Functional Biomolecules Research Articles

Liquid–liquid phase separation in neurodegenerative diseases: An updated understanding

Liquid–liquid phase separation (LLPS), once understood merely as a physicochemical phenomenon, has emerged over the past decade as a critical player in life processes. LLPS provides a novel framework for understanding the structure, function, and spatiotemporal regulation of intracellular biomolecules. Through LLPS, biomolecules within cells can spontaneously assemble into membraneless compartments, which allow precise regulation of biochemical reactions and influence critical cellular processes such as signal transduction and gene expression. Despite its recognized significance in basic biological research, the role of LLPS in human disease is still an area worthy of continued exploration. Currently, the most studied disorders in relation to LLPS are neurodegenerative diseases and cancer. In neurodegenerative diseases, LLPS is closely linked to protein misfolding and aggregation, processes that can lead to the formation of toxic assemblies, ultimately causing neuronal damage and death. In cancer, aberrant LLPS may contribute to the dysregulation of signaling pathways, promoting uncontrolled cell proliferation and metastasis. This review highlights recent advances regarding the role of LLPS in the pathogenesis of neurodegenerative diseases, discussing its function in these pathological conditions and proposing directions for future research. As research progresses, the potential role of LLPS in other human diseases will likely be uncovered, offering new avenues for diagnosis and therapy. Therefore, further investigation into the mechanisms of LLPS and its involvement in disease pathology will be crucial for advancing our understanding of human health and disease.

Unraveling the venom constituents of the endoparasitoid Aphidius gifuensis with an emphasis on the discovery of a novel insecticidal peptide.

Venom serves as a pivotal parasitic factor employed by parasitoid wasps to manipulate their hosts, creating a favorable environment for the successful growth of their progeny, and ultimately kill the host. The bioactive molecules within parasitoid venoms exhibit insecticidal activities with promising prospects for agricultural applications. However, knowledge regarding the venom components of parasitoids and the discovery of functional biomolecules from them remains limited. In this study, 30 venom proteins were identified from the endoparasitoid Aphidius gifuensis through the application of a transcriptomic approach. These proteins were categorized into five groups: hydrolase, molecular chaperone, transferase, other functional protein, and hypothetical protein with unknown function. Particularly noteworthy is the abundant expression of the peptide Vn1 in the venom apparatus of A. gifuensis, indicating its pivotal role in venom activity. Consequently, Vn1 was chosen for further functional analysis, exhibiting insecticidal activity against Tenebrio molitor pupae. Further assessment for revealing its mode of action disclosed that Vn1 impacts genes related to immune response, environmental information processing, metabolism, and response to external stimuli in T. molitor, suggesting its involvement in the intricate parasitoid wasp-host interaction. The findings of this study significantly contribute to our knowledge of the composition and functionality of A. gifuensis venom, establishing a foundation for further investigation into the biological roles of the identified venom constituents. The insecticidal Vn1 isolated from the venom of this parasitoid represents a valuable resource for the development of innovative biocontrol agents with potential applications in agriculture. © 2024 Society of Chemical Industry.

Scalable deoxygenative alkynylation of alcohols via flow photochemistry

Internal alkynes are often contained in bioactive pharmaceuticals and crucial intermediates in material sciences, yet their production methods are often limited and challenging, necessitating the development of more efficient and versatile synthetic routes. Here we report a method of deoxygenative alkynylation of alcohols via flow photochemistry. Formation of N-heterocyclic carbene-alcohol adducts undergoes oxidation by a photocatalyst, generating alkyl radicals. These radicals are subsequently trapped by an alkynylation agent, yielding the desired alkyne. Compared to batch reactions, the strategy using flow photochemistry is practical and efficient to complete the reaction in relatively short time with good yields. A wide range of functional groups were tolerated. The broad application of this method for alkyne synthesis in industry settings is anticipated, supported by the potential in late-stage functionalization of biomolecules and gram-scale synthesis.

Photoclick and Release for Spatiotemporally Localized Theranostics of Single Cells via In Situ Generation of 1,3‐Diaryl‐1H‐benzo[f]indazole‐4,9‐dione

AbstractBioorthogonal click‐release chemistry is a cutting‐edge tool for exploring and manipulating biomolecule functions in native biological systems. However, it is challenging to achieve the precise regulation or therapy of individual cells via click‐release strategies driven by proximity and thermodynamics. Herein, we propose a novel photoclick‐release approach based on a photo‐induced cycloaddition between 4,4′‐bis(N‐arylsydnone) or C‐bithienyl‐diarylsydnone and 2‐arylamino‐naphthoquinone via irradiation with 405 or 485 nm light. It constructs 1,3‐diaryl‐1H‐benzo[f]indazole‐4,9‐dione (BIZON) as a pharmacophore while releases an arylamine for fluorescence turn‐on probing. Both photoclick reagents were tailored by connecting to the triphenyl phosphonium delivery motif for enrichment in the mitochondria of live cells. This enables an intracellular photoclick and release under the control of 405 or 485 nm light. We then discovered that the in situ photo‐generated BIZON is capable of photosensitizing upon 485 or 520 nm light to produce singlet oxygen inside the mitochondria under aerobic conditions. Therefore, we realized wash‐free fluorescence tracking and subsequent anti‐cancer efficacy at single‐cell resolution using global illumination, which provides a foundation for wavelength‐gated single‐cell theranostics.

Photoclick and Release for Spatiotemporally Localized Theranostics of Single Cells via In Situ Generation of 1,3-Diaryl-1H-benzo[f]indazole-4,9-dione.

Bioorthogonal click-release chemistry is a cutting-edge tool for exploring and manipulating biomolecule functions in native biological systems. However, it is challenging to achieve the precise regulation or therapy of individual cells via click-release strategies driven by proximity and thermodynamics. Herein, we propose a novel photoclick-release approach based on a photo-induced cycloaddition between 4,4'-bis(N-arylsydnone) or C-bithienyl-diarylsydnone and 2-arylamino-naphthoquinone via irradiation with 405 or 485 nm light. It constructs 1,3-diaryl-1H-benzo[f]indazole-4,9-dione (BIZON) as a pharmacophore while releases an arylamine for fluorescence turn-on probing. Both photoclick reagents were tailored by connecting to the triphenyl phosphonium delivery motif for enrichment in the mitochondria of live cells. This enables an intracellular photoclick and release under the control of 405 or 485 nm light. We then discovered that the in situ photo-generated BIZON is capable of photosensitizing upon 485 or 520 nm light to produce singlet oxygen inside the mitochondria under aerobic conditions. Therefore, we realized wash-free fluorescence tracking and subsequent anti-cancer efficacy at single-cell resolution using global illumination, which provides a foundation for wavelength-gated single-cell theranostics.

Abstract PR014: DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity

Abstract Cells release extracellular vesicles (EVs) loaded with functional biomolecules that facilitate intercellular communication in health and disease. These EVs contain DNA (EV-DNA), mirroring the mutational profile of parental cells, yet the mechanism of DNA packaging onto EVs and its role in diseases like cancer remain unclear. Using advanced imaging and biochemical techniques, we discovered that EV-DNA predominantly resides on the vesicle surface, bound to modified histones. Through genome-wide CRISPR knockout screens in cancer cell lines, we identified immune-related pathways and genes crucial for EV-DNA loading, such as APAF1 and NCF1. In several cancer models, loss of APAF1 reduced EV-DNA packaging, and enhancing metastasis, thus implicating EV-DNA in immune surveillance against cancer spread. We demonstrated that EV-DNA uptake by liver macrophages triggers DNA damage response, altering cytokine production and promoting immune activation in pre-metastatic sites. Moreover, quantifying EV-DNA in colorectal cancer biopsies revealed its potential as a predictive metastatic risk biomarker. Our findings shed light on EV-DNA packaging mechanisms and its significance, suggesting that elevated levels of tumor-derived EV-DNA bolster anti-tumor immunity and inhibit metastasis. Citation Format: Inbal Wortzel, Han-Sang Kim, David C. Lyden. DNA associated with EVs is uniquely chromatinized and prevents metastasis by enhancing anti-tumor immunity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR014.

Ruminal microbiota, carbohydrate-active enzymes and milk bioactive compounds in Italian Mediterranean dairy buffaloes fed total mixed ration with or without green forage

The aim of the present study was to determine the effect of including green forage in the diet of Italian Mediterranean dairy buffaloes on the ruminal microbiota, CAZymes profile, functional biomolecules and total antioxidant activity in bulk milk. Sixteen buffaloes were randomly assigned according to lactation number and daily milk production to two homogeneous groups, and for 60 days received each: Group 1, a standard total mixed ration (TMR) or group 2, TMR + ryegrass green forage (30% of diet). The diets of the two groups were iso-nitrogenous and iso-energetic and differed only in the proportion of green forage. Buffaloes that received TMR + green feed had a higher (p < .01) representation of bacteria belonging to the orders Veillonellales, Selenomonadales and Bradymonadales compared with buffaloes that received TMR. The former buffaloes also had a greater (p < .01) abundance of CAZymes of the GT class (GHT4, GT14, GT20, GT26, GT39) and AA class (AA1, AA3, AA6). The milk of buffaloes that received TMR + green feed had a higher (p < .01) antioxidant capacity and greater (p < .01) amounts of the functional biomolecules l-carnitine, propionyl-l-carnitine, acetyl-l-carnitine and δ-valerobetaine. The findings have provided evidence for metabolic and biosynthetic pathways that link green forage with rumen bacteria, CAZymes and the synthesis of amino acids and functional biomolecule in buffaloes.

Biofabricated silver nanoparticles from Calotropis procera: A potential antimicrobial solution against pathogenic bacteria

Calotropis procera has the potential as a traditional medicinal plant that offers a diverse array of healing treatments. It was used in conventional medicine to remedy several ailments, including diarrhea and snakebites. This study examines the antibacterial properties of silver nanoparticles derived from the floral and latex components of C. procera. The nanoparticles were tested for their effectiveness against Gram-negative and Gram-positive bacterial strains. For characterization, the synthesized nanoparticles underwent thorough analysis using UV spectroscopy, fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM). After the reduction process, the FTIR analysis revealed that some functional biomolecules found in plants form a coating on the nanoparticles, serving as organic agents that stabilize the nanoparticles. The size of nanoparticles analyzed with SEM falls in the 30 to 130 nm range. Furthermore, the synthesized silver nanoparticles were tested as antimicrobial agents against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Pseudomonas aeruginosa, well-known microorganisms that can produce ulceration phases and severe health consequences. After a comprehensive examination, it was found that the silver nanoparticles have significant antibacterial effectiveness, particularly against S. aureus, a primary bacterium responsible for causing different ailments. The remarkable antibacterial efficacy of the silver nanoparticles synthesized in an environment-friendly manner underscores its potential applications in combating the evolution of antibiotic-resistant strains.

Molecular Engineering of a Tumor-Targeting Thione-Derived Diketopyrrolopyrrole Photosensitizer to Attain NIR Excitation Over 850 nm for Efficient Dual Phototherapy.

Photosensitizers with near-infrared (NIR) excitation, especially above 800 nm which is highly desired for phototherapy, remain rare due to the fast nonradiative relaxation process induced by exciton-vibration coupling. Here, a diketopyrrolopyrrole-derived photosensitizer (DTPA-S) is developed via thionation of carbonyl groups within the diketopyrrolopyrrole skeleton, which results in a large bathochromic shift of 81 nm, endowing the photosensitizer with strong NIR absorption at 712 nm. DTPA-S is then introduced with a functional biomolecule (N3-PEG2000-RGD) via click reaction for the construction of integrin αvβ3 receptor-targeted nano-micelles (NanoDTPA-S/RGD), which endows the photosensitizer with a further superlarge absorption redshift of 138 nm, thus extending the absorption maxima to ≈850 nm. Remarkably, thiocarbonyl substitution increases the nonbonding characters in frontier molecular orbitals, which can effectively suppress the nonradiative vibrational relaxation process via reducing the reorganization energy, enabling efficient reactive oxygen species (ROS) generation under 880 nm excitation. Screened by in vitro and in vivo assays, NanoDTPA-S/RGD with high water solubility, excellent tumor-targeting ability, and photodynamic/photothermal therapy synergistic effect exhibits satisfactory phototherapeutic performance. Overall, this study demonstrates a new design of efficient NIR-triggered diketopyrrolopyrrole photosensitizer with facile installation of functional biomolecules for potential clinical applications.

Flexible substrate-based mass spectrometry platform for in situ non-destructive molecular imaging of living plants.

Monitoringand localizing molecules on living plants is critical for understanding their growth, development and disease. However, current techniques for molecular imaging of living plants often lack spatial information or require tedious pre-labelling. Here, we proposed a novel molecular imaging platform that combines sliver nanowire-doped Ti3C2 MXene (AgNWs@MXene) flexible film substrate with laser desorption/ionization mass spectrometry imaging (AMF-LDI-MSI) to study the spatial distribution of biomolecules on the surface of living plants. This platform overcomes the MSI challenges posed by difficult-to-slice plant tissues (e.g., tough or water-rich roots and fragile flowers) and enables precisely transfer and visualize the molecule. Comparisons of the measurement results to those from matrix-assisted LDI-MSI (MALDI-MSI) technology demonstrate the accuracy and reliability of the platform. Biocompatibility evaluations indicated that the platform without observable adverse effects on the health of living plants. The distribution of growth and disease-associated signalling molecules, such as choline, organic acids and carbohydrates, can be in situ non-destructively detected on the surfaces of living plants, which is important for tracking the health of plants and their diseased areas. AMF-LDI-MSI platform can serve as a promising tool for label-free, in situ and non-destructive monitoring of functional biomolecules and plant growth from a spatial perspective.

An Effective Surface Passivation Assay for Single-Molecule Studies of Chromatin and Topoisomerase II.

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Biomolecular condensates regulate cellular electrochemical equilibria

Control of the electrochemical environment in living cells is typically attributed to ion channels. Here, we show that the formation of biomolecular condensates can modulate the electrochemical environment in bacterial cells, which affects cellular processes globally. Condensate formation generates an electric potential gradient, which directly affects the electrochemical properties of a cell, including cytoplasmic pH and membrane potential. Condensate formation also amplifies cell-cell variability of their electrochemical properties due to passive environmental effect. The modulation of the electrochemical equilibria further controls cell-environment interactions, thus directly influencing bacterial survival under antibiotic stress. The condensate-mediated shift in intracellular electrochemical equilibria drives a change of the global gene expression profile. Our work reveals the biochemical functions of condensates, which extend beyond the functions of biomolecules driving and participating in condensate formation, and uncovers a role of condensates in regulating global cellular physiology.

One-Step fabrication of bioinspired Peptide-Functionalized ice surface for bioanalysis

Solid surfaces modified with functional biomolecules that can selectively capture specific molecules play a crucial role in physical science and biomedical research. However, convenient biomolecule-modified materials are still faced with problems such as harsh synthesis conditions and time-consuming functionalization processes. As an easy-to-prepare solid substance, ice, holds the potential to be developed as an excellent functional material, but its inert nature hampers efficient functionalization with biomolecules. In this study, drawing inspiration from anti-freeze organisms in nature, we endow ice with biorecognition ability by leveraging the natural non-covalent binding between peptides and ice. By exploiting ice as a substrate modified with functional biomolecules, we demonstrate advantages such as high accessibility, one-step modification, and cost-effectiveness compared to conventional biomolecule-modified materials. Additionally, the temperature-controlled melting of ice offers a facile way to release captured biomolecules while maintaining their activity for further studies. The feature is further applied in protein assays, exhibiting satisfactory selectivity and quantitative capabilities with a minimum detectable concentration of approximately 0.1 ng/mL. Overall, our strategy to endow ice with desired biological functions provides a new tool for investigation in physical science and broader applications in the field of biomedicine.

Cyclohexanediamine Triazole (CHDT) Functionalization Enables Labeling of Target Molecules with Al18F/68Ga/111In.

The Al18F-labeling approach offers a one-step access to radiofluorinated biomolecules by mimicking the labeling process for radiometals. Although these labeling conditions are considered to be mild compared to classic radiofluorinations, improvements of the chelating units have led to the discovery of (±)-H3RESCA, which allows Al18F-labeling already at ambient temperature. While the suitability of (±)-H3RESCA for functionalization and radiofluorination of proteins is well established, its use for small molecules or peptides is less explored. Herein, we advanced this acyclic pentadentate ligand by introducing an alkyne moiety for the late-stage functionalization of biomolecules via click chemistry. We show that in addition to Al18F-labeling, the cyclohexanediamine triazole (CHDT) moiety allows stable complexation of 68Ga and 111In. Three novel CHDT-functionalized PSMA inhibitors were synthesized and their Al18F-, 68Ga-, and 111In-labeled analogs were subjected to a detailed in vitro radiopharmacological characterization. Stability studies in vitro in human serum revealed among others a high kinetic inertness of all radiometal complexes. Furthermore, the Al18F-labeled PSMA ligands were characterized for their biodistribution in a LNCaP derived tumor xenograft mouse model by PET imaging. One radioligand, Al[18F]F-CHDT-PSMA-1, bearing a small azidoacetyl linker at the glutamate-urea-lysine motif, provided an in vivo performance comparable to that of [18F]PSMA-1007 but with even higher tumor-to-blood and tumor-to-muscle ratios at 120 min p.i. Overall, our results highlight the suitability of the novel CHDT moiety for functionalization and radiolabeling of small molecules or peptides with Al18F, 68Ga, and 111In and the triazole ring seems to entail favorable pharmacokinetic properties for molecular imaging purposes.

Inflammation, mitochondrial and lysosomal dysfunction as key players in rheumatoid arthritis?

Rheumatoid arthritis (RA) is an inflammatory joint disease characterized by persistent synovitis and inflammation. The exact mechanism of mitochondrial function in the presence of inflammation and dysregulation of autophagic processes in the pathogenesis of RA is still unclear.The aim of our study is to determine mitochondrial function, gene and protein levels of biomolecules related to inflammation (YKL-40) and autophagy (LAMPs) and to search a connection between them in the RA context.Twenty newly diagnosed RA patients and ten healthy individuals were included in the study. Disease severity was assessed by ultrasonography. Conventional clinico-laboratory parameters, immunological markers and protein levels of LAMPs and YKL-40 were examined in plasma. Gene expression analysis for the quantitative measurement of LAMPs and YKL-40 were conducted in white blood cells (WBC). A real-time metabolic analysis was performed to assess mitochondrial function and cell metabolism in peripheral blood mononuclear cells (PBMCs).Increase in spare respiratory capacity in PBMCs of RA patients was detected. Decreased LAMPs plasma protein levels and increased protein levels of YKL-40 in RA patients compared to healthy individuals were measured. No significant differences were found in gene expressions. Correlations between mitochondrial, ultrasonographic and protein levels of the biomarkers related with inflammation and autophagy were established.New data on mitochondrial dysfunction in RA patients and links to inflammation and mitophagy are reported. The relationship between dysregulation of mitophagy and joint diseases deserves to be thoroughly investigated as it would be a promising therapeutic approach.

Solvent effect on the excited state photophysics of Imiquimod: A DFT/TD-DFT and spectroscopic study

Solvation plays an important role in chemistry and biology. The role of the solvent is crucial in any chemical processes such as tautomerization that controls the structure and function of biomolecules. The current study aims to explore how different solvent medium affects the tautomeric forms of an antitumor drug, Imiquimod (IMQ). We have used several spectroscopical methods, including UV–Vis absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy, and quantum mechanical (QM) calculations. Our results revealed that solvent, indeed, plays a significant role in the modulation of the photophysics of the drug. IMQ has three emission bands in protic and aprotic solvents and two bands in non-polar medium associated with different forms of the drug. Using time-resolved technique, and comparing with the predicted lifetimes from QM calculations, we succeed to assign these three forms as cation, tautomer and neutral of IMQ with 1.6 ns, 2.0 ns and 4.0 ns lifetime values, respectively. Since IMQ is a nucleobase analogue and one of the most effective medications for skin tumors; these findings about which specie of IMQ is present in a given medium, and beyond, how the medium alters the photophysics of the molecule may provide deeper insights into its structure and function.

FUCA1: An Underexplored p53 Target Gene Linking Glycosylation and Cancer Progression.

Cancer is a difficult-to-cure disease with high worldwide incidence and mortality, in large part due to drug resistance and disease relapse. Glycosylation, which is a common modification of cellular biomolecules, was discovered decades ago and has been of interest in cancer research due to its ability to influence cellular function and to promote carcinogenesis. A variety of glycosylation types and structures regulate the function of biomolecules and are potential targets for investigating and treating cancer. The link between glycosylation and carcinogenesis has been more recently revealed by the role of p53 in energy metabolism, including the p53 target gene alpha-L-fucosidase 1 (FUCA1), which plays an essential role in fucosylation. In this review, we summarize roles of glycan structures and glycosylation-related enzymes to cancer development. The interplay between glycosylation and tumor microenvironmental factors is also discussed, together with involvement of glycosylation in well-characterized cancer-promoting mechanisms, such as the epidermal growth factor receptor (EGFR), phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and p53-mediated pathways. Glycan structures also modulate cell-matrix interactions, cell-cell adhesion as well as cell migration and settlement, dysfunction of which can contribute to cancer. Thus, further investigation of the mechanistic relationships among glycosylation, related enzymes and cancer progression may provide insights into potential novel cancer treatments.

D-π-A type fluorescent dyes: Effect of π-bridge units on optical and G4 DNA binding properties

Fluorescent solvatochromic dyes that are sensitive to the nature of local microenvironmental, have been explored as probes in applications ranging from the imaging biomolecules to understanding of basic biomolecule functions. To expand the scope of fluorescent solvatochromic dyes for G-quadruplex (G4) DNA structures, and to illustrate the relationship between structure and properties, three newly designed D-π-A type fluorescent dyes were synthesized by introducing diarylimidazole to carbazole skeleton linked to benzene, furan or thiophene π-conjugated bridge and connected with pyridinium acceptor, respectively. Their structural characteristics, optical properties, and G4 DNA binding properties were discussed in detail. In general, the incorporation of furan and thiophene as π-conjugated bridges leads the better conjugation and molecular coplanarity with more efficient intramolecular charge transfer (ICT) effect compared with benzene bridge. The fluorescence intensities induced upon interaction were found that TP-6 with thiophene π-conjugated bridge had the strongest response toward G4 DNAs. In addition, the application of this dye as a fluorescent agent for living cell imaging was also demonstrated.

Enhancement of Orthodontic Tooth Movement by Local Administration of Biofunctional Molecules: A Comprehensive Systematic Review.

Enhancement of orthodontic tooth movement (OTM) through local administration of biofunctional molecules has become increasingly significant, particularly for adult patients seeking esthetic and functional improvements. This comprehensive systematic review analyzes the efficacy of various biofunctional molecules in modulating OTM, focusing on the method of administration and its feasibility, especially considering the potential for topical application. A search across multiple databases yielded 36 original articles of experimental human and animal OTM models, which examined biofunctional molecules capable of interfering with the biochemical reactions that cause tooth movement during orthodontic therapy, accelerating the OTM rate through their influence on bone metabolism (Calcitriol, Prostaglandins, Recombinant human Relaxin, RANKL and RANKL expression plasmid, growth factors, PTH, osteocalcin, vitamin C and E, biocompatible reduced graphene oxide, exogenous thyroxine, sclerostin protein, a specific EP4 agonist (ONO-AE1-329), carrageenan, and herbal extracts). The results indicated a variable efficacy in accelerating OTM, with Calcitriol, Prostaglandins (PGE1 and PGE2), RANKL, growth factors, and PTH, among others, showing promising outcomes. PGE1, PGE2, and Calcitriol experiments had statistically significant outcomes in both human and animal studies and, while other molecules underwent only animal testing, they could be validated in the future for human use. Notably, only one of the animal studies explored topical administration, which also suggests a future research direction. This review concluded that while certain biofunctional molecules demonstrated potential for OTM enhancement, the evidence is not definitive. The development of suitable topical formulations for human use could offer a patient-friendly alternative to injections, emphasizing comfort and cost-effectiveness. Future research should focus on overcoming current methodological limitations and advancing translational research to confirm these biomolecules' efficacy and safety in clinical orthodontic practice.

Gelatin and lipidoid integrate to create gelasomes to enhance siRNA delivery with low toxicity

RNAi therapeutics possess the potential to cure many uncurable human diseases. For instance, RNAi therapeutics using liposomes showed remarkable survival benefits in patients with liver diseases. However, the extension of liposomes to deliver RNA to cure other ailments has largely been unsuccessful. Therefore, researchers are focusing on designing and testing different combinations of materials for versatile RNA delivery applications. Yet, an efficient and safe RNA delivery platform has not been identified. In this work, we have developed a new class of RNA-delivery vehicle called “Gelasomes,” using an incongruous combination of gelatin and lipidoid to exploit each material's unique properties while overcoming their inherent limitations. The low in vivo toxicity of Gelasomes is attributed to the exterior gelatin layers that shield the exposure of cationic lipidoid-siRNA clusters and yet present a biocompatible surface. Indeed, toxicity studies in mice indicate that repeated administration of Gelasomes (up to 48 mg/kg BW) is well-tolerated with no notable changes in body weight, hematology, or serum chemistry. Interestingly, the gelatin outer layer efficiently protects siRNA from serum degradation (48 h), preserving its functionality beyond two months of storage. Notably, Gelasomes possess dual siRNA conjugation modes, i.e., electrostatic binding with lipidoid core and covalent attachment to gelatin surface. The bivalency coupled with lipidoids' high transfection efficiency rendered Gelasomes with remarkably high gene silencing efficiency (>90 %) at very low treatment doses in vitro (40 μg/mL). In vivo studies further confirmed the high gene silencing ability of Gelasomes in non-small cell lung tumor mouse models. This new platform is tunable on all fronts: size, degree of surface coating, and biomolecule functionalization. Truncating the lipidoid C14-tail to a C8-tail yielded Gelasomes of reduced size. As lipidoids with different carbon lengths are synthesizable, we can develop a library of Gelasomes with different sizes. The surface coating with less gelatin resulted in high transfection efficiency at low doses of Gelasomes. The structure of Gelasomes offers chemical handles to couple target-specific molecules like antibodies to tune their properties for efficient biological application.