Dynamic Arrest and Tunable Gelation of Immunogenically Inert POEGMA Nanoparticles

Abstract

Physically cross-linked hydrogels formed through noncovalent interactions offer reversible gelation, injectability, and shear-thinning behavior, making them ideal for biomedical applications. Temperature-responsive systems are particularly attractive, as they enable injection as a solution, and at physiological temperature for minimally invasive delivery. Poly­(oligo­(ethylene glycol) methacrylate) (POEGMA) is a promising nonimmunogenic polymer that combines PEG-like hydration and protein resistance with tunable thermal responsiveness that does not bind pre-existing anti-PEG antibodies that are ubiquitous and elicits a minimal antibody response against itself. Here, we show that diblock POEGMA copolymerscomposed solely of short ethylene glycol side chainsspontaneously self-assemble into nanoparticles and form physically cross-linked hydrogels above a controllable transition temperature, without chemical modification. Using fluorescence recovery after photobleaching and video particle tracking nanorheology, we identify a thermally induced transition from dynamically arrested viscoelastic hydrogels to dynamically arrested liquid coacervates. These results establish a new class of single-component, nonimmunogenic POEGMA hydrogels with tunable phase behavior and mechanical properties, offering a versatile platform for localized drug delivery, sustained release, and regenerative medicine.

Introduction

Physically cross-linked hydrogelsformed through noncovalent interactions such as hydrophobic association, pi–pi interaction, hydrogen bonding, or thermally induced phase transitionshave attracted considerable interest in biomedical research. , Their appeal lies in features like reversible gelation, injectability, shear-thinning behavior, and the avoidance of potentially toxic chemical cross-linkers. Among these, temperature-responsive systems are particularly promising. − They undergo solution-to-gel transitions at physiologically relevant temperatures (i.e., body temperature), enabling in situ formation, minimally invasive delivery, and controlled release of therapeutic agents. −

Polyethylene glycol (PEG) has long been a staple hydrophilic polymer in such systems due to its biocompatibility, high water solubility, and well-established safety profile in humans. − However, emerging evidence challenges the notion that linear PEG is immunologically inert. Repeated administration of PEGylated therapeutics and prolonged exposure to PEG have been shown to elicit anti-PEG antibodies, activate complement pathways, and accelerate blood clearanceultimately compromising therapeutic efficacy and raising safety concerns. − These findings have prompted the search for PEG alternatives that retain its favorable physicochemical properties while minimizing immune recognition.

Poly­(oligo­(ethylene glycol) methacrylate) (POEGMA) has emerged as a compelling substitute to PEG. − Comprising a methacrylate backbone with short oligoethylene glycol (OEG) side chains, POEGMA offers hydration and protein resistance comparable to PEG, along with tunable architecture, side-chain length, graft density, and overall hydrophilicity. These features allow precise control over thermal transition behavior, hydration capacity, and self-assembly while enabling branched or bottlebrush polymer architectures not achievable with linear PEG. Crucially, our previous work has shown that POEGMA can overcome the immunogenicity issues associated with PEG when its ethylene glycol (EG) side chains are limited to three units or fewer. In a recent study, Ozer et al. demonstrated that biologics conjugated to POEGMA exhibited significantly lower immunogenicity than their clinically approved PEGylated counterparts, showing minimal antibody binding and improved pharmacokinetics in vivo. − These findings underscore the role of polymer architecture in reducing immune recognition and position POEGMA as a promising candidate for long-term and repeat biomedical applicationsespecially where PEG-specific immunogenicity is a limiting factor.

Recent efforts have explored the thermoresponsive properties of POEGMA in hydrogel design. For instance, Xu et al. and Liu et al. developed macroporous POEGMA-based hydrogels capable of reversible structural transitions in response to temperature, enabling tunable porosity and mechanical properties. , However, many of these systems rely on chemical cross-linking, PEG incorporation, long EG side chains in POEGMA that are immunogenic, additional monomers, or multicomponent mixtures to form networkscomplicating their design and hindering clinical translation.

The transition temperature (T _t) of POEGMA can be finely tuned by adjusting the side-chain length of its monomers. Shorter EG side chains increase hydrophobicity, resulting in lower T _t. ,,− In previous studies, we demonstrated that POEGMA’s thermal responsiveness can be tailored to biologically relevant conditions using monomers with EG side chains shorter than three unitsmaintaining a nonimmunogenic profile. , Building on this foundation, we now show that diblock POEGMA copolymers composed of these monomers self-assemble into stable nanoparticles in aqueous solution at room temperature and undergo phase separation above a tunable T _t. By carefully modulating hydrophobicity and block architecture, we observed spontaneous formation of physically cross-linked hydrogel networks without the need for chemical cross-linkers.

We investigated molecular to mesoscale dynamics within these networks using fluorescence recovery after photobleaching (FRAP), which revealed dynamic arrest above the T _t. These findings were further supported by video particle tracking (VPT) nanorheology. Notably, heating to temperatures approximately 10 °C above the T _t caused the hydrogel network to collapse into polymer droplets that remained arrested with respect to molecular exchange.

Together, these results demonstrate that both the gelation temperature and mechanical properties of POEGMA hydrogels composed of nonimmunogenic monomers can be precisely engineered. This work lays the groundwork for developing single-component, physically cross-linked, nonimmunogenic POEGMA hydrogels as a next-generation biomaterial platform with strong potential for localized therapeutic delivery, sustained drug release, and tissue engineering applications.

Results and Discussion

Design and Synthesis of Diblock POEGMA with Tunable Micellization and Transition Temperature

We aimed to obtain a POEGMA composition that self-assembles into stable nanoparticles with a critical micellization temperature (CMT) below room temperature (RT) and a micelle to coacervate transition temperature (T _t) below physiological temperature. Such a design would permit subcutaneous or intramuscular injection of a colloidal nanoparticle suspension that subsequently undergoes LCST phase separation in situ, forming a depot that may enable sustained release of nanoparticle-associated drug payloads into systemic circulation. To enable the development of a single-component system capable of undergoing multiple phase transitionsfrom micelles to depotsat biologically relevant conditions, we designed and synthesized a small library of diblock POEGMA copolymers, referred to as the rB series, with carefully tuned amphiphilicity.

We hypothesized that the CMT of diblock POEGMA would be governed primarily by the hydrophobicity of the core-forming block, while the T _t would be dictated by the hydrophilicity of the corona-forming block. Thus, to achieve simultaneous control of both parameters, we systematically varied the hydrophobicity of the hydrophobic block to ensure micellization below room temperature, while the hydrophilicity of the corona-forming block was adjusted to ensure a micelle-coacervate T _t below body temperature. Importantly, to mitigate PEG-associated immunogenicity, we restricted our monomers to EG side-chain lengths of three units or fewer (EG1–EG3). This constraint presented a unique design challenge and required the highly selective modulation of amphiphilicity within a narrow chemical space.

To achieve this, we introduced incremental amounts of the relatively more hydrophobic EG2 monomer into the hydrophilic block, generating a random copolymer of EG3 and EG2 as the corona. By titrating the EG2 content, we aimed to reduce the hydrophilicity of the corona and lower the T _t while preserving the overall amphiphilic balance of the system. The hydrophobic block was designed either as a homopolymer of EG1 or as a random copolymer of EG1 and EG2 to access a range of hydrophobicities that could create micelles at temperatures well below room temperature (Figure a,b). This systematic design allowed us to investigate how fine variations in EG side-chain length and hence hydrophobicity influence both micellization and LCST phase behavior.

1.

Design and synthesis of AB-type diblock POEGMA library. (a,b) Diblock coPOEGMA with a hydrophilic block that is a random copolymer of EG3 and EG2 and a hydrophobic block that is (a) a EG1 homopolymer or (b) a random copolymer of EG1 and EG2 monomers. (c) Two-step polymerization scheme of the diblock POEGMA via the RAFT polymerization reaction. (d) SEC chromatograms showing a narrow molecular weight distribution of the library. (e) Table summarizing the M _n, M _w, and D _m of the diblock coPOEGMA library determined by SEC-MALS. The ID of the diblock POEGMA represents the degree of polymerization (DP) of the individual blocks determined from starting monomer composition and block M _n divided by molecular masses of the respective monomer.

To synthesize the rB diblock POEGMA library, we employed a two-step reversible addition–fragmentation chain-transfer (RAFT) polymerization approach (Figure c). In the first step, a random copolymer of EG3 and EG2 was synthesized to serve as the hydrophilic block. The RAFT reaction was quenched at approximately 80–90% monomer conversion to preserve the chain transfer agent (CTA) end group, ensuring that the resulting polymer could be used as a macro-CTA in the subsequent synthesis of the second block. In the second step, the hydrophobic block was synthesized extending the polymer chain from the macro-CTA using either EG1 as a homopolymer or a mixture of EG1 and EG2 monomers, thus yielding a series of amphiphilic diblock POEGMA copolymers.

The chemical composition and degree of polymerization (DP) of each block were verified by ¹H NMR spectroscopy, which enables calculation of monomer conversion and provides an estimate of the apparent block molecular mass. Purified polymers were further characterized by size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) to determine the number-average molecular weight (M _n), weight-average molecular weight (M _w), and molar mass dispersity (D _m = M _w/M _n) (Figure d). For consistency and to minimize the influence of molecular weight distribution on physical properties of the diblock copolymers, only diblocks with a dispersity of ≤1.2 were retained for subsequent studies. The DP of each block was calculated by dividing the measured M _n of the block by the formal molecular weight of the constituent monomers, and the resulting values along with the starting monomer compositions were used to generate a systematic naming scheme for the library. Each polymer was given a unique ID reflecting the DP of the respective hydrophilic and hydrophobic blocks.

Prior to evaluation of the physical properties of the diblock POEGMAs, the terminal CTA groups introduced during RAFT synthesis were cleaved from all polymers to eliminate potential cytotoxicity associated with the thiocarbonylthio moiety. This step also ensured that subsequent measurements reflected intrinsic polymer behavior without contributions from the residual end-group chemistry.

Physical Characterization of the Diblock POEGMAs

To investigate the thermal phase behavior of the rB-series diblock POEGMAs, we monitored solution turbidity by measuring the absorbance at 600 nm as a function of temperature across a range of polymer concentrations in phosphate-buffered saline (PBS). Heating and cooling cycles were performed to assess the reversibility of the transition, and the T _t was defined as the inflection point corresponding to a sharp increase in absorbance during heating, indicative of polymer phase separation (Figure ). All three diblock POEGMAs in the rB-series exhibited well-defined and reversible lower critical solution temperature (LCST)-type phase behavior, with heating-induced phase separation and resolubilization upon cooling. The measured T _t values at 100 μM concentration are summarized in Table .

2.

LCST phase transition behavior of the diblock POEGMA (a) rB-1, (b) rB-2, (c) rB-3, determined by measuring the absorbance of the polymer at 600 nm at various concentrations as a function of solution temperature. Heating curves are represented by solid lines and cooling curves are represented by dashed lines. A sharp increase in absorbance indicates the T _t of the polymers.

1. Summary of the Physical Properties of the rB Diblock POEGMA.

label	ID	M w (Da)	T t (°C)	CMT (°C)	d (nm) ± SD	R g (nm) ± SE	shape
rB-1	(EG332-co-EG232 )-b-(EG181)	27,740	39	<rt	36 ± 12	11.5 ± 1.2	globular
rB-2	(EG311-co-EG264)-b-(EG28-co-EG145)	24,550	30	<rt	25 ± 10	17.4 ± 3.7	elongated
rB-3	(EG311-co-EG264 )-b-(EG152)	24,960	31.5	<rt	44 ± 18	13.0 ± 2.5	fractal

^aDetermined by SEC-MALS.

^bDetermined by DLS.

^cDetermined by SAXS. rt: room temperature. The T _t and CMT are reported at 100 and 50 μM, respectively.

Consistent with design expectations, the T _t were tuned by varying the EG side-chain length distribution between the hydrophilic and hydrophobic blocks. Notably, all three diblocks in the rB-series displayed a T _t below 37 °C, demonstrating their suitability for in situ depot formation upon subcutaneous injection. These results establish that by modulating block composition, even with short EG side chains (EG1–EG3), the LCST behavior of POEGMA can be precisely controlled without the need for additional components. This tunability provided a foundation for further exploration of self-assembly and thermoresponsive gelation properties of POEGMA for controlled drug delivery and depot formation.

To evaluate the self-assembly behavior of the rB diblock POEGMA copolymers, we performed DLS at temperatures below their T _t, which revealed that all three polymers form stable nanoscale structures in aqueous buffer with a well-defined hydrodynamic diameter (Figure a–c). For example, rB-1 at room temperature at a concentration of 200 μM formed nanostructures with an average hydrodynamic radius of 18 ± 4.8 nm, indicating uniform micellization below its T _t. Even at a much lower concentration of 12.5 μM, stable nanostructures were observed, confirming that the CMT lies well below room temperature. This low CMT is particularly advantageous for biomedical applications, as it ensures consistent nanoparticle formation upon dilution in physiological conditions.

3.

Self-assembly of diblock POEGMA. Hydrodynamic diameter of (a) rB-1, (b) rB-2, and (c) rB-3, determined by DLS at temperatures below their T _t at a low (12.5 μM) and a high (200 μM) concentration. (d–f) SAXS analysis of diblock POEGMA. 1-D SAXS plot and respective model fits for (d) rB-1, (e) rB-2, and (f) rB-3 at 5 mg/mL at room temperature.

To gain deeper insight into the morphology of the self-assembled nanostructures formed by these diblock POEGMA copolymers, SAXS analysis was performed at a concentration of 5 mg/mL at room temperature (Figure d–f). The 1D SAXS profiles of all three polymers displayed scattering that is characteristic of well-defined nanoscale particles. The near-zero slope at low q values for Rb-1 is indicative of spherical nanoparticles (Figure d), whereas the more pronounced negative slope (−1.5) at low q for Rb-2 and Rb-3 (Figure e,f) is indicative of elongated or fractal structures. This is further corroborated by the presence of a pronounced peak in the Kratky plots, indicating a high degree of compactness in the self-assembled structures (Supplementary Figures 1–3). The corresponding Guinier plots show a clear linear region at low q ², allowing for accurate estimation of the radius of gyration (R _g) (Supplementary Figures 1–3). Using R _g = sqrt­(−3 × slope), the calculated R _g value is 11.5 ± 1.21 nm for rB-1. The Guinier analysis of rB-2 and rB-3 yielded an R _g of 17.4 ± 3.69 and 13 ± 2.45 nm, respectively.

Using the R _g values calculated from SAXS and R _h values from DLS, we calculated the form factor (ρ = R _g/R _h) that give an indication of the shape of the nanoparticles. , For rB-1, ρ is 0.48 indicative of a spherical nanoparticle, whereas for rB-2, ρ is 1.33, indicative of cylindrical or elongated micelles. Interestingly, the ρ value for rB-3 is 0.26 which is quite low and does not imply a conclusive shape (Supplementary Figures 1–3).

Further analysis of the SAXS data was performed by model fitting (Figure d–f). The SAXS data for rB-1 was fit to a polymer micelle model that provides form factor for a micelle with a spherical core and Gaussian polymer chains attached to the surface and thus may be applied to block copolymer micelles , (Figure d). The model yielded a core radius of 7.4 ± 0.1 nm with attached Gaussian polymer chains with an R _g of 3 ± 0.5 nm and the aggregation number is 115 ± 4. A cylinder model that describes cylinder with uniform scattering length density was fit to rB-2 (Figure e). − The model yielded an 8 nm radius for the cylinder with an average length of 59.1 nm with a Gaussian distribution of polymer chains. RB-3 was fit to a fractal model that describes scattering from fractal like aggregates of spheres (Figure f). The model yielded a radius of 11.2 nm with a fractal dimension of 2.3 and a cluster correlation length of 14.4 nm. The similarity of the correlation length to the radius indicates that interactions between micelles are weak and that one would expect to see mainly isolated micelles in the fractal aggregate. Any clustering of the micelles is likely due to phase separation of the micelles. , These differences in the self-assembly behavior among the three polymers likely stem from variations in their block composition, which influences chain packing and interfacial curvature. Taken together, the SAXS data support the formation of well-defined nanostructures by all three POEGMA diblock polymers at room temperature (below their T _t).

To directly visualize the morphology of the self-assembled structures formed by rB-1 and rB-3, cryogenic transmission electron microscopy (cryo-TEM) was performed at room temperature. For rB-1, the cryo-TEM micrographs revealed uniform, spherical, and monodisperse nanoparticles. Quantitative image analysis using ImageJ on 50 particles from five independent micrographs yielded an average diameter of 11.3 ± 3 nm (Figure a), consistent with the DLS and SAXS findings and confirming the formation of compact spherical micelles.

4.

cryo-TEM micrographs of (a) rB-1 showing spherical micelles, inset showing a zoomed-in nanoparticle. (b,c) rB-3 shows two distinct morphologies: (b) core–shell spherical nanoparticles, inset showing a zoomed-in nanoparticle, and (c) fractals or worm-like micelles; inset and blue arrows pointing toward spherical nanoparticles budding off from the ends of worm-like micelles.

Interestingly, rB-3 displayed more complex behavior. Two distinct morphologies were observed across different regions of the TEM grid (Figure b,c). First, rB-3 assembled into spherical core–shell nanoparticles with a clearly delineated core and corona structure. ImageJ analysis revealed a core diameter of 11.9 ± 2.5 nm and an average corona radius of 7 ± 1.3 nm. Second, rB-3 also formed fractals (clusters) with internal diameters comparable to those of the spherical particles. This duality is in good agreement with SAXS data. Notably, we were able to capture a rare transition of spherical core–shell nanoparticles budding off from the ends of worm-like micelles (Figure c). This observation provides visual evidence for fractal assemblies that may serve as a reservoir for the generation of monodispersed spherical micelles under certain conditions. Based on DLS, SAXS, and cryo-TEM imaging, we established that all three polymers in the rB-series of diblock POEGMA exist as self-assembled nanostructures before coacervation upon heating above their T _t.

Next, we studied the reversible nature of this transition from micelle to coacervate phase to evaluate if the micellar structures are maintained during coacervation and if the coacervates dissolve back into micelles upon cooling below the T _t or if the polymer chains are reorganized into a different arrangement (Figure a). We encapsulated a small hydrophobic molecule Doxorubicin (Dox) into the rB-3 POEGMA nanoparticles and used the intrinsic fluorescence of Dox to track the fate of self-assembly. Briefly, the rB-3 nanoparticles encapsulating Dox were heated above its T _t to trigger the coacervation and the polymer phase was collected at the bottom of tube by centrifugation. The bright red color of the Dox was observed within the pellet at this step, indicating that the Dox is still present within the polymer. The supernatant was removed, and the polymer phase was dissolved back into solution by adding cold aqueous buffer and hence reversing the coacervation. This process of heating to trigger the coacervation and cooling to reverse it was repeated two additional times, and the resulting single cold phase was analyzed by agarose gel reverse-electrophoresis to visualize if the Dox was still encapsulated within the polymer nanoparticles (Figure b). The positively charged free Dox migrates toward the negative electrode in an agarose gel, whereas its charge is masked when encapsulated in the neutral polymeric nanoparticles, and hence, the encapsulated Dox stays in the well. A diblock POEGMA'polymer A’ that shows LCST phase behavior (Supplementary Figure 4) but does not self-assemble below its T _twas used as a negative control. We observed that the Dox remained encapsulated within the rB-3 nanoparticles and not in polymer A after going through three rounds of coacervation, which is indicative of preservation of rB-3 nanostructures during coacervation that are dissolved back into the aqueous phase upon cooling below the T _t.

5.

Reversible nature of micelle to coacervate phase transition. (a) Schematics of temperature-induced micelle to depot transition that is reversible. (b) Agarose gel showing that the fluorescent small moleculeDoxis still encapsulated within the polymer rB-3 nanoparticles, and not in a control polymer A, after going through three rounds of heating and cooling cycles. Polymer A is used as a negative control because it coacervates at a similar temperature as rB-3 but does not self-assemble.

To study the phase transition of the rB-diblock POEGMA series from a nanoparticle to a coacervate phase, we visualized these polymers by low-resolution light microscopy above their T _t in the presence of thioflavin T (ThT) dye. ThT is a fluorescent dye that is typically used to visualize amyloid structures due to its enhanced fluorescence when bound to protein aggregates. However, we used ThT here for its nonspecific increase in fluorescence when present in a hydrophobic environment to visualize the phase transition of the rB-series by fluorescence microscopy. The green fluorescence of ThT above the T _t of the polymers is indicative of an increase in the local hydrophobicity upon the phase transition of the polymer into a coacervate phase. Notably, we observed a strong ThT fluorescence for rB-2 and rB-3 above their respective T _t values, likely indicative of hydrophobic interactions between the polymer chains (Figure ).

6.

Light microscopy of the rB-series above their T _t in the presence of Thioflavin T (ThT) dye. POEGMA (a) rB-1 at 38 °C, (b) rB-2 at 35 °C, (c) rB-3 at 40 °C.

To get deeper insights into the phase behavior and visualize the strong hydrophobic interactions of the structurally complex rB-2 above its T _t, we covalently labeled the polymer with Alexa Fluor 488. The confocal microscopy of fluorescently labeled rB-2 revealed a network-like structure for rB-2 at 30 °C in contrast to liquid droplets for polymers exhibiting liquid–liquid phase separation behavior (Figure a). We included a simple diblock EG3-EG2 POEGMA “polymer A” as a control that exhibits liquid–liquid phase separation. As expected, confocal imaging of the fluorescently labeled polymer A revealed the classic LCST phase behavior of this control polymer showing spherical liquid-like droplets that fuse together with time to minimize the surface tension (Figure c).

7.

Confocal microscopy (a–c) and fluorescence recovery after bleaching (FRAP) (d–f) for AF488 conjugated complex diblock POEGMArB-2 at 30 and 35 °C and the control POEGMAA at 31 °C.

To investigate the physical properties of the unique network-like phase of the rB-2 polymer upon heating, we used Fluorescence Recovery After Photobleaching (FRAP), a technique that provides insights into fluidity and molecular mobility of the polymer upon phase transition. The control diblock polymer A that shows liquid–liquid LCST phase behavior and has a similar T _t as rB-2 was selected as a control for the FRAP experiment as well. For both labeled polymers, a few spots were locally photobleached using the laser, and the fluorescence recovery was monitored as a function of time. The fluorescence intensity of the control polymer at 31 °C quickly recovered (Figure f), whereas the fluorescence intensity of polymer rB-2 at 30 °C stayed at the same low level as immediately after bleaching, revealing that the molecular exchange in rB-2 is arrested and that the coacervate is more solid-like (Figure d). Interestingly, rB-2 at 35 °C visually does not exhibit the micronetwork assembly and looks like liquid dropletslike the liquid droplets of the control polymer A, but the fluorescence recovery after bleaching suggests that molecular exchange is arrested at 35 °C and the coacervate is more solid-like despite its spherical morphology (Figure b,e).

To understand the material property of the self-assembled states of the polymer, we next employed video particle tracking (VPT) nanorheology , (Figure a), wherein the passive motion of 200 nm sized beads embedded inside the polymer networks and droplets was monitored. The trajectory of the beads inside the condensates was analyzed to estimate their mean-squared displacement (MSD) (Figure b). The bead diffusion within the coacervate phases revealed terminally viscous behavior of the material with a diffusivity parameter, α = 1, signifying the Brownian nature of the bead motion. This allowed us to use the Stokes–Einstein relation to estimate the viscosity of these coacervates. Our analysis revealed that the control POEGMA ‘A’ condensates have a viscosity of 6.35 ± 0.63 Pa·s (Figure c).

8.

(a) Schematic representation of the video particle tracking (VPT) nanorheology used to quantify the material property of the polymer A and POEGMA rB-2 condensates and their different phases. The left panel displays a schematic of beads embedded inside a sample, accompanied by an enlarged view of a bead’s trajectory. The right panel shows the epifluorescence image of a polymer A condensate with beads embedded inside, along with the tracked trajectory of a bead. (b) Ensemble-average mean-squared displacement (MSD) of the beads performing Brownian motion inside the condensates. The terminal region of the MSD has a diffusivity parameter, α = 1, signifying Brownian motion of beads inside the condensate, allowing the estimation of zero-shear viscosity of the condensates. (c) Mean viscosity of polymer A condensates quantified over three different samples. (d) Epifluorescence images of the rB-2 POEGMA when heated from 15 to 85 °C. The temperatures were chosen to capture different transition states of the polymer. (e) Epifluorescence images of the same sample when cooled down to 15 °C from 85 °C, at similar transition temperatures. (f) Schematic showing the reduced extent of bead motion when the beads are dynamically arrested. Temperature-controlled VPT nanorheology was performed to estimate the ensemble-averaged MSDs of the beads inside the coacervate phases observed at different transition temperatures when the POEGMA rB-2 was (g) heated from 15 to 85 °C and (h) subsequently cooled from 15 to 85 °C. In the figure, α = 1 and α < 1 depict the normal diffusion and subdiffusion of the beads, respectively, in the phases observed at T _t’s as indicated. α plotted for different coacervate phases observed at the T _t’s when rB-2 POEGMA was (i) heated and (j) subsequently cooled. Panels (i) and (j) also have the viscosities plotted for the phases for which α = 1, as indicated in the figure.

In contrast to the spherical coacervates observed for the control polymer A, we found that the rB-2 polymer network undergoes shape relaxation, accompanied by a LCST phase transition, when heated from 15 to 80 °C (Figure d). Below the T _t, the polymer remains soluble, and fluorescence is diffuse. When the temperature was increased to 23 °C, a mesh-like network formed with particles on the order of ∼1 μm (Figure a and Supplementary Figure 5a,b). As the temperature was increased, the network underwent shape relaxation, indicating that the network is extensively percolated, and by 32 °C, larger 5–10 μm structures were dominant. Droplets fully relaxed, becoming completely spherical if left at or above 80 °C for more than 10 min (Supplementary Figure 5b). An additional transition is observed reversibly between 32 and 34 °C, where micelles formed inside the condensed phase (Supplementary Figure 5c). Upon cooling to 15 °C, we observed that the droplets swell until they disperse back into the dilute phase (Figure e). Unlike the percolation transition reported for RNA, where percolation traps the RNA molecules in the condensed state, we observed no similar hysteresis in the phase behavior of POEGMA rB-2. Additionally, we performed spatial autocorrelation (SAC) analysis of these coacervate phases which showed that a rapid transition back into the solution-phase upon cooling with the starting and ending states is indistinguishable (Supplementary Figure 6).

To map the changes in the material properties during temperature-dependent transitions observed in Figure d,e, we used temperature-controlled VPT nanorheology , (Figure f–j). We quantified MSDs of the beads inside the structures observed at the T _t while heating and cooling the rB-2 polymer solution (Figure g,h). At 15 °C, the polymer rB-2 was completely soluble, and beads were highly dynamic in it, resulting in a high MSD (Figure g). However, when the temperature increased to 23 °C, the beads were dynamically arrested in the mesh-like structures that emerged upon heating, resulting in a dramatic shift in the MSDs to a lower value. This shows that the mesh-like structures are more dynamically arrested compared to the starting state at 15 °C. Interestingly, a further increase in the temperature resulted in faster diffusion of beads compared to the mesh-like state observed at 23 °C, as observed with an increase in the MSDs with an increase in the temperature to 40 °C and subsequently to 85 °C (Figure g). However, when the system was cooled down to 40 °C, it went into a dynamically arrested state, indicated by the decrease in the MSDs (Figure h). Decreasing the temperature resulted in the swelling of the droplets, but with no appreciable change in the bead diffusion when the temperature was subsequently changed to 32 °C. Finally, when the temperature was further decreased to 15 °C, the coacervates dissolved, and a solution phase was again observed with highly dynamic bead motion, like the starting phase of the sample before heating.

We next quantified the material state of these distinct phases during heating using the diffusivity parameter, α. Values of α were extracted from the MSD profiles at each transition temperature. During heating, we observe that α transitions from 0.99 ± 0.06 at 15 °C to 0.33 ± 0.16 at 23 °C where the mesh-like structures were formed. This transition in α indicates a transition of beads from Brownian motion to subdiffusive motion in the polymer phases at different temperatures, depicting a dynamically arrested state at 23 °C. The diffusivity parameter showed an increase in temperature from 23 to 32 °C and from 32 to 40 °C, where relaxation of the mesh-like structure was observed (Figure i). Note that at temperatures 23, 32, and 40 °C, α still indicates subdiffusive behavior of the beads inside the polymer phases, and thus, viscosity for these phases could not be quantified. However, when the polymer mesh was completely relaxed into a spherical droplet at 85 °C, α = 0.99 ± 0.02 depicted the Brownian motion of beads inside the polymer phases with a viscosity of 6.19 ± 0.49 Pa·s (Figure i).

We subsequently cooled down the same sample, starting with 40 °C, and tracked the bead motion to understand how the transition upon cooling affects the bead dynamics (Figure j). We observed that the polymer transitioned back into a dynamically arrested state with α = 0.69 ± 0.08. Decreasing the temperature further to 32 °C showed a decrease in α to 0.43 ± 0.07, suggesting a relatively more arrested state compared to 40 °C. Changing the temperature further to 23 °C did not change the material state of the polymer phase significantly with α = 0.48 ± 0.06. However, decreasing the temperature to 15 °C led to a transition of the dynamically arrested polymer phase into a single polymer soluble phase with α = 0.99 ± 0.02 and viscosity of 3.73 ± 0.05 mPa.s, similar to the reversibility shown in Supplementary Figures 5 and 6.

Next, we evaluated the potential recognition of the diblock POEGMA nanoparticles by anti-PEG antibodies by using an indirect ELISA assay. Plates coated with either PEG (positive control) or POEGMA were incubated with commercially available anti-PEG IgG or IgM antibodies over a range of concentrations, and binding was quantified by measuring the absorbance at 450 nm. PEG exhibited strong concentration-dependent binding to both anti-PEG IgG and IgM, as expected. In contrast, POEGMA nanoparticles showed negligible binding to either antibody class, even at the highest antibody concentrations tested (Supporting Information Figure 8), demonstrating that the diblock POEGMA system effectively evades recognition by anti-PEG antibodies.

Conclusions

In this work, we explored the potential of structurally versatile and thermoresponsive POEGMA-based block copolymers to create self-assembled nanoparticles and physically cross-linked hydrogels. We designed and synthesized a series of diblock POEGMA polymers (rB-series) that are composed of side-chain lengths limited to three EG units or less. We characterized their thermoresponsive behavior and self-assembly using UV–vis spectroscopy, DLS, SAXS, and cryo-TEM. Our results reveal that all three polymers exhibit LCST phase transitions and form well-defined nanostructures below their T _t’s. SAXS and cryo-TEM analyses confirmed that rB-1 self-assembles into spherical micelles, while rB-2 and rB-3 form more elongated structures.

The behavior of rB-2 is particularly strikingit forms a distinct network-like structure upon heating, rather than undergoing the classical liquid–liquid coacervation observed in typical LCST polymers. Confocal microscopy and FRAP experiments provided compelling evidence that this arrested network-like coacervate is markedly less dynamic than conventional liquid coacervates and behaves more like a solid gel. Even at higher temperatures where rB-2 visually appears more liquid-like, molecular mobility remains restricted, suggesting persistent hydrophobic interactions or local microstructural order that governs its physical state. Taken together, these results highlight the unique capabilities of POEGMA polymers to access a wide array of self-assembled morphologies and phase behaviors simply by tuning block composition and architecture.

Looking forward, this work opens several exciting avenues for future exploration. The distinct arrested phase behavior of rB-2 warrants a deeper mechanistic investigation to unravel the underlying interactions and network topology. Additionally, the tunable nanoparticle and hydrogel systems described here can be further functionalized with targeting ligands or responsive motifs (e.g., pH- or enzyme-sensitive groups) to enable precision delivery and smart responsiveness.

The ability of these polymers to form stable nanoparticles under ambient conditions and undergo temperature-triggered phase separation near physiological temperature positions them as attractive candidates for injectable depot systems, where in situ gelation can enable sustained and localized delivery of therapeutic payloads. The absence of chemical cross-linkers, combined with reversible gelation and shear-thinning behavior, further supports their potential use in minimally invasive administration and tissue engineering applications. In addition, the observed dynamic arrest and unique transition to arrested coacervate states may also provide new opportunities to regulate molecular diffusion and retention within the material, which could be leveraged to control the drug release kinetics.

Importantly, the stealth properties of POEGMA in the context of anti-PEG antibodies offer a strategic path for the translation of self-POEGMA nanoparticles into clinically relevant delivery platforms, particularly for patients sensitized to PEG. In conclusion, this work establishes that precise control over block copolymer architecture enables tunable structure, dynamics, and function in thermoresponsive POEGMA systems, providing a robust foundation for the development of next-generation polymer materials for drug delivery, injectable depots, and soft biomaterial engineering.

Methods

Materials

All reagents for the synthesis of the block coPOEGMA were purchased from Sigma-Aldrich. The monomerstriethylene glycol methyl ether methacrylate (EG3MA, CAS No. 24493-59-2), diethylene glycol methyl ether methacrylate (EG2MA, CAS No. 45103-58-0), and ethylene glycol methyl ether methacrylate (EG1MA, CAS No. 6976-93-8)were passed through a column of activated basic aluminum oxide (CAS No. 1344-28-1, Sigma # 199443) to remove inhibitors before polymerization reactions. 4-Cyano-4-(thiobenzoylthio) pentanoic acid (CAS 201611-92-9, Sigma # 722995), 2,2′-Azobis (2-methyl propionitrile) (AIBN) (CAS 78-67-1, Sigma # 441090), and solvents were used as received.

Synthesis of Diblock POEGMA Copolymers

POEGMA diblock copolymers were synthesized in a two-step sequence of reversible addition–fragmentation chain-transfer (RAFT) polymerization employing 4-Cyano-4-(thiobenzoylthio) pentanoic acid as the chain transfer agent (CTA) and AIBN as the free radical initiator. The first POEGMA block (random copolymer of EG3MA and EG2MA) was polymerized by mixing the EG3MA and EG2MA monomers with CTA and AIBN at a 4x:4:1 mol equiv ratio, respectively, in toluene as a solvent, where x is 1.2 times the desired degree of polymerization (DP) of the combined monomers. The reaction mixture was prepared in a round-bottom flask on an ice bath, and the reaction flask was deoxygenated by inserting a stream of nitrogen gas directly bubbled into the reaction mixture using an inlet needle accompanied by an outlet needle for 40 min before initiating the polymerization at 70 °C for a specified time with continuous nitrogen purging. The reaction time ranged from 1 to 3 h depending on the target molecular weight and the initial monomer concentrations. The reaction was quenched by submersion of the round-bottom flask in liquid nitrogen, and the resulting random EG3-co-EG2 POEGMA was purified by polymer precipitation in a bad solvent. The polymer was forced to precipitate out of the reaction mixture by addition of 5 to 8 times hexane (by volume) in the reaction mixture. The supernatant containing any unreacted monomer was discarded, and the precipitated polymer was dissolved in 1 mL of acetonitrile. The process of polymer precipitation in hexane and dissolving in acetonitrile was repeated 3-times to get a pure product. The purified polymer was freeze-dried and stored at −20 °C until further use. To complete the synthesis of the diblock copolymer, the second blocka random copolymer of EG2MA and EG1MA or just EG1MAwas polymerized by utilizing the first POEGMA block as a macromolecular CTA. To do so, the first polymer block was resuspended in toluene, and the EG2MA and EG1MA monomers and AIBN were added to the RB flask on an ice bath. The reaction conditions and purification procedure were identical to those used for the first block. The exact mol ratios and volumes used for the synthesis of rB-2 diblock POEGMA are listed in Supplementary Tables 1 and 2, as an example.

CTA End Group Removal

The active partthiobenzoylthioof the CTA present as an end group on RAFT synthesized polymers was cleaved off from the polymer chain by treatment with a 20-fold molar excess of AIBN at 80 °C for 3 h in an inert environment, resulting in a colorless solution. The CTA-free polymer was purified by three rounds of hexane precipitation.

Chemical Characterization of POEGMA

The polymerization reactions were monitored with proton nuclear magnetic resonance (¹H NMR) spectroscopy on a 500 MHz NMR spectrometer (Varian) to determine the percent monomer to polymer conversion. After quenching the polymerization reaction, a small amount of the mixture was dissolved in deuterated chloroform (CDCl₃) containing 1% (v/v) tetramethylsilane (TMS) as an internal reference to record the ¹H NMR spectrum. The NMR data was analyzed using MNOVA software. All purified polymers were additionally characterized for number-average and weight-average molecular weightsM _n and M _w, respectivelyand molar mass distribution (D _m) by size exclusion chromatography in line with multi angle light scattering (SEC-MALS) using an Agilent 1260 Infinity HPLC equipped with a DAWN HELEOS II MALS detector and an Optilab T-rEX refractive index detector (Wyatt Technology). The polymers were resuspended in HPLC grade tetrahydrofuran (THF) at a concentration of 2–5 mg/mL and filtered through 0.2 μm syringe filters (Whatman). Each sample (50 μL) was injected into dual Agilent gel columns (7.5 × 300 mm, 1 79911GP-503 (103 Å) and a 1 79911GP-504 (104 Å) and run at room temperature using THF (containing 100 ppm butylated hydroxytoluene (BHT) as a stabilizer) as the mobile phase at a flow rate of 1 mL/min. The light scattering data was analyzed for M _n and M _w using ASTRA software (Wyatt) by inputting the refracting index increment (dn/dc) = 0.07 for POEGMA in THF. The dn/dc value for POEGMA was calculated using the refractive index detector with an online method for a known concentration of the polymer sample.

LCST Phase Behavior

The polymers were resuspended in 1x PBS at 200 μM concentration at 4 °C, and a serial dilution in PBS was performed resulting in 200, 100, 50, 25, 12.5, 6.25 μM as the concentrations to be tested. The optical density (OD) of all polymers was monitored at 600 nm as a function of temperature on a temperature-controlled UV–vis spectrophotometer (Cary 300 Bio, Varian instruments). Starting at 20 °C, the temperature of the samples in the cuvette was increased at a rate of 0.3 °C/min until ∼45 °C and was then cooled back to 20 °C at a rate of 0.3 °C/min. The absorbance was recorded at each 0.6 °C intervals. A sharp increase in the OD with temperature is indicative of the phase transition, and the temperature at the inflection point of the optical density is defined as the T _t. The OD vs temperature was plotted and the temperature at the inflection point that defines the T _t was determined by finding the maximum of the first derivative of the OD versus temperature using GraphPad Prism software.

Dynamic Light Scattering

The size distribution of nanostructures of diblock POEGMA were measured using a Malvern NanoZS Zetasizer equipped with a 173° backward scatter detector. The DLS measurements were performed in triplicate at a range of temperature and concentration in a low-volume quartz cuvette (Malvern #ZEN2112). Data were analyzed by a regularization fit for Raleigh spheres using Malvern sizing software.

Small-Angle X-ray Scattering

SAXS data were collected at the 16ID-LiX Beamline of the National Synchrotron Light Source II (NSLS-II), Brookhaven National Laboratory, Upton, NY. The X-ray wavelength was set to 0.8189 Å, and both small- and wide-angle X-ray scattering (SAXS and WAXS) setups were utilized simultaneously to cover scattering q ranges of 0.006 ≤ q ≤ 3.19 Å^–1, where q = (4π/λ)­sinθ, with 2θ representing the scattering angle and λ indicating the X-ray wavelength. The sample solution and matching buffer (PBS) were measured in a capillary flow cell at room temperature, and data frames (10–16) were recorded with a 0.5 s exposure time per frame while the sample flowed in a single direction. The 2D scattering images collected by the SAXS and WAXS detectors were converted into 1D scattering profiles and merged. Transmission corrections and background subtraction were applied to minimize the water peak at ∼2.0 Å^–1. The final scattering profiles were obtained by averaging the repeated measurements after eliminating outliers and subtracting contributions from the matching buffer. Data processing was performed using LiXTools (https://github.com/NSLS-II-LIX/lixtools) and the Python package py4xs (https://github.com/NSLS-II-LIX/py4xs) in NSLS-II Jupyter Notebook environment. Final scattering profiles were also manually validated using the Irena package for analysis of small-angle scattering data. To calculate the radius of gyration (R _g), we employed the Guinier approximation that at low q, the scattering profile can be approximated as I(q) ≈ I(0)e(−q ² R _g ²/3), where I(0) is the intensity at zero scattering angle. A linear fit to a plot of ln­(I) vs q ² (Guinier plot) in the linear fitting region gives the formula R _g = sqrt­(−3 × a), where ‘a’ is the slope of the Guinier fit. Model fitting was performed using SasView-5.0.6 software.

Cryo-TEM

POEGMA samples were resuspended in 1× PBS at concentrations of 1–5 mg/mL and imaged by TEM at cryogenic temperature for nanoparticle structure analysis below the T_t of the polymerroom temperature. Vitrified specimens were prepared on a copper grid coated with a perforated lacey carbon 300 mesh (Ted Pella Inc.). A 3 μL drop from the solution was applied to the grid in a controlled environment and blotted with a filter paper to form a thin liquid film of solution. The blotted samples were immediately plunged into liquid ethane at its freezing point (−183 °C). The procedure was performed automatically in the Plunger (Leica EM GP). The vitrified specimens were then transferred into liquid nitrogen for storage. The samples were studied using a FEI Talos F200C TEM, 200 kV maintained at liquid nitrogen temperature, and images were recorded by an FEI Ceta16 M camera (4k × 4k CMOS sensor) at low dose conditions, to minimize electron beam radiation damage. The particle size was analyzed manually by measuring the diameters of nanoparticles in ImageJ. Briefly, the scale bar on the TEM micrograph was used as the reference for setting the scale from pixel to nm using the line tool. Then a line was drawn for an imaginary diameter of a nanoparticle on the micrograph and measure functionality under analyze was used to calculate its diameter in nm.

Light Microscopy

The polymers were resuspended in PBS at around 10 mg/mL for envisioning them under a light microscope. A 50× Thioflavin T (ThT, 2 mM) dye stock solution in PBS was added to polymer samples at a 1:50 v/v ratio to visualize polymer assemblies due to fluorescence in high-viscosity microenvironments. For imaging, 3 μL of the sample was placed on a glass slide with double-sided sticky tape lining the sample area to allow sufficient space for phase separation to occur between the slide and coverslip. The samples were imaged on a benchtop Zeiss microscope (Axio Imager.D2m) with a custom heating insert, at a temperature below and above the T _t of the polymer. Brightfield and green fluorescence images were taken under 20× magnification and overlaid in ImageJ.

Fabrication and Imaging of Water-in-Oil Droplets

Water-in-oil emulsion droplets were generated by coinjecting two immiscible liquid phases into a microfluidic droplet generator (Droplet Junction Chip, Dolomite Microfluidics) using precision syringe pumps. The dispersed phase consisted of an aqueous solution of POEGMA in PBS, while the continuous phase comprised an oil–surfactant mixture provided in the Micellula DNA Emulsion and Purification Kit (EURx). The flow rates of both phases were carefully adjusted to achieve droplet formation in the dripping regime, with the continuous organic phase maintained at a constant flow rate of 300 μL/h and the aqueous dispersed phase at 150 μL/h. During droplet formation, the entire microfluidic setup was kept at a low temperature to preserve emulsion stability and avoid temperature triggered phase transition of POEGMA. Droplet generation was visualized and monitored in real time using a 5× objective lens on a Leica inverted microscope, equipped with a Lumenera Infinity 3-1 CCD digital camera.

The formed water-in-oil droplets were collected onto concavity microscope slides for imaging. Fluorescence microscopy was performed using an upright Zeiss Axio Imager D2 microscope equipped with a 20× objective and a GFP-specific filter set (excitation at 485 nm, emission at 518 nm). Temperature control during imaging was maintained using a precision Peltier heating/cooling stage (Linkam LTS120), operated through a Linkam PE95 digital temperature controller.

Confocal Microscopy and Fluorescence Recovery after Photobleaching (FRAP)

The polymers were labeled with AlexaFluor 488 amine (Lumniprobe # 118C0) utilizing the available terminal −COOH group on the POEGMA and employing EDC-NHS chemistry. Unreacted free dye was removed by extensive dialysis against PBS. The Alexafluor 488-labeled polymer was mixed with unlabeled polymer in PBS for a final polymer concentration of 200 μM and labeling percentage of 1–2%. Confocal imaging was performed on a Zeiss 710 inverted confocal microscope heated to slightly above the T _t of the polymer with a custom incubator box. 30 μL of each sample was added to a black 384-well glass-bottom Greiner plate and topped with 50 μL mineral oil immediately to prevent evaporation during imaging. FRAP was performed following the methods and principles outlined elsewhere. Briefly, a small rectangular region of a droplet or gel globule was photobleached to 20–30% of its original intensity using the Argon/2 488 nm laser line at 100% power under 63× magnification. The bleached region was then imaged at 5 frames per second for 40 s to monitor fluorescence recovery. Image analysis was performed using ImageJ and Microsoft Excel, with correction for photofading based on an unbleached reference region. Fluorescence intensity was normalized such that the prebleach level was set to 100% and the immediate postbleach level to 0%. Recovery curves were generated by averaging three regions per sample and plotted using GraphPad Prism.

Sample Preparation for Temperature-Controlled Microscopy and VPT Nanorheology

POEGMA polymer samples were prepared for imaging by diluting the 30 mg/mL stock to 10 mg/mL in 1× PBS. For the POEGMA A polymer, 150 mM (NH₄)₂SO₄ was used to induce the phase separation. In the case of POEGMA rB-2 samples, no salt was used, and the changes in the polymer phases were only due to temperature changes. The samples were prepared in a 10 μL volume and were placed on an 18 × 18 mm microscope coverslip, which was coated with Tween20 solution (20% v/v, Fisher Bioreagents, CAS 9005-64-5). The coverslip coating was done by immersing the coverslips in the 20% Tween20 solution for 30 min, subsequently drying by compressed air, and incubating overnight at 37 °C. The sample was then sandwiched with a 75 × 25 × 1 mm thick microscope glass slide using five layers of double-sided tape (Scotch 3M). To prevent evaporation, ∼175 μL of mineral oil was injected into the chamber to surround the sample.

Temperature-Controlled Microscopy

Samples were prepared as described above. Imaging was conducted as previously described. In summary, for fluorescence images a temperature-controlled stage (Instec) mounted to an Axio Observer microscope (Zeiss) equipped with a Kinetix22 camera (Teledyne FLIR) was used for imaging. This was used to sweep temperatures between 0 to 90 °C while continuously imaging with a 100× oil objective (Zeiss). The temperature was manually set to ramp between each temperature point at a rate of ∼5 °C/min with ∼5 min between ramps to allow for relaxation of the condensates. A K-type thermocouple was positioned within the sample chamber to contact the flow chamber near the imaging position. The temperatures were recorded by a USB digital-to-analog converter (Measurement Computing), and images were acquired with Micromanager at 1s intervals. All experiments were performed with at least three technical replicates. Temperatures were aligned to the images via custom code.

Brightfield images were acquired using the same temperature-controlled stage mounted to a Primovert microscope (Zeiss) with a 40× air objective and a Blackfly S camera (Teledyne FLIR). In this case, the temperature was swept between 0 and 90 °C. We note that the air objective does not function as a heat sink in the same way that the oil immersion objective does allowing for higher temperatures to be reached.

Video Particle Tracking Nanorheology

Samples of POEGMA A were prepared as described above and then placed onto the Zeiss Primovert microscope described in the previous section with a 100× oil-immersion objective lens and the same camera. Condensates were allowed to settle on the glass slide for ∼20 min prior to the experiment start. The microscope was focused on a particular focal plane of the condensates for imaging. 200 nm yellow-green carboxylate-modified polystyrene microspheres (Invitrogen # F8811) partitioned into the condensates were monitored, and a video was collected for 1000 frames at a rate of 10 frames per second (with exposure time set to 100 ms). Measurements were made for three independent sample preparations.

VPT nanorheology was performed as previously reported. , In brief, the movement of the beads partitioned into the condensates is monitored to get the bead trajectories, which can be further analyzed to get the mean square displacement (MSD) of the beads. Parameters such as diffusion coefficients, nature of the diffusion, and the viscosity can be extracted by analyzing the MSDs of the beads embedded inside the condensates. We first used the TrackMate plugin of the ImageJ FIJI software for tracking to extract the diffusion coefficients of beads and produce two-dimensional trajectories of each bead within the condensates. By computing the center of mass vector R at each time point, we calculate the diffusion of the center of mass of the beads. The calculation of the center of mass vector was done using particle velocities according to the following equation:

$$R_{\mathrm{COM}}(k)=R_0+\sum _{k=0}^k{\stackrel{\circledR }{v}}_k=R_0+\sum _{k=0}^k\left(\frac{1}{N_k}\sum _{i=1}^N{v}_{i,k}\right)\mathrm{\Delta }k$$ 1

here, R ₀ is the initial center of mass vector calculated by averaging the coordinates of all beads in the first frame (k = 1), ${\stackrel{\circledR }{v}}_k$ is the mean velocity of all particles in the frame k, v _i,k is the velocity of the particle i in frame k in units of μm/frame, and N _k is the number of particles in the frame k. The quantity Δk is the frame difference, which is 1 in the present case. To ensure the absence of any sample drift in the data, the center of mass is calculated and subtracted from the individual bead trajectories. This was used to calculate the ensemble-averaged mean-squared displacement (MSD) using

$$\mathrm{MSD}(\tau )=⟨R(t+\tau )-R(t)⟩{|}_{t,N}$$ 2

In eq , τ is the lag time. Next, we extracted the diffusion coefficient of the beads by fitting the MSD to the following equation:

$$\mathrm{MSD}(\tau )=4D{\tau }^{\alpha }+N$$ 3

here, D is the diffusion coefficient, α is the diffusivity exponent, and N is a term to account for the tracking noise. For fluids with terminally viscous behavior, α approaches 1 at long lag-times, which allows for the calculation of the terminal viscosity through the Stokes–Einstein equation:

$$\eta =\frac{k_{\mathrm{B}}T}{6\pi Dr}$$ 4

here, r is the particle radius, k _B is the Boltzmann coefficient, and T is the temperature in Kelvin. The averaged value was reported for the viscosity from different condensates over three independently prepared samples of POEGMA A. The error was estimated as the standard deviation.

Temperature-Controlled Video Particle Tracking Nanorheology

POEGMA rB-2 samples were prepared as described in the sample preparation section. The sandwich was then placed on a temperature-controlled stage as described in the previous section. To enable tracking in the sol-phase, 1 μm yellow-green carboxylate-modified polystyrene microspheres (Invitrogen) were used instead of 200 nm beads which were too fast to track in the single-phase regime. These 1 μm beads were mixed with the polymer when the POEGMA rB-2 was in a soluble state to ensure homogeneous mixing of the beads in the sample before imaging. The temperature stage was initially set to 15 °C, at which the polymer is in a single-phase regime. Temperatures were chosen based on prior heating and cooling ramps of the sample shown in Supplementary Figure 5 which also corresponded to the temperature-dependent turbidity plot. The specific temperatures were 23, 32, 40, and 85 °C. At each temperature, the sample was allowed to equilibrate for 10 min before acquiring videos of the bead motion. The same sample was subsequently cooled to the same temperatures in reverse order. To start imaging, the microscope objective was focused on a particular condensate. A time-lapse video was collected for 1000 frames at a rate of 10 frames per second (exposure time is set to 100 ms). Three trials were done for three independent sample preparations of POEGMA rB-2.

Spatial Autocorrelation (SAC) Analysis

To estimate the change in the size of the droplets during the sol–gel transition, the time-lapse fluorescence images were collected using an Axio Observer microscope (Zeiss) with a temperature-controlled stage (Instec) mounted to it and equipped with a Kinetix22 camera (Teledyne FLIR) as described in the temperature microscopy section. The fluorescence images corresponding to different temperatures in the transition were used as inputs for the analysis. The images were subsequently transformed into the power spectral density via the square of the result of the fast Fourier transformation (FFT) using the Weiner–Khinchin theorem and the inverse FFT to estimate the 2D spatial autocorrelation functions, as we have reported previously. The SAC profiles corresponding to the X axis were further fitted with a 2D-Gaussian function to estimate the size of the droplets at that temperature. The analysis was performed for transitions observed across 3 independently prepared samples.

ELISA Assay

Binding of POEGMA to anti-PEG antibodies was assessed using an indirect ELISA. MaxiSorp 96-well plates (Nunc, ThermoScientific #439454) were coated with either methoxy-PEG (mPEG, 20 kDa, Creative PEGWorks) or a diblock POEGMA at a concentration of 200 μg mL^–1 in PBS and incubated for 18 h at 4 °C. Plates were washed four times with PBS and blocked overnight with 5% (w/v) skim milk powder in PBS. Serial dilutions of anti-PEG IgG (mouse monoclonal, Life Diagnostics #5D6-3) or anti-PEG IgM (mouse monoclonal, GenScript #A01795), spanning concentrations from 2 to 0.0325 μg mL^–1, were prepared in 5% skim milk and added to the plates in duplicate for 1 h at room temperature. Plates were then washed twice with 0.1% CHAPS in PBS, followed by four washes with PBS. Biotin-SP-conjugated antimouse IgM (Jackson ImmunoResearch #115-065-075) or biotin-conjugated goat antimouse light-chain IgG (Sigma #AP200B) was added at 0.2 μg mL^–1 and incubated for 1 h at room temperature. After washing, streptavidin–horseradish peroxidase (BioLegend #405210) was added at 0.2 μg mL^–1 in 5% skim milk. Plates were washed again and developed using 100 μL of premixed 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend #421101) for 10 min at room temperature. The reaction was stopped with 100 μL of stop solution (BioLegend #423001), and absorbance was measured at 450 nm using a Wallac 1420 Victor3 plate reader (PerkinElmer). Background absorbance was determined using wells lacking antigen or antibody, and all measurements were corrected accordingly.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acspolymersau.6c00033.

• Experimental procedure such as polymer reaction recipe and characterization details such as additional turbidity data, DLS measurements, SAXS analysis, anti-PEG antibody binding to POEGMA, etc. (PDF)

The authors declare the following competing financial interest(s): P.S., B.S., and A.C. have an international patent application pending titled Block copolymer nanoparticles for sustained drug delivery and methods of making and using same (Application No. PCT/US2024/050476). The technology described herein has been licensed by Veil Therapeutics from Duke University and P.S. and A.C. have a financial interest in Veil Therapeutics.