Self-assembly is ubiquitous in nature. [1][2][3][4][5][6] Natural molecules such as amino acids, peptides, proteins, and DNA can form cooperative self-assemblies that execute vital biological functions. [7][8][9][10][11] Such self-assembly can be considered a versatile bottom-up synthesis method and has led to a myriad of functional hierarchical structures in life science and materials engineering. [12][13][14][15][16] Spherulites are one type of self-assembled systems and polycrystalline structures in which acicular crystals radiate from a common center and grow nearly synchronously, leading to a spherical structure. Indeed, spherulites can be obtained from a wide range of materials (e.g., metals, minerals, organic molecules, proteins, and synthetic polymers) and have garnered extensive attention in drug delivery, tissue engineering, and sensing. [17][18][19][20][21][22][23] Moreover, manipulating the formation of spherulites is an important topic in biological systems. For example, the formation of spherulites by amyloid fibers has been linked to neurodegenerative pathologies (e.g., Alzheimer's diseases). 24,25 Controlling spherulite morphology and exploring the mechanism of spherulite growth are vital steps in enhancing material performance and achieving effective disease treatment. [26][27][28] The most common method for preparing organic spherulites is cooling crystallization, [29][30][31] which involves heating a polymer solution to make it supersaturated and then cooling it rapidly to induce crystallization or directly cooling the polymer melt to form crystals. The nucleation and growth of spherulites are affected by temperature, concentration, and solvent qualities; therefore, precise control of the annealing process and solution supersaturation is critical in preparing spherulites. [32][33][34][35][36][37][38] For instance, neat poly(1, 6-hexamethylene adipate) spherulites transformed from negative-type spherulites (radial refractive index > tangential refractive index) at low crystallization temperatures to positive-type spherulites (tangential refractive index > radial refractive index) at high crystallization temperatures. 39 Porous spherulites were formed with isothermal crystallization of poly(L-lactide) in diethyl phthalate and glycerol tri-n-propionate but not in less viscous solvents like dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). 40 While there are numerous examples of spherulite formation from supersaturated polymer solutions and polymer melts, 41,42 spherulite formation in dilute solutions remains a rare phenomenon. Recently, Winnik and co-workers reported the first example of spherulite formation via the self-assembly of a block copolymer in a dilute solution. They then investigated the assembly mechanism. Of note, this system still required the presynthesized nuclei and delicate control of annealing and aging. 43,44 Understanding the morphological changes, structural evolution, and crystallization mechanisms involved in spherulite formation is a challenge due to the demanding conditions required for their growth. 45,46 We recently found that benzene-1,4-dithiol (BDT) can self-assemble into uniform supramolecular nanoparticles (PBDT) via π-π stacking with disassembly in organic solvents, e.g., DMF. 47 Intriguingly, PBDT nanoparticles rapidly dissolved in DMF and then formed spherulites at room temperature within 1 min. This process did not necessitate intricate temperature control or presynthesized complex polymers. This finding motivated us to further employ this system for spherulite design. Moreover, to the best of our knowledge, the synthesis of homopolymeric spherulites from small molecules in solution remains quite rare.
Here, we report the first example of spherulite formation by the self-assembly of small molecules at room temperature (Figure 1). Specifically, BDT can self-polymerize through disulfide bonds to form polymerized uniform PBDT nanoparticles with tunable size in aqueous environment. These PBDT nanoparticles rapidly dissolve in DMF (i.e., generating dispersive PBDT molecules) and then experience step-growth polymerization via disulfide bonds while assembling into spherulites (rPBDT) within 10 min. The dynamic growth of the rPBDT spherulites indicated the unique evolution of this insoluble spherulite: the disulfide-mediated elongation of the chains from PBDT nanoparticles gives the rPBDT spherulites a higher molecular weight that then precipitates into a crystal spherulite. The rPBDT spherulites have broad luminescence performance probably due to the clusterization-triggered emission of PBDT molecules with different molecular weights. They exhibited obvious fluorescence in various media including at extreme pH conditions (pH 1 or 13). We further validated rPBDT spherulites as a biocompatible and traceable particle system. Our findings not only provide a mild fabrication strategy for uniform functional spherulites but also highlight the role of the covalent bond in spherulitic growth which has been largely overlooked.
Homopolymeric PBDT nanoparticles were first synthesized. In bicine buffer (10 mM, pH 8.5), BDT molecules formed PBDT molecules via disulfide bonds, and the increased hydrophobicity led to the self-assembly of PBDT molecules into uniform nanoparticles. Transmission electron microscopy (TEM) result showed that the resulting nanoparticles were ∼70 nm in diameter (Figure S1) and had a smooth spherical morphology (Figure 2a). Interestingly, the nanoparticle size was tunable via seed-mediated growth, [48][49][50] and these PBDT nanoparticles can be modulated from 90 to 450 nm by repeating this protocol (Figure 2b-h). The PBDT nanoparticles prepared by seedmediated growth maintained a smooth spherical morphology and uniform size (Figure 2i,j). It is notable that the size distribution of the products was affected by the quantity of seeds added in the growing step (Figure S2). If a low concentration of seeds (0.1 mL) was used (i.e., high ratio of BDT precursor to seed), then the nucleation and growth of free PBDT nanoparticles would occur, leading to the poor size distribution of the products. Energy-dispersive X-ray spectroscopy (EDX) results revealed that PBDT nanoparticles were mainly composed of S and C elements (Figure 2k) confirming that only BDT was involved in the formation of PBDT nanoparticles. This homogeneous system significantly streamlines this BDT workflow versus our previous system where nanoparticles comprised PBDT and tannic acid. 51 The PBDT nanoparticles were a soft material that deformed during drying (Figures 2l,m, and Figure S3).
Next, the assembly process of PBDT nanoparticles into rPBDT spherulite was investigated. In a typical process, the as- prepared PBDT nanoparticles (15 mg mL -1 ) of 75 μL were added into 1 mL of DMF. The initial state of the mixture dispersion was turbid due to the nanoparticle light scattering (i.e., the Tyndall effect). The material became transparent shortly due to the disassembly of nanoparticles into PBDT molecules. The sample then formed a milky dispersion again in 60 s (Figure 3a and Movie S1), thus indicating the formation of spherulites. High-angle annular dark-field (HAADF) and TEM results showed that rPBDT formed a branched spherulite with a rough surface topography and radial architecture (Figure 3bd). Of note, the obtained spherulites were composed of S (Figure 3e), suggestive of the identical composition with PBDT nanoparticles. In the X-ray diffraction (XRD) patterns (Figure 3f), rPBDT spherulites showed peaks at 2θ = 16.5, 18.2, 19.3, 21.3, 23.4, and 26.4°, respectively, corresponding to a d-spacing of 0.53 0.49, 0.45, 0.41, 0.38, and 0.33 nm, while PBDT nanoparticles did not exhibit obvious peaks. These indicated that the dissolution and step-growth polymerization process transformed amorphous PBDT nanoparticles into crystalline rPBDT spherulites. Selected-area electron diffraction (SAED) from spikes on the rPBDT spherulites surface further confirmed the XRD structural analysis. rPBDT spherulites were crystalline with a dense core and spiky surface appearing as sharp spots in the SAED pattern (Figure 3g,h).
We further investigated the dynamic assembly of the process. Different morphologies were observed after different reaction times. rPBDT spherulites grew from hedrite to sheaf and finally became spherulite (Figures 4a-c andS4). The whole process was simple and rapid (<10 min) without complex temperature control (Figure 4d). The morphology of the spherulites was also modulated by varying the volume of the PBDT nanoparticle solution (Figures 4e-h andS5-S7). With an increase in the number of PBDT nanoparticles in the initial solution, rPBDT spherulites became smaller with denser branches. This is probably due to the generation of more spherulite nuclei at high PBDT molecules. Spherulites prepared from larger nanoparticles displayed increased dimensions and elongated aciculae, which suggests that fewer nuclei were produced when using large PBDT nanoparticles (Figure S8). This hierarchy structure endows rPBDT spherulites with a large specific surface area, which is beneficial for drug delivery application. Notably, rPBDT spherulites showed improved mechanical and colloidal properties compared to PBDT nanoparticles (Figure S9). By recording the TEM images at different tilting angles (Figure 4i), the interface between the bottom of spikelike spherulite and the underlying substrate (i.e., TEM grid) clearly showed intact boundary of the spherulites. This indicated that the PBDT nanoparticles became rigid materials after the spherulitic growth. These spherulites also exhibited excellent stability when incubated with tetrahydrofuran (THF) (Figure 4j), where the spherulites retained similar morphology during THF washing cycles, suggesting their poor solubility in THF at the current state.
The key question underneath this system is what drives the dissolved PBDT nanoparticles in DMF to form the rPBDT spherulites. Raman spectra evidenced the formation of disulfide bonds in PBDT nanoparticles and rPBDT spherulites (Figure 5a), with the disappearance of -SH (2590 cm -1 ) and the formation of disulfide (480 cm -1 ). Due to the poor solubility of the rPBDT spherulites in common solvent including DMF and THF, we used mass spectrometry to identify PBDT fragments after treating rPBDT with dithiothreitol (Figure S10), which also confirmed the presence of disulfide bonds. Moreover, the thermal stability of rPBDT spherulites was significantly higher than that of PBDT nanoparticles (i.e., 150 vs 300 °C) (Figures 5b andS11), which suggested a higher molecular weight of rPBDT spherulites. 52,53 We, therefore, hypothesize that the incubation of PBDT nanoparticles in DMF leads to the disruption of π-π stacking and produced PBDT oligomers with active thiol terminal groups in the solution. These free thiols can form disulfide and bridge two or more oligomers into elongated rPBDT molecules, which makes rPBDT insoluble in DMF and crystallizes into spherulites. This phenomenon suggests that covalent polymerization can be employed for spherulite development, avoiding complicated assembly control (e.g., cooling and aging).
We then attempted to verify this covalent-bond-driven spherulitic formation mechanism. The formation of -S-Sdepends on the oxidation of -SH by oxidant (i.e., O 2 ). The nitrogen atmosphere prevented the solution from becoming cloudy suggesting that the formation of spherulites is linked to the O 2 present in the system (Figure S12a,c). If the -SH groups were oxidized to -SO 3 H groups by H 2 O 2 , 54 the formation of rPBDT spherulites did not occur, indicating the pivotal role of reactive thiols in the formation of -S-Sfor subsequent rPBDT spherulites (Figure S12b,c). Furthermore, we compared the disassembly behavior of rPBDT spherulites to non-crosslinked (low molecular weight) and cross-linked (high molecular weight) polystyrene (PS). The non-cross-linked PS particles, regardless of their size, were immediately dissolved in THF (Figures S13 andS14), while the morphology of the cross-linked PS particles remained basically unchanged (Figure S15). In contrast, rPBDT spherulites remained intact in THF (Figures S16 andS17). This indicates that the high molecular weight of rPBDT spherulites resists dissolution in organic solvent.
Interestingly, rPBDT spherulites exhibited obvious luminescence (quantum yield: 4.6%) after ultraviolet light irradiation. As the irradiation pretreatment time increases, the luminescence intensity reached a maximum level (Figure 5c and Movie S2). We speculate that the luminescence properties of rPBDT spherulites are related to the formation of rigid disulfide hindering the rotation of benzene rings and the supramolecular structure of molecular chains. [55][56][57][58][59] The necessity for UV pretreatment can be linked to the insufficiently dense arrangement of PBDT molecules. This lack of density stems from the rapid progression of step-growth polymerization and selfassembly processes, and UV irradiation may disrupt partial disulfide bonds, 54 which leads to the reorganization of PBDT molecules within the spherulite for a more tightly packed structure. This increased structural rigidity constrains the rotation of the benzene rings within the molecular chains, allowing for the increased release of energy primarily through the fluorescence. 60 Therefore, the fluorescence intensity of rPBDT increases with prolonged irradiation time and eventually stabilizes. The optical characteristics of rPBDT spherulites without ultraviolet irradiation (Figure 5d) and PS particles (Figure S18) ruled out the potential scattering effects. Notably, these rPBDT spherulites exhibited broad emission from blue, green, to red light as they can be excited in different fluorescence channels (Figures 5e andS19). Specifically, they emitted light ranging from 430 to 750 nm (Figure 5f). We also confirmed that both PBDT nanoparticles (amorphous) and rPBDT spherulites (crystalline) exhibited similar fluorescence while the BDT molecules had no fluorescence properties (Figures S20-S23). These results collectively suggested that the crystalline degree had minor effect on the fluorescence properties. The luminescence characteristics of rPBDT spherulites are stable in diverse media even in various organic solvents (e.g., DMSO, DMF, EtOH, and THF) and harsh conditions (e.g., pH 1 or 13) (Figures 5g andS24). The LUMO and HOMO orbitals of rPBDT spherulites in different media were calculated by Gaussian simulation (Figure S25). Although the values in various solvents are different, the energy band gap is almost the same, which led to a similar luminescence performance. The broad-spectrum, stable, and uniform emission characteristics of the spherulites hold great potential for emerging white-light emission materials. 61 We confirmed the biocompatibility of rPBDT spherulites in vitro (Figure S26). Specifically, both PBDT nanoparticles and rPBDT spherulites showed negligible cytotoxicity. This may be because of their inert and robust structure in aqueous media. Significant cellular uptake of spherulites was observed after 24 h incubation with raw cells (Figure 5h). The high cell internalization was ascribed to the spike structure of the spherulites, which facilitates the active engulfment of cells. 62,63 Moreover, the fluorescence of rPBDT was observed in the cytosol of cells. Collectively, such fluorescent spherulites exhibited great potential for traceable drug delivery due to their good biocompatibility, high porosity, superior cellular uptake, and imaging capability.
In summary, we report a facile synthesis of homopolymeric luminescent spherulites by a unique dissolution and reaggregation strategy. Specifically, this synthesis relies on the formation of disulfide bonds, which make shorter PBDT chains into longer rPBDT chains and then precipitate into rPBDT spherulites. The morphology of the spherulites was modulated by varying the reaction time and PBDT concentration at room temperature, avoiding complicated temperature control. The obtained spherulites exhibit excellent structural stability and tolerate various solvents. Moreover, the broad luminescence, good biocompatibility, and branched structures, combined with superior cellular uptake, make these materials attractive for intracellular drug delivery and fluorescence imaging. The simple molecule-driven spherulitic growth not only creates multifunctional hierarchical organic structures but also provides new insights for designing biomimetic architectures.
* sı Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c08356.
Detailed experimental procedures for PBDT nanoparticles, detailed experimental method for preparing PBDT nanoparticles by seed-mediated growth, characterization of PBDT nanoparticles, detailed preparation of rPBDT spherulites with different sizes and morphologies, and characterization of rPBDT spherulites (PDF)
https://doi.org/10.1021/jacs.3c08356 J. Am. Chem. Soc. 2023, 145, 25664-25672