Efficient Synthesis of Single-Chain Polymer Nanoparticles via Amide Formation

Single-chain technology (SCT) allows the transformation of individual polymer chains to folded/collapsed unimolecular soft nanoparticles. In this work we contribute to the enlargement of the SCT toolbox by demonstrating the efficient synthesis of single-chain polymer nanoparticles (SCNPs) via intrachain amide formation. In particular, we exploit cross-linking between active methylene groups and isocyanate moieties as powerful “click” chemistry driving force for SCNP construction. By employing poly(methyl methacrylate)(PMMA-) based copolymers bearing β-ketoester units distributed randomly along the copolymer chains and bifunctional isocyanate cross-linkers, SCNPs were successfully synthesized at r.t. under appropriate reaction conditions. Characterization of the resulting SCNPs was carried out by means of a combination of techniques including size exclusion chromatography (SEC), infrared (IR) spectroscopy, proton nuclear magnetic resonance (H NMR) spectroscopy, dynamic light scattering (DLS), and elemental analysis (EA).


Introduction
Single-chain technology (SCT) is a new paradigm for the future construction of smart and autonomous soft nanodevices [1].Nowadays, SCT allows the transformation of individual polymer chains to folded/collapsed unimolecular soft nanoparticles, the so-called single-chain polymer nanoparticles (SCNPs) [2][3][4][5].The compaction of synthetic polymers to SCNPs has some resemblance to the folding of proteins to their native state, although it is still far from the perfection found in natural macromolecules [6].Significant effort has been spent in recent years to endow SCNPs with bioinspired functions trying to mimic those found in natural polypeptides (both folded proteins and intrinsically disordered proteins) such as enzymatic activity and selectivity [7][8][9], or multibinding activity and transport properties [10,11].Currently, SCNPs are synthesized through one of the following techniques: (i) intrachain homocoupling, (ii) intrachain heterocoupling, or (iii) cross-linker mediated collapse [4].Concerning the nature of the intrachain crosslinking reaction, it can be irreversible (covalent bonds) or reversible (noncovalent or dynamic covalent bonds) [3].Irreversible SCNPs have been synthesized by means of different intrachain cross-linking reactions .
First reports about the synthesis of permanent SCNPs by means of the intrachain homocoupling technique under high dilution conditions by Mecerreyes et al. relied on the use of poly(styren)-, poly(alkyl methacrylate)-, and poly(caprolactone)-based precursors containing vinyl reactive functional groups [12].Unsaturated functional groups were also used for synthesizing poly(4-N-Boc-aminostyrene)-and poly(carbonate)-based SCNPs by Jiang and Thayumanavan and Cherian et al., respectively [13,14].Poly(styrene)and poly(alkyl methacrylate)-based unimolecular nanoparticles were synthesized in multigram quantities by Harth and coworkers using benzocyclobutene containing precursors via intramolecular Diels-Alder reactions at very high temperature (250 ∘ C) [15].Later, benzosulfone-decorated poly(styrene)-and poly(cyclohexyl acrylate)-based precursors were synthesized by Harth and coworkers for the preparation of individual unimolecular nanoparticles via quinodimethane formation, although cross-linking reaction conditions were still very harsh (250 ∘ C) [16,17].Poly(methyl methacrylate)-based SCNPs have been obtained by Zhu et al. via intramolecular Bergman cyclization at relatively lower temperatures (150 ∘ C) [18,19].The synthesis of poly(styrene)and poly(alkyl methacrylate)-based unimolecular nanoparticles via intramolecular cross-linking of sulfonyl azide- [20] and benzoxazine- [21] functionalized polymers still required very high temperatures (190-200 ∘ C).To solve the potential problems of precursor and nanoparticle thermal degradation, a new route was introduced by Sanchez-Sanchez et al.where naked propargylic-decorated single-chain nanoparticle precursors synthesized via redox-initiated reversible additionfragmentation chain transfer (RAFT) polymerization were intrachain collapsed at room temperature under normal air conditions by means of Glaser-Hay coupling (C-C "click" chemistry) [22].Recently, the B(C 6 F 5 ) 3 -promoted collapse of linear polymer precursors containing epoxide groups to SCNPs endowed with catalytic activity has been carried out by Perez-Baena et al. [23].Using a similar approach, Wong et al. have developed biocompatible SCNPs which were found to be nontoxic toward human embryonic kidney cells [24].Poly(styrene)-based unimolecular nanoparticles have been prepared by Dirlam et al. through intrachain crosslinking via oxidative polymerization of 3,4-propylenedioxythiophene functional groups at 50 ∘ C [25].More recently, a facile strategy for preparation of SCNPs by intramolecular photo-cross-linking of azide polymers has been reported by Li et al. [26], and fluorescence-labeled hydrophilic SCNPs have been synthesized by Wang et al. via single-chain folding of precursors containing fluorescent anthracene and crosslinkable oxazine groups [27].
The fabrication of SCNPs has been also carried out using the intrachain heterocoupling technique.A representative example of the use of the intrachain heterocoupling technique was the construction of permanent SCNPs through highly efficient azide-alkyne "click" chemistry (i.e., copper-catalyzed [3 + 2] cycloaddition of alkynes and azides, CuAAC) [28].By employing this technique, biofunctionalized poly(methyl methacrylate)-based unimolecular nanoparticles were prepared at room temperature in high yield based on azide-and protected alkyne-decorated polymer precursors by Ruiz de Luzuriaga et al. [28].The technique was further simplified by Oria et al. starting with copolymers containing protected alkyne groups as well as chloromethyl groups that were transformed rapidly and quantitatively to azidomethyl groups by means of a simple substitution reaction using sodium azide [29].The intrachain heterocoupling technique has been also used to prepare thermoresponsive SCNPs by Ormategui et al. [30].More recently, photo-induced Diels-Alder ligation and nitrile imine mediated tetrazole-ene cycloaddition have been used by Barner-Kowollik and colleagues to synthesize welldefined functional and fluorescent SCNPs, respectively [31,32].
Bifunctional cross-linker mediated collapse of polymer chains is another efficient and straightforward method for obtaining SCNPs.Water-soluble poly(-glutamic acid)-based SCNPs were fabricated by Radu et al. through the crosslinker-induced collapse technique by using a biosynthetic poly(-glutamic acid) precursor and 2,2  -(ethylenedioxy)diethylamine as bifunctional cross-linker in the presence of carbodiimide as catalyst [33].The synthesis of poly(methyl methacrylate)-and poly(styrene)-based SCNPs by intramolecular cross-linking of isocyanate-functionalized copolymers with diamines via urethane formation was reported by Beck et al. [34].Additionally, the high versatility of the crosslinker induced collapse method via intrachain CuAAC was demonstrated by Ruiz de Luzuriaga et al. in 2010 during the facile synthesis of SCNPs of very different chemical nature [35].Michael addition reaction was employed by Sanchez-Sanchez et al. to synthesize poly(methyl methacrylate)-based SCNPs mimicking transient-binding disordered proteins under mild reaction conditions (at room temperature and in the presence of oxygen) [10].More recently, unimolecular polymer nanoparticles have been formed by Hansell et al. by employing polystyrenes decorated with pendent norbornenes and a bifunctional tetrazine cross-linker, demonstrating that the formation of SCNPs can be carried out via tetrazinenorbornene coupling [36].
In this work, we contribute to the enlargement of the toolbox of single-chain technology by demonstrating the efficient synthesis of SCNPs via amide formation.In particular, we exploit, as powerful driving force for SCNP construction, the intrachain cross-linking through amide formation between active methylene groups and isocyanate moieties (see Figure 1).SCNP synthesis has been carried out by employing the technique of bifunctional cross-linker mediated collapse.The resulting SCNPs have been characterized by a combination of complementary techniques including SEC, IR spectroscopy, 1 H NMR spectroscopy, DLS, and EA.

Results and Discussion
The synthetic procedure followed in this work towards SCNPs via intrachain amide formation is illustrated schematically in Figure 2.
As SCNP precursors, we targeted random copolymers of methyl methacrylate (MMA) and 2-acetoacetoxy ethyl methacrylate (AEMA) containing around 30 mol% of ketoester units and two different molecular weights (denoted by P1 and P2 in Table 1).It has been shown recently that by increasing the amount of functional groups above 30 mol% no significant increase in SCNP compaction is obtained [38,39].The precursors were synthesized by RAFT polymerization employing 2-cyanoprop-2-yl-dithiobenzoate (CPDB) as chain transfer agent and 2,2  -azobis(2-methylpropionitrile) (AIBN) as free-radical initiator.RAFT is a very convenient polymerization technique for obtaining nanoparticle precursors with low molecular-weight polydispersity (Đ =   /  ) and hence nanoparticles with uniform size [4].The main characteristics of the random P(MMA-co-AEMA) copolymers investigated in this work are summarized in Table 1, as determined by size exclusion chromatography with absolute molar mass characterization (SEC/SLS), proton nuclear magnetic resonance ( 1 H NMR) spectroscopy, and dynamic light scattering (DLS).Well-defined copolymers displaying relatively low Đ values were obtained.The lower yield of P1 when compared to P2 can be attributed to the higher amount of chain transfer agent employed in the former case, since polymerization time was identical for both precursors (18 h).
A random placement of the -ketoester moieties along the P(MMA-co-AEMA) chains is expected, due to the similar reactivity ratios of MMA and AEMA ( MMA = 0.90  AEMA = 0.95) during RAFT copolymerization, which are indicative of a statistical copolymerization process.SCNPs were synthesized in THF at r.t., under anhydrous conditions, at high dilution (1 mg P(MMA-co-AEMA)/mL of THF), by addition of ca.0.5 equivalents of the bifunctional isocyanate compound (HMDI) per equivalent of AEMA in the presence of DBN as catalyst (see Figure 2 and Table 1).Samples taken periodically from the reaction media were analyzed by SEC/SLS in THF.A progressive increase in SEC retention time and, hence, a concomitant reduction in P(MMA-co-AEMA) hydrodynamic size [40] were observed as a function of reaction time, pointing to the successful formation of SCNPs via intrachain amide formation (Figure 3).The same behavior was observed by employing P2 instead of P1 as SCNP precursor (data not shown).Direct evidence of amide formation between active methylene groups and isocyanate moieties was obtained by infrared (IR) spectroscopy.Figure 4 shows the appearance of four new IR vibration bands located at 1673, 1587, 1306, and 649 cm −1 upon SCNP formation, which according to previous data reported in the literature [41] can be assigned to amide I, II, III, and V bands, respectively.In the IR spectrum of the SCNPs, also the presence of an -NH-vibration band located between 3100 and 3700 cm −1 is observed (inset in Figure 4), which is very broad due to hydrogen bonding between amide groups.Similarly, the 1 H NMR spectrum of the SCNPs (Figure 5) shows characteristic signatures of amide formation [42]: (i) a peak located at 7.0 ppm coming from the protons of the new formed amide groups (-CO-NH-), (ii) a peak at 3.75 ppm from the methine protons (=CH-), (iii) a peak located at 3.  methylene protons of the HMDI cross-linker next to the new formed amide group (-CO-NH-CH 2 -), and (iv) a broad peak centered around 1.5 ppm assigned to internal methylene protons from the HMDI cross-linker (-CO-NH-CH 2 -CH 2 -).Further support of chain compaction upon SCNP formation was obtained from DLS measurements (see Table 1), which provided a measure of the hydrodynamic size of both the precursor and the SCNPs.As an example, a reduction in hydrodynamic size form 9.5 nm (precursor P1) to 6.4 nm (SCNP N1) was found upon SCNP formation, as illustrated in Figure 6.This significant reduction in size is in good agreement with the SEC/SLS results previously shown in Figure 3. Finally, elemental analysis (EA) was used to obtain a rough estimation of the amount of HMDI crosslinker incorporated into the SCNPs.A comparison of the theoretical composition by assuming a degree of amide formation of 100% and the corresponding experimental EA composition is shown in Table 2. From the ratio of the experimental to theoretical N content, a degree of intrachain cross-linking of ca.97% was estimated.

Conclusions
This work contributes to enlarging the toolbox of single-chain technology by demonstrating the efficient synthesis of SCNPs via intrachain amide formation.First, well-defined random are provided in Table 1.
copolymers of MMA and AEMA were synthesized by RAFT polymerization, showing low molecular-weight polydispersity and random placement of -ketoester moieties along the P(MMA-co-AEMA) chains.Next, intramolecular collapse of these AEMA-containing copolymers as SCNP precursors was performed by the reaction at high dilution of AEMA -ketoester moieties with hexamethylene diisocyanate crosslinkers in THF at r.t.Analysis of SEC/SLS, IR spectroscopy, 1 H NMR spectroscopy, DLS, and EA results demonstrated the efficient synthesis of SCNPs via intrachain amide formation.

Figure 1 :
Figure 1: Schematic illustration of amide formation upon the "click" chemistry reaction at r.t. between a -ketoester moiety and an isocyanate compound (P = polymer chain; R = alkyl substituent).

Figure 5 :
Figure5: (a) 1 H NMR spectra of P(MMA-co-AEMA) copolymer P1 (for complete peak assignment, please refer to[37]).(b) 1 H NMR spectra of SCNPs N1.New peaks observed in the 1 H NMR spectrum of SCNPs N1 upon amide formation are indicated (see text for assignment).Signals coming from traces of catalyst are indicated by asterisk.

Figure 6 :
Figure 6: Hydrodynamic size distributions corresponding to P(MMA-co-AEMA) copolymer P1 (dotted line) and SCNPs N1 (continuous line), as determined by DLS measurements.For clarity reasons, only results for P1 and N1 are included.Data for P2 and N2 are provided in Table1.

Table 1 :
Characteristics of precursors and SCNPs synthesized in this work by RAFT polymerization.
a As determined by SEC/SLS.b As determined by 1 H NMR spectroscopy.c As determined by DLS.

Table 2 :
Comparison of the theoretical and experimental composition of P(MMA-co-AEMA) copolymers and SCNPs.
a As determined by EA. b Obtained as O% = 100 − (C% + H% + N%).c By assuming a degree of amide formation of 100%.