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Simultaneous visualization and concentration quantification of molecules in biological tissue is an important though challenging goal. The advantages of fluorescence lifetime imaging microscopy (FLIM) for visualization, and electron paramagnetic resonance (EPR) spectroscopy for quantification are complementary. Their combination in a multiplexed approach promises a successful but ambitious strategy because of spin label‐mediated fluorescence quenching. Here, we solved this problem and present the molecular design of a dual label (DL) compound comprising a highly fluorescent dye together with an EPR spin probe, which also renders the fluorescence lifetime to be concentration sensitive. The DL can easily be coupled to the biomolecule of choice, enabling in vivo and in vitro applications. This novel approach paves the way for elegant studies ranging from fundamental biological investigations to preclinical drug research, as shown in proof‐of‐principle penetration experiments in human skin ex vivo., A novel multi‐label synthetic platform for a functional dual fluorescence–spin label probe enables the non‐destructive simultaneous quantification and visualization of molecules in biological tissue. Multiplexed FLIM and EPR spectroscopy avoids analytical inconsistencies between both techniques. Beside tissue applications, molecular spectroscopic studies of biomolecular conformation, structure, dynamics, and microenvironment are feasible.

Polymer capsules fabricated via the layer-by-layer (LbL) approach have emerged as promising biomedical systems for the release of a wide variety of therapeutic agents, owing to their tunable and controllable structure and the possibility to include several functionalities in the polymeric membrane during the fabrication process. However, the limitation of the capsules with a single functionality to overcome the challenges involved in the treatment of complex pathologies denotes the need to develop multifunctional capsules capable of targeting several mediators and/or mechanisms. Oxidative stress is caused by the accumulation of reactive oxygen species [e.g., hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and superoxide anion radicals (•O2-)] in the cellular microenvironment and is a key modulator in the pathology of a broad range of inflammatory diseases. The disease microenvironment is also characterized by the presence of proinflammatory cytokines, increased levels of matrix metalloproteinases, and acidic pH, all of which could be exploited to trigger the release of therapeutic agents. In the present work, multifunctional capsules were fabricated via the LbL approach. Capsules were loaded with an antioxidant enzyme (catalase) and functionalized with a model drug (doxorubicin), which was conjugated to an amine-containing dendritic polyglycerol through a pH-responsive linker. These capsules efficiently scavenge H2O2 from solution, protecting cells from oxidative stress, and release the model drug in acidic microenvironments. Accordingly, in this work, a polymeric microplatform is presented as an unexplored combinatorial approach applicable for multiple targets of inflammatory diseases, in order to perform controlled spatiotemporal enzymatic reactions and drug release in response to biologically relevant stimuli.

Pyrimidine-based cationic amphiphiles (PCAms), i.e., di-trifluoroacetic acid salts of N1-[1′-(1″,3″-diglycinatoxy-propane-2″-yl)-1′,2′,3′-triazole-4′-yl]methyl-N3-alkylpyrimidines have been synthesized utilizing naturally occurring biocompatible precursors, like glycerol, glycine, and uracil/ thymine in good yields. Synthesized PCAms consist of a hydrophilic head group comprising TFA salt of glyceryl 1,3-diglycinate and hydrophobic tail comprising of C-7 and C-12 N3-alkylated uracil or thymine conjugated via a 4-methylene-1,2,3-triazolyl linker. The physicochemical properties of all PCAms, such as critical aggregation concentration, hydrodynamic diameter, shape, and zeta potential (surface charge) were analyzed. These PCAms were also evaluated for their anti-proliferative and anti-tubercular activities. One of the synthesized PCAm exhibited 4- to 75-fold more activity than first-line anti-tubercular drugs streptomycin and isoniazid, respectively, against the multidrug resistant clinical isolate 591 of Mycobacterium tuberculosis.

Magnetoplasmonic nanomaterials, which combine light and magnetic field responsiveness in an advantageous manner, are attractive candidates for bio-nanoapplications. However, the synthetic access to such hybrid particles has been limited by the incompatibility of the iron- and gold-based lattices. In this work, we provide the first insights into a new synthetic strategy for developing magnetoplasmonic anisotropic nanocomposites with prominent phototransducing properties. In our approach, magnetic nanocubes based on an alloy of iron oxide, zinc, and silver were constructed. In a key second stage, the galvanic replacement of silver with gold atoms yielded satellite-like magnetoplasmonic anisotropic structures. Superior magnetic and photoconverting properties were observed for the novel magnetoplasmonic nanocomposites when compared with the pure parent structures. Moreover, the synergy between the magnetic and optical stimuli was examined, showing shape-dependent contributions in the magnetization experiments. More importantly, an excellent cell ablation capability upon laser irradiation was observed for the magnetoplasmonic nanocomposites compared to the pure magnetic or plasmonic controls. Further demonstration of these novel theragnostic agents as MRI contrast agents is also reported even during the light-irradiation event. Thus, the described particles showed promising properties for bioapplications emerging from the novel synthetic methodology.

The use of penetration enhancers (chemical or physical) has been proven to dramatically improve the penetration of therapeutics. Nevertheless, their use poses great risks, as they can lead to permanent damage of the skin, reduce its barrier efficiency, and result in the intrusion of harmful substances. Among the most used skin penetration enhancers, water is greatly accepted because skin quickly recovers from its exposure. Nanocapsules (NCs) represent a promising combination of the carrier system and penetration enhancer because their water-containing void combined with their polymer-based shell can be used to induce high local skin hydration, while simultaneously aiding the transport of drugs across the skin barrier. In this study, NCs were synthesized with a void core of 100 nm in diameter, a thermoresponsive shell based on different ratios of poly(N-isopropylacrylamide) and poly(N-isopropylmethacrylamide) as thermoresponsive polymers, and dendritic polyglycerol as a macromolecular cross-linker. These NCs can shrink or swell upon a thermal trigger, which was used to induce the release of the entrapped water in a controlled fashion. The interactions and effects of thermoresponsive NCs on the stratum corneum of excised human skin were investigated using fluorescence microscopy, high-resolution optical microscopy, and stimulated Raman spectromicroscopy. It could be observed that the thermoresponsive NCs increase the amount of deuterated water that penetrated into the viable epidermis. Moreover, NCs increased the skin penetration of a high-molecular weight dye (Atto Oxa12 NHS ester, MW = 835 g/mol) with respect to formulations in water or 30% DMSO, emphasizing the features of the NCs as a skin penetration enhancer.

Singlet oxygen can severely damage biological tissue, which is exploited in photodynamic therapy (PDT). In PDT, the effective range is limited by the distribution of the photosensitizer (PS) and the illuminated area. However, no distinction is made between healthy and pathological tissue, which can cause undesired damage. This encouraged us to exploit the more acidic pH of cancerous tissue and design pH-controllable singlet oxygen-generating boron-dipyrromethene (BODIPY) dyes. A pH sensitivity of the dyes is achieved by the introduction of an electronically decoupled, photoinduced electron transfer (PET)-capable subunit in meso-position of the BODIPY core. To favor triplet-state formation as required for singlet oxygen generation, iodine substituents were introduced at the chromophore core. The resulting pH-controlled singlet oxygen-generating dyes with pKa values in the physiological range were subsequently assessed regarding their potential as pH-controlled PS for PDT. Using HeLa cells, we could successfully demonstrate markedly different pH-dependent cytotoxicities upon illumination.

Biomimetic magnetite nanoparticles (BMNPs) synthesized in the presence of MamC, a magnetosome-associated protein from Magnetoccus marinus MC-1, have gained interest for biomedical applications because of their unique magnetic properties. However, their behavior in biological systems, like their interaction with proteins, still has to be evaluated prior to their use in clinics. In this study, doxorubicin (DOXO) as a model drug was adsorbed onto BMNPs to form nanoassemblies. These were incubated with human plasma to trigger protein corona (PC) formation. Proteins from the human plasma stably attached to either BMNPs or DOXO-BMNP nanoassemblies. In particular, fibrinogen was detected as the main component in the PC of DOXO-BMNPs that potentially provides advantages, e.g. protecting the particles from phagocytosis, thus prolonging their circulation time. Adsorption of PC to the BMNPs did not alter their magnetic properties but improved their colloidal stability, thus reducing their toxicity in human macrophages. In addition, PC formation enhanced cellular internalization and did not interfere with DOXO activity. Overall, our data indicate that the adsorption of PC onto DOXO-BMNPs in biological environment even increases their efficiency as drug carrier systems.

The combination of magnetic nanoparticles and thermoresponsive nanogels represents an appealing strategy for the development of theranostic probes. These hybrid nanocarriers present several advantages such as outstanding properties for guided therapy, magnetic resonance imaging, and triggered release of encapsulated cargoes. Most magnetic thermoresponsive nanogels are built with strategies that comprise a physical interaction of particles with the polymeric network or the covalent attachment of a single particle to the linear polymer. Herein, we report a facile synthetic approach for the synthesis of magnetic and thermoresponsive nanogels that allows the controlled incorporation of multiple superparamagnetic inorganic cores as covalent cross-linkers. An ultrasonication-assisted precipitation-polymerization afforded nanogels with sizes in the nanometric range and similar magnetization and light transduction properties compared to the discrete magnetic nanoparticles. The theranostic capability of these nanocarriers was further investigated both in vitro and in vivo. In vivo experiments demonstrated the capacity of these materials as nanocarriers for near-infrared (NIR) triggered chemotherapy and highlighted the relevance of the correct concentration/dose in this antitumoral modality to achieve a superior therapeutic efficacy. Fil: Biglione, Catalina. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Bergueiro, Julian. Freie Universität Berlin; Alemania Fil: Wedepohl, Stefanie. Freie Universität Berlin; Alemania Fil: Klemke, Bastian. No especifíca; Fil: Strumia, Miriam Cristina. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Calderón, Marcelo. Freie Universität Berlin; Alemania

The mechanical properties of nanoparticles, especially those designed for biomedical purposes, have a large impact on their performance and have been scarcely studied. Thermoresponsive polymer-base...

Skin penetration of active molecules for treatment of diverse diseases is a major field of research owing to the advantages associated with the skin like easy accessibility, reduced systemic-derived side effects, and increased therapeutic efficacy. Despite these advantages, dermal drug delivery is generally challenging due to the low skin permeability of therapeutics. Although various methods have been developed to improve skin penetration and permeation of therapeutics, they are usually aggressive and could lead to irreversible damage to the stratum corneum. Nanosized carrier systems represent an alternative approach for current technologies, with minimal damage to the natural barrier function of skin. In this Review, the use of nanoparticles to deliver drug molecules, genetic material, and vaccines into the skin is discussed. In addition, nanotoxicology studies and the recent clinical development of nanoparticles are highlighted to shed light on their potential to undergo market translation. We are grateful to Maria Angela Motta and Oliver Etzold for their valuable scientific inputs and corrections in the review. We are thankful to MINECO (RTI2018-098951-B-I00), IKERBASQUE-Basque Foundation for Science, Basque Government (Elkartek projects KK-2019/00086 and KK2020/00010; proyectos de investigacion basica/aplicada PIBA_2020_1_0056), Gipuzkoa Provincial Council (2019CIEN-000075-01), and to Polymat, Basque Centre for Macromolecular Design and Engineering for funding. A.S. acknowledges financial support from CONICET, ANPCyT, Universidad Nacional del Litoral. H.W. is thankful for the financial support received from China Scholarship Council (File no. 201804910606).

Skin, being the largest organ of the body, has attracted a lot of attention in recent years as a vector to deliver awide spectrum of cargo molecules to treat multiple conditions, including genetic disorders, infections by pathogens(bacteria, virus, fungus), inflammatory diseases such as psoriasis and atopic dermatitis, and skin cancer. In order todeliver active molecules across the skin layers, it is crucial to understand the morphology and properties of skin. Ahealthy skin is associated with a highly efficient barrier that prevents invasion of foreign particles or microbes fromthe external environment. As a consequence, the outermost layer of the epidermis, also called the stratum corneum(SC), prevents penetration of molecules that are larger than 500 Da [1]. This represents an immense challenge fordelivery of bigger active molecules into the skin tissues via passive diffusion. Various formulations such as creams,gels and ointments have been studied to overcome the skin protective barrier but they mainly intend to have localeffect rather than systemic action. To enhance penetration of active therapeutics across the skin, several techniqueshas been developed. This includes chemicals such as surfactants, alcohols, amines ? among others, or physicaldisruption of the SC using methods such as sonoporation, iontophoresis, electroporation and microneedles [2].Although penetration enhancers have proven to be effective for delivery of active therapeutics, they could leadto long-term or irreparable damage of the lipid structure of the SC. Nanogels, being cross-linked polymers withnanometer dimensions, provide an alternative approach to existing technologies with minimal damage to thenatural barrier function of the skin. Furthermore, nanogels possess certain desirable features such as solubility andstabilization of hydrophobic drugs or proteins and the ability to target encapsulated moieties to specific cell types,with control over release profiles. In addition, nanogels that respond to various stimuli such as pH and temperatureare shown to enhance the penetration of cargo molecules in the skin by interacting with the SC, followed by thetriggered release of cargo molecules [3?5]. Fil: Tiwari, Neha. Universidad del País Vasco; España Fil: Sonzogni, Ana Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Calderon, Marcelo. Universidad del País Vasco; España

The current limitations in the use of nanocarriers to treat constantly evolving diseases call for the design of novel and smarter drug delivery systems (DDS). Nanogels (NGs) are three-dimensional crosslinked polymers with dimensions on the nanoscale and with a great potential for use in the biomedical field. Particular interest focuses on their application as DDS to minimize severe toxic effects and increase the therapeutic index of drugs. They have recently gained attention, since they can include responsive modalities within their structure, which enable them to excerpt a therapeutic function on demand. Their bigger sizes and controlled architecture and functionality, when compared to non-crosslinked polymers, make them particularly interesting to explore novel modalities to cross biological barriers. The present review summarizes the most significant developments of NGs as smart carriers, with focus on smart modalities to cross biological barriers such as cellular membrane, tumor stroma, mucose, skin, and blood brain barrier. We discuss the properties of each barrier and highlight the importance that the NG design has on their capability to overcome them and deliver the cargo at the site of action.

Graphite electrodes were modified with triangular (AuNTrs) or spherical (AuNPs) nanoparticles and further modified with fructose dehydrogenase (FDH). The present study reports the effect of the shape of these nanoparticles (NPs) on the catalytic current of immobilized FDH pointing out the different contributions on the mass transfer–limited and kinetically limited currents. The influence of the shape of the NPs on the mass transfer–limited and the kinetically limited current has been proved by using two different methods: a rotating disk electrode (RDE) and an electrode mounted in a wall jet flow-through electrochemical cell attached to a flow system. The advantages of using the wall jet flow system compared with the RDE system for kinetic investigations are as follows: no need to account for substrate consumption, especially in the case of desorption of enzyme, and studies of product-inhibited enzymes. The comparison reveals that virtually identical results can be obtained using either of the two techniques. The heterogeneous electron transfer (ET) rate constants (kS) were found to be 3.8 ± 0.3 s−1 and 0.9 ± 0.1 s−1, for triangular and spherical NPs, respectively. The improvement observed for the electrode modified with AuNTrs suggests a more effective enzyme-NP interaction, which can allocate a higher number of enzyme molecules on the electrode surface. Graphical abstractThe shape of gold nanoparticles has a crucial effect on the catalytic current related to the oxidation of D-(-)-fructose to 5-keto-D-(-)-fructose occurring at the FDH-modified electrode surface. In particular, AuNTrs have a higher effect compared with the spherical one. Electronic supplementary material The online version of this article (10.1007/s00216-019-01944-6) contains supplementary material, which is available to authorized users.

Macromolecular bioactives, like proteins and peptides, emerged as highly efficient therapeutics. The main limitation for their clinical application is their instability and potential immunogenicity. Thus, controlled delivery systems able protect the proteins prior release are highly on demand. In the present study, we developed hydrophilic thermo-responsive nanogels with tunable volume phase transition temperatures (VPTTs) and suitable features for controlled protein delivery by the use of multifunctional, dendritic polyglycerol (dPG) as macromolecular cross-linker and temperature-sensitive polymers poly(N-isopropylacrylamide) (NIPAM) and poly(N-isopropylacrylmethacrylate) as linear counterpart. We comprehensively studied the impact of the initiator, monomers and cross-linker on the nanogel structure during the synthesis. Careful analysis of the polymerization process revealed importance of balanced reactions kinetics to form particles with diameters in the range 100–200 nm and low polydispersity. We can control the cross-linking density of the nanogels mainly by the dPG feed and its degree of acrylation. In addition, our screenings revealed that the hydrophilic character of dPG enables it to stabilize the growing particles during the polymerization and thereby reduces final particle size. Co-polymerization of NIPAM and NIPMAM allows precise tuning of the VPTT of the nanogels in the desired range of 34–47 °C. Our nanogels showed outstanding high protein encapsulation efficiency and triggered cargo release upon a temperature change. The delivery efficiency of these nanogels was investigated on excised human skin demonstrating efficient dermal penetration of encapsulated proteins dependent on a temperature triggered release mechanism.

A major drawback of chemotherapy is that cancer cells can develop multi-drug resistance, leading to failure of anticancer treatments. To overcome this limitation, we report the preparation of environment-sensitive polylactide (PLA)-based nanoparticles, assembled by multiple hydrogen bonds. These nanoparticles showed sizes in the 100–150 nm range, a spherical morphology, and low toxicity in cell culture. In addition, their 3D supramolecular polymer network was sensitive to both pH and temperature and yielded dynamically reversible networks that could recognize the intrinsic differences between cancer and normal tissues. Release studies of the encapsulated anticancer drug doxorubicin (DOX) demonstrated that the nanoparticles exhibited high stability at physiological conditions. However, when the pH was reduced from 7.4 to 5, an accelerated release was observed. Most importantly, by applying this supramolecular linked nanocarrier for the delivery of DOX, the drug-resistance of KB-V1 cells could be overcome. All these features demonstrate the versatility of the proposed modular approach and their great potential for anticancer therapy.

After several decades of development in the field of near-infrared (NIR) dyes for photothermal therapy (PTT), indocyanine green (ICG) still remains the only FDA-approved NIR contrast agent. However, upon NIR light irradiation ICG can react with molecular oxygen to form reactive oxygen species and degrade the ICG core, losing the convenient dye properties. In this work, we introduce a new approach for expanding the application of ICG in nanotheranostics, which relies on the confinement of self-organized J-type aggregates in hydrophobic protein domains acting as monomer depots. Upon the fast photobleaching, while the dye is irradiated, this strategy permits the equilibrium-driven monomer replacement after each irradiation cycle that radically increases the systems' effectivity and applicability. Gadolinium-doped casein micelles were designed to prove this novel concept at the same time as endowing the nanosystems with further magnetic resonance imaging (MRI) ability for dual-modal imaging-guided PTT. By teaching a new trick to a very old dog, the clinical prospect of ICG will undoubtedly be boosted laying the foundation for novel therapeutics. It is anticipated that future research could be expanded to other relevant J-aggregates-forming cyanine dyes or nanocrystal formulations of poorly water-soluble photosensitizers. Fil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Bergueiro, Julian. Freie Universität Berlin; Alemania Fil: Wedepohl, Stefanie. Freie Universität Berlin; Alemania Fil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Calderón, Marcelo. Polymat; España

Mucosal surfaces pose a challenging environment for efficient drug delivery. Various delivery strategies such as nanoparticles have been employed so far; yet, still yielding limited success. To address the need of efficient transmucosal drug delivery, this report presents the synthesis of novel disulfide-containing dendritic polyglycerol (dPG)-based nanogels and their preclinical testing. A bifunctional disulfide-containing linker is coupled to dPG to act as a macromolecular crosslinker for poly-N-isopropylacrylamide (PNIPAM) and poly-N-isopropylmethacrylamide (PNIPMAM) in a precipitation polymerization process. A systematic analysis of the polymerization reveals the importance of a careful polymer choice to yield mucus-degradable nanogels with diameters between 100 and 200 nm, low polydispersity, and intact disulfide linkers. Absorption studies in porcine intestinal tissue and human bronchial epithelial models demonstrate that disulfide-containing nanogels are highly efficient in overcoming mucosal barriers. The nanogels efficiently degrade and deliver the anti-inflammatory biomacromolecule etanercept into epithelial tissues yielding local anti-inflammatory effects. Over the course of this work, several problems are encountered due to a limited availability of valid test systems for mucosal drug-delivery systems. Hence, this study also emphasizes how critical a combined and multifaceted approach is for the preclinical testing of mucosal drug-delivery systems, discusses potential pitfalls, and provides suggestions for solutions.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 μm. We describe a variation of the conventional approach, wherein the Si3N4 windows are separated by a 10-μm-thick spacer, thus providing room for gas flow inside the liquid specimen enclosure. Adjusting the pressure and flow speed of humid air inside this environmental liquid cell (ELC) creates a stable liquid layer of controllable thickness on the bottom window, thus facilitating high-resolution observations of low mass-thickness contrast objects at low electron doses. We demonstrate controllable liquid thicknesses in the range 160 ± 34 to 340 ± 71 nm resulting in corresponding edge resolutions of 0.8 ± 0.06 to 1.7 ± 0.8 nm as measured for immersed gold nanoparticles. Liquid layer thickness 40 ± 8 nm allowed imaging of low-contrast polystyrene particles. Hydration effects in the ELC have been studied using poly-N-isopropylacrylamide nanogels with a silica core. Therefore, ELC can be a suitable tool for in situ investigations of liquid specimens.

Advanced materials and chemo-specific designs at the nano/micrometer-scale have ensured revolutionary progress in next-generation clinically relevant technologies. For example, isolating a rare population of cells, like circulating tumor cells (CTCs) from the blood amongst billions of other blood cells, is one of the most complex scientific challenges in cancer diagnostics. The chemical tunability for achieving this degree of exceptional specificity for extra-cellular biomarker interactions demands the utility of advanced entities and multistep reactions both in solution and in the insoluble state. Thus, this review delineates the chemo-specific substrates, chemical methods, and structure–activity relationships (SARs) of chemical platforms used for isolation and enumeration of CTCs in advancing the relevance of liquid biopsy in cancer diagnostics and disease management. We highlight the synthesis of cell-specific, tumor biomarker-based, chemo-specific substrates utilizing functionalized linkers through chemistry-based conjugation strategies. The capacity of these nano/micro substrates to enhance the cell interaction specificity and efficiency with the targeted tumor cells is detailed. Furthermore, this review accounts for the importance of CTC capture and other downstream processes involving genotypic and phenotypic CTC analysis in real-time for the detection of the early onset of metastases progression and chemotherapy treatment response, and for monitoring progression free-survival (PFS), disease-free survival (DFS), and eventually overall survival (OS) in cancer patients.

Preprint Treatment of triple negative breast cancer (TNBC)-associated metastasis represents an unmet clinical need, and we lack effective therapeutics for a disease that exhibits high relapse rates and associates with poor patient outcomes. Advanced nanosized drug delivery systems may enhance the efficacy of first-line chemotherapeutics by altering drug pharmacokinetics and enhancing tumor/metastasis targeting to signif-icantly improve efficacy and safety. Herein, we propose the application of injectable poly-amino acid-based nanogels (NGs) as a versatile hydrophilic drug delivery platform for the treatment of TNBC lung metastasis. We prepared biocompatible and biodegradable cross-linked NGs from polyglutamic acid (PGA) loaded with the chemotherapeutic agent doxorubicin (DOX). Our optimized synthetic procedures generated NGs of ~100 nm in size and 25 wt% drug loading content that became rapidly internalized in TNBC cell lines and displayed IC50 values comparable to the free form of DOX. Importantly, PGA-DOX NGs significantly inhibited lung metastases and almost completely suppressed lymph node metastases in a spontaneously metastatic orthotopic mouse TNBC model. Overall, our newly developed PGA-DOX NGs represent a potentially effective therapeutic strategy for the treatment of TNBC metastases. A.S-H thanks MC IEF actions (Project 302717). We thank Dr. M. A. Molina for AFM experiments, Ser-vicio SEM Cordoba (Argentina), and Mario Soriano Navarro from the electron microscopy service for Cryo-TEM pictures at CIPF. The authors would also like to thank Dr. Stuart P. Atkinson for his collabo-ration in the revision of the manuscript. This work has been supported by the European Research Council (grant ERC-CoG-2014-648831 “MyNano”), by the Spanish Ministry of Science and Innovation (SAF2013-44848-R, SAF2016-80427-R, RTI2018-099227-B-I00), by a Marie Curie IEF (Project 302717), and the Bundesministerium für Bildung und Forschung (BMBF) through the NanoMatFutur award (13N12561). Part of the equipment employed in this work has been funded by Generalitat Valen-ciana and co-financed with FEDER funds (PO FEDER of Comunitat Valenciana 2014–2020)

Nanomedicine for cancer therapy is a successful tool to diminish the side effect of chemotherapeutics such as paclitaxel (PTX). In this regard, Abraxane®, a human serum albumin (HSA)-based nanomedicine system has shown lesser side effects than Taxol®. However, the large-scale production of HSA protein is limited and expensive, which is traduced in a high cost of the treatments in clinical applications. Thus, the use of easily-available alternative nanocarriers could increment the accessibility of patients to nanomedicine for cancer treatments. Casein is a low-cost protein able to self-assemble into micelles which could efficiently encapsulate PTX into their structure. In this work, the synthesis of chemically crosslinked casein micelles (CCM), used to prepare PTX-based nanoformulations, is presented. CCM@PTX nanoformulations showed promising results in vitro to be applied as nanomedicine for cancer therapy. Thus, the obtained nanoformulations are great candidates to be parenterally administered, accumulate in tumor by passive targeting without leakage of PTX in plasma, and release the drug within the tumor microenvironment, in response to overexpressed proteases such as trypsin. Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Ronco, Ludmila Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Calderón, Marcelo. Polymat; España Fil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

In this work, we report the synthesis of graft copolymers based on casein and N-isopropylacrylamide, which can self-assemble into biodegradable micelles of approximately 80 nm at physiological conditions. The obtained copolymers were degraded by trypsin, an enzyme that is overexpressed in several malignant tumors. Moreover, graft copolymers were able to load doxorubicin (Dox) by ionic interaction with the casein component. In vitro release experiments showed that the in situ assembled micelles can maintain the cargo at plasma conditions but release Dox immediately after their exposition at pH 5.0 and trypsin. Cellular uptake and cytotoxicity assays revealed the efficient delivery to the nucleus and antiproliferative efficacy of Dox in the breast cancer cell line MDA231. Both delivery and therapeutic activity were enhanced in presence of trypsin. Overall, the prepared micelles hold a great potential for their utilization as dual responsive trypsin/pH drug delivery system. Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Ambrosioni, Franco Exequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Nicola, Maribel. Fundación Para El Progreso de la Medicina; Argentina Fil: Jimenez Kairuz, Alvaro Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Unidad de Investigación y Desarrollo en Tecnología Farmacéutica; Argentina Fil: Gatti, Gerardo Alberto. Fundación Para El Progreso de la Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Calderon, Marcelo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Alvarez Igarzabal, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of the combination therapy to mice bearing intracranial murine glioblastoma resulted in marginal therapeutic outcomes, probably due to brain delivery challenges, p53 mutation status, and the aggressive nature of the selected cell line. Nevertheless, this study provides a proof of concept for exploiting regulators of tumor dormancy for glioblastoma therapy. This therapeutic strategy can be exploited for future investigations using a variety of therapeutic entities that manipulate the expression of dormancy-associated genes in glioblastoma as well as in other cancer types.-Tiram, G., Ferber, S., Ofek, P., Eldar-Boock, A., Ben-Shushan, D., Yeini, E., Krivitsky, A., Blatt, R., Almog, N., Henkin, J., Amsalem, O., Yavin, E., Cohen, G., Lazarovici, P., Lee, J. S., Ruppin, E., Milyavsky, M., Grossman, R., Ram, Z., Calderón, M., Haag, R., Satchi-Fainaro, R. Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Hair follicles can serve as an effective reservoir for dermal drug delivery upon the topical application of particulate substances. Here, the follicular penetration of an indodicarbocyanine-labelled thermoresponsive nanogel (189 nm) having a cloud point temperature of 34°C and linked via an acid-labile linker to the model drug indocarbocyanine was investigated. In total, 227 hair follicles of porcine ear skin were examined after topical application of the thermoresponsive nanogels at room temperature (21°C), physiological skin surface temperature (32°C) and core body temperature (37°C) for the follicular penetration depths of indodicarbocyanine and indocarbocyanine using confocal laser scanning microscopy. The results showed a significantly increased mean follicular penetration of the carrier to a depth of 298.8±85.8 μm after incubation at 37°C compared to samples incubated at 21°C and 32°C with mean follicular penetration depths of 202.7±81.7 μm and 219.4±52.9 μm, respectively (p

Despite exciting advances in gene editing, their clinical translation is still hampered by the lack of delivery systems that can encapsulate and deliver gene editing tools like CRISPR-Cas9 or prime editors to the target side. This is particularly challenging in human epithelia, such as the skin and the lung; the latter of which being a mucosal surface that is covered by a mucus layer. In this perspective, the design and biological assessment of delivery systems for gene editing tools like CRISPR in skin and mucosal surfaces are discussed. The current state-of-the-art, current knowledge, and translational gaps, and guide toward improved translation are highlighted.

The thermoresponsive behavior of an elastin-based polymer can be altered by the polymeric macromolecular conformation. Thus, when the elastin basic amino acid sequence VPGVG is used as a pendant group of a poly(phenylacetylene) (PPA) its thermoresponsive behavior in water can be remotely detected through conformational changes on the formed helix. Circular dichroism at different temperatures shows an inversion of the first Cotton effect (450 nm) at 25.8 °C that matches with the cloud point temperature. The elastin-based side-chain poly(phenylacetylene) shows an upper critical solution temperature with low pH and concentration dependency, not expected in elastin-based polymers. It was found that the polymer self-assembles in water into spherical nanoparticles with hydrodynamic diameters of 140 nm at the hydrophobic state. Fundación Segundo Gil Dávila Dahlem Research School, Freie Universität Berlin. Grant Number: Dahlem International PostDocs fellowship Ministerio de Ciencia e Innovación. Grant Number: CTQ2015-70519-P Xunta de Galicia. Grant Number: Centro Singular de Investigación de Galicia Acreditación 2016-2019 European Regional Development Fund. Grant Number: ERDF Bundesministerium für Bildung und Forschung. Grant Number: Nanomatfutur award, 13N12561, Thermonanogele SI

Nanoscaled soft particles, such as nanogels, can be designed to incorporate different types of compounds and release them in a controlled and triggered manner. Thermoresponsive nanogels (tNG), releasing their cargo above a defined temperature, are promising carrier systems for inflammatory skin diseases, where the temperature of diseased skin differs from that of healthy skin areas. In this study a polyglycerol-based tNG with diameter of 156nm was investigated for penetration and release properties upon topical application on ex vivo human skin with intact or disrupted barrier. Furthermore, temperature-triggered effects and the internalization of tNG by skin cells upon translocation to the viable skin layers were analyzed. The investigated tNG were tagged with indodicarbocyanine and loaded with fluorescein, so that fluorescent microscopy and flow cytometry could be used to evaluate simultaneously particle penetration and release of the fluorochrome. Topically applied tNG penetrated into the SC of both intact and disrupted skin explants. Only in barrier-disrupted skin significant amounts of released fluorochrome and tNG penetrated in the epidermis and dermis 2h after topical application. When a thermal trigger was applied by infrared radiation (30s, 3.9mJ/cm2), a significantly higher penetration of tNG in the SC and release of the dye in the epidermis were detected with respect to non-triggered samples. Penetrated tNG particles were internalized by skin cells in both epidermis and dermis. Only few CD1a-positive Langerhans cells associated with tNG were found in the epidermis. However, in the dermis a significant percentage of cells associated with tNG were identified to be antigen presenting cells, i.e. HLA-DR+and CD206+cells. Thus, tNG represent promising carrier systems for the treatment of inflammatory skin diseases, not only because of their improved penetration and controlled release properties, but also because of their ability to effectively reach dermal dendritic cells in barrier-disrupted skin.

Engineered nanogels are of high value for a targeted and controlled transport of compounds due to the ability to change their chemical properties by external stimuli. As it has been indicated that nanogels possess a high ability to penetrate the stratum corneum, it cannot be excluded that nanogels interact with dermal dendritic cells, especially in diseased skin. In this study the potential crosstalk of the thermore-sponsive nanogels (tNGs) with the dendritic cells of the skin was investigated with the aim to determine the immunotoxicological properties of the nanogels. The investigated tNGs were made of dendritic polyglycerol (dPG) and poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)), as polymer conferring thermoresponsive properties. Although the tNGs were taken up, they displayed neither cytotoxic and genotoxic effects nor any induction of reactive oxygen species in the tested cells. Interestingly, specific uptake mechanisms of the tNGs by the dendritic cells were depending on the nanogels cloud point temperature (Tcp), which determines the phase transition of the nanoparticle. The study points to caveolae-mediated endocytosis as being the major tNGs uptake mechanism at 37 degrees C, which is above the Tcp of the tNGs. Remarkably, an additional uptake mechanism, beside caveolae-mediated endocytosis, was observed at 29 degrees C, which is the Tcp of the tNGs. At this temperature, which is characterized by two different states of the tNGs, macropinocytosis was involved as well. In summary, our study highlights the impact of thermoresponsivity on the cellular uptake mechanisms which has to be taken into account if the tNGs are used as a drug delivery system.

Organic macromolecules with dendrimeric architectures are polymeric materials potentially useful as nanocarriers for therapeutic drugs. In this work, we evaluate a series of Newkome-type dendrons in Langmuir and Langmuir-Blodgett films as platforms capable of interacting with a potential antitumoral agent. The nanocomposite is proposed as model for the development of surface mediated drug delivery systems. We were successful in the formation and characterization of pure (dendrons) and composite (drug-dendron) stable and reproducible monolayers, and their transfer to solid substrates. A detailed study of topographic characteristics of the generated surfaces by atomic force microscopy was conducted. Furthermore, we probed dendron monolayer films as anchorage surfaces for mammalian cells. Normal cell attachment and proliferation on the surfaces were observed. No evident cytotoxic effects were detected, demonstrating the adequate biocompatibility of the surfaces. Fil: Dib, Nahir. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química y Física; Argentina Fil: Reviglio, Ana Lucía. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química y Física; Argentina Fil: Fernández, Luciana. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química y Física; Argentina Fil: Morales, Gustavo. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química y Física; Argentina Fil: Santo, Marisa Rosana. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química y Física; Argentina Fil: Otero, Luis Alberto. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Química y Física; Argentina Fil: Alustiza, Fabrisio Eduardo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Liaudat, Ana Cecilia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Bosch, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina Fil: Calderon, Marcelo. Universität zu Berlin; Alemania. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Orgánica; Argentina Fil: Martinelli, Marisa. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina Fil: Strumia, Miriam Cristina. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; Argentina

The adsorption of biomolecules to the surface of nanoparticles (NPs) following administration into biological environments is widely recognized. In particular, the "protein corona" is well understood in terms of formation kinetics and impact upon the biological interactions of NPs. Its presence is an essential consideration in the design of therapeutic NPs. In the present study, the protein coronas of six polymeric nanoparticles of prospective therapeutic use were investigated. These included three colloidal NPs-soft core-multishell (CMS) NPs, plus solid cationic Eudragit RS (EGRS), and anionic ethyl cellulose (EC) nanoparticles-and three nanogels (NGs)-thermoresponsive dendritic-polyglycerol (dPG) nanogels (NGs) and two amino-functionalized dPG-NGs. Following incubation with human plasma, protein coronas were characterized and their biological interactions compared with pristine NPs. All NPs demonstrated protein adsorption and increased hydrodynamic diameters, although the solid EGRS and EC NPs bound notably more protein than the other tested particles. Shifts toward moderately negative surface charges were also observed for all corona bearing NPs, despite varied zeta potentials in their pristine states. While the uptake and cellular adhesion of the colloidal NPs in primary human keratinocytes and human umbilical vein endothelial cells were significantly decreased when bearing the protein corona, no obvious impact was seen in the NGs. By contrast, corona bearing NGs induced marked increases in cytokine release from primary human macrophages not seen with corona bearing colloidal NPs. Despite this, no apparent enhancement to in vitro toxicity was noted. Finally, drug release from EGRS and EC NPs was assessed, where a decrease was seen in the EGRS NPs alone. Together these results provide a direct comparison of the physical and biological impact the protein corona has on NPs of widely varied character and in particular highlights a distinction between the corona's effects on NGs and colloidal NPs.

Nanoparticles (NPs) are widely explored for various biomedical applications to make more efficient therapeutics and to develop advanced diagnostic tools. The majority of NP-based systems that have been proven to successfully achieve therapeutic efficacy in vitro did not pass the in vivo conditions because of adverse effects, which have led to systemic toxicity and an unpredicted long-term outcome. Therefore, several NP-based therapeutic systems face challenges for their applicability in clinical trials. These discrepancies in the biological outcome could originate from the binding of proteins on the surface of NPs, thereby achieving a brand new biological identity. It is fundamentally important to understand the so-called "protein corona" around NPs for the development of successful products for therapeutics as well as in other biomedical applications. This review will focus on studies of protein corona formation onto the soft, organic-based NPs, upon incubation in biological media such as human plasma or serum and their physicochemical characteristics. These studies aim to describe these supramolecular structures in relationship with the resultant effects at the interface that might impact the therapeutic efficacy of the designed NPs.

Versatile, multifunctional nanomaterials for theranostic approaches in cancer treatment are highly on demand in order to increase therapeutic outcomes. Here, we developed thermo-responsive nanogels equipped with the efficient near-infrared (NIR) transducing polymer polypyrrole (PPY) for combinational photothermal and chemotherapeutic therapy along with photoacoustic imaging ability. Long-term stability and water-dispersibility of PPY was achieved using semi-interpenetration method for in situ polymerization of PPY into hydrophilic thermo-responsive nanogels. The semi-interpenetrated nanogels of spherical shape and with hydrodynamic sizes of around 200 nm retained the temperature response behaviour and exhibit excellent photothermal transducing abilities in the NIR region. The PPY nanogels served as photoacoustic contrast agents, which allowed determination of biodistribution profiles ex vivo. In addition, we developed a new method for biodistribution determination based on the photothermal response of the nanogels with an accuracy down to 12.5 μg/mL. We examined the ability of the nanogels as photothermal agents and drug delivery systems in vitro and in vivo. We showed that they efficiently inhibit tumor growth with combinational effects of chemotherapeutics and photothermal treatment. Our work encourages further exploration of nanogels as functional stabilizing matrix for photothermal transducers and their application as drug delivery devices in combination with photothermal therapy and imaging. Fil: Theune, Loryn E.. Freie Universität Berlin; Alemania Fil: Buchmann, Jens. Martin-universität Halle-wittenberg; Alemania Fil: Wedepohl, Stefanie. Freie Universität Berlin; Alemania Fil: Molina, María Alejandra. Freie Universität Berlin; Alemania. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados. - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Tecnologías Energéticas y Materiales Avanzados; Argentina Fil: Laufer, Jan. Martin-universität Halle-wittenberg; Alemania Fil: Calderón, Marcelo. Freie Universität Berlin; Alemania

Physiological barriers inside of tumor tissue often result in poor interstitial penetration and heterogeneous intratumoral distribution of nanoparticle-based drug delivery systems (DDS). Novel, matrix metalloproteinase (MMP)-sensitive peptide-crosslinked nanogels (pNGs) as multistage DDS are reported with a beneficial size reduction property to promote the process of deep tissue penetration. Methods: The presented pNGs are based on a dendritic polyglycerol (dPG) scaffold crosslinked by a modified MMP-sensitive fluorogenic peptide. The crosslinker integrates degradability in response to proteases present in the tumor microenvironment. Surfactant-free, inverse nanoprecipitation is employed to prepare the nanogels using strain-promoted click chemistry. The size and crosslinking density of the pNGs are controlled by the functionalization degree of dPG with cyclooctyne groups and by the peptide crosslinker fraction. The intrinsic reporter moiety of the crosslinker was used to study the influence of pNG compositions on the degradation profile. The therapeutic drug Doxorubicin was conjugated through a pH-sensitive linkage to dPG to form a multistage DDS. The penetration behavior of the pNGs was studied using agarose matrix and multicellular tumor spheroids (MCTS). Results: Nanogel sizes were controlled in the range of 150-650 nm with narrow size distributions and varying degrees of crosslinking. The pNGs showed stability in PBS and cell media but were readily degraded in the presence of MMP-7. The crosslinking density influenced the degradation kinetic mediated by MMP-7 or cells. Stable conjugation of DOX at physiological pH and controlled drug release at acidic pH were observed. The digestions of nanogels lead to a size reduction to polymer-drug fragments which efficiently penetrated into agarose gels. Moreover, the degradable multistage pNGs demonstrated deeper penetration into MCTS as compared to their non-degradable counterparts. Thus, degradable pNGs were able to deliver their cargo and efficiently reduce the cell viability in MCTS. Conclusion: The triggered size reduction of the pNGs by enzymatic degradation can facilitate the infiltration of the nanocarrier into dense tissue, and thereby promote the delivery of its cargo.

The development of polymers with low toxicity and efficient gene delivery remains a significant barrier of nonviral gene therapy. Modification and tuning of chemical structures of carriers is an attractive strategy for efficient nucleic acid delivery. Here, polyplexes consisting of plasmid DNA (pDNA) and dodecylated or non-dodecylated polysuccinimide (PSI)-based polycations are designed, and their transfection ability into HeLa cells is investigated by green fluorescent protein (GFP) expressing cells quantification. All cationic polymers show lower cytotoxicity than those of branched polyethyleneimine (bPEI). PSI and bPEI-based polyplexes have comparable physicochemical properties such as size and charge. Interestingly, a strong interaction between dodecylated polycations and pDNA caused by the hydrophobic moiety is observed in dodecylated PSI derivatives. Moreover, the decrease of GFP expression is associated with lower dissociation of pDNA from polyplexes according to the heparin displacement assay. Besides, a hydrophobization of PSI cationic derivatives with dodecyl side chains can modulate the integrity of polyplexes by hydrophobic interactions, increasing the binding between the polymer and the DNA. These results provide useful information for designing polyplexes with lower toxicity and greater stability and transfection performance. This work was developed within the framework of National Institute of Science and Technology of Pharmaceutical Nanotechnology (INCT-Nanofarma), which is supported by "Fundacao de Amparo a Pesquisa do Estado de Sao Paulo" (FAPESP, Brazil, Grant #14/14/50928-2) and "Conselho Nacional de Pesquisa" (CNPQ, Brazil, Grant #465687/2014-8). M.H.K. was the recipient of a CAPES and DAAD scholarship. Technical support by University of Sao Paulo and Freie Universitat Berlin is acknowledged.

Transdermal immunization is highly attractive because of the skin's accessibility and unique immunological characteristics. However, it remains a relatively unexplored route of administration because of the great difficulty of transporting antigens past the outermost layer of skin, the stratum corneum. In this article, the abilities of three poly(N-vinylcaprolactam) (PVCL)-based thermoresponsive assemblies - PVCL hydrogels and nanogels plus novel film forming PVCL/acrylic nanogels - to act as protein delivery systems were investigated. Similar thermal responses were observed in all systems, with transition temperatures close to 32 °C, close to that of the skin surface. The investigated dermal delivery systems showed no evidence of cytotoxicity in human fibroblasts and were able to load and release ovalbumin (OVA), a well-studied antigen, in a temperature-dependent manner in vitro. The penetration of OVA into ex vivo human skin following topical application was evaluated, where enhanced skin delivery was seen for the OVA-loaded PVCL systems relative to administration of the protein alone. The distinct protein release and skin penetration profiles observed for the different PVCL assemblies were here discussed on the basis of their structural differences. Fil: Sonzogni, Ana Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Yealland, Guy. Universidad Libre de Berlin; Alemania Fil: Kar, Mrityunjoy. Universidad Libre de Berlin; Alemania Fil: Wedepohl, Stefanie. Universidad Libre de Berlin; Alemania Fil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: González, Verónica Doris Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Hedtrich, Sarah. Universidad Libre de Berlin; Alemania Fil: Calderon, Marcelo. Universidad Libre de Berlin; Alemania Fil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

Semi-interpenetrated nanogels (NGs) able to release and sense diclofenac (DIC) have been designed to act as photothermal agents with the possibility to ablate cancer cells using mild-temperatures (45 °C). Combining mild heat treatments with simultaneous chemotherapy appears as a very promising therapeutic strategy to avoid heat resistance or damaging the surrounding tissues. Particularly, NGs consisted on a poly(N-isopropylacrylamide) (PNIPAM) and dendritic polyglycerol (dPG) mesh containing a semi-interpenetrating network (SIPN) of poly(hydroxymethyl 3,4-ethylenedioxythiophene) (PHMeEDOT). The PHMeEDOT acted as photothermal and conducting agent, while PNIPAM-dPG NG provided thermoresponsivity and acted as stabilizer. We studied how semi-interpenetration modified the physicochemical characteristics of the thermoresponsive SIPN NGs and selected the best condition to generate a multifunctional photothermal agent. The thermoswitchable conductiveness of the multifunctional NGs and the redox activity of DIC could be utilized for its electrochemical detection. Besides, as proof of the therapeutic concept, we investigated the combinatorial effect of photothermal therapy (PTT) and DIC treatment using the HeLa cancer cell line in vitro. Within 15 min NIR irradiation without surpassing 45 °C we were able to kill 95% of the cells, demonstrating the potential of SIPN NGs as drug carriers, sensors and agents for mild PTT.

Glioblastoma Multiforme (GBM) is one of the most aggressive forms of all cancers. The median survival with current standard-of-care radiation and chemotherapy is about 14months. GBM is difficult to treat due to heterogeneity in cancer cell population. MicroRNA-based drugs have rapidly become a vast and burgeoning field due to the ability of a microRNA (miRNA) to target many genes involved in key cellular pathways. However, in vivo delivery of miRNA remains a crucial challenge for its therapeutic success. To bypass this shortcoming, we designed polymeric nanogels (NGs), which are based on a polyglycerol-scaffold, as a new strategy of miRNA delivery for GBM therapy. We focused on miR-34a, which is known for its key role in important oncogenic pathways and its tumor suppression ability in GBM and other cancers. We evaluated the capability of six NG derivatives to complex with miR-34a, neutralize its negative charge and deliver active miRNA to the cell cytoplasm. Human U-87 MG GBM cells treated with our NG-miR-34a nano-polyplexes showed remarkable downregulation of miR-34a target genes, which play key roles in the regulation of apoptosis and cell cycle arrest, and induce inhibition of cells proliferation and migration. Administration of NG-miR-34a nano-polyplexes to human U-87 MG GBM-bearing SCID mice significantly inhibited tumor growth as opposed to treatment with NG-negative control miR polyplex or saline. The comparison between different polyplexes highlighted the key features for the rational design of polymeric delivery systems for oligonucleotides. Taken together, we expect that this new therapeutic approach will pave the way for safe and efficient therapies for GBM.

Various specially designed poly(N-isopropylacrylamide-co-acrylic acid) (NIPA-co-AAc) nanogels (NGs) with different NIPA/AAc molar composition were synthesized by precipitation/dispersion polymerization and evaluated as carriers for drug delivery systems (DDSs) of doxorubicin hydrochloride (DOXOHCl) for cancer therapies. The NGs presented excellent dispersability in physiological environments (pH 7.4 and 5 at 37 C), as shown by dynamic light scattering (DLS). Moreover, the NGs exhibited high drug loading capacity and efficiency due to the ionic interaction of the cationic drug with the anionic NGs. NGDOXO HCl formulation presented excellent dispersability in water and minimal leakageof the cargo at plasma simulated medium (pH 7.4 and 0.14 M NaCl) at 37 C and a triggered release at lysosomal simulated medium (pH 5 and 0.14 M NaCl). This release behavior together with their size and the low cytotoxicity determined by the MTT assay converts these NGs in great candidates for their application as carriers in cancer therapies based on the enhanced permeability and retention effect (EPR) with drug pH-triggered release after endocytosis in tumor cells. Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina Fil: Molina, Maria. Freie Universität Berlin. Institut für Chemie und Biochemie; Alemania Fil: Wedepohl, Stefanie. Freie Universität Berlin. Institut für Chemie und Biochemie; Alemania Fil: Alvarez Igarzabal, Cecilia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentina Fil: Calderon, Marcelo. Freie Universität Berlin. Institut für Chemie und Biochemie; Alemania Fil: Gugliotta, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentina

The presence of dormant, microscopic cancerous lesions poses a major obstacle for the treatment of metastatic and recurrent cancers. While it is well-established that microRNAs play a major role in tumorigenesis, their involvement in tumor dormancy has yet to be fully elucidated. We established and comprehensively characterized pairs of dormant and fast-growing human osteosarcoma models. Using these pairs of mouse tumor models, we identified three novel regulators of osteosarcoma dormancy: miR-34a, miR-93, and miR-200c. This report shows that loss of these microRNAs occurs during the switch from dormant avascular into fast-growing angiogenic phenotype. We validated their downregulation in patients' tumor samples compared to normal bone, making them attractive candidates for osteosarcoma therapy. Successful delivery of miRNAs is a challenge; hence, we synthesized an aminated polyglycerol dendritic nanocarrier, dPG-NH2, and designed dPG-NH2-microRNA polyplexes to target cancer. Reconstitution of these microRNAs using dPG-NH2 polyplexes into Saos-2 and MG-63 cells, which generate fast-growing osteosarcomas, reduced the levels of their target genes, MET proto-oncogene, hypoxia-inducible factor 1α, and moesin, critical to cancer angiogenesis and cancer cells' migration. We further demonstrate that these microRNAs attenuate the angiogenic capabilities of fast-growing osteosarcomas in vitro and in vivo. Treatment with each of these microRNAs using dPG-NH2 significantly prolonged the dormancy period of fast-growing osteosarcomas in vivo. Taken together, these findings suggest that nanocarrier-mediated delivery of microRNAs involved in osteosarcoma tumor-host interactions can induce a dormant-like state.