Epoxy functionalized small particle size superparamagnetic iron oxide nanoparticles for CD4+T cell sorting

Epoxy functionalized small particle size superparamagnetic iron oxide nanoparticles for CD4+T cell sorting | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Epoxy functionalized small particle size superparamagnetic iron oxide nanoparticles for CD4+T cell sorting Yiyang Zhou, Wei Dong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6039824/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Iron oxide nanoparticles have gained increasing attention in various biomedical and industrial sectors due to their physicochemical and magnetic properties. In this study, superparamagnetic Fe 3 O 4 nanoparticles with small particle size were synthesized by co-precipitation method, coated with dextran, and modified by epoxy functional groups with 1,4-butanediol diglycidyl ether. The prepared composites was characterized by transmission electron microscopy, thermogravimetry, fourier transform infrared spectroscopy, X-ray diffraction,and vibrating sample magnetometry. The rat serum albumin protein(rSA) was then coupled to the epoxide group on the nanoparticle and the ability of the epoxide functional group to conjugate the Protein was tested by BCA Protein Assay Kit. At the same time, we used CD4 antibody coupled with epoxy group on the nanoparticles to detect the sorting ability of T cells by flow cytometry. This work shows that our epoxy-modified iron oxide nanoparticles have excellent potential applications in biology. Iron Oxide Nanoparticles Co-precipitation Epoxy Groups Cell Sorting Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Iron oxide nanoparticles(IONPs) are the most promising materials for various applications due to their unique properties[ 1 ].Magnetite (Fe 3 O 4 ), hematite (α-Fe 2 O 3 ), magnetite (γ-Fe 2 O 3 ) and mixed ferrite are considered to be the main representatives of iron oxide nanoparticles[ 2 ].Due to their good biocompatibility, good biodegradability, low toxicity and strong magnetic properties,IONPs have been widely used in biomedical fields,such as magnetic resonance imaging(MRI),targeted drug delivery,cancer immuno- therapy and hyperthermia media[ 3 , 4 , 5 ].Among them,Fe 3 O 4 magnetic nanoparticles are the most promising materials in iron oxide nanoparticles because of their superparamagnetism, magnetothermal effect and non-toxicity.The properties of Fe 3 O 4 magnetic nanoparticles affected by many factors, including particle size, shape, agglomeration and coating[ 6 , 7 , 8 , 9 ].When considering the application of Fe 3 O 4 magnetic nanoparticles in humans, their dispersion and stability need to be kept at a high level to minimize potential adverse effects on normal tissues. However, the highly dispersible and stable Fe 3 O 4 magnetic nanoparticles usually need to be prepared by complex methods, such as hydrothermal methods[ 10 , 11 ]and solvothermal methods[ 12 , 13 , 14 ].These methods usually require high temperature and pressure or use a large number of organic solvents,which are high cost and are not suitable for large-scale production of Fe 3 O 4 magnetic nanoparticles.Co-precipitation method is to precipitate Fe 2+ and Fe 3+ aqueous solution by adding weak base or strong base at the same time, which is one of the most simple and effective synthesis methods. Dextran is a natural biodegradable exopolysacc The co- precipitation method [ 15 , 16 , 17 ] has the advantages of short reaction time, mild reaction conditions and convenient raw material acquisition.At the same time, the exposed Fe 3 O 4 NPs usually has a high chemical activity and is particularly prone to oxidation, which often leads to a decrease in magnetic properties. In order to increase the biocompatibility and stability of the iron oxide nanoparticles, an organic coating can be applied.haride consisting of glucose subunits that is biosynthesized by the nonpathogenic Leuconostoc mesenteroides bacterium[ 18 , 19 ].Dextran is generally known for its anti- inflammatory and antithrombotic properties, and the functional hydroxyl groups within its structure provide a facile means for conjugations with other substances [ 20 ], so dextran can be grafted with various functional groups and components, thus further interacting with proteins, enzymes, antibodies and other biomolecules.Dextran coatings are very popular due to their solubility in water, biocompatibility, and effective stabilization of colloidal forms in water due to electrostatic repulsion[ 21 ].It is widely used to prepare polymeric nanoparticles[ 22 ],hydrogels[ 23 ],microgels[ 24 ],and nanogels[ 25 ], and for functionalizing magnetic nanoparticles for biomedical applications[ 26 ].Dextran can be strongly adsorbed to magnetite nanoparticles in solution through the non-covalent interaction of rich hydroxyl group, resulting in the formation of mesh nanoparticle core [ 27 ].Therefore, the stability of Fe 3 O 4 magnetic nanoparticles is improved. In this study, the co-precipitation method was adopted, with EDTA and citric acid monohydrate serving as stabilizers to mitigate particle aggregation and reduce the particle size of IONPs[ 28 ]. Subsequently, 40,000 Da dextran was coated on the surface of IONPs, and 1,4-butanediol diglycidyl ether was added to form epoxy functional groups. In biological applications, rat serum albumin (rSA) was coupled to the epoxy groups on dextran, and the protein coupling capacity of epoxy-functionalized magnetic beads was evaluated using the micro BCA protein assay kit. Finally, the epoxy-functionalized magnetic beads were coupled with CD4 antibodies, and their ability to sort T cells was detected by flow cytometry. Scheme 1 describes the process of IONPs surface functionalization and sorting CD4 + T cells. 2. Materials and methods 2.1 Chemicals FeSO 4 ·7H 2 O,NaOH,HCl,citric acid monohydrate,EDTA and dextran (Mw ≈ 40000) were purchased from Sinopharm Chemical Reagent Co.,Ltd;1,4-butanediol diglycidyl ether was purchased from Shanghai Macklin Biochemical Co., Ltd.;FeCl 3 ·6H 2 O was purchased from Nanjing Chemical Reagent Co.,Ltd;NaBH 4 was purchased from Guangdong Guanghua Sci-tech Co.,Ltd; Ethanol absolute was purchased from Onde Biochemical Technology (Shanghai) Co., Ltd;Ls columns and Ls columns were purchased from Miltenyi Biotec;Micro BCA Protein Assay Kit was purchased from Sangon Biotech(Shanghai) Co., Ltd;Human peripheral blood mononuclear cells(hPBMCs) were purchased from Hope Biotechnology (Sichuan) Co.,Ltd.;Tonbo™ FITC Anti-Human CD4 was purchased from Beijing Biomex Biotechnology Co., LTD.; Human CD4 antibody was purchased from Biointron Biological Inc.;RPMI 1640 medium and PBS(1×) were purchased from Pimi Biotechnology(Shenzhen) Co.,LTD. Bovine Serum Albumin(BSA) was purchased from Shanghai Aladdin Biochemical Technology Co.,Ltd.;Rat serum albumin (rSA) was purchased from Hangzhou Nuptec Biotechnology Co., LTD.;Dialysis bag (Mw ≈ 300000) was purchased from Yibo Biotechnology(Hunan) Co.,LTD. 2.2 Preparation of Dextran-coated Fe 3 O 4 Nanoparticles(Dex-IONPs) Dextran-coated Fe 3 O 4 nanoparticles were obtained by an improved co-precipitation method.2.16 g FeCl 3 •6H 2 O and 1.11 g FeSO 4 ·7H 2 O, with a molar ratio of Fe 2+ /Fe 3+ =0.5 were dissolved in 30 mL DI water.Prepare a solution containing 100ml of 0.6M NaOH, 20ml anhydrous ethanol, and 20ml 0.1M EDTA, and stir vigorously. Gradually introduce the Fe 2+ /Fe 3+ solution into the mixture, resulting in the formation of a black precipitate. Then,add 2.5g citric acid monohydrate,continuing stirring for 6 hours, followed by centrifugation to remove the supernatant and readjusting the volume to 200ml. Introduce 10ml of a solution containing 0.4g dextran into the adjusted solution and stir vigorously for another 6 hours. Subsequently, transfer the solution into a dialysis bag (Mw ≈ 300000) and conduct dialysis for 24 hours. 2.3 Dex-IONPs Modified with Epoxy Groups(Epoxy-IONPs) 100 ml of the dialyzed solution is diluted to a total volume of 250ml,adding 80mg of NaBH 4 and 6 ml of 1,4-Butanediol diglycidyl ether. The mixture is vigorously stirred for 12 hours and then being placed in a dialysis bag (Mw ≈ 300000) and dialysis for 24 hours. 2.4 Determination the Amount of Protein Coupling Introduce 3 ml of magnetic beads into the LS column, wash three times with DI water, and transfer with 3ml of DI water. Subsequently, add a certain amount of rSA and conduct rotational mixing at 37°C for 1 hour. One hour later, introduce 300 µl of rSA-coupled magnetic beads into the LS column. After the supernatant has flowed out, conduct a single wash with 300 µl of DI water. Subsequently, add another 300 µl of DI water, extrude the Epoxy-IONPs using the push rod and collect them in a centrifuge tube. Add 100 µl of BCA working solution and 100 µl of the washed Epoxy-IONPs to a 96-well plate and incubate at 37°C for 30 minutes. Finally, measure the absorbance values at the primary wavelength of 570 nm and the secondary wavelength of 595 nm. The amount of Epoxy-IONPs protein coupling was calculated according to the instructions on the micro BCA protein assay kit.Then conduct another blank experiment on Dex-IONPs. 2.5 Preparation of CD4-Epoxy-IONPs and Sorting CD4 + T Cells Take 500 µL of Epoxy-IONPs, add 0.2 mg of human CD4 antibody and blend thoroughly. Incubate the mixture at 37°C for 1 hour to obtain CD4-Epoxy- IONPs.Place RPMI 1640 medium and hPBMCs in a 37°C water bath for preheating.Once the hPBMCs are dissolved, take 1 ml of hPBMCs and add 9 ml of the medium for thorough mixing. Centrifuge at 300g for 5 minutes and remove the supernatant. Repeat the above steps once more. Use a cell counting plate for cell counting. Add a certain amount of the medium for thorough blending to ensure a concentration of 1.25×10 8 cells/ml. Take 80 µL of the volumetrically adjusted hPBMCs cell suspension and add 40 µL of CD4-Epoxy-IONPs for thorough mixing. Incubate at 4°C for 20 minutes and then add to the MS Column. After the cell suspension in the MS Column has flowed out, wash the MS Column with 1500 µL of cell buffer (1×PBS containing 0.5% bovine serum albumin and 2 mM EDTA). Remove the MS Column, add 1 mL of cell buffer, and immediately elute the magnetically labeled cells through the push rod. 2.6 Sample preparation for flow cytometry Centrifuge at 300g for 5 minutes and remove the supernatant. Add 1 ml of 1×PBS to re-suspend the cells,centrifuge at 300-500g for 5 minutes and remove the supernatant. Repeat this process once more and re-suspend the cells with 100 µL of cell buffer. Add 5 µl of Tonbo™ FITC Anti-Human CD4 and incubate at 4°C in the dark for 15–20 minutes. After the incubation is complete, add 1 ml of 1×PBS, centrifuge at 500g for 5 minutes and remove the supernatant. Repeat this process once more and re-suspend with 400 µl of 1×PBS. Use flow cytometry to detect the cell sorting efficiency. Conduct another blank experiment without CD4-Epoxy-IONPs. Then, a set of blank experiments without adding CD4-Epoxy-IONPs was conducted. As the cells would flow out of the MS Column, the effluent cell suspension was collected for the preparation of flow cytometry sample. 2.7 Characterization The morphology and structure of IONPs were examined by a JEM-F 200 transmission electron microscopy (TEM) instrument.Hydrodynamic diameter and zeta potential were measured by using a dynamic light scattering (DLS) particle size analyzer (OMEC NS-90Z) at scattering angle of 90°.Power X-ray diffraction (XRD) patterns were recorded on a Bruker D8 Advanced diffractometer (Bruker D8 Super Speed) with Cu Kα radiation, and the scanning angle ranged from 5 to 80° of 2θ at 40 kV.Fourier transform infrared (FT-IR) spectra were recorded on a NICOLETIS 20 spectrometer. Thermo gravimetric analysis (TGA) of nanoparticles was performed on Model TGA55(TA Instruments, USA) with a heating rate of 20°C/min under nitrogen atmosphere.The magnetic properties were checked in fields between ± 15 kOe at room temperature by a vibrating sample magnetometer (VSM, Lake Shore 735).The absorbance of the samples was obtained by HBS-1096A enzyme label analyzer.Flow cytometry was performed on the BD FACS Aria II Cell Sorter. 3. Results and Discussion 3.1 Microscopic Morphology and Hydrodynamic Diameter Figure 1 (a) and Fig. 1 (b) show the TEM images of Fe 3 O 4 IONPs.The uncoated Fe 3 O 4 IONPs exhibit serious agglomeration.The grain sizes obtained from TEM images of uncoated Fe 3 O 4 IONPs is about 11 nm. Figure 1 (c) demonstrates the apparent zeta potential findings, indicating a surface charge of -27.4 mV, indicating that citric acid and EDTA are involved in the complexation. Figure 1 (d) shows the hydrodynamic diameter distribution of epoxy group coated Fe 3 O 4 IONPs, and the corresponding average hydrodynamic diameters are 80.81nm with a polydispersity index (PDI) of 0.143, indicating a relatively uniform distribution of Epoxy-IONPs particles. 3.2 Thermogravimetric Analysis As shown in Fig. 2 , the weight loss of IONPs within the temperature range of 50 to 300°C represents the decomposition of water. The weight loss observed for Dex-IONPs between 240 and 325°C is attributed to the degradation of dextran. Following this, a gradual weight loss occurs, with a notable inflection point identified between 618 and 695°C, which relates to further dextran decomposition. Beyond 700°C, the sample's weight stabilizes. As for Epoxy-IONPs, the weight loss within the temperature range of 235 to 380°C is related to the decomposition of epoxy groups and dextran, and the range from 680 to 730°C corresponds to the further decomposition of dextran. Finally, the Epoxy-IONPs sample contains approximately 25% Fe 3 O 4 and almost no moisture. 3.3 FTIR and XRD Analysis Figure 3 (a) illustrate the FTIR spectra of IONPs and Epoxy-IONPs.The peaks observed in the range of 2900–3700 cm⁻¹ correspond to the solvent peaks of water.The peak at 1637 cm⁻¹ is attributed to the stretching vibration of the C = O bond from residual citric acid and EDTA present in the solution. The peaks at 1367 cm⁻¹ represent the stretching vibrations of the C-N bond and C-C bond, respectively, also associated with residual EDTA in solution. Additionally, a characteristic absorption peak for Fe-O is noted at 576 cm⁻¹.A series of absorption peaks within the range of 1000–1500 cm⁻¹ reflects a combination of stretching vibrations from C-C bonds, C-O bonds, C-O-C bond, as well as in-plane bending vibrations from both C-H and -OH bond. The crystalline structure of the Epoxy-IONPs was characterized by XRD.As shown in Fig. 3 (b), it is found that all the reflection peaks at (111),(220),(311),(400),(422), (511),and (533) can be well indexed to the inverse cubic spinel structure of Fe 3 O 4 (JCPDS card no.26-1136) according to the reflection peak positions and relative intensities, which confirms that the nanoparticles synthesized in this study are the Fe 3 O 4 nanoparticles. 3.4 Protein Coupling Amount The Fig. 4 illustrates that as the amount of rSA increases, there is a corresponding rise in the protein conjugation levels for both Dex-IONPs and Epoxy-IONPs. For Dex-IONPs, as the concentration of rSA in the solution increases, rSA molecules can better adsorb onto the hydroxyl sites of dextran, thereby leading to an increase in protein coupling content. However, the protein coupling content of Dex-IONPs remains very low, because hydroxyl groups themselves lack reactivity and have less surface adsorption. They need to be transformed into other activated groups to enhance the protein coupling ability. After Dex-IONPs are transformed into Epoxy-IONPs through epoxy surface functionalization, the epoxy groups show greater reactivity than the hydroxyl groups on dextran. At this point, the increase in protein coupling content is not due to physical adsorption, but rather to the covalent binding of epoxy groups with amino groups on proteins, which promotes the increase in protein coupling content. 3.5 Flow Cytometry It can be observed from the circled part in Fig. 5 (a) that 59.9% of CD4 + T cells displayed fluorescence in the absence of CD4-Epoxy-IONPs addition. The remaining 40.1% comprised B lymphocytes, natural killer cells, stem cells, etc. There might also be some unlabeled CD4 + T cells. In Fig. 5 (b), upon the addition of CD4-Epoxy-IONPs, 97.7% of CD4 + T cells were sorted out, leaving only a very small portion of unlabeled cells. This indicates that the majority of non-CD4 + cells present in hPBMCs have been removed during various centrifugation, resuspension, and elution processes. Meanwhile, the CD4 + T cells bound to CD4-Epoxy-IONPs were relatively stable and consumed minimally. It is demonstrated that CD4-Epoxy-IONPs can enrich the CD4 + T cells in hPBMCs to a very high purity, further validating the biological application potential of the Epoxy-IONPs we synthesized. Moreover, the sorted CD4 + T cells can be utilized in some downstream biological experiments. 3.6 Hysteresis Loop Using a vibrating sample magnetometer, the magnetic hysteresis loops were measured at room temperature to study the magnetic properties of IONPs and Epoxy-IONPs.The magnetic hysteresis loops of the samples are shown in Fig. 6 (a).The results show that the IONPs and Epoxy-IONPs prepared at room temperature have superparamagnetic behavior induced by thermal energy, and the magnetic hysteresis loop can be ignored.This thermal- induced superparamagnetic behavior overcomes the anisotropic potential barrier and randomizes the magnetic moment. This property is very important for biomedical applications because the magnetic moment is zero when the external magnetic field is removed. The saturation magnetization values of IONPs and Epoxy-IONPs are 68.21 and 4.64 emu/g, respectively. The saturation magnetization value of the original nanoparticles is lower than 90 emu/g due to surface spin tilt and smaller particle size effects. In addition to the above reasons, the lower saturation magnetization value of Epoxy-IONPs may be due to the influence of magnetic nanoparticles surface polymer.Dextran and 1,4-Butanediol diglycidyl ether molecules produce a magnetic dead layer on the surface of magnetite nanoparticles, quenching the surface magnetic moment. The results clearly show that the non-magnetic layer of the polymer reduces the magnetization of Fe 3 O 4 nanoparticles and confirm the surface wrapping of Fe 3 O 4 nanoparticles. Figure 6 (b) also confirms the changes in saturation magnetization intensity values for IONPs and Epoxy-IONPs.After being coated with dextran and epoxy-based modification, Epoxy-IONPs can no longer be magnetically attracted by a permanent magnet and can only be separated using a Miltenyi® LS column. 4. Conclusions In this project, we have synthesized, for the first time, the surface-functionalized iron oxide nanocomposites coated with epoxy group-modified dextran. This kind of composite material possesses ultra-small particle size and superparamagnetism. In the experiment for determining the protein coupling content, epoxy groups demonstrated better reactive performance than hydroxyl groups, revealing a higher protein coupling amount. After combining with the CD4 antibody, CD4-Epoxy-IONPs exhibited outstanding sorting performance of CD4 + T lymphocytes, and the purity of sorted CD4 + T lymphocytes reached 97.7%. This indicates that the small-sized surface-functionalized iron oxide nanocomposites synthesized by us have considerable potential for biological applications. Declarations Conflicts of interest There are no conflicts to declare. Funding Declaration This work is supported by the National Natural Science Foundation of China (51573078). Author Contribution W. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6039824","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":418236021,"identity":"a60ded70-eb3a-4a1a-a337-c9fdc655e960","order_by":0,"name":"Yiyang 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Dong","suffix":""}],"badges":[],"createdAt":"2025-02-16 07:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6039824/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6039824/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77207124,"identity":"ae679c14-aafc-48dd-a163-d317b61b3a54","added_by":"auto","created_at":"2025-02-26 08:24:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":606236,"visible":true,"origin":"","legend":"\u003cp\u003e(a-b)TEM images of IONPs.(c) apparent zeta potential of Epoxy-IONPs.(d) hydrodynamic diameters of Epoxy-IONPs.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/d0d922be6a14e315df343bf5.png"},{"id":77207112,"identity":"1825a521-dadd-4450-9acf-07b02426e7f2","added_by":"auto","created_at":"2025-02-26 08:24:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27267,"visible":true,"origin":"","legend":"\u003cp\u003eTGA of IONPs, IONPs and Epoxy-IONPs.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/7d9c7b6ce680e6c1616d9962.png"},{"id":77207115,"identity":"c1a8d89e-26bc-4314-a8e6-badc11c91097","added_by":"auto","created_at":"2025-02-26 08:24:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113284,"visible":true,"origin":"","legend":"\u003cp\u003eFTIR spectra of (a)IONPs and Epoxy-IONPs and XRD pattern of (b)Epoxy-IONPs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/019785481c4e76153dcc6f35.png"},{"id":77207602,"identity":"7f5be594-ca23-4269-b36d-701d3264c0a7","added_by":"auto","created_at":"2025-02-26 08:32:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":96161,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of rSA addition on protein conjugation content.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/5e27b77fa9bb6a970eca3f22.png"},{"id":77207605,"identity":"15380b37-c558-49ba-9cdc-26fd759843d0","added_by":"auto","created_at":"2025-02-26 08:32:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":203689,"visible":true,"origin":"","legend":"\u003cp\u003eBlank experimental cell sorting efficiency (a) and (b)CD4 Epoxy IONPs added cell sorting efficiency.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/11d9607540387b2132feb0f8.png"},{"id":77207113,"identity":"0aede8d3-d4a4-4130-9837-fb868d061db8","added_by":"auto","created_at":"2025-02-26 08:24:09","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":210795,"visible":true,"origin":"","legend":"\u003cp\u003e(a)VSM analysis of IONPs and Epoxy-IONPs and 6(b) Magnetic response of IONPs and Epoxy-IONPs.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/350d38c4ba9d00958a5b75b6.png"},{"id":77207607,"identity":"396896b2-51ac-422f-b4fc-bf3da0f8b87d","added_by":"auto","created_at":"2025-02-26 08:32:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1928513,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/daa3d4ad-c917-4dde-af1e-403a0ec1c8a5.pdf"},{"id":77207123,"identity":"29dd2d1d-116a-4c16-9363-37465575f732","added_by":"auto","created_at":"2025-02-26 08:24:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":168431,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6039824/v1/8cbe8aa292d769376cccf8d3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Epoxy functionalized small particle size superparamagnetic iron oxide nanoparticles for CD4+T cell sorting","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIron oxide nanoparticles(IONPs) are the most promising materials for various applications due to their unique properties[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].Magnetite (Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e), hematite (α-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e), magnetite (γ-Fe\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e) and mixed ferrite are considered to be the main representatives of iron oxide nanoparticles[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].Due to their good biocompatibility, good biodegradability, low toxicity and strong magnetic properties,IONPs have been widely used in biomedical fields,such as magnetic resonance imaging(MRI),targeted drug delivery,cancer immuno- therapy and hyperthermia media[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].Among them,Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e magnetic nanoparticles are the most promising materials in iron oxide nanoparticles because of their superparamagnetism, magnetothermal effect and non-toxicity.The properties of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e magnetic nanoparticles affected by many factors, including particle size, shape, agglomeration and coating[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].When considering the application of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e magnetic nanoparticles in humans, their dispersion and stability need to be kept at a high level to minimize potential adverse effects on normal tissues. However, the highly dispersible and stable Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e magnetic nanoparticles usually need to be prepared by complex methods, such as hydrothermal methods[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]and solvothermal methods[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].These methods usually require high temperature and pressure or use a large number of organic solvents,which are high cost and are not suitable for large-scale production of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e magnetic nanoparticles.Co-precipitation method is to precipitate Fe\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e aqueous solution by adding weak base or strong base at the same time, which is one of the most simple and effective synthesis methods.\u003c/p\u003e \u003cp\u003eDextran is a natural biodegradable exopolysacc The co- precipitation method [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] has the advantages of short reaction time, mild reaction conditions and convenient raw material acquisition.At the same time, the exposed Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e NPs usually has a high chemical activity and is particularly prone to oxidation, which often leads to a decrease in magnetic properties. In order to increase the biocompatibility and stability of the iron oxide nanoparticles, an organic coating can be applied.haride consisting of glucose subunits that is biosynthesized by the nonpathogenic Leuconostoc mesenteroides bacterium[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].Dextran is generally known for its anti- inflammatory and antithrombotic properties, and the functional hydroxyl groups within its structure provide a facile means for conjugations with other substances [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], so dextran can be grafted with various functional groups and components, thus further interacting with proteins, enzymes, antibodies and other biomolecules.Dextran coatings are very popular due to their solubility in water, biocompatibility, and effective stabilization of colloidal forms in water due to electrostatic repulsion[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].It is widely used to prepare polymeric nanoparticles[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e],hydrogels[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e],microgels[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e],and nanogels[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and for functionalizing magnetic nanoparticles for biomedical applications[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].Dextran can be strongly adsorbed to magnetite nanoparticles in solution through the non-covalent interaction of rich hydroxyl group, resulting in the formation of mesh nanoparticle core [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].Therefore, the stability of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e magnetic nanoparticles is improved.\u003c/p\u003e \u003cp\u003eIn this study, the co-precipitation method was adopted, with EDTA and citric acid monohydrate serving as stabilizers to mitigate particle aggregation and reduce the particle size of IONPs[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Subsequently, 40,000 Da dextran was coated on the surface of IONPs, and 1,4-butanediol diglycidyl ether was added to form epoxy functional groups. In biological applications, rat serum albumin (rSA) was coupled to the epoxy groups on dextran, and the protein coupling capacity of epoxy-functionalized magnetic beads was evaluated using the micro BCA protein assay kit. Finally, the epoxy-functionalized magnetic beads were coupled with CD4 antibodies, and their ability to sort T cells was detected by flow cytometry. Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e describes the process of IONPs surface functionalization and sorting CD4\u0026thinsp;+\u0026thinsp;T cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e \u003cp\u003eFeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO,NaOH,HCl,citric acid monohydrate,EDTA and dextran (Mw\u0026thinsp;\u0026asymp;\u0026thinsp;40000) were purchased from Sinopharm Chemical Reagent Co.,Ltd;1,4-butanediol diglycidyl ether was purchased from Shanghai Macklin Biochemical Co., Ltd.;FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO was purchased from Nanjing Chemical Reagent Co.,Ltd;NaBH\u003csub\u003e4\u003c/sub\u003e was purchased from Guangdong Guanghua Sci-tech Co.,Ltd; Ethanol absolute was purchased from Onde Biochemical Technology (Shanghai) Co., Ltd;Ls columns and Ls columns were purchased from Miltenyi Biotec;Micro BCA Protein Assay Kit was purchased from Sangon Biotech(Shanghai) Co., Ltd;Human peripheral blood mononuclear cells(hPBMCs) were purchased from Hope Biotechnology (Sichuan) Co.,Ltd.;Tonbo\u0026trade; FITC Anti-Human CD4 was purchased from Beijing Biomex Biotechnology Co., LTD.; Human CD4 antibody was purchased from Biointron Biological Inc.;RPMI 1640 medium and PBS(1\u0026times;) were purchased from Pimi Biotechnology(Shenzhen) Co.,LTD. Bovine Serum Albumin(BSA) was purchased from Shanghai Aladdin Biochemical Technology Co.,Ltd.;Rat serum albumin (rSA) was purchased from Hangzhou Nuptec Biotechnology Co., LTD.;Dialysis bag (Mw\u0026thinsp;\u0026asymp;\u0026thinsp;300000) was purchased from Yibo Biotechnology(Hunan) Co.,LTD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of Dextran-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e Nanoparticles(Dex-IONPs)\u003c/h2\u003e \u003cp\u003eDextran-coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles were obtained by an improved co-precipitation method.2.16 g FeCl\u003csub\u003e3\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO and 1.11 g FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO, with a molar ratio of Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e=0.5 were dissolved in 30 mL DI water.Prepare a solution containing 100ml of 0.6M NaOH, 20ml anhydrous ethanol, and 20ml 0.1M EDTA, and stir vigorously. Gradually introduce the Fe\u003csup\u003e2+\u003c/sup\u003e/Fe\u003csup\u003e3+\u003c/sup\u003e solution into the mixture, resulting in the formation of a black precipitate. Then,add 2.5g citric acid monohydrate,continuing stirring for 6 hours, followed by centrifugation to remove the supernatant and readjusting the volume to 200ml. Introduce 10ml of a solution containing 0.4g dextran into the adjusted solution and stir vigorously for another 6 hours. Subsequently, transfer the solution into a dialysis bag (Mw\u0026thinsp;\u0026asymp;\u0026thinsp;300000) and conduct dialysis for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Dex-IONPs Modified with Epoxy Groups(Epoxy-IONPs)\u003c/h2\u003e \u003cp\u003e100 ml of the dialyzed solution is diluted to a total volume of 250ml,adding 80mg of NaBH\u003csub\u003e4\u003c/sub\u003e and 6 ml of 1,4-Butanediol diglycidyl ether. The mixture is vigorously stirred for 12 hours and then being placed in a dialysis bag (Mw\u0026thinsp;\u0026asymp;\u0026thinsp;300000) and dialysis for 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination the Amount of Protein Coupling\u003c/h2\u003e \u003cp\u003eIntroduce 3 ml of magnetic beads into the LS column, wash three times with DI water, and transfer with 3ml of DI water. Subsequently, add a certain amount of rSA and conduct rotational mixing at 37\u0026deg;C for 1 hour. One hour later, introduce 300 \u0026micro;l of rSA-coupled magnetic beads into the LS column. After the supernatant has flowed out, conduct a single wash with 300 \u0026micro;l of DI water. Subsequently, add another 300 \u0026micro;l of DI water, extrude the Epoxy-IONPs using the push rod and collect them in a centrifuge tube. Add 100 \u0026micro;l of BCA working solution and 100 \u0026micro;l of the washed Epoxy-IONPs to a 96-well plate and incubate at 37\u0026deg;C for 30 minutes. Finally, measure the absorbance values at the primary wavelength of 570 nm and the secondary wavelength of 595 nm. The amount of Epoxy-IONPs protein coupling was calculated according to the instructions on the micro BCA protein assay kit.Then conduct another blank experiment on Dex-IONPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Preparation of CD4-Epoxy-IONPs and Sorting CD4\u0026thinsp;+\u0026thinsp;T Cells\u003c/h2\u003e \u003cp\u003eTake 500 \u0026micro;L of Epoxy-IONPs, add 0.2 mg of human CD4 antibody and blend thoroughly. Incubate the mixture at 37\u0026deg;C for 1 hour to obtain CD4-Epoxy- IONPs.Place RPMI 1640 medium and hPBMCs in a 37\u0026deg;C water bath for preheating.Once the hPBMCs are dissolved, take 1 ml of hPBMCs and add 9 ml of the medium for thorough mixing. Centrifuge at 300g for 5 minutes and remove the supernatant. Repeat the above steps once more. Use a cell counting plate for cell counting. Add a certain amount of the medium for thorough blending to ensure a concentration of 1.25\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/ml. Take 80 \u0026micro;L of the volumetrically adjusted hPBMCs cell suspension and add 40 \u0026micro;L of CD4-Epoxy-IONPs for thorough mixing. Incubate at 4\u0026deg;C for 20 minutes and then add to the MS Column. After the cell suspension in the MS Column has flowed out, wash the MS Column with 1500 \u0026micro;L of cell buffer (1\u0026times;PBS containing 0.5% bovine serum albumin and 2 mM EDTA). Remove the MS Column, add 1 mL of cell buffer, and immediately elute the magnetically labeled cells through the push rod.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Sample preparation for flow cytometry\u003c/h2\u003e \u003cp\u003eCentrifuge at 300g for 5 minutes and remove the supernatant. Add 1 ml of 1\u0026times;PBS to re-suspend the cells,centrifuge at 300-500g for 5 minutes and remove the supernatant. Repeat this process once more and re-suspend the cells with 100 \u0026micro;L of cell buffer. Add 5 \u0026micro;l of Tonbo\u0026trade; FITC Anti-Human CD4 and incubate at 4\u0026deg;C in the dark for 15\u0026ndash;20 minutes. After the incubation is complete, add 1 ml of 1\u0026times;PBS, centrifuge at 500g for 5 minutes and remove the supernatant. Repeat this process once more and re-suspend with 400 \u0026micro;l of 1\u0026times;PBS. Use flow cytometry to detect the cell sorting efficiency. Conduct another blank experiment without CD4-Epoxy-IONPs. Then, a set of blank experiments without adding CD4-Epoxy-IONPs was conducted. As the cells would flow out of the MS Column, the effluent cell suspension was collected for the preparation of flow cytometry sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Characterization\u003c/h2\u003e \u003cp\u003eThe morphology and structure of IONPs were examined by a JEM-F 200 transmission electron microscopy (TEM) instrument.Hydrodynamic diameter and zeta potential were measured by using a dynamic light scattering (DLS) particle size analyzer (OMEC NS-90Z) at scattering angle of 90\u0026deg;.Power X-ray diffraction (XRD) patterns were recorded on a Bruker D8 Advanced diffractometer (Bruker D8 Super Speed) with Cu Kα radiation, and the scanning angle ranged from 5 to 80\u0026deg; of 2θ at 40 kV.Fourier transform infrared (FT-IR) spectra were recorded on a NICOLETIS 20 spectrometer. Thermo gravimetric analysis (TGA) of nanoparticles was performed on Model TGA55(TA Instruments, USA) with a heating rate of 20\u0026deg;C/min under nitrogen atmosphere.The magnetic properties were checked in fields between \u0026plusmn;\u0026thinsp;15 kOe at room temperature by a vibrating sample magnetometer (VSM, Lake Shore 735).The absorbance of the samples was obtained by HBS-1096A enzyme label analyzer.Flow cytometry was performed on the BD FACS Aria II Cell Sorter.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.1 Microscopic Morphology and Hydrodynamic Diameter\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a) and Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(b) show the TEM images of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e IONPs.The uncoated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e IONPs exhibit serious agglomeration.The grain sizes obtained from TEM images of uncoated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e IONPs is about 11 nm. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(c) demonstrates the apparent zeta potential findings, indicating a surface charge of -27.4 mV, indicating that citric acid and EDTA are involved in the complexation. Figure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(d) shows the hydrodynamic diameter distribution of epoxy group coated Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e IONPs, and the corresponding average hydrodynamic diameters are 80.81nm with a polydispersity index (PDI) of 0.143, indicating a relatively uniform distribution of Epoxy-IONPs particles.\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.2 Thermogravimetric Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, the weight loss of IONPs within the temperature range of 50 to 300\u0026deg;C represents the decomposition of water. The weight loss observed for Dex-IONPs between 240 and 325\u0026deg;C is attributed to the degradation of dextran. Following this, a gradual weight loss occurs, with a notable inflection point identified between 618 and 695\u0026deg;C, which relates to further dextran decomposition. Beyond 700\u0026deg;C, the sample\u0026apos;s weight stabilizes. As for Epoxy-IONPs, the weight loss within the temperature range of 235 to 380\u0026deg;C is related to the decomposition of epoxy groups and dextran, and the range from 680 to 730\u0026deg;C corresponds to the further decomposition of dextran. Finally, the Epoxy-IONPs sample contains approximately 25% Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e and almost no moisture.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 FTIR and XRD Analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(a) illustrate the FTIR spectra of IONPs and Epoxy-IONPs.The peaks observed in the range of 2900\u0026ndash;3700 cm⁻\u0026sup1; correspond to the solvent peaks of water.The peak at 1637 cm⁻\u0026sup1; is attributed to the stretching vibration of the C\u0026thinsp;=\u0026thinsp;O bond from residual citric acid and EDTA present in the solution. The peaks at 1367 cm⁻\u0026sup1; represent the stretching vibrations of the C-N bond and C-C bond, respectively, also associated with residual EDTA in solution. Additionally, a characteristic absorption peak for Fe-O is noted at 576 cm⁻\u0026sup1;.A series of absorption peaks within the range of 1000\u0026ndash;1500 cm⁻\u0026sup1; reflects a combination of stretching vibrations from C-C bonds, C-O bonds, C-O-C bond, as well as in-plane bending vibrations from both C-H and -OH bond. The crystalline structure of the Epoxy-IONPs was characterized by XRD.As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(b), it is found that all the reflection peaks at (111),(220),(311),(400),(422), (511),and (533) can be well indexed to the inverse cubic spinel structure of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e (JCPDS card no.26-1136) according to the reflection peak positions and relative intensities, which confirms that the nanoparticles synthesized in this study are the Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Protein Coupling Amount\u003c/h2\u003e\n \u003cp\u003eThe Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates that as the amount of rSA increases, there is a corresponding rise in the protein conjugation levels for both Dex-IONPs and Epoxy-IONPs. For Dex-IONPs, as the concentration of rSA in the solution increases, rSA molecules can better adsorb onto the hydroxyl sites of dextran, thereby leading to an increase in protein coupling content. However, the protein coupling content of Dex-IONPs remains very low, because hydroxyl groups themselves lack reactivity and have less surface adsorption. They need to be transformed into other activated groups to enhance the protein coupling ability. After Dex-IONPs are transformed into Epoxy-IONPs through epoxy surface functionalization, the epoxy groups show greater reactivity than the hydroxyl groups on dextran. At this point, the increase in protein coupling content is not due to physical adsorption, but rather to the covalent binding of epoxy groups with amino groups on proteins, which promotes the increase in protein coupling content.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Flow Cytometry\u003c/h2\u003e\n \u003cp\u003eIt can be observed from the circled part in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(a) that 59.9% of CD4\u0026thinsp;+\u0026thinsp;T cells displayed fluorescence in the absence of CD4-Epoxy-IONPs addition. The remaining 40.1% comprised B lymphocytes, natural killer cells, stem cells, etc. There might also be some unlabeled CD4\u0026thinsp;+\u0026thinsp;T cells. In Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e(b), upon the addition of CD4-Epoxy-IONPs, 97.7% of CD4\u0026thinsp;+\u0026thinsp;T cells were sorted out, leaving only a very small portion of unlabeled cells. This indicates that the majority of non-CD4\u0026thinsp;+\u0026thinsp;cells present in hPBMCs have been removed during various centrifugation, resuspension, and elution processes. Meanwhile, the CD4\u0026thinsp;+\u0026thinsp;T cells bound to CD4-Epoxy-IONPs were relatively stable and consumed minimally. It is demonstrated that CD4-Epoxy-IONPs can enrich the CD4\u0026thinsp;+\u0026thinsp;T cells in hPBMCs to a very high purity, further validating the biological application potential of the Epoxy-IONPs we synthesized. Moreover, the sorted CD4\u0026thinsp;+\u0026thinsp;T cells can be utilized in some downstream biological experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6 Hysteresis Loop\u003c/h2\u003e\n \u003cp\u003eUsing a vibrating sample magnetometer, the magnetic hysteresis loops were measured at room temperature to study the magnetic properties of IONPs and Epoxy-IONPs.The magnetic hysteresis loops of the samples are shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e(a).The results show that the IONPs and Epoxy-IONPs prepared at room temperature have superparamagnetic behavior induced by thermal energy, and the magnetic hysteresis loop can be ignored.This thermal- induced superparamagnetic behavior overcomes the anisotropic potential barrier and randomizes the magnetic moment. This property is very important for biomedical applications because the magnetic moment is zero when the external magnetic field is removed. The saturation magnetization values of IONPs and Epoxy-IONPs are 68.21 and 4.64 emu/g, respectively. The saturation magnetization value of the original nanoparticles is lower than 90 emu/g due to surface spin tilt and smaller particle size effects. In addition to the above reasons, the lower saturation magnetization value of Epoxy-IONPs may be due to the influence of magnetic nanoparticles surface polymer.Dextran and 1,4-Butanediol diglycidyl ether molecules produce a magnetic dead layer on the surface of magnetite nanoparticles, quenching the surface magnetic moment. The results clearly show that the non-magnetic layer of the polymer reduces the magnetization of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles and confirm the surface wrapping of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles. Figure \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e(b) also confirms the changes in saturation magnetization intensity values for IONPs and Epoxy-IONPs.After being coated with dextran and epoxy-based modification, Epoxy-IONPs can no longer be magnetically attracted by a permanent magnet and can only be separated using a Miltenyi\u0026reg; LS column.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this project, we have synthesized, for the first time, the surface-functionalized iron oxide nanocomposites coated with epoxy group-modified dextran. This kind of composite material possesses ultra-small particle size and superparamagnetism. In the experiment for determining the protein coupling content, epoxy groups demonstrated better reactive performance than hydroxyl groups, revealing a higher protein coupling amount. After combining with the CD4 antibody, CD4-Epoxy-IONPs exhibited outstanding sorting performance of CD4\u0026thinsp;+\u0026thinsp;T lymphocytes, and the purity of sorted CD4\u0026thinsp;+\u0026thinsp;T lymphocytes reached 97.7%. This indicates that the small-sized surface-functionalized iron oxide nanocomposites synthesized by us have considerable potential for biological applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by the National Natural Science Foundation of China (51573078).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eW. D. participated in the study conceptualization;Y. Z. participated in the experiment, conducted the data analysis and wrote the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFatimah I, Fadillah G and Yudha S P 2021 Synthesis of iron-based magnetic nanocomposites:a review Arab. J. Chem. 14 103301\u003c/li\u003e\n \u003cli\u003eChen Y and Hou S 2023 Recent progress in the effect of magnetic iron oxide nanoparticles on cells and extracellular vesicles Cell Death Discov. 9 195\u003c/li\u003e\n \u003cli\u003eYang Y, Liu Y, Song L, Cui X, Zhou J, Jin G, Boccaccini A R and Virtanen S 2023 Iron oxide nanoparticle-based nanocomposites in biomedical application Trends Biotechnol. 41 1471-87\u003c/li\u003e\n \u003cli\u003eDash S, Das T, Patel P, Panda P K, Suar M and Verma S K 2022 Emerging trends in the nanomedicine applications of functionalized magnetic nanoparticles as novel therapies for acute and chronic diseases J. Nanobiotechnology 20 393\u003c/li\u003e\n \u003cli\u003eSimnani F Z, Singh D, Patel P, Choudhury A, Sinha A, Nandi A, Samal S K, Verma S K, Panda P K 2023 Nanocarrier vaccine therapeutics for global infectious and chronic diseases Mater Today 66 371-408\u003c/li\u003e\n \u003cli\u003eNemati Z,Alonso J, Martinez L M, Khurshid H, Garaio E, Garcia J A, Phan M H and Srikanth H 2016 Enhanced magnetic hyperthermia in iron oxide nano-octopods: size and anisotropy effects J. Phys. Chem. C. 120\u003c/li\u003e\n \u003cli\u003eNemati Z, Alonso J, Rodrigo I, Das R, Garaio E, Garc\u0026iacute;a J \u0026Aacute;, Orue I, Phan M-H and Srikanth H 2018 Improving the heating efficiency of iron oxide nanoparticles by tuning their shape and size J. Phys. Chem. C. 122 81\u003c/li\u003e\n \u003cli\u003eSomvanshi S B, Kharat P B, Khedkar M V and Jadhav K M 2020 Hydrophobic to hydrophilic surface transformation of nano-scale zinc ferrite via oleic acid coating: magnetic hyperthermia study towards biomedical applications Ceram. Int. 46 7642-53\u003c/li\u003e\n \u003cli\u003eDas R, Alonso J, Porshokouh Z N, Kalappattil V, Torres D, Phan M-H, Garaio E, Garc\u0026iacute;a J \u0026Aacute;, Llamazares J L S and Srikanth H 2016 Tunable high aspect ratio iron oxide nanorods for enhanced hyperthermia J. Phys. Chem. C. 120 10086-93\u003c/li\u003e\n \u003cli\u003eFayazzadeh S, Khodaei M, Arani M, Mahdavi S R, Nizamov T and Majouga A 2020 Magnetic properties and magnetic hyperthermia of cobalt ferrite nanoparticles synthesized by hydrothermal method J. Supercond. Nov. Magn. 33 2227-33\u003c/li\u003e\n \u003cli\u003eGahrouei Z E, Labbaf S and Kermanpur A 2020 Cobalt doped magnetite nanoparticles: synthesis, characterization, optimization and suitability evaluations for magnetic hyperthermia applications Physica E. 116 113759\u003c/li\u003e\n \u003cli\u003eElsayed W E M, Al-Hazmi F S, Memesh L S and Bronstein L M 2017 A novel approach for rapid green synthesis of nearly mono-disperse iron oxide magnetic nanocubes with remarkable surface magnetic anisotropy density for enhancing hyperthermia performance Colloid Surf. A-Physicochem. Eng. Asp. 529 239-45\u003c/li\u003e\n \u003cli\u003eZhao H, Cui H-J and Fu M-L 2016 A general and facile method for improving carbon coat on magnetic nanoparticles with a thickness control J. Colloid Interface Sci. 461 20-4\u003c/li\u003e\n \u003cli\u003eDuong H D T, Nguyen D T and Kim K-S 2021 Effects of process variables on properties of CoFe2O4 nanoparticles prepared by solvothermal process Nanomaterials 11 3056\u003c/li\u003e\n \u003cli\u003ePatade S R, Andhare D D, Somvanshi S B, Jadhav S A, Khedkar M V and Jadhav K M 2020 Self-heating evaluation of superparamagnetic MnFe2O4 nanoparticles for magnetic fluid hyper-thermia application towards cancer treatment Ceram. Int. 46 25576-83\u003c/li\u003e\n \u003cli\u003eKusigerski V, Illes E, Blanusa J, Gyergyek S, Boskovic M, Perovic M and Spasojevic V 2019 Magnetic properties and heating efficacy of magnesium doped magnetite nanoparticles obtained by co-precipitation method J. Magn. Magn. Mater. 475 470-8\u003c/li\u003e\n \u003cli\u003eRajan A, Sharma M and Sahu N K 2020 Assessing magnetic and inductive thermal properties of various surfactants functionalised Fe3O4 nanoparticles for hyperthermia Sci Rep. 10 15045\u003c/li\u003e\n \u003cli\u003eUthaman S, Muthiah M, Park I K and Cho C S 2016 Fabrication and development of magnetic particles for gene therapy Polymers and Nanomaterials for Gene Therapy (Woodhead Publishing) 215-30\u003c/li\u003e\n \u003cli\u003eEncyclopedia of Food Sciences and Nutrition ed B Caballero (Academic Press) 1772-3\u003c/li\u003e\n \u003cli\u003eGaspar V M, Moreira A F, Melo-Diogo D, Costa E C, Queiroz J A, Sousa F, Pichon C and Correia I J 2016 Multifunctional nanocarriers for codelivery of nucleic acids and chemotherapeutics to cancer cells Nanobiomaterials in Medical Imaging (William Andrew Publishing) 163-207\u003c/li\u003e\n \u003cli\u003eLinh P H, Phuc N X, Hong L V, Uyen L L, Chien N V, Nam P H, Quy N T, Nhung H T M, Phong P T and Lee I-J 2018 Dextran coated magnetite high susceptibility nanoparticles for hyperthermia applications J. Magn. Magn. Mater. 460 128-136\u003c/li\u003e\n \u003cli\u003eMaingret V, Chartier C, Six J-L, Schmitt V and H\u0026eacute;roguez V 2022 Pickering emulsions stabilized by biodegradable dextran-based nanoparticles featuring enzyme responsiveness and co-encapsulation of actives Carbohydr. Polym. 284 119146\u003c/li\u003e\n \u003cli\u003eTomme S R V and Hennink W E 2007 Biodegradable dextran hydrogels for protein delivery applications Expert Rev. Med. Devices. 4 147-64\u003c/li\u003e\n \u003cli\u003eSu H, Jia Q, Shan S 2016 Synthesis and characterization of schiff base contained dextran microgels in water-in-oil inverse microemulsion Carbohydr. Polym. 152 156-62\u003c/li\u003e\n \u003cli\u003eCurcio M, Diaz-Gomez L, Cirillo G, Concheiro A, Iemma F and Alvarez-Lorenzo C 2017 pH/redox dual-sensitive dextran nanogels for enhanced intracellular drug delivery Eur. J. Pharm. Biopharm. 117 324-32\u003c/li\u003e\n \u003cli\u003eMajewski P and Thierry B 2007 Functionalized magnetite nanoparticles synthesis, properties, and bioapplications Critical Reviews in Solid State and Materials Sciences 32 203-15\u003c/li\u003e\n \u003cli\u003eHauser A K, Mathias R, Anderson K W and Hilt J Z 2015 The effects of synthesis method on the physical and chemical properties of dextran coated iron oxide nanoparticles Mater. Chem. Phys. 160 177-86.\u003c/li\u003e\n \u003cli\u003eDaoush W M 2017 Co-Precipitation and magnetic properties of magnetite nanoparticles for potential biomedical applications Journal of Nanomedicine Research 5 0011\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Iron Oxide Nanoparticles, Co-precipitation, Epoxy Groups, Cell Sorting","lastPublishedDoi":"10.21203/rs.3.rs-6039824/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6039824/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIron oxide nanoparticles have gained increasing attention in various biomedical and industrial sectors due to their physicochemical and magnetic properties. In this study, superparamagnetic Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e nanoparticles with small particle size were synthesized by co-precipitation method, coated with dextran, and modified by epoxy functional groups with 1,4-butanediol diglycidyl ether. The prepared composites was characterized by transmission electron microscopy, thermogravimetry, fourier transform infrared spectroscopy, X-ray diffraction,and vibrating sample magnetometry. The rat serum albumin protein(rSA) was then coupled to the epoxide group on the nanoparticle and the ability of the epoxide functional group to conjugate the Protein was tested by BCA Protein Assay Kit. At the same time, we used CD4 antibody coupled with epoxy group on the nanoparticles to detect the sorting ability of T cells by flow cytometry. This work shows that our epoxy-modified iron oxide nanoparticles have excellent potential applications in biology.\u003c/p\u003e","manuscriptTitle":"Epoxy functionalized small particle size superparamagnetic iron oxide nanoparticles for CD4+T cell sorting","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-26 08:23:29","doi":"10.21203/rs.3.rs-6039824/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"50b0ffdc-5fd5-466c-9d73-00af728c1219","owner":[],"postedDate":"February 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-26T08:23:29+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-26 08:23:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6039824","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6039824","identity":"rs-6039824","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}