Rubber-like DNA hydrogel enabled by fast-shrinking-induced entanglement

Domain: neuroscience
Published: 2026-01-28
Editorial outcome: minor_revision

Abstract
Biomass DNA holds immense potential as a sustainable material, but the scalable production of robust DNA materials with sufficient mechanical strength remains an essential challenge. Herein, we report a Fast-Shrinking-Induced Entanglement (FaSIE) process to achieve rubber-like hydrogels solely composed of DNA (stiffness > 800 kPa, toughness > 5 MJ/m3, and stretchability > 1000%). Fast shrinking kinetic restricts the chain relaxation, while the ultra-long chain feature of biomass DNA further suppress chain reptation. This synergistic effect enables substantial enhancement of entanglement density, exceeding the threshold required for high mechanical strength. We highlight the broad applicability of this strategy for high-resolution 3D printing of mechanically robust DNA hydrogels and the fabrication of DNA based soft magnetic robots. This approach paves the way for the large-scale production of resilient hydrogel materials derived from biomass DNA for versatile applications.

Introduction
The unprecedented accumulation of synthetic materials poses a serious threat to the biosphere, which has led to a critical need in developing sustainable materials from biomass based resources 1–3. Over the years, the researches on sustainable materials are mainly focused on polysaccharides and proteins 4–7. On the other hand, DNA is an inexhaustible biopolymer, which can be extracted from any organisms including plants, animals, and microorganisms. There are ~50 billion metric tons of biomass DNA on the Earth, and utilizing ~0.7% of it would be sufficient to replace the total annual production of synthetic polymers 8. However, developing biomass DNA as sustainable materials is still in its infancy and significantly underestimated. A major obstacle is the lack of bulk production methods for preparing DNA materials with sufficient mechanical strength 9. For instance, conventional approaches via strand hybridization and chemical crosslinking result in soft jelly-like DNA hydrogels with elastic moduli and tensile strengths below 50 kPa (Fig. 1A) 10,11. Therefore, although DNA hydrogels have demonstrated attractive biocompatibility and bioactivity for applications such as cell culture matrices, injectable fillings, and localized drug delivery 12–14, their broader utilization as robust biomaterials are significantly constrained. Enhancing the mechanical properties of DNA hydrogels could significantly expand the application scope of biomass DNA.

Recent studies have introduced a fascinating approach to enhance the strength and resilience of hydrogels by inducing dense polymer entanglements 15–18. Unlike covalent crosslinking, these entanglements function as slip crosslinks within the hydrogel 19, increasing stiffness while maintaining toughness. When the hydrogel is stretched, tension is transmitted along the chain and to multiple other chains via entanglements, storing elastic energy within deformed conformations of the polymer strands before chain breakage. This mechanism could reinforce hydrogels and enable remarkable elasticity. Highly entangled synthetic polymers were produced by in situ polymerization at exceptionally high monomer concentrations, leading to extraordinary stiffness and toughness 15 (Fig. 1B). Meanwhile, the denaturation and refolding of proteins at high concentration could fabricate densely entangled protein hydrogels 16. However, these strategies cannot be adapted to introduce dense entanglements for biomass DNA. Therefore, although the exceptional chain length of biomass DNA (normally in the range of 107-1012 Da) 20 is favourable for entanglement engineering, and studies have revealed that entanglements generally exist in DNA hydrogel 21,22, the fabrication of entanglement dominant DNA hydrogels remains exceptionally challenging. Methods to engage dense entanglements into biomass DNA might promote revolutionary strategies for producing high-strength DNA hydrogels.

In this study, we demonstrate a fast-shrinking-induced entanglement (FaSIE) strategy that facilitates the fabrication of densely entangled, rubber-like hydrogels from biomass DNA. The underlying mechanism stems from distinct polymer chain dynamics under varying dehydration rates: slow solvent evaporation allows sufficient chain relaxation and entropy-driven disentanglement, producing soft polymeric materials, while rapid dehydration kinetically traps non-equilibrium chain conformations through constrained relaxation, leading to increased entanglement density and mechanical rigidity. Building upon this phenomenon, we designed a dehydrating phase separation process for DNA solutions surpassing the entanglement threshold concentration, which triggers rapid volumetric shrinkage within seconds and generates ultrahigh-density topological constraints. The exceptionally long chains of biomass DNA further stabilize these non-equilibrium entanglements through pronounced reptation hindrance (Fig. 1C). This synergistic integration of kinetic control and macromolecular architecture enables instantaneous formation of entanglement-dominated hydrogels, demonstrating tens-to hundreds-fold increase in stiffness (E > 800 kPa) and toughness (W > 5 MJ/m3) relative to chemically crosslinked counterparts at equivalent water content (60 wt%). Notably, slow evaporation of DNA solutions to identical water content produces only viscous liquids, underscoring the pivotal role of dehydration kinetics. Comprehensive analysis identifies three fundamental requirements for hydrogel formation: high initial DNA concentration, rapid shrinking kinetic, and ultra-long chains. While challenging to satisfy with most synthetic polymers, these criteria are readily applicable to other natural biopolymers with extended chain lengths, such as giant polysaccharides. The FaSIE mechanism further empowers advanced material processing, including high-resolution 3D printing of intricate hydrogel architectures and seamless integration with functional components (e.g., magnetic nanoparticles). We validate this multifunctional capacity through prototype magnetoactive soft robots. Overall, the FaSIE strategy facilitated robust fabrication of DNA hydrogels with rubber-like mechanical strength that outperform all existing DNA-only hydrogels, thereby broadening the application horizons of DNA based materials.

Results
The entangled DNA hydrogel (E-Gel) prepared by fast-shrinking-induced entanglement (FaSIE)
To demonstrate the FaSIE process, the commercially available salmon sperm DNA was utilized as the model system. It consists of double-stranded DNA with an average molecular weight of 13200 kDa (N ~ 20000 base pairs (bp)) 23. The contour length could reach about 6.8 μm, which is comparable to 600 kDa fully extended straight-chain polyethylene glycol that is rare achieved synthetically. The 5% (w/v) salmon sperm DNA solution was prepared and a range of solvents was screened to induce dehydrating phase separation to this solution (Supplementary Fig. 1 and Fig. 3). Among these trials, the combination of glacial acetic acid (AA) and Triton X-100 (TX) was identified as the most effective (Supplementary Fig. 2). When the 1:1 mixture of AA and TX (v/v) was added, the volume of the DNA solution could shrink to ~50% within seconds, leading to the formation of a hydrogel instantly (Fig. 1D, Supplementary Video. 1). Further equilibration in the AA/TX solution would lead to additional shrinking of the hydrogel for hours. The resultant hydrogel, named the Entangled DNA Hydrogel (E-Gel), retained ~60% water content after equilibration. The infrared (IR) spectrum showed that the hydrogel primarily consisted of DNA without detectable residues of AA and TX after washing (Supplementary Fig. 4), indicating that AA and TX do not function as crosslinkers. Moreover, GoldViewTM staining confirmed the predominance of double-stranded DNA, and no obvious dissociation of double strands was observed even after stretching (Supplementary Figs. 5, 6). These data indicate the DNA structures were well maintained in the E-Gel, and no chemical changes likely occurred.

The E-Gel demonstrated an average total elongation (TE) at fracture of 1107.0 ± 104.2%, Young’s modulus (E) of 60.48 ± 5.65 kPa and an ultimate tensile strength (UTS) exceeding 0.4 MPa (Fig. 2A, C, and Supplementary Video. 3). When subjected to cyclic stretching at various amplitudes and a constant rate, the hysteresis of E-Gel was minimal (Supplementary Fig. 7). Such excellent elasticity is a feature of entanglement dominant hydrogel that is consistent with previous reports 24,25. In contrast, a conventional chemically crosslinked hydrogel (C-Gel) was also prepared using the same salmon sperm DNA at the same concentration but crosslinked with PEG-DE (Poly(ethylene glycol) diglycidyl ether) as per prior methods (detailed preparation in SI) 26. It displayed significantly weaker mechanical properties (TE < 250%, UTS < 20 kPa, and E < 3 kPa, Fig. 2A, Supplementary Video. 2), even with the same water content (Supplementary Fig. 8). The compressive modulus (Y) of the E-Gel could reach 18.65 ± 2.46 kPa, which is also significantly higher than that of the C-Gel (Y = 1.52 ± 0.76 kPa) (Fig. 2B). Upon compression to 90%, the E-Gel could swiftly regain its original shape within seconds and sustain a stress as 1.304 ± 0.188 MPa without fracture, while the C-Gel collapsed entirely at 70% with a much lower stress of 20.19 ± 7,34 kPa (Fig. 2D). Scanning electron microscopy (SEM) of the E-Gel revealed a uniform structure without obvious pores (Fig. 2E), and E-Gel exhibited a homogeneous morphology throughout its volume (Supplementary Fig. 9). In contrast, immersing the E-Gel in water would result in enormously swelling, reaching up to 350 times of its original weight within hours, and a clearly porous structure could be observed in the SEM after swelling (Supplementary Fig. 10, and Fig. 2E). If left in ample water for over 30 days, the E-Gel could eventually dissolve which is a phenomenon consistent with dynamic interactions in the gel (Supplementary Fig. 10). The E-Gel could fully degrade upon treatment with DNase (Fig. 2F, and Supplementary Fig. 11A), consistent with the features of the other known DNA hydrogels. However, it exhibited resistance to heat up to 100 °C and is stable in a wider range of pH from 2 to 10, which is distinctive compared to strand-hybridized DNA hydrogels (Supplementary Fig. 11B–E).

The elastic modulus and ultimate rupture stretch of E-Gel fit well to the physical model for entanglement dominant hydrogel. In a highly entangled network, the Young’s modulus is intricately linked to the entanglement density through the equation E = 3ρRT/Me (Eq. 1) 27, where ρ represents polymer density, R is the gas constant, T is the absolute temperature, and Me corresponds to the molecular weight of polymer strands between entanglements 28. Consequently, the calculated Me is 61817 ± 5889 g/mol, indicating an average of 214 entanglements per DNA chain. Based on these data, we estimate the rupture strain to be ~1000% using a freely-jointed model (see more details in SI) 24,29, which aligns well with the experimental data (Fig. 2A).

Furthermore, when the DNA solution was initially crosslinked with PEG-DE and subsequently treated with AA/TX for several hours—until the pre-crosslinked DNA hydrogel reached a water content comparable to that of E-Gel—a weak hydrogel was yielded, exhibiting properties similar to the C-Gel (Supplementary Fig. 12), suggesting that pre-chemical crosslinking hindered DNA chain mobility and impeded chain entanglement.

Shrinking kinetic, initial concentration and chain length are the key factors for FaSIE based hydrogel
The FaSIE phenomenon is in general existing for all polymers. However, for most synthetic polymers, such entanglement is not stable to support sufficient mechanical strength. Entropy drive disentanglement could easily occur to reduce the entanglement density 30. In case of long chain nature biopolymers, like biomass DNA, significant chain interpenetrations are existing in solution with a practically high concentration. The AA/TX induce phase separation and fast dehydration of the DNA solution, thus immediately compress the volume of the interpenetrated DNA network. The extraordinary chain length and pre-existing entanglements prohibit the disentanglement tendency 31, and the fast shrinking kinetic further reinforced the entanglements and constrained the DNA chains into the highly entangled network 32. Based on this hypothesis, we further verified the influence of the dehydration time, the initial DNA concentration and the chain length toward the physical properties of the resulting hydrogels.

To compare with the solvent induced fast shrinking, 5% salmon sperm DNA was exposed to gradual dehydration with mild heating until reaching 60% water content as E-Gel. However, no gelation occurred and a viscous paste was obtained. By assessing the plateau elastic modulus (E_plateau) (Supplementary Fig. 13) in rheological test, the Me of this solution was determined to be 861764 g/mol according to E_plateau = ρRT/Me (Eq. 2), indicating an average of only 15 entanglement points on each DNA molecule. This supported that dense entanglements in the hydrogel arise from a kinetically driven process. Slow water evaporation allowed sufficient time for DNA chain disentanglement, resulting in insufficient entanglement density for gelation 33. Consistently, if the E-Gel is immersed in salted water for 2 days to prevent swelling while still permitting adequate water exchange, chain relaxation will also occur, thus leading to a notable weakening of the hydrogel (Supplementary Fig. 14). However, storing the hydrogel in a 4 °C fridge at 40% humidity for over one week does not visibly weaken it (Supplementary Fig. 15), indicating that E-Gel can maintain stability in air.

The initial DNA concentration (Ci) and the chain length of the DNA also significantly influent the FaSIE process. At low Ci, the AA/TX is miscible with the DNA solution, resulting in a clear liquid. As the Ci increases, DNA aggregation and precipitation become noticeable after AA/TX treatment (the concentration at which precipitation occurs is defined as Cp). Gelation requires an even higher concentration, termed the gelation concentration (Cg) (Supplementary Fig. 16, 17). To investigate these phenomena, we estimated the critical entanglement concentration (Ce) of DNA with varying molecular weights by monitoring the onset of the plateau elastic modulus and viscosity (see Supplementary Figs. 18, 19) 34,35. We found a strong correlation between Ce and Cp, emphasizing the importance of pre-existing chain interpenetration in the DNA solution, which is further reinforced by AA/TX-induced phase separation (Supplementary Table. 1). When Ci significantly exceeds Ce, a complete entangled network emerges throughout the DNA solution, leading to gelation. For high molecular weight DNA (e.g., 20,000 bp), Ce is relatively low (0.5%-1%), permitting the formation of stable hydrogels with robust mechanical properties within a practical range of Ci (5% to 15%). Conversely, as molecular weight decreases, Ce increases. When the DNA length is below 1000 bp, it becomes impractical to obtain a hydrogel within the above concentration ranges (Fig. 2G).

The mechanical properties of the hydrogels correlate closely with DNA molecular weight and Ci: higher molecular weights yield increased G’ and G” at a fixed Ci (Supplementary Fig. 20), while an increase in Ci significantly enhances stiffness, strength, and toughness (Fig. 2H, and Supplementary Fig. 21). For instance, in uniaxial tensile tests, increasing Ci from 5% to 12.5% boosted stiffness from 60 kPa to 800 kPa (Supplementary Fig. 22B), and tensile strength increased from 0.4 MPa to over 1.2 MPa (Supplementary Fig. 22C). Stretchability decreased with higher Ci (Supplementary Fig. 22D), consistent with increased entanglement density. By fitting these data with the Mooney-Rivlin model and non-affine tube model (Supplementary Fig. 23–24) 28, we found almost all of the modulus were contributed by the entanglements rather than by crosslinking. These results again supported that the E-Gel is an entanglement-dominated network. Compression tests also reflected this trend (Fig. 2I): at Ci = 10%, the compressive modulus (Y) reached 138.50 ± 28.89 kPa, and compressive strength rose to 6.83 ± 0.24 MPa (Supplementary Fig. 25), comparable to typical stiff and tough double network hydrogels made from synthetic polymers (Supplementary Table. 4). We observed a decline of compressive modulus and strength at an initial concentration of 12.5%, likely due to this concentration having already exceeded the saturation limit (Supplementary Fig. 26).

Stabilization by electrostatic interaction and chemical crosslinking
E-Gel is prepared in the non-swollen state and exhibits limited mechanical stability in aqueous solutions due to disentanglement, making it suitable for use in non-aqueous environments. To enhance the applicability of E-Gel in aqueous environments, Mg2+ and PEG-DE could be added post-gelation to introduce additional electrostatic interactions and chemical crosslinks (exemplified by Ci = 5% E-Gel with relatively low DNA content and representative mechanical properties) (Fig. 3A, and Supplementary Fig. 27). Both the Mg2+-stabilized hydrogel (E-Mg-Gel) and the Mg2+/PEG-DE-stabilized hydrogel (E-Mg-PEG-Gel) could prevent the incessant swelling and obtain an equilibrate hydrogel with a water content of 70–80%. An increase in crosslinking density was also observed after Mg²⁺ and PEG-DE treatment (Supplementary Fig. 28). The UTS of the fully swelled hydrogels exceeded 1 MPa, with TE of ~1200% and a stiffness of 150 kPa (Fig. 3C). These hydrogels displayed more hysteresis comparing to E-Gel during cyclic stretching and compression, indicating increased energy dissipation due to sacrificial bonds (Supplementary Fig. 29–31). Nevertheless, the hydrogel could rapid recover from stretching and compression (Fig. 3D, Supplementary Fig. 31). At large strains (100%), the original energy dissipation capability can be recovered right after unloading, and the remaining can be recovered following a single-exponential kinetics, with k of 0.051 s−1 (E) Stress-strain curves of different samples under compression tests. F–H ashby diagrams of stiffness versus ultimate tensile strength (F), ultimate tensile strength versus toughness (G) and compressive modulus versus compressive strength (H) of E-Gel, E-Mg-PEG-Gel, regular biopolymer hydrogels and reported entangled hydrogels. The data used are summarized in Supplementary Table. 3-4.

High resolution 3D printing of biomass DNA hydrogel
The concentrated DNA solution exhibited high viscosity and shear-thinning behaviour, which is ideal for extrusion-based direct ink writing (DIW) 3D printing (Supplementary Fig. 19). The FaSIE process could be used as a post-printing treatment to obtain DNA hydrogel with micrometer scale resolution. The fast gelation process maintained the original shape of the DNA ink and the uniformed shrinkage significantly increased the structure resolution. For instance, the as-designed DNA frame structure was printed in four layers, with an original size of 10 mm × 10 mm and an original filament diameter of 647.0 ± 71.3 μm (Fig. 4D). After treatment with AA/TX, the gel quickly formed along with drastic volume shrinkage, resulting in a final DNA frame structure with a filament diameter of 66.1 ± 10.8 μm (Fig. 4A–D). Such resolution is among the best resolutions for extrusion-based hydrogel 3D printing 53–62, and it has never been achieved by DNA 3D printing (Supplementary Fig. 33). The different layers were also clearly distinguishable on SEM, supporting the excellent structure fidelity (Fig. 4C). The zoom-in images revealed the microstrutctures of the filaments were as same as E-Gel (Supplementary Fig. 34). By this way, the DNA hydrogels with various structures were printed, such as a cube, star, snowflake, and HUST (Fig. 4E). The FaSIE based 3D printing showed great shape fidelity, while the chemical crosslinking method (see details in supplementary discussion) could barely maintain its shape and quickly collapsed due to the time-consuming gelation process (Fig. 4E). The statistical shrinkage factor was found to be 4.39 ± 0.98 in the lateral dimension and 4.01 ± 0.69 in the axial dimension (Fig. 4F), indicating a favourable uniformed shrinkage.

Functionalization of biomass DNA hydrogel and extension of FaSIE to other biopolymers
The FaSIE process offers versatility for integrating with various functional components. The high viscosity of the initial DNA solution, combined with the rapid gelation process, also facilitates the fabrication of functional hydrogels with varying components in specific areas. For instance, FaSIE treatment of a DNA solution mixed with Fe3O4 nanoparticles could result in a magnetic DNA hydrogel. Thus, a magnetic DNA soft robot could be prepared by fusion the magnetic forks at both ends with a non-magnetic handle in the middle, which is capable of directional movement and can lift objects in response to a magnetic field (Fig. 4G, and Supplementary Video. 4). Additionally, the hydrogel retains the feature of sequence-specific hybridization inherent in DNA-based materials. When a fluorescein-labeled ssDNA probe with a complementary sequence to salmon sperm DNA is incubated with the E-Mg-PEG-Gel, stable integration of green fluorescence is achieved even after thorough washing with PBS. In contrast, free fluorescein dye lacking a DNA probe or with an uncomplimentary DNA probe would be readily washed away by PBS (Supplementary Fig. 35). Moreover, both E-Mg-Gel and E-Mg-PEG-Gel exhibited excellent biocompatibility with mammalian cells (Supplementary Fig. 36). Together, these results highlight the broad application potential of DNA hydrogels.

In addition to Salmon Sperm DNA, we have applied this strategy to another type of biomass DNA, namely Calf Thymus DNA, which exhibited similar mechanical properties to Salmon Sperm DNA (Supplementary Fig. 37, Supplementary Table. 5), demonstrating the generality of this strategy for various long-strand biomass DNAs. With the success of the FaSIE process in DNA hydrogels, the potential for reinforce other biopolymer hydrogels was also explored. Similar trend was observed with sodium alginate, sodium hyaluronate and carboxymethyl cellulose (Supplementary Fig. 38, Supplementary Table. 6). The FaSIE process is able to achieve hydrogels with stiffness and toughness 10 to 270 times enhanced comparing to conventional chemical crosslinking. Notably, these results were obtained with the same protocol used for DNA hydrogels. Additional optimization of the dehydration solution is expected to further enhance the FaSIE performance for individual biopolymers. Nevertheless, the preliminary data have already demonstrated the promising potential of FaSIE to enhance the mechanical properties of various biopolymers.

Methods
Materials
Salmon sperm DNA is purchased from Solarbio. Herring sperm DNA and DNA (Calf thymus) are purchased from Sigma-Aldrich. Acetic acid is purchased from Sinopharm. Triton-X 100 is purchased from Sangon. Magnesium chloride is purchased from Sangon. PEGDE (average MW 500) is purchased from TCI.

Synthesis of highly entangled DNA hydrogel (E-Gel) induced by fast shrinking
5% (w/v) salmon sperm DNA was dissolved in 1× PBS (Sangon) buffer, for instance, 10 mg DNA with 200 μL PBS, mixed well until completely dissolved. The 5% salmon sperm DNA is highly viscous and exhibits semi-solid-like properties, causing it to flow slowly. It was then carefully poured into pre-designed molds, and the surface of the solution was flattened. 1 volume fold 1:1 (v/v) of AA/TX mixture was added into the mold right away to cover the DNA solution. The hydrogel would form quickly in seconds, and then the mold was kept in the oven at 37 °C for 2 h for further shrinking and equilibration with AA/TX.

Synthesis of E-Mg-Gel and E-Mg-PEG-Gel
To obtain the E-Mg-Gel, E-Gel was immersed in a high concentration of MgCl2 (2.5 M) for 5 minutes, repeated three times. The E-Mg-Gel remained stable in PBS buffer and swelled to a final water content of 70-80%. To further strengthen and stiffen the hydrogel, E-Mg-Gel was chemically crosslinked with PEGDE. E-Mg-Gel, without swelling, was taken out of the tube and placed onto a plate for crosslinking; the hydrogel should not be in a solution environment. Then, 5% (w/v) PEGDE (original volume of the DNA solution) was added to the surface of the hydrogel, allowing the penetration of the crosslinker for 15-20 minutes. After that, 0.44% (w/v) TEMED was added to catalyze the reaction for 2 h, resulting in the E-Mg-PEG-Gel, then the gel was immersed in the 1× PBS solution for at least 12 h to swell, which could swell to a final water content of 70-80%.

Synthesis of C-Gel and C-Mg-Gel
The same concentration, 5% DNA, was used to fabricate chemically crosslinked DNA hydrogel. As described, 5% (w/v) PEGDE was added to the DNA solution, along with 0.44% (w/v) TEMED, and the mixture was stirred well to obtain a uniform DNA solution. Then, the DNA solution was carefully poured into the mold, and the mold was left in the oven at 42 °C for 3 hours and at room temperature overnight for the complete reaction. To obtain the C-Mg-Gel, the same process as with E-Gel was followed: the C-Gel was placed in a high concentration MgCl2 solution for 5 minutes, repeated three times.

Functionalization of biomass DNA hydrogel
Various functional components, such as Fe3O4, can be easily introduced into the hydrogel. To fabricate a locally magnetic-responsive DNA hydrogel, a 5% DNA solution was prepared and then poured into the mold. For the magnetic-responsive part, 2-5% (w/w) Fe3O4 was added to the DNA solution and stirred well. The mixed DNA solution could then be added at any desired location in the mold. The DNA solution and magnetic-responsive DNA solution were left to fusion for 5 minutes; the added component did not diffuse due to the high viscosity of the DNA solution. Then, AA/TX was added to fabricate the hydrogel.

Synthesis of crosslinked biopolymer hydrogels
6% sodium alginate was dissolved in the pure water and then immersed into 20 mM CaCl2 solution for 30 min to obtained crosslinked alginate hydrogel. 5% sodium hyaluronate was dissolved in the 0.25 M NaOH solution and then 5% (w/v) PEGDE was added to the solution, the mixture was placed at 50 °C for 4 h to complete the gelation. 5% carboxymethyl cellulose was dissolved in the 0.4 M NaOH solution and then 5% (w/v) PEGDE was added to the solution, the mixture was placed at 50 °C for 4 h to complete the gelation, the hydrogel was collected and stored at 4 °C for use.

Synthesis of highly entangled biopolymer hydrogels
Highly entangled hydrogels were fabricated using solutions containing 6% sodium alginate, 5% sodium hyaluronate, and 5% carboxymethyl cellulose. An equal volume of the AA/TX mixture (1:1, v/v) was added to the mold containing the biopolymer solution. Rapid gelation occurred within seconds, after which the mold was incubated at 37 °C for 2 h to allow for further shrinking and equilibration.

Tensile tests
Various hydrogels were made into rectangular shaped species, with E-Gel, E-Mg-Gel and E-Mg-PEG-Gel of 15 mm × 5 mm × 1 mm. uniaxial tensile tests were performed on a commercial tensile machine (CTM6050) at room temperature, both ends of the samples were glued with a layer of sandpaper to reduce the slippage. The tensile tests were performed at a loading rate of 30 mm min-1. The nominal stress and the elongation in the tensile direction were measured and calculated by machine. The Young’s modulus was calculated from the initial slope of the stress-strain curve. Toughness was determined by integrating stress–strain curves where specimens were loaded directly to failure.

Cyclic Tensile tests
Cyclic tensile tests were also performed using the same experimental setup. The samples were firstly stretched to a certain strain and then immediately unload, and they were stretched to an increased strain and unloaded again with the constant loading and unloading rates of 50 mm min-1. The mechanical hysteresis was calculated by integrating loop area between loading and unloading stress–strain curves. In deformation recovery experiments, a pulling rate of 50 mm min−1 was used. The same hydrogel was loaded and unloaded with various time intervals. The nominal stress and strain were recorded.

Determination of the fracture energy Gc
Fracture tests were performed using the classical single edge notch test. A notch of 1 mm length in the middle of the hydrogel was made which the total width of the hydrogel was about 5 mm. the hydrogel was clamped and stretched with recording the nominal stress and strain with a constant rates of 30 mm min-1. The fracture energy Gc was calculated using the methodology as described previously reported 36.

Compression and cyclic tests
Uniaxial compression tests were performed on cylinder-shaped hydrogels with a diameter of 6 mm and a height 6 mm using the same mechanical machine. The compression tests were performed at a constant rate of 5 mm min-1. And the stress-strain curve was measured and recorded. The compressive modulus was measured at a strain of 10–20%. The ultimate stress was determined at 90% strain. Energy dissipation was calculated by integrating the loop area between compressing and relaxing stress–strain curves. In hysteresis recovery experiments, a compression rate of 50 mm min−1 was used.

Water contents of hydrogels
The water contents of hydrogels were calculated from the weights of the gel before and after drying, the water content, denoted as Ww, using the following equation:
Ww = 1 - Wdry / Wwet (2)

Swelling ratio
The initial weight of E-Gel was recorded as W0, and the E-Gel was immersed in different buffer solutions for different time, at selected time intervals, the hydrogels were taken out and wiped with filter paper to remove excess water from the gel surface and then weighted Ws, the swelling ratio was calculated using the following equation:
Swelling ratio = Ws / W0 (3)

DNA hydrogel staining
The hydrogel was stained by GelRed (Mei5 Bio) to check whether the hydrogel was formed by DNA. To study the DNA conformation in the hydrogel, the DNA hydrogel was stained by GoldView (Biosharp), a dye could emit red color fluorescence when bind with single strand DNA and green for double strand DNA 63.

DNA hydrogel digestion by DNase I
The hydrogel was put into the DNase I solution (0.2 U/μL, Sangon) supplied with Mg2+ and Ca2+ for 24 h, the hydrogel was pictured at different time intervals for evaluating the digestion condition of DNA hydrogel.

Attenuated total reflection-Fourier transform infrared spectrometer (ATR-FTIR)
ATR-FTIR spectra were measured using Thermo Scientific Nicolet iS5 from 600 cm−1 to 4000 cm−1. For AA, Triton-X 100, and AA/TX, the solution was applied to the surface of the instrument for detection. The pure salmon DNA was a fiber-like solid; the probe of the spectrometer was pressed onto the surface of the DNA fiber, and the spectrum was measured. The E-Gel was immersed in PBS solution and washed several times to remove excess AA/TX residues. It was then freeze-dried to obtain a lyophilized hydrogel for ATR-FTIR detection.

Gel electrophoresis characterization
The different molecular weight of DNA was measured by 2% agarose gel. the gel was run in iced 1× TBE buffer at 90 V for 2 h, stained by GelRed, and pictured on the Bio-Rad Gel Doc XR.

Scanning electron microscopy (SEM) imaging
To observe the micro-structure of the E-Gel before and after swelling, the hydrogels were shock-frozen in liquid nitrogen and then quickly transferred into the freeze drier for at least 24 h to obtain the lyophilized hydrogel. and the hydrogel was coated by sputtering gold onto the surface before scanning. The measurement was performed with HITACHI SU8010. 3D printed DNA structure was directly scanned.

Confocal laser scanning microscopy (CLSM)
CLSM images were obtained to visualize the homogeneity of the hydrogels. 10 μL of 10× GoldView was added into 5% DNA solution and then used to form the hydrogel. The CLSM images were obtained with a Olympus FV3000 using an air lens. The three-dimensional images were rendered at 5 μm intervals with a range 500 μm.

Small angle X-ray scattering (SAXS) measurements
The SAXS measurement was performed at BL19U2 beamline of Shanghai Synchrotron Radiation Facility (SSRF, China). The X-ray energy used was 12 keV, and the distance between sample and detector was 5353 mm. A Mar 165 CCD detector (2048 × 2048 pixels with pixel size of 172 μm) was used to collect two-dimensional (2D) scattering patterns with a data acquisition time of 10 s per frame.

Rheology measurements
The elastic modulus and loss modulus of DNA solution, DNA hydrogel was measured by using a rheometer (Anton Paar MCR102) equipped with 8 mm diameter steel plate requiring as low as 50 μL of samples. To measure the entanglement concentration of different chain length DNA, the frequency dependence experiments were performed. The frequency sweeping was carried out in the 0.01-10 rad s-1 range at a constant shear strain of 1%, DNA was dissolved in pure water at different concentration. To compare the elastic modulus of different DNA hydrogels, the frequency sweeping was kept in the range of 0.01-10 Hz at a constant shear strain of 1%.

Viscosity measurements
Viscosity could also help to identify the entanglement concentration. The dynamic viscosity behavior of salmon sperm DNA was studied through flow measurement by rheometer equipped with a 20 mm diameter steel cone and an angle of 2°, with the concentration from the dilute regime through semi-dilute regime to entanglement regime. Simple shear steady state measurements were performed in a shear rate range from 10-3 to 1000 s-1, using five points per decade.

High-resolution 3D printing DNA hydrogel
The printing DNA ink was prepared by dissolving 4-5% (w/v) salmon sperm DNA in PBS solution. The DNA ink was mounted on the self-made 3D printer and extruded by pressure. To print the high-resolution DNA frame structure, a 250 μm diameter nozzle was used with a print speed of 20 mm/s, yielding a 600 μm diameter DNA line. When the DNA frame structure was finished, AA/TX (combination with 1 M MgCl2) was added to trigger the gelation of the DNA solution. After treatment for a few minutes, the DNA frame was transferred into AA/TX solution and immersed for 24 hours to obtain a high-resolution 3D-printed DNA structure, the high-resolution frame structure was imaged by Camera (Sony), Super-Depth-of-Field Microscopy (VHX-1000E) and SEM (HITACHI). Other structures were also printed using a 500 μm diameter nozzle and subjected to the same treatment as the DNA frame. For comparison with chemical crosslinking post-treatment 3D printing, 4-5% DNA was dissolved in the PBS solution with 5% (w/v) PEGDE and 0.44% (w/v) TEMED and then mounted on the 3D printer, the printing process was the same with E-Gel, after the printing, the chemical crosslinking ink was put aside at 40 °C 2 h for crosslinking to obtain the printed hydrogel. Each printing model was built with 3D modeling software (Blender) and then sent to slicer software (Simplify3D) to obtain the print path (G-code).

Cytotoxicity of biomass DNA hydrogel
Cell Counting Kit-8 (CCK-8) (Sangon, China) was used to assess cell viability following the manufacturer’s instructions. Briefly, 1 × 10^4 L929 cells were seeded in each well of a 96-well plate and incubated under standard conditions (37 °C, 5% CO2) for 24 h. In one group, 5% E-Mg-Gel or E-Mg-PEG-Gel of different masses was added to the cells, and the mixtures were incubated under 5% CO2 at 37 °C for 4 h. The hydrogels were then carefully removed from the wells using sterile tweezers. Subsequently, 100 μL of DMEM mixed with 10 μL of CCK-8 solution was added to each well and incubated at 37 °C for 4 h. The absorbance of the medium in each well was measured at 450 nm using a microplate reader. In another group, 400 mg of 5% E-Mg-Gel or E-Mg-PEG-Gel was incubated with DMEM for 24 h. The resulting extract solutions were then added to the cells at volumes adjusted to maintain the same DNA-to-medium mass ratio as in group one, and the mixtures were incubated under 5% CO2 at 37 °C for 24 h.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents an innovative strategy for fabricating DNA hydrogels. Currently, most known DNA hydrogels are\nprimarily formed through strand hybridization and chemical crosslinking, exhibiting limited mechanical properties. While\nchain entanglement is recognized as a potential enhancer of mechanical properties in DNA hydrogels, the entanglement\ndensity has been insufficient to form robust, tough hydrogels. The authors developed a \"fast-shrinking-induced\nentanglement\" strategy that enables the production of DNA hydrogels with remarkably high mechanical properties. This\nprocess was never reported and is conceptually interesting and innovative. They identified an optimal dehydration phase\nseparation solvent to induce rapid shrinking of pre-entangled DNA solutions. The study\ndemonstrates that the fast shrinking kinetics, combined with the extremely long DNA\nchain length, restricts chain reptation and entanglement relaxation, thereby resulting in\nhigh-density entanglement within the resultant DNA hydrogel. Remarkably, relying\nsolely on this entanglement mechanism, the resulting DNA hydrogel exhibits rubber-\nlike material characteristics with high stiffness and toughness. Additional chemical\ncrosslinking can further stabilize this hydrogel material. The authors have\nsystematically investigated key factors influencing the gelation process, confirming that\nchain length, shrinking rate, and initial DNA concentration are primary determinants - findings that strongly support their proposed entanglement-based mechanism. The observed elastic modulus and stretchability characteristics are also consistent with an\nentanglement-dominated hydrogel. They further demonstrated the potential application\nof this strategy in 3D printing and mold casting for magnetic soft robots. This approach\ncould significantly broaden the potential applications of DNA hydrogels and may\nrepresent a milestone in DNA hydrogel development. The manuscript is well-written,\nand the data are convincing. I strongly recommend its publication in\nNature Communications.\nSome minor technique comments:\n1. Previous studies have utilized rolling circle amplification (RCA) to generate long-chain DNA polymers, investigated chain\nentanglement as one of the key mechanisms for DNA hydrogel formation. Although these RCA-produced DNA hydrogels\nexhibit relatively soft and weak mechanical properties, they represent pioneering work in investigating the entanglement\nphenomenon for DNA hydrogel fabrication. These foundational studies (Nature Nanotechnology 2012, 7(12), 816–820;\nAngewandte Chemie International Edition 2020, 59(6), 2490–2495) should be appropriately cited and discussed in this\ncontext.\n2. On Page 4, Line 32, the authors describe the hydrogel as stable in a \"moist atmosphere,\" but this term lacks precision. To\nstrengthen the study's reproducibility, the authors should clearly specify the humidity and temperature ranges defining this\ncondition, and provide quantitative data demonstrating how the hydrogel's mechanical\nproperties (e.g., elasticity, tensile\nstrength) evolve over time under these storage parameters.\n3. In Fig. 2H-I and Supplementary Fig. 20, the authors examine how initial DNA concentration (5%-12.5%) affects the\nmechanical properties of DNA hydrogels. While higher concentrations generally enhance properties, the unexpected\ndecline at 12.5% needs explanation. Additionally, the compression data in Fig. 2I only covers 5%-10% concentrations,\ncreating an inconsistency that requires justification regarding the exclusion of 12.5% data.\n4. The entire manuscript only employed salmon sperm DNA for demonstration, raising questions about sequence specificity.\nIt remains unclear whether this represents a general strategy applicable to biomass DNA from other sources. The authors\nshould validate the method's generality by testing it with various biomass DNA exhibiting different sequences.\n5. The authors hypothesize that the effectiveness of this strategy in forming the entanglement hydrogel lies in the ultra-long\nchains of biomass DNA which could prohibit the disentanglement during the fast shrinkage while enhance the\ninterpenetration. I wonder whether this strategy could be applied to other pre-existing long chain biopolymers to enhance\ntheir mechanical properties, which could be of great interest.\n6. In the caption of Figure 3, the capital letter \"E\" appears twice, please correct it.\n7. The illustration of soft robot fabrication in Fig. 4G seams misaligned, please check out and revise it.\n8. There are some typos in the manuscript, such as on Page 2, Line 32, “W >5 MJ/m3”, instead of “W >5 MJ/m3”, and in\nSupplementary Fig. 12a, “same water content”, instead of “smae water content”, the authors should carefully revise and\ncorrect these errors.", "reviewer_2": "In this manuscript, the authors report a novel Fast-Shrinking-Induced Entanglement (FaSIE) process for engineering DNA\nhydrogels with outstanding mechanical properties, including high stiffness, toughness, and stretchability. They present a\nthorough and systematic investigation of various processing parameters and successfully establish their relationship with\nthe mechanical performance of the hydrogels. Additionally, the utility of the hydrogels in 3D printing is also demonstrated. I\nrecommend publication after the authors address the following comments and concerns:\n1) While the reported mechanical properties are impressive, it must be clearly stated that many of these values are based on\nnon-swollen hydrogels. This distinction is critical, as it may mislead readers regarding the actual performance in practical\nconditions (for example, in aqueous solution). In particular, the E-gel is not stable upon swelling and requires chemical\ncrosslinking to form E-Mg-gel or E-Mg-PEG-gel. The mechanical properties of these chemically crosslinked hydrogels are\nsignificantly reduced compared to their non-swollen counterparts. The manuscript should reflect this nuance and include\nmore detailed comparisons of the mechanical properties in both states.\n2) The FaSIE method relies on the diffusion of acrylamide (AA) and Triton X (TX) into a DNA solution. This raises questions\nabout the homogeneity of the resulting hydrogel network. The authors should discuss how uniform the gel structure is across\nits volume and whether any characterization (e.g., microscopy, fluorescence, or mechanical mapping) has been performed to\nassess spatial uniformity.\n3) Given the importance of chemical crosslinking density in determining mechanical properties, the authors should estimate\nthe crosslinking density for both E-Mg and E-Mg-PEG hydrogels. Furthermore, the manuscript mentions that certain divalent\nions such as Mn, Fe, Cu, and Zn weaken the hydrogel. It would be valuable to clarify whether this weakening effect is\nconsistent across different ion concentrations or if a threshold or dose-dependent response is observed.\n4) The gels' visual appearance appears to vary, as shown in Fig. S21—where the 5% gel is opaque, while the 10% gel is\ntransparent. This observation should be systematically described and explained. The authors should discuss the underlying\ncauses of this difference—e.g., variations in polymer concentration, phase separation, microstructure,—and whether these\noptical differences correlate with any structural or mechanical changes.\n5) The manuscript claims that the FaSIE approach is general and potentially applicable to polysaccharides. However, no\nexperimental data are provided to support this assertion. To strengthen this claim, preliminary results or at least a more\ndetailed discussion of the feasibility, potential challenges, and future plans for extending FaSIE to other biopolymers would\nbe helpful.", "reviewer_3": "The manuscript titled “Rubber-like DNA hydrogel enabled by fast-\nshrinking-induced entanglement” reports the fabrication of a DNA-based hydrogel\nusing a fast-shrinking induced entanglement technique. The study presents substantial\nexperimental work to support the findings; however, several suggestions are provided\nbelow that may help further improve the manuscript.\n1. The DNA concentration used in the study is 5% (w/v). While line 183\ndiscusses the influence of initial DNA concentration on mechanical properties, the\nrationale for selecting this specific value remains unclear. The authors are encouraged\nto clarify the reasoning behind this choice.\n2. Figure 2G presents a schematic of the DNA hydrogel fabrication process, which is\nalready shown in greater detail in Figure 1C. It is recommended to remove this\npanel from Figure 2 to avoid redundancy.\n3. Given the immense biomedical relevance of DNA-based hydrogels, it is\nrecommended to include cytocompatibility testing on human cells (of any type) to\nevaluate potential toxicity of the synthesized formulations.\n4. ATR-FTIR analysis is mentioned in the Supplementary Information (Line 137)\nand presented in Supplementary Fig. 4, with only a brief discussion provided in the\nmain text (Line 101). It is suggested that the authors include a more detailed\nexplanation of these results.\n5. The figure captions for Supplementary Figs. 26 and 27 appear to be out of order. The\ncaptions should be revised to match the sequence of the corresponding images."}, "author_response": "Response to Reviewer’s comments:\nReviewer #1:\nComment: This manuscript presents an innovative strategy for fabricating DNA\nhydrogels. Currently, most known DNA hydrogels are primarily formed through strand\nhybridization and chemical crosslinking, exhibiting limited mechanical properties.\nWhile chain entanglement is recognized as a potential enhancer of mechanical\nproperties in DNA hydrogels, the entanglement density has been insufficient to form\nrobust, tough hydrogels. The authors developed a \"fast-shrinking-induced\nentanglement\" strategy that enables the production of DNA hydrogels with remarkably\nhigh mechanical properties. This process was never reported and is conceptually\ninteresting and innovative. They identified an optimal dehydration phase separation\nsolvent to induce rapid shrinking of pre-entangled DNA solutions. The study\ndemonstrates that the fast shrinking kinetics, combined with the extremely long DNA\nchain length, restricts chain reptation and entanglement relaxation, thereby resulting in\nhigh-density entanglement within the resultant DNA hydrogel. Remarkably, relying\nsolely on this entanglement mechanism, the resulting DNA hydrogel exhibits rubber-\nlike material characteristics with high stiffness and toughness. Additional chemical\ncrosslinking can further stabilize this hydrogel material. The authors have\nsystematically investigated key factors influencing the gelation process, confirming that\nchain length, shrinking rate, and initial DNA concentration are primary determinants -\nfindings that strongly support their proposed entanglement-based mechanism. The\nobserved elastic modulus and stretchability characteristics are also consistent with an\nentanglement-dominated hydrogel. They further demonstrated the potential application\nof this strategy in 3D printing and mold casting for magnetic soft robots. This approach\ncould significantly broaden the potential applications of DNA hydrogels and may\nrepresent a milestone in DNA hydrogel development. The manuscript is well-written,\nand the data are convincing. I strongly recommend its publication in Nature\nCommunications.\nThank you for highlighting the significance of our work and the valuable suggestions\nto help us improve the manuscript.\nComment 1: Previous studies have utilized rolling circle amplification (RCA) to\ngenerate long-chain DNA polymers, investigated chain entanglement as one of the key\nmechanisms for DNA hydrogel formation. Although these RCA-produced DNA\nhydrogels exhibit relatively soft and weak mechanical properties, they represent\npioneering work in investigating the entanglement phenomenon for DNA hydrogel\nfabrication. These foundational studies (Nature Nanotechnology 2012, 7(12), 816–820;\nAngewandte Chemie International Edition 2020, 59(6), 2490–2495) should be\nappropriately cited and discussed in this context.\nThank you for suggesting the additional reference, we cite the papers in the revised\nintroduction:\n“Therefore, although the exceptional chain length of biomass DNA (normally in the\nrange of 107-1012 Da)20 is favourable for entanglement engineering, and studies have\nrevealed that entanglements generally exist in DNA hydrogel21,22, the fabrication of\nentanglement dominant DNA hydrogels remains exceptionally challenging.”\nComment 2: On Page 4, Line 32, the authors describe the hydrogel as stable in a \"moist\natmosphere,\" but this term lacks precision. To strengthen the study's reproducibility, the\nauthors should clearly specify the humidity and temperature ranges defining this\ncondition, and provide quantitative data demonstrating how the hydrogel's mechanical\nproperties (e.g., elasticity, tensile strength) evolve over time under these storage\nparameters.\nThank you for your suggestion. We have tested the mechanical stability of E-Gel in a\n4 ℃ fridge with 40% humidity over the course of one week and found that the\nmechanical performance of E-Gel remained unchanged during this time. We have added\nthis new experiment into the manuscript.\nRevisions in the manuscript:\n“However, storing the hydrogel in a 4 ℃ fridge at 40% humidity for over one week\ndoes not visibly weaken it (Supplementary Fig.15), indicating that E-Gel can maintain\nstability in air.”\nThe following paragraph is added to Supplementary Information:\nSupplementary Fig. 15\nMechanical stability of E-Gel stored at moist atmosphere over time. E-Gel was\nprepared and then stored in a 4 ℃ fridge at 40% humidity for 0, 3 and 7 days. The\ntensile properties of E-Gel were tested and the results showed that the storage in a moist\natmosphere for 7 days didn’t obviously weaken E-Gel which indicates the stability of\nE-Gel in air.\nComment 3: In Fig. 2H-I and Supplementary Fig. 20, the authors examine how initial\nDNA concentration (5%-12.5%) affects the mechanical properties of DNA hydrogels.\nWhile higher concentrations generally enhance properties, the unexpected decline at\n12.5% needs explanation. Additionally, the compression data in Fig. 2I only covers 5%-\n10% concentrations, creating an inconsistency that requires justification regarding the\nexclusion of 12.5% data.\nThank you for highlighting this issue. We have now included the compression data for\nthe 12.5% hydrogel in Figure 2I. Our observations indicate that increasing the initial\nDNA concentration from 10% to 12.5% did not enhance the mechanical strength of the\nhydrogel; in fact, some parameters (e.g., compressive strength and modulus) even\nshowed a decrease. We speculate that this decline in mechanical strength may be\nattributed to the inhomogeneity of the precursor DNA solution at higher concentrations.\nWe noted that the precursor DNA solutions from 5% to 10% are homogeneous and\ntransparent, whereas the 12.5% DNA solution appears more turbid (Supplementary Fig.\n27a), suggesting it may have exceeded the saturation concentration. Furthermore, the\nabsorbance spectrum reveals a significant increase across the entire wavelength range,\nconfirming reduced transparency. This heterogeneity likely hindered the formation of\nentanglement hydrogels and reduced their mechanical properties.\nWe have updated Figure 2I and Figure S27 in the revised manuscript, along with an\nexplanation addressing this phenomenon. Additionally, a new Figure S24 has been\nadded to the supplementary information to illustrate the appearance of the DNA\nprecursor solution.\nRevisions in the manuscript:\n“Compression tests also reflected this trend (Figure.2I): at C = 10%, the compressive\ni\nmodulus (Y) reached 138.50 ± 28.89 kPa, and compressive strength rose to 6.83 ± 0.24\nMPa (Supplementary Fig.26), comparable to typical stiff and tough double network\nhydrogels made from synthetic polymers (Supplementary Table.2). We observed a\ndecline of compressive modulus and strength at an initial concentration of 12.5%,\nlikely\ndue to this concentration having already exceeded the saturation limit (Supplementary\nFig. 27). ”\nFig. 2I The stress-strain curves of E-Gel with different Ci under compression tests\nThe following paragraph is added to Supplementary Information:\nSupplementary Fig. 26\nCompressive properties of DNA hydrogel with different initial DNA concentration.\nSimilar with tensile tests, the compressive modulus and strength increased with the\ninitial DNA concentration. (a) compressive modulus increased from 18 kPa to near 150\nkPa, while (b) compressive strength increased from 1.6 MPa to 7 MPa (data are mean\n± SD, N = 3).\nSupplementary Fig. 27\nTransmittance of DNA solutions with concentrations ranging from 5% to 12.5%.\na) Photographs of DNA solutions (5–12.5%), where samples at 5–10% appear\ntranslucent, while the 12.5% sample appears blurry. b) UV–Vis spectrum (5–12.5%)\nfurther confirm that the transparency of DNA solutions gradually decreases with\nincreasing concentration, followed by a sharp decline at 12.5%, indicating\noversaturation of DNA at this concentration.\nComment 4: The entire manuscript only employed salmon sperm DNA for\ndemonstration, raising questions about sequence specificity. It remains unclear whether\nthis represents a general strategy applicable to biomass DNA from other sources. The\nauthors should validate the method's generality by testing it with various biomass DNA\nexhibiting different sequences.\nWe appreciate your suggestion. To validate the generality of our method, we have tested\nthe tensile mechanical properties of another biomass DNA, namely Calf Thymus DNA.\nThe results demonstrated that the hydrogels obtained from Calf Thymus DNA exhibited\nsimilar mechanical properties to Salmon Sperm DNA. The differences in Young’s\nmodulus and stretchability of these hydrogels are attributed to differences in chain\nlength and/or sequence. Through this experiment, we have confirmed that our strategy\nis applicable to various biomass DNAs, not limited to Salmon Sperm DNA. We have\nupdated the manuscript accordingly.\nRevisions in the manuscript:\n“In addition to Salmon Sperm DNA, we have also applied this strategy to Calf Thymus\nDNA. Hydrogels could be obtained with similar mechanical properties (Supplementary\nFig.38), demonstrating the generality of this strategy for various long-strand biomass\nDNAs.”\nThe following paragraph is added to Supplementary Information:\nSupplementary Fig. 38\nFast shrinking induced entanglement gelation could be applied to various biomass\nDNA. a) Calf Thymus DNA was chosen and the tensile test of resultant hydrogel was\nperformed, the Strain-Stress curve was plotted compared with E-Gel made by salmon\nsperm DNA. d) Mechanical properties of entangled DNA hydrogels made by Calf\nThymus DNA (data are mean ± SD, N = 3).\nComment 5: The authors hypothesize that the effectiveness of this strategy in forming\nthe entanglement hydrogel lies in the ultra-long chains of biomass DNA which could\nprohibit the disentanglement during the fast shrinkage while enhance the\ninterpenetration. I wonder whether this strategy could be applied to other pre-existing\nlong chain biopolymers to enhance their mechanical properties, which could be of great\ninterest.\nWe appreciate your suggestion. To validate the generality of our method, we have\napplied our strategy to various pre-existing long-chain polymers, including sodium\nalginate (SA), sodium hyaluronate (HA), and carboxymethyl cellulose (CMC), all of\nwhich are commercially available. We found that after treatment with AA/TX, both\npolymers could form hydrogels similar to DNA. We then tested the mechanical\nproperties of these entangled biopolymer hydrogels. Compared to biopolymer\nhydrogels formed by conventional chemical crosslinking, we found that the mechanical\nproperties of the entangled biopolymer hydrogels were significantly improved with\nstiffness and toughness 10 to 270 times enhanced. It should be noted that the\nbiopolymer hydrogels were formed following the same procedure as the DNA\nhydrogels, but the dehydration phase separation reagents may not be optimal for these\nbiopolymers. Optimization of the screening process may further strengthen the\nmechanical properties of these biopolymer hydrogels. Nevertheless, the preliminary\nresults have shown the potential of our strategy to enhance the mechanical properties\nof pre-existing long-chain biopolymers. We have updated the manuscript, method, and\nsupporting information accordingly.\nRevisions in the manuscript:\n“With the success of the FaSIE process in DNA hydrogels, the potential for reinforce\nother biopolymer hydrogels was also explored. Similar trend was observed with sodium\nalginate, sodium hyaluronate and carboxymethyl cellulose (Supplementary Fig.39).\nThe FaSIE process is able to achieve hydrogels with stiffness and toughness 10 to 270\ntimes enhanced comparing to conventional chemical crosslinking. Notably, these\nresults were obtained with the same protocol used for DNA hydrogels. Additional\noptimization of the dehydration solution is expected to further enhance the FaSIE\nperformance for individual biopolymers. Nevertheless, the preliminary data have\nalready demonstrated the promising potential of FaSIE to enhance the mechanical\nproperties of various biopolymers.”\nSupplementary Fig. 39\nFast-shrinking-induced entanglement gelation could be applied to various existed\nlong chain polymers. a) alginate could form hydrogel after treatment with AA/TX. b)\nsodium hyaluronate could also form hydrogel, and the stretchability is exceptionally high,\nwhich could reach 60 times of its original length. c) also with carboxymethyl cellulose (CMC).\nd) mechanical properties of entangled hydrogels from other resources (data are mean ± SD, N\n= 3). e) Mechanical performance of the entangled sodium alginate (entangled SA), entangled\nhyaluronic acid (entangled HA) and entangled carboxymethyl cellulose (entangled CMC)\ncomparing to conventional calcium crosslinked alginate (SA), PEG-DE crosslinked hyaluronic\nacid (HA) and PEG-DE crosslinked carboxymethyl cellulose (CMC). The stiffness,\nstretchability, tensile strength and toughness are plotted as a radar map.\nComment 6: In the caption of Figure 3, the capital letter \"E\" appears twice, please\ncorrect it.\nThank you, we have thoroughly checked the manuscript and typos have been corrected.\nComment 7: The illustration of soft robot fabrication in Fig. 4G seems misaligned,\nplease check out and revise it.\nThank you for bringing this to our attention. We have replaced the Fig. 4G image with\nthe correct one.\nComment 7: There are some typos in the manuscript, such as on Page 2, Line 32,\n“W >5 MJ/m3”, instead of “W >5 MJ/m3”, and in Supplementary Fig. 12a, “same water\ncontent”, instead of “smae water content”, the authors should carefully revise and\ncorrect these errors.\nThank you, we have thoroughly checked the manuscript and typos have been corrected.\nReviewer #2:\nComment: In this manuscript, the authors report a novel Fast-Shrinking-Induced\nEntanglement (FaSIE) process for engineering DNA hydrogels with outstanding\nmechanical properties, including high stiffness, toughness, and stretchability. They\npresent a thorough and systematic investigation of various processing parameters and\nsuccessfully establish their relationship with the mechanical performance of the\nhydrogels. Additionally, the utility of the hydrogels in 3D printing is also demonstrated.\nI recommend publication after the authors address the following comments and\nconcerns:\nThank you for carefully reviewing our manuscript and proving the insightful comments\nto improve our paper.\nComment 1: While the reported mechanical properties are impressive, it must be\nclearly stated that many of these values are based on non-swollen hydrogels. This\ndistinction is critical, as it may mislead readers regarding the actual performance in\npractical conditions (for example, in aqueous solution). In particular, the E-gel is not\nstable upon swelling and requires chemical crosslinking to form E-Mg-gel or E-Mg-\nPEG-gel. The mechanical properties of these chemically crosslinked hydrogels are\nsignificantly reduced compared to their non-swollen counterparts. The manuscript\nshould reflect this nuance and include more detailed comparisons of the mechanical\nproperties in both states.\nThank you for your suggestions. We have clarified that the mechanical properties of E-\nGel were measured in a non-swollen state immediately after preparation. Even without\nswelling, the water content of E-Gel already reaches approximately 60%. Under these\nconditions, the mechanical properties (total elongation ~1107%, Young’s modulus ~60\nkPa, ultimate tensile strength >0.4 MPa, and compressive modulus ~19 kPa) are\nsignificantly higher than those of traditional chemically crosslinked DNA hydrogels,\nhighlighting the advantages of the entangled DNA hydrogel produced by the FaSIE\nprocess. Once fully swollen, E-Gel will eventually dissolve; therefore, the mechanical\nproperties of the swollen E-Gel cannot be evaluated. Nevertheless, we anticipate that\nE-Gel remains suitable for applications in air and non-aqueous environments.\nBy introducing additional electrostatic interactions and chemical crosslinking, E-Mg-\nGel can be formed, which swells and retains stability in aqueous solutions. For E-Mg-\nGel, all mechanical properties are measured after complete swelling. The water content\nof fully swollen E-Mg-Gel ranges from 70% to 80%, slightly higher than that of non-\nswollen E-Gel. The ultimate tensile strength of the swollen E-Mg-Gel exceeds 1 MPa,\nwith total elongation of ~1200%, stiffness of 150 kPa and compressive modulus ~222\nkPa, all of which are significantly higher than those of non-swollen E-Gel. These\nproperties demonstrate the promising application potential of E-Mg-Gel even in\naqueous environments.\nIn the revised manuscript, we have clearly mentioned the swollen status and water\ncontent of each hydrogel when discussing mechanical properties, and we have added\nexplanations regarding the application limitations for both types of hydrogels.\nRevisions in the manuscript:\n“E-Gel is prepared in the non-swollen state and exhibits limited mechanical stability in\naqueous solutions due to disentanglement, making it suitable for use in non-aqueous\nenvironments. To enhance the applicability of E-Gel in aqueous environments, Mg2+\nand PEG-DE could be added post-gelation to introduce additional electrostatic\ninteractions and chemical crosslinks.”\nThe swollen status was also labeled in Figure 3F-H:\nComment 2: The FaSIE method relies on the diffusion of acrylamide (AA) and Triton\nX (TX) into a DNA solution. This raises questions about the homogeneity of the\nresulting hydrogel network. The authors should discuss how uniform the gel structure\nis across its volume and whether any characterization (e.g., microscopy, fluorescence,\nor mechanical mapping) has been performed to assess spatial uniformity.\nThank you for highlighting this important issue. We agree that the diffusion of AA/TX\ncan influence the homogeneity of the resulting hydrogel. However, for the hydrogels\nprepared in our experiments (6 mm * 6 mm volume), we observed overall homogeneity.\nTo confirm this, we conducted three additional experiments. Firstly, we obtained\nscanning electron microscopy (SEM) images from both the center and edge areas of the\nhydrogels, revealing similar morphologies that indicate macroscopic homogeneity\n(Figure S9a). Secondly, we performed confocal laser scanning microscopy (CLSM) on\nhydrogels stained with GoldView DNA stain, recording a consistent green color across\na z-scan extending to 500 µm. Thirdly, to evaluate the nanoscale homogeneity of E-Gel,\nwe conducted Small Angle X-ray Scattering (SAXS) with a q range of 0.0029 to 0.2107,\ncorresponding to a size range of 4.7 to 344.8 nm. The resulting 2D SAXS pattern\ndisplayed no scattering rings, and the 1D intensity profile showed no correlation peaks,\nindicating a uniform structure throughout this size range. Overall, we conclude that the\nhydrogels produced under our experimental conditions are homogeneous.\nIt is important to note that achieving such homogeneity requires adequate amounts of\nAA/TX to ensure efficiently rapid dehydration. If insufficient AA/TX is used, SEM\nanalysis (Figure S9b) demonstrates that the pores in the center become significantly\nlarger than those at the edges, indicating heterogeneity.\nWe have now addressed the homogeneity issue in the revised main text, and the new\ndata have been included in the supporting information along with a comprehensive\nexplanatory paragraph.\nRevisions in the manuscript:\n“Scanning electron microscopy (SEM) of the E-Gel revealed a uniform structure\nwithout obvious pores (Fig.2E), and E-Gel exhibited a homogeneous morphology\nthroughout its volume (Supplementary Fig. 9).”\nSupplementary Fig. 9\nHomogeneity of resultant E-Gel. A larger quantity of AA/TX resulted in a) a uniformly\nhomogeneous structure. While a small volume of AA/TX applied to the DNA solution resulted\nin b) a heterogeneous structure of the DNA hydrogel, where the center area exhibited large\npores with a diameter of around 100 µm, while the edge area showed more packed and smaller\npores with a diameter of around 5 µm. c) E-Gel was stained with GoldView and imaged\nusing confocal laser microscopy with Z-scan mode. d) Small Angle X-ray Scattering\n(SAXS) was performed to assess the homogeneity of E-Gel. Both confocal images and\nSAXS plots revealed the uniformity and homogeneity of E-Gel throughout its volume.\nComment 3: Given the importance of chemical crosslinking density in determining\nmechanical properties, the authors should estimate the crosslinking density for both E-\nMg and E-Mg-PEG hydrogels. Furthermore, the manuscript mentions that certain\ndivalent ions such as Mn, Fe, Cu, and Zn weaken the hydrogel. It would be valuable to\nclarify whether this weakening effect is consistent across different ion concentrations\nor if a threshold or dose-dependent response is observed.\nThank you for your suggestions. Following your advice, we used Mooney-Rivlin\nmethod (J. Appl. Polym. Sci, 2013, 127(6): 4537-4541) to estimate the crosslinking-\ndensity of E-Mg-Gel and E-Mg-PEG-Gel. The crosslinking-density was found to be 51\nmol/m3 for E-Mg-Gel and 95 mol/m3 for E-Mg-PEG-Gel.\nWe would like to clarify that the effect of divalent ions occurs only at high\nconcentrations, specifically at a concentration of 2 M in this case. At lower\nconcentrations, swelling and disentanglement dominate the process, resulting in poor\nmechanical properties. In contrast, at high concentrations, the elevated osmotic pressure\nand potential salting-out effects inhibit hydrogel swelling (Advanced Materials, 2021,\n33(11): 2007829), which is critical for effectively incorporating ionic interactions into\nthe network.\nRegarding the weakening of the DNA hydrogel by Fe2⁺, Mn²⁺, Cu²⁺, and Zn²⁺ ions,\nwe\nspeculate that these ions act as chelating agents that interact too strongly with DNA\n(Nanoscale, 2022, 14(39): 14613-14622). The toughening effect of entanglement in the\nhydrogel network depends on its capacity to distribute stress across neighboring\npolymer strands. Unlike Mg²⁺ and Ca²⁺ ions, which interact with the phosphate\nbackbone of DNA to stabilize the hydrogel without significantly hindering chain\nreptation, these strong chelating ions immobilize the chains and restrict effective stress\ndistribution. This restriction undermines the advantages of chain entanglement, thereby\nweakening the hydrogel.\nWe have mentioned the crosslinking density in the main text and added the calculation\ndetails in the supporting information. The explanation of the weakening effect was also\nadded to the paragraph discussing ionic effects in the supporting information.\nRevisions in the manuscript:\n“Mg2+ and PEG-DE could be added post-gelation to introduce additional electrostatic\ninteractions and chemical crosslinks…... An increase in crosslinking density was also\nobserved after Mg²⁺ and PEG-DE treatment (Supplementary Fig. 29).”\nThe following figure and the method for crosslinking density calculation was added to\nsupplementary information:\nSupplementary Fig. 29\nEstimation of crosslinking-density in E-Mg-Gel and E-Mg-PEG-Gel. Mooney-\nRivlin model was used to estimate the crosslinking density of E-Mg-Gel and E-Mg-\nPEG-Gel. The fitting parameters C1 was found to be 63.21 kPa for E-Mg-Gel and 117.7\nkPa for E-Mg-PEG-Gel. and corresponding crosslinking-density was calculated as 44\nmol/m³ for E-Mg-Gel and 95 mol/m³ for E-Mg-PEG-Gel\nComment 4: The gels' visual appearance appears to vary, as shown in Fig. S21—where\nthe 5% gel is opaque, while the 10% gel is transparent. This observation should be\nsystematically described and explained. The authors should discuss the underlying\ncauses of this difference—e.g., variations in polymer concentration, phase separation,\nmicrostructure—and whether these optical differences correlate with any structural or\nmechanical changes.\nWe sincerely appreciate your suggestions and apologize for the misleading\nrepresentation of the two images. In fact, the visual appearance of the 5% E-Gel and\n10% E-Gel is similar; any apparent differences resulted from variations in photographic\nconditions. We have reproduced the hydrogels and captured images under identical\nconditions with the same background, and we measured their transmittance. Both\nhydrogels exhibited approximately 40% transparency, with no discernible difference\nbetween them. The updated images have been included in the Supplementary\nInformation as follows.\nSupplementary Fig. 23a\nThe optical appearance of 5% and 10% E-Gel. ~40% Transmittance was observed for\nboth hydrogels.\nComment 5: The manuscript claims that the FaSIE approach is general and potentially\napplicable to polysaccharides. However, no experimental data are provided to support\nthis assertion. To strengthen this claim, preliminary results or at least a more detailed\ndiscussion of the feasibility, potential challenges, and future plans for extending FaSIE\nto other biopolymers would be helpful.\nWe appreciate your suggestion. To validate the generality of our method, we have\napplied our strategy to various pre-existing long-chain polymers, including sodium\nalginate (SA), sodium hyaluronate (HA), and carboxymethyl cellulose (CMC), all of\nwhich are commercially available. We found that after treatment with AA/TX, both\npolymers could form hydrogels similar to DNA. We then tested the mechanical\nproperties of these entangled biopolymer hydrogels. Compared to biopolymer\nhydrogels formed by conventional chemical crosslinking, we found that the mechanical\nproperties of the entangled biopolymer hydrogels were significantly improved with\nstiffness and toughness 10 to 270 times enhanced. It should be noted that the\nbiopolymer hydrogels were formed following the same procedure as the DNA\nhydrogels, but the dehydration phase separation reagents may not be optimal for these\nbiopolymers. Optimization of the screening process may further strengthen the\nmechanical properties of these biopolymer hydrogels. Nevertheless, the preliminary\nresults have shown the potential of our strategy to enhance the mechanical properties\nof pre-existing long-chain biopolymers. We have added these new data into the\nmanuscript and the supporting information.\nRevisions in the manuscript:\n“With the success of the FaSIE process in DNA hydrogels, the potential for reinforce\nother biopolymer hydrogels was also explored. Similar trend was observed with sodium\nalginate, sodium hyaluronate and carboxymethyl cellulose (Supplementary Fig.39).\nThe FaSIE process is able to achieve hydrogels with stiffness and toughness 10 to 270\ntimes enhanced comparing to conventional chemical crosslinking. Notably, these\nresults were obtained with the same protocol used for DNA hydrogels. Additional\noptimization of the dehydration solution is expected to further enhance the FaSIE\nperformance for individual biopolymers. Nevertheless, the preliminary data have\nalready demonstrated the promising potential of FaSIE to enhance the mechanical\nproperties of various biopolymers.”\nThe following paragraph is added to supplementary information:\nSupplementary Fig. 39\nFast-shrinking-induced entanglement gelation could be applied to various existed\nlong chain polymers. a) alginate could form hydrogel after treatment with AA/TX. b)\nsodium hyaluronate could also form hydrogel, and the stretchability is exceptionally high,\nwhich could reach 60 times of its original length. c) also with carboxymethyl cellulose (CMC).\nd) mechanical properties of entangled hydrogels from other resources (data are mean ± SD, N\n= 3). e) Mechanical performance of the entangled sodium alginate (entangled SA), entangled\nhyaluronic acid (entangled HA) and entangled carboxymethyl cellulose (entangled CMC)\ncomparing to conventional calcium crosslinked alginate (SA), PEG-DE crosslinked hyaluronic\nacid (HA) and PEG-DE crosslinked carboxymethyl cellulose (CMC). The stiffness,\nstretchability, tensile strength and toughness are plotted as a radar map.\nReviewer #3:\nComment: The manuscript titled “Rubber-like DNA hydrogel enabled by fast-\nshrinking-induced entanglement” reports the fabrication of a DNA-based hydrogel\nusing a fast-shrinking induced entanglement technique. The study presents substantial\nexperimental work to support the findings; however, several suggestions are provided\nbelow that may help further improve the manuscript.\nThank you for your generous comments to help us improve this manuscript.\nComment 1: The DNA concentration used in the study is 5% (w/v). While line 183\ndiscusses the influence of initial DNA concentration on mechanical properties, the\nrationale for selecting this specific value remains unclear. The authors are encouraged\nto clarify the reasoning behind this choice.\nWe sincerely appreciate your suggestion. We chose 5% (w/v) for detailed studies due\nto a balance between sample cost and material performance. Although increasing the\ninitial DNA concentration of E-Gel would further enhance Yong’s modulus and\ncompression modulus, but it also compromise the total elongation. Moreover, we also\nfound that the mechanical properties of 10% E-Mg-PEG-Gel and 5% E-Mg-PEG-Gel\nare appeared to be similar, which indicating further increasing the DNA content only\ngain limiting mechanical benefit when there are additional crosslinking. Therefore, to\nreduce the sample cost and present representative material properties, 5% (w/v) was\nchosen as example for the whole manuscript. We have added an explanation of this\nchoice in the manuscript.\nRevisions in the manuscript:\n“To enhance the applicability of E-Gel in aqueous environments, Mg2+ and PEG-DE\ncould be added post-gelation to introduce additional electrostatic interactions and\nchemical crosslinks (exemplified by Ci = 5% E-Gel with relative low DNA content and\nrepresentative mechanical properties).”\nComment 2: Figure 2G presents a schematic of the DNA hydrogel fabrication process,\nwhich is already shown in greater detail in Figure 1C. It is recommended to remove this\npanel from Figure 2 to avoid redundancy.\nWe sincerely appreciate your suggestion. Upon review, we realized that Fig. 2G may\nappear ambiguous and potentially misleading. To clarify, Fig. 2G is not intended to\nrepresent the schematic fabrication process of the DNA hydrogel. Rather, this panel\naims to illustrate the correlation between DNA chain length and its ability to undergo\ngelation. As shown in Supplementary Fig. 16, both chain length and initial DNA\nconcentration play a crucial role in regulating the FASIE process. Specifically, three\ndistinct phases were observed: solution, precipitate, and hydrogel. Short chain lengths\nand low initial concentrations yielded only a solution. With increasing chain length and\nconcentration, precipitation occurred, and we identified a strong correlation between\nthe precipitation concentration and the entanglement concentration. Further increases\nin chain length and/or initial concentration ultimately led to hydrogel formation.\nTherefore, the purpose of Fig. 2G is to depict these phase transitions across the process.\nTo avoid potential misunderstanding, we have revised the graph for better illustration.\nFig. 2G\nThe chain length and concentration dependency of FaSIE process. The phase diagram was\nplotted using the data in Figure S14.\nComment 3: Given the immense biomedical relevance of DNA-based hydrogels, it is\nrecommended to include cytocompatibility testing on human cells (of any type) to\nevaluate potential toxicity of the synthesized formulations.\nWe sincerely appreciate your suggestion. We evaluated the cytotoxicity of E-Mg-Gel\nand E-Mg-PEG-Gel using the fibroblast cell line L929, and the results demonstrated\ntheir high biocompatibility. First, different mass ratios of DNA hydrogels (1–10%\nhydrogel-to-medium) were directly added to the cells and incubated for 4 h. Both E-\nMg-Gel and E-Mg-PEG-Gel maintained cell viability above 90% across the 1–7.5%\nrange, and viability slightly decreased at concentration of 10%. This reduction was\nlikely due to the large mass hydrogel sinking to the bottom of the wells, imposing\nmechanical stress on the cells and leading to cell death. Subsequently, we employed\nanother strategy to assess cytotoxicity: E-Mg-Gel and E-Mg-PEG-Gel were first\nincubated with cell culture medium at a 40% hydrogel-to-medium mass ratio for at least\n24 h. The resulting extract medium was then added to cells at equivalent DNA-to-\nmedium mass ratios and incubated for another 24 h. The results indicate that both\nhydrogels exhibited no cytotoxicity and may even promote cell proliferation. These\nfindings confirm the high biocompatibility of E-Mg-Gel and E-Mg-PEG-Gel,\nsuggesting their potential for further biomedical applications. These data have been\nadded to the manuscript.\nRevisions in the manuscript:\n“Moreover, both E-Mg-Gel and E-Mg-PEG-Gel exhibited excellent biocompatibility\nwith mammalian cells (Supplementary Fig.37). Together, these results highlight the\nbroad application potential of DNA hydrogels.”\nThe following paragraph is added to supplementary information:\nSupplementary Fig. 36\nCell viability of DNA hydrogels. E-Mg-Gel and E-Mg-PEG-Gel were synthesized and\nincubated with the normal cell line L929 to evaluate the cytotoxicity of DNA hydrogels.\na) DNA hydrogels were directly incubated with cells for 4 h, and cell viability was\nassessed using a CCK-8 kit. The DNA concentration in this experiment was defined as\nthe mass ratio of DNA hydrogel to cell culture medium. b) DNA hydrogels were\nincubated with cell culture medium for at least 24 h, after which the extract medium\nwas collected and incubated with cells for another 24 h. The DNA concentration was\ndefined as the mass ratio of the DNA extract to the cell culture medium.\nComment 4: ATR-FTIR analysis is mentioned in the Supplementary Information\n(Line 137) and presented in Supplementary Fig. 4, with only a brief discussion provided\nin the main text (Line 101). It is suggested that the authors include a more detailed\nexplanation of these results.\nThank you for your suggestion. We have added detailed explanations about all the major\nFTIR peaks in the supporting information.\nComment 5: The figure captions for Supplementary Figs. 26 and 27 appear to be out\nof order. The captions should be revised to match the sequence of the corresponding\nimages.\nThank you for bringing this to our attention. We have corrected the captions to match\nthe corresponding images.\nReviewer Comments\nThe manuscript titled “Rubber-like DNA hydrogel enabled by fast-shrinking-induced\nentanglement” reports the fabrication of a DNA-based hydrogel using a fast-shrinking-\ninduced entanglement technique. The study presents substantial experimental work to\nsupport the findings; however, several suggestions are provided below that may help further\nimprove the manuscript.\n1. The DNA concentration used in the study is 5% (w/v). While line 183 discusses the\ninfluence of initial DNA concentration on mechanical properties, the rationale for\nselecting this specific value remains unclear. The authors are encouraged to clarify\nthe reasoning behind this choice.\n2. Figure 2G presents a schematic of the DNA hydrogel fabrication process, which is\nalready shown in greater detail in Figure 1C. It is recommended to remove this panel\nfrom Figure 2 to avoid redundancy.\n3. Given the immense biomedical relevance of DNA-based hydrogels, it is\nrecommended to include cytocompatibility testing on human cells (of any type) to\nevaluate potential toxicity of the synthesized formulations.\n4. ATR-FTIR analysis is mentioned in the Supplementary Information (Line 137) and\npresented in Supplementary Fig. 4, with only a brief discussion provided in the main\ntext (Line 101). It is suggested that the authors include a more detailed explanation of\nthese results.\n5. The figure captions for Supplementary Figs. 26 and 27 appear to be out of order. The\ncaptions should be revised to match the sequence of the corresponding images."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have made adequate revisions to address the comments and concerns raised during the review process. The\nimprovements made in the manuscript significantly enhance the clarity and robustness of the study's findings.Given the high\nquality of the revisions and the importance of the research, I recommend its acceptance in Nature Communications.", "reviewer_2": "The authors have adequately addressed my comments. I am happy to recommend its publication in Nature\nCommunications.", "reviewer_3": "The authors have provided satisfactory responses in revised manuscript."}, "author_response": ""}]

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