XCR1 expression distinguishes human conventional dendritic cell type 1 with full effector functions from their immediate precursors.

Dendritic cells (DCs) are major regulators of innate and adaptive immune responses. DCs can be classified into plasmacytoid DCs and conventional DCs (cDCs) type 1 and 2. Murine and human cDC1 share the mRNA expression of XCR1. Murine studies indicated a specific role of the XCR1-XCL1 axis in the induction of immune responses. Here, we describe that human cDC1 can be distinguished into XCR1- and XCR1+ cDC1 in lymphoid as well as nonlymphoid tissues. Steady-state XCR1+ cDC1 display a preactivated phenotype compared to XCR1- cDC1. Upon stimulation, XCR1+ cDC1, but not XCR1- cDC1, secreted high levels of inflammatory cytokines as well as chemokines. This was associated with enhanced activation of NK cells mediated by XCR1+ cDC1. Moreover, XCR1+ cDC1 excelled in inhibiting replication of Influenza A virus. Further, under DC differentiation conditions, XCR1- cDC1 developed into XCR1+ cDC1. After acquisition of XCR1 expression, XCR1- cDC1 secreted comparable level of inflammatory cytokines. Thus, XCR1 is a marker of terminally differentiated cDC1 that licenses the antiviral effector functions of human cDC1, while XCR1- cDC1 seem to represent a late immediate precursor of cDC1.

3D-Shaped Binders of Unfolded Proteins Inducing Cancer Cell-Specific Endoplasmic Reticulum Stress In Vitro and In Vivo.

The amount of unfolded proteins is increased in cancer cells, leading to endoplasmic reticulum (ER) stress. Therefore, cancer cells are sensitive to drugs capable of further enhancing ER stress. Examples of such drugs include the clinically approved proteosome inhibitors bortezomib and carfilzomib. Unfortunately, the known ER stress inducers exhibit dose-limiting side effects that justify the search for better, more cancer-specific drugs of this type. Herein, we report on FeC 2, which binds to unfolded proteins prevents their further processing, thereby leading to ER stress and ROS increase in cancer cells, but not in normal cells. FeC 2 exhibits low micromolar toxicity toward human acute promyelocytic leukemia HL-60, Burkitt's lymphoma BL-2, T-cell leukemia Jurkat, ovarian carcinoma A2780, lung cancer SK-MES-1, and murine lung cancer LLC1 cells. Due to the cancer-specific mode of action, 2 is not toxic in vivo up to the dose of 147 mg/kg, does not affect normal blood and bone marrow cells at the therapeutically active dose, but strongly suppresses both primary tumor growth (confirmed in Nemeth-Kellner lymphoma and LLC1 lung cancer models of murine tumor) and spreading of metastases (LLC1).

An in silico model of the capturing of magnetic nanoparticles in tumour spheroids in the presence of flow.

One of the main challenges in improving the efficacy of conventional chemotherapeutic drugs is that they do not reach the cancer cells at sufficiently high doses while at the same time affecting healthy tissue and causing significant side effects and suffering in cancer patients. To overcome this deficiency, magnetic nanoparticles as transporter systems have emerged as a promising approach to achieve more specific tumour targeting. Drug-loaded magnetic nanoparticles can be directed to the target tissue by applying an external magnetic field. However, the magnetic forces exerted on the nanoparticles fall off rapidly with distance, making the tumour targeting challenging, even more so in the presence of flowing blood or interstitial fluid. We therefore present a computational model of the capturing of magnetic nanoparticles in a test setup: our model includes the flow around the tumour, the magnetic forces that guide the nanoparticles, and the transport within the tumour. We show how a model for the transport of magnetic nanoparticles in an external magnetic field can be integrated with a multiphase tumour model based on the theory of porous media. Our approach based on the underlying physical mechanisms can provide crucial insights into mechanisms that cannot be studied conclusively in experimental research alone. Such a computational model enables an efficient and systematic exploration of the nanoparticle design space, first in a controlled test setup and then in more complex in vivo scenarios. As an effective tool for minimising costly trial-and-error design methods, it expedites translation into clinical practice to improve therapeutic outcomes and limit adverse effects for cancer patients.

Adjusting Degree of Modification and Composition of gelAGE-Based Hydrogels Improves Long-Term Survival and Function of Primary Human Fibroblasts and Endothelial Cells in 3D Cultures.

This study aimed to develop a suitable hydrogel-based 3D platform to support long-term culture of primary endothelial cells (ECs) and fibroblasts. Two hydrogel systems based on allyl-modified gelatin (gelAGE), G1MM and G2LH, were cross-linked via thiol-ene click reaction with a four-arm thiolated polyethylene glycol (PEG-4-SH). Compared to G1MM, the G2LH hydrogel was characterized by the lower polymer content and cross-linking density with a softer matrix and homogeneous and open porosity. Cell viability in both hydrogels was comparable, although the G2LH-based platform supported better F-actin organization, cell-cell interactions, and collagen and fibronectin production. In co-cultures, early morphogenesis leading to tubular-like structures was observed within 2 weeks. Migration of fibroblasts out of spheroids embedded in the G2LH hydrogels started after 5 days of incubation. Taken together, the results demonstrated that the G2LH hydrogel fulfilled the demands of both ECs and fibroblasts to enable long-term culture and matrix remodeling.

Detection of viral antibodies in camel sera using magnetic particle spectroscopy.

Pandemics like SARS-Cov-2 very frequently have their origin in different animals and in particular herds of camels could be a source of zoonotic diseases. This study took advantage on a highly sensitive and adaptable method for the fast and reliable detection of viral antibodies in camels using low-cost equipment. Magnetic nanoparticles (MNP) have high variability in their functionalization with different peptides and proteins. We confirm that 3-aminopropyl triethoxysilane (APTES)-coated MNP could be functionalized with viral proteins. The protein loading could be confirmed by simple loading controls using FACS-analysis (p < 0.05). Complementary combination of antigen and antibody yields in a significant signal increase could be proven by both FACS and COMPASS. However, COMPASS needs only a few seconds for the measurement. In COMPASS, the phase φn on selected critical point of the fifth higher harmonic (n = 5th). Here, positive sera display highly significant signal increase over the control or negative sera. Furthermore, a clear distinction could be made in antibody detection as an immune response to closely related viruses (SARS-CoV2 and MERS). Using modified MNPs along with COMPASS offers a fast and reliable method that is less cost intensive than current technologies and offers the possibility to be quickly adapted in case of new occurring viral infections. KEY POINTS: • COMPASS (critical offset magnetic particle spectroscopy) allows the fast detection of antibodies. • Magnetic nanoparticles can be adapted by exchange of the linked bait molecule. • Antibodies could be detected in camel sera without washing steps within seconds.

Endothelialization of Whey Protein Isolate-Based Scaffolds for Tissue Regeneration.

BACKGROUND: Whey protein isolate (WPI) is a by-product from the dairy industry, whose main component is ß-lactoglobulin. Upon heating, WPI forms a hydrogel which can both support controlled drug delivery and enhance the proliferation and osteogenic differentiation of bone-forming cells. This study makes a novel contribution by evaluating the ability of WPI hydrogels to support the growth of endothelial cells, which are essential for vascularization, which in turn is a pre-requisite for bone regeneration. METHODS: In this study, the proliferation and antioxidant levels in human umbilical vascular endothelial cells (HUVECs) cultured with WPI supplementation were evaluated using real-time cell analysis and flow cytometry. Further, the attachment and growth of HUVECs seeded on WPI-based hydrogels with different concentrations of WPI (15%, 20%, 30%, 40%) were investigated. RESULTS: Supplementation with WPI did not affect the viability or proliferation of HUVECs monitored with real-time cell analysis. At the highest used concentration of WPI (500 µg/mL), a slight induction of ROS production in HUVECs was detected as compared with control samples, but it was not accompanied by alterations in cellular thiol levels. Regarding WPI-based hydrogels, HUVEC adhered and spread on all samples, showing good metabolic activity. Notably, cell number was highest on samples containing 20% and 30% WPI. CONCLUSIONS: The demonstration of the good compatibility of WPI hydrogels with endothelial cells in these experiments is an important step towards promoting the vascularization of hydrogels upon implantation in vivo, which is expected to improve implant outcomes in the future.

Plasmid-DNA Delivery by Covalently Functionalized PEI-SPIONs as a Potential 'Magnetofection' Agent.

Nanoformulations for delivering nucleotides into cells as vaccinations as well as treatment of various diseases have recently gained great attention. Applying such formulations for a local treatment strategy, e.g., for cancer therapy, is still a challenge, for which improved delivery concepts are needed. Hence, this work focuses on the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) for a prospective "magnetofection" application. By functionalizing SPIONs with an active catechol ester (CafPFP), polyethyleneimine (PEI) was covalently bound to their surface while preserving the desired nanosized particle properties with a hydrodynamic size of 86 nm. When complexed with plasmid-DNA (pDNA) up to a weight ratio of 2.5% pDNA/Fe, no significant changes in particle properties were observed, while 95% of the added pDNA was strongly bound to the SPION surface. The transfection in A375-M cells for 48 h with low amounts (10 ng) of pDNA, which carried a green fluorescent protein (GFP) sequence, resulted in a transfection efficiency of 3.5%. This value was found to be almost 3× higher compared to Lipofectamine (1.2%) for such low pDNA amounts. The pDNA-SPION system did not show cytotoxic effects on cells for the tested particle concentrations and incubation times. Through the possibility of additional covalent functionalization of the SPION surface as well as the PEI layer, Caf-PEI-SPIONs might be a promising candidate as a magnetofection agent in future.

[Abscesstonsillectomy: Uni- or bilateral?] / Abszesstonsillektomie: Uni- oder bilateral?

OBJECTIVE: In addition to an empirical use of antibiotics for treatment of a peritonsillar abscess (PTA) there is a drainage of pus or the abscess tonsillectomy. Postoperative bleeding after abscesstonsillectomy (ABTE) is this surgery's most feared complication which can rarely lead to patients' deaths. The objective of this study was to compare bleeding complications of ABTE with and without contralateral tonsillectomy (TE) and to analyze the occurrence of a metachronous PTA at the contralateral side. METHODS: Retrospective study of n= 655 patients undergoing ABTE with and without TE of the contralateral side from 2004 to 2019. Bleeding complications needing surgical hemostasis were analyzed regarding demographic and surgical parameters. In addition, occurrence of PTA and need for ABTE of the contralateral side after unilateral ABTE were evaluated. RESULTS: Overall, 10/655 (1.5 %) patients presented with postoperative bleeding after ABTE. In 404/655 an ABTE with contralateral TE was performed. Here, 8/404 (1.98 %) patients showed contra- or bilateral bleeding. Only in 2/251 (0.7 %) patients occurred a bleeding complication after unilateral ABTE. Therefore, bleeding after unilateral ABTE was significantly lower than ABTE with contralateral TE (1.98 % vs. 0.7 %, p= 0.001). In 0.8 % of the patients a contralateral ABTE was necessary due to a metachronous PTA. CONCLUSION: Overall, the rate of postoperative bleeding after ABTE (1.5 %) was low. Unilateral ABTE showed significantly lower postoperative bleeding rates compared to ABTE with contralateral TE. Consequently, the indication of a contralateral TE must be very strict.

Hydroxyapatite-Coated SPIONs and Their Influence on Cytokine Release.

Hydroxyapatite- or calcium phosphate-coated iron oxide nanoparticles have a high potential for use in many biomedical applications. In this study, a co-precipitation method for the synthesis of hydroxyapatite-coated nanoparticles (SPIONHAp), was used. The produced nanoparticles have been characterized by dynamic light scattering, X-ray diffraction, vibrating sample magnetometry, Fourier transform infrared spectrometry, atomic emission spectroscopy, scanning electron microscopy, transmission electron microscopy, selected area diffraction, and energy-dispersive X-ray spectroscopy. The results showed a successful synthesis of 190 nm sized particles and their stable coating, resulting in SPIONHAp. Potential cytotoxic effects of SPIONHAp on EL4, THP-1, and Jurkat cells were tested, showing only a minor effect on cell viability at the highest tested concentration (400 µg Fe/mL). The results further showed that hydroxyapatite-coated SPIONs can induce minor TNF-α and IL-6 release by murine macrophages at a concentration of 100 µg Fe/mL. To investigate if and how such particles interact with other substances that modulate the immune response, SPIONHAp-treated macrophages were incubated with LPS (lipopolysaccharides) and dexamethasone. We found that cytokine release in response to these potent pro- and anti-inflammatory agents was modulated in the presence of SPIONHAp. Knowledge of this behavior is important for the management of inflammatory processes following in vivo applications of this type of SPIONs.

Differential Responses to Bioink-Induced Oxidative Stress in Endothelial Cells and Fibroblasts.

A hydrogel system based on oxidized alginate covalently crosslinked with gelatin (ADA-GEL) has been utilized for different biofabrication approaches to design constructs, in which cell growth, proliferation and migration have been observed. However, cell-bioink interactions are not completely understood and the potential effects of free aldehyde groups on the living cells have not been investigated. In this study, alginate, ADA and ADA-GEL were characterized via FTIR and NMR, and their effect on cell viability was investigated. In the tested cell lines, there was a concentration-dependent effect of oxidation degree on cell viability, with the strongest cytotoxicity observed after 72 h of culture. Subsequently, primary human cells, namely fibroblasts and endothelial cells (ECs) were grown in ADA and ADA-GEL hydrogels to investigate the molecular effects of oxidized material. In ADA, an extremely strong ROS generation resulting in a rapid depletion of cellular thiols was observed in ECs, leading to rapid necrotic cell death. In contrast, less pronounced cytotoxic effects of ADA were noted on human fibroblasts. Human fibroblasts had higher cellular thiol content than primary ECs and entered apoptosis under strong oxidative stress. The presence of gelatin in the hydrogel improved the primary cell survival, likely by reducing the oxidative stress via binding to the CHO groups. Consequently, ADA-GEL was better tolerated than ADA alone. Fibroblasts were able to survive the oxidative stress in ADA-GEL and re-entered the proliferative phase. To the best of our knowledge, this is the first report that shows in detail the relationship between oxidative stress-induced intracellular processes and alginate di-aldehyde-based bioinks.

An Endoplasmic Reticulum Specific Pro-amplifier of Reactive Oxygen Species in Cancer Cells.

The folding and export of proteins and hydrolysis of unfolded proteins are disbalanced in the endoplasmic reticulum (ER) of cancer cells, leading to so-called ER stress. Agents further augmenting this effect are used as anticancer drugs including clinically approved proteasome inhibitors bortezomib and carfilzomib. However, these drugs can affect normal cells, which also rely strongly on ER functions, leading, for example, to accumulation of reactive oxygen species (ROS). To address this problem, we have developed ER-targeted prodrugs activated only in cancer cells in the presence of elevated ROS amounts. These compounds are conjugates of cholic acid with N-alkylaminoferrocene-based prodrugs. We confirmed their accumulation in the ER of cancer cells, their anticancer efficacy, and cancer cell specificity. These prodrugs induce ER stress, attenuate mitochondrial membrane potential, and generate mitochondrial ROS leading to cell death via necrosis. We also demonstrated that the new prodrugs are activated in vivo in Nemeth-Kellner lymphoma (NK/Ly) murine model.

Brave new world revisited: Focus on nanomedicine.

Nanomedicine is at a crossroads: with relatively few success stories in terms of clinical translation despite more and more research on increasingly sophisticated nanomaterials, it is important to consider whether we are on the right track. Indeed, it is crucial that we address the fact that while considerable efforts are being made to overcome barriers to translation from the bench to the clinic, scientists are still struggling to decipher fundamental aspects of nanomaterial interactions with biological systems. We believe that a key to the successful adoption of nanomedicines in oncology and beyond lies in a deeper understanding of underlying biological processes and in decoding interactions between engineered nanomaterials and biological systems. Here we provide an overview of progress in nanomedicine during the past 5 years.

Shedding Light on Metal-Based Nanoparticles in Zebrafish by Computed Tomography with Micrometer Resolution.

Metal-based nanoparticles are clinically used for diagnostic and therapeutic applications. After parenteral administration, they will distribute throughout different organs. Quantification of their distribution within tissues in the 3D space, however, remains a challenge owing to the small particle diameter. In this study, synchrotron radiation-based hard X-ray tomography (SRµCT) in absorption and phase contrast modes is evaluated for the localization of superparamagnetic iron oxide nanoparticles (SPIONs) in soft tissues based on their electron density and X-ray attenuation. Biodistribution of SPIONs is studied using zebrafish embryos as a vertebrate screening model. This label-free approach gives rise to an isotropic, 3D, direct space visualization of the entire 2.5 mm-long animal with a spatial resolution of around 2 µm. High resolution image stacks are available on a dedicated internet page (http://zebrafish.pharma-te.ch). X-ray tomography is combined with physico-chemical characterization and cellular uptake studies to confirm the safety and effectiveness of protective SPION coatings. It is demonstrated that SRµCT provides unprecedented insights into the zebrafish embryo anatomy and tissue distribution of label-free metal oxide nanoparticles.

Small Dimension-Big Impact! Nanoparticle-Enhanced Non-Invasive and Intravascular Molecular Imaging of Atherosclerosis In Vivo.

Extensive translational research has provided considerable progress regarding the understanding of atherosclerosis pathophysiology over the last decades. In contrast, implementation of molecular in vivo imaging remains highly limited. In that context, nanoparticles represent a useful tool. Their variable shape and composition assure biocompatibility and stability within the environment of intended use, while the possibility of conjugating different ligands as well as contrast dyes enable targeting of moieties of interest on a molecular level and visualization throughout various imaging modalities. These characteristics have been exploited by a number of preclinical research approaches aimed at advancing understanding of vascular atherosclerotic disease, in order to improve identification of high-risk lesions prior to oftentimes fatal thromboembolic events. Furthermore, the combination of these targeted nanoparticles with therapeutic agents offers the potential of site-targeted drug delivery with minimized systemic secondary effects. This review gives an overview of different groups of targeted nanoparticles, designed for in vivo molecular imaging of atherosclerosis as well as an outlook on potential combined diagnostic and therapeutic applications.

N-Alkylaminoferrocene-Based Prodrugs Targeting Mitochondria of Cancer Cells.

Intracellular concentration of reactive oxygen species (e.g., H2O2) in cancer cells is elevated over 10-fold as compared to normal cells. This feature has been used by us and several other research groups to design cancer specific prodrugs, for example, N-alkylaminoferrocene (NAAF)-based prodrugs. Further improvement of the efficacy of these prodrugs can be achieved by their targeting to intracellular organelles containing elevated reactive oxygen species (ROS) amounts. For example, we have previously demonstrated that lysosome-targeted NAAF-prodrugs exhibit higher anticancer activity in cell cultures, in primary cells and in vivo (Angew. Chem. Int. Ed. 2017, 56, 15545). Mitochondrion is an organelle, where electrons can leak from the respiratory chain. These electrons can combine with O2, generating O2-• that is followed by dismutation with the formation of H2O2. Thus, ROS can be generated in excess in mitochondria and targeting of ROS-sensitive prodrugs to these organelles could be a sensible possibility for enhancing their efficacy. We have previously reported on NAAF-prodrugs, which after their activation in cells, are accumulated in mitochondria (Angew. Chem. Int. Ed. 2018, 57, 11943). Now we prepared two hybrid NAAF-prodrugs directly accumulated in mitochondria and activated in these organelles. We studied their anticancer activity and mode of action. Based on these data, we concluded that ROS produced by mitochondria is not available in sufficient quantities for activation of the ROS-responsive prodrugs. The reason for this can be efficient scavenging of ROS by antioxidants. Our data are important for the understanding of the mechanism of action of ROS-activatable prodrugs and will facilitate their further development.

Synthesis and Characterization of Citrate-Stabilized Gold-Coated Superparamagnetic Iron Oxide Nanoparticles for Biomedical Applications.

Surface-functionalized gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) may be a useful tool in various biomedical applications. To obtain Au-SPIONs, gold salt was precipitated onto citrate-stabilized SPIONs (Cit-SPIONs) using a simple, aqueous one-pot technique inspired by the Turkevich method of gold nanoparticle synthesis. By the further stabilization of the Au-SPION surface with additional citrate (Cit-Au-SPIONs), controllable and reproducible Z-averages enhanced long-term dispersion stability and moderate dispersion pH values were achieved. The citrate concentration of the reaction solution and the gold/iron ratio was found to have a major influence on the particle characteristics. While the gold-coating reduced the saturation magnetization to 40.7% in comparison to pure Cit-SPIONs, the superparamagnetic behavior of Cit-Au-SPIONs was maintained. The formation of nanosized gold on the SPION surface was confirmed by X-ray diffraction measurements. Cit-Au-SPION concentrations of up to 100 µg Fe/mL for 48 h had no cytotoxic effect on Jurkat cells. At a particle concentration of 100 µg Fe/mL, Jurkat cells were found to take up Cit-Au-SPIONs after 24 h of incubation. A significantly higher attachment of thiol-containing L-cysteine to the particle surface was observed for Cit-Au-SPIONs (53%) in comparison to pure Cit-SPIONs (7%).

Magnetically responsive composites: electron beam assisted magnetic nanoparticle arrest in gelatin hydrogels for bioactuation.

As emerging responsive materials, ferrogels have become highly attractive for biomedical and technical applications in terms of soft actuation, tissue engineering or controlled drug release. In the present study, bioderived ferrogels were fabricated and successfully deformed within moderate, heterogeneous magnetic fields. Synthesis was realized by arresting iron oxide nanoparticles in porcine gelatin by introduction of covalent crosslinks via treatment with energetic electrons for mesh refinement. This approach also allows for tuning thermal and mechanical stability of the gelatin matrix. Operating the bioferrogel in compression, magnetic forces on the nanoparticles are counterbalanced by the stiffness of the hydrogel matrix that is governed by a shift in thermodynamic equilibrium of swelling, as derived in the framework of osmosis. As gelatin and iron oxide nanoparticles are established as biocompatible constituents, these findings promise potential for in vivo use as contactless mechanical transducers.

Magnetic Accumulation of SPIONs under Arterial Flow Conditions: Effect of Serum and Red Blood Cells.

Magnetic drug targeting utilizes an external magnetic field to target superparamagnetic iron oxide nanoparticles (SPIONs) and their cargo to the diseased vasculature regions. In the arteries, the flow conditions affect the behavior of magnetic particles and the efficacy of their accumulation. In order to estimate the magnetic capture of SPIONs in more physiological-like settings, we previously established an ex vivo model based on human umbilical cord arteries. The artery model was employed in our present studies in order to analyze the effects of the blood components on the efficacy of magnetic targeting, utilizing 2 types of SPIONs with different physicochemical characteristics. In the presence of freshly isolated human plasma or whole blood, a strong increase in iron content measured by AES was observed for both particle types along the artery wall, in parallel with clotting activation due to endogenous thrombin generation in plasma. Subsequent studies therefore utilized SPION suspensions in serum and washed red blood cells (RBCs) at hematocrit 50%. Interestingly, in contrast to cell culture medium suspensions, magnetic accumulation of circulating SPION-3 under the external magnet was achieved in the presence of RBCs. Taken together, our data shows that the presence of blood components affects, but does not prevent, the magnetic accumulation of circulating SPIONs.

Investigation of phonatory characteristics using ex vivo rabbit larynges.

Quantitative analysis of phonatory characteristics of rabbits has been widely neglected. However, preliminary studies established the rabbit larynx as a potential model of human phonation. This study reports quantitative data on phonation using ex vivo rabbit larynx models to achieve more insight into dependencies of three main components of the phonation process, including airflow, vocal fold dynamics, and the acoustic output. Sustained phonation was induced in 11 ex vivo rabbit larynges. For 414 phonatory conditions, vocal fold vibrations, acoustic, and aerodynamic parameters were analyzed as functions of longitudinal vocal fold pre-stress, applied air flow, and glottal closure insufficiency. Dimensions of the vocal folds were measured and histological data were analyzed. Glottal closure characteristics improved for increasing longitudinal pre-stress and applied airflow. For the subglottal pressure signal only the cepstral peak prominence showed dependency on glottal closure. In contrast, vibrational, acoustic, and aerodynamic parameters were found to be highly dependent on the degree of glottal closure: The more complete the glottal closure during phonation, the better the aerodynamic and acoustic characteristics. Hence, complete or at least partial glottal closure appears to enhance acoustic signal quality. Finally, results validate the ex vivo rabbit larynx as an effective model for analyzing the phonatory process.

ROS-Responsive N-Alkylaminoferrocenes for Cancer-Cell-Specific Targeting of Mitochondria.

Mitochondrial membrane potential is more negative in cancer cells than in normal cells, allowing cancer targeting by delocalized lipophilic cations (DLCs). However, as the difference is rather small, these drugs affect also normal cells. Now a concept of pro-DLCs is proposed based on an N-alkylaminoferrocene structure. These prodrugs are activated by the reaction with reactive oxygen species (ROS) forming ferrocenium-based DLCs. Since ROS are overproduced in cancer, the high-efficiency cancer-cell-specific targeting of mitochondria could be achieved as demonstrated by fluorescence microscopy in combination with two fluorogenic pro-DLCs in vitro and in vivo. We prepared a conjugate of another pro-DLC with a clinically approved drug carboplatin and confirmed that its accumulation in mitochondria was higher than that of the free drug. This was reflected in the substantially higher anticancer effect of the conjugate.