Fabrication and biological investigation of a novel star polymer based on magnetic cyclic aromatic polyimide chains

Herein, a novel nanostructure based on cyclic aromatic polyimide with statistical star polymer structure was synthesized via the functionalization of the CuFe2O4 MNPs surface. The polymerization process on the functionalized surface of CuFe2O4 MNPs was performed with pyromellitic dianhydride and phenylenediamine derivatives. All analytical methods such as Fourier-transform infrared (FT-IR) spectroscopy, thermogravimetric (TG) analysis, X-ray diffraction (XRD) pattern, energy-dispersive X-ray (EDX), field-emission scanning electron microscope (FE-SEM), vibrating-sample magnetometer (VSM) were performed to characterize the structure of CuFe2O4@SiO2-polymer nanomagnetic. The cytotoxicity of CuFe2O4@SiO2-Polymer was investigated for biomedical application by MTT test. The results proved that this nanocmposite was biocompatible with HEK293T healthy cells. Also, the evaluation antibacterial property of CuFe2O4@SiO2-Polymer showed that its MIC in Gram-negative and Gram-positive bacteria were 500–1000 µg/mL, so it had antibacterial activity.


Scientific Reports
| (2023) 13:9598 | https://doi.org/10.1038/s41598-023-36619-x www.nature.com/scientificreports/ These nanoparticles have high permeability and good saturation magnetism and are easily magnetized and lose their magnetic properties and are also electrically insulating. Copper ferrite (CuFe 2 O 4 ) nanoparticles are one of the important ferrites that show phase transfer, change semiconductor properties and electrical switch and quadrilateral changes under different conditions 7,8 . In addition to suitable magnetic, electrical, and thermal stability, these nanoparticles have a wide range of applications in catalysts 9 , lithium-ion batteries 10 , bioprocessing 11 , color imaging 12 , and gas sensing 13 . These nanoparticles also have great potential for use in biomedical application 14,15 , for example in diagnostic imaging 16,17 , drug delivery 18,19 , hyperthermia therapy 15,[20][21][22][23][24][25] , and cell labeling 26 . So far, not much information is available about the biological response of the Copper ferrite in combination with other materials, and this has made the use of these nanoparticles in biomedicine a challenge.
Recently, in order to activate the surfaces of magnetic nanoparticles 27 , increase biocompatibility 28 and colloidal stability 29 in environmentally friendly environments and prevent their accumulation due to magnetic forces between particles, the surface of these nanoparticles is covered with natural and synthetic polymers such as cellulose, pectin, agar, chitosan, alginate, polypyrrole, and polyvinylidene fluoride 4 . In this regard, the polymerization reaction and polymer growth have been performed on the surface of magnetic nanoparticles CuFe 2 O 4 MNPs. Under these conditions, magnetic nanoparticles act as the core of the star-shaped polymer and form magnetic star-shaped structures.
A general strategy for presenting functional molecules and the polymerization process at the surface of magnetic nanoparticles is to use anchor molecules. In the following, the polymerization process is performed radically on the surface of the modified nanoparticles using an initiator. Initiators can react with a monomer to form an intermediate compound capable of sequentially bonding a large number of other monomers to a polymeric compound. In this work, a new nanostructure was synthesized.
The CuFe 2 O 4 as a central core was functionalized by tetraethyl orthosilicate (TEOS), (3-Chloropropyl)trimethoxysilane (CPTMS), and phenylenediamine derivatives, step by step. Finally, the polymerization reaction of pyromellitic dianhydride on the surface of functionalized CuFe 2 O 4 MNPs was performed by different phenylenediamine derivatives. All analysis were done to characterize the structural of the novel magnetic hybrid CuFe 2 O 4 @SiO 2 -cyclic aromatic polyimide. Also, its biological properties were investigated.
The result showed that the toxicity of CuFe 2 O 4 @SiO 2 -Polymer at the highest concentration was 12.64% and this nanostructure is biocompatible with HEK293T cells. Also, minimal inhibitory concentration and minimal bactericidal concentrations of CuFe 2 O 4 @SiO 2 -Polymer in comparison two control antibiotics (Penicillin and Streptomycin) was investigation against a Gram-positive bacteria (Staphylococcus aureus) and two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). The result proved that prepared nanostructure has acceptable antibacterial activity.
A significant breakthrough has been achieved by synthesizing star polymers with a magnetic center for the first time, a feat that has been accomplished only by this particular research group. In this study, not only focused on the magnetic center but also on the arms of these star polymers, which were made up of polyimides. The combination of these two components led to a novel structure that has never been seen before. CuFe 2 O 4 was used as the magnetic center of the star polymer, a unique aspect of this research. The novelty of the structure is not limited to the use of CuFe 2 O 4 alone, but also in the fact that aromatic polyimide chains were used to decorate the magnetic center in the form of a star polymer. This pioneering development marks the first time that CuFe 2 O 4 has been decorated with polyimide chains in the form of a star polymer.
In previous studies of star polymers, the antibacterial property was not attributed to the center. However, this research found that the use of CuFe 2 O 4 as the center of the star polymer led to the antibacterial property. This discovery highlights the importance of the center in determining the properties of star polymers.
Despite the promising benefits of using CuFe 2 O 4 as the magnetic center, it is important to note that it could also be toxic. However, this issue was overcome by decorating it with polyimide arms, which acted as a protective layer.
Overall, this new structure of star polymers with a magnetic center decorated with polyimide arms is a significant innovation in the field. It opens up new avenues of research and has the potential for practical applications in various fields, including biomedicine and environmental science.

Experimental
General. All chemicals used as solvents or reagents with high purities were provided by international Sigma Aldrich and Merck companies. Various analyzes were performed to investigate the structure of magnetic star polymers and confirm the synthesis and growth of the polymer on the modified surface of the central core nanoparticles. For example, the FT-IR (Fourier-transform infrared) spectra were performed by using the KBr pellets method (Perkin Elmer spectrum RX1). The XRD (X-ray diffraction) pattern was taken by using Bruker device (D8 advance model). EDX (Energy dispersive X-ray) analysis was implemented TESCAN MIRA II Xmax device. FE-SEM (field-emission scanning electron microscope) was used to evaluation the structure, morphology, and size of designed magnetic nanostructures by TESCAN MIRA III device. The TGA (thermogravimetric analysis) and VSM (vibrating-sample magnetometer) analysis was taken to evaluate its thermogravimetric and magnetic behavior by Bahr-STA 504 under the argon atmosphere and the rate of 10 °C/min and LBKFB model-magnetic Kashan kavir (Iran) (5000 Oe) devices, respectively. MTT assay. MTT assay was used to measure the toxicity and biocompatibility of the synthesized CuFe 2 O 4 @ SiO 2 -Polymer. First, HEK293T cells (human embryonic kidney cell line) were prepared from the Pasteur Institute of Iran and cultured at 1 × 10 5 cell/well in 96 well plate under optimal conditions (37 °C, 5% CO 2 in humidified incubator). Next, the growth media (10% FBS) was removed and the cells were washed two times with phosphate buffer saline (PBS). New maintenance Roswell Park Memorial Institute Medium (RPMI) medium (10% FBS) containing 0.5, 5, 50, 500, and 1000 µg/mL of synthesized nanostructure was added and the cells were incubated for 24, 48, and 72 h. Quintet wells were analyzed for each concentration and column elution buffer was used as the control. A 10 μL solution of freshly prepared 5 mg/mL MTT in PBS was added to each well and allowed to incubate for an additional 4 h. The media was removed and isopropanol was added at 100 µL/well. Plates were shaken gently to facilitate formazan crystal solubilization. The absorbance was measured at 545 nm using a microplate reader (STAT FAX 2100, BioTek, Winooski, USA). The percentage of toxicity and cell viability was calculated according to these formulas 14,30 .

Results and discussion
The intended nanostructure was synthesized for applying in biological application. The CuFe 2 O 4 nanoparticle was chosen as the central core. According to reports, copper ferrite nanoparticles in combination with other metals have shown significant antibacterial properties 33 , but these nanoparticles can cause dose-dependent cytotoxicity to healthy cells 34 . Functionalizing the surface of these nanoparticles and forming a star-shaped polymer coating minimized the toxicity of these nanoparticles and formed a biocompatible nanostructure. As stated in the experimental section, the surface of these nanoparticles was functionalized by TEOS, CPTMS and phenylenediamine derivatives in three stages. Then, polyamidic acid was performed by pyromellitic dianhydride and phenylenediamine derivatives on the surface of these functionalized nanoparticles. Finally, the polyimide acid was imidized into cyclic aromatic polyimide by in-situ annealing treatment at 160 °C. All synthesis steps of CuFe 2 O 4 @SiO 2 -cyclic aromatic polyimide and the polymerization process are well shown in Fig. 1. The types of analytical and spectral analyses were performed to characterize the structural of CuFe 2 O 4 @SiO 2 -polymer nanostructure. The new functional groups was evaluated by FT-IR spectrum. The TGA analysis was studied to evaluate the thermal behavior. The elemental composition was investigated by EDX spectrum. The XRD patterns and VSM analyses were perused to specify the crystalline phase and magnetic properties of the prepared nanostructure. Finally, the cytotoxicity and antimicrobial properties of CuFe 2 O 4 @SiO 2 -polymer nanostructures were evaluated to apply for biomedical application. The intensity and position of these absorption bands can provide valuable information about the degree of polymerization, the extent of imidization, and the overall chemical structure of the polyimide material. Therefore, the identification and interpretation of these C-N bending peaks in the FTIR spectrum is an important tool for the analysis and characterization of polyimides 39,40 . Also, in the imidization process, the stretching vibrations of C=C double bond of benzene ring and C-N-C bond were appeared around 1458 cm −1 and 1330 cm −1 , respectively [41][42][43] . The two peaks appearing at frequencies 2854 and 2924 cm −1 were as a result of the C-H symmetric and asymmetric stretching bonds in the aliphatic hydrocarbons 44 . In addition, an absorbance peak around 3060 cm −1 probably attributed to sp 2 C-H symmetric and asymmetric stretching bonds 42 . The FT-IR spectrums of the other phenylenediamine derivatives have been shown in Fig. S1 and attached in the supplementary information. The thermal stability of CuFe 2 O 4 @SiO 2 -cyclic aromatic polyimide nanostructure was evaluated by thermogravimetric analysis in a thermal range of 50-600 °C, shown in Fig. 2b. As shown, three distinct reduction peaks is observed in the TGA diagram. The first reduction peak (12%) appeared at 191 °C to 230 °C range temperature which it can be related to the adsorbed solvent molecules and impurities in the structure of magnetic nanostructure star polymer. The second reduction peak (10%) occurred at 230 °C to about 470 °C and it is due to decomposition of organic part of the molecule and grafted linkers 45 . Finally, the third reduction peak (20%) was seen at 470 °C to 600 °C range temperature and it is assigned to the decomposition of fabricated polymer structure 14 . Also, in Fig. S2 Fig. S3 (supporting information). The characteristic magnetic of prepared nanostructure was investigated by VSM analysis by applying a magnetic field between − 15 < k Oe < + 15 showing in Fig. 3b. Several parameters including the crystalline structure of magnetic nanoparticles, core size, shell thickness, and the distance between particles are effective in the magnetization property of core-shell structures. According previous works, the value of saturation magnetization of CuFe 2 O 4 MNPs was between range of 20 and 30 emu g −123 . Figure 3b displays the value saturation magnetization of CuFe 2 O 4 @SiO 2 -cyclic aromatic polyimide nanostructure star polymer, which is decreased to 2.13 emu. g -1 due  Figure 4 contains FES-EM images depicting various stages of the synthesis and resulting products. Figure 4a shows the presence of spherical nanoparticles with an average diameter of 150 nm, which belong to the pure copper ferrite nanoparticles synthesized. In the next stage, after the deposition of silica layers, the average diameter of nanoparticles increased to 300-400 nm, as shown in Fig. 4b. This stage is the main reason for the reduction in toxicity of raw copper ferrite nanoparticles in the final structure. Subsequently, after functionalizing the structure with phenylenediamine derivatives, the average diameter of nanoparticles further increased. As evident in Fig. 4c, the average diameter of the nanostructure exceeded 500 nm. Further, FE-SEM images was used to investigate the morphology and polymer growth around the CuFe 2 O 4 central core and it is shown in Fig. 4d and e. As shown, the grown magnetic nanoparticles have a spherical morphology. The increase in the size of the spherical particles is due to the progressive growth of the cyclic aromatic polyimide chains  www.nature.com/scientificreports/ around the magnetic cores. Additionally, it is noteworthy that the average diameter of the spherical nanostructures has exceeded 1 μm. In order to check the quality of the constituent elements of the designed nanostructure, EDX spectrum was prepared. As seen in Fig. 4f, the three Fe, Cu, and O peaks are related to the CuFe 2 O 4 MNPs as central core. In addition, the presence of Si and N peaks is attributed to the functionalization of magnetic nanoparticles with SiO 2 and CPTMS. The C and N peaks in the spectrum confirmed of the implementation the polymerization process of cyclic aromatic polyimide chains in the presence of phenylenediamine derivatives.

FE-EEM and EDX analyses.
Cytotoxicity. The toxicity and cell viability of CuFe 2 O 4 @SiO 2 -Polymer at the highest concentration (1000 μg/ mL) were 12.64% and 87.36%, respectively (Fig. 5)   www.nature.com/scientificreports/ the bacterial strain S. aureus at a concentration of approximately 1000 μg/mL. Reports have also been presented regarding composite materials containing iron nanoparticles coated with a silicate structure, with an MIC concentration of 1250 μg/ml reported for the P. aeruginosa bacterial strain 48 . Overall, the antibacterial activity and high percentage of cell viability of the nanostructure make it a promising candidate for biomedical applications, such as wound healing, infection control, drug delivery systems and tissue engineering. Therefore, the synthesized nanostructure holds great potential for various biomedical applications due to its superior properties. Further investigations are needed to explore and expand the potential applications of this novel nanostructure in biomedicine.

Conclusions
In summary, a new nanostructure has been prepared based on CuFe 2 O 4 MNPs and pyromellitic dianhydride. In this nanostructure, CuFe 2 O 4 nanoparticles were used as the central core and were functionalized with TEOS, CPTMS, and phenylenediamine derivatives in separate steps. The polymerization reaction of pyromellitic dihydride was carried out on the functionalized surface of CuFe2O4 nanoparticles with phenylenediamine derivatives. Various analytical techniques such as FT-IR, TGA, EDX, FE-SEM, XRD, and VSM confirmed the synthesis of CuFe 2 O 4 @SiO 2 -cyclic aromatic polyamide nanostructure. Finally, the results of the MTT test on HEK293T healthy cells indicated that this nanostructure is biocompatible with a cell viability of 87.36% and can be suitable for in vivo use. Additionally, the antibacterial test on three bacteria strains of S. aureus, E. coli, and P. aeruginosa showed that the prepared nanostructure has antibacterial properties, and its MIC in Gram-negative and Grampositive bacteria is between 500 and 1000 μg/mL. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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