In-Hydrogel Cell-Free Protein Expression System as Biocompatible and Implantable Biomaterial.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

Decellularization of xenografted tumors provides cell-specific in vitro 3D environment.

In vitro cell culture studies are common in the cancer research field, and reliable biomimetic 3D models are needed to ensure physiological relevance. In this manuscript, we hypothesized that decellularized xenograft tumors can serve as an optimal 3D substrate to generate a top-down approach for in vitro tumor modeling. Multiple tumor cell lines were xenografted and the formed solid tumors were recovered for their decellularization by several techniques and further characterization by histology and proteomics techniques. Selected decellularized tumor xenograft samples were seeded with the HCC1806 human triple-negative breast cancer (TNBC) basal-like subtype cell line, and cell behavior was compared among them and with other control 2D and 3D cell culture methods. A soft treatment using Freeze-EDTA-DNAse allows proper decellularization of xenografted tumor samples. Interestingly, proteomic data show that samples decellularized from TNBC basal-like subtype xenograft models had different extracellular matrix (ECM) compositions compared to the rest of the xenograft tumors tested. The in vitro recellularization of decellularized ECM (dECM) yields tumor-type-specific cell behavior in the TNBC context. Data show that dECM derived from xenograft tumors is a feasible substrate for reseeding purposes, thereby promoting tumor-type-specific cell behavior. These data serve as a proof-of-concept for further potential generation of patient-specific in vitro research models.

A carbon-based nanocarrier for efficient gene delivery.

Aim: Several types of nanocarriers, most of which show significant cytotoxicity, have been developed to overcome the problem of gene-delivery barriers. Biocompatibility, low toxicity and water solubility of carbon nanodots (CNDs) are major advantages that recommend them as delivery systems. Materials & methods: We present a simple method to produce positively charged CNDs. Ethanolamine, ethylenediamine and hydrogen peroxide were utilized to synthesize these CNDs. Results & conclusion: Our results indicated that delivery of the CND-siGFP complex led to significant switching-off of the fluorescence of the GFP-expressing A549 cell. Next, the A549 cells were transfected with siRNA against BiP, which is a pivotal protein in the chemotherapy resistance of cancer cells. The expression levels of BiP decreased remarkably.

Metal Nanoparticles/MoS2 Surface-Enhanced Raman Scattering-Based Sandwich Immunoassay for α-Fetoprotein Detection.

The detection of cancer biomarkers at an early stage of tumor development is vital for effective diagnosis and treatment of cancer. Current diagnostic tools can often detect cancer only when the biomarker levels are already too high, so that the tumors have spread and treatments are less effective. It is urgent therefore to develop highly sensitive assays for the detection of such biomarkers at the lowest possible concentration. In this context, we developed a sandwich immunoassay based on surface-enhanced Raman scattering (SERS) for the ultrasensitive detection of α-fetoprotein (AFP), which is typically present in human serum as a biomarker indicative of early stages of hepatocellular carcinoma. In the immunoassay design, molybdenum disulfide (MoS2) modified with a monoclonal antibody was used as a capture probe for AFP. A secondary antibody linked to an SERS-encoded nanoparticle was employed as the Raman signal reporter, that is, the transducer for AFP detection. The sandwich immunocomplex "capture probe/target/SERS tag" was deposited on a silicon wafer and decorated with silver-coated gold nanocubes to increase the density of "hot spots" on the surface of the immunosensor. The developed SERS immunosensor exhibits a wide linear detection range (1 pg mL-1 to 10 ng mL-1) with a limit of detection as low as 0.03 pg mL-1 toward AFP with good reproducibility (RSD < 6%) and stability. These parameters demonstrate that the proposed immunosensor has the potential to be used as an analytical platform for the detection of early-stage cancer biomarkers in clinical applications.

Enhancement of cisplatin sensitivity in human breast cancer MCF-7 cell line through BiP and 14-3-3ζ co-knockdown.

Cisplatin treatment confers the relative resistance to MCF-7 cells as compared to other breast cancer cell lines. One principal reason is that chemotherapeutic agents induce autophagy in these cells to inhibit apoptosis. Binding immunoglobulin protein (BiP), a master regulator of unfolded protein response (UPR) and 14-3-3ζ are two critical proteins upregulated in breast cancer rendering resistance to anticancer drugs. They also play pivotal roles in autophagy with crosstalk with the apoptotic pathways of UPR through certain regulators. Thus, BiP and 14-3-3ζ were selected as the candidate targets to enhance cell death and apoptosis. First, cisplatin resistance was induced and determined by MTT assay and qPCR in MCF-7 cells. Then, the apoptosis axis of UPR was activated by knocking down either BiP or 14-3-3ζ and overactivated by co-knockdown of BiP and 14-3-3ζ. Apoptosis assays were performed using flow cytometry, TUNEL assays utilized confocal microscopy followed by western blot analysis and caspase-3 and JNK activities were investigated to assess the outcomes. Finally, an autophagy assay followed by western blotting was performed to study the effects of co-knockdown genes on cell autophagy in the presence and absence of cisplatin. The present data indicated the enhancement of cisplatin sensitivity in MCF-7 cells co-knocked down in BiP and 14-3-3ζ compared with either gene knockdown. Upregulation of JNK and cleaved-PARP1 protein levels as well as caspase-3 and JNK overactivation confirmed the results. A marked attenuation of autophagy and Beclin1 as well as ATG5 downregulation were detected in co-knockdown cells compared to knockdown with either BiP or 14-3-3ζ. Cisplatin sensitization of MCF-7 cells through double-knockdown of BiP and 14-3-3ζ highlights the potential of targeting UPR and autophagy factors to increase the effect of chemotherapy.

Toward Spontaneous Neuronal Differentiation of SH-SY5Y Cells Using Novel Three-Dimensional Electropolymerized Conductive Scaffolds.

Neuroblastoma-derived SH-SY5Y cells have become an excellent model for nervous system regeneration to treat neurodegenerative disorders. Many approaches achieved a mature population of derived neurons in in vitro plates. However, the importance of the third dimension in tissue regeneration has become indispensable to achieve a potential implant to replace the damaged tissue. Therefore, we have prepared porous 3D structures composed uniquely of carbon nanotubes (CNT) and poly(3,4-ethylenedioxythiophene) (PEDOT) that show great potential in the tridimensional differentiation of SH-SY5Y cells into mature neurons. The scaffolds have been manufactured through electropolymerization by applying 1.2 V in a three-electrode cell using a template of sucrose/CNT as a working electrode. By this method, PEDOT/CNT 3D scaffolds were obtained with homogeneous porosities and high conductivity. In vitro analyses showed that an excellent biocompatibility of the scaffold and the presence of high amount of ß-tubulin class III and MAP-II target proteins that mainly expresses in neurons, suggesting the differentiation into neuronal cells already after a week of incubation.

Electrochemically controlled cleavage of imine bonds on a graphene platform: towards new electro-responsive hybrids for drug release.

Graphene-based materials are particularly suitable platforms for the development of new systems able to release drugs upon the application of controlled electrochemical stimuli. Herein, we report a new electro-responsive graphene carrier functionalised with aldehydes (as drug models) through imine-based linkers. We explore a new type of drug loading/release combination based on the formation of a covalent bond and its cleavage upon electrolysis. The new graphene-drug model hybrid is stable under physiological conditions and displays a fast drug release upon the application of low voltages.

Tailored Methodology Based on Vapor Phase Polymerization to Manufacture PEDOT/CNT Scaffolds for Tissue Engineering.

Three-dimensional (3D) scaffolds with tailored stiffness, porosity, and conductive properties are particularly important in tissue engineering for electroactive cell attachment, proliferation, and vascularization. Carbon nanotubes (CNTs) and poly(3,4-ethylenedioxythiophene) (PEDOT) have been extensively used separately as neural interfaces showing excellent results. Herein, we combine both the materials and manufacture 3D structures composed exclusively of PEDOT and CNTs using a methodology based on vapor phase polymerization of PEDOT onto a CNT/sucrose template. Such a strategy presents versatility to produce porous scaffolds, after leaching out the sucrose grains, with different ratios of polymer/CNTs, and controllable and tunable electrical and mechanical properties. The resulting 3D structures show Young's modulus typical of soft materials (20-50 kPa), as well as high electrical conductivity, which may play an important role in electroactive cell growth. The conductive PEDOT/CNT porous scaffolds present high biocompatibility after 3 and 6 days of C8-D1A astrocyte incubation.

Triple-Labeling of Polymer-Coated Quantum Dots and Adsorbed Proteins for Tracing their Fate in Cell Cultures.

Colloidal CdSe/ZnS quantum dots were water solubilized by overcoating with an amphiphilic polymer. Human serum albumin (HSA) as a model protein was either adsorbed or chemically linked to the surface of the polymer-coated quantum dots. As the quantum dots are intrinsically fluorescent, and as the polymer coating and the HSA were fluorescent labeled, the final nanoparticle had three differently fluorescent components: the quantum dot core, the polymer shell, and the human serum albumin corona. Cells were incubated with these hybrid nanoparticles, and after removal of non-internalized nanoparticles, exocytosis of the three components of the nanoparticles was observed individually by flow cytometry and confocal microscopy. The data indicate that HSA is partly transported with the underlying polymer-coated quantum dots into cells. Upon desorption of proteins, those initially adsorbed to the quantum dots remain longer inside cells compared to free proteins. Part of the polymer shell is released from the quantum dots by enzymatic degradation, which is on a slower time scale than protein desorption. Data are quantitatively analyzed, and experimental pitfalls, such as the impact of cell proliferation and fluorescence quenching, are discussed.

Three-Dimensional Conductive Scaffolds as Neural Prostheses Based on Carbon Nanotubes and Polypyrrole.

Three-dimensional scaffolds for cellular organization need to enjoy a series of specific properties. On the one hand, the morphology, shape and porosity are critical parameters and eventually related with the mechanical properties. On the other hand, electrical conductivity is an important asset when dealing with electroactive cells, so it is a desirable property even if the conductivity values are not particularly high. Here, we construct three-dimensional (3D) porous and conductive composites, where C8-D1A astrocytic cells were incubated to study their biocompatibility. The manufactured scaffolds are composed exclusively of carbon nanotubes (CNTs), a most promising material to interface with neuronal tissue, and polypyrrole (PPy), a conjugated polymer demonstrated to reduce gliosis, improve adaptability, and increase charge-transfer efficiency in brain-machine interfaces. We developed a new and easy strategy, based on the vapor phase polymerization (VPP) technique, where the monomer vapor is polymerized inside a sucrose sacrificial template containing CNT and an oxidizing agent. After removing the sucrose template, a 3D porous scaffold was obtained and its physical, chemical, and electrical properties were evaluated. The obtained scaffold showed very low density, high and homogeneous porosity, electrical conductivity, and Young's Modulus similar to the in vivo tissue. Its high biocompatibility was demonstrated even after 6 days of incubation, thus paving the way for the development of new conductive 3D scaffolds potentially useful in the field of electroactive tissues.

Effective cancer immunotherapy in mice by polyIC-imiquimod complexes and engineered magnetic nanoparticles.

Encouraging results are emerging from systems that exploit Toll like receptor (TLR) signaling, nanotechnology, checkpoint inhibition and molecular imaging for cancer immunotherapy. A major remaining challenge is developing effective, durable and tumour-specific immune responses without systemic toxicity. Here, we report a simple and versatile system based on synergistic activation of immune responses and direct cancer cell killing by combined TLR ligation using polyIC as TLR3 and imiquimod (R837) as TLR7 agonist, in combination with the model antigen ovalbumin (OVA) and phospholipid micelles loaded with zinc-doped iron oxide magnetic nanoparticles (MNPs). The combination of TLR agonists triggered a strong innate immune response in the lymph nodes (LNs) without systemic release of pro-inflammatory cytokines. The vaccines showed excellent efficacy against aggressive B16-F10 melanoma cells expressing OVA, which was improved with immune checkpoint abrogation of the immunosuppressive programmed death-ligand 1 (PD-L1) at the level of the cancer cells. By magnetic resonance (MR) and nuclear imaging we could track the vaccine migration from the site of injection to LNs and tumour. Overall, we show this synergistic TLR agonists and their combination with MNPs and immune checkpoint blockade to have considerable potential for preclinical and clinical development of vaccines for cancer immunotherapy.

Nitrogen-doped carbon nanodots for bioimaging and delivery of paclitaxel.

Carbon nanodots (CNDs) hold great potential in imaging and drug delivery applications. In this study, nitrogen-doped CNDs (NCNDs) were coupled to the anticancer agent paclitaxel (PTX) through a labile ester bond. NCNDs showed excellent cell viability and endowed the NCND-PTX conjugate with good water solubility. The hybrid integrates the optical properties of the nanodots with the anticancer function of the drug into a single unit. Cytotoxicity was evaluated in breast, cervix, lung, and prostate cancer cell lines by the MTT assay while the cellular uptake was monitored using confocal microscopy. NCND-PTX induced apoptosis in cancer cells exhibiting slightly better anticancer activity compared to the drug alone. Moreover, the course of the NCND-PTX interaction with cancer cells was monitored using an xCELLigence system. The NCND-based conjugate represents a promising platform for bioimaging and drug delivery.

Novel fluorinated ligands for gold nanoparticle labelling with applications in 19F-MRI.

Novel fluorinated ligands for gold nanoparticle labelling have been designed and synthesised. Several types of gold nanoparticles have been prepared in the presence of these fluorinated ligands alone, or in combination with non-fluorinated ligands. Their colloidal stability in water and other solvents was tested and the magnetic resonance properties of the so-obtained nanoparticles were also assessed in detail. 1H and 19F-NMR spectra were evaluated and MRI phantoms of the most promising nanoparticles were successfully measured in 19F-MRI. The MRI signal to noise ratio was related to the fluorine concentration and compared with ICP-MS data to correlate the real concentration of fluorine grafted onto the nanoparticles with the actually active fluorine in MRI.

Sugar/gadolinium-loaded gold nanoparticles for labelling and imaging cells by magnetic resonance imaging.

Targeted magnetic resonance imaging (MRI) probes for selective cell labelling and tracking are highly desired. We here present biocompatible sugar-coated paramagnetic Gd-based gold nanoparticles (Gd-GNPs) and test them as MRI T1 reporters in different cellular lines at a high magnetic field (11.7 T). With an average number of 20 Gd atoms per nanoparticle, Gd-GNPs show relaxivity values r1 ranging from 7 to 18 mM-1 s-1 at 1.41 T. The multivalent presentation of carbohydrates on the Gd-GNPs enhances the avidity of sugars for carbohydrate-binding receptors at the cell surface and increases the local concentration of the probes. A large reduction in longitudinal relaxation times T1 is achieved with both fixed cells and live cells. Differences in cellular labelling are obtained by changing the type of sugar on the gold surface, indicating that simple monosaccharides and disaccharides are able to modulate the cellular uptake. These results stress the benefits of using sugars to produce nanoparticles for cellular labelling. To the best of our knowledge this is the first report on labelling and imaging cells with Gd-based gold nanoparticles which use simple sugars as receptor reporters.

Cellular uptake of gold nanoparticles bearing HIV gp120 oligomannosides.

Dendritic cells are the most potent of the professional antigen-presenting cells which display a pivotal role in the generation and regulation of adaptive immune responses against HIV-1. The migratory nature of dendritic cells is subverted by HIV-1 to gain access to lymph nodes where viral replication occurs. Dendritic cells express several calcium-dependent C-type lectin receptors including dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN), which constitute a major receptor for HIV-1. DC-SIGN recognizes N-linked high-mannose glycan clusters on HIV gp120 through multivalent and Ca(2+)-dependent protein-carbohydrate interactions. Therefore, mimicking the cluster presentation of oligomannosides from the virus surface is a strategic approach for carbohydrate-based microbicides. We have shown that gold nanoparticles (mannoGNPs) displaying multiple copies of structural motifs (di-, tri-, tetra-, penta-, or heptaoligomanosides) of the N-linked high-mannose glycan of viral gp120 are efficient inhibitors of DC-SIGN-mediated trans-infection of human T cells. We have now prepared the corresponding fluorescent-labeled glyconanoparticles (FITC-mannoGNPs) and studied their uptake by DC-SIGN expressing Burkitt lymphoma cells (Raji DC-SIGN cell line) and monocyte-derived immature dendritic cells (iDCs) by flow cytometry and confocal laser scanning microscopy. We demonstrate that the 1.8 nm oligomannoside coated nanoparticles are endocytosed following both DC-SIGN-dependent and -independent pathways and part of them colocalize with DC-SIGN in early endosomes. The blocking and sequestration of DC-SIGN receptors by mannoGNPs could explain their ability to inhibit HIV-1 trans-infection of human T cells in vitro.

A novel method to identify and characterise peptide mimotopes of heat shock protein 70-associated antigens.

The heat shock protein, Hsp70, has been shown to play an important role in tumour immunity. Vaccination with Hsp70-peptide complexes (Hsp70-PCs), isolated from autologous tumour cells, can induce protective immune responses. We have developed a novel method to identify synthetic mimic peptides of Hsp70-PCs and to test their ability to activate T-cells. Peptides (referred to as "recognisers") that bind to Hsp70-PCs from the human breast carcinoma cell line, MDA-MB-231, were identified by bio-panning a random peptide M13 phage display library. Synthetic recogniser peptides were subsequently used as bait in a reverse bio-panning experiment to identify potential Hsp70-PC mimic peptides. The ability of the recogniser and mimic peptides to prime human lymphocyte responses against tumour cell antigens was tested by stimulating lymphocytes with autologous peptide-loaded monocyte-derived dendritic cells (DCs). Priming and subsequent stimulation with either the recogniser or mimic peptide resulted in interferon-gamma (IFN-gamma) secretion by the lymphocytes. Furthermore, DCs loaded with Hsp70, Hsp70-PC or the recogniser or the mimic peptide primed the lymphocytes to respond to soluble extracts from breast cells. These results highlight the potential application of synthetic peptide-mimics of Hsp70-PCs, as modulators of the immune response against tumours.