Disulfide Bond Cross-linking Research Articles

Enhancing Tumor Cell Affinity and Inhibiting Growth With Albumin‐Coated Crosslinked Chitosan Nanoparticle Micelles

ABSTRACTChitosan (CS)‐based drug delivery systems often face challenges in obtaining clinical approval. Enhancing the binding affinity of CS‐based carriers to target cells, such as tumor cells, is crucial for their translation from bench to bedside. Strategies like crosslinking (NHS‐disulfide‐NHS) and utilizing materials like albumin have proven effective in strengthening interactions between CS‐based nanoparticles and tumor cells, thereby enhancing cellular uptake (cellular avidity). This study presents a novel approach using crosslinked CS nanoparticles loaded with doxorubicin, featuring reduction‐responsive drug release and albumin coating. The albumin‐coated crosslinked chitosan (ACCC) was assembled via reaction between amino of CS/albumin and NHS‐disulfide‐NHS. The resulting ACCC nanoparticle micelles demonstrate enhanced stability of the micelles. The hybrid crosslinked network ensures stability while providing ample space for drug loading and achieves reduction‐responsive drug release through disulfide bond crosslinking. Moreover, the proposed system possesses excellent biocompatibility. Compared to uncoated crosslinked chitosan nanoparticle micelles, ACCC nanoparticle micelles exhibit potent cytotoxicity against tumor cells, particularly in reducing environment. Fluorescence imaging and flow cytometry analyses confirm the enhanced cellular affinity conferred by albumin coating. These findings underscore the potential of ACCC nanoparticles in improving tumor cell adhesion and anti‐tumor efficacy, thus offering promising prospects for translational medicine in CS‐based drug delivery.

Magnetic relaxation switch biosensor for detection of Vibrio parahaemolyticus based on photocleavable hydrogel

BackgroundFoodborne pathogens, particularly Vibrio parahaemolyticus (VP) found in seafood, pose significant health risks, including abdominal pain, nausea, and even death. Rapid, accurate, and sensitive detection of these pathogens is crucial for food safety and public health. However, existing detection methods often require complex sample pretreatment, which limits their practical application. This study aims to overcome these limitations by developing a label-free magnetic relaxation switch (MRS) biosensor for the detection of VP, utilizing a photocleavable sol-gel phase transition system for improved efficiency and accuracy. ResultsIn this work, a tag-free magnetic relaxation switch (MRS) biosensor was designed for the detection of Vibrio parahaemolyticus (VP), based on a photocleavable sol-gel phase transition system. A large amount of lithium acyl hypophosphite (LAP), gold nanoparticles (AuNPs), and single-stranded DNA (ssDNA) loaded on the surface of Ti3C2Tx MXene acted as the signal unit LAP-MXene@AuNPs-ssDNA. The pipette tip served as a reaction vessel, and when VP was present, Apt specifically captured VP and released the signal units. The released signal units were then injected into the low-field nuclear magnetic resonance (LF-NMR) test solution, a gel formed by crosslinking of disulfide bonds. The gel was cleaved by LAPs on the signal units under ultraviolet (UV) irradiation, triggering a gel-sol phase transition, which increased transverse relaxation time (T2), thus enabling the detection of VP. Under the optimal experimental conditions, the linear range and detection limit for VP were 102 ∼ 108 CFU/mL and 10 CFU/mL, respectively. Significance and noveltyThe simplified biometric identification process in the pipette tip reduces errors from multiple sample transfers, enhancing efficiency. The use of photocleavable hydrogel for signal output eliminates issues associated with magnetic material aggregation, significantly improving detection precision. The assay is of good selectivity, stability reproducibility, and convenience, having a broad application prospect in the rapid detection of pathogenic bacteria in the field.

Fabrication of pea protein isolate-stabilized oil-in-water emulsions with high freeze-thaw stability: Effect of high intensity ultrasonic on emulsions and interfacial protein structure

The poor freeze-thaw stability of plant protein-based emulsions posed a major challenge for their application in the cold chain foods. The present study successfully prepared oil-in-water emulsions stabilized solely with pea protein isolate (PPI) with excellent freeze-thaw stability using a facile high intensity ultrasonic (HIU)-assisted method. The optimized emulsion can endure three freeze-thaw cycles without stratification or oil leakage, maintaining oil droplet sizes at the nanometer scale. Furthermore, changes of interfacial protein structure induced by HIU were systematically investigated to reveal the stabilizing mechanism. The freeze-thaw stability of emulsions treated under varying intensities was evaluated based on changes in the appearance and oil drop size distribution. The results showed that the oil drop size of emulsions decreased from micron to nanometer and the freeze-thaw stability was significantly enhanced with the increase of HIU intensity. The emulsion exhibited optimized freeze-thaw stability under 800W. Subsequently, the effect of HIU on the structure of interfacial protein was analyzed through SDS-PAGE, –SH/S–S contents, UV spectrum, surface hydrophobicity, protein adsorption percentage, emulsifying activity and interfacial dilatational rheology. It was found that HIU triggered crosslinking of disulfide bonds among interfacial PPI molecules and enhanced the strength of the interface layer. Additionally, HIU promoted the unfolding of interfacial PPI structure, resulting in the improvement of surface hydrophobicity, emulsifying activity, and interfacial adsorption percentage of PPI. The study demonstrated the feasibility of using HIU to improve the freeze-thaw stability of oil-in-water emulsions stabilized with PPI and provided references for the fabricating of plant protein-based frozen emulsion foods.

Alkali-Induced Protein Structural, Foaming, and Air-Water Interfacial Property Changes and Quantitative Proteomic Analysis of Buckwheat Sourdough Liquor.

In this study, the protein structural, foaming, and air-water interfacial properties in dough liquor (DL) ultracentrifugated from buckwheat sourdough with different concentrations of an alkali (1.0-2.5% of sodium bicarbonate) were investigated. Results showed that the alkali led to the cross-linking of protein disulfide bonds through the oxidation of free sulfhydryl groups in DL. The alterations in protein secondary and tertiary structures revealed that the alkali caused the proteins in DL to fold, decreased the hydrophobicity, and led to a less flexible but compact structure. The alkali accelerated the diffusion of proteins and decreased the surface tension of DL. In addition, the alkali notably improved the foam stability by up to 34.08% at 2.5% concentration, mainly by increasing the net charge, reducing the bubble size, and strengthening the viscoelasticity of interfacial protein films. Quantitative proteomic analysis showed that histones and puroindolines of wheat and 13S globulin of buckwheat were closely related to the changes in the alkali-induced foaming properties. This study sheds light on the mechanism of alkali-induced improvement in gas cell stabilization and the buckwheat sourdough steamed bread quality from the aspect of the liquid lamella.

Dynamic changes of tenderness, moisture and protein in marinated chicken: the effect of different steaming temperatures.

The steam processing characteristics of chicken are a key factor in the simplicity and versatility of steamed chicken dishes. The aim of this study was to investigate in depth the changes in tenderness and water retention of marinated chicken at different slow steaming endpoint temperatures, and to further explore the effect of the evolution of protein conformations on the water status. The results showed that chicken samples' shear force peaked at 80 °C and decreased rapidly at 90 °C. As the steaming endpoint temperature increased between 50 and 90 °C, T21, T22, moisture content and centrifugal loss decreased, but P21, P22 and myofibril water-holding capacity showed regular changes. The electrophoretic bands and protein conformation changes showed that protein in marinated chicken underwent different degrees of denaturation, degradation and aggregation. And at 70 °C, with an increase of hydrophobic groups and crosslinking of disulfide bonds as well as an increase in the number of denatured sarcoplasmic proteins, the intermolecular network was enhanced, thus affecting the water retention. Water status of chicken meat heated at different steaming temperatures is closely related to the evolution of protein conformations. The present study serves as a robust theoretical foundation for enhancing the quality of steamed chicken products at an industrial scale. © 2024 Society of Chemical Industry.

Effect of Microwave Treatment on Protease Activity, Dough Properties and Protein Quality in Sprouted Wheat.

In this study, the effects of microwave treatment on protease activity, dough properties and protein quality in sprouted wheat were investigated. Microwave treatment led to a significant (p < 0.05) reduction in protease activity in sprouted wheat. Proteases with a pH optimum of 4.4 (cysteine proteinases) were more susceptible to microwave heating, which contributed mostly to protease inactivation. Significant improvements (p < 0.05) in the dough properties and gluten quality of sprouted wheat were observed, which are probably attributable to the synergistic effectiveness of protease inactivation and heat-induced gluten cross-linking. After microwave treatment, the decrease in the solubility and extractability of protein in sprouted wheat indicated protein polymerization, which was induced by intermolecular disulfide bond cross-linking. The changes in gliadin were less pronounced due to the relatively low temperature of the microwave treatment. The cross-linking in sprouted wheat that occurred after microwave treatment seemed to mainly involve glutenin, especially B/C low-molecular-weight glutenin subunits (B/C-LMW-GSs) in the range of 30-50 kD.

Transparent, Tough, and Self-Healable Elastomer Based on Dynamic Dual Cross-Linking of Coordination and Disulfide Bonds

Transparent, Tough, and Self-Healable Elastomer Based on Dynamic Dual Cross-Linking of Coordination and Disulfide Bonds

Glutenin-chitosan 3D porous scaffolds with tunable stiffness and systematized microstructure for cultured meat model

A glutenin (G)-chitosan (CS) complex (G-CS) was cross-linked by water annealing with aim to prepare structured 3D porous cultured meat scaffolds (CMS) here. The CMS has pore diameters ranging from 18 to 67 μm and compressive moduli from 16.09 to 60.35 kPa, along with the mixing ratio of G/CS. SEM showed the porous organized structure of CMS. FTIR and CD showed the increscent content of α-helix and β-sheet of G and strengthened hydrogen-bondings among G-CS molecules, which strengthened the stiffness of G-CS. Raman spectra exhibited an increase of G concentration resulted in higher crosslinking of disulfide-bonds in G-CS, which aggrandized the bridging effect of G-CS and maintained its three-dimensional network. Cell viability assay and immuno-fluorescence staining showed that G-CS effectively facilitated the growth and myogenic differentiation of porcine skeletal muscle satellite cells (PSCs). CLSM displayed that cells first occupied the angular space of hexagon and then ring-growth circle of PSCs were orderly formed on G-CS. The texture and color of CMS which loaded proliferated PSCs were fresh-meat like. These results showed that physical cross-linked G-CS scaffolds are the biocompatible and stable adaptable extracellular matrix with appropriate architectural cues and natural micro-environment for structured CM models.

Combined effects of high-intensity ultrasound treatment and hydrogen peroxide addition on the thermal stabilities of myofibrillar protein emulsions at low ionic strengths

In this study, the effects of high-intensity ultrasound (HIU) treatment combined with hydrogen peroxide (H2O2) addition on the thermal stability of myofibrillar protein (MP)–stabilized emulsions in low-salt conditions were investigated. Results showed that compared to using either HIU or H2O2 treatment alone, HIU treatment combined with H2O2 was most effective in enhancing the physical stability of emulsions. Moreover, the emulsion stabilized by MPs co-treated with HIU and H2O2 exhibited the most uniform distribution, highest absolute zeta potential, and optimal rheological properties upon heating. This combination effect during heating was caused by the inhibition of disulfide bond cross-linking of myosin heads by H2O2 and the dissociation of filamentous myosin structures using the HIU treatment. In addition, the results of oxidative stability analysis indicated that the addition of H2O2 increased the content of oxidation products; however, the overall influence on the oxidative stability of emulsions was not significant. In conclusion, the combination of HIU and H2O2 treatment is a promising approach to suppress heat-induced MP aggregation and improve the thermal stability of corresponding emulsions.

In Vivo Disulfide-Bond Crosslinking to Study β-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded β-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of β-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of β-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between β-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.

Cryo-EM structure of a 16.5-kDa small heat-shock protein from Methanocaldococcus jannaschii

The small heat-shock protein (sHSP) from the archaea Methanocaldococcus jannaschii, MjsHSP16.5, functions as a broad substrate ATP-independent holding chaperone protecting misfolded proteins from aggregation under stress conditions. This protein is the first sHSP characterized by X-ray crystallography, thereby contributing significantly to our understanding of sHSPs. However, despite numerous studies assessing its functions and structures, the precise arrangement of the N-terminal domains (NTDs) within this sHSP cage remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of MjsHSP16.5 at 2.49-Å resolution. The subunits of MjsHSP16.5 in the cryo-EM structure exhibit lesser compaction compared to their counterparts in the crystal structure. This structural feature holds particular significance in relation to the biophysical properties of MjsHSP16.5, suggesting a close resemblance to this sHSP native state. Additionally, our cryo-EM structure unveils the density of residues 24–33 within the NTD of MjsHSP16.5, a feature that typically remains invisible in the majority of its crystal structures. Notably, these residues show a propensity to adopt a β-strand conformation and engage in antiparallel interactions with strand β1, both intra- and inter-subunit modes. These structural insights are corroborated by structural predictions, disulfide bond cross-linking studies of Cys-substitution mutants, and protein disaggregation assays. A comprehensive understanding of the structural features of MjsHSP16.5 expectedly holds the potential to inspire a wide range of interdisciplinary applications, owing to the renowned versatility of this sHSP as a nanoscale protein platform.

High-intensity ultrasound combined with glycation enhances the thermal stability and in vitro digestion behaviors of myofibrillar protein aqueous solution

The low thermal stability of myofibrillar proteins (MPs) is a technological barrier to them being applied in beverage formulas. In this study, we investigated the effect of high-intensity ultrasound (HIU) pretreatment combined with glycation on the thermal stability, structural characteristics, and in vitro digestion behavior of MPs in water. The results indicated that HIU pretreatment combined with glycation significantly inhibited thermal aggregation and reduced the particle size of MPs compared to using either HIU or glycation treatments individually. The grafting of dextran (DX) shielded the sulfhydryl (–SH) and hydrophobic groups and inhibited disulfide bond cross-linking and hydrophobic association. Moreover, HIU pretreatment facilitated the shielding effect of glycation by destroying the filamentous myosin structure and exposing the internal –SH and hydrophobic groups as well as the grafting sites, maximally inhibiting thermal aggregation. In addition, the smaller protein particles and more flexible structure caused by HIU pretreatment combined with glycation increased their binding affinity toward protease. Overall, these findings can promote the technological development of modulating the MP structure–digestion for formulating novel meat protein-based products.

Physicochemical properties and digestibility of thermally induced ovalbumin–oil emulsion gels: Effect of interfacial film type and oil droplets size

To investigate the effect of oil droplets surface film type and droplets size on the physicochemical properties and digestibility of thermally induced ovalbumin–oil emulsion gels, Tween 80 and ovalbumin were selected as individual emulsifiers for the emulsification of sunflower oil at different homogenization speeds (6000, 8000, and 10000 rpm), and the formed emulsions were mixed with ovalbumin matrix solution and then heated to form heat-induced ovalbumin emulsion gels (OEG). The OEG with ovalbumin as emulsifier had higher hardness, water holding capacity (WHC), and surface hydrophobicity, and lower digestibility, spin−spin relaxation time ( T 2 ) and free sulfhydryl content than those of the OEG with Tween 80 as the emulsifier ( p < 0.05). As the oil droplets size decreased, the hardness increased, WHC increased, the amide A band shifted to lower wavenumbers, digestibility decreased, T 2 decreased, free sulfhydryl content decreased, and surface hydrophobicity increased for both OEG ( p < 0.05). These results collectively indicated that the physicochemical properties of the OEG are influenced by the property of the oil interfacial film and the size of the oil droplets. The oil droplets surrounded by the interfacial protein film improved the performance of the OEG greatly, and the proteins within its OEG formed a dense gel network structure through hydrophobic aggregation, disulfide bond crosslinking, and hydrogen bonding. The oil droplets with smaller sizes favored the enhancement in the physicochemical properties of the OEG. Changes in the size of oil droplets and the type of film wrapped around them regulated the digestibility of the OEG. • The OEG-O with the small size of oil droplets had the highest hardness. • Small oil droplets filled better in the gel network to improve the hardness of OEG. • IPF-encapsulated oil droplets as active filler to increase the hardness of OEG. • Effect of filler on hardness depends on the heat stability of its interfacial layer. • Changes in oil droplet size and interfacial layer type regulated OEG digestibility.

Formation of mucus-permeable nanoparticles from soy protein isolate by partial enzymatic hydrolysis coupled with thermal and pH-shifting treatment

Modulating the size and surface charge of nanocarriers provides an efficacious strategy to enhance bioavailability of encapsulated cargos through increased mucus penetration. In this study, mucus-permHeable soy protein nanoparticles (SPNPs) were successfully fabricated via gastrointestinal proteolysis coupled with heating and pH-shifting treatment. Results showed that treatment at 65 °C and 75 °C after proteolysis induced the assembly of α, ά, and β subunits, forming a relatively loose structure. This facilitated further assembly upon pH-shifting, forming smaller-sized and less electronegative nanoparticles, which showed enhanced mucus permeability. However, treatment at 85 °C and 95 °C promoted stronger hydrophobic interactions and induced disulfide bond cross-linking between B and β subunits, forming compact macro-aggregates with high β-sheet structure. These larger-sized aggregates were less influenced by pH-shifting treatment, demonstrating limited mucus diffusion. This study provides a potential alternative to fabricate mucus-permeable nanoparticles, and established a relationship between protein subunit assembly behavior and its mucus permeability.

Design of asymmetric-adhesion lignin-reinforced hydrogels based on disulfide bond crosslinking for strain sensing application

Soft and elastic polymer hydrogel materials are booming in the fields of wearable biomimetic skin, sensors, robotics, and bioelectrodes. Currently, many researchers are exploring new chemistries for the preparation of hydrogels to improve their performance. In the present study, we design and develop a strategy to prepare lignin reinforced hydrogels based on disulfide bond crosslinking mechanisms, and resultant hydrogels exhibit excellent stretchability, with tensile strain of up to 1085.4%, and high adhesion (with the highest T-peel strength of up to 432.2 N/m to pigskin). The underlying mechanism is based on the disulfide bonds that act as crosslinkers in the as-prepared hydrogel, and they can be easily cleaved and re-formed under mild conditions. Thanks to the presence of lignin, the as-obtained hydrogels also have excellent UV shielding effect. When assembled into a strain sensor, they can output stable and sensitive sensing signals, with gauge factor (GF) of 2.72 (strain: 0–72.8%). Furthermore, a simple and effective strategy to construct asymmetric adhesive hydrogels was adopted, which is based on directional soaking of the top portion of the hydrogel in a high-concentrated calcium chloride solution. The asymmetric hydrogel strain sensor transmits accurate and stable signals without the interference of various contaminants.

The conformational rearrangement and microscopic properties of wheat gluten following superheated steam treatment

Superheated steam treatment (SST) can improve cereal-based foodstuff quality by altering the gluten properties. In the current study we investigate the effect of SST on the polymerization behavior, conformational rearrangement and microscopic characteristics of gluten, which contribute to determining the molecular mechanisms of SST that impact the gluten properties. Compared with native wheat gluten, SST was observed to induce an increase in sodium dodecyl sulfate (SDS) insoluble gluten. This indicates that SST promoted the polymerization behavior of gluten via the cross-linking of disulfide (SS) bonds. In addition, hydrogen bonds were observed to develop from the micro-environment variation of tryptophan (Trp) residue in the gluten. This consequently had a synergistic effect on the secondary structure, increasing the proportion of intermolecular β-sheets and α-helices. Furthermore, SST enhanced the molecular surface roughness and weakened the gluten network structure. This study could contribute to insight into the improvement of flour quality following SST.

Preventing the browning of fresh wet noodle sheets by aqueous ozone mixing: Browning and physicochemical properties

The formation and prevention mechanism of the dark spots in fresh wet noodle sheets (FWNS) has been discussed in our previous study, while the factors which cause FWNS browning are very complicated. In this work, the browning of FWNS was prevented through aqueous ozone mixing. Meanwhile, low field nuclear magnetic resonance (LF-NMR), size-exclusion high-performance liquid chromatography (SE-HPLC), dynamic rheometer, and scanning electron microscope (SEM) were used to measure the physicochemical properties and microstructure of the FWNS. The relationship between browning and the physicochemical properties was discussed. The results showed that aqueous ozone mixing improved the color of FWNS, in which physical factors and non-covalent bonds played a key role. The results of the LF-NMR tests showed that aqueous ozone mixing reduced the moisture degree of freedom. The results of the SE-HPLC tests showed that the moderate oxidizing capability of aqueous ozone promoted the cross-linking of disulfide bonds in FWNS. The results of the dynamic rheology tests and SEM observation showed that aqueous ozone mixing strengthened the gluten network structure of FWNS. Thus, aqueous ozone mixing made FWNS more compact and uniform, and indirectly reduced the browning rate.

Combination of high-intensity ultrasound and hydrogen peroxide treatment suppresses thermal aggregation behaviour of myofibrillar protein in water

This study was aimed at evaluating the potential of high-intensity ultrasound (HIU, 450 W for 10 min) combined with hydrogen peroxide (H2O2) having various concentrations (0, 50, 100, 200, 400, 800 μmol/g protein) to inhibit the thermal aggregation behavior of myofibrillar proteins (MPs) in water. The results indicated that the addition of H2O2 interfered with the intermolecular sulfhydryl-disulfide interchange and inhibited the disulfide bond cross-linking. The H2O2-mediated conversion of cysteine to thiol derivatives appeared to be the primary mechanism of this effect. The HIU combined with H2O2, especially at the H2O2 concentration of 200 μmol/g, corresponded to a more significant inhibitory effect than that of only H2O2, which attributed to the dissociation of the filamentous myosin structure that led to an enhanced accessibility of the buried sulfhydryl groups. In conclusion, these findings provide direct evidence for the role of HIU combined with H2O2 in improving the thermal stability of MPs.

Mechanically Promoted Synthesis of Polymer Organogels via Disulfide Bond Cross-Linking.

Mechanically adaptive polymers could significantly improve the life-cycle of current materials. Piezo-polymerization is a novel approach that harnesses vibrational mechanical energy through piezoelectric nanoparticles to generate chemical promoters for linear polymerization and cross-linking reactions. However, the available piezo-polymerization systems rely on reactions forming irreversible covalent bonds. Dynamic covalent linkages could impart further adaptability to these polymeric systems. Here we show the first example of the piezoelectrochemical synthesis of disulfide bonds to form organogels from polymers with thiol side groups. We demonstrate that the reaction proceeds via piezo-oxidation of the thiol to disulfide in the presence of ZnO nanoparticles and iodide anions under mechanical agitation. We use mechanical energy in the form of ultrasound (40 kHz) and low frequency vibrations (2 kHz) to synthesize a variety of organogels from common synthetic polymers. Additionally, we show that the polymers in these gels can be chemically recycled with a reducing agent. Finally, we study the thermal and mechanical properties of the composites obtained after drying the gels. We believe this new system adds to the piezo-polymerization repertoire and serves as the basis to fabricate mechanically adaptive polymeric materials via dynamic covalent bonds.

Size- and Surface- Dual Engineered Small Polyplexes for Efficiently Targeting Delivery of siRNA.

Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes’ size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.