Effect of Micellar Morphology on the Temperature-Induced Structural Evolution of ABC Polypeptoid Triblock Terpolymers into Two-Compartment Hydrogel Network

We investigated the temperature-dependent structural evolution of thermoreversible triblock terpolypeptoid hydrogels, namely poly(N-allyl glycine)-b-poly(N-methyl glycine)-b-poly(N-decyl glycine) (AMD), using small-angle neutron scattering (SANS) with contrast matching in conjunction with X-ray scattering and cryogenic transmission electron microscopy (cryo-TEM) techniques. At room temperature, A100M101D10 triblock terpolypeptoids self-assemble into core–corona-type spherical micelles in aqueous solution. Upon heating above the critical gelation temperature (Tgel), SANS analysis revealed the formation of a two-compartment hydrogel network comprising distinct micellar cores composed of dehydrated A blocks and hydrophobic D blocks. At T ≳ Tgel, the temperature-dependent dehydration of A block further leads to the gradual rearrangement of both A and D domains, forming well-ordered micellar network at higher temperatures. For AMD polymers with either longer D block or shorter A block, such as A101M111D21 and A43M92D9, elongated nonspherical micelles with a crystalline D core were observed at T < Tgel. Although these enlarged crystalline micelles still undergo a sharp sol-to-gel transition upon heating, the higher aggregation number of chains results in the immediate association of the micelles into ordered aggregates at the initial stage, followed by a disruption of the spatial ordering as the temperature further increases. On the other hand, fiber-like structures were also observed for AMD with longer A block, such as A153M127D10, due to the crystallization of A domains. This also influences the assembly pathway of the two-compartment network. Our findings emphasize the critical impact of initial micellar morphology on the structural evolution of AMD hydrogels during the sol-to-gel transition, providing valuable insights for the rational design of thermoresponsive hydrogels with tunable network structures at the nanometer scale.


Materials and Methods for Synthesis of N-Decyl-d21-Amine (Scheme S1, conducted at CNMS)
All reagents were used as received from the suppliers without further purification unless otherwise noted.Decanoic-d19 acid (Lot X-383; stated 98.6 atom %D) was purchased from CDN Isotopes, Quebec, Canada.Lithium aluminum deuteride (98 atom %D, 90 % stated chemical purity) was obtained from the ISOTEC ® Stable Isotopes division of MilliporeSigma.Proton and carbon Nuclear Magnetic Resonance spectra were obtained on a Varian VNMRS 500 NMR spectrometer at the Center for Nanophase Materials Sciences, operating at 499.715 MHz for proton, and were recorded at room temperature in CDCl3 (7.27 ppm 1 H reference and 77.23 ppm 13 C reference).Carbon NMR spectra were obtained using inverse-gated decoupling with a recycle delay of 20 s.

Synthesis of 2-(decyl-d21
)isoindoline-1,3-dione.The Mitsunobu S1 reaction was conducted in a manner similar to that previously described.S2 The crude decan-d21-1-ol product from the previous reaction (ca.0.103 mol) was dissolved in dry ethyl ether (100 mL), and combined with recrystallized triphenyl phosphine (27.02 g, 0.103 mol), and phthalimide (99%, 15.30 g, nominally 0.103 mol), in a 500-mL round bottom flask under nitrogen.The flask was fitted with a 125-mL pressure-equalizing addition funnel, containing diisopropylazodicarboxylate (DIAD, 94%, 22.2 g, nominally 0.103 mol) dissolved in dry ethyl ether (35 mL).Under nitrogen flow, the flask was cooled in an ice-water bath, and the DIAD solution added dropwise over a period of 45 min.The ice bath was then removed, and the slurry stirred at room temperature for 24 h.The slurry was then filtered and the precipitate washed portionwise with ethyl ether until the precipitate cake was white and the filtrate colorless (4 x 15 mL).
The solvent was removed from the filtrate by rotary evaporation to afford as a thick yellow-orange oil, which solidifies upon standing.The solid (42.59 g) is a mixture of mostly the product, triphenyl phosphine oxide, and diisopropyl hydrazidodicarboxylate, and was used in the next step without further purification. 1H NMR (CDCl3):  7.82 (m, 2H, Ar), 7.69 (m, 2H, Ar). 13 C{ 1 H} NMR (CDCl3):

Synthesis of Decan-d21-1-amine (N-decyl-d21-amine).
The crude 2-(decyl-d21)isoindoline-1,3dione product mixture was dissolved in absolute ethanol (70 mL), and an excess of hydrazine monohydrate (7.08 g, 0.14 mol) was added dropwise under nitrogen.The solution was then refluxed with stirring under nitrogen for 22 h, during which an ivory-colored spongy mass form.At this time, 6 M HCl (42 mL) was added to the contents and heating continued for an addition 30 min.The reaction mixture was then allowed to cool to lab temperature, then further cooled in an ice bath.The suspension was then filtered, and the solids washed with cold deionized water (2 × 30 mL).The filtrate and washings were combined, and most of the ethanol removed by rotary evaporation.The concentrated slurry was then cooled in an ice bath, and 3N NaOH added until the solution was alkaline.The alkaline solution was then extracted with diethyl ether (3 × 160 mL).The combined ether extracts were dried (Na2SO4) and the volatiles removed by rotary evaporation to afford the crude product as a turbid brown liquid (28.35 g).This mixture was allowed to stand for several days to allow the triphenyl phosphine oxide and diisopropyl hydrazidodicarboxylate that was carried over to crystallize out as a 1:1 adduct.

CD3(CD2)7CD2CD2-).
Theoretical Scattering Model for Polymer Micelles with a Spherical Core.According to the early description by Pedersen and coworkers, S3, 4 the scattering form factor of a single micelle with a homogeneous core surrounded by Gaussian corona chains contains four different terms: the selfcorrelation term of the core, the self-correlation term of the corona chains, the cross term between the core and corona chains, and the cross term between different chains in the corona.The equation can be written as: In this equation, q is the magnitude of the scattering vector, Nagg is the aggregation number of the micelle, βcore and βcorona are the total excess scattering lengths of the core block and the corona block, respectively.Assuming the core is completely dry, βcore and βcorona are defined as: βcore = (ρcoreρsolvent)Vcore and βcorona = (ρcorona -ρsolvent)Vcorona, where ρcore, ρcorona and ρsolvent are the scattering length densities of the core-forming D block, corona-forming A and M blocks and solvent (D2O), respectively; Vcore and Vcorona are the molecular volumes of a single D block in the core and an A-b-M chain in the corona, respectively.Based on the reported bulk densities of poly(N-allyl glycine), poly(N-methyl glycine) and poly(N-decyl glycine), S5-S8 the ρcore and ρcorona values can be obtained via the equation: ρ = (Σinibi)/V, where bi is the bound coherent scattering length of atomic species i, ni is the number of atoms of i per chain, V is the molecular volume of a single chain that is defined by V = (M0/ρmass)/NA, where M0 is the molar mass of the chain, ρmass is the mass density of the polymeric material, and NA is Avogadro's number.
For a spherical core with a core radius of Rc, the aggregation number (Nagg) is expressed by Nagg = 4Rc 3 /3Vcore., assuming the core contains no solvent.The self-correlation term of the core, Pcore(q), in eq.S1 can be written as: where where R c is the radius of the spherical core.
For the Gaussian chains with a radius of gyration of Rg in the corona, the self-correlation term of the corona chains, Pcorona(q), is therefore given by the Debye function: S9 According to the description, the Gaussian chains are uniformly distributed at a distance dintRg away from the surface of the disk core, where dint is close to unity as to mimic non-penetration of the corona chains into the core region.S3, S4 The interference cross term between the spherical core and the corona chains, S core-corona (q), and the interference cross term between the corona chains, S corona-corona (q), are then written as: The form factor of the polymer micelles with a spherical core can be then obtained by inserting the above terms into eq.S1.
The interparticle structure factor, S(q), which describes the positional correlation of polymer micelles relative to one another, is included in the SANS analysis when interparticle interactions contribute significantly in the scattering profile.For simplicity, the S(q) is described by a hard-sphere interaction that considers short-range repulsive potentials between particles, which depends on the hard-sphere interaction radius (RHS) and hard-sphere volume fraction (ηHS).S10-S13 The detailed analytical expression of S(q) has been described elsewhere.S12, S13 Note that RHS can be described as RHS = Rc + ∆RHS, where ∆RHS is related to the width of the corona region and is approximately 2Rg.S10, S11 The final expression for the scattering intensity using the decoupling approximation is then given by: I(q) = n(P mic (q) + A mic (q) 2 (S(q) − 1)) + I inc (eq.S7) where n is the number density of micelles in the system, Amic(q) is the form factor amplitude of the radial scattering length distribution of the micelle, and Iinc is a q-independent term accountable the incoherent scattering of solvent and hydrogen atoms in the sample.In case where there is little to no interparticle interaction observed, i.e., S(q) ≈ 1, the expression for I(q) is then given by: I(q) = nP mic (q) + I inc (eq.S8) The polydispersity of the core radius can also be considered during the model fitting, assuming a Gaussian number distribution for the core radii, where the Gaussian distribution function is given by: where 〈Rc〉 is the mean radius of the core and σR c is the standard deviation of the distribution truncated at Rc = 0.
Theoretical Scattering Model for Polymer Micelles with a Rod-Shaped Core.For polymer micelles with a rod-shaped core, the form factor has a similar description to the spherical micelle model described above, except for the difference in the geometrical shape of the micellar core.For a micelle with a rod-shaped core, the self-correlation term of the core, Pcore(q), is given by: S4 where where α is the angle between q and the axis of the cylinder parallel to Lc, and J1(x) is the first order Bessel function of the first kind.
When Lc >> Rc + dintRg, Pcore(q) can be simplified as: where Si(x) = ∫ t -1 sin t dt x 0 (eq.S13) The self-correlation term of the corona chains, Pcorona(q), maintains the same expression as described in eq.S4.In case of Lc >> Rc + dintRg, the two cross terms, Score-corona(q) and Scorona-corona(q), can be approximated by: (eq.S14) where J0(x) is the zeroth order Bessel function of the first kind.
Note that the polydispersity of the radius of rod-shaped core is considered during the model fitting, assuming a LogNormal distribution for the core radii, where the LogNormal distribution function is given by: where 〈Rc〉 is the mean radius of the core and σR c is the standard deviation of the distribution truncated at Rc = 0.
It is worth to mention that for polymer micelles with an elongated, rod-shaped core, quantifying intermicellar interactions via small-angle scattering can be challenging due to their anisotropic nature.
Here, we applied the hard-sphere interaction model S12, S13 using the decoupling approximation (eq.S7) for a rough estimation of the intermicellar interactions.In this case, the hard-sphere interaction radius, RHS, serves as an "effective" parameter due to the assumption of centrosymmetric interactions, which may not be able to fully present the actual interactions of elongated micelles.S14

Theoretical Scattering Model for a Binary Mixture of Hard Spheres with Sticky Hard
Sphere Interaction.For the two-compartment hydrogel network, scattering contribution from the two distinct spherical domains can be described by form factors for a binary mixture of hard spheres, while structure factors elucidate the sticky hard-sphere interactions between them.The corresponding expression for the scattering intensity is given by: where n1 and n2 are number densities, A1(q) and A2(q) are the form factor amplitudes for binary hardspheres, which are described by the total volume fraction of the polymer (fp), the hard-sphere volume fractions, micellar radii, and scattering length density contrast between the two types of spheres.In the equation, S11(q), S12(q), and S22(q) are the sticky hard-sphere structure factors that describe the interactions between 1-1, 1-2, and 2-2 spheres by considering both repulsive and attractive interactions.
For the hydrophobic D domain, which is anticipated to be completely dehydrated at all temperatures, its number density, n1, can be described through the relationship between its volume fraction and the volume of an individual spherical domain.This relationship is given by: where fAMD is the volume fraction corresponding to the concentration of the triblock terpolymer in aqueous solution, fD is the volume fraction of the D block within the polymer, Rc, D is the radius of the spherical D domain.
For the A domain, which is thermoresponsive, its hydration state varies with temperature.At higher temperatures, the A domain undergoes dehydration but may still contain residual water.In this case, its number density, n2, is described by the effective volume fraction of the A domain after accounting for the volume fraction of the retained solvent within the A domain.Therefore, n2 is given by: where ηwater, A is the fraction of water content within the A domain, fA is the volume fraction of the A block within the polymer, Rc, A is the radius of the spherical A domain.
The form factor amplitude for the D and A domains, A1(q) and A2(q), is given by: Where ρ1 and ρ2 are the scattering length densities (SLDs) of the D and A spherical domains, respectively.ρM+water is the SLD of the surrounding medium, consisting of solvated M blocks and water within the gel phase.To account for the potential smeared interface between the spherical domains and the surrounding medium, an additional term exp(−q 2 ω 2 /2) has been included, where ωD and ωA are the widths of the diffused interfaces for the D and A domains, respectively.
For the D domain, ρ1 is simply given by ρ1 = ρD, where ρD is the SLD of the core-forming D block.
For the A domain, ρ2 is given by: where ρA is the SLD of the thermoresponsive A block.
For the SLD of the surrounding medium which is consisted of solvated M block and water within the gel phase, ρM+water is given by: where ηwater, gel is the fraction of water content within the gel phase.
For the two-compartment hydrogel networks comprised of ABC-type triblock terpolymers, previous studies have demonstrated that the spherical domains of types 1 and 2 display a non-random, alternating arrangement, which is mediated by the hydrophilic mid-blocks that bridge them.S13 Therefore, it is reasonable to employ a "sticky" hard-sphere model for the structure factor.S15-S17 In this model, the short-range repulsion between the spheres is described by the hard-sphere diameter, which gives a minimum distance that the centers of two spheres can approach each other.On the other hand, a narrow attractive well term is introduced to account for the short-range attractions between alternating types of spheres, thus favoring 1-2 contacts over 1-1 and 2-2 contacts.For a binary mixture of hard spheres, the interaction potential as a function of the distance r between the centers of two spheres is described by a square-well potential, which is given by: where Di,j is the average hard-sphere diameter of spheres of type i and j, which is defined as the mean of their diameters Di and Dj, i.e., Di,j = (Di + Dj)/2.Here, ϵi,j is the depth of the attractive potential well, and ∆i,j is the width of the square well, which represents the range of the adhesive interaction between the i and j spheres.The depth ϵi,j can be expressed in terms of the "stickiness" parameter τ using the equation: where kB is the Boltzmann constant and T is the temperature.By varying the depth and width of the attractive well, the "stickiness" τ can be adjusted, thereby modulating the interaction strength between the spheres.In this study, the perturbation parameter, ∆i,j/(Di,j + ∆i,j), is fixed to 0.05 for simplicity.S16 The interparticle structure factors S11(q), S12(q), and S22(q) can be then estimated as functions of the volume fractions and hard-sphere diameters (or number densities) of the two types of spheres within the gel phase, as well as the stickiness parameter τ that quantifies the strength of interaction between them.S13                 three terms), the low-q scattering term, the high-q scattering term, and the q-independent incoherent background.All SAXS profiles exhibit a primary peak near q = 0.035 Å -1 , which is distinct from the primary peak position observed in the SANS profiles for A100M101D10 in D2O (Figure 2a in the main text), while shows a closer position to that observed for A95M99dD9 in D2O/H2O under the dD contrast matched condition (Figure 2b in the main text).The discrepancy can be attributed to distinct interactions of Xrays and neutrons with matter, resulting in variations in contrast for different regions of the hydrogel sample.The SLD values (ρx-ray) for the A, M and D blocks under X-ray were estimated to be 8.8  10 - 4 , 12.8  10 -4 and 9.0  10 -4 nm -2 , respectively, using the equation ρx-ray = neNAρmassre/M, where ne is the number of electrons per block, NA is Avogadro's number, ρmass is the mass density of the polymer, re = 2.82  10 -15 m is classical electron radius, and M is the molar mass of the block.The SLD of water under X-ray is 9.44  10 -4 nm -2 .As seen from the above plots, the changes in the scattering profiles are primarily temperature-dependent rather than time-dependent under isothermal condition.

Figure S8 .
Figure S8.A comparison between (a) 1 H-NMR and (b) 2 H-NMR spectra of the De-d21-NCA monomer in CDCl3.In the 1 H NMR spectrum, only the -COCH2-protons appear at 4.11 ppm.The deuterated N-C10D21 group, invisible in the 1 H NMR spectrum, is clearly observed in the 2 H NMR spectrum.

Figure S13 .
Figure S13.A comparison between 1 H NMR spectra of (a) A100M101D10 and (b) A95M99dD9 triblock terpolymers in CD2Cl2.Note that the methods for calculating DPn (A) and DPn (M), based on the end group analysis, are the same for both AMD and AMdD terpolymers: DPn (A) = (5  integration of -CH=) / (1  integration of -C6H5); DPn (M) = (5  integration of a-CH3) / (3  integration of -C6H5),where "a" refers to the methyl protons in the M block.By contrast, the method to calculate DPn (D) for AMD and DPn (dD) for AMdD are different.For AMD terpolymers, DPn (D) = (5  integration of b-CH3) / (3  integration of -C6H5), where "b" refers to the methyl protons in the D block.For AMdD terpolymers, the deuteriums on the side chain of the dD segment are invisible in the 1 H NMR spectrum, while the -CH2-on the backbone are hydrogenated, which can be used to determine the DPn (dD) in 1 H NMR. Therefore, DPn (dD) = (5  (integration of -COCH2N-) / (2  integration of -C6H5) − DPn(A) − DPn(M)).

Figure S17 .
Figure S17.SANS profiles of 1 wt.%A100M101D10 in D2O measured at 60 °C.The black line corresponds to the best fit of the data using eq. 2 along with the parameters listed in Table 2 in the main text.The colored lines represent the individual scattering contributions corresponding to different component of eq.2: the binary sticky hard-sphere model contributions from A and D domains (the first

Figure S18 .
Figure S18.Dependence of contrast factor ∆ρ 2 on the volume percent of H2O of the H2O/D2O mixture for (a) the A, M, D, and dD individual blocks and (b) the AMdD polymers.The contrast factor ∆ρ 2 is defined as ∆ρ 2 = (ρp -ρs) 2 , where ρp represents the SLD of an individual block or the averaged SLD of the entire triblock terpolymer, and ρs is the SLD of H2O/D2O mixture.Consequently, the scattering signal from an individual block or the entire polymer, which is proportional to ∆ρ 2 , is minimized when ρp ≈ ρs, i.e., ∆ρ 2 ≈ 0. The dip in the curve indicates the condition where the SLD of the solvent is made equal to that of an individual block, or the averaged SLD of the entire polymer.Hence, two different contrast matching conditions can be achieved by adjusting the D2O/H2O ratio: the "dD contrast matched condition", where the dD block is contrast matched out by the solvent, and the "zero mean contrast condition", where the entire AMdD is contrast matched out by the solvent.

Figure S19 .
Figure S19.SANS profiles and the corresponding fitting curves for (a) 1 wt.%A95M99dD9 in D2O/H2O with 93.8 vol.%D2O (dD contrast matched condition) and (b) 1 wt.%A100M101D10 in D2O at 60 °C.The open black circles represent experimental data and the colored lines represent model fittings using eq. 2 or eq. 3 with varied  and  values.

Figure S20 .
Figure S20.SAXS profiles of the (a) 1 wt.% and (b) 5 wt.%A100M101D10 hydrogels measured at different waiting times after being heated to 40 °C.SAXS profiles of the (c) 1 wt.% and (d) 5 wt.%A100M101D10 hydrogels measured at different temperatures during the heating process.For the temperature-dependent measurements, samples were equilibrated for 30 min before data acquisition.

Figure S21 .
Figure S21.SANS profiles of 1 wt.%A95M99dD9 in D2O/H2O with 93.8 vol.%D2O (dD contrast matched condition) measured during heating process at T ≳ Tgel.The solid black curves correspond to the best fits of the SANS based on eq. 3 described in the main text.All profiles were shifted vertically for clarity by multiplying a factor of 5.

Figure S22 .
Figure S22.(a) SAXS profiles of the 1 wt.%A101M107D21 hydrogels measured at different waiting times after being heated to 40 °C.(b) SAXS profiles of the 1 wt.%A101M107D21 hydrogels measured at different temperatures during the heating process.(c) WAXS profiles of the 1 wt.%A101M107D21hydrogels near q = 0.25 Å -1 measured at different temperatures during the heating process.The scattering contribution from the pure water was subtracted from all the WAXS profiles.For the temperature-dependent measurements, samples were equilibrated for 30 min before data acquisition.

Figure S23 .
Figure S23.(a) SANS profiles of 1 wt.%A96M105dD18 in D2O/H2O with 93.8 vol.%D2O (dD contrast matched condition) measured during heating process at T ≳ Tgel.The solid black curves correspond to the best fits of the SANS based on eq. 3 described in the main text.All profiles were shifted vertically for clarity by multiplying a factor of 5. (b) Changes of core radius (Rc, A) and fraction of water content (water, A) of A domain in A96M105dD18 as a function of temperature.

Figure S24 .
Figure S24.SANS profiles of 5 wt.%A43M92D9 in D2O measured during heating process.As compared to the SANS profiles for 1 wt.%A43M92D9 (Figure5in the main text), the transition point to hydrogel network is clearly decreased to lower temperature at higher polymer concentration, evidenced by the abrupt change in the SANS profile from 25 to 30 °C.At T < Tgel, the SANS profiles exhibit a pronounced scattering shoulder near q = 0.015 Å -1 due to strong intermicellar interactions.Nevertheless, the SANS profiles at T ≳ Tgel display the characteristic scattering peaks with nearly identical peak positions and similar temperature dependent structural evolution to those observed at lower concentration.

Figure S25 .
Figure S25.Representative atomic force microscopy (AFM) height images of the A153M127D10 selfassemblies formed after aging the 1 wt.% aqueous solution at 20 °C for (a) 1 h, (b) 24 h, and (c) 96 h, under stirring at 300 rpm.For AFM sample preparation, the 1 wt.% solution was diluted for ten times and then immediately deposited onto cleaned silicon substrates via spin-coating.AFM measurements were performed using a Dimension Icon AFM (Bruker, Hamburg, Germany) in peak-force tapping mode (i.e., ScanAsyst mode) under ambient conditions, using a ScanAsyst-air probe with a frequency of ~ 70 kHz and a spring constant of 0.4 N/m.The images were captured with a scan rate of ~ 1 Hz and a scanning density of 256 lines per frame.

Figure S26 .
Figure S26.Representative cryo-TEM image of 1 wt.%A153M127D10 in aqueous solution vitrified from 20 °C after the solution been aged at 20 °C by stirring under 300 rpm for about a week.