Monomer 1 was prepared according to our previous published procedure [46]. Epoxy monomer 1 (0.23 g, 1.2 mmol) in DMF was added dropwise to a suspension of 3,5-dimethylpyrazole (0.107 g, 1.11 mmol) and anhydrous potassium carbonate (0.166 g, 1.2 mmol) in 10 mL of anhydrous DMF. The reaction was stirred at 100 °C for 9 h until the disappearance of starting materials (followed by TLC). It was then cooled to room temperature, the salt was filtered off, and the solvent was removed under reduced pressure. A wheatish creamy solid (0.15 g, 55% yield) was obtained after column chromatography (1:1 hexane:ethyl acetate on silica) followed by vacuum drying overnight. ¹H NMR (500 MHz, CDCl₃) δ:6.14–6.03 (m, 1.5H, HC=CH), 5.87 (m, 0.5H, HC=CH), 5.74 (s, 1H-Py ring), 4.52 (b, 1H, -OH), 4.17–3.85 (m, 6H), 3.15 (s, 0.5H), 2.98–2.90 (m, 1H), 2.89–2.86 (m, 1H), 2.20–2.13 (m, 6H, 2 -CH₃ in Py ring), 1.88–1.84 (s, 1H), 1.69–1.19 (m, 3H). ¹³C NMR (125 MHz, CDCl₃) δ: 176.05, 174.54, (C=O exo and endo) 148.24, 139.80, 138.14, 137.96, 137.92, 135.64, 132.26, 105.12 (=C-CH=C), 69.24, 65.19, 65.17, 65.04, 64.97, 49.72, 46.67, 46.36, 45.80, 45.77, 43.26, 43.04, 42.53, 41.64, 30.45, 29.31, 13.43 (Py-CH₃), 10.95 (Py-CH₃) IR (neat): 3349.48 (b, -OH), 3025.81 (=C-H stretch), 2925.08, 1732.36 (vs., C=O), 1601.25 (-C=C- stretch),1553.78 (-C=N-), 1492.89, 1452.06, 1333.81, 1272.80, 1232.39, 1180.06, 1029.02, 906.84, 756.10, 620.99 (-C=C- bend), 540.10 cm⁻¹. Anal. calcd for C₁₆H₂₂N₂O₃: C, 66.18, H, 7.64. Found: C, 65.96, H, 7.65.

ROMP of monomer 2 with Grubbs second generation catalyst 3 was performed as shown in Scheme 1. The glassware was dried and purged with vacuum and N₂ in a Schlenk line several times prior to conducting the polymerization reaction. A solution (0.2 M) of monomer 2 (120 mg, 4.13 × 10⁻⁴ M, 175 eq) was prepared in dry CH₂Cl₂ under N₂. The catalyst solution was prepared by dissolving the catalyst in anhydrous CH₂Cl₂ under N₂ in a glovebox. The catalyst solution (2 mg, 2.36 × 10⁻⁶ M in 0.5 mL CH₂Cl₂, 1 eq) was added to the reaction mixture and stirred for 8 h at 30 °C. A norbornene solution (38 mg, 4.13 × 10⁻⁴ M, 175 eq) was injected and stirred for another 9 h. The polymerization reaction mixture was terminated with excess ethyl vinyl ether (300 eq relative to catalyst) and stirred for another 1 h. The reaction mixture was then poured into cold methanol and stirred, purified, and dried under vacuum, yielding a flaky white solid in 72% yield. ¹H NMR (500 MHz, CDCl₃) δ: 5.82 (b, CH, Py ring), 5.35–5.22 (b, -HC=CH-), 4.17–3.85, 3.18, 2.96, 2.44, 2.23–2.19, 1.87–1.27(b).

Preparation of monodisperse maghemite nanoparticles within copolymer matrices was accomplished by our previously reported method [30] using cyclohexanone as solvent or others as required.

120 mg of F127 was dissolved in 20 mL (the concentration is higher than critical micelle concentrations (CMC); CMC of Pluronic F127 is 0.007 g/cm³ at room temperature) [36,38,39,47,48] of water by vigorously stirring for 3 hrs. Maghemite-polymer nanocomposites (7 mg) were dispersed in CH₂Cl₂. Equal volume of these two mixtures were added together and stirred under N₂ at 40 °C (waterbath) for until all CH₂Cl₂ evaporated in a fume hude and nanocomposite covered by the F127 coating was prepared in water. Then, it was subjected to centrifuge and finally, the light brown colored supernatant was collected which contains block copolymer-maghemite nanocomposites entrapped in pluronic micelles.

The monomer and polymers were designed and prepared by exploiting the versatility of epoxy precursor 1. In the first step, pyrazole-containing monomer 2 was synthesized from 1 [46], by opening the epoxide ring with 3,5-dimethyl pyrazole in the presence of base. Next, both a homopolymer 4 and a 1:1 diblock copolymer 5 were synthesized according to Scheme 1 via ROMP using Grubbs second generation catalyst (3, bis(tricyclohexylphosphine)-benzylideneruthenium dichloride). Copolymer 5 was prepared by sequential addition of monomer 2 and nobornene. The molecular weight and polydispersity of the polymers (Table 1) were controlled on the basis of a time-dependent study, monitoring monomer consumption by ¹H NMR and controlling the [M]/[I] ratio, where M is monomer and I is initiator.

The diblock copolymer template 5 contained the ligand system in one block that can chelate through both N-(from pyrazole) and O-donors (from ring-opened hydroxy and/or carboxylate groups) with the metal oxide core, while the second block contained the steric stabilizing group to disperse the nanoparticle composite and reduce interaction between nanoparticles. The pyrazole ligands played a vital role in stabilizing the maghemite nanoparticles, affording a supramolecular metal-organic hybrid. The polymers were characterized by ¹H NMR (Figure 1) and FTIR. In the ¹H NMR, distinct peaks at 6.14–5.87 ppm for -CH=CH- vinyl protons, 5.74 ppm for -H pyrazole ring and a broad -OH proton peak at 4.52 ppm were present for the monomer (Figure 1a) while the vinyl protons disappear and a new peak appears at 5.35–5.22 ppm for new alkene protons for the polymers Figure 1(b,c).The ratio between the alkene proton vs. pyrazole ring proton present in polymer determined the block ratio (m:n). The molecular weights of the polymers were experimentally determined by standard GPC (Figure 2) method calibrated with PS standards correspond to apparent Mn values, shown in Table1. The relatively high polydispersity index of the copolymer is possibly due to the presence of heteroatom (N, O) in the pendant group functionality.

The glass transition temperature and thermal decomposition temperature were determined by DSC and TGA analyses, respectively (Table 1). The 1:1 block copolymer 5 was more thermally stable (decomposition at 217 °C) compare to the pyrazole group containing homopolymer 4. Because of the thermally labile bond at the side chain, it was noticed, that the thermal stability of the polymer in general decreases with respect to homopolynorbornene (450 °C) or epoxy containing homopolymer (370 °C) as reported before [30]. The glass transition temperature of the polynorbornene, homopolymer, 4, and copolymer, 5 (were approximately 31, 46, and 39 °C respectively), were in close range and broad in nature.

The second step was to generate relatively uniform, highly crystalline maghemite nanoparticles in situ from Fe(CO)₅ precursor in the presence of diblock copolymer 5 (0.68% molar wt of polymer compared to ironpentacarbonyl feed) in cyclohexanone. Supported by our previous study [31], 1:1 block copolymer ratio of the polymer was chosen to stabilize the nanopartcle stabilization at the optimum level. Trimethylamine N-oxide was used as an oxidizer to prepare the maghemite nanocomposite in a similar manner to our previously reported method [30,31]. Briefly, the block copolymer and iron-pentacarbonyl were added in cyclohexanone and refluxed under inert atmosphere until the solution turned to black. Thereafter, solution was cooled to room temperature and oxidizer was added to it, followed by reflux for another few hours, and then, purified as mentioned in literature. Finally, the nanocomposite was dispersed in solvent as required.

The block copolymer-stabilized nanocomposites were characterized by TEM, SEM, XRD, and FTIR. TEM (Figure 3a) confirmed generation of relatively uniform and spherical iron oxide nanoparticles. The size distribution (Figure 3b) of the nanoparticles was in the 3–6 nm range with an average size of 4.3 nm. Evidence of formation of a crystalline structure of the Fe₂O₃ nanoparticles was obtained by SAED and XRD (Figure 3(c,d)), revealing cubic maghemite-C type structures of the nanoparticles, corresponding well with standard data [49]. The average diameter (d) of the singular γ-Fe₂O₃ nanocrystallites, estimated using the Scherrer equation [50], was 4 nm, corresponding to the strongest reflection (311) of Fe₂O₃ nanoparticles at 2θ value of 35.72°. This was in good agreement with TEM results. SEM data revealed the bulk morphology of the 1:1 block copolymer and nanocomposite. FTIR spectroscopic analysis provided compelling evidence of ligand-mediated binding of iron oxide with the pendant chelating group of the diblock copolymer (Figure 3e). The presence of a single, broad band at about 574 cm⁻¹ in the FTIR spectrum of the nanocomposite is characteristic of Fe-O stretching for less than 8 nm nanoparticles [51]. Also, the shift from 1732 cm⁻¹ to 1690 cm⁻¹, corresponding to C=O stretching in the polymer and nanocomposite, respectively, supported interaction between the iron oxide core and the diblock copolymer.

Magnetization (M) measurement as a function of temperature (T) was performed on γ-Fe₂O₃-diblock copolymer nanocomposites in powder form using SQUID. Figure 4a shows the zero field cooled (ZFC) and field cooled (FC) vs. temperature profile, where the blocking temperature (T_B) of 9 K is denoted by the peak of the ZFC curve. The low and sharp blocking temperature of the nanocomposite is consistent with the small and uniform size of the maghemite nanoparticles, respectively. Uniform distribution of the magnetic nanoparticles within the polymer matrix is further strengthened by very close superimposition of the ZFC and FC curves after passing the T_B. The magnetic hysteresis loop at 4.2 K, below T_B, is shown in Figure 4b. The coercive field was of the order of 100 Oe. Above T_B the coercive field was zero and the data collapse onto the same curve when plotted as a function of H/T. This H/T scaling behavior indicates that the sample consists of non-interacting superparamagnetic particles. The magnetization data above T_B are fit to a Langevin function, and a saturated moment of approximately 73 emu/g is determined from the fit.

For potential biomedical applications, the stable dispersion in aqueous and physiological media, and biocompatibility of the pyrazole-containing block copolymer-stabilized maghemite nanocomposites was assessed. The organic soluble maghemite-polymer nanocomposites were dispersed in water using Pluoronic F127 (PEO-PPO-PEO), using to a solvent evaporation method. The Pluronic-coated, water-dispersible nanocomposite was generated by mixing an equal volume of the nanocomposite in CH₂Cl₂ with an aqueous solution of Pluronic F127, followed by vigorous stirring and evaporation of the CH₂Cl₂. The outer hydrophilic (PEO) blocks of Pluronic F127 help to form micelles and dispersion in water while the hydrophobic block (PPO) attracts the polymer-coated maghemite nanoparticles. An aqueous dispersion of micelle-encapsulated superparamagnetic nanocomposites was stable for weeks and retained their magnetic properties.

The morphology of the Pluronic-pyrazole-maghemite nanocomposites in water was studied by TEM (Figure 5a and Figure 5b), indicating that the size of the core maghemite nanoparticles was similar before and after dispersion in water (confirming that the particles were stable and did not aggregate). The observed stability of the Pluronic-coated magnetic nanocomposites in water is important for biological use and was consistent to literature reports [45,52].

For potential biomedical applications, the cytotoxicity of Pluronic™-pyrazole block copolymer-coated superparamagnetic nanoparticles was assessed using a cell viability assay with Hela cells. Hela cells were incubated for 20 h with varying concentrations of the nanocomposites (0–50 µM), exhibiting high cell viability, 90–100%, depending on concentration. Qin et al. reported a novel type ferrogel (SPIONS coated with oleic acid and PF127) to release drug for magnetically controlled release of hydrophobic drug very efficiently [42]. Recently, Zhao et al. also demonstrated a RGD modified ferrogel, where of SPION is encapsulated in PF127, alginate and AAD, for sustain release of cells and other bioactive agents under magnetic field [53]. Thus, the Pluronic encapsulated pyrazole block copolymer stabilized iron oxide nanocomposite also provides a promising tool to encapsulate hydrophobic drug or optical contrast agent and holds the potential for dual imaging modalities or theranostic applications in biomedical field.