Template‐free nanostructured particle growth via a one‐pot continuous gradient nanoprecipitation

Engineered nanoparticles have emerged as new types of materials for a wide range of applications from therapeutics to energy. Still, fabricating nanomaterials presenting complex inner morphologies and shapes in a simple manner remains a great challenge. Herein, we report the template‐free one‐pot continuous gradient nanoprecipitation of different types of non‐compatible polymers to spontaneously form nanostructured particles. The continuous addition of antisolvent induces precipitation and (re)organization of polymer chains at the forming particle interface, ultimately and naturally developing complex inner morphologies and shapes while particle grows. This low‐energy‐cost bottom‐up assembly approach applies to various functional polymers, possibly embedded with metal nanoparticles, for continuous growth into well‐organized nanoparticles. UV crosslinking of the particles and core removal allows both confirming the building process and leading to hollow or multivoid nanomaterials.

templates. [19,20]Besides, polymer deposition and purification normally required for nanomaterial production are timeconsuming.[23] Macromolecular incompatibility triggers spontaneous mass transfer within the particles toward thermodynamic equilibrium or, while playing with thermal transitions, out-of-equilibrium states. [24,25]This kinetical pathway offers a variety of periodic molecular organization within the nanoparticles, [22] but final colloidal morphologies and shapes highly depend on the specific structures of polymers, which limits the versatile translation of the technique to alternative functional polymers. [26]anoprecipitation (also called "Ouzo effect") is a kinetically controlled molecular assembly technique that does not need a precursor emulsion.[35] In general, a rapid solvent shifting promotes high supersaturation of the polymers and ultrafast precipitation into onion-like, lamellar, Janus or core-shell particles made of homopolymers or block copolymers separately (mono-component) or together (multi-component). [36,37]Practically, batch, microfluidic, and flash mixing technologies are commonly applied to carry out the solvent-shifting step. [38]41] Here, we report on a template-free particle growth approach to original nanostructured particles through a one-pot continuous gradient nanoprecipitation of polymeric (co)solutes.We show that the continuous increase of antisolvent content induces a spontaneous polymer aggregation at the growing particle/water interface into various inner morphologies and shapes depending on different experimental parameters.Different homopolymers and copolymers are involved, as well as gold nanoparticles for surface functionalization.Finally, a genuine UV-induced crosslinking followed by solvent extraction allows unraveling the inner structuration of the colloids and producing hollow and multivoid nanomaterials.

Mono-component particle growth
To initially prove the concept we derived here, poly(phenyl methacrylate) (PPMA, M n,GPC = 22.9 kg/mol, Đ = 1.20) was first synthesized as a substrate for mono-component continuous gradient nanoprecipitation (Figure 1A).The critical precipitation limit (CPL) of PPMA (solute) in an acetone (solvent)/water (antisolvent) mixed system was determined by a conventional titration process of polymer solution by water until seeing the onset of precipitation (Figure 1B).We designed a continuous solvent-shifting process by progressively adding the antisolvent (0.8 g of water) to the polymer solution (0.5 wt% in 0.2 g of acetone) using a syringe pump (speed: 800 μL/min).We monitored the real-time compositions of the nanoparticles by comparing the ratio of the aggregated polymers (collected by centrifugation) to the initial substrates at different acetone/water ratios using nuclear magnetic resonance (NMR) spectroscopy (Figure 1C).In the region above CPL (set at 0.88 mass fraction of acetone), the polymer was completely dissolved in the solution.Right after crossing the boundary, ∼47% of the polymer chains were precipitated into nanoparticles of 73 nm in diameter (determined by dynamic light scattering (DLS), Figure 1D).Further continuous addition of antisolvent led to the progressive precipitation of remaining polymer chains at the nanoparticle/water interface in a continuous growth manner.At the acetone mass fraction reaching 0.7, all the polymer was precipitated.The particle growth region (green) was shown in Figures 1B-D.The particle diameters grew up to 152 nm (DLS) with a polydispersity index (PDI) of 0.076 (Figure 1D,E).A similar behavior was also observed when precipitating poly(methyl methacrylate) (PMMA) using the same process (CPL: 0.68 mass fraction of acetone, Figure S2).

2.2
Co-substrate-mediated template-free biocomponent particle growth Next, we turned our attention to multi-component systems.In general, simultaneous nanoprecipitation of two different populations of polymers causes random aggregation of nuclei into mixed bicomponent nanoparticles, before reorganization/phase separation in the confined particles possibly occurs. [31,42]To visualize the morphological evolution of a bicomponent system during continuous particle growth, we first used the PPMA/PMMA couple (1/1, wt/wt, feed polymer concentration: 1 wt% in acetone).PMMA exhibited a lower hydrophobicity than PPMA, as measured by contact angle (see Table S1).Such results agreed with the shifting of the CPL to the high antisolvent content region in the phase diagram (Figure S3).The real-time particle composition evolution with antisolvent addition indicated that PPMA nanoparticles were first formed after crossing the first boundary and continued growing with increasing water content (Figure 2A-C).Around 7% of PMMA was incorporated during the first particle growth without any change in morphologies and shapes (compared with mono-component PPMA nanoparticles).Since PMMA exhibited a much lower CPL, the incorporation of few chains of PMMA may be attributed to their encapsulation by the PPMA during its precipitation; this behavior is comparable to what has been reported for drug delivery systems. [43]Once crossing the second boundary, the 93% remaining PMMA was progressively precipitated at the nanoparticle interface to form a new external polymeric layer, resulting in classical coreshell-structured nanoparticles (D z = 218 nm, PDI = 0.133).Compared to a sequential two-step solvent-shifting procedure (Figure S4), [41,44] the continuous particle growth process allowed producing similar a core-shell morphology but with a larger shell thickness and bigger final particle size.Indeed, lower initial supersaturation leads to less nanoparticle generation while crossing the first CPL [45] ; then the subsequent progressive polymer packing at the interface leads to the growing of the nanoparticles.An unsmooth surface was observed for these colloids by scanning electron microscope (SEM), which may be assigned to partial swelling of the PMMA shell by solvent prior to grid drying (see Figure S5).
We subsequently tested a series of poly(methyl methacrylate-co-2-hydroxyethyl methacrylate) prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization (see the experimental part for synthesis and characterization details).In the following, we named the copolymer as P(MMA x -co-HEMA y ), with x = 9, 6, and 4, and y = 1, 4, and 6; these values represented the average relative ratios of units along chains, the total units were deemed as 10.The hydrophobicity of all polymers was first evaluated by contact angle measurement.The increasing incorporation of HEMA units along the polymer chains significantly shifted their CPLs to lower acetone mass fractions (Figure S3).The first system, PPMA/P(MMA 9 -co-HEMA 1 ), behaved similarly to the PPMA/PMMA system to generate core-shell particles with thick outer layer (Figure S6).When the co-substrate P(MMA 6 -co-HEMA 4 ) was introduced with PPMA (1/1, wt/wt, feed polymer concentration: 1 wt% in acetone), it was interesting to see that more than 20% of the copolymer chains were engaged in the polymer aggregation together with 47% of PPMA after crossing the first CPL (Figure 2D), even though the antisolvent ratio a priori seemed too low to trigger its precipitation (no formation of nano-object was observed with these copolymers under such solvent-shifting conditions).The explanation for that was to be found in the amphiphilicity of the copolymer (see Table S1), so that spontaneous adsorption at the nanoparticle/water interface occurred.This was confirmed by the presence of PPMA core and thin copolymer shell in the TEM image (Figure 2E, image 1) and SEM (Figure S7, image 1).Upon decreasing the acetone content to a mass fraction of 0.78, all the PPMA chains were precipitated into the particles with the incorporation of 26% of copolymers that remained stable with antisolvent addition.The PPMA/P(MMA 6 -co-HEMA 4 ) hybrid layer resulted in a marigold flower-like morphologies with wrinkled surfaces (see, e.g., Figure 2E, image 2). [46]After crossing the second CPL, the copolymer chains remaining in the solution started precipitating and depositing to grow an external thick copolymer shell with folded surface (D z = 222 nm, PDI = 0.076) (Figure 2E, images 3 and 4; Figure S7, images 3-5 and Figure S8).A progressive shell growth was observed with the continuous antisolvent addition (Figure S9).Note that we also observed similar morphologies for the PPMA/P(MMA 4 -co-HEMA 6 ) system (Figure S6).Although the increase in HEMA content led to larger particle size, it did not show a significant impact on the relative shell thickness (shell thickness/particle diameter) in the particles.
This particular folding of the polymer chains at the interface was surprising and prompted us to look for an explanation.First, we performed a conventional batch nanoprecipitation by rapidly adding water into the acetone solution of both polymers, leading to simultaneous precipitation of these two populations into nanoparticles.The different water affinity induced spontaneous polymer organization within the particles into classic core-shell nanostructures (Figure S10), confirming the distinctive molecular assembly process of the continuous particle growth process.Also, a two-step sequential nanoprecipitation process produced smooth core-shell particles (Figure S11).It was thus likely that this interesting shape did not arise from the collapse of the swollen copolymer shell during TEM sample preparation.Another possibility could be that copolymer chains of different microstructures would precipitate sequentially depending on the antisolvent ratio, and thus would affect the microstructure of the shell as well.However, the real-time monitoring of the copolymer compositions within the growing colloids by NMR showed a constant molar ratio of [MMA]/[HEMA] (Figure S12).
To test the influence of the polymer compatibility on final microstructures, we made use of a model system, a nonionic dispersant Pluronic F68 and PPMA (full description in supporting information, Figures S13-S15).F68 blended with PPMA gave homogeneous mixtures (Figure S13), and thus batch nanoprecipitation of F68/PPMA only led to plain particles (Figure S14); Whereas, continuous solvent shifting generated core-shell particles with a smooth surface (Figure S15).On the other hand, blend of PPMA and P(MMA 6 -co-HEMA 4 ) macro-phase separated (Figure S13) owing to the strong incompatibility between the two polymers.Thus, in the continuous shifting process, simultaneous co-aggregation of polymers at the adsorption stage, together with the immiscibility of these two populations, would be a priori responsible for the reorganization of the hybrid layer into folded structures.

Process variations
To further gain insight into particle formation, we worked on process parameters that can be independently varied.The PPMA/P(MMA 6 -co-HEMA 4 ) system was used here as an example.First of all, the impact of temperature was investigated by performing continuous solvent shifting at 4, 25, and 50 • C (Figure S16; note that CPLs were similar at 4 and 25 • C, and could not be tested at 50 • C because of rapid sol-vent evaporation issues, not shown).Similar nanoparticles were obtained suggesting that the formation of the inner morphology and shape was independent of temperature.Then, solvent evaporation after nanoparticle preparation was performed at different temperatures (0, 20, 40, and 80 • C, Figure S17).Again, the morphologies and shapes of the particles were the same, so that thermodynamic rearrangement cannot be invoked here. [21,36]esides, the impact of the solvent was also investigated by using tetrahydrofuran (THF) or dimethyl sulfoxide (DMSO) instead of acetone in the continuous solvent-shifting process (Figure S18).Although the CPLs of the polymers were significantly different according to the acetone/water systems, a similar molecular packing process leading to folded shells was observed; the size variation observed here was tentatively attributed to the difference in solvent affinity. [47]he influence of overall feed concentrations was then studied and obtained morphologies are shown in Figure 3A and Figure S19 (substrate/co-substrate = 1/1 wt/wt, pump rate: 800 μL/min).An increase of the feed concentration from 0.05 to 1 wt% in acetone led to an increase of the particle diameter from 116 to 212 nm.However, the solvent shifting at low concentration only afforded smooth core-shell nanoparticles.It may be attributed to the insufficient content of copolymers for regulating the inner morphologies through coprecipitation.To prove this, we looked at the impact of the compositions by altering the PPMA/P(MMA 6 -co-HEMA 4 ) ratio from 10/1 to 1/10 wt/wt (Figure 3B and Figure S20, feed concentration of polymer mixtures: 1 wt% in acetone, pump rate: 800 μL/min).At low copolymer ratio (10/1), the adsorbed P(MMA 6 -co-HEMA 4 ) content was insufficient to modulate the inner morphologies and shapes, which only afforded classical core-shell nanoparticles.The increase of the copolymer ratio effectively regulated the inner organization of the nanoparticles, resulting in an obvious strengthening of the folded morphologies with increased copolymer volumes.Combining these two parameters, concentrations and compositions (Figure 3C), we can conclude that the critical feed concentration of P(MMA 6 -co-HEMA 4 ) to generate folded nanoparticles was around ∼0.125 wt% in acetone and above.
The impact of the particle growth rate was further explored by tuning the syringe pump from 160 to 9900 μL/min (Figure 3D and Figure S21).As mentioned earlier, in the continuous particle growth process, the co-aggregation of the two polymers started when crossing the first CPL and ends after the complete consuming of the soluble PPMA (orange region in Figure 2D).The duration of this hybrid-layer building period can be dictated by the antisolvent addition rate.High pump rate (>1400 μL/min) caused a very short duration (≤2 s), suggesting a rapid solvent shifting with instantaneous bulk aggregation.These conditions, similar to a sequential two-step shifting, resulted in classic core-shell nanostructures.For periods above 3.5 s (pump rate ≤800 μL/min), PPMA aggregation was sufficiently slow to favor copolymer incorporation that regulated the inner morphology and shape.Note also that the lower the feed rate, the larger the particle sizes and shell thicknesses.
with PPMA (Figure 5A).A surface-folded morphology close to those obtained with previous P(MMA-co-HEMA) polymers was observed (D z = 226 nm, PDI = 0.161).However here, we believe that PCHMA's slight miscibility with phenyl-pendant polymers would favor its partial wetting and penetration in the PPMA phase during the adsorption, ultimately forming ellipsoids. [50]Meanwhile, we also tested an anionically charged copolymer, poly(MMA-co-methacrylic acid) (P(MMA 5 -co-MAA 5 ), that showed similar adsorption and precipitation behavior (Figure 5B).The continuous solvent shifting produced concentric nanospheres with strongly negatively charged surfaces (ζ-potential = −46.1 mV, D z = 326 nm, PDI = 0.108).The presence of strongly charged copolymers generated a thick intermediate hybrid layer that certainly hampers the folding of the copolymer phase at their precipitation stage.Finally, P(PMA 7 -co-HEMA 3 ) and PMMA, that exhibited very close CPLs, were engaged in the continuous solvent-shifting process (Figure 5C).The PPMA-based copolymers were not completely precipitated before PMMA supersaturation.Therefore, the two polymers were simultaneously precipitated and randomly deposited at the interface after crossing the second CPL, affording coprecipitated layer with diffuse boundaries.After consuming the copolymers, the PMMA shell progressively grew to produce a core-shell morphology (D z = 226 nm, PDI = 0.115).All these results demonstrate that continuous gradient nanoprecipitation enables one to produce particles of various inner morphologies and shapes.Most likely, the features of the co-substrates are key to generate these nanostructures.In the particle growth process, the co-substrates would get involved in the progressive precipitation of the substrates and continuously co-aggregate with substrates at the interface to form the growing hybrid phase.The substrate miscibility of the co-substrates together with specific intermolecular interaction would effectively regulate the molecular aggregation behaviors of substrate/cosubstrate at the forming particle interface, resulting in a variety of tailorable molecular organization patterns within the particles.

Nanostructure disclosure by substrate crosslinking/solvent extraction
Hollow nanoparticles with controlled interior mono-or multivoid spaces have shown recent outcomes as nanoreactors, [51] F I G U R E 5 Co-substrate variations.TEM images of nanoparticles using P(CHMA-co-HEMA) (A), P(PMMA-co-MAA) (B) and evolution of aggregated polymers and zoomed nanostructure of poly(phenyl methacrylate)/P(PMMA-co-MAA) nanoparticles.(C) Composition evolution and TEM images of P(PMA 7co-HEMA 3 )/poly(methyl methacrylate) nanoparticles prepared by continuous gradient nanoprecipitation.All nanoparticles were stained by RuO 4 .
drug delivery systems, [13,52] energy storage materials, [53,54] to cite a few.The versatility of particle growth process could be a way to produce hollow polymer nanoparticles with tailorable internal cavity.Specifically here, we designed a continuous production process to solidify the nanoparticle growing interfaces by UV-assisted photopolymerization (Figure 6A).Methacrylate-functionalized P(PMA 5 -co-HEMA 5 ) (grafting ratio: ∼15% of hydroxyl groups, CPL: 0.71 mass fraction of acetone, Figure S23) was used as UV-curable substrate together with P(MMA 6 -co-HEMA 4 ) as co-substrate.We have selected here a water-soluble photoinitiator (PI) Irgacure 2959 that shows a good solubility both in water and water/acetone mixture.The aqueous solution containing the PI was progressively added within a minute to the acetone solution of substrate/co-substrate programmed by using a syringe pump (800 μL/min).A 365 nm LED light with a laser power of 60 mW/cm 2 was used to irradiate the solution and induce crosslinking reactions between pendent methacrylate units during the continuous solvent shifting.Because of the good solubility of PI in water/acetone mixture, the molecules would preferentially dissolve in the solution rather than being entrapped in the hydrophobic polymer matrix during the continuous aggregation.The crosslinking reactions mainly occurred at the interface for solidifying the hybrid layer during the continuous particle growth (Figure S24).The NMR monitoring confirmed the consumption of the vinyl groups in the course of the nanoprecipitation process (Figure 6B).The UV-assisted polymerization was too slow compared to the precipitation process to ensure quantitative conversion of the vinyl groups and the photopolymerization stopped after full precipitation of the methacrylate-functionalized copolymers (Figure 6C).Meanwhile, the photocuring reaction did not interfere with the molecular packing pattern (Figure 6D).Therefore, the noncrosslinked substrate in the inner part of the NPs and the co-substrate in the shell can be regarded as sacrificial phases for templating the 3D geometry of the nanomaterials.After removal by dissolution in acetone, centrifugation, and redispersion in water, nanocages with a diameter of 516 nm (PDI = 0.160) were recovered (Figure 6E).We verified that the inner core substrate phase was destroyed in these conditions, confirming that the photo-crosslinking mainly occurred at the interface.
In this case, the backbone of the nanocages would almost reflect the structuration pattern of the substrates within the hybrid layer during continuous particle growth.For instance, the relative void sizes (void diameters/particle diameters) depend on the molecular aggregation and/or continual interface polymerization.Both decreasing the pump rates (at a laser power of 60 mW/cm 2 ) and strengthening the UV power (at pump rate of 800 μL/min) produced a tighter network within the particles, resulting in 3D network nanocages with smaller void sizes (Figure 6F and Figure S25).In stark contrast, co-nanoprecipitation of substrate and co-substrate led to empty nanocapsules (D z = 445 nm, PDI = 0.134) with a thin polymer shell (Figure 6G).In the same line, the anionic P(MMA 5 -co-MAA 5 )-mediated fabrication of concentric nanospheres produced hollow spheres with a diameter of 303 nm (PDI = 0.159) after subsequent template removal (Figure 6H).The significant shift of zeta potential from −52.3 to −23.0 mV confirmed the successful elimination of most of the anionic template.Finally, 3D network nanocages with gold nanoparticle-based backbones (D z = 587 nm, PDI = 0.206) were robustly produced by using methacrylate-functionalized P(PMA 5 -co-HEMA 5 )grafted gold nanoparticles as substrate (Figure 6I,J).The resulting light-harvesting nanomaterials showed a broadband and strong absorption (Figure S26).

CONCLUSION
In conclusion, we reported a template-free particle growth strategy to fabricate nanostructured particles through a one-pot continuous gradient nanoprecipitation process.The continuous increase of antisolvent controlled the organization of substrate/co-substrate at the growing particle/solvent interface, leading to the spontaneous formation of original inner morphologies and shapes mediated by the nature of cosubstrate mainly.This template-free and unconfined assembly process allowed conveniently tailoring colloidal nanostructures by modulating substrate/co-substrate contents and miscibility, growth rates and solubility.This process is applicable to a large range of functional substrates, namely from functional polymers to inorganic metal nanoparticles.UVassisted continuous interface crosslinking was also a straightforward way to prepare hollow and multivoid nanoparticles by simple solvent post-extraction of uncrosslinked materials.Concerning potential applications, the programmable formation process of these nanostructures with tailorable versatility (i.e., particle size, shell thickness, surface topologies, interior architectures, and exterior geometries) would allow modulating mass transfer within the nanoparticles and superficial interaction with surrounding matters, [55][56][57] of value in biomedicines, [58] sensing, [55] catalysis, [59] and energy. [60]63][64][65] In contrast to classic nanoprecipitation which the solute should be precipitated together and simultaneously engaged in particle formation, continuous gradient nanoprecipitation can program the molecular precipitation and aggregation, achieving the fine and ordered construction of the polymer matrix with functionalizable potential.In addition, compared with confined self-assembly, the resulting inner morphologies and shapes showed a lower sensitivity to the polymer structures, which allowed well customizing the functional nanostructures for a variety of substrates.Meanwhile, the molecular aggregation pattern can be modulated by varying co-substrates' characteristics, which conferred the technique with sufficient convenience for diversifying the nanostructuration.We believe this low-energy-cost bottom-up approach could offer new opportunities to develop functional nanomaterials.

F I G U R E 1
Mono-component particle growth by continuous solvent shifting.(A) Illustration of particle growth of mono-component polymer system through continuous gradient nanoprecipitation.(B) Phase diagram of poly(phenyl methacrylate), the green area indicates the region where particle growth proceeds.(C) Composition evolution of aggregated polymers and dissolved solutes during the solvent-shifting process.Diameter measurements (D) and transmission electron microscope (TEM) images (E) of particles at various stages.

F I G U R E 3
Process variations.Impact of feed concentrations in acetone (A), substrate/co-substrate weight ratios (feed concentration: 1 wt% in acetone) (B) and pump rates (D) on the formation of the nanoparticles through particle growth process.(C) NPs morphologies versus antisolvent feed concentrations (wt% in acetone) and polymer compositions.

F I G U R E 6
Unraveling nanostructuration of the particles.(A) Illustration of concomitant fabrication of nanoparticles through particle growth process and UV crosslinking reaction.(B) Pseudo-first-order kinetics for continuous interface polymerization of vinyl-functionalized substrate polymers in response to UV irradiation (365 nm, 60 mW/cm 2 ).(C) Variation of soluble substrate polymers and vinyl groups in the polymers with continuous solvent shifting and UV irradiation.(D) TEM images of UV-cured methacrylate-functionalized P(PMA 5 -co-HEMA 5 )/P(MMA 6 -co-HEMA 4 ) nanoparticles using a pump rate of 800 μL/min.(E) TEM images of same nanoparticles after solvent extraction showing a multivoid morphology.(F) Impact of pump rates and laser powers on relative void sizes of nanoparticles after solvent extraction.(G) TEM image of empty nanocapsules using poly(methyl methacrylate) as co-substrates after solvent extraction.(H) TEM images of hollow nanospheres using P(MMA 5 -co-MAA 5 ) as co-substrates after solvent extraction.TEM (I), scanning transmission electron microscopy images, energy dispersive -ray spectroscopy mapping, and element distribution (J) of final multivoid gold nanoparticle aggregates.In TEM, nanoparticles were stained by RuO 4 .