Immunoadjuvant-Modified Rhodobacter sphaeroides Potentiate Cancer Photothermal Immunotherapy.

Photothermal immunotherapy has become a promising strategy for tumor treatment. However, the intrinsic drawbacks like light instability, poor immunoadjuvant effect, and poor accumulation of conventional inorganic or organic photothermal agents limit their further applications. Based on the superior carrying capacity and active tumor targeting property of living bacteria, an immunoadjuvant-intensified and engineered tumor-targeting bacterium was constructed to achieve effective photothermal immunotherapy. Specifically, immunoadjuvant imiquimod (R837)-loaded thermosensitive liposomes (R837@TSL) were covalently decorated onto Rhodobacter sphaeroides (R.S) to obtain nanoimmunoadjuvant-armed bacteria (R.S-R837@TSL). The intrinsic photothermal property of R.S combined R837@TSL to achieve in situ near-infrared (NIR) laser-controlled release of R837. Meanwhile, tumor immunogenic cell death (ICD) caused by photothermal effect of R.S-R837@TSL, synergizes with released immunoadjuvants to promote maturation of dendritic cells (DCs), which enhance cytotoxic T lymphocytes (CTLs) infiltration for further tumor eradication. The photosynthetic bacteria armed with immunoadjuvant-loaded liposomes provide a strategy for immunoadjuvant-enhanced cancer photothermal immunotherapy.

How to Design Catechol-Containing Hydrogels for Cell Encapsulation Despite Catechol Toxicity.

Catechol (cat) is a highly adhesive diphenol that can be chemically grafted to polymers such as chitosan (CH) to make them adhesive as well. However, catechol-containing materials experimentally show a large variability of toxicity, especially in vitro. While it is unclear how this toxicity emerges, most concerns are directed toward the oxidation of catechol into quinone that releases reactive oxygen species (ROS) which can, in turn, cause cell apoptosis through oxidative stress. To better understand the mechanisms at play, we examined the leaching profiles, hydrogen peroxide (H2O2) production, and in vitro cytotoxicity of several cat-chitosan (cat-CH) hydrogels that were prepared with different oxidation levels and cross-linking methods. To create cat-CH with different propensities toward oxidation, we grafted either hydrocaffeic acid (HCA, more prone to oxidation) or dihydrobenzoic acid (DHBA, less prone to oxidation) to the backbone of CH. Hydrogels were cross-linked either covalently, using sodium periodate (NaIO4) to trigger oxidative cross-linking, or physically, using sodium bicarbonate (SHC). While using NaIO4 as a cross-linker increased the oxidation levels of the hydrogels, it also significantly reduced in vitro cytotoxicity, H2O2 production, and catechol and quinone leaching in the media. For all gels tested, cytotoxicity could be directly related to the release of quinones rather than H2O2 production or catechol release, showing that oxidative stress may not be the main reason for catechol cytotoxicity, as other pathways of quinone toxicity come into play. Results also suggest that the indirect cytotoxicity of cat-CH hydrogels fabricated through carbodiimide chemistry can be reduced if (i) catechol groups are chemically bound to the polymer backbone to prevent leaching or (ii) the chosen cat-bearing molecule has a high resistance to oxidation. Coupled with the use of other cross-linking chemistries or more efficient purification methods, these strategies can be adopted to synthesize various types of cytocompatible cat-containing scaffolds.

Carbon Dot Emission Enhancement in Covalent Complexes with Plasmonic Metal Nanoparticles.

Carbon dots can be used for the fabrication of colloidal multi-purpose complexes for sensing and bio-visualization due to their easy and scalable synthesis, control of their spectral responses over a wide spectral range, and possibility of surface functionalization to meet the application task. Here, we developed a chemical protocol of colloidal complex formation via covalent bonding between carbon dots and plasmonic metal nanoparticles in order to influence and improve their fluorescence. We demonstrate how interactions between carbon dots and metal nanoparticles in the formed complexes, and thus their optical responses, depend on the type of bonds between particles, the architecture of the complexes, and the degree of overlapping of absorption and emission of carbon dots with the plasmon resonance of metals. For the most optimized architecture, emission enhancement reaching up to 5.4- and 4.9-fold for complexes with silver and gold nanoparticles has been achieved, respectively. Our study expands the toolkit of functional materials based on carbon dots for applications in photonics and biomedicine to photonics.

Photochemical approach for multiplexed biofunctionalisation of gallium arsenide.

The optoelectronic properties of gallium arsenide (GaAs) hold great promise in biosensing applications, currently being held back by the lack of methodologies reporting the spatially selective functionalisation of this material with multiple biomolecules. Here, we exploit the use of a photoreactive crosslinker - a diazirine derivative - for spatially selective covalent immobilisation of multiple bioreceptors on the GaAs surface. As a proof of principle we show the immobilisation of two proteins: neutravidin and endosulfine alpha protein. X-ray photoelectron spectroscopy results showed the presence of the biomolecules on the GaAs regions selectively exposed to ultraviolet light. The approach presented here is applicable to the covalent attachment of other biomolecules, paving the way for using GaAs as a platform for multiplexed biosensing.

Preparation of 10-(hexylcarbamoyl)pyranomalvidin-3-glucoside from 10-carboxypyranomalvidin-3-glucoside using carbodiimide chemistry.

Anthocyanins and pyranoanthocyanins are appealing natural pigments for replacement of synthetic ones. However, due to instability and solubility issues, lipophilization process of anthocyanins has raised as a valuable and efficient strategy to extend their stability and affinity into liposoluble formulations and enhance their unique physicochemical and biological properties. In this work, 10-carboxypyranomalvidin-3-glucoside was functionalized with hexylamine via carbodiimide chemistry compatible with the absence of hydroxyl protecting groups. A new amide conjugate attached to an alkyl chain with better hydrophobic features was obtained and isolated from its precursor. Mass spectrometry, FTIR, and NMR spectroscopy confirmed that the lipophilization site took place at the carboxyl group and the octanol-water partition coefficient determined by UV-Vis revealed its superior affinity for non-polar media. Overall, we reported a new pyranoanthocyanin lipophilic derivative for the first time which encourage further investigation for this novel class of compounds towards their incorporation into lipid-based foods and cosmetic formulations.

Biofunctionalisation of gallium arsenide with neutravidin.

Gallium arsenide (GaAs) is a promising candidate as a platform for optical biosensing devices due to its enabling optoelectronic properties. However, the biofunctionalisation of the GaAs surface has not received much attention compared to gold, carbon and silicon surfaces. Here we report a study presenting a physicochemical surface characterisation of the GaAs surface along the functionalisation with a high-affinity bioconjugation pair widely explored in the life sciences - biotin and neutravidin. Combined X-ray photoelectron spectroscopy (XPS), wettability measurements and spectroscopic ellipsometry were used for a reliable characterisation of the surface functionalisation process. The results suggest that a film with a thickness lower than 10 nm was formed, with a neutravidin to biotin ratio of 1:25 on the GaAs surface. Reduction of non-specific binding of the protein to the surface was achieved by optimising the protein buffer and rinsing steps. This study shows the feasibility of using GaAs as a platform for specific biomolecular recognition, paving the way to a new generation of optoelectronic biosensors.

Optimization of Critical Parameters for Carbodiimide Mediated Production of Highly Modified Chitosan.

An optimization of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and hydroxy benzotriazole mediated conjugation of the polysaccharide chitosan with functional carboxylic acids was shown. Optimal parameters that enable resource-efficient synthesis of highly functionalized chitosan were identified. In particular, use of only catalytic instead of stoichiometric amounts of hydroxy benzotriazole and tight control of pH in reaction mixture resulted in highly efficient incorporation of the desired moieties as side chains in chitosan. As a result, the model reactant 4-azidobenzoic acid was incorporated resulting in a degree of substitution of over 30% with very high coupling efficacy of up to 90%. Similar results were obtained with other carboxylic acids such as methacrylic acid, 3-(2-furyl) propionic acid and 3-maleimido propionic acid, highlighting the broad applicability of our findings for the functionalization of chitosan.

Development of amine-functionalized superparamagnetic iron oxide nanoparticles anchored graphene nanosheets as a possible theranostic agent in cancer metastasis.

The major objective of the present investigation was to assess the targeting potential of a designed system for breast cancer at metastatic phases with imaging ability. In a nutshell, we have developed surface-engineered graphene oxide (GO) nanosheets by covalent linking with amine-functionalized iron oxide nanoparticles (IONPs) (GOIOIs). Gefitinib (Gf) was selected as a model drug and entrapped in between exfoliated GO sheets (GOIGF) via π-π* stacking before functionalization with IONPs. Preliminary characterization of GO, IONPs, GOIOI, and GOIGF was performed using UV-visible and Fourier transform infrared spectroscopy. Scanning and transmission electron microscopy studies confirmed successful surface engineering of GO with IONPs. The in vitro drug release study demonstrated sustained release of Gf. The magnetic behavior of IONPs and GOIOI demonstrated a sigmoidal-shaped hysteresis loop with superparamagnetic properties. The in vitro cell cytotoxicity assay was carried out on MDA-MB-231 breast cancer adenocarcinoma cell lines. The cell cytotoxicity assay showed 61.18% inhibition of cell growth with 30 ppm concentration containing 64% of the drug, whereas 100% of the pure drug revealed only 56% of inhibition. In the near future, GOIOI could be tailored further for theranostic research, especially for metastatic cancers. Graphical abstract.

Functionalizing nanoparticles with cancer-targeting antibodies: A comparison of strategies.

Standard cancer therapies sometimes fail to deliver chemotherapeutic drugs to tumor cells in a safe and effective manner. Nanotechnology takes the lead in providing new therapeutic options for cancer due to major potential for selective targeting and controlled drug release. Antibodies and antibody fragments are attracting much attention as a source of targeting ligands to bind specific receptors that are overexpressed on cancer cells. Therefore, researchers are devoting time and effort to develop targeting strategies based on nanoparticles functionalized with antibodies, which hold great promise to enhance therapeutic efficacy and circumvent severe side effects. Several methods have been described to immobilize antibodies on the surface of nanoparticles. However, selecting the most appropriate for each application is challenging but also imperative to preserve antigen binding ability and yield stable antibody-conjugated nanoparticles. From this perspective, we aim to provide considerable knowledge on the most widely used methods of functionalization that can be helpful for decision-making and design of conjugation protocols as well. This review summarizes adsorption, covalent conjugation (carbodiimide, maleimide and "click" chemistries) and biotin-avidin interaction, while discussing the advantages, limitations and relevant therapeutic approaches currently under investigation.

Plasma Treatment of Polymer Powder as an Effective Tool to Functionalize Polymers: Case Study Application on an Amphiphilic Polyurethane.

Plasma treatment is a widely applied, easy, fast, and highly reproducible surface modification technique. In this work powder plasma treatment was exploited to expose carboxylic groups along the backbone of a water soluble polymer. Specifically, a custom-made amphiphilic poly(ether urethane) containing Poloxamer® 407 blocks (Mw = 54,000 Da) was first synthesized and its powders were plasma treated in the presence of Acrylic Acid vapor. To maximize -COOH group exposure while preventing polymer degradation, different Ar gas flow rates (i.e., 10, 30, and 50 sccm) were investigated. Upon gas flow increase, significant polymer degradation was observed, with a 35% molecular weight reduction at 50 sccm Ar flow rate. On the other hand, the highest number of exposed carboxylic groups (5.3 × 1018 ± 5.5 × 1017 units/gpolymer) was obtained by setting gas flow at 10 sccm. Hence, a gas flow of 10 sccm turned out to be the best set-up to maximize -COOH exposure while preventing degradation phenomena. Additionally, upon plasma treatment, no detrimental effects were observed in the thermoresponsiveness of polymer aqueous solutions, which was ensured by Poloxamer® 407 blocks. Therefore, the newly developed technology here applied on an amphiphilic poly(ether urethane) could pave the way to the tailored design of a plethora of different multifunctional hydrogels.

Biodegradable Nanoparticles Containing Mechanism Based Peptide Inhibitors Reduce Polyglutamine Aggregation in Cell Models and Alleviate Motor Symptoms in a Drosophila Model of Huntington's Disease.

Detailed study of the molecular mechanism behind the pathogenesis of Huntington's disease (HD) suggests that polyglutamine aggregation is one of the fundamental reasons for HD. Despite the discovery of many potential molecules, HD therapy is still limited to symptomatic relief. Among these molecules, few mechanism based peptide inhibitors of polyglutamine aggregation (QBP1, NT17 and PGQ9P2) have shown promising activity; however, poor blood-brain barrier (BBB) penetration, low bioavailability, and low half-life may hinder their therapeutic potential. Hence, to deliver them to the brain for assessing their efficacy, we have designed and synthesized peptide loaded poly-d,l-lactide- co-glycolide (PLGA) nanoparticles of less than 200 nm in size by carbodiimide chemistry and nanoprecipitation protocols. For brain delivery, PLGA nanoparticles were coated with polysorbate 80 which aids receptor mediated internalization. Using the in vitro BBB model of Madin-Darby canine kidney cells and healthy mice, the translocation of polysorbate 80 coated fluorescent nanoparticles was confirmed. Moreover, QBP1, NT17, and PGQ9P2 loaded PLGA nanoparticles showed dose dependent inhibition of polyglutamine aggregation in cell models of HD (Neuro 2A and PC12 cells) and improved motor performance in Drosophila model of HD. Additionally, no toxicity in cells and animals confirmed biocompatibility of the nanoparticulate formulations. Based on this work, future studies can be designed in higher animal models to test peptide loaded nanoparticles in HD and other polyglutamine expansion related diseases.

Carbodiimide-mediated immobilization of acidic biomolecules on reversed-charge zwitterionic sensor chip surfaces.

The carbodiimide-mediated amine coupling of protein ligands to sensor chips coated with anionic polycarboxylate hydrogels, such as carboxymethyl dextran, is the predominant covalent immobilization procedure utilized in optical biosensors, namely surface plasmon resonance (SPR) biosensors. Usually, electrostatic interactions at a slightly acidic pH and low ionic strength are employed to efficiently accumulate neutral and basic ligands on the chip surface, which are then covalently coupled by surface-bound active N-hydroxysuccinimide (NHS) esters. Unfortunately, this approach is not suitable for acidic proteins or other ligands with low isoelectric points (IEPs), such as nucleic acids, because the charge density of the polycarboxylates is greatly reduced at acidic pH or because electrostatic attraction cannot be achieved. To overcome these drawbacks, we have established a charge-reversal approach that allows the preconcentration of acidic proteins above their IEPs. A precisely controlled amount of tertiary amines is applied to reverse the previous anionic surface charge while maintaining carbodiimide compatibility with future protein immobilization. The mechanism of this reversed-charge immobilization approach was demonstrated employing protein A as a model protein and using attenuated total reflectance Fourier transform infrared spectroscopy, dynamic contact angle measurements, colorimetric quantification, and SPR analysis to characterize surface derivatization. Furthermore, even though it had previously proven impossible to preconcentrate DNA electrostatically and to covalently couple it to polyanionic chip surfaces, we demonstrated that our approach allowed DNA to be preconcentrated and immobilized in good yields. Graphical abstract Principle of the covalent immobilization of acidic ligands on reversed-charge zwitterionic sensor chip surfaces.

Covalent Immobilization of Enzymes on Eupergit® Supports: Effect of the Immobilization Protocol.

A selection of best combination of adequate immobilization support and efficient immobilization method is still a key requirement for successful application of immobilized enzymes on an industrial level. Eupergit® supports exhibit good mechanical and chemical properties and allow establishment of satisfactory hydrodynamic regime in enzyme reactors. This is advantageous for their wide application in enzyme immobilization after finding the most favorable immobilization method. Methods for enzyme immobilization that have been previously reported as efficient considering the obtained activity of immobilized enzyme are presented: direct binding to polymers via their epoxy groups, binding to polymers via a spacer made from ethylene diamine/glutaraldehyde, and coupling the periodate-oxidized sugar moieties of the enzymes to the polymer beads. The modification of the conventionally immobilized enzyme with ethylenediamine via the carbodiimide route seems to be a powerful tool to improve its stability and catalytic activity.

Lactoferrin-Immobilized Surfaces onto Functionalized PLA Assisted by the Gamma-Rays and Nitrogen Plasma to Create Materials with Multifunctional Properties.

Both cold nitrogen radiofrequency plasma and gamma irradiation have been applied to activate and functionalize the polylactic acid (PLA) surface and the subsequent lactoferrin immobilization. Modified films were comparatively characterized with respect to the procedure of activation and also with unmodified sample by water contact angle measurements, mass loss, X-ray photoelectron spectroscopy (XPS), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), atomic force microscopy (AFM), and chemiluminescence measurements. All modified samples exhibit enhanced surface properties mainly those concerning biocompatibility, antimicrobial, and antioxidant properties, and furthermore, they are biodegradable and environmentally friendly. Lactoferrin deposited layer by covalent coupling using carbodiimide chemistry showed a good stability. It was found that the lactoferrin-modified PLA materials present significantly increased oxidative stability. Gamma-irradiated samples and lactoferrin-functionalized samples show higher antioxidant, antimicrobial, and cell proliferation activity than plasma-activated and lactoferrin-functionalized ones. The multifunctional materials thus obtained could find application as biomaterials or as bioactive packaging films.

Nanoscale modification of porous gelatin scaffolds with chondroitin sulfate for corneal stromal tissue engineering.

Recent studies reflect the importance of using naturally occurring biopolymers as three-dimensional corneal keratocyte scaffolds and suggest that the porous structure of gelatin materials may play an important role in controlling nutrient uptake. In the current study, the authors further consider the application of carbodiimide cross-linked porous gelatin as an alternative to collagen for corneal stromal tissue engineering. The authors developed corneal keratocyte scaffolds by nanoscale modification of porous gelatin materials with chondroitin sulfate (CS) using carbodiimide chemistry. Scanning electron microscopy/energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy showed that the amount of covalently incorporated polysaccharide was significantly increased when the CS concentration was increased from 0% to 1.25% (w/v). In addition, as demonstrated by dimethylmethylene blue assays, the CS content in these samples was in the range of 0.078-0.149 nmol per 10 mg scaffold. When compared with their counterparts without CS treatment, various CS-modified porous gelatin membranes exhibited higher levels of water content, light transmittance, and amount of permeated nutrients but possessed lower Young's modulus and resistance against protease digestion. The hydrophilic and mechanical properties of scaffolds modified with 0.25% CS were comparable with those of native corneas. The samples from this group were biocompatible with the rabbit corneal keratocytes and showed enhanced proliferative and biosynthetic capacity of cultured cells. In summary, the authors found that the nanoscale-level modification has influence on the characteristics and cell-material interactions of CS-containing gelatin hydrogels. Porous membranes with a CS content of 0.112 ± 0.003 nmol per 10 mg scaffold may hold potential for use in corneal stromal tissue engineering.