagy and induce local protection by alleviating neuronal apoptosis. Our results suggest that rGOT can be safely used immediately in patients suspected of having a stroke. This study requires further validation in clinical stroke populations.

Nanoscale metal–organic frameworks (NMOFs) exhibit unique properties for drug delivery, including ultrahigh storage capabilities, biocompatibility, biodegradability, and sustained release of encapsulated cargo. However, due to their localized electronic states, MOFs are nonresponsive to external stimuli such as light or magnetic fields. This study investigates the integration of light‐responsive nanoantennas into NMOFs to enhance their application as smart drug delivery nanosystems. By integrating gold bipyramid nanoantennas within ZIF‐8 and NU‐1000 NMOFs, core@shell nanosystems are created with photothermal capabilities. Utilizing cresyl violet as a model drug, the loading and release dynamics of these nanosystems are analyzed, demonstrating controlled drug release under near‐infrared (NIR) light stimulation. Photothermal release studies conducted in living cells reveal the potential of these nanocomposites for spatiotemporal targeted, light‐activated drug delivery. Further evaluation of the NU‐1000 nanocomposite loaded with chemotherapeutics—doxorubicin, carboplatin, and oxaliplatin—in both 2D and 3D cell cultures shows the nanosystem effectiveness in cell internalization and therapeutic NIR activation. The findings demonstrate that the incorporation of stimuli‐responsive elements into NMOFs offers a promising approach for developing advanced drug delivery platforms.

While stroke represents one of the main causes of death worldwide, available effective drug treatment options remain limited to classic thrombolysis with recombinant tissue plasminogen activator (rtPA) for arterial-clot occlusion. Following stroke, multiple pathways become engaged in producing a vicious proinflammatory cycle through the release of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1) and heat shock protein 70 kDa (HSP72). HMGB1, in particular, can activate proinflammatory cytokine production when acetylated (AcHMGB1), a form that prefers cytosolic localization and extracellular release. This study aimed at determining how HMGB1 and HSP72 are modulated and affected following treatment with the anti-inflammatory compound resveratrol and novel platelet membrane-derived nanocarriers loaded with rtPA (CSM@rtPA) recently developed by our group for ischemic artery recanalization. Under ischemic conditions of oxygen-glucose deprivation (OGD), nuclear abundance of HMGB1 and AcHMGB1 in microglia and macrophages decreased, whereas treatment with CSM@rtPA did not alter nuclear or cytosolic abundance. Resveratrol treatment markedly increased the cytosolic abundance of HSP72 in microglia. Using proximity ligation assays, we determined that HSP72 interacted with HMGB1 and with acetylated HMGB1. The interaction was differentially affected under the OGD conditions. Resveratrol treatment under the OGD further decreased HSP72-HMGB1 interactions, whereas, in contrast, treatment increased HSP72-AcHMGB1 interactions in microglia. This study points out a salient molecular interaction suited for a two-pronged nanotherapeutic intervention in stroke: enhancement of rtPA's thrombolytic activity and modulation of cytosolic interactions between HMGB1 and HSP72 by resveratrol.

Metal-organic frameworks (MOFs) are extremely versatile materials, which serve to create platforms with exceptional porosity and specific reactivities. The production of MOFs at the nanoscale (NMOFs) offers the possibility of creating innovative materials for bioapplications as long as they maintain the properties of their larger counterparts. Due to their inherent chemical versatility, synthetic methods to produce them at the nanoscale can be combined with inorganic nanoparticles (NPs) to create nanocomposites (NCs) with one-of-a-kind features. These systems can be remotely controlled and can catalyze abiotic reactions in living cells, which have the potential to stimulate further research on these nanocomposites as tools for advanced therapies.

A surface-engineered cell-derived nanocarrier was developed for efficient cytosolic delivery of encapsulated biologically active molecules inside living cells. Thus, a combination of aromatic-labeled and cationic lipids, instrumental in providing fusogenic properties, was intercalated into the biomimetic shell of self-assembled nanocarriers formed from cell membrane extracts. The nanocarriers were loaded, as a proof of concept, with either bisbenzimide molecules, a fluorescently labeled dextran polymer, the bicyclic heptapeptide phalloidin, fluorescently labeled polystyrene nanoparticles or a ribonucleoprotein complex (Cas9/sgRNA). The demonstrated nanocarrieŕs fusogenic behavior relies on the fusogen-like properties imparted by the intercalated exogenous lipids, which allows for circumventing lysosomal storage, thereby leading to efficient delivery into the cytosolic milieu where cargo regains function.

The engagement with the immune system is one of the main cornerstones in the development of nanotechnologies for therapy and diagnostics. Recent advances have made possible the tuning of features like size, shape and biomolecular modifications that influence such interactions, however, the capabilities for immune modulation of nanoparticles are still not well defined and exploited. This review focuses on recent advances made in preclinical research for the application of nanoparticles to modulate immune responses, and the main features making them relevant for such applications. We review and discuss newest evidence in the field, which include in vivo experiments with an extensive physicochemical characterization as well as detailed study of the induced immune response. We emphasize the need of incorporating knowledge about immune response development and regulation in the design and application of nanoparticles, including the effect by parameters such as the administration route and the differential interactions with immune subsets.

Abstract Background Ischemic stroke is the most common cerebrovascular disease and is caused by interruption of blood supply to the brain. To date, recombinant tissue plasminogen activator (rtPA) has been the main pharmacological treatment in the acute phase. However, this treatment has some drawbacks, such as a short half-life, low reperfusion rate, risk of hemorrhagic transformations, and neurotoxic effects. To overcome the limitations of rtPA and improve its effectiveness, we recently designed sonosensitive sub-micrometric capsules (SCs) loaded with rtPA with a size of approximately 600 nm, synthesized using the layer-by-layer (LbL) technique, and coated with gelatine for clot targeting. In this study, we evaluated the rtPA release of ultrasound (US)-responsive SCs in healthy mice and the therapeutic effect in a thromboembolic stroke model. Results In healthy mice, SCs loaded with rtPA 1 mg/kg responded properly to external US exposure, extending the half-life of the drug in the blood stream more than the group treated with free rtPA solution. The gelatine coating also contributed to stabilizing the encapsulation and maintaining the response to US. When the same particles were administered in the stroke model, these SCs appeared to aggregate in the ischemic brain region, probably generating secondary embolisms and limiting the thrombolytic effect of rtPA. Despite the promising results of these thrombolytic particles, at least under the dose and size conditions used in this study, the administration of these capsules represents a risk factor for stroke. Conclusions This is the first study to report the aggregation risk of a drug carrier in neurological pathologies such as stroke. Biocompatibility analysis related to the use of nano-and microparticles should be deeply studied to anticipate the limitations and orientate the design of new nanoparticles for translation to humans. Graphical Abstract

The synthesis of nanosized metal-organic frameworks (NMOFs) is requisite for their application as injectable drug delivery systems (DDSs) and other biorelevant purposes. Herein, we have critically examined the role of different synthetic parameters leading to the production of UiO-66 crystals smaller than 100 nm. Of note, we demonstrate the co-modulator role conferred by halide ions, not only to produce NMOFs with precise morphology and size, but also to significantly improve the reaction yield. The resulting NMOFs are highly crystalline and exhibit sustained colloidal stability in different biologically relevant media. As a proof of concept, these NMOFs were loaded with Rhodamine 6G (R6G), which remained trapped in most common biologically relevant media. When incubated with living mammalian cells, the R6G-loaded NMOFs were efficiently internalized and did not impair cell viability even at relatively high doses.

Nanoparticles have now long demonstrated capabilities that make them attractive to use in biology and medicine. Some of them, such as lipid nanoparticles (SARS-CoV-2 vaccines) or metallic nanoparticles (contrast agents) are already approved for their use in the clinic. However, considering the constantly growing body of different formulations and the huge research around nanomaterials the number of candidates reaching clinical trials or being commercialized is minimal. The reasons behind being related to the “synthetic” and “foreign” character of their surface. Typically, nanomaterials aiming to develop a function or deliver a cargo locally, fail by showing strong off-target accumulation and generation of adverse responses, which is connected to their strong recognition by immune phagocytes primarily. Therefore, rendering in negligible numbers of nanoparticles developing their intended function. While a wide range of coatings has been applied to avoid certain interactions with the surrounding milieu, the issues remained. Taking advantage of the natural cell membranes, in an approach that resembles a cell transfer, the use of cell-derived surfaces has risen as an alternative to artificial coatings or encapsulation methods. Biomimetic technologies are based on the use of isolated natural components to provide autologous properties to the nanoparticle or cargo being encapsulated, thus, improving their therapeutic behavior. The main goal is to replicate the (bio)-physical properties and functionalities of the source cell and tissue, not only providing a stealthy character to the core but also taking advantage of homotypic properties, that could prove relevant for targeted strategies. Such biomimetic formulations have the potential to overcome the main issues of approaches to provide specific features and identities synthetically. In this review, we provide insight into the challenges of nano-biointerfaces for drug delivery; and the main applications of biomimetic materials derived from specific cell types, focusing on the unique strengths of the fabrication of novel nanotherapeutics in cancer therapy.

Despite chemotherapy, residual tumors often rely on so-called drug tolerant persister (DTP) cells, which evade treatment to give rise to therapy-resistant relapse and refractory disease. Detection of residual tumor cells proves to be challenging because of the rarity and heterogeneity of DTP cells. In the framework of a H2020 project, REAP will gather researchers and engineers from six countries, who will combine their expertise in biology, chemistry, oncology, material sciences, photonics, and electrical and biomedical engineering in the hope of revealing DTPs in cancer using contrast enhanced multimodal optical imaging. Laser sources for photoacoustic microscopy, photoacoustic tomography, and optical coherence tomography will be developed to enable the design of a two-photon laser scanning optical coherence photoacoustic microscopy system and an optical coherence photoacoustic tomography system. Furthermore, novel photoacoustic detectors using micro-ring resonator will be designed and fabricated, granting improved sensitivity and easier integration of multiple optical imaging modalities into a single system. Innovative algorithms will be developed to reconstruct and analyze the images quickly and automatically. With successful implementation of this four-year project, we can not only gain insight into the mechanisms governing DTPs, but also significantly advance the technology readiness level of contrast agents, lasers, sensors, and image analysis software through joint efforts.

Ischemic stroke, caused by the interruption of blood flow to the brain and subsequent neuronal death, represents one of the main causes of disability in developed countries. Therapeutic methods such as recanalization approaches, neuroprotective drugs, or recovery strategies have been widely developed to improve the patient's outcome; however, important limitations such as a narrow therapeutic window, the ability to reach brain targets, or drug side effects constitute some of the main aspects that limit the clinical applicability of the current treatments. Nanotechnology has emerged as a promising tool to overcome many of these drug limitations and improve the efficacy of treatments for neurological diseases such as stroke. The use of nanoparticles as a contrast agent or as drug carriers to a specific target are some of the most common approaches developed in nanomedicine for stroke. Throughout this review, we have summarized our experience of using nanotechnology tools for the study of stroke and the search for novel therapies.

We describe a microporous plasmonic nanoreactor to carry out designed near-infrared (NIR)-driven photothermal cyclizations inside living cells. As a proof of concept, we chose an intramolecular cyclization that is based on the nucleophilic attack of a pyridine onto an electrophilic carbon, a process that requires high activation energies and is typically achieved in bulk solution by heating at ∼90 °C. The core-shell nanoreactor (NR) has been designed to include a gold nanostar core, which is embedded within a metal-organic framework (MOF) based on a polymer-stabilized zeolitic imidazole framework-8 (ZIF-8). Once accumulated inside living cells, the MOF-based cloak of NRs allows an efficient diffusion of reactants into the plasmonic chamber, where they undergo the transformation upon near-IR illumination. The photothermal-driven reaction enables the intracellular generation of cyclic fluorescent products that can be tracked using fluorescence microscopy. The strategy may find different type of applications, such as for the spatio-temporal activation of prodrugs.

Self-assembled proteins are privileged building blocks for the bottom-up organization of matter at the nanoscale. However, since most proteins are very large, they have to be produced by recombinant expression, which is less versatile and flexible than chemical synthesis. Here, we show that we can bridge the potential of proteins for nanofabrication with the simplicity and versatility of solid-phase peptide synthesis by relying on the self-assembly of the viral protein gp23.1, a small 50-residue protein that oligomerizes in solution to form a stable toroidal hexamer. We report the chemical synthesis and basic biophysical characterization of a gp23.1 mutant and show that its self-assembled hexamer templates the formation of highly monodisperse gold nanoclusters of about 1.3 nm inside its central cavity. The resulting nanoclusters show catalytic activity in cycloisomerization reactions as well as luminescence emission. This work demonstrates the versatility of this small self- assembled ring protein for a variety of nanotechnological applications.