Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE18 Mar 2022Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain Fang Zhu, Jian Zhong, Junfei Hu, Peng Yang, Jianhua Zhang, Minghua Zhang, Yiwen Li and Zhipeng Gu Fang Zhu College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 , Jian Zhong State Key Laboratory of Biotherapy and Cancer, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041 , Junfei Hu College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 , Peng Yang College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 , Jianhua Zhang College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 , Minghua Zhang College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 , Yiwen Li *Corresponding authors: E-mail Address: [email protected]; E-mail Address: [email protected] College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 and Zhipeng Gu *Corresponding authors: E-mail Address: [email protected]; E-mail Address: [email protected] College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan 610065 https://doi.org/10.31635/ccschem.022.202101696 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Although considerable progress has been achieved in treating iron-overload diseases with deferoxamine (DFO)-based biomaterials, high DFO loading and multifunctional integration in one system are still grand challenges. Herein, a series of carrier-free, high DFO-loading (∼80%), uniform spherical nanoparticles (NPs) assisted by polyphenols have been facilely developed with both efficient iron and reactive oxygen species-scavenging properties. Interestingly, those DFO-based NPs have demonstrated excellent scavenging performance in iron-overloaded cell model and energetically exhibited brain cell protection in vivo in intracerebral hemorrhage animal models. This study could provide a promising strategy to significantly improve the curative effect of DFO delivery systems for iron-overload diseases. Download figure Download PowerPoint Introduction Deferoxamine (DFO), the first U.S. Food and Drug Administration-approved clinical iron scavenger, has been widely incorporated into the treatment of iron-overload diseases (e.g., intracerebral hemorrhage (ICH), thalassemia transfusion, and hereditary hemochromatosis), which has demonstrated fairly good therapeutic outcomes due to its robust iron-binding efficacy. During the past decades, its awkward pharmacokinetic behavior has been the main limitation of DFO (plasma half-life only ∼20 min). To effectively improve the therapeutic effects of DFO, many strategies, including using DFO-loaded nanocarriers,1–3 surface-active agent protection, or conjugating DFO with polymers,4–6 have been designed to ameliorate the toxic dose concentration, circulation time, and bioavailability. However, the DFO content in these systems still has much room for improvement after the introduction of extra nontherapeutic materials.7,8 Besides, iron-overload diseases commonly not only bring iron cytotoxicity to tissues but also endow damage to lipids, nucleic acids, and proteins after iron-overloading involving free radical high expression.9 For instance, ICH exerts serious destruction on the brain through complicated, cascade-related pathological processes, among which overload irons and accompanying reactive oxygen species (ROS) are two significant factors.10 Cells surrounding the diseased region would release more metal ions (such as Fe2+ and Cu2+), which continually increase the severity of oxidative stress, and a vicious cycle inevitably occurs.11 Therefore, the development of a high DFO-loading system such as a carrier-free system with an effective, self-therapeutic function is urgently needed.12–15 Polyphenols are a kind of fascinating carrier materials and have been efficiently applied to the delivery of proteins, nucleic acids, and drugs.16 In view of their superior benefits, they can serve as structural motifs and/or functional motifs in various biomaterials.17–20 Specifically, the catechol and pyrogallol moieties in the polyphenols can be chemically crosslinked by oxidation, and the oxidized quinone can also be designed to react with nucleophilic groups such as –NH2 and –SH.21 Besides, polyphenols can also form hydrogen bonding, electrostatic interactions, catechol-metal ion coordination, π–π electron interactions, and cation-π interactions with polyphenols and/or other molecules.22 It is easy to integrate therapeutic reagents with polyphenols to build a considerable variety of biomaterials benefitting from these abundant structural characteristics.23–25 Moreover, polyphenols featuring antibacterial, anti-inflammatory properties, especially with the excellent ROS-scavenging capacity that has been widely utilized to endow the designed biomaterials with multifunctionality.16,26,27 Based on such considerations, we propose the design and screening of the perfect polyphenol to construct a carrier-free polyphenol-DFO nanoplatform, which capable of tackling the iron-overload and oxidative stress issues to overwhelm therapeutic outcomes in iron-overload diseases. Herein, we have facilely designed a series of high DFO-loading and multifunctional integration polyphenol-DFO nanoparticles (NPs), featuring superior iron- and ROS-scavenging properties via the carrier-free construction concept (Figure 1a). In these systems, the loading of DFO is at least up to 78.5%, in which polyphenols not only restrict the fast metabolic clearance of DFO but also supplement the DFO's additional excellent ROS-scavenging function. DFO and polyphenols, as the significant functional ingredients of the platform, also facilitate the formation of the self-assemblies in the role of structural moieties without introducing extra excipients and without any therapeutic effects at all. Then, these carrier-free DFO-based NPs were evaluated for their structure–activity relationship and iron scavenging and ROS scavenging capacities. In this strategy, the NPs were demonstrated to energetically exhibit brain cell protection in vivo in the ICH animal model. Through systematic investigation, our study will provide prospects for the design of more efficient DFO-based NPs for the treatment of iron-overload diseases, such as tumors,28–31 bacterial infections,32 and wounds.33,34 Figure 1 | (a) Schematic illustration of construction concept of polyphenol-DFO NP. (b) 1H NMR spectra of DFO conjugations in DMSO-d6 only treated with centrifugal washing operation (left) as well as size, structure, and DFO-loading results of these DFO conjugations (right). (c) TEM photographs and DLS data of Mi (i = 1–6) NPs. (Scale bar: 200 nm). (d) 1H–1H NOESY spectra of M6 in D2O. Download figure Download PowerPoint Experimental Methods Synthesis of DFO conjugates 1 mmol benzoic acid, 1 mmol DFO·CH3SO3H, 1 mmol 1-hydroxybenzotriazole monohydrate (HOBt·H2O), 1.1 mmol N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), 0.08 mmol Et3N were dissolved in 50 mL dimethylformamide (DMF) under stirring. 12 h later, the DMF was removed by rotary evaporator, and the obtained product was dispersed in distilled water with the aid of ultrasound. The precipitate was collected after centrifugation, which purification method was utilized to remove unreacted reactants and by-products. Then, the washing product containing a small amount of water was lyophilized for 24 h to obtain light powder; namely benzoic acid-DFO (M1, yield > 90%). 4-Hydroxybenzoic acid-DFO (M2), 3,4-dihydroxybenzoic acid-DFO (M3), 3,5-dihydroxybenzoic acid-DFO (M4), 2,5-dihydroxybenzoic acid-DFO (M5), and gallic acid-DFO (M6) were synthesized in a similar way (yield > 90%). Electrospray ionization mass spectrometry (ESI-MS) (LCMS-2020, Shimadzu, Japan) and nuclear magnetic resonance (NMR) (AV III HD 400 MHz, Bruker, Germany) were used to confirm the successful synthesis of DFO conjugates. Self-assembly of DFO conjugates M1 solution in dimethyl sulfoxide (DMSO) (5 mg/mL) was added to reverse osmosis (RO) water dropwise at 25 °C under violent agitation following dialysis in 4 °C RO water for 72 h. Then M1 NPs were obtained after being concentrated with a rotary evaporator under reduced pressure. 2 mL of concentrated M1 NPs was lyophilized and weighed to determine the concentration. M1 NPs of known concentration were stored at 4 °C for later use. M2 NPs, M3 NPs, M4 NPs, M5 NPs, and M6 NPs were acquired using a similar strategy. Iron-mediated oxidation of hemoglobin experiment Venous blood anticoagulated with heparin sodium (3 mL) was obtained from West China Hospital, and relevant research has been approved by the West China Hospital of Sichuan University Biomedical Research Ethics Committee (2020172A). The upper plasma was discarded after centrifuging at 3000 rpm for 5 min, and the lower red blood cells were washed with normal saline three times following repeated freezing and thawing to obtain hemoglobin (HbA) solution. Before use, HbA solution was diluted with normal saline to 22 μM HbA. 10 μL samples and 50 μL Fe3+ (1.6 mM) were incubated for 10 min following 140 μL HbA solution was quickly added, and the absorbance at 560, 576, 630, and 700 nm was detected immediately. Critical micelle concentration test 25 μL rhodamine red solution (solvent: acetone, 0.16 mM) was added to 4 mL self-assemblies with different concentrations (1 ng/mL to 100 μg/mL). After the acetone volatilized overnight, all samples were monitored with a fluorescence spectrometer after ultrasonic excitation for 2 h (excitation: 570 nm, emission: 660 nm). DPPH assay 100 μg samples were mixed with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution (0.1 mM) and incubated for 30 min, and a UV–vis spectrophotometer (Lambda 650, PerkinElmer, America) was utilized to keep track of changes at 517 nm over time. ABTS assay 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS·+) solution was prepared through the reaction between potassium persulfate (6.62 mg) and ABTS (36.03 mg) in 10 mL deionized water for 12 h in the dark. Then, the resulting ABTS·+ solution was diluted 30 times with water and mixed with the testing sample (100 μg). Meanwhile, the absorbance of mixed solution at 734 nm was monitored with a UV–vis spectrophotometer (Lambda 650, PerkinElmer) for 30 min. Cell culture RAW 264.7 cells were purchased from American Type Culture Collection (Manassas, VA, United States), cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) containing 1% penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin) and 10% fetal bovine serum under a humidified atmosphere with 5% CO2 at 37 °C. alamarBlue assay Resazurin was dissolved in sterile water to obtain alamarBlue solution (2 mg/mL). 3000/vial RAW 264.7 cells were seeded in a 96-well plate, and 24 h later different concentrations of samples were incubated with cells for 48 h. After the medium was replaced with fresh cell culture medium (phenol red free), 10 μL alamarBlue solution was added, incubating for 2–4 h accompanied by the appearance of a pink color. Then the culture medium was transferred to a black 96-well plate to measure the fluorescence intensity (excitation: 550 nm, emission: 590 nm) under multimode microplate reading. Similarly, digested brain cells (dispersed in 100 μL saline) were incubated with alamarBlue (10 μL) in a 96-well plate for 2 h, and then the solution (100 μL) was transferred into a black 96-well plate for testing. Malondialdehyde assay 500 g trichloroacetic acid was dissolved in 227 mL ultrapure water to obtain 100% (w/v) trichloroacetic acid solution. 40 mg 2-thiobarbituric acid was dissolved in 8 mL trichloroacetic acid solution (5%, w/v) to acquire malondialdehyde (MDA) working fluid. A certain amount of cells (RAW 264.7, or mouse brain cells) were lysed with superoxide dismutase (SOD) preparation solution from an SOD assay kit. The 100 μL cell lysate supernatant was mixed with 200 μL MDA working fluid, and then the supernatant of the mixture after centrifugation was tested at 432 nm following a boiling water bath for 15 min. Inductively coupled plasma tests Digested brain cells were lyophilized to acquire the dry weight, and then 200 μL nitric acid (68%) and 50 μL hydrogen peroxide (30%) were added. That mixture was subjected to 100 °C for 30 min. The obtained solution was diluted 25 times before inductively coupled plasma (ICP) testing. Hemocompatibility study Washed red blood cell (RBC) suspension (10% hematocrit) incubated with DFO and M6 NPs of different concentrations was utilized to determine RBC lysis according to the Drabkin method.1 As shown in Supporting Information Figure S16a, similar to normal saline and DFO, M6 NPs have not caused hemolysis at any set concentration. In contrast, Triton X100 surfactants have led to severe hemolysis. Under desktop scanning electron microscopy, no integrated RBC was found in the RBC sample treated with Triton X100. Simultaneously, we discovered that the morphology of RBC treated with M6 NPs (0.6 mg/mL) presented a double concave round cake shape, similar to a normal saline control and equal in concentration to DFO ( Supporting Information Figure S16b). Coagulation blood for examination is of critical significance for the investigation of the coagulation function. Herein, activated partial thromboplastin time, prothrombin time, thrombin time (TT), and concentration of fibrinogen were measured in mouse plasma, and the M6 NPs did not affect the coagulation processes involved in the study comparing it with normal saline ( Supporting Information Figure S17). Based on this investigation, M6 NPs featured satisfactory blood compatibility, which is convenient for the following animal experimental study. Animal studies All animal studies comply with the regulations of the Chinese National Regulations for the care and use of laboratory animals and are approved by the Animal Ethics Committee of West China Hospital of Sichuan University (2020172A). 8-week-old BALB/C mice (25–30 g) purchased from Dashuo Laboratory Animal Co., Ltd. (Chengdu, China) were randomly divided into four groups (normal, model, DFO, and M6 NPs). These mice were anesthetized with sodium amobarbital (3%) and fixed in a stereotaxic apparatus. The scalp skin was cut apart to expose the skull, and specific coordinates (coordinates: 0.2 mm anterior, 3.5 mm ventral, and 2.3 mm lateral to the bregma) were definitely settled with the bregma position as the zero axis. After successful drilling with the help of a cranial drill, 0.075 U collagenase VII (dissolved in 0.5 μL saline) was slowly injected into the right basal ganglia (0.1 μL/min) for model, DFO, and M6 NPs groups, and 0.5 μL saline was infused into certain position as the control (normal group). The cranial hole was sealed with bone wax, and the surgical wound was carefully sewn up after injection. The temperature of the mice was maintained at 37 °C using an electric blanket throughout the operation. In vivo security study To further investigate the potential toxicity of M6 NPs in vivo, histopathological examination and blood biochemistry analyses were conducted. The harvested major organs, including the heart, liver, spleen, lung, and kidneys of the mice injected with normal saline, DFO, and M6 NPs were histologically analyzed after hematoxylin and eosin (H&E) staining, and no noticeable organ damage, inflammation, or abnormal changes were observed in the main organs of the M6 NPs treatment groups, showing no evidence of tissue damage after M6 NPs treatment ( Supporting Information Figure S20). Otherwise, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), lactate dehydrogenase (LDH), and total protein (TP) were similar to the normal mice without any treatment, indicating the unaffected liver function of these ICH mice injected with M6 NPs ( Supporting Information Figures S21a–S21f). Also, the level of uric acid (UA), blood urea nitrogen (BUN), and creatinine (CRE) were located in the normal physiological range, demonstrating no adverse effects to the renal function of M6 NPs-treated mice ( Supporting Information Figures S21g–S21i). Taken together, M6 NPs exhibited satisfactory treatment security in vivo. Statistical analysis All data presented were displayed as mean ± standard errors. For the comparison of different groups, Student's t-test was performed. SPSS software was utilized for statistical analysis (*p < 0.05, **p < 0.01, ***p < 0.001). Results and Discussion Preparation and characterization of DFO-based NPs To systematically investigate the structure–activity relationship between polyphenols and DFO, six benzoic acid derivatives with different structures of phenolic hydroxyl groups (benzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, and gallic acid) were conjugated with the primary amine of aDFO molecule to obtain six synthetic derivatives of DFO (M1, M2, M3, M4, M5, and M6). As shown in Figure 1a, the polyphenols can conjugate with DFO based on the efficient reaction of the carboxyl group with the amino group. In this section, M1–M6 of high yield and purity were all successfully synthesized on the gram scale through "one-pot" EDC- and HOBt-involved amidation reactions without additional chromatographic purification and then were fully characterized using 1H NMR and ESI-MS (Figure 1b and Supporting Information Figures S1–S3). The presence of 6.5–8.0 ppm signals in 1H NMR spectra assigned to the protons of in the the successful of polyphenols and DFO through the Interestingly, all these polyphenol-DFO systems can into NPs through a strategy ( Supporting Information Figures and and uniform spherical NPs with an of 30 nm ± nm for M1 NPs, ± nm for M2 NPs, ± nm for M3 NPs, ± nm for M4 NPs, ± nm for M5 NPs, and ± nm for M6 were observed in electron 1b and and the by light to nm, which be as the of self-assemblies with water these NPs could under high concentration without any For M6 NPs could at 10 concentration and exhibit good in various ( Supporting Information Figure all the DFO-based NPs featured an DFO-loading (Figure All these results that a polyphenol-DFO carrier-free system is and and can be for To further the of the behavior of the resulting M6 was also by NMR in and 1H–1H nuclear effect is used to provide the the of which protons are to other in or in The of hydrogen on the DFO and on the was presented in the 1H–1H NOESY spectra M6 dispersed in that the and the DFO are in (Figure Otherwise, the not in DMSO-d6 ( Supporting Information Figure Simultaneously, the 1H of = was as the into from DMSO-d6 ( Supporting Information Figure Taken together, we can that M6 into the M6 NPs through the caused by hydrogen of DFO, characterized by between and the DFO of integration of DFO-based NPs As the DFO content in our system from to DFO was conjugated polyphenols to form which all the results in the study (Figure the high content of DFO and in various we can that these DFO-based carrier-free systems would not only improve the circulation time of DFO, but also significantly increase the loading of In this the capacity of DFO-based NPs could be The of polyphenols would also improve the and ROS scavenging of NPs. As to their these NPs all after with Fe2+ ( Supporting Information Figure indicating the of DFO was we added different concentrations of Fe2+ into the M6 NPs and 12 h later the mixture in a featuring a specific at nm without of iron-binding capacity to DFO (Figure and Supporting Information Figure The absorbance of nm only the formation of DFO and Fe3+ will into Fe3+ in DFO solution after the potential of polyphenols with it was to at the that the M6 NPs have more iron-binding capacity to In DFO has been as a free radical Herein, we the DPPH and ABTS·+ free effects of DFO-based carrier-free NPs. Supporting Information Figure that DFO can be with a effect only at high that is to under a normal the of DFO is the of our polyphenols would a DFO-based extra ROS scavenging to with oxidative To confirm both DPPH assay and ABTS assay were to the free radical scavenging of these DFO-based NPs ( Supporting Information Figures and Figure 2 | (a) DFO-loading content in this (b) UV–vis spectra of DFO and M6 NPs after with Fe2+ solution mM) for 12 h. (c) of and ABTS·+ after being incubated with DFO, M1–M6 NPs (100 for 30 min, The scavenging of M1–M6 NPs (100 the with (d) and ABTS·+ free for 30 min, of Fe3+ mM) oxidation of HbA of M6 NPs. and absorbance spectra of HbA under different of oxidation of HbA by DFO, M6 NPs in for 5 min. HbA HbA Fe3+ DFO, HbA Fe3+ DFO, HbA Fe3+ DFO, HbA Fe3+ DFO, HbA Fe3+ M6 NPs, HbA Fe3+ M6 NPs, HbA Fe3+ M6 NPs, HbA Fe3+ M6 NPs. The of after with different samples after 5 min. Download figure Download PowerPoint M3 NPs, M5 NPs, and M6 NPs, the of the DPPH free radical different from the other samples (Figure the ROS-scavenging capacity in the of M6 NPs > M5 NPs > M3 NPs (Figure which be as the hydrogen to the and position of phenolic hydroxyl that the and and at a lower potential which that the M3 NPs and M5 NPs can effects to the M4 NPs. M6 NPs the ROS-scavenging capacity due to the presence of a high of phenolic hydroxyl The scavenging behavior of these NPs the free radical in the water its behavior DPPH free in the The of the of after with different NPs the of M6 NPs, M5 NPs, and M3 NPs M6 NPs were utilized as the carrier-free NPs to investigate the effects of iron and ROS-scavenging As we oxidation of lipids, proteins, and nucleic from the Fe3+ and the six of Fe3+ are by the acid group of DFO, the destruction of the Therefore, HbA is commonly utilized as a model protein to the of biomaterials to HbA from being by In this the HbA test was using M6 NPs with HbA under an oxidative stress As shown in Figure HbA from red to after being incubated with Fe3+ at and nm and a at nm of presented after and M6 NPs could more significantly the of the oxidation of HbA the DFO, not only in of the but also the of the HbA (Figure and Supporting Information Figure monitored the HbA after Fe3+ for 5 min, and no sample was set as control (Figure Also, the HbA after 5 min the more capacity of M6 NPs HbA from the oxidation with an equal dose of DFO (Figure Taken together, these DFO-based NPs effectively the ROS-scavenging of DFO they did not the All these results that DFO-based carrier-free NPs can serve as a treatment system for iron-overload diseases. performance of DFO-based NPs As we RAW 264.7 cells are for iron from and can be used as the cell for the following iron-overload cell to further their iron- and ROS-scavenging capacity at the Before we did we the of M6 NPs with the RAW 264.7 cell As in Figure after being with DFO for 48 h, the RAW 264.7 was under μM DFO, no cytotoxicity was for M6 NPs. at the concentration of 100 μM DFO containing M6 NPs, the cell was After with 2 mM) for h following staining, a noticeable region was observed in the RAW 264.7 cell group control with the group without any treatment control Figure The significantly in a after DFO M6 software was utilized to the which demonstrated that M6 NPs have iron with the DFO (Figure These results can be