In contrast to noble metal absorbers, its absorption rates and resonance frequencies can be dynamically regulated by controlling the Fermi level and relaxation time of graphene. In addition, the device can maintain high absorptivity at 0~50° in TE polarization and 0~40° in TM polarization. The maximum refractive index sensitivity can reach SV = 1.75 THz/RIU, and the maximum figure of merit (FOM) can reach FOMV = 12.774 RIU−1. In conclusion, our design has the properties of dynamic tunability, polarization independence, wide-incident-angle absorption, and fine refractive index sensitivity. We believe that the device has potential applications in photodetectors, active optoelectronic devices, sensors, and other related fields.This paper presents a new theoretical proposal for a surface plasmon resonance (SPR) terahertz metamaterial absorber with five narrow absorption peaks. The overall structure comprises a sandwich stack consisting of a gold bottom layer, a silica medium, and a single-layer patterned graphene array on top. COMSOL simulation represents that the five absorption peaks under TE polarization are at  This exceptional frequency band holds paramount significance across diverse domains, encompassing optical sensing [Terahertz (THz) waves constitute a distinctive subset within the electromagnetic spectrum, spanning a frequency range of 0.1~10 THz , fⅡ = 6.00 THz (98.47%), fⅢ = 7.37 THz (98.72%), fIV = 8.47 THz (99.87%), and fV = 9.38 THz (97.20%). This absorption closely aligns with that observed under TM polarization. Subsequently, a detailed analysis of the absorptivity mechanism is carried out, employing transmission line theory and the equivalent impedance matching theory, supplemented by the visualization of surface electric field distribution diagrams. Moreover, the dynamic tunability of the absorber is showcased by manipulating the Fermi level and relaxation time of the graphene metasurface. Additionally, the influence of geometric parameters within the graphene pattern on absorptivity is studied. The findings underscore the robustness of the graphene’s geometry, particularly concerning its capacity to sustain resonance frequencies. Furthermore, the absorber’s absorptivity is evaluated across a range of incident angles. The results suggest that the device effectively sustains significant absorption efficiency for both TE polarization in the range of 0° to 50° and TM polarization spanning from 0° to 40°. Finally, the absorber’s potential for refractive index sensing within environmental contexts is demonstrated. The results suggest that the maximum sensitivity and FOM are SV = 1.75 THz/RIU and FOMV = 12.774 RIU−1.In this study, a novel THz graphene-based SPR metamaterial absorber is introduced. Numerical simulations reveal the emergence of penta-band absorption peaks under TE polarization at the frequencies tm = 1 μm is significantly greater than the skin depth. The device is deposited onto a silicon (Si) substrate.The specific structure of the device is depicted in When an electromagnetic wave impacts the device, the graphene surface becomes excited, giving rise to the generation of surface plasmon waves (SPWs). If the frequency, wave vector, and propagation direction of the electromagnetic wave align with those of the SPW, the energy carried by photons is transferred to the free electrons of the SPW . This phR denotes reflectance, and T signifies transmittance. These quantities can also be represented using the S parameters, specifically the reflectance rate S11 and the transmittance rate S21. In order to achieve T = |S21|2 = 0, the thickness of the Au layer must surpass its skin depth to inhibit the transmission of incident light. The skin depth of Au is defined as the distance at which the amplitude of the electromagnetic wave has attenuated to 1/e (approximately 37%) of its value at the surface [Here,  surface :(4)e−αδ=μ = μ0 = 4π × 10−7 N/A2 and σ = 4.56 × 107 S/m. Consequently, the maximum skin depth within the frequency range of f (1~10 THz) is calculated to be 7.4531 × 10−2 μm, which is much smaller than tm (1 μm).In this study, for gold (Au), the values used are R = |S11|2 = 0. According to the equivalent impedance matching theory, the equivalent impedance of the device is described as follows [In this context, transmission line theory and equivalent impedance matching theory are employed to analyze the objective of achieving  follows :(5)Zeff=S11 = 0, Zeff = 1, that is, when Re(Zeff) = 1 and Im(Zeff) = 0. In fact, S11 and R can be expressed according to Equation (5) as follows:Therefore, perfect absorption is achieved when Zin = Zeff·Z0 signifies the input impedance of the graphene layer, where Z0 represents the intrinsic impedance of free space. When Zin equals Z0, indicating that the input impedance of the absorber matches the intrinsic impedance of free space, perfect absorption can be attained [Here, attained . EquatioAn equivalent circuit model can be established for the device , as showThe impedance of the graphene layer is given byWhen SPR occurs at a certain incident electromagnetic wave frequency, the entire circuit is in a resonant state, exhibiting resistive behavior. Therefore,At this point, the input impedance of it is as follows:The input impedance of the circuit isA = 1 − R can be described as follows: only when Zin = Z0, A reaches its maximum value.Hence, f), the dielectric layer’s material (εd) and thickness (td), as well as the impedance of graphene (Zg). In terms of the dielectric layer, silica was selected due to its exceptional hardness and resistance to abrasion, which render it particularly well suited to the demanding operational conditions. The determination of the appropriate thickness for this layer will be explored in the subsequent section. The impedance of graphene is intricately tied to its relative permittivity, which is expressed as follows [tg represents the thickness of the graphene layer. In this study, a value of tg = 1 nm was selected for ease of calculation purposes.In conclusion, achieving perfect absorption through the alignment of the absorber’s input impedance with the intrinsic impedance of free space necessitates a meticulous choice of several key factors. These factors include the incident frequency ( follows :(14)εg=1σinter(ω) denotes the interband conductivity of graphene, and σintra(ω) represents the intraband conductivity. These can be expressed using the classical Kubo formula [The total surface conductivity of graphene is defined as follows :(15)σg(ω formula :(16)σinte represents the elementary charge,FE signifies the Fermi energy level of graphene,ω stands for the angular frequency of the incident electromagnetic wave,τ is the relaxation time of graphene,kB denotes the Boltzmann constant,T represents the ambient temperature in Kelvin.FE is much greater than σinter(ω) can be disregarded, leading to the simplification of σg(ω) using the Drude model [According to the Pauli exclusion principle, within the terahertz range, where de model  as folloσg(ω) can be modulated by manipulating the frequency of incident light (ω or f = 2πω), the Fermi level (FE), and the relaxation time (τ) of graphene. These dynamics are visually depicted in FE at τ = 2.0 ps, as well as concerning the incident frequency and τ at FE = 0.8 eV. This provides a theoretical foundation for subsequent simulation results.Based on the discussions presented above, it is evident that This section will explain the building of the proposed simulation model of a graphene terahertz absorber in COMSOL Multiphysics 5.6 software.Following the construction of the model using the parameters listed in In the x–y plane, periodic boundary conditions were implemented, while perfect match layers were established in the z-direction to absorb the excess scattered waves. The transition boundary condition was applied to the graphene layer. The incident electromagnetic wave frequency was set between 1 and 10 THz. The excitation port was set above the analyte to simulate the incident electromagnetic wave, and the port was set below the gold layer to extract the S parameters. The “Type of port” was selected as “Periodic”.For meshing, the sequence type was configured as “User-controlled mesh”. A new “Free triangular” mesh was created for the graphene layer, with the specified “Size” parameters. The “Maximum element size” was set to 0.2 μm, and the “Minimum element size” was set to 0.1 μm. The remaining meshing settings were left as default.Distinct tests were conducted to evaluate the absorption performance of the absorber under both TE and TM polarizations, as depicted in Z) is close to 1 and Im(Z) is close to 0, especially for Mode IV (Z) = 0.9958, and Im(Z) = −0.0769, indicating a remarkably close approximation to perfect absorption. These experimental results align very closely with the theoretical derivations discussed earlier.The corresponding equivalent impedance matching diagrams for each mode were mapped as shown in  Mode IV d with anfI = 1.99 THz, fⅡ = 6.00 THz, fⅢ = 7.37 THz, fIV = 8.47 THz, and fV = 9.38 THz. The corresponding absorption rates for these modes were 95.82%, 98.47%, 98.72%, 99.87%, and 97.20%, respectively.As illustrated in To ascertain the precise positions at which LSPR occurs on the graphene patterns for each mode, electric field monitors were strategically positioned on the x–y plane. The resultant findings are visually depicted in In Mode Ⅰ, localized surface plasmon resonance (LSPR) was predominantly concentrated in the proximity of the ring and the Celtic cross, extending along the cross’s edges in the y-direction. This behavior can be attributed to the coupling with neighboring periodic graphene patterns. Furthermore, regions along the ring’s edges exhibited a relatively weaker LSPR intensity. In Mode II, robust resonance was localized near the Celtic cross’s edges in the x-direction and the adjacent region of the ring. In contrast, other positions displayed subdued responses. Moreover, a relatively weaker surface plasmon resonance was discernible along the inner edge of the ring.In Mode III, significant LSPR contributions were evident near the circular segment of the Celtic cross, particularly in proximity to the ring. Additionally, pronounced LSPR effects were observed along the Celtic cross’s edges in the y-direction. For Mode IV, LSPR was conspicuous across several positions of the pattern, primarily along both sides of the ring’s edges and the edges of the Celtic cross, coupled with adjacent periodic patterns.In Mode V, LSPR predominantly emerged within the gaps between the Celtic cross and the ring. These gaps encompass the spaces between the circle and the ring, between the Celtic cross and the ring, and between the Celtic cross and the adjacent periodic cross.θ) is set at 0 degrees. Notably, in this study, it became apparent that the gaps between the subpatterns and their edges played a pivotal role in the occurrence of surface plasmon resonance [Owing to the inherent symmetry of the graphene pattern, the regions in which LSPR is excited maintain symmetry as well . This chesonance ,48. Therr2, as it scales with r1). Hence, this study focused solely on variations in the overall size of the ring. And the results are depicted in Numerical analyses were performed on the geometric parameters of the graphene pattern (excluding d presents the thickness of the dielectric layer (td in this paper). When light is incident on the absorber, the graphene surface generates LSPR, and the light waves undergo multiple reflections within the Fabry–Perot cavity. By carefully selecting the appropriate material and thickness for the dielectric layer, the interference of the reflected light from different orders can be effectively suppressed. This leads to the occurrence of Fabry–Perot resonance, where the Fabry–Perot resonance and the LSPR on the graphene surface are strongly coupled. As a result, the electromagnetic waves are efficiently absorbed. After multiple experiments, it was determined that the average highest absorption rate for the five modes was achieved when td = 4.2 μm.Furthermore, the impact of the thickness of the silica layer on absorption performances was tested, as depicted in FE) and relaxation time (τ) of graphene. The effect of varying the Fermi level (FE) under a relaxation time (τ) of 2.0 ps on the absorption performance of the absorber was initially examined. As shown in FE, the resonance frequencies of the five modes all experienced a blue shift. This shift occurs because the wavevector of surface plasmon polaritons (SPPs) satisfies the following relationship [α0 is fine structure constant and rf is resonance frequency. Hence, there exists a relationship between the Fermi level and resonance frequency:Next, the dynamic tunability of the device was demonstrated by varying the Fermi level (tionship :(20)ksppFE, and the modulation bandwidth at higher frequencies will also be larger. There is a relationship between the Fermi level and the applied voltage [Therefore, the resonance frequency will increase with an increase in  voltage ,(22)EF=FV is the Fermi velocity, with a typical value of approximately 106 m/s,ε0 and rε are the vacuum permittivity and relative permittivity, respectively,Vg is the applied external voltage,e0 is the elementary charge,td is the thickness of the dielectric (SiO2) layer.FE can be controlled through the manipulation of the applied external voltage (Vg). In practical applications, once the device is fabricated, its absorption performance remains fixed. By applying a gate voltage, the capacity to dynamically adjust the Fermi level of graphene is gained, thereby influencing its conductivity. This dynamic capability allows the device to dynamically fine-tune both the absorption rate and resonance frequency, thereby ensuring optimal performance.It is apparent that, by maintaining a constant thickness of the dielectric layer, the magnitude of τ) at a fixed Fermi level (FE) of 0.8 eV. The outcomes of this investigation are depicted in τ increased from 1.6 ps to 2.4 ps, the resonance frequencies of each mode remained constant, with only the absorption rate being modulated. This observation is consistent with the connection between the relaxation time of graphene and the Fermi level, as illustrated by the following relationship [v represents the carrier mobility of graphene. With τ increasing from 1.6 ps to 2.4 ps, the resonant frequencies of each mode remained unaffected, while only the absorption rate underwent modulation. Since FE remains constant, alterations in τ induce variations in the concentration of graphene carriers, subsequently influencing the intensity of plasmonic oscillations [Subsequently, the changes in absorber performance were examined in relation to the relaxation time (tionship :(23)τ=EFllations ,56. Thisθ represents the incident angle of the electromagnetic wave, and n denotes the effective refractive index of the metamaterial absorber. Taking TE polarization as an example, when the electromagnetic wave is normally incident, that is, θ = 0°, the expression for RTE in Equation (23) can be written as follows:According to the Fresnel equations, the transverse electric and transverse magnetic reflectances of the metamaterial absorber are, respectively, expressed as follows ,58:(24){This finding correlates with Equation (7). Moreover, Equation (23) emphasizes that the effect of graphene’s impedance is not solely limited to polarization modes; the incident angle of the electromagnetic wave also holds significant influence. Consequently, it is essential to conduct a thorough investigation into how the incident angle affects the absorption performance of the absorber. To address this, an incident angle ranging from 0° to 50° for TE polarization and from 0° to 40° for TM polarization was set up. The simulation outcomes are graphically presented in Under TE polarization, as the incident angle varied from 0 to 50 degrees, slight shifts in resonant frequencies were observed for each mode, while the absorption rates consistently remained above 85%. Similarly, in the case of TM polarization, in which the incident angle spanned from 0 to 40 degrees, each mode displayed minor resonant frequency shifts, while the absorption rates consistently stayed above 82%. This observation underscores the device’s capability to maintain substantial absorption rates even at larger incident angles. This phenomenon can be attributed to two key factors. Firstly, the device’s dimensions are smaller than the vacuum wavelength of the incident light, allowing it to effectively interact with a wide range of angles ,60. AddiIn summation, our design exhibited remarkable insensitivity to incident angles, encompassing a range from 0 to 50 degrees in the TE mode and 0 to 40 degrees in the TM mode. For practical applications, the incorporation of concave structures positioned above the absorber’s surface could be explored ,62. Thisn) range of 1.00 to 1.08 was selected, and the resulting shifts in the resonant frequencies and absorption rates were explored. The outcomes are visualized in A pivotal performance aspect of the metamaterial SPR absorber is its capability for refractive index (RI) sensing. An analyte refractive index .The ranges of variation for these shifts were Δ follows :(27)S=ΔfThe figure of merit (FOM) is another crucial factor for assessing the sensing performance of the absorber. It is defined as follows ,65:(28)F−1. This underscores the absorber’s strong sensing capabilities and resonance frequency selectivity. Lastly, in order to provide a comprehensive assessment of the absorber’s performance, a comparative analysis between the device and other metamaterial SPR absorbers was conducted. The results are presented in From the results, it can be inferred that the absorber achieved a minimum FHWM of 137 GHz and a maximum FOM of 12.774 RIUIt is clear that our design, when compared with previous studies, exhibits a higher number of absorption peaks and a substantial improvement in refractive index sensing performance (FOM). This strongly indicates the device’s considerable potential for application in fields like biochemistry and sensing.In summation, this study presents a new SPR terahertz absorber based on a single-layer, patterned graphene metasurface. Through rigorous theoretical exploration and meticulous numerical simulations, the penta-band absorption, dynamic tunability, geometric robustness, polarization independence, wide-angle absorption, and great sensing capacity of the absorber were elucidated. Compared with other works, the device features a simple structure that not only widens the number of absorption peaks but also ensures a high absorption rate and great refractive index sensing performance. Our work promotes diversity in the design of graphene-based metamaterial absorbers, offering fresh design inspiration. Consequently, we believe this novel SPR metamaterial absorber holds promising potential for applications in active optoelectronic devices, modulators, optical stealth, RI sensors, and other related fields.


The iron chelating orphan drug deferiprone (L1), discovered over 40 years ago, has been used daily by patients across the world at high doses (75–100 mg/kg) for more than 30 years with no serious toxicity. The level of safety and the simple, inexpensive synthesis are some of the many unique properties of L1, which played a major role in the contribution of the drug in the transition of thalassaemia from a fatal to a chronic disease. Other unique and valuable clinical properties of L1 in relation to pharmacology and metabolism include: oral effectiveness, which improved compliance compared to the prototype therapy with subcutaneous deferoxamine; highly effective iron removal from all iron-loaded organs, particularly the heart, which is the major target organ of iron toxicity and the cause of mortality in thalassaemic patients; an ability to achieve negative iron balance, completely remove all excess iron, and maintain normal iron stores in thalassaemic patients; rapid absorption from the stomach and rapid clearance from the body, allowing a greater frequency of repeated administration and overall increased efficacy of iron excretion, which is dependent on the dose used and also the concentration achieved at the site of drug action; and its ability to cross the blood–brain barrier and treat malignant, neurological, and microbial diseases affecting the brain. Some differential pharmacological activity by L1 among patients has been generally shown in relation to the absorption, distribution, metabolism, elimination, and toxicity (ADMET) of the drug. Unique properties exhibited by L1 in comparison to other drugs include specific protein interactions and antioxidant effects, such as iron removal from transferrin and lactoferrin; inhibition of iron and copper catalytic production of free radicals, ferroptosis, and cuproptosis; and inhibition of iron-containing proteins associated with different pathological conditions. The unique properties of L1 have attracted the interest of many investigators for drug repurposing and use in many pathological conditions, including cancer, neurodegenerative conditions, microbial conditions, renal conditions, free radical pathology, metal intoxication in relation to Fe, Cu, Al, Zn, Ga, In, U, and Pu, and other diseases. Similarly, the properties of L1 increase the prospects of its wider use in optimizing therapeutic efforts in many other fields of medicine, including synergies with other drugs. The development of new pharmaceuticals is usually undertaken by large, multinational companies, and the process requires many years and great expenditure to fulfil the necessary drug regulatory requirements. In contrast, the repurposing of already regulatory approved drugs requires less effort, but it is also a very challenging task, especially in research investigations for the development of therapies for serious and life-threatening diseases and also for emergency medicines ,2,3,4,5.Some of the major categories of diseases affecting humans are those related to the abnormal metabolism of iron, an essential metal required by all cells of the body and also by microbes and cancer cells. Iron is also required for many biological processes involving a large number of iron-containing enzymes participating in a variety of metabolic pathways and also for essential physiological processes including respiration, energy transduction, haemopoiesis, growth, and development ,14,15,16Billions of people are known to be suffering from diseases of iron metabolic imbalance, including iron deficiency anaemia and iron overload ,23,24,25The iron chelating drugs deferiprone (L1), deferasirox (DFRA), and deferoxamine (DF), which are widely used in the treatment of transfusional iron loading conditions, are included in the orphan drug category and have the prospect of being repurposed for the treatment of many other clinical conditions ,33,34,35General limitations of drug repurposing include pharmacological and toxicological investigations, which may be related to the proposed drug’s properties or the disease characteristics. Some of these aspects include, for example, drug posology and protocols, toxicity concerns, compliance with the method of administration, special factors and limitations related to the underlying disease, and the overall risk/benefit assessment in comparison to existing therapies.One of the major concerns regarding chelating drug repurposing is related to the toxicity limitations identified in the cases of DFRA and DF in different categories of patients with normal iron stores, where it was recommended by the manufacturers that these drugs should not be administered to patients with serum ferritin lower than 0.5 mg/L ,39,40. IThe chemical, pharmacological, and other properties of drugs are crucial for identifying their mode of action, toxicity, and therapeutic characteristics in each disease ,35. In tThe prime objective of this review is to identify the unique properties and other characteristics of L1, which helped in the transition of thalassaemia from a fatal to a chronic disease, and to apply  these for repurposing in other diseases. The prospect of wider use of L1 in different diseases, taking into consideration the limitations of the mode of activity, low cost, and other drug characteristics, could benefit many patients, including different orphan disease patient categories who do not receive effective drug treatments at present .Deferiprone, discovered about forty years ago (1981), was the first oral iron chelating drug to be introduced for the treatment of iron overload in thalassaemia and other transfusional iron loading conditions worldwide, following approval by the regulatory authorities for clinical use  ,44,45,46In addition to the inexpensive chemical synthesis and high compliance, several other factors contributed to the wider availability of L1 in comparison to DF before registration, including its high efficacy in iron removal and its low toxicity at the identified efficient selected doses ,49. RegaIn more than 30 years of clinical use and monitoring of L1 in different categories of patients, all the reported toxic side effects of L1 are considered manageable and reversible. The most serious toxic side effects of L1 include agranulocytosis and neutropenia, affecting less than 1% and 5% of patients, respectively. Less serious toxic side effects include gastric intolerance, musculoskeletal and joint pains, and zinc deficiency ,63,64,65The efficacy of L1 in removing sufficient amounts of excess iron for achieving a negative iron balance in transfusional iron-loaded patients has been recognised since the initial clinical trials, where stepwise increments of doses from 10 mg/kg/day to a maximum of 110 mg/kg/day were used ,50. The Most thalassaemia patients are born in developing countries where health resources are scarce, and the majority of patients receive insufficient or no effective treatment. The life expectancy of thalassaemia patients without any treatment is limited to about 2–3 years as a result of ineffective erythropoiesis and severe anaemia ,20,21,22Following the introduction of L1 and the L1/DF combinations, increased survival and a reduction in mortality were reported in thalassaemia patients treated in Cyprus and other countries ,80,81. MAnother major finding in relation to the diagnostic use of MRI T2 and T2* and other iron load diagnostics is the monitoring of the rate of iron removal effects of chelating drugs. In particular, by utilising these diagnostic techniques, it was possible to identify iron chelation protocols, such as the International Committee on Chelation (ICOC) protocol, which can efficiently remove all excess iron in chronically transfused thalassaemia patients ,99,100. The ICOC protocols are based on personalised chelation therapy options of either L1 (75–100 mg/kg/day) monotherapy or a combination therapy of L1 (75–100 mg/kg/day) and DF (40–60 mg/kg at least three times per week) ,103,104.The efficacy and toxicity of L1 and also of other drugs are mostly defined by their pharmacological and metabolic properties, which are also influenced by their molecular and structural characteristics. In the case of L1, the small size, heteroaromatic structure, and hydrophilic properties appear to facilitate its rapid gastrointestinal absorption following oral administration ,109,110.Following iron mobilisation, a characteristic orange/red-coloured urine is excreted in the case of iron-loaded patients receiving L1, which has the same colour as the L1 iron complex . In contMost of the administered L1 in patients and normal volunteers is metabolised to a glucuronide conjugate, which has no chelation properties and is also excreted in the urine. It should be noted that the 3-hydroxyl group of L1 is involved both in iron chelation and also during glucuronic acid conjugation. Overall, it appears that iron binding by L1 prevents glucuronidation and vice versa. In this context, the rate of iron chelation is faster than glucuronidation, and the presence of more chelatable iron forms in an individual reduces the rate of glucuronidation of L1 ,116,117 The same effects on glucuronidation are expected in the chelation of other metals by L1, such as aluminium ,118,119.Pharmacokinetic studies indicate that following oral administration, the clearance of L1 from blood circulation can take about 6 h, and clearance of its glucuronide conjugate takes about 9 h ,110,116.Pharmacological and metabolic properties are important parameters influencing the efficacy of drugs including L1 . SimilarThere are many interactions of L1 with iron-containing and other proteins and different associated effects, all of which appear to influence its therapeutic activity and toxicity. Some of these effects have been discovered in the early screening studies for the identification of L1′s mode of action and also in comparison to DF and other chelators  42,111],11142,11One of the most important and unique properties of L1 not found in any other drugs is the ability to remove iron from the iron transport protein transferrin found in blood, which delivers iron to all the cells of the body, including microbial and cancer cells ,132,133.Another unique property associated with L1 but not with any other drugs is its ability to remove iron from lactoferrin, the sister protein of transferrin, which is found in secretory fluids, such as milk, saliva, tears, and nasal and vaginal secretions. Lactoferrin is also a component of the immune system found in the secondary granules of neutrophils. It has antimicrobial properties and plays a major role in infectious and inflammatory diseases ,142,143.In vitro and clinical studies in thalassaemia patients treated with L1 have shown that iron removal from diferric transferrin is L1-concentration-dependent and can only be accomplished if the L1 concentration is higher than about 0.15 mM in blood  144,145145. ConvThe main sources of excess iron deposition and iron removal by L1 in iron-loaded patients and also in patients with the anaemia of chronic disease are intracellular stored haemosiderin and ferritin polynuclear iron ,151,152.Different forms of interaction of L1 have also been reported with many other iron-containing proteins, thereby affecting their function or associated metabolic pathways. These forms of interactions may not involve iron removal from the proteins but rather other aspects of the iron-containing proteins, including the associated intracellular iron pool from which iron molecules are “in transit” and utilised for the turnover of related iron proteins ,157. ForIn addition to ribonucleotide reductase, several other proteins and transcription factors involved in cancer progression have been targeted by L1. These include the modulation of the hypoxia-inducible factor (HIF) related to hypoxia, the function of newly identified molecular species, such as the “six-transmembrane epithelial antigen of prostate, family member 4” (STEAP4) metalloreductase, and the metastasis suppressor N-MYC downstream-regulated gene-1 (NDRG1) ,167,168.Free radicals, reactive oxygen species, and related by-products are continuously generated in humans and other organisms in normal physiological conditions and also in response to different stimuli, causes, and factors ,175,176.In particular, free radical toxicity and cascades through iron catalysis in the Fenton reaction have been shown to cause oxidative damage to all known biomolecules, including lipids, sugars, proteins, and DNA, and also tissue damage in almost all pathological conditions ,178,179.It should be noted that many different antioxidants, mostly in the form of nutraceuticals supported by a multi-billion industry, are sold daily over the pharmacy counter and other shops. Despite their popular demand and wide use, nutraceuticals are not regulated by drug authorities . SimilarThe possibility of using iron chelators as antioxidants and the potent antioxidant activity of L1 were identified at the early stages (1987) of its development when a three-screening model system was used to examine and compare the redox properties and free radical toxicity effects of different iron chelators and chelating drugs . During There have been further developments in the biological role of iron and free radicals, which have attracted the attention of a wide spectrum of biological and clinical investigators in the last 10 years following the discovery of ferroptosis, a newly identified form of programmed cell death, which is different from apoptosis and necrosis ,189. AmoSeveral other unique characteristics in relation to the antioxidant pharmaceutical potential of L1, in addition to the prevention of iron and copper free radical toxicity, include the rapid drug antioxidant activity effects and its wide accessibility to most organs. In this context, L1 has been shown to inhibit the pro-oxidant effects of vitamin C, or ascorbate, a widely used nutraceutical and a key dietary molecule involved in redox reactions under normal and disease conditions  212,213213. SimiA typical example of the clinical iron chelation–antioxidant potential of L1 is related to its widely studied beneficial effects on the hearts of iron-loaded patients, which was the major cause of mortality in thalassaemia and other transfusional iron-loaded conditions before the introduction of the drug. In extensive clinical investigations, L1 was shown to remove all excess toxic iron from the heart, resulting in the substantial improvement of cardiac function ,100,101.Overall, it appears, in general, that L1 could prevent, delay, or reverse oxidative-stress-toxicity-related tissue damage caused by iron and copper catalytic action . New strThe search for new pharmaceuticals for the treatment of many diseases with no current effective therapies, such as many types of cancer, Alzheimer’s disease, and Parkinson’s disease, as well as many orphan diseases, such as malaria, is a priority for the patients affected, their relatives, and society as a whole. Similarly, the length of timing for the introduction of new pharmaceuticals for such diseases is also very important, because their speedy introduction may save millions of lives each year. In this context, the repurposing of existing drugs for the treatment of such diseases may offer a much-needed solution in contrast to the development of new drugs, which usually takes much longer for the fulfilment of related regulatory requirements . Some ofThe drug repurposing prospects of L1 for use in diseases other than transfusional iron overload began within a few years of the initiation of clinical trials with L1 and continue until now, involving different categories of patients with normal iron stores ,230,231.A major encouragement for initiating the repurposing of L1 in non-iron-loaded conditions was mostly in relation to the safety and efficacy of L1 observed in many iron-loaded categories of patients with various underlying conditions and different drug treatments . Among tThe prospects for the use of L1 in non-iron-loaded categories of patients increased substantially following the achievement of normal iron stores in iron-loaded thalassaemia patients using L1 and L1/DF combinations and their maintenance using L1 monotherapy for more than 100 patient years. This achievement was based on personalised drafted dose protocols, which signalled a new era in the complete treatment of transfusional iron overload in thalassaemia using chelation therapy ,100,101.The same diagnostic criteria of normal serum ferritin and also liver and cardiac MRI T2* also applied in the many categories of non-iron-loaded patients with different underlying pathologies but with normal iron diagnostic indices, such as serum ferritin. However, the iron toxicity and tissue damage identified in many non-iron-loaded categories of patients concern the presence of focal iron deposits, which were detected by MRI T2* and are of major significance in many clinical conditions . In partSignificant clinical improvements have been noted in almost all the different categories of non-iron-loaded patients treated with L1, particularly when appropriate effective doses were selectively used . For exaPromising therapeutic effects have also been observed in other non-iron-loaded categories of patients treated with higher doses of L1. In particular, L1 administered at 50 mg/kg/day for 6–9 months in 53 diabetic and non-diabetic glomerular disease patients has shown a persistent drop in the mean albumin/creatinine ratio and stable renal function in diabetic patients. In contrast, a non-significant reduction in urinary protein with no significant changes in serum creatinine was observed in non-diabetic patients .Much higher doses of L1 have been used in non-iron-loaded categories of HIV-1-infected asymptomatic patients and also patients with malaria. In the former case, seven patients received 33 mg/kg three times daily, and seven patients, as well as six normal volunteers, received a dose of L1 of 50 mg/kg three times daily, with 86% of the subjects tolerating the 99 mg/kg daily dose and 61% the 150 mg/kg daily dose. It was estimated that the L1 threshold at about 150 μM did not allow a viral breakthrough for up to 35 days on-drug and also at least 87 days off-drug for a viral rebound. These studies suggested that L1 is the first low-molecular-weight drug that offered the prospect of reducing the pool of cells that harbour infection-relevant HIV-1 DNA . HaematoA major section of pharmaceutical applications of L1 involves many categories of patients affected by metal intoxication in addition to iron ,257. TheClinical experience of the use of L1 for over 30 years and its high level of safety in thousands of iron-loaded and non-iron-loaded patients increase the prospects of L1′s wider application in many other clinical conditions, whether as monotherapy or in combination therapies with other drugs. Most of the new potential clinical applications of L1 stem from or are related to its unique properties described above and mostly involve metal binding and the modulation of activity, metabolism, and toxicity.One of the many other areas of potential clinical development and application of L1 is related to metal ion modulation, metabolism, and toxicity associated with transferrin, which is known to affect the transport of about 40 metal ions, including essential metals, e.g., Fe, Zn, Cu, and Co, diagnostic metals, e.g., In, Ga, Gd, Tc, Ru, and Sc, and other xenobiotic metals, e.g., Al, U, Pu, and Am ,138,257.There are many possibilities for new drug development or applications of L1 and other drugs by identifying therapeutic targets based on the modulation of key iron and other metal-containing enzymes, as well as associated metabolic pathways or biomolecules involved in many different diseases, including cancer, inflammatory and infectious diseases ,268,269.The expanding field of free radicals and many associated diseases of free radical pathology, including those associated with ferroptosis and cuproptosis, is the subject of many investigations and also a major challenge for the introduction of new pharmaceuticals, including iron chelating drugs  179]. A. A179]. Drug interactions, drug combinations, and synergistic and antagonistic effects are some of the issues affecting the general pharmacological, toxicological, and therapeutic activity of drugs, including chelation therapy and L1. Furthermore, the modulation of activity of drugs and also of natural dietary or other molecules with iron binding properties has a major impact on therapeutic outcomes ,215,270.There are several unexplored combination therapy options in the use of L1 with chelating or other drugs in different conditions, which may offer advantages over existing therapies. Examples of such L1 combinations may involve EDTA, which is widely used in general metal detoxification and also other conditions ,280,281;The optimisation of therapies through modulation of the mode of action and the toxicity of widely used drugs with iron binding properties could also be envisaged from advantages in the use of the unique properties of L1. For example, in the case of the anticancer drug doxorubicin, where iron is thought to be implicated in its cardiotoxic effects, the chelator prodrug dexrazoxane, which is a derivative of EDTA, has been widely used in cancer patients for cardioprotection against doxorubicin and similar drug cardiotoxicity ,286,287.There are many other areas of potential clinical use of L1 where effective therapies for different conditions have not been found and initial in vitro and in vivo findings are very encouraging. In particular, the anticancer potential activity of L1 has been identified in prostate, breast, neuroblastoma, and many other cancer types, as well as the prospect of inhibition of universal mechanisms identified in cancer progression, metastasis, and drug resistance by L1 ,299,300.The unique properties of L1, its safety record, and its antioxidant, anticancer, antimicrobial, metal detoxifying, iron, and other metal metabolism modulating and other effects are very encouraging for drug repurposing. They are also important for considering and planning the design of new therapeutic strategies, including its potential use for the treatment of many new iron-loading and other non-iron-loading diseases, as well in medical diagnostics and other applications  301,302,314,315.Deferiprone has played a major role in the transition of thalassaemia from a fatal to a chronic disease, where the rate of survival of L1-treated patients is approaching that of normal individuals. In this context, many thalassaemia patients have become grandparents, a situation unthinkable before the introduction of L1 over 30 years ago. Similarly, selected dose protocols of L1 and also combination therapies with DF have succeeded for the first time in chelation therapy history to achieve and maintain normal iron stores in chronically transfused thalassaemia patients. The unique molecular and other properties of L1, as well as its mode of antioxidant, antimicrobial, anticancer, and metal detoxifying activity and other effects, increase the prospect of its wider evaluation and use in many clinical conditions and in medicine in general. The therapeutic effects and lack of toxicity of L1 observed in so many different clinical conditions thus far encourages, in particular, its selection and potential clinical use in many conditions with no effective treatments and high rates of morbidity and mortality, including orphan drug diseases and orphan drug patients.


Caryocar brasiliense Cambess is a plant species typical of the Cerrado, a Brazilian biome. The fruit of this species is popularly known as pequi, and its oil is used in traditional medicine. However, an important factor hindering the use of pequi oil is its low yield when extracted from the pulp of this fruit. Therefore, in this study, with aim of developing a new herbal medicine, we an-alyzed the toxicity and anti-inflammatory activity of an extract of pequi pulp residue (EPPR), fol-lowing the mechanical extraction of the oil from its pulp. For this purpose, EPPR was prepared and encapsulated in chitosan. The nanoparticles were analyzed, and the cytotoxicity of the encapsu-lated EPPR was evaluated in vitro. After confirming the cytotoxicity of the encapsulated EPPR, the following evaluations were performed with non-encapsulated EPPR: in vitro anti-inflammatory activity, quantification of cytokines, and acute toxicity in vivo. Once the anti-inflammatory activity and absence of toxicity of EPPR were verified, a gel formulation of EPPR was developed for topical use and analyzed for its in vivo anti-inflammatory potential, ocular toxicity, and previous stability assessment. EPPR and the gel containing EPPR showed effective anti-inflammatory activity and lack of toxicity. The formulation was stable. Thus, a new herbal medicine with anti-inflammatory activity can be developed from discarded pequi residue. The skin is the largest organ of the human body, and it acts as a physical barrier against the external environment. The protective function of the skin and its annexes may be damaged by aggressive factors, which may lead to various types of injuries, such as physical injuries caused by cuts and sunburn, chemical burns from organic solvents, or viral or fungal infections .In response to these different types of traumas, an inflammatory process is induced in the skin as a defense mechanism to repair damaged tissues . This prIn clinical medicine, non-steroidal anti-inflammatory drugs (NSAIDs) are the first choice of treatment for reducing the exaggerated and inappropriate inflammatory response, followed by glucocorticoid anti-inflammatory drugs. However, NSAIDs and glucocorticoids are not effective in many chronic inflammatory processes, and anti-inflammatory drugs such as ketoprofen and ibuprofen generally generate many side effects. Thus, the search for new options in traditional medicine is important for the treatment of inflammation. In particular, medicinal plants can be used as a source of new active ingredients for the development of anti-inflammatory drugs .Caryocar brasiliense Cambess. It is the main tree of the Cerrado, the second-largest Brazilian biome. The economic and cultural importance of the pequi fruit for the Cerrado population is well known, especially for those who work in family farming [A striking example of such a medicinal plant is the pequi tree,  farming . The peq farming .Additionally, in traditional medicine, pequi pulp oil from the pequi fruit is used in the treatment of various conditions resulting from the inflammatory response . The theHowever, an important factor that hinders the use of pequi oil as an anti-inflammatory drug is the low yield of oil extracted from the pulp of this fruit . NeverthNew technologies for drug delivery systems based on plant extracts have been established, including polymeric nanoparticles, which have been widely explored in the development of formulations for topical use. The advantages of nanocarriers compared to conventional topical preparations have been proven; these advantages include improved solubility, pharmacological activity, skin absorption, and formulation stability, in addition to decreased toxicity ,13,14.Based on these considerations, with the aim of developing a new herbal medicine which presents good efficacy and low adverse effects, EPPR was initially developed. EPPR was encapsuled in chitosan (CTS), and the cytotoxicity of encapsulated EPPR was evaluated. Because of the cytotoxicity of encapsulated EPPR, analyses of its anti-inflammatory and toxicological potential were performed using non-encapsulated EPPR as well as a gel containing EPPR, which did not present cytotoxicity in a previous study .After oil extraction from pequi pulp, the residue generated is normally discarded. The composition of the flavonoids remaining in the EPPR was verified .Barreto et al.  showed tp-coumaric acid, coumaroylquinic acid, caffeic acid glycoside, and sugars .Other phenolic compounds of EPPR were identified by Pegorin Brasil et al.  via ultrAdditionally, a comparison was made between the total phenolic compounds in EPPR /g) determined using spectrophotometry by Pegorin Brasil et al.  and dataThe difference in the content of phenolic compounds and flavonoids in the pequi residue can probably be attributed to the processes used to remove the oil from the pulp, the solvents used in the preparation of the extracts, and the use of different parts of the fruit in the extraction process.Furthermore, the total phenolic compound content of different extracts of pequi pulp was lower than that reported by Pegorin Brasil et al. , MagalhãThese results revealed the high added value of the pequi pulp residue, because the phenolic compounds were preserved even after oil processing.CTS/tripolyphosphate (TPP)-hydroethanolic EPPR was characterized using three different techniques: dynamic light scattering (DLS), nano tracking analysis (NTA), and atomic force microscopy (AFM) .10 ± 4 × 109 nanoparticles/mL, which is consistent with the DLS results. AFM images , which the authors attributed to the presence of polyphenol from the extract on the nanoparticle surface.The results obtained in this study showed nanoparticles with the same characteristics of size, surface charge, and morphology as that described in the literature for bio-compounds such as essential oils or plant extracts . AccordiOwing to the amino groups in the CTS structure, CTS nanoparticles present a positive charge, which can increase or decrease according to the pH. The nanoparticle surface is an important characteristic of nanoformulation stability. A high zeta potential value increases repulsion between the nanoparticles, preventing aggregation and precipitation over time .The nanoparticles prepared in this study showed good colloidal characteristics, and they could be used as a nanocarrier system for EPPR.The encapsulated EPPR, at all concentrations used in this assay, produced a significantly lower cell viability than the negative control (NC) after 24, 48, and 72 h .Analysis of the cellular cytotoxicity of a plant extract by measuring its viability guarantees the safety of the final product . In the The results of the MTT test showed that the encapsulated EPPR was cytotoxic to fibroblast cells , even atIn contrast, Pegorin Brasil et al.  reportedTo understand the cytotoxic effect of the encapsulated EPPR, acetic acid was analyzed by MTT assay at the same concentrations that were used for EPPR encapsulated with CTS. In this analysis, pure acetic acid showed the same cytotoxicity profile as the encapsulated EPPR .The results obtained in this study suggest that acetic acid may be responsible for the toxicity of the extract in fibroblast cells. Doughty et al.  showed tThe effects of all treatments were significantly different from that of the NC, saline . EPPR inMacrophages exert immunomodulatory effects on skin wound repair, which is a critical process for restoring skin integrity . Tissue Sigmadocia extract produced a low level of phagocytosis.The results obtained in this study indicate that EPPR inhibited macrophage phagocytosis in a similar manner to the commercial anti-inflammatory drug dexamethasone 100 μg/mL (PC). EPPR promoted greater inhibition of phagocytosis than PC, even at concentrations of 400 and 600 μg/mL. The literature has no reports on the anti-inflammatory activity of pequi plant extracts. Athira and Keerthi  observedSpreading is generally defined as an unsuccessful attempt at phagocytosis. However, as no substance or microorganism is present to be phagocytosed, spreading is the action of a responsive cell with the ability to adhere to the slide and emit microvilli .p < 0.05) (All treatments used in this assay produced significantly different effects compared with the NC ( < 0.05) . EPPR prThe human red blood cell stabilization method was used to analyze the anti-inflammatory activity of the plant extracts . Accordip < 0.05). EPPR promoted the protection of the red blood cell membrane in the following proportions: 18.71 ± 2.01% at 200 μg/mL; 22.85 ± 0.20% at 400 μg/mL; and 76.67 ± 0.22% at 600 μg/mL. Protection from hemolysis promoted by the PC, dexamethasone, was 99.11 ± 0.32%.Skimmia anquetilia, originally from India; the extract exhibited a protection of 68.40% at a concentration of 400 mg/mL. Dias [Lippia pedunculosa and observed 100% inhibition of hemolysis at concentrations of 10, 50, 100, and 250 µg/mL. Sousa et al. [Pavonia glazioviana Gürke  at 500 and 1000 µg/mL and reported a protective effect of less than 60%.Kumar et al. , using tmL. Dias  analyzeda et al.  evaluateOther analyses have shown that pequi oil protects against inflammation ; howeverStudies have established that lipopolysaccharides (LPS) induce the secretion of pro-inflammatory cytokines such as TNF-α and interleukins . Cytokinp < 0.05) in the production of IL-6 (p < 0.05) prevented LPS-induced increase in interleukin concentrations.The results of the present analysis, as expected, showed that the group treated with LPS as the PC showed a significant increase   .According to Di Santo et al. , when thThe paw edema test in rodents has been well established for the evaluation of new drugs. In this test, the development of carrageenan-induced paw edema occurs in two phases. In the initial phase (0–1 h), serotonin, histamine, bradykinin, and substance P are released. The second phase (after 1 h) involves invasion of mainly neutrophils at the site of inflammation and production of large amounts of pro-inflammatory mediators, such as PGE2, and various cytokines, such as IL-6 and IL-10 .p < 0.05) reduction in carrageenan-induced paw edema when compared with the NC. This reduction was observed from the second hour onwards and remained until the sixth hour after the application of carrageenan. More specifically, in the second hour, the NC, PC, and EPPR gels showed, respectively, the following edema values: 0.08 ± 0.005 mL, 0.04 ± 0.002 mL, and 0.04 ± 0.006 mL. In addition, the gels containing EPPR and dexamethasone inhibited edema by 49.95% and 53.33%, respectively (EPPR gel (5 mg/g) and dexamethasone gel  reduced carrageenan-induced edema, which was determined by measuring the paw volume (mL) of the animals. The reduction of edema was considered an anti-inflammatory activity. Gels containing dexamethasone and EPPR showed a significant (ectively .p ˂ 0.05) and remained constant. The NC, PC, and EPPR gel treatments resulted in the following edema volumes per hour: fourth hour  and sixth hour . The PC and EPPR gel treatments, respectively, inhibited edema by similar extents at the fourth and sixth hours: 70.27% and 67.79%.Anti-inflammatory in vivo tests using formulations containing pequi oil are well established in the literature. However, nothing is known regarding the anti-inflammatory effects of topical products containing EPPR. Diniz  found a The final classification of the degree of irritation of the gels was based on the average of the sum of the values obtained by the three samples per group. The results of the in vitro irritability test  showed aFinally, with considerations for the possibility of developing an herbal medicine according to the demands of the international consumer market, the stability of the formulations was evaluated. In the centrifugation test, the base gel and EPPR gel did not show any changes. The thermal stress test verified that none of the samples of the gel presented separation into phases upon reaching a temperature of 80 °C. The pH values of the base gel (the gel without the extract) and the EPPR gel did not present any statistically significant differences. The EPPR gel showed a mean pH of 5.91 ± 0.05, and the base gel showed a mean pH of 5.54 ± 0.06. The values were within the threshold range of compatibility with the physiological pH of the skin, which ranges from 5.5 to 7.3.In conclusion, the chromatographic quantification of EPPR showed a significant presence of flavonoids, suggesting a relationship between the pharmacological activity of this extract and its phytochemical composition. The nanoparticles prepared in this study with CTS showed good colloidal characteristics and could be used as a nanocarrier system for EPPR. However, in the MTT assay, the encapsulated EPPR showed cytotoxicity due to acetic acid. EPPR, a non-encapsulated extract, showed anti-inflammatory activity in vitro. In addition, it significantly reduced the concentration of the inflammatory cytokines IL-6 and IL-10 and showed no toxicity in vivo. Furthermore, in vivo testing of the EPPR gel showed anti-inflammatory activity similar to that of the PC, dexamethasone, an anti-inflammatory agent widely used in clinical medicine. Lastly, the formulation of the EPPR gel showed stability and a lack of toxicity in the ocular test. In conclusion, it is possible to develop a new herbal medicine from pequi residue that is normally discarded for the treatment of inflammatory skin diseases.Caryocar brasiliense Cambess in the Herbarium of the Universidade Estadual Paulista (UNESP), where a voucher specimen was deposited under the number 1998. This study was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under license no. A23C398.Pequi fruits were obtained from the city of Mirabela, state of Minas Gerais, in the southeast region of Brazil . The fruits were first washed and peeled and subsequently packed in plastic bags and stored under refrigeration −18 °C; . The speFruits were washed, peeled, manually cut into small pieces, and subjected to the oil extraction process by pulp cold pressing. The residue resulting from the pulp pressing was dried in an oven (36 °C).v/v), as shown in ®, São Paulo, SP, Brazil) under reduced pressure at 70 °C to eliminate the alcohol and placed in an oven  to dry at 36 °C for the complete elimination of water and to obtain a constant weight of the dried EPPR [The hydroethanolic EPPR was prepared at a proportion of 1 g of dry pequi pulp residue per 10 mL of ethanol solution . The experiments were performed in triplicates. The results were expressed in mg QE/g.The flavonoid content of EPPR was determined using the method described by Serdar et al. . An anal®, St. Louis, MO, USA). Encapsulation was performed according to the protocol proposed by Calvo et al. with modifications [CTS used in this study was a commercial product  with a scattering detection angle of 90°. The analysis was performed in triplicates at 25 °C.The nanoparticles were characterized by DLS to obtain their hydrodynamic size (nm) and PDI. The analyses were performed using ZetaSizer . The analysis was performed in triplicates at 25 °C.The nanoparticle surface charge  was determined using electrophoresis. Analyses were performed using ZetaSizer equipment . The samples were diluted 100× in ultrapure water, the analyses were performed five times, and 400 nanoparticles were counted for each measurement. The samples were stored at 25 °C.The size distribution and the nanoparticle concentration were determined by NTA . The samples were scanned in contact mode using TapAl-G cantilevers . The nanoparticles were diluted 1000× in ultrapure water. The size of the nanoparticles was determined by analyzing the images using the ImageJ® software .Images were obtained using the easyScan 2 basic AFM system (Nanosurf® CRL-1658™) were inoculated into 96-well microtiter plates and incubated in culture medium for 24 h at 37 °C under 5% carbon dioxide (CO2). After reaching approximately 75% confluence (24 h), these cells were exposed to five different concentrations of the encapsulated EPPR . In addition, to understand the cytotoxicity of the encapsulated EPPR, acetic acid was evaluated at the same proportions used in the encapsulation process . For the NC, the extract was replaced with a physiological solution, and for the PC, it was replaced with 2% (v/v) Tween 80. The treatment times were 24, 48, and 72 h.The MTT cytotoxicity assay was performed as described previously by Tsuboy et al. . For thiEPPR was not included in this screening because Pegorin Brasil et al.  analyzedIn all anti-inflammatory tests, the samples analyzed were EPPR. The dry EPPR was dissolved in distilled water at the following concentrations: 200, 400, and 600 μg/mL. For the PC, the extract doses were replaced with dexamethasone (100 μg/mL), and the NC was replaced with saline solution. In the hemolysis stabilization test, the saline solution was replaced with a hyposaline solution (0.18%) to induce hemolysis.2. Cells were grown to 70–80% confluence.Murine macrophages of the Raw 264.7 (ATCC TIB-71) strain were thawed and cultured in a cell culture flask with Dulbecco’s Modified Eagle Medium (DMEM) Ham’s F-12 culture medium at 37 °C under 5% COCells were harvested using a cell scraper, counted in a Neubauer chamber, and centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in culture medium to reach the desired concentration for each experiment.The method described by Azedo et al.  was usedInhibition of phagocytosis (IP) was calculated using the following formula:The method described by Bastos et al.  was usedThe inhibition of spreading was calculated using the following formula:The human red blood cell membrane stabilization (HRBC) test was performed according to the method proposed by Ananthi and Chitra .The test reaction was performed by adding 2 mL of hyposaline solution (0.18%), 1 mL of sodium phosphate buffer , 1 mL of the analyzed samples, and 0.5 mL of HRBC solution. The hemoglobin content in the suspension was estimated using a spectrophotometer at 560 nm.The percentage of protection can hence be calculated from the equation given below:5 cells/well) and stimulated with LPS from Escherichia coli (Sigma-Aldrich) at a concentration of 500 mg/mL. After 3 h, the cells were washed with 1× phosphate-buffered saline and treated with EPPR (600 µg/mL) for 18 h. The supernatant was collected, and the cytokines were measured by enzyme-linked immunosorbent assay (ELISA) using a mouse IL-6 and IL-10 kit (R&D Quantikine ELISA) according to the manufacturer’s instructions.For cytokine determination, bone-marrow-derived macrophages (BMDMs) from C57BL/6 mice were prepared as previously described by the Organization for Economic Co-operation and Development (OECD) . BMDMs wTwelve-week-old male Swiss mice weighing 31–40 g were housed in polypropylene cages . Food (Nuvilab CR-1 kibble) and water were provided ad libitum. The vivarium was maintained at a controlled temperature (23 ± 2 °C) and humidity (55% ± 10%) with an artificial lighting program that corresponded to 50 lx (lights on at 7:00 a.m. and off at 7:00 p.m.). The study was approved by the local Ethics Committee on the Use of Animals of Universidade Estadual Paulista “Júlio Mesquita Filho”, Assis campus, São Paulo, Brazil  and conducted according to the Brazilian Legal Framework for the Scientific Use of Animals.This analysis was performed in accordance with the OECD Guideline No. 423 . Male Sww/w) carbopol and 0.1% (w/w) methylparaben, a preservative, were dissolved in water, and the mixture was allowed to rest for 24 h. Subsequently, the mixture was stirred, and dry EPPR was added at a concentration of 5 mg/g. The pH of the solution was adjusted to 5.5.To prepare the formulation, 1%  before the first carrageenan administration and at 0, 2, 4, and 6 h after administration , PC (sodium hydroxide 0.1 N), base gel (gel without the extract), and EPPR gel 5 mg/g.This test was performed according to the methodology described in the Journal Officiel de la République Française . Four feOn the tenth day of incubation, the treatments were applied to the MCA, and the presence or absence of irritating effects was observed. After visual analysis, a thiopental solution was injected into the fertilized eggs. The graduation of each phenomenon was determined in 5 min and graduated in numerical values  depending on time . Visual The analyzed samples were classified according to the mean value of the sum of the scores of three independent tests (n = 3), and the degree of irritation was divided into four categories: between 0.0 to 0.99, non-irritating (NI); 1.0 to 4.99, mild irritant (MI); 5.0 to 8.99, moderate irritant (MI); and 9.0 to 21, severe irritant (SI) .The formulations with non-encapsulated EPPR were evaluated at a concentration of 5 mg/g. The gel without the extract, the denominated base gel, was used as the negative control. These tests were performed in triplicates.®, New York, NY, USA) was performed at room temperature with a rotational speed of 210× g for 30 min. Subsequently, the formulation was macroscopically analyzed by its appearance.Five grams of each formulation was weighed into centrifuge tubes. The centrifugation test  at temperatures ranging from 40 to 80 °C, with an increase of 10 °C every 30 min up to 80 °C. The formulation was analyzed macroscopically by its appearance after reaching room temperature (25 °C).Tubes containing 5 g of each formulation were subjected to thermal stress in a thermostat water bath .The pH of the formulation was evaluated weekly for 30 days using a calibrated pH meter  was performed according to the experimental protocol, followed by Tukey’s multiple comparison test. For all analyses, a The patent for the EPPR gel was granted by Instituto Nacional da Propriedade Industrial  on 25 November 2020 with the process number BR 10 2020 024093 5.


Popular press coverage of the Medicare Part D coverage gap is based largely on research conducted using retrospective analyses of administrative claims data. These datasets are incomplete because they lack information about methods of obtaining medication that are commonly used by seniors, including free samples, generic drug discount programs, over-the-counter substitution, and patient assistance programs. As a result, evidence about the effects of 100% cost sharing on seniors is limited and suboptimal. Although the current deficit of information about the coverage gap is not entirely unexpected because the Medicare Part D program is relatively new, reliance on claims-based analyses to inform questions that claims data cannot possibly address accurately has tended to mislead and politicize rather than produce constructive policy guidance. Numerous important health policy questions remain unaddressed. These questions are becoming especially important as optimal approaches to providing health care to seniors are the subject of an increasingly vigorous debate.


Titanium (Ti) is now widely used as implant material due to its excellent mechanical properties and superior biocompatibilities, while its inert bioactivities might lead to insufficient osseointegration, and limit its performance in dental applications.Eucommia Ulmoides Polysaccharide (EUP-Sr) complexes are then introduced to the polydopamine-coated porous titanium. Multiple morphological and physiochemical characterizations are employed for material evaluation, while cell proliferation and gene expression tests using macrophages, primary alveolar bone osteoblasts, and vascular endothelial cells are used to provide an overall insight into the functions of the product. The significances of statistical differences were analyzed using student’s t-test.We introduced a robust and simple approach of modifying titanium surfaces with polysaccharide complexes. Titanium samples were subjected to hydrothermal treatment to create a uniform porous structure on the surface, followed by coating with a bioinspired and self-assembly polydopamine layer. Strontium in vivo evaluations.Microscopic and spectrometric characterizations confirmed that the Ti surface formed a porous structure with an adequate amount of EUP-Sr loading. The attachment was attributed to hydrogen bonding between the ubiquitous glycosidic linkage of the polysaccharide complex and the ring structure of polydopamine, yet the loaded EUP-Sr complex can be gradually released, consequently benefiting the neighboring microenvironment. Cell experiments showed no cytotoxicity of the material, and the product showed promising anti-inflammation, osseointegration, and angiogenesis properties, which were further confirmed by We believe the EUP-Sr modified titanium implant is a promising candidate to be used in dental applications with notable osteoimmunomodulation and angiogenesis functions. And the novel technique proposed in this study would benefit the modification of metal/inorganic surfaces with polysaccharides for future research.The online version contains supplementary material available at 10.1186/s40824-023-00361-2. Teeth are often impaired by major traumas, external microbes, and inherited disorders. As a necessary part of teeth replacement, titanium implants have been regarded by a considerably large number of dentists and researchers as a quite mature selection in dentistry with high reliability. But in dental surgery, traumas caused by the implantation procedure often induce inflammation in surrounding tissues. Moreover, recent studies have proven that titanium implants might induce inflammation in surrounding tissues over time, causing local and systemic health issues . The titThe surface modification of titanium focused on improving the biocompatibility and durability of the material where there have been multiple studies investigating the method to create covalent bonds between titanium and the coating material via multiple complicated chemical reactions –7. ExcepEucommia Ulmoides polysaccharides (EUP) is a well-characterized carbohydrate, whose function in regulating the immune system has been revealed [Eucommia Ulmoides polysaccharides (EUP-Sr) were synthesized and investigated in a previous work. The polysaccharide complex demonstrated no cytotoxicity in the cell proliferation test, while significantly inhibited multiple proinflammatory factors in the gene expression of macrophages. It was also proven that EUP-Sr could potentially induce bone regeneration by upregulating osteoblastogenesis gene expression while suppressing osteoclastogenesis [Surface treatment by natural or modified polysaccharides is a promising means to fight against implant-associated problems . Many tyrevealed . Combineogenesis . In thisin vitro biological studies were performed to evaluate the cytocompatibility, cell inflammatory response, angiogenic and osteogenic performances of EUP-Sr modified titanium. In vivo assays confirmed that the EUP-Sr loaded onto the implant could endure surgical manipulations when the surface was rubbed with the alveolar bone, while the polysaccharide complex can actively and consistently benefit the neighboring microenvironment in the recovery process. With this novel and pervasive approach, titanium implants could be loaded with various polysaccharide complexes according to specific clinical needs including anti-inflammation and osteoinduction. It is hopeful that our proposal could potentially spark a way to suppress the inflammatory responses of the patient post dental implantation, as well as reduce the recovery time.In this study, we hypothesize that (i) the PDA coating method suggested in this study, which is feasible for a wide genre of polysaccharide complex, is an economic and facile surface modification method to improve the biophysical and biochemical properties of titanium surface by the coating of polysaccharide complex; and (ii) the introduction of EUP-Sr on titanium surface possesses the capability to significantly increase the anti-inflammation, angiogenic and osteogenic of titanium, leading to better osseointegration effects. Systematic materials characterizations were performed to verify the successful attachment of EUP-Sr on the titanium surface. Various in vivo study were purchased from Shanghai Shuangshen Medical Instrument Co., Ltd. Dopamine (AR) was purchased from Aladdin (China). EUP was bought from Xi’an Haochen Biological Technology Co., Ltd. NaHCO3 (AR) was purchased from Shanghai Lingfeng Chemical Reagents Co., Ltd. NaOH and SrCl2 (AR) were purchased from Aladdin (China). All reactants were used as received unless specified otherwise. Deionized (DI) water (> 18.2 MΩ·cm) was used when water is involved.Titanium (purity > 99.5%) was purchased from Taizhou Zenno Material Technology Co., Ltd. The clinically-available Ti implants (series: HE 1.5 mm) used in the #, 600#, 800#, 1000#, and 2000# waterproof abrasive paper. After polishing by diamond powder paste, the titanium slices were ultrasonically cleaned using ethanol, acetone, and water before surface modification. Supplemental Fig. 1 shows the sketch to modify Ti surface with EUP-Sr. Nano-sized porous structure was introduced onto the Ti surface via hydrothermal alkaline treatment [oC for 2 h. After being cooled to room temperature , the treated Ti slices were washed with extensively NaHCO3 solution (5%) and kept in NaHCO3 solution for at least 12 h to fully neutralize the possible residual alkaline in the pores on the surface. Lastly, to remove the salt residue, the Ti slices were boiled in water for 1 h and rinsed 3 times with water.Ti was cut to slices (1 mm thick), then progressively polished with 240reatment , in whicAn aqueous solution of dopamine was used to attach PDA onto the Ti surface according to a previous study: the dopamine was dissolved in Tris buffer for a concentration of 2 mg/mL pH = 8.5). The treated Ti slices were then immediately immersed in the dopamine solution for 24 h to ensure it reaches the maximum thickness .5. The t was dissScanning electron microscopy  with an accelerating voltage of 10.0 kV was used to observe the surface morphology of different samples , while the energy dispersive spectrometer (EDS) was applied to measure the composition of Sr element on the sample surfaces. For each group, 3 randomly selected locations were detected in EDS measurement. To directly confirm and visualize the EUP-Sr coating, the sample was cut and the cross-sectional surface was observed with SEM. All samples mentioned were sputtered with gold before observation and examination.− 1. By ATR-FTIR analysis, the underlying mechanism for the attachment of the polysaccharide complex would be revealed.Attenuated total reflectance-Fourier transform infrared spectroscopy  was employed to validate the presence of functional groups in EUP-Sr, Ti-PDA, Ti-EUP-Sr-12 H, and Ti-EUP-Sr-24 H in the spectral range of 4000 to 400 cmoC to mimic the in vivo environment. In the time range of 0 to 72 h, 500 µL of the solution was sampled at the interval of 0.5 h upon immersion for two of the samples, followed by adding 500 µL saline back to the release system per sampling. The samples were respectively added with nitric acid (SP) (v/v = 1/4) and kept at 180  oC for 20 min. Then the nitrified sample was diluted to 5 mL with water. The samples were therewith examined using the inductively coupled plasma optical emission spectrometer .The samples Ti-EUP-Sr-12 H and Ti-EUP-Sr-24 H were immersed in 15 mL saline, then incubated and shook at 37 The surface hydrophilicity of the different substrates  was analyzed on a contact angle system . Three samples from each group were selected in the measurement with 5 points on each sample were selected (center and four points near four angles).3) and placed in the culture flask at around 5 mm intervals. After being kept still in the incubator for 1 h, the tissue fragments started to adhere to the flask. The complete culture medium used for HABOBs was HyClone Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% FBS and 2% Penicillin-Streptomycin at primary culture. Before continue to incubate, the complete culture medium was gently and carefully added into the flask to immerse the tissue fragments once the fragments were attached to the flask relatively tight. After 2 weeks of culture, osteoblasts-like cells were observed adjacent to the tissue fragment. Then, the medium was changed every 2–3 days hereafter using HyClone Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% FBS. Once the cells covered more than 75% of the flask, subculture was performed. The primary cells used in this study were within passage 4.HABOBs, the osteoblasts dwelling in the alveolar bone that respond to regulating the formation and regeneration of alveolar bone, are harvested from the disposed tissue of teeth after tooth extraction surgery (orthodontic). The experimental protocol was ethically reviewed by Guangzhou University of Chinese Medicine-Shenzhen Hospital (File # F-GZSY-LL-GZCX-14-2-1). To keep the vitality of the cells, the tissue should be immediately transferred to the primary culture after extraction. A thin layer of pre-warmed fetal bovine serum  was added to a culture flask. The tissue was cut into small fragments  was used to measure the absorbance at 490 nm.The modified Ti samples were prepared according to the size of the 96-well plate in advance. RAW264.7 cells (macrophages) were plated with a concentration of 2000/well onto the samples that were previously sterilized and placed into the 96-well plates, while Human Umbilical Vein Endothelial Cells (HUVECs) and HABOBs were 1000/well. The culture medium for HUVECs and HABOBs was DMEM supplemented with 15% FBS, while the medium for RAW264.7 was DMEM supplemented with 5% FBS. The cells were incubated at 37 oC for 15 min, 85 oC for 5 s, and kept at 4 oC. The resultant cDNA samples were analyzed using SYBR Green Pro Taq HS qPCR Kit  following the instructions provided by the supplier. The targeted genes were normalized against GAPDH  with reference to the cells cultured with no Ti-EUP-Sr samples (control). The target genes of RAW264.7 tested were: interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), and Inducible Nitric Oxide Synthase (iNOS). The target genes of HUVECs were Vascular Endothelial Growth Factor A (VEGFA) and Epidermal Growth Factor Like-domain 6 (EGFL6). And the target gene tested in HABOBs were Alkaline Phosphotase (ALP), osteopontin (OPN), osteocalcin (OCN), and Runx2. The relative expression of each gene was processed and analyzed with the 2− ddCt method [RAW264.7 cells and HUVECs were cultured on the sterilized samples placed in 6-well plates for 24 or 72 h, while HABOBs were cultured for 3 or 7 days before they were cleaved using TRIZOL. Chloroform was later utilized to remove redundant substances other than RNA from water. After being precipitated and rinsed with 75% ethanol (DEPC treated water solution), the RNA was resuspended in DEPC treated water and the concentration was measured on NanoDrop 2000 tester. The total RNA was then reverse-transcribed to complementary DNA (cDNA) using Evo M-MLV RT Premix  at 37 t method .The ELISA assay kits used in this study  were bought from Beyotime Biotechnology. Assays were conducted according to supplier’s instructions came with each kit. Briefly, after culturing RAW264.7, HUVECs, and HABOBs for 1, 3, or 7 days on EUP-Sr modified Ti samples, cell supernatant was obtained and diluted before adding 100 µL samples from each group to a 96 well plate. Standard curves  were obtained with every assay proceeded. After adding biotinylated antibodies, streptavidin labeling solution, and TMB solution stepwisely according to the product instruction (5 times of washing are required before adding each solutions mentioned), the well plates were incubated in dark for 20 min and supplemented with 50µL/well termination solution. A450 values were measured using a microplate reader .t-test. All values involved in the study were denoted as mean ± standard deviation (SD). p < 0.05 was regarded as the appearance of a significant difference in the data.The significant difference in experiment data was analyzed with Student’s in vivo experiment of this study was approved and conducted in Southern University of Science and Technology , where New Zealand rabbits  were purchased from Guangdong Medical Laboratory Animal Center. The animals were restrained and anesthetized using isoflurane with an increasing concentration gradient, followed by hair removal and iodine sterilization at the surgical area (back right tibia). 2% of lidocaine was used at the surgical area to enhance regional anesthesia followed by further iodine sterilization and a ~ 5 cm longitudinal incision until the upper-middle region of the tibia was exposed. Periosteum and deep muscular tissue were identified and carefully dragged towards the side to expose the tibia surface. Pre-cooled (4 oC) saline was used to rinse the implantation region, and a 0.5 mm round bur was used to create a ~ 0.5 mm × 0.5 mm locating region on the cortical bone. Commercialized Ti implants or EUP-Sr modified Ti implants were then inserted through the locating region while screwing. The surgical region was then closed and the animal was recovered. After euthanasia, implants were removed from the tibiae after bone extraction followed by SEM/EDS analysis of the implantation region. The tibiae of the other animals were extracted for micro-CT imaging (150 kV) and reconstructed using Imaris software.The The typical SEM pictures of hydrothermal alkaline treated Ti, Ti-PDA, and Ti-EUP-Sr are shown in Fig. To compare the strontium content on the surface of samples, the EDS tests were carried out on the samples Fig. G. The reTo further confirm the formation of Ti-EUP-Sr on the surface of samples, Fourier transform infrared spectroscopy (ATR-FTIR) was performed. Figure − 1 can be attributed to the -OH stretching vibration. It was evident that the peak area of Ti-EUP-Sr-12 H and -24 H, comparing with Ti-PDA, increased significantly with the addition of EUP-Sr. The weak absorption peak at 2929 cm− 1 resulted from the stretching vibration of the C-H bond [− 1 indicate the symmetric and non-symmetric OCO- stretching vibrations. No absorption peaks were found nearly at 1740 and 1250 cm− 1, confirming the absence of uronic acid and sulfate groups. And the peak at around 1100 cm− 1 corresponds to the ring vibrations overlapping with C-OH side groups and the stretching vibration of the glycosidic linkage (C-O-C) vibration. For the other three groups investigating Ti modification, the characteristic peak of N-H bending and stretching vibration was detected at 1619 cm− 1 in all three groups [− 1 was ascribed to the C = C bonds from the indole structure of PDA, which is also an indicator of successful polymerization from dopamine to PDA [− 1 corresponds to the stretching vibration of catechol hydroxyl C-O and/or C-N [− 1 represented the vibrations of C-O in EUP-Sr [− 1 when EUP-Sr attached to Ti-PDA. This phenomenon can be ascribed to the formation of hydrogen bonding between the glycosidic linkage of EUP-Sr and the ring structure of PDA, which confirmed our initial hypothesis of PDA and the polysaccharide complex can interact through intermolecular forces.The strong and wide absorption peak at 3360 cmC-H bond , 20. Thee groups . The smae to PDA , 22. Thed/or C-N . InteresThe ICP-OES analysis was shown in Fig. As reported in previous studies, hydrophilic surface will facilitate cell adhesion and spreading . Thus, tBased on the former results, the Ti-EUP-Sr-24 H group showed a uniformly porous surface with a higher loading amount of EUP-Sr, which also endows it with a higher hydrophilicity. It could also optimize the neighboring microenvironment by releasing the polysaccharide complex, resulting in a satisfying concentration. Therefore, the 24 h modified Ti-EUP-Sr was selected to be further investigated in the cell experiments hereafter.vitro tests were performed using RAW264.7 cells, HABOBs and HUVECs. Each type of cell was seeded on the Ti-EUP-Sr surfaces with a control group using untreated Ti representing traditional titanium dental implants. After 1, 3 and 5 days of cell culture, the proliferation assay was performed. The results shown in Fig. To investigate cytocompatibility of the immobilized EUP-Sr, in vitro response of macrophages activated by Ti-EUP-Sr, the expression change of inflammation-suppression gene (IL-10) and inflammation-promotion genes (TNF-α and iNOS) between the control group (untreated Ti were used as control in the qPCR study) and Ti-EUP-Sr groups were analyzed by RT-qPCR and the results were demonstrated in Fig. To investigate the in We further tested the osteogenic-related gene expression levels of ALP, OPN, OCN, and Runx2 after culturing HABOBs on Ti-EUP-Sr. As the RT-PCR results suggested Fig. B, the exAngiogenesis is another important factor in evaluating a biomaterial substitute, thus, the gene expressions of HUVECs were investigated focusing on vascular endothelial growth factor A (VEGFA) and EGF-like domain multiple 6 (EGFL6). As shown in Fig. To provide a down-stream evaluation of gene expressions of the mentioned cells in response to EUP-Sr loaded titanium implants, ELISA targeting at vascular endothelial growth factor (VEGF), OCN, and IL-10 were conducted using HUVECs, HABOBs, and RAW264.7 cells, respectively. As shown in Fig. After the implantation surgery, we (1) immediately sacrificed the animal, harvest the surgical region of the tibia, and removed the inserted implants for SEM/EDX tests, and also (2) performed micro-CT on the experimental animal to confirm the osseointegration. Figure Astragalus membranaceus has been well evaluated for pharmacological utilizations including immunomodulatory, angiogenetic, and antitumor functions [2, Al2O3, etc.), and non-metallic materials  [Due to the superior biocompatibility and beneficial bioactivities, polysaccharides and their complexes were intensively investigated for several decades. To the best of our knowledge, it is the first attempt that to introduce polysaccharides on an inert metal surface via biofriendly methods. The presented results suggested that surface coating with bioactive polysaccharides is effective in improving the bioactivity of inert metals, and therefore, further exploring their biomedical applications. Moreover, the use of ATR-FTIR in this research has revealed the underlying mechanism of attaching EUP-Sr onto the PDA film (described in Sect. 3.1.2). As is mentioned, the linkage was via the hydrogen bond formation between the glycosidic linkage of EUP-Sr and the ring structure of PDA, it becomes deducible that our proposed modification approach is valid for other types of carbohydrate polymers (or their modified complexes) with glycosidic linkages. Recently, the field of investigating and applying carbohydrates as medicine has been constantly thriving, with many traditional herbs characterized for their functional content . For example, Astragalus polysaccharide (APS) extracted and purified from unctions –31. The 2, etc.) .in vivo synthesis of massive nitric oxide (NO), which could be highly pro-inflammatory [As is shown in the results of gene expression analysis, IL-10, iNOS, and TNF-α all demonstrated the tendency of suppressing inflammation. It has been previously shown that IL-10 could inhibit the release of inflammatory cytokine and the expression of major histocompatibility complex II, which is the presenter of antigen to T cells . Combiniammatory . The decammatory , 36.The expression of osteogenic genes also demonstrated promising results. OPN has been considered essential in bone formation and was proven to be tightly associated with tooth root development and dental mineralization , 38. OCNin vivo effects of EUP-Sr-loaded titanium implants, we performed implantation surgeries on rabbits and confirmed the stability of the coating. This is considered essential among contemporary research attempting to modify the surfaces of dental or orthopedic implants, because the mechanical rubbing during the surgery might damage and/or remove the coating, making the modification design completely lost. After confirming that the EUP-Sr coating was maintained after the surgery, as well as its satisfying osseointegration effects, we may anticipate that the polysaccharide complex on the titanium surface will be gradually released to affect its surrounding microenvironments. According to the drug release test conducted in this study, titanium dental implant modified with EUP-Sr can gradually unload the polysaccharide, benefiting the neighboring microenvironment. This design accords with many previously reported surface modification agents for Ti aiming to actively optimize the extracellular microenvironment by gradually releasing certain substances [Focusing on the bstances –47. ThisTherefore, we are confident that titanium dental implants modified with EUP-Sr would provide an opportunity to patients with better post-operational performance and a shortened recovery time compared to traditional implants.In this work, we proposed a facile approach for the Ti surface modification and employed it to introduce EUP-Sr to Ti surface with the anti-inflammation and osseointegration properties. The successful modification and the great improvement in surface properties were confirmed by combined techniques of SEM/EDS, FTIR, ICP-OES and contact angle measurements. The cell proliferation tests demonstrate that such surface modification was beneficial to improve the cytocompatibility of titanium dental implants. The gene expression analysis reveals that Ti-EUP-Sr displayed positive effects on inflammation-suppression, osteogenesis and angiogenesis. Thus, we confirmed that the approach of modifying titanium surface with polysaccharide complex was valid and that the modification of EUP-Sr to Ti surface could further optimize current dental implants in all three aspects mentioned above. We therefore believed that the Ti-EUP-Sr implant we fabricated here should be promising materials that be used in dental applications and the PDA coating method proposed should be a facile and useful approach to introduce various bioactive contents to Ti surface.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4Supplementary Material 5Supplementary Material 6