Core-Shell Nanostructured Fe3O4-Poly(styrene-co-vinylbenzyl chloride) Grafted PPI Dendrimers Stabilized with AuNPs/PdNPs for Efficient Nuclease Activity.

Four different novel magnetic core-shell nanocomposites stabilized with Au/Pd nanoparticles (NPs) were prepared by a simple procedure and demonstrated their catalytic activity for effective cleavage of pBR322 DNA. Initially, the Fe3O4-poly(styrene-divinylbenzene-vinylbenzyl chloride) (ST-DVB-VBC) matrix functionalized with 3-aminobenzoic acid was prepared and grafted with PPI-G(2) and PPI-G(3) dendrimers. Each core-shell matrix was immobilized with AuNPs and PdNPs separately. The resulting composites were characterized by FT-IR, UV-vis, SEM, TEM, XRD, VSM, XPS, Raman, and TGA. The magnetic core-shell nanocomposites at concentrations from 30 to 50 μM were employed separately to study DNA cleavage by agarose gel electrophoresis. Among the four magnetic core-shell nanocomposites, Fe3O4-poly(ST-DVB-VBC)-PPI-G(3)-AuNPs showed higher activity than others for DNA cleavage, and formed Form-II and -III DNA. When the concentration of Fe3O4-poly(ST-DVB-VBC)-PPI-G(3)-AuNPs was increased from 40 to 45 and 45 to 50 μM, Form-III (linear) DNA was observed with 10 and 22%, respectively, in addition to Form-II. This observation suggests formation of linear DNA from the supercoiled DNA via nicked DNA-intermediated consecutive cleaving process. The magnetic core-shell nanocomposites were stable and monodispersed, and exhibited rapid magnetic response. These properties are crucial for their application in biomolecular separations and targeted drug-delivery in the future.

Introduction

Over the last few decades, identification of small molecules capable of cleaving DNA has attracted much interest owing to their application in biotechnology, nanotechnology, and therapeutic studies. DNA is an important drug target, and it regulates many biochemical processes that occur in the cellular system. The different loci present in the DNA are involved in various regulatory processes such as gene expression, gene transcription, mutagenesis, carcinogenesis, and so forth.[1] Many small molecules exert their anticancer activities by binding with DNA, thereby altering its replication and inhibiting the growth of tumor cells. The nucleic bases and deoxyribose sugar moiety are not modified, and additional reagents are not necessary when they are hydrolytically cleaved which allows the cleaved fragments to be relegated enzymatically. DNA cleavage is important in gene therapy, gene engineering, nucleic acid structure detection, and so forth.[2−4] The synthesis of novel nucleases is an important aspect of biotechnology, drug design[5,6] and molecular biology.[7] Synthetic nucleases with high efficiency and selectivity are largely demanded, bcause DNA is sensitive to oxidative cleavage, many studies have been focused on molecules capable of cleaving DNA oxidatively.[8] Such molecules induce oxidative cleavage of DNA photolytically with redox cofactors, hydrogen peroxide,[9] ascorbic acid,[10] mercaptopropionic acid,[11] or potassium monopersulfate.[12] These molecules bear some advantages over conventional enzymatic nucleases because of the small size which facilitates accessibility to even sterically hindered regions of a macromolecule.

Transition-metal complexes have been extensively studied for their nuclease-like activity using the redox properties of the metal and dioxygen to produce reactive oxygen species to promote DNA cleavage.[13] Therefore, designing new metal complexes which are capable of cleaving DNA in aqueous medium has received considerable attention. The hydrolytic cleavage of DNA finds use in the fields of molecular biology and biotechnology.

The core–shell composite materials are in the frontiers of advanced research in which the core induces optical, catalytic, and magnetic properties, and the shell the surface properties of the particles. Various cores and diverse shells have been already exploited, for example, various inorganic materials such as zirconium phosphate sheets, silica, alumina, and various metal oxides are used as core moiety, and dextran, chitosan, gelatin, poly(ethylene glycol), poly(d,l-lactide), poly(glycolide) and so forth are used as shells.[14,15] Generally, the shell protects the core from oxidation, enhances its stability and compatibility,[16] becomes a platform for surface functionalization,[17] and provides a natural vehicle for obtaining the hybrid-multifunctional materials.[18] Over the past few years, Au nanoparticles (NPs) have been coated on various magnetic core–shell nanocomposites such as Fe3O4–polymer@Au, γ-Fe2O3–polymer@Au. They have low reactivity, high chemical stability, and biocompatibility. Recently, magnetic adsorbents such as Fe3O4NPs and amidoxime-functionalized Fe3O4@SiO2NPs have been reported to remove U(VI), WOX/C to remove Pb2+ and methylene blue, and Fe3O4/polydopamine hollow spheres to remove Eu(III) ions.[19−22] These magnetic core–shell nanocomposites have been used for protein separation,[23] catalysis,[24] cell separation,[25] drug delivery,[26] detection,[27] biological sensing, and probing.[28,29]

Polymers coated on magnetic NPs can prevent grain growth and agglomeration, and facilitate binding of other NPs.[30] Polymer coating has an additional advantage of providing functionality. Similarly, it is our expectation that the effective dispersability of core–shell Fe3O4–polymer in organic/aqueous phases would provide plenty of biological and catalytic applications. Hence, researchers have strived their efforts to develop core–shell magnetic nanocomposites which have the properties of more stability and effective dispersability in organic/aqueous phase without losing the magnetic saturation property. Further, to enhance the stability and dispersability of magnetic nanocomposite, it is essential to identify the relevant functional polymer coated with Fe3O4 NPs and the relevant shell or functional polymer. More specifically, dendrimers grown onto the solid matrix including poly(styrene) bead,[31] magnetic NPs,[32] carbon black,[33] and silica[34] have been the recently reported techniques for the design of innovative dendritic-based catalysts. Dendrimers are an attractive symmetrical supramolecules having excellent efficiency to generate metal NPs via encapsulation and stabilization processes. The salient merits of these dendrimer-supported catalysts include easy recovery and recycling and easy handling of odorous and toxic substances.[35]

Gold and platinum metal NPs were studied for DNA degradation.[36,37] Midander and co-workers studied single-stranded breakage in the cultured human lung cells with CuNPs.[38] CuNPs are potent cytotoxic and genotoxic and exhibit toxicological activities in vivo[39] and in cultured cancer cell lines.[40] The application of metal NPs in DNA cleavage leads to provide an advantage such as generation of singlet oxygen species.[41,42] Recently, Geddes and co-workers have demonstrated enhancement of singlet oxygen generation using metal NPs.[43] Metal NPs in conjunction with photosensitizers can also enhance singlet oxygen generation.[44] Although a number of copper(II) complexes exhibited DNA degradation,[45] use of magnetic core–shell nanocomposites containing dendrimer-stabilized gold NPs (AuNPs) and palladium NPs (PdNPs) as singlet oxygen generators were reported in the literature to the best of our knowledge. It is in this background, we described a novel experimental strategy for synthesis of core–shell magnetic nanocomposites via integration of magnetic NPs, polymers, and 3-D dendrimers. In the present study, we report the synthesis of new magnetic core–shell nanocomposites using Fe3O4–poly(styrene-divinylbenzene-vinylbenzyl chloride) (ST-DVB-VBC) matrix grafted with PPI-G(2) and PPI-(G3) dendrimers, and these two matrices in turn were used to stabilize AuNPs and PdNPs to form four types of magnetic core–shell nanocomposites. These magnetic core–shell nanocomposites were then examined for nuclease activities with pBR322 DNA by gel electrophoresis technique.

Results and Discussions

Synthesis and Characterization of Magnetic Core–Shell Nanocomposites Fe3O4–Poly(ST-DVB-VBC)–PPIG(2)/PPI(G3) Stabilized AuNPs and PdNPs

Characterization by Fourier Transform Infrared (FT-IR) Spectroscopy

In Figure a, the FT-IR spectrum of Fe3O4NPs showed a characteristic absorption peak at 589 cm–1 due to magnetic Fe3O4 NPs. The FT-IR spectrum of OA–Fe3O4NPs (Figure b) showed peaks at 1610, 1660, 2853 and 2924, and 3446 cm–1, and they are assigned to C=C, C=O, −CH3 and −CH2, and OH groups, respectively. Therefore, oleic acid caps Fe3O4NPs. The magnetic Fe3O4–poly(ST-DVB-VBC) nanocomposite was prepared by suspension polymerization technique: OA–Fe3O4NPs were coated with poly(styrene) copolymer formed by copolymerization of styrene (ST), VBC, and DVB. In Figure c, the FT-IR spectrum of Fe3O4–poly(ST-DVB-VBC) showed peaks due to C=O at 1728, C=C of benzene at 1603, C–O–C at 1269 and 1147, and C–H at 2924 cm–1. It confirms formation of Fe3O4–poly(ST-DVB-VBC). It was again functionalized with 3-aminobenzoic acid. The functionalization was confirmed by its FT-IR spectrum in comparison with the FT-IR spectrum of Fe3O4–poly(ST-DVB-VBC) (control) (Figure c,d). The peaks at 1664 cm–1 due to C=O (COOH) and at 3425 cm–1 due to −OH (COOH) confirm functionalization with 3-aminobenzoic acid. In addition, the intensity of the peak due to C–Cl stretching at 698 cm–1 in Figure c significantly reduced confirming nucleophilic substitution on it with 3-aminobenzoic acid.

Figure 1

FT–IR spectra of (a) Fe3O4, (b) Fe3O4–OA, (c) Fe3O4–poly(ST-DVB-VBC), (d) Fe3O4–poly(ST-DVB-VBC)–COOH, (e) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2), and (f) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3).

Further, the separate grafting of dendrimers, PPI-G(2), and PPI-G(3) onto the Fe3O4–poly(ST-DVB-VBC)–COOH matrix was also established by their FT-IR spectra. Comparison of the FT-IR spectra of Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) (Figure e) and Fe3O4–poly(ST-DVBVBC)–PPI-G(3) (Figure f) with that of Figure d reveals that the characteristic peak due to C=O group, observed at 1738 cm–1 in Figure d, is shifted to 1645 cm–1 in Figure e,f. It confirms the formation a −CONH– linkage. The appearance of −CONH– groups confirms the grafting of PPI-G(2) and PPI-G(3) dendrimers on the respective matrix. The FT-IR spectra derived from all of the catalysts (Figure S1a-d) reveal the peak due to −NH2 between 3000 and 3200 cm–1 (Figure e,f) completely disappeared, confirming stabilization PdNPs and AuNPs.

Characterization by UV–Visible Spectroscopy

The immobilization of PdNPs and AuNPs onto the Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and −PPI-G(3) nanocomposites is established through UV–vis spectroscopy. Two types of Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and −PPI-G(3), separately dispersed in ethanol was treated with a solution of K2PdCl4 in water. The magnetic NPs became brownish yellow, thus confirming bonding of Pd2+ with the composite. The composites were then converted into Pd0NPs by slow addition of NaBH4 solution. The brown color was changed to dark brown, confirming immobilization of PdNPs onto the respective composite. To establish the immobilization of PdNPs, the composites were dispersed separately in ethanol and characterized by UV–vis spectroscopy. The surface plasmon resonance band was observed at 248 nm (λmax) for each solution (Figure S2a). Therefore, it confirms formation of PdNPs in both the composites. Similarly, the formation of AuNPs in Fe3O4–poly(ST-DVBVBC)–PPI-G(2)–AuNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs was also confirmed by UV–vis spectral analysis. Both composites gave surface plasmon resonance band with λmax at 545 nm (Figure S2b), thus confirming immobilization of AuNPs in the composites.

Characterization by Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)

The SEM and TEM images of Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs, and −PPI-G(3)–AuNPs are shown in Figure a–d. The SEM and TEM images showed spherical particles with rough surface, and there were two layers: OA–Fe3O4 forms the internal layer and it is the core of the composite, and poly(ST-DVB-VBC) grafted with PPI-G(2) and G(3) dendrimers form the external layer, called shell. The thickness of the shell increased with the increase in the generation number of PPI dendrimer which in turn led to stabilize more number of AuNPs and PdNPs. It was observed maintaining of the spherical shape of the first level matrix viz., OA–Fe3O4 even after the multistep synthesis and metal NP formation.

Figure 2

SEM and TEM images of (a,b) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)/PPI-G(3)–PdNPs and (c,d) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)/PPI-G(3)–AuNPs.

Characterization by X-ray Diffraction (XRD) Studies

The XRD patterns of Fe3O4, Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs and −PPI-G(3)–AuNPs are shown in Figure S3a–e. The bare Fe3O4 (Figure S3a) showed peaks at 30.1°, 35.5°, 43.2°, 57.1°, and 62.7° (2θ). They are due to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1), and (4 4 0), respectively. The diffraction patterns support the formation of cubic spinel phase. The XRD patterns of all four magnetic core–shell nanocomposites showed two reflections at 7–10° and 19–23° (2θ). They are assigned to the polymeric network existing in semicrystalline form. The Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs (Figure S3b) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs (Figure S3c) core–shell nanocomposites showed three reflections due to (1 1 1), (2 0 0), and (2 2 0). The reflections reveal that PdNPs have face-centered cubic symmetry. Similarly both Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs (Figure S3d) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs (Figure S3e) also showed similar reflections at 38.1°, 44.3°, and 64.4° (2θ) due to (1 1 1), (2 0 0), and (2 2 0). Hence, AuNPs also have a face-centered cubic symmetry. The average crystal size of AuNPs and PdNPs was calculated by substituting the respective 2θ values of the reflection of (1 1 1) into the Scherrer equation, and it was equal to 14 and 9 nm for PdNPs present in Fe3O4–poly(STDVB-VBC)–PPI-G(2)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs, respectively. Similarly, the size of the AuNPs in Fe3O4–poly(STDVB-VBC)–PPI-G(2)–AuNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs was calculated, and it was equal to 5 and 3.8 nm, respectively. The formation of such low-size AuNPs and PdNPs on Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) is due to the influence of higher generation PPI-G(3) dendrimer templates grafted onto the Fe3O4–poly(ST-DVBVBC) nanocomposite.

Characterization by Vibrating Sample Magnetometer (VSM)

The magnetic properties of Fe3O4, OA–Fe3O4, Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs and −PPI-G(3)–AuNPs were determined by VSM, and the corresponding M–H curves are depicted in Figure a–f. The saturation magnetization (Ms) was equal to 72.7 and 64.92 emu/g for Fe3O4NPs and OA–Fe3O4, respectively, 43.72 and 28.06 for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and −PPI-G(3)–PdNPs, respectively, 39.07 and 24.39 emu/g for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and −PPI-G(3)–AuNPs, respectively. A comparison of VSM curves reveals that the Ms values of Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVBVBC)–PPI-G(3)–AuNPs are lower than that of the respective magnetic nanocomposites derived from lower generation number (PPI-G(2)) matrix, Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs. It is due to Fe3O4–poly(ST-DVB-VBC) matrix grafted with PPI-G(3) dendrimer containing large number of −NH2 groups and adding much organic load to the shell. The formation of such a thick shell around Fe3O4 can reduce magnetic attraction on the outer layer. Similarly, the decreased Ms value for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)-AuNPs and −PPI-(G3)–AuNPs compared to Fe3O4–poly(STDVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs is due to oxidation of the PdNPs more easily than AuNPs because of their lower reduction potential. Hence, the magnetic core–shell nanocomposites containing gold may provide a shell with thick wall compared to that of the core–shell matrix nanocomposites containing PdNPs. Therefore, the AuNP composite gives lower Ms value than the PdNP composite. The percentage of Fe3O4NPs of each composite was calculated using the Ms value of bare-Fe3O4NPs, and it was equal to about 89.29% in the OA–Fe3O4, 60.14 and 38.6% for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs, respectively, and 53.7 and 33.5% for Fe3O4–poly(ST-DVBVBC)–PPI-G(2)–AuNPs and −PPI-G(3)–AuNPs nanocomposites, respectively. Because the percentage of the Fe3O4 content was calculated from the Ms value, whatever explanation offered earlier regarding magnetic impact with respect to Ms value goes well with the present values of the percentage of Fe3O4.

Figure 3

VSM curves of (a,b) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)/PPI-G(3)–PdNPs and (c,d) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)/PPI-G(3)–AuNPs.

X-ray Photoelectron Spectroscopy (XPS) Analysis

It was carried out to verify the oxidation state of the iron in iron oxide, and immobilization of AuNPs and PdNPs onto the four magnetic core–shell nanocomposites. The XPS spectra of Fe3O4–poly(STDVB-VBC)–PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs are shown in Figure a,b, respectively. The XPS spectrum of Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs showed the peaks due to Fe2+ (Fe 2p3/2) and Fe3+ (Fe 2p1/2) at the binding energies of 712.4 and 725.6 eV, respectively. Figure a shows the appearance of two peaks for Pd at the binding energy of 335.5 (3d5/2) and 341.4 eV (3d3/2). The decreased binding energy is due to the interaction between the nitrogen atoms of the PPI dendrimer with PdNPs. Further, the grafting of PPI dendrimer onto the Fe3O4–polymer nanocomposite is confirmed by the appearance of N(1s) peak at the binding energy of 402.3 eV. All of the above observations confirm the core, Fe3O4, existing in Fe2+ and Fe3+ states. The grafting of PPI-G(3) dendrimer is confirmed based on the appearance of nitrogen peak. The appearance of 3d level peaks for Pd and 4f level peaks for Au confirms immobilization in Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs core–shell composites, respectively. Similarly, the XPS spectrum of Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs (Figure b) showed C(1s) signal at 284.70 eV, Fe2+ (Fe 2p3/2) and Fe3+ (Fe 2p1/2) at 714.2 and 724.5 eV, respectively. The binding energies are in agreement with the reported values.[46] Similarly, the grafting of PPI-G(3) and immobilization of AuNPs are confirmed by the characteristic peaks of N(1s), O(1s), C(1s), and Au(4f) levels at 401.2, 530.94, 287.7, 82.8, and 86.6 eV, respectively.

Figure 4

XPS spectra of (a) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs and (b) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs.

Characterization by Raman Spectroscopy

Raman spectroscopy is an important tool to distinguish iron oxides in different structural phases. The Raman spectra of the four nanocomposites are shown in Figure S4a–d. The Fe3O4 core showed a broad and intense peak for magnetite at 662 cm–1. It is due to the symmetric stretch of Fe–O bonds (A1g). In contrast, the Raman spectra of the four core–shell Fe3O4–polymer nanocomposites (Figure S4a–d) yielded an intense peak due to T2g(2) mode at 545 cm–1. The appearance of T2g(2) mode is obviously due to the laser irradiation (1.95 mW), which transforms magnetite nanocrystals partially into hematite or maghemite nanocrystals because of meta stability of magnetite. A similar observation was also reported.[47,48]

Thermogravimetric Analysis (TGA)

It is used to determine the weight percentage of Fe3O4 content in the four nanocomposites. The thermal behavior of nanocomposites was examined from 30 to 600 °C. The TGA curves for Fe3O4–poly(STDVB-VBC)–PPI-G(2)–AuNPs, −PPI-G(3)–AuNPs, and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs are shown in Figure S5a–d. The results showed a rapid weight loss between 350 and 400 °C and above 480 °C. The loss in weight occurred above 480 °C is attributed to degradation and desorption of the polymer from the outer layer of the Fe3O4 core. The magnetite content in each nanocomposites was estimated from the residual mass percentage, and it was equal to 42.2 and 8.6% for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs and −PPI-G(3)–AuNPs composites, respectively, and 43.1 and 9.4% for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPI-G(3)–PdNPs composites, respectively.

Comparative Studies of Magnetic Core–Shell Nanocomposites for DNA Cleavage Studies

The nanocomposites were employed separately for cleavage of plasmid pBR322 DNA using H2O2 as a free radical scavenger under similar reaction condition. The cleavage of the supercoiled pBR322 plasmid DNA was probed using agarose gel electrophoresis. A rapid migration of supercoiled form (Form-I) was noticed. In general, if one strand is cleaved, the supercoil will relax to produce a slower-moving nicked circular form (Form-II). If both strands are cleaved, a linear form (Form-III) will be generated that migrates between Form-I and Form-II. In our case, irrespective of the core–shell nanocomposites, the PdNPs and AuNPs reacted with H2O2 and produced •OH radicals and –OH ions. The •OH radicals then cleaved the pBR322 plasmid DNA. The degree of DNA cleavage was quantitatively estimated from the results of electrophoresis. Five different concentrations were fixed between 30 and 50 μM, and the DNA cleavage was studied. The DNA cleavage pattern obtained for core–shell nanocomposites viz., Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs, −PPI-G(3)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs, −PPI-G(3)–AuNPs are shown in Figure a–d, and the corresponding bar diagrams are shown in Figure a–d. The DNA cleavage pattern and their bar diagrams showed an increase in the efficiency of DNA cleavage with an increase in the concentration of the nanocomposite. As discussed above, the cleavage of DNA occurred due to •OH radical generated in the reaction mixture. The PdNPs/AuNPs immobilized in the nanocomposite reacted with H2O2, and generated the •OH radicals and hydroxyl ions. The number of •OH radicals generated in the reaction mixture depends on the amount of H2O2 decomposed by PdNPs/AuNPs. In other words, the more the number of PdNPs/AuNPs, the higher is the amount of H2O2 decomposed, and the more the number of •OH radicals formed, the higher is the degree of cleavage of supercoiled plasmid DNA. The efficiency of DNA cleavage was in the order Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs > Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs > Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs > Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs. Although equal amount of the precursors of PdNPs/AuNPs was added in the preparation of all four magnetic core–shell nanocomposites, the amount of PdNPs/AuNPs immobilized on composites derived from PPI-G(2) matrices was less than that of PPI-G(3). It is due to less number of −NH2 surface functional groups available in PPI-G(2). Therefore, the amount of H2O2 decomposed to generate •OH radicals was less. Therefore, the number of DNA molecules cleaved was also less. Similarly, among four types of nanocomposites, the Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs (50% of Form-II and 22% of Form-III) showed more active DNA cleavage than Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs (45% of Form-II), Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs (62% of Form-II), and Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs (99% of Form-II). In the DNA cleavage studies, in the presence of core shell composite, the Form-I DNA was cleaved into Form-II. The percentage of Form-II is usually accounted to ascertain the performance of the core–shell composite. In certain circumstances especially in the presence of efficient core–shell composite, the Form-I DNA directly gets cleaved and gives both Form-II and -III. This indicates that a particular core–shell composite is extraordinarily accelerating the DNA cleavage and producing both Form-II and -III. Hence, the Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs rapidly cleaved DNA and thus produced both Form-II and -III DNA. When the concentration of the Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs was increased from 40 to 45 and then to 50 μM, Form-III DNA was observed. This observation suggests that the linear DNA is formed from the supercoiled DNA via nicked DNA-intermediated consecutive cleaving process. This is because, the composites having PPI-G(3) contain more number of −NH2 surface functional groups than PPI-G(2). Therefore, the metal content of PPI-G(3) is higher than PPI-G(2) grafted nanocomposites. The more number of PdNPs/AuNPs available in PPI-G(3) nanocomposite increased DNA cleavage. Although in Fe3O4–poly(STDVB-VBC)–PPI-G(3)–AuNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs, same amount of Fe3O4–poly(ST-DVB-VBC) matrix and PPI-G(3) were used as a common stabilizing agents, because of high reduction potential of Au3+ to Au (+0.152 V), the AuNPs were produced kinetically at a rapid rate and thus yielded smaller size AuNPs (3.8 nm) with high surface area. However, in the case of PdNPs formation, the standard reduction potential of Pd2+ to Pd is low (+0.915 V), and hence, the rate of growth of PdNPs was lower than that of the AuNPs and hence yielded a larger size PdNPs (9 nm) with low surface area for DNA cleavage. Therefore, it confirms higher activity of Fe3O4–poly(ST-DVB VBC)–PPI-G(3)–AuNPs than Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs.

Figure 5

DNA cleavage patterns of (a) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs, (b) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs, (c) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs, and (d) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs.

Figure 6

Bar diagram for DNA cleavage studies of (a) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs, (b) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs, (c) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs, and (d) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs.

Conclusions

We have developed four types of novel, magnetically recoverable core–shell nanocomposites using Fe3O4 as a core and poly(MS-DVB-VBC) as a shell. The disappearance of −NH2 peaks between 3000 and 3200 cm–1 in the FTIR spectra and appearance of SPR peak at 547 and 210 nm in UV–vis spectra confirms the formation of AuNPs and PdNPs. The SEM images showed smooth-to-rough surface with increased white patches on PPI-G(3)-stabilized AuNPs/PdNPs. VSM studies revealed that the percentage of Fe3O4NPs was equal to 95.5% in OA–Fe3O4, 60.14 and 38.6% in Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and −PPIG(3)–PdNPs, respectively, and 53.7 and 33.5% in Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)- AuNPs and −PPI-G(3)–AuNPs, respectively. The XPS results showed all of the elements present in the core: the N(1s), O(1s), C(1s), Fe2+ and Fe3+, and Au(4f) levels were observed respectively at 401.2, 530.94, 287.7, 714.2 and 724.5, and 82.8 and 86.6 eV for Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs. Similarly, two peaks at 335.5 (3d5/2) and 341.4 eV (3d3/2) due to PdNPs were observed for Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs. The integration of Fe3O4, polystyrene, dendrimer, and Au/PdNPs produced an efficient magnetic core–shell nanocomposite for effective cleavage of DNA. The DNA cleavage was in the order Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs (100%) > Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs (99%) > Fe3O4–poly(ST-DVB-VBC)–PPI G(3)–PdNPs (68%) > Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs (45%). In the case of Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs nanocomposite, there is co-existence of all three forms of DNA, suggesting the formation of linear DNA from the supercoiled DNA via nicked DNA-intermediated consecutive cleaving process. Although the same amount of Type-II Fe3O4–poly(ST-DVB-VBC) matrix and PPI-G(3) was used as a stabilizing agent, because of high reduction potential of Au3+ to Au (+0.152 V), the AuNPs was produced kinetically at a rapid rate and thus yielded small size AuNPs (3.8 nm) with high surface area for DNA cleavage compared with PdNPs with low reduction potential (+0.915 V). Hence, it is worth to claim that the Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs is a new addition to the literature for effective cleavage of DNA. These new magnetic nanocomposites could offer new approaches and opportunities in chemistry, biology, and medicine in the future. Similar approaches can be used for the development of certain new fluorescent-magnetic nanocomposites with low toxicity. A significant part of the future work in this area must be focused on the investigation of the toxicity and improvement of biocompatibility of multimodal nanocomposites.

Experimental Methods

Synthesis of Fe3O4 Magnetic NPs (Fe3O4NPs)

Fe3O4 magnetic particles (Fe3O4NPs) were synthesized by modifying the reported procedure (Scheme ) through the co-precipitation method.[49] The dried magnetic Fe3O4NPs were characterized by FT-IR (Figure a).

Scheme 1

Synthesis of Fe3O4–Poly(ST-DVB-VBC)–PPI-(G2)–Pd/AuNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-(G3)–Pd/AuNPs

Preparation of Magnetic Fe3O4–Poly(ST-DVB-VBC) Nanocomposite

The Fe3O4–poly(ST-DVB-VBC) polymer-coated magnetic nanocomposite was prepared by following the below mentioned procedure (Scheme ).

One gram of OA–Fe3O4NPs dispersed in 50 mL of ethanol in a 250 mL RB flask was ultrasonicated for 15 min at 3 eV output. Two grams of poly(ethylene glycol) dissolved in 10 mL of hot water, 3 mL (0.007 mol) of ST, 3 mL of (0.007 mol) DVB, and 6 mL of (0.014 mol) VBC were mixed and stirred. Then, both the solutions were transferred to the OA–Fe3O4NPs dispersion. Benzoyl peroxide (0.4 g) dissolved in 2 mL of ethanol was added dropwise under vigorous stirring using a mechanical stirrer. The mixture was stirred at 80 °C for 4 h. The resulting nanocomposite was isolated using a bar magnet and washed with water and ethanol. It was then dried under vacuum for 24 h to obtain core–shell nanocomposite Fe3O4–poly(ST-DVB-VBC). It was characterized by FT-IR and the spectrum is shown in Figure c.

Acid Functionalization of Fe3O4–Poly(ST-DVB-VBC) Nanocomposite

In a 100 mL RB flask, 1 g of the magnetic core–shell nanocomposite containing the chloride groups, Fe3O4–poly(ST-DVB-VBC) was dispersed in 20 mL of ethanol ultrasonication for 15 min (Scheme ). 3-Aminobenzoic acid (0.75 g) dissolved in 5 mL of ethanol was added to it, and the reaction mixture was stirred mechanically for 2 h at 50 °C. The resulting magnetic nanocomposite was centrifuged and the residue was washed repeatedly with ethanol. Then, the magnetic nanocomposite was stirred with 10% of KOH for 1 h, filtered and washed with water and finally with water–ethanol mixture. It was then dried, and Fe3O4–poly(ST-DVB-VBC)–COOH matrix was obtained. The dried matrix was characterized by FT-IR (Figure d). This magnetic core–shell nanocomposite Fe3O4–poly(ST-DVB-VBC)–COOH was then used as a common matrix for grafting of stabilizing agents such as PPI-G(2) and PPI-G(3) dendrimers.

Grafting of PPI-(G2) and PPI-(G3) Dendrimers onto the Fe3O4–Poly(ST-DVB-VBC)–COOH Matrix

The grafting of PPI (G2) dendrimer was performed by the following procedure: in a 100 mL RB flask, 1 g of Fe3O4–poly(ST-DVB-VBC)–COOH was dispersed in 10 mL of dimethylformamide by ultrasonication for 15 min (Scheme ). PPI-G(2) (100 mg (0.1293 mmol)), 0.04 g (0.15 mmol) of dicyclohexyl carbodiimide, and 0.02 g (0.15 mmol) of dry dimethylaminopyridine were added to it. The reaction mixture was stirred for 72 h at ambient temperature under nitrogen atmosphere, and the resulting product was filtered and washed with hot chloroform, and the byproducts were removed. It was dried under vacuum to obtain Fe3O4–poly(ST-DVB-VBC)–PPI-G(2). In a similar manner, the PPIG(3) was grafted by taking equal amount of reagent/solvent mentioned in the earlier procedure, and another magnetic core–shell matrix Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) was obtained. These two different magnetic core–shell nanocomposites Fe3O4–poly(ST-DVB-VBC)–PPIG(2) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) were characterized by FT-IR (Figure e,f).

Preparation of Magnetic Core–Shell Nanocomposites Fe3O4–Poly(ST-DVB-VBC)–PPI-G(2)/PPI-G(3)–PdNPs/AuNPs

The immobilization of PdNPs and AuNPs onto the magnetic core–shell nanocomposite Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) were performed separately by taking 3.5 × 10–3 mmol of K2PdCl4 and HAuCl4 as metal precursors. By using NaBH4 as a reducing agent the magnetic core–shell nanocomposites were obtained (Scheme ). Magnetic core–shell dendrimer matrices (0.5 g) viz., Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) were taken separately in a 50 mL RB flask, and ultrasonication was done for 15 min in 20 mL ethanol (Scheme ). K2PdCl4 (1.5 mg (3.5 × 10–3 mol)) in 5 mL of ethanol solution was added to it slowly at room temperature, and then, the respective reaction mixture was stirred with mechanical stirrer for 1 h. As a result, the corresponding Fe3O4–polymer-coated magnetic matrices changed from brownish yellow to dark brown. It indicates the immobilization of Pd2+ in PPI-G(2) and PPI-G(3) dendrimer grafted Fe3O4–poly(ST-DVB-VBC) composite. After 2 h, the respective Fe3O4–poly(ST-DVB-VBC)-PPI-G(2) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) complexed with Pd2+ was filtered and washed with water and methanol until the supernatant was free of Pd2+ ions, and then both the composites were dried in a vacuum oven at 60 °C for 12 h. Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–Pd2+ and Fe3O4–poly(ST-DVBVBC)–PPI-G(3)–Pd2, taken separately in 50 mL RB flask, were reduced by adding 2 mmol of freshly prepared aqueous NaBH4 (5 mL) under vigorous stirring for about 2 h at room temperature. After the reduction, the light yellowish brown color of the particles became dark brown. The resulting magnetic nanocomposite was filtered and washed repeatedly with water and ethanol. It was then dried under vacuum at 60 °C for 24 h to obtain Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs. Similarly, the nanocomposites containing AuNPs were prepared using the same two matrices in presence of HAuCl4. To confirm the immobilization of PdNPs/AuNPs onto the Fe3O4–poly(ST-DVB-VBC)–PPI-G(2) and Fe3O4–poly(ST-DVB-VBC)–PPI-G(3) magnetic nanocomposites, they were characterized by FT-IR and UV–vis spectroscopy. The spectra are shown in Figures S1 and S2.

Comparative Efficiency of Magnetic Core–Shell Nanocomposites in DNA Cleavage Studies

The efficiency of four types polymer-coated magnetic core–shell nanocomposites viz., (i) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs, (ii) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–PdNPs, (iii) Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–AuNPs, and (iv) Fe3O4–poly(ST-DVB-VBC)–PPI-G(3)–AuNPs was studied separately in the cleavage of supercoiled pBR322 DNA through agarose gel electrophoresis. The supercoiled pBR322 DNA (0.020 mg mL–1) was taken separately in a test tube and mixed with 50 mM solution of Tris-HCl/NaCl buffer (pH 7.2) and H2O2 (40 μL). Each magnetic core–shell nanocomposite was added separately to it by fixing five different concentrations from 30 to 50 μM, irrespective of the composite. All of the samples were incubated for 30 min at 37 °C followed by their addition to the loading buffer containing 25% bromo phenol blue, 0.25% xylene cyanol, and 30% glycerol (3 mL). Finally, the respective mixture was loaded on 0.8% agarose gel-containing ethidium bromide (1 mg mL–1) and then, the electrophoresis was carried out at 50 V for 1 h in TBE buffer (45 mM Tris, 45 mM H3BO3, 1 mM EDTA, pH 8.3). The bands observed for Fe3O4–poly(ST-DVB-VBC)–PPI-G(2)–PdNPs/AuNPs and PPI-G(3)–PdNPs/AuNPs when visualized with UV light were photographed (Figure a–d).