Search Results (26)

For complexation of mRNA into polyplexes, double-pH-responsive lipo-xenopeptides (XP), comprising tetraethylene pentamino succinic acid (Stp) and lipoamino fatty acids (LAFs), were combined with PEGylated lipids, either DMG-PEG 2 kDa (DMG-PEG) or azido-group-containing DSPE-PEG 2 kDa (DSPE-PEG-N3), to increase colloidal stability and to facilitate ligand-mediated targeted mRNA delivery. LAF-XPs mixed with DMG-PEG at low (1.5% and 3%) molar ratios improved colloidal stability and retained transfection efficiency. PEGylation also enabled the formulation of otherwise unstable carrier complexes and prevented aggregation induced by salt, proteins, and serum. PEGylation of more positively charged Stp-LAF₂ mRNA polyplexes decreased fibrinogen adsorption. More neutral, LAF-rich Stp-LAF₄ polyplexes exhibited low fibrinogen binding without PEGylation. Intravenous administration of these stabilized mRNA complexes demonstrated enhanced biosafety while preserving transfection efficiency. DSPE-PEG-N3 was selected for cell targeting after strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated click-coupling of DBCO-modified ligands. Higher PEG ratios (10% and 20%) provided effective shielding but reduced transfection efficiency, a drawback known as the “PEG dilemma”. Functionalization with an EGFR-targeting ligand restored transfection in EGFR-positive cell lines in a ligand-specific manner. High transfection efficiency is consistent with a lipophilic-to-hydrophilic polarity switch of LAF-XP carriers upon endosomal protonation, triggering dissociation of the PEG lipids and deshielding of the polyplex. Full article

Strain-promoted azide–alkyne cycloaddition (SPAAC) is widely used in solution-phase bioconjugation. However, its application in surface chemistry remains limited because substrate-independent azide films that remain stable upon reaction with bulky strained alkynes have not yet been developed. In this study, we address this challenge using a melanin-inspired coating based on tyrosine–azide derivatives with different linkers. In particular, we investigated how differences in linker length and hydrophilicity affect the hydrophobic interactions within the film network and, ultimately, determine film stability. Specifically, Tyr-3-N₃, a tyrosine–azide derivative having an azide group tethered to tyrosine through a short three-carbon alkyl linker, was identified as optimal, forming azide-presenting films via tyrosinase-mediated oxidation and retaining integrity during SPAAC with external dibenzocyclooctyne (DBCO) ligands. The optimized poly(Tyr-3-N₃) coatings enabled efficient methoxypolyethylene glycol (mPEG) immobilization, thereby exhibiting excellent antifouling performance against protein adsorption, and further supported spatially controlled protein patterning through soft lithography techniques such as micromolding in capillaries (MIMIC) and microcontact printing (µCP). The approach was broadly applicable with a range of inorganic and polymeric substrates, as well as living cell surfaces; even after encapsulation and SPAAC-based functionalization, the cells remained viable. Collectively, these findings establish a substrate-independent and biocompatible coating platform that preserves film stability through SPAAC functionalization, supporting applications in antifouling coatings, biosensing, and cell surface engineering. Full article

Background/Objectives: Nanogels (NGs) are promising carriers for drug delivery due to their tunable size, biocompatibility, and capability to encapsulate sensitive molecules. However, conventional batch synthesis often lacks control over key parameters, such as size distribution and encapsulation efficiency. This study aimed to develop a microfluidic platform for the reproducible synthesis of poly(α-glutamic acid) (PGA)-based NGs using strain-promoted azide–alkyne cycloaddition (SPAAC) click chemistry and to investigate the effects of flow parameters on the physicochemical properties of nanogels. Methods: Functionalized PGAs (with azide and DBCO) were co-injected into a microfluidic system within a flux of acetone to form NGs via SPAAC. Flow rate ratios (FRR) and total flow rates were systematically screened at 25 °C, with tests at 50 °C. We evaluated the particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency (EE%) of doxorubicin-loaded NGs. Results: NGs with tunable sizes ranging from ~50 nm to >170 nm and low PDI (<0.1 in optimal conditions) were obtained. Higher FRR and total flow rates yielded smaller and more uniform NGs. Doxorubicin loading did not affect the nanogel size and uniformity, and in some cases, it improved them. The EE% reached up to ~65%, and ~40% for the best formulations. Elevated temperature improved the characteristics of drug-loaded nanogels at intermediate solvent ratios. Compared to batch synthesis, the microfluidic process offers enhanced reproducibility and size control. Conclusions: Microfluidic SPAAC synthesis enables precise and scalable fabrication of PGA NGs with controllable size and drug loading. This platform supports future integration of on-chip purification and monitoring for clinical nanomedicine applications. Full article

The synthesis of novel biodegradable polymers as non-viral vectors remains one of the challenging tasks in the field of gene delivery. In this study, the synthesis of the polysaccharide-g-polypeptide copolymers, namely, hyaluronic acid-g-polylysine (HA-g-PLys), using a copper-free strain-promoted azide-alkyne cycloaddition reaction was proposed. For this purpose, hyaluronic acid was modified with dibenzocyclooctyne moieties, and poly-L-lysine with a terminal azido group was obtained using ring-opening polymerization of N-carboxyanhydride of the corresponding protected amino acid, initiated with the amino group azido-PEG₃-amine. Two HA-g-PLys samples with different degrees of grafting were synthesized, and the structures of all modified and synthesized polymers were confirmed using ¹H NMR and FTIR spectroscopy. The HA-g-PLys samples obtained were able to form nanoparticles in aqueous media due to self-assembly driven by electrostatic interactions. The binding of DNA and model siRNA by copolymers to form polyplexes was analyzed using ethidium bromide, agarose gel electrophoresis, and SybrGreen I assays. The hydrodynamic diameter of polyplexes was ˂300 nm (polydispersity index, PDI ˂ 0.3). The release of a model fluorescently-labeled oligonucleotide in the complex biological medium was significantly higher in the case of HA-g-PLys as compared to that in the case of PLys-based polyplexes. In addition, the cytotoxicity in normal and cancer cells, as well as the ability of HA-g-PLys to facilitate intracellular delivery of anti-GFP siRNA to NIH-3T3/GFP+ cells, were evaluated. Full article

Combination chemotherapy is preferred for the treatment of ovarian cancer (OC). Systemic toxicity, however, frequently limits the effectiveness of treatment. Polymer–drug conjugates (PDCs) containing synergistic combinations of chemotherapeutic drugs can be used to enhance therapeutic efficacy. We earlier reported the use of a strain-promoted [3 + 2] azide–alkyne cycloaddition (SPAAC)-mediated polymerization method for the preparation of single-drug PDCs. In this report, the polymerization method was used to prepare gemcitabine–doxorubicin combination PDC. The PDC had a high molecular weight (M_w 1360 kDa) and high drug loading (36.6% weight gemcitabine; 7.0% weight doxorubicin). It demonstrated cathepsin B-catalyzed drug release at pH 5.0 and good hydrolytic stability at pH 7.4. The combination index analysis of free gemcitabine and free doxorubicin showed a concentration-dependent synergism (combination index < 1) in OVCAR-3 OC cells. Compared to individual gemcitabine PDC (the concentration that inhibited 50% growth (IC₅₀) > 50 µg/mL) and doxorubicin PDC (IC₅₀ = 1.79 µg/mL), the combination PDC (IC₅₀ = 0.99 µg/mL) showed greater cytotoxicity against OVCAR-3 cells and was less cytotoxic than the equivalent free drug combination (IC₅₀ = 0.11 µg/mL). The gemcitabine–doxorubicin combination PDC is promising for targeted combination chemotherapy of OC. Full article

Since its conceptualization, click chemistry in all its variants has proven to be a superior synthesis protocol, compared to conventional methods, for forming new covalent bonds under mild conditions, orthogonally, and with high yields. If a term like reactive resilience could be established, click reactions would be good examples, as they perform better under increasingly challenging conditions. Particularly, highly hindered couplings that perform poorly with conventional chemistry protocols—such as those used to conjugate biomacromolecules (e.g., proteins and aptamers) or multiple drugs onto macromolecular platforms—can be more easily achieved using click chemistry principles, while also promoting high stereoselectivity in the products. In this review, three molecular platforms relevant in the field of nanomedicine are considered: polymers/copolymers, cyclodextrins, and fullerenes, whose functionalization poses a challenge due to steric hindrance, either from the intrinsic bulk behavior (as in polymers) or from the proximity of confined reactive sites, as seen in cyclodextrins and fullerenes. Their functionalization with biologically active groups (drugs or biomolecules), primarily through copper-catalyzed azide–alkyne cycloaddition (CuAAC), strain-promoted azide–alkyne cycloaddition (SPAAC), inverse electron-demand Diels–Alder (IEDDA) and thiol–ene click reactions, has led to the development of increasingly sophisticated systems with enhanced specificity, multifunctionality, bioavailability, delayed clearance, multi-targeting, selective cytotoxicity, and tracking capabilities—all essential in the field of nanomedicine. Full article

Background: The incorporation of radionuclides into peptides and larger biomolecules requires efficient and sometimes biorthogonal reaction conditions, to which click chemistry provides a convenient approach. Methods: Traditionally, click-based radiolabeling techniques have focused on classical click chemistry, such as copper(I)-catalyzed alkyne-azide [3+2] cycloaddition (CuAAC), strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), traceless Staudinger ligation, and inverse electron demand Diels–Alder (IEDDA). Results: However, newly emerging click-based radiolabeling techniques, including tyrosine-click, sulfo-click, sulfur(VI) fluoride exchange (SuFEx), thiol-ene click, azo coupling, hydrazone formations, oxime formations, and RIKEN click offer valuable alternatives to classical click chemistry. Conclusions: This review will discuss the applications of these techniques in peptide radiochemistry. Full article

For successful therapeutic interventions in cancer immunotherapy, strong antigen-specific immune responses are required. To this end, immunostimulating cues must be combined with antigens to simultaneously arrive at antigen-presenting cells and initiate cellular immune responses. Recently, imidazoquinolines have shown their vast potential as small molecular Toll-like receptor 7/8 (TLR7/8) agonists for immunostimulation when delivered by nanocarriers. At the same time, peptide antigens are promising antigen candidates but require combination with immune-stimulating adjuvants to boost their immunogenicity and exploit their full potential. Consequently, we herein present biodegradable polycarbonate nanogels as versatile delivery system for adjuvants within the particles’ core as well as for peptide antigens by surface decoration. For that purpose, orthogonally addressable multifunctional polycarbonate block copolymers were synthesized, enabling adjuvant conjugation through reactive ester chemistry and peptide decoration by strain-promoted alkyne-azide cycloaddition (SPAAC). In preparation for SPAAC, CD4⁺-specific peptide sequences of the model protein antigen ovalbumin were equipped with DBCO-moieties by site-selective modification at their N-terminal cysteine. With their azide groups exposed on their surface, the adjuvant-loaded nanogels were then efficiently decorated with DBCO-functional CD4⁺-peptides by SPAAC. In vitro evaluation of the adjuvant-loaded peptide-decorated gels then confirmed their strong immunostimulating properties as well as their high biocompatibility. Despite their covalent conjugation, the CD4⁺-peptide-decorated nanogels led to maturation of primary antigen-presenting cells and the downstream priming of CD4⁺-T cells. Subsequently, the peptide-decorated nanogels loaded with TLR7/8 agonist were successfully processed by antigen-presenting cells, enabling potent immune responses for future application in antigen-specific cancer immunotherapy. Full article

The recently developed compound, tetramethylthiocycloheptyne sulfoximine (TMTHSI), has shown to be a promising strained alkyne for strain-promoted azide–alkyne cycloaddition (SPAAC), metal-free click chemistry. This research explores the properties of TMTHSI-based compounds via three aspects: (1) large-scale production, (2) unique stability in acidic conditions and its subsequent use in peptide synthesis, and (3) the functionalization of antibodies. Here, it is shown that (1) scale-up is achieved on a scale of up to 100 g. (2) TMTHSI is remarkably stable against TFA allowing for the site-specific functionalization of peptides on resin. Finally, (3) the functionalization of an antibody with a model payload is very efficient, with antibody conjugation demonstrating more beneficial features such as a high yield and limited hydrophobicity as compared to other alkyne reagent conjugates. These results illustrate the high potential of TMTHSI for diverse bioconjugation applications, with production already being GMP-compatible and a highly efficient conversion resulting in attractive costs of goods. Full article

Cyclooctyne molecules have found wide applications in the strain-promoted azide–alkyne cycloaddition (SPAAC) reactions, which avoid the biotoxicity caused by the use of Cu(I) catalysts. Among the various cyclooctyne systems, dibenzocyclooctyne (DBCO) series have displayed the highest reaction activity. However, the synthesis processes of such structures are time-consuming, which to some extent limit their large-scale development and application. This review has summarized current synthesis routes of two DBCO molecules, aza-dibenzocyclooctyne (DIBAC) and biarylazacyclooctynone (BARAC). Full article

We report herein on a new platform for synthesizing stable, inert, and dispersible metal-free single-chain nanoparticles (SCNPs) via intramolecular metal-traceless azide–alkyne click chemistry. It is well known that SCNPs synthesized via Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) often experience metal-induced aggregation issues during storage. Moreover, the presence of metal traces limits its use in a number of potential applications. To address these problems, we selected a bifunctional cross-linker molecule, sym-dibenzo-1,5-cyclooctadiene-3,7-diyne (DIBOD). DIBOD has two highly strained alkyne bonds that allow for the synthesis of metal-free SCNPs. We demonstrate the utility of this new approach by synthesizing metal-free polystyrene (PS)-SCNPs without significant aggregation issues during storage, as demonstrated by small-angle X-ray scattering (SAXS) experiments. Notably, this method paves the way for the synthesis of long-term-dispersible, metal-free SCNPs from potentially any polymer precursor decorated with azide functional groups. Full article

Here we present an approach to functionalize silanized single-walled carbon nanotubes (SWNTs) through copper-free click chemistry for the assembly of inorganic and biological nanohybrids. The nanotube functionalization route involves silanization and strain-promoted azide–alkyne cycloaddition reactions (SPACC). This was characterized by X-ray photoelectron spectroscopy, scanning electron microscopy, transmission electron microscopy, Raman spectroscopy and Fourier transform infra-red spectroscopy. Silane–azide-functionalized SWNTs were immobilized from solution onto patterned substrates through dielectrophoresis (DEP). We demonstrate the general applicability of our strategy for the functionalization of SWNTs with metal nanoparticles (gold nanoparticles), fluorescent dyes (Alexa Fluor 647) and biomolecules (aptamers). In this regard, dopamine-binding aptamers were conjugated to the functionalized SWNTs to perform real-time detection of dopamine at different concentrations. Additionally, the chemical route is shown to selectively functionalize individual nanotubes grown on the surface of silicon substrates, contributing towards future nano electronic device applications. Full article

The development of ⁶⁴Cu-based immuno-PET radiotracers requires the use of copper-specific bifunctional chelators (BFCs) that contain functional groups allowing both convenient bioconjugation and stable copper complexes to limit in vivo bioreduction, transmetallation and/or transchelation. The excellent in vivo kinetic inertness of the pentaazamacrocyclic [⁶⁴Cu]Cu-15-5 complex prompted us to investigate its potential for the ⁶⁴Cu-labelling of monoclonal antibodies (mAbs), compared with the well-known NODAGA and DOTA chelators. To this end, three NODAGA, DOTA and 15-5-derived BFCs, containing a pendant azadibenzocyclooctyne moiety, were synthesised and a robust methodology was determined to form covalent bonds between them and azide-functionalised trastuzumab, an anti-HER2 mAb, using strain-promoted azide-alkyne cycloaddition. Unlike the DOTA derivative, the NODAGA- and 15-5-mAb conjugates were radiolabelled with ⁶⁴Cu, obtaining excellent radiochemical yields, under mild conditions. Although all the radioimmunoconjugates showed excellent stability in PBS or mouse serum, [⁶⁴Cu]Cu-15-5- and [⁶⁴Cu]Cu-NODAGA-trastuzumab presented higher resistance to transchelation when challenged by EDTA. Finally, the immunoreactive fraction of the radioimmunoconjugates (88–94%) was determined in HER-2 positive BT474 human breast cancer cells, confirming that the bioconjugation and radiolabelling processes implemented had no significant impact on antigen recognition. Full article

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo–enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo–enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels–Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar K_D values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts. Full article

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5). Full article