Functionalization with doxorubicin and trastuzumab of medium-entropy MgFeGdDy layered double hydroxides with magnetic resonance imaging contrast activity

Functionalization with doxorubicin and trastuzumab of medium-entropy MgFeGdDy layered double hydroxides with magnetic resonance imaging contrast activity | 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 Functionalization with doxorubicin and trastuzumab of medium-entropy MgFeGdDy layered double hydroxides with magnetic resonance imaging contrast activity Gregorio Guadalupe Carbajal-Arízaga, Norma Alejandra González-Rojas This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7633707/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Journal of Nanoparticle Research → Version 1 posted 11 You are reading this latest preprint version Abstract Layered double hydroxides (LDH) with the MgAl and ZnAl cation combination are well-known as drug-delivery vehicles. Aluminum provides structural stability, enabling the intercalation and functionalization of materials to produce multifunctional materials for biomedical applications. However, aluminum is not desirable as it is a possible promoter of neurodegenerative diseases. In this work, we explore some procedures to obtain aluminum-free LDHs composed of MgFe, additionally including Gd(III) and Dy (III) cations. The resulting medium-entropy material was further functionalized with doxorubicin (Dox), a chemotherapeutic drug, and trastuzumab (Tzmb), an antibody used to confer selectivity to breast cancer cells. The stability of the medium-entropy structure was assessed against the functionalization processes by X-ray diffraction (XRD), infrared spectroscopy (FTIR), and the Z-potential technique. Finally, the functionalized particles were active as magnetic resonance imaging (MRI) contrast agents, proving the potential use of the MgFeGdDy LDH as theragnostic particles. Layered double hydroxide Lanthanides Doxorubicin Trastuzumab Functionalization MRI Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Please have a look at courier new font provided for text in article. Layered double hydroxides (LHD) are compounds of interest in materials science due to their anion exchange capacity, catalytic capacity, and applications as drug carriers, among others [ 1 , 2 ]. In their most basic composition, the structures are formed by a combination of M(II)/M(III) metal cations [ 3 ]. For biomedical purposes, the M(II) cations are usually magnesium and zinc, and the M(III) cation is Al 3+ . The most widely accepted rule is that LDH structures are formed when M(II) and M(III) cations meet an ionic radius between 0.67 and 0.93 Å, since the octahedral structure is usually stabilized due to Paulli's first law between these dimensions [ 4 ]. Complying with these dimensions, paramagnetic cations can be selected to present spontaneous ferromagnetic couplings [ 5 ]and produce LDH-based contrast agents (CA) in magnetic resonance imaging (MRI), such as manganese-doped LDHs [ 6 , 7 ]. Currently, commercial CAs contain gadolinium because of its high magnetic moment related to contrast effectiveness; however, the commercial CAs are formulated with gadolinium complexes, which could present some adverse effects due to long-term toxicity, the need for high doses, and particle agglomeration, among others [ 8 – 10 ]. Such disadvantages can be mitigated by incorporating gadolinium cations into an LDH lattice. Another lanthanide that exhibits this activity, albeit to a lesser extent, is Dysprosium (Dy). It offers the additional advantage of being chemically inert and can enhance its properties when is in oxide [ 11 , 12 ]. Both elements are of interest in MRI, as they may possess the ability to demagnetize protons in water molecules at two types of spin relaxation in MRI, thereby generating a contrast effect; Gd 3+ at longitudinal relaxation (T 1 ) and Dy 3+ at transversal relaxation (T 2 ) [ 11 , 13 ]. Although the large atomic radius of paramagnetic cations such as Gd 3+ (1.03 Å), or Dy 3+ (0.99 Å), would not form a stable LDH structures, in recent years, several researchers have successfully incorporated these cations in MgAl [ 14 ]and ZnAl LDHs [ 15 ]and contrast activity was obtained in MRI assays. On the other hand, while nanoparticles containing aluminum have been shown to be toxic to human cells and rodents [ 16 ], this element could be associated with the appearance of neurodegenerative diseases in humans [ 17 ], then it would be desirable to remove it from the LDH structures. Therefore, this work proposes to replace Al(III) with Fe(III) cations, leading to a combination of a biocompatible LDH lattice Mg-Fe dopped with two lanthanides Gd 3+ and Dy 3+ with T 1 and T 2 MRI activity cations that would involve the formation of a medium-entropy structure [ 18 ], where the presence of iron could form poorly crystalline LDH particles. Medium-entropy structures consist of a solid phase composed of three or four metals and have a value of ∆S mix between 0.69R and 1.61R according to the equation: \(\:\varDelta\:{S}_{mix}=-R{\sum\:}_{i}^{n}{c}_{i}\bullet\:\text{ln}{c}_{i}\) (Eq. 1) where c i is the atomic percentage of the i-th cation and R is the gas constant [ 19 , 20 ]. In this work, we explored different synthesis processes combining sequences of addition and treatments of reagents to obtain a medium-entropy MgFeGdDy LDH structure capable of being functionalized with doxorubicin and trastuzumab, which are therapeutic and targeting agents, respectively, desirable in theragnostic assays. The MRI contrast activity of the medium-entropy LDHs and the functionalization derivatives was also evaluated. 2. Methodology 2.1. Synthesis The reagents used in this experiment were Mg(NO 3 ) 2 •6H 2 O (98.0-102.0%, Golden Bell), Fe(NO 3 ) 3 •9H 2 O (≥ 98%, Sigma-Aldrich), Gd(NO 3 ) 3 •6H 2 O (99.99%-Sigma-Aldrich), Dy(NO 3 ) 3 •xH 2 O (99.9%, Sigma-Aldrich), NaOH (≥ 98%, JT Baker), doxorubicin (≥ 90-≤100%, Sigma-Aldrich), trastuzumab (≥ 90%, Sigma-Aldrich), and phosphate buffer solution (pH 7.2–7.6, Sigma-Aldrich). To obtain the LDH with a nominal composition Mg 2.0 Fe 0.5 Gd 0.25 Dy 0.25 , four methods were designed combining treatments of reagents and sequences of addition as summarized in Table 1 and detailed below: Method A The cation salts were added in the same flask with 50 mL of water, the solution was sonicated for 3 minutes and a 1M NaOH was added quickly until pH 13 ± 0.5 was reached, the suspension was shaken for 20 minutes and then left to age for 72 hours in a closed container isolated from light. Method B Separate solutions of each salt were prepared. Each solution was sonicated for 3 minutes and then mixed abruptly with a 1 M NaOH solution in a vessel to obtain a suspension with a pH of 13 ± 0.5. The mixture was agitated for 18 hours and aged in a closed container away from light for 24 hours. Method C A solution with gadolinium, magnesium, and dysprosium salts was prepared separately from the iron salt solution. These two solutions were mixed with a 1M NaOH solution at the same time to reach a pH 13 ± 0.5. The suspension was stirred for 18 hours and aged for 24 hours. Method D The salts were added at the same time in a mortar, and 15 mL of NaOH 1 M was added. The mixture was macerated for 20 minutes and aged for 24 hours in a closed container isolated from light. In all the methods, after the aging period, the suspensions were centrifuged, discarding the supernatant and washing with deionized water until the pH of the liquid phase was 7. The solids were dried at 25°C. Table 1 Conditions of the synthesis methods to obtain a MgFeGdDy LDH structure. Method Addition of M(II) and M(III) salts Ultrasound Final pH Agitation after alkalinization Aging (h) A All the cations in the same solution Yes 13 20 h 72 B One solution per cation Yes 13 18 h 24 C One solution with Mg, Gd, and Dy. A second solution with Fe No 13 18 h 24 D All the cations mixed in a mortar No - 20 min 24 2.2. Functionalization with doxorubicin and trastuzumab 2.2.1. Doxorubicin-functionalized LDH LDH-Dox was prepared by dissolving 12.5 mg of doxorubicin in 1 mL of double distilled water, once the doxorubicin was dissolved, 37.5 mg of previously dried and pulverized LDH was added [ 21 ]. The mixture was kept under constant agitation for 24 hours. Subsequently, half of the mixture was dried for characterization, and the other part was kept wet for relaxometry evaluation. 2.2.2. trastuzumab-functionalized LDH For the Tzmb-LDH, 80 µg of the antibody were dispersed in 1.0 mL of phosphate-buffered saline (PBS) solution and mixed with 37.5 mg of the LDH [ 21 ]. The suspension was shaken for 24 h in closed vial protected from light. Subsequently, half of the suspension was dried for characterization and the other half was kept wet for relaxometry assays. 2.2.3. Trastuzumab and doxorubicin functionalized LDH To obtain LDH-Dox-Tzmb, 80 µg of antibody was dissolved in 1.0 mL of PBS. Then, 2 mL of a 4 mg/mL aqueous solution of Dox was added and mixed. Finally, 37.5 mg of LDH was added and stirred for 24 h in a closed vial protected from light. One half of the suspension was dried for characterization, and the second liquid fraction was stored for the relaxometry evaluation. 2.3 Characterization The infrared spectroscopy (FTIR) analyses were conducted with the dried samples in a Thermo Fisher Scientific instrument, model iS50 ATR, with an ATR module. The spectra were averaged after collecting 15 scans at a resolution of 4 cm-1. For X-ray diffraction (XRD) analyses, 15 mg of the ground and dried samples were placed onto a glass holder and read in a PANalytical diffractometer model EMPYREAN at a step of 0.02°, and a speed of 30 s per step. The Cu-Kα radiation was produced with 45 kV and 35 mA. The particle size in aqueous suspension and its stability were determined by dynamic light scattering (DLS) and Z-potential in Malvern ZetaSizer equipment. Samples were prepared by suspending 1 mg of the powdered samples in 1.5 ml of double distilled water and sonicated for 3 minutes before measurement. The diffraction coefficient used was 1.6 corresponding to a LDH [ 22 ]. The elemental analysis was performed by energy dispersive spectroscopy (EDS) with an OXFORD EDS detector coupled to a scanning electron microscope (SEM), model JEOL JSM-6610LV. The pulverized and perfectly dried material was placed onto a carbon tape and introduced into the vacuum chamber to be treated with an electron beam at 15 kV. The morphology was assessed using transmission electron microscopy (TEM) on a JEOL JEM1010 microscope operating at 90 kV. Solid samples were dispersed in water, and a drop of the resulting suspension was placed onto a copper grid for imaging. Relaxometry studies using magnetic resonance imaging (MRI) were performed with a Bruker PHARMASCAN 70/16US 7T system. A solution with 8.0 g of grenetine and 10 mL of hot water was prepared. This solution was used to dilute a 10,000 ppm aqueous solution of LDH to obtain concentrations of 1250, 2500, 5000, and 7500 ppm. The jelly samples were read in T 1 and T 2 modes, and relaxation times were obtained through the Bruker Paravision 6.0.1 software. 3. Results and discussion 3.1. LDH synthesis and characterization The products of the four methods were fine white powders with a talc appearance. The infrared spectra (Fig. 1 a) showed similar signals in the four products associated with typical bands in LDH, such as the broad ~ 3000 cm − 1 signal characteristic of the O-H stretching in hydroxyl ions [ 23 ]. Besides, a sharp band close to 3600 cm − 1 appears, and it is found sometimes in well-crystallized single hydroxide structures of Mg(OH) 2 or Gd(OH) 3 [ 24 ]. Signals from the asymmetric stretching of the nitrate ion are also found at 1300 and 1600 cm − 1 [ 25 ]. Finally, all the spectra contain intense signals between 700 and 500 cm − 1 , associated with vibrations of the metal-oxygen bond [ 26 ]. ~ On the other hand, the X-ray diffractograms (Fig. 1 b) have a low signal-to-noise ratio, especially for the products of methods B, C, and D, which do not exhibit clear signals indicating low or no crystallinity. The product from method A presented signals at 10.9° 22.4° 59.4°, and 61.6° 2theta, which are sharp and defined despite their low intensity. These signals correspond to the (003), (006), (110) and (113) planes in an LDH structure [ 27 ]. The low intensity of the reflection at 10.9° implies the possible delamination of LDHs particles [ 28 ], while the overall low intensity is likely related to the high deformation of medium-entropy LDHs [ 29 , 30 ]. In the case of the products from the B, C and D methods, signals are not clear, and a wide halo predominates at 10 degrees from the glass holder, representing a profile of amorphous powders. Then, the treatment of the reagents and the sequence of addition play a crucial role in obtaining a ME LDH, as the mixture of all the cations in the same solution was the only successful procedure. Once the product from method A corresponds to the desirable MgFeGdDy LDH structure, it was used for further analyses and functionalization reactions. The distribution of the cations in the powder was explored by mapping the emitted electron energy from each element [ 31 ]. The map in Fig. 2 revealed the presence and homogeneous distribution of nitrogen, oxygen, magnesium, iron, gadolinium and dysprosium, these elements are part of the same product. Regarding the map of carbon, the distribution seems irregular since the sample was thinner in some regions, and the carbon from the adhesive tape was detected. The mass percent of metals quantified by the spectrometer was 10.4% Mg, 7.4% Fe, 15.3% Gd, and 14.7% Dy. Converting these values, the molar relationship of cations in the LDH is Mg 2.0 Fe 0.6 Gd 0.3 Dy 0.4 , which is close to the theoretical composition Mg 2.0 Fe 0.5 Gd 0.25 Dy 0.25 , established for the synthesis. According to Eq. (1); the composition Mg 2.0 Fe 0.6 Gd 0.3 Dy 0.4 possesses ∆S mix = 1.09R, associated with a medium-entropy materials, including hydroxides[ 19 , 32 ] The particle size study by DLS showed two size populations (Fig. 3 a), one of 164 nm and the other of 825 nm, which are probably aggregates of the first group. Zeta potential (Fig. 3 b) showed a value of -6.0 ± 1.2 mV, suggesting a low affinity for the solvent, since a high affinity would generate values between ± 16 and ± 40 mV [ 33 ]. One of the most important data extracted from the Zeta potential is the sign, related to the charge density of the particles, which in this case is negative [ 33 ], indicating the predominant anionic charge due to the nitrate ions surrounding the layered structure. 3.2. Functionalization of MgFeGdDy with doxorubicin and trastuzumab The medium-entropy MgFeGdDY LDH was used to produce the functionalization products LDH-Dox, LDH-Tzmb, and LDH-Dox-Tzmb. In the case of LDH-Dox, a dark purple viscous fluid was obtained, LDH-Tzmb resulted in a yellowish white fluid, and LDH-Dox-Tzmb presented a dark red color. The infrared spectra of the three functionalized compounds (Fig. 4 a) exhibited signals from the O-H stretching mode at 3377 cm − 1 and nitrate signals at approximately 1352 cm − 1 . These signals are more intense in LDH-Dox and LDH-Dox-Tzmb than in LDH-Tzmb, due to the presence of OH- groups in the doxorubicin molecule. On the other hand, new signals associated with doxorubicin are observed in LDH-Dox and LDH-Dox-Tzmb, such as the stretching of C-H bonds at 2910 cm − 1 , the C = C stretching in the aromatic rings at 1642 cm − 1 , and the C-O-C stretching at 1069 cm − 1 [ 34 ]. For LDH-Tzmb the signal around 1069cm − 1 is related to stretching C = N and has C = C stretching around 1642cm − 1 less intense than LDH-Dox and LDH-Dox-Tzmb[ 35 ]. Regarding the X-ray diffractograms, the reflection profile of the LDH structure is retained, indicating that the particles remain structurally stable after functionalization (Fig. 4 b). A slight change was observed in the reflection of the (003) plane, going from 10.9° in the pristine MgFeGdDy LDH (Method A in Fig. 1 b) to 11.4° in all the functionalized products (Fig. 4 b). Converting the angles to interplanar distances with the Bragg equation [ 36 ], the d (003) is 0.81 nm in the MgFeGdDy LDH, in agreement with the presence of nitrate ions in the interlayer space [ 37 , 38 ]. After functionalization, the distances decrease to 0.78 nm; this reduction occurs probably due to partial intercalation of carbonate from the environment, as carbonate tends to approximate the layers due to its double negative charge [ 39 ]. Then the Dox molecules and Tzmb would only cover the external surface of the LDH particles and do not produce any intercalation reaction, as it occurs with anionic drugs. The integrity of the layers can be confirmed with the appearance of two reflections above 60 degrees [ 40 ], confirming the stability of the MgFeGdDy LDH structure. Another change is the relative intensity of the 10.8 degree reflection compared to 59.1 and 61.1 degree signals. This low intensity can be associated with a reduced number of hydroxylated layers per particle [ 41 ], indicating the functionalization processes promote partial delamination, which is an advantage for functionalization, as more surface area could be produced. The particle size and Zeta potential of the functionalized LDH presented in Fig. 5 reveals a size of 275.25 ± 2.19 for LDH-Dox, 9015.50 ± 289.21 for LDH-Tzmb, and 5262.00 ± 199.40 nm for LDH-Dox-Tzmb. Considering that the pristine MgFeGdDy LDH is formed by particles of 164 nm and aggregates of 825 nm, the functionalization with Dox avoids aggregation producing particles of 275 nm in LDH-DOX, whereas the LDH-Tzmb increases the size to 9015 nm, as a result of the protein aggregation, in agreement with the extremely low zeta potential value, 1.3 mV (Fig. 5 b). When the particle is simultaneously functionalized, with Dox and Tzmb, the size presents middle values of 5262.00 ± 199.40nm in LDH-Dox-Tzmb. Then, the presence of Dox avoids aggregation by increasing the zeta potential, in agreement with the bimodal profile, with a value of -23 mV being one of the observed values associated with the stability of the suspension [ 33 ]. The negative value could be associated with the deamination of amino acids in the antibody[ 42 ]. The LDH-Dox sample also exhibited a bimodal zeta potential, with the highest value at 41.7 mV, indicating an enhanced interaction between the particles and the solvent, which led to a dispersion of the particles. The positive value in LDH-DOX could be related to the complete covering of the particles by Dox molecules with a positive charge due to the protonation of the amino groups [ 43 ]. Despite the relatively low zeta potential values, these suspensions are stable for one day, and even if a precipitate is formed, it is easily resuspended by hand shaking. 3.3. Magnetic resonance imaging The contrast activity of MgFeGdDy LDH and the functionalization products was evaluated using magnetic resonance imaging, which measures the longitudinal (spin-lattice) and transverse (spin-spin) relaxation times, designated as T1 and T2, respectively. Among contrast agents, the positive contrast is related to the longitudinal relaxation time (T 1 ) and increases the brightness of the tissues; on the other hand, the negative contrast agents darken the tissues and are related to the transverse recovery time (T 2 ) [ 44 ]. Figure 6 a contains the MRI images of the products read in the T1 and T2 modes. The brightness of the LDH-Dox and LDH-Dox-Tzmb samples gradually increases as the concentration increases; this trend is confirmed by the reciprocal relaxation time versus concentration curves (Fig. 6 b), which present a positive slope in LDH-Dox (r 1 = 1.3x10 − 3 ppm − 1 s − 1 ) and LDH-Dox-Tzmb (r 1 = 7.3x10 − 4 ppm − 1 s − 1 ). When only Tzmb is assembled onto the LDH, the images of the LDH-Tzmb darken at a concentration of 5000 ppm and above (Fig. 6 a), which implies a loss of positive contrast, confirmed in Fig. 6 b with a negative slope (r 1 =-2.2x10 − 4 ppm − 1 s − 1 ). The second row in Fig. 6 a shows the activity as a negative contrast agent in the T 2 mode, the increase of the negative contrast with respect to the concentration in the three compounds is noticeable and coherent with the relaxation against concentration (Fig. 6 b) with a positive slope in LDH-Dox (r 2 = 1.7 x10 − 2 ppm − 1 s − 1 ), LDH-Dox-Tzmb (r 2 = 1.1 x10 − 2 ppm − 1 s − 1 ), and LDH-Tzmb (r 2 = 9.0 x10 − 4 ppm − 1 s − 1 ). The higher r 2 values compared to r 1 indicate that the compounds LDH-DOx, LDH-Dox-Tzmb, and LDH-Tzmb are more efficient as negative contrasts in T2 readings, which is associated with the effective demagnetization caused by Fe 3+ and Dy 3+ cations in the nanoparticles [ 8 , 10 ]. Then, despite the functionalization, the LDH particles are active, especially when the functionalization involves Dox (LDH-Dox and LDH-Dox-Tzmb) molecules, in contrast to particles where trastuzumab is used exclusively (LDH-Tzmb). The poor performance of LDH-Tzmb indicates low access of water molecules to the LDH surface, which is explained by the particles agglutination that occurs within the trastuzumab matrix as observed by TEM micrographs (Fig. 7 ). The microscopy images are consistent with the DLS size data. Doxorubicin and trastuzumab form globular structures when they are immobilized over nanoparticles[ 45 , 46 ] This morphology is observed in the micrographs collected with 25k magnification for both doxorubicin-containing LDHs. When the magnification increases to 250k, particles with well-defined edges appear, similar to the typical hexagonal morphology of LDHs [4,47]. In all of the cases, the predominant size of the dark LDH particles is below 80 nm and they agglutinate, aided by the presence of the functionalizing agent. The aggregates reach sizes of 327.13 ± 59.59 nm in LDH-Dox, 442.75 ± 93.61 nm in LDH-Dox-Tzmb, and 2123.33 ± 732.28 nm in LDH-Tzmb. The trend in the aggregate sizes is the same as that detected in the DLS measurement, where the LDH-Tzmb material has the larger size, here, the protein matrix envelopes the LDH particles, preventing water from reaching their surface and inhibiting contrast in MRI. While the ME LDH particles could be functionalized with Dox and Tzmb, their use as contrast agents in MRI is limited when functionalized exclusively with Tzmb. 4. Conclusion The method A proposed in this work enabled the obtention of an aluminum-free LDH with composition Mg 2.0 Fe 0.6 Gd 0.3 Dy 0.4 , which is close to the theoretical value. Such composition corresponds to a medium-entropy LDH (∆S mix = 1.09R) with a low crystalline lattice influenced by the presence or large ionic radii Gd(III) and Dy(III) cations. The crystal structure of these particles is stable against the functionalization of doxorubicin and trastuzumab. The particle size of the LDH was below 80 nm and forms aggregates in solution, when functionalized with Dox, the drug promotes the disaggregation; however, after functionalization with Tzmb, large protein aggregates are formed, covering the LDH particles and impeding the approximation to water molecules, thus the contrast activity in MRI assays is inhibited. Although these LDH-Tzmb particles require further studies to obtain efficient targeted contrast agents, the functionalization with Dox enabled the MRI contrast activity, generating materials with both diagnostic and therapeutic capabilities (LDH-Dox) as well as diagnostic, therapeutic, and targeting capabilities (LDH-Dox-Tzmb). Declarations Conflict of interest Authors declare no conflicts of interest in this work. Contributions N.A.G.R. experiments, data organization, data curation, draft and discussion. G.G.C.A. Fund acquisition, data curation, discussion, draft and revise the article. Acknowledgment Authors are thankful to Dr. Juan Ortiz for operating and collecting the IMR data at the "Laboratorio Nacional de Imagenología por Resonancia Magnética, LANIERM", and to the Transdisciplinary Institute for Research and Services (ITRANS)for access to the EDS/SEM service. Funding NAGR received a fellowship from "Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI)", Mexico, formerly "Consejo Nacional de Humanidades, Ciencia y Tecnología (CONAHCYT)". Funds were obtained from the Project "Ciencia de Frontera CF-2023-I-885" sponsored by the formerly "Consejo Nacional de Humanidades, Ciencias y Tecnologías, CONAHCYT", now SECIHTI, Mexico. Data availability Data is available on request Ethics statement Ethical approval is not needed for these experiments. References S. Muráth, A. Szerlauth, D. Sebők, I. 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16:39:57","extension":"html","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127878,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/d56adbb1521248c0398d0abc.html"},{"id":96654028,"identity":"cb0ccb5f-eaca-48d9-9e18-a9e0d633d52c","added_by":"auto","created_at":"2025-11-24 16:39:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":156417,"visible":true,"origin":"","legend":"\u003cp\u003ea) Infrared spectra of the products obtained from each synthesis method b) and their X-ray diffraction profiles.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/631f9d85d9eb568d6b1f81d1.png"},{"id":96654030,"identity":"d80c5047-ff0b-4017-87f4-347edff185ad","added_by":"auto","created_at":"2025-11-24 16:39:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1194216,"visible":true,"origin":"","legend":"\u003cp\u003eElemental mapping by EDS of the MgFeGdDy LDH sample.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/46bf1e557e875fdf72b4df1e.png"},{"id":96654027,"identity":"1202c46f-720a-4990-9e52-b7d37ebf90fe","added_by":"auto","created_at":"2025-11-24 16:39:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30888,"visible":true,"origin":"","legend":"\u003cp\u003ea) Particle size determined by DLS and b) zeta potential of the MgFeGdDy LDH particles synthesized by method A. The Zeta potential plot was recorded as a figure and transformed to numerical values with https://automeris.io/.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/448fa5628eb876933eb2a0a4.png"},{"id":96709467,"identity":"0afb5f57-f23e-4b4b-bad8-23374652da45","added_by":"auto","created_at":"2025-11-25 10:09:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":173353,"visible":true,"origin":"","legend":"\u003cp\u003ea) Infrared spectra of the MgFeGdDy LDH functionalized with doxorubicin and trastuzumab b) their X-ray diffraction profiles.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/80630b3d19a2e58deafa3d03.png"},{"id":96709635,"identity":"09a28d48-0c1d-42b8-be56-f891ffeb008f","added_by":"auto","created_at":"2025-11-25 10:09:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":42157,"visible":true,"origin":"","legend":"\u003cp\u003ea) DLS particle size and b) Zeta potential of the functionalized products LDH-Dox, LDH-Tzmb and LDH-Dox-Tzmb. The Zeta potential plot was recorded as a figure and transformed to numerical values with https://automeris.io/.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/e4232d49cab26ec959abf747.png"},{"id":96654035,"identity":"1b02ae4d-7f42-4a8a-a2e9-a0ac67c61362","added_by":"auto","created_at":"2025-11-24 16:39:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":319852,"visible":true,"origin":"","legend":"\u003cp\u003ea) MRI images of the MgFeGdDy LDH functionalized products obtained from the T1 and T2 modes (B: Blank; N1:1250 ppm; N2:2500 ppm; N3:5000 ppm; N4:7500 ppm; N5:10000 ppm). b) and the reciprocal relaxivity time as a function of the concentration.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/65144ae25c6c227026bfb5d8.png"},{"id":96709532,"identity":"83ea9374-e7bb-4abf-973e-48bdcd228d45","added_by":"auto","created_at":"2025-11-25 10:09:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":758721,"visible":true,"origin":"","legend":"\u003cp\u003eTEM micrographs for LDH-Dox, LDH-Tzmb and LDH-Dox-Tzmb.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/42e154bbd9bad949889d039c.png"},{"id":103765489,"identity":"d685e857-32b9-4e91-9f58-62158afcc575","added_by":"auto","created_at":"2026-03-02 16:03:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3327091,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/b51f33b4-e298-4a00-b614-96ae2ae35b7e.pdf"},{"id":96654032,"identity":"f449d438-0d81-4f27-a30b-cbce84821af0","added_by":"auto","created_at":"2025-11-24 16:39:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1009807,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7633707/v1/87791cf727274cdd66a4a4a6.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Functionalization with doxorubicin and trastuzumab of medium-entropy MgFeGdDy layered double hydroxides with magnetic resonance imaging contrast activity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cspan fontcategory=\"NonProportional\" class=\"\" name=\"Emphasis\"\u003ePlease have a look at courier new font provided for text in article.\u003c/span\u003e\u003c/p\u003e\u003cp\u003eLayered double hydroxides (LHD) are compounds of interest in materials science due to their anion exchange capacity, catalytic capacity, and applications as drug carriers, among others [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In their most basic composition, the structures are formed by a combination of M(II)/M(III) metal cations [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. For biomedical purposes, the M(II) cations are usually magnesium and zinc, and the M(III) cation is Al\u003csup\u003e3+\u003c/sup\u003e. The most widely accepted rule is that LDH structures are formed when M(II) and M(III) cations meet an ionic radius between 0.67 and 0.93 \u0026Aring;, since the octahedral structure is usually stabilized due to Paulli's first law between these dimensions [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Complying with these dimensions, paramagnetic cations can be selected to present spontaneous ferromagnetic couplings [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]and produce LDH-based contrast agents (CA) in magnetic resonance imaging (MRI), such as manganese-doped LDHs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Currently, commercial CAs contain gadolinium because of its high magnetic moment related to contrast effectiveness; however, the commercial CAs are formulated with gadolinium complexes, which could present some adverse effects due to long-term toxicity, the need for high doses, and particle agglomeration, among others [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Such disadvantages can be mitigated by incorporating gadolinium cations into an LDH lattice. Another lanthanide that exhibits this activity, albeit to a lesser extent, is Dysprosium (Dy). It offers the additional advantage of being chemically inert and can enhance its properties when is in oxide [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Both elements are of interest in MRI, as they may possess the ability to demagnetize protons in water molecules at two types of spin relaxation in MRI, thereby generating a contrast effect; Gd\u003csup\u003e3+\u003c/sup\u003e at longitudinal relaxation (T\u003csub\u003e1\u003c/sub\u003e) and Dy\u003csup\u003e3+\u003c/sup\u003e at transversal relaxation (T\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Although the large atomic radius of paramagnetic cations such as Gd\u003csup\u003e3+\u003c/sup\u003e (1.03 \u0026Aring;), or Dy\u003csup\u003e3+\u003c/sup\u003e (0.99 \u0026Aring;), would not form a stable LDH structures, in recent years, several researchers have successfully incorporated these cations in MgAl [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]and ZnAl LDHs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]and contrast activity was obtained in MRI assays. On the other hand, while nanoparticles containing aluminum have been shown to be toxic to human cells and rodents [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], this element could be associated with the appearance of neurodegenerative diseases in humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], then it would be desirable to remove it from the LDH structures. Therefore, this work proposes to replace Al(III) with Fe(III) cations, leading to a combination of a biocompatible LDH lattice Mg-Fe dopped with two lanthanides Gd\u003csup\u003e3+\u003c/sup\u003e and Dy\u003csup\u003e3+\u003c/sup\u003e with T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e MRI activity cations that would involve the formation of a medium-entropy structure [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], where the presence of iron could form poorly crystalline LDH particles. Medium-entropy structures consist of a solid phase composed of three or four metals and have a value of ∆S\u003csub\u003emix\u003c/sub\u003e between 0.69R and 1.61R according to the equation:\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varDelta\\:{S}_{mix}=-R{\\sum\\:}_{i}^{n}{c}_{i}\\bullet\\:\\text{ln}{c}_{i}\\)\u003c/span\u003e\u003c/span\u003e (Eq.\u0026nbsp;1)\u003c/p\u003e\u003cp\u003ewhere c\u003csub\u003ei\u003c/sub\u003e is the atomic percentage of the i-th cation and R is the gas constant [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this work, we explored different synthesis processes combining sequences of addition and treatments of reagents to obtain a medium-entropy MgFeGdDy LDH structure capable of being functionalized with doxorubicin and trastuzumab, which are therapeutic and targeting agents, respectively, desirable in theragnostic assays. The MRI contrast activity of the medium-entropy LDHs and the functionalization derivatives was also evaluated.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Synthesis\u003c/h2\u003e\u003cp\u003eThe reagents used in this experiment were Mg(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO (98.0-102.0%, Golden Bell), Fe(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026bull;9H\u003csub\u003e2\u003c/sub\u003eO (\u0026ge;\u0026thinsp;98%, Sigma-Aldrich), Gd(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026bull;6H\u003csub\u003e2\u003c/sub\u003eO (99.99%-Sigma-Aldrich), Dy(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e3\u003c/sub\u003e\u0026bull;xH\u003csub\u003e2\u003c/sub\u003eO (99.9%, Sigma-Aldrich), NaOH (\u0026ge;\u0026thinsp;98%, JT Baker), doxorubicin (\u0026ge;\u0026thinsp;90-\u0026le;100%, Sigma-Aldrich), trastuzumab (\u0026ge;\u0026thinsp;90%, Sigma-Aldrich), and phosphate buffer solution (pH 7.2\u0026ndash;7.6, Sigma-Aldrich).\u003c/p\u003e\u003cp\u003eTo obtain the LDH with a nominal composition Mg\u003csub\u003e2.0\u003c/sub\u003eFe\u003csub\u003e0.5\u003c/sub\u003eGd\u003csub\u003e0.25\u003c/sub\u003eDy\u003csub\u003e0.25\u003c/sub\u003e, four methods were designed combining treatments of reagents and sequences of addition as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and detailed below:\u003c/p\u003e\u003cp\u003eMethod A\u003c/p\u003e\u003cp\u003eThe cation salts were added in the same flask with 50 mL of water, the solution was sonicated for 3 minutes and a 1M NaOH was added quickly until pH 13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 was reached, the suspension was shaken for 20 minutes and then left to age for 72 hours in a closed container isolated from light.\u003c/p\u003e\u003cp\u003eMethod B\u003c/p\u003e\u003cp\u003eSeparate solutions of each salt were prepared. Each solution was sonicated for 3 minutes and then mixed abruptly with a 1 M NaOH solution in a vessel to obtain a suspension with a pH of 13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5. The mixture was agitated for 18 hours and aged in a closed container away from light for 24 hours.\u003c/p\u003e\u003cp\u003eMethod C\u003c/p\u003e\u003cp\u003eA solution with gadolinium, magnesium, and dysprosium salts was prepared separately from the iron salt solution. These two solutions were mixed with a 1M NaOH solution at the same time to reach a pH 13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5. The suspension was stirred for 18 hours and aged for 24 hours.\u003c/p\u003e\u003cp\u003eMethod D\u003c/p\u003e\u003cp\u003eThe salts were added at the same time in a mortar, and 15 mL of NaOH 1 M was added. The mixture was macerated for 20 minutes and aged for 24 hours in a closed container isolated from light.\u003c/p\u003e\u003cp\u003eIn all the methods, after the aging period, the suspensions were centrifuged, discarding the supernatant and washing with deionized water until the pH of the liquid phase was 7. The solids were dried at 25\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eConditions of the synthesis methods to obtain a MgFeGdDy LDH structure.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMethod\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAddition of M(II) and M(III) salts\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUltrasound\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFinal pH\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAgitation after alkalinization\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAging (h)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAll the cations in the same solution\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOne solution per cation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOne solution with Mg, Gd, and Dy. A second solution with Fe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e18 h\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAll the cations mixed in a mortar\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNo\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Functionalization with doxorubicin and trastuzumab\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. Doxorubicin-functionalized LDH\u003c/h2\u003e\u003cp\u003eLDH-Dox was prepared by dissolving 12.5 mg of doxorubicin in 1 mL of double distilled water, once the doxorubicin was dissolved, 37.5 mg of previously dried and pulverized LDH was added [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The mixture was kept under constant agitation for 24 hours. Subsequently, half of the mixture was dried for characterization, and the other part was kept wet for relaxometry evaluation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2. trastuzumab-functionalized LDH\u003c/h2\u003e\u003cp\u003eFor the Tzmb-LDH, 80 \u0026micro;g of the antibody were dispersed in 1.0 mL of phosphate-buffered saline (PBS) solution and mixed with 37.5 mg of the LDH [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The suspension was shaken for 24 h in closed vial protected from light. Subsequently, half of the suspension was dried for characterization and the other half was kept wet for relaxometry assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3. Trastuzumab and doxorubicin functionalized LDH\u003c/h2\u003e\u003cp\u003eTo obtain LDH-Dox-Tzmb, 80 \u0026micro;g of antibody was dissolved in 1.0 mL of PBS. Then, 2 mL of a 4 mg/mL aqueous solution of Dox was added and mixed. Finally, 37.5 mg of LDH was added and stirred for 24 h in a closed vial protected from light. One half of the suspension was dried for characterization, and the second liquid fraction was stored for the relaxometry evaluation.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Characterization\u003c/h2\u003e\u003cp\u003eThe infrared spectroscopy (FTIR) analyses were conducted with the dried samples in a Thermo Fisher Scientific instrument, model iS50 ATR, with an ATR module. The spectra were averaged after collecting 15 scans at a resolution of 4 cm-1. For X-ray diffraction (XRD) analyses, 15 mg of the ground and dried samples were placed onto a glass holder and read in a PANalytical diffractometer model EMPYREAN at a step of 0.02\u0026deg;, and a speed of 30 s per step. The Cu-Kα radiation was produced with 45 kV and 35 mA. The particle size in aqueous suspension and its stability were determined by dynamic light scattering (DLS) and Z-potential in Malvern ZetaSizer equipment. Samples were prepared by suspending 1 mg of the powdered samples in 1.5 ml of double distilled water and sonicated for 3 minutes before measurement. The diffraction coefficient used was 1.6 corresponding to a LDH [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The elemental analysis was performed by energy dispersive spectroscopy (EDS) with an OXFORD EDS detector coupled to a scanning electron microscope (SEM), model JEOL JSM-6610LV. The pulverized and perfectly dried material was placed onto a carbon tape and introduced into the vacuum chamber to be treated with an electron beam at 15 kV. The morphology was assessed using transmission electron microscopy (TEM) on a JEOL JEM1010 microscope operating at 90 kV. Solid samples were dispersed in water, and a drop of the resulting suspension was placed onto a copper grid for imaging.\u003c/p\u003e\u003cp\u003eRelaxometry studies using magnetic resonance imaging (MRI) were performed with a Bruker PHARMASCAN 70/16US 7T system. A solution with 8.0 g of grenetine and 10 mL of hot water was prepared. This solution was used to dilute a 10,000 ppm aqueous solution of LDH to obtain concentrations of 1250, 2500, 5000, and 7500 ppm. The jelly samples were read in T\u003csub\u003e1\u003c/sub\u003e and T\u003csub\u003e2\u003c/sub\u003e modes, and relaxation times were obtained through the Bruker Paravision 6.0.1 software.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1. LDH synthesis and characterization\u003c/h2\u003e\u003cp\u003eThe products of the four methods were fine white powders with a talc appearance. The infrared spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) showed similar signals in the four products associated with typical bands in LDH, such as the broad\u0026thinsp;~\u0026thinsp;3000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e signal characteristic of the O-H stretching in hydroxyl ions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Besides, a sharp band close to 3600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e appears, and it is found sometimes in well-crystallized single hydroxide structures of Mg(OH)\u003csub\u003e2\u003c/sub\u003e or Gd(OH)\u003csub\u003e3\u003c/sub\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Signals from the asymmetric stretching of the nitrate ion are also found at 1300 and 1600 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Finally, all the spectra contain intense signals between 700 and 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, associated with vibrations of the metal-oxygen bond [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. ~\u003c/p\u003e\u003cp\u003eOn the other hand, the X-ray diffractograms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) have a low signal-to-noise ratio, especially for the products of methods B, C, and D, which do not exhibit clear signals indicating low or no crystallinity. The product from method A presented signals at 10.9\u0026deg; 22.4\u0026deg; 59.4\u0026deg;, and 61.6\u0026deg; 2theta, which are sharp and defined despite their low intensity. These signals correspond to the (003), (006), (110) and (113) planes in an LDH structure [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The low intensity of the reflection at 10.9\u0026deg; implies the possible delamination of LDHs particles [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], while the overall low intensity is likely related to the high deformation of medium-entropy LDHs [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In the case of the products from the B, C and D methods, signals are not clear, and a wide halo predominates at 10 degrees from the glass holder, representing a profile of amorphous powders. Then, the treatment of the reagents and the sequence of addition play a crucial role in obtaining a ME LDH, as the mixture of all the cations in the same solution was the only successful procedure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eOnce the product from method A corresponds to the desirable MgFeGdDy LDH structure, it was used for further analyses and functionalization reactions.\u003c/p\u003e\u003cp\u003eThe distribution of the cations in the powder was explored by mapping the emitted electron energy from each element [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The map in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e revealed the presence and homogeneous distribution of nitrogen, oxygen, magnesium, iron, gadolinium and dysprosium, these elements are part of the same product. Regarding the map of carbon, the distribution seems irregular since the sample was thinner in some regions, and the carbon from the adhesive tape was detected.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe mass percent of metals quantified by the spectrometer was 10.4% Mg, 7.4% Fe, 15.3% Gd, and 14.7% Dy. Converting these values, the molar relationship of cations in the LDH is Mg\u003csub\u003e2.0\u003c/sub\u003eFe\u003csub\u003e0.6\u003c/sub\u003eGd\u003csub\u003e0.3\u003c/sub\u003eDy\u003csub\u003e0.4\u003c/sub\u003e, which is close to the theoretical composition Mg\u003csub\u003e2.0\u003c/sub\u003eFe\u003csub\u003e0.5\u003c/sub\u003eGd\u003csub\u003e0.25\u003c/sub\u003eDy\u003csub\u003e0.25\u003c/sub\u003e, established for the synthesis. According to Eq.\u0026nbsp;(1); the composition Mg\u003csub\u003e2.0\u003c/sub\u003eFe\u003csub\u003e0.6\u003c/sub\u003eGd\u003csub\u003e0.3\u003c/sub\u003eDy\u003csub\u003e0.4\u003c/sub\u003e possesses ∆S\u003csub\u003emix\u003c/sub\u003e= 1.09R, associated with a medium-entropy materials, including hydroxides[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eThe particle size study by DLS showed two size populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), one of 164 nm and the other of 825 nm, which are probably aggregates of the first group. Zeta potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb) showed a value of -6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 mV, suggesting a low affinity for the solvent, since a high affinity would generate values between \u0026plusmn;\u0026thinsp;16 and \u0026plusmn;\u0026thinsp;40 mV [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. One of the most important data extracted from the Zeta potential is the sign, related to the charge density of the particles, which in this case is negative [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], indicating the predominant anionic charge due to the nitrate ions surrounding the layered structure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Functionalization of MgFeGdDy with doxorubicin and trastuzumab\u003c/h2\u003e\u003cp\u003eThe medium-entropy MgFeGdDY LDH was used to produce the functionalization products LDH-Dox, LDH-Tzmb, and LDH-Dox-Tzmb. In the case of LDH-Dox, a dark purple viscous fluid was obtained, LDH-Tzmb resulted in a yellowish white fluid, and LDH-Dox-Tzmb presented a dark red color.\u003c/p\u003e\u003cp\u003eThe infrared spectra of the three functionalized compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) exhibited signals from the O-H stretching mode at 3377 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and nitrate signals at approximately 1352 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These signals are more intense in LDH-Dox and LDH-Dox-Tzmb than in LDH-Tzmb, due to the presence of OH- groups in the doxorubicin molecule.\u003c/p\u003e\u003cp\u003eOn the other hand, new signals associated with doxorubicin are observed in LDH-Dox and LDH-Dox-Tzmb, such as the stretching of C-H bonds at 2910 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, the C\u0026thinsp;=\u0026thinsp;C stretching in the aromatic rings at 1642 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and the C-O-C stretching at 1069 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For LDH-Tzmb the signal around 1069cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is related to stretching C\u0026thinsp;=\u0026thinsp;N and has C\u0026thinsp;=\u0026thinsp;C stretching around 1642cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e less intense than LDH-Dox and LDH-Dox-Tzmb[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRegarding the X-ray diffractograms, the reflection profile of the LDH structure is retained, indicating that the particles remain structurally stable after functionalization (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). A slight change was observed in the reflection of the (003) plane, going from 10.9\u0026deg; in the pristine MgFeGdDy LDH (Method A in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) to 11.4\u0026deg; in all the functionalized products (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Converting the angles to interplanar distances with the Bragg equation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], the d\u003csub\u003e(003)\u003c/sub\u003e is 0.81 nm in the MgFeGdDy LDH, in agreement with the presence of nitrate ions in the interlayer space [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. After functionalization, the distances decrease to 0.78 nm; this reduction occurs probably due to partial intercalation of carbonate from the environment, as carbonate tends to approximate the layers due to its double negative charge [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Then the Dox molecules and Tzmb would only cover the external surface of the LDH particles and do not produce any intercalation reaction, as it occurs with anionic drugs. The integrity of the layers can be confirmed with the appearance of two reflections above 60 degrees [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], confirming the stability of the MgFeGdDy LDH structure.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnother change is the relative intensity of the 10.8 degree reflection compared to 59.1 and 61.1 degree signals. This low intensity can be associated with a reduced number of hydroxylated layers per particle [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], indicating the functionalization processes promote partial delamination, which is an advantage for functionalization, as more surface area could be produced.\u003c/p\u003e\u003cp\u003eThe particle size and Zeta potential of the functionalized LDH presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e reveals a size of 275.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.19 for LDH-Dox, 9015.50\u0026thinsp;\u0026plusmn;\u0026thinsp;289.21 for LDH-Tzmb, and 5262.00\u0026thinsp;\u0026plusmn;\u0026thinsp;199.40 nm for LDH-Dox-Tzmb. Considering that the pristine MgFeGdDy LDH is formed by particles of 164 nm and aggregates of 825 nm, the functionalization with Dox avoids aggregation producing particles of 275 nm in LDH-DOX, whereas the LDH-Tzmb increases the size to 9015 nm, as a result of the protein aggregation, in agreement with the extremely low zeta potential value, 1.3 mV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). When the particle is simultaneously functionalized, with Dox and Tzmb, the size presents middle values of 5262.00\u0026thinsp;\u0026plusmn;\u0026thinsp;199.40nm in LDH-Dox-Tzmb. Then, the presence of Dox avoids aggregation by increasing the zeta potential, in agreement with the bimodal profile, with a value of -23 mV being one of the observed values associated with the stability of the suspension [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The negative value could be associated with the deamination of amino acids in the antibody[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe LDH-Dox sample also exhibited a bimodal zeta potential, with the highest value at 41.7 mV, indicating an enhanced interaction between the particles and the solvent, which led to a dispersion of the particles. The positive value in LDH-DOX could be related to the complete covering of the particles by Dox molecules with a positive charge due to the protonation of the amino groups [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Despite the relatively low zeta potential values, these suspensions are stable for one day, and even if a precipitate is formed, it is easily resuspended by hand shaking.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Magnetic resonance imaging\u003c/h2\u003e\u003cp\u003eThe contrast activity of MgFeGdDy LDH and the functionalization products was evaluated using magnetic resonance imaging, which measures the longitudinal (spin-lattice) and transverse (spin-spin) relaxation times, designated as T1 and T2, respectively. Among contrast agents, the positive contrast is related to the longitudinal relaxation time (T\u003csub\u003e1\u003c/sub\u003e) and increases the brightness of the tissues; on the other hand, the negative contrast agents darken the tissues and are related to the transverse recovery time (T\u003csub\u003e2\u003c/sub\u003e) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea contains the MRI images of the products read in the T1 and T2 modes. The brightness of the LDH-Dox and LDH-Dox-Tzmb samples gradually increases as the concentration increases; this trend is confirmed by the reciprocal relaxation time versus concentration curves (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), which present a positive slope in LDH-Dox (r\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.3x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e ppm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) and LDH-Dox-Tzmb (r\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.3x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e ppm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). When only Tzmb is assembled onto the LDH, the images of the LDH-Tzmb darken at a concentration of 5000 ppm and above (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), which implies a loss of positive contrast, confirmed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb with a negative slope (r\u003csub\u003e1\u003c/sub\u003e =-2.2x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e ppm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe second row in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea shows the activity as a negative contrast agent in the T\u003csub\u003e2\u003c/sub\u003e mode, the increase of the negative contrast with respect to the concentration in the three compounds is noticeable and coherent with the relaxation against concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) with a positive slope in LDH-Dox (r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.7 x10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e ppm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), LDH-Dox-Tzmb (r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.1 x10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e ppm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), and LDH-Tzmb (r\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.0 x10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e ppm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The higher r\u003csub\u003e2\u003c/sub\u003e values compared to r\u003csub\u003e1\u003c/sub\u003e indicate that the compounds LDH-DOx, LDH-Dox-Tzmb, and LDH-Tzmb are more efficient as negative contrasts in T2 readings, which is associated with the effective demagnetization caused by Fe\u003csup\u003e3+\u003c/sup\u003e and Dy\u003csup\u003e3+\u003c/sup\u003e cations in the nanoparticles [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Then, despite the functionalization, the LDH particles are active, especially when the functionalization involves Dox (LDH-Dox and LDH-Dox-Tzmb) molecules, in contrast to particles where trastuzumab is used exclusively (LDH-Tzmb). The poor performance of LDH-Tzmb indicates low access of water molecules to the LDH surface, which is explained by the particles agglutination that occurs within the trastuzumab matrix as observed by TEM micrographs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe microscopy images are consistent with the DLS size data. Doxorubicin and trastuzumab form globular structures when they are immobilized over nanoparticles[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] This morphology is observed in the micrographs collected with 25k magnification for both doxorubicin-containing LDHs. When the magnification increases to 250k, particles with well-defined edges appear, similar to the typical hexagonal morphology of LDHs [4,47]. In all of the cases, the predominant size of the dark LDH particles is below 80 nm and they agglutinate, aided by the presence of the functionalizing agent. The aggregates reach sizes of 327.13\u0026thinsp;\u0026plusmn;\u0026thinsp;59.59 nm in LDH-Dox, 442.75\u0026thinsp;\u0026plusmn;\u0026thinsp;93.61 nm in LDH-Dox-Tzmb, and 2123.33\u0026thinsp;\u0026plusmn;\u0026thinsp;732.28 nm in LDH-Tzmb. The trend in the aggregate sizes is the same as that detected in the DLS measurement, where the LDH-Tzmb material has the larger size, here, the protein matrix envelopes the LDH particles, preventing water from reaching their surface and inhibiting contrast in MRI. While the ME LDH particles could be functionalized with Dox and Tzmb, their use as contrast agents in MRI is limited when functionalized exclusively with Tzmb.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe method A proposed in this work enabled the obtention of an aluminum-free LDH with composition Mg\u003csub\u003e2.0\u003c/sub\u003eFe\u003csub\u003e0.6\u003c/sub\u003eGd\u003csub\u003e0.3\u003c/sub\u003eDy\u003csub\u003e0.4\u003c/sub\u003e, which is close to the theoretical value. Such composition corresponds to a medium-entropy LDH (∆S\u003csub\u003emix\u003c/sub\u003e = 1.09R) with a low crystalline lattice influenced by the presence or large ionic radii Gd(III) and Dy(III) cations. The crystal structure of these particles is stable against the functionalization of doxorubicin and trastuzumab. The particle size of the LDH was below 80 nm and forms aggregates in solution, when functionalized with Dox, the drug promotes the disaggregation; however, after functionalization with Tzmb, large protein aggregates are formed, covering the LDH particles and impeding the approximation to water molecules, thus the contrast activity in MRI assays is inhibited. Although these LDH-Tzmb particles require further studies to obtain efficient targeted contrast agents, the functionalization with Dox enabled the MRI contrast activity, generating materials with both diagnostic and therapeutic capabilities (LDH-Dox) as well as diagnostic, therapeutic, and targeting capabilities (LDH-Dox-Tzmb).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflicts of interest in this work. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eN.A.G.R. experiments, data organization, data curation, draft and discussion. G.G.C.A. Fund acquisition, data curation, discussion, draft and revise the article.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors are thankful to Dr. Juan Ortiz for operating and collecting the IMR data at the \u0026quot;Laboratorio Nacional de Imagenolog\u0026iacute;a por Resonancia Magn\u0026eacute;tica, LANIERM\u0026quot;, and to the Transdisciplinary Institute for Research and Services (ITRANS)for access to the EDS/SEM service.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNAGR received a fellowship from \u0026quot;Secretar\u0026iacute;a de Ciencia, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (SECIHTI)\u0026quot;, Mexico, formerly \u0026quot;Consejo Nacional de Humanidades, Ciencia y Tecnolog\u0026iacute;a (CONAHCYT)\u0026quot;. Funds were obtained from the Project \u0026quot;Ciencia de Frontera CF-2023-I-885\u0026quot; sponsored by the formerly \u0026quot;Consejo Nacional de Humanidades, Ciencias y Tecnolog\u0026iacute;as, CONAHCYT\u0026quot;, now SECIHTI, Mexico. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available on request\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval is not needed for these experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eS. Mur\u0026aacute;th, A. Szerlauth, D. Sebők, I. Szil\u0026aacute;gyi, Layered double hydroxide nanoparticles to overcome the hydrophobicity of ellagic acid: An antioxidant hybrid material, Antioxidants 9 (2020). https://doi.org/10.3390/antiox9020153.\u003c/li\u003e\n\u003cli\u003eS. Tang, Y. Yao, T. Chen, D. Kong, W. Shen, H.K. 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Li, Layered double hydroxide membrane with high hydroxide conductivity and ion selectivity for energy storage device, Nat Commun 12 (2021) 3409. https://doi.org/10.1038/s41467-021-23721-9.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Layered double hydroxide, Lanthanides, Doxorubicin, Trastuzumab, Functionalization, MRI","lastPublishedDoi":"10.21203/rs.3.rs-7633707/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7633707/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLayered double hydroxides (LDH) with the MgAl and ZnAl cation combination are well-known as drug-delivery vehicles. Aluminum provides structural stability, enabling the intercalation and functionalization of materials to produce multifunctional materials for biomedical applications. However, aluminum is not desirable as it is a possible promoter of neurodegenerative diseases. In this work, we explore some procedures to obtain aluminum-free LDHs composed of MgFe, additionally including Gd(III) and Dy (III) cations. The resulting medium-entropy material was further functionalized with doxorubicin (Dox), a chemotherapeutic drug, and trastuzumab (Tzmb), an antibody used to confer selectivity to breast cancer cells. The stability of the medium-entropy structure was assessed against the functionalization processes by X-ray diffraction (XRD), infrared spectroscopy (FTIR), and the Z-potential technique. Finally, the functionalized particles were active as magnetic resonance imaging (MRI) contrast agents, proving the potential use of the MgFeGdDy LDH as theragnostic particles.\u003c/p\u003e","manuscriptTitle":"Functionalization with doxorubicin and trastuzumab of medium-entropy MgFeGdDy layered double hydroxides with magnetic resonance imaging contrast activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 16:39:52","doi":"10.21203/rs.3.rs-7633707/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-15T20:13:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T01:28:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T11:31:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102348405277308835570146032519336708898","date":"2025-11-15T12:11:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T04:23:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232235175449404521640903500089030502716","date":"2025-11-13T03:53:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246921296172286439642538873419503395166","date":"2025-11-13T01:22:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T15:18:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-20T20:00:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-17T00:23:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Nanoparticle Research","date":"2025-09-16T20:29:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-nanoparticle-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nano","sideBox":"Learn more about [Journal of Nanoparticle Research](http://link.springer.com/journal/11051)","snPcode":"11051","submissionUrl":"https://submission.nature.com/new-submission/11051/3","title":"Journal of Nanoparticle Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3d76546a-e9d6-4237-aebe-170c09217ff6","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:01:08+00:00","versionOfRecord":{"articleIdentity":"rs-7633707","link":"https://doi.org/10.1007/s11051-026-06584-3","journal":{"identity":"journal-of-nanoparticle-research","isVorOnly":false,"title":"Journal of Nanoparticle Research"},"publishedOn":"2026-02-24 15:57:19","publishedOnDateReadable":"February 24th, 2026"},"versionCreatedAt":"2025-11-24 16:39:52","video":"","vorDoi":"10.1007/s11051-026-06584-3","vorDoiUrl":"https://doi.org/10.1007/s11051-026-06584-3","workflowStages":[]},"version":"v1","identity":"rs-7633707","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7633707","identity":"rs-7633707","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}