PET Imaging of Self-Assembled 18 F-Labelled Pd2 L4 Metallacages for Anticancer Drug Delivery.

To advance the design of self-assembled metallosupramolecular architectures as new generation theranostic agents, the synthesis of 18 F-labelled [Pd2 L4 ]4+ metallacages is reported. Different spectroscopic and bio-analytical methods support the formation of the host-guest cage-cisplatin complex. The biodistribution profiles of one of the cages, alone or encapsulating cisplatin have been studied by PET/CT imaging in healthy mice in vivo, in combination to ICP-MS ex vivo.

Nanoparticles for Triple Drug Release for Combined Chemo- and Photodynamic Therapy.

A pH-responsive drug delivery system (DDS) based on mesoporous silica nanoparticles (MSNs) has been prepared for the delivery of three anticancer drugs with different modes of action. The novelty of this system is its ability to combine synergistic chemotherapy and photodynamic therapy. A photoactive conjugate of a phthalocyanine (Pc) and a topoisomerase I inhibitor (topo-I), namely camptothecin (CPT), linked by a poly(ethylene glycol) (PEG) chain has been synthesized and then loaded into the mesopores of MSNs. Doxorubicin (DOX), which is a topoisomerase II inhibitor (topo-II), has also been covalently anchored to the outer surface of the MSNs through a dihydrazide PEG linker. In the acidic environment of tumor cells, selective release of the three drugs takes place. In vitro studies have demonstrated the endocytosis of the system into HeLa and HepG2 cells, and the subsequent release of the three drugs into the cytoplasm and nucleus. Furthermore, the cytotoxic effect of DOX, CPT and Pc has been assessed in vitro before and upon light irradiation.

Analytical Parameters of a Novel Glucose Biosensor Based on Grafted PFM as a Covalent Immobilization Technique.

Bioanalytical methods, in particular electrochemical biosensors, are increasingly used in different industrial sectors due to their simplicity, low cost, and fast response. However, to be able to reliably use this type of device, it is necessary to undertake in-depth evaluation of their fundamental analytical parameters. In this work, analytical parameters of an amperometric biosensor based on covalent immobilization of glucose oxidase (GOx) were evaluated. GOx was immobilized using plasma-grafted pentafluorophenyl methacrylate (pgPFM) as an anchor onto a tailored HEMA-co-EGDA hydrogel that coats a titanium dioxide nanotubes array (TiO2NTAs). Finally, chitosan was used to protect the enzyme molecules. The biosensor offered outstanding analytical parameters: repeatability (RSD = 1.7%), reproducibility (RSD = 1.3%), accuracy (deviation = 4.8%), and robustness (RSD = 2.4%). In addition, the Ti/TiO2NTAs/ppHEMA-co-EGDA/pgPFM/GOx/Chitosan biosensor showed good long-term stability; after 20 days, it retained 89% of its initial sensitivity. Finally, glucose concentrations of different food samples were measured and compared using an official standard method (HPLC). Deviation was lower than 10% in all measured samples. Therefore, the developed biosensor can be considered to be a reliable analytical tool for quantification measurements.

Continuous-flow, microfluidic, qRT-PCR system for RNA virus detection.

One of the main challenges in the diagnosis of infectious diseases is the need for rapid and accurate detection of the causative pathogen in any setting. Rapid diagnosis is key to avoiding the spread of the disease, to allow proper clinical decisions to be made in terms of patient treatment, and to mitigate the rise of drug-resistant pathogens. In the last decade, significant interest has been devoted to the development of point-of-care reverse transcription polymerase chain reaction (PCR) platforms for the detection of RNA-based viral pathogens. We present the development of a microfluidic, real-time, fluorescence-based, continuous-flow reverse transcription PCR system. The system incorporates a disposable microfluidic chip designed to be produced industrially with cost-effective roll-to-roll embossing methods. The chip has a long microfluidic channel that directs the PCR solution through areas heated to different temperatures. The solution first travels through a reverse transcription zone where RNA is converted to complementary DNA, which is later amplified and detected in real time as it travels through the thermal cycling area. As a proof of concept, the system was tested for Ebola virus detection. Two different master mixes were tested, and the limit of detection of the system was determined, as was the maximum speed at which amplification occurred. Our results and the versatility of our system suggest its promise for the detection of other RNA-based viruses such as Zika virus or chikungunya virus, which constitute global health threats worldwide. Graphical abstract Photograph of the RT-PCR thermoplastic chip.

Stable 5,5'-Substituted 2,2'-Bipyrroles: Building Blocks for Macrocyclic and Materials Chemistry.

The preparation and characterization of a family of stable 2,2'-bipyrroles substituted at positions 5 and 5' with thienyl, phenyl, TMS-ethynyl, and vinyl groups is reported herein. The synthesis of these new bipyrroles comprises three steps: formation of the corresponding 5,5'-unsubstituted bipyrrole, bromination, and Stille or Suzuki coupling. The best results in the coupling are obtained using the Stille reaction under microwave irradiation. The new compounds have been fully characterized by UV-vis absorption, fluorescence, and IR spectroscopies and cyclic voltammetry. X-ray single-crystal analysis of four of the synthesized bipyrroles indicates a trans coplanar geometry of the pyrrole rings. Furthermore, the substituents at positions 5,5' remain coplanar to the central rings. This particular geometry extends the π-conjugation of the systems, which is in agreement with a red-shifting observed for the λmax of the substituted molecules compared to the unsubstituted bipyrrole. All of these new compounds display a moderate fluorescence. In contrast with unsubstituted bipyrroles, these bipyrroles are endowed with a high chemical and thermal stability and solubility in organic solvents.

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations.

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols.

BACKGROUND: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. METHODS: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. RESULTS: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. CONCLUSION: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology.

Low-cost, real-time, continuous flow PCR system for pathogen detection.

In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device.

Novel 18F labeling strategy for polyester-based NPs for in vivo PET-CT imaging.

Drug-loaded nanocarriers and nanoparticulate systems used for drug release require a careful in vivo evaluation in terms of physicochemical and pharmacokinetic properties. Nuclear imaging techniques such as positron emission tomography (PET) are ideal and noninvasive tools to investigate the biodistribution and biological fate of the nanostructures, but the incorporation of a positron emitter is required. Here we describe a novel approach for the (18)F-radiolabeling of polyester-based nanoparticles. Our approach relies on the preparation of the radiolabeled active agent 4-[(18)F]fluorobenzyl-2-bromoacetamide ([(18)F]FBBA), which is subsequently coupled to block copolymers under mild conditions. The labeled block copolymers are ultimately incorporated as constituent elements of the NPs by using a modified nano coprecipitation method. This strategy has been applied in the current work to the preparation of peptide-functionalized NPs with potential applications in drug delivery. According to the measurements of particle size and zeta potential, the radiolabeling process did not result in a statistically significant alteration of the physicochemical properties of the NPs. Moreover, radiochemical stability studies showed no detachment of the radioactivity from NPs even at 12 h after preparation. The radiolabeled NPs enabled the in vivo quantification of the biodistribution data in rats using a combination of imaging techniques, namely, PET and computerized tomography (CT). Low accumulation of the nanoparticles in the liver and their elimination mainly via urine was found. The different biodistribution pattern obtained for the "free" radiolabeled polymer suggests chemical and radiochemical integrity of the NPs under investigation. The strategy reported here may be applied to any polymeric NPs containing polymers bearing a nucleophile, and hence our novel strategy may find application for the in vivo and noninvasive investigation of a wide range of NPs.

Tailoring the LCST of thermosensitive hydrogel thin films deposited by iCVD.

Using the iCVD (initiated chemical vapor deposition) polymerization technique, we generated a library of thermosensitive thin film hydrogels in the physiological temperature range. The library shows how a specific hydrogel with a desired temperature response can be synthesized via the copolymerization of three main components: (a) the main thermosensitive monomer, which determines the temperature range of the LCST; (b) the comonomer, which modulates the temperature according to its hydrophilic/hydrophobic behavior; and (c) the cross-linker, which determines the swelling degree and the polymer chain mobility of the resulting hydrogel. The thermosensitive thin films included in the library have been characterized by the water contact angle (WCA), revealing a switchable hydrophobic/hydrophilic behavior depending on the temperature and a decrease in the WCA with the incorporation of hydrophilic moieties. Moreover, a more accurate characterization by quartz crystal microbalance (QCM) is performed. With temperature and flow control, the switchable swelling properties of the thermosensitive thin films (due to the polymer mixture transition) can be recorded and analyzed in order to study the effects of the comonomer moieties on the lower critical solution temperature (LCST). Thus, the LCST tailoring method has been successfully used in this paper, and thermoresponsive thin films (50 nm in thickness) have been deposited by iCVD, exhibiting LCSTs in the 32-49 °C range. Due to the presented method's ability to tailor the LCST in the physiological temperature range, the developed thermoresponsive films present potential biosensing and drug delivery applications in the biomedical field.

Development of high drug loaded and customizing novel nanoparticles for modulated and controlled release of Paclitaxel.

PURPOSE: The objective of this study was to develop a custom-tailored polymeric drug delivery system for paclitaxel, employing a novel biodegradable block co-polymer (P), intended to be intravenously administered, capable of improving therapeutic index of the drug and devoid of the adverse effect of an uncontrolled release. METHODS: Paclitaxel loaded nanoparticles (PTX-NPs) were prepared by a modified nanoprecipitation method and emulsification-solvent evaporation method. Our approach involves a focusing on the formulation parameters that can be modified in order to obtain completely customized NPs in terms of size, zeta-potential, drug content and release profile. The biocompatibility and anti-proliferative efficiency of PTX-NPs against glioblastoma cell line were evaluated in vitro by MTS. RESULTS: All formulations showed spherical nanometric (<200 nm), monodisperse (~0.1), Poly (Ethylene Glycol) (PEG)-coated and negatively charged particles. Selected NPs revealed higher PTX content (up to 24%) in comparison with polyester-based NPs. The release behaviour of PTX from the developed NPs exhibited an approximately first-order profile, without initial burst and characterized by a slow and constant release. Hydrophobic character of the NPs can be set in order to achieve a slower and more controlled release for a prolonged period of time. PTX-NPs were hemocompatible and had significant in vitro anti-tumoral activity against human primary glioblastoma cell line (U-87 MG); cytotoxicity was in time- and drug concentration- dependent manner. CONCLUSIONS: The developed drug delivery system proved to be suitable for intravenous administration. NPs characteristics can be customized to obtain high PTX loaded NPs that can improve therapeutic index and avoid an uncontrolled release.

PExM: polyplex expansion microscopy for cell trafficking studies.

Nanomedicine is a field at the intersection of nanotechnology and medicine, promising due to its potential to revolutionize healthcare. Despite its long trajectory, there is still a long road ahead for its full development, and smart design of nanomedicines is still a challenge. Among other problems, this is due to the scarcity of tools available for the precise visualization and comprehension of nano-bio interactions, impeding progress towards the clinical phase. One of the developed tools that stands out to be a strong nanoscopy technique for studying nano-delivery systems within cellular environments is expansion microscopy (ExM). This technique was used for tissue and cell expansion and most recently for lipid molecule expansion inside cells. Herein, we present for the first time polyplex expansion microscopy (PExM); a comprehensive examination of ExM as an already developed technique, but adapted for expanding polymer based nanocarriers, in particular polyplexes within cells, allowing the analysis of their trafficking. With our method set up, PExM will be extensively used for the study of polyplex nanoparticle cell trafficking, becoming a high-resolution technique which can also be applied to primary amine containing polymeric nanoparticles without requiring expensive super-resolution microscopes.

Nucleic acid-loaded poly(beta-aminoester) nanoparticles for cancer nano-immuno therapeutics: the good, the bad, and the future.

Immunotherapy has emerged as a promising approach to cancer treatment, offering improved survival rates and enhanced patients' quality of life. However, realizing the full potential of immunotherapy in clinical practice remains a challenge, as there is still plenty of room for modulating the complexity of the human immune system in favor of an antitumor immunogenicity. Nanotechnology, with its unique properties, holds promise in augmenting the efficacy of cancer immunotherapies in biotherapeutic protection and site- and time-controlled delivery of the immune modulator biologicals. Polymeric nanoparticles are promising biomaterials among different nanocarriers thanks to their robustness, versatility, and cost-efficient design and production. This perspective paper overviews critical concepts in nanometric advanced delivery systems applied to cancer immunotherapy. We focus on a detailed exploration of the current state of the art and trends in using poly(beta-aminoester) (pBAE) polymers for nucleic acid-based antitumor immunotherapies. Through different examples of the use of pBAE polymers reported in the literature, we revise the main advantages these polymers offer and some challenges to overcome. Finally, the paper provides insights and predictions on the path toward the clinical implementation of cancer nano-immunotherapies, highlighting the potential of pBAE polymers for advancements in this field.

Stealth mRNA nanovaccines to control lymph node trafficking.

mRNA-based vaccines symbolize a new paradigm shift in personalized medicine for the treatment of infectious and non-infectious diseases. However, the reactogenicity associated with the currently approved formulations limits their applicability in autoinflammatory disorders, such as tumour therapeutics. In this study, we present a delivery system showing controlled immunogenicity and minimal non-specific inflammation, allowing for selective delivery of mRNA to antigen presenting cells (APCs) within the medullary region of the lymph nodes. Our platform offers precise control over the trafficking of nanoparticles within the lymph nodes by optimizing stealth and targeting properties, as well as the subsequent opsonization process. By targeting specific cells, we observed a potent adaptive and humoral immune response, which holds promise for preventive and therapeutic anti-tumoral vaccines. Through spatial programming of nanoparticle distribution, we can promote robust immunization, thus improving and expanding the utilization of mRNA vaccines. This innovative approach signifies a remarkable step forward in the field of targeted nanomedicine.

Designing Polyelectrolyte Microneedles Based on Borylated Poly(ß-aminoester) Polymers To Enhance Transdermal pH-Controlled Delivery of Nucleic Acids.

The use of transdermal delivery for nucleic acid administration is an interesting approach to overcoming limitations of systemic administration routes, such as first-pass effects, the painful needle injection, or their poor biodistribution. Thus, the use of a microneedle-based patch could represent a turning point for nucleic acid delivery, thanks to the possibility of self-administration of the actives in a painless and easy procedure. However, the design of transdermal systems with a higher degree of precision release is a clear need that has not been fully resolved. Committed to tackling this challenge, we present here a microneedle patch that involves a smart delivery system supported by the well-established ability of boronic acid to interact with carbohydrates in a pH-dependent manner. This system builds up a multilayer structure over a solid microneedle platform whose surface has been modified to immobilize glucosamine units that are able to interact with an oligopeptide-end terminated poly(ß-aminoester) that presents a 4-carboxy-3-fluorophenylboronic acid (Bor-pBAE). Thus, sequential layers of the Bor-pBAE and plasmid DNA have been assembled, thanks to the ability of the polymer to interact with the nucleic acid at a basic pH and then gradually release the plasmid under two different conditions of pH (the physiological pH = 7.4 and the acidic pH = 5.1). We set up the design and implementation of this first proof of concept while demonstrating microneedles' safety and functionality. Additionally, we have shown the efficacy of the construct to express the encoded genes in model cell lines. In conclusion, we have established the basis to confirm that this generation of borylated poly(ß-aminoesters) holds great promise as a transdermal local nucleic acid delivery system.

Liver-targeted nanoparticles delivering nitric oxide reduce portal hypertension in cirrhotic rats.

Nitric oxide (NO) is a small vasodilator playing a key role in the pathogenesis of portal hypertension. Here, we assessed the potential therapeutic effect of a NO donor targeted to the liver by poly(beta-amino ester) nanoparticles (pBAE NPs) in experimental cirrhosis. Retinol-functionalized NO donor pBAE NPs (Ret pBAE NPs) were synthetized with the aim of actively targeting the liver. Administration of Ret pBAE NPs resulted in uptake and transfection by the liver and spleen. NPs were not found in other organs or the systemic circulation. Treatment with NO donor Ret pBAE NPs (30 mg/ kg body weight) significantly decreased aspartate aminotransferase, lactate dehydrogenase and portal pressure (9.75 ± 0.64 mmHg) compared to control NPs (13.4 ± 0.53 mmHg) in cirrhotic rats. There were no effects on mean arterial pressure and cardiac output. Liver-targeted NO donor NPs reduced collagen fibers and steatosis, activation of hepatic stellate cells and mRNA expression of profibrogenic and proinflammatory genes. Finally, Ret pBAE NPs displayed efficient transfection in human liver slices. Overall, liver-specific NO donor NPs effectively target the liver and mitigated inflammation and portal hypertension in cirrhotic rats. The use of Ret pBAE may prove to be an effective therapeutic strategy to treat advanced liver disease.

In Vivo Efficacy of an Adhesive Bioresorbable Patch to Treat Aortic Dissections.

Endovascular repair of aortic dissection still presents significant limitations. Preserving the mechanical and biological properties set by the aortic microstructure is critical to the success of implantable grafts. In this paper, we present the performance of an adhesive bioresorbable patch designed to cover the entry tear of aortic dissections. We demonstrate the power of using a biomimetic scaffold in a vascular environment.

Comparison of two different plasma surface-modification techniques for the covalent immobilization of protein monolayers.

The immobilization of biologically active species is crucial for the fabrication of smart bioactive surfaces. For this purpose, plasma polymerization is frequently used to modify the surface nature without affecting the bulk properties of the material. Thus, it is possible to create materials with surface functional groups that can promote the anchoring of all kinds of biomolecules. Different methodologies in protein immobilization have been developed in recent years, although some drawbacks are still not solved, such as the difficulties that some procedures involve and/or the denaturalization of the protein due to the immobilization process. In this work, two different strategies to covalently attach bovine serum albumin (BSA) protein are developed. Both techniques are compared in order to understand how the nature of the surface modification affects the conformation of the protein upon immobilization.

Poly(ß-amino ester)s-Based Delivery Systems for Targeted Transdermal Vaccination.

Nucleic acid vaccines have become a transformative technology to fight emerging infectious diseases and cancer. Delivery of such via the transdermal route could boost their efficacy given the complex immune cell reservoir present in the skin that is capable of engendering robust immune responses. We have generated a novel library of vectors derived from poly(ß-amino ester)s (PBAEs) including oligopeptide-termini and a natural ligand, mannose, for targeted transfection of antigen presenting cells (APCs) such as Langerhans cells and macrophages in the dermal milieu. Our results reaffirmed terminal decoration of PBAEs with oligopeptide chains as a powerful tool to induce cell-specific transfection, identifying an outstanding candidate with a ten-fold increased transfection efficiency over commercial controls in vitro. The inclusion of mannose in the PBAE backbone rendered an additive effect and increased transfection levels, achieving superior gene expression in human monocyte-derived dendritic cells and other accessory antigen presenting cells. Moreover, top performing candidates were capable of mediating surface gene transfer when deposited as polyelectrolyte films onto transdermal devices such as microneedles, offering alternatives to conventional hypodermic administration. We predict that the use of highly efficient delivery vectors derived from PBAEs could advance clinical translation of nucleic acid vaccination over protein- and peptide-based strategies.

Improving Glass Transition Temperature and Toughness of Epoxy Adhesives by a Complex Room-Temperature Curing System by Changing the Stoichiometry.

The glass transition temperature (Tg) of room-temperature curing epoxy adhesives is limited by the temperature used during curing. It is already known that the excess of epoxy groups can undergo a homopolymerization reaction initiated by tertiary amines at elevated temperatures, resulting in an increase in Tg. However, there is no evidence of this reaction occurring at room temperature. In the present work, the influence of formulation stoichiometry on Tg and mechanical properties was investigated. Dynamomechanical, rheological and mechanical properties of epoxy adhesives were determined by DSC, DMA, rheometer and tensile and shear strength testing. It has been probed that an excess of epoxy resin combined with a complex curing system composed of a primary amine, a polymercaptan and a tertiary amine leads to an increase in Tg up to 70 °C due to the homopolymerization reaction that takes place at room temperature. However, as the excess of epoxy resin is increased, gel time becomes slower. Regarding mechanical properties, it has been proven that an excess of epoxy resin provides a tighter and tougher material but maintains flexibility of the stoichiometric formulation, which is meant to enhance the resistance to impact-type forces, thermal shock and thermal cycling.