Search Results (119)

Nanodiamonds (NDs) are an innovative material in biomedical applications based on their excellent biocompatibility, nanoscale dimensions, and high surface area. In this study, we evaluated the potential of ND-in-oil emulsion to induce potent antibody responses in animals immunized with cobra venom. NDs demonstrated the capacity to bind complex venom proteins as stable conjugates, well dispersed in aqueous solution. Immunization of mice with cobra venom incorporated with ND-in-oil emulsion adjuvant (ND/venom) elicited strong venom-specific antibody responses with titers comparable to those induced by venom formulation with conventional Freund’s adjuvants (FA/venom). IgG subclass analysis revealed that ND- and FA-based formulations induced a Th2-biased immune response in mice. Moreover, antibodies elicited by ND/venom or FA/venom immunization specifically recognized the epitopes of the lethal component of short-chain neurotoxin and conferred full protection against lethal cobra venom challenge (3LD₅₀). Further, ND/venom hyperimmunization was capable of inducing high levels of neutralizing antibodies in larger animals, rabbits, highlighting the potential for antivenom manufacturing. Notably, there were no obvious lesions at the injection sites of animals that received ND/venom, in contrast to those that received FA/venom. These findings indicated NDs as an effective and safe additive in venom formulation for antivenom production. Full article

Antibody–drug conjugates (ADCs) are a rapidly advancing class of targeted cancer therapeutics that couple the antigen specificity of monoclonal antibodies (mAbs) with the potent cytotoxicity of small-molecule drugs. In their core design, a tumor-targeting antibody is covalently linked to a cytotoxic payload via a chemical linker, enabling the selective delivery of highly potent agents to malignant cells while sparing normal tissues, thereby improving the therapeutic index. Humanized and fully human immunoglobulin G1(IgG1) antibodies are the most common ADC backbones due to their stability in systemic circulation, robust Fcγ receptor engagement for immune effector functions, and reduced immunogenicity. Antibody selection requires balancing tumor specificity, internalization rate, and binding affinity to avoid barriers to tissue penetration, such as the binding-site barrier effect, while emerging designs exploit tumor-specific antigen variants or unique post-translational modifications to further enhance selectivity. Advances in antibody engineering, linker chemistry, and payload innovation have reinforced the clinical success of ADCs, with more than a dozen agents FDA approved for hematologic malignancies and solid tumors and over 200 in active clinical trials. This review critically examines established and emerging conjugation strategies, including lysine- and cysteine-based chemistries, enzymatic tagging, glycan remodeling, non-canonical amino acid incorporation, and affinity peptide-mediated methods, and discusses how conjugation site, drug-to-antibody ratio (DAR) control, and linker stability influence pharmacokinetics, efficacy, and safety. Innovations in site-specific conjugation have improved ADC homogeneity, stability, and clinical predictability, though challenges in large-scale manufacturing and regulatory harmonization remain. Furthermore, novel ADC architectures such as bispecific ADCs, conditionally active (probody) ADCs, immune-stimulating ADCs, protein-degrader ADCs, and dual-payload designs are being developed to address tumor heterogeneity, drug resistance, and off-target toxicity. By integrating mechanistic insights, preclinical and clinical data, and recent technological advances, this work highlights current progress and future directions for next-generation ADCs aimed at achieving superior efficacy, safety, and patient outcomes, especially in treating refractory cancers. Full article

Background/Objectives: Intramuscular immunization elicits systemic IgG and is the primary route of vaccine administration in humans. However, there is growing interest in utilizing other routes of administration to tailor antibody profiles, increase immunity at primary sites of infection, simplify administration, and eliminate needle waste. Here, we investigated the antibody profiles elicited by immunization with bacteriophage virus-like particle vaccine platforms at various routes of administration. Methods: We chose two model bacteriophage vaccines for investigation: bacteriophage MS2 virus-like particles (VLPs) recombinantly displaying a short, conserved peptide from Chlamydia trachomatis major outer membrane protein (MS2) and bacteriophage Qβ VLPs displaying oxycodone through chemical conjugation (Qβ). We comprehensively characterized the antibodies elicited systemically and at various mucosal sites when the vaccines were administered intramuscularly, intranasally or periocularly with or without an intramuscular prime using various prime/boost schemes. Results: Intranasal and periocular immunization elicited robust mucosal and systemic IgA responses for both MS2 and Qβ. The intramuscular prime followed by intranasal or periocular boosts elicited broad antibody responses, and increased antibodies titers at certain anatomical sites. Conclusions: These findings demonstrate the tractability of bacteriophage VLP-based vaccines in generating specific antibody profiles based on the prime–boost regimen and route of administration. Full article

Recombinant protein hydrogels have emerged as transformative biomaterials that overcome the bioinertness and unpredictable degradation of traditional synthetic systems by leveraging genetically engineered backbones, such as elastin-like polypeptides, SF, and resilin-like polypeptides, to replicate extracellular matrix (ECM) dynamics and enable programmable functionality. Constructed through a hierarchical crosslinking strategy, these hydrogels integrate reversible physical interactions with covalent crosslinking approaches, collectively endowing the system with mechanical strength, environmental responsiveness, and controlled degradation behavior. Critically, molecular engineering strategies serve as the cornerstone for functional precision: domain-directed self-assembly exploits coiled-coil or β-sheet motifs to orchestrate hierarchical organization, while modular fusion of bioactive motifs through genetic encoding or site-specific conjugation enables dynamic control over cellular interactions and therapeutic release. Such engineered designs underpin advanced applications, including immunomodulatory scaffolds for diabetic wound regeneration, tumor-microenvironment-responsive drug depots, and shear-thinning bioinks for vascularized bioprinting, by synergizing material properties with biological cues. By uniting synthetic biology with materials science, recombinant hydrogels deliver unprecedented flexibility in tuning physical and biological properties. This review synthesizes emerging crosslinking paradigms and molecular strategies, offering a framework for engineering next-generation, adaptive biomaterials poised to address complex challenges in regenerative medicine and beyond. Full article

The site-directed immobilization of enzymes has demonstrated significant potential in industrial applications due to its ability to minimize enzyme heterogeneity and maximize retained activity. However, existing approaches often require the introduction of unnatural amino acids or excessive specific ligase to achieve this goal. In this study, a self-catalyzed protein capture system (i.e., the SnoopCatcher/SnoopTag pair) was utilized for the directed immobilization of lipase on magnetic carriers. By tagging the Pseudomonas fluorescens lipase (PFL) with a SnoopTag at the C-terminal, the fused lipase PFL-SnoopTag (PSNT) readily conjugated with the SnoopCatcher partner via a spontaneously formed isopeptide bond between them. Novel magnetic particles functionalized by SnoopCatcher proteins were prepared using a co-precipitation method, achieving a loading capacity of around 0.8 mg/g carrier for the SnoopCatcher. This functionalized magnetic carrier enabled the site-directed immobilization of lipase PSNT at 81.4% efficiency, while the enzyme loading capacity reached 3.04 mg/g carriers. To further assess the practical performance of site-directed immobilized lipases, they were applied in biodiesel production and achieved a yield of 88.5%. Our results demonstrate a universal platform for the site-directed immobilization of enzymes with high performance, which offers significant advantages, e.g., single-step purification and catalyst-free immobilization of engineered enzymes, as well as easy recovery, highlighting its potential for industrial applications. Full article

The immunofluorescence assay is widely used for cellular biology and diagnosis applications. Such an antigen–antibody detection system enables the assessment and visualization of the expression and localization of target proteins. In the classical indirect immunofluorescence assay, secondary antibodies are conjugated to fluorophores. However, conventional secondary antibodies have limited applications due to their large size (150 kDa). Moreover, as animal-derived products, secondary antibodies are associated with ethical concerns and batch-to-batch variability. In this study, we developed fluorescence-labeled recombinant nanobodies as secondary antibodies by utilizing previously established anti–mouse and anti–rabbit IgG secondary nanobodies in combination with the self-labeling SNAP-tag. Nanobodies, which are significantly smaller (15 kDa), are capable to detect primary antibodies produced in mice and rabbits. The SNAP-tag (20 kDa) enables site-specific binding of various O⁶-benzylguanine (BG)-modified fluorophores to the recombinant nanobodies. These recombinant nanobodies were produced using mammalian cell expression system, and their specific binding to mouse or rabbit antibodies was validated using flow cytometry and multi-color fluorescence microscopy. The low cost, easy of expression, purification and site-specific conjugation procedures for these anti–mouse and anti–rabbit IgG secondary nanobodies make them an attractive alternative to traditional secondary antibodies for indirect immunofluorescence assays. Full article

The genes that encode the universal stress protein (USP) family domain (pfam00582) aid the survival of bacteria in specific host or habitat-induced stress conditions. Genome sequencing revealed that the genome of Helicobacter pylori, a gastric cancer pathogen, typically contains one USP gene, while related helicobacters have one or two distinct USP genes. However, insights into the functions of Helicobacteraceae (Helicobacter and Wolinella) USP genes are still limited to inferences from large-scale genome sequencing. Thus, we have combined bioinformatics and visual analytics approaches to conduct a more comprehensive data investigation of a set of 1045 universal stress protein sequences encoded in 1014 genomes including 785 Helicobacter pylori genomes. The study generated a representative set of 183 USP sequences consisting of 180 Helicobacter sequences, two Wolinella succinogenes sequences, and a sequence from a related campylobacteria. We used the amino acid residues and positions of the 12 possible functional sites in 1030 sequences to identify 25 functional sites patterns for guiding studies on functional interactions of Helicobacteraceae USPs with ATP and other molecules. Genomic context searches and analysis identified USP genes of gastric and enterohepatic helicobacters that are adjacent or in operons with genes for proteins responsive to DNA-damaging oxidative stress (ATP-dependent proteases: ClpS and ClpA); and DNA uptake proteins (natural competence for transformation proteins: ComB6, ComB7, ComB8, ComB9, ComB10, ComBE, and conjugative transfer signal peptidase TraF). Since transcriptomic evidence indicates that oxidative stress and the presence of virulence-associated genes regulate the transcription of H. pylori USP gene, we recommend further research on Helicobacter USP genes and their neighboring genes in oxidative stress response and virulence of helicobacters. To facilitate the reuse of data and research, we produced interactive analytics resources of a dataset composed of values for variables including phylogeography of H. pylori strains, protein sequence features, and gene neighborhood. Full article

Background/Objectives: Paclitaxel (PTX) is a potent anticancer drug that is poorly soluble in water. To enhance its delivery efficiency in aqueous environments, amphiphilic polymer micelles are often used as nanocarriers for PTX in clinical settings. However, the hydrophilic polymer segments on the surface of these micelles may possess potential immunogenicity, posing risks in clinical applications. To address this issue, nanomicelles based on human serum albumin (HSA)–hydrophobic polymer conjugates constructed via site-specific in situ polymerization-induced self-assembly (SI-PISA) are considered a promising alternative. The HSA shell not only ensures good biocompatibility but also enhances cellular uptake because of endogenous albumin trafficking pathways. Moreover, compared to traditional methods of creating protein–hydrophobic polymer conjugates, SI-PISA demonstrates higher reaction efficiency and better preservation of protein functionality. Methods: We synthesized HSA-PMEMA nanomicelles via SI-PISA using HSA and methoxyethyl methacrylate (MEMA)—a novel hydrophobic monomer with a well-defined and stable chemical structure. The protein activity and the PTX intracellular delivery efficiency of HSA-PMEMA nanomicelles were evaluated. Results: The CD spectra of HSA and HSA-PMEMA exhibited similar shapes, and the relative esterase-like activity of HSA-PMEMA was 94% that of unmodified HSA. Flow cytometry results showed that Cy7 fluorescence intensity in cells treated with HSA-PMEMA-Cy7 was approximately 1.35 times that in cells treated with HSA-Cy7; meanwhile, HPLC results indicated that, under the same conditions, the PTX loading per unit protein mass on HSA-PMEMA was approximately 1.43 times that of HSA. These collectively contributed to a 1.78-fold overall PTX intracellular delivery efficiency of HSA-PMEMA compared to that of HSA. Conclusions: In comparison with HSA, HSA-PMEMA nanomicelles exhibit improved cellular uptake and higher loading efficiency for PTX, effectively promoting the intracellular delivery of PTX. Tremendous potential lies in these micelles for developing safer and more efficient next-generation PTX formulations for tumor treatment. Full article

Fluorination of carbohydrates is a promising strategy to produce glycomimetics with improved pharmacological properties, such as increased metabolic stability, bioavailability and protein-binding affinity. Fluoroglycans are not only of interest as inhibitors and chemical probes but are increasingly being used to develop potential synthetic vaccine candidates for cancer, HIV and bacterial infections. Despite their attractiveness, the synthesis of fluorinated oligosaccharides is still challenging, emphasizing the need for efficient protocols that allow for the site-specific incorporation of fluorine atoms (especially at late stages of the synthesis). This is particularly true for the development of fully synthetic vaccine candidates, whose (modified) carbohydrate antigen structures (glycotopes) per se comprise multistep synthesis routes. Based on a known minimal protective epitope from the capsular polysaccharide of S. pneumoniae serotype 8, a panel of six novel F-glycotope mimetics was synthesized, equipped with amine linkers for subsequent conjugation to immunogens. Next to the stepwise assembly via fluorinated building blocks, the corresponding 6F-substituted derivatives could be obtained by microwave-assisted, nucleophilic late-stage fluorination of tri- and tetrasaccharidic precursors in high yields. The described synthetic strategy allowed for preparation of the targeted fluorinated oligosaccharides in sufficient quantities for future immunological studies. Full article

CD44, a pivotal cell surface molecule, plays a crucial role in many cellular functions, including cell-cell interactions, adhesion, and migration. It serves as a receptor for hyaluronic acid and is involved in lymphocyte activation, recirculation, homing, and hematopoiesis. Moreover, CD44 is a commonly used cancer stem cell marker associated with tumor progression and metastasis. The development of CD44 aptamers that specifically target CD44 can be utilized to target CD44-positive cells, including cancer stem cells, and for drug delivery. Building on the primary sequences of our previously selected thioaptamers (TAs) and observed variations, we developed a bead-based X-aptamer (XA) library by conjugating drug-like ligands (X) to the 5-positions of certain uridines on a complete monothioate backbone. From this, we selected an XA with high affinity to the CD44 hyaluronic acid binding domain (HABD) from a large combinatorial X-aptamer library modified with N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (ADDA). This XA demonstrated an enhanced binding affinity for the CD44 protein up to 23-fold. The selected CD44 X-aptamers (both amine form and ADDA form) also showed enhanced binding affinity to CD44-overexpressing human ovarian cancer IGROV cells. Secondary structure predictions of CD44 using MFold identified several binding motifs and smaller constructs of various stem-loop regions. Among our identified binding motifs, X-aptamer motif 3 and motif 5 showed enhanced binding affinity to CD44-overexpressing human ovarian cancer IGROV cells with ADDA form, compared to the binding affinities with amine form and scrambled sequence. The effect of ADDA as a binding affinity enhancer was not uniform within the aptamer, highlighting the importance of optimal ligand positioning. The incorporation of ADDA not only broadened the XA’s chemical diversity but also increased the binding surface area, offering enhanced specificity. Therefore, the strategic use of site-directed modifications allows for fine-tuning aptamer properties and offers a flexible, generalizable framework for developing high-performance aptamers that target a wide range of molecules. Full article

In this study, a novel rapid immunochromatographic (IC) test for African swine fever virus (ASFV) antibodies is presented. An immunochromatographic test (IC) is a detection technique that combines membrane chromatography with immunolabeling. This approach saves time for antibody preparation, resulting in a shorter production cycle. p54 is an important structural protein of African swine fever, and an ideal protein for serotype diagnosis. Gold nanoparticles are attached to the ASFV p54-Fc fusion protein, and the ASFV-specific antigen p54 and Staphylococcus aureus protein A (SPA) are labeled on a nitrocellulose membrane, at positions T and C, respectively. We developed a SPA double sandwich IC test strip, and assessed its feasibility using ASFV p54 and p54-Fc fusion proteins as antigens. ASFV p54 and p54-Fc fusion proteins were expressed and purified. A sandwich cross-flow detection method for p54, which is the primary structural protein of ASFV, was established, using colloidal gold conjugation. Our method can detect ASFV antibodies in field serum samples in about 15 min using a portable colloidal gold detector, demonstrating high specificity and sensitivity (1:320), and the coincidence rate was 98% using a commercial ELISA kit. The dilution of the serum sample can be determined by substituting the absorbance (T-line) interpreted by portable devices into the calibration curve function formula of an African swine fever virus standard serum. In summary, our method is rapid, cost-effective, precise, and highly selective. Additionally, it introduces a new approach for constructing IC test strips using SPA protein without antibody preparation, making it a reliable on-site antibody test for ASFV. 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

The ability of bacteria to recycle exogenous amino acid-based peptides and amino sugars for peptidoglycan biosynthesis was extensively investigated using optical imaging. In particular, fluorescent AeK–NBD was effectively utilized to study the peptidoglycan recycling pathway in Gram-negative bacteria. Based on these promising results, we were inspired to develop the radioactive AeK conjugate [⁶⁸Ga]Ga-DOTA-AeK for the in vivo localization of bacterial infection using PET/CT. An easy-to-implement radiolabeling procedure for DOTA-AeK with [⁶⁸Ga]GaCI₃ followed by solid-phase purification was successfully established to obtain [⁶⁸Ga]Ga-DOTA-AeK with a radiochemical purity of ≥95%. [⁶⁸Ga]Ga-DOTA-AeK showed good stability over time with less protein binding under physiological conditions. The bacterial incorporation of [⁶⁸Ga]Ga-DOTA-AeK and its fluorescent Aek-NBD analog were investigated in live and heat-killed Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Unfortunately, no conclusive in vitro intracellular uptake of [⁶⁸Ga]Ga-DOTA-AeK was observed for E. coli or S. aureus live and heat-killed bacterial strains (p > 0.05). In contrast, AeK-NBD showed significantly higher intracellular incorporation in live bacteria compared to the heat-killed control (p < 0.05). Preliminary biodistribution studies of [⁶⁸Ga]Ga-DOTA-AeK in a dual-model of chronic infection and inflammation revealed limited localization at the infection site with non-specific accumulation in response to inflammatory markers. Finally, our study demonstrates proof that the intracellular incorporation of AeK is necessary for successful bacteria-specific imaging using PET/CT. Therefore, Ga-68 was not a suitable radioisotope for tracing the bacterial uptake of AeK tripeptide, as it required chelation with a bulky metal chelator such as DOTA, which may have limited its active membrane transportation. An alternative for optimization is to explore diverse chemical structures of AeK that would allow for radiolabeling with ¹⁸F or ¹¹C. Full article

Nano-encapsulation and conjugation are the main strategies employed for drug delivery. Nanoparticles help improve encapsulation and targeting efficiency, thus optimizing therapeutic efficacy. Through nanoparticle technology, replacement of a defective gene or delivery of a new gene into a patient’s genome has become possible. Lipid nanoparticles (LNPs) loaded with genetic materials are designed to be delivered to specific target sites to enable gene therapy. The lipid shells protect the fragile genetic materials from degradation, then successfully release the payload inside of the cells, where it can integrate into the patient’s genome and subsequently express the protein of interest. This review focuses on the development of LNPs and nano-pharmaceutical techniques for improving the potency of gene therapies, reducing toxicities, targeting specific cells, and releasing genetic materials to achieve therapeutic effects. In addition, we discuss preparation techniques, encapsulation efficiency, and the effects of conjugation on the efficacy of LNPs in delivering nucleic acid materials. Full article

Lipids are primarily transported in the bloodstream by lipoproteins, which are macromolecules of lipids and conjugated proteins also known as apolipoproteins. The processes of lipoprotein assembly, secretion, transportation, modification, and clearance are crucial components of maintaining a healthy lipid metabolism. Disruption in any of these steps results in pathophysiological abnormalities such as dyslipidemia, obesity, insulin resistance, inflammation, atherosclerosis, peripheral artery disease, and cardiovascular diseases. By studying these genetic mutations, researchers can gain valuable insights into the underlying mechanisms that govern the relationship between protein structure and its physiological role. These lipoproteins, including HDL, LDL, lipoprotein(a), and VLDL, mainly serve the purpose of transporting lipids between tissues and organs. However, studies have provided evidence that apo(a) also possesses protective properties against pathogens. In the future, the field of study will be significantly influenced by the integration of recombinant DNA technology and human site-specific mutagenesis for treating hereditary disorders. Several medications are available for the treatment of dyslipoproteinemia. These include statins, fibrates, ezetimibe, niacin, PCSK9 inhibitors, evinacumab, DPP 4 inhibitors, glucagon-like peptide-1 receptor agonists GLP1RAs, GLP-1, and GIP dual receptor agonists, in addition to SGLT2 inhibitors. This current review article exhibits, for the first time, a comprehensive reflection of the available body of publications concerning the impact of lipoproteins on metabolic well-being across various pathological states. Full article