One-step construction of robust protocells and prototissues in water

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: minor_revision

Abstract
Bottom-up assembly of protocell building blocks into self-supporting, macroscopic, and robust prototissues that are stable in water and exhibit biomimetic behaviors remains a fundamental challenge. Here, we present a gas-liquid microfluidics-assisted diffusion-inhibited complexation strategy that enables low-cost, high-throughput fabrication of protocells and precise, large-scale (>10 cm) 3D construction of prototissues. The resulting prototissues display good mechanical integrity in both water and air and resist disintegration under external perturbations. By modulating the surrounding matrix or integrating bioactive components into the prototissue framework, programmable deformation/motion can be achieved through chemo-mechanical transduction. This strategy provides a platform for modeling the intricate behaviors of living systems and may facilitate applications in synthetic biology and bioengineering.

Introduction
Protocells are miniature biomimetic systems that mimic the structure and function of natural cells, serving as simplified models for investigating complex physiological processes1. By integrating multiple protocells into interconnected 3D networks, hierarchical tissue-like structures, termed prototissues, can be further constructed, holding significant promise for bottom-up synthetic biology2–4, tissue engineering5, and bioinspired soft machines6. Various protocell models, including vesicles7–10, proteinosomes11–13, polymersomes14, colloidosomes15,16, coacervates17–19, and microdroplets20–22 have been explored for prototissue construction. In a series of pioneering studies, Bayley et al20 developed a strategy using droplet interface bilayers (DIBs) to create prototissues, with 3D printing facilitating spatial organization of lipid-coated water-in-oil droplets to generate millimeter-scale architectures with complex geometries. However, the DIB technique relies on an oil-phase medium, and the resulting prototissues exhibit poor stability under variations in pH, ionic strength, and temperature2. Recent advancements by Mann et al11–13 have introduced a water/oil Pickering emulsion approach, employing bioorthogonal chemistry to programmably assemble proteinosomes into prototissues. While this method offers advantages over lipid-based systems, including enhanced protocellular adhesion, prolonged prototissue stability, and improved permeability, some drawbacks persist. For example, the synthetic chemistry may require harsh conditions or toxic reagents, the protocell organization lacks spatial control, and the removal of the oil phase complicates manufacturing processes. Currently, developing robust, self-supporting, and long-term-stable protocells/prototissues in aqueous media, coupled with the achievement of scalability and low cost, has emerged as a critical challenge in this field2,23.

In this work, we propose an electrostatically mediated diffusion-inhibited complexation (DIC) strategy to address this challenge by using negatively charged cellulose nanofibril (CNF) and a polycation (poly(diallyldimethylammonium chloride), PDDA) as building blocks to construct protocells and prototissues (Fig. 1). Gas-liquid microfluidic technology enables the efficient production of CNF/PDDA microcapsule-based protocells by spraying CNF-containing aqueous droplets into a PDDA-containing aqueous solution. During diffusion, CNF interacts electrostatically with PDDA, forming a robust and dense CNF/PDDA membrane that stabilizes the droplet, thereby generating protocells. CNF not only serves as a component of the membrane, but also acts as a framework that supports the internal cavity of the protocells, similar to the cytoskeleton in natural cells, endowing the protocells with self-supporting properties. By integrating graphene oxide (GO) nanosheets into the membrane, the mechanical strength and permeability of protocells can be controlled, allowing an enhanced elastic modulus and diverse diffusion behaviors of neutral polymers, polyanions, and polycations across the membrane. Importantly, the use of electrolytes to weaken the electrostatic interactions between CNF and PDDA induces adhesion between protocells, leading to the one-step construction of prototissues in aqueous media. Due to the robustness of the protocells and their strong inter-protocell adhesion, the resulting prototissues display high structural stability and scalability, reaching sizes up to the decimeter scale. Finally, we demonstrate the precise 3D construction of prototissues via a molding strategy, and highlight their chemo-mechanical transduction behaviors, such as osmotic pressure-induced deformation and buoyancy-driven motion.

Results
CNF/PDDA microcapsules for protocell modeling

By extruding an aqueous phase containing CNF (0.6 wt%) into an aqueous collection bath containing PDDA (0.5 wt%, Mw: 200 ~ 350 kDa) and Triton X-100 (0.1 wt%) at an extrusion rate of 30 mL h-1 and a compressed gas flow rate of 1.0 L min-1, CNF/PDDA microcapsules with diameters of ~ 800 μm were prepared at a rate of ~ 120 per minute (Fig. 2a,b, Supplementary Fig. 1, 2 and Supplementary Movie 1; the extruded aqueous phase was stained with Amaranth Red to visualize microcapsule formation of microcapsules). Triton X-100 was used to reduce the air–water surface tension of the collection bath, thereby allowing droplets to pass through the surface and transform into microcapsules by DIC24. Without Triton X-100, or when its concentration was very low, the high interfacial tension prevented the formation of intact CNF/PDDA microcapsules (Supplementary Fig. 3,4 and Supplementary Movie 2). In addition to Triton X-100, surfactants including Pluronic F68, Pluronic F127, Tween-20, and Solutol HS-15 also enabled successful microcapsule formation (Supplementary Fig. 5). The produced CNF/PDDA microcapsules were washed with deionized water 10 times, then dispersed in deionized water for further experiments. The effects of concentration, pH, extrusion rate and gas flow rate on microcapsule formation and size were systematically investigated (Supplementary Fig. 6-8). CNF/PDDA microcapsules exhibited mechanical robustness and thermal stability, allowing them to withstand compression without rupture, remain self-supporting in air, and not disassemble in boiling water for 30 min (Fig. 2c, Supplementary Fig. 9,10). By mixing fluorescein isothiocyanate (FITC)-labelled CNF (CNF-FITC) into the droplets, microcapsules were visualized using confocal laser scanning microscopy (CLSM). 2D and reconstructed 3D fluorescence images revealed that CNF was distributed both on the membrane and inside the microcapsules, playing a role similar to that of the cytoskeleton, which is crucial for enhancing the mechanical properties of microcapsules (Fig. 2d-f). Using rhodamine B isothiocyanate (RITC)-labelled PDDA (PDDA-RITC) as a fluorescent probe, it was observed that PDDA was fully confined to the membrane and did not permeate into the interior (Supplementary Fig. 11). Scanning electron microscopy (SEM) characterization of freeze-dried CNF/PDDA microcapsules confirmed the dense CNF/PDDA membrane and 3D CNF network inside (Fig. 2g,h). Fluorescence recovery after photobleaching (FRAP) measurements showed that CNF had poor fluidity within the membrane and inside the microcapsules, as the fluorescence intensity exhibited almost no recovery after photobleaching in either region (Fig. 2i-k).

To assess the potential of CNF/PDDA microcapsules as protocell models, we investigated the membrane permeability. By dispersing CNF/PDDA microcapsules in hypertonic solutions of 1 M glucose or 1 wt% dextran of different molecular weights ranging from 10 to 500 kDa, no shrinkage of microcapsules was observed under an optical microscope (Fig. 2l,m and Supplementary Fig. 12). This indicated that the membrane of CNF/PDDA microcapsules did not selectively block glucose or dextran, and instead both solutes diffused into the microcapsules, leading to rapid equilibrium of osmotic pressure across the membrane25,26. Using FITC-labelled dextran (Dex-FITC) as a fluorescent probe, CLSM imaging showed that Dex-FITC permeated across the membrane and diffused into the interior of microcapsules (Supplementary Fig. 13). In addition to neutral hypertonic solutions of glucose or dextran, microcapsules were also dispersed in a polyanion aqueous solution containing 1 wt% poly(acrylic acid) (PAA-2k, Mw: ~ 2 kDa). Due to the electrostatic repulsion between PAA-2k and CNF, PAA-2k could not diffuse into the microcapsule, while the water inside the microcapsules could diffuse out, leading to microcapsule shrinkage (Supplementary Fig. 14). When the microcapsules were redispersed in water, they returned to spherical shape to balance the osmotic pressure. These results demonstrate the elastic nature of the membrane. Consequently, regulating the permeability of CNF/PDDA microcapsules was key to constructing protocells for further biomimetic applications.

CNF-GO/PDDA microcapsules for protocell modeling

To address the above issue, negatively charged GO nanosheets were incorporated into the CNF/PDDA membrane due to their large aspect ratio and specific surface area27,28. The addition of 0.2 wt% GO into the extruded aqueous phase did not interfere with the preparation of microcapsules, producing CNF-GO/PDDA microcapsules with a narrow size distribution, mechanical robustness, and biocompatibility (Supplementary Fig. 15-17, and Supplementary Movie 3). Using CNF-FITC and pyrene-labelled GO (GO-Pyrene) as fluorescent probes (Supplementary Fig. 18-20), CLSM images showed that CNF was distributed on the membrane and in the cavity, similar to the case of CNF/PDDA microcapsules (Fig. 3a-c and Fig. 2d-f). GO, on the other hand, was mainly distributed on the membrane, as the fluorescent signal inside the microcapsule was weak (Fig. 3c). This behavior could be attributed to the amphiphilic nature of GO, which possessed a hydrophilic surface and a hydrophobic basal plane29,30, making it more likely to accumulate at the air–water interface during gas shearing and form a CNF-GO/PDDA membrane by DIC. It was also difficult to observe GO nanosheets within CNF networks inside the microcapsules by SEM (Fig. 3d,e).

CNF-GO/PDDA microcapsules exhibited completely different permeability behavior compared to CNF/PDDA microcapsules. In a hypertonic solution of 1 M glucose, CNF-GO/PDDA microcapsules first shrank due to the difference in osmotic pressure, then gradually returned to their original shape (spherical) within 60 min as a result of the increased concentration of glucose and osmotic pressure inside the microcapsules (Fig. 3f,g). By analyzing the spherical microcapsule percentage (SMP, defined as the ratio of the number of spherical microcapsules to the total number of microcapsules) over time, a sigmoidal curve was obtained that reflects the kinetics of glucose diffusion (Fig. 3h)31–33. The diffusion rate of glucose could be well controlled by varying the concentration of GO in the extrusion droplet. The lower the concentration of GO, the faster the diffusion rate. These results demonstrated that the incorporation of GO significantly reduced the porosity of the membrane (in the following discussions, the GO concentration in the extrusion droplets was fixed at 0.2 wt% unless otherwise noted). When dispersing microcapsules in a hypertonic solution of dextran, a much longer time was required for the transmembrane diffusion of dextran, due to its larger hydrodynamic volume. Taking dextran with molecular weights of 100 and 500 kDa (termed Dex-100k and Dex-500k) as examples: for Dex-100k, the SMP reached 100% after 20 h, whereas for Dex-500k, the SMP only reached 40% after 40 h (Fig. 3i and Supplementary Fig. 21,22). By encapsulating FITC-labelled Dex-100k (Dex-100k-FITC) and rhodamine B isothiocyanate (RITC)-labelled Dex-500k (Dex-500k-RITC) in CNF-GO/PDDA microcapsules and aging for 8 h in water, CLSM was used to probe the distribution of dextran. The fluorescence intensity of Dex-100k was similar outside and inside the microcapsule, while the fluorescence intensity of Dex-500k inside the microcapsule was stronger than outside, confirming the selective permeability of CNF-GO/PDDA membrane (Fig. 3j,k).

In addition to neutral glucose and dextran, we further investigated the effects of hypertonic solutions of polyanions and polycations on CNF-GO/PDDA microcapsules. For the polyanion PAA-2k, CNF-GO/PDDA microcapsules exhibited behavior similar to that of CNF/PDDA microcapsules but with reduced collapse, indicating enhanced mechanical strength of CNF-GO/PDDA microcapsules due to the incorporation of GO nanosheets (Fig. 4a,b and Supplementary Fig. 14). By dispersing CNF-GO/PDDA microcapsules in an aqueous solution of the polycation PDDA (PDDA-100k, Mw: 100 ~ 200 kDa), shrinkage of microcapsules was observed. However, when redispersed in water, the microcapsules remained shriveled and lost the ability to undergo elastic recovery (Fig. 4c,d). This could be attributed to the transmembrane diffusion of PDDA-100k, which crosslinked the CNF/GO inside the microcapsules, thereby inhibiting the swelling of microcapsules induced by osmotic pressure. A control experiment using higher molecular weight PDDA (PDDA-400k, Mw: 400 ~ 500 kDa) showed typical shrink-swell behavior. Due to its larger hydrodynamic volume, PDDA-400k was unable to penetrate the membrane to crosslink the interior of the microcapsules, allowing microcapsules to swell upon redispersion in water. Notably, compared to their initial state, the surface of microcapsules produced by secondary swelling exhibited discontinuous dark microdomains, suggesting a certain degree of cross-linking between PDDA-400k and the membrane (Fig. 4e,f and Supplementary Fig. 23). When polyethyleneimine with very low molecular weight of 0.6 kDa (PEI-0.6k) was used, an interesting aggregation behavior was observed inside the microcapsules (Fig. 4g,h and Supplementary Fig. 24). PEI-0.6k was able to diffuse into the microcapsules and electrostatically interact with CNF/GO. However, in contrast to PDDA-100k, which bore permanently charged quaternary ammonium groups, the protonatable amine groups along the PEI-0.6k chains provided weaker electrostatic interactions. In addition, the much lower molecular weight and shorter chain length of PEI-0.6k limited the spatial extent of electrostatic crosslinking with CNF/GO, resulting in a more localized and mechanically weaker network. Consequently, the PEI-0.6k-induced crosslinked structure was unable to resist osmotic swelling upon redispersion of microcapsules in pure water, leading to network disruption, fragmentation, and the formation of small fragments that gradually associated into larger aggregates. Using FITC-labelled PEI-0.6k as a fluorescent probe and characterizing the aggregates by FRAP, we observed almost no fluorescence recovery after photobleaching, indicating the limited mobility of PEI (Supplementary Fig. 25).

Chemical communication in CNF-GO/PDDA protocells

Having established the membrane permeability of CNF-GO/PDDA microcapsules, we employed an enzymatic cascade to demonstrate the biomimetic potential of this protocell system. Two enzymes, horseradish peroxidase (HRP) and glucose oxidase (GOx), were incorporated into the CNF-GO/PDDA protocells by mixing them into the extrusion droplets. Due to the negative charge of both HRP and GOx under neutral conditions, they participated in membrane formation via DIC, as evidenced by the strong fluorescence signals of FITC-labelled HRP and RITC-labelled GOx on membrane (Fig. 5a,b). HRP and GOx were also distributed inside the protocells, with a lower content of HRP detected. Compared to GOx, HRP contained more hydrophobic groups, enhancing its interfacial activity at the air–water interface and facilitating the DIC process, similar to the case of GO-containing protocells34. CNF-GO/PDDA protocells effectively retained HRP and GOx, with no leakage observed for 5 days (Supplementary Fig. 26). The enzymatic cascade was triggered by the addition of glucose and benzoyl leuco methylene blue (BLMB) to the continuous phase (Fig. 5c). Glucose diffused into the protocells and was catalyzed by GOx to release hydrogen peroxide (H2O2) first; subsequently, the non-fluorescent BLMB that had diffused into the protocells was oxidized by H2O2 under HRP catalysis, producing fluorescent methylene blue (MB). CLSM imaging showed that MB initially concentrated near the membrane due to enzyme enrichment at membrane, and gradually diffused into the interior and exterior of the protocells over time (Fig. 5d,e). To investigate the chemical signaling between protocells, HRP and GOx were separately loaded in two populations of CNF-GO/PDDA protocells. In the cascade reactions, H2O2 was produced by GOx-containing protocells, then diffused into HRP-containing protocells, where it triggered HRP-catalyzed oxidation of BLMB to MB (Supplementary Fig. 27a). Tracking the fluorescence signal of MB showed that MB first appeared in the region of HRP-containing protocells, before gradually diffusing to the region of GOx-containing protocells (Supplementary Fig. 27b). In comparison to protocells co-encapsulating HRP and GOx, the reaction kinetics were slower due to the longer diffusion time of chemicals between different protocells.

One-step construction of 3D prototissues by protocells

The robustness and oil-free production of CNF-GO/PDDA protocells enabled the construction of prototissues. Here, we propose a straightforward strategy to achieve the assembly of protocells by adding 0.3 M NaCl into the extruded aqueous phase. As the droplets entered the collection bath to form CNF-GO/PDDA protocells, they were not dispersed in water but instead adhered to each other, creating a self-supporting tissue-like structure at the centimeter scale, which could be further transferred to air. The protocells exhibited sufficiently strong adhesion, enabling the prototissue to withstand needle-induced disturbances without disintegration (Fig. 6a and Supplementary Movie 4). Freeze-drying the CNF-GO/PDDA prototissue yielded a fully dehydrated, tissue-like material that retained its structural integrity and was capable of supporting external loads (Supplementary Fig. 28). The constructed conical prototissue could be scaled up to a height of > 6.5 cm and a diameter of > 5 cm, comprising > 20,000 protocells (Fig. 6b).

The primary mechanism underlying microcapsule-microcapsule adhesion should be attributed to the electrolyte-induced screening of electrostatic repulsion35. Without electrolyte, CNF/GO and PDDA assembled into well-stratified structures via strong electrostatic interactions, with a predominantly positively charged outer surface and a negatively charged interior. This charge asymmetry led to strong electrostatic repulsion between individual microcapsules, preventing adhesion. Upon addition of electrolyte, increased ionic strength weakened the electrostatic complexation between CNF/GO and PDDA, thereby enhancing the mobility of components within the membrane and leading to the formation of a more homogeneous membrane. This membrane contained a uniform distribution of positive and negative charges, enabling microcapsules to adhere upon contact. Meanwhile, the weakened electrostatic interactions also promoted interdiffusion between CNF/GO and PDDA during complexation, which may lead to membrane thickening. Using CNF-FITC as a fluorescent probe, CLSM imaging visualized the thickened CNF-GO/PDDA membranes and protocell adhesion (Fig. 6d and Supplementary Fig. 29). SEM analysis of a freeze-dried prototissue demonstrated that the protocells were tightly interconnected via CNF/GO networks (Supplementary Fig. 30).

To further clarify the role of ionic strength in microcapsule adhesion, CNF-GO/PDDA prototissues were prepared at different NaCl concentrations (Supplementary Fig. 31). At low NaCl concentrations (0.1 M), relatively strong electrostatic repulsion prevented microcapsule adhesion and prototissue formation. Stable prototissues were formed at intermediate NaCl concentrations (0.2 and 0.3 M), where effective microcapsule adhesion occurred. At higher NaCl concentration (0.4 and 0.5 M), electrostatic interactions between CNF/GO and PDDA were excessively weakened, leading to extensive interdiffusion between the CNF/GO-containing extruded aqueous phase and the PDDA-containing collection bath, thereby inhibiting the formation of stable complex membranes and microcapsules. In control experiments, 2D macroscopic CNF-GO/PDDA membranes were prepared under different NaCl concentrations and characterized by atomic force microscopy (AFM). The results showed that the membrane thickness increased with higher NaCl concentrations, confirming the proposed mechanism (Supplementary Fig. 32). Beyond NaCl, other electrolytes, including KCl, CaCl2, MgCl2, Na2SO4, as well as neutral or basic buffer systems were also effective in regulating protocell adhesion (Supplementary Fig. 33).

Having achieved the stable construction of macroscopic prototissues, we investigated their osmotic responsiveness in polyanion solutions to determine whether the behavior of individual protocells could be integrated into the prototissues. By immersing the conical prototissue composed of CNF-GO/PDDA protocells into a polyanion solution of 1 wt% PAA-2k, a pronounced volume shrinkage was observed within 30 min due to water loss from the individual protocells (volume shrinkage ratio: ~ 45%, calculated as 1 − V/V₀ based on cone volume before and after shrinkage). Upon replacing the PAA-2k solution with deionized water, the prototissue reabsorbed water and recovered to its original volume within 30 min as a result of decreased osmotic pressure (Fig. 6e and Supplementary Movie 5). When the concentration of PAA-2k was further increased to 3 wt% and 5 wt%, the elevated osmotic pressure led to more significant water loss from the prototissue, with shrinkage ratios reaching 60% and 80%, respectively. Owing to the robust nature of CNF-GO/PDDA protocells, the prototissue was able to undergo multiple shrinkage–swelling cycles without rupture or disintegration (Fig. 6f).

The integration of gas-liquid microfluidic technology with molding enabled the precise 3D construction of prototissues. Patterned molds were pre-positioned in the collection bath, and protocells were deposited into the molds through manual control of the needle during the gas-spraying process (Fig. 6g and Supplementary Movie 6). After demolding, prototissues with well-defined shapes such as humanoid, cloverleaf, and Mickey Mouse-like geometries were readily obtained. The molded prototissues remained intact under vigorous agitation, further confirming the strong inter-protocell adhesion between protocells (Supplementary Fig. 34 and Supplementary Movie 7). Moreover, by alternately stacking CNF-GO/PDDA and CNF/PDDA protocells, a Janus-type starfish-shaped prototissue was fabricated (Fig. 6i). When placed in a 1 wt% PDDA-100k aqueous solution, both layers of the prototissue experienced water loss and shrinkage. However, due to the incorporation of GO nanosheets, the bottom layer composed of CNF-GO/PDDA protocells exhibited higher mechanical strength and resisted deformation under osmotic pressure. In contrast, the top layer composed of CNF/PDDA protocells, with a softer membrane structure, collapsed more significantly upon dehydration (Supplementary Fig. 35). Owing to their different mechanical properties and osmotic responsiveness, the two layers underwent unequal magnitudes and rates of volume contraction under the same hypertonic conditions, thereby driving the bending of bilayer structure (Fig. 6j,k and Supplementary Movie 8).

Programmable chemo-mechanical transduction of prototissues

By mixing 0.02 wt% catalase into the extruded aqueous phase to generate CNF-GO/PDDA protocells, we employed enzymatic reactions to demonstrate the lifelike potential of protocells and prototissues33. For individual protocells, CLSM imaging confirmed that FITC-labelled catalase was distributed both on the membrane and inside the protocells, suggesting its involvement in membrane formation via DIC, owing to its negative charge under neutral conditions (Fig. 7a). Upon the addition of hydrogen peroxide (H2O2, 0.3 wt%) to the surrounding water, oxygen bubbles were generated inside the protocells and as oxygen accumulated, the resulting buoyant force overcame gravity, causing the protocells to rise (Fig. 7b,c and Supplementary Movie 9). Prototissues exhibited similar buoyancy-driven motion, which could be harnessed for chemo-mechanical transduction (Fig. 7d and Supplementary Movie 10). By varying the concentration of H2O2 or catalase, the buoyancy velocity of prototissues could be tuned (Supplementary Fig. 36).

By assembling catalase-loaded and unloaded CNF-GO/PDDA protocells into a single prototissue, we investigated the programmable chemo-mechanical transduction and complex motion behavior36–38. A strip-shaped, unloaded prototissue was first fabricated via molding. When catalase-loaded protocells were deposited at one end, the subsequent addition of H2O2 to the surrounding water led to localized oxygen generation, resulting in asymmetric buoyancy and upward bending of the prototissue (Fig. 7e and Supplementary Movie 11). When catalase-loaded protocells were deposited at both ends or in the middle of the prototissue, different bending behaviors were observed, forming either concave or convex shapes depending on the location of buoyancy (Fig. 7f, Supplementary Fig. 37 and Supplementary Movie 12,13). Furthermore, by tuning the catalase concentrations in the protocells at both ends (0.2 wt% on the left and 0.1 wt% on the right), 4D motion/deformation could be achieved, where asymmetric reaction rates caused one end to rise earlier than the other (Fig. 7g and Supplementary Movie 14). A cross-shaped prototissue was also fabricated, with catalase-loaded protocells deposited at its four ends. Upon exposure to H2O2, buoyancy induced upward bending of the four arms, transforming the prototissue into a cage-like structure capable of capturing hollow plastic spheres (Supplementary Movie 15). By further incorporating protocells containing NdFeB ferromagnetic microparticles into the central region, the resulting soft gripper could be directed to translate and rotate under an external magnetic field (Fig. 7h-k and Supplementary Movie 16).

Finally, we investigated a buoyancy-driven oscillatory system. A small CNF–GO/PDDA prototissue encapsulating 0.1 wt% catalase and 0.1 wt% GOx was prepared and placed in a water-filled glass tube (diameter = 2 cm, Fig. 7l). An agarose hydrogel containing 3 wt% H2O2 was fixed at the bottom of tube, while an agarose hydrogel containing 18 wt% glucose was fixed at the top. H2O2 and glucose gradually diffused from the hydrogels into the surrounding water. When the prototissue approached the bottom H2O2 reservoir, catalase decomposed H2O2 into O2, generating gas bubbles inside the protocells and increasing buoyancy, which caused the prototissue to rise. Upon reaching the top glucose reservoir, GOx consumed the encapsulated O2, leading to a decrease in buoyancy and causing the prototissue to sink back toward the bottom. In this manner, the prototissue underwent repeated up-down motion between the two agarose hydrogels until the H2O2 and glucose were largely depleted, after which the prototissue gradually ceased motion and settled at the bottom. Under the experimental conditions used, the prototissue exhibited three reproducible oscillation cycles (Fig. 7m,n and Supplementary Movie S17).

Methods
Materials

All materials were used without further purification. Cellulose nanofibril (CNF) aqueous dispersion (1.0 wt%, from cotton) was purchased from Guilin Qihong Tech. Ltd. Poly(diallyldimethylammonium chloride) (PDDA, 20 wt% aqueous solution, Mw: 100-200 kDa, 200-350 kDa, 400-500 kDa) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The hydrodynamic sizes of three PDDA samples, measured by dynamic light scattering in dilute aqueous solution, are ~ 23.4, 29.4, and 40.5 nm, respectively. Triton X-100 (CBR), hydrogen peroxide (H2O2) (30 wt% aqueous solution), N-(1-Pyrenyl) maleimide (≥ 98%), thionyl chloride (≥ 99%), L-Cysteine (≥ 98%), triethylamine (≥ 98%), and polyethyleneimine (Mw: 600 Da) were purchased from Aladdin. Fluorescein 5(6)-isothiocyanate (FITC, ≥ 96%, mixture of 5- and 6- isomers), rhodamine B isothiocyanate (RITC, ≥ 96%, mixture of isomers), dextran (Mw: 10 kDa, 40 kDa, 70 kDa, 100 kDa, 500 kDa), tris(2-carboxyethyl)phosphine (TCEP, ≥ 98%), and poly(acrylic acid) (PAA, 40 wt% aqueous solution, Mw: 2 kDa) were purchased from J&K. β-D-glucose (≥ 99%) was purchased from TCI. Graphene oxide (GO) was purchased from XFNANO. Catalase (1000 U mg-1), carbonate buffer (0.1 M, pH 9.2), PBS buffer (0.1 M, pH 7.4), and agarose (≥ 99%) were purchased from Shanghai Yuanye. Sodium chloride (≥ 99.5%), potassium chloride (≥ 99.5%), calcium chloride (≥ 96%), magnesium chloride (≥ 98%), and anhydrous sodium sulfate (≥ 99%) were purchased from Fuchen Chemical. Milli-Q water (18.2 MΩ∙cm) was used in the preparation of all aqueous solutions.

Characterizations

The preparation, self-supporting, and chemo-mechanical transduction behaviors of protocells and prototissues were recorded as images or movies with a digital camera (SONY Alpha 6400 equipped with Tamron 17-70 mm F/2.8). The morphology and deformation of protocells were characterized by polarized optical microscopy (ZEISS Imager.A2). The surface characteristics of protocells and prototissues were characterized using scanning electron microscopy (SEM, JSM-7900F, JEOL). 2D films were characterized by atomic force microscopy (AFM, Bruker-Fastscan DMFASTSCAN2-SYS). The fluorescence images were obtained using a Leica SP8 laser scanning microscope attached to a Leica DMi 8 inverted fluorescence microscope. The following emission wavelengths (λem) were monitored for the corresponding probes: FITC λem = 525 nm, RITC λem = 566 nm, Pyrene λem = 485 nm. Image analysis was performed with ImageJ software. The fluorescence recovery after photobleaching (FRAP) assays were conducted using a FRAP module. One pre-bleach frame (1024 × 1024 pixels) of the labeled CNF/PDDA microcapsule was first recorded at 5% laser power with a 525-nm laser. Afterward, bleaching was performed for 400 s at 100% power of the 525-nm laser by defining the specified region in the FRAP module. The fluorescence recovery was recorded for 300 post-bleach frames (each frame with an interval of 4 s) at 5% laser power with the 525-nm laser. The intensity of fluorescence was determined by LASX software (Leica). Fourier transform infrared (FTIR) spectra of GO and GO-Pyrene were recorded on a Nicolet 6700 FTIR spectrometer. Raman spectra were acquired on a Renishaw inVia Reflex Raman microscope using a 514 nm excitation laser. Hydrodynamic diameters of PDDA samples were measured by dynamic light scattering (DLS) on a Malvern Zetasizer Nano ZS90 at 25 °C. X-ray photoelectron spectroscopy (XPS) measurements were performed on a Thermo Scientific ESCALAB 250 spectrometer using Al Kα radiation.

Preparation of protocells and prototissues

For the preparation of protocells, typically, an aqueous phase of 0.6 wt% CNF (or 0.6 wt% CNF + 0.2 wt% GO) was pumped through the inner needle of a coaxial needle at a flow rate of 30 mL h⁻¹ using a syringe pump. Compressed nitrogen gas, regulated by a rotameter to a flow rate of 1.0 L min⁻¹, was connected to the outer needle of the coaxial setup. The shear force exerted by the outer gas flow fragmented the extruded phase into uniform microdroplets. These microdroplets subsequently fell into the aqueous collecting bath containing 0.5 wt% PDDA and 0.1 wt% Triton X-100 to form CNF/PDDA microcapsules (or CNF-GO/PDDA microcapsules). The produced microcapsules were transferred into water and washed thoroughly, with the water being replaced repeatedly over 10 cycles to ensure complete removal of residual components.

For the preparation of prototissues, an aqueous phase containing 0.6 wt% CNF (or 0.6 wt% CNF + 0.2 wt% GO) and 0.3 M NaCl was extruded into an aqueous collection bath containing 0.5 wt% PDDA and 0.1 wt% Triton X-100 to produce CNF/PDDA microcapsules (or CNF-GO/PDDA microcapsules) (extrusion rate = 30 mL h-1, gas flow rate = 1.0 L min-1). In the absence of molds, when the needle position was fixed, the microcapsules settled to the bottom of collection bath, where they spontaneously accumulated and adhered to one another to form a macroscopic, self-supporting conical prototissue. Arbitrary shaped prototissues could also be constructed by manually moving the needle during gas-spraying, but the precision of resulting shapes was relatively limited. When molds were used, a patterned mold was pre-positioned in collection bath, and microcapsules were deposited into the mold by manually moving the needle during gas-spraying process, where they adhered to each other. After depositing the required amount of microcapsules, the mold was lifted to yield prototissues that faithfully replicated the mold geometry. Regardless of mold use, the constructed prototissues were transferred to deionized water and washed by repeated water replacement for 10 cycles to remove residual components.

All patterned molds used in this work were commercially fabricated open-frame molds, consisting of a hollow frame with a defined height and open top/bottom. The molds can be made from polytetrafluoroethylene (PTFE), polypropylene (PP), polystyrene (PS), high-density polyethylene (HDPE), stainless steel, aluminum alloy, or hydrophobically modified glass. Owing to the weak interfacial interactions between these mold materials and prototissues, demolding was readily achieved by simply lifting the mold, allowing the prototissues inside to retain their shape intact.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors present a novel diffusion-inhibited complexation strategy that enables low-cost, high-throughput fabrication of protocells and precise, large-scale 3D construction of prototissues. This simple, fully aqueous method yields protocells and prototissues with excellent mechanical strength. The resulting prototissues reach sizes exceeding 10 cm, significantly larger than the millimeter-scale structures reported previously, addressing a long-standing challenge in the field. The permeability and chemical communication of the protocells, as well as the osmotic-pressure-induced deformation and buoyancy-driven motion of the prototissues were thoroughly investigated. This work provides a promising platform for future applications in areas such as soft robotics, biomedical devices and tissue engineering. Therefore, I recommend the publication of the manuscript in Nature Communications after minor revisions.\n1. The system used by the authors appears to be quite different from conventional aqueous two-phase systems (ATPS), such as the widely studied PEG-dextran system. In comparison to ATPS, what are the advantages of achieving compartmentalization in pure water? It would also be helpful if the authors could clarify whether spontaneous phase separation occurs upon direct mixing of CNF and PDDA aqueous solutions.\n2. While the effects of gas flow rate and extrusion rate on the size of the microcapsules have been investigated. It is recommended to assess the influence of CNF and PDDA concentrations on microcapsule formation and size.\n3. The prepared prototissues exhibit excellent mechanical strength in both water and air, which is very impressive. Would the prototissues still maintain their structural stability after complete removal of water from both the internal and external prototissues?\n4. The pH values of both CNF and PDDA solutions should be stated in the manuscript, as the pH is a critical role in the strength of electrostatic interactions between CNF and PDDA. In addition, the influence of pH on microcapsule formation is recommended to be explored.\n5. Morphological characterization of CNF such as AFM or TEM should be provided.", "reviewer_2": "In this work, Weixiao Feng et al. present a microfluidic method to construct protocells and assemble prototissues with complex 3D architecture. While the methodology is novel and interesting, several aspects of its functioning principles remain unclear and require more thorough investigation before publication. Moreover, beyond the novelty of the production method for protocells and prototissues, the work does not present a significant conceptual advancement in the protocell/prototissue engineering field, as it primarily reproduces results that have already been demonstrated by established leaders in the field such as Bayley, Mann, and Han. Given the limited conceptual progress, issues with the methodology's working principles, and inadequate scholarly presentation, I do not recommend publication of this manuscript in Nature Communications.\nBelow are specific comments that may help improve the rigor and significance of the work:\nMaterials Characterization and Methodology\n1. Essential details regarding CNF and PDDA are missing, which is fundamental for result reproduction. The authors should report the molecular weight and dispersity of all polymers used, their hydrodynamic radius in the working solvent, and degree of branching. Additionally, why is cellulose negatively charged? Since cellulose and PDDA interact electrostatically, how many charges per polymer chain do they carry?\n2. The authors refer to their objects as \"microcapsules,\" but \"microspheres\" would be more appropriate since they are not hollow structures.\n3. The role of Triton-X should be clarified: what concentration was used, and what surface tension is required to achieve microcapsule formation? Can other surfactants be employed, particularly those that are completely biocompatible?\n4. The statement \"By varying extrusion rate or gas flow rate, the size of microcapsule could be controlled\" contradicts Supplementary Figure 3a, which clearly shows that extrusion rate does not influence microcapsule size. This discrepancy should be corrected.\n5. The microcapsule structure requires further exploration. Where does PDDA localize? Does it permeate and form a network inside the microsphere, which could explain their resilience and reduced polymer chain mobility?\nPermeability Studies\n6. The statement \"This suggested the poor semi-permeability of membrane, leading to a rapid equilibrium of osmotic pressure across the microcapsules\" lacks clarity. It is unclear how membrane permeability can be assessed by placing microcapsules in high-concentration glucose or dextran solutions and observing shrinkage behavior. This requires further explanation.\n7. For the permeability experiment using dextran (Supplementary Figure S7), the partition coefficient of FITC-tagged dextran should be provided. Complete characterization of the dextran (molecular weight, dispersity, degree of labeling) is also necessary. Importantly, a molecular weight cut-off for the microspheres should be estimated.\n8. There is confusion regarding the statement \"It should be noted that if the microcapsules were sufficiently rigid, the shrinkage induced by the hypertonic solution could be suppressed\" when earlier the authors mention \"no shrinkage of microcapsules was observed within 24 h.\" This apparent contradiction requires clarification.\nCharacterization Issues\n9. The authors tagged GO with pyrene but did not explain the attachment method or characterize the product. How can they confirm that pyrene is covalently bound rather than physisorbed on GO, which might detach in complex solutions containing diverse polymers and salts? Control experiments are required to validate the conclusions regarding GO localization in microspheres.\n10. There is an apparent contradiction regarding GO's effect on permeability. The authors report that GO hinders glucose permeability, with smaller GO amounts leading to faster glucose permeation. However, in earlier experiments (lines 106107), they state that permeability is poor in GO's absence. This needs clarification.\nCoacervate Formation and Prototissue Assembly\n11. In the discussion from line 213, when using polyethyleneimine, how can the authors confirm coacervate formation rather than simple polymer aggregates formed by electrostatic interactions in a molecularly crowded environment? This claim requires supporting data. Additionally, what is an \"egg-shaped coacervate\"?\n12. The prototissue preparation lacks sufficient detail. The Materials and Methods section only states: \"For the preparation of prototissues, 0.3 M electrolyte (e.g., NaCl) was added into the extrusion solution, while other parameters remained unchanged.\" No explanation of how different shapes are achieved is provided. The term \"extrusion droplets\" requires definition. Details about the molds (material, fabrication process, filling method, removal procedure) are absent.\n13. The nature of microsphere-microsphere adhesion remains unclear. Why does a thicker, more relaxed membrane promote adhesion? While the authors attempt an explanation, this fundamental aspect requires precise understanding supported by experimental data.\nCharacterization and Applications\n14. The authors demonstrate various possibilities that have already been reported in the literature using their new prototissue types but provide insufficient characterization. Detailed mechanical property analysis of protocells and prototissues is necessary to support claims about mechanical properties and bending mechanics of layered prototissues. What forces are exerted during bending?\n15. For the buoyant prototissue: what is the intended purpose and biological relevance? What is the buoyancy rate and its dependence on experimental parameters?\nBiocompatibility Claims\n16. The authors claim biocompatibility in their conclusions without providing supporting data. Triton-X usage could be problematic in this regard. While an in vitro cytotoxicity assay is mentioned in the experimental section, no data are presented in the main text.\nRecommendation\nThe manuscript requires substantial improvements in experimental design, data presentation, and mechanistic understanding before it can meet the publication standards of Nature Communications.", "reviewer_3": "The authors describe the construction of prototissues via adhesive interaction between microgel compartments fabricated from PDDA and CNF. The membrane permeability of the compartments were studied and modulated by inclusion of GO. They also illustrated how osmotic changes can help regulate the morphology of the microgel compartments irreversibly via cross linking of the interior or reversibly via delayed permeability. They further went on to study a two-enzyme cascade within the CNF-GO/PDDA microcapsules and show chemomechanical actuation of the prototissues mediated by osmotic changes and bubble mediated buoyancy.\nThe comments are as follows:\n1) The primary concern is regarding novelty – as a significant part of the paper mostly deals with preparation of the protocells and their osmotic response along with enzyme cascade studies. These are routine studies in protocell field and the authors do well to thoroughly characterize their new protocell model/chasis but these do not add significant novelty to the work.\n2) The advantage of a prototissue is that it has compartments which can host a reaction to induce morphological or functional output. In the current design, the authors have used the osmotic response of the protocells and bubble mediated buoyancy to produce actuation. The osmotic pressure mediated actuation due to differential response in the two layer tissue was also illustrated in earlier studies (G. Villar et al. Science 2013, 340, 48-52). The buoyancy-based actuation of prototissues which the authors have shown is quite interesting as it is mediated by reactions within the protocells. However, this reviewer would like to see not just actuation but also recovery and re-actuation over multiple cycles which would then constitute a significant advance in the field.\n3) One suggestion would be to use Glucose oxidase mediated consumption of the oxygen bubble to execute multiple cycles of actuation or formulate a different strategy for dissolution of the bubble for recovery of the actuated tissue.\n4) The authors have studied the shrinkage of the CNF-GO/PDDA protocells with PEI and PDDA. It is seen that PDDA 100 kDa permeates and cross links but does not form a yolk structure but PEI forms a yolk structure which constitutes a highly cross linked network. Author could conduct FRAP studies to understand the condensation mediated changes in fluidity of the interior and also clarify the why there is difference in morphological outcomes for PEI and PDDA.\n5) the facile construction of the prototissue due to sticking interactions at high electrolyte concentrations has the limitation of lacking programmability of the internal structure of the prototissue – in terms of self organization of different protocells to form complex hierarchical structures. This reviewer would like to see the potential of this prototissue design in terms of soft robotics, where these prototissues can manipulate macroscopic objects or these actuatable tissues can be used as artificial muscle to operate a gripper like structure. These experiments would add significant novelty to the manuscript.\n6) Another general comment would be that it may be investigated what is the advantage of using a prototissue over an actuatable gel for carrying out these tasks."}, "author_response": "Manuscript ID: NCOMMS-25-49805A-Z\nTitle: One-Step Construction of Robust Protocells and Prototissues in Water\nAuthors: Weixiao Feng, Peifan Li, Xin Li, Ziwei Wang, Min Chen, Yang Hu, Fu-Jian Xu*, Shaowei\nShi*\nDear editor:\nWe would like to thank the reviewers for their detailed feedback to improve our submission. We\nhave considered each comment carefully and revised our manuscript to address the issues raised.\nResponses to Reviewer #1\nReviewer #1 (Remarks to the Author):\nIn this study, the authors present a novel diffusion-inhibited complexation strategy that enables low-\ncost, high-throughput fabrication of protocells and precise, large-scale 3D construction of\nprototissues. This simple, fully aqueous method yields protocells and prototissues with excellent\nmechanical strength. The resulting prototissues reach sizes exceeding 10 cm, significantly larger\nthan the millimeter-scale structures reported previously, addressing a long-standing challenge in the\nfield. The permeability and chemical communication of the protocells, as well as the osmotic-\npressure-induced deformation and buoyancy-driven motion of the prototissues were thoroughly\ninvestigated. This work provides a promising platform for future applications in areas such as soft\nrobotics, biomedical devices and tissue engineering. Therefore, I recommend the publication of the\nmanuscript in Nature Communications after minor revisions.\n1. The system used by the authors appears to be quite different from conventional aqueous two-\nphase systems (ATPS), such as the widely studied PEG-dextran system. In comparison to ATPS,\nwhat are the advantages of achieving compartmentalization in pure water? It would also be helpful\nif the authors could clarify whether spontaneous phase separation occurs upon direct mixing of CNF\nand PDDA aqueous solutions.\nResponse: We appreciate the comment from the reviewer. Conventional ATPS is a mixture of two\npolymers, or a polymer and a salt, which phase-separates to form two immiscible aqueous phases. To\nachieve stable compartmentalized microcapsules, it is usually necessary to introduce additional solid\nparticles or oppositely charged polyelectrolytes to stabilize the interface (Langmuir 2012, 28, 5921-\n5926; ACS Macro Lett. 2016, 5, 666-670). In contrast, our system enables the one-step fabrication of\nmicrocapsules based on two aqueous solutions using the DIC method. This strategy\ngreatly simplifies the preparation process, requires fewer components, and reduces overall cost.\nWe compared the mixing behaviors of the dextran-PEG ATPS and the CNF-PDDA DIC system.\nAs shown in Fig. R1, in the ATPS, mixing 20 wt% dextran solution with 15 wt% PEG solution results\nin spontaneous separation into immiscible dextran-rich and PEG-rich phases. After vigorous shaking\nto homogenize the mixture, it re-separates into two phases to minimize interfacial energy. In the DIC\nsystem, when 0.5 wt% PDDA solution is brought into contact with 0.8 wt% CNF solution,\nelectrostatic complexation occurs instantaneously, forming a thin film that suppresses further\ndiffusion and mixing. After vigorous shaking, the system undergoes polyelectrolyte complex\nflocculation rather than forming the two aqueous phases observed in the ATPS, indicating that\ncompartmentalization in our system originates from interfacial film formation rather than from bulk\nphase separation.\nFig. R1. Mixing behaviors of (a) dextran-PEG ATPS and (b) CNF-PDDA DIC system. Scale bar,\n0.5 cm.\n2. While the effects of gas flow rate and extrusion rate on the size of the microcapsules have been\ninvestigated. It is recommended to assess the influence of CNF and PDDA concentrations on\nmicrocapsule formation and size.\nResponse: We appreciate the comment from the reviewer. We systematically investigated the\ninfluence of CNF and PDDA concentrations on microcapsule formation at a fixed extrusion rate of\n30 mL h-1 and a compressed gas flow rate of 1.0 L min-1. As shown in Fig. R2, when the CNF\nconcentration is fixed, increasing the PDDA concentration raises the viscosity of the collection bath,\nwhich prevents CNF-containing droplets from entering the bath smoothly and thus hinders\nmicrocapsule formation. On the other hand, at a fixed PDDA concentration, increasing the CNF\nconcentration enhances the mechanical strength of microcapsules, thereby facilitating their\nformation. Within the concentration range that yields microcapsules, the size remains nearly constant\nat approximately 800 μm, indicating that concentration does not significantly influence microcapsule\ndimensions.\nWe have added Fig. R2 in the revised Supplementary Information as Supplementary Fig. 6, along\nwith a brief discussion.\nFig. R2. Influence of CNF and PDDA concentrations on microcapsule formation. [Triton X-100] =\n0.1 wt%; extrusion rate = 30 mL h-1; gas flow rate = 1.0 L min-1.\n3. The prepared prototissues exhibit excellent mechanical strength in both water and air, which is\nvery impressive. Would the prototissues still maintain their structural stability after complete\nremoval of water from both the internal and external prototissues?\nResponse: We appreciate the reviewer’s comment. As shown in Fig. R3, freeze-drying the CNF–\nGO/PDDA prototissues yields a highly dehydrated, tissue-like material that retains its structural\nintegrity. Even under a 200 g load, the structure remains intact, demonstrating the excellent\nmechanical stability of the prototissue.\nWe have added a brief discussion in the revised manuscript as follows: “Freeze-drying the CNF–\nGO/PDDA prototissue yielded a fully dehydrated, tissue-like material that retains its structural integrity\nand was capable of supporting external loads”. Fig. R3 has been included in the revised\nSupplementary Information as Supplementary Fig. 27.\nFig. R3. Digital images showing the excellent mechanical stability of freeze-dried CNF–GO/PDDA\nprototissue. Scale bar, 1 cm.\n4. The pH values of both CNF and PDDA solutions should be stated in the manuscript, as the pH is\na critical role in the strength of electrostatic interactions between CNF and PDDA. In addition, the\ninfluence of pH on microcapsule formation is recommended to be explored.\nResponse: We appreciate the comment from the reviewer. We have stated the pH values in the revised\nSupplementary Information, where the 0.6 wt% CNF and 0.5 wt% PDDA solutions exhibit pH values\nof approximately 7.0 and 5.0, respectively. In fact, we did not intentionally adjust the pH of either\nsolution during the experiments. We fully agree with the reviewer that pH plays a critical role in\ngoverning the strength of electrostatic interactions between CNF and PDDA, and we have\nsystematically investigated the influence of pH (1.0-13) on microcapsule formation. As shown in Fig.\nR4, microcapsules can be successfully produced over a wide pH range. At extremely low pH (pH =\n1.0), however, extensive protonation of the carboxyl groups on CNF weakens electrostatic interactions\nwith PDDA, resulting in failure of microcapsule formation. Within the pH range that yields\nmicrocapsules, the capsule size remains nearly constant at approximately 800 μm, indicating that pH\ndoes not significantly influence microcapsule dimensions.\nWe have added Fig. R4 in the revised Supplementary Information as Supplementary Fig. 7, along\nwith a brief discussion.\nFig. R4. Influence of pH on microcapsule formation. [CNF] = 0.6 wt%; [PDDA] = 0.5 wt%;\nextrusion rate = 30 mL h-1; gas flow rate = 1.0 L min-1.\n5. Morphological characterization of CNF such as AFM or TEM should be provided.\nResponse: We appreciate the comment from the reviewer, and have provided TEM characterization\nof CNF. As shown in Fig. R5, CNF is a fibrous, one-dimensional nanomaterial with diameters of\n10-20 nm and lengths of several micrometers. Fig. R5 has been included in the revised\nSupplementary Information as Supplementary Fig. 1.\nFig. R5. TEM images of CNF.\nResponses to Reviewer #2\nReviewer #2 (Remarks to the Author):\nIn this work, Weixiao Feng et al. present a microfluidic method to construct protocells and assemble\nprototissues with complex 3D architecture. While the methodology is novel and interesting, several\naspects of its functioning principles remain unclear and require more thorough investigation before\npublication. Moreover, beyond the novelty of the production method for protocells and prototissues,\nthe work does not present a significant conceptual advancement in the protocell/prototissue engineering\nfield, as it primarily reproduces results that have already been demonstrated by established leaders in\nthe field such as Bayley, Mann, and Han. Given the limited conceptual progress, issues with the\nmethodology's working principles, and inadequate scholarly presentation, I do not recommend\npublication of this manuscript in Nature Communications. The manuscript requires substantial\nimprovements in experimental design, data presentation, and mechanistic understanding before it can\nmeet the publication standards of Nature Communications.\nResponse: We appreciate the reviewer’s comment and recognition of our method for preparing\nprotocells and prototissues. As discussed in the introduction, beyond the novelty of the production\nmethod, this work also addresses several key challenges in this field, including the large-scale\npreparation of protocells/prototissues in aqueous media, achieving excellent mechanical strength and\nstability, and enabling precise shaping of prototissues. We fully acknowledge the pioneering\ncontributions of Bayley, Mann and Han, but we believe our study makes substantive progress on\nseveral long-standing issues. In the revised manuscript, we have added substantial experimental data\nand discussion, addressing all reviewer questions and including additional results such as the periodic\nmotion of prototissues, the preparation of gripper-like prototissues, and their ability to manipulate\nmacroscopic objects, which further strengthen the significance of our work. We hope that the revised\nmanuscript meets the reviewer’s high expectations.\nTo further clarify the novelty of our contribution, we summarize below several bottlenecks and\nchallenges identified in review articles by multiple groups, including Prof. Han’s.\nProf. Gobbo et al. (Biochem. Soc. Trans. 2020, 48, 2579–2589; Prof. Gobbo was a postdoctoral\nfellow in the research group of Professor Stephen Mann):\n “To date, prototissues have been assembled in the millimetre and submillimetre size range\nusing a variety of techniques...A materials chemistry approach would allow the advancement\nof current prototissue construction technologies and enable the precise assembly of protocells\ninto large-scale materials (>1 cm in size) that are robust, free-standing, and stable in water\nmedia. This is critical for future technological applications of prototissues and would represent\na major achievement in the field.”\n• “To date, research has been focusing on the development of methodologies to assemble\nprotocells together in a controlled and reproducible manner. However, if we want to develop\nprototissue-based technologies, it is of paramount importance that we start advancing the\nprototissue’s mechanical properties. In this regard, current studies have been limited to the\ncharacterisation of the prototissue’s tensile strength and elastic modulus...It is now critical to\nconstruct protocellular materials with a range of mechanical properties that mimic those of\nliving tissues and perhaps even outperform them.”\nProf. Han et al. (Ac/v. Funct. Mater. 2024, 34, 2405823):\n• “Although prototissues with precise spatial structures can be assembled with micrometer and\nmillimeter ranges, they are still small compared to tissues for tissue transplantation. To\nassemble large-scale prototissues, the long-distance signaling, synergistic effects between\nartificial cells, and collective functions will face enormous challenges.”\nProf. Lin et al. (Ac/v. Healthcare Mater. 2025, 14, 2500376):\n• “Despite significant progress in the development of prototissues, several challenges remain... 2)\nNatural tissues exhibit a broad range of mechanical strength due to the ECM composition (e.g.,\ncollagen, elastin) and cellular interactions. The mechanical stability of prototissues or protocell\nassemblies is typically low; as such, the incorporation of synthetic hydrogels, scaffolds, or\nartificial ECM needs to be considered for structural support ...5) Engineering protocell\nassemblies into large-scale, functional 3D structures with batch-to-batch reproducibility remains\nchallenging. Current fabrication techniques, including microfluidics and 3D printing, struggle to\nprecisely control spatial organization at a tissue-like scale.”\nBelow are specific comments that may help improve the rigor and significance of the work:\nMaterials Characterization and Methodology\n1. Essential details regarding CNF and PDDA are missing, which is fundamental for result\nreproduction. The authors should report the molecular weight and dispersity of all polymers used,\ntheir hydrodynamic radius in the working solvent, and degree of branching. Additionally, why is\ncellulose negatively charged? Since cellulose and PDDA interact electrostatically, how many\ncharges per polymer chain do they carry?\nResponse: We appreciate the comment from the reviewer. CNF is not traditional linear polymer but\ncellulose-based polysaccharide nanomaterial that contain both crystalline and amorphous regions.\nTherefore, parameters such as molecular weight, dispersity, hydrodynamic radius, and degree of\nbranching are not appropriate descriptors for CNF. Here we characterized CNF using TEM. As\nshown in Fig. R6a,b, CNF are fibrous, one-dimensional nanomaterials with diameters of 10-20 nm\nand lengths of several micrometers. The negative charge of CNFs arises from the 2,2,6,6-\ntetramethylpiperidinyl-1-oxyl (TEMPO) oxidation process used in their preparation, during which a\nfraction of the primary C6 hydroxyl groups on the cellulose backbone is converted into carboxyl\ngroups (–COOH) (Biomacromolecules 2007, 8, 2485-2491; Nanoscale 2011, 3, 71-85). According\nto the supplier’s specifications, the carboxylate content of the CNFs is 1.2-3.0 mmol COOH per\ngram of CNF.\nPDDA, on the other hand, is a linear polymer without branching (Fig. R6c). In this work, three\nPDDA samples with different molecular weights (M : 100-200 kDa, 200-350 kDa, 400-500 kDa) were\nw\nused, all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Detailed information on\nmolecular weights is provided at the end of the reply letter. Because commercial PDDA typically has a\nbroad molecular-weight distribution, the dispersity is of limited significance and generally not reported\nby suppliers. The hydrodynamic sizes of PDDA in deionized water (25 oC) were measured by dynamic\nlight scattering (DLS) at 0.01 mg mL-1, yielding values of 23.4, 29.4, and 40.5 nm for the three samples\n(Fig. R7). Moreover, since each repeat unit carries one charged quaternary ammonium group, the\nnumber of charges per polymer chain corresponds to the degree of polymerization, where the weight-\naverage degrees of polymerization for the three PDDA samples are ~600-1200, ~1200-2200, and\n~2500-3100, respectively.\nWe have stated the molecular weights and hydrodynamic sizes of PDDA in Materials section.\nFig. R6 has been included in the revised Supplementary Information as Supplementary Fig. 1.\nFig. R6 a,b TEM images of CNF. c Chemical structure of PDDA.\nFig. R7 Characterization of PDDA hydrodynamic sizes in water using DLS.\n2. The authors refer to their objects as \"microcapsules,\" but \"microspheres\" would be more\nappropriate since they are not hollow structures.\nResponse: We appreciate the reviewer’s comment. We prefer the term “microcapsules” rather than\n“microspheres” because the internal and surface structures of the CNF (or CNF-GO)/PDDA\nassemblies differ significantly. Taking CNF/PDDA microcapsules as an example, CLSM and SEM\nimages show that CNF is mainly distributed at the surface and electrostatically complexes with\nPDDA to form a dense membrane, while the interior consists of a loose CNF network (Fig. R8a-d).\nAlthough this structure is not completely hollow, its shrinkage behavior under osmotic pressure\nclosely resembles that of conventional microcapsules.\nAs a comparison, we fabricated sodium alginate–Ca2+ hydrogel microspheres using a gas–liquid\nmicrofluidic approach by extruding sodium alginate droplets into a CaCl-containing aqueous bath (Adv.\nis labeled with rhodamine-B-isothiocyanate (RITC), CLSM shows uniform fluorescence throughout\nboth the interior and the surface, indicating a homogeneous structure. SEM images of\nfreeze-dried microspheres further confirm structural uniformity from surface to interior. These\nresults confirm that the assemblies formed from CNF (or CNF-GO) and PDDA are more accurately\nclassified as microcapsules rather than microspheres.\nFig. R8. a, CLSM images of a CNF/PDDA microcapsule containing CNF-FITC and corresponding\nfluorescence intensity profile (b, dashed line in a). Scale bar, 200 tim. c,d, SEM images of a ruptured,\nfreeze-dried CNF/PDDA microcapsule, showing the membrane (c, scale bar, 100 tim) and the internal\nfibrillar network (d, scale bar, 10 tim). e, CLSM image of Sodium alginate–Ca2+ hydrogel microspheres\ncontaining Alg-RITC and corresponding fluorescence intensity profile (f, dashed line in e). Scale bar,\n200 tim. g,h, Freeze-dried SEM images reveal a continuous, uniform gel network from surface to\ninterior. Scale bar in g, 100 tim, scale bar in h, 10 tim.\n3. The role of Triton-X should be clarified: what concentration was used, and what surface tension\nis required to achieve microcapsule formation? Can other surfactants be employed, particularly those\nthat are completely biocompatible?\nResponse: We appreciate the comment from the reviewer. Triton X-100 serves to reduce the air-water\nsurface tension of the collection bath, enabling the droplets to pass through the interface and undergo DIC\nto form microcapsules. Without Triton X-100, or when its concentration is very low, intact CNF/PDDA\nmicrocapsules cannot be produced. The concentration of Triton X-100 used in our work is 0.1 wt%. To\nclarify the relationship between surface tension and microcapsule formation, we measured the surface\ntension of Triton X-100 solutions at different concentrations and prepared microcapsules accordingly. As\nshown in Fig. R9, microcapsules can be successfully formed when the surface tension is below ~ 45 mN\n×\nm-1 (corresponding to a Triton X-100 concentration of ~ 6\n10-4 wt%). When the surface tension exceeds 45 mN m-1, CNF-containing droplets are unable to\npass through the interface and therefore fail to transform into microcapsules.\nIn addition to Triton X-100, we also tested four widely used, nonionic and biocompatible\nsurfactants,including Pluronic F68, Pluronic F127, Tween-20, and Solutol HS-15. All of them\nenabled successful microcapsule formation at a concentration of 0.1 wt%, indicating that the type\nof surfactant does not affect the ability to produce microcapsules (Fig. R10).\nWe have made a brief discussion in the revised manuscript as follows: “Triton X-100 was used to\nreduce the air-water surface tension of the collection bath, facilitating droplets passing through the\nsurface and transforming into microcapsules by DIC. Without Triton X-100, or when its\nconcentration was very low, the high interfacial tension prevented the formation of intact\nCNF/PDDA microcapsules. In addition to Triton X-100, surfactants including Pluronic F68,\nPluronic F127, Tween-20, and Solutol HS-15 also enabled successful microcapsule formation”.\nFig. R9 and Fig. R10 have been included in the revised Supplementary Information as\nSupplementary Fig. 3 and 5.\nFig. R9. Equilibrium air-water surface tension of the collection bath as a function of Triton X-100\nconcentration and the resulting effect on microcapsule formation.\nFig. R10. a-d Digital images showing the successful fabrication of CNF/PDDA microcapsules\nusing four different surfactants including Pluronic F68, Pluronic F127, Tween-20, and Solutol HS-\n15. [CNF] = 0.6 wt%; [PDDA] = 0.5 wt%; [Surfactant] = 0.1 wt%; extrusion rate = 30 mL h-1;\ngas flow rate = 1.0 L min-1. Scale bar, 1 cm.\n4. The statement \"By varying extrusion rate or gas flow rate, the size of microcapsule could be\ncontrolled\" contradicts Supplementary Figure 3a, which clearly shows that extrusion rate does not\ninfluence microcapsule size. This discrepancy should be corrected.\nResponse: We appreciate the comment from the reviewer. We have removed the inaccurate\ndescription from the manuscript and have made a brief discussion in the revised Supplementary\nInformation as follows: “At a fixed gas flow rate of 1.0 L min-1, varying the extrusion rate from 5.0\nto 50 mL h-1 produces microcapsules with nearly constant sizes of ~ 800 µm. In contrast, at a fixed\nextrusion rate of 30 mL h-1, increasing the gas flow rate from 0.8 to 1.6 L min-1 reduces the\nmicrocapsule size from ~1000 to ~ 400 µm. These results indicate that microcapsule size is\ngoverned primarily by the gas flow rate rather than the extrusion rate.”\n5. The microcapsule structure requires further exploration. Where does PDDA localize? Does it\npermeate and form a network inside the microsphere, which could explain their resilience and\nreduced polymer chain mobility?\nResponse: We appreciate the comment from the reviewer. PDDA is primarily localized at the\nmembrane of the microcapsules rather than within their interiors. To directly visualize the distribution\nof PDDA, we labeled PDDA with rhodamine-B-isothiocyanate (RITC) and used it to produce\nCNF/PDDA microcapsules. As shown in Fig. R11, the CLSM image reveals that PDDA is\npredominantly concentrated on the microcapsule surface and does not permeate or form a network\ninside the capsule. Therefore, the resilience of the microcapsules and the reduced polymer chain\nmobility arise mainly from the internal CNF network and the composite film formed between CNF\n(or CNF-GO) and PDDA.\nWe have added a brief discussion in the revised manuscript as follows: “Using rhodamine-B-\nisothiocyanate (RITC)-labelled PDDA (PDDA-RITC) as a fluorescent probe, it was observed that\nPDDA was fully concentrated on the membrane and did not permeate into the interior.” The synthesis\nof PDDA-RITC has been incorporated into the Supplementary Methods section (Synthesis of\nfluorescently labelled dextran, CNF, enzymes and PDDA). Fig. R11 has been included in the revised\nSupplementary Information as Supplementary Fig. 11.\nFig. R11. CLSM images of a CNF/PDDA microcapsule containing PDDA-RITC and corresponding\nfluorescence intensity profile along the dashed line. Scale bar, 200 μm.\nPermeability Studies\n6. The statement \"This suggested the poor semi-permeability of membrane, leading to a rapid\nequilibrium of osmotic pressure across the microcapsules\" lacks clarity. It is unclear how membrane\npermeability can be assessed by placing microcapsules in high-concentration glucose or dextran\nsolutions and observing shrinkage behavior. This requires further explanation.\nResponse: We appreciate the comment from the reviewer. The reviewer is correct that membrane\npermeability is not affected by the surrounding high-concentration solution. Our intention was to\nclarify that the membrane of CNF/PDDA microcapsules has relatively large pores and does not\nselectively block small molecules or polymers, e.g., glucose or dextran. In other words, the\nCNF/PDDA membrane does not behave as a semipermeable membrane. As a result, when the\nmicrocapsules are placed in a high-concentration glucose or dextran solution, although an osmotic\npressure difference initially exists, both water molecules and solutes (glucose or dextran) can freely\ndiffuse across the membrane. This rapid diffusion leads to fast equilibration of osmotic pressure\ninside and outside the microcapsule, and no shrinkage behavior is observed. In contrast, if the\nmembrane were semipermeable and able to block glucose or dextran, only water would diffuse\noutward while the solute remained outside, resulting in microcapsule shrinkage.\nWe have revised the manuscript as follows: “This indicates that the membrane of the CNF/PDDA\nmicrocapsule does not selectively block glucose or dextran, and instead both solutes freely diffuse into\nthe microcapsule, leading to rapid equilibrium of osmotic pressure across the membrane.\n7. For the permeability experiment using dextran (Supplementary Figure S7), the partition\ncoefficient of FITC-tagged dextran should be provided. Complete characterization of the dextran\n(molecular weight, dispersity, degree of labeling) is also necessary. Importantly, a molecular weight\ncut-off for the microspheres should be estimated.\nResponse: We appreciate the comment from the reviewer. Because the concentration of FITC-labeled\ndextran (Dex-FITC) inside and outside the CNF/PDDA microcapsules is proportional to the\nfluorescence intensity, the partition coefficient (PC) can be determined from the ratio of internal to\nexternal fluorescence intensity. As shown in Fig. R12, using PC = C in/C out ≈ I in/I out, the PC values of\nDex-FITC with molecular weights of 10, 40, 70, 100, and 500 kDa are 0.37, 0.34, 0.34, 0.34, and\n0.22, respectively. It should be noted that, although the CNF/PDDA microcapsule membrane does\nnot block Dex-FITC, the interior is filled with a loose CNF network that reduces the accessible free\nvolume for dextran. In addition, negatively charged CNF introduces electrostatic repulsion against\nthe anionic FITC label. Both steric exclusion and electrostatic effects lead to a lower Dex-FITC\ncontent inside the microcapsules compared to the exterior.\nWe have added Fig. R12 to the revised Supplementary Information as Supplementary Fig. 13,\nalong with a brief discussion.\nFig. R12. a-e, Confocal fluorescence images of CNF/PDDA microcapsules after equilibration for 4\nh in dextran solutions containing trace amounts of FITC-dextran. Scale bars, 200 μm. f, Summary of\npartition coefficients (PCs) of dextran, determined using FITC-dextran as a fluorescent tracer, where\nthe PC is defined as the ratio of internal to external fluorescence intensity. Error bars represent the\nstandard deviation (n = 3).\nFive dextran samples with different molecular weights (M : 10, 40, 70, 100, and 500 kDa) were\nw\npurchased from J&K Scientific. Detailed information on molecular weights is provided at the end of\nreply letter. Commercial dextran is a natural polysaccharide produced via microbial fermentation and\ninherently exhibits a broad molecular-weight distribution. Therefore, suppliers typically provide only\nthe average molecular weight rather than dispersity values. Dex-FITC was prepared by covalently\ngrafting FITC onto commercially purchased dextran. Based on 1H NMR spectra (Fig. R13), the FITC\ngrafting ratios for the five dextran samples were determined to be 0.0526, 0.0469, 0.0358, 0.0281,\nand 0.0226 mol FITC per mol glucose (mol%), respectively. As Dex-FITC serves solely as a\nfluorescent tracer in this study, the grafting density of FITC is not a critical parameter for the main\nconclusions. Therefore, we provide the detailed grafting ratios in the reply letter rather than\nincorporating them into the revised manuscript.\nFor the molecular-weight cut-off of CNF/PDDA microcapsules, the fact that even the largest\ndextran sample (M : 500 kDa) can permeate the membrane indicates that CNF/PDDA microcapsules\nw\nare not suitable as protocell models. Consequently, estimating a molecular-weight cut-off is not\nparticularly meaningful, as the membrane does not impose effective size exclusion. This is also why\nwe further incorporated graphene oxide into the membrane to enhance molecular selectivity.\nFig. R13. a, Chemical structure of FITC-dextran. b–f, 1H NMR spectra of FITC-dextran (400 MHz,\nD O, 25 °C).\n2\n8. There is there confusion regarding the statement \"It should be noted that if the microcapsules were\nsufficiently rigid, the shrinkage induced by the hypertonic solution could be suppressed\" when earlier\nthe authors mention \"no shrinkage of microcapsules was observed within 24 h.\" This apparent\ncontradiction requires clarification.\nResponse: We appreciate the comment from the reviewer. As discussed in the manuscript,\nCNF/PDDA microcapsules do not shrink when placed in a neutral glucose or dextran hypertonic\nsolution, but they do shrink in a negatively charged poly(acrylic acid) hypertonic solution. Our\nintention was to use this shrinkage behavior to illustrate the elastic nature of the membrane. However,\nas the reviewer pointed out, the original wording could lead to confusion. Therefore, in the revised\nmanuscript, we have removed the sentence “It should be noted that if the microcapsules were\nsufficiently rigid, the shrinkage induced by the hypertonic solution could be suppressed” and instead\ndiscuss the behaviors in glucose/dextran and poly(acrylic acid) solutions separately.\nCharacterization Issues\n9. The authors tagged GO with pyrene but did not explain the attachment method or characterize the\nproduct. How can they confirm that pyrene is covalently bound rather than physisorbed on GO,\nwhich might detach in complex solutions containing diverse polymers and salts? Control\nexperiments are required to validate the conclusions regarding GO localization in microspheres.\nResponse: We appreciate the comment from the reviewer. The synthesis of pyrene-labelled GO\n(GO-pyrene) has been incorporated into the Methods section (Synthesis of fluorescently labelled\nnanomaterials and polymers) as follows:\n“Synthesis of GO-Pyrene\nGraphene oxide (GO, 100 mg) was refluxed in thionyl chloride (50 mL) for 12 h, and excess thionyl\nchloride was removed by rotary evaporation to afford GO–COCl (91 mg). GO–COCl was then reacted\nwith cysteine (50 mg) in DMF (50 mL) in the presence of triethylamine (0.5 mL) at room temperature\nfor 24 h to yield GO–Cys (80 mg) after filtration, washing (DMF, deionized water, and ethanol), and\nvacuum drying. GO–Cys was dispersed in DMF/HO (50 mL, v/v ≈ 4:1), treated with TCEP (50 mg)\n2\nfor 12 h, and then reacted with pyrene–maleimide (50 mg) at room temperature for 24 h. The product\nwas collected and washed with DMF, deionized water, and ethanol, followed by repeated ultrasonication\nin fresh ethanol to remove physically adsorbed pyrene–maleimide, and finally vacuum-dried to afford\nGO–Pyrene (52 mg).”\nFor the characterization of GO-pyrene, FTIR and Raman spectroscopy were employed to\ndemonstrate that pyrene is covalently bound to GO:\nFTIR Analysis. Pristine GO shows the expected bands of oxygenated graphene oxide, including a\nbroad O–H stretching band (~3440 cm-1), a C=O stretching band of –COOH groups (~1730 cm-1),\nand C–O/C–O–C vibrations (~1385 cm-1). After functionalization, the spectrum of GO–pyrene\ndisplays new features in the amide I/II region (~1648 and ~1546 cm-1), consistent with the formation\nof amide linkages. The carbonyl region of GO–pyrene therefore reflects contributions from both\ncarboxyl and amide C=O groups, giving an altered band shape compared to pristine GO. These\nchanges are consistent with the introduction of cysteine–pyrene moieties covalently attached to the\nGO surface.\nFig. R14. FTIR spectra of pristine GO (red) and GO–pyrene (grey).\nRaman Analysis. In the Raman spectra, both GO and GO–pyrene exhibit the characteristic D (~1350\ncm-1) and G (~1590 cm-1) bands of graphene oxide. Upon conversion to GO–pyrene, the I D/I G ratio\nincreases from 0.85 (GO) to 1.12 (GO–pyrene), which is consistent with an increased defect\ndensity/disorder in the sp2 carbon framework after chemical modification.\nFig. R15. Raman spectra of pristine GO (grey) and GO–pyrene (red) (excitation: 514 nm).\nXPS Analysis. The XPS results are consistent with successful formation of GO-Pyrene. In the survey\nspectrum of GO-Pyrene, in addition to the dominant C 1s and O 1s peaks characteristic of GO, clear\nN 1s (~400 eV) and S 2p (~163–169 eV) signals are observed. The high-resolution N 1s spectrum\ncan be deconvoluted into components centred at ~399.6 and ~400.2 eV, which are characteristic of\namide/amine-type nitrogen introduced by the pyrene linker. The S 2p region shows a low-binding-\nenergy doublet at ~164.1 and ~165.5 eV, assignable to C–S / C–S–C species, together with a higher-\nbinding-energy component near ~168.4 eV that indicates a small fraction of oxidized sulfur, likely\narising from partial oxidation of the sulfur-containing linker. The appearance of these N and S signals\nin the expected binding-energy ranges, absent in pristine GO, strongly supports that pyrene groups\nhave been covalently grafted onto the GO sheets.\nFig. R16. XPS analysis of GO–Pyrene. a, Survey spectrum of GO–Pyrene. b, High-resolution N 1s\nspectrum. c, High-resolution S 2p spectrum.\nTo further clarify the localization of GO in microspheres, we dispersed CNF–GO/PDDA\nmicrocapsules in dextran and NaCl solutions, using pyrene-labelled GO for fluorescence\nvisualization. As shown in Fig. R17, CLSM images indicate that GO remains predominantly localized\nat the membrane in both solutions, as evidenced by the weak fluorescence signal inside the\nmicrocapsules. This behavior is consistent with that observed in pure water, demonstrating that the\nexternal solution environment does not affect the localization of GO.\nFig. R14, Fig. R15 and Fig. R16 have been included in the revised Supplementary Information\nas Supplementary Fig. 18, 19 and 20. Since incorporating control experiments in Fig. R17 into the\nmain text or Supporting Information would substantially disrupt the overall structure and narrative\nflow of the manuscript. Therefore, we provide these results and analysis in the reply letter for the\nreviewer’s evaluation.\nFig. R17. Confocal fluorescence images (left) and corresponding intensity profiles along the dashed\nlines (right) for CNF-GO/PDDA microcapsules containing GO–pyrene. a,b As-prepared\nmicrocapsules. c,d Microcapsules after incubation for 48 h in 1 M NaCl solution. e,f Microcapsules\nafter incubation for 48 h in 1 wt% of dextran-100k. Scale bars, 200 μm.\n10. There is an apparent contradiction regarding GO's effect on permeability. The authors report that\nGO hinders glucose permeability, with smaller GO amounts leading to faster glucose permeation.\nHowever, in earlier experiments (lines 106-107), they state that permeability is poor in GO's absence.\nThis needs clarification.\nResponse: We appreciate the reviewer’s comment and apologize for the misunderstanding caused\nby our wording (lines 106–107). In fact, the membrane of CNF/PDDA microcapsules has relatively\nlarge pores and does not selectively block glucose or dextran. As clarified in our response to\nComment 6, we have revised the manuscript as follows: “This indicates that the membrane of\nCNF/PDDA microcapsules does not selectively block glucose or dextran, and instead both solutes\nfreely diffuse into the microcapsule, leading to rapid equilibrium of osmotic pressure across the\nmembrane.”\nCoacervate Formation and Prototissue Assembly\n11. In the discussion from line 213, when using polyethyleneimine, how can the authors confirm\ncoacervate formation rather than simple polymer aggregates formed by electrostatic interactions in\na molecularly crowded environment? This claim requires supporting data. Additionally, what is an\n\"egg-shaped coacervate\"?\nResponse: We thank the reviewer for this important comment. The reviewer is correct that when\npositively charged polyethyleneimine (PEI) diffuses into the CNF-GO/PDDA microcapsules, its\nelectrostatic interactions with negatively charged CNF/GO lead to the formation of solid-like\naggregates, rather than liquid-like coacervates.\nTo verify this conclusion, we synthesized FITC-labelled PEI and characterized the spherical\nstructures formed inside the microcapsules using fluorescence recovery after photobleaching\n(FRAP). As shown in Fig. R18, the fluorescence intensity displayed almost no recovery after\nphotobleaching, indicating that PEI exhibits very limited molecular mobility, which is a hallmark of\nsolid-like aggregates. In contrast, if liquid-like coacervates had formed, the internal fluidity would\nhave resulted in a rapid fluorescence recovery.\nWe have removed the incorrect and potentially misleading wording in the revised manuscript, such\nas “egg-shaped coacervate”. In addition, we have added a new discussion on the formation and\ncharacterization of the aggregates, as follows: “PEI-0.6k was able to diffuse into the microcapsules and\nelectrostatically interact with CNF/GO. However, in contrast to PDDA-100k, which bears permanently\ncharged quaternary ammonium groups, the protonatable amine groups along the PEI-0.6k chains\nprovided weaker electrostatic interactions. In addition, the much lower molecular weight and shorter\nchain length of PEI-0.6k limited the spatial extent of electrostatic crosslinking with CNF/GO, resulting\nin a more localized and mechanically weaker network. Consequently, the PEI-\n0.6k–induced crosslinked structure was unable to resist osmotic swelling upon redispersion of the\nmicrocapsules in pure water, leading to network disruption, fragmentation, and the formation of\nsmall fragments that gradually associated into larger aggregates. Using FITC-labelled PEI-0.6k as a\nfluorescent probe and characterizing the aggregates by FRAP, we observed almost no fluorescence\nrecovery after photobleaching, indicating the limited mobility of PEI.” Fig. R18 has been included\nin the revised Supplementary Information as Supplementary Fig. 24.\nFig. R18. a, Confocal fluorescence images of a CNF-GO/PDDA microcapsule containing PEI–\nFITC before bleaching (-1 s), immediately after bleaching (0 s) and after 1200 s recovery, with the\nbleached region indicated by the arrow. Scale bar, 200 μm. b, The corresponding fluorescence\nintensity in the bleached region.\n12. The prototissue preparation lacks sufficient detail. The Materials and Methods section only states:\n\"For the preparation of prototissues, 0.3 M electrolyte (e.g., NaCl) was added into the extrusion solution,\nwhile other parameters remained unchanged.\" No explanation of how different shapes are achieved is\nprovided. The term \"extrusion droplets\" requires definition. Details about the molds (material,\nfabrication process, filling method, removal procedure) are absent.\nResponse: We appreciate the comment from the reviewer. We have added a detailed description of\nthe prototissue preparation process including the details about the molds in the Methods section as\nfollows:\n“For the preparation of prototissues, an aqueous phase containing 0.6 wt% CNF (or 0.6 wt% CNF +\n0.2 wt% GO) and 0.3 M NaCl was extruded into an aqueous collection bath containing 0.5 wt% PDDA\nand 0.1 wt% Triton X-100 to produce CNF/PDDA microcapsules (or GO-CNF/PDDA microcapsules)\n(extrusion rate = 30 mL h-1, gas flow rate = 1.0 L min-1). In the absence of molds, when the needle\nposition was fixed, the microcapsules settled to the bottom of collection bath, where they spontaneously\naccumulated and adhered to one another to form a macroscopic, self-supporting\nconical prototissue. Arbitrary shaped prototissues could also be constructed by manually moving\nthe needle during gas-spraying, but the precision of resulting shapes was relatively limited. When\nmolds were used, a patterned mold was pre-positioned in collection bath, and microcapsules were\ndeposited into the mold by manually moving the needle during gas-spraying process, where they\nadhered to each other. After depositing the required amount of microcapsules, the mold was lifted\nto yield prototissues that faithfully replicated the mold geometry. Regardless of mold use, the\nconstructed prototissues were transferred to deionized water and washed by repeated water\nreplacement for 10 cycles to remove residual components.\nAll patterned molds used in this work were commercially fabricated open-frame molds, consisting\nof a hollow frame with a defined height and open top and bottom. The molds can be made from\npolytetrafluoroethylene (PTFE), polypropylene (PP), polystyrene (PS), high-density polyethylene\n(HDPE), stainless steel, aluminum alloy, or hydrophobically modified glass. Owing to the weak\ninterfacial interactions between these mold materials and prototissues, demolding was readily\nachieved by simply lifting the mold, allowing the prototissues inside to retain their shape intact.”\nThe term “extrusion droplets” was intended to describe droplets formed when the aqueous phase\nis extruded and subsequently gas-sheared. However, to avoid confusion, in the revised manuscript\nwe have replaced “extrusion droplets” with “extruded aqueous phase,” which more clearly\ndescribes the process without introducing unnecessary terminology.\n13. The nature of microsphere-microsphere adhesion remains unclear. Why does a thicker, more\nrelaxed membrane promote adhesion? While the authors attempt an explanation, this fundamental\naspect requires precise understanding supported by experimental data.\nResponse: We thank the reviewer for this important comment. We recognize that explaining\nmicrocapsule adhesion by membrane thickening is not appropriate. The primary mechanism underlying\nmicrocapsule–microcapsule adhesion should be attributed to the electrolyte-induced screening of\nelectrostatic repulsion (ACS Nano 2022, 16, 21087–21097). Without electrolyte, CNF/GO and PDDA\nassemble into well-stratified structures via strong electrostatic interactions, with a predominantly\npositively charged outer surface and a negatively charged interior. This charge asymmetry leads to strong\nelectrostatic repulsion between individual microcapsules, preventing\nadhesion. Upon addition of electrolyte, increased ionic strength weakens the electrostatic\ncomplexation between CNF/GO and PDDA, thereby enhancing the mobility of components within\nthe membrane and leading to the formation of a homogeneous membrane. This membrane contains\na uniform distribution of positive and negative charges, enabling microcapsules to adhere upon\ncontact. Meanwhile, the weakened electrostatic interactions also promote interdiffusion between\nCNF/GO and PDDA during complexation, which can lead to membrane thickening.\nTo further clarify the role of ionic strength in microcapsule adhesion, we prepared CNF–\nGO/PDDA prototissues at different NaCl concentrations. As shown in Fig. R19, at low NaCl\nconcentrations (0.1 M), relatively strong electrostatic repulsion prevented microcapsule adhesion and\nprototissue formation. Stable prototissues were formed at intermediate NaCl concentrations (0.2 and\n0.3 M), where effective microcapsule adhesion occured. At a higher NaCl concentration (0.4 and 0.5\nM), electrostatic interactions between CNF/GO and PDDA were excessively weakened, leading to\nextensive interdiffusion between the CNF/GO-containing extruded aqueous phase and the PDDA-\ncontaining collection bath, thereby inhibiting the formation of stable complex membranes and\nmicrocapsules.\nWe have added the above discussion in the revised manuscript. Fig. R19 has been included in the\nrevised Supplementary Information as Supplementary Fig. 30.\nFig. R19. Influence of ionic strength on the construction of CNF-GO/PDDA prototissues by adding\nNaCl at 0.1 M (a), 0.2 M (b), 0.3 M (c), 0.4 M (d), and 0.5 M (e) into the extrusion droplets. Scale\nbar, 1 cm (left), 0.2 cm (right).\nCharacterization and Applications\n14. The authors demonstrate various possibilities that have already been reported in the literature using\ntheir new prototissue types but provide insufficient characterization. Detailed mechanical property\nanalysis of protocells and prototissues is necessary to support claims about mechanical properties and\nbending mechanics of layered prototissues. What forces are exerted during bending? Response: We\nappreciate the comment from the reviewer. The layered prototissue consists of two stacked layers: a\nCNF/PDDA prototissue and a CNF–GO/PDDA prototissue. To quantitatively compare the mechanical\nstrength of CNF/PDDA and CNF–GO/PDDA prototissues, we separately prepared conical\nCNF/PDDA and CNF–GO/PDDA prototissues and immersed them in a 1 wt% PDDA-100k aqueous\nsolution. The time-dependent volume shrinkage of each prototissue was recorded and quantified as the\nvolume shrinkage ratio, calculated as 1 − V/V0 based on the cone\nvolume before and after shrinkage. As shown in Fig. R20, CNF/PDDA prototissue exhibits both a\nfaster and larger volume reduction than CNF–GO/PDDA prototissue. After 1 hour, the volume\nshrinkage ratios of the CNF/PDDA and CNF–GO/PDDA prototissues reach 77% and 56%,\nrespectively. These results indicate that CNF/PDDA prototissues possess lower mechanical strength\nand are more susceptible to osmotic-pressure-induced shrinkage.\nBending of the layered prototissue is not driven by an external mechanical load but arises from\ninternal stress generated by asymmetric osmotic shrinkage between the two layers. Owing to their\ndifferent mechanical strength and osmotic responsiveness, the CNF/PDDA and CNF–GO/PDDA\nprototissues undergo unequal magnitudes and rates of volumetric contraction under the same\nhypertonic conditions. This mismatch produces a through-thickness strain gradient, which drives\nbending of the bilayer structure.\nWe have added a brief discussion in the revised manuscript. Fig. R20 has been included in the\nrevised Supplementary Information as Supplementary Fig. 34, along with a brief discussion.\nFig. R20. a, Side-view images of a CNF/PDDA prototissue before (left) and after 60 min immersion in\n1 wt% PDDA-100k solution (right). b, Side-view images of a CNF–GO/PDDA prototissue before (left)\nand after 60 min immersion in 1 wt% PDDA-100k solution (right). c, Time evolution of the prototissue\nshrinkage for the two compositions. Error bars represent the standard deviation (n = 3). Scale bars, 5\nmm.\n15. For the buoyant prototissue: what is the intended purpose and biological relevance? What is the\nbuoyancy rate and its dependence on experimental parameters?\nResponse: We appreciate the comment from the reviewer. The purpose of the buoyant prototissue\nis to demonstrate that our DIC-assembled, large-scale prototissues can be endowed with enzyme-\npowered, buoyancy-controlled actuation. In this way, they can achieve vertical displacement, gas-\ndriven shape editing, and cargo manipulation. Biologically, this design provides a minimal tissue-\nlevel analogue of gas-filled, buoyancy-regulating and shape-modulating structures in living systems\ne.g., gas vesicles in microorganisms or swim bladders in fish (Nat. Rev. Microbiol. 2008, 6, 466– 476;\nNat. Rev. Microbiol. 2012, 10, 705–715), and also illustrates how multicompartment prototissues can\nfunction as programmable, enzyme-driven actuator modules that combine buoyancy control with\nreconfigurable morphology for potential applications in dynamic, bioinspired materials (Small 2024,\n25, 2308580; Angew. Chem. Int. Ed. 2024, 6, e202311556).\nIn the revised manuscript we now quantify the buoyancy rate and its dependence on experimental\nparameters (Fig. R20). Here, the buoyancy rate is defined as the vertical ascent velocity of the\nprototissue, obtained from the slope of the normalized distance-time curves during the\napproximately linear rising stage after the induction period. As shown in Fig. R20 b,c, when the\ncatalase content in the CNF–GO/PDDA prototissue is fixed at 0.1 wt%, increasing the H O\n2 2\nconcentration from 3 wt% to 6 wt% accelerates bubble generation and leads to a higher ascent\nvelocity, whereas further increasing the H O concentration slows down the motion because of\n2 2\npartial inhibition of catalase at excessive substrate levels. As shown in Fig. R20 d,e, when the\nexternal H O concentration is fixed at 3 wt%, reducing the amount of encapsulated catalase leads\n2 2\nto a systematic decrease in the buoyancy rate, thus the prototissue rises more slowly.\nThese data demonstrate that the buoyancy-driven actuation of the prototissue can be quantitatively\ntuned by both the external fuel concentration and the internal enzyme loading. Fig. R21 has been\nincluded in the revised Supplementary Information as Supplementary Fig. 35, along with a brief\ndiscussion.\nFig. R21. a, Digital images showing a catalase-loaded CNF–GO/PDDA prototissue rising in aqueous\nHO after an induction period. Scale bar, 1 cm. b, Height–time traces for prototissues with fixed\n2 2\ncatalase content (0.1 wt%) in H O solutions of different concentrations (3, 6 and 9 wt%). c,\n2 2\nCorresponding buoyancy rates, defined as the vertical ascent velocity obtained from the slope of the\napproximately linear rising segment of the curves in b. d, Normalized distance–time traces for\nprototissues with different catalase loadings at a fixed HO concentration (3 wt%). e, Buoyancy rates\n2 2\nas a function of catalase loading. Error bars represent the standard deviation (n = 3).\nBiocompatibility Claims\n16. The authors claim biocompatibility in their conclusions without providing supporting data.\nTriton-X usage could be problematic in this regard. While an in vitro cytotoxicity assay is mentioned\nin the experimental section, no data are presented in the main text.\nResponse: We appreciate the comment from the reviewer. For the biocompatibility of\nmicrocapsules, we performed an in vitro CCK-8 assay. As shown in Fig. R22, L929 cells were\nincubated with extracts from CNF–GO/PDDA microcapsules for 72 h, and cell viability was\nassessed by the CCK-8 assay. Over the tested concentration range, cell viability remained above\n95%, indicating excellent biocompatibility. Regarding the reviewer’s concern about the\nbiocompatibility of Triton X-100, it can be readily replaced with biocompatible surfactants. As noted\nin our response to Comment 3, in addition to Triton X-100, microcapsules can also be successfully\nprepared using Pluronic F68, Pluronic F127, Tween-20, and Solutol HS-15. CNF– GO/PDDA\nmicrocapsules were also prepared using Pluronic F127 and Pluronic F68, and cytotoxicity assays\nlikewise demonstrated that these CNF–GO/PDDA microcapsules exhibit excellent biocompatibility.\nWe have added the corresponding results to the revised Supplementary Information as\nSupplementary Fig. 17 with a brief discussion.\nFig. R22. a, Live/dead staining images of L929 cells after incubation with the corresponding\nmaterial extracts (Control, Triton X-100, Pluronic F127, and Pluronic F68); live cells are stained\ngreen (Calcein-AM) and dead cells are stained red (PI), with merged images shown in the right\ncolumn. Scale bar, 100 μm. b, CCK-8 assay results (OD value at 450 nm) of L929 cells cultured\nwith the corresponding extracts for 1–3 days. Error bars represent the standard deviation (n = 3), and\nP values are indicated. [Surfactant] = 10-3 wt%.\nResponses to Reviewer #3\nReviewer #3 (Remarks to the Author):\nThe authors describe the construction of prototissues via adhesive interaction between microgel\ncompartments fabricated from PDDA and CNF. The membrane permeability of the compartments\nwere studied and modulated by inclusion of GO. They also illustrated how osmotic changes can help\nregulate the morphology of the microgel compartments irreversibly via cross linking of the interior or\nreversibly via delayed permeability. They further went on to study a two-enzyme cascade within the\nCNF-GO/PDDA microcapsules and show chemomechanical actuation of the prototissues mediated\nby osmotic changes and bubble mediated buoyancy.\n1) The primary concern is regarding novelty – as a significant part of the paper mostly deals with\npreparation of the protocells and their osmotic response along with enzyme cascade studies. These\nare routine studies in protocell field and the authors do well to thoroughly characterize their new\nprotocell model/chasis but these do not add significant novelty to the work.\nResponse: We appreciate the reviewer’s comment. In this work, we employed a new strategy to\nconstruct protocells using a new system. We agree with the reviewer that osmotic response and\nenzymatic reactions are well-established approaches in protocell research; however, owing to the\nfundamentally different nature of our system, a thorough understanding of these newly developed\nprotocells is essential for subsequent prototissue construction and functional implementation.\nBeyond the novelty of the production method, our work addresses several key challenges in this\nfield, including the large-scale preparation of protocell/prototissue in aqueous media, achieving\nexcellent mechanical strength and stability, and enabling precise shaping of prototissues. In the\nrevised manuscript, we have added substantial experimental data and discussion, addressing all\nreviewer questions and including additional results such as the periodic motion of prototissues,\nthe preparation of gripper-like prototissues, and their ability to manipulate macroscopic objects,\nwhich further strengthen the significance of our work. We hope that the revised manuscript meets the\nreviewer’s high expectations.\nTo further clarify the novelty of our contribution, we summarize below several bottlenecks and\nchallenges identified in review articles by multiple groups.\nProf. Gobbo et al. (Biochem. Soc. Trans. 2020, 48, 2579–2589):\n• “To date, prototissues have been assembled in the millimetre and submillimetre size range using\na variety of techniques...A materials chemistry approach would allow the advancement of current\nprototissue construction technologies and enable the precise assembly of protocells into large-\nscale materials (>1 cm in size) that are robust, free-standing, and stable in water media. This is\ncritical for future technological applications of prototissues and would represent a major\nachievement in the field.”\n• “To date, research has been focusing on the development of methodologies to assemble\nprotocells together in a controlled and reproducible manner. However, if we want to develop\nprototissue-based technologies, it is of paramount importance that we start advancing the\nprototissue’s mechanical properties. In this regard, current studies have been limited to the\ncharacterisation of the prototissue’s tensile strength and elastic modulus...It is now critical to\nconstruct protocellular materials with a range of mechanical properties that mimic those of\nliving tissues and perhaps even outperform them.”\nProf. Han et al. (Ac/v. Funct. Mater. 2024, 34, 2405823):\n• “Although prototissues with precise spatial structures can be assembled with micrometer and\nmillimeter ranges, they are still small compared to tissues for tissue transplantation. To\nassemble large-scale prototissues, the long-distance signaling, synergistic effects between\nartificial cells, and collective functions will face enormous challenges.”\nProf. Lin et al. (Ac/v. Healthcare Mater. 2025, 14, 2500376):\n• “Despite significant progress in the development of prototissues, several challenges remain... 2)\nNatural tissues exhibit a broad range of mechanical strength due to the ECM composition (e.g.,\ncollagen, elastin) and cellular interactions. The mechanical stability of prototissues or protocell\nassemblies is typically low; as such, the incorporation of synthetic hydrogels, scaffolds, or\nartificial ECM needs to be considered for structural support ...5) Engineering protocell\nassemblies into large-scale, functional 3D structures with batch-to-batch reproducibility\nremains challenging. Current fabrication techniques, including microfluidics and 3D printing,\nstruggle to precisely control spatial organization at a tissue-like scale.”\n2) The advantage of a prototissue is that it has compartments which can host a reaction to induce\nmorphological or functional output. In the current design, the authors have used the osmotic\nresponse of the protocells and bubble mediated buoyancy to produce actuation. The osmotic pressure\nmediated actuation due to differential response in the two layer tissue was also illustrated in earlier\nstudies (G. Villar et al. Science 2013, 340, 48-52). The buoyancy-based actuation of prototissues\nwhich the authors have shown is quite interesting as it is mediated by reactions within the protocells.\nHowever, this reviewer would like to see not just actuation but also recovery and re-actuation over\nmultiple cycles which would then constitute a significant advance in the field.\n3) One suggestion would be to use Glucose oxidase mediated consumption of the oxygen bubble to\nexecute multiple cycles of actuation or formulate a different strategy for dissolution of the bubble\nfor recovery of the actuated tissue.\nResponse: We appreciate the reviewer’s constructive suggestions. We achieved this goal by\nemploying glucose oxidase (GOx)-mediated consumption of oxygen bubbles. As shown in Fig. R23,\na small CNF–GO/PDDA prototissue encapsulating 0.1 wt% catalase and 0.1 wt% GOx was prepared\nand placed in a water-filled glass tube (diameter = 2 cm). An agarose hydrogel containing 3 wt%\nH O was fixed at the bottom of the tube, while an agarose hydrogel containing 18 wt% glucose was\n2 2\nfixed at the top. H O and glucose gradually diffused from the hydrogels into surrounding water.\n2 2\nWhen the prototissue approached the bottom H O reservoir, catalase decomposed H O into O ,\n2 2 2 2 2\ngenerating gas bubbles inside the protocells and increasing buoyancy, which caused the prototissue\nto rise. Upon reaching the top glucose reservoir, GOx consumed the encapsulated O , leading to a\n2\ndecrease in buoyancy and causing the prototissue to sink back toward the bottom. In this manner, the\nprototissue underwent repeated up-down motion between the two agarose hydrogels until the H O\n2 2\nand glucose were largely depleted, after which the prototissue gradually ceased motion and settled at\nthe bottom. Under experimental conditions used, the prototissue exhibited three reproducible\noscillation cycles.\nWe have added a discussion in the revised manuscript, and Fig. R23 is included as Fig. 7. l-m.\nA supplementary movie has also been provided as Supplementary Movie 17.\nFig. R23. Schematic illustration (a) and digital images (b) showing the vertical oscillatory motion\nand displacement-time trace (c) of CNF-GO/PDDA prototissue. Scale bar, 1 cm.\n4) The authors have studied the shrinkage of the CNF-GO/PDDA protocells with PEI and PDDA. It\nis seen that PDDA 100 kDa permeates and cross links but does not form a yolk structure but PEI\nforms a yolk structure which constitutes a highly cross linked network. Author could conduct FRAP\nstudies to understand the condensation mediated changes in fluidity of the interior and also clarify\nthe why there is difference in morphological outcomes for PEI and PDDA.\nResponse: We thank the reviewer for this important suggestion and have performed fluorescence\nrecovery after photobleaching (FRAP) experiments using FITC-labelled PEI-0.6k and FITC-labelled\nPDDA-100k as fluorescent probes. As shown in Fig. R24, almost no fluorescence recovery was\nobserved after photobleaching, indicating that both PDDA and PEI exhibit limited mobility. These\nresults also indicate that the yolk structure inside the microcapsules formed by PEI/CNF/GO are solid-\nlike aggregates rather than liquid-like coacervates (We note that after treatment with PDDA-100k, the\nmicrocapsules irreversibly collapse into crumpled shells. During confocal imaging, only the folded shell\nedges intersect the focal plane, resulting in a bright fluorescent rim and an apparently dark center that\narises from out-of-focus effects rather than the absence of PDDA).\nThe distinct morphological outcomes observed for PEI-0.6k and PDDA-100k arise from two primary\nfactors. First, the permanently charged quaternary ammonium groups on PDDA-100k interact more\nstrongly with negatively charged CNF/GO than the protonatable amine groups on PEI-0.6k, leading to\na more stable and robust electrostatically crosslinked network. Second, the much higher molecular\nweight and longer chain length of PDDA-100k allow electrostatic crosslinking with CNF/GO over a\nlarger internal volume of the microcapsules, favoring the formation of a more extended and continuous\nnetwork. Consequently, when shrunken CNF–GO/PDDA protocells\nformed in PDDA-100k or PEI-0.6k solutions are redispersed in pure water, the stronger PDDA-\n100k/CNF-GO network effectively suppresses reswelling. In contrast, the weaker and more localized\nnetwork formed with PEI-0.6k cannot withstand osmotic swelling, resulting in network disruption\nand the formation of small fragments that gradually associate into larger aggregates.\nWe have added a discussion in the revised manuscript, and Fig. R24c,d is included as\nSupplementary Fig. 24.\nFig. R24. a, Confocal fluorescence images of a CNF–GO/PDDA protocell containing PDDA–FITC\nbefore bleaching (-1 s), immediately after bleaching (0 s) and after 1200 s recovery, with the bleached\nregion indicated by the arrow. Scale bar, 200 μm. b, The corresponding fluorescence intensity in the\nbleached region. c, Confocal fluorescence images of a CNF–GO/PDDA protocell containing PEI–\nFITC before bleaching (-1 s), immediately after bleaching (0 s) and after 1200 s recovery, with the\nbleached region indicated by the arrow. Scale bar, 200 μm. d, The corresponding fluorescence intensity\nin the bleached region.\n5) the facile construction of the prototissue due to sticking interactions at high electrolyte\nconcentrations has the limitation of lacking programmability of the internal structure of the\nprototissue – in terms of self-organization of different protocells to form complex hierarchical\nstructures. This reviewer would like to see the potential of this prototissue design in terms of soft\nrobotics, where these prototissues can manipulate macroscopic objects or these actuatable tissues\ncan be used as artificial muscle to operate a gripper like structure. These experiments would add\nsignificant novelty to the manuscript.\nResponse: We appreciate the reviewer’s comment. In the original manuscript, we have already\ndemonstrated two approaches to program the internal structure and functionality of prototissues: (1)\nby alternately stacking CNF–GO/PDDA and CNF/PDDA protocells, a Janus-type starfish-shaped\nprototissue was fabricated, which exhibited bending behavior under osmotic stimulation; (2) by\nintegrating catalase-containing CNF–GO/PDDA protocells with catalase-free CNF–GO/PDDA\nprotocells, different buoyancy-driven motion modes of prototissues were achieved.\nFollowing the reviewer’s constructive suggestion, we designed and fabricated a soft gripper\nprototissue. As shown in Fig. R25, a cross-shaped CNF–GO/PDDA prototissue was first\nconstructed. CNF–GO/PDDA protocells containing NdFeB ferromagnetic microparticles were then\ndeposited in the central region of the cross, while CNF–GO/PDDA protocells containing catalase\nwere deposited at the ends of the four arms. Upon addition of H2O2 to the aqueous environment,\noxygen was generated inside the catalase-containing protocells, producing buoyancy that induced\nupward bending of the four arms and enabled the gripper to capture three hollow plastic spheres.\nFurthermore, directional movement and rotation of the loaded soft gripper could be achieved by\napplying an external magnetic field.\nWe have added a discussion in the revised manuscript, and Fig. R25 is included as Fig. 7j,k. A\nsupplementary movie has also been provided as Supplementary Movie 16.\nFig. R25. a,b, Schematic illustration (a) and digital images (b) showing the cross-shaped prototissue\nfunctioning as a soft gripper. Scale bar, 2 cm.\n6) Another general comment would be that it may be investigated what is the advantage of using a\nprototissue over an actuatable gel for carrying out these tasks.\nResponse: We thank the reviewer for this important comment. The choice of a protocell-based\nprototissue over a monolithic actuatable hydrogel is motivated by several complementary advantages\nin transport, spatial programmability and architecture, which are directly relevant to the\ntasks demonstrated in our work.\nFirst, in a bulk hydrogel actuator the response time is typically limited by diffusive transport of\nwater and solutes through a continuous polymer network, so that the characteristic time increases\nstrongly with size and can reach tens of minutes to hours for centimetre-scale gels (Mater. Today\n2014, 17, 494–503). In our DIC-assembled prototissues, the material is instead built from many thin-\nshelled, highly permeable CNF/PDDA protocells. Fuel molecules and osmolytes only need to diffuse\nshort distances to reach the active regions. As a result, even prototissues with characteristic\ndimensions up to ~10 cm can undergo pronounced osmotic reshaping and enzyme-powered,\nbuoyancy-mediated motion on the timescale of a few minutes in most cases, whereas an equally large\nhomogeneous gel would respond much more slowly under comparable conditions.\nSecond, achieving spatially programmed, multi-module behaviour in a single continuous hydrogel\nusually requires sophisticated patterning technologies such as multimaterial 3D printing or multi-\nstep photo-patterning of orthogonal chemistries, which demand specialized equipment and elaborate\nformulations (Matter 2023, 7, 2419-2438; Front. Robot. AI 2021, 8, 673533). By contrast, the\ninternal architecture of our prototissues is encoded simply by mixing and placing different protocell\npopulations (e.g., catalase-loaded, magnetic, passive) at defined locations during assembly, without\nchanging the base chemistry or fabrication setup.\nThird, each protocell acts as a separate but communicating reaction compartment. This allows\ndifferent microenvironments and reaction networks to coexist within a single prototissue, while the\npermeable shells support controlled exchange of substances with the external medium. Such a\nmulticompartment design is more closely analogous to the hierarchical organisation of living tissues\nthan a monolithic hydrogel (Cell Biomater. 2025, 9, 100097)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The questions have been well addressed and it is recommedated for publication.", "reviewer_3": "The authors have significantly addressed many of the points raised by this reviewer which has increased the scope of the work. One key distinguishing point noted in the revised manuscript is the mechanical strength of the fabricated prototissues which is quite commendable.\nMinor points of concern remain which are discussed below:\n1) The key focus of the manuscript is fabrication of prototissues and in order to mimic prototissues it is essential to have a membrane permeability cut-off which can encapsulate enzymes. The higher permeability of the CNF-GO/PDDA microcapsules fabricated can only entrap small enzymes such as Horse Radish Peroxidase (44 kDa) as also seen in their data where the peroxidase is only found entrapped in the membrane not in the lumen. The permeability of the microcapsules has been adjusted by adding the GO – can this be taken forward more to get more stringent molecular weight cut-off to be able to encapsulate enzymes? If yes, add relevant data or these limitations and potential ways to overcome this may be discussed in the conclusion.\n2) The authors have fabricated a soft gripper powered by buoyant forces. The limitation of such design is that the forces are only acting along the vertical axis. The osmotic actuation is not enzyme driven and hence not as interesting, but the buoyant design seems limited in its outlook. These limitations may be discussed and future plan for improvements may be discussed in the conclusion."}, "author_response": "Manuscript ID: NCOMMS-25-49805B\nTitle: One-Step Construction of Robust Protocells and Prototissues in Water\nAuthors: Weixiao Feng, Peifan Li, Xin Li, Ziwei Wang, Min Chen, Yang Hu, Fu-Jian\nXu*, Shaowei Shi*\nDear editor:\nWe would like to thank the reviewers for their detailed feedback to improve our submission.\nWe have considered each comment carefully and revised our manuscript to address the issues\nraised.\nResponses to Reviewer #1\nReviewer #1 (Remarks to the Author):\nThe questions have been well addressed and it is recommedated for publication.\n[Editorial note: Reviewer #1 addressed authors' responses to the concerns of Reviewer #2 and\nfinds them adequately and satisfactorily addressed.]\nResponse:We appreciate the reviewer for the positive evaluation and for recognizing our\nefforts in addressing the concerns.\nResponses to Reviewer #3\nReviewer #3 (Remarks to the Author):\nThe authors have significantly addressed many of the points raised by this reviewer which\nhas increased the scope of the work. One key distinguishing point noted in the revised\nmanuscript is the mechanical strength of the fabricated prototissues which is quite\ncommendable. Response:We appreciate the reviewer for the positive evaluation and for\nrecognizing our efforts in addressing the concerns.\nMinor points of concern remain which are discussed below:\n1) The key focus of the manuscript is fabrication of prototissues and in order to mimic\nprototissues it is essential to have a membrane permeability cut-off which can encapsulate\nenzymes. The higher permeability of the CNF-GO/PDDA microcapsules fabricated can\nonly entrap small enzymes such as Horse Radish Peroxidase (44 kDa) as also seen in their\ndata where the peroxidase is only found entrapped in the membrane not in the lumen. The\npermeability of the microcapsules has been adjusted by adding the GO – can this be taken\nforward more to get more stringent molecular weight cut-off to be able to encapsulate\nenzymes? If yes, add relevant data or these limitations and potential ways to overcome this\nmay be discussed in the conclusion.\nResponse:We thank the reviewer for this comment. We note that the apparent enrichment of\nhorseradish peroxidase (HRP) in the membrane mainly arises from its participation in diffusion-\ninhibited complexation (DIC) via electrostatic interactions during microcapsule formation,\nrather than from the high permeability of CNF–GO/PDDA microcapsules. We have clarified\nthis in the revised manuscript as follows: “Due to the negative charge of both HRP and GOx\nunder neutral conditions, they participated in membrane formation via DIC, as evidenced by the\nstrong fluorescence signals of FITC-labelled HRP and RITC-labelled GOx on the membrane.\nHRP and GOx were also distributed inside the protocells, with a lower content of HRP detected.\nCompared to GOx, HRP contained more hydrophobic groups, enhancing its interfacial activity\nat the air–water interface and facilitating the DIC process, similar to the case of GO-containing\nprotocell.”\nWe fully agree with the reviewer that tuning membrane composition (e.g., adjusting GO\ncontent) could further regulate permeability and enable a more stringent molecular-weight cut-\noff for broader enzyme encapsulation. However, such tuning may affect not only permeability\nbut also mechanical strength and structural stability of microcapsules, and requires systematic\ninvestigation. We therefore added a brief statement in the conclusion: “Despite these advances,\nseveral aspects remain to be explored. For example, further tuning of membrane composition\nand/or post-modification of the membrane could enable more precise control over permeability\nand a tighter molecular-weight cut-off, which are important for broadening the range of\nencapsulation.”\n2) The authors have fabricated a soft gripper powered by buoyant forces. The limitation of\nsuch design is that the forces are only acting along the vertical axis. The osmotic actuation is\nnot enzyme driven and hence not as interesting, but the buoyant design seems limited in its\noutlook. These limitations may be discussed and future plan for improvements may be\ndiscussed in the conclusion.\nResponse:We thank the reviewer for this comment. As described in the revised manuscript\n(Figure 7j,k and Supplementary Video 16), in addition to buoyancy- and osmosis-driven\nactuation, we also demonstrated magnetic-field actuation: “By further incorporating protocells\ncontaining NdFeB ferromagnetic microparticles into the central region, the resulting soft gripper\ncould be directed to translate and rotate under an external magnetic field.” Accordingly,\nintegrating additional actuation mechanisms (e.g., light and temperature) is expected to enable\nmore complex and programmable motion and deformation of prototissues. We have added this\noutlook in the conclusion."}]

Decision letter