Toward Long-Term-Dispersible, Metal-Free Single-Chain Nanoparticles

We report herein on a new platform for synthesizing stable, inert, and dispersible metal-free single-chain nanoparticles (SCNPs) via intramolecular metal-traceless azide–alkyne click chemistry. It is well known that SCNPs synthesized via Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) often experience metal-induced aggregation issues during storage. Moreover, the presence of metal traces limits its use in a number of potential applications. To address these problems, we selected a bifunctional cross-linker molecule, sym-dibenzo-1,5-cyclooctadiene-3,7-diyne (DIBOD). DIBOD has two highly strained alkyne bonds that allow for the synthesis of metal-free SCNPs. We demonstrate the utility of this new approach by synthesizing metal-free polystyrene (PS)-SCNPs without significant aggregation issues during storage, as demonstrated by small-angle X-ray scattering (SAXS) experiments. Notably, this method paves the way for the synthesis of long-term-dispersible, metal-free SCNPs from potentially any polymer precursor decorated with azide functional groups.

Among the different techniques developed to synthesize permanent SCNPs endowed with robust intra-chain covalent bonds, those based on "click" chemistry are regarded as attractive [26]. Since its discovery by Sharpless [27] and Meldal [28] in 2002, Cu(I)-catalyzed azide-alkyne coupling (CuAAC)-the archetypical "click" reaction [29]-has gained enormous interest in a broad number of fields, including pharmaceutical science and materials science. One of the authors of this paper pioneered the use of CuAAC for SCNP construction a few years after its discovery [30][31][32]. CuAAC is a rapid, high-yielding reaction that can be conducted at room temperature (r.t.) using diluted concentrations of reactants, making it compatible with many other functional groups. However, the resulting triazole science and materials science. One of the authors of this paper pioneered the use of Cu-AAC for SCNP construction a few years after its discovery [30][31][32]. CuAAC is a rapid, high-yielding reaction that can be conducted at room temperature (r.t.) using diluted concentrations of reactants, making it compatible with many other functional groups. However, the resulting triazole group formed on CuAAC is prone to complex Cu ions from the catalyst. In this sense, the removal of metal traces from polymers and SCNPs is significantly more complicated than in the case of water-soluble low-molecular-weight substances and, consequently, severely limits practical applications due to potential toxicity, coloration, and stability issues in the resulting materials [33,34]. On the one hand, the interference of Cu ions with many biological processes (for instance, metal ions can strongly bind to guanosine bases, disrupting the double-helical structure of DNA) [35] and various electrical/optoelectronic phenomena [36] is well-known. On the other hand, metal ions in some macromolecule-metal complexes tend to aggregate, forming nanoscale ionic domains due to their electrostatic or dipole-dipole interactions [37]. In the case of SCNPs, these effects lead to the irreversible aggregation of SCNPs into multi-chain nanoparticles during storage in the solid state [38]. Consequently, when SCNPs containing limited amounts of Cu ions are dissolved after storage, aggregates with significantly larger sizes than individual SCNPs are observed.
A promising approach to avoid any residual metal ions while retaining the advantages of click chemistry is to use metal-free versions, such as the strain-promoted azide−alkyne cycloaddition (SPAAC) developed by Bertozzi [39] for covalent modification of biomolecules in living systems. SPAAC utilizes strained cyclooctynes that have a decreased activation energy to react with azides in a click-like fashion, eliminating the need for a Cu(I) catalyst. This type of metal-free click chemistry has been applied in several fields, from dynamic in vivo imaging [40] to cellulose modification [41] and the functionalization of surfaces [42], among others. In this work, in order to synthesize metal-free SCNPs that are free from aggregation issues during storage in the solid state, we turn our attention to a bifunctional cross-linker molecule, sym-dibenzo-1,5-cyclooctadiene-3,7diyne (DIBOD), with two highly strained alkyne bonds( Figure 1). DIBOD was first synthesized by Sondheimer et al. in 1974 [43]. The use of DIBOD for fluorescence labeling of azido-glycoconjugates on cell surfaces was pioneered by Hosoya et al. in 2010 [44]. More recently, this bifunctional cross-linker molecule has been employed for the efficient construction of various rings [45] and cage-shaped polymers [46]. However, to the best of our knowledge, DIBOD has not yet been investigated as an intra-chain cross-linker for metalfree SCNP construction. To illustrate the utility of DIBOD as an external bifunctional cross-linker molecule for the synthesis of metal-free SCNPs via SPAAC, we first synthetized a polystyrene (PS)based precursor decorated with azide functional groups. Next, we investigated the reaction conditions to produce PS-SCNPs using DIBOD molecules as intra-chain cross-linkers. Through small-angle X-ray scattering (SAXS) experiments, we found that this new approach leads to metal-free PS-SCNPs without significant aggregation issues during storage. Remarkably, the new synthetic method reported in this work is useful for the preparation of long-term-dispersible, metal-free SCNPs from potentially any polymer precursor decorated with azide functional groups. To illustrate the utility of DIBOD as an external bifunctional cross-linker molecule for the synthesis of metal-free SCNPs via SPAAC, we first synthetized a polystyrene (PS)-based precursor decorated with azide functional groups. Next, we investigated the reaction conditions to produce PS-SCNPs using DIBOD molecules as intra-chain crosslinkers. Through small-angle X-ray scattering (SAXS) experiments, we found that this new approach leads to metal-free PS-SCNPs without significant aggregation issues during storage. Remarkably, the new synthetic method reported in this work is useful for the preparation of long-term-dispersible, metal-free SCNPs from potentially any polymer precursor decorated with azide functional groups.

Results and Discussion
Scheme 1 illustrates the procedure followed in this work for the synthesis of metal-free PS-SCNPs via SPAAC using DIBOD molecules as intra-chain cross-linkers.

Results and Discussion
Scheme 1 illustrates the procedure followed in this work for the synthesis of metalfree PS-SCNPs via SPAAC using DIBOD molecules as intra-chain cross-linkers. Scheme 1. Illustration of the synthesis of metal-free PS-SCNPs, 3, at r.t. from a PS-based precursor decorated with azide functional groups (2).

Synthesis and Characterization of the Polystyrene-Based Precursor Decorated with Azide Functional Groups (2)
In the first step, we synthetized a random copolymer of styrene (S) and chloromethyl styrene (CMS) via reversible addition-fragmentation chain-transfer (RAFT) polymerization (see Scheme 1) [47]. The resulting poly(S-co-CMS) copolymer, 1, containing 27 mol% of CMS units-as estimated by proton nuclear magnetic resonance ( 1 H NMR)-showed a weight-average molecular weight (Mw) of 100.5 kDa and a narrow dispersity (Ɖ) of 1.15, as determined by size exclusion chromatography (SEC) equipped with triple detection (RI, MALS, and VIS, see Section 3.2.1). The SEC/RI trace of 1 is illustrated in Figure 2  Subsequently, 1 was subjected to an azidation procedure [47] (NaN3, DFMF, 24 h) at 80 °C to give the polystyrene (PS)-based precursor decorated with -N3 (instead -Cl) functional groups: poly(S-co-AMS), 2 (see Scheme 1). The SEC/RI trace of 2 is shown in Figure  2

Synthesis and Characterization of the Polystyrene-Based Precursor Decorated with Azide Functional Groups (2)
In the first step, we synthetized a random copolymer of styrene (S) and chloromethyl styrene (CMS) via reversible addition-fragmentation chain-transfer (RAFT) polymerization (see Scheme 1) [47]. The resulting poly(S-co-CMS) copolymer, 1, containing 27 mol% of CMS units-as estimated by proton nuclear magnetic resonance ( 1 H NMR)-showed a weight-average molecular weight (M w ) of 100.5 kDa and a narrow dispersity (Ð) of 1.15, as determined by size exclusion chromatography (SEC) equipped with triple detection (RI, MALS, and VIS, see Section 3.2.1). The SEC/RI trace of 1 is illustrated in Figure 2. The hydrodynamic radius (R h ) of 1 in THF was R h (VIS) = 8.2 nm.

Results and Discussion
Scheme 1 illustrates the procedure followed in this work for the synthesis of metalfree PS-SCNPs via SPAAC using DIBOD molecules as intra-chain cross-linkers.

Synthesis and Characterization of the Polystyrene-Based Precursor Decorated with Azide Functional Groups (2)
In the first step, we synthetized a random copolymer of styrene (S) and chloromethyl styrene (CMS) via reversible addition-fragmentation chain-transfer (RAFT) polymerization (see Scheme 1) [47]. The resulting poly(S-co-CMS) copolymer, 1, containing 27 mol% of CMS units-as estimated by proton nuclear magnetic resonance ( 1 H NMR)-showed a weight-average molecular weight (Mw) of 100.5 kDa and a narrow dispersity (Ɖ) of 1.15, as determined by size exclusion chromatography (SEC) equipped with triple detection (RI, MALS, and VIS, see Section 3.2.1). The SEC/RI trace of 1 is illustrated in Figure 2. The hydrodynamic radius (Rh) of 1 in THF was Rh (VIS) = 8.2 nm. Subsequently, 1 was subjected to an azidation procedure [47] (NaN3, DFMF, 24 h) at 80 °C to give the polystyrene (PS)-based precursor decorated with -N3 (instead -Cl) functional groups: poly(S-co-AMS), 2 (see Scheme 1). The SEC/RI trace of 2 is shown in Figure  2 Subsequently, 1 was subjected to an azidation procedure [47] (NaN 3 , DFMF, 24 h) at 80 • C to give the polystyrene (PS)-based precursor decorated with -N 3 (instead -Cl) functional groups: poly(S-co-AMS), 2 (see Scheme 1). The SEC/RI trace of 2 is shown in Figure 2, with values of M w (MALS) = 129.1 kDa, Ð = 1.18, and R h (VIS) = 9.2 nm. We attributed the slight increase in R h to steric effects along the copolymer chain induced by the replacement of the -Cl groups with -N 3 moieties. The total replacement of -Cl moieties with -N 3 functional groups was confirmed by both 1 H and 13  the slight increase in Rh to steric effects along the copolymer chain induced by the replacement of the -Cl groups with -N3 moieties. The total replacement of -Cl moieties with -N3 functional groups was confirmed by both 1 H and 13 C NMR spectroscopy, as shown in Fig

Synthesis and Characterization of Metal-Free Single-Chain Nanoparticles (3) from the Polystyrene-Based Precursor Decorated with Azide Functional Groups (2)
Metal-free PS-SCNPs were synthesized at r.t. via SPAAC using DIBOD molecules as intra-chain cross-linkers, as depicted in Scheme 1. We used a slow addition technique, first introduced by Hawker et al. [48], in which a solution of the precursor (i.e., 2) was added at a low addition rate to a solution of the cross-linker (i.e., DIBOD). After the complete

Synthesis and Characterization of Metal-Free Single-Chain Nanoparticles (3) from the Polystyrene-Based Precursor Decorated with Azide Functional Groups (2)
Metal-free PS-SCNPs were synthesized at r.t. via SPAAC using DIBOD molecules as intra-chain cross-linkers, as depicted in Scheme 1. We used a slow addition technique, first introduced by Hawker et al. [48], in which a solution of the precursor (i.e., 2) was added at a low addition rate to a solution of the cross-linker (i.e., DIBOD). After the complete

Synthesis and Characterization of Metal-Free Single-Chain Nanoparticles (3) from the Polystyrene-Based Precursor Decorated with Azide Functional Groups (2)
Metal-free PS-SCNPs were synthesized at r.t. via SPAAC using DIBOD molecules as intra-chain cross-linkers, as depicted in Scheme 1. We used a slow addition technique, first introduced by Hawker et al. [48], in which a solution of the precursor (i.e., 2) was added at a low addition rate to a solution of the cross-linker (i.e., DIBOD). After the complete addition of 2 in ca. 12 h, the mixture remained (additionally) for 12 h under stirring to allow the SPAAC to go to completion. Then, an appropriate amount of BzA was added to end-cap any potential unreacted alkyne group, and the mixture was left under stirring for an additional 24 h. After concentrating the reaction mixture, 3 was obtained by precipitation in cold EtOH. Figure 5 shows the SEC/MALS traces of 2, 3 before precipitation after 48 h of reaction, and 3 after precipitation, drying, and re-dissolution. No change in the SEC trace of the PS-SCNPs, 3, was observed from the crude to the dried (and re-dissolved) 5 of 13 samples. The PS-SCNPs, 3, exhibited values of M w (MALS) = 195.8 kDa, Ð = 1.21, and R h (VIS) = 6.9 nm. The increase in M w observed was due to the incorporation of the intra-chain cross-linker DIBOD. The SEC results confirmed the successful formation of SCNPs with a hydrodynamic size (R h (VIS) = 6.9 nm) smaller than that of the precursor copolymer (R h (VIS) = 9.2 nm). We can exclude the presence of multi-chain aggregates since the SEC/MALS technique is highly sensitive to their presence, and they should be observed at a low SEC retention time (<7 min.). Additionally, dynamic light scattering (DLS) measurements also showed a significant reduction in size (R h (DLS) = 7.5 nm) upon the formation of metal-free PS-SCNPs via SPAAC using DIBOD molecules as intra-chain cross-linkers, as shown in Figure 6. addition of 2 in ca. 12 h, the mixture remained (additionally) for 12 h under stirring to allow the SPAAC to go to completion. Then, an appropriate amount of BzA was added to end-cap any potential unreacted alkyne group, and the mixture was left under stirring for an additional 24 h. After concentrating the reaction mixture, 3 was obtained by precipitation in cold EtOH. Figure 5 shows the SEC/MALS traces of 2, 3 before precipitation after 48 h of reaction, and 3 after precipitation, drying, and re-dissolution. No change in the SEC trace of the PS-SCNPs, 3, was observed from the crude to the dried (and re-dissolved) samples. The PS-SCNPs, 3, exhibited values of Mw (MALS) = 195.8 kDa, Ð = 1.21, and Rh (VIS) = 6.9 nm. The increase in Mw observed was due to the incorporation of the intrachain cross-linker DIBOD. The SEC results confirmed the successful formation of SCNPs with a hydrodynamic size (Rh (VIS) = 6.9 nm) smaller than that of the precursor copolymer (Rh (VIS) = 9.2 nm). We can exclude the presence of multi-chain aggregates since the SEC/MALS technique is highly sensitive to their presence, and they should be observed at a low SEC retention time (<7 min.). Additionally, dynamic light scattering (DLS) measurements also showed a significant reduction in size (Rh (DLS) = 7.5 nm) upon the formation of metal-free PS-SCNPs via SPAAC using DIBOD molecules as intra-chain crosslinkers, as shown in Figure 6.  addition of 2 in ca. 12 h, the mixture remained (additionally) for 12 h under stirring to allow the SPAAC to go to completion. Then, an appropriate amount of BzA was added to end-cap any potential unreacted alkyne group, and the mixture was left under stirring for an additional 24 h. After concentrating the reaction mixture, 3 was obtained by precipitation in cold EtOH. Figure 5 shows the SEC/MALS traces of 2, 3 before precipitation after 48 h of reaction, and 3 after precipitation, drying, and re-dissolution. No change in the SEC trace of the PS-SCNPs, 3, was observed from the crude to the dried (and re-dissolved) samples. The PS-SCNPs, 3, exhibited values of Mw (MALS) = 195.8 kDa, Ð = 1.21, and Rh (VIS) = 6.9 nm. The increase in Mw observed was due to the incorporation of the intrachain cross-linker DIBOD. The SEC results confirmed the successful formation of SCNPs with a hydrodynamic size (Rh (VIS) = 6.9 nm) smaller than that of the precursor copolymer (Rh (VIS) = 9.2 nm). We can exclude the presence of multi-chain aggregates since the SEC/MALS technique is highly sensitive to their presence, and they should be observed at a low SEC retention time (<7 min.). Additionally, dynamic light scattering (DLS) measurements also showed a significant reduction in size (Rh (DLS) = 7.5 nm) upon the formation of metal-free PS-SCNPs via SPAAC using DIBOD molecules as intra-chain crosslinkers, as shown in Figure 6.  The incorporation of DIBOD molecules as intra-chain cross-linkers was confirmed by 1 H NMR and 13 C NMR spectroscopy, as illustrated in Figures 7 and 8, respectively. Additionally, the disappearance of the infra-red (IR) vibration band corresponding to the azide functional group located near 2100 cm −1 was clearly visible in the FTIR spectrum of the metal-free PS-SCNPs, 3, as illustrated in Figure 9.
The incorporation of DIBOD molecules as intra-chain cross-linkers was confirmed by 1 H NMR and 13 C NMR spectroscopy, as illustrated in Figure 7 and Figure 8, respectively. Additionally, the disappearance of the infra-red (IR) vibration band corresponding to the azide functional group located near 2100 cm −1 was clearly visible in the FTIR spectrum of the metal-free PS-SCNPs, 3, as illustrated in Figure 9.   The incorporation of DIBOD molecules as intra-chain cross-linkers was confirmed by 1 H NMR and 13 C NMR spectroscopy, as illustrated in Figure 7 and Figure 8, respectively. Additionally, the disappearance of the infra-red (IR) vibration band corresponding to the azide functional group located near 2100 cm −1 was clearly visible in the FTIR spectrum of the metal-free PS-SCNPs, 3, as illustrated in Figure 9.    Figure 10a illustrates the thermal stability of precursor 2 and the PS-SCNPs, 3, as determined by TGA measurements. The initial decomposition temperature of 2 was 220 °C, corresponding to the extrusion of dinitrogen molecules from the azide groups. The initial decomposition temperature of 3 was found to be 153 °C. We attributed the lower  Figure 10a illustrates the thermal stability of precursor 2 and the PS-SCNPs, 3, as determined by TGA measurements. The initial decomposition temperature of 2 was 220 • C, corresponding to the extrusion of dinitrogen molecules from the azide groups. The initial decomposition temperature of 3 was found to be 153 • C. We attributed the lower thermal stability of 3 to the strained nature of the 1,10-dihydrodibenzo [3,4:7,8]cycloocta [1,2-d:5,6d']bis( [1,2,3]triazole) cross-linking units (see Figure 10b), which promoted their thermal decomposition when heating above 150 • C. Below this limiting temperature, the PS-SCNPs showed no weight loss so one can consider 3 to be a stable material up to 150 • C. Figure 9. Disappearance of the azide IR vibration band of precursor 2 upon reaction with DIBOD and the formation of metal-free SCNPs, 3. Figure 10a illustrates the thermal stability of precursor 2 and the PS-SCNPs, 3, as determined by TGA measurements. The initial decomposition temperature of 2 was 220 °C, corresponding to the extrusion of dinitrogen molecules from the azide groups. The initial decomposition temperature of 3 was found to be 153 °C. We attributed the lower thermal stability of 3 to the strained nature of the 1,10-dihydrodibenzo [3,4:7,8]cycloocta [1,2-d:5,6-d ]bis([1,2,3]triazole) cross-linking units (see Figure 10b), which promoted their thermal decomposition when heating above 150 °C. Below this limiting temperature, the PS-SCNPs showed no weight loss so one can consider 3 to be a stable material up to 150 °C.

Long-Term Stability against Aggregation of Metal-Free Polystyrene-Single-Chain Nanoparticles (3)
The long-term stability against aggregation of the metal-free PS-SCNPs was investigated using small angle X-ray scattering (SAXS) measurements in THF solution at high dilution. Reference SAXS measurements were taken to determine the form factor of precursor 2 and the metal-free PS-SCNPs, 3, after synthesis (see Figure 11). Analysis of the data shown in Figure 11 in terms of the generalized Gaussian coil function [49] provided the values of the radius of gyration (R g ) and scaling exponent (ν) of 2 and 3 in solution at high dilution. Precursor 2 showed a size of R g = 14.1 nm and ν = 0.59, a scaling exponent value that was in agreement with the expected Flory value for a linear chain in good solvent (ν F ≈ 3/5). On the other hand, the freshly synthesized metal-free PS-SCNPs exhibited a reduction in both the size and scaling exponent: R g = 9.1 nm and ν = 0.46. These values were in agreement with previously reported data on the single-chain folding of synthetic polymers to SCNPs [1,38]. the values of the radius of gyration (Rg) and scaling exponent (ν) of 2 and 3 in solution at high dilution. Precursor 2 showed a size of Rg = 14.1 nm and ν = 0.59, a scaling exponent value that was in agreement with the expected Flory value for a linear chain in good solvent (νF ≈ 3/5). On the other hand, the freshly synthesized metal-free PS-SCNPs exhibited a reduction in both the size and scaling exponent: Rg = 9.1 nm and ν = 0.46. These values were in agreement with previously reported data on the single-chain folding of synthetic polymers to SCNPs [1,38].  Figure 12 shows the SAXS results obtained for the SCNPs, 3, in THF dilute solution after 2 months of storage. Figure 12a corresponds to storage in the same solution, whereas Figure 12b corresponds to storage in the solid state and the redispersion of the SCNPs in THF. Analysis of the data in terms of a generalized Gaussian coil function revealed no significant changes (within the experimental uncertainties) in Rg orν with respect to the values observed for the PS-SCNPs, 3, as synthetized (Figure 11b). Only in the case of storage in the solid state was a minor increase in Rg observed, although the scaling exponent did not change significantly. Hence, unlike the case of CuAAC in which large aggregates are typically found after prolonged storage, the synthesis of the metal-free SCNPs via SPAAC using DIBOD molecules as intra-chain cross-linkers can prevent irreversible multi-chain aggregation issues.  Figure 12 shows the SAXS results obtained for the SCNPs, 3, in THF dilute solution after 2 months of storage. Figure 12a corresponds to storage in the same solution, whereas Figure 12b corresponds to storage in the solid state and the redispersion of the SCNPs in THF. Analysis of the data in terms of a generalized Gaussian coil function revealed no significant changes (within the experimental uncertainties) in R g orν with respect to the values observed for the PS-SCNPs, 3, as synthetized (Figure 11b). Only in the case of storage in the solid state was a minor increase in R g observed, although the scaling exponent did not change significantly. Hence, unlike the case of CuAAC in which large aggregates are typically found after prolonged storage, the synthesis of the metal-free SCNPs via SPAAC using DIBOD molecules as intra-chain cross-linkers can prevent irreversible multi-chain aggregation issues. We are currently investigating other azide-containing precursors besides PS to evaluate the scope of the SPAAC method using DIBOD molecules as intra-chain cross-linkers for the synthesis of long-term-dispersible, metal-free SCNPs. The results will be reported at another time. We are currently investigating other azide-containing precursors besides PS to evaluate the scope of the SPAAC method using DIBOD molecules as intra-chain cross-linkers for the synthesis of long-term-dispersible, metal-free SCNPs. The results will be reported at another time.

Nuclear Magnetic Resonance (NMR)
1 H NMR and 13 C NMR spectra were recorded at room temperature on Bruker (Madrid, Spain) spectrometers operating at 400 MHz, using CDCl 3 as solvent.

Dynamic Light Scattering (DLS)
DLS measurements (number distribution) were taken at room temperature on a Malvern Zetasizer Nano ZS (Cambridge, United Kingdom) apparatus.

Fourier Transform Infra-Red (FTIR) Spectroscopy
FTIR spectra were recorded at room temperature on a JASCO 3600 (Madrid, Spain) FTIR spectrometer.

Thermogravimetric Analysis (TGA)
TGA measurements were performed on a Q500-TA Instruments (Cerdanyola del Valles, Spain) apparatus at a heating rate of 10 • C/min under a nitrogen atmosphere.

Small-Angle X-ray Scattering (SAXS)
SAXS experiments were conducted on the Rigaku (Barcelona, Spain) 3-pinhole PSAXS-L equipment of the Materials Physics Center, operating at 45 kV and 0.88 mA. The MicroMax-002+ X-ray Generator System is composed of a microfocus sealed tube source module and an integrated X-ray generator unit, which produces CuKα transition photons of wavelength λ = 1.54 Å. The flight path and the sample chamber in this equipment are under vacuum. The scattered X-rays are detected on a two-dimensional multiwire X-ray Detector (Gabriel (Barcelona, Spain) design, 2D-200X) and converted to one-dimensional scattering curves by radial averaging. This gas-filled proportional type detector offers a 200 mm diameter active area with ca. 200-micron resolution. After radial integration, the scattered intensities were obtained as a function of momentum transfer Q = 4πλ −1 sinθ, where θ is half the scattering angle. Reciprocal space calibration was performed using silver behenate as standard. The sample-to-detector distance was 2 m, covering a Q-range between 0.008 and 0.20 Å −1 . The measurements were taken at r.t. on the THF solutions in boron-rich capillaries of 2 mm thickness, with counting times of 1 h. The concentration was 1 mg/mL in order to avoid interference effects between different macromolecules. The data were carefully corrected for background scattering (due to the capillary and solvent) and measured for each sample on the same capillary for the same time. Scattering cross-sections were obtained in absolute units using water as the calibration standard. The generalized Gaussian coil function [49] was employed for a precise determination of the values of the radius of gyration, R g , and scaling exponent, ν. Poly(S-co-AMS), 2, (50 mg, 0.106 mmol of N 3 , 1 eq) was dissolved in THF (25 mL) in a flask that was sealed and degassed. In a second flask that was also sealed, degassed, and protected from light with aluminum foil, DIBOD (12 mg, 0.53 eq) was dissolved in 75 mL of THF. The solution containing 2 was injected into the DIBOD solution (both at r.t.) using an infusion pump at an addition rate of 2.1 mL/h. After 24 h under stirring, BzA (20 µL, 1.6 eq) was added and the solution was subjected to stirring at r.t. for an additional 24 h. The product was precipitated in cold EtOH, filtrated, and dried at 35 • C under a vacuum overnight. Its parameter values were yield = 89%; M w (SEC/MALS) = 195.8 kDa; Ð (SEC) = 1.21; and R h (SEC/VIS) = 6.9 nm.

Conclusions
We report on a new platform for the synthesis of stable, inert, dispersible, metal-free single-chain nanoparticles (SCNPs) via intramolecular metal-traceless azide-alkyne click chemistry at r.t. The use of the strain-promoted azide−alkyne cycloaddition (SPAAC) technique with sym-dibenzo-1,5-cyclooctadiene-3,7-diyne (DIBOD) as an external bifunctional cross-linker molecule, in combination with azide-containing polymeric precursors, provides a new method for synthesizing metal-free SCNPs through intramolecular folding/collapse. We highlight the possibilities of this new synthesis technique by preparing metal-free polystyrene (PS) SCNPs from a copolymer precursor containing 27 mol% of azide functional groups. The successful formation of metal-free PS-SCNPs was confirmed through a powerful combination of techniques, including SEC with triple detection (RI, MALS, and VIS), 1 H and 13 C NMR spectroscopy, DLS, FTIR, TGA, and SAXS measurements. In addition to the advantages of these metal-free SCNPs, including their potential use in applications in which the total absence of metal traces is essential, the resulting nanoparticles exhibited significant long-term stability against irreversible aggregation. Finally, it is worth mentioning the potential versatility of this method for the synthesis of long-term-dispersible, metal-free SCNPs from any polymer precursor decorated with azide functional groups.