Multifunctional Iron Oxide Magnetic Nanoparticles for Biomedical Applications: A Review

Due to their good magnetic properties, excellent biocompatibility, and low price, magnetic iron oxide nanoparticles (IONPs) are the most commonly used magnetic nanomaterials and have been extensively explored in biomedical applications. Although magnetic IONPs can be used for a variety of applications in biomedicine, most practical applications require IONP-based platforms that can perform several tasks in parallel. Thus, appropriate engineering and integration of magnetic IONPs with different classes of organic and inorganic materials can produce multifunctional nanoplatforms that can perform several functions simultaneously, allowing their application in a broad spectrum of biomedical fields. This review article summarizes the fabrication of current composite nanoplatforms based on integration of magnetic IONPs with organic dyes, biomolecules (e.g., lipids, DNAs, aptamers, and antibodies), quantum dots, noble metal NPs, and stimuli-responsive polymers. We also highlight the recent technological advances achieved from such integrated multifunctional platforms and their potential use in biomedical applications, including dual-mode imaging for biomolecule detection, targeted drug delivery, photodynamic therapy, chemotherapy, and magnetic hyperthermia therapy.


Introduction
Nanoparticles (NPs) have attracted substantial scientific attention because they offer novel structural, optical, and electronic properties that are distinct from those of individual molecules or bulk materials. Currently, scientists can design and prepare exotic NPs with controllable sizes, morphologies, and compositions for various applications [1][2][3][4][5]. Among the various types of NPs, magnetic NPs are a promising nanoscale tool in the current biomedical field [5]. For example, the capacity of NPs to generate magnetic fields and influence their local environment has led to their use as contrast agents in magnetic resonance imaging (MRI) techniques [6,7]. Furthermore, their capacity to be manipulated via an external magnetic field makes them attractive candidates for use as drug-delivery vehicles and in cell separation/purification and cell tracking [8,9]. Additionally, their capacity to produce heat when subjected to an oscillating magnetic field makes them suitable as antitumor therapeutic agents [7,9]. Due to their good magnetic properties, excellent biocompatibility, and low cast, magnetic iron oxide nanoparticles (IONPs) are the most commonly used magnetic nanomaterials and have been extensively explored in a wide range of fields, including biomedical, sensing, environmental science, energy storage, and electronic devices [5,6,8,10]. Although magnetic IONPs can be used for a variety of applications in biomedicine, most practical applications require IONP platforms that perform several tasks in parallel. This parallel activity can be achieved by appropriate engineering and integration of magnetic IONPs with suitable conjugates, rendering them practical for use in a broad spectrum of biomedical fields. Moreover, attaching appropriate organic molecules (e.g., dyes, polymers, proteins, and/or antibodies, or other nanomaterials, such as quantum dots and noble metal NPs) to magnetic IONPs allows new biological applications, including protein purification and biosensing [1,8]. In this review, we focus on the progress in current composite NPs based on the integration of magnetic IONPs with different classes of organic and inorganic materials. The integrated materials include organic dyes, biomolecules (e.g., lipids, DNAs, and folic acid), quantum dots, noble metal NPs (including Au and Ag NPs), and stimuli-responsive polymers. We will give a brief overview and highlight the recent technological advances achieved in these integrated multifunctional platforms and their potential use in biomedical applications, as shown in Figure 1. range of fields, including biomedical, sensing, environmental science, energy storage, and electronic devices [5,6,8,10]. Although magnetic IONPs can be used for a variety of applications in biomedicine, most practical applications require IONP platforms that perform several tasks in parallel. This parallel activity can be achieved by appropriate engineering and integration of magnetic IONPs with suitable conjugates, rendering them practical for use in a broad spectrum of biomedical fields. Moreover, attaching appropriate organic molecules (e.g., dyes, polymers, proteins, and/or antibodies, or other nanomaterials, such as quantum dots and noble metal NPs) to magnetic IONPs allows new biological applications, including protein purification and biosensing [1,8]. In this review, we focus on the progress in current composite NPs based on the integration of magnetic IONPs with different classes of organic and inorganic materials. The integrated materials include organic dyes, biomolecules (e.g., lipids, DNAs, and folic acid), quantum dots, noble metal NPs (including Au and Ag NPs), and stimuli-responsive polymers. We will give a brief overview and highlight the recent technological advances achieved in these integrated multifunctional platforms and their potential use in biomedical applications, as shown in Fig
In 2019, a multifunctional nanotheranostic agent, SPIO@DSPE-PEG/DOX/ICG nanoparticles, was developed by Dai and coworkers, who simultaneously loaded the traditional chemotherapeutic doxorubicin (DOX) and the organic dye indocyanine green (ICG) into 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]coated SPIONs [17]. The hydrophobic SPIONs were coated with DSPE-PEG 2000 to form a phospholipid shell layer surrounding the SPION core, with the DSPE moieties and hydrophilic outside surfaces derived from the PEG moieties of DSPE-PEG 200. While the lipid shell layer enabled high loading of hydrophobic ICG and DOX, the hydrophilic outside surface ensured high water solubility, excellent biocompatibility, and increased cellular uptake of the NPs [17]. Both in vivo near-infrared (NIR) fluorescence imaging and MRI showed slow and sustained release of DOX from the SPIO@DSPE-PEG/DOX/ICG NPs within tumor cells, resulting in high antitumor efficacy against C6 glioma in rats, without obvious side effects. Figure 4 illustrates the preparation and utilization of SPIO@DSPE-PEG/DOX/ICG NPs in MR/NIR fluorescence dual-modal imaging and chemotherapy of glioma through intravenous injection.  Iron oxides and organic dyes can also be integrated into micelle-based nanoparticles for biomedical applications. Specifically, Yan et al. reported the fabrication of Fe3O4-encapsulated polymeric micelles via self-assembly of fluorine-containing amphiphilic poly(2,2,3,4,4,4-hexafluorobutyl methacrylate-co-9-(4-vinylbenzyl)-9H-carbazole)-gpoly(ethylene glycol) copolymers and oleic acid-modified Fe3O4 NPs for dual-modality magnetic resonance and optical imaging [19]. Due to the presence of the magnetic core and fluorescent carbazole dyes in the polymeric shell, the Fe3O4@poly(HFMA-co-VBK)-g-  In addition to polymer-based structures, silica-based [28] and silica/polymer-based NPs [23,24] have been integrated with organic dyes and magnetic iron oxides for biomedical applications. Hyeon and collaborators described the synthesis of uniform mesoporous dye-doped silica NPs decorated with multiple Fe3O4 nanocrystals for use as contract agents for simultaneous enhanced MRI, FI, and delivery of the anticancer drug doxorubicin (DOX) [23]. Specifically, the surface of the rhodamine B (RITC)-or fluorescein isothiocyanate (FITC)-doped mesoporous silica NPs was functionalized with APS before reaction with 2-bromo-2-methyl propionic acid-coated Fe3O4 NPs, followed by surface modification with methoxy poly(ethylene glycol) succinimidyl glutarate (mPEG-SG) and methoxy poly(ethylene glycol) amine (mPEG-AM) to form PEG-coated Fe3O4-MSNs. DOX was subsequently loaded into the pores of the PEG-coated Fe3O4-MSNs to generate DOXloaded PEG-coated Fe3O4-MSNs. Scheme 3 illustrates the synthesis of the DOX-loaded PEG-coated Fe3O4-MSNs [23]. In addition to polymer-based structures, silica-based [28] and silica/polymer-based NPs [23,24] have been integrated with organic dyes and magnetic iron oxides for biomedical applications. Hyeon and collaborators described the synthesis of uniform mesoporous dyedoped silica NPs decorated with multiple Fe 3 O 4 nanocrystals for use as contract agents for simultaneous enhanced MRI, FI, and delivery of the anticancer drug doxorubicin (DOX) [23]. Specifically, the surface of the rhodamine B (RITC)-or fluorescein isothiocyanate (FITC)doped mesoporous silica NPs was functionalized with APS before reaction with 2-bromo-2methyl propionic acid-coated Fe 3 O 4 NPs, followed by surface modification with methoxy poly(ethylene glycol) succinimidyl glutarate (mPEG-SG) and methoxy poly(ethylene glycol) amine (mPEG-AM) to form PEG-coated Fe 3 O 4 -MSNs. DOX was subsequently loaded into the pores of the PEG-coated Fe 3 O 4 -MSNs to generate DOX-loaded PEG-coated Fe 3 O 4 -MSNs. Scheme 3 illustrates the synthesis of the DOX-loaded PEG-coated Fe 3 O 4 -MSNs [23].
The synthesized dye-doped PEG-coated Fe 3 O 4 -MSNs exhibited high stability in aqueous solutions and did not show detectable effects on the viability and proliferation of cells. The composite NPs accumulated at the tumor site after intravenous injection. Importantly, the presence of a multitude of magnetic Fe 3 O 4 NPs on silica surfaces resulted in strongly enhanced MR signals, and the integrated dye molecules in the silica frameworks allowed FI functionality of the dye-doped PEG-coated Fe 3 O 4 -MSNs in vivo [23], as shown in Figure 6. Moreover, DOX was successfully delivered by the platform to the tumor site and induced efficient cancer cell death [23].
The highlighted examples and composite NP systems summarized in Table 1 demonstrate that the integration of iron oxide NPs and organic dyes offers important advantages. Specifically, these platforms can provide better diagnostic information with a more complementary dataset obtained from dual-diagnostic MRI/FI modalities than the corresponding isolated imaging techniques. While the magnetic component can facilitate localization in tumor tissues for MRI in the preoperative stage, the resection of tumors during surgery can be guided by fluorescence imaging. Thus, the combination of magnetic iron oxide NPs and organic dyes can improve the accuracy of diagnosis and reduce damage to healthy tissue, the time needed for testing, the expense, and the uncertainty associated with multiple agents. Moreover, simultaneous multifunctional platforms that perform several tasks in parallel, such as dual-mode imaging and photodynamic therapy or drug delivery, can be achieved by integration of numerous organic dyes and iron-oxide-based MNPs. The highlighted examples and composite NP systems summarized in Table 1 demonstrate that the integration of iron oxide NPs and organic dyes offers important advantages. Specifically, these platforms can provide better diagnostic information with a more complementary dataset obtained from dual-diagnostic MRI/FI modalities than the corresponding isolated imaging techniques. While the magnetic component can facilitate localization in tumor tissues for MRI in the preoperative stage, the resection of tumors during surgery can be guided by fluorescence imaging. Thus, the combination of magnetic iron oxide NPs and organic dyes can improve the accuracy of diagnosis and reduce damage to healthy tissue, the time needed for testing, the expense, and the uncertainty associated with multiple agents. Moreover, simultaneous multifunctional platforms that perform several tasks in parallel, such as dual-mode imaging and photodynamic therapy or drug delivery, can be achieved by integration of numerous organic dyes and iron-oxide-based MNPs. Reproduced with permission from reference [23]. Copyright 2010 American Chemical Society.

Integration of Biomolecules and Magnetic Iron Oxide Nanoparticles
High biocompatibility, low cytotoxicity, and low risk of immune rejection make biomolecules good candidates for combination with magnetic iron oxide nanoparticles (IONPs) to develop highly targeted platforms for diagnosis, therapy, and theranostics for many notorious diseases. Depending on targeting applications, biomolecules (such as aptamers, antibodies, and even stem cells) can be integrated with magnetic IONPs to achieve desired results. Table 2 summarizes important recent studies on biomolecule-functionalized magnetic iron oxide nanoparticles for biomedical applications covered in this section. In recent years, aptamer-enabled technologies have attracted significant attention in the research community as a sensitive, reliable, and convenient method for biomarker detection in various critical human diseases [48]. Combining aptamers and IONPs offers a new set of approaches to develop more effective medical technologies for both diagnostic and therapeutic applications. Taheri and coworkers recently demonstrated that silica-coated IONPs conjugated with DNA aptamers (Ap-SiMNPs) can efficiently remove toxic serum albumin prefibrillar amyloid aggregates (AA20) as a potential method to overcome complications related to diabetes [34]. Fe 3 O 4 magnetic nanoparticles (MNPs) were synthesized using the coprecipitation method, coated with silica, and then conjugated with GRA33 DNA aptamers, as shown in Scheme 4.
The selective binding of AA20 to the GRA33 DNA aptamer (Figure 7a) was confirmed by an electrophoretic mobility shift assay (EMSA), as shown in Figure 7b. Specifically, GRA33 mobility was strongly retarded after incubation with AA20 compared to GRA33 without AA20. Surface plasmon resonance (SPR) assay results (Figure 7c) also showed the binding of GRA33 to AA20 at various aptamer concentrations. The inset in Figure 7c plots the equilibrium responses used to determine the dissociation constant K D = 3.4 × 10 −9 M, which represents a high-affinity interaction between GRA33 and AA20. This work provides a promising approach to solve amyloidosis-related complications in diabetic patients and other amyloid disorders.
Conventional chemotherapeutic agents used in various medical procedures can cause side effects in patients during and after treatment [49]. Therefore, effective methods to remove excess chemicals from the circulatory system are badly needed to make chemotherapy safer for medical treatment. In 2018, Grubbs and coworkers showed the capture of common chemotherapy agents, including cisplatin, epirubicin (EPI), and doxorubicin (DOX), from biological solutions using genomic DNA-conjugated IONPs [35]. Up to 98% capture of DOX, a commonly used cancer treatment agent, was achieved in human serum within 10 min (Figure 8a). The DNA-conjugated IONPs also captured 20% cisplatin and 68% EPI within 30 min and 25 min, respectively. The authors also demonstrated that their DNA-conjugated nanoparticles could rescue cultured cardiac myoblasts from lethal levels of DOX more efficiently than Dowex, which has previously been shown to reduce DOX levels in vivo ( Figure 8b). These results indicate the promising applicability of genomic DNA-conjugated IONPs for drug capture applications to reduce the side effects of chemotherapies. The selective binding of AA20 to the GRA33 DNA aptamer (Figure 7a) was confirmed by an electrophoretic mobility shift assay (EMSA), as shown in Figure 7b. Specifically, GRA33 mobility was strongly retarded after incubation with AA20 compared to GRA33 without AA20. Surface plasmon resonance (SPR) assay results (Figure 7c) also showed the binding of GRA33 to AA20 at various aptamer concentrations. The inset in Figure 7c plots the equilibrium responses used to determine the dissociation constant KD = 3.4 × 10 −9 M, which represents a high-affinity interaction between GRA33 and AA20. This work provides a promising approach to solve amyloidosis-related complications in diabetic patients and other amyloid disorders.   The selective binding of AA20 to the GRA33 DNA aptamer (Figure 7a) was confirmed by an electrophoretic mobility shift assay (EMSA), as shown in Figure 7b. Specifically, GRA33 mobility was strongly retarded after incubation with AA20 compared to GRA33 without AA20. Surface plasmon resonance (SPR) assay results (Figure 7c) also showed the binding of GRA33 to AA20 at various aptamer concentrations. The inset in Figure 7c plots the equilibrium responses used to determine the dissociation constant KD = 3.4 × 10 −9 M, which represents a high-affinity interaction between GRA33 and AA20. This work provides a promising approach to solve amyloidosis-related complications in diabetic patients and other amyloid disorders.  within 10 min (Figure 8a). The DNA-conjugated IONPs also captured 20% cisplatin and 68% EPI within 30 min and 25 min, respectively. The authors also demonstrated that their DNA-conjugated nanoparticles could rescue cultured cardiac myoblasts from lethal levels of DOX more efficiently than Dowex, which has previously been shown to reduce DOX levels in vivo (Figure 8b). These results indicate the promising applicability of genomic DNA-conjugated IONPs for drug capture applications to reduce the side effects of chemotherapies. The high toxicity and low efficiency of traditional cancer therapies have been problematic, and nanotechnologies can help with novel solutions. Recently, Kolovskaya et al. reported a magnetodynamic therapy to selectively eliminate tumor cells in vivo [36]. In this work, magnetite nanoparticles functionalized with fibronectin (AS-14), heat shock cognate 71 kDa protein (AS-42) aptamers, and arabinogalactan (AG) polysaccharide were applied for selective cancer cell targeting and cellular internalization. Specifically, the IONPs functionalized with AS aptamers and fragmented AG polysaccharide (AS-FrFeAG) were tested in vivo as a contrast agent for magnetic resonance imaging (MRI) of Ehrlich carcinoma in mice. For tumors in the mouse leg, AS-FrFeAG nanoparticles provided a contrast similar to that of the Omniscan contrast agent (Figure 9a(1-3),b(1-3)). However, the AS-FrFeAG nanoparticles penetrated the blood-brain barrier to reach the brain tumor and yielded a greater contrast than OmniScan (Figure 9c(2,3)), suggesting the potential use of AS-FrFeAG as a contrast agent for brain tumors. The high toxicity and low efficiency of traditional cancer therapies have been problematic, and nanotechnologies can help with novel solutions. Recently, Kolovskaya et al. reported a magnetodynamic therapy to selectively eliminate tumor cells in vivo [36]. In this work, magnetite nanoparticles functionalized with fibronectin (AS-14), heat shock cognate 71 kDa protein (AS-42) aptamers, and arabinogalactan (AG) polysaccharide were applied for selective cancer cell targeting and cellular internalization. Specifically, the IONPs functionalized with AS aptamers and fragmented AG polysaccharide (AS-FrFeAG) were tested in vivo as a contrast agent for magnetic resonance imaging (MRI) of Ehrlich carcinoma in mice. For tumors in the mouse leg, AS-FrFeAG nanoparticles provided a contrast similar to that of the Omniscan contrast agent (Figure 9a(1-3),b(1-3)). However, the AS-FrFeAG nanoparticles penetrated the blood-brain barrier to reach the brain tumor and yielded a greater contrast than OmniScan (Figure 9c(2,3)), suggesting the potential use of AS-FrFeAG as a contrast agent for brain tumors. . AS-FrFeAG functional nanoparticles used as a contrast agent for magnetic resonance imaging of tumors. A mouse with a tumor transplanted in the right leg (a,b) or brain (c) imaged without a contrast agent (1), with OmniScan as a contrast agent (2), or with AS-FrFeAG as a contrast agent (3). Reproduced from reference [36].
AS-FrFeAG nanoparticles were also evaluated for their effect on tumorous cells in the presence of a low-frequency (50 Hz) alternating magnetic field (LFAMF). Compared to untreated tumors (Figure 10a), tumors treated with aptamers alone (Figure 10b), and tumors treated with FrFeAG/LFAMF but without the AS aptamer to target the nanoparti- Figure 9. AS-FrFeAG functional nanoparticles used as a contrast agent for magnetic resonance imaging of tumors. A mouse with a tumor transplanted in the right leg (a,b) or brain (c) imaged without a contrast agent (1), with OmniScan as a contrast agent (2), or with AS-FrFeAG as a contrast agent (3). Reproduced from reference [36].
AS-FrFeAG nanoparticles were also evaluated for their effect on tumorous cells in the presence of a low-frequency (50 Hz) alternating magnetic field (LFAMF). Compared to untreated tumors (Figure 10a), tumors treated with aptamers alone (Figure 10b), and tumors treated with FrFeAG/LFAMF but without the AS aptamer to target the nanoparticles to the cancer cell (Figure 10c,d), an enhanced reduction in the bulk of the tumor cells was observed for tumors treated with AS-FrFeAG nanoparticles (Figure 10e,f). This work demonstrates the potential of aptamer-functionalized magnetite nanoparticles for noninvasive targeted cancer therapy and contrast enhancement in MRI. Figure 9. AS-FrFeAG functional nanoparticles used as a contrast agent for magnetic resonance imaging of tumors. A mouse with a tumor transplanted in the right leg (a,b) or brain (c) imaged without a contrast agent (1), with OmniScan as a contrast agent (2), or with AS-FrFeAG as a contrast agent (3). Reproduced from reference [36].
AS-FrFeAG nanoparticles were also evaluated for their effect on tumorous cells in the presence of a low-frequency (50 Hz) alternating magnetic field (LFAMF). Compared to untreated tumors (Figure 10a), tumors treated with aptamers alone (Figure 10b), and tumors treated with FrFeAG/LFAMF but without the AS aptamer to target the nanoparticles to the cancer cell (Figure 10c,d), an enhanced reduction in the bulk of the tumor cells was observed for tumors treated with AS-FrFeAG nanoparticles (Figure 10e,f). This work demonstrates the potential of aptamer-functionalized magnetite nanoparticles for noninvasive targeted cancer therapy and contrast enhancement in MRI.  Moreover, with proper aptamer conjugates, magnetic nanoparticles have also been used to enhance the performance of various biomedical applications. Katz and coworkers reported the ability of DNA-modified magnetite nanoparticles to selectively detect and induce downregulation of up to 99% of target mRNA in cultured MCF-7 cancer cells [37]. In a separate work, Fe 3 O 4 @SiO 2 functionalized with a specific ssDNA aptamer showed highly selective targeting, with a recovery of 87% toward alpha fetoprotein (AFP), a pregnancy biomarker [38]. Zeng and coworkers developed a one-step method to detect thrombin using DNA aptamer-conjugated magnetic nanospheres, with a detection limit of 97 pM [39].
In addition to aptamers, antibodies are another type of biomolecule that is regularly used for many biomedical purposes. Combining various antibodies with magnetic nanoparticles in an appropriate manner can yield a significantly good performance in diagnostics, therapeutics, and theranostics [50]. In a recent report, Timur and coworkers described a biosensor for JWH-073 cannabinoid detection using anti-K2 antibody-immobilized iron oxide magnetic nanoparticles (MNP-K2), as outlined in Figure 11 [40].
In addition to aptamers, antibodies are another type of biomolecule that is regularly used for many biomedical purposes. Combining various antibodies with magnetic nanoparticles in an appropriate manner can yield a significantly good performance in diagnostics, therapeutics, and theranostics [50]. In a recent report, Timur and coworkers described a biosensor for JWH-073 cannabinoid detection using anti-K2 antibody-immobilized iron oxide magnetic nanoparticles (MNP-K2), as outlined in Figure 11 [40]. Testing of the sensing device showed a linear relationship between the response signal and the concentration of JWH-073 used in the test run, as shown in Figure 12a. The high linearity of the signal vs. concentration relationship enables reliable and reproducible detection results, which are important in sensing applications. Moreover, a strong selectivity for JWH-073 by the MNP-K2 sensor was also observed in the presence of possible interferents, such as benzoylecgonine (BE), methamphetamine (METH), nicotine, and cotinine ( Figure 12b).  Testing of the sensing device showed a linear relationship between the response signal and the concentration of JWH-073 used in the test run, as shown in Figure 12a. The high linearity of the signal vs. concentration relationship enables reliable and reproducible detection results, which are important in sensing applications. Moreover, a strong selectivity for JWH-073 by the MNP-K2 sensor was also observed in the presence of possible interferents, such as benzoylecgonine (BE), methamphetamine (METH), nicotine, and cotinine ( Figure 12b).
In addition to aptamers, antibodies are another type of biomolecule that is regularly used for many biomedical purposes. Combining various antibodies with magnetic nanoparticles in an appropriate manner can yield a significantly good performance in diagnostics, therapeutics, and theranostics [50]. In a recent report, Timur and coworkers described a biosensor for JWH-073 cannabinoid detection using anti-K2 antibody-immobilized iron oxide magnetic nanoparticles (MNP-K2), as outlined in Figure 11 [40]. Testing of the sensing device showed a linear relationship between the response signal and the concentration of JWH-073 used in the test run, as shown in Figure 12a. The high linearity of the signal vs. concentration relationship enables reliable and reproducible detection results, which are important in sensing applications. Moreover, a strong selectivity for JWH-073 by the MNP-K2 sensor was also observed in the presence of possible interferents, such as benzoylecgonine (BE), methamphetamine (METH), nicotine, and cotinine ( Figure 12b).  Adapted with permission from reference [40]. Copyright 2020 American Chemical Society.
In addition to biomarker detection, antibody-functionalized IONPs have also been used for tumor-targeted drug delivery, as reported in a study by Oltolina et al. [41]. MNPs were functionalized with doxorubicin (DOX, a common chemotherapeutic drug, and with anti-Met/HGFR-positive antibody (mAb), which targets Met/HGFR-positive GTL-16 xenotumor cells. The mAb-functionalized MNPs displayed selective adsorption toward GTL-16 cells compared to Met/HGFR-negative Huh7 cells, as shown in both optical ( Figure 13a) and fluorescence (Figure 13b) micrographs.
Due to the GTL-16-specific binding of the mAb, the presence of functionalized MNPs (mAb-DOX-MNPs) significantly reduced the viability of Met/HGFR-positive GTL-16 cells (Figure 14a), but no similar decrease in cell viability was observed in Met/HGFR-negative Huh7 cells (Figure 14b). Although the cell viability was roughly similar in the presence of free DOX and mAb-DOX-MNPs, the targeted interaction provided by the mAb can reduce the damaging side effects that are typically observed when using DOX in cancer chemotherapeutic therapies.
In addition to biomarker detection, antibody-functionalized IONPs have also been used for tumor-targeted drug delivery, as reported in a study by Oltolina et al. [41]. MNPs were functionalized with doxorubicin (DOX, a common chemotherapeutic drug, and with anti-Met/HGFR-positive antibody (mAb), which targets Met/HGFR-positive GTL-16 xenotumor cells. The mAb-functionalized MNPs displayed selective adsorption toward GTL-16 cells compared to Met/HGFR-negative Huh7 cells, as shown in both optical (  Due to the GTL-16-specific binding of the mAb, the presence of functionalized MNPs (mAb-DOX-MNPs) significantly reduced the viability of Met/HGFR-positive GTL-16 cells (Figure 14a), but no similar decrease in cell viability was observed in Met/HGFR-negative Huh7 cells (Figure 14b). Although the cell viability was roughly similar in the presence of free DOX and mAb-DOX-MNPs, the targeted interaction provided by the mAb can reduce the damaging side effects that are typically observed when using DOX in cancer chemotherapeutic therapies. Remarkable results in biomedical applications have also been achieved with various antibody-functionalized iron oxide magnetic nanoparticle-based materials. Ramos-Gómez and coworkers demonstrated specific detection of Alzheimer's disease biomarkers using a nanoconjugate of IONPs and antiferritin antibodies at a nontoxic level [42]. In another work, Wong et al. modified the surface of dextran-coated Fe3O4 nanoparticles with anti-fetal fibronectin (fFN) antibody to detect the biomarker fFN for sensitive and accurate predictions of the risks of preterm birth [43].
In addition to aptamers and antibodies, various biomolecules have been conjugated with IONPs in research efforts to develop better biomedical technologies or expand the capability of current techniques. In an effort to create a better magnetic resonance imaging (MRI) contrast agent for pancreatic cancer, Zhu et al. grafted a pancreatic cancer-targeting CKAAKN peptide-functionalized amphiphilic hyaluroric acid-vitamin E succinate polymer (HA-VES) onto ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, as illustrated in Figure 15 [44]. Remarkable results in biomedical applications have also been achieved with various antibody-functionalized iron oxide magnetic nanoparticle-based materials. Ramos-Gómez and coworkers demonstrated specific detection of Alzheimer's disease biomarkers using a nanoconjugate of IONPs and antiferritin antibodies at a nontoxic level [42]. In another work, Wong et al. modified the surface of dextran-coated Fe 3 O 4 nanoparticles with antifetal fibronectin (fFN) antibody to detect the biomarker fFN for sensitive and accurate predictions of the risks of preterm birth [43].
In addition to aptamers and antibodies, various biomolecules have been conjugated with IONPs in research efforts to develop better biomedical technologies or expand the capability of current techniques. In an effort to create a better magnetic resonance imaging (MRI) contrast agent for pancreatic cancer, Zhu et al. grafted a pancreatic cancer-targeting CKAAKN peptide-functionalized amphiphilic hyaluroric acid-vitamin E succinate polymer (HA-VES) onto ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, as illustrated in Figure 15 [44].
Gómez and coworkers demonstrated specific detection of Alzheimer's disease biomarkers using a nanoconjugate of IONPs and antiferritin antibodies at a nontoxic level [42]. In another work, Wong et al. modified the surface of dextran-coated Fe3O4 nanoparticles with anti-fetal fibronectin (fFN) antibody to detect the biomarker fFN for sensitive and accurate predictions of the risks of preterm birth [43].
In addition to aptamers and antibodies, various biomolecules have been conjugated with IONPs in research efforts to develop better biomedical technologies or expand the capability of current techniques. In an effort to create a better magnetic resonance imaging (MRI) contrast agent for pancreatic cancer, Zhu et al. grafted a pancreatic cancer-targeting CKAAKN peptide-functionalized amphiphilic hyaluroric acid-vitamin E succinate polymer (HA-VES) onto ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, as illustrated in Figure 15 [44]. The modified CKAAKN-HA-VES@USPIO nanoparticles preferentially internalized into CKAAKN-positive BxPC-3 cells and led to a decrease in MRI signal intensity compared with CKAAKN-negative HPDE6-C7 cells ( Figure 16a). As shown in the relative intensity chart in Figure 16b, CKAAKN-HA-VES@USPIO nanoparticles were able to clearly distinguish between BxPC-3 and HPDE6-C7 cells, which led to an enhanced imaging contrast. The work also studied the cytotoxicity of the modified nanoparticles, which resulted in a cell survival rate of over 80% for both BxPC-3 and HPDE6C7 cells after 48 h of incubation with the CKAAKN-HA-VES@USPIO nanoparticles. These results suggest that The modified CKAAKN-HA-VES@USPIO nanoparticles preferentially internalized into CKAAKN-positive BxPC-3 cells and led to a decrease in MRI signal intensity compared with CKAAKN-negative HPDE6-C7 cells ( Figure 16a). As shown in the relative intensity chart in Figure 16b, CKAAKN-HA-VES@USPIO nanoparticles were able to clearly distinguish between BxPC-3 and HPDE6-C7 cells, which led to an enhanced imaging contrast. The work also studied the cytotoxicity of the modified nanoparticles, which resulted in a cell survival rate of over 80% for both BxPC-3 and HPDE6C7 cells after 48 h of incubation with the CKAAKN-HA-VES@USPIO nanoparticles. These results suggest that CKAAKN-HA-VES@USPIO nanoparticles have the potential as an enhanced MRI contrast agent with high specificity and low toxicity for pancreatic cancer diagnosis.  In another report, Ilyas, Mathur, and coworkers grafted biotin (BT) and folic acid (FA) onto Fe3O4 magnetic nanoparticles for potential use as biocompatible carriers for anticancer drug delivery or targeting treatment utilizing hypothermal effects from the Fe3O4 core [45]. The structure combined the targeting abilities of BT and FA with IONPs into a single platform that can molecularly recognize and bind to target cancer cells, as illustrated in Figure 17. Due to molecular identification and internalization by the cell mem- In another report, Ilyas, Mathur, and coworkers grafted biotin (BT) and folic acid (FA) onto Fe 3 O 4 magnetic nanoparticles for potential use as biocompatible carriers for anticancer drug delivery or targeting treatment utilizing hypothermal effects from the Fe 3 O 4 core [45]. The structure combined the targeting abilities of BT and FA with IONPs into a single platform that can molecularly recognize and bind to target cancer cells, as illustrated in Figure 17. Due to molecular identification and internalization by the cell membrane, the modified nanoparticles can enter tumor cells more easily during diagnosis or treatment processes. Figure 16. (a) In vitro magnetic resonance imaging (MRI) and (b) relative MRI signal intensities calculated with ImageJ software of BxPC-3 and HPDE6-C7 cells incubated with magnetic nanoparticles modified with and without CKAAKN (*: ImageJ p value < 0.05). Reproduced from reference [44].
In another report, Ilyas, Mathur, and coworkers grafted biotin (BT) and folic acid (FA) onto Fe3O4 magnetic nanoparticles for potential use as biocompatible carriers for anticancer drug delivery or targeting treatment utilizing hypothermal effects from the Fe3O4 core [45]. The structure combined the targeting abilities of BT and FA with IONPs into a single platform that can molecularly recognize and bind to target cancer cells, as illustrated in Figure 17. Due to molecular identification and internalization by the cell membrane, the modified nanoparticles can enter tumor cells more easily during diagnosis or treatment processes. Figure 17. Internalization of the modified magnetic nanoparticles into cancer cells based on multireceptor recognition. Reproduced with permission from reference [45]. Copyright 2020 American Chemical Society.  Figure 18a reveals that significantly more of the nanoparticles modified with biotin and folic acid than nonmodified nanoparticles were taken up by both cancer cell lines. The functionalized particles also showed higher cell uptake with increasing incubation time ( Figure 18b) and low cytotoxicity, with 98% cell viability after 48 h, indicating the high biocompatibility of the particles reported. Separately, iron oxide-lipidoid core-shell nanoparticles were reported by Clauson et al. for MRI-trackable delivery to lymph nodes in mice and can potentially be used as image-guided immunotherapy agents [46]. Kuo et al. reported peptide-functionalized aluminum-oxide-coated IONPs for capture of Staphylococcus aureus, a Gram-positive pathogenic bacterium that can cause food poisoning or infectious disease [47]. Hence, various recent works have demonstrated many designs and combinations of various biomolecules and magnetic iron oxide nanoparticles for advanced biomedical applications.
reported. Separately, iron oxide-lipidoid core-shell nanoparticles were reported by Clauson et al. for MRI-trackable delivery to lymph nodes in mice and can potentially be used as image-guided immunotherapy agents [46]. Kuo et al. reported peptide-functionalized aluminum-oxide-coated IONPs for capture of Staphylococcus aureus, a Gram-positive pathogenic bacterium that can cause food poisoning or infectious disease [47]. Hence, various recent works have demonstrated many designs and combinations of various biomolecules and magnetic iron oxide nanoparticles for advanced biomedical applications.

Integration of Quantum Dots and Magnetic Iron Oxide Nanoparticles
Quantum dots (QDs), such as CdSe, ZnS, and CdS QDs, and carbon dots (CDs) have been recognized for their unique optical properties. For example, their resistance to photobleaching and their molar extinction coefficient are greater than those of traditional organic dyes; thus, they offer long lifetimes and high brightness [51][52][53][54]. Importantly, their emission spectra can be tuned from the visible to NIR regions by controlling their size and chemical composition [51][52][53][54]. These unique properties have led to great interest in the use of QDs in biomedical applications. To enhance their functionality, QDs are often incorporated with magnetic NPs to produce composite NPs that exhibit both fluorescence and magnetism [55][56][57][58]. These integrated properties allow the development of suitable platforms, which can be examined in vitro before moving to in vivo tests [58,59]. Table 3 lists representative studies on QD-integrated magnetic iron oxide nanoparticles for biomedical applications covered in this section. Recently, the Namazi group developed gelatin-coated Fe 3 O 4 /graphene QD hybrid microspheres (Fe 3 O 4 /GQDs@GM) for anticancer drug delivery, as shown in Figure 19a [64]. The authors used carbon-based graphene quantum dots (GQDs) due to their excellent biocompatibility, high crystallinity, and fluorescence properties combined with their high density of hydroxyl (−OH) and carboxylic (−COOH) groups on the QD surface. Consequently, the combination of Fe 3 O 4 and GQDs provides opportunities for loading of abundant types of anticancer drugs in suitably designed microspheres. According to Figure 19b, the synthesized hybrid Fe 3 O 4 /GQDs@GMs revealed a higher loading capability of the well-known anticancer drug curcumin (CUR) than the pure gelatin microspheres (GMs). CUR release studies for both materials are presented in Figure 19c, showing that the release of CUR depended on the pH. The data also showed that the Fe 3 O 4 /GQDs@GMs are better than pure GMs for CUR delivery at both of the studied pH conditions. Ou et al. reported magnetic Fe 3 O 4 /SiO 2 /graphene-CdTe QDs/chitosan nanocomposites (FGQCs) as a promising multifunctional drug delivery system for biological and medical applications [61]. The CdTe QDs embedded inside the graphene provided the fluorescence capability for traceable imaging to track and diagnose the effectiveness of treatments. While the graphene shells showed good drug-loading capability due to their noncovalent π-π stacking, the SiO 2 layers prevented fluorescence quenching by blocking direct contact of fluorophores with the magnetic iron oxide NPs.
The morphologies of both Fe 3 O 4 @SiO 2 and FGQCs are presented in Figure 20a,b. The size of FGQC increased to~460 nm after coating the~220 nm Fe 3 O 4 @SiO 2 NPs with graphene-CdTe QD shells. Fluorouracil (5-FU) was used as the drug sample in this study and showed 70% loading content and 50% entrapment efficiency. By comparing the results presented in Figure 20c,d, it can be observed that significant growth inhibition of the hepatoma cell line SMMC-7721 occurred. The authors believe that the growth inhibition of the hepatoma cell line SMMC-7721 was induced by 5-FU-FGQCs at~1 µg/mL (the IC 50 concentration was 50% compared to only 10% of the same amount of free 5-FU). Hence, FGQCs, with magnetic and fluorescence characteristics, have potential for drug delivery applications. Ou et al. reported magnetic Fe3O4/SiO2/graphene-CdTe QDs/chitosan nanocomposites (FGQCs) as a promising multifunctional drug delivery system for biological and medical applications [61]. The CdTe QDs embedded inside the graphene provided the fluorescence capability for traceable imaging to track and diagnose the effectiveness of treatments. While the graphene shells showed good drug-loading capability due to their noncovalent π-π stacking, the SiO2 layers prevented fluorescence quenching by blocking direct contact of fluorophores with the magnetic iron oxide NPs.
The morphologies of both Fe3O4@SiO2 and FGQCs are presented in Figure 20a,b. The size of FGQC increased to ~460 nm after coating the ~220 nm Fe3O4@SiO2 NPs with graphene-CdTe QD shells. Fluorouracil (5-FU) was used as the drug sample in this study and showed 70% loading content and 50% entrapment efficiency. By comparing the results Despite these promising results, we note that the main concern regarding the use of many magnetic-QD platforms in biomedical applications is their inherent toxicity, which arises from the heavy metals in QDs (e.g., Cd, Se, Pb, As, and/or In) [65,66]. Consequently, applications involving these magnetic-QD platforms are currently restricted to in vitro and animal studies. presented in Figure 20c,d, it can be observed that significant growth inhibition of the hepatoma cell line SMMC-7721 occurred. The authors believe that the growth inhibition of the hepatoma cell line SMMC-7721 was induced by 5-FU-FGQCs at ~1 μg/mL (the IC50 concentration was 50% compared to only 10% of the same amount of free 5-FU). Hence, FGQCs, with magnetic and fluorescence characteristics, have potential for drug delivery applications. Despite these promising results, we note that the main concern regarding the use of many magnetic-QD platforms in biomedical applications is their inherent toxicity, which arises from the heavy metals in QDs (e.g., Cd, Se, Pb, As, and/or In) [65,66]. Consequently, applications involving these magnetic-QD platforms are currently restricted to in vitro and animal studies.

Integration of Noble Metal Nanoparticles and Magnetic Iron Oxide Nanoparticles
Noble metal nanoparticles exhibit remarkable optical properties that arise from surface plasmon resonances, which can be controlled from the ultraviolet to the near-infrared (NIR) regions of the electronic spectrum by optimizing the size, composition, shape, and topology of noble metal NPs [67][68][69][70]. As NIR light can penetrate deeply into human tissue [71], the absorption of light in the NIR regions by noble metal NPs makes them great candidates for use as contrast imaging agents to visualize organ tissue [72] and as therapeutic agents for tumor ablation and/or drug delivery [73]. The combination of noble metal NPs and magnetic NPs into one platform offers multiple diagnostic and therapeutic modalities simultaneously, which can improve the accuracy and clarity of diagnostic images while

Integration of Noble Metal Nanoparticles and Magnetic Iron Oxide Nanoparticles
Noble metal nanoparticles exhibit remarkable optical properties that arise from surface plasmon resonances, which can be controlled from the ultraviolet to the near-infrared (NIR) regions of the electronic spectrum by optimizing the size, composition, shape, and topology of noble metal NPs [67][68][69][70]. As NIR light can penetrate deeply into human tissue [71], the absorption of light in the NIR regions by noble metal NPs makes them great candidates for use as contrast imaging agents to visualize organ tissue [72] and as therapeutic agents for tumor ablation and/or drug delivery [73]. The combination of noble metal NPs and magnetic NPs into one platform offers multiple diagnostic and therapeutic modalities simultaneously, which can improve the accuracy and clarity of diagnostic images while reducing time and expense [74]. Table 4 summarizes representative studies on the integration of noble metal NPs and magnetic NPs for biomedical applications covered in this section. Due to their optical properties, stability, and biocompatibility, gold nanoparticles (Au NPs) have been used in various biological applications. Iron oxide and gold-based nanostructures for medical applications have been reviewed in the literature [81]. In general, magnetoplasmonic nanoassembly hybrid systems combine the advantages of the magnetic and plasmonic properties of Fe 3 O 4 and Au NPs. For example, Li et al. reported the use of Fe 3 O 4 @Au-mPEG-PEI core-shell composite nanoparticles for dualmode MR/CT imaging applications [77]. In this system, the Fe 3 O 4 @Au NPs were stabilized with polyethyleneimine (PEI) and poly(ethylene glycol) monomethyl ether (mPEG). A hemolytic assay showed excellent hemocompatibility of Fe 3 O 4 @Au-mPEG-PEI core-shell nanoparticles in the concentration range of 0−400 µg/mL. Additionally, the nanoparticles showed good cytocompatibility at concentrations up to 100 µg/mL. Importantly, the composite NPs exhibited a relatively high r 2 relaxivity of~146 mM −1 S −1 and good X-ray attenuation properties. Consequently, the nanoparticles were successfully used as a contrast agent for dual-mode MR/computed tomography (CT) imaging of tumor cells in mouse and rat livers.
Coating of gold on magnetic nanoparticles has been reported to not only enhance biocompatibility but also maintain the magnetic properties of the original NPs. Moreover, the high stability of gold can prevent decomposition of the magnetic nanoparticles [82]. Recently, Multari et al. synthesized hybrid nanoparticles of iron oxide decorated with gold (Fe 3 O 4 -Au) nanoparticles using tannic acid as a reducing agent [75]. The nanoparticles exhibited superparamagnetic behavior and a plasmonic peak at 560 nm, which is suitable for use in biomedical applications. The hybrid Fe 3 O 4 -Au nanoparticles showed good photothermal therapy ability against cancer cells under laser irradiation. Other shapes of gold-based magnetoplasmonic nanoparticles have been developed for use in biological applications. For example, Zhou et al. reported an in vivo study of spiky Fe 3 O 4 @AuNPs [76]. Specifically, spiky Fe 3 O 4 @AuNPs with different branch lengths exhibited different SPR peak positions. While short-branched NPs with 51.4 nm cone-shaped AuNPs on spherical Fe 3 O 4 surfaces showed an SPR peak at 575 nm, long-branched NPs exhibited two extinction peaks of transverse and longitudinal modes at 540 and 745 nm, respectively [76]. Moreover, both short-and long-branched spiky Fe 3 O 4 @AuNPs exhibited low toxicity and good biocompatibility, which are important for applications of nanoparticles as theragnostic agents.
Hou and coworkers synthesized theranostic agents based on Fe3O4@SiO2@GNS-PEG (PMGNS) nanoparticles [78]. The PMGNS nanoparticles showed imaging contrast abilities in both magnetic resonance (MR) and computed tomography (CT) at different NP concentrations. Specifically, the photothermal properties of the aforementioned nanoparticles were evaluated by testing the temperature increase profiles of PMGNS solutions at different concentrations (Figure 21a) and different power densities (Figure 21b) under NIR laser irradiation. The results from both sets of photothermal heating curves showed the highest temperature of 79.1 • C at a concentration of 320 µg/mL. Moreover, the temperature profiles after five cycles of heating were obtained to test the thermal stability of the PMGNS nanoparticles. As shown in Figure 21c, the temperature of the PMGNS aqueous solution still increased after five heating cycles. The magnetization curves obtained using a vibrating sample magnetometer (VSM) showed a saturation magnetization (Ms) value of 0.52 emu g −1 for these PMGNSs, as illustrated in Figure 21d. applications. For example, Zhou et al. reported an in vivo study of spiky Fe3O4@AuNPs [76]. Specifically, spiky Fe3O4@AuNPs with different branch lengths exhibited different SPR peak positions. While short-branched NPs with 51.4 nm cone-shaped AuNPs on spherical Fe3O4 surfaces showed an SPR peak at 575 nm, long-branched NPs exhibited two extinction peaks of transverse and longitudinal modes at 540 and 745 nm, respectively [76]. Moreover, both short-and long-branched spiky Fe3O4@AuNPs exhibited low toxicity and good biocompatibility, which are important for applications of nanoparticles as theragnostic agents.
Hou and coworkers synthesized theranostic agents based on Fe3O4@SiO2@GNS-PEG (PMGNS) nanoparticles [78]. The PMGNS nanoparticles showed imaging contrast abilities in both magnetic resonance (MR) and computed tomography (CT) at different NP concentrations. Specifically, the photothermal properties of the aforementioned nanoparticles were evaluated by testing the temperature increase profiles of PMGNS solutions at different concentrations (Figure 21a) and different power densities (Figure 21b) under NIR laser irradiation. The results from both sets of photothermal heating curves showed the highest temperature of 79.1 °C at a concentration of 320 μg/mL. Moreover, the temperature profiles after five cycles of heating were obtained to test the thermal stability of the PMGNS nanoparticles. As shown in Figure 21c, the temperature of the PMGNS aqueous solution still increased after five heating cycles. The magnetization curves obtained using a vibrating sample magnetometer (VSM) showed a saturation magnetization (Ms) value of 0.52 emu g −1 for these PMGNSs, as illustrated in Figure 21d. In addition to gold nanoparticles, silver nanoparticles (Ag NPs) have also been utilized in various biomedical applications, especially as anticancer or antibacterial agents In addition to gold nanoparticles, silver nanoparticles (Ag NPs) have also been utilized in various biomedical applications, especially as anticancer or antibacterial agents [83]. Various magnetite-silver hybrid nanoparticles, including core-shell, multishell core-shell, and heteromeric structures, have been reported [79,80]. For example, magnetite-silver hybrid NPs, both core-shell (Fe 3 O 4 @Ag-PAA) and heteromeric (Fe 3 O 4 -Ag-PAA) nanoparticles, were developed for tumor magnetic hyperthermia treatment [79]. Both the Fe 3 O 4 @Ag-PAA and Fe 3 O 4 -Ag-PAA nanoparticles exhibited higher hyperthermia efficiency than bare mag-netic nanoparticles. As shown in Figure 22, TEM images of SMMC-7721 cells treated with Fe 3 O 4 -PAA NPs (Figure 22a,d) showed significantly less magnetic hyperthermia efficiency than SMMC-7721 cells treated with Fe 3 O 4 @Ag-PAA (Figure 22b,e) and Fe 3 O 4 -Ag-PAA hybrid NPs (Figure 22c,f) in the presence of an alternating-current magnetic field. [83]. Various magnetite-silver hybrid nanoparticles, including core-shell, multishell coreshell, and heteromeric structures, have been reported [79,80]. For example, magnetite-silver hybrid NPs, both core-shell (Fe3O4@Ag-PAA) and heteromeric (Fe3O4-Ag-PAA) nanoparticles, were developed for tumor magnetic hyperthermia treatment [79]. Both the Fe3O4@Ag-PAA and Fe3O4-Ag-PAA nanoparticles exhibited higher hyperthermia efficiency than bare magnetic nanoparticles. As shown in Figure 22, TEM images of SMMC-7721 cells treated with Fe3O4-PAA NPs (Figure 22a,d) showed significantly less magnetic hyperthermia efficiency than SMMC-7721 cells treated with Fe3O4@Ag-PAA (Figure 22b,e) and Fe3O4-Ag-PAA hybrid NPs (Figure 22c,f) in the presence of an alternating-current magnetic field. In a separate study, Marmoudi and Serpooshan reported superparamagnetic iron oxide nanoparticle (SPION) core-ultrathin silver shell structures with polymeric ligand gaps for use as multimodal antibacterial agents [80]. Interestingly, SPION-silver core-shell, SPION-gold core-shell, and SPION-gold-silver core-intermediate shell-shell nanoparticles were synthesized, and the properties of the silver and gold shells were compared for therapeutic applications. The silver-coated SPIONs and gold-silver-coated SPIONs showed high therapeutic indices against Staphylococcus epidermidis and Staphylococcus aureus infections, as determined by live/dead assays. Moreover, the silver-coated SPIONs exhibited less toxicity in a human liver carcinoma cell line (HepG2), confirming the biocompatibility of these functionalized silver nanoparticles.
The examples shown here demonstrate the fabrication and utility of various types of magnetic iron oxide NP nanocomposites integrated with different noble metal NPs. The functionality of the nanocomposites can be tuned by varying the size, composition, shape, and topology of the NPs. Moreover, the examples presented here illustrate that rational engineering and design can transform nanomaterials into practical tools for future biomedical applications. In a separate study, Marmoudi and Serpooshan reported superparamagnetic iron oxide nanoparticle (SPION) core-ultrathin silver shell structures with polymeric ligand gaps for use as multimodal antibacterial agents [80]. Interestingly, SPION-silver core-shell, SPIONgold core-shell, and SPION-gold-silver core-intermediate shell-shell nanoparticles were synthesized, and the properties of the silver and gold shells were compared for therapeutic applications. The silver-coated SPIONs and gold-silver-coated SPIONs showed high therapeutic indices against Staphylococcus epidermidis and Staphylococcus aureus infections, as determined by live/dead assays. Moreover, the silver-coated SPIONs exhibited less toxicity in a human liver carcinoma cell line (HepG2), confirming the biocompatibility of these functionalized silver nanoparticles.
The examples shown here demonstrate the fabrication and utility of various types of magnetic iron oxide NP nanocomposites integrated with different noble metal NPs. The functionality of the nanocomposites can be tuned by varying the size, composition, shape, and topology of the NPs. Moreover, the examples presented here illustrate that rational engineering and design can transform nanomaterials into practical tools for future biomedical applications.

Integration of Stimuli-Responsive Polymers and Magnetic Iron Oxide Nanoparticles
Integration of magnetic NPs with polymers can expand the scope of their application in a variety of ways. Polymers have been widely used to improve the stability, aqueous dispersion, biocompatibility, and bioavailability of magnetic NPs for in vivo applications [84]. A common example of this is embedding magnetic NPs inside a shell composed of the US FDA-approved polymer poly(lactic-co-glycolic acid) (PLGA) and its derivatives [84][85][86]. However, the potential of magnetic nanoparticles is truly realized when integrated with stimuli-responsive polymers. Stimuli-responsive polymers can exhibit dramatic property changes in response to one or more external stimuli, such as temperature, pH, light, chemical, electrical, and mechanical stress [86]. Table 5 lists representative studies on stimuli-responsive polymer-integrated magnetic nanoparticles for biomedical applications covered in this section.

Integration of Thermo-Responsive Polymers
By coupling the unique properties of magnetic NPs and thermally responsive polymers, the emergence of a new paradigm in controlled drug delivery and novel nanoscale therapeutic agents is now a reality. In general, these developed platforms are prepared by the encapsulation/deposition of magnetic NPs within stimuli-responsive polymer networks [98][99][100][101][102][103][104]. The polymeric layer serves as a "smart" container that can encapsulate/load and subsequently release drugs/substances from the polymer network upon external adjustment of the local temperature above the lower critical solution temperature (LCST) of the stimuli-responsive polymer [98][99][100][101][102][103][104]. The magnetic component acts as an energy converter to transfer received magnetic energy to heat via hysteresis losses and Néel and Brownian relaxation effects [105,106]. Hence, an oscillating magnetic field can increase the temperature of polymer networks near magnetic NPs, thereby enabling re-motely modulated drug delivery [100,[102][103][104]. The process of releasing a drug from these polymer-MNP composites is illustrated in Figure 23.
the encapsulation/deposition of magnetic NPs within stimuli-responsive polymer networks [98][99][100][101][102][103][104]. The polymeric layer serves as a "smart" container that can encapsulate/load and subsequently release drugs/substances from the polymer network upon external adjustment of the local temperature above the lower critical solution temperature (LCST) of the stimuli-responsive polymer [98][99][100][101][102][103][104]. The magnetic component acts as an energy converter to transfer received magnetic energy to heat via hysteresis losses and Néel and Brownian relaxation effects [105,106]. Hence, an oscillating magnetic field can increase the temperature of polymer networks near magnetic NPs, thereby enabling remotely modulated drug delivery [100,[102][103][104]. The process of releasing a drug from these polymer-MNP composites is illustrated in Figure 23. Poly(N-isopropylacrylamide) (PNIPAm) is one of the most commonly used thermally responsive polymers, with dimensions that can be altered upon stimulation by temperature [107]. Above a certain temperature in aqueous solutions, the hydrogen bonds in such hydrogel polymers are broken, and the polymer undergoes a reversible phase transition from a swollen hydrated state to a collapsed dehydrated state. The temperature at which the phase transition occurs is defined as the lower critical solution temperature (LCST) [108][109][110][111]. The LCST of PNIPAm is approximately 32 °C [112,113]. Localized heating of PNIPAm integrated with plasmonic metal nanostructures has been widely exploited for remote-controlled drug delivery triggered by NIR light [85]. NIR light can pass Figure 23. Illustration of the reversible swelling-collapse behavior and drug release from thermoresponsive polymer-magnetic NP composites upon heating or application of an alternating magnetic field (AMF). Adapted with permission from reference [89]. Copyright 2010 Elsevier.
Poly(N-isopropylacrylamide) (PNIPAm) is one of the most commonly used thermally responsive polymers, with dimensions that can be altered upon stimulation by temperature [107]. Above a certain temperature in aqueous solutions, the hydrogen bonds in such hydrogel polymers are broken, and the polymer undergoes a reversible phase transition from a swollen hydrated state to a collapsed dehydrated state. The temperature at which the phase transition occurs is defined as the lower critical solution temperature (LCST) [108][109][110][111]. The LCST of PNIPAm is approximately 32 • C [112,113]. Localized heating of PNIPAm integrated with plasmonic metal nanostructures has been widely exploited for remote-controlled drug delivery triggered by NIR light [85]. NIR light can pass through human tissues and couple with NIR-responsive nanoparticles, which in turn generate heat in the integrated PNIPAm materials [85]. NIR light-triggered delivery of anticancer drugs has also been achieved using Fe 3 O 4 @PNIPAm yolk-shell NPs [90].
Magnetic nanoparticles offer another important mechanism for remote heating of PIN-PAM nanoparticles. Heat is generated in magnetic nanoparticles when they are subjected to an alternating magnetic field (AMF), which can lead to deswelling of integrated polymers and the release of encapsulated drugs. Ramanujan and coworkers were among the first groups to integrate PNIPAm with IONPs for targeted release of the anticancer drug doxorubicin (DOX) [88,89]. Preliminary studies showed that DOX can be loaded within a magnetic PNIPAm nanoparticle and subsequently released by either adjusting the local temperature above the LCST or using a rapidly alternating magnetic field [88]. During the course of exposure to a magnetic field (47 min), approximately 14% of the drug was released, and the temperature of the colloidal solution was rapidly increased from room temperature to 41-48 • C. The ability to generate heat in the range of 41-48 • C when subjected to an external magnetic field allows this platform to potentially be used as a therapeutic agent that can simultaneously release drugs and generate heat for cell ablation (e.g., tumor eradication). In a related study, drug release from a PNIPAm-based core-shell magnetic NP was confirmed to be much higher above the LCST, with 78% release of the loaded drug within 29 h [89]. In another example, Jaber et al. prepared IONPs embedded in mesoporous SiO 2 shells (IONPs@mSiO 2 ) and then loaded PNIPAm containing the anticancer drug H 3 PMo 12 O 40 in the pores of the mesoporous SiO 2 shells for controlled drug release under an alternating current magnetic field [114]. However, in most of these studies, the LCST of PNIPAm itself was approximately 34-35 • C and was unchanged when integrated with IONPs. Given that the physiological temperature of the human body is approximately 37 • C, a higher LCST is desired to prevent undesired drug release in the absence of an AMF.
The LCST of PNIPAm can be increased by increasing the hydrophilic character of the polymer. Chen et al. grew a PNIPAm shell around an IONP@carbon core-shell NP [87]. The presence of multiple functional groups on the carbon shell displayed a ζ-potential of up to -33.0 mV and increased the LCST of the PNIPAm networks to 45 • C. These nanoparticles collapsed from a size of 280 nm to 257 nm under an AMF, effectively releasing the loaded hydrophilic epithelial anticancer drug 5-fluorouracil. Thus, application of an AMF can serve as an "on-off switch" for drug release. Higher LCSTs can also be achieved by cografting other monomers into the PNIPAm chains. Kakwere et al. incorporated the hydrophilic polymer polyethylene-glycolmethyl-ether-acrylate with PNIPAm (PNIPAm-co-PEGMEA) to increase the LCST above 37 • C (see Figure 24a) [92]. In this study, the authors used cubic IONPs, which possess a high specific absorption rate compared to spherical IONPs; as a result, lower doses of IONPs could be applied in vivo to reach therapeutic temperatures. Other commonly used comonomers for increasing the LCST of hydrogel polymers are acryl amide (-co-AAm) and acrylic acid (-co-AAc); the LCST of PNIPAm-co-AAc can be adjusted in a range of~30-60 • C [115].
Magnetic NPs also have an intrinsic ability to kill cancer cells via magnetic hyperthermia therapy (MHT). AMF-induced MHT is preferred because the tumor temperature can be easily regulated by adjusting the magnetic field strength (H) and frequency (f) [116]. Additionally, AMF can penetrate and heat deep tumors without damaging normal hypodermal tissues [116]. Modern therapeutic materials have been designed to simultaneously exploit both the anticancer drug release and MHT properties of polymer-integrated IONPs, with targeted temperatures above~45 • C [116]. Aoyagi and coworkers designed DOXand MNP-embedded hyperthermia nanofibers consisting of a copolymer of NIPAm and N-hydroxymethylacrylamide (HMAAm), named poly(NIPAm-co-HMAAm) [91]. Localized heating to a temperature of 45 • C was achieved, triggering a synergistic activity of DOX release and hyperthermia; as a result, 70% cell death was observed within 5 min. The HMAAm copolymer cross-links provided better stability to the system and prevented MHT side effects from eluted MNPs. Nearly all of the loaded DOX (>90%) was released after four AMF 'on' cycles, while only negligible amounts were released during the cooling 'off' process (see Figure 24b).
Another important benefit of MNPs is their ability to be viewed via magnetic resonance imaging (MRI), which can enable visual tracking of polymer-integrated MNPs in vivo. Magnetic resonance imaging (MRI)-guided MHT can not only monitor therapeutic outcomes but also measure the tumor temperature without insertion of a thermal probe [93]. Jaiswal et al. embedded PEG-functionalized Fe 3 O 4 nanostructures in PNIPAm hydrogels, ensuring 95% cancer cell death via the synergistic effects of heating and DOX drug release, as demonstrated in Figure 25 [94]. Importantly, MRI monitoring allowed the authors to study the accumulation of NPs in mouse livers, lungs, and hearts. released after four AMF 'on' cycles, while only negligible amounts were released during the cooling 'off' process (see Figure 24b). Another important benefit of MNPs is their ability to be viewed via magnetic resonance imaging (MRI), which can enable visual tracking of polymer-integrated MNPs in vivo. Magnetic resonance imaging (MRI)-guided MHT can not only monitor therapeutic outcomes but also measure the tumor temperature without insertion of a thermal probe [93]. Jaiswal et al. embedded PEG-functionalized Fe3O4 nanostructures in PNIPAm hydrogels, ensuring 95% cancer cell death via the synergistic effects of heating and DOX drug release, as demonstrated in Figure 25 [94]. Importantly, MRI monitoring allowed the authors to study the accumulation of NPs in mouse livers, lungs, and hearts. Reproduced with permission from reference [92]. Copyright 2015 American Chemical Society. (b) 'Onoff' switchable and reversible heat profile and swelling ratio of MNPs with crosslinked poly(NIPAmco-HMAAm) nanofibers and the DOX release profile corresponding to the reversible swell-shrink property in response to temperature changes. DOX release = D released at X/D total × 100 (%); M released at X is the cumulative amount of released DOX at the X cycle of AMF alternation; and M total is the total amount of incorporated DOX in MNP-loaded nanofibers. Swelling ratio = (W swell − W dry )/W dry , where W swell is the weight of swelled MNP-loaded nanofibers and W dry is the weight of dried MNP-nanofibers. Reproduced with permission from reference [91]. Copyright 2013 John Wiley and Sons.
Although PNIPAm is the most popular polymer for AMF-triggered drug release, other polymers have also been incorporated with MNPs to perform similar functions. Liu et al. utilized block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEO-b-PPOb-PEO) combined with MNPs to trigger an 80% 'burst' drug release at 45 • C [95]. The hybrid NPs showed very little leakage at room temperature (25 • C) and physiological temperature (37 • C) [95]. Integrated polymers can also provide additional functions in thermoresponsive magnetic systems, such as long-term retention, cell targeting, and enhanced stability. Zhang et al. used SPION-loaded nanocapsule hydrogels of poly(organophosphazene) (PPZ) to impart long-term retention in tumors (80% after 6 days) for multiple MHT treatments (temp.~45 • C) while also serving as an MRI contrast agent to guide the therapeutic process [93]. The long-term retention in tumors of PPZ allowed multiple MHT treatments without the need for additional injections, increasing the efficacy compared to a single MHT process (see Figure 26).

Figure 25.
Schematic illustration of the anticancer activity of magnetic nanostructure (MNS) hydrogels loaded with the chemotherapeutic agent doxorubicin (DOX). The MNS was functionalized with nitro-dopamine-PEG to achieve aqueous stability. After cellular uptake, the applied external RF field activates the MNS hydrogels and stimulates DOX release by rupturing the polymeric hydrogel. Due to the presence of the MNS, the hydrogel also acts as an MRI contrast agent. Reproduced with permission from reference [94]. Copyright 2015 Elsevier.
Although PNIPAm is the most popular polymer for AMF-triggered drug release, other polymers have also been incorporated with MNPs to perform similar functions. Liu et al. utilized block copolymers of poly(ethylene oxide) and poly(propylene oxide) (PEOb-PPO-b-PEO) combined with MNPs to trigger an 80% 'burst' drug release at 45 °C [95]. The hybrid NPs showed very little leakage at room temperature (25 °C) and physiological temperature (37 °C) [95]. Integrated polymers can also provide additional functions in thermoresponsive magnetic systems, such as long-term retention, cell targeting, and enhanced stability. Zhang et al. used SPION-loaded nanocapsule hydrogels of poly(organophosphazene) (PPZ) to impart long-term retention in tumors (80% after 6 days) for multiple MHT treatments (temp. ~ 45 °C) while also serving as an MRI contrast agent to guide the therapeutic process [93]. The long-term retention in tumors of PPZ allowed multiple MHT treatments without the need for additional injections, increasing the efficacy compared to a single MHT process (see Figure 26). Figure 25. Schematic illustration of the anticancer activity of magnetic nanostructure (MNS) hydrogels loaded with the chemotherapeutic agent doxorubicin (DOX). The MNS was functionalized with nitro-dopamine-PEG to achieve aqueous stability. After cellular uptake, the applied external RF field activates the MNS hydrogels and stimulates DOX release by rupturing the polymeric hydrogel. Due to the presence of the MNS, the hydrogel also acts as an MRI contrast agent. Reproduced with permission from reference [94]. Copyright 2015 Elsevier.

Integration of pH-Responsive Polymers
With a pH of~6.8, the extracellular medium of a tumor is more acidic than that of blood and normal tissues (with a pH of 7.4), and lysosomes are even more acidic (pH~5.0-5.5) [97]. The three-dimensional structure of pH-responsive polymers can expand under basic conditions and collapse under acidic conditions [107]. Consequently, pH-responsive polymers have been investigated by many researchers for possible applications in anticancer drug delivery [117]. For example, diblock copolymers of PEG and 2-(diisopropylamino)ethanol grafted poly(L-aspartic acid) (PEG-PAsp(DIP)) integrated with SPIONS showed significantly higher release of DOX (80% after 2 h and 90% after 24 h) at pH 5 compared to pH 7.4 (<20% after 24 h) [96]. After 2 h of incubation with cells at 37 • C, the drug-loaded nanovesicles were taken up by endocytosis and entrapped inside lysosomes. Importantly, the authors observed that DOX accumulated inside the nuclei, indicating release and migration of DOX from the nanovesicles at lysosomal pH. Moreover, integration with magnetic nanoparticles enabled monitoring of this chemotherapeutic process using MRI.

Integration of pH-Responsive Polymers
With a pH of ~6.8, the extracellular medium of a tumor is more acidic than that of blood and normal tissues (with a pH of 7.4), and lysosomes are even more acidic (pH ~ 5.0-5.5) [97]. The three-dimensional structure of pH-responsive polymers can expand under basic conditions and collapse under acidic conditions [107]. Consequently, pH-responsive polymers have been investigated by many researchers for possible applications in anticancer drug delivery [117]. For example, diblock copolymers of PEG and 2-(diisopropylamino)ethanol grafted poly(L-aspartic acid) (PEG-PAsp(DIP)) integrated with SPI-ONS showed significantly higher release of DOX (80% after 2 h and 90% after 24 h) at pH 5 compared to pH 7.4 (<20% after 24 h) [96]. After 2 h of incubation with cells at 37 °C, the drug-loaded nanovesicles were taken up by endocytosis and entrapped inside lysosomes. Importantly, the authors observed that DOX accumulated inside the nuclei, indicating release and migration of DOX from the nanovesicles at lysosomal pH. Moreover, integration with magnetic nanoparticles enabled monitoring of this chemotherapeutic process using MRI. Recent designs combine both the thermoresponsive and pH-responsive properties of polymers with the MHT and MRI capabilities of IONPs [94,97]. Dutta et al. grafted poly((Nisopropylacrylamide-ran-poly(ethylene glycol) methyl ether acrylate)-block-poly(acrylic acid) (P(NIPAm-r-PEGMEA)-b-PAAc) polymers onto IONPs and investigated the effects of polymer composition, temperature, and pH on DOX release [97]. The results showed a much higher drug release at pH 5.0 than at pH 7.4, while an increase in the number of PEGMEA units restricted drug release at temperatures of 37 and 40 • C (see Figure 27). Thus, temperature-controlled release of DOX was achieved at 45 • C, preferentially in lysosomes of cancer cells at pH 5.0. Overall, the above examples demonstrate that integration of magnetic NPs and stimuli-responsive polymers allows the development of new nanotechnologybased therapeutic strategies. These platforms might contribute to future controlled drugrelease systems, novel therapeutics, and new contrast agents in which the drugs or agents can be released by exposure to an oscillating magnetic field, while the therapeutic results can be synergistically monitored and diagnosed using MRI techniques. tially in lysosomes of cancer cells at pH 5.0. Overall, the above examples demonstrate that integration of magnetic NPs and stimuli-responsive polymers allows the development of new nanotechnology-based therapeutic strategies. These platforms might contribute to future controlled drug-release systems, novel therapeutics, and new contrast agents in which the drugs or agents can be released by exposure to an oscillating magnetic field, while the therapeutic results can be synergistically monitored and diagnosed using MRI techniques.

Integration of Multiple Conjugates with Magnetic Iron Oxide Nanoparticles
In addition to NP-based systems that integrate magnetic iron oxide NPs with one of the five conjugate types mentioned above, numerous studies have developed platforms for biomedical applications by combining magnetic IONs with multiple conjugates, such as an organic dye and a biomolecule or a biomolecule with a stimuli-responsive polymer. These systems can offer various advantages due to their ability to perform several tasks in parallel, such as cancer cell targeting, dual-mode imaging, drug delivery, and therapy [118][119][120][121][122]. Table 6 provides a summary of multiconjugate integrated magnetic iron oxide NP-based platforms for biomedical applications.  IONPs mPEG-b-P (DPA-DE)LG -~120 pH-triggered MRI probes [148] SPIONs p(HEMA-co-DMA) Spherical~20 DDS (pH) for BTZ, and MHT [149] Abbreviations Very recently, the Bao group developed lipid-encapsulated Fe 3 O 4 nanoparticles as a contrast agent in multimodal MRI/FI [15]. Specifically, magnetite NPs with a well-controlled size distribution were synthesized via thermodecomposition before being coated with copolymers of phospholipids and phospholipid-PEG to generate water-soluble MNPs. Then, these polymer-coated MNPs were integrated with an organic dye dialkylcarbocyanine, such as DiO, DiI, DiD, or DiR, via hydrophobic interactions of the organic dyes with the lipid layer of the shell of the MNPs. Finally, nanoprobes with desired sizes and optical and magnetic properties were achieved by conjugation of the bioactive ligands cyclic RGD or cyclic RAD peptides to the nanoprobes [15]. Figure 28 presents an illustration of the design of these lipid-encapsulated organic dye-doped iron oxide MNPs, which exhibited high stability in biomedical media and good biocompatibility. Moreover, the conjugated peptides greatly increased the uptake of these nanoprobes by cells. In vitro and in vivo experiments revealed good fluorescence signals and MRI contrast, demonstrating potential application of these MNPs in biomedical fields [15]. Very recently, the Bao group developed lipid-encapsulated Fe3O4 nanoparticles as a contrast agent in multimodal MRI/FI [15]. Specifically, magnetite NPs with a well-controlled size distribution were synthesized via thermodecomposition before being coated with copolymers of phospholipids and phospholipid-PEG to generate water-soluble MNPs. Then, these polymer-coated MNPs were integrated with an organic dye dialkylcarbocyanine, such as DiO, DiI, DiD, or DiR, via hydrophobic interactions of the organic dyes with the lipid layer of the shell of the MNPs. Finally, nanoprobes with desired sizes and optical and magnetic properties were achieved by conjugation of the bioactive ligands cyclic RGD or cyclic RAD peptides to the nanoprobes [15]. Figure 28 presents an illustration of the design of these lipid-encapsulated organic dye-doped iron oxide MNPs, which exhibited high stability in biomedical media and good biocompatibility. Moreover, the conjugated peptides greatly increased the uptake of these nanoprobes by cells. In vitro and in vivo experiments revealed good fluorescence signals and MRI contrast, demonstrating potential application of these MNPs in biomedical fields [15]. , which indicate early cancer development [133]. Due to the characteristic recognition of the antibodies, the dual-antibody functionalized F-MNPs can selectively bind to CTCs before being isolated using an external magnetic field and identified under a fluorescence microscope, as shown in Figure 29a. The magnetite magnetic core was encapsulated with a fluorescent dye DiI-decorated silica shell before being modified with poly(carboxybetaine methacrylate) (pCBMA), streptavidin, and antibodies, as illustrated in Figure 29b. , which indicate early cancer development [133]. Due to the characteristic recognition of the antibodies, the dual-antibody functionalized F-MNPs can selectively bind to CTCs before being isolated using an external magnetic field and identified under a fluorescence microscope, as shown in Figure 29a. The magnetite magnetic core was encapsulated with a fluorescent dye DiI-decorated silica shell before being modified with poly(carboxybetaine methacrylate) (pCBMA), streptavidin, and antibodies, as illustrated in Figure 29b.
The authors evaluated the MCF-7 human breast cancer cell line capture efficiency of F-MNPs at various modification steps. The results shown in Figure 30a suggest the highest capture efficiency of 98.8% for F-MNPs functionalized with both anti-EpCAM and anti-N-cadherin antibodies. The modified magnetic nanoparticles were also evaluated for their recognition performance, with MCF-7, HeLa, and CCRF-CEM cells used as cell models for epithelial, mesenchymal CTCs, and blood cells, respectively. The dual-antibodymodified F-MNPs showed higher capture efficiencies for both MCF-7 and HeLa cells than single-antibody-modified nanoparticles, as shown in Figure 30b. Additionally, the selective CTC identification and capture capability of the modified F-MNPs was shown by the near-zero capture efficiency of the human T lymphocytic leukemia cell line CCRF-CEM, suggesting that the antibody-modified magnetic nanoparticles have potential for efficient early cancer detection. In a separate work, White et al. fabricated magnetic-plasmonic ultrasmall superparamagnetic iron oxide (USPIO)-gold hybrid nanoparticles with anti-MG1 antibodies for targeted photothermal ablation of colorectal liver metastases [134]. Moreover, Dong and coworkers prepared fluorescent magnetic mesoporous silica nanoparticles (M-MSNs) conjugated with fluorescein isothiocyanate (FITC) before modifying them with EpCAM antibody for efficient detection of circulating tumor cells (CTCs) [135]. The authors evaluated the MCF-7 human breast cancer cell line capture efficiency of F-MNPs at various modification steps. The results shown in Figure 30a suggest the highest capture efficiency of 98.8% for F-MNPs functionalized with both anti-EpCAM and anti-N-cadherin antibodies. The modified magnetic nanoparticles were also evaluated for their recognition performance, with MCF-7, HeLa, and CCRF-CEM cells used as cell models for epithelial, mesenchymal CTCs, and blood cells, respectively. The dual-antibody-modified F-MNPs showed higher capture efficiencies for both MCF-7 and HeLa cells than single-antibody-modified nanoparticles, as shown in Figure 30b. Additionally, the selective CTC identification and capture capability of the modified F-MNPs was shown by the nearzero capture efficiency of the human T lymphocytic leukemia cell line CCRF-CEM, suggesting that the antibody-modified magnetic nanoparticles have potential for efficient early cancer detection. In a separate work, White et al. fabricated magnetic-plasmonic ultrasmall superparamagnetic iron oxide (USPIO)-gold hybrid nanoparticles with anti-MG1 antibodies for targeted photothermal ablation of colorectal liver metastases [134]. Moreover, Dong and coworkers prepared fluorescent magnetic mesoporous silica nanoparticles (M-MSNs) conjugated with fluorescein isothiocyanate (FITC) before modifying them with EpCAM antibody for efficient detection of circulating tumor cells (CTCs) [135]. Wang et al. developed improved magnetic-core@dual QD-shell nanoparticles (Fe3O4@DQDs) as multifunctional fluorescent labels for fluorescence lateral flow detection of bacteria [136]. In this study, mercapto-propionic acid-functionalized QDs (CdSe/ZnS-MPA) were chosen as dual QDs due to their outstanding and stable fluorescence properties. Interestingly, the QD-adhering step was carried out two times to obtain the final targeted Fe3O4@DQDs. Compared to Fe3O4@QDs, the Fe3O4@DQDs had a higher number of QDs, leading to fluorescence enhancement of the whole nanostructure. For ultrasensitive bacteria detection, the combination of the magnetic property of Fe3O4 and the high fluorescence of DQDs led to a highly sensitive bacteria detection LFA strip.
Recently, the Lee group reported a nanotriplex particle Fe3O4@Ag/graphene quantum dot (GQD) as an electrochemical immunosensor for tuberculosis [140]. Because of the  [136]. In this study, mercapto-propionic acid-functionalized QDs (CdSe/ZnS-MPA) were chosen as dual QDs due to their outstanding and stable fluorescence properties. Interestingly, the QD-adhering step was carried out two times to obtain the final targeted Fe 3 O 4 @DQDs. Compared to Fe 3 O 4 @QDs, the Fe 3 O 4 @DQDs had a higher number of QDs, leading to fluorescence enhancement of the whole nanostructure. For ultrasensitive bacteria detection, the combination of the magnetic property of Fe 3 O 4 and the high fluorescence of DQDs led to a highly sensitive bacteria detection LFA strip.
Recently, the Lee group reported a nanotriplex particle Fe 3 O 4 @Ag/graphene quantum dot (GQD) as an electrochemical immunosensor for tuberculosis [140]. Because of the Fe 3 O 4 core, the nanomaterial had excellent magnetic properties and good water solubility, as well as a large surface area due to its nanomorphology. Ag NPs were incorporated into the nanomaterial to increase conductivity. For electrochemical immunosensor applications, graphene quantum dots (GQDs) were integrated because they are great electron donors and acceptors, which is rationalized by the large surface area and the presence of multiple types of functional groups on the QD surface. The preparation of Fe 3 O 4 @Ag/GQDs is presented in Figure 31a, and Figure 31b shows TEM images of the nanotriplex, revealing a diameter of~270 nm. As shown in Figure 31c, the differential pulse voltammetry (DPV) peak was enhanced when the concentration of the analyte culture filtrate protein CFP-10 increased during immunocomplex formation. The system showed a robust performance and high selectivity for CFP-10 in the presence of antigen 85 complexes (Ag85), HspX protein of Mtb (16 kDa), and bovine serum albumin (BSA), as shown in Figure 31d. Irudayaraj and coworkers fabricated a novel "nanopearl-necklace" structure that consists of a single gold nanorod (Aurod) decorated with multiple "pearls" of 15 nm Fe3O4 magnetic NPs for use in simultaneous targeting, bimodal imaging, and photothermal ablation of cancer cells [121]. In vitro assays of this system revealed that Aurod-(Fe3O4) exhibited a stronger MR signal than bare Fe3O4 at an equivalent iron concentration due to the magnetic coupling between the Fe3O4 particles assembled on the Aurod core. Conjugates derived from covalent attachment of Herceptin to the Aurod-(Fe3O4) nanocomplexes were observed to bind specifically to cancer cell surfaces and were internalized into the cytoplasm, which was verified by MR and transmission electron microscopy (TEM) images. An additional advantage of these nanocomposites is that the inherent fluorescence of the Irudayaraj and coworkers fabricated a novel "nanopearl-necklace" structure that consists of a single gold nanorod (Au rod ) decorated with multiple "pearls" of 15 nm Fe 3 O 4 magnetic NPs for use in simultaneous targeting, bimodal imaging, and photothermal ablation of cancer cells [121]. In vitro assays of this system revealed that Au rod -(Fe 3 O 4 ) exhibited a stronger MR signal than bare Fe 3 O 4 at an equivalent iron concentration due to the magnetic coupling between the Fe 3 O 4 particles assembled on the Au rod core. Conju-gates derived from covalent attachment of Herceptin to the Au rod -(Fe 3 O 4 ) nanocomplexes were observed to bind specifically to cancer cell surfaces and were internalized into the cytoplasm, which was verified by MR and transmission electron microscopy (TEM) images. An additional advantage of these nanocomposites is that the inherent fluorescence of the Au rod component allows FI analysis. These studies indicate that such nanocomposites can potentially serve as contrast agents for both MRI and FI modalities. Furthermore, the use of these Herceptin-conjugated Au rod -(Fe 3 O 4 ) NPs as therapeutic agents was also demonstrated. Once the Herceptin-conjugated Au rod -(Fe 3 O 4 ) NPs were ingested and allowed to accumulate in the internal vesicles and cytoplasm of cancer cells, the NPs were irradiated with a 785 nm NIR laser. The absorbed energy heated the Au rod , allowing local destruction of cancer cells and illustrating efficient photothermal ablation.
Gold nanocages functionalized with Fe 3 O 4 nanoparticles (F-AuNC@Fe 3 O 4 ) have also been studied for multimodal imaging of tumors ( Figure 32) [141]. In this study, nanocomposites were used as multimodal contrast agents for MRI/computed tomography (CT) multimodal imaging. The aforementioned nanoparticles were conjugated with folic acid to selectively bind with targeted folate receptor-overexpressing cancer cells. The results from an in vivo CT imaging study showed that F-AuNC@Fe 3 O 4 can enhance CT imaging in the circulatory system. As shown in Figure 32b, the average signal intensity in kidney and tumor tissues was enhanced from 0.5 h to 6 h, while the signal in other organs showed fluctuation. Additionally, to study the biocompatibility of F-AuNC@Fe 3 O 4 nanoparticles, the nanoparticles were tested via a pathological assay (H&E staining). As shown in Figure 32c-h, the results demonstrated that F-AuNC@Fe 3 O 4 nanoparticles were present in the kidney, bladder, and tumor at 6 h after injection. However, the nanoparticles were absent in the liver and heart after the same period of time. Additionally, no morphological changes were observed in the organs, suggesting that the nanoparticles were biocompatible.
Separate studies have described the fabrication of nanocomplexes composed of spherical gold nanoshell/silica core structures that were subsequently coated with 10 nm magnetic NPs and an outer SiO 2 layer doped with an NIR dye [142]. In this geometric structure, the gold nanoshell absorbs light in NIR regions and can also significantly enhance the fluorescence of the adjacent NIR dye molecules, thus significantly improving the resolution of fluorescence images. Furthermore, incorporation of magnetic NPs on the gold nanoshell surfaces led to increased magnetic interactions among the particles, thereby enhancing the relaxivity and improving the MR signals. Additionally, the silica outer layer can be readily modified with biomolecular entities for cell or protein targeting. Furthermore, the ability to absorb NIR light and produce heat from the gold nanoshell component enables these hybrid NPs to be used for photothermal ablation. Consequently, these integrated properties allowed the use of this platform in multiple diagnostic and therapeutic modalities.
Surface-enhanced Raman spectroscopy (SERS)-encoded MNPs and AgNP-embedded SiO 2 -coated Fe 3 O 4 NPs (M-SERS) were reported as multifunctional materials for cancer cell targeting and separation [143]. In this study, M-SERS nanoparticles were functionalized with various types of thiol-group-containing organic compounds to demonstrate the enhancement of Raman scattering. Additionally, due to the presence of selected aromatic compounds, the aforementioned nanoparticles exhibited strong SERS signals. To demonstrate the magnetic properties of the M-SERS nanoparticles, an external magnetic force was applied to the nanoparticles after conjugation with antibodies and targeted molecules. The targeted cells with the M-SERS nanoparticles moved toward the magnet under an external magnetic field. These results confirmed that the M-SERS nanoparticles are promising materials for detection and separation of biomolecules.
In another study, Li et al. used a thermally sensitive and hepatic tumor-cell-targeting peptide-A54-modified polymer, A54-poly(ethylene glycol)-g-poly(acrylamide-co-acrylonitrile) (A54-PEG-g-p(AAm-co-AN)), assembled into 80 nm sized micelles, allowing DOX drug transport and release and augmenting microwave hyperthermia at 43 • C [144]. Instead of LCST, the operation of these polymers was centered around their upper critical solution temperature (UCST). Other thermally responsive polymers include those with glass transition temperatures (T g ) that coincide with typical MHT temperatures. For example, IONP-embedded DOX-containing carboxylic polypyrrole systems soften above their T g of 44 • C, leading to the release of DOX [116]. These NPs were further modified with PEG functionalized with folic acid (FA), and FA specifically binds to folate receptors overexpressed in cancer cells. Tumor temperatures of 44 • C were reached within 7 min of AMF exposure, killing cancer cells throughout the entire tumor.
Materials 2022, 15, x FOR PEER REVIEW 39 of 50 in the circulatory system. As shown in Figure 32b, the average signal intensity in kidney and tumor tissues was enhanced from 0.5 h to 6 h, while the signal in other organs showed fluctuation. Additionally, to study the biocompatibility of F-AuNC@Fe3O4 nanoparticles, the nanoparticles were tested via a pathological assay (H&E staining). As shown in Figure  32c-h, the results demonstrated that F-AuNC@Fe3O4 nanoparticles were present in the kidney, bladder, and tumor at 6 h after injection. However, the nanoparticles were absent in the liver and heart after the same period of time. Additionally, no morphological changes were observed in the organs, suggesting that the nanoparticles were biocompatible. Separate studies have described the fabrication of nanocomplexes composed of spherical gold nanoshell/silica core structures that were subsequently coated with 10 nm magnetic NPs and an outer SiO2 layer doped with an NIR dye [142]. In this geometric structure, the gold nanoshell absorbs light in NIR regions and can also significantly enhance the fluorescence of the adjacent NIR dye molecules, thus significantly improving the resolution of fluorescence images. Furthermore, incorporation of magnetic NPs on the gold nanoshell surfaces led to increased magnetic interactions among the particles, thereby enhancing the relaxivity and improving the MR signals. Additionally, the silica outer layer can be readily modified with biomolecular entities for cell or protein targeting. Furthermore, the ability to absorb NIR light and produce heat from the gold nanoshell component enables these hybrid NPs to be used for photothermal ablation. Consequently, As mentioned earlier, the inclusion of folic acid (FA) moieties can improve tumorspecific delivery of pH-responsive integrated nanocarriers via attachment to folate receptors overexpressed in cancer cells. Yang et al. designed triblock polymers with varying M w consisting of (folate (FA) or methoxy)-poly(ethylene glycol) (Mw: 5000)-poly(glutamate hydrozone doxorubicin)-poly(ethylene glycol) (M w : 2000)-acrylate (i.e., R (FA or methoxy)-PEG 114 -P(Glu-Hyd-DOX)-PEG 46 -acrylate) [145]. The longer PEG chains containing FA segregated toward the outer layer of the vesicles and facilitated targeted endocytic delivery, while the shorter PEG block segregated to the inner region of the vesicles and facilitated cross-linking and enhanced the in vivo stability of the nanovesicles. In another design, hollow SiO 2 NPs integrated with hydrophobic polymers containing folic acid and SPIONs demonstrated drug release in an acidic environment in cancer cells; 70% of DOX was released at pH 5.0, whereas less than 5% was released in a neutral environment after 150 h (see Figure 33a) [146]. The hollow SiO 2 core enabled high drug loading efficiency, and targeted delivery was tracked via MRI-active SPIONs. The MRI contrast of Fe-based nanoparticles can be further enhanced by combination with Gd-based compounds, as shown by Sun et al. with their Fe 3 O 4 @Gd 2 O 3 yolk-shell NPs functionalized with PEG and FA [122]. The yolk-shell design with a porous Gd 2 O 3 shell improved MRI contrast, increased drug loading, and enabled pH-induced release of the anticancer drug cisplatin inside tumor cells (see Figure 33b-d). This targeted drug delivery nanosystem also demonstrated a marked reduction in damage to vital organs compared with that observed with free cisplatin. Folate-conjugated, pH-sensitive poly(β-aminoester) self-assembled micelles with hydrophobic oleic acid-modified IONPs delivering DOX were shown to facilitate the treatment of advanced gastric cancer via apoptosis of cancerous cells [147]. As seen in Figure  34a-c, once again, the folate conjugates showed much greater suppression of tumor growth without affecting the overall body weight and can be safely monitored via MRI. Yang et al. also used methyloxy-poly(ethylene glycol)-block-poly[dopamine-2-(dibutylamino) ethylamine-L-glutamate] (mPEG-b-P(DPA-DE)LG) micelles [148]. Dopamine has a high affinity for IONPs, enhancing stability at physiological pH, while the mPEG moiety enhances dispersion in aqueous media. These nanoparticles acted as pH-sensitive MRI probes, releasing IONPs at an acidic pH. Sasikala et al. used a unique copolymer with dopamine, poly(2-hydroxyethyl methacrylate codopamine methacrylamide) p(HEMAco-DMA) to surface functionalize IONPs, taking advantage of dopamine's affinity for IONPs [149]. The catechol groups in dopamine were also exploited to conjugate the borate-containing anticancer drug bortezomib (BTZ). In this study, the researchers exploited the MHT capabilities of SPIONs along with pH-sensitive drug release for synergistic thermochemotherapy. The synergistic effect of BTZ and MHT therapy increased the apoptosis of cancer cells by nearly 4-fold compared to BTZ release or MHT alone (Figure 34d,e). Folate-conjugated, pH-sensitive poly(β-aminoester) self-assembled micelles with hydrophobic oleic acid-modified IONPs delivering DOX were shown to facilitate the treatment of advanced gastric cancer via apoptosis of cancerous cells [147]. As seen in Figure 34a-c, once again, the folate conjugates showed much greater suppression of tumor growth without affecting the overall body weight and can be safely monitored via MRI. Yang et al. also used methyloxy-poly(ethylene glycol)-block-poly[dopamine-2-(dibutylamino) ethylamine-L-glutamate] (mPEG-b-P(DPA-DE)LG) micelles [148]. Dopamine has a high affinity for IONPs, enhancing stability at physiological pH, while the mPEG moiety enhances dispersion in aqueous media. These nanoparticles acted as pH-sensitive MRI probes, releasing IONPs at an acidic pH. Sasikala et al. used a unique copolymer with dopamine, poly(2hydroxyethyl methacrylate codopamine methacrylamide) p(HEMA-co-DMA) to surface functionalize IONPs, taking advantage of dopamine's affinity for IONPs [149]. The catechol groups in dopamine were also exploited to conjugate the borate-containing anticancer drug bortezomib (BTZ). In this study, the researchers exploited the MHT capabilities of SPIONs along with pH-sensitive drug release for synergistic thermochemotherapy. The synergistic effect of BTZ and MHT therapy increased the apoptosis of cancer cells by nearly 4-fold compared to BTZ release or MHT alone (Figure 34d,e).

Summary and Perspectives
This review examines a variety of current composite magnetic IONPs that have been integrated with different classes of organic and inorganic materials for biomedical applications. Research efforts have focused on integrating iron oxide MNPs and organic dyes into single platforms for use as bimodal imaging agents for both in vitro and in vivo imaging and to produce multifunctional platforms that simultaneously perform several tasks in parallel, such as photodynamic therapy and dual-mode imaging for biomolecule detection. Importantly, these platforms can provide better diagnostic information and a more complementary dataset due to their dual-diagnostic MRI/FI modalities than the corresponding isolated imaging techniques. Due to their high biocompatibility, low cytotoxicity, and low risk of immune rejection, various biomolecules, such as aptamers, antibodies, and even stem cells, can be integrated with magnetic IONPs to develop highly targeted techniques for diagnosis, therapy, and theranostics for many notorious diseases, and the results have been promising.
Quantum dots (QDs), such as CdSe, ZnS, and CdS, and carbon dots (CDs), are recognized for their unique optical properties, such as resistance to photobleaching, high molar extinction coefficient, and tunable emission ranges. Consequently, QDs are often incorporated with magnetic NPs to produce composite NPs that exhibit both fluorescence and magnetism, and the effects of these composite NPs can be examined in vitro before progressing to in vivo tests. However, the use of magnetic-QD platforms in biomedical applications is limited due to the inherent toxicity of many current QDs. Thus, more biocompatible QDs are needed for broader use of magnetic-QD platforms in biomedical applica- Figure 34. (a) The mRNA expression of Caspase 3, an apoptosis-associated gene, was examined via RT-PCR upon exposure to PBS buffer, free DOX, or IONPs integrated with poly(β-aminoester) (P-DOX) and folate (F-P-DOX). (b) Tumor growth curves and (c) body weight curves demonstrating the improved antitumor efficacy of F−P-DOX in a GC xenograft model, without obvious adverse effects. Each experiment was conducted in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 vs PBS group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs DOX group; § p < 0.05, § § p < 0.01 vs P-DOX group. Reproduced with permission from reference [147].

Summary and Perspectives
This review examines a variety of current composite magnetic IONPs that have been integrated with different classes of organic and inorganic materials for biomedical applications. Research efforts have focused on integrating iron oxide MNPs and organic dyes into single platforms for use as bimodal imaging agents for both in vitro and in vivo imaging and to produce multifunctional platforms that simultaneously perform several tasks in parallel, such as photodynamic therapy and dual-mode imaging for biomolecule detection. Importantly, these platforms can provide better diagnostic information and a more complementary dataset due to their dual-diagnostic MRI/FI modalities than the corresponding isolated imaging techniques. Due to their high biocompatibility, low cytotoxicity, and low risk of immune rejection, various biomolecules, such as aptamers, antibodies, and even stem cells, can be integrated with magnetic IONPs to develop highly targeted techniques for diagnosis, therapy, and theranostics for many notorious diseases, and the results have been promising.
Quantum dots (QDs), such as CdSe, ZnS, and CdS, and carbon dots (CDs), are recognized for their unique optical properties, such as resistance to photobleaching, high molar extinction coefficient, and tunable emission ranges. Consequently, QDs are often incorporated with magnetic NPs to produce composite NPs that exhibit both fluorescence and magnetism, and the effects of these composite NPs can be examined in vitro before progressing to in vivo tests. However, the use of magnetic-QD platforms in biomedical applications is limited due to the inherent toxicity of many current QDs. Thus, more biocompatible QDs are needed for broader use of magnetic-QD platforms in biomedical applications. In addition to QDs, combining noble metal NPs and magnetic NPs into one platform offers multiple diagnostic and therapeutic modalities simultaneously, which can improve the accuracy and clarity of diagnostic images while reducing time and expense. Magnetic-noble metal NP-based platforms combine the magnetic properties of IONPs with remarkable optical properties that arise from surface plasmon resonances, which can be controlled from the ultraviolet to the near-infrared (NIR) regions of the electronic spectrum by optimizing the size, composition, shape, and topology of noble metal NPs.
Moreover, integrating magnetic nanoparticles with polymers allows for fabrication of multifunctional systems. Encapsulation and cross-linking with polymers provide improved stability, circulation, biocompatibility, and pH-sensitive magnetic resonance imaging. An alternating magnetic field can be applied to heat magnetic nanoparticles in vivo, which can be used to stimulate thermosensitive and pH-sensitive polymers for targeted drug delivery and chemotherapy. These advanced anticancer drug delivery capabilities can also be coupled with magnetic hyperthermia therapy to kill cancer cells more effectively. These chemotherapy and magnetic hyperthermia therapy processes can also be simultaneously monitored by utilizing the magnetic resonance imaging capabilities of magnetic nanoparticle-polymer conjugates. Thus, coupling all of these unique properties of polymers and magnetic nanoparticles enables the design and fabrication of more effective biomedical materials. In addition, appropriate engineering and integration of magnetic IONPs with multiple conjugates, such as an organic dye and a biomolecule or a biomolecule with a stimuli-responsive polymer, can generate multifunctional nanoplatforms that can perform multiple tasks simultaneously and be used in a broad range of biomedical fields.