Delivery of peptide coacervates to form stable interaction hubs in cells

Domain: neuroscience
Published: 2026-02-02
Editorial outcome: minor_revision

Abstract
Cells contain membrane-bound and membraneless organelles that operate as spatially distinct biochemical niches. However, these reaction centers lose fidelity due to aging or diseases. A grand challenge for biomedicine is restoring or augmenting cellular functionalities. An excited strategy is the delivery of protein-based materials that can directly interact with cellular biological networks. In this study, we sought to develop long-lasting materials capable of cellular uptake, akin to intracellular interaction hubs. We develop a delivery method to efficiently transplant stable micron-size peptide-based compartments into living cells. By loading coacervates with nanobodies and bioPROTACs, we demonstrate successful target sequestration of natively expressed GFP to our synthetic hubs, and function as bioreactors to selectively degrade GFP inside human cells. These results represent an important step toward the development of synthetic organelles that can be fabricated in vitro and taken up by cells for applications in cell engineering and regenerative medicine.

Introduction
A grand challenge for biomedical science is achieving predictable control over cellular functions by controlling protein networks inside cells1–3. In nature, cells utilize spatial partitioning via anchoring and scaffold proteins and leverage subcellular compartmentalization as key strategies to enhance the efficiency of biochemical processes4–6. We previously demonstrated the feasibility of controlling cellular processes and cell behavior by expressing proteins capable of forming synthetic membraneless organelles intracellularly and targeting endogenous enzymes7,8. However, to date this approach has required gene delivery using viruses or nanoparticles. A major advance would be the ability to fabricate compartments ex vivo and deliver them into cells to stably function as designer synthetic hubs or organelles.

Cells control the rate and fidelity of biochemical reactions by compartmentalizing components in membrane-bound and membraneless organelles. For example, oxidative phosphorylation is caried out in lipid bilayer containing mitochondria9, and membraneless organelles such as the nucleolus specifically govern rRNA processing10–12. Stress granules sequester factors to downregulate protein translation13 and Nck-N-WASP condensates14,15 recruit Sos1 and Arp2/3 to form a reaction hub16. Membraneless compartments are formed by the self-assembly of proteins and RNAs into micron-size condensates driven via multivalent interactions6,17,18. Similar ideology can be adopted to generate new designer condensates or compartments to programmably control reaction pathways in a cell7,19,20. Often, the proteins that participate in this self-assembly and condensation contain large disordered or low-complexity polypeptide sequences6,21. We previously created synthetic condensates by expressing a disordered scaffold protein in cells and used them to reprogram cell behaviors. For example, we built compartments that functioned as insulators, turning signal flow on or off, to regulate growth and division or to polarize cytoskeleton organization7. Separately, synthetic condensates can be used as bioreactors to enhance signaling between components or generate new signaling cascades among factors that normally only weakly interact20,22. However, there are limitations to the use of these approaches for clinical applications: gene delivery is required, gene expression has a maximum23 that limits the amount of condensed material (expression relative to csat)8, and native targets must be tagged to drive their localization to the synthetic condensate7,8. We sought to overcome these hurdles using a strategy for protein-based delivery via peptide coacervates.

There are a variety of strategies to achieve protein or nucleic acid delivery to cells. Commonly, the RNA for a target of interest is delivered via lipid nanoparticles, which then drives expression inside a cell24. However, it is not feasible to achieve very high protein expression with these methods23,25, which limits their utility for delivering materials to form protein condensates that function as synthetic organelles. Cell permeable peptides exist in nature and have been designed to be capable of crossing the plasma membrane26–28. Although useful for drug delivery, these short peptide sequences cannot simply be appended to target proteins of interest to drive their cellular uptake. Recently, a promising strategy for protein delivery has emerged based on identification of peptide coacervate particles capable of crossing the plasma membrane for cellular uptake29,30. A 26 amino acid disordered peptide HBpep was identified and shown capable of self-assembly into micron-size coacervates through liquid-liquid phase separation29,31,32. These particles readily cross the cell membrane for cytosolic delivery. These coacervates can be co-assembled or loaded with cargos, including proteins and RNAs and will partially disassemble upon entering the cells, highlighting their utility for efficiency cytosolic delivery of macromolecules29,33,34. Variants of HBpep were developed, such as HBpep-SA and HBpep-SP, which are sensitive to redox state and partially disassemble in the cytosol of a cell29. Although primarily designed for drug delivery, we reason that the HBpep platform could be adapted to create stable protein-based hubs or organelles in cells, creating a toolbox to augment or revitalize cellular functions.

In this study, we create a platform to form large peptide coacervates capable of uptake and stable maintenance inside living cells. We develop strategies to enhance protein cargo loading and topological organization in coacervates, moving beyond passive loading and uniform partitioning. In contrast to previous controlled release systems, we develop strategies to deliver micron-size coacervates to cells that can serve as synthetic hubs or compartments, stable for days. Further, we embed nanobodies within these coacervates and demonstrate that they are capable of interfacing with protein targets in vitro and in cells. Finally, by co-loading bioPROTACs, we demonstrate that these deliverable intracellular coacervates can function as reaction centers for degradation. Delivery of synthetic hubs capable of targeting native enzymes and signaling pathways provides a powerful strategy in cellular engineering. By eliminating the requirement to deliver genetic material, this modular platform promises to fast-track therapeutic development, for example, through formation of degradosomes or via compartments that rewire aberrant signaling responses.

Results
Parameter optimization to form stable micron-size protein coacervates
Building from a previous peptide coacervate platform, HBpep31,33, we sought to develop a protocol to form gel-like particles that range in size from 1 to 5 µm diameter for cargo loading and cellular uptake, and that could ultimately serve as stable synthetic hubs or organelles in a cell. We chose to promote nucleation phase separation using two different methods: pH shift and temperature shift. HBpep contains five histidines and is largely soluble in acidic buffer, such as pH 4.5, but will phase separate at physiological pH 7.4 under standard conditions33. Further, HBpep displays upper critical solution temperature behavior and LLPS can be induced by shifting the temperature from 50 °C to room temperature, 23 °C. We also chose to test both the standard HBpep, which has only limited cargo release capability and a redox sensitive form HBpep-SA, that will release cargo into the cytoplasm of a cell29.

We first measured coacervation via pH shift for HBpep and HBpep-SA over a range of peptide concentrations from 0.1 to 2 mg/mL, corresponding to approximately 35–700 micromolar. We found coacervate size and number depended on peptide concentration (Fig. 1A–C), as expected35. Although smaller particles have utility for drug delivery applications, we were interested in formation of larger hubs that would be stably maintained in cells. At concentrations of 0.5 mg/mL or higher, the average HBpep particle diameter was larger than 2 µm and could range from 1 to 6 µm (Fig. 1B). Similar results were attained with HBpep-SA, although the sizes were slightly smaller. A higher fraction of the HBpep-SA peptide partitioned to the coacervates as measured by sedimentation of particles (Supplementary Fig. 1A, B), although in both cases at majority of the scaffold peptide was condensed when assembled at a concentration of 0.5 mg/mL. We separately tested induction of LLPS via temperature shift and observed similar size and numbers of particles compared to induction via pH shift (Supplementary Fig. 1C–E). During protocol optimization, we found that the free peptides in solutions at pH 7.4 were actually quite sticky and would foul surfaces of test tubes or imaging chambers over time. Particle production via pH shift is somewhat more reliable at creating dose-dependent coacervation and indicates use of acidic buffers for storage and handling of HBpep and HBpep-SA prior to assembly.

For coacervates to be useful for non-genetic delivery of ‘hubs’ to cells, they need to show gel-like material properties and not require free peptide present in the continuous phase to maintain their assembly. In contrast, liquid-like condensates of disordered proteins formed inside cells from gene expression can have high turnover because the protein is continuously expressed and maintained by a pool of material in the cytosol36. We thus tested the extent to which coacervates were in a gel-like or more liquid-like state by measuring labeled peptide diffusion following fluorescence recovery after photobleaching (FRAP). Particles were co-assembled with tracer amounts of a FITC labeled HBpep (FITC-HBpep) (Supplementary Fig. 1F) peptides and bleached using a 405 nm laser. Fluorescence recovery was quite limited over time, for both types of peptides, indicating very slow diffusion and suggesting that these particles are gels (Fig. 1D, E), as expected34. Recovery was slow but detectable over 10 min when photobleaching a portion of the particle to measured diffusion internally within the coacervate (Supplementary Fig. 1G, H). Fluorescence recovery from full photobleaching of particles was nearly undetectable, suggesting minimal recovery via diffusion from the continuous phase. We conclude it is feasible to generate particles of 1–5 µm in diameter using either method to induce LLPS and that peptide concentration can be varied to tune average coacervate size. Given their gel-phase properties, we expected it would be feasible to dilute them into new solutions without providing additional material in continuous phase and that they would remain stable.

Enhanced cargo loading into peptide coacervates via selective partitioning
We wondered whether it would be feasible to enhance selectivity and overall amount of cargo loaded into coacervates by tagging the cargo. Previous studies demonstrated non-specific entrapment or loading of cargos such as protein and RNA during assembly of HBpep and HBpep-SA coacervates29. However, loading efficiency varies for different cargos and depends on HBpep peptide concentration29. In this study, we explored increasing the specificity of cargo loading without having to adapt the cargo or change HBpep peptide concentration. We used GFP as a model folded protein cargo for this proof-of-concept test. To promote selective loading, we added a 25-amino-acid motif from HBpep (lacking tryptophan): GHGVYGHGVYGHGPYGHGPYGHGLY to the C-terminus of GFP, which we term GFP-HBP (Fig. 2A), and we generated the protein via recombinant expression and purification. We co-assembled the cargo via LLPS of HBpep and measured extent of loading. We compared loading of GFP vs GFP-HBP both for HBpep and HBpep-SA particles, in which particles were formed by pH shift. We found that HBpep enhanced cargo loading into coacervates (Fig. 2B–D), without changing coacervate sizes. We observed an early two to three fold increase in internal concentration of cargo as measured by fluorescence microscopy (Fig. 2C, D) and total partitioning as measured via sedimentation (Supplementary Fig. 2A, B). We conclude it is feasible to enhance the overall magnitude of loading and internal concentration of cargo by fusion of an HBP tag to a protein of interest. Under conditions in which cargos would compete, tagging would provide enhanced selectivity.

We next wondered whether it would be feasible to control the topology of cargo loading, dictating its spatial partitioning in the coacervate. Cargo loading appears relatively spatially uniform for coacervates formed via pH shift method, and in general cargos reach a higher internal concentration in particles comparing LLPS of HBpep via pH versus temperature for HBpep (Fig. 2B). Tagging greatly increases the partitioning of GFP to particles formed via temperature shift (Fig. 2C). Interestingly, the HBP tag also dictates the topology of cargo partitioning. GFP alone as a cargo shows relatively spatial uniform partitioning within particles, whereas GFP-HBP enriches at the particle surface (Fig. 2B, C). We conclude it is possible to specify a core-shell organization of scaffold and cargo that would enable spatially partitioning discrete cargos and would be broadly useful for a variety of applications in drug delivery and cell engineering. Nonetheless, for the remainder of this study, we utilize pH shift to generate particles with uniform loading.

Delivering micron-size or larger coacervates to cells and characterizing their stability
A previous study focused on delivery of particles smaller than 1 µm to cells and for the purpose of cytosolic particle dissociation and cargo release29. However, in this study, we were interested in using the HBpep and HBpep-SA platforms as stable synthetic hubs or organelles inside cells. As a result, we wanted to test the feasibility of delivering larger condensates to cells and their stability following uptake inside cells.

We sought to identify conditions for highly efficient uptake of large particles in a variety of cell types. This included a transformed human cell line for osteosarcoma, U2OS, a mouse melanoma cell line, B16-F10, and primary human immune cells—monocytes (Fig. 3A). We pre-assembled particles at 0.5 mg/mL, diluted them in cell media from 1:10 to 1:3 and measured particle uptake after 4 h and 24 h. Cells were washed extensively after particle addition to remove those not delivered to cells. We observed generally highly efficient uptake by cells for both particles of HBpep and HBpep-SA, in a variety of cell types (Fig. 3A). These particles were present in the cytosol, but not the nucleus (Fig. 3A), and showed diffusive motion (Supp Movie 1). A recent study suggests these coacervates cross the cell membrane via macropinocytosis, which enables the uptake of large portions of extracellular material32. At 1 day, we measured effective and robust uptake of particles formed at both 0.2 and 2 mg/mL, averaging uptake of nearly 40 particles per cell at higher concentrations (Fig. 3B–E). Particle formulations include a range of sizes, although those formed at 0.2 mg/mL, the mode for the distribution of diameters was less than 1 µm and those formed at 2 mg/mL were generally larger than 1 µm. Importantly, there was a long tail in the distribution of particle sizes at higher concentrations (2 mg/mL formulation), and cells given these formulations contained nearly tenfold higher numbers of larger particles, greater than 2 µm diameter (Fig. 3D, E). In exemplary cases we found it possible to fill up the cytoplasm of a cell with approximately 100 particles (Supplementary Fig. 3E and Supp Movie 2), a feat not possible from genetic expression of a coacervating protein. Therefore, it is feasible for larger coacervates to be efficiently taken up by cells.

In an effort to build stable hubs that can be delivered to cells, we were interested in the stability of the particles inside cells over time. Previous efforts focused on use of particles <1 µm diameter for drug delivery that would subsequently partially disassemble in the cytoplasm29. We sought to test the stability of the intracellular coacervates, comparing formulations better suited for drug delivery versus those ideal for making stable hubs (Supplementary Fig. 3A, B). Particles were generally more stable at day 5 for coacervates assembled at 2 mg/mL vs. 0.2 mg/mL, likely related to their larger sizes (Fig. 3B, D and Supplementary Fig. 3C). Indeed, if when we quantified only larger particles (those >2 µm diameter), the number per cell was relatively constant over time (Fig. 3E). In contrast smaller particles (<2 µm diameter) were lost from cells for the 0.2 mg/mL formulation. We suspect the disassembly is caused by protonated or reduced HBpep-SA peptides diffusing away. This whole process is kinetically slow, especially for large coacervates, making larger coacervates more “stable.” We conclude that our large coacervates are efficiently taken up by cells, and that these particles are stable intracellularly for at least 5 days. These results provide a roadmap toward delivery and maintenance of synthetic ‘hubs’ within living cells without the need for gene transfer.

Selective targeting of proteins to pre-formed coacervates using nanobodies in vitro
To be useful as a designer organelle or hub, coacervates must be functionalized with molecules that enable them to interface with native protein interaction networks or signaling pathways. Native membraneless organelles can function as insulators13—sequestering components—or as bioreactor compartments that co-localize components at elevated concentrations15. We previously demonstrated that synthetic condensates could be formed via expression of a scaffold protein in cells, and that they could function as insulators, targeting and partitioning native enzymes that were tagged with a complementary coiled coil7. In this study, we wanted to go further: to deliver synthetic hubs via extracellular uptake rather than via gene transfer and expression, and to embed nanobodies within them that could bind to a target of interest without needing to tag the target. It would then be possible to create a designer biochemical niche inside a cell through the delivery of a one-pot peptide coacervate platform.

To start, we tested the feasibility of targeting proteins to pre-assembled peptide coacervates in vitro comparing those containing or lacking a nanobody (Fig. 4A). As a proof-of-concept, we tested co-assembly and embedding of the nanobody VHHGFP4, which binds with nanomolar affinity to GFP37. This nanobody is highly useful because many stable cell lines have been created that contain a GFP-fusion to a target protein of interest. To track nanobody loading using fluorescence microscopy, we fused it to mCherry (VHHGFP4-mCherry), or also to the HBP targeting sequence (VHHGFP4-mCherry-HBP). To form particles, these purified proteins were added a 1:5 mass ratio into formulations of 0.5 mg/mL HBpep and HBpep-SA. Both nanobody cargos enriched within the HBpep and HBpep-SA particles after co-assembly (Supplementary Fig. 4A). We also observed somewhat elevated loading of VHHGFP4-mCherry-HBP into HBpep particles (Supplementary Fig. 4B), consistent with the results described above in Fig. 2, that HBP-tagging of proteins enhances cargo loading during co-assembly with HBpep or HBpep-SA peptide.

Once particles were formed, we quantitatively measured partitioning of GFP target protein added to pre-formed coacervates in an imaging microwell. Particles lacking a VHHGFP4 nanobody did not enrich GFP and instead partially excluded it (Fig. 4B, C); note partition coefficient of less than 1. Thus, cargo uptake is almost non-existent following peptide coacervate formation. In contrast particles containing VHHGFP4 nanobody enriched the GFP target at their interface (Fig. 4B) and this enrichment or partition coefficient at the surface was approximately twofold (Fig. 4C and Supplementary Fig. 4C, D), dependent on the type of peptide and whether HBP was present or not on the nanobody. This enrichment could be further enhanced to four to six fold by diluting the particles (Fig. 4D, E), which is consistent with excess nanobody free in solution competing with recruitment to the hubs. Note that the nanobody loading is relatively uniform throughout particles, and thus the surface targeting is not caused by spatial partitioning of nanobody to periphery. It was recently shown that these gel-like particles of HBpep and HBpep-SA have limited permeability for macromolecules larger than 2–10 kDa38. At 27 kDa in molecular weight, the target GFP would be expected to have limited diffusion inside the coacervate in the time course of our experiments, consistent with the ring-like GFP partitioning to our VHHGFP4 containing coacervates (Fig. 4B, D and Supplementary Fig. 4C).

Although mostly surface recruitment was achieved for this in vitro test using VHHGFP4 and GFP, it should be possible to achieve higher internal partitioning of molecules with smaller molecular weights, or by increasing the porosity of the coacervates. Notably for drug delivery, the cargo can be co-assembled with the particle as we show in Fig. 2. To conclude, in this initial test, we sought and found proof of feasibility for selective binding of target proteins to coacervates using nanobodies. Next, we wanted to test these particles for targeting in living cells.

Target enrichment at synthetic protein hubs inside cells
It has been suggested that HBpep particles are taken up by cells through actin-mediated macropinocytosis and phagocytosis32. In such a process, the coacervates will be encapsulated by the endosomal lipid membrane, which limits accessibility and thus binding of target protein proteins. Based on published results and our own GFP delivery experiments using HBpep-SA particles (Supplementary Fig. 3D), some GFP can be released into the cytosol, which implies that there may be some stochastic escape from membrane encapsulation. However, to further promote endosomal escape, we included TAT-HA2 assist peptide39 in co-assembly of HBpep-SA particles loaded with VHHGFP4 nanobody or a control protein. This TAT-HA2 assist peptide has been shown to create nanopores in the endosomal membranes that promote endosomal escape26,39,40. As a test case, we used a U2OS human cell line that was virally transduced to stably express GFP as a target protein.

To determine whether coacervates could function as hubs in cells, we wanted to compare target GFP binding to control mCherry-containing HBpep-SA particles versus those containing the VHHGFP4-mCherry nanobody. In cells loaded with control particles, we observed that most coacervates excluded the target GFP (Fig. 5A, C), showing a partition coefficient less than 1. In contrast for VHHGFP4-mCherry group, although many coacervates did not sequester the target (likely due to presence of membrane), we saw a large significant jump in target binding (Fig. 5B, D). The partition coefficients achieved both for scaffold and target are on par with our previous study, which used coiled-coil recruitment of targets into RGG condensates7. In cells, enrichment indices below 2 are often non-specific. For control mCherry particles target GFP enrichment was below this value in nearly every case; we observed it <1% of time (Fig. 5E). In contrast hubs containing VHHGFP4-mCherry and TAT-HA2 assist peptide showed target GFP partitioning as high as 15-fold above cytosolic levels (Fig. 5D and Supplementary Fig. 5A). The percentage of condensates with GFP enrichment >2 was approximately 11.5% for particles co-assembled with VHHGFP4-mCherry and TAT-HA2, representing an order of magnitude increase in target enrichment compared to control particles, and comparable to the rates of efficiency of endosomal escape for some LNPs41,42. There is also a general trend of increasing average GFP enrichment among condensates with GFP enrichment >1 with VHHGFP4-mCherry and TAT-HA2 loaded compared to control group (Supplementary Fig. 5B). At a single-cell level, the fraction of cells that show at least one hub with significant target recruitment is 7–10% in control hubs and as high as 47–60% in hubs co-assembled with the VHHGFP4-mchery nanobody (Fig. 5F). These results demonstrated initial feasibility of the platform and also hinted that additional refinements were needed to control or enhance the target recruitment process.

With Nanobody Without Nanobody
To optimize the hub system and methodology, we decided to generate a stable monoclonal human cell line expressing the GFP target and explored additional mediators of endosomal escape. Using the monoclonal cell line with more uniform expression we found reduced non-specific recruitment to the control hubs lacking nanobody; 0% hubs showing recruitment (Supplementary Fig. 5D) and 0% of cells with a recruiting hub (Supplementary Fig. 5E). Whereas nanobody hubs still selectively bound to target: 13% of hubs showed targeting and 81% of cells had at least one recruiting hub (Supplementary Fig. 5D, E). Second, we tested use of chloroquine (CQ), a molecule can partition to endosomes and act as a proton sponge that causes overuse of proton pumps and endosomal membrane rupture43. We added to the cell media during hub delivery and then imaged cells 1 day later. We found that addition of CQ had a number of useful effects related to target binding to hubs. First, it caused a large increase in the fraction of hubs that could recruit target protein to the surface or interior (Fig. 5G). Interestingly, most of the recruitment was to the surface, similar to our in vitro data in Figs. 4 and 5H. Targeting was still highly selective, as control hubs showed de-enrichment at the surface, whereas hubs co-assembled with VHHGFP4 nanobody showed target surface enrichment, with an enrichment index for GFP of up to 2–4 (Supplementary Fig. 5F–H). Combining surface and internal target binding to our hubs, we observed that 44.6% of hubs showed GFP targeting (Fig. 5G), a value that exceeds most reported values of LNP endosomal escape41,42. Nanobody loaded hubs are stable in cells over time, in terms of number and size, up to 5 days (Supplementary Fig. 5I, J). Interestingly, if waited up to 5 days after hub delivery with CQ, we saw that most of GFP recruitment to the hubs was now internal and was very efficient: up to 40% of hubs showed target enrichment (Supplementary Fig. 5K). These data show that with increased endosomal escape and time, hub targeting can be highly selectively and highly efficient.

Given that hubs delivered to cells could show both topologies of target recruitment—surface and internal—we wanted to re-examine target recruitment in vitro. In our in vitro experiments (Fig. 4), VHHGFP4-mCherry loaded condensates were only able to recruit GFP to the surface, likely limited by the small coacervate pore size38. In cells, HBpep-SA condensates may partially disassemble inside acidifying endosomes or upon escape to the reducing environment of the cytosol, likely due to increased porosity, which would allow larger macromolecules such as GFP to diffuse in. To test this, we pre-assembled nanobody-coacervates in vitro and imaged GFP target recruitment to hubs following addition of glutathione (GSH) for reducing, or acetate pH 4.5. Both conditions promoted reconfiguration of target recruitment, from entirely at the hub surface, to now also inside the compartment (Supplementary Fig. 5L). Combined, these data show surface and internal recruitment are feasible both in vitro or in cells in vivo. Notably, the partial disassembly of HBpep-SA coacervates through GSH reduction is slow when GSH is at similar concentration to that in the cytosolic (10 mM), taking hours, further explaining the high stability and long duration of observation of HBpep-SA coacervates delivered to cells.

Hubs as functional degradosomes from Co-assembly with bioPROTACs
We next wanted to test the feasibility of hubs to serve as reaction centers to regulate intracellular targets. We sought to assemble degradation hubs, in which bioPROTACs were pre-loaded as cargoes inside HBpep-SA coacervates. BioPROTACs are fusion domains that are able to recruit target proteins and an E3 ligase complex in proximity to one another for ubiquitination and degradation44. However, bioPROTACs cannot readily cross the cell membrane and face challenges with efficiency when simply genetically expressed in the cytosol45. It has been suggested that multimerizing bioPROTACs by forming condensates can increase degradation efficiency46. Therefore, we reasoned that bioPROTACs could be more functional when housed within an HBpep microcompartment delivered to cells.

We selected a proven GFP bioPROTAC (VHHGFP4-SPOP 167–374) for co-assembly in HBpep-SA coacervates and delivered them to our U2OS monoclonal GFP-expressing cell line to test the efficiency of target degradation (Fig. 6A)47. When co-assembled with the bioPROTAC construct (VHHGFP4-SPOP 167–374), HBpep-SA coacervates efficiently recruited GFP into hubs and decreased cytosolic GFP levels by ~78% compared to control coacervates (Fig. 6B, C) after one day. Hubs containing only the GFP nanobody VHHGFP4 alone did not decrease the cytosolic GFP level, indicating the decreased in cytosolic GFP signal in cells containing hub-loaded bioPROTAC is due to GFP degradation and not simply GFP sequestration. When we loaded just the E3 adaptor portion (SPOP 167–374) of the bioPROTAC, we observed modest GFP degradation, presumably by non-specific ubiquitination and degradation of targets. We noticed target enrichment to hubs still occurred when loaded with bioPROTAC (Fig. 6B), even though overall cytosolic signal was greatly reduced, suggesting that ubiquitylated targets may be protected from degradation inside the hub. As noted above, we do not believe this is a partitioning effect alone as the nanobody-loaded hubs did not show lowered cytosolic signal (Fig. 6C). Rather, we posit that when targets are ubiquitylated at hubs and dissociate back into the cytosol, they are then degraded by proteosome. Overall, these data shows that bioPROTAC-loaded HBpep-SA coacervates can function as ‘degradosomes’ that selectively target protein for degradation, offering a potential generalizable strategy for target and pathway modulation in cells.

We conclude it is feasible to deliver nanobody coacervates to cells that can function for days as stable hubs, capable of targeting endogenously expressed proteins to the microcompartment. These results demonstrate a one-pot solution toward remodeling protein interaction networks in living cells using synthetic hubs or organelles and represent a unique opportunity for cell engineering without the requirement for gene delivery.

Methods
Peptides and proteins
HBpep and FITC-HBpep peptides were customize ordered from Genescript
HBpep-SA peptides were ordered from MCE (MedChemExpress, (HY-000)) and TargetMol (Catalog No. TP2797)
HBpep amino acid sequence29: GHGVY GHGVY GHGPY GHGPY GHGLYW
Tag-free GFP was purchased from Abcam (ab84191, Recombinant A. victoria GFP protein)

Oligonucleotides and proteins
Oligonucleotide and protein sequences are listed in supplementary Tables T1 and T2.

Cells
U2OS cells were acquired from ATCC as U-2OS HTB-96TM. B16 cells were gift from Andy Minn’s lab, which were purchased from ATCC as B16-F10 CRL-6475TM. Human Primary Monocytes were purchased from Human Immunology Core at the Perelman School of Medicine at the University of Pennsylvania from a female 27-year-old donor.

Temperature shift method to induce LLPS
In this method, HBpep peptide powder was dissolved in 1× phosphate buffered saline (PBS) pH 7.4, aliquoted as a high concentration stock (~10 mg/ml) and stored at −80 °C. To make coacervates, HBpep peptide and cargo were added to 1× PBS solution at 50 °C and allowed to thoroughly mix, with all reagents in 0.5 ml Eppendorf tube in a bead bath. Following 10 min of incubation, the tube was shifted to room temperature (~23 °C) for 15 min to induce formation of coacervates.

pH shift method to induce LLPS
In this method, HBpep and HBpep-SA peptide powder were dissolved in 50 mM acetate buffer at pH 4.5, aliquoted as high concentration stocks (~15 mg/ml), and stored at −80 °C. To generate coacervates, dissolved HBpep or HBpep-SA peptides had added to them sequentially water, 1 M NaCl salt and cargo, and after thorough mixing, 10× phosphate buffer was added to shift the pH to ~7.4. We waited 15 min at room temperature for coacervate particles to formation and gelate, following the pH shift. The final solution is expected to have 100 ml NaCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, and 5 mM Acetate besides peptide and cargo.

Delivery of condensates to cells
Cells were seeded at 50% confluency one day before delivery in glass-bottom 96-well plates (Cellvis P96-1-N). At this time, a final concentration of 10 µg/mL DAPI was added in the culture to stain nucleus. On the day of coacervate delivery, cells were first washed with growth medium: EMEM for U2OS cells, DMEM for B16-F10 melanoma cells, and RPMI 1640 for primary human monocytes. For coacervate delivery, growth medium was replaced by a 100 µl solution of Opti-MEM combined with a volume of 10–30 µl of preformed coacervates. Particle uptake was detectable within 4 h and cells. We generally quantified at 24 h after coacervate addition, following extensive washing of the wells to remove and external particles. Membranes were stained by Abcam Cytopainter Cell Membrane Staining Kit in the experiment in Fig. 3A. For stability tests in Fig. 3B–E and Supplementary Fig. 5I, J, U2OS cells were cultured in EMEM for 5 days. For Fig. 5G, H, Chloroquine was added in growth medium at 100 µM final concentration. For Supplementary Fig. 5K, Chloroquine was added in the growth medium for 5 days at 20 µM final concentration.

Partial disassembly of HBpep-SA coacervates in vitro by Acid and GSH in vitro
To test feasibility of partially disassembling HBpep-SA coacervates by acidification and redox state, we tested titration to imaging wells of acetate pH 4.5 and GSH. We first added 30 µl of preformed HBpep-SA coacervates (assembled at 0.5 mg/mL) loaded with 0.1 mg/mL VHHGFP4-mCherry on top of 100 µl Opti-MEM media in glass-bottom 96-well plates. After waiting 15 min and confirming visible formation of coacervates via microscopy, we then titrated the solution by adding 500 mM Acetate pH 4.5 in 1 µl volume increments (pipetted on top of the well solution). Generally, partial disassembly of HBpep-SA coacervates—detectable by internal recruitment of GFP target—was observed after ~1 or 2 µl titration and within minutes. For testing the effect of GSH on partial disassemble HBpep-SA coacervates, we performed a similar set of steps. We added 30 µl of preformed HBpep-SA coacervates (assembled at 0.5 mg/mL) loaded with 0.1 mg/mL VHHGFP4-mCherry on top of 100 µl Opti-MEM in glass-bottom 96-well plates. After waiting 15 min, we added GSH to a final concentration at 10 mM and incubated the plates at 37 °C for ~2.5 h. Partial disassembled HBpep-SA coacervates—detectable by internal recruitment of GFP target—were observed primarily among smaller coacervates.

Cloning
Codon-optimized 6his-GFPHBP, mCherry-6his, 6his-VHHGFP4-mCherry, 6his-VHHGFP4-mCherry-HBP, 6his-VHHGFP4-SPOP 167–374, and 6his-SPOP 167–374 constructs were cloned into pET expression vectors between NdeI and XhoI sites and assembled via In-Fusion ligation (Takara Bio) for transformation into Stellar competent cells. For stable cell lines, GFP was cloned into pLJM1 vector between AgeI and EcoRI sites and assembled in Stbl3 competent cells by In-Fusion ligation (Takara Bio).

Protein purification
In all cases, recombinant His-tagged proteins were purified from E. coli lysates using Ni-NTA agarose beads and affinity chromatography. First, pET plasmids were transformed into Rosetta 2 Escherichia coli cells (Sigma Aldrich). Bacterial cultures were grown in Luria Broth supplemented with kanamycin and chloramphenicol at 37 °C to an OD600 of 0.6–0.8, and expression was induced by addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside. Cultured cells were grown overnight a 16 °C overnight (18 h). Cell cultures were pelleted by centrifugation at 1960×g and the resulting pellets were stored at −80 °C. To generate cell lysates, bacterial pellets were thawed, then resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol) along with complete EDTA-free protease inhibitor cocktail (Roche). Cells were lysed with 2 min of sonication at 50% power using a Branson Sonifier. Lysates were clarified by centrifugation at 13,250×g for 20 min in a Sorvall RC6+ centrifuge using a F21S-8×50y rotor (Thermo Fisher Scientific) at 4 °C and the supernatant was collected. For soluble proteins the supernatant was incubated with Ni–NTA beads (Thermo Fisher Scientific) while rotating at 4 °C for 1 h. Beads were then washed three times. Proteins were eluted with elution buffer (20 mM Tris-HCl, pH 7.5, 500 mM imidazole, 1 mM dithiothreitol (DTT), and 1 M NaCl). Eluted proteins were dialyzed overnight into PBS buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4) using a Slide-A-Lyzer membrane cassette (Thermo Fisher Scientific) with a 10-kDa size cutoff at room temperature48. Protein aliquots were flash frozen and stored at −80 °C.

For 6his-VHHGFP4-SPOP 167–374, which was insoluble, the pellet after centrifugation was dissolved in urea, the protein purified via affinity on Ni-NTA beads and then slowly refolded. Pellets were resuspended in buffer (6 M Urea, 50 mM Tris-HCl, pH 7.5, 1 M NaCl, 20 mM imidazole) and this re-suspension solution was centrifuged at 13,250×g for 20 min. The collected supernatant was then incubated with Ni-NTA beads while rotating at 4 °C for 1 h. Beads were then washed three times. Proteins were eluted by elution buffer (20 mM Tris-HCl, pH 7.5, 500 mM imidazole, 1 mM DTT, and 1 M NaCl, 6 M Urea). The elution solution was then dialyzed for a total of 3 days with steady graded reduce ction urea concentration from 6 M to 4 M, 2 M, 1 M, 0.5 M, and 0 M (in PBS buffer) and finally dialyzed into PBS buffer.

Mammalian cell procedures
U2OS cells were cultured in Eagle’s minimal essential medium (EMEM, Quality Biological) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 10 U mL−1 penicillin-streptomycin (Gibco). B16 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 10 U mL−1 penicillin-streptomycin (Gibco). Cells are maintained at 37 °C in a humidified atmosphere with 5% CO2. Cells were split in 1:5 ratio every three days and had been passaged for less than two months, were negative for known infection. Frozen human primary monocytes were obtained from Penn Human Immunology Core, then thawed and cultured in RPMI 1640 with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 10 U mL−1 penicillin-streptomycin (Gibco).

Generating stable cell lines
At day 0, wild type U2OS cells were seeded in 24-well plates at 70-90% confluence in 500 µl Eagle’s minimal essential medium (EMEM, Quality Biological) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 10 U mL−1 penicillin-streptomycin (Gibco). At day 1, growth medium was replaced with 500 µl GFP lentivirus stock. At day 2, growth medium was replaced back to EMEM with 10% FBS, 2 mM L-glutamine, and 10 U mL−1 penicillin-streptomycin. At Day 4, cells were trypsinized and collected for Fluorescence-activated cell sorting (FACS) to select GFP-expression cells in the middle of the expression distribution for generating polyclonal U2OS cell line. This polyclonal cell line was expanded for 2 weeks and stored in EMEM with 10% DMSO in nitrogen tank.

To generate monoclonal GFP-expressing U2OS cell line, the polyclonal cell line in culture was trypsinized and collected for FACS for medium GFP expression cells. Ninety-six single U2OS cells were sorted in 96-well plates (one cell per well). After 2 weeks of expansion, 4 monoclonal cell lines were generated and stored in EMEM with 10% DMSO in nitrogen tank. A monoclonal line expressing low-medium GFP levels was chosen for experiments in Fig. 5H, Supplementary Fig. 5C–K, and Fig. 6.

Microscopy
Images were collected on an Olympus IX81 inverted confocal microscope (Olympus Life Science) equipped with a Yokogawa CSU-X1 spinning disk, a mercury lamp, 405-488- and 561-nm laser launches, and an iXon3 electron-multiplying charge-coupled device camera (Andor). Multidimensional acquisition was controlled by MetaMorph software (Molecular Devices, version 7.8.10.0). Samples were illuminated using a 405-nm laser and/or 488-nm laser and/or a 561-nm laser and were imaged through a ×100, 1.4-NA oil immersion objective and/or a ×20, 0.75 NA air objective. Z stacks were collected in 0.3-μm steps.

For in vitro experiments, HBpep and HBpep-SA condensates were imaged in custom-fabricated acrylic gasket chambers adhered to glass coverslips (#1.5 glass thickness; Corning Inc.) The glass bottom was passivated by 10 mg/mL BSA overnight. For in vivo experiments, cells were seeded in glass bottom 96-well plates (Cellvis P96-1-N).

For FRAP experiments on FITC-HBpep labeled HBpep and HBpep-SA condensates, a Roper iLas2 photoactivation system controlling a 405-nm laser was used. An area containing multiple condensates were selected and photobleached, and fluorescence recovery in the bleached region was analyzed in ImageJ after normalizing the fluorescence intensity from 0 to 100 based on the florescence intensity before FRAP and right after FRAP. Images were collected to visualize the condensates at the 488-nm wavelength and bright field using a ×100, 1.4-NA oil immersion objective.

Image analysis
Image segmentation and analysis were performed in FIJI and Excel. “Analyze Particle” function in FIJI was used to count and measure the size of condensates in Fig. 1 and Supplementary Fig. 1 and measure the average fluorescence intensity in condensates in Fig. 2 (except for temperature based assembly with GFP). In other quantitation, measures of condensate size, number, and fluoresce intensity were measured manually in FIJI. Calculation of Enrichment Index in Fig. 2 for cargo loading of GFP and GFPHBP was calculated by measuring mean fluorescence intensity of whole coacervate divided by average fluorescence intensity in the continuous phase outside coacervate. EI = (Intensity coacervate / Intensity solution). For line scans, such as in Fig. 4, these were performed using FIJI in the 488nm channel. Maximum fluorescence intensity was recorded and divided by the average fluorescence intensity in the liquid phase outside condensates to determine ring enrichment index. To measure target partitioning and enrichment to hubs, in Fig. 5E, F and Supplementary Fig. 5A, B, line scans were performed in FIJI in the 488nm channel. The maximum fluorescence intensity was recorded and divided by the average fluorescence intensity in the cell cytosol to generate the maximum enrichment index. EI = (Max. Intensity coacervate / Average. Intensity cytosol). Similarly, for Fig. 5G and Supplementary Fig. 5C, D, the mean enrichment index was calculated by dividing average coacervate GFP fluorescence intensity was divided by the mean fluorescence intensity recorded in FIJI. For target surface enrichment calculations in Fig. 5G and Supplementary Fig. 5G, the average surface GFP fluorescence intensity derived from a line scan was divided by the mean cytosol GFP fluorescence intensity using FIJI. The 3D reconstruction in the Supplement Movie 2 was rendered in Imaris. For measurements of degradation activity in Fig. 6C, the average cytosol GFP fluorescence intensity was measured in FIJI by selecting multiple random cytosolic areas, excluding hubs and nucleus.

Statistics and reproducibility
The experiments were reproducible. All statistical analyses were performed in GraphPad Prism 9. To test the significance of two categories, an unpaired one-tailed Welch’s t-test was used. To test the significance of more than two categories, a one-way Brown–Forsythe ANOVA was used. In all cases, NS indicates not significant (P>0.05), *P≤0.05, **P≤0.01, ***P≤0.001, and ****P≤0.0001. The sample size is determined by the ability to acquire and assess corresponding images. The sample images are randomly taken by avoiding seeing 488 nm laser channel. In quantitative analysis, coacervates on the edge of images were excluded. The Investigators were not blinded to allocation during experiments and outcome assessment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Comments on “Delivery of Peptide Coacervates to Form Stable Interaction Hubs in Cells” by Matt Good and colleagues\nIn this concise study, Tu et al. established a peptide-derived gel-like condensates for cellular delivery and cytosolic\nexposure. A large portion of large delivered condensates (> 2 micron) remain stable in cells up to at least 5 days.\nFunctionalized condensates are able to recruit a cellular target via nanobody binding. This is an interesting technological\ndevelopment with plenty of applications. However, the authors need to substantiate their study with some further\nexperiments before I can suggest its acceptance for publication in Nature Communications.\nMajor comments:\n1)It is pivotal demonstrate cytosolic exposed condensates can indeed trigger endogenous signaling. For instance the\nauthors can functionalize the condensates with LC3-interaction regions to see the recruitment LC3-labeled membrane for\nautophagosome formation. They can also functionalize the condensates with apoptosis-inducing domains to see whether\nthe condensates are sufficient to induce apoptosis. Etc......\n2)The authors mentioned that previous papers reported that these condensates are engulfed into cells via micropinocytosis.\nIt is still instrumental to observe the internalization of the condensates via long-term imaging.\n3)They showed that about 10% condensates can actually recruite target protein (Figure 5E). It is also important to determine\nwhat percentage of cells lack of any condensates that can recruite target protein. This issue is related to the future\napplication of this technology.\n4)It is also important to know which type of condensates is more stable, the ones entrapped in endosome or those escaped\nfrom endosome.\nMinor comments:\nFigure 1D, 1E, the time for FRAP is only 5 minutes. Longer is desirable.\nThere are many typos and inconsistent nomenclatures. To name a few below: 1) in Figure 1B, the unit for diameter is um. It\nshould be μm. 2) “using either\nmethod for to induce LLPS”, “for” is not needed. 3) “In an effort to build stable hubs than can be delivered to cells”, “than” is\n“that”. 4) “We conclude that cells our large coacervates”, “cells” is not needed. 5) “for macromolecules larger than 2-10 kDA”,\nkDA? 6) many protein names are not consistent in text versus in figures, e.g. GFP-HBP is used in text while GFPHBP is\nused in Figure 2; VHH4-mCherry is used in text while VHH4 and mCherry were just stacked in Figure 4.", "reviewer_2": "The authors describe the use of peptide coacervates as internal artificial organelles capable of promoting the targeted\nlocalization of a protein (GFP) in cells. The results suggest that peptide coacervates can be explored as a viable platform for\nthe design of synthetic organelles and cargo delivery systems to cells. The results are interesting and raise questions that I\nbelieve are needed to improve the impact and clarity of the work. The paper is well written and easy to read. I hope my\ncomments and questions below will be helpful.\nThe work is based on the use of HBpep and HBpep-SA, which are peptides that have been previously reported and also\nused for cargo delivery to cells. A substantial part of the manuscript is dedicated to the characterization of the coacervates\nobtained using these peptides, which reproduces some of the main conclusions demonstrated in previous publications.\nHowever, there are a few points that I feel need attention, as follows:\n1. What is the rationale for using HBpep-SA vs. HBpep? The main experiments in the paper, showing the functionality of the\nhubs in the cells, only use BHpep-SA and do not really explain the unique properties of this peptide compared to HBpep.\nMore information on this is recommended for clarity.\n2. One of the conclusions from the GFP partitioning experiments is that the peptide coacervates could be used for a variety\nof applications in drug delivery and engineering. I agree with this statement. Is it possible to demonstrate a more general\neffect where different relevant model molecules are encapsulated using the described procedure? This would enrich the\nmanuscript and support the generality statement.\n3. The tagging approach to increase the partitioning of GFP is really interesting. However, the paper does not provide\nenough information about the rationale for the particular tag used and the properties and effects observed when the tag was\nused. Why does the tag work? Can it be applied to other types of molecules or does it only work with GFP?\n4. The manuscript says that the particles were generally more stable when they were larger. What is the hypothesis for this\nsize-dependent stability? Is it a kinetic effect? What is the stability of the droplets outside the cells? Is it comparable to the\nobserved stability inside the cells? Is there something special in the cytosol that contributes to the observed stability?\n5. Figure 4B. The labels can be confusing. It could give the impression that VHH4 was only tested with mCHerry (not GFP).\nI recommend revising the labels.\n6. Figure 4B shows data for HBpep-SA only. Why not both peptides (HBpep vs. HBpep-SA)? How does peptide structure\naffect nanobody partitioning?\n7. Figure 4B. GFP is enriched at the interface. Why is that? Figure 2B shows that GFP partitions homogeneously in HBpep\ndroplets (pH shift). Does this suggest that the nanobodies are not homogeneously distributed in the droplets? It would be\nrecommended to determine the distribution of nanobodies.\n8. The manuscript states that it should be possible to achieve higher internal partitioning of molecules with lower molecular\nweights or by allowing incubation with coacervates for longer periods of time. A more detailed study showing the generality\nof the approach for encapsulation and delivery of various cargoes is recommended (and quite interesting if successfully\ndemonstrated).\n9. Does the TAT-HA2 peptide promote endosomal escape in this paper? The manuscript says so, but I could not find a\ncontrol experiment showing that the peptide works as expected. A control showing the effect of this peptide is recommended.\n10. The authors mention membranization of the coacervate droplets. This is not clearly explained. What is the nature of the\nmembrane? How can it be demonstrated that a membrane actually surrounds the droplets? Does the surface charge of the\nmembranized coacervates change? How does this affect the uptake of the coacervates and the stability of the droplets in the\ncells?\n11. Figure 5B shows GFP homogeneously distributed in the droplets, while Figure 4B shows a different result (surface\naccumulation). A more detailed explanation seems to be necessary. What causes the differences in GFP distribution?\n12. The conclusion of the paper states that the nanobody coacervates can function as stable hubs for days. However, this\ndata is not provided. Given the importance of this conclusion to the paper, I recommend that the stability of the droplets in\ncells be investigated and the data included in the manuscript or supporting information."}, "author_response": "We thank the referee for their careful review of our study and for their positive comments that our\nplatform is innovative technically.\nMajor comments:\n1) It is pivotal demonstrate cytosolic exposed condensates can indeed trigger endogenous signaling.\nFor instance the authors can functionalize the condensates with LC3-interaction regions to see the\nrecruitment LC3-labeled membrane for autophagosome formation. They can also functionalize the\ncondensates with apoptosis-inducing domains to see whether the condensates are sufficient to induce\napoptosis. Etc.\nThis is a thoughtful point. Following reviewer advice, we sought to generate reactive hubs – and\nsettled on a strategy to specifically degrade intracellular targets by co-assembly and delivery of a\nbioRPOTAC in HBpepSA coacervates. This bioPROTAC, comprised of a VHHGFP4 nanobody with\npartial SPOP (an E3 ubiquitin ligase), when delivered to cells selectively degraded an endogenously\nexpressed GFP target protein (new Fig. 6, see below). Delivery of SPOP enzyme alone did not\nefficiently degrade targets; instead, showing it must be linked to the VHHGFP4 nanobody (Fig. 6C).\nNote, bioPROTACs such as these have very limited ability to cross the plasma membrane on their\nown. By coupling it to peptide coacervate based cell permeation, we demonstrate the utility of these\ndeliverable hubs to serve as hubs not only for binding cellular targets but also as reaction centers to\ncontrol target turnover.\nFigure 6A, C. bioPROTAC-loaded hubs function as degradation centers for target GFP in cells\n2) The authors mentioned that previous papers reported that these condensates are engulfed into\ncells via m(a)cropinocytosis. It is still instrumental to observe the internalization of the condensates via\nlong-term imaging.\nWe note that the uptake of coacervates has been previously established as a non-canonical pathway\nintegrating features of macropinocytosis and phagocytosis by the Miserez lab in a recent publication1\n(Shebanova, Advanced Science, 2024, DOI: 10.1002/advs.). In that study, three stages of\ncoacervates uptake were observed and characterized: membrane adhesion, cytoskeleton\nreorganization and internalization. Additionally, transmission electron microscopy and live imaging\nwere provided in that study to illustrate each process and showed membrane surrounding the\nengulfed particles after internalization. Furthermore, they tested a potent macropinocytosis inhibitor\nEIPA, which decreased coacervate uptake by nearly 70%. This study utilized the same HBpep\ncoacervate platform in cell lines and we feel it is redundant re-establish the process of uptake.\nInstead, our study focuses on long-term stability of hubs, functionalization with nanobodies and ability\nto interface with native targets in the cell, areas previously not investigated.\n3) They showed that about 10% condensates can actually recruit target protein (Figure 5E). It is also\nimportant to determine what percentage of cells lack of any condensates that can recruit target\nprotein. This issue is related to the future application of this technology.\nWe believe the reviewer may be asking for clarification on two points: what % of cells show\nany significant target recruitment to hubs and what is the background level of target recruitment when\nonly hubs are delivered without the co-assembled nanobody. First, the percentage of cells that show\nsignificant to recruitment to internal hubs is now quantified in Fig. 5F and Supp Fig 5E. This shows\nup to 60-80% of cells are positive for hub recruitment when the nanobody is present in hubs, whereas\nonly 0-7% are in the control when no nanobody is present (control: cells containing hubs loaded with\nmCherry alone).\nTo the second point, we note that there can be no target recruitment when hubs are absent\nfrom cells, because there is no ‘body’ or ‘locus’ for which we can measure enrichment. Notably GFP is\nwell-dispersed within the cytosol of this stable cell line in the absence of hubs (Supp Fig. 5C).\nHowever, we also note that there is very little target recruitment to hubs that are delivered without co-\nassembled VHHGFP4 nanobody (Fig 5A-F, and Supp Fig 5D). This demonstrates that the\nbackground non-specific binding of targets to hubs is very low or absent. Notably in a new stable\nmonoclonal cell line expressing GFP, we observed 0% of control hubs with significant target\nrecruitment and 0% of cells containing any significant target recruitment to cells (Supp Fig 5D,E).\nThis demonstrates the specificity of the nanobody hub platform and why it has broad utility.\nAdditionally, in new experiments we found that 5 days after delivery, up to 40% of all hubs\nshowed target recruitment when chloroquine was added to promote endosomal escape, which\nexceeds typical rate of endosomal escape rates of LNPs (Wang, Materials Horizons, 2025, DOI:\n10.1039/D4MH01781A)(Beach, ACS Applied Materials & Interfaces, 2021, DOI:\n10.1021/acsami.1c18359) (Supp Fig 5K). This further demonstrates the potential of these hubs to\nefficiently interact with native protein targets. These data are shown below.\n4)It is also important to know which type of condensates is more stable, the ones entrapped in\nendosome or those escaped from endosome.\nThe reviewer asks an interesting question. To date, it has not been reported whether hubs are stable\nfor days within endosomes or when free and cytosolic. To address this question, we used target\nrecruitment to hubs as a proxy for hub release and followed the fraction of hubs that did or did not\nrecruit target from day 1 up through day 5 (Supp Fig S5K). Using HBpep-SA coacevates co-\nassembled with nanobody VHHGFP4, we found similar but increased percentages of hubs that\nshowed target GFP recruitment at Day 5 (19%) compared to Day 1 (12%). This suggest that hubs\nhave similar stability when entrapped in endosome or perhaps are slightly more stable when free in\nthe cytosol. However, this is only a proxy metric for release, and we believe it is beyond the scope of\nthe current study to track both entrapped and free populations and to pulse-chase them to measure\nturnover rates. Because it is possible that the kinetics of endosomal release and degradation are not\nmatched, with the current data we cannot definitively determine whether a similar proportion of hubs\nare recruiting because of similar degradation rates or because of fast release and intermediate\ndegradation which may result in a similar steady state to what we observe. Notably, the number of\nlarge hubs is not decreasing dramatically over 5 days (Fig 3E), so degradation rates appear slow in\ngeneral, for either population.\nK\n50\n40 28.0588\n30 19.5422\n20\n11.6922 11.1496\n10\n0\n20µM CQ\nMinor comments:\nFigure 1D, 1E, the time for FRAP is only 5 minutes. Longer is desirable.\nAs requested, we have repeated FRAP out to 10 min and show the data now in Fig 1D,E. We also\nnote now in manuscript that recovery rates are very slow but measurable internally (from partial\nbleaching of particle). Recovery is essentially undetectable when fully bleaching particle which\nsuggests very little turnover with the continuous phase. Both results are consistent with gel-like nature\nof these coacervate particles.\nThere are many typos and inconsistent nomenclatures. To name a few below: 1) in Figure 1B, the unit\nfor diameter is um. It should be μm. 2) “using either method for to induce LLPS”, “for” is not needed.\n3) “In an effort to build stable hubs than can be delivered to cells”, “than” is “that”. 4) “We conclude\nthat cells our large coacervates”, “cells” is not needed. 5) “for macromolecules larger than 2-10 kDA”,\nkDA? 6) many protein names are not consistent in text versus in figures, e.g. GFP-HBP is used in text\nwhile GFPHBP is used in Figure 2; VHH4-mCherry is used in text while VHH4 and mCherry were just\nstacked in Figure 4.\nWe thank the reviewer for catching these errors, which we have now corrected.\n2>IE\netasnednoC\nfo\negatnecreP\n24hr\n120hr\n-CQ\nFigureS5K Percentage of Recruiting\nCoacervates Over Time\nWe thank the referee for their favorable assessment of our work along with their thoughtful\nsuggestions for improvement.\nThe work is based on the use of HBpep and HBpep-SA, which are peptides that have been previously\nreported and also used for cargo delivery to cells. A substantial part of the manuscript is dedicated to\nthe characterization of the coacervates obtained using these peptides, which reproduces some of the\nmain conclusions demonstrated in previous publications. However, there are a few points that I feel\nneed attention, as follows:\n1. What is the rationale for using HBpep-SA vs. HBpep? The main experiments in the paper, showing\nthe functionality of the hubs in the cells, only use HBpep-SA and do not really explain the unique\nproperties of this peptide compared to HBpep. More information on this is recommended for clarity.\nTo address this comment, we have included more information on the utility of these variants in the\nbackground/introduction. First, nearly identical peptides have been assembled into and tested as\ncoacervates previously and show differing levels of stability (Sun et al, Nature Chem 2022). HBpep is\nless insensitive to reducing environments whereas HBpepSA partially disassembles in the reducing\ncondition of the cell cytosol, which we find helps with internal target recruitment (see Fig S5F,L). We\nhave added text to the introduction to better clarify the rationale and we highlight their differences\nthroughout the results section.\n2. One of the conclusions from the GFP partitioning experiments is that the peptide coacervates could\nbe used for a variety of applications in drug delivery and engineering. I agree with this statement. Is it\npossible to demonstrate a more general effect where different relevant model molecules are\nencapsulated using the described procedure? This would enrich the manuscript and support the\ngenerality statement.\nWe note that the delivery of various types of cargos using HBpep coacervates has been\ndemonstrated previously in multiple previous publications. This includes (1) RNA2.3 (Sun, Nature\nChemistry, 2022) (Sun, ACS Nano, 2023) (2) many different proteins including fluorophores2 (Sun,\nNature Chemistry, 2022), enzymes4 (Cerrato, Molecular Pharmaceutics, 2025), CAS93 (Sun, ACS\nNano, 2023) for editing, etc. In this study, we further expanded the deliverable protein types to\nnanobodies and bioPROTACs. Nonetheless, the focus of our study was to develop stable interactive\nhubs – rather the controlled release particles – which is why we honed in on testing nanobodies and\nbioPROTACs and their ability to interact with native proteins as opposed to delivery per se\n(experiments in Figures 5, 6).\n3. The tagging approach to increase the partitioning of GFP is really interesting. However, the paper\ndoes not provide enough information about the rationale for the particular tag used and the properties\nand effects observed when the tag was used. Why does the tag work? Can it be applied to other\ntypes of molecules or does it only work with GFP?\nWe thank the reviewer for raising this question, which we clarify below. The use of disordered\nsequences to promote partitioning to a condensate has been demonstrated in a number of different\nsystems and contexts. Our lab has previously demonstrated attachment of the disordered RGG IDP\nsequence to various target proteins to promote partitioning to LAF-1 RGG condensates in vitro5\n(Schuster et al, Nature Comm 2018) and in vivo6 (Garabedian et al, Nature Chem Biol 2021). Other\ngroups have used the low complexity domain of FUS-LC to promote partitioning with opto-droplets7\n(Zhao and Toettcher, Nature Chem Biol 2019). Indeed, this is a generalizable, modular strategy to tag\na soluble cytosolic protein and promote its uptake or co-partition with a condensate. In this study we\nused the HBpep tag attached to cargos to enhance partitioning of GFP as well as nanobody VHH4-\nmCherry (Fig. 2 and Supp Fig 4C). Thus, this strategy works for both protein targets in our study and\nwell as many other protein targets (enzymes) used in previous studies. Note: RNA-binding domains\nhave been added to IDPs previously to recruit target RNAs8 (Nakamura et al, Nature Materials 2018).\n4. The manuscript says that the particles were generally more stable when they were larger. What is\nthe hypothesis for this size-dependent stability? Is it a kinetic effect? What is the stability of the\ndroplets outside the cells? Is it comparable to the observed stability inside the cells? Is there\nsomething special in the cytosol that contributes to the observed stability?\nThe referee highlights an interesting question. Disassembly of particles is thought to be a kinetic\nprocess although the mechanism has not been definitively established. A previous study showed that\nfor HBpepSA particles, the reducing environment of cell cytosol is likely destabilizing the particle2 (Sun\nand Miserez, Nature Chem 2022). In vitro, for HBpepSA particles, 60% of the peptide was reduced in\npresence of GSH at 24 hrs. In just buffer in vitro, we observe stability of particles across multiple days\nin vitro. Thus, we do not believe the cytosol is stabilizing the particle. Rather, in case of HBpepSA\nparticles, the reducing environment is likely destabilizing the particle as noted above. Additionally, in\nnew data, we found that addition of reduced glutathione (GSH) or low pH (4.5) is capable of altering\ncoacervate permeability in vitro to allow for enhanced internal partitioning of protein targeted by the\nVHH4 nanobody (Supp Fig S5L). This is consistent with a model in which endosomal acidification\nand escape into the reducing environment of the cytosol can partially disassemble particles, and\nincreasing their permeability. Ultimately, we feel quantitative measurement of disassembly or\ndegradation kinetics in cells is beyond the scope of the current study. However, we added a sentence\nto the discussion of the results for Fig 3 noting that larger particles may be stable for longer related to\nthe kinetics of disassembly.\n5. Figure 4B. The labels can be confusing. It could give the impression that VHH4 was only tested\nwith mCherry (not GFP). I recommend revising the labels.\nThank you for this note. We have edited the labels accordingly to prevent misunderstanding. We\nchanged the name of nanobody to its full name VHHGFP4, which is a nanobody that binds to GFP.\nTo clarify, in Figure 4B, VHHGFP4 nanobody was covalently attached to mCherry as one fusion\nprotein for convenient tracking by fluorescence microscopy.\n6. Figure 4B shows data for HBpep-SA only. Why not both peptides (HBpep vs. HBpep-SA)? How\ndoes peptide structure affect nanobody partitioning?\nWe indeed provide data for both. The data on target recruitment to HBpep coacervates is present in\nSupp Fig S4B,C. The data on HBpep-SA is present in Fig 4B,C (and also new Fig 4D,E)\n7. Figure 4B. GFP is enriched at the interface. Why is that? Figure 2B shows that GFP partitions\nhomogeneously in HBpep droplets (pH shift). Does this suggest that the nanobodies are not\nhomogeneously distributed in the droplets? It would be recommended to determine the distribution of\nnanobodies.\nWe would like to clarify the spatial differences in partitioning relate to the type of experiment and\nwhether the ‘cargo’ is co-assembled with the peptide into a coacervate, or whether the ‘target’ is\nrecruited after coacervate assembly has already occurred. When co-loading GFP or VHH4 nanobody\nwith HBpep into coacervates the loading appears uniform (see Fig 2B and Supp Fig 4A). In Figure\n4B, the GFP is added into solution after coacervate formation and is being targeted by the nanobody\npre-assembled in the coacervate. In this way, for the second half of the paper (Fig 4-6), we are\nmeasuring target recruitment to pre-formed hubs, rather than co-assembly of cargo. This is an\nimportant distinction because the goal of our study was to target cargos to stable hubs in cells, as\nopposed to controlled re-lease of co-assembled cargos.\n8. The manuscript states that it should be possible to achieve higher internal partitioning of molecules\nwith lower molecular weights or by allowing incubation with coacervates for longer periods of time. A\nmore detailed study showing the generality of the approach for encapsulation and delivery of various\ncargoes is recommended (and quite interesting if successfully demonstrated).\nThe reviewer brings up a number of interesting points. First, the porosity of these HBpep coacervates\nhas been established in a previous study9 (Lim, bioRxiv, 2024, doi.org/10.1101/2024.07.06.602323).\nSecond the stability of the coacervates can be controlled by addition of GSH and acidity, which we\nshow in new data in Supp Fig S5L. In this way we can now observe internal partitioning of target in\nvitro, similar to what is observed in cells. In terms of higher target partitioning to hubs inside cells, we\nnow show up at day 5 after hub delivery and with the addition of chloroquine (CQ) to promote\nendosomal escape that up to 40% of all hubs show significant target enrichment (new Supp Fig 5k).\nLastly in terms of generality, the delivery of a variety of types of cargos, including RNA and different\nproteins/enzymes has been establish in recent publications (see response to referee point #2 above)\nK\nL\nAcetate pH4.5 Titration\n50\n40 28.0588\n30 19.5422\nGSH Incubation\n20\n11.6922 11.1496\n10\n0 561nm 488nm Merge\n20µM CQ\nFigureS5L Internal Target Recruitment After\nGSH or Acid Mediated Partial Disassembly\n9. Does the TAT-HA2 peptide promote endosomal escape in this paper? The manuscript says so, but\nI could not find a control experiment showing that the peptide works as expected. A control showing\nthe effect of this peptide is recommended.\nEndosomal compartmentalization of HBpep coacervates was established in recent work2 (Shebanova,\nAdvanced Science 2024). We purchased a commercial TAT-HA2 peptide which has been used as an\nassist peptide for endosomal escape of LNPs10,11 (Wadia, Nature Med, 2004)(Bulmus, ACS Omega,\n2024). We also want to highlight for the reviewer our data in Fig 5E, experiments +/- TAT-HA2.\nThis assist peptide increases % of hubs showing recruitment (from 8.82% to 11.56%). We also note we are\nnot directly measuring escape but inferring escape from target recruitment to hubs via microscopy.\nNotably, this increases from almost zero in control hubs to nearly 10-15% when VHH4 nanobody is\nco-loaded, suggesting that this % of hubs are no longer membrane encapsulated and instead has\nphysical access to the cytoplasm where the GFP target resides.\n10. The authors mention membranization of the coacervate droplets. This is not clearly explained.\nWhat is the nature of the membrane? How can it be demonstrated that a membrane actually\nsurrounds the droplets? Does the surface charge of the membranized coacervates change? How\ndoes this affect the uptake of the coacervates and the stability of the droplets in the cells?\nA previous study demonstrated membrane encapsulation of these HBpep coacervates as a non-\ncanonical pathway integrating features of macropinocytosis and phagocytosis2 (Shebanova,\nAdvanced Science, 2024). Their work described three stages of coacervates uptake: membrane\nadhesion, cytoskeleton reorganization and internalization. Additionally, transmission electron\nmicroscopy and live imaging illustrated each process and showed membrane surrounding the\nengulfed coavervate particles after internalization.\nIn terms of particle stability inside and outside of the endosomes, we have included new data on the\n% of hubs that recruit target (a proxy for having been released from endosome) on Day 1 vs Day 5\nafter particle (Supp Fig 5K). These data show that the % of particles that have escaped endosome\nstays relatively constant, slightly increasing (Day 1: 11%, Day 5: 19%), suggesting that stability of\ncoacervates is similar inside the endosome or when released from endosome into the cytosol.\nHowever, we note that it is not possible to make this conclusion without careful pulse-chase\nexperiments and endosomal labeling; experiments that are beyond the scope of the current study.\nK\n50\n40 28.0588\n30 19.5422\n20\n11.6922 11.1496\n10\n0\n20µM CQ\n11. Figure 5B shows GFP homogeneously distributed in the droplets, while Figure 4B shows a\ndifferent result (surface accumulation). A more detailed explanation seems to be necessary. What\ncauses the differences in GFP distribution?\nThe reviewer raises an excellent point. To address this, we performed two new sets of experiments.\nWe demonstrate in vitro that addition of GSH and acid enables internal recruitment of cargos (Supp\nFig S5L), as expected from the effect of endosome entrapment and cytosolic release. The target\nrecruitment to hub surface (visible as a ring) occurs because of the limited porosity of these gel\ncoacervates9 (Lim, bioRxiv, 2024, doi.org/10.1101/2024.07.06.602323), which prevents internal\nrecruitment to nanobodies in absence of reducing agent and acid. Second, we also now demonstrate\nthe formation of ring-like target recruitment in cells in vivo, under conditions in which prevents\nacidification of the endosomal compartments through addition of chloroquine (CQ) (Figure 5H). Thus,\nboth topologies can be observed both in vivo and in vitro under defined sets of chemical conditions.\nData is summarized in figures below\n2>IE\netasnednoC\nfo\negatnecreP\n24hr\n120hr\n-CQ\nFigureS5K Percentage of Recruiting\nCoacervates Over Time\n12. The conclusion of the paper states that the nanobody coacervates can function as stable hubs for\ndays. However, this data is not provided. Given the importance of this conclusion to the paper, I\nrecommend that the stability of the droplets in cells be investigated and the data included in the\nmanuscript or supporting information.\nWe agree with the reviewer’s thoughtful comment. As requested, we have now measured the stability\nof the hubs containing nanobodies (Supp Fig S5I,J). These data are similar at 5 days to coacervates\nthat contain no cargo.\n64\n32 28.3 22.2\n16 9.7\n8\n4\n2\nFigureS5I, J Quantification of Coacervate Number\nand Size over 5 Days with Coassembled Nanobody\nlleC\nreP\nrebmuN\netasnednoC\n8\n6\n4\n2\nmµ\nretemaiD\netasnednoC\nL Acetate pH4.5 Titration H\nVHHGFP4-mCherry\nGSH Incubation\n-CQ +CQ\n561nm 488nm Merge\nFigureS5L Internal Target Recruitment After FigureH Representative Images Showing Both Internal\nGSH or Acid Mediated Partial Disassembly and Ring Recruitment Topologies -/+ Chloroquine\nJ\nI\nns"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors fully addressed my concerns. Especially they demonstrated that the deliverable hubs to serve as hubs not only\nfor binding cellular targets but also as reaction centers to control target turnover.\nTwo typos: In Figure S1A, the term \"Supernatent\" should be corrected to \"Supernatant\"; “We sought to identify conditions to\nfor highly efficient uptake”, “for” is not needed.", "reviewer_2": "The authors have satisfactorily addressed all the comments and questions raised in my original report."}, "author_response": "We have corrected these in the revised text and figure."}]

Decision letter